Note: Descriptions are shown in the official language in which they were submitted.
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COMPOSITIONS AND METHODS FOR RECOMBINANT AAV PRODUCTION
TECHNICAL FIELD
[0001] The present disclosure relates to recombinant polynucleotides encoding
helper functions
and their use in a method of producing recombinant adeno-associated virus
(rAAV) particles.
CROSS-REFRENCE TO RELATED APPLICATIONS
[0002] This application claims the benefit of U.S. application no. 63/252,585,
filed October 5,
2021 and U.S. application no. 63/320,335, filed March 16, 2022, each of which
is incorporated
herein by reference in its entirety.
REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY
[0003] The content of the electronically submitted sequence
listing (Name:
6728_1802_Sequence_Listing.xml; Size: 376,980 bytes; and Date of Creation:
September 13,
2022) filed with the application is incorporated herein by reference in its
entirety.
BACKGROUND
[0004] Recombinant adeno-associated virus (AAV)-based vectors are currently
the most widely
used gene therapy products in development. The preferred use of rAAV vector
systems is due, in
part, to the lack of disease associated with the wild-type virus, the ability
of AAV to transduce
non-dividing as well as dividing cells, and the resulting long-term robust
transgene expression
observed in clinical trials and that indicate great potential for delivery in
gene therapy indications.
Additionally, different naturally occurring and recombinant rAAV vector
serotypes, specifically
target different tissues, organs, and cells, and help evade any pre-existing
immunity to the vector,
thus expanding the therapeutic applications of AAV-based gene therapies.
Before replication
defective virus, for example, AAV based gene therapies can be more widely
adopted for late
clinical stage and commercial use, new methods for large scale production of
recombinant virus
particles need to be developed.
[0005] Thus, there is a need in the art to improve the productivity and yield
of methods for the
large scale production of rAAV particles.
BRIEF SUMMARY
[0006] In one aspect, the disclosure provides an isolated recombinant
polynucleotide comprising
one or more of a) a nucleotide sequence encoding an adenovirus E2A DNA binding
protein
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(DBP) operably linked to a first promoter and to a first polyA signal; b) a
nucleotide sequence
encoding an adenovirus E4 ORF6 and ORF7 polypeptide operably linked to a
second promoter
and a second polyA signal; and c) a nucleotide sequence encoding an adenovirus
VA RNA I,
wherein the isolated recombinant polynucleotide does not comprise a nucleotide
sequence
encoding an adenovirus 1TR sequence, L3 23K endoprotease, L5 pVI/fibre, and/or
L4
pVIII/hexon-associated precursor.
[0007] In some embodiments, the isolated recombinant polynucleotide comprises
the nucleotide
sequence encoding the adenovirus E2A DBP, the nucleotide sequence encoding the
adenovirus
E4 ORF6 and ORF7 polypeptide and the nucleotide sequence encoding the
adenovirus VA RNA
I. In some embodiments, the nucleotide sequence encoding the adenovirus VA RNA
I encodes
VA RNA T and VA RNA II.
[0008] In some embodiments, the isolated recombinant polynucleotide comprises
the nucleotide
sequence encoding the adenovirus E2A DBP, the nucleotide sequence encoding the
adenovirus
E4 ORF6 and ORF7 polypeptide and the nucleotide sequence encoding the
adenovirus VA RNA
I, wherein the nucleotide sequence encoding the adenovirus E2A DBP and the
nucleotide
sequence encoding the adenovirus E4 ORF6 and ORF7 are in opposite 5' to 3'
orientation.
[0009] In some embodiments, the isolated recombinant polynucleotide is a
plasmid comprising a
bacterial replication origin and a selectable marker gene.
[0010] In some embodiments, the isolated recombinant polynucleotide comprises
a nucleotide
sequence having at least 80%, at least 85%, at least 90%, at least 95%, at
least 97%, at least 98%,
at least 99% or 100 % identity to SEQ ID NO: 10, 11, 12, 13, 14, 15, 16, 17,
18, 19, 20, 25, 26,
27, 28, 29, 30, 31, 32, 33, 34, 37, 38, 39, 40, 41, 42, 43 or 51.
[Nil] In somc embodiments, the isolated recombinant polynucleotide comprises
the nucleotide
sequence of SEQ ID NO: 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 26, 27,
28, 29, 30, 31, 32,
33, 34, 37, 38, 39, 40, 41, 42, 43 or 51.
[0012] In one aspect, the disclosure provides a host cell comprising an
isolated recombinant
polynucleotide described herein. In some embodiments, the host cell is a
bacterial cell. In some
embodiments, the host cell is a eukaryotic cell. In some embodiments, the host
cell is a HEK293
cell, HEK derived cell, CHO cell, CHO derived cell, HeLa cell, SF-9 cell, BHK
cell, Vero cell,
CAP cell, or PerC6 cell.
2
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[0013] In one aspect, the disclosure provides a method of producing an
isolated recombinant
polynucleotide described herein comprising incubating under suitable
conditions a host cell
described herein.
[0014] In one aspect, the disclosure provides a method of producing
recombinant adeno-
associated virus (rAAV) particles comprising culturing a cell capable of
producing the rAAV
particles, wherein the cell comprises i) a polynucleotide encoding an AAV
capsid protein; ii) a
polynucleotide encoding a functional rep gene; iii) polynucleotide comprising
a genome
comprising at least one AAV inverted terminal repeat (ITR) and a non-AAV
nucleic acid
sequence encoding a gene product operably linked to sequences which direct
expression of the
gene product in a target cell; and iv) one or more polynucleotides comprising
sufficient helper
functions to permit packaging of the genome into the AAV capsid protein under
conditions which
permit packaging of the genome into the AAV capsid, wherein the one or more
polynucleotides
comprising sufficient helper functions independently comprise an isolated
recombinant
polynucleotide described herein.
[0015] In one aspect, the disclosure provides a method of producing rAAV
particles, comprising
a) providing a cell culture comprising a cell; b) introducing into the cell
one or more
polynucleotides comprising i) a polynucleotide encoding an AAV capsid protein:
ii) a
polynucleotide encoding a functional rep gene; iii) polynucleotide comprising
a genome
comprising at least one AAV inverted terminal repeat (ITR) and a non-AAV
nucleic acid
sequence encoding a gene product operably linked to sequences which direct
expression of the
gene product in a target cell; and iv) one or more polynucleotides comprising
sufficient helper
functions to permit packaging of the genome into the AAV capsid protein under
conditions which
permit packaging of the genome into thc AAV capsid, wherein the one or more
polynucleotides
comprising sufficient helper functions independently comprise an isolated
recombinant
polynucleotide described herein, and c) maintaining the cell culture under
conditions that allow
production of the rAAV particles.
[0016] In some embodiments, the disclosure provides:
[1.] An isolated recombinant polynucleotide comprising one or more
of
a) a nucleotide sequence encoding an adenovirus E2A DNA binding
protein (DBP)
operably linked to a first promoter and to a first polyA signal;
3
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b) a nucleotide sequence encoding an adenovirus E4 ORF6 and ORF7
polypeptide operably
linked to a second promoter and a second polyA signal; and
c) a nucleotide sequence encoding an adenovirus VA RNA I,
wherein the isolated recombinant polynucleotide does not comprise a nucleotide
sequence
encoding an adenovirus 1TR sequence, L3 23K endoprotease, L5 pVI/fibre, and/or
L4
pVIII/hexon-associated precursor;
[2.] the isolated recombinant polynucleotide of [1], wherein the
isolated recombinant
polynucleotide comprises:
a) the nucleotide sequence encoding the adenovirus E2A DBP, the nucleotide
sequence
encoding the adenovirus E4 ORF6 and ORF7 polypeptide and the nucleotide
sequence encoding
the adenovirus VA RNA I;
b) the nucleotide sequence encoding the adenovirus E2A DBP, and the
nucleotide sequence
encoding the adenovirus E4 ORF6 and ORF7 polypeptide;
c) the nucleotide sequence encoding the adenovirus E2A DBP, and the
nucleotide sequence
encoding the adenovirus VA RNA I;
d) the nucleotide sequence encoding the adenovirus E4 ORF6 and ORF7
polypeptide and
the nucleotide sequence encoding the adenovirus VA RNA I;
e) the nucleotide sequence encoding the adenovirus E2A DBP;
0 the nucleotide sequence encoding the adenovirus E4 ORF6 and
ORF7 polypeptide; or
the nucleotide sequence encoding the adenovirus VA RNA I;
LI] the isolated recombinant polynucleotide of [1] comprising the
nucleotide sequence
encoding the adenovirus E2A DBP, the nucleotide sequence encoding the
adenovirus E4 ORF6
and ORF7 polypeptide and the nucleotide sequence encoding the adenovirus VA
RNA I, wherein
the nucleotide sequence encoding the adenovirus E2A DBP and the nucleotide
sequence encoding
the adenovirus E4 ORF6 and ORF7 are in opposite 5' to 3' orientation;
[4.] the isolated recombinant polynucleotide of any one of [1] to
[3], wherein the nucleotide
sequence encoding the adenovirus E2A DBP has at least 80%, at least 85%, at
least 90%, at least
95%, at least 97%, at least 98%, at least 99% or 100 % identity to SEQ ID NO:
1;
115.1 the isolated recombinant polynucleotide of any one of [1] to
[3], wherein the nucleotide
sequence encoding the adenovirus E2A DBP comprises SEQ ID NO: 1;
4
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[6.1 the isolated recombinant polynucleotide of any one of [11 to
[51, wherein the adenovirus
E2A DBP comprises an amino acid sequence having at least 80%, at least 85%, at
least 90%, at
least 95%, at least 97%, at least 98%, at least 99% or 100 % identity to SEQ
ID NO: 45;
LT] the isolated recombinant polynucleotide of any one of [1] to
[5], wherein the adenovirus
E2A DBP comprises the amino acid sequence of SEQ ID NO: 45;
118.1 the isolated recombinant polynucleotide of any one of [1] to
[7], wherein the nucleotide
sequence encoding the adenovirus E4 ORF6 and ORF7 polypeptide has at least
80%, at least
85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or
100 % identity to SEQ
Ill NO: 8;
119-] the isolated recombinant polynucleotide of any one of [1] to
[7], wherein the nucleotide
sequence encoding the adenovirus E4 ORF6 and ORF7 polypeptide comprises SEQ ID
NO: 8;
[10.] the isolated recombinant polynucleotide of any one of [1] to [9],
wherein the adenovirus
E4 ORF6 and ORF7 polypeptide comprises an amino acid sequence having at least
80%, at least
85%, at least 90%, at least 95%, at least 97%. at least 98%, at least 99% or
100 % identity to SEQ
ID NO: 46;
[11.] the isolated recombinant polynucleotide of any one of [1] to [9],
wherein the adenovirus
E4 ORF6 and ORF7 polypeptide comprises the amino acid sequence of SEQ ID NO:
46;
[12j the isolated recombinant polynucleotide of any one of [1] to
[11], wherein the nucleotide
sequence encoding the adenovirus VA RNA I comprises a nucleotide sequence
having at least
80%, at least 85%, at least 90%, at least 95%. at least 97%, at least 98%, at
least 99% or 100 %
identity to SEQ ID NO: 54;
[13.] the isolated recombinant polynucleotide of any one of [1] to
[11], wherein the nucleotide
sequence encoding the adenovirus VA RNA I encodes VA RNA I and VA RNA II, and
optionally comprises a nucleotide sequence having at least 80%, at least 85%,
at least 90%, at
least 95%, at least 97%, at least 98%, at least 99% or 100 % identity to SEQ
ID NO: 9;
[14.1 the isolated recombinant polynucleotide of any one of [11 to
1131, wherein the first
promoter and second promoter are different promoters;
[15.] the isolated recombinant polynucleotide of any one of [1] to [14],
wherein the first
promoter is an adenovirus E2A promoter, a CMV promoter, or a CMV derived
promoter;
[16.] the isolated recombinant polynucleotide of [15], wherein the first
promoter is an
adenovirus E2A promoter;
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[17.1 the isolated recombinant polynucleotide of [16], wherein the adenovirus
E2A promoter
comprises a nucleotide sequence having at least 80%, at least 85%, at least
90%, at least 95%, at
least 97%, at least 98%, at least 99% or 100 % identity to SEQ ID NO: 2;
[18.] the isolated recombinant polynucleotide of [16], wherein the adenovirus
E2A promoter
comprises the nucleotide sequence of SEQ ID NO: 2;
[19.] the isolated recombinant polynucleotide of any one of [15] to [18],
wherein the
nucleotide sequence encoding the adenovirus E2A promoter and E2A DBP comprises
a
nucleotide sequence having at least 80%, at least 85%, at least 90%, at least
95%, at least 97%, at
least 98%, at least 99% or 100 % identity to SEQ Ill NO: 3 or 4;
[20.] the isolated recombinant polynucleotide of any one of [15] to [18],
wherein the
nucleotide sequence encoding the adenovirus E2A promoter and E2A DBP comprises
SEQ ID
NO: 3 or 4;
[21.] the isolated recombinant polynucleotide of any one of [15] to [18],
wherein the
nucleotide sequence encoding the adenovirus E2A promoter and E2A DBP comprises
a
nucleotide sequence having at least 80%, at least 85%, at least 90%, at least
95%, at least 97%, at
least 98%, at least 99% or 100 % identity to SEQ ID NO: 22 or 23;
[22.] the isolated recombinant polynucleotide of any one of [15] to [18],
wherein the
nucleotide sequence encoding the adenovirus E2A promoter and E2A DBP comprises
SEQ ID
NO: 22 or 23;
[23.] the isolated recombinant polynucleotide of any one of [1] to [14],
wherein the first
promoter is an inducible promoter;
[24.] the isolated recombinant polynucleotide of any one of [1] to [23],
wherein the second
promoter is an adenovirus E4 promoter, a CMV promoter, or a CMV derived
promoter;
[25.] the isolated recombinant polynucleotide of [24], wherein the second
promoter is an
adenovirus E4 promoter;
126.] the isolated recombinant polynucleotide of 125], wherein the adenovirus
E4 promoter
comprises a nucleotide sequence having at least 80%, at least 85%, at least
90%, at least 95%, at
least 97%, at least 98%, at least 99% or 100 % identity to SEQ ID NO: 5;
[27.1 the isolated recombinant polynucleotide of [25], wherein the adenovirus
E4 promoter
comprises the nucleotide sequence of SEQ ID NO: 5;
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[28.] the isolated recombinant polynucleotide of any one of [1] to [23],
wherein the second
promoter is an inducible promoter;
[29.] the isolated recombinant polynucleotide of any one of [1] to [28]
comprising a nucleotide
sequence having at least 80%, at least 85%, at least 90%, at least 95%, at
least 97%, at least 98%,
at least 99% or 100 % identity to SEQ ID NO: 10;
[30.] the isolated recombinant polynucleotide of any one of [1] to [28]
comprising the
nucleotide sequence of SEQ ID NO: 10;
[31.] the isolated recombinant polynucleotide of any one of [1] to [28
comprising a nucleotide
sequence having at least 80%, at least 85%, at least 90%, at least 95%, at
least 97%, at least 98%,
at least 99% or 100 % identity to SEQ ID NO: 11;
[32.] the isolated recombinant polynucleotide of any one of [1] to [28]
comprising the
nucleotide sequence of SEQ ID NO: 11;
[33.] the isolated recombinant polynucleotide of any one of [1] to [28]
further comprising a
nucleotide sequence encoding a Boca virus NP1 and NS2 polypeptides operably
linked to a third
promoter and to a third polyA signal;
[34.] the isolated recombinant polynucleotide of [33], wherein the nucleotide
sequence
encoding the Boca virus NP1 and NS2 polypeptides have at least 80%, at least
85%, at least 90%,
at least 95%, at least 97%, at least 98%, at least 99% or 100 % identity to
SEQ ID NO: 12;
[35.] the isolated recombinant polynucleotide of [33], wherein the nucleotide
sequence
encoding the Boca virus NP1 and NS2 polypeptides comprises SEQ ID NO: 12;
[36.] the isolated recombinant polynucleotide of any one of [33] to [35],
wherein the third
promoter is a CMV promoter;
[37.] the isolated recombinant polynucleotide of any one of [33] to [35]
comprising a
nucleotide sequence having at least 80%, at least 85%, at least 90%, at least
95%, at least 97%, at
least 98%, at least 99% or 100 % identity to SEQ ID NO: 13;
[38.1 the isolated recombinant polynucleotide of any one of [331 to [351
comprising the
nucleotide sequence of SEQ ID NO: 13;
[39.] the isolated recombinant polynucleotide of any one of [33] to [38]
comprising a
nucleotide sequence having at least 80%, at least 85%, at least 90%, at least
95%, at least 97%, at
least 98%, at least 99% or 100 % identity to SEQ ID NO: 14;
7
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140.1 the isolated recombinant polynucleotide of any one of [33] to [38]
comprising the
nucleotide sequence of SEQ ID NO: 14;
[41.] the isolated recombinant polynucleotide of any one of [I] to [28]
further comprising a
nucleotide sequence encoding a adeno-associated virus (AAV) assembly-
activating protein
(AAP) operably linked to a third promoter and to a third polyA signal;
[42.] the isolated recombinant polynucleotide of [41], wherein the nucleotide
sequence
encoding the AAV AAP have at least 80%, at least 85%, at least 90%, at least
95%, at least 97%,
at least 98%, at least 99% or 100 % identity to SEQ Ill NO: 15;
143.] the isolated recombinant polynucleotide of 141], wherein the nucleotide
sequence
encoding the AAV AAP comprises SEQ ID NO: 15;
[44.] the isolated recombinant polynucleotide of any one of [41] to [43],
wherein the third
promoter is a CMV promoter;
[45.] the isolated recombinant polynucleotide of any one of [41] to [44]
comprising a
nucleotide sequence having at least 80%, at least 85%, at least 90%, at least
95%, at least 97%, at
least 98%, at least 99% or 100 % identity to SEQ ID NO: 16;
[46.] the isolated recombinant polynucleotide of any one of [41] to [44]
comprising the
nucleotide sequence of SEQ ID NO: 16;
[47.] the isolated recombinant polynucleotide of any one of [41] to 1146
comprising a nucleotide
sequence having at least 80%, at least 85%, at least 90%, at least 95%, at
least 97%, at least 98%,
at least 99% or 100 % identity to SEQ ID NO: 17;
[48.] the isolated recombinant polynucleotide of any one of [41] to [46]
comprising the
nucleotide sequence of SEQ ID NO: 17;
[49.] the isolated recombinant polynucleotide of any one of [I] to [28]
further comprising a
nucleotide sequence encoding an adenovirus ElA polypeptide operably linked to
a third promoter
and to a third polyA signal;
1_50.] the isolated recombinant polynucleotide of 149], wherein the nucleotide
sequence
encoding the adenovirus ElA polypeptide has at least 80%, at least 85%, at
least 90%, at least
95%, at least 97%, at least 98%, at least 99% or 100 % identity to SEQ ID NO:
18;
151.] the isolated recombinant polynucleotide of 149], wherein the nucleotide
sequence
encoding the adenovirus ElA polypeptide comprises SEQ ID NO: 18;
8
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[52.1 the isolated recombinant polynucleotide of 149], wherein the adenovirus
E1A polypeptide
comprises an amino acid sequence having at least 80%, at least 85%, at least
90%, at least 95%,
at least 97%, at least 98%, at least 99% or 100 % identity to SEQ ID NO: 51;
[53.] the isolated recombinant polynucleotide of [49], wherein the adenovirus
El A polypeptide
comprises the amino acid sequence of SEQ ID NO: 51;
[54.] the isolated recombinant polynucleotide of any one of [49] to [53],
wherein the third
promoter is a CMV promoter;
[55.] the isolated recombinant polynucleotide of any one of 149] to [54]
comprising a
nucleotide sequence having at least 80%, at least 85%, at least 90%, at least
95%, at least 97%, at
least 98%, at least 99% or 100 % identity to SEQ ID NO: 19;
[56.] the isolated recombinant polynucleotide of any one of [49] to [54]
comprising the
nucleotide sequence of SEQ ID NO: 19;
[57.] the isolated recombinant polynucleotide of any one of [49] to [56]
comprising a
nucleotide sequence having at least 80%, at least 85%, at least 90%, at least
95%, at least 97%, at
least 98%, at least 99% or 100 % identity to SEQ ID NO: 20;
[58.] the isolated recombinant polynucleotide of any one of [49] to [56]
comprising the
nucleotide sequence of SEQ ID NO: 20;
[59.] the isolated recombinant polynucleotide of any one of [1] to [27],
[33] to [36], [41] to
[44] and [49] to [54, wherein the isolated recombinant polynucleotide
comprises a nucleotide
sequence encoding the E2A promoter, L4 22K/33K polypeptides and promoter, L4
100k/hexon
assembly polypeptide comprising an N terminal deletion and the E2A DBP,
wherein the N-
terminal deletion of the L4 100k/hexon assembly polypeptide corresponds to the
nucleotide
sequence of SEQ ID NO: 21;
[60.] the isolated recombinant polynucleotide of [59], wherein the nucleotide
sequence
encoding the E2A promoter, L4 22K/33K polypeptides and promoter, L4 100k/hexon
assembly
polypeptide comprising an N terminal deletion and the E2A DBP comprises a
nucleotide
sequence having at least 80%, at least 85%, at least 90%, at least 95%, at
least 97%, at least 98%,
at least 99% or 100% identity to SEQ ID NO: 22;
[61.1 the isolated recombinant polynucleotide of 159], wherein the nucleotide
sequence
encoding the E2A promoter, L4 22K/33K polypeptides and promoter, L4 100k/hexon
assembly
polypeptide comprising an N terminal deletion and the E2A DBP comprises SEQ ID
NO: 22;
9
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[62.] the isolated recombinant polynucleotide of any one of [1] to [27],
[33] to [36], [41] to
[44] and [49] to [54], wherein the isolated recombinant polynucleotide
comprises a nucleotide
sequence encoding the E2A promoter, L4 22K/33K polypeptides and promoter, L4
100k/hexon
assembly polypeptide comprising a mutation in its start codon and the E2A DBP;
[63.] the isolated recombinant polynucleotide of [62], wherein the nucleotide
sequence
encoding the E2A promoter, L4 22K/33K polypeptides and promoter, L4 100k/hexon
assembly
polypeptide comprising a mutation in its start codon and the E2A DBP comprises
a nucleotide
sequence having at least 80%, at least 85%, at least 90%, at least 95%, at
least 97%, at least 98%,
at least 99% or 100% identity to SEQ Ill NO: 23;
[64.] the isolated recombinant polynucleotide of [62], wherein the nucleotide
sequence
encoding the E2A promoter, L4 22K/33K polypeptides and promoter, L4 100k/hexon
assembly
polypeptide comprising a mutation in its start codon and the E2A DBP comprises
SEQ ID NO:
23;
[65.] the isolated recombinant polynucleotide of any one of [1] to [27],
[33] to [36], [41] to
[44] and [49] to [54], wherein the isolated recombinant polynucleotide
comprises a nucleotide
sequence encoding the E2A promoter, L4 22K/33K polypeptides and promoter, L4
100k/hexon
assembly polypeptide comprising an N terminal deletion and the E2A DBP,
wherein the N-
terminal deletion of the L4 100k/hexon assembly polypeptide encompasses the
start codon of L4
100k/hexon assembly but does not encompass the start codon of the L4 22K133K
polypeptides;
[66.] the isolated recombinant polynucleotide of any one of [1] to [27],
[33] to [36], [41] to
[44] and [49] to [54], wherein the isolated recombinant polynucleotidc
comprises a nucleotide
sequence encoding the E2A promoter, L4 22K/33K polypeptides and promoter, L4
100k/hexon
assembly polypeptide comprising an N terminal deletion and the E2A DBP,
wherein all or part of
the L4 100k/hexon assembly polypeptide is deleted without disruption of the L4
22K/33K start
codon;
[67.] the isolated recombinant polynucleotide of any one of [1] to 127],
[33] to [36], 141] to
[44] and [49] to [54], and [45] to [50], wherein the isolated recombinant
polynucleotide
comprises a nucleotide sequence encoding the E2A promoter, L4 22K/33K
polypeptides and
promoter, L4 100k/hexon assembly polypeptide comprising an N terminal deletion
and the E2A
DBP, wherein the N-terminal deletion of the L4 100k/hexon assembly starts at
the start codon of
L4 100k/hexon assembly and ends immediately adjacent to the L4 22K/33K
promoter;
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[68.] the isolated recombinant polynucleotide of any one of [1] to [27],
[33] to [36], [41] to
[44] and [49] to [54], and [59] to [67] comprising a nucleotide sequence
having at least 80%, at
least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least
99% or 100 % identity to
SEQ ID NO: 25, 27, 29, 31 or 33;
[69.] the isolated recombinant polynucleotide of any one of [1] to [27],
[33] to [36], [41] to
[44] and [49] to [54], and [59] to [67] comprising the nucleotide sequence of
SEQ ID NO: 25, 27,
29, 31 or 33;
170.] the isolated recombinant polynucleotide of any one of [1] to
[27], [33] to [36], [41] to
144] and 149] to [54]. and [59] to [69] comprising a nucleotide sequence
having at least 80%, at
least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least
99% or 100 % identity to
SEQ ID NO: 26, 28, 30, 32 or 34;
[71.] the isolated recombinant polynucleotide of any one of [1] to
[27], [33] to [36], [41] to
[44] and [49] to [54], and [59] to [69] comprising the nucleotide sequence of
SEQ ID NO: 26, 28,
30, 32 or 34;
[72.] the isolated recombinant polynucleotide of any one of [1]-[71],
wherein the isolated
recombinant polynucleotide comprises the nucleotide sequence encoding the
adenovirus E2A
DBP, the nucleotide sequence encoding the adenovirus E4 ORF6 and ORF7
polypeptide and the
nucleotide sequence encoding the adenovirus VA RNA I;
[73.] the isolated recombinant polynucleotide of any one of [1]-[71],
wherein the isolated
recombinant polynucleotide comprises the nucleotide sequence encoding the
adenovirus E2A
DBP, and the nucleotide sequence encoding the adenovirus E4 ORF6 and ORF7
polypeptide;
[74.] the isolated recombinant polynucleotide of any one of [1]-[71],
wherein the isolated
recombinant polynucleotide comprises thc nucleotide sequence cncoding the
adenovirus E2A
DBP, and the nucleotide sequence encoding the adenovirus VA RNA I;
175.] the isolated recombinant polynucleotide of any one of [1]-
171], wherein the isolated
recombinant polynucleotide comprises the nucleotide sequence encoding the
adenovirus E4
ORF6 and ORF7 polypeptide and the nucleotide sequence encoding the adenovirus
VA RNA I;
[76.] the isolated recombinant polynucleotide of any one of [1]-
[71], wherein the isolated
recombinant polynucleotide comprises the nucleotide sequence encoding the
adenovirus E2A
DBP;
11
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177.1 the isolated recombinant polynucleotide of any one of [11-
1711, wherein the isolated
recombinant polynucleotide comprises the nucleotide sequence encoding the
adenovirus E4
ORF6 and ORF7 polypeptide;
[78.] the isolated recombinant polynucleotide of any one of [1]-[71],
wherein the isolated
recombinant polynucleotide comprises the nucleotide sequence encoding the
adenovirus VA
RNA I;
[79.] the isolated recombinant polynucleotide of any one of [1] to [78],
wherein the isolated
recombinant polynucleotide is a plasmid comprising a bacterial replication
origin and a selectable
marker gene;
[80.] the isolated recombinant polynucleotide of [79], wherein the
bacterial replication origin is
a ColE1 origin;
[81.] the isolated recombinant polynucleotide of [79] or [80], wherein the
selectable marker
gene is a drug resistance gene;
[82.] the isolated recombinant polynucleotide of [81], wherein the
selectable marker gene is a
kanamycin resistance gene;
[83.] the isolated recombinant polynucleotide of any one of [1] to [82]
comprising a nucleotide
sequence having at least 80%, at least 85%, at least 90%, at least 95%, at
least 97%, at least 98%,
at least 99% or 100 % identity to SEQ ID NO: 37-42 or 43;
[84.] the isolated recombinant polynucleotide of any one of [1] to [82]
comprising the
nucleotide sequence of SEQ ID NO: 37-42 or 43;
[85.] the isolated recombinant polynucleotide of any one of [1] to [82]
comprising a nucleotide
sequence having at least 80%, at least 85%, at least 90%, at least 95%, at
least 97%, at least 98%,
at least 99% or 100 % identity to SEQ ID NO: 37;
[86.] the isolated recombinant polynucleotide of any one of [1] to [82]
comprising the
nucleotide sequence of SEQ ID NO: 37.
[87.1 A host cell comprising the isolated recombinant
polynucleotide of any one of [11 to [86_1;
[88.] the host cell of [87], wherein the host cell is a bacterial cell;
[89.] the host cell of [87], wherein the host cell is an E. coli cell;
[90.1 the host cell of 1871, wherein the host cell is a eukaryotic
cell;
[91.] the host cell of [87], wherein the host cell is a mammalian
cell;
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[92.] the host cell of [87], wherein the host cell is a HEK293 cell. HEK
derived cell. CHO cell,
CHO derived cell, HeLa cell, SF-9 cell, BHK cell, Vero cell, CAP cell, or
PerC6 cell.
[93.] A method of producing the isolated recombinant polynucleotide of any one
of [I] to [86]
comprising incubating under suitable conditions the host cell of any of [87]
to [92];
[94.] the method of [93] comprising incubating under suitable conditions the
host cell of [88]
or [89].
[95.] A method of producing recombinant adeno-associated virus (rAAV)
particles comprising
culturing a cell capable of producing the rAAV particles, wherein the cell
comprises
i. a polynucleotide encoding an AAV capsid protein;
a polynucleotide encoding a functional rep gene;
a polynucleotide comprising a genome comprising at least one AAV inverted
terminal
repeat (ITR) and a non-AAV nucleic acid sequence encoding a gene product
operably linked to
sequences which direct expression of the gene product in a target cell; and
iv. one or more polynucleotides comprising sufficient helper
functions to permit packaging
of the genome into the AAV capsid protein under conditions which permit
packaging of the
genome into the AAV capsid, wherein the one or more polynucleotides comprising
sufficient
helper functions independently comprise the isolated recombinant
polynucleotide of any one of
[1] to [86];
[96.] the method of [95], wherein the one or more polynucleotides comprising
sufficient helper
functions comprise the isolated polynucleotide comprising the nucleotide
sequence encoding the
adenovirus E2A DBP, the nucleotide sequence encoding the adenovirus E4 ORF6
and ORF7
polypeptide and the nucleotide sequence encoding the adenovirus VA RNA I.
[97.] A method of producing rAAV particles, comprising
a) providing a cell culture comprising a cell;
b) introducing into the cell one or more polynucleotides comprising
i. a polynucleotide encoding an AAV capsid protein;
a polynucleotide encoding a functional rep gene;
a polynucleotide comprising a genome comprising at least one AAV inverted
terminal repeat (ITR) and a non-AAV nucleic acid sequence encoding a gene
product
operably linked to sequences which direct expression of the gene product in a
target cell;
and
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iv. one or more polynucleotides comprising sufficient
helper functions to permit
packaging of the genome into the AAV capsid protein under conditions which
permit
packaging of the genome into the AAV capsid, wherein the one or more
polynucleotides
comprising sufficient helper functions independently comprise the
polynucleotide of any
one of [1] to [86], and
c) maintaining the cell culture under conditions that allow
production of the rAAV particles;
[98.] the method of [97], wherein the one or more polynucleotides comprising
sufficient helper
functions comprise the isolated polynucleotide comprising the nucleotide
sequence encoding the
adenovirus E2A DBP, the nucleotide sequence encoding the adenovirus E4 ORF6
and ORF7
polypeptide and the nucleotide sequence encoding the adenovirus VA RNA I;
[99.] the method of [97] or [98], comprising introducing into the cell a
polynucleotide
encoding an AAV capsid protein and a functional rep gene;
[100.] the method of any one of [97] to [99], wherein the introducing of the
one or more
polynucleotides into the cell is by transfection;
[101.] the method of any one of [95] to [100], wherein the cell is a mammalian
cell;
[102.] the method of any one of [95] to [100], wherein the cell is an insect
cell;
[103.] the method of any one of 11951 to [100], wherein the cell is a HEK293
cell, HEK derived
cell, CHO cell, CHO derived cell, HeLa cell, SF-9 cell, BHK cell, Vero cell,
CAP cell or PerC6
cell;
[104.] the method of any one of [95] to [100], wherein the cell is a HEK293
cell;
[105.] the method of any one of [95] to [104], wherein the cell culture is a
suspension culture or
an adherent culture;
[106.] the method of any one of [95] to [105], further comprising recovering
the rAAV
particles;
[107.] the method of any one of 11951 to [105], wherein the method produces
more rAAV
particles measured as GC/ml than a reference method using a polynucleotide
comprising helper
functions comprising the nucleotide sequence of SEQ ID NO: 44;
[108.] the method of any one of [95] to [105], wherein the method produces at
least about twice
as many rAAV particles measured as GC/ml than a reference method using a
polynucleotide
comprising helper functions comprising the nucleotide sequence of SEQ ID NO:
44;
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[109.] the method of any one of [951 to [105], wherein the method produces a
population of
rAAV particles comprising more full capsids than a reference method using a
polynucleotide
comprising helper functions comprising the nucleotide sequence of SEQ ID NO:
44;
[110.] the method of any one of [95] to [109], wherein the cell culture has a
volume between
about 50 liters and about 20,000 liters;
[111.] the method of any one of [95] to [110], wherein the rAAV particles
comprise a capsid
protein of the AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10,
AAV11, AAV12, AAV13, AAV14, AAV15 and AAV16, AAV.rh8, AAV.rh10, AAV.rh20,
AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8,
AAV.PHP.B, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAVMYO, MyoAAV.1A,
MyoAAV1C, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6,
AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10 , AAV.HSC11, AAV.HSC12,
AAV.HSC13, AAV.HSC14, AAV.HSC15, or AAV.HSC16 serotype;
[112.] the method of any one of [95] to [110], wherein the rAAV particles
comprise a capsid
protein of the AAV8, AAV9, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1,
or
AAV.hu37 serotype;
[113.] the method of any one of 11951 to [110], wherein the rAAV particles
comprise a capsid
protein of the AAV8 or AAV9 serotype;
[114.] the method of any one of [95] to [110], wherein the gene product is a
polypeptide or a
double stranded RNA molecule;
[115.] the method of [114], wherein the gene product is a polypeptide;
[116.] the method of [115], wherein the gene product is anti-VEGF Fab, anti-
kallikrein
antibody, anti-TNF antibody, microdystrophin, minidystrophin, iduronidasc
(IDUA), iduronatc 2-
sulfatase (IDS), low-density lipoprotein receptor (LDLR), tripeptidyl
peptidase 1 (TPP1), or non-
membrane associated splice variant of VEGF receptor 1 (sFlt-1);
[117.] the method of [115], wherein the gene product is an gamma-sarcoglycan,
Rab Escort
Protein 1 (REP1/CHM), retinoid isomerohydrolase (RPE65), cyclic nucleotide
gated channel
alpha 3 (CNGA3), cyclic nucleotide gated channel beta 3 (CNGB3), aromatic L-
amino acid
decarboxylase (AADC), lysosome-associated membrane protein 2 isoform B
(LAMP2B), Factor
VIII, Factor IX, retinitis pigmentosa GTPase regulator (RPGR), retinoschisin
(RS1),
sarcoplasmic reticulum calcium ATPase (SERCA2a), aflibercept, battenin (CLN3),
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transmembrane ER protein (CLN6), glutamic acid decarboxylase (GAD), Glial cell
line-derived
neurotrophic factor (GDNF), aquaporin 1 (AQP1), dystrophin, myotubularin 1
(MTM1),
follistatin (FST), glucose-6-phosphatase (G6Pase), apolipoprotein A2 (AP0A2),
uridine
diphosphate glucuronosyl transferase 1A1 (UGT1A1), arylsulfatase B (ARSB), N-
acetyl-alpha-
glucosaminidase (NAGLU), alpha-glucosidase (GAA), alpha-galactosidase (GLA),
beta-
galactosidasc (GLB1), lipoprotein lipase (LPL), alpha 1-antitrypsin (AAT),
phosphodiesterase 6B
(PDE6B), ornithine carbamoyltransferase 90TC), survival motor neuron (SMN1),
survival motor
neuron (SMN2), neurturin (NRTN), Neurotrophin-3 (NT-3/NTF3), porphobilinogen
deaminase
(PBGD), nerve growth factor (NGF), mitochondrially encoded NADH:ubiquinone
oxidoreductase core subunit 4 (MT-ND4), protective protein cathepsin A (PPCA),
dysferlin,
MER proto-oncogene, tyrosine kinase (MERTK), cystic fibrosis transmembrane
conductance
regulator (CFTR), or tumor necrosis factor receptor (TNFR)-immunoglobulin
(IgG1) Fc fusion;
[118.] the method of [115], wherein the gene product is a dystrophin or a
microdystrophin;
[119.] the method of [114], wherein the gene product is a microRNA.
[0017] Still other features and advantages of the compositions and methods
described herein will
become more apparent from the following detailed description when read in
conjunction with the
accompanying drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
[0018] Figure 1. The pAdDeltaF6 reference helper plasmid.
[0019] Figure 2. Helper plasmid #1 map.
[0020] Figure 3. Helper #1 improved AAV titers. Fold change in rAAV production
titer relative
to titer obtained using pAdDeltaF6 Original/Old helper and clone 1-P8 is
shown.
[0021] Figure 4. Helper plasmid #2 map.
[0022] Figure 5. Helper #2 plasmid improved AAV titers. Fold change in rAAV
production titer
relative to titer obtained using pAdDeltaF6 Original/Old helper and clone 1 is
shown.
[0023] Figure 6. Screening of E4 variants. Fold change in rAAV production
titer relative to titer
obtained using a helper comprising whole E4 is shown.
[0024] Figure 7. Helper #3 plasmid map.
[0025] Figure 8. Helper #3 further improved AAV titers. Fold change in rAAV
production titer
relative to titer obtained using pAdDeltaF6 Original/Old helper and clone 1
(5e6) is shown.
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[0026] Figure 9. Helper #3 further improved AAV titers. Fold change in rAAV
production titer
relative to titer obtained using pAdDeltaF6 Original/Old helper and clone 1
(5e6) is shown.
[0027] Figure 10. Addition of Boca virus genes NP1 and NS2 to helper plasmid
#2.
[0028] Figure 11. Addition of Boca virus helper genes did not improve AAV
titers. Fold change
in rAAV production titer relative to titer obtained using pAdDeltaF6
Original/Old helper and
clone 1 is shown.
[0029] Figure 12. Addition of AAP to helper #3.
[0030] Figure 13. Helper plasmid #4 map.
[0031] Figure 14. Effect of adding AAP and El A on the virus titers. Fold
change in rAAV
production titer relative to titer obtained using pAdDeltaF6 Original/Old
helper and clone 1 is
shown.
[0032] Figure 15. Effect of mutations in hexon assembly and L4 22K/33K
sequences on AAV
titers. Fold change in rAAV production titer relative to titer obtained using
New Helper #3 and
clone 1 is shown.
DETAILED DESCRIPTION
[0033] In one aspect, provided herein are improved recombinant polynucleotides
and plasmids
encoding helper functions suitable for use in the production of recombinant
AAV particles. In
some embodiments, the recombinant polynucleotides and plasmids encode one or
more of an
adenovirus E2A DNA binding protein, a nucleotide sequence encoding an
adenovirus E4 ORF6
and ORF7 polypeptide and a nucleotide sequence encoding an adenovirus VA RNA
I. In some
embodiments, the polynucleotides and plasmids do not comprise a nucleotide
sequence encoding
an adenovirus ITR sequence, L3 23K endoprotease, L5 pVI/fibre, and/or L4
pVIII/hexon-
associated precursor. In some embodiments, the polynucleotides and plasmids
are smaller than
previously available polynucleotides and plasmids encoding helper functions
suitable for use in
the production of recombinant AAV particles. In some embodiments, use of the
improved
polynucleotides and plasmids described herein in the production of recombinant
AAV particles
results in increased rAAV yield.
DEFINITIONS
[0034] Unless defined otherwise, 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 disclosure is
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related. To facilitate an understanding of the disclosed methods, a number of
terms and phrases
are defined below.
[0035] "AAV" is an abbreviation for adeno-associated virus, and may be used to
refer to the
virus itself or modifications, derivatives, or pseudotypes thereof. The term
covers all subtypes and
both naturally occurring and recombinant forms, except where required
otherwise. The
abbreviation "rAAV" refers to recombinant adeno-associated virus. The term
"AAV" includes
AAV type 1 (AAV1), AAV type 2 (AAV2), AAV type 3 (AAV3), AAV type 4 (AAV4),
AAV
type 5 (AAV5), AAV type 6 (AAV6), AAV type 7 (AAV7), AAV type 8 (AAV8), AAV
type 9
(AAV9), avian AAV, bovine AAV, canine AAV, equine AAV, primate AAV, non-
primate AAV,
and ovine AAV, and modifications, derivatives, or pseudotypes thereof.
"Primate AAV" refers to
AAV that infect primates, "non-primate AAV' refers to AAV that infect non-
primate mammals,
"bovine AAV" refers to AAV that infect bovine mammals, etc.
[0036] "Recombinant" , as applied to an AAV particle means that the AAV
particle is the product
of one or more procedures that result in an AAV particle construct that is
distinct from an AAV
particle in nature.
[0037] A recombinant adeno-associated virus particle "rAAV particle" refers to
a viral particle
composed of at least one AAV capsid protein and an encapsidated polynucleotide
rAAV vector
genome comprising a heterologous polynucleotide (i.e. a polynucleotide other
than a wild-type
AAV genome such as a transgene to be delivered to a mammalian cell). The rAAV
particle may
be of any AAV serotype, including any modification, derivative or pseudotype
(e.g., AAV1,
AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, or AAV10, or
derivatives/modifications/pseudotypes thereof). Such AAV serotypes and
derivatives/modifications/pseudotypes, and methods of producing such
serotypes/derivatives/modifications/ pseudotypes are known in the art (see,
e.g., Asokan et al.,
Mol. Ther. 20(4):699-708 (2012).
[0038] The rAAV particles of the disclosure may be of any serotype, or any
combination of
serotypes, (e.g., a population of rAAV particles that comprises two or more
serotypes, e.g.,
comprising two or more of rAAV2, rAAV8, and rAAV9 particles). In some
embodiments, the
rAAV particles are rAAV1, rAAV2, rAAV3, rAAV4, rAAV5, rAAV6, rAAV7, rAAV8,
rAAV9,
rAAV10, or other rAAV particles, or combinations of two or more thereof). In
some
embodiments, the rAAV particles are rAAV8 or rAAV9 particles.
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[0039] In some embodiments, the rAAV particles have an AAV capsid protein of a
serotype
selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6,
AAV7,
AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15 and AAV16 or a
derivative,
modification, or pseudotype thereof. In some embodiments, the rAAV particles
have an AAV
capsid protein of a serotype of AAV8, AAV9, or a derivative, modification, or
pseudotype
thereof.
[0040] The term "cell culture," refers to cells grown adherent or in
suspension, bioreactors, roller
bottles, hyperstacks, microspheres, macrospheres, flasks and the like, as well
as the components
of the supernatant or suspension itself, including but not limited to rAAV
particles, cells, cell
debris, cellular contaminants, colloidal particles, biomolecules, host cell
proteins, nucleic acids,
and lipids, and flocculants. Large scale approaches, such as bioreactors,
including suspension
cultures and adherent cells growing attached to microcarriers or macrocarriers
in stirred
bioreactors, are also encompassed by the term "cell culture." Cell culture
procedures for both
large and small-scale production of proteins are encompassed by the present
disclosure. In some
embodiments, the term "cell culture" refers to cells grown in suspension. In
some embodiments,
the term "cell culture" refers to adherent cells grown attached to
microcarriers or macrocarriers in
stirred bioreactors. In some embodiments, the term "cell culture" refers to
cells grown in a
perfusion culture. In some embodiments, the term "cell culture" refers to
cells grown in an
alternating tangential flow (ATF) supported high-density perfusion culture.
[0041] The terms "purifying", "purification", "separate", "separating",
"separation", "isolate",
"isolating", or "isolation", as used herein, refer to increasing the degree of
purity of a target
product, e.g., rAAV particles and rAAV genome from a sample comprising the
target product and
one or more impurities. Typically, the degree of purity of the target product
is increased by
removing (completely or partially) at least one impurity from the sample. In
some embodiments,
the degree of purity of the rAAV in a sample is increased by removing
(completely or partially)
one or more impurities from the sample by using a method described herein.
[0042] "About" modifying, for example, the quantity of an ingredient in the
compositions,
concentration of an ingredient in the compositions, flow rate, rAAV particle
yield, feed volume,
salt concentration, and like values, and ranges thereof, employed in the
methods provided herein,
refers to variation in the numerical quantity that can occur, for example,
through typical
measuring and handling procedures used for making concentrates or use
solutions; through
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inadvertent error in these procedures; through differences in the manufacture,
source, or purity of
the ingredients employed to make the compositions or carry out the methods;
and like
considerations. The term "about" also encompasses amounts that differ due to
aging of a
composition with a particular initial concentration or mixture. The term
"about" also encompasses
amounts that differ due to mixing or processing a composition with a
particular initial
concentration or mixture. Whether or not modified by the term "about" the
claims include
equivalents to the quantities. In some embodiments, the term ''about" refers
to ranges of
approximately 10-20% greater than or less than the indicated number or range.
In further
embodiments, "about" refers to plus or minus 10% of the indicated number or
range. For
example, "about 10%" indicates a range of 9% to 11%.
[0043] As used in the present disclosure and claims, the singular forms "a",
"an" and "the"
include plural forms unless the context clearly dictates otherwise.
[0044] It is understood that wherever embodiments are described herein with
the language
"comprising" otherwise analogous embodiments described in terms of "consisting
of' and/or
"consisting essentially of" are also provided. It is also understood that
wherever embodiments are
described herein with the language "consisting essentially of" otherwise
analogous embodiments
described in terms of "consisting of" are also provided.
[0045] The term "and/or" as used in a phrase such as "A and/or B" herein is
intended to include
both A and B; A or B; A (alone); and B (alone). Likewise, the term "and/or" as
used in a phrase
such as "A, B, and/or C" is intended to encompass each of the following
embodiments: A, B, and
C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B
(alone); and C
(alone).
[0046] Where embodiments of the disclosure arc described in tcrms of a Markush
group or other
grouping of alternatives, the disclosed method encompasses not only the entire
group listed as a
whole, but also each member of the group individually and all possible
subgroups of the main
group, and also the main group absent one or more of the group members. The
disclosed methods
also envisage the explicit exclusion of one or more of any of the group
members in the disclosed
methods.
RECOMBINANT POLYNUCLEOTIDES
[0047] In some embodiments, the disclosure provides an isolated recombinant
polynucleotide
encoding one or more helper functions that are capable of promoting production
of recombinant
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AAV particles in a host cell, e.g., an HEK cell. In some embodiments, an
isolated recombinant
polynucleotide described herein comprises one or more of (a) a nucleotide
sequence encoding an
adenovirus E2A DNA binding protein (DBP); (b) a nucleotide sequence encoding
an adenovirus
E4 ORF6 and ORF7 polypeptide; and (c) a nucleotide sequence encoding an
adenovirus VA
RNA I. In some embodiments, the nucleotide sequence encoding an adenovirus VA
RNA I
encodes an adcnovirus VA RNA I and VA RNA II. In some embodiments, the
isolated
recombinant polynucleotide does not comprise a nucleotide sequence encoding an
adenovirus
ITR sequence, L3 23K endoprotease, L5 pVI/fibre, and/or L4 pVIII/hexon-
associated precursor.
In some embodiments, the nucleotide sequence encoding the adenovirus ITR
sequence, L3 23K
endoprotease, L5 pVI/fibre, and/or L4 pVIII/hexon-associated precursor is the
corresponding
nucleotide sequence of pAdDeltaF6. In some embodiments, a nucleotide sequence
encoding a
protein or polypeptide (e.g., E2A DBP or E4 ORF6 and ORF7), or RNA (e.g., VA
RNA I)
comprises a promoter operably linked to a nucleotide sequence comprising the
coding region for
the protein or polypeptide, or RNA. In some embodiments, a nucleotide sequence
encoding a
protein or polypeptide comprises a promoter and a polyA signal operably linked
to a nucleotide
sequence comprising the coding region.
[0048] In some embodiments, an isolated recombinant polynucleotide described
herein
comprises a nucleotide sequence encoding an adenovirus E2A DBP, a nucleotide
sequence
encoding an adenovirus E4 ORF6 and ORF7 polypeptide and a nucleotide sequence
encoding an
adenovirus VA RNA I. In some embodiments, an isolated recombinant
polynucleotide described
herein comprises a nucleotide sequence encoding an adenovirus E2A DBP, and a
nucleotide
sequence encoding an adenovirus E4 ORF6 and ORF7 polypeptide. In some
embodiments, an
isolated recombinant polynucleotide described herein comprises a nucleotide
sequence encoding
an adenovirus E2A DBP, and a nucleotide sequence encoding an adenovirus VA RNA
I. In some
embodiments, an isolated recombinant polynucleotide described herein comprises
a nucleotide
sequence encoding an adenovirus E4 ORF6 and ORF7 polypeptide and a nucleotide
sequence
encoding an adenovirus VA RNA I. In some embodiments, an isolated recombinant
polynucleotide described herein comprises a nucleotide sequence encoding an
adenovirus E2A
DBP. In some embodiments, an isolated recombinant polynucleotide described
herein comprises
a nucleotide sequence encoding an adenovirus E4 ORF6 and ORF7 polypeptide. In
some
embodiments, an isolated recombinant polynucleotide described herein comprises
a nucleotide
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sequence encoding an adenovirus VA RNA I. In some embodiments, the nucleotide
sequence
encoding an adenovirus VA RNA I encodes an adenovirus VA RNA I and VA RNA IL
In some
embodiments, the isolated recombinant polynucleotide does not comprise a
nucleotide sequence
encoding an adenovirus ITR sequence, L3 23K endoprotease, L5 pVI/fibre, and/or
L4
pVIII/hexon-associated precursor. In some embodiments, the nucleotide sequence
encoding the
adcnovirus ITR sequence, L3 23K endoprotease, L5 pVI/fibre, and/or L4
pVIII/hexon-associated
precursor is the corresponding nucleotide sequence of pAdDeltaF6. In some
embodiments of the
isolated recombinant polynucleotide, the nucleotide sequence encoding the
adenovirus E2A DBP
and the nucleotide sequence encoding the adenovirus E4 ORF6 and ORF7 are in
opposite 5' to 3'
orientation. In some embodiments of the isolated recombinant polynucleotide,
the nucleotide
sequence encoding the adenovirus E2A DBP and the nucleotide sequence encoding
the
adenovirus E4 ORF6 and ORF7 are in the same 5' to 3 orientation.
[0049] In some embodiments, an isolated recombinant polynucleotide described
herein
comprises a nucleotide sequence encoding an adenovirus E2A DBP, a nucleotide
sequence
encoding an adenovirus E4 ORF6 and ORF7 polypeptide and a nucleotide sequence
encoding an
adenovirus VA RNA I, wherein the nucleotide sequence encoding the adenovirus
E2A DBP and
the nucleotide sequence encoding the adenovirus E4 ORF6 and ORF7 are in
opposite 5' to 3'
orientation, and wherein the isolated recombinant polynucleotide does not
comprise a nucleotide
sequence encoding an adenovirus ITR sequence, L3 23K endoprotease, L5
pVI/fibre, and/or L4
pVIII/hexon-associated precursor. In some embodiments, the nucleotide sequence
encoding an
adenovirus VA RNA I encodes an adenovirus VA RNA I and VA RNA II.
Adenovirus E2A DNA binding protein
[0050] In some embodiments, the nucleotide sequence encoding an adenovirus E2A
DBP
comprises a nucleotide sequence having at least 80%, at least 85%, at least
90%, at least 95%, at
least 97%, at least 98%, at least 99% or 100 % identity to SEQ ID NO: 1. In
some embodiments,
the nucleotide sequence encoding an adenovirus E2A DBP comprises a nucleotide
sequence
having at least 90% identity to SEQ ID NO: 1. In some embodiments, the
nucleotide sequence
encoding an adenovirus E2A DBP comprises a nucleotide sequence having at least
95% identity
to SEQ ID NO: 1. In some embodiments, the nucleotide sequence encoding an
adenovirus E2A
DBP comprises a nucleotide sequence having at least 98% identity to SEQ ID NO:
1. In some
embodiments, the nucleotide sequence encoding an adenovirus E2A DBP comprises
SEQ ID NO:
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1. In some embodiments, the adenovirus E2A DBP polypeptide comprises an amino
acid
sequence having at least 80%, at least 85%, at least 90%, at least 95%, at
least 97%, at least 98%,
at least 99% or 100 % identity to SEQ ID NO: 45. In some embodiments, the
adenovirus E2A
DBP polypeptide comprises an amino acid sequence having at least 90% identity
to SEQ ID NO:
45. In some embodiments, the adenovirus E2A DBP polypeptide comprises an amino
acid
sequence having at least 95% identity to SEQ ID NO: 45. In some embodiments,
the adenovirus
E2A DBP polypeptide comprises an amino acid sequence having at least 98%
identity to SEQ ID
NO: 45. In some embodiments, the adenovirus E2A DBP polypeptide comprises the
amino acid
sequence of SEQ Ill NO: 45. In some embodiments, the nucleotide sequence
encoding an
adenovirus E2A DBP is operably linked to a promoter and to a polyA signal.
[0051] In some embodiments, the nucleotide sequence encoding an adenovirus E2A
DBP
comprises a nucleotide sequence encoding a polypeptide comprising an amino
acid sequence
having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%,
at least 98%, at least
99% or 100 % identity to SEQ ID NO: 45. In sonic embodiments, the adenovirus
E2A DBP
polypeptide comprises an amino acid sequence having at least 90% identity to
SEQ ID NO: 45. In
some embodiments, the adenovirus E2A DBP polypeptide comprises an amino acid
sequence
having at least 95% identity to SEQ ID NO: 45. In some embodiments, the
adenovirus E2A DBP
polypeptide comprises an amino acid sequence having at least 98% identity to
SEQ ID NO: 45. In
some embodiments, the adenovirus E2A DBP polypeptide comprises the amino acid
sequence of
SEQ ID NO: 45. In some embodiments, the nucleotide sequence encoding an
adenovirus E2A
DBP is operably linked to a promoter and to a polyA signal.
[0052] In some embodiments, the nucleotide sequence encoding an adenovirus E2A
DBP is
operably linked to an adenovirus E2A promoter. In some embodiments, the
adenovirus E2A
promoter comprises a nucleotide sequence comprising at least 80%, at least
85%, at least 90%, at
least 95%, at least 97%, at least 98%, at least 99% or 100 % identity to SEQ
ID NO: 2. In some
embodiments, the adenovirus E2A promoter comprises a nucleotide sequence
comprising at least
95% identity to SEQ ID NO: 2. In some embodiments, the adenovirus E2A promoter
comprises
the nucleotide sequence of SEQ ID NO: 2. In some embodiments, the nucleotide
sequence
encoding an adenovirus E2A DBP is operably linked to a promoter that is not an
adenovirus E2A
promoter.
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[0053] In some embodiments, the nucleotide sequence encoding an adenovirus E2A
DBP
operably linked to an adenovirus E2A promoter, and optionally a polyA signal,
encompasses,
from 3' to 5', the adenovirus E2A promoter, an adenovirus L4 22K/33K gene, an
adenovirus L4
100k/hexon assembly gene, the nucleotide sequence encoding an adenovirus E2A
DBP and
optionally the polyA signal. In some embodiments, the relative orientation of
the adenovirus E2A
promoter, adenovirus L4 22K/33K gene, adenovirus L4 100k/hexon assembly gene,
nucleotide
sequence encoding an adenovirus E2A DBP and optional polyA signal is the same
as in
pAdDeltaF6. In some embodiments, the nucleotide sequence encoding an
adenovirus E2A DBP
operably linked to an adenovirus E2A promoter comprises a nucleotide sequence
having at least
80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at
least 99% or 100 %
identity to SEQ ID NO: 3. In some embodiments, the nucleotide sequence
encoding an
adenovirus E2A DBP operably linked to an adenovirus E2A promoter comprises a
nucleotide
sequence having at least 90% identity to SEQ ID NO: 3. In some embodiments,
the nucleotide
sequence encoding an adenovirus E2A DBP operably linked to an adenovirus E2A
promoter
comprises a nucleotide sequence having at least 95% identity to SEQ ID NO: 3.
In some
embodiments, the nucleotide sequence encoding an adenovirus E2A DBP operably
linked to an
adenovirus E2A promoter comprises a nucleotide sequence having at least 98%
identity to SEQ
ID NO: 3. In some embodiments, the nucleotide sequence encoding an adenovirus
E2A DBP
operably linked to an adenovirus E2A promoter comprises the nucleotide
sequence of SEQ ID
NO: 3. In some embodiments, the nucleotide sequence encoding an adenovirus E2A
DBP
operably linked to an adenovirus E2A promoter and polyA signal comprises a
nucleotide
sequence having at least 80%, at least 85%, at least 90%, at least 95%, at
least 97%, at least 98%,
at least 99% or 100 % identity to SEQ ID NO: 4. In some embodiments, the
nucleotide sequence
encoding an adenovirus E2A DBP operably linked to an adenovirus E2A promoter
and polyA
signal comprises a nucleotide sequence having at least 90% identity to SEQ ID
NO: 4. In some
embodiments, the nucleotide sequence encoding an adenovirus E2A DBP operably
linked to an
adenovirus E2A promoter and polyA signal comprises a nucleotide sequence
having at least 95%
identity to SEQ ID NO: 4. In some embodiments, the nucleotide sequence
encoding an
adenovirus E2A DBP operably linked to an adenovirus E2A promoter and polyA
signal
comprises a nucleotide sequence having at least 98% identity to SEQ ID NO: 4.
In some
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embodiments, the nucleotide sequence encoding an adenovirus E2A DBP operably
linked to an
adenovirus E2A promoter and polyA signal comprises the nucleotide sequence of
SEQ ID NO: 4.
[0054] In some embodiments, the nucleotide sequence encoding an adenovirus E2A
DBP
operably linked to an adenovirus E2A promoter encompasses, from 3' to 5', the
adenovirus E2A
promoter, an adenovirus L4 22K/33K gene, an adenovirus L4 100k/hexon assembly
gene, the
nucleotide sequence encoding an adenovirus E2A DBP, wherein the adenovirus L4
100k/hexon
assembly gene comprises an N terminal deletion of the L4 100k/hexon assembly
polypeptide. In
some embodiments, the N terminal deletion does not affect the L4 100k/hexon
assembly
promoter. In some embodiments, the N terminal deletion corresponds to the
sequence of SEQ ID
NO: 21. In some embodiments, the relative orientation of the adenovirus E2A
promoter,
adenovirus L4 22K/33K gene, adenovirus L4 100k/hexon assembly gene and
nucleotide sequence
encoding an adenovirus E2A DBP is the same as in pAdDeltaF6. In some
embodiments, the
nucleotide sequence encoding an adenovirus E2A DBP operably linked to an
adenovirus E2A
promoter comprises a nucleotide sequence having at least 80%, at least 85%, at
least 90%, at least
95%, at least 97%, at least 98%, at least 99% or 100 % identity to SEQ ID NO:
22. In some
embodiments, the nucleotide sequence encoding an adenovirus E2A DBP operably
linked to an
adenovirus E2A promoter comprises a nucleotide sequence having at least 90%
identity to SEQ
ID NO: 22. In some embodiments, the nucleotide sequence encoding an adenovirus
E2A DBP
operably linked to an adenovirus E2A promoter comprises a nucleotide sequence
having at least
95% identity to SEQ ID NO: 22. In some embodiments, the nucleotide sequence
encoding an
adenovirus E2A DBP operably linked to an adenovirus E2A promoter comprises a
nucleotide
sequence having at least 98% identity to SEQ ID NO: 22. In some embodiments,
the nucleotide
sequence encoding an adenovirus E2A DBP operably linked to an adenovirus E2A
promoter
comprises the nucleotide sequence of SEQ ID NO: 22. In some embodiments, the
nucleotide
sequence encoding an adenovirus E2A DBP and adenovirus E2A promoter further
comprises an
operably linked polyA signal.
[0055] In some embodiments, the nucleotide sequence encoding an adenovirus E2A
DBP
operably linked to an adenovirus E2A promoter encompasses, from 3' to 5', the
adenovirus E2A
promoter, an adenovirus L4 22K/33K gene, an adenovirus L4 100k/hexon assembly
gene, the
nucleotide sequence encoding an adenovirus E2A DBP, wherein the adenovirus L4
100k/hexon
assembly gene comprises a mutation in the start codon of the L4 100k/hexon
assembly
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polypeptide. In some embodiments, the relative orientation of the adenovirus
E2A promoter,
adenovirus L4 22K133K gene, adenovirus L4 100k/hexon assembly gene and
nucleotide sequence
encoding an adenovirus E2A DBP is the same as in pAdDeltaF6. In some
embodiments, the
nucleotide sequence encoding an adenovirus E2A DBP operably linked to an
adenovirus E2A
promoter comprises a nucleotide sequence having at least 80%, at least 85%, at
least 90%, at least
95%, at least 97%, at least 98%, at least 99% or 100 % identity to SEQ ID NO:
23. In some
embodiments, the nucleotide sequence encoding an adenovirus E2A DBP operably
linked to an
adenovirus E2A promoter comprises a nucleotide sequence having at least 90%
identity to SEQ
Ill NO: 23. In some embodiments, the nucleotide sequence encoding an
adenovirus E2A DBP
operably linked to an adenovirus E2A promoter comprises a nucleotide sequence
having at least
95% identity to SEQ ID NO: 23. In some embodiments, the nucleotide sequence
encoding an
adenovirus E2A DBP operably linked to an adenovirus E2A promoter comprises a
nucleotide
sequence having at least 98% identity to SEQ ID NO: 23. In some embodiments,
the nucleotide
sequence encoding an adenovirus E2A DBP operably linked to an adenovirus E2A
promoter
comprises the nucleotide sequence of SEQ ID NO: 23. In some embodiments, the
nucleotide
sequence encoding an adenovirus E2A DBP and adenovirus E2A promoter further
comprises an
operably linked polyA signal.
[0056] In some embodiments, the nucleotide sequence encoding an adenovirus E2A
DBP
operably linked to an adenovirus E2A promoter encompasses, from 3' to 5', the
adenovirus E2A
promoter, an adenovirus L4 22K/33K gene, an adenovirus L4 100k/hexon assembly
gene, the
nucleotide sequence encoding an adenovirus E2A DBP, wherein the adenovirus L4
22K/33K
gene comprises a mutation in the start codon of the L4 22K/33K polypeptide. In
some
embodiments, the relative orientation of the adenovirus E2A promoter,
adenovirus L4 22K/33K
gene, adenovirus L4 100k/hexon assembly gene and nucleotide sequence encoding
an adenovirus
E2A DBP is the same as in pAdDeltaF6. In some embodiments, the nucleotide
sequence
encoding an adenovirus E2A DBP operably linked to an adenovirus E2A promoter
comprises a
nucleotide sequence having at least 80%, at least 85%, at least 90%, at least
95%, at least 97%, at
least 98%, at least 99% or 100 % identity to SEQ ID NO: 24. In some
embodiments, the
nucleotide sequence encoding an adenovirus E2A DBP operably linked to an
adenovirus E2A
promoter comprises a nucleotide sequence having at least 90% identity to SEQ
ID NO: 24. In
some embodiments, the nucleotide sequence encoding an adenovirus E2A DBP
operably linked
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to an adenovirus E2A promoter comprises a nucleotide sequence having at least
95% identity to
SEQ ID NO: 24. In some embodiments, the nucleotide sequence encoding an
adenovirus E2A
DBP operably linked to an adenovirus E2A promoter comprises a nucleotide
sequence having at
least 98% identity to SEQ ID NO: 24. In some embodiments, the nucleotide
sequence encoding
an adenovirus E2A DBP operably linked to an adenovirus E2A promoter comprises
the
nucleotide sequence of SEQ ID NO: 24. In some embodiments, the nucleotide
sequence encoding
an adenovirus E2A DBP and adenovirus E2A promoter further comprises an
operably linked
polyA signal.
[0057] In some embodiments, the nucleotide sequence encoding an adenovirus E2A
DBP
operably linked to an adenovirus E2A promoter encompasses, from 3' to 5', the
adenovirus E2A
promoter, an adenovirus L4 22K/33K gene, an adenovirus L4 100k/hexon assembly
gene, the
nucleotide sequence encoding an adenovirus E2A DBP, wherein the L4 100k/hexon
assembly
gene comprises an N terminal deletion of the L4 100k/hexon assembly
polypeptide that
encompasses the start codon of L4 100k/hexon assembly polypeptide but does not
encompass the
start codon of the L4 22K/33K polypeptide. In some embodiments, the nucleotide
sequence
encoding an adenovirus E2A DBP operably linked to an adenovirus E2A promoter
encompasses,
from 3' to 5', the adenovirus E2A promoter, an adenovirus L4 22K/33K gene, an
adenovirus L4
100k/hexon assembly gene, the nucleotide sequence encoding an adenovirus E2A
DBP, wherein
the L4 100k/hexon assembly gene comprises an N terminal deletion of the L4
100k/hexon
assembly polypeptide, wherein all or part of the L4 100k/hexon assembly
polypeptide is deleted
without disrupting the L4 22K/33K start codon. In some embodiments, the
nucleotide sequence
encoding an adenovirus E2A DBP operably linked to an adenovirus E2A promoter
encompasses,
from 3' to 5', the adenovirus E2A promoter, an adenovirus L4 22K/33K gene, an
adenovirus L4
100k/hexon assembly gene, the nucleotide sequence encoding an adenovirus E2A
DBP, wherein
the L4 100k/hexon assembly gene comprises an N terminal deletion of the L4
100k/hexon
assembly polypeptide that encompasses the start codon of L4 100k/hexon
assembly polypeptide
but does not encompass the L4 22K/33K promoter. In some embodiments, the
nucleotide
sequence encoding an adenovirus E2A DBP operably linked to an adenovirus E2A
promoter
encompasses, from 3' to 5', the adenovirus E2A promoter, an adenovirus L4
22K/33K gene, an
adenovirus L4 100k/hexon assembly gene, the nucleotide sequence encoding an
adenovirus E2A
DBP, wherein the L4 100k/hexon assembly gene comprises an N terminal deletion
of the L4
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100k/hexon assembly polypeptide that starts at the start codon of L4
100k/hexon assembly
polypeptide and ends immediately adjacent to the L4 22K/33K promoter. In some
embodiments,
the relative orientation of the adenovirus E2A promoter, adenovirus L4 22K/33K
gene,
adenovirus L4 100k/hexon assembly gene and nucleotide sequence encoding an
adenovirus E2A
DBP is the same as in pAdDeltaF6. In some embodiments, the nucleotide sequence
encoding an
adenovirus E2A DBP and adenovirus E2A promoter further comprises an operably
linked polyA
signal.
[0058] In some embodiments, the nucleotide sequence encoding an adenovirus E2A
DBP is
operably linked to a CMV immediate early promoter. In some embodiments, the
nucleotide
sequence encoding an adenovirus E2A DBP is operably linked to an engineered
CMV immediate
early promoter, or a transcriptionally active fragment or portion thereof.
[0059] In some embodiments, the nucleotide sequence encoding an adenovirus E2A
DBP is
operably linked to an inducible promoter.
Adenovirus E4 ORF6 and ORF7 polypeptide
[0060] In some embodiments, the nucleotide sequence encoding an adenovirus E4
ORF6 and
ORF7 polypeptide comprises a nucleotide sequence having at least 80%, at least
85%, at least
90%, at least 95%, at least 97%, at least 98%, at least 99% or 100 % identity
to SEQ ID NO: 8. In
some embodiments, the nucleotide sequence encoding an adenovirus E4 ORF6 and
ORF7
polypeptide comprises a nucleotide sequence having at least 90% identity to
SEQ ID NO: 8. In
some embodiments, the nucleotide sequence encoding an adenovirus E4 ORF6 and
ORF7
polypeptide comprises a nucleotide sequence having at least 95% identity to
SEQ ID NO: 8. In
some embodiments, the nucleotide sequence encoding an adenovirus E4 ORF6 and
ORF7
polypeptide comprises a nucleotide sequence having at least 98% identity to
SEQ Ill NO: 8. In
some embodiments, the nucleotide sequence encoding an adenovirus E4 ORF6 and
ORF7
polypeptide comprises SEQ ID NO: 8. In some embodiments, the adenovirus E4
ORF6 and
ORF7 polypeptide comprises an amino acid sequence having at least 80%, at
least 85%, at least
90%, at least 95%, at least 97%, at least 98%, at least 99% or 100 % identity
to SEQ ID NO: 46.
In some embodiments, the adenovirus E4 ORF6 and ORF7 polypeptide comprises an
amino acid
sequence having at least 90% identity to SEQ ID NO: 46. In some embodiments,
the adenovirus
E4 ORF6 and ORF7 polypeptide comprises an amino acid sequence having at least
95% identity
to SEQ ID NO: 46. In some embodiments, the adenovirus E4 ORF6 and ORF7
polypeptide
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comprises an amino acid sequence having at least 98% identity to SEQ ID NO:
46. In some
embodiments, the adenovirus E4 ORF6 and ORF7 polypeptide comprises the amino
acid
sequence of SEQ ID NO: 46. In some embodiments, the nucleotide sequence
encoding an
adenovirus E4 ORF6 and ORF7 polypeptide is operably linked to a promoter and
to a polyA
signal.
[0061] In some embodiments, the nucleotide sequence encoding an adenovirus E4
ORF6 and
ORF7 polypeptide comprises a nucleotide sequence encoding a polypeptide
comprising an amino
acid sequence having at least 80%, at least 85%, at least 90%, at least 95%,
at least 97%, at least
98%, at least 99% or 100 % identity to SEQ Ill NO: 46. In some embodiments,
the adenovirus E4
ORF6 and ORF7 polypeptide comprises an amino acid sequence having at least 90%
identity to
SEQ ID NO: 46. In some embodiments, the adenovirus E4 ORF6 and ORF7
polypeptide
comprises an amino acid sequence having at least 95% identity to SEQ ID NO:
46. In some
embodiments, the adenovirus E4 ORF6 and ORF7 polypeptide comprises an amino
acid sequence
having at least 98% identity to SEQ ID NO: 46. In some embodiments, the
adenovirus E4 ORF6
and ORF7 polypeptide comprises the amino acid sequence of SEQ ID NO: 46. In
some
embodiments, the nucleotide sequence encoding an adenovirus E4 ORF6 and ORF7
polypeptide
is operably linked to a promoter and to a polyA signal.
[0062] In some embodiments, the nucleotide sequence encoding an adenovirus E4
ORF6 and
ORF7 polypeptide is operably linked to an adenovirus E4 promoter. In some
embodiments, the
adenovirus E4 promoter comprises a nucleotide sequence comprising at least
80%, at least 85%,
at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100 %
identity to SEQ ID
NO: 5. In some embodiments, the adenovirus E4 promoter comprises a nucleotide
sequence
comprising at least 95% identity to SEQ ID NO: 5. In some embodiments, the
adenovirus E4
promoter comprises the nucleotide sequence of SEQ ID NO: 5. In some
embodiments, the
nucleotide sequence encoding an adenovirus E4 ORF6 and ORF7 polypeptide is
operably linked
to a promoter that is not an adenovirus E4 promoter.
[0063] In some embodiments, the nucleotide sequence encoding an adenovirus E4
ORF6 and
ORF7 polypeptide is operably linked to a CMV immediate early promoter. In some
embodiments, the nucleotide sequence encoding an adenovirus E4 ORF6 and ORF7
polypeptide
is operably linked to an engineered CMV immediate early promoter, or a
transcriptionally active
fragment or portion thereof.
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[0064] In some embodiments, the nucleotide sequence encoding an adenovirus E4
ORF6 and
ORF7 polypeptide is operably linked to an inducible promoter.
Adenovirus VA RNA
[0065] In some embodiments, the nucleotide sequence encoding an adenovirus VA
RNA I
comprises a nucleotide sequence having at least 80%, at least 85%, at least
90%, at least 95%, at
least 97%, at least 98%, at least 99% or 100 % identity to SEQ ID NO: 54. In
some embodiments,
the nucleotide sequence encoding an adenovirus VA RNA I comprises a nucleotide
sequence
having at least 90 % identity to SEQ ID NO: 54. In some embodiments, the
nucleotide sequence
encoding an adenovirus VA RNA I comprises a nucleotide sequence having at
least 95 % identity
to SEQ ID NO: 54. In some embodiments, the nucleotide sequence encoding an
adenovirus VA
RNA I comprises a nucleotide sequence having at least 98 % identity to SEQ ID
NO: 54. In some
embodiments, the nucleotide sequence encoding an adenovirus VA RNA I comprises
SEQ ID
NO: 54.
[0066] In some embodiments, the nucleotide sequence encoding an adenovirus VA
RNA II
comprises a nucleotide sequence having at least 80%, at least 85%, at least
90%, at least 95%, at
least 97%, at least 98%, at least 99% or 100 % identity to SEQ ID NO: 55. In
some embodiments,
the nucleotide sequence encoding an adenovirus VA RNA II comprises a
nucleotide sequence
having at least 90 % identity to SEQ ID NO: 55. In some embodiments, the
nucleotide sequence
encoding an adenovirus VA RNA II comprises a nucleotide sequence having at
least 95 %
identity to SEQ ID NO: 55. In some embodiments, the nucleotide sequence
encoding an
adenovirus VA RNA II comprises a nucleotide sequence having at least 98 %
identity to SEQ ID
NO: 55. In some embodiments, the nucleotide sequence encoding an adenovirus VA
RNA II
comprises SEQ Ill NO: 55.
[0067] In some embodiments, the nucleotide sequence encoding an adenovirus VA
RNA I and
VA RNA II comprises a nucleotide sequence having at least 80%, at least 85%,
at least 90%, at
least 95%, at least 97%, at least 98%, at least 99% or 100 % identity to SEQ
ID NO: 9. In some
embodiments, the nucleotide sequence encoding an adenovirus VA RNA I and VA
RNA II
comprises a nucleotide sequence having at least 90 % identity to SEQ ID NO: 9.
In sonic
embodiments, the nucleotide sequence encoding an adenovirus VA RNA I and VA
RNA II
comprises a nucleotide sequence having at least 95 % identity to SEQ ID NO: 9.
In some
embodiments, the nucleotide sequence encoding an adenovirus VA RNA I and VA
RNA II
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comprises a nucleotide sequence having at least 98 % identity to SEQ ID NO: 9.
In some
embodiments, the nucleotide sequence encoding an adenovirus VA RNA I and VA
RNA II
comprises SEQ ID NO: 9.
Polynuclentides encoding E2A DRP, E4 0RF6/7 and VA RNA
[0068] In some embodiments, an isolated recombinant polynucleotide described
herein
comprises a nucleotide sequence encoding an adenovirus E2A DBP, a nucleotide
sequence
encoding an adenovirus E4 ORF6 and ORF7 polypeptide and a nucleotide sequence
encoding an
adenovirus VA RNA I. In some embodiments, the nucleotide sequence encoding an
adenovirus
VA RNA I encodes an adenovirus VA RNA I and VA RNA II. In some embodiments,
the
promoter expressing the adenovirus E2A DBP and the promoter expressing the
adenovirus E4
ORF6 and ORF7 polypeptide are the same. In some embodiments, the promoter
expressing the
adenovirus E2A DBP and the promoter expressing the adenovirus E4 ORF6 and ORF7
polypeptide are different.
[0069] In some embodiments, an isolated recombinant polynucleotide described
herein
comprising a nucleotide sequence encoding an adenovirus E2A DBP, a nucleotide
sequence
encoding an adenovirus E4 ORF6 and ORF7 polypeptide and a nucleotide sequence
encoding an
adenovirus VA RNA I and VA RNA II comprises a nucleotide sequence having at
least 80%, at
least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least
99% or 100 % identity to
SEQ ID NO: 10. In some embodiments, the isolated recombinant polynucleotide
described
herein comprises a nucleotide sequence having at least 90% identity to SEQ ID
NO: 10. In some
embodiments, the isolated recombinant polynucleotide described herein
comprises a nucleotide
sequence having at least 95% identity to SEQ ID NO: 10. In some embodiments,
the isolated
recombinant polynucleotide described herein comprises a nucleotide sequence
having at least
98% identity to SEQ ID NO: 10. In some embodiments, the isolated recombinant
polynucleotide
described herein comprises the nucleotide sequence of SEQ ID NO: 10.
[0070] In some embodiments, an isolated recombinant polynucleotide described
herein
comprising a nucleotide sequence encoding an adenovirus E2A DBP, a nucleotide
sequence
encoding an adenovirus E4 ORF6 and ORF7 polypeptide and a nucleotide sequence
encoding an
adenovirus VA RNA I and VA RNA II comprises a nucleotide sequence having at
least 80%, at
least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least
99% or 100 % identity to
SEQ ID NO: 11. In some embodiments, the isolated recombinant polynucleotide
described
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herein comprises a nucleotide sequence having at least 90% identity to SEQ ID
NO: 11. In some
embodiments, the isolated recombinant polynucleotide described herein
comprises a nucleotide
sequence having at least 95% identity to SEQ ID NO: 11. In some embodiments,
the isolated
recombinant polynucleotide described herein comprises a nucleotide sequence
having at least
98% identity to SEQ ID NO: 11. In some embodiments, the isolated recombinant
polynucleotide
described herein comprises the nucleotide sequence of SEQ ID NO: 11.
[0071] In some embodiments, an isolated recombinant polynucleotide described
herein
comprising a nucleotide sequence encoding an adenovirus E2A DBP, a nucleotide
sequence
encoding an adenovirus E4 ORF6 and ORF7 polypeptide and a nucleotide sequence
encoding an
adenovirus VA RNA I and VA RNA II comprises a nucleotide sequence having at
least 80%, at
least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least
99% or 100 % identity to
SEQ ID NO: 56. In some embodiments, the isolated recombinant polynucleotide
described
herein comprises a nucleotide sequence having at least 90% identity to SEQ ID
NO: 56. In some
embodiments, the isolated recombinant polynucleotide described herein
comprises a nucleotide
sequence having at least 95% identity to SEQ ID NO: 56. In some embodiments,
the isolated
recombinant polynucleotide described herein comprises a nucleotide sequence
having at least
98% identity to SEQ ID NO: 56. In some embodiments, the isolated recombinant
polynucleotide
described herein comprises the nucleotide sequence of SEQ ID NO: 56.
[0072] In some embodiments, an isolated recombinant polynucleotide described
herein
comprising a nucleotide sequence encoding an adenovirus E2A DBP, a nucleotide
sequence
encoding an adenovirus E4 ORF6 and ORF7 polypeptide and a nucleotide sequence
encoding an
adenovirus VA RNA I and VA RNA II comprises a nucleotide sequence having at
least 80%, at
least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least
99% or 100 % identity to
SEQ ID NO: 57. In some embodiments, the isolated recombinant polynucleotide
described
herein comprises a nucleotide sequence having at least 90% identity to SEQ ID
NO: 57. In some
embodiments, the isolated recombinant polynucleotide described herein
comprises a nucleotide
sequence having at least 95% identity to SEQ ID NO: 57. In some embodiments,
the isolated
recombinant polynucleotide described herein comprises a nucleotide sequence
having at least
98% identity to SEQ ID NO: 57. In some embodiments, the isolated recombinant
polynucleotide
described herein comprises the nucleotide sequence of SEQ ID NO: 57.
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[0073] In some embodiments, an isolated recombinant polynucleotide described
herein
comprising a nucleotide sequence encoding an adenovirus E2A DBP, a nucleotide
sequence
encoding an adenovirus E4 ORF6 and ORF7 polypeptide and a nucleotide sequence
encoding an
adenovirus VA RNA I and VA RNA II comprises a nucleotide sequence having at
least 80%, at
least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least
99% or 100 % identity to
SEQ ID NO: 25. In some embodiments, thc isolated recombinant polynucleotide
described
herein comprises a nucleotide sequence having at least 90% identity to SEQ ID
NO: 25. In some
embodiments, the isolated recombinant polynucleotide described herein
comprises a nucleotide
sequence having at least 95% identity to SEQ Ill NO: 25. In some embodiments,
the isolated
recombinant polynucleotide described herein comprises a nucleotide sequence
having at least
98% identity to SEQ ID NO: 25. In some embodiments, the isolated recombinant
polynucleotide
described herein comprises the nucleotide sequence of SEQ ID NO: 25.
[0074] In some embodiments, an isolated recombinant polynucleotide described
herein
comprising a nucleotide sequence encoding an adenovirus E2A DBP, a nucleotide
sequence
encoding an adenovirus E4 ORF6 and ORF7 polypeptide and a nucleotide sequence
encoding an
adenovirus VA RNA I and VA RNA II comprises a nucleotide sequence having at
least 80%, at
least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least
99% or 100 % identity to
SEQ ID NO: 26. In some embodiments, the isolated recombinant polynucleotide
described
herein comprises a nucleotide sequence having at least 90% identity to SEQ ID
NO: 26. In some
embodiments, the isolated recombinant polynucleotide described herein
comprises a nucleotide
sequence having at least 95% identity to SEQ ID NO: 26. In some embodiments,
the isolated
recombinant polynucleotide described herein comprises a nucleotide sequence
having at least
98% identity to SEQ ID NO: 26. In some embodiments, the isolated recombinant
polynucleotide
described herein comprises the nucleotide sequence of SEQ ID NO: 26.
[0075] In some embodiments, an isolated recombinant polynucleotide described
herein
comprising a nucleotide sequence encoding an adenovirus E2A DBP, a nucleotide
sequence
encoding an adenovirus E4 ORF6 and ORF7 polypeptide and a nucleotide sequence
encoding an
adenovirus VA RNA I and VA RNA II comprises a nucleotide sequence having at
least 80%, at
least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least
99% or 100 % identity to
SEQ ID NO: 27. In some embodiments, the isolated recombinant polynucleotide
described
herein comprises a nucleotide sequence having at least 90% identity to SEQ ID
NO: 27. In sonic
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embodiments, the isolated recombinant polynucleotide described herein
comprises a nucleotide
sequence having at least 95% identity to SEQ ID NO: 27. In some embodiments,
the isolated
recombinant polynucleotide described herein comprises a nucleotide sequence
having at least
98% identity to SEQ ID NO: 27. In some embodiments, the isolated recombinant
polynucleotide
described herein comprises the nucleotide sequence of SEQ ID NO: 27.
[0076] In somc embodiments, an isolated recombinant polynucleotide described
herein
comprising a nucleotide sequence encoding an adenovirus E2A DBP, a nucleotide
sequence
encoding an adenovirus E4 ORF6 and ORF7 polypeptide and a nucleotide sequence
encoding an
adenovirus VA RNA I and VA RNA 11 comprises a nucleotide sequence having at
least 80%, at
least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least
99% or 100 % identity to
SEQ ID NO: 28. In some embodiments, the isolated recombinant polynucleotide
described
herein comprises a nucleotide sequence having at least 90% identity to SEQ ID
NO: 28. In some
embodiments, the isolated recombinant polynucleotide described herein
comprises a nucleotide
sequence having at least 95% identity to SEQ ID NO: 28. In some embodiments,
the isolated
recombinant polynucleotide described herein comprises a nucleotide sequence
having at least
98% identity to SEQ ID NO: 28. In some embodiments, the isolated recombinant
polynucleotide
described herein comprises the nucleotide sequence of SEQ ID NO: 28.
[0077] In some embodiments, an isolated recombinant polynucleotide described
herein
comprising a nucleotide sequence encoding an adenovirus E2A DBP, a nucleotide
sequence
encoding an adenovirus E4 ORF6 and ORF7 polypeptide and a nucleotide sequence
encoding an
adenovirus VA RNA I and VA RNA II comprises a nucleotide sequence having at
least 80%, at
least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least
99% or 100 % identity to
SEQ ID NO: 29. In some embodiments, thc isolated recombinant polynucleotide
described
herein comprises a nucleotide sequence having at least 90% identity to SEQ ID
NO: 29. In some
embodiments, the isolated recombinant polynucleotide described herein
comprises a nucleotide
sequence having at least 95% identity to SEQ Ill NO: 29. In some embodiments,
the isolated
recombinant polynucleotide described herein comprises a nucleotide sequence
having at least
98% identity to SEQ ID NO: 29. In some embodiments, the isolated recombinant
polynucleotide
described herein comprises the nucleotide sequence of SEQ ID NO: 29.
[0078] In some embodiments, an isolated recombinant polynucleotide described
herein
comprising a nucleotide sequence encoding an adenovirus E2A DBP, a nucleotide
sequence
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encoding an adenovirus E4 ORF6 and ORF7 polypeptide and a nucleotide sequence
encoding an
adenovirus VA RNA I and VA RNA II comprises a nucleotide sequence having at
least 80%, at
least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least
99% or 100 % identity to
SEQ ID NO: 30. In some embodiments, the isolated recombinant polynucleotide
described
herein comprises a nucleotide sequence having at least 90% identity to SEQ ID
NO: 30. In some
embodiments, the isolated recombinant polynucleotide described herein
comprises a nucleotide
sequence having at least 95% identity to SEQ ID NO: 30. In some embodiments,
the isolated
recombinant polynucleotide described herein comprises a nucleotide sequence
having at least
98% identity to SEQ ID NO: 30. In some embodiments, the isolated recombinant
polynucleotide
described herein comprises the nucleotide sequence of SEQ ID NO: 30.
[0079] In some embodiments, an isolated recombinant polynucleotide described
herein
comprising a nucleotide sequence encoding an adenovirus E2A DBP, a nucleotide
sequence
encoding an adenovirus E4 ORF6 and ORF7 polypeptide and a nucleotide sequence
encoding an
adenovirus VA RNA I and VA RNA II comprises a nucleotide sequence having at
least 80%, at
least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least
99% or 100 % identity to
SEQ ID NO: 31. In some embodiments, the isolated recombinant polynucleotide
described
herein comprises a nucleotide sequence having at least 90% identity to SEQ ID
NO: 31. In some
embodiments, the isolated recombinant polynucleotide described herein
comprises a nucleotide
sequence having at least 95% identity to SEQ ID NO: 31. In some embodiments,
the isolated
recombinant polynucleotide described herein comprises a nucleotide sequence
having at least
98% identity to SEQ ID NO: 31. In some embodiments, the isolated recombinant
polynucleotide
described herein comprises the nucleotide sequence of SEQ ID NO: 31.
[0080] In somc embodiments, an isolated recombinant polynucleotide described
herein
comprising a nucleotide sequence encoding an adenovirus E2A DBP, a nucleotide
sequence
encoding an adenovirus E4 ORF6 and ORF7 polypeptide and a nucleotide sequence
encoding an
adenovirus VA RNA 1 and VA RNA 11 comprises a nucleotide sequence having at
least 80%, at
least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least
99% or 100 % identity to
SEQ ID NO: 32. In some embodiments, the isolated recombinant polynucleotide
described
herein comprises a nucleotide sequence having at least 90% identity to SEQ ID
NO: 32. In some
embodiments, the isolated recombinant polynucleotide described herein
comprises a nucleotide
sequence having at least 95% identity to SEQ ID NO: 32. In some embodiments,
the isolated
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recombinant polynucleotide described herein comprises a nucleotide sequence
having at least
98% identity to SEQ ID NO: 32. In some embodiments, the isolated recombinant
polynucleotide
described herein comprises the nucleotide sequence of SEQ ID NO: 32.
[0081] In some embodiments, an isolated recombinant polynucleotide described
herein
comprising a nucleotide sequence encoding an adenovirus E2A DBP, a nucleotide
sequence
encoding an adcnovirus E4 ORF6 and ORF7 polypeptide and a nucleotide sequence
encoding an
adenovirus VA RNA I and VA RNA II comprises a nucleotide sequence having at
least 80%, at
least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least
99% or 100 % identity to
SEQ Ill NO: 33. In some embodiments, the isolated recombinant polynucleotide
described
herein comprises a nucleotide sequence having at least 90% identity to SEQ ID
NO: 33. In some
embodiments, the isolated recombinant polynucleotide described herein
comprises a nucleotide
sequence having at least 95% identity to SEQ ID NO: 33. In some embodiments,
the isolated
recombinant polynucleotide described herein comprises a nucleotide sequence
having at least
98% identity to SEQ ID NO: 33. In some embodiments, the isolated recombinant
polynucleotide
described herein comprises the nucleotide sequence of SEQ ID NO: 33.
[0082] In some embodiments, an isolated recombinant polynucleotide described
herein
comprising a nucleotide sequence encoding an adenovirus E2A DBP, a nucleotide
sequence
encoding an adenovirus E4 ORF6 and ORF7 polypeptide and a nucleotide sequence
encoding an
adenovirus VA RNA I and VA RNA II comprises a nucleotide sequence having at
least 80%, at
least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least
99% or 100 % identity to
SEQ ID NO: 34. In some embodiments, the isolated recombinant polynucleotide
described
herein comprises a nucleotide sequence having at least 90% identity to SEQ ID
NO: 34. In some
embodiments, the isolated recombinant polynucleotide described herein
comprises a nucleotide
sequence having at least 95% identity to SEQ ID NO: 34. In some embodiments,
the isolated
recombinant polynucleotide described herein comprises a nucleotide sequence
having at least
98% identity to SEQ ID NO: 34. In some embodiments, the isolated recombinant
polynucleotide
described herein comprises the nucleotide sequence of SEQ ID NO: 34.
Boca virus NP1 and NS2 polypeptides
[0083] In some embodiments, an isolated recombinant polynucleotide described
herein
comprising a nucleotide sequence encoding an adenovirus E2A DBP, a nucleotide
sequence
encoding an adenovirus E4 ORF6 and ORF7 polypeptide and a nucleotide sequence
encoding an
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adenovirus VA RNA I, and optionally VA RNA II, further comprises a nucleotide
sequence
encoding a Boca virus NP1 and NS2 polypeptides. In some embodiments, the
nucleotide
sequence encoding the Boca virus NP1 and NS2 polypeptides has at least 80%, at
least 85%, at
least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100 %
identity to SEQ ID NO:
12. In some embodiments, the nucleotide sequence encoding the Boca virus NP1
and NS2
polypeptides has at least 90% identity to SEQ ID NO: 12. In some embodiments,
the nucleotide
sequence encoding the Boca virus NP1 and NS2 polypeptides has at least 95%
identity to SEQ ID
NO: 12. In some embodiments, the nucleotide sequence encoding the Boca virus
NP1 and NS2
polypeptides has at least 98% identity to SEQ Ill NO: 12. In some embodiments,
the nucleotide
sequence encoding the Boca virus NP1 and NS2 polypeptides comprises SEQ ID NO:
12. In
some embodiments, the Boca virus NP1 and NS2 polypeptides comprise an amino
acid sequence
having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%,
at least 98%, at least
99% or 100 % identity to SEQ ID NO: 52. In some embodiments, the Boca virus
NP1 and N52
polypeptides comprise an amino acid sequence having at least 90% identity to
SEQ ID NO: 52. In
some embodiments, the Boca virus NP1 and NS2 polypeptides comprise an amino
acid sequence
having at least 95% identity to SEQ ID NO: 52. In some embodiments, the Boca
virus NP1 and
NS2 polypeptides comprise an amino acid sequence having at least 98% identity
to SEQ ID NO:
52. In some embodiments, the Boca virus NP1 and NS2 polypeptides comprise the
amino acid
sequence of SEQ ID NO: 52. In some embodiments, the nucleotide sequence
encoding the Boca
virus NP1 and NS2 polypeptides comprises a CMV promoter. In some embodiments,
the
nucleotide sequence encoding the Boca virus NP1 and N52 polypeptides comprises
an engineered
CMV inunediate early promoters.
[0084] In some embodiments, an isolated recombinant polynucleotide described
herein
comprising a nucleotide sequence encoding an adenovirus E2A DBP, a nucleotide
sequence
encoding an adenovirus E4 ORF6 and ORF7 polypeptide, a nucleotide sequence
encoding an
adenovirus VA RNA 1 and VA RNA 11 and a Boca virus NP1 and NS2 polypeptides
comprises a
nucleotide sequence having at least 80%, at least 85%, at least 90%, at least
95%, at least 97%, at
least 98%, at least 99% or 100 % identity to SEQ ID NO: 13. In some
embodiments, the isolated
recombinant polynucleotide described herein comprises a nucleotide sequence
having at least
90% identity to SEQ ID NO: 13. In some embodiments, the isolated recombinant
polynucleotide
described herein comprises a nucleotide sequence having at least 95% identity
to SEQ ID NO: 13.
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In some embodiments, the isolated recombinant polynucleotide described herein
comprises a
nucleotide sequence having at least 98% identity to SEQ ID NO: 13. In some
embodiments, the
isolated recombinant polynucleotide described herein comprises the nucleotide
sequence of SEQ
ID NO: 13.
[0085] In some embodiments, an isolated recombinant polynucleotide described
herein
comprising a nucleotide sequence encoding an adenovirus E2A DBP, a nucleotide
sequence
encoding an adenovirus E4 ORF6 and ORF7 polypeptide, a nucleotide sequence
encoding an
adenovirus VA RNA I and VA RNA 11 and a Boca virus NP1 and NS2 polypeptides
comprises a
nucleotide sequence having at least 80%, at least 85%, at least 90%, at least
95%, at least 97%, at
least 98%, at least 99% or 100 % identity to SEQ ID NO: 14. In some
embodiments, the isolated
recombinant polynucleotide described herein comprises a nucleotide sequence
having at least
90% identity to SEQ ID NO: 14. In some embodiments, the isolated recombinant
polynucleotide
described herein comprises a nucleotide sequence having at least 95% identity
to SEQ ID NO: 14.
In some embodiments, the isolated recombinant polynucleotide described herein
comprises a
nucleotide sequence having at least 98% identity to SEQ ID NO: 14. In some
embodiments, the
isolated recombinant polynucleotide described herein comprises the nucleotide
sequence of SEQ
ID NO: 14.
Adeno-associated virus assembly-activating protein
[0086] In some embodiments, an isolated recombinant polynucleotide described
herein
comprising a nucleotide sequence encoding an adenovirus E2A DBP, a nucleotide
sequence
encoding an adenovirus E4 ORF6 and ORF7 polypeptide and a nucleotide sequence
encoding an
adenovirus VA RNA I, and optionally VA RNA II, further comprises a nucleotide
sequence
encoding an adeno-associated virus (AAV) assembly-activating protein (AAP). A
skilled artisan
understands that the AAV AAP ORF overlaps with the AAV capsid ORF in the wild
type virus,
and consequently there are AAV serotype specific A APs, e.g., AAP 1 to 13
corresponding to
AAV serotypes 1 to 13. Sonntag et al., Journal of Virology, 85: 12686-12697
(2011). In some
embodiments the AAP is AAP 1, AAP 2, AAP 3B, AAP 4, AAP 5, AAP 6, AAP 7, AAP
8, AAP
9, AAP 10, AAP 11, AAP 12 or AAV 13. In some embodiments, the AAP isotype
matches the
capsid isotype of the recombinant AAV being produced. In some embodiments, the
AAP is AAP
8. In some embodiments, the AAP is AAP 9. In some embodiments, the AAP
comprises an
amino acid sequence having at least 80%, at least 85%, at least 90%, at least
95%, at least 97%, at
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least 98%, at least 99% or 100 % identity to SEQ ID NO: 53. In some
embodiments, the AAP
comprises the amino acid sequence of SEQ ID NO: 53. In some embodiments, the
nucleotide
sequence encoding the AAV AAP has at least 80%, at least 85%, at least 90%, at
least 95%, at
least 97%, at least 98%, at least 99% or 100 % identity to SEQ ID NO: 15. In
some embodiments,
the nucleotide sequence encoding the AAV AAP has at least 90% identity to SEQ
ID NO: 15. In
some embodiments, the nucleotide sequence encoding the AAV AAP has at least
95% identity to
SEQ ID NO: 15. In some embodiments, the nucleotide sequence encoding the AAV
AAP has at
least 98% identity to SEQ Ill NO: 15. In some embodiments, the nucleotide
sequence encoding
the AAV AAP comprises SEQ Ill NO: 15. In some embodiments, the AAV AAP
comprises an
amino acid sequence having at least 80%, at least 85%, at least 90%, at least
95%, at least 97%, at
least 98%, at least 99% or 100 % identity to SEQ ID NO: 53. In some
embodiments, the AAV
AAP comprises an amino acid sequence having at least 90% identity to SEQ ID
NO: 53. In some
embodiments, the AAV AAP comprises an amino acid sequence having at least 95%
identity to
SEQ ID NO: 53. In sonic embodiments, the AAV AAP comprises an amino acid
sequence having
at least 98% identity to SEQ ID NO: 53. In some embodiments, the AAV AAP
comprises the
amino acid sequence of SEQ ID NO: 53. In some embodiments, the nucleotide
sequence
encoding the AAV AAP comprises a CMV promoter. In some embodiments, the
nucleotide
sequence encoding the AAV AAP comprises an engineered CMV immediate early
promoters.
[0087] In some embodiments, an isolated recombinant polynucleotide described
herein
comprising a nucleotide sequence encoding an adenovirus E2A DBP, a nucleotide
sequence
encoding an adenovirus E4 ORF6 and ORF7 polypeptide, a nucleotide sequence
encoding an
adenovirus VA RNA I and VA RNA II and an adeno-associated virus (AAV) assembly-
activating
protein (AAP) comprises a nucleotide sequence having at least 80%, at least
85%, at least 90%, at
least 95%, at least 97%, at least 98%, at least 99% or 100 % identity to SEQ
ID NO: 16. In some
embodiments, the isolated recombinant polynucleotide described herein
comprises a nucleotide
sequence having at least 90% identity to SEQ Ill NO: 16. In some embodiments,
the isolated
recombinant polynucleotide described herein comprises a nucleotide sequence
having at least
95% identity to SEQ ID NO: 16. In some embodiments, the isolated recombinant
polynucleotide
described herein comprises a nucleotide sequence having at least 98% identity
to SEQ ID NO: 16.
In some embodiments, the isolated recombinant polynucleotide described herein
comprises the
nucleotide sequence of SEQ ID NO: 16.
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[0088] In some embodiments, an isolated recombinant polynucleotide described
herein
comprising a nucleotide sequence encoding an adenovirus E2A DBP, a nucleotide
sequence
encoding an adenovirus E4 ORF6 and ORF7 polypeptide, a nucleotide sequence
encoding an
adenovirus VA RNA I and VA RNA II and an adeno-associated virus (AAV) assembly-
activating
protein (AAP) comprises a nucleotide sequence having at least 80%, at least
85%, at least 90%, at
least 95%, at least 97%, at least 98%, at least 99% or 100 % identity to SEQ
ID NO: 17. In some
embodiments, the isolated recombinant polynucleotide described herein
comprises a nucleotide
sequence having at least 90% identity to SEQ Ill NO: 17. In some embodiments,
the isolated
recombinant polynucleotide described herein comprises a nucleotide sequence
having at least
95% identity to SEQ ID NO: 17. In some embodiments, the isolated recombinant
polynucleotide
described herein comprises a nucleotide sequence having at least 98% identity
to SEQ ID NO: 17.
In some embodiments, the isolated recombinant polynucleotide described herein
comprises the
nucleotide sequence of SEQ ID NO: 17.
Adenovirus ElA polypeptide
[0089] In some embodiments, an isolated recombinant polynucleotide described
herein
comprising a nucleotide sequence encoding an adenovirus E2A DBP, a nucleotide
sequence
encoding an adenovirus E4 ORF6 and ORF7 polypeptide and a nucleotide sequence
encoding an
adenovirus VA RNA I, and optionally VA RNA II, further comprises a nucleotide
sequence
encoding an adenovirus ElA polypeptide. In some embodiments, the nucleotide
sequence
encoding the adenovirus ElA polypeptide has at least 80%, at least 85%, at
least 90%, at least
95%, at least 97%, at least 98%, at least 99% or 100 % identity to SEQ ID NO:
18. In some
embodiments, the nucleotide sequence encoding the adenovirus ElA polypeptide
has at least 90%
identity to SEQ Ill NO: 18. In some embodiments, the nucleotide sequence
encoding the
adenovirus ElA polypeptide has at least 95% identity to SEQ ID NO: 18. In some
embodiments,
the nucleotide sequence encoding the adenovirus El A polypeptide has at least
98% identity to
SEQ ID NO: 18. In some embodiments, the nucleotide sequence encoding the
adenovirus ElA
polypeptide comprises SEQ ID NO: 18. In some embodiments, the adenovirus ElA
polypeptide
comprises an amino acid sequence having at least 80%, at least 85%, at least
90%, at least 95%,
at least 97%, at least 98%, at least 99% or 100 % identity to SEQ ID NO: 51.
In some
embodiments, the adenovirus ElA polypeptide comprises an amino acid sequence
having at least
90% identity to SEQ ID NO: 51. In some embodiments, the adenovirus ElA
polypeptide
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comprises an amino acid sequence having at least 95% identity to SEQ ID NO:
51. In some
embodiments, the adenovirus ElA polypeptide comprises an amino acid sequence
having at least
98% identity to SEQ ID NO: 51. In some embodiments, the adenovirus ElA
polypeptide
comprises the amino acid sequence of SEQ ID NO: 51. In some embodiments, the
nucleotide
sequence encoding the adenovirus ElA polypeptide comprises a CMV promoter. In
some
embodiments, the nucleotide sequence encoding the adenovirus ElA polypeptide
comprises an
engineered CMV immediate early promoters.
[0090] In some embodiments, an isolated recombinant polynucleotide described
herein
comprising a nucleotide sequence encoding an adenovirus E2A DBP, a nucleotide
sequence
encoding an adenovirus E4 ORF6 and ORF7 polypeptide, a nucleotide sequence
encoding an
adenovirus VA RNA I and VA RNA II and an adenovirus El A polypeptide comprises
a
nucleotide sequence having at least 80%, at least 85%, at least 90%, at least
95%, at least 97%, at
least 98%, at least 99% or 100 % identity to SEQ ID NO: 19. In some
embodiments, the isolated
recombinant polynucleotide described herein comprises a nucleotide sequence
having at least
90% identity to SEQ ID NO: 19. In some embodiments, the isolated recombinant
polynucleotide
described herein comprises a nucleotide sequence having at least 95% identity
to SEQ ID NO: 19.
In some embodiments, the isolated recombinant polynucleotide described herein
comprises a
nucleotide sequence having at least 98% identity to SEQ ID NO: 19. In some
embodiments, the
isolated recombinant polynucleotide described herein comprises the nucleotide
sequence of SEQ
ID NO: 19.
[0091] In some embodiments, an isolated recombinant polynucleotide described
herein
comprising a nucleotide sequence encoding an adenovirus E2A DBP, a nucleotide
sequence
encoding an adenovirus E4 ORF6 and ORF7 polypeptide, a nucleotide sequence
encoding an
adenovirus VA RNA I and VA RNA II and an adenovirus ElA polypeptide comprises
a
nucleotide sequence having at least 80%, at least 85%, at least 90%, at least
95%, at least 97%, at
least 98%, at least 99% or 100 % identity to SEQ Ill NO: 20. In some
embodiments, the isolated
recombinant polynucleotide described herein comprises a nucleotide sequence
having at least
90% identity to SEQ ID NO: 20. In some embodiments, the isolated recombinant
polynucleotide
described herein comprises a nucleotide sequence having at least 95% identity
to SEQ ID NO: 20.
In some embodiments, the isolated recombinant polynucleotide described herein
comprises a
nucleotide sequence having at least 98% identity to SEQ ID NO: 20. In some
embodiments, the
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isolated recombinant polynucleotide described herein comprises the nucleotide
sequence of SEQ
ID NO: 20.
PLASMIDS
[0092] In some embodiments, the disclosure provides a plasmid comprising a
recombinant
polynucleotide described herein wherein the plasmid encodes one or more helper
functions that
are capable of promoting production of recombinant AAV particles in a host
cell, e.g., an HEK
cell. In some embodiments, a plasmid described herein comprises a recombinant
polynucleotide
comprising one or more of (a) a nucleotide sequence encoding an adenovirus E2A
DNA binding
protein (DBP); (b) a nucleotide sequence encoding an adenovirus E4 ORF6 and
ORF7
polypeptide; and (c) a nucleotide sequence encoding an adenovirus VA RNA I. In
some
embodiments, the nucleotide sequence encoding an adenovirus VA RNA I encodes
an adenovirus
VA RNA I and VA RNA II. In some embodiments, the plasmid does not comprise a
nucleotide
sequence encoding an adenovirus ITR sequence, L3 23K endoprotease, L5
pVI/fibre, and/or L4
pVIII/hexon-associated precursor. In some embodiments, the plasmid is a
bacterial plasmid.
[0093] In some embodiments, a plasmid described herein comprises a bacterial
replication origin
capable of propagating the plasmid in a bacterial host cell, e.g., E. coli
host cell. In some
embodiments, the bacterial replication origin is a ColE1 origin.
[0094] In some embodiments, a plasmid described herein comprises a selectable
marker gene. In
some embodiments, the selectable marker gene is a drug resistance gene. In
some embodiments,
the selectable marker gene is a kanamycin resistance gene. In some
embodiments, the selectable
marker gene is an ampicillin resistance gene.
[0095] In some embodiments, a plasmid described herein comprises a bacterial
replication origin
and a selectable marker gene.
[0096] In some embodiments, a plasmid described herein comprises a recombinant
polynucleotide described herein comprising a nucleotide sequence encoding an
adenovirus E2A
DBP, a nucleotide sequence encoding an adenovirus E4 ORF6 and ORF7 polypeptide
and a
nucleotide sequence encoding an adenovirus VA RNA I. In some embodiments, a
plasmid
described herein comprises a recombinant polynucleotide described herein
comprising a
nucleotide sequence encoding an adenovirus E2A DBP, and a nucleotide sequence
encoding an
adenovirus E4 ORF6 and ORF7 polypeptide. In some embodiments, a plasmid
described herein
comprises a recombinant polynucleotide described herein comprising a
nucleotide sequence
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encoding an adenovirus E2A DBP, and a nucleotide sequence encoding an
adenovirus VA RNA
I. In some embodiments, a plasmid described herein comprises a recombinant
polynucleotide
described herein comprising a nucleotide sequence encoding an adenovirus E4
ORF6 and ORF7
polypeptide and a nucleotide sequence encoding an adenovirus VA RNA I. In some
embodiments, a plasmid described herein comprises a recombinant polynucleotide
described
herein comprising a nucleotide sequence cncoding an adenovirus E2A DBP. In
some
embodiments, a plasmid described herein comprises a recombinant polynucleotide
described
herein comprising a nucleotide sequence encoding an adenovirus E4 ORF6 and
ORF7
polypeptide. In some embodiments, a plasmid described herein comprises a
recombinant
polynucleotide described herein comprising a nucleotide sequence encoding an
adenovirus VA
RNA T. In some embodiments, the nucleotide sequence encoding an adenovirus VA
RNA I
encodes an adenovirus VA RNA I and VA RNA II. In some embodiments, the plasmid
does not
comprise a nucleotide sequence encoding an adenovirus ITR sequence, L3 23K
endoprotease, L5
pVI/fibre, and/or L4 pVIII/hexon-associated precursor. In some embodiments,
the nucleotide
sequence encoding the adenovirus ITR sequence, L3 23K endoprotease, L5
pVI/fibre, and/or L4
pVIII/hexon-associated precursor is the corresponding nucleotide sequence of
pAdDe1taF6.
[0097] In some embodiments, a plasmid described herein comprises a recombinant
polynucleotide described herein comprising a nucleotide sequence encoding an
adenovirus E2A
DBP, a nucleotide sequence encoding an adenovirus E4 ORF6 and ORF7 polypeptide
and a
nucleotide sequence encoding an adenovirus VA RNA I. In some embodiments, the
nucleotide
sequence encoding an adenovirus VA RNA I encodes an adenovirus VA RNA I and VA
RNA II.
[0098] In some embodiments, a plasmid described herein comprises a nucleotide
sequence
having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%,
at least 98%, at least
99% or 100 % identity to SEQ ID NO: 10. In some embodiments, a plasmid
described herein
comprises the nucleotide sequence of SEQ ID NO: 10.
[0099] In some embodiments, a plasmid described herein comprises a nucleotide
sequence
having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%,
at least 98%, at least
99% or 100 % identity to SEQ ID NO: 11. In some embodiments, a plasmid
described herein
comprises the nucleotide sequence of SEQ ID NO: 11.
[00100] In some embodiments, a plasmid described herein comprises a nucleotide
sequence
having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%,
at least 98%, at least
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99% or 100 % identity to SEQ ID NO: 25-34, 58 or 59. In some embodiments, a
plasmid
described herein comprises the nucleotide sequence of SEQ ID NO: 25-34, 58 or
59.
[00101] In some embodiments, a plasmid described herein is less than 15,000 bp
long. In some
embodiments, a plasmid described herein is less than 12,000 bp long. In some
embodiments, a
plasmid described herein is between 9,000 and 12,000 bp long.
[00102] In some embodiments, a plasmid described herein comprises a nucleotide
sequence
having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%,
at least 98%, at least
99% or 100 % identity to SEQ Ill NO: 35. In some embodiments, the plasmid
described herein
comprises a nucleotide sequence having at least 90% identity to SEQ Ill NO:
35. In some
embodiments, the plasmid described herein comprises a nucleotide sequence
having at least 95%
identity to SEQ ID NO: 35. In some embodiments, the plasmid described herein
comprises a
nucleotide sequence having at least 98% identity to SEQ ID NO: 35. In some
embodiments, the
plasmid described herein comprises the nucleotide sequence of SEQ ID NO: 35.
[00103] In sonic embodiments, a plasmid described herein comprises a
nucleotide sequence
having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%,
at least 98%, at least
99% or 100 % identity to SEQ ID NO: 36. In some embodiments, the plasmid
described herein
comprises a nucleotide sequence having at least 90% identity to SEQ ID NO: 36.
In some
embodiments, the plasmid described herein comprises a nucleotide sequence
having at least 95%
identity to SEQ ID NO: 36. In some embodiments, the plasmid described herein
comprises a
nucleotide sequence having at least 98% identity to SEQ ID NO: 36. In some
embodiments, the
plasmid described herein comprises the nucleotide sequence of SEQ ID NO: 36.
[00104] In some embodiments, a plasmid described herein comprises a nucleotide
sequence
having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%,
at least 98%, at least
99% or 100 % identity to SEQ ID NO: 37. In some embodiments, the plasmid
described herein
comprises a nucleotide sequence having at least 90% identity to SEQ ID NO: 37.
In some
embodiments, the plasmid described herein comprises a nucleotide sequence
having at least 95%
identity to SEQ ID NO: 37. In some embodiments, the plasmid described herein
comprises a
nucleotide sequence having at least 98% identity to SEQ ID NO: 37. In some
embodiments, the
plasmid described herein comprises the nucleotide sequence of SEQ ID NO: 37.
[00105] In some embodiments, a plasmid described herein comprises a nucleotide
sequence
having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%,
at least 98%, at least
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99% or 100 % identity to SEQ ID NO: 38. In some embodiments, the plasmid
described herein
comprises a nucleotide sequence having at least 90% identity to SEQ ID NO: 38.
In some
embodiments, the plasmid described herein comprises a nucleotide sequence
having at least 95%
identity to SEQ ID NO: 38. In some embodiments, the plasmid described herein
comprises a
nucleotide sequence having at least 98% identity to SEQ ID NO: 38. In some
embodiments, the
plasmid described herein comprises the nucleotide sequence of SEQ ID NO: 38.
[00106] In some embodiments, a plasmid described herein comprises a nucleotide
sequence
having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%,
at least 98%, at least
99% or 100 % identity to SEQ Ill NO: 39. In some embodiments, the plasmid
described herein
comprises a nucleotide sequence having at least 90% identity to SEQ ID NO: 39.
In some
embodiments, the plasmid described herein comprises a nucleotide sequence
having at least 95%
identity to SEQ ID NO: 39. In some embodiments, the plasmid described herein
comprises a
nucleotide sequence having at least 98% identity to SEQ ID NO: 39. In some
embodiments, the
plasmid described herein comprises the nucleotide sequence of SEQ ID NO: 39.
[00107] In some embodiments, a plasmid described herein comprises a nucleotide
sequence
having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%,
at least 98%, at least
99% or 100 % identity to SEQ ID NO: 40. In some embodiments, the plasmid
described herein
comprises a nucleotide sequence having at least 90% identity to SEQ ID NO: 40.
In some
embodiments, the plasmid described herein comprises a nucleotide sequence
having at least 95%
identity to SEQ ID NO: 40. In some embodiments, the plasmid described herein
comprises a
nucleotide sequence having at least 98% identity to SEQ ID NO: 40. In some
embodiments, the
plasmid described herein comprises the nucleotide sequence of SEQ ID NO: 40.
[00108] In some embodiments, a plasmid described herein comprises a nucleotide
sequence
having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%,
at least 98%, at least
99% or 100 % identity to SEQ ID NO: 41. In some embodiments, the plasmid
described herein
comprises a nucleotide sequence having at least 90% identity to SEQ Ill NO:
41. In some
embodiments, the plasmid described herein comprises a nucleotide sequence
having at least 95%
identity to SEQ ID NO: 41. In some embodiments, the plasmid described herein
comprises a
nucleotide sequence having at least 98% identity to SEQ ID NO: 41. In some
embodiments, the
plasmid described herein comprises the nucleotide sequence of SEQ ID NO: 41.
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[00109]In some embodiments, a plasmid described herein comprises a nucleotide
sequence
having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%,
at least 98%, at least
99% or 100 % identity to SEQ ID NO: 42. In some embodiments, the plasmid
described herein
comprises a nucleotide sequence having at least 90% identity to SEQ ID NO: 42.
In some
embodiments, the plasmid described herein comprises a nucleotide sequence
having at least 95%
identity to SEQ ID NO: 42. In some embodiments, the plasmid described herein
comprises a
nucleotide sequence having at least 98% identity to SEQ ID NO: 42. In some
embodiments, the
plasmid described herein comprises the nucleotide sequence of SEQ Ill NO: 42.
[00110]In some embodiments, a plasmid described herein comprises a nucleotide
sequence
having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%,
at least 98%, at least
99% or 100 % identity to SEQ ID NO: 43. In some embodiments, the plasmid
described herein
comprises a nucleotide sequence having at least 90% identity to SEQ ID NO: 43.
In some
embodiments, the plasmid described herein comprises a nucleotide sequence
having at least 95%
identity to SEQ ID NO: 43. In some embodiments, the plasmid described herein
comprises a
nucleotide sequence having at least 98% identity to SEQ ID NO: 43. In some
embodiments, the
plasmid described herein comprises the nucleotide sequence of SEQ ID NO: 43.
HOST CELLS
[00111]In some embodiments, the disclosure provides a host cell comprising a
recombinant
polynucleotide or a plasmid described herein. In some embodiments, the host
cell is a prokaryotic
cell capable of propagating a recombinant polynucleotide or a plasmid
described herein. In some
embodiments, the prokaryotic host cell is a bacterial cell. In some
embodiments, the prokaryotic
host cell is E. coll. In some embodiments, the host cell is a eukaryotic cell
capable of producing
recombinant AAV particles. In some embodiments, the eukaryotic host cell is a
mammalian cell.
In some embodiments, the eukaryotic host cell is a 11EK293 cell, HEK derived
cell, CHO cell.
CHO derived cell, HeLa cell, SF-9 cell, BHK cell, Vero cell, CAP cell, or
PerC6 cell.
[00112]In some embodiments, a host cell described herein comprises a
recombinant
polynucleotide comprising one or more of (a) a nucleotide sequence encoding an
adenovirus E2A
DNA binding protein (DBP); (b) a nucleotide sequence encoding an adenovirus E4
ORF6 and
ORF7 polypeptide; and (c) a nucleotide sequence encoding an adenovirus VA RNA
T. In some
embodiments, the nucleotide sequence encoding an adenovirus VA RNA I encodes
an adenovirus
VA RNA I and VA RNA II. In some embodiments, the recombinant polynucleotide
does not
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comprise a nucleotide sequence encoding an adenovirus ITR sequence, L3 23K
endoprotease, L5
pVI/fibre, and/or L4 pVIII/hexon-associated precursor. In some embodiments,
the plasmid is a
bacterial plasmid.
[00113] In some embodiments, a host cell described herein comprises a plasmid
described herein
comprising a nucleotide sequence having at least 80%, at least 85%, at least
90%, at least 95%, at
least 97%, at least 98%, at least 99% or 100 % identity to SEQ ID NO: 11. In
some
embodiments, the plasmid described herein comprises a nucleotide sequence
having at least 90%
identity to SEQ Ill NO: 11. In some embodiments, the plasmid described herein
comprises a
nucleotide sequence having at least 95% identity to SEQ Ill NO: 11. In some
embodiments, the
plasmid described herein comprises a nucleotide sequence having at least 98%
identity to SEQ ID
NO: 11. In some embodiments, the plasmid described herein comprises the
nucleotide sequence
of SEQ ID NO: 11.
[00114] In some embodiments, a host cell described herein comprises a plasmid
described herein
comprising a nucleotide sequence having at least 80%, at least 85%, at least
90%, at least 95%, at
least 97%, at least 98%, at least 99% or 100 % identity to SEQ ID NO: 37. In
some
embodiments, the plasmid described herein comprises a nucleotide sequence
having at least 90%
identity to SEQ ID NO: 37. In some embodiments, the plasmid described herein
comprises a
nucleotide sequence having at least 95% identity to SEQ ID NO: 37. In some
embodiments, the
plasmid described herein comprises a nucleotide sequence having at least 98%
identity to SEQ ID
NO: 37. In some embodiments, the plasmid described herein comprises the
nucleotide sequence
of SEQ ID NO: 37.
[00115] In some embodiments, a host cell described herein comprises a plasmid
described herein
comprising a nucleotide sequence having at least 80%, at least 85%, at least
90%, at least 95%, at
least 97%, at least 98%, at least 99% or 100 % identity to SEQ ID NO: 10, 11,
25-34, 58 or 59. In
some embodiments, the plasmid described herein comprises the nucleotide
sequence of SEQ ID
NO: 10, 11. 25-34, 58 or 59.
[00116] In some embodiments, a host cell described herein comprises a plasmid
described herein
comprising a nucleotide sequence having at least 80%, at least 85%, at least
90%, at least 95%, at
least 97%, at least 98%, at least 99% or 100 % identity to SEQ ID NO: 35-43.
In some
embodiments, the plasmid described herein comprises the nucleotide sequence of
SEQ ID NO:
35-43.
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[00117]In some embodiments, the disclosure provides a method of producing a
recombinant
polynucleotide described herein or a plasmid described herein comprising
incubating a host cell
described herein under suitable conditions to produce the recombinant
polynucleotide or a
plasmid. In some embodiments, the host cell is a prokaryotic cell capable of
propagating a
plasmid described herein. In some embodiments, the prokaryotic host cell is a
bacterial cell. In
some embodiments, the prokaryotic host cell is E. coli. In some embodiments,
thc recombinant
polynucleotide comprises a nucleotide sequence having at least 80%, at least
85%, at least 90%,
at least 95%, at least 97%, at least 98%, at least 99% or 100 % identity to
SEQ Ill NO: 10, 11,
25-34, 58 or 59. In some embodiments, the recombinant polynucleotide comprises
the nucleotide
sequence of SEQ ID NO: 10, 11, 25-34, 58 or 59. In some embodiments, the
plasmid comprises a
nucleotide sequence having at least 80%, at least 85%, at least 90%, at least
95%, at least 97%, at
least 98%, at least 99% or 100 % identity to SEQ ID NO: 35-43. In some
embodiments, the
plasmid comprises the nucleotide sequence of SEQ ID NO: 35-43.
METHODS OF PRODUCING A RECOMBINANT VIRAL PARTICLE
[00118] In one aspect, the disclosure provides a method of producing
recombinant adeno-
associated virus (rAAV) particles in a eukaryotic host cell by using a
recombinant polynucleotide
or plasmid described herein to provide one or more helper functions that are
capable of promoting
production of recombinant AAV particles. In some embodiments, the method
further comprises
recovering the rAAV particles.
[00119]In some embodiments, a method of producing rAAV particles described
herein comprises
the use of a recombinant polynucleotide or plasmid comprising a nucleotide
sequence having at
least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least
98%, at least 99% or 100
% identity to SEQ ID NO: 11. In some embodiments, the recombinant
polynucleotide or plasmid
comprises a nucleotide sequence having at least 90% identity to SEQ ID NO: 11.
In some
embodiments, the recombinant polynucleotide or plasmid comprises a nucleotide
sequence
having at least 95% identity to SEQ ID NO: 11. In some embodiments, the
recombinant
polynucleotide or plasmid comprises a nucleotide sequence having at least 98%
identity to SEQ
ID NO: 11. In some embodiments, the recombinant polynucleotide or plasmid
comprises the
nucleotide sequence of SEQ ID NO: 11.
[00120]In some embodiments, a method of producing rAAV particles described
herein comprises
the use of a recombinant polynucleotide or plasmid comprising a nucleotide
sequence having at
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least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least
98%, at least 99% or 100
% identity to SEQ ID NO: 37. In some embodiments, the recombinant
polynucleotide or plasmid
comprises a nucleotide sequence having at least 90% identity to SEQ ID NO: 37.
In some
embodiments, the recombinant polynucleotide or plasmid comprises a nucleotide
sequence
having at least 95% identity to SEQ ID NO: 37. In some embodiments, the
recombinant
polynucleotide or plasmid comprises a nucleotide sequence having at least 98%
identity to SEQ
ID NO: 37. In some embodiments, the recombinant polynucleotide or plasmid
comprises the
nucleotide sequence of SEQ ID NO: 37.
[00121] In some embodiments, a method of producing rAAV particles described
herein comprises
the use of a recombinant polynucleotide or plasmid comprising a nucleotide
sequence having at
least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least
98%, at least 99% or 100
% identity to SEQ ID NO: 10, 11, 25-34, 58 or 59. In some embodiments, the
recombinant
polynucleotide or plasmid comprises the nucleotide sequence of SEQ ID NO: 10,
11, 25-34, 58 or
59.
[00122] In some embodiments, a method of producing rAAV particles described
herein comprises
the use of a recombinant polynucleotide or plasmid comprising a nucleotide
sequence having at
least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least
98%, at least 99% or 100
% identity to SEQ ID NO: 35-43. In some embodiments, the recombinant
polynucleotide or
plasmid comprises the nucleotide sequence of SEQ ID NO: 35-43.
[00123] In some embodiments, the disclosure provides a method of producing
recombinant
adeno-associated virus (rAAV) particles comprising culturing a cell capable of
producing the
rAAV particles, wherein the cell comprises (i) a polynucleotide encoding an
AAV capsid protein;
(ii) a polynucleotide encoding a functional rep gene; (iii) a polynucleotide
comprising a genome
comprising at least one AAV inverted terminal repeat (ITR) and a non-AAV
nucleic acid
sequence encoding a gene product operably linked to sequences which direct
expression of the
gene product in a target cell; and (iv) one or more polynucleotides comprising
sufficient helper
functions to permit packaging of the genome into the AAV capsid protein under
conditions which
permit packaging of the genome into the AAV capsid, wherein the one or more
polynucleotides
comprising sufficient helper functions independently comprise a recombinant
polynucleotide
described herein or a plasmid described herein. In some embodiments, the one
or more
polynucleotides comprising sufficient helper functions comprise a nucleotide
sequence encoding
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the adenovirus E2A DBP, the nucleotide sequence encoding the adenovirus E4
ORF6 and ORF7
polypeptide and the nucleotide sequence encoding the adenovirus VA RNA I. In
some
embodiments, the nucleotide sequence encoding an adenovirus VA RNA I encodes
an adenovirus
VA RNA I and VA RNA II. In some embodiments, the one or more polynucleotides
comprising
sufficient helper functions comprise the nucleotide sequence of SEQ ID NO: 10.
In some
embodiments, the one or more polynucleotides comprising sufficient helper
functions comprise
the nucleotide sequence of SEQ ID NO: 37. In some embodiments, the one or more
polynucleotides comprising sufficient helper functions comprise a nucleotide
sequence having at
least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least
98%, at least 99% or 100
% identity to SEQ ID NO: 35-43. In some embodiments, the one or more
polynucleotides
comprising sufficient helper functions comprise a nucleotide sequence having
at least 80%, at
least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least
99% or 100 % identity to
SEQ ID NO: 10, 11, 25-34, 58 or 59. In some embodiments, the method further
comprises
recovering the rAAV particles. In some embodiments, the cell comprises one
polynucleotide
encoding the cap and rep genes, one polynucleotide disclosed herein that
encodes adenovirus
helper functions necessary for packaging (e.g., adenovirus El a gene, Elb
gene, E4 gene, E2a
gene, and VA gene), and one polynucleotide encoding the rAAV genome to be
packaged. In
some embodiments, the rAAV particles are A AV8 or A AV9 particles. In some
embodiments, the
rAAV particles have an AAV capsid protein of a serotype selected from the
group consisting of
AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37,
AAV.PHB, and AAV.7m8. In some embodiments, the rAAV particles have an AAV
capsid
protein with high sequence homology to AAV8 or AAV9 such as, AAV.rh10,
AAV.rh20,
AAV.rh39, AAV.Rh74, AAV.RHM4-1, and AAV.hu37. In some embodiments, the cell
culture is
a suspension culture. In some embodiments, the cell culture comprises HEK293
cells adapted for
growth in suspension culture. In some embodiments, the cell culture has a
volume of between
about 400 liters and about 5,000 liters.
[00124] In some embodiments, the disclosure provides a method of producing
recombinant
adeno-associated virus (rAAV) particles comprising (a) providing a cell
culture comprising a cell;
(b) introducing into the cell one or more polynucleotides comprising (i) a
polynucleotide
encoding an AAV capsid protein; (ii) a polynucleotide encoding a functional
rep gene; (iii) a
polynucleotide comprising a genome comprising at least one AAV inverted
terminal repeat (ITR)
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and a non-AAV nucleic acid sequence encoding a gene product operably linked to
sequences
which direct expression of the gene product in a target cell; and (iv) one or
more polynucleotides
comprising sufficient helper functions to permit packaging of the genome into
the AAV capsid
protein under conditions which permit packaging of the genome into the AAV
capsid, wherein
the one or more polynucleotides comprising sufficient helper functions
independently comprise a
recombinant polynucleotide described herein or a plasmid described herein, and
(c) maintaining
the cell culture under conditions that allow production of the rAAV particles.
In some
embodiments, the one or more polynucleotides comprising sufficient helper
functions comprise a
nucleotide sequence encoding the adenovirus E2A DBP, the nucleotide sequence
encoding the
adenovirus E4 ORF6 and ORF7 polypeptide and the nucleotide sequence encoding
the
adenovirus VA RNA I/II gene. In some embodiments, the nucleotide sequence
encoding an
adenovirus VA RNA I encodes an adenovirus VA RNA I and VA RNA II. In some
embodiments,
the one or more polynucleotides comprising sufficient helper functions
comprise the nucleotide
sequence of SEQ ID NO: 10. In some embodiments, the one or more
polynucleotides comprising
sufficient helper functions comprise the nucleotide sequence of SEQ ID NO: 37.
In some
embodiments, the one or more polynucleotides comprising sufficient helper
functions comprise a
nucleotide sequence having at least 80%, at least 85%, at least 90%, at least
95%, at least 97%, at
least 98%, at least 99% or 100 % identity to SEQ ID NO: 35-43. In some
embodiments, the one
or more polynucleotides comprising sufficient helper functions comprise a
nucleotide sequence
having at least 80%, at least 85%, at least 90%, at least 95%. at least 97%,
at least 98%, at least
99% or 100 % identity to SEQ ID NO: 10, 11, 25-34, 58 or 59. In some
embodiments, the method
further comprises recovering the rAAV particles. In some embodiments, the one
or more
polynucleotides introduced into the cell comprise a mixture of three
polynucleotides: one
encoding the cap and rep genes, one polynucleotide disclosed herein that
encodes adenovirus
helper functions necessary for packaging (e.g., adenovirus El a gene, Elb
gene, E4 gene, E2a
gene, and VA gene), and one encoding the rAAV genome to be packaged. In some
embodiments,
the rAAV particles are AAV8 or AAV9 particles. In some embodiments, the rAAV
particles have
an AAV capsid protein of a serotype selected from the group consisting of
AAV.rh8. AAV.rh10,
AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.PHB, and AAV.7m8. In
some embodiments, the rAAV particles have an AAV capsid protein with high
sequence
homology to AAV8 or AAV9 such as, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74,
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AAV.RHM4-1, and AAV.hu37. In some embodiments, the cell culture is a
suspension culture. In
some embodiments, the cell culture comprises HEK293 cells adapted for growth
in suspension
culture. In some embodiments, the cell culture has a volume of between about
400 liters and
about 5,000 liters.
[00125] In some embodiments, a method disclosed herein comprises introducing
into the cell a
polynucleotide encoding an AAV capsid protein and a functional rep gene.
[00126] In some embodiments, the introducing of the one or more
polynucleotides into the cell is
by transfection.
[00127] In some embodiments, the cell is a mammalian cell. In some
embodiments, the cell is an
insect cell. In some embodiments, the cell is a HEK293 cell, HEK derived cell,
CHO cell, CHO
derived cell, HeLa cell, SF-9 cell, BHK cell, Vero cell CAP cell or PerC6
cell. In some
embodiments, the cell is a HEK293 cell.
[00128] In some embodiments, the cell culture is a suspension culture or an
adherent culture. In
some embodiments, the cell culture is a suspension culture.
[00129] In some embodiments, the cell culture has a volume between about 50
liters and about
20,000 liters.
[00130] In some embodiments, a method described herein produces more rAAV
particles
measured as GC/ml than a reference method. In some embodiments, the reference
method uses a
polynucleotide comprising helper functions that comprises the nucleotide
sequence of SEQ ID
NO: 35. In some embodiments, the reference method uses a polynucleotide
comprising helper
functions that comprises the nucleotide sequence of SEQ ID NO: 44. In some
embodiments, the
method described herein produces at least about 10% more rAAV particles
measured as GC/nil
than the reference method. In some embodiments, the method described herein
produces at least
about 10% more rAAV particles measured as GC/ml than the reference method. In
some
embodiments, the method described herein produces at least about 20% more rAAV
particles
measured as GC/nil than the reference method. In some embodiments, the method
described
herein produces at least about 30% more rAAV particles measured as GC/ml than
the reference
method. In some embodiments, the method described herein produces at least
about 40% more
rAAV particles measured as GC/ml than the reference method. In some
embodiments, the method
described herein produces at least about 50% more rAAV particles measured as
GC/ml than the
reference method. In some embodiments, the method described herein produces at
least about
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70% more rAAV particles measured as GC/m1 than the reference method. In some
embodiments,
the method described herein produces at least about 90% more rAAV particles
measured as
GC/ml than the reference method. In some embodiments, the method described
herein produces
at least about twice as many rAAV particles measured as GC/ml than the
reference method. In
some embodiments, the method produces at least about three times as many rAAV
particles
measured as GC/nil than the reference method. In some embodiments, the method
produces at
least about four times as many rAAV particles measured as GC/ml than the
reference method.
[00131] In some embodiments, the method produces a population of rAAV
particles comprising
more full capsids than a reference method. In some embodiments, the reference
method uses a
polynucleotide comprising helper functions that comprises the nucleotide
sequence of SEQ ID
NO: 35. In some embodiments, the reference method uses a polynucleotide
comprising helper
functions that comprises the nucleotide sequence of SEQ ID NO: 44.
[00132] In some embodiments, the rAAV particles comprise a capsid protein of
the AAV1,
AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12,
AAV13, AAV14, AAV15, AAV16, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74,
AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B, AAV2.5,
AAV2tYF, AAV3B, AAV.LK03, AAVMYO, MyoAAV.1A, MyoAAV1C, AAV.HSC1,
AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8,
AAV.HSC9, AAV.HSC10 , AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14,
AAV.HSC15, or AAV.HSC16 serotype. In some embodiments, the rAAV particles
comprise a
capsid protein of the AAV8, AAV9, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74,
AAV.RHM4-1, or AAV.hu37 serotype. In some embodiments, the rAAV particles
comprise a
capsid protein of the AAV8 scrotype. In some embodiments, the rAAV particles
comprise a
capsid protein of the AAV9 serotype.
1001331In some embodiments, the rAAV particle comprises a transgene encoding a
gene product.
In some embodiments, the gene product is a polypeptide or a double stranded
RNA molecule. In
some embodiments, the gene product is a polypeptide. In some embodiments, the
transgene
encodes an antibody or antigen-binding fragment thereof, fusion protein, Fc-
fusion polypeptide,
immunoadhesin, immunoglobulin, engineered protein, protein fragment or enzyme.
In some
embodiments, the transgene comprises a regulatory element operatively
connected to a
polynucleotide encoding the gene product.
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[00134] In some embodiments, the gene product is anti-VEGF Fab, anti-
kallikrein antibody, anti-
TNF antibody, microdystrophin, minidystrophin, iduronidase (IDUA), iduronate 2-
sulfatase
(IDS), low-density lipoprotein receptor (LDLR), tripeptidyl peptidase 1
(TPP1), or non-
membrane associated splice variant of VEGF receptor 1 (sFlt-1). In some
embodiments, the gene
product is an gamma-sarcoglycan, Rab Escort Protein 1 (REP1/CHM), retinoid
isomerohydrolase
(RPE65), cyclic nucleotide gated channel alpha 3 (CNGA3), cyclic nucleotide
gated channel beta
3 (CNGB3), aromatic L-amino acid decarboxylase (AADC), lysosome-associated
membrane
protein 2 isoform B (LAMP2B), Factor VIII, Factor IX, retinitis pigmentosa
GTPase regulator
(RPGR), retinoschisin (RS1), sarcoplasmic reticulum calcium ATPase (SERCA2a),
aflibercept,
battenin (CLN3), transmembrane ER protein (CLN6), glutamic acid decarboxylase
(GAD), Glial
cell line-derived neurotrophic factor (GDNF), aquaporin 1 (AQP1), dystrophin,
myotubularin 1
(MTM1), follistatin (FST), glucose-6-phosphatase (G6Pase), apolipoprotein A2
(AP0A2),
uridine diphosphate glucuronosyl transferase 1A1 (UGT1A1), arylsulfatase B
(ARSB), N-acetyl-
alpha-glucosaminidase (NAGLU), alpha-glucosidase (GAA), alpha-galactosidase
(GLA), beta-
galactosidase (GLB1), lipoprotein lipase (LPL), alpha 1-antitrypsin (AAT),
phosphodiesterase 6B
(PDE6B), ornithine carbamoyltransferase 90TC), survival motor neuron (SMN1),
survival motor
neuron (SMN2), neurturin (NRTN), Neurotrophin-3 (NT-3/NTF3), porphobilinogen
deaminase
(PBGD), nerve growth factor (NGF), mitochondri ally encoded NADH:ubiquinone
oxidoreductase core subunit 4 (MT-ND4), protective protein cathepsin A (PPCA),
dysferlin,
MER proto-oncogene, tyrosine Idnase (MERTK), cystic fibrosis transmembrane
conductance
regulator (CFTR), or tumor necrosis factor receptor (TNFR)-immunoglobulin
(IgG1) Fc fusion.
In some embodiments, the gene product is a dystrophin or a microdystrophin. In
some
embodiments, the gene product is a microRNA.
[00135] In some embodiments, a method described herein increases production of
rAAV particles
while maintaining or improving the quality attributes of the rAAV particles
and compositions
comprising thereof. In some embodiments, the quality of rAAV particles and
compositions
comprising thereof is assessed by determining the concentration of rAAV
particles (e.g., GC/ml),
the percentage of particles comprising a copy of the rAAV genome; the ratio of
particles without
a genome, infectivity of the rAAV particles, stability of rAAV particles,
concentration of residual
host cell proteins, or concentration of residual host cell nucleic acids
(e.g., host cell genomic
DNA, plasmid encoding rep and cap genes, plasmid encoding helper functions,
plasmid encoding
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rAAV genome). In some embodiments, the quality of rAAV particles produced by a
method
described herein or compositions comprising thereof is the same as that of
rAAV particles or
compositions produced by a reference method using a helper plasmid comprising
the nucleotide
sequence of SEQ ID NO: 35 or 44. In some embodiments, the quality of rAAV
particles produced
by a method described herein or compositions comprising thereof is better than
the quality of
rAAV particles or compositions produced by a reference method using a helper
plasmid
comprising the nucleotide sequence of SEQ ID NO: 35 or 44.
[00136] Numerous cell culture based systems are known in the art for
production of rAAV
particles, any of which can be used to practice a method described herein.
rAAV production
cultures for the production of rAAV virus particles require; (1) suitable host
cells, including, for
example, human-derived cell lines such as HeLa, A549, or HEK293 cells and
their derivatives
(HEK293T cells, HEK293F cells), or mammalian cell lines such as Vero,
amniocyte-derived cells
such as CAP cells, CHO cells or CHO-derived cells; (2) suitable helper virus
function, provided
by wild type or mutant adenovirus (such as temperature sensitive adenovirus),
herpes virus,
baculovirus, or a plasmid construct providing helper functions; (3) AAV rep
and cap genes and
gene products; (4) a transgene (such as a therapeutic transgene) flanked by
AAV ITR sequences;
and (5) suitable media and media components to support rAAV production.
[00137] A skilled artisan is aware of the numerous methods by which AAV rep
and cap genes,
AAV helper genes (e.g., adenovirus El a gene, Elb gene, E4 gene, E2a gene, and
VA gene), and
rAAV genomes (comprising one or more genes of interest flanked by inverted
terminal repeats
(ITRs)) can be introduced into cells to produce or package rAAV. The phrase
"adenovirus helper
functions" refers to a number of viral helper genes expressed in a cell (as
RNA or protein) such
that the AAV grows efficiently in the cell. The skilled artisan understands
that helper viruses,
including adenovirus and herpes simplex virus (HSV), promote AAV replication
and certain
genes have been identified that provide the essential functions, e.g. the
helper may induce
changes to the cellular environment that facilitate such AAV gene expression
and replication. In
some embodiments of a method described herein, AAV rep and cap genes, helper
genes, and
rAAV genomes are introduced into cells by transfection of one or more plasmid
vectors encoding
the AAV rep and cap genes, helper genes, and rAAV genome.
[00138]Molecular biology techniques to develop plasmid or viral vectors
encoding the AAV rep
and cap genes, helper genes, and/or rAAV genome are commonly known in the art.
In some
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embodiments, AAV rep and cap genes are encoded by one plasmid vector. In some
embodiments,
AAV helper genes (e.g., adenovirus Ela gene, Elb gene, E4 gene, E2a gene, and
VA gene) are
encoded by one plasmid vector. In some embodiments, the Ela gene or Elb gene
is stably
expressed by the host cell, and the remaining AAV helper genes are introduced
into the cell by
transfection by one viral vector. In some embodiments, the Ela gene and Elb
gene are stably
expressed by the host cell, and the E4 gene, E2a gene, and VA gene arc
introduced into the cell
by transfection by one plasmid vector. In some embodiments, one or more helper
genes are stably
expressed by the host cell, and one or more helper genes are introduced into
the cell by
transfection by one plasmid vector. In some embodiments, the helper genes are
stably expressed
by the host cell. In some embodiments, AAV rep and cap genes are encoded by
one viral vector.
In some embodiments, AAV helper genes (e.g., adenovirus Ela gene, El b gene,
E4 gene, E2a
gene, and VA gene) are encoded by one viral vector. In some embodiments, the
Ela gene or Elb
gene is stably expressed by the host cell, and the remaining AAV helper genes
are introduced into
the cell by transfection by one viral vector. In some embodiments. the Ela
gene and Elb gene are
stably expressed by the host cell, and the E4 gene, E2a gene, and VA gene are
introduced into the
cell by transfection by one viral vector. In some embodiments, one or more
helper genes are
stably expressed by the host cell, and one or more helper genes are introduced
into the cell by
transfection by one viral vector. In some embodiments, the AAV rep and cap
genes, the
adenovirus helper functions necessary for packaging, and the rAAV genome to be
packaged are
introduced to the cells by transfection with one or more polynucleotides,
e.g., vectors. In some
embodiments, a method described herein comprises transfecting the cells with a
mixture of three
polynucleotides: one encoding the cap and rep genes, one encoding adenovirus
helper functions
necessary for packaging (e.g., adenovirus Ela gene, Elb gene, E4 gene, E2a
gene, and VA gene),
and one encoding the rAAV genome to be packaged. In some embodiments, the AAV
cap gene is
an AAV8 or AAV9 cap gene. In some embodiments, the AAV cap gene is an AAV.rh8,
AAV sh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.PHB, or
AAV.7m8 cap gene. In some embodiments, the AAV cap gene encodes a capsid
protein with
high sequence homology to AAV8 or AAV9 such as, AAV.rh10, AAV.rh20, AAV.rh39,
AAV.Rh74, AAV.RHM4-1, and AAV.hu37. In some embodiments, the vector encoding
the
rAAV genome to be packaged comprises a gene of interest flanked by AAV ITRs.
In some
embodiments, the AAV ITRs are from AAV1, AAV2, rAAV3, AAV4, AAV5, AAV6. AAV7,
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AAV8, AAV9, AAVIO, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16. AAV.rh8,
AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.Anc80,
AAV.Anc80L65. AAV.7m8, AAV.PHP.B, AAV2.5, AAV2tYF, AAV3B, AAV.LK03,
AAVMYO, MyoAAV.1A, MyoAAV1C, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4,
AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10 , AAV.HSC11,
AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, or AAV.HSC16 or other AAV
serotype.
[00139] Any combination of vectors can be used to introduce AAV rep and cap
genes, AAV
helper genes, and rAAV genome to a cell in which rAAV particles are to be
produced or
packaged. In some embodiments of a method described herein, a first plasmid
vector encoding an
rAAV genome comprising a gene of interest flanked by AAV inverted terminal
repeats (ITRs), a
second vector encoding AAV rep and cap genes, and a third vector encoding
helper genes can be
used. In some embodiments, a mixture of the three vectors is co-transfected
into a cell.
[00140] In some embodiments, a combination of transfection and infection is
used by using both
plasmid vectors as well as viral vectors.
[00141] In some embodiments, one or more of rep and cap genes, and AAV helper
genes are
constitutively expressed by the cells and does not need to be transfected or
transduced into the
cells. In some embodiments, the cell constitutively expresses rep and/or cap
genes. In some
embodiments, the cell constitutively expresses one or more AAV helper genes.
In some
embodiments, the cell constitutively expresses El a. In some embodiments, the
cell comprises a
stable transgene encoding the rAAV genome.
[00142] In some embodiments, AAV rep, cap, and helper genes (e.g., Ela gene,
Elb gene, E4
gene, E2a gene, or VA gene) can be of any AAV scrotypc. Similarly, AAV ITRs
can also be of
any AAV serotype. For example, in some embodiments, AAV ITRs are from AAV1,
AAV2,
rAAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13,
AAV14, AAV15, AAV16, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74,
AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B, AAV2.5,
AAV2tYF, AAV3B, AAV.LK03, AAVMYO, MyoAAV.1A, MyoAAV1C, AAV.HSC1,
AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8,
AAV.HSC9, AAV.HSC10 , AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14,
AAV.HSC15, or AAV.HSC16 or other AAV serotypes (e.g., a hybrid serotype
harboring
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sequences from more than one serotype). In some embodiments, AAV cap gene is
from AAV9 or
AAV8 cap gene. In some embodiments, an AAV cap gene is from AAV1, AAV2, rAAV3,
AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14,
AAV15, AAV16, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1,
AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B, AAV2.5, AAV2tYF,
AAV3B, AAV.LK03, AAVMYO, MyoAAV.1A, MyoAAV1C, AAV.HSC1, AAV.HSC2,
AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9,
AAV.HSC10 , AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, or
AAV.HSC16 or other AAV serotypes (e.g., a hybrid serotype harboring sequences
from more
than one serotype). In some embodiments, AAV rep and cap genes for the
production of a rAAV
particle is from different serotypes. For example, the rep gene is from AAV2
whereas the cap
gene is from AAV9.
[00143] Any suitable media known in the art can be used for the production of
recombinant virus
particles (e.g., rAAV particles) according to a method described herein. These
media include,
without limitation, media produced by Hyclone Laboratories and JRH including
Modified Eagle
Medium (MEM), Dulbecco's Modified Eagle Medium (DMEM), and Sf-900 II SFM media
as
described in U.S. Pat. No. 6,723,551, which is incorporated herein by
reference in its entirety. In
some embodiments, the medium comprises DynamiSTM Medium, FreeStyleTM 293
Expression
Medium, or Expi293Tm Expression Medium from Invitrogen/ ThermoFisher. In some
embodiments, the medium comprises DynamisTM Medium. In some embodiments, a
method
described herein uses a cell culture comprising a serum-free medium, an animal-
component free
medium, or a chemically defined medium. In some embodiments, the medium is an
animal-
component free medium. In some embodiments, the medium comprises scrum. In
some
embodiments, the medium comprises fetal bovine serum. In some embodiments, the
medium is a
glutamine-free medium. In some embodiments, the medium comprises glutamine. In
some
embodiments, the medium is supplemented with one or more of nutrients, salts,
buffering agents,
and additives (e.g., antifoam agent). In some embodiments, the medium is
supplemented with
glutamine. In some embodiments, the medium is supplemented with serum. In some
embodiments, the medium is supplemented with fetal bovine serum. In some
embodiments, the
medium is supplemented with poloxamer, e.g., Kolliphor P 188 Bio. In some
embodiments, a
medium is a base medium. In some embodiments, the medium is a feed medium.
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[00144] Recombinant virus (e.g., rAAV) production cultures can routinely be
grown under a
variety of conditions (over a wide temperature range, for varying lengths of
time, and the like)
suitable to the particular host cell being utilized. As is known in the art,
virus production cultures
include suspension-adapted host cells such as HeLa cells, HEK293 cells, HEK293
derived cells
(e.g., HEK293T cells, HEK293F cells), Vero cells, CAP cells, CHO cells, CHO-Kl
cells, CHO
derived cells, EB66 cells, BSC cells, HcpG2 cells, LLC-MK cells, CV-1 cells,
COS cells, MDBK
cells, MDCK cells, CRFK cells, RAF cells, RK cells, TCMK-1 cells, LLCPK cells,
PK15 cells,
LLC-RK cells, MDOK cells, BHK cells, BHK-21 cells, NS-1 cells, MRC-5 cells, W1-
38 cells,
BHK cells, 3T3 cells, 293 cells, RK cells, Per.C6 cells, chicken embryo cells
and SF-9 cells
which can be cultured in a variety of ways including, for example, spinner
flasks, stirred tank
bioreactors, and disposable systems such as the Wave bag system. Numerous
suspension cultures
are known in the art for production of rAAV particles, including for example,
the cultures
disclosed in U.S. Patent Nos. 6,995,006, 9,783,826, and in U.S. Pat. Appl.
Pub. Nos.
20070111312 and 20120122155, each of which is incorporated herein by reference
in its entirety.
[00145] Any cell or cell line that is known in the art to produce a
recombinant virus particles (e.g.,
rAAV particles) can be used in any one of the methods described herein. In
some embodiments, a
method of producing recombinant virus particles (e.g., rAAV particles) or
increasing the
production of recombinant virus particles (e.g., a rAAV particles) described
herein uses HeLa
cells, HEK293 cells, HEK293 derived cells (e.g., HEK293T cells, HEK293F
cells), Vero cells,
CAP cells, CHO cells, CHO-K1 cells. CHO derived cells, EB66 cells. LLC-MK
cells, MDCK
cells, RAF cells, RK cells, TCMK-1 cells, PK15 cells, BHK cells, BHK-21 cells,
NS-1 cells,
BHK cells, 293 cells, RK cells, Per.C6 cells, chicken embryo cells or SF-9
cells. In some
embodiments, a method described herein uses mammalian cells. In some
embodiments, a method
described herein uses insect cells, e.g., SF-9 cells. In some embodiments, a
method described
herein uses cells adapted for growth in suspension culture. In some
embodiments, a method
described herein uses HEK293 cells adapted for growth in suspension culture.
[00146] In some embodiments, a cell culture described herein is a suspension
culture. In some
embodiments, a large scale suspension cell culture described herein comprises
HEK293 cells
adapted for growth in suspension culture. In some embodiments, a cell culture
described herein
comprises a serum-free medium, an animal-component free medium, or a
chemically defined
medium. In sonic embodiments, a cell culture described herein comprises a
serum-free medium.
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In some embodiments, suspension-adapted cells are cultured in a shaker flask,
a spinner flask, a
cell bag, or a bioreactor.
[00147] In some embodiments, a cell culture described herein comprises a serum-
free medium, an
animal-component free medium, or a chemically defined medium. In some
embodiments, a cell
culture described herein comprises a serum-free medium.
[00148] In somc embodiments, a large scale suspension culture cell culture
described herein
comprises a high density cell culture. In some embodiments, the culture has a
total cell density of
between about 1x10E+06 cells/int and about 30x10E+06 cells/ml. In some
embodiments, more
than about 50% of the cells are viable cells. In some embodiments, the cells
are HeLa cells,
HEK293 cells, HEK293 derived cells (e.g., HEK293T cells, HEK293F cells), Vero
cells, CAP
cells, or SF-9 cells. In further embodiments, the cells are HEK293 cells.
[00149] Methods described herein can be used in the production of rAAV
particles comprising a
capsid protein from any AAV capsid serotype. In some embodiments, the rAAV
particles
comprise a capsid protein from an AAV capsid serotype selected from AAV1,
AAV2, rAAV3,
AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14,
AAV15, AAV16, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1,
AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B, AAV2.5, AAV2tYF,
AAV3B, AAV.LK03, AAVMYO, MyoAAV.1 A, MyoAAV1C, AAV.HSC1, AAV.HSC2,
AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9,
AAV.HSC10 , AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, and
AAV.HSC16. In some embodiments, the rAAV particles comprise a capsid protein
that is a
derivative, modification, or pseudotype of AAV1, AAV2, rAAV3, AAV4, AAV5,
AAV6,
AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV.rh8,
AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.Anc80,
AAV.Anc80L65, AAV.7m8, AAV.PHP.B, AAV2.5, AAV2tYF, AAV3B, AAV.LK03,
AAVMYO, MyoAAV.1A, MyoAAV1C, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4,
AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10 , AAV.HSC11,
AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, or AAV.HSC16 capsid protein.
[00150] In some embodiments, the rAAV particles comprise a capsid protein from
an AAV
capsid serotype selected from AAV8 and AAV9. In some embodiments, the rAAV
particles have
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an AAV capsid serotype of AAV8. In some embodiments, the rAAV particles have
an AAV
capsid serotype of AAV9.
[00151] In some embodiments, the rAAV particles comprise a capsid protein from
an AAV
capsid serotype selected from the group consisting of AAV.rh8, AAV.rh10,
AAV.rh20,
AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.PHB, and AAV.7m8. In some
embodiments, the rAAV particles comprise a capsid protein with high sequence
homology to
AAV8 or AAV9 such as, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, and
AAV.hu37.
[00152] In some embodiments, the rAAV particles comprise a capsid protein that
is a derivative,
modification, or pseudotype of AAV8 or AAV9 capsid protein. In some
embodiments, the rAAV
particles comprise a capsid protein that has an AAV8 capsid protein at least
80% or more
identical, e.g., 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%,
99%, 99.5%, etc., i.e. up to 100% identical, to the VP1, VP2 and/or VP3
sequence of AAV8
capsid protein.
[00153] In some embodiments, the rAAV particles comprise a capsid protein that
is a derivative,
modification, or pseudotype of AAV9 capsid protein. In some embodiments, rAAV
particles
comprise a capsid protein that has an AAV9 capsid protein at least 80% or more
identical, e.g.,
85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,
99.5%,
etc., i.e. up to 100% identical, to the VP1, VP2 and/or VP3 sequence of AAV9
capsid protein.
[00154] In some embodiments, the rAAV particles comprise a capsid protein that
has at least 80%
or more identity, e.g., 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%,
98%, 99%, 99.5%, etc., i.e. up to 100% identity, to the VP1, VP2 and/or VP3
sequence of
AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37,
AAV.PHB, or AAV.7m8 capsid protein. In some embodiments, the rAAV particles
comprise a
capsid protein that has at least 80% or more identity, e.g., 85%, 85%, 87%,
88%, 89%, 90%,
91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, etc., i.e. up to 100%
identity, to the
VP1, VP2 and/or VP3 sequence of an AAV capsid protein with high sequence
homology to
AAV8 or AAV9 such as, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, and
AAV.hu37.
[00155] In additional embodiments, the rAAV particles comprise a mosaic
capsid. In additional
embodiments, the rAAV particles comprise a pseudotyped rAAV particle. In
additional
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embodiments, the rAAV particles comprise a capsid containing a capsid protein
chimera of two
or more AAV capsid serotypes.
rAAV PARTICLES
[00156]The provided methods are suitable for use in the production of any
isolated recombinant
AAV particles. As such, the rAAV can be of any serotype, modification, or
derivative, known in
the art, or any combination thereof (e.g., a population of rAAV particles that
comprises two or
more serotypes, e.g., comprising two or more of rAAV2, rAAV8, and rAAV9
particles) known in
the art. In some embodiments, the rAAV particles are AAV1, AAV2, rAAV3, AAV4,
AAV5,
AAV6, AAV7,AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16,
AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37,
AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B, AAV2.5, AAV2tYF, AAV3B,
AAV.LK03, AAVMYO, MyoAAV.1A, MyoAAV1C, AAV.HSC1, AAV.HSC2, AAV.HSC3,
AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10 ,
AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, or AAV.HSC16 or other
rAAV particles, or combinations of two or more thereof.
[00157]In some embodiments, rAAV particles have a capsid protein from an AAV
serotype
selected from AAV1, AAV1, AAV2, rAAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9,
AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV.rh8, AAV.rh10, AAV.rh20,
AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8,
AAV.PHP.B, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAVMYO, MyoAAV.1A,
MyoAAV1C, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6,
AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10 , AAV.HSC11, AAV.HSC12,
AAV.HSC13, AAV.HSC14, AAV.HSC15, or AAV.HSC16 or a derivative, modification,
or
pseudotype thereof. In some embodiments, rAAV particles comprise a capsid
protein at least 80%
or more identical, e.g., 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,
95%, 96%,
97%, 98%, 99%, 99.5%, etc., i.e. up to 100% identical, to e.g., VP1, VP2
and/or VP3 sequence of
an AAV capsid scrotype selected from AAV1, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6,
AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV.rh8,
AAV.rhl 0, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.Anc80,
rAAV.Anc80L65, AAV.7m8, AAV.PHRB, AAV2.5, AAV2tYF, AAV3B, AAV.LK03,
AAVMYO, MyoAAV.1A, MyoAAV1C, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4,
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AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSCIO , AAV.HSCII,
AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, or AAV.HSC16.
[00158] In some embodiments, rAAV particles comprise a capsid protein from an
AAV capsid
serotype selected from AAV1, AAV1, AAV2, rAAV3, AAV4, AAV5, AAV6, AAV7, AAV8,
AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV.rh8, AAV.rh10,
AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAV.Anc80L65,
AAV.7m8, AAV.PHP.B, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAVMYO, MyoAAV.1A,
MyoAAV1C, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6,
AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10 AAV.HSC11, AAV.HSC12,
AAV.HSC13, AAV.HSC14, AAV.HSC15, or AAV.HSC16, or a derivative, modification,
or
pseudotype thereof. In some embodiments, rAAV particles comprise a capsid
protein at least
80% or more identical, e.g., 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,
95%, 96%,
97%, 98%, 99%, 99.5%, etc., i.e. up to 100% identical, to e.g., VP1, VP2
and/or VP3 sequence
of an AAV capsid serotype selected from AAV1, AAV1, AAV2, AAV3, AAV4, AAV5,
AAV6,
AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV.rh8,
AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.Anc80,
AAV.Anc80L65. AAV.7m8, AAV.PHP.B, AAV2.5, AAV2tYF, AAV3B, AAV.LK03,
AAVMYO, MyoAAV.1A, MyoAAV1C, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4,
AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10 , AAV.HSC11,
AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, or AAV.HSC16.
[00159] In some embodiments, rAAV particles comprise the capsid
of Anc80 or
Anc80L65, as described in Zinn et al., 2015, Cell Rep. 12(6): 1056-1068, which
is incorporated
by reference in its entirety. In certain embodiments, the rAAV particles
comprise the capsid with
one of the following amino acid insertions: LGETTRP or LALGETTRP, as described
in United
States Patent Nos. 9,193,956; 9458517; and 9,587,282 and US patent application
publication no.
2016/0376323, each of which is incorporated herein by reference in its
entirety. In some
embodiments, rAAV particles comprise the capsid of AAV.7m8, as described in
United States
Patent Nos. 9,193,956; 9,458,517; and 9,587,282 and US patent application
publication no.
2016/0376323, each of which is incorporated herein by reference in its
entirety. In some
embodiments, rAAV particles comprise any AAV capsid disclosed in United States
Patent No.
9,585,971, such as AAVPHP.B. In some embodiments, rAAV particles comprise any
AAV
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capsid disclosed in United States Patent No. 9,840,719 and WO 2015/013313,
such as
AAV.Rh74 and RHM4-1, each of which is incorporated herein by reference in its
entirety. In
some embodiments, rAAV particles comprise any AAV capsid disclosed in WO
2014/172669,
such as AAV rh.74, which is incorporated herein by reference in its entirety.
In some
embodiments, rAAV particles comprise the capsid of AAV2/5, as described in
Georgiadis et al.,
2016, Gene Therapy 23: 857-862 and Gcorgiadis et al., 2018, Gene Therapy 25:
450, each of
which is incorporated by reference in its entirety. In some embodiments, rAAV
particles
comprise any AAV capsid disclosed in WO 2017/070491, such as AAV2tY1-', which
is
incorporated herein by reference in its entirety. In some embodiments, rAAV
particles comprise
the capsids of AAVLKO3 or AAV3B, as described in Puzzo et al., 2017, Sci.
Transl. Med. 29(9):
418, which is incorporated by reference in its entirety. In some embodiments,
r AAV particles
comprise any AAV capsid disclosed in US Pat Nos. 8,628,966; US 8,927,514; US
9,923,120 and
WO 2016/049230, such as HSC1, HSC2, HSC3, HSC4, HSC5, HSC6, HSC7, HSC8, HSC9,
HSC10 , HSC11, HSC12, HSC13, HSC14, HSC15, or HSC16, each of which is
incorporated by
reference in its entirety. In other embodiments, rAAV particles comprise
capsids having enhanced
tropism to muscle tissue, such capsids being engineered by inserting a RGD-
containing peptide
into the parental capsid of interest. Such exemplary capsids are AAVMYO (AAV9-
RGDLGLS,
MyoAAV.1A (AAV9-RGDLTTP), and MyoAAV1C (AAV9-RGDLSTP) (peptide inserted after
residue Q588 of AAV9). In some embodiments, rAAV particles comprise any AAV
capsid
disclosed in PCT International Publication Nos. W02019/207132, W02020/206189,
W02021/072197, W02021/050974, W02021/077000, and WO 2022/020616.
[00160] In some embodiments, rAAV particles comprise an AAV capsid disclosed
in any of the
following patents and patent applications, each of which is incorporated
herein by reference in its
entirety: United States Patent Nos. 7,282,199; 7,906,111; 8,524,446;
8,999,678; 8,628,966;
8,927,514; 8,734,809; US 9,284,357; 9,409,953; 9,169,299; 9,193,956; 9458517;
and 9,587,282;
US patent application publication nos. 2015/0374803; 2015/0126588;
2017/0067908;
2013/0224836; 2016/0215024; 2017/0051257; and International Patent Application
Nos.
PCT/U52015/034799; PCT/EP2015/053335. In some embodiments, rAAV particles have
a
capsid protein at least 80% or more identical, e.g., 85%, 85%, 87%, 88%, 89%,
90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, etc., i.e. up to 100% identical, to
the VP1, VP2
and/or VP3 sequence of an AAV capsid disclosed in any of the following patents
and patent
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applications, each of which is incorporated herein by reference in its
entirety: United States
Patent Nos. 7,282,199; 7,906,111; 8,524,446; 8,999,678; 8,628,966; 8,927,514;
8,734,809; US
9,284,357; 9,409,953; 9,169,299; 9,193,956; 9458517; and 9,587,282; US patent
application
publication nos. 2015/0374803; 2015/0126588; 2017/0067908; 2013/0224836;
2016/0215024;
2017/0051257; and International Patent Application Nos. PCT/US2015/034799;
PCT/EP2015/053335.
[00161] In some embodiments, rAAV particles have a capsid protein disclosed in
Intl. Appl. Publ.
No. WO 2003/052051 (see, e.g., SEQ Ill NO: 2), WO 2005/033321 (see, e.g., SEQ
ID NOs: 123
and 88), WO 03/042397 (see, e.g., SEQ ID NOs: 2, 81, 85, and 97), WO
2006/068888 (see, e.g.,
SEQ ID NOs: 1 and 3-6), WO 2006/110689, (see, e.g., SEQ ID NOs: 5-38)
W02009/104964
(see, e.g., SEQ ID NOs: 1-5, 7, 9, 20, 22, 24 and 31), WO 2010/127097 (see,
e.g., SEQ ID NOs:
5-38), and WO 2015/191508 (see, e.g., SEQ ID NOs: 80-294), and U.S. Appl.
Publ. No.
20150023924 (see, e.g., SEQ ID NOs: 1, 5-10), the contents of each of which is
herein
incorporated by reference in its entirety. In some embodiments, rAAV particles
have a capsid
protein at least 80% or more identical, e.g., 85%, 85%, 87%, 88%, 89%, 90%,
91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, 99%, 99.5%, etc., i.e. up to 100% identical, to the
VP1, VP2 and/or
VP3 sequence of an AAV capsid disclosed in Intl. Appl. Publ. No. WO
2003/052051 (see, e.g.,
SEQ ID NO: 2), WO 2005/033321 (see, e.g., SEQ ID NOs: 123 and 88), WO
03/042397 (see,
e.g., SEQ ID NOs: 2, 81, 85, and 97), WO 2006/068888 (see, e.g., SEQ ID NOs: 1
and 3-6), WO
2006/110689 (see, e.g., SEQ ID NOs: 5-38) W02009/104964 (see, e.g., SEQ ID
NOs: 1-5, 7, 9,
20, 22, 24 and 31), WO 2010/127097 (see, e.g., SEQ ID NOs: 5-38), and WO
2015/191508 (see,
e.g., SEQ ID NOs: 80-294), and U.S. Appl. Publ. No. 20150023924 (see, e.g.,
SEQ ID NOs: 1, 5-
10).
[00162] Nucleic acid sequences of AAV based viral vectors and methods of
making recombinant
AAV and AAV capsids are taught, for example, in United States Patent Nos.
7,282,199;
7,906,111; 8,524,446; 8,999,678; 8,628,966; 8,927,514; 8,734,809; US
9,284,357; 9,409,953;
9,169,299; 9,193,956; 9458517; and 9,587,282; US patent application
publication nos.
2015/0374803; 2015/0126588; 2017/0067908; 2013/0224836; 2016/0215024;
2017/0051257;
International Patent Application Nos. PCT/US2015/034799; PCT/EP2015/053335; WO
2003/052051, WO 2005/033321, WO 03/042397, WO 2006/068888, WO 2006/110689,
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W02009/104964, WO 2010/127097, and WO 2015/191508, and U.S. Appl. Publ. No.
20150023924.
[00163] The provided methods are suitable for use in the production of
recombinant AAV
encoding a transgene. In certain embodiments, the transgene is from Tables 1A-
1C. In some
embodiments, the rAAV genome comprises a vector comprising the following
components: (1)
AAV inverted terminal repeats that flank an expression cassette; (2)
regulatory control elements,
such as a) promoter/enhancers, b) a polyA signal, and c) optionally an intron;
and (3) nucleic acid
sequences coding for a transgene. In other embodiments for expressing an
intact or substantially
intact monoclonal antibody (mAb), the rAAV genome comprises a vector
comprising the
following components: (1) AAV inverted terminal repeats that flank an
expression cassette; (2)
regulatory control elements, such as a) promoter/enhancers, b) a polyA signal,
and c) optionally
an intron; and (3) nucleic acid sequences coding for the light chain Fab and
heavy chain Fab of
the antibody, or at least the heavy chain or light chain Fab, and optionally a
heavy chain Fe
region. In still other embodiments for expressing an intact or substantially
intact mAb, the rAAV
genome comprises a vector comprising the following components: (1) AAV
inverted terminal
repeats that flank an expression cassette; (2) regulatory control elements,
such as a)
promoter/enhancers, b) a polyA signal, and c) optionally an intron; and (3)
nucleic acid sequences
coding for the heavy chain Fab of an anti-VEGF (e.g., sevacizumab,
ranibizumab, bevacizumab,
and brolucizumab), anti-EpoR (e.g., LKA-651, ), anti-ALK1 (e.g.,
ascrinvacumab), anti-05 (e.g.,
tesidolumab and eculizumab), anti-CD105 (e.g., carotuximab), anti-CC1Q (e.g.,
ANX-007), anti-
TNFa (e.g., adalimumab, infliximab, and golimumab), anti-RGMa (e.g.,
ciczanumab), anti-TTR
(e.g., NI-301 and PRX-004), anti-CTGF (e.g., panu-evlumab), anti-IL6R (e.g.,
satralizumab and
sarilumab), anti-IL4R (e.g., dupilumab), anti-IL17A (e.g., ixckizumab and
sccukinumab), anti-
IL-5 (e.g., mepolizumab), anti-IL12/IL23 (e.g., ustekinumab), anti-CD19 (e.g.,
inebilizumab),
anti-ITGF7 mAb (e.g., etrolizumab), anti-SOST mAb (e.g., romosozumab), anti-
pKal mAb (e.g.,
lanadelumab), anti-1TGA4 (e.g., natalizumab), anti-1TGA4B7 (e.g.,
vedolizumab), anti-BLyS
(e.g., belimumab), anti-PD-1 (e.g., nivolumab and pembrolizumab), anti-RANKL
(e.g.,
densomab), anti-PCSK9 (e.g., alirocumab and evolocumab), anti-ANGPTL3 (e.g.,
evinacumab*),
anti-OxPL (e.g., E06), anti-fD (e.g., lampalizumab), or anti-MMP9 (e.g.,
andecaliximab);
optionally an Fc polypeptide of the same isotype as the native form of the
therapeutic antibody,
such as an IgG isotype amino acid sequence IgGl, IgG2 or IgG4 or modified Fe
thereof; and the
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light chain of an anti-VEGF (e.g., sevacizumab, ranibizumab, bevacizumab, and
brolucizumab),
anti-EpoR (e.g., LKA-651, ), anti-ALK1 (e.g., ascrinvacumab), anti-05 (e.g.,
tesidolumab and
eculizumab), anti-CD105 or anti-ENG (e.g., carotuximab). anti-CC1Q (e.g., ANX-
007), anti-
TNFa (e.g., adalimumab, infliximab, and golimumab), anti-RGMa (e.g.,
elezanumab), anti-TTR
(e.g., N1-301 and PRX-004). anti-CTGF (e.g., pamrevlumab), anti-IL6R (e.g.,
satralizumab and
sarilumab), anti-IL4R (e.g., dupilumab), anti-IL17A (e.g., ixckizumab and
sccukinumab), anti-
IL-5 (e.g., mepolizumab), anti-IL12/IL23 (e.g., ustekinumab), anti-CD19 (e.g.,
inebilizumab),
anti-1TGF7 inAb (e.g., etrolizumab), anti-SOST inAb (e.g., romosozumab), anti-
pKal inAb (e.g.,
lanadelumab), anti-1TGA4 (e.g., natalizumab), anti-1TGA4B7 (e.g.,
vedolizumab), anti-BLyS
(e.g., belimumab), anti-PD-1 (e.g., nivolumab and pembrolizumab), anti-RANKL
(e.g.,
densomab), anti-PCSK9 (e.g., alirocumab and evolocumab), anti-ANGPTL3 (e.g.,
evinacumab),
anti-OxPL (e.g., E06), anti-fD (e.g., lampalizumab), or anti-MMP9 (e.g.,
andecaliximab);
wherein the heavy chain (Fab and optionally Fe region) and the light chain are
separated by a
self-cleaving furin (F)/F2A or flexible linker, ensuring expression of equal
amounts of the heavy
and the light chain polypeptides.
[00164] In other embodiments for expressing an mRNA, such as an antisense RNA
in the context
of a guide RNA (antisense strand) and/or a passenger RNA (sense strand) as in
miRNA and
shRNA structures, the rA AV genome comprises a vector comprising the following
components:
(1) AAV inverted terminal repeats that flank an expression cassette; (2)
regulatory control
elements, such as a) promoter/enhancers, b) a polyA signal, and c) optionally
an intron; and (3)
nucleic acid sequences coding for the mRNA. In some embodiments, the transgene
(nucleic acid
sequences coding for the mRNA) comprises or consists of microRNA, shRNA, or U7-
snRNA
encoding sequences.
Table 1A
Disease Transgene
MPS I alpha-L-iduronidase (IDUA)
MPS II (Hunter Syndrome) iduronate-2-sulfatase (IDS)
ceroid lipofuscinosis (Batten disease) (CLN1, CLN2, CLN10, CLN13), a
soluble
lysosomal protein (CLN5), a protein in the
secretory pathway (CLN11), two cytoplasmic
proteins that also peripherally associate with
membranes (CLN4, CLN14), and many
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Disease Transgene
transmembrane proteins with different
subcellular locations (CLN3, CLN6, CLN7,
CLN8, CLN12)
MPS Ma (Sanfilippo type A Syndrome) heparan sulfate sulfatase (also
called N-
sulfoglucosamine sulfohydrolase (SGSH))
MPS HIS (Sanfilippo type B Syndrome) N-acetyl-alpha-D-glucosaminidase
(NAGLU)
MPS VI (Maroteaux-Lamy Syndrome) arylsulfatase B
Gaucher disease (type 1, II and III) Glucocerebrosidase, GBA1
Parkinson's Disease Glucocerebrosidase; GB A 1
Parkinson's Disease dopamine decarboxylase
Pompe acid maltase; GAA
Metachromatic leukodystrophy Aryl sulfatase A
MPS VII (Sly syndrome) beta-glucuronidase
MPS VIII glucosamine-6-sulfate sulfatase
MPS IX Hyaluronidase
Niemann-Pick disease Sphingomyelinase
Niemann-Pick disease without a npcl gene encoding a
sphingomyelinase deficiency cholesterol metabolizing enzyme
Tay-Sachs disease Alpha subunit of beta-
hexosaminidase
Sandhoff disease both alpha and beta subunit of
beta-
hexosaminidase
Fabry Disease alpha-galactosidase
Fucosidosis Fucosidase (FUCA1 gene)
Alpha-mannosidosis alpha-mannosidase
Beta-mannosidosis Beta-mannosidase
Wolman disease cholesterol ester hydrolase
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Disease Transgene
Parkinson's disease Neurturin
Parkinson's disease glial derived growth factor
(GDGF)
Parkinson's disease tyrosine hydroxylase
Parkinson's disease glutamic acid decarboxylase.
Parkinson's disease fibroblast growth factor-2 (FGF-
2)
Parkinson's disease brain derived growth factor
(BDGF)
No disease listed (Galactosialidosis neuraminidase deficiency with
betagalactosidase
(Goldberg syndrome)) deficiency
Spinal Muscular Atrophy (SMA) SMN
Friedreich's ataxia Frataxin
Amyotrophic lateral sclerosis (ALS) SOD1
Glycogen Storage Disease la Glucose-6-phosphatase
XLMTM MTM1
Crigler Najjar UGTIA1
CPVT CASQ2
Rett syndrome MECP2
Achromatopsia CNGB3, CNGA3, GNAT2, PDE6C
Choroidermia CDM
Danon Disease LAMP2
Cystic Fibrosis CFTR
Duchenne Muscular Dystrophy Mini-Dystrophin or
Microdystrophin Gene
Limb Girdle Muscular Dystrophy Type human-alpha-sarcoglycan
2C1Gamma-sarcoglycanopathy
Advanced Heart Failure SERCA2a
Rheumatoid Arthritis TNFR:Fc Fusion Gene
Leber Congenital Amaurosis GAA
Limb Girdle Muscular Dystrophy Type gamma-sarcoglycan
2C1Gamma-sarcoglycanopathy
Retinitis Pigmentosa hMERTK
Age-Related Macular Degeneration sFLT01
Becker Muscular Dystrophy and Sporadic huFollistatin344
Inclusion Body Myositis
Parkinson's Disease GDNF
Metachromatic Leukodystrophy (MLD) cuARSA
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Disease Transgene
Hepatitis C anti-HCV shRNA
Limb Girdle Muscular Dystrophy Type 2D hSGCA
Human Immunodeficiency Virus PG9DP
Infections; HIV Infections (HIV-1)
Acute Intermittant Porphyria PBGD
Leber's Hereditary Optical Neuropathy P1ND4v2
Alpha-1 Antitrypsin Deficiency alphalAT
Pompe Disease hGAA
X-linked Retinoschisis RS1
Choroideremia hCHM
Giant Axonal Neuropathy JeT-GAN
X-linked Retinoschisis hRS1
Squamous Cell Head and Neck Cancer; hAQP1
Radiation Induced Xerostomia
Hemophilia B Factor IX
Homozygous FH hLDLR
Dysferlinopathies dysferlin transgene (e.g.
rAAVrh74.MHCK7.DYSF.DV)
Hemophilia B AAV6 ZFP nuclease
MPS I AAV6 ZFP nuclease
Rheumatoid Arthritis NF-kB.IFN-f3
Batten / CLN6 CLN6
Sanfilippo Disease Type A hSGSH
Osteoarthritis 5IL-1Ra
Achromatopsia CNGA3
Achromatopsia CNGB3
Ornithine Transcarbamylase (OTC) OTC
Deficiency
Hemophilia A Factor VIII
Mucopolysaccharidosis II ZFP nuclease
Hemophilia A ZFP nuclease
Wet AMD anti-VEGF
X-Linked Retinitis Pigmentosa RPGR
Mucopolysaccharidosis Type VI hARSB
Leber Hereditary Optic Neuropathy ND4
X-Linked Myotubular Myopathy MTM1
Crigler-Najjar Syndrome UGT1A1
Achromatopsia CNGB3
Retinitis Pigmentosa hPDE6B
X-Linked Retinitis Pigmentosa RPGR
Mucopolysaccharidosis Type 3 B hNAGLU
Duchenne Muscular Dystrophy GALGT2
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Disease Transgene
Arthritis, Rheumatoid; Arthritis, TNFR:Fc Fusion Gene
Psoriatic; Ankylosing Spondylitis
Idiopathic Parkinson's Disease Neurturin
Alzheimer's Disease NGF
Human Immunodeficiency Virus tgAAC09
Infections; HIV Infections (HIV-1)
Familial Lipoprotein Lipase Deficiency LPL
Idiopathic Parkinson's Disease Neurturin
Alpha-1 Antitrypsin Deficiency hAAT
Leber Congenital Amaurosis (LCA) 2 hRPE65v2
Batten Disease; Late Infantile Neuronal CLN2
Lipofuscinosis
Parkinson's Disease GAD
Sanfilippo Disease Type Al N-sulfoglucosamine sulfohydrolase
(SGSH) gene
Mucopolysaccharidosis Type IIIA
Congestive Heart Failure SERC2a
Becker Muscular Dystrophy and Sporadic Follistatin (e.g.
rAAV.CMV.huFollistatin344)
Inclusion Body Myositis
Parkinson's Disease hAADC-2
Choroideremia REP1
CEA Specific A AV-DC-CTL Treatment in CEA
Stage IV Gastric Cancer
Gastric Cancer MUCl-peptide-DC-CTL
Leber's Hereditary Optical Neuropathy scAAV2-PiND4v2
Aromatic Amino Acid Decarboxylase hAADC
Deficiency
Hemophilia B Factor IX
Parkinson's Disease AADC
Leber Hereditary Optic Neuropathy Genetic: GS0101Drug: Placebo
SMA - Spinal Muscular AtrophylGene SMN
Therapy
Hemophilia A B-Domain Deleted Factor VIII
MPS I IDUA
MPS II IDS
CLN3-Related Neuronal Ceroid- CLN3
Lipotuscinosis (Batten)
Limb-Girdle Muscular Dystrophy, Type hSGCB
2E
Alzheimer Disease APOE2
Retinitis Pigmentosa hMERKTK
Retinitis Pigmentosa RLBP I
Wet AMD or diabetic retinopathy Anti-VEGF antibody or Anti-VEGF
trap (e.g.
one or more extracellular domains of VEGFR-1
and/or VEGFR-2; e.g. afliberccpt)
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Table 1B
AN ANTIBODIES INDICATIONS
(TRANSGENE)
Amylaid beta Solanezumab
Alzheimer' s Disease
(A,8 or Abeta)
GSK933776
peptides derived
from APP
Nervous System
Sortilin AL-001 Frontotemporal
dementia
Targets
(FTD)
Tau protein ABBV-8E12 Alzheimer's,
Progressive
supranuclear palsy.
UCB-0107
frontotemporal demential,
NI-105 (BIIB076) chronic
traumatic
encephalopathy, Pick's
complex, primary age-
related taupathy
Semaphorin-4D VX15/2503
Huntington's disease,
(SEMA4D)
juvenile Huntington' s
disease
alpha-synucicin Prasinczumab
Parkinson's disease,
NI-202 (BIIB054)
synucleinopathies
MED-1341
superoxide NI-204 ALS, Alzheimer's
Disease
dismutase-1
(SOD-I)
CGRP Receptor eptinezumab, Migraines,
Cluster
headaches
fremanezumab
galcanezumab
Sevacizumab diabetic
retinopathy (DR),
myopic choroidal
Ocular Anti- VEGF
neovascularization
Angiogenic
(mCNV), age-related
Targets
macular degeneration
(AMD), macular edema
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VEGF ranibizumab Wet AMD
(LUCENTIS )
bevacizumab
(AVASTIN )
brolucizumab
erythropoietin LKA-651 retinal vein
occlusion
receptor (RVO), wet AMD,
macular
edema
A myloid beta Sol anezumab Dry AMD
(A,6 or Abeta)
GSK933776
peptides derived
from APP
activin receptor ascrinvacumab neovascular age-
related
like kin use 1 macular degeneration
(ALK1)
complement tesidolumab dry AMD, uveitis
component 5
(C5)
endoglin (END carotuximab wet AMD and other
retinal
or CD105) disorders caused by
increased vascularization
complement ANX-007 glaucoma
component 1Q
(C1Q)
adalimumab uveitis
(HUMIRA )
TNP-alpha
infliximab
(REMICADE )
golimumab
Repulsive guidance molecule-A elezanumab multiple sclerosis
Transthyretin ('TTR) NI-301 amyl oidosi
s
PRX-004
73
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Connective tissue growth factor pamrevlumab fibrotic
diseases, e.g.
(CTGF) diabetic
nephropathy, liver
fibrosis, idiopathic
pulmonary fibrosis
Neuromyelitis interleukin Satralizumab NMO, DR,
DME, uveitis
optica receptor 6
(NMO)/Uveitis (IL6R) sarilumab
targets
CD19 incbilizumab NMO
Integrin beta 7 etrolizumab ulcerative
colitis, Crohn's
disease
Sclerostin romosozumab
Osteoporosis, abnormal
(EVENITY ) bone loss or weakness
Table 1C
ANTIGENS ANTIBODIES INDICATIONS
(TRANSGENE)
Amyloid beta (A,8 Aducanumab Alzheimer' s Disease
or Abeta) peptides
crenezumab
gantenerumab
Nervous System
Targets
Tau protein anti-TAU Alzheimer's,
Progressive
supranuclear palsy,
frontotemporal demential,
chronic traumatic
encephalopathy, Pick's
complex, primary age-
related taupathy
CGRP Receptor erenumab Migraine
(AIMOVIGTm)
ixekizumab Plaque psoriasis, psoriatic
(TALTZ ) arthritis, ankylosing
IL-17A sponylitis
secukinumab
(COSENTYX )
74
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IL-5 mepolizumab Asthma
(NUCALA )
Interleukins or
interleukin IL-12/IL-23 ustekinumab Psoriasis &
Crohn's disease
receptors (STELARA )
IL-4R dupilumab Atopic
dermatitis
vcdolizumab Ulcerative
colitis &
(ENTYVIO ) Crohn's
disease
Integrin
Natalizumah (anti- Multiple
sclerosis &
integrin alpha 4) Crohn's
disease
PCSK9 alirocumab HeFH & HoFH
(PRALUENT )
Cardiovascular
evolueomab
Targets
(REPATHA )
ANGPTL3 evinacumab HoFH & severe
forms of
dyslipidema
Proinflammatory/ E06-scFv Cardiovascular diseases
proatherogenic such as
atherosclerosis
phospholipids
denosumab Osteoporosis,
increasing
RANKL (XGEVA and bone mass in
breast and
PROLIA ) prostate cancer
patients, &
preventing skeletal-related
events due to bone
metastasis
PD-I, or PD-Li or PD-L2 nivolumab Metastatic melanoma,
(OPDIVO ) lymphoma, non-
small cell
lung carcinoma
pembrolizumab
(KEYTRUDA )
BLyS (B-lymphocyte stimulator, also belimumab Systemic lupus
known as B-cell activating factor (BENLYSTA ) erythromatosis
(BAFF))
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lampalizumab Dry AMD
Ocular Targets Factor D
MMP9 andecaliximab Dry AMD
adalimumab Rheumatoid
arthritis,
(HUMIRA ) and psoriatic
arthritis,
TNF-alpha
askylosing spondylitis,
infliximab
(REMICADE ) Crohn's disease,
plaque
psoriasis, ulcerative colitis
eculi zumab Paroxysmal
nocturnal
(SOLIRIS ) hemoglobinuria,
atypical
hemolytic uremic
Plasma Protein CS, CSa syndrome, complement-
targets mediated
thrombotic
microangiopathy
Plasma kallikrein lanadelumab Hereditary
angioedema
(HAE)
[00165] In some embodiments, the rAAV particles are rAAV viral vectors
encoding an anti-
VEGF Fab. In specific embodiments, the rAAV particles are rAAV8-based viral
vectors
encoding an anti-VEGF Fab. hi more specific embodiments, the rAAV particles
are rAAV8-
based viral vectors encoding ranibizumab. In some embodiments, the rAAV
particles are rAAV
viral vectors encoding iduronidase (IDUA). In specific embodiments, the rAAV
particles are
rAAV9-based viral vectors encoding IDUA. In some embodiments, the rAAV
particles are rAAV
viral vectors encoding iduronate 2-sulfatase (IDS). In specific embodiments,
the rAAV particles
are rAAV9-based viral vectors encoding IDS. In some embodiments, the rAAV
particles are
rAAV viral vectors encoding a low-density lipoprotein receptor (LDLR). In
specific
embodiments, the rAAV particles arc rAAV8-based viral vectors cncoding LDLR.
In somc
embodiments, the rAAV particles are rAAV viral vectors encoding tripeptidyl
peptidase 1 (TPP1)
protein. In specific embodiments, the rAAV particles are rAAV9-based viral
vectors encoding
TPPl. In some embodiments, the rAAV particles are rAAV viral vectors encoding
non-
membrane associated splice variant of VEGF receptor 1 (sFlt-1). In some
embodiments, the
rAAV particles are rAAV viral vectors encoding gamma-sarcoglycan, Rab Escort
Protein 1
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(REP1/CHM), retinoid isomerohydrolase (RPE65), cyclic nucleotide gated channel
alpha 3
(CNGA3), cyclic nucleotide gated channel beta 3 (CNGB3), aromatic L-amino acid
decarboxylase (AADC), lysosome-associated membrane protein 2 isoform B
(LAMP2B), Factor
VIII, Factor IX, retinitis pigmentosa GTPase regulator (RPGR), retinoschisin
(RS1), sarcoplasmic reticulum calcium ATPase (SERCA2a), aflibercept, battenin
(CLN3),
transmembrane ER protein (CLN6), glutamic acid decarboxylase (GAD), Glial cell
line-derived
neurotrophic factor (GDNF), aquaporin 1 (AQP1), dystrophin, microdystrophin,
myotubularin 1
(MTM1), follistatin (FST), glucose-6-phosphatase (G6Pase), apolipoprotein A2
(AP0A2),
uridine diphosphate glucuronosyl transferase 1A1 (UGT1A1), arylsulfatase B
(ARSB), N-acetyl-
alpha-glucosaminidase (NAGLU), alpha-glucosidase (GAA), alpha-galactosidase
(GLA), beta-
gal actosidase (GLB1), lipoprotein lipase (LPL), alpha 1-antitrypsin (A AT),
phosphodiesterase 6B
(PDE6B), ornithine carbamoyltransferase 90TC), survival motor neuron (SMN1),
survival motor
neuron (SMN2), neurturin (NRTN), Neurotrophin-3 (NT-3/NTF3), porphobilinogen
deaminase
(PBGD), nerve growth factor (NGF), mitochondrially encoded NADH:ubiquinone
oxidoreductase core subunit 4 (MT-ND4), protective protein cathepsin A (PPCA),
dysferlin,
MER proto-oncogene, tyrosine Idnase (MERTK), cystic fibrosis transmembrane
conductance
regulator (CFTR), or tumor necrosis factor receptor (TNFR)-immunoglobulin
(IgG1) Fe fusion.
[00166] In additional embodiments, rAAV particles comprise a pseudotyped AAV
capsid. In
some embodiments, the pseudotyped AAV capsids are rAAV2/8 or rAAV2/9
pseudotyped AAV
capsids. Methods for producing and using pseudotyped rAAV particles are known
in the art (see,
e.g., Duan et al., J. Virol., 75:7662-7671 (2001); Halbert et al., J. Virol.,
74:1524-1532 (2000);
Zolotukhin et al.. Methods 28:158-167 (2002); and Auricchio et al., Hum.
Molec. Genet.
10:3075-3081, (2001).
[00167] In additional embodiments, rAAV particles comprise a capsid containing
a capsid protein
chimeric of two or more AAV capsid serotypes. In some embodiments, the capsid
protein is a
chimeric of 2 or more AAV capsid proteins from AAV serotypes selected from
AAV1, AAV1,
AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12,
AAV13, AAV14, AAV15 and AAV16, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39,
AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8,
AAV.PHP.B, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAVMYO, MyoAAV.1A,
MyoAAV1C, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6,
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AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSCIO , AAV.HSC11, AAV.HSC12,
AAV.HSC13, AAV.HSC14, AAV.HSC15, or AAV.HSC16.
[00168] In certain embodiments, a single-stranded AAV (ssAAV) can be used. In
certain
embodiments, a self-complementary vector, e.g., scAAV, can be used (see, e.g.,
Wu, 2007,
Human Gene Therapy, 18(2):171-82, McCarty et al, 2001, Gene Therapy, Vol. 8,
Number 16,
Pages 1248-1254; and U.S. Patent Nos. 6,596,535; 7,125,717; and 7,456,683,
each of which is
incorporated herein by reference in its entirety).
[00169] In some embodiments, the rAAV particles comprise a capsid protein from
an AAV
capsid serotype selected from AAV8 or AAV9. In some embodiments, the rAAV
particles have
an AAV capsid serotype of AAV8. In some embodiments, the rAAV particles have
an AAV
capsid serotype of AAV9.
[00170] In some embodiments, the rAAV particles comprise a capsid protein that
is a derivative,
modification, or pseudotype of AAV8 or AAV9 capsid protein. In some
embodiments, the rAAV
particles comprise a capsid protein that has an AAV8 capsid protein at least
80% or more
identical, e.g., 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%,
99%, 99.5%, etc., i.e. up to 100% identical, to the VP1, VP2 and/or VP3
sequence of AAV8
capsid protein.
[00171] In some embodiments, the rAAV particles comprise a capsid protein that
is a derivative,
modification, or pseudotype of AAV9 capsid protein. In some embodiments, the
rAAV particles
comprise a capsid protein that has an AAV9 capsid protein at least 80% or more
identical, e.g.,
85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,
99.5%,
etc., i.e. up to 100% identical, to the VP1. VP2 and/or VP3 sequence of AAV9
capsid protein.
[00172] In additional embodiments, the rAAV particles comprise a mosaic
capsid. Mosaic AAV
particles are composed of a mixture of viral capsid proteins from different
serotypes of AAV. In
some embodiments, the rAAV particles comprise a mosaic capsid containing
capsid proteins of a
serotype selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9,
AAV10, AAV11, AAV12, AAV13, AAV14, AAV15 and AAV16, AAV.rh8, AAV.rh10,
AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAV.Anc80L65,
AAV.7m8, AAV.PHP.B, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAVMYO, MyoAAV.1A,
MyoAAV1C, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6,
AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10 , AAV.HSC11, AAV.HSC12,
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AAV.HSC13, AAV.HSC14, AAV.HSC15, and AAV.HSC16. In some embodiments, the rAAV
particles comprise a mosaic capsid containing capsid proteins of a serotype
selected from AAV1,
AAV2, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAVrh.8, AAVrh.10, AAVhu.37,
AAVrh.20, and AAVrh.74.
[00173] In additional embodiments, the rAAV particles comprise a pseudotyped
rAAV particle.
In some embodiments, the pseudotyped rAAV particle comprises (a) a nucleic
acid vector
comprising AAV ITRs and (b) a capsid comprised of capsid proteins derived from
AAVx (e.g.,
AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12,
AAV13, AAV14, AAV15 and AAV16, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39,
AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8,
AAV.PHP.B, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAVMYO, MyoAAV.1A,
MyoAAV1C, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6,
AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10 , AAV.HSC11, AAV.HSC12,
AAV.HSC13, AAV.HSC14, AAV.HSC15, and AAV.HSC16). In additional embodiments,
the
rAAV particles comprise a pseudotyped rAAV particle comprised of a capsid
protein of an AAV
serotype selected from AAV1, AAV2, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10,
AAVrh.8,
and AAVrh.10. AAVhu.37, AAVrh.20, and AAVrh.74. In additional embodiments, the
rAAV
particles comprise a pseudotyped rAAV particle containing AAV8 capsid protein.
In additional
embodiments, the rAAV particles comprise a pseudotyped rAAV particle is
comprised of AAV9
capsid protein. In some embodiments, the pseudotyped rAAV8 or rAAV9 particles
are rAAV2/8
or rAAV2/9 pseudotyped particles. Methods for producing and using pseudotyped
rAAV
particles are known in the art (see, e.g., Duan et al., J. Virol., 75:7662-
7671 (2001); Halbert et al.,
J. Virol., 74:1524-1532 (2000); Zolotukhin et al., Methods 28:158-167 (2002);
and Auricchio et
al., Hum. Molec. Genet. 10:3075-3081, (2001).
[00174] In additional embodiments, the rAAV particles comprise a capsid
containing a capsid
protein chimeric of two or more AAV capsid serotypes. In some embodiments, the
rAAV
particles comprise an AAV capsid protein chimeric of AAV8 capsid protein and
one or more
AAV capsid proteins from an AAV serotype selected from AAV1, AAV2, AAV3, AAV4,
AAV5, AAV6, AAV7, AAV8, AAV9, AAVIO, AAV11, AAV12, AAV13, AAV14, AAV15 and
AAV16, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37,
AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B, AAV2.5, AAV2tYF, AAV3B,
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AAV.LK03, AAVMYO, MyoAAV.1A, MyoAAV1C, AAV.HSC1, AAV.HSC2, AAV.HSC3,
AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10 ,
AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, and AAV.HSC16. In
some embodiments, the rAAV particles comprise an AAV capsid protein chimeric
of AAV8
capsid protein and one or more AAV capsid proteins from an AAV serotype
selected from
AAV1, AAV2, AAV5, AAV6, AAV7, AAV9, AAV10, rAAVrh10, AAVrh.8, AAVrh.10,
AAVhu.37, AAVrh.20, and AAVrh.74. In some embodiments, the rAAV particles
comprise an
AAV capsid protein chimeric of AAV9 capsid protein the capsid protein of one
or more AAV
capsid serotypes selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8,
AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15 and AAV16, AAV.rh8, AAV.rh10,
AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAV.Anc80L65,
AAV.7m8, AAV.PHP.B, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAVMYO, MyoAAV.1A,
MyoAAV1C, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6,
AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10 , AAV.HSC11, AAV.HSC12,
AAV.HSC13, AAV.HSC14, AAV.HSC15, and AAV.HSC16. In some embodiments, the rAAV
particles comprise an AAV capsid protein chimeric of AAV9 capsid protein the
capsid protein of
one or more AAV capsid serotypes selected from AAV1, AAV2, AAV3, AAV4, AAV5,
AA6,
AAV7, AAV8, AAV9, AAVrh.8, AAVrh.10, AAVhu.37, AAVrh.20, and AAVrh.74.
METHODS FOR ISOLATING rAAV PARTICLES
[00175] In some embodiments, the disclosure provides methods for producing
recombinant
adeno-associated virus (rAAV) particles, comprising isolating rAAV particles
from a feed
comprising an impurity (for example, rAAV production culture). In some
embodiments, a method
for producing recombinant adeno-associated virus (rAAV) particles described
herein comprises
(a) isolating rAAV particles from a feed comprising an impurity (for example,
rAAV production
culture), and (b) formulating the isolated rAAV particles to produce the
formulation.
[00176] In some embodiments, the disclosure further provides methods for
producing a
pharmaceutical unit dosage of a formulation comprising isolated recombinant
adeno-associated
virus (rAAV) particles, comprising isolating rAAV particles from a feed
comprising an impurity
(for example, rAAV production culture), and formulating the isolated rAAV
particles.
[00177] Isolated rAAV particles can be isolated using methods known in the
art. In some
embodiments, methods of isolating rAAV particles comprises downstream
processing such as, for
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example, harvest of a cell culture, clarification of the harvested cell
culture (e.g., by
centrifugation or depth filtration), tangential flow filtration, affinity
chromatography, anion
exchange chromatography, cation exchange chromatography, size exclusion
chromatography,
hydrophobic interaction chromatography, hydroxylapatite chromatography,
sterile filtration, or
any combination(s) thereof. In some embodiments, downstream processing
includes at least 2, at
least 3, at least 4, at least 5 or at least 6 of: harvest of a cell culture,
clarification of the harvested
cell culture (e.g., by centrifugation or depth filtration), tangential flow
filtration, affinity
chromatography, anion exchange chromatography, cation exchange chromatography,
size
exclusion chromatography, hydrophobic interaction chromatography,
hydroxylapatite
chromatography, and sterile filtration. In some embodiments, downstream
processing comprises
harvest of a cell culture, clarification of the harvested cell culture (e.g.,
by depth filtration), sterile
filtration, tangential flow filtration, affinity chromatography, and anion
exchange
chromatography. In some embodiments, downstream processing comprises
clarification of a
harvested cell culture, sterile filtration, tangential flow filtration,
affinity chromatography, and
anion exchange chromatography. In some embodiments, downstream processing
comprises
clarification of a harvested cell culture by depth filtration, sterile
filtration, tangential flow
filtration, affinity chromatography, and anion exchange chromatography. In
some embodiments,
clarification of the harvested cell culture comprises sterile filtration. In
some embodiments,
downstream processing does not include centrifugation. In some embodiments,
the rAAV
particles comprise a capsid protein of the AAV8 serotype. In some embodiments,
the rAAV
particles comprise a capsid protein of the AAV9 scrotypc.
[00178] In some embodiments, a method of isolating rAAV particles produced
according to a
method described herein comprises harvest of a cell culture, clarification of
the harvested cell
culture (e.g., by depth filtration), a first sterile filtration, a first
tangential flow filtration, affinity
chromatography, anion exchange chromatography (e.g., monolith anion exchange
chromatography or AEX chromatography using a quaternary amine ligand), a
second tangential
flow filtration, and a second sterile filtration. In some embodiments, a
method of isolating rAAV
particles described herein comprises harvest of a cell culture, clarification
of the harvested cell
culture (e.g., by depth filtration), a first sterile filtration, affinity
chromatography, anion exchange
chromatography (e.g., monolith anion exchange chromatography or AEX
chromatography using
a quaternary amine ligand), a tangential flow filtration, and a second sterile
filtration. In some
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embodiments, a method of isolating rAAV particles produced according to a
method described
herein comprises clarification of a harvested cell culture, a first sterile
filtration, a first tangential
flow filtration, affinity chromatography, anion exchange chromatography (e.g.,
monolith anion
exchange chromatography or AEX chromatography using a quaternary amine
ligand), a second
tangential flow filtration, and a second sterile filtration. In some
embodiments, a method of
isolating rAAV particles described herein comprises clarification of a
harvested cell culture, a
first sterile filtration, affinity chromatography, anion exchange
chromatography (e.g., monolith
anion exchange chromatography or AEX chromatography using a quaternary amine
ligand),
tangential flow filtration, and a second sterile filtration. In some
embodiments, a method of
isolating rAAV particles produced according to a method described herein
comprises clarification
of a harvested cell culture by depth filtration, a first sterile filtration, a
first tangential flow
filtration, affinity chromatography, anion exchange chromatography (e.g.,
monolith anion
exchange chromatography or AEX chromatography using a quaternary amine
ligand), a second
tangential flow filtration, and a second sterile filtration. In some
embodiments, a method of
isolating rAAV particles described herein comprises clarification of a
harvested cell culture by
depth filtration, a first sterile filtration, affinity chromatography, anion
exchange chromatography
(e.g., monolith anion exchange chromatography or AEX chromatography using a
quaternary
amine ligand), tangential flow filtration, and a second sterile filtration. In
some embodiments, the
method does not include centrifugation. In some embodiments, clarification of
the harvested cell
culture comprises sterile filtration. In some embodiments, the rAAV particles
comprise a capsid
protein of the AAV8 scrotype. In some embodiments, the rAAV particles comprise
a capsid
protein of the AAV9 serotype.
[00179] Numerous methods are known in the art for production of rAAV
particles, including
transfection, stable cell line production, and infectious hybrid virus
production systems which
include adenovirus-AAV hybrids, herpesvirus-AAV hybrids and baculovirus-AAV
hybrids.
rAAV production cultures for the production of rAAV virus particles all
require; (1) suitable host
cells, including, for example, human-derived cell lines such as HeLa, A549, or
HEK293 cells and
their derivatives (HEK293T cells, HEK293F cells), mammalian cell lines such as
Vero, and
amniocyte-derived cells such as CAP cells, or insect-derived cell lines such
as SF-9 in the case of
baculovirus production systems; (2) suitable helper virus function, provided
by wild type or
mutant adenovirus (such as temperature sensitive adenovirus), herpes virus,
baculovirus, or a
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plasmid construct providing helper functions; (3) AAV rep and cap genes and
gene products; (4)
a transgene (such as a therapeutic transgene) flanked by AAV ITR sequences;
and (5) suitable
media and media components to support rAAV production. In some embodiments,
the suitable
helper virus function is provided by a recombinant polynucleotide described
herein or a plasmid
described herein. Suitable media known in the art may be used for the
production of rAAV
vectors. These media include, without limitation, media produced by Hyclonc
Laboratories and
JRH including Modified Eagle Medium (MEM), Dulbecco's Modified Eagle Medium
(DMEM),
and Sf-900 11 SFM media as described in U.S. Pat. No. 6,723,551, which is
incorporated herein
by reference in its entirety.
[00180] rAAV production cultures can routinely be grown under a variety of
conditions (over a
wide temperature range, for varying lengths of time, and the like) suitable to
the particular host
cell being utilized. As is known in the art, rAAV production cultures include
attachment-
dependent cultures which can be cultured in suitable attachment-dependent
vessels such as, for
example, roller bottles, hollow fiber filters, microcarriers, and packed-bed
or fluidized-bed
bioreactors. rAAV vector production cultures may also include suspension-
adapted host cells
such as HeLa cells, HEK293 cells, HEK293 derived cells (e.g., HEK293T cells,
HEK293F cells),
Vero cells, CAP cells, CHO cells, CHO-Kl cells, CHO derived cells, EB66 cells,
BSC cells,
HepG2 cells, LLC-MK cells, CV-1 cells, COS cells, MDBK cells, MDCK cells, CRFK
cells,
RAF cells, RK cells, TCMK-1 cells, LLCPK cells, PK15 cells, LLC-RK cells, MDOK
cells,
BHK cells, BHK-21 cells, NS-1 cells, MRC-5 cells, WI-38 cells, BHK cells, 3T3
cells, 293 cells,
RK cells, Per.C6 cells, chicken embryo cells or SF-9 cells which can be
cultured in a variety of
ways including, for example, spinner flasks, stirred tank bioreactors, and
disposable systems such
as the Wave bag system. In some embodiments, the cells are HEK293 cells. In
some
embodiments, the cells are HEK293 cells adapted for growth in suspension
culture. Numerous
suspension cultures are known in the art for production of rAAV particles,
including for example,
the cultures disclosed in U.S. Patent Nos. 6,995,006, 9,783,826, and in U.S.
Pat. Appl. Pub. No.
20120122155, each of which is incorporated herein by reference in its
entirety.
[00181] In some embodiments, the rAAV production culture comprises a high
density cell
culture. In some embodiments, the culture has a total cell density of between
about 1x10E+06
cells/ml and about 30x10E+06 cells/ml. In some embodiments, more than about
50% of the cells
are viable cells. In some embodiments, the cells are HeLa cells, HEK293 cells,
HEK293 derived
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cells (e.g., HEK293T cells, HEK293F cells), Vero cells, CAP cells, or SF-9
cells. In further
embodiments, the cells are HEK293 cells. In further embodiments, the cells are
HEK293 cells
adapted for growth in suspension culture.
[00182] In additional embodiments of the provided method the rAAV production
culture
comprises a suspension culture comprising rAAV particles. Numerous suspension
cultures are
known in the art tor production of rAAV particles, including for example, the
cultures disclosed
in U.S. Patent Nos. 6,995,006, 9,783,826, and in U.S. Pat. Appl. Pub. No.
20120122155, each of
which is incorporated herein by reference in its entirety. In some
embodiments, the suspension
culture comprises a culture of mammalian cells or insect cells. In some
embodiments, the
suspension culture comprises a culture of HeLa cells, HEK293 cells, HEK293
derived cells (e.g.,
HEK293T cells, HEK293F cells), Vero cells, CAP cells, CHO cells, CHO-Kl cells,
CHO derived
cells, EB66 cells, BSC cells, HepG2 cells, LLC-MK cells, CV-1 cells, COS
cells, MDBK cells,
MDCK cells, CRFK cells, RAF cells, RK cells, TCMK-1 cells, LLCPK cells, PK15
cells, LLC-
RK cells, MDOK cells, BHK cells, BHK-21 cells, NS-1 cells, MRC-5 cells, WI-38
cells, BHK
cells, 313 cells, 293 cells, RK cells, Per.C6 cells, chicken embryo cells or
SF-9 cells. In some
embodiments, the suspension culture comprises a culture of HEK293 cells.
[00183] In some embodiments, methods for the production of rAAV particles
encompasses
providing a cell culture comprising a cell capable of producing rAAV; adding
to the cell culture a
histone deacetylase (HDAC) inhibitor to a final concentration between about
0.1 m1V1 and about
20 mNI; and maintaining the cell culture under conditions that allows
production of the rAAV
particles. In some embodiments, the HDAC inhibitor comprises a short-chain
fatty acid or salt
thereof. In some embodiments, the HDAC inhibitor comprises butyrate (e.g.,
sodium butyrate),
valproatc (e.g., sodium valproatc), propionate (e.g., sodium propionate), or a
combination thereof.
[00184] In some embodiments, rAAV particles are produced as disclosed in WO
2020/033842,
which is incorporated herein by reference in its entirety.
[00185] Recombinant AAV particles can be harvested from rAAV production
cultures by harvest
of the production culture comprising host cells or by harvest of the spent
media from the
production culture, provided the cells are cultured under conditions known in
the art to cause
release of rAAV particles into the media from intact host cells. Recombinant
AAV particles can
also be harvested from rAAV production cultures by lysis of the host cells of
the production
culture. Suitable methods of lysing cells are also known in the art and
include for example
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multiple freeze/thaw cycles, sonication, microfluidization, and treatment with
chemicals, such as
detergents and/or proteases.
[00186] At harvest, rAAV production cultures can contain one or more of the
following: (1) host
cell proteins; (2) host cell DNA; (3) plasmid DNA; (4) helper virus; (5)
helper virus proteins; (6)
helper virus DNA; and (7) media components including, for example, serum
proteins, amino
acids, transfcrrins and other low molecular weight proteins. rAAV production
cultures can further
contain product-related impurities, for example, inactive vector forms, empty
viral capsids,
aggregated viral particles or capsids, mis-folded viral capsids, degraded
viral particle.
[00187] In some embodiments, the rAAV production culture harvest is clarified
to remove host
cell debris. In some embodiments, the production culture harvest is clarified
by filtration through
a series of depth filters. Clarification can also be achieved by a variety of
other standard
techniques known in the art, such as, centrifugation or filtration through any
cellulose acetate
filter of 0.2 mm or greater pore size known in the art. In some embodiments,
clarification of the
harvested cell culture comprises sterile filtration. In some embodiments, the
production culture
harvest is clarified by centrifugation. In some embodiments, clarification of
the production
culture harvest does not included centrifugation.
[00188] In some embodiments, harvested cell culture is clarified using
filtration. In some
embodiments, clarification of the harvested cell culture comprises depth
filtration. In some
embodiments, clarification of the harvested cell culture further comprises
depth filtration and
sterile filtration. In some embodiments, harvested cell culture is clarified
using a filter train
comprising one or more different filtration media. In some embodiments, the
filter train
comprises a depth filtration media. In some embodiments, the filter train
comprises one or more
depth filtration media. In some embodiments, the filter train comprises two
dcpth filtration media.
In some embodiments, the filter train comprises a sterile filtration media. In
some embodiments,
the filter train comprises 2 depth filtration media and a sterile filtration
media. In some
embodiments, the depth filter media is a porous depth filter. In some
embodiments, the filter train
comprises Claris lve 20MS, Millistak+0 COHC, and a sterilizing grade filter
media. In some
embodiments, the filter train comprises Claris lye 20MS, Millistak+C) COHC,
and SartoporeCD
2 XLG 0.2 nm. In some embodiments, the harvested cell culture is pretreated
before contacting it
with the depth filter. In some embodiments, the pretreating comprises adding a
salt to the
harvested cell culture. In sonic embodiments, the pretreating comprises adding
a chemical
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flocculent to the harvested cell culture. In some embodiments, the harvested
cell culture is not
pre-treated before contacting it with the depth filter.
[00189] In some embodiments, the production culture harvest is clarified by
filtration are
disclosed in WO 2019/212921, which is incorporated herein by reference in its
entirety.
[00190] In some embodiments, the rAAV production culture harvest is treated
with a nuclease
(e.g., Benzonase0) or endonuclease (c.g., endonuclease from Scrratia
marcesccns) to digest high
molecular weight DNA present in the production culture. The nuclease or
endonuclease digestion
can routinely be performed under standard conditions known in the art. For
example, nuclease
digestion is performed at a final concentration of 1-2.5 units/nil of
Benzonase0 at a temperature
ranging from ambient to 37 C for a period of 30 minutes to several hours.
[00191] Sterile filtration encompasses filtration using a sterilizing grade
filter media. In some
embodiments, the sterilizing grade filter media is a 0.2 or 0.22 tm pore
filter. In some
embodiments, the sterilizing grade filter media comprises polyethersulfone
(PES). In some
embodiments, the sterilizing grade filter media comprises polyvinylidene
fluoride (PVDF). In
some embodiments, the sterilizing grade filter media has a hydrophilic
heterogeneous double
layer design. In some embodiments, the sterilizing grade filter media has a
hydrophilic
heterogeneous double layer design of a 0.8 pm pre-filter and 0.2 pm final
filter membrane. In
some embodiments, the sterilizing grade filter media has a hydrophilic
heterogeneous double
layer design of a 1.2 pm pre-filter and 0.2 pm final filter membrane. In some
embodiments, the
sterilizing grade filter media is a 0.2 or 0.22 pm pore filter. In further
embodiments, the
sterilizing grade filter media is a 0.2 pm pore filter. In some embodiments,
the sterilizing grade
filter media is a Sartopore0 2 XLG 0.2 pm, DuraporeTM PVDF Membranes 0.45pm,
or
Sartoguard0 PES 1.2 pm + 0.2 lam nominal pore size combination. In some
embodiments, the
sterilizing grade filter media is a Sartoporee 2 XLG 0.2 pm.
[00192] In some embodiments, the clarified feed is concentrated via tangential
flow filtration
("TFF") before being applied to a chromatographic medium, for example,
affinity
chromatography medium. Large scale concentration of viruses using TFF
ultrafiltration has been
described by Paul et al., Human Gene Therapy 4:609-615 (1993). TFF
concentration of the
clarified feed enables a technically manageable volume of clarified feed to be
subjected to
chromatography and allows for more reasonable sizing of columns without the
need for lengthy
recirculation times. In some embodiments, the clarified feed is concentrated
between at least two-
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fold and at least ten-fold. In some embodiments, the clarified feed is
concentrated between at
least ten-fold and at least twenty-fold. In some embodiments, the clarified
feed is concentrated
between at least twenty-fold and at least fifty-fold. In some embodiments, the
clarified feed is
concentrated about twenty-fold. One of ordinary skill in the art will also
recognize that TFF can
also be used to remove small molecule impurities (e.g., cell culture
contaminants comprising
media components, scrum albumin, or other scrum proteins) form the clarified
feed via
diafiltration. In some embodiments, the clarified feed is subjected to
diafiltration to remove small
molecule impurities. In some embodiments, the diafiltration comprises the use
of between about 3
and about 10 diafiltration volume of buffer. In some embodiments, the
diafiltration comprises the
use of about 5 diafiltration volume of buffer. One of ordinary skill in the
art will also recognize
that TFF can also be used at any step in the purification process where it is
desirable to exchange
buffers before performing the next step in the purification process. In some
embodiments, the
methods for isolating rAAV from the clarified feed described herein comprise
the use of TFF to
exchange buffers.
[001931 Affinity chromatography can be used to isolate rAAV particles from a
composition. In
some embodiments, affinity chromatography is used to isolate rAAV particles
from the clarified
feed. In some embodiments, affinity chromatography is used to isolate rAAV
particles from the
clarified feed that has been subjected to tangential flow filtration. Suitable
affinity
chromatography media are known in the art and include without limitation, AVB
SepharoseTM,
POROSTM CaptureSelectTM AAVX affinity resin, POROSTM CaptureSelectTM AAV9
affinity
resin, and POROSTM CapturcScicctTM AAV8 affinity resin. In some embodiments,
the affinity
chromatography media is POROSTm CaptureSelectTM AAV9 affinity resin. In some
embodiments, the affinity chromatography media is POROSTm CaptureSelcctTM AAV8
affinity
resin. In some embodiments, the affinity chromatography media is POROSTM
CaptureSelectTm
AAVX affinity resin.
1100194] Anion exchange chromatography can be used to isolate rAAV particles
from a
composition. In some embodiments, anion exchange chromatography is used after
affinity
chromatography as a final concentration and polish step. Suitable anion
exchange
chromatography media are known in the art and include without limitation,
UNOsphereTm Q
(Biorad, Hercules, Calif.), and N-charged amino or imino resins such as e.g.,
POROSTM 50 PI, or
any DEAE, TMAE, tertiary or quaternary amine, or PEI-based resins known in the
art (U.S. Pat.
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No. 6,989,264; Brument et al., Mol. Therapy 6(5):678-686 (2002); Gao et al..
Hum. Gene
Therapy 11:2079-2091(2000)). In some embodiments, the anion exchange
chromatography
media comprises a quaternary amine. In some embodiments, the anion exchange
media is a
monolith anion exchange chromatography resin. In some embodiments, the
monolith anion
exchange chromatography media comprises glycidylmethacrylate-
ethylenedimethacrylate or
styrenc-divinylbenzene polymers. In some embodiments, the monolith anion
exchange
chromatography media is selected from the group consisting of CIMmultusTm QA-1
Advanced
Composite Column (Quaternary amine), CIIVImultus'm DEAE-1 Advanced Composite
Column
(Diethylamino), CIM QA Disk (Quaternary amine), CIM DEAL, and CIM EDA Disk
(Ethylene diamino). In some embodiments, the monolith anion exchange
chromatography media
is CIMmultusTm QA-1 Advanced Composite Column (Quaternary amine). In some
embodiments,
the monolith anion exchange chromatography media is CIM QA Disk (Quaternary
amine). In
some embodiments, the anion exchange chromatography media is CIM QA (BIA
Separations,
Slovenia). In some embodiments, the anion exchange chromatography media is BIA
CIM QA-
80 (Column volume is 80mL). One of ordinary skill in the art can appreciate
that wash buffers of
suitable ionic strength can be identified such that the rAAV remains bound to
the resin while
impurities, including without limitation impurities which may be introduced by
upstream
purification steps are stripped away.
[00195] In some embodiments, anion exchange chromatography is performed
according to a
method disclosed in WO 2019/241535, which is incorporated herein by reference
in its entirety.
[00196] In some embodiments, a method of isolating rAAV particles comprises
determining the
vector genome titer, capsid titer, and/or the ratio of full to empty capsids
in a composition
comprising the isolated rAAV particles. In some embodiments, the vector genome
titer is
determined by quantitative PCR (qPCR) or digital PCR (dPCR) or droplet digital
PCR (ddPCR).
In some embodiments, the capsid titer is determined by serotype-specific
ELISA. In some
embodiments, the ratio of full to empty capsids is determined by Analytical
Ultracentrifugation
(AUC) or Transmission Electron Microscopy (TEM).
[00197] In some embodiments, the vector genome titer, capsid titer, and/or the
ratio of full to
empty capsids is determined by spectrophotometry, for example, by measuring
the absorbance of
the composition at 260 nm; and measuring the absorbance of the composition at
280 nm. In some
embodiments, the rAAV particles are not denatured prior to measuring the
absorbance of the
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composition. In some embodiments, the rAAV particles are denatured prior to
measuring the
absorbance of the composition. In some embodiments, the absorbance of the
composition at 260
nm and 280 nm is determined using a spectrophotometer. In some embodiments,
the absorbance
of the composition at 260 nm and 280 nm is determined using a HPLC. In some
embodiments,
the absorbance is peak absorbance. Several methods for measuring the
absorbance of a
composition at 260 nm and 280 nm arc known in the art. Methods of determining
vector gcnomc
titer and capsid titer of a composition comprising the isolated recombinant
rAAV particles are
disclosed in WO 2019/212922, which is incorporated herein by reference in its
entirety.
[00198] In additional embodiments the disclosure provides compositions
comprising isolated
rAAV particles produced according to a method described herein. In some
embodiment, the
composition is a pharmaceutical composition comprising a pharmaceutically
acceptable carrier.
[00199] As used herein the term "pharmaceutically acceptable means a
biologically acceptable
formulation, gaseous, liquid or solid, or mixture thereof, which is suitable
for one or more routes
of administration, in vivo delivery or contact. A "pharmaceutically
acceptable" composition is a
material that is not biologically or otherwise undesirable, e.g., the material
may be administered
to a subject without causing substantial undesirable biological effects. Thus,
such a
pharmaceutical composition may be used, for example in administering rAAV
isolated according
to the disclosed methods to a subject. Such compositions include solvents
(aqueous or non-
aqueous), solutions (aqueous or non-aqueous), emulsions (e.g., oil-in-water or
water-in-oil),
suspensions, syrups, elixirs, dispersion and suspension media, coatings,
isotonic and absorption
promoting or delaying agents, compatible with pharmaceutical administration or
in vivo contact
or delivery. Aqueous and non-aqueous solvents, solutions and suspensions may
include
suspending agents and thickcning agents. Such pharmaceutically acceptable
carriers include
tablets (coated or uncoated), capsules (hard or soft), microbeads, powder,
granules and crystals.
Supplementary active compounds (e.g., preservatives, antibacterial, antiviral
and antifungal
agents) can also be incorporated into the compositions. Pharmaceutical
compositions can be
formulated to be compatible with a particular route of administration or
delivery, as set forth
herein or known to one of skill in the art. Thus, pharmaceutical compositions
include carriers,
diluents, or excipients suitable for administration by various routes.
Pharmaceutical compositions
and delivery systems appropriate for rAAV particles and methods and uses of
the invention are
known in the art (see, e.g., Remington: The Science and Practice of Pharmacy
(2003) 20th ed.,
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Mack Publishing Co., Easton, Pa.; Remington's Pharmaceutical Sciences (1990)
18th ed., Mack
Publishing Co., Easton, Pa.; The Merck Index (1996) 12th ed., Merck Publishing
Group,
Whitehouse, N.J.; Pharmaceutical Principles of Solid Dosage Forms (1993),
Technonic
Publishing Co., Inc., Lancaster, Pa.; Ansel and Stoklosa, Pharmaceutical
Calculations (2001) 11th
ed., Lippincott Williams & Wilkins, Baltimore, Md.; and Poznansky et al., Drug
Delivery
Systems (1980), R. L. Juliano, ed., Oxford, N.Y., pp. 253-315).
[00200] In some embodiments, the composition
is a
pharmaceutical unit dose. A "unit dose" refers to a physically discrete unit
suited as a unitary
dosage for the subject to be treated; each unit containing a predetermined
quantity optionally in
association with a pharmaceutical carrier (excipient, diluent, vehicle or
filling agent) which, when
administered in one or more doses, is calculated to produce a desired effect
(e.g., prophylactic or
therapeutic effect). Unit dose forms may be within, for example, ampules and
vials, which may
include a liquid composition, or a composition in a freeze-dried or
lyophilized state; a sterile
liquid carrier, for example, can be added prior to administration or delivery
in vivo. Individual
unit dose forms can be included in multi-dose kits or containers. Recombinant
vector (e.g.. AAV)
sequences, plasmids, vector genomes, and recombinant virus particles, and
pharmaceutical
compositions thereof can be packaged in single or multiple unit dose form for
ease of
administration and uniformity of dosage. In some embodiments, the composition
comprises
rAAV particles comprising an AAV capsid protein from an AAV capsid serotype
selected from
AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12,
AAV13, AAV14, AAV15 and AAV16, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39,
AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8,
AAV.PHP.B, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAVMYO, MyoAAV.1A,
MyoAAV1C, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6,
AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10 , AAV.HSC11, AAV.HSC12,
AAV.HSC13, AAV.HSC14. AAV.HSC15, and AAV.HSC16. In some embodiments, the AAV
capsid serotype is AAV8. In some embodiments, the AAV capsid serotype is AAV9.
EXAMPLES
Example 1. Development of improved helper plasmids.
[00201]
Plasmid pAdDeltaF6 was constructed by Dr. James M. Wilson and colleagues
at
UPenn. pAdDe1taF6 is 15770 bp in size. The plasmid contains the regions of
adenovirus genome
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that are important for AAV replication, namely E2A (DNA binding protein), E4,
and VA RNAI
but does not contain other adenovirus replication genes. This plasmid was
derived from an El, E3
deleted molecular clone of Ad5 (pBHG10, a pBR322 based plasmid). Deletions
were introduced
in the Ad5 DNA to remove expression of unnecessary adenovirus genes and reduce
the amount of
adenovirus DNA from 32 kb to 12 kb (Figure 1, A). Finally, the ampicillin
resistance gene was
replaced by the kanamycin resistance gene to give pAdDe1taF6 (Figure 1, B).
The functional
elements of the E2A. E4 and VA RNAI adenoviral genes necessary for AAV vector
production
remain in this plasmid. The adenoviral El essential gene functions are
supplied by the HEK293
cells. There are also some remnant genes/elements that were resulted from
partial digestion of
pBHG10. These include the promoterless L3 23K/viral endoprotease, L4
100K/hexon assembly
gene, L4 pVIII/hexon-associated precursor and L5 pVI/fiber genes in the map.
Figure 1 C). In
pAdDeltaF6 plasmid, these genes are not transcribed due to the deletion of
their promoter MLP
(Major Late Promoter). Biasiotto et al., Int. J. Mol. Sci., 16: 2893-2912;
doi:10.3390/ijms16022893 (2015). Since some of these genes including L4 100K
and L4 pVIII
overlap with E2A region, deletion of these genes may impact the production of
the essential
helper protein E2A as described below during the sequential reconfiguration of
the helper
plasmid. Furthermore, there is a L4 22K/33K gene with its own intact promoter
located at this
region. This gene encodes the L4 22K and L4 33K proteins involved in
Adenovirus 5 packaging.
The promoter of the L4 22K/33K gene also overlaps with E2A region. Therefore,
deletion of the
promoter may impact the production of E2A. There is a partial adenoviral
inverted terminal
repeat in thc plasmid map that also resulted from partial digestion of pBHG10.
However, due to
the deletion of the essential DNA polymerase gene (E2 region) for Adenovirus 5
DNA
replication, no infectious adenovirus is expected to be generated. DNA plasmid
sequencing was
performed by Qiagen Genomic Services and revealed 100% homology with the
following
important functional elements of the reference sequence pAdDeltaF6 p1707FH-Q:
E4 ORF6
3692-2808 bp; E2A DNA binding protein 11784-10194 bp; VA RNAI region 12426-
13378 bp.
The sequence is confirmed at Aldevron, as part of the manufacturing process.
[00202] New helper plasmid #1 The new helper plasmid #1 (Figure 2) was
constructed based on
Ad5 sequence where E2A and E4 orientations were re-configured to express them
bidirectionally.
The rationale behind this was to avoid possible interference from E4 strong
promoter which could
result in lowering the expression from E2A promoter located downstream. The
new helper
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plasmid #1 genes were synthesized by Genscript and cloned into EcoRI/NotI
sites of pUC57
vector that was freely available from Genscript. In this new designed plasmid,
some nonessential
remnant genes (Ad5 structural genes) and elements that include the ITR
sequence (Ad5 inverted
terminal repeat) next to E4 promoter, L3 23K/viral endoprotease, L5 pVI/fibre,
and L4
pVIII/hexon-associated precursor sequences were removed. On the other hand,
the L4 33K/L4
100K hcxon assembly gene was kept since the E2A transcription starting sites
(TSS) arc located
at that region and their removal may impact E2A expression. The virus
associated (VA) RNA
was further modified by incorporating VA RNAII to VA RNA1. VA RNA is known to
stimulate
viral protein synthesis in infected cells and antagonizes the interferon-
induced cellular defense
system by regulating innate cellular response (Ma et al., Journal of Virology,
Aug. 1996, p 5083-
5099). The new plasmid has the size of 11,484 bp.
[00203] The new helper plasmid #1 improved AAV titers and
performed well on different
transgenes as shown in Figure 3. rAAV production titers were assessed using
the clone 1, 2, 3, 4,
and 5 HEK293-derived host cells.
[00204] New helper plasmid #2 The new helper plasmid #2 (Figure
4) was designed
based on the new helper #1. In this new design, the E4 region was dissected by
sequential
deletion and the impact of the deletions on AAV production was investigated.
E4 Orf 1 and 2
were deleted based on results indicating that deletion of E4 Orf 1 and 2
improved AAV titers
(data not shown). It is known in the field that the promoter controlling E4
region is active at
earlier phase of adenovirus infection and continues to the late phases. The E4
region has the
potential to transcribe and encode for 7 different proteins that are resulted
from differential
splicing of a single primary transcript (Orf1, 2, 3, 3/4, 4, 6, 6/7) generated
by this promoter. The
pattern of differential splicing for this transcript changes during the phases
of viral infection with
some appearing only in early phases and other in late phase (Dix et al.,
Journal of General
Virology (1995), 76, 1051-1055). The encoded protein products of Orfl, 0rf2,
0rf3, 0r14, 0rf6,
and 0rf6/7 were reported to exist in infected cells except for 0rf3/4, which
might be absent or
expressed below detection limit (Tauber et al., Gene 278 (2001) 1-23). Orfl
encoded protein is
expressed in the late phase and target a family of cellular proteins that play
a role in cell signaling
and signal transfection. There is no functional information about E4 product
encoded by 0rf2.
Furthermore, Ad5 mutants in which E4 0rf2 were deleted, were about to grow to
wild-type levels
(Tauber et al., Gene 278 (2001) 1-23). The deletion of Orfl and 2 did not
impact AAV production
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but improved its titer which indicated that E4 Orfl and 2 are not essential
(Figure 5). rAAV
production titers were assessed using the clone 1, 2, 4, and 6 HEK293-derived
host cells.
[00205] New helper plasmid #3 During helper plasmid #3 design,
the E4 region was
further dissected by sequential deletion. Different E4 variants with E4 native
promoter and CMV
promoter were screened for AAV production (Figure 6). Those E4 variants with
E4 0rf6-7 only
gave the highest titers. E4 0rf3-4 was further removed from helper #2 to
generate helper #3
(Figure 7). To further explain the rationale behind removing 0rf3 and 0rf4, it
appears that 0rf3
and 0rf6 can partially or totally compensate for each other's defects. 0rf3
and 0rf6 have
redundant functions and independently amplify viral DNA replication, late
viral protein synthesis,
shut-off of host protein synthesis, and prevent concatemer formation of viral
genomes (Tauber et
al., Gene 278 (2001) 1-23). E4 0rf4 also downregulates E4 transcription by
inhibiting El A-
mediated transactivation of the E4 promoter through its interaction with the
serine/threonine
protein phosphatase 2A (PP2A), an enzyme that plays an important role on
numerous cellular
processes. This autoregulatory loop may be required to limit the cytotoxic
effects of E4 gene
products during the early phase of infection, where E4 0rf4 can induce
apoptosis through caspase
activation in a cell line-specific manner. Therefore, further removal of E4
0rf5 resulted in
prevention of this cytotoxic effect (Tauber et al., Gene 278 (2001) 1-23).
[00206] The helper #3 improved AAV titers including A AV8 and A
AV9, and different
transgenes (Figures 8 and 9). rAAV production titers were assessed using the
clone 1 and clone 4
HEK293-derived host cells.
[00207] New helper plasmid #4 The possibility of adding other
genes to the new helper
plasmid to further improve AAV titers was investigated. Incorporation of
selected genes from
Boca virus helper that were reported to have positive impact on AAV production
(Wang et al.,
Molecular Therapy: Methods & Clinical Development Vol.11 December 2018),
addition of a
copy of El A gene and AAP (assembly-activating protein derived from trans
plasmid) under
CMV promoter were explored. The addition of Boca virus selected genes NP1 and
NS2 genes to
helper plasmid #2 (Figure 10) had no impact on AAV titers (Figure 11). It is
known in the field
that the assembly activating protein encoded by AAV capsid can provide
increased capsid protein
stability when expressed in trans (Maurer et al., 2018, Cell Reports 23, 1817-
1830; Maurer et al.,
Journal Virology, 2019 Volume 93 Issue 7 e02013-18). The addition of AAP gene
expressed in
trans for AAV8 (Figure 12) had a negative impact on AAV titers (Figure 14).
ElA is known to
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start AAV virus replication by enhancing the transcription from the rep gene
promoters, P5 and
P19 and by activating E2A and E4 adenovirus promoters. ElA is also known to
control the host
cell cycle to accommodate for AAV viral DNA replication. A potential drawback
from
overexpressing ElA is that it is known to stabilize p53, which can lead to
apoptosis. This can be
overcome by the E1B55K and the E4Orf6 proteins that will form a complex with
p53 and cause it
to be degraded (Matsushita et al., Journal of General Virology (2004), 85,
2209-2214; Meier et
al., Viruses 2020, 12, 662;). A copy of ElA under the control of CMV promoter
was added to the
helper plasmid #3 to create helper plasmid #4 (Figure 13). The location of ElA
was between E4
and VA RNA I/11. The results indicated that helper #4 further improved AAV
titers as shown in
Figure 14. rAAV production titers were assessed using the clone 1 and 4 HEK293-
derived host
cells.
[00208] New helper plasmids #5, #6, #7, #8 and #9 It is known that E2A, E4 and
VA RNA I/II
microRNA are essential helper components for AAV production (Meier et al.,
Viruses 2020, 12,
662; doi:10.3390/v12060662). In the current helper plasmids #1-4, L4
100K/hexon assembly and
L4 22K/33K were kept in the helper plasmid #3 because their genes are located
between the E2A
promoter and E2A open reading frame. This region might be important since two
E2A
transcription starting sites (TSS) are located at this region as documented
from the long-read
direct RNA sequencing study of Donovan-Banfield et al., (Communication Biology
(2020)
3:124). To test whether these two sequences could be removed while maintaining
high titer,
several mutations were generated based on helper #3 (Table 2). The analysis of
all these
mutations indicated that helper #5 and helper #8 gave similar titers or
slightly higher titers than
the helper plasmid #3 (Figure 15). rAAV production titers were assessed using
the clone 1 and 4
HEK293-derived host cells. In the helper plasmid #5, N-terminal region of
encoded hexon
assembly was removed, while in helper plasmid #8 the start codon was mutated
for the hexon
assembly region. On the other hand, all mutants in which L4 22K/33K start
codon was mutated
showed decrease in titers indicating that L4 22K/33K might be important for
AAV production.
These findings accord with the reported effect of L4 22K deletion, which
resulted in continuous
increase in E2A (DBP) expression in later phases and subsequently had a
negative impact on E4
expression (Wu et al., Journal of Virology (2012) p.10474-10483; Guimet et
al., Journal of
Virology (2013) p.7688-7699).
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[00209] The new helper plasmids also improve the quality of rAAV particles
produced.
Compared to a production run performed with original helper in the
transfection process
transferred to a 200L production bioreactor, viral vector encoding transgene A
production
performed with a Helper #5 transfection process resulted in significantly
increased % full capsids
(compare 36.2% to 71.9% full, as measured by AUC).
Table 2. Mutation of hexon assembly and L4 22K/33K gene based on helper
plasmid #3
Plasmid Size Hexon Assembly L4
22K/33K
Helper #5 8.2Kb Partial deletion
Helper #6 8.2Kb Partial deletion
Mutation (stop codon)
Helper #7 10.0Kb
Mutation (stop codon)
Helper #8 10.0Kb Mutation (stop codon)
Helper #9 10Kb Mutation (stop codon)
Mutation (stop codon)
[00210] While the disclosed methods have been described in
connection with what is
presently considered to be the most practical and preferred embodiments, it is
to be understood
that the methods encompassed by the disclosure are not to be limited to the
disclosed
cmbodimcnts, but on the contrary, is intcndcd to cover various modifications
and equivalent
arrangements included within the spirit and scope of the appended claims.
[00211] All publications, patents, patent applications,
internet sites, and accession
numbers/database sequences including both polynucleotide and polypeptide
sequences cited
herein are hereby incorporated by reference herein in their entirety for all
purposes to the same
extent as if each individual publication, patent, patent application, internet
site, or accession
number/database sequence were specifically and individually indicated to be so
incorporated by
reference.
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