Note: Descriptions are shown in the official language in which they were submitted.
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CAPSID VARIANTS AND METHODS OF USING THE SAME
CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims priority to U.S. Provisional Application No.
63/196.554, filed
June 3. 2021, U.S. Provisional Application No, 63/331,627, filed April 15,
2022, and U.S.
Provisional Application No. 63/342,455, filed May 16, 2022, each of which is
hereby
incorporated by reference in their entirety.
BACKGROUND
Dependoparvoviruses, e.g. adeno-associated dependoparvovirnses, e.g. adeno-
associated
viruses (AAVs), are of interest as vectors for delivering various payloads to
cells, including in
human subjects.
SUMMARY
The present disclosure provides, in part, improved variant dependoparvovirus
capsid
proteins (e.g. AAV2), such as VP1, methods of producing a dependoparvovirus,
compositions
for use in the same, as well as viral particles produced by the same. In some
embodiments, the
viral particles that are produced have increased ocular transduction as
compared to viral particles
without the mutations in the capsid proteins.
In some embodiments, the disclosure is directed, in part, to a nucleic acid
comprising a
sequence encoding a variant capsid protein as provided for herein. In some
embodiments, the
dependoparvovirus is an adeno-associated dependoparvovirus (AAV). In some
embodiments, the
AAV is AAV2.
In some embodiments, the disclosure is directed, in part, to a capsid
polypeptide
described herein.
In some embodiments, the disclosure is directed, in part, to a
dependoparvovirus particle
comprising a nucleic acid described herein.
In some embodiments, the disclosure is directed, in part, to a vector, e.g., a
plasmid,
comprising a nucleic acid described herein.
In some embodiments, the disclosure is directed, in part, to a
dependoparvovirus particle
comprising a nucleic acid described herein (e.g., a nucleic acid comprising a
sequence encoding
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a capsid polypeptide, such as VP1, wherein the encoding sequence comprises a
change or
mutation as provided herein.
In some embodiments, the disclosure is directed, in part, to dependoparvovirus
particle
comprising a variant capsid polypeptide comprising a polypeptide that has at
least 85, 90, 91, 92,
93, 94, 95, 96, 97, 98, 99%, or 100% identity to a VP1, VP2, or VP3 sequence
of SEQ ID NO: 2,
SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID
NO: 8,
SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ
ID
NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO:
20,
SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ
ID
NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO:
31,
SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ
ID
NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41. SEQ ID NO:
42.
SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, or SEQ ID NO: 46.
In some embodiments, the variant capsid polypeptide comprises a mutation
selected from
a mutation associated with any of VAR-1 to VAR-2, and a mutation selected from
a mutation
associated with any of VAR-3 to VAR-16; a mutation selected from a mutation
associated with
any of VAR-1 to VAR-2 and a mutation selected from a mutation associated with
any of VAR-
17 to VAR-45; or a mutation selected from a mutation associated with any of
VAR-3 to VAR-16
and a mutation selected from a mutation associated with any of VAR-17 to VAR-
45.
In some embodiments, the disclosure is directed, in part, to a nucleic acid
molecule
comprising SEQ ID NO: 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60,
61, 62, 63, 64, 65,
66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84,
85, 86, 87, 88, 89, 90. or
91, a fragment thereof, or a variant thereof having at least 85, 90, 91, 92,
93, 94, 95, 96, 97, 98,
99, or 100% sequence identity thereto.
In some embodiments, the disclosure is directed, in part, to a vector
comprising a nucleic
acid described herein, e.g., a nucleic acid comprising a sequence encoding a
capsid polypeptide,
e.g. a VP1 polypeptide, wherein the encoding sequence comprises a change or
mutation as
provided for herein.
In some embodiments, the disclosure is directed, in part, to a cell, cell-free
system, or
other translation system comprising a nucleic acid or vector described herein,
e.g., comprising a
sequence encoding capsid polypeptide, such as VP1, wherein the capsid
polypeptide encoding
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sequence comprises a change or mutation as provided for herein in the encoding
sequence. In
some embodiments, the cell, cell-free system, or other translation system
comprises a
dependoparvovirus particle described herein, e.g., wherein the particle
comprises a nucleic acid
comprising a sequence encoding a capsid polypeptide, such as a VP1
polypeptide, wherein the
encoding sequence comprises a change or mutation as provided for herein.
In some embodiments, the disclosure is directed, in part, to a cell, cell-free
system, or
other translation system comprising a polypeptide described herein, wherein
the polypeptide
encoding sequence comprises a change or mutation as provided for herein.. In
some
embodiments, the cell, cell-free system, or other translation system comprises
a
dependoparvovirus particle described herein, e.g., wherein the particle
comprises a nucleic acid
comprising a sequence encoding a VP1 polypeptide, wherein the VP1 encoding
sequence
comprises a change or mutation corresponding such as provided for herein.
In some embodiments, the disclosure is directed, in part, to a method of
delivering a
payload to a cell comprising contacting the cell with a dependoparvovirus
particle comprising a
nucleic acid described herein. In some embodiments, the disclosure is
directed, in part, to a
method of delivering a payload to a cell comprising contacting the cell with a
dependoparvovirus
particle comprising a capsid polypeptide described herein.
In some embodiments, the disclosure is directed, in part, to a method of
making a
dependoparvovirus particle, comprising providing a cell, cell-free system, or
other translation
system, comprising a nucleic acid described herein (e.g., a nucleic acid
comprising a sequence
encoding an AAV2 capsid variant as provided for herein); and cultivating the
cell, cell-free
system, or other translation system, under conditions suitable for the
production of the
dependoparvovirus particle, thereby making the dependoparvovirus particle. In
some
embodiments, the disclosure is directed, in part, to a method of making a
dependoparvovirus
particle described herein.
In some embodiments, the disclosure is directed, in part, to a method of
making a
dependoparvovirus particle, comprising providing a cell, cell-free system, or
other translation
system, comprising a polypeptide described herein; and cultivating the cell,
cell-free system, or
other translation system, under conditions suitable for the production of the
dependoparvovirus
particle, thereby making the dependoparvovirus particle. In some embodiments,
the disclosure is
directed, in part, to a method of making a dependoparvovirus particle
described herein.
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In some embodiments, the disclosure is directed, in part, to a
dependoparvovirus particle
made in a cell, cell-free system, or other translation system, wherein the
cell, cell-free system, or
other translation system comprises a nucleic acid encoding a dependoparvovirus
comprising an
capsid variant as provided for herein.
In some embodiments, the disclosure is directed, in part, to a method of
treating a disease
or condition in a subject, comprising administering to the subject a
dependoparvovirus particle
described herein in an amount effective to treat the disease or condition.
The invention is further described with reference to the following numbered
embodiments.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG.1. Diagram of tissues collected in each region of the eye. In the retina
(left and center
figures), peripheral and central retina samples from each of the superior,
nasal, inferior and
temporal regions of the retina were separately collected, macula was also
separately collected. In
each region, neural retina and choroid/RPE layers (center figure) were
separately collected. In
the TM/SC region (right figure), superior, temporal, nasal and inferior
samples were separately
collected.
Multisequence alignment of representative reference capsid VP1 polypeptides.
Such
alignment can be used to determine the amino acid positions which correspond
to positions
within different reference capsid polypeptides.
FIG.3. Data from variants included in the medium throughput study and injected
via an
intravitreal (IVT) route of administration. All values 10g2 relative to AAV2
wild-type. Values of
"-10" indicate variant was not measured.
FIG.4. Data from variants included in the medium throughput study and injected
via an
intracameral (IC) route of administration. All values 1og2 relative to AAV2
wild-type. Values of
"-10" indicate variant was not measured.
FIG.5A-5C. Data from bulk tissue processed from the medium throughput study
for variants
injected via the IVT route of administration. All values log2 relative to AAV2
wild-type. Values
of "-10" indicate variant was not measured.
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FIG.6A-6C. Data from bulk tissue processed from the medium throughput study
for variants
injected via the IC route of administration. All values log2 relative to AAV2
wild-type. Values
of "-10" indicate variant was not measured.
FIG.7. Plot of bulk trabecular meshwork transduction (IC admin) vs. retina
transduction (IVT
admin) from Library Experiment 1. Each dot is a unique variant. Variants in
dark black were
selected for inclusion in the medium throughput experiment. All values log2
relative to AAV2
wild-type.
FIG.8. Correlation of neural retina transduction (IVT admin) for variants from
Library
Experiment 1 (x-axis) and medium throughput experiment (y-axis). All values
log2 relative to
AAV2 wild-type.
FIG.9. single nuclear RNA sequencing results for variants from posterior eye
tissue samples
from the medium throughput study, reporting number of unique transduction
events for each
variant. All results are normalized to the amount of each variant in the input
test article. Variants
labeled -VAR-05-n" correspond to "VAR-n" in Table 1, Table 2 and Table 3.
FIG.10A-53. Relative biodistribution and transduction measurements for the
indicated variants.
IVT-MT-BD and IC-MT-BD plot bulk biodistribution rate measurements for the
viral vector
comprising the variant capsid identified in the FIGs.10A (VAR-1), 11A (VAR-2),
12A (VAR-3),
13A (VAR-4), 14A (VAR-5), 17A (VAR-8), 18A (VAR-9), 20A (VAR-11), 21A (VAR-
12),
22A (VAR-13), 23A (VAR-14), 25A (VAR-17), 27A (VAR-19), 28A (VAR-20), 29A (VAR-
21), 30A (VAR-22), 31A (VAR-23), 32A (VAR-24), 34A (VAR-26), 37A (VAR-29), 38A
(VAR-30), 39A (VAR-31), 41A (VAR-33), 42A (VAR-34), 43A (VAR-35), 44A (VAR-
36),
45A (VAR-37), 46A (VAR-38), 47A (VAR-39), 48A (VAR-40), 49A (VAR-41), 50A (VAR-
42), and 51A (VAR-43) from intravitreal injection ("IVT") and intracameral
injection ("IC")
respectively, from each of the ocular tissue samples collected in the medium-
throughput
experiment. IVT-MT-TD and IC-MT-TD plot bulk transduction rate measurements
for the viral
vector comprising the variant capsid identified in the FIGs.10A (VAR-1), 11A
(VAR-2), 12A
(VAR-3), 13A (VAR-4), 14A (VAR-5), 17A (VAR-8), 18A (VAR-9), 20A (VAR-11), 21A
(VAR-12), 22A (VAR-13), 23A (VAR-14), 25A (VAR-17), 27A (VAR-19), 28A (VAR-
20),
29A (VAR-21), 30A (VAR-22), 31A (VAR-23), 32A (VAR-24), 34A (VAR-26), 37A (VAR-
29), 38A (VAR-30), 39A (VAR-31), 41A (VAR-33), 42A (VAR-34), 43A (VAR-35), 44A
(VAR-36), 45A (VAR-37), 46A (VAR-38), 47A (VAR-39), 48A (VAR-40), 49A (VAR-
41),
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50A (VAR-42), and 51A (VAR-43) from intravitreal injection ("IVT") and
intracameral
injection ("IC") respectively, from each of the ocular tissue samples
collected in the medium-
throughput experiment. All values are plotted as log(2) variant rate relative
to wild-type AAV2
rate. Points represent means of barcode replicate rates (n=8). Error bars are
95% confidence
intervals (+/- 1.96 * standard error of the mean ("SEM")). The aggregated bulk
transduction rates
were computed by combining observed counts across all constituent eye regions
of a tissue
(choroid/RPE, neural retina, neural retina non-macula). The choroid/RPE rate
was computed by
combining all choroid/RPE samples collected; The neural retina rate was
computed by
combining all neural retina layer samples collected, including macula. Neural
retina non-macula
was computed by combining all neural retina layer non-macula retina samples.
"Comparison"
panels summarize bulk transduction and biodistribution rate measurements (as
indicated in the
panel) for the viral vector comprising the variant capsid identified in the
FIGs.10B (VAR-1),
11B (VAR-2), 12B (VAR-3), 13B (VAR-4), 14B (VAR-5), 15 (VAR-6), 16 (VAR-7),
17B
(VAR-8), 18B (VAR-9), 19 (VAR-10), 20B (VAR-11), 21B (VAR-12), 22B (VAR-13),
23B
(VAR-14), 24 (VAR-15), 25B (VAR-17). 26 (VAR-18), 27B (VAR-19), 28B (VAR-20),
29B
(VAR-21), 30B (VAR-22), 31B (VAR-23), 32B (VAR-24), 33 (VAR-25), 34B (VAR-26),
35
(VAR-27), 36 (VAR-28), 37B (VAR-29). 38B (VAR-30), 39B (VAR-31), 40 (VAR-32),
41B
(VAR-33), 42B (VAR-34), 43B (VAR-35), 44B (VAR-36), 45B (VAR-37), 46B (VAR-
38), 47B
(VAR-39), 48B (VAR-40), 49B (VAR-41), 50B (VAR-42), 51B (VAR-43), 52 (VAR-44),
and
53 (VAR-45) collected in the medium-throughput (MT) experiment Library
Experiment 1 arid
Library Experiment 2 via either IVT or IC administration, as indicated in the
panel. All values
plotted as log(2) variant rate relative to wild-type AAV2 rate. For Library
Experiment 1, points
represent 1og2-transformed mean of the calculated Bayesian posterior
distribution relative to the
wild-type AAV2 (see Example section). Error bars represent the 95% confidence
intervals using
the posterior distribution (+/- 1.96 * standard deviation ("SD")). For Library
Experiment 2,
points represent means of biological replicate rates (n=4 eyes). Error bars
are 95% confidence
intervals (+/- 1.96 * SEM). For the MT experiment, points represent means of
barcode replicate
rates (n=8). Error bars are 95% confidence intervals (+/- 1.96 * SEM). The
aggregated bulk
transduction rates for a tissue (e.g. choroid/RPE, neural retina, TM/SC) were
computed by
combining observed counts across all collected samples for regions of a
tissue. Absence of a
point indicates the data was not collected.
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ENUMERATED EMBODIMENTS
1. A variant capsid polypeptide comprising a polypeptide that has at least
85, 90, 91, 92, 93,
94, 95, 96, 97, 98, 99%, or 100% identity to a VP1, VP2, or VP3 sequence of
SEQ ID NO: 37,
SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID
NO: 7,
SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 13, SEQ
ID NO:
14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19,
SEQ
ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID
NO:
25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30.
SEQ
ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID
NO:
36, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42,
SEQ
ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, or SEQ ID NO: 46.
2. The variant capsid polypeptide of embodiment 1, wherein the polypeptide
comprises:
a mutation selected from a mutation associated with any of VAR-1 to VAR-2, and
a mutation selected from a mutation associated with any of VAR-3 to VAR-16;
a mutation selected from a mutation associated with any of VAR-1 to VAR-2 and
a mutation selected from a mutation associated with any of VAR-17 to VAR-45;
or
a mutation selected from a mutation associated with any of VAR-3 to VAR-16
and a mutation selected from a mutation associated with any of VAR-17 to VAR-
45.
3. The variant capsid polypeptide of embodiment 2, wherein:
the mutation associated with any of VAR-1 to VAR-2 comprises mutations
corresponding to residues 1-36 as compared to SEQ ID NO: 1;
the mutation associated with any of VAR-3 to VAR-16 comprises mutations
corresponding to residues 431-466 as compared to SEQ ID NO: 1; and
the mutation associated with any of VAR-17 to VAR45 comprises mutations
corresponding to residues 552-617 as compared to SEQ ID NO: 1.
4. The variant capsid polypeptide of any of the preceding embodiments,
wherein the
polypeptide comprises a variant of SEQ ID NO: 1, wherein the variant capsid
polypeptide
comprises a mutation selected from a mutation associated with any of VAR-1 to
VAR-2 and a
mutation associated with any of VAR-3 to VAR-16.
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5. The variant capsid polypeptide of any of the preceding embodiments,
wherein the
polypeptide comprises a variant of SEQ ID NO: 1, wherein the variant capsid
polypeptide
comprises a mutation selected from a mutation associated with any of VAR-1 to
VAR-2 and a
mutation associated with any of VAR-17 to VAR-45.
6. The variant capsid polypeptide of any of the preceding embodiments,
wherein the
polypeptide comprises a variant of SEQ ID NO: 1, wherein the variant capsid
polypeptide
comprises a mutation selected from a mutation associated with any of VAR-3 to
VAR-16 and a
mutation associated with any of VAR-17 to VAR-45.
7. The variant capsid polypeptide of any of the preceding embodiments,
wherein the
polypeptide comprises a variant of SEQ ID NO: 1, wherein the variant capsid
polypeptide
comprises a mutation that corresponds to a mutation at position 3, 6, 11, 12,
14, 15, 19, 21, 23,
24, 25, 29, 31. 33, 446, 449, 450. 451, 452, 454, 455, 456. 457. 458, 459,
460. 461, 464, 552,
554, 555, 556, 557, 558, 559, 561, 566, 575, 578, 580, 581, 585, 586, 587,
588, 589, 590, 591,
593, 594, 597, or 600, an insertion between positions 446 and 447, 447 and
448, 448 and 449,
449 and 450, 451 and 452, 453 and 454, 581 and 582, 583 and 584, 584 and 585,
585 and 586,
586 and 587, 587 and 588, 588 and 589, 589 and 590, 590 and 591, 591 and 592,
or 592 and 593
according to SEQ ID NO: 1, optionally wherein the mutation comprises an
insertion, a deletion
or a substitution.
8. The variant capsid polypeptide of any of the preceding embodiments,
wherein the
polypeptide comprises a variant of SEQ ID NO: 1, wherein the variant capsid
polypeptide
comprises a mutation that corresponds to a mutation at position 3, 6, 11, 12,
14, 15, 19, 21, 23,
24, 25, 29, 31, 33, or any combination thereof according to SEQ ID NO: 1, and
wherein the
mutation comprises a deletion or a substitution.
9. The variant capsid polypeptide of any of the preceding embodiments,
wherein the
polypeptide comprises a variant of SEQ ID NO: 1, wherein the variant capsid
polypeptide
comprises a mutation that corresponds to a mutation at position 446, 448, 449,
450, 451, 452,
453, 454, 455, 456, 457, 458, 459. 460, 461, 464, or any combination thereof,
an insertion
between positions 446 and 447, 447 and 448, 448 and 449, 449 and 450, 451 and
452, 453 and
454, or any combination thereof according to SEQ ID NO: 1, and wherein the
mutation
comprises an insertion, a deletion or a substitution.
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10. The variant capsid polypeptide of any of the preceding embodiments,
wherein the
polypeptide comprises a variant of SEQ ID NO: 1, wherein the variant capsid
polypeptide
comprises a mutation that corresponds to a mutation at position 552, 554, 555,
556, 557, 558,
559, 561, 566, 575, 578, 580, 581, 585, 586, 587, 588, 589, 590, 591, 593,
594, 597, 600 or any
combination thereof, an insertion between positions 583 and 584, 584 and 585,
585 and 586. 586
and 587, 587 and 588, 588 and 589, 589 and 590, 590 and 591, 591 and 592, 592
and 593, or any
combination thereof according to SEQ ID NO: 1, and wherein the mutation
comprises an
insertion, a deletion or a substitution.
11. A variant capsid polypeptide comprising a variant of SEQ ID NO: 1
comprising a
mutation selected from a mutation associated with any of VAR-3 to VAR-16,
wherein the
mutation is:
an insertion, e.g., an insertion of 4 or more amino acids. e.g., 4-5 amino
acids,
e.g., 4-6 amino acids, e.g., 4-7 amino acids. e.g., 7 amino acids, that
corresponds to an insertion
between positions 446 and 454 as compared to SEQ ID NO: 1, and wherein the
insertion
comprises a polypeptide that has at least 60%, 70%, 80%, 90%, or 100% identity
to SEQ ID NO:
93-122;
a substitution, e.g., a substitution of at least 2 or more residues, e.g., at
least 6-10
residues, e.g., at least 7-10 residues, e.g., at least 8-10 residues, e.g., at
least 9-10 residues, e.g.,
at least 10 residues that correspond to a substitution at positions between
445 and 465 as
compared to SEQ ID NO: 1; and
any combination thereof;
and wherein the variant comprises at least 3 mutations selected from:
T455G/L/Q, T456G,
Q457T, S458Q, R459G/T; and further comprises at least 3 other mutations
between positions
445-465 as compared to SEQ ID NO: 1, and wherein the mutations are
substitutions, insertions,
or deletions.
12. A variant capsid polypeptide comprising a variant of SEQ ID NO: 1
comprising a
mutation selected from a mutation associated with any of VAR-17 to VAR-45,
wherein the
mutation is between positions 552 and 588 as compared to SEQ ID NO:1, and
wherein the
mutation comprises:
an insertion, e.g., an insertion of 4 or more amino acids, e.g., 4-5 amino
acids,
e.g., 4-6 amino acids, that corresponds to an insertion between positions 583
and 588 as
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compared to SEQ ID NO: 1, and wherein the insertion comprises a polypeptide
that has at least
60%. 70%, 80%, 90%, or 100% identity to SEQ ID NO: 93-122 or comprises a
fragment of 4 or
more amino acids of SEQ ID NO: 93-122;
a substitution, e.g., a substitution of at least 2 or more residues, e.g., at
least 3-9
residues, e.g., at least 4-9 residues, e.g., at least 5-9 residues, e.g., at
least 6-9 residues, e.g., at
least 7-9 residues, e.g., at least 8-9 residues, e.g., at least 9 residues
that correspond to a
substitution at positions between 552 and 588 as compared to SEQ ID NO: 1; and
any combination thereof.
13. A variant capsid polypeptide comprising a variant of SEQ ID NO: 1
comprising a
mutation selected from a mutation associated with any of VAR-17 to VAR-45,
wherein the
mutation is between positions 552 and 566 as compared to SEQ ID NO: 1, and
wherein the
mutation comprises:
(a) a substitution comprising the following consensus formula:
A-n-L-S/A-D/R/N-L-L-L-n-S-n-n-n-n-K/A (SEQ ID NO: 132)
wherein n is wild type residue as set forth in SEQ ID NO: 1;
(b) a substitution comprising
1, 2, 3, 4, 5. 6, or 7 of the non-naturally occurring amino acids of the
consensus; or
(c) a substitution at any of positions 557, 558 or 559 as compared to SEQ ID
NO: 1, and
wherein the substitution introduces a leucine at any of positions 557, 558.
559, or any
combination thereof.
14. A variant capsid polypeptide comprising a variant of SEQ ID NO: 1
further comprising a
mutation selected from a mutation associated with any of VAR-17 to VAR-45,
wherein the
mutation is between positions 575 and 588 as compared to SEQ ID NO: 1, and
wherein the
mutation comprises:
(a) a substitution comprising the following consensus formula:
E-n-n-A/I/D/V-n-A-A/D-n-n-n-S/del-R/Q-S (SEQ ID NO: 133)
wherein n is wild type residue as set forth in SEQ ID NO: 1 and del is a
deletion;
(b) a substitution comprising
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1-5 non-naturally occurring amino acids of the consensus formula E-n-n-A/I/D/V-
n-A-A/D-n-n-n-S/del-R/Q-S (SEQ ID NO: 133); or
(c) a substitution at any of positions 578, 580 or 581 as compared to SEQ ID
NO: 1, and
wherein the substitution at position 578 is isoleucine, substitution at
position 580 is alanine, and
substitution at position 581 is aspartic acid.
15. A variant capsid polypeptide comprising a variant of SEQ ID NO:
1 further comprising a
mutation selected from a mutation associated with any of VAR-17 to VAR-45,
wherein the
mutation is between positions 552 and 588 as compared to SEQ ID NO: 1, and
wherein
the mutation is a substitution between positions 552 and 566 as compared to
SEQ
ID NO: 1 comprising:
(a) a substitution comprising the following consensus formula:
A-n-L-S/A-D/R/N-L-L-L-n-S-n-n-n-n-K/A (SEQ ID NO: 132)
wherein n is wild type residue as set forth in SEQ ID NO: 1;
(b) a substitution comprising
1, 2. 3, 4, 5. 6, or 7 of the non-naturally occurring amino acids of
the consensus; or
(c) a substitution at any of positions 557, 558 or 559 as compared to SEQ
ID NO: 1, and wherein the substitution introduces a leucine at any of
positions 557. 558, 559, or
any combination thereof; and
the mutation is a substitution between positions 575 and 588 as compared to
SEQ
ID NO: 1 comprising:
(a) a substitution comprising the following consensus formula:
E-n-n-A/I/D/V-n-A-AID-n-n-n-S/del-R/Q-S (SEQ ID NO: 133)
wherein n is wild type residue as set forth in SEQ ID NO: 1 and
del is a deletion;
(b) a substitution comprising
1-5 non-naturally occurring amino acids of the consensus formula
E-n-n-A/I/D/V-n-A-A/D-n-n-n-S/del-R/Q-S (SEQ ID NO: 133); or
(c) a substitution at any of positions 578, 580 or 581 as compared to SEQ
ID NO: 1, and wherein the substitution at position 578 is isoleucine,
substitution at position 580
is alanine, and substitution at position 581 is aspartic acid.
It
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16. A variant capsid polypeptide comprising a variant of SEQ ID NO: 1
comprising a
mutation, wherein the mutation is between positions 584 and 617 as compared to
SEQ ID NO:1,
and wherein the mutation comprises:
an insertion, e.g., an insertion of 1 or more amino acids, e.g., 1-5 amino
acids,
e.g., 5-8 amino acids, e.g., 7-10 amino acids that corresponds to an insertion
between positions
584 and 593 as compared to SEQ ID NO: 1, and wherein the insertion comprises a
polypeptide
that has at least 60%, 70%, 80%, 90%, or 100% identity to SEQ ID NO: 93-122,
or a single
residue selected from: F, I, P, G, or a fragment of at least 4 amino acids of
any of SEQ ID NO:
93-122;
a substitution, e.g., a substitution of at least 1 or more residues, e.g., at
least 1-2
residues, e.g., at least 2-7 residues, e.g., at least 2-8 residues that
correspond to a substitution at
positions between 584 and 617 as compared to SEQ ID NO: 1; and
any combination thereof.
17. The variant capsid polypeptide of embodiment 17, wherein the insertion
comprises 7-10
amino acids and has at least 60%, 70%, 80%, 90%, or 100% identity to
LALGETTRPA (SEQ ID
NO: 93) or comprises a fragment of at least 4, at least 5, at least 6, or at
least 7 amino acids of
LALGETTRPA (SEQ ID NO: 93), e.g., comprises LGETTRP (SEQ ID NO: 94).
18. The variant capsid polypeptide of any of the preceding embodiments,
wherein the capsid
polypeptide comprises a mutation that corresponds to a mutation at position
14, 15, 19, and 24 as
compared to SEQ ID NO: 1.
19. The variant capsid polypeptide of any of the preceding embodiments,
wherein the capsid
polypeptide comprises a mutation that corresponds to a mutation at position 3,
6, 11, 12, 21, 23,
25, 29, 31, and 33 as compared to SEQ ID NO: 1.
20. The variant capsid polypeptide of any of the preceding embodiments,
wherein the capsid
polypeptide comprises a mutation that corresponds to a mutation at position
449, 450, 451, 455,
456, 457, 458, and 459 as compared to SEQ ID NO: 1.
21. The variant capsid polypeptide of any of the preceding embodiments,
wherein the capsid
polypeptide comprises a mutation that corresponds to a mutation at position
449, 450, 452, 456,
457, and 459 as compared to SEQ ID NO: 1.
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22. The variant capsid polypeptide of any of the preceding embodiments,
wherein the capsid
polypeptide comprises a mutation that corresponds to a mutation at position
457 and 458 as
compared to SEQ ID NO: 1.
23. The variant capsid polypeptide of any of the preceding embodiments,
wherein the capsid
polypeptide comprises a mutation that corresponds to a mutation at position
446 as compared to
SEQ ID NO: 1.
24. The variant capsid polypeptide of any of the preceding embodiments,
wherein the capsid
polypeptide comprises a mutation that corresponds to a mutation at position
449 as compared to
SEQ ID NO: 1.
25. The variant capsid polypeptide of any of the preceding embodiments,
wherein the capsid
polypeptide comprises a mutation that corresponds to a mutation at position
451, 455, 456, 457,
458, 459, and 461 as compared to SEQ ID NO: 1.
26. The variant capsid polypeptide of any of the preceding embodiments,
wherein the capsid
polypeptide comprises a mutation that corresponds to a mutation at position
448 as compared to
SEQ ID NO: 1.
27. The variant capsid polypeptide of any of the preceding embodiments,
wherein the capsid
polypeptide comprises a mutation that corresponds to a mutation at position
453 as compared to
SEQ ID NO: 1.
28. The variant capsid polypeptide of any of the preceding embodiments,
wherein the capsid
polypeptide comprises a mutation that corresponds to a mutation at position
456, 457, 458, 459,
460, and 461, as compared to SEQ ID NO: 1.
29. The variant capsid polypeptide of any of the preceding embodiments,
wherein the capsid
polypeptide comprises a mutation that corresponds to a mutation at position
449, 450, 451, 454,
455, 456, 458, 459, 461, and 464 as compared to SEQ ID NO: 1.
30. The variant capsid polypeptide of any of the preceding embodiments,
wherein the capsid
polypeptide comprises a mutation that corresponds to a mutation at position
446, 448, 449, 451,
453, 455, 456. 457, and 459 as compared to SEQ ID NO: 1.
31. The variant capsid polypeptide of any of the preceding embodiments,
wherein the capsid
polypeptide comprises a mutation that corresponds to a mutation at position
447 as compared to
SEQ ID NO: 1.
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32. The variant capsid polypeptide of any of the preceding embodiments,
wherein the capsid
polypeptide comprises a mutation that corresponds to a mutation at position
581 as compared to
SEQ ID NO: 1.
33. The variant capsid polypeptide of any of the preceding embodiments,
wherein the capsid
polypeptide comprises a mutation that corresponds to a mutation at position
584 as compared to
SEQ ID NO: 1.
34. The variant capsid polypeptide of any of the preceding embodiments,
wherein the capsid
polypeptide comprises a mutation that corresponds to a mutation at position
555, 561, 566, 578,
580, 581, and 585 as compared to SEQ ID NO: 1.
35. The variant capsid polypeptide of any of the preceding embodiments,
wherein the capsid
polypeptide comprises a mutation that corresponds to a mutation at position
557 and 578 as
compared to SEQ ID NO: 1.
36. The variant capsid polypeptide of any of the preceding embodiments,
wherein the capsid
polypeptide comprises a mutation that corresponds to a mutation at position
556, 558, 578, and
580 as compared to SEQ ID NO: 1.
37. The variant capsid polypeptide of any of the preceding embodiments,
wherein the capsid
polypeptide comprises a mutation that corresponds to a mutation at position
554, 556, 559, 561,
566, 581, 585, 586, and 587 as compared to SEQ ID NO: 1.
38. The variant capsid polypeptide of any of the preceding embodiments,
wherein the capsid
polypeptide comprises a mutation that corresponds to a mutation at position
552, 555, 556, 559,
561, 566, 578, 581, and 586 as compared to SEQ ID NO: 1.
39. The variant capsid polypeptide of any of the preceding embodiments,
wherein the capsid
polypeptide comprises a mutation that corresponds to a mutation at position
575 and 578 as
compared to SEQ ID NO: 1.
40. The variant capsid polypeptide of any of the preceding embodiments,
wherein the capsid
polypeptide comprises a mutation that corresponds to a mutation at position
583 as compared to
SEQ ID NO: 1.
41. The variant capsid polypeptide of any of the preceding embodiments,
wherein the capsid
polypeptide comprises a mutation that corresponds to a mutation at position
585, 588, 589, 590,
591, 593, 597, and 600 as compared to SEQ ID NO: 1.
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42. The variant capsid polypeptide of any of the preceding embodiments,
wherein the capsid
polypeptide comprises a mutation that corresponds to a mutation at position
592 as compared to
SEQ ID NO: 1.
43. The variant capsid polypeptide of any of the preceding embodiments,
wherein the capsid
polypeptide comprises a mutation that corresponds to a mutation at position
585 as compared to
SEQ ID NO: 1.
44. The variant capsid polypeptide of any of the preceding embodiments,
wherein the capsid
polypeptide comprises a mutation that corresponds to a mutation at position
586 as compared to
SEQ ID NO: 1.
45. The variant capsid polypeptide of any of the preceding embodiments,
wherein the capsid
polypeptide comprises a mutation that corresponds to a mutation at position
589 as compared to
SEQ ID NO: 1.
46. The variant capsid polypeptide of any of the preceding embodiments,
wherein the capsid
polypeptide comprises a mutation that corresponds to a mutation at position
587 as compared to
SEQ ID NO: 1.
47. The variant capsid polypeptide of any of the preceding embodiments,
wherein the capsid
polypeptide comprises a mutation that corresponds to a mutation at position
590 as compared to
SEQ ID NO: 1.
48. The variant capsid polypeptide of any of the preceding embodiments,
wherein the capsid
polypeptide comprises a mutation that corresponds to a mutation at position
585, 586, 587, 591,
594, 597, and 600 as compared to SEQ ID NO: 1.
49. The variant capsid polypeptide of any of the preceding embodiments,
wherein the capsid
polypeptide comprises a mutation that corresponds to a mutation at position
591 as compared to
SEQ ID NO: 1.
50. The variant capsid polypeptide of any of the preceding embodiments,
wherein the capsid
polypeptide comprises a mutation that corresponds to a mutation at position
588 as compared to
SEQ ID NO: 1.
51. The variant capsid polypeptide of any of the preceding embodiments,
wherein the capsid
polypeptide comprises an insertion, e.g., an insertion of 1 or more amino
acids, e.g., 1-10 amino
acids, e.g., 2-8 amino acids, e.g., 3-7 amino acids, e.g., 7 amino acids, that
corresponds to an
insertion between positions 446 and 447, 447 and 448, 448 and 449, 449 and
450, 451, and 452,
Is
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453 and 454, 581 and 582, 583 and 584, 584 and 585, 585 and 586, 586 and 587,
587 and 588,
588 and 589, 589 and 590, 590 and 591, 591 and 592, or 592 and 593, as
compared to SEQ ID
NO: 1.
52. The variant capsid polypeptide of any of the preceding embodiments,
wherein the capsid
polypeptide comprises a mutation that corresponds to a T14L, L15V, 119A. and
1(24H mutation
as compared to SEQ ID NO: 1.
53. The variant capsid polypeptide of any of the preceding embodiments,
wherein the capsid
polypeptide comprises a mutation that corresponds to a A3V, Y6F, Ll1F, E12Q,
Q21L, W23I,
L25C, P29A, P3 IN, and K33R mutation as compared to SEQ ID NO: 1.
54. The variant capsid polypeptide of any of the preceding embodiments,
wherein the capsid
polypeptide comprises a mutation that corresponds to a N449Q, T450M, P451T,
T455L, T456G,
Q457T, S458Q, and R459M mutation as compared to SEQ ID NO: 1.
55. The variant capsid polypeptide of any of the preceding embodiments,
wherein the capsid
polypeptide comprises a mutation that corresponds to a N449Q, residue P451
deletion, S452T,
T456G, Q457T, and R459G mutation as compared to SEQ ID NO: 1.
56. The variant capsid polypeptide of any of the preceding embodiments,
wherein the capsid
polypeptide comprises a mutation that corresponds to a Q457F and S458C
mutation as compared
to SEQ ID NO: 1.
57. The variant capsid polypeptide of any of the preceding embodiments,
wherein the capsid
polypeptide comprises a mutation that corresponds to an insertion between
residues 446 and 447
as compared to SEQ ID NO: I, wherein the insertion comprises a polypeptide of
KCQEGMA
(SEQ ID NO: 95) or a fragment of at least 4, at least 5, or at least 6 amino
acids thereof.
58. The variant capsid polypeptide of any of the preceding embodiments,
wherein the capsid
polypeptide comprises a mutation that corresponds to an insertion between
residues 449 and 450
as compared to SEQ ID NO: 1, wherein the insertion comprises a polypeptide of
LMVDRLG
(SEQ ID NO: 96) or a fragment of at least 4, at least 5, or at least 6 amino
acids thereof.
59. The variant capsid polypeptide of any of the preceding embodiments,
wherein the capsid
polypeptide comprises a mutation that corresponds to a P45 1T, T455G, T456G,
Q457T, 5458Q,
R459T, and Q461A mutation as compared to SEQ ID NO: 1.
60. The variant capsid polypeptide of any of the preceding embodiments,
wherein the capsid
polypeptide comprises a mutation that corresponds to an insertion between
residues 448 and 449
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as compared to SEQ ID NO: 1, wherein the insertion comprises a polypeptide of
HCQECPI
(SEQ ID NO: 97) or a fragment of at least 4, at least 5, or at least 6 amino
acids thereof.
61. The variant capsid polypeptide of any of the preceding embodiments,
wherein the capsid
polypeptide comprises a mutation that corresponds to an insertion between
residues 453 and 454
as compared to SEQ ID NO: 1, wherein the insertion comprises a polypeptide of
FSGLEN (SEQ
ID NO: 98) or a fragment of at least 5 amino acids thereof.
62. The variant capsid polypeptide of any of the preceding embodiments,
wherein the capsid
polypeptide comprises a mutation that corresponds to a residue deletion at
positions T456, Q457,
S458, R459, L460, and Q461 as compared to SEQ ID NO: 1.
63. The variant capsid polypeptide of any of the preceding embodiments,
wherein the capsid
polypeptide comprises a mutation that corresponds to a N449I, T450N, P451G,
residue T454
deletion, T455Q, T456N, S458Q, R459T, Q461K, and Q464V mutation as compared to
SEQ ID
NO: 1.
64. The variant capsid polypeptide of any of the preceding embodiments,
wherein the capsid
polypeptide comprises a mutation that corresponds to a S446A, residue T448 and
N449 deletion,
P451Q, G453T, T455G, T456G, Q457T, and R459G mutation as compared to SEQ ID
NO: 1.
65. The variant capsid polypeptide of any of the preceding embodiments,
wherein the capsid
polypeptide comprises a mutation that corresponds to a N449Q, T450S, S452G,
T455A, Q457F,
S458M, R459D, and an insertion between residues 451 and 452 as compared to SEQ
ID NO: 1,
wherein the insertion comprises an amino acid Y.
66. The variant capsid polypeptide of any of the preceding embodiments,
wherein the capsid
polypeptide comprises a mutation that corresponds to an insertion between
residues 447 and 448
as compared to SEQ ID NO: 1, wherein the insertion comprises a polypeptide of
HETEFNF
(SEQ ID NO: 99) or a fragment of at least 4, at least 5, or at least 6 amino
acids thereof.
67. The variant capsid polypeptide of any of the preceding embodiments,
wherein the capsid
polypeptide comprises a mutation that corresponds to a P45 1T, T455G, T456G,
Q457T, S458Q,
R4591, and Q461A mutation as compared to SEQ ID NO: 1.
68. The variant capsid polypeptide of any of the preceding embodiments,
wherein the capsid
polypeptide comprises a mutation that corresponds to an insertion between
residues 581 and 582
as compared to SEQ ID NO: 1, wherein the insertion comprises a polypeptide of
FALMEP (SEQ
ID NO: 100) or a fragment of at least 4, or at least 5 amino acids thereof.
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69. The variant capsid polypeptide of any of the preceding embodiments,
wherein the capsid
polypeptide comprises a mutation that corresponds to an insertion between
residues 584 and 585
as compared to SEQ ID NO: 1, wherein the insertion comprises a polypeptide of
RAYNPD
(SEQ ID NO: 101) or a fragment of at least 4, or at least 5 amino acids
thereof.
70. The variant capsid polypeptide of any of the preceding embodiments,
wherein the capsid
polypeptide comprises a mutation that corresponds to a residue R585 deletion
and a E555A,
D461S, R566K, S578V, S580A, and T581A mutation as compared to SEQ ID NO: 1.
71. The variant capsid polypeptide of any of the preceding embodiments,
wherein the capsid
polypeptide comprises a mutation that corresponds to a V557L and S578D
mutation as compared
to SEQ ID NO: 1.
72. The variant capsid polypeptide of any of the preceding embodiments,
wherein the capsid
polypeptide comprises a mutation that corresponds to a K556N, M558L. S578I,
and S580A
mutation as compared to SEQ ID NO: 1.
73. The variant capsid polypeptide of any of the preceding embodiments,
wherein the capsid
polypeptide comprises a mutation that corresponds to a I554L, K556R, I559L,
D561S, R566A,
T581D, R585S, G586R, and N587S mutation as compared to SEQ ID NO: 1.
74. The variant capsid polypeptide of any of the preceding embodiments,
wherein the capsid
polypeptide comprises a mutation that corresponds to a V552A, E5555, K556D,
I559L, D5615,
R566K, S578I, T581D. and G586Q mutation as compared to SEQ ID NO: 1.
75. The variant capsid polypeptide of any of the preceding embodiments,
wherein the capsid
polypeptide comprises a mutation that corresponds to a Q575E and S578A
mutation as compared
to SEQ ID NO: 1.
76. The variant capsid polypeptide of any of the preceding embodiments,
wherein the capsid
polypeptide comprises a mutation that corresponds to an insertion between
residues 583 and 584
as compared to SEQ ID NO: 1, wherein the insertion comprises a polypeptide of
LNWTAE
(SEQ ID NO: 102) or a fragment of at least 4, or at least 5 amino acids
thereof.
77. The variant capsid polypeptide of any of the preceding embodiments,
wherein the capsid
polypeptide comprises a mutation that corresponds to a R585S, R588T, Q589N,
A590P, A591I,
A593G, T5975, and V600A mutation as compared to SEQ ID NO: 1.
78. The variant capsid polypeptide of any of the preceding embodiments,
wherein the capsid
polypeptide comprises a mutation that corresponds to a R585N and G586A
mutation, and an
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insertion between residues 584 and 585 as compared to SEQ ID NO: 1, wherein
the insertion
comprises a polypeptide of LAKEFTTR (SEQ ID NO: 103) or a fragment of at least
4, at least 5,
at least 6, or at least 7 amino acids thereof (e.g., a fragment comprising or
consisting of KEFTTR
(SEQ ID NO: 104)).
79. The variant capsid polypeptide of any of the preceding embodiments,
wherein the capsid
polypeptide comprises a mutation that corresponds to an insertion between
residues 592 and 593
as compared to SEQ ID NO: 1, wherein the insertion comprises a polypeptide of
LHPLE (SEQ
ID NO: 105) or a fragment of at least 4 amino acids thereof.
80. The variant capsid polypeptide of any of the preceding embodiments,
wherein the capsid
polypeptide comprises a mutation that corresponds to an insertion between
residues 585 and 586
as compared to SEQ ID NO: 1, wherein the insertion comprises an amino acid F.
81. The variant capsid polypeptide of any of the preceding embodiments,
wherein the capsid
polypeptide comprises a mutation that corresponds to an insertion between
residues 586 and 587
as compared to SEQ ID NO: 1, wherein the insertion comprises a polypeptide of
DQDFKNR
(SEQ ID NO: 106) or a fragment of at least 4, at least 5, or at least 6 amino
acids thereof.
82. The variant capsid polypeptide of any of the preceding embodiments,
wherein the capsid
polypeptide comprises a mutation that corresponds to an insertion between
residues 589 and 590
as compared to SEQ ID NO: 1, wherein the insertion comprises an amino acid I.
83. The variant capsid polypeptide of any of the preceding embodiments,
wherein the capsid
polypeptide comprises a mutation that corresponds to an insertion between
residues 587 and 588
as compared to SEQ ID NO: 1, wherein the insertion comprises a polypeptide of
LAIEQTRPA
(SEQ ID NO: 107) or a fragment of at least 4, at least 5,at least 6, at least
7, or at least 8 amino
acids thereof (e.g., a fragment comprising or consisting of IEQTRPA (SEQ ID
NO: 108)).
84. The variant capsid polypeptide of any of the preceding embodiments,
wherein the capsid
polypeptide comprises a mutation that corresponds to a G586P and N587A
mutation, and an
insertion between residues 584 and 585 as compared to SEQ ID NO: 1, wherein
the insertion
comprises a polypeptide of RARLDETT (SEQ ID NO: 109) or a fragment of at least
4, at least 5,
at least 6, or at least 7 amino acids thereof (e.g., a fragment comprising or
consisting of RLDETT
(SEQ ID NO: 110).
85. The variant capsid polypeptide of any of the preceding embodiments,
wherein the capsid
polypeptide comprises a mutation that corresponds to a N587A mutation, and an
insertion
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between residues 586 and 587 as compared to SEQ ID NO: 1, wherein the
insertion comprises a
polypeptide of LALAEITRP (SEQ ID NO: 111) or a fragment of at least 4, at
least 5, at least 6,
at least 7, or at least 8 amino acids thereof (e.g., a fragment comprising or
consisting of
LAEITRP (SEQ ID NO: 112)).
86. The variant capsid polypeptide of any of the preceding embodiments,
wherein the capsid
polypeptide comprises a mutation that corresponds to a N587A mutation, and an
insertion
between residues 586 and 587 as compared to SEQ ID NO: 1, wherein the
insertion comprises a
polypeptide of LANGEQTRP (SEQ ID NO: 113) or a fragment of at least 4, at
least 5, at least 6,
at least 7, or at least 8 amino acids thereof (e.g., a fragment comprising or
consisting of
NGEQTRP (SEQ ID NO: 114)).
87. The variant capsid polypeptide of any of the preceding embodiments,
wherein the capsid
polypeptide comprises a mutation that corresponds to an insertion between
residues 586 and 587
as compared to SEQ ID NO: 1, wherein the insertion comprises a polypeptide of
ATDTKT (SEQ
ID NO: 115) or a fragment of at least 4, or at least 5 amino acids thereof.
88. The variant capsid polypeptide of any of the preceding embodiments,
wherein the capsid
polypeptide comprises a mutation that corresponds to an insertion between
residues 590 and 591
as compared to SEQ ID NO: 1, wherein the insertion comprises an amino acid P.
89. The variant capsid polypeptide of any of the preceding embodiments,
wherein the capsid
polypeptide comprises a mutation that corresponds to an insertion between
residues 587 and 588
as compared to SEQ ID NO: 1, wherein the insertion comprises a polypeptide of
APGETTRPA
(SEQ ID NO: 116) or a fragment of at least 4, at least 5, at least 6, at least
7, or at least 8 amino
acids thereof.
90. The variant capsid polypeptide of any of the preceding embodiments,
wherein the capsid
polypeptide comprises a mutation that corresponds to an insertion between
residues 589 and 590
as compared to SEQ ID NO: 1, wherein the insertion comprises an amino acid P.
91. The variant capsid polypeptide of any of the preceding embodiments,
wherein the capsid
polypeptide comprises a mutation that corresponds to a R585S, G586S, N587A,
A591E, D594R,
7597A, and V6001 mutation as compared to SEQ ID NO: 1.
92. The variant capsid polypeptide of any of the preceding embodiments,
wherein the capsid
polypeptide comprises a mutation that corresponds to an insertion between
residues 586 and 587
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as compared to SEQ ID NO: 1, wherein the insertion comprises a polypeptide of
FHNEGKY
(SEQ ID NO: 118) or a fragment of at least 4, at least 5, or at least 6 amino
acids thereof.
93. The variant capsid polypeptide of any of the preceding embodiments,
wherein the capsid
polypeptide comprises a mutation that corresponds to an insertion between
residues 591 and 592
as compared to SEQ ID NO: 1, wherein the insertion comprises an amino acid G.
94. The variant capsid polypeptide of any of the preceding embodiments,
wherein the capsid
polypeptide comprises a mutation that corresponds to an insertion between
residues 588 and 589
as compared to SEQ ID NO: 1, wherein the insertion comprises a polypeptide of
QPWEPDK
(SEQ ID NO: 119) or a fragment of at least 4, at least 5, or at least 6 amino
acids thereof.
95. The variant capsid polypeptide of any of the preceding embodiments,
wherein the capsid
polypeptide comprises a mutation that corresponds to an insertion between
residues 592 and 593
as compared to SEQ ID NO: 1, wherein the insertion comprises a polypeptide of
ALALSTTN
(SEQ ID NO: 120) or a fragment of at least 4, at least 5, at least 6, or at
least 7 amino acids
thereof (e.g., a fragment comprising or consisting of ALSTTN (SEQ ID NO:
121)).
96. The variant capsid polypeptide of any of the preceding embodiments,
wherein the capsid
polypeptide comprises a mutation that corresponds to an insertion between
residues 585 and 586
as compared to SEQ ID NO: 1, wherein the insertion comprises a polypeptide of
PWGTAG
(SEQ ID NO: 122) or a fragment of at least 4, or at least 5 amino acids
thereof.
97. A variant capsid polypeptide, comprising (a) a polypeptide of any one
of SEQ ID NO: 2,
SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID
NO: 8,
SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ
ID
NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO:
19,
SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ
ID
NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO:
30,
SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ
ID
NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO:
41,
SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, or SEQ ID NO: 46,
(b)
comprising the VP2 or VP3 sequence of any one of SEQ ID NO: 2, SEQ ID NO: 3,
SEQ ID NO:
4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ
ID NO:
10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15,
SEQ
ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID
NO:
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21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26,
SEQ
ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID
NO:
32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37,
SEQ
ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID
NO:
43, SEQ ID NO: 44, SEQ ID NO: 45, or SEQ ID NO: 46; ; (c) a polypeptide
comprising a
sequence having at least 90%, at least 95%, at least 96%, at least 97%, at
least 98%, or at least
99% identity thereto, wherein said sequence comprises at least one (e.g., one,
two, three or more,
e.g., all) of the mutation differences associated with any of SEQ ID NO: 2
through SEQ ID NO:
46, relative to SEQ ID NO: 1; or (d) a polypeptide having at least 1, but no
more than 20, no
more than 19, no more than 18, no more than 17, no more than 16, no more than
1 5, no more
than 14, no more than 13, no more than 12, no more than 10, no more than 9, no
more than 8, no
more than 7, no more than 6, no more than 5, no more than 3, or no more than 2
amino acid
mutations relative to the polypeptide of (a) or (b), wherein said polypeptide
comprises at least
one (e.g., one, two, three or more, e.g., all) of the mutation differences
associated with any of
SEQ ID NO: 2 through SEQ ID NO: 46, relative to SEQ ID NO: 1.
98. The variant capsid polypeptide of any of the preceding embodiments,
wherein the variant
capsid polypeptide is a VP1 polypeptide, a VP2 polypeptide or a VP3
polypeptide.
99. A variant capsid polypeptide comprising a mutation that corresponds to
a leucine at a
position corresponding to 554 of SEQ ID NO: 1 (e.g., comprising I554L as
compared to SEQ ID
NO: 1).
100. The variant capsid polypeptide of any of the preceding embodiments,
wherein the capsid
polypeptide has up to 12 additional mutations, e.g., between 8 and 12
mutations, as compared to
SEQ ID NO: 1.
101. The variant capsid polypeptide of any of the preceding embodiments,
wherein the capsid
polypeptide has up to 16 additional mutations, e.g., between 11 and 16
mutations, as compared to
VAR-22 (SEQ ID NO: 23).
102. The variant capsid polypeptide of any of the preceding embodiments,
further comprising
a mutation that corresponds to a serine at a position corresponding to 561 of
SEQ ID NO: 1 (e.g.,
comprising D561S as compared to SEQ ID NO: 1).
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103. The variant capsid polypeptide of any of the preceding embodiments,
further comprising
a mutation that corresponds to an aspartic acid at a position corresponding to
581 of SEQ ID NO:
1 (e.g., comprising T581D as compared to SEQ ID NO: 1).
104. A variant capsid polypeptide comprising a mutation that corresponds to a
leucine at a
position corresponding to 559 of SEQ ID NO: 1 (e.g., comprising I559L as
compared to SEQ ID
NO: 1).
105. The variant capsid polypeptide of any of the preceding embodiments,
wherein the capsid
polypeptide has up to 13 additional mutations, e.g., between 4 and 13
mutations, as compared to
SEQ ID NO: 1.
106. The variant capsid polypeptide of any of the preceding embodiments,
wherein the capsid
polypeptide has between 8 and 19 mutations as compared to VAR-22 (SEQ ID NO:
23).
107. The variant capsid polypeptide of any of the preceding embodiments,
wherein the capsid
polypeptide has up to 20 additional mutations, e.g., between 5 and 20
mutations, as compared to
VAR-23 (SEQ ID NO: 24).
108. The variant capsid polypeptide of any of the preceding embodiments,
further comprising
a mutation that corresponds to a serine at a position corresponding to 561 of
SEQ ID NO: 1 (e.g.,
comprising D561S as compared to SEQ ID NO: 1), optionally wherein capsid
polypeptide
further comprises an aspartic acid at a position corresponding to 581 of SEQ
ID NO: 1 (e.g.,
comprising a T581D as compared to SEQ ID NO: 1).
109. A variant capsid polypeptide comprising a mutation that corresponds to
asparagine at a
position corresponding to 556 of SEQ ID NO: 1 (e.g., comprising 1(556N as
compared to SEQ ID
NO: 1).
110. The variant capsid polypeptide of any of the preceding embodiments,
wherein the capsid
polypeptide has up to 12 additional mutations, e.g., between 4 and 12
mutations, as compared to
SEQ ID NO: 1.
111. The variant capsid polypeptide of any of the preceding embodiments,
wherein the capsid
polypeptide has up to 12 additional mutations, e.g., between 9 and 12
mutations, as compared to
VAR-21 (SEQ ID NO: 22).
112. A variant capsid polypeptide comprising a mutation that corresponds to a
serine at a
position corresponding to 561 of SEQ ID NO: 1 (e.g., comprising D561S as
compared to SEQ
ID NO: 1).
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113. The variant capsid polypeptide of any of the preceding embodiments,
wherein the capsid
polypeptide has up to 12 additional mutations, e.g., between 7 and 12
mutations, as compared to
SEQ ID NO: 1.
114. The variant capsid polypeptide of any of the preceding embodiments,
wherein the capsid
polypeptide has up to 14 additional mutations, e.g., between 11 and 14
mutations, as compared to
VAR-19 (SEQ ID NO: 20).
115. The variant capsid polypeptide of any of the preceding embodiments,
wherein the capsid
polypeptide has up to 14 additional mutations, e.g., between 10 and 14
mutations, as compared to
VAR-22 (SEQ ID NO: 23).
116. The variant capsid polypeptide of any of the preceding embodiments,
wherein the capsid
polypeptide has up to 14 additional mutations, e.g., between 9 and 14
mutations, as compared to
VAR-23 (SEQ ID NO: 24).
117. The variant capsid polypeptide of any of the preceding embodiments,
further comprising
a mutation that corresponds to an aspartic acid at a position corresponding to
581 of SEQ ID
NO:1 (e.g., comprising T581D as compared to SEQ ID NO: 1).
118. A capsid polypeptide comprising a mutation at a position corresponding to
587 of SEQ ID
NO: 1, optionally wherein the mutation is to an alanine (e.g., wherein the
mutation is N587A
according to SEQ ID NO: 1), optionally wherein the capsid polypeptide further
comprises an
insertion (e.g., an insertion of 4 or more amino acids, optionally an
insertion of 7 or more amino
acids, optionally an insertion of 7-10 amino acids, optionally an insertion of
7, 8 or 9 amino
acids) between two adjacent amino acids between 580 and 587 (optionally
between 586 and 587)
according to SEQ ID NO: 1.
119. A nucleic acid molecule comprising sequence encoding a variant capsid
polypeptide of
any one of embodiments 1-118.
120. The nucleic acid molecule of embodiment 119, comprising one or more
regulatory
elements operably linked to the sequence encoding the variant capsid
polypeptide.
121. The nucleic acid molecule of any of embodiments 119-120. comprising SEQ
ID NO: 47,
48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66,
67, 68, 69, 70, 71, 72, 73,
74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, or a
fragment thereof, or a
variant thereof having at least 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or
100% sequence
identity thereto.
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122. A virus particle (e.g., adeno-associated virus ("AAV") particle)
comprising the variant
capsid polypeptide of any one of embodiments 1-118 or comprising a variant
capsid polypeptide
encoded by the nucleic acid molecule of any one of embodiments 119-121.
123. The virus particle of embodiment 122, comprising a nucleic acid
comprising a
heterologous transgene and one or more regulatory elements.
124. A virus particle of any of embodiments 122-123 comprising the variant
capsid
polypeptide of any one of embodiments 1-118, wherein said virus particle, or a
virus particle
comprising said variant capsid polypeptide or a virus particle comprising a
variant capsid
polypeptide encoded by a nucleic acid molecule of any one of embodiments 119-
121 exhibits
increased ocular transduction, e.g., as measured in a mouse or in NHP, e.g.,
as described herein,
relative to wild-type AAV2 (e.g., a virus particle comprising capsid
polypeptides of SEQ ID NO:
1 or encoded by SEQ ID NO: 92).
125. The nucleic acid molecule of any one of embodiments 119-121, wherein the
nucleic acid
molecule is double-stranded or single-stranded, optionally wherein the nucleic
acid molecule is
linear or circular, e.g., wherein the nucleic acid molecule is a plasmid.
126. A method of producing a virus particle comprising a variant AAV2 capsid
polypeptide,
said method comprising introducing a nucleic acid molecule of any one of
embodiments 119-121
or 125 into a cell (e.g., a HEK293 cell), and harvesting said virus particle
therefrom.
127. A method of delivering a payload (e.g., a nucleic acid) to a cell
comprising contacting the
cell with a dependoparvovirus particle comprising a variant capsid polypeptide
of any one of
embodiments 1-118 or the virus particle of any of embodiments 123-124 and a
payload.
128. The method of embodiment 127, wherein the cell is an ocular cell.
129. The method of embodiment 128, wherein the ocular cell is in the retina,
the macula, or
the trabecular meshwork.
130. A method of delivering a payload (e.g., a nucleic acid) to a subject
comprising
administering to the subject a dependoparvovirus particle comprising a variant
capsid
polypeptide of any one of embodiments 1-118 and the payload, or administering
to the subject
the virus particle of any one of embodiments 122-124.
131. The method of embodiment 130, wherein the particle delivers the payload
to the eye.
132. The method of embodiment 131, wherein the particle delivers the payload
to the retina,
the macular, or the trabecular meshwork.
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133. The method of any one of embodiments 130-132, wherein the particle
delivers the
payload to the eye with increased transduction in one or more regions of the
eye as compared to
a virus particle comprising capsid polypeptides of SEQ ID NO: 1.
134. The method of embodiment 133, wherein the one or more regions of the eye
is selected
from the retina, the macula, the trabecular meshwork, or any combination
thereof.
135. The method of embodiment 133, wherein the retina comprises non-macular
retina.
136. The variant capsid polypeptide of any of embodiments 1-118, the virus
particle of any of
embodiments 122-124 or the method of any one of embodiments 130-135, wherein
the particle
(e.g., particle comprising the variant capsid polypeptide) delivers the
payload to the eye with
increased transduction in one or more regions of the eye as compared to a
virus particle
comprising capsid polypeptides of SEQ ID NO: 1, and wherein the increase in
transduction is at
least 2-times, 4-times, 8-times. 16-times, 32-times, 64-times, 100-times. or
150-times as
compared to a virus particle comprising capsid polypeptides of SEQ ID NO: 1.
137. The variant capsid polypeptide of any of embodiments 1-118, the virus
particle of any of
embodiments 122-124 or the method of any one of embodiments 130-136, wherein
the particle
(e.g., particle comprising the variant capsid polypeptide) delivers the
payload to the eye with
increased transduction specificity in one or more regions of the eye as
compared to a virus
particle comprising capsid polypeptides of SEQ ID NO: 1, wherein the increase
in transduction
is at least 2-times. 4-times, 8-times, 16-times, 32-times, 64-times, 100-
times, 200-times, 500-
times, or 1000-times as compared to a virus particle comprising capsid
polypeptides of SEQ ID
NO: 1, and wherein the increase in transduction is specific to non-macular
retina tissue relative
to macular tissue.
138. The variant capsid polypeptide of any of embodiments 1-118, the virus
particle of any of
embodiments 122-124 or the method of any one of embodiments 130-137, wherein
the particle
(e.g., particle comprising the variant capsid polypeptide) delivers the
payload to the eye with
increased transduction specificity in one or more regions of the eye as
compared to a virus
particle comprising cap sid polypeptides of SEQ ID NO: 1, wherein the increase
in transduction
is at least 2-times. 4-times, 8-times, 16-times, 32-times, 64-times, 100-
times, 200-times, 500-
times, or 1000-times as compared to a virus particle comprising capsid
polypeptides of SEQ ID
NO: 1, and wherein the increase in transduction is specific to macular tissue
relative to non-
macular retina tissue.
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139. The variant capsid polypeptide of any of embodiments 1-118, the virus
particle of any of
embodiments 122-124 or the method of any one of embodiments 130-138, wherein
the particle
(e.g., particle comprising the variant capsid polypeptide) delivers the
payload to the eye with
increased transduction specificity in one or more regions of the eye as
compared to a virus
particle comprising capsid polypeptides of SEQ ID NO: 1, wherein the increase
in transduction
is at least 2-times. 4-times, 8-times, 16-times, 32-times, 64-times, 100-
times, 200-times, 500-
times, or 1000-times as compared to a virus particle comprising capsid
polypeptides of SEQ ID
NO: 1, and wherein the increase in transduction is specific to macular tissue
relative to trabecular
meshwork tissue.
140. The variant capsid polypeptide of any of embodiments 1-118, the virus
particle of any of
embodiments 122-124 or the method of any one of embodiments 130-139, wherein
the particle
(e.g., particle comprising the variant capsid polypeptide) delivers the
payload to the eye with
increased transduction specificity in one or more regions of the eye as
compared to a virus
particle comprising capsid polypeptides of SEQ ID NO: 1, wherein the increase
in transduction
is at least 2-times. 4-times, 8-times, 16-times, 32-times, 64-times, 100-
times, 200-times, 500-
times, or 1000-times as compared to a virus particle comprising capsid
polypeptides of SEQ ID
NO: 1, and wherein the increase in transduction is specific to non-macular
retina tissue relative
to trabecular meshwork tissue.
141. The variant capsid polypeptide of any of embodiments 1-118, the virus
particle of any of
embodiments 122-124 or the method of any one of embodiments 130-140, wherein
the (e.g.,
particle comprising the variant capsid polypeptide) particle delivers the
payload to the eye with
increased transduction specificity in one or more regions of the eye as
compared to a virus
particle comprising capsid polypeptides of SEQ ID NO: 1, wherein the increase
in transduction
is at least 2-times. 4-times, 8-times, 16-times, 32-times, 64-times, 100-
times, 200-times, 500-
times, or 1000-times as compared to a virus particle comprising capsid
polypeptides of SEQ ID
NO: 1, and wherein the increase in transduction is specific to macular tissue
and non-macular
retina tissue relative to trabecular meshwork tissue.
142. The variant capsid polypeptide of any of embodiments 1-118, the virus
particle of any of
embodiments 122-124 or the method of any one of embodiments 130-141, wherein
the particle
(e.g., particle comprising the variant capsid polypeptide) delivers the
payload to the eye with
increased transduction specificity in one or more regions of the eye as
compared to a virus
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particle comprising capsid polypeptides of SEQ ID NO: 1, wherein the increase
in transduction
is at least 2-times, 4-times, 8-times, 16-times, 32-times, 64-times, 100-
times, 200-times, 500-
times, or 1000-times as compared to a virus particle comprising capsid
polypeptides of SEQ ID
NO: 1, and wherein the increase in transduction is specific to trabecular
meshwork tissue relative
to macular tissue and non-macular retina tissue.
143. The variant capsid polypeptide of any of embodiments 1-118, the virus
particle of any of
embodiments 122-124 or the method of any one of embodiments 130-142, wherein
the particle
(e.g., particle comprising the variant capsid polypeptide) delivers the
payload to the eye with
increased transduction specificity in one or more regions of the eye as
compared to a virus
particle comprising capsid polypeptides of SEQ ID NO: 1, wherein the increase
in transduction
is at least 2-times, 4-times, 8-times, 16-times, 32-times, 64-times, 100-
times, 200-times, 500-
times, or 1000-times as compared to a virus particle comprising capsid
polypeptides of SEQ ID
NO: 1, and wherein the increase in transduction is specific to trabecular
meshwork tissue relative
to macular tissue.
144. The variant capsid polypeptide of any of embodiments 1-118, the virus
particle of any of
embodiments 122-124 or the method of any one of embodiments 130-143, wherein
the particle
(e.g., particle comprising the variant capsid polypeptide) delivers the
payload to the eye with
increased transduction specificity in one or more regions of the eye as
compared to a virus
particle comprising capsid polypeptides of SEQ ID NO: 1, wherein the increase
in transduction
is at least 2-times, 4-times, 8-times, 16-times, 32-times, 64-times, 100-
times, 200-times, 500-
times, or 1000-times as compared to a virus particle comprising capsid
polypeptides of SEQ ID
NO: 1, and wherein the increase in transduction is specific to trabecular
meshwork tissue relative
to non-macular retina tissue.
145. The variant capsid polypeptide of any of embodiments 1-118, the virus
particle of any of
embodiments 122-124 or the method of any one of embodiments 130-144, wherein
the particle
(e.g., particle comprising the variant capsid polypeptide) delivers the
payload to the eye with
increased transduction specificity in one or more regions of the eye as
compared to a virus
particle comprising capsid polypeptides of SEQ ID NO: 1, wherein the increase
in transduction
is at least 2-times, 4-times, 8-times, 16-times, 32-times, 64-times, 100-
times, 200-times, 500-
times, or 1000-times as compared to a virus particle comprising capsid
polypeptides of SEQ ID
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NO: 1, and wherein the increase in transduction is specific to trabecular
meshwork tissue,
macular tissue, and non-macular retina tissue.
146. The variant capsid polypeptide of any of embodiments 1-118, the virus
particle of any of
embodiments 122-124 or the method of any one of embodiments 130-145, wherein
the particle
(e.g., particle comprising the variant capsid polypeptide) delivers the
payload to the eye with
increased transduction specificity in one or more regions of the eye as
compared to a virus
particle comprising capsid polypeptides of SEQ ID NO: 1 without increased
biodistribution in
one or more regions of the eye as compared to a virus particle comprising
capsid polypeptides of
SEQ ID NO: 1.
147. The method of any one of embodiments 130-146, wherein the administration
to the
subject is via an intravitreal injection, or an intracameral injection.
148. A method of treating a disease or condition in a subject, comprising
administering to the
subject a dependoparvovirus particle in an amount effective to treat the
disease or condition,
wherein the dependoparvovirus particle is a particle comprising a capsid
polypeptide of any one
of embodiments 1-118 and 136-146, or encoded by the nucleic acid of any one of
embodiments
119-121 or 125, or is a virus particle of any one of embodiments 122-124.
149. A cell, cell-free system, or other translation system, comprising the
capsid polypeptide,
nucleic acid molecule, or virus particle of any one of embodiments 1-125 or
136-146.
150. A method of making a dependoparvovirus (e.g., an adeno-associated
dependoparvovirus
(AAV) particle, comprising:
providing a cell, cell-free system, or other translation system, comprising a
nucleic acid
of any of embodiments 119-121 or 125; and
cultivating the cell, cell-free system, or other translation system, under
conditions suitable
for the production of the dependoparvovirus particle,
thereby making the dependoparvovirus particle.
151. The method of embodiment 150, wherein the cell, cell-free system, or
other translation
system comprises a second nucleic acid molecule and said second nucleic acid
molecule is
packaged in the dependoparvovirus particle.
152. The method of embodiment 150, wherein the second nucleic acid comprises a
payload,
e.g., a heterologous nucleic acid sequence encoding a therapeutic product.
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153. The method of any one of embodiments 150-152, wherein the nucleic acid of
any of
embodiments 119-121 or 125 mediates the production of a dependoparvovirus
particle which
does not include said nucleic acid of any of embodiments 119-121 or 125.
154. The method of any one of embodiments 150-153, wherein the nucleic acid of
any of
embodiments 119-121 or 125 mediates the production of a dependoparvovirus
particle at a level
at least 10%, at least 20%, at least 50%, at least 100%õ at least 200% or
greater than the
production level mediated by the nucleic acid of SEQ ID NO: 92.
155. A composition, e.g., a pharmaceutical composition, comprising a virus
particle of any one
of embodiments 122-124 or a virus particle produced by the method of any one
of embodiments
126 or 150-154, and a pharmaceutically acceptable carrier.
156. The variant capsid polypeptide of any of embodiments 1-118 and 136-146,
the nucleic acid
molecule of any of embodiments 119-121 or 125, or the virus particle of any of
embodiments
122-124 and 136-146 for use in treating a disease or condition in a subject.
157. The variant capsid polypeptide of any of embodiments 1-118 and 136-146,
the nucleic acid
molecule of any of claims 119-121 or 125, or the virus particle of any of
claims 122-124 and
136-146 for use in the manufacture of a medicament for use in treating a
disease or condition in a
subject.
DETAILED DESCRIPTION
The present disclosure is directed, in part, to the variant capsid variants
that can be used
to generate dependoparvovirus particles. In some embodimenis, the particles
have increased
ocular transduction that can be used to deliver a transgene or molecule of
interest to an eye with
higher transduction efficiency in the eye as compared to a dependoparvovirus
particle without
the variant capsid polypeptides. Accordingly, provided herein are variant
capsid polypeptides,
nucleic acid molecules encoding the same, viral particles comprising the
variant capsid
polypeptides, and methods of using the same.
Definitions
A, An, The: As used herein, the singular forms "a," "an" and "the" include
plural
referents unless the context clearly dictates otherwise.
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About, Approximately: As used herein, the terms "about" and "approximately"
shall
generally mean an acceptable degree of error for the quantity measured given
the nature or
precision of the measurements. Exemplary degrees of error are within 15
percent (%), typically,
within 10%, and more typically, within 5% of a given value or range of values.
Dependoparvovirus capsid: As used herein, the term "dependoparvovirus capsid"
refers
to an assembled viral capsid comprising dependoparvovirus polypeptides. In
some embodiments,
a dependoparvovirus capsid is a functional dependoparvovirus capsid, e.g., is
fully folded and/or
assembled, is competent to infect a target cell, or remains stable (e.g.,
folded/assembled and/or
competent to infect a target cell) for at least a threshold time.
Dependoparvovirus particle: As used herein, the term "dependoparvovirus
particle"
refers to an assembled viral capsid comprising dependoparvovirus polypeptides
and a packaged
nucleic acid. e.g., comprising a payload, one or more components of a
dependoparvovirus
genome (e.g., a whole dependoparvovirus genome), or both. In some embodiments,
a
dependoparvovirus particle is a functional dependoparvovirus particle, e.g.,
comprises a desired
payload, is fully folded and/or assembled, is competent to infect a target
cell, or remains stable
(e.g., folded/assembled and/or competent to infect a target cell) for at least
a threshold time.
Dependoparvovirus X particle/capsid: As used herein, the term
"dependoparvovirus X
particle/capsid" refers to a dependoparvovirus particle/capsid comprising at
least one polypeptide
or polypeptide encoding nucleic acid sequence derived from a naturally
occurring
dependoparvovirus X species. For example, a dependoparvovirus B particle
refers to a
dependoparvovirus particle comprising at least one polypeptide or polypeptide
encoding nucleic
acid sequence derived from a naturally occurring dependoparvovirus B sequence.
Derived from,
as used in this context, means having at least 80, 85, 90, 91, 92, 93, 94, 95,
96, 97, 98, 99, or
100% identity to the sequence in question. Correspondingly, an AAVX
particle/capsid, as used
herein, refers to an AAV particle/caspid comprising at least one polypeptide
or polypeptide
encoding nucleic acid sequence derived from a naturally occurring AAV X
serotype. For
example, an AAV2 particle refers to an AAV particle comprising at least one
polypeptide or
polypeptide encoding nucleic acid sequence derived from a naturally occurring
AAV2 sequence.
Exogenous: As used herein, the term "exogenous" refers to a feature, sequence,
or
component present in a circumstance (e.g., in a nucleic acid, polypeptide, or
cell) that does not
naturally occur in said circumstance. For example, a nucleic acid sequence
comprising a mutant
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capsid polypeptide or a nucleic acid molecule encoding the same may comprise
an capsid
polypeptide. Use of the term exogenous in this fashion means that the
polypeptide or the nucleic
acid molecule encoding a polypeptide comprising the mutation in question at
this position does
not occur naturally, e.g., is not present in AAV2, e.g., is not present in SEQ
ID NO: 1.
Functional: As used herein in reference to a polypeptide component of a
dependoparvovirus capsid (e.g., Cap (e.g., VP1, VP2, and/or VP3) or Rep), the
term "functional"
refers to a polypeptide which provides at least 50, 60, 70, 80, 90, or 100% of
the activity of a
naturally occurring version of that polypeptide component (e.g., when present
in a host cell). For
example, a functional VP1 polypeptide may stably fold and assemble into a
dependoparvovirus
capsid (e.g., that is competent for packaging and/or secretion). As used
herein in reference to a
dependoparvovirus capsid or particle, "functional" refers to a capsid or
particle comprising one
or more of the following production characteristics: comprises a desired
payload, is fully folded
and/or assembled, is competent to infect a target cell, or remains stable
(e.g., folded/assembled
and/or competent to infect a target cell) for at least a threshold time.
Nucleic acid: As used herein, in its broadest sense, the term "nucleic acid"
refers to any
compound and/or substance that is or can be incorporated into an
oligonucleotide chain. In some
embodiments, a nucleic acid is a compound and/or substance that is or can be
incorporated into
an oligonucleotide chain via a phosphodiester linkage. As will be clear from
context, in some
embodiments, "nucleic acid" refers to an individual nucleic acid monomer
(e.g., a nucleotide
and/or nucleoside); in some embodiments, "nucleic acid" refers to an
oligonucleotide chain
comprising individual nucleic acid monomers or a longer polynucleotide chain
comprising many
individual nucleic acid monomers. In some embodiments, a "nucleic acid" is or
comprises RNA;
in some embodiments, a "nucleic acid" is or comprises DNA. In some
embodiments, a nucleic
acid is, comprises, or consists of one or more natural nucleic acid residues.
In some
embodiments, a nucleic acid is, comprises, or consists of one or more nucleic
acid analogs. In
some embodiments, a nucleic acid is, comprises, or consists of one or more
modified, synthetic,
or non-naturally occurring nucleotides. In some embodiments, a nucleic acid
analog differs from
a nucleic acid in that it does not utilize a phosphodiester backbone. For
example, in some
embodiments, a nucleic acid is, comprises, or consists of one or more "peptide
nucleic acids",
which are known in the art and have peptide bonds instead of phosphodiester
bonds in the
backbone, are considered within the scope of the present invention.
Alternatively or
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additionally, in some embodiments, a nucleic acid has one or more
phosphorothioate and/or 5'-
N-phosphoramidite linkages rather than phosphodiester bonds. In some
embodiments, a nucleic
acid has a nucleotide sequence that encodes a functional gene product such as
an RNA or
protein. In some embodiments, a nucleic acid is partly or wholly single
stranded; in some
embodiments, a nucleic acid is partly or wholly double stranded.
Variant: As used herein, a "variant capsid polypeptide" refers to a
polypeptide that
differs from a reference sequence (e.g. SEQ ID NO: 1). The variant can, for
example, comprise
a mutation (e.g. substitution, deletion, or insertion). In some embodiments,
the variant is about,
or at least, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%., 97%,
98%. or
99% identical to the reference sequence. In some embodiments, the reference
sequence is a
polypeptide comprising SEQ ID NO: 1.
Capsid Polypeptides and Nucleic Acids Encoding the Same
The disclosure is directed, in part, to a nucleic acid comprising a sequence
encoding an a
variant capsid polypeptide comprising a mutation (insertion, deletion, or
substitution) as
compared to the wild-type sequence. In some embodiments, the wild-type
sequence is SEQ ID
NO: 1. The disclosure is directed, in part, to a variant capsid polypeptides
comprising SEQ ID
NO: 1 with one or more mutations as compared to SEQ ID NO: 1. The mutation can
be, for
example, an insertion, deletion, or substitution as compared to the wild-type
sequence. In some
embodiments, the wild-type sequence is SEQ ID NO: 1.
In some embodiments, the nucleic acid molecule encodes a capsid polypeptide
that
comprises a mutation selected from Table 2, Table 3, or Table 4. In some
embodiments, the
nucleic acid molecule encodes a capsid polypeptide that comprises a mutation
selected from
Table 2 and Table 3; Table 2 and Table 4. or Table 3 and Table 4. In some
embodiments, the
nucleic acid molecule encodes a capsid polypeptide that comprises a mutation
selected from
Table 2 and Table 3. In some embodiments, the nucleic acid molecule encodes a
capsid
polypeptide that comprises a mutation selected from Table 2 and Table 4. In
some embodiments,
the nucleic acid molecule encodes a capsid polypeptide that comprises a
mutation selected from
Table 3 and Table 4. In some embodiments, Table 2 comprises mutations
corresponding to
residues 1-36 as compared to SEQ ID NO: 1. In some embodiments, Table 3
comprises
mutations corresponding to residues 431-466 as compared to SEQ ID NO: 1. In
some
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embodiments, Table 4 comprises mutations corresponding to residues 552-617 as
compared to
SEQ ID NO: 1. In some embodiments, the nucleic acid molecule encodes a capsid
polypeptide
that comprises a mutation within the 1-36 amino acid region of SEQ ID NO: 1.
In some
embodiments, the nucleic acid molecule encodes a capsid polypeptide that
comprises a mutation
within the 431-466 amino acid region of SEQ ID NO: 1. In some embodiments, the
nucleic acid
molecule encodes a capsid polypeptide that comprises a mutation within the 552-
617 amino acid
region of SEQ ID NO: 1. In some embodiments, the nucleic acid molecule encodes
a capsid
polypeptide that comprises a mutation within the 1-36, 431-466, and 552-617
amino acid region
of SEQ ID NO: 1. In some embodiments, the nucleic acid molecule encodes a
capsid
polypeptide that comprises a mutation selected from Table 3. In some
embodiments, the
mutation selected from Table 3 is an insertion, e.g., an insertion of 4 or
more amino acids, e.g..
4-5 amino acids, e.g., 4-6 amino acids, e.g.. 4-7 amino acids, e.g., 7 amino
acids, that
corresponds to an insertion between positions 446 and 454 as compared to SEQ
ID NO: 1, and
wherein the insertion comprises a polypeptide that has at least 60%, 70%, 80%,
90%, or 100%
identity to SEQ ID NO: 93-122; a substitution, e.g., a substitution of at
least 2 or more residues,
e.g., at least 6-10 residues, e.g., at least 7-10 residues, e.g., at least 8-
10 residues, e.g., at least 9-
residues, e.g., at least 10 residues that correspond to a substitution at
positions between 445
and 465 as compared to SEQ ID NO: 1; and the variant comprises at least 3
mutations selected
from: T455G/L/Q, T456G, Q457T, S458Q. R459G/T; and further comprises at least
3 other
mutations between positions 445-465 as compared to SEQ ID NO: 1, and wherein
the mutations
are substitutions, insertions, or deletions. In some embodiments, the nucleic
acid molecule
encodes a capsid polypeptide that comprises a mutation selected from Table 4.
In some
embodiments, the mutation selected from Table 4 is an insertion, e.g., an
insertion of 4 or more
amino acids, e.g., 4-5 amino acids, e.g., 4-6 amino acids, that corresponds to
an insertion
between positions 583 and 588 as compared to SEQ ID NO: 1, and wherein the
insertion
comprises a polypeptide that has at least 60%, 70%, 80%, 90%, or 100% identity
to SEQ ID NO:
93-122; and a substitution, e.g., a substitution of at least 2 or more
residues, e.g., at least 3-9
residues, e.g., at least 4-9 residues, e.g., at least 5-9 residues, e.g., at
least 6-9 residues, e.g., at
least 7-9 residues, e.g., at least 8-9 residues, e.g., at least 9 residues
that correspond to a
substitution at positions between 552 and 588 as compared to SEQ ID NO: 1. In
some
embodiments, the mutation selected from Table 4 is a mutation between
positions 552 and 566 as
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compared to SEQ ID NO: 1, and the mutation is a substitution comprising the
following
consensus formula:
A-n-L-S/A-D/R/N-L-L-L-n-S-n-n-n-n-K/A (SEQ ID NO: 132)
wherein n is wild type residue as set forth in SEQ ID NO: 1; or the mutation
comprises a
substitution comprising at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 of the non-
wild type amino acids of
the consensus formula A-n-L-S/A-D/R/N-L-L-L-n-S-n-n-n-n-KJA (SEQ ID NO: 132);
or the
variant comprises a substitution at any of positions 557, 558 or 559 as
compared to SEQ ID NO:
1, and wherein the substitution introduces a leucine at any of positions 557,
558, 559, or any
combination thereof.
In some embodiments, the mutation selected from Table 4 is a mutation between
positions 575
and 588 as compared to SEQ ID NO: 1, and the mutation is a substitution
comprising the
following consensus formula:
E-n-n-A/I/D/V-n-A-A/D-n-n-n-S/del-R/Q-S (SEQ ID NO: 133)
wherein n is wild type residue as set forth in SEQ ID NO: 1 ("der is a
deletion); or the mutation
comprises a substitution comprising at least 1-7 of the non-wild-type amino
acids of the
consensus formula E-n-n-A/I/D/V-n-A-AID-n-n-n-S/del-R/Q-S (SEQ ID NO: 133); or
the
variant comprises a substitution at position 578, 580 or 581 as compared to
SEQ ID NO: 1, and
wherein the substitution at position 578 is isoleucine, substitution at
position 580 is alanine, and
substitution at position 581 is aspartic acid.
In some embodiments, the mutation selected from Table 4 is a mutation between
positions 552
and 588 as compared to SEQ ID NO: 1, and the mutation is a substitution
between positions 552
and 566 comprising the following consensus formula:
A-n-L-S/A-D/R/N-L-L-L-n-S-n-n-n-n-K/A (SEQ lD NO: 132)
wherein n is wild type residue as set forth in SEQ ID NO: 1; 1, 2, 3, 4, 5, 6,
or 7 amino acids of
the consensus ; the variant further comprises a substitution at any of
positions 557, 558 or 559 as
compared to SEQ ID NO: 1, and wherein the substitution introduces a leucine at
any of positions
557, 558, 559, or any combination thereof; and the mutation is a substitution
between positions
575 and 588 comprising the following consensus formula:
E-n-n-A/I/D/V-n-A-AID-n-n-n-S/del-R/Q-S (SEQ ID NO: 133)
wherein n is wild type residue as set forth in SEQ ID NO: 1; 1-6 amino acids
of the consensus;
del is a deletion; the variant further comprises a substitution at position
578, 580 or 581 as
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compared to SEQ ID NO: 1, and wherein the substitution at position 578 is
isoleucine,
substitution at position 580 is alanine, and substitution at position 581 is
aspartic acid.
In some embodiments, the mutation selected from Table 4 is a mutation between
positions 584
and 617 as compared to SEQ ID NO:1, and wherein the mutation comprises:
an insertion, e.g., an insertion of 1 or more amino acids, e.g., 1-5 amino
acids,
e.g., 5-8 amino acids, e.g., 7-10 amino acids that corresponds to an insertion
between positions
584 and 593 as compared to SEQ ID NO: 1, and wherein the insertion comprises a
polypeptide
that has at least 60%, 70%, 80%, 90%, or 100% identity to SEQ ID NO: 93-122,
or a single
residue selected from: F, I, P, G:
a substitution, e.g., a substitution of at least 1 or more residues, e.g., at
least 1-2
residues, e.g., at least 2-7 residues, e.g., at least 2-8 residues that
correspond to a substitution at
positions between 584 and 617 as compared to SEQ ID NO: 1; and any combination
thereof.
In some embodiments, the insertion comprises 7-10 amino acids and has at least
60%, 70%,
80%, 90%, or 100% identity to LALGETTRPA (SEQ ID NO: 93).
In some embodiments, the nucleic acid molecule encodes a capsid polypeptide
that
comprises a mutation that corresponds to a mutation at position 3, 6, 11, 12,
14, 15, 19, 21, 23,
24, 25, 29, 31, 33, 446, 449, 450, 451, 452, 454, 455, 456, 457, 458, 459,
460, 461, 464, 552,
554, 555, 556, 557, 558, 559, 561, 566, 575, 578, 580, 581, 585, 586, 587,
588, 589, 590, 591,
593, 594, 597, 600, or any combination thereof, an insertion between positions
446 and 447, 447
and 448, 448 and 449, 449 and 450, 451, and 452, 453 and 454, 581 and 582, 583
and 584, 584
and 585, 585 and 586, 586 and 587, 587 and 588, 588 and 589, 589 and 590, 590
and 591, 591
and 592, 592 and 593, or any combination thereof according to SEQ ID NO: 1,
optionally
wherein the mutation comprises an insertion, a deletion or a substitution.
In some embodiments, the nucleic acid molecule encodes a capsid polypeptide
that
comprises a mutation that corresponds to a mutation at position 3 as compared
to SEQ ID NO: 1.
In some embodiments, the nucleic acid molecule encodes a capsid polypeptide
that comprises a
mutation that corresponds to a mutation at position 6 as compared to SEQ ID
NO: 1. In some
embodiments, the nucleic acid molecule encodes a capsid polypeptide that
comprises a mutation
that corresponds to a mutation at position 11 as compared to SEQ ID NO: 1. In
some
embodiments, the nucleic acid molecule encodes a capsid polypeptide that
comprises a mutation
that corresponds to a mutation at position 12 as compared to SEQ ID NO: 1. In
some
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embodiments, the nucleic acid molecule encodes a capsid polypeptide that
comprises a mutation
that corresponds to a mutation at position 14 as compared to SEQ ID NO: 1. In
some
embodiments, the nucleic acid molecule encodes a capsid polypeptide that
comprises a mutation
that corresponds to a mutation at position 15 as compared to SEQ ID NO: 1. In
some
embodiments, the nucleic acid molecule encodes a capsid polypeptide that
comprises a mutation
that corresponds to a mutation at position 19 as compared to SEQ ID NO: 1. In
some
embodiments, the nucleic acid molecule encodes a capsid polypeptide that
comprises a mutation
that corresponds to a mutation at position 21 as compared to SEQ ID NO: 1. In
some
embodiments, the nucleic acid molecule encodes a capsid polypeptide that
comprises a mutation
that corresponds to a mutation at position 23 as compared to SEQ ID NO: 1. In
some
embodiments, the nucleic acid molecule encodes a capsid polypeptide that
comprises a mutation
that corresponds to a mutation at position 24 as compared to SEQ ID NO: 1. In
some
embodiments, the nucleic acid molecule encodes a capsid polypeptide that
comprises a mutation
that corresponds to a mutation at position 25 as compared to SEQ ID NO: 1. In
some
embodiments, the nucleic acid molecule encodes a capsid polypeptide that
comprises a mutation
that corresponds to a mutation at position 29 as compared to SEQ ID NO: 1. In
some
embodiments, the nucleic acid molecule encodes a capsid polypeptide that
comprises a mutation
that corresponds to a mutation at position 31 as compared to SEQ ID NO: 1. In
some
embodiments, the nucleic acid molecule encodes a capsid polypeptide that
comprises a mutation
that corresponds to a mutation at position 33 as compared to SEQ ID NO: 1. In
some
embodiments, the nucleic acid molecule encodes a capsid polypeptide that
comprises a mutation
that corresponds to a mutation at position 446 as compared to SEQ ID NO: 1. In
some
embodiments, the nucleic acid molecule encodes a capsid polypeptide that
comprises a mutation
that corresponds to a mutation at position 449 as compared to SEQ ID NO: 1. In
some
embodiments, the nucleic acid molecule encodes a capsid polypeptide that
comprises a mutation
that corresponds to a mutation at position 450 as compared to SEQ ID NO: 1. In
some
embodiments, the nucleic acid molecule encodes a capsid polypeptide that
comprises a mutation
that corresponds to a mutation at position 451 as compared to SEQ ID NO: 1. In
some
embodiments, the nucleic acid molecule encodes a capsid polypeptide that
comprises a mutation
that corresponds to a mutation at position 452 as compared to SEQ ID NO: 1. In
some
embodiments, the nucleic acid molecule encodes a capsid polypeptide that
comprises a mutation
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that corresponds to a mutation at position 454 as compared to SEQ ID NO: 1. In
some
embodiments, the nucleic acid molecule encodes a capsid polypeptide that
comprises a mutation
that corresponds to a mutation at position 455 as compared to SEQ ID NO: 1. In
some
embodiments, the nucleic acid molecule encodes a capsid polypeptide that
comprises a mutation
that corresponds to a mutation at position 456 as compared to SEQ ID NO: 1. In
some
embodiments, the nucleic acid molecule encodes a capsid polypeptide that
comprises a mutation
that corresponds to a mutation at position 457 as compared to SEQ ID NO: 1. In
some
embodiments, the nucleic acid molecule encodes a capsid polypeptide that
comprises a mutation
that corresponds to a mutation at position 458 as compared to SEQ ID NO: 1. In
some
embodiments, the nucleic acid molecule encodes a capsid polypeptide that
comprises a mutation
that corresponds to a mutation at position 459 as compared to SEQ ID NO: 1. In
some
embodiments, the nucleic acid molecule encodes a capsid polypeptide that
comprises a mutation
that corresponds to a mutation at position 460 as compared to SEQ ID NO: 1. In
some
embodiments, the nucleic acid molecule encodes a capsid polypeptide that
comprises a mutation
that corresponds to a mutation at position 461 as compared to SEQ ID NO: 1. In
some
embodiments, the nucleic acid molecule encodes a capsid polypeptide that
comprises a mutation
that corresponds to a mutation at position 464 as compared to SEQ ID NO: 1. In
some
embodiments, the nucleic acid molecule encodes a capsid polypeptide that
comprises a mutation
that corresponds to a mutation at position 552 as compared to SEQ ID NO: 1. In
some
embodiments, the nucleic acid molecule encodes a capsid polypeptide that
comprises a mutation
that corresponds to a mutation at position 554 as compared to SEQ ID NO: 1. In
some
embodiments, the nucleic acid molecule encodes a capsid polypeptide that
comprises a mutation
that corresponds to a mutation at position 555 as compared to SEQ ID NO: 1. In
some
embodiments, the nucleic acid molecule encodes a capsid polypeptide that
comprises a mutation
that corresponds to a mutation at position 556 as compared to SEQ ID NO: 1. In
some
embodiments, the nucleic acid molecule encodes a capsid polypeptide that
comprises a mutation
that corresponds to a mutation at position 557 as compared to SEQ ID NO: 1. In
some
embodiments, the nucleic acid molecule encodes a capsid polypeptide that
comprises a mutation
that corresponds to a mutation at position 558 as compared to SEQ ID NO: 1. In
some
embodiments, the nucleic acid molecule encodes a capsid polypeptide that
comprises a mutation
that corresponds to a mutation at position 559 as compared to SEQ ID NO: 1. In
some
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embodiments, the nucleic acid molecule encodes a capsid polypeptide that
comprises a mutation
that corresponds to a mutation at position 561 as compared to SEQ ID NO: 1. In
some
embodiments, the nucleic acid molecule encodes a capsid polypeptide that
comprises a mutation
that corresponds to a mutation at position 566 as compared to SEQ ID NO: 1. In
some
embodiments, the nucleic acid molecule encodes a capsid polypeptide that
comprises a mutation
that corresponds to a mutation at position 575 as compared to SEQ ID NO: 1. In
some
embodiments, the nucleic acid molecule encodes a capsid polypeptide that
comprises a mutation
that corresponds to a mutation at position 578 as compared to SEQ ID NO: 1. In
some
embodiments, the nucleic acid molecule encodes a capsid polypeptide that
comprises a mutation
that corresponds to a mutation at position 580 as compared to SEQ ID NO: 1. In
some
embodiments, the nucleic acid molecule encodes a capsid polypeptide that
comprises a mutation
that corresponds to a mutation at position 581 as compared to SEQ ID NO: 1. In
some
embodiments, the nucleic acid molecule encodes a capsid polypeptide that
comprises a mutation
that corresponds to a mutation at position 585 as compared to SEQ ID NO: 1. In
some
embodiments, the nucleic acid molecule encodes a capsid polypeptide that
comprises a mutation
that corresponds to a mutation at position 586 as compared to SEQ ID NO: 1. In
some
embodiments, the nucleic acid molecule encodes a capsid polypeptide that
comprises a mutation
that corresponds to a mutation at position 587 as compared to SEQ ID NO: 1. In
some
embodiments, the nucleic acid molecule encodes a capsid polypeptide that
comprises a mutation
that corresponds to a mutation at position 588 as compared to SEQ ID NO: 1. In
some
embodiments, the nucleic acid molecule encodes a capsid polypeptide that
comprises a mutation
that corresponds to a mutation at position 589 as compared to SEQ ID NO: 1. In
some
embodiments, the nucleic acid molecule encodes a capsid polypeptide that
comprises a mutation
that corresponds to a mutation at position 590 as compared to SEQ ID NO: 1. In
some
embodiments, the nucleic acid molecule encodes a capsid polypeptide that
comprises a mutation
that corresponds to a mutation at position 591 as compared to SEQ ID NO: 1. In
some
embodiments, the nucleic acid molecule encodes a capsid polypeptide that
comprises a mutation
that corresponds to a mutation at position 593 as compared to SEQ ID NO: 1. In
some
embodiments, the nucleic acid molecule encodes a capsid polypeptide that
comprises a mutation
that corresponds to a mutation at position 594 as compared to SEQ ID NO: 1. In
some
embodiments, the nucleic acid molecule encodes a capsid polypeptide that
comprises a mutation
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that corresponds to a mutation at position 597 as compared to SEQ ID NO: 1. In
some
embodiments, the nucleic acid molecule encodes a capsid polypeptide that
comprises a mutation
that corresponds to a mutation at position 600 as compared to SEQ ID NO: 1.
In some embodiments, the nucleic acid molecule encodes a capsid polypeptide
that
comprises a mutation that corresponds to an insertion at position between
positions 446 and 447
as compared to SEQ ID NO: 1. In some embodiments, the nucleic acid molecule
encodes a
capsid polypeptide that comprises a mutation that corresponds to an insertion
at position between
positions 447 and 448 as compared to SEQ ID NO: 1. In some embodiments, the
nucleic acid
molecule encodes a capsid polypeptide that comprises a mutation that
corresponds to an insertion
at position between positions 448 and 449 as compared to SEQ ID NO: 1. In some
embodiments, the nucleic acid molecule encodes a capsid polypeptide that
comprises a mutation
that corresponds to an insertion at position between positions 449 and 450 as
compared to SEQ
ID NO: 1. In some embodiments, the nucleic acid molecule encodes a capsid
polypeptide that
comprises a mutation that corresponds to an insertion at position between
positions 451 and 452
as compared to SEQ ID NO: 1. In some embodiments, the nucleic acid molecule
encodes a
capsid polypeptide that comprises a mutation that corresponds to an insertion
at position between
positions 453 and 454 as compared to SEQ ID NO: 1. In some embodiments, the
nucleic acid
molecule encodes a capsid polypeptide that comprises a mutation that
corresponds to an insertion
at position between positions 581 and 582 as compared to SEQ ID NO: 1. In some
embodiments, the nucleic acid molecule encodes a capsid polypeptide that
comprises a mutation
that corresponds to an insertion at position between positions 583 and 584 as
compared to SEQ
ID NO: 1. In some embodiments, the nucleic acid molecule encodes a capsid
polypeptide that
comprises a mutation that corresponds to an insertion at position between
positions 584 and 585
as compared to SEQ ID NO: 1. In some embodiments, the nucleic acid molecule
encodes a
capsid polypeptide that comprises a mutation that corresponds to an insertion
at position between
positions 585 and 586 as compared to SEQ ID NO: 1. In some embodiments, the
nucleic acid
molecule encodes a capsid polypeptide that comprises a mutation that
corresponds to an insertion
at position between positions 586 and 587 as compared to SEQ ID NO: 1. In some
embodiments,
the nucleic acid molecule encodes a capsid polypeptide that comprises a
mutation that
corresponds to an insertion at position between positions 587 and 588 as
compared to SEQ ID
NO: 1. In some embodiments, the nucleic acid molecule encodes a capsid
polypeptide that
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comprises a mutation that corresponds to an insertion at position between
positions 588 and 589
as compared to SEQ ID NO: 1. In some embodiments, the nucleic acid molecule
encodes a
capsid polypeptide that comprises a mutation that corresponds to an insertion
at position between
positions 589 and 590 as compared to SEQ ID NO: 1. In some embodiments, the
nucleic acid
molecule encodes a capsid polypeptide that comprises a mutation that
corresponds to an insertion
at position between positions 590 and 591 as compared to SEQ ID NO: 1. In some
embodiments, the nucleic acid molecule encodes a capsid polypeptide that
comprises a mutation
that corresponds to an insertion at position between positions 591 and 592 as
compared to SEQ
ID NO: 1. In some embodiments, the nucleic acid molecule encodes a capsid
polypeptide that
comprises a mutation that corresponds to an insertion at position between
positions 592 and 593
as compared to SEQ ID NO: 1.
In some embodiments, the nucleic acid molecule encodes a capsid polypeptide
that
comprises a mutation that corresponds to a mutation at position 3, 6, 11, 12,
14, 15, 19, 21, 23,
24, 25, 29, 31, 33, or any combination thereof according to SEQ ID NO: 1, and
wherein the
mutation comprises a deletion or a substitution.
In some embodiments, the nucleic acid molecule encodes a capsid polypeptide
that
comprises a mutation that corresponds to a mutation at position 446, 448, 449,
450, 451, 452,
453, 454, 455, 456, 457, 458, 459, 460, 461, 464, or any combination thereof,
an insertion
between positions 446 and 447, 447 and 448, 448 and 449, 449 and 450, 451 and
452, 453 and
454, or any combination thereof according to SEQ ID NO: 1, and wherein the
mutation
comprises an insertion, a deletion or a substitution.
In some embodiments, the nucleic acid molecule encodes a capsid polypeptide
that
comprises a mutation that corresponds to a mutation at position 552, 554, 555,
556, 557, 558,
559, 561, 566, 575, 578, 580, 581, 585, 586, 587, 588, 589, 590, 591, 593,
594, 597, 600 or any
combination thereof, an insertion between positions 583 and 584, 584 and 585,
585 and 586. 586
and 587, 587 and 588, 588 and 589, 589 and 590, 590 and 591, 591 and 592, 592
and 593, or any
combination thereof according to SEQ ID NO: 1, and wherein the mutation
comprises an
insertion, a deletion or a substitution.
In some embodiments, the nucleic acid molecule encodes a capsid polypeptide
that
comprises a mutation that corresponds to a mutation at position 14, 15, 19,
and 24 as compared
to SEQ ID NO: 1.
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In some embodiments, the nucleic acid molecule encodes a capsid polypeptide
that
comprises a mutation that corresponds to a mutation at position 3, 6, 11, 12,
21, 23, 25, 29, 31,
and 33 as compared to SEQ ID NO: 1.
In some embodiments, the nucleic acid molecule encodes a capsid polypeptide
that
comprises a mutation that corresponds to a mutation at position 449, 450, 451,
455, 456, 457,
458, and 459 as compared to SEQ ID NO: 1.
In some embodiments, the nucleic acid molecule encodes a capsid polypeptide
that
comprises a mutation that corresponds to a mutation at position 449, 450, 452,
456, 457, and 459
as compared to SEQ ID NO: 1.
In some embodiments, the nucleic acid molecule encodes a capsid polypeptide
that
comprises a mutation that corresponds to a mutation at position 457 and 458 as
compared to SEQ
ID NO: 1.
In some embodiments, the nucleic acid molecule encodes a capsid polypeptide
that
comprises a mutation that corresponds to a mutation at position 446 as
compared to SEQ ID NO:
1.
In some embodiments, the nucleic acid molecule encodes a capsid polypeptide
that
comprises a mutation that corresponds to a mutation at position 449 as
compared to SEQ ID NO:
1.
In some embodiments, the nucleic acid molecule encodes a capsid polypeptide
that
comprises a mutation that corresponds to a mutation at position 451, 455, 456,
457, 458, 459,
and 461 as compared to SEQ ID NO: 1.
In some embodiments, the nucleic acid molecule encodes a capsid polypeptide
that
comprises a mutation that corresponds to a mutation at position 448 as
compared to SEQ ID NO:
1.
In some embodiments, the nucleic acid molecule encodes a capsid polypeptide
that
comprises a mutation that corresponds to a mutation at position 453 as
compared to SEQ ID NO:
1.
In some embodiments, the nucleic acid molecule encodes a capsid polypeptide
that
comprises a mutation that corresponds to a mutation at position 456, 457, 458,
459, 460, and
461, as compared to SEQ ID NO: 1.
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In some embodiments, the nucleic acid molecule encodes a capsid polypeptide
that
comprises a mutation that corresponds to a mutation at position 449, 450, 451,
454, 455, 456,
458, 459, 461, and 464 as compared to SEQ ID NO: 1.
In some embodiments, the nucleic acid molecule encodes a capsid polypeptide
that
comprises a mutation that corresponds to a mutation at position 446, 448, 449,
451, 453, 455,
456, 457, and 459 as compared to SEQ ID NO: 1.
In some embodiments, the nucleic acid molecule encodes a capsid polypeptide
that
comprises a mutation that corresponds to a mutation at position 447 as
compared to SEQ ID NO:
1.
In some embodiments, the nucleic acid molecule encodes a capsid polypeptide
that
comprises a mutation that corresponds to a mutation at position 581 as
compared to SEQ ID NO:
1.
In some embodiments, the nucleic acid molecule encodes a capsid polypeptide
that
comprises a mutation that corresponds to a mutation at position 584 as
compared to SEQ ID NO:
1.
In some embodiments, the nucleic acid molecule encodes a capsid polypeptide
that
comprises a mutation that corresponds to a mutation at position 555, 561, 566,
578, 580, 581,
and 585 as compared to SEQ ID NO: 1.
In some embodiments, the nucleic acid molecule encodes a capsid polypeptide
that
comprises a mutation that corresponds to a mutation at position 557 and 578 as
compared to SEQ
ID NO: 1.
In some embodiments, the nucleic acid molecule encodes a capsid polypeptide
that
comprises a mutation that corresponds to a mutation at position 556, 558, 578,
and 580 as
compared to SEQ ID NO: 1.
In some embodiments, the nucleic acid molecule encodes a capsid polypeptide
that
comprises a mutation that corresponds to a mutation at position 554, 556, 559,
561, 566, 581,
585, 586, and 587 as compared to SEQ ID NO: 1.
In some embodiments, the nucleic acid molecule encodes a capsid polypeptide
that
comprises a mutation that corresponds to a mutation at position 552, 555, 556,
559, 561, 566,
578, 581, and 586 as compared to SEQ ID NO: 1.
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In some embodiments, the nucleic acid molecule encodes a capsid polypeptide
that
comprises a mutation that corresponds to a mutation at position 575 and 578 as
compared to SEQ
ID NO: 1.
In some embodiments, the nucleic acid molecule encodes a capsid polypeptide
that
comprises a mutation that corresponds to a mutation at position 583 as
compared to SEQ ID NO:
1.
In some embodiments, the nucleic acid molecule encodes a capsid polypeptide
that
comprises a mutation that corresponds to a mutation at position 585, 588, 589,
590, 591, 593,
597, and 600 as compared to SEQ ID NO: 1.
In some embodiments, the nucleic acid molecule encodes a capsid polypeptide
that
comprises a mutation that corresponds to a mutation at position 592 as
compared to SEQ ID NO:
1.
In some embodiments, the nucleic acid molecule encodes a capsid polypeptide
that
comprises a mutation that corresponds to a mutation at position 585 as
compared to SEQ ID NO:
1.
In some embodiments, the nucleic acid molecule encodes a capsid polypeptide
that
comprises a mutation that corresponds to a mutation at position 586 as
compared to SEQ ID NO:
1.
In some embodiments, the nucleic acid molecule encodes a capsid polypeptide
that
comprises a mutation that corresponds to a mutation at position 589 as
compared to SEQ ID NO:
1.
In some embodiments, the nucleic acid molecule encodes a capsid polypeptide
that
comprises a mutation that corresponds to a mutation at position 587 as
compared to SEQ ID NO:
1.
In some embodiments, the nucleic acid molecule encodes a capsid polypeptide
that
comprises a mutation that corresponds to a mutation at position 590 as
compared to SEQ ID NO:
1.
In some embodiments, the nucleic acid molecule encodes a capsid polypeptide
that
comprises a mutation that corresponds to a mutation at position 585, 586, 587,
591, 594, 597,
and 600 as compared to SEQ ID NO: 1.
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In some embodiments, the nucleic acid molecule encodes a capsid polypeptide
that
comprises a mutation that corresponds to a mutation at position 591 as
compared to SEQ ID NO:
1.
In some embodiments, the nucleic acid molecule encodes a capsid polypeptide
that
comprises a mutation that corresponds to a mutation at position 588 as
compared to SEQ ID NO:
1.
In some embodiments, the nucleic acid molecule encodes a capsid polypeptide
that
comprises a mutation that comprises an insertion, e.g., an insertion of 1 or
more amino acids,
e.g., 1-10 amino acids, e.g., 2-8 amino acids, e.g., 3-7 amino acids, e.g., 7
amino acids, that
corresponds to an insertion between positions 446 and 447, 447 and 448, 448
and 449, 449 and
450, 451, and 452, 453 and 454, 581 and 582, 583 and 584, 584 and 585, 585 and
586, 586 and
587, 587 and 588. 588 and 589, 589 and 590, 590 and 591, 591 and 592, or 592
and 593, as
compared to SEQ ID NO: 1.
In some embodiments, the nucleic acid molecule encodes a capsid polypeptide
that
comprises a mutation that comprises an insertion between positions 446 and 447
as compared to
SEQ ID NO: 1.
In some embodiments, the nucleic acid molecule encodes a capsid polypeptide
that
comprises a mutation that comprises an insertion between positions 447 and 448
as compared to
SEQ ID NO: 1.
In some embodiments, the nucleic acid molecule encodes a capsid polypeptide
that
comprises a mutation that comprises an insertion between positions 448 and 449
as compared to
SEQ ID NO: 1.
In some embodiments, the nucleic acid molecule encodes a capsid polypeptide
that
comprises a mutation that comprises an insertion between positions 449 and 450
as compared to
SEQ ID NO: 1.
In some embodiments, the nucleic acid molecule encodes a capsid polypeptide
that
comprises a mutation that comprises an insertion between positions 451, and
452 as compared to
SEQ ID NO: 1.
In some embodiments, the nucleic acid molecule encodes a capsid polypeptide
that
comprises a mutation that comprises an insertion between positions 453 and 454
as compared to
SEQ ID NO: 1.
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In some embodiments, the nucleic acid molecule encodes a capsid polypeptide
that
comprises a mutation that comprises an insertion between positions 581 and 582
as compared to
SEQ ID NO: 1.
In some embodiments, the nucleic acid molecule encodes a capsid polypeptide
that
comprises a mutation that comprises an insertion between positions 583 and 584
as compared to
SEQ ID NO: 1.
In some embodiments, the nucleic acid molecule encodes a capsid polypeptide
that
comprises a mutation that comprises an insertion between positions 584 and 585
as compared to
SEQ ID NO: 1.
In some embodiments, the nucleic acid molecule encodes a capsid polypeptide
that
comprises a mutation that comprises an insertion between positions 585 and 586
as compared to
SEQ ID NO: 1.
In some embodiments, the nucleic acid molecule encodes a capsid polypeptide
that
comprises a mutation that comprises an insertion between positions 586 and 587
as compared to
SEQ ID NO: 1.
In some embodiments, the nucleic acid molecule encodes a capsid polypeptide
that
comprises a mutation that comprises an insertion between positions 587 and 588
as compared to
SEQ ID NO: 1.
In some embodiments, the nucleic acid molecule encodes a capsid polypeptide
that
comprises a mutation that comprises an insertion between positions 588 and 589
as compared to
SEQ ID NO: 1.
In some embodiments, the nucleic acid molecule encodes a capsid polypeptide
that
comprises a mutation that comprises an insertion between positions 589 and 590
as compared to
SEQ ID NO: 1.
In some embodiments, the nucleic acid molecule encodes a capsid polypeptide
that
comprises a mutation that comprises an insertion between positions 590 and 591
as compared to
SEQ ID NO: 1.
In some embodiments, the nucleic acid molecule encodes a capsid polypeptide
that
comprises a mutation that comprises an insertion between positions 591 and 592
as compared to
SEQ ID NO: 1.
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In some embodiments, the nucleic acid molecule encodes a capsid polypeptide
that
comprises a mutation that comprises an insertion between positions 592 and 593
as compared to
SEQ ID NO: 1.
In some embodiments, the nucleic acid molecule encodes a capsid polypeptide
that
comprises a mutation that corresponds to a mutation at position 449, 450, 452,
455, 457, 458,
459, and an insertion between positions 451 and 452 as compared to SEQ ID NO:
1.
In some embodiments, the nucleic acid molecule encodes a capsid polypeptide
that
comprises a mutation that corresponds to a mutation at position 585 and 586
and an insertion
between positions 584 and 585 as compared to SEQ ID NO: 1.
In some embodiments, the nucleic acid molecule encodes a capsid polypeptide
that
comprises a mutation that corresponds to a mutation at position 586 and 587
and an insertion
between positions 584 and 585 as compared to SEQ ID NO: 1.
In some embodiments, the nucleic acid molecule encodes a capsid polypeptide
that
comprises a mutation that corresponds to a mutation at position 587 and an
insertion between
positions 586 and 587 as compared to SEQ ID NO: 1.
In some embodiments, the nucleic acid molecule encodes a capsid polypeptide
that
comprises a mutation that corresponds to a T14L, L15V, 119A, and K24H mutation
as compared
to SEQ ID NO: 1.
In some embodiments, the nucleic acid molecule encodes a capsid polypeptide
that
comprises a mutation that corresponds to a A3V. Y6F, L11F, E12Q, Q21L, W23I,
L25C, P29A.
P3 1N, and K33R mutation as compared to SEQ ID NO: 1.
In some embodiments, the nucleic acid molecule encodes a capsid polypeptide
that
comprises a mutation that corresponds to a N449Q, T450M, P45 1T, T455L, T456G,
Q457T,
S458Q, and R459M mutation as compared to SEQ ID NO: 1.
In some embodiments, the nucleic acid molecule encodes a capsid polypeptide
that
comprises a mutation that corresponds to a N449Q, residue 451 deletion, S452T,
T456G, Q457T,
and R459G mutation as compared to SEQ ID NO: 1.
In some embodiments, the nucleic acid molecule encodes a capsid polypeptide
that
comprises a mutation that corresponds to a Q457F and S458C mutation as
compared to SEQ ID
NO: 1.
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In some embodiments, the nucleic acid molecule encodes a capsid polypeptide
that
comprises a mutation that corresponds to an insertion between residues 446 and
447 as compared
to SEQ ID NO: 1, wherein the insertion comprises, e.g., consists of, a
polypeptide of
KCQEGMA (SEQ ID NO: 95). In some embodiments, the insertion comprises a
polypeptide that
has at least 57.1%, 71.4%, 85.7%, or 100% identity to KCQEGMA (SEQ ID NO: 95).
In some
embodiments, the insertion comprises a polypeptide that has at least 1, 2, or
3 mutations as
compared to KCQEGMA (SEQ ID NO: 95). In some embodiments, the insertion
comprises a
polypeptide comprising a fragment of at least 4 amino acids of KCQEGMA (SEQ ID
NO: 95).
In some embodiments, the insertion comprises a polypeptide comprising a
fragment of at least 5
amino acids of KCQEGMA (SEQ ID NO: 95). In some embodiments, the insertion
comprises a
polypeptide comprising a fragment of at least 6 amino acids of KCQEGMA (SEQ ID
NO: 95).
In some embodiments, the nucleic acid molecule encodes a capsid polypeptide
that
comprises a mutation that corresponds to an insertion between residues 449 and
450 as compared
to SEQ ID NO: 1, wherein the insertion comprises, e.g., consists of, a
polypeptide of
LMVDRLG (SEQ ID NO: 96). In some embodiments, the insertion comprises a
polypeptide that
has at least 57.1%, 71.4%, 85.7%, or 100% identity to LMVDRLG (SEQ ID NO: 96).
In some
embodiments, the insertion comprises a polypeptide that has at least 1, 2, or
3 mutations as
compared to LMVDRLG (SEQ ID NO: 96). In some embodiments, the insertion
comprises a
polypeptide comprising a fragment of at least 4 amino acids of LMVDRLG (SEQ ID
NO: 96). In
some embodiments, the insertion comprises a polypeptide comprising a fragment
of at least 5
amino acids of LMVDRLG (SEQ ID NO: 96). In some embodiments, the insertion
comprises a
polypeptide comprising a fragment of at least 6 amino acids of LMVDRLG (SEQ ID
NO: 96).
In some embodiments, the nucleic acid molecule encodes a capsid polypeptide
that
comprises a mutation that corresponds to a P45 1T. T455G, T456G, Q457T, S458Q,
R459T, and
Q461A mutation as compared to SEQ ID NO: 1.
In some embodiments, the nucleic acid molecule encodes a capsid polypeptide
that
comprises a mutation that corresponds to an insertion between residues 448 and
449 as compared
to SEQ ID NO: 1, wherein the insertion comprises, e.g., consists of, a
polypeptide of HCQECPI
(SEQ ID NO: 97). In some embodiments, the insertion comprises a polypeptide
that has at least
57.1%, 71.4%, 85.7%, or 100% identity to HCQECPI (SEQ ID NO: 97). In some
embodiments,
the insertion comprises a polypeptide that has at least 1, 2, or 3 mutations
as compared to
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HCQECPI (SEQ ID NO: 97). In some embodiments, the insertion comprises a
polypeptide
comprising a fragment of at least 4 amino acids of HCQECPI (SEQ ID NO: 97). In
some
embodiments, the insertion comprises a polypeptide comprising a fragment of at
least 5 amino
acids of HCQECPI (SEQ ID NO: 97). In some embodiments, the insertion comprises
a
polypeptide comprising a fragment of at least 6 amino acids of HCQECPI (SEQ ID
NO: 97).
In some embodiments, the nucleic acid molecule encodes a capsid polypeptide
that
comprises a mutation that corresponds to an insertion between residues 453 and
454 as compared
to SEQ ID NO: 1, wherein the insertion comprises, e.g., consists of, a
polypeptide of FSGLEN
(SEQ ID NO: 98). In some embodiments, the insertion comprises a polypeptide
that has at least
50%. 66.67%, 83.3%, or 100% identity to FSGLEN (SEQ ID NO: 98). In some
embodiments,
the insertion comprises a polypeptide that has at least 1, 2, or 3 mutations
as compared to
FSGLEN (SEQ ID NO: 98). In some embodiments, the insertion comprises a
polypeptide
comprising a fragment of at least 4 amino acids of FSGLEN (SEQ ID NO: 98). In
some
embodiments, the insertion comprises a polypeptide comprising a fragment of at
least 3 amino
acids of FSGLEN (SEQ ID NO: 98).
In some embodiments, the nucleic acid molecule encodes a capsid polypeptide
that
comprises a mutation that corresponds to a residue deletion at positions 456,
457, 458, 459. 460,
and 461 as compared to SEQ ID NO: 1.
In some embodiments, the nucleic acid molecule encodes a capsid polypeptide
that
comprises a mutation that corresponds to a N449I, T450N, P451G, residue 454
deletion, T455Q,
T456N, S458Q, R459T, Q461K, and Q464V mutation as compared to SEQ ID NO: 1.
In some embodiments, the nucleic acid molecule encodes a capsid polypeptide
that
comprises a mutation that corresponds to a S446A, residue 448 and 449
deletion, P451Q,
G453T, T455G, T456G, Q457T, and R459G mutation as compared to SEQ ID NO: 1.
In some embodiments, the nucleic acid molecule encodes a capsid polypeptide
that
comprises a mutation that corresponds to a N449Q, T450S, 5452G, T455A, Q457F,
S458M.
R459D, and an insertion between residues 451 and 452 as compared to SEQ ID NO:
1, wherein
the insertion comprises an amino acid Y.
In some embodiments, the nucleic acid molecule encodes a capsid polypeptide
that
comprises a mutation that corresponds to an insertion between residues 447 and
448 as compared
to SEQ ID NO: 1, wherein the insertion comprises, e.g., consists of, a
polypeptide of HETEFNF
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(SEQ ID NO: 99). In some embodiments, the insertion comprises a polypeptide
that has at least
57.1%, 71.4%, 85.7%, or 100% identity to HETEFNF (SEQ ID NO: 99). In some
embodiments,
the insertion comprises a polypeptide that has at least 1, 2, or 3 mutations
as compared to
HETEFNF (SEQ ID NO: 99). In some embodiments, the insertion comprises a
polypeptide
comprising a fragment of at least 4 amino acids of HETEFNF (SEQ ID NO: 99). In
some
embodiments, the insertion comprises a polypeptide comprising a fragment of at
least 5 amino
acids of HETEFNF (SEQ ID NO: 99). In some embodiments, the insertion comprises
a
polypeptide comprising a fragment of at least 6 amino acids of HETEFNF (SEQ ID
NO: 99).
In some embodiments, the nucleic acid molecule encodes a capsid polypeptide
that
comprises a mutation that corresponds to a P45 1T. T455G, T456G, Q457T, 5458Q,
R459T, and
Q461A mutation as compared to SEQ ID NO: 1.
In some embodiments, the nucleic acid molecule encodes a capsid polypeptide
that
comprises a mutation that corresponds to an insertion between residues 581 and
582 as compared
to SEQ ID NO: 1, wherein the insertion comprises, e.g., consists of, a
polypeptide of FALMEP
(SEQ ID NO: 100). In some embodiments, the insertion comprises a polypeptide
that has at least
50%. 66.67%, 83.3%, or 100% identity to FALMEP (SEQ ID NO: 100). In some
embodiments,
the insertion comprises a polypeptide that has at least 1, 2, or 3 mutations
as compared to
FALMEP (SEQ ID NO: 100). In some embodiments, the insertion comprises a
polypeptide
comprising a fragment of at least 4 amino acids of FALMEP (SEQ ID NO: 100). In
some
embodiments, the insertion comprises a polypeptide comprising a fragment of at
least 5 amino
acids of FALMEP (SEQ ID NO: 100).
In some embodiments, the nucleic acid molecule encodes a capsid polypeptide
that
comprises a mutation that corresponds to an insertion between residues 584 and
585 as compared
to SEQ ID NO: 1, wherein the insertion comprises, e.g., consists of, a
polypeptide of RAYNPD
(SEQ ID NO: 101). In some embodiments, the insertion comprises a polypeptide
that has at least
50%. 66.67%, 83.3%, or 100% identity to RAYNPD (SEQ ID NO: 101). In some
embodiments,
the insertion comprises a polypeptide that has at least 1, 2, or 3 mutations
as compared to
RAYNPD (SEQ ID NO: 101). In some embodiments, the insertion comprises a
polypeptide
comprising a fragment of at least 4 amino acids of RAYNPD (SEQ ID NO: 101). In
some
embodiments, the insertion comprises a polypeptide comprising a fragment of at
least 5 amino
acids of RAYNPD (SEQ ID NO: 101).
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In some embodiments, the nucleic acid molecule encodes a capsid polypeptide
that
comprises a mutation that corresponds to a residue 585 deletion and a E555A,
D461S, R566K,
S578V, S580A, and T581A mutation as compared to SEQ ID NO: 1.
In some embodiments, the nucleic acid molecule encodes a capsid polypeptide
that
comprises a mutation that corresponds to a V557L and S578D mutation as
compared to SEQ ID
NO: 1.
In some embodiments, the nucleic acid molecule encodes a capsid polypeptide
that
comprises a mutation that corresponds to a K556N, M558L, S578I, and S580A
mutation as
compared to SEQ ID NO: 1.
In some embodiments, the nucleic acid molecule encodes a capsid polypeptide
that
comprises a mutation that corresponds to a 1554L, K556R, I559L, D561S, R566A,
T581D,
R585S, G586R, and N587S mutation as compared to SEQ ID NO: 1.
In some embodiments, the nucleic acid molecule encodes a capsid polypeptide
that
comprises a mutation that corresponds to a V552A, E555S, K556D, 1559L, D561S,
R566K,
S578I, T581D, and G586Q mutation as compared to SEQ ID NO: 1.
In some embodiments, the nucleic acid molecule encodes a capsid polypeptide
that
comprises a mutation that corresponds to a Q575E and 5578A mutation as
compared to SEQ ID
NO: 1.
In some embodiments, the nucleic acid molecule encodes a capsid polypeptide
that
comprises a mutation that corresponds to an insertion between residues 583 and
584 as compared
to SEQ ID NO: 1, wherein the insertion comprises, e.g., consists of, a
polypeptide of LNWTAE
(SEQ ID NO: 102). In some embodiments, the insertion comprises a polypeptide
that has at least
50%. 66.67%, 83.3%, or 100% identity to LNWTAE (SEQ ID NO: 102). In some
embodiments,
the insertion comprises a polypeptide that has at least 1, 2, or 3 mutations
as compared to
LNWTAE (SEQ ID NO: 102). In some embodiments, the insertion comprises a
polypeptide
comprising a fragment of at least 4 amino acids of LNWTAE (SEQ ID NO: 102). In
some
embodiments, the insertion comprises a polypeptide comprising a fragment of at
least 5 amino
acids of LNWTAE (SEQ ID NO: 102).
In some embodiments, the nucleic acid molecule encodes a capsid polypeptide
that
comprises a mutation that corresponds to a R585S, R588T, Q589N, A590P, A591I,
A593G,
T597S, and V600A mutation as compared to SEQ ID NO: 1.
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In some embodiments, the nucleic acid molecule encodes a capsid polypeptide
that
comprises a mutation that corresponds to a R585N and G586A mutation, and an
insertion
between residues 584 and 585 as compared to SEQ ID NO: 1, wherein the
insertion comprises,
e.g., consists of, a polypeptide of LAKEFTTR (SEQ ID NO: 103). In some
embodiments, the
insertion comprises a polypeptide that has at least 50%, 62.5%, 75%, 87.5%, or
100% identity to
LAKEFTTR (SEQ ID NO: 103). In some embodiments, the insertion comprises a
polypeptide
that has at least 1, 2, 3, or 4 mutations as compared to LAKEFTTR (SEQ ID NO:
103). In some
embodiments, the insertion comprises a polypeptide comprising a fragment of at
least 4 amino
acids of LAKEFTTR (SEQ ID NO: 103). In some embodiments, the insertion
comprises a
polypeptide comprising a fragment of at least 5 amino acids of LAKEFTTR (SEQ
ID NO: 103).
In some embodiments, the insertion comprises a polypeptide comprising a
fragment of at least 6
amino acids of LAKEFTTR (SEQ ID NO: 103). In some embodiments, the insertion
comprises a
polypeptide comprising a fragment of at least 7 amino acids of LAKEFTTR (SEQ
ID NO: 103).
In some embodiments, the insertion comprises, e.g., consists of, KEFTTR (SEQ
ID NO: 104).
In some embodiments, the nucleic acid molecule encodes a capsid polypeptide
that
comprises a mutation that corresponds to an insertion between residues 592 and
593 as compared
to SEQ ID NO: 1, wherein the insertion comprises, e.g., consists of, a
polypeptide of LHPLE
(SEQ ID NO: 105). In some embodiments, the insertion comprises a polypeptide
that has at least
60%. 80%, or 100% identity to LHPLE (SEQ ID NO: 105). In some embodiments, the
insertion
comprises a polypeptide that has at least 1, or 2 mutations as compared to
LHPLE (SEQ ID NO:
105). In some embodiments, the insertion comprises a polypeptide comprising a
fragment of at
least 4 amino acids of LHPLE (SEQ ID NO: 105).
In some embodiments, the nucleic acid molecule encodes a capsid polypeptide
that
comprises a mutation that corresponds to an insertion between residues 585 and
586 as compared
to SEQ ID NO: 1, wherein the insertion comprises an amino acid F.
In some embodiments, the nucleic acid molecule encodes a capsid polypeptide
that
comprises a mutation that corresponds to an insertion between residues 586 and
587 as compared
to SEQ ID NO: 1, wherein the insertion comprises, e.g., consists of, a
polypeptide of DQDFKNR
(SEQ ID NO: 106). In some embodiments, the insertion comprises a polypeptide
that has at least
57.1%, 71.4%, 85.7%, or 100% identity to DQDFKNR (SEQ ID NO: 106). In some
embodiments, the insertion comprises a polypeptide that has at least 1, 2, or
3 mutations as
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compared to DQDFKNR (SEQ ID NO: 106). In some embodiments, the insertion
comprises a
polypeptide comprising a fragment of at least 4 amino acids of DQDFKNR (SEQ ID
NO: 106).
In some embodiments, the insertion comprises a polypeptide comprising a
fragment of at least 5
amino acids of DQDFKNR (SEQ ID NO: 106). In some embodiments, the insertion
comprises a
polypeptide comprising a fragment of at least 6 amino acids of DQDFKNR (SEQ ID
NO: 106).
In some embodiments, the nucleic acid molecule encodes a capsid polypeptide
that
comprises a mutation that corresponds to an insertion between residues 589 and
590 as compared
to SEQ ID NO: 1, wherein the insertion comprises an amino acid I.
In some embodiments, the nucleic acid molecule encodes a capsid polypeptide
that
comprises a mutation that corresponds to an insertion between residues 587 and
588 as compared
to SEQ ID NO: 1, wherein the insertion comprises, e.g., consists of, a
polypeptide of
LAIEQTRPA (SEQ ID NO: 107). In some embodiments, the insertion comprises a
polypeptide
that has at least 55.5%, 66.6%, 77.7%, 88.8%, or 100% identity to LAIEQTRPA
(SEQ ID NO:
107). In some embodiments, the insertion comprises a polypeptide that has at
least 1, 2, 3, or 4
mutations as compared to LAIEQTRPA (SEQ ID NO: 107). In some embodiments, the
insertion
comprises a polypeptide comprising a fragment of at least 4 amino acids of
LAIEQTRPA (SEQ
ID NO: 107). In some embodiments, the insertion comprises a polypeptide
comprising a
fragment of at least 5 amino acids of LAIEQTRPA (SEQ ID NO: 107). In some
embodiments,
the insertion comprises a polypeptide comprising a fragment of at least 6
amino acids of
LAIEQTRPA (SEQ ID NO: 107). In some embodiments, the insertion comprises a
polypeptide
comprising a fragment of at least 7 amino acids of LAIEQTRPA (SEQ ID NO: 107).
In some
embodiments, the insertion comprises a polypeptide comprising a fragment of at
least 8 amino
acids of LAIEQTRPA (SEQ ID NO: 107). In some embodiments, the insertion
comprises, e.g.,
consists of, IEQTRPA (SEQ ID NO: 108).
In some embodiments, the nucleic acid molecule encodes a capsid polypeptide
that
comprises a mutation that corresponds to a G586P and N587A mutation, and an
insertion
between residues 584 and 585 as compared to SEQ ID NO: I, wherein the
insertion comprises,
e.g., consists of, a polypeptide of RARLDETT (SEQ ID NO: 109). In some
embodiments, the
insertion comprises a polypeptide that has at least 50%, 62.5%, 75%, 87.5%, or
100% identity to
RARLDETT (SEQ ID NO: 109). In some embodiments, the insertion comprises a
polypeptide
that has at least 1, 2, 3, or 4 mutations as compared to RARLDETT (SEQ ID NO:
109). In some
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embodiments, the insertion comprises a polypeptide comprising a fragment of at
least 4 amino
acids of RARLDETT (SEQ ID NO: 109). In some embodiments, the insertion
comprises a
polypeptide comprising a fragment of at least 5 amino acids of RARLDETT (SEQ
ID NO: 109).
In some embodiments, the insertion comprises a polypeptide comprising a
fragment of at least 6
amino acids of RARLDETT (SEQ ID NO: 109). In some embodiments, the insertion
comprises a
polypeptide comprising a fragment of at least 7 amino acids of RARLDETT (SEQ
ID NO: 109).
In some embodiments, the insertion comprises, e.g., consists of, RLDETT (SEQ
ID NO: 110).
In some embodiments, the nucleic acid molecule encodes a capsid polypeptide
that
comprises a mutation that corresponds to a N587A mutation, and an insertion
between residues
586 and 587 as compared to SEQ ID NO: 1, wherein the insertion comprises,
e.g., consists of, a
polypeptide of LALAEITRP (SEQ ID NO: 111). In some embodiments, the insertion
comprises
a polypeptide that has at least 55.5%, 66.6%, 77.7%. 88.8%, or 100% identity
to LALAEITRP
(SEQ ID NO: 111). In some embodiments, the insertion comprises a polypeptide
that has at least
1, 2, 3, or 4 mutations as compared to LALAEITRP (SEQ ID NO: 111). In some
embodiments,
the insertion comprises a polypeptide comprising a fragment of at least 4
amino acids of
LALAEITRP (SEQ ID NO: 111). In some embodiments, the insertion comprises a
polypeptide
comprising a fragment of at least 5 amino acids of LALAEITRP (SEQ ID NO: 111).
In some
embodiments, the insertion comprises a polypeptide comprising a fragment of at
least 6 amino
acids of LALAEITRP (SEQ ID NO: 111). In some embodiments, the insertion
comprises a
polypeptide comprising a fragment of at least 7 amino acids of LALAEITRP (SEQ
ID NO: 111).
In some embodiments, the insertion comprises a polypeptide comprising a
fragment of at least 8
amino acids of LALAEITRP (SEQ ID NO: 111). In some embodiments, the insertion
comprises,
e.g., consists of, LAEITRP (SEQ ID NO: 112).
In some embodiments, the nucleic acid molecule encodes a capsid polypeptide
that
comprises a mutation that corresponds to a N587A mutation, and an insertion
between residues
586 and 587 as compared to SEQ ID NO: 1, wherein the insertion comprises,
e.g., consists of, a
polypeptide of LANGEQTRP (SEQ ID NO: 113). In some embodiments, the insertion
comprises
a polypeptide that has at least 55.5%, 66.6%, 77.7%, 88.8%, or 100% identity
to LANGEQTRP
(SEQ ID NO: 113). In some embodiments, the insertion comprises a polypeptide
that has at least
1, 2, 3, or 4 mutations as compared to LANGEQTRP (SEQ ID NO: 113). In some
embodiments,
the insertion comprises a polypeptide comprising a fragment of at least 4
amino acids of
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LANGEQTRP (SEQ ID NO: 113). In some embodiments, the insertion comprises a
polypeptide
comprising a fragment of at least 5 amino acids of LANGEQTRP (SEQ ID NO: 113).
In some
embodiments, the insertion comprises a polypeptide comprising a fragment of at
least 6 amino
acids of LANGEQTRP (SEQ ID NO: 113). In some embodiments, the insertion
comprises a
polypeptide comprising a fragment of at least 7 amino acids of LANGEQTRP (SEQ
ID NO:
113). In some embodiments, the insertion comprises a polypeptide comprising a
fragment of at
least 8 amino acids of LANGEQTRP (SEQ ID NO: 113). In some embodiments, the
insertion
comprises, e.g., consists of, NGEQTRP (SEQ ID NO: 114).
In some embodiments, the nucleic acid molecule encodes a capsid polypeptide
that
comprises a mutation that corresponds to an insertion between residues 586 and
587 as compared
to SEQ ID NO: 1, wherein the insertion comprises, e.g., consists of, a
polypeptide of ATDTKT
(SEQ ID NO: 115). In some embodiments, the insertion comprises a polypeptide
that has at least
50%. 66.67%, 83.3%, or 100% identity to ATDTKT (SEQ ID NO: 115). In some
embodiments,
the insertion comprises a polypeptide that has at least 1, 2, or 3 mutations
as compared to
ATDTKT (SEQ ID NO: 115). In some embodiments, the insertion comprises a
polypeptide
comprising a fragment of at least 4 amino acids of ATDTKT (SEQ ID NO: 115). In
some
embodiments, the insertion comprises a polypeptide comprising a fragment of at
least 5 amino
acids of ATDTKT (SEQ ID NO: 115).
In some embodiments, the nucleic acid molecule encodes a capsid polypeptide
that
comprises a mutation that corresponds to an insertion between residues 590 and
591 as compared
to SEQ ID NO: 1, wherein the insertion comprises an amino acid P.
In some embodiments, the nucleic acid molecule encodes a capsid polypeptide
that
comprises a mutation that corresponds to an insertion between residues 587 and
588 as compared
to SEQ ID NO: 1, wherein the insertion comprises, e.g., consists of, a
polypeptide of
APGETTRPA (SEQ ID NO: 116). In some embodiments, the insertion comprises a
polypeptide
that has at least 55.5%, 66.6%, 77.7%, 88.8%, or 100% identity to APGETTRPA
(SEQ ID NO:
116). In some embodiments, the insertion comprises a polypeptide that has at
least 1, 2, 3, or 4
mutations as compared to APGETTRPA (SEQ ID NO: 116). In some embodiments, the
insertion
comprises a polypeptide comprising a fragment of at least 4 amino acids of
APGETTRPA (SEQ
ID NO: 116). In some embodiments, the insertion comprises a polypeptide
comprising a
fragment of at least 5 amino acids of APGETTRPA (SEQ ID NO: 116). In some
embodiments,
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the insertion comprises a polypeptide comprising a fragment of at least 6
amino acids of
APGETTRPA (SEQ ID NO: 116). In some embodiments, the insertion comprises a
polypeptide
comprising a fragment of at least 7 amino acids of APGETTRPA (SEQ ID NO: 116).
In some
embodiments, the insertion comprises a polypeptide comprising a fragment of at
least 8 amino
acids of APGETTRPA (SEQ ID NO: 116). In some embodiments, the insertion
comprises, e.g.,
consists of, GETTRP (SEQ ID NO: 117).
In some embodiments, the nucleic acid molecule encodes a capsid polypeptide
that
comprises a mutation that corresponds to an insertion between residues 589 and
590 as compared
to SEQ ID NO: 1, wherein the insertion comprises an amino acid P.
In some embodiments, the nucleic acid molecule encodes a capsid polypeptide
that
comprises a mutation that corresponds to a R585S, G586S, N587A, A591E, D594R,
T597A, and
V600I mutation as compared to SEQ ID NO: 1.
In some embodiments, the nucleic acid molecule encodes a capsid polypeptide
that
comprises a mutation that corresponds to an insertion between residues 586 and
587 as compared
to SEQ ID NO: 1, wherein the insertion comprises, e.g.. consists of, a
polypeptide of FHNEGKY
(SEQ ID NO: 118). In some embodiments, the insertion comprises a polypeptide
that has at least
57.1%, 71.4%, 85.7%, or 100% identity to FHNEGKY (SEQ ID NO: 118). In some
embodiments, the insertion comprises a polypeptide that has at least 1, 2, or
3 mutations as
compared to FHNEGKY (SEQ ID NO: 118). In some embodiments, the insertion
comprises a
polypeptide comprising a fragment of at least 4 amino acids of FHNEGKY (SEQ ID
NO: 118).
In some embodiments, the insertion comprises a polypeptide comprising a
fragment of at least 5
amino acids of FHNEGKY (SEQ ID NO: 118). In some embodiments, the insertion
comprises a
polypeptide comprising a fragment of at least 6 amino acids of FHNEGKY (SEQ ID
NO: 118).
In some embodiments, the nucleic acid molecule encodes a capsid polypeptide
that
comprises a mutation that corresponds to an insertion between residues 591 and
592 as compared
to SEQ ID NO: 1, wherein the insertion comprises an amino acid G.
In some embodiments, the nucleic acid molecule encodes a capsid polypeptide
that
comprises a mutation that corresponds to an insertion between residues 588 and
589 as compared
to SEQ ID NO: 1, wherein the insertion comprises, e.g., consists of, a
polypeptide of QPWEPDK
(SEQ ID NO: 119). In some embodiments, the insertion comprises a polypeptide
that has at least
57.1%, 71.4%, 85.7%, or 100% identity to QPWEPDK (SEQ ID NO: 119). In some
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embodiments, the insertion comprises a polypeptide that has at least 1, 2, or
3 mutations as
compared to QPWEPDK (SEQ ID NO: 119). In some embodiments, the insertion
comprises a
polypeptide comprising a fragment of at least 4 amino acids of QPWEPDK (SEQ ID
NO: 119).
In some embodiments, the insertion comprises a polypeptide comprising a
fragment of at least 5
amino acids of QPWEPDK (SEQ ID NO: 119). In some embodiments, the insertion
comprises a
polypeptide comprising a fragment of at least 6 amino acids of QPWEPDK (SEQ ID
NO: 119).
In some embodiments, the nucleic acid molecule encodes a capsid polypeptide
that
comprises a mutation that corresponds to an insertion between residues 592 and
593 as compared
to SEQ ID NO: 1, wherein the insertion comprises, e.g., consists of, a
polypeptide of
ALALSTTN (SEQ ID NO: 120). In some embodiments, the insertion comprises a
polypeptide
that has at least 50%, 62.5%, 75%, 87.5%, or 100% identity to ALALSTTN (SEQ ID
NO: 120).
In some embodiments, the insertion comprises a polypeptide that has at least
1, 2, 3, or 4
mutations as compared to ALALSTTN (SEQ ID NO: 120). In some embodiments, the
insertion
comprises a polypeptide comprising a fragment of at least 4 amino acids of
ALALSTTN (SEQ
ID NO: 120). In some embodiments, the insertion comprises a polypeptide
comprising a
fragment of at least 5 amino acids of ALALSTTN (SEQ ID NO: 120). In some
embodiments, the
insertion comprises a polypeptide comprising a fragment of at least 6 amino
acids of
ALALSTTN (SEQ ID NO: 120). In some embodiments, the insertion comprises a
polypeptide
comprising a fragment of at least 7 amino acids of ALALSTTN (SEQ ID NO: 120).
In some
embodiments, the insertion comprises, e.g., consists of, ALSTTN (SEQ ID NO:
121).
In some embodiments, the nucleic acid molecule encodes a capsid polypeptide
that
comprises a mutation that corresponds to an insertion between residues 585 and
586 as compared
to SEQ ID NO: 1, wherein the insertion comprises, e.g., consists of, a
polypeptide of PWGTAG
(SEQ ID NO: 122). In some embodiments, the insertion comprises a polypeptide
that has at least
50%. 66.7%, 83.3%. or 100% identity to PWGTAG (SEQ ID NO: 122). In some
embodiments,
the insertion comprises a polypeptide that has at least 1, 2, or 3 mutations
as compared to
PWGTAG (SEQ ID NO: 122). In some embodiments, the insertion comprises a
polypeptide
comprising a fragment of at least 4 amino acids of PWGTAG (SEQ ID NO: 122). In
some
embodiments, the insertion comprises a polypeptide comprising a fragment of at
least 5 amino
acids of PWGTAG (SEQ ID NO: 122).
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In some embodiments, the nucleic acid molecule encodes a capsid polypeptide as
provided herein. In some embodiments, the nucleic acid molecule encodes a
capsid polypeptide
that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, 99%,
or 100% identical to a capsid polypeptide as provided herein.
In some embodiments, a capsid polypeptide is provided that comprises a capsid
polypeptide that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%,
98%. 99%, or 100% identical to a capsid polypeptide as provided herein.
In some embodiments, the capsid polypeptide, or the reference polypeptide for
purposes
of % identity, comprises a sequence of SEQ ID NOs: 2, 3, 4, 5, 6, 7, 8, 9, 10,
11, 12, 13, 14, 15,
16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,
35, 36, 37, 38, 39, 40, 41,
42, 43, 44, 45, or 46. In some embodiments, the capsid polypeptide, or the
reference polypeptide
for purposes of % identity, comprises a sequence of SEQ ID NO: 2. In some
embodiments, the
capsid polypeptide, or the reference polypeptide for purposes of % identity,
comprises a
sequence of SEQ ID NO: 3. In some embodiments, the capsid polypeptide, or the
reference
polypeptide for purposes of % identity, comprises a sequence of SEQ ID NO: 4.
In some
embodiments, the capsid polypeptide, or the reference polypeptide for purposes
of % identity,
comprises a sequence of SEQ ID NO: 5. In some embodiments, the capsid
polypeptide, or the
reference polypeptide for purposes of % identity, comprises a sequence of SEQ
ID NO: 6. In
some embodiments, the capsid polypeptide, or the reference polypeptide for
purposes of %
identity, comprises a sequence of SEQ ID NO: 7. In some embodiments, the
capsid polypeptide,
or the reference polypeptide for purposes of % identity, comprises a sequence
of SEQ ID NO: 8.
In some embodiments, the capsid polypeptide, or the reference polypeptide for
purposes of %
identity, comprises a sequence of SEQ ID NO: 9. In some embodiments, the
capsid polypeptide,
or the reference polypeptide for purposes of % identity, comprises a sequence
of SEQ ID NO:
10. In some embodiments, the capsid polypeptide, or the reference polypeptide
for purposes of
% identity, comprises a sequence of SEQ ID NO: 11. In some embodiments, the
capsid
polypeptide, or the reference polypeptide for purposes of % identity,
comprises a sequence of
SEQ ID NO: 12. In some embodiments, the capsid polypeptide, or the reference
polypeptide for
purposes of % identity, comprises a sequence of SEQ ID NO: 13. In some
embodiments, the
capsid polypeptide, or the reference polypeptide for purposes of % identity,
comprises a
sequence of SEQ ID NO: 14. In some embodiments, the capsid polypeptide, or the
reference
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polypeptide for purposes of c/c identity, comprises a sequence of SEQ ID NO:
15. In some
embodiments, the capsid polypeptide, or the reference polypeptide for purposes
of % identity,
comprises a sequence of SEQ ID NO: 16. In some embodiments, the capsid
polypeptide, or the
reference polypeptide for purposes of % identity, comprises a sequence of SEQ
ID NO: 17. In
some embodiments, the capsid polypeptide, or the reference polypeptide for
purposes of %
identity, comprises a sequence of SEQ ID NO: 18. In some embodiments, the
capsid
polypeptide, or the reference polypeptide for purposes of % identity,
comprises a sequence of
SEQ ID NO: 19. In some embodiments, the capsid polypeptide, or the reference
polypeptide for
purposes of % identity, comprises a sequence of SEQ ID NO: 20. In some
embodiments, the
capsid polypeptide, or the reference polypeptide for purposes of % identity,
comprises a
sequence of SEQ ID NO: 21. In some embodiments, the capsid polypeptide, or the
reference
polypeptide for purposes of % identity, comprises a sequence of SEQ ID NO: 22.
In some
embodiments, the capsid polypeptide, or the reference polypeptide for purposes
of % identity,
comprises a sequence of SEQ ID NO: 23. In some embodiments, the capsid
polypeptide, or the
reference polypeptide for purposes of % identity, comprises a sequence of SEQ
ID NO: 24. In
some embodiments, the capsid polypeptide, or the reference polypeptide for
purposes of %
identity, comprises a sequence of SEQ ID NO: 25. In some embodiments, the
capsid
polypeptide, or the reference polypeptide for purposes of % identity,
comprises a sequence of
SEQ ID NO: 26. In some embodiments, the capsid polypeptide, or the reference
polypeptide for
purposes of % identity, comprises a sequence of SEQ ID NO: 27. In some
embodiments, the
capsid polypeptide, or the reference polypeptide for purposes of % identity,
comprises a
sequence of SEQ ID NO: 28. In some embodiments, the capsid polypeptide, or the
reference
polypeptide for purposes of % identity, comprises a sequence of SEQ ID NO: 29.
In some
embodiments, the capsid polypeptide, or the reference polypeptide for purposes
of % identity,
comprises a sequence of SEQ ID NO: 30. In some embodiments, the capsid
polypeptide, or the
reference polypeptide for purposes of % identity, comprises a sequence of SEQ
ID NO: 31. In
some embodiments, the capsid polypeptide, or the reference polypeptide for
purposes of %
identity, comprises a sequence of SEQ ID NO: 32. In some embodiments, the
capsid
polypeptide, or the reference polypeptide for purposes of % identity,
comprises a sequence of
SEQ ID NO: 33. In some embodiments, the capsid polypeptide, or the reference
polypeptide for
purposes of % identity, comprises a sequence of SEQ ID NO: 34. In some
embodiments, the
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capsid polypeptide, or the reference polypeptide for purposes of % identity,
comprises a
sequence of SEQ ID NO: 35. In some embodiments, the capsid polypeptide, or the
reference
polypeptide for purposes of % identity, comprises a sequence of SEQ ID NO: 36.
In some
embodiments, the capsid polypeptide, or the reference polypeptide for purposes
of % identity,
comprises a sequence of SEQ ID NO: 37. In some embodiments, the capsid
polypeptide, or the
reference polypeptide for purposes of % identity, comprises a sequence of SEQ
ID NO: 38. In
some embodiments, the capsid polypeptide, or the reference polypeptide for
purposes of %
identity, comprises a sequence of SEQ ID NO: 39. In some embodiments, the
capsid
polypeptide, or the reference polypeptide for purposes of % identity,
comprises a sequence of
SEQ ID NO: 40. In some embodiments, the capsid polypeptide, or the reference
polypeptide for
purposes of % identity, comprises a sequence of SEQ ID NO: 41. In some
embodiments, the
capsid polypeptide, or the reference polypeptide for purposes of % identity,
comprises a
sequence of SEQ ID NO: 42. In some embodiments, the capsid polypeptide, or the
reference
polypeptide for purposes of % identity, comprises a sequence of SEQ ID NO: 43.
In some
embodiments, the capsid polypeptide, or the reference polypeptide for purposes
of % identity,
comprises a sequence of SEQ ID NO: 44. In some embodiments, the capsid
polypeptide, or the
reference polypeptide for purposes of % identity, comprises a sequence of SEQ
ID NO: 45. In
some embodiments, the capsid polypeptide, or the reference polypeptide for
purposes of %
identity, comprises a sequence of SEQ ID NO: 46.
In some embodiments, the capsid polypeptide, or the reference polypeptide for
purposes
of % identity, comprises a nucleotide sequence of SEQ ID NOs: 47, 48, 49, 50,
51, 52, 53, 54,
55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73,
74, 75, 76, 77, 78, 79, 80,
81, 82, 83, 84, 85, 86, 87, 88, 89, 90, or 91. In some embodiments, the capsid
polypeptide, or the
reference polypeptide for purposes of % identity, comprises a nucleotide
sequence of SEQ ID
NO: 47. In some embodiments, the capsid polypeptide, or the reference
polypeptide for
purposes of % identity, comprises a nucleotide sequence of SEQ ID NO: 48. In
some
embodiments, the capsid polypeptide, or the reference polypeptide for purposes
of % identity,
comprises a nucleotide sequence of SEQ ID NO: 49. In some embodiments, the
capsid
polypeptide, or the reference polypeptide for purposes of % identity,
comprises a nucleotide
sequence of SEQ ID NO: 50. In some embodiments, the capsid polypeptide, or the
reference
polypeptide for purposes of % identity, comprises a nucleotide sequence of SEQ
ID NO: 51. In
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some embodiments, the capsid polypeptide, or the reference polypeptide for
purposes of %
identity, comprises a nucleotide sequence of SEQ ID NO: 52. In some
embodiments, the capsid
polypeptide, or the reference polypeptide for purposes of % identity,
comprises a nucleotide
sequence of SEQ ID NO: 53. In some embodiments, the capsid polypeptide, or the
reference
polypeptide for purposes of % identity, comprises a nucleotide sequence of SEQ
ID NO: 54. In
some embodiments, the capsid polypeptide, or the reference polypeptide for
purposes of %
identity, comprises a nucleotide sequence of SEQ ID NO: 55. In some
embodiments, the capsid
polypeptide, or the reference polypeptide for purposes of % identity,
comprises a nucleotide
sequence of SEQ ID NO: 56. In some embodiments, the capsid polypeptide, or the
reference
polypeptide for purposes of % identity, comprises a nucleotide sequence of SEQ
ID NO: 57.1n
some embodiments, the capsid polypeptide, or the reference polypeptide for
purposes of %
identity, comprises a nucleotide sequence of SEQ ID NO: 58. In some
embodiments, the capsid
polypeptide, or the reference polypeptide for purposes of % identity,
comprises a nucleotide
sequence of SEQ ID NO: 59. In some embodiments, the capsid polypeptide, or the
reference
polypeptide for purposes of % identity, comprises a nucleotide sequence of SEQ
ID NO: 60. In
some embodiments, the capsid polypeptide, or the reference polypeptide for
purposes of %
identity, comprises a nucleotide sequence of SEQ ID NO: 61. In some
embodiments, the capsid
polypeptide, or the reference polypeptide for purposes of % identity,
comprises a nucleotide
sequence of SEQ ID NO: 62. In some embodiments, the capsid polypeptide, or the
reference
polypeptide for purposes of % identity, comprises a nucleotide sequence of SEQ
ID NO: 63. In
some embodiments, the capsid polypeptide, or the reference polypeptide for
purposes of %
identity, comprises a nucleotide sequence of SEQ ID NO: 64. In some
embodiments, the capsid
polypeptide, or the reference polypeptide for purposes of % identity,
comprises a nucleotide
sequence of SEQ ID NO: 65. In some embodiments, the capsid polypeptide, or the
reference
polypeptide for purposes of % identity, comprises a nucleotide sequence of SEQ
ID NO: 66. In
some embodiments, the capsid polypeptide, or the reference polypeptide for
purposes of %
identity, comprises a nucleotide sequence of SEQ ID NO: 67. In some
embodiments, the capsid
polypeptide, or the reference polypeptide for purposes of % identity,
comprises a nucleotide
sequence of SEQ ID NO: 68. In some embodiments, the capsid polypeptide, or the
reference
polypeptide for purposes of % identity, comprises a nucleotide sequence of SEQ
ID NO: 69. In
some embodiments, the capsid polypeptide, or the reference polypeptide for
purposes of %
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identity, comprises a nucleotide sequence of SEQ ID NO: 70. In some
embodiments, the capsid
polypeptide, or the reference polypeptide for purposes of % identity,
comprises a nucleotide
sequence of SEQ ID NO: 71. In some embodiments, the capsid polypeptide, or the
reference
polypeptide for purposes of % identity, comprises a nucleotide sequence of SEQ
ID NO: 72. In
some embodiments, the capsid polypeptide, or the reference polypeptide for
purposes of %
identity, comprises a nucleotide sequence of SEQ ID NO: 73. In some
embodiments, the capsid
polypeptide, or the reference polypeptide for purposes of % identity,
comprises a nucleotide
sequence of SEQ ID NO: 74. In some embodiments, the capsid polypeptide, or the
reference
polypeptide for purposes of % identity, comprises a nucleotide sequence of SEQ
ID NO: 75. In
some embodiments, the capsid polypeptide, or the reference polypeptide for
purposes of %
identity, comprises a nucleotide sequence of SEQ ID NO: 76. In some
embodiments, the capsid
polypeptide, or the reference polypeptide for purposes of % identity.
comprises a nucleotide
sequence of SEQ ID NO: 77. In some embodiments, the capsid polypeptide, or the
reference
polypeptide for purposes of % identity, comprises a nucleotide sequence of SEQ
ID NO: 78. In
some embodiments, the capsid polypeptide. or the reference polypeptide for
purposes of %
identity, comprises a nucleotide sequence of SEQ ID NO: 79. In some
embodiments, the capsid
polypeptide, or the reference polypeptide for purposes of % identity,
comprises a nucleotide
sequence of SEQ ID NO: 80. In some embodiments, the capsid polypeptide, or the
reference
polypeptide for purposes of % identity, comprises a nucleotide sequence of SEQ
ID NO: 81. In
some embodiments, the capsid polypeptide, or the reference polypeptide for
purposes of %
identity, comprises a nucleotide sequence of SEQ ID NO: 82. In some
embodiments, the capsid
polypeptide, or the reference polypeptide for purposes of % identity,
comprises a nucleotide
sequence of SEQ ID NO: 83. In some embodiments, the capsid polypeptide, or the
reference
polypeptide for purposes of % identity, comprises a nucleotide sequence of SEQ
ID NO: 84. In
some embodiments, the capsid polypeptide, or the reference polypeptide for
purposes of %
identity, comprises a nucleotide sequence of SEQ ID NO: 85. In some
embodiments, the capsid
polypeptide, or the reference polypeptide for purposes of % identity,
comprises a nucleotide
sequence of SEQ ID NO: 86. In some embodiments, the capsid polypeptide, or the
reference
polypeptide for purposes of % identity, comprises a nucleotide sequence of SEQ
ID NO: 87. In
some embodiments, the capsid polypeptide, or the reference polypeptide for
purposes of %
identity, comprises a nucleotide sequence of SEQ ID NO: 88. In some
embodiments, the capsid
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polypeptide, or the reference polypeptide for purposes of % identity,
comprises a nucleotide
sequence of SEQ ID NO: 89. In some embodiments, the capsid polypeptide, or the
reference
polypeptide for purposes of % identity, comprises a nucleotide sequence of SEQ
ID NO: 90. In
some embodiments, the capsid polypeptide, or the reference polypeptide for
purposes of %
identity, comprises a nucleotide sequence of SEQ ID NO: 91.
In some embodiments, the capsid polypeptide, or the reference polypeptide for
purposes
% identity, comprises a nucleotide sequence of SEQ ID NOs: 47, 48, 49, 50, 51,
52, 53, 54,
55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73,
74, 75, 76, 77, 78, 79, 80,
81, 82, 83, 84, 85, 86, 87, 88, 89, 90, or 91, that encodes a sequence of SEQ
ID NOs: 2, 3, 4, 5,
6, 7, 8, 9, 10, 11, 12. 13, 14, 15, 16, 17, 18, 19, 20. 21. 22, 23, 24, 25,
26, 27, 28, 29, 30, 31, 32,
33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, or 46. In some
embodiments, the capsid
polypeptide, or the reference polypeptide for purposes of % identity.
comprises a nucleotide
sequence of SEQ ID NO: 47 that encodes a sequence of SEQ ID NO: 2. In some
embodiments,
the capsid polypeptide, or the reference polypeptide for purposes of %
identity, comprises a
nucleotide sequence of SEQ ID NO: 48 that encodes a sequence of SEQ ID NO: 3.
In some
embodiments, the capsid polypeptide, or the reference polypeptide for purposes
of % identity,
comprises a nucleotide sequence of SEQ ID NO: 49 that encodes a sequence of
SEQ ID NO: 4.
In some embodiments, the capsid polypeptide, or the reference polypeptide for
purposes of %
identity, comprises a nucleotide sequence of SEQ ID NO: 50 that encodes a
sequence of SEQ ID
NO: 5. In some embodiments, the capsid polypeptide, or the reference
polypeptide for purposes
of % identity, comprises a nucleotide sequence of SEQ ID NO: 51 that encodes a
sequence of
SEQ ID NO: 6. In some embodiments, the capsid polypeptide, or the reference
polypeptide for
purposes of % identity, comprises a nucleotide sequence of SEQ ID NO: 52 that
encodes a
sequence of SEQ ID NO: 7. In some embodiments, the capsid polypeptide, or the
reference
polypeptide for purposes of % identity, comprises a nucleotide sequence of SEQ
ID NO: 53 that
encodes a sequence of SEQ ID NO: 8. In some embodiments, the capsid
polypeptide, or the
reference polypeptide for purposes of % identity, comprises a nucleotide
sequence of SEQ ID
NO: 54 that encodes a sequence of SEQ ID NO: 9. In some embodiments, the
capsid
polypeptide, or the reference polypeptide for purposes of % identity,
comprises a nucleotide
sequence of SEQ ID NO: 55 that encodes a sequence of SEQ ID NO: 10. In some
embodiments,
the capsid polypeptide, or the reference polypeptide for purposes of %
identity, comprises a
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nucleotide sequence of SEQ ID NO: 56 that encodes a sequence of SEQ ID NO: 11.
In some
embodiments, the capsid polypeptide, or the reference polypeptide for purposes
of % identity,
comprises a nucleotide sequence of SEQ ID NO: 57 that encodes a sequence of
SEQ ID NO: 12.
In some embodiments, the capsid polypeptide, or the reference polypeptide for
purposes of %
identity, comprises a nucleotide sequence of SEQ ID NO: 58 that encodes a
sequence of SEQ ID
NO: 13. In some embodiments, the capsid polypeptide, or the reference
polypeptide for purposes
of % identity, comprises a nucleotide sequence of SEQ ID NO: 59 that encodes a
sequence of
SEQ ID NO: 14. In some embodiments, the capsid polypeptide, or the reference
polypeptide for
purposes of % identity, comprises a nucleotide sequence of SEQ ID NO: 60 that
encodes a
sequence of SEQ ID NO: 15. In some embodiments, the capsid polypeptide, or the
reference
polypeptide for purposes of % identity, comprises a nucleotide sequence of SEQ
ID NO: 61 that
encodes a sequence of SEQ ID NO: 16. In some embodiments, the capsid
polypeptide, or the
reference polypeptide for purposes of % identity, comprises a nucleotide
sequence of SEQ ID
NO: 62 that encodes a sequence of SEQ ID NO: 17. In some embodiments, the
capsid
polypeptide, or the reference polypeptide for purposes of % identity,
comprises a nucleotide
sequence of SEQ ID NO: 63 that encodes a sequence of SEQ ID NO: 18. In some
embodiments,
the capsid polypeptide, or the reference polypeptide for purposes of %
identity, comprises a
nucleotide sequence of SEQ ID NO: 64 that encodes a sequence of SEQ ID NO: 19.
In some
embodiments, the capsid polypeptide, or the reference polypeptide for purposes
of % identity,
comprises a nucleotide sequence of SEQ ID NO: 65 that encodes a sequence of
SEQ ID NO: 20.
In some embodiments, the capsid polypeptide, or the reference polypeptide for
purposes of %
identity, comprises a nucleotide sequence of SEQ ID NO: 66 that encodes a
sequence of SEQ ID
NO: 21. In some embodiments, the capsid polypeptide, or the reference
polypeptide for
purposes of % identity, comprises a nucleotide sequence of SEQ ID NO: 67 that
encodes a
sequence of SEQ ID NO: 22. In some embodiments, the capsid polypeptide, or the
reference
polypeptide for purposes of % identity, comprises a nucleotide sequence of SEQ
ID NO: 68 that
encodes a sequence of SEQ ID NO: 23. In some embodiments, the capsid
polypeptide, or the
reference polypeptide for purposes of % identity, comprises a nucleotide
sequence of SEQ ID
NO: 69 that encodes a sequence of SEQ ID NO: 24. In some embodiments, the
capsid
polypeptide, or the reference polypeptide for purposes of % identity,
comprises a nucleotide
sequence of SEQ ID NO: 70 that encodes a sequence of SEQ ID NO: 25. In some
embodiments,
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the capsid polypeptide, or the reference polypeptide for purposes of %
identity, comprises a
nucleotide sequence of SEQ ID NO: 71 that encodes a sequence of SEQ ID NO: 26.
In some
embodiments, the capsid polypeptide, or the reference polypeptide for purposes
of % identity,
comprises a nucleotide sequence of SEQ ID NO: 72 that encodes a sequence of
SEQ ID NO: 27.
In some embodiments, the capsid polypeptide, or the reference polypeptide for
purposes of %
identity, comprises a nucleotide sequence of SEQ ID NO: 73 that encodes a
sequence of SEQ ID
NO: 28. In some embodiments, the capsid polypeptide, or the reference
polypeptide for
purposes of % identity, comprises a nucleotide sequence of SEQ ID NO: 74 that
encodes a
sequence of SEQ ID NO: 29. In some embodiments, the capsid polypeptide, or the
reference
polypeptide for purposes of % identity, comprises a nucleotide sequence of SEQ
ID NO: 75 that
encodes a sequence of SEQ ID NO: 30. In some embodiments, the capsid
polypeptide, or the
reference polypeptide for purposes of ck identity, comprises a nucleotide
sequence of SEQ ID
NO: 76 that encodes a sequence of SEQ ID NO: 31. In some embodiments, the
capsid
polypeptide, or the reference polypeptide for purposes of % identity,
comprises a nucleotide
sequence of SEQ ID NO: 77 that encodes a sequence of SEQ ID NO: 32. In some
embodiments,
the capsid polypeptide, or the reference polypeptide for purposes of %
identity, comprises a
nucleotide sequence of SEQ ID NO: 78 that encodes a sequence of SEQ ID NO: 33.
In some
embodiments, the capsid polypeptide, or the reference polypeptide for purposes
of % identity,
comprises a nucleotide sequence of SEQ ID NO: 79 that encodes a sequence of
SEQ ID NO: 34.
In some embodiments, the capsid polypeptide, or the reference polypeptide for
purposes of %
identity, comprises a nucleotide sequence of SEQ ID NO: 80 that encodes a
sequence of SEQ ID
NO: 35. In some embodiments, the capsid polypeptide, or the reference
polypeptide for
purposes of % identity, comprises a nucleotide sequence of SEQ ID NO: 81 that
encodes a
sequence of SEQ ID NO: 36. In some embodiments, the capsid polypeptide, or the
reference
polypeptide for purposes of % identity, comprises a nucleotide sequence of SEQ
ID NO: 82 that
encodes a sequence of SEQ ID NO: 37. In some embodiments, the capsid
polypeptide, or the
reference polypeptide for purposes of % identity, comprises a nucleotide
sequence of SEQ ID
NO: 83 that encodes a sequence of SEQ ID NO: 38. In some embodiments, the
capsid
polypeptide, or the reference polypeptide for purposes of % identity,
comprises a nucleotide
sequence of SEQ ID NO: 84 that encodes a sequence of SEQ ID NO: 39. In some
embodiments,
the capsid polypeptide, or the reference polypeptide for purposes of %
identity, comprises a
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nucleotide sequence of SEQ ID NO: 85 that encodes a sequence of SEQ ID NO: 40.
In some
embodiments, the capsid polypeptide, or the reference polypeptide for purposes
of % identity,
comprises a nucleotide sequence of SEQ ID NO: 86 that encodes a sequence of
SEQ ID NO: 41.
In some embodiments, the capsid polypeptide, or the reference polypeptide for
purposes of %
identity, comprises a nucleotide sequence of SEQ ID NO: 87 that encodes a
sequence of SEQ ID
NO: 42. In some embodiments, the capsid polypeptide, or the reference
polypeptide for
purposes of % identity, comprises a nucleotide sequence of SEQ ID NO: 88 that
encodes a
sequence of SEQ ID NO: 43. In some embodiments, the capsid polypeptide, or the
reference
polypeptide for purposes of % identity, comprises a nucleotide sequence of SEQ
ID NO: 89 that
encodes a sequence of SEQ ID NO: 44. In some embodiments, the capsid
polypeptide, or the
reference polypeptide for purposes of % identity, comprises a nucleotide
sequence of SEQ ID
NO: 90 that encodes a sequence of SEQ ID NO: 45. In some embodiments, the
capsid
polypeptide, or the reference polypeptide for purposes of % identity,
comprises a nucleotide
sequence of SEQ ID NO: 91 that encodes a sequence of SEQ ID NO: 46.
In some embodiments, the capsid polypeptide, or the reference polypeptide for
purposes
of % identity, comprises a sequence of SEQ ID NOs: 2, 3, 4, 5, 6, 7, 8, 9, 10,
11, 12, 13, 14, 15,
16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,
35, 36, 37, 38, 39, 40, 41,
42, 43, 44, 45, or 46 that is encoded by a nucleotide sequence of SEQ ID NOs:
47, 48, 49, 50, 51,
52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70,
71, 72, 73, 74, 75, 76, 77,
78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, or 91. In some
embodiments, the capsid
polypeptide, or the reference polypeptide for purposes of % identity,
comprises a sequence of
SEQ ID NO: 2 that is encoded by a nucleotide sequence of SEQ ID NO: 47. In
some
embodiments, the capsid polypeptide, or the reference polypeptide for purposes
of % identity,
comprises a sequence of SEQ ID NO: 3 that is encoded by a nucleotide sequence
of SEQ ID NO:
48. In some embodiments, the capsid polypeptide, or the reference polypeptide
for purposes of
% identity, comprises a sequence of SEQ ID NO: 4 that is encoded by a
nucleotide sequence of
SEQ ID NO: 49. In some embodiments, the capsid polypeptide, or the reference
polypeptide for
purposes of % identity, comprises a sequence of SEQ ID NO: 5 that is encoded
by a nucleotide
sequence of SEQ ID NO: 50. In some embodiments, the capsid polypeptide, or the
reference
polypeptide for purposes of % identity, comprises a sequence of SEQ ID NO: 6
that is encoded
by a nucleotide sequence of SEQ ID NO: 51. In some embodiments, the capsid
polypeptide, or
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the reference polypeptide for purposes of % identity, comprises a sequence of
SEQ ID NO: 7
that is encoded by a nucleotide sequence of SEQ ID NO: 52. In some
embodiments, the capsid
polypeptide, or the reference polypeptide for purposes of % identity,
comprises a sequence of
SEQ ID NO: 8 that is encoded by a nucleotide sequence of SEQ ID NO: 53. In
some
embodiments, the capsid polypeptide, or the reference polypeptide for purposes
of % identity,
comprises a sequence of SEQ ID NO: 9 that is encoded by a nucleotide sequence
of SEQ ID NO:
54. In some embodiments, the capsid polypeptide, or the reference polypeptide
for purposes of
% identity, comprises a sequence of SEQ ID NO: 10 that is encoded by a
nucleotide sequence of
SEQ ID NO: 55. In some embodiments, the capsid polypeptide, or the reference
polypeptide for
purposes of % identity, comprises a sequence of SEQ ID NO: 11 that is encoded
by a nucleotide
sequence of SEQ ID NO: 56. In some embodiments, the capsid polypeptide, or the
reference
polypeptide for purposes of % identity, comprises a sequence of SEQ ID NO: 12
that is encoded
by a nucleotide sequence of SEQ ID NO: 57. In some embodiments, the capsid
polypeptide, or
the reference polypeptide for purposes of % identity, comprises a sequence of
SEQ ID NO: 13
that is encoded by a nucleotide sequence of SEQ ID NO: 58. In some
embodiments, the capsid
polypeptide, or the reference polypeptide for purposes of % identity,
comprises a sequence of
SEQ ID NO: 14 that is encoded by a nucleotide sequence of SEQ ID NO: 59. In
some
embodiments, the capsid polypeptide, or the reference polypeptide for purposes
of % identity,
comprises a sequence of SEQ ID NO: 15 that is encoded by a nucleotide sequence
of SEQ ID
NO: 60. In some embodiments, the capsid polypeptide, or the reference
polypeptide for
purposes of % identity, comprises a sequence of SEQ ID NO: 16 that is encoded
by a nucleotide
sequence of SEQ ID NO: 61. In some embodiments, the capsid polypeptide, or the
reference
polypeptide for purposes of % identity, comprises a sequence of SEQ ID NO: 17
that is encoded
by a nucleotide sequence of SEQ ID NO: 62. In some embodiments, the capsid
polypeptide, or
the reference polypeptide for purposes of % identity, comprises a sequence of
SEQ ID NO: 18
that is encoded by a nucleotide sequence of SEQ ID NO: 63. In some
embodiments, the capsid
polypeptide, or the reference polypeptide for purposes of % identity,
comprises a sequence of
SEQ ID NO: 19 that is encoded by a nucleotide sequence of SEQ ID NO: 64. In
some
embodiments, the capsid polypeptide, or the reference polypeptide for purposes
of % identity,
comprises a sequence of SEQ ID NO: 20 that is encoded by a nucleotide sequence
of SEQ ID
NO: 65. In some embodiments, the capsid polypeptide, or the reference
polypeptide for purposes
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of % identity, comprises a sequence of SEQ ID NO: 21 that is encoded by a
nucleotide sequence
of SEQ ID NO: 66. In some embodiments, the capsid polypeptide, or the
reference polypeptide
for purposes of % identity, comprises a sequence of SEQ ID NO: 22 that is
encoded by a
nucleotide sequence of SEQ ID NO: 67. In some embodiments, the capsid
polypeptide, or the
reference polypeptide for purposes of % identity, comprises a sequence of SEQ
ID NO: 23 that is
encoded by a nucleotide sequence of SEQ ID NO: 68. In some embodiments, the
capsid
polypeptide, or the reference polypeptide for purposes of % identity,
comprises a sequence of
SEQ ID NO: 24 that is encoded by a nucleotide sequence of SEQ ID NO: 69. In
some
embodiments, the capsid polypeptide, or the reference polypeptide for purposes
of % identity,
comprises a sequence of SEQ ID NO: 25 that is encoded by a nucleotide sequence
of SEQ ID
NO: 70. In some embodiments, the capsid polypeptide, or the reference
polypeptide for
purposes of % identity, comprises a sequence of SEQ ID NO: 26 that is encoded
by a nucleotide
sequence of SEQ ID NO: 71. In some embodiments, the capsid polypeptide, or the
reference
polypeptide for purposes of % identity, comprises a sequence of SEQ ID NO: 27
that is encoded
by a nucleotide sequence of SEQ ID NO: 72. In some embodiments, the capsid
polypeptide, or
the reference polypeptide for purposes of % identity, comprises a sequence of
SEQ ID NO: 28
that is encoded by a nucleotide sequence of SEQ ID NO: 73. In some
embodiments, the capsid
polypeptide, or the reference polypeptide for purposes of % identity,
comprises a sequence of
SEQ ID NO: 29 that is encoded by a nucleotide sequence of SEQ ID NO: 74. In
some
embodiments, the capsid polypeptide, or the reference polypeptide for purposes
of % identity,
comprises a sequence of SEQ ID NO: 30 that is encoded by a nucleotide sequence
of SEQ ID
NO: 75. In some embodiments, the capsid polypeptide, or the reference
polypeptide for
purposes of % identity, comprises a sequence of SEQ ID NO: 31 that is encoded
by a nucleotide
sequence of SEQ ID NO: 76. In some embodiments, the capsid polypeptide, or the
reference
polypeptide for purposes of % identity, comprises a sequence of SEQ ID NO: 32
that is encoded
by a nucleotide sequence of SEQ ID NO: 77. In some embodiments, the capsid
polypeptide, or
the reference polypeptide for purposes of % identity, comprises a sequence of
SEQ ID NO: 33
that is encoded by a nucleotide sequence of SEQ ID NO: 78. In some
embodiments, the capsid
polypeptide, or the reference polypeptide for purposes of % identity,
comprises a sequence of
SEQ ID NO: 34 that is encoded by a nucleotide sequence of SEQ ID NO: 79.
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In some embodiments, the capsid polypeptide, or the reference polypeptide for
purposes
of % identity, comprises a sequence of SEQ ID NO: 33 that is encoded by a
nucleotide sequence
of SEQ ID NO: 78. In some embodiments, the capsid polypeptide, or the
reference polypeptide
for purposes of % identity, comprises a sequence of SEQ ID NO: 34 that is
encoded by a
nucleotide sequence of SEQ ID NO: 79. In some embodiments, the capsid
polypeptide, or the
reference polypeptide for purposes of % identity, comprises a sequence of SEQ
ID NO: 35 that is
encoded by a nucleotide sequence of SEQ ID NO: 80. In some embodiments, the
capsid
polypeptide, or the reference polypeptide for purposes of % identity,
comprises a sequence of
SEQ ID NO: 36 that is encoded by a nucleotide sequence of SEQ ID NO: 81. In
some
embodiments, the capsid polypeptide, or the reference polypeptide for purposes
of % identity,
comprises a sequence of SEQ ID NO: 37 that is encoded by a nucleotide sequence
of SEQ ID
NO: 82. In some embodiments, the capsid polypeptide, or the reference
polypeptide for
purposes of % identity, comprises a sequence of SEQ ID NO: 38 that is encoded
by a nucleotide
sequence of SEQ ID NO: 83. In some embodiments, the capsid polypeptide, or the
reference
polypeptide for purposes of % identity, comprises a sequence of SEQ ID NO: 39
that is encoded
by a nucleotide sequence of SEQ ID NO: 84. In some embodiments, the capsid
polypeptide, or
the reference polypeptide for purposes of % identity, comprises a sequence of
SEQ ID NO: 40
that is encoded by a nucleotide sequence of SEQ ID NO: 85. In some
embodiments, the capsid
polypeptide, or the reference polypeptide for purposes of % identity,
comprises a sequence of
SEQ ID NO: 41 that is encoded by a nucleotide sequence of SEQ ID NO: 86. In
some
embodiments, the capsid polypeptide, or the reference polypeptide for purposes
of % identity,
comprises a sequence of SEQ ID NO: 42 that is encoded by a nucleotide sequence
of SEQ ID
NO: 87. In some embodiments, the capsid polypeptide, or the reference
polypeptide for
purposes of % identity, comprises a sequence of SEQ ID NO: 43 that is encoded
by a nucleotide
sequence of SEQ ID NO: 88. In some embodiments, the capsid polypeptide, or the
reference
polypeptide for purposes of % identity, comprises a sequence of SEQ ID NO: 44
that is encoded
by a nucleotide sequence of SEQ ID NO: 89. In some embodiments, the capsid
polypeptide, or
the reference polypeptide for purposes of % identity, comprises a sequence of
SEQ ID NO: 45
that is encoded by a nucleotide sequence of SEQ ID NO: 90. In some
embodiments, the capsid
polypeptide, or the reference polypeptide for purposes of % identity,
comprises a sequence of
SEQ ID NO: 46 that is encoded by a nucleotide sequence of SEQ ID NO: 91.
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In some embodiments, the capsid polypeptide comprises a sequence that includes
all of
the mutation differences associated with any one of VAR-1 through VAR-45
(e.g., as indicated
in Table 1, column 7), and further includes no more than 30, no more than 20,
no more than 10,
no more than 9, no more than 8, no more than 7, no more than 6, no more than
5, no more than 4,
no more than 3, no more than 2 or no more than 1 additional mutations relative
to SEQ ID NO:
1.
In some embodiments, the capsid polypeptide is a VP1 capsid polypeptide. In
embodiments, the capsid polypeptide is a VP2 capsid polypeptide. In
embodiments, the capsid
polypeptide is a VP3 capsid polypeptide. With respect to reference sequence
SEQ ID NO: 1, a
VP1 capsid polypeptide comprises amino acids 1-724 of SEQ ID NO: 1. With
respect to
reference sequence SEQ ID NO: 1, a VP2 capsid polypeptide comprises amino
acids 138-724 of
SEQ ID NO: 1. With respect to reference sequence SEQ ID NO: 1, a VP3 capsid
polypeptide
comprises amino acids 203-724 of SEQ ID NO: 1.
Table 1 lists information regarding exemplary variant dependoparvovirus
particles
comprising nucleic acids comprising the variant capsid regarding the ocular
transduction
properties and production characteristics of said non-limiting exemplary
variants. Exemplary
sequences of capsid polypeptides and nucleic acid molecules encoding the same
are provided in
Table 2, Table 3, and Table 4.
Table 1. Transduction and virus production of exemplary variant
dependoparvovirus (e.g., AVV)
particles comprising variant capsid polypeptides. Macular and retinal
transduction are as
measured following IVT injection; trabecular transduction is as measured
following IC injection.
Substitutions are notated as n##-#N where "N" is the final amino acid, "n" is
the reference amino
acid and "###" is the reference amino acid position of SEQ ID NO:1; deletions
are notated as
n###- where "-" indicates the deletion of "n" at position "###" of the
reference sequence SEQ ID
NO: 1; insertions are notated as ### Naa_###_(n)y, where "###" are the amino
acid positions in
the reference sequence SEQ ID NO: 1 between which the insertion occurs, "Naa"
refers to the
length of the insertion (having "N" aminio acids) and "(n)y" providing the
sequence of the
insertion). Each individual Mutation Difference (e.g., within a row, each
mutation in quotations
(") in column 7) and combinations of such individual mutation differences is
sometimes referred
to herein as a "mutation associated with VAR-X", where VAR-X is the variant
identifier listed in
the "Name column."
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Table 1
Name SEQ ID Macular Retinal Trabecular Virus
Mutation
NO: of Transduction Transduction Transduction Production as Differences as
VP1 as compared as compared as compared compared to
compared to SEQ
capsid to wild-type to wild-type to wild-type wild-type SEQ ID NO: 1
poly- SEQ ID NO: SEQ ID NO: 1SEQ ID NO: 1 ID NO: 1
(Collectively the
peptide 1 (Log2) (Log2) (Log2)
"Mutation Set")
(Log2)
VAR-1 2 -0.623109184 -3.703538304 1.725067698 -0.970942107
['T14L', 'L15V',
9A', 'K2411']
VAR-2 3 -0.048551556 -2.614465707 2.659319064 -3.169014336
['A3V', 'Y6F',
'L 1 1F', 'E 12Q,
'Q21L', 'W231',
'L25C', 'P29A',
'P3 1N', 'K33R']
VAR-3 4 5.790478596 3.036979883 0.395414334 -1.00780873
['N449Q',
'T450M',
'P45 1T', 'T455L',
'T456G',
'Q457T', 'S458Q',
'R459M']
VAR-4 5 5.614983257 4.644634839 1.892617279 0.250924135
['N449Q',
P451-
','S452T',
'T456G',
'Q457T',
'R459G1
VAR-5 6 3.88486946 -2.450075546 -1.634402766 -1.233590205
['Q457F,
'S458C1
VAR-6 7 2.853842111 -1.792540736 -2.923205527 -1.532404634
['446 7aa_447_K
CQEGMA']
VAR-7 8 2.970149877 -1.540722356 -1.140865448 -3.184814278
[449_7aa 450 L
MVDRLG']
VAR-8 9 5.582852851 5.427442711 2.525830415 1.385285762
[P4511', 7455G',
'T456G', 'Q457T',
'S458Q', 'R459T',
'Q461A1
VAR-9 10 4.669316134 3.927282285 -3.376530605 0.001142983 [448
7aa 449 H
CQECPF]
VAR- 11 3.292909035 3.163362277 -1.978073355 1.240848456 ['453
6aa 454 F
SGLEN']
VAR- 12 -0.576693086 3.596174251 3.834588967 -2.671820443
[T456-', 'Q457-',
11 'S458-
', 'R459-',
'L460-', 'Q461-']
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VAR- 13 -0.11342336711.044477801 3.674269913 -3.189453528
[N4491',
12
'T450N', 'P451G',
T454-'T455Q',
7456N', 'S458Q',
'R459T',
'Q461K'.
'Q464V']
VAR- 14 3.165665208 3.89324002 1100397565 0.842518983 [S446A',
'T448-',
13
*1\1449-*, *P451Q,
'G453T',
'T455G',
'T4566',
'Q457T',
R459G1
VAR- 15 2.075188868 3.289224002 3.023792623 -0.475270261
[N449Q',
14
'T450S',
'451 laa 452 Y'
, 'S452G',
'T455A', 'Q457F,
'S458M'.
R459D]
VAR- 16 -0.376914592-3.500304156 1.324154172 -2.458171594
['447 7aa 448 H
15
ETEFNF]
VAR- 17 5.582852851 5.427442711 2.525830415 1.385285762
[P451T,
16
'T455G',
'T456G',
'Q457T', 'S458Q',
'R459T',
'Q461A']
VAR- 18 2.655350309 -3.112635564 -3.049750846 -2.601297654
['581 6aa 582 F
17
ALMEP']
VAR- 19 4.005041582 4.890414229 -2.277159374 0.742690925 ['584
6aa 585 R
18
AYNPD']
VAR- 20 -0.533212469 0.980979298 3.697444744 0.090864036
[E555A`,
19
'D561S',
'R566K',
'S578V', 'S580A',
'T581A', 'R585-']
VAR- 21 2.556229802 1.402914506 3.663931804 -1.767018218
[V557L',
20
'S578D']
VAR- 22 0.208673258 3.174958358 3.619270294 0.907816878
[K556N',
21
'M558L', 'S578I',
'S580A']
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VAR- 23 -1.16330231510.807138873 2.946707073 Ø724043413
[1554L',
22
'K556R', '1559L',
'D561S',
'R566A',
'T581D', 'R585S',
'G586R',
'N587S']
VAR- 24 -0.603741502 -3.96314209 2.195605843 -1.54313115
['V552A',
23
*E555S*, *K5561)*,
'1559L', 'D561S',
'R566K', 'S578I',
7581D',
'G586Q']
VAR- 25 -0.32559336 -2.523773182 3.018751224 -2.351824168
['Q575E',
24
'S578A']
VAR- 26 -0.764009051 -3.347174497 1.221512579 -2.743972813
['583 6aa 584 L
25
NWTAE']
VAR- 27 6.543457411 4.274472882 Not measured 2.647908003
[R585S',
26 'R588T,
'Q589N'.
'A590P', 'A591I',
'A593G'. 'T597S',
'V600A']
VAR- 28 2.378409668 -2.554934829 -3.294730082 -1.661215309
['584 8aa 585 L
27
AKEFTTR,
'R585N',
'G586A1
VAR- 29 3.179099645 -1.033272432 -3.418068966 -0.287722121
['592 5aa 593 L
28 HPLE*1
VAR- 30 0.106530846 5.978252156 -1.307152207 -1.835575632
[585_1aa_586_F
29 ']
VAR- 31 2.388880748 5.767603532 -0.598614376 -1.37771365 [586
7aa 587 D
30
QDFICNR]
VAR- 32 1.231859812 5.703988038 -0.109487377 -0.461783742 [589
laa 590 I'
31
VAR- 33 7.17868729 5.676618671 1.847520944 1.47820717 ['587
9aa 588 L
32
AIEQTRPAI
VAR- 34 6.718793621 5.633141613 0.915715523 0.089517923 ['584
8aa 585 R
33
ARLDETT',
'G586P',
'N587A']
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VAR- 35 7.147097174 15.009347977 2.548103321 .-0.417075
['586 9aa 587 L
34
ALAEITRP',
'N587A']
VAR- 36 7.144189999 4.822397494 0.295037676 0.656194018 ['586
9aa 587 L
35
ANGEQTRP',
'N587A]
VAR- 37 5.69301826 5.642147204 1.165262957 1.961105857 ['586
6aa 587 A
36
TDTKT']
VAR- 38 5.426413987 5.646189636 2.526214031 0.606956149 ['590
laa 591 P
37
VAR- 39 5.256650619 5.61442957 3.562509958 1.182074152 ['587
9aa 588 A
38
PGETTRPA1
VAR- 40 5.560027745 5.255320701 2.631680376 1.835598668 ['589
laa 590 P
39 ']
VAR- 41 5.342177909 5.237000927 -0.042394845 0.280289485
['R585S',
40
'G586S',
'N 87A'
'A591E',
D594R',
7597A', 'V6001]
VAR- 42 6.012003904 2.91024213 -2.709889212 -1.935204488 ['586
7aa 587 F
41
HNEGKY']
VAR- 43 4.249223885 4.499692005 -2.506762655 0.70070977
['591 laa 592 G
42 ']
VAR- 44 0.482055709 1.250967689 3.121559452 -0.741624797
[588_7aa_589_Q
43
PWEPDK']
VAR- 45 -0.029969745 -1.721769787 1.831873906 -2.556026398
['592 8aa 593 A
44
LALSTTN]
VAR- 46 0.8687088
-1.294220763 2.096329284 -1.272924619 ['585 6aa 586 P
45
WGTAG']
Table 2
Cap sid Amino Acid Sequence of VP1 Exemplary Nucleic Acid Molecule
Sequence(SEQ
Variant capsid polypeptide (SEQ ID ID NO)
NO; starting amino acid of
VP2 is underlined; starting
amino acid of VP3 is in bold.
VAR-1 MAADGYLPDWLEDLVSE ATGGCTGCCGATGGTTATCTTCCAGATTGG
GARQWWHLKPGPPPPKP CTCGAGGACCTGGTATCTGAAGGAGCGAG
AERHKDDSRGLVLPGYKY ACAGTGGTGGCATCTCAAACCTGGCCCAC
LGPFNGLDKGEPVNEADA CACCACCAAAGCCCGCAGAGCGGCATAAG
74
CA 03220810 2023- 11- 29 SUBSTITUTE SHEET (RULE 26)
WO 2022/256557
PCT/US2022/032004
AALEHDKAYDRQLDSGD GACGACAGCAGGGGTCTTGTGCTTCCTGG
NPYLKYNHADAEFQERLK GTACAAGTACCTCGGACCCTTCAACGGACT
EDTSFGGNLGRAVFQAKK CGACAAGGGAGAGCCGGTCAACGAGGCAG
RVLEPLGLVEEPVKTAPG ACGCCGCGGCCCTCGAGCACGACAAAGCC
KKRPVEHSPVEPDSSSGTG TACGACCGGCAGCTCGACAGCGGAGACAA
KAGQQPARKRLNFGQTG CCCGTACCTCAAGTACAACCACGCCGACG
DADS VPDPQPLGQPPAAP CGGAGTTTCAGGAGCGCCTTAAAGAAGAT
SGLGTNTMATGSGAPMA ACGTCTTTTGGGGGCAACCTCGGACGAGC
DNNEGADGVGNSSGNWH AGTCTTCCAGGCGAAAAAGAGGGTTCTTG
CDSTWMGDRVITTSTRTW AACCTCTGGGCCTGGTTGAGGAACCTGTTA
ALPTYNNHLYKQISSQSG AGACGGCTCCGGGAAAAAAGAGGCCGGTA
ASNDNHYFGYSTPWGYF GAGCACTCTCCTGTGGAGCCAGACTCCTCC
DFNRFHCHFSPRDWQRLI TCGGGAACCGGAAAGGCGGGCCAGCAGCC
NNNWGFRPKRLNFKLFNI TGCAAGAAAAAGATTGAATTTTGGTCAGA
QVKEVTQNDGTTTIANNL CTGGAGACGCAGACTCAGTACCTGACCCC
TSTVQVFTDSEYQLPYVL CAGCCTCTCGGACAGCCACCAGCAGCCCC
GSAHQGCLPPFPADVFMV CTCTGGTCTGGGAACTAATACGATGGCTAC
PQYGYLTLNNGSQAVGRS AGGCAGTGGCGCACCAATGGCAGACAAT A
SFYCLEYFPSQMLRTGNN ACGAGGGCGCCGACGGAGTGGGTAATTCC
FTFSYTFEDVPFHSSYAHS TCGGGAAATTGGCATTGCGATTCCACATGG
QSLDRLMNPLIDQYLYYL ATGGGCGACAGAGTCATCACCACCAGCAC
SRTNTPSGTTTQSRLQFSQ CCGAACCTGGGCCCTGCCCACCTACAACA
AGASDIRDQSRNWLPGPC ACC ACCTCTAC A A AC A AATTTCCAGCCAAT
YRQQRVSKTSADNNNSEY CAGGAGCCTCGAACGACAATCACTACTTT
SWTGATKYHLNGRDSLV GGCTACAGCACCCCTTGGGGGTATTTTGAC
NPGPAMASHKDDEEKFFP TTCAACAGATTCCACTGCCACTTTTCACCA
QSGVLIFGKQGSEKTNVDI CGTGACTGGCAAAGACTCATCAACAACAA
EKVMITDEEEIRTTNP VAT CTGGGGATTCCGACCCAAGAGACTCAACT
EQYGSVSTNLQRGNRQAA TCAAGCTCTTTAACATTCAAGTCAAAGAGG
TADVNTQGVLPGMVWQD TCACGCAGAATGACGGTACGACGACGATT
RDVYLQGPIWAKIPHTDG GCCAATAACCTTACCAGCACGGTTCAGGT
HFHPSPLMGGFGLKHPPP GTTTACTGACTCGGAGTACCAGCTCCCGTA
QILIKNTPVPANPSTTFSAA CGTCCTCGGCTCGGCGCATCAAGGATGCCT
KFASFITQYSTGQVSVEIE CCCGCCGTTCCCAGCAGACGTCTTCATGGT
WELQKENSKRWNPEIQYT GCCACAGTATGGATACCTCACCCTGAACA
SNYNKSVNVDFTVDTNG ACGGGAGTCAGGCAGTAGGACGCTCTTCA
VYSEPRPIGTRYLTRNL" TTTTACTGCCTGGAGTACTTTCCTTCTCAG
(SEQ ID NO: 2)
ATGCTGCGTACCGGAAACAACTTTACCTTC
AGCTACACTTTTGAGGACGTTCCTTTCCAC
AGCAGCTACGCTCACAGCCAGAGTCTGGA
CCGTCTCATGAATCCTCTCATCGACCAGTA
CCTGTATTACTTGAGCAGAACAAACACTCC
AAGTGGAACCACCACGCAGTCAAGGCTTC
AGTTTTCTCAGGCCGGAGCGAGTGACATTC
GGGACCAGTCTAGGAACTGGCTTCCTGGA
CCCTGTTACCGCCAGCAGCGAGTATCAAA
CA 03220810 2023- 11- 29 SUBSTITUTE SHEET (RULE 26)
WO 2022/256557
PCT/US2022/032004
GACATCTGCGGATAACAACAACAGTGAAT
ACTCGTGGACTGGAGCTACCAAGTACCAC
CTCAATGGCAGAGACTCTCTGGTGAATCCG
GGCCCGGCCATGGCAAGCCACAAGGACGA
TGAAGAAAAGTTTTTTCCTCAGAGCGGGGT
TCTCATCTTTGGGAAGCAAGGCTCAGAGA
AAACAAATGTGGACATTGAAAAGGTCATG
ATTACAGACGAAGAGGAAATCAGGACAAC
CAATCCCGTGGCTACGGAGCAGTATGGTTC
TGTATCTACCAACCTCCAGAGAGGCAACA
GACAAGCAGCTACCGCAGATGTCAACACA
CAAGGCGTTCTTCCAGGCATGGTCTGGCAG
GACAGAGATGTGTACCTTCAGGGGCCCAT
CTGGGCAAAGATTCCACACACGGACGGAC
ATTTTCACCCCTCTCCCCTCATGGGTGGAT
TCGGACTTAAACACCCTCCTCCACAGATTC
TCATCAAGAACACCCCGGTACCTGCGAAT
CCTTCGACCACCTTCAGTGCGGCAAAGTTT
GCTTCCTTCATCACACAGTACTCCACGGGA
CAGGTCAGCGTGGAGATCGAGTGGGAGCT
GCAGAAGGAAAACAGCAAACGCTGGAATC
CCGAAATTCAGTACACTTCCAACTACAACA
AGTCTGTTA ATGTGGACTTTACTGTGGAC A
CTAATGGCGTGTATTCAGAGCCTCGCCCCA
TTGGCACCAGATACCTGACTCGTAATCTGT
AA (SEQ ID NO: 47)
VAR-2 MAVDGFLPDWFQDTLSE ATGGCTGTTGATGGTTTTCTTCCAGATTGG
GIRLWIKCKPGAPNPRPAE TTTCAAGACACTCTCTCTGAAGGAATAAGA
RHKDDSRGLVLPGYKYLG TTATGGATCAAGTGCAAACCTGGCGCGCC
PFNGLDKGEPVNEADAAA AAACCCACGCCCCGCAGAGCGGCATAAGG
LEHDKAYDRQLDSGDNP ACGACAGCAGGGGTCTTGTGCTTCCTGGGT
YLKYNHADAEFQERLKED ACAAGTACCTCGGACCCTTCAACGGACTC
TSFGGNLGRAVFQAKKRV GACAAGGGAGAGCCGGTCAACGAGGCAG
LEPLGLVEEPVKTAPGKK ACGCCGCGGCCCTCGAGCACGACAAAGCC
RPVEHSPVEPDSSSGTGKA TACGACCGGCAGCTCGACAGCGGAGACAA
GQQPARKRLNFGQTGDA CCCGTACCTCAAGTACAACCACGCCGACG
DSVPDPQPLGQPPAAPSGL CGGAGTTTCAGGAGCGCCTT A A AGA AGAT
GTNTMATGSGAPMADNN ACGTCTTTTGGGGGCAACCTCGGACGAGC
EGADGVGNSSGNWHCDS AGTCTTCCAGGCGAAAAAGAGGGTTCTTG
TWMGDRVITTSTRTWALP AACCTCTGGGCCTGGTTGAGGAACCTGTTA
TYNNHLYKQISSQSGASN AGACGGCTCCGGGAAAAAAGAGGCCGGTA
DNHYEGYSTPWGYFDENR GAGCACTCTCCTGTGGAGCCAGACTCCTCC
FHCHFSPRDWQRLINNNW TCGGGAACCGGAAAGGCGGGCCAGCAGCC
GFRPKRLNFKLFNIQVKEV TGCAAGAAAAAGATTGAATTTTGGTCAGA
TQNDGTTTIANNLTSTVQ CTGGAGACGCAGACTCAGTACCTGACCCC
VFTDSEYQLPYVLGSAHQ CAGCCTCTCGGACAGCCACCAGCAGCCCC
76
CA 03220810 2023- 11- 29 SUBSTITUTE SHEET (RULE 26)
WO 2022/256557 PCT/US2022/032004
GCLPPFPADVFMVPQYGY CTCTGGTCTGGGAACTAATACGATGGCTAC
LTLNNGS QAVGRSS FYCL AGGCAGTGGCGCACCAATGGCAGACAAT A
EYFPS QMLRTGNNFTFSY ACGAGGGCGCCGACGGAGTGGGTAATTCC
TFEDVPFHSSYAHSQSLDR TCGGGAAATTGGCATTGCGATTCCACATGG
LMNPLIDQYLYYLSRTNT ATGGGCGACAGAGTCATCACCACCAGCAC
PS GTTTQSRLQFSQAGASD CCGAACCTGGGCCCTGCCCACCTACAACA
IRDQSRNWLPGPCYRQQR ACC ACCTCTACAAACAAATTTCCAGCCAAT
VS KTS ADNNNSEYSWTGA CAGGAGCCTCGAACGACAATCACTACTTT
TKYHLNGRDSLVNPGPA GGCTACAGCACCCCTTGGGGGTATTTTGAC
MASHKDDEEKFFPQSGVL TTCAACAGATTCCACTGCCACTTTTCACCA
IFGKQGSEKTNVDIEKVMI CGTGACTGGCAAAGACTCATCAACAACAA
TDEEEIRTTNPVATEQYGS CTGGGGATTCCGACCCAAGAGACTCAACT
VSTNLQRGNRQAATADV TCAAGCTCTTTAACATTCAAGTCAAAGAGG
NTQGVLPGMVWQDRDVY TCACGCAGAATGACGGTACGACGACGATT
LQGPIWAKIPHTDGHFHPS GCCAATAACCTTACCAGCACGGTTCAGGT
PLMGGFGLKHPPPQILIKN GTTTACTGACTCGGAGTACCAGCTCCCGTA
TPVPANPSTTFSAAKFASF CGTCCTCGGCTCGGCGCATCAAGGATGCCT
ITQYSTGQVSVEIEWELQK CCCGCCGTTCCCAGCAGACGTCTTCATGGT
ENS KRWNPEIQYT SNYNK GCCACAGTATGGATACCTCACCCTGAACA
SVNVDFTVDTNGVYSEPR ACGGGAGTCAGGCAGTAGGACGCTCTTCA
PIGTRYLTRNL* (SEQ ID TTTTACTGCCTGGAGTACTTTCCTTCTCAG
NO: 3)
ATGCTGCGTACCGGAAACAACTTTACCTTC
AGCT AC ACTTTTGAGGACGTTCCTTTCC AC
AGCAGCTACGCTCACAGCCAGAGTCTGGA
CCGTCTCATGAATCCTCTCATCGACCAGTA
CCTGTATTACTTGAGCAGAACAAACACTCC
AAGTGGAACCACCACGCAGTCAAGGCTTC
AGTTTTCTCAGGCCGGAGCGAGTGACATTC
GGGACCAGTCTAGGAACTGGCTTCCTGGA
CCCTGTTACCGCCAGCAGCGAGTATCAAA
GACATCT GC GGATAACAACAACAGTGAAT
ACTCGTGGACTGGAGCTACCAAGTACCAC
CTCAATGGCAGAGACTCTCTGGTGAATCCG
GGCCCGGCCATGGCAAGCCACAAGGACGA
TGAAGAAAAGTTTTTTCCTCAGAGCGGGGT
TCTCATCTTTGGGAAGCAAGGCTCAGAGA
AAACAAATGTGGACATTGAAAAGGTCATG
ATTACAGACGAAGAGGAAATCAGGACAAC
CAATCCCGTGGCTACGGAGCAGTATGGTTC
TGTATCTACCAACCTCCAGAGAGGCAACA
GACAAGCAGCTACCGCAGATGTCAACACA
CAAGGCGTTCTTCCAGGCATGGTCTGGCAG
GACAGAGATGTGTACCTTCAGGGGCCCAT
CTGGGCAAAGATTCCACACACGGACGGAC
ATTTTCACCCCTCTCCCCTCATGGGTGGAT
TCGGACTT A A AC ACCCTCCTCC AC A GATTC
77
CA 03220810 2023- 11- 29 SUBSTITUTE SHEET (RULE 26)
WO 2022/256557
PCT/US2022/032004
TCATCAAGAACACCCCGGTACCTGCGAAT
CCTTCGACCACCTTCAGTGCGGCAAAGTTT
GCTTCCTTCATCACACAGTACTCCACGGGA
CAGGTCAGCGTGGAGATCGAGTGGGAGCT
GCAGAAGGAAAACAGCAAACGCTGGAATC
CCGAAATTCAGTACACTTCCAACTACAACA
AGTCTGTTAATGTGGACTTTACTGTGGACA
CTAATGGCGTGTATTCAGAGCCTCGCCCCA
TTGGCACCAGATACCTGACTCGTAATCTGT
AA (SEQ ID NO: 48)
Table 3
Capsid Amino Acid Sequence of VP1 Exemplary Nucleic Acid Molecule
Sequence(SEQ
Variant capsid polypeptide (SEQ ID ID NO)
NO; starting amino acid of
VP2 is underlined; starting
amino acid of VP3 is in bold.
VAR-3 MAADGYLPDWLEDTLSE ATGGCTGCCGATGGTTATCTTCCAGATTGG
GIRQWWKLKPGPPPPKPA CTCGAGGACACTCTCTCTGAAGGAATAAG
ERHKDDSRGLVLPGYKYL ACAGTGGTGGAAGCTCAAACCTGGCCCAC
GPFNGLDKGEPVNEADAA CACC ACC AAAGCCCGC AGAGCGGCAT AAG
ALEHDKAYDRQLDSGDN GACGACAGCAGGGGTCTTGTGCTTCCTGG
PYLKYNHADAEFQERLKE GTACAAGTACCTCGGACCCTTCAACGGACT
DTSFGGNLGRAVFQAKKR CGACAAGGGAGAGCCGGTCAACGAGGCAG
VLEPLGLVEEPVKTAPGK ACGCCGCGGCCCTCGAGCACGACAAAGCC
KRPVEHSPVEPDSSSGTGK TACGACCGGCAGCTCGACAGCGGAGAC AA
AGQQPARKRLNFGQTGD CCCGTACCTCAAGTACAACCACGCCGACG
ADS VPDPQPLGQPPAAPS CGGAGTTTCAGGAGCGCCTTAAAGAAGAT
GLGTNTMATGSGAPMAD ACGTCTTTTGGGGGCAACCTCGGACGAGC
NNEGADGVGNSSGNWHC AGTCTTCCAGGCGAAAAAGAGGGTTCTTG
DSTWMGDRVITTSTRTWA AACCTCTGGGCCTGGTTGAGGAACCTGTTA
LPTYNNHLYKQISSQSGAS AGACGGCTCCGGGAAAAAAGAGGCCGGTA
NDNHYFGYSTPWGYFDF GAGCACTCTCCTGTGGAGCCAGACTCCTCC
NRFHCHFSPRDWQRLINN TCGGGAACCGGAAAGGCGGGCCAGCAGCC
NWGFRPKRLNFKLFNIQV TGCAAGAAAAAGATTGAATTTTGGTCAGA
KEVTQNDGTTTIANNLTS CTGGAGACGCAGACTCAGTACCTGACCCC
TVQVFTDSEYQLPYVLGS CAGCCTCTCGGACAGCCACCAGCAGCCCC
AHQGCLPPFPADVFMVPQ CTCTGGTCTGGGAACTAATACGATGGCTAC
YGYLTLNNGS QAVGRSSF AGGCAGTGGCGC ACC AATGGC AGAC AAT A
YCLEYFPSQMLRTGNNFT ACGAGGGCGCCGACGGAGTGGGTAATTCC
FSYTFEDVPFHSSYAHS QS TCGGGAAATTGGCATTGCGATTCCACATGG
LDRLMNPLIDQYLYYLSR ATGGGCGACAGAGTCATCACCACCAGCAC
TQMTSGTLGTQMLQFS QA CCGAACCTGGGCCCTGCCCACCTACAACA
GASDIRDQSRN WLPGPC Y ACC ACCTCTACAAACAAATTTCCAGCCAAT
78
CA 03220810 2023- 11- 29 SUBSTITUTE SHEET (RULE 26)
WO 2022/256557
PCT/US2022/032004
RQQRVSKTSADNNNSEYS CAGGAGCCTCGAACGACAATCACTACTTT
WTGATKYHLNGRDSLVN GGCTACAGCACCCCTTGGGGGTATTTTGAC
PGPAMASHKDDEEKFFPQ TTCAACAGATTCCACTGCCACTTTTCACCA
SGVLIFGKQGSEKTNVDIE CGTGACTGGCAAAGACTCATCAACAACAA
KVMITDEEEIRTTNPVATE CTGGGGATTCCGACCCAAGAGACTCAACT
QYGSVSTNLQRGNRQAAT TCAAGCTCTTTAACATTCAAGTCAAAGAGG
ADVNTQGVLPGMVWQDR TCACGCAGAATGACGGTACGACGACGATT
DVYLQGPIWAIUPHTDGH GCCAATAACCTTACCAGCACGGTTCAGGT
FHPSPLMGGFGLKHPPPQI GTTTACTGACTCGGAGTACCAGCTCCCGTA
LIKNTPVPANPSTTFSAAK CGTCCTCGGCTCGGCGCATCAAGGATGCCT
FASFITQYSTGQVSVEIEW CCCGCCGTTCCCAGCAGACGTCTTCATGGT
ELQKENSKRWNPEIQYTS GCCACAGTATGGATACCTCACCCTGAACA
NYNKSVNVDFTVDTNGV ACGGGAGTCAGGCAGTAGGACGCTCTTCA
YSEPRPIGTRYLTRNL*
TTTTACTGCCTGGAGTACTTTCCTTCTCAG
(SEQ ID NO: 4)
ATGCTGCGTACCGGAAACAACTTTACCTTC
AGCTACACTTTTGAGGACGTTCCTTTCCAC
AGCAGCTACGCTCACAGCCAGAGTCTGGA
CCGGCTCATGAATCCTCTCATCGACCAGTA
CCTGTATTACTTGAGCAGAACACAGATGA
CCAGTGGAACCTTGGGTACACAGATGCTTC
AGTTTTCTCAGGCCGGAGCGAGTGACATTC
GGGACCAGTCTAGGAACTGGCTTCCTGGA
CCCTGTTACCGCCAGCAGCGAGTATCAAA
GACATCTGCGGATAACAACAACAGTGAAT
ACTCGTGGACTGGAGCTACCAAGTACCAC
CTCAATGGCAGAGACTCTCTGGTGAATCCG
GGCCCGGCCATGGCAAGCCACAAGGACGA
TGAAGAAAAGTTTTTTCCTCAGAGCGGGGT
TCTCATCTTTGGGAAGCAAGGCTCAGAGA
AAACAAATGTGGACATTGAAAAGGTCATG
ATTACAGACGAAGAGGAAATCAGGACAAC
CAATCCCGTGGCTACGGAGCAGTATGGTTC
TGTATCTACCAACCTCCAGAGAGGCAACA
GACAAGCAGCTACCGCAGATGTCAACACA
CAAGGCGTTCTTCCAGGCATGGTCTGGCAG
GACAGAGATGTGTACCTTCAGGGGCCCAT
CTGGGCAAAGATTCCACACACGGACGGAC
ATTTTCACCCCTCTCCCCTCATGGGTGGAT
TCGGACTTAAACACCCTCCTCCACAGATTC
TCATCAAGAACACCCCGGTACCTGCGAAT
CCTTCGACCACCTTCAGTGCGGCAAAGTTT
GCTTCCTTCATCACACAGTACTCCACGGGA
CAGGTCAGCGTGGAGATCGAGTGGGAGCT
GCAGAAGGAAAACAGCAAACGCTGGAATC
CCGAAATTCAGTACACTTCCAACTACAACA
AGTCTGTTAATGTGGACTTTACTGTGGAC A
79
CA 03220810 2023- 11- 29 SUBSTITUTE SHEET (RULE 26)
WO 2022/256557
PCT/US2022/032004
CTAATGGCGTGTATTCAGAGCCTCGCCCCA
TTGGCACCAGATACCTGACTCGTAATCTGT
AA (SEQ ID NO: 49)
VAR-4 MAADGY LPDWLEDTLSE ATGGCT GC C GATGGTTATCTTCCAGATT GG
GIRQWWKLKPGPPPPKPA CTCGAGGACACTCTCTCTGAAGGAATAAG
ERHKDDSRGLVLPGYKYL ACAGTGGTGGAAGCTCAAACCTGGCCCAC
GPFNGLDKGEPVNEADAA CACCACCAAAGCCCGCAGAGCGGCATAAG
ALEHDKAYDRQLDS GDN GACGACAGCAGGGGTCTTGTGCTTCCTGG
PYLKYNHADAEFQERLKE GTACAAGTACCTCGGACCCTTCAACGGACT
DTSFGGNLGRAVFQAKKR CGACAAGGGAGAGCCGGTCAACGAGGCAG
VLEPLGLVEEPVKTAPGK ACGCCGCGGCCCTCGAGCACGACAAAGCC
KRPVEHS PVEPDS S S GTGK TACGACCGGCAGCTCGACAGCGGAGAC AA
AGQQPARKRLNFGQTGD CCCGTACCTCAAGTACAACCACGCCGACG
ADS VPDPQPLGQPPAAPS CGGAGTTTCAGGAGCGCCTTAAAGAAGAT
GLGTNTIVIATGSGAPMAD ACGTCTTTTGGGGGCAACCTCGGACGAGC
NNEGADGVGNSSGNWHC AGTCTTCCAGGCGAAAAAGAGGGTTCTTG
DSTWMGDRVITTSTRTWA AACCTCTGGGCCTGGTTGAGGAACCTGTTA
LPTYNNHLY KQIS S QS GAS AGACGGCTCCGGGAAAAAAGAGGCCGGTA
NDNHYFGYSTPWGYFDF GAGCACTCTCCTGTGGAGCCAGACTCCTCC
NRFHCHFS PRD WQRLINN TCGGGAACCGGAAAGGCGGGCCAGCAGCC
NWGFRPKRLNFKLFNIQV TGCAAGAAAAAGATTGAATTTTGGTCAGA
KE VTQN DGTTT I ANN LTS CTGG AG ACGC AG ACTC AGT ACCTGACCCC
TVQVFTDS EYQLPYVLGS CAGCCTCTCGGACAGCCACCAGCAGCCCC
AH QGC LPP FP AD V FM V PQ CTCTGGTCTGGG A ACT A AT ACGATGGCT AC
YGYLTLNNGS QAVGRS SF AGGCAGT GGC GC ACC AAT GGC AGAC AAT A
YCLEYFPSQMLRTGNNFT ACGAGGGCGCCGACGGAGTGGGTAATTCC
FSYTFEDVPFHSSYAHS QS TCGGGAAATTGGCATTGCGATTCCACATGG
LDRLMNPLIDQYLYYLSR ATGGGCGACAGAGTCATCACCACCAGCAC
TQTTGTTGTS GLQFS QAG CCGAACCTGGGCCCTGCCCACCTACAACA
ASDIRDQSRNWLPGPCYR ACCACCTCTACAAACAAATTTCCAGCCAAT
QQRVS KTSADNNNSEYS CAGGAGCCTCGAACGACAATCACTACTTT
WTGATKYHLNGRDSLVN GGCTACAGCACCCCTTGGGGGTATTTTGAC
PGPAMASHKDDEEKFFPQ TTCAACAGATTCCACTGCCACTTTTCACCA
S GVLIFGKQGSEKTNVDIE CGTGACTGGCAAAGACTCATCAACAACAA
KVMITDEEEIRTTNPVATE CTGGGGATTCCGACCCAAGAGACTCAACT
QYGSVSTNLQRGNRQAAT TCAAGCTCTTTAACATTCAAGTCAAAGAGG
ADVNTQGVLPGMVWQDR TCACGCAGAATGACGGTACGACGACGATT
DVYLQGPIWAKIPHTDGH GCCAATAACCTTACCAGCACGGTTCAGGT
FHPSPLMGGFGLKHPPPQI GTTTACTGACTCGGAGTACCAGCTCCCGTA
LIKNTPVPANPSTTFSAAK CGTCCTCGGCTCGGCGCATCAAGGATGCCT
FAS FITQYSTGQVS VEIEW CCCGCCGTTCCCAGCAGACGTCTTCATGGT
ELQKENS KRWNPEIQYTS GCCACAGTATGGATACCTCACCCTGAACA
NYNKSVNVDFTVDTNGV ACGGGAGTCAGGCAGTAGGACGCTCTTCA
YSEPRPIGTRYLTRNL* TTTTACTGCCTGGAGTACTTTCCTTCTCAG
(SEQ ID NO: 5) ATGCTGCGTACCGGAAACAACTTTACCTTC
AGCTACACTTTTGAGGACGTTCCTTTCCAC
CA 03220810 2023- 11- 29 SUBSTITUTE SHEET (RULE 26)
WO 2022/256557
PCT/US2022/032004
AGCAGCTACGCTCACAGCCAGAGTCTGGA
CAGACTCATGAATCCTCTCATCGACCAGTA
CCTGTATTACTTGAGCAGAACACAAACTAC
TGGAACCACCGGTACCTCAGGACTTCAGTT
TTCTCAGGCCGGAGCGAGTGACATTCGGG
ACCAGTCTAGGAACTGGCTTCCTGGACCCT
GTTACCGCCAGCAGCGAGTATCAAAGACA
TCTGCGGATAACAACAACAGTGAATACTC
GTGGACTGGAGCTACCAAGTACCACCTCA
ATGGCAGAGACTCTCTGGTGAATCCGGGC
CCGGCCATGGCAAGCCACAAGGACGATGA
AGAAAAGTTTTTTCCTCAGAGCGGGGTTCT
CATCTTTGGGAAGCAAGGCTCAGAGAAAA
CAAATGTGGACATTGAAAAGGTCATGATT
ACAGACGAAGAGGAAATCAGGACAACCA
ATCCCGTGGCTACGGAGCAGTATGGTTCTG
TATCTACCAACCTCCAGAGAGGCAACAGA
CAAGCAGCTACCGCAGATGTCAACACACA
AGGCGTTCTTCCAGGCATGGTCTGGCAGG
ACAGAGATGTGTACCTTCAGGGGCCCATCT
GGGCAAAGATTCCACACACGGACGGACAT
TTTCACCCCTCTCCCCTCATGGGTGGATTC
GGACTT A A AC ACCCTCCTCC ACAGATTCTC
ATCAAGAACACCCCGGTACCTGCGAATCC
TTCGACCACCTTCAGTGCGGCAAAGTTTGC
TTCCTTCATCACACAGTACTCCACGGGACA
GGTCAGCGTGGAGATCGAGTGGGAGCTGC
AGAAGGAAAACAGCAAACGCTGGAATCCC
GAAATTCAGTACACTTCCAACTACAACAA
GTCTGTTAATGTGGACTTTACTGTGGACAC
TAATGGCGTGTATTCAGAGCCTCGCCCCAT
TGGCACCAGATACCTGACTCGTAATCTGTA
A (SEQ ID NO: 50)
VAR-5 MAADGYLPDWLEDTLSE ATGGCTGCCGATGGTTATCTTCCAGATTGG
GIRQWWKLKPGPPPPKPA CTCGAGGACACTCTCTCTGAAGGAATAAG
ERHKDDSRGLVLPGYKYL ACAGTGGTGGAAGCTCAAACCTGGCCCAC
GPFNGLDKGEPVNEAD A A CACC ACC A A AGCCCGC AGAGCGGC AT A AG
ALEHDKAYDRQLDSGDN GACGACAGCAGGGGTCTTGTGCTTCCTGG
PYLKYNHADAEFQERLKE GTACAAGTACCTCGGACCCTTCAACGGACT
DTSFGGNLGRAVFQAKKR CGACAAGGGAGAGCCGGTCAACGAGGCAG
VLEPLGLVEEPVKTAPGK ACGCCGCGGCCCTCGAGCACGACAAAGCC
KRPVEHSPVEPDSSSGTGK TACGACCGGCAGCTCGACAGCGGAGACAA
AGQQPARKRLNFGQTGD CCCGTACCTCAAGTACAACCACGCCGACG
ADS VPDPQPLGQPPAAPS CGGAGTTTCAGGAGCGCCTTAAAGAAGAT
GLGTNTIVIATGSGAPMAD ACGTCTTTTGGGGGCAACCTCGGACGAGC
NNEGADGVGNSSGNWHC AGTCTTCCAGGCGAAAAAGAGGGTTCTTG
81
CA 03220810 2023- 11- 29 SUBSTITUTE SHEET (RULE 26)
WO 2022/256557
PCT/US2022/032004
DSTWMGDRVITTSTRTWA AACCTCTGGGCCTGGTTGAGGAACCTGTTA
LPTYNNHLYKQISSQSGAS AGACGGCTCCGGGAAAAAAGAGGCCGGTA
NDNHYFGYSTPWGYFDF GAGCACTCTCCTGTGGAGCCAGACTCCTCC
NRFHCHFSPRDWQRLINN TCGGGAACCGGAAAGGCGGGCCAGCAGCC
NWGFRPKRLNFKLFNIQV TGCAAGAAAAAGATTGAATTTTGGTCAGA
KEVTQNDGTTTIANNLTS CTGGAGACGCAGACTCAGTACCTGACCCC
TVQVFTDSEYQLPYVLGS CAGCCTCTCGGACAGCCACCAGCAGCCCC
AHQGCLPPFPADVFMVPQ CTCTGGTCTGGGAACTAATACGATGGCTAC
YGYLTLNNGS QAVGRSSF AGGCAGTGGCGCACCAATGGCAGACAAT A
YCLEYFPSQMLRTGNNFT ACGAGGGCGCCGACGGAGTGGGTAATTCC
FSYTFEDVPFHSSYAHSQS TCGGGAAATTGGCATTGCGATTCCACATGG
LDRLMNPLIDQYLYYLSR ATGGGCGACAGAGTCATCACCACCAGCAC
TNTPSGTTTFCRLQFS QAG CCGAACCTGGGCCCTGCCCACCTACAACA
ASDIRDQSRNWLPGPCYR ACCACCTCTACAAACAAATTTCCAGCCAAT
QQRVSKTSADNNNSEYS CAGGAGCCTCGAACGACAATCACTACTTT
WTGATKYHLNGRDSLVN GGCTACAGCACCCCTTGGGGGTATTTTGAC
PGPAMASHKDDEEKFFPQ TTCAACAGATTCCACTGCCACTTTTCACCA
SGVLIFGKQGSEKTNVDIE CGTGACTGGCAAAGACTCATCAACAACAA
KVMITDEEEIRTTNPVATE CTGGGGATTCCGACCCAAGAGACTCAACT
QYGSVSTNLQRGNRQAAT TCAAGCTCTTTAACATTCAAGTCAAAGAGG
ADVNTQGVLPGMVWQDR TCACGCAGAATGACGGTACGACGACGATT
DVYLQGPIWAKIPHTDGH GCCAATAACCTTACCAGCACGGTTCAGGT
FHPSPLMGGFGLKHPPPQI GTTTACTGACTCGGA GT ACC AGCTCCCGT A
LIKNTPVPANPSTTFSAAK CGTCCTCGGCTCGGCGCATCAAGGATGCCT
FASFITQYSTGQVSVEIEW CCCGCCGTTCCCAGCAGACGTCTTCATGGT
ELQKENSKRWNPEIQYTS GCCACAGTATGGATACCTCACCCTGAACA
NYNKSVNVDFTVDTNGV ACGGGAGTCAGGCAGTAGGACGCTCTTCA
YSEPRPIGTRYLTRNL*
TTTTACTGCCTGGAGTACTTTCCTTCTCAG
(SEQ ID NO: 61)
ATGCTGCGTACCGGAAACAACTTTACCTTC
AGCTACACTTTTGAGGACGTTCCTTTCCAC
AGCAGCTACGCTCACAGCCAGAGTCTGGA
CCGTCTTATGAATCCTCTCATCGACCAGTA
CCTGTATTACTTGAGCAGAACAAACACTCC
AAGTGGAACCACCACGTTTTGTAGGCTTCA
GTTTTCTCAGGCCGGAGCGAGTGACATTCG
GGACCAGTCTAGGAACTGGCTTCCTGGAC
CCTGTTACCGCCAGCAGCGAGTATCAAAG
ACATCTGCGGATAACAACAACAGTGAATA
CTCGTGGACTGGAGCTACCAAGTACCACCT
CAATGGCAGAGACTCTCTGGTGAATCCGG
GCCCGGCCATGGCAAGCCACAAGGACGAT
GAAGAAAAGTTTTTTCCTCAGAGCGGGGTT
CTCATCTTTGGGAAGCAAGGCTCAGAGAA
AACAAATGTGGACATTGAAAAGGTCATGA
TTACAGACGAAGAGGAAATCAGGACAACC
A ATCCCGT GGCT ACGGAGCAGT ATGGTTCT
82
CA 03220810 2023- 11- 29 SUBSTITUTE SHEET (RULE 26)
WO 2022/256557
PCT/US2022/032004
GTATCTACCAACCTCCAGAGAGGCAACAG
ACAAGCAGCTACCGCAGATGTCAACACAC
AAGGCGTTCTTCCAGGCATGGTCTGGCAG
GACAGAGATGTGTACCTTCAGGGGCCCAT
CTGGGCAAAGATTCCACACACGGACGGAC
ATTTTCACCCCTCTCCCCTCATGGGTGGAT
TCGGACTTAAACACCCTCCTCCACAGATTC
TCATCAAGAACACCCCGGTACCTGCGAAT
CCTTCGACCACCTTCAGTGCGGCAAAGTTT
GCTTCCTTCATCACACAGTACTCCACGGGA
CAGGTCAGCGTGGAGATCGAGTGGGAGCT
GCAGAAGGAAAACAGCAAACGCTGGAATC
CCGAAATTCAGTACACTTCCAACTACAACA
AGTCTGTTAATGTGGACTTTACTGTGGACA
CTAATGGCGTGTATTCAGAGCCTCGCCCCA
TTGGCACCAGATACCTGACTCGTAATCTGT
AA (SEQ ID NO: 51)
VAR-6 MAADGY LPDWLEDTLSE ATGGCT GC C GATGGTTATCTTCCAGATT GG
GIRQWWKLKPGPPPPKPA CTCGAGGACACTCTCTCTGAAGGAATAAG
ERHKDDSRGLVLPGY KYL ACAGTGGTGGAAGCTCAAACCTGGCCCAC
GPFNGLDKGEPVNEADAA CACCACCAAAGCCCGCAGAGCGGCATAAG
ALEN KAYD DRQ LDS GDN GACGACAGCAGGGGTCTTGTGCTTCCTGG
PYLKYNHADAEFQERLKE GTACAAGTACCTCGGACCCTTCAACGGACT
DTS FGGNLGR AV FQ A K KR CGAC A AGGGAG AGCCGGTC A AC G AGGC AG
VLEPLGLVEEPVKTAPGK ACGCCGCGGCCCTCGAGCACGACAAAGCC
KRPVEHS PVEPDS S S GTGK TACGACCGGCAGCTCGACAGCGGAGAC AA
AGQQPARKRLNFGQTGD CCCGTACCTCAAGTACAACCACGCCGACG
ADS VPDPQPLGQPPAAPS CGGAGTTTCAGGAGCGCCTTAAAGAAGAT
GLGTNTIVIATGSGAPMAD ACGTCTTTTGGGGGCAACCTCGGACGAGC
NNEGADGVGNSSGNWHC AGTCTTCCAGGCGAAAAAGAGGGTTCTTG
DSTWMGDRVITTSTRTWA AACCTCTGGGCCTGGTTGAGGAACCTGTTA
LPTYNNHLYKQISSQSGAS AGACGGCTCCGGGAAAAAAGAGGCCGGTA
NDNHYFGYSTPWGYFDF GAGCACTCTCCTGTGGAGCCAGACTCCTCC
NRFHCHFS PRDWQRLINN TCGGGAACCGGAAAGGCGGGCCAGCAGCC
NWGFRPKRLNEKLENIQV TGCAAGAAAAAGATTGAATTTTGGTCAGA
KEVTQNDGTTTIANNLTS CTGGAGACGCAGACTCAGTACCTGACCCC
TVQVFTDS EYQLPYVLGS CAGCCTCTCGG AC AGCC ACC A GC AGCCCC
AHQGCLPPFPADVFMVPQ CTCTGGTCTGGGAACTAATACGATGGCTAC
YGYLTLNNGS QAVGRS SF AGGCAGT GGC GC ACC AAT GGC AGAC AAT A
YCLEYFPSQMLRTGNNFT ACGAGGGCGCCGACGGAGTGGGTAATTCC
FSYTFEDVPFHSSYAHS QS TCGGGAAATTGGCATTGCGATTCCACATGG
LDRLMNPLIDQYLYYLSK ATGGGCGACAGAGTCATCACCACCAGCAC
CQEGMARTNTPSGTTTQS CCGAACCTGGGCCCTGCCCACCTACAACA
RLQFS QAGAS DIRDQS RN ACC ACCTCTACAAACAAATTTCCAGCCAAT
WLPGPCYRQQRVS KTS AD CAGGAGCCTCGAACGACAATCACTACTTT
NNNSEYSWTGATKYHLN GGCTACAGCACCCCTTGGGGGTATTTTGAC
83
CA 03220810 2023- 11- 29 SUBSTITUTE SHEET (RULE 26)
WO 2022/256557
PCT/US2022/032004
GRDSLVNPGPAMASHKD TTCAACAGATTCCACTGCCACTTTTCACCA
DEEKFFPQSGVLIFGKQGS CGTGACTGGCAAAGACTCATCAACAACAA
EKTNVDIEKVMITDEEEIR CTGGGGATTCCGACCCAAGAGACTCAACT
TTNPVATEQYGSVSTNLQ TCAAGCTCTTTAACATTCAAGTCAAAGAGG
RGNRQAATADVNTQGVL TCACGCAGAATGACGGTACGACGACGATT
PGMVWQDRDVYLQGPIW GCCAATAACCTTACCAGCACGGTTCAGGT
AKIPHTDGHFHPSPLMGG GTTTACTGACTCGGAGTACCAGCTCCCGTA
FGLKHPPPQILIKNTPVPA CGTCCTCGGCTCGGCGCATCAAGGATGCCT
NPSTTFSAAKFASFITQYS CCCGCCGTTCCCAGCAGACGTCTTCATGGT
TGQVSVEIEWELQKENSK GCCACAGTATGGATACCTCACCCTGAACA
RWNPEIQYTSNYNKSVNV ACGGGAGTCAGGCAGTAGGACGCTCTTCA
DFTVDTNGVYSEPRPIGTR TTTTACTGCCTGGAGTACTTTCCTTCTCAG
YLTRNLI- (SEQ ID NO: 7) ATGCTGCGTACCGGAAACAACTTTACCTTC
AGCTACACTTTTGAGGACGTTCCTTTCCAC
AGCAGCTACGCTCACAGCCAGAGTCTGGA
CCGTCTGATGAATCCTCTCATCGACCAGTA
CCTGTATTACTTGAGCAAGTGCCAGGAAG
GAATGGCTAGAACAAACACTCCAAGTGGA
ACCACCACGCAGTCAAGGCTTCAGTTTTCT
CAGGCCGGAGCGAGTGACATTCGGGACCA
GTCTAGGAACTGGCTTCCTGGACCCTGTTA
CCGCCAGCAGCGAGTATCAAAGACATCTG
CGGATAACAACAACAGTGAATACTCGTGG
ACTGGAGCTACCAAGTACCACCTCAATGG
CAGAGACTCTCTGGTGAATCCGGGCCCGG
CCATGGCAAGCCACAAGGACGATGAAGAA
AAGTTTTTTCCTCAGAGCGGGGTTCTCATC
TTTGGGAAGCAAGGCTCAGAGAAAACAAA
TGTGGACATTGAAAAGGTCATGATTACAG
ACGAAGAGGAAATCAGGACAACCAATCCC
GTGGCTACGGAGCAGTATGGTTCTGTATCT
ACCAACCTCCAGAGAGGCAACAGACAAGC
AGCTACCGCAGATGTCAACACACAAGGCG
TTCTTCCAGGCATGGTCTGGCAGGACAGA
GATGTGTACCTTCAGGGGCCCATCTGGGCA
AAGATTCCACACACGGACGGACATTTTCA
CCCCTCTCCCCTCATGGGTGGATTCGGACT
TAAACACCCTCCTCCACAGATTCTCATCAA
GAACACCCCGGTACCTGCGAATCCTTCGAC
CACCTTCAGTGCGGCAAAGTTTGCTTCCTT
CATCACACAGTACTCCACGGGACAGGTCA
GCGTGGAGATCGAGTGGGAGCTGCAGAAG
GAAAACAGCAAACGCTGGAATCCCGAAAT
TCAGTACACTTCCAACTACAACAAGTCTGT
TAATGTGGACTTTACTGTGGACACTAATGG
CGTGT ATTCAG A GCCTCGCCCCATTGGCAC
84
CA 03220810 2023- 11- 29 SUBSTITUTE SHEET (RULE 26)
WO 2022/256557
PCT/US2022/032004
CAGATACCTGACTCGTAATCTGTAA (SEQ
ID NO: 52)
VAR-7 MAADGYLPDWLEDTLSE ATGGCTGCCGATGGTTATCTTCCAGATTGG
GIRQWWKLKPGPPPPKPA CTCGAGGACACTCTCTCTGAAGGAATAAG
ERHKDDSRGLVLPGYKYL ACAGTGGTGGAAGCTCAAACCTGGCCCAC
GPFNGLDKGEPVNEADAA CACCACCAAAGCCCGCAGAGCGGCATAAG
ALEHDKAYDRQLDSGDN GACGACAGCAGGGGTCTTGTGCTTCCTGG
PYLKYNHADAEFQERLKE GTACAAGTACCTCGGACCCTTCAACGGACT
DTSFGGNLGRAVFQAKKR CGACAAGGGAGAGCCGGTCAACGAGGCAG
VLEPLGLVEEPVKTAPGK ACGCCGCGGCCCTCGAGCACGACAAAGCC
KRPVEHSPVEPDSSSGTGK TACGACCGGCAGCTCGACAGCGGAGACAA
AGQQPARKRLNFGQTGD CCCGTACCTCAAGTACAACCACGCCGACG
ADS VPDPQPLGQPPAAPS CGGAGTTTCAGGAGCGCCTTAAAGAAGAT
GLGTNTIVIATGSGAPMAD ACGTCTTTTGGGGGCAACCTCGGACGAGC
NNEGADGVGNSSGNWHC AGTCTTCCAGGCGAAAAAGAGGGTTCTTG
DSTWMGDRVITTSTRTWA AACCTCTGGGCCTGGTTGAGGAACCTGTTA
LPTYNNHLYKQISSQSGAS AGACGGCTCCGGGAAAAAAGAGGCCGGTA
NDNHYFGYSTPWGYFDF GAGCACTCTCCTGTGGAGCCAGACTCCTCC
NRFHCHFSPRDWQRLINN TCGGGAACCGGAAAGGCGGGCCAGCAGCC
NWGFRPKRLNFKLFNIQV TGCAAGAAAAAGATTGAATTTTGGTCAGA
KEVTQNDGTTTIANNLTS CTGGAGACGCAGACTCAGTACCTGACCCC
TVQVFTDSEYQLPYVLGS CAGCCTCTCGGACAGCCACCAGCAGCCCC
AHQGCLPPFPADVFMVPQ CTCTGGTCTGGGAACTAATACGATGGCTAC
YGYLTLNNGSQAVGRSS F AGGCAGTGGCGC ACC A ATGGC AG AC A AT A
YCLEYFPSQMLRTGNNFT ACGAGGGCGCCGACGGAGTGGGTAATTCC
FSYTFEDVPFHSSYAHSQS TCGGGAAATTGGCATTGCGATTCCACATGG
LDRLMNPLIDQYLYYLSR ATGGGCGACAGAGTCATCACCACCAGCAC
TNLMVDRLGTPSGTTTQS CCGAACCTGGGCCCTGCCCACCTACAACA
RLQFSQAGASDIRDQSRN ACCACCTCTACAAACAAATTTCCAGCCAAT
WLPGPCYRQQRVSKTSAD CAGGAGCCTCGAACGACAATCACTACTTT
NNNSEYSWTGATKYHLN GGCTACAGCACCCCTTGGGGGTATTTTGAC
GRDSLVNPGPAMASHKD TTCAACAGATTCCACTGCCACTTTTCACCA
DEEKFFPQSGVLIFGKQGS CGTGACTGGCAAAGACTCATCAACAACAA
EKTNVDIEKVMITDFEFIR CTGGGGATTCCGACCCAAGAGACTCAACT
TTNPVATEQYGSVSTNLQ TCAAGCTCTTTAACATTCAAGTCAAAGAGG
RGNRQAATADVNTQGVL TCACGCAGAATGACGGTACGACGACGATT
PGMVWQDRDVYLQGPIW GCC A AT A ACCTT ACC AGC ACGGTTC AGGT
AKIPHTDGHFHPSPLMGG GTTTACTGACTCGGAGTACCAGCTCCCGTA
FGLKHPPPQILIKNTPVPA CGTCCTCGGCTCGGCGCATCAAGGATGCCT
NPSTTFSAAKFASFITQYS CCCGCCGTTCCCAGCAGACGTCTTCATGGT
TGQVSVEIEWELQKENSK GCCACAGTATGGATACCTCACCCTGAACA
RWNPEIQYTSNYNKSVNV ACGGGAGTCAGGCAGTAGGACGCTCTTCA
DFTVDTNGVYSEPRPIGTR TTTTACTGCCTGGAGTACTTTCCTTCTCAG
YLTRNL* (SEQ ID NO: 8) ATGCTGCGTACCGGAAACAACTTTACCTTC
AGCTACACTTTTGAGGACGTTCCTTTCCAC
AGCAGCTACGCTCACAGCCAGAGTCTGGA
CA 03220810 2023- 11- 29 SUBSTITUTE SHEET (RULE 26)
WO 2022/256557
PCT/US2022/032004
CCGTCTAATGAATCCTCTCATCGACCAGTA
CCTGTATTACTTGAGCAGAACAAACCTCAT
GGTTGACCGACTTGGGACTCCAAGTGGAA
CCACCACGCAGTCAAGGCTTCAGTTTTCTC
AGGCCGGAGCGAGTGACATTCGGGACCAG
TCTAGGAACTGGCTTCCTGGACCCTGTTAC
CGCCAGCAGCGAGTATCAAAGACATCTGC
GGATAACAACAACAGTGAATACTCGTGGA
CTGGAGCTACCAAGTACCACCTCAATGGC
AGAGACTCTCTGGTGAATCCGGGCCCGGC
CATGGCAAGCCACAAGGACGATGAAGAAA
AGTTTTTTCCTCAGAGCGGGGTTCTCATCT
TTGGGAAGCAAGGCTCAGAGAAAACAAAT
GTGGACATTGAAAAGGTCATGATTACAGA
CGAAGAGGAAATCAGGACAACCAATCCCG
TGGCTACGGAGCAGTATGGTTCTGTATCTA
CCAACCTCCAGAGAGGCAACAGACAAGCA
GCTACCGCAGATGTCAACACACAAGGCGT
TCTTCCAGGCATGGTCTGGCAGGACAGAG
ATGTGTACCTTCAGGGGCCCATCTGGGCAA
AGATTCCACACACGGACGGACATTTTCACC
CCTCTCCCCTCATGGGTGGATTCGGACTTA
AACACCCTCCTCCACAGATTCTCATCAAGA
ACACCCCGGTACCTGCGAATCCTTCGACCA
CCTTCAGTGCGGCAAAGTTTGCTTCCTTCA
TCACACAGTACTCCACGGGACAGGTCAGC
GTGGAGATCGAGTGGGAGCTGCAGAAGGA
AAACAGCAAACGCTGGAATCCCGAAATTC
AGTACACTTCCAACTACAACAAGTCTGTTA
ATGTGGACTTTACTGTGGACACTAATGGCG
TGTATTCAGAGCCTCGCCCCATTGGCACCA
GATACCTGACTCGTAATCTGTAA (SEQ ID
NO: 53)
VAR-8 MAADGYLPDWLEDTLSE ATGGCTGCCGATGGTTATCTTCCAGATTGG
GIRQWWKLKPGPPPPKPA CTCGAGGACACTCTCTCTGAAGGAATAAG
ERHKDDSRGLVLPGYKYL ACAGTGGTGGAAGCTCAAACCTGGCCCAC
GPFNGLDKGEPVNEADAA CACCACCAAAGCCCGCAGAGCGGCAT A AG
ALEHDKAYDRQLDSGDN GACGACAGCAGGGGTCTTGTGCTTCCTGG
PYLKYNHADAEFQERLKE GTACAAGTACCTCGGACCCTTCAACGGACT
DTSFGGNLGRAVFQAKKR CGACAAGGGAGAGCCGGTCAACGAGGCAG
VLEPLGLVEEPVKTAPGK ACGCCGCGGCCCTCGAGCACGACAAAGCC
KRPVEHSPVEPDSSSGTGK TACGACCGGCAGCTCGACAGCGGAGACAA
AGQQPARKRLNFGQTGD CCCGTACCTCAAGTACAACCACGCCGACG
ADS VPDPQPLGQPPAAPS CGGAGTTTCAGGAGCGCCTTAAAGAAGAT
GLGTNT1VIATGSGAPMAD ACGTCTTTTGGGGGCAACCTCGGACGAGC
NNEGADGVGNSSGNWHC AGTCTTCCAGGCGAAAAAGAGGGTTCTTG
86
CA 03220810 2023- 11- 29 SUBSTITUTE SHEET (RULE 26)
WO 2022/256557
PCT/US2022/032004
DSTWMGDRVITTSTRTWA AACCTCTGGGCCTGGTTGAGGAACCTGTTA
LPTYNNHLYKQISSQSGAS AGACGGCTCCGGGAAAAAAGAGGCCGGTA
NDNHYFGYSTPWGYFDF GAGCACTCTCCTGTGGAGCCAGACTCCTCC
NRFHCHFSPRDWQRLINN TCGGGAACCGGAAAGGCGGGCCAGCAGCC
NWGFRPKRLNFKLFNIQV TGCAAGAAAAAGATTGAATTTTGGTCAGA
KEVTQNDGTTTIANNLTS CTGGAGACGCAGACTCAGTACCTGACCCC
TVQVFTDSEYQLPYVLGS CAGCCTCTCGGACAGCCACCAGCAGCCCC
AHQGCLPPFPADVFMVPQ CTCTGGTCTGGGAACTAATACGATGGCTAC
YGYLTLNNGS QAVGRSSF AGGCAGTGGCGCACCAATGGCAGACAAT A
YCLEYFPSQMLRTGNNFT ACGAGGGCGCCGACGGAGTGGGTAATTCC
FSYTFEDVPFHSSYAHSQS TCGGGAAATTGGCATTGCGATTCCACATGG
LDRLMNPLIDQYLYYLSR ATGGGCGACAGAGTCATCACCACCAGCAC
TNTTS GT GGTQTLAFS QA CCGAACCTGGGCCCTGCCCACCTACAACA
GASDIRDQSRNWLPGPCY ACCACCTCTACAAACAAATTTCCAGCCAAT
RQQRVSKTSADNNNSEYS CAGGAGCCTCGAACGACAATCACTACTTT
WTGATKYHLNGRDSLVN GGCTACAGCACCCCTTGGGGGTATTTTGAC
PGPAMASHKDDEEKFFPQ TTCAACAGATTCCACTGCCACTTTTCACCA
SGVLIFGKQGSEKTNVDIE CGTGACTGGCAAAGACTCATCAACAACAA
KVMITDEEEIRTTNPVATE CTGGGGATTCCGACCCAAGAGACTCAACT
QYGSVSTNLQRGNRQAAT TCAAGCTCTTTAACATTCAAGTCAAAGAGG
ADVNTQGVLPGMVWQDR TCACGCAGAATGACGGTACGACGACGATT
DVYLQGPIWAKIPHTDGH GCCAATAACCTTACCAGCACGGTTCAGGT
FHPSPLMGGFGLKHPPPQI GTTTACTGACTCGGA GT ACC AGCTCCCGT A
LIKNTPVPANPSTTFSAAK CGTCCTCGGCTCGGCGCATCAAGGATGCCT
FASFITQYSTGQVSVEIEW CCCGCCGTTCCCAGCAGACGTCTTCATGGT
ELQKENSKRWNPEIQYTS GCCACAGTATGGATACCTCACCCTGAACA
NYNKSVNVDFTVDTNGV ACGGGAGTCAGGCAGTAGGACGCTCTTCA
YSEPRPIGTRYLTRNL*
TTTTACTGCCTGGAGTACTTTCCTTCTCAG
(SEQ ID NO: 91)
ATGCTGCGTACCGGAAACAACTTTACCTTC
AGCTACACTTTTGAGGACGTTCCTTTCCAC
AGCAGCTACGCTCACAGCCAGAGTCTGGA
CCGACTCATGAATCCTCTCATCGACCAGTA
CCTGTATTACTTGAGCAGAACAAACACTAC
AAGTGGAACCGGAGGAACACAGACGCTTG
CTTTTTCTCAGGCCGGAGCGAGTGACATTC
GGGACCAGTCTAGGAACTGGCTTCCTGGA
CCCTGTTACCGCCAGCAGCGAGTATCAAA
GACATCTGCGGATAACAACAACAGTGAAT
ACTCGTGGACTGGAGCTACCAAGTACCAC
CTCAATGGCAGAGACTCTCTGGTGAATCCG
GGCCCGGCCATGGCAAGCCACAAGGACGA
TGAAGAAAAGTTTTTTCCTCAGAGCGGGGT
TCTCATCTTTGGGAAGCAAGGCTCAGAGA
AAACAAATGTGGACATTGAAAAGGTCATG
ATTACAGACGAAGAGGAAATCAGGACAAC
CA ATCCCGT GGCT ACGGAGC AGT ATGGTTC
87
CA 03220810 2023- 11- 29 SUBSTITUTE SHEET (RULE 26)
WO 2022/256557
PCT/US2022/032004
TGTATCTACCAACCTCCAGAGAGGCAACA
GACAAGCAGCTACCGCAGATGTCAACACA
CAAGGCGTTCTTCCAGGCATGGTCTGGCAG
GACAGAGATGTGTACCTTCAGGGGCCCAT
CTGGGCAAAGATTCCACACACGGACGGAC
ATTTTCACCCCTCTCCCCTCATGGGTGGAT
TCGGACTTAAACACCCTCCTCCACAGATTC
TCATCAAGAACACCCCGGTACCTGCGAAT
CCTTCGACCACCTTCAGTGCGGCAAAGTTT
GCTTCCTTCATCACACAGTACTCCACGGGA
CAGGTCAGCGTGGAGATCGAGTGGGAGCT
GCAGAAGGAAAACAGCAAACGCTGGAATC
CCGAAATTCAGTACACTTCCAACTACAACA
AGTCTGTTAATGTGGACTTTACTGTGGACA
CTAATGGCGTGTATTCAGAGCCTCGCCCCA
TTGGCACCAGATACCTGACTCGTAATCTGT
AA (SEQ ID NO: 54)
VAR-9 MAADGY LPDWLEDTLSE ATGGCT GC C GATGGTTATCTTCCAGATT GG
GIRQWWKLKPGPPPPKPA CTCGAGGACACTCTCTCTGAAGGAATAAG
ERHKDDSRGLVLPGY KYL ACAGTGGTGGAAGCTCAAACCTGGCCCAC
GPFNGLDKGEPVNEADAA CACCACCAAAGCCCGCAGAGCGGCATAAG
ALEN KAYD DRQ LDS GDN GACGACAGCAGGGGTCTTGTGCTTCCTGG
PYLKYNHADAEFQERLKE GTACAAGTACCTCGGACCCTTCAACGGACT
DTS FGGNLGR AV FQ A K KR CGAC A AGGGAG AGCCGGTC A AC G AGGC AG
VLEPLGLVEEPVKTAPGK ACGCCGCGGCCCTCGAGCACGACAAAGCC
KRPVEHS PVEPDS S S GTGK TACGACCGGCAGCTCGACAGCGGAGAC AA
AGQQPARKRLNFGQTGD CCCGTACCTCAAGTACAACCACGCCGACG
ADS VPDPQPLGQPPAAPS CGGAGTTTCAGGAGCGCCTTAAAGAAGAT
GLGTNTIVIATGSGAPMAD ACGTCTTTTGGGGGCAACCTCGGACGAGC
NNEGADGVGNSSGNWHC AGTCTTCCAGGCGAAAAAGAGGGTTCTTG
DSTWMGDRVITTSTRTWA AACCTCTGGGCCTGGTTGAGGAACCTGTTA
LPTYNNHLYKQISSQSGAS AGACGGCTCCGGGAAAAAAGAGGCCGGTA
NDNHYFGYSTPWGYFDF GAGCACTCTCCTGTGGAGCCAGACTCCTCC
NRFHCHFS PRDWQRLINN TCGGGAACCGGAAAGGCGGGCCAGCAGCC
NWGFRPKRLNEKLENIQV TGCAAGAAAAAGATTGAATTTTGGTCAGA
KEVTQNDGTTTIANNLTS CTGGAGACGCAGACTCAGTACCTGACCCC
TVQVFTDS EYQLPYVLGS CAGCCTCTCGG AC AGCC ACC A GC AGCCCC
AHQGCLPPFPADVFMVPQ CTCTGGTCTGGGAACTAATACGATGGCTAC
YGYLTLNNGS QAVGRS SF AGGCAGT GGC GC ACC AAT GGC AGAC AAT A
YCLEYFPSQMLRTGNNFT ACGAGGGCGCCGACGGAGTGGGTAATTCC
FSYTFEDVPFHSSYAHS QS TCGGGAAATTGGCATTGCGATTCCACATGG
LDRLMNPLIDQYLYYLSR ATGGGCGACAGAGTCATCACCACCAGCAC
THCQECPINTPS GTTTQSR CCGAACCTGGGCCCTGCCCACCTACAACA
LQFS QAGASDIRDQSRNW ACC ACCTCTACAAACAAATTTCCAGCCAAT
LPGPCYRQQRVS KTSADN CAGGAGCCTCGAACGACAATCACTACTTT
NNSEYSWTGATKYHLNG GGCTACAGCACCCCTTGGGGGTATTTTGAC
88
CA 03220810 2023- 11- 29 SUBSTITUTE SHEET (RULE 26)
WO 2022/256557
PCT/US2022/032004
RDSLVNPGPAMASHKDDE TTCAACAGATTCCACTGCCACTTTTCACCA
EKFFPQSGVLIFGKQGSEK CGTGACTGGCAAAGACTCATCAACAACAA
TNVDIEKVMITDEEEIRTT CTGGGGATTCCGACCCAAGAGACTCAACT
NPVATEQYGSVSTNLQRG TCAAGCTCTTTAACATTCAAGTCAAAGAGG
NRQAATADVNTQGVLPG TCACGCAGAATGACGGTACGACGACGATT
MVWQDRDVYLQGPIWAK GCCAATAACCTTACCAGCACGGTTCAGGT
IPHTDGHFHPSPLMGGFGL GTTTACTGACTCGGAGTACCAGCTCCCGTA
KHPPPQILIKNTPVPANPST CGTCCTCGGCTCGGCGCATCAAGGATGCCT
TFSAAKFASFITQYSTGQV CCCGCCGTTCCCAGCAGACGTCTTCATGGT
SVEIEWELQKENSKRWNP GCCACAGTATGGATACCTCACCCTGAACA
EIQYTSNYNKSVNVDFTV ACGGGAGTCAGGCAGTAGGACGCTCTTCA
DTNGVYSEPRPIGTRYLTR TTTTACTGCCTGGAGTACTTTCCTTCTCAG
NU- (SEQ ID NO. 10)
ATGCTGCGTACCGGAAACAACTTTACCTTC
AGCTACACTTTTGAGGACGTTCCTTTCCAC
AGCAGCTACGCTCACAGCCAGAGTCTGGA
CCGTCTGATGAATCCTCTCATCGACCAGTA
CCTGTATTACTTGAGCAGAACACATTGTCA
GGAGTGTCCAATTAACACTCCAAGTGGAA
CCACCACGCAGTCAAGGCTTCAGTTTTCTC
AGGCCGGAGCGAGTGACATTCGGGACCAG
TCTAGGAACTGGCTTCCTGGACCCTGTTAC
CGCCAGCAGCGAGTATCAAAGACATCTGC
GGATAACAACAACAGTGAATACTCGTGGA
CTGGAGCTACCAAGTACCACCTCAATGGC
AGAGACTCTCTGGTGAATCCGGGCCCGGC
CATGGCAAGCCACAAGGACGATGAAGAAA
AGTTTTTTCCTCAGAGCGGGGTTCTCATCT
TTGGGAAGCAAGGCTCAGAGAAAACAAAT
GTGGACATTGAAAAGGTCATGATTACAGA
CGAAGAGGAAATCAGGACAACCAATCCCG
TGGCTACGGAGCAGTATGGTTCTGTATCTA
CCAACCTCCAGAGAGGCAACAGACAAGCA
GCTACCGCAGATGTCAACACACAAGGCGT
TCTTCCAGGCATGGTCTGGCAGGACAGAG
ATGTGTACCTTCAGGGGCCCATCTGGGCAA
AGATTCCACACACGGACGGACATTTTCACC
CCTCTCCCCTCATGGGTGGATTCGGACTTA
AACACCCTCCTCCACAGATTCTCATCAAGA
ACACCCCGGTACCTGCGAATCCTTCGACCA
CCTTCAGTGCGGCAAAGTTTGCTTCCTTCA
TCACACAGTACTCCACGGGACAGGTCAGC
GTGGAGATCGAGTGGGAGCTGCAGAAGGA
AAACAGCAAACGCTGGAATCCCGAAATTC
AGTACACTTCCAACTACAACAAGTCTGTTA
ATGTGGACTTTACTGTGGACACTAATGGCG
TGTATTCAGAGCCTCGCCCCATTGGCACCA
89
CA 03220810 2023- 11- 29 SUBSTITUTE SHEET (RULE 26)
WO 2022/256557
PCT/US2022/032004
GATACCTGACTCGTAATCTGTAA (SEQ ID
NO: 55)
VAR-10 MAADGYLPDWLEDTLSE ATGGCTGCCGATGGTTATCTTCCAGATTGG
GIRQWWKLKPGPPPPKPA CTCGAGGACACTCTCTCTGAAGGAATAAG
ERHKDDSRGLVLPGYKYL ACAGTGGTGGAAGCTCAAACCTGGCCCAC
GPFNGLDKGEPVNEADAA CACCACCAAAGCCCGCAGAGCGGCATAAG
ALEHDKAYDRQLDSGDN GACGACAGCAGGGGTCTTGTGCTTCCTGG
PYLKYNHADAEFQERLKE GTACAAGTACCTCGGACCCTTCAACGGACT
DTSFGGNLGRAVFQAKKR CGACAAGGGAGAGCCGGTCAACGAGGCAG
VLEPLGLVEEPVKTAPGK ACGCCGCGGCCCTCGAGCACGACAAAGCC
KRPVEHSPVEPDSSSGTGK TACGACCGGCAGCTCGACAGCGGAGACAA
AGQQPARKRLNFGQTGD CCCGTACCTCAAGTACAACCACGCCGACG
ADS VPDPQPLGQPPAAPS CGGAGTTTCAGGAGCGCCTTAAAGAAGAT
GLGTNTIVIATGSGAPMAD ACGTCTTTTGGGGGCAACCTCGGACGAGC
NNEGADGVGNSSGNWHC AGTCTTCCAGGCGAAAAAGAGGGTTCTTG
DSTWMGDRVITTSTRTWA AACCTCTGGGCCTGGTTGAGGAACCTGTTA
LPTYNNHLYKQISSQSGAS AGACGGCTCCGGGAAAAAAGAGGCCGGTA
NDNHYFGYSTPWGYFDF GAGCACTCTCCTGTGGAGCCAGACTCCTCC
NRFHCHFSPRDWQRLINN TCGGGAACCGGAAAGGCGGGCCAGCAGCC
NWGFRPKRLNFKLFNIQV TGCAAGAAAAAGATTGAATTTTGGTCAGA
KEVTQNDGTTTIANNLTS CTGGAGACGCAGACTCAGTACCTGACCCC
TVQVFTDSEYQLPYVLGS CAGCCTCTCGGACAGCCACCAGCAGCCCC
AHQGCLPPFPADVFMVPQ CTCTGGTCTGGGAACTAATACGATGGCTAC
YGYLTLNNGSQAVGRSS F AGGCAGTGGCGC ACC A ATGGC AG AC A AT A
YCLEYFPSQMLRTGNNFT ACGAGGGCGCCGACGGAGTGGGTAATTCC
FSYTFEDVPFHSSYAHSQS TCGGGAAATTGGCATTGCGATTCCACATGG
LDRLMNPLIDQYLYYLSR ATGGGCGACAGAGTCATCACCACCAGCAC
TNTPSGFSGLENTTTQSRL CCGAACCTGGGCCCTGCCCACCTACAACA
QFSQAGASDIRDQSRNWL ACCACCTCTACAAACAAATTTCCAGCCAAT
PGPCYRQQRVSKTSADNN CAGGAGCCTCGAACGACAATCACTACTTT
NSEYSWTGATKYHLNGR GGCTACAGCACCCCTTGGGGGTATTTTGAC
DSLVNPGPAMASHKDDEE TTCAACAGATTCCACTGCCACTTTTCACCA
KFFPQSGVLIFGKQGSEKT CGTGACTGGCAAAGACTCATCAACAACAA
NVDIEKVMITDEEFIRTTN CTGGGGATTCCGACCCAAGAGACTCAACT
PVATEQYGSVSTNLQRGN TCAAGCTCTTTAACATTCAAGTCAAAGAGG
RQAATADVNTQGVLPGM TCACGCAGAATGACGGTACGACGACGATT
VWQDRDVYLQGPIW AMP GCC A AT A ACCTT ACC AGC ACGGTTC AGGT
HTDGHFHPSPLMGGFGLK GTTTACTGACTCGGAGTACCAGCTCCCGTA
HPPPQILIKNTPVPANPSTT CGTCCTCGGCTCGGCGCATCAAGGATGCCT
FS AAKFASFITQYSTGQVS CCCGCCGTTCCCAGCAGACGTCTTCATGGT
VEIEWELQKENS KRWNPE GCCACAGTATGGATACCTCACCCTGAACA
IQYTSNYNKSVNVDFTVD ACGGGAGTCAGGCAGTAGGACGCTCTTCA
TNGVYSEPRPIGTRYLTRN TTTTACTGCCTGGAGTACTTTCCTTCTCAG
L* (SEQ ID NO: 11) ATGCTGCGTACCGGAAACAACTTTACCTTC
AGCTACACTTTTGAGGACGTTCCTTTCCAC
AGCAGCTACGCTCACAGCCAGAGTCTGGA
CA 03220810 2023- 11- 29 SUBSTITUTE SHEET (RULE 26)
WO 2022/256557
PCT/US2022/032004
CCGTCTAATGAATCCTCTCATCGACCAGTA
CCTGTATTACTTGAGCAGAACAAACACTCC
AAGTGGATTCTCTGGCTTGGAGAACACCA
CCACGCAGTCAAGGCTTCAGTTTTCTCAGG
CCGGAGCGAGTGACATTCGGGACCAGTCT
AGGAACTGGCTTCCTGGACCCTGTTACCGC
CAGCAGCGAGTATCAAAGACATCTGCGGA
TAACAACAACAGTGAATACTCGTGGACTG
GAGCTACCAAGTACCACCTCAATGGCAGA
GACTCTCTGGTGAATCCGGGCCCGGCCATG
GCAAGCCACAAGGACGATGAAGAAAAGTT
TTTTCCTCAGAGCGGGGTTCTCATCTTTGG
GAAGCAAGGCTCAGAGAAAACAAATGTGG
ACATTGAAAAGGTCATGATTACAGACGAA
GAGGAAATCAGGACAACCAATCCCGTGGC
TACGGAGCAGTATGGTTCTGTATCTACCAA
CCTCCAGAGAGGCAACAGACAAGCAGCTA
CCGCAGATGTCAACACACAAGGCGTTCTTC
CAGGCATGGTCTGGCAGGACAGAGATGTG
TACCTTCAGGGGCCCATCTGGGCAAAGATT
CCACACACGGACGGACATTTTCACCCCTCT
CCCCTCATGGGTGGATTCGGACTTAAACAC
CCTCCTCCACAGATTCTCATCAAGAACACC
CCGGTACCTGCGAATCCTTCGACCACCTTC
AGTGCGGCAAAGTTTGCTTCCTTCATCACA
CAGTACTCCACGGGACAGGTCAGCGTGGA
GATCGAGTGGGAGCTGCAGAAGGAAAACA
GCAAACGCTGGAATCCCGAAATTCAGTAC
ACTTCCAACTACAACAAGTCTGTTAATGTG
GACTTTACTGTGGACACTAATGGCGTGTAT
TCAGAGCCTCGCCCCATTGGCACCAGATAC
CTGACTCGTAATCTGTAA (SEQ ID NO: 56)
VAR-11 MAADGYLPDWLEDTLSE ATGGCTGCCGATGGTTATCTTCCAGATTGG
GIRQWWKLKPGPPPPKPA CTCGAGGACACTCTCTCTGAAGGAATAAG
ERHKDDSRGLVLPGYKYL ACAGTGGTGGAAGCTCAAACCTGGCCCAC
GPFNGLDKGEPVNEADAA CACCACCAAAGCCCGCAGAGCGGCATAAG
ALEHDKAYDRQLDSGDN GACGACAGCAGGGGTCTTGTGCTTCCTGG
PYLKYNHADAEFQERLKE GTACAAGTACCTCGGACCCTTCAACGGACT
DTSFGGNLGRAVFQAKKR CGACAAGGGAGAGCCGGTCAACGAGGCAG
VLEPLGLVEEPVKTAPGK ACGCCGCGGCCCTCGAGCACGACAAAGCC
KRPVEHSPVEPDSSSGTGK TACGACCGGCAGCTCGACAGCGGAGACAA
AGQQPARKRLNFGQTGD CCCGTACCTCAAGTACAACCACGCCGACG
ADS VPDPQPLGQPPAAPS CGGAGTTTCAGGAGCGCCTTAAAGAAGAT
GLGTNTMATGSGAPMAD ACGTCTTTTGGGGGCAACCTCGGACGAGC
NNEGADGVGNSSGNWHC AGTCTTCCAGGCGAAAAAGAGGGTTCTTG
DSTWMGDRVITTSTRTWA AACCTCTGGGCCTGGTTGAGGAACCTGTTA
91
CA 03220810 2023- 11- 29 SUBSTITUTE SHEET (RULE 26)
WO 2022/256557
PCT/US2022/032004
LPTYNNHLYKQISSQSGAS AGACGGCTCCGGGAAAAAAGAGGCCGGTA
NDNHYFGYSTPWGYFDF GAGCACTCTCCTGTGGAGCCAGACTCCTCC
NRFHCHFSPRDWQRLINN TCGGGAACCGGAAAGGCGGGCCAGCAGCC
NWGFRPKRLNFKLFNIQV TGCAAGAAAAAGATTGAATTTTGGTCAGA
KEVTQNDGTTTIANNLTS CTGGAGACGCAGACTCAGTACCTGACCCC
TVQVFTDSEYQLPYVLGS CAGCCTCTCGGACAGCCACCAGCAGCCCC
AHQGCLPPFPADVFMVPQ CTCTGGTCTGGGAACTAATACGATGGCTAC
YGYLTLNNGSQAVGRSSF AGGCAGTGGCGCACCAATGGCAGACAATA
YCLEYFPSQMLRTGNNFT ACGAGGGCGCCGACGGAGTGGGTAATTCC
FSYTFEDVPFHSSYAHSQS TCGGGAAATTGGCATTGCGATTCCACATGG
LDRLMNPLIDQYLYYLSR ATGGGCGACAGAGTCATCACCACCAGCAC
TNTPSGTTFSQAGASDIRD CCGAACCTGGGCCCTGCCCACCTACAACA
QSRNWLPGPCYRQQRVSK ACCACCTCTACAAACAAATTTCCAGCCAAT
TSADNNNSEYSWTGATK CAGGAGCCTCGAACGACAATCACTACTTT
YHLNGRDSLVNPGPAMAS GGCTACAGCACCCCTTGGGGGTATTTTGAC
HKDDEEKFFPQSGVLIFGK TTCAACAGATTCCACTGCCACTTTTCACCA
QGSEKTNVDIEKVMITDE CGTGACTGGCAAAGACTCATCAACAACAA
EEIRTTNPVATEQYGSVST CTGGGGATTCCGACCCAAGAGACTCAACT
NLQRGNRQAATADVNTQ TCAAGCTCTTTAACATTCAAGTCAAAGAGG
GVLPGMVWQDRDVYLQG TCACGCAGAATGACGGTACGACGACGATT
PIWAKIPHTDGHFHPSPLM GCCAATAACCTTACCAGCACGGTTCAGGT
GGFGLKHPPPQILIKNTPV GTTTACTGACTCGGAGTACCAGCTCCCGTA
PANPSTTFS A AKFASFITQ CGTCCTCGGCTCGGCGC ATC A AGGATGCCT
YSTGQVSVEIEWELQKEN CCCGCCGTTCCCAGCAGACGTCTTCATGGT
SKRWNPEIQYTSNYNKS V GCCACAGTATGGATACCTCACCCTGAACA
NVDFTVDTNGVYSEPRPI ACGGGAGTCAGGCAGTAGGACGCTCTTCA
GTRYLTRNL* (SEQ ID NO: TTTTACTGCCTGGAGTACTTTCCTTCTCAG
12)
ATGCTGCGTACCGGAAACAACTTTACCTTC
AGCTACACTTTTGAGGACGTTCCTTTCCAC
AGCAGCTACGCTCACAGCCAGAGTCTGGA
CAGACTCATGAATCCTCTCATCGACCAGTA
CCTGTATTACTTGAGCAGAACAAACACTCC
AAGTGGAACCACCTTTTCTCAGGCCGGAG
CGAGTGACATTCGGGACCAGTCTAGGAAC
TGGCTTCCTGGACCCTGTTACCGCCAGCAG
CGAGTATCAAAGACATCTGCGGATAACAA
CAACAGTGAATACTCGTGGACTGGAGCTA
CCAAGTACCACCTCAATGGCAGAGACTCT
CTGGTGAATCCGGGCCCGGCCATGGCAAG
CCACAAGGACGATGAAGAAAAGTTTTTTC
CTCAGAGCGGGGTTCTCATCTTTGGGAAGC
AAGGCTCAGAGAAAACAAATGTGGACATT
GAAAAGGTCATGATTACAGACGAAGAGGA
AATCAGGACAACCAATCCCGTGGCTACGG
AGCAGTATGGTTCTGTATCTACCAACCTCC
AGAGAGGCAACAGACAAGCAGCTACCGCA
92
CA 03220810 2023- 11- 29 SUBSTITUTE SHEET (RULE 26)
WO 2022/256557
PCT/US2022/032004
GATGTCAACACACAAGGCGTTCTTCCAGG
CATGGTCTGGCAGGACAGAGATGTGTACC
TTCAGGGGCCCATCTGGGCAAAGATTCCA
CACACGGACGGACATTTTCACCCCTCTCCC
CTCATGGGTGGATTCGGACTTAAACACCCT
CCTCCACAGATTCTCATCAAGAACACCCCG
GTACCTGCGAATCCTTCGACCACCTTCAGT
GCGGCAAAGTTTGCTTCCTTCATCACACAG
TACTCCACGGGACAGGTCAGCGTGGAGAT
CGAGTGGGAGCTGCAGAAGGAAAACAGCA
AACGCTGGAATCCCGAAATTCAGTACACTT
CC AACT AC AAC AAGTCTGTTAATGTGGACT
TTACTGTGGACACTAATGGCGTGTATTCAG
AGCCTCGCCCCATTGGCACCAGATACCTGA
CTCGTAATCTGTAA (SEQ ID NO: 57)
VAR-12 MAADGYLPDWLEDTLSE ATGGCTGCCGATGGTTATCTTCCAGATTGG
GIRQWWKLKPGPPPPKPA CTCGAGGACACTCTCTCTGAAGGAATAAG
ERHKDDSRGLVLPGYKYL ACAGTGGTGGAAGCTCAAACCTGGCCCAC
GPFNGLDKGEPVNEADAA CACCACCAAAGCCCGCAGAGCGGCATAAG
ALEHDKAYDRQLDSGDN GACGACAGCAGGGGTCTTGTGCTTCCTGG
PYLKYNHADAEFQERLKE GTACAAGTACCTCGGACCCTTCAACGGACT
DTS FGGNLGR AV FQ A K KR CGACAAGGGAGAGCCGGTCAACGAGGCAG
VLEPLGLVEEPVKTAPGK ACGCCGCGGCCCTCGAGCACGACAAAGCC
KRPVEHS P V E PDS S S GTGK TACG ACCGGC A GCTCGAC AGCGGAGAC A A
AGQQPARKRLNFGQTGD CCCGTACCTCAAGTACAACCACGCCGACG
ADS VPDPQPLGQPPAAPS CGGAGTTTCAGGAGCGCCTTAAAGAAGAT
GLGTNTMATGSGAPMAD ACGTCTTTTGGGGGCAACCTCGGACGAGC
NNEGADGVGNSSGNWHC AGTCTTCCAGGCGAAAAAGAGGGTTCTTG
DSTWMGDRVITTSTRTWA AACCTCTGGGCCTGGTTGAGGAACCTGTTA
LPTYNNHLYKQISSQSGAS AGACGGCTCCGGGAAAAAAGAGGCCGGTA
NDNHYFGYSTPWGYFDF GAGCACTCTCCTGTGGAGCCAGACTCCTCC
NRFHCHFSPRDWQRLINN TCGGGAACCGGAAAGGCGGGCCAGCAGCC
NWGFRPKRLNFKLFNIQV TGCAAGAAAAAGATTGAATTTTGGTCAGA
KFVTQNDGTTTIANNLTS CTGGAGACGCAGACTCAGTACCTGACCCC
TVQVFTDSEYQLPYVLGS CAGCCTCTCGGACAGCCACCAGCAGCCCC
AHQGCLPPFPADVFMVPQ CTCTGGTCTGGGAACTAATACGATGGCTAC
YGYLTLNNGS QAVGRS SF AGGCAGTGGCGC ACC A ATGGC AG AC A AT A
YCLEYFPSQMLRTGNNFT ACGAGGGCGCCGACGGAGTGGGTAATTCC
FSYTFEDVPFHSSYAHS QS TCGGGAAATTGGCATTGCGATTCCACATGG
LDRLMNPLIDQYLYYLSR ATGGGCGACAGAGTCATCACCACCAGCAC
TINGSGQNQQTLKFSVAG CCGAACCTGGGCCCTGCCCACCTACAACA
ASDIRDQSRNWLPGPCYR ACCACCTCTACAAACAAATTTCCAGCCAAT
QQRVS KTSADNNNSEYS CAGGAGCCTCGAACGACAATCACTACTTT
WTGATKYHLNGRDSLVN GGCTACAGCACCCCTTGGGGGTATTTTGAC
PGPAMASHKDDEEKFFPQ TTCAACAGATTCCACTGCCACTTTTCACCA
SGVLIFGKQGSEKTNVDIE CGTGACTGGCAAAGACTCATCAACAACAA
93
CA 03220810 2023- 11- 29 SUBSTITUTE SHEET (RULE 26)
WO 2022/256557
PCT/US2022/032004
KVMITDEEEIRTTNPVATE CTGGGGATTCCGACCCAAGAGACTCAACT
QYGSVSTNLQRGNRQAAT TCAAGCTCTTTAACATTCAAGTCAAAGAGG
ADVNTQGVLPGMVWQDR TCACGCAGAATGACGGTACGACGACGATT
DVYLQGPIWAKIPHTDGH GCCAATAACCTTACCAGCACGGTTCAGGT
FHPSPLMGGFGLKHPPPQI GTTTACTGACTCGGAGTACCAGCTCCCGTA
LIKNTPVPANPSTTFSAAK CGTCCTCGGCTCGGCGCATCAAGGATGCCT
FASFITQYSTGQVSVEIEW CCCGCCGTTCCCAGCAGACGTCTTCATGGT
ELQKENS KRWNPEIQYTS GCCACAGTATGGATACCTCACCCTGAACA
NYNKSVNVDFTVDTNGV ACGGGAGTCAGGCAGTAGGACGCTCTTCA
YSEPRPIGTRYLTRNL* TTTTACTGCCTGGAGTACTTTCCTTCTCAG
(SEQ ID NO: 13) ATGCTGCGTACCGGAAACAACTTTACCTTC
AGCTACACTTTTGAGGACGTTCCTTTCCAC
AGCAGCTACGCTCACAGCCAGAGTCTGGA
CCGTCTGATGAATCCTCTCATCGACCAGTA
CCTGTATTACTTGAGCAGAACAATCAACG
GGAGTGGACAGAACCAGCAAACTCTTAAG
TTTTCTGTGGCCGGAGCGAGTGACATTCGG
GACCAGTCTAGGAACTGGCTTCCTGGACCC
TGTTACCGCCAGCAGCGAGTATCAAAGAC
ATCTGCGGATAACAACAACAGTGAATACT
CGTGGACTGGAGCTACCAAGTACCACCTC
AATGGCAGAGACTCTCTGGTGAATCCGGG
CCCGGCCATGGCAAGCCACAAGGACGATG
AAGAAAAGTTTTTTCCTCAGAGCGGGGTTC
TCATCTTTGGGAAGCAAGGCTCAGAGAAA
ACAAATGTGGACATTGAAAAGGTCATGAT
TACAGACGAAGAGGAAATCAGGACAACCA
ATCCCGTGGCTACGGAGCAGTATGGTTCTG
TATCTACCAACCTCCAGAGAGGCAACAGA
CAAGCAGCTACCGCAGATGTCAACACACA
AGGCGTTCTTCCAGGCATGGTCTGGCAGG
ACAGAGATGTGTACCTTCAGGGGCCCATCT
GGGCAAAGATTCCACACACGGACGGACAT
TTTCACCCCTCTCCCCTCATGGGTGGATTC
GGACTTAAACACCCTCCTCCACAGATTCTC
ATCAAGAACACCCCGGTACCTGCGAATCC
TTCGACCACCTTCAGTGCGGCAAAGTTTGC
TTCCTTCATCACACAGTACTCCACGGGACA
GGTCAGCGTGGAGATCGAGTGGGAGCTGC
AGAAGGAAAACAGCAAACGCTGGAATCCC
GAAATTCAGTACACTTCCAACTACAACAA
GTCTGTTAATGTGGACTTTACTGTGGACAC
TAATGGCGTGTATTCAGAGCCTCGCCCCAT
TGGCACCAGATACCTGACTCGTAATCTGTA
A (SEQ ID NO: 58)
94
CA 03220810 2023- 11- 29 SUBSTITUTE SHEET (RULE 26)
WO 2022/256557
PCT/US2022/032004
VAR-13 MAADGYLPDWLEDTLSE ATGGCTGCCGATGGTTATCTTCCAGATTGG
GIRQWWKLKPGPPPPKPA CTCGAGGACACTCTCTCTGAAGGAATAAG
ERHKDDSRGLVLPGYKYL ACAGTGGTGGAAGCTCAAACCTGGCCCAC
GPFNGLDKGEPVNEADAA CACC ACC AAAGCCCGC AGAGCGGCAT AAG
ALEHDKAYDRQLDSGDN GACGACAGCAGGGGTCTTGTGCTTCCTGG
PYLKYNHADAEFQERLKE GTACAAGTACCTCGGACCCTTCAACGGACT
DTSFGGNLGRAVFQAKKR CGAC AAGGGAGAGCCGGTC AAC GAGGC AG
VLEPLGLVEEPVKTAPGK ACGCCGCGGCCCTCGAGCACGACAAAGCC
KRPVEHSPVEPDSSSGTGK TACGACCGGCAGCTCGACAGCGGAGAC AA
AGQQPARKRLNFGQTGD CCCGTACCTCAAGTACAACCACGCCGACG
ADS VPDPQPLGQPPAAPS CGGAGTTTCAGGAGCGCCTTAAAGAAGAT
GLGTNTMATGSGAPMAD ACGTCTTTTGGGGGCAACCTCGGACGAGC
NNEGADGVGNSSGNWHC AGTCTTCCAGGCGAAAAAGAGGGTTCTTG
DS TWMGDRVITTS TRTWA AACCTCTGGGCCTGGTTGAGGAACCTGTTA
LPTYNNHLYKQISSQSGAS AGACGGCTCCGGGAAAAAAGAGGCCGGTA
NDNHYFGYSTPWGYFDF GAGCACTCTCCTGTGGAGCCAGACTCCTCC
NRFHCHFSPRDWQRLINN TCGGGAACCGGAAAGGCGGGCCAGCAGCC
NWGFRPKRLNFKLFNIQV TGCAAGAAAAAGATTGAATTTTGGTCAGA
KEVTQNDGTTTIANNLTS CTGGAGACGCAGACTCAGTACCTGACCCC
TVQVFTDSEYQLPYVLGS CAGCCTCTCGGACAGCCACCAGCAGCCCC
AHQGCLPPFPADVFMVPQ CTCTGGTCTGGGAACTAATACGATGGCTAC
YGYLTLNNGS QAVGRSSF AGGCAGT GGCGC ACC AAT GGC AGAC AAT A
YCLEYFPS QMLRTGNNFT ACGAGGGCGCCGACGGAGTGGGT A ATTCC
FSYTFEDVPFHSSYAHS QS TCGGGAAATTGGCATTGCGATTCCACATGG
LDRLMNPLIDQYLYYLAR AT GGGCGAC AGAGTC ATC ACCACC AGC AC
TQS TT GGT S GLQFSQAGA CCGAACCTGGGCCCTGCCCACCTACAACA
SDIRDQSRNWLPGPCYRQ ACC ACCTCTACAAACAAATTTCCAGCCAAT
QRVSKTS ADNNNSEYSWT CAGGAGCCTCGAACGACAATCACTACTTT
GATKYHLNGRDSLVNPGP GGCTACAGCACCCCTTGGGGGTATTTTGAC
AMASHKDDEEKFFPQSGV TTCAACAGATTCCACTGCCACTTTTCACCA
LIFGKQGSEKTNVDIEKV CGT GACT GGC AAAGACTCATC AAC AAC AA
MITDEEEIRTTNPVATEQY CTGGGGATTCCGACCCAAGAGACTCAACT
GS VSTNLQRGNRQAATAD TCAAGCTCTTTAACATTCAAGTCAAAGAGG
VNTQGVLPGMVWQDRDV TCACGCAGAATGACGGTACGACGACGATT
YLQGPIWAKIPHTDGHFH GCCAATAACCTTACCAGCACGGTTCAGGT
PSPLMGGFGLKHPPPQILI GTTTACTGACTCGGAGTACCAGCTCCCGTA
KNTPVPANPSTTFSAAKF CGTCCTCGGCTCGGCGCATCAAGGATGCCT
AS FITQYS TGQVS VE1EWE CCCGCCGTTCCCAGCAGACGTCTTCATGGT
LQKENSKRWNPEIQYTSN GCCACAGTATGGATACCTCACCCTGAACA
YNKS VN VDFTVDTNGVY ACGGGAGTCAGGCAGTAGGACGCTCTTCA
SEPRPIGTRYLTRNL* (SEQ TTTTACTGCCTGGAGTACTTTCCTTCTCAG
ID NO: 14)
ATGCTGCGTACCGGAAACAACTTTACCTTC
AGCTACACTTTTGAGGACGTTCCTTTCCAC
AGCAGCTACGCTCACAGCCAGAGTCTGGA
CCGTCTTATGAATCCTCTCATCGACCAGTA
CCTGT ATT ACTTGGCT AGA ACTC A A A GTAC
CA 03220810 2023- 11- 29 SUBSTITUTE SHEET (RULE 26)
WO 2022/256557
PCT/US2022/032004
AACCGGGGGTACCTCAGGGCTTCAGTTTTC
TCAGGCCGGAGCGAGTGACATTCGGGACC
AGTCTAGGAACTGGCTTCCTGGACCCTGTT
ACCGCCAGCAGCGAGTATCAAAGACATCT
GCGGATAACAACAACAGTGAATACTCGTG
GACTGGAGCTACCAAGTACCACCTCAATG
GCAGAGACTCTCTGGTGAATCCGGGCCCG
GCCATGGCAAGCCACAAGGACGATGAAGA
AAAGTTTTTTCCTCAGAGCGGGGTTCTCAT
CTTTGGGAAGCAAGGCTCAGAGAAAACAA
ATGTGGACATTGAAAAGGTCATGATTACA
GACGAAGAGGAAATCAGGACAACCAATCC
CGTGGCTACGGAGCAGTATGGTTCTGTATC
TACCAACCTCCAGAGAGGCAACAGACAAG
CAGCTACCGCAGATGTCAACACACAAGGC
GTTCTTCCAGGCATGGTCTGGCAGGACAG
AGATGTGTACCTTCAGGGGCCCATCTGGGC
AAAGATTCCACACACGGACGGACATTTTC
ACCCCTCTCCCCTCATGGGTGGATTCGGAC
TTAAACACCCTCCTCCACAGATTCTCATCA
AGAACACCCCGGTACCTGCGAATCCTTCG
ACCACCTTCAGTGCGGCAAAGTTTGCTTCC
TTCATC AC AC A GTACTCC ACGGGACAGGTC
AGCGTGGAGATCGAGTGGGAGCTGCAGAA
GGAAAACAGCAAACGCTGGAATCCCGAAA
TTCAGTACACTTCCAACTACAACAAGTCTG
TTAATGTGGACTTTACTGTGGACACTAATG
GCGTGTATTCAGAGCCTCGCCCCATTGGCA
CCAGATACCTGACTCGTAATCTGTAA (SEQ
ID NO: 59)
VAR-14 MAADGYLPDWLEDTLSE ATGGCTGCCGATGGTTATCTTCCAGATTGG
GIRQWWKLKPGPPPPKPA CTCGAGGACACTCTCTCTGAAGGAATAAG
ERHKDDSRGLVLPGYKYL ACAGTGGTGGAAGCTCAAACCTGGCCCAC
GPFNGLDKGEPVNEADAA CACCACCAAAGCCCGCAGAGCGGCATAAG
ALEHDKAYDRQLDSGDN GACGACAGCAGGGGTCTTGTGCTTCCTGG
PYLKYNHADAEFQERLKE GTACAAGTACCTCGGACCCTTCAACGGACT
DTSFGGNLGRAVFQAKKR CGACAAGGGAGAGCCGGTC A ACGAGGC AG
VLEPLGLVEEPVKTAPGK ACGCCGCGGCCCTCGAGCACGACAAAGCC
KRPVEHSPVEPDSSSGTGK TACGACCGGCAGCTCGACAGCGGAGACAA
AGQQPARKRLNFGQTGD CCCGTACCTCAAGTACAACCACGCCGACG
ADS VPDPQPLGQPPAAPS CGGAGTTTCAGGAGCGCCTTAAAGAAGAT
GLGTNTMATGSGAPMAD ACGTCTTTTGGGGGCAACCTCGGACGAGC
NNEGADGVGNSSGNWHC AGTCTTCCAGGCGAAAAAGAGGGTTCTTG
DSTWMGDRVITTSTRTWA AACCTCTGGGCCTGGTTGAGGAACCTGTTA
LPTYNNHLYKQISSQSGAS AGACGGCTCCGGGAAAAAAGAGGCCGGTA
NDNHYFGYSTPWGYFDF GAGCACTCTCCTGTGGAGCCAGACTCCTCC
96
CA 03220810 2023- 11- 29 SUBSTITUTE SHEET (RULE 26)
WO 2022/256557
PCT/US2022/032004
NRFHCHFSPRDWQRLINN TCGGGAACCGGAAAGGCGGGCCAGCAGCC
NWGFRPKRLNFKLFNIQV TGCAAGAAAAAGATTGAATTTTGGTCAGA
KEVTQNDGTTTIANNLTS CTGGAGACGCAGACTCAGTACCTGACCCC
TVQVFTDSEYQLPYVLGS CAGCCTCTCGGACAGCCACCAGCAGCCCC
AHQGCLPPFPADVFMVPQ CTCTGGTCTGGGAACTAATACGATGGCTAC
YGYLTLNNGSQAVGRSSF AGGCAGTGGCGCACCAATGGCAGACAAT A
YCLEYFPSQMLRTGNNFT ACGAGGGCGCCGACGGAGTGGGTAATTCC
FSYTFEDVPFHSSYAHSQS TCGGGAAATTGGCATTGCGATTCCACATGG
LDRLMNPLIDQYLYYLSR ATGGGCGACAGAGTCATCACCACCAGCAC
TQSPYGGTATFMDLQFSQ CCGAACCTGGGCCCTGCCCACCTACAACA
AGAS DIRD QS RNWLPGPC ACC ACCTCTACAAACAAATTTCCAGCCAAT
YRQQRVSKTSADNNNSEY CAGGAGCCTCGAACGACAATCACTACTTT
SWTGATKYHLNGRDSLV GGCTACAGCACCCCTTGGGGGTATTTTGAC
NPGPAMASHKDDEEKFFP TTCAACAGATTCCACTGCCACTTTTCACCA
QSGVLIFGKQGSEKTNVDI CGTGACTGGCAAAGACTCATCAACAACAA
EKVMITDEEEIRTTNP VAT CTGGGGATTCCGACCCAAGAGACTCAACT
EQYGSVSTNLQRGNRQAA TCAAGCTCTTTAACATTCAAGTCAAAGAGG
TADVNTQGVLPGMVWQD TCACGCAGAATGACGGTACGACGACGATT
RDVYLQGPIWAKIPHTDG GCCAATAACCTTACCAGCACGGTTCAGGT
HFHPSPLMGGFGLKHPPP GTTTACTGACTCGGAGTACCAGCTCCCGTA
QILIKNTPVPANPSTTFSAA CGTCCTCGGCTCGGCGCATCAAGGATGCCT
KFASFITQYSTGQVSVEIE CCCGCCGTTCCCAGCAGACGTCTTCATGGT
WELQKENS KRWNPEIQYT GCC AC AGT ATGGAT ACCTC ACCCTGA AC A
SNYNKSVNVDFTVDTNG ACGGGAGTCAGGCAGTAGGACGCTCTTCA
VYSEPRPIGTRYLTRNL
TTTTACTGCCTGGAGTACTTTCCTTCTCAG
(SEQ ID NO: 15)
ATGCTGCGTACCGGAAACAACTTTACCTTC
AGCTACACTTTTGAGGACGTTCCTTTCCAC
AGCAGCTACGCTCACAGCCAGAGTCTGGA
CCGCCTCATGAATCCTCTCATCGACCAGTA
CCTGTATTACTTGAGCAGAACACAATCGCC
ATACGGGGGAACCGCCACGTTCATGGACC
TTCAGTTTTCTCAGGCCGGAGCGAGTGACA
TTCGGGACCAGTCTAGGAACTGGCTTCCTG
GACCCTGTTACCGCCAGCAGCGAGTATCA
AAGACATCTGCGGATAACAACAACAGTGA
ATACTCGTGGACTGGAGCTACCAAGTACC
ACCTCAATGGCAGAGACTCTCTGGTGAATC
CGGGCCCGGCCATGGCAAGCCACAAGGAC
GATGAAGAAAAGTTTTTTCCTCAGAGCGG
GGTTCTCATCTTTGGGAAGCAAGGCTCAGA
GAAAACAAATGTGGACATTGAAAAGGTCA
TGATTACAGACGAAGAGGAAATCAGGACA
ACCAATCCCGTGGCTACGGAGCAGTATGG
TTCTGIATCTACCAACCTCCAGAGAGGCAA
CAGACAAGCAGCTACCGCAGATGTCAACA
CAC A AGGCGTTCTTCC AGGC ATGGTCTGGC
97
CA 03220810 2023- 11- 29 SUBSTITUTE SHEET (RULE 26)
WO 2022/256557
PCT/US2022/032004
AGGACAGAGATGTGTACCTTCAGGGGCCC
ATCTGGGCAAAGATTCCACACACGGACGG
ACATTTTCACCCCTCTCCCCTCATGGGTGG
ATTCGGACTTAAACACCCTCCTCCACAGAT
TCTCATCAAGAACACCCCGGTACCTGCGA
ATCCTTCGACCACCTTCAGTGCGGCAAAGT
TTGCTTCCTTCATCACACAGTACTCCACGG
GACAGGTCAGCGTGGAGATCGAGTGGGAG
CTGCAGAAGGAAAACAGCAAACGCTGGAA
TCCCGAAATTCAGTACACTTCCAACTACAA
CAAGTCTGTTAATGTGGACTTTACTGTGGA
CACTAATGGCGTGTATTCAGAGCCTCGCCC
CATTGGCACCAGATACCTGACTCGTAATCT
GTAA (SEQ ID NO: 601)
VAR- 15 MAADGYLPDWLEDTLSE ATGGCTGCCGATGGTTATCTTCCAGATTGG
GIRQWWKLKPGPPPPKPA CTCGAGGACACTCTCTCTGAAGGAATAAG
ERHKDDSRGLVLPGYKYL ACAGTGGTGGAAGCTCAAACCTGGCCCAC
GPFNGLDKGEPVNEADAA CACCACCAAAGCCCGCAGAGCGGCATAAG
ALEHDKAYDRQLDSGDN GACGACAGCAGGGGTCTTGTGCTTCCTGG
PYLKYNHADAEFQERLKE GTACAAGTACCTCGGACCCTTCAACGGACT
DTSEGGNLGRAVFQAKKR CGACAAGGGAGAGCCGGTCAACGAGGCAG
VLEPLGLV EEPV KT APGK ACGCCGCGGCCCTCGAGCACGAC A A AGCC
KRPVEHSPVEPDSSSGTGK TACGACCGGCAGCTCGACAGCGGAGACAA
AGQQP A R KR LN FGQTGD CCCGT ACCTC A A GTACA ACC ACGCCGACG
ADS VPDPQPLGQPPAAPS CGGAGTTTCAGGAGCGCCTTAAAGAAGAT
GLGTNT1VIATGSGAPMAD ACGTCTTTTGGGGGCAACCTCGGACGAGC
NNEGADGVGNSSGNWHC AGTCTTCCAGGCGAAAAAGAGGGTTCTTG
DSTWMGDRVITTSTRTWA AACCTCTGGGCCTGGTTGAGGAACCTGTTA
LPTYNNHLYKQISSQSGAS AGACGGCTCCGGGAAAAAAGAGGCCGGTA
NDNHYFGYSTPWGYFDF GAGCACTCTCCTGTGGAGCCAGACTCCTCC
NRFHCHFSPRDWQRLINN TCGGGAACCGGAAAGGCGGGCCAGCAGCC
NWGFRPKRLNFKLFNIQV TGCAAGAAAAAGATTGAATTTTGGTCAGA
KEVTQNDGTTTIANNLTS CTGGAGACGCAGACTCAGTACCTGACCCC
TVQVFTDSEYQLPYVLGS CAGCCTCTCGGACAGCCACCAGCAGCCCC
AHQGCLPPFPADVFMVPQ CTCTGGTCTGGGAACTAATACGATGGCTAC
YGYLTLNNGS QAVGRS SF AGGCAGTGGCGCACCAATGGCAGACAAT A
YCLEYFPSQMLRTGNNFT ACGAGGGCGCCGACGGAGTGGGT A ATTCC
FSYTFEDVPFHSSYAHS QS TCGGGAAATTGGCATTGCGATTCCACATGG
LDRLMNPLIDQYLYYLSR ATGGGCGACAGAGTCATCACCACCAGCAC
HETEFNFTNTPSGTTTQSR CCGAACCTGGGCCCTGCCCACCTACAACA
LQFSQAGASDIRDQSRNW ACCACCTCTACAAACAAATTTCCAGCCAAT
LPGPCYRQQRVS KTSADN CAGGAGCCTCGAACGACAATCACTACTTT
NNSEYSWTGATKYHLNG GGCTACAGCACCCCTTGGGGGTATTTTGAC
RDSLVNPGPAMASHKDDE TTCAACAGATTCCACTGCCACTTTTCACCA
EKFFPQSGVLIFGKQGSEK CGTGACTGGCAAAGACTCATCAACAACAA
TNVDIEKVMITDEEEIRTT CTGGGGATTCCGACCCAAGAGACTCAACT
98
CA 03220810 2023- 11- 29 SUBSTITUTE SHEET (RULE 26)
WO 2022/256557
PCT/US2022/032004
NPVATEQYGSVSTNLQRG TCAAGCTCTTTAACATTCAAGTCAAAGAGG
NRQAATADVNTQGVLPG TCACGCAGAATGACGGTACGACGACGATT
MVWQDRDVYLQGPIWAK GCC AAT AACCTT ACCAGC AC GGTTCAGGT
IPHTDGHFHPSPLMGGFGL GTTTACTGACTCGGAGTACCAGCTCCCGTA
KHPPPQILIKNTPVPANPST CGTCCTCGGCTCGGCGCATCAAGGATGCCT
TES AAKFAS FITQYS TGQV CCCGCCGTTCCCAGCAGACGTCTTCATGGT
SVEIEWELQKENS KRWNP GCCACAGTATGGATACCTCACCCTGAACA
EIQYTSNYNKSVNVDFTV ACGGGAGTCAGGCAGTAGGACGCTCTTCA
DTNGVYSEPRPIGTRYLTR TTTTACTGCCTGGAGTACTTTCCTTCTCAG
NL* (SEQ ID NO: 16)
ATGCTGCGTACCGGAAACAACTTTACCTTC
AGCTACACTTTTGAGGACGTTCCTTTCCAC
AGCAGCTACGCTCACAGCCAGAGTCTGGA
CCGTTTGATGAATCCTCTCATCGACCAGTA
CCTGTATTACTTGAGCAGACACGAAACCG
AATTCAATTTTACAAACACTCCAAGTGGAA
CCACCACGCAGTCAAGGCTTCAGTTTTCTC
AGGCCGGAGCGAGTGACATTCGGGACCAG
TCTAGGAACTGGCTTCCTGGACCCTGTTAC
CGCCAGCAGCGAGTATCAAAGACATCTGC
GGATAACAACAACAGTGAATACTCGTGGA
CTGGAGCTACCAAGTACCACCTCAATGGC
AGAGACTCTCTGGTGAATCCGGGCCCGGC
CATGGCAAGCCACAAGGACGATGAAGAAA
AGTTTTTTCCTCAGAGCGGGGTTCTCATCT
TTGGGAAGCAAGGCTCAGAGAAAACAAAT
GTGGACATTGAAAAGGTCATGATTACAGA
CGAAGAGGAAATCAGGACAACCAATCCCG
TGGCTACGGAGCAGTATGGTTCTGTATCTA
CCAACCTCCAGAGAGGCAACAGACAAGCA
GCTACCGCAGATGTCAACACACAAGGCGT
TCTTCCAGGCATGGTCTGGCAGGACAGAG
ATGTGTACCTTCAGGGGCCCATCTGGGCAA
AGATTCCACACACGGACGGACATTTTCACC
CCTCTCCCCTCATGGGTGGATTCGGACTTA
AACACCCTCCTCCACAGATTCTCATCAAGA
ACACCCCGGTACCTGCGAATCCTTCGACCA
CCTTCAGTGCGGCAAAGTTTGCTTCCTTCA
TCACACAGTACTCCACGGGACAGGTCAGC
GTGGAGATCGAGTGGGAGCTGCAGAAGGA
AAACAGCAAACGCTGGAATCCCGAAATTC
AGTACACTTCCAACTACAACAAGTCTGTTA
ATGTGGACTTTACTGTGGACACTAATGGCG
TGTATTCAGAGCCTCGCCCCATTGGCACCA
GATACCTGACTCGTAATCTGTAA (SEQ ID
NO: 61)
99
CA 03220810 2023- 11- 29 SUBSTITUTE SHEET (RULE 26)
WO 2022/256557
PCT/US2022/032004
VAR-16 MAADGYLPDWLEDTLSE ATGGCTGCCGATGGTTATCTTCCAGATTGG
GIRQWWKLKPGPPPPKPA CTCGAGGACACTCTCTCTGAAGGAATAAG
ERHKDDSRGLVLPGYKYL ACAGTGGTGGAAGCTCAAACCTGGCCCAC
GPFNGLDKGEPVNEADAA CACCACCAAAGCCCGCAGAGCGGCATAAG
ALEHDKAYDRQLDSGDN GACGACAGCAGGGGTCTTGTGCTTCCTGG
PYLKYNHADAEFQERLKE GTACAAGTACCTCGGACCCTTCAACGGACT
DTSFGGNLGRAVFQAKKR CGACAAGGGAGAGCCGGTCAACGAGGCAG
VLEPLGLVEEPVKTAPGK ACGCCGCGGCCCTCGAGCACGACAAAGCC
KRPVEHSPVEPDSSSGTGK TACGACCGGCAGCTCGACAGCGGAGACAA
AGQQPARKRLNFGQTGD CCCGTACCTCAAGTACAACCACGCCGACG
ADS VPDPQPLGQPPAAPS CGGAGTTTCAGGAGCGCCTTAAAGAAGAT
GLGTNTMATGSGAPMAD ACGTCTTTTGGGGGCAACCTCGGACGAGC
NNEGADGVGNSSGNWHC AGTCTTCCAGGCGAAAAAGAGGGTTCTTG
DSTWMGDRVITTSTRTWA AACCTCTGGGCCTGGTTGAGGAACCTGTTA
LPTYNNHLYKQISSQSGAS AGACGGCTCCGGGAAAAAAGAGGCCGGTA
NDNHYFGYSTPWGYFDF GAGCACTCTCCTGTGGAGCCAGACTCCTCC
NRFHCHFSPRDWQRLINN TCGGGAACCGGAAAGGCGGGCCAGCAGCC
NWGFRPKRLNFKLFNIQV TGCAAGAAAAAGATTGAATTTTGGTCAGA
KEVTQNDGTTTIANNLTS CTGGAGACGCAGACTCAGTACCTGACCCC
TVQVFTDSEYQLPYVLGS CAGCCTCTCGGACAGCCACCAGCAGCCCC
AHQGCLPPFPADVFMVPQ CTCTGGTCTGGGAACTAATACGATGGCTAC
YGYLTLNNGS QAVGRS SF AGGCAGTGGCGCACCAATGGCAGACAAT A
YCLEYFPSQMLRTGNNFT ACGAGGGCGCCGACGGAGTGGGT A ATTCC
FSYTFEDVPFHSSYAHS QS TCGGGAAATTGGCATTGCGATTCCACATGG
LDRLMNPLIDQYLYYLSR ATGGGCGACAGAGTCATCACCACCAGCAC
TNTTS GTGGTQTLAFSQA CCGAACCTGGGCCCTGCCCACCTACAACA
GAS DIRDQSRNWLPGPCY ACC ACCTCTACAAACAAATTTCCAGCCAAT
RQQRVS KTSADNNNSEYS CAGGAGCCTCGAACGACAATCACTACTTT
WTGATKYHLNGRDSLVN GGCTACAGCACCCCTTGGGGGTATTTTGAC
PGPAMASHKDDEEKFFPQ TTCAACAGATTCCACTGCCACTTTTCACCA
SGVLIFGKQGSEKTNVDIE CGTGACTGGCAAAGACTCATCAACAACAA
KVMITDEEEIRTTNPVATE CTGGGGATTCCGACCCAAGAGACTCAACT
QYGSVSTNLQRGNRQAAT TCAAGCTCTTTAACATTCAAGTCAAAGAGG
ADVNTQGVLPGMVWQDR TCACGCAGAATGACGGTACGACGACGATT
DVYLQGPIWAKIPHTDGH GCCAATAACCTTACCAGCACGGTTCAGGT
FHPSPLMGGFGLKHPPPQI GTTTACTGACTCGGAGT ACCAGCTCCCGT A
LIKNTPVPANPSTTFSAAK CGTCCTCGGCTCGGCGCATCAAGGATGCCT
FAS FITQYSTGQVS VEIEW CCCGCCGTTCCCAGCAGACGTCTTCATGGT
ELQKENS KRWNPEIQYTS GCCACAGTATGGATACCTCACCCTGAACA
NYNKS VN VDFTVDTNGV ACGGGAGTCAGGCAGTAGGACGCTCTTCA
YSEPRPIGTRYLTRNL*
TTTTACTGCCTGGAGTACTTTCCTTCTCAG
(SEQ ID NO: 17)
ATGCTGCGTACCGGAAACAACTTTACCTTC
AGCTACACTTTTGAGGACGTTCCTTTCCAC
AGCAGCTACGCTCACAGCCAGAGTCTGGA
CCGACTCATGAATCCTCTCATCGACCAGTA
CCTGTATTACTTGAGCAGAACAAACACTAC
100
CA 03220810 2023- 11- 29 SUBSTITUTE SHEET (RULE 26)
WO 2022/256557
PCT/US2022/032004
AAGTGGAACCGGAGGAACACAGACGCTTG
CTTTTTCTCAGGCCGGAGCGAGTGACATTC
GGGACCAGTCTAGGAACTGGCTTCCTGGA
CCCTGTTACCGCCAGCAGCGAGTATCAAA
GACATCTGCGGATAACAACAACAGTGAAT
ACTCGTGGACTGGAGCTACCAAGTACCAC
CTCAATGGCAGAGACTCTCTGGTGAATCCG
GGCCCGGCCATGGCAAGCCACAAGGACGA
TGAAGAAAAGTTTTTTCCTCAGAGCGGGGT
TCTCATCTTTGGGAAGCAAGGCTCAGAGA
AAACAAATGTGGACATTGAAAAGGTCATG
ATTACAGACGAAGAGGAAATCAGGACAAC
CAATCCCGTGGCTACGGAGCAGTATGGTTC
TGTATCTACCAACCTCCAGAGAGGCAACA
GACAAGCAGCTACCGCAGATGTCAACACA
CAAGGCGTTCTTCCAGGCATGGTCTGGCAG
GACAGAGATGTGTACCTTCAGGGGCCCAT
CTGGGCAAAGATTCCACACACGGACGGAC
ATTTTCACCCCTCTCCCCTCATGGGTGGAT
TCGGACTTAAACACCCTCCTCCACAGATTC
TCATCAAGAACACCCCGGTACCTGCGAAT
CCTTCGACCACCTTCAGTGCGGCAAAGTTT
GCTTCCTTCATCACACAGTACTCCACGGGA
CAGGTCAGCGTGGAGATCGAGTGGGAGCT
GCAGAAGGAAAACAGCAAACGCTGGAATC
CCGAAATTCAGTACACTTCCAACTACAACA
AGTCTGTTAATGTGGACTTTACTGTGGACA
CTAATGGCGTGTATTCAGAGCCTCGCCCCA
TTGGCACCAGATACCTGACTCGTAATCTGT
AA (SEQ ID NO: 62)
Table 4
Capsid Amino Acid Sequence of Exemplary Nucleic Acid Molecule
Sequence(SEQ ID
Variant VP1 capsid polypeptide NO)
(SEQ ID NO; starting
amino acid of VP2 is
underlined; starting amino
acid of VP3 is in bold.
VAR-17 MAADGYLPDWLEDTL ATGGCTGCCGATGGTTATCTTCCAGATTGGCT
SEGIRQWWKLKPGPPP CGAGGACACTCTCTCTGAAGGAATAAGACAG
PKPAERHKDDSRGLVL TGGTGGAAGCTCAAACCTGGCCCACCACCACC
PGYKYLGPFNGLDKGE AAAGCCCGCAGAGCGGCATAAGGACGACAGC
PVNEADAAALEHDKA AGGGGTCTTGTGCTTCCTGGGTACAAGTACCT
YDRQLDSGDNPYLKY CGGACCCTTCAACGGACTCGACAAGGGAGAG
1()1
CA 03220810 2023- 11- 29 SUBSTITUTE SHEET (RULE 26)
WO 2022/256557
PCT/US2022/032004
NHADAEFQERLKEDTS CCGGTCAACGAGGCAGACGCCGCGGCCCTCG
FGGNLGRAVFQAKKR AGC ACGAC AAAGCCTACGACCGGC AGCTC GA
VLEPLGLVEEPVKTAP CAGCGGAGACAACCCGTACCTCAAGTACAAC
GKKRPVEHSPVEPDSS CACGCCGACGCGGAGTTTCAGGAGCGCCTTA
SGTGKAGQQPARKRL AAGAAGATACGTCTTTTGGGGGCAACCTCGG
NFGQTGDADSVPDPQP ACGAGCAGTCTTCCAGGCGAAAAAGAGGGTT
LGQPPAAPSGLGTNTM CTTGAACCTCTGGGCCTGGTTGAGGAACCTGT
ATGSGAPMADNNEGA TAAGACGGCTCCGGGAAAAAAGAGGCCGGTA
DGVGNSSGNWHCDST GAGCACTCTCCTGTGGAGCCAGACTCCTCCTC
WMGDRVITTSTRTWA GGGAACCGGAAAGGCGGGCCAGCAGCCTGCA
LPT YNNHLYKQIS S QS AGAAAAAGATTGAATTTTGGTCAGACTGGAG
GASNDNHYFGYSTPW ACGCAGACTCAGTACCTGACCCCCAGCCTCTC
GYFDFNRFHCHFSPRD GGACAGCCACCAGCAGCCCCCTCTGGTCTGGG
WQRLINNNWGFRPKR AACTAATACGATGGCTACAGGCAGTGGCGCA
LNFKLFNIQVKEVTQN CCAATGGCAGACAATAACGAGGGCGCCGACG
DGTTTIANNLTSTVQV GAGTGGGTAATTCCTCGGGAAATTGGCATTGC
FTDSEYQLPYVLGS AH GATTCCACATGGATGGGCGACAGAGTCATCA
QGCLPPFPADVFMVPQ CCACCAGCACCCGAACCTGGGCCCTGCCCACC
YGYLTLNNGS QAVGR TAC AAC AACC ACCTCT AC AAAC AAATTTCCAG
SSFYCLEYFPS QMLRT CCAATCAGGAGCCTCGAACGACAATCACTACT
GNNFTFSYTFEDVPFH TTGGCTACAGCACCCCTTGGGGGTATTTTGAC
SSYAHS QSLDRLMNPL TTCAACAGATTCCACTGCCACTTTTCACCACG
IDQYLYYLSRTNTPSG TGACTGGC A A A GACTC ATC A AC A AC A ACTGG
TTTQSRLQFS QAGASDI GGATTCCGACCCAAGAGACTCAACTTCAAGCT
RDQSRNWLPGPCYRQ CTTTAAC ATTCAAGTC AAAGAGGTC ACGC AGA
QRVSKTS ADNNNSEYS ATGACGGTACGACGACGATTGCCAATAACCTT
WTGATKYHLNGRDSL ACCAGCACGGTTCAGGTGTTTACTGACTCGGA
VNPGPAMASHKDDEE GTACCAGCTCCCGTACGTCCTCGGCTCGGC GC
KFFPQSGVLIFGKQGSE ATCAAGGATGCCTCCCGCCGTTCCCAGCAGAC
KTNVDIEKVMITDEEEI GTCTTCATGGTGCCACAGTATGGATACCTCAC
RTTNPVATEQYGS VS T CCTGAACAACGGGAGTCAGGCAGTAGGACGC
FALMEPNLQRGNRQA TCTTCATTTTACTGCCTGGAGTACTTTCCTTCT
AT ADVNTQGVLPGMV CAGATGCTGCGTACCGGAAACAACTTTACCTT
WQDRDVYLQGPIWAK CAGCTACACTTTTGAGGACGTTCCTTTCCACA
IPHTDGHFHPSPLMGG GCAGCTACGCTCACAGCCAGAGTCTGGACCGT
FGLKHPPPQILIKNTPV CTCATGAATCCTCTCATCGACCAGTACCTGTA
PANPSTTFSAAKFASFI TTACTTGAGCAGAACAAACACTCCAAGTGGA
TQYSTGQVS VEIEWEL ACCACCACGCAGTCAAGGCTTCAGTTTTCTCA
QKENS KRWNPEIQYTS GGCCGGAGCGAGTGACATTCGGGACCAGTCT
NYNKS VN VDFTVDTN AGGAACTGGCTTCCTGGACCCTGTTACCGCCA
GVYSEPRPIGTRYLTR GC AGCGAGT ATC AAAGAC ATCT GCGGAT AAC
NL* (SEQ ID NO: 18)
AACAACAGTGAATACTCGTGGACTGGAGCTA
CCAAGTACCACCTCAATGGCAGAGACTCTCTG
GTGAATCCGGGCCCGGCCATGGCAAGCCACA
AGGAC GAT GAAGAAAAGTTTTTTCCTCAGAGC
GGGGTTCTCATCTTTGGGAAGC A A GGCTC AGA
102
CA 03220810 2023- 11- 29 SUBSTITUTE SHEET (RULE 26)
WO 2022/256557
PCT/US2022/032004
GAAAACAAATGTGGACATTGAAAAGGTCATG
ATTACAGACGAAGAGGAAATCAGGACAACCA
ATCCCGTGGCTACGGAGCAGTATGGTTCTGTA
TCTACCTTTGCCCTCATGGAGCCGAACCTCCA
GAGAGGCAACAGACAAGCAGCTACCGCAGAT
GTCAACACACAAGGCGTTCTTCCAGGCATGGT
CTGGCAGGACAGAGATGTGTACCTTCAGGGG
CCCATCTGGGCAAAGATTCCACACACGGACG
GACATTTTCACCCCTCTCCCCTCATGGGTGGA
TTCGGACTTAAACACCCTCCTCCACAGATTCT
CATCAAGAACACCCCGGTACCTGCGAATCCTT
CGACCACCTTCAGTGCGGCAAAGTTTGCTTCC
TTCATCACACAGTACTCCACGGGACAGGTCAG
CGTGGAGATCGAGTGGGAGCTGCAGAAGGAA
AACAGCAAACGCTGGAATCCCGAAATTCAGT
ACACTTCCAACTACAACAAGTCTGTTAATGTG
GACTTTACTGTGGACACTAATGGCGTGTATTC
AGAGCCTCGCCCCATTGGCACCAGATACCTGA
CTCGTAATCTGTAA (SEQ ID NO: 63)
VAR- 18 MAADGYLPDWLEDTL ATGGCTGCCGATGGTTATCTTCCAGATTGGCT
SEGIRQWWKLKPGPPP CGAGGACACTCTCTCTGAAGGAATAAGACAG
PKPAERH KDDSRGLV L TGGTGGA AGCTC A A ACCTGGCCC ACC ACCACC
PGYKYLGPFNGLDKGE AAAGCCCGCAGAGCGGCATAAGGACGACAGC
PVNEAD A A A LEH DK A AGGGGTCTTGTGCTTCCTGGGTACA AGT ACCT
YDRQLDSGDNPYLKY CGGACCCTTCAACGGACTCGACAAGGGAGAG
NHADAEFQERLKEDTS CCGGTCAACGAGGCAGACGCCGCGGCCCTCG
FGGNLGRAVFQAKKR AGCACGACAAAGCCTACGACCGGCAGCTC GA
VLEPLGLVEEPVKTAP CAGCGGAGACAACCCGTACCTCAAGTACAAC
GKKRPVEHSPVEPDSS CACGCCGACGCGGAGTTTCAGGAGCGCCTTA
SGTGKAGQQPARKRL AAGAAGATACGTCTTTTGGGGGCAACCTCGG
NFGQTGDADSVPDPQP ACGAGCAGTCTTCCAGGCGAAAAAGAGGGTT
LGQPPAAPSGLGTNTM CTTGAACCTCTGGGCCTGGTTGAGGAACCTGT
ATGSGAPMADNNEGA TAAGACGGCTCCGGGAAAAAAGAGGCCGGTA
DGVGNSSGNWHCDST GAGCACTCTCCTGTGGAGCCAGACTCCTCCTC
WMGDRVITTSTRTWA GGGAACCGGAAAGGCGGGCCAGCAGCCTGCA
LPTYNNHLYKQISSQS AGAAAAAGATTGAATTTTGGTCAGACTGGAG
GASNDNHYFGYSTPW ACGC AG ACTC AGT ACCTGACCCCCAGCCTCTC
GYFDFNRFHCHFSPRD GGACAGCCACCAGCAGCCCCCTCTGGTCTGGG
WQRLINNNWGFRPKR AACTAATACGATGGCTACAGGCAGTGGCGCA
LNFKLFN1QVKEVTQN CCAATGGCAGACAATAACGAGGGCGCCGACG
DGTTTIANNLTSTVQV GAGTGGGTAATTCCTCGGGAAATTGGCATTGC
FTDSEYQLPYVLGSAH GATTCCACATGGATGGGCGACAGAGTCATCA
QGCLPPFPADVFMVPQ CCACCAGCACCCGAACCTGGGCCCTGCCCACC
YGYLTLNNGSQAVGR TACAACAACC ACCTCT AC AAACAAATTTCCAG
SSFYCLEYFPSQMLRT CCAATCAGGAGCCTCGAACGACAATCACTACT
GNNFTFSYTFEDVPFH TTGGCTACAGCACCCCTTGGGGGTATTTTGAC
103
CA 03220810 2023- 11- 29 SUBSTITUTE SHEET (RULE 26)
WO 2022/256557
PCT/US2022/032004
S SYAHS QSLDRLMNPL TTCAACAGATTCCACTGCCACTTTTCACCACG
IDQYLYYLSRTNTPSG TGACTGGCAAAGACTCATCAACAACAACTGG
TTTQS RLQFS QAGASDI GGATTCCGACCCAAGAGACTCAACTTCAAGCT
RDQSRNWLPGPCYRQ CTTTAACATTCAAGTCAAAGAGGTCACGCAGA
QRVSKTS ADNNNSEYS ATGACGGTACGACGACGATTGCCAATAACCTT
WTGATKYHLNGRDSL ACCAGCACGGTTCAGGTGTTTACTGACTCGGA
VNPGPAMASHKDDEE GTACCAGCTCCCGTACGTCCTCGGCTCGGC GC
KFFPQSGVLIFGKQGSE ATCAAGGATGCCTCCCGCCGTTCCCAGCAGAC
KTNVDIEKVMITDEEEI GTCTTCATGGTGCCACAGTATGGATACCTCAC
RTTNPVATEQYGS VS T CCTGAACAAC GGGAGTCAGGCAGTAGGAC GC
NLQRAYNPDRGNRQA TCTTCATTTTACTGCCTGGAGTACTTTCCTTCT
ATADVNTQGVLPGMV CAGATGCTGCGTACCGGAAACAACTTTACCTT
WQDRDVYLQGPIWAK CAGCTACACTTTTGAGGACGTTCCTTTCCACA
IPHTDGHFHPSPLMGG GCAGCTACGCTCACAGCCAGAGTCTGGACCGT
FGLKHPPPQILIKNTPV CTCATGAATCCTCTCATCGACCAGTACCTGTA
PANPSTTFSAAKFASFI TTACTTGAGCAGAACAAACACTCCAAGTGGA
TQYSTGQVSVEIEWEL ACCACCACGCAGTCAAGGCTTCAGTTTTCTCA
QKENS KRWNPEIQYTS GGCCGGAGCGAGTGACATTCGGGACCAGTCT
NYNKSVNVDFTVDTN AGGAACTGGCTTCCTGGACCCTGTTACCGCCA
GVYSEPRPIGTRYLTR GCAGCGAGTATCAAAGACATCTGCGGATAAC
NL* (SEQ ID NO: 19) AACAACAGTGAATACTCGTGGACTGGAGCTA
CCAAGTACCACCTCAATGGCAGAGACTCTCTG
GTGA ATCCGGGCCCGGCCATGGCAAGCCACA
AGGACGATGAAGAAAAGTTTTTTCCTCAGAGC
GGGGTTCTCATCTTTGGGAAGCAAGGCTCAGA
GAAAACAAATGTGGACATTGAAAAGGTCATG
ATTACAGACGAAGAGGAAATCAGGACAACCA
ATCCCGTGGCTACGGAGCAGTATGGTTCTGTA
TCTACCAACCTCCAGCGTGCGTACAATCCTGA
TAGAGGCAACAGACAAGCAGCTACCGCAGAT
GTCAACACACAAGGCGTTCTTCCAGGCATGGT
CTGGCAGGACAGAGATGTGTACCTTCAGGGG
CCCATCTGGGCAAAGATTCCACACACGGACG
GACATTTTCACCCCTCTCCCCTCATGGGTGGA
TTCGGACTTAAACACCCTCCTCCACAGATTCT
CATCAAGAACACCCCGGTACCTGCGAATCCTT
CGACCACCTTCAGTGCGGCAAAGTTTGCTTCC
TTCATCACACAGTACTCCACGGGACAGGTCAG
CGTGGAGATCGAGTGGGAGCTGCAGAAGGAA
AACAGCAAACGCTGGAATCCCGAAATTCAGT
ACACTTCCAACTACAACAAGTCTGTTAATGTG
GACTTTACTGTGGACACTAATGGCGTGTATTC
AGAGCCTCGCCCCATTGGCACCAGATACCTGA
CTCGTAATCTGTAA (SEQ ID NO: 64)
VAR-19 MAADGYLPDWLEDTL ATGGCTGCCGATGGTTATCTTCCAGATTGGCT
SEGIRQWWKLKPGPPP CGAGGACACTCTCTCTGAAGGAATAAGACAG
104
CA 03220810 2023- 11- 29 SUBSTITUTE SHEET (RULE 26)
WO 2022/256557
PCT/US2022/032004
PKPAERHKDDSRGLVL TGGTGGAAGCTCAAACCTGGCCCACCACCACC
PGYKYLGPFNGLDKGE AAAGCCCGCAGAGCGGCATAAGGACGACAGC
PVNEADAAALEHDKA AGGGGT C TT GT GCTTCCT GGGTACAAGT AC C T
YDRQLDSGDNPYLKY CGGACCCTTCAACGGACTCGACAAGGGAGAG
NHADAEFQERLKEDTS CCGGTCAACGAGGCAGACGCCGCGGCCCTCG
FGGNLGRAVFQAKKR AGC AC G AC AAAGC CTAC GAC C GGC AGCTC GA
VLEPLGLVEEPVKT AP CAGCGGAGACAACCCGTACCTCAAGTACAAC
GKKRPVEHSPVEPDSS CACGCCGAC GC GGAGTTTCAGGAGCGCCTTA
SGTGKAGQQPARKRL AAGAAGATACGTCTTTTGGGGGCAACCTCGG
NFGQTGDADSVPDPQP AC GAGC AGTCTTCCAGGCGAAAAAGAGGGTT
LGQPPAAPSGLGTNTM CTTGAACCTCTGGGCCTGGTTGAGGAACCTGT
ATGSGAPMADNNEGA TAAGACGGCTCCGGGAAAAAAGAGGCCGGTA
DGVGNSSGNWHCDST GAGCACTCTCCTGTGGAGCCAGACTCCTCCTC
WMGDRVITTSTRTWA GGGAACC GGAAAGGC GGGCC AG C AGC CTGCA
LPT YNNHLY KQIS S QS AGAAAAAGATTGAATTTTGGTCAGACTGGAG
GASNDNHYFGYSTPW AC GCAGACTC AGT ACCT GACCCCCAGCCTCTC
GYFDFNRFHCHFSPRD GGACAGCCACCAGCAGCCCCCTCTGGTCTGGG
WQRLINNNWGFRPKR AACTAAT AC G AT G GCTAC AGGC AGT GGC GCA
LNFKLFNIQVKEVTQN CCAATGGCAGACAATAACGAGGGCGCCGACG
DGTTTIANNLTSTVQV GAGTGGGTAATTCCTCGGGAAATTGGCATTGC
FTDSEYQLPYVLGS AH GATTCCACATGGATGGGCGACAGAGTCATCA
QGCLPPFPADVFMVPQ CCACCAGCAC CC GAACCTGGGCCCTGCCCACC
YGYLTLNNGS QAVGR T AC A AC A ACC A CCTCT AC A A AC A A ATTTCCAG
SSFYCLEYFPS QMLRT CCAATCAGGAGCCTCGAACGACAATCACTACT
GNNFTFSYTFEDVPFH TTGGCTACAGCACCCCTTGGGGGT AT TTTGAC
SS YAHS QSLDRLMNPL TTCAACAGATTCCACTGCCACTTTTCACCACG
IDQYLYYLSRTNTPSG TGACTGGCAAAGACTCATCAACAACAACTGG
TTTQSRLQFS QAGASDI GGATTCCGACCCAAGAGACTCAACTTCAAGCT
RD QS RNWLPGPCYRQ CTTTAAC ATTCAAGTC AAAGAGGTC AC GC AGA
QRVSKTS ADNNNSEYS ATGACGGT AC G ACGAC GATTGCCAATAACCTT
WTGATKYHLNGRDSL ACCAGCACGGTTCAGGTGTTTACTGACTCGGA
VNPGPAMASHKDDEE GTACCAGCTCCCGTACGTCCTCGGCTCGGC GC
KFFPQSGVLIFGKQGSE ATCAAGGAT GC CTCCC GCC GTTCCCAGC AGAC
KTNVDIAKVMITSEEEI GTCTTCATGGTGCCACAGTATGGATACCTCAC
KTTNPVATEQYGVVA CCTGAACAAC GGGAGTCAGGCAGTAGGAC GC
ANLQGNRQAATADVN TCTTCATTTTACTGCCTGGAGTACTTTCCTTCT
T Q GVLPGMVWQD RD CAGAT GCT GC GTACCGGAAAC AACTTTAC CTT
V YLQGPIWAKIPHTDG CAGCTACACTTTTGAGGACGTTCCTTTCCACA
HFHPSPLMGGFGLKHP GCAGCTACGCTCACAGCCAGAGTCTGGACCGT
PPQILIKNTPVPANPST CTCATGAATCCTCTCATCGACCAGTACCTGTA
TFSAAKFASFITQYS TG TTACTTGAGCAGAACAAACACTCCAAGTGGA
QVS VEIEWELQKENS K ACCACCACGCAGTCAAGGCTTCAGTTTTCTCA
RWNPEIQYTSNYNKS V GGCCGGAGCGAGTGACATTCGGGACCAGTCT
NVDFTVDTNGV YSEPR AGGAACTGGCTTCCTGGACCCTGTTACCGCCA
PIGTRYLTRNL* (SEQ GCAGCGAGTATCAAAGACATCTGCGGATAAC
ID NO: 20)
AACA AC AGTGA AT ACTCGTGGACTGGAGCTA
105
CA 03220810 2023- 11- 29 SUBSTITUTE SHEET (RULE 26)
WO 2022/256557
PCT/US2022/032004
CCAAGTACCACCTCAATGGCAGAGACTCTCTG
GTGAATCCGGGCCCGGCCATGGCAAGCCACA
AGGAC GAT GAAGAAAAGTTTTTTCCTCAGAGC
GGGGTTCTCATCTTTGGGAAGCAAGGCTCAGA
GAAAACAAATGTGGACATTGCAAAGGTCATG
ATTACATCCGAAGAGGAAATCAAGACAACCA
ATCCCGTGGCTACGGAGCAGTATGGTGTGGTA
GCTGCTAACCTCCAGGGCAACAGACAAGCAG
CTACCGCAGATGTCAACACACAAGGCGTTCTT
CCAGGCATGGTCTGGCAGGACAGAGATGT GT
ACCTTCAGGGGCCCATCTGGGCAAAGATTCCA
CACACGGACGGACATTTTCACCCCTCTCCCCT
CATGGGTGGATTCGGACTTAAACACCCTCCTC
CACAGATTCTCATCAAGAACACCCCGGTACCT
GCGAATCCTTCGACCACCTTCAGTGCGGCAAA
GTTTGCTTCCTTCATCACACAGTACTCCACGG
GACAGGTCAGCGTGGAGATCGAGTGGGAGCT
GC AGAAGGAAAACAGCAAACGC T GGAATCCC
GAAATTCAGTACACTTCCAACTACAACAAGTC
TGTTAATGTGGACTTTACTGTGGACACTAATG
GCGTGTATTCAGAGCCTCGCCCCATTGGC ACC
AGATACCTGACTCGTAATCTGTAA (SEQ ID
NO: 65)
V AR-20 MA A DG Y LPDWLEDTL ATGGCTGCCGATGGTT ATCTTCC AG ATTGGCT
SEGIRQWWKLIKPGPPP CGAGGACACTCTCTCTGAAGGAATAAGACAG
PKPAERHKDDSRGLVL TGGTGGAAGCTCAAACCTGGCCCACCACCACC
PGYKYLGPFNGLDKGE AAAGCCCGCAGAGCGGCATAAGGACGACAGC
PVNEADAAALEHDKA AGGGGTCTTGTGCTTCCTGGGTACAAGTACCT
YDRQLDSGDNPYLKY CGGACCCTTCAACGGACTCGACAAGGGAGAG
NHADAEFQERLKEDTS CCGGTCAACGAGGCAGACGCCGCGGCCCTCG
FGGNLGRAVFQAKKR AGCACGACAAAGCCTACGACCGGCAGCTC GA
VLEPLGLVEEPVKTAP CAGCGGAGACAACCCGTACCTCAAGTACAAC
GKKRPVEHSPVEPDSS CACGCCGACGCGGAGTTTCAGGAGCGCCTTA
S GTGKAGQQPARKRL AAGAAGATACGTCTTTTGGGGGCAACCTCGG
NFGQTGDADSVPDPQP ACGAGCAGTCTTCCAGGCGAAAAAGAGGGTT
LGQPPAAPSGLGTNTM CTTGAACCTCTGGGCCTGGTTGAGGAACCTGT
ATGS G APM ADNNEG A TA AG ACGGCTCCGGG A A A A A AG AGGCCGGT A
DGVGNSSGNWHCDST GAGCACTCTCCTGTGGAGCCAGACTCCTCCTC
WMGDRVITTSTRTWA GGGAACCGGAAAGGCGGGCCAGCAGCCTGCA
LPTYNNHLYKQISSQS AGAAAAAGATTGAATTTTGGTCAGACTGGAG
GASNDNHYFGYSTPW ACGCAGACTCAGTACCTGACCCCCAGCCTCTC
GYFDFNRFHCHFSPRD GGACAGCCACCAGCAGCCCCCTCTGGTCTGGG
WQRLINNNWGFRPKR AACTAAT AC G AT GGCTAC AGGC AGT GGC GCA
LNFKLFNIQVKEVTQN CCAATGGCAGACAATAACGAGGGCGCCGACG
DGTTTIANNLTSTVQV GAGTGGGTAATTCCTCGGGAAATTGGCATTGC
FTDSEYQLPYVLGSAH GATTCCACATGGATGGGCGACAGAGTCATCA
106
CA 03220810 2023- 11- 29 SUBSTITUTE SHEET (RULE 26)
WO 2022/256557
PCT/US2022/032004
QGCLPPFPADVFMVPQ CCACCAGCACCCGAACCTGGGCCCTGCCCACC
YGYLTLNNGSQAVGR TACAACAACCACCTCTACAAACAAATTTCCAG
S SFYCLEYFPS QMLRT CCAATCAGGAGCCTCGAACGACAATCACTACT
GNNFTFSYTFEDVPFH TTGGCTACAGCACCCCTTGGGGGTATTTTGAC
SSYAHS QSLDRLMNPL TTCAACAGATTCCACTGCCACTTTTCACCACG
IDQYLYYLSRTNTPSG TGACTGGCAAAGACTCATCAACAACAACTGG
TTTQSRLQFS QAGASDI GGATTCCGACCCAAGAGACTCAACTTCAAGCT
RDQSRNWLPGPCYRQ CTTTAACATTCAAGTCAAAGAGGTCACGCAGA
QRVSKTS ADNNNSEYS ATGACGGTACGACGACGATTGCCAATAACCTT
WTGATKYHLNGRDSL ACCAGCACGGTTCAGGTGTTTACTGACTCGGA
VNPGPAMASHKDDEE GTACCAGCTCCCGTACGTCCTCGGCTCGGCGC
KFFPQSGVLIFGKQGSE ATCAAGGATGCCTCCCGCCGTTCCCAGCAGAC
KTNVDIEKLMITDEEEI GTCTTCATGGTGCCACAGTATGGATACCTCAC
RTTNPVATEQYGDVST CCTGAACAACGGGAGTCAGGCAGTAGGACGC
NLQRGNRQAATADVN TCTTCATTTTACTGCCTGGAGTACTTTCCTTCT
TQGVLPGMVWQDRD CAGATGCTGCGTACCGGAAACAACTTTACCTT
VYLQGPIWAKIPHTDG CAGCTACACTTTTGAGGACGTTCCTTTCCACA
HFHPSPLMGGFGLKHP GCAGCTACGCTCACAGCCAGAGTCTGGACCGT
PPQILIKNTPVPANPST CTCATGAATCCTCTCATCGACCAGTACCTGTA
TFSAAKFAS FITQYS TG TTACTTGAGCAGAACAAACACTCCAAGTGGA
QVSVEIEWELQKENSK ACCACCACGCAGTCAAGGCTTCAGTTTTCTCA
RWNPEIQYTS NYNKS V GGCCGGAGCGAGTGACATTCGGGACCAGTCT
NVDFTVDTNGVYSEPR AGGAACTGGCTTCCTGGACCCTGTTACCGCCA
PIGTRYLTRNL* (SEQ GCAGCGAGTATCAAAGACATCTGCGGATAAC
ID NO: 21)
AACAACAGTGAATACTCGTGGACTGGAGCTA
CCAAGTACCACCTCAATGGCAGAGACTCTCTG
GTGAATCCGGGCCCGGCCATGGCAAGCCACA
AGGACGATGAAGAAAAGTTTTTTCCTCAGAGC
GGGGTTCTCATCTTTGGGAAGCAAGGCTCAGA
GAAAACAAATGTGGACATTGAAAAGCTCATG
ATTACAGACGAAGAGGAAATCAGGACAACCA
ATCCCGTGGCTACGGAGCAGTATGGTGATGTA
TCTACCAACCTCCAGAGAGGCAACAGACAAG
CAGCTACCGCAGATGTCAACACACAAGGCGT
TCTTCCAGGCATGGTCTGGCAGGACAGAGATG
TGTACCTTCAGGGGCCCATCTGGGCAAAGATT
CCACACACGGACGGACATTTTCACCCCTCTCC
CCTCATGGGTGGATTCGGACTTAAACACCCTC
CTCCACAGATTCTCATCAAGAACACCCCGGTA
CCTGCGAATCCTTCGACCACCTTCAGTGCGGC
AAAGTTTGCTTCCTTCATCACACAGTACTCCA
CGGGACAGGTCAGCGTGGAGATCGAGTGGGA
GCTGCAGAAGGAAAACAGCAAACGCTGGAAT
CCCGAAATTCAGTACACTTCCAACTACAACAA
GTCTGTTAATGTGGACTTTACTGTGGACACTA
ATGGCGTGTATTCAGAGCCTCGCCCCATTGGC
107
CA 03220810 2023- 11- 29 SUBSTITUTE SHEET (RULE 26)
WO 2022/256557
PCT/US2022/032004
ACCAGATACCTGACTCGTAATCTGTAA (SEQ
ID NO: 66)
VAR-21 MAADGYLPDWLEDTL ATGGCTGCCGATGGTTATCTTCCAGATTGGCT
SEGIRQWWKLKPGPPP CGAGGACACTCTCTCTGAAGGAATAAGACAG
PKPAERHKDDSRGLVL TGGTGGAAGCTCAAACCTGGCCCACCACCACC
PGYKYLGPFNGLDKGE AAAGCCC GCAGAGCGGCATAAGGACGACAGC
PVNEADAAALEHDKA AGGGGT C TT GT GCTTCCT GGGTACAAGT AC C T
YDRQLDSGDNPYLKY CGGACCCTTCAACGGACTCGACAAGGGAGAG
NHADAEFQERLKEDTS CCGGTCAACGAGGCAGACGCCGCGGCCCTCG
FGGNLGRAVFQAKKR AGC AC G AC AAAGC CTAC GAC C GGC AGCTC GA
VLEPLGLVEEPVKT AP CAGCGGAGACAACCCGTACCTCAAGTACAAC
GKKRPVEHSPVEPDSS CACGCCGAC GC GGAGTTTCAGGAGCGCCTTA
SGTGKAGQQPARKRL AAGAAGATACGTCTTTTGGGGGCAACCTCGG
NFGQTGDADSVPDPQP AC GAGC AGTCTTCCAGGCGAAAAAGAGGGTT
LGQPPAAPSGLGTNTM CTTGAACCTCTGGGCCTGGTTGAGGAACCTGT
ATGSGAPMADNNEGA TAAGACGGCTCCGGGAAAAAAGAGGCCGGTA
DGVGNSSGNWHCDST GAGCACTCTCCTGTGGAGCCAGACTCCTCCTC
WMGDRVITT S TRT WA GGGAACC GGAAAGGC GGGCC AG C AGC CTGCA
LPTYNNHLYKQIS S QS AGAAAAAGATTGAATTTTGGTCAGACTGGAG
GASNDNHYFGYSTPW AC GCAGACTC AGT ACCT GACCCCCAGCCTCTC
GYFDFNRFHCHFSPRD GGACAGCCACCAGCAGCCCCCTCTGGTCTGGG
WQRLINNNWGFRPKR AACTA AT ACG ATGGCTACAGGC AGTGGCGCA
LNFKLFNIQVKEVTQN CCAATGGCAGACAATAACGAGGGCGCCGACG
DGTTT I ANN LTSTVQV GAGTGGGT A AT TCCTCGGG A A ATTGGCATTGC
FTDSEYQLPYVLGS AH GATTCCACATGGATGGGCGACAGAGTCATCA
QGCLPPFPADVFMVPQ CCACCAGCAC CC GAACCTGGGCCCTGCCCACC
YGYLTLNNGS QAVGR TAC AAC AAC C AC C TCT AC AAAC AAATTTCCAG
SSFYCLEYFPS QMLRT CCAATCAGGAGCCTCGAACGACAATCACTACT
GNNFTFSYTFEDVPFH TTGGCTACAGCACCCCTTGGGGGT AT TTTGAC
SSYAHS QSLDRLMNPL TTCAACAGATTCCACTGCCACTTTTCACCACG
IDQYLYYLSRTNTPSG TGACTGGCAAAGACTCATCAACAACAACTGG
TTTQSRLQFS QAGASDI GGATTCCGACCCAAGAGACTCAACTTCAAGCT
RD QS RNWLPGPCYRQ CTTTAAC ATTCAAGTC AAAGAGGTC AC GC AGA
QRVSKTS ADNNNSEYS ATGACGGT AC G ACGAC GATTGCCAATAACCTT
WTGATKYHLNGRDSL ACCAGCACGGTTCAGGTGTTTACTGACTCGGA
VNPGPAMASHKDDEE GTACCAGCTCCCGTACGTCCTCGGCTCGGC GC
KFFPQSGVLIFGKQGSE ATC A AGG AT GC CTCCC GCC GTTCCC A GC AG AC
KTNVDIENVLITDEEEI GTCTTCATGGTGCCACAGTATGGATACCTCAC
RTTNPVATEQYGIVAT CCTGAACAAC GGGAGTC AGGCAGTAG GAC GC
NLQRGNRQAAT AD VN TCTTCATTTTACTGCCTGGAGTACTTTCCTTCT
T QGVLPGMVWQD RD CAGAT GCT GC GTACCGGAAAC AACTTTAC CTT
VYLQGPIWAKIPHTDG CAGCTACACTTTTGAGGACGTTCCTTTCCACA
HFHPSPLMGGFGLKHP GCAGCTACGCTCACAGCCAGAGTCTGGACCGT
PPQILIKNTPVPANPST CTCATGAATCCTCTCATCGACCAGTACCTGTA
TFSAAKFASFITQYS TG TTACTTGAGCAGAACAAACACTCCAAGTGGA
QVSVEIEWELQKENS K ACCACCACGCAGTCAAGGCTTCAGTTTTCTCA
108
CA 03220810 2023- 11- 29 SUBSTITUTE SHEET (RULE 26)
WO 2022/256557
PCT/US2022/032004
RWNPEIQYTSNYNKS V GGCCGGAGCGAGTGACATTCGGGACCAGTCT
NVDFTVDTNGVYSEPR AGGAACTGGCTTCCTGGACCCTGTTACCGCCA
PIGTRYLTRNL* (SEQ GCAGCGAGTATCAAAGACATCTGCGGATAAC
ID NO: 22) AACAACAGTGAATACTCGTGGACTGGAGCTA
CCAAGTACCACCTCAATGGCAGAGACTCTCTG
GTGAATCCGGGCCCGGCCATGGCAAGCCACA
AGGACGATGAAGAAAAGTTTTTTCCTCAGAGC
GGGGTTCTCATCTTTGGGAAGCAAGGCTCAGA
GAAAACAAATGTGGACATTGAAAACGTCCTC
ATTACAGACGAAGAGGAAATCAGGACAACCA
ATCCCGTGGCTACGGAGCAGTATGGTATTGTA
GCTACCAACCTCCAGAGAGGCAACAGACAAG
CAGCTACCGCAGATGTCAACACACAAGGCGT
TCTTCCAGGCATGGTCTGGCAGGACAGAGATG
TGTACCTTCAGGGGCCCATCTGGGCAAAGATT
CCACACACGGACGGACATTTTCACCCCTCTCC
CCTCATGGGTGGATTCGGACTTAAACACCCTC
CTCCACAGATTCTCATCAAGAACACCCCGGTA
CCTGCGAATCCTTCGACCACCTTCAGTGCGGC
AAAGTTTGCTTCCTTCATCACACAGTACTCCA
CGGGACAGGTCAGCGTGGAGATCGAGTGGGA
GCTGCAGAAGGAAAACAGCAAACGCTGGAAT
CCCGAAATTCAGTACACTTCCAACTACAACAA
GTCTGTTAATGTGGACTTTACTGTGGACACTA
ATGGCGTGTATTCAGAGCCTCGCCCCATTGGC
ACCAGATACCTGACTCGTAATCTGTAA (SEQ
ID NO: 67)
VAR-22 MAADGYLPDWLEDTL ATGGCTGCCGATGGTTATCTTCCAGATTGGCT
SEGIRQWWKLKPGPPP CGAGGACACTCTCTCTGAAGGAATAAGACAG
PKPAERHKDDSRGLVL TGGTGGAAGCTCAAACCTGGCCCACCACCACC
PGYKYLGPFNGLDKGE AAAGCCCGCAGAGCGGCATAAGGACGACAGC
PVNEADAAALEHDKA AGGGGTCTTGTGCTTCCTGGGTACAAGTACCT
YDRQLDSGDNPYLKY CGGACCCTTCAACGGACTCGACAAGGGAGAG
NHADAEFQERLKEDTS CCGGTCAACGAGGCAGACGCCGCGGCCCTCG
FGGNLGRAVFQAKKR AGCACGACAAAGCCTACGACCGGCAGCTCGA
VLEPLGLVEEPVKTAP CAGCGGAGACAACCCGTACCTCAAGTACAAC
GKKRPVEHSPVEPDS S CACGCCG AC GC GG AGTTTC AGG AGCGCCTTA
S GTGKAGQQPARKRL AAGAAGATACGTCTTTTGGGGGCAACCTCGG
NFGQTGDADSVPDPQP ACGAGCAGTCTTCCAGGCGAAAAAGAGGGTT
LGQPPAAPS GLGTNTM CTTGAACCTCTGGGCCTGGTTGAGGAACCTGT
ATGS GAPMADNNEGA TAAGACGGCTCCGGGAAAAAAGAGGCCGGTA
DGVGNS SGNWHCDST GAGCACTCTCCTGTGGAGCCAGACTCCTCCTC
WMGDRVITTSTRTWA GGGAACCGGAAAGGCGGGCCAGCAGCCTGCA
LPTYNNHLYKQIS S QS AGAAAAAGATTGAATTTTGGTCAGACTGGAG
GASNDNHYFGYSTPW ACGCAGACTCAGTACCTGACCCCCAGCCTCTC
GYFDFNRFHCHFSPRD GGACAGCCACCAGCAGCCCCCTCTGGTCTGGG
109
CA 03220810 2023- 11- 29 SUBSTITUTE SHEET (RULE 26)
WO 2022/256557
PCT/US2022/032004
WQRLINNNWGFRPKR AACTAATACGATGGCTACAGGCAGTGGCGCA
LNFKLFNIQVKEVTQN CCAATGGCAGACAATAACGAGGGCGCCGACG
DGTTTIANNLTSTVQV GAGTGGGTAATTCCTCGGGAAATTGGCATTGC
FTDSEYQLPYVLGSAH GATTCCACATGGATGGGCGACAGAGTCATCA
QGCLPPFPADVFMVPQ CCACCAGCACCCGAACCTGGGCCCTGCCCACC
YGYLTLNNGSQAVGR TACAACAACCACCTCTACAAACAAATTTCCAG
SSFYCLEYFPSQMLRT CCAATCAGGAGCCTCGAACGACAATCACTACT
GNNFTFSYTFEDVPFH TTGGCTACAGCACCCCTTGGGGGTATTTTGAC
SSYAHSQSLDRLMNPL TTCAACAGATTCCACTGCCACTTTTCACCACG
IDQYLYYLSRTNTPSG TGACTGGCAAAGACTCATCAACAACAACTGG
TTTQSRLQFSQAGASDI GGATTCCGACCCAAGAGACTCAACTTCAAGCT
RDQSRNWLPGPCYRQ CTTTAACATTCAAGTCAAAGAGGTCACGCAGA
QRVSKTSADNNNSEYS ATGACGGTACGACGACGATTGCCAATAACCTT
WTGATKYHLNGRDSL ACCAGCACGGTTCAGGTGTTTACTGACTCGGA
VNPGPAMASHKDDEE GTACCAGCTCCCGTACGTCCTCGGCTCGGCGC
KFFPQSGVLIFGKQGSE ATCAAGGATGCCTCCCGCCGTTCCCAGCAGAC
KTNVDLERVMLTSEEE GTCTTCATGGTGCCACAGTATGGATACCTCAC
IATTNPVATEQYGSVS CCTGAACAACGGGAGTCAGGCAGTAGGACGC
DNLQSRSRQAATADV TCTTCATTTTACTGCCTGGAGTACTTTCCTTCT
NTQGVLPGMVWQDR CAGATGCTGCGTACCGGAAACAACTTTACCTT
DVYLQGPIWAKIPHTD CAGCTACACTTTTGAGGACGTTCCTTTCCACA
GHFHPSPLMGGFGLKH GCAGCTACGCTCACAGCCAGAGTCTGGACCGT
PPPQILIKNTPVPANPST CTCATGAATCCTCTCATCGACCAGTACCTGTA
TFSAAKFASFITQYSTG TTACTTGAGCAGAACAAACACTCCAAGTGGA
QVSVEIEWELQKENSK ACCACCACGCAGTCAAGGCTTCAGTTTTCTCA
RWNPEIQYTSN YNKS V GGCCGGAGCGAGTGACATTCGGGACCAGTCT
NVDFTVDTNGVYSEPR AGGAACTGGCTTCCTGGACCCTGTTACCGCCA
PIGTRYLTRNL* (SEQ GCAGCGAGTATCAAAGACATCTGCGGATAAC
ID NO: 23)
AACAACAGTGAATACTCGTGGACTGGAGCTA
CCAAGTACCACCTCAATGGCAGAGACTCTCTG
GTGAATCCGGGCCCGGCCATGGCAAGCCACA
AGGACGATGAAGAAAAGTTTTTTCCTCAGAGC
GGGGTTCTCATCTTTGGGAAGCAAGGCTCAGA
GAAAACAAATGTGGACCTCGAACGTGTCATG
CTTACATCGGAAGAGGAAATCGCCACAACCA
ATCCCGTGGCTACGGAGCAGTATGGTTCTGTA
TCTGACAACCTCCAGTCGAGATCAAGACAAG
CAGCTACCGCAGATGTCAACACACAAGGCGT
TCTTCCAGGCATGGTCTGGCAGGACAGAGATG
TGTACCTTCAGGGGCCCATCTGGGCAAAGATT
CCACACACGGACGGACATTTTCACCCCTCTCC
CCTCATGGGTGGATTCGGACTTAAACACCCTC
CTCCACAGATTCTCATCAAGAACACCCCGGTA
CCTGCGAATCCTTCGACCACCTTCAGTGCGGC
AAAGTTTGCTTCCTTCATCACACAGTACTCCA
CGGGACAGGTCAGCGTGGAGATCGAGTGGGA
110
CA 03220810 2023- 11- 29 SUBSTITUTE SHEET (RULE 26)
WO 2022/256557
PCT/US2022/032004
GCTGCAGAAGGAAAACAGCAAACGCTGGAAT
CCCGAAATTCAGTACACTTCCAACTACAACAA
GTCTGTTAATGTGGACTTTACTGTGGACACTA
ATGGCGTGTATTCAGAGCCTCGCCCCATTGGC
ACCAGATACCTGACTCGTAATCTGTAA (SEQ
ID NO: 68)
VAR-23 MAADGYLPDWLEDTL ATGGCTGCCGATGGTTATCTTCCAGATTGGCT
SEGIRQWWKLKPGPPP CGAGGACACTCTCTCTGAAGGAATAAGACAG
PKPAERHKDDSRGLVL TGGTGGAAGCTCAAACCTGGCCCACCACCACC
PGYKYLGPFNGLDKGE AAAGCCCGCAGAGCGGCATAAGGACGACAGC
PVNEADAAALEHDKA AGGGGTCTTGTGCTTCCTGGGTACAAGT AC CT
YDRQLDSGDNPYLKY CGGACCCTTCAACGGACTCGACAAGGGAGAG
NHADAEFQERLKEDTS CCGGTCAACGAGGCAGACGCCGCGGCCCTCG
FGGNLGRAVFQAKKR AGCACGACAAAGCCTACGACCGGCAGCTC GA
VLEPLGLVEEPVKTAP CAGCGGAGACAACCCGTACCTCAAGTACAAC
GKKRPVEHSPVEPDSS CACGCCGACGCGGAGTTTCAGGAGCGCCTTA
SGTGKAGQQPARKRL AAGAAGATACGTCTTTTGGGGGCAACCTCGG
NFGQTGDADSVPDPQP ACGAGCAGTCTTCCAGGCGAAAAAGAGGGTT
LGQPPAAPSGLGTNTM CTTGAACCTCTGGGCCTGGTTGAGGAACCTGT
ATGSGAPMADNNEGA TAAGACGGCTCCGGGAAAAAAGAGGCCGGTA
DGVGNS SGNWHCDST GAGCACTCTCCTGTGGAGCCAGACTCCTCCTC
WMGDRVITTSTRTWA GGGA ACCGGA A AGGCGGGCC AGC AGCCTGCA
LPTYNNHLYKQISSQS AGAAAAAGATTGAATTTTGGTCAGACTGGAG
GAS NDNH YFGYSTPW ACGC AG ACTC AGT ACCTGACCCCCAGCCTCTC
GYFDENREHCHFSPRD GGACAGCCACCAGCAGCCCCCTCTGGTCTGGG
WQRLINNNWGFRPKR AACTAATACGATGGCTACAGGCAGTGGCGCA
LNFKLFNIQVKEVTQN CCAATGGCAGACAATAACGAGGGCGCCGACG
DGTTTIANNLTSTVQV GAGTGGGTAATTCCTCGGGAAATTGGCATTGC
FTDSEYQLPYVLGSAH GATTCCACATGGATGGGCGACAGAGTCATCA
QGCLPPFPADVFMVPQ CCACCAGCACCCGAACCTGGGCCCTGCCCACC
YGYLTLNNGSQAVGR TACAACAACC ACCTCT AC AAACAAATTTCCAG
SSFYCLEYFPSQMLRT CCAATCAGGAGCCTCGAACGACAATCACTACT
GNNFTFSYTFEDVPFH TTGGCTACAGCACCCCTTGGGGGTATTTTGAC
SSYAHS QSLDRLMNPL TTCAACAGATTCCACTGCCACTTTTCACCACG
IDQYLYYLSRTNTPSG TGACTGGCAAAGACTCATCAACAACAACTGG
TTTQSRLQFSQAGASDI GGATTCCGACCCAAGAGACTCAACTTCAAGCT
RDQSRNWLPGPCYRQ CTTT A AC ATTCA AGTCA A AGAGGTCACGC AGA
QRVSKTS ADNNNSEYS ATGACGGTACGACGACGATTGCCAATAACCTT
WTGATKYHLNGRDSL ACCAGCACGGTTCAGGTGTTTACTGACTCGGA
VNPGPAMASHKDDEE GTACCAGCTCCCGTACGTCCTCGGCTCGGC GC
KFFPQSGVLIFGKQGSE ATCAAGGATGCCTCCCGCCGTTCCCAGCAGAC
KTNADISDVMLTSEEEI GTCTTCATGGTGCCACAGTATGGATACCTCAC
KTTNPVATEQYGIVSD CCTGAACAACGGGAGTCAGGCAGTAGGACGC
NLQRQNRQAATADVN TCTTCATTTTACTGCCTGGAGTACTTTCCTTCT
TQGVLPGMVWQDRD CAGATGCTGCGTACCGGAAACAACTTTACCTT
VYLQGPIWAKIPHTDG CAGCTACACTTTTGAGGACGTTCCTTTCCACA
1 1 1
CA 03220810 2023- 11- 29 SUBSTITUTE SHEET (RULE 26)
WO 2022/256557
PCT/US2022/032004
HFHPSPLMGGFGLKHP GCAGCTACGCTCACAGCCAGAGTCTGGACCGT
PPQILIKNTPVPANPST CTCATGAATCCTCTCATCGACCAGTACCTGTA
TFSAAKFASFITQYS TG TTACTTGAGCAGAACAAACACTCCAAGTGGA
QVSVEIEWELQKENS K ACCACCACGCAGTCAAGGCTTCAGTTTTCTCA
RWNPEIQYTSNYNKS V GGCCGGAGCGAGTGACATTCGGGACCAGTCT
NVDFTVDTNGVYSEPR AGGAACTGGCTTCCTGGACCCTGTTACCGCCA
PIGTRYLTRNL* (SEQ GCAGCGAGTATCAAAGACATCTGCGGATAAC
ID NO: 24) AACAACAGTGAATACTCGTGGACTGGAGCTA
CCAAGTACCACCTCAATGGCAGAGACTCTCTG
GTGAATCCGGGCCCGGCCATGGCAAGCCACA
AGGACGATGAAGAAAAGTTTTTTCCTCAGAGC
GGGGTTCTCATCTTTGGGAAGCAAGGCTCAGA
GAAAACAAATGCTGACATTTCTGACGTCATGC
TTACAAGCGAAGAGGAAATCAAAACAACCAA
TCCCGTGGCTACGGAGCAGTATGGTATTGTAT
CTGACAACCTCCAGAGACAGAACAGACAAGC
AGCTACCGCAGATGTCAACACACAAGGCGTT
CTTCCAGGCATGGTCTGGCAGGACAGAGATGT
GTACCTTCAGGGGCCCATCTGGGCAAAGATTC
CACACACGGACGGACATTTTCACCCCTCTCCC
CTCATGGGTGGATTCGGACTTAAACACCCTCC
TCCACAGATTCTCATCAAGAACACCCCGGTAC
CTGCGA ATCCTTCGACC ACCTTC A GTGCGGC A
AAGTTTGCTTCCTTCATCACACAGTACTCCAC
GGGACAGGTCAGCGTGGAGATCGAGTGGGAG
CTGCAGAAGGAAAACAGCAAACGCTGGAATC
CCGAAATTCAGTACACTTCCAACTACAACAAG
TCTGTTAATGTGGACTTTACTGTGGACACTAA
TGGCGTGTATTCAGAGCCTCGCCCCATTGGCA
CCAGATACCTGACTCGTAATCTGTAA (SEQ ID
NO: 69)
VAR-24 MAADGYLPDWLEDTL ATGGCTGCCGATGGTTATCTTCCAGATTGGCT
SEGIRQWWKLKPGPPP CGAGGACACTCTCTCTGAAGGAATAAGACAG
PKPAERHKDDSRGLVL TGGTGGAAGCTCAAACCTGGCCCACCACCACC
PGYKYLGPFNGLDKGE AAAGCCCGCAGAGCGGCATAAGGACGACAGC
PVNEADAAALEHDKA AGGGGTCTTGTGCTTCCTGGGTACAAGTACCT
YDRQLDSGDNPYLKY CGGACCCTTCAACGGACTCGACAAGGGAGAG
NHADAEFQERLKEDTS CCGGTCAACGAGGCAGACGCCGCGGCCCTCG
FGGNLGRAVFQAKKR AGCACGACAAAGCCTACGACCGGCAGCTCGA
VLEPLGLVEEPVKTAP CAGCGGAGACAACCCGTACCTCAAGTACAAC
GKKRPVEHSPVEPDSS CACGCCGACGCGGAGTTTCAGGAGCGCCTTA
SGTGKAGQQPARKRL AAGAAGATACGTCTTTTGGGGGCAACCTCGG
NFGQTGDADSVPDPQP ACGAGCAGTCTTCCAGGCGAAAAAGAGGGTT
LGQPPAAPSGLGTNTM CTTGAACCTCTGGGCCTGGTTGAGGAACCTGT
ATGSGAPMADNNEGA TAAGACGGCTCCGGGAAAAAAGAGGCCGGTA
DGVGNSSGNWHCDST GAGCACTCTCCTGTGGAGCCAGACTCCTCCTC
112
CA 03220810 2023- 11- 29 SUBSTITUTE SHEET (RULE 26)
WO 2022/256557
PCT/US2022/032004
WMGDRVITTSTRTWA GGGAACCGGAAAGGCGGGCCAGCAGCCTGCA
LPTYNNHLYKQISSQS AGAAAAAGATTGAATTTTGGTCAGACTGGAG
GASNDNHYFGYSTPW ACGCAGACTCAGTACCTGACCCCCAGCCTCTC
GYFDFNRFHCHFSPRD GGACAGCCACCAGCAGCCCCCTCTGGTCTGGG
WQRLINNNWGFRPKR AACTAATACGATGGCTACAGGCAGTGGCGCA
LNFKLFNIQVKEVTQN CCAATGGCAGACAATAACGAGGGCGCCGACG
DGTTTIANNLTSTVQV GAGTGGGTAATTCCTCGGGAAATTGGCATTGC
FTDSEYQLPYVLGSAH GATTCCACATGGATGGGCGACAGAGTCATCA
QGCLPPFPADVFMVPQ CCACCAGCACCCGAACCTGGGCCCTGCCCACC
YGYLTLNNGSQAVGR TACAACAACCACCTCTACAAACAAATTTCCAG
SSFYCLEYFPSQMLRT CCAATCAGGAGCCTCGAACGACAATCACTACT
GNNFTFSYTFEDVPFH TTGGCTACAGCACCCCTTGGGGGTATTTTGAC
SSYAHS QSLDRLMNPL TTCAACAGATTCCACTGCCACTTTTCACCACG
IDQYLYYLSRTNTPSG TGACTGGCAAAGACTCATCAACAACAACTGG
TTTQSRLQFSQAGASDI GGATTCCGACCCAAGAGACTCAACTTCAAGCT
RDQSRNWLPGPCYRQ CTTTAACATTCAAGTCAAAGAGGTCACGCAGA
QRVSKTS ADNNNSEYS ATGACGGTACGACGACGATTGCCAATAACCTT
WTGATKYHLNGRDSL ACCAGCACGGTTCAGGTGTTTACTGACTCGGA
VNPGPAMASHKDDEE GTACCAGCTCCCGTACGTCCTCGGCTCGGCGC
KFFPQSGVLIFGKQGSE ATCAAGGATGCCTCCCGCCGTTCCCAGCAGAC
KTNVDIEKVMITDEEEI GTCTTCATGGTGCCACAGTATGGATACCTCAC
RTTNPVATEEYGAVS T CCTGAACAACGGGAGTCAGGCAGTAGGACGC
NLQRGNRQ A AT ADVN TCTTCATTTTACTGCCTGGAGTACTTTCCTTCT
TQGVLPGMVWQDRD CAGATGCTGCGTACCGGAAACAACTTTACCTT
VYLQGPIWAKIPHTDG CAGCTACACTTTTGAGGACGTTCCTTTCCACA
HFHPSPLMGGFGLKHP GCAGCTACGCTCACAGCCAGAGTCTGGACCGT
PPQILIKNTPVPANPST CTCATGAATCCTCTCATCGACCAGTACCTGTA
TFSAAKFASFITQYS TG TTACTTGAGCAGAACAAACACTCCAAGTGGA
QVSVEIEWELQKENSK ACCACCACGCAGTCAAGGCTTCAGTTTTCTCA
RWNPEIQYTSNYNKS V GGCCGGAGCGAGTGACATTCGGGACCAGTCT
NVDFTVDTNGVYSEPR AGGAACTGGCTTCCTGGACCCTGTTACCGCCA
PIGTRYLTRNL* (SEQ GCAGCGAGTATCAAAGACATCTGCGGATAAC
ID NO: 25)
AACAACAGTGAATACTCGTGGACTGGAGCTA
CCAAGTACCACCTCAATGGCAGAGACTCTCTG
GTGAATCCGGGCCCGGCCATGGCAAGCCACA
AGGACGATGAAGAAAAGTTTTTTCCTCAGAGC
GGGGTTCTCATCTTTGGGAAGCAAGGCTCAGA
GAAAACAAATGTGGACATTGAAAAGGTCATG
ATTACAGACGAAGAGGAAATCAGGACAACCA
ATCCCGTGGCTACGGAGGAGTATGGTGCTGTA
TCTACCAACCTCCAGAGAGGCAACAGACAAG
CAGCTACCGCAGATGTCAACACACAAGGCGT
TCTTCCAGGCATGGTCTGGCAGGACAGAGATG
TGTACCTTCAGGGGCCCATCTGGGCAAAGATT
CCACACACGGACGGACATTTTCACCCCTCTCC
CCTC ATGGGTGGATTCGG ACTT A A AC ACCCTC
113
CA 03220810 2023- 11- 29 SUBSTITUTE SHEET (RULE 26)
WO 2022/256557
PCT/US2022/032004
CTCCACAGATTCTCATCAAGAACACCCCGGTA
CCTGCGAATCCTTCGACCACCTTCAGTGCGGC
AAAGTTTGCTTCCTTCATCACACAGTACTCCA
CGGGACAGGTCAGCGTGGAGATCGAGTGGGA
GCTGCAGAAGGAAAACAGCAAACGCTGGAAT
CCCGAAATTCAGTACACTTCCAACTACAACAA
GTCTGTTAATGTGGACTTTACTGTGGACACTA
ATGGCGTGTATTCAGAGCCTCGCCCCATTGGC
ACCAGATACCTGACTCGTAATCTGTAA (SEQ
ID NO: 70)
VAR-25 MAADGYLPDWLEDTL ATGGCTGCCGATGGTTATCTTCCAGATTGGCT
SEGIRQWWKLKPGPPP CGAGGACACTCTCTCTGAAGGAATAAGACAG
PKPAERHKDDSRGLVL TGGTGGAAGCTCAAACCTGGCCCACCACCACC
PGYKYLGPFNGLDKGE AAAGCCCGCAGAGCGGCATAAGGACGACAGC
PVNEADAAALEHDKA AGGGGTCTTGTGCTTCCTGGGTACAAGTACCT
YDRQLDSGDNPYLKY CGGACCCTTCAACGGACTCGACAAGGGAGAG
NHADAEFQERLKEDTS CCGGTCAACGAGGCAGACGCCGCGGCCCTCG
FGGNLGRAVFQAKKR AGCACGACAAAGCCTACGACCGGCAGCTCGA
VLEPLGLVEEPVKTAP CAGCGGAGACAACCCGTACCTCAAGTACAAC
GKKRPVEHSPVEPDSS CACGCCGACGCGGAGTTTCAGGAGCGCCTTA
SGTGKAGQQPARKRL AAGAAGATACGTCTTTTGGGGGCAACCTCGG
NFGQTGDADSVPDPQP ACGAGCAGTCTTCCAGGCGAAAAAGAGGGTT
LGQPPAAPSGLGTNTM CTTGAACCTCTGGGCCTGGTTGAGGAACCTGT
ATGSGAPMADNNEGA TAAGACGGCTCCGGGAAAAAAGAGGCCGGTA
DGVGNSSGNWHCDST GAGCACTCTCCTGTGGAGCCAGACTCCTCCTC
WMGDRVITTSTRTWA GGGAACCGGAAAGGCGGGCCAGCAGCCTGCA
LPTYNNHLYKQISSQS AGAAAAAGATTGAATTTTGGTCAGACTGGAG
GASNDNHYFGYSTPW ACGCAGACTCAGTACCTGACCCCCAGCCTCTC
GYFDFNRFHCHFSPRD GGACAGCCACCAGCAGCCCCCTCTGGTCTGGG
WQRLINNNWGFRPKR AACTAATACGATGGCTACAGGCAGTGGCGCA
LNEKLENIQVKEVTQN CCAATGGCAGACAATAACGAGGGCGCCGACG
DGTTTIANNLTSTVQV GAGTGGGTAATTCCTCGGGAAATTGGCATTGC
FTDSEYQLPYVLGSAH GATTCCACATGGATGGGCGACAGAGTCATCA
QGCLPPFPADVFMVPQ CCACCAGCACCCGAACCTGGGCCCTGCCCACC
YGYLTLNNGSQAVGR TACAACAACCACCTCTACAAACAAATTTCCAG
SSFYCLEYFPSQMLRT CCAATCAGGAGCCTCGAACGACAATCACTACT
GNNFTESYTFEDVPFH TTGGCT AC AGC ACCCCTTGGGGGT ATTTTGAC
SSYAHS QSLDRLMNPL TTCAACAGATTCCACTGCCACTTTTCACCACG
IDQYLYYLSRTNTPSG TGACTGGCAAAGACTCATCAACAACAACTGG
TTTQSRLQFSQAGASDI GGATTCCGACCCAAGAGACTCAACTTCAAGCT
RDQSRNWLPGPCYRQ CTTTAACATTCAAGTCAAAGAGGTCACGCAGA
QRVSKTSADNNNSEYS ATGACGGTACGACGACGATTGCCAATAACCTT
WTGATKYHLNGRDSL ACCAGCACGGTTCAGGTGTTTACTGACTCGGA
VNPGPAMASHKDDEE GTACCAGCTCCCGTACGTCCTCGGCTCGGCGC
KFFPQSGVLIFGKQGSE ATCAAGGATGCCTCCCGCCGTTCCCAGCAGAC
KTNVDIEKVMITDEEEI GTCTTCATGGTGCCACAGTATGGATACCTCAC
114
CA 03220810 2023- 11- 29 SUBSTITUTE SHEET (RULE 26)
WO 2022/256557
PCT/US2022/032004
RTTNPVATEQYGS VS T CCTGAACAACGGGAGTCAGGCAGTAGGACGC
NLLNWTAEQRGNRQA TCTTCATTTTACTGCCTGGAGTACTTTCCTTCT
ATADVNTQGVLPGMV CAGATGCTGCGTACCGGAAACAACTTTACCTT
WQDRDVYLQGPIWAK CAGCTACACTTTTGAGGACGTTCCTTTCCACA
IPHTDGHFHPSPLMGG GCAGCTACGCTCACAGCCAGAGTCTGGACCGT
FGLKHPPPQILIKNTPV CTCATGAATCCTCTCATCGACCAGTACCTGTA
PANPSTTFSAAKFASFI TTACTTGAGCAGAACAAACACTCCAAGTGGA
TQYSTGQVSVEIEWEL ACCACCACGCAGTCAAGGCTTCAGTTTTCTCA
QKENS ICRWNPEIQYTS GGCCGGAGCGAGTGACATTCGGGACCAGTCT
NYNKSVNVDFTVDTN AGGAACTGGCTTCCTGGACCCTGTTACCGCCA
GVYSEPRPIGTRYLTR GCAGCGAGTATCAAAGACATCTGCGGATAAC
NL* (SEQ ID NO: 26) AACAACAGTGAATACTCGTGGACTGGAGCTA
CCAAGTACCACCTCAATGGCAGAGACTCTCTG
GTGAATCCGGGCCCGGCCATGGCAAGCCACA
AGGACGATGAAGAAAAGTTTTTTCCTCAGAGC
GGGGTTCTCATCTTTGGGAAGCAAGGCTCAGA
GAAAACAAATGTGGACATTGAAAAGGTCATG
ATTACAGACGAAGAGGAAATCAGGACAACCA
ATCCCGTGGCTACGGAGCAGTATGGTTCTGTA
TCTACCAACCTCCTCAATTGGACTGCCGAACA
GAGAGGCAACAGACAAGCAGCTACCGCAGAT
GTCAACACACAAGGCGTTCTTCCAGGCATGGT
CTGGC AGG AC AGA GATGTGTACCTTC AGGGG
CCCATCTGGGCAAAGATTCCACACACGGACG
GACATTTTCACCCCTCTCCCCTCATGGGTGGA
TTCGGACTTAAACACCCTCCTCCACAGATTCT
CATCAAGAACACCCCGGTACCTGCGAATCCTT
CGACCACCTTCAGTGCGGCAAAGTTTGCTTCC
TTCATCACACAGTACTCCACGGGACAGGTCAG
CGTGGAGATCGAGTGGGAGCTGCAGAAGGAA
AACAGCAAACGCTGGAATCCCGAAATTCAGT
ACACTTCCAACTACAACAAGTCTGTTAATGTG
GACTTTACTGTGGACACTAATGGCGTGTATTC
AGAGCCTCGCCCCATTGGCACCAGATACCTGA
CTCGTAATCTGTAA (SEQ ID NO: 71)
VAR-26 MAADGYLPDWLEDTL ATGGCTGCCGATGGTTATCTTCCAGATTGGCT
SEGIRQWWKLKPGPPP CGAGGAC ACTCTCTCTGA A GGA AT A AGAC AG
PKPAERHKDDSRGLVL TGGTGGAAGCTCAAACCTGGCCCACCACCACC
PGYKYLGPFNGLDKGE AAAGCCCGCAGAGCGGCATAAGGACGACAGC
PVNEADAAALEHDKA AGGGGTCTTGTGCTTCCTGGGTACAAGTACCT
YDRQLDSGDNPYLKY CGGACCCTTCAACGGACTCGACAAGGGAGAG
NHADAEFQERLKEDTS CCGGTCAACGAGGCAGACGCCGCGGCCCTCG
FGGNLGRAVFQAKKR AGCACGACAAAGCCTACGACCGGCAGCTCGA
VLEPLGLVEEPVKTAP CAGCGGAGACAACCCGTACCTCAAGTACAAC
GKKRPVEHSPVEPDSS CACGCCGACGCGGAGTTTCAGGAGCGCCTTA
SGTGKAGQQPARKRL AAGAAGATACGTCTTTTGGGGGCAACCTCGG
IS
CA 03220810 2023- 11- 29 SUBSTITUTE SHEET (RULE 26)
WO 2022/256557
PCT/US2022/032004
NFGQTGDADSVPDPQP ACGAGCAGTCTTCCAGGCGAAAAAGAGGGTT
LGQPPAAPSGLGTNTM CTTGAACCTCTGGGCCTGGTTGAGGAACCTGT
ATGSGAPMADNNEGA TAAGACGGCTCCGGGAAAAAAGAGGCCGGTA
DGVGNSSGNWHCDST GAGCACTCTCCTGTGGAGCCAGACTCCTCCTC
WMGDRVITTSTRTWA GGGAACCGGAAAGGCGGGCCAGCAGCCTGCA
LPTYNNHLYKQISSQS AGAAAAAGATTGAATTTTGGTCAGACTGGAG
GASNDNHYFGYSTPW ACGCAGACTCAGTACCTGACCCCCAGCCTCTC
GYFDFNRFHCHFSPRD GGACAGCCACCAGCAGCCCCCTCTGGTCTGGG
WQRLINNNWGFRPKR AACTAATACGATGGCTACAGGCAGTGGCGCA
LNFKLFNIQVKEVTQN CCAATGGCAGACAATAACGAGGGCGCCGACG
DGTTTIANNLTSTVQV GAGTGGGTAATTCCTCGGGAAATTGGCATTGC
FTDSEYQLPYVLGSAH GATTCCACATGGATGGGCGACAGAGTCATCA
QGCLPPFPADVFMVPQ CCACCAGCACCCGAACCTGGGCCCTGCCCACC
YGYLTLNNGSQAVGR TACAACAACCACCTCTACAAACAAATTTCCAG
SSFYCLEYFPSQMLRT CCAATCAGGAGCCTCGAACGACAATCACTACT
GNNFTFSYTFEDVPFH TTGGCTACAGCACCCCTTGGGGGTATTTTGAC
SSYAHS QSLDRLMNPL TTCAACAGATTCCACTGCCACTTTTCACCACG
IDQYLYYLSRTNTPSG TGACTGGCAAAGACTCATCAACAACAACTGG
TTTQSRLQFSQAGASDI GGATTCCGACCCAAGAGACTCAACTTCAAGCT
RDQSRNWLPGPCYRQ CTTTAACATTCAAGTCAAAGAGGTCACGCAGA
QRVSKTS ADNNNSEYS ATGACGGTACGACGACGATTGCCAATAACCTT
WTGATKYHLNGRDSL ACCAGCACGGTTCAGGTGTTTACTGACTCGGA
VNPGPAMASHKDDEE GT ACC A GCTCCCGTACGTCCTCGGCTCGGC GC
KFFPQSGVLIFGKQGSE ATCAAGGATGCCTCCCGCCGTTCCCAGCAGAC
KTNVDIEKVMITDEEEI GTCTTCATGGTGCCACAGTATGGATACCTCAC
RTTNPVATEQYGS VS T CCTGAACAACGGGAGTCAGGCAGTAGGACGC
NLQSGNTNPITGDVNS TCTTCATTTTACTGCCTGGAGTACTTTCCTTCT
QGALPGMVWQDRDV CAGATGCTGCGTACCGGAAACAACTTTACCTT
YLQGPIWAKIPHTDGH CAGCTACACTTTTGAGGACGTTCCTTTCCACA
FHPSPLMGGFGLKHPP GCAGCTACGCTCACAGCCAGAGTCTGGACCGT
PQILIKNTPVPANPSTT CTCATGAATCCTCTCATCGACCAGTACCTGTA
FS AAKFASFITQYSTGQ TTACTTGAGCAGAACAAACACTCCAAGTGGA
VSVEIEWELQKENS KR ACCACCACGCAGTCAAGGCTTCAGTTTTCTCA
WNPEIQYTSNYNKS VN GGCCGGAGCGAGTGACATTCGGGACCAGTCT
VDFTVDTNGVYSEPRP AGGAACTGGCTTCCTGGACCCTGTTACCGCCA
IGTRYLTRNL* (SEQ ID GCAGCGAGTATCAAAGACATCTGCGGATAAC
NO: 27)
AACAACAGTGAATACTCGTGGACTGGAGCTA
CCAAGTACCACCTCAATGGCAGAGACTCTCTG
GTGAATCCGGGCCCGGCCATGGCAAGCCACA
AGGACGATGAAGAAAAGTTTTTTCCTCAGAGC
GGGGTTCTCATCTTTGGGAAGCAAGGCTCAGA
GAAAACAAATGTGGACATTGAAAAGGTCATG
ATTACAGACGAAGAGGAAATCAGGACAACCA
ATCCCGTGGCTACGGAGCAGTATGGTTCTGTA
TCTACCAACCTCCAGTCTGGCAACACAAATCC
TATTACCGGAGATGTC A ACTCGC A AGGCGCGC
116
CA 03220810 2023- 11- 29 SUBSTITUTE SHEET (RULE 26)
WO 2022/256557
PCT/US2022/032004
TTCCAGGCATGGTCTGGCAGGACAGAGATGT
GTACCTTCAGGGGCCCATCTGGGCAAAGATTC
CACACACGGACGGACATTTTCACCCCTCTCCC
CTCATGGGTGGATTCGGACTTAAACACCCTCC
TCCACAGATTCTCATCAAGAACACCCCGGTAC
CTGCGAATCCTTCGACCACCTTCAGTGCGGCA
AAGTTTGCTTCCTTCATCACACAGTACTCCAC
GGGACAGGTCAGCGTGGAGATCGAGTGGGAG
CTGCAGAAGGAAAACAGCAAACGCTGGAATC
CCGAAATTCAGTACACTTCCAACTACAACAAG
TCTGTTAATGTGGACTTTACTGTGGACACTAA
TGGCGTGTATTCAGAGCCTCGCCCCATTGGCA
CCAGATACCTGACTCGTAATCTGTAA (SEQ ID
NO: 72)
VAR-27 MAADGYLPDWLEDTL ATGGCTGCCGATGGTTATCTTCCAGATTGGCT
SEGIRQWWKLKPGPPP CGAGGACACTCTCTCTGAAGGAATAAGACAG
PKPAERHKDDSRGLVL TGGTGGAAGCTCAAACCTGGCCCACCACCACC
PGYKYLGPFNGLDKGE AAAGCCCGCAGAGCGGCATAAGGACGACAGC
PVNEADAAALEHDKA AGGGGTCTTGTGCTTCCTGGGTACAAGTACCT
YDRQLDSGDNPYLKY CGGACCCTTCAACGGACTCGACAAGGGAGAG
NHADAEFQERLKEDTS CCGGTCAACGAGGCAGACGCCGCGGCCCTCG
FGGN LGR AV FQA K KR AGCACG AC A A AGCCTACGACCGGCAGCTCGA
VLEPLGLVEEPVKTAP CAGCGGAGACAACCCGTACCTCAAGTACAAC
GKKRPVEHSPVEPDSS CACGCCGACGCGGAGTTTCAGGAGCGCCTTA
SGTGKAGQQPARKRL AAGAAGATACGTCTTTTGGGGGCAACCTCGG
NFGQTGDADSVPDPQP ACGAGCAGTCTTCCAGGCGAAAAAGAGGGTT
LGQPPAAPSGLGTNTM CTTGAACCTCTGGGCCTGGTTGAGGAACCTGT
ATGSGAPMADNNEGA TAAGACGGCTCCGGGAAAAAAGAGGCCGGTA
DGVGNSSGNWHCDST GAGCACTCTCCTGTGGAGCCAGACTCCTCCTC
WMGDRVITTSTRTWA GGGAACCGGAAAGGCGGGCCAGCAGCCTGCA
LPTYNNHLYKQISSQS AGAAAAAGATTGAATTTTGGTCAGACTGGAG
GASNDNHYFGYSTPW ACGCAGACTCAGTACCTGACCCCCAGCCTCTC
GYFDENREHCHFSPRD GGACAGCCACCAGCAGCCCCCTCTGGTCTGGG
WQRLINNNWGFRPKR AACTAATACGATGGCTACAGGCAGTGGCGCA
LNEKLENIQVKEVTQN CCAATGGCAGACAATAACGAGGGCGCCGACG
DGTTTIANNLTSTVQV GAGTGGGTAATTCCTCGGGAAATTGGCATTGC
FTDSEYQLPYVLGS AH GATTCC AC ATGGATGGGCGAC AGAGTCATCA
QGCLPPFPADVFMVPQ CCACCAGCACCCGAACCTGGGCCCTGCCCACC
YGYLTLNNGSQAVGR TACAACAACCACCTCTACAAACAAATTTCCAG
SSEYCLEYEPSQMLRT CCAATCAGGAGCCTCGAACGACAATCACTACT
GNNFTFSYTFEDVPFH TTGGCTACAGCACCCCTTGGGGGTATTTTGAC
SSYAHS QSLDRLMNPL TTCAACAGATTCCACTGCCACTTTTCACCACG
IDQYLYYLSRTNTPSG TGACTGGCAAAGACTCATCAACAACAACTGG
TTTQSRLQFSQAGASDI GGATTCCGACCCAAGAGACTCAACTTCAAGCT
RDQSRNWLPGPCYRQ CTTTAACATTCAAGTCAAAGAGGTCACGCAGA
QRVSKTSADNNNSEYS ATGACGGTACGACGACGATTGCCAATAACCTT
117
CA 03220810 2023- 11- 29 SUBSTITUTE SHEET (RULE 26)
WO 2022/256557
PCT/US2022/032004
WTGATKYHLNGRDSL ACCAGCACGGTTCAGGTGTTTACTGACTCGGA
VNPGPAMASHKDDEE GTACCAGCTCCCGTACGTCCTCGGCTCGGC GC
KFFPQSGVLIFGKQGSE ATCAAGGATGCCTCCCGCCGTTCCCAGCAGAC
KTNVDIEKVMITDEEEI GTCTTCATGGTGCCACAGTATGGATACCTCAC
RTTNPVATEQYGS VS T CCTGAACAAC G GGAGTCAGGCAGTAG GAC GC
NLQLAKEFTTRNANRQ TCTTCATTTTACTGCCTGGAGTACTTTCCTTCT
AATADVNTQGVLPGM CAGATGCTGCGTACCGGAAACAACTTTACCTT
VWQDRDVYLQGPIWA CAGCTACACTTTTGAGGACGTTCCTTTCCACA
KIPHTDGHFHPSPLMG GCAGCTACGCTCACAGCCAGAGTCTGGACCGT
GEGLICHPPPQILIKNTP CTCATGAATCCTCTCATCGACCAGTACCTGTA
VPANPS TTFS AAKFAS TTACTTGAGCAGAACAAACACTCCAAGTGGA
FITQYSTGQVSVEIEWE ACCACCACGCAGTCAAGGCTTCAGTTTTCTCA
LQKENSKRWNPEIQYT GGCCGGAGCGAGTGACATTCGGGACCAGTCT
SNYNKSVNVDFTVDT AGGAACTGGCTTCCTGGACCCTGTTACCGCCA
NGVYSEPRPIGTRYLT GCAGCGAGTATCAAAGACATCTGCGGATAAC
RNL* (SEQ ID NO: 28) AACAACAGTGAATACTCGTGGACTGGAGCTA
CCAAGTACCACCTCAATGGCAGAGACTCTCTG
GTGAATCCGGGCCCGGCCATGGCAAGCCACA
AGGACGATGAAGAAAAGTTTTTTCCTCAGAGC
GGGGTTCTCATCTTTGGGAAGCAAGGCTCAGA
GAAAACAAATGTGGACATTGAAAAGGTCATG
ATTACAGACGAAGAGGAAATCAGGACAACCA
ATCCCGTGGCT ACGGAGCAGT ATGGTTCTGT A
TCTACCAACCTCCAGTTGGCGAAAGAATTCAC
TACGCGGAATGCGAACAGACAAGCAGCTACC
GCAGATGTCAACACACAAGGCGTTCTTCCAGG
CATGGTCTGGCAGGACAGAGATGTGTACCTTC
AGGGGCCCATCTGGGCAAAGATTCCACACAC
GGACGGACATTTTCACCCCTCTCCCCTCATGG
GTGGATTCGGACTTAAACACCCTCCTCCACAG
ATTCTCATCAAGAACACCCCGGTACCTGCGAA
TCCTTCGACCACCTTCAGTGCGGCAAAGTTTG
CTTCCTTCATCACACAGTACTCCACGGGACAG
GTCAGCGTGGAGATCGAGTGGGAGCTGCAGA
AGGAAAACAGCAAACGCTGGAATCCCGAAAT
TCAGTACACTTCCAACTACAACAAGTCTGTTA
ATGTGGACTTTACTGTGGACACTAATGGCGTG
TATTCAGAGCCTCGCCCCATTGGCACCAGATA
CCTGACTCGTAATCTGTAA (SEQ ID NO: 73)
VAR-28 MAADG Y LPDWLEDTL ATGGCTGCCGATGGTTATCTTCCAGATTGGCT
SEGIRQWWKLKPGPPP CGAGGACACTCTCTCTGAAGGAATAAGACAG
PICPAERHICDDSRGLVL TGGTGGAAGCTCAAACCTGGCCCACCACCACC
PGYKYLGPFNGLDKGE AAAGCCCGCAGAGCGGCATAAGGACGACAGC
PVNEADAAALEHDKA AGGGGTCTTGTGCTTCCTGGGTACAAGTACCT
YDRQLDSGDNPYLKY CGGACCCTTCAACGGACTCGACAAGGGAGAG
NHADAEFQERLKEDTS CCGGTCAACGAGGCAGACGCCGCGGCCCTCG
118
CA 03220810 2023- 11- 29 SUBSTITUTE SHEET (RULE 26)
WO 2022/256557
PCT/US2022/032004
FGGNLGRAVFQAKKR AGCACGACAAAGCCTACGACCGGCAGCTCGA
VLEPLGLVEEPVKTAP CAGCGGAGACAACCCGTACCTCAAGTACAAC
GKKRPVEHSPVEPDSS CACGCCGACGCGGAGTTTCAGGAGCGCCTTA
SGTGKAGQQPARKRL AAGAAGATACGTCTTTTGGGGGCAACCTCGG
NFGQTGDADSVPDPQP ACGAGCAGTCTTCCAGGCGAAAAAGAGGGTT
LGQPPAAPSGLGTNTM CTTGAACCTCTGGGCCTGGTTGAGGAACCTGT
ATGSGAPMADNNEGA TAAGACGGCTCCGGGAAAAAAGAGGCCGGTA
DGVGNSSGNWHCDST GAGCACTCTCCTGTGGAGCCAGACTCCTCCTC
WMGDRVITTSTRTWA GGGAACCGGAAAGGCGGGCCAGCAGCCTGCA
LPTYNNHLYKQISSQS AGAAAAAGATTGAATTTTGGTCAGACTGGAG
GASNDNHYFGYSTPW ACGCAGACTCAGTACCTGACCCCCAGCCTCTC
GYFDFNRFHCHFSPRD GGACAGCCACCAGCAGCCCCCTCTGGTCTGGG
WQRLINNNWGFRPKR AACTAATACGATGGCTACAGGCAGTGGCGCA
LNFKLFNIQVKEVTQN CCAATGGCAGACAATAACGAGGGCGCCGACG
DGTTTIANNLTSTVQV GAGTGGGTAATTCCTCGGGAAATTGGCATTGC
FTDSEYQLPYVLGSAH GATTCCACATGGATGGGCGACAGAGTCATCA
QGCLPPFPADVFMVPQ CCACCAGCACCCGAACCTGGGCCCTGCCCACC
YGYLTLNNGSQAVGR TACAACAACCACCTCTACAAACAAATTTCCAG
SSFYCLEYFPSQMLRT CCAATCAGGAGCCTCGAACGACAATCACTACT
GNNFTFSYTFEDVPFH TTGGCTACAGCACCCCTTGGGGGTATTTTGAC
SSYAHS QSLDRLMNPL TTCAACAGATTCCACTGCCACTTTTCACCACG
IDQYLYYLSRTNTPSG TGACTGGCAAAGACTCATCAACAACAACTGG
TTTQSRLQFSQAGASDI GGATTCCGACCCAAGAGACTCAACTTCAAGCT
RDQSRNWLPGPCYRQ CTTTAACATTCAAGTCAAAGAGGTCACGCAGA
QRVSKTSADNNNSEYS ATGACGGTACGACGACGATTGCCAATAACCTT
WTGATKYHLNGRDSL ACCAGCACGGTTCAGGTGTTTACTGACTCGGA
VNPGPAMASHKDDEE GTACCAGCTCCCGTACGTCCTCGGCTCGGCGC
KFFPQSGVLIFGKQGSE ATCAAGGATGCCTCCCGCCGTTCCCAGCAGAC
KTNVDIEKVMITDEEEI GTCTTCATGGTGCCACAGTATGGATACCTCAC
RTTNPVATEQYGS VS T CCTGAACAACGGGAGTCAGGCAGTAGGACGC
NLQRGNRQAATLHPLE TCTTCATTTTACTGCCTGGAGTACTTTCCTTCT
ADVNTQGVLPGMVW CAGATGCTGCGTACCGGAAACAACTTTACCTT
QDRDVYLQGPIWAKIP CAGCTACACTTTTGAGGACGTTCCTTTCCACA
HTDGHFHPSPLMGGFG GCAGCTACGCTCACAGCCAGAGTCTGGACCGT
LKHPPPQILIKNTPVPA CTCATGAATCCTCTCATCGACCAGTACCTGTA
NPSTTFSAAKFASFITQ TTACTTGAGCAGAACAAACACTCCAAGTGGA
YSTGQVSVEIEWELQK ACCACCACGCAGTCAAGGCTTCAGTTTTCTCA
ENS KRWNPEIQ YT SN Y GGCCGGAGCGAGTGACATTCGGGACCAGTCT
NKSVNVDFTVDTNGV AGGAACTGGCTTCCTGGACCCTGTTACCGCCA
YSEPRPIGTRYLTRNL* GCAGCGAGTATCAAAGACATCTGCGGATAAC
(SEQ ID NO: 29)
AACAACAGTGAATACTCGTGGACTGGAGCTA
CCAAGTACCACCTCAATGGCAGAGACTCTCTG
GTGAATCCGGGCCCGGCCATGGCAAGCCACA
AGGACGATGAAGAAAAGTTTTTTCCTCAGAGC
GGGGTTCTCATCTTTGGGAAGCAAGGCTCAGA
GAAAACAAATGTGGACATTGAAAAGGTCATG
119
CA 03220810 2023- 11- 29 SUBSTITUTE SHEET (RULE 26)
WO 2022/256557
PCT/US2022/032004
ATTACAGACGAAGAGGAAATCAGGACAACCA
ATCCCGTGGCTACGGAGCAGTATGGTTCTGTA
TCTACCAACCTCCAGAGAGGCAACAGACAAG
CAGCTACCTTACATCCGCTGGAGGCAGATGTC
AACACACAAGGCGTTCTTCCAGGCATGGTCTG
GCAGGACAGAGATGTGTACCTTCAGGGGCCC
ATCTGGGCAAAGATTCCACACACGGACGGAC
ATTTTCACCCCTCTCCCCTCATGGGTGGATTCG
GACTTAAACACCCTCCTCCACAGATTCTCATC
AAGAACACCCCGGTACCTGCGAATCCTTCGAC
CACCTTCAGTGCGGCAAAGTTTGCTTCCTTCA
TCACACAGTACTCCACGGGACAGGTCAGCGT
GGAGATCGAGTGGGAGCTGCAGAAGGAAAAC
AGCAAACGCTGGAATCCCGAAATTCAGTACA
CTTCCAACTACAACAAGTCTGTTAATGTGGAC
TTTACTGTGGACACTAATGGCGTGTATTCAGA
GCCTCGCCCCATTGGCACCAGATACCTGACTC
GTAATCTGTAA (SEQ ID NO: 74)
VAR-29 MAADGYLPDWLEDTL ATGGCTGCCGATGGTTATCTTCCAGATTGGCT
SEGIRQWWKLKPGPPP CGAGGACACTCTCTCTGAAGGAATAAGACAG
PKPAERHKDDSRGLVL TGGTGGAAGCTCAAACCTGGCCCACCACCACC
PGY KY LGPFNGLD K GE A A AGCCC GC AG AGCGGC AT A AGGACG AC AGC
PVNEADAAALEHDKA AGGGGTCTTGTGCTTCCTGGGTACAAGTACCT
YDRQLDSGDNPYLKY CGGACCCTTCAACGGACTCGACAAGGGAGAG
NHADAEFQERLKEDTS CCGGTCAACGAGGCAGACGCCGCGGCCCTCG
FGGNLGRAVFQAKKR AGCACGACAAAGCCTACGACCGGCAGCTCGA
VLEPLGLVEEPVKTAP CAGCGGAGACAACCCGTACCTCAAGTACAAC
GKKRPVEHSPVEPDSS CACGCCGACGCGGAGTTTCAGGAGCGCCTTA
SGTGKAGQQPARKRL AAGAAGATACGTCTTTTGGGGGCAACCTCGG
NFGQTGDADSVPDPQP ACGAGCAGTCTTCCAGGCGAAAAAGAGGGTT
LGQPPAAPSGLGTNTM CTTGAACCTCTGGGCCTGGTTGAGGAACCTGT
ATGSGAPMADNNEGA TAAGACGGCTCCGGGAAAAAAGAGGCCGGTA
DGVGNSSGNWHCDST GAGCACTCTCCTGTGGAGCCAGACTCCTCCTC
WMGDRVITTSTRTWA GGGAACCGGAAAGGCGGGCCAGCAGCCTGCA
LPTYNNHLYKQISSQS AGAAAAAGATTGAATTTTGGTCAGACTGGAG
GASNDNHYFGYSTPW ACGCAGACTCAGTACCTGACCCCCAGCCTCTC
GYFDFNRFHCHFSPRD GGAC A GCC ACC A GC AGCCCCCTCTGGTCTGGG
WQRLINNNWGFRPKR AACTAATACGATGGCTACAGGCAGTGGCGCA
LNFKLENIQVIKEVTQN CCAATGGCAGACAATAACGAGGGCGCCGACG
DGTTTIANNLTSTVQV GAGTGGGTAATTCCTCGGGAAATTGGCATTGC
FTDSEYQLPYVLGSAH GATTCCACATGGATGGGCGACAGAGTCATCA
QGCLPPFPADVFMVPQ CCACCAGCACCCGAACCTGGGCCCTGCCCACC
YGYLTLNNGSQAVGR TACAACAACCACCTCTACAAACAAATTTCCAG
SSFYCLEYFPS QMLRT CCAATCAGGAGCCTCGAACGACAATCACTACT
GNNFTFSYTFEDVPFH TTGGCTACAGCACCCCTTGGGGGTATTTTGAC
SSYAHS QSLDRLMNPL TTCAACAGATTCCACTGCCACTTTTCACCACG
120
CA 03220810 2023- 11- 29 SUBSTITUTE SHEET (RULE 26)
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PCT/US2022/032004
IDQYLYYLSRTNTPSG TGACTGGCAAAGACTCATCAACAACAACTGG
TTTQSRLQFSQAGASDI GGATTCCGACCCAAGAGACTCAACTTCAAGCT
RDQSRNWLPGPCYRQ CTTTAACATTCAAGTCAAAGAGGTCACGCAGA
QRVSKTS ADNNNSEYS ATGACGGTACGACGACGATTGCCAATAACCTT
WTGATKYHLNGRDSL ACCAGCACGGTTCAGGTGTTTACTGACTCGGA
VNPGPAMASHKDDEE GTACCAGCTCCCGTACGTCCTCGGCTCGGC GC
KFFPQSGVLIFGKQGSE ATCAAGGATGCCTCCCGCCGTTCCCAGCAGAC
KTNVDIEKVMITDEEEI GTCTTCATGGTGCCACAGTATGGATACCTCAC
RTTNPVATEQYGS VS T CCTGAACAACGGGAGTCAGGCAGTAGGACGC
NLQRFGNRQAATADV TCTTCATTTTACTGCCTGGAGTACTTTCCTTCT
NTQGVLPGMVWQDR CAGATGCTGCGTACCGGAAACAACTTTACCTT
DVYLQGPIWAKIPHTD CAGCTACACTTTTGAGGACGTTCCTTTCCACA
GHFHPSPLMGGFGLKH GCAGCTACGCTCACAGCCAGAGTCTGGACCGT
PPPQILIKNTPVPANPST CTCATGAATCCTCTCATCGACCAGTACCTGTA
TFSAAKFASFITQYS TG TTACTTGAGCAGAACAAACACTCCAAGTGGA
QVSVEIEWELQKENS K ACCACCACGCAGTCAAGGCTTCAGTTTTCTCA
RWNPEIQYTSNYNKS V GGCCGGAGCGAGTGACATTCGGGACCAGTCT
NVDFTVDTNGVYSEPR AGGAACTGGCTTCCTGGACCCTGTTACCGCCA
PIGTRYLTRNL* (SEQ GCAGCGAGTATCAAAGACATCTGCGGATAAC
ID NO: 30) AACAACAGTGAATACTCGTGGACTGGAGCTA
CCAAGTACCACCTCAATGGCAGAGACTCTCTG
GTGAATCCGGGCCCGGCCATGGCAAGCCACA
AGGACGATGAAGAAAAGTTTTTTCCTCAGAGC
GGGGTTCTCATCTTTGGGAAGCAAGGCTCAGA
GAAAACAAATGTGGACATTGAAAAGGTCATG
ATTACAGACGAAGAGGAAATCAGGACAACCA
ATCCCGTGGCTACGGAGCAGTATGGTTCTGTA
TCTACCAACCTCCAGAGATTTGGCAACAGACA
AGCAGCT ACC GC AGATGTCAACACACAAGGC
GTTCTTCCAGGCATGGTCTGGCAGGACAGAGA
TGTGTACCTTCAGGGGCCCATCTGGGCAAAGA
TTCCACACACGGACGGACATTTTCACCCCTCT
CCCCTCATGGGTGGATTCGGACTTAAACACCC
TCCTCCACAGATTCTCATCAAGAACACCCCGG
TACCTGCGAATCCTTCGACCACCTTCAGTGCG
GCAAAGTTTGCTTCCTTCATCACACAGTACTC
CACGGGACAGGTCAGCGTGGAGATCGAGTGG
GAGCTGCAGAAGGAAAACAGCAAACGCTGGA
ATCCCGAAATTCAGTACACTTCCAACTACAAC
AAGTCTGTTAATGTGGACTTTACTGTGGACAC
TAATGGCGTGTATTCAGAGCCTCGCCCCATTG
GCACCAGATACCTGACTCGTAATCTGTAA
(SEQ ID NO: 75)
VAR-30 MAADGYLPDWLEDTL ATGGCTGCCGATGGTTATCTTCCAGATTGGCT
SEGIRQWWKLKPGPPP CGAGGACACTCTCTCTGAAGGAATAAGACAG
PKPAERHKDDSRGLVL TGGTGGAAGCTCAAACCTGGCCCACCACCACC
121
CA 03220810 2023- 11- 29 SUBSTITUTE SHEET (RULE 26)
WO 2022/256557
PCT/US2022/032004
PGYKYLGPFNGLDKGE AAAGCCCGCAGAGCGGCATAAGGACGACAGC
PVNEADAAALEHDKA AGGGGTCTTGTGCTTCCTGGGTACAAGTACCT
YDRQLDSGDNPYLKY CGGACCCTTCAACGGACTCGACAAGGGAGAG
NHADAEFQERLKEDTS CCGGTCAACGAGGCAGACGCCGCGGCCCTCG
FGGNLGRAVFQAKKR AGCACGACAAAGCCTACGACCGGCAGCTCGA
VLEPLGLVEEPVKTAP CAGCGGAGACAACCCGTACCTCAAGTACAAC
GKKRPVEHSPVEPDS S CACGCCGAC GC GGAGTTTCAGGAGCGCCTTA
S GTGKAGQQPARKRL AAGAAGATACGTCTTTTGGGGGCAACCTCGG
NFGQTGDADSVPDPQP ACGAGCAGTCTTCCAGGCGAAAAAGAGGGTT
LGQPPAAPS GLGTNTM CTTGAACCTCTGGGCCTGGTTGAGGAACCTGT
ATGS GAPMADNNEGA TAAGACGGCTCCGGGAAAAAAGAGGCCGGTA
DGVGNS SGNWHCDST GAGCACTCTCCTGTGGAGCCAGACTCCTCCTC
WMGDRVITTSTRTWA GGGAACCGGAAAGGCGGGCCAGCAGCCTGCA
LPTYNNHLYKQIS S QS AGAAAAAGATTGAATTTTGGTCAGACTGGAG
GASNDNHYFGYSTPW ACGCAGACTCAGTACCTGACCCCCAGCCTCTC
GYFDFNRFHCHFSPRD GGACAGCCACCAGCAGCCCCCTCTGGTCTGGG
WQRLINNNWGFRPKR AACTAAT AC G AT GGCTAC AGGC AGT GGC GCA
LNFKLFNIQVKEVTQN CCAATGGCAGACAATAACGAGGGCGCCGACG
DGTTTIANNLTSTVQV GAGTGGGTAATTCCTCGGGAAATTGGCATTGC
FTDSEYQLPYVLGS AH GATTCCACATGGATGGGCGACAGAGTCATCA
QGCLPPFPADVFMVPQ CCACCAGCACCCGAACCTGGGCCCTGCCCACC
YGYLTLNNGS QAVGR TAC AAC AAC C AC C TCT AC AAAC AAATTTCCAG
S SFYCLEYFPS QMLRT CC A ATC AGG A GCCTC G A ACGAC A ATC ACT ACT
GNNFTFSYTFEDVPFH TTGGCTACAGCACCCCTTGGGGGTATTTTGAC
SSYAHS QSLDRLMNPL TTCAACAGATTCCACTGCCACTTTTCACCACG
IDQYLY YLSRTNTPSG TGACTGGCAAAGACTCATCAACAACAACTGG
TTTQS RLQFS QAGASDI GGATTCCGACCCAAGAGACTCAACTTCAAGCT
RDQSRNWLPGPCYRQ CTTTAACATTCAAGTCAAAGAGGTCACGCAGA
QRVSKTS ADNNNSEYS ATGACGGTACGACGACGATTGCCAATAACCTT
WTGATKYHLNGRDSL ACCAGCACGGTTCAGGTGTTTACTGACTCGGA
VNPGPAMASHKDDEE GTACCAGCTCCCGTACGTCCTCGGCTCGGC GC
KFFPQSGVLIFGKQGSE ATCAAGGATGCCTCCCGCCGTTCCCAGCAGAC
KTNVDIEKVMITDEEEI GTCTTCATGGTGCCACAGTATGGATACCTCAC
RTTNPVATEQYGS VS T CCTGAACAAC GGGAGTCAGGCAGTAGGAC GC
NLQRGDQDFKNRNRQ TCTTCATTTTACTGCCTGGAGTACTTTCCTTCT
AATADVNTQGVLPGM CAGATGCTGCGTACCGGAAACAACTTTACCTT
VWQDRDVYLQGPIWA CAGCTACACTTTTGAGGACGTTCCTTTCCACA
KIPHTDGHFHPSPLMG GCAGCTACGCTCACAGCCAGAGTCTGGACCGT
GFGLKHPPPQILIKNTP CTCATGAATCCTCTCATCGACCAGTACCTGTA
VPANPS TTFS AAKFAS TTACTTGAGCAGAACAAACACTCCAAGTGGA
FITQYSTGQVSVEIEWE ACCACCACGCAGTCAAGGCTTCAGTTTTCTCA
LQKENSKRWNPEIQYT GGCCGGAGCGAGTGACATTCGGGACCAGTCT
SNYNKSVNVDFTVDT AGGAACTGGCTTCCTGGACCCTGTTACCGCCA
NGVYSEPRPIGTRYLT GCAGCGAGTATCAAAGACATCTGCGGATAAC
RNL* (SEQ ID NO: 31) AACAACAGTGAATACTCGTGGACTGGAGCTA
CC A AGT ACC A CCTC A AT GGC AG A G ACTCTCTG
122
CA 03220810 2023- 11- 29 SUBSTITUTE SHEET (RULE 26)
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PCT/US2022/032004
GTGAATCCGGGCCCGGCCATGGCAAGCCACA
AGGACGATGAAGAAAAGTTTTTTCCTCAGAGC
GGGGTTCTCATCTTTGGGAAGCAAGGCTCAGA
GAAAACAAATGTGGACATTGAAAAGGTCATG
ATTACAGACGAAGAGGAAATCAGGACAACCA
ATCCCGTGGCTACGGAGCAGTATGGTTCTGTA
TCTACCAACCTCCAGAGAGGCGACCAAGATTT
CAAAAACCGAAACAGACAAGCAGCTACC GCA
GATGTCAACACACAAGGCGTTCTTCCAGGCAT
GGTCTGGCAGGACAGAGATGTGTACCTTCAG
GGGCCCATCTGGGCAAAGATTCCACACACGG
ACGGACATTTTCACCCCTCTCCCCTCATGGGT
GGATTCGGACTTAAACACCCTCCTCCACAGAT
TCTCATCAAGAACACCCCGGTACCTGCGAATC
CTTCGACCACCTTCAGTGCGGCAAAGTTTGCT
TCCTTCATCACACAGTACTCCACGGGACAGGT
CAGCGTGGAGATCGAGTGGGAGCTGCAGAAG
GAAAACAGCAAACGCTGGAATCCCGAAATTC
AGTACACTTCCAACTACAACAAGTCTGTTAAT
GTGGACTTTACTGTGGACACTAATGGC GTGTA
TTCAGAGCCTCGCCCCATTGGCACCAGATACC
TGACTCGTAATCTGTAA (SEQ ID NO: 76)
VAR-31 MAADGYLPDWLEDTL ATGGCTGCCGATGGTTATCTTCCAGATTGGCT
SEGIRQWWKLKPCIPPP CGAGGACACTCTCTCTGAAGGAATAAGACAG
PKPAERHKDDSRGLVL TGGTGGAAGCTCAAACCTGGCCCACCACCACC
PGYKYLGPFNGLDKGE AAAGCCCGCAGAGCGGCATAAGGACGACAGC
PVNEADAAALEHDKA AGGGGTCTTGTGCTTCCTGGGTACAAGTACCT
YDRQLDSGDNPYLKY CGGACCCTTCAACGGACTCGACAAGGGAGAG
NHADAEFQERLKEDTS CCGGTCAACGAGGCAGACGCCGCGGCCCTCG
FGGNLGRAVFQAKKR AGCACGACAAAGCCTACGACCGGCAGCTCGA
VLEPLGLVEEPVKTAP CAGCGGAGACAACCCGTACCTCAAGTACAAC
GKKRPVEHSPVEPDS S CACGCCGAC GC GGAGTTTCAGGAGCGCCTTA
S GTGKAGQQPARKRL AAGAAGATACGTCTTTTGGGGGCAACCTCGG
NFGQTGDADSVPDPQP ACGAGCAGTCTTCCAGGCGAAAAAGAGGGTT
LGQPPAAPS GLGTNTM CTTGAACCTCTGGGCCTGGTTGAGGAACCTGT
ATGS GAPMADNNEGA TAAGACGGCTCCGGGAAAAAAGAGGCCGGTA
DGVGNSSGNWHCDST GAGC ACTCTCCTGTGG A GCC A G ACTCCTCCTC
WMGDRVITTSTRTWA GGGAACCGGAAAGGCGGGCCAGCAGCCTGCA
LPTYNNHLYKQIS S QS AGAAAAAGATTGAATTTTGGTCAGACTGGAG
GASNDNHYFG YSTPW AC GCAG ACTC AGTACCT GACCCCCAGCCTCTC
GYFDENRFECHFSPRD GGACAGCCACCAGCAGCCCCCTCTGGTCTGGG
WQRLINNNWGFRPKR AACTAATACGATGGCTACAGGCAGTGGCGCA
LNEKLENIQVKEVTQN CCAATGGCAGACAATAACGAGGGCGCCGACG
DGTTTIANNLTSTVQV GAGTGGGTAATTCCTCGGGAAATTGGCATTGC
FTDSEYQLPYVLGS AH GATTCCACATGGATGGGCGACAGAGTCATCA
QGCLPPFPADVFMVPQ CCACCAGCACCCGAACCTGGGCCCTGCCCACC
123
CA 03220810 2023- 11- 29 SUBSTITUTE SHEET (RULE 26)
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PCT/US2022/032004
YGYLTLNNGSQAVGR TACAACAACCACCTCTACAAACAAATTTCCAG
SSFYCLEYFPSQMLRT CCAATCAGGAGCCTCGAACGACAATCACTACT
GNNFTFSYTFEDVPFH TTGGCTACAGCACCCCTTGGGGGTATTTTGAC
SSYAHSQSLDRLMNPL TTCAACAGATTCCACTGCCACTTTTCACCACG
IDQYLYYLSRTNTPSG TGACTGGCAAAGACTCATCAACAACAACTGG
TTTQSRLQFSQAGASDI GGATTCCGACCCAAGAGACTCAACTTCAAGCT
RDQSRNWLPGPCYRQ CTTTAACATTCAAGTCAAAGAGGTCACGCAGA
QRVSKTSADNNNSEYS ATGACGGTACGACGACGATTGCCAATAACCTT
WTGATKYHLNGRDSL ACCAGCACGGTTCAGGTGTTTACTGACTCGGA
VNPGPAMASHKDDEE GTACCAGCTCCCGTACGTCCTCGGCTCGGCGC
KFFPQSGVLIFGKQGSE ATCAAGGATGCCTCCCGCCGTTCCCAGCAGAC
KTNVDIEKVMITDEEEI GTCTTCATGGTGCCACAGTATGGATACCTCAC
RTTNPVATEQYGS VS T CCTGAACAACGGGAGTCAGGCAGTAGGACGC
NLQRGNRQIAATADV TCTTCATTTTACTGCCTGGAGTACTTTCCTTCT
NTQGVLPGMVWQDR CAGATGCTGCGTACCGGAAACAACTTTACCTT
DVYLQGPIWAKIPHTD CAGCTACACTTTTGAGGACGTTCCTTTCCACA
GHFHPSPLMGGFGLKH GCAGCTACGCTCACAGCCAGAGTCTGGACCGT
PPPQILIKNTPVPANPST CTCATGAATCCTCTCATCGACCAGTACCTGTA
TFSAAKFASFITQYSTG TTACTTGAGCAGAACAAACACTCCAAGTGGA
QVSVEIEWELQKENSK ACCACCACGCAGTCAAGGCTTCAGTTTTCTCA
RWNPEIQYTSNYNKS V GGCCGGAGCGAGTGACATTCGGGACCAGTCT
NVDFTVDTNGVYSEPR AGGAACTGGCTTCCTGGACCCTGTTACCGCCA
PIGTRYLTRNL* (SEQ GC AGCG AGT ATC A A AGAC ATCTGCGGAT A AC
ID NO: 32)
AACAACAGTGAATACTCGTGGACTGGAGCTA
CCAAGTACCACCTCAATGGCAGAGACTCTCTG
GTGAATCCGGGCCCGGCCATGGCAAGCCACA
AGGACGATGAAGAAAAGTTTTTTCCTCAGAGC
GGGGTTCTCATCTTTGGGAAGCAAGGCTCAGA
GAAAACAAATGTGGACATTGAAAAGGTCATG
ATTACAGACGAAGAGGAAATCAGGACAACCA
ATCCCGTGGCTACGGAGCAGTATGGTTCTGTA
TCTACCAACCTCCAGAGAGGCAACAGACAAA
TCGCAGCTACCGCAGATGTCAACACACAAGG
CGTTCTTCCAGGCATGGTCTGGCAGGACAGAG
ATGTGTACCTTCAGGGGCCCATCTGGGCAAAG
ATTCCACACACGGACGGACATTTTCACCCCTC
TCCCCTCATGGGTGGATTCGGACTTAAACACC
CTCCTCCACAGATTCTCATCAAGAACACCCCG
GTACCTGCGAATCCTTCGACCACCTTCAGTGC
GGCAAAGTTTGCTTCCTTCATCACACAGTACT
CCACGGGACAGGTCAGCGTGGAGATCGAGTG
GGAGCTGCAGAAGGAAAACAGCAAACGCTGG
AATCCCGAAATTCAGTACACTTCCAACTACAA
CAAGTCTGTTAATGTGGACTTTACTGTGGACA
CTAATGGCGTGTATTCAGAGCCTCGCCCCATT
124
CA 03220810 2023- 11- 29 SUBSTITUTE SHEET (RULE 26)
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PCT/US2022/032004
GGCACCAGATACCTGACTCGTAATCTGTAA
(SEQ ID NO: 77)
VAR-32 MAADGYLPDWLEDTL ATGGCTGCCGATGGTTATCTTCCAGATTGGCT
SEGIRQWWKLKPGPPP CGAGGACACTCTCTCTGAAGGAATAAGACAG
PKPAERHKDDSRGLVL TGGTGGAAGCTCAAACCTGGCCCACCACCACC
PGYKYLGPFNGLDKGE AAAGCCCGCAGAGCGGCATAAGGACGACAGC
PVNEADAAALEHDKA AGGGGTCTTGTGCTTCCTGGGTACAAGTACCT
YDRQLDSGDNPYLKY CGGACCCTTCAACGGACTCGACAAGGGAGAG
NHADAEFQERLKEDTS CCGGTCAACGAGGCAGACGCCGCGGCCCTCG
FGGNLGRAVFQAKKR AGCACGACAAAGCCTACGACCGGCAGCTCGA
VLEPLGLVEEPVKTAP CAGCGGAGACAACCCGTACCTCAAGTACAAC
GKKRPVEHSPVEPDSS CACGCCGACGCGGAGTTTCAGGAGCGCCTTA
SGTGKAGQQPARKRL AAGAAGATACGTCTTTTGGGGGCAACCTCGG
NFGQTGDADSVPDPQP ACGAGCAGTCTTCCAGGCGAAAAAGAGGGTT
LGQPPAAPSGLGTNTM CTTGAACCTCTGGGCCTGGTTGAGGAACCTGT
ATGSGAPMADNNEGA TAAGACGGCTCCGGGAAAAAAGAGGCCGGTA
DGVGNSSGNWHCDST GAGCACTCTCCTGTGGAGCCAGACTCCTCCTC
WMGDRVITTSTRTWA GGGAACCGGAAAGGCGGGCCAGCAGCCTGCA
LPTYNNHLYKQISSQS AGAAAAAGATTGAATTTTGGTCAGACTGGAG
GASNDNHYFGYSTPW ACGCAGACTCAGTACCTGACCCCCAGCCTCTC
GYFDFNRFHCHFSPRD GGACAGCCACCAGCAGCCCCCTCTGGTCTGGG
WQRLINNNWGFRPKR AACTAATACGATGGCTACAGGCAGTGGCGCA
LNFKLFNIQVKEVTQN CCAATGGCAGACAATAACGAGGGCGCCGACG
DGTTTI ANN LTSTVQV GAGTGGGT A ATTCCTCGGGA A ATTGGCATTGC
FTDSEYQLPYVLGSAH GATTCCACATGGATGGGCGACAGAGTCATCA
QGCLPPFPADVFMVPQ CCACCAGCACCCGAACCTGGGCCCTGCCCACC
YGYLTLNNGSQAVGR TACAACAACCACCTCTACAAACAAATTTCCAG
SSFYCLEYFPSQMLRT CCAATCAGGAGCCTCGAACGACAATCACTACT
GNNFTFSYTFEDVPFH TTGGCTACAGCACCCCTTGGGGGTATTTTGAC
SSYAHS QSLDRLMNPL TTCAACAGATTCCACTGCCACTTTTCACCACG
IDQYLYYLSRTNTPSG TGACTGGCAAAGACTCATCAACAACAACTGG
TTTQSRLQFSQAGASDI GGATTCCGACCCAAGAGACTCAACTTCAAGCT
RDQSRNWLPGPCYRQ CTTTAACATTCAAGTCAAAGAGGTCACGCAGA
QRVSKTSADNNNSEYS ATGACGGTACGACGACGATTGCCAATAACCTT
WTGATKYHLNGRDSL ACCAGCACGGTTCAGGTGTTTACTGACTCGGA
VNPGPAMASHKDDEE GTACCAGCTCCCGTACGTCCTCGGCTCGGCGC
KFFPQSGVLIFGKQGSE ATC A AGG AT GCCTCCCGCCGTTCCC A GC AGAC
KTNVDIEKVMITDEEEI GTCTTCATGGTGCCACAGTATGGATACCTCAC
RTTNPVATEQYGS VS T CCTGAACAACGGGAGTCAGGCAGTAGGACGC
NLQRGNLAIEQTRPAR TCTTCATTTTACTGCCTGGAGTACTTTCCTTCT
QAATADVNTQGVLPG CAGATGCTGCGTACCGGAAACAACTTTACCTT
MVWQDRDVYLQGPIW CAGCTACACTTTTGAGGACGTTCCTTTCCACA
AKIPHTDGHFHPSPLM GCAGCTACGCTCACAGCCAGAGTCTGGACCGT
GGFGLKHPPPQILIKNT CTCATGAATCCTCTCATCGACCAGTACCTGTA
PVPANPSTTFSAAKFA TTACTTGAGCAGAACAAACACTCCAAGTGGA
SFITQYSTGQVSVEIEW ACCACCACGCAGTCAAGGCTTCAGTTTTCTCA
125
CA 03220810 2023- 11- 29 SUBSTITUTE SHEET (RULE 26)
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ELQKENSKRWNPEIQY GGCCGGAGCGAGTGACATTCGGGACCAGTCT
TSNYNKSVNVDFTVDT AGGAACTGGCTTCCTGGACCCTGTTACCGCCA
NGVYSEPRPIGTRYLT GCAGCGAGTATCAAAGACATCTGCGGATAAC
RNL* (SEQ ID NO: 33) AACAACAGTGAATACTCGTGGACTGGAGCTA
CCAAGTACCACCTCAATGGCAGAGACTCTCTG
GTGAATCCGGGCCCGGCCATGGCAAGCCACA
AGGACGATGAAGAAAAGTTTTTTCCTCAGAGC
GGGGTTCTCATCTTTGGGAAGCAAGGCTCAGA
GAAAACAAATGTGGACATTGAAAAGGTCATG
ATTACAGACGAAGAGGAAATCAGGACAACCA
ATCCCGTGGCTACGGAGCAGTATGGTTCTGTA
TCTACCAACCTCCAGAGAGGCAACCTTGCTAT
CGAACAAACAAGACCAGCGAGACAAGCAGCT
ACCGCAGATGTCAACACACAAGGCGTTCTTCC
AGGCATGGTCTGGCAGGACAGAGATGTGTAC
CTTCAGGGGCCCATCTGGGCAAAGATTCCACA
CACGGACGGACATTTTCACCCCTCTCCCCTCA
TGGGTGGATTCGGACTTAAACACCCTCCTCCA
CAGATTCTCATCAAGAACACCCCGGTACCTGC
GAATCCTTCGACCACCTTCAGTGCGGCAAAGT
TTGCTTCCTTCATCACACAGTACTCCACGGGA
CAGGTCAGCGTGGAGATCGAGTGGGAGCTGC
AGAAGGAAAACAGCAAACGCTGGAATCCCGA
AATTCAGTACACTTCCAACTACAACAAGTCTG
TTAATGTGGACTTTACTGTGGACACTAATGGC
GTGTATTCAGAGCCTCGCCCCATTGGCACCAG
ATACCTGACTCGTAATCTGTAA (SEQ ID NO:
78)
VAR-33 MAADGYLPDWLEDTL ATGGCTGCCGATGGTTATCTTCCAGATTGGCT
SEGIRQWWKLKPGPPP CGAGGACACTCTCTCTGAAGGAATAAGACAG
PKPAERHKDDSRGLVL TGGTGGAAGCTCAAACCTGGCCCACCACCACC
PGYKYLGPFNGLDKGE AAAGCCCGCAGAGCGGCATAAGGACGACAGC
PVNEADAAALEHDKA AGGGGTCTTGTGCTTCCTGGGTACAAGTACCT
YDRQLDSGDNPYLKY CGGACCCTTCAACGGACTCGACAAGGGAGAG
NHADAEFQERLKEDTS CCGGTCAACGAGGCAGACGCCGCGGCCCTCG
FGGNLGRAVFQAKKR AGCACGACAAAGCCTACGACCGGCAGCTCGA
VLEPLGLVEEPVKT AP C A GCGG AGAC A ACCCGT ACCTC A A GT AC A AC
GKKRPVEHSPVEPDSS CACGCCGACGCGGAGTTTCAGGAGCGCCTTA
SGTGKAGQQPARKRL AAGAAGATACGTCTTTTGGGGGCAACCTCGG
NFGQTGDADS VPDPQP ACGAGCAGTCTTCCAGGCGAAAAAGAGGGTT
LGQPPAAPSGLGTNTM CTTGAACCTCTGGGCCTGGTTGAGGAACCTGT
ATGSGAPMADNNEGA TAAGACGGCTCCGGGAAAAAAGAGGCCGGTA
DGVGNSSGNWHCDST GAGCACTCTCCTGTGGAGCCAGACTCCTCCTC
WMGDRVITTSTRTWA GGGAACCGGAAAGGCGGGCCAGCAGCCTGCA
LPT YNNHLYKQIS S QS AGAAAAAGATTGAATTTTGGTCAGACTGGAG
GASNDNHYFGYSTPW ACGCAGACTCAGTACCTGACCCCCAGCCTCTC
126
CA 03220810 2023- 11- 29 SUBSTITUTE SHEET (RULE 26)
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PCT/US2022/032004
GYFDFNRFHCHFSPRD GGACAGCCACCAGCAGCCCCCTCTGGTCTGGG
WQRLINNNWGFRPKR AACTAAT AC G AT GGCTAC AGGC AGT GGC GCA
LNFKLFNIQVKEVTQN CCAATGGCAGACAATAACGAGGGCGCCGACG
DGTTTIANNLTSTVQV GAGTGGGTAATTCCTCGGGAAATTGGCATTGC
FTDSEYQLPYVLGS AH GATTCCACATGGATGGGCGACAGAGTCATCA
QGCLPPFPADVFMVPQ CCACCAGCACCCGAACCTGGGCCCTGCCCACC
YGYLTLNNGS QAVGR TAC AAC AAC C AC C TCT AC AAAC AAATTTCCAG
S SFYCLEYFPS QMLRT CCAATCAGGAGCCTCGAACGACAATCACTACT
GNNFTFSYTFEDVPFH TTGGCTACAGCACCCCTTGGGGGTATTTTGAC
SSYAHS QSLDRLMNPL TTCAACAGATTCCACTGCCACTTTTCACCACG
IDQYLYYLSRTNTPSG TGACTGGCAAAGACTCATCAACAACAACTGG
TTTQS RLQFS QAGASDI GGATTCCGACCCAAGAGACTCAACTTCAAGCT
RDQSRNWLPGPCYRQ CTTTAACATTCAAGTCAAAGAGGTCACGCAGA
QRVSKTS ADNNNSEYS ATGACGGTACGACGACGATTGCCAATAACCTT
WTGATKYHLNGRDSL ACCAGCACGGTTCAGGTGTTTACTGACTCGGA
VNPGPAMASHKDDEE GTACCAGCTCCCGTACGTCCTCGGCTCGGC GC
KFFPQSGVLIFGKQGSE ATCAAGGATGCCTCCCGCCGTTCCCAGCAGAC
KTNVDIEKVMITDEEEI GTCTTCATGGTGCCACAGTATGGATACCTCAC
RTTNPVATEQYGS VS T CCTGAACAAC GGGAGTCAGGCAGTAGGAC GC
NLQRARLDETTRPARQ TCTTCATTTTACTGCCTGGAGTACTTTCCTTCT
AATADVNTQGVLPGM CAGATGCTGCGTACCGGAAACAACTTTACCTT
VWQDRDVYLQGPIWA CAGCTACACTTTTGAGGACGTTCCTTTCCACA
KIPHTDGHFHPSPLMG GC AGCT ACGCTC AC A GCC AGAGTCTGG ACCGT
GFGLKHPPPQILIKNTP CTCATGAATCCTCTCATCGACCAGTACCTGTA
VPANPS TTFS AAKFAS TTACTTGAGCAGAACAAACACTCCAAGTGGA
FITQYSTGQVS VEIE WE ACCACCACGCAGTCAAGGCTTCAGTTTTCTCA
LQKENSKRWNPEIQYT GGCCGGAGCGAGTGACATTCGGGACCAGTCT
SNYNKSVNVDFTVDT AGGAACTGGCTTCCTGGACCCTGTTACCGCCA
NGVYSEPRPIGTRYLT GC AGC G AGT AT C AAAGAC ATCT GC GGAT AAC
RNL* (SEQ ID NO: 34) AACAACAGTGAATACTCGTGGACTGGAGCTA
CCAAGTACCACCTCAATGGCAGAGACTCTCTG
GTGAATCCGGGCCCGGCCATGGCAAGCCACA
AGGACGATGAAGAAAAGTTTTTTCCTCAGAGC
GGGGTTCTCATCTTTGGGAAGCAAGGCTCAGA
GAAAACAAATGTGGACATTGAAAAGGTCATG
ATTACAGACGAAGAGGAAATCAGGACAACCA
ATCCCGTGGCTACGGAGCAGTATGGTTCTGTA
TCTACCAACCTCCAGAGAGCTCGATTGGATGA
GAC AAC AC GCC CT GCTAGAC AAGC AGCT ACC
GCAGATGTCAACACACAAGGCGTTCTTCCAGG
CATGGTCTGGCAGGACAGAGATGTGTACCTTC
AGGGGCCCATCTGGGCAAAGATTCCACACAC
GGACGGACATTTTCACCCCTCTCCCCTCATGG
GTGGATTCGGACTTAAACACCCTCCTCCACAG
ATTCTCATCAAGAACACCCCGGTACCTGCGAA
TCCTTCGACC ACCTTC AGTGCGGC A A A GTTTG
127
CA 03220810 2023- 11- 29 SUBSTITUTE SHEET (RULE 26)
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PCT/US2022/032004
CTTCCTTCATCACACAGTACTCCACGGGACAG
GTCAGCGTGGAGATCGAGTGGGAGCTGCAGA
AGGAAAACAGCAAACGCTGGAATCCCGAAAT
TCAGTACACTTCCAACTACAACAAGTCTGTTA
ATGTGGACTTTACTGTGGACACTAATGGCGTG
TATTCAGAGCCTCGCCCCATTGGCACCAGATA
CCTGACTCGTAATCTGTAA (SEQ ID NO: 79)
VAR-34 MAADGYLPDWLEDTL ATGGCTGCCGATGGTTATCTTCCAGATTGGCT
SEGIRQWWKLKPGPPP CGAGGACACTCTCTCTGAAGGAATAAGACAG
PKPAERHKDDSRGLVL TGGTGGAAGCTCAAACCTGGCCCACCACCACC
PGYKYLGPFNGLDKGE AAAGCCCGCAGAGCGGCATAAGGACGACAGC
PVNEADAAALEHDKA AGGGGTCTTGTGCTTCCTGGGTACAAGTACCT
YDRQLDSGDNPYLKY CGGACCCTTCAACGGACTCGACAAGGGAGAG
NHADAEFQERLKEDTS CCGGTCAACGAGGCAGACGCCGCGGCCCTCG
FGGNLGRAVFQAKKR AGCACGACAAAGCCTACGACCGGCAGCTCGA
VLEPLGLVEEPVKTAP CAGCGGAGACAACCCGTACCTCAAGTACAAC
GKKRPVEHSPVEPDSS CACGCCGACGCGGAGTTTCAGGAGCGCCTTA
SGTGKAGQQPARKRL AAGAAGATACGTCTTTTGGGGGCAACCTCGG
NFGQTGDADSVPDPQP ACGAGCAGTCTTCCAGGCGAAAAAGAGGGTT
LGQPPAAPSGLGTNTM CTTGAACCTCTGGGCCTGGTTGAGGAACCTGT
ATGSGAPMADNNEGA TAAGACGGCTCCGGGAAAAAAGAGGCCGGTA
DGVGNSSGN W H C DST GAGC ACTCTCCTGTGG A GCC A GACTCCTCCTC
WMGDRVITTSTRTWA GGGAACCGGAAAGGCGGGCCAGCAGCCTGCA
LPTYNNHLYKQISSQS AGAAAAAGATTGAATTTTGGTCAGACTGGAG
GASNDNHYFGYSTPW ACGCAGACTCAGTACCTGACCCCCAGCCTCTC
GYFDENREHCHFSPRD GGACAGCCACCAGCAGCCCCCTCTGGTCTGGG
WQRLINNNWGFRPKR AACTAATACGATGGCTACAGGCAGTGGCGCA
LNEKLENIQVKEVTQN CCAATGGCAGACAATAACGAGGGCGCCGACG
DGTTTIANNLTSTVQV GAGTGGGTAATTCCTCGGGAAATTGGCATTGC
FTDSEYQLPYVLGS AH GATTCCACATGGATGGGCGACAGAGTCATCA
QGCLPPFPADVFMVPQ CCACCAGCACCCGAACCTGGGCCCTGCCCACC
YGYLTLNNGSQAVGR TAC AAC AACC ACCTCT AC AAAC AAATTTCCAG
SSFYCLEYFPSQMLRT CCAATCAGGAGCCTCGAACGACAATCACTACT
GNNFTESYTFEDVPFH TTGGCTACAGCACCCCTTGGGGGTATTTTGAC
SSYAHS QSLDRLMNPL TTCAACAGATTCCACTGCCACTTTTCACCACG
IDQYLYYLSRTNTPSG TGACTGGCAAAGACTCATCAACAACAACTGG
TTTQSRLQFSQAGASDI GGATTCCGACCC A A GAGACTC A ACTTCAAGCT
RDQSRNWLPGPCYRQ CTTTAACATTCAAGTCAAAGAGGTCACGCAGA
QRVSKTS ADNNNSEYS ATGACGGTACGACGACGATTGCCAATAACCTT
WTGATKYHLNGRDSL ACCAGCACGGTTCAGGTGTTTACTGACTCGGA
VNPGPAMASHKDDEE GTACCAGCTCCCGTACGTCCTCGGCTCGGC GC
KFFPQSGVLIFGKQGSE ATCAAGGATGCCTCCCGCCGTTCCCAGCAGAC
KTNVDIEKVMITDEEEI GTCTTCATGGTGCCACAGTATGGATACCTCAC
RTTNPVATEQYGS VS T CCTGAACAACGGGAGTCAGGCAGTAGGACGC
NLQRGLALAEITRPAR TCTTCATTTTACTGCCTGGAGTACTTTCCTTCT
QAATADVNTQGVLPG CAGATGCTGCGTACCGGAAACAACTTTACCTT
128
CA 03220810 2023- 11- 29 SUBSTITUTE SHEET (RULE 26)
WO 2022/256557
PCT/US2022/032004
MVWQDRDVYLQGPIW CAGCTACACTTTTGAGGACGTTCCTTTCCACA
AKIPHTDGHFHPSPLM GCAGCTACGCTCACAGCCAGAGTCTGGACCGT
GGEGLKIIPPPQILIKNT CTCATGAATCCTCTCATCGACCAGTACCTGTA
PVPANPSTTFSAAKFA TTACTTGAGCAGAACAAACACTCCAAGTGGA
SFITQYSTGQVSVEIEW ACCACCACGCAGTCAAGGCTTCAGTTTTCTCA
ELQKENS KRWNPEIQY GGCCGGAGCGAGTGACATTCGGGACCAGTCT
TSNYNKSVNVDFTVDT AGGAACTGGCTTCCTGGACCCTGTTACCGCCA
NGVYSEPRPIGTRYLT GCAGCGAGTATCAAAGACATCTGCGGATAAC
RNL* (SEQ ID NO: 35) AACAACAGTGAATACTCGTGGACTGGAGCTA
CCAAGTACCACCTCAATGGCAGAGACTCTCTG
GTGAATCCGGGCCCGGCCATGGCAAGCCACA
AGGACGATGAAGAAAAGTTTTTTCCTCAGAGC
GGGGTTCTCATCTTTGGGAAGCAAGGCTCAGA
GAAAACAAATGTGGACATTGAAAAGGTCATG
ATTACAGACGAAGAGGAAATCAGGACAACCA
ATCCCGTGGCTACGGAGCAGTATGGTTCTGTA
TCTACCAACCTCCAGAGAGGCTTGGCTCTC GC
TGAGATCACCAGACCGGCGAGACAAGCAGCT
ACCGCAGATGTCAACACACAAGGCGTTCTTCC
AGGCATGGTCTGGCAGGACAGAGATGTGTAC
CTTCAGGGGCCCATCTGGGCAAAGATTCCACA
CACGGACGGACATTTTCACCCCTCTCCCCTCA
TGGGTGGATTCGGACTT A A AC ACCCTCCTCC A
CAGATTCTCATCAAGAACACCCCGGTACCTGC
GAATCCTTCGACCACCTTCAGTGCGGCAAAGT
TTGCTTCCTTCATCACACAGTACTCCACGGGA
CAGGTCAGCGTGGAGATCGAGTGGGAGCTGC
AGAAGGAAAACAGCAAACGCTGGAATCCCGA
AATTCAGTACACTTCCAACTACAACAAGTCTG
TTAATGTGGACTTTACTGTGGACACTAATGGC
GTGTATTCAGAGCCTCGCCCCATTGGCACCAG
ATACCTGACTCGTAATCTGTAA (SEQ ID NO:
80)
VAR-35 MAADGYLPDWLEDTL ATGGCTGCCGATGGTTATCTTCCAGATTGGCT
SEGIRQWWKLKPGPPP CGAGGACACTCTCTCTGAAGGAATAAGACAG
PKPAERHKDDSRGLVL TGGTGGAAGCTCAAACCTGGCCCACCACCACC
PGYKYLGPFNGLDKGE AAAGCCCGCAGAGCGGCATAAGGACGACAGC
PVNEADAAALEHDKA AGGGGTCTTGTGCTTCCTGGGTACAAGTACCT
YDRQLDSGDNPYLKY CGGACCCTTCAACGGACTCGACAAGGGAGAG
NHADAEFQERLKEDTS CCGGTCAACGAGGCAGACGCCGCGGCCCTCG
FGGNLGRAVFQAKKR AGCACGACAAAGCCTACGACCGGCAGCTCGA
VLEPLGLVEEPVKTAP CAGCGGAGACAACCCGTACCTCAAGTACAAC
GKKRPVEHSPVEPDSS CACGCCGACGCGGAGTTTCAGGAGCGCCTTA
SGTGKAGQQPARKRL AAGAAGATACGTCTTTTGGGGGCAACCTCGG
NFGQTGDADSVPDPQP ACGAGCAGTCTTCCAGGCGAAAAAGAGGGTT
LGQPPAAPSGLGTNTM CTTGAACCTCTGGGCCTGGTTGAGGAACCTGT
129
CA 03220810 2023- 11- 29 SUBSTITUTE SHEET (RULE 26)
WO 2022/256557
PCT/US2022/032004
ATGSGAPMADNNEGA TAAGACGGCTCCGGGAAAAAAGAGGCCGGTA
DGVGNSSGNWHCDST GAGCACTCTCCTGTGGAGCCAGACTCCTCCTC
WMGDRVITTSTRTWA GGGAACCGGAAAGGCGGGCCAGCAGCCTGCA
LPTYNNHLYKQISSQS AGAAAAAGATTGAATTTTGGTCAGACTGGAG
GASNDNHYFGYSTPW ACGCAGACTCAGTACCTGACCCCCAGCCTCTC
GYFDFNRFHCHFSPRD GGACAGCCACCAGCAGCCCCCTCTGGTCTGGG
WQRLINNNWGFRPKR AACTAATACGATGGCTACAGGCAGTGGCGCA
LNFKLFNIQVKEVTQN CCAATGGCAGACAATAACGAGGGCGCCGACG
DGTTTIANNLTSTVQV GAGTGGGTAATTCCTCGGGAAATTGGCATTGC
FTDSEYQLPYVLGSAH GATTCCACATGGATGGGCGACAGAGTCATCA
QGCLPPFPADVFMVPQ CCACCAGCACCCGAACCTGGGCCCTGCCCACC
YGYLTLNNGSQAVGR TACAACAACCACCTCTACAAACAAATTTCCAG
SSFYCLEYFPSQMLRT CCAATCAGGAGCCTCGAACGACAATCACTACT
GNNFTFSYTFEDVPFH TTGGCTACAGCACCCCTTGGGGGTATTTTGAC
SSYAHS QSLDRLMNPL TTCAACAGATTCCACTGCCACTTTTCACCACG
IDQYLYYLSRTNTPSG TGACTGGCAAAGACTCATCAACAACAACTGG
TTTQSRLQFSQAGASDI GGATTCCGACCCAAGAGACTCAACTTCAAGCT
RDQSRNWLPGPCYRQ CTTTAACATTCAAGTCAAAGAGGTCACGCAGA
QRVSKTS ADNNNSEYS ATGACGGTACGACGACGATTGCCAATAACCTT
WTGATKYHLNGRDSL ACCAGCACGGTTCAGGTGTTTACTGACTCGGA
VNPGPAMASHKDDEE GTACCAGCTCCCGTACGTCCTCGGCTCGGCGC
KFFPQSGVLIFGKQGSE ATCAAGGATGCCTCCCGCCGTTCCCAGCAGAC
KTNVDIEKVMITDEEEI GTCTTCATGGTGCC AC AGT ATGG AT ACCTC AC
RTTNPVATEQYGS VS T CCTGAACAACGGGAGTCAGGCAGTAGGACGC
NLQRGLANGEQTRPA TCTTCATTTTACTGCCTGGAGTACTTTCCTTCT
RQAATADVNTQGVLP CAGATGCTGCGTACCGGAAACAACTTTACCTT
GMVWQDRDVYLQGPI CAGCTACACTTTTGAGGACGTTCCTTTCCACA
WAKIPHTDGHFHPSPL GCAGCTACGCTCACAGCCAGAGTCTGGACCGT
MGGFGLKHPPPQILIK CTCATGAATCCTCTCATCGACCAGTACCTGTA
NTPVPANPSTTFSAAK TTACTTGAGCAGAACAAACACTCCAAGTGGA
FASFITQYSTGQVSVEI ACCACCACGCAGTCAAGGCTTCAGTTTTCTCA
EWELQKENSKRWNPEI GGCCGGAGCGAGTGACATTCGGGACCAGTCT
QYTSNYNKSVNVDFT AGGAACTGGCTTCCTGGACCCTGTTACCGCCA
VDTNGVYSEPRPIGTR GCAGCGAGTATCAAAGACATCTGCGGATAAC
YLTRNL* (SEQ ID NO: AACAACAGTGAATACTCGTGGACTGGAGCTA
361) CCAAGTACCACCTCAATGGCAGAGACTCTCTG
GTGAATCCGGGCCCGGCCATGGCAAGCCACA
AGGACGATGAAGAAAAGTTTTTTCCTCAGAGC
GGGGTTCTCATCTTTGGGAAGCAAGGCTCAGA
GAAAACAAATGTGGACATTGAAAAGGTCATG
ATTACAGACGAAGAGGAAATCAGGACAACCA
ATCCCGTGGCTACGGAGCAGTATGGTTCTGTA
TCTACCAACCTCCAGAGAGGCCTGGCGAACG
GCGAACAAACCAGGCCTGCGAGACAAGCAGC
TACCGCAGATGTCAACACACAAGGCGTTCTTC
C A GGC ATGGTCTGGC AGGAC AGAGATGTGT A
130
CA 03220810 2023- 11- 29 SUBSTITUTE SHEET (RULE 26)
WO 2022/256557
PCT/US2022/032004
CCTTCAGGGGCCCATCTGGGCAAAGATTCCAC
ACACGGACGGACATTTTCACCCCTCTCCCCTC
ATGGGTGGATTCGGACTTAAACACCCTCCTCC
ACAGATTCTCATCAAGAACACCCCGGTACCTG
CGAATCCTTCGACCACCTTCAGTGCGGCAAAG
TTTGCTTCCTTCATCACACAGTACTCCACGGG
ACAGGTCAGCGTGGAGATCGAGTGGGAGCTG
CAGAAGGAAAACAGCAAACGCTGGAATCCCG
AAATTCAGTACACTTCCAACTACAACAAGTCT
GTTAATGTGGACTTTACTGTGGACACTAATGG
CGTGTATTCAGAGCCTCGCCCCATTGGCACCA
GATACCTGACTCGTAATCTGTAA (SEQ ID NO:
81)
VAR-36 MAADGYLPDWLEDTL ATGGCTGCCGATGGTTATCTTCCAGATTGGCT
SEGIRQWWKLKPGPPP CGAGGACACTCTCTCTGAAGGAATAAGACAG
PKPAERHKDDSRGLVL TGGTGGAAGCTCAAACCTGGCCCACCACCACC
PGYKYLGPFNGLDKGE AAAGCCCGCAGAGCGGCATAAGGACGACAGC
PVNEADAAALEHDKA AGGGGTCTTGTGCTTCCTGGGTACAAGTACCT
YDRQLDSGDNPYLKY CGGACCCTTCAACGGACTCGACAAGGGAGAG
NHADAEFQERLKEDTS CCGGTCAACGAGGCAGACGCCGCGGCCCTCG
FGGNLGRAVFQAKKR AGCACGACAAAGCCTACGACCGGCAGCTCGA
VLEPLGLVEEPVKTAP CAGCGGAGACAACCCGTACCTCAAGTACAAC
GKKRPVEHSPVEPDSS CACGCCGACGCGGAGTTTCAGGAGCGCCTTA
SGTGK AGQQPARKRL A AGA AGAT ACGTCTTTTGGGGGC A ACCTCGG
NFGQTGDADSVPDPQP ACGAGCAGTCTTCCAGGCGAAAAAGAGGGTT
LGQPPAAPSGLGTNTM CTTGAACCTCTGGGCCTGGTTGAGGAACCTGT
ATGSGAPMADNNEGA TAAGACGGCTCCGGGAAAAAAGAGGCCGGTA
DGVGNSSGNWHCDST GAGCACTCTCCTGTGGAGCCAGACTCCTCCTC
WMGDRVITTSTRTWA GGGAACCGGAAAGGCGGGCCAGCAGCCTGCA
LPTYNNHLYKQISSQS AGAAAAAGATTGAATTTTGGTCAGACTGGAG
GASNDNHYFGYSTPW ACGCAGACTCAGTACCTGACCCCCAGCCTCTC
GYFDENREHCHFSPRD GGACAGCCACCAGCAGCCCCCTCTGGTCTGGG
WQRLINNNWGFRPKR AACTAATACGATGGCTACAGGCAGTGGCGCA
LNEKLENIQVKEVTQN CCAATGGCAGACAATAACGAGGGCGCCGACG
DGTTTIANNLTSTVQV GAGTGGGTAATTCCTCGGGAAATTGGCATTGC
FTDSEYQLPYVLGSAH GATTCCACATGGATGGGCGACAGAGTCATCA
QGCLPPFPADVFMVPQ CC ACC A GC ACCCG A ACCTGGGCCCTGCCC ACC
YGYLTLNNGSQAVGR TACAACAACCACCTCTACAAACAAATTTCCAG
SSFYCLEYFPSQMLRT CCAATCAGGAGCCTCGAACGACAATCACTACT
GNNFTFSYTFEDVPFH TTGGCTACAGCACCCCTTGGGGGTATTTTGAC
SSYAHS QSLDRLMNPL TTCAACAGATTCCACTGCCACTTTTCACCACG
IDQYLYYLSRTNTPSG TGACTGGCAAAGACTCATCAACAACAACTGG
TTTQSRLQFSQAGASDI GGATTCCGACCCAAGAGACTCAACTTCAAGCT
RDQSRNWLPGPCYRQ CTTTAACATTCAAGTCAAAGAGGTCACGCAGA
QRVSKTSADNNNSEYS ATGACGGTACGACGACGATTGCCAATAACCTT
WTGATKYHLNGRDSL ACCAGCACGGTTCAGGTGTTTACTGACTCGGA
131
CA 03220810 2023- 11- 29 SUBSTITUTE SHEET (RULE 26)
WO 2022/256557
PCT/US2022/032004
VNPGPAMASHKDDEE GTACCAGCTCCCGTACGTCCTCGGCTCGGC GC
KFFPQSGVLIFGKQGSE ATCAAGGATGCCTCCCGCCGTTCCCAGCAGAC
KTNVDIEKVMITDEEEI GTCTTCATGGTGCCACAGTATGGATACCTCAC
RTTNPVATEQYGS VS T CCTGAACAACGGGAGTCAGGCAGTAGGACGC
NLQRGATDTKTNRQA TCTTCATTTTACTGCCTGGAGTACTTTCCTTCT
ATADVNTQGVLPGMV CAGATGCTGCGTACCGGAAACAACTTTACCTT
WQDRDVYLQGPIWAK CAGCTACACTTTTGAGGACGTTCCTTTCCACA
IPHTDGHFHPSPLMGG GCAGCTACGCTCACAGCCAGAGTCTGGACCGT
FGLKHPPPQILIKNTPV CTCATGAATCCTCTCATCGACCAGTACCTGTA
PANPSTTFSAAKFASFI TTACTTGAGCAGAACAAACACTCCAAGTGGA
TQYSTGQVSVEIEWEL ACCACCACGCAGTCAAGGCTTCAGTTTTCTCA
QKENS KRWNPEIQYTS GGCCGGAGCGAGTGACATTCGGGACCAGTCT
NYNKSVNVDFTVDTN AGGAACTGGCTTCCTGGACCCTGTTACCGCCA
GVYSEPRPIGTRYLTR GCAGCGAGTATCAAAGACATCTGCGGATAAC
NL* (SEQ ID NO: 37) AACAACAGTGAATACTCGTGGACTGGAGCTA
CCAAGTACCACCTCAATGGCAGAGACTCTCTG
GTGAATCCGGGCCCGGCCATGGCAAGCCACA
AGGACGATGAAGAAAAGTTTTTTCCTCAGAGC
GGGGTTCTCATCTTTGGGAAGCAAGGCTCAGA
GAAAACAAATGTGGACATTGAAAAGGTCATG
ATTACAGACGAAGAGGAAATCAGGACAACCA
ATCCCGTGGCTACGGAGCAGTATGGTTCTGTA
TCT ACC A ACCTCC A GAG A GGCGCTACGGATAC
TAAGACGAACAGACAAGCAGCTACCGCAGAT
GTCAACACACAAGGCGTTCTTCCAGGCATGGT
CIGGCAGGACAGAGATGTGTACCTICAGGGG
CCCATCTGGGCAAAGATTCCACACACGGACG
GACATTTTCACCCCTCTCCCCTCATGGGTGGA
TTCGGACTTAAACACCCTCCTCCACAGATTCT
CATCAAGAACACCCCGGTACCTGCGAATCCTT
CGACCACCTTCAGTGCGGCAAAGTTTGCTTCC
TTCATCACACAGTACTCCACGGGACAGGTCAG
CGTGGAGATCGAGTGGGAGCTGCAGAAGGAA
AACAGCAAACGCTGGAATCCCGAAATTCAGT
ACACTTCCAACTACAACAAGTCTGTTAATGTG
GACTTTACTGTGGACACTAATGGCGTGTATTC
AGAGCCTCGCCCCATTGGCACCAGATACCTGA
CTCGTAATCTGTAA (SEQ ID NO: 82)
VAR-37 MAADGYLPDWLEDTL ATGGCTGCCGATGGTTATCTTCCAGATTGGCT
SEGIRQWWKLKPGPPP CGAGGACACTCTCTCTGAAGGAATAAGACAG
PKPAERHKDDSRGLVL TGGTGGAAGCTCAAACCTGGCCCACCACCACC
PGYKYLGPFNGLDKGE AAAGCCCGCAGAGCGGCATAAGGACGACAGC
PVNEADAAALEHDKA AGGGGTCTTGTGCTTCCTGGGTACAAGTACCT
YDRQLDSGDNPYLKY CGGACCCTTCAACGGACTCGACAAGGGAGAG
NHADAEFQERLKEDTS CCGGTCAACGAGGCAGACGCCGCGGCCCTCG
FGGNLGRAVFQAKKR AGCACGACAAAGCCTACGACCGGCAGCTCGA
132
CA 03220810 2023- 11- 29 SUBSTITUTE SHEET (RULE 26)
WO 2022/256557
PCT/US2022/032004
VLEPLGLVEEPVKTAP CAGCGGAGACAACCCGTACCTCAAGTACAAC
GKKRPVEHSPVEPDSS CACGCCGACGCGGAGTTTCAGGAGCGCCTTA
SGTGKAGQQPARKRL AAGAAGATACGTCTTTTGGGGGCAACCTCGG
NFGQTGDADSVPDPQP ACGAGCAGTCTTCCAGGCGAAAAAGAGGGTT
LGQPPAAPSGLGTNTM CTTGAACCTCTGGGCCTGGTTGAGGAACCTGT
ATGSGAPMADNNEGA TAAGACGGCTCCGGGAAAAAAGAGGCCGGTA
DGVGNSSGNWHCDST GAGCACTCTCCTGTGGAGCCAGACTCCTCCTC
WMGDRVITTSTRTWA GGGAACCGGAAAGGCGGGCCAGCAGCCTGCA
LPTYNNHLYKQISSQS AGAAAAAGATTGAATTTTGGTCAGACTGGAG
GASNDNHYFGYSTPW ACGCAGACTCAGTACCTGACCCCCAGCCTCTC
GYFDFNRFHCHFSPRD GGACAGCCACCAGCAGCCCCCTCTGGTCTGGG
WQRLINNNWGFRPKR AACTAATACGATGGCTACAGGCAGTGGCGCA
LNFKLFNIQVKEVTQN CCAATGGCAGACAATAACGAGGGCGCCGACG
DGTTTIANNLTSTVQV GAGTGGGTAATTCCTCGGGAAATTGGCATTGC
FTDSEYQLPYVLGSAH GATTCCACATGGATGGGCGACAGAGTCATCA
QGCLPPFPADVFMVPQ CCACCAGCACCCGAACCTGGGCCCTGCCCACC
YGYLTLNNGSQAVGR TACAACAACCACCTCTACAAACAAATTTCCAG
SSFYCLEYFPSQMLRT CCAATCAGGAGCCTCGAACGACAATCACTACT
GNNFTFSYTFEDVPFH TTGGCTACAGCACCCCTTGGGGGTATTTTGAC
SSYAHSQSLDRLMNPL TTCAACAGATTCCACTGCCACTTTTCACCACG
IDQYLYYLSRTNTPSG TGACTGGCAAAGACTCATCAACAACAACTGG
TTTQSRLQFSQAGASDI GGATTCCGACCCAAGAGACTCAACTTCAAGCT
RDQSRNWLPGPCYRQ CTTT A AC ATTCA AGTC A A AGAGGTC ACGC AGA
QRVSKTSADNNNSEYS ATGACGGTACGACGACGATTGCCAATAACCTT
WTGATKYHLNGRDSL ACCAGCACGGTTCAGGTGTTTACTGACTCGGA
VNPGPAMASHKDDEE GTACCAGCTCCCGTACGTCCTCGGCTCGGCGC
KFFPQSGVLIFGKQGSE ATCAAGGATGCCTCCCGCCGTTCCCAGCAGAC
KTNVDIEKVMITDEEEI GTCTTCATGGTGCCACAGTATGGATACCTCAC
RTTNPVATEQYGS VS T CCTGAACAACGGGAGTCAGGCAGTAGGACGC
NLQRGNRQAPATADV TCTTCATTTTACTGCCTGGAGTACTTTCCTTCT
NTQGVLPGMVWQDR CAGATGCTGCGTACCGGAAACAACTTTACCTT
DVYLQGPIWAKIPHTD CAGCTACACTTTTGAGGACGTTCCTTTCCACA
GHFHPSPLMGGFGLKH GCAGCTACGCTCACAGCCAGAGTCTGGACCGT
PPPQILIKNTPVPANPST CTCATGAATCCTCTCATCGACCAGTACCTGTA
TFSAAKFASFITQYSTG TTACTTGAGCAGAACAAACACTCCAAGTGGA
QVSVEIEWELQKENSK ACCACCACGCAGTCAAGGCTTCAGTTTTCTCA
RWNPEIQYTSNYNKS V GGCCGGAGCGAGTGACATTCGGGACCAGTCT
NVDFTVDTNGVYSEPR AGGAACTGGCTTCCTGGACCCTGTTACCGCCA
PIGTRYLTRNL* (SEQ GCAGCGAGTATCAAAGACATCTGCGGATAAC
ID NO: 38)
AACAACAGTGAATACTCGTGGACTGGAGCTA
CCAAGTACCACCTCAATGGCAGAGACTCTCTG
GTGAATCCGGGCCCGGCCATGGCAAGCCACA
AGGACGATGAAGAAAAGTTTTTTCCTCAGAGC
GGGGTTCTCATCTTTGGGAAGCAAGGCTCAGA
GAAAACAAATGTGGACATTGAAAAGGTCATG
ATTACAGACGAAGAGGAAATCAGGACAACCA
133
CA 03220810 2023- 11- 29 SUBSTITUTE SHEET (RULE 26)
WO 2022/256557
PCT/US2022/032004
ATCCCGTGGCTACGGAGCAGTATGGTTCTGTA
TCTACCAACCTCCAGAGAGGCAACAGACAAG
CACCAGCTACCGCAGATGTCAACACACAAGG
CGTTCTTCCAGGCATGGTCTGGCAGGACAGAG
ATGTGTACCTTCAGGGGCCCATCTGGGCAAAG
ATTCCACACACGGACGGACATTTTCACCCCTC
TCCCCTCATGGGTGGATTCGGACTTAAACACC
CTCCTCCACAGATTCTCATCAAGAACACCCCG
GTACCTGCGAATCCTTCGACCACCTTCAGTGC
GGCAAAGTTTGCTTCCTTCATCACACAGTACT
CCACGGGACAGGTCAGCGTGGAGATCGAGTG
GGAGCTGCAGAAGGAAAACAGCAAACGCTGG
AATCCCGAAATTCAGTACACTTCCAACTACAA
CAAGTCTGTTAATGTGGACTTTACTGTGGACA
CTAATGGCGTGTATTCAGAGCCTCGCCCCATT
GGCACCAGATACCTGACTCGTAATCTGTAA
(SEQ ID NO: 83)
VAR-38 MAADGY LPDWLEDTL ATGGCTGCCGATGGTTATCTTCCAGATTGGCT
SEGIRQWWKLKPGPPP CGAGGACACTCTCTCTGAAGGAATAAGACAG
PKPAERHKDDSRGLVL TGGTGGAAGCTCAAACCTGGCCCACCACCACC
PGYKYLGPFNGLDKGE AAAGCCCGCAGAGCGGCATAAGGACGACAGC
PV N EAD A A A LEH DK A AGGGGTCTTGTGCTTCCTGGGT AC A A GT AC CT
YDRQLDSGDNPYLKY CGGACCCTTCAACGGACTCGACAAGGGAGAG
NH AD AE FQERLKEDTS CCGGTC A ACG A GGC AG ACGCCGCGGCCCTCG
FGGNLGRAVFQAKKR AGCACGACAAAGCCTACGACCGGCAGCTCGA
VLEPLGLVEEPVKTAP CAGCGGAGACAACCCGTACCTCAAGTACAAC
GKKRPVEHSPVEPDSS CACGCCGACGCGGAGTTTCAGGAGCGCCTTA
SGTGKAGQQPARKRL AAGAAGATACGTCTTTTGGGGGCAACCTCGG
NFGQTGDADSVPDPQP ACGAGCAGTCTTCCAGGCGAAAAAGAGGGTT
LGQPPAAPSGLGTNTM CTTGAACCTCTGGGCCTGGTTGAGGAACCTGT
ATGSGAPMADNNEGA TAAGACGGCTCCGGGAAAAAAGAGGCCGGTA
DGVGNSSGNWHCDST GAGCACTCTCCTGTGGAGCCAGACTCCTCCTC
WMGDRVITTSTRTWA GGGAACCGGAAAGGCGGGCCAGCAGCCTGCA
LPTYNNHLYKQISSQS AGAAAAAGATTGAATTTTGGTCAGACTGGAG
GASNDNHYFGYSTPW ACGCAGACTCAGTACCTGACCCCCAGCCTCTC
GYFDENREHCHFSPRD GGACAGCCACCAGCAGCCCCCTCTGGTCTGGG
WQRLINNNWGFRPKR A ACT A AT ACG AT GGCTAC AGGC AGTGGC GC A
LNEKLENIQVKEVTQN CCAATGGCAGACAATAACGAGGGCGCCGACG
DGTTTIANNLTSTVQV GAGTGGGTAATTCCTCGGGAAATTGGCATTGC
FTDSEY QLPY VLGS AH GATTCCACATGGATGGGCGACAGAGTCATCA
QGCLPPFPADVFMVPQ CCACCAGCACCCGAACCTGGGCCCTGCCCACC
YGYLTLNNGSQAVGR TACAACAACCACCTCTACAAACAAATTTCCAG
S SFYCLEYFPS QMLRT CCAATCAGGAGCCTCGAACGACAATCACTACT
GNNFTESYTFEDVPFH TTGGCTACAGCACCCCTTGGGGGTATTTTGAC
SSYAHS QSLDRLMNPL TTCAACAGATTCCACTGCCACTTTTCACCACG
IDQYLYYLSRTNTPSG TGACTGGCAAAGACTCATCAACAACAACTGG
134
CA 03220810 2023- 11- 29 SUBSTITUTE SHEET (RULE 26)
WO 2022/256557
PCT/US2022/032004
TTTQS RLQFS QAGASDI GGATTCCGACCCAAGAGACTCAACTTCAAGCT
RDQSRNWLPGPCYRQ CTTTAACATTCAAGTCAAAGAGGTCACGCAGA
QRVSKTS ADNNNSEYS ATGACGGTACGACGACGATTGCCAATAACCTT
WTGATKYHLNGRDSL ACCAGCACGGTTCAGGTGTTTACTGACTCGGA
VNPGPAMASHKDDEE GTACCAGCTCCCGTACGTCCTCGGCTCGGC GC
KFFPQSGVLIFGKQGSE ATCAAGGATGCCTCCCGCCGTTCCCAGCAGAC
KTNVDIEKVMITDEEEI GTCTTCATGGTGCCACAGTATGGATACCTCAC
RTTNPVATEQYGS VS T CCTGAACAAC GGGAGTCAGGCAGTAGGAC GC
NLQRGNAPGETTRPAR TCTTCATTTTACTGCCTGGAGTACTTTCCTTCT
QAATADVNTQGVLPG CAGATGCTGCGTACCGGAAACAACTTTACCTT
MVWQDRDVYLQGPIW CAGCTACACTTTTGAGGACGTTCCTTTCCACA
AKIPHTDGHFHPSPLM GC AGCT AC GCTC AC AGCC AGAGTCT GGACCGT
GGFGLKHPPPQILIKNT CTCATGAATCCTCTCATCGACCAGTACCTGTA
PVPANPSTTFSAAKFA TTACTTGAGCAGAACAAACACTCCAAGTGGA
SF1TQYSTGQVSVEIEW ACCACCACGCAGTCAAGGCTTCAGTTTTCTCA
ELQKENS KRWNPEIQY GGCCGGAGCGAGTGACATTCGGGACCAGTCT
TSNYNKSVNVDFTVDT AGGAACTGGCTTCCTGGACCCTGTTACCGCCA
NGVYSEPRPIGTRYLT GC AGC G AGT AT C AAAGAC ATCT GC GGAT AAC
RNL* (SEQ ID NO: 39) AACAACAGTGAATACTCGTGGACTGGAGCTA
CCAAGTACCACCTCAATGGCAGAGACTCTCTG
GTGAATCCGGGCCCGGCCATGGCAAGCCACA
AGGACGATGAAGAAAAGTTTTTTCCTCAGAGC
GGGGTTCTCATCTTTGGGA AGC A A GGCTC AGA
GAAAACAAATGTGGACATTGAAAAGGTCATG
ATTACAGACGAAGAGGAAATCAGGACAACCA
ATCCCGTGGCTACGGAGCAGTATGGTTCTGTA
TCTACCAACCTCCAGAGAGGCAACGCACCCG
GAGAGACT ACT AGGCC AGC AAGACAAGC A GC
TACCGCAGATGTCAACACACAAGGCGTTCTTC
CAGGCATGGTCTGGCAGGACAGAGATGTGTA
CCTTCAGGGGCCCATCTGGGCAAAGATTCCAC
ACACGGACGGACATTTTCACCCCTCTCCCCTC
ATGGGTGGATTCGGACTTAAACACCCTCCTCC
ACAGATTCTCATCAAGAACACCCCGGTACCTG
CGAATCCTTCGACCACCTTCAGTGCGGCAAAG
TTTGCTTCCTTCATCACACAGTACTCCACGGG
ACAGGTCAGCGTGGAGATCGAGTGGGAGCTG
CAGAAGGAAAACAGCAAACGCTGGAATCCCG
AAATTC AGT AC ACTTCCAACTAC AAC AAGTCT
GTTAATGTGGACTTTACTGTGGACACTAATGG
CGTGTATTCAGAGCCTCGCCCCATTGGCACCA
GATACCTGACTCGTAATCTGTAA (SEQ ID NO:
84)
VAR-39 MAADGYLPDWLEDTL ATGGCTGCCGATGGTTATCTTCCAGATTGGCT
SEGIRQWWKLKPGPPP CGAGGACACTCTCTCTGAAGGAATAAGACAG
PKPAERHKDDSRGLVL TGGTGGAAGCTCAAACCTGGCCCACCACCACC
135
CA 03220810 2023- 11- 29 SUBSTITUTE SHEET (RULE 26)
WO 2022/256557
PCT/US2022/032004
PGYKYLGPFNGLDKGE AAAGCCCGCAGAGCGGCATAAGGACGACAGC
PVNEADAAALEHDKA AGGGGTC TT GT GCTTCCT GGGTACAAGT AC C T
YDRQLDSGDNPYLKY CGGACCCTTCAACGGACTCGACAAGGGAGAG
NHADAEFQERLKEDTS CCGGTCAACGAGGCAGACGCCGCGGCCCTCG
FGGNLGRAVFQAKKR AGCACGACAAAGCCTACGACCGGCAGCTCGA
VLEPLGLVEEPVKTAP CAGCGGAGACAACCCGTACCTCAAGTACAAC
GKKRPVEHSPVEPDSS CACGCCGACGCGGAGTTTCAGGAGCGCCTTA
SGTGKAGQQPARKRL AAGAAGATACGTCTTTTGGGGGCAACCTCGG
NFGQTGDADSVPDPQP ACGAGCAGTCTTCCAGGCGAAAAAGAGGGTT
LGQPPAAPSGLGTNTM CTTGAACCTCTGGGCCTGGTTGAGGAACCTGT
ATGSGAPMADNNEGA TAAGACGGCTCCGGGAAAAAAGAGGCCGGTA
DGVGNSSGNWHCDST GAGCACTCTCCTGTGGAGCCAGACTCCTCCTC
WMGDRVITTSTRTWA GGGAACCGGAAAGGCGGGCCAGCAGCCTGCA
LPT YNNHLYKQIS S QS AGAAAAAGATTGAATTTTGGTCAGACTGGAG
GASNDNHYFGYSTPW ACGCAGACTCAGTACCTGACCCCCAGCCTCTC
GYFDFNRFHCHFSPRD GGACAGCCACCAGCAGCCCCCTCTGGTCTGGG
WQRLINNNWGFRPKR AACTAATACGATGGCTACAGGCAGTGGCGCA
LNFKLFNIQVKEVTQN CCAATGGCAGACAATAACGAGGGCGCCGACG
DGTTTIANNLTSTVQV GAGTGGGTAATTCCTCGGGAAATTGGCATTGC
FTDSEYQLPYVLGSAH GATTCCACATGGATGGGCGACAGAGTCATCA
QGCLPPFPADVFMVPQ CCACCAGCACCCGAACCTGGGCCCTGCCCACC
YGYLTLNNGSQAVGR TAC AAC AACC ACC TCT AC AAAC AAATTTCCAG
SSFYCLEYFPSQMLRT CC A ATCAGGAGCCTCGA ACGAC A ATC ACT ACT
GNNFTFSYTFEDVPFH TTGGCTACAGCACCCCTTGGGGGTATTTTGAC
SSYAHS QSLDRLMNPL TTCAACAGATTCCACTGCCACTTTTCACCACG
IDQYLY YLSRTNTPSG TGACTGGCAAAGACTCATCAACAACAACTGG
TTTQSRLQFSQAGASDI GGATTCCGACCCAAGAGACTCAACTTCAAGCT
RDQSRNWLPGPCYRQ CTTTAAC ATTCAAGTC AAAGAGGTC ACGC AGA
QRVSKTS ADNNNSEYS ATGACGGTACGACGACGATTGCCAATAACCTT
WTGATKYHLNGRDSL ACCAGCACGGTTCAGGTGTTTACTGACTCGGA
VNPGPAMASHKDDEE GTACCAGCTCCCGTACGTCCTCGGCTCGGC GC
KFFPQSGVLIFGKQGSE ATCAAGGATGCCTCCCGCCGTTCCCAGCAGAC
KTNVDIEKVMITDEEEI GTCTTCATGGTGCCACAGTATGGATACCTCAC
RTTNPVATEQYGS VS T CCTGAACAACGGGAGTCAGGCAGTAGGACGC
NLQRGNRQPAATADV TCTTCATTTTACTGCCTGGAGTACTTTCCTTCT
NTQGVLPGMVWQDR CAGATGCTGCGTACCGGAAACAACTTTACCTT
DVYLQGPIWAKIPHTD CAGCTACACTTTTGAGGACGTTCCTTTCCACA
GHFHPSPLMGGFGLKH GCAGCTACGCTCACAGCCAGAGTCTGGACCGT
PPPQILIKNTPVPANPST CTCATGAATCCTCTCATCGACCAGTACCTGTA
TFSAAKFASFITQYSTG TTACTTGAGCAGAACAAACACTCCAAGTGGA
QVSVEIEWELQKENSK ACCACCACGCAGTCAAGGCTTCAGTTTTCTCA
RWNPEIQYTSN YNKS V GGCCGGAGCGAGTGACATTCGGGACCAGTCT
NVDFTVDTNGVYSEPR AGGAACTGGCTTCCTGGACCCTGTTACCGCCA
PIGTRYLTRNL* (SEQ GCAGCGAGTATCAAAGACATCTGCGGATAAC
ID NO: 40) AACAACAGTGAATACTCGTGGACTGGAGCTA
CC A AGT ACC ACCTCA ATGGCAGAGACTCTCTG
136
CA 03220810 2023- 11- 29 SUBSTITUTE SHEET (RULE 26)
WO 2022/256557
PCT/US2022/032004
GTGAATCCGGGCCCGGCCATGGCAAGCCACA
AGGACGATGAAGAAAAGTTTTTTCCTCAGAGC
GGGGTTCTCATCTTTGGGAAGCAAGGCTCAGA
GAAAACAAATGTGGACATTGAAAAGGTCATG
ATTACAGACGAAGAGGAAATCAGGACAACCA
ATCCCGTGGCTACGGAGCAGTATGGTTCTGTA
TCTACCAACCTCCAGAGAGGCAACAGACAAC
CGGCAGCTACCGCAGATGTCAACACACAAGG
CGTTCTTCCAGGCATGGTCTGGCAGGACAGAG
ATGTGTACCTTCAGGGGCCCATCTGGGCAAAG
ATTCCACACACGGACGGACATTTTCACCCCTC
TCCCCTCATGGGTGGATTCGGACTTAAACACC
CTCCTCCACAGATTCTCATCAAGAACACCCCG
GTACCTGCGAATCCTTCGACCACCTTCAGTGC
GGCAAAGTTTGCTTCCTTCATCACACAGTACT
CCACGGGACAGGTCAGCGTGGAGATCGAGTG
GGAGCTGCAGAAGGAAAACAGCAAACGCTGG
AATCCCGAAATTCAGTACACTTCCAACTACAA
CAAGTCTGTTAATGTGGACTTTACTGTGGACA
CTAATGGCGTGTATTCAGAGCCTCGCCCCATT
GGCACCAGATACCTGACTCGTAATCTGTAA
(SEQ ID NO: 85)
VAR-40 MAADGYLPDWLEDTL ATGGCTGCCGATGGTTATCTTCCAGATTGGCT
SEGIRQWWKLKPGPPP CGAGGACACTCTCTCTGAAGGAATAAGACAG
PKPAERHKDDSRGLVL TGGTGGAAGCTCAAACCTGGCCCACCACCACC
PGYKYLGPFNGLDKGE AAAGCCCGCAGAGCGGCATAAGGACGACAGC
PVNEADAAALEHDKA AGGGGTCTTGTGCTTCCTGGGTACAAGTACCT
YDRQLDSGDNPYLKY CGGACCCTTCAACGGACTCGACAAGGGAGAG
NHADAEFQERLKEDTS CCGGTCAACGAGGCAGACGCCGCGGCCCTCG
FGGNLGRAVFQAKKR AGCACGACAAAGCCTACGACCGGCAGCTCGA
VLEPLGLVEEPVKTAP CAGCGGAGACAACCCGTACCTCAAGTACAAC
GKKRPVEHSPVEPDSS CACGCCGACGCGGAGTTTCAGGAGCGCCTTA
SGTGKAGQQPARKRL AAGAAGATACGTCTTTTGGGGGCAACCTCGG
NFGQTGDADSVPDPQP ACGAGCAGTCTTCCAGGCGAAAAAGAGGGTT
LGQPPAAPSGLGTNTM CTTGAACCTCTGGGCCTGGTTGAGGAACCTGT
ATGSGAPMADNNEGA TAAGACGGCTCCGGGAAAAAAGAGGCCGGTA
DGVGNSSGNWHCDST GAGC ACTCTCCTGTGG A GCC A GACTCCTCCTC
WMGDRVITTSTRTWA GGGAACCGGAAAGGCGGGCCAGCAGCCTGCA
LPTYNNHLYKQISSQS AGAAAAAGATTGAATTTTGGTCAGACTGGAG
GASNDNHYFGYSTPW ACGCAGACTCAGTACCTGACCCCCAGCCTCTC
GYFDENREHCHFSPRD GGACAGCCACCAGCAGCCCCCTCTGGTCTGGG
WQRLINNNWGFRPKR AACTAATACGATGGCTACAGGCAGTGGCGCA
LNEKLENIQVKEVTQN CCAATGGCAGACAATAACGAGGGCGCCGACG
DGTTTIANNLTSTVQV GAGTGGGTAATTCCTCGGGAAATTGGCATTGC
FTDSEYQLPYVLGSAH GATTCCACATGGATGGGCGACAGAGTCATCA
QGCLPPFPADVFMVPQ CCACCAGCACCCGAACCTGGGCCCTGCCCACC
137
CA 03220810 2023- 11- 29 SUBSTITUTE SHEET (RULE 26)
WO 2022/256557
PCT/US2022/032004
YGYLTLNNGSQAVGR TACAACAACCACCTCTACAAACAAATTTCCAG
SSFYCLEYFPSQMLRT CCAATCAGGAGCCTCGAACGACAATCACTACT
GNNFTFSYTFEDVPFH TTGGCTACAGCACCCCTTGGGGGTATTTTGAC
SSYAHS QSLDRLMNPL TTCAACAGATTCCACTGCCACTTTTCACCACG
IDQYLYYLSRTNTPSG TGACTGGCAAAGACTCATCAACAACAACTGG
TTTQSRLQFSQAGASDI GGATTCCGACCCAAGAGACTCAACTTCAAGCT
RDQSRNWLPGPCYRQ CTTTAACATTCAAGTCAAAGAGGTCACGCAGA
QRVSKTS ADNNNSEYS ATGACGGTACGACGACGATTGCCAATAACCTT
WTGATKYHLNGRDSL ACCAGCACGGTTCAGGTGTTTACTGACTCGGA
VNPGPAMASHKDDEE GTACCAGCTCCCGTACGTCCTCGGCTCGGCGC
KFFPQSGVLIFGKQGSE ATCAAGGATGCCTCCCGCCGTTCCCAGCAGAC
KTNVDIEKVMITDEEEI GTCTTCATGGTGCCACAGTATGGATACCTCAC
RTTNPVATEQYGS VS T CCTGAACAACGGGAGTCAGGCAGTAGGACGC
NLQSS ARQAETARVN TCTTCATTTTACTGCCTGGAGTACTTTCCTTCT
AQGILPGMVWQDRDV CAGATGCTGCGTACCGGAAACAACTTTACCTT
YLQGPIWAKIPHTDGH CAGCTACACTTTTGAGGACGTTCCTTTCCACA
FHPSPLMGGFGLKHPP GCAGCTACGCTCACAGCCAGAGTCTGGACCGT
PQILIKNTPVPANPSTT CTCATGAATCCTCTCATCGACCAGTACCTGTA
FS AAKFASFITQYSTGQ TTACTTGAGCAGAACAAACACTCCAAGTGGA
VSVEIEWELQKENS KR ACCACCACGCAGTCAAGGCTTCAGTTTTCTCA
WNPEIQYTSNYNKS VN GGCCGGAGCGAGTGACATTCGGGACCAGTCT
VDFTVDTNGVYSEPRP AGGAACTGGCTTCCTGGACCCTGTTACCGCCA
IGTRYLTRNL* (SEQ ID GCAGCGAGTATCAAAGACATCTGCGGATAAC
NO: 41)
AACAACAGTGAATACTCGTGGACTGGAGCTA
CCAAGTACCACCTCAATGGCAGAGACTCTCTG
GTGAATCCGGGCCCGGCCATGGCAAGCCACA
AGGACGATGAAGAAAAGTTTTTTCCTCAGAGC
GGGGTTCTCATCTTTGGGAAGCAAGGCTCAGA
GAAAACAAATGTGGACATTGAAAAGGTCATG
ATTACAGACGAAGAGGAAATCAGGACAACCA
ATCCCGTGGCTACGGAGCAGTATGGTTCTGTA
TCTACCAACCTCCAGTCTTCTGCTAGACAAGC
AGAGACAGCACGGGTCAACGCTCAAGGCATT
CTTCCAGGCATGGTCTGGCAGGACAGAGATGT
GTACCTTCAGGGGCCCATCTGGGCAAAGATTC
CACACACGGACGGACATTTTCACCCCTCTCCC
CTCATGGGTGGATTCGGACTTAAACACCCTCC
TCCACAGATTCTCATCAAGAACACCCCGGTAC
CTGCGAATCCTTCGACCACCTTCAGTGCGGCA
AAGTTTGCTTCCTTCATCACACAGTACTCCAC
GGGACAGGTCAGCGTGGAGATCGAGTGGGAG
CTGCAGAAGGAAAACAGCAAACGCTGGAATC
CCGAAATTCAGTACACTTCCAACTACAACAAG
TCTGTTAATGTGGACTTTACTGTGGACACTAA
TGGCGTGTATTCAGAGCCTCGCCCCATTGGCA
138
CA 03220810 2023- 11- 29 SUBSTITUTE SHEET (RULE 26)
WO 2022/256557
PCT/US2022/032004
CCAGATACCTGACTCGTAATCTGTAA (SEQ ID
NO: 86)
VAR-41 MAADGYLPDWLEDTL ATGGCTGCCGATGGTTATCTTCCAGATTGGCT
SEGIRQWWKLKPGPPP CGAGGACACTCTCTCTGAAGGAATAAGACAG
PKPAERHKDDSRGLVL TGGTGGAAGCTCAAACCTGGCCCACCACCACC
PGYKYLGPFNGLDKGE AAAGCCCGCAGAGCGGCATAAGGACGACAGC
PVNEADAAALEHDKA AGGGGTCTTGTGCTTCCTGGGTACAAGTACCT
YDRQLDSGDNPYLKY CGGACCCTTCAACGGACTCGACAAGGGAGAG
NHADAEFQERLKEDTS CCGGTCAACGAGGCAGACGCCGCGGCCCTCG
FGGNLGRAVFQAKKR AGCACGACAAAGCCTACGACCGGCAGCTCGA
VLEPLGLVEEPVKTAP CAGCGGAGACAACCCGTACCTCAAGTACAAC
GKKRPVEHSPVEPDSS CACGCCGACGCGGAGTTTCAGGAGCGCCTTA
SGTGKAGQQPARKRL AAGAAGATACGTCTTTTGGGGGCAACCTCGG
NFGQTGDADSVPDPQP ACGAGCAGTCTTCCAGGCGAAAAAGAGGGTT
LGQPPAAPSGLGTNTM CTTGAACCTCTGGGCCTGGTTGAGGAACCTGT
ATGSGAPMADNNEGA TAAGACGGCTCCGGGAAAAAAGAGGCCGGTA
DGVGNSSGNWHCDST GAGCACTCTCCTGTGGAGCCAGACTCCTCCTC
WMGDRVITTSTRTWA GGGAACCGGAAAGGCGGGCCAGCAGCCTGCA
LPTYNNHLYKQISSQS AGAAAAAGATTGAATTTTGGTCAGACTGGAG
GASNDNHYFGYSTPW ACGCAGACTCAGTACCTGACCCCCAGCCTCTC
GYFDFNRFHCHFSPRD GGACAGCCACCAGCAGCCCCCTCTGGTCTGGG
WQRLINNNWGFRPKR AACTAATACGATGGCTACAGGCAGTGGCGCA
LNFKLFNIQVKEVTQN CCAATGGCAGACAATAACGAGGGCGCCGACG
DGTTT I ANN LTSTVQV GAGTGGGT A ATTCCTCGGG A A ATTGGCATTGC
FTDSEYQLPYVLGSAH GATTCCACATGGATGGGCGACAGAGTCATCA
QGCLPPFPADVFMVPQ CCACCAGCACCCGAACCTGGGCCCTGCCCACC
YGYLTLNNGSQAVGR TACAACAACCACCTCTACAAACAAATTTCCAG
SSFYCLEYFPS QMLRT CCAATCAGGAGCCTCGAACGACAATCACTACT
GNNFTFSYTFEDVPFH TTGGCTACAGCACCCCTTGGGGGTATTTTGAC
SSYAHS QSLDRLMNPL TTCAACAGATTCCACTGCCACTTTTCACCACG
IDQYLYYLSRTNTPSG TGACTGGCAAAGACTCATCAACAACAACTGG
TTTQSRLQFS QAGASDI GGATTCCGACCCAAGAGACTCAACTTCAAGCT
RDQSRNWLPGPCYRQ CTTTAACATTCAAGTCAAAGAGGTCACGCAGA
QRVSKTSADNNNSEYS ATGACGGTACGACGACGATTGCCAATAACCTT
WTGATKYHLNGRDSL ACCAGCACGGTTCAGGTGTTTACTGACTCGGA
VNPGPAMASHKDDEE GTACCAGCTCCCGTACGTCCTCGGCTCGGCGC
KFFPQSGVLIFGKQGSE ATC A AGG AT GCCTCCCGCCGTTCCC A GC AG AC
KTNVDIEKVMITDEEEI GTCTTCATGGTGCCACAGTATGGATACCTCAC
RTTNPVATEQYGS VS T CCTGAACAAC GGGAGTCAGGCAGTAGGACGC
NLQRGFHNEGKYNRQ TCTTCATTTTACTGCCTGGAGTACTTTCCTTCT
AATADVNTQGVLPGM CAGATGCTGCGTACCGGAAACAACTTTACCTT
VWQDRDVYLQGPIWA CAGCTACACTTTTGAGGACGTTCCTTTCCACA
KIPHTDGHFHPSPLMG GCAGCTACGCTCACAGCCAGAGTCTGGACCGT
GFGLKHPPPQILIKNTP CTCATGAATCCTCTCATCGACCAGTACCTGTA
VPANPS TTFS AAKFAS TTACTTGAGCAGAACAAACACTCCAAGTGGA
FITQYSTGQVSVEIEWE ACCACCACGCAGTCAAGGCTTCAGTTTTCTCA
139
CA 03220810 2023- 11- 29 SUBSTITUTE SHEET (RULE 26)
WO 2022/256557
PCT/US2022/032004
LQKENSKRWNPEIQYT GGCCGGAGCGAGTGACATTCGGGACCAGTCT
SNYNKSVNVDFTVDT AGGAACTGGCTTCCTGGACCCTGTTACCGCCA
NGVYSEPRPIGTRYLT GCAGCGAGTATCAAAGACATCTGCGGATAAC
RNL* (SEQ ID NO: 42) AACAACAGTGAATACTCGTGGACTGGAGCTA
CCAAGTACCACCTCAATGGCAGAGACTCTCTG
GTGAATCCGGGCCCGGCCATGGCAAGCCACA
AGGACGATGAAGAAAAGTTTTTTCCTCAGAGC
GGGGTTCTCATCTTTGGGAAGCAAGGCTCAGA
GAAAACAAATGTGGACATTGAAAAGGTCATG
ATTACAGACGAAGAGGAAATCAGGACAACCA
ATCCCGTGGCTACGGAGCAGTATGGTTCTGTA
TCTACCAACCTCCAGAGAGGCTTCCATAATGA
AGGAAAAT AC AAC AGAC AAGC AGC T ACC GCA
GATGTCAACACACAAGGCGTTCTTCCAGGCAT
GGTCTGGCAGGACAGAGATGTGTACCTTCAG
GGGCCCATCTGGGCAAAGATTCCACACACGG
ACGGACATTTTCACCCCTCTCCCCTCATGGGT
GGATTCGGACTTAAACACCCTCCTCCACAGAT
TCTCATCAAGAACACCCCGGTACCTGCGAATC
CTTCGACCACCTTCAGTGCGGCAAAGTTTGCT
TCCTTCATCACACAGTACTCCACGGGACAGGT
CAGCGTGGAGATCGAGTGGGAGCTGCAGAAG
GAAAACAGCAAACGCTGGAATCCCGAAATTC
AGTACACTTCCAACTACAACAAGTCTGTTAAT
GTGGACTTTACTGTGGACACTAATGGC GTGTA
TTCAGAGCCTCGCCCCATTGGCACCAGATACC
TGACTCGTAATCTGTAA (SEQ ID NO: 87)
VAR-42 MAADGYLPDWLEDTL ATGGCTGCCGATGGTTATCTTCCAGATTGGCT
SEGIRQWWKLKPGPPP CGAGGACACTCTCTCTGAAGGAATAAGACAG
PKPAERHKDDSRGLVL TGGTGGAAGCTCAAACCTGGCCCACCACCACC
PGYKYLGPFNGLDKGE AAAGCCCGCAGAGCGGCATAAGGACGACAGC
PVNEADAAALEHDKA AGGGGTCTTGTGCTTCCTGGGTACAAGTACCT
YDRQLDSGDNPYLKY CGGACCCTTCAACGGACTCGACAAGGGAGAG
NHADAEFQPRLKEDTS CCGGTCAACGAGGCAGACGCCGCGGCCCTCG
FGGNLGRAVFQAKKR AGCACGACAAAGCCTACGACCGGCAGCTCGA
VLEPLGLVEEPVKTAP CAGCGGAGACAACCCGTACCTCAAGTACAAC
GKKRPVEHSPVEPDS S CACGCCG AC GC GG AGTTTC AGG AGCGCCTTA
S GTGKAGQQPARKRL AAGAAGATACGTCTTTTGGGGGCAACCTCGG
NFGQTGDADSVPDPQP ACGAGCAGTCTTCCAGGCGAAAAAGAGGGTT
LGQPPAAPS GLGTNTM CTTGAACCTCTGGGCCTGGTTGAGGAACCTGT
ATGS GAPMADNNEGA TAAGACGGCTCCGGGAAAAAAGAGGCCGGTA
DGVGNS SGNWHCDST GAGCACTCTCCTGTGGAGCCAGACTCCTCCTC
WMGDRVITTSTRTWA GGGAACCGGAAAGGCGGGCCAGCAGCCTGCA
LPTYNNHLYKQIS S QS AGAAAAAGATTGAATTTTGGTCAGACTGGAG
GASNDNHYFGYSTPW ACGCAGACTCAGTACCTGACCCCCAGCCTCTC
GYFDFNRFHCHFSPRD GGACAGCCACCAGCAGCCCCCTCTGGTCTGGG
140
CA 03220810 2023- 11- 29 SUBSTITUTE SHEET (RULE 26)
WO 2022/256557
PCT/US2022/032004
WQRLINNNWGFRPKR AACTAATACGATGGCTACAGGCAGTGGCGCA
LNFKLFNIQVKEVTQN CCAATGGCAGACAATAACGAGGGCGCCGACG
DGTTTIANNLTSTVQV GAGTGGGTAATTCCTCGGGAAATTGGCATTGC
FTDSEYQLPYVLGSAH GATTCCACATGGATGGGCGACAGAGTCATCA
QGCLPPFPADVFMVPQ CCACCAGCACCCGAACCTGGGCCCTGCCCACC
YGYLTLNNGSQAVGR TACAACAACCACCTCTACAAACAAATTTCCAG
SSFYCLEYFPSQMLRT CCAATCAGGAGCCTCGAACGACAATCACTACT
GNNFTFSYTFEDVPFH TTGGCTACAGCACCCCTTGGGGGTATTTTGAC
SSYAHSQSLDRLMNPL TTCAACAGATTCCACTGCCACTTTTCACCACG
IDQYLYYLSRTNTPSG TGACTGGCAAAGACTCATCAACAACAACTGG
TTTQSRLQFSQAGASDI GGATTCCGACCCAAGAGACTCAACTTCAAGCT
RDQSRNWLPGPCYRQ CTTTAACATTCAAGTCAAAGAGGTCACGCAGA
QRVSKTSADNNNSEYS ATGACGGTACGACGACGATTGCCAATAACCTT
WTGATKYHLNGRDSL ACCAGCACGGTTCAGGTGTTTACTGACTCGGA
VNPGPAMASHKDDEE GTACCAGCTCCCGTACGTCCTCGGCTCGGCGC
KFFPQSGVLIFGKQGSE ATCAAGGATGCCTCCCGCCGTTCCCAGCAGAC
KTNVDIEKVMITDEEEI GTCTTCATGGTGCCACAGTATGGATACCTCAC
RTTNPVATEQYGS VS T CCTGAACAACGGGAGTCAGGCAGTAGGACGC
NLQRGNRQAAGTADV TCTTCATTTTACTGCCTGGAGTACTTTCCTTCT
NTQGVLPGMVWQDR CAGATGCTGCGTACCGGAAACAACTTTACCTT
DVYLQGPIWAKIPHTD CAGCTACACTTTTGAGGACGTTCCTTTCCACA
GHFHPSPLMGGFGLKH GCAGCTACGCTCACAGCCAGAGTCTGGACCGT
PPPQILIKNTPVPANPST CTCATGAATCCTCTCATCGACCAGTACCTGTA
TFSAAKFASFITQYSTG TTACTTGAGCAGAACAAACACTCCAAGTGGA
QVSVEIEWELQKENSK ACCACCACGCAGTCAAGGCTTCAGTTTTCTCA
RWNPEIQYTSN YNKS V GGCCGGAGCGAGTGACATTCGGGACCAGTCT
NVDFTVDTNGVYSEPR AGGAACTGGCTTCCTGGACCCTGTTACCGCCA
PIGTRYLTRNL* (SEQ GCAGCGAGTATCAAAGACATCTGCGGATAAC
ID NO: 43)
AACAACAGTGAATACTCGTGGACTGGAGCTA
CCAAGTACCACCTCAATGGCAGAGACTCTCTG
GTGAATCCGGGCCCGGCCATGGCAAGCCACA
AGGACGATGAAGAAAAGTTTTTTCCTCAGAGC
GGGGTTCTCATCTTTGGGAAGCAAGGCTCAGA
GAAAACAAATGTGGACATTGAAAAGGTCATG
ATTACAGACGAAGAGGAAATCAGGACAACCA
ATCCCGTGGCTACGGAGCAGTATGGTTCTGTA
TCTACCAACCTCCAGAGAGGCAACAGACAAG
CAGCTGGGACCGCAGATGTCAACACACAAGG
CGTTCTTCCAGGCATGGTCTGGCAGGACAGAG
ATGTGTACCTTCAGGGGCCCATCTGGGCAAAG
ATTCCACACACGGACGGACATTTTCACCCCTC
TCCCCTCATGGGTGGATTCGGACTTAAACACC
CTCCTCCACAGATTCTCATCAAGAACACCCCG
GTACCTGCGAATCCTTCGACCACCTTCAGTGC
GGCAAAGTTTGCTTCCTTCATCACACAGTACT
CCACGGGACAGGTCAGCGTGGAGATCGAGTG
141
CA 03220810 2023- 11- 29 SUBSTITUTE SHEET (RULE 26)
WO 2022/256557
PCT/US2022/032004
GGAGCTGCAGAAGGAAAACAGCAAACGCTGG
AATCCCGAAATTCAGTACACTTCCAACTACAA
CAAGTCTGTTAATGTGGACTTTACTGTGGACA
CTAATGGCGTGTATTCAGAGCCTCGCCCCATT
GGCACCAGATACCTGACTCGTAATCTGTAA
(SEQ ID NO: 88)
VAR-43 MAADGYLPDWLEDTL ATGGCTGCCGATGGTTATCTTCCAGATTGGCT
SEGIRQWWKLKPGPPP CGAGGACACTCTCTCTGAAGGAATAAGACAG
PKPAERHKDDSRGLVL TGGTGGAAGCTCAAACCTGGCCCACCACCACC
PGYKYLGPFNGLDKGE AAAGCCCGCAGAGCGGCATAAGGACGACAGC
PVNEADAAALEHDKA AGGGGTCTTGTGCTTCCTGGGTACAAGTACCT
YDRQLDSGDNPYLKY CGGACCCTTCAACGGACTCGACAAGGGAGAG
NHADAEFQERLKEDTS CCGGTCAACGAGGCAGACGCCGCGGCCCTCG
FGGNLGRAVFQAKKR AGCACGACAAAGCCTACGACCGGCAGCTCGA
VLEPLGLVEEPVKTAP CAGCGGAGACAACCCGTACCTCAAGTACAAC
GKKRPVEHSPVEPDSS CACGCCGACGCGGAGTTTCAGGAGCGCCTTA
SGTGKAGQQPARKRL AAGAAGATACGTCTTTTGGGGGCAACCTCGG
NFGQTGDADSVPDPQP ACGAGCAGTCTTCCAGGCGAAAAAGAGGGTT
LGQPPAAPSGLGTNTM CTTGAACCTCTGGGCCTGGTTGAGGAACCTGT
ATGSGAPMADNNEGA TAAGACGGCTCCGGGAAAAAAGAGGCCGGTA
DGVGNSSGNWHCDST GAGCACTCTCCTGTGGAGCCAGACTCCTCCTC
W MGDRVITTSTRTWA GGGA ACCGGA A AGGCGGGCC AGC AGCCTGC A
LPTYNNHLYKQISSQS AGAAAAAGATTGAATTTTGGTCAGACTGGAG
GAS NDNH YFGYSTPW ACGC AG ACTC AGT ACCTGACCCCCAGCCTCTC
GYFDFNRFHCHFSPRD GGACAGCCACCAGCAGCCCCCTCTGGTCTGGG
WQRLINNNWGFRPKR AACTAATACGATGGCTACAGGCAGTGGCGCA
LNFKLFNIQVKEVTQN CCAATGGCAGACAATAACGAGGGCGCCGACG
DGTTTIANNLTSTVQV GAGTGGGTAATTCCTCGGGAAATTGGCATTGC
FTDSEYQLPYVLGSAH GATTCCACATGGATGGGCGACAGAGTCATCA
QGCLPPFPADVFMVPQ CCACCAGCACCCGAACCTGGGCCCTGCCCACC
YGYLTLNNGSQAVGR TACAACAACCACCTCTACAAACAAATTTCCAG
SSFYCLEYFPSQMLRT CCAATCAGGAGCCTCGAACGACAATCACTACT
GNNFTFSYTFEDVPFH TTGGCTACAGCACCCCTTGGGGGTATTTTGAC
SSYAHS QSLDRLMNPL TTCAACAGATTCCACTGCCACTTTTCACCACG
IDQYLYYLSRTNTPSG TGACTGGCAAAGACTCATCAACAACAACTGG
TTTQSRLQFSQAGASDI GGATTCCGACCCAAGAGACTCAACTTCAAGCT
RDQSRNWLPGPCYRQ CTTT A AC ATTCA AGTC A A AGAGGTC ACGC AGA
QRVSKTSADNNNSEYS ATGACGGTACGACGACGATTGCCAATAACCTT
WTGATKYHLNGRDSL ACCAGCACGGTTCAGGTGTTTACTGACTCGGA
VNPGPAMASHKDDEE GTACCAGCTCCCGTACGTCCTCGGCTCGGCGC
KFFPQSGVLIFGKQGSE ATCAAGGATGCCTCCCGCCGTTCCCAGCAGAC
KTNVDIEKVMITDEEEI GTCTTCATGGTGCCACAGTATGGATACCTCAC
RTTNPVATEQYGS VS T CCTGAACAACGGGAGTCAGGCAGTAGGACGC
NLQRGNRQPWEPDKQ TCTTCATTTTACTGCCTGGAGTACTTTCCTTCT
AATADVNTQGVLPGM CAGATGCTGCGTACCGGAAACAACTTTACCTT
VWQDRDVYLQGPIWA CAGCTACACTTTTGAGGACGTTCCTTTCCACA
142
CA 03220810 2023- 11- 29 SUBSTITUTE SHEET (RULE 26)
WO 2022/256557
PCT/US2022/032004
KIPHTDGHFHPSPLMG GCAGCTACGCTCACAGCCAGAGTCTGGACCGT
GFGLKHPPPQILIKNTP CTCATGAATCCTCTCATCGACCAGTACCTGTA
VPANPS TTFS AAKFAS TTACTTGAGCAGAACAAACACTCCAAGTGGA
FITQYSTGQVSVEIEWE ACCACCACGCAGTCAAGGCTTCAGTTTTCTCA
LQKENSKRWNPEIQYT GGCCGGAGCGAGTGACATTCGGGACCAGTCT
SNYNKSVNVDFTVDT AGGAACTGGCTTCCTGGACCCTGTTACCGCCA
NGVYSEPRPIGTRYLT GCAGCGAGTATCAAAGACATCTGCGGATAAC
RNL* (SEQ ID NO: 44) AACAACAGTGAATACTCGTGGACTGGAGCTA
CCAAGTACCACCTCAATGGCAGAGACTCTCTG
GTGAATCCGGGCCCGGCCATGGCAAGCCACA
AGGACGATGAAGAAAAGTTTTTTCCTCAGAGC
GGGGTTCTCATCTTTGGGAAGCAAGGCTCAGA
GAAAACAAATGTGGACATTGAAAAGGTCATG
ATTACAGACGAAGAGGAAATCAGGACAACCA
ATCCCGTGGCTACGGAGCAGTATGGTTCTGTA
TCTACCAACCTCCAGAGAGGCAACAGACAAC
CGTGGGAACCGGATAAGCAAGCAGCTACCGC
AGATGTCAACACACAAGGCGTTCTTCCAGGCA
TGGTCTGGCAGGACAGAGATGTGTACCTTCAG
GGGCCCATCTGGGCAAAGATTCCACACACGG
ACGGACATTTTCACCCCTCTCCCCTCATGGGT
GGATTCGGACTTAAACACCCTCCTCCACAGAT
TCTCATCAAGAACACCCCGGTACCTGCGAATC
CTTCGACCACCTTCAGTGCGGCAAAGTTTGCT
TCCTTCATCACACAGTACTCCACGGGACAGGT
CAGCGTGGAGATCGAGTGGGAGCTGCAGAAG
GAAAACAGCAAACGCTGGAATCCCGAAATTC
AGTACACTTCCAACTACAACAAGTCTGTTAAT
GTGGACTTTACTGTGGACACTAATGGCGTGTA
TTCAGAGCCTCGCCCCATTGGCACCAGATACC
TGACTCGTAATCTGTAA (SEQ ID NO: 89)
VAR-44 MAADGYLPDWLEDTL ATGGCTGCCGATGGTTATCTTCCAGATTGGCT
SEGIRQWWKLKPGPPP CGAGGACACTCTCTCTGAAGGAATAAGACAG
PKPAERHKDDSRGLVL TGGTGGAAGCTCAAACCTGGCCCACCACCACC
PGYKYLGPFNGLDKGE AAAGCCCGCAGAGCGGCATAAGGACGACAGC
PVNEADAAALEHDKA AGGGGTCTTGTGCTTCCTGGGTACAAGTACCT
YDRQLDSGDNPYLKY CGGACCCTTCAACGGACTCGACAAGGGAGAG
NHADAEFQERLKEDTS CCGGTCAACGAGGCAGACGCCGCGGCCCTCG
FGGNLGRAVFQAKKR AGCACGACAAAGCCTACGACCGGCAGCTCGA
VLEPLGLVEEPVKTAP CAGCGGAGACAACCCGTACCTCAAGTACAAC
GKKRPVEHSPVEPDSS CACGCCGACGCGGAGTTTCAGGAGCGCCTTA
SGTGKAGQQPARKRL AAGAAGATACGTCTTTTGGGGGCAACCTCGG
NFGQTGDADSVPDPQP ACGAGCAGTCTTCCAGGCGAAAAAGAGGGTT
LGQPPAAPSGLGTNTM CTTGAACCTCTGGGCCTGGTTGAGGAACCTGT
ATGSGAPMADNNEGA TAAGACGGCTCCGGGAAAAAAGAGGCCGGTA
DGVGNSSGNWHCDST GAGCACTCTCCTGTGGAGCCAGACTCCTCCTC
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WMGDRVITTSTRTWA GGGAACCGGAAAGGCGGGCCAGCAGCCTGCA
LPTYNNHLYKQIS S QS AGAAAAAGATTGAATTTTGGTCAGACTGGAG
GASNDNHYFGYSTPW ACGCAGACTCAGTACCTGACCCCCAGCCTCTC
GYFDFNRFHCHFSPRD GGACAGCCACCAGCAGCCCCCTCTGGTCTGGG
WQRLINNNWGFRPKR AACTAATACG AT G GCTACAGGCAGTGGC GCA
LNFKLFNIQVKEVTQN CCAATGGCAGACAATAACGAGGGCGCCGACG
DGTTTIANNLTSTVQV GAGTGGGTAATTCCTCGGGAAATTGGCATTGC
FTDSEYQLPYVLGS AH GATTCCACATGGATGGGCGACAGAGTCATCA
QGCLPPFPADVFMVPQ CCACCAGCACCCGAACCTGGGCCCTGCCCACC
YGYLTLNNGS QAVGR TAC AAC AAC C AC C TCT AC AAAC AAATTTCCAG
S SFYCLEYFPS QMLRT CCAATCAGGAGCCTCGAACGACAATCACTACT
GNNFTFSYTFEDVPFH TTGGCTACAGCACCCCTTGGGGGTATTTTGAC
SSYAHS QSLDRLMNPL TTCAACAGATTCCACTGCCACTTTTCACCACG
IDQYLYYLSRTNTPSG TGACTGGCAAAGACTCATCAACAACAACTGG
TTTQS RLQFS QAGASDI GGATTCCGACCCAAGAGACTCAACTTCAAGCT
RDQSRNWLPGPCYRQ CTTTAACATTCAAGTCAAAGAGGTCACGCAGA
QRVSKTS ADNNNSEYS ATGACGGTACGACGACGATTGCCAATAACCTT
WTGATKYHLNGRDSL ACCAGCACGGTTCAGGTGTTTACTGACTCGGA
VNPGPAMASHKDDEE GTACCAGCTCCCGTACGTCCTCGGCTCGGC GC
KFFPQSGVLIFGKQGSE ATCAAGGATGCCTCCCGCCGTTCCCAGCAGAC
KTNVDIEKVMITDEEEI GTCTTCATGGTGCCACAGTATGGATACCTCAC
RTTNPVATEQYGS VS T CCTGAACAAC GGGAGTCAGGCAGTAGGAC GC
NLQRGNRQ A AT AL AL TCTTCATTTT ACTGCCTGGAGT ACTTTCCTTCT
STTNADVNTQGVLPG CAGATGCTGCGTACCGGAAACAACTTTACCTT
MVWQDRDVYLQGPIW CAGCTACACTTTTGAGGACGTTCCTTTCCACA
AKIPHTDGHFHPSPLM GCAGCTACGCTCACAGCCAGAGTCTGGACCGT
GGFGLKHPPPQILIKNT CTCATGAATCCTCTCATCGACCAGTACCTGTA
PVPANPSTTFSAAKFA TTACTTGAGCAGAACAAACACTCCAAGTGGA
SFITQYSTGQVSVEIEW ACCACCACGCAGTCAAGGCTTCAGTTTTCTCA
ELQKENSKRWNPEIQY GGCCGGAGCGAGTGACATTCGGGACCAGTCT
TSNYNKSVNVDFTVDT AGGAACTGGCTTCCTGGACCCTGTTACCGCCA
NGVYSEPRPIGTRYLT GCAGCGAGTATCAAAGACATCTGCGGATAAC
RNL* (SEQ ID NO: 45) AACAACAGTGAATACTCGTGGACTGGAGCTA
CCAAGTACCACCTCAATGGCAGAGACTCTCTG
GTGAATCCGGGCCCGGCCATGGCAAGCCACA
AGGACGATGAAGAAAAGTTTTTTCCTCAGAGC
GGGGTTCTCATCTTTGGGAAGCAAGGCTCAGA
GAAAACAAATGTGGACATTGAAAAGGTCATG
ATTACAGACGAAGAGGAAATCAGGACAACCA
ATCCCGTGGCTACGGAGCAGTATGGTTCTGTA
TCTACCAACCTCCAGAGAGGCAACAGACAAG
CAGCTACCGCACTGGCGCTTAGTACAACTAAT
GCAGATGTCAACACACAAGGCGTTCTTCCAGG
CATGGTCTGGCAGGACAGAGATGTGTACCTTC
AGGGGCCCATCTGGGCAAAGATTCCACACAC
GGACGGAC ATTTTC AC CCCTCTCCCCTC AT GG
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GTGGATTCGGACTTAAACACCCTCCTCCACAG
ATTCTCATCAAGAACACCCCGGTACCTGCGAA
TCCTTCGACCACCTTCAGTGCGGCAAAGTTTG
CTTCCTTCATCACACAGTACTCCACGGGACAG
GTCAGCGTGGAGATCGAGTGGGAGCTGCAGA
AGGAAAACAGCAAACGCTGGAATCCCGAAAT
TCAGTACACTTCCAACTACAACAAGTCTGTTA
ATGTGGACTTTACTGTGGACACTAATGGCGTG
TATTCAGAGCCTCGCCCCATTGGCACCAGATA
CCTGACTCGTAATCTGTAA (SEQ ID NO: 901)
VAR-45 MAADGYLPDWLEDTL ATGGCTGCCGATGGTTATCTTCCAGATTGGCT
SEGIRQWWKLKPGPPP CGAGGACACTCTCTCTGAAGGAATAAGACAG
PKPAERHKDDSRGLVL TGGTGGAAGCTCAAACCTGGCCCACCACCACC
PGYKYLGPFNGLDKGE AAAGCCCGCAGAGCGGCATAAGGACGACAGC
PVNEADAAALEHDKA AGGGGTCTTGTGCTTCCTGGGTACAAGTACCT
YDRQLDSGDNPYLKY CGGACCCTTCAACGGACTCGACAAGGGAGAG
NHADAEFQERLKEDTS CCGGTCAACGAGGCAGACGCCGCGGCCCTCG
FGGNLGRAVFQAKKR AGCACGACAAAGCCTACGACCGGCAGCTCGA
VLEPLGLVEEPVKTAP CAGCGGAGACAACCCGTACCTCAAGTACAAC
GKKRPVEHSPVEPDSS CACGCCGACGCGGAGTTTCAGGAGCGCCTTA
SGTGKAGQQPARKRL AAGAAGATACGTCTTTTGGGGGCAACCTCGG
NFGQTGD ADS VPDPQP ACGAGC AGTCTTCC AGGCGA A A A AGA GGGTT
LGQPPAAPSGLGTNTM CTTGAACCTCTGGGCCTGGTTGAGGAACCTGT
ATGSGAPMADNNEGA TAAGACGGCTCCGGGAAAAAAGAGGCCGGTA
DGVGNSSGNWHCDST GAGCACTCTCCTGTGGAGCCAGACTCCTCCTC
WMGDRVITTSTRTWA GGGAACCGGAAAGGCGGGCCAGCAGCCTGCA
LPT YNNHLYKQIS S QS AGAAAAAGATTGAATTTTGGTCAGACTGGAG
GASNDNHYFGYSTPW ACGCAGACTCAGTACCTGACCCCCAGCCTCTC
GYFDFNRFHCHFSPRD GGACAGCCACCAGCAGCCCCCTCTGGTCTGGG
WQRLINNNWGFRPKR AACTAATACGATGGCTACAGGCAGTGGCGCA
LNEKLENIQVKEVTQN CCAATGGCAGACAATAACGAGGGCGCCGACG
DGTTTIANNLTSTVQV GAGTGGGTAATTCCTCGGGAAATTGGCATTGC
FTDSEYQLPYVLGS AH GATTCCACATGGATGGGCGACAGAGTCATCA
QGCLPPFPADVFMVPQ CCACCAGCACCCGAACCTGGGCCCTGCCCACC
YGYLTLNNGSQAVGR TACAACAACC ACCTCT AC AAACAAATTTCCAG
SSFYCLEYFPSQMLRT CCAATCAGGAGCCTCGAACGACAATCACTACT
GNNFTESYTFEDVPFH TTGGCT AC AGC ACCCCTTGGGGGT ATTTTGAC
SSYAHS QSLDRLMNPL TTCAACAGATTCCACTGCCACTTTTCACCACG
IDQYLYYLSRTNTPSG TGACTGGCAAAGACTCATCAACAACAACTGG
TTTQSRLQFSQAGASDI GGATTCCGACCCAAGAGACTCAACTTCAAGCT
RDQSRNWLPGPCYRQ CTTTAACATTCAAGTCAAAGAGGTCACGCAGA
QRVSKTS ADNNNSEYS ATGACGGTACGACGACGATTGCCAATAACCTT
WTGATKYHLNGRDSL ACCAGCACGGTTCAGGTGTTTACTGACTCGGA
VNPGPAMASHKDDEE GTACCAGCTCCCGTACGTCCTCGGCTCGGC GC
KFFPQSGVLIFGKQGSE ATCAAGGATGCCTCCCGCCGTTCCCAGCAGAC
KTNVDIEKVMITDEEEI GTCTTCATGGTGCCACAGTATGGATACCTCAC
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RTTNPVATEQYGSVST CCTGAACAACGGGAGTCAGGCAGTAGGACGC
NLQRPWGTAGGNRQA TCTTCATTTTACTGCCTGGAGTACTTTCCTTCT
ATADVNTQGVLPGMV CAGATGCTGCGTACCGGAAACAACTTTACCTT
WQDRDVYLQGPIWAK CAGCTACACTTTTGAGGACGTTCCTTTCCACA
IPHTDGHFHPSPLMGG GCAGCTACGCTCACAGCCAGAGTCTGGACCGT
FGLKHPPPQILIICNTPV CTCATGAATCCTCTCATCGACCAGTACCTGTA
PANPSTTFSAAKFASFI TTACTTGAGCAGAACAAACACTCCAAGTGGA
TQYSTGQVSVEIEWEL ACCACCACGCAGTCAAGGCTTCAGTTTTCTCA
QKENSICRWNPEIQYTS GGCCGGAGCGAGTGACATTCGGGACCAGTCT
NYNKSVNVDFTVDTN AGGAACTGGCTTCCTGGACCCTGTTACCGCCA
GVYSEPRPIGTRYLTR GCAGCGAGTATCAAAGACATCTGCGGATAAC
NL* (SEQ ID NO: 46)
AACAACAGTGAATACTCGTGGACTGGAGCTA
CCAAGTACCACCTCAATGGCAGAGACTCTCTG
GTGAATCCGGGCCCGGCCATGGCAAGCCACA
AGGACGATGAAGAAAAGTTTTTTCCTCAGAGC
GGGGTTCTCATCTTTGGGAAGCAAGGCTCAGA
GAAAACAAATGTGGACATTGAAAAGGTCATG
ATTACAGACGAAGAGGAAATCAGGACAACCA
ATCCCGTGGCTACGGAGCAGTATGGTTCTGTA
TCTACCAACCTCCAGCGGCCTTGGGGCACAGC
TGGAGGCAACAGACAAGCAGCTACCGCAGAT
GTCAACACACAAGGCGTTCTTCCAGGCATGGT
CTGGCAGGACAGAGATGTGTACCTTCAGGGG
CCCATCTGGGCAAAGATTCCACACACGGACG
GACATTTTCACCCCTCTCCCCTCATGGGTGGA
TTCGGACTTAAACACCCTCCTCCACAGATTCT
CATCAAGAACACCCCGGTACCTGCGAATCCTT
CGACCACCTTCAGTGCGGCAAAGTTTGCTTCC
TTCATCACACAGTACTCCACGGGACAGGTCAG
CGTGGAGATCGAGTGGGAGCTGCAGAAGGAA
AACAGCAAACGCTGGAATCCCGAAATTCAGT
ACACTTCCAACTACAACAAGTCTGTTAATGTG
GACTTTACTGTGGACACTAATGGCGTGTATTC
AGAGCCTCGCCCCATTGGCACCAGATACCTGA
CTCGTAATCTGTAA (SEQ ID NO: 91)
In some embodiments, the nucleic acid molecule encodes a capsid polypeptide
that has at
least 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99%, or 100% identity to a VP1,
VP2, or VP3
sequence as provided in Table 2. In some embodiments, the nucleic acid
molecule encodes a
capsid polypeptide that has at least 85, 90, 91, 92, 93, 94, 95, 96, 97, 98,
99%, or 100% identity
to a VP1, VP2, or VP3 sequence as provided in Table 3. In some embodiments,
the nucleic acid
molecule encodes a capsid polypeptide that has at least 85, 90, 91, 92, 93,
94, 95, 96, 97, 98,
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99%. or 100% identity to a VP1, VP2, or VP3 sequence as provided in Table 4.
In some
embodiments, the nucleic acid molecule encodes a capsid polypeptide that has
at least 85, 90, 91,
92, 93, 94, 95, 96, 97, 98, 99%, or 100% identity to a VP1, VP2, or VP3
sequence of SEQ ID
NO: 2. In some embodiments, the nucleic acid molecule encodes a capsid
polypeptide that has at
least 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99%, or 100% identity to a VP1,
VP2, or VP3
sequence of SEQ ID NO: 3. In some embodiments, the nucleic acid molecule
encodes a capsid
polypeptide that has at least 85, 90, 91, 92, 93, 94, 95, 96, 97, 98. 99%, or
100% identity to a
VP1, VP2, or VP3 sequence of SEQ ID NO: 4. In some embodiments, the nucleic
acid molecule
encodes a capsid polypeptide that has at least 85, 90, 91, 92, 93, 94, 95, 96,
97, 98, 99%, or
100% identity to a VP1, VP2, or VP3 sequence of SEQ ID NO: 5. In some
embodiments, the
nucleic acid molecule encodes a capsid polypeptide that has at least 85, 90,
91, 92, 93, 94, 95,
96, 97, 98, 99%, or 100% identity to a VP1, VP2, or VP3 sequence of SEQ ID NO:
6. In some
embodiments, the nucleic acid molecule encodes a capsid polypeptide that has
at least 85, 90, 91,
92, 93, 94, 95, 96, 97, 98, 99%, or 100% identity to a VP1, VP2, or VP3
sequence of SEQ ID
NO: 2. In some embodiments, the nucleic acid molecule encodes a capsid
polypeptide that has at
least 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99%, or 100% identity to a VP1,
VP2, or VP3
sequence of SEQ ID NO: 7. In some embodiments, the nucleic acid molecule
encodes a capsid
polypeptide that has at least 85, 90, 91, 92, 93, 94, 95, 96, 97, 98. 99%, or
100% identity to a
VP1, VP2, or VP3 sequence of SEQ ID NO: 8. In some embodiments, the nucleic
acid molecule
encodes a capsid polypeptide that has at least 85, 90, 91, 92, 93, 94, 95, 96,
97, 98, 99%, or
100% identity to a VP1, VP2, or VP3 sequence of SEQ ID NO: 9. In some
embodiments, the
nucleic acid molecule encodes a capsid polypeptide that has at least 85, 90,
91, 92, 93, 94, 95,
96, 97, 98, 99%, or 100% identity to a VP1, VP2, or VP3 sequence of SEQ ID NO:
10. In some
embodiments, the nucleic acid molecule encodes a capsid polypeptide that has
at least 85, 90, 91,
92, 93, 94, 95, 96, 97, 98, 99%, or 100% identity to a VP1, VP2, or VP3
sequence of SEQ ID
NO: 11. In some embodiments, the nucleic acid molecule encodes a capsid
polypeptide that has
at least 85, 90, 91, 92, 93, 94. 95, 96, 97, 98, 99%, or 100% identity to a
VP1, VP2, or VP3
sequence of SEQ ID NO: 12. In some embodiments, the nucleic acid molecule
encodes a capsid
polypeptide that has at least 85, 90, 91, 92, 93, 94, 95, 96, 97, 98. 99%, or
100% identity to a
VP1, VP2, or VP3 sequence of SEQ ID NO: 13. In some embodiments, the nucleic
acid
molecule encodes a capsid polypeptide that has at least 85, 90, 91, 92, 93,
94, 95, 96, 97, 98,
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99%. or 100% identity to a VP1, VP2, or VP3 sequence of SEQ ID NO: 14. In some
embodiments, the nucleic acid molecule encodes a capsid polypeptide that has
at least 85, 90, 91,
92, 93, 94, 95, 96, 97, 98, 99%, or 100% identity to a VP1, VP2, or VP3
sequence of SEQ ID
NO: 15. In some embodiments, the nucleic acid molecule encodes a capsid
polypeptide that has
at least 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99%, or 100% identity to a
VP1, VP2, or VP3
sequence of SEQ ID NO: 16. In some embodiments, the nucleic acid molecule
encodes a capsid
polypeptide that has at least 85, 90, 91, 92, 93, 94, 95, 96, 97, 98. 99%, or
100% identity to a
VP1, VP2, or VP3 sequence of SEQ ID NO: 17. In some embodiments, the nucleic
acid
molecule encodes a capsid polypeptide that has at least 85, 90, 91, 92, 93,
94, 95, 96, 97, 98,
99%. or 100% identity to a VP1, VP2, or VP3 sequence of SEQ ID NO: 18. In some
embodiments, the nucleic acid molecule encodes a capsid polypeptide that has
at least 85, 90, 91,
92, 93, 94, 95. 96, 97, 98, 99%, or 100% identity to a VP1, VP2, or VP3
sequence of SEQ ID
NO: 19. In some embodiments, the nucleic acid molecule encodes a capsid
polypeptide that has
at least 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99%, or 100% identity to a
VP1, VP2, or VP3
sequence of SEQ ID NO: 20. In some embodiments, the nucleic acid molecule
encodes a capsid
polypeptide that has at least 85, 90, 91, 92, 93, 94, 95, 96, 97, 98. 99%, or
100% identity to a
VP1, VP2, or VP3 sequence of SEQ ID NO: 21. In some embodiments, the nucleic
acid
molecule encodes a capsid polypeptide that has at least 85, 90, 91, 92, 93,
94, 95, 96, 97, 98,
99%. or 100% identity to a VP1, VP2, or VP3 sequence of SEQ ID NO: 22. In some
embodiments, the nucleic acid molecule encodes a capsid polypeptide that has
at least 85, 90, 91,
92, 93, 94, 95, 96, 97, 98, 99%, or 100% identity to a VP1, VP2, or VP3
sequence of SEQ ID
NO: 23. In some embodiments, the nucleic acid molecule encodes a capsid
polypeptide that has
at least 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99%, or 100% identity to a
VP1, VP2, or VP3
sequence of SEQ ID NO: 24. In some embodiments, the nucleic acid molecule
encodes a capsid
polypeptide that has at least 85, 90, 91, 92, 93, 94, 95, 96, 97, 98. 99%, or
100% identity to a
VP1, VP2, or VP3 sequence of SEQ ID NO: 25. In some embodiments, the nucleic
acid
molecule encodes a capsid polypeptide that has at least 85, 90, 91, 92, 93,
94, 95, 96, 97, 98,
99%. or 100% identity to a VP1, VP2, or VP3 sequence of SEQ ID NO: 26. In some
embodiments, the nucleic acid molecule encodes a capsid polypeptide that has
at least 85, 90, 91,
92, 93, 94, 95, 96, 97, 98, 99%, or 100% identity to a VP1, VP2, or VP3
sequence of SEQ ID
NO: 27. In some embodiments, the nucleic acid molecule encodes a capsid
polypeptide that has
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at least 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99%, or 100% identity to a
VP1, VP2, or VP3
sequence of SEQ ID NO: 28. In some embodiments, the nucleic acid molecule
encodes a capsid
polypeptide that has at least 85, 90, 91, 92, 93, 94, 95, 96, 97, 98. 99%, or
100% identity to a
VP1, VP2, or VP3 sequence of SEQ ID NO: 29. In some embodiments, the nucleic
acid
molecule encodes a capsid polypeptide that has at least 85, 90, 91, 92, 93,
94, 95, 96, 97, 98,
99%. or 100% identity to a VP1, VP2, or VP3 sequence of SEQ ID NO: 30. In some
embodiments, the nucleic acid molecule encodes a capsid polypeptide that has
at least 85, 90, 91,
92, 93, 94, 95, 96, 97, 98, 99%, or 100% identity to a VP1, VP2, or VP3
sequence of SEQ ID
NO: 31. In some embodiments, the nucleic acid molecule encodes a capsid
polypeptide that has
at least 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99%, or 100% identity to a
VP1, VP2, or VP3
sequence of SEQ ID NO: 32. In some embodiments, the nucleic acid molecule
encodes a capsid
polypeptide that has at least 85, 90, 91, 92, 93, 94, 95, 96. 97, 98. 99%, or
100% identity to a
VP1, VP2, or VP3 sequence of SEQ ID NO: 33. In some embodiments, the nucleic
acid
molecule encodes a capsid polypeptide that has at least 85, 90, 91, 92, 93,
94, 95, 96, 97, 98,
99%. or 100% identity to a VP1, VP2, or VP3 sequence of SEQ ID NO: 34. In some
embodiments, the nucleic acid molecule encodes a capsid polypeptide that has
at least 85, 90, 91,
92, 93, 94, 95, 96, 97, 98, 99%, or 100% identity to a VP1, VP2, or VP3
sequence of SEQ ID
NO: 35. In some embodiments, the nucleic acid molecule encodes a capsid
polypeptide that has
at least 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99%, or 100% identity to a
VP1, VP2, or VP3
sequence of SEQ ID NO: 36. In some embodiments, the nucleic acid molecule
encodes a capsid
polypeptide that has at least 85, 90, 91, 92, 93, 94, 95, 96, 97, 98. 99%, or
100% identity to a
VP1, VP2, or VP3 sequence of SEQ ID NO: 37. In some embodiments, the nucleic
acid
molecule encodes a capsid polypeptide that has at least 85, 90, 91, 92, 93.
94, 95, 96, 97, 98,
99%. or 100% identity to a VP1, VP2, or VP3 sequence of SEQ ID NO: 38. In some
embodiments, the nucleic acid molecule encodes a capsid polypeptide that has
at least 85, 90, 91,
92, 93, 94, 95, 96, 97, 98, 99%, or 100% identity to a VP1, VP2, or VP3
sequence of SEQ ID
NO: 39. In some embodiments, the nucleic acid molecule encodes a capsid
polypeptide that has
at least 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99%, or 100% identity to a
VP1, VP2, or VP3
sequence of SEQ ID NO: 40. In some embodiments, the nucleic acid molecule
encodes a capsid
polypeptide that has at least 85, 90, 91, 92, 93, 94, 95, 96, 97, 98. 99%, or
100% identity to a
VP1, VP2, or VP3 sequence of SEQ ID NO: 41. In some embodiments, the nucleic
acid
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molecule encodes a capsid polypeptide that has at least 85, 90, 91, 92, 93,
94, 95, 96, 97, 98,
99%. or 100% identity to a VP1, VP2, or VP3 sequence of SEQ ID NO: 42. In some
embodiments, the nucleic acid molecule encodes a capsid polypeptide that has
at least 85, 90, 91,
92, 93, 94, 95, 96, 97, 98, 99%, or 100% identity to a VP1, VP2, or VP3
sequence of SEQ ID
NO: 43. In some embodiments, the nucleic acid molecule encodes a capsid
polypeptide that has
at least 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99%, or 100% identity to a
VP1, VP2, or VP3
sequence of SEQ ID NO: 44. In some embodiments, the nucleic acid molecule
encodes a capsid
polypeptide that has at least 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99%, or
100% identity to a
VP1, VP2, or VP3 sequence of SEQ ID NO: 45. In some embodiments, the nucleic
acid
molecule encodes a capsid polypeptide that has at least 85, 90, 91, 92, 93,
94, 95, 96, 97, 98,
99%. or 100% identity to a VP1, VP2, or VP3 sequence of SEQ ID NO: 46.
In some embodiments, the capsid polypeptide comprises a reference capsid
sequence,
such as SEQ ID NO: 1, and at least, or about, 80%, 85%, 90%, or 95%, or 100%
of the mutations
(insertions, deletions, or substitutions) as shown in the Mutation Differences
column of Table 1
of VAR-1. VAR-2, VAR-3, VAR-4, VAR-5, VAR-6, VAR-7, VAR-8, VAR-9, VAR-10, VAR-
11, VAR-12, VAR-13, VAR-14, VAR-15, VAR-16, VAR-17, VAR-18, VAR-19, VAR-20,
VAR-21, VAR-22, VAR-23, VAR-24, VAR-25, VAR-26, VAR-27, VAR-28, VAR-29, VAR-
30, VAR-31, VAR-32, VAR-33, VAR-34, VAR-35, VAR-36, VAR-37, VAR-38, VAR-39,
VAR-40, VAR-41, VAR-42, VAR-43, VAR-44, or VAR-45. In some embodiments, the
reference capsid sequence comprises at least, about, or exactly, 80% of the
mutations (insertions,
deletions, or substitutions). In some embodiments, the reference capsid
sequence comprises at
least, about, or exactly, 85% of the mutations (insertions, deletions, or
substitutions). In some
embodiments, the reference capsid sequence comprises at least, about, or
exactly, 90% of the
mutations (insertions, deletions, or substitutions). In some embodiments, the
reference capsid
sequence comprises at least, about, or exactly, 95% of the mutations
(insertions, deletions, or
substitutions). In some embodiments, the reference capsid sequence comprises
100% of the
mutations (insertions, deletions, or substitutions).
In some embodiments, the capsid polypeptide comprises a reference capsid
sequence,
such as SEQ ID NO: 1, and at least, or about, or exactly, 80%, 85%, 90%, or
95%, or 100% of
one of the following groups of mutations (the terminology for these groups of
mutations is
provided for in the legend of Table 1 above):
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['T14L', 'L15V', 119A', 'K24H'];
['A3V', 'Y6F', 'Ll1F', 'E 12Q, 'Q21L', 'W23I', 'L25C', 'P29A', 'P3 1N',
'K33R'];
['N449Q', 'T450M', 'P451T', 'T455L', 'T456G', 'Q457T'. 'S458Q', 'R4591\41 ;
['N449Q', 'P451-', 'S452T', 'T456G', 'Q457T'. 'R459G] ;
['Q457F', 'S458C'];
[446_7aa_447_KCQEGMAl;
[449 7aa_450_LMVDRLG'l ;
[P451T', '1455G, '1456G, 'Q4571, 'S458Q, 'R4591, 'Q461A'];
['448 7aa_449_HCQECPI1 ;
[453 6aa_454_FS GLEN] ;
[7456-', 'Q457-', 'S458-', 'R459-', 'L460-', 'Q461-'];
[N4491'. 'T45ON', 'P451G', T454-, 'T455Q', 'T456N', 'S458Q', 'R459T', Q461K',
'Q464 V1 ;
[S446A', 'T448-', 'N449-', 'P451Q', 'G453T', 'T455G', 'T456G', 'Q457T',
R459G1;
['N449Q', 'T450S', '451 1 aa 452 Y. 'S452G', 'T455A', 'Q457F', 'S458M',
'R459D'];
I '447 7aa_448_HETEENF1 ;
[13451T', 'T455G', 'T456G', 'Q457T', 'S458Q1, 'R459T', 'Q461A'];
[581 6aa_582_FALMEF];
[584 6aa_585_RAYNPD'];
[E555A', 'D561S', 'R566K', 'S578V', 'S580A', 'T581A', 'R585-1 ;
['V557L', 'S578D1 ;
['K556N', 'M558L', 'S578I', 'S580A'];
[1554L', 'K556R', 'I559L', 'D5615', 'R566A', 'T581D', R585S', 'G586R',
'N587S'];
['V552A', 'E555S', 'K556D', 'I559L', 'D561S', 'R566K', 'S578I', 'T581D',
'G586Q'];
['Q575E', 'S578A1 ;
['583 6aa_584_LNWTAE'l ;
['R585S', 'R588T', 'Q589N', 'A590P', A591F, 'A593G', 'T597S', 'V600A1;
[584 8aa_585_LAKEFTTR', 'R585N', 'G586A'];
[592 5aa_593_LHPLE1;
11585 1 aa_586_F] ;
['586 7aa_587_DQDFKNW];
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[589 laa_590_11;
['587 9aa_588_LAIEQTRPA'];
[584 8aa_585_RARLDETT', 'G586P', 'N587A'1;
['586 9aa_587_LALAEITRP', 'N587A'];
['586 9aa_587_LANGEQTRP', 'N587A'];
[586_6aa_587_ATDTKT];
[590 laa_591_Pl;
['587 9aa_588_APGETTRPATh
['589 laa_590_131;
[R585S', V-1586S1, 'N587A', 'A591E', 'D594R', 'T597A'. 'V600I'];
['586 7aa_587_FHNEGKY'];
[591 laa_592_G'];
['588 7aa_589_QPWEPDK];
[592 8aa 593 ALALSTTN']; or
[585 6aa 586 PWGTAG'].
In some embodiments, the capsid polypeptide comprises a reference capsid
sequence,
such as SEQ ID NO: 1, and at least, or about, or exactly, 80%, 85%, 90%, or
95%. or 100% of
['A3V', 'Y6F', 'E12Q', 'Q21L', W231', 'L25C', 'P29A', 'P31N',
'K33R']. In some
embodiments, the capsid polypeptide comprises at least 8, 9 or all of the
mutations of [A3V,
'Y6F', 'Ll 1F', 'E 12Q', 'Q21L'. 'W23I', 'L25C', 'P29A', 'P3 1N', 'K33RI] =
In some embodiments, the capsid polypeptide comprises a reference capsid
sequence,
such as SEQ ID NO: 1, and at least, or about, or exactly, 80%, 85%, 90%, or
95%. or 100% of
['N449Q', 'P451-', '5452T', 'T456G', 'Q457T', 'R459G']. In some embodiments,
the capsid
polypeptide comprises at least 5 or all of the mutations of [1\1449Q, 'P451-',
'S452T', 'T456G',
'Q457T', 'R459G'].
In some embodiments, the capsid polypeptide comprises a reference capsid
sequence,
such as SEQ ID NO: 1, and at least, or about, or exactly, 80%, 85%, 90%, or
95%. or 100% of
[446 7aa_447_KCQEGMAl. In some embodiments, the capsid polypeptide comprises
at least
6 or all of the amino acid residues of the seven amino acid insertion.
In some embodiments, the capsid polypeptide comprises a reference capsid
sequence,
such as SEQ ID NO: 1, and at least, or about, or exactly, 80%, 85%, 90%, or
95%, or 100% of
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[449 7aa_450_LMVDRLGT In some embodiments, the capsid polypeptide comprises at
least
6 or all of the amino acid residues of the seven amino acid insertion.
In some embodiments, the capsid polypeptide comprises a reference capsid
sequence,
such as SEQ ID NO: 1, and at least, or about, or exactly, 809c, 85%, 90%, or
95%, or 100% of
[13451T',1T455G', 'T456G', 'Q457T', 'S458Q', 'R459T, 'Q461AT In some
embodiments, the
capsid polypeptide comprises at least 6 or all of the mutations of [13451T',
'T455G', 'T456G',
*Q457T', 'S45 8Q', 'R459T', 'Q461A'[.
In some embodiments, the capsid polypeptide comprises a reference capsid
sequence,
such as SEQ ID NO: 1, and at least, or about, or exactly, 80%, 85%, 90%, or
95%, or 100% of
[448 7aa_449_HCQECP11. In some embodiments, the capsid polypeptide comprises
at least 6
or all of the amino acid residues of the seven amino acid insertion.
In some embodiments, the capsid polypeptide comprises a reference capsid
sequence,
such as SEQ ID NO: 1, and at least, or about, or exactly, 80%, 85%, 90%, or
95%, or 100% of
[453 6aa 454 FSGLEN]. In some embodiments, the capsid polypeptide comprises at
least 5 or
all of the amino acid residues of the six amino acid insertion.
In some embodiments, the capsid polypeptide comprises a reference capsid
sequence,
such as SEQ ID NO: 1, and at least, or about, or exactly, 80%, 85%, 90%, or
95%, or 100% of
[T456-', 'Q457-', 'S458-', 'R459-', 'L460-', 'Q461-1 In some embodiments, the
capsid
polypeptide comprises at least 5 or all of the mutations of [T456-', 'Q457-',
'L460-', 'Q4611.
In some embodiments, the capsid polypeptide comprises a reference capsid
sequence,
such as SEQ ID NO: I, and at least, or about, or exactly, 80%, 85%, 90%, or
95%, or 100% of
[N449I', 'T450N', 'P451G', 'T454-', 'T455Q', 7456N', '5458Q', 'R459T'.
'Q461K',1Q464VT In
some embodiments, the capsid polypeptide comprises at least 8, 9, or all of
the mutations of
['N4491', '1450N', 'P451G', 'T454-', 'T455Q', 'T456N', 'S458Q, 'R459T'.
'Q4611(1, 'Q464VT
In some embodiments, the capsid polypeptide comprises a reference capsid
sequence,
such as SEQ ID NO: I, and at least, or about, or exactly, 80%, 85%, 90%, or
95%. or 100% of
[5446A', 'T448-', 'N449-', 'P451Q', 'G453T', 'T455G', 'T456G', 'Q457T',
'R459GT In some
embodiments, the capsid polypeptide comprises at least 8 or all of the
mutations of [S446A',
'T448-', 'N449-'. 'P451Q'. 'G453T', 'T455G'. 'T456G', 'Q457T', 'R459G1.
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In some embodiments, the capsid polypeptide comprises a reference capsid
sequence,
such as SEQ ID NO: 1, and at least, or about, or exactly, 80%, 85%, 90%, or
95%. or 100% of
[N449Q', 'T450S', '451 laa_452 Y', 'S452G', 'T455A', 'Q457F', 'S458M', 'R459DT
In some
embodiments, the capsid polypeptide comprises at least 7 or all of the
mutations of ['N449Q',
'T450S', '451_1aa_452 Y', 'S452G', 'T455A', 'Q457F, 'S458M', 'R459D1].
In some embodiments, the capsid polypeptide comprises a reference capsid
sequence,
such as SEQ ID NO: 1, and at least, or about, or exactly, 80%, 85%, 90%, or
95%. or 100% of
[1(585S', `G586S', 'N587A1, 1A591E', 'D594R', 'T597A', 'V60011. In some
embodiments, the
capsid polypeptide comprises at least 6 Or all of the mutations of ['R585S',
'G586S', 'N587A',
'A591E', 'D594R', 'T597 A', 'V60011.
In some embodiments, the capsid polypeptide comprises a reference capsid
sequence,
such as SEQ ID NO: 1, and at least, or about, or exactly, 80%, 85%, 90%, or
95%. or 100% of
[13451T', 'T455G', 'T456G', 'Q457T', 'S458Q', 'R459T, 'Q461A']. In some
embodiments, the
capsid polypeptide comprises at least 6 or all of the mutations of [13451T',
'T455G', 'T456G',
'Q457T', 'S45 8Q', 'R459T', 'Q461AT
In some embodiments, the capsid polypeptide comprises a reference capsid
sequence,
such as SEQ ID NO: 1, and at least, or about, or exactly, 80%, 85%, 90%, or
95%. or 100% of
[E555A1, 'D561S', 'R566K', 'S578V', 'S580A', 'T581A', 'R585-']. In some
embodiments, the
capsid polypeptide comprises at least 6 or all of the mutations of [E555A',
'D5615', 'R566K',
'S578V', 'S580A`, 'T581A', 'R585-'].
In some embodiments, the capsid polypeptide comprises a reference capsid
sequence,
such as SEQ ID NO: 1, and at least, or about, or exactly, 80%, 85%, 90%, or
95%. or 100% of
[1554L', 'K556R',1559L', 'D5615', 'R566A', 'T581D', 'R585S', 'G586R', 'N587ST
In some
embodiments, the capsid polypeptide comprises at least 8 or all of the
mutations of [1554L,
'K556R', '1559L', 'D561S', 'R566A', IT581D', 'R585S', 'G586R', 'N587ST
In some embodiments, the capsid polypeptide comprises a reference capsid
sequence,
such as SEQ ID NO: 1, and at least, or about, or exactly, 80%, 85%, 90%, or
95%. or 100% of
[V552A', 'E555S', 'K556D', 'I559L', 'D561S', 'R566K', 'S578I', 'T581D',
'G586Q1. In some
embodiments, the capsid polypeptide comprises at least 8 or all of the
mutations of ['V552A',
'E555S', 'K556D', 'I559L'. 'D561S', 'R566K', 'S578I', 'T581D', U586Q1.
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In some embodiments, the capsid polypeptide comprises a reference capsid
sequence,
such as SEQ ID NO: 1, and at least, or about, or exactly, 80%, 85%, 90%, or
95%, or 100% of
[R585S', 'R588T', 'Q589N', 'A590P', 'A591I', 'A593G', 'T597S', 'V600A'[. In
some
embodiments, the capsid polypeptide comprises at least 7 or all of the
mutations of [R585S'.
'R588T', 'Q589N', 'A590P', 'A591I', 'A593G', 'T597S', 'V600A1.
In some embodiments, the capsid polypeptide comprises a reference capsid
sequence,
such as SEQ ID NO: 1, and at least, or about, or exactly, 80%, 85%, 90%, or
95%. or 100% of
[447 7aa_448_HETEFNF]. In some embodiments, the capsid polypeptide comprises
at least 6
or all of the amino acid residues of the 7 amino acid insertion.
In some embodiments, the capsid polypeptide comprises a reference capsid
sequence,
such as SEQ ID NO: 1, and at least, or about, or exactly, 80%, 85%, 90%, or
95%. or 100% of
[581 6aa_582_FALME11. In some embodiments, the capsid polypeptide comprises at
least 5
or all of the amino acid residues of the 6 amino acid insertion.
In some embodiments, the capsid polypeptide comprises a reference capsid
sequence,
such as SEQ ID NO: 1, and at least, or about, or exactly, 80%, 85%, 90%, or
95%. or 100% of
1'584 6aa_585_RAYNPDT In some embodiments, the capsid polypeptide comprises at
least 5
or all of the amino acid residues of the 6 amino acid insertion.
In some embodiments, the capsid polypeptide comprises a reference capsid
sequence,
such as SEQ ID NO: 1, and at least, or about, or exactly, 80%, 85%, 90%, or
95%. or 100% of
[583 6aa_584_LNWTAEl. In some embodiments, the capsid polypeptide comprises at
least 5
or all of the amino acid residues of the 6 amino acid insertion.
In some embodiments, the capsid polypeptide comprises a reference capsid
sequence,
such as SEQ ID NO: 1, and at least, or about, or exactly, 80%, 85%, 90%, or
95%, or 100% of
[592 5aa_593_LHPLE]. In some embodiments, the capsid polypeptide comprises at
least 4 or
all of the amino acid residues of the 5 amino acid insertion.
In some embodiments, the capsid polypeptide comprises a reference capsid
sequence,
such as SEQ ID NO: 1, and at least, or about, or exactly, 80%, 85%, 90%, or
95%. or 100% of
[586 7aa_587_DQDFKNRT In some embodiments, the capsid polypeptide comprises at
least 6
or all of the amino acid residues of the 7 amino acid insertion.
In some embodiments, the capsid polypeptide comprises a reference capsid
sequence,
such as SEQ ID NO: 1, and at least, or about, or exactly, 80%, 85%, 90%, or
95%, or 100% of
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[587 9aa_588_LAIEQTRPAT In some embodiments, the capsid polypeptide comprises
at least
8 or all of the amino acid residues of the 9 amino acid insertion.
In some embodiments, the capsid polypeptide comprises a reference capsid
sequence,
such as SEQ ID NO: 1, and at least, or about, or exactly, 809c, 85%, 90%, or
95%. or 100% of
[586 6aa_587_ATDTKTT In some embodiments, the capsid polypeptide comprises at
least 5
or all of the amino acid residues of the 6 amino acid insertion.
In some embodiments, the capsid polypeptide comprises a reference capsid
sequence,
such as SEQ ID NO: 1, and at least, or about, or exactly, 80%, 85%, 90%, or
95%, or 100% of
[587 9aa_588_APGETTRPA1 . In some embodiments, the capsid polypeptide
comprises at
least 8 or all of the amino acid residues of the 9 amino acid insertion.
In some embodiments, the capsid polypeptide comprises a reference capsid
sequence,
such as SEQ ID NO: 1, and at least, or about, or exactly, 80%, 85%, 90%, or
95%. or 100% of
[586 7aa_587_FHNEGKY]. In some embodiments, the capsid polypeptide comprises
at least 6
or all of the amino acid residues of the 7 amino acid insertion.
In some embodiments, the capsid polypeptide comprises a reference capsid
sequence.
such as SEQ ID NO: 1, and at least, or about, or exactly, 80%, 85%, 90%, or
95%, or 100% of
[588 7aa_589_QPWEPDKT In some embodiments, the capsid polypeptide comprises at
least 6
or all of the amino acid residues of the 7 amino acid insertion.
In some embodiments, the capsid polypeptide comprises a reference capsid
sequence,
such as SEQ ID NO: 1, and at least, or about, or exactly, 80%, 85%, 90%, or
95%. or 100% of
['592 8aa_593_ALALSTTNT In some embodiments, the capsid polypeptide comprises
at least
7 or all of the amino acid residues of the 8 amino acid insertion.
In some embodiments, the capsid polypeptide comprises a reference capsid
sequence,
such as SEQ ID NO: 1, and at least, or about, or exactly, 80%, 85%, 90%, or
95%. or 100% of
[1585 6aa_586_PWGTAGT In some embodiments, the capsid polypeptide comprises at
least 5
or all of the amino acid residues of the 6 amino acid insertion.
In some embodiments, the capsid polypeptide comprises a reference capsid
sequence,
such as SEQ ID NO: 1, and at least, or about, or exactly, 80%, 85%, 90%, or
95%, or 100% of
[584 8aa_585_LAKEFTTR', 'R585N', 'G586A1. In some embodiments, the capsid
polypeptide
comprises at least 8, 9, or all of the amino acid residues of the insertion
and the point mutations
recited in [584_8aa 585_LAKEFTTR', 'R585N', *G586A'].
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In some embodiments, the capsid polypeptide comprises a reference capsid
sequence,
such as SEQ ID NO: 1, and at least, or about, or exactly, 80%, 85%, 90%, or
95%. or 100% of
[584 8aa_585_RARLDETT', 'G586P', 'N587A1. In some embodiments, the capsid
polypeptide
comprises at least 8, 9, or all of the amino acid residues of the insertion
and the point mutations
recited in [584_8aa 585_RARLDETT', '@58 6P', 'N5 87A].
In some embodiments, the capsid polypeptide comprises a reference capsid
sequence,
such as SEQ ID NO: 1, and at least, or about, or exactly, 80%, 85%, 90%, or
95%. or 100% of
[586 9aa_587_LALAEITRP', 'N587A1. In some embodiments, the capsid polypeptide
comprises at least 8, 9, or all of the amino acid residues of the insertion
and the point mutations
recited in [586_9aa 587_LALAEITRP', 'N587AT
In some embodiments, the capsid polypeptide comprises a reference capsid
sequence,
such as SEQ ID NO: I, and at least, or about, or exactly, 80%, 85%, 90%, or
95%. or 100% of
[586 9aa_587_LANGEQTRP',1\1587A1 In some embodiments, the capsid polypeptide
comprises at least 8, 9, or all of the amino acid residues of the insertion
and the point mutations
recited in [586 9aa 587 LANGEQTRP'. 'N5 87A].
Variant Capsids (Corresponding Positions)
The mutations to capsid polypeptide sequences described herein are described
in relation
to a position and/or amino acid at a position within a reference sequence,
e.g., SEQ ID NO: 1.
Thus, in some embodiments, the capsid polypeptides described herein are
variant capsid
polypeptides of the reference sequence, e.g., SEQ ID NO: 1, e.g., include
capsid polypeptides
comprising at least 80%, at least 85%, at least 90%, at least 91%, at least
92%, at least 93%, at
least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least
99% identity to the
reference capsid polypeptide sequence (e.g., reference capsid polypeptide VP1,
VP2 and/or VP3
sequence), e.g., SEQ ID NO: 1 (or VP2 or VP3 sequence comprised therein) and
further
including one or more mutations described herein.
It will be understood by the skilled artisan, and without being bound by
theory, that each
amino acid position within a reference sequence corresponds to a position
within the sequence of
other capsid polypeptides such as capsid polypeptides derived from
dependoparvoviruses with
different serotypes. Such corresponding positions are identified using
sequence alignment tools
known in the art. A particularly preferred sequence alignment tool is Clustal
Omega (Sievers F.,
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et al., Mol. Syst. Biol. 7:359, 2011, DOI: 10.1038/msb.2011.75, incorporated
herein by reference
in its entirety). An alignment of exemplary reference capsid polypeptides is
shown in FIG.1A-
1C. Thus, in some embodiments, the variant capsid polypeptides of the
invention include
variants of reference capsid polypeptides that include one or more mutations
described herein in
such reference capsid polypeptides at positions corresponding to the position
of the mutation
described herein in relation to a different reference capsid polypeptide.
Thus, for example, a
mutation described as XnnnY relative to SEQ ID NO: 1 (where X is the amino
acid present at
position nnn in SEQ ID NO: 1 and Y is the amino acid mutation at that
position, e.g., described
herein), the disclosure provides variant capsid polypeptides comprising at
least 80%, at least
85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at
least 95%, at least
96%, at least 97%, at least 98%, or at least 99 identity to the reference
capsid polypeptide
sequence (e.g., reference capsid polypeptide VP1, VP2 and/or VP3 sequence)
other than SEQ ID
NO: 1 (or VP2 or VP3 sequence comprised therein) and further comprising the
disclosed
mutation at a position corresponding to position nnn of SEQ ID NO: 1 (e.g.,
comprising Y at the
position in the new variant capsid polypeptide sequence that corresponds to
position nnn of SEQ
ID NO: 1). As described above, such corresponding position is determined using
a sequence
alignment tool, such as, for example, the clustal omega tool described above.
Examples of
corresponding amino acid positions of exemplary known AAV serotypes is
provided in FIG.2A-
C. In some embodiments, the variant is a variant of the AAV2 capsid
polypeptide, which can be
referred to as a "variant AAV2 capsid polypeptide."
Thus, in embodiments, the disclosure provides capsid polypeptide sequences
that are
variants of a reference sequence other than SEQ ID NO: 1, e.g., a reference
sequence other than
SEQ ID NO: 1 as described herein, which include one or more mutation
corresponding to the
mutations described herein. In embodiments, such variants include mutations
corresponding to
all of the mutations associated with any one of VAR-I through VAR-53 as
provided herein.
As used herein, the term "corresponds to" as used in reference to a position
in a sequence,
such as an amino acid or nucleic acid sequence, can be used in reference to an
entire capsid
polypeptide or polynucleotide sequence, such as the full length sequence of
the capsid
polypeptide that comprises a VP1. VP2, and VP3 polypeptide, or a nucleic acid
molecule
encoding the same. In some embodiments, the term "corresponds to" can be used
in reference to
a region or domain of the capsid polypeptide. For example, a position that
corresponds to a
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position in the VP1 section of the reference capsid polypeptide can correspond
to the VP1
portion of the polypeptide of the variant capsid polypeptide. Thus, when
aligning the two
sequences to determine whether a position corresponds to another position the
full length
polypeptide can be used or domains (regions) can be used to determine whether
a position
corresponds to a specific position. In some embodiments, the region is the VP1
polypeptide. In
some embodiments, the region is the VP2 polypeptide. In some embodiments, the
region is the
VP3 polypeptide. In some embodiments, when the reference polypeptide is the
wild-type
sequence (e.g., full length or region) of a certain serotype of AAV, the
variant polypeptide can be
of the same serotype with a mutation made at such corresponding position as
compared to the
reference sequence (e.g., full length or region). In some embodiments, the
variant capsid
polypeptide is a different serotype as compared to the reference sequence.
The variant capsid polypeptides described herein are optionally variants of
reference
capsids serotypes known in the art. Non-limiting examples of such reference
AAV serotypes
include AAV1, AAVrh10, AAV-DJ, AAV-DJ8, AAV5. AAVPHP.B (PHP.B), AAVPHP.A
(PHP.A), AAVG2B-26, AAVG2B-13, AAVTH1.1-32, AAVTH1.1- 35, AAVPHP.B2
(PHP.B2), AAVPHP.B3 (PHP.B3), AAVPHP.N/PHP.B-DGT, AAVPHP.B-EST, AAVPHP.B-
GGT, AAVPHP.B-ATP, AAVPHP.B-ATT-T, AAVPHP.B- DGT-T, AAVPHP.B-GGT-T,
AAVPHP.B-SGS, AAVPHP.B-AQP, AAVPHP.B-QQP, AAVPHP.B-SNP(3), AAVPHP.B-
SNP, AAVPHP.B-QGT, AAVPHP.B-NQT, AAVPHP.B- EGS, AAVPHP.B-SGN, AAVPHP.B-
EGT, AAVPHP.B-DST, AAVPHP.B-DST, AAVPHP.B-STP, AAVPHP.B-PQP, AAVPHP.B-
SQP, AAVPHP.B-QLP, AAVPHP.B-TMP, AAVPHP.B-TTP, AAVPHP.S/G2Al2,
AAVG2A15/G2A3 (G2A3), AAVG2B4 (G2B4), AAVG2B5 (G2B5), PHP.S, AAV2, AAV2G9,
AAV3, AAV3a, AAV3b, AAV3-3, AAV4, AAV4-4, AAV6, AAV6.I, AAV6.2, AAV6.1.2,
AAV7, AAV7.2, AAV8, AAV9.11, AAV9.13, AAV9, AAV9 K449R (or K449R AAV9),
AAV9.16, AAV9.24, AAV9.45, AAV9.47, AAV9.61, AAV9.68, AAV9.84, AAV9.9, AAV10,
AAV11, AAV12, AAV16.3, AAV24.1, AAV27.3, AAV42.12. AAV42- lb, AAV42-2, AAV42-
3a, AAV42-3b, AAV42-4, AAV42-5a, AAV42-5b, AAV42-6b, AAV42-8, AAV42-10, AAV42-
11, AAV42-12, AAV42-13, AAV42-15, AAV42-aa, AAV43-1, AAV43-12, AAV43-20,
AAV43-21, AAV43-23, AAV43-25, AAV43-5, AAV44.1, AAV44.2, AAV44.5, AAV223.1,
AAV223.2, AAV223.4, AAV223.5, AAV223.6, AAV223.7, AAV1-7/rh.48, AAV1-8/rh.49,
AAV2-15/rh.62, AAV2-3/rh.61, AAV2-4/rh.50, AAV2-5/rh.51, AAV3.1/hu.6,
AAV3.1/hu.9,
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AAV3-9/rh.52, AAV3-11/rh.53, AAV4- 8/r11.64, AAV4-9/rh.54, AAV4-19/rh.55, AAV5-
3/rh.57, AAV5-22/rh.58, AAV7.3/hu.7, AAV16.8/hu.10, AAV16.12/hu.11,
AAV29.3/bb.1,
AAV29.5/bb.2, AAV106.1/hu.37, AAV114.3/hu.40, AAV127.2/hu.41, AAV127.5/hu.42,
AAV128.3/hu.44. AAV130.4/hu.48, AAV145.1/hu.53, AAV145.5/hu.54,
AAV145.6/hu.55,
AAV161.10/hu.60, AAV161.6/hu.61, AAV33.12/hu.17, AAV33.4/hu.15, AAV33.8/hu.16,
AAV52/hu.19, AAV52.1/hu.20, AAV58.2/hu.25, AAVA3.3, AAVA3.4, AAVA3.5, AAVA3.7,
AAVC1, AAVC2, AAVC5, AAVF3, AAVF5, AAVH2, AAVrh.72, AAVhu.8, AAVrh.68,
AAVrh.70, AAVpi.1, AAVpi.3, AAVpi.2, AAVrh.60, AAVrh.44, AAVrh.65, AAVrh.55,
AAVrh.47, AAVrh.69, AAVrh.45, AAVrh.59, AAVhu.12, AAVH6, AAVH-1/hu.1, AAVH-
5/hu.3, AAVLG- 10/r11.40, AAVLG-4/rh.38, AAVLG-9/hu.39, AAVN721-8/rh.43,
AAVCh.5,
AAVCh.5R1, AAVcy.2, AAVcy.3, AAVcy.4, AAVcy.5, AAVCy.5R1, AAVCy.5R2,
AAVCy.5R3, AAVCy.5R4, AAVcy.6, AAVhu.1, AAVhu.2, AAVhu.3, AAVhu.4, AAVhu.5,
AAVhu.6, AAVhu.7, AAVhu.9, AAVhu.10, AAVhu.11, AAVhu.13, AAVhu.15, AAVhu.16,
AAVhu.17, AAVhu.18, AAVhu.20, AAVhu.21, AAVhu.22, AAVhu.23.2, AAVhu.24,
AAVhu.25, AAVhu.27, AAVhu.28, AAVhu.29, AAVhu.29R, AAVhu.31, AAVhu.32,
AAVhu.34, AAVhu.35, AAVhu.37, AAVhu.39, AAVhu.40, AAVhu.41, AAVhu.42,
AAVhu.43, AAVhu.44, AAVhu.44R1, AAVhu.44R2, AAVhu.44R3, AAVhu.45, AAVhu.46,
AAVhu.47, AAVhu.48, AAVhu.48R1, AAVhu.48R2, AAVhu.48R3, AAVhu.49, AAVhu.51,
AAVhu.52, AAVhu.54, AAVhu.55, AAVhu.56, AAVhu.57, AAVhu.58, AAVhu.60,
AAVhu.61, AAVhu.63, AAVhu.64, AAVhu.66, AAVhu.67, AAVhu.14/9, AAVhu.t 19,
AAVrh.2, AAVrh.2R, AAVrh.8, AAVrh.8R, AAVrh.10, AAVrh.12, AAVrh.13, AAVrh.13R,
AAVrh.14, AAVrh.17, AAVrh.18, AAVrh.19, AAVrh.20, AAVrh.21, AAVrh.22,
AAVrh.23,
AAVrh.24, AAVrh.25, AAVrh.31, AAVrh.32, AAVrh.33, AAVrh.34, AAVrh.35,
AAVrh.36,
AAVrh.37, AAVrh.37R2, AAVrh.38, AAVrh.39, AAVrh.40, AAVrh.46, AAVrh.48,
AAVrh.48.1, AAVrh.48.1.2, AAVrh.48.2, AAVrh.49, AAVrh.51, AAVrh.52, AAVrh.53,
AAVrh.54, AAVrh.56, AAVrh.57, AAVrh.58, AAVrh.61, AAVrh.64, AAVrh.64R1,
AAVrh.64R2, AAVrh.67, AAVrh.73, AAVrh.74 (also referred to as AAVrh74),
AAVrh8R,
AAVrh8R A586R mutant, AAVrh8R R533A mutant, AAAV, BAAV, caprine AAV, bovine
AAV, AAVhE1.1, AAVhEr1.5, AAVhER1.14, AAVhEr1.8, AAVhEr1.16, AAVhEr1.18,
AAVhEr1.35, AAVhEr1.7, AAVhEr1.36, AAVhEr2.29, AAVhEr2.4, AAVhEr2.16,
AAVhEr2.30, AAVhEr2.31, AAVhEr2.36, AAVhER1.23, AAVhEr3.1, AAV2.5T , AAV-
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PAEC, AAV-LKOI, AAV-LK02, AAV- LK03, AAV-LK04, AAV-LK05, AAV-LK06, AAV-
LK07, AAV-LK08, AAV-LK09, AAV- LK10, AAV-LK11, AAV-LK12, AAV-LK13, AAV-
LK14, AAV-LK15, AAV-LK16, AAV- LK17, AAV-LK18, AAV-LK19, AAV-PAEC2, AAV-
PAEC4, AAV-PAEC6, AAV-PAEC7, AAV-PAEC8, AAV-PAEC11, AAV-PAEC12, AAV-2-
pre-miRNA-101 , AAV-8h, AAV- 8b, AAV-h, AAV-b, AAV SM 10-2 , AAV Shuffle 100-
1,
AAV Shuffle 100-3, AAV Shuffle 100-7, AAV Shuffle 10-2, AAV Shuffle 10-6, AAV
Shuffle
10-8. AAV Shuffle 100- 2, AAV SM 10-1, AAV SM 10-8 . AAV SM 100-3, AAV SM 100-
10,
BNP61 AAV, BNP62 AAV, BNP63 AAV, AAVrh.50, AAVrh.43, AAVrh.62, AAVrh.48,
AAVlau.19, AAVbu.11, AAVIm.53, AAV4-8/rh.64, AAVLG-9/hu.39, AAV54.5/hu.23,
AAVS4.2/hu.22, AAV54.7/hu.24, AAV54.1/hu.21, AAV54.4R/hu.27, AAV46.2/hu.28,
AAV46.6/hu.29, AAV128.1/hu.43, true type AAV (ttAAV), UPENN AAV 10, Japanese
AAV
serotypes, AAV CBr-7.1, AAV CBr-7.10, AAV CBT-7.2, AAV CBr-7.3, AAV CBr-7.4,
AAV CBr-7.5, AAV CBr-7.7, AAV CBr-7.8, AAV CBr-B7.3, AAV CBr-B7.4, AAV CBr-El,
AAV CBr- E2, AAV CBr-E3, AAV CBr-E4, AAV CBr-E5, AAV CBr-e5, AAV CBr-E6, AAV
CBr-E7, AAV CBr-E8, AAV CHt-1, AAV CHt-2, AAV CHt-3, AAV CHt-6.1, AAV CHt-
6.10,
AAV CHt-6.5, AAV CHt-6.6, AAV CHt-6.7, AAV CHt-6.8, AAV CHt-P1, AAV CHt-P2,
AAV
CHt-P5, AAV CHt-P6, AAV CHt-P8, AAV CHt-P9, AAV CKd-1, AAV CKd-10, AAV CKd-2,
AAV CKd-3, AAV CKd-4, AAV CKd-6, AAV CKd-7, AAV CKd-8, AAV CKd-B1, AAV
CKd-B2, AAV CKd-B3, AAV CKd-B4, AAV CKd-B5, AAV CKd-B6, AAV CKd-B7. AAV
CKd-B8, AAV CKd-H1, AAV CK(1-H2, AAV CKd-113, AAV CKd-1-I4, AAV CKd- H5, AAV
CKd-H6, AAV CKd-N3, AAV CKd-N4, AAV CKd-N9, AAV CLg-F1, AAV CLg-F2, AAV
CLg-F3, AAV CLg-F4, AAV CLg-F5, AAV CLg-F6, AAV CLg-F7, AAV CLg-F8, AAV CLy-
1, AAV CLy1-1, AAV Clv1-10, AAV CLy1-2, AAV CLy-12, AAV CLy1-3, AAV CLy-13,
AAV CLy1-4, AAV C1y1-7, AAV C1y1-8, AAV C1y1-9, AAV CLy- 2, AAV CLy-3, AAV CLy-
4, AAV CLy-6, AAV CLy-8, AAV CLy-D1, AAV CLy-D2, AAV CLy-D3, AAV CLy-D4,
AAV CLy-D5, AAV CLv-D6, AAV CLy-D7, AAV CLy-D8, AAV CLy-El, AAV CLy-K1,
AAV CLv-K3, AAV CLv-K6, AAV CLY-L4, AAV CLv-L5. AAV CLy-L6, AAV CLy-Ml,
AAV CLy-M11, AAV CLy-M2, AAV CLy-M5, AAV CLy- M6, AAV CLv-M7, AAV CLy-M8,
AAV CLy-M9, AAV CLy-R1, AAV CLy-R2, AAV CLy-R3, AAV CLY-R4, AAV CLv-R5,
AAV CLy-R6, AAV CLy-R7, AAV CLy-R8, AAV CLy-R9, AAV CSp-1, AAV CSp-10, AAV
CSp-11, AAV CSp-2, AAV CSp-3, AAV CSp-4, AAV CSp-6, AAV CSp-7, AAV CSp-8, AAV
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CSp-8.10, AAV CSp-8.2, AAV CSp-8.4, AAV CSp-8.5, AAV CSp-8.6, AAV CSp-8.7, AAV
CSp-8.8, AAV CSp-8.9, AAV CSp-9, AAV.hu.48R3, AAV.VR-355, AAV3B, AAV4, AAV5,
AAVF1/HSC1, AAVF11/HSC11, AAVF12/HSC12, AAVF13/HSC13, AAVF14/HSC14,
AAVF15/HSC15, AAVF16/HSC16, AAVF17/HSC17, AAVF2/HSC2, AAVF3/HSC3,
AAVF4/HSC4, AAVF5/HSC5, AAVF6/HSC6, AAVF7/HSC7, AAVF8/HSC8. and/or
AAVF9/HSC9, 7m8, Spark100, AAVMYO and variants thereof.
In some embodiments, the reference AAV capsid sequence comprises an AAV2
sequence. In some embodiments, the reference AAV capsid sequence comprises an
AAV5
sequence. In some embodiments, the reference AAV capsid sequence comprises an
AAV8
sequence. In some embodiments, the reference AAV capsid sequence comprises an
AAV9
sequence. In some embodiments, the reference AAV capsid sequence comprises an
AAVrh74
sequence. While not wishing to be bound by theory, it is understood that a
reference AAV capsid
sequence comprises a VP1 region. In certain embodiments, a reference AAV
capsid sequence
comprises a VP1, VP2 and/or VP3 region, or any combination thereof. A
reference VP1
sequence may be considered synonymous with a reference AAV capsid sequence.
An exemplary reference sequence of SEQ ID NO: 1 (wild-type AAV2) is as
follows:
MAADGYLPDWLEDTLSEGIRQWWKLKP GPPPPKPAERHKDDSRGLVLP GYKYLGPFNGLDKGEP
VNEADAAALEHDKAYDRQLD SGDNP YLKYNHADAEFQERLKEDT SFGGNLGRAVFQAKKRVLEP
LGLVEEPVKTAPGKKRPVEHSPVEPDSSSGTGKAGQQPARKRLNFGQTGDADSVPDPQPLGQPP
AAPSGLGTNTMATGSGAPMADNNEGADGVGNSSGNWHCDSTWMGDRVITTSTRTWALPTYNNHL
YKQISSQSGASNDNHYFGYSTPWGYEDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLENIQV
KEVInNDGTTTIANNLTSTVQVFTDSEYnLPYVLGSAHnGCLPPFPADVFMVPQYGYLTLNNGS
QAVGRSSFYCLEYFPSQMLRTGNNFIFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRI
NTPSGITTQSRLQFSQAGASDIRDQSRNWLPGPCYRQQRVSKTSADNNNSEYSWTGATKYHLNG
RDSLVNPGPAMASHKDDEEKFFPQSGVLIFGKQGSEKTNVDIEKVMITDEEEIRTTNPVATEQY
GSVSTNLQRGNKAATADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHIDGHFHPSPLMGGFGLK
HPPPQILIKNTPVPANPSTIFSAAKFASFITQYSTGQVSVEIEWELQKENSKRWMPEIQYTSNY
NKSVNVDFTVDTNGVYSEPRPIGTRYLTRNL.(SEQ ID NO: 1)
Unless otherwise noted, SEQ ID NO: 1 is the reference sequence. In the
sequence above,
the sequence found in VP1, VP2 and VP3 is underlined (e.g.. a VP3 capsid
polypeptide includes,
e.g., consists of, amino acids corresponding to amino acids 203-735 of SEQ ID
NO: 1), the
sequence found in both VP1 and VP2 is in bold (e.g., a VP2 capsid polypeptide
includes, e.g.,
consists of, the sequence corresponding to amino acids 138-735 of SEQ ID NO:
1) and the
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sequence that is not underlined or bold is found only in VP1 (e.g., a VP1
capsid polypeptide
includes, e.g., consists of, amino acids corresponding to amino acids 1-735 of
SEQ ID NO: 1).
An example nucleic acid sequence encoding SEQ ID NO: 1 is SEQ ID NO: 92:
ATGGCTGCCGATGGTTATCTTCCAGATTGG CTCGAGGACACTCTCTCTGAAGGAAT
AAGACAGTGGTGGAAG CTCAAACCTGGCCCACCACCACCAAAGCCCGCAGAG CG
GCATAAGGACGACAGCAGGGGTCTTGTGCTTCCTGGGTACAAGTACCTCGGACCC
TTCAACGGACTCGACAAGGGAGAGCCGGTCAACGAGGCAGACGCCGCGGCCCTC
GAGCACGACAAAGCCTACGACCGGCAGCTCGACAGCGGAGACAACCCGTACCTC
AAGTACAACCACGCCGACG CGGAGTTTCAGGAGCG CCTTAAAGAAGATACGTCTT
TTGGGGGCAACCTCGGACGAGCAGTCTTCCAGG CGAAAAAGAGGGTTCTTGAACC
TCTGGGCCTGGTTGAGGAACCTGTTAAGACGGCTCCGGGAAAAAAGAGGCCGGTA
GAGCACTCTCCTGTGGAG CCAGACTCCTCCTCGGGAACCGGAAAGGCGG GCCAG
CAGCCTGCAAGAAAAAGATTGAATTTTGGTCAGACTG GAGACGCAGACTCAGTACC
TGACCCCCAGCCTCTCGGACAGCCACCAGCAG CCCCCTCTGGTCTGGGAACTAAT
ACGATGGCTACAGGCAGTG GCGCACCAATGG CAGACAATAACGAGGGCG CCGAC
GGAGTGGGTAATTCCTCGGGAAATTGGCATTGCGATTCCACATGGATGGGCGACA
GAGTCATCACCACCAGCACCCGAACCTGGGCCCTG CCCACCTACAACAACCACCT
CTACAAACAAATTTCCAG CCAATCAGGAG CCTCGAACGACAATCACTACTTTGGCT
ACAGCACCCCTTGGGGGTATTTTGACTTCAACAGATTCCACTGCCACTTTTCACCA
CGTGACTGGCAAAGACTCATCAACAACAACTGGGGATTCCGACCCAAGAGACTCA
ACTTCAAG CTCTTTAACATTCAAGTCAAAGAGGTCACGCAGAATGACGGTACGACG
ACGATTGCCAATAACCTTACCAGCACGGTTCAGGTGTTTACTGACTCGGAGTACCA
G CTCCCGTACGTCCTCGG CTCGGCGCATCAAGGATG CCTCCCGCCGTTCCCAG CA
GACGTCTTCATGGTGCCACAGTATGGATACCTCACCCTGAACAACGGGAGTCAGG
CAGTAGGACGCTCTTCATTTTACTGCCTGGAGTACTTTCCTTCTCAGATGCTGCGT
ACCGGAAACAACTTTACCTTCAGCTACACTTTTGAGGACGTTCCTTTCCACAGCAG
CTACGCTCACAGCCAGAGTCTG GACCGTCTCATGAATCCTCTCATCGACCAGTACC
TGTATTACTTGAG CAGAACAAACACTCCAAGTGGAACCACCACGCAGTCAAGG CTT
CAGTTTTCTCAG GCCGGAGCGAGTGACATTCGGGACCAGTCTAGGAACTGGCTTC
CTGGACCCTGTTACCGCCAGCAGCGAGTATCAAAGACATCTGCGGATAACAACAA
CAGTGAATACTCGTGGACTGGAGCTACCAAGTACCACCTCAATGGCAGAGACTCT
CTG GTGAATCCGGGCCCGGCCATGGCAAG CCACAAGGACGATGAAGAAAAGTTTT
TTCCTCAGAGCGGGGTTCTCATCTTTGGGAAGCAAGGCTCAGAGAAAACAAATGT
GGACATTGAAAAGGTCATGATTACAGACGAAGAGGAAATCAGGACAACCAATCCC
GTGGCTACGGAGCAGTATGGTTCTGTATCTACCAACCTCCAGAGAGG CAACAGAC
AAGCAGCTACCGCAGATGTCAACACACAAGGCGTTCTTCCAGGCATGGTCTGGCA
GGACAGAGATGTGTACCTTCAGGGGCCCATCTGGGCAAAGATTCCACACACGGAC
GGACATTTTCACCCCTCTCCCCTCATGGGTGGATTCGGACTTAAACACCCTCCTCC
ACAGATTCTCATCAAGAACACCCCGGTACCTG CGAATCCTTCGACCACCTTCAGTG
CGGCAAAGTTTGCTTCCTTCATCACACAGTACTCCACG GGACAGGTCAGCGTGGA
GATCGAGTGGGAGCTGCAGAAGGAAAACAGCAAACGCTGGAATCCCGAAATTCAG
TACACTTCCAACTACAACAAGTCTGTTAATGTGGACTTTACTGTGGACACTAATGGC
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GTGTATTCAGAGCCTCGCCCCATTGGCACCAGATACCTGACTCGTAATCTGTAA
(SEQ ID NO: 92)
An exemplary reference sequence of wild type AAV5, SEQ ID NO: 123 (wild-type
AAV5), is as follows:
MSFVDHPPDWLEEVGEGLREFLGLEAGPPKPKPNQQHQDQARGLVLPGYNYLGPGNGLDRGEPV
NRADEVAREHDISYNEQLEAGDNPYLKYNHADAEFQEKLADDTSEGGNLGKAVFQAKKRVLEPF
GLVEEGAKTAPTGKRIDDHFPERKKARTEEDSKPSTSSDAEAGPSGSQQLQIPAQPASSLGADT
MSAGGGGPLGDNNWADGVGNASGDWHCDSTWMGDRVVIKSTRTWVLPSYNNHQYREIKSGSVD
GSNANAYEGYSTPWGYFDENREHSHWSPRDWQRLINNYWGFRPRSLRVKIFNIQVKEVTVQDST
TTIANNLTSTVQVFTDDDYQLPYVVGNGTEGCLPAFPPQVFTLPQYGYATLNRDNTENPTERSS
FFCLEYFPSKMERTGNNFEFTYNFEEVPFHSSFAPSQNLFKLANPLVDQYLYRFVSTNNTGGVQ
FNKNLAGRYANTYKNWFPGPMGRTQGWNLGSGVNRASVSAFATTNRMELEGASYQVPPQPNGMT
NNLQGSNTYALENTMIFNSQPANPGTTATYLEGNMLITSESETQPVNRVAYNVGGQMATNNQSS
TTAPATOTYNLQEIVPGSVWMERDVYLQGPIWAKIPETGAHFHPSPAMGGFOLKHPPPMMLIKN
TPVPGNITSFSDVPVSSEITQYSTGQVTVEMEWELKKENSKRWMPEIQYTNNYNDPQFVDFAPD
STGEYRTTRPIGTRYLTRPL (SEQ ID NO: 123)
In the sequence above, the sequence found in VP1, VP2 and VP3 is underlined
(e.g., a
VP3 capsid polypeptide includes, e.g., consists of, amino acids corresponding
to amino acids
193-725 of SEQ ID NO: 123), the sequence found in both VP1 and VP2 is in bold
(e.g., a VP2
capsid polypeptide includes, e.g., consists of, the sequence corresponding to
amino acids 137-
725 of SEQ ID NO: 123) and the sequence that is not underlined or bold is
found only in VP1
(e.g., a VP1 capsid polypeptide includes, e.g., consists of, amino acids
corresponding to amino
acids 1-725 of SEQ ID NO: 123).
An example nucleic acid sequence encoding SEQ ID NO: 123 is SEQ ID NO: 124:
ATGTCTTTTGTTGATCACCCTCCAGATTGGTTGGAAGAAGTTGGTGAAGGTCTTCG
CGAGTTTTTGGGCCTTGAAGCG G GCCCACCGAAACCAAAACCCAATCAGCAGCAT
CAAGATCAAGCCCGTGGTCTTGTG CTGCCTGGTTATAACTATCTCGGACCCG GAAA
CGGGCTCGATCGAG GAGAGCCTGTCAACAG GGCAGACGAGGTCGCGCGAGAG CA
CGACATCTCGTACAACGAGCAGCTTGAGGCGGGAGACAACCCCTACCTCAAGTAC
AACCACGCGGACG CCGAGTTTCAGGAGAAGCTCG CCGACGACACATCCTTCG G G
G GAAACCTCGGAAAGG CAGTCTTTCAGGCCAAGAAAAGGGTTCTCGAACCTTTTG
GCCTGGTTGAAGAGGGTGCTAAGACGGCCCCTACCGGAAAGCGGATAGACGACC
ACTTTCCAAAAAGAAAGAAGGCTCGGACCGAAGAGGACTCCAAGCCTTCCACCTC
GTCAGACGCCGAAGCTGGACCCAGCGGATCCCAGCAGCTGCAAATCCCAGCCCA
ACCAGCCTCAAGTTTGG GAG CTGATACAATGTCTGCGGGAG GTGGCGGCCCATTG
G GCGACAATAACCAAGGTG CCGATGGAGTGG G CAATG CCTCG G GAG ATTGG GATT
G CGATTCCACGTGGATGG GGGACAGAGTCGTCACCAAGTCCACCCGAACCTG GG
TGCTGCCCAGCTACAACAACCACCAGTACCGAGAGATCAAAAGCGGCTCCGTCGA
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CGGAAGCAACGCCAACGCCTACTTTGGATACAGCACCCCCTGGGGGTACTTTGAC
TTTAACCGCTTCCACAGCCACTGGAGCCCCCGAGACTGGCAAAGACTCATCAACA
ACTACTGGGGCTTCAGACCCCGGTCCCTCAGAGTCAAAATCTTCAACATTCAAGTC
AAAGAGGTCACGGTGCAGGACTCCACCACCACCATCGCCAACAACCTCACCTCCA
CCGTCCAAGTGTTTACGGACGACGACTACCAGCTGCCCTACGTCGTCGGCAACGG
GACCGAGGGATGCCTGCCGGCCTTCCCTCCGCAGGTCTTTACGCTGCCGCAGTAC
GGTTACGCGACGCTGAACCGCGACAACACAGAAAATCCCACCGAGAGGAGCAGC
TTCTTCTGCCTAGAGTACTTTCCCAGCAAGATGCTGAGAACGGGCAACAACTTTGA
GTTTACCTACAACTTTGAGGAGGTGCCCTTCCACTCCAGCTTCGCTCCCAGTCAGA
ACCTGTTCAAGCTGGCCAACCCGCTGGTGGACCAGTACTTGTACCGCTTCGTGAG
CACAAATAACACTGGCGGAGTCCAGTTCAACAAGAACCTGGCCGGGAGATACGCC
AACACCTACAAAAACTGGTTCCCGGGGCCCATGGGCCGAACCCAGGGCTGGAAC
CTGGGCTCCGGGGTCAACCGCGCCAGTGTCAGCGCCTTCGCCACGACCAATAGG
ATGGAGCTCGAGGGCGCGAGTTACCAGGTGCCCCCGCAGCCGAACGGCATGACC
AACAACCTCCAGGGCAGCAACACCTATGCCCTGGAGAACACTATGATCTTCAACAG
CCAGCCGGCGAACCCGGGCACCACCGCCACGTACCTCGAGGGCAACATGCTCAT
CACCAGCGAGAGCG AGACGCAGCCGGTGAACCGCGTGGCGTACAACGTCGGCG
GGCAGATGGCCACCAACAACCAGAGCTCCACCACTGCCCCCGCGACCGGCACGT
ACAACCTCCAGGAAATCGTGCCCGGCAGCGTGTGGATGGAGAGGGACGTGTACC
TCCAAGGACCCATCTGGGCCAAGATCCCAGAGACGGGGGCGCACTTTCACCCCTC
TCCGGCCATGGGCGGATTCGGACTCAAACACCCACCGCCCATGATGCTCATCAAG
AACACGCCTGTGCCCGGAAATATCACCAGCTTCTCGGACGTGCCCGTCAGCAGCT
TCATCACCCAGTACAGCACCGGGCAGGTCACCGTGGAGATGGAGTGGGAGCTCA
AGAAGGAAAACTCCAAGAGGTGGAACCCAGAGATCCAGTACACAAACAACTACAA
CGACCCCCAGTTTGTGGACTTTGCCCCGGACAGCACCGGGGAATACAGAACCACC
AGACCTATCGGAACCCGATACCTTACCCGACCCCTTTAA (SEQ ID NO: 124)
An exemplary reference sequence of wild-type AAV8, SEQ ID NO: 125 (wild-type
AAV8), is as follows:
MAADGYLPDWLEDNLSEGIREWWALKPGAPKPKANQQKQDDGRGLVLPGYKYLGPFNGLDKGEP
VNAADAAALEHDKAYDQQLQAGDNPYLRYNHADAEFQERLQEDISFCGNLGRAVFQAKKRVLEP
LGLVEEGAKTAPGKKRPVEPSPQRSPDSSTGIGKKGQQPARKRLNFGQTGDSESVPDPQPLGEP
PAAPSGVGPNTMAAGGGAPMADNNEGADGVGSSSGNWHCDSTWLGDRVITTSTRTWALPTYNNH
LYKQISNGTSGCATNDNTYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWCFRPKRLSFKLFN
IQVKEVTQNEGTKTIANNLTSTIQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMIPQYGYLTLN
NCSQAVCRSSFYCLEYFPSQMLRTCNNFUTYTFEDVPFHSSYAHSQSLDRLMNPLIDULYYL
SRTOTCGTANTQTLCFSQGGPNTMANQAKNWLPGPCYRQQRVSTITGQNNNSNFAWTAGTKYH
L.NRNSLANPG1AMAZHKDDEERFPSNU1LII'GKQNAARDNADYSDVMLISEEE1K11NPVAf
EEYGIVADNLQQQNTAPQIGTVNSQGALPGMVWQNRDVYLQGPIWAKIPHTDGNFHPSPLMGGF
GLKHPPPQILIKNTPVPADPPTTFNQSKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYT
SNYYKSTSVDFAVNTEGVYSEPRPIGTRYLTRNL (SEQ ID NO: 125)
In the sequence above, the sequence found in VP1, VP2 and VP3 is underlined
(e.g., a
VP3 capsid polypeptide includes, e.g., consists of, amino acids corresponding
to amino acids
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204-739 of SEQ ID NO: 1251), the sequence found in both VP1 and VP2 is in bold
(e.g., a VP2
capsid polypeptide includes, e.g., consists of, the sequence corresponding to
amino acids 138-
735 of SEQ ID NO: 125) and the sequence that is not underlined or bold is
found only in VP1
(e.g., a VP1 capsid polypeptide includes, e.g., consists of, amino acids
corresponding to amino
acids 1-739 of SEQ ID NO: 125).
An example nucleic acid sequence encoding SEQ ID NO: 125 is SEQ ID NO: 126:
ATGGCTGCCGATGGTTATCTTCCAGATTGG CTCGAGGACAACCTCTCTGAGGGCA
TTCGCGAGTGGTGGGCGCTGAAACCTGGAGCCCCGAAGCCCAAAGCCAACCAGC
AAAAGCAGGACGACGGCCGGGGTCTGGTGCTTCCTGGCTACAAGTACCTCGGAC
CCTTCAACGGACTCGACAAGG GGGAGCCCGTCAACGCGGCGGACGCAGCGG CC
CTCGAGCACGACAAGGCCTACGACCAGCAGCTGCAGGCGGGTGACAATCCGTAC
CTG CGGTATAACCACGCCGACGCCGAGTTTCAGGAG CGTCTGCAAGAAGATACGT
CTTTTGGGGGCAACCTCGGGCGAGCAGTCTTCCAGGCCAAGAAGCGGGTTCTCG
AACCTCTCGGTCTGGTTGAGGAAG GCGCTAAGACGG CTCCTGGAAAGAAGAGACC
G GTAGAGCCATCACCCCAG CGTTCTCCAGACTCCTCTACGG G CATCGGCAAGAAA
GGCCAACAGCCCGCCAGAAAAAGACTCAATTTTGGTCAGACTGGCGACTCAGAGT
CAGTTCCAGACCCTCAACCTCTCGGAGAACCTCCAGCAGCG CCCTCTGGTGTGGG
ACCTAATACAATGGCTGCAGGCGGTGGCGCACCAATGGCAGACAATAACGAAGGC
G CCGACGGAGTG GGTAGTTCCTCGGGAAATTGG CATTGCGATTCCACATGGCTGG
G CGACAGAGTCATCACCACCAGCACCCGAACCTGG GCCCTGCCCACCTACAACAA
CCACCTCTACAAGCAAATCTCCAACGGGACATCGGGAGGAG CCACCAACGACAAC
ACCTACTTCGGCTACAGCACCCCCTGGGGGTATTTTGACTTTAACAGATTCCACTG
CCACTTTTCACCACGTGACTGGCAGCGACTCATCAACAACAACTGGGGATTCCGG
CCCAAGAGACTCAGCTTCAAGCTCTTCAACATCCAG GTCAAGGAGGTCACG CAGA
ATGAAGGCACCAAGACCATCGCCAATAACCTCACCAGCACCATCCAGGTGTTTAC
GGACTCGGAGTACCAGCTGCCGTACGTTCTCGGCTCTGCCCACCAGGGCTGCCT
GCCTCCGTTCCCGGCGGACGTGTTCATGATTCCCCAGTACGGCTACCTAACACTC
AACAACGGTAGTCAGGCCGTGGGACGCTCCTCCTTCTACTG CCTGGAATACTTTC
CTTCGCAGATGCTGAGAACCGGCAACAACTTCCAGTTTACTTACACCTTCGAGGAC
GTGCCTTTCCACAGCAGCTACGCCCACAGCCAGAGCTTGGACCGGCTGATGAATC
CTCTGATTGACCAGTACCTGTACTACTTGTCTCGGACTCAAACAACAGGAGGCACG
GCAAATACGCAGACTCTGGGCTTCAGCCAAGGTGGGCCTAATACAATGGCCAATC
AG GCAAAGAACTGGCTG CCAGGACCCTGTTACCGCCAACAACGCGTCTCAACGAC
AACCGGGCAAAACAACAATAGCAACTTTGCCTGGACTGCTGGGACCAAATACCATC
TGAATGGAAGAAATTCATTGGCTAATCCTGGCATCGCTATGGCAACACACAAAGAC
GACGAGGAGCGTTTTTTTCCCAGTAACGGGATCCTGATTTTTGGCAAACAAAATGC
TGCCAGAGACAATGCGGATTACAGCGATGTCATGCTCACCAGCGAGGAAGAAATC
AAAAC CACTAACCCTGTGGCTACAGAGGAATACGGTATCGTGGCAGATAACTTG CA
GCAGCAAAACACGGCTCCTCAAATTGGAACTGTCAACAGCCAGGGGGCCTTACCC
GGTATGGTCTGGCAGAACCGGGACGTGTACCTGCAGGGTCCCATCTGGGCCAAG
ATTCCTCACACGGACGGCAACTTCCACCCGTCTCCGCTGATGGG CGGCTTTGGCC
TGAAACATCCTCCGCCTCAGATCCTGATCAAGAACACGCCTGTACCTGCGGATCCT
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CCGACCACCTTCAACCAGTCAAAGCTGAACTCTTTCATCACGCAATACAGCACCGG
ACAGGTCAGCGTGGAAATTGAATGGGAGCTGCAGAAGGAAAACAGCAAGCGCTG
GAACCCCGAGATCCAGTACACCTCCAACTACTACAAATCTACAAGTGTGGACTTTG
CTGTTAATACAGAAGGCGTGTACTCTGAACCCCGCCCCATTGGCACCCGTTACCTC
ACCCGTAATCTGTAA (SEQ ID NO: 126)
An exemplary reference sequence of wild-type AAV9, SEQ ID NO: 127 (wild-type
AAV9), is as follows:
MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLGPGNGLDKGEP
VNAADAAALEHDKAYWOLKAGDNPYLKYNHADAFFOERLKEDTSFGGNLGRAVFQAKKRLLEP
LOLVEFAAKTAPGKKRPVEQSPQEPDSSAGIGKSGAQPAKKRLNFGQTGDTESVPDPQPIGEPP
AAPSGVGSLTMASGGGAPVADNNEGADGVOSSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHL
YKQISNSTSGGSSNDNAYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNI
QVKEVIDNNGVKTIANNLISTVQVFIDSDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLND
GSQAVGRSSFYCLEYFPSQMLRTGNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLIDQYLYYLS
KTINGSGOWTLKFSVAGPSNMAVQGRNYIPGPSYRQQRVSTTVTONNSEFAWPGASSWALN
GRNSLMNPGPAMASHKEGEDRFFPLSGSLIFGKQCTORDNVDADKVMITNEEEIKTTNPVATES
YGQVAINHQSAQAQAQTGWVQNQGILPGMVWQDRDVYLQGPIWAKIPHTDONFHPSPLMOGFGM
KHPPPQILIKNIPVPADPPTAFNKDKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSN
YYKSNNVEFAVNTEGVYSEPRPIGTRYLTRNL (SEQ ID NO: 127)
In the sequence above, the sequence found in VP1, VP2 and VP3 is underlined
(e.g., a
VP3 capsid polypeptide includes, e.g., consists of, amino acids corresponding
to amino acids
203-737 of SEQ ID NO: 127), the sequence found in both VP1 and VP2 is in bold
(e.g., a VP2
capsid polypeptide includes, e.g., consists of, the sequence corresponding to
amino acids 138-
737 of SEQ ID NO: 127) and the sequence that is not underlined or bold is
found only in VP1
(e.g., a VP1 capsid polypeptide includes, e.g., consists of, amino acids
corresponding to amino
acids 1-737 of SEQ ID NO: 127).
An example nucleic acid sequence encoding SEQ ID NO: 127 is SEQ ID NO: 128:
ATGGCTGCCGATGGTTATCTTCCAGATTGGCTCGAGGACAACCTTAGTGAAGGTAT
TCGCGAGTGGTGGGCTTTGAAACCTGGAGCCCCTCAACCCAAGGCAAATCAACAA
CATCAAGACAACGCTCGAGGTCTTGTGCTTCCGGGTTACAAATACCTTGGACCCG
GCAACGGACTCGACAAG GGGGAGCCGGTCAACGCAGCAGACGCGGCGGCCCTC
GAGCACGACAAGGCCTACGACCAGCAGCTCAAG GCCGGAGACAACCCGTACCTC
AAGTACAACCACGCCGACGCCGAGTTCCAGGAGCGGCTCAAAGAAGATACGTCTT
TTGGGGGCAACCTCGGGCGAGCAGTCTTCCAGGCCAAAAAGAGGCTTCTTGAACC
TCTTGGTCTGGTTGAGGAAGCGGCTAAGACGGCTCCTGGAAAGAAGAGGCCTGTA
GAGCAGTCTCCTCAGGAACCGGACTCCTCCGCG G GTATTGGCAAATCGGGTGCAC
AGCCCGCTAAAAAGAGACTCAATTTCGGTCAGACTGGCGACACAGAGTCAGTCCC
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AGACCCTCAACCAATCGGAGAACCTCCCGCAGCCCCCTCAGGTGTGGGATCTCTT
ACAATGGCTTCAGGTGGTGGCGCACCAGTGGCAGACAATAACGAAGGTGCCGATG
GAGTGGGTAGTTCCTCGGGAAATTGGCATTGCGATTCCCAATGGCTGGGGGACAG
AGTCATCACCACCAGCACCCGAACCTGGGCCCTGCCCACCTACAACAATCACCTC
TACAAG CAAATCTCCAACAGCACATCTG GAG GATCTTCAAATGACAACGCCTACTT
CGGCTACAGCACCCCCTG GGGGTATTTTGACTTCAACAGATTCCACTGCCACTTCT
CACCACGTGACTGGCAGCGACTCATCAACAACAACTGGGGATTCCGGCCTAAGCG
ACTCAACTTCAAGCTCTTCAACATTCAGGTCAAAGAGGTTACGGACAACAATGGAG
TCAAGACCATCGCCAATAACCTTACCAGCACGGTCCAGGTCTTCACGGACTCAGA
CTATCAGCTCCCGTACGTGCTCGGGTCGGCTCACGAGGGCTGCCTCCCGCCGTTC
CCAGCGGACGTTTTCATGATTCCTCAGTACG GGTATCTGACGCTTAATGATGGAAG
CCAGGCCGTGG GTCGTTCGTCCTTTTACTGCCTGGAATATTTCCCGTCGCAAATG C
TAAGAACGGGTAACAACTTCCAGTTCAGCTACGAGTTTGAGAACGTACCTTTCCAT
AG CAGCTACGCTCACAGCCAAAGCCTGGACCGACTAATGAATCCACTCATCGACC
AATACTTGTACTATCTCTCAAAGACTATTAACGGTTCTGGACAGAATCAACAAACGC
TAAAATTCAGTGTG GCCGGACCCAG CAACATGGCTGTCCAGG GAAGAAACTACAT
ACCTGGACCCAGCTACCGACAACAACGTGTCTCAACCACTGTGACTCAAAACAACA
ACAGCGAATTTGCTTGGCCTGGAGCTTCTTCTTGGGCTCTCAATGGACGTAATAGC
TTGATGAATCCTGGACCTGCTATGGCCAGCCACAAAGAAGGAGAGGACCGTTTCT
TTCCTTTGTCTGGATCTTTAATTTTTGGCAAACAAGG AACTGGAAGAGACAACGTG
GATGCGGACAAAGTCATGATAACCAACGAAGAAGAAATTAAAACTACTAACCCGGT
AG CAACGGAGTCCTATGGACAAGTGGCCACAAACCACCAGAGTGCCCAAGCACAG
GCGCAGACCGGCTGGGTTCAAAACCAAGGAATACTTCCGGGTATGGTTTGGCAGG
ACAGAGATGTGTACCTGCAAGGACCCATTTGGGCCAAAATTCCTCACACGGACGG
CAACTTTCACCCTTCTCCGCTGATGGGAGGGTTTGGAATGAAGCACCCGCCTCCT
CAGATCCTCATCAAAAACACACCTGTACCTGCG GATCCTCCAACGGCCTTCAACAA
GGACAAGCTGAACTCTTTCATCACCCAGTATTCTACTGGCCAAGTCAGCGTGGAGA
TCGAGTGGGAGCTGCAGAAGGAAAACAGCAAGCGCTGGAACCCGGAGATCCAGT
ACACTTCCAACTATTACAAGTCTAATAATGTTGAATTTGCTGTTAATACTGAAGGTG
TATATAGTGAACCCCGCCCCATTGGCACCAGATACCTGACTCGTAATCTGTAA (SEQ
ID NO: 128)
An exemplary reference sequence of wild-type AAVrh74, SEQ ID NO: 129 (wild-
type
AAVrh74), is as follows:
MAADGYLPDWLEDNLSEGIREWWDLKP GAPKPKANQQKQDNGRGLVLP GYKYLGPFNGLDKGEP
VNAADAAALEHDKAYDQQLQAGDNPYLRYNHADAEFQERLQEDISFGGNLGRAVFQAKKRVLEP
LGLVES P VK TAP GKKRPVEP SPQRSPDS STGI GKKGQQPAKKRLNF GQTGD SE SVP DPQP I GEP
PAGP SGLGSGTMAAGGGAP MADNNE GAD GVGS S S GNWH CD S TWLGDRV IT T S TRIVALP
TYNNH
LYKQ I SNGT SGGSTNDNTYFGYSTPWGYFDFNRFECHF SPRDWQRL INNNWGFRPKRLNFKLFN
IQVKEVTQNEGTKT IANNLT S T IQVFTD SEYQLPYVLGSAHQCCLPPFPADVFMIPQYGYLTLN
NGSQAVGRS SF YCLEYFP S QMLRIGNNFEFS YNFEDVP FHS SYAHS Q S LDRLMNPL I DQYLYYL
SRTQS TCGTAGTQQLLFSQAGPNNMSAQAKNWLPCPCYRQQRVS TTLSQNNNSNFAWTGATKYH
LNGRDSLVNPGVAMATHKDDEERFFP S SGVLMFGKQGAGKDNVDYS SVMLT SEEEIKTTNPVAT
EQYGVVADNLQQQNAAP IVGAVNSQGALPGMVWQNRDVYLQGP IWAKIPHTDGNFHP SP LMGGF
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GLKHPPPQIL IKNTPVPADPP TTFNQAKLASF I TQYS TGQVSVEIEWELQKENSKRWNPEIQYT
SNYYKSINVDFAVNIEGTYSEPRPIGTRYLIRNL (SEQ ID NO: 129)
An alternative exemplary reference sequence of SEQ ID NO: 130 (alternate wild-
type
AAVrh74) is as follows:
MAADGYLPDWLEDNLSEGIREWWDLKPGAPKPKANQQKONGRGLVLPGYKYLGPFNGLDKGEP
VNAADAAALEHDKAYDOOT,OACIDNPYtRYNHADAFFDERTDEDTSFCMNT,C,RAVFOAKKRVMFP
LGLVESPVKTAPGKKRPVEPSPQRSPDSSTGIGKKGQQPAKKRLNFGQTGDSESVPDPQPIGEP
PAGP SGLGSGTMAAGGGAP MADNNE GAD GVGS S SGNWH CD S TWLGDRVI T T STRTWALP TYNNH
LYKQ I SNGISGGSINDNIYEGYSTPWGYFDFNREECHE SPRDWQRL INNNWGFRPKRLNEKLEN
IQVKEVTQNEGTKT IANNLT S TIQVFTD SEYQLPYVLGSAHQGCLPPFPADVFMIPQYGYLTLN
NGSQAVGRSSFYCLEYFPSQMLRIGNNFEFSYNFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYL
SRTQS TGGTAGTQQLLFSQAGPNNMSAQAKNWLPGPCYRQQRVS TTLSQNNNSNFAWTGATKYH
LNGRDSLVNPGVAMATHKDDEERFFPSSGVLMFGKQGAGKDNVDYSSVMLTSEEEIKTTNPVAT
EQYCVVADNLQQQNAAP IVGAVNSQGALPCMVWQNRDVYLQGP IWAKIPHTDCNFHP SP LMCCF
GLKHPPPQILIKNTPVPADPPTTFTKAKLASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYT
SNYYKSTNVDFAVNTEGTYSEPRPIGTRYLTRNL (SEQ ID NO: 130)
In the sequences above (SEQ ID NO: 129 or SEQ ID NO: 130), the sequence found
in
VP1, VP2 and VP3 is underlined (e.g., a VP3 capsid polypeptide includes, e.g.,
consists of,
amino acids corresponding to amino acids 204-739 of SEQ ID NO: 129), the
sequence found in
both VP1 and VP2 is in bold (e.g., a VP2 capsid polypeptide includes, e.g.,
consists of, the
sequence corresponding to amino acids 137-739 of SEQ ID NO: 129) and the
sequence that is
not underlined or bold is found only in VP1 (e.g., a VP1 capsid polypeptide
includes, e.g.,
consists of, amino acids corresponding to amino acids 1-739 of SEQ ID NO:
129).
An example nucleic acid sequence encoding SEQ ID NO: 129 is SEQ ID NO: 131.
ATGGCTGCCGATGGTTATCTTCCAGATTGG CTCGAGGACAACCTCTCTGAGGGCA
TTCGCGAGTGGTGGGACCTGAAACCTGGAGCCCCGAAACCCAAAGCCAACCAGC
AAAAGCAGGACAACGGCCGGGGTCTGGTGCTTCCTGGCTACAAGTACCTCGGACC
CTTCAACGGACTCGACAAGGG GGAGCCCGTCAACGCGGCGGACGCAGCGGCCCT
CGAGCACGACAAGGCCTACGACCAGCAGCTCCAAGCGGGTGACAATCCGTACCT
G CGGTATAATCACGCCGACG CCGAGTTTCAGGAGCGTCTGCAAGAAGATACGTCT
TTTGG GGGCAACCTCGG GCGCGCAGTCTTCCAG GCCAAAAAGCGGGTTCTCGAA
CCTCTGGGCCTGGTTGAATCG CCGGTTAAGACGGCTCCTGGAAAGAAGAGGCCG
GTAGAGCCATCACCCCAGCGCTCTCCAGACTCCTCTACGG GCATCGGCAAGAAAG
GCCAGCAGCCCGCAAAAAAGAGACTCAATTTTGGGCAGACTGGCGACTCAGAGTC
AGTCCCCGACCCTCAACCAATCG GAGAACCACCAGCAGGCCCCTCTGGTCTGG GA
TCTGGTACAATGGCTGCAGG CGGTGGCGCTCCAATG GCAGACAATAACGAAGGC
G CCGACGGAGTG GGTAGTTCCTCAGGAAATTGGCATTGCGATTCCACATGGCTGG
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GCGACAGAGTCATCACCACCAGCACCCGCACCTGGGCCCTGCCCACCTACAACAA
CCACCTCTACAAGCAAATCTCCAACGGGACCTCGGGAGGAAGCACCAACGACAAC
ACCTACTTCGGCTACAGCACCCCCTGGGGGTATTTTGACTTCAACAGATTCCACTG
CCACTTTTCACCACGTGACTGGCAGCGACTCATCAACAACAACTGGGG ATTCCGG
CCCAAGAGGCTCAACTTCAAGCTCTTCAACATCCAAG TCAAGGAGGTCACGCAG A
ATGAAGGCACCAAGACCATCG CCAATAACCTTACCAGCACGATTCAGGTCTTTACG
GACTCGGAATACCAGCTCCCGTACGTGCTCGGCTCGGCGCACCAGGGCTGCCTG
CCTCCGTTCCCGGCGGACGICTTCATGATTCCTCAGTACGGGTACCTGACTCTGAA
CAATGGCAGTCAGGCTGTGGGCCGGTCGTCCTTCTACTGCCTGGAGTACTTTCCT
TCTCAAATGCTGAGAACGGGCAACAACTTTGAATTCAGCTACAACTTCGAGGACGT
GCCCTTCCACAGCAGCTACGCGCACAGCCAGAGCCTGGACCGGCTGATGAACCC
TCTCATCGACCAGTACTTGTACTACCTGTCCCGGACTCAAAGCACGGGCGGTACT
GCAGGAACTCAGCAGTTGCTATTTTCTCAGGCCGGGCCTAACAACATGTCGGCTC
AG GCCAAGAACTGGCTACCCGGTCCCTGCTACCGG CAGCAACGTGTCTCCACGAC
ACTGTCGCAGAACAACAACAGCAACTTTGCCTGGACGGGTG CCACCAAGTATCAT
CTG AATGGCAGAGACTCTCTGG TGAATCCTGGCGTTGCCATGGCTACCCACAAGG
ACGACGAAGAGCGATTTTTTCCATCCAGCG GAGTCTTAATGTTTGGGAAACAGGG A
GCTGGAAAAGACAACGTGGACTATAGCAGCGTGATGCTAACCAGCGAGGAAGAAA
TAAAGACCACCAACCCAGTGGCCACAGAACAGTACGGCGTGGTGGCCGATAACCT
GCAACAGCAAAACGCCGCTCCTATTGTAGGGGCCGTCAATAGTCAAGGAGCCTTA
CCTGGCATGGTGTGGCAGAACCGGGACGTGTACCTGCAGGGTCCCATCTGGGCC
AAGATTCCTCATACGGACGGCAACTTTCATCCCTCGCCGCTGATGGGAGGCTITG
GACTGAAGCATCCGCCTCCTCAGATCCTGATTAAAAACACACCTGTTCCCGCGGAT
CCTCCGACCACCTTCAATCAGGCCAAGCTGGCTTCTTTCATCACGCAGTACAGTAC
CGGCCAGGTCAGCGTGGAGATCGAGTGGGAGCTGCAGAAGGAGAACAGCAAACG
CTGGAACCCAGAGATTCAGTACACTTCCAACTACTACAAATCTACAAATGTGGACTT
TG CTGTCAATACTGAGGGTACTTATTCCGAGCCTCG CCCCATTGGCACCCGTTACC
TCACCCGTAATCTGTAA (SEQ ID NO: 131)
The present disclosure refers to structural capsid proteins (including VP1,
VP2 and VP3)
which are encoded by capsid (Cap) genes. These capsid proteins form an outer
protein structural
shell (i.e. capsid) of a viral vector such as AAV. VP capsid proteins
synthesized from Cap
polynucleotides generally include a methionine as the first amino acid in the
polypeptide
sequence (Met 1), which is associated with the start codon (AUG or ATG) in the
corresponding
Cap nucleotide sequence. However, it is common for a first-rnethionine (Metl)
residue or
generally any first amino acid (AA1) to be cleaved off after or during
polypeptide synthesis by
protein processing enzymes such as Met-aminopeptidases. This "Met/AA-clipping"
process
often correlates with a corresponding acetylation of the second amino acid in
the polypeptide
sequence (e.g., alanine, valine, serine, threonine, etc.). Met-clipping
commonly occurs with VP1
and VP3 capsid proteins but can also occur with VP2 capsid proteins. Where the
Met/AA-
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clipping is incomplete, a mixture of one or more (one, two or three) VP capsid
proteins
comprising the viral capsid can be produced, some of which include a Metl/AA1
amino acid
(Met+/AA+) and some of which lack a Metl/AA1 amino acid as a result of Met/AA-
clipping
(Met-/AA-). For further discussion regarding Met/AA-clipping in capsid
proteins, see Jin, et al.
Direct Liquid Chromatography/Mass Spectrometry Analysis for Complete
Characterization of
Recombinant Adeno-Associated Virus Capsid Proteins. Hum Gene Ther Methods.2017
Oct.28(5):255-267; Hwang, et al. N- Terminal Acetylation of Cellular Proteins
Creates Specific
Degradation Signals. Science. 2010 February 19.327(5968): 973-977; the
contents of which are
each incorporated herein by reference in its entirety. According to the
present disclosure,
references to capsid polypeptides is not limited to either clipped (Met-/AA-)
or unclipped
(Met+/AA+) and, in context, also refer to independent capsid polypeptides,
viral capsids
comprised of a mixture of capsid proteins, and/or polynucleotide sequences (or
fragments
thereof) which encode, describe, produce or result in capsid polypeptides of
the present
disclosure. A direct reference to a -capsid polypeptide" (such as VP1, VP2 or
VP3) also
comprise VP capsid proteins which include a Metl/AA1 amino acid (Met+/AA+) as
well as
corresponding VP capsid polypeptide which lack the Metl/AA1 amino acid e.g. as
a result of
Met/AA-clipping (Met-/AA-). Further according to the present disclosure, a
reference to a
specific SEQ ID NO: (whether a protein or nucleic acid) which comprises or
encodes,
respectively, one or more capsid polypeptides which include a Metl/AA1 amino
acid
(Met+/AA+) should be understood to teach the VP capsid polypeptides which lack
the
Metl/AA1 amino acid as upon review of the sequence, it is readily apparent
that the first listed
amino acid (whether or not Metl/AA1) may be absent. As a non-limiting example,
reference to a
VP1 polypeptide sequence which is 736 amino acids in length and which includes
a "Metl"
amino acid (Met+) encoded by the AUG/ATG start codon is also understood to
teach a VP1
polypeptide sequence which is 735 amino acids in length and which does not
include the "Metl"
amino acid (Met-) of the 736 amino acid Met+ sequence. As a second non-
limiting example,
reference to a VP1 polypeptide sequence which is 736 amino acids in length and
which includes
an "AA1" amino acid (AA1+) encoded by any NNN initiator codon can also be
understood to
teach a VP1 polypeptide sequence which is 735 amino acids in length and which
does not
include the "AA1" amino acid (AA1-) of the 736 amino acid AA1+ sequence.
References to
viral capsids formed from VP capsid proteins (such as reference to specific
AAV capsid
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serotypes), can incorporate VP capsid proteins which include a Metl/AA1 amino
acid
(Met+/AA1+), corresponding VP capsid proteins which lack the Me1l/AA1 amino
acid e.g. as a
result of Met/AA1-clipping (Met-/AA1-), and combinations thereof (Met+/AA1+
and Met-
/AA1-). As a non-limiting example, an AAV capsid serotype can include VP1
(Met+/AA1+),
VP1 (Met-/AA1-), or a combination of VP1 (Met+/AA1+) and VP1 (Met- /AA1-). An
AAV
capsid serotype can also include VP3 (Met+/AA1+), VP3 (Met-/AA1-), or a
combination of VP3
(Met+/AA1+) and VP3 (Met-/AA1-); and can also include similar optional
combinations of VP2
(Met+/AA1) and VP2 (Met-/AA1-).
In some embodiments, the reference AAV capsid sequence comprises an amino acid
sequence with 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%,
63%,
64%. 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%. 74%, 75%, 76%, 77%, 78%,
79%,
80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%. 91%, 92%, 93%, 94%,
95%.
96%, 97%, 98%, 99%, or 100% identity to any of the those described above.
In some embodiments, the reference AAV capsid sequence is encoded by a
nucleotide
sequence with 50%, 51%. 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%. 60%, 61%, 62%,
63%,
64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%,
79%,
80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,
95%,
96%, 97%, 98%, 99%, or 100% identity to any of those described above. In
certain
embodiments, the reference sequence is not an AAV capsid sequence and is
instead a different
vector (e.g., lentivirus, plasmid, etc.).
In some embodiments, a nucleic acid of the disclosure (e.g., encoding an AAV2
variant
capsid protein) comprises conventional control elements or sequences which are
operably linked
to the nucleic acid molecule in a manner which permits transcription,
translation and/or
expression in a cell transfected with the nucleic acid (e.g., a plasmid vector
comprising said
nucleic acid) or infected with a virus comprising said nucleic acid. As used
herein, "operably
linked" sequences include both expression control sequences that are
contiguous with the gene of
interest and expression control sequences that act in trans or at a distance
to control the gene of
interest.
Expression control sequences include efficient RNA processing signals such as
splicing
and polyadenylation (polyA) signals; appropriate transcription initiation,
termination, promoter
and enhancer sequences; sequences that stabilize cytoplasmic mRNA; sequences
that enhance
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protein stability; sequences that enhance translation efficiency (e.g., Kozak
consensus sequence);
and in some embodiments, sequences that enhance secretion of the encoded
transgene product.
Expression control sequences, including promoters which are native,
constitutive, inducible
and/or tissue-specific, are known in the art and may be utilized with the
compositions and
methods disclosed herein.
In some embodiments, the native promoter for the transgene may be used.
Without
wishing to be bound by theory, the native promoter may mimic native expression
of the
transgene, or provide temporal, developmental, or tissue-specific expression,
or expression in
response to specific transcriptional stimuli. In some embodiment, the
transgene may be operably
linked to other native expression control elements, such as enhancer elements,
polyadenylation
sites or Kozak consensus sequences, e.g., to mimic the native expression.
In some embodiments, the transgene is operably linked to a tissue-specific
promoter.
In some embodiments, a vector, e.g., a plasmid, carrying a transgene may also
include a
selectable marker or a reporter gene. Such selectable reporters or marker
genes can be used to
signal the presence of the vector, e.g., plasmid, in bacterial cells. Other
components of the vector,
e.g., plasmid, may include an origin of replication. Selection of these and
other promoters and
vector elements are conventional and many such sequences are available (see,
e.g., Sambrook et
al, and references cited therein).
In some embodiments, the capsid polypeptide present in a viral particle
increases
transduction in the eye as compared to a viral particle with a reference
capsid polypeptide, for
example, with the wild-type capsid polypeptide (SEQ ID NO: 1). In some
embodiments, the
capsid polypeptide present in a viral particle increases transduction in the
retina as compared to a
viral particle with a reference capsid polypeptide, for example, with the wild-
type capsid
polypeptide (SEQ ID NO: 1). In some embodiments, the capsid polypeptide
present in a viral
particle increases transduction in the non-macular retina as compared to a
viral particle with a
reference capsid polypeptide, for example, with the wild-type capsid
polypeptide (SEQ ID NO:
1). In some embodiments, the capsid polypeptide present in a viral particle
increases transduction
in the macula as compared to a viral particle with a reference capsid
polypeptide, for example,
with the wild-type capsid polypeptide (SEQ ID NO: 1). In some embodiments, the
capsid
polypeptide present in a viral particle increases transduction in the
trabecular meshwork as
compared to a viral particle with a reference capsid polypeptide, for example,
with the wild-type
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capsid polypeptide (SEQ ID NO: 1). In some embodiments, the capsid polypeptide
present in a
viral particle increases transduction in the trabecular meshwork relative to
retina as compared to
a viral particle with a reference capsid polypeptide, for example, with the
wild-type capsid
polypeptide (SEQ ID NO: 1). In some embodiments, the capsid polypeptide
present in a viral
particle increases transduction in the trabecular meshwork relative to non-
macular retina as
compared to a viral particle with a reference capsid polypeptide, for example,
with the wild-type
capsid polypeptide (SEQ ID NO: 1). In some embodiments, the capsid polypeptide
present in a
viral particle increases transduction in the trabecular meshwork relative to
macula as compared
to a viral particle with a reference capsid polypeptide, for example, with the
wild-type capsid
polypeptide (SEQ ID NO: 1). Tn some embodiments, the capsid polypeptide
present in a viral
particle increases transduction in the macula relative to retina as compared
to a viral particle with
a reference capsid polypeptide, for example, with the wild-type capsid
polypeptide (SEQ ID NO:
1). In some embodiments, the capsid polypeptide present in a viral particle
increases transduction
in the macula relative to non-macular retina as compared to a viral particle
with a reference
capsid polypeptide, for example, with the wild-type capsid polypeptide (SEQ ID
NO: 1). In some
embodiments, the capsid polypeptide present in a viral particle increases
transduction in the
macula relative to trabecular meshwork as compared to a viral particle with a
reference capsid
polypeptide, for example, with the wild-type capsid polypeptide (SEQ ID NO:
1). In some
embodiments, the capsid polypeptide present in a viral particle increases
transduction in the
macula relative to non-macular retina and trabecular meshwork as compared to a
viral particle
with a reference capsid polypeptide, for example, with the wild-type capsid
polypeptide (SEQ ID
NO: 1). In some embodiments, the capsid polypeptide present in a viral
particle increases
transduction in the macula relative to retina and trabecular meshwork as
compared to a viral
particle with a reference capsid polypeptide, for example, with the wild-type
capsid polypeptide
(SEQ ID NO: 1). In some embodiments, the capsid polypeptide present in a viral
particle
increases transduction in the retina relative to macula and trabecular
meshwork as compared to a
viral particle with a reference capsid polypeptide, for example, with the wild-
type capsid
polypeptide (SEQ ID NO: 1). In some embodiments, the capsid polypeptide
present in a viral
particle increases transduction in the non-macular retina relative to macula
and trabecular
meshwork as compared to a viral particle with a reference capsid polypeptide,
for example, with
the wild-type capsid polypeptide (SEQ ID NO: 1). In some embodiments, the
capsid polypeptide
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present in a viral particle increases transduction in the trabecular meshwork
relative to macula
and retina as compared to a viral particle with a reference capsid
polypeptide, for example, with
the wild-type capsid polypeptide (SEQ ID NO: 1). In some embodiments, the
capsid polypeptide
present in a viral particle increases transduction in the trabecular meshwork
relative to macula
and non-macular retina as compared to a viral particle with a reference capsid
polypeptide, for
example, with the wild-type capsid polypeptide (SEQ ID NO: 1).
In some embodiments, the capsid polypeptide present in a viral particle
increases ocular
transduction at least 1-fold, e.g., as compared to a viral particle with a
reference capsid
polypeptide, for example, with the wild-type capsid polypeptide (SEQ ID NO:
1). In some
embodiments, the capsid polypeptide present in a viral particle increases
ocular transduction at
least 2-fold, e.g., as compared to a viral particle with a reference capsid
polypeptide, for
example, with the wild-type capsid polypeptide (SEQ ID NO: 1). In some
embodiments, the
capsid polypeptide present in a viral particle increases ocular transduction 4-
fold, e.g., as
compared to a viral particle with a reference capsid polypeptide, for example,
with the wild-type
capsid polypeptide (SEQ ID NO: 1). In some embodiments. the capsid polypeptide
present in a
viral particle increases ocular transduction 6-fold, e.g., as compared to a
viral particle with a
reference capsid polypeptide, for example, with the wild-type capsid
polypeptide (SEQ ID NO:
1). In some embodiments, the capsid polypeptide present in a viral particle
increases v
transduction 8-fold, e.g., as compared to a viral particle with a reference
capsid polypeptide, for
example, with the wild-type capsid polypeptide (SEQ ID NO: 1). In some
embodiments, the
capsid polypeptide present in a viral particle increases ocular transduction
10-fold, e.g., as
compared to a viral particle with a reference capsid polypeptide, for example,
with the wild-type
capsid polypeptide (SEQ ID NO: 1). In some embodiments, the capsid polypeptide
present in a
viral particle increases ocular transduction 15-fold, e.g., as compared to a
viral particle with a
reference capsid polypeptide, for example, with the wild-type capsid
polypeptide (SEQ ID NO:
1). In some embodiments, the capsid polypeptide present in a viral particle
increases ocular
transduction 16-fold, e.g., as compared to a viral particle with a reference
capsid polypeptide, for
example, with the wild-type capsid polypeptide (SEQ ID NO: 1). In some
embodiments, the
capsid polypeptide present in a viral particle increases ocular transduction
32-fold, e.g., as
compared to a viral particle with a reference capsid polypeptide, for example,
with the wild-type
capsid polypeptide (SEQ ID NO: 1). In some embodiments, the capsid polypeptide
present in a
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viral particle increases ocular transduction 64-fold, e.g., as compared to a
viral particle with a
reference capsid polypeptide, for example, with the wild-type capsid
polypeptide (SEQ ID NO:
1). In some embodiments, the capsid polypeptide present in a viral particle
increases ocular
transduction 100-fold, e.g., as compared to a viral particle with a reference
capsid polypeptide,
for example, with the wild-type capsid polypeptide (SEQ ID NO: 1). In some
embodiments, the
capsid polypeptide present in a viral particle increases ocular transduction
150-fold, e.g., as
compared to a viral particle with a reference capsid polypeptide, for example,
with the wild-type
capsid polypeptide (SEQ ID NO: 1). In some embodiments, the capsid polypeptide
present in a
viral particle increases ocular transduction 200-fold, e.g., as compared to a
viral particle with a
reference capsid polypeptide, for example, with the wild-type capsid
polypeptide (SEQ ID NO:
1). In some embodiments, the capsid polypeptide present in a viral particle
increases ocular
transduction 500-fold, e.g., as compared to a viral particle with a reference
capsid polypeptide,
for example, with the wild-type capsid polypeptide (SEQ ID NO: 1). In some
embodiments, the
capsid polypeptide present in a viral particle increases ocular transduction
1000-fold, e.g., as
compared to a viral particle with a reference capsid polypeptide, for example,
with the wild-type
capsid polypeptide (SEQ ID NO: 1). In embodiments, increased ocular
transduction is measured
by comparing the level of mRNA in the target tissue (e.g., in a cell or
population of cells of the
target tissue) produced from a nucleic acid packaged in the variant viral
particle with the level of
mRNA in the target tissue (e.g., in a cell or population of cells of the
target tissue) produced
from a nucleic acid packaged in a reference viral particle (e.g., packaged in
a capsid comprising
capsid polypeptides of SEQ ID NO: 1).
In some embodiments, the capsid polypeptide is an isolated or purified
polypeptide (e.g.,
isolated or purified from a cell, other biological component, or contaminant).
In some
embodiments, the variant polypeptide is present in a dependoparvovirus
particle, e.g., described
herein. In some embodiments, the variant capsid polypeptide is present in a
cell, cell-free system,
or translation system, e.g., described herein.
In some embodiments, the capsid polypeptide is present in a dependoparvovirus
B (e.g.,
AAV2) particle. In some embodiments, the capsid particle has increased ocular
transduction.
In some embodiments, a dependoparvovirus particle comprises an amino acid
sequence
that has at least 80, 85, 90, 91, 92, 93, 94. 95, 96, 97, 98, 99, or 100%
identity to the amino acid
sequences provided for herein (e.g., SEQ ID NO: 2-46). In some embodiments,
the variant
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capsid polypeptide comprises an amino acid sequence that differs by no more
than 30, 29, 28, 27,
26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8,7, 6,
5,4, 3,2, or 1 amino
acids from the amino acid sequence of a variant capsid polypeptide provided
for herein.
In some embodiments, the additional alteration improves a production
characteristic of a
dependoparvovirus particle or method of making the same. In some embodiments,
the additional
alteration improves or alters another characteristic of a dependoparvovirus
particle, e.g., tropism.
VP1 Nucleic Acids and Polypeptides
The disclosure is further directed, in part, to a nucleic acid comprising a
sequence
encoding a dependoparvovirus (e.g., dependoparvovirus B, e.g., an AAV2)
polypeptide as
provided for herein, as well as to a VP1 polypeptide encoded by the same. In
some
embodiments, the polypeptide comprises a sequence of SEQ ID NOs: 2, 3, 4, 5,
6, 7, 8, 9, 10, 11,
12, 13, 14, 15, 16, 17, 18, 19, 20. 21, 22, 23, 24, 25, 25, 27, 28. 29, 31,
32, 33. 34, 35, 36, 37, 38,
39, 40, 41, 42, 43, 44, 45, or 46.
Dependoparvovirus Particles
The disclosure is also directed, in part, to a dependoparvovirus particle
(e.g., a functional
dependoparvovirus particle) comprising a nucleic acid or polypeptide described
herein or
produced by a method described herein.
Dependoparvovirus is a single-stranded DNA parvovirus that grows only in cells
in
which certain functions are provided, e.g., by a co-infecting helper virus.
Several species of
dependoparvovirus are known, including dependoparvovirus A and
dependoparvovirus B, which
include serotypes known in the art as adeno-associated viruses (AAV). At least
thirteen serotypes
of AAV that have been characterized. General information and reviews of AAV
can be found in,
for example, Carter, Handbook of Parvoviruses, Vol. 1, pp. 169-228 (1989). and
Berns, Virology, pp. 1743-1764, Raven Press, (New York, 1990). AAV serotypes,
and to a
degree, dependoparvovirus species, are significantly interrelated structurally
and functionally.
(See, for example, Blacklowe, pp. 165-174 of Parvoviruses and Human Disease,
J. R. Pattison,
ed. (1988); and Rose, Comprehensive Virology 3:1-61 (1974)). For example, all
AAV serotypes
apparently exhibit very similar replication properties mediated by homologous
rep genes; and all
bear three related capsid proteins. In addition, heteroduplex analysis reveals
extensive cross-
hybridization between serotypes along the length of the genome, further
suggesting
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interrelatedness. Dependoparvoviruses genomes also comprise self-annealing
segments at the
termini that correspond to "inverted terminal repeat sequences" (ITRs).
The genomic organization of naturally occurring dependoparvoviruses, e.g.. AAV
serotypes, is very similar. For example, the genome of AAV is a linear, single-
stranded DNA
molecule that is approximately 5,000 nucleotides (nt) in length or less.
Inverted terminal repeats
(ITRs) flank the unique coding nucleotide sequences for the non-structural
replication (Rep)
proteins and the structural capsid (Cap) proteins. Three different viral
particle (VP) proteins form
the capsid. The terminal 145 nt are self-complementary and are organized so
that an
energetically stable intramolecular duplex forming a T-shaped hairpin may be
formed. These
hairpin structures function as an origin for viral DNA replication, serving as
primers for the
cellular DNA polymerase complex. The Rep genes encode the Rep proteins: Rep78,
Rep68,
Rep52. and Rep40. Rep78 and Rep68 are transcribed from the p5 promoter, and
Rep 52 and
Rep40 are transcribed from the p19 promoter. The cap genes encode the VP
proteins, VP1, VP2,
and VP3. The cap genes are transcribed from the p40 promoter.
In some embodiments, a dependoparvovirus particle of the disclosure comprises
a nucleic
acid comprising a capsid polypeptide provided for herein. In some embodiments,
the particle
comprises a polypeptide as provided for herein.
In some embodiments, the dependoparvovirus particle of the disclosure may be
an AAV2
particle. In some embodiments, the AAV2 particle comprises a capsid
polypeptide as provided
for herein or a nucleic acid molecule encoding the same.
In some embodiments the dependoparvovirus particle comprises a capsid
comprising a
variant capsid polypeptide described herein. In embodiments, the
dependoparvovirus particle
comprises variant capsid polypeptide described herein and a nucleic acid
molecule. In
embodiments, the dependoparvovirus particle comprises variant capsid
polypeptide described
herein and a nucleic acid molecule comprising one or more inverted terminal
repeat sequences
(ITRs), for example, ITRs derived from an AAV2 dependoparvovirus, one or more
regulatory
elements (for example, a promoter), and a payload (e.g., as described herein).
In embodiments, at
least one of the ITRs is modified. In embodiments, the nucleic acid molecule
is single-stranded.
In embodiments, the nucleic acid molecule is self-complementary.
Increased Ocular Transduction Characteristics
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The disclosure is directed, in part, to nucleic acids, polypeptides, cells,
cell free systems,
translation systems, viral particles, and methods associated with making the
same to produce
virus particles that have increased ocular transduction as compared to a virus
particle having
capsid polypeptides of a reference sequence, e.g., with a wild-type sequence
of SEQ ID NO: 1.
In some embodiments, a use of a viral particle comprising the variant capsid
polypeptide leads to
increased ocular transduction of a transgene in the eye, and, therefore,
expression of the
transgene in the eye. In some embodiments, a use of a viral particle
comprising the variant
capsid polypeptide leads to increased ocular transduction of a transgene in
the retina, and,
therefore, expression of the transgene in the retina. In some embodiments, a
use of a viral
particle comprising the variant capsid polypeptide leads to increased ocular
transduction of a
transgene in the non-macular retina, and, therefore, expression of the
transgene in the non-
macular retina. In some embodiments, a use of a viral particle comprising the
variant capsid
polypeptide leads to increased ocular transduction of a transgene in the
macula, and, therefore,
expression of the transgene in the macula. In some embodiments, a use of a
viral particle
comprising the variant capsid polypeptide leads to increased ocular
transduction of a transgene in
the trabecular meshwork, and, therefore, expression of the transgene in the
trabecular meshwork.
In some embodiments, a use of a viral particle comprising the variant capsid
polypeptide leads to
increased ocular transduction of a transgene in the front third of the eye,
which includes the
structures in front of the vitreous humor. Examples of structures in front of
the vitreous humor,
include the cornea, iris, ciliary body, lens, trabecular meshwork, and
Schlemm's canal.
Accordingly, in some embodiments, use of a viral particle comprising the
variant capsid
polypeptide leads to increased ocular transduction of a transgene in the
cornea, iris, ciliary body,
lens, trabecular meshwork, or Schlemm's canal, or any combination thereof. In
some
embodiments, a use of a viral particle comprising the variant capsid
polypeptide leads to
increased ocular transduction of a transgene posterior to the lens, such as in
the anterior hyaloid
membrane and all of the optical structures behind it, such as the vitreous
humor, retina, choroid
or optic nerve, or any combination thereof. Accordingly, in some embodiments,
use of a viral
particle comprising the variant capsid polypeptide leads to increased ocular
transduction of a
transgene in the anterior hyaloid membrane and all of the optical structures
behind it, such as the
vitreous humor, retina, choroid or optic nerve, or any combination thereof. In
some
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embodiments, a use of a viral particle comprising the variant capsid
polypeptide leads to
increased ocular transduction of a transgene in the front third of the eye and
posterior to the lens.
In some embodiments, the increase in ocular transduction is, on a 10g2 scale,
about 1-5
times better (e.g., about 2-5 times better, e.g.. about 3-5 times better) than
a virus particle having
a reference sequence capsid polypeptide, e.g., having the wild-type capsid
polypeptide SEQ ID
NO: 1.
In some embodiments, the capsid polypeptide present in a viral particle
increases
transduction without increasing the biodistribution of the variant capsid
polypeptide in the eye
relative to SEQ ID NO: 1. In some embodiments, the capsid polypeptide present
in a viral
particle increases transduction without increasing the biodistribution of the
variant capsid
polypeptide in the retina relative to SEQ ID NO: 1. In some embodiments, the
capsid
polypeptide present in a viral particle increases transduction without
increasing the
biodistribution of the variant capsid polypeptide in the trabecular meshwork
relative to SEQ ID
NO: 1.
Table 5 lists information regarding biodistribution of variant
dependoparvovirus particles
comprising capsid polypeptides of the indicated variant capsid in the
different layers, structures,
and/or parts of the eye. Biodistribution in retina is as measured following
IVT injection.
Biodistribution in trabecular meshwork is as measured following IC injection.
Table 5
Name SEQ ID Biodistribution in Biodistribution in
NO: of Retina as compared to Trabecular Meshwork as
VP1 wild-type SEQ ID NO: compared to wild-type
capsid 1 SEQ ID NO: 1
poly- (Log2) (Log2)
peptide
VAR-1 2 0.219554617 -0.129512259
VAR-2 3 0.082776163 -0.093624787
VAR-3 4 1.048865823 -0.097505361
VAR-4 5 1.985458577 0.752795147
VAR-5 6 0.150339954 0.240806265
VAR-6 7 -0.329057077 -0.127768486
VAR-7 8 -0.485573763 0.038632248
VAR-8 9 2.592283234 0.639490423
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VAR-9 10 0.907849174 -0.129048301
VAR-10 11 0.582650953 -0.951767221
VAR-11 12 1.055569616 0.517659804
VAR-12 13 0.38695579 0.536577648
VAR-13 14 1.336116818 1.154005075
VAR-14 15 0.05824263 0.403134632
VAR-15 16 -2.505454013 -0.891040148
VAR-16 17 -1.08115075 0.639490423
VAR-17 18 0.979546949 -0.282516221
VAR-18 19 -0.226638005 -0.219115256
VAR-19 20 0.876781617 0.618096401
VAR-20 21 0.837693238 0.750988668
VAR-21 22 -0.239478568 0.539038877
VAR-22 23 -1.789903254 0.973742845
VAR-23 24 -0.304881561 0.427104122
VAR-24 25 -1.041019591 1.040413058
VAR-25 26 0.901098892 -0.888153107
VAR-26 27 -1.095478038 Not measured
VAR-27 28 -1.157196676 0.001971904
VAR-28 29 0.5459013 -0.238124001
VAR-29 30 1.266904701 -0.49656868
VAR-30 31 0.443890223 0.110293386
VAR-31 32 1.695754653 0.781373819
VAR-32 33 1.970541935 0.194078521
VAR-33 34 1.615256454 -0.347209059
VAR-34 35 1.104478115 1.045899544
VAR-35 36 2.189521186 0.671956827
VAR-36 37 1.720060573 0.031648718
VAR-37 38 1.810695682 1.101034688
VAR-38 39 0.820878902 1.097873642
VAR-39 40 1.282933021 1.250467569
VAR-40 41 0.218752223 0.889665279
VAR-41 42 0.059531262 0.04061276
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VAR-42 43 ¨0.959157062 0.791762522
VAR-43 44 ¨1.779802685 ¨0.20186619
VAR-44 45 ¨0.569765447 0.946089818
VAR-45 46 0.901098892 0.048731959
According to some embodiments, disclosed herein are capsid polypeptides
comprising a
point mutation that corresponds to a leucine at a position corresponding to
544 of SEQ ID NO: 1
(e.g., a I554L as compared to SEQ ID NO: 1). For example, VAR-22 includes a
I554L mutation
as compared to SEQ ID NO: 1 (shown in Table 1) Additional variant capsid
polypeptides
(VAR-46, VAR-47, VAR-48, VAR-49) having a I554L mutation were evaluated, where
each of
these additional variant capsid polypeptides do not have a mutation at the 555
amino acid
position as compared to SEQ ID NO: 1, as in VAR-22. Data for VAR-46, VAR-47,
VAR-48,
and VAR-49 is shown in Table 6, and like VAR-22, virus particles comprising
these capsid
polypeptides comprising the I554L mutation resulted in increased transduction
of trabecular
meshwork tissue, relative to virus particles comprising capsid polypeptides of
SEQ ID NO: 1.
Thus, without being bound by theory, it is surprisingly discovered that the
presence of a I554L
mutation contributes to increased transduction of trabecular meshwork tissue
in the context of
diverse capsid polypeptide sequences, relative to capsid polypeptides which do
not comprise the
mutation. Relative to SEQ ID NO: 1, these additional variant capsid
polypeptides comprising a
1554L mutation have an edit distance in the range of 8-12 (as shown in Table
6). Relative to
VAR-22, these additional variant capsid polypeptides comprising a I554L
mutation have an edit
distance of 11-16 (as shown in Table 6). Thus, as described herein are capsid
polypeptides,
nucleic acid molecules that encode capsid polypeptides, virus particles, and
methods of making
and using the same, comprising a I554L mutation as compared to SEQ ID NO: 1.
In some
embodiments, the capsid polypeptide comprising a I554L mutation has greater
than 95%, greater
than 96%, greater than 97% or optionally greater than 98% or greater than 99%
sequence identity
to SEQ ID NO: 1. In some embodiments, the capsid polypeptide comprising a
I554L mutation
has greater than 95%, greater than 96%, greater than 97% or optionally greater
than 98% or
greater than 99% sequence identity to the capsid polypeptide sequence of VAR-
22. In some
embodiments, the capsid polypeptide comprising a I554L mutation has 1-20,
e.g., 1, 2, 3, 4, 5, 6,
7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 additional mutations
relative to SEQ ID NO: 1.
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Also disclosed, in some embodiments, are nucleic acid molecules that encode a
capsid
polypeptide that comprises a mutation corresponding to a I554L mutation as
compared to SEQ
ID NO: 1. Also disclosed, in some embodiments are virus particles, for example
dependoparvovirus particles, comprising a capsid polypeptide that comprises a
mutation
corresponding to a I554L mutation as compared to SEQ ID NO: 1.
As shown in Table 6, each of these additional variant capsid polypeptides
comprising a
I554L mutation have increased trabecular transduction as compared to SEQ ID
NO: 1.
Accordingly, in some embodiments, a capsid polypeptide, comprising a mutation
that
corresponds to I554L as compared to SEQ ID NO: 1, present in a viral particle
increases
transduction in the trabecular meshwork as compared to a viral particle with a
reference capsid
polypeptide, for example, with the wild-type capsid polypeptide (SEQ ID NO:
1).
Table 6
Library Experiment 1
Name Edit Edit Macular Retinal Trabecular Virus
Production as
Distance Distance Transduction Transduction Transduction
compared to wild-type
to SEQ to VAR- as compared as compared as compared
SEQ ID NO: 1 (Log2)
ID NO: 22 to wild-type to wild-type to wild-type
1 SEQ ID NO: 1 SEQ ID NO: 1 SEQ ID NO: 1
(Log2) (Log2) (Log2)
VAR-22 9 -1.16 0.81 2.95 0.72
Library Experiment 2
Name Edit Edit Macular Retinal Trahecular Virus
Production as
Distance Distance Transduction Transduction Transduction
compared to wild-type
to SEQ to VAR- as compared as compared as compared
SEQ Ill NO: 1 (Log2)
ID NO: 22 to wild-type to wild-type to wild-type
1 SEQ ID NO: 1 SEQ ID NO: 1 SEQ ID NO: 1
Anterior Posterior
(Log2) (Log2) (Log2) eye
library eye library
VAR-22 9 -1.57 Not Measured -0.80 -4.36
1.03
VAR-46 8 11 Not Measured -3.23 2.54 -4.83
0.44
VAR-47 12 13 Not Measured -3.30 3.24 -3.23
2.90
VAR-48 10 15 -3.92 -3.18 2.10 2.55
3.04
VAR-49 11 16 Not Measured -3.52 3.28 1.49
-0.02
In some embodiments, the capsid polypeptide comprising the L at position 554
(e.g., the
I554L) mutation has one or more additional mutations. In some embodiments, the
capsid
polypeptide comprises point mutations that correspond to a leucine at a
position corresponding to
544 in SEQ ID NO: 1 and a serine at a position corresponding to 561 in SEQ ID
NO:1 (e.g.,
comprising a I554L and D561S as compared to SEQ ID NO: 1). For example, VAR-
22, VAR-
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46, and VAR-47 include mutations that correspond to I554L and D561S as
compared to SEQ ID
NO: 1. VAR-46 and VAR-47 have no mutations at the 560, 562, 563, 564, and 565
amino acid
residues as compared to SEQ ID NO: 1, as in VAR-22. Thus, without being bound
by theory, it
is surprisingly discovered that the presence of both I554L and D561S mutations
contribute to
increased transduction of trabecular meshwork tissue in the context of diverse
capsid polypeptide
sequences, relative to capsid polypeptides which do not comprise these
mutations.
In some embodiments, the capsid polypeptide comprises point mutations that
correspond
to I554L and T581D as compared to SEQ ID NO: 1. For example, VAR-22, VAR-46,
and
VAR-47 include mutations that correspond to a leucine at a position
corresponding to 544 in
SEQ ID NO: 1 and a aspartic acid at a position corresponding to 581 in SEQ ID
NO:1 (e.g.,
comprising a I554L and T581D as compared to SEQ ID NO: 1. VAR-46 and VAR-47
comprise
an I554L mutation and a T581D mutation, but have no mutations at the 579. 580,
582, 583, and
584 amino acid residues as compared to SEQ ID NO: 1, as in VAR-22. Thus,
without being
bound by theory, it is surprisingly discovered that the presence of both I554L
and T581D
mutations contribute to increased transduction of trabecular meshwork tissue
in the context of
diverse capsid polypeptide sequences, relative to capsid polypeptides which do
not comprise the
mutation.
In some embodiments, the capsid polypeptide comprises point mutations that
correspond
to a leucine at a position corresponding to 544 in SEQ ID NO: 1, a serine at a
position
corresponding to 561 in SEQ ID NO:1 and an aspartic acid at a position
corresponding to 581 in
SEQ ID NO: 1 (e.g., comprising a to I554L, D561S, and T581D as compared to SEQ
ID NO: 1).
For example, VAR-22, VAR-46, and VAR-47 include mutations that correspond to
I554L,
D561S, and T581D as compared to SEQ ID NO: 1.
Thus, as described herein are capsid polypeptides, nucleic acid molecules that
encode
said capsid polypeptides, virus particles comprising said capsid polypeptides,
and methods of
making and using the same, comprising a leucine at a position corresponding to
554 of SEQ ID
NO: 1 (e.g., comprising a I554L mutation as compared to SEQ ID NO: 1) and one
or both of a
serine at a position corresponding to 561 and a threonine at a positions
corresponding to 581 of
SEQ ID NO: 1 (e.g., D561S and T581D mutations as compared to SEQ ID NO: 1). In
some
embodiments, the capsid polypeptide comprising a I554L mutation and one or
both D561S and
T581D mutations has greater than 95%, greater than 96%, greater than 97% or
optionally greater
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than 98% or greater than 99% sequence identity to SEQ ID NO: 1. In some
embodiments, the
capsid polypeptide comprising a I554L mutation and one or both D561S and T581D
mutations
has greater than 95%, greater than 96%, greater than 97% or optionally greater
than 98% or
greater than 99% sequence identity to the capsid polypeptide sequence of VAR-
22. In some
embodiments, the capsid polypeptide comprising a I554L mutation and one or
both D5615 and
7581D mutations has 1-20, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,
15, 16, 17, 18, 19 or 20
additional mutations relative to SEQ ID NO: 1. In some embodiments, a nucleic
acid molecule
encodes a capsid polypeptide that comprises a mutation corresponding to a
I554L mutation as
compared to SEQ ID NO: 1 and one or both D561S and T581D mutations as compared
to SEQ
ID NO: 1. In some embodiments, a capsid polypeptide, comprising a mutation
that corresponds
to I554L as compared to SEQ ID NO: 1 and one or both D561S and T581D mutations
as
compared to SEQ ID NO: 1, present in a viral particle increases transduction
in the trabecular
meshwork as compared to a viral particle with a reference capsid polypeptide,
for example, with
the wild-type capsid polypeptide (SEQ ID NO: 1).
According to some embodiments, disclosed herein are capsid polypeptides
comprising a
point mutation that corresponds to a leucine at position 559 of SEQ ID NO: 1
(e.g., a 1559L as
compared to SEQ ID NO: 1). For example. VAR-22 and VAR-23 include a I559L
mutation as
compared to SEQ ID NO: 1 (shown in Table 1). Additional variant capsid
polypeptides (VAR-
50, VAR-51, VAR-52) having a I559L mutation were evaluated, where each of
these additional
variant capsid polypeptides have no mutations at the 557, 558, and 560 amino
acid residues as
compared to SEQ ID NO: 1, as in VAR-22 and VAR-23. Data for VAR-50, VAR-51,
and VAR-
52 is shown in Table 7, and like VAR-22 and VAR-23, virus particles comprising
these capsid
polypeptides comprising the I559L mutation resulted in increased transduction
of trabecular
meshwork tissue, relative to virus particles comprising capsid polypeptides of
SEQ ID NO: 1.
Thus, without being bound by theory, it is surprisingly discovered that the
presence of a leucine
at a position corresponding to 559 of SEQ ID NO: 1 (e.g., comprising a I559L
mutation)
contributes to increased transduction of trabecular meshwork tissue in the
context of diverse
capsid polypeptide sequences, relative to capsid polypeptides which do not
comprise the
mutation. Relative to SEQ ID NO: 1, these additional variant capsid
polypeptides comprising a
I559L mutation have an edit distance in the range of 4-13 (as shown in Table
7). Relative to
VAR-22, these additional variant capsid polypeptides comprising a I559L
mutation have an edit
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distance of 8-19 (as shown in Table 7). Relative to VAR-23, these additional
variant capsid
polypeptides comprising a I559L mutation have an edit distance of 5-20 (as
shown in Table 7).
Thus, as described herein are capsid polypeptides, nucleic acid molecules that
encode said capsid
polypeptides, virus particles comprising said capsid polypeptides, and methods
of making and
using the same, comprising a leucine at a position corresponding to 559 of SEQ
ID NO: 1 (e.g., a
I559L mutation as compared to SEQ ID NO: 1). In some embodiments, the capsid
polypeptide
comprising a I559L mutation has greater than 95%, greater than 96%, greater
than 97% or
optionally greater than 98% or greater than 99% sequence identity to SEQ ID
NO: 1. In some
embodiments, the capsid polypeptide comprising a I559L mutation has greater
than 95%, greater
than 96%, greater than 97% or optionally greater than 98% or greater than 99%
sequence identity
to the capsid polypeptide sequence of VAR-22. In some embodiments, the capsid
polypeptide
comprising a I559L mutation has greater than 95%, greater than 96%, greater
than 97% or
optionally greater than 98% or greater than 99% sequence identity to the
capsid polypeptide
sequence of VAR-23. In some embodiments, the capsid polypeptide comprising a
I559L
mutation has 1-20, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,
16, 17, 18, 19 or 20
additional mutations relative to SEQ ID NO: 1. In some embodiments, disclosed
herein are
nucleic acid molecules encoding a capsid polypeptide that comprises a leucine
at a position
corresponding to 559 of SEQ ID NO: 1 (e.g., a mutation corresponding to a
I559L mutation as
compared to SEQ ID NO: 1). Also disclosed herein are virus particles, e.g.,
dependoparvovirus
particles, comprising a capsid polypeptide that comprises a leucine at a
position corresponding to
559 of SEQ ID NO: 1 (e.g., a mutation corresponding to a I559L mutation as
compared to SEQ
ID NO: 1).
As shown in Table 7, each of these additional variant capsid polypeptides
comprising a
I559L mutation have increased trabecular transduction as compared to SEQ ID
NO: 1.
Accordingly, in some embodiments, a capsid polypeptide, comprising a mutation
that
corresponds to I559L as compared to SEQ ID NO: 1, present in a viral particle
increases
transduction in the trabecular meshwork as compared to a viral particle with a
reference capsid
polypeptide, for example, with the wild-type capsid polypeptide (SEQ ID NO:
1).
Table 7
Library Experiment 1
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Name Edit Edit Edit Macular Retinal
Trabccular Virus Production as
Distanc Distanc Distanc Transduction as Transduction
Transduction compared to wild-type
e to e to e to compared to wild- as compared
as compared SEQ ID NO: 1 (Log2)
SEQ ID VAR- VAR- type SEQ ID NO: 1 to wild-type to wild-
type
NO: 1 22 23 (Log2) SEQ ID NO: SEQ ID NO:
1 (Log2) 1 (Log2)
VAR- 9 -1.16 0.81 2.95 0.72
22
VAR- 9 -0.60 -3.96 2.20 -
1.54
23
VAR- 4 8 5 -0.48 2.49 2.85 1.54
51
Library Experiment 2
Name Edit Edit Edit Macular Retinal
Trabecular Virus Production as
Distanc Distanc Distanc Transduction as Transduction
Transduction compared to wild-type
e to e to e to compared to wild- as compared
as compared SEQ ID NO: 1 (Log2)
SEQ ID VAR- VAR- type SEQ ID NO: 1 to wild-type to wild-
type
NO: 1 22 23 (Log2) SEQ ID NO: SEQ ID NO:
Anterior Posterior
(Log2) 1 (Log2) eye eye
library
library
VAR- 9 -1.57 Not -0.80 -
4.36 1.03
22 Measured
VAR- 9 10 9 Not Measured Not 2.68
-2.96 5.60
50 Measured
VAR- 4 8 5 Not Measured Not 1.51
Not -0.25
51 Measured
Measured
VAR- 13 19 20 0.95 -0.04 4.47 1.54
-3.34
52
In some embodiments, the capsid polypeptide comprising the leucine at a
position
corresponding to 559 of SEQ ID NO: 1 (e.g., I559L mutation) has one or more
additional
mutations. In some embodiments, the capsid polypeptide comprises point
mutations that
correspond to leucine at a position corresponding to 559 of SEQ ID NO: 1 and a
serine at a
position corresponding to 561 of SEQ ID NO: 1 (e.2., comprising I559L and
D5615 as compared
to SEQ ID NO: 1). For example, VAR-22, VAR-23, and VAR-50 include mutations
that
correspond to I559L and D5615 as compared to SEQ ID NO: 1. VAR-50 has no
mutations at
the 562, 563, 564, and 565 amino acid residues as compared to SEQ ID NO: 1, as
in VAR-22
and VAR-23. Thus, without being bound by theory, it is surprisingly discovered
that the
presence of both I559L and D5615 mutations contribute to increased
transduction of trabecular
meshwork tissue in the context of diverse capsid polypeptide sequences,
relative to capsid
polypeptides which do not comprise these mutations.
In some embodiments, the capsid polypeptide comprises point mutations that
correspond
to a leucine at a position corresponding to 559 of SEQ ID NO: 1 and aspartic
acid at a position
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corresponding to 581 of SEQ ID NO: 1 (e.g., comprising I559L and T581D as
compared to SEQ
ID NO: 1). For example, VAR-22, VAR-23, VAR-50, and VAR-51 include mutations
that
correspond to I559L and T581D as compared to SEQ ID NO: 1. VAR-50 and VAR-51
have no
mutations at the 579, 582, 583, and 584 amino acid residues as compared to SEQ
ID NO: 1, as in
VAR-22 and VAR-23. Thus, without being bound by theory, it is surprisingly
discovered that
the presence of both I559L and T581D mutations contribute to increased
transduction of
trabecular meshwork tissue in the context of diverse capsid polypeptide
sequences, relative to
capsid polypeptides which do not comprise the mutation.
In some embodiments, the capsid polypeptide comprises point mutations that
correspond
to leucine at a position corresponding to 559 of SEQ ID NO: 1 and a serine at
a position
corresponding to 561 of SEQ ID NO: 1 and an aspartic acid at a position
corresponding to 581 of
SEQ ID NO: 1 (e.g., comprising I5549L, D561S, and T581D as compared to SEQ ID
NO: 1).
For example, as discussed above, VAR-22, VAR-23, and VAR-50 include mutations
that
correspond to I554L, D561S, and T581D as compared to SEQ ID NO: 1. Thus, as
described
herein are capsid polypeptides, nucleic acid molecules that encode capsid
polypeptides, virus
particles, and methods of making and using the same, comprising a I559L
mutation as compared
to SEQ ID NO: 1 and one or both D561S and T581D mutations as compared to SEQ
ID NO: 1.
In some embodiments, the capsid polypeptide comprising a I559L mutation and
one or both
D561S and T581D mutations has greater than 95%, greater than 96%, greater than
97% or
optionally greater than 98% or greater than 99% sequence identity to SEQ ID
NO: 1. In some
embodiments, the capsid polypeptide comprising a I559L mutation and one or
both D561S and
T581D mutations has greater than 95%, greater than 96%, greater than 97% or
optionally greater
than 98% or greater than 99% sequence identity to the capsid polypeptide
sequence of VAR-22.
In some embodiments, the capsid polypeptide comprising a I559L mutation and
one or both
D5615 and T581D mutations has greater than 95%, greater than 96%, greater than
97% or
optionally greater than 98% or greater than 99% sequence identity to the
capsid polypeptide
sequence of VAR-23. In some embodiments, the capsid polypeptide comprising a
I559L
mutation and one or both D561S and T581D mutations has 1-20, e.g., 1, 2, 3, 4,
5, 6, 7, 8, 9, 10,
11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 additional mutations relative to SEQ
ID NO: 1. In some
embodiments, a nucleic acid molecule encodes a capsid polypeptide that
comprises a mutation
corresponding to a I559L mutation as compared to SEQ ID NO: 1 and one or both
D561S and
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T581D mutations as compared to SEQ ID NO: 1. In some embodiments, a capsid
polypeptide,
comprising a mutation that corresponds to I559L as compared to SEQ ID NO: 1
and one or both
D561S and T581D mutations as compared to SEQ ID NO: 1, present in a viral
particle increases
transduction in the trabecular meshwork as compared to a viral particle with a
reference capsid
polypeptide, for example, with the wild-type capsid polypeptide (SEQ ID NO:
1).
In some embodiments, disclosed herein are capsid polypeptides comprising a
I5549L,
D561S, and/or T581D mutation has additionally comprising one or more
additional mutations.
In some embodiments, the capsid polypeptide comprises point mutations that
correspond to
I5549L, D561S, T581D, and a mutation at a position corresponding to 556 of SEQ
ID NO: 1 to
R, D, or N. For example, VAR-22 includes a mutation that corresponds to K556R,
VAR-23
includes a mutation that corresponds to K556D, and VAR-50 includes a mutation
that
corresponds to K556N as compared to SEQ ID NO: 1. Thus, without being bound by
theory, it
is surprisingly discovered that the presence of I5549L. D561S, and/or T581D
mutations, a
mutation at a position corresponding to position 556 of SEQ ID NO: 1 to R, D,
or N contributes
to increased transduction of trabecular meshwork tissue in the context of
diverse capsid
polypeptide sequences, relative to capsid polypeptides which do not comprise
these mutations.
In some embodiments, disclosed herein are capsid polypeptides comprising point
mutations that correspond to I5549L, D561S, and/or T581D, and a mutation at
position
corresponding to 586 of SEQ ID NO: 1 to either R or Q. For example, VAR-22
includes a
mutation that corresponds to G586R, VAR-23 includes a mutation that
corresponds to G586Q,
and VAR-50 includes a mutation that corresponds to G586Q as compared to SEQ ID
NO: L
Thus, without being bound by theory, it is surprisingly discovered that the
presence of I5549L,
D561S, and/or T581D mutations, a mutation at a position corresponding to 586
of SEQ ID NO: 1
to either R or Q contributes to increased transduction of trabecular meshwork
tissue in the
context of diverse capsid polypeptide sequences, relative to capsid
polypeptides which do not
comprise these mutations.
In some embodiments, the capsid polypeptide comprises point mutations that
correspond
to I5549L, D561S, and/or T581D, and a mutation at a position corresponding to
566 of SEQ ID
NO: 1 to either A or K. For example, VAR-22 includes a mutation that
corresponds to R566A,
VAR-23 includes a mutation that corresponds to R566K, and VAR-51 includes a
mutation that
corresponds to R566K as compared to SEQ ID NO: 1. Thus, without being bound by
theory, it
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is surprisingly discovered that the presence of I5549L. D561S, and/or T581D
mutations, a
mutation at a position corresponding to 566 in SEQ ID NO: 1 to either A or K
contributes to
increased transduction of trabecular meshwork tissue in the context of diverse
capsid polypeptide
sequences, relative to capsid polypeptides which do not comprise these
mutations.
According to some embodiments, the capsid polypeptide comprises a point
mutation that
corresponds to an asparagine at a position corresponding to 556 of SEQ ID NO:
1 (e.g.,
comprising K556N as compared to SEQ ID NO: 1). For example, VAR-21 includes a
K556N
mutation as compared to SEQ ID NO: 1 (shown in Table 1). Additional variant
capsid
polypeptides (VAR-47. VAR-49, VAR-50) having a K556N mutation were evaluated,
where
each of these additional variant capsid polypeptides have no mutations at the
555 and 557 amino
acid residues as compared to SEQ ID NO: 1, as in VAR-21. Data for VAR-47, VAR-
49, and
VAR-50 is shown in Table 8, and like VAR-21, virus particles comprising these
capsid
polypeptides comprising the asparagine at a position corresponding to 556 of
SEQ ID NO: 1
(e.g., K556N mutation) resulted in increased transduction of trabecular
meshwork tissue, relative
to virus particles comprising capsid polypeptides of SEQ ID NO: 1. Thus,
without being bound
by theory, it is surprisingly discovered that the presence of an asparagine at
a position
corresponding to 556 of SEQ ID NO: 1 (e.g., the presence of a K556N mutation)
contributes to
increased transduction of trabecular meshwork tissue in the context of diverse
capsid polypeptide
sequences, relative to capsid polypeptides which do not comprise the mutation.
Relative to SEQ
ID NO: 1, these additional variant capsid polypeptides comprising a K556N
mutation have an
edit distance in the range of 4-12 (as shown in Table 8). Relative to VAR-21,
these additional
variant capsid polypeptides comprising a K556N mutation have an edit distance
of 9-12 (as
shown in Table 8). Thus, as described herein are capsid polypeptides, nucleic
acid molecules
that encode said capsid polypeptides, virus particles comprising said capsid
polypeptides, and
methods of making and using the same, comprising an asparagine at a position
corresponding to
556 of SEQ ID NO: 1 (e.g., comprising a K556N mutation as compared to SEQ ID
NO: 1). In
some embodiments, the capsid polypeptide comprising a K556N mutation has
greater than 95%,
greater than 96%, greater than 97% or optionally greater than 98% or greater
than 99% sequence
identity to SEQ ID NO: 1. In some embodiments, the capsid polypeptide
comprising a K556N
mutation has greater than 95%, greater than 96%, greater than 97% or
optionally greater than
98% or greater than 99% sequence identity to the capsid polypeptide sequence
of VAR-21. In
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some embodiments, the capsid polypeptide comprising a K556N mutation has 1-20,
e.g., 1. 2, 3,
4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 additional
mutations relative to SEQ ID
NO: 1.
In some embodiments, disclosed herein are nucleic acid molecules encoding a
capsid
polypeptide that comprises a mutation corresponding to an asparagine at a
position
corresponding to 556 of SEQ ID NO: 1 (e.g., comprising a K556N mutation as
compared to SEQ
ID NO: 1). As shown in Table 8, each of these additional variant capsid
polypeptides
comprising a K556N mutation have increased trabecular transduction as compared
to SEQ ID
NO: 1. Accordingly, in some embodiments, a capsid polypeptide, comprising a
mutation that
corresponds to K556N as compared to SEQ ID NO: 1, present in a viral particle
increases
transduction in the trabecular meshwork as compared to a viral particle with a
reference capsid
polypeptide, for example, with the wild-type capsid polypeptide (SEQ ID NO:
1).
Table 8
Library Experiment 1
Name Edit Edit Macular Retinal Trabecular Virus
Production as
Distance Distance Transduction Transduction Transduction
compared to wild-type
to SEQ to VAR- as compared as compared as compared
SEQ ID NO: 1 (Log2)
ID NO: 21 to wild-type to wild-type to wild-type
1 SEQ ID NO: 1 SEQ ID NO: 1 SEQ ID NO: 1
(Log2) (Log2) (Log2)
VAR-21 4 0.21 3.17 3.62 0.91
Library Experiment 2
Name Edit Edit Macular Retinal Trabecular Virus
Production as
Distance Distance Transduction Transduction Transduction
compared to wild-type
to SEQ to VAR- as compared as compared as compared
SEQ ID NO: 1 (Log2)
ID NO: 21 to wild-type to wild-type to wild-type
1 SEQ ID NO: 1 SEQ ID NO: 1 SEQ ID NO: 1
Anterior Posterior
(Log2) (Log2) (Log2) eye
library eye library
VAR-21 4 Not Measured -4.08 -1.08 -2.66
0.67
VAR-47 12 11 Not Measured -3.30 3.24 -3.23
2.90
VAR-49 11 12 Not Measured -3.52 3.28 1.49
-0.02
VAR-50 9 9 Not Measured Not Measured 2.68
-2.96 5.60
In some embodiments, the capsid polypeptide comprising the asparagine at a
position
corresponding to 556 of SEQ ID NO: 1 (e.g., comprising a K556N mutation) has
one or more
additional mutations. In some embodiments, the capsid polypeptide comprises
the mutation that
corresponds to K556N, and a mutation at a position corresponding to 578 of SEQ
ID NO: 1 to I,
V, or T. For example, VAR-21 includes a mutation that corresponds to S578I,
VAR-46 includes
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a mutation that corresponds to S578V, and VAR-47 includes a mutation that
corresponds to
S578T as compared to SEQ ID NO: 1. Thus, without being bound by theory, it is
surprisingly
discovered that the presence of K556N mutations and a mutation at a position
corresponding to
578 of SEQ ID NO: 1 to I, V, or T contribute to increased transduction of
trabecular meshwork
tissue in the context of diverse capsid polypeptide sequences, relative to
capsid polypeptides
which do not comprise these mutations.
According to some embodiments, the capsid polypeptide comprises a point
mutation that
corresponds to a serine at a position corresponding to 561 of SEQ ID NO: 1
(e.g., comprises
D561S as compared to SEQ ID NO: 1). For example, VAR-19, VAR-22, and VAR-23
include a
D561S mutation as compared to SEQ ID NO: 1 (shown in Table 1). Additional
variant capsid
polypeptides (VAR-46. VAR-47, VAR-50, VAR-53) having a D561S mutation were
evaluated,
where each of these additional variant capsid polypeptides have no mutations
at the 560, 562,
563, 564, and 565 amino acid residues as compared to SEQ ID NO: 1, as in VAR-
19, VAR-22,
and VAR-23. Data for VAR-46, VAR-47, VAR-50. and VAR-53 is shown in Table 9,
and like
VAR-19, VAR-22, and VAR-23, virus particles comprising these capsid
polypeptides
comprising the D561S mutation resulted in increased transduction of trabecular
meshwork tissue,
relative to virus particles comprising capsid polypeptides of SEQ ID NO: 1.
Thus, without being
bound by theory, it is surprisingly discovered that the presence of a serine
at a position
corresponding to 561 of SEQ ID NO: 1 (e.g., a D561S mutation) contributes to
increased
transduction of trabecular meshwork tissue in the context of diverse capsid
polypeptide
sequences, relative to capsid polypeptides which do not comprise the mutation.
Relative to SEQ
ID NO: 1, these additional variant capsid polypeptides comprising a D561S
mutation have an
edit distance in the range of 7-12 (as shown in Table 9). Relative to VAR-19,
these additional
variant capsid polypeptides comprising a D561S mutation have an edit distance
of 11-14 (as
shown in Table 9). Relative to VAR-22, these additional variant capsid
polypeptides comprising
a D561S mutation have an edit distance of 10-14 (as shown in Table 9).
Relative to VAR-23,
these additional variant capsid polypeptides comprising a 11J561S mutation
have an edit distance
of 9-14 (as shown in Table 9). Thus, as described herein are capsid
polypeptides, nucleic acid
molecules that encode said capsid polypeptides, virus particles comprising
said capsid
polypeptides, and methods of making and using the same, comprising a serine at
a position
corresponding to 561 of SEQ ID NO: 1 (e.g., comprising a D561S mutation as
compared to SEQ
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ID NO: 1). In some embodiments, the capsid polypeptide comprising a D561S
mutation has
greater than 95%, greater than 96%, greater than 97% or optionally greater
than 98% or greater
than 99% sequence identity to SEQ ID NO: 1. In some embodiments, the capsid
polypeptide
comprising a D561S mutation has greater than 95%, greater than 96%, greater
than 97% or
optionally greater than 98% or greater than 99% sequence identity to the
capsid polypeptide
sequence of VAR-19. In some embodiments, the capsid polypeptide comprising a
D561S
mutation has greater than 95%, greater than 96%, greater than 97% or
optionally greater than
98% or greater than 99% sequence identity to the capsid polypeptide sequence
of VAR-22. In
some embodiments, the capsid polypeptide comprising a D561S mutation has
greater than 95%,
greater than 96%, greater than 97% or optionally greater than 98% or greater
than 99% sequence
identity to the capsid polypeptide sequence of VAR-23. In some embodiments,
the capsid
polypeptide comprising a D561S mutation has 1-20, e.g., 1, 2, 3, 4, 5, 6, 7,
8, 9, 10, 11. 12, 13,
14, 15, 16, 17, 18, 19 or 20 additional mutations relative to SEQ ID NO: 1. In
some
embodiments, disclosed herein are nucleic acid molecules encoding a capsid
polypeptide that
comprises a mutation corresponding to a serine at a position corresponding to
561 of SEQ ID
NO: 1 (e.g., comprising a D561S mutation as compared to SEQ ID NO: 1). As
shown in Table
9, each of these additional variant capsid polypeptides comprising a D561S
mutation have
increased trabecular transduction as compared to SEQ ID NO: 1. Accordingly, in
some
embodiments, a capsid polypeptide, comprising a mutation that corresponds to
D561S as
compared to SEQ ID NO: 1, present in a viral particle increases transduction
in the trabecular
meshwork as compared to a viral particle with a reference capsid polypeptide,
for example, with
the wild-type capsid polypeptide (SEQ ID NO: 1).
Table 9
Library Experiment 1
Name Edit ED to ED to ED to Macular Retinal Trabecular
.. Virus Production as
Distance VAR- VAR- VAR- Transduction Transduction Transduction compared to
wild-type
(ED) to 19 22 23 as compared as compared
as compared SEQ ID NO: 1 (Log2)
SEQ ID to wild-type to wild-type
to wild-type
NO: 1 SEQ ID NO: SEQ ID NO: SEQ ID NO:
1 (Log2) 1 (Log2) 1 (Log2)
VAR- 7 -0.53 0.98 3.70
0.09
19
VAR- 9 -1.16 0.81 2.95
0.72
22
VAR- 9 -0.60 -3.96 2.20 -
1.54
23
Library Experiment 2
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Name Edit ED to ED to ED to Macular Retinal Tr abccular
Virus Production as
Distance VAR- VAR- VAR- Transduction Transduction Transduction compared to
wild-type
(ED) to 19 22 23 as compared as compared
as compared SEQ ID NO: 1 (Log2)
SEQ ID to wild-type to wild-type
to wild-type
NO: 1 SEQ ID NO: SEQ ID NO: SEQ ID NO:
Anterior Posterior
1 (Log2) 1 (Log2) 1 (Log2)
eye eye
library
library
VAR- 9 -1.57 Not -0.80 -
4.36 1.03
22 Measured
VAR- 8 12 11 13 Not -3.23 2.54 -
4.83 044
46 Measured
VAR- 12 14 13 14 Not -3.30 3.24 -
3.23 2.90
47 Measured
VAR- 9 11 10 9 Not Not 2.68 -
2.96 5.60
50 Measured Measured
VAR- 9 12 14 13 Not Not 4.47 0.14
1.82
53 Measured Measured
In some embodiments, the capsid polypeptide comprising the serine at a
position
corresponding to 561 of SEQ ID NO: 1 (e.g., comprising a D561S mutation) has
one or more
additional mutations. In some embodiments, the capsid polypeptide comprises
point mutations
that correspond to a serine at a position corresponding to 561 of SEQ ID NO: 1
and an aspartic
acid at a position corresponding to 581 of SEQ ID NO: 1 (e.g., comprising a
D561S and T581D
mutation as compared to SEQ ID NO: 1). For example, VAR-22, VAR-23, VAR-46,
VAR-47,
VAR-50, and VAR-53 include mutations that correspond to D561S and T581D as
compared to
SEQ ID NO: 1. Thus, without being bound by theory, it is surprisingly
discovered that the
presence of both D561S and T581D mutations contribute to increased
transduction of trabecular
meshwork tissue in the context of diverse capsid polypeptide sequences,
relative to capsid
polypeptides which do not comprise these mutations. Thus, as described herein
are capsid
polypeptides, nucleic acid molecules that encode said capsid polypeptides,
virus particles
comprising said capsid polypeptides, and methods of making and using the same,
comprising a
serine at a position corresponding to 561 of SEQ ID NO:1 and an aspartic acid
at a position
corresponding to 581 of SEQ ID NO: 1 (e.g., comprising D561S and T581D
mutations as
compared to SEQ ID NO: 1). In some embodiments, the capsid polypeptide
comprising D561S
and T581D mutations has greater than 95%, greater than 96%, greater than 97%
or optionally
greater than 98% or greater than 99% sequence identity to SEQ ID NO: 1. In
some
embodiments, the capsid polypeptide comprising D561S and T581D mutations has
greater than
95%, greater than 96%, greater than 97% or optionally greater than 98% or
greater than 99%
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sequence identity to the capsid polypeptide sequence of VAR-22. In some
embodiments, the
capsid polypeptide comprising D561S and T581D mutations has greater than 95%,
greater than
96%, greater than 97% or optionally greater than 98% or greater than 99%
sequence identity to
the capsid polypeptide sequence of VAR-23. In some embodiments, the capsid
polypeptide
comprising D561S and T581D mutations has 1-20, e.g., 1, 2, 3, 4, 5, 6, 7, 8,
9, 10, 11, 12, 13, 14,
15, 16, 17, 18, 19 or 20 additional mutations relative to SEQ ID NO: 1. In
some embodiments,
disclosed herein are nucleic acid molecules encoding a capsid polypeptide that
comprises a
mutation corresponding to D5615 and T581D mutations as compared to SEQ ID NO:
1. In some
embodiments, a capsid polypeptide, comprising D5615 and T581D mutations as
compared to
SEQ ID NO: 1, present in a viral particle increases transduction in the
trabecular meshwork as
compared to a viral particle with a reference capsid polypeptide, for example,
with the wild-type
capsid polypeptide (SEQ ID NO: 1).
Disclosed herein are capsid polypeptides comprising a mutation at a position
corresponding to 587 of SEQ ID NO: 1. In embodiments, the mutation is to an
alanine. In
embodiments, the capsid polypeptide comprises a N587A mutation according to
SEQ ID NO: 1.
Also disclosed herein are capsid polypeptides comprising the mutation, e.g.,
mutation to alanine,
at a position corresponding to 587 of SEQ ID NO: 1 (e.g., comprising N587A
according to SEQ
ID NO: 1) further comprising an insertion of one or more amino acids, e.g., 4
or more amino
acids, e.g., 7, 8, 9 or more amino acids, e.g., between 7-10 amino acids,
between two adjacent
amino acids N-terminal to the mutation at the position corresponding to 587 of
SEQ ID NO: 1. In
embodiments, the insertion is 7 amino acids. In embodiments, the insertion is
8 amino acids. In
embodiments, the insertion is 9 amino acids. In embodiments, the insertion is
10 amino acids. In
embodiments, the insertion occurs after an amino acid no more than 3 amino
acids N-terminal to
the mutation at the position corresponding to 587 of SEQ ID NO: 1. In
embodiments, the
insertion occurs between two adjacent amino acids selected from the amino
acids corresponding
to 580 to 587 of SEQ ID NO: 1. In embodiments, the insertion is between amino
acids
corresponding to 584 and 585 of SEQ ID NO: 1. In embodiments, the insertion is
between amino
acids corresponding to 586 and 587 of SEQ ID NO: 1. In embodiments, disclosed
herein are
capsid polypeptides comprising a sequence at least 85%, 90%, 95%, 96%, 97%,
98%, or 99%
identical to SEQ ID NO: 1 and comprising an alanine at a position
corresponding to N587 of
SEQ ID NO: 1 and comprising an insertion between amino acids corresponding to
positions 586
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and 587 of SEQ ID NO: 1. Disclosed herein are nucleic acid molecules
comprising sequence
encoding said capsid polypeptides. Also disclosed are virus particles, e.g.,
dependoparvovirus
particles, comprising said capsid polypeptides. In embodiments, such virus
particles, e.g.,
dependoparvovirus particles, have increased transduction of the macula and/or
non-macula retina
tissue of the eye, for example, as measured by quantification of viral RNA
from the target tissue,
relative to virus particles comprising the capsid polypeptides of SEQ ID NO:
1. In embodiments,
such virus particles, e.g., dependoparvovirus particles, have increased
transduction of the anterior
eye tissue (e.g., trabecular meshwork and/or Schlemm's canal tissue), for
example, as measured
by quantification of viral RNA from the target tissue, relative to virus
particles comprising the
capsid polypeptides of SEQ ID NO: 1.
As described herein in Example 3, the subcombinations of mutations present in
each of
the variants described herein were determined by computationally defining all
possible 1-
mutation, 2-mutation, 3-mutation, 4-mutation, etc., up to the total number of
mutations in each
variant (with contiguous strings of inserted amino acids counted as 1 mutation
for purposes of
this analysis only). The unique variants tested in library experiment 1 and
Library Experiment 2
comprising at least those subcombination of mutations and at most 3 additional
mutations not
specified in the subcombination were pooled, and a median whole retina
transduction calculated
for all variants in each pool. The analysis identifies several minimal
structural elements (sets of
mutations) which, when present in a variant capsid polypeptide, result in
better than wtAAV2
median whole retina transduction variants tested in Library Experiment 1 and
Library
Experiment 2. These mutation sets as well as the additional data associated
with each pool are
shown in Table 12 and Table 13, below.
Table 12: Minimum structural elements comprised in the variants described
herein and
associated with increased median retina transduction relative to wild-type
AAV2 identified from
Library Experiment 1. In the second from left column, all amino acid position
numbering is
according to SEQ ID NO: 1. Amino acids are represented by their one-letter
code; deletions are
represented by a --"; insertions are represented by the notation [last wild-
type amino acid
position before the insertion]-[length of insertionLaa-[wild-type amino acid
position after the
insertion]_[identity of the insertion]. A "+" indicates a motif with one or
more additional
positions which can be any amino acid.
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Variant Name Common Mutation Set Number of viral Average Median
whole Number of
(Numbering according to SEQ ID particles with number of retina
mutations from
NO: 1) representing a unique VP1 mutations from
transduction wild type
subcombination of the mutations capsid wild type measurement
AAV2 in the
found in the Variant(s) listed in polypeptide AAV2 of all from
Library Common
the left-most column variants unique VP1 Experiment
1 Mutation Set
comprising the capsici across Set
(for purposes of
Common polypeptides in this
table,
Mutation Set the Set,
contiguous
and up to 3 excluding the amino
acids
additional mutations
inserted at the
mutations from comprised in same
position
wt AAV2 (the the Common are
counted as
"Set") Mutation Set 1)
VAR-11 mutation sets with median whole retina transduction (1og2 relative to
wtAAV2) greater than 1.0
VAR-11 R459-, T456- 3 2.67 3.56 /
VAR-11 R459-, Q457- 6 2.33 3.38 9
VAR-11 S458-, T456-, L460- 3 2.67 3.31 3
VAR-11 R459-, Q457-, T456- 6 2.33 3.19 3
VAR-11 Q457-, L460- 3 2.67 3A9 9
VAR-11 R459-, Q457-, L460- 5 2.20 3.19 3
VAR-11 R459-, S458-, Q457- 9 1.89 3.19 3
VAR-11 S458-, Q457-, T456- 9 2.11 3.08 3
VAR-11 L460-, S458-, R459-, Q457-, 4 1.00 2.92
5
T456-
VAR-11 R459-, S458-, Q457-, L460- 6 1.50 2.86
4
VAR-11 R459-, S458-, Q457-, 1456- 8 1.75 2.80
4
VAR-11 S458-, L460- 4 2.25 2.51 9
VAR-11 R459-, S458-, L460- 6 1.83 2.51 3
VAR-11 R459-, S458- 8 1.88 2.51 9
VAR-11 S458-, Q457- 10 2.30 2.47 /
VAR-11 S458-, T456- 6 2.50 2.38 9
VAR-11 R459- 6 1.83 1.86 1
VAR-11 L460- 3 2.00 1.82 1
VAR-11 Q461-, L460-, S458-, R459-, 2 0.50 1.63
5
Q457-
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VAR-11 S458- 13 2.00 1.36 1
VAR-11 mutation sets with median whole retina transduction (1og2 relative to
wtAAV2) greater than 0 and
less than 1.0
VAR-11 Q461-, L460- 2 1.50 0.68 7
VAR-11 Q457- 22 2.36 0.48 1
VAR-11 Q457-, T456- 13 215 0.37 2
VAR-12 mutation sets with median whole retina transduction (1og2 relative to
wtAAV2) greater than 1.0
VAR-12 S458Q, T455Q, P451G 2 2.50 2.99 3
VAR-12 T456N, R459T 5 2.40 2.57 9
VAR-12 N449I, T456N, R459T, T455Q 2 1.00 2.33 4
VAR-12 R459T, P451G 4 2.25 2.29 7
VAR-12 S458Q, T454-, T450N 2 3.00 2.24 3
VAR-12 R459T, T454- 2 1.00 2.06 7
VAR-12 S458Q, Q461K, R459T, Q464V 3 3.00 2.05 4
VAR-12 T454-, T450N, P451G, Q464V, 3 2.67 1.92 6
R459T, S458Q
VAR-12 Q461K, T450N, P451(1, Q464V, 4 2.75 1.90 7
T456N, R459T, T455Q
VAR-12 Q461K, N449I, 1450N, P451G, 5 2.00 1.83 8
Q464V, T456N, R459T, T455Q
VAR-12 Q461K, N449I, T450N, P451G, 4 2.50 1.79 6
Q464V, R459T
VAR-12 Q461K, N449I, T450N, P451G, 4 2.50 1.77 8
T456N, R459T, S458Q, T455Q
VAR-12 S458Q, T455Q 6 2.50 1.75 7
VAR-12 N449I, T456N, R4591, Q464V 2 2.00 L72 4
VAR-12 Q461K, T450N, P451G, Q464V, 3 2.33 1.72 8
T456N, R459T, S458Q, T455Q
VAR-12 Q461K, N449I, P451G, Q464V, 5 2.80 L72 7
T456N, R459T, T455Q
VAR-12 N449I, T450N, P451G, Q464V, 3 2.33 L65 6
R459T, S458Q
VAR-12 Q461K, T450N, P451G, Q464V, 5 2.40 1.65 6
R4591, S458Q
VAR-12 Q461K, N449I, T450N, P451G, 5 2.00 1.65 7
Q464V, R459T, S458Q
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VAR-12 Q461K, Q464V, 1456N, R459T, 2 2.50 1.65 5
S458Q
VAR-12 T456N, T450N 2 2.50 1.60 9
VAR-12 Q461K, T454-, T450N, P451G, 5 2.00 1.53 6
R459T, S458Q
VAR-12 S458Q, R459T, P451G 3 2.00 1.53 3
VAR-12 Q461K, T450N, P451G, Q464V, 2 2.50 1.52 6
T456N, R459T
VAR-12 T454-, N4491, T450N, P451G, 2 2.50 1.42 6
Q464V, S458Q
VAR-12 Q461K, T454-, P451G, Q464V, 4 2.50 1.42 6
R459T, S458Q
VAR-12 Q461K, T454-, T450N, P451G, 4 2.00 1.42 7
Q464V, R459T, S458Q
VAR-12 S458Q, P451G 17 2.18 1.28 2
VAR-12 Q461K, N4491, 1450N, P451G, 2 3.00 1.28 5
Q464V
VAR-12 Q461K, N4491, Q464V, R459T, 4 2.75 1.28 5
S458Q
VAR-12 Q461K, N4491, P451G, Q464V, 4 2.25 1.28 6
R459T, S458Q
VAR-12 Q461K, N4491, T450N, P451G, 4 2.25 1.28 6
R459T, S458Q
VAR-12 T456N, S458Q 2 2.50 1.11 2
VAR-12 Q461K, T450N, P451G 2 2.50 1.11 3
VAR-12 N4491, T456N, T455Q 3 2.33 1.10 3
VAR-12 T456N, T455Q 3 2.00 1.10 /
VAR-12 R459T, T455Q 3 2.67 1.10 2
VAR-12 N4491, T455Q 3 3.00 1.10 2
VAR-12 Q461K, T450N, P451G, T456N, 2 3.00 1.09 7
R459T, S458Q, T455Q
VAR-12 Q461K, N4491, T450N, P451G, 4 1.75 1.09 9
Q464V, T456N, R459T, S458Q,
T455Q
VAR-12 S458Q, Q461K, R459T 2 1.50 1.01 3
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VAR-12 mutation sets with median whole retina transduction (1og2 relative to
wtAAV2) greater than 0 and
less than 1.0
VAR-12 Q461K, N4491, 1450N, P451G, 2 2.50 0.97 6
R459T, T455Q
VAR-12 Q461K, T450N, P451G, Q464V, 3 2.33 (196 7
T456N, R459T, S458Q
VAR-12 T450N, P451G, Q464V, R459T, 3 3.00 0.96 5
S458Q
VAR-12 Q461K, T450N, P451G, R459T, 5 2.60 0.96 5
S458Q
VAR-12 Q461K, P451G, Q464V, R459T, 4 3.00 0.94 5
S458Q
VAR-12 T454-, N4491, P451G, Q464V, 3 2.33 0.91 6
R459T, S458Q
VAR-12 N4491, P451G, Q464V, R459T, 3 3.00 0.91 5
S458Q
VAR-12 T454-, N4491, T450N, P451G, 3 2.33 0.91
6
R459T, S458Q
VAR-12 Q461K, N4491, T450N, R459T, 3 3.00 0.91 5
S458Q
VAR-12 T454-, T450N, P451G, R459T, 3 2.33 0.91 5
S458Q
VAR-12 Q461K, T454-, N4491, T450N, 3 1.00 0.91 8
P451G, Q464V, R459T, S458Q
VAR-12 T454-, N4491, Q464V, R459T, 2 3.00 0.90 5
S458Q
VAR-12 Q461K, T454-, N4491, P451G, 4 2.25 0.90 6
R459T, S458Q
VAR-12 Q461K, N4491, P451G, R459T, 4 3.00 0.90 5
S458Q
VAR-12 Q461K, T454-, P451G, R459T, 4 2.50 0.90 5
S458Q
VAR-12 Q461K, T454-, N4491, R459T, 3 3.00 0.89 5
S458Q
VAR-12 Q461K, T454-, N4491, P451G, 5 2.00 0.89 7
Q464V, R459T, S458Q
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VAR-12 Q461K, N4491, Q464V, T456N, 3 3.00 0.89 7
R459T, S458Q, T455Q
VAR-12 Q461K, N4491, P451G, Q464V, 5 2.40 0.89 8
T456N, R459T, S458Q, T455Q
VAR-12 R459T, T454-, Q461K, P451G 2 2.50 (188 4
VAR-12 N4491, T456N, T455Q, T450N 2 L50 (181 4
VAR-12 Q461K, T454-, N4491, T450N, 4 1.75 0.69 7
P451G, R459T, S458Q
VAR-12 Q461K, T454-, N4491, P451G, 2 1.00 0.68 9
Q464V, T456N, R459T, S458Q,
T455Q
VAR-12 Q461K, T454-, N4491, P451G, 4 2.75 0.56 5
R459T
VAR-12 P451G 45 2.18 0.48 1
VAR-12 T454- 44 2.00 0.33 1
VAR-12 N4491 13 1.54 0.32 1
VAR-12 T456N 17 L76 0.10 1
VAR-12 Q461K 12 2.00 0.09 1
VAR-13 mutation sets with median whole retina transduction (1og2 relative to
wtAAV2) greater than 1.0
VAR-13 S446A, P451Q, T455G, N449-, 3 2.67 3.17 7
T448-, G453T, Q457T
VAR-13 S446A, P451Q, T456G,1455G, 2 1.50 2.17 8
N449-, T448-, G453T, Q457T
VAR-13 S446A, Q457T 2 3.00 1.79 2
VAR-13 S446A, T456G 2 L50 L53 2
VAR-13 mutation sets with median whole retina transduction (1og2 relative to
wtAAV2) greater than 0 and
less than 1.0
VAR-13 S446A, P451Q, T455G, N449-, 2 2.50 0.95 6
T448-, G453T
VAR-13 S446A 16 1.63 0.94 1
VAR-13 G453T 7 1.43 0.71 1
VAR-13 T448- 9 1.89 0.59 1
VAR-13 P451Q 16 2.00 0.51 1
VAR-13 N449-, T448- 9 1.89 0.42 2
VAR-13 N449- 13 2.31 0.42 1
VAR-14 mutation sets with median whole retina transduction (1og2 relative to
wtAAV2) greater than 1.0
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VAR-14 T455A, T450S, R459D 2 3.00 3.71 3
VAR-14 N449Q, T455A, T450S, R459D 3 2.33 3.63 4
VAR-14 N449Q, T450S, R459D 3 3.00 3.63 3
VAR-14 S452G, T455A, T450S, N449Q, 3 3.00 2.61 5
R459D
VAR-14 N449Q, S452G, T455A, R459D 2 3.00 2.35 4
VAR-14 N449Q, T455A, R459D 8 2.50 2.12 3
VAR-14 N449Q, S452G, R459D 3 3.00 2.05 3
VAR-14 N449Q, R459D 6 1.67 2.02 9
VAR-14 N449Q, S4520, T455A, T450S 18 2.83 1.99 4
VAR-14 R459D 19 1.74 1.67 1
VAR-14 S458M 11 1.27 1.64 1
VAR-14 T455A, R459D 4 1.75 1.60 2
VAR-14 N449Q, S452G, T455A 26 2.65 1.13 3
VAR-14 mutation sets with median whole retina transduction (1og2 relative to
wtAAV2) greater than 0 and
less than 1.0
VAR-14 N449Q, T455A 32 2.47 1.12 2
VAR-14 N449Q, T455A, T450S 23 2.70 0.80 3
VAR-14 T455A 66 2.30 0.52 1
VAR-14 S452G, T455A 18 2.44 0.35 2
VAR-14 N449Q, T4508 42 2.48 0.22 2
VAR-14 S452G 50 2.34 0.22 1
VAR-14 N449Q, S452G, T450S 24 2.92 0.06 3
VAR-14 N449Q, S452G 36 2.61 0.05 2
VAR-14 T450S 80 2.15 0.04 1
VAR-14 S452G, T450S 12 2.58 0.03 /
VAR-19 mutation sets with median whole retina transduction (1og2 relative to
wtAAV2) greater than 0 and
less than 1.0
VAR-19 T581A 2 1.00 0.56 1
VAR-19 R585- 7 2.00 0.53 1
VAR-19 S578V 57 2.28 0.32 1
VAR-19 D561S, S578V, S580A 13 2.54 0.31 3
VAR-19 S578V, S580A 24 2.63 0.28 9
VAR-19 S580A 75 2.33 0.23 1
VAR-19 R566K, S578V, S580A 15 2.40 0.20 3
VAR-19 D561S 59 2.17 0.01 1
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VAR-20 mutation sets with median whole retina transduction (1og2 relative to
wtAAV2) greater than 1.0
VAR-20 5578D 3 0.67 1.98 1
VAR-21 mutation sets with median whole retina transduction (1og2 relative to
wtAAV2) greater than 1.0
VAR-21 55781, M558L 2 L50 L67 /
VAR-21 55781, 5580A 2 2.50 1.16 /
VAR-21 M558L, 5580A 4 1.75 1.03 1
VAR-21 K556N, 55781 3 2.00 1.03 /
VAR-21 mutation sets with median whole retina transduction (1og2 relative to
wtAAV2) greater than 0 and
less than 1.0
VAR-21 K556N, M558L, 5580A 2 1.00 0.61 3
VAR-21 K556N, M558L 10 2.00 0.45 /
VAR-21 K556N 152 2.21 0.06 1
VAR-21 M558L 36 2.17 0.06 1
VAR-21 and VAR-23 common mutation set with median whole retina transduction
(1og2 relative to wtAAV2)
greater than 0 and less than 1.0
VAR-21, 55781 10 L80 0.94 1
VAR-23
VAR-22 mutation sets with median whole retina transduction (log2 relative to
wtAAV2) greater than 0 and
less than 1.0
VAR-22 R5855, D5615, I554L, G586R 2 3.00 0.98 4
VAR-22 N5875 30 9.20 0.47 1
VAR-22 R585S, N587S 8 1.63 0.38 2
VAR-22 R566A 8 1.88 0.22 1
VAR-22 T581D, R5855,15541. 6 3J)0 ft 03 3
VAR-22 I554L 118 2.23 0.02 1
VAR-22 and VAR-23 common mutation set with median whole retina transduction
(1og2 relative to wtAAV2)
greater than 1.0
VAR-22, I559L, T581D 17 2.53 1.01 /
VAR-23
VAR-22 and VAR-23 common mutation set with median whole retina transduction
(1og2 relative to wtAAV2)
greater than 0 and less than 1.0
VAR-22, T581D 54 2.22 0.65 1
VAR-23
VAR-22, T581D, D5615 21 2.14 0.53 9
VAR-23
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VAR-22, I559L, D561S, T581D 15 2.73 0.44 3
VAR-23
VAR-22, VAR-26 and VAR-240 common mutation set with median whole retina
transduction (1og2 relative
to wtAAV2) greater than 0 and less than 1.0
VAR-22, R585S 107 218 0.23 1
VAR-26,
VAR-40
VAR-23 mutation sets with median whole retina transduction (1og2 relative to
wtAAV2) greater than 1.0
VAR-23 R566K, 1559L, G586Q, T581D, 5 2.60 1.16 5
E555S
VAR-23 G586Q, D561S, 1559L, E555S 5 2.20 1.14 4
VAR-23 R566K, I559L, D561S, G586Q, 5 2.40 1.14 5
E555S
VAR-23 1559L, D561S, G586Q, T581D, 7 2.00 1.14 5
E555S
VAR-23 T581D, D561S, E555S 13 2.46 1.14 3
VAR-23 R566K, D561S, I559L, T581D, 6 2.50 1.13 5
E555S
VAR-23 G586Q, 1559L, T581D. E555S 7 1.86 1.11 4
VAR-23 1559L, D561S, T581D, E555S 10 2.00
1.11 4
VAR-23 I559L, T581D, E555S 15 2.33 1.07 3
VAR-23 G586Q, T581D 12 2.17 1.02 /
VAR-23 mutation sets with median whole retina transduction (1og2 relative to
wtAAV2) greater than 0 and
less than 1.0
VAR-23 T581D, E555S 21 2.57 0.90 2
VAR-23 R566K, G586Q, D561S, I559L, 6 2.50 0.83 5
T581D
VAR-23 R566K, D561S, G586Q, T581D, 8 2.50 0.83 5
E555S
VAR-23 R566K, T581D, D561S, E555S 7 2.57 0.80 4
VAR-23 I559L, V552A, T581D 2 2.00 0.79 3
VAR-23 R566K, T581D 15 2.47 0.78 /
VAR-23 I559L, D561S, E555S 10 2.60 0.77 3
VAR-23 R566K, 1559L, D561S, G586Q 4 2.75 0.75 4
VAR-23 R566K, 1559L, T581D, E555S 5 2.20 0.75 4
VAR-23 R566K, T581D, E555S 7 2.43 0.74 3
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VAR-23 G586Q, T581D, E555S 11 2.55 0.74 3
VAR-23 S578I, D561S, T581D, E555S 2 2.50
0.74 4
VAR-23 R566K. G586Q, D561S, S578I. 3 2.00 0.63 5
E555S
VAR-23 G586Q, D561S, T581D, E555S 9 2A4 0.63 4
VAR-23 T581D, V552A 2 2.00 0.58 2
VAR-23 G586Q, D561S, T581D 11 2.45 0.57 3
VAR-23 G586Q, I559L, T581D 13 2.69 0.54 3
VAR-23 G586Q, 1559L, E555S 8 2.25 0.52 3
VAR-23 R566K, I559L, G586Q, E555S 3 2.67 0.52 4
VAR-23 G586Q, D561S, I559L, T581D 8 2.13 0.43 4
VAR-23 R566K, 1559L, G586Q, T581D 8 2.63 0.43 4
VAR-23 R566K. 1559L. G586Q 5 2.60 0.43 3
VAR-23 R566K, 1559L, T581D 12 2.58 0.43 3
VAR-23 G586Q, D561S, I559L 8 2.63 0.42 3
VAR-23 G586Q, D561S, E555S 10 2.20 0.41 3
VAR-23 R566K, T581D, G586Q, E555S 6 2.17 0.41 4
VAR-23 G586Q 63 2.30 0.38 1
VAR-23 R566K, 1559L, D561S, T581D 8 2.50 0.31 4
VAR-23 R566K, 1559L, E555S 6 2.50 0.26 3
VAR-23 R566K, 1559L, D561S, 6586Q, 9 2.44 0.26 6
T581D, E555S
VAR-23 R566K, 1559L, D561S, E555S 6 2.67 0.22 4
VAR-23 R566K, G586Q, E555S 5 2.40 0.19 3
VAR-23 R566K, T581D, G586Q 9 2.67 0.19 3
VAR-23 R566K, G586Q 9 2.44 0.19 2
VAR-23 R566K, T581D, D561S, G586Q 8 2.63 0.14 4
VAR-23 R566K, G586Q, D561S, E555S 9 2.67 0.12 4
VAR-23 G586Q, 1559L 12 2.08 0.10 2
VAR-23 V552A 72 2.19 0.08 1
VAR-23 G586Q, D561S 14 2.36 0.01 2
VAR-24 mutation set with median whole retina transduction (1og2 relative to
wtAAV2) greater than 1.0
VAR-24 Q575E 18 2.06 L10 1
VAR-26 mutation set with median whole retina transduction (1og2 relative to
wtAAV2) greater than 1.0
VAR-26 R588T, R585S, T597S, A591I, 2 3.00 3.59 5
V600A
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VAR-26 R588T, R5855, Q589N, A590P, 2 2.50 3.25 6
T5975, A5911
VAR-26 A593G, R585S, T597S, A5911, 2 3.00 2.88 5
V600A
VAR-26 R588T, R5855, A590P, T597S, 2 2.50 2.50 6
A591I, V600A
VAR-26 R588T, A593G, R5855, Q589N, 2 2.00 2.48 6
A590P, V600A
VAR-26 R588T, A593G, Q589N, A590P, 2 2.50 2.41 5
A591I
VAR-26 R588T, R5855, Q589N, A590P, 2 3.00 2.11 5
T5975
VAR-26 R588T, R5855, Q589N, A590P, 2 2.50 2.03 6
T5975, V600A
VAR-26 T597S, A5911 8 2.38 1.65 2
VAR-26 R5855, T5975 5 2.60 1.58 /
VAR-26 R5855, T597S, V600A 3 2.33 1.54 3
VAR-26 T5975, V600A 16 2.38 1.46 2
VAR-26 A590P, T597S, V600A 16 2.81 1.23 3
VAR-26 T5975, A5911, V600A 10 2.50 1.17 3
VAR-26 A590P, T5975, A5911 9 2.67 1.02 3
VAR-26 A593G, T5975, A5911 10 2.40 1.02 3
VAR-26 R588T, Q589N 2 3.00 1.01 2
VAR-26 R588T, A590P, T5975, A5911, 4 2.75 1.01 5
V600A
VAR-26 mutation sets with median whole retina transduction (1og2 relative to
wtAAV2) greater than 0 and
less than 1.0
VAR-26 A591I, V600A 13 2.31 0.95 /
VAR-26 V600A 93 2.28 0.91 1
VAR-26 R5855, V600A 14 2.29 0.91 2
VAR-26 A593G, T5975, A591I, V600A 5 2.40 0.90 4
VAR-26 R588T, T597S 9 2.33 0.88 /
VAR-26 A593G, T5975, V600A 8 2.25 0.87 3
VAR-26 A593G, A591I, V600A 6 2.67 0.84 3
VAR-26 R588T, T597S, V600A 8 2.75 0.79 3
VAR-26 A593G, A590P, T597S 12 2.58 0.77 3
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VAR-26 A590P, V600A 35 2.40 0.76 9
VAR-26 A590P, T597S, A591I, V600A 6 2.17 0.74 4
VAR-26 A590P, T597S 23 2.22 0.73 /
VAR-26 R588T, A593G, A591I, V600A 5 2.00 0.72 4
VAR-26 R588T, A591I, V600A 7 2.29 0.72 3
VAR-26 A5911 36 2.17 0.68 1
VAR-26 R588T, V600A 36 2.33 0.65 9
VAR-26 T597S 50 2.22 0.63 1
VAR-26 R588T, A593G, R585S, Q589N, 3 3.00 0.60 5
A590P
VAR-26 Q589N 5 1.80 0.59 1
VAR-26 R588T, A590P, T597S 17 2.65 0.57 3
VAR-26 A590P, A5911. V600A 10 2.60 0.51 3
VAR-26 A593G, A590P, A591I, V600A 6 2.50 0.50 4
VAR-26 R588T, T597S, A5911 5 3.00 0.49 3
VAR-26 A593G, T597S 20 2.00 0.49 9
VAR-26 R588T, A593G, T597S, A5911, 5 2.40 0.44 5
V600A
VAR-26 A593G, A5911 11 2.18 0.40 2
VAR-26 A590P 129 2.25 0.38 1
VAR-26 A593G, V600A 33 2.30 0.38 2
VAR-26 A593G, A590P, T597S, A5911, 8 2.63 0.38 5
V600A
VAR-26 R588T 117 219 0_37 1
VAR-26 A590P, A5911 16 2.19 0.33 9
VAR-26 R588T, A5911 10 230 033 9
VAR-26 R588T, A593G, A5911 11 2.45 0.32 3
VAR-26 A593G 82 2.22 0.29 1
VAR-26 A590P, R585S 21 2.43 0.28 2
VAR-26 R588T, A593G, T597S 19 2.79 0.28 3
VAR-26 R588T, A593G, T597S, A5911 9 2.67 0.27 4
VAR-26 A590P, T597S, R585S 9 2.67 0.26 3
VAR-26 R588T, A590P, A5911, T597S 6 3.00 0.24 4
VAR-26 A593G, T597S, A591I, A590P 10 2.70 0.23 4
VAR-26 R588T, A593G, V600A 32 2.63 0.23 3
VAR-26 R588T, A590P 48 2.42 0.21 9
207
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VAR-26 R588T, A593G, R585S, A590P, 5 2.40 0.21 7
T597S, A5911, V600A
VAR-26 R588T, A593G, R585S, A590P, 5 2.20 0.21 6
T597S, A5911
VAR-26 R588T, A593G, R585S, T597S, 5 2.20 0.21 5
A5911
VAR-26 R588T, A593G, A590P, T597S, 11 2.64 0.21 5
A5911
VAR-26 A593G, R585S, V600A 15 2.53 0.20 3
VAR-26 R588T, R585S, V600A 16 2.56 0.20 3
VAR-26 A593G, A590P, T597S, V600A 13 2.92 0.20 4
VAR-26 A590P, R585S, V600A 13 2.77 0.19 3
VAR-26 R588T, A593G, A590P, T597S, 6 2.33 0.19 6
A5911, V600A
VAR-26 R588T, A593G, A590P, A5911 12 2.50 0.19 4
VAR-26 A593G, A590P 34 2.35 0.16 9
VAR-26 A590P, T597S, R585S, V600A 14 2.86 0.15 4
VAR-26 A593G, A590P, V600A 28 2.64 0.15 3
VAR-26 R588T, A590P, A5911 8 2.50 0.14 3
VAR-26 A593G, R585S 17 2.35 0.13 2
VAR-26 R588T, A590P, V600A 37 2.68 0.11 3
VAR-26 R588T, A590P, R585S 38 2.82 0.11 3
VAR-26 A593G, A590P, R585S, V600A 26 2.69 0.11 4
VAR-26 R588T, A590P, R585S, V600A 40 2.78 0.11 4
VAR-26 R588T, A593G, T597S, V600A 17 2.71 0.08 4
VAR-26 R588T, A593G, R585S, V600A 33 2.76 0.08 4
VAR-26 R588T, R585S 23 2.39 0.08 2
VAR-26 R588T, A593G, A590P, R585S 74 2.55 0.08 4
VAR-26 R588T, A593G 44 2.52 0.06 2
VAR-26 R588T, A590P, T597S. V600A 17 2.82 0.03 4
VAR-26 A593G, A590P, R585S 32 2.75 0.02 3
VAR-27 mutation sets with median whole retina transduction (1og2 relative to
wtAAV2) greater than 0 and
less than 1.0
VAR-27 G586A, R585N 4 2.00 0.68 /
VAR-27 G586A 62 2.29 0.29 1
VAR-28 mutation set with median whole retina transduction (1og2 relative to
wtAAV2) greater than 1.0
208
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VAR-28 592 2+aa 593 L+ 2 1.00 2.82 1
VAR-3 mutation set with median whole retina transduction (1og2 relative to
wtAAV2) greater than LO
VAR-3 P45 1T, T456G, T455L, N449Q, 2 2.50 4.16 6
S458Q, Q457T
VAR-3 P45 1T, T456G, T450M, N449Q, 2 2.50 4.07 6
S458Q, Q457T
VAR-3 N449Q, T455L, T456G, S458Q 2 3.00 2.58 4
VAR-3 P45 1T, T455L, N449Q, S458Q, 2 3.00 2.46 5
Q457T
VAR-3 P45 1T, T456G, T455L, N449Q, 2 2.50 2.42 5
Q457T
VAR-3 P45 1T, T456G, R459M, N449Q, 2 3.00 2.09 5
Q457T
VAR-3 P45 1T, T456G, R459M, N449Q, 3 2.67 1.87 5
S458Q
VAR-3 N449Q, P45 1T, Q457T, T450M 2 3.00 1.87 4
VAR-3 N449Q, S458Q, T456G, Q457T 20 2.65 1.65 4
VAR-3 R459M 4 0.75 1.51 1
VAR-3 N449Q, P45 1T, R459M, Q457T 2 2.00 1.49 4
VAR-3 N449Q, P45 1T, T456G 22 2.73 1.15 3
VAR-3 N449Q, P451T, T455L, Q457T 3 3.00 1.15 4
VAR-3 mutation sets with median whole retina transduction (1og2 relative to
wtAAV2) greater than 0 and
less than 1.0
VAR-3 N449Q, P45 1T, T456G, Q457T 35 2.66 0.99 4
VAR-3 N449Q, S458Q, Q457T 29 2.72 0.95 3
VAR-3 P45 1T, T456G, N449Q, S458Q, 23 2.65 0.93 5
Q457T
VAR-3 N449Q, P45 IT, Q457T 46 2.74 0.77 3
VAR-3 N449Q, P45 1T, T456G, R459M 2 3.00 0.67 4
VAR-3 T450M 8 1.25 0.50 1
VAR-3 N449Q, P45 1T, Q457T, S458Q 31 2.68 0.44 4
VAR-3 N449Q, P45 1T, T456G, S458Q 23 2.65 0.43 4
VAR-3 N449Q, P45 1T, S458Q 25 2.80 0.33 3
VAR-3 N449Q, S458Q, T456G 21 2.52 0.22 3
VAR-3 N449Q, P45 1T 36 2.58 0.15 2
VAR-3 N449Q, S458Q 35 2.54 0.09 2
209
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VAR-3 N449Q, T455L 15 2.20 0.06
VAR-3 and VAR-4 common mutation sets with median whole retina transduction
(log2 relative to wtAAV2)
greater than 1.0
VAR-3, VAR- N449Q, T456G 40 2.60 1.51
4
VAR-3, VAR- N449Q, T456G, Q457T 28 2.68 L37 3
4
VAR-3, VAR- N449Q, Q457T 47 2.62 1.03
4
VAR-3, VAR-4 and VAR-14 common mutation set with median whole retina
transduction (1og2 relative to
wtAAV2) greater than 0.0 and less than 1.0
VAR-3, VAR- N449Q 122 2.36 0.32 1
4, VAR-14
VAR-3, VAR-4 and VAR-13 common mutation sets with median whole retina
transduction (1og2 relative to
w1AAV2) greater than 1.0
VAR-3, VAR- T456G 74 2.22 1.27 1
4, VAR-8,
VAR-13
VAR-3, VAR- T456G, Q457T 35 2.60 1.18
4, VAR-8,
VAR-13
VAR-3, VAR-4 and VAR-13 common mutation set with median whole retina
transduction (1og2 relative to
wtAAV2) greater than 0.0 and less than 1.0
VAR-3, VAR- Q457T 77 2.38 0.72 1
4, VAR-8,
VAR-13
VAR-3 and VAR-8 common mutation sets with median whole retina transduction
(10g2 relative to wtAAV2)
greater than 1.0
VAR-3, VAR- P45 1T, S458Q, T456G, Q457T 20 2.75 1.94 4
8
VAR-3, VAR- S458Q, T456G, Q457T 20 255 168 3
8
VAR-3, VAR- S458Q, T456G 30 2.60 1.63
8
VAR-3, VAR- P45 1T, S458Q, T456G 14 2.71 1.23 3
8
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VAR-3, VAR- P45 1T, T456G 14 2.29 1.23 9
8
VAR-3, VAR- P45 1T, T456G, Q457T 19 2.68 1.03 3
8
VAR-3 and VAR-8 common mutation sets with median whole retina transduction
(log2 relative to wtAAV2)
greater than 0.0 and less than 1.0
VAR-3, VAR- S458Q, Q457T 32 2.44 0.91 2
8
VAR-3, VAR- P45 1T, Q457T 19 2.63 0.77 9
8
VAR-3, VAR- P45 1T, S458Q 16 2.63 0.35 -)
8
VAR-3, VAR- S458Q 72 2.13 0.29 1
8
VAR-3, VAR- P45 1T, S458Q, Q457T 18 2.89 0.14 3
8
VAR-32 mutation set with median whole retina transduction (log2 relative to
wtAAV2) greater than 1.0
VAR-32 587_7+aa_588_+EQTRPA 2 3.00 3.43 1
VAR-4 mutation sets with median whole retina transduction (log2 relative to
wtAAV2) greater than IA)
VAR-4 N449Q, T456G, S452T 2 3.00 4.34 3
VAR-4 S452T, T456G, Q457T 2 3.00 3.89 3
VAR-4 T456G, P451-, Q457T, N449Q, 2 1.50 3.74 5
R459G
VAR-4 S452T, P451-, Q457T 4 3.00 3.61 3
VAR-4 N449Q, P451-. T4566, S452T 2 2.50 3.38 4
VAR-4 S452T, P451- 5 2.60 3.38 7
VAR-4 N449Q, Q457T, S452T 3 3.00 2.75 3
VAR-4 N449Q, Q457T, R459G 5 3.00 2.66 3
VAR-4 N449Q, P451-. T456G, Q4571 8 2.88 2.26 4
VAR-4 P451-, R459G 2 2.00 2.23 2
VAR-4 T456G, P451-, S452T, N449Q, 3 2.00 1.87 5
Q457T
VAR-4 N449Q, P451-, S452T 7 2.86 1.83 3
VAR-4 N449Q, T456G, R459G, Q457T 6 2.67 1.81 4
VAR-4 S452T, P451-, T456G, Q457T 3 2.67
1.63 4
VAR-4 N449Q, P451-. Q4571, S452T 5 2.40 1.63 4
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VAR-4 N449Q, P451-, Q457T 8 2.75 1.30 3
VAR-4 P451-, T456G 7 2.14 1.27 2
VAR-4 S452T 6 1.83 1.24 1
VAR-4 P451-, T456G, Q457T 4 2.00 1.06 3
VAR-4 mutation sets with median whole retina transduction (1og2 relative to
wtAAV2) greater than 0.0 and
less than 1.0
VAR-4 P451- 46 2.20 0.75 1
VAR-4 P451-, Q457T 11 2.27 0.60 /
VAR-4 N449Q, P451- 11 2.45 0.53 9
VAR-4 N449Q, P451-, T456G 5 2.40 0.09 3
VAR-4 and VAR-13 common mutation sets with median whole retina transduction
(1og2 relative to wtAAV2)
greater than 1.0
VAR-4, VAR- T456G, R459G, Q457T 2 3.00 2.93 3
13
VAR-4, VAR- R459G 14 2.00 2.42 1
13
VAR-40 mutation sets with median whole retina transduction (1og2 relative to
wtAAV2) greater than 1.0
VAR-40 T597A, R585S, G586S, A591E, 2 2.00 3.67 6
D594R, V6001
VAR-40 R585S, G586S, A591E, D594R, 2 2.50 3.39 5
V6001
VAR-40 A591E, T597A, R585S, D594R 3 2.00 2.93 4
VAR-40 T597A, R585S, N587A, G586S, 2 2.00 2.68 6
D594R, V6001
VAR-40 T597A, R585S, N587A, A591E, 2 1.50 2.54 5
D594R
VAR-40 T597A, R585S, N587A, G586S, 2 2_00 2_54 5
D594R
VAR-40 A591E, T597A, R585S, G586S 2 2.00 2.49 4
VAR-40 R585S, N587A, G586S, A591E, 2 2.50 2.10 5
V6001
VAR-40 A591E, T597A, D594R 2 2.50 1.36 3
VAR-40 A591E, R585S 2 3.00 1.32 2
VAR-40 mutation sets with median whole retina transduction (10g2 relative to
wtAAV2) greater than 0.0 and
less than 1.0
VAR-40 G586S, V6001 2 3.00 0.85 2
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VAR-40 G586S 90 2.22 0.33 1
VAR-40 T597A, R585S 9 2.11 0.31 2
VAR-40 A591E 8 1.50 0.28 1
VAR-40 V6001 17 L88 0.25 1
VAR-40 T597A 23 2.17 0.13 1
VAR-40 R585S, V6001 5 2.60 0.13 1
VAR-40 R585S, G586S 25 2.28 0.07 /
VAR-5 mutation set with median whole retina transduction (1og2 relative to
wtAAV2) greater than 0.0 and
less than 1.0
VAR-5 S458C 6 1.17 0.31 1
VAR-5 and VAR-14 common mutation set with median whole retina transduction
(1og2 relative to wtAAV2)
greater than 0.0 and less than 1.0
VAR-5, VAR- Q457F 5 0.80 0.26 1
14
VAR-8 mutation sets with median whole retina transduction (1og2 relative to
wtAAV2) greater than 1.0
VAR-8 P45 1T, T456G, T455G, R459T, 2 1.00 5.34 6
S458Q, Q457T
VAR-8 T456G, T455G, R459T, S458Q, 3 2.00 5.17 5
Q457T
VAR-8 P45 1T, T456G, T455G, R459T, 3 2.33 5.17 5
Q457T
VAR-8 P45 1T, S458Q, Q457T, T455G 3 3.00 5.17 4
VAR-8 Q461A, R459T, T456G, T455G 3 2.67 5.05 4
VAR-8 S458Q, R459T, T456G, T455G 5 2.80 5.05 4
VAR-8 P45 1T, T456G, R459T, S458Q, 4 2.25 4.67 5
Q457T
VAR-8 P45 1T, S458Q, Q457T, R459T 4 2.75 4.67 4
VAR-8 P45 1T, S458Q, T456G, R459T 4 2.75 4.67 4
VAR-8 T456G, T455G, Q461A, S458Q, 2 2.00 4.54 5
Q457T
VAR-8 P45 1T, T456G, T455G, Q461A, 2 L50 4.47 6
R459T, Q457T
VAR-8 P45 IT, Q461A, R459T, S458Q, 2 2.50 4.42 5
Q4571
VAR-8 S458Q, T456G, T455G, Q457T 4 3.00 4.38 4
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VAR-8 P45 1T, T456G, Q461A, R459T, 2 2.50 4.36 5
S458Q
VAR-8 Q461A, R459T, T455G 4 2.75 4.31 3
VAR-8 T456G, T455G, Q461A, R459T, 4 2.00 4.31 5
S458Q
VAR-8 Q461A, P45 1T, T455G 2 2.50 4.18 3
VAR-8 P45 1T, R459T, T456G, Q457T 5 2.80 4.17 4
VAR-8 5458Q, R459T, T456G, Q457T 5 3.00 4.17 4
VAR-8 R459T, T456G, T455G 2 2.50 4.12 3
VAR-8 S458Q, T456G, R459T 4 2.50 4.03 3
VAR-8 Q461A, R459T, 5458Q, T455G 5 2.80 4.01 4
VAR-8 T456G, T455G, 0461A, R459T, 3 1.67 4.01 6
5458Q, Q457T
VAR-8 T456G, Q461A, R459T, 5458Q, 4 2.50 3.67 5
Q457T
VAR-8 R459T, T456G 7 2.29 3.65 9
VAR-8 P451T, 5458Q, R459T, Q461A 2 2.50 3.61 4
VAR-8 T455G, Q461A, R459T, 5458Q, 3 2.33 3.58 5
Q457T
VAR-8 T456G, T455G, Q461A, R459T, 3 2.33 3.58 5
Q457T
VAR-8 Q461A, T456G, T455G, Q457T 3 2.67 3.58 4
VAR-8 Q461A, R459T, T456G, Q457T 3 3.00 3.58 4
VAR-8 Q461A, 5458Q, R459T 2 3.00 3.38 3
VAR-8 5458Q, R459T, Q457T 5 2.40 3.25 3
VAR-8 R459T, Q457T 9 2.00 3.22 9
VAR-8 R459T, T455G 3 3.00 3.20 2
VAR-8 Q461A, R459T 2 2.00 2.82 9
VAR-8 Q461A, P45 1T, T456G, Q457T 2 2.50 2.79 4
VAR-8 P45 1T, T456G, R459T 3 3.00 2.36 3
VAR-8 P45 1T, 5458Q, R459T 3 3.00 2.36 3
VAR-8 Q461A, T455G 3 3.00 2.27 9
VAR-8 Q461A, T456G, T455G 2 3.00 1.94 3
VAR-8 P45 1T, R459T, Q457T 2 3.00 1.79 3
VAR-8 R459T, T456G, Q457T 2 3.00 1.50 3
VAR-8 Q461A 13 1.46 1.32 1
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VAR-8 mutation set with median whole retina transduction (1og2 relative to
wtAAV2) greater than 0.0 and
less than 1.0
VAR-8 P45 1T, T456G, 1455G 2 2.00 0.90 3
VAR-8 and VAR-12 common mutation sets with median whole retina transduction
(1og2 relative to wtAAV2)
greater than 1.0
VAR-8, VAR- S458Q, R459T 13 2.00 3.25
12
VAR-8, VAR- R459T 37 1.95 2.21 1
12
VAR-8 and VAR-13 common mutation sets with median whole retina transduction
(1og2 relative to wtAAV2)
greater than 1.0
VAR-8, VAR- T456G, T455G 2 2.50 1.41
13
VAR-8, VAR- Q457T, 1455G 6 2.50 1.36
13
Table 13: Minimum structural elements comprised in the variants described
herein and
associated with increased median retina transduction relative to wild-type
AAV2 identified from
Library Experiment 2. In the second from left column, all amino acid position
numbering is
according to SEQ ID NO: 1. Amino acids are represented by their one-letter
code; deletions are
represented by a "-"; insertions are represented by the notation [last wild-
type amino acid
position before the insertionMlength of insertionl_aa-[wild-type amino acid
position after the
insertion]_[identity of the insertion]. A "+" indicates a motif with one or
more additional
positions which can be any amino acid.
Variant Name Common Mutai ton Set Number of viral Average number Median
whole Number of
(Numbering according to SEQ particles with of mutaitons retina
mutations from
ID NO: 1) representing a unique VP1 from wild type
transduction wild type AAV2
subcombination of the capsid AAV2 of all measurement
in the Common
mutations found in the polypeptide unique VP1 from
Library Mutation Set
Variant(s) listed in the left-most variants capsid Experiment 1
(for purposes of
column comprising the polypeptides in across
Set this table,
Common the Set,
ontiguous amino
Mutation Set and excluding the acids
inserted at
up to 3 additional mutations the
same
mutations from comprised in the
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wt AAV2 (the Common
position are
"Set") Mutation Set
counted as 1)
VAR-11 mutation set with median whole retina transduction (1og2 relative to
wtAAV2) greater than 0.0
VAR-11 S458- 8 2.625 0.258 1
VAR-13 mutation sets with median whole retina transduction (1og2 relative to
wtAAV2) greater than 0.0
VAR-13 T456G, Q457T, R459G, T455G2 3.000 0.301 4
VAR-13 P451Q, T455G, N449-, G453T, 4 2.750 0.257 5
Q457T
VAR-13 P451Q, N449-, Q457T 2 3.000 0.206 3
VAR-13 P451Q, T455G, N449-, T448-, 6 2.833 0.199 5
G453T
VAR-13 T455G, N449-, T448-, G453T, 6 2.833 0.199 5
R459G
VAR-13 T456G, P451Q, T455G, N449-, 4 2.750 0.167 6
T448-, G453T
VAR-13 T456G, P451Q, T455G, N449-, 3 1.667 0.083 7
T448-, G453T, R459G
VAR-13 P451Q, T455G, N449-, T448-, 11 2.455 0.083 6
G453T, R459G
VAR-13 N449-, Q457T, T448- 3 3.000 0.076 3
VAR-13 S446A, P451Q, T455G, N449-, 2 3.000 0.057 5
G453T
VAR-13 P451Q, Q457T, T448-, G453T, 6 2.833 0.035 5
R459G
VAR-13 P451Q, T456G, N449-, Q457T 4 3.000 0.030 4
VAR-13 G453T, R459G, Q457T 2 3.000 0.015 3
VAR-13 T456G, T455G, N449-, G453T, 2 3.000 0.015 5
R459G
VAR-13 P451Q, T456G, Q457T, N449-, 8 2.500 0.000 5
R459G
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VAR-13 P451Q, T456G, N449-, T448-, 18 2.278 0.000 5
R4596
VAR-18 mutation set with median whole retina transduction (1og2 relative to
wtAAV2) greater than 0.0
VAR-18 584_5+aa_58.5_R AYN+ 2 3.000 0.358 1
VAR-19 and VAR-23 common mutation set with median whole retina transduction
(1og2 relative to wtAAV2)
greater than 0.0
VAR-19, R566K 3 2.333 0.048 1
VAR-23
VAR-26 mutation set with median whole retina transduction (1og2 relative to
wtAAV2) greater than 0.0
VAR-26 A590P, T597S, V600A 4 2.500 0.093 3
VAR-3 mutation set with median whole retina transduction (1og2 relative to
wtAAV2) greater than 0.0
VAR-3 N449Q, P45 1T 2 1.500 0.680
VAR-3 and VAR-8 common mutation set with median whole retina transduction
(1og2 relative to wtAAV2)
greater than 0.0
VAR-3, P45 IT, S4.58Q, T456G, Q457T 3 3.000 0.045 4
VAR-8
VAR-30 mutation set with median whole retina transduction (1og2 relative to
wtAAV2) greater than 0.0
VAR-30 586_6+aa_587_DQDFK+ 4 2.500 0.113 1
VAR-32 mutation sets with median whole retina transduction (1og2 relative to
wtAAV2) greater than 0.0
VAR-32 587_8+aa_588_LAIEQTR+ 2 2.500 0.175 1
VAR-32 587_7+aa_588_+EQTRPA 9 2.778 0.126 1
VAR-32 587_6+aa_588_+QTRPA 9 3.000 0.036 1
VAR-33 mutation sets with median whole retina transduction (1og2 relative to
wtAAV2) greater than 0.0
VAR-33 584_8+aa_585_RARLDET+, 200 2.980 0.505 2
G586P
VAR-33 584 8+aa 585 RARLDET+, 203 2.980 0.491
N587A
VAR-33 584_8+aa_585_RARLDET+, 268 2.239 0.488 3
G586P, N587A
VAR-33 584_7+aa_585_RARLDE+, 234 2.970 0.446 3
G586P, N587A
VAR-33 584_7+aa_585_RARLDE+, 7 3.000 0.368 2
N587A
VAR-33 G586P, N587A, 8 3.000 0.233 3
584 6+aa 585 RARLD+
VAR-33 584 8+aa 585 RARLDET+ 4 3.000 0.073 1
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VAR-35 mutation sets with median whole retina transduction (1og2 relative to
wtAAV2) greater than 0.0
VAR-35 586_7+aa_587_+GEQTRP 4 3.000 0.736 1
VAR-35 586 6+aa 587 +EQTRP, 16 3.000 0.736 1
N587A
VAR-35 N587A, 7 2.429 0.494 /
586_7+aa_587_+GEQTRP
VAR-36 mutation sets with median whole retina transduction (1og2 relative to
wtAAV2) greater than 0.0
VAR-36 586_5+aa_587_ATDT+ 3 2.333 0.514 1
VAR-36 586_6+aa_587_ATDTK+ 19 2.842 0.143 1
VAR-36 586_6aa_587_ATDTKT 20 1.900 0.111 1
VAR-36 586_6+aa_587_+TDTKT 20 2.750 0.111 1
VAR-4 mutation set with median whole retina transduction (1og2 relative to
wtAAV2) greater than 0.0
VAR-4 S452T, T456G 3 2.667 0.474 /
VAR-8 mutation sets with median whole retina transduction (1og2 relative to
wtAAV2) greater than 0.0
VAR-8 P45 1T, T456G, Q461A, / 2.000 0.252 5
S458Q, Q457T
VAR-8 S458Q, R459T, T455G 9 2.778 0.118 3
VAR-8 Q461A, S458Q, T456G, Q457T 3 3.000 0.045 4
VAR-8 P451T, T456G, 1455G, R459T, 2 1.000 0.043 6
S458Q, Q457T
VAR-8 P45 1T, S458Q, Q457T, R459T 3 3.000 0.041 4
VAR-8 P45 IT, S458Q, T456G, T455G 3 2.667 0.041 4
VAR-8 R459T, T456G, T455G, Q457T 3 3.000 0.041 4
VAR-8 T455G, 0461A, R459T, 4 2 750 0.011 5
S458Q, Q457T
Accordingly, provided herein is a capsid polypeptide that comprises a sequence
at least
90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at
least 96%, at least
97%, at least 98%, or at least 99% identical to a VP1, VP2, or VP3 of SEQ ID
NO: 1, and
comprising a Common Mutation Set listed in Table 12 or Table 13. In
embodiments, a virus
particle comprising said capsid polypeptide exhibits increased retina
transduction, relative to a
virus particle comprising wild-type AAV2, for example, as determined by NGS
sequencing of
viral cDNA from bulk retina tissue as described herein. In embodiments, the
virus particle has at
least 2-fold increased retina transduction relative to wild-type AAV2. for
example, as determined
by NGS sequencing of viral cDNA from bulk retina tissue as described herein.
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Provided herein is a capsid polypeptide, for example, a capsid polypeptide
comprising a
sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at
least 94%, at least 95%,
at least 96%, at least 97%, at least 98%, or at least 99% identical to a VP1,
VP2 or VP3 of SEQ
ID NO: 1, and comprising a Common Mutation Set associated with VAR-11. In
embodiments,
the Common Mutation Set comprises or consists of: (1) R459-. and T456-, (2)
R459-, and Q457-
(3) S458-, T456-, and L460-, (4) R459-, Q457-, and T456-, (5) Q457-, and L460-
, (6) R459-.
Q457-. and L460-, (7) R459-, S458-, and Q457-, (8) S458-. Q457-, and T456-,
(9) L460-, S458-,
R459-, Q457-, and T456-, (10) R459-, S458-, Q457-, and L460-, (11) R459-, S458-
, Q457-, and
T456-, (12) S458-, and L460-, (13) R459-, S458-, L460-, (14) R459-, and S458-,
(15) S458-, and
Q457-, or (16) S458-, and T456-. In embodiments. the capsid polypeptide
comprises fewer than
10, fewer than 9, fewer than 8, fewer than 7, fewer than 6, fewer than 5,
fewer than 4, fewer than
3, fewer than 2 or no additional mutations relative to the VP1, VP2 or VP3
sequence of SEQ ID
NO: 1. In embodiments, a virus particle comprising said capsid polypeptide
exhibits increased
retina transduction, relative to a virus particle comprising wild-type AAV2,
for example, as
determined by NGS sequencing of viral cDNA from bulk retina tissue. In
embodiments, the virus
particle exhibits at least 8-fold or at least 4-fold increased retina
transduction, relative to wild-
type AAV2, for example as determined as described herein.
Provided herein is a capsid polypeptide, for example, a capsid polypeptide
comprising a
sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at
least 94%, at least 95%,
at least 96%, at least 97%, at least 98%, or at least 99% identical to a VP1,
VP2 or VP3 of SEQ
ID NO: 1, and comprising a Common Mutation Set associated with VAR-12. In
embodiments,
the Common Mutation Set comprises or consists of: (1) 5458Q, T455Q, and P451G,
(2) T456N,
and R459T. (3) N449I, T456N, R459T, and T455Q, (4) R459T, and P451G, (5)
5458Q, T454-,
and T450N, (6) R459T, and T454-, or (7) S458Q, Q461K, R459T, and Q464V. In
embodiments,
the capsid polypeptide comprises fewer than 10, fewer than 9, fewer than 8,
fewer than 7, fewer
than 6, fewer than 5, fewer than 4, fewer than 3, fewer than 2 or no
additional mutations relative
to the VP1, VP2 or VP3 sequence of SEQ ID NO: 1. In embodiments, a virus
particle comprising
said capsid polypeptide exhibits increased retina transduction, relative to a
virus particle
comprising wild-type AAV2, for example, as determined by NOS sequencing of
viral cDNA
from bulk retina tissue. In embodiments, the virus particle exhibits at least
4-fold increased retina
transduction, relative to wild-type AAV2, for example as determined as
described herein.
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Provided herein is a capsid polypeptide, for example, a capsid polypeptide
comprising a
sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at
least 94%, at least 95%,
at least 96%, at least 97%, at least 98%, or at least 99% identical to a VP1,
VP2 or VP3 of SEQ
ID NO: 1, and comprising a Common Mutation Set associated with VAR-13. In
embodiments,
the Common Mutation Set comprises or consists of: (1) 5446A, P451Q, T455G,
N449-, T448-,
G453T, and Q457T, (2) S446A, P451Q, T456G, T455G, N449-, T448-, G453T, and
Q457T, (3)
S446A, and Q457T, or (4) 5446A, and T456G. In embodiments, the capsid
polypeptide
comprises fewer than 10, fewer than 9, fewer than 8, fewer than 7, fewer than
6, fewer than 5,
fewer than 4, fewer than 3, fewer than 2 or no additional mutations relative
to the VP1, VP2 or
VP3 sequence of SEQ ID NO: 1. In embodiments, a virus particle comprising said
capsid
polypeptide exhibits increased retina transduction, relative to a virus
particle comprising wild-
type AAV2, for example, as determined by NGS sequencing of viral cDNA from
bulk retina
tissue. In embodiments, the virus particle exhibits at least 8-fold, at least
4-fold or at least 2 fold
increased retina transduction, relative to wild-type AAV2, for example as
determined as
described herein.
Provided herein is a capsid polypeptide, for example, a capsid polypeptide
comprising a
sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at
least 94%, at least 95%,
at least 96%, at least 97%, at least 98%, or at least 99% identical to a VP1,
VP2 or VP3 of SEQ
ID NO: 1, and comprising a Common Mutation Set associated with VAR-14. In
embodiments,
the Common Mutation Set comprises or consists of: (1) T455A, T450S, and R459D,
(2) N449Q.
T455A, T450S, and R459D, (3) N449Q, T450S, and R459D, (4) S452G, T455A, T450S,
N449Q, and R459D, (5) N449Q, S452G, T455A, and R459D, (6) N449Q, T455A. and
R459D,
(7) N449Q, S452G, and R459D, or (8) N449Q, and R459D. In embodiments, the
capsid
polypeptide comprises fewer than 10, fewer than 9, fewer than 8, fewer than 7,
fewer than 6,
fewer than 5, fewer than 4, fewer than 3, fewer than 2 or no additional
mutations relative to the
VP1, VP2 or VP3 sequence of SEQ ID NO: 1. In embodiments, a virus particle
comprising said
capsid polypeptide exhibits increased retina transduction, relative to a virus
particle comprising
wild-type AAV2, for example, as determined by NGS sequencing of viral cDNA
from bulk
retina tissue. In embodiments, the virus particle exhibits at least 8-fold, at
least 4-fold or at least
2-fold increased retina transduction, relative to wild-type AAV2, for example
as determined as
described herein.
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Provided herein is a capsid polypeptide, for example, a capsid polypeptide
comprising a
sequence that is at least 90% identical to a VP1, VP2 or VP3 of SEQ ID NO: 1,
and comprising a
S578D mutation. In embodiments, the capsid polypeptide comprises fewer than
10, fewer than 9,
fewer than 8, fewer than 7, fewer than 6, fewer than 5, fewer than 4, fewer
than 3, fewer than 2
or no additional mutations relative to the VP1, VP2 or VP3 sequence of SEQ ID
NO: 1. In
embodiments, a virus particle comprising said capsid polypeptide exhibits
increased retina
transduction, relative to a virus particle comprising wild-type AAV2, for
example, as determined
by NGS sequencing of viral cDNA from bulk retina tissue.
Provided herein is a capsid polypeptide, for example, a capsid polypeptide
comprising a
sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at
least 94%, at least 95%,
at least 96%, at least 97%, at least 98%, or at least 99% identical to a VP1,
VP2 or VP3 of SEQ
ID NO: 1, and comprising a Common Mutation Set associated with VAR-21. In
embodiments,
the Conunon Mutation Set comprises or consists of: (1) S578I, and M558L, (2)
S578I, and
S580A, (3) M558L, and S580A, (4) K556N, and S578I. In embodiments, the capsid
polypeptide
comprises fewer than 10, fewer than 9, fewer than 8, fewer than 7, fewer than
6, fewer than 5,
fewer than 4, fewer than 3, fewer than 2 or no additional mutations relative
to the VP1, VP2 or
VP3 sequence of SEQ ID NO: 1. In embodiments, a virus particle comprising said
capsid
polypeptide exhibits increased retina transduction, relative to a virus
particle comprising wild-
type AAV2, for example, as determined by NGS sequencing of viral cDNA from
bulk retina
tissue. In embodiments, the virus particle exhibits at least 8-fold, at least
4-fold or at least 2-fold
increased retina transduction, relative to wild-type AAV2, for example as
determined as
described herein.
Provided herein is a capsid polypeptide, for example, a capsid polypeptide
comprising a
sequence that is at least 90% identical to a VP1, VP2 or VP3 of SEQ ID NO: 1,
and comprising a
S578I mutation. In embodiments, the capsid polypeptide comprises fewer than
10, fewer than 9,
fewer than 8, fewer than 7, fewer than 6, fewer than 5, fewer than 4, fewer
than 3, fewer than 2
or no additional mutations relative to the VP1, VP2 or VP3 sequence of SEQ ID
NO: 1. In
embodiments, a virus particle comprising said capsid polypeptide exhibits
increased retina
transduction, relative to a virus particle comprising wild-type AAV2, for
example, as determined
by NGS sequencing of viral cDNA from bulk retina tissue.
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Provided herein is a capsid polypeptide, for example, a capsid polypeptide
comprising a
sequence that is at least 90% identical to a VP1, VP2 or VP3 of SEQ ID NO: 1,
and comprising a
I559L, and T581D mutation. In embodiments, the capsid polypeptide comprises
fewer than 10,
fewer than 9, fewer than 8, fewer than 7, fewer than 6, fewer than 5, fewer
than 4, fewer than 3,
fewer than 2 or no additional mutations relative to the VP1. VP2 or VP3
sequence of SEQ ID
NO: 1. In embodiments, a virus particle comprising said capsid polypeptide
exhibits increased
retina transduction, relative to a virus particle comprising wild-type AAV2,
for example, as
determined by NGS sequencing of viral cDNA from bulk retina tissue.
Provided herein is a capsid polypeptide, for example, a capsid polypeptide
comprising a
sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at
least 94%, at least 95%,
at least 96%, at least 97%, at least 98%, or at least 99% identical to a VP1,
VP2 or VP3 of SEQ
ID NO: 1, and comprising a Common Mutation Set associated with VAR-26. In
embodiments,
the Conunon Mutation Set comprises or consists of: (1) R588T, R585S, T597S,
A591I, and
V600A, (2) R588T, R585S, Q589N, A590P, T597S, and A591I, (3) A593G, R585S,
T597S,
A591I, and V600A, (4) R588T, R585S, A590P, T597S, A591I, and V600A. (5) R588T,
A593G,
R5855, Q589N, A590P, and V600A, (6) R588T, A593G, Q589N, A590P, and A591I. (7)
R588T, R585S, Q589N, A590P, and T597S, or (8) R588T, R585S, Q589N, A590P,
T597S,
andV600A. In embodiments, the capsid polypeptide comprises fewer than 10,
fewer than 9,
fewer than 8, fewer than 7, fewer than 6, fewer than 5, fewer than 4, fewer
than 3, fewer than 2
or no additional mutations relative to the VP1, VP2 or VP3 sequence of SEQ ID
NO: 1. In
embodiments, a virus particle comprising said capsid polypeptide exhibits
increased retina
transduction, relative to a virus particle comprising wild-type AAV2, for
example, as determined
by NGS sequencing of viral cDNA from bulk retina tissue. In embodiments, the
virus particle
exhibits at least 8-fold, at least 4-fold or at least 2-fold increased retina
transduction, relative to
wild-type AAV2, for example as determined as described herein.
Provided herein is a capsid polypeptide, for example, a capsid polypeptide
comprising a
sequence that is at least 90% identical to a VP1, VP2 or VP3 of SEQ ID NO: 1,
and comprising
an insertion at any position between amino acid 580 and amino acid 600 of SEQ
ID NO:1,
preferably after position 592 of SEQ ID NO:1, wherein the insertion comprises
LX1X2, wherein
X1 and X2 are any amino acid or not present. In embodiments, the capsid
polypeptide comprises
fewer than 10, fewer than 9, fewer than 8, fewer than 7, fewer than 6, fewer
than 5, fewer than 4,
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fewer than 3, fewer than 2 or no additional mutations relative to the VP1, VP2
or VP3 sequence
of SEQ ID NO: 1. In embodiments, a virus particle comprising said capsid
polypeptide exhibits
increased retina transduction, relative to a virus particle comprising wild-
type AAV2, for
example, as determined by NGS sequencing of viral cDNA from bulk retina
tissue.
Provided herein is a capsid polypeptide, for example, a capsid polypeptide
comprising a
sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at
least 94%, at least 95%,
at least 96%, at least 97%, at least 98%, or at least 99% identical to a VP1,
VP2 or VP3 of SEQ
ID NO: 1, and comprising a Common Mutation Set associated with VAR-3. In
embodiments, the
Common Mutation Set comprises or consists of: (1) P45 1T. T456G, T455L, N449Q,
S458Q, and
Q457T, (2) P451T, T456G, T450M, N449Q, S458Q, and Q457T, (3) N449Q, T455L,
T456G,
and S458Q. (4) P45 1T, T455L, N449Q, S458Q, and Q457T, (5) P45 1T, T456G,
T455L, N449Q,
and Q4571, (6) P45 1T. T456G, R459M, N449Q, and Q457T. In embodiments, the
capsid
polypeptide comprises fewer than 10, fewer than 9, fewer than 8, fewer than 7,
fewer than 6,
fewer than 5, fewer than 4, fewer than 3, fewer than 2 or no additional
mutations relative to the
VP1, VP2 or VP3 sequence of SEQ ID NO: 1. In embodiments, a virus particle
comprising said
capsid polypeptide exhibits increased retina transduction, relative to a virus
particle comprising
wild-type AAV2, for example, as determined by NGS sequencing of viral cDNA
from bulk
retina tissue. In embodiments, the virus particle exhibits at least 8-fold, at
least 4-fold or at least
2-fold increased retina transduction, relative to wild-type AAV2, for example
as determined as
described herein.
Provided herein is a capsid polypeptide, for example, a capsid polypeptide
comprising a
sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at
least 94%, at least 95%,
at least 96%, at least 97%, at least 98%, or at least 99% identical to a VP1,
VP2 or VP3 of SEQ
ID NO: 1, and comprising a Common Mutation Set associated with VAR-3, VAR-4
VAR-8,
VAR-13 and/or VAR-14. In embodiments, the Common Mutation Set comprises or
consists of:
(1) N449Q, (2) N449Q, and T456G, (3) N449Q, T456G, and Q457T, (4) T456G, (5)
T456G, and
Q457T, (6) Q457T or (7) N449Q, and Q457T. In embodiments, the capsid
polypeptide
comprises fewer than 10, fewer than 9, fewer than 8, fewer than 7, fewer than
6, fewer than 5,
fewer than 4, fewer than 3, fewer than 2 or no additional mutations relative
to the VP1, VP2 or
VP3 sequence of SEQ ID NO: 1. In embodiments, a virus particle comprising said
capsid
polypeptide exhibits increased retina transduction, relative to a virus
particle comprising wild-
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type AAV2, for example, as determined by NGS sequencing of viral cDNA from
bulk retina
tissue. In embodiments, the virus particle exhibits at least 8-fold, at least
4-fold or at least 2-fold
increased retina transduction, relative to wild-type AAV2, for example as
determined as
described herein.
Provided herein is a capsid polypeptide, for example, a capsid polypeptide
comprising a
sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at
least 94%, at least 95%,
at least 96%, at least 97%, at least 98%, or at least 99% identical to a VP1,
VP2 or VP3 of SEQ
ID NO: 1, and comprising an insertion of at least 6 amino acids following any
of amino acid
positions 580-600 of SEQ ID NO: 1, for example, following amino acid 587
according to SEQ
ID NO: 1, wherein the insertion comprises or consists of X1X2EQTRPA (SEQ ID
NO: 134),
wherein X1 and X2 are independently selected from any amino acid or not
present. In
embodiments, the capsid polypeptide comprises fewer than 10, fewer than 9,
fewer than 8, fewer
than 7, fewer than 6, fewer than 5, fewer than 4, fewer than 3, fewer than 2
or no additional
mutations relative to the VP1, VP2 or VP3 sequence of SEQ ID NO: 1. In
embodiments, a virus
particle comprising said capsid polypeptide exhibits increased retina
transduction, relative to a
virus particle comprising wild-type AAV2, for example, as determined by NGS
sequencing of
viral cDNA from bulk retina tissue. In embodiments, the virus particle
exhibits at least 8-fold, at
least 4-fold or at least 2-fold increased retina transduction, relative to
wild-type AAV2, for
example as determined as described herein.
Provided herein is a capsid polypeptide, for example, a capsid polypeptide
comprising a
sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at
least 94%, at least 95%,
at least 96%, at least 97%, at least 98%, or at least 99% identical to a VP1,
VP2 or VP3 of SEQ
ID NO: 1, and comprising a Common Mutation Set associated with VAR-4. In
embodiments, the
Common Mutation Set comprises or consists of: (1) N449Q, T456G, and S452T, (2)
S452T,
T456G, and Q457T, (3) T456G, P451-, Q457T, N449Q, and R459G, (4) S452T, P451-,
and
Q457T, (5) N449Q, P451-, T456G, and S452T, (6) S452T, and P451-, (7) N449Q,
Q457T, and
S452T, (8) N449Q, Q4571, and R459G, (9) N449Q, P451-. T456G, and Q457T. or
(10) P451-,
and R459G. In embodiments, the capsid polypeptide comprises fewer than 10,
fewer than 9,
fewer than 8, fewer than 7, fewer than 6, fewer than 5, fewer than 4, fewer
than 3, fewer than 2
or no additional mutations relative to the VP1, VP2 or VP3 sequence of SEQ ID
NO: 1. In
embodiments, a virus particle comprising said capsid polypeptide exhibits
increased retina
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transduction, relative to a virus particle comprising wild-type AAV2, for
example, as determined
by NGS sequencing of viral cDNA from bulk retina tissue. In embodiments, the
virus particle
exhibits at least 8-fold, at least 4-fold or at least 2-fold increased retina
transduction, relative to
wild-type AAV2, for example as determined as described herein.
Provided herein is a capsid polypeptide, for example, a capsid polypeptide
comprising a
sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at
least 94%, at least 95%,
at least 96%, at least 97%, at least 98%, or at least 99% identical to a VP1,
VP2 or VP3 of SEQ
ID NO: 1, and comprising a Common Mutation Set associated with VAR-4 and VAR-
13. In
embodiments, the Common Mutation Set comprises or consists of: (1) T456G,
R459G, and
Q457T, (2) R459G. In embodiments, the capsid polypeptide comprises fewer than
10, fewer than
9, fewer than 8, fewer than 7, fewer than 6, fewer than 5, fewer than 4, fewer
than 3, fewer than 2
or no additional mutations relative to the VP1, VP2 or VP3 sequence of SEQ ID
NO: 1. In
embodiments, a virus particle comprising said capsid polypeptide exhibits
increased retina
transduction, relative to a virus particle comprising wild-type AAV2, for
example, as determined
by NGS sequencing of viral cDNA from bulk retina tissue. In embodiments, the
virus particle
exhibits at least 8-fold, at least 4-fold or at least 2-fold increased retina
transduction, relative to
wild-type AAV2, for example as determined as described herein.
Provided herein is a capsid polypeptide, for example, a capsid polypeptide
comprising a
sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at
least 94%, at least 95%,
at least 96%, at least 97%, at least 98%, or at least 99% identical to a VP1,
VP2 or VP3 of SEQ
ID NO: 1, and comprising a Common Mutation Set associated with VAR-40. In
embodiments,
the Common Mutation Set comprises or consists of: (1) T597A, R585S, G586S,
A591E, D594R,
and V600I, (2) R585S, G586S, A591E, D594R, and V600I, (3) A591E, T597A. R585S,
and
D594R, (4) T597A, R585S, N587A, G586S, D594R, and V600I, (5) T597A, R585S,
N587A,
A591E, and D594R, (6) T597A, R585S, N587A. G586S, and D594R, (7) A591E, T597A,
R5855, and G586S, or (8) R585S, N587A, G586S, A591E, and V600I. In
embodiments, the
capsid polypeptide comprises fewer than 10, fewer than 9, fewer than 8, fewer
than 7, fewer than
6, fewer than 5, fewer than 4, fewer than 3, fewer than 2 or no additional
mutations relative to
the VP1, VP2 or VP3 sequence of SEQ ID NO: 1. In embodiments, a virus particle
comprising
said capsid polypeptide exhibits increased retina transduction, relative to a
virus particle
comprising wild-type AAV2, for example, as determined by NGS sequencing of
viral cDNA
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from bulk retina tissue. In embodiments, the virus particle exhibits at least
8-fold, at least 4-fold
or at least 2-fold increased retina transduction, relative to wild-type AAV2,
for example as
determined as described herein.
Provided herein is a capsid polypeptide, for example, a capsid polypeptide
comprising a
sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at
least 94%, at least 95%,
at least 96%, at least 97%, at least 98%, or at least 99% identical to a VP1,
VP2 or VP3 of SEQ
ID NO: 1, and comprising a Common Mutation Set associated with VAR-8. In
embodiments, the
Common Mutation Set comprises or consists of: (1) P451T, T456G, T455G, R459T,
S458Q, and
Q457T, (2) T456G, T455G, R459T, S458Q, and Q457T, (3) P451T, T456G, T455G,
R459T, and
Q457T, (4) P451T, S458Q, Q457T, and T455G, (5) Q461A, R459T, T456G, and T455G,
(6)
S458Q, R459T, T456G, and T455G, (7) P451T, T456G, R459T, S458Q, and Q457T, (8)
P451T,
S458Q, Q457T, and R459T, (9) P451T. S458Q, 1456G, and R459T, (10) T456G,
T455G,
Q461A, 5458Q, and Q457T, (11) P451T, T456G, T455G, Q461A, R459T, and Q457T,
(12)
P451T, Q461A, R459T, S458Q, and Q457T, (13) S458Q, T456G, T455G, and Q457T,
(14)
P451T, T456G, Q461A, R459T, and S458Q, (15) Q461A, R459T, and T455G, (16)
T456G,
T455G, Q461A, R459T, and S458Q, (17) Q461A, P451T, and T455G, (18) P451T.
R459T,
T456G, and Q457T, (19) 5458Q, R459T, T456G, and Q457T, (20) R459T, T456G, and
T455G.
(21) S458Q, T456G, and R459T, (22) Q461A, R459T, S458Q, and T455G. (23) T456G,
T455G,
Q461A, R459T, S458Q, and Q457T, (24) T456G, Q461A, R459T, S458Q, and Q4571,
(25)
R459T, and T4566, (26) P451T, S458Q, R459T, and Q461A, (27) T4556, Q461A,
R459T,
S458Q, and Q457T, (28) T456G, T455G, Q461A, R459T, and Q457T, (29) Q461A,
T456G,
T455G, and Q457T, (30) Q461A, R459T. T456G, and Q457T, (31) Q461A, S458Q, and
R459T
(32) 5458Q, R459T, and Q457T, (33) R459T, and Q457T, (34) R459T, and T455G,
(35)
Q461A, and R459T, (36) Q461A, P451T, T456G, and Q457T, (37) P451T, T456G, and
R459T,
(38) P451T, S458Q, and R459T, or (39) Q461A, and T455G. In embodiments, the
capsid
polypeptide comprises fewer than 10, fewer than 9, fewer than 8, fewer than 7,
fewer than 6,
fewer than 5, fewer than 4, fewer than 3, fewer than 2 or no additional
mutations relative to the
VP1, VP2 or VP3 sequence of SEQ ID NO: 1. In embodiments, a virus particle
comprising said
capsid polypeptide exhibits increased retina transduction, relative to a virus
particle comprising
wild-type AAV2, for example, as determined by NGS sequencing of viral cDNA
from bulk
retina tissue. In embodiments, the virus particle exhibits at least 32-fold,
at least 16-fold, at least
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8-fold, at least 4-fold or at least 2-fold increased retina transduction,
relative to wild-type AAV2,
for example as determined as described herein.
Provided herein is a capsid polypeptide, for example, a capsid polypeptide
comprising a
sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at
least 94%, at least 95%,
at least 96%, at least 97%, at least 98%, or at least 99% identical to a VP1,
VP2 or VP3 of SEQ
ID NO: 1, and comprising a Common Mutation Set associated with VAR-8 and VAR-
12 or
VAR-13. In embodiments, the Common Mutation Set comprises or consists of: (1)
S458Q, and
R4591, (2) R4591, (3) T456G, and T455G, or (4) Q457T, and T455G. In
embodiments, the
capsid polypeptide comprises fewer than 10, fewer than 9, fewer than 8, fewer
than 7, fewer than
6, fewer than 5, fewer than 4, fewer than 3, fewer than 2 or no additional
mutations relative to
the VP1, VP2 or VP3 sequence of SEQ ID NO: 1. In embodiments, a virus particle
comprising
said capsid polypeptide exhibits increased retina transduction, relative to a
virus particle
comprising wild-type AAV2, for example, as determined by NGS sequencing of
viral cDNA
from bulk retina tissue. In embodiments, the virus particle exhibits at least
32-fold, at least 16-
fold, at least 8-fold, at least 4-fold or at least 2-fold increased retina
transduction, relative to
wild-type AAV2, for example as determined as described herein.
Provided herein is a capsid polypeptide, for example, a capsid polypeptide
comprising a
sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at
least 94%, at least 95%,
at least 96%, at least 97%, at least 98%, or at least 99% identical to a VP1,
VP2 or VP3 of SEQ
ID NO: 1, and comprising an insertion of at least 4 amino acids following any
of amino acid
positions 580-600 of SEQ ID NO: 1, for example, following amino acid 584
according to SEQ
ID NO: 1, wherein the insertion comprises or consists of RAYNX1X2 (SEQ ID NO:
135),
wherein X1 and X2 are independently selected from any amino acid or not
present. In
embodiments, the capsid polypeptide comprises fewer than 10, fewer than 9,
fewer than 8, fewer
than 7, fewer than 6, fewer than 5, fewer than 4, fewer than 3, fewer than 2
or no additional
mutations relative to the VP1, VP2 or VP3 sequence of SEQ ID NO: 1. In
embodiments, a virus
particle comprising said capsid polypeptide exhibits increased retina
transduction, relative to a
virus particle comprising wild-type AAV2, for example, as determined by NGS
sequencing of
viral cDNA from bulk retina tissue. In embodiments, the virus particle
exhibits at least 8-fold, at
least 4-fold or at least 2-fold increased retina transduction, relative to
wild-type AAV2, for
example as determined as described herein.
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Provided herein is a capsid polypeptide, for example, a capsid polypeptide
comprising a
sequence that is at least 90% identical to a VP1, VP2 or VP3 of SEQ ID NO: 1,
and comprising a
R566K mutation. In embodiments, the capsid polypeptide comprises fewer than
10, fewer than 9,
fewer than 8, fewer than 7, fewer than 6, fewer than 5, fewer than 4, fewer
than 3, fewer than 2
or no additional mutations relative to the VP1, VP2 or VP3 sequence of SEQ ID
NO: 1. In
embodiments, a virus particle comprising said capsid polypeptide exhibits
increased retina
transduction, relative to a virus particle comprising wild-type AAV2, for
example, as determined
by NGS sequencing of viral cDNA from bulk retina tissue.
Provided herein is a capsid polypeptide, for example, a capsid polypeptide
comprising a
sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at
least 94%, at least 95%,
at least 96%, at least 97%, at least 98%, or at least 99% identical to a VP1,
VP2 or VP3 of SEQ
ID NO: 1, and comprising an insertion of at least 4 amino acids following any
of amino acid
positions 580-600 of SEQ ID NO: 1, for example, following amino acid 586
according to SEQ
ID NO: 1, wherein the insertion comprises or consists of DQDFKX1X2 (SEQ ID NO:
136),
wherein X1 and X2 are independently selected from any amino acid or not
present. In
embodiments, the capsid polypeptide comprises fewer than 10, fewer than 9,
fewer than 8, fewer
than 7, fewer than 6, fewer than 5, fewer than 4, fewer than 3, fewer than 2
or no additional
mutations relative to the VP1, VP2 or VP3 sequence of SEQ ID NO: 1. In
embodiments, a virus
particle comprising said capsid polypeptide exhibits increased retina
transduction, relative to a
virus particle comprising wild-type AAV2, for example, as determined by NGS
sequencing of
viral cDNA from bulk retina tissue. In embodiments, the virus particle
exhibits at least 8-fold, at
least 4-fold or at least 2-fold increased retina transduction, relative to
wild-type AAV2, for
example as determined as described herein.
Provided herein is a capsid polypeptide, for example, a capsid polypeptide
comprising a
sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at
least 94%, at least 95%,
at least 96%, at least 97%, at least 98%, or at least 99% identical to a VP1,
VP2 or VP3 of SEQ
ID NO: 1, and comprising an insertion of at least 5 amino acids following any
of amino acid
positions 580-600 of SEQ ID NO: 1, for example, following amino acid 587
according to SEQ
ID NO: 1, wherein the insertion comprises or consists of IEQTRX1X2 (SEQ ID NO:
137) or
LAIEQTRX1X2 (SEQ ID NO: 138), wherein X1 and X2 are independently selected
from any
amino acid or not present. In embodiments, the capsid polypeptide comprises
fewer than 10,
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fewer than 9, fewer than 8, fewer than 7, fewer than 6, fewer than 5, fewer
than 4, fewer than 3,
fewer than 2 or no additional mutations relative to the VP1. VP2 or VP3
sequence of SEQ ID
NO: 1. In embodiments, a virus particle comprising said capsid polypeptide
exhibits increased
retina transduction, relative to a virus particle comprising wild-type AAV2,
for example, as
determined by NOS sequencing of viral cDNA from bulk retina tissue. In
embodiments, the virus
particle exhibits at least 8-fold, at least 4-fold or at least 2-fold
increased retina transduction,
relative to wild-type AAV2, for example as determined as described herein.
Provided herein is a capsid polypeptide, for example, a capsid polypeptide
comprising a
sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at
least 94%, at least 95%,
at least 96%, at least 97%, at least 98%, or at least 99% identical to a VP1,
VP2 or VP3 of SEQ
ID NO: 1, and comprising an insertion of at least 6 amino acids following any
of amino acid
positions 580-600 of SEQ ID NO: 1, for example, following amino acid 587
according to SEQ
ID NO: 1, wherein the insertion comprises or consists of X1X2EQTRPA (SEQ ID
NO: 132),
wherein X1 and X2 are independently selected from any amino acid or not
present. In
embodiments, the capsid polypeptide comprises fewer than 10, fewer than 9,
fewer than 8, fewer
than 7, fewer than 6, fewer than 5, fewer than 4, fewer than 3, fewer than 2
or no additional
mutations relative to the VP1, VP2 or VP3 sequence of SEQ ID NO: 1. In
embodiments, a virus
particle comprising said capsid polypeptide exhibits increased retina
transduction, relative to a
virus particle comprising wild-type AAV2, for example, as determined by NGS
sequencing of
viral cDNA from bulk retina tissue. In embodiments, the virus particle
exhibits at least 8-fold, at
least 4-fold or at least 2-fold increased retina transduction, relative to
wild-type AAV2, for
example as determined as described herein.
Provided herein is a capsid polypeptide, for example, a capsid polypeptide
comprising a
sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at
least 94%, at least 95%,
at least 96%, at least 97%, at least 98%, or at least 99% identical to a VP1,
VP2 or VP3 of SEQ
ID NO: 1, and comprising an insertion of at least 5 amino acids following any
of amino acid
positions 580-600 of SEQ ID NO: 1, for example, following amino acid 587
according to SEQ
ID NO: 1, wherein the insertion comprises or consists of X1X2QTRPA (SEQ ID NO:
139),
wherein X1 and X2 are independently selected from any amino acid or not
present. In
embodiments, the capsid polypeptide comprises fewer than 10, fewer than 9,
fewer than 8, fewer
than 7, fewer than 6, fewer than 5, fewer than 4, fewer than 3, fewer than 2
or no additional
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mutations relative to the VP1, VP2 or VP3 sequence of SEQ ID NO: 1. In
embodiments, a virus
particle comprising said capsid polypeptide exhibits increased retina
transduction, relative to a
virus particle comprising wild-type AAV2, for example, as determined by NGS
sequencing of
viral cDNA from bulk retina tissue. In embodiments, the virus particle
exhibits at least 8-fold, at
least 4-fold or at least 2-fold increased retina transduction, relative to
wild-type AAV2, for
example as determined as described herein.
Provided herein is a capsid polypeptide, for example, a capsid polypeptide
comprising a
sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at
least 94%, at least 95%,
at least 96%, at least 97%, at least 98%, or at least 99% identical to a VP1,
VP2 or VP3 of SEQ
ID NO: 1, and comprising an insertion of at least 5 amino acids following any
of amino acid
positions 580-600 of SEQ ID NO: 1, for example, following amino acid 584
according to SEQ
ID NO: 1, wherein the insertion comprises or consists of RARLDX1X2 (SEQ ID NO:
140),
RARLDEX1X2 (SEQ ID NO: 141) or RARLDETX1X2 (SEQ ID NO: 142), wherein X1 and X2
are independently selected from any amino acid or not present. In embodiments
the capsid
polypeptide further comprises a G586P mutation. In embodiments, the capsid
polypeptide further
comprises a N587A mutation. In embodiments, the capsid polypeptide further
comprises a
G586P and N587A mutation. In embodiments, the capsid polypeptide comprises
fewer than 10,
fewer than 9, fewer than 8, fewer than 7, fewer than 6, fewer than 5, fewer
than 4, fewer than 3,
fewer than 2 or no additional mutations relative to the VP1. VP2 or VP3
sequence of SEQ ID
NO: 1. In embodiments, a virus particle comprising said capsid polypeptide
exhibits increased
retina transduction, relative to a virus particle comprising wild-type AAV2,
for example, as
determined by NGS sequencing of viral cDNA from bulk retina tissue. In
embodiments, the virus
particle exhibits at least 8-fold, at least 4-fold or at least 2-fold
increased retina transduction,
relative to wild-type AAV2, for example as determined as described herein.
Provided herein is a capsid polypeptide, for example, a capsid polypeptide
comprising a
sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at
least 94%, at least 95%,
at least 96%, at least 97%, at least 98%, or at least 99% identical to a VP1,
VP2 or VP3 of SEQ
ID NO: 1, and comprising an insertion of at least 7 amino acids following any
of amino acid
positions 580-600 of SEQ ID NO: 1, for example, following amino acid 584
according to SEQ
ID NO: 1, wherein the insertion comprises or consists of RARLDETX1X2 (SEQ ID
NO: 142),
wherein X1 and X2 are independently selected from any amino acid or not
present. In
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embodiments, the capsid polypeptide comprises fewer than 10, fewer than 9,
fewer than 8, fewer
than 7, fewer than 6, fewer than 5, fewer than 4, fewer than 3, fewer than 2
or no additional
mutations relative to the VP1, VP2 or VP3 sequence of SEQ ID NO: 1. In
embodiments, a virus
particle comprising said capsid polypeptide exhibits increased retina
transduction, relative to a
virus particle comprising wild-type AAV2, for example, as determined by NGS
sequencing of
viral cDNA from bulk retina tissue. In embodiments, the virus particle
exhibits at least 8-fold, at
least 4-fold or at least 2-fold increased retina transduction, relative to
wild-type AAV2, for
example as determined as described herein.
Provided herein is a capsid polypeptide, for example, a capsid polypeptide
comprising a
sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at
least 94%, at least 95%,
at least 96%, at least 97%, at least 98%, or at least 99% identical to a VP1,
VP2 or VP3 of SEQ
ID NO: 1, and comprising an insertion of at least 5 amino acids following any
of amino acid
positions 580-600 of SEQ ID NO: 1, for example, following amino acid 586
according to SEQ
ID NO: 1, wherein the insertion comprises or consists of X1X2GEQTRP (SEQ ID
NO: 143) or
X1X2EQTRP (SEQ ID NO: 144), wherein X1 and X2 are independently selected from
any
amino acid or not present. In embodiments, the capsid polypeptide further
comprises a N587A
mutation. In embodiments, the capsid polypeptide comprises fewer than 10,
fewer than 9, fewer
than 8, fewer than 7, fewer than 6, fewer than 5, fewer than 4, fewer than 3,
fewer than 2 or no
additional mutations relative to the VP1, VP2 or VP3 sequence of SEQ ID NO: 1.
In
embodiments, a virus particle comprising said capsid polypeptide exhibits
increased retina
transduction, relative to a virus particle comprising wild-type AAV2, for
example, as determined
by NGS sequencing of viral eDNA from bulk retina tissue. In embodiments, the
virus particle
exhibits at least 8-fold, at least 4-fold or at least 2-fold increased retina
transduction, relative to
wild-type AAV2, for example as determined as described herein.
Provided herein is a capsid polypeptide, for example, a capsid polypeptide
comprising a
sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at
least 94%, at least 95%,
at least 96%, at least 97%, at least 98%, or at least 99% identical to a VP1,
VP2 or VP3 of SEQ
ID NO: 1, and comprising an insertion of at least 4 amino acids following any
of amino acid
positions 580-600 of' SEQ ID NO: 1, for example, following amino acid 586
according to SEQ
ID NO: 1, wherein the insertion comprises or consists of ATDTX1X2 (SEQ ID NO:
145),
wherein X1 and X2 are independently selected from any amino acid or not
present. In
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embodiments, the capsid polypeptide comprises fewer than 10, fewer than 9,
fewer than 8, fewer
than 7, fewer than 6, fewer than 5, fewer than 4, fewer than 3, fewer than 2
or no additional
mutations relative to the VP1, VP2 or VP3 sequence of SEQ ID NO: 1. In
embodiments, a virus
particle comprising said capsid polypeptide exhibits increased retina
transduction, relative to a
virus particle comprising wild-type AAV2, for example, as determined by NGS
sequencing of
viral cDNA from bulk retina tissue. In embodiments, the virus particle
exhibits at least 8-fold, at
least 4-fold or at least 2-fold increased retina transduction, relative to
wild-type AAV2, for
example as determined as described herein.
Provided herein is a capsid polypeptide, for example, a capsid polypeptide
comprising a
sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at
least 94%, at least 95%,
at least 96%, at least 97%, at least 98%, or at least 99% identical to a VP1,
VP2 or VP3 of SEQ
ID NO: 1, and comprising an insertion of at least 5 amino acids following any
of amino acid
positions 580-600 of SEQ ID NO: 1, for example, following amino acid 586
according to SEQ
ID NO: 1, wherein the insertion comprises or consists of ATDTKX1X2 (SEQ ID NO:
146),
wherein X1 and X2 are independently selected from any amino acid or not
present. In
embodiments, the capsid polypeptide comprises fewer than 10, fewer than 9,
fewer than 8, fewer
than 7, fewer than 6, fewer than 5, fewer than 4, fewer than 3, fewer than 2
or no additional
mutations relative to the VP1, VP2 or VP3 sequence of SEQ ID NO: 1. In
embodiments, a virus
particle comprising said capsid polypeptide exhibits increased retina
transduction, relative to a
virus particle comprising wild-type AAV2, for example, as determined by NGS
sequencing of
viral cDNA from bulk retina tissue. In embodiments, the virus particle
exhibits at least 8-fold, at
least 4-fold or at least 2-fold increased retina transduction, relative to
wild-type AAV2, for
example as determined as described herein.
Provided herein is a capsid polypeptide, for example, a capsid polypeptide
comprising a
sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at
least 94%, at least 95%,
at least 96%, at least 97%, at least 98%, or at least 99% identical to a VP1,
VP2 or VP3 of SEQ
ID NO: 1, and comprising an insertion of at least 6 amino acids following any
of amino acid
positions 580-600 of SEQ ID NO: 1, for example, following amino acid 586
according to SEQ
ID NO: 1, wherein the insertion comprises or consists of ATDTKT (SEQ ID NO:
115), wherein
X1 and X2 are independently selected from any amino acid or not present. In
embodiments, the
capsid polypeptide comprises fewer than 10, fewer than 9, fewer than 8, fewer
than 7, fewer than
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6, fewer than 5, fewer than 4, fewer than 3, fewer than 2 or no additional
mutations relative to
the VP1, VP2 or VP3 sequence of SEQ ID NO: 1. In embodiments, a virus particle
comprising
said capsid polypeptide exhibits increased retina transduction, relative to a
virus particle
comprising wild-type AAV2, for example, as determined by NGS sequencing of
viral cDNA
from bulk retina tissue. In embodiments, the virus particle exhibits at least
8-fold, at least 4-fold
or at least 2-fold increased retina transduction, relative to wild-type AAV2,
for example as
determined as described herein.
Provided herein is a capsid polypeptide, for example, a capsid polypeptide
comprising a
sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at
least 94%, at least 95%,
at least 96%, at least 97%, at least 98%, or at least 99% identical to a VP1,
VP2 or VP3 of SEQ
ID NO: 1, and comprising an insertion of at least 5 amino acids following any
of amino acid
positions 580-600 of SEQ ID NO: I, thr example, following amino acid 586
according to SEQ
ID NO: 1, wherein the insertion comprises or consists of X1X2TDTKT (SEQ ID NO:
147),
wherein X1 and X2 are independently selected from any amino acid or not
present. In
embodiments, the capsid polypeptide comprises fewer than 10, fewer than 9,
fewer than 8, fewer
than 7, fewer than 6, fewer than 5, fewer than 4, fewer than 3, fewer than 2
or no additional
mutations relative to the VP1, VP2 or VP3 sequence of SEQ ID NO: 1. In
embodiments, a virus
particle comprising said capsid polypeptide exhibits increased retina
transduction, relative to a
virus particle comprising wild-type AAV2, for example, as determined by NGS
sequencing of
viral cDNA from bulk retina tissue. In embodiments, the virus particle
exhibits at least 8-fold, at
least 4-fold or at least 2-fold increased retina transduction, relative to
wild-type AAV2, for
example as determined as described herein.
In embodiments, provided herein is a nucleic acid molecule encoding a capsid
polypeptide, for example a VP1, VP2 or VP3 capsid polypeptide, described
above. In
embodiments, provided herein is a virus particle comprising a capsid
polypeptide, for example, a
VP1, VP2 or VP3 capsid polypeptide, described above.
Methods of Making Compositions Described Herein
The disclosure is directed, in part, to a method of making a capsid
polypeptide described
herein or a dependoparvovirus particle, e.g., a dependoparvovirus particle
described herein. In
some embodiments, a method of making dependoparvovirus particle comprises
providing a cell,
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cell-free system, or other translation system, comprising a nucleic acid
described herein encoding
a variant capsid polypeptide provided for herein, or a polypeptide provided
for herein (e.g., a
variant capsid polypeptide); and cultivating the cell, cell-free system, or
other translation system
under conditions suitable for the production of the dependoparvovirus
particle, thereby making
the dependoparvovirus particle.
In some embodiments, providing a cell comprising a nucleic acid described
herein
comprises introducing the nucleic acid to the cell, e.g., transfecting or
transforming the cell with
the nucleic acid. The nucleic acids of the disclosure may be situated as a
part of any genetic
element (vector) which may be delivered to a host cell, e.g., naked DNA, a
plasmid, phage,
transposon, cosmid, episome, a protein in a non-viral delivery vehicle (e.g.,
a lipid-based carrier),
virus, etc. which transfer the sequences carried thereon. Such a vector may be
delivered by any
suitable method, including transfection, liposome delivery, electroporation,
membrane fusion
techniques, viral infection, high velocity DNA- coated pellets, and protoplast
fusion. A person of
skill in the art possesses the knowledge and skill in nucleic acid
manipulation to construct any
embodiment of this invention and said skills include genetic engineering,
recombinant
engineering, and synthetic techniques. See, e.g., Sambrook et al, Molecular
Cloning: A
Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY.
In some embodiments, a vector of the disclosure comprises sequences encoding a
dependoparvovirus variant capsid polypeptide as provided for herein or a
fragment thereof. In
some embodiments, vectors of the disclosure comprises sequences encoding a
dependoparvovirus rep protein or a fragment thereof. In some embodiments, such
vectors may
contain sequence encoding both dependoparvovirus cap (e.g., a variant capsid
polypeptide
described herein) and rep proteins. In vectors in which both AAV rep and cap
are provided, the
dependoparvovirus rep and dependoparvovirus cap sequences may both be of the
same
dependoparvovirus species or serotype origin, such as AAV2. Alternatively, the
present
disclosure also provides vectors in which the rep sequences are from a
dependoparvovirus
species or serotype which differs from that from which the cap sequences are
dervied. In some
embodiments, the rep and cap sequences are expressed from separate sources
(e.g., separate
vectors, or a host cell genome and a vector). In some embodiments, the rep
sequences are fused
in frame to cap sequences of a different dependoparvovirus species or serotype
to form a
chimeric dependoparvovirus vector. In some embodiments, the vectors of the
invention further
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contain a payload, e.g., a minigene comprising a selected transgene (e.g., a
payload as described
herein), e.g., flanked by dependoparvovirus 5' ITR and dependoparvovirus 3'
ITR.
The vectors described herein, e.g., a plasmid, are useful for a variety of
purposes, but are
particularly well suited for use in production of recombinant
dependoparvovirus particles
comprising dependoparvovirus sequences or a fragment thereof, and in some
embodiments, a
payload.
In some embodiments, the disclosure provides a method of making a
dependoparvovirus
particle (e.g., a dependoparvovirus B particle, e.g., an AAV2 particle or
particle comprising a
variant capsid polypeptide as described herein), or a portion thereof. In some
embodiments, the
method comprises culturing a host cell which contains a nucleic acid sequence
encoding a
dependoparvovirus variant capsid polypeptide as provided for herein, or
fragment thereof, ; a
functional rep gene; a payload (e.g., as described herein), e.g., a minigene
comprising
dependoparvovirus inverted terminal repeats (ITRs) and a transgene, optionally
under the control
of a regulatory element such as a promoter; and sufficient helper functions to
promote packaging
of the payload, e.g., minigene, into the dependoparvovirus capsid. The
components necessary to
be cultured in the host cell to package a payload, e.g., minigene, in a
dependoparvovirus capsid
may be provided to the host cell in trans. In some embodiments, any one or
more of the required
components (e.g., payload (e.g., minigene), rep sequences, cap sequences,
and/or helper
functions) may be provided by a host cell which has been engineered to stably
comprise one or
more of the required components using methods known to those of skill in the
art. In some
embodiments, a host cell which has been engineered to stably comprise the
required
component(s) comprises it under the control of an inducible promoter. In some
embodiments, the
required component may be under the control of a constitutive promoter.
Examples of suitable
inducible and constitutive promoters are provided herein and further examples
are known to
those of skill in the art. In some embodiments, a selected host cell which has
been engineered to
stably comprise one or more components may comprise a component under the
control of a
constitutive promoter and another component under the control of one or more
inducible
promoters. For example, a host cell which has been engineered to stably
comprise the required
components may be generated from 293 cells (e.g., which comprise helper
functions under the
control of a constitutive promoter), which comprises the rep and/or cap
proteins under the
control of one or more inducible promoters.
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The payload (e.g., minigene), rep sequences, cap sequences, and helper
functions
required for producing a dependoparvovirus particle of the disclosure may be
delivered to the
packaging host cell in the form of any genetic element which transfers the
sequences carried
thereon (e.g., in a vector or combination of vectors). The genetic element may
be delivered by
any suitable method, including those described herein. Methods used to
construct genetic
elements, vectors, and other nucleic acids of the disclosure are known to
those with skill and
include genetic engineering, recombinant engineering, and synthetic
techniques. See, e.g.,
Sambrook et al, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor
Press, Cold
Spring Harbor, NY. Similarly, methods of generating rAAV virions are well
known and the
selection of a suitable method is not a limitation on the present invention.
See, e.g., K. Fisher et
al, J. Virol, 70:520-532 (1993) and US Patent 5,478.745. Unless otherwise
specified, the
dependoparvovirus ITRs, and other selected dependoparvovirus components
described herein,
may be readily selected from among any dependoparvovirus species and
serotypes, e.g., AAV1,
AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV9. ITRs or other dependoparvovirus
components may be readily isolated using techniques available to those of
skill in the art from a
dependoparvovirus species or serotype. Dependoparvovirus species and serotypes
may be
isolated or obtained from academic, commercial, or public sources (e.g., the
American Type
Culture Collection, Manassas, VA). In some embodiments, the dependoparvovirus
sequences
may be obtained through synthetic or other suitable means by reference to
published sequences
such as are available in the literature or in databases such as, e.g., GenBank
or PubMed.
The dependoparvovirus particles (e.g., including a variant capsid polypeptide
and, for
example, a payload) of the disclosure may be produced using any invertebrate
cell type which
allows for production of dependoparvovirus or biologic products and which can
be maintained in
culture. In some embodiments, an insect cell may be used in production of the
compositions
described herein or in the methods of making a dependoparvovirus particle
described herein. For
example, an insect cell line used can be from Spodoptera frugiperda, such as
Sf9, SF21, SF900+,
drosophila cell lines, mosquito cell lines, e.g., Aedes albopictus derived
cell lines, domestic
silkworm cell lines, e.g. Bombyxmori cell lines, Trichoplusia ni cell lines
such as High Five cells
or Lepidoptera cell lines such as Ascalapha odorata cell lines. In some
embodiments, the insect
cells are susceptible to baculovirus infection, including High Five, Sf9,
Se301, SeIZD2109,
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SeUCR1, SP900+, Sf21, BTI-TN-5B1-4, MG-1, Tn368, HzAml, BM-N, Ha2302, Hz2E5
and
Ao38.
In some embodiments, the methods of the disclosure can be carried out with any
mammalian cell type which allows for replication of dependoparvovirus or
production of
biologic products, and which can be maintained in culture. In some
embodiments, the
mammalian cells used can be 11EK293, HEK293T, HeLa, CHO, NSO, SP2/0, PER.C6,
Vero,
RD, BHK, HT 1080, A549, Cos-7, ARPE-19 or MRC-5 cells. In some embodiments the
culture
is an adherent cell culture. In some embodiments, the culture is a suspension
cell culture.
Methods of expressing proteins (e.g., recombinant or heterologous proteins,
e.g.,
dependoparvovirus polypeptides) in insect cells are well documented, as are
methods of
introducing nucleic acids, such as vectors, e.g., insect-cell compatible
vectors, into such cells and
methods of maintaining such cells in culture. See, for example, METHODS IN
MOLECULAR
BIOLOGY, ed. Richard, Humana Press, N J (1995); O'Reilly et al., BACULOVIRUS
EXPRESSION VECTORS, A LABORATORY MANUAL, Oxford Univ. Press (1994); Samulski
et
al., J. Vir. 63:3822-8 (1989); Kajigaya et al., Proc. Nat'l. Acad. Sci. USA
88:4646-50 (1991);
Ruffing et al., J. Vir. 66:6922-30 (1992); Kirnbauer et al., Vir. 219:37-44
(1996); Zhao et
al., Vir. 272:382-93 (2000); and Samulski et al., U.S. Pat. No. 6,204,059. In
some embodiments,
a nucleic acid construct encoding dependoparvovirus polypeptides (e.g., a
dependoparvovirus
genome) in insect cells is an insect cell-compatible vector. An "insect cell-
compatible vector" as
used herein refers to a nucleic acid molecule capable of productive
transformation or transfection
of an insect or insect cell. Exemplary biological vectors include plasmids,
linear nucleic acid
molecules, and recombinant viruses. Any vector can be employed as long as it
is insect cell-
compatible. The vector may integrate into the insect cell's genome or remain
present extra-
chromosomally. The vector may be present permanently or transiently, e.g., as
an episomal
vector. Vectors may be introduced by any means known in the art. Such means
include but are
not limited to chemical treatment of the cells, electroporation, or infection.
In some
embodiments, the vector is a baculovirus, a viral vector, or a plasmid.
In some embodiments, a nucleic acid sequence encoding an dependoparvovirus
polypeptide is operably linked to regulatory expression control sequences for
expression in a
specific cell type, such as Sf9 or HEK cells. Techniques known to one skilled
in the art for
expressing foreign genes in insect host cells or mammalian host cells can be
used with the
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compositions and methods of the disclosure. Methods for molecular engineering
and expression
of polypeptides in insect cells is described, for example, in Summers and
Smith. A Manual of
Methods for Baculovirus Vectors and Insect Culture Procedures, Texas
Agricultural
Experimental Station Bull. No. 7555, College Station, Tex. (1986); Luckow.
1991. In Prokop et
al., Cloning and Expression of Heterologous Genes in Insect Cells with
Baculovirus Vectors'
Recombinant DNA Technology and Applications, 97-152 (1986); King, L. A. and R.
D.
Possee, The baculovirus expression system, Chapman and Hall, United Kingdom
(1992);
O'Reilly, D. R., L. K. Miller, V. A. Luckow, Baculovirus Expression Vectors: A
Laboratory
Manual, New York (1992); W. H. Freeman and Richardson, C. D., Baculovirus
Expression
Protocols, Methods in Molecular Biology, volume 39 (1995); U.S. Pat. No.
4,745,051;
US2003148506; and WO 03/074714. Promoters suitable for transcription of a
nucleotide
sequence encoding a dependoparvovirus polypeptide include the polyhedronõ p10,
p35 or 1E-1
promoters and further promoters described in the above references are also
contemplated.
In some embodiments, providing a cell comprising a nucleic acid described
herein
comprises acquiring a cell comprising the nucleic acid.
Methods of cultivating cells, cell-free systems, and other translation systems
are known
to those of skill in the art. In some embodiments, cultivating a cell
comprises providing the cell
with suitable media and incubating the cell and media for a time suitable to
achieve viral particle
production.
In some embodiments, a method of making a dependoparvovirus particle further
comprises a purification step comprising isolating the dependoparvovirus
particle from one or
more other components (e.g., from a cell or media component).
In some embodiments, production of the dependoparvovirus particle comprises
one or
more (e.g., all) of: expression of dependoparvovirus polypeptides, assembly of
a
dependoparvovirus capsid (e.g., a capsid comprising a variant capsid
polypeptide provided for
herein), expression (e.g., duplication) of a dependoparvovirus genome, and
packaging of the
dependoparvovirus genome into the dependoparvovirus capsid to produce a
dependoparvovirus
particle. In some embodiments, production of the dependoparvovirus particle
further comprises
secretion of the dependoparvovirus particle.
In some embodiments, and as described elsewhere herein, the nucleic acid
molecule
encoding the variant capsid polypeptide is disposed in a dependoparvovirus
genome. In some
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embodiments, and as described elsewhere herein, the nucleic acid molecule
encoding the variant
capsid polypeptide is packaged into a dependoparvovirus particle along with
the
dependoparvovirus genome as part of a method of making a dependoparvovirus
particle
described herein. In other embodiments, the nucleic acid molecule encoding the
variant capsid
polypeptide is not packaged into a dependoparvovirus particle made by a method
described
herein.
In some embodiments, a method of making a dependoparvovirus particle described
herein produces a dependoparvovirus particle comprising a payload (e.g., a
payload described
herein) and the variant capsid polypeptide. In some embodiments, the payload
comprises a
second nucleic acid (e.g., in addition to the dependoparvovirus genome), and
production of the
dependoparvovirus particle comprises packaging the second nucleic acid into
the
dependoparvovirus particle. In some embodiments, a cell, cell-free system, or
other translation
system for use in a method of making a dependoparvovirus particle comprises
the second nucleic
acid. In some embodiments, the second nucleic acid comprises an exogenous
sequence (e.g.,
exogenous to the dependoparvovirus, the cell, or to a target cell or subject
who will be
administered the dependoparvovirus particle). In some embodiments, the
exogenous sequence
encodes an exogenous polypeptide. In some embodiments, the exogenous sequence
encodes a
therapeutic product.
In some embodiments, a nucleic acid or polypeptide described herein is
produced by a
method known to one of skill in the art. The nucleic acids, polypeptides, and
fragments thereof
of the disclosure may be produced by any suitable means, including recombinant
production,
chemical synthesis, or other synthetic means. Such production methods are
within the knowledge
of those of skill in the art and are not a limitation of the present
invention.
Applications
The disclosure is directed, in part, to compositions comprising a nucleic
acid,
polypeptide, or particles described herein. The disclosure is further
directed, in part, to methods
utilizing a composition, nucleic acid, polypeptide, or particles described
herein. As will be
apparent based on the disclosure, nucleic acids, polypeptides, particles, and
methods disclosed
herein have a variety of utilities.
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The disclosure is directed, in part, to a vector comprising a nucleic acid
described herein,
e.g., a nucleic acid encoding a variant capsid polypeptide. Many types of
vectors are known to
those of skill in the art. In some embodiments, a vector comprises a plasmid.
In some
embodiments, the vector is an isolated vector, e.g., removed from a cell or
other biological
components.
The disclosure is directed, in part to a cell, cell-free system, or other
translation system,
comprising a nucleic acid or vector described herein, e.g., a nucleic acid or
vector comprising a
nucleic acid molecule encoding a variant capsid polypeptide. In some
embodiments, the cell,
cell-free system, or other translation system is capable of producing
dependoparvovirus particles
comprising the variant capsid polypeptides. In some embodiments, the cell,
cell-free system, or
other translation system comprises a nucleic acid comprising a
dependoparvovirus genome or
components of a dependoparvovirus genome sufficient to promote production of
dependoparvovirus particles comprising the variant capsid polypeptides.
In some embodiments, the cell, cell-free system, or other translation system
further
comprises one or more non-dependoparvovirus nucleic acid sequences that
promote
dependoparvovirus particle production and/or secretion. Said sequences are
referred to herein as
helper sequences. In some embodiments, a helper sequence comprises one or more
genes from
another virus, e.g., an adenovirus or herpes virus. In some embodiments, the
presence of a helper
sequence is necessary for production and/or secretion of a dependoparvovirus
particle. In some
embodiments, a cell, cell-free system, or other translation system comprises a
vector, e.g.,
plasmid, comprising one or more helper sequences.
In some embodiments, a cell, cell-free system, or other translation system
comprises a
first nucleic acid and a second nucleic acid, wherein the first nucleic acid
comprises a sequences
encoding one or more dependoparvovirus genes (e.g., a Cap gene, a Rep gene, or
a complete
dependoparvovirus genome) and a helper sequence, and wherein the second
nucleic acid
comprises a payload. In some embodiments, a cell, cell-free system, or other
translation system
comprises a first nucleic acid and a second nucleic acid, wherein the first
nucleic acid comprises
a sequences encoding one or more dependoparvovirus genes (e.g., a Cap gene, a
Rep gene, or a
complete dependoparvovirus genome) and a payload, and wherein the second
nucleic acid
comprises a helper sequence. In some embodiments, a cell, cell-free system, or
other translation
system comprises a first nucleic acid and a second nucleic acid, wherein the
first nucleic acid
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comprises a helper sequence and a payload, and wherein the second nucleic acid
comprises a
sequences encoding one or more dependoparvovirus genes (e.g., a Cap gene, a
Rep gene, or a
complete dependoparvovirus genome). In some embodiments, a cell, cell-free
system, or other
translation system comprises a first nucleic acid, a second nucleic acid, and
a third nucleic acid,
wherein the first nucleic acid comprises a sequences encoding one or more
dependoparvovirus
genes (e.g., a Cap gene, a Rep gene, or a complete dependoparvovirus genome),
the second
nucleic acid comprises a helper sequence, and the third nucleic acid comprises
a payload.
In some embodiments, the first nucleic acid, second nucleic acid, and
optionally third
nucleic acid are situated in separate molecules, e.g., separate vectors or a
vector and genomic
DNA. In some embodiments, one, two, or all of the first nucleic acid, second
nucleic acid, and
optionally third nucleic acid are integrated (e.g., stably integrated) into
the genome of a cell.
A cell of the disclosure may be generated by transfeeting a suitable cell with
a nucleic
acid described herein. In some embodiments, a method of making a
dependoparvovirus particle
comprising a variant capsid polypeptide as provided for herein or improving a
method of making
a dependoparvovirus particle comprises providing a cell described herein. In
some embodiments,
providing a cell comprises transfecting a suitable cell with one or more
nucleic acids described
herein.
In some embodiments, the virus particle comprising the variant capsid is
produced at a
level at least 10%, at least 20%, at least 50%, at least 100%, at least 200%
or greater than the
production level of wt AAV2 from the same producer cell type, e.g., from
HEK293 cells, e.g.,
from adherent culture of HEK293 cells.
Many types and kinds of cells suitable for use with the nucleic acids and
vectors
described herein are known in the art. In some embodiments, the cell is a
human cell. In some
embodiments, the cell is an immortalized cell or a cell from a cell line known
in the art. In some
embodiments, the cell is an HEK293 cell.
Virus particles and Methods of delivering a payload
The disclosure is directed, in part, to a method of delivering a payload to a
cell, e.g., a
cell in a subject or in a sample. In some embodiments, a method of delivering
a payload to a cell
comprises contacting the cell with a dependoparvovirus particle comprising a
variant capsid
polypeptide (e.g., described herein) comprising the payload. In some
embodiments, the
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dependoparvovirus particle is a dependoparvovirus particle described herein
and comprises a
payload described herein. In some embodiments, the cell is an ocular cell. In
some
embodiments, the ocular cell is in the retina, macula, or trabecular meshwork.
In some
embodiments, the ocular cell is in the retina. In some embodiments, the ocular
cell is in the
macula. In some embodiments, the ocular cell is in the trabecular meshwork.
In some embodiments, the ocular cell is in the front third of the eye, which
includes the
structures in front of the vitreous humor. Examples of structures in front of
the vitreous humor,
include the cornea, iris, ciliary body, lens, trabecular meshwork, and
Schlemm's canal.
Accordingly, in some embodiments, the cell is in the cornea, iris, ciliary
body, lens, trabecular
meshwork, or Schlemm's canal, or any combination thereof.
In some embodiments, the ocular cell is posterior to the lens, such as in the
anterior
hyaloid membrane and all of the optical structures behind it, such as the
vitreous humor, retina,
choroid or optic nerve, or any combination thereof. Accordingly, in some
embodiments, the cell
is in the anterior hyaloid membrane and all of the optical structures behind
it, such as the vitreous
humor, retina, choroid or optic nerve, or any combination thereof.
The disclosure is further directed in part to a virus particle comprising a
capsid
polypeptide described herein. In embodiments, the virus particle comprises a
capsid polypeptide
described herein and a nucleic acid expression construct. In embodiments the
nucleic acid
expression construct of the virus particle comprises a payload.
In some embodiments, the payload comprises a transgene. In some embodiments,
the
transgene is a nucleic acid sequence heterologous to the vector sequences
flanking the transgene
which encodes a polypeptide, RNA (e.g., a miRNA or siRNA) or other product of
interest. The
nucleic acid of the transgene may be operatively linked to a regulatory
component in a manner
sufficient to promote transgene transcription, translation, and/or expression
in a host cell.
A transgene may be any polypeptide or RNA encoding sequence and the transgene
selected will depend upon the use envisioned. In some embodiments, a transgene
comprises a
reporter sequence, which upon expression produces a detectable signal. Such
reporter sequences
include, without limitation, DNA sequences encoding colorimetric reporters
(e.g., p-lactamase,
P-galactosidase (LacZ), alkaline phosphatase), cell division reporters (e.g.,
thymidine kinase),
fluorescent or luminescence reporters (e.g., green fluorescent protein (GFP)
or luciferase),
resistance conveying sequences (e.g., chloramphenicol acetyltransferase
(CAT)), or membrane
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bound proteins including to which high affinity antibodies directed thereto
exist or can be
produced by conventional means, e.g., comprising an antigen tag, e.g.,
hemagglutinin or Myc.
In some embodiments, a reporter sequence operably linked with regulatory
elements
which drive their expression, provide signals detectable by conventional
means, including
enzymatic, radiographic, colorimetric, fluorescence or other spectrographic
assays, fluorescent
activating cell sorting assays and immunological assays, including enzyme
linked
immunosorbent assay (ELISA), radioimmunoassay (RIA) and immunohistochemistry.
In some embodiments, the transgene encodes a product which is useful in
biology and
medicine, such as RNA, proteins, peptides, enzymes, dominant negative mutants.
In some
embodiments, the RNA comprises a tRNA, ribosomal RNA, dsRNA, catalytic RNAs,
small
hairpin RNA, siRNA, trans-splicing RNA, and antisense RNAs. In some
embodiments, the RNA
inhibits or abolishes expression of a targeted nucleic acid sequence in a
treated subject (e.g., a
human or animal subject).
In some embodiments, the transgene may be used to correct or ameliorate gene
deficiencies. In some embodiments, gene deficiencies include deficiencies in
which normal
genes are expressed at less than normal levels or deficiencies in which the
functional gene
product is not expressed. In some embodiments, the transgene encodes a
therapeutic protein or
polypeptide which is expressed in a host cell. In some embodiments, a
dependoparvovirus
particle may comprise or deliver multiple transgenes, e.g., to correct or
ameliorate a gene defect
caused by a multi-subunit protein. In some embodiments, a different transgene
(e.g., each
situated/delivered in a different dependoparvovirus particle, or in a single
dependoparvovirus
particle) may be used to encode each subunit of a protein, or to encode
different peptides or
proteins, e.g., when the size of the DNA encoding the protein subunit is
large, e.g., for
immunoglobulin, platelet-derived growth factor, or dystrophin protein. In some
embodiments,
different subunits of a protein may be encoded by the same transgene, e.g., a
single transgene
encoding each of the subunits with the DNA for each subunit separated by an
internal ribozyme
entry site (TRES) or enzymatically cleavable sequence (e.g., a furin cleavage
site). In some
embodiments, the DNA may be separated by sequences encoding a 2A peptide,
which self-
cleaves in a post-translational event. See, e.g., Donnelly et al, J. Gen.
Virol., 78(Pt 1):13-21
(January 1997); Furler, et al, Gene Ther., 8(11):864-873 (June 2001); Klump et
al., Gene
Ther 8(10):811-817 (May 2001).
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In some embodiments, virus particles comprising a genome are provided, wherein
the
genome includes a nucleic acid expression construct. The nucleic acid
expression construct can
include a payload, for example a payload comprising a heterologous transgene
and one or more
regulatory elements.
In some embodiments, the particle delivers the payload to the eye with
increased
transduction in one or more regions of the eye as compared to a virus particle
comprising capsid
polypeptides of SEQ ID NO: 1, and wherein the increase in transduction is at
least 2-times, 4-
times, 8-times, 16-times, 32-times, 64-times, 100-times, or 150-times as
compared to a virus
particle comprising capsid polypeptides of SEQ ID NO: 1. In some embodiments,
the particle
delivers the payload to the eye with increased transduction specificity in one
or more regions of
the eye as compared to a virus particle comprising capsid polypeptides of SEQ
ID NO: 1,
wherein the increase in transduction is at least 2-times, 4-times, 8-times, 16-
times, 32-times, 64-
times, 100-times, 200-times, 500-times, or 1000-times as compared to a virus
particle comprising
capsid polypeptides of SEQ ID NO: 1, and wherein the increase in transduction
is specific to
non-macular retina tissue relative to macular tissue. In some embodiments, the
particle delivers
the payload to the eye with increased transduction specificity in one or more
regions of the eye
as compared to a virus particle comprising capsid polypeptides of SEQ ID NO:
1, wherein the
increase in transduction is at least 2-times, 4-times, 8-times, 16-times, 32-
times, 64-times, 100-
times, 200-times, 500-times, or 1000-times as compared to a virus particle
comprising capsid
polypeptides of SEQ ID NO: 1, and wherein the increase in transduction is
specific to macular
tissue relative to non-macular retina tissue. In some embodiments, the
particle delivers the
payload to the eye with increased transduction specificity in one or more
regions of the eye as
compared to a virus particle comprising capsid polypeptides of SEQ ID NO: 1,
wherein the
increase in transduction is at least 2-times, 4-times, 8-times, 16-times, 32-
times, 64-times, 100-
times, 200-times, 500-times, or 1000-times as compared to a virus particle
comprising capsid
polypeptides of SEQ ID NO: 1, and wherein the increase in transduction is
specific to macular
tissue relative to trabecular meshwork tissue. In some embodiments, the
particle delivers the
payload to the eye with increased transduction specificity in one or more
regions of the eye as
compared to a virus particle comprising capsid polypeptides of SEQ ID NO: 1,
wherein the
increase in transduction is at least 2-times, 4-times, 8-times, 16-times, 32-
times, 64-times, 100-
times, 200-times, 500-times, or 1000-times as compared to a virus particle
comprising capsid
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polypeptides of SEQ ID NO: 1, and wherein the increase in transduction is
specific to non-
macular retina tissue relative to trabecular meshwork tissue. In some
embodiments, the particle
delivers the payload to the eye with increased transduction specificity in one
or more regions of
the eye as compared to a virus particle comprising capsid polypeptides of SEQ
ID NO: 1,
wherein the increase in transduction is at least 2-times, 4-times, 8-times, 16-
times, 32-times, 64-
times, 100-times, 200-times, 500-times, or 1000-times as compared to a virus
particle comprising
capsid polypeptides of SEQ ID NO: 1, and wherein the increase in transduction
is specific to
macular tissue and non-macular retina tissue relative to trabecular meshwork
tissue. In some
embodiments, the particle delivers the payload to the eye with increased
transduction specificity
in one or more regions of the eye as compared to a virus particle comprising
capsid polypeptides
of SEQ ID NO: 1, wherein the increase in transduction is at least 2-times, 4-
times, 8-times, 16-
times, 32-times, 64-times, 100-times, 200-times, 500-times, or 1000-times as
compared to a virus
particle comprising capsid polypeptides of SEQ ID NO: 1, and wherein the
increase in
transduction is specific to trabecular meshwork tissue relative to macular
tissue and non-macular
retina tissue. In some embodiments, the particle delivers the payload to the
eye with increased
transduction specificity in one or more regions of the eye as compared to a
virus particle
comprising capsid polypeptides of SEQ ID NO: 1, wherein the increase in
transduction is at least
2-times, 4-times, 8-times, 16-times, 32-times, 64-times, 100-times, 200-times,
500-times, or
1000-times as compared to a virus particle comprising capsid polypeptides of
SEQ ID NO: 1,
and wherein the increase in transduction is specific to trabecular meshwork
tissue relative to
macular tissue. In some embodiments, the particle delivers the payload to the
eye with increased
transduction specificity in one or more regions of the eye as compared to a
virus particle
comprising capsid polypeptides of SEQ ID NO: 1, wherein the increase in
transduction is at least
2-times, 4-times, 8-times, 16-times, 32-times, 64-times, 100-times, 200-times,
500-times, or
1000-times as compared to a virus particle comprising capsid polypeptides of
SEQ ID NO: 1,
and wherein the increase in transduction is specific to trabecular meshwork
tissue relative to non-
macular retina tissue. In some embodiments, the particle delivers the payload
to the eye with
increased transduction specificity in one or more regions of the eye as
compared to a virus
particle comprising capsid polypeptides of SEQ ID NO: 1, wherein the increase
in transduction
is at least 2-times, 4-times, 8-times, 16-times, 32-times, 64-times, 100-
times, 200-times, 500-
times, or 1000-times as compared to a virus particle comprising capsid
polypeptides of SEQ ID
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NO: 1, and wherein the increase in transduction is specific to trabecular
meshwork tissue,
macular tissue, and non-macular retina tissue. In some embodiments, the
particle delivers the
payload to the eye with increased transduction specificity in one or more
regions of the eye as
compared to a virus particle comprising capsid polypeptides of SEQ ID NO: 1
without increased
biodistribution in one or more regions of the eye as compared to a virus
particle comprising
capsid polypeptides of SEQ ID NO: 1. In any of the aforementioned embodiments,
increased
transduction or biodistribution is as measured as described herein in Example
1 (for example,
with respect to transduction, as measured by quantification of viral cDNA
isolated from the bulk
tissue, e.g., NHP tissue, of interest normalized to prevalence of that virus
particle in the test
article, and with respect to biodistribution, as measured by quantification of
viral DNA isolated
from bulk tissue, e.g., NHP tissue, of interest normalized to prevalence of
that virus particle in
the test article).
In some embodiments, the regulatory elements include a promotor. In some
embodiments, the promoter is a ubiquitous or constitutive promoter active in a
mammalian cell,
for example a human cell, for example, in a human cell type of interest.
In some embodiments, the cell type is an ocular cell such as, for example, a
neural retinal cell, a
photoreceptive retinal ganglion cell, a bipolar cell, a horizontal cell, a
amacrine cell, a
photoreceptor (e.g., a rod or a cone cell), an endothelial cell (e.g., a
retinal pigmented epithelial
cell), and endothelial-like cell, and the like. Examples of ubiquitous
promoters include, but are
not limited, to a CAG promoter (hybrid from a cytomegalovirus early enhancer
element, a
chicken-beta actin promoter, e.g., the first exon and the first intron of the
chicken beta actin
gene, and optionally the splice acceptor of the rabbit beta globin gene),
chicken-beta actin
promoter, CBA promoter, CMV promoter, human PGK promoter, ubiquitin promoter,
human
EF1-alpha promoter and fragments thereof. In some embodiments, the promoter is
a tissue-
specific promoter, for example, a promoter specific in ocular tissue or cells
of the eye. Examples
of ocular tissue-specific promoters include but are not limited to TBG
promoters, hAAT
promoters, CK8 promoters and SPc5-12 promoters, rho promoters, which are
active in rods, or
opsin promoters, which are active in cones. In some embodiments, the
regulatory element
includes a photoreceptor cell-specific regulatory element (e.g., promoter)
such as, e.g., a
rhodopsin promoter; a rhodopsin kinase promoter; a beta phosphodiesterase gene
promoter; a
retinitis pigmentosa gene promoter; an interphotoreceptor retinoid-binding
protein (IRBP) gene
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enhancer; an IRBP gene promoter, an opsin gene promoter, a retinoschisin gene
promoter, a
CRX homeodomain protein gene promoter, a guanine nucleotide binding protein
alpha
transducing activity polypeptide 1 (GNAT1) gene promoter, a neural retina-
specific leucine
zipper protein (NRL) gene promoter, human cone arrestin (hCAR) promoter, and
the PR2.1,
PR1.7, PR1.5, and PR1.1 promoters. In some embodiments, the regulatory element
includes, a
retinal pigment epithelia (RPE) cell-specific regulatory element (e.g., a RPE-
specific promoter),
e.g., a regulatory element that confers selective expression of the operably
linked gene in a RPE
cell, such as, e.g., an RPE65 gene promoter, a cellular retinaldehyde- binding
protein (CRALBP)
gene promoter, a pigment epithelium-derived factor (PEDF aka serpin Fl) gene
promoter, and a
vitelliform macular dystrophy (VMD2) promoter. In some embodiments, the
regulatory element
includes a promoter specific to a glial cell, e.g., a regulatory element that
confers selective
expression of the operably linked payload in a retinal glial cell, such as,
e.g., a glial fibrillary
acidic protein (GFAP) promoter. In some instances, the regulatory element
includes a promoter
that is specific to a bipolar cell (e.g., a bipolar-specific promoter), e.g.,
a regulatory element that
confers selective expression of the operably linked payload in a bipolar cell,
such as, e.g., a
GRM6 promoter, in embodiments, the promoter sequence is between 100 and 1000
nucleotides
in length. In embodiments, the promoter sequence is about 100, about 200,
about 300, about 400,
about 500, about 600, about 700, about 800, about 900 or about 1000
nucleotides in length. As
used in the preceding sentence, "about" refers to a value within 50
nucleotides of the recited
length. Suitable regulatory elements, e.g., promoters, may be readily selected
by persons of skill
in the art, such as those, but not limited to, those described herein.
In some embodiments, the nucleic acid expression construct comprises an
intron. The
intron may be disposed between the promoter and the heterologous transgene. In
some aspects,
the intron is disposed 5 to the heterologous transgene on the expression
construct, for example
immediately 5' to the heterologous transgene or 100 nucleotides or less 5' to
the heterologous
transgene. In some aspects, the intron is a chimeric intron derived from human
b-globin and Ig
heavy chain (also known as b- globin splice donor/immunoglobulin heavy chain
splice acceptor
intron, or b-globin/IgG chimeric intron; Reed, R., et al. Genes and
Development, 1989,
incorporated herein by reference in its entirety). In other aspects, the
intron is a VH4 intron or a
SV40 intron.
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As provided herein, in some embodiments, virus particles comprising a payload,
wherein
the payload includes a nucleic acid that includes a heterologous transgene are
provided. In some
embodiments, the heterologous transgene encodes an RNA interference agent, for
example a
siRNA, shRNA or other interfereing nucleic acid.
In some embodiments, the payload includes a heterologous transgene that
encodes a
therapeutic polypeptide. In some aspects, the heterologous transgene is a
human gene or
fragment thereof. In some aspects, the therapeutic polypeptide is a human
protein. In some
embodiments, the heterologous transgene of the virus particle encodes a
molecule useful in
treating a disease, and the virus particle is administered to a patient in
need thereof to treat said
disease. Examples of diseases (and heterologous transgenes or molecules
encoded by said
heterologous transgenes) according to the present disclosure include: MPSI
(alpha-L-iduronidase
(IDUA)); MPS II ¨ Hunter syndrome (iduronate-2-sulfatase (IDS)); Ceroid
lipotuscinosis-Batten
disease (CLN1, CLN2, CLN10, CLN1 3, CLN5, CLN1 1, CLN4, CNL14, CLN3, CLN6,
CLN7,
CLN8, CLN12); MPS Ilia - Sanfilippo Type A syndrome (heparin sulfate sulfatase
(also called
N-sulfoglucosamine sulfohydrolase (SGSH)); MPS 11113 ¨ Sanfilippo Type b
syndrome (N-
acetyl-alpha-D-glucosaminidase (NAGLU)); MPS VI - Maroteaux-Lamy syndrome
(arylsulfatase B); MPS IV A - Morquio syndrome type A (GALNS); MPS IV B ¨
Morquio
syndrome type B (GLB 1); Osteogenesis Imperfecgta Type I, II, III or IV
(COL1A1 and/or
COL1A2); hereditary angioedema (SERPING1, C1NH); Osteogenesis Imperfecta Type
V
(IFITM5); Osteogenesis Imperfecta Type VI (SERPINF1); Osteogenesis Imperfecta
Type VII
(CRTAP); Osteogenesis Imperfecta Type VIII (LEPRE1 and/or P3H1); Osteogenesis
Imperfecia
Type IX (PPIB); Gaucher disease type I, II and III (Glucocerebrosidase; GBA1);
Parkinson's
Disease (Glucocerebrosidase; GBA1 and/or dopamine decarboxylase); Pompe (acid
maltase;
GAA; hGAA); Metachromatic leukodystrophy (Aryl sulfatase A); MPS VII - Sly
syndrome
(beta-glucuronidase); MPS VIII (glucosamine-6-sulfate sulfatase); MPS IX
(Hyaluronidase);
maple syrup urine disease (BCKDHA, BCKDHB, and/or DBT); Niemann-Pick disease
(Sphingomyelinase); Parkinson's disease (anti-alpha synuclein RNAi);
Alzheimer's disease
(alit-mutant APP RNAi); Niemann-Pick disease without sphingomyelinase
deficiency (NPC1 or
NPC gene encoding a cholesterol metabolizing enzyme); Tay-Sachs disease (alpha
subunit of
beta-hexosaminidase); Sandhoff disease (both alpha and beta subunit of beta-
hexosaminidase);
Fabry Disease (alpha-galactosidase); Fucosidosis (fucosidase (FUCA1)); Alpha-
mannosidosis
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(alpha-mannosidase); Beta-mannosidosis (beta-mannosidase); Wolman disease
(cholesterol ester
hydrolase); Dravet syndrome (SCN1A, SCN1B, SCN2A, GABRG2); Parkinson's disease
(Neurturin); Parkinson's disease (glial derived growth factor (GDGF));
Parkinson's disease
(tyrosine hydroxylase); Parkinson's disease (glutamic acid decarboxylase; FGF-
2; BDGF);
Spinal Muscular Atrophy (SMN, including SMN1 or SMN2); Friedreich's ataxia
(Frataxin);
Amyotrophic lateral sclerosis (ALS) (SOD1 inhibitor, e.g., anti-SOD1 RNAi);
Glycogen Storage
Disease la (Glucose-6-phosphatase); XLMTM (MTM1); Crigler Najjar (UGT1A1);
CPVT
(CASQ2); spinocerebellar ataxia (ATXN2; ATXN3 or other ATXN gene; anti-mutant
Machado-
Joseph disease/SCA3 allele RNAi); Rett syndrome (MECP2 or fragment thereof);
Achromatopsia (CNGB3, CNGA3, GNAT2, PDE6C); Choroiderrnia (CDM); Danon Disease
(LAMP2); Cystic Fibrosis (CFTR or fragment thereof); Duchenne Muscular
Dystrophy (Mini-/
Micro-Dystrophin Gene); SARS-Cov-2 infection (anti-SARS-Cov-2 RNAi, SARS-Cov-2
genome fragments or S protein (including variants)); Limb Girdle Muscular
Dystrophy Type 2C
- Gamma- sarcoglycanopathy (human-alpha-sarcoglycan); Advanced Heart Failure
(SERCA2a);
Rheumatoid Arthritis (TNFR:Fc Fusion; anti-TNF antibody or fragment thereof);
Leber
Congenital Amaurosis (GAA); X-linked adrenoleukodystrophy (ABCD1); Limb Girdle
Muscular Dystrophy Type 2C - Gamma-sarcoglycanopathy (ganama-sarcoglycan);
Angelman
syndrome (UBE3A); Retinitis Pigmentosa (hMERTK); Age-Related Macular
Degeneration
(sFLT01); Phelan-McDermid syndrome (SHANK3; 22q13.3 replacement); Becker
Muscular
Dystrophy and Sporadic Inclusion Body Myositis (huFollistatin344); Parkinson's
Disease
(GDNF); Metachromatic Leukodystrophy ¨ MLD (cuARS A); Hepatitis C (anti-HCV
RNAi);
Limb Girdle Muscular Dystrophy Type 2D (hSGCA); Human Immunodeficiency Virus
Infections; (PG9DP); Acute Intermittant Porphyria (PBGD); Leber's Hereditary
Optical
Neuropathy (PIND4v2); Alpha-1 Antitrypsin Deficiency (alphaIAT); X-linked
Retinoschisis
(RS1); Choroideremia (hCHM); Giant Axonal Neuropathy (GAN); Hemophilia B
(Factor IX);
Homozygous FH (hLDLR); Dysferlinopathies (DYSF); Achromatopsia (CNGA3 or
CNGB3);
Progressive supranuclear palsy (MAPT; anti-Tau; anti-MAPT RNAi); Omithine
Transcarbamylase deficiency (OTC); Hemophilia A (Factor VIII); Age-related
macular
degeneration (AMD), including wetAMD (anti-VEGF antibody or RNAi); X-Linked
Retinitis
Pigmentosa (RPGR); Myotonic dystrophy Type 1 (DMPK; anti-DMPK RNAi, including
anti-
CTG trinucleotide repeat RNAi); Myotonic dystrophy Type 2 (CNBP);
Facioscapulohumeral
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muscular dystrophy (D4Z4 DNA); oculopharynggeal muscular dystrophy (PABPN1;
mutated
PABPN1 inhibitor (e.g., RNAi)); Mucopolysaccharidosis Type VI (hARSB); Leber
Hereditary
Optic Neuropathy (ND4); X-Linked myotubular Myopathy (MTM1); Crigler-Najjar
Syndrome
(UGT1A1); Retinitis Pigmentosa (hPDE6B); Mucopolysaccharidosis Type 3B
(hNAGLU);
Duchenne Muscular Dystrophy (GALGT2); Alzheimer's Disease (NGF; ApoE4; ApoE2;
ApoE3;
Anti-ApoE RNAi); Familial Lipoprotein Lipase Deficiency (LPL); Alpha-1
Antitryp sin
Deficiency (hAAT); Leber Congenital Amaurosis 2 (hRPE65v2); Batten Disease;
Late Infantile
Neuronal Lipofuscinosis (CLN2); Huntington's disease (HTT; anti-HTT RNAi);
Fragile X
syndrome (FMR1); Leber's Hereditary Optical Neuropathy (P1ND4v2); Aromatic
Amino Acid
Decarboxylase Deficiency (hAADC); Retinitis Pigmentosa (hMERKTK); and
Retinitis
Pigmentosa (RLBP1).
In some aspects, the heterologous transgene encodes an antibody or fragment
thereof (for
example an antibody light chain, an antibody heavy chain, a Fab or an scFv).
Examples of
antibodies or fragments thereof that are encoded by the heterologous transgene
include but are
not limited to: and an anti-Ab antibody (e.g. solanezumab, GSK933776, and
lecanemab), anti-
sortilin ( e.g. AL-001), anti-Tau (e.g. ABBV-8E12, UCB-0107. and Ni- 105),
anti-SEMA4D
(e.g. VX15/2503), anti-alpha synuclein (e.g. prasinezumab, NI-202, and MED-
1341), anti-
SOD1 (e.g. NI-204), anti-CGRP receptor (e.g. eptinezumab, fremanezumab, or
galcanezumab),
anti-VEGF (e.g., sevacizumab, ranibizumab, bevacizumab, and brolucizumab),
anti-EpoR (e.g.,
LKA-651, ), anti-ALK1 (e.g., ascrinvacumab), anti-05 (e.g., tesidolumab,
ravulizumab, and
eculizumab), anti-CD105 (e.g., carotuximab), anti-CC1Q (e.g., ANX-007), anti-
TNFa (e.g.,
adalimumab, infliximab, and golimumab), anti-RGMa (e.g., elezanumab), anti-TTR
(e.g., NI-
301 and PRX-004), anti-CTGF (e.g., pamrevlumab), anti- IL6R (e.g.,
satralizumab, tocilizumab,
and sarilumab), anti-IL6 (e.g. siltuximab, clazakizumab, sirukumab,
olokizumab, and
gerilimzumab). anti-IL4R (e.g., dupilumab), anti-IL17A (e.g., ixekizumab and
secukinumab),
anti-IL5R (e.g. reslizumab), anti-IL-5 (e.g., benralizumab and mepolizumab),
anti-IL13 (e.g.
tralokinumab), anti-IL12/IL23 (e.g., ustekinumab), anti-CD 19 (e.g.,
inebilizumab). anti-IL31RA
(e.g. nemolizumab), anti-ITGF7 nnAb (e.g., etrolizumab), anti-SOST mAb (e.g.,
romosozumab),
anti-IgE (e.g. omalizumab). anti-TSLP (e.g. nemolizumab), anti-pKal mAb (e.g.,
lanadelumab),
anti-ITGA4 (e.g., natalizumab), anti- ITGA4B7 (e.g., vedolizumab), anti-BLyS
(e.g.,
belimumab), anti-PD-1 (e.g., nivolumab and pembrolizumab), anti-RANKL (e.g.,
denosumab),
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anti-PCSK9 (e.g., alirocumab and evolocumab), anti-ANGPTL3 (e.g.,
evinacumab*), anti-OxPL
(e.g., E06), anti-ID (e.g., lampalizumab), or anti-MMP9 (e.g., andecaliximab),
optionally
wherein the heavy chain (Fab and Fc region) and the light chain are separated
by a self-cleaving
furin (F)/F2A or furin (F)/T2A, IRES site, or flexible linker, for example,
ensuring expression of
equal amounts of the heavy and the light chain polypeptides.
In embodiments, the payload comprises a nucleic acid encoding a gene product
linked to
a disorder of the eye, or a fragment thereof. Exemplary gene products linked
to a disorder of the
eye include, for example, ADP-ribosylation factor-like 6 (ARL6); BBSome
interacting protein 1
(BBIP1); BBSome protein 1 (BBS1); BBSome protein 2 (BBS2); BBSome protein 4
(BBS4);
BBSome protein 5 (BBS5); BBSome protein 7 (BBS7); BBSome protein 9 (BBS9);
BBSome
protein 10 (BBS10); BBSome protein 12 (BBS12); centrosomal protein 290 kDa
(CEP290);
intraflagellar transport protein 172 (IFT172); intraflagellar transport
protein 27 (IFT27); inositol
polyphosphate-5-phosphatase E (INPP5E); inwardly-rectifying potassium channel
subfamily J
member 13 (KCNJ13); leucine zipper transcription factor like-1 (LZTFL1);
McKusick-Kaufman
syndrome protein (MKKS); Meckel syndrome type 1 protein (MKS1);
nephronophthisis 3
protein (NPHP1); serologically-defined colon cancer antigen 8 (SDCCAG8);
tripartite motif-
containing protein 32 (TRIM32); tetratricopeptide repeat domain 8 (TTC8);
Batten disease
protein (CLN3); cytochrome P450 4V2 (CYP4V2); Rab escort protein 1 (CHM); PR
(positive
regulatory) domain-containing 13 protein (PRDM13); RPE-retinal G protein-
coupled receptor
(RGR); TEA domain family member 1 (TEAD1); arylhydrocarbon-interacting
receptor protein-
like 1 (AIPL1); cone-rod otx-like photoreceptor homeobox transcription factor
(CRX); guanylate
cyclase activating protein lA (GUCA1A); retinal-specific guanylate cyclase
(GUCY2D);
phosphatidylinositol transfer membrane-associated family member 3 (PITPNM3);
prominin 1
(PROM1); peripherin (PRPH); peripherin 2 (PRPH2); regulating synaptic membrane
exocytosis
protein 1 (RIMS1); semaphorin 4A (SEMA4A); human homolog of C. elegans unc119
protein
(UNC119); ATP-binding cassette transporter __ retinal (ABCA4); ADAM
metallopeptidase
domain 9 (ADAM9); activating transcription factor 6 (ATF6); chromosome 21 open
reading
frame 2 (C21orf2); chromosome 8 open reading frame 37 (C8orf37); calcium
channel; voltage-
dependent; alpha 2/delta subunit 4 (CACNA2D4); cadherin-related family member
1
(protocadherin 21) (CDHR1); ceramide kinase-like protein (CERKL); cone
photoreceptor
cGMP-gated cation channel alpha subunit (CNGA3); cone cyclic nucleotide-gated
cation
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channel beta 3 subunit (CNGB3); cyclin M4 (CNNM4); guanine nucleotide binding
protein (G
protein); alpha transducing activity polypeptide 2 (GNAT2); potassium channel
subfamily V
member 2 (KCNV2); Phosphodiesterase 6C (PDE6C); Phosphodiesterase 6H (PDE6H);
proteome of centriole 1 centriolar protein B (POC1B); RAB28 member of RAS
oncogene family
(RAB28); retina and anterior neural fold homeobox 2 transcription factor
(RAX2); 11-cis retinol
dehydrogenase 5 (RDH5); RP GTPase regulator-interacting protein 1 (RPGRIP1);
tubulin
tyrosine ligase-like family member 5 (TTLL5); L-type voltage-gated calcium
channel alpha-1
subunit (CACNA1F); retinitis pigmentosa GTPase regulator (RPGR); rod
transducin alpha
subunit (GNAT1); rod cGMP phosphodiesterase beta subunit (PDE6B); rhodopsin
(RHO);
calcium binding protein 4 (CABP4); G protein-coupled receptor 179 (GPR179);
rhodopsin
kinase (GRK1); metabotropic glutamate receptor 6 (GRM6); leucine-rich repeat
immunoglobulin-like transmembrane domains protein 3 (LRIT3); arrestin (s-
antigen) (SAG);
solute carrier family 24 (SLC24A1); transient receptor potential cation
channel, subfamily M,
member 1 (TRPM1); nyctalopin (NYX); green cone opsin (OPN1LW); red cone opsin
(OPN1MW); blue cone opsin (OPN1SW); frataxin (FXN); inosine monophosphate
dehydrogenase 1 (IMPDH1); orthodenticle homeobox 2 protein (0TX2); crumbs
homolog 1
(CRB1); death domain containing protein 1 (DTHD1); growth differentiation
factor 6 (GDF6);
intraflagellar transport 140 Chlamydomonas homolog protein (IFT140); IQ motif
containing B
protein (IQCB1); lebercilin (LCA5); lecithin retinol acyltransferase (LRAT);
nicotinamide
nucleotide adenylyltransferase 1 (NMNAT1); RD3 protein (RD3); retinol
dehydrogenase 12
(RDH12); retinal pigment epithelium-specific 65 kD protein (RPE65);
spermatogenesis
associated protein 7 (SPATA7); tubby-like protein 1 (TULP1); mitochondrial
genes (KSS,
LHON, MT-ATP6, MT-TH, MT-TL1, MT-TP, MT-TS2, mitochondrially encoded NADH
dehydrogenases [MT-ND]); bestrophin 1 (BEST1); Clq and tumor necrosis-related
protein 5
collagen (C1QTNF5); EGF-containing fibrillin-like extracellular matrix protein
1 (EFEMP1);
elongation of very long fatty acids protein (ELOVL4); retinal fascin homolog
2, actin bundling
protein (FSCN2); guanylate cyclase activating protein 1B (GUCAB); hemicentin 1
(HMCN1);
interphotoreceptor matrix proteoglycan 1 (IMPG1); retinitis pigmentosa 1-like
protein 1
(RP1L1); tissue inhibitor of metalloproteinases-3 (TIMP3); complement factor H
(CFH);
complement factor D (CFD); complement component 2 (C2); complement component
3(C3);
complement factor B (CFB); DNA-damage regulated autophagy modulator 2 (DRAM2);
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chondroitin sulfate proteoglycan 2 (VCAN); mitofusin 2 (MFN2); nuclear
receptor subfamily 2
group F member 1 (NR2F1); optic atrophy 1 (OPA1); transmembrane protein 126A
(TMEM126A); inner mitochondria) membrane translocase 8 homolog A (TIMM8A);
carbonic
anhydrase IV (CA4); hexokinase 1 (HK1); kelch-like 7 protein (KLHL7); nuclear
receptor
subfamily 2 group E3 (NR2E3); neural retina lucine zipper (NRL); olfactory
receptor family 2
subfamily W member 3 (0R2W3); pre-mRNA processing factor 3 (PRPF3); pre-mRNA
processing factor 4 (PRPF4); pre-mRNA processing factor 6 (PRPF6); pre-mRNA
processing
factor 8 (PRPF8); pre-mRNA processing factor 31 (PRPF31); retinal outer
segment membrane
protein 1 (ROM1); retinitis pigmentosa protein 1 (RP1); PIM-kinase associated
protein 1 (RP9);
small nuclear ribonucleoprotein 200 kDa (SNRNP200); secreted phosphoprotein 2
(SPP2);
topoisomerase I binding arginine/serine rich protein (TOPORS); ADP-
ribosylation factor-like 2
binding protein (ARL2BP); chromosome 2 open reading frame 71 (C2or171); clarin-
1 (CLRN1);
rod cGMP-gated channel alpha subunit (CNGA1); rod cGMP-gated channel beta
subunit
(CNGB1); cytochrome P450 4V2 (CYP4V2); dehydrodolichyl diphosphate synthetase
(DHDDS); DEAH box polypeptide 38 (DHX38); ER membrane protein complex subunit
1
(EMC1); eyes shut/spacemaker homolog (EYS); family with sequence similarity
161 member A
(FAM161A); G protein-coupled receptor 125 (GPR125); heparan-alpha-
glucosaminide N-
acetyltransferase (HGSNAT); NAD(+)-specific isocitrate dehydrogenase 3 beta
(IDH3B);
interphotoreceptor matrix proteoglycan 2 (IMPG2); KIAA1549 protein (KIAA1549);
kizuna
centrosomal protein (IUZ); male germ-cell associated kinase (MAK); c-mer
protooncogene
receptor tyrosine kinase (MERTK); mevalonate kinase (MVK); NIMA (never in
mitosis gene
A)-related kinase 2 (NEK2); neuronal differentiation protein 1 (NEUROD1); cGMP
phosphodiesterase alpha subunit (PDE6A); phosphodiesterase 6G cGMP-specific
rod gamma
(PDE6G); progressive rod-cone degeneration protein (PRCD); retinol binding
protein 3 (RBP3);
retinaldehyde-binding protein 1 (RLBP1); solute carrier family 7 member 14
(SLC7A14);
usherin (USH2A); zinc finger protein 408 (ZNF408); zinc finger protein 513
(ZNF513); oral-
facial-digital syndrome 1 protein (OFD1); retinitis pigmentosa 2 (RP2);
retinoschisin (RS1);
abhydrolase domain containing protein 12 (ABHD12); cadherin-like gene 23
(CDH23);
centrosomal protein 250 kDa (CEP250); calcium and integrin binding family
member 2 (CIB2);
whirlin (DFNB31); monogenic audiogenic seizure susceptibility 1 homolog
(GPR98); histidyl-
tRNA synthetase (HARS); myosin VIIA (MY07A); protocadherin 15 (PCDH15);
harmonin
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(USH1C); human homolog of mouse scaffold protein containing ankyrin repeats
and SAM
domain (USH1G); dystrophin (DMD); norrin (NDP); phosphoglycerate kinase
(PGK1); calpain
(CAPN5); frizzled-4 Wnt receptor homolog (FZD4); integral membrane protein 2B
(ITM2B);
low density lipoprotein receptor-related protein 5 (LRP5); micro RNA 204
(MIR204);
retinoblastoma protein 1 (RB1); tetraspanin 12 (TSPAN12); chromosome 12 open
reading frame
65 (C12orf65); cadherin 3 (CDH3); membrane-type frizzled-related protein
(MFRP); ornithine
aminotransferase (OAT); phospholipase A2 group V (PLA2G5); retinol-binding
protein 4
(RBP4); regulator of G-protein signaling 9 (RGS9); regulator of G-protein
signaling 9-binding
protein (RGS9BP); ARMS2; excision repair cross-complementing rodent repair
deficiency
complementation group 6 protein (ERCC6); fibulin 5 (FBLN5); HtrA serine
peptidase 1
(HTRA1); toll-like receptor 3 (TLR3); and toll-like receptor 4 (TLR4), opsin;
rhodopsin; channel
rhodopsin; halo rhodopsin, and the like.
In some embodiments, the virus particle comprises a heterologous transgene
encoding a
genome editing system. Examples include a CRISPR genome editing system (e.g.,
one or more
components of a CRISPR genome editing system such as, for example, a guide RNA
molecule
and/or a RNA-guided nuclease such as a Cas enzyme such as Cas9, Cpfl and the
like), a zinc
finger nuclease genome editing system, a TALEN genome editing system or a
meganuclease
genome editing system. In embodiments, the genome editing system targets a
mammalian, e.g.,
human, genomic target sequence. In embodiments, the virus particle includes a
heterologous
transgene encoding a targetable transcription regulator. Examples include a
CRISPR-based
trascription regulator (for example, one or more components of a CRISPR-based
transcription
regulator, for example, a guide RNA molecule and/or a enzymatically-inactive
RNA-guided
nuclease/transcription factor ("TF") fusion protein such as a dCas9-TF fusion,
dCpfl-TF fusion
and the like), a zinc finger transcription factor fusion protein, a TALEN
transcription regulator or
a meganuclease transcription regulator.
In some embodiments, components of a therapeutic molecule or system are
delivered by
more than one unique virus particle (e.g., a population that includes more
than one unique virus
particles). In other embodiments, the therapeutic molecule or components of a
therapeutic
molecule or system are delivered by a single unique virus particle (e.g., a
population that
includes a single unique virus particle).
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The transgene may also encode any biologically active product or other
product, e.g., a
product desirable for study. Suitable transgenes may be readily selected by
persons of skill in the
art, such as those, but not limited to, those described herein.
Other examples of proteins encoded for by the transgene include, but are not
limited to,
colony stimulating factors (CSF); blood factors, such as 13-globin,
hemoglobin, tissue
plasminogen activator, and coagulation factors; interleukins; soluble
receptors, such as soluble
TNF-cc receptors, soluble VEGF receptors, soluble interleukin receptors (e.g.,
soluble IL-1
receptors and soluble type II IL-1 receptors), or ligand- binding fragments of
a soluble receptor;
growth factors, such as keratinocyte growth factor (KGF), stem cell factor
(SCF), or fibroblast
growth factor (FGF, such as basic FGF and acidic FGF); enzymes; chemokines,;
enzyme
activators, such as tissue plasminogen activator; angiogenic agents, such as
vascular endothelial
growth factors, glioma-derived growth factor, angiogenin. or angiogenin-2;
anti-angiogenic
agents, such as a soluble VEGF receptor; a protein vaccine; neuroactive
peptides, such as nerve
growth factor (NGF) or oxytocin; thrombolytic agents;; tissue factors;
macrophage activating
factors; tissue inhibitors of metalloproteinases; or IL-1 receptor
antagonists.
Accordingly, provided herein is a virus particle comprising a capsid
polypeptide
comprising (a) a VP1, VP2 or VP3 sequence of SEQ ID NO: 2. (b) a VP1. VP2 or
VP3 sequence
comprising the mutation set of VAR-1 and having greater than 80% (for example,
greater than
90% greater than 91%, greater than 92%, greater than 93%, greater than 94%,
greater than 95%,
greater than 96%, greater than 97%, greater than 98%, greater than 99%)
identity to SEQ ID NO:
1, or (c) a VP1, VP2 or VP3 sequence comprising the mutation set of VAR-1 and
having at least
1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 additional mutations, but fewer than 40, 39,
38, 37, 36, 35, 34, 33, 32
or 31 additional mutations relative to SEQ ID NO: 1. In embodiments, the
capsid polypeptide
comprises VP1, VP2 and VP3 sequences of SEQ ID NO: 2. In embodiments, the
virus particle
comprises a nucleic acid molecule comprising a heterologous transgene, for
example a
heterologous transgene encoding a product directed to an ocular disorder. In
embodiments, the
heterologous transgene encodes an anti-VEGF antibody or antibody fragment, an
anti-VEGF
RNA inhibitory molecule, a RPE65 (e.g., human RPE65) protein, a ABCA4 (e.g.,
human
ABCA4) protein or fragment thereof, a RLBP1 (e.g., human RLBP1) protein or
fragment
thereof, a PDE6B (e.g.. human PDE6B) protein or fragment thereof, a RPGR
(e.g., human
RPGR) protein or fragment thereof or a ACHM3A or ACHM3B (e.g., human ACHM3A or
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human ACHM3B) protein or fragment thereof. In embodiments, the nucleic acid
molecule of
the virus particle further comprises one or more regulatory elements, e.g.,
comprises a promoter,
e.g., a promoter operably linked to the heterologous transgene and which
regulates expression
from the heterologous transgene in a tissue of interest. In embodiments, the
nucleic acid
molecule of the virus particle further comprises one or more of (a) a
dependoparvovirus ITR, (b)
an intron, (c) an enhancer or repressor sequence, (d) a stuffer sequence, and
(e) a polyA
sequence.
Accordingly, provided herein is a virus particle comprising a capsid
polypeptide
comprising (a) a VP1, VP2 or VP3 sequence of SEQ ID NO: 3. (b) a VP1. VP2 or
VP3 sequence
comprising the mutation set of VAR-2 and having greater than 80% (for example,
greater than
90% greater than 91%, greater than 92%, greater than 93%, greater than 94%,
greater than 95%,
greater than 96%, greater than 97%, greater than 98%, greater than 99%)
identity to SEQ ID NO:
1, or (c) a VP1, VP2 or VP3 sequence comprising the mutation set of VAR-2 and
having at least
1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 additional mutations, but fewer than 40, 39,
38, 37, 36, 35, 34, 33, 32
or 31 additional mutations relative to SEQ ID NO: 1. In embodiments, the
capsid polypeptide
comprises VP1, VP2 and VP3 sequences of SEQ ID NO: 3. In embodiments, the
virus particle
comprises a nucleic acid molecule comprising a heterologous transgene, for
example a
heterologous transgene encoding a product directed to an ocular disorder. In
embodiments, the
heterologous transgene encodes an anti-VEGF antibody or antibody fragment, an
anti-VEGF
RNA inhibitory molecule, a RPE65 (e.g., human RPE65) protein, a ABCA4 (e.g.,
human
ABCA4) protein or fragment thereof, a RLBP1 (e.g., human RLBP1) protein or
fragment
thereof, a PDE6B (e.g.. human PDE6B) protein or fragment thereof, a RPGR
(e.g., human
RPGR) protein or fragment thereof or a ACHM3A or ACHM3B (e.g., human ACHM3A or
human ACHM3B) protein or fragment thereof. In embodiments, the nucleic acid
molecule of
the virus particle further comprises one or more regulatory elements, e.g.,
comprises a promoter,
e.g., a promoter operably linked to the heterologous transgene and which
regulates expression
from the heterologous transgene in a tissue of interest. In embodiments, the
nucleic acid
molecule of the virus particle further comprises one or more of (a) a
dependoparvovirus ITR, (b)
an intron, (c) an enhancer or repressor sequence, (d) a stuffer sequence, and
(e) a polyA
sequence.
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Accordingly, provided herein is a virus particle comprising a capsid
polypeptide
comprising (a) a VP1, VP2 or VP3 sequence of SEQ ID NO: 4. (b) a VP1. VP2 or
VP3 sequence
comprising the mutation set of VAR-3 and having greater than 80% (for example,
greater than
90% greater than 91%, greater than 92%, greater than 93%, greater than 94%,
greater than 95%,
greater than 96%, greater than 97%, greater than 98%, greater than 99%)
identity to SEQ ID NO:
1, or (c) a VP1, VP2 or VP3 sequence comprising the mutation set of VAR-3 and
having at least
1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 additional mutations, but fewer than 40, 39,
38, 37, 36, 35, 34, 33, 32
or 31 additional mutations relative to SEQ ID NO: 1. In embodiments, the
capsid polypeptide
comprises VP1, VP2 and VP3 sequences of SEQ ID NO: 4. In embodiments, the
virus particle
comprises a nucleic acid molecule comprising a heterologous transgene, for
example a
heterologous transgene encoding a product directed to an ocular disorder. In
embodiments, the
heterologous transgene encodes an anti-VEGF antibody or antibody fragment, an
anti-VEGF
RNA inhibitory molecule, a RPE65 (e.g., human RPE65) protein, a ABCA4 (e.g.,
human
ABCA4) protein or fragment thereof, a RLBP1 (e.g., human RLBP1) protein or
fragment
thereof, a PDE6B (e.g.. human PDE6B) protein or fragment thereof, a RPGR
(e.g., human
RPGR) protein or fragment thereof or a ACHM3A or ACHM3B (e.g., human ACHM3A or
human ACHM3B) protein or fragment thereof. In embodiments, the nucleic acid
molecule of
the virus particle further comprises one or more regulatory elements, e.g.,
comprises a promoter,
e.g., a promoter operably linked to the heterologous transgene and which
regulates expression
from the heterologous transgene in a tissue of interest. In embodiments, the
nucleic acid
molecule of the virus particle further comprises one or more of (a) a
dependoparvovirus ITR, (b)
an intron, (c) an enhancer or repressor sequence, (d) a stuffer sequence, and
(e) a polyA
sequence.
Accordingly, provided herein is a virus particle comprising a capsid
polypeptide
comprising (a) a VP1, VP2 or VP3 sequence of SEQ ID NO: 5. (b) a VP1. VP2 or
VP3 sequence
comprising the mutation set of VAR-4 and having greater than 80% (for example,
greater than
90% greater than 91%, greater than 92%, greater than 93%, greater than 94%,
greater than 95%,
greater than 96%, greater than 97%, greater than 98%, greater than 99%)
identity to SEQ ID NO:
1, or (c) a VP1, VP2 or VP3 sequence comprising the mutation set of VAR-4 and
having at least
1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 additional mutations, but fewer than 40, 39,
38, 37, 36, 35, 34, 33, 32
or 31 additional mutations relative to SEQ ID NO: 1. In embodiments, the
capsid polypeptide
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comprises VP1, VP2 and VP3 sequences of SEQ ID NO: 5. In embodiments, the
virus particle
comprises a nucleic acid molecule comprising a heterologous transgene, for
example a
heterologous transgene encoding a product directed to an ocular disorder. In
embodiments, the
heterologous transgene encodes an anti-VEGF antibody or antibody fragment, an
anti-VEGF
RNA inhibitory molecule, a RPE65 (e.g., human RPE65) protein, a ABCA4 (e.g.,
human
ABCA4) protein or fragment thereof, a RLBP1 (e.g., human RLBP1) protein or
fragment
thereof, a PDE6B (e.g.. human PDE6B) protein or fragment thereof, a RPGR
(e.g., human
RPGR) protein or fragment thereof or a ACHM3A or ACHM3B (e.g., human ACHM3A or
human ACHM3B) protein or fragment thereof. In embodiments, the nucleic acid
molecule of
the virus particle further comprises one or more regulatory elements, e.g.,
comprises a promoter,
e.g., a promoter operably linked to the heterologous transgene and which
regulates expression
from the heterologous transgene in a tissue of interest. In embodiments, the
nucleic acid
molecule of the virus particle further comprises one or more of (a) a
dependoparvovirus ITR, (b)
an intron, (c) an enhancer or repressor sequence, (d) a stuffer sequence, and
(e) a polyA
sequence.
Accordingly, provided herein is a virus particle comprising a capsid
polypeptide
comprising (a) a VP1, VP2 or VP3 sequence of SEQ ID NO: 6. (b) a VP1. VP2 or
VP3 sequence
comprising the mutation set of VAR-5 and having greater than 80% (for example,
greater than
90% greater than 91%, greater than 92%, greater than 93%, greater than 94%,
greater than 95%,
greater than 96%, greater than 97%, greater than 98%, greater than 99%)
identity to SEQ ID NO:
1, or (c) a VP1, VP2 or VP3 sequence comprising the mutation set of VAR-5 and
having at least
1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 additional mutations, but fewer than 40, 39,
38, 37, 36, 35, 34, 33, 32
or 31 additional mutations relative to SEQ ID NO: 1. In embodiments, the
capsid polypeptide
comprises VP1, VP2 and VP3 sequences of SEQ ID NO: 6. In embodiments, the
virus particle
comprises a nucleic acid molecule comprising a heterologous transgene, for
example a
heterologous transgene encoding a product directed to an ocular disorder. In
embodiments, the
heterologous transgene encodes an anti-VEGF antibody or antibody fragment, an
anti-VEGF
RNA inhibitory molecule, a RPE65 (e.g., human RPE65) protein, a ABCA4 (e.g.,
human
ABCA4) protein or fragment thereof, a RLBP1 (e.g., human RLBP1) protein or
fragment
thereof, a PDE6B (e.g.. human PDE6B) protein or fragment thereof, a RPGR
(e.g., human
RPGR) protein or fragment thereof or a ACHM3A or ACHM3B (e.g., human ACHM3A or
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human ACHM3B) protein or fragment thereof. In embodiments, the nucleic acid
molecule of
the virus particle further comprises one or more regulatory elements, e.g.,
comprises a promoter,
e.g., a promoter operably linked to the heterologous transgene and which
regulates expression
from the heterologous transgene in a tissue of interest. In embodiments, the
nucleic acid
molecule of the virus particle further comprises one or more of (a) a
dependoparvovirus ITR, (b)
an intron, (c) an enhancer or repressor sequence, (d) a stuffer sequence, and
(e) a polyA
sequence.
Accordingly, provided herein is a virus particle comprising a capsid
polypeptide
comprising (a) a VP1, VP2 or VP3 sequence of SEQ ID NO: 7. (b) a VP1. VP2 or
VP3 sequence
comprising the mutation set of VAR-6 and having greater than 80% (for example,
greater than
90% greater than 91%, greater than 92%, greater than 93%, greater than 94%,
greater than 95%,
greater than 96%, greater than 97%, greater than 98%, greater than 99%)
identity to SEQ ID NO:
1, or (c) a VP1, VP2 or VP3 sequence comprising the mutation set of VAR-6 and
having at least
1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 additional mutations, but fewer than 40, 39,
38, 37, 36, 35, 34, 33, 32
or 31 additional mutations relative to SEQ ID NO: 1. In embodiments, the
capsid polypeptide
comprises VP1, VP2 and VP3 sequences of SEQ ID NO: 7. In embodiments, the
virus particle
comprises a nucleic acid molecule comprising a heterologous transgene, for
example a
heterologous transgene encoding a product directed to an ocular disorder. In
embodiments, the
heterologous transgene encodes an anti-VEGF antibody or antibody fragment, an
anti-VEGF
RNA inhibitory molecule, a RPE65 (e.g., human RPE65) protein, a ABCA4 (e.g.,
human
ABCA4) protein or fragment thereof, a RLBP1 (e.g., human RLBP1) protein or
fragment
thereof, a PDE6B (e.g.. human PDE6B) protein or fragment thereof, a RPGR
(e.g., human
RPGR) protein or fragment thereof or a ACHM3A or ACHM3B (e.g., human ACHM3A or
human ACHM3B) protein or fragment thereof. In embodiments, the nucleic acid
molecule of
the virus particle further comprises one or more regulatory elements, e.g.,
comprises a promoter,
e.g., a promoter operably linked to the heterologous transgene and which
regulates expression
from the heterologous transgene in a tissue of interest. In embodiments, the
nucleic acid
molecule of the virus particle further comprises one or more of (a) a
dependoparvovirus ITR, (b)
an intron, (c) an enhancer or repressor sequence, (d) a stuffer sequence, and
(e) a polyA
sequence.
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Accordingly, provided herein is a virus particle comprising a capsid
polypeptide
comprising (a) a VP1, VP2 or VP3 sequence of SEQ ID NO: 8. (b) a VP1. VP2 or
VP3 sequence
comprising the mutation set of VAR-7 and having greater than 80% (for example,
greater than
90% greater than 91%, greater than 92%, greater than 93%, greater than 94%,
greater than 95%,
greater than 96%, greater than 97%, greater than 98%, greater than 99%)
identity to SEQ ID NO:
1, or (c) a VP1, VP2 or VP3 sequence comprising the mutation set of VAR-7 and
having at least
1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 additional mutations, but fewer than 40, 39,
38, 37, 36, 35, 34, 33, 32
or 31 additional mutations relative to SEQ ID NO: 1. In embodiments, the
capsid polypeptide
comprises VP1, VP2 and VP3 sequences of SEQ ID NO: 8. In embodiments, the
virus particle
comprises a nucleic acid molecule comprising a heterologous transgene, for
example a
heterologous transgene encoding a product directed to an ocular disorder. In
embodiments, the
heterologous transgene encodes an anti-VEGF antibody or antibody fragment, an
anti-VEGF
RNA inhibitory molecule, a RPE65 (e.g., human RPE65) protein, a ABCA4 (e.g.,
human
ABCA4) protein or fragment thereof, a RLBP1 (e.g., human RLBP1) protein or
fragment
thereof, a PDE6B (e.g.. human PDE6B) protein or fragment thereof, a RPGR
(e.g., human
RPGR) protein or fragment thereof or a ACHM3A or ACHM3B (e.g., human ACHM3A or
human ACHM3B) protein or fragment thereof. In embodiments, the nucleic acid
molecule of
the virus particle further comprises one or more regulatory elements, e.g.,
comprises a promoter,
e.g., a promoter operably linked to the heterologous transgene and which
regulates expression
from the heterologous transgene in a tissue of interest. In embodiments, the
nucleic acid
molecule of the virus particle further comprises one or more of (a) a
dependoparvovirus ITR, (b)
an intron, (c) an enhancer or repressor sequence, (d) a stuffer sequence, and
(e) a polyA
sequence.
Accordingly, provided herein is a virus particle comprising a capsid
polypeptide
comprising (a) a VP1, VP2 or VP3 sequence of SEQ ID NO: 9. (b) a VP1. VP2 or
VP3 sequence
comprising the mutation set of VAR-8 and having greater than 80% (for example,
greater than
90% greater than 91%, greater than 92%, greater than 93%, greater than 94%,
greater than 95%,
greater than 96%, greater than 97%, greater than 98%, greater than 99%)
identity to SEQ ID NO:
1, or (c) a VP1, VP2 or VP3 sequence comprising the mutation set of VAR-8 and
having at least
1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 additional mutations, but fewer than 40, 39,
38, 37, 36, 35, 34, 33, 32
or 31 additional mutations relative to SEQ ID NO: 1. In embodiments, the
capsid polypeptide
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comprises VP1, VP2 and VP3 sequences of SEQ ID NO: 9. In embodiments, the
virus particle
comprises a nucleic acid molecule comprising a heterologous transgene, for
example a
heterologous transgene encoding a product directed to an ocular disorder. In
embodiments, the
heterologous transgene encodes an anti-VEGF antibody or antibody fragment, an
anti-VEGF
RNA inhibitory molecule, a RPE65 (e.g., human RPE65) protein, a ABCA4 (e.g.,
human
ABCA4) protein or fragment thereof, a RLBP1 (e.g., human RLBP1) protein or
fragment
thereof, a PDE6B (e.g.. human PDE6B) protein or fragment thereof, a RPGR
(e.g., human
RPGR) protein or fragment thereof or a ACHM3A or ACHM3B (e.g., human ACHM3A or
human ACHM3B) protein or fragment thereof. In embodiments, the nucleic acid
molecule of
the virus particle further comprises one or more regulatory elements, e.g.,
comprises a promoter,
e.g., a promoter operably linked to the heterologous transgene and which
regulates expression
from the heterologous transgene in a tissue of interest. In embodiments, the
nucleic acid
molecule of the virus particle further comprises one or more of (a) a
dependoparvovirus ITR, (b)
an intron, (c) an enhancer or repressor sequence, (d) a stuffer sequence, and
(e) a polyA
sequence.
Accordingly, provided herein is a virus particle comprising a capsid
polypeptide
comprising (a) a VP1, VP2 or VP3 sequence of SEQ ID NO: 10, (b) a VP1, VP2 or
VP3
sequence comprising the mutation set of VAR-9 and having greater than 80% (for
example,
greater than 90% greater than 91%, greater than 92%, greater than 93%, greater
than 94%,
greater than 95%, greater than 96%, greater than 97%, greater than 98%,
greater than 99%)
identity to SEQ ID NO: 1, or (c) a VP1. VP2 or VP3 sequence comprising the
mutation set of
VAR-9 and having at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 additional
mutations, but fewer than 40,
39, 38, 37, 36, 35, 34, 33, 32 or 31 additional mutations relative to SEQ ID
NO: 1. In
embodiments, the capsid polypeptide comprises VP1, VP2 and VP3 sequences of
SEQ ID NO:
10. In embodiments, the virus particle comprises a nucleic acid molecule
comprising a
heterologous transgene, for example a heterologous transgene encoding a
product directed to an
ocular disorder. In embodiments, the heterologous transgene encodes an anti-
VEGF antibody or
antibody fragment, an anti-VEGF RNA inhibitory molecule, a RPE65 (e.g., human
RPE65)
protein, a ABCA4 (e.g., human ABCA4) protein or fragment thereof, a RLBP1
(e.g., human
RLBP1) protein or fragment thereof, a PDE6B (e.g.. human PDE6B) protein or
fragment thereof,
a RPGR (e.g., human RPGR) protein or fragment thereof or a ACHM3A or ACHM3B
(e.g.,
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human ACHM3A or human ACHM3B) protein or fragment thereof. In embodiments, the
nucleic acid molecule of the virus particle further comprises one or more
regulatory elements,
e.g., comprises a promoter, e.g., a promoter operably linked to the
heterologous transgene and
which regulates expression from the heterologous transgene in a tissue of
interest. In
embodiments, the nucleic acid molecule of the virus particle further comprises
one or more of (a)
a dependoparvovirus ITR, (b) an intron, (c) an enhancer or repressor sequence,
(d) a stuffer
sequence, and (e) a polyA sequence.
Accordingly, provided herein is a virus particle comprising a capsid
polypeptide
comprising (a) a VP1, VP2 or VP3 sequence of SEQ ID NO: 11, (b) a VP1, VP2 or
VP3
sequence comprising the mutation set of VAR-10 and having greater than 80%
(for example,
greater than 90% greater than 91%, greater than 92%, greater than 93%, greater
than 94%,
greater than 95%, greater than 96%, greater than 97%, greater than 98%.
greater than 99%)
identity to SEQ ID NO: 1, or (c) a VP1. VP2 or VP3 sequence comprising the
mutation set of
VAR-10 and having at least 1, 2, 3, 4, 5, 6, 7. 8, 9 or 10 additional
mutations, but fewer than 40,
39, 38, 37, 36, 35, 34, 33, 32 or 31 additional mutations relative to SEQ ID
NO: 1. In
embodiments, the capsid polypeptide comprises VP1, VP2 and VP3 sequences of
SEQ ID NO:
11. In embodiments, the virus particle comprises a nucleic acid molecule
comprising a
heterologous transgene, for example a heterologous transgene encoding a
product directed to an
ocular disorder. In embodiments, the heterologous transgene encodes an anti-
VEGF antibody or
antibody fragment, an anti-VEGF RNA inhibitory molecule, a RPE65 (e.g., human
RPE65)
protein, a ABCA4 (e.g., human ABCA4) protein or fragment thereof, a RLBPI
(e.g., human
RLBP1) protein or fragment thereof, a PDE6B (e.g.. human PDE6B) protein or
fragment thereof,
a RPGR (e.g., human RPGR) protein or fragment thereof or a ACHM3A or ACHM3B
(e.g.,
human ACHM3A or human ACHM3B) protein or fragment thereof. In embodiments, the
nucleic acid molecule of the virus particle further comprises one or more
regulatory elements,
e.g., comprises a promoter, e.g., a promoter operably linked to the
heterologous transgene and
which regulates expression from the heterologous transgene in a tissue of
interest. In
embodiments, the nucleic acid molecule of the virus particle further comprises
one or more of (a)
a dependoparvovirus ITR, (b) an intron, (c) an enhancer or repressor sequence,
(d) a stuffer
sequence, and (e) a polyA sequence.
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Accordingly, provided herein is a virus particle comprising a capsid
polypeptide
comprising (a) a VP1, VP2 or VP3 sequence of SEQ ID NO: 12, (b) a VP1, VP2 or
VP3
sequence comprising the mutation set of VAR-11 and having greater than 80%
(for example,
greater than 90% greater than 91%, greater than 92%, greater than 93%, greater
than 94%,
greater than 95%, greater than 96%, greater than 97%, greater than 98%,
greater than 99%)
identity to SEQ ID NO: 1, or (c) a VP1. VP2 or VP3 sequence comprising the
mutation set of
VAR-11 and having at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 additional
mutations, but fewer than 40,
39, 38, 37, 36, 35, 34, 33, 32 or 31 additional mutations relative to SEQ ID
NO: 1. In
embodiments, the capsid polypeptide comprises VP1, VP2 and VP3 sequences of
SEQ ID NO:
12. In embodiments, the virus particle comprises a nucleic acid molecule
comprising a
heterologous transgene, for example a heterologous transgene encoding a
product directed to an
ocular disorder. In embodiments, the heterologous transgene encodes an anti-
VEGF antibody or
antibody fragment, an anti-VEGF RNA inhibitory molecule, a RPE65 (e.g., human
RPE65)
protein, a ABCA4 (e.g., human ABCA4) protein or fragment thereof, a RLBP1
(e.g., human
RLBP1) protein or fragment thereof, a PDE6B (e.g.. human PDE6B) protein or
fragment thereof,
a RPGR (e.g., human RPGR) protein or fragment thereof or a ACHM3A or ACHM3B
(e.g.,
human ACHM3A or human ACHM3B) protein or fragment thereof. In embodiments, the
nucleic acid molecule of the virus particle further comprises one or more
regulatory elements,
e.g., comprises a promoter, e.g., a promoter operably linked to the
heterologous transgene and
which regulates expression from the heterologous transgene in a tissue of
interest. In
embodiments, the nucleic acid molecule of the virus particle further comprises
one or more of (a)
a dependoparvovirus ITR, (b) an intron, (c) an enhancer or repressor sequence,
(d) a stuffer
sequence, and (e) a polyA sequence.
Accordingly, provided herein is a virus particle comprising a capsid
polypeptide
comprising (a) a VP1, VP2 or VP3 sequence of SEQ ID NO: 13, (b) a VP1, VP2 or
VP3
sequence comprising the mutation set of VAR-12 and having greater than 80%
(for example,
greater than 90% greater than 91%, greater than 92%, greater than 93%, greater
than 94%,
greater than 95%, greater than 96%, greater than 97%, greater than 98%,
greater than 99%)
identity to SEQ ID NO: 1, or (c) a VP1. VP2 or VP3 sequence comprising the
mutation set of
VAR-12 and having at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 additional
mutations, but fewer than 40,
39, 38, 37, 36, 35, 34, 33, 32 or 31 additional mutations relative to SEQ ID
NO: 1. In
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embodiments, the capsid polypeptide comprises VP I, VP2 and VP3 sequences of
SEQ ID NO:
13. In embodiments, the virus particle comprises a nucleic acid molecule
comprising a
heterologous transgene, for example a heterologous transgene encoding a
product directed to an
ocular disorder. In embodiments, the heterologous transgene encodes an anti-
VEGF antibody or
antibody fragment, an anti-VEGF RNA inhibitory molecule, a RPE65 (e.g., human
RPE65)
protein, a ABCA4 (e.g., human ABCA4) protein or fragment thereof, a RLBP1
(e.g., human
RLBP1) protein or fragment thereof, a PDE6B (e.g.. human PDE6B) protein or
fragment thereof,
a RPGR (e.g., human RPGR) protein or fragment thereof or a ACHM3A or ACHM3B
(e.g.,
human ACHM3A or human ACHM3B) protein or fragment thereof. In embodiments, the
nucleic acid molecule of the virus particle further comprises one or more
regulatory elements,
e.g., comprises a promoter, e.g., a promoter operably linked to the
heterologous transgene and
which regulates expression from the heterologous transgene in a tissue of
interest. In
embodiments, the nucleic acid molecule of the virus particle further comprises
one or more of (a)
a dependoparvovirus ITR, (b) an intron, (c) an enhancer or repressor sequence,
(d) a stuffer
sequence, and (e) a polyA sequence.
Accordingly, provided herein is a virus particle comprising a capsid
polypeptide
comprising (a) a VP1, VP2 or VP3 sequence of SEQ ID NO: 14, (b) a VP1, VP2 or
VP3
sequence comprising the mutation set of VAR-13 and having greater than 80%
(for example,
greater than 90% greater than 91%, greater than 92%, greater than 93%, greater
than 94%,
greater than 95%, greater than 96%, greater than 97%, greater than 98%,
greater than 99%)
identity to SEQ ID NO: 1, or (c) a VP1. VP2 or VP3 sequence comprising the
mutation set of
VAR-13 and having at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 additional
mutations, but fewer than 40,
39, 38, 37, 36, 35, 34, 33, 32 or 31 additional mutations relative to SEQ ID
NO: 1. In
embodiments, the capsid polypeptide comprises VP1, VP2 and VP3 sequences of
SEQ ID NO:
14. In embodiments, the virus particle comprises a nucleic acid molecule
comprising a
heterologous transgene, for example a heterologous transgene encoding a
product directed to an
ocular disorder. In embodiments, the heterologous transgene encodes an anti-
VEGF antibody or
antibody fragment, an anti-VEGF RNA inhibitory molecule, a RPE65 (e.g., human
RPE65)
protein, a ABCA4 (e.g., human ABCA4) protein or fragment thereof, a RLBP1
(e.g., human
RLBP1) protein or fragment thereof, a PDE6B (e.g.. human PDE6B) protein or
fragment thereof,
a RPGR (e.g., human RPGR) protein or fragment thereof or a ACHM3A or ACHM3B
(e.g.,
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human ACHM3A or human ACHM3B) protein or fragment thereof. In embodiments, the
nucleic acid molecule of the virus particle further comprises one or more
regulatory elements,
e.g., comprises a promoter, e.g., a promoter operably linked to the
heterologous transgene and
which regulates expression from the heterologous transgene in a tissue of
interest. In
embodiments, the nucleic acid molecule of the virus particle further comprises
one or more of (a)
a dependoparvovirus ITR, (b) an intron, (c) an enhancer or repressor sequence,
(d) a stuffer
sequence, and (e) a polyA sequence.
Accordingly, provided herein is a virus particle comprising a capsid
polypeptide
comprising (a) a VP1, VP2 or VP3 sequence of SEQ ID NO: 15, (b) a VP1, VP2 or
VP3
sequence comprising the mutation set of VAR-14 and having greater than 80%
(for example,
greater than 90% greater than 91%, greater than 92%, greater than 93%, greater
than 94%,
greater than 95%, greater than 96%, greater than 97%, greater than 98%.
greater than 99%)
identity to SEQ ID NO: 1, or (c) a VP1. VP2 or VP3 sequence comprising the
mutation set of
VAR-14 and having at least 1, 2, 3, 4, 5, 6, 7. 8, 9 or 10 additional
mutations, but fewer than 40,
39, 38, 37, 36, 35, 34, 33, 32 or 31 additional mutations relative to SEQ ID
NO: 1. In
embodiments, the capsid polypeptide comprises VP1, VP2 and VP3 sequences of
SEQ ID NO:
15. In embodiments, the virus particle comprises a nucleic acid molecule
comprising a
heterologous transgene, for example a heterologous transgene encoding a
product directed to an
ocular disorder. In embodiments, the heterologous transgene encodes an anti-
VEGF antibody or
antibody fragment, an anti-VEGF RNA inhibitory molecule, a RPE65 (e.g., human
RPE65)
protein, a ABCA4 (e.g., human ABCA4) protein or fragment thereof, a RLBPI
(e.g., human
RLBP1) protein or fragment thereof, a PDE6B (e.g.. human PDE6B) protein or
fragment thereof,
a RPGR (e.g., human RPGR) protein or fragment thereof or a ACHM3A or ACHM3B
(e.g.,
human ACHM3A or human ACHM3B) protein or fragment thereof. In embodiments, the
nucleic acid molecule of the virus particle further comprises one or more
regulatory elements,
e.g., comprises a promoter, e.g., a promoter operably linked to the
heterologous transgene and
which regulates expression from the heterologous transgene in a tissue of
interest. In
embodiments, the nucleic acid molecule of the virus particle further comprises
one or more of (a)
a dependoparvovirus ITR, (b) an intron, (c) an enhancer or repressor sequence,
(d) a stuffer
sequence, and (e) a polyA sequence.
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Accordingly, provided herein is a virus particle comprising a capsid
polypeptide
comprising (a) a VP1, VP2 or VP3 sequence of SEQ ID NO: 16, (b) a VP1, VP2 or
VP3
sequence comprising the mutation set of VAR-15 and having greater than 80%
(for example,
greater than 90% greater than 91%, greater than 92%, greater than 93%, greater
than 94%,
greater than 95%, greater than 96%, greater than 97%, greater than 98%,
greater than 99%)
identity to SEQ ID NO: 1, or (c) a VP1. VP2 or VP3 sequence comprising the
mutation set of
VAR-15 and having at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 additional
mutations, but fewer than 40,
39, 38, 37, 36, 35, 34, 33, 32 or 31 additional mutations relative to SEQ ID
NO: 1. In
embodiments, the capsid polypeptide comprises VP1, VP2 and VP3 sequences of
SEQ ID NO:
14. In embodiments, the virus particle comprises a nucleic acid molecule
comprising a
heterologous transgene, for example a heterologous transgene encoding a
product directed to an
ocular disorder. In embodiments, the heterologous transgene encodes an anti-
VEGF antibody or
antibody fragment, an anti-VEGF RNA inhibitory molecule, a RPE65 (e.g., human
RPE65)
protein, a ABCA4 (e.g., human ABCA4) protein or fragment thereof, a RLBP1
(e.g., human
RLBP1) protein or fragment thereof, a PDE6B (e.g.. human PDE6B) protein or
fragment thereof,
a RPGR (e.g., human RPGR) protein or fragment thereof or a ACHM3A or ACHM3B
(e.g.,
human ACHM3A or human ACHM3B) protein or fragment thereof. In embodiments, the
nucleic acid molecule of the virus particle further comprises one or more
regulatory elements,
e.g., comprises a promoter, e.g., a promoter operably linked to the
heterologous transgene and
which regulates expression from the heterologous transgene in a tissue of
interest. In
embodiments, the nucleic acid molecule of the virus particle further comprises
one or more of (a)
a dependoparvovirus ITR, (b) an intron, (c) an enhancer or repressor sequence,
(d) a stuffer
sequence, and (e) a polyA sequence.
Accordingly, provided herein is a virus particle comprising a capsid
polypeptide
comprising (a) a VP1, VP2 or VP3 sequence of SEQ ID NO: 17, (b) a VP1, VP2 or
VP3
sequence comprising the mutation set of VAR-16 and having greater than 80%
(for example,
greater than 90% greater than 91%, greater than 92%, greater than 93%, greater
than 94%,
greater than 95%, greater than 96%, greater than 97%, greater than 98%,
greater than 99%)
identity to SEQ ID NO: 1, or (c) a VP1. VP2 or VP3 sequence comprising the
mutation set of
VAR-16 and having at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 additional
mutations, but fewer than 40,
39, 38, 37, 36, 35, 34, 33, 32 or 31 additional mutations relative to SEQ ID
NO: 17. In
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embodiments, the capsid polypeptide comprises VP1, VP2 and VP3 sequences of
SEQ ID NO:
5. In embodiments, the virus particle comprises a nucleic acid molecule
comprising a
heterologous transgene, for example a heterologous transgene encoding a
product directed to an
ocular disorder. In embodiments, the heterologous transgene encodes an anti-
VEGF antibody or
antibody fragment, an anti-VEGF RNA inhibitory molecule, a RPE65 (e.g., human
RPE65)
protein, a ABCA4 (e.g., human ABCA4) protein or fragment thereof, a RLBP1
(e.g., human
RLBP1) protein or fragment thereof, a PDE6B (e.g.. human PDE6B) protein or
fragment thereof,
a RPGR (e.g., human RPGR) protein or fragment thereof or a ACHM3A or ACHM3B
(e.g.,
human ACHM3A or human ACHM3B) protein or fragment thereof. In embodiments, the
nucleic acid molecule of the virus particle further comprises one or more
regulatory elements,
e.g., comprises a promoter, e.g., a promoter operably linked to the
heterologous transgene and
which regulates expression from the heterologous transgene in a tissue of
interest. In
embodiments, the nucleic acid molecule of the virus particle further comprises
one or more of (a)
a dependoparvovirus ITR, (b) an intron, (c) an enhancer or repressor sequence,
(d) a stuffer
sequence, and (e) a polyA sequence.
Accordingly, provided herein is a virus particle comprising a capsid
polypeptide
comprising (a) a VP1, VP2 or VP3 sequence of SEQ ID NO: 18, (b) a VP1, VP2 or
VP3
sequence comprising the mutation set of VAR-17 and having greater than 80%
(for example,
greater than 90% greater than 91%, greater than 92%, greater than 93%, greater
than 94%,
greater than 95%, greater than 96%, greater than 97%, greater than 98%,
greater than 99%)
identity to SEQ ID NO: 1, or (c) a VP1. VP2 or VP3 sequence comprising the
mutation set of
VAR-17 and having at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 additional
mutations, but fewer than 40,
39, 38, 37, 36, 35, 34, 33, 32 or 31 additional mutations relative to SEQ ID
NO: 1. In
embodiments, the capsid polypeptide comprises VP1, VP2 and VP3 sequences of
SEQ ID NO:
18. In embodiments, the virus particle comprises a nucleic acid molecule
comprising a
heterologous transgene, for example a heterologous transgene encoding a
product directed to an
ocular disorder. In embodiments, the heterologous transgene encodes an anti-
VEGF antibody or
antibody fragment, an anti-VEGF RNA inhibitory molecule, a RPE65 (e.g., human
RPE65)
protein, a ABCA4 (e.g., human ABCA4) protein or fragment thereof, a RLBP1
(e.g., human
RLBP1) protein or fragment thereof, a PDE6B (e.g.. human PDE6B) protein or
fragment thereof,
a RPGR (e.g., human RPGR) protein or fragment thereof or a ACHM3A or ACHM3B
(e.g.,
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human ACHM3A or human ACHM3B) protein or fragment thereof. In embodiments, the
nucleic acid molecule of the virus particle further comprises one or more
regulatory elements,
e.g., comprises a promoter, e.g., a promoter operably linked to the
heterologous transgene and
which regulates expression from the heterologous transgene in a tissue of
interest. In
embodiments, the nucleic acid molecule of the virus particle further comprises
one or more of (a)
a dependoparvovirus ITR, (b) an intron, (c) an enhancer or repressor sequence,
(d) a stuffer
sequence, and (e) a polyA sequence.
Accordingly, provided herein is a virus particle comprising a capsid
polypeptide
comprising (a) a VP1, VP2 or VP3 sequence of SEQ ID NO: 19, (b) a VP1, VP2 or
VP3
sequence comprising the mutation set of VAR-18 and having greater than 80%
(for example,
greater than 90% greater than 91%, greater than 92%, greater than 93%, greater
than 94%,
greater than 95%, greater than 96%, greater than 97%, greater than 98%.
greater than 99%)
identity to SEQ ID NO: 1, or (c) a VP1. VP2 or VP3 sequence comprising the
mutation set of
VAR-18 and having at least 1, 2, 3, 4, 5, 6, 7. 8, 9 or 10 additional
mutations, but fewer than 40,
39, 38, 37, 36, 35, 34, 33, 32 or 31 additional mutations relative to SEQ ID
NO: 1. In
embodiments, the capsid polypeptide comprises VP1, VP2 and VP3 sequences of
SEQ ID NO:
19. In embodiments, the virus particle comprises a nucleic acid molecule
comprising a
heterologous transgene, for example a heterologous transgene encoding a
product directed to an
ocular disorder. In embodiments, the heterologous transgene encodes an anti-
VEGF antibody or
antibody fragment, an anti-VEGF RNA inhibitory molecule, a RPE65 (e.g., human
RPE65)
protein, a ABCA4 (e.g., human ABCA4) protein or fragment thereof, a RLBPI
(e.g., human
RLBP1) protein or fragment thereof, a PDE6B (e.g.. human PDE6B) protein or
fragment thereof,
a RPGR (e.g., human RPGR) protein or fragment thereof or a ACHM3A or ACHM3B
(e.g.,
human ACHM3A or human ACHM3B) protein or fragment thereof. In embodiments, the
nucleic acid molecule of the virus particle further comprises one or more
regulatory elements,
e.g., comprises a promoter, e.g., a promoter operably linked to the
heterologous transgene and
which regulates expression from the heterologous transgene in a tissue of
interest. In
embodiments, the nucleic acid molecule of the virus particle further comprises
one or more of (a)
a dependoparvovirus ITR, (b) an intron, (c) an enhancer or repressor sequence,
(d) a stuffer
sequence, and (e) a polyA sequence.
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Accordingly, provided herein is a virus particle comprising a capsid
polypeptide
comprising (a) a VP1, VP2 or VP3 sequence of SEQ ID NO: 20, (b) a VP1, VP2 or
VP3
sequence comprising the mutation set of VAR-19 and having greater than 80%
(for example,
greater than 90% greater than 91%, greater than 92%, greater than 93%, greater
than 94%,
greater than 95%, greater than 96%, greater than 97%, greater than 98%,
greater than 99%)
identity to SEQ ID NO: 1, or (c) a VP1. VP2 or VP3 sequence comprising the
mutation set of
VAR-19 and having at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 additional
mutations, but fewer than 40,
39, 38, 37, 36, 35, 34, 33, 32 or 31 additional mutations relative to SEQ ID
NO: 1. In
embodiments, the capsid polypeptide comprises VP1, VP2 and VP3 sequences of
SEQ ID NO:
20. In embodiments, the virus particle comprises a nucleic acid molecule
comprising a
heterologous transgene, for example a heterologous transgene encoding a
product directed to an
ocular disorder. In embodiments, the heterologous transgene encodes an anti-
VEGF antibody or
antibody fragment, an anti-VEGF RNA inhibitory molecule, a RPE65 (e.g., human
RPE65)
protein, a ABCA4 (e.g., human ABCA4) protein or fragment thereof, a RLBP1
(e.g., human
RLBP1) protein or fragment thereof, a PDE6B (e.g.. human PDE6B) protein or
fragment thereof,
a RPGR (e.g., human RPGR) protein or fragment thereof or a ACHM3A or ACHM3B
(e.g.,
human ACHM3A or human ACHM3B) protein or fragment thereof. In embodiments, the
nucleic acid molecule of the virus particle further comprises one or more
regulatory elements,
e.g., comprises a promoter, e.g., a promoter operably linked to the
heterologous transgene and
which regulates expression from the heterologous transgene in a tissue of
interest. In
embodiments, the nucleic acid molecule of the virus particle further comprises
one or more of (a)
a dependoparvovirus ITR, (b) an intron, (c) an enhancer or repressor sequence,
(d) a stuffer
sequence, and (e) a polyA sequence.
Accordingly, provided herein is a virus particle comprising a capsid
polypeptide
comprising (a) a VP1, VP2 or VP3 sequence of SEQ ID NO: 21, (b) a VP1, VP2 or
VP3
sequence comprising the mutation set of VAR-20 and having greater than 80%
(for example,
greater than 90% greater than 91%, greater than 92%, greater than 93%, greater
than 94%,
greater than 95%, greater than 96%, greater than 97%, greater than 98%,
greater than 99%)
identity to SEQ ID NO: 1, or (c) a VP1. VP2 or VP3 sequence comprising the
mutation set of
VAR-20 and having at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 additional
mutations, but fewer than 40,
39, 38, 37, 36, 35, 34, 33, 32 or 31 additional mutations relative to SEQ ID
NO: 1. In
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embodiments, the capsid polypeptide comprises VP I, VP2 and VP3 sequences of
SEQ ID NO:
21. In embodiments, the virus particle comprises a nucleic acid molecule
comprising a
heterologous transgene, for example a heterologous transgene encoding a
product directed to an
ocular disorder. In embodiments, the heterologous transgene encodes an anti-
VEGF antibody or
antibody fragment, an anti-VEGF RNA inhibitory molecule, a RPE65 (e.g., human
RPE65)
protein, a ABCA4 (e.g., human ABCA4) protein or fragment thereof, a RLBP1
(e.g., human
RLBP1) protein or fragment thereof, a PDE6B (e.g.. human PDE6B) protein or
fragment thereof,
a RPGR (e.g., human RPGR) protein or fragment thereof or a ACHM3A or ACHM3B
(e.g.,
human ACHM3A or human ACHM3B) protein or fragment thereof. In embodiments, the
nucleic acid molecule of the virus particle further comprises one or more
regulatory elements,
e.g., comprises a promoter, e.g., a promoter operably linked to the
heterologous transgene and
which regulates expression from the heterologous transgene in a tissue of
interest. In
embodiments, the nucleic acid molecule of the virus particle further comprises
one or more of (a)
a dependoparvovirus ITR, (b) an intron, (c) an enhancer or repressor sequence,
(d) a stuffer
sequence, and (e) a polyA sequence.
Accordingly, provided herein is a virus particle comprising a capsid
polypeptide
comprising (a) a VP1, VP2 or VP3 sequence of SEQ ID NO: 22, (b) a VP1, VP2 or
VP3
sequence comprising the mutation set of VAR-21 and having greater than 80%
(for example,
greater than 90% greater than 91%, greater than 92%, greater than 93%, greater
than 94%,
greater than 95%, greater than 96%, greater than 97%, greater than 98%,
greater than 99%)
identity to SEQ ID NO: 1, or (c) a VP1. VP2 or VP3 sequence comprising the
mutation set of
VAR-21 and having at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 additional
mutations, but fewer than 40,
39, 38, 37, 36, 35, 34, 33, 32 or 31 additional mutations relative to SEQ ID
NO: 1. In
embodiments, the capsid polypeptide comprises VP1, VP2 and VP3 sequences of
SEQ ID NO:
22. In embodiments, the virus particle comprises a nucleic acid molecule
comprising a
heterologous transgene, for example a heterologous transgene encoding a
product directed to an
ocular disorder. In embodiments, the heterologous transgene encodes an anti-
VEGF antibody or
antibody fragment, an anti-VEGF RNA inhibitory molecule, a RPE65 (e.g., human
RPE65)
protein, a ABCA4 (e.g., human ABCA4) protein or fragment thereof, a RLBP1
(e.g., human
RLBP1) protein or fragment thereof, a PDE6B (e.g.. human PDE6B) protein or
fragment thereof,
a RPGR (e.g., human RPGR) protein or fragment thereof or a ACHM3A or ACHM3B
(e.g.,
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human ACHM3A or human ACHM3B) protein or fragment thereof. In embodiments, the
nucleic acid molecule of the virus particle further comprises one or more
regulatory elements,
e.g., comprises a promoter, e.g., a promoter operably linked to the
heterologous transgene and
which regulates expression from the heterologous transgene in a tissue of
interest. In
embodiments, the nucleic acid molecule of the virus particle further comprises
one or more of (a)
a dependoparvovirus ITR, (b) an intron, (c) an enhancer or repressor sequence,
(d) a stuffer
sequence, and (e) a polyA sequence.
Accordingly, provided herein is a virus particle comprising a capsid
polypeptide
comprising (a) a VP1, VP2 or VP3 sequence of SEQ ID NO: 23, (b) a VP1, VP2 or
VP3
sequence comprising the mutation set of VAR-22 and having greater than 80%
(for example,
greater than 90% greater than 91%, greater than 92%, greater than 93%, greater
than 94%,
greater than 95%, greater than 96%, greater than 97%, greater than 98%.
greater than 99%)
identity to SEQ ID NO: 1, or (c) a VP1. VP2 or VP3 sequence comprising the
mutation set of
VAR-22 and having at least 1, 2, 3, 4, 5, 6, 7. 8, 9 or 10 additional
mutations, but fewer than 40,
39, 38, 37, 36, 35, 34, 33, 32 or 31 additional mutations relative to SEQ ID
NO: 1. In
embodiments, the capsid polypeptide comprises VP1, VP2 and VP3 sequences of
SEQ ID NO:
23. In embodiments, the virus particle comprises a nucleic acid molecule
comprising a
heterologous transgene, for example a heterologous transgene encoding a
product directed to an
ocular disorder. In embodiments, the heterologous transgene encodes an anti-
VEGF antibody or
antibody fragment, an anti-VEGF RNA inhibitory molecule, a RPE65 (e.g., human
RPE65)
protein, a ABCA4 (e.g., human ABCA4) protein or fragment thereof, a RLBPI
(e.g., human
RLBP1) protein or fragment thereof, a PDE6B (e.g.. human PDE6B) protein or
fragment thereof,
a RPGR (e.g., human RPGR) protein or fragment thereof or a ACHM3A or ACHM3B
(e.g.,
human ACHM3A or human ACHM3B) protein or fragment thereof. In embodiments, the
nucleic acid molecule of the virus particle further comprises one or more
regulatory elements,
e.g., comprises a promoter, e.g., a promoter operably linked to the
heterologous transgene and
which regulates expression from the heterologous transgene in a tissue of
interest. In
embodiments, the nucleic acid molecule of the virus particle further comprises
one or more of (a)
a dependoparvovirus ITR, (b) an intron, (c) an enhancer or repressor sequence,
(d) a stuffer
sequence, and (e) a polyA sequence.
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Accordingly, provided herein is a virus particle comprising a capsid
polypeptide
comprising (a) a VP1, VP2 or VP3 sequence of SEQ ID NO: 24, (b) a VP1, VP2 or
VP3
sequence comprising the mutation set of VAR-23 and having greater than 80%
(for example,
greater than 90% greater than 91%, greater than 92%, greater than 93%, greater
than 94%,
greater than 95%, greater than 96%, greater than 97%, greater than 98%,
greater than 99%)
identity to SEQ ID NO: 1, or (c) a VP1. VP2 or VP3 sequence comprising the
mutation set of
VAR-23 and having at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 additional
mutations, but fewer than 40,
39, 38, 37, 36, 35, 34, 33, 32 or 31 additional mutations relative to SEQ ID
NO: 1. In
embodiments, the capsid polypeptide comprises VP1, VP2 and VP3 sequences of
SEQ ID NO:
24. In embodiments, the virus particle comprises a nucleic acid molecule
comprising a
heterologous transgene, for example a heterologous transgene encoding a
product directed to an
ocular disorder. In embodiments, the heterologous transgene encodes an anti-
VEGF antibody or
antibody fragment, an anti-VEGF RNA inhibitory molecule, a RPE65 (e.g., human
RPE65)
protein, a ABCA4 (e.g., human ABCA4) protein or fragment thereof, a RLBP1
(e.g., human
RLBP1) protein or fragment thereof, a PDE6B (e.g.. human PDE6B) protein or
fragment thereof,
a RPGR (e.g., human RPGR) protein or fragment thereof or a ACHM3A or ACHM3B
(e.g.,
human ACHM3A or human ACHM3B) protein or fragment thereof. In embodiments, the
nucleic acid molecule of the virus particle further comprises one or more
regulatory elements,
e.g., comprises a promoter, e.g., a promoter operably linked to the
heterologous transgene and
which regulates expression from the heterologous transgene in a tissue of
interest. In
embodiments, the nucleic acid molecule of the virus particle further comprises
one or more of (a)
a dependoparvovirus ITR, (b) an intron, (c) an enhancer or repressor sequence,
(d) a stuffer
sequence, and (e) a polyA sequence.
Accordingly, provided herein is a virus particle comprising a capsid
polypeptide
comprising (a) a VP1, VP2 or VP3 sequence of SEQ ID NO: 25, (b) a VP1, VP2 or
VP3
sequence comprising the mutation set of VAR-24 and having greater than 80%
(for example,
greater than 90% greater than 91%, greater than 92%, greater than 93%, greater
than 94%,
greater than 95%, greater than 96%, greater than 97%, greater than 98%,
greater than 99%)
identity to SEQ ID NO: 1, or (c) a VP1. VP2 or VP3 sequence comprising the
mutation set of
VAR-24 and having at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 additional
mutations, but fewer than 40,
39, 38, 37, 36, 35, 34, 33, 32 or 31 additional mutations relative to SEQ ID
NO: 1. In
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embodiments, the capsid polypeptide comprises VP I, VP2 and VP3 sequences of
SEQ ID NO:
25. In embodiments, the virus particle comprises a nucleic acid molecule
comprising a
heterologous transgene, for example a heterologous transgene encoding a
product directed to an
ocular disorder. In embodiments, the heterologous transgene encodes an anti-
VEGF antibody or
antibody fragment, an anti-VEGF RNA inhibitory molecule, a RPE65 (e.g., human
RPE65)
protein, a ABCA4 (e.g., human ABCA4) protein or fragment thereof, a RLBP1
(e.g., human
RLBP1) protein or fragment thereof, a PDE6B (e.g.. human PDE6B) protein or
fragment thereof,
a RPGR (e.g., human RPGR) protein or fragment thereof or a ACHM3A or ACHM3B
(e.g.,
human ACHM3A or human ACHM3B) protein or fragment thereof. In embodiments, the
nucleic acid molecule of the virus particle further comprises one or more
regulatory elements,
e.g., comprises a promoter, e.g., a promoter operably linked to the
heterologous transgene and
which regulates expression from the heterologous transgene in a tissue of
interest. In
embodiments, the nucleic acid molecule of the virus particle further comprises
one or more of (a)
a dependoparvovirus ITR, (b) an intron, (c) an enhancer or repressor sequence,
(d) a stuffer
sequence, and (e) a polyA sequence.
Accordingly, provided herein is a virus particle comprising a capsid
polypeptide
comprising (a) a VP1, VP2 or VP3 sequence of SEQ ID NO: 26, (b) a VP1, VP2 or
VP3
sequence comprising the mutation set of VAR-25 and having greater than 80%
(for example,
greater than 90% greater than 91%, greater than 92%, greater than 93%, greater
than 94%,
greater than 95%, greater than 96%, greater than 97%, greater than 98%,
greater than 99%)
identity to SEQ ID NO: 1, or (c) a VP1. VP2 or VP3 sequence comprising the
mutation set of
VAR-25 and having at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 additional
mutations, but fewer than 40,
39, 38, 37, 36, 35, 34, 33, 32 or 31 additional mutations relative to SEQ ID
NO: 1. In
embodiments, the capsid polypeptide comprises VP1, VP2 and VP3 sequences of
SEQ ID NO:
26. In embodiments, the virus particle comprises a nucleic acid molecule
comprising a
heterologous transgene, for example a heterologous transgene encoding a
product directed to an
ocular disorder. In embodiments, the heterologous transgene encodes an anti-
VEGF antibody or
antibody fragment, an anti-VEGF RNA inhibitory molecule, a RPE65 (e.g., human
RPE65)
protein, a ABCA4 (e.g., human ABCA4) protein or fragment thereof, a RLBP1
(e.g., human
RLBP1) protein or fragment thereof, a PDE6B (e.g.. human PDE6B) protein or
fragment thereof,
a RPGR (e.g., human RPGR) protein or fragment thereof or a ACHM3A or ACHM3B
(e.g.,
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human ACHM3A or human ACHM3B) protein or fragment thereof. In embodiments, the
nucleic acid molecule of the virus particle further comprises one or more
regulatory elements,
e.g., comprises a promoter, e.g., a promoter operably linked to the
heterologous transgene and
which regulates expression from the heterologous transgene in a tissue of
interest. In
embodiments, the nucleic acid molecule of the virus particle further comprises
one or more of (a)
a dependoparvovirus ITR, (b) an intron, (c) an enhancer or repressor sequence,
(d) a stuffer
sequence, and (e) a polyA sequence.
Accordingly, provided herein is a virus particle comprising a capsid
polypeptide
comprising (a) a VP1, VP2 or VP3 sequence of SEQ ID NO: 27, (b) a VP1, VP2 or
VP3
sequence comprising the mutation set of VAR-26 and having greater than 80%
(for example,
greater than 90% greater than 91%, greater than 92%, greater than 93%, greater
than 94%,
greater than 95%, greater than 96%, greater than 97%, greater than 98%.
greater than 99%)
identity to SEQ ID NO: 1, or (c) a VP1. VP2 or VP3 sequence comprising the
mutation set of
VAR-26 and having at least 1, 2, 3, 4, 5, 6, 7. 8, 9 or 10 additional
mutations, but fewer than 40,
39, 38, 37, 36, 35, 34, 33, 32 or 31 additional mutations relative to SEQ ID
NO: 1. In
embodiments, the capsid polypeptide comprises VP1, VP2 and VP3 sequences of
SEQ ID NO:
27. In embodiments, the virus particle comprises a nucleic acid molecule
comprising a
heterologous transgene, for example a heterologous transgene encoding a
product directed to an
ocular disorder. In embodiments, the heterologous transgene encodes an anti-
VEGF antibody or
antibody fragment, an anti-VEGF RNA inhibitory molecule, a RPE65 (e.g., human
RPE65)
protein, a ABCA4 (e.g., human ABCA4) protein or fragment thereof, a RLBPI
(e.g., human
RLBP1) protein or fragment thereof, a PDE6B (e.g.. human PDE6B) protein or
fragment thereof,
a RPGR (e.g., human RPGR) protein or fragment thereof or a ACHM3A or ACHM3B
(e.g.,
human ACHM3A or human ACHM3B) protein or fragment thereof. In embodiments, the
nucleic acid molecule of the virus particle further comprises one or more
regulatory elements,
e.g., comprises a promoter, e.g., a promoter operably linked to the
heterologous transgene and
which regulates expression from the heterologous transgene in a tissue of
interest. In
embodiments, the nucleic acid molecule of the virus particle further comprises
one or more of (a)
a dependoparvovirus ITR, (b) an intron, (c) an enhancer or repressor sequence,
(d) a stuffer
sequence, and (e) a polyA sequence.
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Accordingly, provided herein is a virus particle comprising a capsid
polypeptide
comprising (a) a VP1, VP2 or VP3 sequence of SEQ ID NO: 28, (b) a VP1, VP2 or
VP3
sequence comprising the mutation set of VAR-27 and having greater than 80%
(for example,
greater than 90% greater than 91%, greater than 92%, greater than 93%, greater
than 94%,
greater than 95%, greater than 96%, greater than 97%, greater than 98%,
greater than 99%)
identity to SEQ ID NO: 1, or (c) a VP1. VP2 or VP3 sequence comprising the
mutation set of
VAR-27 and having at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 additional
mutations, but fewer than 40,
39, 38, 37, 36, 35, 34, 33, 32 or 31 additional mutations relative to SEQ ID
NO: 1. In
embodiments, the capsid polypeptide comprises VP1, VP2 and VP3 sequences of
SEQ ID NO:
28. In embodiments, the virus particle comprises a nucleic acid molecule
comprising a
heterologous transgene, for example a heterologous transgene encoding a
product directed to an
ocular disorder. In embodiments, the heterologous transgene encodes an anti-
VEGF antibody or
antibody fragment, an anti-VEGF RNA inhibitory molecule, a RPE65 (e.g., human
RPE65)
protein, a ABCA4 (e.g., human ABCA4) protein or fragment thereof, a RLBP1
(e.g., human
RLBP1) protein or fragment thereof, a PDE6B (e.g.. human PDE6B) protein or
fragment thereof,
a RPGR (e.g., human RPGR) protein or fragment thereof or a ACHM3A or ACHM3B
(e.g.,
human ACHM3A or human ACHM3B) protein or fragment thereof. In embodiments, the
nucleic acid molecule of the virus particle further comprises one or more
regulatory elements,
e.g., comprises a promoter, e.g., a promoter operably linked to the
heterologous transgene and
which regulates expression from the heterologous transgene in a tissue of
interest. In
embodiments, the nucleic acid molecule of the virus particle further comprises
one or more of (a)
a dependoparvovirus ITR, (b) an intron, (c) an enhancer or repressor sequence,
(d) a stuffer
sequence, and (e) a polyA sequence.
Accordingly, provided herein is a virus particle comprising a capsid
polypeptide
comprising (a) a VP1, VP2 or VP3 sequence of SEQ ID NO: 29, (b) a VP1, VP2 or
VP3
sequence comprising the mutation set of VAR-28 and having greater than 80%
(for example,
greater than 90% greater than 91%, greater than 92%, greater than 93%, greater
than 94%,
greater than 95%, greater than 96%, greater than 97%, greater than 98%,
greater than 99%)
identity to SEQ ID NO: 1, or (c) a VP1. VP2 or VP3 sequence comprising the
mutation set of
VAR-28 and having at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 additional
mutations, but fewer than 40,
39, 38, 37, 36, 35, 34, 33, 32 or 31 additional mutations relative to SEQ ID
NO: 1. In
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embodiments, the capsid polypeptide comprises VP I, VP2 and VP3 sequences of
SEQ ID NO:
29. In embodiments, the virus particle comprises a nucleic acid molecule
comprising a
heterologous transgene, for example a heterologous transgene encoding a
product directed to an
ocular disorder. In embodiments, the heterologous transgene encodes an anti-
VEGF antibody or
antibody fragment, an anti-VEGF RNA inhibitory molecule, a RPE65 (e.g., human
RPE65)
protein, a ABCA4 (e.g., human ABCA4) protein or fragment thereof, a RLBP1
(e.g., human
RLBP1) protein or fragment thereof, a PDE6B (e.g.. human PDE6B) protein or
fragment thereof,
a RPGR (e.g., human RPGR) protein or fragment thereof or a ACHM3A or ACHM3B
(e.g.,
human ACHM3A or human ACHM3B) protein or fragment thereof. In embodiments, the
nucleic acid molecule of the virus particle further comprises one or more
regulatory elements,
e.g., comprises a promoter, e.g., a promoter operably linked to the
heterologous transgene and
which regulates expression from the heterologous transgene in a tissue of
interest. In
embodiments, the nucleic acid molecule of the virus particle further comprises
one or more of (a)
a dependoparvovirus ITR, (b) an intron, (c) an enhancer or repressor sequence,
(d) a stuffer
sequence, and (e) a polyA sequence.
Accordingly, provided herein is a virus particle comprising a capsid
polypeptide
comprising (a) a VP1, VP2 or VP3 sequence of SEQ ID NO: 30, (b) a VP1, VP2 or
VP3
sequence comprising the mutation set of VAR-29 and having greater than 80%
(for example,
greater than 90% greater than 91%, greater than 92%, greater than 93%, greater
than 94%,
greater than 95%, greater than 96%, greater than 97%, greater than 98%,
greater than 99%)
identity to SEQ ID NO: 1, or (c) a VP1. VP2 or VP3 sequence comprising the
mutation set of
VAR-29 and having at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 additional
mutations, but fewer than 40,
39, 38, 37, 36, 35, 34, 33, 32 or 31 additional mutations relative to SEQ ID
NO: 1. In
embodiments, the capsid polypeptide comprises VP1, VP2 and VP3 sequences of
SEQ ID NO:
31. In embodiments, the virus particle comprises a nucleic acid molecule
comprising a
heterologous transgene, for example a heterologous transgene encoding a
product directed to an
ocular disorder. In embodiments, the heterologous transgene encodes an anti-
VEGF antibody or
antibody fragment, an anti-VEGF RNA inhibitory molecule, a RPE65 (e.g., human
RPE65)
protein, a ABCA4 (e.g., human ABCA4) protein or fragment thereof, a RLBP1
(e.g., human
RLBP1) protein or fragment thereof, a PDE6B (e.g.. human PDE6B) protein or
fragment thereof,
a RPGR (e.g., human RPGR) protein or fragment thereof or a ACHM3A or ACHM3B
(e.g.,
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human ACHM3A or human ACHM3B) protein or fragment thereof. In embodiments, the
nucleic acid molecule of the virus particle further comprises one or more
regulatory elements,
e.g., comprises a promoter, e.g., a promoter operably linked to the
heterologous transgene and
which regulates expression from the heterologous transgene in a tissue of
interest. In
embodiments, the nucleic acid molecule of the virus particle further comprises
one or more of (a)
a dependoparvovirus ITR, (b) an intron, (c) an enhancer or repressor sequence,
(d) a stuffer
sequence, and (e) a polyA sequence.
Accordingly, provided herein is a virus particle comprising a capsid
polypeptide
comprising (a) a VP1, VP2 or VP3 sequence of SEQ ID NO: 31, (b) a VP1, VP2 or
VP3
sequence comprising the mutation set of VAR-30 and having greater than 80%
(for example,
greater than 90% greater than 91%, greater than 92%, greater than 93%, greater
than 94%,
greater than 95%, greater than 96%, greater than 97%, greater than 98%,
greater than 99%)
identity to SEQ ID NO: 1, or (c) a VP1. VP2 or VP3 sequence comprising the
mutation set of
VAR-30 and having at least 1, 2, 3, 4, 5, 6, 7. 8, 9 or 10 additional
mutations, but fewer than 40,
39, 38, 37, 36, 35, 34, 33, 32 or 31 additional mutations relative to SEQ ID
NO: 1. In
embodiments, the capsid polypeptide comprises VP1, VP2 and VP3 sequences of
SEQ ID NO:
31. In embodiments, the virus particle comprises a nucleic acid molecule
comprising a
heterologous transgene, for example a heterologous transgene encoding a
product directed to an
ocular disorder. In embodiments, the heterologous transgene encodes an anti-
VEGF antibody or
antibody fragment, an anti-VEGF RNA inhibitory molecule, a RPE65 (e.g., human
RPE65)
protein, a ABCA4 (e.g., human ABCA4) protein or fragment thereof, a RLBPI
(e.g., human
RLBP1) protein or fragment thereof, a PDE6B (e.g.. human PDE6B) protein or
fragment thereof,
a RPGR (e.g., human RPGR) protein or fragment thereof or a ACHM3A or ACHM3B
(e.g.,
human ACHM3A or human ACHM3B) protein or fragment thereof. In embodiments, the
nucleic acid molecule of the virus particle further comprises one or more
regulatory elements,
e.g., comprises a promoter, e.g., a promoter operably linked to the
heterologous transgene and
which regulates expression from the heterologous transgene in a tissue of
interest. In
embodiments, the nucleic acid molecule of the virus particle further comprises
one or more of (a)
a dependoparvovirus ITR, (b) an intron, (c) an enhancer or repressor sequence,
(d) a stuffer
sequence, and (e) a polyA sequence.
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Accordingly, provided herein is a virus particle comprising a capsid
polypeptide
comprising (a) a VP1, VP2 or VP3 sequence of SEQ ID NO: 32, (b) a VP1, VP2 or
VP3
sequence comprising the mutation set of VAR-31 and having greater than 80%
(for example,
greater than 90% greater than 91%, greater than 92%, greater than 93%, greater
than 94%,
greater than 95%, greater than 96%, greater than 97%, greater than 98%,
greater than 99%)
identity to SEQ ID NO: 1, or (c) a VP1. VP2 or VP3 sequence comprising the
mutation set of
VAR-31 and having at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 additional
mutations, but fewer than 40,
39, 38, 37, 36, 35, 34, 33, 32 or 31 additional mutations relative to SEQ ID
NO: 1. In
embodiments, the capsid polypeptide comprises VP1, VP2 and VP3 sequences of
SEQ ID NO:
32. In embodiments, the virus particle comprises a nucleic acid molecule
comprising a
heterologous transgene, for example a heterologous transgene encoding a
product directed to an
ocular disorder. In embodiments, the heterologous transgene encodes an anti-
VEGF antibody or
antibody fragment, an anti-VEGF RNA inhibitory molecule, a RPE65 (e.g., human
RPE65)
protein, a ABCA4 (e.g., human ABCA4) protein or fragment thereof, a RLBP1
(e.g., human
RLBP1) protein or fragment thereof, a PDE6B (e.g.. human PDE6B) protein or
fragment thereof,
a RPGR (e.g., human RPGR) protein or fragment thereof or a ACHM3A or ACHM3B
(e.g.,
human ACHM3A or human ACHM3B) protein or fragment thereof. In embodiments, the
nucleic acid molecule of the virus particle further comprises one or more
regulatory elements,
e.g., comprises a promoter, e.g., a promoter operably linked to the
heterologous transgene and
which regulates expression from the heterologous transgene in a tissue of
interest. In
embodiments, the nucleic acid molecule of the virus particle further comprises
one or more of (a)
a dependoparvovirus ITR, (b) an intron, (c) an enhancer or repressor sequence,
(d) a stuffer
sequence, and (e) a polyA sequence.
Accordingly, provided herein is a virus particle comprising a capsid
polypeptide
comprising (a) a VP1, VP2 or VP3 sequence of SEQ ID NO: 33, (b) a VP1, VP2 or
VP3
sequence comprising the mutation set of VAR-32 and having greater than 80%
(for example,
greater than 90% greater than 91%, greater than 92%, greater than 93%, greater
than 94%,
greater than 95%, greater than 96%, greater than 97%, greater than 98%,
greater than 99%)
identity to SEQ ID NO: 1, or (c) a VP1. VP2 or VP3 sequence comprising the
mutation set of
VAR-32 and having at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 additional
mutations, but fewer than 40,
39, 38, 37, 36, 35, 34, 33, 32 or 31 additional mutations relative to SEQ ID
NO: 1. In
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embodiments, the capsid polypeptide comprises VP I, VP2 and VP3 sequences of
SEQ ID NO:
33. In embodiments, the virus particle comprises a nucleic acid molecule
comprising a
heterologous transgene, for example a heterologous transgene encoding a
product directed to an
ocular disorder. In embodiments, the heterologous transgene encodes an anti-
VEGF antibody or
antibody fragment, an anti-VEGF RNA inhibitory molecule, a RPE65 (e.g., human
RPE65)
protein, a ABCA4 (e.g., human ABCA4) protein or fragment thereof, a RLBP1
(e.g., human
RLBP1) protein or fragment thereof, a PDE6B (e.g.. human PDE6B) protein or
fragment thereof,
a RPGR (e.g., human RPGR) protein or fragment thereof or a ACHM3A or ACHM3B
(e.g.,
human ACHM3A or human ACHM3B) protein or fragment thereof. In embodiments, the
nucleic acid molecule of the virus particle further comprises one or more
regulatory elements,
e.g., comprises a promoter, e.g., a promoter operably linked to the
heterologous transgene and
which regulates expression from the heterologous transgene in a tissue of
interest. In
embodiments, the nucleic acid molecule of the virus particle further comprises
one or more of (a)
a dependoparvovirus ITR, (b) an intron, (c) an enhancer or repressor sequence,
(d) a stuffer
sequence, and (e) a polyA sequence.
Accordingly, provided herein is a virus particle comprising a capsid
polypeptide
comprising (a) a VP1, VP2 or VP3 sequence of SEQ ID NO: 34, (b) a VP1, VP2 or
VP3
sequence comprising the mutation set of VAR-33 and having greater than 80%
(for example,
greater than 90% greater than 91%, greater than 92%, greater than 93%, greater
than 94%,
greater than 95%, greater than 96%, greater than 97%, greater than 98%,
greater than 99%)
identity to SEQ ID NO: 1, or (c) a VP1. VP2 or VP3 sequence comprising the
mutation set of
VAR-33 and having at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 additional
mutations, but fewer than 40,
39, 38, 37, 36, 35, 34, 33, 32 or 31 additional mutations relative to SEQ ID
NO: 1. In
embodiments, the capsid polypeptide comprises VP1, VP2 and VP3 sequences of
SEQ ID NO:
34. In embodiments, the virus particle comprises a nucleic acid molecule
comprising a
heterologous transgene, for example a heterologous transgene encoding a
product directed to an
ocular disorder. In embodiments, the heterologous transgene encodes an anti-
VEGF antibody or
antibody fragment, an anti-VEGF RNA inhibitory molecule, a RPE65 (e.g., human
RPE65)
protein, a ABCA4 (e.g., human ABCA4) protein or fragment thereof, a RLBP1
(e.g., human
RLBP1) protein or fragment thereof, a PDE6B (e.g.. human PDE6B) protein or
fragment thereof,
a RPGR (e.g., human RPGR) protein or fragment thereof or a ACHM3A or ACHM3B
(e.g.,
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human ACHM3A or human ACHM3B) protein or fragment thereof. In embodiments, the
nucleic acid molecule of the virus particle further comprises one or more
regulatory elements,
e.g., comprises a promoter, e.g., a promoter operably linked to the
heterologous transgene and
which regulates expression from the heterologous transgene in a tissue of
interest. In
embodiments, the nucleic acid molecule of the virus particle further comprises
one or more of (a)
a dependoparvovirus ITR, (b) an intron, (c) an enhancer or repressor sequence,
(d) a stuffer
sequence, and (e) a polyA sequence.
Accordingly, provided herein is a virus particle comprising a capsid
polypeptide
comprising (a) a VP1, VP2 or VP3 sequence of SEQ ID NO: 35, (b) a VP1, VP2 or
VP3
sequence comprising the mutation set of VAR-34 and having greater than 80%
(for example,
greater than 90% greater than 91%, greater than 92%, greater than 93%, greater
than 94%,
greater than 95%, greater than 96%, greater than 97%, greater than 98%.
greater than 99%)
identity to SEQ ID NO: 1, or (c) a VP1. VP2 or VP3 sequence comprising the
mutation set of
VAR-34 and having at least 1, 2, 3, 4, 5, 6, 7. 8, 9 or 10 additional
mutations, but fewer than 40,
39, 38, 37, 36, 35, 34, 33, 32 or 31 additional mutations relative to SEQ ID
NO: 1. In
embodiments, the capsid polypeptide comprises VP1, VP2 and VP3 sequences of
SEQ ID NO:
35. In embodiments, the virus particle comprises a nucleic acid molecule
comprising a
heterologous transgene, for example a heterologous transgene encoding a
product directed to an
ocular disorder. In embodiments, the heterologous transgene encodes an anti-
VEGF antibody or
antibody fragment, an anti-VEGF RNA inhibitory molecule, a RPE65 (e.g., human
RPE65)
protein, a ABCA4 (e.g., human ABCA4) protein or fragment thereof, a RLBPI
(e.g., human
RLBP1) protein or fragment thereof, a PDE6B (e.g.. human PDE6B) protein or
fragment thereof,
a RPGR (e.g., human RPGR) protein or fragment thereof or a ACHM3A or ACHM3B
(e.g.,
human ACHM3A or human ACHM3B) protein or fragment thereof. In embodiments, the
nucleic acid molecule of the virus particle further comprises one or more
regulatory elements,
e.g., comprises a promoter, e.g., a promoter operably linked to the
heterologous transgene and
which regulates expression from the heterologous transgene in a tissue of
interest. In
embodiments, the nucleic acid molecule of the virus particle further comprises
one or more of (a)
a dependoparvovirus ITR, (b) an intron, (c) an enhancer or repressor sequence,
(d) a stuffer
sequence, and (e) a polyA sequence.
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Accordingly, provided herein is a virus particle comprising a capsid
polypeptide
comprising (a) a VP1, VP2 or VP3 sequence of SEQ ID NO: 36, (b) a VP1, VP2 or
VP3
sequence comprising the mutation set of VAR-35 and having greater than 80%
(for example,
greater than 90% greater than 91%, greater than 92%, greater than 93%, greater
than 94%,
greater than 95%, greater than 96%, greater than 97%, greater than 98%,
greater than 99%)
identity to SEQ ID NO: 1, or (c) a VP1. VP2 or VP3 sequence comprising the
mutation set of
VAR-35 and having at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 additional
mutations, but fewer than 40,
39, 38, 37, 36, 35, 34, 33, 32 or 31 additional mutations relative to SEQ ID
NO: 1. In
embodiments, the capsid polypeptide comprises VP1, VP2 and VP3 sequences of
SEQ ID NO:
36. In embodiments, the virus particle comprises a nucleic acid molecule
comprising a
heterologous transgene, for example a heterologous transgene encoding a
product directed to an
ocular disorder. In embodiments, the heterologous transgene encodes an anti-
VEGF antibody or
antibody fragment, an anti-VEGF RNA inhibitory molecule, a RPE65 (e.g., human
RPE65)
protein, a ABCA4 (e.g., human ABCA4) protein or fragment thereof, a RLBP1
(e.g., human
RLBP1) protein or fragment thereof, a PDE6B (e.g.. human PDE6B) protein or
fragment thereof,
a RPGR (e.g., human RPGR) protein or fragment thereof or a ACHM3A or ACHM3B
(e.g.,
human ACHM3A or human ACHM3B) protein or fragment thereof. In embodiments, the
nucleic acid molecule of the virus particle further comprises one or more
regulatory elements,
e.g., comprises a promoter, e.g., a promoter operably linked to the
heterologous transgene and
which regulates expression from the heterologous transgene in a tissue of
interest. In
embodiments, the nucleic acid molecule of the virus particle further comprises
one or more of (a)
a dependoparvovirus ITR, (b) an intron, (c) an enhancer or repressor sequence,
(d) a stuffer
sequence, and (e) a polyA sequence.
Accordingly, provided herein is a virus particle comprising a capsid
polypeptide
comprising (a) a VP1, VP2 or VP3 sequence of SEQ ID NO: 37, (b) a VP1, VP2 or
VP3
sequence comprising the mutation set of VAR-36 and having greater than 80%
(for example,
greater than 90% greater than 91%, greater than 92%, greater than 93%, greater
than 94%,
greater than 95%, greater than 96%, greater than 97%, greater than 98%,
greater than 99%)
identity to SEQ ID NO: 1, or (c) a VP1. VP2 or VP3 sequence comprising the
mutation set of
VAR-36 and having at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 additional
mutations, but fewer than 40,
39, 38, 37, 36, 35, 34, 33, 32 or 31 additional mutations relative to SEQ ID
NO: 1. In
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embodiments, the capsid polypeptide comprises VP I, VP2 and VP3 sequences of
SEQ ID NO:
37. In embodiments, the virus particle comprises a nucleic acid molecule
comprising a
heterologous transgene, for example a heterologous transgene encoding a
product directed to an
ocular disorder. In embodiments, the heterologous transgene encodes an anti-
VEGF antibody or
antibody fragment, an anti-VEGF RNA inhibitory molecule, a RPE65 (e.g., human
RPE65)
protein, a ABCA4 (e.g., human ABCA4) protein or fragment thereof, a RLBP1
(e.g., human
RLBP1) protein or fragment thereof, a PDE6B (e.g.. human PDE6B) protein or
fragment thereof,
a RPGR (e.g., human RPGR) protein or fragment thereof or a ACHM3A or ACHM3B
(e.g.,
human ACHM3A or human ACHM3B) protein or fragment thereof. In embodiments, the
nucleic acid molecule of the virus particle further comprises one or more
regulatory elements,
e.g., comprises a promoter, e.g., a promoter operably linked to the
heterologous transgene and
which regulates expression from the heterologous transgene in a tissue of
interest. In
embodiments, the nucleic acid molecule of the virus particle further comprises
one or more of (a)
a dependoparvovirus ITR, (b) an intron, (c) an enhancer or repressor sequence,
(d) a stuffer
sequence, and (e) a polyA sequence.
Accordingly, provided herein is a virus particle comprising a capsid
polypeptide
comprising (a) a VP1, VP2 or VP3 sequence of SEQ ID NO: 38, (b) a VP1, VP2 or
VP3
sequence comprising the mutation set of VAR-37 and having greater than 80%
(for example,
greater than 90% greater than 91%, greater than 92%, greater than 93%, greater
than 94%,
greater than 95%, greater than 96%, greater than 97%, greater than 98%,
greater than 99%)
identity to SEQ ID NO: 1, or (c) a VP1. VP2 or VP3 sequence comprising the
mutation set of
VAR-37 and having at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 additional
mutations, but fewer than 40,
39, 38, 37, 36, 35, 34, 33, 32 or 31 additional mutations relative to SEQ ID
NO: 1. In
embodiments, the capsid polypeptide comprises VP1, VP2 and VP3 sequences of
SEQ ID NO:
38. In embodiments, the virus particle comprises a nucleic acid molecule
comprising a
heterologous transgene, for example a heterologous transgene encoding a
product directed to an
ocular disorder. In embodiments, the heterologous transgene encodes an anti-
VEGF antibody or
antibody fragment, an anti-VEGF RNA inhibitory molecule, a RPE65 (e.g., human
RPE65)
protein, a ABCA4 (e.g., human ABCA4) protein or fragment thereof, a RLBP1
(e.g., human
RLBP1) protein or fragment thereof, a PDE6B (e.g.. human PDE6B) protein or
fragment thereof,
a RPGR (e.g., human RPGR) protein or fragment thereof or a ACHM3A or ACHM3B
(e.g.,
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human ACHM3A or human ACHM3B) protein or fragment thereof. In embodiments, the
nucleic acid molecule of the virus particle further comprises one or more
regulatory elements,
e.g., comprises a promoter, e.g., a promoter operably linked to the
heterologous transgene and
which regulates expression from the heterologous transgene in a tissue of
interest. In
embodiments, the nucleic acid molecule of the virus particle further comprises
one or more of (a)
a dependoparvovirus ITR, (b) an intron, (c) an enhancer or repressor sequence,
(d) a stuffer
sequence, and (e) a polyA sequence.
Accordingly, provided herein is a virus particle comprising a capsid
polypeptide
comprising (a) a VP1, VP2 or VP3 sequence of SEQ ID NO: 39, (b) a VP1, VP2 or
VP3
sequence comprising the mutation set of VAR-38 and having greater than 80%
(for example,
greater than 90% greater than 91%, greater than 92%, greater than 93%, greater
than 94%,
greater than 95%, greater than 96%, greater than 97%, greater than 98%,
greater than 99%)
identity to SEQ ID NO: 1, or (c) a VP1. VP2 or VP3 sequence comprising the
mutation set of
VAR-38 and having at least 1, 2, 3, 4, 5, 6, 7. 8, 9 or 10 additional
mutations, but fewer than 40,
39, 38, 37, 36, 35, 34, 33, 32 or 31 additional mutations relative to SEQ ID
NO: 1. In
embodiments, the capsid polypeptide comprises VP1, VP2 and VP3 sequences of
SEQ ID NO:
39. In embodiments, the virus particle comprises a nucleic acid molecule
comprising a
heterologous transgene, for example a heterologous transgene encoding a
product directed to an
ocular disorder. In embodiments, the heterologous transgene encodes an anti-
VEGF antibody or
antibody fragment, an anti-VEGF RNA inhibitory molecule, a RPE65 (e.g., human
RPE65)
protein, a ABCA4 (e.g., human ABCA4) protein or fragment thereof, a RLBPI
(e.g., human
RLBP1) protein or fragment thereof, a PDE6B (e.g.. human PDE6B) protein or
fragment thereof,
a RPGR (e.g., human RPGR) protein or fragment thereof or a ACHM3A or ACHM3B
(e.g.,
human ACHM3A or human ACHM3B) protein or fragment thereof. In embodiments, the
nucleic acid molecule of the virus particle further comprises one or more
regulatory elements,
e.g., comprises a promoter, e.g., a promoter operably linked to the
heterologous transgene and
which regulates expression from the heterologous transgene in a tissue of
interest. In
embodiments, the nucleic acid molecule of the virus particle further comprises
one or more of (a)
a dependoparvovirus ITR, (b) an intron, (c) an enhancer or repressor sequence,
(d) a stuffer
sequence, and (e) a polyA sequence.
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Accordingly, provided herein is a virus particle comprising a capsid
polypeptide
comprising (a) a VP1, VP2 or VP3 sequence of SEQ ID NO: 40, (b) a VP1, VP2 or
VP3
sequence comprising the mutation set of VAR-39 and having greater than 80%
(for example,
greater than 90% greater than 91%, greater than 92%, greater than 93%, greater
than 94%,
greater than 95%, greater than 96%, greater than 97%, greater than 98%,
greater than 99%)
identity to SEQ ID NO: 1, or (c) a VP1. VP2 or VP3 sequence comprising the
mutation set of
VAR-39 and having at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 additional
mutations, but fewer than 40,
39, 38, 37, 36, 35, 34, 33, 32 or 31 additional mutations relative to SEQ ID
NO: 1. In
embodiments, the capsid polypeptide comprises VP1, VP2 and VP3 sequences of
SEQ ID NO:
40. In embodiments, the virus particle comprises a nucleic acid molecule
comprising a
heterologous transgene, for example a heterologous transgene encoding a
product directed to an
ocular disorder. In embodiments, the heterologous transgene encodes an anti-
VEGF antibody or
antibody fragment, an anti-VEGF RNA inhibitory molecule, a RPE65 (e.g., human
RPE65)
protein, a ABCA4 (e.g., human ABCA4) protein or fragment thereof, a RLBP1
(e.g., human
RLBP1) protein or fragment thereof, a PDE6B (e.g.. human PDE6B) protein or
fragment thereof,
a RPGR (e.g., human RPGR) protein or fragment thereof or a ACHM3A or ACHM3B
(e.g.,
human ACHM3A or human ACHM3B) protein or fragment thereof. In embodiments, the
nucleic acid molecule of the virus particle further comprises one or more
regulatory elements,
e.g., comprises a promoter, e.g., a promoter operably linked to the
heterologous transgene and
which regulates expression from the heterologous transgene in a tissue of
interest. In
embodiments, the nucleic acid molecule of the virus particle further comprises
one or more of (a)
a dependoparvovirus ITR, (b) an intron, (c) an enhancer or repressor sequence,
(d) a stuffer
sequence, and (e) a polyA sequence.
Accordingly, provided herein is a virus particle comprising a capsid
polypeptide
comprising (a) a VP1, VP2 or VP3 sequence of SEQ ID NO: 41, (b) a VP1, VP2 or
VP3
sequence comprising the mutation set of VAR-40 and having greater than 80%
(for example,
greater than 90% greater than 91%, greater than 92%, greater than 93%, greater
than 94%,
greater than 95%, greater than 96%, greater than 97%, greater than 98%,
greater than 99%)
identity to SEQ ID NO: 1, or (c) a VP1. VP2 or VP3 sequence comprising the
mutation set of
VAR-40 and having at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 additional
mutations, but fewer than 40,
39, 38, 37, 36, 35, 34, 33, 32 or 31 additional mutations relative to SEQ ID
NO: 1. In
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embodiments, the capsid polypeptide comprises VP I, VP2 and VP3 sequences of
SEQ ID NO:
41. In embodiments, the virus particle comprises a nucleic acid molecule
comprising a
heterologous transgene, for example a heterologous transgene encoding a
product directed to an
ocular disorder. In embodiments, the heterologous transgene encodes an anti-
VEGF antibody or
antibody fragment, an anti-VEGF RNA inhibitory molecule, a RPE65 (e.g., human
RPE65)
protein, a ABCA4 (e.g., human ABCA4) protein or fragment thereof, a RLBP1
(e.g., human
RLBP1) protein or fragment thereof, a PDE6B (e.g.. human PDE6B) protein or
fragment thereof,
a RPGR (e.g., human RPGR) protein or fragment thereof or a ACHM3A or ACHM3B
(e.g.,
human ACHM3A or human ACHM3B) protein or fragment thereof. In embodiments, the
nucleic acid molecule of the virus particle further comprises one or more
regulatory elements,
e.g., comprises a promoter, e.g., a promoter operably linked to the
heterologous transgene and
which regulates expression from the heterologous transgene in a tissue of
interest. In
embodiments, the nucleic acid molecule of the virus particle further comprises
one or more of (a)
a dependoparvovirus ITR, (b) an intron, (c) an enhancer or repressor sequence,
(d) a stuffer
sequence, and (e) a polyA sequence.
Accordingly, provided herein is a virus particle comprising a capsid
polypeptide
comprising (a) a VP1, VP2 or VP3 sequence of SEQ ID NO: 42, (b) a VP1, VP2 or
VP3
sequence comprising the mutation set of VAR-41 and having greater than 80%
(for example,
greater than 90% greater than 91%, greater than 92%, greater than 93%, greater
than 94%,
greater than 95%, greater than 96%, greater than 97%, greater than 98%,
greater than 99%)
identity to SEQ ID NO: 1, or (c) a VP1. VP2 or VP3 sequence comprising the
mutation set of
VAR-41 and having at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 additional
mutations, but fewer than 40,
39, 38, 37, 36, 35, 34, 33, 32 or 31 additional mutations relative to SEQ ID
NO: 1. In
embodiments, the capsid polypeptide comprises VP1, VP2 and VP3 sequences of
SEQ ID NO:
42. In embodiments, the virus particle comprises a nucleic acid molecule
comprising a
heterologous transgene, for example a heterologous transgene encoding a
product directed to an
ocular disorder. In embodiments, the heterologous transgene encodes an anti-
VEGF antibody or
antibody fragment, an anti-VEGF RNA inhibitory molecule, a RPE65 (e.g., human
RPE65)
protein, a ABCA4 (e.g., human ABCA4) protein or fragment thereof, a RLBP1
(e.g., human
RLBP1) protein or fragment thereof, a PDE6B (e.g.. human PDE6B) protein or
fragment thereof,
a RPGR (e.g., human RPGR) protein or fragment thereof or a ACHM3A or ACHM3B
(e.g.,
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human ACHM3A or human ACHM3B) protein or fragment thereof. In embodiments, the
nucleic acid molecule of the virus particle further comprises one or more
regulatory elements,
e.g., comprises a promoter, e.g., a promoter operably linked to the
heterologous transgene and
which regulates expression from the heterologous transgene in a tissue of
interest. In
embodiments, the nucleic acid molecule of the virus particle further comprises
one or more of (a)
a dependoparvovirus ITR, (b) an intron, (c) an enhancer or repressor sequence,
(d) a stuffer
sequence, and (e) a polyA sequence.
Accordingly, provided herein is a virus particle comprising a capsid
polypeptide
comprising (a) a VP1, VP2 or VP3 sequence of SEQ ID NO: 43, (b) a VP1, VP2 or
VP3
sequence comprising the mutation set of VAR-42 and having greater than 80%
(for example,
greater than 90% greater than 91%, greater than 92%, greater than 93%, greater
than 94%,
greater than 95%, greater than 96%, greater than 97%, greater than 98%.
greater than 99%)
identity to SEQ ID NO: 1, or (c) a VP1. VP2 or VP3 sequence comprising the
mutation set of
VAR-42 and having at least 1, 2, 3, 4, 5, 6, 7. 8, 9 or 10 additional
mutations, but fewer than 40,
39, 38, 37, 36, 35, 34, 33, 32 or 31 additional mutations relative to SEQ ID
NO: 1. In
embodiments, the capsid polypeptide comprises VP1, VP2 and VP3 sequences of
SEQ ID NO:
43. In embodiments, the virus particle comprises a nucleic acid molecule
comprising a
heterologous transgene, for example a heterologous transgene encoding a
product directed to an
ocular disorder. In embodiments, the heterologous transgene encodes an anti-
VEGF antibody or
antibody fragment, an anti-VEGF RNA inhibitory molecule, a RPE65 (e.g., human
RPE65)
protein, a ABCA4 (e.g., human ABCA4) protein or fragment thereof, a RLBPI
(e.g., human
RLBP1) protein or fragment thereof, a PDE6B (e.g.. human PDE6B) protein or
fragment thereof,
a RPGR (e.g., human RPGR) protein or fragment thereof or a ACHM3A or ACHM3B
(e.g.,
human ACHM3A or human ACHM3B) protein or fragment thereof. In embodiments, the
nucleic acid molecule of the virus particle further comprises one or more
regulatory elements,
e.g., comprises a promoter, e.g., a promoter operably linked to the
heterologous transgene and
which regulates expression from the heterologous transgene in a tissue of
interest. In
embodiments, the nucleic acid molecule of the virus particle further comprises
one or more of (a)
a dependoparvovirus ITR, (b) an intron, (c) an enhancer or repressor sequence,
(d) a stuffer
sequence, and (e) a polyA sequence.
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Accordingly, provided herein is a virus particle comprising a capsid
polypeptide
comprising (a) a VP1, VP2 or VP3 sequence of SEQ ID NO: 44, (b) a VP1, VP2 or
VP3
sequence comprising the mutation set of VAR-43 and having greater than 80%
(for example,
greater than 90% greater than 91%, greater than 92%, greater than 93%, greater
than 94%,
greater than 95%, greater than 96%, greater than 97%, greater than 98%,
greater than 99%)
identity to SEQ ID NO: 1, or (c) a VP1. VP2 or VP3 sequence comprising the
mutation set of
VAR-43 and having at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 additional
mutations, but fewer than 40,
39, 38, 37, 36, 35, 34, 33, 32 or 31 additional mutations relative to SEQ ID
NO: 1. In
embodiments, the capsid polypeptide comprises VP1, VP2 and VP3 sequences of
SEQ ID NO:
44. In embodiments, the virus particle comprises a nucleic acid molecule
comprising a
heterologous transgene, for example a heterologous transgene encoding a
product directed to an
ocular disorder. In embodiments, the heterologous transgene encodes an anti-
VEGF antibody or
antibody fragment, an anti-VEGF RNA inhibitory molecule, a RPE65 (e.g., human
RPE65)
protein, a ABCA4 (e.g., human ABCA4) protein or fragment thereof, a RLBP1
(e.g., human
RLBP1) protein or fragment thereof, a PDE6B (e.g.. human PDE6B) protein or
fragment thereof,
a RPGR (e.g., human RPGR) protein or fragment thereof or a ACHM3A or ACHM3B
(e.g.,
human ACHM3A or human ACHM3B) protein or fragment thereof. In embodiments, the
nucleic acid molecule of the virus particle further comprises one or more
regulatory elements,
e.g., comprises a promoter, e.g., a promoter operably linked to the
heterologous transgene and
which regulates expression from the heterologous transgene in a tissue of
interest. In
embodiments, the nucleic acid molecule of the virus particle further comprises
one or more of (a)
a dependoparvovirus ITR, (b) an intron, (c) an enhancer or repressor sequence,
(d) a stuffer
sequence, and (e) a polyA sequence.
Accordingly, provided herein is a virus particle comprising a capsid
polypeptide
comprising (a) a VP1, VP2 or VP3 sequence of SEQ ID NO: 45, (b) a VP1, VP2 or
VP3
sequence comprising the mutation set of VAR-44 and having greater than 80%
(for example,
greater than 90% greater than 91%, greater than 92%, greater than 93%, greater
than 94%,
greater than 95%, greater than 96%, greater than 97%, greater than 98%,
greater than 99%)
identity to SEQ ID NO: 1, or (c) a VP1. VP2 or VP3 sequence comprising the
mutation set of
VAR-44 and having at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 additional
mutations, but fewer than 40,
39, 38, 37, 36, 35, 34, 33, 32 or 31 additional mutations relative to SEQ ID
NO: 1. In
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embodiments, the capsid polypeptide comprises VP I, VP2 and VP3 sequences of
SEQ ID NO:
45. In embodiments, the virus particle comprises a nucleic acid molecule
comprising a
heterologous transgene, for example a heterologous transgene encoding a
product directed to an
ocular disorder. In embodiments, the heterologous transgene encodes an anti-
VEGF antibody or
antibody fragment, an anti-VEGF RNA inhibitory molecule, a RPE65 (e.g., human
RPE65)
protein, a ABCA4 (e.g., human ABCA4) protein or fragment thereof, a RLBP1
(e.g., human
RLBP1) protein or fragment thereof, a PDE6B (e.g.. human PDE6B) protein or
fragment thereof,
a RPGR (e.g., human RPGR) protein or fragment thereof or a ACHM3A or ACHM3B
(e.g.,
human ACHM3A or human ACHM3B) protein or fragment thereof. In embodiments, the
nucleic acid molecule of the virus particle further comprises one or more
regulatory elements,
e.g., comprises a promoter, e.g., a promoter operably linked to the
heterologous transgene and
which regulates expression from the heterologous transgene in a tissue of
interest. In
embodiments, the nucleic acid molecule of the virus particle further comprises
one or more of (a)
a dependoparvovirus ITR, (b) an intron, (c) an enhancer or repressor sequence,
(d) a stuffer
sequence, and (e) a polyA sequence.
Accordingly, provided herein is a virus particle comprising a capsid
polypeptide
comprising (a) a VP1, VP2 or VP3 sequence of SEQ ID NO: 46, (b) a VP1, VP2 or
VP3
sequence comprising the mutation set of VAR-45 and having greater than 80%
(for example,
greater than 90% greater than 91%, greater than 92%, greater than 93%, greater
than 94%,
greater than 95%, greater than 96%, greater than 97%, greater than 98%,
greater than 99%)
identity to SEQ ID NO: 1, or (c) a VP1. VP2 or VP3 sequence comprising the
mutation set of
VAR-45 and having at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 additional
mutations, but fewer than 40,
39, 38, 37, 36, 35, 34, 33, 32 or 31 additional mutations relative to SEQ ID
NO: 1. In
embodiments, the capsid polypeptide comprises VP1, VP2 and VP3 sequences of
SEQ ID NO:
46. In embodiments, the virus particle comprises a nucleic acid molecule
comprising a
heterologous transgene, for example a heterologous transgene encoding a
product directed to an
ocular disorder. In embodiments, the heterologous transgene encodes an anti-
VEGF antibody or
antibody fragment, an anti-VEGF RNA inhibitory molecule, a RPE65 (e.g., human
RPE65)
protein, a ABCA4 (e.g., human ABCA4) protein or fragment thereof, a RLBP1
(e.g., human
RLBP1) protein or fragment thereof, a PDE6B (e.g.. human PDE6B) protein or
fragment thereof,
a RPGR (e.g., human RPGR) protein or fragment thereof or a ACHM3A or ACHM3B
(e.g.,
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human ACHM3A or human ACHM3B) protein or fragment thereof. In embodiments, the
nucleic acid molecule of the virus particle further comprises one or more
regulatory elements,
e.g., comprises a promoter, e.g., a promoter operably linked to the
heterologous transgene and
which regulates expression from the heterologous transgene in a tissue of
interest. In
embodiments, the nucleic acid molecule of the virus particle further comprises
one or more of (a)
a dependoparvovirus ITR, (b) an intron, (c) an enhancer or repressor sequence,
(d) a stuffer
sequence, and (e) a polyA sequence.
The disclosure is further directed, in part, to a method of delivering a
payload to a
subject, e.g., an animal or human subject. In some embodiments, a method of
delivering a
payload to a subject comprises administering to the subject a
dependoparvovirus particle
comprising a variant polypeptide (e.g., described herein) comprising the
payload, e.g., in a
quantity and for a time sufficient to deliver the payload. In some
embodiments, the
dependoparvovirus particle is a dependoparvovirus particle described herein
and comprises a
payload described herein. In some embodiments, the particle delivers the
payload to the eye. In
some embodiments, the delivery to the eye is increased as compared to a
particle without the
variant capsid polypeptide or as compared to a wild-type capsid polypeptide.
Methods of treatment
The disclosure is directed, in part, to a method of treating a disease or
condition in a
subject, e.g., an animal or human subject. As used herein, the term "treating
a disease or
condition" refers to treating a manifest disease or condition, for example,
where the subject is
already suffering from one or more symptoms of the disease or condition, or
refers to treating a
pre-manifest disease or condition, for example, where the subject is
identified as having a disease
or condition but is not yet exhibiting one or more symptoms of the disease or
condition. Pre-
manifest conditions may be identified by, for example, genetic testing. In
some embodiments, a
method of treating a disease or condition in a subject comprises administering
to the subject a
dependoparvovirus particle comprising a variant polypeptide described herein,
e.g., comprising a
payload described herein. In some embodiments, the dependoparvovirus particle,
which
comprises a variant polypeptide, comprising a payload described herein is
administered in an
amount and/or time effective to treat the disease or condition. In some
embodiments, the payload
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is a therapeutic product. In some embodiments, the payload is a nucleic acid,
e.g., encoding an
exogenous polypeptide.
The dependoparvovirus particles comprising a variant polypeptide described
herein or
produced by the methods described herein can be used to express one or more
therapeutic
proteins to treat various diseases or disorders. In some embodiments, the
disease or disorder is a
cancer, e.g., a cancer such as carcinoma, sarcoma, leukemia, lymphoma; or an
autoimmune
disease, e.g., multiple sclerosis. Non-limiting examples of carcinomas include
esophageal
carcinoma; bronchogenic carcinoma; colon carcinoma; colorectal carcinoma;
gastric carcinoma;
laepatocellular carcinoma; basal cell carcinoma, squamous cell carcinoma
(various tissues);
bladder carcinoma, including transitional cell carcinoma; lung carcinoma,
including small cell
carcinoma and non-small cell carcinoma of the lung; adrenocortical carcinoma;
sweat gland
carcinoma; sebaceous gland carcinoma; thyroid carcinoma; pancreatic carcinoma;
breast
carcinoma; ovarian carcinoma; prostate carcinoma; adenocarcinoma; papillary
carcinoma;
papillary adenocarcinoma; cystadenocarcinoma; medullary carcinoma; renal cell
carcinoma;
uterine carcinoma; testicular carcinoma; osteogenic carcinoma; ductal
carcinoma in situ or bile
duct carcinoma; choriocarcinoma; seminoma; embryonal carcinoma; Wilm's tumor;
cervical
carcinoma; epithelieal carcinoma; and nasopharyngeal carcinoma. Non-limiting
examples of
sarcomas include fibro sarcoma, myxosarcoma, lipo sarcoma, angiosarcoma,
endothelio sarcoma,
lymphangio sarcoma, chondrosarcoma, chordoma, osteogenic sarcoma,
osteosarcoma,
lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's sarcoma,
leiomyosarcoma,
rhabdomyosarcoma, and other soft tissue sarcomas. Non-limiting examples of
solid tumors
include ependymoma, pinealoma, hemangioblastoma, acoustic neuroma,
oligodendroglioma,
glioma, astrocytoma, medulloblastoma, craniopharyngioma, menangioma, melanoma,
neuroblastoma, and retinoblastoma. Non-limiting examples of leukemias include
chronic
myeloproliferative syndromes; T-cell CLL prolymphocytic leukemia, acute
myelogenous
leukemias; chronic lymphocytic leukemias, including B-cell CLL, hairy cell
leukemia; and acute
lymphoblastic leukemias. Examples of lymphomas include, but are not limited
to, B-cell
lymphomas, such as Burkitt's lymphoma; and Hodgkin's lymphoma.
In some embodiments, the disease or disorder is a genetic disorder. In some
embodiments, the genetic disorder is sickle cell anemia, Glycogen storage
diseases (GSD, e.g.,
GSD types I, II, III, IV, V, VI, VII, VIII, IX, X, XI, XII, XIII, and XIV),
cystic fibrosis,
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lysosomal acid lipase (LAL) deficiency 1, Tay-Sachs disease, Phenylketonuria,
Mucopolysaccharidoses, Galactosemia, muscular dystrophy (e.g., Duchenne
muscular
dystrophy), hemophilia such as hemophilia A (classic hemophilia) or hemophilia
B (Christmas
Disease), Wilson's disease, Fabry Disease, Gaucher Disease hereditary
angioedema (HAE), and
alpha 1 antitrypsin deficiency. Examples of other diseases or disorders are
provided above in the
"Methods of delivering a payload" section.
The dependoparvo virus particles comprising a variant polypeptide described
herein or
produced by the methods described herein can be used to express one or more
therapeutic
proteins to treat various diseases or disorders. In some embodiments, the
disease or disorder is a
disease or disorder of the eye, for example, retinitis pigmentosa; macular
degeneration (e.g.; wet
age-related macular degeneration), optic neuritis; Leber's congenital
amaurosis; Leber's
hereditary optic neuropathy; achromatopsia; X-linked retinoschisis; optic
neuritis;
choroideremia; optic atrophy; retinal cone dystrophy; retinopathy;
retinoblastoma; glaucoma;
Bardet-Biedl syndrome; Usher syndrome; aniridia; Friedreich's ataxia;
vitelliform macular
dystrophy; retinoblastoma; Stargardt disease; Charcot-Marie-Tooth disease;
Fuch's dystrophy;
propionic acidemia; or color blindness; corneal dystrophy; keratoconus; night
blindness; dry eye;
Bardet-Biedl syndrome; Batten's Disease; Bietti's Crystalline Dystrophy;
chorioretinal atrophy;
chorioretinal degeneration; cone or cone-rod dystrophies (autosomal dominant,
autosomal
recessive, and X-linked), congenital stationary night blindness (autosomal
dominant, autosomal
recessive, and X-linked); disorders of color vision, including achromatopsia
(including ACHM2,
ACHM3, ACHM4, and ACHM5), protanopia, deuteranopia, and tritanopia;
Friedreich's ataxia;
Leber's congenital amaurosis (autosomal dominant and autosomal recessive),
including, but not
limited to, LCA1, LCA2, LCA3, LCA4, LCA6, LCA7, LCA8, LCA12, and LCA15;
Leber's
Hereditary Optic Neuropathy; macular dystrophy (autosomal dominant and auto
somal recessive),
including, but not limited to, acute macular degeneration, Best vitelliform
macular dystrophy,
pattern dystrophy, North Carolina Macular Dystrophy, inherited drusen,
Sorsby's fundus
dystrophy, malattia levantanese, and genetically-determined retinopathy of
prematurity; ocular-
retinal developmental disease; ocular albinism; optic atrophies (autosomal
dominant, autosomal
recessive, and X-linked); retinitis pigmentosa (autosomal dominant, autosomal
recessive, X-
linked, and mitochondrially- inherited traits), examples of which include RP1,
RP2, RP3, RP10,
RP20, RP38, RP40, and RP43; X-linked retinoschisis; Stargardt disease; and
Usher syndrome,
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including, but not limited to, USH1B, USH1C, USH1D, USH IF, USH1G, USH2A,
USH2C,
USH2D, AND USH3. Examples of complex genetic diseases include, but are not
limited to,
glaucoma (open angle, angle-closure, low-tension, normal-tension, congenital,
neovascular,
pigmentary, pseudoexfoliation); age-related and other forms of macular
degeneration, both
exudative and non-exudative forms (autosomal dominant and autosomal
recessive), such as acute
macular degeneration, vitelliform macular degeneration; retinopathy of
prematurity; and Vogt
Koyanagi-Harada (VKH) syndrome. Examples of acquired diseases include, but are
not limited
to, acute macular neuroretinopathy; anterior ischemic optic neuropathy and
posterior ischemic
optic neuropathy; Behcet's disease; branch retinal vein occlusion; choroidal
neovascularization;
diabetic retinopathy, including proliferative diabetic retinopathy and
associated complications;
diabetic uveitis; edema, such as macular edema, cystoid macular edema and
diabetic macular
edema; epiretinal membrane disorders; macular telangiectasia; multithcal
choroiditis; non-
retinopathy diabetic retinal dysfunction; ocular tumors; optic atrophies;
retinal detachment;
retinal disorders, such as central retinal vein occlusion, proliferative
vitreoretinopathy (PVR),
retinal arterial and venous occlusive disease, vascular occlusion, uveitic
retinal disease; uveal
effusion; retinal infective and infiltrative disease; optic nerve diseases
such as acquired optic
atrophy. Examples of traumatic injuries include, but are not limited to,
histoplasmosis; optic
nerve trauma; ocular trauma which affects a posterior ocular site or location;
retinal trauma; viral
infection of the eye; viral infection of the optic nerve; a posterior ocular
condition caused by or
influenced by an ocular laser treatment; posterior ocular conditions caused by
or influenced by a
photodynamic therapy; photocoagulation, radiation retinopathy; and sympathetic
ophthalmia.
In some embodiments, administration of a dependoparvovirus particle comprising
a
variant polypeptide and comprising a payload (e.g., a transgene) to a subject
induces expression
of the payload (e.g., transgene) in a subject. In some embodiments, the
expression is induced in
the eye. In some embodiments, the production is increased in the eye as
compared to a similar
particle with the wild-type capsid protein. The amount of a payload, e.g.,
transgene, e.g.,
heterologous protein, e.g., therapeutic polypeptide, expressed in a subject
(e.g., the serum of the
subject) can vary. For example, in some embodiments the payload, e.g., protein
or RNA product
of a transgene, can be expressed in the serum of the subject in the amount of
less than about 5
jig/ml. For example, in some embodiments the payload, e.g., protein or RNA
product of a
transgene, can be expressed in the serum of the subject in the amount of at
least about 9 pg/ml, at
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least about 10 pg/ml, at least about 50 pg/ml, at least about 100 g/ml, at
least about 200 jig/ml,
at least about 300 g/ml, at least about 400 jig/ml, at least about 500
jig/ml, at least about 600
pg/ml, at least about 700 pg/ml, at least about 800 g/ml, at least about 900
jig/ml, or at least
about 1000 jig/mi. In some embodiments, the payload, e.g., protein or RNA
product of a
transgene, is expressed in the serum of the subject in the amount of about 9
jig/nil, about 10
g/ml. about 50 jig/ml, about 100 jig/ml, about 200 g/ml, about 300 g/ml,
about 400 g/ml,
about 500 g/ml, about 600 g/ml. about 700 jig/ml, about 800 jig/ml, about
900 g/ml, about
1000 jig/ml, about 1500 g/ml, about 2000 g/ml, about 2500 Rg/ml, or a range
between any two
of these values.
In some embodiments, the a dependoparvovirus particle comprising a variant
polypeptide
and comprising a payload (e.g., a transgene) is administered to a subject via
an injection. In some
embodiments, the injection is a systemic injection, for example, intravenous,
intraarterial,
intramuscular, or subcutaneous injection. In some embodiments, the injection
is an injection to
the eye. In some embodiments, the injection is an intravitreal injection,
intraorbital injection,
retro-orbital injection, suprachoroidal injection, subretinal injection,
subconjunctivital injection,
or intracameral injection. In some embodiments, the injection is an
intravitreal injection. In some
embodiments, the injection is an intraorbital injection. In some embodiments,
the injection is a
retro-orbital injection. In some embodiments, the injection is a
suprachoroidal injection. In some
embodiments, the injection is a subretinal injection. In some embodiments, the
injection is a
subconjunctivital injection. In some embodiments, the injection is an
intracameral injection.
Sequences disclosed herein may be described in terms of percent identity. A
person of
skill will understand that such characteristics involve alignment of two or
more sequences.
Alignments may be performed using any of a variety of publicly or commercially
available
Multiple Sequence Alignment Programs, such as "Clustal W", accessible via the
Internet. As
another example, nucleic acid sequences may be compared using FASTA, a program
in GCG
Version 6.1. FASTA provides alignments and percent sequence identity of the
regions of the best
overlap between the query and search sequences. For instance, percent identity
between nucleic
acid sequences may be determined using FASTA with its default parameters as
provided in GCG
Version 6.1, herein incorporated by reference. Similar programs are available
for amino acid
sequences, e.g., the "Clustal X" program. Additional sequence alignment tools
that may be used
are provided by (protein sequence alignment:
(http://www.ebi.ac.uk/Tools/psa/emboss needle/))
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and (nucleic acid alignment; http://www.ebi.ac.uk/Tools/psa/emboss
neecile/nucleotide.html)).
Generally, any of these programs may be used at default settings, although one
of skill in the art
can alter these settings as needed. Alternatively, one of skill in the art can
utilize another
algorithm or computer program which provides at least the level of identity or
alignment as that
provided by the referenced algorithms and programs. Sequences disclosed herein
may further be
described in terms of edit distance. The minimum number of sequence edits
(i.e., additions,
substitutions, or deletions of a single base or nucleotide) which change one
sequence into another
sequence is the edit distance between the two sequences. In some embodiments,
the distance
between two sequences is calculated as the Levenshtein distance.
All publications, patent applications, patents, and other publications and
references (e.g.,
sequence database reference numbers) cited herein are incorporated by
reference in their entirety.
For example, all GenBank, Unigene, and Entrez sequences referred to herein,
e.g., in any Table
herein, are incorporated by reference. Unless otherwise specified, the
sequence accession
numbers specified herein, including in any Table herein, refer to the database
entries current as
of August 21, 2020. When one gene or protein references a plurality of
sequence accession
numbers, all of the sequence variants are encompassed.
The invention is further illustrated by the following examples. The examples
are provided
for illustrative purposes only and are not to be construed as limiting the
scope or content of the
invention in any way.
EXAMPLES
Example 1
Library Creation
A library of 2.5E5 capsid variants of wild-type AAV2 were designed and cloned
into
plasmids to create a library of plasmids encoding the capsid variants (library
for Library
Experiment 1). Experimental results from Library Experiment 1 were assessed
and machine
learning models trained on this and other data, and used to design two
separate libraries with 1E8
capsid variants of wild-type AAV2 each (libraries for Library Experiment 2),
These libraries are
significantly more diverse than the library tested in Library Experiment I.
Variants in one library
were designed to maximize posterior eye transduction (including for example
retina, macula,
non-macular retina, neural retina and ehoroid/RPE) (posterior eye library) and
variants in the
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other library were designed to maximize anterior eye transduction (including
for example tissues
of the trabecular meshwork. and Schtempi' s canal) (anterior eye library).
13otia libraries were
designed to include variants with which would produce virus particles. A
library of AAV variant
genomes encoding each variant's capsid and a unique capsid variant barcode
identifier was
cloned into three 'TR plasmid backbones as described previously (Ogden et in.
2019). Each
plasmid backbone contained a unique genomic identifier enabling analysis of
biodistribution and
transduction efficiencies via different routes of administration. The
libraries were produced via
transient triple transfection of adherent HEK293T followed by iodixanol
gradient purification.
In Vitro Evaluation of Library
Data was prepared as described below. To measure each variant's packaging
efficiency
(or "production"), barcodes from. vector genomes in the plasmid and produced
AAV library were
prepared for illumina sequencing using two rounds of PCR. Production
efficiency, normalized
for presence in the input plasmid library, for each variant is expressed by
comparing bareode
sequencing levels for each variant in the produced vector pool to the bareode
sequence levels for
each variant in the input plasmid library used to create the vector pool. The
measurements of
variant frequency in the vector library also enable downstream normalization
of biodistribution
and transduction measurements by variant frequency in the input vector
library. Production
efficiency for variants in Library Experiment 1 is reported in Table 1, and
each reported value is
reported as the 1og2 production relative to the production of wild-type AAV2.
Production
efficiency for variants in the posterior eye library and anterior eye library
for Library Experiment
2 are reported in Tables 6-9 and Figure 3 and Figure 4, and each reported
value is reported as the
1og2 production relative to the production of wild-type AAV2.
In Vivo Evaluation of Library in Non-Human Primate
Library Experiment 1
All NHP experiments were conducted in accordance with institutional policies
and NIH
guidelines. One young adult male and one young adult female cynomolgus macaque
(Macaca
fascicular's) weighing 2.4-2.9 kg seronegative for anti-AAV2 neutralizing
antibodies (serum
NAb titers <1:20 based on in vitro NAb assay) were selected for the study.
Prior to test article
administrations samples of blood, aqueous humor (50 pL) and vitreous humor (up
to 50 plr)
were collected. The animals were anesthetized with ketamine and
dexmedetomidine and received
intravitreal (IVT; 4.8E11 vg/eye in 50 pL), intracameral (IC; 8.5E11 vg/eye in
50 pL) and
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intravenous (IV; 1.8-2.5E13 vg/kg) injections of the vector libraries. During
the in-life period the
animals were monitored for signs of ocular inflammation via indirect
ophthalmoscopy and slit-
lamp biomicroscopy and treated with weekly IM injections of steroids
(methylprednisolone, 40-
80 mg) and topical steroids (Durezol), and atropine as needed according to the
animal facility's
SOPs and recommendations from the veterinarian. Serum samples were collected
at 1 h, 4 h and
24 h, and weekly after the injections. The animals were sacrificed 4 weeks
after the injections
and tissues were collected for biodistribution and transduction analyses.
Library Experiment 2
The evaluations of anterior eye library variants and posterior eye library
variants in
Library Experiment 2 were carried out in Cynomolgus macaques. Both eyes of two
non-naive
animals with low serum anti-AAV2 NAb titers (<1:10) received intravitreal
injections of the
posterior eye library and intracameral injections of the anterior eye library
at total doses of
7.3E11 vg/eye (3.9E11 vg for IC, 3.4E11 vg for IVT), with both libraries
injected via both routes
of administration in each eye. Ophthalmic examinations were performed weekly
during the 4
week in-life period to monitor levels of ocular inflammation.
Tissue Processing and Data Analysis for Library Experiment 1 and Library
Experiment 2
Retinas and trabecular meshwork were dissected as shown in FIG.1. A list of
other tissue
samples collected is shown in Table 10. All samples were collected into
RNAlater (Sigma-
Aldrich) and incubated overnight at RT, after which the RNAlater was drained
and samples
were frozen at -80 C. In addition, samples of aqueous humor, vitreous humor,
serum, and
cerebrospinal fluid were collected at necropsy and stored at -80 C.
Table 10. List of tissues collected.
Tissue
Adrenal gland
brain (cortical slices) coronal axis
dorsal root ganglion (cervical)
dorsal root ganglion (thoracic)
dorsal root ganglion (lumbar)
gonad (testes and ovaries)
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heart, basal (left atrium)
heart, apex
heart, right ventricle
kidney
liver
Lung (superior lobe)
lymph nodes, cervical
skeletal muscle, bicep brachii
skeletal muscle, diaphragm
skeletal muscle, quadriceps
spinal cord (cervical)
spinal cord (thoracic)
spinal cord (lumbar)
spleen
For biodistribution and transduction analyses, total DNA and RNA was extracted
from
tissue samples with Trizol/chloroform and isopropanol precipitation. RNA
samples were treated
with TURBO DNase (Invitrogen). Reverse transcription was done with Protoscript
II Reverse
Transcriptase (NEB) with primers that were specific to the vector transgene
and included unique
molecular identifiers (UMIs). Control reactions lacking the reverse
transcriptase enzyme (-RT
control) were also prepared. Quantification of biodistribution and
transduction was done with
Luna Universal Probe qPCR Master Mix (NEB) using primers and probes specific
to the
transgene construct. Finally, samples were prepared for next-generation
sequencing by
amplifying the transgene barcode regions with primers compatible with Illumina
NOS platform
and sequenced with NextSeq 550 (I1lumina).
After sequencing, the barcode tags were extracted from reads with the expected
amplicon
structure, and the abundance (number of reads or number of UMIs) of each
barcode was
recorded. Analyses were restricted to the set of barcodes that were present in
the input plasmid
sample and that did not contain errors in the variant sequence, as measured by
a separate
sequencing assay that targeted the variant regions of the input plasmid
sample.
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To aggregate packaging replicates, the read counts from replicate virus
production
samples were summed. To aggregate transduction samples, the UMI counts from
samples from
the same tissue were summed.
Virus packaging, biodistribution and transduction of tissue were calculated
using a
Bayesian model with aggregated production, biodistribution and/or transduction
samples as the
input. Briefly, probabilistic programming and stochastic variational inference
were used to
model the measurement process and sources of decoupling (e.g., cross-
packaging, template
switching, and errors in DNA synthesis) between the actual test virus
particles and their designed
sequences, and to calculate virus production, biodistribution and transduction
(in various tissue
samples), and error rates. The output was the 1og2-transformed mean of the
calculated
distribution relative to the wild-type (WT) AAV2. Thus, positive values
indicate better
performance than WT for the measured property, and negative values indicate
worse-than-WT
performance. Transduction and biodistribution for Library Experiment 1 is
reported in Table 1
(transduction) and Table 5 (biodistribution). Transduction and biodistribution
for Library
Experiment 2 is reported in Tables 6-9. Where indicated, macula transduction
and biodistribution
refers to measurements taken from tissues consisting of the neural retina
layer of the macula.
Retina or non-macula retina transduction and biodistribution refers to
measurements taken from
tissues consisting of the neural retina layer of the non-macular retina areas
of the eye.
Measurements including the choroid and/or RPE are taken from tissues
consisting of the choroid
layer of the whole retina. Without being bound by theory, because the
complexity of the libraries
Ihr this experiment were high relative to the total overall dose (1E8 variants
with total doses of
approximately 7E11 vg/eye), relative transduction and biodistribution values
from Library
Experiment 2 are compressed and, therefore, represent an underrepresentation
of the relative
transduction and biodistribution rates. The results demonstrate however, that
the relative rank
ordering of the variants that were included in both Library Experiment 1 and
Library Experiment
2 was consistent (Spearman correlation = 0.72), confirming the top variants as
having significant
transduction (for example, better than wild-type AA.V2) improvements. Variants
from this
experiment are included in a follow-on library experiment of similar
complexity to Library
Experiment 1 (e.g., 1-2E5 variants per library), and as d.escribed in Example
2, and properties are
confirmed as described herein for Library Experiment 1 and in Example 2.
Library Experiment I Results
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To assess efficiency of retinal transduction, measurements were obtained in
both eyes of
two animals across retinal regions. Data quality was ascertained through the
segregation of
negative controls and correlation of measurements between tissue replicates
and animals.
Aggregated (across eyes and animals) biodistribution and transduction
measurements in eye
regions of interest for virus particles comprising the capsid polypeptides
described herein are
provided in Table 1 and Table 5. Of note, variants with 20-60x improved
transduction rates in
target retinal and/or trabecular meshwork tissue relative to WT controls were
discovered.
Interestingly, some improved retina transducers also displayed improved
transduction of the
trabecular meshwork following intracameral administration, but did not display
enhanced
transduction of the liver following TV administration, indicating a
specificity for tissues of the
eye. A subset of variants with improvements specifically in peripheral retina
or macula
transduction were also identified. In addition, the library was assessed for
other relevant
parameters, such as productivity of individual variants, transduction
efficiency in non-ocular
tissues, and biodistribution in ocular and non-ocular tissues. In conclusion,
these results
demonstrate the power of machine learning models in the design of AAV variants
with improved
clinically relevant properties.
Library Experiment 2 Results
Production, transduction and biodistribution measurements for variants from
Library Experiment
1 that were included in Library Experiment 2 are shown in Figure 3 (IVT
administration) and
Figure 4 (IC administration). All values are log(2) relative to wt_AAV2.
Columns labeled "_std"
provide the 95% confidence intervals for the associated biodistribution or
transduction
measurements reported immediately to the left of the "_std" columns.
"Retina_macula" provides
measurements from aggregation of the neural retina layer of all macula samples
collected.
"Retina_non macula" provides measurements from aggregation of the neural
retina layer of all
non-macular posterior eye regions collected. In Figure 4, "choroid" and
"retina" provide
measurements from aggregation of the choroid layer or the neural retina layer
across all posterior
eye samples.
Example 2: Medium ThrouPhput Experiment
In vivo Evaluation of Medium-Throughput Library in Non-Human Primate
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The virus particles comprising a selection of the variant capsids provided in
Table 2
(sequences) are produced individually via transient triple transfection of
adherent HEK293T
followed by iodixanol gradient purification. Representation of individual
variants within the final
pooled test article were balanced to be within 10-fold range where possible.
Each variant capsid
was produced with a genome encoding a unique barcode and a fluorescent
reporter gene under
the control of a ubiquitous promoter (cbh). In all, each variant was produced
with separate
genomes comprising 8 unique barcocles, providing a measure of biological
replicates within the
study. Variants were selected from the ocular NHP dataset from Library
Experiment 1 based on
an algorithm that optimizes for capsid performance while balancing diversity
and measurement
certainty. In one arm of the experiment, variants were selected based on their
transduction
performance in posterior eye (including for example retina, macula, non-
macular retina, neural
retina and choroid/RPE) and anterior eye (including for example tissues of the
trabecular
meshwork and Schlenun's canal) via intravitreal (IVT) delivery. In the other
arm of the
experiment, variants were selected based on their transduction performance in
the posterior eye
via intracameral (IC) delivery. Figure 7 shows plots all measured variants
from Library
Experiment 1 as a function of relative trabecular meshwork transduction by IC
administration (y-
axis) and relative retina (non-macula) transduction via IVT administration (x-
axis). Dots
represent individual capsid variants from Library Experiment 1. Dark black
dots are variants
chosen for this medium throughput study. As shown in Figure 7 and as described
herein, variants
with high transduction across both trabecular meshwork and retina were
identified, as well as
variants with specificity for the retina or specificity for the trabecular
meshwork tissue. Without
being bound by theory, capsids with specificity for one region of the eye
could have benefit for
gene therapies by providing increased targeting to the tissue of interest. The
study also included
variants that contain stop codons in VP1 and VP2 as transduction negative
controls (expected to
produce virus but not transduce cells), and containing vp3 stop codons as
production negative
controls (not expected to produce virus).
Production efficiency is assessed as described above. Equivalent amounts (vg)
of each
virus particle are pooled (approximately 50-100 variants total) in equimolar
amounts, and
injected into AGM or other non-human primate, for example, Cynomolgus macaque
at doses
used in Example 1. Virus properties, including biodistribution and tissue
transduction are
assessed, for example, as described in Example 1.
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We focused our study on variants identified in Library Experiment 1 (for
example,
variants described herein) datasets with improved transduction of the retina
and trabecular
meshwork. First, we selected variants from the ocular NHP dataset from Library
Experiment 1
based on an algorithm that optimizes for capsid performance while balancing
diversity and
measurement certainty. Next, we synthesized 86 variants and benchmarks, and
produced these
separately with co-purification, balancing representation of individual
variants to be within a
defined range of abundance in the final vector preparations. Variants were
paired with genomes
bearing identifying barcode sets as well as diverse random sequence IDs for
quantification of
transduction events in bulk data.
All NHP experiments were conducted in accordance with institutional policies
and NIH
guidelines. Two young adult male cynomolgus macaques (Macaca fascicularis)
weighing 2.8-3
kg, one seronegative (serum NAb titers <1:20 based on in vitro NAb assay) and
one seropositive
(1:128) for anti-AAV2 neutralizing antibodies were selected for the study.
Prior to test article
administrations samples of blood, aqueous humor (50 !IL) and vitreous humor
(up to 50 [tL)
were collected. The animals were anesthetized with ketamine and
dexmedetomidine and received
intravitreal (IVT; 2.63E11 vg/eye in 50 pL) and intracameral (IC; 1.11E11
vg/eye in 50 [IL)
injections of the vector libraries. During the in-life period the animals were
monitored for signs
of ocular inflammation via indirect ophthalmoscopy and slit-lamp biomicroscopy
and treated
with weekly 1M injections of steroids (methylprednisolone, 80 mg) and topical
steroids
(Durezol), and atropine as needed according to the animal facility's SOPs and
recommendations
from the veterinarian. Confocal scanning laser ophthalmoscopy (cSLO) with
green fluorescent
protein (GFP) imaging using the Heidelberg Spectralis HRA/OCT system was used
to take
fluorescent images of each eye prior to necropsy. The animals were sacrificed
4 weeks after the
injections and tissues were collected for biodistribution and transduction
analyses.
Retinas and trabecular meshwork were dissected as shown in FIG.1. All Ocular
tissues
were weighed and flash frozen on dry ice following dissection. A list of all
Ocular tissues
collected is shown in Table 11.
Table 11. Samples from Medium Throughput Study
Front of the Eye Samples
Sample # Region
1 Iris/ciliary body (superior)
2 Iris/ciliary body (nasal)
3 Iris/ciliary body (inferior)
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4 Iris! ciliary body (temporal)
Region containing Trabecula/Schlemm's (superior)
6 Region containing Trabecula/Schlernm's (nasal)
7 Region containing Trabecula/Schlemm's (inferior)
8 Region containing Trabecula/Schlen-un's (temporal)
9 Cornea (combined)
Remaining limbal tissue (combined)
No significant deviations were reported during the in-life period of the
study, and no
major ocular inflammation was observed. 4 weeks following injection,
transduction was
measured by bulk barcode-seq as well as snRNA-seq. The data enabled us to
observe
correlations between Library Experiment 1 measurements and property
measurements from this
experiment, to compare transduction efficiency between bulk and single-cell
measurements, and
to determine transduction rates for high-performing variants across major cell
types within the
eye. In addition to identifying highly promising capsids for improved ocular
gene therapy
delivery, this high-resolution dataset provides validation of Library
Experiment 1 measurement
quality and valuable input data for future machine-guided design directed at
improving cell
transduction, specificity and tissue distribution. Results from bulk tissue
for variants included in
this medium throughput study described in this Example 2 arc shown in Figures
5A-5C (IVT
administration) and Figures 6A-6C (IC administration). All values are log(2)
relative to wild-
type AAV2. Figure 8 plots the measured Library Experiment 1 neural retina
transduction (x-axis)
against the medium throughput measured retina transduction for the same
variant from this
experiment (y-axis). As shown, both the retinal transduction measurements, as
well as the rank
ordering amongst all variants tested, was highly correlated across the two
studies (Pearson =
0.789; Spearman = 0.757).
Single-cell RNA sequencing has been previously demonstrated to allow
characterization
of cell-type specific tropism of barcoded rAAVs (Brown et al., Front.
Tmmunol., 2021).
However, obtaining single cell suspension from certain tissue types and/or
flash frozen samples
from externally-sourced non human primate (NHP) studies can be extremely
challenging. We
developed an approach that combines single-nuclei RNA sequencing (snRNA-Seq)
with targeted
amplicon sequencing to reliably detect cell-type specific transduction from up
to 50-100
barcoded rAAVs with minimal sequencing depth, with an initial focus on tissues
of the eye, and
applied these methods to the tissues collected from the experiment described
in this example. To
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implement this approach we have: 1) developed optimized protocols for
isolation of high quality
single nuclei suspensions from flash frozen NHP retina, 2) used the 10x
Genomics Chromium
platform to encapsulate these nuclei and generate gene expression libraries
for reliable
identification of cell types, and 3) leveraged the 10x feature barcode kit to
selectively amplify
(for sequencing) barcoded viral transcripts that were captured using the 10x
CS1 feature
designed into the viral genomes. Using this approach we investigated cell-type
specific tropism
of multiple rAAVs in the NHP (cynomolgus macaque) retina. Our snRNA-seq gene
expression
analysis identified all the major retina cell types including therapeutically
relevant cells such as
Rods, Cones and Retinal ganglion cells. Viral transduction events, as assessed
from our targeted
library sequencing, were detected in almost all clusters and we could
successfully quantify
differences in transduction rates between rAAVs and benchmarks. Overall, we
demonstrate that
snRNA sequencing can be used to both effectively determine cell-type specific
tropism of
barcoded rAAVs, and quantitate relative transduction between multiple rAAVs in
a single
experiment. These developments open up opportunities for further designing and
validating
rAAVs capable of cell-type specific targeting for gene therapy. Additionally,
this approach
enables a medium throughput identification of rAAVs with desired properties
for further study.
Details of the single nuclear experimental workflow that was applied to the
medium
throughput study described in this example are below:
Materials
EZ lysis buffer + 0.2 U/p1 of murine RNAse inhibitor
1X PBS + 9% BSA + 0.2 U/p1 murine RNAse inhibitor
1X PBS + 5% BSA + 0.2 U/jd of murine RNAse inhibitor/Wash
IX PBS + 2% BSA + 0.2 U/ 1 of murine RNAse inhibitor
Two 15 ml tubes pre-coated with 2% BSA+lx PBS+0.2 U/p1RNAse inhibitor
Six 1.5 ml protein lobind tubes pre-coated with 2% BSA+lx PBS+0.2 U/p1 RNAse
inhibitor
One 2 ml dounce homogenizer + Pestle A and Pestle B
2x 0.4 pm filters
2x 0.7 jtm filters
1 ml wide bore pipette tips
Hemocytometer
4-6 0.5 ml protein lobind tube with 15 pl of 1xPBS+5%BSA+
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Single nuclei dissociation from Retina and Trabecular Meshwork
Mincing: For the retina, the tissue sample was placed in a tube on ice and 100
}al of EZ lysis
buffer + RNAse inhibitor was added. The tissue was minced with a pair of
microscissors for
about 1 min while holding the tube on ice. 50 jd of the minced sample was
transferred to a 2 nil
dounce homogenizer. 1-2 nil of Trizol was added to the rest of the sample for
paired bulk RNA
extraction and sequencing.
For the trabecular meshwork, the tissue was transferred to a new 1.5 ml tube
and 50 jd of EZ
lysis buffer + RNAse inhibitor was added. The tissue was minced with a pair of
micro scissors for
about 1 min while holding the tube on ice. The minced tissue was transferred
to a 2 ml dounce
homogenizer.
Dounce homogenization: More EZ lysis buffer + RNAse inhibitor was added to the
dounce
homogenizer with sample to make up the volume to 2m1. A loose fitting pestle
(Pestle A) was
used to dounce the sample with 10 steady strokes (about 1 stroke per second).
The sample was
allowed to stand on ice for 20 seconds and then was dounced with a tight
fitting pestle (Pestle B)
with 5 steady strokes. The sample was again allowed to stand on ice for 20
seconds and douneed
for another 5 strokes with Pestle B.
Filtration and clean up: Post douce the sample (2 ml) was immediately
transferred to a 15 ml
falcon tube containing 2 ml of 9.5%+lx PBS+RNAse Inhibitor. The sample was
mixed and first
filtered through a 70 micron filter and then through a 40 micron filter. A
small (5 W) aliquot of
the filtered sample was diluted 1:4 in 1xPBS+2% BSA+propidium iodide (PI) for
counting.
The sample (about 4 ml now) was split equally into four 1.5 ml protein lobind
centrifuge
tubes precoated with 2% BSA+1xPBS+RNAseInhibitor. The samples were centrifuged
at 200
RCF for 5 mins at 4 C. The supernatant was discarded and the pellet was
resuspended in 5%
BSA+1xPB S +RNA se-Inhibitor+PI .
The nuclei were then sorted on a WOLF sorter. We gated for intact nuclei that
were
positively stained for PI. We also discarded any doublets by gating with the
area under the curse
for PI as a proxy for doublets. The FACS cleaned nuclei were centrifuged at
200 RFC for 5 mins
at 4C. The pellet was resuspended in 2% BSA+1xPBS+RNAse-inhibitor and counted.
Final
nuclei concentration was adjusted as needed for 10x encapsulation.
10x Encapsulation and library preparation: We then used the 10X chromium
platform for single
cell encapsulation as per the manufacturer's standard instructions. Reverse
transcription was
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performed as per 10x protocols. cDNA amplification was performed using the 10X
feature
barcode cDNA amplification kit with a Dyno viral transcript specific forward
primer spiked in.
Post cDNA amplification a portion of the cDNA library was used to generate
gene
expression library as per 10x standard protocol. A small portion of the same
cDNA library was
used to generate targeted libraries by PCR amplifying the Dyno barcode region.
Primers binding
to either the Nextera Handle or the TruSeq Handle, in combination with a viral
transcript specific
primer, were used for the targeted amplification. When amplifying using the
TruSeq-Handle
primer, a gel extraction step was performed right after the targeted
amplification to select out the
product of interest from the larger linearly amplified background. Once the
targeted
amplification product was purified we performed pre-indexing and indexing PCRs
and
sequenced the libraries using an illumina Next Seq sequencer.
Results
Control experiments comprising a mixture of 5% HEK293 cells transduced with
the library of
variants and 95% untransduced NHP liver cells were performed to determine the
sensitivity of
the assay. After processing and data analysis, we were able to determine that
6% of all nuclei
processed were HEK-293 nuclei and detected viral transcripts from the library
in approximately
20% of the HEK-293 nuclei. This sensitivity is sufficient to identify and
characterize viral
vectors comprising variant capsid polypeptides from the medium throughput
study described
herein. Next, we sequenced data from nuclei isolated from NHP posterior eye
and anterior eye
tissue. We plotted RNA transcript data on a UMAP plot (Leiden clustering) to
show major cell
types when projected onto an annotated eye reference dataset using PCA (Swamy,
VS et al.,
Gigascience 2021, incorporated herein by reference). This allowed us to
annotated major cell
types of the eye, including Amacrine cells, bipolar cells, cones, horizontal
cells, microglia,
Muller glia, retinal ganglion cells, and rods. Using cell-type specific
markers from the literature
(Menon M et al., Nature Comm. 2019; Peng Y et al., Cell, 2019, both
incorporated herein by
reference) we confirmed expected expression patterns from these cell clusters.
Figure 9 shows
the cell type transduction for variants from the medium throughput study from
samples collected
from the posterior eye. The error bars show 90% confidence interval, estimated
by randomly
resampling the cells 2000 times. As shown in Figure 9, variants described
herein have enhanced
transduction of rods and cones, among other cells, relative to AAV2 wild-type.
In addition,
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variants described herein are capable of transducing Muller glia and Amacrine
cells which
showed no detectable wild-type AAV2 transduction.
Example 3
In order to identify structure-function relationships between the mutation
sets (i.e., groups
of two or more mutations) associated with the variant capsid polypeptides
described herein and
increased retina transduction, all mutations relative to wild-type AAV2
present in each of the
variants in Table 2 were decomposed into their constituent mutations such that
each substitution
or deletion counted as an individual mutation, each insertion sequence counted
as a single
mutation, and all possible prefixes and suffixes of an insertion counted as
individual mutations.
For example, an insertion ABC at position n would be decomposed into both ABC
_n to represent
the full insertion and also IAB+_n. A+_n, +BC_n, +C n] where `+' represents
one or more of
any amino acid in that insertion position.
Next, for all possible subcombinations of mutations (referred to as "Common
Mutation
Sets") generated as described above, we identified and grouped all variants
tested in either
Library Experiment 1 or Library Experiment 2 that contained the same mutations
present in the
Common Mutation Set and had an edit distance of at most 3 from a sequence
containing only the
Common Mutation Set present in the specific subcombination. To calculate this,
we first reduced
mutation sets containing redundant insertion prefixes or suffixes to the
minimal descriptive set of
mutations (for example, the set {AB+_n, ABC_n} is reduced to ABC_n} and the
set {+BC_n,
+C _n} is reduced to {+BC_n1). For reduced mutation sets containing insertion
mutations
including a `+' we subtracted one when calculating edit distance to allow a
single wildcard. We
then calculated the median neural retina transduction measurements (10g2
relative to wild-type
AAV2) across all variants associated with each Common Mutation Set. We chose
the median
because it is a robust statistic that is more resilient to outliers than the
mean.
The resulting Common Mutation Sets of minimal structural elements (i.e.,
subcombinations of mutations found in the variants described herein) were
filtered according to
one of the following two criteria: (1) at least 5 unique capsid polypeptide
variants comprising the
submotif present in the library experiment and median 1og2 relative to wild-
type AAV2 whole
retina transduction greater than 0 or (2) less than 5 unique capsid
polypeptide variants
comprising the submotif and median 1og2 relative to wild-type AAV2 whole
retina transduction
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greater than 0.5. The resulting Common Mutation Sets are those that the data
indicate are
associated with increased whole retinal transduction and thus comprise unique
structure-function
relationships identified and described herein. Results are shown in Table 12
(submotifs identified
from an analysis of Library Experiment 1 variants) and Table 13 (submotifs
identified from an
analysis of Library Experiment 2 variants).
References:
Ogden Pi, Kelsic ED, Sinai S, Church GM. Comprehensive AAV capsid fitness
landscape
reveals a viral gene and enables machine-guided design. Science. 2019 Nov
29:366(6469):1139-
1143. doi: 10.1126/science.aaw2900. PMI D: 31780559; PMC I D: PM C7197022.
The results show that variant capsid polypeptides provided for herein produce
virus
particles that have increased packaging, increased biodistribution, increased
transduction and/or
increased expression of a transgene (payload) in various regions of the eye
relative to a wild-type
AAV2 upon intravitreal or intracameral injection. In addition, the variant
capsid polypeptides
described herein provide selective biodistribution and/or expression in
regions of the eye that
include target cell populations for gene therapy (for example, macula-
selectivity, non-macula
retina selectivity, macula/retina selectivity and/or trabecular meshwork
selectivity). Without
limitation, the capsid polypeptides, nucleic acids and virus particles
described herein are used to
deliver therapeutics to the eye, e.g., to certain cell types of the eye, and
are used to treat disorders
of the eye as described herein, with higher efficiency.
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