Note: Descriptions are shown in the official language in which they were submitted.
RATIONAL POLYPLOID ADENO-ASSOCIATED VIRUS VECTORS AND
METHODS OF MAKING AND USING THE SAME
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims benefit under 35 U.S.C. 119(e) of U.S.
Provisional
Application Nos. 62/668,056 filed May 7, 2018; and 62/678,675 filed May 31,
2018, the
contents of each of which are incorporated herein by reference in their
entireties.
STATEMENT OF GOVERNMENT SUPPORT
[0002] This invention was made with government support under Grant Numbers
DK084033, A1117408, AI072176, CA016086, CA151652, HL125749, and HL112761
awarded by the National Institutes of Health. The government has certain
rights in the
invention.
STATEMENT REGARDING ELECTRONIC FILING OF A SEQUENCE LISTING
[0003] A Sequence Listing in ASCII text format, submitted under 37 C.F.R.
1.821,
entitled 5470-786W02 ST25.txt, 111,597 bytes in size, generated on July 31,
2018 and filed
via EFS-Web, is provided in lieu of a paper copy. This Sequence Listing is
hereby
incorporated herein by reference into the specification for its disclosures.
TECHNICAL FIELD
[0004] The present invention is directed to methods for production of rational
polyploid
virions with desired properties, the virions, substantially homogenous
populations of such
virions, methods of producing substantially homogenous populations, and uses
thereof.
BACKGROUND OF THE INVENTION
[0005] Adeno-associated virus (AAV) vector has been used in over 100 clinical
trials with
promising results, in particular, for the treatment of blindness and
hemophilia B. AAV is
non-pathogenic, has a broad tissue tropism, and can infect dividing or non-
dividing cells.
More importantly, AAV vector transduction has induced long-term therapeutic
transgene
expression in pre-clinical and clinical trials. Currently there are 12
serotypes of AAV
isolated for gene delivery. Among them, AAV8 has been shown to be the best for
mouse
liver targeting. Extensive studies in pre-clinical animals with FIX deficiency
and Phase I/II
clinical trials have been carried out using AAV2 and AAV8 in patients with
hemophilia B.
The results from these trials are very promising; however, the FIX expression
from patients
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= I
receiving AAV/FIX was not proportional to what has been achieved in animal
models even
though the same vector dosage/kg was used. When 1x10'1 particles of AAV8
encoding FIX
were used in FIX knock out mice for systemic administration, 160% of normal
level FIX was
detected in blood. However, when 2x1011 particles of AAV8/FIX were
administered, only
40% of FIX was achieved in primates and less than 1% of FIX was found in
human. The
inconsistent FIX expression following AAV vector transduction among these
species may be
due to altered hepatocyte tropism in different species. Another interesting
finding from AAV
FIX clinical trials is the capsid specific cytotoxic T lymphocyte (CTL)
response that
eradicates AAV transduced hepatocytes, resulting in therapeutic failure. This
phenomenon
has not been seen in animal models following AAV delivery, which points out
another
variation between preclinical and clinical studies. When a much higher dose of
AAV/FIX
vector was used, FIX expression was detected in both clinical trials using
either AAV2 or
AAV8; however the blood FIX level decreased at week 4 or 9 post injection,
respectively.
Further studies suggested that AAV vector infection elicited a capsid specific
CTL response,
which appeared to eliminate AAV transduced hepatocytes. Therefore, the results
from these
clinical trials highlight the necessity to explore effective approaches for
enhancement of
AAV transduction without increasing vector capsid burden. Any vector
improvement that
reduces AAV capsid antigen effect will also impact the daunting vector
production concerns
and be a welcome addition to viable gene therapy drug development.
[0006] Adeno-associated virus (AAV), a non-pathogenic-dependent parvovirus
that needs
helper viruses for efficient replication, is utilized as a virus vector for
gene therapy because of
its safety and simplicity. AAV has a broad host and cell type tropism capable
of transducing
both dividing and non-dividing cells. To date, 12 AAV serotypes and more than
100 variants
have been identified. Different serotype capsids have different infectivity in
tissues or culture
cells, which depend on the primary receptor and co-receptors on the cell
surface or the
intracellular trafficking pathway itself. The primary receptors of some
serotypes of AAV
have been determined, such as heparin sulfate proteoglycan (HSPG) for AAV2 and
AAV3,
and N-linked sialic acid for AAV5, while the primary receptor of AAV7 and AAV8
has not
been identified. Interestingly, AAV vector transduction efficiency in cultured
cells may not
always be translated into that in animals. For instance, AAV8 induces much
higher transgene
expression than other serotypes in mouse liver, but not in culture cell lines.
[0007] Of the above-mentioned 12 serotypes, several AAV serotypes and variants
have
been used in clinical trials. As the first characterized capsid, AAV2 has been
most widely
used in gene delivery such as RPE 65 for Leber congenital amaurosis and Factor
IX (FIX) for
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hemophilia B. Although the application of AAV vectors has been proven safe and
therapeutic effect has been achieved in these clinical trials, one of the
major challenges of
AAV vector is its low infectivity that requires relatively huge numbers of
virus genomes.
AAV8 vector is another vector which has been used in several clinical trials
in patients with
hemophilia B. The results from AAV8/FIX liver- targeted delivery have
demonstrated that
there are distinct species-specific differences in transgene expression
between mice, non-
human primates and humans. While 1010 vg of AAV8 with FIX gene could reach
supra-
physiologic levels (>100%) of FIX expression in FIX knock-out mice, only high
doses (2 x
1012 vg/kg of body weight) could induce detectable FIX expression in humans.
Based on
these results described above, the development of effective strategies to
enhance AAV
transduction is still necessary.
[0008] The majority of people have been naturally exposed to AAVs. As a
result, a large
portion of the population has developed neutralizing antibodies (Nabs) in the
blood and other
bodily fluids against certain serotype AAVs. The presence of Nabs poses
another major
challenge for broader AAV applications in future clinical trials. Many
approaches have been
explored to enhance AAV transduction or evade Nab activity, especially genetic
modification
of the AAV capsid based on rational design and directed evolution. Although
several AAV
mutants have demonstrated high transduction in vitro or in animal models,
along with the
capacity to escape Nabs, the modification of the capsid composition provides
an ability to
alter the cell tropisms of parental AAVs.
[0009] The present invention addresses a need in the art for AAV vectors with
combined
desirable features.
SUMMARY OF THE INVENTION
[0010] Our previous studies have shown that the capsids from different AAV
serotypes
(AAV1 to AAV5) were compatible to assemble AAV virions (the terms virions,
capsids,
viral particles, and particles are used interchangeably in this application)
and most isolated
AAV monoclonal antibodies recognized several sites located on different AAV
subunits.
Additionally, the studies from chimeric AAV capsids demonstrated that higher
transduction
can be achieved with introduction of a domain for a primary receptor or tissue-
specific
domain from other serotypes. Introduction of AAV9 glycan receptor into AAV2
capsid
enhances AAV2 transduction. Substitution of a 100 aa domain from AAV6 into
AAV2
capsid increases muscle tropism. We discovered that polyploid AAV vectors
which are
composed of capsids from two or more AAV serotypes might take advantages from
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individual serotypes for higher transduction but not in certain embodiments
eliminate the
tropism from the parents. Moreover, these polyploid viruses might have the
ability to escape
the neutralization by Nabs since the majority of Nab recognize conformational
epitopes and
polyploid virions can have changed its surface structure.
[0011] One approach for generating rAAV with mixed or mosaic capsid shells has
been to
add AAV helper plasmids encoding the capsid proteins (VP1, VP2, and VP3) from
a mixture
of AAV serotypes. This methodology is sometimes referred to as cross-dressing.
In certain
embodiments it can change the antigenic patterns of certain virions. However,
a wide range
of virions are produced. Moreover, the virions produced are mosaics that have
a mixture of
serotypes. Accordingly, the population of virions produced retains some
particles that will
elicit an antigen response. Thus, obtaining a substantially homogenous
population of
predetermined virions would be desirable.
[0012] We have now discovered methodology that permits the rational design and
production of such chimeric or shuffled virions. The resultant virions are
sometimes referred
to as polyploid, haploid, or triploid to refer to the fact that the capsid
proteins VP I, VP2, and
VP3 come from at least two different serotypes. The capsids can be from any of
the AAV
serotype, including the 12 serotypes of AAV isolated for gene therapy, other
species, mutant
serotypes, shuffled serotypes of such genes, e.g., AAV2, VP1.5 and AAV4 VP2,
AAV4 VP3,
or any other AAV serotype desired. This method permits production of
infectious virus of
only the virion desired which results in substantially homogenous populations
of the virion.
[0013] The AAV virion has T=1 icosahedral symmetry and is composed of the
three
structural viral proteins, VP!, VP2, and VP3. 60 copies of the three viral
proteins in a ratio of
1:1:8 to 10 (VP1:VP2:VP3, respectively) form the virion (Rayaprolu, V., et
al., J. Virol.
87(24): 13150-13160 (2013).
[0014] In one embodiment, the AAV virion is an isolated virion that has at
least one of the
viral structural proteins, VP1, VP2, and VP3 from a different serotype than
the other VPs,
and each VP is only from one serotype. For example, the VP1 is only from AAV2,
the VP2 is
only from AAV4, and the VP3 is only from AAV8.
[0015] In an alternative embodiment, a virion particle can be constructed
wherein at least one
viral protein from the group consisting of AAV capsid proteins, VP1, VP2 and
VP3, is
different from at least one of the other viral proteins, required to form the
virion particle
capable of encapsidating an AAV genome. For each viral protein present (VP1,
VP2, and/or
VP3), that protein is the same type (e.g., all AAV2 VP1). In one instance, at
least one of the
viral proteins is a chimeric viral protein and at least one of the other two
viral proteins is not a
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chimeric. In one embodiment VP1 and VP2 are chimeric and only VP3 is non-
chimeric. For
example, only the viral particle composed of VP1NP2 from the chimeric AAV2/8
(the N-
terminus of AAV2 and the C-terminus of AAV8) paired with only VP3 from AAV2;
or only
the chimeric VP1/VP2 28m-2P3 (the N-terminal from AAV8 and the C-terminal from
AAV2
without mutation of VP3 start codon) paired with only VP3 from AAV2. In
another
embodiment only VP3 is chimeric and VP1 and VP2 are non-chimeric. In another
embodiment at least one of the viral proteins is from a completely different
serotype. For
example, only the chimeric VP1/VP2 28m-2P3 paired with VP3 from only AAV3. In
another
example, no chimeric is present.
[0016] In one embodiment an AAV virion that encapsidates an AAV genome
(including a
heterologous gene between 2 AAV ITRs) can be formed with only two of the viral
structural
proteins, VP1 and VP3. In one embodiment this virion is conformationally
correct, i.e., has
T=1 icosahedral symmetry. In one embodiment the virions are infectious.
[0017] The population is at least 101 virions, at least 102 virions, at least
103 virions, at least
104 virions, at least 105 virions,... at least 1010 virions, at least 1011
virions, at least 1012
virions, at least 1015 virions, at least 1017 virions. In one embodiment, the
population is at
least 100 viral particles. In one embodiment, the population is from 109 to
1012 virions
[0018] In one embodiment, the population is at least 1 x 104 viral genomes
(vg) /ml, is at least
1 x 105 viral genomes (vg) /ml, is at least 1 x 106 viral genomes (vg) /ml, at
least I x 107 viral
genomes (vg) /ml, at least 1 x 108 viral genomes (vg) /ml, at least 1 x 109
viral genomes (vg)
/ml, at least 1 X 101 vg/ per ml, at least 1 X 1011 vg/ per ml, at least 1 X
1012 vg/ per
ml. In one embodiment, the population ranges from about 1 x 105 vg/ml to about
1 x 1013
vg/ml.
[0019] A substantially homogenous population is at least 90% of only the
desired virion, at
least 91%, at least 93%, at least 95%, at least 97%, at least 99%, at least
99.5%, or at least
99.9%. In one embodiment, the population is completely homogenous.
[0020] AAV2 and AAV8 have been used for clinical application. In one
embodiment, we
first characterized the haploid AAV virus from AAV2 and AAV8 for transduction
efficiency
in vitro and in vivo, as well as Nab escape ability, i.e., the immune response
such as an
antigenic response. In that study, we found that the virus yield of the
haploid vector was not
compromised and the heparin binding profile was related to the incorporation
of AAV2
capsid subunit proteins. The haploid vectors AAV2/8 initiated a higher
transduction in
mouse muscle and liver. When applied to a mouse model with FIX deficiency,
higher FIX
expression and improved bleeding phenotypic correction were observed in
haploid vector-
CA 3054600 2019-09-06
treated mice compared to AAV8 group. Importantly, the haploid virus AAV2/8 had
low
binding affinity to A20 and was able to escape the neutralization from anti-
AAV2 serum.
The next polyploid virus AAV2/8/9 was made from capsids of three serotypes
(AAV2, 8 and
9). It was demonstrated that the neutralizing antibody escape ability of
haploid AAV2/8/9
was significantly improved against sera immunized with parental serotypes.
[0021] Thus, in one embodiment, the present invention provides an adeno-
associated virus
(AAV) capsid, wherein the capsid comprises capsid protein VP!, wherein said
capsid protein
VP1 is from one or more than one first AAV serotype and capsid protein VP3,
wherein said
capsid protein VP3 is from one or more than one second AAV serotype and
wherein at least
one of said first AAV serotype is different from at least one of said second
AAV serotype, in
any combination. Preferably such population is substantially homogenous. In
some
embodiments, the capsid of this invention comprises capsid protein VP2,
wherein said capsid
protein VP2 is from one or more than one third AAV serotype, wherein at least
one of said
one or more than one third AAV serotype is different from said first AAV
serotype and/or
said second AAV serotype, in any combination.
[0022] In some embodiments the AAV virion can be formed by more than the
typical 3 viral
structural proteins, VP1, VP2, and VP3 (see e.g., Wang, Q. et al., "Syngeneic
AAV Pseudo-
particles Potentiate Gene Transduction of AAV Vectors," Molecular Therapy:
Methods and
Clinical Development, Vol. 4, 149-158 (2017)). Such viral capsids also fall
within the present
invention. For example, an isolated AAV virion having viral capsid structural
proteins
sufficient to form an AAV virion that encapsidates an AAV genome, wherein at
least one of
the viral capsid structural proteins is different from the other viral capsid
structural proteins,
and wherein each viral capsid structural protein is only of the same type. In
a further
embodiment the isolated AAV virion has at least two viral structural proteins
from the group
consisting of AAV capsid proteins, VP1, VP2, VP1.5 and VP3, wherein the two
viral
proteins are sufficient to form an AAV virion that encapsidates an AAV genome,
and
wherein at least one of the viral structural proteins present is from a
different serotype than
the other viral structural protein, and wherein the VP1 is only from one
serotype, the VP2 is
only from one serotype, the VP1.5 is only from one serotype, and the VP3 is
only from one
serotype. For example, the VP1.5 can be from AAV serotype 2 and the VP3 can be
from
AAV serotype 8.
[0023] In some embodiments, the capsid of this invention comprises capsid
protein VP1.5,
wherein said capsid protein VP1.5 is from one or more than one fourth AAV
serotype,
wherein at least one of said one or more than one fourth AAV serotype is
different from said
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first AAV serotype and/or said second AAV serotype, in any combination. In
some
embodiments, the AAV capsid protein described herein can comprise capsid
protein VP2.
[0024] Thus, in certain embodiments the at least one of the viral structural
proteins can be a
chimeric viral structural protein, i.e., can contain segments from more than
one protein. In
one embodiment the chimeric viral structural protein is all from the same
serotype. In another
embodiment, the chimeric viral structural protein is made up of components
from a more than
one serotype, but these serotypes are different from at least one other
serotype. In one
embodiment, the viral structural proteins are not chimeric. In one embodiment,
the chimeric
AAV structural protein does not comprise structural amino acids from canine
parvovirus. In
one embodiment, the chimeric AAV structural protein does not comprise
structural amino
acids from b19 parvovirus. In one embodiment, the chimeric AAV structural
protein does not
comprise structural amino acids from canine parvovirus or b19 parvovirus. In
one
embodiment, the chimeric AAV structural protein only comprises structural
amino acids from
AAV.
[0025] In some embodiments only virions that contain at least one viral
protein that is
different than the other viral proteins is produced. For example, VP1 and VP2
from the same
serotype and VP3 from an alternative serotype, only. In other embodiments, the
VP1 is from
one serotype and the VP2 and VP3 are from another serotype, only. In another
embodiment,
only particles where VP1 is from one serotype, VP2 is from a second serotype,
and VP3 is
from yet another serotype are produced.
[0026] This can be done by, for example, site specific deletions, and/or
additions, changing
splice donor sites, splice acceptor sites, start codons and combinations
thereof.
[0027] Using AAV serotype 2 as an exemplary virus, MI1 is the VP1 start codon,
M138 is
the VP2 start codon, and M203 is the VP3 start codon. While deletion of the
start codon,
typically by a substitution of M1 1 and M138 will render expression of VP1 and
VP2
inoperative, a similar deletion of the VP3 start codon is not sufficient. This
is because the
viral capsid ORF contains numerous ATG codons with varying strengths as
initiation codons.
Thus, in designing a construct that will not express VP3 care must be taken to
insure that an
alternative VP3 species is not produced. With respect to VP3 either
elimination of M138 is
necessary or if VP2 is desired, but not VP3, then deletion of M211 and 235 in
addition to
M203 is typically the best approach (Warrington, K. H. Jr., et al., J. of
Virol. 78(12): 6595-
6609 (June 2004)). This can be done by mutations such as substitution or other
means known
in the art. The corresponding start codons in other serotypes can readily be
determined as
well as whether additional ATG sequences such as in VP3 can serve as
alternative initiation
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codons.
[0028] This permits methods for producing populations of substantially
homogenous
populations of the polyploid virions ¨ such as the haploid or triploid viral
particles.
[0029] The present invention also provides an AAV capsid wherein the capsid
comprises
capsid protein VP1, wherein said capsid protein VP1 is from one or more than
one first AAV
serotype, and capsid protein VP2, wherein said capsid protein VP2 is from one
or more than
one second AAV serotype, and wherein at least one of said first AAV serotype
is different
from at least one of said second AAV serotype, in any combination.
[0030] In some embodiments, the capsid comprises capsid protein VP3, wherein
said
capsid protein VP3 is from one or more than one third AAV serotype, wherein at
least one of
said one or more than one third AAV serotype is different from said first AAV
serotype
and/or said second AAV serotype, in any combination. In some embodiments, the
AAV
capsid described herein can comprise capsid protein VP1.5.
[0031] The present invention further provides an adeno-associated virus (AAV)
capsid,
wherein the capsid comprises capsid protein VP1, wherein said capsid protein
VP1 is from
one or more than one first AAV serotype, and capsid protein VP1.5, wherein
said capsid
protein VP1.5 is from one or more than one second AAV serotype, and wherein at
least one
of said first AAV serotype is different from at least one of said second AAV
serotype, in any
combination.
[0032] In additional embodiments, the present invention provides a virus
vector
comprising: (a) an AAV capsid of this invention; and (b) a nucleic acid
comprising at least
one terminal repeat sequence, wherein the nucleic acid is encapsidated by the
AAV capsid.
The virus vector can be an AAV particle and the capsid protein, capsid, virus
vector and/or
AAV particle of this invention can be present in a composition that further
comprises a
pharmaceutically acceptable carrier.
[0033] Further provided herein is a method of making an AAV particle
comprising the
AAV capsid of any preceding claim, comprising: (a) transfecting a host cell
with one or more
plasmids that provide, in combination all functions and genes needed to
assemble AAV
particles; (b) introducing one or more nucleic acid constructs into a
packaging cell line or
producer cell line to provide, in combination all functions and genes needed
to assemble
AAV particles; (c) introducing into a host cell one or more recombinant
baculovirus vectors
that provide in combination all functions and genes needed to assemble AAV
particles;
and/or (d) introducing into a host cell one or more recombinant herpesvirus
vectors that
provide in combination all functions and genes needed to assemble AAV
particles.
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[0034] In further embodiments, the present invention provides a method of
administering a
nucleic acid to a cell, the method comprising contacting the cell with the
virus vector of this
invention and/or a composition of this invention.
[0035] Also provided herein is a method of delivering a nucleic acid to a
subject, the
method comprising administering to the subject the virus vector and/or a
composition of this
invention.
[0036] Additionally, provided herein is the capsid protein, capsid, virus
vector, AAV
particle and/or composition of this invention for use as a medicament in the
beneficial
treatment of a disorder or disease.
[0037] These and other aspects of the invention are addressed in more detail
in the
description of the invention set forth below.
BRIEF DESCRIPTION OF THE DRAWINGS
[0038] Fig. 1: Transduction profiles of the haploid viruses in vitro. Haploid
or parental
viruses were added to Huh7 or C2C12 cells at 104 vg/cell. Cells were lysed for
luciferase
assay at 48 h post-transduction. The data represent an average of three
separate infections,
with the standard deviation indicated by an error bar.
[0039] Fig. 2: Transduction of the haploid viruses in mouse muscle. 1 X 101
vg of the
haploid viruses, parental viruses or viruses mixed with AAV2 and AAV8 were
injected into
C57BL/6 mice via direct muscular injection. Each group included 4 mice. (Panel
A) After
one week, luciferase gene expression was imaged by IVIS imaging system. (Panel
B) The
photon signal was measured and calculated. The data represent an average of
luciferase gene
expression values for the 4 injected mice in each group, with the standard
deviation indicated
by an error bar. Face up: left leg-AAV8 or haploid or mixture viruses, right
leg-AAV2.
[0040] Fig. 3: Transduction of the haploid viruses in mouse liver. 3 X 1010 vg
of the
haploid virus was administered via intravenous injection. At week 1 post-
injection, luciferase
expression was imaged by IVIS imaging system (Panel A), and the photon signal
was
measured and calculated (Panel B). At week 2 post-injection, mice were
euthanized and their
livers were harvested for DNA extraction AAV genome copy in the liver was
measured by
ciPCR ((Panel C) and relatively luciferase expression per AAV genome copy
number was
calculated (Panel D). The data represent the average and standard deviation
from 4 mice.
[0041] Fig. 4: Therapeutic level of fix via haploid virus delivery. FIX
knockout mice were
injected with 1 X 101 vg of each vector via tail vein. At 1, 2 and 4 weeks
post-injection,
blood samples were collected. (Panel A) hFIX protein levels were tested by
enzyme-linked
9
CA 3054600 2019-09-06
immunosorbent assay. (Panel B) hFIX function was tested by the hFIX-specific
one stage
clotting assay. At week 6 post-injection, blood loss was determined by
measuring the
absorbance at A575 of hemoglobin content in the saline solution (Panel C). The
data
represent the average and standard deviations from 5 mice (knock-out mice and
normal mice,
without AAV treatment, as controls) or 8 mice (AAV8 FIX or AAV2/8 1:3/FIX
treated
groups).
[0042] Fig. 5: Transduction of haploid AAV82 from AAV2 and AAV8. Panel A. The
composition of AAV capsid subunits. Panel B. Western blot for haploid viruses.
Panel. C.
Representative imaging and the quantitation of liver transduction. Panel D.
Representative
imagin and the quantification of muscle transduction.
[0043] Fig. 6: Liver transduction with the triploid virus AAV2/8/9. 3 X 1010
vg of the
haploid viruses were injected via retro-orbital vein. At week 1 post-
injection, luciferase gene
expression was imaged by IVIS imaging system (Panel A), and the photon signal
was
measured and calculated (Panel B). The data represent the average and standard
deviation
from 5 mice.
[0044] Fig. 7: AAV stability against heating.
[0045] Fig. 8: Haploid design by mutating start codons of capsid protein VP1.
[0046] Fig. 9: Haploid design by mutating the Splice Acceptor Site A2.
[0047] Fig. 10: Haploid design by mutating the Splice Acceptor Site Al.
[0048] Fig. 11: Haploid design by mutating the start codons of capsid proteins
for
VP2/VP3 and the Splice Acceptor Site A2.
[0049] Fig. 12: Haploid design by mutating the start codon of capsid protein
VP1 and the
Splice Acceptor Site Al.
[0050] Fig. 13: Haploid vector production using two plasmids.
[0051] Fig. 14: Haploid vector production using three plasmids.
[0052] Fig. 15: Haploid vector production using four plasm ids.
[0053] Fig.16: A schematic showing the use of DNA shuffling to obtain virions
having
desired characteristics.
[0054] Fig. 17: Plasmid including DNA sequence (SEQ ID NO:139) for AAV2 capsid
proteins wherein the start codons for VP1 and VP2 have been mutated.
[0055] Fig. 18: Plasmid including DNA sequence (SEQ ID NO:140) for AAV2 capsid
proteins wherein the start codon for VP1 has been mutated.
[0056] Fig. 19: Plasmid including DNA sequence (SEQ ID NO:141) for AAV2 capsid
proteins wherein the start codons for VP2 and VP3 have been mutated.
CA 3054600 2019-09-06
[0057] Fig. 20: Plasmid including DNA sequence (SEQ ID NO:142) for AAV2 capsid
proteins wherein the start codon for VP2 has been mutated.
[0058] Fig. 21: Single or multiple subunits substituted to generate a novel
polyploid AAV
capsid.
[0059] Figs. 22A-C: Liver transduction of haploid vector H-AAV82.
(22A) the
composition of AAV capsid subunits. Haploid AAV viruses were produced from co-
transfection of two plasmids (one encoding VP1 and VP2, another one for VP3).
(22B)
3x101 particles of AAV vector were injected into C57BL mice via retro-orbital
vein. The
imaging was performed one week later. (22C) The quantitation of liver
transduction. The
data represented the average of 5 mice and standard deviations.
[0060] Figs. 23A-B: Muscle transduction of haploid vector H-AAV82. 1x109
particles of
AAV/luc were injected into mouse hind leg muscle. At week 3 post injection,
the imaging
was taken for 3 min. Face up: left leg-haploid AAV, right leg-AAV2. (23A)
Representative
imaging. (23B) Data from 4 mice after muscular injection. The fold increase of
transduction
was calculated by transduction from haploid AAV to AAV2.
[0061] Figs. 24A-C: Liver transduction of haploid vector H-AAV92.
(24A) the
composition of AAV capsid subunit. Haploid AAV viruses were produced from co-
transfection of two plasmids (one encoding AAV9 VP1 and VP2, another one for
AAV2
VP3). (24B) 3x101 particles of AAV vector were injected into C57BL mice via
retro-orbital
vein. The imaging was performed one week later. (24C) The quantitation of
liver
transduction. The data represented the average of 5 mice and standard
deviations.
[0062] Figs. 25A-C: Liver transduction of haploid vector H-AAV82G9. (25A) the
composition of AAV capsid subunit. Haploid AAV viruses were produced from co-
transfection of two plasmids (one encoding AAV8 VP1 and VP2, another one for
AAV2G9
VP3). (25B) 3x101 particles of AAV vector were injected into C57BL mice via
retro-orbital
vein. At week 1 post AAV administration, the imaging was carried out. (25C)
The
quantitation of liver transduction. The data represented the average of 5 mice
and standard
deviations.
[0063] Figs. 26A-D: Liver transduction of haploid AAV83, AAV93 and AAVrh10-3.
(26A) The composition of AAV capsid subunits. (26B) Representative imaging.
(26C) The
quantification of liver transduction. (26D) The quantification of viral genome
in the
indicated organ, as compared to mouse lamin (internal control for expression
levels).
[0064] Figs. 27A-D: Transduction of haploid AAV82 from AAV2 and AAV8. (27A)
The
composition of AAV capsid subunits. (27B) Western blot for haploid viruses.
(27C)
11
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Representative imaging and the quantitation of liver transduction. (27D)
Representative
imaging and the quantitation of muscle transduction.
[0065] Fig. 28: Analysis of haploid abilities for binding and trafficking.
[0066] Fig. 29: AAV stability against heating.
[0067] Fig. 30: Detection of N-terminus exposure under different pH.
DETAILED DESCRIPTION OF THE INVENTION
[0068] The present invention will now be described with reference to the
accompanying
drawings, in which representative embodiments of the invention are shown. This
invention
may, however, be embodied in different forms and should not be construed as
limited to the
embodiments set forth herein. Rather, these embodiments are provided so that
this disclosure
will be thorough and complete, and will fully convey the scope of the
invention to those
skilled in the art.
[0069] Unless otherwise defined, all technical and scientific terms used
herein have the
same meaning as commonly understood by one of ordinary skill in the art to
which this
invention belongs. The terminology used in the description of the invention
herein is for the
purpose of describing particular embodiments only and is not intended to be
limiting of the
invention. All publications, patent applications, patents, accession numbers
and other
references mentioned herein are incorporated by reference herein in their
entirety.
[0070] The designation of all amino acid positions in the AAV capsid viral
structural
proteins in the description of the invention and the appended claims is with
respect to VP1
capsid subunit numbering (native AAV2 VP1 capsid protein: GenBank Accession
No. AAC03780 or YP680426). It will be understood by those skilled in the art
that the
modifications described herein if inserted into the AAV cap gene may result in
modifications
in the structural viral proteins VP!, VP2 and/or VP3 which make up the capsid
subunits.
Alternatively, the capsid subunits can be expressed independently to achieve
modification in
only one or two of the capsid subunits (VP1, VP2, VP3, VP1 + VP2, VP1 +VP3, or
VP2
+VP3).
Definitions
[0071] The following terms are used in the description herein and the appended
claims:
[0072] The singular forms "a," "an" and "the" are intended to include the
plural forms as
well, unless the context clearly indicates otherwise.
12
CA 3054600 2019-09-06
[0073] Furthermore, the term "about," as used herein when referring to a
measurable value
such as an amount of the length of a polynucleotide or polypeptide sequence,
dose, time,
temperature, and the like, is meant to encompass variations of 20%, 10%, +
5%, 1%,
0.5%, or even 0.1% of the specified amount.
[0074] Also as used herein, "and/or" refers to and encompasses any and all
possible
combinations of one or more of the associated listed items, as well as the
lack of
combinations when interpreted in the alternative ("or").
[0075] As used herein, the transitional phrase "consisting essentially of'
means that the
scope of a claim is to be interpreted to encompass the specified materials or
steps recited in
the claim, "and those that do not materially affect the basic and novel
characteristic(s)" of the
claimed invention. See, In re Herz, 537 F.2d 549, 551-52, 190 USPQ 461,463
(CCPA 1976)
(emphasis in the original); see also MPEP 2111.03. Thus, the term
"consisting essentially
of' when used in a claim of this invention is not intended to be interpreted
to be equivalent to
"comprising." Unless the context indicates otherwise, it is specifically
intended that the
various features of the invention described herein can be used in any
combination.
[0076] Moreover, the present invention also contemplates that in some
embodiments of the
invention, any feature or combination of features set forth herein can be
excluded or omitted.
[0077] To illustrate further, if, for example, the specification indicates
that a particular
amino acid can be selected from A, G, I, L and/or V, this language also
indicates that the
amino acid can be selected from any subset of these amino acid(s) for example
A, G, I or L;
A, G, I or V; A or G; only L; etc. as if each such subcombination is expressly
set forth herein.
Moreover, such language also indicates that one or more of the specified amino
acids can be
disclaimed (e.g., by negative proviso). For example, in particular embodiments
the amino
acid is not A, G or I; is not A; is not G or V; etc. as if each such possible
disclaimer is
expressly set forth herein.
[0078] As used herein, the terms "reduce," "reduces," "reduction" and similar
terms mean a
decrease of at least about 25%, 35%, 50%, 75%, 80%, 85%, 90%, 95%, 97% or
more.
[0079] As used herein, the terms "enhance," "enhances," "enhancement" and
similar terms
indicate an increase of at least about 25%, 50%, 75%, 100%, 150%, 200%, 300%,
400%,
500% or more.
[0080] The term "parvovirus" as used herein encompasses the family
Parvoviridae,
including autonomously replicating parvoviruses and dependoviruses. The
autonomous
parvoviruses include members of the genera Parvovirus, Erythrovirus,
Densovirus,
Iteravirus, and Contravirus. Exemplary autonomous parvoviruses include, but
are not limited
13
CA 3054600 2019-09-06
to, minute virus of mouse, bovine parvovirus, canine parvovirus, chicken
parvovirus, feline
panleukopenia virus, feline parvovirus, goose parvovirus, H1 parvovirus,
Muscovy duck
parvovirus, B19 virus, and any other autonomous parvovirus now known or later
discovered.
Other autonomous parvoviruses are known to those skilled in the art. See,
e.g., BERNARD
N. FIELDS et al., VIROLOGY, volume 2, chapter 69 (4th ed., Lippincott-Raven
Publishers).
[0081] As used herein, the term "adeno-associated virus" (AAV), includes but
is not
limited to, AAV type 1, AAV type 2, AAV type 3 (including types 3A and 3B),
AAV type 4,
AAV type 5, AAV type 6, AAV type 7, AAV type 8, AAV type 9, AAV type 10, AAV
type
11, avian AAV, bovine AAV, canine AAV, equine AAV, ovine AAV, and any other
AAV
now known or later discovered. See, e.g., BERNARD N. FIELDS et al., VIROLOGY,
volume 2, chapter 69 (4th ed., Lippincott-Raven Publishers). A number of
relatively new
AAV serotypes and clades have been identified (see, e.g., Gao et al., (2004)
J. Virology
78:6381-6388; Moris et al., (2004) Virology 33-:375- 383; and Table 3).
[0082] The genomic sequences of various serotypes of AAV and the autonomous
parvoviruses, as well as the sequences of the native terminal repeats (TRs),
Rep proteins, and
capsid subunits are known in the art. Such sequences may be found in the
literature or in
public databases such as GenBank. See, e.g., GenBank Accession Numbers NC
002077,
NC 001401, NC 001729, NC 001863, NC 001829, NC 001862, NC 000883, NC 001701,
NC 001510, NC 006152, NC 006261, AF063497, U89790, AF043303, AF028705,
AF028704, J02275, J01901, J02275, X01457, AF288061, AH009962, AY028226,
AY028223, NC 001358, NC 001540, AF513851, AF513852, AY530579; the disclosures
of
which are incorporated by reference herein for teaching parvovirus and AAV
nucleic acid
and amino acid sequences. See also, e.g., Srivistava et al., (1983) 1 Virology
45:555;
Chiarini et al., (1998) 1 Virology 71:6823; Chiarini et al., (1999) 1 Virology
73:1309;
Bantel-Schaal et al., (1999) 1 Virology 73:939; Xiao et al., (1999) 1 Virology
73:3994;
Muramatsu et al., (1996) Virology 221:208; Shade et al., (1986) J. ViroL
58:921; Gao et al.,
(2002) Proc. Nat. Acad. ScL USA 99:11854; Moris et al., (2004) Virology 33-
:375- 383;
international patent publications WO 00/28061, WO 99/61601, WO 98/11244; and
U.S.
Patent No. 6,156,303; the disclosures of which are incorporated by reference
herein for
teaching parvovirus and AAV nucleic acid and amino acid sequences. See also
Table 1.
[0083] The capsid structures of autonomous parvoviruses and AAV are described
in more
detail in BERNARD N. FIELDS et al., VIROLOGY, volume 2, chapters 69 & 70 (4th
ed.,
Lippincott-Raven Publishers). See also, description of the crystal structure
of AAV2 (Xie et
al., (2002) Proc. Nat. Acad. ScL 99:10405-10), AAV4 (Padron et al., (2005) 1
ViroL 79:
14
CA 3054600 2019-09-06
5047-58), AAV5 (Walters et al., (2004)J. ViroL 78: 3361-71) and CPV (Xie et
al., (1996)1
Mol. Biol. 6:497-520 and Tsao et al., (1991) Science 251: 1456-64).
[0084] The term "tropism" as used herein refers to preferential entry of the
virus into
certain cells or tissues, optionally followed by expression (e.g.,
transcription and, optionally,
translation) of a sequence(s) carried by the viral genome in the cell, e.g.,
for a recombinant
virus, expression of a heterologous nucleic acid(s) of interest.
[0085] As used here, "systemic tropism" and "systemic transduction" (and
equivalent
terms) indicate that the virus capsid or virus vector of the invention
exhibits tropism for
and/or transduces tissues throughout the body (e.g., brain, lung, skeletal
muscle, heart, liver,
kidney and/or pancreas). In embodiments of the invention, systemic
transduction of the
central nervous system (e.g., brain, neuronal cells, etc.) is observed. In
other embodiments,
systemic transduction of cardiac muscle tissues is achieved.
[0086] As used herein, "selective tropism" or "specific tropism" means
delivery of virus
vectors to and/or specific transduction of certain target cells and/or certain
tissues.
[0087] Unless indicated otherwise, "efficient transduction" or "efficient
tropism," or similar
terms, can be determined by reference to a suitable control (e.g., at least
about 50%, 60%,
70%, 80%, 85%, 90%, 95%, 100%, 125%, 150%, 175%, 200%, 250%, 300%, 350%, 400%,
500% or more of the transduction or tropism, respectively, of the control). In
particular
embodiments, the virus vector efficiently transduces or has efficient tropism
for neuronal
cells and cardiomyocytes. Suitable controls will depend on a variety of
factors including the
desired tropism and/or transduction profile.
[0088] Similarly, it can be determined if a virus "does not efficiently
transduce" or "does
not have efficient tropism" for a target tissue, or similar terms, by
reference to a suitable
control. In particular embodiments, the virus vector does not efficiently
transduce (i.e., has
does not have efficient tropism) for liver, kidney, gonads and/or germ cells.
In particular
embodiments, transduction (e.g., undesirable transduction) of tissue(s) (e.g.,
liver) is 20% or
less, 10% or less, 5% or less, 1% or less, 0.1% or less of the level of
transduction of the
desired target tissue(s) (e.g., skeletal muscle, diaphragm muscle, cardiac
muscle and/or cells
of the central nervous system).
[0089] In some embodiments of this invention, an AAV particle comprising a
capsid of this
invention can demonstrate multiple phenotypes of efficient transduction of
certain
tissues/cells and very low levels of transduction (e.g., reduced transduction)
for certain
tissues/cells, the transduction of which is not desirable.
[0090] As used herein, the term "polypeptide" encompasses both peptides and
proteins,
CA 3054600 2019-09-06
unless indicated otherwise.
[0091] A "polynucleotide" is a sequence of nucleotide bases, and may be RNA,
DNA or
DNA-RNA hybrid sequences (including both naturally occurring and non-naturally
occurring
nucleotides), but in representative embodiments are either single or double
stranded DNA
sequences.
[0092] As used herein, an "isolated" polynucleotide (e.g., an "isolated DNA"
or an
"isolated RNA") means a polynucleotide at least partially separated from at
least some of the
other components of the naturally occurring organism or virus, for example,
the cell or viral
structural components or other polypeptides or nucleic acids commonly found
associated
with the polynucleotide. In representative embodiments an "isolated"
nucleotide is enriched
by at least about 10-fold, 100-fold, 1000-fold, 10,000-fold or more as
compared with the
starting material.
[0093] Likewise, an "isolated" polypeptide means a polypeptide that is at
least partially
separated from at least some of the other components of the naturally
occurring organism or
virus, for example, the cell or viral structural components or other
polypeptides or nucleic
acids commonly found associated with the polypeptide. In representative
embodiments an
"isolated" polypeptide is enriched by at least about 10-fold, 100-fold, 1000-
fold, 10,000-fold
or more as compared with the starting material.
[0094] An "isolated cell" refers to a cell that is separated from other
components with
which it is normally associated in its natural state. For example, an isolated
cell can be a cell
in culture medium and/or a cell in a pharmaceutically acceptable carrier of
this invention.
Thus, an isolated cell can be delivered to and/or introduced into a subject.
In some
embodiments, an isolated cell can be a cell that is removed from a subject and
manipulated as
described herein ex vivo and then returned to the subject.
[0095] A population of virions can be generated by any of the methods
described herein.
In one embodiment, the population is at least 10' virions. In one embodiment,
the population
is at least 102 virions, at least 103, virions, at least 104 virions, at least
105 virions, at least 106
virions, at least 107 virions, at least 108 virions, at least 109 virions, at
least 1010 virions, at
least 1011 virions, at least 1012 virions, at least 1013 virions, at least
1014 virions, at least 1015
virions, at least 1016 virions, or at least 1017 virions. A population of
virions can be
heterogeneous or can be homogeneous (e.g., substantially homogeneous or
completely
homogeneous).
[0096] A "substantially homogeneous population" as the term is used herein,
refers to a
population of virions that are mostly identical, with few to no contaminant
virions (those that
16
CA 3054600 2019-09-06
are not identical) therein. A
substantially homogeneous population is at least 90% of
identical virions (e.g., the desired virion), and can be at least 91%, at
least 92%, at least 93%,
at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least
99%, at least
99.5%, at least 99.9% of identical virions.
[0097] A population of virions that is completely homogeneous contains only
identical
virions.
[0098] As used herein, by "isolate" or "purify" (or grammatical equivalents) a
virus vector
or virus particle or population of virus particles, it is meant that the virus
vector or virus
particle or population of virus particles is at least partially separated from
at least some of the
other components in the starting material. In representative embodiments an
"isolated" or
"purified" virus vector or virus particle or population of virus particles is
enriched by at least
about 10-fold, 100-fold, 1000-fold, 10,000-fold or more as compared with the
starting
material.
[0099] A "therapeutic polypeptide" is a polypeptide that can alleviate,
reduce, prevent,
delay and/or stabilize symptoms that result from an absence or defect in a
protein in a cell or
subject and/or is a polypeptide that otherwise confers a benefit to a subject,
e.g., anti-cancer
effects or improvement in transplant survivability or induction of an immune
response.
[00100] By the terms "treat," "treating," or "treatment of' (and grammatical
variations
thereof) it is meant that the severity of the subject's condition is reduced,
at least partially
improved or stabilized and/or that some alleviation, mitigation, decrease or
stabilization in at
least one clinical symptom is achieved and/or there is a delay in the
progression of the disease
or disorder.
[00101] The terms "prevent," "preventing" and "prevention" (and grammatical
variations
thereof) refer to prevention and/or delay of the onset of a disease, disorder
and/or a clinical
symptom(s) in a subject and/or a reduction in the severity of the onset of the
disease, disorder
and/or clinical symptom(s) relative to what would occur in the absence of the
methods of the
invention. The prevention can be complete, e.g., the total absence of the
disease, disorder
and/or clinical symptom(s). The prevention can also be partial, such that the
occurrence of
the disease, disorder and/or clinical symptom(s) in the subject and/or the
severity of onset is
substantially less than what would occur in the absence of the present
invention.
[00102] A "treatment effective" amount as used herein is an amount that is
sufficient to
provide some improvement or benefit to the subject. Alternatively stated, a
"treatment
effective" amount is an amount that will provide some alleviation, mitigation,
decrease or
stabilization in at least one clinical symptom in the subject. Those skilled
in the art will
17
CA 3054600 2019-09-06
appreciate that the therapeutic effects need not be complete or curative, as
long as some
benefit is provided to the subject.
[00103] A "prevention effective" amount as used herein is an amount that is
sufficient to
prevent and/or delay the onset of a disease, disorder and/or clinical symptoms
in a subject
and/or to reduce and/or delay the severity of the onset of a disease, disorder
and/or clinical
symptoms in a subject relative to what would occur in the absence of the
methods of the
invention. Those skilled in the art will appreciate that the level of
prevention need not be
complete, as long as some preventative benefit is provided to the subject.
[00104] The terms "heterologous nucleotide sequence" and "heterologous nucleic
acid
molecule" are used interchangeably herein and refer to a nucleic acid sequence
that is not
naturally occurring in the virus. Generally, the heterologous nucleic acid
molecule or
heterologous nucleotide sequence comprises an open reading frame that encodes
a
polypeptide and/or nontranslated RNA of interest (e.g., for delivery to a cell
and/or subject).
[00105] As used herein, the terms "virus vector," "vector" or "gene delivery
vector" refer to
a virus (e.g., AAV) particle that functions as a nucleic acid delivery
vehicle, and which
comprises the vector genome (e.g., viral DNA [vDNA]) packaged within a virion.
Alternatively, in some contexts, the term "vector" may be used to refer to the
vector
genome/vDNA alone.
[00106] A "rAAV vector genome" or "rAAV genome" is an AAV genome (i.e., vDNA)
that
comprises one or more heterologous nucleic acid sequences. rAAV vectors
generally require
only the terminal repeat(s) (TR(s)) in cis to generate virus. All other viral
sequences are
dispensable and may be supplied in trans (Muzyczka, (1992) Curr. Topics
Microbiol.
Immunol. 158:97). Typically, the rAAV vector genome will only retain the one
or more TR
sequence so as to maximize the size of the transgene that can be efficiently
packaged by the
vector. The structural and non- structural protein coding sequences may be
provided in trans
(e.g., from a vector, such as a plasmid, or by stably integrating the
sequences into a
packaging cell). In embodiments of the invention the rAAV vector genome
comprises at
least one TR sequence (e.g., AAV TR sequence), optionally two TRs (e.g., two
AAV TRs),
which typically will be at the 5' and 3' ends of the vector genome and flank
the heterologous
nucleic acid, but need not be contiguous thereto. The TRs can be the same or
different from
each other.
[00107] The term "terminal repeat" or "TR" includes any viral terminal repeat
or synthetic
sequence that forms a hairpin structure and functions as an inverted terminal
repeat (i.e.,
mediates the desired functions such as replication, virus packaging,
integration and/or
18
CA 3054600 2019-09-06
provirus rescue, and the like). The TR can be an AAV TR or a non-AAV TR. For
example,
a non-AAV TR sequence such as those of other parvoviruses (e.g., canine
parvovirus (CPV),
mouse parvovirus (MVM), human parvovirus B-19) or any other suitable virus
sequence
(e.g., the SV40 hairpin that serves as the origin of SV40 replication) can be
used as a TR,
which can further be modified by truncation, substitution, deletion, insertion
and/or addition.
Further, the TR can be partially or completely synthetic, such as the "double-
D sequence" as
described in United States Patent No. 5,478,745 to Samulski et al.
[00108] An "AAV terminal repeat" or "AAV TR" may be from any AAV, including
but not
limited to serotypes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 or any other AAV
now known or later
discovered (see, e.g., Table 1). An AAV terminal repeat need not have the
native terminal
repeat sequence (e.g., a native AAV TR sequence may be altered by insertion,
deletion,
truncation and/or missense mutations), as long as the terminal repeat mediates
the desired
functions, e.g., replication, virus packaging, integration, and/or provirus
rescue, and the like.
[00109] AAV proteins VP1, VP2 and VP3 are capsid proteins that interact
together to form
an AAV capsid of an icosahedral symmetry. VP! .5 is an AAV capsid protein
described in
US Publication No. 2014/0037585.
[00110] The virus vectors of the invention can further be "targeted" virus
vectors (e.g.,
having a directed tropism) and/or a "hybrid" parvovirus (i.e., in which the
viral TRs and viral
capsid are from different parvoviruses) as described in international patent
publication WO
00/28004 and Chao et al., (2000) Molecular Therapy 2:619.
[00111] The virus vectors of the invention can further be duplexed parvovirus
particles as
described in international patent publication WO 01/92551 (the disclosure of
which is
incorporated herein by reference in its entirety). Thus, in some embodiments,
double
stranded (duplex) genomes can be packaged into the virus capsids of the
invention.
[00112] Further, the viral capsid or genomic elements can contain other
modifications,
including insertions, deletions and/or substitutions.
[00113] A "chimeric" viral structural protein as used herein means an AAV
viral structural
protein (capsid) that has been modified by substitutions in one or more (e.g.,
2, 3, 4, 5, 6, 7, 8,
9, 10, etc.) amino acid residues in the amino acid sequence of the capsid
protein relative to
wild type, as well as insertions and/or deletions of one or more (e.g., 2, 3,
4, 5, 6, 7, 8, 9, 10,
etc.) amino acid residues in the amino acid sequence relative to wild type. In
some
embodiments, complete or partial domains, functional regions, epitopes, etc.,
from one AAV
serotype can replace the corresponding wild type domain, functional region,
epitope, etc. of a
different AAV serotype, in any combination, to produce a chimeric capsid
protein of this
19
CA 3054600 2019-09-06
invention. In other embodiments the substitutions are all from the same
serotype. In other
embodiments the substitutions are all from AAV or synthetic. Production of a
chimeric
capsid protein can be carried out according to protocols well known in the art
and a large
number of chimeric capsid proteins are described in the literature as well as
herein that can be
included in the capsid of this invention.
[00114] In an alternative embodiment, a virion particle can be constructed
wherein at least
one viral protein from the group consisting of AAV capsid proteins, VP1, VP2
and VP3, is
different from at least one of the other viral proteins, required to form the
virion particle
capable of encapsidating an AAV genome. For each viral protein present (VP1,
VP2, and/or
VP3), that protein is the same type (e.g.,all AAV2 VP!). In one instance, at
least one of the
viral proteins is a chimeric viral protein and at least one of the other two
viral proteins is not a
chimeric. In one embodiment VP! and VP2 are chimeric and only VP3 is non-
chimeric. For
example, only the viral particle composed of VP1NP2 from the chimeric AAV2/8
(the N-
terminus of AAV2 and the C-terminus of AAV8) paired with only VP3 from AAV2;
or only
the chimeric VP1NP2 28m-2P3 (the N-terminal from AAV8 and the C-terminal from
AAV2
without mutation of VP3 start codon) paired with only VP3 from AAV2. In
another
embodiment only VP3 is chimeric and VP! and VP2 are non-chimeric. In another
embodiment at least one of the viral proteins is from a completely different
serotype. For
example, only the chimeric VP1/VP2 28m-2P3 paired with VP3 from only AAV3. In
another
example, no chimeric is present.
[00115] As used herein, the term "amino acid" encompasses any naturally
occurring amino
acid, modified forms thereof, and synthetic amino acids.
[00116] Naturally occurring, levorotatory (L-) amino acids are shown in Table
2.
[00117] Alternatively, the amino acid can be a modified amino acid residue
(nonlimiting
examples are shown in Table 4) and/or can be an amino acid that is modified by
post-
translation modification (e.g., acetylation, amidation, formylation,
hydroxylation,
methylation, phosphorylation or sulfatation).
[00118] Further, the non-naturally occurring amino acid can be an "unnatural"
amino acid as
described by Wang et al., Annu Rev Biophys Biomol Struct. 35:225-49 (2006).
These
unnatural amino acids can advantageously be used to chemically link molecules
of interest to
the AAV capsid protein.
[00119] As used herein, the term "homologous recombination" means a type of
genetic
recombination in which nucleotide sequences are exchanged between two similar
or identical
molecules of DNA. Homologous recombination also produces new combinations of
DNA
CA 3054600 2019-09-06
sequences. These new combinations of DNA represent genetic variation.
Homologous
recombination is also used in horizontal gene transfer to exchange genetic
material between
different strains and species of viruses.
[00120] As used herein, the term "gene editing," "Genome editing," or "genome
engineering" means a type of genetic engineering in which DNA is inserted,
deleted or
replaced in the genome of a living organism using engineered nucleases, or
"molecular
scissors." These nucleases create site-specific double-strand breaks (DSBs) at
desired
locations in the genome.
[00121] As used herein, the term "gene delivery" means a process by which
foreign DNA is
transferred to host cells for applications of gene therapy.
[00122] As used herein, the term "CRISPR" stands for Clustered Regularly
Interspaced
Short Palindromic Repeats, which are the hallmark of a bacterial defense
system that forms
the basis for CRISPR-Cas9 genome editing technology.
[00123] As used herein, the term "zinc finger" means a small protein
structural motif that is
characterized by the coordination of one or more zinc ions, in order to
stabilize the fold.
[00124] In some embodiments, the AAV particle of this invention can be
synthetic viral
vector designed to display a range of desirable phenotypes that are suitable
for different in
vitro and in vivo applications. Thus, in one embodiment, the present invention
provides an
AAV particle comprising an adeno-associated virus (AAV).
[00125] The present invention provides an array of synthetic viral vectors
displaying a range
of desirable phenotypes that are suitable for different in vitro and in vivo
applications. In
particular, the present invention is based on the unexpected discovery that
combining capsid
proteins from different AAV serotypes in an individual capsid allows for the
development of
improved AAV capsids that have multiple desirable phenotypes in each
individual capsid.
Such chimeric or shuffled virions are sometimes referred to as polyploid,
haploid, or triploid
to refer to the fact that the capsid proteins VP!, VP2, and VP3 come from at
least two
different serotypes. New methods for producing such virions are described
herein. By
preventing the translation of undesired open reading frames these methods
result in the
production of homogeneous populations of the generated virions.
[00126] The ability to generate a homogeneous (e.g., substantially or
completely) population
of recombinant virions dramatically reduces or eliminates carryover of
properties of
undesired/contaminating virions (e.g., transduction specificity or
antigenicity).
[00127] The AAV virion has T=1 icosahedral symmetry and is composed of the
three
structural viral proteins, VP1, VP2, and VP3. 60 copies of the three viral
proteins in a ratio of
21
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1:1:8 to 10 (VP1:VP2:VP3, respectively) form the virion (Rayaprolu, V., et
al., J. Virol.
87(24): 13150-13160(2013).
[00128] In one embodiment, the AAV virion is an isolated virion that has at
least one of the
viral structural proteins, VP1, VP2, and VP3 from a different serotype than
the other VPs,
and each VP is only from one serotype. For example, the VP1 is only from AAV2,
the VP2 is
only from AAV4, and the VP3 is only from AAV8.
[00129] In one embodiment an AAV virion that encapsidates an AAV genome
including a
heterologous gene between 2 AAV ITRs can be formed with only two of the viral
structural
proteins, VP1 and VP3. In one embodiment this virion is conformationally
correct, i.e., has
T=1 icosahedral symmetry. In one embodiment the virions are infectious.
[00130] Infectious virions include VP1/VP3 VP1/VP2/VP3. Typically VP2/VP3 and
VP3
only virions are not infectious.
[00131] The viral structural proteins used to generate these populations of
virions can be
from any of the 12 serotypes of AAV isolated for gene therapy, other species,
mutant
serotypes, shuffled serotypes of such genes, e.g., AAV2, VP1.5 and AAV4 VP2,
AAV4 VP3,
or any other AAV serotype desired.
[00132] For example, triploid AAV2/8/9 vector described herein, which is
produced by co-
transfection of AAV helper plasmids from serotypes 2, 8 and 9, has a much
higher mouse
liver transduction than AAV2, similar to AAV8. Importantly, triploid AAV2/8/9
vector has
an improved ability to escape neutralizing antibodies from sera immunized with
parental
serotypes. Although AAV3 is less efficient in transducing the whole mouse body
after
systemic administration, the haploid vectors H-AAV83 or H-AAV93 or H-rh10-3
described
herein, in which VP3 is from AAV3 and VP1/VP2 from AAV8, 9 or rh10, induce
whole
body transduction, as well as much higher transduction in the liver and other
tissues,
compared to AAV3.
[00133] Thus, in one embodiment, the present invention provides an adeno-
associated virus
(AAV) with a viral capsid, wherein the capsid comprises the protein VP1,
wherein said VP1
is from one or more than one first AAV serotype and capsid protein VP3,
wherein said capsid
protein VP3 is from one or more than one second AAV serotype and wherein at
least one of
said first AAV serotype is different from at least one of said second AAV
serotype, in any
combination. When at least one viral structural protein is from more than one
serotype we are
referring to the phenomenon sometimes referred to as crossdressing, which
results in a
mosaic capsid. On the other hand when the viral capsid proteins are each from
the same
serotype, even though at least one of the viral proteins is from a different
serotype, a mosaic
22
CA 3054600 2019-09-06
capsid does not result. For example VP! from AAV2, VP2 from AAV6, and VP3 from
AAV8.
[00134] In some embodiments, the capsid of this invention comprises capsid
protein VP2,
wherein said capsid protein VP2 is from one or more than one third AAV
serotype, wherein
at least one of said one or more than one third AAV serotype is different from
said first AAV
serotype and/or said second AAV serotype, in any combination. In some
embodiments, the
AAV capsid described herein can comprise capsid protein VP1.5. VP1.5 is
described in U.S.
Patent Publication No. 2014/0037585 and the amino acid sequence of VP1.5 is
provided
herein.
[00135] In some embodiments only virions that contain at least one viral
protein that is
different than the other viral proteins are produced. For example, VP1 and VP2
from the
same serotype and VP3 from an alternative serotype, only. In other
embodiments, the VP1 is
from one serotype and the VP2 and VP3 are from another serotype, only. In
another
embodiment, only particles where VP1 is from one serotype, VP2 is from a
second serotype,
and VP3 is from yet another serotype are produced.
[00136] This can be done by, for example, site specific deletions, and/or
additions, changing
splice donor sites, splice acceptor sites, start codons and combinations
thereof.
[00137] This permits methods for producing populations of substantially
homogenous
populations of the polyploid virions ¨ such as the haploid or triploid viral
particles.
[00138] In some embodiments the AAV virion can be formed by more than the
typical 3
viral structural proteins, VP!, VP2, and VP3 (see e.g., Wang, Q. et al.,
"Syngeneic AAV
Pseudo-particles Potentiate Gene Transduction of AAV Vectors," Molecular
Therapy:
Methods and Clinical Development, Vol. 4, 149-158 (2017)). Such viral capsids
also fall
within the present invention. For example, an isolated AAV virion having viral
capsid
structural proteins sufficient to form an AAV virion that encapsidates an AAV
genome,
wherein at least one of the viral capsid structural proteins is different from
the other viral
capsid structural proteins, and wherein each viral capsid structural protein
is only of the same
type. In a further embodiment the isolated AAV virion has at least two viral
structural
proteins from the group consisting of AAV capsid proteins, VP!, VP2, VP1.5 and
VP3,
wherein the two viral proteins are sufficient to form an AAV virion that
encapsidates an
AAV genome, and wherein at least one of the viral structural proteins present
is from a
different serotype than the other viral structural protein, and wherein the
VP1 is only from
one serotype, the VP2 is only from one serotype, the VP1.5 is only from one
serotype, and
the VP3 is only from one serotype. For example, the VP1.5 can be from AAV
serotype 2 and
23
CA 3054600 2019-09-06
the VP3 can be from AAV serotype 8.
[00139] In some embodiments, the capsid of this invention comprises capsid
protein VP1.5,
wherein said capsid protein VP1.5 is from one or more than one fourth AAV
serotype,
wherein at least one of said one or more than one fourth AAV serotype is
different from said
first AAV serotype and/or said second AAV serotype, in any combination. In
some
embodiments, the AAV viral structural protein described herein can comprise
viral structural
protein VP2.
[00140] The present invention also provides an AAV capsid wherein the capsid
comprises
capsid protein VP1, wherein said capsid protein VP1 is from one or more than
one first AAV
serotype and capsid protein VP2, wherein said capsid protein VP2 is from one
or more than
one second AAV serotype and wherein at least one of said first AAV serotype is
different
from at least one of said second AAV serotype, in any combination. In some
embodiments no
chimeric viral structural protein is present in the virion.
[00141] In some embodiments, the AAV particle of this invention can comprise a
capsid that
comprises capsid protein VP3, wherein said capsid protein VP3 is from one or
more than one
third AAV serotype, wherein at least one of said one or more than one third
AAV serotype is
different from said first AAV serotype and/or said second AAV serotype, in any
combination. In some embodiments, the AAV capsid described herein can comprise
capsid
protein VP1.5.
[00142] The present invention further provides an AAV particle that comprises
an adeno-
associated virus (AAV) capsid, wherein the capsid comprises capsid protein
VP1, wherein
said capsid protein VP1 is from one or more than one first AAV serotype and
capsid protein
VP1.5, wherein said capsid protein VP1.5 is from one or more than one second
AAV
serotype and wherein at least one of said first AAV serotype is different from
at least one of
said second AAV serotype, in any combination.
[00143] In some embodiments, the capsid comprises capsid protein VP3, wherein
said
capsid protein VP3 is from one or more than one third AAV serotype, wherein at
least one of
said one or more than one third AAV serotype is different from said first AAV
serotype
and/or said second AAV serotype, in any combination. In some embodiments, the
AAV
capsid described herein can comprise capsid protein VP1.5.
[00144] The present invention further provides an adeno-associated virus (AAV)
capsid,
wherein the capsid comprises capsid protein VP1, wherein said capsid protein
VP1 is from
one or more than one first AAV serotype and capsid protein VP1.5, wherein said
capsid
protein VP1.5 is from one or more than one second AAV serotype and wherein at
least one of
24
CA 3054600 2019-09-06
said first AAV serotype is different from at least one of said second AAV
serotype, in any
combination.
[00145] In some embodiments, the AAV capsid of this invention comprises capsid
protein
VP3, wherein said capsid protein VP3 is from one or more than one third AAV
serotype,
wherein at least one of said one or more than one third AAV serotype is
different from said
first AAV serotype and/or said second AAV serotype, in any combination. In
some
embodiments, the AAV capsid protein described herein can comprise capsid
protein VP2.
[00146] In some embodiments of the capsid of this invention, said one or more
than one first
AAV serotype, said one or more than one second AAV serotype, said one or more
than one
third AAV serotype and said one or more than one fourth AAV serotype are
selected from the
group consisting of the AAV serotypes listed in Table 1, in any combination.
[00147] In some embodiments of this invention, the AAV capsid described herein
lacks
capsid protein VP2.
[00148] In some embodiments of the capsid of this invention comprises a
chimeric capsid
VP1 protein, a chimeric capsid VP2 protein, a chimeric capsid VP3 protein
and/or a chimeric
capsid VP1.5 protein.
[00149] In some embodiments, the AAV capsid of this invention can be AAV
AAV2/8/9, H-
AAV82, H-AAV92, H-AAV82G9, AAV2/8 3:1, AAV2/8 1:1, AAV2/8 1:3, or AAV8/9, all
of which are described in the EXAMPLES section provided herein.
[00150] Nonlimiting examples of AAV capsid proteins that can be included in
the capsid of
this invention in any combination with other capsid proteins described herein
and/or with
other capsid proteins now known or later developed, include LK3, LK01-19, AAV-
DJ,
Olig001, rAAV2-retro, AAV-LiC, AAVOKeral, AAV-Kera2, AAV-Kera3, AAV 7m8,
AAV1,9, AAVr3.45, AAV clone 32, AAV clone 83, AAV-U87R7-05, AAV ShH13, AAV
ShH19, AAV L1-12, AAV HAE-1, AAV HAE-2, AAV variant ShH10, AAV2.5T, AAV
LS1-4, AAV Lsm, AAV1289, AAVHSC 1-17, AAV2 Rec 1-4, AAV8BP2, AAV-B1, AAV-
PHP.B, AAV9.45, AAV9.61, AAV9.47, AAVM41, AAV2 displayed peptides, AAV2-GMN,
AAV9-peptide displayed, AAV8 and AAV9 peptide displayed, AAVpo2.1, AAVpo4,
AAVpo5, AAVpo6, AAV rh, AAV Hu, AAV-Go.1, AAV-mo.1, BAAV, AAAV, AAV8
K137R, AAV Anc80L65, AAV2G9, AAV2 265 insertion-AAV2/265D, AAV2.5, AAV3
SASTG, AAV2i8, AAV8G9, AAV2 tyrosine mutants AAV2 Y-F, AAV8 Y-F, AAV9 Y-F,
AAV6 Y-F, AAV6.2 and any combination thereof.
[00151] As a nonlimiting example, the AAV capsid proteins and virus capsids of
this
invention can be chimeric in that they can comprise all or a portion of a
capsid subunit from
CA 3054600 2019-09-06
another virus, optionally another parvovirus or AAV, e.g., as described in
international patent
publication WO 00/28004.
[00152] The following publications describe chimeric or variant capsid
proteins that can be
incorporated into the AAV capsid of this invention in any combination with
wild type capsid
proteins and/or other chimeric or variant capsid proteins now known or later
identified.
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Rajalingam S, Ramya V, Nair SC, Srinivasan N, Srivastava A, Jayandharan GR.
Targeted
modifications in adeno-associated virus serotype 8 capsid improves its hepatic
gene transfer
efficiency in vivo. Hum Gene Ther Methods. 2013 Apr: 24(2):104-16. (AAV8
K137R).
[00194] Li B, Ma W, Ling C, Van Vliet K, Huang LY, Agbandje-McKenna M,
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A, Aslanidi GV. Site-Directed Mutagenesis of Surface-Exposed Lysine Residues
Leads to
Improved Transduction by AAV2, But Not AAV8, Vectors in Murine Hepatocytes In
Vivo.
Hum Gene Ther Methods. 2015 Dec: 26(6):211-20.
[00195] Gabriel N, Hareendran S, Sen D, Gadkari RA, Sudha G, Selot R, Hussain
M,
Dhaksnamoorthy R, Samuel R, Srinivasan N, et al. Bioengineering of AAV2 capsid
at
specific serine, threonine, or lysine residues improves its transduction
efficiency in vitro and
in vivo. Hum Gene Ther Methods. 2013 Apr: 24(2):80-93.
[00196] Zinn E, Pacouret S, Khaychuk V, Turunen HT, Carvalho LS, Andres-Mateos
E,
Shah S, Shelke R, Maurer AC, Plovie E, Xiao R, Vandenberghe LH. In Silico
Reconstruction
of the Viral Evolutionary Lineage Yields a Potent Gene Therapy Vector. Cell
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[00197] Shen S, Horowitz ED, Troupes AN, Brown SM, Pulicherla N, Samulski RJ,
Agbandje-McKenna M, Asokan A. Engraftment of a galactose receptor footprint
onto adeno-
associated viral capsids improves transduction efficiency. J Biol Chem. 2013
Oct 4:
288(40):28814-23. (AAV2G9).
[00198] Li C, Diprimio N, Bowles DE, Hirsch ML, Monahan PE, Asokan A,
Rabinowitz J,
Agbandje-McKenna M, Samulski RJ. Single amino acid modification of adeno-
associated
virus capsid changes transduction and humoral immune profiles. I Virol. 2012
Aug:
86(15):7752-9. (AAV2 265 insertion-AAV2/265D).
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[00199] Bowles DE, McPhee SW, Li C, Gray SJ, Samulski JJ, Camp AS, Li J, Wang
B,
Monahan PE, Rabinowitz JE, et al. Phase 1 gene therapy for Duchenne muscular
dystrophy
using a translational optimized AAV vector. Mol. Ther. 2012 Feb: 20(2):443-55.
(AAV2.5).
[00200] Messina EL, Nienaber J, Daneshmand M, Villamizar N, Samulski J, Milano
C,
Bowles DE. Adeno-associated viral vectors based on serotype 3b use components
of the
fibroblast growth factor receptor signaling complex for efficient
transduction. Hum. Gene
Ther. 2012 Oct: 23(10):1031-42. (AAV3 SASTG).
[00201] Asokan A, Conway JC, Phillips JL, Li C, Hegge J, Sinnott R, Yadav S,
DiPrimio N,
Nam HJ, Agbandje-McKenna M, McPhee S, Wolff J, Samulski RJ. Reengineering a
receptor footprint of adeno-associated virus enables selective and systemic
gene transfer to
muscle. Nat Biotechnol. 2010 Jan: 28(1):79-82. (AAV2i8).
[00202] Vance M, Llanga T, Bennett W, Woodard K, Murlidharan G, Chungfat N,
Asokan
A, Gilger B, Kurtzberg J, Samulski RJ, Hirsch ML. AAV Gene Therapy for MPS1-
associated
Corneal Blindness. Sci Rep. 2016 Feb 22: 6:22131. (AAV8G9).
[00203] Zhong L, Li B, Mah CS, Govindasamy L, Agbandje-McKenna M, Cooper M,
Herzog RW, Zolotukhin I, Warrington KH Jr, Weigel-Van Aken KA, Hobbs JA,
Zolotukhin
S, Muzyczka N, Srivastava A. Next generation of adeno-associated virus 2
vectors: point
mutations in tyrosines lead to high-efficiency transduction at lower doses.
Proc Natl Acad Sci
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[00204] Petrs-Silva H, Dinculescu A, Li Q, Min SH, Chiodo V, Pang JJ, Zhong L,
Zolotukhin S, Srivastava A, Lewin AS, Hauswirth WW. High-efficiency
transduction of the
mouse retina by tyrosine-mutant AAV serotype vectors. Mol. Ther. 2009 Mar:
17(3):463-71.
(AAV8 V-F and AAV9 V-F).
[00205] Qiao C, Zhang W, Yuan Z, Shin JH, Li J, Jayandharan GR, Zhong L,
Srivastava A,
Xiao X, Duan D. Adeno-associated virus serotype 6 capsid tyrosine-to-
phenylalanine
mutations improve gene transfer to skeletal muscle. Hum Gene Ther. 2010 Oct:
21(10):1343-
8 (AAV6 V-F).
[00206] Carlon M, Toelen J, Van der Perren A, Vandenberghe LH, Reumers V,
Sbragia L,
Gijsbers R, Baekelandt V, Himmelreich U, Wilson JM, Deprest J, Debyser Z.
Efficient gene
transfer into the mouse lung by fetal intratracheal injection of rAAV2/6.2.
MoL Ther. 2010
Dec: 18(12):2130-8. (AAV6.2).
[00207] PCT Publication No. W02013158879A1. (lysine mutants).
[00208] The following biological sequence files listed in the file wrappers of
USPTO issued
patents and published applications describe chimeric or variant capsid
proteins that can be
31
CA 3054600 2019-09-06
incorporated into the AAV capsid of this invention in any combination with
wild type capsid
proteins and/or other chimeric or variant capsid proteins now known or later
identified (for
demonstrative purposes, U.S. Patent Application No. 11/486,254 corresponds to
U.S. Patent
Application No. 11/486,254): 11486254.raw, 11932017.raw, 12172121.raw,
12302206.raw,
12308959.raw, 12679144.raw, 13036343.raw, 13121532.raw, 13172915.raw,
13583920.raw,
13668120.raw, 13673351.raw, 13679684.raw, 14006954.raw, 14149953.raw,
14192101.raw,
14194538.raw, 14225821.raw, 14468108.raw, 14516544.raw, 14603469.raw,
14680836.raw,
14695644.raw, 14878703.raw, 14956934.raw, 15191357.raw, 15284164.raw,
15368570.raw,
15371188.raw, 15493744.raw, 15503120.raw, 15660906.raw, and 15675677.raw.
[00209] It would be understood that any combination of VP1 and VP3, and when
present,
VP1.5 and VP2 from any combination of AAV serotypes can be employed to produce
the
AAV capsids of this invention. For example, a VP1 protein from any combination
of AAV
serotypes can be combined with a VP3 protein from any combination of AAV
serotypes and
the respective VP1 proteins can be present in any ratio of different serotypes
and the
respective VP3 proteins can be present in any ratio of different serotypes and
the VP1 and
VP3 proteins can be present in any ratio of different serotypes. It would be
further
understood that, when present, a VP! .5 and/or VP2 protein from any
combination of AAV
serotypes can be combined with VP1 and VP3 protein from any combination of AAV
serotypes and the respective VP1 .5 proteins can be present in any ratio of
different serotypes
and the respective VP2 proteins can be present in any ratio of different
serotypes and the
respective VP1 proteins can be present in any ratio of different serotypes and
the respective
VP3 proteins can be present in any ratio of different serotypes and the VP! .5
and/or VP2
proteins can be present in combination with VP1 and VP3 proteins in any ratio
of different
serotypes.
[00210] For example, the respective viral proteins and/or the respective AAV
serotypes can
be combined in any ratio, which can be a ratio of A:B, A:B:C, A:B:C:D,
A:B:C:D:E,
A:B:C:D:E:F, A:B:C:D:E:F:G, A:B:C:D:E:F:G:H, A:B:C:D:E:F:G:H:I
or
A:B:C:D:E:F:G:H:I:J, wherein A can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,
13, 14, 15, 20, 25,
30, 35, 40, 50, 60, 70, 80, 90, 100, etc.; B can be 1, 2, 3, 4, 5, 6, 7, 8, 9,
10, 11, 12, 13, 14, 15,
20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, etc.; C can be 1, 2, 3, 4, 5, 6,
7, 8, 9, 10, 11, 12, 13,
14, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, etc.; D can be 1, 2, 3,
4, 5, 6, 7, 8, 9, 10, 11,
12, 13, 14, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, etc.; E can be 1,
2, 3, 4, 5, 6, 7, 8, 9,
10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, etc.; F
can be 1, 2, 3, 4, 5,6,
7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100,
etc.; G can be 1,2, 3,
32
CA 3054600 2019-09-06
4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80,
90, 100, etc.; H can be
1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 50, 60,
70, 80, 90, 100, etc.; I
can be 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40,
50, 60, 70, 80, 90, 100,
etc.; and J can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25,
30, 35, 40, 50, 60, 70,
80, 90, 100, etc.
[00211] It would also be understood that any of the VP1, VP1.5, VP2 and/or VP3
capsid
proteins can be present in a capsid of this invention as a chimeric capsid
protein, in any
combination and ratio relative to the same protein type and/or relative to the
different capsid
proteins.
[00212] In further embodiments, the present invention further provides a virus
vector
comprising, consisting essentially of and/or consisting of (a) the AAV capsid
of this
invention; and (b) a nucleic acid molecule comprising at least one terminal
repeat sequence,
wherein the nucleic acid molecule is encapsidated by the AAV capsid. In
some
embodiments, the virus vector can be an AAV particle.
[00213] In some embodiments, the virus vector of this invention can have
systemic or
selective tropism for skeletal muscle, cardiac muscle and/or diaphragm muscle.
In some
embodiments, the virus vector of this invention can have reduced tropism for
liver.
[00214] The present invention further provides a composition, which can be a
pharmaceutical formulation, comprising the capsid protein, capsid, virus
vector, AAV
particle composition and/or pharmaceutical formulation of this invention and a
pharmaceutically acceptable carrier.
[00215] In some nonlimiting examples, the present invention provides AAV
capsid proteins
(VP1, VP1.5, VP2 and/or VP3) comprising a modification in the amino acid
sequence in the
three-fold axis loop 4 (Opie et al., J. Viral. 77: 6995-7006 (2003)) and virus
capsids and virus
vectors comprising the modified AAV capsid protein. The inventors have
discovered that
modifications in this loop can confer one or more desirable properties to
virus vectors
comprising the modified AAV capsid protein including without limitation (i)
reduced
transduction of liver, (ii) enhanced movement across endothelial cells, (iii)
systemic
transduction; (iv) enhanced transduction of muscle tissue (e.g., skeletal
muscle, cardiac
muscle and/or diaphragm muscle), and/or (v) reduced transduction of brain
tissues (e.g.,
neurons). Thus, the present invention addresses some of the limitations
associated with
conventional AAV vectors. For example, vectors based on AAV8 and rAAV9 vectors
are
attractive for systemic nucleic acid delivery because they readily cross the
endothelial cell
barrier; however, systemic administration of rAAV8 or rAAV9 results in most of
the vector
33
CA 3054600 2019-09-06
being delivered to the liver, thereby reducing transduction of other important
target tissues
such as skeletal muscle.
[00216] In an embodiment, the modified AAV capsid can be comprised of a VP1, a
VP2
and/or a VP3 that is created through DNA shuffling to develop cell type
specific vectors
through directed evolution. DNA shuffling with AAV is generally descried in
Li, W. et al.,
Mol. Ther. 16(7): 1252 ¨ 12260 (2008), which is incorporated herein by
reference. In an
embodiment, DNA shuffling can be used to create a VP1, a VP2 and/or a VP3
using the
DNA sequence for the capsid genes from two or more different AAV serotypes,
AAV
chimerics or other AAV. In an embodiment, a haploid AAV can be comprised of a
VP1
created by DNA shuffling, a VP2 created by DNA shuffling and/or a VP3 created
by DNA
shuffling.
[00217] In an embodiment, a VP1 from a haploid AAV could be created by
randomly
fragmenting the capsid genomes of AAV2, AAV8 and AAV9 using a restriction
enzyme
and/or DNase to generate a VP1 capsid protein library comprised of portions of
AAV2/8/9.
In this embodiment, the AAV2/8/9 VP1 capsid protein created by DNA shuffling
could be
combined with a VP2 and/or a VP3 protein from a different serotype, in an
embodiment,
from AAV3. This would result in a haploid AAV wherein the capsid is comprised
of a VP1
that includes amino acids from AAV2, AAV8 and AAV9 that are joined together
randomly
through DNA shuffling and the VP2 and/or VP3 comprise only amino acids from a
native,
AAV3 VP2 and/or VP3. In an embodiment, the donor to create a VP1, VP2 and/or a
VP3
can be any AAV, including, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8,
AAV9, AAV10, AAV11, AAV chimerics or other AAV, or those selected from Table 1
or
Table 3. In certain embodiments, the shuffled VP1 expresses e.g.,only VP1, or
only
VP1/VP2, or only VP3.
[00218] In another embodiment, the nucleic acid encoding VP1, VP2 and/or VP3
can be
created through DNA shuffling. In one embodiment, a first nucleic acid created
by DNA
shuffling would encode VP1. In this same embodiment, a second nucleic acid
created by
DNA shuffling would encode VP2 and VP3. In another embodiment, a first nucleic
acid
created by DNA shuffling would encode VP1. In this same embodiment, a second
nucleic
acid created by DNA shuffling would encode VP2 and a third nucleic acid would
encode
VP3. In a further embodiment, a first nucleic acid created by DNA shuffling
would encode
VP1 and VP2 and a second nucleic acid created by DNA shuffling would encode
VP3. In an
additional embodiment, a first nucleic acid created by DNA shuffling would
encode VP1 and
VP3 and a second nucleic acid created by DNA shuffling would encode VP2.
34
CA 3054600 2019-09-06
[00219] In embodiments of the invention, transduction of cardiac muscle and/or
skeletal
muscle (determined on the basis of an individual skeletal muscle, multiple
skeletal muscles,
or the whole range of skeletal muscles) is at least about five-fold, ten-fold,
50-fold, 100-fold,
1000-fold or higher than transduction levels in liver.
[00220] In particular embodiments, the modified AAV capsid protein of the
invention
comprises one or more modifications in the amino acid sequence of the three-
fold axis loop 4
(e.g., amino acid positions 575 to 600 [inclusive] of the native AAV2 VP1
capsid protein or
the corresponding region of a capsid protein from another AAV). As used
herein, a
"modification" in an amino acid sequence includes substitutions, insertions
and/or deletions,
each of which can involve one, two, three, four, five, six, seven, eight,
nine, ten or more
amino acids. In particular embodiments, the modification is a substitution.
For example, in
particular embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,
17, 18, 19, 20, 21,
22, 23, 24, 25 or more amino acids from the three-fold axis loop 4 from one
AAV can be
substituted into amino acid positions 575-600 of the native AAV2 capsid
protein or the
corresponding positions of the capsid protein from another AAV. However, the
modified
virus capsids of the invention are not limited to AAV capsids in which amino
acids from
one AAV capsid are substituted into another AAV capsid, and the substituted
and/or inserted
amino acids can be from any source, and can further be naturally occurring or
partially or
completely synthetic.
[00221] As described herein, the nucleic acid and amino acid sequences of the
capsid
proteins from a number of AAV are known in the art. Thus, the amino acids
"corresponding"
to amino acid positions 575 to 600 (inclusive) or amino acid positions 585 to
590 (inclusive)
of the native AAV2 capsid protein can be readily determined for any other AAV
(e.g., by
using sequence alignments).
[00222] In some embodiments, the invention contemplates that the modified
capsid proteins
of the invention can be produced by modifying the capsid protein of any AAV
now known or
later discovered. Further, the AAV capsid protein that is to be modified can
be a naturally
occurring AAV capsid protein (e.g., an AAV2, AAV3a or 3b, AAV4, AAV5, AAV8,
AAV9,
AAV10, AAV11, or AAV12 capsid protein or any of the AAV shown in Table 3) but
is not
so limited. Those skilled in the art will understand that a variety of
manipulations to the
AAV capsid proteins are known in the art and the invention is not limited to
modifications of
naturally occurring AAV capsid proteins. For example, the capsid protein to be
modified
may already have alterations as compared with naturally occurring AAV (e.g.,
is derived
from a naturally occurring AAV capsid protein, e.g., AAV2, AAV3a, AAV3b, AAV4,
CA 3054600 2019-09-06
AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11 and/or AAV12 or any other AAV
now known or later discovered). Such AAV capsid proteins are also within the
scope of the
present invention.
[00223] For example, in some embodiments, the AAV capsid protein to be
modified can
comprise an amino acid insertion directly following amino acid 264 of the
native AAV2
capsid protein sequence (see, e.g., PCT Publication WO 2006/066066) and/or can
be an AAV
with an altered HI loop as described in PCT Publication WO 2009/108274 and/or
can be an
AAV that is modified to contain a poly-His sequence to facilitate
purification. As another
illustrative example, the AAV capsid protein can have a peptide targeting
sequence
incorporated therein as an insertion or substitution. Further, the AAV capsid
protein can
comprise a large domain from another AAV that has been substituted and/or
inserted into the
capsid protein.
[00224] Thus, in particular embodiments, the AAV capsid protein to be modified
can be
derived from a naturally occurring AAV but further comprise one or more
foreign sequences
(e.g., that are exogenous to the native virus) that are inserted and/or
substituted into the
capsid protein and/or has been altered by deletion of one or more amino acids.
[00225] Accordingly, when referring herein to a specific AAV capsid protein
(e.g., an
AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11 or AAV12
capsid protein or a capsid protein from any of the AAV shown in Table 1,
etc.), it is intended
to encompass the native capsid protein as well as capsid proteins that have
alterations other
than the modifications of the invention. Such alterations include
substitutions, insertions
and/or deletions. In particular embodiments, the capsid protein comprises 1,
2, 3, 4, 5, 6, 7,
8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20, less than 20, less than
30, less than 40 less
than 50, less than 60, or less than 70 amino acids inserted therein (other
than the insertions of
the present invention) as compared with the native AAV capsid protein
sequence. In
embodiments of the invention, the capsid protein comprises 1, 2, 3, 4, 5, 6,
7, 8, 9, 10, 11, 12,
13, 14, 15, 16, 17, 18, 19 or 20, less than 20, less than 30, less than 40
less than 50, less than
60, or less than 70 amino acid substitutions (other than the amino acid
substitutions according
to the present invention) as compared with the native AAV capsid protein
sequence. In
embodiments of the invention, the capsid protein comprises a deletion of 1, 2,
3, 4, 5, 6, 7, 8,
9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20, more than 20, more than 30,
more than 40,
more than 50, more than 60, or more than 70 amino acids (other than the amino
acid deletions
of the invention) as compared with the native AAV capsid protein sequence.
[00226] Using AAV serotype 2 as an exemplary virus, Mll is the VP1 start
codon, M138 is the
36
CA 3054600 2019-09-06
VP2 start codon, and M203 is the VP3 start codon. While deletion of the start
codon, typically by a
substitution of Ml! and M138 will render expression of VP1 and VP2
inoperative, a similar deletion
of the VP3 start codon is not sufficient. This is because the viral capsid ORF
contains numerous ATG
codons with varying strengths as initiation codons. Thus, in designing a
construct that will not express
VP3 care must be taken to insure that an alternative VP3 species is not
produced. With respect to VP3
either elimination of M138 is necessary or if VP2 is desired, but not VP3,
then deletion of M211 and
235 in addition to M203 is typically the best approach (Warrington, K. H. Jr.,
et al., J. of Virol.
78(12): 6595-6609 (June 2004)). This can be done by mutations such as
substitution or other means
known in the art. The corresponding start codons in other serotypes can
readily be determined as well
as whether additional ATG sequences such as in VP3 can serve as alternative
initiation codons.
[00227] Thus, for example, the term "AAV2 capsid protein" includes AAV capsid
proteins
having the native AAV2 capsid protein sequence (see GenBank Accession No.
AAC03780)
as well as those comprising substitutions, insertions and/or deletions (as
described in the
preceding paragraph) in the native AAV2 capsid protein sequence.
[00228] In particular embodiments, the AAV capsid protein has the native AAV
capsid
protein sequence or has an amino acid sequence that is at least about 70%,
75%, 80%, 85%,
90%, 95%, 97%, 98% or 99% similar or identical to a native AAV capsid protein
sequence.
For example, in particular embodiments, an "AAV2" capsid protein encompasses
the native
AAV2 capsid protein sequence as well as sequences that are at least about 75%,
80%< 85%,
90%, 95%, 97%, 98% or 99% similar or identical to the native AAV2 capsid
protein
sequence.
[00229] Methods of determining sequence similarity or identity between two or
more amino
acid sequences are known in the art. Sequence similarity or identity may be
determined using
standard techniques known in the art, including, but not limited to, the local
sequence identity
algorithm of Smith & Waterman, Adv. App!. Math. 2,482 (1981), by the sequence
identity
alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48,443 (1970), by the
search for
similarity method of Pearson & Lipman, Proc. Natl. Acad. ScL USA 85, 2444
(1988), by
computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and
TFASTA
in the Wisconsin Genetics Software Package, Genetics Computer Group, 575
Science Drive,
Madison, WI), the Best Fit sequence program described by Devereux et at.,
Nucl. Acid Res.
12, 387-395 (1984), or by inspection.
[00230] Another suitable algorithm is the BLAST algorithm, described in
Altschul et al., J.
Mol. Biol. 215, 403-410, (1990) and Karlin et al., Proc. Natl. Acad. Sci. USA
90, 5873-5787
(1993). A particularly useful BLAST program is the WU-BLAST-2 program which
was
37
CA 3054600 2019-09-06
obtained from Altschul et al., Methods in Enzymology, 266, 460-480 (1996);
http://blast.wustl/edu/blast/README.html. WU-BLAST-2 uses several search
parameters,
which are optionally set to the default values. The parameters are dynamic
values and are
established by the program itself depending upon the composition of the
particular sequence
and composition of the particular database against which the sequence of
interest is being
searched; however, the values may be adjusted to increase sensitivity.
[00231] Further, an additional useful algorithm is gapped BLAST as reported by
Altschul et
al., (1997) Nucleic Acids Res. 25, 3389-3402.
[00232] In some embodiments of the invention, a modification can be made in
the region of
amino acid positions 585 to 590 (inclusive) of the native AAV2 capsid protein
(using VP1
numbering) or the corresponding positions of other AAV (native AAV2 VP1 capsid
protein:
GenBank Accession No. AAC03780 or YP680426), i.e., at the amino acids
corresponding to
amino acid positions 585 to 590 (VP1 numbering) of the native AAV2 capsid
protein. The
amino acid positions in other AAV serotypes or modified AAV capsids that
"correspond to"
positions 585 to 590 of the native AAV2 capsid protein will be apparent to
those skilled in
the art and can be readily determined using sequence alignment techniques
(see, e.g., Figure 7
of WO 2006/066066) and/or crystal structure analysis (Padron et al., (2005) 1
ViroL 79:
5047-58).
[00233] To illustrate, the modification can be introduced into an AAV capsid
protein that
already contains insertions and/or deletions such that the position of all
downstream
sequences is shifted. In this situation, the amino acid positions
corresponding to amino acid
positions 585 to 590 in the AAV2 capsid protein would still be readily
identifiable to those
skilled in the art. To illustrate, the capsid protein can be an AAV2 capsid
protein that
contains an insertion following amino acid position 264 (see, e.g., WO
2006/066066). The
amino acids found at positions 585 through 590 (e.g., RGNRQA (SEQ ID NO:1) in
the native
AAV2 capsid protein) would now be at positions 586 through 591 but would still
be
identifiable to those skilled in the art.
[00234] The invention also provides a virus capsid comprising, consisting
essentially of, or
consisting of the modified AAV capsid proteins of the invention. In particular
embodiments,
the virus capsid is a parvovirus capsid, which may further be an autonomous
parvovirus
capsid or a dependovirus capsid. Optionally, the virus capsid is an AAV
capsid. In particular
embodiments, the AAV capsid is an AAV1, AAV2, AAV3a, AAV3b, AAV4, AAV5, AAV6,
AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, or any other AAV shown in Table 1 or
otherwise known or later discovered, and/or is derived from any of the
foregoing by one or
38
CA 3054600 2019-09-06
more insertions, substitutions and/or deletions.
[00235] In embodiments of the present invention, the isolated AAV virion or
substantially
homogenous population of AAV virions is not a product of expression of a
mixture of one
nucleic acid helper plasmid that express VP1, VP2 and VP3 of one serotype with
another
nucleic acid helper plasmid that express VP1, VP2 and VP3 of another serotype,
such
expression being termed "cross-dressing."
[00236] In embodiments of the present invention, the isolated AAV virion does
not comprise
a mosaic capsid and the substantially homogenous population of AAV virions
does not
comprise a substantially homogenous population of mosaic capsids.
[00237] To the extent that any disclosure in PCT/US18/22725 filed March 15,
2018 falls
within the invention as defined in any one or more of the claims of this
application, or within
any invention to be defined in amended claims that may in the future be filed
in this
application or in any patent derived therefrom, and to the extent that the
laws of any relevant
country or countries to which that or those claims apply provide that the
disclosure of
PCT/US18/22725 is part of the state of the art against that or those claims in
or for that or
those countries, we hereby reserve the right to disclaim the said disclosure
from the claims of
the present application or any patent derived therefrom to the extent
necessary to prevent
invalidation of the present application or any patent derived therefrom.
[00238] For example, and without limitation, we reserve the right to disclaim
any one or
more of the following subject-matters from any claim of the present
application, now or as
amended in the future, or any patent derived therefrom:
[00239] A. any subject-matter disclosed in Example 9 of PCT/US18/22725; or
[00240] B. vector virions, termed polyploid vector virions, which are
produced or
producible by transfection of two AAV helper plasmids or three plasmids to
produce
individual polyploid vector virions composed of different capsid subunits from
different
serotypes; or
[00241] C. .. vector virions, termed polyploid vector virions, which are
produced or
producible by transfection of two AAV helper plasmids which are AAV2 and AAV8
or
AAV9 to produce individual polyploid vector virions composed of different
capsid subunits
from different serotypes; or
[00242] D. vector virions, termed polyploid vector virions, which are produced
or
producible by transfection of three AAV helper plasmids which are AAV2, AAV8
and
AAV9 to produce individual polyploid vector virions composed of different
capsid subunits
from different serotypes; or
39
CA 3054600 2019-09-06
[00243] E. vector virions, termed haploid vectors, with VP1/VP2 from one AAV
vector
capsid or AAV serotype and VP3 from an alternative one, for example VP1/VP2
from (the
capsid of) only one AAV serotype and VP3 from only one alternative AAV
serotype; or
[00244] F. any one or more AAV vector virion(s) selected from:
[00245] a vector which is generated by transfection of AAV2 helper and AAV8
helper
plasmids (termed haploid AAV2/8) and which has VP1 capsid subunit from AAV8
and
VP2/VP3 capsid subunits from AAV2; or
[00246] a vector which is generated by transfection of AAV2 helper and AAV8
helper
plasmids (termed haploid AAV2/8 or haploid AAV8/2 or haploid AAV82 or H-AAV82)
and
which has VP1/VP2 capsid subunits from AAV8 and VP3 capsid subunit from AAV2;
or
[00247] a vector in which VP1/VP2 is derived from different serotypes; or
[00248] a vector (termed haploid AAV92 or H-AAV92) which has VP1/VP2 capsid
subunits
from AAV9 and VP3 capsid subunit from AAV2; or
[00249] a vector (termed haploid AAV2G9 or H-AAV2G9) which has VP1/VP2 capsid
subunits from AAV8 and VP3 capsid subunit from AAV2G9, in which AAV9 glycan
receptor binding site was engrafted into AAV2; or
[00250] a vector (termed haploid AAV83 or H-AAV83) which has VP1/VP2 capsid
subunits
from AAV8 and VP3 capsid subunit from AAV3; or
[00251] a vector (termed haploid AAV93 or H-AAV93) which has VP1/VP2 capsid
subunits
from AAV9 and VP3 capsid subunit from AAV3; or
[00252] a vector (termed haploid AAVrh10-3 or H-AAVrh10-3) which has VP1/VP2
capsid
subunits from AAVrh10 and VP3 capsid subunit from AAV3; or
[00253] a vector which is generated by transfection of AAV2 helper and AAV8
helper
plasmids (termed haploid AAV2/8) and which has VPI capsid subunit from AAV2
and
VP2/VP3 capsid subunits from AAV8; or
[00254] a vector which is generated by transfection of AAV2 helper and AAV8
helper
plasmids (termed haploid AAV2/8) and which has VP1/VP2 capsid subunit from
AAV2 and
VP3 capsid subunits from AAV8; or
[00255] a vector which is generated by transfection of AAV2 helper and AAV8
helper
plasmids (termed haploid AAV2/8) and which has VP1 capsid subunit from AAV8
and VP3
capsid subunit from AAV2; or
[00256] a vector which is generated by transfection of AAV2 helper and AAV8
helper
plasmids (termed haploid AAV2/8) and which has VPI capsid subunit from AAV2
and VP3
capsid subunits from AAV8; or
CA 3054600 2019-09-06
[00257] a vector which is generated by transfection of AAV2 helper and AAV8
helper
plasmids (termed haploid AAV2/8) and which has VP1/VP2/VP3 capsid subunits
from
AAV2; or
[00258] a vector which is generated by transfection of AAV2 helper and AAV8
helper
plasmids (termed haploid AAV2/8) and which has VP1/VP2/VP3 capsid subunits
from
AAV 8 ; or
[00259] a vector termed 28m-2VP3 or haploid 2m-2VP3 or haploid vector 28m-2VP3
in
which chimeric VP1/VP2 capsid subunits have N-terminal from AAV2 and C-
terminal from
AAV8, and the VP3 capsid subunit is from AAV2; or
[00260] a vector termed chimeric AAV8/2 or chimeric AAV82 in which chimeric
VP1/VP2
capsid subunits have N-terminal from AAV8 and C-terminal from AAV2 without
mutation of
the VP3 start codon, and the VP3 capsid subunit is from AAV2; or
[00261] a vector in which chimeric VP1/VP2 capsid subunits have N-terminal
from AAV2
and C-terminal from AAV8; or
[00262] G. a population, for example a substantially homogenous population,
for example
a population of 1010 particles, for example a substantially homogenous
population of 1010
particles, of any one of the vectors of F; or
[00263] H. a method of producing any one of the vectors or populations of
vectors of A
and/or B and/or C and/or D and/or E and/or F and/or G; or
[00264] I. any combination thereof.
[00265] Without limitation, we state that the above reservation of a right of
disclaimer
applies at least to claims 1-30 as appended to this application and paragraphs
1-83 as set forth
at [00437]. The modified virus capsids can be used as "capsid vehicles," as
has been
described, for example, in U.S. Patent No. 5,863,541. Molecules that can be
packaged by the
modified virus capsid and transferred into a cell include heterologous DNA,
RNA,
polypeptides, small organic molecules, metals, or combinations of the same.
[00266] Heterologous molecules are defined as those that are not naturally
found in an AAV
infection, e.g., those not encoded by a wild-type AAV genome. Further,
therapeutically
useful molecules can be associated with the outside of the virus capsid for
transfer of the
molecules into host target cells. Such associated molecules can include DNA,
RNA, small
organic molecules, metals, carbohydrates, lipids and/or polypeptides. In one
embodiment of
the invention, the therapeutically useful molecule is covalently linked (i.e.,
conjugated or
chemically coupled) to the capsid proteins. Methods of covalently linking
molecules are
known by those skilled in the art.
41
CA 3054600 2019-09-06
[00267] The modified virus capsids of the invention also find use in raising
antibodies
against the novel capsid structures. As a further alternative, an exogenous
amino acid
sequence may be inserted into the modified virus capsid for antigen
presentation to a cell,
e.g., for administration to a subject to produce an immune response to the
exogenous amino
acid sequence.
[00268] In other embodiments, the virus capsids can be administered to block
certain
cellular sites prior to and/or concurrently with (e.g., within minutes or
hours of each other)
administration of a virus vector delivering a nucleic acid encoding a
polypeptide or functional
RNA of interest. For example, the inventive capsids can be delivered to block
cellular
receptors on liver cells and a delivery vector can be administered
subsequently or
concurrently, which may reduce transduction of liver cells, and enhance
transduction of other
targets (e.g., skeletal, cardiac and/or diaphragm muscle).
[00269] According to representative embodiments, modified virus capsids can be
administered to a subject prior to and/or concurrently with a modified virus
vector according
to the present invention. Further, the invention provides compositions and
pharmaceutical
formulations comprising the inventive modified virus capsids; optionally, the
composition
also comprises a modified virus vector of the invention.
[00270] The invention also provides nucleic acid molecules (optionally,
isolated nucleic acid
molecules) encoding the modified virus capsids and capsid proteins of the
invention. Further
provided are vectors, comprising the nucleic acid molecules and cells (in vivo
or in culture),
comprising the nucleic acid molecules and/or vectors of the invention.
Suitable vectors
include without limitation viral vectors (e.g., adenovirus, AAV, herpesvirus,
alphaviruses,
vaccinia, poxviruses, baculoviruses, and the like), plasmids, phage, YACs,
BACs, and the
like. Such nucleic acid molecules, vectors and cells can be used, for example,
as reagents
(e.g., helper packaging constructs or packaging cells) for the production of
modified virus
capsids or virus vectors as described herein.
[00271] Virus capsids according to the invention can be produced using any
method known
in the art, e.g., by expression from a baculovirus (Brown et al., (1994)
Virology 198:477-
488).
[00272] In some embodiments, the modifications to the AAV capsid protein of
this
invention are "selective" modifications. This approach is in contrast to
previous work with
whole subunit or large domain swaps between AAV serotypes (see, e.g.,
international patent
publication WO 00/28004 and Hauck et al., (2003)1 Virology 77:2768-2774). In
particular
embodiments, a "selective" modification results in the insertion and/or
substitution and/or
42
CA 3054600 2019-09-06
deletion of less than about 20, 18, 15, 12, 10, 9, 8, 7, 6, 5, 4, 3 or 2
contiguous amino acids.
[00273] The modified capsid proteins and capsids of the invention can further
comprise any
other modification, now known or later identified.
[00274] The virus capsid can be a targeted virus capsid comprising a targeting
sequence
(e.g., substituted or inserted in the viral capsid) that directs the virus
capsid to interact with
cell-surface molecules present on a desired target tissue(s) (see, e.g.,
International Patent
Publication No. WO 00/28004 and Hauck et al., (2003)1 Virology 77:2768-2774);
Shi et al.,
Human Gene Therapy 17:353-361 (2006) [describing insertion of the integrin
receptor
binding motif RGD at positions 520 and/or 584 of the AAV capsid subunit]; and
U.S. Patent
No. 7,314,912 [describing insertion of the P1 peptide containing an RGD motif
following
amino acid positions 447, 534, 573 and 587 of the AAV2 capsid subunit]). Other
positions
within the AAV capsid subunit that tolerate insertions are known in the art
(e.g., positions
449 and 588 described by Grifman et al., Molecular Therapy 3:964-975 (2001)).
[00275] For example, some of the virus capsids of the invention have
relatively inefficient
tropism toward most target tissues of interest (e.g., liver, skeletal muscle,
heart, diaphragm
muscle, kidney, brain, stomach, intestines, skin, endothelial cells, and/or
lungs). A targeting
sequence can advantageously be incorporated into these low-transduction
vectors to thereby
confer to the virus capsid a desired tropism and, optionally, selective
tropism for particular
tissue(s). AAV capsid proteins, capsids and vectors comprising targeting
sequences are
described, for example in international patent publication WO 00/28004. As
another
possibility one or more non-naturally occurring amino acids as described by
Wang et al.,
Annu Rev Biophys Biomol Struct. 35:225-49 (2006)) can be incorporated into the
AAV capsid
subunit at an orthogonal site as a means of redirecting a low-transduction
vector to a desired
target tissue(s). These unnatural amino acids can advantageously be used to
chemically link
molecules of interest to the AAV capsid protein including without limitation:
glycans
(mannose-dendritic cell targeting); RGD, bombesin or a neuropeptide for
targeted delivery to
specific cancer cell types; RNA aptamers or peptides selected from phage
display targeted to
specific cell surface receptors such as growth factor receptors, integrins,
and the like.
Methods of chemically modifying amino acids are known in the art (see, e.g.,
Greg T.
Hermanson, Bioconjugate Techniques, 1st edition, Academic Press, 1996).
[00276] In representative embodiments, the targeting sequence may be a virus
capsid
sequence (e.g., an autonomous parvovirus capsid sequence, AAV capsid sequence,
or any
other viral capsid sequence) that directs infection to a particular cell
type(s).
[00277] As another nonlimiting example, a heparin binding domain (e.g., the
respiratory
43
CA 3054600 2019-09-06
syncytial virus heparin binding domain) may be inserted or substituted into a
capsid subunit
that does not typically bind HS receptors (e.g., AAV 4, AAV5) to confer
heparin binding to
the resulting mutant.
[00278] B19 infects primary erythroid progenitor cells using globoside as its
receptor
(Brown et al., (1993) Science 262:114). The structure of B19 has been
determined to 8 A
resolutions (Agbandje-McKenna et al., (1994) Virology 203:106). The region of
the B19
capsid that binds to globoside has been mapped between amino acids 399- 406
(Chapman et
al., (1993) Virology 194:419), a looped out region between 13-barrel
structures E and F.
(Chipman et al., (1996) Proc. Nat. Acad. Sci. USA 93:7502). Accordingly, the
globoside
receptor binding domain of the B19 capsid may be substituted into the AAV
capsid protein to
target a virus capsid or virus vector comprising the same to erythroid cells.
[00279] In representative embodiments, the exogenous targeting sequence may be
any
amino acid sequence encoding a peptide that alters the tropism of a virus
capsid or virus
vector comprising the modified AAV capsid protein. In particular embodiments,
the
targeting peptide or protein may be naturally occurring or, alternately,
completely or partially
synthetic. Exemplary targeting sequences include ligands and other peptides
that bind to cell
surface receptors and glycoproteins, such as RGD peptide sequences,
bradykinin, hormones,
peptide growth factors (e.g., epidermal growth factor, nerve growth factor,
fibroblast growth
factor, platelet- derived growth factor, insulin-like growth factors I and II,
etc.), cytokines,
melanocyte stimulating hormone (e.g., a, P or 7), neuropeptides and
endorphins, and the like,
and fragments thereof that retain the ability to target cells to their cognate
receptors. Other
illustrative peptides and proteins include substance P, keratinocyte growth
factor,
neuropeptide Y, gastrin releasing peptide, interleukin 2, hen egg white
lysozyme,
erythropoietin, gonadoliberin, corticostatin, 13-endorphin, leu-enkephalin,
rimorphin, a-neo-
enkephalin, angiotensin, pneumadin, vasoactive intestinal peptide,
neurotensin, motilin, and
fragments thereof as described above. As yet a further alternative, the
binding domain from a
toxin (e.g., tetanus toxin or snake toxins, such as a-bungarotoxin, and the
like) can be
substituted into the capsid protein as a targeting sequence. In a yet further
representative
embodiment, the AAV capsid protein can be modified by substitution of a
"nonclassical"
import/export signal peptide (e.g., fibroblast growth factor-1 and -2,
interleukin 1, HIV-1 Tat
protein, herpes virus VP22 protein, and the like) as described by Cleves
(Current Biology
7:R318 (1997)) into the AAV capsid protein. Also encompassed are peptide
motifs that
direct uptake by specific cells, e.g., a FVFLP peptide motif triggers uptake
by liver cells.
44
CA 3054600 2019-09-06
[00280] Phage display techniques, as well as other techniques known in the
art, may be used
to identify peptides that recognize any cell type of interest.
[00281] The targeting sequence may encode any peptide that targets to a cell
surface binding
site, including receptors (e.g., protein, carbohydrate, glycoprotein or
proteoglycan).
Examples of cell surface binding sites include, but are not limited to,
heparan sulfate,
chondroitin sulfate, and other glycosaminoglycans, sialic acid moieties found
on mucins,
glycoproteins, and gangliosides, MHC I glycoproteins, carbohydrate components
found on
membrane glycoproteins, including, mannose, N-acetyl-galactosamine, N-acetyl-
glucosamine, fucose, galactose, and the like.
[00282] In particular embodiments, a heparan sulfate (HS) or heparin binding
domain is
substituted into the virus capsid (for example, in an AAV that otherwise does
not bind to HS
or heparin). It is known in the art that HS/heparin binding is mediated by a
"basic patch" that
is rich in arginines and/or lysines. In exemplary embodiments, a sequence
following the
motif BXXB, where "B" is a basic residue and X is neutral and/or hydrophobic.
As one
nonlimiting example, BXXB is RGNR. In particular embodiments, BXXB is
substituted for
amino acid positions 262 through 265 in the native AAV2 capsid protein or the
corresponding position in the capsid protein of another AAV.
[00283] Other nonlimiting examples of suitable targeting sequences include the
peptides
targeting coronary artery endothelial cells identified by Mailer et al.,
Nature Biotechnology
21:1040-1046 (2003) (consensus sequences NSVRDLG/S ( SEQ ID NO:2), PRSVTVP
(SEQ
ID NO:3), NSVSSXS/A (SEQ ID NO:4)); tumor-targeting peptides as described by
Grifman
et al., Molecular Therapy 3:964-975 (2001) (e.g., NGR, NGRAHA (SEQ ID NO:5));
lung or
brain targeting sequences as described by Work et al., Molecular Therapy
13:683-693 (2006)
(QPEHSST (SEQ ID NO:6), VNTANST (SEQ ID NO:7), HGPMQKS (SEQ ID NO:8),
PHKPPLA (SEQ ID NO:9), IKNNEMW (SEQ ID NO:10), RNLDTPM (SEQ ID NO:11),
VDSHRQS (SEQ ID NO:12), YDSKTKT (SEQ ID NO:13), SQLPHQK (SEQ ID NO:14),
STMQQNT (SEQ ID NO:15), TERYMTQ (SEQ ID NO:16), QPEHSST (SEQ ID NO:6),
DASLSTS (SEQ ID NO:17), DLPNKKT (SEQ ID NO:18), DLTAARL (SEQ ID NO:19),
EPHQFNY (SEQ ID NO:20), EPQSNHT (SEQ ID NO:21), MSSWPSQ (SEQ ID NO:22),
NPKHNAT (SEQ ID NO:23), PDGMRTT (SEQ ID NO:24), PNNNKTT (SEQ ID NO:25),
QSTTHDS (SEQ ID NO:26), TGSKQKQ (SEQ ID NO:27), SLKHQAL (SEQ ID NO:28)
and SPIDGEQ (SEQ ID NO:29)); vascular targeting sequences described by Hajitou
et al.,
TCM 16:80-88 (2006) (WIFPWIQL (SEQ ID NO:30), CDCRGDCFC (SEQ ID NO:31),
CNGRC (SEQ ID NO:32), CPRECES (SEQ ID NO:33), GSL, CTTHWGFTLC (SEQ ID
CA 3054600 2019-09-06
NO:34), CGRRAGGSC (SEQ ID NO:35), CKGGRAKDC (SEQ ID NO:36), and
CVPELGHEC (SEQ ID NO:37)); targeting peptides as described by Koivunen et al.,
J. Nucl.
Med. 40:883-888 (1999) (CRRETAWAK (SEQ ID NO:38), KGD, VSWFSHRYSPFAVS
(SEQ ID NO:39), GYRDGYAGPILYN (SEQ ID NO:40), XXXY*XXX [where Y* is
phospho-Tyr] (SEQ ID NO:41), Y*E/MNW (SEQ ID NO:42), RPLPPLP (SEQ ID NO:43),
APPLPPR (SEQ ID NO:44), DVFYPYPY ASGS (SEQ ID NO:45), MYWYPY (SEQ ID
NO:46), DITWDQL WDLMK (SEQ ID NO:47), CWDDG/L WLC (SEQ ID NO:48),
EWCEYLGGYLRCY A (SEQ ID NO:49), YXCXXGPXTWXCXP (SEQ ID NO:50),
IEGPTLRQWLAARA (SEQ ID NO:51), LWXXY/W/F/H (SEQ ID NO:52), XFXXYLW
(SEQ ID NO:53), SSIISHFRWGLCD (SEQ ID NO:54), MSRPACPPNDKYE (SEQ ID
NO:55), CLRSGRGC (SEQ ID NO:56), CHWMFSPWC (SEQ ID NO:57), WXXF (SEQ ID
NO:58), CSSRLDAC (SEQ ID NO:59), CLPVASC (SEQ ID NO:60), CGFECVRQCPERC
(SEQ ID NO:61), CVALCREACGEGC (SEQ ID NO:62), SWCEPGWCR (SEQ ID NO:63),
YSGKWGW (SEQ ID NO:64), GLSGGRS (SEQ ID NO:65), LMLPRAD (SEQ ID NO:66),
CSCFRDVCC (SEQ ID NO:67), CRDVVSVIC (SEQ ID NO:68), CNGRC (SEQ ID
NO:32), and GSL); and tumor targeting peptides as described by Newton &
Deutscher, Phage
Peptide Display in Handbook of Experimental Pharmacology, pages 145-163,
Springer-
Verlag, Berlin (2008) (MARSGL (SEQ ID NO:69), MARAKE (SEQ ID NO:70), MSRTMS
(SEQ ID NO:71), KCCYSL (SEQ ID NO:72), WRR, WKR, WVR, WVK, WIK, WTR,
WVL, WLL, WRT, WRG, WVS, WVA, MYWGDSHWLQYWYE (SEQ ID NO:73),
MQLPLAT (SEQ ID NO:74), EWLS (SEQ ID NO:75), SNEW (SEQ ID NO:76), TNYL
(SEQ ID NO:77), WIFPWIQL (SEQ ID NO:30), WDLAWMFRLPVG (SEQ ID NO:78),
CTVALPGGYVRVC (SEQ ID NO:79), CVPELGHEC (SEQ ID NO:37), CGRRAGGSC
(SEQ ID NO:35), CVAYCIEHHCWTC (SEQ ID NO:80), CVFAHNYDYL VC (SEQ ID
NO:81), and CVFTSNYAFC (SEQ ID NO:82), VHSPNKK (SEQ ID NO:83),
CDCRGDCFC (SEQ ID NO:31), CRGDGWC (SEQ ID NO:84), XRGCDX (SEQ ID
NO:85), P:XXS/T (SEQ ID NO:86), CTTHWGFTLC (SEQ ID NO:34), SGKGPRQITAL
(SEQ ID NO:87), A9A/Q)(N/A)(L/Y)(TN/M/R)(R/K) (SEQ ID NO:88), VYMSPF (SEQ ID
NO:89), MQLPLAT (SEQ ID NO:74), ATWLPPR (SEQ ID NO:90), HTMYYHHYQHHL
(SEQ ID NO:91), SEVGCRAGPLQWLCEKYFG (SEQ ID NO:92),
CGLLPVGRPDRNVWRWLC (SEQ ID NO:93), CKGQCDRFKGLPWEC (SEQ ID
NO:94), SGRSA (SEQ ID NO:95), WGFP (SEQ ID NO:96), LWXXAr [Ar=Y, W, F, H)
(SEQ ID NO:97), XF:XXYLW (SEQ ID NO:98), AEPMPHSLNFSQYLWYT (SEQ ID
NO:99), WAY(W/F)SP (SEQ ID NO:100), IELLQAR (SEQ ID NO:101),
46
CA 3054600 2019-09-06
DITWDQLWDLMK (SEQ ID NO:102), AYTKCSRQWRTCMTTH (SEQ ID NO:103),
PQNSKIPGPTFLDPH (SEQ ID NO:104), SMEPALPDWWWKMFK (SEQ ID NO:105),
ANTPCGPYTHDCPVKR (SEQ ID NO:106), TACHQHVRMVRP (SEQ ID NO:107),
VPWMEPAYQRFL (SEQ ID NO:108), DPRATPGS (SEQ ID NO:109),
FRPNRAQDYNTN (SEQ ID NO:110), CTKNSYLMC (SEQ ID NO:111),
C(R/Q)URT(G/N)XXG(AN)GC (SEQ ID NO:112), CPIEDRPMC (SEQ ID NO:113),
HEWSYLAPYPWF (SEQ ID NO:114), MCPKHPLGC (SEQ ID NO:115),
RMWPSSTVNLSAGRR (SEQ ID NO:116), SAKTAVSQRVWLPSHRGGEP (SEQ ID
NO:117), KSREHVNNSACPSKRITAAL (SEQ ID NO:118), EGFR (SEQ ID NO:119),
RVS, AGS, AGLGVR (SEQ ID NO:120), GGR, GGL, GSV, GVS,
GTRQGHTMRLGVSDG (SEQ ID NO:121), IAGLATPGWSHWLAL (SEQ ID NO:122),
SMSIARL (SEQ ID NO:123), HTFEPGV (SEQ ID NO:124), NTSLKRISNKRIRRK (SEQ
ID NO:125), LRIKRKRRKRKKTRK (SEQ ID NO:126), GGG, GFS, LWS, EGG, LLV,
LSP, LBS, AGG, GRR, GGH and GTV).
[00284] As yet a further alternative, the targeting sequence may be a peptide
that can be
used for chemical coupling (e.g., can comprise arginine and/or lysine residues
that can be
chemically coupled through their R groups) to another molecule that targets
entry into a cell.
[00285] As another option, the AAV capsid protein or virus capsid of the
invention can
comprise a mutation as described in WO 2006/066066. For example, the capsid
protein can
comprise a selective amino acid substitution at amino acid position 263, 705,
708 and/or 716
of the native AAV2 capsid protein or a corresponding change(s) in a capsid
protein from
another AAV. Additionally, or alternatively, in representative embodiments,
the capsid
protein, virus capsid or vector comprises a selective amino acid insertion
directly following
amino acid position 264 of the AAV2 capsid protein or a corresponding change
in the capsid
protein from other AAV. By "directly following amino acid position X" it is
intended that
the insertion immediately follows the indicated amino acid position (for
example, "following
amino acid position 264" indicates a point insertion at position 265 or a
larger insertion, e.g.,
from positions 265 to 268, etc.). The foregoing embodiments of the invention
can be used to
deliver a heterologous nucleic acid to a cell or subject as described herein.
For example, the
modified vector can be used to treat a lysosomal storage disorder such as a
mucopolysaccharidosis disorder (e.g., Sly syndrome [P-glucuronidase], Hurler
Syndrome [a-
L-iduronidase], Scheie Syndrome [a-L-iduronidase], Hurler-Scheie Syndrome [a-L-
iduronidase], Hunter's Syndrome [iduronate sulfatase], Sanfilippo Syndrome A
[heparan
sulfam idase], B [N-acetylglucosaminidase], C
[acetyl-CoA:a-glucosaminide
47
CA 3054600 2019-09-06
acetyltransferase], D [N- acetylglucosamine 6-sulfatase], Morquio Syndrome A
[galactose-6-
sulfate sulfatase], B [P-galactosidase], Maroteaux-Lamy Syndrome [N-
acetylgalactosamine-
4-sulfatase], etc.), Fabry disease (a-galactosidase), Gaucher's disease
(glucocerebrosidase), or
a glycogen storage disorder (e.g., Pompe disease; lysosomal acid a-
glucosidase) as described
here in.
[00286] Those skilled in the art will appreciate that for some AAV capsid
proteins the
corresponding modification will be an insertion and/or a substitution,
depending on whether
the corresponding amino acid positions are partially or completely present in
the virus or,
alternatively, are completely absent. Likewise, when modifying AAV other than
AAV2, the
specific amino acid position(s) may be different than the position in AAV2
(see, e.g., Table
3). As discussed elsewhere herein, the corresponding amino acid position(s)
will be readily
apparent to those skilled in the art using well-known techniques.
[00287] In representative embodiments, the insertion and/or substitution
and/or deletion in
the capsid protein(s) results in the insertion, substitution and/or
repositioning of an amino
acid that (i) maintains the hydrophilic loop structure in that region; (ii) an
amino acid that
alters the configuration of the loop structure; (iii) a charged amino acid;
and/or (iv) an amino
acid that can be phosphorylated or sulfated or otherwise acquire a charge by
post-
translational modification (e.g., glycosylation) following 264 in an AAV2
capsid protein or a
corresponding change in a capsid protein of another AAV. Suitable amino acids
for
insertion/substitution include aspartic acid, glutamic acid, valine, leucine,
lysine, arginine,
threonine, serine, tyrosine, glycine, alanine, proline, asparagine,
phenylalanine, tyrosine or
glutamine. In particular embodiments, a threonine is inserted or substituted
into the capsid
subunit. Nonlimiting examples of corresponding positions in a number of other
AAV are
shown in Table 3 (Position 2). In particular embodiments, the amino acid
insertion or
substitution is a threonine, aspartic acid, glutamic acid or phenylalanine
(excepting AAV that
have a threonine, glutamic acid or phenylalanine, respectively, at this
position).
[00288] According to this aspect of the invention, in some embodiments the AAV
capsid
protein comprises an amino acid insertion following amino acid position 264 in
an AAV2,
AAV3a or AAV3b capsid protein(s) or in the corresponding position in an AAV2,
AAV3a or
AAV3b capsid protein that has been modified to comprise non-AAV2, AAV3a or
AAV3b
sequences, respectively, and/or has been modified by deletion of one or more
amino acids
(i.e., is derived from AAV2, AAV3a or AAV3b). The amino acid corresponding to
position
264 in an AAV2 (or AAV3a or AAV3b) capsid subunit(s) will be readily
identifiable in the
starting virus that has been derived from AAV2 (or AAV3a or AAV3b), which can
then be
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CA 3054600 2019-09-06
further modified according to the present invention. Suitable amino acids for
insertion
include aspartic acid, glutamic acid, valine, leucine, lysine, arginine,
threonine, serine,
tyrosine, glycine, alanine, proline, asparagine, phenylalanine, tyrosine or
glutamine.
[00289] In other embodiments, the AAV capsid protein comprises an amino acid
substitution at amino acid position 265 in an AAV1 capsid protein(s), at amino
acid position
266 in an AAV8 capsid protein, or an amino acid substitution at amino acid
position 265 in
an AAV9 capsid protein or in the corresponding position in an AAV1, AAV8 or
AAV9
capsid protein that has been modified to comprise non-AAV1, non-AAV8 or non-
AAV9
sequences, respectively, and/or has been modified by deletion of one or more
amino acids
(i.e., is derived from AAV1, AAV8 or AAV9). The amino acid corresponding to
position
265 in an AAV1 and AAV9 capsid subunit(s) and position 266 in the AAV8 capsid
subunit(s) will be readily identifiable in the starting virus that has been
derived from AAV I,
AAV8 or AAV9, which can then be further modified according to the present
invention.
Suitable amino acids for insertion include aspartic acid, glutamic acid,
valine, leucine, lysine,
arginine, threonine, serine, tyrosine, glycine, alanine, proline, asparagine,
phenylalanine,
tyrosine or glutamine.
[00290] In representative embodiments of the invention, the capsid protein
comprises a
threonine, aspartic acid, glutamic acid, or phenylalanine following amino acid
position 264 of
the AAV2 capsid protein (i.e., an insertion) or the corresponding position of
another capsid
protein.
[00291] In other representative embodiments, the modified capsid proteins or
virus capsids
of the invention further comprise one or more mutations as described in WO
2007/089632
(e.g., an E7K mutation at amino acid position 531 of the AAV2 capsid protein
or the
corresponding position of the capsid protein from another AAV).
[00292] In further embodiments, the modified capsid protein or capsid can
comprise a
mutation as described in WO 2009/108274.
[00293] As another, possibility, the AAV capsid protein can comprise a
mutation as
described by Zhong et al. (Virology 381: 194-202 (2008); Proc. Nat. Acad. Sci.
105: 7827-32
(2008)). For example, the AAV capsid protein can comprise an YF mutation at
amino acid
position 730.
[00294] The modifications described above can be incorporated into the capsid
proteins or
capsids of the invention in combination with each other and/or with any other
modification
now known or later discovered.
[00295] The invention also encompasses virus vectors comprising the modified
capsid
49
CA 3054600 2019-09-06
proteins and capsids of the invention. In particular embodiments, the virus
vector is a
parvovirus vector (e.g., comprising a parvovirus capsid and/or vector genome),
for example,
an AAV vector (e.g., comprising an AAV capsid and/or vector genome). In
representative
embodiments, the virus vector comprises a modified AA V capsid comprising a
modified
capsid protein subunit of the invention and a vector genome.
[00296] For example, in representative embodiments, the virus vector
comprises: (a) a
modified virus capsid (e.g., a modified AAV capsid) comprising a modified
capsid protein of
the invention; and (b) a nucleic acid comprising a terminal repeat sequence
(e.g., an AAV
TR), wherein the nucleic acid comprising the terminal repeat sequence is
encapsidated by the
modified virus capsid. The nucleic acid can optionally comprise two terminal
repeats (e.g.,
two AAV TRs).
[00297] In representative embodiments, the virus vector is a recombinant virus
vector
comprising a heterologous nucleic acid encoding a polypeptide or functional
RNA of interest.
Recombinant virus vectors are described in more detail below.
[00298] In some embodiments, the virus vectors of the invention (i) have
reduced
transduction of liver as compared with the level of transduction by a virus
vector without the
modified capsid proteins of this invention; (ii) exhibit enhanced systemic
transduction by the
virus vector in an animal subject as compared with the level observed by a
virus vector
without the modified capsid proteins of this invention; (iii) demonstrate
enhanced movement
across endothelial cells as compared with the level of movement by a virus
vector without the
modified capsid proteins of this invention, and/or (iv) exhibit a selective
enhancement in
transduction of muscle tissue (e.g., skeletal muscle, cardiac muscle and/or
diaphragm
muscle), and/or (v) reduced transduction of brain tissues (e.g., neurons) as
compared with the
level of transduction by a virus vector without the modified capsid proteins
of this invention.
In some embodiments, the virus vector has systemic transduction toward muscle,
e.g., it
transduces multiple skeletal muscle groups throughout the body and optionally
transduces
cardiac muscle and/or diaphragm muscle.
[00299] Further, in some embodiments of the invention, the modified virus
vectors
demonstrate efficient transduction of target tissues.
[00300] It will be understood by those skilled in the art that the modified
capsid proteins,
virus capsids, virus vectors and AAV particles of the invention exclude those
capsid proteins,
capsids, virus vectors and AAV particles as they would be present or found in
their native
state.
CA 3054600 2019-09-06
Methods of Producing Virus Vectors
[00301] The present invention further provides methods of producing the
inventive virus
vectors of this invention as AAV particles. Thus, the present invention
provides a method
of making an AAV particle comprising the AAV capsid of this invention,
comprising: (a)
transfecting a host cell with one or more plasmids that provide, in
combination all functions
and genes needed to assemble AAV particles; (b) introducing one or more
nucleic acid
constructs into a packaging cell line or producer cell line to provide, in
combination, all
functions and genes needed to assemble AAV particles; (c) introducing into a
host cell one or
more recombinant baculovirus vectors that provide in combination all functions
and genes
needed to assemble AAV particles; and/or (d) introducing into a host cell one
or more
recombinant herpesvirus vectors that provide in combination all functions and
genes needed
to assemble AAV particles. The conditions for formation of an AAV virion are
the standard
conditions for production of AAV vectors in cells (e.g.,mammalian or insect
cells), which
includes as a nonlimiting example transfection of cells in the presence of an
Ad helper
plasmid, or other helper virus such as HSV.
[00302] Nonlimiting examples of various methods of making the virus vectors of
this
invention are described in Clement and Grieger ("Manufacturing of recombinant
adeno-
associated viral vectors for clinical trials" Mol. Ther. Methods Clin Dev.
3:16002 (2016)) and
in Grieger et al. ("Production of recombinant adeno-associated virus vectors
using suspension
HEK293 cells and continuous harvest of vector from the culture media for GMP
FIX and
FLT1 clinical vector" Mol Ther 24(2):287-297 (2016)), the entire contents of
which are
incorporated by reference herein.
[00303] In one representative embodiment, the present invention provides a
method of
producing a virus vector, the method comprising providing to a cell: (a) a
nucleic acid
template comprising at least one TR sequence (e.g., AAV TR sequence), and (b)
AAV
sequences sufficient for replication of the nucleic acid template and
encapsidation into AAV
capsids (e.g., AAV rep sequences and AAV cap sequences encoding the AAV
capsids of the
invention). Optionally, the nucleic acid template further comprises at least
one heterologous
nucleic acid sequence. In particular embodiments, the nucleic acid template
comprises two
AAV ITR sequences, which are located 5' and 3' to the heterologous nucleic
acid sequence (if
present), although they need not be directly contiguous thereto.
[00304] The nucleic acid template and AAV rep and cap sequences are provided
under
conditions such that virus vector comprising the nucleic acid template
packaged within the
AAV capsid is produced in the cell. The method can further comprise the step
of collecting
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CA 3054600 2019-09-06
the virus vector from the cell. The virus vector can be collected from the
medium and/or by
lysing the cells.
[00305] In one embodiment, the nucleic acid template is altered so that the
cap sequences
cannot express all three viral structural proteins, VP!, VP2, and VP3 from a
nucleic acid
sequence only from one serotype (first nucleic acid sequence). This alteration
can be by, for
example, eliminating start codons for at least one of the viral structural
proteins. The template
will also contain at least one additional nucleic acid sequence (second
nucleic acid sequence)
from a different serotype encoding and capable of expressing the viral
structural protein not
capable of being expressed by the first nucleic acid sequence, wherein the
second nucleic
acid sequence is not capable of expressing the viral structural protein
capable of expression
by the first nucleic acid sequence. In one embodiment, the first nucleic acid
sequence is
capable of expressing two of the viral structural proteins whereas the second
nucleic acid
sequence is capable of expressing only the remaining viral sequence. For
example, the first
nucleic acid sequence is capable of expression of VP! and VP2 but not VP3 from
one
serotype and the second nucleic acid sequence is capable of expression of VP3
from an
alternative serotype, but not VP! or VP2. The template is not capable of
expressing any other
of the three viral structural proteins. In one embodiment the first nucleic
acid sequence is
only capable of expressing one of the three viral structural proteins, the
second nucleic acid
sequence is capable of expressing only the other two viral structural
proteins, but not the first.
[00306] In another embodiment there is a third nucleic acid sequence from a
third serotype.
In this embodiment each of the three nucleic acid sequences is only capable of
expressing one
of the three capsid viral structural proteins, VP!, VP2, and VP3, and each
does not express a
viral structural protein expressed by another of the sequences so that
collectively a capsid is
produced containing VP!, VP2, and VP3, wherein each of the viral structural
proteins in the
capsid are all from the same serotype only and in this embodiment VP1, VP2,
and VP3 are all
from different serotypes.
[00307] The alteration to prevent expression can be by any means known in the
art. For
example, eliminating start codons, splice acceptors, splice donors, and
combinations thereof.
Deletions and additions can be use as well as site specific changes to change
reading frames.
Nucleic acid sequences can also be synthetically produced. These helper
templates typically
do not contain ITRs.
[00308] The cell can be a cell that is permissive for AAV viral replication.
Any suitable cell
known in the art may be employed. In particular embodiments, the cell is a
mammalian cell.
As another option, the cell can be a trans-complementing packaging cell line
that provides
52
CA 3054600 2019-09-06
functions deleted from a replication-defective helper virus, e.g., 293 cells
or other Ela trans-
complementing cells.
[00309] The AAV replication and capsid sequences may be provided by any method
known
in the art. Current protocols typically express the AAV rep/cap genes on a
single plasmid.
The AAV replication and packaging sequences need not be provided together,
although it
may be convenient to do so. The AAV rep and/or cap sequences may be provided
by any
viral or non-viral vector. For example, the rep/cap sequences may be provided
by a hybrid
adenovirus or herpesvirus vector (e.g., inserted into the Ela or E3 regions of
a deleted
adenovirus vector). EBV vectors may also be employed to express the AAV cap
and rep
genes. One advantage of this method is that EBV vectors are episomal, yet will
maintain a
high copy number throughout successive cell divisions (i.e., are stably
integrated into the cell
as extra-chromosomal elements, designated as an "EBV based nuclear episome,"
see
Margolski, (1992) Curr. Top. Microbiol. Immun. 158:67).
[00310] As a further alternative, the rep/cap sequences may be stably
incorporated into a
cell. Typically the AAV rep/cap sequences will not be flanked by the TRs, to
prevent rescue
and/or packaging of these sequences.
[00311] The nucleic acid template can be provided to the cell using any method
known in
the art. For example, the template can be supplied by a non-viral (e.g.,
plasmid) or viral
vector. In particular embodiments, the nucleic acid template is supplied by a
herpesvirus or
adenovirus vector (e.g., inserted into the Ela or E3 regions of a deleted
adenovirus). As
another illustration, Palombo et al., (1998) 1 Virology 72:5025, describes a
baculovirus
vector carrying a reporter gene flanked by the AAV TRs. EBV vectors may also
be employed
to deliver the template, as described above with respect to the rep/cap genes.
[00312] In another representative embodiment, the nucleic acid template is
provided by a
replicating rAAV virus. In still other embodiments, an AAV provirus comprising
the nucleic
acid template is stably integrated into the chromosome of the cell.
[00313] To enhance virus titers, helper virus functions (e.g., adenovirus or
herpesvirus) that
promote a productive AAV infection can be provided to the cell. Helper virus
sequences
necessary for AAV replication are known in the art. Typically, these sequences
will be
provided by a helper adenovirus or herpesvirus vector. Alternatively, the
adenovirus or
herpesvirus sequences can be provided by another non-viral or viral vector,
e.g., as a non-
infectious adenovirus miniplasmid that carries all of the helper genes that
promote efficient
AAV production as described by Ferrari et al., (1997) Nature Med. 3:1295, and
U.S. Patent
Nos. 6,040,183 and 6,093,570.
53
CA 3054600 2019-09-06
[00314] Further, the helper virus functions may be provided by a packaging
cell with the
helper sequences embedded in the chromosome or maintained as a stable
extrachromosomal
element. Generally, the helper viruses sequences cannot be packaged into AAV
virions, e.g.,
are not flanked by TRs.
[00315] Those skilled in the art will appreciate that it may be advantageous
to provide the
AAV replication and capsid sequences and the helper virus sequences (e.g.,
adenovirus
sequences) on a single helper construct. This helper construct may be a non-
viral or viral
construct. As one nonlimiting illustration, the helper construct can be a
hybrid adenovirus or
hybrid herpesvirus comprising the AAV rep/cap genes.
[00316] In one particular embodiment, the AAV rep/cap sequences and the
adenovirus
helper sequences are supplied by a single adenovirus helper vector. This
vector further can
further comprise the nucleic acid template. The AAV rep/cap sequences and/or
the rAAV
template can be inserted into a deleted region (e.g., the El a or E3 regions)
of the adenovirus.
[00317] In a further embodiment, the AAV rep/cap sequences and the adenovirus
helper
sequences are supplied by a single adenovirus helper vector. According to this
embodiment,
the rAAV template can be provided as a plasmid template.
[00318] In another illustrative embodiment, the AAV rep/cap sequences and
adenovirus
helper sequences are provided by a single adenovirus helper vector, and the
rAAV template is
integrated into the cell as a provirus. Alternatively, the rAAV template is
provided by an
EBV vector that is maintained within the cell as an extrachromosomal element
(e.g., as an
EBV based nuclear episome).
[00319] In a further exemplary embodiment, the AAV rep/cap sequences and
adenovirus
helper sequences are provided by a single adenovirus helper. The rAAV template
can be
provided as a separate replicating viral vector. For example, the rAAV
template can be
provided by a rAAV particle or a second recombinant adenovirus particle.
[00320] According to the foregoing methods, the hybrid adenovirus vector
typically
comprises the adenovirus 5' and 3' cis sequences sufficient for adenovirus
replication and
packaging (i.e., the adenovirus terminal repeats and PAC sequence). The AAV
rep/cap
sequences and, if present, the rAAV template are embedded in the adenovirus
backbone and
are flanked by the 5' and 3' cis sequences, so that these sequences may be
packaged into
adenovirus capsids. As described above, the adenovirus helper sequences and
the AAV
rep/cap sequences are generally not flanked by TRs so that these sequences are
not packaged
into the AAV virions.
[00321] Zhang et at., ((2001) Gene Ther. 18:704-12) describe a chimeric helper
comprising
54
CA 3054600 2019-09-06
both adenovirus and the AAV rep and cap genes.
[00322] Herpesvirus may also be used as a helper virus in AAV packaging
methods.
[00323] Hybrid herpesviruses encoding the AAV Rep protein(s) may
advantageously
facilitate scalable AAV vector production schemes. A hybrid herpes simplex
virus type I
(HSV-1) vector expressing the AAV-2 rep and cap genes has been described
(Conway et al.,
(1999) Gene Therapy 6:986 and WO 00/17377.
[00324] As a further alternative, the virus vectors of the invention can be
produced in insect
cells using baculovirus vectors to deliver the rep/cap genes and rAAV template
as described,
for example, by Urabe etal., (2002) Human Gene Therapy 13:1935-43.
[00325] AAV vector stocks free of contaminating helper virus may be obtained
by any
method known in the art. For example, AAV and helper virus may be readily
differentiated
based on size. AAV may also be separated away from helper virus based on
affinity for a
heparin substrate (Zolotukhin et al. (1999) Gene Therapy 6:973). Deleted
replication-
defective helper viruses can be used so that any contaminating helper virus is
not replication
competent. As a further alternative, an adenovirus helper lacking late gene
expression may
be employed, as only adenovirus early gene expression is required to mediate
packaging of
AAV virus. Adenovirus mutants defective for late gene expression are known in
the art (e.g.,
tslOOK and ts149 adenovirus mutants).
Recombinant Virus Vectors
[00326] The present invention provides a method of administering a nucleic
acid molecule
to a cell, the method comprising contacting the cell with the virus vector,
the AAV particle
and/or the composition or pharmaceutical formulation of this invention.
[00327] The present invention further provides a method of delivering a
nucleic acid to a
subject, the method comprising administering to the subject the virus vector,
the AAV
particle and/or the composition or pharmaceutical formulation of this
invention.
[00328] In particular embodiments, the subject is human, and in some
embodiments, the
subject has or is at risk for a disorder that can be treated by gene therapy
protocols.
Nonlimiting examples of such disorders include a muscular dystrophy including
Duchenne or
Becker muscular dystrophy, hemophilia A, hemophilia B, multiple sclerosis,
diabetes
mellitus, Gaucher disease, Fabry disease, Pompe disease, cancer, arthritis,
muscle wasting,
heart disease including congestive heart failure or peripheral artery disease,
intimal
hyperplasia, a neurological disorder including: epilepsy, Huntington's
disease, Parkinson's
disease or Alzheimer's disease, an autoimmune disease, cystic fibrosis,
thalassemia, Hurler's
CA 3054600 2019-09-06
Syndrome, Sly syndrome, Scheie Syndrome, Hurler-Scheie Syndrome, Hunter's
Syndrome,
Sanfilippo Syndrome A, B, C, D, Morquio Syndrome, Maroteaux-Lamy Syndrome,
Krabbe's
disease, phenylketonuria, Batten's disease, spinal cerebral ataxia, LDL
receptor deficiency,
hyperammonemia, anemia, arthritis, a retinal degenerative disorder including
macular
degeneration, adenosine deaminase deficiency, a metabolic disorder, and cancer
including
tumor-forming cancers.
[00329] In some embodiments of the methods of this invention, the virus
vector, the AAV
particle and/or the composition or pharmaceutical formulation of this
invention can be
administered to skeletal muscle, cardiac muscle and/or diaphragm muscle.
[00330] In the methods described herein, the virus vector, the AAV particle
and/or the
composition or pharmaceutical formulation of this invention can be
administered/delivered to
a subject of this invention via a systemic route (e.g., intravenously,
intraarterially,
intraperitoneally, etc.). In some embodiments, the virus vector and/or
composition can be
administered to the subject via an intracerebroventrical, intracisternal,
intraparenchymal,
intracranial and/or intrathecal route. In particular embodiments, the virus
vector and/or
pharmaceutical formulation of this invention are administered intravenously.
[00331] The virus vectors of the present invention are useful for the delivery
of nucleic acid
molecules to cells in vitro, ex vivo, and in vivo. In particular, the virus
vectors can be
advantageously employed to deliver or transfer nucleic acid molecules to
animal cells,
including mammalian cells.
[00332] Any heterologous nucleic acid sequence(s) of interest may be delivered
in the virus
vectors of the present invention. Nucleic acid molecules of interest include
nucleic acid
molecules encoding polypeptides, including therapeutic (e.g., for medical or
veterinary uses)
and/or immunogenic (e.g., for vaccines) polypeptides.
[00333] Therapeutic polypeptides include, but are not limited to, cystic
fibrosis
transmembrane regulator protein (CFTR), dystrophin (including mini- and micro-
dystrophins,
see, e.g., Vincent et al., (1993) Nature Genetics 5:130; U.S. Patent
Publication No.
2003/017131; International Patent Publication No. WO/2008/088895, Wang et al.,
Proc.
Natl. Acad. Sci. USA 97:13714-13719 (2000); and Gregorevic et al., Mol. Ther.
16:657-64
(2008)), myostatin propeptide, follistatin, activin type II soluble receptor,
IGF-1, anti-
inflammatory polypeptides such as the Ikappa B dominant mutant, sarcospan,
utrophin
(Tinsley et al., (1996) Nature 384:349), mini-utrophin, clotting factors
(e.g., Factor VIII,
Factor IX, Factor X, etc.), erythropoietin, angiostatin, endostatin, catalase,
tyrosine
hydroxylase, superoxide dismutase, leptin, the LDL receptor, lipoprotein
lipase, ornithine
56
CA 3054600 2019-09-06
transcarbamylase, 3-globin, a-globin, spectrin, ai-antitrypsin, adenosine
deaminase,
hypoxanthine guanine phosphoribosyl transferase, glucocerebrosidase,
sphingomyelinase,
lysosomal hexosaminidase A, branched-chain keto acid dehydrogenase, RP65
protein,
cytokines (e.g., a-interferon, 3-interferon, interferon-y, interleukin-2,
interleukin-4,
granulocyte-macrophage colony stimulating factor, lymphotoxin, and the like),
peptide
growth factors, neurotrophic factors and hormones (e.g., somatotropin,
insulin, insulin-like
growth factors 1 and 2, platelet derived growth factor, epidermal growth
factor, fibroblast
growth factor, nerve growth factor, neurotrophic factor-3 and -4, brain-
derived neurotrophic
factor, bone morphogenic proteins [including RANKL and VEGF], glial derived
growth
factor, transforming growth factor-a and -p, and the like), lysosomal acid a-
glucosidase, a-
galactosidase A, receptors (e.g., the tumor necrosis growth factor-a soluble
receptor),
S100A1, parvalbumin, adenylyl cyclase type 6, a molecule that modulates
calcium handling
(e.g., SERCA2A, Inhibitor 1 of PP1 and fragments thereof [e.g., WO 2006/029319
and
WO 2007/100465]), a molecule that effects G- protein coupled receptor kinase
type 2
knockdown such as a truncated constitutively active bARKct, anti-inflammatory
factors such
as TRAP, anti-myostatin proteins, aspartoacylase, monoclonal antibodies
(including single
chain monoclonal antibodies; an exemplary Mab is the Herceptine Mab),
neuropeptides and
fragments thereof (e.g., galanin, Neuropeptide Y (see, U.S. Patent No.
7,071,172),
angiogenesis inhibitors such as Vasohibins and other VEGF inhibitors (e.g.,
Vasohibin 2 [see,
WO JP2006/073052]). Other illustrative heterologous nucleic acid sequences
encode suicide
gene products (e.g., thymidine kinase, cytosine deaminase, diphtheria toxin,
and tumor
necrosis factor), proteins conferring resistance to a drug used in cancer
therapy, tumor
suppressor gene products (e.g., p53, Rb, Wt-1), TRAIL, FAS-ligand, and any
other
polypeptide that has a therapeutic effect in a subject in need thereof. AAV
vectors can also
be used to deliver monoclonal antibodies and antibody fragments, for example,
an antibody
or antibody fragment directed against myostatin (see, e.g., Fang et al.,
Nature Biotechnology
23:584-590 (2005)).
[00334] Heterologous nucleic acid sequences encoding polypeptides include
those encoding
reporter polypeptides (e.g., an enzyme). Reporter polypeptides are known in
the art and
include, but are not limited to, Green Fluorescent Protein (GFP), luciferase,
3-galactosidase,
alkaline phosphatase, luciferase, and chloramphenicol acetyltransferase gene.
[00335] Optionally, the heterologous nucleic acid molecule encodes a secreted
polypeptide
(e.g., a polypeptide that is a secreted polypeptide in its native state or
that has been
57
CA 3054600 2019-09-06
engineered to be secreted, for example, by operable association with a
secretory signal
sequence as is known in the art).
[00336] Alternatively, in particular embodiments of this invention, the
heterologous nucleic
acid molecule may encode an antisense nucleic acid molecule, a ribozyme (e.g.,
as described
in U.S. Patent No. 5,877,022), RNAs that effect spliceosome-mediated trans-
splicing (see,
Puttaraju et al., (1999) Nature Biotech. 17:246; U.S. Patent No. 6,013,487;
U.S. Patent No.
6,083,702), interfering RNAs (RNAi) including siRNA, shRNA or miRNA that
mediate gene
silencing (see, Sharp et al., (2000) Science 287:2431), and other non-
translated RNAs, such
as "guide" RNAs (Gorman et al., (1998) Proc. Nat. Acad. Sci. USA 95:4929; U.S.
Patent No.
5,869,248 to Yuan et al.), and the like. Exemplary untranslated RNAs include
RNAi against a
multiple drug resistance (MDR) gene product (e.g., to treat and/or prevent
tumors and/or for
administration to the heart to prevent damage by chemotherapy), RNAi against
myostatin
(e.g., for Duchenne muscular dystrophy), RNAi against VEGF (e.g., to treat
and/or prevent
tumors), RNAi against phospholamban (e.g., to treat cardiovascular disease,
see, e.g., Andino
et al., J Gene Med. 10:132-142 (2008) and Li et al., Acta Pharmacol Sin. 26:51-
55 (2005));
phospholamban inhibitory or dominant-negative molecules such as phospholamban
S 1 6E
(e.g., to treat cardiovascular disease, see, e.g., Hoshijima et al. Nat. Med.
8:864-871 (2002)),
RNAi to adenosine kinase (e.g., for epilepsy), and RNAi directed against
pathogenic
organisms and viruses (e.g., hepatitis B and/or C virus, human
immunodeficiency virus,
CMV, herpes simplex virus, human papilloma virus, etc.).
[00337] Further, a nucleic acid sequence that directs alternative splicing can
be delivered.
To illustrate, an antisense sequence (or other inhibitory sequence)
complementary to the 5'
and/or 3' splice site of dystrophin exon 51 can be delivered in conjunction
with a Ul or U7
small nuclear (sn) RNA promoter to induce skipping of this exon. For example,
a DNA
sequence comprising a U 1 or U7 snRNA promoter located 5' to the
antisense/inhibitory
sequence(s) can be packaged and delivered in a modified capsid of the
invention.
[00338] The virus vector may also comprise a heterologous nucleic acid
molecule that
shares homology with and recombines with a locus on a host cell chromosome.
This
approach can be utilized, for example, to correct a genetic defect in the host
cell.
[00339] The present invention also provides virus vectors that express an
immunogenic
polypeptide, peptide and/or epitope, e.g., for vaccination. The nucleic acid
molecule may
encode any immunogen of interest known in the art including, but not limited
to,
immunogens from human immunodeficiency virus (HIV), simian immunodeficiency
virus
(Sly), influenza virus, HIV or SIV gag proteins, tumor antigens, cancer
antigens, bacterial
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CA 3054600 2019-09-06
antigens, viral antigens, and the like.
[00340] The use of parvoviruses as vaccine vectors is known in the art (see,
e.g., Miyamura
et al., (1994) Proc. Nat. Acad. Sci USA 91:8507; U.S. Patent No. 5,916,563 to
Young et al.,
U.S. Patent No. 5,905,040 to Mazzara et al., U.S. Patent No. 5,882,652, and
U.S. Patent No.
5,863,541 to Samulski et al.). The antigen may be presented in the parvovirus
capsid.
Alternatively, the immunogen or antigen may be expressed from a heterologous
nucleic acid
molecule introduced into a recombinant vector genome. Any immunogen or antigen
of
interest as described herein and/or as is known in the art can be provided by
the virus vector
of the present invention.
[00341] An immunogenic polypeptide can be any polypeptide, peptide, and/or
epitope
suitable for eliciting an immune response and/or protecting the subject
against an infection
and/or disease, including, but not limited to, microbial, bacterial,
protozoal, parasitic, fungal
and/or viral infections and diseases. For example, the immunogenic polypeptide
can be an
orthomyxovirus immunogen (e.g., an influenza virus immunogen, such as the
influenza virus
hemagglutinin (HA) surface protein or the influenza virus nucleoprotein, or an
equine
influenza virus immunogen) or a lentivirus immunogen (e.g., an equine
infectious anemia
virus immunogen, a Simian Immunodeficiency Virus (STY) immunogen, or a Human
Immunodeficiency Virus (HIV) immunogen, such as the HIV or SIV envelope GP160
protein, the HIV or STY matrix/capsid proteins, and the HIV or STY gag, poi
and env gene
products). The immunogenic polypeptide can also be an arenavirus immunogen
(e.g., Lassa
fever virus immunogen, such as the Lassa fever virus nucleocapsid protein and
the Lassa
fever envelope glycoprotein), a poxvirus immunogen (e.g., a vaccinia virus
immunogen, such
as the vaccinia Ll or L8 gene products), a flavivirus immunogen (e.g., a
yellow fever virus
immunogen or a Japanese encephalitis virus immunogen), a filovirus immunogen
(e.g., an
Ebola virus immunogen, or a Marburg virus immunogen, such as NP and GP gene
products),
a bunyavirus immunogen (e.g., RVFV, CCHF, and/or SFS virus immunogens), or a
coronavirus immunogen (e.g., an infectious human coronavirus immunogen, such
as the
human coronavirus envelope glycoprotein, or a porcine transmissible
gastroenteritis virus
immunogen, or an avian infectious bronchitis virus immunogen). The immunogenic
polypeptide can further be a polio immunogen, a herpes immunogen (e.g., CMV,
EBV, HSV
immunogens) a mumps immunogen, a measles immunogen, a rubella immunogen, a
diphtheria toxin or other diphtheria immunogen, a pertussis antigen, a
hepatitis (e.g., hepatitis
A, hepatitis B, hepatitis C, etc.) immunogen, and/or any other vaccine
immunogen now
known in the art or later identified as an immunogen.
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CA 3054600 2019-09-06
[00342] Alternatively, the immunogenic polypeptide can be any tumor or cancer
cell
antigen. Optionally, the tumor or cancer antigen is expressed on the surface
of the cancer
cell. Exemplary cancer and tumor cell antigens are described in S.A. Rosenberg
(Immunity
10:281 (1991)). Other illustrative cancer and tumor antigens include, but are
not limited to:
BRCA I gene product, BRCA2 gene product, gp100, tyrosinase, GAGE-1/2, BAGE,
RAGE,
LAGE, NY-ESO-1, CDK-4, 13-catenin, MUM- 1, Caspase-8, KIAA0205, HPVE, SART-1,
PRAME, p15, melanoma tumor antigens (Kawakami et al., (1994) Proc. Natl. Acad.
Sci.
USA 91:3515; Kawakami et al., (1994) J. Exp. Med., 180:347; Kawakami et al.,
(1994)
Cancer Res. 54:3124), MART-1, gp100 MAGE-1, MAGE-2, MAGE-3, CEA, TRP-1, TRP-
2, P-15, tyrosinase (Brichard et al., (1993)1 Exp . Med. 178:489); HER-2/neu
gene product
(U.S. Pat. No. 4,968,603), CA 125, LK26, FB5 (endosialin), TAG 72, AFP, CA19-
9, NSE,
DU-PAN-2, CA50, SPan-1, CA72-4, HCG, STN (sialyl Tn antigen), c-erbB-2
proteins, PSA,
L-CanAg, estrogen receptor, milk fat globulin, p53 tumor suppressor protein
(Levine, (1993)
Ann. Rev. Biochem. 62:623); mucin antigens (International Patent Publication
No. WO
90/05142); telomerases; nuclear matrix proteins; prostatic acid phosphatase;
papilloma virus
antigens; and/or antigens now known or later discovered to be associated with
the following
cancers: melanoma, adenocarcinoma, thymoma, lymphoma (e.g., non-Hodgkin's
lymphoma,
Hodgkin's lymphoma), sarcoma, lung cancer, liver cancer, colon cancer,
leukemia, uterine
cancer, breast cancer, prostate cancer, ovarian cancer, cervical cancer,
bladder cancer, kidney
cancer, pancreatic cancer, brain cancer and any other cancer or malignant
condition now
known or later identified (see, e.g., Rosenberg, (1996) Ann. Rev. Med. 47:481-
91).
[00343] As a further alternative, the heterologous nucleic acid molecule can
encode any
polypeptide, peptide and/or epitope that is desirably produced in a cell in
vitro, ex vivo, or in
vivo. For example, the virus vectors may be introduced into cultured cells and
the expressed
gene product isolated therefrom.
[00344] It will be understood by those skilled in the art that the
heterologous nucleic acid
molecule(s) of interest can be operably associated with appropriate control
sequences. For
example, the heterologous nucleic acid molecule can be operably associated
with expression
control elements, such as transcription/translation control signals, origins
of replication,
polyadenylation signals, internal ribosome entry sites (IRES), promoters,
and/or enhancers,
and the like.
[00345] Further, regulated expression of the heterologous nucleic acid
molecule(s) of
interest can be achieved at the post-transcriptional level, e.g., by
regulating selective splicing
CA 3054600 2019-09-06
of different introns by the presence or absence of an oligonucleotide, small
molecule and/or
other compound that selectively blocks splicing activity at specific sites
(e.g., as described in
WO 2006/119137).
[00346] Those skilled in the art will appreciate that a variety of
promoter/enhancer elements
can be used depending on the level and tissue-specific expression desired. The
promoter/enhancer can be constitutive or inducible, depending on the pattern
of expression
desired. The promoter/enhancer can be native or foreign and can be a natural
or a synthetic
sequence. By foreign, it is intended that the transcriptional initiation
region is not found in
the wild-type host into which the transcriptional initiation region is
introduced.
[00347] In particular embodiments, the promoter/enhancer elements can be
native to the
target cell or subject to be treated. In representative embodiments, the
promoters/enhancer
element can be native to the heterologous nucleic acid sequence.
[00348] The promoter/enhancer element is generally chosen so that it functions
in the target
cell(s) of interest. Further, in particular embodiments the promoter/enhancer
element is a
mammalian promoter/enhancer element. The promoter/enhancer element may be
constitutive
or inducible.
[00349] Inducible expression control elements are typically advantageous in
those
applications in which it is desirable to provide regulation over expression of
the heterologous
nucleic acid sequence(s). Inducible promoters/enhancer elements for gene
delivery can be
tissue-specific or -preferred promoter/enhancer elements, and include muscle
specific or
preferred (including cardiac, skeletal and/or smooth muscle specific or
preferred), neural
tissue specific or preferred (including brain-specific or preferred), eye
specific or preferred
(including retina-specific and cornea-specific), liver specific or preferred,
bone marrow
specific or preferred, pancreatic specific or preferred, spleen specific or
preferred, and lung
specific or preferred promoter/enhancer elements. Other inducible
promoter/enhancer
elements include hormone-inducible and metal-inducible elements. Exemplary
inducible
promoters/enhancer elements include, but are not limited to, a Tet on/off
element, a RU486-
inducible promoter, an ecdysone-inducible promoter, a rapamycin-inducible
promoter, and a
metallothionein promoter.
[00350] In embodiments wherein the heterologous nucleic acid sequence(s) is
transcribed
and then translated in the target cells, specific initiation signals are
generally included for
efficient translation of inserted protein coding sequences. These exogenous
translational
control sequences, which may include the ATG initiation codon and adjacent
sequences, can
be of a variety of origins, both natural and synthetic.
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CA 3054600 2019-09-06
[00351] The virus vectors according to the present invention provide a means
for delivering
heterologous nucleic acid molecules into a broad range of cells, including
dividing and non-
dividing cells. The virus vectors can be employed to deliver a nucleic acid
molecule of
interest to a cell in vitro, e.g., to produce a polypeptide in vitro or for ex
vivo or in vivo gene
therapy. The virus vectors are additionally useful in a method of delivering a
nucleic acid to
a subject in need thereof, e.g., to express an immunogenic or therapeutic
polypeptide or a
functional RNA. In this manner, the polypeptide or functional RNA can be
produced in vivo
in the subject. The subject can be in need of the polypeptide because the
subject has a
deficiency of the polypeptide.
[00352] Further, the method can be practiced because the production of the
polypeptide or
functional RNA in the subject may impart some beneficial effect.
[00353] The virus vectors can also be used to produce a polypeptide of
interest or functional
RNA in cultured cells or in a subject (e.g., using the subject as a bioreactor
to produce the
polypeptide or to observe the effects of the functional RNA on the subject,
for example, in
connection with screening methods).
[00354] In general, the virus vectors of the present invention can be employed
to deliver a
heterologous nucleic acid molecule encoding a polypeptide or functional RNA to
treat and/or
prevent any disorder or disease state for which it is beneficial to deliver a
therapeutic
polypeptide or functional RNA. Illustrative disease states include, but are
not limited to:
cystic fibrosis (cystic fibrosis transmembrane regulator protein) and other
diseases of the
lung, hemophilia A (Factor VIII), hemophilia B (Factor IX), thalassemia (13-
globin), anemia
(erythropoietin) and other blood disorders, Alzheimer's disease (GDF;
neprilysin), multiple
sclerosis (13-interferon), Parkinson's disease (glial-cell line derived
neurotrophic factor
[GDNF]), Huntington's disease (RNAi to remove repeats), amyotrophic lateral
sclerosis,
epilepsy (galanin, neurotrophic factors), and other neurological disorders,
cancer (endostatin,
angiostatin, TRAIL, FAS-ligand, cytokines including interferons; RNAi
including RNAi
against VEGF or the multiple drug resistance gene product, mir-26a [e.g., for
hepatocellular
carcinoma]), diabetes mellitus (insulin), muscular dystrophies including
Duchenne
(dystrophin, mini-dystrophin, insulin-like growth factor I, a sarcoglycan
[e.g., a, 13, RNAi
against myostatin, myostatin propeptide, follistatin, activin type II soluble
receptor, anti-
inflammatory polypeptides such as the Ikappa B dominant mutant, sarcospan,
utrophin, mini-
utrophin, antisense or RNAi against splice junctions in the dystrophin gene to
induce exon
skipping [see, e.g., WO 2003/095647], antisense against U7 snRNAs to induce
exon skipping
[see, e.g., WO 2006/021724], and antibodies or antibody fragments against
myostatin or
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CA 3054600 2019-09-06
myostatin propeptide) and Becker, Gaucher disease (glucocerebrosidase),
Hurler's disease (a-
L-iduronidase), adenosine deaminase deficiency (adenosine deaminase), glycogen
storage
diseases (e.g., Fabry disease [a-galactosidase] and Pompe disease [lysosomal
acid a-
glucosidase]) and other metabolic disorders, congenital emphysema (al-
antitrypsin), Lesch-
Nyhan Syndrome (hypoxanthine guanine phosphoribosyl transferase), Niemann-Pick
disease
(sphingomyelinase), Tay-Sachs disease (lysosomal hexosaminidase A), Maple
Syrup Urine
Disease (branched-chain keto acid dehydrogenase), retinal degenerative
diseases (and other
diseases of the eye and retina; e.g., PDGF for macular degeneration and/or
vasohibin or other
inhibitors of VEGF or other angiogenesis inhibitors to treat/prevent retinal
disorders, e.g., in
Type I diabetes), diseases of solid organs such as brain (including
Parkinson's Disease
[GDNF], astrocytomas [endostatin, angiostatin and/or RNAi against VEGF],
glioblastomas
[endostatin, angiostatin and/or RNAi against VEGF]), liver, kidney, heart
including
congestive heart failure or peripheral artery disease (PAD) (e.g., by
delivering protein
phosphatase inhibitor I (I-1) and fragments thereof (e.g., I1C), serca2a, zinc
finger proteins
that regulate the phospholamban gene, Barkct, [32-adrenergic receptor, 132-
adrenergic receptor
kinase (BARK), phosphoinositide-3 kinase (PI3 kinase), S100A1S100A1,
parvalbumin,
adenylyl cyclase type 6, a molecule that effects G-protein coupled receptor
kinase type 2
knockdown such as a truncated constitutively active bARKct; calsarcin, RNAi
against
phospholamban; phospholamban inhibitory or dominant-negative molecules such as
phospholamban S16E, etc.), arthritis (insulin-like growth factors), joint
disorders (insulin-like
growth factor 1 and/or 2), intimal hyperplasia (e.g., by delivering enos,
inos), improve
survival of heart transplants (superoxide dismutase), AIDS (soluble CD4),
muscle wasting
(insulin-like growth factor I), kidney deficiency (erythropoietin), anemia
(erythropoietin),
arthritis (anti-inflammatory factors such as TRAP and TNFa soluble receptor),
hepatitis (a-
interferon), LDL receptor deficiency (LDL receptor), hyperammonemia (ornithine
transcarbamylase), Krabbe's disease (galactocerebrosidase), Batten's disease,
spinal cerebral
ataxias including SCA1, SCA2 and SCA3, phenylketonuria (phenylalanine
hydroxylase),
autoimmune diseases, and the like. The invention can further be used following
organ
transplantation to increase the success of the transplant and/or to reduce the
negative side
effects of organ transplantation or adjunct therapies (e.g., by administering
immunosuppressant agents or inhibitory nucleic acids to block cytokine
production). As
another example, bone morphogenic proteins (including BNP 2, 7, etc., RANKL
and/or
VEGF) can be administered with a bone allograft, for example, following a
break or surgical
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CA 3054600 2019-09-06
removal in a cancer patient.
[00355] The invention can also be used to produce induced pluripotent stem
cells (iPS). For
example, a virus vector of the invention can be used to deliver stem cell
associated nucleic
acid(s) into a non-pluripotent cell, such as adult fibroblasts, skin cells,
liver cells, renal cells,
adipose cells, cardiac cells, neural cells, epithelial cells, endothelial
cells, and the like.
Nucleic acids encoding factors associated with stem cells are known in the
art. Nonlimiting
examples of such factors associated with stem cells and pluripotency include
Oct-3/4, the
SOX family (e.g., SOX1, SOX2, SOX3 and/or SOX15), the Klf family (e.g., Klfl,
Klf2, Klf4
and/or Klf5), the Myc family (e.g., C-myc, L-myc and/or N-myc), NANOG and/or
LIN28.
[00356] The invention can also be practiced to treat and/or prevent a
metabolic disorder
such as diabetes (e.g., insulin), hemophilia (e.g., Factor IX or Factor VIII),
a lysosomal
storage disorder such as a mucopolysaccharidosis disorder (e.g., Sly syndrome
[13-
glucuronidase], Hurler Syndrome [a-L-iduronidase], Scheie Syndrome [a-L-
iduronidase],
Hurler-Scheie Syndrome [a-L-iduronidase], Hunter's Syndrome [iduronate
sulfatase],
Sanfilippo Syndrome A [heparan sulfamidase], B [N-acetylglucosaminidase], C
[acetyl-
CoA:a-glucosaminide acetyltransferase], D [N-acetylglucosamine 6-sulfatase],
Morquio
Syndrome A [galactose-6-sulfate sulfatase], B [0-galactosidase], Maroteaux-
Lamy Syndrome
[N-acetylgalactosamine-4-sulfatase], etc.), Fabry disease (a-galactosidase),
Gaucher's disease
(glucocerebrosidase), or a glycogen storage disorder (e.g., Pompe disease;
lysosomal acid a-
glucosidase).
[00357] Gene transfer has substantial potential use for understanding and
providing therapy
for disease states. There are a number of inherited diseases in which
defective genes are
known and have been cloned. In general, the above disease states fall into two
classes:
deficiency states, usually of enzymes, which are generally inherited in a
recessive manner,
and unbalanced states, which may involve regulatory or structural proteins,
and which are
typically inherited in a dominant manner. For deficiency state diseases, gene
transfer can be
used to bring a normal gene into affected tissues for replacement therapy, as
well as to create
animal models for the disease using antisense mutations. For unbalanced
disease states, gene
transfer can be used to create a disease state in a model system, which can
then be used in
efforts to counteract the disease state. Thus, virus vectors according to the
present invention
permit the treatment and/or prevention of genetic diseases.
[00358] The virus vectors according to the present invention may also be
employed to
provide a functional RNA to a cell in vitro or in vivo. Expression of the
functional RNA in
the cell, for example, can diminish expression of a particular target protein
by the cell.
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CA 3054600 2019-09-06
Accordingly, functional RNA can be administered to decrease expression of a
particular
protein in a subject in need thereof. Functional RNA can also be administered
to cells in
vitro to regulate gene expression and/or cell physiology, e.g., to optimize
cell or tissue culture
systems or in screening methods.
[00359] In addition, virus vectors according to the instant invention find use
in diagnostic
and screening methods, whereby a nucleic acid of interest is transiently or
stably expressed in
a cell culture system, or alternatively, a transgenic animal model.
[00360] The virus vectors of the present invention can also be used for
various non-
therapeutic purposes, including but not limited to use in protocols to assess
gene targeting,
clearance, transcription, translation, etc., as would be apparent to one
skilled in the art. The
virus vectors can also be used for the purpose of evaluating safety (spread,
toxicity,
immunogenicity, etc.). Such data, for example, are considered by the United
States Food and
Drug Administration as part of the regulatory approval process prior to
evaluation of clinical
efficacy.
[00361] As a further aspect, the virus vectors of the present invention may be
used to
produce an immune response in a subject. According to this embodiment, a virus
vector
comprising a heterologous nucleic acid sequence encoding an immunogenic
polypeptide can
be administered to a subject, and an active immune response is mounted by the
subject
against the immunogenic polypeptide.
Immunogenic polypeptides are as described
hereinabove. In some embodiments, a protective immune response is elicited.
[00362] Alternatively, the virus vector may be administered to a cell ex vivo
and the altered
cell is administered to the subject. The virus vector comprising the
heterologous nucleic acid
is introduced into the cell, and the cell is administered to the subject,
where the heterologous
nucleic acid encoding the immunogen can be expressed and induce an immune
response in
the subject against the immunogen. In particular embodiments, the cell is an
antigen-
presenting cell (e.g., a dendritic cell).
[00363] An "active immune response" or "active immunity" is characterized by
"participation of host tissues and cells after an encounter with the
immunogen. It involves
differentiation and proliferation of immunocompetent cells in lymphoreticular
tissues, which
lead to synthesis of antibody or the development of cell-mediated reactivity,
or both."
Herbert B. Herscowitz, Immunophysiology: Cell Function and Cellular
Interactions in
Antibody Formation, in IMMUNOLOGY: BASIC PROCESSES 117 (Joseph A. Bellanti
ed.,
1985). Alternatively stated, an active immune response is mounted by the host
after exposure
to an immunogen by infection or by vaccination. Active immunity can be
contrasted with
CA 3054600 2019-09-06
passive immunity, which is acquired through the "transfer of preformed
substances (antibody,
transfer factor, thymic graft, and interleukin-2) from an actively immunized
host to a non-
immune host." Id.
[00364] A "protective" immune response or "protective" immunity as used herein
indicates
that the immune response confers some benefit to the subject in that it
prevents or reduces the
incidence of disease. Alternatively, a protective immune response or
protective immunity
may be useful in the treatment and/or prevention of disease, in particular
cancer or tumors
(e.g., by preventing cancer or tumor formation, by causing regression of a
cancer or tumor
and/or by preventing metastasis and/or by preventing growth of metastatic
nodules). The
protective effects may be complete or partial, as long as the benefits of the
treatment
outweigh any disadvantages thereof.
[00365] In particular embodiments, the virus vector or cell comprising the
heterologous
nucleic acid molecule can be administered in an immunogenically effective
amount, as
described below.
[00366] The virus vectors of the present invention can also be administered
for cancer
immunotherapy by administration of a virus vector expressing one or more
cancer cell
antigens (or an immunologically similar molecule) or any other immunogen that
produces an
immune response against a cancer cell. To illustrate, an immune response can
be produced
against a cancer cell antigen in a subject by administering a virus vector
comprising a
heterologous nucleic acid encoding the cancer cell antigen, for example to
treat a patient with
cancer and/or to prevent cancer from developing in the subject. The virus
vector may be
administered to a subject in vivo or by using ex vivo methods, as described
herein.
Alternatively, the cancer antigen can be expressed as part of the virus capsid
or be otherwise
associated with the virus capsid (e.g., as described above).
[00367] As another alternative, any other therapeutic nucleic acid (e.g.,
RNAi) or
polypeptide (e.g., cytokine) known in the art can be administered to treat
and/or prevent
cancer.
[00368] As used herein, the term "cancer" encompasses tumor-forming cancers.
[00369] Likewise, the term "cancerous tissue" encompasses tumors. A "cancer
cell antigen"
encompasses tumor antigens.
[00370] The term "cancer" has its understood meaning in the art, for example,
an
uncontrolled growth of tissue that has the potential to spread to distant
sites of the body (i.e.,
metastasize). Exemplary cancers include, but are not limited to melanoma,
adenocarcinoma,
thymoma, lymphoma (e.g., non-Hodgkin's lymphoma, Hodgkin's lymphoma), sarcoma,
lung
66
CA 3054600 2019-09-06
cancer, liver cancer, colon cancer, leukemia, uterine cancer, breast cancer,
prostate cancer,
ovarian cancer, cervical cancer, bladder cancer, kidney cancer, pancreatic
cancer, brain
cancer and any other cancer or malignant condition now known or later
identified. In
representative embodiments, the invention provides a method of treating and/or
preventing
tumor-forming cancers.
[00371] The term "tumor" is also understood in the art, for example, as an
abnormal mass of
undifferentiated cells within a multicellular organism. Tumors can be
malignant or benign.
In representative embodiments, the methods disclosed herein are used to
prevent and treat
malignant tumors.
[00372] By the terms "treating cancer," "treatment of cancer" and equivalent
terms it is
intended that the severity of the cancer is reduced or at least partially
eliminated and/or the
progression of the disease is slowed and/or controlled and/or the disease is
stabilized. In
particular embodiments, these terms indicate that metastasis of the cancer is
prevented or
reduced or at least partially eliminated and/or that growth of metastatic
nodules is prevented
or reduced or at least partially eliminated.
[00373] By the terms "prevention of cancer" or "preventing cancer" and
equivalent terms it
is intended that the methods at least partially eliminate or reduce and/or
delay the incidence
and/or severity of the onset of cancer. Alternatively stated, the onset of
cancer in the subject
may be reduced in likelihood or probability and/or delayed.
[00374] In particular embodiments, cells may be removed from a subject with
cancer and
contacted with a virus vector expressing a cancer cell antigen according to
the instant
invention. The modified cell is then administered to the subject, whereby an
immune
response against the cancer cell antigen is elicited. This method can be
advantageously
employed with immunocompromised subjects that cannot mount a sufficient immune
response in vivo (i.e., cannot produce enhancing antibodies in sufficient
quantities).
[00375] It is known in the art that immune responses may be enhanced by
immunomodulatory cytokines (e.g., a-interferon, 13-interferon, y-interferon,
co-interferon, t-
interferon, interleukin-la, interleukin-1 p, interleukin-2, interleukin-3,
interleukin-4,
interleukin-5, interleukin-6, interleukin-7, interleukin-8, interleukin-9,
interleukin-10,
interleukin-11, interleukin-12, interleukin-13, interleukin-14, interleukin-
18, B cell Growth
factor, CD40 Ligand, tumor necrosis factor-a, tumor necrosis factor-13,
monocyte
chemoattractant protein-1, granulocyte-macrophage colony stimulating factor,
and
lymphotoxin). Accordingly, immunomodulatory cytokines (preferably, CTL
inductive
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CA 3054600 2019-09-06
cytokines) may be administered to a subject in conjunction with the virus
vector.
[00376] Cytokines may be administered by any method known in the art.
Exogenous
cytokines may be administered to the subject, or alternatively, a nucleic acid
encoding a
cytokine may be delivered to the subject using a suitable vector, and the
cytokine produced in
vivo.
Subjects, Pharmaceutical Formulations, and Modes of Administration
[00377] Virus vectors, AAV particles and capsids according to the present
invention find use
in both veterinary and medical applications. Suitable subjects include both
avians and
mammals. The term "avian" as used herein includes, but is not limited to,
chickens, ducks,
geese, quail, turkeys, pheasant, parrots, parakeets, and the like. The term
"mammal" as used
herein includes, but is not limited to, humans, non-human primates, bovines,
ovines, caprines,
equines, felines, canines, lagomorphs, etc.
[00378] Human subjects include neonates, infants, juveniles, adults and
geriatric subjects.
[00379] In representative embodiments, the subject is "in need of' the methods
of the
invention.
[00380] In particular embodiments, the present invention provides a
pharmaceutical
composition comprising a virus vector and/or capsid and/or AAV particle of the
invention in
a pharmaceutically acceptable carrier and, optionally, other medicinal agents,
pharmaceutical
agents, stabilizing agents, buffers, carriers, adjuvants, diluents, etc. For
injection, the carrier
will typically be a liquid. For other methods of administration, the carrier
may be either solid
or liquid. For inhalation administration, the carrier will be respirable, and
optionally can be
in solid or liquid particulate form. For administration to a subject or for
other pharmaceutical
uses, the carrier will be sterile and/or physiologically compatible.
[00381] By "pharmaceutically acceptable" it is meant a material that is not
toxic or
otherwise undesirable, i.e., the material may be administered to a subject
without causing any
undesirable biological effects.
[00382] One aspect of the present invention is a method of transferring a
nucleic acid
molecule to a cell in vitro. The virus vector may be introduced into the cells
at the
appropriate multiplicity of infection according to standard transduction
methods suitable for
the particular target cells. Titers of virus vector to administer can vary,
depending upon the
target cell type and number, and the particular virus vector, and can be
determined by those
of skill in the art without undue experimentation. In representative
embodiments, at least
about 103 infectious units, optionally at least about 105 infectious units are
introduced to the
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cell.
[00383] The cell(s) into which the virus vector is introduced can be of any
type, including
but not limited to neural cells (including cells of the peripheral and central
nervous systems,
in particular, brain cells such as neurons and oligodendrocytes), lung cells,
cells of the eye
(including retinal cells, retinal pigment epithelium, and corneal cells),
epithelial cells (e.g.,
gut and respiratory epithelial cells), muscle cells (e.g., skeletal muscle
cells, cardiac muscle
cells, smooth muscle cells and/or diaphragm muscle cells), dendritic cells,
pancreatic cells
(including islet cells), hepatic cells, myocardial cells, bone cells (e.g.,
bone marrow stem
cells), hematopoietic stem cells, spleen cells, keratinocytes, fibroblasts,
endothelial cells,
prostate cells, germ cells, and the like. In representative embodiments, the
cell can be any
progenitor cell. As a further possibility, the cell can be a stem cell (e.g.,
neural stem cell,
liver stem cell). As still a further alternative, the cell can be a cancer or
tumor cell.
Moreover, the cell can be from any species of origin, as indicated above.
[00384] The virus vector can be introduced into cells in vitro for the purpose
of
administering the modified cell to a subject. In particular embodiments, the
cells have been
removed from a subject, the virus vector is introduced therein, and the cells
are then
administered back into the subject. Methods of removing cells from subject for
manipulation
ex vivo, followed by introduction back into the subject are known in the art
(see, e.g., U.S.
patent No. 5,399,346). Alternatively, the recombinant virus vector can be
introduced into
cells from a donor subject, into cultured cells, or into cells from any other
suitable source,
and the cells are administered to a subject in need thereof (L e., a
"recipient" subject).
[00385] Suitable cells for ex vivo nucleic acid delivery are as described
above. Dosages of
the cells to administer to a subject will vary upon the age, condition and
species of the
subject, the type of cell, the nucleic acid being expressed by the cell, the
mode of
administration, and the like. Typically, at least about 102 to about 108 cells
or at least about
103 to about 106 cells will be administered per dose in a pharmaceutically
acceptable carrier.
In particular embodiments, the cells transduced with the virus vector are
administered to the
subject in a treatment effective or prevention effective amount in combination
with a
pharmaceutical carrier.
[00386] In some embodiments, the virus vector is introduced into a cell and
the cell can be
administered to a subject to elicit an immunogenic response against the
delivered polypeptide
(e.g., expressed as a transgene or in the capsid). Typically, a quantity of
cells expressing an
immunogenically effective amount of the polypeptide in combination with a
pharmaceutically acceptable carrier is administered. An "immunogenically
effective amount"
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CA 3054600 2019-09-06
is an amount of the expressed polypeptide that is sufficient to evoke an
active immune
response against the polypeptide in the subject to which the pharmaceutical
formulation is
administered. In particular embodiments, the dosage is sufficient to produce a
protective
immune response (as defined above).
[00387] The degree of protection conferred need not be complete or permanent,
as long as
the benefits of administering the immunogenic polypeptide outweigh any
disadvantages
thereof.
[00388] A further aspect of the invention is a method of administering the
virus vector
and/or virus capsid to subjects. Administration of the virus vectors and/or
capsids according
to the present invention to a human subject or an animal in need thereof can
be by any means
known in the art. Optionally, the virus vector and/or capsid is delivered in a
treatment
effective or prevention effective dose in a pharmaceutically acceptable
carrier.
[00389] The virus vectors and/or capsids of the invention can further be
administered to
elicit an immunogenic response (e.g., as a vaccine). Typically, immunogenic
compositions
of the present invention comprise an immunogenically effective amount of virus
vector
and/or capsid in combination with a pharmaceutically acceptable carrier.
Optionally, the
dosage is sufficient to produce a protective immune response (as defined
above). The degree
of protection conferred need not be complete or permanent, as long as the
benefits of
administering the immunogenic polypeptide outweigh any disadvantages thereof.
Subjects
and immunogens are as described above.
[00390] Dosages of the virus vector and/or capsid to be administered to a
subject depend
upon the mode of administration, the disease or condition to be treated and/or
prevented, the
individual subject's condition, the particular virus vector or capsid, and the
nucleic acid to be
delivered, and the like, and can be determined in a routine manner. Exemplary
doses for
achieving therapeutic effects are titers of at least about 105, 106, 107, 108,
109, 1010, 10", 1012,
iv, 1014, 10'5 transducing units, optionally about 108 to about 1013
transducing units.
[00391] In particular embodiments, more than one administration (e.g., two,
three, four, five,
six, seven, eight, nine, ten, etc., or more administrations) may be employed
to achieve the
desired level of gene expression over a period of various intervals, e.g.,
hourly, daily, weekly,
monthly, yearly, etc. Dosing can be single dosage or cumulative (serial
dosing), and can be
readily determined by one skilled in the art. For instance, treatment of a
disease or disorder
may comprise a one-time administration of an effective dose of a
pharmaceutical composition
virus vector disclosed herein. Alternatively, treatment of a disease or
disorder may comprise
multiple administrations of an effective dose of a virus vector carried out
over a range of time
CA 3054600 2019-09-06
periods, such as, e.g., once daily, twice daily, trice daily, once every few
days, or once
weekly. The timing of administration can vary from individual to individual,
depending upon
such factors as the severity of an individual's symptoms. For example, an
effective dose of a
virus vector disclosed herein can be administered to an individual once every
six months for
an indefinite period of time, or until the individual no longer requires
therapy. A person of
ordinary skill in the art will recognize that the condition of the individual
can be monitored
throughout the course of treatment and that the effective amount of a virus
vector disclosed
herein that is administered can be adjusted accordingly.
[00392] In an embodiment, the period of administration of a virus vector is
for I day, 2 days,
3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12
days, 13 days, 14
days, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks,
11 weeks,
12 weeks, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10
months, 11
months, 12 months, or more. In a further embodiment, a period of during which
administration is stopped is for 1 day, 2 days, 3 days, 4 days, 5 days, 6
days, 7 days, 8 days, 9
days, 10 days, 11 days, 12 days, 13 days, 14 days, 3 weeks, 4 weeks, 5 weeks,
6 weeks, 7
weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 4 months, 5 months, 6
months, 7
months, 8 months, 9 months, 10 months, 11 months, 12 months, or more.
[00393] Exemplary modes of administration include oral, rectal, transmucosal,
intranasal,
inhalation (e.g., via an aerosol), buccal (e.g., sublingual), vaginal,
intrathecal, intraocular,
transdermal, in utero (or in ovo), parenteral (e.g., intravenous,
subcutaneous, intradermal,
intramuscular [including administration to skeletal, diaphragm and/or cardiac
muscle],
intradermal, intrapleural, intracerebral, and intraarticular), topical (e.g.,
to both skin and
mucosal surfaces, including airway surfaces, and transdermal administration),
intralymphatic,
and the like, as well as direct tissue or organ injection (e.g., to liver,
skeletal muscle, cardiac
muscle, diaphragm muscle or brain). Administration can also be to a tumor
(e.g., in or near a
tumor or a lymph node). The most suitable route in any given case will depend
on the nature
and severity of the condition being treated and/or prevented and on the nature
of the
particular vector that is being used.
[00394] Administration to skeletal muscle according to the present invention
includes but is
not limited to administration to skeletal muscle in the limbs (e.g., upper
arm, lower arm,
upper leg, and/or lower leg), back, neck, head (e.g., tongue), thorax,
abdomen,
pelvis/perineum, and/or digits. Suitable skeletal muscles include but are not
limited to
abductor digiti minimi (in the hand), abductor digiti minimi (in the foot),
abductor hallucis,
abductor ossis metatarsi quinti, abductor pollicis brevis, abductor pollicis
longus, adductor
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brevis, adductor hallucis, adductor longus, adductor magnus, adductor
pollicis, anconeus,
anterior scalene, articularis genus, biceps brachii, biceps femoris,
brachialis, brachioradialis,
buccinator, coracobrachialis, corrugator supercilii, deltoid, depressor anguli
oris, depressor
labii inferioris, digastric, dorsal interossei (in the hand), dorsal
interossei (in the foot),
extensor carpi radialis brevis, extensor carpi radialis longus, extensor carpi
ulnaris, extensor
digiti minimi, extensor digitorum, extensor digitorum brevis, extensor
digitorum longus,
extensor hallucis brevis, extensor hallucis longus, extensor indicis, extensor
pollicis brevis,
extensor pollicis longus, flexor carpi radialis, flexor carpi ulnaris, flexor
digiti minimi brevis
(in the hand), flexor digiti minimi brevis (in the foot), flexor digitorum
brevis, flexor
digitorum longus, flexor digitorum profundus, flexor digitorum superficialis,
flexor hallucis
brevis, flexor hallucis longus, flexor pollicis brevis, flexor pollicis
longus, frontalis,
gastrocnemius, geniohyoid, gluteus maximus, gluteus medius, gluteus minimus,
gracilis,
iliocostalis cervicis, iliocostalis lumborum, iliocostalis thoracis, illiacus,
inferior gemellus,
inferior oblique, inferior rectus, infraspinatus, interspinal is,
intertransversi, lateral pterygoid,
lateral rectus, latissimus dorsi, levator anguli oris, levator labii
superioris, levator labii
superioris alaeque nasi, levator palpebrae superioris, levator scapulae, long
rotators,
longissimus capitis, longissimus cervicis, longissimus thoracis, longus
capitis, longus colli,
lumbricals (in the hand), lumbricals (in the foot), masseter, medial
pterygoid, medial rectus,
middle scalene, multifidus, mylohyoid, obliquus capitis inferior, obliquus
capitis superior,
obturator externus, obturator internus, occipitalis, omohyoid, opponens digiti
minimi,
opponens pollicis, orbicularis oculi, orbicularis oris, palmar interossei,
palmaris brevis,
palmaris longus, pectineus, pectoralis major, pectoralis minor, peroneus
brevis, peroneus
longus, peroneus tertius, piriformis, plantar interossei, plantaris, platysma,
popliteus,
posterior scalene, pronator quadratus, pronator teres, psoas major, quadratus
femoris,
quadratus plantae, rectus capitis anterior, rectus capitis lateralis, rectus
capitis posterior
major, rectus capitis posterior minor, rectus femoris, rhomboid major,
rhomboid minor,
risorius, sartorius, scalenus minimus, semimembranosus, semispinalis capitis,
semispinalis
cervicis, semispinalis thoracis, semitendinosus, serratus anterior, short
rotators, soleus,
spinalis capitis, spinalis cervicis, spinalis thoracis, splenius capitis,
splenius cervicis,
sternocleidomastoid, sternohyoid, sternothyroid, stylohyoid, subclavius,
subscapularis,
superior gemellus, superior oblique, superior rectus, supinator,
supraspinatus, temporalis,
tensor fascia lata, teres major, teres minor, thoracis, thyrohyoid, tibialis
anterior, tibialis
posterior, trapezius, triceps brachii, vastus intermedius, vastus lateralis,
vastus medialis,
zygomaticus major, and zygomaticus minor, and any other suitable skeletal
muscle as known
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CA 3054600 2019-09-06
in the art.
[00395] The virus vector and/or capsid can be delivered to skeletal muscle by
intravenous
administration, intra-arterial administration, intraperitoneal administration,
limb perfusion,
(optionally, isolated limb perfusion of a leg and/or arm; see, e.g.,Arruda et
al., (2005) Blood
105: 3458-3464), and/or direct intramuscular injection. In particular
embodiments, the virus
vector and/or capsid is administered to a limb (arm and/or leg) of a subject
(e.g., a subject
with muscular dystrophy such as DMD) by limb perfusion, optionally isolated
limb perfusion
(e.g., by intravenous or intra-articular administration). In embodiments of
the invention, the
virus vectors and/or capsids of the invention can advantageously be
administered without
employing "hydrodynamic" techniques. Tissue delivery (e.g., to muscle) of
prior art vectors
is often enhanced by hydrodynamic techniques (e.g., intravenous/intravenous
administration
in a large volume), which increase pressure in the vasculature and facilitate
the ability of the
vector to cross the endothelial cell barrier. In particular embodiments, the
viral vectors
and/or capsids of the invention can be administered in the absence of
hydrodynamic
techniques such as high volume infusions and/or elevated intravascular
pressure (e.g., greater
than normal systolic pressure, for example, less than or equal to a 5%, 10%,
15%, 20%, 25%
increase in intravascular pressure over normal systolic pressure). Such
methods may reduce
or avoid the side effects associated with hydrodynamic techniques such as
edema, nerve
damage and/or compartment syndrome.
[00396] Administration to cardiac muscle includes administration to the left
atrium, right
atrium, left ventricle, right ventricle and/or septum. The virus vector and/or
capsid can be
delivered to cardiac muscle by intravenous administration, intra-arterial
administration such
as intra-aortic administration, direct cardiac injection (e.g., into left
atrium, right atrium, left
ventricle, right ventricle), and/or coronary artery perfusion.
[00397] Administration to diaphragm muscle can be by any suitable method
including
intravenous administration, intra-arterial administration, and/or intra-
peritoneal
administration.
[00398] Delivery to a target tissue can also be achieved by delivering a depot
comprising the
virus vector and/or capsid. In representative embodiments, a depot comprising
the virus
vector and/or capsid is implanted into skeletal, cardiac and/or diaphragm
muscle tissue or the
tissue can be contacted with a film or other matrix comprising the virus
vector and/or capsid.
Such implantable matrices or substrates are described in U.S. Patent No.
7,201,898.
[00399] In particular embodiments, a virus vector and/or virus capsid
according to the
present invention is administered to skeletal muscle, diaphragm muscle and/or
cardiac muscle
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CA 3054600 2019-09-06
(e.g., to treat and/or prevent muscular dystrophy, heart disease [for example,
PAD or
congestive heart failure]).
[00400] In representative embodiments, the invention is used to treat and/or
prevent
disorders of skeletal, cardiac and/or diaphragm muscle.
[00401] In a representative embodiment, the invention provides a method of
treating and/or
preventing muscular dystrophy in a subject in need thereof, the method
comprising:
administering a treatment or prevention effective amount of a virus vector of
the invention to
a mammalian subject, wherein the virus vector comprises a heterologous nucleic
acid
encoding dystrophin, a mini-dystrophin, a micro-dystrophin, myostatin
propeptide, follistatin,
activin type II soluble receptor, IGF-1, anti-inflammatory polypeptides such
as the Ikappa B
dominant mutant, sarcospan, utrophin, a micro-dystrophin, laminin-a2, a-
sarcoglycan, 13-
sarcoglycan, y-sarcoglycan, 8-sarcoglycan, IGF-1, an antibody or antibody
fragment against
myostatin or myostatin propeptide, and/or RNAi against myostatin. In
particular
embodiments, the virus vector can be administered to skeletal, diaphragm
and/or cardiac
muscle as described elsewhere herein.
[00402] Alternatively, the invention can be practiced to deliver a nucleic
acid to skeletal,
cardiac or diaphragm muscle, which is used as a platform for production of a
polypeptide
(e.g., an enzyme) or functional RNA (e.g., RNAi, microRNA, antisense RNA) that
normally
circulates in the blood or for systemic delivery to other tissues to treat
and/or prevent a
disorder (e.g., a metabolic disorder, such as diabetes [e.g., insulin],
hemophilia [e.g., Factor
IX or Factor VIII], a mucopolysaccharide disorder [e.g., Sly syndrome, Hurler
Syndrome,
Scheie Syndrome, Hurler-Scheie Syndrome, Hunter's Syndrome, Sanfilippo
Syndrome A, B,
C, D, Morquio Syndrome, Maroteaux-Lamy Syndrome, etc.] or a lysosomal storage
disorder
such as Gaucher's disease [glucocerebrosidase] or Fabry disease [a-
galactosidase A] or a
glycogen storage disorder such as Pompe disease [lysosomal acid a
glucosidase]). Other
suitable proteins for treating and/or preventing metabolic disorders are
described herein. The
use of muscle as a platform to express a nucleic acid of interest is described
in U.S. Patent
publication US 2002/0192189.
[00403] Thus, as one aspect, the invention further encompasses a method of
treating and/or
preventing a metabolic disorder in a subject in need thereof, the method
comprising:
administering a treatment or prevention effective amount of a virus vector of
the invention to
skeletal muscle of a subject, wherein the virus vector comprises a
heterologous nucleic acid
encoding a polypeptide, wherein the metabolic disorder is a result of a
deficiency and/or
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CA 3054600 2019-09-06
defect in the polypeptide. Illustrative metabolic disorders and heterologous
nucleic acids
encoding polypeptides are described herein. Optionally, the polypeptide is
secreted (e.g., a
polypeptide that is a secreted polypeptide in its native state or that has
been engineered to be
secreted, for example, by operable association with a secretory signal
sequence as is known
in the art). Without being limited by any particular theory of the invention,
according to this
embodiment, administration to the skeletal muscle can result in secretion of
the polypeptide
into the systemic circulation and delivery to target tissue(s). Methods of
delivering virus
vectors to skeletal muscle are described in more detail herein.
[00404] The invention can also be practiced to produce antisense RNA, RNAi or
other
functional RNA (e.g., a ribozyme) for systemic delivery.
[00405] The invention also provides a method of treating and/or preventing
congenital heart
failure or PAD in a subject in need thereof, the method comprising
administering a
treatment or prevention effective amount of a virus vector of the invention to
a mammalian
subject, wherein the virus vector comprises a heterologous nucleic acid
encoding, for
example, a sarcoplasmic endoreticulum Ca2+-ATPase (SERCA2a), an angiogenic
factor,
phosphatase inhibitor 1(1-1) and fragments thereof (e.g., I1C), RNAi against
phospholamban;
a phospholamban inhibitory or dominant-negative molecule such as phospholamban
S16E, a
zinc finger protein that regulates the phospholamban gene, 132-adrenergic
receptor, 132-
adrenergic receptor kinase (BARK), PI3 kinase, calsarcan, a P-adrenergic
receptor kinase
inhibitor (PARKet), inhibitor 1 of protein phosphatase 1 and fragments thereof
(e.g., I1C),
S100A1, parvalbumin, adenylyl cyclase type 6, a molecule that effects G-
protein coupled
receptor kinase type 2 knockdown such as a truncated constitutively active
bARKet, Pim-1,
PGC-1 a, SOD-1, SOD-2, EC-SOD, kallikrein, HIF, thymosin-134, mir-1, mir- 133,
mir-206,
mir-208 and/or mir-26a.
[00406] Injectables can be prepared in conventional forms, either as liquid
solutions or
suspensions, solid forms suitable for solution or suspension in liquid prior
to injection, or as
emulsions. Alternatively, one may administer the virus vector and/or virus
capsids of the
invention in a local rather than systemic manner, for example, in a depot or
sustained-release
formulation. Further, the virus vector and/or virus capsid can be delivered
adhered to a
surgically implantable matrix (e.g., as described in U.S. Patent Publication
No.
US2004/0013645. The
virus vectors and/or virus capsids disclosed herein can be
administered to the lungs of a subject by any suitable means, optionally by
administering an
aerosol suspension of respirable particles comprised of the virus vectors
and/or virus capsids,
which the subject inhales. The respirable particles can be liquid or solid.
Aerosols of liquid
CA 3054600 2019-09-06
particles comprising the virus vectors and/or virus capsids may be produced by
any suitable
means, such as with a pressure-driven aerosol nebulizer or an ultrasonic
nebulizer, as is
known to those of skill in the art. See, e.g., U.S. Patent No. 4,501,729.
Aerosols of solid
particles comprising the virus vectors and/or capsids may likewise be produced
with any
solid particulate medicament aerosol generator, by techniques known in the
pharmaceutical
art.
[00407] The virus vectors and virus capsids can be administered to tissues of
the CNS (e.g.,
brain, eye) and may advantageously result in broader distribution of the virus
vector or capsid
than would be observed in the absence of the present invention.
[00408] In particular embodiments, the delivery vectors of the invention may
be
administered to treat diseases of the CNS, including genetic disorders,
neurodegenerative
disorders, psychiatric disorders and tumors. Illustrative diseases of the CNS
include, but are
not limited to Alzheimer's disease, Parkinson's disease, Huntington's disease,
Canavan
disease, Leigh's disease, Refsum disease, Tourette syndrome, primary lateral
sclerosis,
amyotrophic lateral sclerosis, progressive muscular atrophy, Pick's disease,
muscular
dystrophy, multiple sclerosis, myasthenia gravis, Binswanger's disease, trauma
due to spinal
cord or head injury, Tay-Sachs disease, Lesch-Nyan disease, epilepsy, cerebral
infarcts,
psychiatric disorders including mood disorders (e.g., depression, bipolar
affective disorder,
persistent affective disorder, secondary mood disorder), schizophrenia, drug
dependency
(e.g., alcoholism and other substance dependencies), neuroses (e.g., anxiety,
obsessional
disorder, somatoform disorder, dissociative disorder, grief, post-partum
depression),
psychosis (e.g., hallucinations and delusions), dementia, paranoia, attention
deficit disorder,
psychosexual disorders, sleeping disorders, pain disorders, eating or weight
disorders (e.g.,
obesity, cachexia, anorexia nervosa, and bulemia) and cancers and tumors
(e.g., pituitary
tumors) of the CNS.
[00409] Disorders of the CNS include ophthalmic disorders involving the
retina, posterior
tract, and optic nerve (e.g., retinitis pigmentosa, diabetic retinopathy and
other retinal
degenerative diseases, uveitis, age-related macular degeneration, glaucoma).
[00410] Most, if not all, ophthalmic diseases and disorders are associated
with one or more
of three types of indications: (1) angiogenesis, (2) inflammation, and (3)
degeneration. The
delivery vectors of the present invention can be employed to deliver anti-
angiogenic factors;
anti-inflammatory factors; factors that retard cell degeneration, promote cell
sparing, or
promote cell growth and combinations of the foregoing.
[00411] Diabetic retinopathy, for example, is characterized by angiogenesis.
Diabetic
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CA 3054600 2019-09-06
retinopathy can be treated by delivering one or more anti-angiogenic factors
either
intraocularly (e.g., in the vitreous) or periocularly (e.g., in the sub-
Tenon's region). One or
more neurotrophic factors may also be co-delivered, either intraocularly
(e.g., intravitreally)
or periocularly.
[00412] Uveitis involves inflammation. One or more anti-inflammatory factors
can be
administered by intraocular (e.g., vitreous or anterior chamber)
administration of a delivery
vector of the invention.
[00413] Retinitis pigmentosa, by comparison, is characterized by retinal
degeneration. In
representative embodiments, retinitis pigmentosa can be treated by intraocular
(e.g., vitreal
administration) of a delivery vector encoding one or more neurotrophic
factors.
[00414] Age-related macular degeneration involves both angiogenesis and
retinal
degeneration. This disorder can be treated by administering the inventive
deliver vectors
encoding one or more neurotrophic factors intraocularly (e.g., vitreous)
and/or one or more
anti-angiogenic factors intraocularly or periocularly (e.g., in the sub-
Tenon's region).
[00415] Glaucoma is characterized by increased ocular pressure and loss of
retinal ganglion
cells. Treatments for glaucoma include administration of one or more
neuroprotective agents
that protect cells from excitotoxic damage using the inventive delivery
vectors. Such agents
include N-methyl-D-aspartate (NMDA) antagonists, cytokines, and neurotrophic
factors,
delivered intraocularly, optionally intravitreally.
[00416] In other embodiments, the present invention may be used to treat
seizures, e.g., to
reduce the onset, incidence or severity of seizures. The efficacy of a
therapeutic treatment for
seizures can be assessed by behavioral (e.g., shaking, ticks of the eye or
mouth) and/or
electrographic means (most seizures have signature electrographic
abnormalities). Thus, the
invention can also be used to treat epilepsy, which is marked by multiple
seizures over time.
[00417] In one representative embodiment, somatostatin (or an active fragment
thereof) is
administered to the brain using a delivery vector of the invention to treat a
pituitary tumor.
According to this embodiment, the delivery vector encoding somatostatin (or an
active
fragment thereof) is administered by microinfusion into the pituitary.
Likewise, such
treatment can be used to treat acromegaly (abnormal growth hormone secretion
from the
pituitary). The nucleic acid (e.g., GenBank Accession No. J00306) and amino
acid (e.g.,
GenBank Accession No. P01166; contains processed active peptides somatostatin-
28 and
somatostatin-14) sequences of somatostatins are known in the art.
[00418] In particular embodiments, the vector can comprise a secretory signal
as described
in U.S. Patent No. 7,071,172.
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CA 3054600 2019-09-06
[00419] In representative embodiments of the invention, the virus vector
and/or virus capsid
is administered to the CNS (e.g., to the brain or to the eye). The virus
vector and/or capsid
may be introduced into the spinal cord, brainstem (medulla oblongata, pons),
midbrain
(hypothalamus, thalamus, epithalamus, pituitary gland, substantia nigra,
pineal gland),
cerebellum, telencephalon (corpus striatum, cerebrum including the occipital,
temporal,
parietal and frontal lobes, cortex, basal ganglia, hippocampus and
portaamygdala), limbic
system, neocortex, corpus striatum, cerebrum, and inferior colliculus. The
virus vector
and/or capsid may also be administered to different regions of the eye such as
the retina,
cornea and/or optic nerve.
[00420] The virus vector and/or capsid may be delivered into the cerebrospinal
fluid (e.g.,
by lumbar puncture) for more disperse administration of the delivery vector.
[00421] The virus vector and/or capsid may further be administered
intravascularly to the
CNS in situations in which the blood-brain barrier has been perturbed (e.g.,
brain tumor or
cerebral infarct).
[00422] The virus vector and/or capsid can be administered to the desired
region(s) of the
CNS by any route known in the art, including but not limited to, intrathecal,
intra- ocular,
intracerebral, intraventricular, intravenous (e.g., in the presence of a sugar
such as mannitol),
intranasal, intra-aural, intra-ocular (e.g., intra-vitreous, sub-retinal,
anterior chamber) and
pen-ocular (e.g., sub-Tenon's region) delivery as well as intramuscular
delivery with
retrograde delivery to motor neurons.
[00423] In particular embodiments, the virus vector and/or capsid is
administered in a liquid
formulation by direct injection (e.g., stereotactic injection) to the desired
region or
compartment in the CNS. In other embodiments, the virus vector and/or capsid
may be
provided by topical application to the desired region or by intra-nasal
administration of an
aerosol formulation. Administration to the eye may be by topical application
of liquid
droplets. As a further alternative, the virus vector and/or capsid may be
administered as a
solid, slow-release formulation (see, e.g., U.S. Patent No. 7,201,898).
[00424] In yet additional embodiments, the virus vector can used for
retrograde transport to
treat and/or prevent diseases and disorders involving motor neurons (e.g.,
amyotrophic lateral
sclerosis (ALS); spinal muscular atrophy (SMA), etc.). For example, the virus
vector can be
delivered to muscle tissue from which it can migrate into neurons.
[00425] In other aspects of this embodiment, a virus vector reduces the
severity of a disease
or disorder by, e.g., at least 10%, at least 15%, at least 20%, at least 25%,
at least 30%, at
least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least
60%, at least 65%, at
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least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least
95%. In yet other
aspects of this embodiment, a virus vector reduces the severity of a disease
or disorder from,
e.g., about 5% to about 100%, about 10% to about 100%, about 20% to about
100%, about
30% to about 100%, about 40% to about 100%, about 50% to about 100%, about 60%
to
about 100%, about 70% to about 100%, about 80% to about 100%, about 10% to
about 90%,
about 20% to about 90%, about 30% to about 90%, about 40% to about 90%, about
50% to
about 90%, about 60% to about 90%, about 70% to about 90%, about 10% to about
80%,
about 20% to about 80%, about 30% to about 80%, about 40% to about 80%, about
50% to
about 80%, or about 60% to about 80%, about 10% to about 70%, about 20% to
about 70%,
about 30% to about 70%, about 40% to about 70%, or about 50% to about 70%.
[00426] A virus vector disclosed herein may comprise a solvent, emulsion or
other diluent in
an amount sufficient to dissolve a virus vector disclosed herein. In other
aspects of this
embodiment, a virus vector disclosed herein may comprise a solvent, emulsion
or a diluent in
an amount of, e.g., less than about 90% (v/v), less than about 80% (v/v), less
than about 70%
(v/v), less than about 65% (v/v), less than about 60% (v/v), less than about
55% (v/v), less
than about 50% (v/v), less than about 45% (v/v), less than about 40% (v/v),
less than about
35% (v/v), less than about 30% (v/v), less than about 25% (v/v), less than
about 20% (v/v),
less than about 15% (v/v), less than about 10% (v/v), less than about 5%
(v/v), or less than
about 1% (v/v). In other aspects of this embodiment, a virus vector disclosed
herein may
comprise a solvent, emulsion or other diluent in an amount in a range of,
e.g., about 1% (v/v)
to 90% (v/v), about 1% (v/v) to 70% (v/v), about 1% (v/v) to 60% (v/v), about
1% (v/v) to
50% (v/v), about 1% (v/v) to 40% (v/v), about 1% (v/v) to 30% (v/v), about 1%
(v/v) to 20%
(v/v), about 1% (v/v) to 10% (v/v), about 2% (v/v) to 50% (v/v), about 2%
(v/v) to 40% (v/v),
about 2% (v/v) to 30% (v/v), about 2% (v/v) to 20% (v/v), about 2% (v/v) to
10% (v/v),
about 4% (v/v) to 50% (v/v), about 4% (v/v) to 40% (v/v), about 4% (v/v) to
30% (v/v),
about 4% (v/v) to 20% (v/v), about 4% (v/v) to 10% (v/v), about 6% (v/v) to
50% (v/v),
about 6% (v/v) to 40% (v/v), about 6% (v/v) to 30% (v/v), about 6% (v/v) to
20% (v/v),
about 6% (v/v) to 10% (v/v), about 8% (v/v) to 50% (v/v), about 8% (v/v) to
40% (v/v),
about 8% (v/v) to 30% (v/v), about 8% (v/v) to 20% (v/v), about 8% (v/v) to
15% (v/v), or
about 8% (v/v) to 12% (v/v).
[00427] Aspects of the present specification disclose, in part, treating an
individual suffering
from a disease or disorder. As used herein, the term "treating," refers to
reducing or
eliminating in an individual a clinical symptom of the disease or disorder; or
delaying or
preventing in an individual the onset of a clinical symptom of a disease or
disorder. For
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example, the term "treating" can mean reducing a symptom of a condition
characterized by a
disease or disorder, by, e.g., at least 20%, at least 25%, at least 30%, at
least 35%, at least
40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at
least 70%, at least
75%, at least 80%, at least 85%, at least 90% at least 95%, or at least 100%.
The actual
symptoms associated with a specific disease or disorder are well known and can
be
determined by a person of ordinary skill in the art by taking into account
factors, including,
without limitation, the location of the disease or disorder, the cause of the
disease or disorder,
the severity of the disease or disorder, and/or the tissue or organ affected
by the disease or
disorder. Those of skill in the art will know the appropriate symptoms or
indicators
associated with a specific type of disease or disorder and will know how to
determine if an
individual is a candidate for treatment as disclosed herein.
[00428] In aspects of this embodiment, a therapeutically effective amount of a
virus vector
disclosed herein reduces a symptom associated with a disease or disorder by,
e.g., at least
10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at
least 40%, at least
45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at
least 75%, at least
80%, at least 85%, at least 90%, at least 95% or at least 100%. In other
aspects of this
embodiment, a therapeutically effective amount of a virus vector disclosed
herein reduces a
symptom associated with a disease or disorder by, e.g., at most 10%, at most
15%, at most
20%, at most 25%, at most 30%, at most 35%, at most 40%, at most 45%, at most
50%, at
most 55%, at most 60%, at most 65%, at most 70%, at most 75%, at most 80%, at
most 85%,
at most 90%, at most 95% or at most 100%. In yet other aspects of this
embodiment, a
therapeutically effective amount of a virus vector disclosed herein reduces a
symptom
associated with disease or disorder by, e.g., about 10% to about 100%, about
10% to about
90%, about 10% to about 80%, about 10% to about 70%, about 10% to about 60%,
about
10% to about 50%, about 10% to about 40%, about 20% to about 100%, about 20%
to about
90%, about 20% to about 80%, about 20% to about 20%, about 20% to about 60%,
about
20% to about 50%, about 20% to about 40%, about 30% to about 100%, about 30%
to about
90%, about 30% to about 80%, about 30% to about 70%, about 30% to about 60%,
or about
30% to about 50%.
[00429] In one embodiment, a virus vector disclosed herein is capable of
increasing the level
and/or amount of a protein encoded in the virus vector that is administered to
a patient by,
e.g., at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at
least 35%, at least
40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at
least 70%, at least
75%, at least 80%, at least 85%, at least 90% or at least 95% as compared to a
patient not
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receiving the same treatment. In other aspects of this embodiment, virus
vector is capable of
reducing the severity of a disease or disorder in an individual suffering from
the disease or
disorder by, e.g., about 10% to about 100%, about 20% to about 100%, about 30%
to about
100%, about 40% to about 100%, about 50% to about 100%, about 60% to about
100%,
about 70% to about 100%, about 80% to about 100%, about 10% to about 90%,
about 20% to
about 90%, about 30% to about 90%, about 40% to about 90%, about 50% to about
90%,
about 60% to about 90%, about 70% to about 90%, about 10% to about 80%, about
20% to
about 80%, about 30% to about 80%, about 40% to about 80%, about 50% to about
80%, or
about 60% to about 80%, about 10% to about 70%, about 20% to about 70%, about
30% to
about 70%, about 40% to about 70%, or about 50% to about 70% as compared to a
patient
not receiving the same treatment.
[00430] In aspects of this embodiment, a therapeutically effective amount of a
virus vector
disclosed herein increases the amount of protein that is encoded within the
virus vector in an
individual by, e.g., at least 10%, at least 15%, at least 20%, at least 25%,
at least 30%, at least
35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at
least 65%, at least
70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or
at least 100% as
compared to an individual not receiving the same treatment. In other aspects
of this
embodiment, a therapeutically effective amount of a virus vector disclosed
herein reduces the
severity of a disease or disorder or maintains the severity of a disease or
disorder in an
individual by, e.g., at most 10%, at most 15%, at most 20%, at most 25%, at
most 30%, at
most 35%, at most 40%, at most 45%, at most 50%, at most 55%, at most 60%, at
most 65%,
at most 70%, at most 75%, at most 80%, at most 85%, at most 90%, at most 95%
or at most
100%. In yet other aspects of this embodiment, a therapeutically effective
amount of a virus
vector disclosed herein reduces or maintains the severity of a disease or
disorder in an
individual by, e.g., about 10% to about 100%, about 10% to about 90%, about
10% to about
80%, about 10% to about 70%, about 10% to about 60%, about 10% to about 50%,
about
10% to about 40%, about 20% to about 100%, about 20% to about 90%, about 20%
to about
80%, about 20% to about 20%, about 20% to about 60%, about 20% to about 50%,
about
20% to about 40%, about 30% to about 100%, about 30% to about 90%, about 30%
to about
80%, about 30% to about 70%, about 30% to about 60%, or about 30% to about
50%.
[00431] A virus vector is administered to an individual or a patient. An
individual or a
patient is typically a human being, but can be an animal, including, but not
limited to, dogs,
cats, birds, cattle, horses, sheep, goats, reptiles and other animals, whether
domesticated or
not.
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[00432] In an embodiment, a virus vector of the present invention can be used
to create an
AAV that targets a specific tissue including, but not limited to, the central
nervous system,
retina, heart, lung, skeletal muscle and liver. These targeted virus vectors
can be used to treat
diseases that are tissue specific, or for the production of proteins that are
endogenously
produced in a specific normal tissue, such as a Factor IX (FIX), Factor VIII,
FVIII and other
proteins known in the art.
Diseases of the Central Nervous System
[00433] In an embodiment, diseases of the central nervous system can be
treated using an
AAV, wherein the AAV comprises a recipient AAV that can be any AAV serotype
and a
donor capsid that is selected from one or more of AAV1, AAV2, AAV3, AAV4,
AAV5,
AAV7, AAV8, AAV9 or AAV10. In one embodiment, the recipient AAV is an AAV2 and
the donor capsid that is selected from one or more of AAV1, AAV2, AAV3, AAV4,
AAV5,
AAV7, AAV8, AAV9 or AAVIO. In another embodiment, the recipient AAV is AAV3
and
the donor capsid that is selected from one or more of AAV I, AAV2, AAV3, AAV4,
AAV5,
AAV7, AAV8, AAV9 or AAV10.
Diseases of the Retina
[00434] In an embodiment, diseases of the retina can be treated using an AAV,
wherein the
AAV comprises a recipient AAV that can be any AAV serotype and a donor capsid
that is
selected from one or more of AAV1, AAV2, AAV3, AAV4, AAV5, AAV7, AAV8, AAV9
or AAV10. In one embodiment, the recipient AAV is an AAV2 and the donor capsid
that is
selected from one or more of AAV1, AAV2, AAV3, AAV4, AAV5, AAV7, AAV8, AAV9
or AAV10. In another embodiment, the recipient AAV is AAV3 and the donor
capsid is
selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV7, AAV8, AAV9 or AAV 10.
Diseases of the Heart
[00435] In a further embodiment, diseases of the heart can be treated using an
AAV,
wherein the AAV comprises a recipient AAV that can be any AAV serotype and the
donor
capsid that is selected from one or more of AAV1, AAV3, AAV4, AAV6 or AAV9. In
an
additional embodiment, the recipient AAV is an AAV2 and the donor capsid that
is selected
from one or more of AAV1, AAV3, AAV4, AAV6 or AAV9. In another embodiment, the
recipient AAV is an AAV3, and the donor capsid that is selected from one or
more of AAV1,
AAV3, AAV4, AAV6 or AAV9.
Diseases of the Lung
[00436] In an embodiment, diseases of the lung can be treated using an AAV,
wherein the
AAV serotype comprises a recipient AAV that can be any AAV serotype and the
donor
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capsid that is selected from one or more of AAV1, AAV5, AAV6, AAV9 or AAV10.
In
another embodiment, the recipient AAV is AAV2 and the donor capsid that is
selected from
one or more of AAV1, AAV5, AAV6, AAV9 or AAV10. In a further embodiment, the
recipient AAV is AAV3 and the donor capsid is selected from that is selected
from one or
more of AAV1, AAV5, AAV6, AAV9 or AAV10.
Diseases of the Skeletal Muscle
[00437] In a further embodiment, diseases of the skeletal muscles can be
treated using an
AAV, wherein the AAV serotype comprises a recipient AAV that can be any AAV
serotype
and the donor capsid that is selected from one or more of AAV1, AAV2, AAV6,
AAV7,
AAV8, or AAV9. In another embodiment, the recipient AAV is AAV2 and the donor
capsid
that is selected from one or more of AAV1, AAV2, AAV6, AAV7, AAV8, or AAV9. In
an
embodiment, the recipient AAV is AAV3 and the donor capsid that is selected
from one or
more of AAV1, AAV2, AAV6, AAV7, AAV8, or AAV9.
Diseases of the Liver
[00438] In an embodiment, diseases of the liver can be treated using an AAV,
wherein the
AAV serotype comprises a recipient AAV that can be any AAV and the donor
capsid that is
selected from one or more of AAV2, AAV3, AAV6, AAV7, AAV8, or AAV9. In an
additional embodiment, the recipient AAV is AAV2 and the donor capsid that is
selected
from one or more of AAV2, AAV3, AAV6, AAV7, AAV8, or AAV9. In a further
embodiment, the recipient AAV is AAV3 and the donor capsid that is selected
from one or
more of AAV2, AAV3, AAV6, AAV7, AAV8, or AAV9.
[00439] In some embodiments, the present application may be defined in any of
the
following paragraphs:
1. An isolated AAV virion having at least two viral structural proteins from
the group
consisting of AAV capsid proteins, VP1, VP2, and VP3, wherein the two viral
proteins are
sufficient to form an AAV virion that encapsidates an AAV genome, and wherein
at least one
of the viral structural proteins present is from a different serotype than the
other viral
structural protein, and wherein the VPI is only from one serotype, the VP2 is
only from one
serotype and the VP3 is only from one serotype.
2. The
isolated AAV virion of paragraph 1, wherein all three viral structural
proteins are
present.
3. The isolated AAV virion of paragraph 2, wherein all three viral structural
proteins are
from different serotypes.
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4. The isolated AAV virion of paragraph 2, wherein only one of the three
structural
proteins is from a different serotype.
5. The isolated AAV virion of paragraph 4, wherein the one viral structural
protein
different from the other two viral structural proteins is VP1.
6. The isolated AAV virion of paragraph 4, wherein the one viral structural
protein
different from the other two viral structural proteins is VP2.
7. The isolated AAV virion of paragraph 4, wherein the one viral structural
protein
different from the other two viral structural proteins is VP3.
8. A substantially homogenous population of virions of paragraphs 1-7, wherein
the
population is at least 101 virions.
9. The substantially homogenous population of virions of paragraph 8, wherein
the
population is at least 107 virions.
10. The substantially homogenous population of virions of paragraph 8, wherein
the
population is at least 107 to 1015 virions.
11. The substantially homogenous population of virions of paragraph 8, wherein
the
population is at least 109 virions.
12. The substantially homogenous population of virions of paragraph 8, wherein
the
population is at least 1010 virions.
13. The substantially homogenous population of virions of paragraph 8, wherein
the
population is at least 1011 virions.
14. The substantially homogenous population of virions of paragraph 10, where
population of virions is at least 95% homogenous.
15. The substantially homogenous population of virions of paragraph 10, where
population of virions is at least 99% homogenous.
16. A method to create an adeno-associated virus (AAV) virion comprising
contacting
cells, under conditions for formation of AAV virions, with a first nucleic
acid sequence and a
second nucleic acid sequence, wherein the AAV virion is formed from at least
VP1, and VP3
viral structural proteins, wherein the first nucleic acid encodes VP1 from a
first AAV
serotype only but is not capable of expressing VP3 and the second nucleic acid
sequence
encodes VP3 from a second AAV serotype only that is different than the first
AAV serotype
and further is not capable of expressing VP1, and wherein, the AAV virion
comprises VP1
from the first serotype only and VP3 from the second serotype only, and
wherein if VP2 is
expressed, it is only from one serotype.
17. The method of paragraph 16, wherein the first nucleic acid has mutations
in the start
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codons of VP2 and VP3 that prevent translation of VP2 and VP3 from an RNA
transcribed
from the first nucleic acid and further wherein, the second nucleic acid has
mutations in the
start codon of VP1 that prevent translation of VP1 from an RNA transcribed
from the second
nucleic acid.
18. The method of paragraph 16, wherein VP2 from only one serotype is
expressed.
19. The method of paragraph 18, wherein VP2 is from a different serotype than
VP1 and
a different serotype than VP3.
20. The method of paragraph 18, wherein VP2 is from the same serotype as VP1.
21. The method of paragraph 18, wherein VP2 is from the same serotype as VP3.
22. The method of paragraph 16, wherein the first AAV serotype is AAV1, AAV2,
AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV 11, or an AAV selected
from Table 1 or Table 3, or any chimeric of each AAV.
23. The method of paragraph 16, wherein the second AAV serotype is AAV!, AAV2,
AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV 10 or AAV11, or an AAV
selected from Table 1 or Table 3, or any chimeric of each AAV.
24. The method of paragraph 18 wherein an AAV virion is formed from VP!, VP2
and
VP3 capsid proteins, wherein the viral structural proteins are encoded in the
first nucleic acid
from a first AAV serotype only and a second nucleic acid from a second AAV
serotype only
that is different than the first AAV serotype and further wherein, the first
nucleic acid has
mutations in the A2 Splice Acceptor Site and further wherein, the second
nucleic acid has
mutations in the Al Splice Acceptor Site, and wherein, the polyploid AAV
virion comprises
VP1 from the first serotype only and VP2 and VP3 from the second serotype
only.
25. The method of paragraph 24, wherein the first AAV serotype is AAV1, AAV2,
AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10 or AAV11, or an AAV
selected from Table 1 or Table 3, or any chimeric of each AAV.
26. The method of paragraph 24, wherein the second AAV serotype is AAV1, AAV2,
AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10 or AAV11, or an AAV
selected from Table 1 or Table 3, or any chimeric of each AAV.
27. The method of paragraph 18, wherein the viral structural proteins are
encoded in the
first nucleic acid sequence from a first AAV serotype only, that is different
from the second
and third serotypes, the second nucleic acid sequence from a second AAV
serotype only that
is different than the first and third AAV serotypes and the third nucleic acid
sequence from a
third AAV serotype only that is different from the first and second AAV
serotypes and
further wherein, the first nucleic acid sequence has mutations in the start
codons of VP2 and
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VP3 that prevent translation of VP2 and VP3 from an RNA transcribed from the
first nucleic
acid and further wherein, the second nucleic acid sequence has mutations in
the start codons
of VP1 and VP3 that prevent translation of VP1 and VP3 from an RNA transcribed
from the
second nucleic acid sequence and further wherein, the third nucleic acid
sequence has
mutations in the start codons of VP1 and VP2 that prevent translation of VP1
and VP2 form
an RNA transcribed from the third nucleic acid, and wherein, the AAV virion
comprises VP1
form the first serotype only, VP2 from the second serotype only, and VP3 from
the third
serotype only.
28. The method of paragraph 27, wherein the first AAV serotype is AAV1, AAV2,
AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10 or AAV11, or an AAV
selected from Table 1 or Table 3, or any chimeric of each AAV.
29. The method of paragraph 27, wherein the second AAV serotype is AAV1, AAV2,
AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVIO or AAV11, or an AAV
selected from Table 1 or Table 3, or any chimeric of each AAV.
30. The method of paragraph 27, wherein the third AAV serotype is AAV1, AAV2,
AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10 or AAV11, or an AAV
selected from Table 1 or Table 3, or any chimeric of each AAV.
31. The method of paragraph 18 wherein, the first nucleic acid sequence has
mutations in
the start codons of VP2 and VP3 that prevent translation of VP2 and VP3 from
an RNA
transcribed from the first nucleic acid sequence and a mutation in the A2
Splice Acceptor Site
and further wherein, the second nucleic acid sequence has mutations in the
start codon of
VP1 that prevent translation of VP1 from an RNA transcribed from the second
nucleic acid
sequence and a mutation in the Al Splice Acceptor Site, and wherein, the AAV
polyploid
capsid comprises VP1 form the first serotype only and VP2 and VP3 from the
second
serotype only.
32. The method of paragraph 31, wherein the first AAV serotype is AAV1, AAV2,
AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10 or AAV11, or an AAV
selected from Table 1 or Table 3, or any chimeric of each AAV.
33. The method of paragraph 31, wherein the second AAV serotype is AAV1, AAV2,
AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV 10 or AAV11, or an AAV
selected from Table 1 or Table 3, or any chimeric of each AAV.
34. The method of paragraph 18, wherein the viral structural proteins are
encoded in the
first nucleic acid sequence are created through DNA shuffling of two or more
different AAV
serotypes and further wherein, the start codons for VP2 and VP3 are mutated
such that VP2
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and VP3 cannot be translated from an RNA transcribed from the first nucleic
acid sequence,
and further wherein, the capsid proteins are encoded in the second nucleic
acid from a single
AAV serotype only, wherein the second nucleic acid has mutations in the start
codon of VP1
that prevent translation of VP1 from an RNA transcribed from the second
nucleic acid, and
wherein, the polyploid AAV capsid comprises VP1 form the first nucleic acid
sequence
created through DNA shuffling and VP2 and VP3 from the second serotype only.
35. The method of paragraph 18, wherein the viral structural proteins are
encoded in the
first nucleic acid sequence are created through DNA shuffling of two or more
different AAV
serotypes and further wherein, the start codons for VP2 and VP3 are mutated
such that VP2
and VP3 cannot be translated from an RNA transcribed from the first nucleic
acid and the A2
Splice Acceptor Site of the first nucleic acid is mutated, and further
wherein, the capsid
proteins are encoded in the second nucleic acid sequence from a single AAV
serotype only,
wherein the second nucleic action has mutations in the start codon of VP1 that
prevent
translation of VP1 from an RNA transcribed from the second nucleic acid and a
mutation in
the Al Splice Acceptor Site, and wherein, the polyploid AAV capsid comprises
VP1 form the
first nucleic acid created through DNA shuffling and VP2 and VP3 from the
second serotype
only.
36. The virion of paragraph 15, wherein the AAV serotype is selected from the
group
consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVIO,
AAV11, an AAV selected from Table 1 or Table 3, and any chimeric of each AAV.
37. A substantially homogenous population of virions produced by the method of
paragraph 16.
38. A substantially homogenous population of virions produced by the method of
paragraph 18.
39. The AAV virion of paragraph 38, wherein the heterologous gene encodes a
protein to
treat a disease.
40. The AAV virion of paragraph 39, wherein the disease is selected from a
lysosomal
storage disorder such as a mucopolysaccharidosis disorder (e.g., Sly syndrome[
-
glucuronidase], Hurler Syndrome [a-L-iduronidase], Scheie Syndrome [a-L-
iduronidase],
Hurler-Scheie Syndrome [a-L-iduronidase], Hunter's Syndrome [iduronate
sulfatase],
Sanfilippo Syndrome A [heparan sulfamidase], B [N- acetylglucosaminidase], C
[acetyl-
CoA:a-glucosaminide acetyltransferase], D [N- acetylglucosamine 6-sulfatase],
Morquio
Syndrome A [galactose-6-sulfate sulfatase], B [ -galactosidase], Maroteaux-
Lamy Syndrome
[N-acetylgalactosamine-4-sulfatase], etc.), Fabry disease (a-galactosidase),
Gaucher's disease
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(glucocerebrosidase), or a glycogen storage disorder (e.g., Pompe disease;
lysosomal acid a-
glucosidase).
41. The isolated AAV virion of paragraphs 1-7, wherein at least one of the
viral structural
proteins is a chimeric viral structural protein.
42. The isolated AAV virion of paragraph 41, wherein the chimeric viral
structural
protein is from AAV serotypes, but different from the other viral structural
proteins.
43. The isolate AAV virion of paragraphs 1-7, wherein none of the viral
structural
proteins are chimeric viral structural proteins.
44. The isolated AAV virion of paragraph 41, wherein there is no overlap in
serotypes
between the chimeric viral structural protein and at least one other viral
structural protein.
45. A method of modulating transduction using the method of paragraphs 16-35.
46. The method of paragraph 45, wherein the method enhances transduction.
47. A method of changing tropism of an AAV virion comprising using the method
of
paragraphs 16-35.
48. A method of changing immunogenicity of an AAV virion comprising using the
method of paragraphs 16-35.
49. A method of increasing vector genome copy number in tissues comprising
using the
method of paragraphs 16-35.
50. A method for increasing transgene expression comprising using the method
of
paragraphs 16-35.
51. A method of treating a disease comprising administering an effective
amount of the
virion of paragraphs 1-7, 36, 43, and 44, the substantially homogenous
population of virions
of paragraphs 8-15, 37-42, and 44, or the virions made by the method of
paragraphs 16-35,
wherein the heterologous gene encodes a protein to treat a disease suitable
for treatment by
gene therapy to a subject having the disease.
52. The method of paragraph 51, wherein the disease is selected from genetic
disorders,
cancers, immunological diseases, inflammation, autoimmune diseases and
degenerative
diseases.
53. The method of paragraphs 51 and 52, wherein multiple administrations are
made.
54. The method of paragraph 53, wherein different polyploid virions are used
to evade
neutralizing antibodies formed in response to a prior administration.
55. A method of increasing at least one of transduction, copy number, and
transgene
expression over an AAV vector having a particle having all its viral
structural proteins from
only one serotype comprising administering the AAV virion of paragraphs 1-15
and 36-44.
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56. An isolated AAV virion having viral capsid structural proteins sufficient
to form an
AAV virion that encapsidates an AAV genome, wherein at least one of the viral
capsid
structural proteins is different from the other viral capsid structural
proteins, and wherein the
virion only contains the same type of each of the structural proteins.
57. The isolated AAV virion of paragraph 56 having at least two viral
structural proteins
from the group consisting of AAV capsid proteins, VP1, VP2, and VP3, wherein
the two
viral proteins are sufficient to form an AAV virion that encapsidates an AAV
genome, and
wherein at least one of the other viral structural proteins present is
different than the other
viral structural protein, and wherein the virion contains only the same type
of each structural
protein.
58. The isolated AAV virion of paragraph 57, wherein all three viral
structural proteins
are present.
59. The isolated AAV virion of paragraph 58, further comprising a fourth AAV
structural
protein.
60. The isolated AAV virion of paragraph 56 having at least two viral
structural proteins
from the group consisting of AAV capsid proteins, VP!, VP2, VP1.5 and VP3,
wherein the
two viral proteins are sufficient to form an AAV virion that encapsidates an
AAV genome,
and wherein at least one of the viral structural proteins present is from a
different serotype
than the other viral structural protein, and wherein the VP1 is only from one
serotype, the
VP2 is only from one serotype, the VP1.5 is only from one serotype, and the
VP3 is only
from one serotype.
61. The isolated AAV virion of paragraphs 57-60, wherein at least one of the
viral
structural proteins is a chimeric protein that is different from at least one
of the other viral
structural proteins.
62. The virion of paragraph 61, wherein only VP3 is chimeric and VP1 and VP2
are non-
chimeric.
63. The virion of paragraph 61, wherein only VP! and VP2 are chimeric and only
VP3 is
non-chimeric.
64. The virion of paragraph 63 wherein the chimeric is comprised of subunits
from AAV
serotypes 2 and 8 and VP3 is from AAV serotype 2.
65. The isolated AAV virion of paragraphs 56-64, wherein all the viral
structural proteins
are from different serotypes.
66. The isolated AAV virion of paragraphs 56-64, wherein only one of the
structural
proteins is from a different serotype.
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67. A substantially homogenous population of virions of paragraphs 56-66,
wherein the
population is at least 107 virions.
68. The substantially homogenous population of virions of paragraph 67,
wherein the
population is at least 107 to 1015 virions.
69. The substantially homogenous population of virions of paragraph 67,
wherein the
population is at least 109 virions.
70. The substantially homogenous population of virions of paragraph 67,
wherein the
population is at least 1010 virions.
71. The substantially homogenous population of virions of paragraph 67,
wherein the
population is at least 1011 virions.
72. The substantially homogenous population of virions of paragraphs 67-71,
where
population of virions is at least 95% homogenous.
73. The substantially homogenous population of virions of paragraph 72, where
population of virions is at least 99% homogenous.
74. The virion of paragraphs 56-73, wherein the AAV serotype is selected from
the group
consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10,
AAV11, an AAV selected from Table 1 or Table 3, and any chimeric of each AAV.
75. A substantially homogenous population of virions of paragraph 73.
76. The AAV virion of paragraphs 56-74, wherein the heterologous gene encodes
a
protein to treat a disease.
77. The AAV virion of paragraph 76, wherein the disease is selected from a
lysosomal
storage disorder such as a mucopolysaccharidosis disorder (e.g., Sly syndrome[
-
glucuronidase], Hurler Syndrome [a-L-iduronidase], Scheie Syndrome [a-L-
iduronidase],
Hurler-Scheie Syndrome [a-L-iduronidase], Hunter's Syndrome [iduronate
sulfatase],
Sanfilippo Syndrome A [heparan sulfamidase], B [N- acetylglucosaminidase], C
[acetyl-
CoA:a-glucosaminide acetyltransferase], D [N- acetylglucosamine 6-sulfatase],
Morquio
Syndrome A [galactose-6-sulfate sulfatase], B [ -galactosidase], Maroteaux-
Lamy Syndrome
[N-acetylgalactosamine-4-sulfatase], etc.), Fabry disease (a-galactosidase),
Gaucher's disease
(glucocerebrosidase), or a glycogen storage disorder (e.g., Pompe disease;
lysosomal acid a-
glucosidase).
78. The isolated AAV virion of paragraphs 56-60 and 66-77, wherein none of the
viral
structural proteins are chimeric viral structural proteins.
79. The isolated AAV virion of paragraphs 57-78, wherein there is no overlap
in
serotypes between the chimeric viral structural protein and at least one other
viral structural
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protein.
80. A method of treating a disease comprising administering an effective
amount of the
virion of paragraphs 56-66, 74, 76-79, or the substantially homogenous
population of virions
of paragraphs 67-73 and 75, wherein the heterologous gene encodes a protein to
treat a
disease suitable for treatment by gene therapy to a subject having the
disease.
81. The method of paragraph 80, wherein the disease is selected from genetic
disorders,
cancers, immunological diseases, inflammation, autoimmune diseases and
degenerative
diseases.
82. The method of paragraphs 80 and 81, wherein multiple administrations are
made.
83. The method of paragraph 82, wherein different polyploid virions are used
to evade
neutralizing antibodies formed in response to a prior administration.
84. The isolated AAV virion of paragraphs 1-7, 36, 39-44, 56-66, 74, 76-79,
the
substantially homogenous population of paragraphs 8-15, 37-38, 67-73, 75 and
methods of
16-35, 45-55, and 80-83, wherein applicants disclaim as follows: To the extent
that any
disclosure in PCT/US18/22725 filed March 15, 2018 falls within the invention
as defined in
any one or more of the claims of this application, or within any invention to
be defined in
amended claims that may in the future be filed in this application or in any
patent derived
therefrom, and to the extent that the laws of any relevant country or
countries to which that or
those claims apply provide that the disclosure of PCT/US18/22725 is part of
the state of the
art against that or those claims in or for that or those countries, we hereby
reserve the right to
disclaim the said disclosure from the claims of the present application or any
patent derived
therefrom to the extent necessary to prevent invalidation of the present
application or any
patent derived therefrom.
For example, and without limitation, we reserve the right to disclaim any one
or
more of the following subject-matters from any claim of the present
application, now or as
amended in the future, or any patent derived therefrom:
A. any subject-matter disclosed in Example 9 of PCT/US18/22725; or
B. vector virions, termed polyploid vector virions, which are produced or
producible
by transfection of two AAV helper plasmids or three plasmids to produce
individual
polyploid vector virions composed of different capsid subunits from different
serotypes;
or
C. vector virions, termed polyploid vector virions, which are produced or
producible
by transfection of two AAV helper plasmids which are AAV2 and AAV8 or AAV9 to
produce individual polyploid vector virions composed of different capsid
subunits from
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different serotypes; or
D. vector virions, termed polyploid vector virions, which are produced or
producible
by transfection of three AAV helper plasmids which are AAV2, AAV8 and AAV9 to
produce individual polyploid vector virions composed of different capsid
subunits from
different serotypes; or
E. vector virions, termed haploid vectors, with VP1NP2 from one AAV vector
capsid
or AAV serotype and VP3 from an alternative one, for example VP1NP2 from (the
capsid of) only one AAV serotype and VP3 from only one alternative AAV
serotype; or
F. any one or more AAV vector virion(s) selected from:
a vector which is generated by transfection of AAV2 helper and AAV8 helper
plasmids
(termed haploid AAV2/8) and which has VP1 capsid subunit from AAV8 and VP2NP3
capsid subunits from AAV2; or
a vector which is generated by transfection of AAV2 helper and AAV8 helper
plasmids
(termed haploid AAV2/8 or haploid AAV8/2 or haploid AAV82 or H-AAV82) and
which
has VP1NP2 capsid subunits from AAV8 and VP3 capsid subunit from AAV2; or
a vector in which VP1/VP2 is derived from different serotypes; or
a vector (termed haploid AAV92 or H-AAV92) which has VP1/VP2 capsid subunits
from
AAV9 and VP3 capsid subunit from AAV2; or
a vector (termed haploid AAV2G9 or H-AAV2G9) which has VP1NP2 capsid subunits
from AAV8 and VP3 capsid subunit from AAV2G9, in which AAV9 glycan receptor
binding site was engrafted into AAV2; or
a vector (termed haploid AAV83 or H-AAV83) which has VP1/VP2 capsid subunits
from
AAV8 and VP3 capsid subunit from AAV3; or
a vector (termed haploid AAV93 or H-AAV93) which has VP1/VP2 capsid subunits
from
AAV9 and VP3 capsid subunit from AAV3; or
a vector (termed haploid AAVrh10-3 or H-AAVrh10-3) which has VP1NP2 capsid
subunits from AAVrh10 and VP3 capsid subunit from AAV3; or
a vector which is generated by transfection of AAV2 helper and AAV8 helper
plasmids
(termed haploid AAV2/8) and which has VP1 capsid subunit from AAV2 and VP2/VP3
capsid subunits from AAV8; or
a vector which is generated by transfection of AAV2 helper and AAV8 helper
plasmids
(termed haploid AAV2/8) and which has VP1/VP2 capsid subunit from AAV2 and VP3
capsid subunits from AAV8; or
a vector which is generated by transfection of AAV2 helper and AAV8 helper
plasmids
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(termed haploid AAV2/8) and which has VPI capsid subunit from AAV8 and VP3
capsid
subunit from AAV2; or
a vector which is generated by transfection of AAV2 helper and AAV8 helper
plasmids
(termed haploid AAV2/8) and which has VPI capsid subunit from AAV2 and VP3
capsid
subunits from AAV8; or
a vector which is generated by transfection of AAV2 helper and AAV8 helper
plasmids
(termed haploid AAV2/8) and which has VP I/VP2/VP3 capsid subunits from AAV2;
or
a vector which is generated by transfection of AAV2 helper and AAV8 helper
plasmids
(termed haploid AAV2/8) and which has VP1/VP2/VP3 capsid subunits from AAV8;
or
a vector termed 28m-2VP3 or haploid 2m-2VP3 or haploid vector 28m-2VP3 in
which
chimeric VP1/VP2 capsid subunits have N-terminal from AAV2 and C-terminal from
AAV8, and the VP3 capsid subunit is from AAV2; or
a vector termed chimeric AAV8/2 or chimeric AAV82 in which chimeric VPI/VP2
capsid subunits have N-terminal from AAV8 and C-terminal from AAV2 without
mutation of the VP3 start codon and the VP3 capsid subunit is from AAV2; or
a vector in which chimeric VP INP2 capsid subunits have N-terminal from AAV2
and C-
terminal from AAV8; or
G. a population, for example a substantially homogenous population, for
example a
population of 1010 particles, for example a substantially homogenous
population of 1010
particles, of any one of the vectors of F; or
H. a method of producing any one of the vectors or populations of vectors
of A and/or
B and/or C and/or D and/or E and/or F and/or G; or
I. any combination thereof.
Without limitation, we state that the above reservation of a right of
disclaimer applies at
least to claims 1-30 as appended to this application and paragraphs 1-83 as
set forth at
[00437]. The modified virus capsids can be used as "capsid vehicles," as has
been
described, for example, in U.S. Patent No. 5,863,541. Molecules that can be
packaged by
the modified virus capsid and transferred into a cell include heterologous
DNA, RNA,
polypeptides, small organic molecules, metals, or combinations of the same.
[00440] In some embodiments, the present application may be defined in any of
the
following paragraphs:
1. An isolated AAV virion having three viral structural proteins from the
group
consisting of AAV capsid proteins, VP1, VP2, and VP3, wherein the viral
proteins are
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sufficient to form an AAV virion that encapsidates an AAV genome, and wherein
the VP1
and VP2 viral structural proteins present are from the same serotype and the
VP3 serotype is
from an alternative serotype, and wherein the VP1 and VP2 are only from a
single serotype,
and the VP3 is only from a single serotype.
2. The isolated AAV virion of paragraph 1 wherein VP1 and VP2 are from AAV
serotype 8 or 9 and VP3 is from AAV serotype 3 or 2.
3. The isolated AAV virion of paragraph 1 wherein VP1 and VP2 are from AAV
serotype 8 and VP3 is from AAV serotype 2G9.
4. An isolated AAV virion having three viral structural proteins from the
group
consisting of AAV capsid proteins, VP1, VP2, and VP3, wherein the viral
proteins are
sufficient to form an AAV virion that encapsidates an AAV genome, and wherein
the VP1
and VP2 viral structural proteins present are from the same chimeric serotype
and the VP3
serotype is not a chimeric serotype, and wherein the VP1 and VP2 are only from
a single
chimeric serotype, and the VP3 is only from a single serotype, wherein VP1 and
VP2 are
from chimeric AAV serotype 28m and VP3 is from AAV serotype 2.
5. The isolated AAV virion of paragraph 1 wherein VP1 and VP2 are from AAV
serotype AAV rh10 and VP3 is from AAV serotype 2G9.
6. A method to create an adeno-associated virus (AAV) virion comprising
contacting
cells, under conditions for formation of AAV virions, with a first nucleic
acid sequence and a
second nucleic acid sequence, wherein the AAV virion is formed from VP1, VP2,
and VP3
viral structural proteins, wherein the first nucleic acid encodes VP1 and VP2
from a first
AAV serotype only but is not capable of expressing VP3 and the second nucleic
acid
sequence encodes VP3 from an alternative AAV serotype that is different than
the first AAV
serotype and further is not capable of expressing VP1 or VP2, and wherein, the
AAV virion
comprises VP1 and VP2 only from the first serotype and VP3 only from the
second serotype.
7. The AAV virion produced by the method of paragraph 6.
8. The method of paragraph 2, wherein VP1 and VP2 are from AAV serotype 8 or 9
and
VP3 is from AAV serotype 3 or 2.
9. The method of paragraph 2, wherein VP1 and VP2 are from AAV serotype 8 and
VP3
is from AAV serotype 2G9.
10. A method to create an adeno-associated virus (AAV) virion comprising
contacting
cells, under conditions for formation of AAV virions, with a first nucleic
acid sequence and a
second nucleic acid sequence, wherein the AAV virion is formed from VP1, VP2,
and VP3
viral structural proteins, wherein the first nucleic acid encodes VP1 and VP2
from a first
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chimeric AAV serotype only but is not capable of expressing VP3 and the second
nucleic
acid sequence encodes VP3 from an alternative AAV serotype and further is not
capable of
expressing VP1 or VP2, wherein VP1 and VP2 are from AAV serotype 28m and VP3
is from
AAV serotype 2.
11. The method of paragraph 2, wherein VP1 and VP2 are from AAV serotype AAV
rh10
and VP3 is from AAV serotype 2G9.
12. A haploid vector with VP1/VP2 from one AAV vector capsid and VP3 from an
alternative one.
13. A haploid vector AAV82 (H-AAV82) with VP1/VP2 from AAV8 and VP3 from
AAV2.
14. A haploid vector AAV92 (H-AAV92) with VP1NP2 from AAV9 and VP3 from
AAV2.
15. A haploid vector AAV82 G9 (H-AAV82G9) in which VP1/VP2 is from AAV8 and
VP3 is from AAV2G9, wherein AAV2G9 has engrafted AAV9 glycan receptor binding
sites
into AAV2.
16. A haploid vector AAV83 (H-AAV83), wherein VP1/VP2 is from AAV8 and VP3 is
from AAV3.
17. A haploid vector AAV93 (H-AAV93), wherein VP1/VP2 is from AAV9 and VP3 is
from AAV3.
18. A haploid vector AAVrh10-3 (H-AAVrh10-3), wherein VP1/VP2 is from AAVrhl 0
and VP3 is from AAV3.
19. A vector 28m-2VP3 (H-28m-2VP3) in which chimeric VP1/VP2 capsid subunits
have
N-terminal from AAV2 and C-terminal from AAV8, and the VP3 capsid subunit is
from
AAV2.
20. A vector termed chimeric AAV8/2 or chimeric AAV82 in which chimeric
VP1/VP2
capsid subunits have N-terminal from AAV8 and C-terminal from AAV2 without
mutation of
the VP3 start codon and the VP3 capsid subunit is from AAV2.
[00441] In some embodiments, the present application may be defined in any of
the
following paragraphs:
1. A
method to create a polyploid adeno-associated virus (AAV) capsid
comprising contacting cells, under conditions for formation of AAV virions,
with a first
nucleic acid sequence and a second nucleic acid sequence, wherein an AAV
capsid is formed
from VP!, VP2 and VP3 capsid proteins, wherein the capsid proteins are encoded
in the first
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nucleic acid from a first AAV serotype only and the second nucleic acid from a
second AAV
serotype only that is different than the first AAV serotype and further
wherein, the first
nucleic acid has mutations in the start codons of VP2 and VP3 that prevent
translation of VP2
and VP3 from an RNA transcribed from the first nucleic acid and further
wherein, the second
nucleic acid has mutations in the start codon of VP1 that prevent translation
of VP1 from an
RNA transcribed from the second nucleic acid, and wherein, the polyploid AAV
capsid
comprises VP1 from the first serotype only and VP2 and VP3 from the second
serotype only.
2. The method of paragraph 1, wherein the first AAV serotype is AAV I,
AAV2,
AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, or an AAV
selected from Table 1 or Table 3, or any chimeric of each AAV.
3. The method of paragraph 1, wherein the second AAV serotype is AAV1,
AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10 or AAV1 1, or an
AAV selected from Table 1 or Table 3, or any chimeric of each AAV.
4. A method to create a polyploid adeno-associated virus (AAV) capsid
comprising contacting cells, under conditions for formation of AAV virions,
with a first
nucleic acid sequence, and a second nucleic acid sequence, wherein an AAV
capsid is formed
from VP1, VP2 and VP3 capsid proteins, wherein the capsid proteins are encoded
in the first
nucleic acid from a first AAV serotype only and a second nucleic acid from a
second AAV
serotype only that is different than the first AAV serotype and further
wherein, the first
nucleic acid has mutations in the A2 Splice Acceptor Site and further wherein,
the second
nucleic acid has mutations in the Al Splice Acceptor Site, and wherein, the
polyploid AAV
capsid comprises VP1 from the first serotype only and VP2 and VP3 from the
second
serotype only.
5. The method of paragraph 4, wherein the first AAV serotype is AAV1, AAV2,
AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV 10 or AAV1 1, or an AAV
selected from Table 1 or Table 3, or any chimeric of each AAV.
6. The method of paragraph 4, wherein the second AAV serotype is AAV1,
AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10 or AAV11, or an
AAV selected from Table 1 or Table 3, or any chimeric of each AAV.
7. A method to create a polyploid adeno-associated virus (AAV) capsid
comprising contacting cells, under conditions for formation of AAV virions,
with a first
nucleic acid sequence, a second nucleic acid sequence, and a third nucleic
acid sequence,
wherein an AAV capsid is formed from VP1, VP2 and VP3 capsid proteins, wherein
the
capsid proteins are encoded in the first nucleic acid from a first AAV
serotype only that is
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different from the second and third serotypes, the second nucleic acid from a
second AAV
serotype only that is different than the first and third AAV serotypes and the
third nucleic
acid from a third AAV serotype only that is different from the first and
second AAV
serotypes and further wherein, the first nucleic acid has mutations in the
start codons of VP2
and VP3 that prevent translation of VP2 and VP3 from an RNA transcribed from
the first
nucleic acid and further wherein, the second nucleic acid has mutations in the
start codons of
VP1 and VP3 that prevent translation of VP1 and VP3 from an RNA transcribed
from the
second nucleic acid and further wherein, the third nucleic acid has mutations
in the start
codons of VP1 and VP2 that prevent translation of VPI and VP2 form an RNA
transcribed
from the third nucleic acid, and wherein, the polyploid AAV capsid comprises
VPI form the
first serotype only, VP2 from the second serotype only and VP3 from the third
serotype only.
8. The method of paragraph 7, wherein the first AAV serotype is
AAV1, AAV2,
AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV 10 or AAV11, or an AAV
selected from Table 1 or Table 3, or any chimeric of each AAV.
9. The method of paragraph 7, wherein the second AAV serotype is
AAV I,
AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10 or AAV11, or an
AAV selected from Table 1 or Table 3, or any chimeric of each AAV.
10. The method of paragraph 7, wherein the third AAV serotype is
AAV1, AAV2,
AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV 10 or AAV11, or an AAV
selected from Table 1 or Table 3, or any chimeric of each AAV.
11. A method to create a polyploid adeno-associated virus (AAV)
capsid
comprising contacting cells, under conditions for formation of AAV virions,
with a first
nucleic acid sequence and a second nucleic acid sequence, wherein an AAV
capsid is
constructed from VP1, VP2 and VP3 capsid proteins, wherein the capsid proteins
are
encoded in the first nucleic acid from a first AAV serotype only and the
second nucleic acid
from a second AAV serotype only that is different than the first AAV serotype
and further
wherein, the first nucleic acid has mutations in the start codons of VP2 and
VP3 that prevent
translation of VP2 and VP3 from an RNA transcribed from the first nucleic acid
and a
mutation in the A2 Splice Acceptor Site and further wherein, the second
nucleic acid has
mutations in the start codon of VPI that prevent translation of VP I from an
RNA transcribed
from the second nucleic acid and a mutation in the Al Splice Acceptor Site,
and wherein, the
AAV polyploid capsid comprises VP1 form the first serotype only and VP2 and
VP3 from
the second serotype only.
12. The method of paragraph 11, wherein the first AAV serotype is
AAV I,
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AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10 or AAV11, or an
AAV selected from Table 1 or Table 3, or any chimeric of each AAV.
13. The method of paragraph 11, wherein the second AAV serotype is AAV1,
AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10 or AAV11, or an
AAV selected from Table 1 or Table 3, or any chimeric of each AAV.
14. A method to create a polyploid adeno-associated virus (AAV) capsid,
comprising contacting cells, under conditions for formation of AAV virions,
with a first
nucleic acid and a second nucleic acid, wherein an AAV capsid is formed from
VP1, VP2
and VP3 capsid proteins, wherein the capsid proteins are encoded in the first
nucleic acid that
is created through DNA shuffling of two or more different AAV serotypes and
further
wherein, the start codons for VP2 and VP3 are mutated such that VP2 and VP3
cannot be
translated from an RNA transcribed from the first nucleic acid, and further
wherein, the
capsid proteins are encoded in the second nucleic acid from a single AAV
serotype only,
wherein the second nucleic acid has mutations in the start codon of VP1 that
prevent
translation of VP1 from an RNA transcribed from the second nucleic acid, and
wherein, the
polyploid AAV capsid comprises VP1 form the first nucleic acid created through
DNA
shuffling and VP2 and VP3 from the second serotype only.
15. A method to create a polyploid adeno-associated virus (AAV) capsid
comprising contacting cells, under conditions for formation of AAV virions,
with a first
nucleic acid and a second nucleic acid, wherein an AAV capsid is formed from
VP1, VP2
and VP3 capsid proteins, wherein the capsid proteins are encoded in the first
nucleic acid that
is created through DNA shuffling of two or more different AAV serotypes and
further
wherein, the start codons for VP2 and VP3 are mutated such that VP2 and VP3
cannot be
translated from an RNA transcribed from the first nucleic acid and the A2
Splice Acceptor
Site of the first nucleic acid is mutated, and further wherein, the capsid
proteins are encoded
in the second nucleic acid from a single AAV serotype only, wherein the second
nucleic
action has mutations in the start codon of VP1 that prevent translation of VP1
from an RNA
transcribed from the second nucleic acid and a mutation in the Al Splice
Acceptor Site, and
wherein, the polyploid AAV capsid comprises VP1 form the first nucleic acid
created
through DNA shuffling and VP2 and VP3 from the second serotype only.
16. The method of paragraphs 14 and 15, wherein the AAV serotype is AAV1,
AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10 or AAV11, or an
AAV selected from Table 1 or Table 3, or any chimeric of each AAV.
17. The method of any of paragraphs 1 - 16, wherein the AAV capsid has
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substantially homogenous capsid proteins.
18. The method of paragraph 17, wherein the polyploid adeno-associated
virus
(AAV) substantially homogenous capsid protein is VP 1 .
19. The method of paragraph 17, wherein the substantially homogenous capsid
protein is VP2.
20. The method of paragraph 17, wherein the substantially homogenous capsid
protein is VP3.
21. The method of paragraph 17, wherein the substantially homogenous capsid
protein is VP1 and VP2, VPI and VP3, VP2 and VP3, or VP I and VP2 and VP3.
22. The method of any of paragraphs 1 ¨ 21, wherein the polyploid adeno-
associated virus (AAV) is in a substantially homogenous population of AAV
capsids.
23. The method of paragraph 22, wherein the polyploid adeno-associated
virus
(AAV) is in a substantially homogenous population of AAV virions comprising
capsid
protein VP1 of only one serotype.
24. The method of paragraph 22, The method of paragraph 17, wherein the
polyploid adeno-associated virus (AAV) is in a substantially homogenous
population of AAV
virions comprising capsid protein VP2 of only one serotype.
25. The method of paragraph 22, wherein the polyploid adeno-associated
virus
(AAV) is in a substantially homogenous population of AAV virions comprising
capsid
protein VP3 of only one serotype.
26. The method of paragraph 22, wherein the polyploid adeno-associated
virus
(AAV) is in a substantially homogenous population of AAV virions comprising
capsid
protein VP1 and VP2 of only one serotype, or VP1 and VP3 of only one serotype,
or VP2 and
VP3 of only one serotype, or VP1 of only one serotype.
27. A polyploid AAV, wherein the polyploid AAV is prepared using the method
of any of paragraphs I -26.
28. The polyploid AAV of any of paragraphs 1-27, wherein the polyploid AAV
is
constructed from VP1 and VP3 only.
29. A polyploid AAV, wherein the polyploid AAV is prepared using the method
of any of paragraphs 1 -28 and further wherein, the polyploid AAV includes a
heterologous
gene.
30. The polyploid AAV of paragraph 29, wherein the heterologous gene
encodes a
protein to treat a disease.
31. The polyploid AAV of paragraph 30, wherein the disease is selected from
a
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lysosomal storage disorder such as a mucopolysaccharidosis disorder (e.g., Sly
syndrome[ -
glucuronidase], Hurler Syndrome [a-L-iduronidase], Scheie Syndrome [a-L-
iduronidase],
Hurler-Scheie Syndrome [a-L-iduronidase], Hunter's Syndrome [iduronate
sulfatase],
Sanfilippo Syndrome A [heparan sulfamidase], B [N- acetylglucosaminidase], C
[acetyl-
CoA:a-glucosaminide acetyltransferase], D [N- acetylglucosamine 6-sulfatase],
Morquio
Syndrome A [galactose-6-sulfate sulfatase], B [ -galactosidase], Maroteaux-
Lamy Syndrome
[N-acetylgalactosamine-4-sulfatase], etc.), Fabry disease (a-galactosidase),
Gaucher's disease
(glucocerebrosidase), or a glycogen storage disorder (e.g., Pompe disease;
lysosomal acid a-
glucosidase).
[00442] In some embodiments, the present application may be defined in any of
the
following paragraphs:
1. An isolated AAV virion having at least two viral structural proteins from
the group
consisting of AAV capsid proteins, VP!, VP2, and VP3, wherein the two viral
proteins are
sufficient to form an AAV virion that encapsidates an AAV genome, and wherein
at least one
of the other viral structural proteins present is different than the other
viral structural protein,
and wherein the virion contains only the same type of each structural protein.
2. The isolated AAV virion of paragraph 1, wherein all three viral structural
proteins are
present.
3. The isolated AAV virion of paragraphs 1 and 2, wherein at least one of the
viral
structural proteins is a chimeric protein that is different from at least one
of the other viral
structural proteins.
4. The virion of paragraph 3, wherein only VP3 is chimeric and VP1 and VP2 are
non-
chimeric.
5. The virion of paragraph 3, wherein only VP! and VP2 are chimeric and only
VP3 is
non-chimeric.
6. The virion of paragraph 5 wherein the chimeric is comprised of subunits
from AAV
serotypes 2 and 8 and VP3 is from AAV serotype 2.
7. The isolated AAV virion of paragraphs 1-6, wherein all three viral
structural proteins
are from different serotypes.
8. The isolated AAV virion of paragraphs 1-6, wherein only one of the three
structural
proteins is from a different serotype.
9. The substantially homogenous population of virions of paragraph 8, wherein
the
population is at least 107 virions.
10. The substantially homogenous population of virions of paragraph 8, wherein
the
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population is at least 107 to 1015 virions.
11. The substantially homogenous population of virions of paragraph 8, wherein
the
population is at least 109 virions.
12. The substantially homogenous population of virions of paragraph 8, wherein
the
population is at least 1010 virions.
13. The substantially homogenous population of virions of paragraph 8, wherein
the
population is at least 1011 virions.
14. The substantially homogenous population of virions of paragraphs 9-13,
where
population of virions is at least 95% homogenous.
15. The substantially homogenous population of virions of paragraph 14, where
population of virions is at least 99% homogenous.
16. The virion of paragraphs 1-15, wherein the AAV serotype is AAV1, AAV2,
AAV3,
AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10 or AAV11, or an AAV selected from
Table 1 or Table 3, or any chimeric of each AAV.
17. A substantially homogenous population of virions of paragraph 16.
18. The AAV virion of paragraphs 1-17, wherein the heterologous gene encodes a
protein
to treat a disease.
19. The AAV virion of paragraph 18, wherein the disease is selected from a
lysosomal
storage disorder such as a mucopolysaccharidosis disorder (e.g., Sly syndrome[
-
glucuronidase], Hurler Syndrome [a-L-iduronidase], Scheie Syndrome [a-L-
iduronidase],
Hurler-Scheie Syndrome [a-L-iduronidase], Hunter's Syndrome [iduronate
sulfatase],
Sanfilippo Syndrome A [heparan sulfamidase], B [N- acetylglucosaminidase], C
[acetyl-
CoA:a-glucosaminide acetyltransferase], D [N- acetylglucosamine 6-sulfatase],
Morquio
Syndrome A [galactose-6-sulfate sulfatase], B [ -galactosidase], Maroteaux-
Lamy Syndrome
[N-acetylgalactosamine-4-sulfatase], etc.), Fabry disease (a-galactosidase),
Gaucher's disease
(glucocerebrosidase), or a glycogen storage disorder (e.g., Pompe disease;
lysosomal acid a-
glucosidase).
20. The isolated AAV virion of paragraphs 1-2 and 8-19, wherein none of the
viral
structural proteins are chimeric viral structural proteins.
21. The isolated AAV virion of paragraphs 1-19, wherein there is no overlap in
serotypes
between the chimeric viral structural protein and at least one other viral
structural protein.
22. A method of treating a disease comprising administering an effective
amount of the
virion of paragraphs 1-9, 16, 18-21, or the substantially homogenous
population of virions of
paragraphs 10-15 and 17, wherein the heterologous gene encodes a protein to
treat a disease
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suitable for treatment by gene therapy to a subject having the disease.
23. The method of paragraph 22, wherein the disease is selected from genetic
disorders,
cancers, immunological diseases, inflammation, autoimmune diseases and
degenerative
diseases.
24. The method of paragraphs 22 and 23, wherein multiple administrations are
made.
25. The method of paragraph 24, wherein different polyploid virions are used
to evade
neutralizing antibodies formed in response to a prior administration.
26. The isolated AAV virion of paragraphs 1-25, wherein applicants disclaim as
follows:
To the extent that any disclosure in PCT/US18/22725 filed March 15, 2018 falls
within the
invention as defined in any one or more of the claims of this application, or
within any
invention to be defined in amended claims that may in the future be filed in
this application
or in any patent derived therefrom, and to the extent that the laws of any
relevant country or
countries to which that or those claims apply provide that the disclosure of
PCT/US18/22725
is part of the state of the art against that or those claims in or for that or
those countries, we
hereby reserve the right to disclaim the said disclosure from the claims of
the present
application or any patent derived therefrom to the extent necessary to prevent
invalidation of
the present application or any patent derived therefrom.
For example, and without limitation, we reserve the right to disclaim any one
or
more of the following subject-matters from any claim of the present
application, now or
as amended in the future, or any patent derived therefrom:
A. any subject-matter disclosed in Example 9 of PCT/US18/22725; or
B. vector virions, termed polyploid vector virions, which are produced or
producible
by transfection of two AAV helper plasmids or three plasmids to produce
individual
polyploid vector virions composed of different capsid subunits from different
serotypes;
or
C. vector virions, termed polyploid vector virions, which are produced or
producible
by transfection of two AAV helper plasmids which are AAV2 and AAV8 or AAV9 to
produce individual polyploid vector virions composed of different capsid
subunits from
different serotypes; or
D. vector virions, termed polyploid vector virions, which are produced or
producible
by transfection of three AAV helper plasmids which are AAV2, AAV8 and AAV9 to
produce individual polyploid vector virions composed of different capsid
subunits from
different serotypes; or
E. vector virions, termed haploid vectors, with VP1NP2 from one AAV vector
capsid
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or AAV serotype and VP3 from an alternative one, for example VP1/VP2 from (the
capsid of) only one AAV serotype and VP3 from only one alternative AAV
serotype; or
F. any one or more AAV vector virion(s) selected from:
a vector which is generated by transfection of AAV2 helper and AAV8 helper
plasmids
(termed haploid AAV2/8) and which has VP1 capsid subunit from AAV8 and VP2NP3
capsid subunits from AAV2; or
a vector which is generated by transfection of AAV2 helper and AAV8 helper
plasmids
(termed haploid AAV2/8 or haploid AAV8/2 or haploid AAV82 or H-AAV82) and
which
has VP1/VP2 capsid subunits from AAV8 and VP3 capsid subunit from AAV2; or
a vector in which VP1/VP2 is derived from different serotypes; or
a vector (termed haploid AAV92 or H-AAV92) which has VP1/VP2 capsid subunits
from
AAV9 and VP3 capsid subunit from AAV2; or
a vector (termed haploid AAV2G9 or H-AAV2G9) which has VP1/VP2 capsid subunits
from AAV8 and VP3 capsid subunit from AAV2G9, in which AAV9 glycan receptor
binding site was engrafted into AAV2; or
a vector (termed haploid AAV83 or H-AAV83) which has VP1/VP2 capsid subunits
from
AAV8 and VP3 capsid subunit from AAV3; or
a vector (termed haploid AAV93 or H-AAV93) which has VP1/VP2 capsid subunits
from
AAV9 and VP3 capsid subunit from AAV3; or
a vector (termed haploid AAVrh10-3 or H-AAVrh10-3) which has VP1/VP2 capsid
subunits from AAVrh10 and VP3 capsid subunit from AAV3; or
a vector which is generated by transfection of AAV2 helper and AAV8 helper
plasmids
(termed haploid AAV2/8) and which has VP1 capsid subunit from AAV2 and VP2NP3
capsid subunits from AAV8; or
a vector which is generated by transfection of AAV2 helper and AAV8 helper
plasmids
(termed haploid AAV2/8) and which has VP1/VP2 capsid subunit from AAV2 and VP3
capsid subunits from AAV8; or
a vector which is generated by transfection of AAV2 helper and AAV8 helper
plasmids
(termed haploid AAV2/8) and which has VP1 capsid subunit from AAV8 and VP3
capsid
subunit from AAV2; or
a vector which is generated by transfection of AAV2 helper and AAV8 helper
plasmids
(termed haploid AAV2/8) and which has VP1 capsid subunit from AAV2 and VP3
capsid
subunits from AAV8; or
a vector which is generated by transfection of AAV2 helper and AAV8 helper
plasmids
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(termed haploid AAV2/8) and which has VP1NP2/VP3 capsid subunits from AAV2; or
a vector which is generated by transfection of AAV2 helper and AAV8 helper
plasmids
(termed haploid AAV2/8) and which has VP1NP2/VP3 capsid subunits from AAV8; or
a vector termed 28m-2VP3 or haploid 2m-2VP3 or haploid vector 28m-2VP3 in
which
chimeric VP1NP2 capsid subunits have N-terminal from AAV2 and C-terminal from
AAV8, and the VP3 capsid subunit is from AAV2; or
a vector termed chimeric AAV8/2 or chimeric AAV82 in which chimeric VP1/VP2
capsid subunits have N-terminal from AAV8 and C-terminal from AAV2 without
mutation of the VP3 start codon, and the VP3 capsid subunit is from AAV2; or
a vector in which chimeric VP1NP2 capsid subunits have N-terminal from AAV2
and C-
terminal from AAV8; or
G. a population, for example a substantially homogenous population, for
example a
population of 1010 particles, for example a substantially homogenous
population of 1010
particles, of any one of the vectors of F; or
H. a method of producing any one of the vectors or populations of vectors
of A and/or
B and/or C and/or D and/or E and/or F and/or G; or
I. any combination thereof.
Without limitation, we state that the above reservation of a right of
disclaimer applies at
least to claims 1-30 as appended to this application and paragraphs 1-83 as
set forth at
[00437]. The modified virus capsids can be used as "capsid vehicles," as has
been
described, for example, in U.S. Patent No. 5,863,541. Molecules that can be
packaged by
the modified virus capsid and transferred into a cell include heterologous
DNA, RNA,
polypeptides, small organic molecules, metals, or combinations of the same.
EXAMPLES
Example 1: Application of polyploid adeno-associated virus vector for
transduction
enhancement and neutralizing antibody evasion
[00443] Adeno-associated virus (AAV) vectors have been successfully used in
clinical trials
in patients with hemophilia and blindness. Exploration of effective strategies
to enhance
AAV transduction and escape neutralizing antibody activity is still
imperative. Previous
studies have shown the compatibility of capsids from AAV serotypes and
recognition sites of
AAV Nab located on different capsid subunits of one virion. In this study, we
co-transfected
AAV2 and AAV8 helper plasmids at different ratios (3:1, 1:1 and 1:3) to
assemble haploid
capsids and study their transduction and Nab escape activity. The haploid
virus yield was
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similar to the parental ones and the heparin sulfate binding ability was
positively correlated
with AAV2 capsid input. To determine whether the tropism of these haploid
vectors was
changed by mixing the capsid protein, the transduction efficacy of the haploid
viruses was
analyzed by transducing human Huh7 and mouse C2C12 cell lines (Figure 1).
Although the
haploid vector transduction was lower than AAV2 in Huh7 cells, haploid vector
AAV2/8 3:1
induced a 3-fold higher transduction that that of AAV2 in C2C12 cells.
[00444] After muscular injection, all of the haploid viruses induced higher
transduction than
parental AAV vectors (2- to 9-fold over AAV2) with the highest of these being
the haploid
vector AAV2/8 1:3. After systemic administration, 4-fold higher transduction
in the liver
was observed with haploid AAV2/8 1:3 than that with AAV8 alone. Haploid
AAV2/89 and
their parental vectors were directly injected into the muscle of the hind legs
in C57B16 mice.
As controls, the mixtures of AAV2 and AAV8 viruses at ratios of 3:1, 1:1 and
1:3 were also
investigated. For a convenient comparison, one leg was injected with AAV2 and
the opposite
leg with haploid vector. Compared to AAV2, a similar muscular transduction was
achieved
for the parental AAV8 capsid (Figure 2). Contrary to the results in C2C12
cells, an enhanced
muscular transduction was observed form all of the haploid viruses (Figure 2).
The haploid
vectors AAV2/9 1:1 and AAV2/8 1:3 achieved a 4-fold and a 2-fold higher
transduction than
AAV2, respectively. Notably, the muscular transduction of the haploid vector
AAV2/8 3:1
was over 6-fold higher than that of AAV2. All of the controls (injections that
were a result of
physically mixing parental vectors), however, had similar transduction
efficiencies as the
AAV2 vector.
[00445] Further, we packaged the therapeutic factor IX cassette into haploid
AAV2/8 1:3
capsids and injected them into FIX knockout mice via tail vein. Higher FIX
expression and
improved phenotypic correction were achieved with haploid AAV2/8 1:3 virus
vector
compared to that of AAV8. Additionally, haploid virus AAV2/8 1:3 was able to
escape
AAV2 neutralization and had very low Nab cross-reactivity with AAV2.
[00446] To improve Nab evasion ability of polyploid virus, we produced
triploid vector
AAV2/8/9 vector by co-transfecting AAV2, AAV8 and AAV9 helper plasmids at the
ratio of
1:1:1. After systemic administration, 2-fold higher transduction in the liver
was observed
with triploid vector AAV2/8/9 than that with AAV8. Neutralizing antibody
analysis
demonstrated that AAV2/8/9 vector was able to escape neutralizing antibody
activity from
mouse sera immunized with parental serotypes. These results indicate that
polyploid virus
might potentially acquire advantage from parental serotypes for enhancement of
transduction
and evasion of Nab recognition. This strategy should be explored in future
clinical trials in
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patients with positive neutralizing antibodies.
[00447] The number of helper plasmids with different cap genes is not limited
and can be
mixed and matched based on the specific requirements of a particular treatment
regimen.
[00448] Cell lines. HEK293 cells, Huh7 cells and C2C12 cells were maintained
at 37 C in
5% CO2 in Dulbecco's Modified Eagle's Medium with 10% fetal bovine serum and
10%
penicillin-streptomycin.
[00449] Recombinant AAV virus production. Recombinant AAV was produced by a
triple-
plasmid transfection system. A 15-cm dish of HEK293 cells was transfected with
9 !At of
AAV transgene plasmid pTR/CBA-Luc, 12 lig of AAV helper plasmid, and 1511g of
Ad
helper plasmid XX680. To generate triploid AAV2/8 virions, the amount of each
helper
plasmid for AAV2 or AAV8 transfected was co-transfected at three different
ratios of 1:1,
1:3 and 3:1. To make haploid AAV2/8/9 vectors, the ratio of helper plasmid for
each
serotype was 1:1:1. Sixty hours post-transfection, HEK293 cells were collected
and lysed.
Supernatant was subjected to CsC1 gradient ultra-centrifugation. Virus titer
was determined
by quantitative PCR.
[00450] Western and Immune-blot. According to the virus titer, the same amount
of virions
were loaded in each lane, followed by electrophoresis on a NuPage 4-10%
polyacrylamide
Bis-Tris gel (Invitrogen, Carlsbad, CA) and then transferred to PVDF membrane
via iBlot 2
Dry Blotting System (Invitrogen, Carlsbad, CA). The membrane was incubated
with the B1
antibody specific to AAV capsid proteins.
[00451] A native immunoblot assay was carried out as previously described.
Briefly,
purified capsids were transferred to a Hybond-ECL membrane (Amersham,
Piscataway, NJ)
by using vacuum dot-blotter. The membranes were blocked for 1 h in 10% milk
PBS and
then incubated with monoclonal antibody A20 or ADK8. The membranes were
incubated
with a peroxidase-coupled goat anti-mouse antibody for 1 hr. The proteins were
visualized by
Amersham Imager 600 (GE Healthcare Biosciences, Pittsburg, PA).
[00452] In vitro transduction assay. Huh7 and C2C12 cells were transduced by
recombinant viruses with 1 x 104 vg/cell in a flat-bottom, 24-well plate.
Forty-eight hours
later, cells were harvested and evaluated by a luciferase assay system
(Promega, Madison,
WI).
[00453] Heparin inhibition assays. The ability of soluble heparin to inhibit
the binding of
recombinant viruses to Huh7 or C2C12 cells was assayed. Briefly, AAV2, AAV8,
haploid
viruses AAV2/8 1:1, AAV2/8 1:3 and AAV2/8 3:1 were incubated in DMEM in the
presence, or absence, of soluble HS for 1 hat 37 C. After the pre-incubation,
the mixture of
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recombinant viruses and soluble HS were added into Huh7 or C2C12 cells. At 48
h post-
transduction, cells were harvested and evaluated by luciferase assay.
[00454] The antigen presentation from the haploid AAV capsid is similar to
that of AAV8
in vivo. To study the efficacy of the capsid antigen presentation, we produced
a haploid
AAV2/8 OVA 1:3 vector by the transfection of pXR2-OVA and pXR8-OVA at the
ratio of
1:3. 1x1011 vg of AAV2/8-OVA and AAV8-OVA vectors were administered via retro-
orbital
injection in the C57BL/6 mice. Three days later, CFSE-labeled OT-1 mouse
spleen cells
were transferred into the C57BL/6 mice. At day 10 post-transferring OT-1
spleen cells, T
cell proliferation was measured by flow cytometry. OT-1 T cell proliferation
was
significantly increased in mice receiving AAV2/8-OVA 1:3 or AAV8-OVA when
compared
to control mice without AAV vector administration (Figure 5). There was no
difference,
however, for OT-1 cell proliferation between the AAV2/8-OVA 1:3 and AAV8-OVA
groups.
[00455] Animal study. Animal experiments performed in this study were
conducted with
C57BL/6 mice and FIX-/- mice. The mice were maintained in accordance to NIH
guidelines,
as approved by the UNC Institutional Animal Care and Use Committee (IACUC).
Six-
week-old female C57BL/6 mice were injected with 3 x 1010 vg of recombinant
viruses via
retro-orbital injection. Luciferase expression was imaged one week post-
injection using a
Xenogen IVIS Lumina (Caliper Lifesciences, Waltham, MA) following i.p.
injection of D-
luciferin substrate (Nanolight Pinetop, AZ). Bioluminescent images were
analyzed using
Living Image (PerkinElmer, Waltham, MA). For muscle transduction, 1 x 1010
particles of
AAV/Luc were injected into the gastrocnemius of 6-week-old C57BL/6 females.
Mice were
imaged at the indicated time points.
[00456] Next, the transduction efficiency of haploid viruses in the mouse
liver was
evaluated. The mixtures of AAV2 and AAV8 viruses were also injected as
controls. A dose
of C57BL/6 mice were injected with 3 x 101 vg of recombinant viruses via the
retro-orbital
vein and the imaging was carried out at day 3 post-AAV injection. The haploid
virus
AAV2/8 1:3 induced the highest transduction efficiency even over the other
haploid
combinations, the mixtures of parental viruses and the parental AAV8 in mouse
livers (Figure
3A and 3B). The transduction efficiency of the haploid vector AAV2/8 1:3 was
about 4-fold
higher than that of AAV8 (Figure 3 B). The liver transduction from the other
haploid viruses
was lower than that from the parental AAV8, but higher than that of AAV2
(Figure 3A and
3B). At day 7 post-injection, the mice were sacrificed, the livers were
harvested, and the
genomic DNA was isolated. The luciferase gene copy number in the liver was
determined by
ciPCR. Different from the results for liver transduction efficiency, a similar
AAV vector
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genome copy number was found in the liver regardless of virus composition
(Figure 3C).
When transgene expression was normalized to gene copy number, the haploid
vector
AAV2/8 1:3 induced the highest relative transgene expression than any other
haploid vector
combination or parental serotypes (Figure 3D).
[00457] FIX knockout male mice (FIX KO mice) received 1 x 1010 vg via tail
vein injection.
At various time points after injection, blood was collected from the retro-
orbital plexus. At
week 6, mouse bleeding analysis was performed.
[00458] Quantitation of luciferase expression in the liver Animals utilized
for imaging
studies were sacrificed at week 4 after recombinant virus injection, and the
livers were
collected. Livers were minced and homogenized in passive lysis buffer. After
the liver
lysates were centrifuged, luciferase activity in supernatant was detected.
Total protein
concentration in tissue lysates were measured using the Bradford assay
(BioRad, Hercules,
CA).
[00459] Detection of AAV genome copy number in the liver. The minced livers
were
treated by Protease K. The total genome DNA was isolated by PureLink Genomic
DNA mini
Kit (Invitrogen, Carlsbad, CA). The luciferase gene was detected by qPCR
assay. The
mouse lamin gene served as an internal control.
[00460] Human FIX expression, function and tail-bleeding time assays. The
human FIX
expression, one-stage hFIX activity assay and tail-bleeding time assay were
performed as
previously described. Neutralization assay Huh7 cells were seeded in a 48-well
plate at a
density of 105 cells for each well. Two-fold dilutions of the mouse antibody
were incubated
with AAV-Luc (1x108 vg) for 1 hr 37 C. The mixture was added into cells and
incubated
for 48 hers at 37 C. Cells were lysed with passive lysis buffer (Promega,
Madison, WI) and
luciferase activity was measured. Nab titers were defined as the highest
dilution for which
luciferase activity was 50% lower than serum-free controls.
[00461] Statistical analysis. The data were presented as mean SD. The
Student t test was
used to carry out all statistical analyses. P values< 0.05 were considered a
statistically
significant difference.
[00462] An AAV2/8 1:3 was tested to determine if it would increase the
therapeutic
transgene expression in an animal disease model. A human FIX (hFIX or human
Factor IX)
was used as a therapeutic gene and injected the haploid vector AAV2/8 1:3/hFIX
into FIX
knockout (KO) mice via tail vein at a dose of 1x10' vg/mouse. The haploid
vector encodes
the human-optimized FIX transgene and is driven by the liver specific
promoter, TTR. At
week 1, 2, and 4 post-injection, ELISA and one-stage factor activity analyzed
the hFIX
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expression and activity in circulation, respectively. At week 6, the blood
loss for in vivo
hFIX function was evaluated using a tail clipping assay. Consistent with the
observation of
high liver transduction with the haploid AAV vectors in wild-type C57BL/6
mice, the haploid
vector AAV2/8 1:3 liver targeting produced much more hFIX than an AAV8 vector
after 2
weeks post-injection (Figure 4A). The higher hFIX protein expression of AAV2/8
1:3
correlated as predicted with high FIX activity (Figure 4B). The blood loss for
the mice with
AAV2/8 1:3/hFIX injection was similar to that of wild-type C57BL/6 mice, and
much less
than that of KO mice (Figure 4C). Although there was no significant difference
of the blood
loss between the mice with AAV8 and AAV2/8 1:3/hFIX injection in statistics,
the AAV8
mice had a little more blood loss that that of AAV2/8 1:3 mice (Figure 4C).
[00463] Ability of the haploid viruses AAV2/8 to escape Nab. To study whether
the haploid
virus is able to escape Nabs generated in response to a parental vector, a Nab
binding assay
was performed using monoclonal antibodies by an immune-blot assay. Three
dilutions of
virus-genome-containing particles were adsorbed to a nitrocellulose membrane
and probed
with Nab A20 or ADK8, which recognizes intact AAV2 or AAV8 respectively. The
neutralization profiles of the haploid viruses against A20 and ADK9 were
similar to the data
from a native immune-blot. (Table 5). The haploid AAV2/8 1:3 almost completely
escaped
the AAV2 serum and A20 neutralization, which suggest that this haploid virus
has the
potential to be used for individuals who have anti-AAV2 Nabs (Table 5).
[00464] Characterization of haploid viruses in vitro. Our previous study has
demonstrated
the capsid compatibility among AAV1, 2, 3 and 5 capsids The haploid viruses
were
produced by transfection of AAV helper plasmids from two serotypes at the
different ratios
with AAV transgene and adenovirus helper pXX6-80. The enhanced transduction
from
haploid virus was observed in some cell lines compared to the parental
vectors. AAV2 is
well characterized for its biology and as a gene delivery vehicle and AAV8 has
attracted a lot
of attention due to high transduction in mouse liver. Both serotypes have been
utilized in
several clinical trials in patients with hemophilia. To investigate the
possibility of AAV
serotype 2 and 8 capsid to form haploid virus and their transduction profile,
we transfected
the helper plasmids of AAV2 and AAV8 at the ratios of 3:1, 1:1 and 1:3 to make
haploid
vectors. All of the haploid viruses were purified using cesium gradient and
tittered by Q-
PCR. There was no significant difference in virus yield between the haploid
viruses and the
parental AAV2 or AAV8. To determine whether the capsid proteins of haploid
viruses were
expressed, Western blot analysis was performed on equivalent virus genomes
from purified
haploid viruses using monoclonal antibody B1 which recognizes the capsid
proteins of AAV2
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and AAV8. In all haploid viruses, the mixture of VP2 capsids from AAV2 and
AAV8 was
observed, the intensity of VP2 capsid from AAV2 or AAV8 in haploid viruses was
related to
the ratio of two helper plasmids. These results suggested that the capsids
from AAV 2 and
AAV8 were compatible and able to be ensemble into AAV virions.
[00465] To determine whether the tropism of haploid virus was changed by
mixing the
capsid proteins, the transduction efficacy of haploid viruses was analyzed by
transducing
human Huh7 and mouse C2C12 cell lines. The transduction efficiency of AAV8 was
much
lower than AAV2 in both of the cell lines. The transduction from all haploid
vectors was
higher than that from AAV8, and the efficiency was positively correlated with
addition of
AAV2 capsid in both cell lines. Although haploid vector transduction was lower
than AAV2
in Huh7 cells, haploid vector AAV2/8 3:1 induced 3-fold higher transduction
than AAV2 in
C2C12 cells.
[00466] This in vitro transduction data supports that the virus preparation is
composed of
haploid vectors but not the mixture of individual serotype vector and indicate
that haploid
vector may enhance AAV transduction. Heparin sulfate proteoglycan has been
identified as
the primary receptor of AAV2 Next, we investigated whether inhibition of
heparin binding
ability changed transduction of haploid viruses. Pre-incubation of AAV vectors
with soluble
heparin blocked AAV2 transduction by nearly 100% in both Huh7 and C2C12 cells,
and
blocked AAV8 transduction by 37% and 56% in Huh7 and C2C12 cells,
respectively. The
inhibition of haploid vector transduction by soluble heparin was dependent on
the input of
AAV2 capsid in both cell lines. Higher inhibition of transduction was observed
with more
AAV2 capsid input. This result suggests that haploid viruses may use both
primary receptors
from parental vectors for effective transduction [Figure 1].
[00467] Increased muscular transduction of haploid viruses. As described
above, the
transduction efficiency of haploid virus AAV2/8 3:1 is higher than that of
AAV2 and AAV8
in the muscle cell line C2C12. Next we studied whether the high transduction
in vitro was
translated into mouse muscle tissues. AAV2/8 haploid and parental vectors were
directly
injected into muscle of hind legs in C57BL/6 mouse. As controls, the mixtures
of AAV2 and
AAV8 viruses at the ratios of 3:1, 1:1 and 1:3 were also investigated. For
convenient
comparison, one leg was injected with AAV2 and the other one with tested
vector. A total
vector of I x 1010 vg for each virus was administered. Compared to AAV2,
similar muscular
transduction was achieved for AAV8. Contrary to the result in C2C12 cells,
enhanced
muscular transduction was observed from all of the haploid viruses [Figure 2].
[00468] Haploid vectors AAV2/8 1:1 and AAV2/8 1:3 achieved 4- and 2-fold
higher
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transduction than AAV2, respectively. Notably, the muscular transduction of
haploid vector
AAV2/8 3:1 was over 6-fold higher than that of AAV2. However, all of the
mixture viruses
had similar transduction efficiencies to AAV2. These results suggest that
haploid virus is
able to increase muscular transduction and further supports that viruses
produced from co-
transfection of two capsid plasmids are haploid.
[00469] Enhanced liver transduction of haploid viruses. AAV2 and AAV8 have
been used
for liver targeting in several clinical trials in patients with hemophilia B.
We also evaluated
the transduction efficiency of haploid viruses in mouse liver. The viruses
mixed with AAV2
and AAV8 were also injected as controls. A dose of 3 x 1010 vg of AAV/luc
vector was
administered in C57BL mice via retro-orbital vein; the imaging was carried out
at day 3 post-
AAV injection. The haploid virus AAV2/8 1:3 induced the highest transduction
efficiency
than other haploid, mixture viruses and even parental AAV8 in mouse livers
[Figure 3A and
3B]. The transduction efficiency of haploid vector AAV2/8 1:3 was about 4-fold
higher than
that of AAV8 [Figure 3B]. The liver transduction from other haploid viruses
was lower than
that from the parental vector AAV8 but higher than AAV2 [Figure 3A and 3B]. At
day 7
post-injection, the mice were sacrificed, the livers were harvested and the
genomic DNA was
isolated. The luciferase gene copy number in the liver was determined by qPCR.
Different
from the result for liver transduction efficiency, similar AAV vector genome
copy number
was found in the liver regardless of haploid viruses or AAV serotypes 2 and 8
[Figure 3C].
When transgene expression was normalized to gene copy number, consistent to
transgene
expression in the liver, haploid vector AAV2/8 1:3 induced the highest
relative transgene
expression than any other haploid vectors and serotypes [Figure 3D]. The
transduction
profile of haploid viruses in the liver was different from that in muscle
transduction, in which
all haploid viruses induced higher transgene expression than that from
parental serotypes,
with the best from AAV2/8 3:1.
[00470] Augmented therapeutic FIX expression and improved bleeding phenotypic
correction with haploid vector in a hemophilia B mouse model. Based on the
above results,
haploid vector AAV2/8 1:3 induced much higher liver transduction than AAV8.
Next, we
further tested whether the haploid vector AAV2/8 1:3 could increase the
therapeutic
transgene expression in an animal disease model. We used human FIX (hFIX) as a
therapeutic gene and injected haploid vector AAV2/8 1:3/hFIX, which encoded
human-
optimized FIX transgene, and driven by the liver-specific promoter, TTR, into
FIX knockout
(KO) mice via tail vein at a dose of 1 x 1010 vg/mouse. At week 1, 2 and 4
post-injection, the
hFIX expression and activity in circulation were analyzed by ELISA and one-
stage factor
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activity, respectively. At week 6, the blood loss for in vivo hFIX function
was evaluated
using a tail clipping assay. Consistent to the observation of high liver
transduction with
haploid AAV vector in wide-type C57BL/6 mice, haploid vector AAV2/8 1:3 liver
targeting
produced much more hFIX than AAV8 vector after 2 weeks post-injection [Figure
4A]. The
higher hFIX protein expression of AAV2/8 1:3 was closely related to high FIX
activity
[Figure 4B]. The blood loss for the mice with AAV2/8 1:3/hFIX injection was
similar to that
of wild-type C57BL/6 mice and less than that of KO mice [Figure 4C]. However,
AAV8-
treated mice had more blood loss than that in wild type mice [Figure 4C).
These data show
that haploid vector AAV2/8 1:3 increases therapeutic transgene expression from
the liver and
improves disease phenotypic correction.
[00471] The ability of haploid viruses AAV2/8 to escape neutralizing antibody.
Each
individual haploid virus virion is composed of 60 subunits from different AAV
serotype
capsids. Insertion of some capsid subunits from one serotype into other capsid
subunits from
a different serotype may change the virion surface structure. It is well known
that most AAV
monoclonal antibodies recognize residues on the different subunits of one
single virion. To
study whether haploid virus is able to escape Nabs generated from parental
vector, first we
performed Nab binding assay using monoclonal antibodies by an immune-blot
assay. Three
dilutions of virus- genome-containing particles were adsorbed to a
nitrocellulose membrane
and probed with Nab A20 or ADK8, which recognizes intact AAV2 or AAV8,
respectively.
All of the haploid viruses and virus with mixture of AAV2 and AAV8 were
recognized by
monoclonal antibody ADK8 or A20. The reactivity of haploid viruses with A20
was
increased by incorporation of more AAV2 capsids into haploid virus virion.
However, there
was no obvious change for the recognition of anti-AAV8 Nab ADK8 among the
haploid
viruses, regardless of capsid ratios. Notably, the binding of haploid AAV2/8
1:3 to A20 was
much weaker than those of parental AAV2 and the virus with mixture of AAV2 and
8 at the
ratio 1:3, which indicated that A20 binding sites are depleted on the haploid
AAV2/8 1:3
virion surface.
[00472] Next we analyzed the immunological profile of haploid viruses against
sera from
AAV-immunized mice. Nab titers were used to evaluate the ability of serum to
inhibit vector
transduction. Sera were collected from mice treated with parental viruses at
week 4 post-
injection. As shown in Table 5, the neutralization profiles of the haploid
viruses against A20
or ADK8 were similar to the data from native immune-blot. There was no Nab
cross-
reactivity between AAV8 and AAV2. It is interesting to note that AAV8-
immunized mouse
sera had similar neutralizing activity against AAV8 virus and all of the
haploid viruses,
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regardless of the amount of AAV8 capsid incorporation, but not the viruses
mixed with
AAV2 and AAV8. No inhibition of AAV8 serum on mixture viruses may be explained
by
the superior transduction from AAV2 to AAV8 in tested cell line. However,
haploid viruses
partially escaped the neutralization from AAV2 serum. The transduction of
haploid AAV2/8
1:1 got a 16-fold decrease than parental AAV2 after incubation of virus and
anti-AAV2
serum. The ability to escape AAV2 serum Nab for haploid viruses was much
higher than that
for viruses mixed with AAV2 and AAV8. Strikingly, the haploid AAV2/8 1:3
almost
completely escaped the AAV2 serum and A20 neutralization, suggesting that the
haploid
virus has the potential to be used for the individuals who have the anti-AAV2
Nab (Table 5).
[00473] Improved neutralizing antibody evasion ability with triploid vector
made from
three serotypes. Our data described above demonstrated that haploid AAV2/8
viruses were
not able to escape AAV8 neutralizing antibody activity, but had the capacity
to evade AAV2
neutralizing antibody, which depended on the amount of capsid integration from
AAV8. To
study whether the polyploid virus made from more serotypes capsids improved
the Nab
escaping ability, we made the triploid virus AAV2/8/9 with the ratio of 1:1:1.
After injection
of the triploid vector AAV2/8/9 into mice, compared to AAV2, triploid virus
AAV2/8/9
induced 2 fold higher transduction in the liver than AAV8. No difference in
liver
transduction was observed among AAV8 and haploid vectors AAV2/9 and AAV8/9 in
which
the triploid vector was made from two AAV helper plasmids at ratio of 1:1. It
was noted that
AAV9 systemic administration induced higher liver transduction than AAV8. When
neutralizing antibody assay was performed, haploid AAV2/8/9 vector improved
its Nab
escape ability by about 20 fold, 32 fold and 8 fold, respectively when
compared to AAV2, 8
and 9 (Table 6).
[00474] In this study, polyploid AAV virions were assembled from capsids of 2
serotypes or
3 serotypes. The binding ability of haploid viruses to AAV2 primary receptor
heparin was
dependent on the amount of AAV2 capsid input. All of the haploid viruses
achieved higher
transduction efficacy than parental AAV2 vector in mouse muscle and liver,
while haploid
virus AAV2/8 1:3 had a significant enhancement of liver transduction than
parental AAV8
vector. Compared to AAV8, systemic administration of the haploid virus AAV2/8
1:3 to
deliver human FIX induced much higher FIX expression and improved hemophilia
phenotypic correction in FIX-/- mice. Importantly, the haploid virus AAV2/8
1:3 was able to
escape the neutralization of anti-AAV2 serum. Integration of AAV9 capsid into
haploid
AAV2/8 virions further improved neutralizing antibody escape capacity.
[00475] The primary receptor of AAV2 is HSPG, while the primary receptor of
AAV8 is
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still unclear. To study whether haploid viruses could use receptors from both
AAV2 and
AAV8, we performed heparin inhibition assay to test the ability of haploid
viruses to binding
heparin receptor motif. The heparin inhibition results, in Huh7 and C2C12 cell
lines, support
that haploid viruses use the heparin receptor motif of AAV2 capsids for
effective
transduction. To some extent, AAV8 also showed decreased transduction
efficiency in the
presence of heparin, but the transduction efficiency is still higher than that
of AAV2.
[00476] One of the most challenging aspects of efficient transduction in
clinical trials is
broad prevalence of neutralizing antibodies to AAV vector. Nab-mediated
clearance of AAV
vectors has become a limited factor for repeating administration of AAV gene
transfer.
Several studies have explored genetically modifying AAV capsids for Nab
evasion by
rational mutation of neutralizing antibody recognizing sites or directed
evolution approaches.
Capsid mutation may change AAV tropism and transduction efficiency.
Additionally, the
identification of Nab binding sites on AAV virions is far behind vector
application in clinical
trials, and it is impossible to figure out all Nab binding sites from poly
sera. Previous studies
have demonstrated that the recognition sites of several AAV monoclonal
antibodies are spun
on the different subunits of one virion. When AAV8 capsid is introduced into
AAV2 virion,
the A20 binding ability and neutralizing activity from AAV2-immunized sera
were
dramatically decreased for haploid viruses. Integration of AAV2 capsids into
AAV8 virions
did not reduce the capacity to bind intact AAV8 monoclonal antibody ADK8 and
did not
escape the neutralizing activity of anti-AAV8 sera (Table 5). This suggests
that all Nab
recognition sites from poly-sera may be located on the same subunit of AAV8
virion. Also,
the result suggests that the AAV8 capsids integrated into AAV2 virions may
play a major
role in virus intracellular trafficking.
[00477] When triploid virus was made from capsids of three serotypes AAV2, 8
and 9,
different from triploid vectors AAV2/8, haploid AAV2/8/9 virus has an ability
to escape
neutralizing antibody activity sera from AAV2, 8 or 9 immunized mice, which
suggests that
AAV8 and AAV9 share the similar transduction pathway.
[00478] Several lines of evidences from this study support the polyploid
virion assembly
from transfection of two or three AAV helper plasmids. (1) Two VP2 bands of
different
sizes were displayed from haploid viruses using western blot analysis. These
VP2s match the
size from different serotypes. (2) The transduction profiles were different in
C2C12 versus
Huh7 cells. Haploid AAV2/8 3:1 vector, in particular, demonstrated lower
transduction than
that with AAV2 in Huh7 cells, but higher in C2C12 cells. (3) Higher muscle
transduction
was demonstrated with all haploid AAV2/8 viruses as compared with parental
vectors AAV2
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and AAV8, as well as the viruses with a mixture of AAV2 and AAV8. (4) Triploid
virus
AAV2/8 1:3 had enhanced liver tropism when compared to AAV8. (5) The binding
pattern
of haploid viruses to A20 and ADK8 is different from the viruses with a
mixture of AAV2
and AAV8. (6) The profile of AAV2 serum neutralizing activity is different
between haploid
viruses and mixture viruses. (7) Triploid AAV2/8/9 virus evades neutralizing
antibody
activity of sera from mice immunized with any parental serotypes.
[00479] These polyploid viruses enhance the transduction efficiency in vitro
and in vivo, and
even escape neutralization from parental vector immunized sera. Application of
the
polyploid virus to deliver a therapeutic transgene FIX was able to increase
FIX expression
and improve hemophilia phenotypic correction in mice with FIX deficiency.
These results
indicate that haploid AAV vectors have the ability to enhance transduction and
evade Nabs.
Example 2: Enhanced AAV transduction from haploid AAV vectors by assembly of
AAV
virions with VP1/VP2 from one AAV vector and VP3 from an alternative one by
application
of rational polyploid methodology
[00480] In above studies, we have demonstrated that increased AAV transduction
has been
achieved using polyploid vectors which are produced by transfection of two AAV
helper
plasmids (AAV2 and AAV8 or AAV9) or three plasmids (AAV2, AAV8 and AAV9).
These
individual polyploid vector virions may be composed of different capsid
subunits from
different serotypes. For example, haploid AAV2/8, which is generated by
transfection of
AAV2 helper and AAV8 helper plasmids, may have capsid subunits with different
combinations in one virion for effective transduction: VP1 from AAV8 and
VP2/VP3 from
AAV2, or VP1/VP2 from AAV8 and VP3 from AAV2, or VP1 from AAV2 and VP2/VP3
from AAV8, or VP1/VP2 from AAV2 and VP3 from AAV8, or VP1 from AAV8 and VP3
from AAV2, or VP1 from AAV2 and VP3 from AAV8, or VP!/VP2/VP3 from AAV2, or
VP1/VP2/VP3 from AAV8. In the following studies, we found that enhanced
transduction
could be achieved from haploid vectors with VP1/VP2 from one AAV vector capsid
and VP3
from an alternative one.
[00481] The generation of VP1, VP2 and VP3 by different AAV serotypes offers
two
different strategies for producing these different proteins. Interestingly,
the VP proteins are
translated from a single CAP nucleotide sequence with overlapping sequences
for VP1, VP2
and VP3.
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p40
CAP
VP1
P2
VP
[00482] The Cap gene encodes for 3 proteins ¨ VP1, VP2 and VP3. As shown in
the above
figure, VP1 contains the VP2 and VP3 proteins, and VP2 contains the VP3
protein.
Therefore, the Cap gene has 3 segments, start of VP1 ¨ start of VP2 ¨ start of
VP3 ¨ end of
all 3 VP proteins.
[00483] In the case of sourcing the Cap genes from two different AAV serotypes
(designated
as A and B), there are 6 possible combinations of the three Cap proteins. In
one case, the
VP1 identified as serotype A, which can be any serotype (or chimeric or other
nonnaturally
occurring AAV) is only from a first serotype A and the VP2/VP3 identified as
serotype B, is
only from serotype B, and is a serotype that is different from the serotype
(or chimeric or
other nonnaturally occurring AAV) of VP1. In one case both VP! and VP2 are
only from a
first serotype A, and VP3 is only from serotype B. Methods to create a VP1 of
a first serotype
and VP2/VP3 of a second serotype; or VP1NP2 from a first serotype and VP3 form
a second
serotype, are disclosed in the Examples set forth herein. In one case, VP1 and
VP3 are only
from a first serotype and VP2 is only from a second serotype.
VP! VP2 VP3
A
A B A
A A
A
A
A A
[00484] In the case of sourcing the Cap genes from three different AAV
serotypes
(designated as A, B and C), there are 6 possible combination of the three Cap
proteins. In
this case, the VP1 identified as serotype A, which can be any serotype (or
chimeric or other
nonnaturally occurring AAV) is from a first serotype that is different from
the serotype of
VP2 and VP3; the VP2 identified as serotype B, which is a serotype that is
different from the
serotype (or chimeric or other nonnaturally occurring AAV) of VP! and VP3, is
from a
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second serotype; and, the serotype of VP3 identified as serotype C, which is a
serotype that is
different from the serotype (or chimeric or other nonnaturally occurring AAV)
of VP1 and
the serotype of VP2, is from a third serotype. Methods to create a VP1 of a
first serotype, a
VP2 of a second serotype and a VP3 of a third serotype are disclosed in the
Examples set
forth herein.
VP1 VP2 VP3
A B C
A C B
B A C
B C A
C A B
C B A
[00485] In an embodiment, when VP1 is identified as a first serotype A and VP2
and VP3
are identified as a second serotype B, it is understood that in one
embodiment, this would
mean that VP1 is only from serotype A and that VP2 and VP3 is only from
serotype B. In
another embodiment, when VP1 is identified as a first serotype A, VP2 as a
second serotype
B and VP3 as a third serotype C, it is understood that in one embodiment, this
this would
mean that VP1 is only from serotype A; that VP2 is only from serotype B; and
VP3 is only
from serotype C. As described in more detail in the Examples below, in one
embodiment, to
create a haploid vector using two different serotypes you could include a
nucleotide sequence
for VP1 from serotype A (or chimeric or other nonnaturally occurring AAV) that
expresses
only VP1 from serotype A and a second nucleotide sequence for VP2 and/or VP3
only from a
second serotype, or alternatively VP2 only from a second serotype, and VP3
only from a
third serotype (see for example, Figures 13 ¨ 15). In one embodiment, VP1/VP2
are only
from a first serotype and VP3 is only from a second serotype.
[00486] In the case of 3 different Cap genes, the helper plasmid can be
generated with a full
copy of the nucleotide sequence for the particular VP protein from the three
AAV serotypes.
The individual Cap genes will generate the VP proteins associated with that
particular AAV
serotype (designated as A, B and C).
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VP! VP2 VP3
A
A
A
A
A
A
[00487] In an embodiment, when VPI is identified as a first serotype A and VP2
is
identified as a second serotype B and VP3 is identified as a third serotype C,
it is understood
that in one embodiment, this would mean that VP1 is only from serotype A; that
VP2 is only
from serotype B and VP3 is only from serotype C. As described in more detail
in the
Examples below, to create such a haploid vector would include a nucleotide
sequence for
VP1 from serotype A that expresses only VPI from serotype A and not VP2 or VP3
from
serotype A; a second nucleotide sequence that expresses VP2 of serotype B and
not VP3 of
serotype B; and a third nucleotide sequence that expresses VP3 of serotype C.
[00488] In certain embodiments, the haploid virions comprise only VP! and VP3
capsid
proteins. In certain embodiments, the haploid virions comprise VP!, VP2, and
VP3 capsid
proteins.
[00489] It should be noted that in each of these embodiments of various
combinations of
VP1 with VP3 to form a haploid virion; or various serotype combinations of
VP1NP2NP3
to from a haploid virion, the nucleotide sequences that express the capsid
proteins can be
expressed from one or more vector, e.g.,plasmid. In one embodiment, the
nucleic acid
sequences that express VP1, or VP2, or VP3, are codon optimized so that
recombination
between the nucleotide sequences is significantly reduced, particularly when
expressed from
one vector, e.g.,plasmid etc.
[00490] Rational Haploid vector with C-terminal of VP1/VP2 from AAV8 and VP3
from
AAV2 enhances AAV transduction. It has been demonstrated that haploid vectors
AAV2/8
at any ratio of AAV2 capsid to AAV8 capsid induced higher liver transduction
than AAV2 or
the viruses with mixture of AAV2 vectors and AAV8 vectors at the same ratio.
To elucidate
which AAV subunits in individual haploid AAV2/8 vector contributes to higher
transduction
than AAV2, we made different constructs which expressed AAV8 VP1/VP2 only,
AAV2
VP3 only, chimeric VP!/VP2 (28m-2VP3) with N-terminal from AAV2 and C-terminal
from
AAV8, or chimeric AAV8/2 with N-terminal from AAV8 and C-terminal from AAV2
without mutation of VP3 start codon. These plasmids were used to produce
haploid AAV
vector with different combination. After injection ofl x101 particles of
these haploid vectors
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in mice via retro-orbital vein, the liver transduction efficiency was
evaluated. Chimeric
AAV82 vector (AAV82) induced a little higher liver transduction than AAV2.
However,
haploid AAV82 (H-AAV82) had much higher liver transduction than AAV2. A
further
increase in liver transduction with haploid vector 28m-2vp3 was observed. We
also
administered these haploid vectors into the muscles of mice. For easy
comparison, the right
leg was injected with AAV2 vector and the left leg was injected with haploid
vector when the
mouse was face up. At week 3 after AAV injection, the images were taken.
Consistent to
observation in the liver, all haploid vectors and chimeric vectors had higher
muscular
transduction with the best from haploid vector 28m-2vp3. This result indicates
that the
chimeric VP1/VP2 with N-terminal from AAV2 and C-terminal from AAV8 attributes
to
high liver transduction of haploid AAV82 vectors.
[00491] Enhanced AAV liver transduction from haploid vector with VP1/VP2 from
other
serotypes and VP3 from AAV2. We have shown that haploid vector AAV82 with
VP1/VP2
from AAV8 and VP3 from AAV2 increases the liver transduction as described
above. Next,
we would like to examine whether other haploid virions, in which VP1NP2 is
derived from
different serotypes, also increases transduction. In preclinical studies, AAV9
has been shown
to efficiently transduce different tissues. We have made a haploid AAV92
vector (H-
AAV92) in which VP1/VP2 was from AAV9 and VP3 from AAV2. After systemic
administration, the imaging was performed at week 1. About 4- fold higher
liver transduction
was achieved with 1-1-AAV92 than that with AAV2. This data indicates that
VP1/VP2 from
other serotypes is also capable of increasing AAV2 transduction.
[00492] Enhanced AAV liver transduction from haploid vector with VP3 from AAV2
mutant or other serotypes. AAV9 uses glycan as primary receptor for effective
transduction.
In our previous studies, we have engrafted AAV9 glycan receptor binding site
into AAV2 to
make AAV2G9 and found that AAV2G9 has higher liver tropism than AAV2. Herein
we
made haploid vector (H-AAV82G9) in which VP1/VP2 from AAV8 and VP3 from
AAV2G9. After systemic injection into mice, compared to AAV2G9, more than 10
fold
higher liver transduction was observed at both week 1 and week 2 post H-
AAV82G9
application. To study haploid vectors in which VP3 from other serotypes and
VP1/VP2 from
different serotypes or variants, we cloned other constructs: AAV3 VP3 only,
AAV rh10
VP1/VP2 only, and made different haploid vectors with various combination (H-
AAV83, H-
AAV93 and H-AAVrh10-3). After systemic injection into mice, the imaging was
carried out
at week 1. Consistent to the results obtained from other haploid vectors,
higher liver
transduction was achieved with haploid vectors (H-AAV83, H-AAV93 and H-AAVrh10-
3)
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than that with AAV3. It is interesting to note that these haploid vectors also
induced a whole
body transduction based on imaging profile, which is different from the
results from haploid
vectors 5 with VP3 from AAV2, which only transduced the liver efficiently.
Collectively,
haploid vectors with VP1/VP2 from one serotype and VP3 from an alternative one
are able to
enhance transduction and perhaps change tropism.
[00493] Haploid vector with VP1/VP3 from one AAV serotype and VP2 from another
AAV serotype enhances AAV transduction and escapes antibody neutralization. To
study
haploid vectors in which VP2 is from one serotype and VP1/VP3 from a different
serotype,
several constructs will be generated. A construct that expresses AAV2 VP2 only
will be
generated. This will be accomplished by incorporation of a mutation of the
AAV2 VP1 start
scodon and/or a mutation of the AAV2 VP1 splice acceptor site e.g.,shown in
Figure 10,
combined with a mutation of the VP3 start codon. A construct that expresses
AAV8 VP1/3
only will also be generated. This will be accomplished by incorporation of a
mutation of the
AAV8 VP2 start codon. Similarly a construct that expresses AAV2 VP1/3 only,
and a
construct that expresses AAV8 VP2 only will be generated.
[00494] A substantially homogeneous population of haploid vectors encoding a
luciferase
transgene and having either AAV2VP1 and AAV8VP1/3, or having AAV8VP1 and AAV2
VP1/3, will be made from these constructs using the appropriate plasmids and
helper virus. 1
x101 particles of these haploid vectors will be injected into mice via retro-
orbital vein, and
the liver transduction efficiency evaluated by imaging after 1 week. It is
expected that higher
liver transduction will be achieved with the homogeneous population of the
haploid vector
than with AAV2, and that far lower Nab cross-reactivity will be seen with the
haploid vector,
compared to activity with AAV2 or AAV8. Further, the homogeneous haploid
vector
population may also induce a whole body transduction (e.g., as identified
based on an
imaging profile), which differs from the results using either AAV2 or AAV8.
[00495] In these examples, we demonstrate that the haploid viruses made from
the VP1/VP2
and VP3s from compatible serotypes also increase transduction. After systemic
injection of
2x101 vg of AAV vectors into mice, it was found that haploid AAV vectors
composed of
VP1/VP2 from serotypes 7, 8, 9, and rh10 and VP3 from AAV2 or AAV3 display a 2-
to 7-
fold increase in transduction across multiple tissue types, including liver,
heart, and brain,
when compared to AAV2-only and AAV3-only capsids. These tissues additionally
had
higher vector genome copy numbers in these tissues, indicating that an
incorporation of non-
cognate VP1/VP2 can influence AAV receptor binding and intracellular
trafficking. In
addition, chimeric and haploid capsids were created with either AAV2 or AAV8
VP1/VP2
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combined with AAV2 or AAV8 VP3. When these haploid AAV vectors were injected
into
mice, the haploid AAV vectors composed of AAV8 VP!/2 and AAV2 VP3 had a 5-fold
higher transduction than viruses composed solely of AAV2 VPs. Remarkably,
haploid
vectors composed of VP INP2 from the chimeric AAV2/8 (the N-terminus of AAV2
and the
C-terminus of AAV8) paired with VP3 from AAV2 had a 50-fold increase in
transgene
expression compared to capsids composed of AAV8 VP1/VP2 paired with AAV2 VP3.
Given the same proportion of the capsid coming from AAV8 VP3, the difference
lies in the
VP1/2 N-terminal region between AAV2 and AAV8, which may indicate a
'communication'
between the VP1/2 N-terminus of AAV2 with its cognate VP3. Collectively, work
presented
herein provides insight into current AAV production strategies that can
increase transduction
across multiple tissue types.
[00496] The haploid vectors will also be injected into the muscles of mice.
For easy
comparison, the right leg will be injected with AAV2 vector and the left leg
will be injected
with haploid vector when the mouse is face up. At week 3 after AAV injection,
the images
will be taken. Enhanced transduction in muscle by the haploid vectors is also
expected.
[00497] The ability of homogeneous population of haploid viruses to escape
neutralizing
antibody. To study whether haploid virus is able to escape Nabs generated from
parental
vector, an Nab binding assay will be performed using monoclonal antibodies by
an immune-
blot assay. Three dilutions of virus- genome-containing particles will be
adsorbed to a
nitrocellulose membrane and probed with Nab A20 or ADK8, which recognizes
intact AAV2
or AAV8, respectively. It is expected that the homogeneous population of
haploid viruses
will have much reduced to undetectable recognition by monoclonal antibody ADK8
or A20.
[00498] Next, the immunological profile of the homogeneous population of
haploid viruses
using sera from AAV-immunized mice will be generated. Nab titers will be used
to evaluate
the ability of serum to inhibit vector transduction. Sera will be collected
from mice treated
with parental viruses at week 4 post-injection. The neutralization profiles of
the haploid
viruses against A20 or ADK8 will be compared, and are expected to be similar
to the data
obtained from a native immune-blot. No Nab cross-reactivity is expected to be
seen between
AAV8 and AAV2. The homogeneous population of haploid viruses are expected to
at least
partially, and perhaps completely escape the neutralization from either AV2
serum or AAV8
serum.
[00499] Haploid vector with VP2/VP3 from one AAV serotype and VP1 from another
AAV
serotype enhances AAV transduction and escapes antibody neutralization. To
study
haploid vectors in which VP1 is from one serotype and VP2/VP3 from a different
serotype,
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several constructs will be generated. A construct that expresses AAV2 VP1 only
will be
generated. This will be accomplished by incorporation of a mutation of the
AAV2 VP2 start
codon, a mutation of the AAV2 VP3 start codon e.g.,as shown in Figure 7 and
Figure 21, or a
mutation of the VP2 and VP3 splice acceptor site e.g., as shown in Figure 9,
or mutation of both
e.g., as shown in Figure 11. A construct that expresses AAV8 VP2/3 only will
be generated.
This will be accomplished by incorporation of a mutation of the AAV8 VP1 start
codon, e.g.,
see Figure 21, and/or the splice acceptor site e.g., see Figure 12. Similarly,
a construct that
expresses AAV2 VP2/3 only will be generated, and a construct that expresses
AAV8 VP1
only will be generated.
[00500] A substantially homogeneous population of haploid vectors encoding a
luciferase
transgene and having either AAV2VP1 and AAV8VP2/3, or having AAV8VP1 and AAV2
VP2/3, will be made from these constructs using the appropriate plasmids and
helper virus. 1
x101 particles of these haploid vectors will be injected into mice via retro-
orbital vein, and
the liver transduction efficiency evaluated by imaging after 1 week. It is
expected that higher
liver transduction will be achieved with the homogeneous population of the
haploid vector
than with AAV2, and that far lower Nab cross-reactivity will be seen with the
haploid vector,
compared to activity with AAV2 or AAV8. Further, the homogeneous haploid
vector
population may also induce a whole body transduction (e.g., as identified
based on an
imaging profile), which differs from the results using either AAV2 or AAV8.
[00501] The haploid vectors will also be injected into the muscles of mice.
For easy
comparison, the right leg will be injected with AAV2 vector and the left leg
will be injected
with haploid vector when the mouse is face up. At week 3 after AAV injection,
the images
will be taken. Enhanced transduction in muscle by the haploid vectors is also
expected.
[00502] The ability of homogeneous population of haploid viruses to escape
neutralizing
antibody. To study whether haploid virus is able to escape Nabs generated from
parental
vector, an Nab binding assay will be performed using monoclonal antibodies by
an immune-
blot assay. Three dilutions of virus- genome-containing particles will be
adsorbed to a
nitrocellulose membrane and probed with Nab A20 or ADK8, which recognizes
intact AAV2
or AAV8, respectively. It is expected that the homogeneous population of
haploid viruses
will have much reduced to undetectable recognition by monoclonal antibody ADK8
or A20.
[00503] Next, the immunological profile of the homogeneous population of
haploid viruses
using sera from AAV-immunized mice will be generated. Nab titers will be used
to evaluate
the ability of serum to inhibit vector transduction. Sera will be collected
from mice treated
with parental viruses at week 4 post-injection. The neutralization profiles of
the haploid
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viruses against A20 or ADK8 will be compared, and are expected to be similar
to the data
obtained from a native immune-blot. No Nab cross-reactivity is expected to be
seen between
AAV8 and AAV2. The homogeneous population of haploid viruses are expected to
at least
partially, and perhaps completely escape the neutralization from either AV2
serum or AAV8
serum.
[00504] Triploid vector with VP1 from one AAV serotype, VP2 from another AA V
serotype, and VP3 from a third AAV serotype enhances AAV transduction and
escapes
antibody neutralization.
[00505] To study triploid vectors in which VP1, VP2 and VP3 are each from a
different
AAV serotype, several constructs will be generated. A construct that expresses
AAV2 VP1
only will be generated. This will be accomplished by incorporation of either a
mutation of
the AAV2 VP2 start codon and mutation of the VP3 start codon e.g., as shown in
Figure 7, or
incorporation of a mutation of the splice acceptor site for VP2/3 e.g., as
shown in Figure 9. A
construct that expresses AAV9 VP2 only will be generated. This will be
accomplished by
incorporation of a mutation in the AAV9 VP1 start codon and/or incorporation
of a mutation
in the AAV9 VP1 splice acceptor site, and mutation of the VP3 start codon.
Alternatively,
this will be accomplished by synthesizing a fragment of the AAV9 Cap coding
sequence that
omits the upstream coding sequences for VP1, and mutation of the VP3 start
codon. A
construct that expresses AAV8 VP3 only will be generated. This will be
accomplished by
incorporatin of a mutation in the AAV8 VP1 start codon and/or splice acceptor
site, and
incorporation of a mutation in the AAV8 VP2 start codon. Alternatively, this
will be
accomplished by synthesizing a fragment of the AAV8 Cap coding sequence that
omits the
upstream coding sequences for VP1 and VP2.
[00506] A substantially homogeneous population of triploid vectors encoding a
luciferase
transgene and having AAV2 VP1, AAV9 VP2 , and AAV8 VP3, will be made from
these
constructs using the appropriate plasmids and helper virus (e.g., see Figure
13, 14, and 15). I
x101 particles of these triploid vectors will be injected into mice via retro-
orbital vein, and
the liver transduction efficiency evaluated by imaging after 1 week. It is
expected that higher
liver transduction will be achieved with the homogeneous population of the
triploid vector
than with AAV2, AAV9 or AAV8, and that far lower Nab cross-reactivity will be
seen with
the triploid vector, compared to activity with either AAV2, AAV8 or AAV8.
Further, the
homogeneous triploid vector population may also induce a whole body
transduction (e.g., as
identified based on an imaging profile).
[00507] The triploid vectors will also be injected into the muscles of mice.
For easy
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comparison, the right leg will be injected with AAV2 vector, AAV9 vector or
AAV8 vector,
and the left leg will be injected with triploid vector when the mouse is face
up. At week 3
after AAV injection, the images will be taken. Enhanced transduction in muscle
by the
triploid vectors is expected.
[00508] The ability of homogeneous population of triploid viruses to escape
neutralizing
antibody. Each individual haploid virus virion is composed of 60 subunits from
the
respective different AAV serotype capsids. Combining serotype capsid proteins
derived from
three different serotypes is expected to change the virion surface structure.
It is well known
that most AAV monoclonal antibodies recognize residues on the different
subunits of one
single virion. To study whether triploid virus is able to escape Nabs
generated from parental
vector, an Nab binding assay will be performed using monoclonal antibodies by
an immune-
blot assay. Three dilutions of virus- genome-containing particles will be
adsorbed to a
nitrocellulose membrane and probed with Nab A20 or ADK8, which recognizes
intact AAV2
or AAV8, respectively. It is expected that the homogeneous population of
triploid viruses
will have much reduced to undetectable recognition by monoclonal antibody ADK8
or A20.
[00509] Next, the immunological profile of the homogeneous population of
triploid viruses
using sera from AAV-immunized mice will be generated. Nab titers will be used
to evaluate
the ability of serum to inhibit vector transduction. Sera will be collected
from mice treated
with parental viruses at week 4 post-injection. The neutralization profiles of
the triploid
viruses against A20 or ADK8 will be compared, and are expected to be similar
to the data
obtained from a native immune-blot. No Nab cross-reactivity is expected to be
seen between
AAV8 and AAV2. The homogeneous population of triploid viruses are expected to
at least
partially, and perhaps completely escape the neutralization from either AAV2
serum, AAV9
serum, or AAV8 serum.
Example 3: Polvploid Adeno-Associated Virus Vectors Enhance Transduction and
Escape
Neutralizing Antibody
[00510] Adeno-associated virus (AAV) vectors have been successfully used in
clinical trials
in patients with hemophilia and blindness. Although the application of AAV
vectors has
proven safe and shown therapeutic effect in these clinical trials, one of the
major challenges
is its low infectivity that requires relatively large amount of virus genomes.
Additionally, a
large portion of the population has neutralizing antibodies (Nabs) against
AAVs in the blood
and other bodily fluids. The presence of Nabs poses another major challenge
for broader
AAV applications in future clinical trials. Effective strategies to enhance
AAV transduction
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and escape neutralizing antibody activity are highly demanded. Previous
studies have shown
the compatibility of capsids from AAV serotypes and recognition sites of AAV
Nab located
on different capsid subunits of one virion. In this study, we propose to study
whether
polyploid AAV viruses produced from co-transfection of different AAV helper
plasmids
have the ability for enhanced AAV transduction and escape of Nabs. We co-
transfected
AAV2 and AAV8 helper plasmids at different ratios (3:1, 1:1 and 1:3) to
assemble haploid
capsids. The haploid virus yield was similar to the parental ones, suggesting
that these two
AAV capsids were compatible. In Huh7 and C2C12 cell lines, the transduction
efficiency of
AAV8 was much lower than those from AAV2; however, the transduction from all
haploid
vectors was higher than that from AAV8. The transduction efficiency and the
heparin sulfate
binding ability for haploid vectors were positively correlated with amount of
integrated
AAV2 capsid. These results indicate that the haploid virus vectors retain
their parental virus
properties and take advantage of the parental vectors for enhanced
transduction. After
muscular injection, all of the haploid viruses induced higher transduction
than parental AAV
vectors (2- to 9-fold over AAV2) with the highest of these being the haploid
vector AAV2/8
3:1.
[00511] After systemic administration, 4-fold higher transduction in the liver
was observed
with haploid vector AAV2/8 1:3 than that with AAVS alone. Importantly, we
packaged the
therapeutic factor IX cassette into haploid vector AAV2/8 1:3 capsids and
injected them into
FIX knockout mice via tail vein. Higher FIX expression and improved phenotypic
correction
were achieved with haploid vector AAV2/8 1:3 virus vector compared to that of
AAVS.
Strikingly, haploid virus AAV2/8 1:3 was able to escape AAV2 neutralization
and had very
low Nab cross-reactivity with AAV2. But AAVS neutralizing antibody can inhibit
haploid
vector AAV2/8 transduction the same efficiency as AAV8. Next, we produced
triploid
vector AAV2/8/9 vector by co-transfecting AAV2, AAV8 and AAV9 helper plasmids
at the
ratio of 1:1:1. After systemic administration, 2-fold higher transduction in
the liver was
observed with triploid vector AAV2/8/9 than that with AAV8 (Figure 6).
Neutralizing
antibody analysis demonstrated that AAV2/8/9 vector was able to escape
neutralizing
antibody activity from mouse sera immunized with parental serotype, different
from AAV2/8
triploid vector. The results indicate that polyploid virus might potentially
acquire advantage
from parental serotypes for enhancement of transduction and has ability for
evasion of Nab
recognition. This strategy should be explored in future clinical trials in
patients with positive
neutralizing antibodies.
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Example 4: Substitution of AAV capsid subunits enhances transduction and
escapes
neutralizing antibody
[00512] Therapeutic effect has been achieved in clinical trials in patients
with blood diseases
and blind disorders using adeno-associated virus (AAV) vector. However, two
concerns
restrict broadening AAV vector application: AAV capsid specific cytotoxic T
cell (CTL) and
neutralizing antibodies (Nabs). Enhancing AAV transduction with low dose of
AAV vector
will potentially decrease capsid antigen load and hopefully ablate capsid CTL
mediated
clearance of AAV transduced target cells without compromise of transgene
expression.
Currently, 12 serotypes and over 100 variants or mutants have been explored
for gene
delivery due to their different tissue tropism and transduction efficiency. It
has been
demonstrated that there is compatibility of capsid among AAV serotypes, and
integration of
specific amino acids from one serotype into another AAV capsid enhances AAV
transduction. By taking advantage of different mechanisms for effective AAV
transduction
from different serotypes, enhanced AAV transduction was achieved using mosaic
virus in
which AAV capsid subunits are derived from different serotypes in vitro and in
vivo. The
recent structural studies on interaction of AAV vectors with monoclonal
neutralizing
antibodies demonstrated that Nab binds to residues on several different
subunits of one virion
surface, which suggests that change of subunit assembly of AAV virion may
ablate the AAV
Nab binding site and then escape Nab activity. We have demonstrated that the
mosaic AAV
vector is able to evade Nab activity. These results indicate that substitution
of AAV capsid
subunits has the potential to enhance AAV transduction and the ability of
neutralizing
antibody evasion.
[00513] Adeno-associated virus (AAV) vector has been successfully applied in
clinical trials
in patients with blood diseases and blind disorders. Two concerns restrict
broad AAV vector
application: AAV capsid specific cytotoxic T cell (CTL) response mediated
elimination of
AAV transduced target cells and neutralizing antibodies (Nabs) mediated
blocking of AAV
transduction. It has been demonstrated that capsid antigen presentation is
dose-dependent,
which indicates that enhancing AAV transduction with low dose of AAV vector
will
potentially decrease capsid antigen load and hopefully ablate capsid CTL
mediated clearance
of AAV transduced target cells without compromise of transgene expression.
Several
approaches have been explored for this purpose including: optimization of
transgene cassette,
modification of AAV capsid and interference of AAV trafficking with
pharmacological
agents. Modification of AAV capsid may change AAV tropism; especially AAV
transduction efficiency is unknown in human tissues. Though several clinical
trials have
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been ongoing, the AAV vector was empirically chosen based on observation from
animal
models. Pharmacological reagents for enhancing AAV transduction usually have
unwanted
side effects. It is imperative to develop ideal strategies to enhance AAV
transduction but
without changing its tropism from modification of capsids and no side effects
from
pharmacological treatment. Currently, there are 12 serotypes and over 100
variants or
mutants which have been explored for gene delivery. Effective AAV transduction
involves
following steps including: binding on the target cell surface via receptors
and co-receptors,
endocytosis into endosomes, escape from endosomes, nuclear entrance, AAV
virion
uncoating followed by transgene expression. To rationally design novel AAV
vectors for
enhanced transduction, we have developed chimeric viruses: AAV2.5 (in which
AAV2
mutant with 5 aa substitution from AAV1) and AAV2G9 (in which galactose
receptor from
AAV9 is engrafted into AAV2 capsid). Both chimeric mutants induce a much
higher
transduction than AAV2 in mouse muscle and liver, respectively. These
observations
indicate that these chimeric viruses may use properties from both AAV
serotypes for
enhanced transduction (for example, AAV2G9 uses two primary receptors-heparin
and
galactose for effective cell surface binding). Based on the compatibility
among capsid
subunits from different AAV serotypes for virus assembly and our preliminary
results, which
demonstrated that integration of specific amino acids from other serotypes (1
or 9) into AAV
serotype 2 enhanced AAV2 transduction in muscle and the liver, we reason that
substitution
of some capsid subunits from other serotypes is able to enhance AAV
transduction by taking
advantage of different mechanisms for effective AAV transduction from
different serotypes.
In addition, pre-existing antibodies to naturally occurring AAV have impacted
success for
hemophilia B and other AAV gene transfer studies. In the general human
population, around
50% carry neutralizing antibodies. Several approaches have been considered to
design NAb-
evading AAV vectors, including chemical modification, different serotype of
AAV vector,
rational design and combinatorial mutagenesis of the capsid in situ as well as
biological
depletion of NAb titer (empty capsid utilization, B cell depletion and plasma-
apheresis).
These approaches have low efficiency or side-effect or change of AAV tropism.
The recent
structural studies on interaction of AAV vectors with monoclonal neutralizing
antibodies
demonstrated that Nab binds to residues on several different subunits of one
virion surface,
which suggests that change of subunit assembly of AAV virion may ablate the
AAV Nab
binding site and then escape Nab activity. We have results strongly supporting
the notion
that novel mosaic AAV vectors have potential to enhance transduction in
various tissues and
are able to escape neutralizing antibody activity.
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[00514] Treatment of Diseases
[00515] In each of the following Examples 5-6 for treatment of diseases: e.g.,
of the central
nervous system, heart, lung, skeletal muscle, and liver; including e.g.,
Parkinson's disease,
Alzheimer's disease, cystic fibrosis, ALS, Duchenne Muscular Dystrophy, limb
girdle
muscular dystrophy, Myasthenia Gravis, and Hemophilia A or B; the capsid
virion described
therein that is generated using the specified AAV serotypes and mosaicism is
alternatively
generated using the rational polyploid method of Example 2, to generate a
haploid capsid
where VP1 is only from the first serotype, and VP2 and/or VP3 is only from the
second
serotype; or e.g., where VP!, VP2 and VP3 are each from a different serotype.
Alternative
methods for creating such virions are also, e.g., described in Examples 7-15.
Example 5: Treatment of diseases of the central nervous system (CNS) with
VP1/VP2/VP3
from two or more different AAV serotypes
[00516] In a first experiment, two helper plasmids are used. The first helper
plasmid has the
Rep and Cap genes from AAV2 and the second helper plasm id has the Rep gene
from AAV2
and the Cap gene from AAV4. A third plasmid encodes for the nucleotide
sequence for
Glutamic Acid Decarboxylase 65 (GAD65) and/or Glutamic Acid Decarboxylase 67
(GAD67), which nucleotide sequence is inserted between two ITRs. A polyploid
virion can
be used to encapsidate the therapeutic GAD65 and/or GAD67 containing nucleic
acid
sequence. In the following examples, the capsid can be prepared using for
example the
rational polyploid method of Example 2 to produce, for example, a haploid
capsid where VP1
is only from one serotype, VP3 is only from an alternative serotype, and VP2
may or may not
be present. When VP2 is present it is only from one serotype that may be the
same as either
VP1 or VP3, or can be from a third serotype or the capsid can be prepared by
the cross-
dressing methodology described above that results in mosaic haploid capsids.
The haploid
AAV generated from the three plasmids contains the nucleotide sequence for
GAD65 and/or
GAD67protein to treat Parkinson's disease, in part by increasing the
specificity for central
nervous system tissues associated with Parkinson's disease through the use of
multiple AAV
serotypes to source the proteins that code for VP1, VP2 and VP3 according to
the methods of
the present invention. In fact, the haploid virus created by this method to
treat Parkinson's
disease can have a higher specificity for the relevant tissue than a virus
vector comprised of
only AAV2 or AAV4.
[00517] In a further experiment, two helper plasmids are again used with
different AAV
serotypes as the source for the Rep and Cap genes. The first helper plasmid
has the Rep and
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Cap genes from AAV3 and the second helper plasmid has the Rep gene from AAV3
and the
Cap gene from AAV5. A third plasmid encodes the nucleotide sequence for CLN2
to treat
Batten's disease is contained in a third plasmid and has been inserted between
two ITRs. The
haploid AAV generated from the three plasmids contains the nucleotide sequence
to treat
Batten's disease, in part by increasing the specificity for central nervous
system tissues
associated with Parkinson's disease through the use of multiple AAV serotypes
to source the
proteins that code for VP1, VP2 and VP3 according to the methods of the
present invention.
In fact, the haploid virus created by this method to treat Batten's disease
has a higher
specificity for the relevant central nervous system tissue than a virus vector
comprised of
only AAV3 or AAV5.
[00518] In another experiment, three helper plasmids are used with different
AAV serotypes
as the source for the Rep and Cap genes. The first helper plasmid has the Rep
and Cap genes
from AAV3 and the second helper plasmid has the Rep gene from AAV3 and the Cap
gene
from AAV4. A third helper plasmid has the Rep gene from AAV3 and the Cap gene
from
AAV5. A fourth plasmid encodes the nucleotide sequence for Nerve Growth Factor
(NGF)
to treat Alzheimer's disease is contained in a third plasmid and has been
inserted between two
ITRs. The triploid AAV generated from the four plasmids contains the
nucleotide sequence
to treat Alzheimer's disease, in part by increasing the specificity for
central nervous system
tissues associated with Alzheimer's disease through the use of multiple AAV
serotypes (e.g.,
AAV3, AAV4 and AAV5) to source the proteins that code for VP1, VP2 and VP3
according
to the methods of the present invention. In fact, the triploid virus created
by this method to
treat Alzheimer's disease has a higher specificity for the relevant central
nervous system
tissue than a virus vector comprised of only AAV3, AAV4 or AAV5.
[00519] In another experiment, one helper plasmid is used with different AAV
serotypes as
the source for the Rep and Cap genes. The helper plasmid has the Rep from AAV2
and VP1
from AAV2, VP2 from AAV4 and VP3 from AAV5. A second plasmid encodes the
nucleotide sequence for AAC inserted between two ITRs to treat Canavan's
disease. The
triploid AAV generated from the two plasmids contains the nucleotide sequence
to treat
Canavan's disease, in part by increasing the specificity for central nervous
system tissues
associated with Canavan's disease through the use of multiple AAV serotypes
(e.g., AAV2,
AAV4 and AAV5) to source the proteins that code for VP1, VP2 and VP3 according
to the
methods of the present invention. In fact, the triploid virus created by this
method to treat
Canavan's disease has a higher specificity for the relevant central nervous
system tissue than
a virus vector comprised of only AAV2, AAV4 or AAV5.
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[00520] Treatment of Diseases of Heart with VP1/VP2/VP3 from Two or More
Different
AAV Serotypes. In an experiment, two helper plasmids are used with different
AAV
serotypes as the source for the Rep and Cap genes. The first helper plasmid
has the Rep and
Cap genes from AAV2 and the second helper plasmid has the Rep gene from AAV2
and the
Cap gene from AAV6. A third plasmid encodes the nucleotide sequence for the
protein to
treat heart disease is contained in a third plasmid and has been inserted
between two ITRs.
The haploid AAV generated from the three plasmids contains the nucleotide
sequence to treat
heart disease, in part by increasing the specificity heart tissue associated
with heart's disease
through the use of multiple AAV serotypes to source the proteins that code for
VP!, VP2 and
VP3 according to the methods of the present invention. In fact, the haploid
virus created by
this method to treat heart disease has a higher specificity for the relevant
heart tissue than a
virus vector comprised of only AAV2 or AAV6.
[00521] In a further experiment, two helper plasmids are used with different
AAV serotypes
as the source for the Rep and Cap genes. The first helper plasmid has the Rep
and Cap genes
from AAV3 and the second helper plasmid has the Rep gene from AAV3 and the Cap
gene
from AAV9. A third plasmid encodes the nucleotide sequence for the protein to
treat heart
disease is contained in a third plasmid and has been inserted between two
ITRs. The haploid
AAV generated from the three plasmids contains a nucleotide sequence encoding
a protein to
treat heart disease, in part by increasing the specificity heart tissue
associated with heart's
disease through the use of multiple AAV serotypes to source the proteins that
code for VP1,
VP2 and VP3 according to the methods of the present invention. In fact, the
haploid virus
created by this method to treat heart disease has a higher specificity for the
relevant heart
tissue than a virus vector comprised of only AAV3 or AAV9.
[00522] In an experiment, three helper plasmids are used with different AAV
serotypes as
the source for the Rep and Cap genes. The first helper plasmid has the Rep and
Cap genes
from AAV3 and the second helper plasmid has the Rep gene from AAV3 and the Cap
gene
from AAV6. A third helper plasmid has the Rep gene from AAV3 and the Cap gene
from
AAV9. A fourth plasmid contains a nucleotide sequence that encodes a protein
to treat heart
disease is contained in a third plasmid and has been inserted between two
ITRs. The triploid
AAV generated from the four plasmids contains the nucleotide sequence to treat
heart
disease, in part by increasing the specificity for heart tissue associated
with heart disease
through the use of multiple AAV serotypes (e.g., AAV3, AAV6 and AAV9) to
source the
proteins that code for VP!, VP2 and VP3 according to the methods of the
present invention.
In fact, the triploid virus created by this method to treat heart disease has
a higher specificity
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for the relevant heart tissue than a virus vector comprised of only AAV3, AAV6
or AAV9.
[00523] In another experiment, one helper plasmid is used with different AAV
serotypes as
the source for the Rep and Cap genes. The helper plasmid has the Rep from AAV2
and VP1
from AAV2, VP2 from AAV3 and VP3 from AAV9. A second plasmid contains a
nucleotide sequence encoding a protein to treat heart disease inserted between
two ITRs. The
triploid AAV generated from the two plasmids encodes the nucleotide sequence
to treat heart
disease, in part by increasing the specificity for heart tissues associated
with heart disease
through the use of multiple AAV serotypes (e.g., AAV2, AAV3 and AAV9) to
source the
proteins that code for VP1, VP2 and VP3 according to the methods of the
present invention.
In fact, the triploid virus created by this method to treat heart disease has
a higher specificity
for the relevant heart tissue than a virus vector comprised of only AAV2, AAV3
or AAV9.
[00524] In another experiment, one helper plasmid is used with different AAV
serotypes as
the source for the Rep and Cap genes. The helper plasm id has the Rep from
AAV3 and VP1
from AAV3, VP2 from AAV6 and VP3 from AAV6. A second plasmid contains a
nucleotide sequence encoding a protein to treat heart disease inserted between
two ITRs. The
haploid AAV generated from the two plasmids encodes the nucleotide sequence to
treat heart
disease, in part by increasing the specificity for heart tissues associated
with heart disease
through the use of multiple AAV serotypes (e.g., AAV3 and AAV6) to source the
proteins
that code for VP1, VP2 and VP3 according to the methods of the present
invention. In fact,
the haploid virus created by this method to treat heart disease has a higher
specificity for the
relevant heart tissue than a virus vector comprised of only AAV2 or AAV6.
[00525] In another experiment, one helper plasmid is used with different AAV
serotypes as
the source for the Rep and Cap genes. The helper plasmid has the Rep from AAV3
and VP1
from AAV3, VP2 from AAV6 and VP3 from AAV9. A second plasmid contains a
nucleotide sequence encoding a protein to treat heart disease inserted between
two ITRs. The
triploid AAV generated from the two plasmids encodes the nucleotide sequence
to treat heart
disease, in part by increasing the specificity for heart tissues associated
with heart disease
through the use of multiple AAV serotypes (e.g., AAV3, AAV6 and AAV9) to
source the
proteins that code for VP1, VP2 and VP3 according to the methods of the
present invention.
In fact, the triploid virus created by this method to treat heart disease has
a higher specificity
for the relevant heart tissue than a virus vector comprised of only AAV3, AAV6
or AAV9.
[00526] Treatment of Diseases of the Lung with VP1/VP2/VP3 from Two or More
Different AAV Serotypes. In an experiment, two helper plasmids are again used
with
different AAV serotypes as the source for the Rep and Cap genes. The first
helper plasmid
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has the Rep and Cap genes from AAV2 and the second helper plasmid has the Cap
gene from
AAV9. A third plasmid encodes for the nucleotide sequence for CFTR to treat
Cystic
Fibrosis is inserted between two ITRs. The haploid AAV generated from the
three plasmids
contains the nucleotide sequence for CFTR to treat Cystic Fibrosis, in part by
increasing the
specificity for lung tissue associated with Cystic Fibrosis through the use of
multiple AAV
serotypes to source the proteins that code for VP1, VP2 and VP3 according to
the methods of
the present invention. In fact, the haploid virus created by this method to
treat Cystic Fibrosis
has a higher specificity for the relevant tissue than a virus vector comprised
of only AAV2 or
AAV9.
[00527] In an experiment, two helper plasmids are again used with different
AAV serotypes
as the source for the Rep and Cap genes. The first helper plasmid has the Rep
and Cap genes
from AAV3 and the second helper plasmid has the Rep from AAV3 and the Cap gene
from
AAV 1 O. A third plasmid encodes for the nucleotide sequence for CFTR to treat
Cystic
Fibrosis is inserted between two ITRs. The haploid AAV generated from the
three plasmids
contains the nucleotide sequence for CFTR to treat Cystic Fibrosis, in part by
increasing the
specificity for lung tissue associated with Cystic Fibrosis through the use of
multiple AAV
serotypes to source the proteins that code for VP1, VP2 and VP3 according to
the methods of
the present invention. In fact, the haploid virus created by this method to
treat Cystic Fibrosis
has a higher specificity for the relevant tissue than a virus vector comprised
of only AAV3 or
AAV10.
[00528] In an experiment, three helper plasmids are used with different AAV
serotypes as
the source for the Rep and Cap genes. The first helper plasmid has the Rep and
Cap genes
from AAV3 and the second helper plasmid has the Rep gene from AAV3 and the Cap
gene
from AAV9. A third helper plasmid has the Rep gene from AAV3 and the Cap gene
from
AAV 10. A fourth plasmid encodes a nucleotide sequence for CFTR to treat
Cystic Fibrosis
is contained in a third plasmid and has been inserted between two ITRs. The
triploid AAV
generated from the four plasmids contains the nucleotide sequence for CFTR to
treat Cystic
Fibrosis, in part by increasing the specificity for lung tissue associated
with Cystic Fibrosis
through the use of multiple AAV serotypes (e.g., AAV3, AAV9 and AAV10) to
source the
proteins that code for VP!, VP2 and VP3 according to the methods of the
present invention.
In fact, the triploid virus created by this method to treat Cystic Fibrosis
has a higher
specificity for the relevant tissue than a virus vector comprised of only
AAV3, AAV9 or
AAVIO.
[00529] In another experiment, one helper plasmid is used with different AAV
serotypes as
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CA 3054600 2019-09-06
the source for the Rep and Cap genes. The helper plasmid has the Rep from AAV2
and VP1
from AAV2, VP2 from AAV9 and VP3 from AAV9. A second plasmid encodes the
nucleotide sequence for CFTR inserted between two ITRs to treat Cystic
Fibrosis. The
haploid AAV generated from the two plasmids contains the nucleotide sequence
to treat
Cystic Fibrosis, in part by increasing the specificity for central nervous
system tissues
associated with Cystic Fibrosis through the use of multiple AAV serotypes
(e.g., AAV2 and
AAV9) to source the proteins that code for VP1, VP2 and VP3 according to the
methods of
the present invention. In fact, the haploid virus created by this method to
treat Cystic Fibrosis
has a higher specificity for the relevant tissue than a virus vector comprised
of only AAV2 or
AAV9.
[00530] In a further experiment, one helper plasmid is used with different AAV
serotypes as
the source for the Rep and Cap genes. The helper plasmid has the Rep from AAV3
and VP1
from AAV2, VP2 from AAV10 and VP3 from AAV 10. A second plasmid encodes the
nucleotide sequence for CFTR inserted between two ITRs to treat Cystic
Fibrosis. The
haploid AAV generated from the two plasmids contains the nucleotide sequence
to treat
Cystic Fibrosis, in part by increasing the specificity for central nervous
system tissues
associated with Cystic Fibrosis through the use of multiple AAV serotypes
(e.g., AAV3 and
AAV10) to source the proteins that code for VP1, VP2 and VP3 according to the
methods of
the present invention. In fact, the haploid virus created by this method to
treat Cystic Fibrosis
has a higher specificity for the relevant tissue than a virus vector comprised
of only AAV3 or
AAV10.
[00531] In another experiment, one helper plasmid is used with different AAV
serotypes as
the source for the Rep and Cap genes. The helper plasmid has the Rep from AAV2
and VP1
from AAV2, VP2 from AAV9 and VP3 from AAVIO. A second plasmid encodes the
nucleotide sequence for CFTR inserted between two ITRs to treat Cystic
Fibrosis. The
triploid AAV generated from the two plasmids contains the nucleotide sequence
to treat
Cystic Fibrosis, in part by increasing the specificity for central nervous
system tissues
associated with Canavan's disease through the use of multiple AAV serotypes
(e.g., AAV2,
AAV9 and AAV 10) to source the proteins that code for VP1, VP2 and VP3
according to the
methods of the present invention. In fact, the triploid virus created by this
method to treat
Cystic Fibrosis has a higher specificity for the relevant tissue than a virus
vector comprised of
only AAV2, AAV9 or AAV10.
[00532] Treatment of Diseases of the Skeletal Muscle with VP1/VP2/VP3 from Two
or
More Different AAV Serotypes. For the following experiments, the skeletal
muscle disease
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can be, but is not limited to, Duchene Muscular Dystrophy, Limb Girdle
Muscular
Dystrophy, Cerebral Palsy, Myasthenia Gravis and Amyotrophic Lateral Sclerosis
(ALS).
[00533] In an experiment, two helper plasmids are again used with different
AAV serotypes
as the source for the Rep and Cap genes. The first helper plasmid has the Rep
and Cap genes
from AAV2 and the second helper plasmid has the Rep from AAV2 and the Cap gene
from
AAV8. A third plasmid encodes for the nucleotide sequence for a protein to
treat a disease of
the skeletal muscle that is inserted between two ITRs. The haploid AAV
generated from the
three plasmids contains the nucleotide sequence for a protein to treat a
disease of the skeletal
muscle, in part by increasing the specificity for skeletal muscle associated
with a disease of
the skeletal muscle through the use of multiple AAV serotypes to source the
proteins that
code for VP1, VP2 and VP3 according to the methods of the present invention.
In fact, the
haploid virus created by this method to treat a skeletal muscle disease has a
higher specificity
for the relevant skeletal muscle tissue than a virus vector comprised of only
AAV2 or AAV8.
[00534] In an experiment, two helper plasmids are again used with different
AAV serotypes
as the source for the Rep and Cap genes. The first helper plasmid has the Rep
and Cap genes
from AAV3 and the second helper plasmid has the Rep from AAV3 and the Cap gene
from
AAV9. A third plasmid encodes for the nucleotide sequence for a protein to
treat a disease of
the skeletal muscle that is inserted between two ITRs. The haploid AAV
generated from the
three plasmids contains the nucleotide sequence for a protein to treat a
disease of the skeletal
muscle, in part by increasing the specificity for skeletal muscle associated
with a disease of
the skeletal muscle through the use of multiple AAV serotypes to source the
proteins that
code for VP1, VP2 and VP3 according to the methods of the present invention.
In fact, the
haploid virus created by this method to treat a skeletal muscle disease has a
higher specificity
for the relevant skeletal muscle tissue than a virus vector comprised of only
AAV3 or AAV9.
[00535] In an experiment, three helper plasmids are used with different AAV
serotypes as
the source for the Rep and Cap genes. The first helper plasmid has the Rep and
Cap genes
from AAV3 and the second helper plasmid has the Rep gene from AAV3 and the Cap
gene
from AAV8. A third helper plasmid has the Rep gene from AAV3 and the Cap gene
from
AAV9. A fourth plasmid encodes for the nucleotide sequence for a protein to
treat a disease
of the skeletal muscle that is inserted between two ITRs. The triploid AAV
generated from
the four plasmids contains the nucleotide sequence for a protein to treat a
skeletal muscle
disease, in part by increasing the specificity for skeletal muscle associated
with a disease of
the skeletal muscle through the use of multiple AAV serotypes (e.g., AAV3,
AAV8 and
AAV9) to source the proteins that code for VP1, VP2 and VP3 according to the
methods of
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CA 3054600 2019-09-06
the present invention. In fact, the triploid virus created by this method to
treat a skeletal
muscle disease has a higher specificity for the relevant tissue than a virus
vector comprised of
only AAV3, AAV8 or AAV9.
[00536] In another experiment, one helper plasmid is used with different AAV
serotypes as
the source for the Rep and Cap genes. The helper plasmid has the Rep from AAV3
and VP!
from AAV3, VP2 from AAV9 and VP3 from AAV9. A second plasmid encodes for the
nucleotide sequence for a protein to treat a disease of the skeletal muscle
that is inserted
between two ITRs. The haploid AAV generated from the two plasmids contains the
nucleotide sequence to treat a disease of the skeletal muscle that, in part by
increasing the
specificity for skeletal muscle tissues associated with a skeletal muscle
disease through the
use of multiple AAV serotypes (e.g., AAV3 and AAV9) to source the proteins
that code for
VP1, VP2 and VP3 according to the methods of the present invention. In fact,
the haploid
virus created by this method to treat a skeletal muscle disease has a higher
specificity for the
relevant skeletal muscle tissue than a virus vector comprised of only AAV3 or
AAV9.
[00537] In another experiment, one helper plasmid is used with different AAV
serotypes as
the source for the Rep and Cap genes. The helper plasmid has the Rep from AAV3
and VP1
from AAV3, VP2 from AAV8 and VP3 from AAV8. A second plasmid encodes for the
nucleotide sequence for a protein to treat a disease of the skeletal muscle
that is inserted
between two ITRs. The haploid AAV generated from the two plasmids contains the
nucleotide sequence to treat a disease of the skeletal muscle that, in part by
increasing the
specificity for skeletal muscle tissues associated with a skeletal muscle
disease through the
use of multiple AAV serotypes (e.g., AAV3 and AAV8) to source the proteins
that code for
VP!, VP2 and VP3 according to the methods of the present invention. In fact,
the haploid
virus created by this method to treat a skeletal muscle disease has a higher
specificity for the
relevant skeletal muscle tissue than a virus vector comprised of only AAV3 or
AAV8.
[00538] In another experiment, one helper plasmid is used with different AAV
serotypes as
the source for the Rep and Cap genes. The helper plasmid has the Rep from AAV3
and VP!
from AAV3, VP2 from AAV8 and VP3 from AAV9. A second plasmid encodes for the
nucleotide sequence for a protein to treat a disease of the skeletal muscle
that is inserted
between two ITRs. The triploid AAV generated from the two plasmids contains
the
nucleotide sequence to treat a disease of the skeletal muscle that, in part by
increasing the
specificity for skeletal muscle tissues associated with a skeletal muscle
disease through the
use of multiple AAV serotypes (e.g., AAV3, AAV8 and AAV9) to source the
proteins that
code for VP!, VP2 and VP3 according to the methods of the present invention.
In fact, the
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CA 3054600 2019-09-06
triploid virus created by this method to treat a skeletal muscle disease has a
higher specificity
for the relevant skeletal muscle tissue than a virus vector comprised of only
AAV3, AAV8 or
AAV9.
[00539] Treatment of Diseases of the Liver with VP1/VP2/VP3 from Two or More
Different AAV Serotypes. In an experiment, two helper plasmids are again used
with
different AAV serotypes as the source for the Rep and Cap genes. The first
helper plasmid
has the Rep and Cap genes from AAV2 and the second helper plasmid has the Rep
from
AAV2 and the Cap gene from AAV6. A third plasmid encodes for the nucleotide
sequence
for a Factor IX (FIX) to treat Hemophilia B that is inserted between two ITRs.
The haploid
AAV generated from the three plasmids contains the nucleotide sequence for a
protein to
treat a disease of the skeletal muscle, in part by increasing the specificity
for FIX associated
with Hemophilia B through the use of multiple AAV serotypes to source the
proteins that
code for VP1, VP2 and VP3 according to the methods of the present invention.
In fact, the
haploid virus created by this method to treat liver tissue in a patient
suffering from
Hemophilia B has a higher specificity for the relevant tissue than a virus
vector comprised of
only AAV2 or AAV6.
[00540] In an experiment, two helper plasmids are again used with different
AAV serotypes
as the source for the Rep and Cap genes. The first helper plasmid has the Rep
and Cap genes
from AAV2 and the second helper plasmid has the Rep from AAV3 and the Cap gene
from
AAV7. A third plasmid encodes for the nucleotide sequence for a Factor IX
(FIX) to treat
Hemophilia B that is inserted between two ITRs. The haploid AAV generated from
the three
plasmids contains the nucleotide sequence for a protein to treat a disease of
the skeletal
muscle, in part by increasing the specificity for FIX associated with
Hemophilia B through
the use of multiple AAV serotypes to source the proteins that code for VP1,
VP2 and VP3
according to the methods of the present invention. In fact, the haploid virus
created by this
method to treat liver tissue in a patient suffering from Hemophilia B has a
higher specificity
for the relevant tissue than a virus vector comprised of only AAV3 or AAV7.
[00541] In an experiment, three helper plasmids are used with different AAV
serotypes as
the source for the Rep and Cap genes. The first helper plasmid has the Rep and
Cap genes
from AAV3 and the second helper plasmid has the Rep gene from AAV3 and the Cap
gene
from AAV6. A third helper plasmid has the Rep gene from AAV3 and the Cap gene
from
AAV7. A fourth plasmid encodes for the nucleotide sequence for a Factor IX
(FIX) to treat
Hemophilia B that is inserted between two ITRs. The triploid AAV generated
from the four
plasmids contains the nucleotide sequence for a protein to treat Hemophilia B,
in part by
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CA 3054600 2019-09-06
increasing the specificity for liver tissue associated with Hemophilia B
through the use of
multiple AAV serotypes (e.g., AAV3, AAV6 and AAV7) to source the proteins that
code for
VP!, VP2 and VP3 according to the methods of the present invention. In fact,
the triploid
virus created by this method to treat liver tissue in a patient suffering from
Hemophilia B has
a higher specificity for the relevant tissue than a virus vector comprised of
only AAV3,
AAV6 or AAV7.
[00542] In another experiment, one helper plasmid is used with different AAV
serotypes as
the source for the Rep and Cap genes. The helper plasmid has the Rep from AAV2
and VP!
from AAV2, VP2 from AAV6 and VP3 from AAV6. A second plasmid encodes for the
nucleotide sequence for FIX to treat Hemophilia B that is inserted between two
ITRs. The
haploid AAV generated from the two plasmids contains the nucleotide sequence
to treat
Hemophilia B that, in part by increasing the specificity for liver tissues
associated with
Hemophilia B through the use of multiple AAV serotypes (e.g., AAV2 and AAV6)
to source
the proteins that code for VP1, VP2 and VP3 according to the methods of the
present
invention. In fact, the haploid virus created by this method to treat liver
tissue in a patient
suffering from Hemophilia B has a higher specificity for the relevant tissue
than a virus
vector comprised of only AAV2 or AAV6.
[00543] In another experiment, one helper plasmid is used with different AAV
serotypes as
the source for the Rep and Cap genes. The helper plasmid has the Rep from AAV2
and VP1
from AAV3, VP2 from AAV7 and VP3 from AAV7. A second plasmid encodes for the
nucleotide sequence for FIX to treat Hemophilia B that is inserted between two
ITRs. The
haploid AAV generated from the two plasmids contains the nucleotide sequence
to treat
Hemophilia B that, in part by increasing the specificity for liver tissues
associated with
Hemophilia B through the use of multiple AAV serotypes (e.g., AAV3 and AAV7)
to source
the proteins that code for VP!, VP2 and VP3 according to the methods of the
present
invention. In fact, the haploid virus created by this method to treat liver
tissue in a patient
suffering from Hemophilia B has a higher specificity for the relevant tissue
than a virus
vector comprised of only AAV3 or AAV7.
[00544] In another experiment, one helper plasmid is used with different AAV
serotypes as
the source for the Rep and Cap genes. The helper plasmid has the Rep from AAV2
and VP!
from AAV3, VP2 from AAV6 and VP3 from AAV7. A second plasmid encodes for the
nucleotide sequence for FIX to treat Hemophilia B that is inserted between two
ITRs. The
triploid AAV generated from the two plasmids contains the nucleotide sequence
to treat
Hemophilia B that, in part by increasing the specificity for liver tissues
associated with
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CA 3054600 2019-09-06
Hemophilia B through the use of multiple AAV serotypes (e.g., AAV3, AAV6 and
AAV7) to
source the proteins that code for VP1, VP2 and VP3 according to the methods of
the present
invention. In fact, the triploid virus created by this method to treat liver
tissue in a patient
suffering from Hemophilia B has a higher specificity for the relevant tissue
than a virus
vector comprised of only AAV3, AAV6 or AAV7.
[00545] In an experiment, two helper plasmids are again used with different
AAV serotypes
as the source for the Rep and Cap genes. The first helper plasmid has the Rep
and Cap genes
from AAV2 and the second helper plasmid has the Rep from AAV2 and the Cap gene
from
AAV6. A third plasmid encodes for the nucleotide sequence for a Factor VIII
(FVIII) to treat
Hemophilia A that is inserted between two ITRs. The haploid AAV generated from
the three
plasmids contains the nucleotide sequence for a protein to treat a disease of
the skeletal
muscle, in part by increasing the specificity for FVIII associated with
Hemophilia A through
the use of multiple AAV serotypes to source the proteins that code for VP1,
VP2 and VP3
according to the methods of the present invention. In fact, the haploid virus
created by this
method to treat liver tissue in a patient suffering from Hemophilia A has a
higher specificity
for the relevant tissue than a virus vector comprised of only AAV2 or AAV6.
[00546] In an experiment, two helper plasmids are again used with different
AAV serotypes
as the source for the Rep and Cap genes. The first helper plasmid has the Rep
and Cap genes
from AAV2 and the second helper plasmid has the Rep from AAV3 and the Cap gene
from
AAV7. A third plasmid encodes for the nucleotide sequence for a FVIII to treat
Hemophilia
A that is inserted between two ITRs. The haploid AAV generated from the three
plasmids
contains the nucleotide sequence for a protein to treat a disease of the
skeletal muscle, in part
by increasing the specificity for FVIII associated with Hemophilia A through
the use of
multiple AAV serotypes to source the proteins that code for VP I , VP2 and VP3
according to
the methods of the present invention. In fact, the haploid virus created by
this method to treat
liver tissue in a patient suffering from Hemophilia A has a higher specificity
for the relevant
tissue than a virus vector comprised of only AAV3 or AAV7.
[00547] In an experiment, three helper plasmids are used with different AAV
serotypes as
the source for the Rep and Cap genes. The first helper plasmid has the Rep and
Cap genes
from AAV3 and the second helper plasmid has the Rep gene from AAV3 and the Cap
gene
from AAV6. A third helper plasmid has the Rep gene from AAV3 and the Cap gene
from
AAV7. A fourth plasmid encodes for the nucleotide sequence for a FVIII to
treat Hemophilia
A that is inserted between two ITRs. The triploid AAV generated from the four
plasmids
contains the nucleotide sequence for a FVIII protein to treat Hemophilia A, in
part by
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CA 3054600 2019-09-06
increasing the specificity for liver tissue associated with Hemophilia B
through the use of
multiple AAV serotypes (e.g., AAV3, AAV6 and AAV7) to source the proteins that
code for
VP1, VP2 and VP3 according to the methods of the present invention. In fact,
the triploid
virus created by this method to treat liver tissue in a patient suffering from
Hemophilia A has
a higher specificity for the relevant tissue than a virus vector comprised of
only AAV3,
AAV6 or AAV7.
[00548] In another experiment, one helper plasmid is used with different AAV
serotypes as
the source for the Rep and Cap genes. The helper plasmid has the Rep from AAV2
and VP1
from AAV2, VP2 from AAV6 and VP3 from AAV6. A second plasmid encodes for the
nucleotide sequence for FVIII to treat Hemophilia B that is inserted between
two ITRs. The
haploid AAV generated from the two plasmids contains the nucleotide sequence
for FVIII to
treat Hemophilia A that, in part by increasing the specificity for liver
tissues associated with
Hemophilia A through the use of multiple AAV serotypes (e.g., AAV2 and AAV6)
to source
the proteins that code for VP1, VP2 and VP3 according to the methods of the
present
invention. In fact, the haploid virus created by this method to treat liver
tissue in a patient
suffering from Hemophilia A has a higher specificity for the relevant tissue
than a virus
vector comprised of only AAV2 or AAV6.
[00549] In another experiment, one helper plasmid is used with different AAV
serotypes as
the source for the Rep and Cap genes. The helper plasmid has the Rep from AAV2
and VP1
from AAV3, VP2 from AAV7 and VP3 from AAV7. A second plasmid encodes for the
nucleotide sequence for FVIII to treat Hemophilia A that is inserted between
two ITRs. The
haploid AAV generated from the two plasmids contains the nucleotide sequence
for FVIII to
treat Hemophilia A that, in part by increasing the specificity for liver
tissues associated with
Hemophilia B through the use of multiple AAV serotypes (e.g., AAV3 and AAV7)
to source
the proteins that code for VP I, VP2 and VP3 according to the methods of the
present
invention. In fact, the haploid virus created by this method to treat liver
tissue in a patient
suffering from Hemophilia A has a higher specificity for the relevant tissue
than a virus
vector comprised of only AAV3 or AAV7.
[00550] In another experiment, one helper plasmid is used with different AAV
serotypes as
the source for the Rep and Cap genes. The helper plasmid has the Rep from AAV2
and VP1
from AAV3, VP2 from AAV6 and VP3 from AAV7. A second plasmid encodes for the
nucleotide sequence for FVIII to treat Hemophilia A that is inserted between
two ITRs. The
triploid AAV generated from the two plasmids contains the nucleotide sequence
for FVIII to
treat Hemophilia B that, in part by increasing the specificity for liver
tissues associated with
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CA 3054600 2019-09-06
Hemophilia A through the use of multiple AAV serotypes (e.g., AAV3, AAV6 and
AAV7) to
source the proteins that code for VP1, VP2 and VP3 according to the methods of
the present
invention. In fact, the triploid virus created by this method to treat liver
tissue in a patient
suffering from Hemophilia A has a higher specificity for the relevant tissue
than a virus
vector comprised of only AAV3, AAV6 or AAV7.
Example 6: Use of AAVs of the instant invention to treat a disease
[00551] Treatment of Parkinson's Disease. A male patient of 45 years of age
suffering from
Parkinson's disease is treated with an AAV generated from a cell line, such as
the isolated
HEK293 cell line with ATCC No. PTA 13274 (see e.g., U.S. Patent No.
9,441,206), which
contains a first helper plasmid that has the Rep and Cap genes from AAV2 and a
second
helper plasmid that has the Rep gene from AAV2 and the Cap gene from AAV4 and
a third
plasmid that encodes for the nucleotide sequence for Glutamic Acid
Decarboxylase 65
(GAD65) and/or Glutamic Acid Decarboxylase 67 (GAD67), which nucleotide
sequence is
inserted between two ITRs. The haploid AAV generated from the three plasmids
contains
the nucleotide sequence for GAD65 and/or GAD67 protein to treat Parkinson's
disease. The
AAV is administered to the patient, who shortly after administration shows a
reduction in the
frequency of tremors and an improvement in the patient's balance. Over time
the patient also
sees a reduction in the number and severity of hallucinations and delusions
that the patient
suffered from prior to administration of the AAV.
[00552] Treatment of Batten Disease. A male patient of 8 years of age
suffering from
Batten disease is treated with an AAV generated from a cell line, such as the
isolated
HEK293 cell line with ATCC No. PTA 13274 (see e.g., U.S. Patent No.
9,441,206), which
contains a first helper plasmid that has the Rep and Cap genes from AAV3 and a
second
helper plasmid that has the Rep gene from AAV3 and the Cap gene from AAV5. A
third
plasmid encodes the nucleotide sequence for CLN2 to treat Batten's disease,
wherein the
CLN 2 gene has been inserted between two ITRs. The haploid AAV generated from
the
three plasmids contains the nucleotide sequence to treat Batten's disease. The
AAV is
administered to the patient, who shortly after administration shows an
increase in mental
acuity. Additionally, the patient sees a reduction in seizures and improvement
in sign and
motor skills that the patient suffered from prior to administration of the
AAV.
[00553] Treatment of Alzheimers Disease. A female patient of 73 years
suffering from
Alzheimer's disease is treated with an AAV generated from a cell line, such as
the isolated
HEK293 cell line with ATCC No. PTA 13274 (see e.g., U.S. Patent No.
9,441,206), which
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contains a first helper plasmid that has the Rep and Cap genes from AAV3; a
second helper
plasmid that has the Rep gene from AAV3 and the Cap gene from AAV4; and, a
third helper
plasmid that has the Rep gene from AAV3 and the Cap gene from AAV5. A fourth
plasmid
encodes the nucleotide sequence for Nerve Growth Factor (NGF) to treat
Alzheimer's
disease, wherein NGF has been inserted between two ITRs. The triploid AAV is
administered to the patient, who shortly after administration shows an
increase in mental
acuity and short-term memory. The patient also is able to better communicate
with others
and begins to function more independently than prior to administration of the
AAV.
[00554] Treatment of Heart Disease. A male patient of 63 years suffering from
heart
disease is treated with an AAV generated from a cell line, such as the
isolated HEK293 cell
line with ATCC No. PTA 13274 (see, e.g., U.S. Patent No. 9,441,206), which
contains either:
(1) a first helper plasmid that has the Rep and Cap genes from AAV2; a second
helper
plasmid that has the Rep gene from AAV2 and the Cap gene from AAV6; and, a
third plasmid encodes the nucleotide sequence for the protein to treat heart
disease
that is contained in a third plasmid and has been inserted between two ITRs;
(2) a first helper plasmid that has the Rep and Cap genes from AAV3; a second
helper plasmid that has the Rep gene from AAV3 and the Cap gene from AAV9;
and, a third plasmid encodes the nucleotide sequence for the protein to treat
heart
disease that is contained in a third plasmid and has been inserted between two
ITRs;
(3) a first helper plasmid that has the Rep and Cap genes from AAV3; a second
helper
plasmid that has the Rep gene from AAV3 and the Cap gene from AAV6; a third
helper plasmid that has the Rep gene from AAV3 and the Cap gene from AAV9;
and, a fourth plasmid contains a nucleotide sequence that encodes a protein to
treat heart disease is contained in a third plasmid and has been inserted
between
two ITRs;
(4) a helper plasmid that has the Rep from AAV2 and VP1 from AAV2, VP2 from
AAV3 and VP3 from AAV9; and, a second plasmid that contains a nucleotide
sequence encoding a protein to treat heart disease inserted between two ITRs;
(5) a helper plasmid that has the Rep from AAV3 and VP1 from AAV3, VP2 from
AAV6 and VP3 from AAV6; and, a second plasmid contains a nucleotide
sequence encoding a protein to treat heart disease inserted between two ITRs;
or,
(6) a helper plasmid that has the Rep from AAV3 and VP1 from AAV3, VP2 from
AAV6 and VP3 from AAV9; and, a second plasmid contains a nucleotide
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sequence encoding a protein to treat heart disease inserted between two ITRs,
wherein
the polyploid AAV is administered to the patient, who shortly after
administration shows a
reduction in the symptoms associated with heart disease and shows a
commensurate
improvement in the patient's heart health.
[00555] Treatment of Cystic Fibrosis. A 19 year old female suffering from
Cystic Fibrosis
is treated with an AAV generated from a cell line, such as the isolated HEK293
cell line with
ATCC No. PTA 13274 (see e.g., U.S. Patent No. 9,441,206), which contains
either:
(1) a first helper plasmid that has the Rep and Cap genes from AAV3; a second
helper
plasmid that has the Rep from AAV3 and the Cap gene from AAV 10; and, a third
plasmid that encodes for the nucleotide sequence for CFTR that is inserted
between two ITRs;
(2) a first helper plasmid that has the Rep and Cap genes from AAV3; a second
helper
plasmid that has the Rep gene from AAV3 and the Cap gene from AAV9; a third
helper plasmid that has the Rep gene from AAV3 and the Cap gene from AAV10;
and a fourth plasmid that encodes a nucleotide sequence for CFTR that has been
inserted between two ITRs;
(3) a helper plasmid that has the Rep from AAV2 and VP1 from AAV2, VP2 from
AAV9 and VP3 from AAV9; and a second plasmid that encodes the nucleotide
sequence for CFTR inserted between two ITRs;
(4) a helper plasmid that has the Rep from AAV3 and VP1 from AAV2, VP2 from
AAV10 and VP3 from AAVIO; and, a second plasmid that encodes the nucleotide
sequence for CFTR inserted between two ITRs; or,
(7) a helper plasmid that has the Rep from AAV2 and VP1 from AAV2, VP2 from
AAV9 and VP3 from AAV10; and, a second plasmid encodes the nucleotide
sequence for CFTR inserted between two ITRs, wherein
the AAV is administered to the patient, who shortly after administration shows
a slowing in
the increase of damage to the patient's lung; a reduction in the increase in
the loss of lung
function and a reduction in the speed by which the liver is damaged and a
slowdown in the
increase in the severity of liver cirrhosis. The same patient also sees a
reduction in the
severity of the Cystic Fibrosis-related diabetes that the patient had begun to
suffer.
[00556] Treatment of Skeletal Muscle Disease ¨ Amyotrophic Lateral Sclerosis
(ALS).
A male of 33 years of age who is suffering from Amyotrophic Lateral Sclerosis
(ALS) is
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treated with an AAV generated from a cell line, such as the isolated HEK293
cell line with
ATCC No. PTA 13274 (see e.g., U.S. Patent No. 9,441,206), which contains
either:
(1) a first helper plasmid that has the Rep and Cap genes from AAV2; a second
helper
plasmid that has the Rep from AAV2 and the Cap gene from AAV8; and, a third
plasmid that encodes for the nucleotide sequence for superoxide dismutase 1
(SOD1) that is inserted between two ITRs;
(2) a first helper plasmid that has the Rep and Cap genes from AAV3; a second
helper
plasmid that has the Rep from AAV3 and the Cap gene from AAV9; and, a third
plasmid that encodes for the nucleotide sequence for SOD1 that is inserted
between two ITRs;
(3) a first helper plasmid that has the Rep and Cap genes from AAV3; a second
helper
plasmid that has the Rep gene from AAV3 and the Cap gene from AAV8; a third
helper plasmid that has the Rep gene from AAV3 and the Cap gene from AAV9;
and, a fourth plasmid that encodes for the nucleotide sequence for SOD1 that
is
inserted between two ITRs;
(4) a helper plasmid that has the Rep from AAV3 and VP! from AAV3, VP2 from
AAV9 and VP3 from AAV9; and, a second plasmid that encodes for the
nucleotide sequence for SOD1 that is inserted between two ITRs;
(5) a helper plasmid that has the Rep from AAV3 and VP1 from AAV3, VP2 from
AAV8 and VP3 from AAV8; and, a second plasmid encodes for the nucleotide
sequence for SOD1 that is inserted between two ITRs; or,
(6) a helper plasmid that has the Rep from AAV3 and VP! from AAV3, VP2 from
AAV8 and VP3 from AAV; and, a second plasmid encodes for the nucleotide
sequence for SOD1 that is inserted between two ITRs, wherein
the AAV is administered to the patient, who shortly after administration shows
a reduction in
the symptoms associated with ALS, including a slow down or stop in the
progression of
damage to motor neurons in the brain and the spinal cord and the maintenance
of
communication between the brain and the muscles of the patient.
[00557] Treatment of Duchenne Muscular Dystrophy. A male of 5 years of age who
is
suffering from Duchenne Muscular Dystrophy (DMD) is treated with an AAV
generated
from a cell line, such as the isolated HEK293 cell line with ATCC No. PTA
13274, which
contains either:
(1) a first helper plasmid that has the Rep and Cap genes from AAV2; a second
helper
plasmid that has the Rep from AAV2 and the Cap gene from AAV8; and, a third
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plasmid that encodes for the nucleotide sequence for dystrophin that is
inserted
between two ITRs;
(2) a first helper plasmid that has the Rep and Cap genes from AAV3; a second
helper
plasmid that has the Rep from AAV3 and the Cap gene from AAV9; and, a third
plasmid that encodes for the nucleotide sequence for dystrophin that is
inserted
between two ITRs;
(3) a first helper plasmid that has the Rep and Cap genes from AAV3; a second
helper
plasmid that has the Rep gene from AAV3 and the Cap gene from AAV8; a third
helper plasmid that has the Rep gene from AAV3 and the Cap gene from AAV9;
and, a fourth plasmid that encodes for the nucleotide sequence for dystrophin
that
is inserted between two ITRs;
(4) a helper plasmid that has the Rep from AAV3 and VP1 from AAV3, VP2 from
AAV9 and VP3 from AAV9; and, a second plasmid that encodes for the
nucleotide sequence for dystrophin that is inserted between two ITRs;
(5) a helper plasmid that has the Rep from AAV3 and VP1 from AAV3, VP2 from
AAV8 and VP3 from AAV8; and, a second plasmid encodes for the nucleotide
sequence for dystrophin that is inserted between two ITRs; or,
(6) a helper plasmid that has the Rep from AAV3 and VP1 from AAV3, VP2 from
AAV8 and VP3 from AAV; and, a second plasmid encodes for the nucleotide
sequence for dystrophin that is inserted between two ITRs, wherein
the AAV is administered to the patient, who shortly after administration shows
a slowing in
the increase of damage and wasting to the patient's skeletal muscles, as well
a slowing or
stoppage to the damage suffered by heart and lung as a result of Duchene
Muscular
Dystrophy.
[00558] Treatment of Myasthenia Gravis. A female of 33 years of age who is
suffering
from Myasthenia Gravis (MG) is treated with an AAV generated from a cell line,
such as the
isolated HEK293 cell line with ATCC No. PTA 13274, which contains either:
(1) a first helper plasmid that has the Rep and Cap genes from AAV2; a second
helper
plasmid that has the Rep from AAV2 and the Cap gene from AAV8; and, a third
plasmid that encodes the nucleotide sequence for the gene such that the
patient
will no longer suffer from MG that is inserted between two ITRs;
(2) a first helper plasmid that has the Rep and Cap genes from AAV3; a second
helper
plasmid that has the Rep from AAV3 and the Cap gene from AAV9; and, a third
plasmid that encodes for the gene such that the patient will no longer suffer
from
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MG that is inserted between two ITRs;
(3) a first helper plasmid that has the Rep and Cap genes from AAV3; a second
helper
plasmid that has the Rep gene from AAV3 and the Cap gene from AAV8; a third
helper plasmid that has the Rep gene from AAV3 and the Cap gene from AAV9;
and, a fourth plasmid that encodes for the gene such that the patient will no
longer
suffer from MG that is inserted between two ITRs;
(4) a helper plasmid that has the Rep from AAV3 and VP1 from AAV3, VP2 from
AAV9 and VP3 from AAV9; and, a second plasmid that encodes for the gene
such that the patient will no longer suffer from MG that is inserted between
two
ITRs;
(5) a helper plasmid that has the Rep from AAV3 and VP1 from AAV3, VP2 from
AAV8 and VP3 from AAV8; and, a second plasmid encodes for the gene such
that the patient will no longer suffer from MG that is inserted between two
ITRs;
or,
(6) a helper plasmid that has the Rep from AAV3 and VP1 from AAV3, VP2 from
AAV8 and VP3 from AAV; and, a second plasmid encodes for the gene such that
the patient will no longer suffer from MG that is inserted between two ITRs,
wherein
the AAV is administered to the patient, who shortly after administration shows
a slowing in
the increase breakdown in the communication between muscles and the nerves of
the
patient's body, resulting in a slow down or stoppage in the severity in the
loss of muscle
control. The patient's mobility stabilizes and no longer worsens after
administration of the
AAV and the patient's breathing also does not worsen after administration of
the AAV.
[00559] Treatment of Limb Girdle Muscular Dystrophy. A male of 13 years of age
who is
suffering from Limb Girdle Muscular Dystrophy (LGMD) is treated with an AAV
generated
from a cell line, such as the isolated HEK293 cell line with ATCC No. PTA
13274, which
contains either:
(1) a first helper plasmid that has the Rep and Cap genes from AAV2; a second
helper
plasmid that has the Rep from AAV2 and the Cap gene from AAV8; and, a third
plasmid that encodes for the nucleotide sequence for one of the fifteen genes
with
a mutation associated with LGMD, including, but not limited to myotilin,
telethonin, calpain-3, alpha-sarcoglycan and beta-sarcoglycan that is inserted
between two ITRs;
(2) a first helper plasmid that has the Rep and Cap genes from AAV3; a second
helper
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plasmid that has the Rep from AAV3 and the Cap gene from AAV9; and, a third
plasmid that encodes for the nucleotide sequence for one of the fifteen genes
with
a mutation associated with LGMD, including, but not limited to myotilin,
telethonin, calpain-3, alpha-sarcoglycan and beta-sarcoglycan that is inserted
between two ITRs;
(3) a first helper plasmid that has the Rep and Cap genes from AAV3; a second
helper
plasmid that has the Rep gene from AAV3 and the Cap gene from AAV8; a third
helper plasmid that has the Rep gene from AAV3 and the Cap gene from AAV9;
and, a fourth plasmid that encodes for the nucleotide sequence for one of the
fifteen genes with a mutation associated with LGMD, including, but not limited
to
myotilin, telethonin, calpain-3, alpha-sarcoglycan and beta-sarcoglycan that
is
inserted between two ITRs;
(4) a helper plasmid that has the Rep from AAV3 and VP! from AAV3, VP2 from
AAV9 and VP3 from AAV9; and, a second plasmid that encodes for the
nucleotide sequence for one of the fifteen genes with a mutation associated
with
LGMD, including, but not limited to myotilin, telethonin, calpain-3, alpha-
sarcoglycan and beta-sarcoglycan that is inserted between two ITRs;
(5) a helper plasmid that has the Rep from AAV3 and VP1 from AAV3, VP2 from
AAV8 and VP3 from AAV8; and, a second plasmid encodes for the nucleotide
sequence for one of the fifteen genes with a mutation associated with LGMD,
including, but not limited to myotilin, telethonin, calpain-3, alpha-
sarcoglycan and
beta-sarcoglycan that is inserted between two ITRs; or,
(6) a helper plasmid that has the Rep from AAV3 and VP! from AAV3, VP2 from
AAV8 and VP3 from AAV; and, a second plasmid encodes for the nucleotide
sequence for one of the fifteen genes with a mutation associated with LGMD,
including, but not limited to myotilin, telethonin, calpain-3, alpha-
sarcoglycan and
beta-sarcoglycan that is inserted between two ITRs, wherein
one or more of the AAV's, each encoding one of the 15 different genes
associated with
LGMD is administered to the patient, who shortly after administration shows a
slowing or
stoppage in additional muscle wasting and atrophy.
[00560] Treatment of Diseases of the Liver ¨ Hemophilia B. A male of 9 years
of age who
is suffering from a Hemophilia B resulting from a deficiency of Factor IX
(FIX) is treated
with an AAV generated from a cell line, such as the isolated HEK293 cell line
with ATCC
No. PTA 13274, which contains either:
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(1) a first helper plasmid that has the Rep and Cap genes from AAV2; a second
helper
plasmid that has the Rep from AAV2 and the Cap gene from AAV6; and, a third
plasmid that encodes for the nucleotide sequence for FIX to treat Hemophilia B
that is inserted between two ITRs;
(2) a first helper plasmid that has the Rep and Cap genes from AAV2; a second
helper
plasmid that has the Rep from AAV3 and the Cap gene from AAV7; and a third
plasmid that encodes for the nucleotide sequence for FIX to treat Hemophilia B
that is inserted between two ITRs;
(3) a first helper plasmid that has the Rep and Cap genes from AAV3; a second
helper
plasmid that has the Rep gene from AAV3 and the Cap gene from AAV6; a third
helper plasmid that has the Rep gene from AAV3 and the Cap gene from AAV7;
and a fourth plasmid that encodes for the nucleotide sequence for FIX that is
inserted between two ITRs;
(4) a helper plasmid that has the Rep from AAV2 and VP1 from AAV2, VP2 from
AAV6 and VP3 from AAV6; and a second plasmid that encodes for the
nucleotide sequence for FIX that is inserted between two ITRs;
(5) a helper plasmid that has the Rep from AAV2 and VP! from AAV3, VP2 from
AAV7 and VP3 from AAV7; and a second plasmid that encodes for the
nucleotide sequence for FIX that is inserted between two ITRs; or,
(6) a helper plasmid that has the Rep from AAV2 and VP! from AAV3, VP2 from
AAV6 and VP3 from AAV7' and a second plasmid encodes for the nucleotide
sequence for FIX that is inserted between two ITRs, wherein
the AAV is administered to the patient, who shortly after administration shows
a reduction in
the severity of the Hemophilia B, including a reduction in bleeding episodes.
[00561] Treatment of Hemophilia A. A male of 8 years of age who is suffering
from a
Hemophilia A resulting from a deficiency of Factor VIII (FVIII) is treated
with an AAV
generated from a cell line, such as the isolated HEK293 cell line with ATCC
No. PTA 13274,
which contains either:
(1) a first helper plasmid that has the Rep and Cap genes from AAV2; a second
helper
plasmid that has the Rep from AAV2 and the Cap gene from AAV6; and, a third
plasmid that encodes for the nucleotide sequence for FVIII that is inserted
between two ITRs;
(2) a first helper plasmid that has the Rep and Cap genes from AAV2; a second
helper
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plasmid that has the Rep from AAV3 and the Cap gene from AAV7; and a third
plasmid that encodes for the nucleotide sequence for FVIII that is inserted
between two ITRs;
(3) a first helper plasmid that has the Rep and Cap genes from AAV3; a second
helper
plasmid that has the Rep gene from AAV3 and the Cap gene from AAV6; a third
helper plasmid that has the Rep gene from AAV3 and the Cap gene from AAV7;
and a fourth plasmid that encodes for the nucleotide sequence for FVIII that
is
inserted between two ITRs;
(4) a helper plasmid that has the Rep from AAV2 and VP! from AAV2, VP2 from
AAV6 and VP3 from AAV6; and a second plasmid that encodes for the
nucleotide sequence for FVIII that is inserted between two ITRs;
(5) a helper plasmid that has the Rep from AAV2 and VP! from AAV3, VP2 from
AAV7 and VP3 from AAV7; and a second plasmid that encodes for the
nucleotide sequence for FVIII that is inserted between two ITRs; or,
(6) a helper plasmid that has the Rep from AAV2 and VP1 from AAV3, VP2 from
AAV6 and VP3 from AAV7' and a second plasmid encodes for the nucleotide
sequence for FVIII that is inserted between two ITRs, wherein
the AAV is administered to the patient, who shortly after administration shows
a reduction in
the severity of the Hemophilia A, including a reduction in bleeding episodes.
Example 7. Creation of haploid capsids from two different serotypes and
mutation of start
codons.
[00562] In this example, polyploid AAV virions are assembled from capsids of
two different
serotypes. The nucleotide sequence for VP1, VP2 and VP3 from a first AAV
serotype only
are ligated into a helper plasmid and the nucleotide sequence for VP!, VP2 and
VP3 from a
second AAV serotype only is ligated into the same or different helper plasmid,
such that the
helper plasmid/s include/s the nucleic acid sequences for VP1, VP2 and VP3
capsid proteins
from two different serotypes. Either prior to ligation, or following ligation
of the first and
second serotype nucleotide sequences coding for VP!, VP2 and VP3 capsid
proteins into the
helper plasmid, the capsid nucleotide sequences are altered to provide a VP1
from a first
serotype only and a VP2 and VP3 from a second serotype only. In this example,
the VP1
nucleotide sequence of the first serotype has been altered by mutating the
start codons for
VP2 and VP3 capsid proteins as shown in Figure 7. In this example, the ACG
start site of
VP2 and the three ATG start sites of VP3 are mutated such that these codons
cannot initiate
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the translation of the RNA transcribed from the nucleotide sequence of the VP2
and VP3
capsid proteins from the first serotype. Similarly, as shown in Figure 8, the
ATG start site of
VP1 is mutated in the nucleotide sequence coding for the capsid proteins of
the second
serotype such that this codon cannot initiate the translation of the RNA
coding for VP1, but
translation can be initiated for both VP2 and VP3. Thus, in this example, a
polypoid AAV
virion is created that includes a VP1, but not VP2 or VP3 from a first
serotype only and a
VP2 and VP3, but not a VP! from a second serotype only.
[00563] In applying this technique of creating a polyploid AAV virion through
mutation of
start codons, the start codons of VP2 and VP3 of AAV2 were mutated as shown
with
highlights in Figure 19, such that only VP1 is translated from an RNA
transcribed from the
plasmid set forth in Figure 19. In the further application of this technique,
the start codon of
VP1 of AAV2 were mutated as shown with highlights in Figure 18 such that VP2
and VP3,
but not VP1 is translated from an RNA transcribed from the plasmid set forth
in Figure 19.
Thus, mutation of the start codons provides a method of knocking out the
expression of one
or more of VP1, VP2 and VP3.
Example 8. Creation of haploid capsids from two different serotypes and
mutation of start
codons.
[00564] In this example, polyploid AAV virions are assembled from capsids of
two different
serotypes. The nucleotide sequence for VP1, VP2 and VP3 from a first AAV
serotype only
are ligated into a helper plasmid and the VP1, VP2 and VP3 from a second AAV
serotype
only is ligated into the same or different helper plasmid, such that the
helper plasmid/s
include the VP1, VP2 and VP3 capsid proteins from two different serotypes.
Either prior to
ligation or following ligation of the first and second serotype nucleotide
sequences coding for
VP1, VP2 and VP3 capsid proteins into the helper plasmid, the capsid
nucleotide sequences
are altered to provide a VP1 and VP3 from a first serotype only and a VP2 from
a second
serotype only. In this example, the ACG start site of VP2 is mutated such that
this codon
cannot initiate the translation of the RNA transcribed from the nucleotide
sequence of the
VP2 capsid protein from the first serotype. Similarly, the ATG start site of
VP1 and VP3 is
mutated in the nucleotide sequence coding for the capsid proteins of the
second serotype such
that these codons cannot initiate the translation of the RNA coding for VP1
and VP3, but
translation can be initiated for both VP2. Thus, in this example, a polypoid
AAV virion is
created that includes VP1 and VP3, but not VP2 from a first serotype only and
a VP2, but not
VP1 and VP3 from a second serotype only.
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[00565] In applying this technique of creating a polyploid AAV virion through
mutation of
start codons, the start codon of VP2 of AAV2 were mutated as shown with
highlights in
Figure 20, such that VP1 and VP3 are translated from an RNA transcribed from
the plasmid
set forth in Figure 20. Thus, mutation of the start codons provides a method
of knocking out
the expression of one or more of VP1, VP2 and VP3.
Example 9. Creation of haploid capsids from two different serotypes and
mutation of splice
acceptor sites.
[00566] In this example, polyploid AAV virions are assembled from capsids of
two different
serotypes. The nucleotide sequence for VP1, VP2 and VP3 from a first AAV
serotype only
are ligated into a helper plasmid and the VP1, VP2 and VP3 from a second AAV
serotype
only is ligated into the same or different helper plasmid, such that the
helper plasmid/s
include the VP1, VP2 and VP3 capsid proteins from two different serotypes.
Either prior to
ligation or following ligation of the first and second serotype nucleotide
sequences coding for
VP1, VP2 and VP3 capsid proteins into the helper plasmid/s, the capsid
nucleotide sequences
are altered to provide a VP1 from a first serotype only and a VP2 and VP3 from
a second
serotype only. In this example, the nucleotide sequence of the first serotype
has been altered
by mutating the A2 Splice Acceptor Site as shown in Figure 9. In this example,
by mutating
the A2 Splice Acceptor Site, the VP2 and VP3 capsid proteins from the first
serotype are not
produced. Similarly, as shown in Figure 10, by mutating the Al Splice Acceptor
Site, the
VP1 capsid protein from the second serotype is not produced, while VP2 and VP3
capsid
proteins are produced. Thus, in this example, a polypoid AAV virion is created
that includes
a VP1, but not VP2 or VP3 from a first serotype only and a VP2 and VP3, but
not a VPI
from a second serotype only.
Example 10. Creation of haploid capsids from two different serotypes and
mutation of start
codons and splice acceptor sites.
[00567] In this example, polyploid AAV virions are assembled from capsids of
two different
serotypes. The nucleotide sequence for VP1, VP2 and VP3 from a first AAV
serotype only
are ligated into a helper plasmid and the VP1, VP2 and VP3 from a second AAV
serotype
only are ligated into a same or different plasmid, such that the helper
plasmid/s include/s the
VP1, VP2 and VP3 capsid proteins from two different serotypes. Either prior to
ligation or
following ligation of the first and second serotype nucleotide sequences
coding for VP1, VP2
and VP3 capsid proteins into the helper plasmid, the capsid nucleotide
sequences are altered
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to provide a VP1 from a first serotype only and a VP2 and VP3 from a second
serotype only.
In this example, the nucleotide sequence of the first serotype has been
altered by mutating the
start codons for the VP2 and VP3 capsid proteins and mutating the A2 Splice
Acceptor Site
as shown in Figure 11. In this example, the ACG start site of VP2 and the
three ATG start
sites of VP3 along with the A2 Splice Acceptor Site are mutated. As a result,
only the VPI
capsid protein of the first serotype is produced. Neither the VP2 or VP3
capsid proteins from
the first serotype are produced. Similarly, as shown in Figure 12, the ATG
start site of VP1
is mutated along with the Al Splice Acceptor Site. As a result, only the VP2
and VP3 capsid
proteins of the second serotype are produced. VP1 capsid protein form the
second serotype is
not produced. Thus, in this example, a polypoid AAV virion is created that
includes VP!, but
not VP2 or VP3 from a first serotype only and VP2 and VP3, but not VP1 from a
second
serotype only.
Example 11. Creation of haploid capsids from two different serotypes using two
plasmids.
[00568] In this example, a haploid AAV virion comprising VPI from AAV5 and
VP2/VP3
from AAV9 is created using two plasmids. As shown in Figure 13, a helper
plasmid is
created that includes a plasmid backbone along with Ad Early Genes andRep
(e.g., from
AAV2). This helper plasmid has ligated into it the nucleotide sequence coding
for the capsid
proteins from AAV5 only and a separate nucleotide sequence coding for the
capsid proteins
of AAV9 only. With regard to the nucleotide sequence coding for the capsid
proteins of
AAV5, this nucleotide sequence has had either the start codons for VP2/VP3
mutated to
prevent translation and/or the A2 Splice Acceptor Site has been mutated to
prevent splicing.
With regard to the nucleotide sequence coding for the capsid proteins of AAV9,
this
nucleotide sequence has had either the start codon for VP I mutated to prevent
translation
and/or the Al Splice Acceptor Site has been mutated to prevent splicing. The
helper plasmid,
along with a plasmid encoding the transgene with two ITRs are transfected into
HEK293 cell
line with ATCC No. PTA 13274 (see e.g., U.S. Patent No. 9,441,206). The virus
is purified
from the supernatant and characterized. As shown in Figure 13, the viral
capsid includes
VP2/VP3 of AA9 (shown in light grey) and VP1 of AAV5 (shown in dark grey) as
seen in
the virions set forth at the bottom of Figure 13.
Example 12. Creation of haploid capsids from two different serotypes using
three plasmids.
[00569] In this example, a haploid AAV virion comprising VP1 from AAV5 and
VP2/VP3
from AAV9 is created using three plasmids. As shown in Figure 14, a first
helper plasmid is
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created that includes the Ad Early Genes. A second helper plasmid is created
that includes a
plasmid backbone along with Rep (e.g., AAV2). This second helper plasmid has
ligated into
it the nucleotide sequence coding for the capsid proteins from AAV5 only and a
separate
nucleotide sequence coding for the capsid proteins of AAV9 only. With regard
to the
nucleotide sequence coding for the capsid proteins of AAV5, this nucleotide
sequence has
had either the start codons for VP2/VP3 mutated to prevent translation and/or
the A2 Splice
Acceptor Site has been mutated to prevent splicing. With regard to the
nucleotide sequence
coding for the capsid proteins of AAV9, this nucleotide sequence has had
either the start
codon for VP1 mutated to prevent translation and/or the Al Splice Acceptor
Site has been
mutated to prevent splicing. The helper plasmids, along with a plasmid
encoding the
transgene with two ITRs are transfected into HEK293 cell line with ATCC No.
PTA 13274
(see e.g., U.S. Patent No. 9,441,206). The
virus is purified form the supernatant and
characterized. As shown in Figure 14, the viral capsid includes VP2/VP3 of
AAV9 (shown
in light grey) and VP1 of AAV5 (shown in dark grey) as seen in the virions set
forth at the
bottom of Figure 13.
Example 13. Creation of haploid capsids from two different serotypes using
four plasmids.
[00570] In this example, a haploid AAV virion comprising VP1 from AAV5 and
VP2/VP3
from AAV9 is created using four plasmids. As shown in Figure 15, a first
helper plasmid is
created that includes the Ad Early Genes. A second helper plasmid is created
that includes a
plasmid backbone along with Rep (e.g., AAV2). This second helper plasmid has
ligated into
it the nucleotide sequence coding for the capsid proteins from AAV5 only. A
third helper
plasmid is created that includes a plasmid backbone along with the Rep. This
third helper
plasmid has ligated into it the nucleotide sequence coding for the capsid
proteins of AAV9
only. A fourth plasmid includes the transgene and two ITRs. With regard to the
nucleotide
sequence coding for the capsid proteins of AAV5, this nucleotide sequence has
had either the
start codons for VP2/VP3 mutated to prevent translation and/or the A2 Splice
Acceptor Site
has been mutated to prevent splicing. With regard to the nucleotide sequence
coding for the
capsid proteins of AAV9, this nucleotide sequence has had either the start
codon for VP1
mutated to prevent translation and/or the Al Splice Acceptor Site has been
mutated to
prevent splicing. The helper plasmids, along with a plasmid encoding the
transgene with two
ITRs are transfected into HEK293 cell line with ATCC No. PTA 13274 (see e.g.,
U.S. Patent
No. 9,441,206). The virus is purified form the supernatant and characterized.
As shown in
Figure 14, the viral capsid includes VP2/VP3 of AA9 (shown in light grey) and
VP1 of
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AAV5 (shown in dark grey) as seen in the virions set forth at the bottom of
Figure 13.
Example 14. Creation of haploid capsids from three different serotypes and
mutation of start
codons.
[00571] In this example, polyploid AAV virions are assembled from capsids of
three
different serotypes. A helper plasmid is constructed so that the nucleotide
sequence for VP1,
VP2 and VP3 from a first AAV serotype only, the VP1, VP2 and VP3 from a second
AAV
serotype only and the VP1, VP2 and VP3 from a third AAV serotype only are
ligated into a
helper plasmid/s, such that the helper plasmid/s include/s the nucleic acid
sequences for VP1,
VP2 and VP3 capsid proteins from three different serotypes. Either prior to
ligation or
following ligation of the nucleotide sequences coding for VP1, VP2 and VP3
capsid proteins
from each of the three different serotypes into the helper plasmid, the capsid
nucleotide
sequences are altered to provide VP1 from the first serotype only, VP2 from
the second
serotype only and VP3 from the third serotype only. In this example, the VP1
nucleotide
sequence of the first serotype has been altered by mutating the start codons
for the VP2 and
VP3 capsid proteins. In this example, the ACG start codon of VP2 and the three
ATG start
codons of VP3 are mutated such that these codons cannot initiate the
translation of the RNA
transcribed from the nucleotide sequence of the VP2 and VP3 capsid proteins
from the first
serotype. Similarly, the VP1 and VP3 nucleotide sequence of the second
serotype have been
altered by mutating the start codons for the VP1 and VP3 capsid proteins. In
this example,
the ATG start site of VP1 and the three ATG start codons of VP3 are mutated
such that these
codons cannot initiate the translation of the RNA transcribed from the
nucleotide sequence of
the VP1 and VP3 capsid proteins. Further, the VP1 and VP2 nucleotide sequence
of the third
serotype have been altered by mutating the start codons for the VP1 and VP2
capsid proteins.
In this example, the ATG start codon of VP1 and the ACG start codon of VP2 are
mutated
such that these codons cannot initiate the translation of the RNA transcribed
from the
nucleotide sequence of the VP1 and VP2 capsid proteins. Thus, in this example,
a polypoid
AAV virion is created that includes a VP1, but not VP2, nor VP3 from a first
serotype only; a
VP2, but not a VP1, nor VP2 from a second serotype only; and, VP3, but not
VP1, nor VP2
from a third serotype only.
Example 15. Creation of haploid capsids from two different serotypes using DNA
shuffling
[00572] In this experiment, polyploid AAV virions are created from AAV capsid
proteins
from one AAV serotype only and from a nucleic acid created from DNA shuffling
of three
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different AAV serotypes. In this example, the nucleotide capsid protein
sequences for
AAV!, AAV2 and AAV8 are subjected to treatment with one or more restriction
enzymes
and/or DNase and the DNA is cleaved into DNA fragments of 50 ¨ 100 bp in
length. The
mixture of DNA fragments is then subject to polymerase chain reaction (PCR)
without
primers. The PCR is repeated multiple times or until the DNA molecules created
by PCR
reach the size of the nucleic acid coding for the capsid genes. At this point,
another round of
PCR is conducted wherein primers are added that include sequences for
restriction enzyme
recognition sites to allow for ligation of the newly created DNA into a helper
plasmid. Prior
to ligation into a helper plasmid, the AAV!/2/8 nucleotide sequence is
sequenced and any
start codons within the nucleotide sequence that could start translation of
VP2 and VP3
capsid proteins from an RNA transcribed from this sequence are mutated to
prevent
translation. In this manner, the AAV1/2/8 can only produce VP1 and the
AAV1/2/8
nucleotide sequence is ligated into a helper plasmid. In this experiment, the
nucleotide
sequence coding for the capsid proteins (VP1, VP2 and VP3) of AAV9 is also
ligated into the
same of different helper plasmid. To create the polyploid AAV virion with VP1
from the
AAV1/2/8 nucleotide sequence created by DNA shuffling and VP2 and VP3 from
AAV9
only, the ATG start codon of VP1 of AAV9 is mutated such that an RNA encoding
VP1
cannot be translated. Thus, in this example, a polypoid AAV virion is created
that includes
VP1, but not VP2 or VP3 from a nucleotide sequence created by DNA shuffling
the capsid
protein nucleotide sequences of AAV1/2/8 and VP2 and VP3, but not VP1 from
AAV9 only.
[00573] An example of DNA shuffling is set forth in Figure 16, that starts
with the nucleic
acid coding for VP1, VP2 and VP3 from eight AAV serotypes and processes the
nucleic acid,
first through DNase I fragmentation, which is followed by assembly and
amplification of the
various fragments of the nucleic acid from eight AAVs. The DNA shuffled
nucleic acids that
are generated encode for the AAV capsid proteins, which are then expressed to
create a
library of capsids. These capsids are then tested on animals to screen for
those capsids that
show specific tissue tropism and/or reduced immunogenicity and those that show
promise are
selected for further development (Figure 16).
Example 16. Liver transduction of haploid vector H-AAV829.
[00574] An experiment was conducted with three AAVs. In Figure 22 A. the
composition
of AAV capsid subunits is shown. A hybrid AAV is shown that combines the VP1
only
amino acids from AAV8 with those coding for VP2 and VP3 from AAV2 (AAV82). Two
haploid AAV viruses were produced from co-transfection of two plasmids (one
encoding
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VP1 and VP2, another one for VP3) into HEK293 cells. The three AAVs, AAV82,
28m-
2vp3 and H-AAV82, along with an AAV2 parental control were injected in C57BL6
mice via
the retro-orbital vein at a dose of 3x1019 particles (Figure 22B). The imaging
was performed
one week later (Figure 22B). Liver transduction was quantitated based on data
that
represented the average of 5 mice and standard deviations (Figure 22C).
Example 17. Muscle transduction of haploid vector H-AAV82.
[00575] The three AAVs from Example 23 (AAV82, H-AAV82 and 28m-vp3) were next
injected into mouse hind leg muscle at a dose of 1x109 particles of AAV/luc.
At week 3 post
injection, imaging was conducted for a period of 3 minutes as seen in Figure
23A. The
imaging was conducted face up: left leg- AAV82, H-AAV82 or 28m-vp3 and right
leg-
AAV2 parental AAV. Figure 23B provides the data from 4 mice after the muscular
injection
with the fold increase of transduction calculated by transduction from AAV82,
H-AAV82 or
28m-vp3 to the parental AAV2.
Example 18. Liver transduction of haploid vector H-AAV92.
[00576] In this experiment a haploid AAV92 is created wherein the VP1 and VP2
are from
AAV9 only and the VP3 is from AAV3 only (Figure 24A). The H-AAV92 was produced
from co-transfection of two plasmids (one encoding AAV9 VP1 and VP2, another
one for
AAV2 VP3) into HEK293 cells. H-AAV92 and parental AAV2 were injected into
C57BL6
mice via the retro-orbital vein at a dose of 3x101 particles (Figure 24B).
Imaging was
performed one week later (Figure 24B). Liver transduction was quantitated
based on data
that represented the average of 5 mice and standard deviations (Figure 24C).
Example 19. Liver transduction of haploid vector H-AAV82G9.
[00577] In this experiment a haploid AAV82G9 is created wherein the VP1 and
VP2 are
from AAV8 only and the VP3 is from AAV2G9 only (Figure 25A). The H-AAV82G9 was
produced from co-transfection of two plasmids (one encoding AAV8 VP1 and VP2,
another
one for AAV2G9 VP3) into HEK293 cells. H-AAV82G9 and AAV2G9 were injected into
C57BL6 mice via the retro-orbital vein at a dose of 3x101 particles (Figure
25B). Imaging
was performed one week later (Figure 25 B). Liver transduction was quantitated
based on
data that represented the average of 5 mice and standard deviations (Figure
25C).
[00578] In closing, it is to be understood that although aspects of the
present specification
are highlighted by referring to specific embodiments, one skilled in the art
will readily
appreciate that these disclosed embodiments are only illustrative of the
principles of the
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subject matter disclosed herein. Therefore, it should be understood that the
disclosed subject
matter is in no way limited to a particular methodology, protocol, and/or
reagent, etc.,
described herein. As such, various modifications or changes to or alternative
configurations
of the disclosed subject matter can be made in accordance with the teachings
herein without
departing from the spirit of the present specification. Lastly, the
terminology used herein is
for the purpose of describing particular embodiments only, and is not intended
to limit the
scope of the present invention, which is defined solely by the claims.
Accordingly, the
present invention is not limited to that precisely as shown and described.
[00579] Certain embodiments of the present invention are described herein,
including the
best mode known to the inventors for carrying out the invention. Of course,
variations on
these described embodiments will become apparent to those of ordinary skill in
the art upon
reading the foregoing description. The inventor expects skilled artisans to
employ such
variations as appropriate, and the inventors intend for the present invention
to be practiced
otherwise than specifically described herein. Accordingly, this invention
includes all
modifications and equivalents of the subject matter recited in the claims
appended hereto as
permitted by applicable law. Moreover, any combination of the above-described
embodiments in all possible variations thereof is encompassed by the invention
unless
otherwise indicated herein or otherwise clearly contradicted by context.
[00580] Groupings of alternative embodiments, elements, or steps of the
present invention
are not to be construed as limitations. Each group member may be referred to
and claimed
individually or in any combination with other group members disclosed herein.
It is
anticipated that one or more members of a group may be included in, or deleted
from, a group
for reasons of convenience and/or patentability. When any such inclusion or
deletion occurs,
the specification is deemed to contain the group as modified thus fulfilling
the written
description of all Markush groups used in the appended claims.
[00581] Unless otherwise indicated, all numbers expressing a characteristic,
item, quantity,
parameter, property, term, and so forth used in the present specification and
claims are to be
understood as being modified in all instances by the term "about." As used
herein, the term
"about" means that the characteristic, item, quantity, parameter, property, or
term so qualified
encompasses a range of plus or minus ten percent above and below the value of
the stated
characteristic, item, quantity, parameter, property, or term. Accordingly,
unless indicated to
the contrary, the numerical parameters set forth in the specification and
attached claims are
approximations that may vary. At the very least, and not as an attempt to
limit the application
of the doctrine of equivalents to the scope of the claims, each numerical
indication should at
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least be construed in light of the number of reported significant digits and
by applying
ordinary rounding techniques. Notwithstanding that the numerical ranges and
values setting
forth the broad scope of the invention are approximations, the numerical
ranges and values
set forth in the specific examples are reported as precisely as possible. Any
numerical range
or value, however, inherently contains certain errors necessarily resulting
from the standard
deviation found in their respective testing measurements. Recitation of
numerical ranges of
values herein is merely intended to serve as a shorthand method of referring
individually to
each separate numerical value falling within the range. Unless otherwise
indicated herein,
each individual value of a numerical range is incorporated into the present
specification as if
it were individually recited herein.
[00582] All methods described herein can be performed in any suitable order
unless
otherwise indicated herein or otherwise clearly contradicted by context. The
use of any and
all examples, or exemplary language (e.g., "such as") provided herein is
intended merely to
better illuminate the present invention and does not pose a limitation on the
scope of the
invention otherwise claimed. No language in the present specification should
be construed as
indicating any non-claimed element essential to the practice of the invention.
[00583] Specific embodiments disclosed herein may be further limited in the
claims using
consisting of or consisting essentially of language. When used in the claims,
whether as filed
or added per amendment, the transition term "consisting of' excludes any
element, step, or
ingredient not specified in the claims. The transition term "consisting
essentially of' limits
the scope of a claim to the specified materials or steps and those that do not
materially affect
the basic and novel characteristic(s). Embodiments of the present invention so
claimed are
inherently or expressly described and enabled herein.
[00584] All patents, patent publications, and other publications referenced
and identified in
the present specification are individually and expressly incorporated herein
by reference in
their entirety for the purpose of describing and disclosing, for example, the
compositions and
methodologies described in such publications that might be used in connection
with the
present invention. These publications are provided solely for their disclosure
prior to the
filing date of the present application. Nothing in this regard should be
construed as an
admission that the inventors are not entitled to antedate such disclosure by
virtue of prior
invention or for any other reason. All statements as to the date or
representation as to the
contents of these documents is based on the information available to the
applicants and does
not constitute any admission as to the correctness of the dates or contents of
these documents.
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Example 20. Chimeric capsid proteins and AAV haploid virus vector
transduction.
[00585] As explained above, a series of constructs for AAV helper plasmids
were made with
mutants in start codes of capsid ORFs, in which only one or two viral VP
proteins would be
expressed. Chimeric AAV helper constructs in which VP1/2 protein was driven
from two
different serotypes (AAV2 and AAV8) were also made. These constructs were used
to
produce a bunch of haploid virus vectors and evaluate their transduction
efficacy in mice. It
was found that enhanced transduction was achieved from haploid vectors with
VP1/VP2 from
serotypes 7, 8, 9, and rh10, and VP3 from AAV2 or AAV3 when compared to AAV2-
only
and AAV3-only vectors. It was further shown that AAV vectors made from the
chimeric
VP1/VP2 capsid with N-terminus from AAV2 and C-terminus from AAV8 and VP3 from
AAV2 induced much higher transduction. The data provided herein show a simple
and
effective method that enhances AAV transduction for further application of AAV
vectors.
[00586] Haploid vector with VP1/VP2 from other serotypes and VP3 from AAV2
enhance
AAV liver transduction.
[00587] The haploid virus was produced by co-transfecting the plasmids
expressed AAV8
VP1/2 and AAV2 VP3 at the ratio of 1:1. The results showed that haploid vector
AAV82
with VP1/VP2 from AAV8 and VP3 from AAV2 increased the liver transduction
(Figure
22B and 22C).
[00588] A haploid AAV92 vector (1-1-AAV92) was produced using VP1/VP2 of AAV9
and
VP3 of AAV2 (Figure 24A). After systemic administration, the imaging was
performed at
week I. About 4-fold higher liver transduction was achieved with H-AAV92 than
that with
AAV2 (Figure 24B and 24C). This data indicates that VP11VP2 from other
serotype is able to
increase AAV2 transduction.
[00589] Enhanced AAV liver transduction from haploid vector with VP3 from AAV2
mutant.
[00590] AAV9 vectors use glycan as primary receptor for their effective
transduction. In
previous studies, AAV9 glycan receptor binding site were engrafted into the
AAV2 capsid to
make AAV2G9 vector and it was found that AAV2G9 has higher liver tropism than
AAV2.
Described herein is a haploid vector (H-AAV82G9) in which VP1/VP2 from AAV8
and VP3
from AAV2G9 (Figure 25A). After systemic injection into mice, compared to
AAV2G9,
more than 10 fold higher liver transduction was observed at both week 1 and
week 2 post H-
AAV82G9 application (Figure 25B and 25C). This data indicates that the
integration of
VP1/VP2 from other serotype into AAV2 mutant VP3was able to increase liver
transduction.
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[00591] Enhanced AAV liver transduction from haploid vector with VP3 from
AAV3.
[00592] Haploid vectors in which VP3 is from other serotypes and VP1/VP2 from
different
serotypes or variants where the start codes were mutated and the VP proteins
constructs were
made to express AAV3 VP3 only or AAV rh10 VP1/VP2 only. The different haploid
H-
AAV83 (VP1/VP2 from AAV8 and VP3 from AAV3), H-AAV93 (VP1/VP2 from AAV9
and VP3 from AAV3) and H-AAVrh10-3 (VP1/VP2 from AAV rh10 and VP3 from AAV3)
vectors were produced (Figure 26A) and injected into mice via systemic
administration. The
imaging was carried out at week 1. As shown in Figure 26B and 26C, higher
liver
transduction was achieved with haploid vectors (H-AAV83, H-AAV93 and H-AAVrh10-
3)
than that with AAV3. This is consistent to the results obtained from other
haploid vectors.
Furthermore, these haploid vectors also enhanced the transduction from other
tissues as
shown in Figure 26B and 26D. Interestingly, these haploid vectors also induced
a whole body
transduction based on imaging profile, which is different from the results
from haploid
vectors with VP3 from AAV2, which only transduced the liver efficiently
(Figures 22 and
24). Collectively, haploid vectors with VP1/VP2 from one serotype and VP3 from
an
alternative one were able to enhance transduction and perhaps change their
tropism.
[00593] Haploid vector with C-terminus of VP1/VP2 from AAV8 and VP3 from AAV2
enhances AAV transduction.
[00594] A series of constructs which expressed AAV8 VP1/VP2 only, AAV2 VP3
only,
chimeric VP1/VP2 (28m-2VP3) with N-terminal from AAV2 and C-terminal from
AAV8, or
chimeric AAV8/2 with N-terminal from AAV8 and C-terminal from AAV2 without
mutation
of VP3 start codon were generated (Figure 27A). These plasmids were used to
produce
haploid AAV vector with different combination at a plasmid ratio of 1:1
(Figure 27B). After
injection of lx101 particles of these haploid vectors in mice via retro-
orbital vein, the liver
transduction efficiency was evaluated (Figure 27C). Chimeric AAV82 vector
(AAV82)
induced a little higher liver transduction than AAV2. However, haploid AAV82
(H-AAV82)
had much higher liver transduction than AAV2. A further increase in liver
transduction with
haploid vector 28m-2vp3 was observed. These haploid vectors were administered
into the
muscles of mice. For easy comparison, the right leg was injected with AAV2
vector and the
left leg was injected with haploid vector when the mouse was face up. At week
3 after AAV
injection, the images were taken. Consistent to observation in the liver, all
haploid vectors
and chimeric vectors had higher muscular transduction with the best from
haploid vector
28m-2vp3 (Figure 27D). This result indicates that the chimeric VP1/VP2 with N-
terminal
from AAV2 and C-terminal from AAV8 attributes to high liver transduction of
haploid
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AAV82 vectors.
[00595] Increased virion trafficking to the nucleus from chimeric haploid
vectors.
[00596] AAV transduction involves many steps. Upon binding, AAV virions are
taken up into
the endosome via endocytosis. After escape from the endosomes, AAV virions
travel to the
nucleus for transgene expression. It was determined which steps result in the
high transduction
from the haploid vectors. First, AAV vector binding assay was performed and
less 28m-2VP3
virions was found bound to Huh7 cells than other vectors (Figure 28). Next,
the AAV genome
copy number was detected in the nucleus and no difference was found between
different AAV
vectors. It is interesting to note, when compared the AAV genome copy number
to bound
virion, more AAV virions were observed in the nucleus (Figure 28). These
results indicate that
AAV vector 28m-2VP3 is more efficient for trafficking.
[00597] High transduction of haploid AAV vector does not result from virion
stability.
[00598] The following experiments were performed by heating the virus virions.
The viruses
were heated at different temperature for half hour and then applied for
western blot using the
primary antibodies A20 ADK8 or B1 to recognize intact or un-intact virions. As
shown in
Figure 29, when viruses were heated at 70 C, all virus virions fell apart.
There was no
different for stability against heating between AAV haploid vectors regardless
of different
temperature except for AAV82 vectors. This data indicates that the enhanced
transduction
may not related to haploid virion stability.
[00599] The effect of acidic condition on VP1 N-terminus exposure of haploid
vector.
[00600] It has been demonstrated that VP1NP2 N-terminus is exposed on virion
surface in
the acidic endosome after endocytosis of AAV vectors. VP1NP2 terminus contains
the
phosophlipase A2 and NLS domains for AAV vector which help AAV viruses escape
from
the endosome and travel to the nucleus. AAV haploid vectors were incubated
with PBS at
different pH values for 30 minutes, then applied to Western blot analysis to
detect N-terminus
of VP1 using antibody Al. The result showed that no any VP1 N-terminus was
exposed when
virus was treated with different pH (Figure 30).
[00601] The data presented herein show that enhanced transduction could be
achieved from
haploid vectors with VP1NP2 from one AAV vector capsid and VP3 from an
alternative
one.
[00602] Plasmids and site-directed mutagenesis. All of the plasmids that were
used to
express VP12 and VP3 were made by site-directed mutagenesis. Mutagenesis was
performed
using QuikChange II XL Site-Directed mutagenesis Kit (Agilent) according to
the
manufacturer's manual. The fragment that contained the N-terminus (1201 aa) of
AAV2
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capsid and C-terminus of AAV8 capsid was generated by overlapping PCR. Then,
the
fragment was cloned into the SwaI and NotI sites of pXR. All of the mutations
and constructs
were verified by DNA sequencing.
[00603] Virus production. Recombinant AAV was produced by a triple-plasmid
transfection
system. A 15-cm dish of HEI(293 cells was transfected with 9 ug of AAV
transgene plasmid
pTR/CBA-Luc, 12ug of AAV helper plasmid containing AAV Rep and Cap genes, and
15ug of
Ad helper plasmid pXX6-80. Sixty hours post-transfection, HEK293 cells were
collected and
lysed. Supernatant was subjected to CsCl gradient ultra-centrifugation. Virus
titer was
determined by quantitative PCR.
[00604] In vitro transduction assay. Huh7 and C2C12 cells were transduced by
recombinant viruses with 1 x 104 vg/cell in a flat-bottom, 24-well plate.
Forty-eight hours
later, cells were harvested and evaluated by a luciferase assay system
(Promega, Madison,
WI).
[00605] Animal study. Animal experiments performed in this study were
conducted with
C57BL/6 mice and FIX-/- mice. The mice were maintained in accordance to NIH
guidelines, as
approved by the UNC Institutional Animal Care and Use Committee (IACUC). Six-
week-old
female C57BL/6 mice were injected with 1 x 1010 vg of recombinant viruses via
retro-orbital
injection. Luciferase expression was imaged 1 week post-injection using a
Xenogen IVIS
Lumina (Caliper Lifesciences, Waltham, MA) following i.p. injection of D-
luciferin substrate
(Nanolight Pinetop, AZ). Bioluminescent images were analyzed using Living
Image
(PerkinElmer, Waltham, MA). For muscle transduction, 5 x 109 particles of
AAV/Luc were
injected into the gastrocnemius of 6-week-old C57BL/6 females. Mice were
imaged at the
indicated time points.
[00606] Detection of AAV genome copy number in the liver. The minced livers
were
treated with Protease K and total genomic DNA was isolated by the Pure Link
Genomic
DNA mini Kit (Invitrogen, Carlsbad, CA). The luciferase gene was detected by
qPCR assay.
The mouse lamin gene served as an internal control.
[00607] Statistical analysis. The data were presented as mean SD. The
Student t test was
used to carry out all statistical analyses. P values of < 0.05 were considered
a statistically
significant difference.
[00608] References
1. Srivastava A, Lusby EW, Berns KI. 1983. Nucleotide sequence and
organization of
the adeno-associated virus 2 genome. Journal of Virology 45:555-564.
161
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2. Srivastava A. 2016. In vivo tissue-tropism of adeno-associated viral
vectors. Current
Opinion in Virology 21:75-80.
3. Manno CS, Chew AJ, Hutchison S, Larson PJ, Herzog RW, Arruda VR, Tai SJ,
Ragni
MV, Thompson A, Ozelo M, Couto LB, Leonard DGB, Johnson FA, McClelland A,
Scallan C, Skarsgard E, Flake AW, Kay MA, High KA, Glader B. 2003. AAV-
mediated factor IX gene transfer to skeletal muscle in patients with severe
hemophilia
B. Blood 101:29632972.
4. Lisowski L, Tay SS, Alexander IE. 2015. Adeno-associated virus serotypes
for gene
therapeutics. Current Opinion in Pharmacology 24:59-67.
5. Boye SE, Boye SL, Lewin AS, Hauswirth WW. 2013. A Comprehensive Review of
Retinal Gene Therapy. Mol Ther 21:509-519.
6. Smalley E. 2017. First AAV gene therapy poised for landmark approval.
Nature
Biotechnology 35:998.
7. Nathwani AC, Reiss UM, Tuddenham EGD, Rosales C, Chowdary P, McIntosh J,
Della Peruta M, Lheriteau E, Patel N, Raj D, Riddell A, Pie J, Rangarajan S,
Bevan D,
Recht M, Shen YM, Halka KG, Basner-Tschakarjan E, Mingozzi F, High KA, Allay
J, Kay MA, Ng CYC, Zhou J, Cancio M, Morton CL, Gray JT, Srivastava D,
Nienhuis AW, Davidoff AM. 2014. Long-Term Safety and Efficacy of Factor IX
Gene Therapy in Hemophilia B. The New England journal of medicine 371:1994-
2004.
8. Nathwani AC, Tuddenham EGD, Rangarajan S, Rosales C, McIntosh J, Linch DC,
Chowdary P, Riddell A, Pie AJ, Harrington C, O'Beirne J, Smith K, Pasi J,
Glader B,
Rustagi P, Ng CYC, Kay MA, Zhou J, Spence Y, Morton CL, Allay J, Coleman J,
Sleep S, Cunningham JM, Srivastava D, Basner-Tschakarjan E, Mingozzi F, High
KA, Gray JT, Reiss UM, Nienhuis AW, Davidoff AM. 2011. Adenovirus-Associated
Virus Vector¨Mediated Gene Transfer in Hemophilia B. New England Journal of
Medicine 365:2357-2365.
9. Simioni P, Tormene D, Tognin G, Gavasso S, Bulato C, Iacobelli NP, Finn JD,
Spiezia L, Radu C, Arruda VR. 2009. X-Linked Thrombophilia with a Mutant
Factor
IX (Factor IX Padua). New England Journal of Medicine 361:1671-1675.
10. Saraiva J, Nobre RJ, Pereira de Almeida L. 2016. Gene therapy for the CNS
using
AAVs: The impact of systemic delivery by AAV9. Journal of Controlled Release
241:94-109.
162
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11. Chai Z, Sun J, Rigsbee KM, Wang M, Samulski RJ, Li C. 2017. Application of
polyploid adeno-associated virus vectors for transduction enhancement and
neutralizing antibody evasion. Journal of Controlled Release 262:348-356.
163
CA 3054600 2019-09-06
Table 1:
GenBank GenBank
GenBank
Accession Accession Accession
Number Number
Number
Complete Genomes HuS17 AY695376 Hu66
AY530626
Adeno-associated NC 002077, HuT88 AY695375 Hu42
AY530605
virus 1 AF063497
Adeno-associated NC 001401 HuT71 AY695374 Hu67
AY530627
virus 2
Adeno-associated NC 001729 HuT70 AY695373 Hu40
AY530603
virus 3
Adeno-associated NC 001863 HuT40 AY695372 Hu41
AY530604
virus 3B
Adeno-associated NC 001829 Hu T32 AY695371 Hu37
AY530600
virus 4
Adeno-associated Y18065, Hu T17 AY695370 Rh40
AY530559
virus 5 AF085716
Adeno-associated NC 001862 Hu LG15 AY695377 Rh2
AY243007
virus 6
Avian AAVA TCC AY186198, Clade C Bbl AY243023
VR-865 AY629583 ,
NC_004828
Avian AAV strain NC 006263, Hu9 AY530629 Bb2
AY243022
DA-1 AY629583
Bovine AAV NC_005889, Hu JO AY530576
AY388617,
AAR26465
AAVIJ AAT46339, Hull AY530577 Rh10 AY243015
AY631966
AAV12 AB116639, Hui? AY530582
DQ813647
Clade A Hu53 AY530615 Hu6
AY530621
AAVI NC 002077, Hu55 AY530617 Rh25
AY530557
AF063497
AAV6 NC 001862 Hu54 AY530616 Pi2
AY530554
Hu.48 AY530611 Hu7 AY530628 Pil
AY530553
Hu43 AY530606 Hul8 AY530583 Pi3 AY530555
Hu 44 AY530607 Hu IS AY530580 Rh57
AY530569
Hu 46 AY530609 Hul6 AY530581 Rh50
AY530563
Clade B Hu25 AY530591 RM9 AY530562
Hu19 AY530584 Hu60 AY530622 Hu39 AY530601
Hu20 AY530586 Ch5 AY243021 Rh58 AY530570
Hu23 AY530589 Hu3 AY530595 Rh61 AY530572
Hu22 AY530588 Hui AY530575 Rh52 AY530565
Hu24 AY530590 Hu4 AY530602 Rh53 AY530566
Hu21 AY530587 Hu2 AY530585 RhSI AY530564
Hu27 AY530592 Hu61 AY530623 Rh64 AY530574
Hu28 AY530593 Clade D Rh43 AY530560
164
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Hu 29 AY530594 Rh62 AY530573 AAV8
AF513852
Hu63 AY530624 RMB AY530561 Rh8 AY242997
Hu64 AY530625 Rh54 AY5 30567 Rhl
AY530556
Hul3 AY530578 Rh55 AY530568 Clade F
Hu56 AY530618 Cy2 AY243020 Hu14 AY530579
(AAV9)
Hu57 AY530619 AAV7 AF5J3851 Hu31 AY530596
I-Iu49 AY530612 Rh35 AY243000 Hu32 AY530597
Hu58 AY530620 Rh37 AY242998 Clonal
Isolate
Hu34 AY530598 Rh36 AY242999 AAVS Y18065,
AF085716
Hu35 AY530599 Cy6 AY243016 AAV3
NC 001729
AAV2 NC_001401 Cy4 AY243018
AAV3B NC_001863
Hu45 AY530608 Cy3 AY243019 AAV4 NC_001829
Hu47 AY5306J0 Cy5 AY243017 Rh34 AY243001
Hu51 AY530613 RJ1(3 AY243013 Rh33 AY243002
Hu52 AY530614 Clade E Rh32
AY243003
HuT41 AY695378 Rh38 AY530558
165
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Table 2: Amino acid residues and abbreviations
Abbreviation
Amino Acid Residue
Three-Letter Code One-Letter Code
Alanine Ala A
Arginine Arg R
Asparagine Asn N
Aspartic acid (Aspartate) Asp D
Cysteine Cys C
Glutam ine Gin Q
Glutamic acid (Glutamate) Glu E
Glycine Gly G
Histidine His H
Isoleucine Ile I
Leucine Leu L
Lysine Lys K
Methionine Met M
Phenylalanine Phe F
Praline Pro P
Serine Ser S
Threonine Thr T
Tryptophan Trp W
Tyrosine Tyr Y
Valine Val V
Table 3.
Serotype Position 1 Position 2
AAVI A263X T265X
AAV2 Q263X -265X
AAV3a Q263X -265X
AAV3b Q263X -265X
AAV4 S257X -259X
AAV5 G253X V255X
AAV6 A263X T265X
AAV7 E264X A266X
AAV8 G264X S266X
AAV9 S263X S265X
Where, (X)¨> mutation to any amino acid
(-) --> insertion of any amino acid
Note: Position 2 inserts are indicated by the site of insertion
166
CA 3054600 2019-09-06
Table 4.
Modified Amino Acid Residue
Abbreviation
Amino Acid Residue Derivatives
2-Aminoadipic acid Aad
3-Aminoadipic acid bAad
beta-Alanine, beta-Aminoproprionic acid bAla
2-Aminobutyric acid Abu
4-Aminobutyric acid, Piperidinic acid 4Abu
6-Aminocaproic acid Acp
2-Aminoheptanoic acid Ahe
2-Aminoisobutyric acid Aib
3-Aminoisobutyric acid bAib
2-Aminopimelic acid Apm
t-butylalanine t-BuA
Citrulline Cit
Cyclohexylalanine Cha
2,4-Diaminobutyric acid Dbu
Desmosine Des
2,2'-Diaminopimelic acid Dpm
2,3-Diaminoproprionic acid Dpr
N-Ethylglycine EtGly
N-Ethylasparagine EtAsn
Homoarginine hArg
Homocysteine hCys
Homoserine hSer
Hydroxylysine Hyl
Al lo-Hydroxylysine aHyl
3-Hydroxyproline 3Hyp
4-Hydroxyproline 4Hyp
Isodesmosine Ide
allo-Isoleucine alle
Methionine sulfoxide MSO
N-Methylglycine, sarcosine MeGly
N-Methylisoleucine MeIle
6-N-Methyllysine MeLys
N-Methylvaline MeVal
2-Naphthylalanine 2-Nal
Norval ine Nva
Norleucine Nle
Ornithine Orn
4-Chlorophenylalanine Phe(4-C1)
2-Fluorophenylalanine Phe(2-F)
3-Fluorophenylalanine Phe(3-F)
4-Fluorophenylalanine Phe(4-F)
Phenylglycine Phg
Beta-2-thienylalanine Thi
167
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Table 5: Neutralization antibody titer and cross-reactivity for triploid virus
AAV2/8
Vector
AAV2 Haploid virus Mixture
virus AAV2 AAV8
AAV2/8 and AAV8
3:1 1:1 1:3 3:1 1:1 1:3
mAb A20 512 2048 32 <2 ND
ND ND <2
ADK8 <2 512 512 1024 ND ND ND 1024
serum AAV2 4096 1024 256 8 4096 2048 1024 --
<2
AAV8 <2 256 256 512 <2 <2 <2 512
Table 6: Neutralization antibody titer and cross-reactivity for haploid virus
AAV2/8/9
AAV2 AAV8 AAV9 AAV2/9 AAV8/9 AAV2/8/9
SerumAAV2 >2048 <2 512 128
SerumAAV8 <2 128 32 4
SerumAAV9 <2 16 2048 512 256
Serum AAV2/8/9 8 128 128 64 512 128
168
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Sequences
AAV1 (SEQ ID NO:127)
1 ctctcccccc tgtcgcgttc gctcgctcgc tggctcgttt gggggggtgg cagctcaaag
61 agctgccaga cgacggccct ctggccgtcg cccccccaaa cgagccagcg agcgagcgaa
121 cgcgacaggg gggagagtgc cacactctca agcaaggagg ttttgtaagt ggtgatgtca
181 tatagttgtc acgcgatagt taatgattaa cagtcaggtg atgtgtgtta tccaatagga
241 tgaaagcgcg cgcatgagtt ctcgcgagac ttccggggta taaaggggtg agtgaacgag
301 cccgccgcca ttctctgctc tgaactgcta gaggaccctc gctgccatgg ctaccttcta
361 cgaagtcatt gttcgcgtcc catttgacgt ggaggaacat ctgcctggaa tttctgacag
421 ctttgtggac tgggtaactg gtcaaatttg ggagctgcct cccgagtcag atttgaattt
481 gactctgatt gagcagcctc agctgacggt tgctgacaga attcgccgcg tgttcctgta
541 cgagtggaac aaattttcca agcaggaatc caaattcttt gtgcagtttg aaaagggatc
601 tgaatatttt catctgcaca cgcttgtgga gacctccggc atctatcca tggtcctagg
661 ccgctacgtg agtcagattc gcgcccagct ggtgaaagtg gtcttccagg gaatcgagcc
721 acagatcaac gactgggtcg ccatcaccaa ggtaaagaag ggcggagcca ataaggtggt
781 ggattctggg tatattcccg cctacctgct gccgaaggtc caaccggagc ttcagtgggc
841 gtggacaaac ctggacgagt ataaattggc cgccctgaac ctggaggagc gcaaacggct
901 cgtcgcgcag tttctggcag aatcctcgca gcgctcgcag gaggcggctt cgcagcgtga
961 gttcteggct gacccggtca tcaaaagcaa gacttcccag aaatacatgg cgctcgtcaa
1021 ctggctcgtg gagcacggca tcacttccga gaagcagtgg atccaggaga atcaggagag
1081 ctacctctcc ttcaactcca cgggcaactc tcggagccaa atcaaggccg cgctcgacaa
1141 cgcgaccaaa atcatgagtc tgacaaaaag cgcggtggac tacctcgtgg ggagctccgt
1201 tcccgaggac atttcaaaaa acagaatctg gcaaattttt gagatgaacg gctacgaccc
1261 ggcctacgcg ggatccatcc tctacggctg gtgtcagcgc tccttcaaca agaggaacac
1321 cgtctggctc tacggacccg ccacgaccgg caagaccaac atcgcggagg ccatcgccca
1381 cactgtgccc ttttacggct gcgtgaactg gaccaatgaa aactttccct ttaatgactg
1441 tgtggacaaa atgctcattt ggtgggagga gggaaagatg accaacaagg tggttgaatc
1501 cgccaaggcc atcctggggg gctccaaggt gegggtcgat cagaaatgta aatcctctgt
1561 tcaaattgat tctacccccg tcattgtaac ttccaataca aacatgtgtg tggtggtgga
1621 tgggaattcc acgacctttg aacaccagca gccgctggag gaccgcatgt tcaaatttga
1681 actgactaag cggctcccgc cagattttgg caagattact aagcaggaag tcaaagactt
1741 ttttgcttgg gcaaaggtca atcaggtgcc ggtgactcac gagtttaaag ttcccaggga
1801 attggcggga actaaagggg cggagaaatc tctaaaacgc ccactgggtg acgtcaccaa
1861 tactagctat aaaagtccag agaagcgggc ccggctctca tttgttcccg agacgcctcg
1921 cagttcagac gtgactgtcg atcccgctcc tctgcgaccg ctcaattgga attcaaggta
1981 tgattgcaaa tgtgaccatc atgctcaatt tgacaacatt tctgacaaat gtgatgaatg
2041 tgaatatttg aatcggggca aaaatggatg tatctgtcac aatgtaactc actgtcaaat
2101 ttgtcacggg attccccect gggagaagga aaacttgtca gattttgggg attttgacga
2161 tgccaataaa gaacagtaaa taaagcgagt agtcatgtct tttgttgatc accctccaga
2221 ttggttggaa gaagttggtg aaggtcttcg cgagtttttg ggccttgaag cgggcccacc
2281 gaaaccgaaa cccaatcagc agcatcaaga tcaagcccgt ggtcttgtgc tgcctggtta
2341 taactatctc ggacccggaa acggtacga tcgaggagag cctgtcaaca gggcagacga
2401 ggtcgcgcga gagcacgaca tctcgtacaa cgagcagctt gaggcgggag acaaccccta
2461 cctcaagtac aaccacgcgg acgccgagtt tcaggagaag ctcgccgacg acacatcctt
2521 cgggggaaac ctcggaaagg cagtctttca ggccaagaaa agggttctcg aaccttttgg
2581 cctggttgaa gagggtgcta agacggcccc taccggaaag cggatagacg accactttcc
2641 aaaaagaaag aaggctcgga ccgaagagga ctccaagcct tccacctcgt cagacgccga
2701 agctggaccc agcggatccc agcagctgca aatcccagca caaccagcct caagtttggg
169
CA 3054600 2019-09-06
2761 agctgataca atgtctgcgg gaggtggcgg cccattgggc gacaataacc aaggtgccga
2821 tggagtgggc aatgcctcgg gagattggca ttgcgattcc acgtggatgg gggacagagt
2881 cgtcaccaag tccacccgca cctgggtgct gcccagctac aacaaccacc agtaccgaga
2941 gatcaaaagc ggctccgtcg acggaagcaa cgccaacgcc tactttggat acagcacccc
3001 ctgggggtac tttgacttta accgcttcca cagccactgg agcccccgag actggcaaag
3061 actcatcaac aactattggg gcttcagacc ccggtctctc agagtcaaaa tcttcaacat
3121 ccaagtcaaa gaggtcacgg tgcaggactc caccaccacc atcgccaaca acctcacctc
3181 caccgtccaa gtgtttacgg acgacgacta ccaactcccg tacgtcgtcg gcaacgggac
3241 cgagggatgc ctgccggcct tccccccgca ggtctttacg ctgccgcagt acggctacgc
3301 gacgctgaac cgagacaacg gagacaaccc gacagagcgg agcagcttct tttgcctaga
3361 gtactttccc agcaagatgc tgaggacggg caacaacttt gagtttacct acagctttga
3421 agaggtgccc ttccactgca gcttcgcccc gagccagaac ctctttaagc tggccaaccc
3481 gctggtggac cagtacctgt accgcttcgt gagcacctcg gccacgggcg ccatccagtt
3541 ccaaaagaac ctggcgggca gatacgccaa cacctacaaa aactggttcc cggggcccat
3601 gggccgaacc cagggctgga acacgagctc tggcagcagc accaacagag tcagcgtcaa
3661 caacttttcc gtctcaaacc ggatgaacct ggagggggcc agctaccaag tgaaccccca
3721 gcccaacggg atgacaaaca cgctccaagg cagcaaccgc tacgcgctgg aaaacaccat
3781 gatcttcaac gctcaaaacg ccacgccggg aactacctcg gtgtacccag aggacaatct
3841 actgctgacc agcgagagcg agactcagcc cgtcaaccgg gtggcttaca acacgggcgg
3901 tcagatggcc accaacgccc agaacgccac cacggctccc acggtcggga cctacaacct
3961 ccaggaagtg cttcctggca gcgtatggat ggagagggac gtgtacctcc aaggacccat
4021 ctgggccaag atcccagaga cgggggcgca ctttcacccc tctccggcca tgggcggatt
4081 cggactcaaa cacccgccgc ccatgatgct catcaaaaac acgccggtgc ccggcaacat
4141 caccagcttc tcggacgtgc ccgtcagcag cttcatcacc cagtacagca ccgggcaggt
4201 caccgtggag atggaatggg agctcaaaaa ggaaaactcc aagaggtgga acccagagat
4261 ccagtacacc aacaactaca acgaccccca gtttgtggac tttgctccag acggctccgg
4321 cgaatacaga accaccagag ccatcggaac ccgatacctc acccgacccc tttaacccat
4381 tcatgtcgca taccctcaat aaaccgtgta ttcgtgtcag tgaaatactg cctcttgtgg
4441 tcattcaatg aacatcagct tacaacatct acaaaacccc cttgcttgag agtgtggcac
4501 tctcccccct gtcgcgttcg ctcgctcgct ggctcgtttg ggggggtggc agctcaaaga
4561 gctgccagac gacggccctc tggccgtcgc ccccccaaac gagccagcga gcgagcgaac
4621 gcgacagggg ggagag
170
CA 3054600 2019-09-06
AAV2 (SEQ ID NO:128)
1 ttggccactc cctctctgcg cgctcgctcg ctcactgagg ccgggcgacc aaaggtcgcc
61 cgacgcccgg gctttgcccg ggcggcctca gtgagcgagc gagcgcgcag agagggagtg
121 gccaactcca tcactagggg ttcctggagg ggtggagtcg tgacgtgaat tacgtcatag
181 ggttagggag gtcctgtatt agaggtcacg tgagtgtttt gcgacatttt gcgacaccat
241 gtggtcacgc tgggtattta agcccgagtg agcacgcagg gtctccattt tgaagcggga
301 ggtttgaacg cgcagccgcc atgccggggt tttacgagat tgtgattaag gtccccagcg
361 accttgacga gcatctgccc ggcatttctg acagctttgt gaactgggtg gccgagaagg
421 aatgggagtt gccgccagat tctgacatgg atctgaatct gattgagcag gcacccctga
481 ccgtggccga gaagctgcag cgcgactttc tgacggaatg gcgccgtgtg agtaaggccc
541 cggaggccct tttctttgtg caatttgaga agggagagag ctacttccac atgcacgtgc
601 tcgtggaaac caccggggtg aaatccatgg ttttgggacg tttcctgagt cagattcgcg
661 aaaaactgat tcagagaatt taccgcggga tcgagccgac tttgccaaac tggttcgcgg
721 tcacaaagac cagaaatggc gccggaggcg ggaacaaggt ggtggatgag tgctacatcc
781 ccaattactt gctccccaaa acccagcctg agctccagtg ggcgtggact aatatggaac
841 agtatttaag cgcctgtttg aatctcacgg agcgtaaacg gttggtggcg cagcatctga
901 cgcacgtgtc gcagacgcag gagcagaaca aagagaatca gaatcccaat tctgatgcgc
961 cggtgatcag atcaaaaact tcagccaggt acatggagct ggtcgggtgg ctcgtggaca
1021 aggggattac ctcggagaag cagtggatcc aggaggacca ggcctcatac atctccttca
1081 atgcggcctc caactcgcgg tcccaaatca aggctgcctt ggacaatgcg ggaaagatta
1141 tgagcctgac taaaaccgcc cccgactacc tggtgggcca gcagcccgtg gaggacattt
1201 ccagcaatcg gatttataaa attttggaac taaacgggta cgatccccaa tatgcggctt
1261 ccgtctttct gggatgggcc acgaaaaagt tcggcaagag gaacaccatc tggctgtttg
1321 ggcctgcaac taccgggaag accaacatcg cggaggccat agcccacact gtgcccttct
1381 acgggtgcgt aaactggacc aatgagaact ttcccttcaa cgactgtgtc gacaagatgg
1441 tgatctggtg ggaggagggg aagatgaccg ccaaggtcgt ggagtcggcc aaagccattc
1501 tcggaggaag caaggtgcgc gtggaccaga aatgcaagtc ctcggcccag atagacccga
1561 ctcccgtgat cgtcacctcc aacaccaaca tgtgcgccgt gattgacggg aactcaacga
1621 ccttcgaaca ccagcagccg ttgcaagacc ggatgttcaa atttgaactc acccgccgtc
1681 tggatcatga ctttgggaag gtcaccaagc aggaagtcaa agactttttc cggtgggcaa
1741 aggatcacgt ggttgaggtg gagcatgaat tctacgtcaa aaagggtgga gccaagaaaa
1801 gacccgcccc cagtgacgca gatataagtg agcccaaacg ggtgcgcgag tcagttgcgc
1861 agccatcgac gtcagacgcg gaagcttcga tcaactacgc agacaggtac caaaacaaat
1921 gttctcgtca cgtgggcatg aatctgatgc tgtttccctg cagacaatgc gagagaatga
1981 atcagaattc aaatatctgc ttcactcacg gacagaaaga ctgtttagag tgctttcccg
2041 tgtcagaatc tcaacccgtt tctgtcgtca aaaaggcgta tcagaaactg tgctacattc
2101 atcatatcat gggaaaggtg ccagacgctt gcactgcctg cgatctggtc aatgtggatt
2161 tggatgactg catctttgaa caataaatga tttaaatcag gtatggctgc cgatggttat
2221 cttccagatt ggctcgagga cactctctct gaaggaataa gacagtggtg gaagctcaaa
2281 cctggcccac caccaccaaa gcccgcagag cggcataagg acgacagcag gggtcttgtg
2341 cttcctgggt acaagtacct cggacccttc aacggactcg acaagggaga gccggtcaac
2401 gaggcagacg ccgcggccct cgagcacgac aaagcctacg accggcagct cgacagcgga
2461 gacaacccgt acctcaagta caaccacgcc gacgcggagt ttcaggagcg ccttaaagaa
2521 gatacgtctt ttgggggcaa cctcggacga gcagtcttcc aggcgaaaaa gagggttctt
2581 gaacctctgg gcctggttga ggaacctgtt aagacggctc cgggaaaaaa gaggccggta
2641 gagcactctc ctgtggagcc agactcctcc tcgggaaccg gaaaggcggg ccagcagcct
2701 gcaagaaaaa gattgaattt tggtcagact ggagacgcag actcagtacc tgacccccag
2761 cctctcggac agccaccagc agccccctct ggtctgggaa ctaatacgat ggctacaggc
2821 agtggcgcac caatggcaga caataacgag ggcgccgacg gagtgggtaa ttcctcggga
171
CA 3054600 2019-09-06
2881 aattggcatt gcgattccac atggatgggc gacagagtca tcaccaccag cacccgaacc
2941 tgggccctgc ccacctacaa caaccacctc tacaaacaaa tttccagcca atcaggagcc
3001 tcgaacgaca atcactactt tggctacagc accccaggg ggtattttga cttcaacaga
3061 ttccactgcc acttttcacc acgtgactgg caaagactca tcaacaacaa ctggggattc
3121 cgacccaaga gactcaactt caagctcttt aacattcaag tcaaagaggt cacgcagaat
3181 gacggtacga cgacgattgc caataacctt accagcacgg ttcaggtgtt tactgactcg
3241 gagtaccagc tcccgtacgt cctcggctcg gcgcatcaag gatgcctccc gccgttccca
3301 gcagacgtct tcatggtgcc acagtatgga tacctcaccc tgaacaacgg gagtcaggca
3361 gtaggacgct cttcatttta ctgcctggag tactttcctt ctcagatgct gcgtaccgga
3421 aacaacttta ccttcagcta cacttttgag gacgttcctt tccacagcag ctacgctcac
3481 agccagagtc tggaccgtct catgaatcct ctcatcgacc agtacctgta ttacttgagc
3541 agaacaaaca ctccaagtgg aaccaccacg cagtcaaggc ttcagttttc tcaggccgga
3601 gcgagtgaca ttcgggacca gtctaggaac tggcttcctg gaccctgtta ccgccagcag
3661 cgagtatcaa agacatctgc ggataacaac aacagtgaat actcgtggac tggagctacc
3721 aagtaccacc tcaatggcag agactctctg gtgaatccgg gcccggccat ggcaagccac
3781 aaggacgatg aagaaaagtt ttttcctcag agcggggttc tcatctttgg gaagcaaggc
3841 tcagagaaaa caaatgtgga cattgaaaag gtcatgatta cagacgaaga ggaaatcagg
3901 acaaccaatc ccgtggctac ggagcagtat ggttctgtat ctaccaacct ccagagaggc
3961 aacagacaag cagctaccgc agatgtcaac acacaaggcg ttcttccagg catggtctgg
4021 caggacagag atgtgtacct tcaggggccc atctgggcaa agattccaca cacggacgga
4081 cattttcacc cctctcccct catgggtgga ttcggactta aacaccctcc tccacagatt
4141 ctcatcaaga acaccccggt acctgcgaat ccttcgacca ccttcagtgc ggcaaagttt
4201 gettecttca tcacacagta ctccacggga caggtcagcg tggagatcga gtgggagctg
4261 cagaaggaaa acagcaaacg ctggaatccc gaaattcagt acacttccaa ctacaacaag
4321 tctgttaatg tggactttac tgtggacact aatggcgtgt attcagagcc tcgccccatt
4381 ggcaccagat acctgactcg taatctgtaa ttgcttgtta atcaataaac cgtttaattc
4441 gtttcagttg aactttggtc tctgcgtatt tctttcttat ctagtttcca tggctacgta
4501 gataagtagc atggcgggtt aatcattaac tacaaggaac ccctagtgat ggagttggcc
4561 actccctctc tgcgcgctcg ctcgctcact gaggccgggc gaccaaaggt cgcccgacgc
4621 ccgggctttg cccgggcggc ctcagtgagc gagcgagcgc gcagagaggg agtggccaa
172
CA 3054600 2019-09-06
AAV3 (SEQ ID NO:129)
1 ttggccactc cctctatgcg cactcgctcg ctcggtgggg cctggcgacc aaaggtcgcc
61 agacggacgt gctttgcacg tccggcccca ccgagcgagc gagtgcgcat agagggagtg
121 gccaactcca tcactagagg tatggcagtg acgtaacgcg aagcgcgcga agcgagacca
181 cgcctaccag ctgcgtcagc agtcaggtga cccttttgcg acagtttgcg acaccacgtg
241 gccgctgagg gtatatattc tcgagtgagc gaaccaggag ctccattttg accgcgaaat
301 ttgaacgagc agcagccatg ccggggttct acgagattgt cctgaaggtc ccgagtgacc
361 tggacgagcg cctgccgggc atttctaact cgtttgttaa ctgggtggcc gagaaggaat
421 gggacgtgcc gccggattct gacatggatc cgaatctgat tgagcaggca cccctgaccg
481 tggccgaaaa gcttcagcgc gagttcctgg tggagtggcg ccgcgtgagt aaggccccgg
541 aggccctctt ttttgtccag ttcgaaaagg gggagaccta cttccacctg cacgtgctga
601 ttgagaccat cggggtcaaa tccatggtgg tcggccgcta cgtgagccag attaaagaga
661 agctggtgac ccgcatctac cgcggggtcg agccgcagct tccgaactgg ttcgcggtga
721 ccaaaacgcg aaatggcgcc gggggcggga acaaggtggt ggacgactgc tacatcccca
781 actacctgct ccccaagacc cagcccgagc tccagtgggc gtggactaac atggaccagt
841 atttaagcgc ctgtttgaat ctcgcggagc gtaaacggct ggtggcgcag catctgacgc
901 acgtgtcgca gacgcaggag cagaacaaag agaatcagaa ccccaattct gacgcgccgg
961 tcatcaggtc aaaaacctca gccaggtaca tggagctggt cgggtggctg gtggaccgcg
1021 ggatcacgtc agaaaagcaa tggattcagg aggaccaggc ctcgtacatc tccttcaacg
1081 ccgcctccaa ctcgcggtcc cagatcaagg ccgcgctgga caatgcctcc aagatcatga
1141 gcctgacaaa gacggctccg gactacctgg tgggcagcaa cccgccggag gacattacca
1201 aaaatcggat ctaccaaatc ctggagctga acgggtacga tccgcagtac gcggcctccg
1261 tcttcctggg ctgggcgcaa aagaagttcg ggaagaggaa caccatctgg ctctttgggc
1321 cggccacgac gggtaaaacc aacatcgcgg aagccatcgc ccacgccgtg cccttctacg
1381 gctgcgtaaa ctggaccaat gagaactttc ccttcaacga ttgcgtcgac aagatggtga
1441 tctggtggga ggagggcaag atgacggcca aggtcgtgga gagcgccaag gccattctgg
1501 gcggaagcaa ggtgcgcgtg gaccaaaagt gcaagtcatc ggcccagatc gaacccactc
1561 ccgtgatcgt cacctccaac accaacatgt gcgccgtgat tgacgggaac agcaccacct
1621 tcgagcatca gcagccgctg caggaccgga tgtttgaatt tgaacttacc cgccgtttgg
1681 accatgactt tgggaaggtc accaaacagg aagtaaagga ctttttccgg tgggcttccg
1741 atcacgtgac tgacgtggct catgagttct acgtcagaaa gggtggagct aagaaacgcc
1801 ccgcctccaa tgacgcggat gtaagcgagc caaaacggga gtgcacgtca cttgcgcagc
1861 cgacaacgtc agacgcggaa gcaccggcgg actacgcgga caggtaccaa aacaaatgtt
1921 ctcgtcacgt gggcatgaat ctgatgcttt ttccctgtaa aacatgcgag agaatgaatc
1981 aaatttccaa tgtctgtttt acgcatggtc aaagagactg tggggaatgc ttccctggaa
2041 tgtcagaatc tcaacccgtt tctgtcgtca aaaagaagac ttatcagaaa ctgtgtccaa
2101 ttcatcatat cctgggaagg gcacccgaga ttgcctgttc ggcctgcgat ttggccaatg
2161 tggacttgga tgactgtgtt tctgagcaat aaatgactta aaccaggtat ggctgctgac
2221 ggttatcttc cagattggct cgaggacaac ctttctgaag gcattcgtga gtggtgggct
2281 ctgaaacctg gagtccctca acccaaagcg aaccaacaac accaggacaa ccgtcggggt
2341 cttgtgcttc cgggttacaa atacctcgga cccggtaacg gactcgacaa aggagagccg
2401 gtcaacgagg cggacgcggc agccctcgaa cacgacaaag cttacgacca gcagctcaag
2461 gccggtgaca acccgtacct caagtacaac cacgccgacg ccgagtttca ggagcgtat
2521 caagaagata cgtcttttgg gggcaacctt ggcagagcag tatccaggc caaaaagagg
2581 atccttgagc ctcttggtct ggttgaggaa gcagctaaaa cggctcctgg aaagaagggg
2641 gctgtagatc agtctcctca ggaaccggac tcatcatctg gtgttggcaa atcgggcaaa
2701 cagcctgcca gaaaaagact aaatttcggt cagactggag actcagagtc agtcccagac
2761 cctcaacctc tcggagaacc accagcagcc cccacaagtt tgggatctaa tacaatggct
2821 tcaggcggtg gcgcaccaat ggcagacaat aacgagggtg ccgatggagt gggtaattcc
173
CA 3054600 2019-09-06
2881 tcaggaaatt ggcattgcga ttcccaatgg ctgggcgaca gagtcatcac caccagcacc
2941 agaacctggg ccctgcccac ttacaacaac catctctaca agcaaatctc cagccaatca
3001 ggagcttcaa acgacaacca ctactttggc tacagcaccc cttgggggta ttttgacttt
3061 aacagattcc actgccactt ctcaccacgt gactggcagc gactcattaa caacaactgg
3121 ggattccggc ccaagaaact cagcttcaag ctcttcaaca tccaagttag aggggtcacg
3181 cagaacgatg gcacgacgac tattgccaat aaccttacca gcacggttca agtgtttacg
3241 gactcggagt atcagctccc gtacgtgctc gggtcggcgc accaaggctg tctcccgccg
3301 tttccagcgg acgtcttcat ggtccctcag tatggatacc tcaccctgaa caacggaagt
3361 caagcggtgg gacgctcatc cttttactgc ctggagtact tcccttcgca gatgctaagg
3421 actggaaata acttccaatt cagctatacc ttcgaggatg taccttttca cagcagctac
3481 gctcacagcc agagtttgga tcgcttgatg aatcctctta ttgatcagta tctgtactac
3541 ctgaacagaa cgcaaggaac aacctctgga acaaccaacc aatcacggct gctttttagc
3601 caggctgggc ctcagtctat gtctttgcag gccagaaatt ggctacctgg gccctgctac
3661 cggcaacaga gactttcaaa gactgctaac gacaacaaca acagtaactt tccttggaca
3721 gcggccagca aatatcatct caatggccgc gactcgctgg tgaatccagg accagctatg
3781 gccagtcaca aggacgatga agaaaaattt ttccctatgc acggcaatct aatatttggc
3841 aaagaaggga caacggcaag taacgcagaa ttagataatg taatgattac ggatgaagaa
3901 gagattcgta ccaccaatcc tgtggcaaca gagcagtatg gaactgtggc aaataacttg
3961 cagagctcaa atacagctcc cacgactgga actgtcaatc atcagggggc cttacctggc
4021 atggtgtggc aagatcgtga cgtgtacctt caaggaccta tctgggcaaa gattcctcac
4081 acggatggac actttcatcc ttctcctctg atgggaggct ttggactgaa acatccgcct
4141 cctcaaatca tgatcaaaaa tactccggta ccggcaaatc ctccgacgac tttcagcccg
4201 gccaagtttg cttcatttat cactcagtac tccactggac aggtcagcgt ggaaattgag
4261 tgggagctac agaaagaaaa cagcaaacgt tggaatccag agattcagta cacttccaac
4321 tacaacaagt ctgttaatgt ggactttact gtagacacta atggtgttta tagtgaacct
4381 cgccctattg gaacccggta tctcacacga aacttgtgaa tcctggttaa tcaataaacc
4441 gtttaattcg tttcagttga actttggctc ttgtgcactt ctttatcttt atcttgtttc
4501 catggctact gcgtagataa gcagcggcct gcggcgcttg cgcttcgcgg tttacaactg
4561 ctggttaata tttaactctc gccatacctc tagtgatgga gttggccact ccctctatgc
4621 gcactcgctc gctcggtggg gcctggcgac caaaggtcgc cagacggacg tgctttgcac
4681 gtccggcccc accgagcgag cgagtgcgca tagagggagt ggccaa
174
CA 3054600 2019-09-06
AAV4 (SEQ ID NO:130)
1 ttggccactc cctctatgcg cgctcgctca ctcactcggc cctggagacc aaaggtctcc
61 agactgccgg cctctggccg gcagggccga gtgagtgagc gagcgcgcat agagggagtg
121 gccaactcca tcatctaggt ttgcccactg acgtcaatgt gacgtcctag ggttagggag
181 gtccctgtat tagcagtcac gtgagtgtcg tatttcgcgg agcgtagcgg agcgcatacc
241 aagctgccac gtcacagcca cgtggtccgt ttgcgacagt ttgcgacacc atgtggtcag
301 gagggtatat aaccgcgagt gagccagcga ggagctccat tttgcccgcg aattttgaac
361 gagcagcagc catgccgggg ttctacgaga tcgtgctgaa ggtgcccagc gacctggacg
421 agcacctgcc cggcatttct gactcttttg tgagctgggt ggccgagaag gaatgggagc
481 tgccgccgga ttctgacatg gacttgaatc tgattgagca ggcacccctg accgtggccg
541 aaaagctgca acgcgagttc ctggtcgagt ggcgccgcgt gagtaaggcc ccggaggccc
601 tcttctttgt ccagttcgag aagggggaca gctacttcca cctgcacatc ctggtggaga
661 ccgtgggcgt caaatccatg gtggtgggcc gctacgtgag ccagattaaa gagaagctgg
721 tgacccgcat ctaccgcggg gtcgagccgc agcttccgaa ctggttcgcg gtgaccaaga
781 cgcgtaatgg cgccggaggc gggaacaagg tggtggacga ctgctacatc cccaactacc
841 tgctccccaa gacccagccc gagctccagt gggcgtggac taacatggac cagtatataa
901 gcgcctgttt gaatctcgcg gagcgtaaac ggctggtggc gcagcatctg acgcacgtgt
961 cgcagacgca ggagcagaac aaggaaaacc agaaccccaa ttctgacgcg ccggtcatca
1021 ggtcaaaaac ctccgccagg tacatggagc tggtcgggtg gctggtggac cgcgggatca
1081 cgtcagaaaa gcaatggatc caggaggacc aggcgtccta catctccttc aacgccgcct
1141 ccaactcgcg gtcacaaatc aaggccgcgc tggacaatgc ctccaaaatc atgagcctga
1201 caaagacggc tccggactac ctggtgggcc agaacccgcc ggaggacatt tccagcaacc
1261 gcatctaccg aatcctcgag atgaacgggt acgatccgca gtacgcggcc tccgtcttcc
1321 tgggctgggc gcaaaagaag ttcgggaaga ggaacaccat ctggctcttt gggccggcca
1381 cgacgggtaa aaccaacatc gcggaagcca tcgcccacgc cgtgcccttc tacggctgcg
1441 tgaactggac caatgagaac tttccgttca acgattgcgt cgacaagatg gtgatctggt
1501 gggaggaggg caagatgacg gccaaggtcg tagagagcgc caaggccatc ctgggcggaa
1561 gcaaggtgcg cgtggaccaa aagtgcaagt catcggccca gatcgaccca actcccgtga
1621 tcgtcacctc caacaccaac atgtgcgcgg tcatcgacgg aaactcgacc accttcgagc
1681 accaacaacc actccaggac cggatgttca agttcgagct caccaagcgc ctggagcacg
1741 actttggcaa ggtcaccaag caggaagtca aagacttttt ccggtgggcg tcagatcacg
1801 tgaccgaggt gactcacgag ttttacgtca gaaagggtgg agctagaaag aggcccgccc
1861 ccaatgacgc agatataagt gagcccaagc gggcctgtcc gtcagttgcg cagccatcga
1921 cgtcagacgc ggaagctccg gtggactacg cggacaggta ccaaaacaaa tgttctcgtc
1981 acgtgggtat gaatctgatg ctttttccct gccggcaatg cgagagaatg aatcagaatg
2041 tggacatttg cttcacgcac ggggtcatgg actgtgccga gtgatcccc gtgtcagaat
2101 ctcaacccgt gtctgtcgtc agaaagcgga cgtatcagaa actgtgtccg attcatcaca
2161 tcatggggag ggcgcccgag gtggcctgct cggcctgcga actggccaat gtggacttgg
2221 atgactgtga catggaacaa taaatgactc aaaccagata tgactgacgg ttaccttcca
2281 gattggctag aggacaacct ctctgaaggc gttcgagagt ggtgggcgct gcaacctgga
2341 gcccctaaac ccaaggcaaa tcaacaacat caggacaacg ctcggggtct tgtgcttccg
2401 ggttacaaat acctcggacc cggcaacgga ctcgacaagg gggaacccgt caacgcagcg
2461 gacgcggcag ccctcgagca cgacaaggcc tacgaccagc agctcaaggc cggtgacaac
2521 ccctacctca agtacaacca cgccgacgcg gagttccagc ageggatca gggcgacaca
2581 tcgtttgggg gcaacctcgg cagagcagtc ttccaggcca aaaagagggt tcttgaacct
2641 cttggtctgg ttgagcaagc gggtgagacg gctcctggaa agaagagacc gttgattgaa
2701 tccccccagc agcccgactc ctccacgggt atcggcaaaa aaggcaagca gccggctaaa
2761 aagaagctcg ttttcgaaga cgaaactgga gcaggcgacg gaccccctga gggatcaact
2821 tccggagcca tgtctgatga cagtgagatg cgtgcagcag ctggcggagc tgcagtcgag
175
CA 3054600 2019-09-06
2881 ggcggacaag gtgccgatgg agtgggtaat gcctcgggtg attggcattg cgattccacc
2941 tggtctgagg gccacgtcac gaccaccagc accagaacct gggtcttgcc cacctacaac
3001 aaccacctct acaagcgact cggagagagc ctgcagtcca acacctacaa cggattctcc
3061 accccctggg gatactttga cttcaaccgc ttccactgcc acttctcacc acgtgactgg
3121 cagcgactca tcaacaacaa ctggggcatg cgacccaaag ccatgcgggt caaaatcttc
3181 aacatccagg tcaaggaggt cacgacgtcg aacggcgaga caacggtggc taataacctt
3241 accagcacgg ttcagatctt tgcggactcg tcgtacgaac tgccgtacgt gatggatgcg
3301 ggtcaagagg gcagcctgcc tectittccc aacgacgtct ttatggtgcc ccagtacggc
3361 tactgtggac tggtgaccgg caacacttcg cagcaacaga ctgacagaaa tgccttctac
3421 tgcctggagt actttccttc gcagatgctg cggactggca acaactttga aattacgtac
3481 agttttgaga aggtgccttt ccactcgatg tacgcgcaca gccagagcct ggaccggctg
3541 atgaaccctc tcatcgacca gtacctgtgg ggactgcaat cgaccaccac cggaaccacc
3601 ctgaatgccg ggactgccac caccaacttt accaagctgc ggcctaccaa cttttccaac
3661 tttaaaaaga actggctgcc cgggccttca atcaagcagc agggcttctc aaagactgcc
3721 aatcaaaact acaagatccc tgccaccggg tcagacagtc tcatcaaata cgagacgcac
3781 agcactctgg acggaagatg gagtgccctg acccccggac ctccaatggc cacggctgga
3841 cctgcggaca gcaagttcag caacagccag ctcatctttg cggggcctaa acagaacggc
3901 aacacggcca ccgtacccgg gactctgatc ttcacctctg aggaggagct ggcagccacc
3961 aacgccaccg atacggacat gtggggcaac ctacctggcg gtgaccagag caacagcaac
4021 ctgccgaccg tggacagact gacagccttg ggagccgtgc ctggaatggt ctggcaaaac
4081 agagacattt actaccaggg tcccatttgg gccaagattc ctcataccga tggacacttt
4141 cacccctcac cgctgattgg tgggtttggg ctgaaacacc cgcctcctca aatttttatc
4201 aagaacaccc cggtacctgc gaatcctgca acgaccttca gctctactcc ggtaaactcc
4261 ttcattactc agtacagcac tggccaggtg tcggtgcaga ttgactggga gatccagaag
4321 gagcggtcca aacgctggaa ccccgaggtc cagtttacct ccaactacgg acagcaaaac
4381 tctctgttgt gggctcccga tgcggctggg aaatacactg agcctagggc tatcggtacc
4441 cgctacctca cccaccacct gtaataacct gttaatcaat aaaccggttt attcgtttca
4501 gttgaacttt ggtctccgtg tecttatat cttatctcgt ttccatggct actgcgtaca
4561 taagcagcgg cctgcggcgc ttgcgcttcg cggtttacaa ctgccggtta atcagtaact
4621 tctggcaaac cagatgatgg agttggccac attagctatg cgcgctcgct cactcactcg
4681 gccctggaga ccaaaggtct ccagactgcc ggcctctggc cggcagggcc gagtgagtga
4741 gcgagcgcgc atagagggag tggccaa
176
CA 3054600 2019-09-06
AAV5 (SEQ ID NO:131)
1 ctctcccccc tgtcgcgttc gctcgctcgc tggctcgttt gggggggtgg cagctcaaag
61 agctgccaga cgacggccct ctggccgtcg cccccccaaa cgagccagcg agcgagcgaa
121 cgcgacaggg gggagagtgc cacactctca agcaaggggg ttttgtaagc agtgatgtca
181 taatgatgta atgcttattg tcacgcgata gttaatgatt aacagtcatg tgatgtgttt
241 tatccaatag gaagaaagcg cgcgtatgag ttctcgcgag acttccgggg tataaaagac
301 cgagtgaacg agcccgccgc cattctttgc tctggactgc tagaggaccc tcgctgccat
361 ggctaccttc tatgaagtca ttgttcgcgt cccatttgac gtggaggaac atctgcctgg
421 aatttctgac agctttgtgg actgggtaac tggtcaaatt tgggagctgc ctccagagtc
481 agatttaaat ttgactctgg ttgaacagcc tcagttgacg gtggctgata gaattcgccg
541 cgtgttcctg tacgagtgga acaaattttc caagcaggag tccaaattct ttgtgcagtt
601 tgaaaaggga tctgaatatt ttcatctgca cacgcttgtg gagacctccg gcatctatc
661 catggtcctc ggccgctacg tgagtcagat tcgcgcccag ctggtgaaag tggtcttcca
721 gggaattgaa ccccagatca acgactgggt cgccatcacc aaggtaaaga agggcggagc
781 caataaggtg gtggattctg ggtatattcc cgcctacctg ctgccgaagg tccaaccgga
841 gcttcagtgg gcgtggacaa acctggacga gtataaattg gccgccctga atctggagga
901 gcgcaaacgg ctcgtcgcgc agtttctggc agaatcctcg cagcgctcgc aggaggcggc
961 ttcgcagcgt gagttctcgg ctgacccggt catcaaaagc aagacttccc agaaatacat
1021 ggcgctcgtc aactggctcg tggagcacgg catcacttcc gagaagcagt ggatccagga
1081 aaatcaggag agctacctct ccttcaactc caccggcaac tctcggagcc agatcaaggc
1141 cgcgctcgac aacgcgacca aaattatgag tctgacaaaa agcgcggtgg actacctcgt
1201 ggggagctcc gttcccgagg acatttcaaa aaacagaatc tggcaaattt ttgagatgaa
1261 tggctacgac ccggcctacg cgggatccat cctctacggc tggtgtcagc gctccttcaa
1321 caagaggaac accgtctggc tctacggacc cgccacgacc ggcaagacca acatcgcgga
1381 ggccatcgcc cacactgtgc ccttttacgg ctgcgtgaac tggaccaatg aaaactttcc
1441 ctttaatgac tgtgtggaca aaatgctcat ttggtgggag gagggaaaga tgaccaacaa
1501 ggtggttgaa tccgccaagg ccatcctggg gggctcaaag gtgcgggtcg atcagaaatg
1561 taaatcctct gttcaaattg attctacccc tgtcattgta acttccaata caaacatgtg
1621 tgtggtggtg gatgggaatt ccacgacctt tgaacaccag cagccgctgg aggaccgcat
1681 gttcaaattt gaactgacta agcggctccc gccagatttt ggcaagatta ctaagcagga
1741 agtcaaggac ttttttgctt gggcaaaggt caatcaggtg ccggtgactc acgagtttaa
1801 agttcccagg gaattggcgg gaactaaagg ggcggagaaa tctctaaaac gcccactggg
1861 tgacgtcacc aatactagct ataaaagtct ggagaagcgg gccaggctct catttgttcc
1921 cgagacgcct cgcagttcag acgtgactgt tgatcccgct cctctgcgac cgctcaattg
1981 gaattcaagg tatgattgca aatgtgacta tcatgctcaa tttgacaaca tttctaacaa
2041 atgtgatgaa tgtgaatatt tgaatcgggg caaaaatgga tgtatctgtc acaatgtaac
2101 tcactgtcaa atttgtcatg ggattccccc ctgggaaaag gaaaacttgt cagattttgg
2161 ggattttgac gatgccaata aagaacagta aataaagcga gtagtcatgt cttttgttga
2221 tcaccctcca gattggttgg aagaagttgg tgaaggtctt cgcgagtttt tgggccttga
2281 agcgggccca ccgaaaccaa aacccaatca gcagcatcaa gatcaagccc gtggtcttgt
2341 gctgcctggt tataactatc tcggacccgg aaacggtctc gatcgaggag agcctgtcaa
2401 cagggcagac gaggtcgcgc gagagcacga catctcgtac aacgagcagc ttgaggcggg
2461 agacaacccc tacctcaagt acaaccacgc ggacgccgag tttcaggaga agctcgccga
2521 cgacacatcc ttcgggggaa acctcggaaa ggcagtcttt caggccaaga aaagggttct
2581 cgaacctttt ggcctggttg aagagggtgc taagacggcc cctaccggaa agcggataga
2641 cgaccacttt ccaaaaagaa agaaggctcg gaccgaagag gactccaagc cttccacctc
2701 gtcagacgcc gaagctggac ccagcggatc ccagcagctg caaatcccag cccaaccagc
2761 ctcaagtttg ggagctgata caatgtctgc gggaggtggc ggcccattgg gcgacaataa
2821 ccaaggtgcc gatggagtgg gcaatgcctc gggagattgg cattgcgatt ccacgtggat
177
CA 3054600 2019-09-06
2881 gggggacaga gtcgtcacca agtccacccg aacctgggtg ctgcccagct acaacaacca
2941 ccagtaccga gagatcaaaa gcggctccgt cgacggaagc aacgccaacg cctactttgg
3001 atacagcacc ccctgggggt actttgactt taaccgcttc cacagccact ggagcccccg
3061 agactggcaa agactcatca acaactactg gggcttcaga ccccggtccc tcagagtcaa
3121 aatcttcaac attcaagtca aagaggtcac ggtgcaggac tccaccacca ccatcgccaa
3181 caacctcacc tccaccgtcc aagtgtttac ggacgacgac taccagctgc cctacgtcgt
3241 cggcaacggg accgagggat gcctgccggc cttccctccg caggtcttta cgctgccgca
3301 gtacggttac gcgacgctga accgcgacaa cacagaaaat cccaccgaga ggagcagctt
3361 cttctgccta gagtactttc ccagcaagat gctgagaacg ggcaacaact ttgagtttac
3421 ctacaacttt gaggaggtgc ccttccactc cagcttcgct cccagtcaga acctgttcaa
3481 gctggccaac ccgctggtgg accagtactt gtaccgcttc gtgagcacaa ataacactgg
3541 cggagtccag ttcaacaaga acctggccgg gagatacgcc aacacctaca aaaactggtt
3601 cccggggccc atgggccgaa cccagggctg gaacctgggc tccggggtca accgcgccag
3661 tgtcagcgcc ttcgccacga ccaataggat ggagctcgag ggcgcgagtt accaggtgcc
3721 cccgcagccg aacggcatga ccaacaacct ccagggcagc aacacctatg ccctggagaa
3781 cactatgatc ttcaacagcc agccggcgaa cccgggcacc accgccacgt acctcgaggg
3841 caacatgctc atcaccagcg agagcgagac gcagccggtg aaccgcgtgg cgtacaacgt
3901 cggcgggcag atggccacca acaaccagag ctccaccact gcccccgcga ccggcacgta
3961 caacctccag gaaatcgtgc ccggcagcgt gtggatggag agggacgtgt acctccaagg
4021 acccatctgg gccaagatcc cagagacggg ggcgcacttt cacccctctc cggccatggg
4081 cggattcgga ctcaaacacc caccgcccat gatgctcatc aagaacacgc ctgtgcccgg
4141 aaatatcacc agcttctcgg acgtgcccgt cagcagcttc atcacccagt acagcaccgg
4201 gcaggtcacc gtggagatgg agtgggagct caagaaggaa aactccaaga ggtggaaccc
4261 agagatccag tacacaaaca actacaacga cccccagttt gtggactttg ccccggacag
4321 caccggggaa tacagaacca ccagacctat cggaacccga taccttaccc gaccccttta
4381 acccattcat gtcgcatacc ctcaataaac cgtgtattcg tgtcagtaaa atactgcctc
4441 ttgtggtcat tcaatgaata acagcttaca acatctacaa aacctccttg cttgagagtg
4501 tggcactctc ccccctgtcg cgttcgctcg ctcgctggct cgtttggggg ggtggcagct
4561 caaagagctg ccagacgacg gccctctggc cgtcgccccc ccaaacgagc cagcgagcga
4621 gcgaacgcga caggggggag ag
178
CA 3054600 2019-09-06
AAV6 (SEQ ID NO:132)
1 ttggccactc cctctctgcg cgctcgctcg ctcactgagg ccgggcgacc aaaggtcgcc
61 cgacgcccgg gctttgcccg ggcggcctca gtgagcgagc gagcgcgcag agagggagtg
121 gccaactcca tcactagggg ttcctggagg ggtggagtcg tgacgtgaat tacgtcatag
181 ggttagggag gtcctgtatt agaggtcacg tgagtgtttt gcgacatttt gcgacaccat
241 gtggtcacgc tgggtattta agcccgagtg agcacgcagg gtctccattt tgaagcggga
301 ggtttgaacg cgcagcgcca tgccggggtt ttacgagatt gtgattaagg tccccagcga
361 ccttgacgag catctgcccg gcatttctga cagctttgtg aactgggtgg ccgagaagga
421 atgggagttg ccgccagatt ctgacatgga tctgaatctg attgagcagg cacccctgac
481 cgtggccgag aagctgcagc gcgacttcct ggtccagtgg cgccgcgtga gtaaggcccc
541 ggaggccctc ttctttgttc agttcgagaa gggcgagtcc tacttccacc tccatattct
601 ggtggagacc acgggggtca aatccatggt gctgggccgc ttcctgagtc agattaggga
661 caagctggtg cagaccatct accgcgggat cgagccgacc ctgcccaact ggttcgcggt
721 gaccaagacg cgtaatggcg ccggaggggg gaacaaggtg gtggacgagt gctacatccc
781 caactacctc ctgcccaaga ctcagcccga gctgcagtgg gcgtggacta acatggagga
841 gtatataagc gcgtgtttaa acctggccga gcgcaaacgg ctcgtggcgc acgacctgac
901 ccacgtcagc cagacccagg agcagaacaa ggagaatctg aaccccaatt ctgacgcgcc
961 tgtcatccgg tcaaaaacct ccgcacgcta catggagctg gtcgggtggc tggtggaccg
1021 gggcatcacc tccgagaagc agtggatcca ggaggaccag gcctcgtaca tctccttcaa
1081 cgccgcctcc aactcgcggt cccagatcaa ggccgctctg gacaatgccg gcaagatcat
1141 ggcgctgacc aaatccgcgc ccgactacct ggtaggcccc gctccgcccg ccgacattaa
1201 aaccaaccgc atttaccgca tcctggagct gaacggctac gaccctgcct acgccggctc
1261 cgtctttctc ggctgggccc agaaaaggtt cggaaaacgc aacaccatct ggctgtttgg
1321 gccggccacc acgggcaaga ccaacatcgc ggaagccatc gcccacgccg tgcccttcta
1381 cggctgcgtc aactggacca atgagaactt tcccttcaac gattgcgtcg acaagatggt
1441 gatctggtgg gaggagggca agatgacggc caaggtcgtg gagtccgcca aggccattct
1501 cggcggcagc aaggtgcgcg tggaccaaaa gtgcaagtcg tccgcccaga tcgatcccac
1561 ccccgtgatc gtcacctcca acaccaacat gtgcgccgtg attgacggga acagcaccac
1621 cttcgagcac cagcagccgt tgcaggaccg gatgttcaaa tttgaactca cccgccgtct
1681 ggagcatgac tttggcaagg tgacaaagca ggaagtcaaa gagttcttcc gctgggcgca
1741 ggatcacgtg accgaggtgg cgcatgagtt ctacgtcaga aagggtggag ccaacaagag
1801 acccgccccc gatgacgcgg ataaaagcga gcccaagcgg gcctgcccct cagtcgcgga
1861 tccatcgacg tcagacgcgg aaggagctcc ggtggacttt gccgacaggt accaaaacaa
1921 atgttctcgt cacgcgggca tgcttcagat gctgtttccc tgcaaaacat gcgagagaat
1981 gaatcagaat ttcaacattt gcttcacgca cgggaccaga gactgttcag aatgtttccc
2041 cggcgtgtca gaatctcaac cggtcgtcag aaagaggacg tatcggaaac tctgtgccat
2101 tcatcatctg ctggggcggg ctcccgagat tgcttgctcg gcctgcgatc tggtcaacgt
2161 ggatctggat gactgtgttt ctgagcaata aatgacttaa accaggtatg gctgccgatg
2221 gttatcttcc agattggctc gaggacaacc tctctgaggg cattcgcgag tggtgggact
2281 tgaaacctgg agccccgaaa cccaaagcca accagcaaaa gcaggacgac ggccggggtc
2341 tggtgcttcc tggctacaag tacctcggac ccttcaacgg actcgacaag ggggagcccg
2401 tcaacgcggc ggatgcagcg gccctcgagc acgacaaggc ctacgaccag cagctcaaag
2461 cgggtgacaa tccgtacctg cggtataacc acgccgacgc cgagtttcag gagcgtctgc
2521 aagaagatac gtcttttggg ggcaacctcg ggcgagcagt cttccaggcc aagaagaggg
2581 ttctcgaacc ttttggtctg gttgaggaag gtgctaagac ggctcctgga aagaaacgtc
2641 cggtagagca gtcgccacaa gagccagact cctcctcggg cattggcaag acaggccagc
2701 agcccgctaa aaagagactc aattttggtc agactggcga ctcagagtca gtccccgacc
2761 cacaacctct cggagaacct ccagcaaccc ccgctgctgt gggacctact acaatggctt
2821 caggcggtgg cgcaccaatg gcagacaata acgaaggcgc cgacggagtg ggtaatgcct
179
CA 3054600 2019-09-06
2881 caggaaattg gcattgcgat tccacatggc tgggcgacag agtcatcacc accagcaccc
2941 gaacatgggc cttgcccacc tataacaacc acctctacaa gcaaatctcc agtgcttcaa
3001 cgggggccag caacgacaac cactacttcg gctacagcac cccctggggg tattttgatt
3061 tcaacagatt ccactgccat ttctcaccac gtgactggca gcgactcatc aacaacaatt
3121 ggggattccg gcccaagaga ctcaacttca agctcttcaa catccaagtc aaggaggtca
3181 cgacgaatga tggcgtcacg accatcgcta ataaccttac cagcacggtt caagtcttct
3241 cggactcgga gtaccagttg ccgtacgtcc tcggctctgc gcaccagggc tgcctccctc
3301 cgttcccggc ggacgtgttc atgattccgc agtacggcta cctaacgctc aacaatggca
3361 gccaggcagt gggacggtca tccttttact gcctggaata tttcccatcg cagatgctga
3421 gaacgggcaa taactttacc ttcagctaca ccttcgagga cgtgcctttc cacagcagct
3481 acgcgcacag ccagagcctg gaccggctga tgaatcctct catcgaccag tacctgtatt
3541 acctgaacag aactcagaat cagtccggaa gtgcccaaaa caaggacttg ctgtttagcc
3601 gggggtctcc agctggcatg tctgttcagc ccaaaaactg gctacctgga ccctgttacc
3661 ggcagcagcg cgtttctaaa acaaaaacag acaacaacaa cagcaacttt acctggactg
3721 gtgcttcaaa atataacctt aatgggcgtg aatctataat caaccctggc actgctatgg
3781 cctcacacaa agacgacaaa gacaagttct ttcccatgag cggtgtcatg atttttggaa
3841 aggagagcgc cggagcttca aacactgcat tggacaatgt catgatcaca gacgaagagg
3901 aaatcaaagc cactaacccc gtggccaccg aaagatttgg gactgtggca gtcaatctcc
3961 agagcagcag cacagaccct gcgaccggag atgtgcatgt tatgggagcc ttacctggaa
4021 tggtgtggca agacagagac gtatacctgc agggtcctat ttgggccaaa attcctcaca
4081 cggatggaca ctttcacccg tctcctctca tgggeggctt tggacttaag cacccgcctc
4141 ctcagatcct catcaaaaac acgcctgttc ctgcgaatcc tccggcagag ttttcggcta
4201 caaagtttgc ttcattcatc acccagtatt ccacaggaca agtgagcgtg gagattgaat
4261 gggagctgca gaaagaaaac agcaaacgct ggaatcccga agtgcagtat acatctaact
4321 atgcaaaatc tgccaacgtt gatttcactg tggacaacaa tggactttat actgagcctc
4381 gccccattgg cacccgttac ctcacccgtc ccctgtaatt gtgtgttaat caataaaccg
4441 gttaattcgt gtcagttgaa ctttggtctc atgtcgttat tatcttatct ggtcaccata
4501 gcaaccggtt acacattaac tgcttagttg cgcttcgcga atacccctag tgatggagtt
4561 gcccactccc tctatgcgcg ctcgctcgct cggtggggcc ggcagagcag agctctgccg
4621 tctgcggacc tttggtccgc aggccccacc gagcgagcga gcgcgcatag agggagtggg
4681 caa
180
CA 3054600 2019-09-06
AAV7 (SEQ ID NO:133)
1 ttggccactc cctctatgcg cgctcgctcg ctcggtgggg cctgcggacc aaaggtccgc
61 agacggcaga gctctgctct gccggcccca ccgagcgagc gagcgcgcat agagggagtg
121 gccaactcca tcactagggg taccgcgaag cgcctcccac gctgccgcgt cagcgctgac
181 gtaaatcacg tcatagggga gtggtcctgt attagctgtc acgtgagtgc ttttgcgaca
241 ttttgcgaca ccacgtggcc atttgaggta tatatggccg agtgagcgag caggatctcc
301 attttgaccg cgaaatttga acgagcagca gccatgccgg gtttctacga gatcgtgatc
361 aaggtgccga gcgacctgga cgagcacctg ccgggcattt ctgactcgtt tgtgaactgg
421 gtggccgaga aggaatggga gctgcccccg gattctgaca tggatctgaa tctgatcgag
481 caggcacccc tgaccgtggc cgagaagctg cagcgcgact tcctggtcca atggcgccgc
541 gtgagtaagg ccccggaggc cctgttcttt gttcagttcg agaagggcga gagctacttc
601 caccttcacg ttctggtgga gaccacgggg gtcaagtcca tggtgctagg ccgcttcctg
661 agtcagattc gggagaagct ggtccagacc atctaccgcg gggtcgagcc cacgctgccc
721 aactggttcg cggtgaccaa gacgcgtaat ggcgccggcg gggggaacaa ggtggtggac
781 gagtgctaca tccccaacta cctcctgccc aagacccagc ccgagctgca gtgggcgtgg
841 actaacatgg aggagtatat aagcgcgtgt ttgaacctgg ccgaacgcaa acggctcgtg
901 gcgcagcacc tgacccacgt cagccagacg caggagcaga acaaggagaa tctgaacccc
961 aattctgacg cgcccgtgat caggtcaaaa acctccgcgc gctacatgga gctggtcggg
1021 tggctggtgg accggggcat cacctccgag aagcagtgga tccaggagga ccaggcctcg
1081 tacatctcct tcaacgccgc ctccaactcg cggtcccaga tcaaggccgc gctggacaat
1141 gccggcaaga tcatggcgct gaccaaatcc gcgcccgact acctggtggg gccctcgctg
1201 cccgcggaca ttaaaaccaa ccgcatctac cgcatcctgg agctgaacgg gtacgatcct
1261 gcctacgccg gctccgtctt tctcggctgg gcccagaaaa agttcgggaa gcgcaacacc
1321 atctggctgt ttgggcccgc caccaccggc aagaccaaca ttgcggaagc catcgcccac
1381 gccgtgccct tctacggctg cgtcaactgg accaatgaga actttccctt caacgattgc
1441 gtcgacaaga tggtgatctg gtgggaggag ggcaagatga cggccaaggt cgtggagtcc
1501 gccaaggcca ttctcggcgg cagcaaggtg cgcgtggacc aaaagtgcaa gtcgtccgcc
1561 cagatcgacc ccacccccgt gatcgtcacc tccaacacca acatgtgcgc cgtgattgac
1621 gggaacagca ccaccttcga gcaccagcag ccgttgcagg accggatgtt caaatttgaa
1681 ctcacccgcc gtctggagca cgactttggc aaggtgacga agcaggaagt caaagagttc
1741 ttccgctggg ccagtgatca cgtgaccgag gtggcgcatg agttctacgt cagaaagggc
1801 ggagccagca aaagacccgc ccccgatgac gcggatataa gcgagcccaa gcgggcctgc
1861 ccctcagtcg cggatccatc gacgtcagac gcggaaggag ctccggtgga ctttgccgac
1921 aggtaccaaa acaaatgttc tcgtcacgcg ggcatgattc agatgctgtt tccctgcaaa
1981 acgtgcgaga gaatgaatca gaatttcaac atttgcttca cacacggggt cagagactgt
2041 ttagagtgtt tccccggcgt gtcagaatct caaccggtcg tcagaaaaaa gacgtatcgg
2101 aaactctgcg cgattcatca tctgctgggg cgggcgcccg agattgcttg ctcggcctgc
2161 gacctggtca acgtggacct ggacgactgc gtttctgagc aataaatgac ttaaaccagg
2221 tatggctgcc gatggttatc ttccagattg gctcgaggac aacctctctg agggcattcg
2281 cgagtggtgg gacctgaaac ctggagcccc gaaacccaaa gccaaccagc aaaagcagga
2341 caacggccgg ggtctggtgc ttcctggcta caagtacctc ggaccettca acggactcga
2401 caagggggag cccgtcaacg cggcggacgc agcggccctc gagcacgaca aggcctacga
2461 ccagcagctc aaagcgggtg acaatccgta cctgcggtat aaccacgccg acgccgagtt
2521 tcaggagcgt ctgcaagaag atacgtcatt tgggggcaac ctcgggcgag cagtcttcca
2581 ggccaagaag cgggttctcg aacctctcgg tctggttgag gaaggcgcta agacggctcc
2641 tgcaaagaag agaccggtag agccgtcacc tcagcgttcc cccgactcct ccacgggcat
2701 cggcaagaaa ggccagcagc ccgccagaaa gagactcaat ttcggtcaga ctggcgactc
2761 agagtcagtc cccgaccctc aacctctcgg agaacctcca gcagcgccct ctagtgtggg
2821 atctggtaca gtggctgcag gcggtggcgc accaatggca gacaataacg aaggtgccga
181
CA 3054600 2019-09-06
2881 cggagtgggt aatgcctcag gaaattggca ttgcgattcc acatggctgg gcgacagagt
2941 cattaccacc agcacccgaa cctgggccct gcccacctac aacaaccacc tctacaagca
3001 aatctccagt gaaactgcag gtagtaccaa cgacaacacc tacttcggct acagcacccc
3061 ctgggggtat tttgacttta acagattcca ctgccacttc tcaccacgtg actggcagcg
3121 actcatcaac aacaactggg gattccggcc caagaagctg cggttcaagc tcttcaacat
3181 ccaggtcaag gaggtcacga cgaatgacgg cgttacgacc atcgctaata accttaccag
3241 cacgattcag gtattctcgg actcggaata ccagctgccg tacgtcctcg gctctgcgca
3301 ccagggctgc ctgcctccgt tcccggcgga cgtcttcatg attcctcagt acggctacct
3361 gactctcaac aatggcagtc agtctgtggg acgttcctcc ttctactgcc tggagtactt
3421 cccctctcag atgctgagaa cgggcaacaa ctttgagttc agctacagct tcgaggacgt
3481 gcctttccac agcagctacg cacacagcca gagcctggac cggctgatga atcccctcat
3541 cgaccagtac ttgtactacc tggccagaac acagagtaac ccaggaggca cagctggcaa
3601 tcgggaactg cagttttacc agggcgggcc ttcaactatg gccgaacaag ccaagaattg
3661 gttacctgga ccttgatcc ggcaacaaag agtctccaaa acgctggatc aaaacaacaa
3721 cagcaacttt gcttggactg gtgccaccaa atatcacctg aacggcagaa actcgttggt
3781 taatcccggc gtcgccatgg caactcacaa ggacgacgag gaccgctttt tcccatccag
3841 cggagtcctg atttttggaa aaactggagc aactaacaaa actacattgg aaaatgtgtt
3901 aatgacaaat gaagaagaaa ttcgtcctac taatcctgta gccacggaag aatacgggat
3961 agtcagcagc aacttacaag cggctaatac tgcagcccag acacaagttg tcaacaacca
4021 gggagcctta cctggcatgg tctggcagaa ccgggacgtg tacctgcagg gtcccatctg
4081 ggccaagatt cctcacacgg atggcaactt tcacccgtct cctttgatgg gcggctttgg
4141 acttaaacat ccgcctcctc agatcctgat caagaac act cccgttcccg ctaatcctcc
4201 ggaggtgttt actcctgcca agtttgcttc gttcatcaca cagtacagca ccggacaagt
4261 cagcgtggaa atcgagtggg agctgcagaa ggaaaacagc aagcgctgga acccggagat
4321 tcagtacacc tccaactttg aaaagcagac tggtgtggac tttgccgttg acagccaggg
4381 tgtttactct gagcctcgcc ctattggcac tcgttacctc acccgtaatc tgtaattgca
4441 tgttaatcaa taaaccggtt gattcgtttc agttgaactt tggtctcctg tgcttcttat
4501 cttatcggtt tccatagcaa ctggttacac attaactgct tgggtgcgct tcacgataag
4561 aacactgacg tcaccgcggt acccctagtg atggagttgg ccactccctc tatgcgcgct
4621 cgctcgctcg gtggggcctg cggaccaaag gtccgcagac ggcagagctc tgctctgccg
4681 gccccaccga gcgagcgagc gcgcatagag ggagtggcca a
182
CA 3054600 2019-09-06
AAV8 (SEQ ID NO:134)
1 cagagaggga gtggccaact ccatcactag gggtagcgcg aagcgcctcc cacgctgccg
61 cgtcagcgct gacgtaaatt acgtcatagg ggagtggtcc tgtattagct gtcacgtgag
121 tgcttttgcg gcattttgcg acaccacgtg gccatttgag gtatatatgg ccgagtgagc
181 gagcaggatc tccattttga ccgcgaaatt tgaacgagca gcagccatgc cgggcttcta
241 cgagatcgtg atcaaggtgc cgagcgacct ggacgagcac ctgccgggca tttctgactc
301 gtttgtgaac tgggtggccg agaaggaatg ggagctgccc ccggattctg acatggatcg
361 gaatctgatc gagcaggcac ccctgaccgt ggccgagaag ctgcagcgcg acttcctggt
421 ccaatggcgc cgcgtgagta aggccccgga ggccctcttc tttgttcagt tcgagaaggg
481 cgagagctac tttcacctgc acgttctggt cgagaccacg ggggtcaagt ccatggtgct
541 aggccgcttc ctgagtcaga ttcgggaaaa gcttggtcca gaccatctac ccgcggggtc
601 gagccccacc ttgcccaact ggttcgcggt gaccaaagac gcggtaatgg cgccggcggg
661 ggggaacaag gtggtggacg agtgctacat ccccaactac ctcctgccca agactcagcc
721 cgagctgcag tgggcgtgga ctaacatgga ggagtatata agcgcgtgct tgaacctggc
781 cgagcgcaaa cggctcgtgg cgcagcacct gacccacgtc agccagacgc aggagcagaa
841 caaggagaat ctgaacccca attctgacgc gcccgtgatc aggtcaaaaa cctccgcgcg
901 ctatatggag ctggtcgggt ggctggtgga ccggggcatc acctccgaga agcagtggat
961 ccaggaggac caggcctcgt acatctcctt caacgccgcc tccaactcgc ggtcccagat
1021 caaggccgcg ctggacaatg ccggcaagat catggcgctg accaaatccg cgcccgacta
1081 cctggtgggg ccctcgctgc ccgcggacat tacccagaac cgcatctacc gcatcctcgc
1141 tctcaacggc tacgaccctg cctacgccgg ctccgtcttt ctcggctggg ctcagaaaaa
1201 gttcgggaaa cgcaacacca tctggctgtt tggacccgcc accaccggca agaccaacat
1261 tgcggaagcc atcgcccacg ccgtgccctt ctacggctgc gtcaactgga ccaatgagaa
1321 ctttcccttc aatgattgcg tcgacaagat ggtgatctgg tgggaggagg gcaagatgac
1381 ggccaaggtc gtggagtccg ccaaggccat tctcggcggc agcaaggtgc gcgtggacca
1441 aaagtgcaag tcgtccgccc agatcgaccc cacccccgtg atcgtcacct ccaacaccaa
1501 catgtgcgcc gtgattgacg ggaacagcac caccttcgag caccagcagc ctctccagga
1561 ccggatgttt aagttcgaac tcacccgccg tctggagcac gactttggca aggtgacaaa
1621 gcaggaagtc aaagagttct tccgctgggc cagtgatcac gtgaccgagg tggcgcatga
1681 gttttacgtc agaaagggcg gagccagcaa aagacccgcc cccgatgacg cggataaaag
1741 cgagcccaag cgggcctgcc cctcagtcgc ggatccatcg acgtcagacg cggaaggagc
1801 tccggtggac tttgccgaca ggtaccaaaa caaatgttct cgtcacgcgg gcatgcttca
1861 gatgctgttt ccctgcaaaa cgtgcgagag aatgaatcag aatttcaaca tttgcttcac
1921 acacggggtc agagactgct cagagtgttt ccccggcgtg tcagaatctc aaccggtcgt
1981 cagaaagagg acgtatcgga aactctgtgc gattcatcat ctgctggggc gggctcccga
2041 gattgcttgc tcggcctgcg atctggtcaa cgtggacctg gatgactgtg tttctgagca
2101 ataaatgact taaaccaggt atggctgccg atggttatct tccagattgg ctcgaggaca
2161 acctctctga gggcattcgc gagtggtggg cgctgaaacc tggagccccg aagcccaaag
2221 ccaaccagca aaagcaggac gacggccggg gtctggtgct tcctggctac aagtacctcg
2281 gacccttcaa cggactcgac aagggggagc ccgtcaacgc ggcggacgca gcggccctcg
2341 agcacgacaa ggcctacgac cagcagctgc aggcgggtga caatccgtac ctgcggtata
2401 accacgccga cgccgagttt caggagcgtc tgcaagaaga tacgtctttt gggggcaacc
2461 tcgggcgagc agtcttccag gccaagaagc gggttctcga acctctcggt ctggttgagg
2521 aaggcgctaa gacggctcct ggaaagaaga gaccggtaga gccatcaccc cagcgttctc
2581 cagactcctc tacgggcatc ggcaagaaag gccaacagcc cgccagaaaa agactcaatt
2641 ttggtcagac tggcgactca gagtcagttc cagaccctca acctctcgga gaacctccag
2701 cagcgccctc tggtgtggga cctaatacaa tggctgcagg cggtggcgca ccaatggcag
2761 acaataacga aggcgccgac ggagtgggta gttcctcggg aaattggcat tgcgattcca
2821 catggctggg cgacagagtc atcaccacca gcacccgaac ctgggccctg cccacctaca
183
CA 3054600 2019-09-06
2881 acaaccacct ctacaagcaa atctccaacg ggacatcggg aggagccacc aacgacaaca
2941 cctacttcgg ctacagcacc ccctgggggt attttgactt taacagattc cactgccact
3001 tttcaccacg tgactggcag cgactcatca acaacaactg gggattccgg cccaagagac
3061 tcagcttcaa gctcttcaac atccaggtca aggaggtcac gcagaatgaa ggcaccaaga
3121 ccatcgccaa taacctcacc agcaccatcc aggtgtttac ggactcggag taccagctgc
3181 cgtacgttct cggctctgcc caccagggct gcctgcctcc gttcccggcg gacgtgttca
3241 tgattcccca gtacggctac ctaacactca acaacggtag tcaggccgtg ggacgctcct
3301 ccttctactg cctggaatac tttccttcgc agatgctgag aaccggcaac aacttccagt
3361 ttacttacac cttcgaggac gtgcctttcc acagcagcta cgcccacagc cagagcttgg
3421 accggctgat gaatcctctg attgaccagt acctgtacta cttgtctcgg actcaaacaa
3481 caggaggcac ggcaaatacg cagactctgg gcttcagcca aggtgggcct aatacaatgg
3541 ccaatcaggc aaagaactgg ctgccaggac cctgttaccg ccaacaacgc gtctcaacga
3601 caaccgggca aaacaacaat agcaactttg cctggactgc tgggaccaaa taccatctga
3661 atggaagaaa ttcattggct aatcctggca tcgctatggc aacacacaaa gacgacgagg
3721 agcgatttt tcccagtaac gggatcctga tttttggcaa acaaaatgct gccagagaca
3781 atgcggatta cagcgatgtc atgctcacca gcgaggaaga aatcaaaacc actaaccctg
3841 tggctacaga ggaatacggt atcgtggcag ataacttgca gcagcaaaac acggctcctc
3901 aaattggaac tgtcaacagc cagggggcct tacccggtat ggtctggcag aaccgggacg
3961 tgtacctgca gggtcccatc tgggccaaga ttcctcacac ggacggcaac ttccacccgt
4021 ctccgctgat gggcggcttt ggcctgaaac atcctccgcc tcagatcctg atcaagaaca
4081 cgcctgtacc tgcggatcct ccgaccacct tcaaccagtc aaagctgaac tctttcatca
4141 cgcaatacag caccggacag gtcagcgtgg aaattgaatg ggagctgcag aaggaaaaca
4201 gcaagcgctg gaaccccgag atccagtaca cctccaacta ctacaaatct acaagtgtgg
4261 actttgctgt taatacagaa ggcgtgtact ctgaaccccg ccccattggc acccgttacc
4321 tcacccgtaa tctgtaattg cctgttaatc aataaaccgg ttgattcgtt tcagttgaac
4381 tttggIctct gcg
184
CA 3054600 2019-09-06
AAV9 (SEQ ID NO:135)
1 gcccaatacg caaaccgcct ctccccgcgc gttggccgat tcattaatgc agctggcgta
61 atagcgaaga ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg aatggcgaat
121 ggcgattccg ttgcaatggc tggcggtaat attgttctgg atattaccag caaggccgat
181 agtttgagtt cttctactca ggcaagtgat gttattacta atcaaagaag tattgcgaca
241 acggttaatt tgcgtgatgg acagactctt ttactcggtg gcctcactga ttataaaaac
301 acttctcagg attctggcgt accgttcctg tctaaaatcc ctttaatcgg cctcctgttt
361 agctcccgct ctgattctaa cgaggaaagc acgttatacg tgctcgtcaa agcaaccata
421 gtacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac
481 cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc
541 cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt
601 tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt cacgtagtgg
661 gccatcgccc tgatagacgg atttcgccc tttgacgttg gagtccacgt tctttaatag
721 tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt cttttgattt
781 ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt
841 taacgcgaat tttaacaaaa tattaacgct tacaatttaa atatttgctt atacaatctt
901 cctgtttttg gggcttttct gattatcaac cggggtacat atgattgaca tgctagtttt
961 acgattaccg ttcatcgccc tgcgcgctcg ctcgctcact gaggccgccc gggcaaagcc
1021 cgggcgtcgg gcgacctttg gtcgcccggc ctcagtgagc gagcgagcgc gcagagaggg
1081 agtggaattc acgcgtggat ctgaattcaa ttcacgcgtg gtacctctgg tcgttacata
1141 acttacggta aatggcccgc ctggctgacc gcccaacgac ccccgcccat tgacgtcaat
1201 aatgacgtat gttcccatag taacgccaat agggactttc cattgacgtc aatgggtgga
1261 gtatttacgg taaactgccc acttggcagt acatcaagtg tatcatatgc caagtacgcc
1321 ccctattgac gtcaatgacg gtaaatggcc cgcctggcat tatgcccagt acatgacctt
1381 atgggacttt cctacttggc agtacatcta ctcgaggcca cgttctgat cactctcccc
1441 atctcccccc cctccccacc cccaattttg tatttattta ttttttaatt attttgtgca
1501 gcgatggggg cggggggggg gggggggcgc gcgccaggcg gggcggggcg gggcgagggg
1561 cggggcgggg cgaggcggag aggtgcggcg gcagccaatc agagcggcgc gctccgaaag
1621 tttcctttta tggcgaggcg gcggcggcgg cggccctata aaaagcgaag cgcgcggcgg
1681 gcgggagcgg gatcagccac cgcggtggcg gcctagagtc gacgaggaac tgaaaaacca
1741 gaaagttaac tggtaagttt agtattttg tatttattt caggtcccgg atccggtggt
1801 ggtgcaaatc aaagaactgc tcctcagtgg atgttgcctt tacttctagg cctgtacgga
1861 agtgttactt ctgctctaaa agctgcggaa ttgtacccgc ggccgatcca ccggtccgga
1921 attcccggga tatcgtcgac ccacgcgtcc gggccccacg ctgcgcaccc gcgggtttgc
1981 tatggcgatg agcagcggcg gcagtggtgg cggcgtcccg gagcaggagg attccgtgct
2041 gttccggcgc ggcacaggcc agagcgatga ttctgacatt tgggatgata cagcactgat
2101 aaaagcatat gataaagctg tggcttcatt taagcatgct ctaaagaatg gtgacatttg
2161 tgaaacttcg ggtaaaccaa aaaccacacc taaaagaaaa cctgctaaga agaataaaag
2221 ccaaaagaag aatactgcag cttccttaca acagtggaaa gttggggaca aatgttctgc
2281 catttggtca gaagacggtt gcatttaccc agctaccatt gcttcaattg attttaagag
2341 agaaacctgt gttgtggttt acactggata tggaaataga gaggagcaaa atctgtccga
2401 tctactttcc ccaatctgtg aagtagctaa taatatagaa cagaatgctc aagagaatga
2461 aaatgaaagc caagtttcaa cagatgaaag tgagaactcc aggtctcctg gaaataaatc
2521 agataacatc aagcccaaat ctgctccatg gaactattt ctccctccac caccccccat
2581 gccagggcca agactgggac caggaaagcc aggtctaaaa ttcaatggcc caccaccgcc
2641 accgccacca ccaccacccc acttactatc atgctggctg cctccatttc cttctggacc
2701 accaataatt cccccaccac ctcccatatg tccagattct cttgatgatg ctgatgcttt
2761 gggaagtatg ttaatttcat ggtacatgag tggctatcat actggctatt atatgggttt
2821 tagacaaaat caaaaagaag gaaggtgctc acattcctta aattaaggag aaatgctggc
185
CA 3054600 2019-09-06
2881 atagagcagc actaaatgac accactaaag aaacgatcag acagatctag aaagcttatc
2941 gataccgtcg actagagctc gctgatcagc ctcgactgtg ccttctagtt gccagccatc
3001 tgttgtttgc ccctcccccg tgccttcctt gaccctggaa ggtgccactc ccactgtcct
3061 ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg
3121 gggtggggtg gggcaggaca gcaaggggga ggattgggaa gacaatagca ggcatgctgg
3181 ggagagatcg atctgaggaa cccctagtga tggagttggc cactccctct ctgcgcgctc
3241 gctcgctcac tgaggccggg cgaccaaagg tcgcccgacg cccgggcttt gcccgggcgg
3301 cctcagtgag cgagcgagcg cgcagagagg gagtggcccc cccccccccc cccccggcga
3361 ttctcttgtt tgctccagac tctcaggcaa tgacctgata gcctttgtag agacctctca
3421 aaaatagcta ccctctccgg catgaattta tcagctagaa cggttgaata tcatattgat
3481 ggtgatttga ctgtctccgg cctttctcac ccgtttgaat ctttacctac acattactca
3541 ggcattgcat ttaaaatata tgagggttct aaaaattttt atccttgcgt tgaaataaag
3601 gcttctcccg caaaagtatt acagggtcat aatgtttttg gtacaaccga tttagcttta
3661 tgctctgagg ctttattgct taattttgct aattctttgc cttgcctgta tgatttattg
3721 gatgttggaa tcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc
3781 gcatatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc cagccccgac
3841 acccgccaac actatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc
3901 agccccgaca cccgccaaca cccgctgacg cgccctgacg ggcttgtctg ctcccggcat
3961 ccgcttacag acaagctgtg accgtctccg ggagctgcat gtgtcagagg ttttcaccgt
4021 catcaccgaa acgcgcgaga cgaaagggcc tcgtgatacg cctattttta taggttaatg
4081 tcatgataat aatggtttct tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa
4141 cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac
4201 cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg
4261 tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc
4321 tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg
4381 atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga
4441 gcacttttaa agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc
4501 aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag
4561 aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga
4621 gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg
4681 cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga
4741 atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt
4801 tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact
4861 ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt
4921 ttattgctga taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg
4981 ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta
5041 tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac
5101 tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat ttttaattta
5161 aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt
5221 tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt
5281 tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt
5341 gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc
5401 agataccaaa tactgttctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg
5461 tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg
5521 ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt
5581 cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac
5641 tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg
5701 acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg
5761 gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat
5821 ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt
186
CA 3054600 2019-09-06
5881 tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg
5941 attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa
6001 cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga gc
187
CA 3054600 2019-09-06
AAV10 (SEQ ID NO:136)
1 atgccgggct tctacgagat cgtgatcaag gtgccgagcg acctggacga gcacctgccg
61 ggcatttctg actcgtttgt gaactgggtg gccgagaagg aatgggagct gcccccggat
121 tctgacatgg atcggaatct gatcgagcag gcacccctga ccgtggccga gaagctgcag
181 cgcgacttcc tggtccactg gcgccgcgtg agtaaggccc cggaggccct cttctttgtt
241 cagttcgaga agggcgagtc ctactttcac ctgcacgttc tggtcgagac cacgggggtc
301 aagtccatgg tcctgggccg cacctgagt cagatcagag acaggctggt gcagaccatc
361 taccgcgggg tagagcccac gctgcccaac tggttcgcgg tgaccaagac gcgaaatggc
421 gccggcgggg ggaacaaggt ggtggacgag tgctacatcc ccaactacct cctgcccaag
481 acgcagcccg agctgcagtg ggcgtggact aacatggagg agtatataag cgcgtgtctg
541 aacctcgcgg agcgtaaacg gctcgtggcg cagcacctga cccacgtcag ccagacgcag
601 gagcagaaca aggagaatct gaacccgaat tctgacgcgc ccgtgatcag gtcaaaaacc
661 tccgcgcgct acatggagct ggtcgggtgg ctggtggacc ggggcatcac ctccgagaag
721 cagtggatcc aggaggacca ggcctcgtac atctccttca acgccgcctc caactcgcgg
781 tcccagatca aggccgcgct ggacaatgcc ggaaagatca tggcgctgac caaatccgcg
841 cccgactacc tggtaggccc gtccttaccc gcggacatta aggccaaccg catctaccgc
901 atcctggagc tcaacggcta cgaccccgcc tacgccggct ccgtcttcct gggctgggcg
961 cagaaaaagt tcggtaaaag gaatacaatt tggctgttcg ggcccgccac caccggcaag
1021 accaacatcg cggaagccat cgcccacgcc gtgcccttct acggctgcgt caactggacc
1081 aatgagaact ttccatcaa cgattgcgtc gacaagatgg tgatctggtg ggaggagggc
1141 aagatgaccg ccaaggtcgt ggagtccgcc aaggccattc tgggcggaag caaggtgcgc
1201 gtcgaccaaa agtgcaagtc ctcggcccag atcgacccca cgcccgtgat cgtcacctcc
1261 aacaccaaca tgtgcgccgt gatcgacggg aacagcacca ccttcgagca ccagcagccc
1321 ctgcaggacc gcatgttcaa gttcgagctc acccgccgtc tggagcacga ctttggcaag
1381 gtgaccaagc aggaagtcaa agagttcttc cgctgggctc aggatcacgt gactgaggtg
1441 acgcatgagt tctacgtcag aaagggcgga gccaccaaaa gacccgcccc cagtgacgcg
1501 gatataagcg agcccaagcg ggcctgcccc tcagttgcgg agccatcgac gtcagacgcg
1561 gaagcaccgg tggactttgc ggacaggtac caaaacaaat gttctcgtca cgcgggcatg
1621 cttcagatgc tgatccctg caagacatgc gagagaatga atcagaattt caacgtctgc
1681 ttcacgcacg gggtcagaga ctgctcagag tgcttccccg gcgcgtcaga atctcaacct
1741 gtcgtcagaa aaaagacgta tcagaaactg tgcgcgattc atcatctgct ggggcgggca
1801 cccgagattg cgtgttcggc ctgcgatctc gtcaacgtgg acttggatga ctgtgtttct
1861 gagcaataaa tgacttaaac caggtatggc tgctgacggt tatcttccag attggctcga
1921 ggacaacctc tctgagggca ttcgcgagtg gtgggacctg aaacctggag cccccaagcc
1981 caaggccaac cagcagaagc aggacgacgg ccggggtctg gtgcttcctg gctacaagta
2041 cctcggaccc ttcaacggac tcgacaaggg ggagcccgtc aacgcggcgg acgcagcggc
2101 cctcgagcac gacaaggcct acgaccagca gctcaaagcg ggtgacaatc cgtacctgcg
2161 gtataaccac gccgacgccg agtttcagga gcgtctgcaa gaagatacgt cttttggggg
2221 caacctcggg cgagcagtct tccaggccaa gaagegggtt ctcgaacctc tcggtctggt
2281 tgaggaagct gctaagacgg ctcctggaaa gaagagaccg gtagaaccgt cacctcagcg
2341 ttcceccgac tcctccacgg gcatcggcaa gaaaggccag cagcccgcta aaaagagact
2401 gaactttggg cagactggcg agtcagagtc agtccccgac cctcaaccaa tcggagaacc
2461 accagcaggc ccctctggtc tgggatctgg tacaatggct gcaggcggtg gcgctccaat
2521 ggcagacaat aacgaaggcg ccgacggagt gggtagttcc tcaggaaatt ggcattgcga
2581 ttccacatgg ctgggcgaca gagtcatcac caccagcacc cgaacctggg ccctgcccac
2641 ctacaacaac cacctctaca agcaaatctc caacgggaca tcgggaggaa gcaccaacga
2701 caacacctac ttcggctaca gcaccccctg ggggtatttt gacttcaaca gattccactg
2761 ccacttctca ccacgtgact ggcagcgact catcaacaac aactggggat tccggccaaa
2821 aagactcagc ttcaagctct tcaacatcca ggtcaaggag gtcacgcaga atgaaggcac
188
CA 3054600 2019-09-06
2881 caagaccatc gccaataacc ttaccagcac gattcaggta tttacggact cggaatacca
2941 gctgccgtac gtcctcggct ccgcgcacca gggctgcctg cctccgttcc cggcggatgt
3001 cttcatgatt ccccagtacg gctacctgac actgaacaat ggaagtcaag ccgtaggccg
3061 ttcctccttc tactgcctgg aatattttcc atctcaaatg ctgcgaactg gaaacaattt
3121 tgaattcagc tacaccttcg aggacgtgcc tttccacagc agctacgcac acagccagag
3181 cttggaccga ctgatgaatc ctctcattga ccagtacctg tactacttat ccagaactca
3241 gtccacagga ggaactcaag gtacccagca attgttattt tctcaagctg ggcctgcaaa
3301 catgtcggct caggccaaga actggctgcc tggaccttgc taccggcagc agcgagtctc
3361 cacgacactg tcgcaaaaca acaacagcaa ctttgcttgg actggtgcca ccaaatatca
3421 cctgaacgga agagactctc tggtgaatcc cggtgtcgcc atggcaaccc acaaggacga
3481 cgaggaacgc ttcttcccgt cgagcggagt cctgatgttt ggaaaacagg gtgctggaag
3541 agacaatgtg gactacagca gcgttatgct aacaagcgaa gaagaaatta aaaccactaa
3601 ccctgtagcc acagaacaat acggcgtggt ggctgacaac ttgcagcaag ccaatacagg
3661 gcctattgtg ggaaatgtca acagccaagg agccttacct ggcatggtct ggcagaaccg
3721 agacgtgtac ctgcagggtc ccatctgggc caagattcct cacacggacg gcaactttca
3781 cccgtctcct ctgatgggcg gctttggact taaacacccg cctccacaga tcctgatcaa
3841 gaacacgccg gtacctgcgg atcctccaac aacgttcagc caggcgaaat tggcttcctt
3901 catcacgcag tacagcaccg gacaggtcag cgtggaaatc gagtgggagc tgcagaagga
3961 gaacagcaaa cgctggaacc cagagattca gtacacttca aactactaca aatctacaaa
4021 tgtggacttt gctgtcaata cagagggaac ttattctgag cctcgcccca ttggtactcg
4081 ttatctgaca cgtaatctgt aa
189
CA 3054600 2019-09-06
AAV11 (SEQ ID NO:137)
1 atgccgggct tctacgagat cgtgatcaag gtgccgagcg acctggacga gcacctgccg
61 ggcatttctg actcgtttgt gaactgggtg gccgagaagg aatgggagct gcccccggat
121 tctgacatgg atcggaatct gatcgagcag gcacccctga ccgtggccga gaagctgcag
181 cgcgacttcc tggtccactg gcgccgcgtg agtaaggccc cggaggccct cttctttgtt
241 cagttcgaga agggcgagtc ctacttccac ctccacgttc tcgtcgagac cacgggggtc
301 aagtccatgg tcctgggccg cttcctgagt cagatcagag acaggctggt gcagaccatc
361 taccgcgggg tcgagcccac gctgcccaac tggttcgcgg tgaccaagac gcgaaatggc
421 gccggcgggg ggaacaaggt ggtggacgag tgctacatcc ccaactacct cctgcccaag
481 acccagcccg agctgcagtg ggcgtggact aacatggagg agtatataag cgcgtgtcta
541 aacctcgcgg agcgtaaacg gctcgtggcg cagcacctga cccacgtcag ccagacgcag
601 gagcagaaca aggagaatct gaacccgaat tctgacgcgc ccgtgatcag gtcaaaaacc
661 tccgcgcgct acatggagct ggtcgggtgg ctggtggacc ggggcatcac ctccgagaag
721 cagtggatcc aggaggacca ggcctcgtac atctccttca acgccgcctc caactcgcgg
781 tcccagatca aggccgcgct ggacaatgcc ggaaagatca tggcgctgac caaatccgcg
841 cccgactacc tggtaggccc gtccttaccc gcggacatta aggccaaccg catctaccgc
901 atcctggagc tcaacggcta cgaccccgcc tacgccggct ccgtcttcct gggctgggcg
961 cagaaaaagt tcggtaaacg caacaccatc tggctgtttg ggcccgccac caccggcaag
1021 accaacatcg cggaagccat agcccacgcc gtgcccttct acggctgcgt gaactggacc
1081 aatgagaact ttcccttcaa cgattgcgtc gacaagatgg tgatctggtg ggaggagggc
1141 aagatgaccg ccaaggtcgt ggagtccgcc aaggccattc tgggcggaag caaggtgcgc
1201 gtggaccaaa agtgcaagtc ctcggcccag atcgacccca cgcccgtgat cgtcacctcc
1261 aacaccaaca tgtgcgccgt gatcgacggg aacagcacca ccttcgagca ccagcagccg
1321 ctgcaggacc gcatgttcaa gttcgagctc acccgccgtc tggagcacga ctttggcaag
1381 gtgaccaagc aggaagtcaa agagttcttc cgctgggctc aggatcacgt gactgaggtg
1441 gcgcatgagt tctacgtcag aaagggcgga gccaccaaaa gacccgcccc cagtgacgcg
1501 gatataagcg agcccaagcg ggcctgcccc tcagttccgg agccatcgac gtcagacgcg
1561 gaagcaccgg tggactttgc ggacaggtac caaaacaaat gttctcgtca cgcgggcatg
1621 cttcagatgc tgtaccctg caagacatgc gagagaatga atcagaattt caacgtctgc
1681 ttcacgcacg gggtcagaga ctgctcagag tgcttccccg gcgcgtcaga atctcaaccc
1741 gtcgtcagaa aaaagacgta tcagaaactg tgcgcgattc atcatctgct ggggcgggca
1801 cccgagattg cgtgttcggc ctgcgatctc gtcaacgtgg acttggatga ctgtgtttct
1861 gagcaataaa tgacttaaac caggtatggc tgctgacggt tatcttccag attggctcga
1921 ggacaacctc tctgagggca ttcgcgagtg gtgggacctg aaacctggag ccccgaagcc
1981 caaggccaac cagcagaagc aggacgacgg ccggggtctg gtgcttcctg gctacaagta
2041 cctcggaccc ttcaacggac tcgacaaggg ggagcccgtc aacgcggcgg acgcagcggc
2101 cctcgagcac gacaaggcct acgaccagca gctcaaagcg ggtgacaatc cgtacctgcg
2161 gtataaccac gccgacgccg agtttcagga gcgtctgcaa gaagatacgt cttttggggg
2221 caacctcggg cgagcagtct tccaggccaa gaagagggta ctcgaacctc tgggcctggt
2281 tgaagaaggt gctaaaacgg ctcctggaaa gaagagaccg ttagagtcac cacaagagcc
2341 cgactcctcc tcgggcatcg gcaaaaaagg caaacaacca gccagaaaga ggctcaactt
2401 tgaagaggac actggagccg gagacggacc ccctgaagga tcagatacca gcgccatgtc
2461 ttcagacatt gaaatgcgtg cagcaccggg cggaaatgct gtcgatgcgg gacaaggttc
2521 cgatggagtg ggtaatgcct cgggtgattg gcattgcgat tccacctggt ctgagggcaa
2581 ggtcacaaca acctcgacca gaacctgggt cttgcccacc tacaacaacc acttgtacct
2641 gcgtctcgga acaacatcaa gcagcaacac ctacaacgga ttctccaccc cctggggata
2701 ttttgacttc aacagattcc actgtcactt ctcaccacgt gactggcaaa gactcatcaa
2761 caacaactgg ggactacgac caaaagccat gcgcgttaaa atcttcaata tccaagttaa
2821 ggaggtcaca acgtcgaacg gcgagactac ggtcgctaat aaccttacca gcacggttca
190
CA 3054600 2019-09-06
2881 gatatttgcg gactcgtcgt atgagctccc gtacgtgatg gacgctggac aagaggggag
2941 cctgcctcct ttccccaatg acgtgttcat ggtgcctcaa tatggctact gtggcatcgt
3001 gactggcgag aatcagaacc aaacggacag aaacgctttc tactgcctgg agtattttcc
3061 ttcgcaaatg ttgagaactg gcaacaactt tgaaatggct tacaactttg agaaggtgcc
3121 gttccactca atgtatgctc acagccagag cctggacaga ctgatgaatc ccctcctgga
3181 ccagtacctg tggcacttac agtcgactac ctctggagag actctgaatc aaggcaatgc
3241 agcaaccaca tttggaaaaa tcaggagtgg agactttgcc ttttacagaa agaactggct
3301 gcctgggcct tgtgttaaac agcagagatt ctcaaaaact gccagtcaaa attacaagat
3361 tcctgccagc gggggcaacg ctctgttaaa gtatgacacc cactatacct taaacaaccg
3421 ctggagcaac atcgcgcccg gacctccaat ggccacagcc ggaccttcgg atggggactt
3481 cagtaacgcc cagcttatat tccctggacc atctgttacc ggaaatacaa caacttcagc
3541 caacaatctg ttgtttacat cagaagaaga aattgctgcc accaacccaa gagacacgga
3601 catgtttggc cagattgctg acaataatca gaatgctaca actgctccca taaccggcaa
3661 cgtgactgct atgggagtgc tgcctggcat ggtgtggcaa aacagagaca tttactacca
3721 agggccaatt tgggccaaga tcccacacgc ggacggacat tttcatcctt caccgctgat
3781 tggtgggttt ggactgaaac acccgcctcc ccagatattc atcaagaaca ctcccgtacc
3841 tgccaatcct gcgacaacct tcactgcagc cagagtggac tctttcatca cacaatacag
3901 caccggccag gtcgctgttc agattgaatg ggaaattgaa aaggaacgct ccaaacgctg
3961 gaatcctgaa gtgcagttta cttcaaacta tgggaaccag tcttctatgt tgtgggctcc
4021 tgatacaact gggaagtata cagagccgcg ggttattggc tctcgttatt tgactaatca
4081 tttgtaa
191
CA 3054600 2019-09-06
AAV12 (SEQ ID NO:138)
1 ttgcgacagt ttgcgacacc atgtggtcac aagaggtata taaccgcgag tgagccagcg
61 aggagctcca ttttgcccgc gaagtttgaa cgagcagcag ccatgccggg gttctacgag
121 gtggtgatca aggtgcccag cgacctggac gagcacctgc ccggcatttc tgactccttt
181 gtgaactggg tggccgagaa ggaatgggag ttgcccccgg attctgacat ggatcagaat
241 ctgattgagc aggcacccct gaccgtggcc gagaagctgc agcgcgagtt cctggtggaa
301 tggcgccgag tgagtaaatt tctggaggcc aagtttatg tgcagtttga aaagggggac
361 tcgtactttc atttgcatat tctgattgaa attaccggcg tgaaatccat ggtggtgggc
421 cgctacgtga gtcagattag ggataaactg atccagcgca tctaccgcgg ggtcgagccc
481 cagctgccca actggttcgc ggtcacaaag acccgaaatg gcgccggagg cgggaacaag
541 gtggtggacg agtgctacat ccccaactac ctgctcccca aggtccagcc cgagcttcag
601 tgggcgtgga ctaacatgga ggagtatata agcgcctgtt tgaacctcgc ggagcgtaaa
661 cggctcgtgg cgcagcacct gacgcacgtc tcccagaccc aggagggcga caaggagaat
721 ctgaacccga attctgacgc gccggtgatc cggtcaaaaa cctccgccag gtacatggag
781 ctggtcgggt ggctggtgga caagggcatc acgtccgaga agcagtggat ccaggaggac
841 caggcctcgt acatctcctt caacgcggcc tccaactccc ggtcgcagat caaggcggcc
901 ctggacaatg cctccaaaat catgagcctc accaaaacgg ctccggacta tctcatcggg
961 cagcagcccg tgggggacat taccaccaac cggatctaca aaatcctgga actgaacggg
1021 tacgaccccc agtacgccgc ctccgtcttt ctcggctggg cccagaaaaa gtttggaaag
1081 cgcaacacca tctggctgtt tgggcccgcc accaccggca agaccaacat cgcggaagcc
1141 atcgcccacg cggtcccctt ctacggctgc gtcaactgga ccaatgagaa ctaccatc
1201 aacgactgcg tcgacaaaat ggtgatttgg tgggaggagg gcaagatgac cgccaaggtc
1261 gtagagtccg ccaaggccat tctgggcggc agcaaggtgc gcgtggacca aaaatgcaag
1321 gcctctgcgc agatcgaccc cacccccgtg atcgtcacct ccaacaccaa catgtgcgcc
1381 gtgattgacg ggaacagcac caccttcgag caccagcagc ccctgcagga ccggatgttc
1441 aagtttgaac tcacccgccg cctcgaccac gactttggca aggtcaccaa gcaggaagtc
1501 aaggactttt tccggtgggc ggctgatcac gtgactgacg tggctcatga gttttacgtc
1561 acaaagggtg gagctaagaa aaggcccgcc ccctctgacg aggatataag cgagcccaag
1621 cggccgcgcg tgtcatttgc gcagccggag acgtcagacg cggaagctcc cggagacttc
1681 gccgacaggt accaaaacaa atgttctcgt cacgcgggta tgctgcagat gctctttccc
1741 tgcaagacgt gcgagagaat gaatcagaat tccaacgtct gcttcacgca cggtcagaaa
1801 gattgcgggg agtgctttcc cgggtcagaa tctcaaccgg tttctgtcgt cagaaaaacg
1861 tatcagaaac tgtgcatcct tcatcagctc cggggggcac ccgagatcgc ctgctctgct
1921 tgcgaccaac tcaaccccga tttggacgat tgccaatttg agcaataaat gactgaaatc
1981 aggtatggct gctgacggtt atcttccaga ttggctcgag gacaacctct ctgaaggcat
2041 tcgcgagtgg tgggcgctga aacctggagc tccacaaccc aaggccaacc aacagcatca
2101 ggacaacggc aggggtcttg tgcttcctgg gtacaagtac ctcggaccct tcaacggact
2161 cgacaaggga gagccggtca acgaggcaga cgccgcggcc ctcgagcacg acaaggccta
2221 cgacaagcag ctcgagcagg gggacaaccc gtatctcaag tacaaccacg ccgacgccga
2281 gttccagcag cgcttggcga ccgacacctc ttttgggggc aacctcgggc gagcagtat
2341 ccaggccaaa aagaggattc tcgagcctct gggtctggtt gaagagggcg ttaaaacggc
2401 tcctggaaag aaacgcccat tagaaaagac tccaaatcgg ccgaccaacc cggactctgg
2461 gaaggccccg gccaagaaaa agcaaaaaga cggcgaacca gccgactctg ctagaaggac
2521 actcgacttt gaagactctg gagcaggaga cggaccccct gagggatcat cttccggaga
2581 aatgtctcat gatgctgaga tgcgtgcggc gccaggcgga aatgctgtcg aggcgggaca
2641 aggtgccgat ggagtgggta atgcctccgg tgattggcat tgcgattcca cctggtcaga
2701 gggccgagtc accaccacca gcacccgaac ctgggtccta cccacgtaca acaaccacct
2761 gtacctgcga atcggaacaa cggccaacag caacacctac aacggattct ccaccccctg
2821 gggatacttt gactttaacc gcttccactg ccacttttcc ccacgcgact ggcagcgact
192
CA 3054600 2019-09-06
2881 catcaacaac aactggggac tcaggccgaa atcgatgcgt gttaaaatct tcaacataca
2941 ggtcaaggag gtcacgacgt caaacggcga gactacggtc gctaataacc ttaccagcac
3001 ggttcagatc tttgcggatt cgacgtatga actcccatac gtgatggacg ccggtcagga
3061 ggggagcttt cctccgtttc ccaacgacgt ctttatggtt ccccaatacg gatactgcgg
3121 agttgtc act ggaaaaaacc agaaccagac agacagaaat gccttttact gcctggaata
3181 ctttccatcc caaatgctaa gaactggcaa caattttgaa gtcagttacc aatttgaaaa
3241 agttcctttc cattcaatgt acgcgcacag ccagagcctg gacagaatga tgaatccttt
3301 actggatcag tacctgtggc atctgcaatc gaccactacc ggaaattccc ttaatcaagg
3361 aacagctacc accacgtacg ggaaaattac cactggagac tttgcctact acaggaaaaa
3421 ctggttgcct ggagcctgca ttaaacaaca aaaattttca aagaatgcca atcaaaacta
3481 caagattccc gccagcgggg gagacgccct tttaaagtat gacacgcata ccactctaaa
3541 tgggcgatgg agtaacatgg ctcctggacc tccaatggca accgcaggtg ccggggactc
3601 ggattttagc aacagccagc tgatctttgc cggacccaat ccgagcggta acacgaccac
3661 atcttcaaac aatttgttgt ttacctcaga agaggagatt gccacaacaa acccacgaga
3721 cacggacatg tttggacaga ttgcagataa taatcaaaat gccaccaccg cccctcacat
3781 cgctaacctg gacgctatgg gaattgttcc cggaatggtc tggcaaaaca gagacatcta
3841 ctaccagggc cctatttggg ccaaggtccc tcacacggac ggacactttc acccttcgcc
3901 gctgatggga ggatttggac tgaaacaccc gcctccacag attttcatca aaaacacccc
3961 cgtacccgcc aatcccaata ctacctttag cgctgcaagg attaattat ttctgacgca
4021 gtacagcacc ggacaagttg ccgttcagat cgactgggaa attcagaagg agcattccaa
4081 acgctggaat cccgaagttc aatttacttc aaactacggc actcaaaatt ctatgctgtg
4141 ggctcccgac aatgctggca actaccacga actccgggct attgggtccc gtacctcac
193
CA 3054600 2019-09-06
