U.S. patent application number 17/466237 was filed with the patent office on 2022-04-07 for adeno-associated virus vector.
The applicant listed for this patent is ICAHN SCHOOL OF MEDICINE AT MOUNT SINAI, KING'S COLLEGE LONDON. Invention is credited to Ralph Michael LINDEN.
Application Number | 20220106612 17/466237 |
Document ID | / |
Family ID | 1000006027620 |
Filed Date | 2022-04-07 |
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United States Patent
Application |
20220106612 |
Kind Code |
A1 |
LINDEN; Ralph Michael |
April 7, 2022 |
ADENO-ASSOCIATED VIRUS VECTOR
Abstract
Disclosed herein is a recombinant adeno-associated virus (AAV)
vector comprising (a) a variant AAV2 capsid protein, wherein the
variant AAV2 capsid protein comprises at least four amino acid
substitutions with respect to a wild type AAV2 capsid protein;
wherein the at least four amino acid substitutions are present at
the following positions in an AAV2 capsid protein sequence: 457,
492, 499 and 533; and (b) a heterologous nucleic acid comprising a
nucleotide sequence encoding a gene product.
Inventors: |
LINDEN; Ralph Michael;
(London, GB) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
KING'S COLLEGE LONDON
ICAHN SCHOOL OF MEDICINE AT MOUNT SINAI |
London
New York |
NY |
GB
US |
|
|
Family ID: |
1000006027620 |
Appl. No.: |
17/466237 |
Filed: |
September 3, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15570687 |
Oct 30, 2017 |
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PCT/EP2015/053335 |
Feb 17, 2015 |
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17466237 |
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61940639 |
Feb 17, 2014 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61P 25/00 20180101;
A61P 1/16 20180101; A61P 27/02 20180101; C12N 15/86 20130101; A61K
48/0008 20130101; C12N 2750/14143 20130101; A61P 27/06 20180101;
C07K 14/005 20130101; C12N 2750/14122 20130101; C12N 2750/14142
20130101 |
International
Class: |
C12N 15/86 20060101
C12N015/86; A61K 48/00 20060101 A61K048/00; A61P 25/00 20060101
A61P025/00; C07K 14/005 20060101 C07K014/005; A61P 27/02 20060101
A61P027/02; A61P 1/16 20060101 A61P001/16; A61P 27/06 20060101
A61P027/06 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 3, 2014 |
GB |
1403684.2 |
Claims
1. A recombinant adeno-associated virus (AAV) vector comprising:
(a) a variant AAV2 capsid protein, wherein the variant AAV2 capsid
protein comprises at least four amino acid substitutions with
respect to a wild type AAV2 capsid protein; wherein the at least
four amino acid substitutions are present at the following
positions in an AAV2 capsid protein sequence: 457, 492, 499 and
533; and (b) a heterologous nucleic acid comprising a nucleotide
sequence encoding a gene product.
2. A recombinant AAV vector according to claim 1, wherein (i) the
variant AAV capsid protein comprises a sequence of SEQ ID NO:2, or
a sequence having at least 95% sequence identity thereto; and/or
(ii) the wild type AAV capsid protein comprises a sequence of SEQ
ID NO:1.
3. A recombinant AAV vector according to claim 1, wherein the
variant AAV2 capsid protein comprises one or more of the following
residues: M457, A492, D499 and Y533.
4. (canceled)
5. A recombinant AAV vector according claim 1, wherein the variant
AAV2 capsid protein further comprises one or more amino acid
substitutions with respect to the wild type AAV capsid protein at
the following positions in the AAV2 capsid protein sequence: 125,
151, 162 and 205.
6. (canceled)
7. (canceled)
8. A recombinant AAV vector according to claim 1, wherein the
variant AAV2 capsid protein further comprises one or more amino
acid substitutions with respect to the wild type AAV capsid protein
at the following positions in the AAV2 capsid protein sequence: 585
and 588.
9. (canceled)
10. (canceled)
11. A recombinant AAV vector according to claim 1, wherein the
variant AAV2 capsid protein further comprises one or more amino
acid substitutions with respect to the wild type AAV capsid protein
at the following positions in the AAV2 capsid protein sequence:
546, 548 and 593.
12. (canceled)
13. (canceled)
14. A recombinant AAV vector according to claim 1, wherein the
variant AAV2 capsid protein comprises the residue N312.
15. A recombinant adeno-associated virus (AAV) vector comprising:
(a) a variant AAV8 capsid protein, wherein the variant AAV8 capsid
protein comprises an amino acid substitution with respect to a wild
type AAV8 capsid protein at position 315 in an AAV8 capsid protein
sequence; and (b) a heterologous nucleic acid comprising a
nucleotide sequence encoding a gene product.
16. A recombinant AAV vector according to claim 15, wherein (i) the
variant AAV capsid protein comprises a sequence having at least 95%
sequence identity to SEQ ID NO:6; and/or (ii) the wild type AAV
capsid protein comprises a sequence of SEQ ID NO:6.
17. A recombinant AAV vector according to claim 15, wherein the
variant AAV8 capsid protein comprises the amino acid substitution
S315N with respect to a wild type AAV8 capsid protein.
18. A recombinant AAV vector according to claim 15, further
comprising one or more amino acid substitution present at one or
more of the following positions in the AAV8 capsid protein
sequence: 125, 151, 163, 206, 460, 495, 502, 536, 549, 551, 588,
591 and/or 596.
19. (canceled)
20. A recombinant adeno-associated virus (AAV) vector comprising:
(a) a variant AAV3B capsid protein, wherein the variant AAV3B
capsid protein comprises an amino acid substitution with respect to
a wild type AAV3B capsid protein at position 312 in an AAV3B capsid
protein sequence; and (b) a heterologous nucleic acid comprising a
nucleotide sequence encoding a gene product.
21. A recombinant AAV vector according to claim 20, wherein (i) the
variant AAV3B capsid protein comprises a sequence having at least
95% sequence identity to SEQ ID NO:11; and/or (ii) the wild type
AAV capsid protein comprises a sequence of SEQ ID NO:11.
22. A recombinant AAV vector according to claim 20, wherein the
variant AAV3B capsid protein comprises the amino acid substitution
S312N with respect to a wild type AAV3B capsid protein.
23. A recombinant adeno-associated virus (AAV) vector comprising:
(a) a variant AAV-LK03 capsid protein, wherein the variant AAV-LK03
capsid protein comprises an amino acid substitution at position 312
with respect to a AAV-LK03 capsid protein sequence as defined in
SEQ ID NO:12; and (b) a heterologous nucleic acid comprising a
nucleotide sequence encoding a gene product.
24. A recombinant AAV vector according to claim 23, wherein the
variant AAV-LK03 capsid protein comprises a sequence having at
least 95% sequence identity to SEQ ID NO:12.
25. (canceled)
26. (canceled)
27. A recombinant AAV vector according to claim 1, wherein the gene
product comprises an interfering RNA, an aptamer, a polypeptide, a
neuroprotective polypeptide, an anti-angiogenic polypeptide, a
polypeptide that enhances function of a neuronal or retinal cell,
glial derived neurotrophic factor, fibroblast growth factor, nerve
growth factor, brain derived neurotrophic factor, rhodopsin,
retinoschisin, RPE65 or peripherin.
28. (canceled)
29. (canceled)
30. (canceled)
31. A pharmaceutical composition comprising: (a) a recombinant AAV
vector according to claim 1; and (b) a pharmaceutically acceptable
excipient.
32. A method for delivering a gene product to a tissue in a
subject, the method comprising administering to the subject a
recombinant AAV vector or pharmaceutical composition according to
claim 1.
33. (canceled)
34. (canceled)
35. A method for treating a disorder in a subject, the method
comprising administering to the subject a recombinant AAV vector or
pharmaceutical composition according to claim 1.
36. (canceled)
37. (canceled)
38. (canceled)
39. (canceled)
Description
CROSS REFERENCE TO RELATED APPLICATION
[0001] This application is a continuation of U.S. patent
application Ser. No. 15/570,687, filed Oct. 30, 2017, which is a
371 of International Application No. PCT/EP2015/053335 filed on
Feb. 17, 2015, which claims the benefit of U.S. Provisional Patent
Application No. 61/940,639 filed on Feb. 17, 2014, the entire
content and disclosure of which is incorporated herein by
reference.
INCORPORATION BY REFERENCE OF SEQUENCE LISTING
[0002] The instant applications contains a Sequence Listing which
has been submitted via EFS-Web and is hereby incorporated by
reference in its entirety. Said ASCII copy, created on Dec. 15,
2021, is named 00890048US3SL.txt, and is 78,696 bytes in size.
FIELD OF THE INVENTION
[0003] The present invention relates to the field of recombinant
viral vectors. In particular, the invention relates to recombinant
viral vectors which are suitable for the delivery of therapeutic
genes in vivo.
BACKGROUND TO THE INVENTION
[0004] To date, adeno-associated virus remains one of the most
promising vectors for the delivery of therapeutic genes. A
significant number of preclinical and clinical studies have firmly
established that this approach is suitable for the development of
gene-based drugs that can reach market approval.
[0005] Since the beginning of the development of AAV2 as a vector
for gene therapy in the 1980s much progress has been made in
optimizing this platform for a variety of applications and target
tissues. Among those developments, possibly the most consequential
has been the discovery of a wide variety of serotypes of which ten
to twelve are now commonly explored. Among the most prominent
characteristics of these various serotypes are their respective
relative tissue tropism and--in some cases --the ability of
neuronal retrograde transport. Of these serotypes, AAV1-10 are
broadly used for pre-clinical and clinical purposes.
[0006] A newer platform has been developed that involves processes
that allow for the targeting and de-targeting of specific tissues
and cell sub-types in patients. The core technology of these
approaches is based on trial and error evaluation of existing AAV
variants (serotypes) and in vivo selection of randomly introduced
AAV capsid mutants. Together, these two promising approaches
provide tens--if not hundreds of potential vectors with different
transduction behaviour.
[0007] The most intriguing aspect of AAV serotypes is their ability
to efficiently transduce specific tissues in animal models and in
man. To date, comprehensive molecular understanding of the
underlying mechanisms for the tissue tropism has yet to be put
forward and it is thus generally assumed that the available
tissue-specific receptors for each serotype play a central role in
the efficient transduction by the various serotypes.
[0008] Accordingly there is still a need for additional AAV
vectors, which have improved properties in terms of in vivo
transgene expression and tissue specificity. In particular, such
vectors have the potential to provide greatly enhanced benefits for
gene delivery to various target tissues in humans.
SUMMARY OF THE INVENTION
[0009] In one aspect, the present invention provides a recombinant
adeno-associated virus (AAV) vector comprising: (a) a variant AAV2
capsid protein, wherein the variant AAV2 capsid protein comprises
at least four amino acid substitutions with respect to a wild type
AAV2 capsid protein; wherein the at least four amino acid
substitutions are present at the following positions in an AAV2
capsid protein sequence: 457, 492, 499 and 533; and (b) a
heterologous nucleic acid comprising a nucleotide sequence encoding
a gene product.
[0010] In one embodiment, the variant AAV capsid protein comprises
a sequence of SEQ ID NO:2, or a sequence having at least 95%
sequence identity thereto. In another embodiment, the wild type AAV
capsid protein comprises a sequence of SEQ ID NO:1.
[0011] In one embodiment, the variant AAV2 capsid protein comprises
one or more of the following residues: M457, A492, D499 and Y533.
In a preferred embodiment, the variant AAV2 capsid protein
comprises one or more of the following amino acid substitutions
with respect to a wild type AAV2 capsid protein: Q457M, S492A,
E499D and F533Y.
[0012] In one embodiment, the variant AAV2 capsid protein further
comprises one or more amino acid substitutions with respect to the
wild type AAV capsid protein at the following positions in the AAV2
capsid protein sequence: 125, 151, 162 and 205. In a preferred
embodiment, the variant AAV2 capsid protein comprises one or more
of one or more of the following residues: I125, A151, S162 and
S205. In another preferred embodiment, the variant AAV2 capsid
protein comprises one or more of the following amino acid
substitutions with respect to a wild type AAV2 capsid protein:
V125I, V151A, A162S and T205S.
[0013] In one embodiment, the variant AAV2 capsid protein further
comprises one or more amino acid substitutions with respect to the
wild type AAV capsid protein at the following positions in the AAV2
capsid protein sequence: 585 and 588. Preferably the variant AAV2
capsid protein comprises one or more of one or more of the
following residues: S585 and T588. More preferably the variant AAV2
capsid protein comprises one or more of the following amino acid
substitutions with respect to a wild type AAV2 capsid protein:
R585S and R588T.
[0014] In one embodiment, the variant AAV2 capsid protein further
comprises one or more amino acid substitutions with respect to the
wild type AAV capsid protein at the following positions in the AAV2
capsid protein sequence: 546, 548 and 593. Preferably the variant
AAV2 capsid protein comprises one or more of one or more of the
following residues: D546, G548, and S593. More preferably the
variant AAV2 capsid protein comprises one or more of the following
amino acid substitutions with respect to a wild type AAV2 capsid
protein: G546D, E548G and A593S.
[0015] In one embodiment, the variant AAV2 capsid protein comprises
the residue N312, i.e. the residue which is present in the wild
type AAV2 capsid protein at position 312. In this embodiment, the
variant AAV2 capsid protein is not mutated at position 312 compared
to the wild type AAV2 capsid protein sequence.
[0016] In another aspect, the present invention provides a
recombinant adeno-associated virus (AAV) vector comprising: (a) a
variant AAV8 capsid protein, wherein the variant AAV8 capsid
protein comprises an amino acid substitution with respect to a wild
type AAV8 capsid protein at position 315 in an AAV8 capsid protein
sequence; and (b) a heterologous nucleic acid comprising a
nucleotide sequence encoding a gene product.
[0017] In one embodiment, the variant AAV capsid protein comprises
a sequence having at least 95% sequence identity to SEQ ID NO:6. In
another embodiment, the wild type AAV capsid protein comprises a
sequence of SEQ ID NO:6.
[0018] In one embodiment, the variant AAV8 capsid protein comprises
the amino acid substitution S315N with respect to a wild type AAV8
capsid protein. Preferably the AAV8 capsid protein sequence
comprises one or more amino acid substitution present at one or
more of the following positions: 125, 151, 163, 206, 460, 495, 502,
536, 549, 551, 588, 591 and/or 596.
[0019] In a preferred embodiment, the variant AAV8 capsid protein
comprises one or more of the following amino acid substitutions
with respect to a wild type AAV8 capsid protein: (a) V125I, Q151A,
K163S, A206S, T460M, T495A, N502D, F536Y, N549D, A551G, Q588S
and/or G596S; and/or (b) T591R.
[0020] In another aspect, the present invention provides a
recombinant adeno-associated virus (AAV) vector comprising: (a) a
variant AAV3B capsid protein, wherein the variant AAV3B capsid
protein comprises an amino acid substitution with respect to a wild
type AAV3B capsid protein at position 312 in an AAV3B capsid
protein sequence; and (b) a heterologous nucleic acid comprising a
nucleotide sequence encoding a gene product.
[0021] In one embodiment, the variant AAV3B capsid protein
comprises a sequence having at least 95% sequence identity to SEQ
ID NO:11. In another embodiment, the wild type AAV capsid protein
comprises a sequence of SEQ ID NO:11.
[0022] In one embodiment, the variant AAV3B capsid protein
comprises the amino acid substitution S312N with respect to a wild
type AAV3B capsid protein.
[0023] In another aspect, the present invention provides a
recombinant adeno-associated virus (AAV) vector comprising (a) a
variant AAV-LK03 capsid protein, wherein the variant AAV-LK03
capsid protein comprises an amino acid substitution at position 312
with respect to a AAV-LK03 capsid protein sequence as defined in
SEQ ID NO:12; and (b) a heterologous nucleic acid comprising a
nucleotide sequence encoding a gene product.
[0024] In one embodiment, the variant AAV-LK03 capsid protein
comprises a sequence having at least 95% sequence identity to SEQ
ID NO:12.
[0025] In another aspect, the present invention provides a
recombinant adeno-associated virus (AAV) vector comprising: (a) a
variant AAV capsid protein, wherein the variant AAV capsid protein
comprises at least one amino acid substitution with respect to a
wild type AAV capsid protein at a position corresponding to one or
more of the following positions in an AAV2 capsid protein sequence:
125, 151, 162, 205, 312, 457, 492, 499, 533, 546, 548, 585, 588
and/or 593; and (b) a heterologous nucleic acid comprising a
nucleotide sequence encoding a gene product.
[0026] In one embodiment, the at least one amino acid substitution
is present at one or more of the following positions in an AAV2
capsid protein sequence: 125, 151, 162, 205, 312, 457, 492, 499,
533, 546, 548, 585, 588 and/or 593; or at one or more corresponding
positions in an alternative AAV capsid protein sequence.
[0027] In one embodiment, the vector comprises a variant AAV2
capsid protein. In another embodiment, the variant AAV capsid
protein comprises a sequence of SEQ ID NO:2, or a sequence having
at least 95% sequence identity thereto. In another embodiment, the
wild type AAV capsid protein is from AAV2. In another embodiment,
the wild type AAV capsid protein comprises a sequence of SEQ ID
NO:1.
[0028] In one embodiment, the variant AAV2 capsid protein comprises
one or more of the following residues: I125, A151, S162, S205,
S312, M457, A492, D499, Y533, D546, G548, S585, T588 and/or S593.
In a preferred embodiment, the variant AAV2 capsid protein
comprises one or more of the following amino acid substitutions
with respect to a wild type AAV2 capsid protein: V125I, V151A,
A162S, T205S, N312S, Q457M, S492A, E499D, F533Y, G546D, E548G,
R585S, R588T and/or A593S.
[0029] In further embodiments, the variant AAV capsid protein is
from AAV1, AAV5, AAV6, AAV8, AAV9 or AAV10.
[0030] In one embodiment, the vector comprises a variant AAV1
capsid protein. In another embodiment, the variant AAV capsid
protein comprises a sequence having at least 95% sequence identity
to SEQ ID NO:3. In another embodiment, the wild type AAV capsid
protein is from AAV1. In another embodiment, the wild type AAV
capsid protein comprises a sequence of SEQ ID NO:3.
[0031] In one embodiment, at least one amino acid substitution is
present at one or more of the following positions in the AAV1
capsid protein sequence: 125, 151, 162, 205, 313, 458, 493, 500,
534, 547, 549, 586, 589 and/or 594. In a preferred embodiment, the
variant AAV1 capsid protein comprises one or more of the following
amino acid substitutions with respect to a wild type AAV1 capsid
protein: V125I, Q151A, T162S, N313S, N458M, K493A, N500D, F534Y,
S547D, and/or G594S. In an alternative embodiment, the variant AAV1
capsid protein comprises one or more of the following amino acid
substitutions with respect to a wild type AAV1 capsid protein:
S205T, G549E, S586R and/or T589R.
[0032] In one embodiment, the vector comprises a variant AAV5
capsid protein. In another embodiment, the variant AAV capsid
protein comprises a sequence having at least 95% sequence identity
to SEQ ID NO:4. In another embodiment, the wild type AAV capsid
protein is from AAV5. In another embodiment, the wild type AAV
capsid protein comprises a sequence of SEQ ID NO:4.
[0033] In one embodiment, at least one amino acid substitution is
present at one or more of the following positions in the AAV5
capsid protein sequence: 124, 150, 153, 195, 303, 444, 479, 486,
520, 533, 537, 575, 578 and/or 583. In a preferred embodiment, the
variant AAV5 capsid protein comprises one or more of the following
amino acid substitutions with respect to a wild type AAV5 capsid
protein: V124I, K150A, K153S, A195S, R303S, T444M, S479A, V486D,
T520Y, P533D, and/or G583S. In an alternative embodiment, the
variant AAV5 capsid protein comprises one or more of the following
amino acid substitutions with respect to a wild type AAV5 capsid
protein: G537E, S575R and/or T578R.
[0034] In one embodiment, the vector comprises a variant AAV6
capsid protein. In another embodiment, the variant AAV capsid
protein comprises a sequence having at least 95% sequence identity
to SEQ ID NO:5. In another embodiment, the wild type AAV capsid
protein is from AAV6. In another embodiment, the wild type AAV
capsid protein comprises a sequence of SEQ ID NO:5.
[0035] In one embodiment, at least one amino acid substitution is
present at one or more of the following positions in the AAV6
capsid protein sequence: 125, 151, 162, 205, 313, 458, 493, 500,
534, 547, 549, 586, 589 and/or 594. In a preferred embodiment, the
variant AAV6 capsid protein comprises one or more of the following
amino acid substitutions with respect to a wild type AAV6 capsid
protein: V125I, Q151A, T162S, N313S, N458M, K493A, N500D, F534Y,
S547D, and/or G594S. In an alternative embodiment, the variant AAV6
capsid protein comprises one or more of the following amino acid
substitutions with respect to a wild type AAV6 capsid protein:
S205T, G549E, S586R and/or T589R.
[0036] In one embodiment, the vector comprises a variant AAV8
capsid protein. In another embodiment, the variant AAV capsid
protein comprises a sequence having at least 95% sequence identity
to SEQ ID NO:6. In another embodiment, the wild type AAV capsid
protein is from AAV8. In another embodiment, the wild type AAV
capsid protein comprises a sequence of SEQ ID NO:6.
[0037] In one embodiment, at least one amino acid substitution is
present at one or more of the following positions in the AAV8
capsid protein sequence: 125, 151, 163, 206, 315, 460, 495, 502,
536, 549, 551, 588, 591 and/or 596. In a preferred embodiment, the
variant AAV8 capsid protein comprises one or more of the following
amino acid substitutions with respect to a wild type AAV8 capsid
protein: V125I, Q151A, K163S, A206S, T460M, T495A, N502D, F536Y,
N549D, A551G, Q588S and/or G596S. In an alternative embodiment, the
variant AAV8 capsid protein comprises one or more of the following
amino acid substitutions with respect to a wild type AAV8 capsid
protein: S315N and/or T591R.
[0038] In one embodiment, the vector comprises a variant AAV9
capsid protein. In another embodiment, the variant AAV capsid
protein comprises a sequence having at least 95% sequence identity
to SEQ ID NO:7. In another embodiment, the wild type AAV capsid
protein is from AAV9. In another embodiment, the wild type AAV
capsid protein comprises a sequence of SEQ ID NO:7.
[0039] In one embodiment, at least one amino acid substitution is
present at one or more of the following positions in the AAV9
capsid protein sequence: 125, 151, 162, 205, 314, 458, 493, 500,
534, 547, 549, 586, 589 and/or 594. In a preferred embodiment, the
variant AAV9 capsid protein comprises one or more of the following
amino acid substitutions with respect to a wild type AAV9 capsid
protein: L125I, Q151A, N314S, Q458M, V493A, E500D, F534Y, G547D,
A589T and/or G594S. In an alternative embodiment, the variant AAV9
capsid protein comprises one or more of the following amino acid
substitutions with respect to a wild type AAV9 capsid protein:
S162A, S205T, G549E and/or S586R.
[0040] In one embodiment, the vector comprises a variant AAV10
capsid protein. In another embodiment, the variant AAV capsid
protein comprises a sequence having at least 95% sequence identity
to SEQ ID NO:8. In another embodiment, the wild type AAV capsid
protein is from AAV10. In another embodiment, the wild type AAV
capsid protein comprises a sequence of SEQ ID NO: 8.
[0041] In one embodiment, at least one amino acid substitution is
present at one or more of the following positions in the AAV10
capsid protein sequence: 125, 151, 163, 206, 315, 460, 495, 502,
536, 549, 551, 588, 591 and/or 596. In a preferred embodiment, the
variant AAV10 capsid protein comprises one or more of the following
amino acid substitutions with respect to a wild type AAV10 capsid
protein: V125I, Q151A, K163S, A206S, N315S, T460M, L495A, N502D,
F536Y, G549D, Q588S, A591T and/or G596S. In an alternative
embodiment, the variant AAV10 capsid protein comprises the
following amino acid substitution with respect to a wild type AAV10
capsid protein: G551E.
[0042] In one embodiment, the recombinant AAV vector exhibits
increased transduction of a neuronal or retinal tissue compared to
an AAV vector comprising a corresponding wild type AAV capsid
protein.
[0043] In another embodiment, the recombinant AAV vector exhibits
increased transduction of liver tissue compared to a corresponding
wild type AAV capsid protein.
[0044] In one embodiment, the gene product comprises an interfering
RNA or an aptamer. In another embodiment, the gene product
comprises a polypeptide. Preferably the gene product comprises a
neuroprotective polypeptide, an anti-angiogenic polypeptide, or a
polypeptide that enhances function of a neuronal or retinal cell.
In preferred embodiments, the gene product comprises glial derived
neurotrophic factor, fibroblast growth factor, nerve growth factor,
brain derived neurotrophic factor, rhodopsin, retinoschisin, RPE65
or peripherin.
[0045] In another aspect, the present invention provides a
pharmaceutical composition comprising: (a) a recombinant AAV vector
as defined above; and (b) a pharmaceutically acceptable
excipient.
[0046] In another aspect, the present invention provides a method
for delivering a gene product to a tissue in a subject, the method
comprising administering to the subject a recombinant AAV vector or
pharmaceutical composition as defined above.
[0047] In some embodiments, the tissue is selected from blood, bone
marrow, muscle tissue, neuronal tissue, retinal tissue, pancreatic
tissue, liver tissue, kidney tissue, lung tissue, intestinal tissue
or heart tissue. Preferably the tissue is neuronal, retinal or
liver tissue.
[0048] In another aspect, the present invention provides a method
for treating a disorder in a subject, the method comprising
administering to the subject a recombinant AAV vector or
pharmaceutical composition as defined above. In some embodiments,
the disorder is a neurological, ocular or hepatic disorder.
[0049] In another aspect, the present invention provides a
recombinant AAV vector or pharmaceutical composition as defined
above, for use in treating a disorder in a subject. In some
embodiments, the disorder is a neurological, ocular or hepatic
disorder. Preferably the neurological disorder is a
neurodegenerative disease. In an alternative embodiment, the ocular
disorder is glaucoma, retinitis pigmentosa, macular degeneration,
retinoschisis or diabetic retinopathy.
[0050] In another aspect, the present invention provides an
isolated variant AAV capsid protein, wherein the variant AAV capsid
protein comprises at least one amino acid substitution with respect
to a wild type AAV capsid protein; wherein the at least one amino
acid substitution is present at one or more of the following
positions in an AAV2 capsid protein sequence: 125, 151, 162, 205,
312, 457, 492, 499, 533, 546, 548, 585, 588 and/or 593; or at one
or more corresponding positions in an alternative AAV capsid
protein sequence.
[0051] In another aspect, the present invention provides an
isolated nucleic acid comprising a nucleotide sequence that encodes
a variant AAV capsid protein as defined above.
[0052] In another aspect, the present invention provides an
isolated host cell comprising a nucleic acid as defined above.
BRIEF DESCRIPTION OF THE DRAWINGS
[0053] This patent or application file contains at least one
drawing executed in color.
[0054] Copies of this patent or patent application publication with
color drawing(s) will be provided
by the United States Patent and Trademark Office upon request and
payment of the necessary fee.
[0055] FIG. 1 shows the amino acid sequence of wild-type
adeno-associated virus 2 capsid protein VP1 (SEQ ID NO:1; NCBI
Reference Sequence: NC_001401). Residues V125, V151, A162, T205,
N312, Q457, S492, E499, F533, G546, E548, R585, R588 and A593 are
highlighted.
[0056] FIG. 2 shows the amino acid sequence of true-type
adeno-associated virus 2 (ttAAV2) capsid protein VP1 (SEQ ID NO:2).
Residues I125, A151, S162, S205, S312, M457, A492, D499, Y533,
D546, G548, S585, T588, S593 differ compared to wild-type AAV2 VP1
(SEQ ID NO:1) and are highlighted.
[0057] FIG. 3 shows the amino acid sequence of wild-type
adeno-associated virus 1 capsid protein VP1 (SEQ ID NO:3; NCBI
Reference Sequence: NC_002077). Highlighted residues: S205 (aligns
with S205 in ttAAV2 (SEQ ID NO:2))--G549 (aligns with G548 in
ttAAV2)--S586 (aligns with S585 in ttAAV2)--T589 (aligns with T588
in ttAAV2).
[0058] FIG. 4 shows the amino acid sequence of wild-type
adeno-associated virus 5 capsid protein VP1 (SEQ ID NO:4; NCBI
Reference Sequence: AF085716). Highlighted residues: G537 (aligns
with G548 in ttAAV2)--S575 (aligns with S585 in ttAAV2)--T578
(aligns with T588 in ttAAV2).
[0059] FIG. 5 shows the amino acid sequence of wild-type
adeno-associated virus 6 capsid protein VP1 (SEQ ID NO:5; NCBI
Reference Sequence: AF028704). Highlighted residues: S205 (aligns
with S205 in ttAAV2)--G549 (aligns with G548 in ttAAV2)--S586
(aligns with S585 in ttAAV2)--T589 (aligns with T588 in
ttAAV2).
[0060] FIG. 6 shows the amino acid sequence of wild-type
adeno-associated virus 8 capsid protein VP1 (SEQ ID NO:6; NCBI
Reference Sequence: NC_006261). Highlighted residues: S315 (aligns
with S312 in ttAAV2)--T591 (aligns with T588 in ttAAV2).
[0061] FIG. 7 shows the amino acid sequence of wild-type
adeno-associated virus 9 capsid protein VP1 (SEQ ID NO:7; NCBI
Reference Sequence: AY530579). Highlighted residues: S162 (aligns
with S162 in ttAAV2)--S205 (aligns with S205 in ttAAV2)--G549
(aligns with G548 in ttAAV2)--S586 (aligns with S585 in
ttAAV2).
[0062] FIG. 8 shows the amino acid sequence of wild-type
adeno-associated virus 10 capsid protein VP1 (SEQ ID NO:8).
Highlighted residue: G551 (aligns with G548 in ttAAV2).
[0063] FIGS. 9A and 9B shows an alignment of AAV capsid protein VP1
amino acid sequences.
[0064] FIG. 10 The plasmid used to produce AAV2 vectors was the
packaging plasmid pDG. Above: pDG with the wild-type AAV2 genes.
Below: pDG-ttAAV2 with the true-type AAV2 genes, highlighted are
the two key mutations in the heparan binding domains at positions
585 and 588. MMTV: promoter driving AAV rep expression, E2a, E4ORF6
and VA are the genes expressing adenovirus helper factors.
[0065] FIG. 11 Quantification of viral titres of rAAV2 true-type
(TT) and wild-type (WT) for in vivo injections by SDS-PAGE showing
Krypton staining for separated proteins, and scanned using an
infrared-fluorescence scanner (Odyssey Imaging systems). A: 10
.mu.l of AAV2 virus particles, and 62.5 ng-500 ng of BSA were
separated on a 12% separating gel containing SDS and stained with
Krypton Protein Stain. The image was converted to grayscale. The
capsid gene proteins VP1, VP2, VP 3 are labelled on the left. B:
Table showing titres from qPCR (vector genome [vg/ml]) and SDS-Page
(capsid titre [capsid/ml]).
[0066] FIG. 12 A. Representative examples of rat brain sections
stained with a GFP-specific antibody are shown. The vector was
injected into the striatum as shown by the arrow. B. representative
example of an injection into the substantia nigra is shown.
[0067] FIG. 13 GFP transduction of the eye using ttAAV2 and wtAAV2
is shown. A. Retina in a transverse section is shown after ttAAV2
(top) and wtAAV2 (bottom) vector administration is shown. B.
Magnifications of the dashed boxes in A are shown.
[0068] FIG. 14 Transduction of mouse brains after neonatal vector
injection. i.v., intra-venous vector administration; i.c.,
intra-cranial injection; AAV-2, wtAAV2; AAV-TT, ttAAV2.
[0069] FIG. 15 Three-dimensional representation of the AAV2 capsid.
The highlighted residues correspond to the amino acid changes
between ttAAV2 and wild-type particles, grouped by colour depending
on their position.
[0070] FIG. 16 Representation of a threefold spike on the AAV2
capsid. The highlighted residues correspond to the amino acid
changes between True-type and Wild-type particles. The heparin
binding site residues are highlighted in green.
[0071] FIG. 17 Representation of the internal side of the AAV2
capsid. The highlighted residues in light-blue correspond to the
single amino acid change in ttAAV2 that is located on the internal
side of the capsid.
[0072] FIG. 18 Representation of a threefold spike on the AAV2
capsid. The residues highlighted in beige correspond to two amino
acid changes in the True-type vector that are spatially close and
located in the groove between two threefold-proximal peaks on the
AAV capsid.
[0073] FIG. 19 Representation of a threefold spike on the AAV2
capsid. The residue highlighted in brown corresponds to a single
isolated amino acid change (S593) in the True-type vector that is
located in the groove between threefold-proximal peaks
[0074] FIG. 20 Representation of a threefold spike on the AAV2
capsid. The four amino acids highlighted in pink are involved in
receptor binding and closely situated on the threefold spikes.
[0075] FIG. 21 Three-dimensional representation of an alignment
between VP1 capid monomer from AAV2 (light blue) and VP1 monomer
from AAV1 (orange). The highlighted residues in the middle-left of
the picture correspond to G549 in AAV1 (orange spheres) and E548 in
AAV2 (cyan sphere). The highlighted residues in the top-right of
the picture correspond to S586 and T589 in AAV1 (orange spheres)
and R585 and R588 in AAV2 (cyan sphere).
[0076] FIG. 22 Three-dimensional representation of an alignment
between VP1 capsid monomer from AAV2 (light blue) and VP1 monomer
from AAV5 (purple). The highlighted residues in the middle of the
picture correspond to G537 in AAV5 (purple spheres) and E548 in
AAV2 (cyan sphere). The highlighted residues in the top-right of
the picture correspond to S575 and T578 in AAV5 (purple spheres)
and R585 and R588 in AAV2 (cyan sphere).
[0077] FIG. 23 Three-dimensional representation of an alignment
between VP1 capsid monomer from AAV2 (light blue) and VP1 monomer
from AAV6 (yellow). The highlighted residues in the bottom of the
picture correspond to G549 in AAV6 (orange spheres) and E548 in
AAV2 (cyan sphere). The highlighted residues in the top-right of
the picture correspond to S586 and T589 in AAV6 (orange spheres)
and R585 and R588 in AAV2 (cyan sphere).
[0078] FIG. 24 Three-dimensional representation of an alignment
between VP1 capsid monomer from AAV2 (light blue) and VP1 monomer
from AAV8 (pink). The highlighted residues in the top-left of the
picture correspond to S315 in AAV8 (red spheres) and N312 in AAV2
(cyan sphere). The highlighted residues in the bottom-right of the
picture correspond to T591 in AAV8 (red spheres) and R588 in AAV2
(cyan sphere).
[0079] FIG. 25 Three-dimensional representation of an alignment
between VP1 capsid monomer from AAV2 (light blue) and VP1 monomer
from AAV9 (green). The highlighted residues in the middle of the
picture correspond to G549 in AAV9 (yellow spheres) and E548 in
AAV2 (cyan sphere). The highlighted residues in the bottom-left of
the picture correspond to S586 in AAV9 (yellow spheres) and R585 in
AAV2 (cyan sphere).
[0080] FIG. 26 Analysis of rAAV2 TT and WT expression in the
parafascicularis nucleus after striatal injection in rat brain. A:
Representative images of rat brain sections showing the rostral
side on the left and the caudal side on the right. The site of
injection in the striatum is indicated, and the area of projection
in the hypothalamus observed in B and C is shown (parafascicularis
nucleus, pf). B and C: High magnification images of the GFP
expression detected in the parafascicularis nucleus (pf) after
striatal injection of rAAV2 WT (B) or TT (C).
[0081] FIG. 27 Overview of intracranial injections of rAAV2 TT and
WT in neonatal mice. Representative examples of neonate brain
sections stained with a GFP-specific antibody are shown.
5.times.10.sup.10 vg of rAAV2 TT (top) or rAAV2 WT (middle) were
injected into the lateral ventricle of neonatal mouse brains. An
uninjected brain from a neonatal mouse, stained simultaneously, is
represented as a negative control (NT, non transduced).
[0082] FIG. 28 High magnification pictures of neonatal mouse brain
sections after intracranial injections of rAAV2 TT or WT. Neonate
brain sections stained with a GFP-specific antibody are shown.
5.times.10.sup.10 vg of rAAV2 TT (left panels) or rAAV2 WT (right
panels) were injected into the lateral ventricle of neonatal mouse
brains. S1BF: barrel field primary somatosensory cortex.
[0083] FIG. 29 Overview of brain transduction after systemic
injection of rAAV2 TT and WT in neonatal mice. Representative
examples of neonate brain sections stained with a GFP-specific
antibody are shown. 2.times.10'' vg of rAAV2 TT (top) or rAAV2 WT
(bottom) were injected into the jugular veins of neonatal mice.
[0084] FIG. 30 High magnification pictures of neonatal mouse brain
sections after systemic injections of rAAV2 TT or WT. Neonate brain
sections stained with a GFP-specific antibody are shown.
2.times.10'' vg of rAAV2 TT (left panels) or rAAV2 WT (right
panels) were injected into the jugular veins of neonatal mice.
S1BF: barrel field primary somatosensory cortex.
[0085] FIG. 31 High magnification pictures of neonatal mouse tissue
sections after systemic injections of rAAV2 TT or WT.
2.times.10.sup.11 vg of rAAV2 TT or rAAV2 WT were injected into the
jugular veins of neonatal mice. Uninjected mouse organs were used
as negative controls.
[0086] FIG. 32 High magnification images of adult rat brain
sections after striatal injections of rAAV2 TT, WT and HBnull.
Representative examples of rat brain sections stained with a
GFP-specific antibody are shown. 3.5.times.10.sup.9 vg of rAAV2 WT
(left), TT (right) or AAV2-HBnull (middle) were injected into the
striatum of adult rat brains and representative pictures were taken
in the thalamus or in the substantia nigra (SN).
[0087] FIG. 33 Overview of intracranial injections of the full
AAV-TT compared with various TT mutants in neonatal mice.
Representative examples of neonate brain sections stained with a
GFP-specific antibody are shown. 5.times.10.sup.19 vg of rAAV2 TT,
TT-S312N, TT-S593A or TT-D546G/G548E (TT-DG) were injected into the
lateral ventricle of neonatal mouse brains. An uninjected brain
from a neonatal mouse, stained simultaneously, is represented as a
negative control (NT).
[0088] FIG. 34 High magnification pictures of neonatal mouse brain
sections after intracranial injections of various TT mutant
vectors. Neonate brain sections stained with a GFP-specific
antibody are shown. 5.times.10.sup.19 vg of vectors were injected
into the lateral ventricle of neonatal mouse brains. TT-DG:
TT-D546G/G548E.
[0089] FIG. 35 Overview of neonatal mice intracranial injections of
the full AAV-TT compared with the TT-S312N mutant and the potential
final TT vector containing 10 mutations. Representative examples of
neonate brain sections stained with a GFP-specific antibody are
shown. 5.times.10.sup.09 vg of rAAV2 TT, TT-S312N, TT or
TT-S312N-D546G/G548E-S593A (TT-S312N-DG-S593A) were injected into
the lateral ventricle of neonatal mouse brains. An uninjected brain
from a neonatal mouse, stained simultaneously, is represented as a
negative control (NT).
[0090] FIG. 36 High magnification pictures of neonatal mouse brain
sections after intracranial injections of various TT mutant
vectors. Neonate brain sections stained with a GFP-specific
antibody are shown. 5.times.10.sup.09 vg of vectors were injected
into the lateral ventricle of neonatal mouse brains.
[0091] FIG. 37 ELISA quantification of GFP protein in neonatal mice
brains injected with the full AAV-TT, the TT-S312N mutant or the
TT-S312N-DG-S593A. 5.times.10.sup.09 vg of vectors were injected
into the lateral ventricle of neonatal mouse brains and total
proteins were extracted from whole harvested brains. A GFP-specific
antibody was used to detect the GFP expression in each brain sample
and a standard GFP protein was used for quantification. N=5 animals
per condition. Error bars represent the mean.+-.SEM
[0092] FIG. 38 Amino acid sequence of the VP1 capsid protein of
AAV3B. The highlighted residues represent the residues that are
identical to the ones in AAV-tt at corresponding positions. The
internal serine residue at position 312 is underlined.
[0093] FIGS. 39A and 39B Amino acid sequence of the VP1 capsid
protein of AAV-LK03.
LIST OF SEQUENCES
[0094] SEQ ID NO:1 is the amino acid sequence of wild-type
adeno-associated virus 2 capsid protein VP1 (see FIG. 1).
[0095] SEQ ID NO:2 is the amino acid sequence of true-type
adeno-associated virus 2 (ttAAV2) capsid protein (see FIG. 2).
[0096] SEQ ID NO:3 is the amino acid sequence of wild-type
adeno-associated virus 1 capsid protein VP1 (see FIG. 3).
[0097] SEQ ID NO:4 is the amino acid sequence of wild-type
adeno-associated virus 5 capsid protein VP1 (see FIG. 4).
[0098] SEQ ID NO:5 is the amino acid sequence of wild-type
adeno-associated virus 6 capsid protein VP1 (see FIG. 5).
[0099] SEQ ID NO:6 is the amino acid sequence of wild-type
adeno-associated virus 8 capsid protein VP1 (see FIG. 6).
[0100] SEQ ID NO:7 is the amino acid sequence of wild-type
adeno-associated virus 9 capsid protein VP1 (see FIG. 7).
[0101] SEQ ID NO:8 is the amino acid sequence of wild-type
adeno-associated virus 10 Upenn capsid protein VP1 (see FIG.
8).
[0102] SEQ ID NO:9 is the amino acid sequence of wild-type
adeno-associated virus 10 japanese capsid protein VP1 (see FIG.
9).
[0103] SEQ ID NO:10 is the consensus amino acid sequence for
adeno-associated viruses shown in FIG. 9.
[0104] SEQ ID NO:11 is the amino acid sequence of wild-type
adeno-associated virus 3B capsid protein VP1 (see FIG. 38).
[0105] SEQ ID NO:12 is the amino acid sequence of adeno-associated
virus LK-03 capsid protein VP1 (see FIG. 39).
DETAILED DESCRIPTION OF THE INVENTION
[0106] In one aspect, the present invention relates to a
recombinant adeno-associated virus (AAV) vector. The rAAV vector
typically comprises a variant capsid protein which differs compared
to a wild-type AAV capsid protein. The variant capsid protein may
advantageously confer enhanced infectivity of the vector in brain
and/or eye, making the vector particularly suited to delivery of
therapeutic agents by gene therapy into these tissues.
Recombinant AAV Vector
[0107] The present disclosure provides a recombinant
adeno-associated virus (rAAV) vector. "AAV" is an abbreviation for
adeno-associated virus, and may be used to refer to the virus
itself or derivatives 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, also referred to as a recombinant AAV
vector (or "rAAV vector"). The term "AAV" includes, for example,
AAV type 1 (AAV-1), AAV type 2 (AAV-2), AAV type 3 (AAV-3), AAV
type 4 (AAV-4), AAV type 5 (AAV-5), AAV type 6 (AAV-6), AAV type 7
(AAV-7), AAV type 8 (AAV-8), AAV type 9 (AAV-9), AAV type 10
(AAV-10, including AAVrh10), AAV type 12 (AAV-12), avian AAV,
bovine AAV, canine AAV, equine AAV, primate AAV, non-primate AAV,
and ovine AAV. "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, and so
on.
[0108] The genomic sequences of various serotypes of AAV, as well
as the sequences of the native terminal repeats (TRs), Rep
proteins, and capsid subunits are known in the art. Such sequences
may be found in the literature or in public databases such as
GenBank. See, e.g., GenBank Accession Numbers NC-002077 (AAV-1),
AF063497 (AAV-1), NC-001401 (AAV-2), AF043303 (AAV-2), NC-001729
(AAV-3), NC-001829 (AAV-4), U89790 (AAV-4), NC-006152 (AAV-5),
AF513851 (AAV-7), AF513852 (AAV-8), and NC-006261 (AAV-8); the
disclosures of which are incorporated by reference herein. See
also, e.g., Srivistava et al. (1983) J. Virology 45:555; Chiorini
et al. (1998) J. Virology 71:6823; Chiorini et al. (1999) J.
Virology 73: 1309; Bantel-Schaal et al. (1999) J. Virology 73:939;
Xiao et al. (1999) J. Virology 73:3994; Muramatsu et al. (1996)
Virology 221:208; Shade et al., (1986) J. Virol. 58:921; Gao et al.
(2002) Proc. Nat. Acad. Sci. USA 99: 11854; Moris et al. (2004)
Virology 33:375-383; international patent publications WO 00/28061,
WO 99/61601, WO 98/11244; and U.S. Pat. No. 6,156,303.
[0109] An "rAAV vector" as used herein refers to an AAV vector
comprising a polynucleotide sequence not of AAV origin (i.e., a
polynucleotide heterologous to AAV), typically a sequence of
interest for the genetic transformation of a cell. In some
embodiments, the heterologous polynucleotide may be flanked by at
least one, and sometime sby two, AAV inverted terminal repeat
sequences (ITRs). The term rAAV vector encompasses both rAAV vector
particles and rAAV vector plasmids. An rAAV vector may either be
single-stranded (ssAAV) or self-complementary (scAAV).
[0110] An "AAV virus" or "AAV viral particle" or "rAAV vector
particle" refers to a viral particle composed of at least one AAV
capsid protein (typically by all of the capsid proteins of a
wild-type AAV) and an encapsidated polynucleotide rAAV vector. If
the particle comprises 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), it is typically
referred to as an "rAAV vector particle" or simply an "rAAV
vector". Thus, production of rAAV particle necessarily includes
production of rAAV vector, as such a vector is contained within an
rAAV particle.
[0111] "Recombinant," as used herein means that the vector,
polynucleotide, polypeptide or cell is the product of various
combinations of cloning, restriction or ligation steps (e.g.
relating to a polynucleotide or polypeptide comprised therein),
and/or other procedures that result in a construct that is distinct
from a product found in nature. A recombinant virus or vector is a
viral particle comprising a recombinant polynucleotide. The terms
respectively include replicates of the original polynucleotide
construct and progeny of the original virus construct.
Variant AAV Capsid Proteins
[0112] The rAAV vectors described herein comprise a variant AAV
capsid protein. By "variant" it is meant that the AAV capsid
protein differs from a corresponding wild type AAV capsid protein
of the same serotype. For instance, the variant AAV capsid protein
may comprise one or more amino acid substitutions with respect to
the corresponding wild type AAV capsid protein. In this context,
"corresponding" refers to a capsid protein of the same serotype,
i.e. a variant AAV1 capsid protein comprises one or more amino acid
substitutions with respect to the corresponding wild type AAV1
capsid protein, a variant AAV2 capsid protein comprises one or more
amino acid substitutions with respect to the corresponding wild
type AAV2 capsid protein, and so on.
[0113] The variant AAV capsid protein may comprise, for example, 1
to 50, 1 to 30, 1 to 20 or 1 to 15 amino acid substitutions with
respect to the wild type AAV capsid protein. Preferably the variant
AAV capsid protein comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,
13 or 14 amino acid substitutions with respect to the corresponding
wild type AAV capsid protein. In preferred embodiments, the variant
AAV capsid protein retains at least 70%, at least 80%, at least
90%, at least 95%, at least 96%, at least 97%, at least 98% or at
least 99% sequence identity to the wild type capsid protein.
[0114] In embodiments of the present invention, the variant AAV
capsid protein comprises at least one amino acid substitution with
respect to a wild type AAV capsid protein at a position
corresponding to one or more of the following positions in an AAV2
capsid protein sequence: 125, 151, 162, 205, 312, 457, 492, 499,
533, 546, 548, 585, 588 and/or 593. In this context,
"corresponding" refers to a position in any AAV capsid protein
sequence (e.g. in an AAV2 protein sequence or a non-AAV2 capsid
protein sequence) which corresponds to one of the above positions
in AAV2 capsid protein. In one embodiment, the at least one amino
acid substitution is present at one or more of the following
positions in an AAV2 capsid protein sequence: 125, 151, 162, 205,
312, 457, 492, 499, 533, 546, 548, 585, 588 and/or 593; or at one
or more corresponding positions in an alternative AAV capsid
protein sequence.
[0115] In general, AAV capsid proteins include VP1, VP2 and VP3. In
a preferred embodiment, the capsid protein comprises AAV capsid
protein VP1.
Nucleic Acid and Amino Acid Sequences and Sequence Identity
[0116] The term "polynucleotide" refers to a polymeric form of
nucleotides of any length, including deoxyribonucleotides or
ribonucleotides, or analogs thereof. A polynucleotide may comprise
modified nucleotides, such as methylated nucleotides and nucleotide
analogs, and may be interrupted by non-nucleotide components. If
present, modifications to the nucleotide structure may be imparted
before or after assembly of the polymer. The term polynucleotide,
as used herein, refers interchangeably to double- and
single-stranded molecules. Unless otherwise specified or required,
any embodiment of the invention described herein that is a
polynucleotide encompasses both the double-stranded form and each
of two complementary single-stranded forms known or predicted to
make up the double-stranded form.
[0117] The terms "polypeptide," "peptide," and "protein" are used
interchangeably herein to refer to polymers of amino acids of any
length. The terms also encompass an amino acid polymer that has
been modified; for example, disulfide bond formation,
glycosylation, lipidation, phosphorylation, or conjugation with a
labeling component. Polypeptides such as anti-angiogenic
polypeptides, neuroprotective polypeptides, and the like, when
discussed in the context of delivering a gene product to a
mammalian subject, and compositions therefor, refer to the
respective intact polypeptide, or any fragment or genetically
engineered derivative thereof, which retains the desired
biochemical function of the intact protein. Similarly, references
to nucleic acids encoding anti-angiogenic polypeptides, nucleic
acids encoding neuroprotective polypeptides, and other such nucleic
acids for use in delivery of a gene product to a mammalian subject
(which may be referred to as "transgenes" to be delivered to a
recipient cell), include polynucleotides encoding the intact
polypeptide or any fragment or genetically engineered derivative
possessing the desired biochemical function.
[0118] A polynucleotide or polypeptide has a certain percent
"sequence identity" to another polynucleotide or polypeptide,
meaning that, when aligned, that percentage of bases or amino acids
are the same when comparing the two sequences. Sequence similarity
can be determined in a number of different manners. To determine
sequence identity, sequences can be aligned using the methods and
computer programs, including BLAST, available over the world wide
web at ncbi.nlm.nih.gov/BLAST/. Another alignment algorithm is
FASTA, available in the Genetics Computing Group (GCG) package,
from Madison, Wis., USA, a wholly owned subsidiary of Oxford
Molecular Group, Inc. Other techniques for alignment are described
in Methods in Enzymology, vol. 266: Computer Methods for
Macromolecular Sequence Analysis (1996), ed. Doolittle, Academic
Press, Inc., a division of Harcourt Brace & Co., San Diego,
Calif., USA. Of particular interest are alignment programs that
permit gaps in the sequence. The Smith-Waterman is one type of
algorithm that permits gaps in sequence alignments. See Meth. Mol.
Biol. 70: 173-187 (1997). Also, the GAP program using the Needleman
and Wunsch alignment method can be utilized to align sequences. See
J. Mol. Biol. 48: 443-453 (1970)
[0119] Of interest is the BestFit program using the local homology
algorithm of Smith and Waterman (Advances in Applied Mathematics 2:
482-489 (1981) to determine sequence identity. The gap generation
penalty will generally range from 1 to 5, usually 2 to 4 and in
many embodiments will be 3. The gap extension penalty will
generally range from about 0.01 to 0.20 and in many instances will
be 0.10. The program has default parameters determined by the
sequences inputted to be compared. Preferably, the sequence
identity is determined using the default parameters determined by
the program. This program is available also from Genetics Computing
Group (GCG) package, from Madison, Wis., USA.
[0120] Another program of interest is the FastDB algorithm. FastDB
is described in Current Methods in Sequence Comparison and
Analysis, Macromolecule Sequencing and Synthesis, Selected Methods
and Applications, pp. 127-149, 1988, Alan R. Liss, Inc. Percent
sequence identity is calculated by FastDB based upon the following
parameters:
Mismatch Penalty: 1.00;
Gap Penalty: 1.00;
Gap Size Penalty: 0.33; and
Joining Penalty: 30.0.
Variant AAV2 Capsid Protein
[0121] In one embodiment, the vector comprises a variant AAV2
capsid protein. In this embodiment, the variant AAV2 capsid protein
comprises at least one amino acid substitution at one or more of
the following positions in an AAV2 capsid protein sequence: 125,
151, 162, 205, 312, 457, 492, 499, 533, 546, 548, 585, 588 and/or
593.
[0122] The sequence of wild type AAV2 capsid protein VP1 is known,
and is shown in FIG. 1 (SEQ ID NO:1). Wild type AAV2 capsid protein
sequences are also available from database accession no.s:
NC-001401; UniProt P03135; NCBI Reference Sequence: YP_680426.1;
GenBank: AAC03780.1.
[0123] Preferably the variant AAV2 capsid protein has at least 70%,
at least 80%, at least 90%, at least 95%, at least 96%, at least
97%, at least 98% or at least 99% sequence identity to SEQ ID NO:1.
In a preferred embodiment, the variant AAV2 capsid protein
comprises a sequence of SEQ ID NO:2, or a sequence having at least
70%, at least 80%, at least 90%, at least 95%, at least 96%, at
least 97%, at least 98% or at least 99% sequence identity
thereto.
[0124] In one embodiment, the variant AAV2 capsid protein comprises
one or more of the following residues: I125, A151, S162, S205,
S312, M457, A492, D499, Y533, D546, G548, S585, T588 and/or S593.
In a preferred embodiment, the variant AAV2 capsid protein
comprises one or more of the following amino acid substitutions
with respect to a wild type AAV2 capsid protein: V125I, V151A,
A162S, T205S, N312S, Q457M, S492A, E499D, F533Y, G546D, E548G,
R585S, R588T and/or A593S.
Combinations of Mutations in AAV2 Capsid Protein
[0125] The variant AAV2 capsid protein may comprise any combination
of the above amino acid substitutions. Therefore in particular
embodiments, the variant AAV2 capsid protein comprises 1, 2, 3, 4,
5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 amino acid substitutions
selected from the list above. In one embodiment, the variant AAV2
capsid protein comprises all 14 amino acid substitutions disclosed
above, e.g. the variant AAV2 capsid protein comprises a sequence of
SEQ ID NO:2 (i.e. ttAAV2 or AAV2-TT as referred to herein).
[0126] In further embodiments, the variant AAV2 capsid protein may
comprise a sub-set of the above 14 mutations. Without being bound
by theory, in individual embodiments, the variant AAV2 capsid
protein may comprise the following residues, which are divided
below into functional groups:
1) S585 and/or T588; these residues may be associated with
decreased heparin binding and increased spread of the virus in
heparin sulphate proteoglycan-rich brain tissue; 2) S312; this
internal serine residue may play a role in capsid-DNA interactions;
3) D546 and/or G548; these residues may be involved in interactions
with neutralizing antibodies and thus contribute to in vivo
transduction characteristics; 4) S593; this residue is located in
the groove between threefold-proximal spikes; 5) M457, A492, D499
and/or Y533; these four amino acids may be involved in receptor
binding and are closely situated on the threefold spikes; 6) I125,
A151, S162 and/or S205; these residues may be associated with PLA2
activity and/or trafficking of the incoming virus.
[0127] It will be appreciated that also contemplated herein are
corresponding sub-groups comprising mutations corresponding to the
above residues when present at corresponding positions in further
AAV serotypes (see below).
[0128] In preferred embodiments, the variant AAV2 capsid protein
comprises four or more mutations at the positions mentioned above
which may be associated with receptor binding, i.e. residues 457,
492, 499 and 533. Thus it is particularly preferred that the
variant AAV2 capsid protein comprises the following residues M457,
A492, D499 and Y533.
[0129] In some preferred embodiments, the variant AAV2 capsid
protein is not mutated with respect to the wild type AAV2 capsid
protein at position 312, e.g. the variant AAV2 capsid protein
comprises the residue N312 (which is present in the wild type AAV2
capsid protein). Thus in some embodiments, the variant AAV2 capsid
protein may comprise 1 to 13 of the specific mutations mentioned
above, but not the mutation N312S.
Variant AAV Capsid Proteins from Other Serotypes
[0130] In further embodiments, the variant AAV capsid protein is
from an alternative AAV serotype, i.e. an AAV serotype other than
AAV2. For instance, the variant AAV capsid protein may be derived
from an AAV1, AAV3B, AAV-LK03, AAV5, AAV6, AAV8, AAV9 or AAV10
(e.g. AAVrh10) capsid protein.
[0131] In these embodiments, the variant AAV capsid protein
comprises at least one amino acid substitution at one or more
positions corresponding to those described above with respect to
AAV2. In other words, the variant AAV capsid protein comprises at
least one amino acid substitution at a position in an alternative
(i.e. non-AAV2) AAV capsid protein sequence which corresponds to
positions 125, 151, 162, 205, 312, 457, 492, 499, 533, 546, 548,
585, 588 and/or 593 in an AAV2 capsid protein sequence.
[0132] Those skilled in the art would know, based on a comparison
of the amino acid sequences of capsid proteins of various AAV
serotypes, how to identify positions in capsid proteins from
alternative AAV serotypes which correspond to positions 125, 151,
162, 205, 312, 457, 492, 499, 533, 546, 548, 585, 588 and/or 593 in
an AAV2 capsid protein. In particular, such positions can easily be
identified by sequence alignments as known in the art and described
herein. For instance, one such sequence alignment is provided in
FIG. 9.
[0133] Of particular relevance in this context are positions in
alternative AAV capsid protein sequences which correspond in
three-dimensional space to positions 125, 151, 162, 205, 312, 457,
492, 499, 533, 546, 548, 585, 588 and/or 593 in an AAV2 capsid
protein. Methods for three-dimensional modelling and alignment of
protein structures are well known in the art, and can be used to
identify such corresponding positions in non-AAV2 capsid protein
sequences. Exemplary 3D alignments of AAV2 capsid protein sequences
with capsid protein sequences of alternative AAV serotypes (e.g.
AAV1, AAV5, AAV6, AAV8 and AAV9) are shown in FIGS. 21 to 25 and
discussed below. A skilled person can perform similar 3D alignments
with capsid proteins from further serotypes, e.g. AAV2, AAV3, AAV7,
AAV10 and AAV12), and identify positions in such sequences which
correspond with to the positions defined above in AAV2.
Variant AAV1 Capsid Protein
[0134] In one embodiment, the vector comprises a variant AAV1
capsid protein. In this embodiment, the variant AAV1 capsid protein
comprises at least one amino acid substitution at one or more of
the following positions in the AAV1 capsid protein sequence: 125,
151, 162, 205, 313, 458, 493, 500, 534, 547, 549, 586, 589 and/or
594. These positions in AAV1 capsid protein VP1 correspond to those
disclosed above in relation to AAV2.
[0135] The sequence of wild type AAV1 capsid protein VP1 is known,
and is shown in FIG. 3 (SEQ ID NO:3). A wild type AAV1 capsid
protein sequences is also available from database accession no.:
NC-002077. Preferably the variant AAV1 capsid protein has at least
70%, at least 80%, at least 90%, at least 95%, at least 96%, at
least 97%, at least 98% or at least 99% sequence identity to SEQ ID
NO:3.
[0136] Wild type AAV1 capsid protein VP1 already contains the
following residues at positions which correspond to amino acid
residues which are present in the variant AAV2 capsid protein
disclosed above (SEQ ID NO:2, ttAAV2), but not wild type AAV2 (SEQ
ID NO:1): S205 (aligns with S205 in ttAAV2); G549 (aligns with G548
in ttAAV2); S586 (aligns with S585 in ttAAV2); and T589 (aligns
with T588 in ttAAV2). Accordingly, in a preferred embodiment, the
variant AAV1 capsid protein comprises one or more of the following
amino acid substitutions with respect to a wild type AAV1 capsid
protein: V125I, Q151A, T162S, N313S, N458M, K493A, N500D, F534Y,
S547D, and/or G594S. Typically such a variant AAV1 capsid protein
may share one or more functional properties with the variant AAV2
capsid protein (SEQ ID NO:2, ttAAV2), e.g. may confer increased
infectivity and/or transduction of neuronal of retinal tissue
compared to wild type AAV1 capsid protein.
[0137] In alternative embodiments, the variant AAV1 capsid protein
comprises one or more amino acid substitutions which correspond to
reversions of mutations present in ttAAV2 back to the wild type
AAV2 sequence. For instance, the variant AAV1 capsid protein may
comprise one or more of the following substitutions: S205T, G549E,
S586R and/or T589R. Typically such a variant AAV1 capsid protein
may share one or more functional properties with the wild type AAV2
capsid protein (SEQ ID NO:1), e.g. may confer reduced infectivity
and/or transduction of neuronal of retinal tissue compared to wild
type AAV1 capsid protein.
Variant AAV5 Capsid Protein
[0138] In one embodiment, the vector comprises a variant AAV5
capsid protein. In this embodiment, the variant AAV5 capsid protein
comprises at least one amino acid substitution at one or more of
the following positions in the AAV5 capsid protein sequence: 124,
150, 153, 195, 303, 444, 479, 486, 520, 533, 537, 575, 578 and/or
583. These positions in AAV5 capsid protein VP1 correspond to those
disclosed above in relation to AAV2.
[0139] The sequence of wild type AAV5 capsid protein VP1 is known,
and is shown in FIG. 4 (SEQ ID NO:4). A wild type AAV5 capsid
protein sequences is also available from database accession no.:
AF085716. Preferably the variant AAV5 capsid protein has at least
70%, at least 80%, at least 90%, at least 95%, at least 96%, at
least 97%, at least 98% or at least 99% sequence identity to SEQ ID
NO:4.
[0140] Wild type AAV5 capsid protein VP1 already contains the
following residues at positions which correspond to amino acid
residues which are present in the variant AAV2 capsid protein
disclosed above (SEQ ID NO:2, ttAAV2), but not wild type AAV2 (SEQ
ID NO:1): G537 (aligns with G548 in ttAAV2); S575 (aligns with S585
in ttAAV2); T578 (aligns with T588 in ttAAV2). Accordingly, in a
preferred embodiment, the variant AAV5 capsid protein comprises one
or more of the following amino acid substitutions with respect to a
wild type AAV5 capsid protein: V124I, K150A, K153S, A195S, R303S,
T444M, S479A, V486D, T520Y, P533D, and/or G583S. Typically such a
variant AAV5 capsid protein may share one or more functional
properties with the variant AAV2 capsid protein (SEQ ID NO:2,
ttAAV2), e.g. may confer increased infectivity and/or transduction
of neuronal of retinal tissue compared to wild type AAV5 capsid
protein.
[0141] In alternative embodiments, the variant AAV5 capsid protein
comprises one or more amino acid substitutions which correspond to
reversions of mutations present in ttAAV2 back to the wild type
AAV2 sequence. For instance, the variant AAV5 capsid protein may
comprise one or more of the following substitutions: G537E, S575R
and/or T578R. Typically such a variant AAV5 capsid protein may
share one or more functional properties with the wild type AAV2
capsid protein (SEQ ID NO:1), e.g. may confer reduced infectivity
and/or transduction of neuronal of retinal tissue compared to wild
type AAV5 capsid protein.
Variant AAV6 Capsid Protein
[0142] In one embodiment, the vector comprises a variant AAV6
capsid protein. In this embodiment, the variant AAV6 capsid protein
comprises at least one amino acid substitution at one or more of
the following positions in the AAV6 capsid protein sequence: 125,
151, 162, 205, 313, 458, 493, 500, 534, 547, 549, 586, 589 and/or
594. These positions in AAV6 capsid protein VP1 correspond to those
disclosed above in relation to AAV2.
[0143] The sequence of wild type AAV6 capsid protein VP1 is known,
and is shown in FIG. 5 (SEQ ID NO:5). A wild type AAV6 capsid
protein sequences is also available from database accession no.:
AF028704. Preferably the variant AAV6 capsid protein has at least
70%, at least 80%, at least 90%, at least 95%, at least 96%, at
least 97%, at least 98% or at least 99% sequence identity to SEQ ID
NO:5.
[0144] Wild type AAV6 capsid protein VP1 already contains the
following residues at positions which correspond to amino acid
residues which are present in the variant AAV2 capsid protein
disclosed above (SEQ ID NO:2, ttAAV2), but not wild type AAV2 (SEQ
ID NO:1): S205 (aligns with S205 in ttAAV2); G549 (aligns with G548
in ttAAV2); S586 (aligns with S585 in ttAAV2); T589 (aligns with
T588 in ttAAV2). Accordingly, in a preferred embodiment, the
variant AAV6 capsid protein comprises one or more of the following
amino acid substitutions with respect to a wild type AAV6 capsid
protein: V125I, Q151A, T162S, N313S, N458M, K493A, N500D, F534Y,
S547D, and/or G594S. Typically such a variant AAV6 capsid protein
may share one or more functional properties with the variant AAV2
capsid protein (SEQ ID NO:2, ttAAV2), e.g. may confer increased
infectivity and/or transduction of neuronal of retinal tissue
compared to wild type AAV6 capsid protein.
[0145] In alternative embodiments, the variant AAV6 capsid protein
comprises one or more amino acid substitutions which correspond to
reversions of mutations present in ttAAV2 back to the wild type
AAV2 sequence. For instance, the variant AAV6 capsid protein may
comprise one or more of the following substitutions: S205T, G549E,
S586R and/or T589R. Typically such a variant AAV6 capsid protein
may share one or more functional properties with the wild type AAV2
capsid protein (SEQ ID NO:1), e.g. may confer reduced infectivity
and/or transduction of neuronal of retinal tissue compared to wild
type AAV6 capsid protein.
Variant AAV8 Capsid Protein
[0146] In one embodiment, the vector comprises a variant AAV8
capsid protein. In this embodiment, the variant AAV8 capsid protein
comprises at least one amino acid substitution at one or more of
the following positions in the AAV8 capsid protein sequence: 125,
151, 163, 206, 315, 460, 495, 502, 536, 549, 551, 588, 591 and/or
596. These positions in AAV8 capsid protein VP1 correspond to those
disclosed above in relation to AAV2.
[0147] The sequence of wild type AAV8 capsid protein VP1 is known,
and is shown in FIG. 6 (SEQ ID NO:6). A wild type AAV8 capsid
protein sequences is also available from database accession no.:
NC_006261. Preferably the variant AAV8 capsid protein has at least
70%, at least 80%, at least 90%, at least 95%, at least 96%, at
least 97%, at least 98% or at least 99% sequence identity to SEQ ID
NO:6.
[0148] Wild type AAV8 capsid protein VP1 already contains the
following residues at positions which correspond to amino acid
residues which are present in the variant AAV2 capsid protein
disclosed above (SEQ ID NO:2, ttAAV2), but not wild type AAV2 (SEQ
ID NO:1): S315 (aligns with S312 in ttAAV2); T591 (aligns with T588
in ttAAV2). Accordingly, in a preferred embodiment, the variant
AAV8 capsid protein comprises one or more of the following amino
acid substitutions with respect to a wild type AAV8 capsid protein:
V125I, Q151A, K163S, A206S, T460M, T495A, N502D, F536Y, N549D,
A551G, Q588S and/or G596S. Typically such a variant AAV8 capsid
protein may share one or more functional properties with the
variant AAV2 capsid protein (SEQ ID NO:2, ttAAV2), e.g. may confer
increased infectivity and/or transduction of neuronal of retinal
tissue compared to wild type AAV8 capsid protein.
[0149] In alternative embodiments, the variant AAV8 capsid protein
comprises one or more amino acid substitutions which correspond to
reversions of mutations present in ttAAV2 back to the wild type
AAV2 sequence. For instance, the variant AAV8 capsid protein may
comprise one or more of the following substitutions: S315N and/or
T591R. Typically such a variant AAV8 capsid protein may share one
or more functional properties with the wild type AAV2 capsid
protein (SEQ ID NO:1), e.g. may confer reduced infectivity and/or
transduction of neuronal of retinal tissue compared to wild type
AAV8 capsid protein.
[0150] In one embodiment, the variant AAV8 capsid protein comprises
an amino acid substitution with respect to a wild type AAV8 capsid
protein at position 315 in an AAV8 capsid protein sequence. For
instance, the variant AAV8 capsid protein may comprise the residue
N315. Thus in one embodiment the variant AAV8 capsid protein
comprises the amino acid substitution S315N with respect to a wild
type AAV8 capsid protein.
Variant AAV9 Capsid Protein
[0151] In one embodiment, the vector comprises a variant AAV9
capsid protein. In this embodiment, the variant AAV9 capsid protein
comprises at least one amino acid substitution at one or more of
the following positions in the AAV9 capsid protein sequence: 125,
151, 162, 205, 314, 458, 493, 500, 534, 547, 549, 586, 589 and/or
594. These positions in AAV9 capsid protein VP1 correspond to those
disclosed above in relation to AAV2.
[0152] The sequence of wild type AAV9 capsid protein VP1 is known,
and is shown in FIG. 7 (SEQ ID NO:7). A wild type AAV9 capsid
protein sequences is also available from database accession no.:
AY530579. Preferably the variant AAV9 capsid protein has at least
70%, at least 80%, at least 90%, at least 95%, at least 96%, at
least 97%, at least 98% or at least 99% sequence identity to SEQ ID
NO:7.
[0153] Wild type AAV9 capsid protein VP1 already contains the
following residues at positions which correspond to amino acid
residues which are present in the variant AAV2 capsid protein
disclosed above (SEQ ID NO:2, ttAAV2), but not wild type AAV2 (SEQ
ID NO:1): S162 (aligns with S162 in ttAAV2); S205 (aligns with S205
in ttAAV2); G549 (aligns with G548 in ttAAV2); S586 (aligns with
S585 in ttAAV2). Accordingly, in a preferred embodiment, the
variant AAV9 capsid protein comprises one or more of the following
amino acid substitutions with respect to a wild type AAV9 capsid
protein: L125I, Q151A, N314S, Q458M, V493A, E500D, F534Y, G547D,
A589T and/or G594S. Typically such a variant AAV9 capsid protein
may share one or more functional properties with the variant AAV2
capsid protein (SEQ ID NO:2, ttAAV2), e.g. may confer increased
infectivity and/or transduction of neuronal of retinal tissue
compared to wild type AAV9 capsid protein.
[0154] In alternative embodiments, the variant AAV9 capsid protein
comprises one or more amino acid substitutions which correspond to
reversions of mutations present in ttAAV2 back to the wild type
AAV2 sequence. For instance, the variant AAV9 capsid protein may
comprise one or more of the following substitutions: S162A, S205T,
G549E and/or S586R. Typically such a variant AAV9 capsid protein
may share one or more functional properties with the wild type AAV2
capsid protein (SEQ ID NO:1), e.g. may confer reduced infectivity
and/or transduction of neuronal of retinal tissue compared to wild
type AAV9 capsid protein.
Variant AAV10 Capsid Protein
[0155] In one embodiment, the vector comprises a variant AAV10
capsid protein. As used herein, "AAV10" includes AAVrh10. In this
embodiment, the variant AAV10 (e.g. AAVrh10) capsid protein
comprises at least one amino acid substitution at one or more of
the following positions in the AAV10 capsid protein sequence: 125,
151, 163, 206, 315, 460, 495, 502, 536, 549, 551, 588, 591 and/or
596. These positions in AAV10 capsid protein VP1 correspond to
those disclosed above in relation to AAV2.
[0156] The sequence of wild type AAV10 capsid protein VP1 is known,
and is shown in FIG. 8 (SEQ ID NO:8). Preferably the variant AAV10
capsid protein has at least 70%, at least 80%, at least 90%, at
least 95%, at least 96%, at least 97%, at least 98% or at least 99%
sequence identity to SEQ ID NO:8.
[0157] Wild type AAV10 capsid protein VP1 already contains the
following residue at a position which corresponds to an amino acid
residue which is present in the variant AAV2 capsid protein
disclosed above (SEQ ID NO:2, ttAAV2), but not wild type AAV2 (SEQ
ID NO:1): G551 (aligns with G548 in ttAAV2). Accordingly, in a
preferred embodiment, the variant AAV10 capsid protein comprises
one or more of the following amino acid substitutions with respect
to a wild type AAV10 capsid protein: V125I, Q151A, K163S, A206S,
N315S, T460M, L495A, N502D, F536Y, G549D, Q588S, A591T and/or
G596S. Typically such a variant AAV10 capsid protein may share one
or more functional properties with the variant AAV2 capsid protein
(SEQ ID NO:2, ttAAV2), e.g. may confer increased infectivity and/or
transduction of neuronal of retinal tissue compared to wild type
AAV10 capsid protein.
[0158] In alternative embodiments, the variant AAV10 capsid protein
comprises an amino acid substitution which corresponds to a
reversion of a mutations present in ttAAV2 back to the wild type
AAV2 sequence. For instance, the variant AAV10 capsid protein may
comprise the following substitution: G551E. Typically such a
variant AAV10 capsid protein may share one or more functional
properties with the wild type AAV2 capsid protein (SEQ ID NO:1),
e.g. may confer reduced infectivity and/or transduction of neuronal
of retinal tissue compared to wild type AAV10 capsid protein.
Variant AAV3B Capsid Protein
[0159] In one embodiment, the vector comprises a variant AAV3B
capsid protein. In this embodiment, the variant AAV3B capsid
protein may comprise an amino acid substitution with respect to a
wild type AAV3B capsid protein at position 312. For instance, the
variant AAV3B capsid protein may comprise the residue N312. Thus in
one embodiment the variant AAV8 capsid protein comprises the amino
acid substitution S312N with respect to a wild type AAV8 capsid
protein. In further embodiments, the variant AAV3B capsid protein
may comprise one or more additional mutations at positions which
correspond to those disclosed above in relation to AAV2.
[0160] The sequence of wild type AAV3B capsid protein VP1 is known,
and is shown in FIG. 38 (SEQ ID NO:11). A wild type AAV3B capsid
protein sequence is also available from NCBI database accession no.
AF028705. Preferably the variant AAV3B capsid protein has at least
70%, at least 80%, at least 90%, at least 95%, at least 96%, at
least 97%, at least 98% or at least 99% sequence identity to SEQ ID
NO:11.
Variant AAV-LK03 Capsid Protein
[0161] In one embodiment, the vector comprises a variant AAV-LK03
capsid protein. In this embodiment, the variant AAV-LK03 capsid
protein may comprise an amino acid substitution at position 312
with respect to a AAV-LK03 capsid protein sequence as defined in
SEQ ID NO:12. For instance, the variant AAV-LK03 capsid protein may
comprise the residue N312. Thus in one embodiment the variant
AAV-LK03 capsid protein comprises the amino acid substitution S312N
with respect to a AAV-LK03 capsid protein sequence as defined in
SEQ ID NO:12. In further embodiments, the variant AAV-LK03 capsid
protein may comprise one or more additional mutations at positions
which correspond to those disclosed above in relation to AAV2.
[0162] The sequence of wild type AAV-LK03 capsid protein VP1 is
known, and is shown in FIG. 39 (SEQ ID NO:12). A AAV-LK03 capsid
protein sequence is also disclosed in WO 2013/029030 as sequence
number 31 therein. Preferably the variant AAV-LK03 capsid protein
has at least 70%, at least 80%, at least 90%, at least 95%, at
least 96%, at least 97%, at least 98% or at least 99% sequence
identity to SEQ ID NO:12.
Gene Products
[0163] In one embodiment, the rAAV further comprises a heterologous
nucleic acid comprising a nucleotide sequence encoding a gene
product. A "gene" refers to a polynucleotide containing at least
one open reading frame that is capable of encoding a particular
protein after being transcribed and translated. A "gene product" is
a molecule resulting from expression of a particular gene. Gene
products include, e.g., a polypeptide, an aptamer, an interfering
RNA, an mRNA, and the like.
[0164] "Heterologous" means derived from a genotypically distinct
entity from that of the rest of the entity to which it is being
compared. For example, a polynucleotide introduced by genetic
engineering techniques into a plasmid or vector derived from a
different species is a heterologous polynucleotide. A promoter
removed from its native coding sequence and operatively linked to a
coding sequence with which it is not naturally found linked is a
heterologous promoter. Thus, for example, an rAAV that includes a
heterologous nucleic acid encoding a heterologous gene product is
an rAAV that includes a nucleic acid not normally included in a
naturally-occurring, wild-type AAV, and the encoded heterologous
gene product is a gene product not normally encoded by a
naturally-occurring, wild-type AAV.
[0165] In some embodiments, the gene product is an interfering RNA.
In some embodiments, the gene product is an aptamer. In some
embodiments, the gene product is a polypeptide.
Interfering RNA
[0166] Where the gene product is an interfering RNA (RNAi),
suitable RNAi include RNAi that decrease the level of an apoptotic
or angiogenic factor in a cell. For example, an RNAi can be an
shRNA or siRNA that reduces the level of a gene product that
induces or promotes apoptosis in a cell. Genes whose gene products
induce or promote apoptosis are referred to herein as
"pro-apoptotic genes" and the products of those genes (mRNA;
protein) are referred to as "pro-apoptotic gene products."
Pro-apoptotic gene products include, e.g., Bax, Bid, Bak, and Bad
gene products. See, e.g., U.S. Pat. No. 7,846,730.
[0167] Interfering RNAs could also be against an angiogenic
product, for example VEGF (e.g., Cand5; see, e.g., U.S. Patent
Publication No. 2011/0143400; U.S. Patent Publication No.
2008/0188437; and Reich et al. (2003) Mol. Vis. 9:210), VEGFR1
(e.g., Sirna-027; see, e.g., Kaiser et al. (2010) Am. J.
Ophthalmol. 150:33; and Shen et al. (2006) Gene Ther. 13:225), or
VEGFR2 (Kou et al. (2005) Biochem. 44: 15064). See also, U.S. Pat.
Nos. 6,649,596, 6,399,586, 5,661,135, 5,639,872, and 5,639,736; and
7,947,659 and 7,919,473.
[0168] A "small interfering" or "short interfering RNA" or siRNA is
a RNA duplex of nucleotides that is targeted to a gene interest (a
"target gene"). An "RNA duplex" refers to the structure formed by
the complementary pairing between two regions of a RNA molecule.
siRNA is "targeted" to a gene in that the nucleotide sequence of
the duplex portion of the siRNA is complementary to a nucleotide
sequence of the targeted gene. In some embodiments, the length of
the duplex of siRNAs is less than 30 nucleotides. In some
embodiments, the duplex can be 29, 28, 27, 26, 25, 24, 23, 22, 21,
20, 19, 18, 17, 16, 15, 14, 13, 12, 11 or 10 nucleotides in length.
In some embodiments, the length of the duplex is 19-25 nucleotides
in length. The RNA duplex portion of the siRNA can be part of a
hairpin structure. In addition to the duplex portion, the hairpin
structure may contain a loop portion positioned between the two
sequences that form the duplex. The loop can vary in length. In
some embodiments the loop is 5, 6, 7, 8, 9, 10, 11, 12 or 13
nucleotides in length. The hairpin structure can also contain 3' or
5' overhang portions. In some embodiments, the overhang is a 3' or
a 5' overhang 0, 1, 2, 3, 4 or 5 nucleotides in length.
[0169] A "short hairpin RNA," or shRNA, is a polynucleotide
construct that can be made to express an interfering RNA such as
siRNA.
Aptamers
[0170] Where the gene product is an aptamer, exemplary aptamers of
interest include an aptamer against vascular endothelial growth
factor (VEGF). See, e.g., Ng et al. (2006) Nat. Rev. Drug Discovery
5: 123; and Lee et al. (2005) Proc. Natl. Acad. Sci. USA 102:
18902. Also suitable for use is a PDGF-specific aptamer, e.g.,
E10030; see, e.g., Ni and Hui (2009) Ophthalmologica 223:401; and
Akiyama et al. (2006) J. Cell Physiol. 207:407).
Polypeptides
[0171] In one embodiment, the gene product is a therapeutic
protein. A "therapeutic" peptide or protein is a peptide or protein
that may alleviate or reduce symptoms that result from an absence
or defect in a protein in a cell or subject. Alternatively, a
"therapeutic" peptide or protein is one that otherwise confers a
benefit to a subject, e.g., anti-degenerative effects.
[0172] Where the gene product is a polypeptide, the polypeptide is
generally a polypeptide that enhances function of a cell, for
example a cell present in neuronal, retinal or liver tissue, e.g.,
a hepatocyte, a neuron, a glial cell, a rod or cone photoreceptor
cell, a retinal ganglion cell, a Muller cell, a bipolar cell, an
amacrine cell, a horizontal cell, or a retinal pigmented epithelial
cell.
[0173] Exemplary polypeptides include neuroprotective polypeptides
(e.g., GDNF, CNTF, NT4, NGF, and NTN); anti-angiogenic polypeptides
(e.g., a soluble vascular endothelial growth factor (VEGF)
receptor; a VEGF-binding antibody; a VEGF-binding antibody fragment
(e.g., a single chain anti-VEGF antibody); endostatin; tumstatin;
angiostatin; a soluble Fit polypeptide (Lai et al. (2005) Mol.
Ther. 12:659); an Fc fusion protein comprising a soluble Fit
polypeptide (see, e.g., Pechan et al. (2009) Gene Ther. 16: 10);
pigment epithelium-derived factor (PEDF); a soluble Tie-2 receptor;
etc.); tissue inhibitor of metalloproteinases-3 (TIMP-3); a
light-responsive opsin, e.g., a rhodopsin; anti-apoptotic
polypeptides (e.g., Bcl-2, Bcl-X1); and the like. Suitable
polypeptides include, but are not limited to, glial derived
neurotrophic factor (GDNF); fibroblast growth factor 2; neurturin
(NTN); ciliary neurotrophic factor (CNTF); nerve growth factor
(NGF); neurotrophin-4 (NT4); brain derived neurotrophic factor
(BDNF); epidermal growth factor; rhodopsin; X-linked inhibitor of
apoptosis; and Sonic hedgehog. Suitable polypeptides are disclosed,
for example, in WO WO 2012/145601.
[0174] Exemplary polypeptides for gene deliver to the liver
include, for example, PBGD (porphobilinogen deaminase) IDUA
(iduronidase) Fah (fumarylacetoacetate hydrolyase) A1AT
(alpha(1)-antitrypsin), 1A1 (hUGT1A1) (uridine disphoshate
glucuronyltransferase), HCCS1 (hepatocellular carcinoma suppressor
1), CD (cytosine deaminase), SOCS3 (suppressor of cytokine
signaling 3), TNF (tumor necrosis factor), thymidine kinase, IL-24
(interleukin-24), IL-12 (interleukin-12), and TRAIL (tumor necrosis
factor-related apoptosis-inducing ligand).
Regulatory Sequences
[0175] In some embodiments, a nucleotide sequence encoding a gene
product of interest is operably linked to a constitutive promoter.
In other embodiments, a nucleotide sequence encoding a gene product
of interest is operably linked to an inducible promoter. In some
instances, a nucleotide sequence encoding a gene product of
interest is operably linked to a tissue specific or cell type
specific regulatory element.
[0176] For example, in some instances, a nucleotide sequence
encoding a gene product of interest is operably linked to a
hepatocyte-specific, neuron-specific or photoreceptor-specific
regulatory element (e.g., a photoreceptor-specific promoter), e.g.,
a regulatory element that confers selective expression of the
operably linked gene in a neuron or photoreceptor cell. Suitable
photoreceptor-specific regulatory elements include, e.g., a
rhodopsin promoter; a rhodopsin kinase promoter (Young et al.
(2003) Ophthalmol. Vis. Sci. 44:4076); a beta phosphodiesterase
gene promoter (Nicoud et al. (2007) J. Gene Med. 9: 1015); a
retinitis pigmentosa gene promoter (Nicoud et al. (2007) supra); an
interphotoreceptor retinoid-binding protein (IRBP) gene enhancer
(Nicoud et al. (2007) supra); an IRBP gene promoter (Yokoyama et
al. (1992) Exp Eye Res. 55:225). Suitable neuronal-specific
promoters include neuron-specific enolase (NSE) promoter, Andersen
et al. Cell. Mol. Neurobiol., 13:503-15 (1993; neurofilament
light-chain gene promoter, Piccioli et al., Proc. Natl. Acad. Sci.
USA, 88:561 1-5 (1991); and the neuron-specific vgf gene promoter,
Piccioli et al., Neuron, 15:373-84 (1995)]; among others. Suitable
hepatocyte-specific promoters include an albumin promoter (Heard et
al., Mol Cell Biol 1987; 7: 2425) or an alpha 1-antitrypsin
promoter (Hafenrichter et al. Blood 1994; 84, 3394-404).
[0177] A "control element" or "control sequence" is a nucleotide
sequence involved in an interaction of molecules that contributes
to the functional regulation of a polynucleotide, including
replication, duplication, transcription, splicing, translation, or
degradation of the polynucleotide. The regulation may affect the
frequency, speed, or specificity of the process, and may be
enhancing or inhibitory in nature. Control elements known in the
art include, for example, transcriptional regulatory sequences such
as promoters and enhancers. A promoter is a DNA region capable
under certain conditions of binding RNA polymerase and initiating
transcription of a coding region usually located downstream (in the
3' direction) from the promoter.
[0178] "Operatively linked" or "operably linked" refers to a
juxtaposition of genetic elements, wherein the elements are in a
relationship permitting them to operate in the expected manner. For
instance, a promoter is operatively linked to a coding region if
the promoter helps initiate transcription of the coding sequence.
There may be intervening residues between the promoter and coding
region so long as this functional relationship is maintained.
[0179] The term "promoters" or "promoter" as used herein can refer
to a DNA sequence that is located adjacent to a DNA sequence that
encodes a recombinant product. A promoter is preferably linked
operatively to an adjacent DNA sequence. A promoter typically
increases an amount of recombinant product expressed from a DNA
sequence as compared to an amount of the expressed recombinant
product when no promoter exists. A promoter from one organism can
be utilized to enhance recombinant product expression from a DNA
sequence that originates from another organism. For example, a
vertebrate promoter may be used for the expression of jellyfish GFP
in vertebrates. In addition, one promoter element can increase an
amount of recombinant products expressed for multiple DNA sequences
attached in tandem. Hence, one promoter element can enhance the
expression of one or more recombinant products. Multiple promoter
elements are well-known to persons of ordinary skill in the
art.
[0180] The term "enhancers" or "enhancer" as used herein can refer
to a DNA sequence that is located adjacent to the DNA sequence that
encodes a recombinant product. Enhancer elements are typically
located upstream of a promoter element or can be located downstream
of or within a coding DNA sequence (e.g., a DNA sequence
transcribed or translated into a recombinant product or products).
Hence, an enhancer element can be located 100 base pairs, 200 base
pairs, or 300 or more base pairs upstream or downstream of a DNA
sequence that encodes recombinant product. Enhancer elements can
increase an amount of recombinant product expressed from a DNA
sequence above increased expression afforded by a promoter element.
Multiple enhancer elements are readily available to persons of
ordinary skill in the art.
Pharmaceutical Compositions
[0181] The present disclosure provides a pharmaceutical composition
comprising: a) a rAAV vector, as described above; and b) a
pharmaceutically acceptable carrier, diluent, excipient, or buffer.
In some embodiments, the pharmaceutically acceptable carrier,
diluent, excipient, or buffer is suitable for use in a human.
[0182] Such excipients, carriers, diluents, and buffers include any
pharmaceutical agent that can be administered without undue
toxicity. Pharmaceutically acceptable excipients include, but are
not limited to, liquids such as water, saline, glycerol and
ethanol.
[0183] Pharmaceutically acceptable salts can be included therein,
for example, mineral acid salts such as hydrochlorides,
hydrobromides, phosphates, sulfates, and the like; and the salts of
organic acids such as acetates, propionates, malonates, benzoates,
and the like. Additionally, auxiliary substances, such as wetting
or emulsifying agents, pH buffering substances, and the like, may
be present in such vehicles. A wide variety of pharmaceutically
acceptable excipients are known in the art and need not be
discussed in detail herein. Pharmaceutically acceptable excipients
have been amply described in a variety of publications, including,
for example, A. Gennaro (2000) "Remington: The Science and Practice
of Pharmacy," 20th edition, Lippincott, Williams, & Wilkins;
Pharmaceutical Dosage Forms and Drug Delivery Systems (1999) H. C.
Ansel et al., eds., 7(th) ed., Lippincott, Williams, & Wilkins;
and Handbook of Pharmaceutical Excipients (2000) A. H. Kibbe et
al., eds., 3 rd ed. Amer. Pharmaceutical Assoc.
[0184] In particular embodiments, the present invention provides a
pharmaceutical composition comprising a rAAV vector as described
above in a pharmaceutically-acceptable carrier or other medicinal
agents, pharmaceutical agents, carriers, adjuvants, diluents, etc.
For injection, the carrier will typically be a liquid. For other
methods of administration, the carrier may be either solid or
liquid, such as sterile, pyrogen-free water or sterile pyrogen-free
phosphate-buffered saline solution. For inhalation administration,
the carrier will be respirable, and will preferably be in solid or
liquid particulate form. As an injection medium, it is preferred to
use water that contains the additives usual for injection
solutions, such as stabilizing agents, salts or saline, and/or
buffers.
[0185] By "pharmaceutically acceptable" it is meant a material that
is not biologically or otherwise undesirable, e.g., the material
may be administered to a subject without causing any undesirable
biological effects. Thus, such a pharmaceutical composition may be
used, for example, in transfection of a cell ex vivo or in
administering a viral particle or cell directly to a subject.
Methods of Delivering a Gene Product to a Tissue or Cell (for
Example a Hepatic, Neuronal or Retinal Tissue or Cell) and
Treatment Methods
[0186] The methods of the present invention provide a means for
delivering heterologous nucleic acid sequences into a host tissue
or cell, including both dividing and non-dividing cells. The
vectors and other reagents, methods and pharmaceutical formulations
of the present invention are additionally useful in a method of
administering a protein or peptide to a subject in need thereof, as
a method of treatment or otherwise. In this manner, the protein or
peptide may thus be produced in vivo in the subject. The subject
may be in need of the protein or peptide because the subject has a
deficiency of the protein or peptide, or because the production of
the protein or peptide in the subject may impart some therapeutic
effect, as a method of treatment or otherwise, and as explained
further below.
[0187] As used herein, the terms "treatment," "treating," and the
like, refer to obtaining a desired pharmacologic and/or physiologic
effect. The effect may be prophylactic in terms of completely or
partially preventing a disease or symptom thereof and/or may be
therapeutic in terms of a partial or complete cure for a disease
and/or adverse effect attributable to the disease. "Treatment," as
used herein, covers any treatment of a disease in a mammal,
particularly in a human, and includes: (a) preventing the disease
from occurring in a subject which may be predisposed to the disease
or at risk of acquiring the disease but has not yet been diagnosed
as having it; (b) inhibiting the disease, i.e., arresting its
development; and (c) relieving the disease, i.e., causing
regression of the disease.
[0188] In general, the present invention may be employed to deliver
any foreign nucleic acid with a biological effect to treat or
ameliorate the symptoms associated with any disorder related to
gene expression in any organ, tissue or cell, especially those
associated with e.g. the liver, brain or eye. Illustrative disease
states include, but are not limited to: lysosomal storage disease,
acute intermittent porphyria, ornithine transcarbamylase
deficiency, alpha(1)-antitrypsin deficiency, acute liver failure,
Pompe disease, Tyrosinemia, Crigler-Najjar syndrome, hepatitis,
cirrhosis, hepatocellular carcinoma, AIDS, Alzheimer's disease,
Parkinson's disease, Huntington's disease, amyotrophic lateral
sclerosis, epilepsy, and other neurological disorders, cancer (e.g.
brain cancer), retinal degenerative diseases and other diseases of
the eye.
[0189] Gene transfer has substantial potential use in understanding
and providing therapy for disease states. There are a number of
inherited diseases in which defective genes are known and have been
cloned. In some cases, the function of these cloned genes is known.
In general, the above disease states fall into two classes:
deficiency states, usually of enzymes, which are generally
inherited in a recessive manner, and unbalanced states, at least
sometimes involving regulatory or structural proteins, which are
inherited in a dominant manner. For deficiency state diseases, gene
transfer could be used to bring a normal gene into affected tissues
for replacement therapy, as well as to create animal models for the
disease using antisense mutations. For unbalanced disease states,
gene transfer could be used to create a disease state in a model
system, which could then be used in efforts to counteract the
disease state. Thus the methods of the present invention permit the
treatment of genetic diseases. As used herein, a disease state is
treated by partially or wholly remedying the deficiency or
imbalance that causes the disease or makes it more severe. The use
of site-specific integration of nucleic sequences to cause
mutations or to correct defects is also possible.
[0190] In one aspect the present invention provides a method of
delivering a gene product to a tissue or cell (e.g. a hepatic,
neuronal or retinal tissue or cell) in a subject, the method
comprising administering to the subject a rAAV vector as described
above. The gene product can be a polypeptide or an interfering RNA
(e.g., an shRNA, an siRNA, and the like), or an aptamer, e.g. as
described above. The cell may, for example, be a blood cell, stem
cell, bone marrow (e.g. hematopoietic) cell, liver cell, cancer
cell, vascular cell, pancreatic cell, neural cell, glial cell,
ocular or retinal cell, epithelial or endothelial cell, dendritic
cell, fibroblast, lung cell, muscle cell, cardiac cell, intestinal
cell or renal cell. Similarly the tissue may, for example, be
selected from blood, bone marrow, muscle tissue (e.g. skeletal
muscle, cardiac muscle or smooth muscle including vascular smooth
muscle), central or peripheral nervous system tissue (e.g. brain,
neuronal tissue or retinal tissue), pancreatic tissue, liver
tissue, kidney tissue, lung tissue, intestinal tissue or heart
tissue.
[0191] Delivering a gene product to a retinal cell can provide for
treatment of a retinal disease. The retinal cell can be a
photoreceptor, a retinal ganglion cell, a Muller cell, a bipolar
cell, an amacrine cell, a horizontal cell, or a retinal pigmented
epithelial cell. In some cases, the retinal cell is a photoreceptor
cell, e.g., a rod or cone cell. Similarly, delivering a gene
product to a neuronal tissue or cell can provide for treatment of a
neurological disorder. The gene product may be delivered to various
cell types present in neuronal tissue, e.g. neurons or glial cells
(e.g. astrocytes, oligodendrocytes and so on). Delivering a gene
product to the liver may provide treatment for a hepatic disorder.
The gene product may be delivered to, for example, hepatocytes.
[0192] The present disclosure provides a method of treating a
disease (e.g. a hepatic, neurological or ocular disease), the
method comprising administering to an individual in need thereof an
effective amount of a rAAV vector as described above. A subject
rAAV vector can be administered via intracranial injection,
intracerebral injection, intraocular injection, by intravitreal
injection, retinal injection, sub-retinal injection, intravenous
injection or by any other convenient mode or route of
administration.
[0193] Further exemplary modes of administration include oral,
rectal, transmucosal, topical, transdermal, inhalation, parenteral
(e.g., intravenous, subcutaneous, intradermal, intramuscular, and
intraarticular) administration, and the like, as well as direct
tissue or organ injection, alternatively, intrathecal, direct
intramuscular, intraventricular, intravenous, intraperitoneal,
intranasal, or intraocular injections. Injectables can be prepared
in conventional forms, either as liquid solutions or suspensions,
solid forms suitable for solution or suspensions in liquid prior to
injection, or as emulsions. Alternatively, one may administer the
virus in a local rather than systemic manner, for example in a
depot or sustained-release formation.
[0194] Recombinant virus vectors are preferably administered to the
subject in an amount that is sufficient to result in infection (or
transduction) and expression of the heterologous nucleic acid
sequence in cells (e.g. liver, neuronal or retinal cells) of the
subject. Preferably the target cells are hepatocytes, neural cells
(including cells of the central and peripheral nervous systems, in
particular, brain cells) or retinal cells. In some cases, the
retinal cell is a photoreceptor cell (e.g., rods and/or cones). In
other cases, the retinal cell is an RGC cell. In other cases, the
retinal cell is an RPE cell. In other cases, retinal cells may
include amacrine cells, bipolar cells, and horizontal cells.
[0195] Preferably the vector is administered in a therapeutically
effective amount. A "therapeutically-effective" amount as used
herein is an amount of that is sufficient to alleviate (e.g.,
mitigate, decrease, reduce) at least one of the symptoms associated
with a disease state. Alternatively stated, a
"therapeutically-effective" amount is an amount that is sufficient
to provide some improvement in the condition of the subject. A
"therapeutically effective amount" will fall in a relatively broad
range that can be determined through experimentation and/or
clinical trials. For example, for in vivo injection, a
therapeutically effective dose will be on the order of from about
10.sup.6 to about 10.sup.15 of rAAV virions, e.g., from about
10.sup.8 to 10.sup.12 rAAV virions. For in vitro transduction, an
effective amount of rAAV virions to be delivered to cells will be
on the order of from about 10.sup.8 to about 10.sup.13 of the rAAV
virions. Other effective dosages can be readily established by one
of ordinary skill in the art through routine trials establishing
dose response curves.
[0196] In some embodiments, more than one administration (e.g.,
two, three, four or more administrations) may be employed to
achieve the desired level of gene expression over a period of
various intervals, e.g., daily, weekly, monthly, yearly, etc.
[0197] Neurological diseases which may be treated include any
disease associated with the brain or CNS, including psychiatric
diseases. Diseases of the brain fall into two general categories:
(a) pathologic processes such as infections, trauma and neoplasm;
and (b) diseases unique to the nervous system which include
diseases of myelin and degeneration of neurons. Disease from either
category may be treated. For example, the neurological disease may
be selected from neurodegenerative diseases such as Alzheimer's
Disease, Parkinson's Disease, amyotrophic lateral sclerosis (ALS),
spinal muscular atrophy and cerebella degeneration; schizophrenia;
epilepsy; ischemia-related disease and stroke; demyelinating
diseases such as multiple sclerosis, perivenous encephalitis,
leukodystrophies such as metachromatic leukodystrophy due to
deficiency of arylsulfatase A, Krabbe's disease due to deficiency
of gal actocerebroside beta-galactosidase, adrenoleukodystrophy and
adrenomyeloneuropathy; post-viral diseases such as progressive
multifocal leukoencephalopathy, acute disseminated
encephalomyelitis, acute necrotizing hemorrhagic leukoencephalitis;
mitochondrial encephalomyopathies; neurological cancers, such as
primary brain tumors including glioma, meningioma, neurinoma,
pituitary adenoma, medulloblastoma, craniopharyngioma, hemangioma,
epidermoid, sarcoma and intracranial metastasis from other tumor
sources; neurological infections or neurological inflammatory
conditions.
[0198] Ocular diseases that can be treated using a subject method
include, but are not limited to, acute macular neuroretinopathy;
Behcet's disease; choroidal neovascularization; diabetic uveitis;
histoplasmosis; macular degeneration, such as acute macular
degeneration, non-exudative age related macular degeneration and
exudative age related macular degeneration; edema, such as macular
edema, cystoid macular edema and diabetic macular edema; multifocal
choroiditis; ocular trauma which affects a posterior ocular site or
location; ocular tumors; retinal disorders, such as central retinal
vein occlusion, diabetic retinopathy (including proliferative
diabetic retinopathy), proliferative vitreoretinopathy (PVR),
retinal arterial occlusive disease, retinal detachment, uveitic
retinal disease; sympathetic opthalmia; Vogt Koyanagi-Harada (VKH)
syndrome; uveal diffusion; a posterior ocular condition caused by
or influenced by an ocular laser treatment; posterior ocular
conditions caused by or influenced by a photodynamic therapy;
photocoagulation, radiation retinopathy; epiretinal membrane
disorders; branch retinal vein occlusion; anterior ischemic optic
neuropathy; non-retinopathy diabetic retinal dysfunction;
retinoschisis; retinitis pigmentosa; glaucoma; Usher syndrome,
cone-rod dystrophy; Stargardt disease (fundus flavimaculatus);
inherited macular degeneration; chorioretinal degeneration; Leber
congenital amaurosis; congenital stationary night blindness;
choroideremia; Bardet-Biedl syndrome; macular telangiectasia;
Leber's hereditary optic neuropathy; retinopathy of prematurity;
and disorders of color vision, including achromatopsia, protanopia,
deuteranopia, and tritanopia.
[0199] Diseases of the liver which may be treated include, for
example, lysosomal storage diseases, e.g. acute intermittent
porphyria, ornithine transcarbamylase deficiency, Wilson's disease,
mucopolysaccharidoses (e.g. MPS type I or MPS type VI), Sly
syndrome, Pompe disease, tyrosinemia, alpha(1)-antitrypsin
deficiency, Crigler-Najjar syndrome; hepatitis A, B or C; liver
cirrhosis; liver cancer, e.g. hepatocellular carcinoma; or acute
liver failure.
[0200] The present invention finds use in both veterinary and
medical applications. Suitable subjects include both avians and
mammals, with mammals being preferred. The term "avian" as used
herein includes, but is not limited to, chickens, ducks, geese,
quail, turkeys and pheasants. The term "mammal" as used herein
includes, but is not limited to, humans, bovines, ovines, caprines,
equines, felines, canines, lagomorphs, etc. Human subjects are the
most preferred. Human subjects include fetal, neonatal, infant,
juvenile and adult subjects.
Transduction of Tissue (for Example Hepatic, Neuronal or Retinal
Tissue)
[0201] In some embodiments, the rAAV vectors disclosed herein
exhibit increased transduction of a tissue (e.g. hepatic, neuronal
and/or retinal tissues), e.g. compared to a corresponding AAV
vector (from the same serotype) comprising a wild type AAV capsid
protein. For example, the rAAV vector may exhibit at least 10%,
50%, 100%, 500% or 1000% increased infectivity, compared to the
infectivity by an AAV virion comprising the corresponding wild type
AAV capsid protein.
[0202] In further embodiments, the rAAV vectors disclosed herein
may selectively or specifically infect a tissue (e.g. hepatic,
neuronal or retinal tissues), e.g. show increased transduction of
hepatic, neuronal or retinal cells compared to other cell types.
For instance, the rAAV vector may exhibit at least 10%, 50%, 100%,
500% or 1000% increased infectivity of a particular cell type (e.g.
hepatic, neuronal or retinal cells), compared to another cell type
(e.g. non-hepatic, non-neuronal and/or non-retinal cells). For
instance, the rAAV vector may selectively infect hepatocytes,
neurons and/or photoreceptor cells compared to cells outside the
liver, brain and/or eye.
[0203] Where the recombinant AAV vector exhibits increased
transduction of a neuronal or retinal tissue, e.g. where the vector
is used to treat a neurological or ocular disorder, the vector
preferably comprises a variant AAV2 capsid protein.
[0204] Where the recombinant AAV vector exhibits increased
transduction of liver tissue, e.g. where the vector is used to
treat a hepatic disorder, the vector preferably comprises a variant
AAV3B, AAV-LK03 or AAV8 capsid protein.
Nucleic Acids and Host Cells
[0205] The present disclosure provides an isolated nucleic acid
comprising a nucleotide sequence that encodes a variant
adeno-associated virus (AAV) capsid protein as described above. The
isolated nucleic acid can be comprised in an AAV vector, e.g., a
recombinant AAV vector.
[0206] A recombinant AAV vector comprising such a variant AAV
capsid protein-encoding sequence can be used to generate a
recombinant AAV virion (i.e. a recombinant AAV vector particle).
Thus, the present disclosure provides a recombinant AAV vector
that, when introduced into a suitable cell, can provide for
production of a recombinant AAV virion.
[0207] The present invention further provides host cells, e.g.,
isolated (genetically modified) host cells, comprising a subject
nucleic acid. A subject host cell can be an isolated cell, e.g., a
cell in in vitro culture. A subject host cell is useful for
producing a subject rAAV virion, as described below. Where a
subject host cell is used to produce a subject rAAV virion, it is
referred to as a "packaging cell." In some embodiments, a subject
host cell is stably genetically modified with a subject nucleic
acid. In other embodiments, a subject host cell is transiently
genetically modified with a subject nucleic acid.
[0208] A subject nucleic acid is introduced stably or transiently
into a host cell, using established techniques, including, but not
limited to, electroporation, calcium phosphate precipitation,
liposome-mediated transfection, and the like. For stable
transformation, a subject nucleic acid will generally further
include a selectable marker, e.g., any of several well-known
selectable markers such as neomycin resistance, and the like.
[0209] A subject host cell is generated by introducing a subject
nucleic acid into any of a variety of cells, e.g., mammalian cells,
including, e.g., murine cells, and primate cells (e.g., human
cells). Suitable mammalian cells include, but are not limited to,
primary cells and cell lines, where suitable cell lines include,
but are not limited to, 293 cells, COS cells, HeLa cells, Vero
cells, 3T3 mouse fibroblasts, C3H10T1/2 fibroblasts, CHO cells, and
the like. Non-limiting examples of suitable host cells include,
e.g., HeLa cells (e.g., American Type Culture Collection (ATCC) No.
CCL-2), CHO cells (e.g., ATCC Nos. CRL9618, CCL61, CRL9096), 293
cells (e.g., ATCC No. CRL-1573), Vero cells, NIH 3T3 cells (e.g.,
ATCC No. CRL-1658), Huh-7 cells, BHK cells (e.g., ATCC No. CCL10),
PC12 cells (ATCC No. CRL1721), COS cells, COS-7 cells (ATCC No.
CRL1651), RATI cells, mouse L cells (ATCC No. CCLI.3), human
embryonic kidney (HEK) cells (ATCC No. CRL1573), HLHepG2 cells, and
the like. A subject host cell can also be made using a baculovirus
to infect insect cells such as Sf9 cells, which produce AAV (see,
e.g., U.S. Pat. No. 7,271,002; U.S. patent application Ser. No.
12/297,958).
[0210] In some embodiments, a subject genetically modified host
cell includes, in addition to a nucleic acid comprising a
nucleotide sequence encoding a variant AAV capsid protein, as
described above, a nucleic acid that comprises a nucleotide
sequence encoding one or more AAV rep proteins. In other
embodiments, a subject host cell further comprises an rAAV vector.
An rAAV virion can be generated using a subject host cell. Methods
of generating an rAAV virion are described in, e.g., U.S. Patent
Publication No. 2005/0053922 and U.S. Patent Publication No.
2009/0202490.
[0211] As used herein, "packaging" refers to a series of
intracellular events that result in the assembly and encapsidation
of an AAV particle. AAV "rep" and "cap" genes refer to
polynucleotide sequences encoding replication and encapsidation
proteins of adeno-associated virus. AAV rep and cap are referred to
herein as AAV "packaging genes." Assembly associated protein (AAP)
is the product of an open reading frame within the cap gene, and
may also be required for packaging.
[0212] A "helper virus" for AAV refers to a virus that allows AAV
(e.g. wild-type AAV) to be replicated and packaged by a mammalian
cell. A variety of such helper viruses for AAV are known in the
art, including adenoviruses, herpesviruses and poxviruses such as
vaccinia. The adenoviruses encompass a number of different
subgroups, although Adenovirus type 5 of subgroup C is most
commonly used. Numerous adenoviruses of human, non-human mammalian
and avian origin are known and available from depositories such as
the ATCC. Viruses of the herpes family include, for example, herpes
simplex viruses (HSV) and Epstein-Barr viruses (EBV), as well as
cytomegaloviruses (CMV) and pseudorabies viruses (PRV); which are
also available from depositories such as ATCC.
[0213] "Helper virus function(s)" refers to function(s) encoded in
a helper virus genome which allow AAV replication and packaging (in
conjunction with other requirements for replication and packaging
described herein). As described herein, "helper virus function" may
be provided in a number of ways, including by providing helper
virus or providing, for example, polynucleotide sequences encoding
the requisite function(s) to a producer cell in trans. For example,
a plasmid or other expression vector comprising nucleotide
sequences encoding one or more adenoviral proteins is transfected
into a producer cell along with an rAAV vector.
[0214] An "infectious" virus or viral particle is one that
comprises a competently assembled viral capsid and is capable of
delivering a polynucleotide component into a cell for which the
viral species is tropic. The term does not necessarily imply any
replication capacity of the virus. Assays for counting infectious
viral particles are described elsewhere in this disclosure and in
the art. Viral infectivity can be expressed as the ratio of
infectious viral particles to total viral particles. Methods of
determining the ratio of infectious viral particle to total viral
particle are known in the art. See, e.g., Grainger et al. (2005)
Mol. Ther. I1:S337 (describing a TCID50 infectious titer assay);
and Zolotukhin et al. (1999) Gene Ther. 6:973.
[0215] A "replication-competent" virus (e.g. a
replication-competent AAV) refers to a phenotypically wild-type
virus that is infectious, and is also capable of being replicated
in an infected cell (i.e. in the presence of a helper virus or
helper virus functions). In the case of AAV, replication competence
generally requires the presence of functional AAV packaging genes.
In general, rAAV vectors as described herein are
replication-incompetent in mammalian cells (especially in human
cells) by virtue of the lack of one or more AAV packaging genes.
Typically, such rAAV vectors lack any AAV packaging gene sequences
in order to minimize the possibility that replication competent AAV
are generated by recombination between AAV packaging genes and an
incoming rAAV vector. In many embodiments, rAAV vector preparations
as described herein are those which contain few if any replication
competent AAV (rcAAV, also referred to as RCA) (e.g., less than
about 1 rcAAV per 10.sup.2 rAAV particles, less than about 1 rcAAV
per 10.sup.4 rAAV particles, less than about 1 rcAAV per 10.sup.8
rAAV particles, less than about 1 rcAAV per 10.sup.12 rAAV
particles, or no rcAAV).
[0216] An "isolated" nucleic acid, vector, virus, virion, host
cell, or other substance refers to a preparation of the substance
devoid of at least some of the other components that may also be
present where the substance or a similar substance naturally occurs
or is initially prepared from. Thus, for example, an isolated
substance may be prepared by using a purification technique to
enrich it from a source mixture. Enrichment can be measured on an
absolute basis, such as weight per volume of solution, or it can be
measured in relation to a second, potentially interfering substance
present in the source mixture. Increasing enrichments of the
embodiments of this disclosure are increasingly more isolated. An
isolated nucleic acid, vector, virus, host cell, or other substance
is in some embodiments purified, e.g., from about 80% to about 90%
pure, at least about 90% pure, at least about 95% pure, at least
about 98% pure, or at least about 99%, or more, pure.
[0217] 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 invention belongs. Although
any methods and materials similar or equivalent to those described
herein can also be used in the practice or testing of the present
invention, the preferred methods and materials are now described.
All publications mentioned herein are incorporated herein by
reference to disclose and describe the methods and/or materials in
connection with which the publications are cited.
[0218] It is appreciated that certain features of the invention,
which are, for clarity, described in the context of separate
embodiments, may also be provided in combination in a single
embodiment. Conversely, various features of the invention, which
are, for brevity, described in the context of a single embodiment,
may also be provided separately or in any suitable sub-combination.
All combinations of the embodiments pertaining to the invention are
specifically embraced by the present invention and are disclosed
herein just as if each and every combination was individually and
explicitly disclosed. In addition, all sub-combinations of the
various embodiments and elements thereof are also specifically
embraced by the present invention and are disclosed herein just as
if each and every such sub-combination was individually and
explicitly disclosed herein.
[0219] The following example is provided to illustrate certain
embodiments of the invention. It is not intended to limit the
invention in any way.
EXAMPLES
Example 1
[0220] In this Example, the inventors designed and constructed a
novel AAV2 vector designated ttAAV2 (as in true-type). In addition,
the novel vector was tested in a number of animal models (rats,
mice and neonatal mice) in order to evaluate whether ttAAV2 behaved
differently as compared to the tissue culture adapted (wild type)
AAV2. The inventors demonstrated that ttAAV2 has advantages for
gene delivery over AAV2, and is particularly useful for in vivo
transduction of brain or eye tissues with heterologous
sequences.
Methods
[0221] 1. Cloning: The capsid gene of wtAAV2 was taken from our
producer plasmid pDG (FIG. 10). This plasmid contains wtAAV2 rep
and cap genes. Subfragments of the capsid gene (pDG nucleotides
A:3257-3759, B:4025-4555, C:4797-5287 and D:5149-5425,
respectively) were subcloned into pBS for subsequent mutagenesis.
Four mutations were introduced into fragment A via site-directed
mutagenesis resulting in a construct that encodes for amino acid
(AA) changes V125I, V151A, A162S and T205S. Fragment B was mutated
to encode the single AA change, N312S. Fragment C was mutated to
encode the AA exchanges Q457M, S492A, E499D, F533Y, G546D, E548G,
R585S, R588T, and A593S. Upon confirmation of successful
mutagenesis fragments A-C were re-cloned into pDG, resulting in a
producer plasmid (pDG-ttAAV2) that would support the production of
a recombinant virus that is encapsidated by ttAAV2 capsid. 2.
ttAAV2-GFP viral vector production purification and titration.
[0222] Vector production was established following the standard
protocols employing co-transfection of rAAV plasmids with pDG,
which provides both the Ad helper functions as well as the AAV rep
and cap genes. A variety of rAAV plasmids were used to generate
recombinant plasmids.
[0223] pTR-UF11 (CAG-GFP) was used as the rAAV plasmid.
8.times.10.sup.8 293 cells were seeded per cell factory (CF10).
14-18 hours later, the cells were transfected with pDG or
pDG-rrAAV2 and prAAV (e.g. pTR-UF11) using the CaPO.sub.4
co-precipitation method. After 72 hours the cells were harvested
and resuspended in lysis buffer (20mMTris-HCl, pH8, 150 mM NaCl,
0.5% deoxycholate). The cell pellets were lysed by four cycles of
freeze and thaw to release the virus, where each cycle consists of
30 minutes at -80.degree. C. followed by 30 minutes at 37.degree.
C. After the last thaw the lysate was treated with benzonase at a
concentration of 50 U/ml and incubated for 30 min at 37.degree. C.
The recombinant virus was purified using gravity flow columns.
Purification. As a first step, the crude lysate was clarified by
centrifugation at 4000 g for 15 minutes and applied to the
pre-formed iodixanol step gradient. The viral fraction was then
collected and re-buffered into Lactate Ringer's solution as well as
concentrated using Amicon centrifugation filters.
[0224] Subsequently, purity of the viral preparations were assessed
by SDS polyacrylamide electrophoresis and titered using real time
PCR methods. The crude extract contained 4.5.times.10.sup.12
particles; the collected viral fractions contained
1.5.times.10.sup.12 particles. At this point the purification
method recovered ca. 33% of the virus present in the crude
extract.
3. rAAV vector production and purification (alternative method)
[0225] In an alternative embodiment to that at point 2 above, the
rAAV2 vector is produced as follows. To produce rAAV2 virions,
5.times.10.sup.8 293T cells were seeded per cell factory (CF10).
14-18 hours later, the cells were double transfected with the
GFP-containing vector PD10-pST2-CMV-GFP, and either the pDG or the
pDG capsid mutant (pDG-ttAAV2) to produce AAV2-CMV-GFP wild-type or
true-type vectors, respectively. The double transfections were
realised using PEI-max from Polysciences at a ratio of 3.5 ml of
PEI per mg of DNA. The cells were harvested after 72 hours of
incubation at 37.degree. C. by centrifugating the media and cells
at 2200 rpm for 10 minutes at 4.degree. C. The supernatant was
removed and kept for further treatment, and the cells pellets were
resuspended in lysis buffer (0.15 M NaCl, 50 mM Tris-HCl [pH
8.8]).
[0226] The cell pellets were then lysed by 4 cycles of freeze and
thaw to release the virus, where each cycle consists of 30 minutes
at -80.degree. C. followed by 30 minutes at 37.degree. C. After the
last thaw the lysate was treated with benzonase at a concentration
of 150 U/ml and incubated at 37.degree. for 30 minutes. The lysate
was then spun at 2000 rpm for 20 minutes to clarify the lysate. The
supernatant was filtered using a 0.22 .mu.m cellulose acetate
filter and the recombinant AAV2 virus preparations were purified by
FPLC using the AKTApurifier chromatography system (GE Healthcare)
and an AVB sepharose affinity column (bfr. A: PBS, pH 8; bfr B:
0.5M glycine, pH2.7). The collected fractions were dialysed against
PBS overnight and the viral preparations were then titered by SDS
polyacrylamide electrophoresis and real time PCR methods.
Results
[0227] In Vivo Transduction and Spread of ttAAV2
[0228] The ttAAV2 vector was tested in vivo in order to assess the
bioactivity of the modified virus in such a context. Samples of
AAV2-CMV-GFP WT and TT viruses were prepared for injections into a
number of in vivo models. For this purpose we concentrated the
viruses, as only limited volumes of vectors can be injected in
vivo. We then performed a qPCR and SDS-PAGE to assess the new
titres of the concentrated vectors (FIG. 11).
[0229] After qPCR and protein gel analysis we obtained the
following new titres: AAV2-CMV-GFP TT at 1.33.times.10.sup.12 viral
genomes/ml and AAV2-CMV-GFP WT at 1.25.times.10.sup.12 viral
genomes/ml. The capsid titres were as follow: AAV2-CMV-GFP TT at
8.89.times.10.sup.12 capsids/ml and AAV2-CMV-GFP WT at
7.83.times.10.sup.12 capsids/ml. The titres differ between the
genome copies and the capsid copies as the SDS-PAGE also shows
empty capsids, which are normally generated during recombinant AAV
vectors production, hence the capsid titre is higher than the viral
genome titre obtained from the qPCR.
Transduction in Rat Brain.
[0230] The rAAV2 TT and WT viruses were injected in the substantia
nigra or in the striatum of wild-type rats, with 3 rats being
injected per condition, at a dose of 2.times.10.sup.9 vg or
3.5.times.10.sup.9 vg per injection. After 28 days brains were
dissected and tissue sections were prepared for immunofluorescence
analysis. The primary data are shown in FIG. 12 and FIG. 26.
[0231] Both the rAAV2 TT and WT viruses were able to transduce
neuronal and glial cells from each injection site, albeit with
varying efficiencies. By comparison, we observed that the TT vector
transduced brain tissues more efficiently and spread more from the
site of injection than the WT vector. Furthermore, we observed the
presence of transduced neurons in the parafascicularis nucleus, an
area of the hypothalamus, after striatal injection of the rAAV2 TT.
This indicates that the TT vector was able to travel from the
transduced cell bodies at the site of injection to the hypothalamus
by active transport along the neuron projections, highlighting a
strong ability for retrograde transport. This retrograde transport
ability has been lost in the tissue-culture adapted WT rAAV2 vector
as no transduced cells could be observed in the same area (see FIG.
26).
[0232] Taken together, in rat brains these results indicate a
significantly increased spread and transduction efficiency by
ttAAV2 as compared to a titre-matched wtAAV2. Furthermore, AAV2 TT
displays evidence for very good retrograde transport ability, which
has been lost in the AAV2 WT virus.
Transduction in a Mouse Eye Model.
[0233] ttAAV2 and wt-AAV2 from the same batch as was used for our
rat brain studies was injected into adult mouse eyes at a dose of
2.times.10.sup.9 vg per eye. To avoid animal to animal variability,
each mouse received an injection of rAAV2 TT in one eye and an
injection of rAAV2 WT in the contra-lateral eye. Three different
routes of intra-ocular injections were analysed: intra-cameral,
intra-vitreal and sub-retinal. The animals were harvested and GFP
expression was assessed by immunofluorescence after 6 weeks. The
results are shown in FIG. 13. Together, these data indicate a
marked enhancement of transduction of photoreceptor cells by ttAAV2
following sub-retinal injection, in terms of both level and numbers
of photoreceptor cells transduced, if compared to wtAAV2 (which was
used in the successful RPE65 clinical trial).
Transduction in Neonatal Mouse Model.
[0234] In summary, both ttAAV2 and wtAAV2 GFP vectors were injected
into mouse neonates. Two routes of injections were tested,
intra-venous injection and intra-cranial injections. After 4 weeks,
the animals were sacrificed and all tissues were harvested from all
mice. We have analysed the brain, which after harvesting was
visualised by direct fluorescence of the organ on a fluorescence
microscope. The results are shown in FIG. 14. The results of
intracranial and systemic injections are discussed in more detail
below.
Intracranial Injections
[0235] 5.times.10.sup.10 vg of either vector were injected in the
lateral ventricle of P1 neonates. The animals were sacrificed 4
weeks post-injection and the brains were dissected, sectioned and
stained using an anti-GFP antibody. The results are shown in FIG.
27.
[0236] As observed in adult rat brains, these data indicate that
AAV2 TT displays enhanced transduction of mouse brain tissues and
higher spread after intracranial injection as compared to the AAV2
WT vector. When observing the stained sections at a higher
magnification, the differences in transduction efficiency between
both vectors were further highlighted: the TT vector performed
better both in terms of level of expression and of number of cells
transduced. AAV2 TT and WT seem to have the same cell type
affinity, each displaying transduction of neuronal as well as glial
cells, suggesting that the differences observed are differences in
efficiency rather than in cell-type specificity (FIG. 28). Taken
together these data indicate that ttAAV2 shows much enhanced
transduction of mouse brain tissues after i.c. injection as
compared to wtAAV2-based vectors. In addition, some evidence is
suggestive for transduction of the ependymal cell layer lining the
ventricles when ttAAV2 vectors are used. This phenomenon is not
visible with the wtAAV2 vector.
Systemic Injections
[0237] Intrajugular injections of 2.times.10.sup.11 vg of either
vector were done in P1 neonates. The animals were sacrificed 4
weeks post-injection and various organs were harvested and assessed
for GFP transduction by immunohistochemistry using an anti-GFP
antibody (brain, liver, heart, muscle, lungs, spleen and kidney).
Results of the brains staining are shown in FIG. 29 and high
magnification pictures are presented in FIG. 30.
[0238] We observed good transgene expression in the CNS after
systemic injection of AAV2 TT. The AAV2 WT vector performed worse
in comparison, with only few transduced neurons observed.
[0239] In order to assess the overall biodistribution of the AAV2
TT vector we assessed the level of transduction obtained in various
tissues after systemic injection. The harvested organs were fixed,
paraffin embedded, sectioned and stained for GFP expression (FIG.
31).
[0240] These data indicate that the AAV-TT vector doesn't seem to
have a strong affinity for other organs but instead displays
specificity mainly for neuronal tissues. This observation could
prove beneficial for the treatment of neuronal genetic disorders by
intravenous injections of AAV as it ensures that the vector will
not transduce non-target peripheral organs but mainly only the
brain via this injection route.
[0241] Together, our in vivo data suggests that ttAAV2 has
extraordinary transduction characteristics in eye and brain
tissues, displaying specificity for neuronal tissues almost
exclusively.
Example 2
Additional Considerations
[0242] Without being bound by theory, it is believed that the
mutations present in ttAAV2 compared to wtAAV2 comprise the
following functional groups:
1) heparin binding residues located on the AAV2 capsid threefold
spikes (S585 and T588); it is believed that these residues are
responsible for heparin binding of the wtAAV2 capsid. In ttAAV2
these are replaced and we assume that this replacement supports the
spread of the virus in heparan sulphate proteoglycan-rich brain
tissue. 2) the single amino acid change in ttAAV2 that is located
on the internal side of the capsid (S312); this internal serine
residue might play a role in capsid-DNA interactions, thereby
potentially contributing to, either virus stability, genome
packaging or genome release during infection. 3) two spatially
close amino acids (D546 and G548) located in the groove between the
threefold-proximal spikes on AAV capsid structure; these residues
might be involved in interactions with neutralizing antibodies and
thus contribute to in vivo transduction characteristics. 4) a
single isolated amino acid change (S593) located in the groove
between threefold-proximal spikes; 5) four amino acids believed to
be involved in receptor binding and closely situated on the
threefold spikes (M457, A492, D499 and Y533); 6) the four remaining
amino acid changes situated in VP1/VP2 primary sequence (I125,
A151, S162 and S205); these residues are within the capsid region
that is not part of VP3. It is known that VP1/VP2 specific regions
within the virus capsid contain PLA2 activity which might be
involved in trafficking of the incoming virus. Changes in this
region have been shown to influence viral infectivity.
[0243] The three-dimensional positions of these mutations in the
AAV2 capsid protein are known and are shown in FIGS. 15 to 20.
Corresponding positions can be identified in the AAV capsid
proteins from other serotypes (see below). To further characterize
the ttAAV2 and the role of individual amino acid changes that are
responsible for the improved ttAAV2 phenotype, the ttAAV2 capsid
can be mutated in order to reverse individual chosen amino acids
(or groups of amino acids, e.g. based on groups 1 to 6 discussed
above) to their corresponding sequence in the wild-type AAV2
capsid. Each mutant vector can then be analysed using methods as
described in the examples above.
[0244] For instance the various mutant vectors, expressing GFP, can
be submitted to a first screen in an animal model by intracranial
(IC) injection in CD1 neonatal mice. The GFP signal obtained in the
injected brains from each mutant vector is then observed by
fluorescence microscopy and compared to that obtained from the
original GFP-expressing wtAAV2 and ttAAV2 vectors. Upon
identification of a new phenotype (i.e. the abolition of the
ttAAV2-specific strong GFP expression when mutating a particular
amino acid group), the injected brains are then further sectioned
and analysed by immunohistochemistry.
[0245] In parallel, these selected mutant vectors are submitted to
a second screen by adult rat intracranial injections. Additionally,
relevant mutant combinations are analysed by intravenous (IV)
injections into neonatal mice in order to evaluate the
biodistribution of the vectors. Selected organs (heart, lung,
liver, spleen, kidney, muscle) are then processed for
immunohistochemistry and evaluation of GFP expression.
Analysis of the Contribution of Each TT-Specific Residue to the
Efficiency and Biodistribution of the Vector
[0246] To further characterize the AAV2 TT and pinpoint the amino
acid changes that are responsible for the improved TT phenotype,
the AAV2 TT capsid was mutated step by step in order to reverse the
chosen amino acids to their corresponding sequence in the wild-type
AAV2 capsid. This strategy enabled us to discern more specifically
the contribution of each of the 14 amino acid mutations towards the
phenotype of AAV2 TT and to define a minimal true-type vector,
containing only the necessary mutations.
[0247] As discussed above, we grouped the 14 TT-specific residues
into groups based on their positions on the AAV capsid and their
associated potential contributions to the transduction profile. The
various AAV-TT mutants were screened by intracranial injections in
neonatal mouse brains or in adult rat brains in order to observe
whether the reversion of some TT-specific residues to the WT
equivalents was associated with a loss of phenotype, thereby
identifying the important amino acid changes amongst the 14 TT
residues.
The Heparin Binding Site (HBS)
[0248] It has been shown that residues 585 and 588 are responsible
for heparin binding of the AAV2 WT capsid. In AAV-TT these are
replaced and we assumed that this replacement support the spread of
the virus in heparin sulphate proteoglycan-rich brain tissue. These
two residues are situated on the three-fold proximal spikes of the
AAV2 capsid structure (see FIG. 16).
[0249] We abolished the AAV2 WT heparin binding ability by
engineering the changes R585S and R588T on the WT capsid
(AAV2-HBnull), i.e. mutating the residues to the true-type
equivalents. This first analysis was performed in order to
ascertain that AAV-TT was not merely an AAV2 without heparin
binding site, able to spread more, but that some of the other 12
amino acid changes also play a role in the improved AAV-TT
transduction profile.
Intracranial Injections in Adult Rat
[0250] Titer-matched AAV2-TT, AAV2-WT and AAV2-HBnull vectors were
injected in the substantia nigra or in the striatum of wild-type
rats at a dose of 3.5.times.10.sup.9 vg per injection. After 28
days brains were dissected and tissue sections were prepared for
immunohistochemistry analysis of GFP expression (FIG. 32).
[0251] We observed a strong GFP expression in the thalamus and in
the substantia nigra after striatum injection of AAV-TT virus,
highlighting the strong retrograde transport ability of the vector.
In comparison, AAV2-HBnull and AAV2 WT displayed much less--if
any--retrograde transport than AAV-TT as very few cell bodies were
transduced in these area after striatal injections. This
observation showed that AAV2-HBnull is different to AAV-TT and that
the absence of heparin binding ability on the AAV2 capsid
contributes to the good spread of the true-type vector in the
brain. However it is not sufficient to explain its improved
transduction profile.
The Residues S312, D546-G548 and S593
[0252] The S312 residue is the only TT-specific change that is
located on the internal side of the AAV2 capsid. Our assumption was
that this internal residue might play a role in capsid-DNA
interactions, thereby potentially contributing to either virus
stability, genome packaging or genome release during infection.
[0253] The D and G residues at positions 546 and 548, respectively,
are located in the groove between the proximal 3-fold peaks.
[0254] A single isolated serine, S593, is situated in the groove
between threefold-proximal spikes.
[0255] The positions of these TT-specific residues on the
three-dimensional structure of the AAV2 capsid are presented in
FIGS. 17 to 19. Given their less prominent position in the
structure we hypothesised that these residues might not contribute
to the ttAAV2 transduction phenotype.
Neonatal Mice Intracranial Injections
[0256] The mutation S312N was engineered on the AAV2-TT capsid to
create the TT-S312N mutant. The mutations D546G and G548E were
engineered on the AAV2-TT capsid to generate the AAV-TT-DG mutant.
The mutation S593A was engineered on the AAV2-TT capsid to create
the AAV-TT-S593A mutant.
[0257] By reverting these chosen TT amino acids to their
corresponding sequence in the wild-type AAV2 capsid we aimed to
determine the contribution of these residues to the improved AAV-TT
phenotype.
[0258] 5.times.10.sup.10 vg of each mutant vector were injected in
the lateral ventricle of P1 neonates. The animals were sacrificed 4
weeks post-injection and the brains were dissected, sectioned and
stained using an anti-GFP antibody. The results are shown in FIG.
33.
[0259] Interestingly, these data suggest that the AAV TT-S312N
displays enhanced transduction of mouse brain tissues as compared
to the full AAV2 TT vector. On the other hand, the amino acid
changes S593A or D546E/G548D did not seem to affect the TT
phenotype as similar transduction profiles could be observed
throughout the brains. When observing the stained sections at a
higher magnification, the differences in transduction efficiency
were further highlighted (FIG. 34).
[0260] From the high magnification pictures, we could observe that
the AAV TT-S312 seems to transduce neuronal tissues with higher
efficiency than the original AAV-TT with 14 amino acid changes. In
particular, we could see stronger transgene expression in the
rostral side of the brain (cortex, striatum, hippocampus) after
TT-S312N vector injection, both in terms of level and of number of
cells transduced. Despite the high variability in injected neonatal
brains due to the difficulty associated with targeting the
injection site, this observation was confirmed in all the animals
analysed. On the other hand, the reversions S593A or D546E/G548D
did not seem to have much impact on the TT vector transduction
phenotype.
Example 3
[0261] Targeted Amino Acid Mutations on the AAV2 True-Type Capsid,
Selected from Results Obtained with the Mutant Combinations in
Example 2
[0262] Based on the results from amino acid group mutations on the
full AAV TT capsid, we could determine that the mutation S312N
seems to be beneficial for the TT phenotype, further increasing its
transduction efficiency in the brain. Furthermore, we observed that
the reversions S593A and D546G/G548E did not seem to affect the
neuronal phenotype of AAV-TT. We therefore hypothesised that the
TT-specific residues S593, D546 and G548 could be excluded from the
True-type capsid sequence, leaving instead the AAV2 WT residues at
these positions to obtain a final TT vector with only 10 amino acid
changes.
[0263] In order to verify these hypotheses, we engineered the
TT-S312N-D546G-G548D-S593A vector and tested its transduction
efficiency by neonatal mouse brain injections. Because the last
neonate intracranial injections seemed to lead to a saturated
signal in the GFP expression detected, we decided to also inject
the TT and the TT-S312N vectors alongside this "pre-final" TT,
using a 10 times lower dose than used previously. By using this
lower dose we aimed to avoid reaching saturating levels of GFP
staining in the transduced brains and avoid difficulties in
transduction efficiency comparison between different mutants.
[0264] 5.times.10.sup.09 vg of each mutant vector were injected in
the lateral ventricle of P1 neonates. The animals were sacrificed 4
weeks post-injection and the brains were dissected, sectioned and
stained using an anti-GFP antibody. The results are shown in FIG.
35.
[0265] As previously observed, these data suggest that the AAV
TT-S312N displays enhanced transduction of mouse brain tissues as
compared to the full AAV2 TT vector. On the other hand, the minimal
TT-S312N-D546G/G548E-S593A vector did not seem to reach these
higher levels of transduction even though it also contained the
internal S312N mutation. This suggests that one or more of the
amino acid changes plays some role in the TT phenotype. By
reverting these residues back to AAV2 WT equivalents, we lost some
of the increased transduction ability. When observing the stained
sections at a higher magnification, these observations were
confirmed (FIG. 36).
[0266] We decided to further investigate the transduction
efficiency obtained by each of these vectors by quantifying the
total GFP expression obtained in injected brains by enzyme-linked
immunosorbent assay (ELISA) on full brain protein extracts.
Briefly, 4 weeks after injection of 5.times.10.sup.09 vg of
vectors, the animals were sacrificed, the whole brains were harvest
and lysed, and total brain proteins were extracted. Using a GFP
protein standard, we could then quantify the amounts of GFP protein
expressed in each injected brain (FIG. 37).
[0267] We could confirm that the TT-S312N internal mutant
transduces mouse brains with more efficiency than the full TT
vector as it leads to more GFP expression overall in all the
injected brains. On the other hand the minimal TT vector,
TT-S312N-D546G/G548E-S593A, seemed to lead to lower levels of
transduction: although the average amount of GFP expressed per
brain seems higher on this graph, this was due to extreme GFP
values measured in one of the brains as illustrated by the high
error bar for this condition. With this minimal TT vector, the
variability between animals was very high, with only one animal out
of five performing better than the animals transduced with
TT-S312N. This high variability led us to consider this provisional
minimal TT vector with caution, especially since the
immunohistochemistry analyses also showed that the TT-S312N variant
performed better than the TT-S312N-DG-S593A.
[0268] We therefore selected the TT-S312N variant as our most
preferred AAV TT vector, which is composed of the following 13
amino acid mutations compared to the wild-type AAV2: V125I, V151A,
A162S, T205S, Q457M, S492A, E499D, F533Y, G546D, E548G, R585S,
R588T, and A593S.
[0269] Although the above studies suggest the TT-S312N as the most
preferred AAV-TT vector, these studies illustrate the individual
function and contribution of a number TT-specific residues. In
particular, four amino acids closely situated on the threefold
spike of the capsid are likely involved in receptor binding. In
some embodiments, these residues are reverted in the AAV-TT back to
the AAV2 WT corresponding residues. For instance, the mutations
M457Q, A492S, D499E and Y533F may be engineered on the AAV-TT
capsid and this mutant vector analysed as previously described, in
order to illustrate the role of these residues. In further
embodiments, four amino acids situated in VP1/VP2 primary sequence,
which are likely to be involved in trafficking of the virus, may be
reverted in the AAV-TT back to AAV2 WT corresponding residues
(I125V, A151V, S162A, S205T) and analysed similarly.
Example 4
Construction of Variant AAV Vectors in Other Serotypes
[0270] The function of the ttAAV2-specific amino acid changes in
the context of other, non-AAV2 serotypes can also be
determined.
[0271] The capsid amino acid sequences of the main adeno-associated
viruses (AAV), namely AAV1, 5, 6, 8, 9 and rh10, can be aligned
with the one from ttAAV2, e.g. as shown in FIG. 9. This enables the
identification of which ttAAV2-specific amino acids are already
present at the same positions in other serotypes. The relevant
residues in the various serotypes are then mutated into the
corresponding residues in wtAAV2. These changes demonstrate the
importance of the ttAAV2-specific residues for the efficiency and
biodistribution of each of the serotype.
[0272] As discussed above, the various mutant vectors, expressing
GFP, are then submitted to a first screen by intracranial (IC)
injection in CD1 neonatal mice. The GFP signal obtained in the
injected brains is then compared to that obtained from the
appropriate GFP-expressing serotype controls. The diminution or
increase of GFP expression when mutating the identified amino acids
into their corresponding AAV2 residues demonstrates the importance
of these particular residues at these specific positions. Where
applicable, the injected brains are then further sectioned and
analyzed by immunohistochemistry. Additionally, chosen mutant
serotypes are analyzed by intravenous (IV) injections into neonatal
mice in order to evaluate the biodistribution of the vectors and
compare it to the original, non-mutated counterparts.
[0273] In further embodiments, the relevant amino acids identified
in the ttAAV2 are inserted as key mutations into the other
prominent serotypes at the relevant positions. The insertion of
ttAAV2-specific residues in other AAV subtypes enables us to
improve the transduction and biodistribution profiles of each
serotype.
Example 5
[0274] Modification of ttAAV2-Specific Residues Conserved in Other
AAV Serotypes into the Corresponding AAV2 Residues.
[0275] A comparative analysis of the capsid amino acid sequences of
existing adeno-associated serotypes with the one from ttAAV2 first
enabled us to identify ttAAV2-specific residues that are conserved
in other serotypes (see FIG. 9). These residues consist of S162,
S205, S312, G548, S585 and T588. Each non-AAV2 serotype contains
one or a combination of several of these residues at a
corresponding amino acid position in its sequence.
[0276] In specific embodiments, each of these residues in the
various serotypes are converted into the corresponding wild-type
AAV2 amino-acid(s) and the transduction efficiency of these new
mutants is tested.
1) Modification of the AAV1 Serotype
[0277] AAV1 contains the residues S205, G549, S586 and T589 which
correspond to the following residues in ttAAV2: S205, G548, S585
and T588. When the VP1 monomer from AAV1 was aligned
three-dimensionally with VP1 from AAV2 we could verify that the
corresponding residues in wtAAV2, namely T205, E548, R585 and R588,
are at perfectly matching positions on the 3D structure (FIG. 21).
We thus concluded that it would be significant to convert each of
the ttAAV2-specific residue(s) in AAV1 into the corresponding
wild-type AAV2 counterparts without affecting the three-dimensional
structure of the protein. In particular embodiments the following
mutations are made in AAV1 capsid sequence: S205T, G549E, S586R,
T589R.
2) Modification of the AAV5 Serotype
[0278] AAV5 contains the residues G537, S575 and T578 which
correspond to the following residues in ttAAV2: G548, S585 and
T588. The R585 and R588 residues in AAV2 are at matching positions
with S575 and T578 in AAV5 on the 3D structure. Although the
residue E548 in AAV2 did not perfectly match the residue G537 in
AAV5 according to the three-dimensional structure (FIG. 22), we
still decided to include it in the study as both residues are
relatively spatially close. Therefore in particular embodiments the
following mutations are made in AAV5 capsid sequence: G537E, S575R,
T578R.
3) Modification of the AAV6 Serotype
[0279] AAV6 contains the residues S205, G549, S586 and T589 which
correspond to the following residues in ttAAV2: S205, G548, S585
and T588. The corresponding residues in wtAAV2, namely T205, E548,
R585 and R588 (FIG. 23), are at perfectly matching positions on the
VP1 3D structures. Therefore in particular embodiments the
following mutations are made in AAV6 capsid sequence: S205T, G549E,
S586R, T589R.
4) Modification of the AAV8 Serotype
[0280] AAV8 contains the residues S315 and T591 which correspond to
the following residues in ttAAV2: S312 and T588. The corresponding
residues in wtAAV2, namely N312 and R588, are at perfectly matching
positions on the VP1 3D structures (FIG. 24). Therefore in
particular embodiments the following mutations are made in AAV8
capsid sequence: S315N, T591R.
[0281] In one embodiment, the improved transduction efficiency
imparted by the S312N mutation in TT AAV2 may be transferred to the
AAV8 serotype by applying the amino acid change S315N.
[0282] We mutated the AAV8 capsid sequence by site-directed
mutagenesis and thereby created the AAV8-S315N vector plasmid. This
plasmid was used to produce recombinant AAV8-S315N vectors
expressing an ITR-containing CMV-GFP transgene by double
transfection of 293T cells. The vector was then purified from the
cell lysate and from the harvested culture supernatant by FPLC
affinity chromatography, using an AVB sepharose resin. The capsid
titer and vector genome titer were assessed by SDS-PAGE and qPCR,
respectively.
[0283] The mutant AAV8-S315N vector, expressing GFP, is screened by
systemic injections in CD1 neonatal mice. A titer-matched AAV8
vector is used as a control. GFP expression obtained in various
organs after intra-jugular injection of 2.times.10.sup.11 vg is
then analysed, primarily focusing on the liver where AAV8 has
previously shown some strong transduction efficiency.
5) Modification of the AAV9 Serotype
[0284] AAV9 contains the residues S162, S205, G549 and S586 which
correspond to the following residues in ttAAV2: S162, S205, G548
and S585. The corresponding residues in AAV2, namely A162, T205,
E548 and R585, are at perfectly matching positions on the VP1 3D
structures (FIG. 25). Therefore in particular embodiments the
following mutations are made in AAV8 capsid sequence: S162A, S205T,
G549E, and S586R.
6) Modification of the AAVrh10 Serotype
[0285] AAVrh10 contains the residue G551 which corresponds with
G548 with ttAAV2. Considering how conserved this residue and
position appears among the various serotypes, we assume that G551
in AAVrh10 will align with E548 in wtAAV2 three-dimensionally.
Therefore in one embodiment the following mutation is made in
AAVrh10 capsid sequence: G551E.
7) Modification of the AAV3B and AAV-LK03 Serotypes
[0286] Similarly to the AAV8 serotype, we observed that the AAV3B
serotype also contains an internal serine at position 312 after
aligning the capsid protein VP1 sequence with the one from AAV-TT
(see AAV3B capsid sequence in FIG. 38).
[0287] The newly described LK03 AAV vector, a chimeric capsid
composed of five different parental AAV capsids engineered by M. A.
Kay by DNA-shuffling, also contains the residue S312 in the
internal side of the capsid (see Lisowski et al., Selection and
evaluation of clinically relevant AAV variants in a xenograft liver
model, Nature 506, 382-386 (2014)). The capsid sequence of AAV-LK03
is disclosed in WO2013/029030 and shown in FIG. 39.
[0288] Therefore in further embodiments, the AAV3B and the AAV-LK03
vectors are mutated by applying the amino-acid change S312N in both
vectors. These new mutants, and their corresponding AAV control
serotypes, are also tested by intra-jugular injections in neonatal
mice. The GFP expression obtained in various harvested organs is
then analysed.
Example 6
[0289] Identification of ttAAV2-Specific Amino Acids that are
Transferable Between AAV Serotypes
[0290] In further embodiments, the key amino acids identified
during the ttAAV2 characterization are inserted into the other
prominent serotypes at the relevant positions. The newly engineered
vectors are then tested using the appropriate non-mutated serotypes
as controls. This validates the importance of individual amino
acids at specific positions on AAV capsids, independently of the
serotype.
1) Residues S585, T588, S312, D546, G548 and S593
[0291] AAV1, AAV5 and AAV6 naturally contain the same amino acid
residue at positions in their capsid protein sequences
corresponding to G548, S585 and T588 in ttAAV2. Therefore in
further embodiments, the capsid proteins in these serotypes are
mutated at matching positions to include the other residues S312,
D546, and S593 present in ttAAV2. Similarly AAV8, which already
contains the same amino acid residue as in ttAAV2 at positions
corresponding to S312 and T588 in ttAAV2, is further mutated to
contain residues corresponding to S585, D546, G548 and S593 in
ttAAV2. AAV9, that already contains corresponding residues to G548
and S585 in ttAAV2, is mutated to include residues corresponding to
T588, S312, D546 and S593 in ttAAV2. Finally AAV10, which already
contains a residue corresponding to G548, is further modified to
also contain residues corresponding to S585, T588, S312, G548 and
S593.
2) Residues I125, A151, S162, S205, M457, A492, D499 and Y533
[0292] AAV1, and AAV6 already naturally contain the residue S205.
Therefore in further embodiments these serotypes are mutated at
positions corresponding to the residues I125, A151, S162, M457,
A492, D499 and Y533 in ttAAV2. Similarly AAV9, that already
contains the residues S162 and S205, is further mutated to contain
residues corresponding to I125, A151, M457, A492, D499 and Y533 in
ttAAV2. Finally, AAV5, 8 and 10 are modified to display residues
corresponding to I125, A151, S162, S205, M457, A492, D499 and Y533
in ttAAV2.
[0293] The positions the mutations present in ttAAV2 and the
corresponding residues present in the wild type capsid protein VP1
sequences of other AAV serotypes are shown in Table 1 below. In
general, variant non-AAV2 vectors can be constructed by mutating
any of the residues shown in Table 1 for these serotypes. The
residues shown in italics are residues which are already present in
ttAAV2 at a corresponding position. In preferred embodiment, the
non-AAV2 serotypes are mutated at one or more the residues shown in
non-italic script. In this ways, the advantageous properties shown
by ttAAV2 can be transferred into alternative AAV serotypes.
TABLE-US-00001 TABLE 1 ttAAV2 AAV1 AAV5 AAV6 AAV8 AAV9 AAV10 I125
V125 V124 V125 V125 L125 V125 A151 Q151 K150 Q151 Q151 Q151 Q151
S162 T162 K153 T162 K163 S162 K163 S205 S205 A195 S205 A206 S205
A206 S312 N313 R303 N313 S315 N314 N315 M457 N458 T444 N458 T460
Q458 T460 A492 K493 S479 K493 T495 V493 L495 D499 N500 V486 N500
N502 E500 N502 Y533 F534 T520 F534 F536 F534 F536 D546 S547 P533
S547 N549 G547 G549 G548 G549 G537 G549 A551 G549 G551 S585 S586
S575 S586 Q588 S586 Q588 T588 T589 T578 T589 T591 A589 A591 S593
G594 G583 G594 G596 G594 G596
Further Embodiments of the Invention
[0294] The invention also relates additional aspects, as defined in
the following summary paragraphs:
1. A recombinant adeno-associated virus (AAV) vector comprising;
(a) a variant AAV capsid protein, wherein the variant AAV capsid
protein comprises at least one amino acid substitution with respect
to a wild type AAV capsid protein; wherein the at least one amino
acid substitution is present at a position corresponding to one or
more of the following positions in an AAV2 capsid protein sequence:
125, 151, 162, 205, 312, 457, 492, 499, 533, 546, 548, 585, 588
and/or 593; and (b) a heterologous nucleic acid comprising a
nucleotide sequence encoding a gene product. 2. A recombinant AAV
vector according to paragraph 1, wherein (i) the vector comprises a
variant AAV2 capsid protein; (ii) the variant AAV capsid protein
comprises a sequence of SEQ ID NO:2, or a sequence having at least
95% sequence identity thereto; (iii) the wild type AAV capsid
protein is from AAV2; and/or (iv) the wild type AAV capsid protein
comprises a sequence of SEQ ID NO:1. 3. A recombinant AAV vector
according to paragraph 2, wherein the variant AAV2 capsid protein
comprises one or more of the following residues: I125, A151, S162,
S205, S312, M457, A492, D499, Y533, D546, G548, S585, T588 and/or
S593. 4. A recombinant AAV vector according to paragraph 2 or
paragraph 3, wherein the variant AAV2 capsid protein comprises one
or more of the following amino acid substitutions with respect to a
wild type AAV2 capsid protein: V125I, V151A, A162S, T205S, N312S,
Q457M, S492A, E499D, F533Y, G546D, E548G, R585S, R588T and/or
A593S. 5. A recombinant AAV vector according to any of paragraphs 1
to 3, wherein the variant AAV capsid protein is from AAV1, AAV5,
AAV6, AAV8, AAV5 or AAV10. 6. A recombinant AAV vector according to
paragraph 6, wherein (i) the vector comprises a variant AAV1 capsid
protein, (ii) the variant AAV capsid protein comprises a sequence
having at least 95% sequence identity to SEQ ID NO:3; (iii) the
wild type AAV capsid protein is from AAV1; and/or (iv) the wild
type AAV capsid protein comprises a sequence of SEQ ID NO:3; and
wherein at least one amino acid substitution is present at one or
more of the following positions in the AAV1 capsid protein
sequence: 125, 151, 162, 205, 313, 458, 493, 500, 534, 547, 549,
586, 589 and/or 594. 7. A recombinant AAV vector according to
paragraph 6, wherein the variant AAV1 capsid protein comprises one
or more of the following amino acid substitutions with respect to a
wild type AAV1 capsid protein: (a) V125I, Q151A, T162S, N313S,
N458M, K493A, N500D, F534Y, S547D, and/or G594S; and/or (b) S205T,
G549E, S586R and/or T589R. 8. A recombinant AAV vector according to
paragraph 6, wherein (i) the vector comprises a variant AAV5 capsid
protein, (ii) the variant AAV capsid protein comprises a sequence
having at least 95% sequence identity to SEQ ID NO:4; (iii) the
wild type AAV capsid protein is from AAV5; and/or (iv) the wild
type AAV capsid protein comprises a sequence of SEQ ID NO:4; and
wherein at least one amino acid substitution is present at one or
more of the following positions in the AAV5 capsid protein
sequence: 124, 150, 153, 195, 303, 444, 479, 486, 520, 533, 537,
575, 578 and/or 583. 9. A recombinant AAV vector according to
paragraph 8, wherein the variant AAV5 capsid protein comprises one
or more of the following amino acid substitutions with respect to a
wild type AAV5 capsid protein: (a) V124I, K150A, K153S, A195S,
R303S, T444M, S479A, V486D, T520Y, P533D, and/or G583S; and/or (b)
G537E, S575R and/or T578R. 10. A recombinant AAV vector according
to paragraph 5, wherein (i) the vector comprises a variant AAV6
capsid protein, (ii) the variant AAV capsid protein comprises a
sequence having at least 95% sequence identity to SEQ ID NO:5;
(iii) the wild type AAV capsid protein is from AAV6; and/or (iv)
the wild type AAV capsid protein comprises a sequence of SEQ ID
NO:5; and wherein at least one amino acid substitution is present
at one or more of the following positions in the AAV6 capsid
protein sequence: 125, 151, 162, 205, 313, 458, 493, 500, 534, 547,
549, 586, 589 and/or 594. 11. A recombinant AAV vector according to
paragraph 10, wherein the variant AAV6 capsid protein comprises one
or more of the following amino acid substitutions with respect to a
wild type AAV6 capsid protein: (a) V125I, Q151A, T162S, N313S,
N458M, K493A, N500D, F534Y, S547D, and/or G594S; and/or (b) S205T,
G549E, S586R and/or T589R. 12. A recombinant AAV vector according
to paragraph 5, wherein (i) the vector comprises a variant AAV8
capsid protein, (ii) the variant AAV capsid protein comprises a
sequence having at least 95% sequence identity to SEQ ID NO:6;
(iii) the wild type AAV capsid protein is from AAV8; and/or (iv)
the wild type AAV capsid protein comprises a sequence of SEQ ID
NO:6; and wherein at least one amino acid substitution is present
at one or more of the following positions in the AAV8 capsid
protein sequence: 125, 151, 163, 206, 315, 460, 495, 502, 536, 549,
551, 588, 591 and/or 596. 13. A recombinant AAV vector according to
paragraph 12, wherein the variant AAV8 capsid protein comprises one
or more of the following amino acid substitutions with respect to a
wild type AAV8 capsid protein: (a) V125I, Q151A, K163S, A206S,
T460M, T495A, N502D, F536Y, N549D, A551G, Q588S and/or G596S;
and/or (b) S315N and/or T591R. 14. A recombinant AAV vector
according to paragraph 5, wherein (i) the vector comprises a
variant AAV9 capsid protein, (ii) the variant AAV capsid protein
comprises a sequence having at least 95% sequence identity to SEQ
ID NO:7; (iii) the wild type AAV capsid protein is from AAV9;
and/or (iv) the wild type AAV capsid protein comprises a sequence
of SEQ ID NO:7; and wherein at least one amino acid substitution is
present at one or more of the following positions in the AAV9
capsid protein sequence: 125, 151, 162, 205, 314, 458, 493, 500,
534, 547, 549, 586, 589 and/or 594. 15. A recombinant AAV vector
according to paragraph 14, wherein the variant AAV9 capsid protein
comprises one or more of the following amino acid substitutions
with respect to a wild type AAV9 capsid protein: (a) L125I, Q151A,
N314S, Q458M, V493A, E500D, F534Y, G547D, A589T and/or G594S;
and/or (b) S162A, S205T, G549E and/or S586R. 16. A recombinant AAV
vector according to paragraph 5, wherein (i) the vector comprises a
variant AAVrh10 capsid protein, (ii) the variant AAV capsid protein
comprises a sequence having at least 95% sequence identity to SEQ
ID NO:8; (iii) the wild type AAV capsid protein is from AAVrh10;
and/or (iv) the wild type AAV capsid protein comprises a sequence
of SEQ ID NO: 8; and wherein at least one amino acid substitution
is present at one or more of the following positions in the AAV10
capsid protein sequence: 125, 151, 163, 206, 315, 460, 495, 502,
536, 549, 551, 588, 591 and/or 596. 17. A recombinant AAV vector
according to paragraph 16, wherein the variant AAVrh10 capsid
protein comprises one or more of the following amino acid
substitutions with respect to a wild type AAVrh10 capsid protein:
(a) V125I, Q151A, K163S, A206S, N315S, T460M, L495A, N502D, F536Y,
G549D, Q588S, A591T and/or G596S; and/or
(b) G551E.
[0295] 18. A recombinant AAV vector according to any preceding
paragraph, wherein the recombinant AAV vector exhibits increased
transduction of a neuronal or retinal tissue compared to an AAV
vector comprising a corresponding wild type AAV capsid protein. 19.
A recombinant AAV vector according to any preceding paragraph,
wherein the gene product comprises an interfering RNA or an
aptamer. 20. A recombinant AAV vector according to any of
paragraphs 1 to 18, wherein the gene product comprises a
polypeptide. 21. A recombinant AAV vector according to paragraph
20, wherein the gene product comprises a neuroprotective
polypeptide, an anti-angiogenic polypeptide, or a polypeptide that
enhances function of a neuronal or retinal cell. 22. A recombinant
AAV vector according to paragraph 21, wherein the gene product
comprises glial derived neurotrophic factor, fibroblast growth
factor, nerve growth factor, brain derived neurotrophic factor,
rhodopsin, retinoschisin, RPE65 or peripherin. 23. A pharmaceutical
composition comprising: (a) a recombinant AAV vector according to
any preceding paragraph; and (b) a pharmaceutically acceptable
excipient. 24. A method for delivering a gene product to a neuronal
or retinal tissue in a subject, the method comprising administering
to the subject a recombinant AAV vector or pharmaceutical
composition according to any preceding paragraph. 25. A method for
treating a neurological or ocular disorder, the method comprising
administering to the subject a recombinant AAV vector or
pharmaceutical composition according to any preceding paragraph.
26. A recombinant AAV vector or pharmaceutical composition
according to any of paragraphs 1 to 23, for use in treating a
neurological or ocular disorder. 27. A method, recombinant AAV
vector or pharmaceutical composition for use according to any of
paragraphs 24 to 26, wherein the neurological disorder is a
neurodegenerative disease. 28. A method, recombinant AAV vector or
pharmaceutical composition for use according to any of paragraphs
24 to 26, wherein the ocular disorder is glaucoma, retinitis
pigmentosa, macular degeneration, retinoschisis or diabetic
retinopathy. 29. An isolated variant AAV capsid protein, wherein
the variant AAV capsid protein comprises at least one amino acid
substitution with respect to a wild type AAV capsid protein;
wherein the at least one amino acid substitution is present at one
or more of the following positions in an AAV2 capsid protein
sequence: 125, 151, 162, 205, 312, 457, 492, 499, 533, 546, 548,
585, 588 and/or 593; or at one or more corresponding positions in
an alternative AAV capsid protein sequence. 30. An isolated nucleic
acid comprising a nucleotide sequence that encodes a variant AAV
capsid protein as defined in paragraph 29. 31. An isolated host
cell comprising a nucleic acid as defined in paragraph 30.
Sequence CWU 1
1
121735PRTAdeno-associated virus-2 1Met Ala Ala Asp Gly Tyr Leu Pro
Asp Trp Leu Glu Asp Thr Leu Ser1 5 10 15Glu Gly Ile Arg Gln Trp Trp
Lys Leu Lys Pro Gly Pro Pro Pro Pro 20 25 30Lys Pro Ala Glu Arg His
Lys Asp Asp Ser Arg Gly Leu Val Leu Pro 35 40 45Gly Tyr Lys Tyr Leu
Gly Pro Phe Asn Gly Leu Asp Lys Gly Glu Pro 50 55 60Val Asn Glu Ala
Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp65 70 75 80Arg Gln
Leu Asp Ser Gly Asp Asn Pro Tyr Leu Lys Tyr Asn His Ala 85 90 95Asp
Ala Glu Phe Gln Glu Arg Leu Lys Glu Asp Thr Ser Phe Gly Gly 100 105
110Asn Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg Val Leu Glu Pro
115 120 125Leu Gly Leu Val Glu Glu Pro Val Lys Thr Ala Pro Gly Lys
Lys Arg 130 135 140Pro Val Glu His Ser Pro Val Glu Pro Asp Ser Ser
Ser Gly Thr Gly145 150 155 160Lys Ala Gly Gln Gln Pro Ala Arg Lys
Arg Leu Asn Phe Gly Gln Thr 165 170 175Gly Asp Ala Asp Ser Val Pro
Asp Pro Gln Pro Leu Gly Gln Pro Pro 180 185 190Ala Ala Pro Ser Gly
Leu Gly Thr Asn Thr Met Ala Thr Gly Ser Gly 195 200 205Ala Pro Met
Ala Asp Asn Asn Glu Gly Ala Asp Gly Val Gly Asn Ser 210 215 220Ser
Gly Asn Trp His Cys Asp Ser Thr Trp Met Gly Asp Arg Val Ile225 230
235 240Thr Thr Ser Thr Arg Thr Trp Ala Leu Pro Thr Tyr Asn Asn His
Leu 245 250 255Tyr Lys Gln Ile Ser Ser Gln Ser Gly Ala Ser Asn Asp
Asn His Tyr 260 265 270Phe Gly Tyr Ser Thr Pro Trp Gly Tyr Phe Asp
Phe Asn Arg Phe His 275 280 285Cys His Phe Ser Pro Arg Asp Trp Gln
Arg Leu Ile Asn Asn Asn Trp 290 295 300Gly Phe Arg Pro Lys Arg Leu
Asn Phe Lys Leu Phe Asn Ile Gln Val305 310 315 320Lys Glu Val Thr
Gln Asn Asp Gly Thr Thr Thr Ile Ala Asn Asn Leu 325 330 335Thr Ser
Thr Val Gln Val Phe Thr Asp Ser Glu Tyr Gln Leu Pro Tyr 340 345
350Val Leu Gly Ser Ala His Gln Gly Cys Leu Pro Pro Phe Pro Ala Asp
355 360 365Val Phe Met Val Pro Gln Tyr Gly Tyr Leu Thr Leu Asn Asn
Gly Ser 370 375 380Gln Ala Val Gly Arg Ser Ser Phe Tyr Cys Leu Glu
Tyr Phe Pro Ser385 390 395 400Gln Met Leu Arg Thr Gly Asn Asn Phe
Thr Phe Ser Tyr Thr Phe Glu 405 410 415Asp Val Pro Phe His Ser Ser
Tyr Ala His Ser Gln Ser Leu Asp Arg 420 425 430Leu Met Asn Pro Leu
Ile Asp Gln Tyr Leu Tyr Tyr Leu Ser Arg Thr 435 440 445Asn Thr Pro
Ser Gly Thr Thr Thr Gln Ser Arg Leu Gln Phe Ser Gln 450 455 460Ala
Gly Ala Ser Asp Ile Arg Asp Gln Ser Arg Asn Trp Leu Pro Gly465 470
475 480Pro Cys Tyr Arg Gln Gln Arg Val Ser Lys Thr Ser Ala Asp Asn
Asn 485 490 495Asn Ser Glu Tyr Ser Trp Thr Gly Ala Thr Lys Tyr His
Leu Asn Gly 500 505 510Arg Asp Ser Leu Val Asn Pro Gly Pro Ala Met
Ala Ser His Lys Asp 515 520 525Asp Glu Glu Lys Phe Phe Pro Gln Ser
Gly Val Leu Ile Phe Gly Lys 530 535 540Gln Gly Ser Glu Lys Thr Asn
Val Asp Ile Glu Lys Val Met Ile Thr545 550 555 560Asp Glu Glu Glu
Ile Arg Thr Thr Asn Pro Val Ala Thr Glu Gln Tyr 565 570 575Gly Ser
Val Ser Thr Asn Leu Gln Arg Gly Asn Arg Gln Ala Ala Thr 580 585
590Ala Asp Val Asn Thr Gln Gly Val Leu Pro Gly Met Val Trp Gln Asp
595 600 605Arg Asp Val Tyr Leu Gln Gly Pro Ile Trp Ala Lys Ile Pro
His Thr 610 615 620Asp Gly His Phe His Pro Ser Pro Leu Met Gly Gly
Phe Gly Leu Lys625 630 635 640His Pro Pro Pro Gln Ile Leu Ile Lys
Asn Thr Pro Val Pro Ala Asn 645 650 655Pro Ser Thr Thr Phe Ser Ala
Ala Lys Phe Ala Ser Phe Ile Thr Gln 660 665 670Tyr Ser Thr Gly Gln
Val Ser Val Glu Ile Glu Trp Glu Leu Gln Lys 675 680 685Glu Asn Ser
Lys Arg Trp Asn Pro Glu Ile Gln Tyr Thr Ser Asn Tyr 690 695 700Asn
Lys Ser Val Asn Val Asp Phe Thr Val Asp Thr Asn Gly Val Tyr705 710
715 720Ser Glu Pro Arg Pro Ile Gly Thr Arg Tyr Leu Thr Arg Asn Leu
725 730 7352735PRTArtificial sequenceSynthetic sequence Amino acid
sequence of true-type adeno-associated virus 2 (ttAAV2) capsid
protein VP1 2Met Ala Ala Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp
Thr Leu Ser1 5 10 15Glu Gly Ile Arg Gln Trp Trp Lys Leu Lys Pro Gly
Pro Pro Pro Pro 20 25 30Lys Pro Ala Glu Arg His Lys Asp Asp Ser Arg
Gly Leu Val Leu Pro 35 40 45Gly Tyr Lys Tyr Leu Gly Pro Phe Asn Gly
Leu Asp Lys Gly Glu Pro 50 55 60Val Asn Glu Ala Asp Ala Ala Ala Leu
Glu His Asp Lys Ala Tyr Asp65 70 75 80Arg Gln Leu Asp Ser Gly Asp
Asn Pro Tyr Leu Lys Tyr Asn His Ala 85 90 95Asp Ala Glu Phe Gln Glu
Arg Leu Lys Glu Asp Thr Ser Phe Gly Gly 100 105 110Asn Leu Gly Arg
Ala Val Phe Gln Ala Lys Lys Arg Ile Leu Glu Pro 115 120 125Leu Gly
Leu Val Glu Glu Pro Val Lys Thr Ala Pro Gly Lys Lys Arg 130 135
140Pro Val Glu His Ser Pro Ala Glu Pro Asp Ser Ser Ser Gly Thr
Gly145 150 155 160Lys Ser Gly Gln Gln Pro Ala Arg Lys Arg Leu Asn
Phe Gly Gln Thr 165 170 175Gly Asp Ala Asp Ser Val Pro Asp Pro Gln
Pro Leu Gly Gln Pro Pro 180 185 190Ala Ala Pro Ser Gly Leu Gly Thr
Asn Thr Met Ala Ser Gly Ser Gly 195 200 205Ala Pro Met Ala Asp Asn
Asn Glu Gly Ala Asp Gly Val Gly Asn Ser 210 215 220Ser Gly Asn Trp
His Cys Asp Ser Thr Trp Met Gly Asp Arg Val Ile225 230 235 240Thr
Thr Ser Thr Arg Thr Trp Ala Leu Pro Thr Tyr Asn Asn His Leu 245 250
255Tyr Lys Gln Ile Ser Ser Gln Ser Gly Ala Ser Asn Asp Asn His Tyr
260 265 270Phe Gly Tyr Ser Thr Pro Trp Gly Tyr Phe Asp Phe Asn Arg
Phe His 275 280 285Cys His Phe Ser Pro Arg Asp Trp Gln Arg Leu Ile
Asn Asn Asn Trp 290 295 300Gly Phe Arg Pro Lys Arg Leu Ser Phe Lys
Leu Phe Asn Ile Gln Val305 310 315 320Lys Glu Val Thr Gln Asn Asp
Gly Thr Thr Thr Ile Ala Asn Asn Leu 325 330 335Thr Ser Thr Val Gln
Val Phe Thr Asp Ser Glu Tyr Gln Leu Pro Tyr 340 345 350Val Leu Gly
Ser Ala His Gln Gly Cys Leu Pro Pro Phe Pro Ala Asp 355 360 365Val
Phe Met Val Pro Gln Tyr Gly Tyr Leu Thr Leu Asn Asn Gly Ser 370 375
380Gln Ala Val Gly Arg Ser Ser Phe Tyr Cys Leu Glu Tyr Phe Pro
Ser385 390 395 400Gln Met Leu Arg Thr Gly Asn Asn Phe Thr Phe Ser
Tyr Thr Phe Glu 405 410 415Asp Val Pro Phe His Ser Ser Tyr Ala His
Ser Gln Ser Leu Asp Arg 420 425 430Leu Met Asn Pro Leu Ile Asp Gln
Tyr Leu Tyr Tyr Leu Ser Arg Thr 435 440 445Asn Thr Pro Ser Gly Thr
Thr Thr Met Ser Arg Leu Gln Phe Ser Gln 450 455 460Ala Gly Ala Ser
Asp Ile Arg Asp Gln Ser Arg Asn Trp Leu Pro Gly465 470 475 480Pro
Cys Tyr Arg Gln Gln Arg Val Ser Lys Thr Ala Ala Asp Asn Asn 485 490
495Asn Ser Asp Tyr Ser Trp Thr Gly Ala Thr Lys Tyr His Leu Asn Gly
500 505 510Arg Asp Ser Leu Val Asn Pro Gly Pro Ala Met Ala Ser His
Lys Asp 515 520 525Asp Glu Glu Lys Tyr Phe Pro Gln Ser Gly Val Leu
Ile Phe Gly Lys 530 535 540Gln Asp Ser Gly Lys Thr Asn Val Asp Ile
Glu Lys Val Met Ile Thr545 550 555 560Asp Glu Glu Glu Ile Arg Thr
Thr Asn Pro Val Ala Thr Glu Gln Tyr 565 570 575Gly Ser Val Ser Thr
Asn Leu Gln Ser Gly Asn Thr Gln Ala Ala Thr 580 585 590Ser Asp Val
Asn Thr Gln Gly Val Leu Pro Gly Met Val Trp Gln Asp 595 600 605Arg
Asp Val Tyr Leu Gln Gly Pro Ile Trp Ala Lys Ile Pro His Thr 610 615
620Asp Gly His Phe His Pro Ser Pro Leu Met Gly Gly Phe Gly Leu
Lys625 630 635 640His Pro Pro Pro Gln Ile Leu Ile Lys Asn Thr Pro
Val Pro Ala Asn 645 650 655Pro Ser Thr Thr Phe Ser Ala Ala Lys Phe
Ala Ser Phe Ile Thr Gln 660 665 670Tyr Ser Thr Gly Gln Val Ser Val
Glu Ile Glu Trp Glu Leu Gln Lys 675 680 685Glu Asn Ser Lys Arg Trp
Asn Pro Glu Ile Gln Tyr Thr Ser Asn Tyr 690 695 700Asn Lys Ser Val
Asn Val Asp Phe Thr Val Asp Thr Asn Gly Val Tyr705 710 715 720Ser
Glu Pro Arg Pro Ile Gly Thr Arg Tyr Leu Thr Arg Asn Leu 725 730
7353736PRTAdeno-associated virus-1 3Met Ala Ala Asp Gly Tyr Leu Pro
Asp Trp Leu Glu Asp Asn Leu Ser1 5 10 15Glu Gly Ile Arg Glu Trp Trp
Asp Leu Lys Pro Gly Ala Pro Lys Pro 20 25 30Lys Ala Asn Gln Gln Lys
Gln Asp Asp Gly Arg Gly Leu Val Leu Pro 35 40 45Gly Tyr Lys Tyr Leu
Gly Pro Phe Asn Gly Leu Asp Lys Gly Glu Pro 50 55 60Val Asn Ala Ala
Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp65 70 75 80Gln Gln
Leu Lys Ala Gly Asp Asn Pro Tyr Leu Arg Tyr Asn His Ala 85 90 95Asp
Ala Glu Phe Gln Glu Arg Leu Gln Glu Asp Thr Ser Phe Gly Gly 100 105
110Asn Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg Val Leu Glu Pro
115 120 125Leu Gly Leu Val Glu Glu Gly Ala Lys Thr Ala Pro Gly Lys
Lys Arg 130 135 140Pro Val Glu Gln Ser Pro Gln Glu Pro Asp Ser Ser
Ser Gly Ile Gly145 150 155 160Lys Thr Gly Gln Gln Pro Ala Lys Lys
Arg Leu Asn Phe Gly Gln Thr 165 170 175Gly Asp Ser Glu Ser Val Pro
Asp Pro Gln Pro Leu Gly Glu Pro Pro 180 185 190Ala Thr Pro Ala Ala
Val Gly Pro Thr Thr Met Ala Ser Gly Gly Gly 195 200 205Ala Pro Met
Ala Asp Asn Asn Glu Gly Ala Asp Gly Val Gly Asn Ala 210 215 220Ser
Gly Asn Trp His Cys Asp Ser Thr Trp Leu Gly Asp Arg Val Ile225 230
235 240Thr Thr Ser Thr Arg Thr Trp Ala Leu Pro Thr Tyr Asn Asn His
Leu 245 250 255Tyr Lys Gln Ile Ser Ser Ala Ser Thr Gly Ala Ser Asn
Asp Asn His 260 265 270Tyr Phe Gly Tyr Ser Thr Pro Trp Gly Tyr Phe
Asp Phe Asn Arg Phe 275 280 285His Cys His Phe Ser Pro Arg Asp Trp
Gln Arg Leu Ile Asn Asn Asn 290 295 300Trp Gly Phe Arg Pro Lys Arg
Leu Asn Phe Lys Leu Phe Asn Ile Gln305 310 315 320Val Lys Glu Val
Thr Thr Asn Asp Gly Val Thr Thr Ile Ala Asn Asn 325 330 335Leu Thr
Ser Thr Val Gln Val Phe Ser Asp Ser Glu Tyr Gln Leu Pro 340 345
350Tyr Val Leu Gly Ser Ala His Gln Gly Cys Leu Pro Pro Phe Pro Ala
355 360 365Asp Val Phe Met Ile Pro Gln Tyr Gly Tyr Leu Thr Leu Asn
Asn Gly 370 375 380Ser Gln Ala Val Gly Arg Ser Ser Phe Tyr Cys Leu
Glu Tyr Phe Pro385 390 395 400Ser Gln Met Leu Arg Thr Gly Asn Asn
Phe Thr Phe Ser Tyr Thr Phe 405 410 415Glu Glu Val Pro Phe His Ser
Ser Tyr Ala His Ser Gln Ser Leu Asp 420 425 430Arg Leu Met Asn Pro
Leu Ile Asp Gln Tyr Leu Tyr Tyr Leu Asn Arg 435 440 445Thr Gln Asn
Gln Ser Gly Ser Ala Gln Asn Lys Asp Leu Leu Phe Ser 450 455 460Arg
Gly Ser Pro Ala Gly Met Ser Val Gln Pro Lys Asn Trp Leu Pro465 470
475 480Gly Pro Cys Tyr Arg Gln Gln Arg Val Ser Lys Thr Lys Thr Asp
Asn 485 490 495Asn Asn Ser Asn Phe Thr Trp Thr Gly Ala Ser Lys Tyr
Asn Leu Asn 500 505 510Gly Arg Glu Ser Ile Ile Asn Pro Gly Thr Ala
Met Ala Ser His Lys 515 520 525Asp Asp Glu Asp Lys Phe Phe Pro Met
Ser Gly Val Met Ile Phe Gly 530 535 540Lys Glu Ser Ala Gly Ala Ser
Asn Thr Ala Leu Asp Asn Val Met Ile545 550 555 560Thr Asp Glu Glu
Glu Ile Lys Ala Thr Asn Pro Val Ala Thr Glu Arg 565 570 575Phe Gly
Thr Val Ala Val Asn Phe Gln Ser Ser Ser Thr Asp Pro Ala 580 585
590Thr Gly Asp Val His Ala Met Gly Ala Leu Pro Gly Met Val Trp Gln
595 600 605Asp Arg Asp Val Tyr Leu Gln Gly Pro Ile Trp Ala Lys Ile
Pro His 610 615 620Thr Asp Gly His Phe His Pro Ser Pro Leu Met Gly
Gly Phe Gly Leu625 630 635 640Lys Asn Pro Pro Pro Gln Ile Leu Ile
Lys Asn Thr Pro Val Pro Ala 645 650 655Asn Pro Pro Ala Glu Phe Ser
Ala Thr Lys Phe Ala Ser Phe Ile Thr 660 665 670Gln Tyr Ser Thr Gly
Gln Val Ser Val Glu Ile Glu Trp Glu Leu Gln 675 680 685Lys Glu Asn
Ser Lys Arg Trp Asn Pro Glu Val Gln Tyr Thr Ser Asn 690 695 700Tyr
Ala Lys Ser Ala Asn Val Asp Phe Thr Val Asp Asn Asn Gly Leu705 710
715 720Tyr Thr Glu Pro Arg Pro Ile Gly Thr Arg Tyr Leu Thr Arg Pro
Leu 725 730 7354724PRTAdeno-associated virus-5 4Met Ser Phe Val Asp
His Pro Pro Asp Trp Leu Glu Glu Val Gly Glu1 5 10 15Gly Leu Arg Glu
Phe Leu Gly Leu Glu Ala Gly Pro Pro Lys Pro Lys 20 25 30Pro Asn Gln
Gln His Gln Asp Gln Ala Arg Gly Leu Val Leu Pro Gly 35 40 45Tyr Asn
Tyr Leu Gly Pro Gly Asn Gly Leu Asp Arg Gly Glu Pro Val 50 55 60Asn
Arg Ala Asp Glu Val Ala Arg Glu His Asp Ile Ser Tyr Asn Glu65 70 75
80Gln Leu Glu Ala Gly Asp Asn Pro Tyr Leu Lys Tyr Asn His Ala Asp
85 90 95Ala Glu Phe Gln Glu Lys Leu Ala Asp Asp Thr Ser Phe Gly Gly
Asn 100 105 110Leu Gly Lys Ala Val Phe Gln Ala Lys Lys Arg Val Leu
Glu Pro Phe 115 120 125Gly Leu Val Glu Glu Gly Ala Lys Thr Ala Pro
Thr Gly Lys Arg Ile 130 135 140Asp Asp His Phe Pro Lys Arg Lys Lys
Ala Arg Thr Glu Glu Asp Ser145 150 155 160Lys Pro Ser Thr Ser Ser
Asp Ala Glu Ala Gly Pro Ser Gly Ser Gln 165 170 175Gln Leu Gln Ile
Pro Ala Gln Pro Ala Ser Ser Leu Gly Ala Asp Thr 180 185 190Met Ser
Ala Gly Gly Gly Gly Pro Leu Gly Asp Asn Asn Gln Gly Ala 195 200
205Asp Gly Val Gly Asn Ala Ser Gly Asp Trp His Cys Asp Ser Thr Trp
210 215 220Met Gly Asp Arg Val Val Thr Lys Ser Thr Arg Thr Trp Val
Leu Pro225
230 235 240Ser Tyr Asn Asn His Gln Tyr Arg Glu Ile Lys Ser Gly Ser
Val Asp 245 250 255Gly Ser Asn Ala Asn Ala Tyr Phe Gly Tyr Ser Thr
Pro Trp Gly Tyr 260 265 270Phe Asp Phe Asn Arg Phe His Ser His Trp
Ser Pro Arg Asp Trp Gln 275 280 285Arg Leu Ile Asn Asn Tyr Trp Gly
Phe Arg Pro Arg Ser Leu Arg Val 290 295 300Lys Ile Phe Asn Ile Gln
Val Lys Glu Val Thr Val Gln Asp Ser Thr305 310 315 320Thr Thr Ile
Ala Asn Asn Leu Thr Ser Thr Val Gln Val Phe Thr Asp 325 330 335Asp
Asp Tyr Gln Leu Pro Tyr Val Val Gly Asn Gly Thr Glu Gly Cys 340 345
350Leu Pro Ala Phe Pro Pro Gln Val Phe Thr Leu Pro Gln Tyr Gly Tyr
355 360 365Ala Thr Leu Asn Arg Asp Asn Thr Glu Asn Pro Thr Glu Arg
Ser Ser 370 375 380Phe Phe Cys Leu Glu Tyr Phe Pro Ser Lys Met Leu
Arg Thr Gly Asn385 390 395 400Asn Phe Glu Phe Thr Tyr Asn Phe Glu
Glu Val Pro Phe His Ser Ser 405 410 415Phe Ala Pro Ser Gln Asn Leu
Phe Lys Leu Ala Asn Pro Leu Val Asp 420 425 430Gln Tyr Leu Tyr Arg
Phe Val Ser Thr Asn Asn Thr Gly Gly Val Gln 435 440 445Phe Asn Lys
Asn Leu Ala Gly Arg Tyr Ala Asn Thr Tyr Lys Asn Trp 450 455 460Phe
Pro Gly Pro Met Gly Arg Thr Gln Gly Trp Asn Leu Gly Ser Gly465 470
475 480Val Asn Arg Ala Ser Val Ser Ala Phe Ala Thr Thr Asn Arg Met
Glu 485 490 495Leu Glu Gly Ala Ser Tyr Gln Val Pro Pro Gln Pro Asn
Gly Met Thr 500 505 510Asn Asn Leu Gln Gly Ser Asn Thr Tyr Ala Leu
Glu Asn Thr Met Ile 515 520 525Phe Asn Ser Gln Pro Ala Asn Pro Gly
Thr Thr Ala Thr Tyr Leu Glu 530 535 540Gly Asn Met Leu Ile Thr Ser
Glu Ser Glu Thr Gln Pro Val Asn Arg545 550 555 560Val Ala Tyr Asn
Val Gly Gly Gln Met Ala Thr Asn Asn Gln Ser Ser 565 570 575Thr Thr
Ala Pro Ala Thr Gly Thr Tyr Asn Leu Gln Glu Ile Val Pro 580 585
590Gly Ser Val Trp Met Glu Arg Asp Val Tyr Leu Gln Gly Pro Ile Trp
595 600 605Ala Lys Ile Pro Glu Thr Gly Ala His Phe His Pro Ser Pro
Ala Met 610 615 620Gly Gly Phe Gly Leu Lys His Pro Pro Pro Met Met
Leu Ile Lys Asn625 630 635 640Thr Pro Val Pro Gly Asn Ile Thr Ser
Phe Ser Asp Val Pro Val Ser 645 650 655Ser Phe Ile Thr Gln Tyr Ser
Thr Gly Gln Val Thr Val Glu Met Glu 660 665 670Trp Glu Leu Lys Lys
Glu Asn Ser Lys Arg Trp Asn Pro Glu Ile Gln 675 680 685Tyr Thr Asn
Asn Tyr Asn Asp Pro Gln Phe Val Asp Phe Ala Pro Asp 690 695 700Ser
Thr Gly Glu Tyr Arg Thr Thr Arg Pro Ile Gly Thr Arg Tyr Leu705 710
715 720Thr Arg Pro Leu5736PRTAdeno-associated virus-6 5Met Ala Ala
Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Asn Leu Ser1 5 10 15Glu Gly
Ile Arg Glu Trp Trp Asp Leu Lys Pro Gly Ala Pro Lys Pro 20 25 30Lys
Ala Asn Gln Gln Lys Gln Asp Asp Gly Arg Gly Leu Val Leu Pro 35 40
45Gly Tyr Lys Tyr Leu Gly Pro Phe Asn Gly Leu Asp Lys Gly Glu Pro
50 55 60Val Asn Ala Ala Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr
Asp65 70 75 80Gln Gln Leu Lys Ala Gly Asp Asn Pro Tyr Leu Arg Tyr
Asn His Ala 85 90 95Asp Ala Glu Phe Gln Glu Arg Leu Gln Glu Asp Thr
Ser Phe Gly Gly 100 105 110Asn Leu Gly Arg Ala Val Phe Gln Ala Lys
Lys Arg Val Leu Glu Pro 115 120 125Phe Gly Leu Val Glu Glu Gly Ala
Lys Thr Ala Pro Gly Lys Lys Arg 130 135 140Pro Val Glu Gln Ser Pro
Gln Glu Pro Asp Ser Ser Ser Gly Ile Gly145 150 155 160Lys Thr Gly
Gln Gln Pro Ala Lys Lys Arg Leu Asn Phe Gly Gln Thr 165 170 175Gly
Asp Ser Glu Ser Val Pro Asp Pro Gln Pro Leu Gly Glu Pro Pro 180 185
190Ala Thr Pro Ala Ala Val Gly Pro Thr Thr Met Ala Ser Gly Gly Gly
195 200 205Ala Pro Met Ala Asp Asn Asn Glu Gly Ala Asp Gly Val Gly
Asn Ala 210 215 220Ser Gly Asn Trp His Cys Asp Ser Thr Trp Leu Gly
Asp Arg Val Ile225 230 235 240Thr Thr Ser Thr Arg Thr Trp Ala Leu
Pro Thr Tyr Asn Asn His Leu 245 250 255Tyr Lys Gln Ile Ser Ser Ala
Ser Thr Gly Ala Ser Asn Asp Asn His 260 265 270Tyr Phe Gly Tyr Ser
Thr Pro Trp Gly Tyr Phe Asp Phe Asn Arg Phe 275 280 285His Cys His
Phe Ser Pro Arg Asp Trp Gln Arg Leu Ile Asn Asn Asn 290 295 300Trp
Gly Phe Arg Pro Lys Arg Leu Asn Phe Lys Leu Phe Asn Ile Gln305 310
315 320Val Lys Glu Val Thr Thr Asn Asp Gly Val Thr Thr Ile Ala Asn
Asn 325 330 335Leu Thr Ser Thr Val Gln Val Phe Ser Asp Ser Glu Tyr
Gln Leu Pro 340 345 350Tyr Val Leu Gly Ser Ala His Gln Gly Cys Leu
Pro Pro Phe Pro Ala 355 360 365Asp Val Phe Met Ile Pro Gln Tyr Gly
Tyr Leu Thr Leu Asn Asn Gly 370 375 380Ser Gln Ala Val Gly Arg Ser
Ser Phe Tyr Cys Leu Glu Tyr Phe Pro385 390 395 400Ser Gln Met Leu
Arg Thr Gly Asn Asn Phe Thr Phe Ser Tyr Thr Phe 405 410 415Glu Asp
Val Pro Phe His Ser Ser Tyr Ala His Ser Gln Ser Leu Asp 420 425
430Arg Leu Met Asn Pro Leu Ile Asp Gln Tyr Leu Tyr Tyr Leu Asn Arg
435 440 445Thr Gln Asn Gln Ser Gly Ser Ala Gln Asn Lys Asp Leu Leu
Phe Ser 450 455 460Arg Gly Ser Pro Ala Gly Met Ser Val Gln Pro Lys
Asn Trp Leu Pro465 470 475 480Gly Pro Cys Tyr Arg Gln Gln Arg Val
Ser Lys Thr Lys Thr Asp Asn 485 490 495Asn Asn Ser Asn Phe Thr Trp
Thr Gly Ala Ser Lys Tyr Asn Leu Asn 500 505 510Gly Arg Glu Ser Ile
Ile Asn Pro Gly Thr Ala Met Ala Ser His Lys 515 520 525Asp Asp Lys
Asp Lys Phe Phe Pro Met Ser Gly Val Met Ile Phe Gly 530 535 540Lys
Glu Ser Ala Gly Ala Ser Asn Thr Ala Leu Asp Asn Val Met Ile545 550
555 560Thr Asp Glu Glu Glu Ile Lys Ala Thr Asn Pro Val Ala Thr Glu
Arg 565 570 575Phe Gly Thr Val Ala Val Asn Leu Gln Ser Ser Ser Thr
Asp Pro Ala 580 585 590Thr Gly Asp Val His Val Met Gly Ala Leu Pro
Gly Met Val Trp Gln 595 600 605Asp Arg Asp Val Tyr Leu Gln Gly Pro
Ile Trp Ala Lys Ile Pro His 610 615 620Thr Asp Gly His Phe His Pro
Ser Pro Leu Met Gly Gly Phe Gly Leu625 630 635 640Lys His Pro Pro
Pro Gln Ile Leu Ile Lys Asn Thr Pro Val Pro Ala 645 650 655Asn Pro
Pro Ala Glu Phe Ser Ala Thr Lys Phe Ala Ser Phe Ile Thr 660 665
670Gln Tyr Ser Thr Gly Gln Val Ser Val Glu Ile Glu Trp Glu Leu Gln
675 680 685Lys Glu Asn Ser Lys Arg Trp Asn Pro Glu Val Gln Tyr Thr
Ser Asn 690 695 700Tyr Ala Lys Ser Ala Asn Val Asp Phe Thr Val Asp
Asn Asn Gly Leu705 710 715 720Tyr Thr Glu Pro Arg Pro Ile Gly Thr
Arg Tyr Leu Thr Arg Pro Leu 725 730 7356738PRTAdeno-associated
virus-8 6Met Ala Ala Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Asn
Leu Ser1 5 10 15Glu Gly Ile Arg Glu Trp Trp Ala Leu Lys Pro Gly Ala
Pro Lys Pro 20 25 30Lys Ala Asn Gln Gln Lys Gln Asp Asp Gly Arg Gly
Leu Val Leu Pro 35 40 45Gly Tyr Lys Tyr Leu Gly Pro Phe Asn Gly Leu
Asp Lys Gly Glu Pro 50 55 60Val Asn Ala Ala Asp Ala Ala Ala Leu Glu
His Asp Lys Ala Tyr Asp65 70 75 80Gln Gln Leu Gln Ala Gly Asp Asn
Pro Tyr Leu Arg Tyr Asn His Ala 85 90 95Asp Ala Glu Phe Gln Glu Arg
Leu Gln Glu Asp Thr Ser Phe Gly Gly 100 105 110Asn Leu Gly Arg Ala
Val Phe Gln Ala Lys Lys Arg Val Leu Glu Pro 115 120 125Leu Gly Leu
Val Glu Glu Gly Ala Lys Thr Ala Pro Gly Lys Lys Arg 130 135 140Pro
Val Glu Pro Ser Pro Gln Arg Ser Pro Asp Ser Ser Thr Gly Ile145 150
155 160Gly Lys Lys Gly Gln Gln Pro Ala Arg Lys Arg Leu Asn Phe Gly
Gln 165 170 175Thr Gly Asp Ser Glu Ser Val Pro Asp Pro Gln Pro Leu
Gly Glu Pro 180 185 190Pro Ala Ala Pro Ser Gly Val Gly Pro Asn Thr
Met Ala Ala Gly Gly 195 200 205Gly Ala Pro Met Ala Asp Asn Asn Glu
Gly Ala Asp Gly Val Gly Ser 210 215 220Ser Ser Gly Asn Trp His Cys
Asp Ser Thr Trp Leu Gly Asp Arg Val225 230 235 240Ile Thr Thr Ser
Thr Arg Thr Trp Ala Leu Pro Thr Tyr Asn Asn His 245 250 255Leu Tyr
Lys Gln Ile Ser Asn Gly Thr Ser Gly Gly Ala Thr Asn Asp 260 265
270Asn Thr Tyr Phe Gly Tyr Ser Thr Pro Trp Gly Tyr Phe Asp Phe Asn
275 280 285Arg Phe His Cys His Phe Ser Pro Arg Asp Trp Gln Arg Leu
Ile Asn 290 295 300Asn Asn Trp Gly Phe Arg Pro Lys Arg Leu Ser Phe
Lys Leu Phe Asn305 310 315 320Ile Gln Val Lys Glu Val Thr Gln Asn
Glu Gly Thr Lys Thr Ile Ala 325 330 335Asn Asn Leu Thr Ser Thr Ile
Gln Val Phe Thr Asp Ser Glu Tyr Gln 340 345 350Leu Pro Tyr Val Leu
Gly Ser Ala His Gln Gly Cys Leu Pro Pro Phe 355 360 365Pro Ala Asp
Val Phe Met Ile Pro Gln Tyr Gly Tyr Leu Thr Leu Asn 370 375 380Asn
Gly Ser Gln Ala Val Gly Arg Ser Ser Phe Tyr Cys Leu Glu Tyr385 390
395 400Phe Pro Ser Gln Met Leu Arg Thr Gly Asn Asn Phe Gln Phe Thr
Tyr 405 410 415Thr Phe Glu Asp Val Pro Phe His Ser Ser Tyr Ala His
Ser Gln Ser 420 425 430Leu Asp Arg Leu Met Asn Pro Leu Ile Asp Gln
Tyr Leu Tyr Tyr Leu 435 440 445Ser Arg Thr Gln Thr Thr Gly Gly Thr
Ala Asn Thr Gln Thr Leu Gly 450 455 460Phe Ser Gln Gly Gly Pro Asn
Thr Met Ala Asn Gln Ala Lys Asn Trp465 470 475 480Leu Pro Gly Pro
Cys Tyr Arg Gln Gln Arg Val Ser Thr Thr Thr Gly 485 490 495Gln Asn
Asn Asn Ser Asn Phe Ala Trp Thr Ala Gly Thr Lys Tyr His 500 505
510Leu Asn Gly Arg Asn Ser Leu Ala Asn Pro Gly Ile Ala Met Ala Thr
515 520 525His Lys Asp Asp Glu Glu Arg Phe Phe Pro Ser Asn Gly Ile
Leu Ile 530 535 540Phe Gly Lys Gln Asn Ala Ala Arg Asp Asn Ala Asp
Tyr Ser Asp Val545 550 555 560Met Leu Thr Ser Glu Glu Glu Ile Lys
Thr Thr Asn Pro Val Ala Thr 565 570 575Glu Glu Tyr Gly Ile Val Ala
Asp Asn Leu Gln Gln Gln Asn Thr Ala 580 585 590Pro Gln Ile Gly Thr
Val Asn Ser Gln Gly Ala Leu Pro Gly Met Val 595 600 605Trp Gln Asn
Arg Asp Val Tyr Leu Gln Gly Pro Ile Trp Ala Lys Ile 610 615 620Pro
His Thr Asp Gly Asn Phe His Pro Ser Pro Leu Met Gly Gly Phe625 630
635 640Gly Leu Lys His Pro Pro Pro Gln Ile Leu Ile Lys Asn Thr Pro
Val 645 650 655Pro Ala Asp Pro Pro Thr Thr Phe Asn Gln Ser Lys Leu
Asn Ser Phe 660 665 670Ile Thr Gln Tyr Ser Thr Gly Gln Val Ser Val
Glu Ile Glu Trp Glu 675 680 685Leu Gln Lys Glu Asn Ser Lys Arg Trp
Asn Pro Glu Ile Gln Tyr Thr 690 695 700Ser Asn Tyr Tyr Lys Ser Thr
Ser Val Asp Phe Ala Val Asn Thr Glu705 710 715 720Gly Val Tyr Ser
Glu Pro Arg Pro Ile Gly Thr Arg Tyr Leu Thr Arg 725 730 735Asn
Leu7736PRTAdeno-associated virus-9 7Met Ala Ala Asp Gly Tyr Leu Pro
Asp Trp Leu Glu Asp Asn Leu Ser1 5 10 15Glu Gly Ile Arg Glu Trp Trp
Ala Leu Lys Pro Gly Ala Pro Gln Pro 20 25 30Lys Ala Asn Gln Gln His
Gln Asp Asn Ala Arg Gly Leu Val Leu Pro 35 40 45Gly Tyr Lys Tyr Leu
Gly Pro Gly Asn Gly Leu Asp Lys Gly Glu Pro 50 55 60Val Asn Ala Ala
Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp65 70 75 80Gln Gln
Leu Lys Ala Gly Asp Asn Pro Tyr Leu Lys Tyr Asn His Ala 85 90 95Asp
Ala Glu Phe Gln Glu Arg Leu Lys Glu Asp Thr Ser Phe Gly Gly 100 105
110Asn Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg Leu Leu Glu Pro
115 120 125Leu Gly Leu Val Glu Glu Ala Ala Lys Thr Ala Pro Gly Lys
Lys Arg 130 135 140Pro Val Glu Gln Ser Pro Gln Glu Pro Asp Ser Ser
Ala Gly Ile Gly145 150 155 160Lys Ser Gly Ala Gln Pro Ala Lys Lys
Arg Leu Asn Phe Gly Gln Thr 165 170 175Gly Asp Thr Glu Ser Val Pro
Asp Pro Gln Pro Ile Gly Glu Pro Pro 180 185 190Ala Ala Pro Ser Gly
Val Gly Ser Leu Thr Met Ala Ser Gly Gly Gly 195 200 205Ala Pro Val
Ala Asp Asn Asn Glu Gly Ala Asp Gly Val Gly Ser Ser 210 215 220Ser
Gly Asn Trp His Cys Asp Ser Gln Trp Leu Gly Asp Arg Val Ile225 230
235 240Thr Thr Ser Thr Arg Thr Trp Ala Leu Pro Thr Tyr Asn Asn His
Leu 245 250 255Tyr Lys Gln Ile Ser Asn Ser Thr Ser Gly Gly Ser Ser
Asn Asp Asn 260 265 270Ala Tyr Phe Gly Tyr Ser Thr Pro Trp Gly Tyr
Phe Asp Phe Asn Arg 275 280 285Phe His Cys His Phe Ser Pro Arg Asp
Trp Gln Arg Leu Ile Asn Asn 290 295 300Asn Trp Gly Phe Arg Pro Lys
Arg Leu Asn Phe Lys Leu Phe Asn Ile305 310 315 320Gln Val Lys Glu
Val Thr Asp Asn Asn Gly Val Lys Thr Ile Ala Asn 325 330 335Asn Leu
Thr Ser Thr Val Gln Val Phe Thr Asp Ser Asp Tyr Gln Leu 340 345
350Pro Tyr Val Leu Gly Ser Ala His Glu Gly Cys Leu Pro Pro Phe Pro
355 360 365Ala Asp Val Phe Met Ile Pro Gln Tyr Gly Tyr Leu Thr Leu
Asn Asp 370 375 380Gly Ser Gln Ala Val Gly Arg Ser Ser Phe Tyr Cys
Leu Glu Tyr Phe385 390 395 400Pro Ser Gln Met Leu Arg Thr Gly Asn
Asn Phe Gln Phe Ser Tyr Glu 405 410 415Phe Glu Asn Val Pro Phe His
Ser Ser Tyr Ala His Ser Gln Ser Leu 420 425 430Asp Arg Leu Met Asn
Pro Leu Ile Asp Gln Tyr Leu Tyr Tyr Leu Ser 435 440 445Lys Thr Ile
Asn Gly Ser Gly Gln Asn Gln Gln Thr Leu Lys Phe Ser 450 455 460Val
Ala Gly Pro Ser Asn Met Ala Val Gln Gly Arg Asn Tyr Ile Pro465 470
475 480Gly Pro Ser Tyr Arg Gln Gln Arg Val Ser Thr Thr Val Thr Gln
Asn 485 490
495Asn Asn Ser Glu Phe Ala Trp Pro Gly Ala Ser Ser Trp Ala Leu Asn
500 505 510Gly Arg Asn Ser Leu Met Asn Pro Gly Pro Ala Met Ala Ser
His Lys 515 520 525Glu Gly Glu Asp Arg Phe Phe Pro Leu Ser Gly Ser
Leu Ile Phe Gly 530 535 540Lys Gln Gly Thr Gly Arg Asp Asn Val Asp
Ala Asp Lys Val Met Ile545 550 555 560Thr Asn Glu Glu Glu Ile Lys
Thr Thr Asn Pro Val Ala Thr Glu Ser 565 570 575Tyr Gly Gln Val Ala
Thr Asn His Gln Ser Ala Gln Ala Gln Ala Gln 580 585 590Thr Gly Trp
Val Gln Asn Gln Gly Ile Leu Pro Gly Met Val Trp Gln 595 600 605Asp
Arg Asp Val Tyr Leu Gln Gly Pro Ile Trp Ala Lys Ile Pro His 610 615
620Thr Asp Gly Asn Phe His Pro Ser Pro Leu Met Gly Gly Phe Gly
Met625 630 635 640Lys His Pro Pro Pro Gln Ile Leu Ile Lys Asn Thr
Pro Val Pro Ala 645 650 655Asp Pro Pro Thr Ala Phe Asn Lys Asp Lys
Leu Asn Ser Phe Ile Thr 660 665 670Gln Tyr Ser Thr Gly Gln Val Ser
Val Glu Ile Glu Trp Glu Leu Gln 675 680 685Lys Glu Asn Ser Lys Arg
Trp Asn Pro Glu Ile Gln Tyr Thr Ser Asn 690 695 700Tyr Tyr Lys Ser
Asn Asn Val Glu Phe Ala Val Asn Thr Glu Gly Val705 710 715 720Tyr
Ser Glu Pro Arg Pro Ile Gly Thr Arg Tyr Leu Thr Arg Asn Leu 725 730
7358738PRTAdeno-associated virus-10 8Met Ala Ala Asp Gly Tyr Leu
Pro Asp Trp Leu Glu Asp Asn Leu Ser1 5 10 15Glu Gly Ile Arg Glu Trp
Trp Asp Leu Lys Pro Gly Ala Pro Lys Pro 20 25 30Lys Ala Asn Gln Gln
Lys Gln Asp Asp Gly Arg Gly Leu Val Leu Pro 35 40 45Gly Tyr Lys Tyr
Leu Gly Pro Phe Asn Gly Leu Asp Lys Gly Glu Pro 50 55 60Val Asn Ala
Ala Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp65 70 75 80Gln
Gln Leu Lys Ala Gly Asp Asn Pro Tyr Leu Arg Tyr Asn His Ala 85 90
95Asp Ala Glu Phe Gln Glu Arg Leu Gln Glu Asp Thr Ser Phe Gly Gly
100 105 110Asn Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg Val Leu
Glu Pro 115 120 125Leu Gly Leu Val Glu Glu Gly Ala Lys Thr Ala Pro
Gly Lys Lys Arg 130 135 140Pro Val Glu Pro Ser Pro Gln Arg Ser Pro
Asp Ser Ser Thr Gly Ile145 150 155 160Gly Lys Lys Gly Gln Gln Pro
Ala Lys Lys Arg Leu Asn Phe Gly Gln 165 170 175Thr Gly Asp Ser Glu
Ser Val Pro Asp Pro Gln Pro Ile Gly Glu Pro 180 185 190Pro Ala Gly
Pro Ser Gly Leu Gly Ser Gly Thr Met Ala Ala Gly Gly 195 200 205Gly
Ala Pro Met Ala Asp Asn Asn Glu Gly Ala Asp Gly Val Gly Ser 210 215
220Ser Ser Gly Asn Trp His Cys Asp Ser Thr Trp Leu Gly Asp Arg
Val225 230 235 240Ile Thr Thr Ser Thr Arg Thr Trp Ala Leu Pro Thr
Tyr Asn Asn His 245 250 255Leu Tyr Lys Gln Ile Ser Asn Gly Thr Ser
Gly Gly Ser Thr Asn Asp 260 265 270Asn Thr Tyr Phe Gly Tyr Ser Thr
Pro Trp Gly Tyr Phe Asp Phe Asn 275 280 285Arg Phe His Cys His Phe
Ser Pro Arg Asp Trp Gln Arg Leu Ile Asn 290 295 300Asn Asn Trp Gly
Phe Arg Pro Lys Arg Leu Asn Phe Lys Leu Phe Asn305 310 315 320Ile
Gln Val Lys Glu Val Thr Gln Asn Glu Gly Thr Lys Thr Ile Ala 325 330
335Asn Asn Leu Thr Ser Thr Ile Gln Val Phe Thr Asp Ser Glu Tyr Gln
340 345 350Leu Pro Tyr Val Leu Gly Ser Ala His Gln Gly Cys Leu Pro
Pro Phe 355 360 365Pro Ala Asp Val Phe Met Ile Pro Gln Tyr Gly Tyr
Leu Thr Leu Asn 370 375 380Asn Gly Ser Gln Ala Val Gly Arg Ser Ser
Phe Tyr Cys Leu Glu Tyr385 390 395 400Phe Pro Ser Gln Met Leu Arg
Thr Gly Asn Asn Phe Glu Phe Ser Tyr 405 410 415Gln Phe Glu Asp Val
Pro Phe His Ser Ser Tyr Ala His Ser Gln Ser 420 425 430Leu Asp Arg
Leu Met Asn Pro Leu Ile Asp Gln Tyr Leu Tyr Tyr Leu 435 440 445Ser
Arg Thr Gln Ser Thr Gly Gly Thr Ala Gly Thr Gln Gln Leu Leu 450 455
460Phe Ser Gln Ala Gly Pro Asn Asn Met Ser Ala Gln Ala Lys Asn
Trp465 470 475 480Leu Pro Gly Pro Cys Tyr Arg Gln Gln Arg Val Ser
Thr Thr Leu Ser 485 490 495Gln Asn Asn Asn Ser Asn Phe Ala Trp Thr
Gly Ala Thr Lys Tyr His 500 505 510Leu Asn Gly Arg Asp Ser Leu Val
Asn Pro Gly Val Ala Met Ala Thr 515 520 525His Lys Asp Asp Glu Glu
Arg Phe Phe Pro Ser Ser Gly Val Leu Met 530 535 540Phe Gly Lys Gln
Gly Ala Gly Lys Asp Asn Val Asp Tyr Ser Ser Val545 550 555 560Met
Leu Thr Ser Glu Glu Glu Ile Lys Thr Thr Asn Pro Val Ala Thr 565 570
575Glu Gln Tyr Gly Val Val Ala Asp Asn Leu Gln Gln Gln Asn Ala Ala
580 585 590Pro Ile Val Gly Ala Val Asn Ser Gln Gly Ala Leu Pro Gly
Met Val 595 600 605Trp Gln Asn Arg Asp Val Tyr Leu Gln Gly Pro Ile
Trp Ala Lys Ile 610 615 620Pro His Thr Asp Gly Asn Phe His Pro Ser
Pro Leu Met Gly Gly Phe625 630 635 640Gly Leu Lys His Pro Pro Pro
Gln Ile Leu Ile Lys Asn Thr Pro Val 645 650 655Pro Ala Asp Pro Pro
Thr Thr Phe Ser Gln Ala Lys Leu Ala Ser Phe 660 665 670Ile Thr Gln
Tyr Ser Thr Gly Gln Val Ser Val Glu Ile Glu Trp Glu 675 680 685Leu
Gln Lys Glu Asn Ser Lys Arg Trp Asn Pro Glu Ile Gln Tyr Thr 690 695
700Ser Asn Tyr Tyr Lys Ser Thr Asn Val Asp Phe Ala Val Asn Thr
Asp705 710 715 720Gly Thr Tyr Ser Glu Pro Arg Pro Ile Gly Thr Arg
Tyr Leu Thr Arg 725 730 735Asn Leu9738PRTAdeno-associated virus 10
9Met Ala Ala Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Asn Leu Ser1 5
10 15Glu Gly Ile Arg Glu Trp Trp Asp Leu Lys Pro Gly Ala Pro Lys
Pro 20 25 30Lys Ala Asn Gln Gln Lys Gln Asp Asp Gly Arg Gly Leu Val
Leu Pro 35 40 45Gly Tyr Lys Tyr Leu Gly Pro Phe Asn Gly Leu Asp Lys
Gly Glu Pro 50 55 60Val Asn Ala Ala Asp Ala Ala Ala Leu Glu His Asp
Lys Ala Tyr Asp65 70 75 80Gln Gln Leu Lys Ala Gly Asp Asn Pro Tyr
Leu Arg Tyr Asn His Ala 85 90 95Asp Ala Glu Phe Gln Glu Arg Leu Gln
Glu Asp Thr Ser Phe Gly Gly 100 105 110Asn Leu Gly Arg Ala Val Phe
Gln Ala Lys Lys Arg Val Leu Glu Pro 115 120 125Leu Gly Leu Val Glu
Glu Ala Ala Lys Thr Ala Pro Gly Lys Lys Arg 130 135 140Pro Val Glu
Pro Ser Pro Gln Arg Ser Pro Asp Ser Ser Thr Gly Ile145 150 155
160Gly Lys Lys Gly Gln Gln Pro Ala Lys Lys Arg Leu Asn Phe Gly Gln
165 170 175Thr Gly Glu Ser Glu Ser Val Pro Asp Pro Gln Pro Ile Gly
Glu Pro 180 185 190Pro Ala Gly Pro Ser Gly Leu Gly Ser Gly Thr Met
Ala Ala Gly Gly 195 200 205Gly Ala Pro Met Ala Asp Asn Asn Glu Gly
Ala Asp Gly Val Gly Ser 210 215 220Ser Ser Gly Asn Trp His Cys Asp
Ser Thr Trp Leu Gly Asp Arg Val225 230 235 240Ile Thr Thr Ser Thr
Arg Thr Trp Ala Leu Pro Thr Tyr Asn Asn His 245 250 255Leu Tyr Lys
Gln Ile Ser Asn Gly Thr Ser Gly Gly Ser Thr Asn Asp 260 265 270Asn
Thr Tyr Phe Gly Tyr Ser Thr Pro Trp Gly Tyr Phe Asp Phe Asn 275 280
285Arg Phe His Cys His Phe Ser Pro Arg Asp Trp Gln Arg Leu Ile Asn
290 295 300Asn Asn Trp Gly Phe Arg Pro Lys Arg Leu Ser Phe Lys Leu
Phe Asn305 310 315 320Ile Gln Val Lys Glu Val Thr Gln Asn Glu Gly
Thr Lys Thr Ile Ala 325 330 335Asn Asn Leu Thr Ser Thr Ile Gln Val
Phe Thr Asp Ser Glu Tyr Gln 340 345 350Leu Pro Tyr Val Leu Gly Ser
Ala His Gln Gly Cys Leu Pro Pro Phe 355 360 365Pro Ala Asp Val Phe
Met Ile Pro Gln Tyr Gly Tyr Leu Thr Leu Asn 370 375 380Asn Gly Ser
Gln Ala Val Gly Arg Ser Ser Phe Tyr Cys Leu Glu Tyr385 390 395
400Phe Pro Ser Gln Met Leu Arg Thr Gly Asn Asn Phe Glu Phe Ser Tyr
405 410 415Thr Phe Glu Asp Val Pro Phe His Ser Ser Tyr Ala His Ser
Gln Ser 420 425 430Leu Asp Arg Leu Met Asn Pro Leu Ile Asp Gln Tyr
Leu Tyr Tyr Leu 435 440 445Ser Arg Thr Gln Ser Thr Gly Gly Thr Gln
Gly Thr Gln Gln Leu Leu 450 455 460Phe Ser Gln Ala Gly Pro Ala Asn
Met Ser Ala Gln Ala Lys Asn Trp465 470 475 480Leu Pro Gly Pro Cys
Tyr Arg Gln Gln Arg Val Ser Thr Thr Leu Ser 485 490 495Gln Asn Asn
Asn Ser Asn Phe Ala Trp Thr Gly Ala Thr Lys Tyr His 500 505 510Leu
Asn Gly Arg Asp Ser Leu Val Asn Pro Gly Val Ala Met Ala Thr 515 520
525His Lys Asp Asp Glu Glu Arg Phe Phe Pro Ser Ser Gly Val Leu Met
530 535 540Phe Gly Lys Gln Gly Ala Gly Arg Asp Asn Val Asp Tyr Ser
Ser Val545 550 555 560Met Leu Thr Ser Glu Glu Glu Ile Lys Thr Thr
Asn Pro Val Ala Thr 565 570 575Glu Gln Tyr Gly Val Val Ala Asp Asn
Leu Gln Gln Ala Asn Thr Gly 580 585 590Pro Ile Val Gly Asn Val Asn
Ser Gln Gly Ala Leu Pro Gly Met Val 595 600 605Trp Gln Asn Arg Asp
Val Tyr Leu Gln Gly Pro Ile Trp Ala Lys Ile 610 615 620Pro His Thr
Asp Gly Asn Phe His Pro Ser Pro Leu Met Gly Gly Phe625 630 635
640Gly Leu Lys His Pro Pro Pro Gln Ile Leu Ile Lys Asn Thr Pro Val
645 650 655Pro Ala Asp Pro Pro Thr Thr Phe Ser Gln Ala Lys Leu Ala
Ser Phe 660 665 670Ile Thr Gln Tyr Ser Thr Gly Gln Val Ser Val Glu
Ile Glu Trp Glu 675 680 685Leu Gln Lys Glu Asn Ser Lys Arg Trp Asn
Pro Glu Ile Gln Tyr Thr 690 695 700Ser Asn Tyr Tyr Lys Ser Thr Asn
Val Asp Phe Ala Val Asn Thr Glu705 710 715 720Gly Thr Tyr Ser Glu
Pro Arg Pro Ile Gly Thr Arg Tyr Leu Thr Arg 725 730 735Asn
Leu10744PRTArtificial sequenceConsensus sequenceSITE(148)..(148)Xaa
indicates no overall consensus with any amino
acidSITE(202)..(202)Xaa indicates no overall consensus with any
amino acidSITE(207)..(207)Xaa indicates no overall consensus with
any amino acidSITE(270)..(270)Xaa indicates no overall consensus
with any amino acidSITE(458)..(458)Xaa indicates no overall
consensus with any amino acidSITE(462)..(462)Xaa indicates no
overall consensus with any amino acidSITE(465)..(465)Xaa indicates
no overall consensus with any amino acidSITE(474)..(474)Xaa
indicates no overall consensus with any amino
acidSITE(496)..(496)Xaa indicates no overall consensus with any
amino acidSITE(498)..(498)Xaa indicates no overall consensus with
any amino acidSITE(500)..(500)Xaa indicates no overall consensus
with any amino acidSITE(538)..(538)Xaa indicates no overall
consensus with any amino acidSITE(557)..(557)Xaa indicates no
overall consensus with any amino acidSITE(562)..(563)Xaa indicates
no overall consensus with any amino acidSITE(565)..(565)Xaa
indicates no overall consensus with any amino
acidSITE(570)..(570)Xaa indicates no overall consensus with any
amino acidSITE(587)..(587)Xaa indicates no overall consensus with
any amino acidSITE(598)..(598)Xaa indicates no overall consensus
with any amino acidSITE(675)..(675)Xaa indicates no overall
consensus with any amino acid 10Met Ala Ala Asp Gly Tyr Leu Pro Asp
Trp Leu Glu Asp Asn Leu Ser1 5 10 15Glu Gly Ile Arg Glu Trp Trp Asp
Leu Lys Pro Gly Ala Pro Lys Pro 20 25 30Lys Ala Asn Gln Gln Lys Gln
Asp Asp Gly Arg Gly Leu Val Leu Pro 35 40 45Gly Tyr Lys Tyr Leu Gly
Pro Phe Asn Gly Leu Asp Lys Gly Glu Pro 50 55 60Val Asn Ala Ala Asp
Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp65 70 75 80Gln Gln Leu
Lys Ala Gly Asp Asn Pro Tyr Leu Arg Tyr Asn His Ala 85 90 95Asp Ala
Glu Phe Gln Glu Arg Leu Gln Glu Asp Thr Ser Phe Gly Gly 100 105
110Asn Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg Val Leu Glu Pro
115 120 125Leu Gly Leu Val Glu Glu Gly Ala Lys Thr Ala Pro Gly Lys
Lys Arg 130 135 140Pro Val Glu Xaa Ser Pro Gln Arg Glu Pro Asp Ser
Ser Ser Gly Ile145 150 155 160Gly Lys Lys Gly Gln Gln Pro Ala Lys
Lys Arg Leu Asn Phe Gly Gln 165 170 175Thr Gly Asp Ser Glu Ser Val
Pro Asp Gly Pro Gln Pro Leu Gly Glu 180 185 190Pro Pro Ala Ala Pro
Ser Gly Leu Gly Xaa Asn Thr Met Ala Xaa Gly 195 200 205Gly Gly Ala
Pro Met Ala Asp Asn Asn Glu Gly Ala Asp Gly Val Gly 210 215 220Asn
Ser Ser Gly Asn Trp His Cys Asp Ser Thr Trp Leu Gly Asp Arg225 230
235 240Val Ile Thr Thr Ser Thr Arg Thr Trp Ala Leu Pro Thr Tyr Asn
Asn 245 250 255His Leu Tyr Lys Gln Ile Ser Ser Gly Ser Ser Gly Gly
Xaa Ser Asn 260 265 270Asp Asn His Tyr Phe Gly Tyr Ser Thr Pro Trp
Gly Tyr Phe Asp Phe 275 280 285Asn Arg Phe His Cys His Phe Ser Pro
Arg Asp Trp Gln Arg Leu Ile 290 295 300Asn Asn Asn Trp Gly Phe Arg
Pro Lys Arg Leu Asn Phe Lys Leu Phe305 310 315 320Asn Ile Gln Val
Lys Glu Val Thr Gln Asn Asp Gly Thr Thr Thr Ile 325 330 335Ala Asn
Asn Leu Thr Ser Thr Val Gln Val Phe Thr Asp Ser Glu Tyr 340 345
350Gln Leu Pro Tyr Val Leu Gly Ser Ala His Gln Gly Cys Leu Pro Pro
355 360 365Phe Pro Ala Asp Val Phe Met Ile Pro Gln Tyr Gly Tyr Leu
Thr Leu 370 375 380Asn Arg Asp Asn Gly Ser Gln Ala Val Gly Arg Ser
Ser Phe Tyr Cys385 390 395 400Leu Glu Tyr Phe Pro Ser Gln Met Leu
Arg Thr Gly Asn Asn Phe Thr 405 410 415Phe Ser Tyr Thr Phe Glu Asp
Val Pro Phe His Ser Ser Tyr Ala His 420 425 430Ser Gln Ser Leu Asp
Arg Leu Met Asn Pro Leu Ile Asp Gln Tyr Leu 435 440 445Tyr Tyr Leu
Ser Arg Thr Gln Asn Thr Xaa Gly Thr Ala Xaa Thr Gln 450 455 460Xaa
Leu Leu Phe Ser Gln Ala Gly Pro Xaa Asn Met Ser Val Gln Ala465 470
475 480Lys Asn Trp Leu Pro Gly Pro Cys Tyr Arg Gln Gln Arg Val Ser
Xaa 485 490 495Thr Xaa Thr Xaa Asn Asn Asn Ser Asn Phe Ala Trp Thr
Gly Ala Thr 500 505 510Lys Tyr His Leu Asn Gly Arg Asp Ser Leu Val
Asn Pro Gly Pro Ala 515 520 525Met Ala Ser His Lys Asp Asp Glu Glu
Xaa Phe Phe Pro Ser Ser Gly 530
535 540Val Leu Ile Phe Gly Lys Gln Gly Ala Asn Pro Gly Xaa Asp Asn
Val545 550 555 560Asp Xaa Xaa Gly Xaa Val Met Ile Thr Xaa Glu Glu
Glu Ile Lys Thr 565 570 575Thr Asn Pro Val Ala Thr Glu Gln Tyr Gly
Xaa Val Ala Thr Asn Leu 580 585 590Gln Ser Ser Asn Thr Xaa Pro Ala
Thr Gly Asp Val Asn Ser Gln Gly 595 600 605Ala Leu Pro Gly Met Val
Trp Gln Asp Arg Asp Val Tyr Leu Gln Gly 610 615 620Pro Ile Trp Ala
Lys Ile Pro His Thr Asp Gly His Phe His Pro Ser625 630 635 640Pro
Leu Met Gly Gly Phe Gly Leu Lys His Pro Pro Pro Gln Ile Leu 645 650
655Ile Lys Asn Thr Pro Val Pro Ala Asn Pro Pro Thr Thr Phe Ser Ala
660 665 670Ala Lys Xaa Ala Ser Phe Ile Thr Gln Tyr Ser Thr Gly Gln
Val Ser 675 680 685Val Glu Ile Glu Trp Glu Leu Gln Lys Glu Asn Ser
Lys Arg Trp Asn 690 695 700Pro Glu Ile Gln Tyr Thr Ser Asn Tyr Tyr
Lys Ser Thr Asn Val Asp705 710 715 720Phe Ala Val Asp Thr Asn Gly
Val Tyr Ser Glu Pro Arg Pro Ile Gly 725 730 735Thr Arg Tyr Leu Thr
Arg Asn Leu 74011736PRTAdeno-associated virus 3B 11Met Ala Ala Asp
Gly Tyr Leu Pro Asp Trp Leu Glu Asp Asn Leu Ser1 5 10 15Glu Gly Ile
Arg Glu Trp Trp Ala Leu Lys Pro Gly Val Pro Gln Pro 20 25 30Lys Ala
Asn Gln Gln His Gln Asp Asn Arg Arg Gly Leu Val Leu Pro 35 40 45Gly
Tyr Lys Tyr Leu Gly Pro Gly Asn Gly Leu Asp Lys Gly Glu Pro 50 55
60Val Asn Glu Ala Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp65
70 75 80Gln Gln Leu Lys Ala Gly Asp Asn Pro Tyr Leu Lys Tyr Asn His
Ala 85 90 95Asp Ala Glu Phe Gln Glu Arg Leu Gln Glu Asp Thr Ser Phe
Gly Gly 100 105 110Asn Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg
Ile Leu Glu Pro 115 120 125Leu Gly Leu Val Glu Glu Ala Ala Lys Thr
Ala Pro Gly Lys Lys Arg 130 135 140Pro Val Asp Gln Ser Pro Gln Glu
Pro Asp Ser Ser Ser Gly Val Gly145 150 155 160Lys Ser Gly Lys Gln
Pro Ala Arg Lys Arg Leu Asn Phe Gly Gln Thr 165 170 175Gly Asp Ser
Glu Ser Val Pro Asp Pro Gln Pro Leu Gly Glu Pro Pro 180 185 190Ala
Ala Pro Thr Ser Leu Gly Ser Asn Thr Met Ala Ser Gly Gly Gly 195 200
205Ala Pro Met Ala Asp Asn Asn Glu Gly Ala Asp Gly Val Gly Asn Ser
210 215 220Ser Gly Asn Trp His Cys Asp Ser Gln Trp Leu Gly Asp Arg
Val Ile225 230 235 240Thr Thr Ser Thr Arg Thr Trp Ala Leu Pro Thr
Tyr Asn Asn His Leu 245 250 255Tyr Lys Gln Ile Ser Ser Gln Ser Gly
Ala Ser Asn Asp Asn His Tyr 260 265 270Phe Gly Tyr Ser Thr Pro Trp
Gly Tyr Phe Asp Phe Asn Arg Phe His 275 280 285Cys His Phe Ser Pro
Arg Asp Trp Gln Arg Leu Ile Asn Asn Asn Trp 290 295 300Gly Phe Arg
Pro Lys Lys Leu Ser Phe Lys Leu Phe Asn Ile Gln Val305 310 315
320Lys Glu Val Thr Gln Asn Asp Gly Thr Thr Thr Ile Ala Asn Asn Leu
325 330 335Thr Ser Thr Val Gln Val Phe Thr Asp Ser Glu Tyr Gln Leu
Pro Tyr 340 345 350Val Leu Gly Ser Ala His Gln Gly Cys Leu Pro Pro
Phe Pro Ala Asp 355 360 365Val Phe Met Val Pro Gln Tyr Gly Tyr Leu
Thr Leu Asn Asn Gly Ser 370 375 380Gln Ala Val Gly Arg Ser Ser Phe
Tyr Cys Leu Glu Tyr Phe Pro Ser385 390 395 400Gln Met Leu Arg Thr
Gly Asn Asn Phe Gln Phe Ser Tyr Thr Phe Glu 405 410 415Asp Val Pro
Phe His Ser Ser Tyr Ala His Ser Gln Ser Leu Asp Arg 420 425 430Leu
Met Asn Pro Leu Ile Asp Gln Tyr Leu Tyr Tyr Leu Asn Arg Thr 435 440
445Gln Gly Thr Thr Ser Gly Thr Thr Asn Gln Ser Arg Leu Leu Phe Ser
450 455 460Gln Ala Gly Pro Gln Ser Met Ser Leu Gln Ala Arg Asn Trp
Leu Pro465 470 475 480Gly Pro Cys Tyr Arg Gln Gln Arg Leu Ser Lys
Thr Ala Asn Asp Asn 485 490 495Asn Asn Ser Asn Phe Pro Trp Thr Ala
Ala Ser Lys Tyr His Leu Asn 500 505 510Gly Arg Asp Ser Leu Val Asn
Pro Gly Pro Ala Met Ala Ser His Lys 515 520 525Asp Asp Glu Glu Lys
Phe Phe Pro Met His Gly Asn Leu Ile Phe Gly 530 535 540Lys Glu Gly
Thr Thr Ala Ser Asn Ala Glu Leu Asp Asn Val Met Ile545 550 555
560Thr Asp Glu Glu Glu Ile Arg Thr Thr Asn Pro Val Ala Thr Glu Gln
565 570 575Tyr Gly Thr Val Ala Asn Asn Leu Gln Ser Ser Asn Thr Ala
Pro Thr 580 585 590Thr Arg Thr Val Asn Asp Gln Gly Ala Leu Pro Gly
Met Val Trp Gln 595 600 605Asp Arg Asp Val Tyr Leu Gln Gly Pro Ile
Trp Ala Lys Ile Pro His 610 615 620Thr Asp Gly His Phe His Pro Ser
Pro Leu Met Gly Gly Phe Gly Leu625 630 635 640Lys His Pro Pro Pro
Gln Ile Met Ile Lys Asn Thr Pro Val Pro Ala 645 650 655Asn Pro Pro
Thr Thr Phe Ser Pro Ala Lys Phe Ala Ser Phe Ile Thr 660 665 670Gln
Tyr Ser Thr Gly Gln Val Ser Val Glu Ile Glu Trp Glu Leu Gln 675 680
685Lys Glu Asn Ser Lys Arg Trp Asn Pro Glu Ile Gln Tyr Thr Ser Asn
690 695 700Tyr Asn Lys Ser Val Asn Val Asp Phe Thr Val Asp Thr Asn
Gly Val705 710 715 720Tyr Ser Glu Pro Arg Pro Ile Gly Thr Arg Tyr
Leu Thr Arg Asn Leu 725 730 73512736PRTArtificial
sequenceRecombinant sequence 12Met Ala Ala Asp Gly Tyr Leu Pro Asp
Trp Leu Glu Asp Asn Leu Ser1 5 10 15Glu Gly Ile Arg Glu Trp Trp Ala
Leu Gln Pro Gly Ala Pro Lys Pro 20 25 30Lys Ala Asn Gln Gln His Gln
Asp Asn Ala Arg Gly Leu Val Leu Pro 35 40 45Gly Tyr Lys Tyr Leu Gly
Pro Gly Asn Gly Leu Asp Lys Gly Glu Pro 50 55 60Val Asn Ala Ala Asp
Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp65 70 75 80Gln Gln Leu
Lys Ala Gly Asp Asn Pro Tyr Leu Lys Tyr Asn His Ala 85 90 95Asp Ala
Glu Phe Gln Glu Arg Leu Lys Glu Asp Thr Ser Phe Gly Gly 100 105
110Asn Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg Leu Leu Glu Pro
115 120 125Leu Gly Leu Val Glu Glu Ala Ala Lys Thr Ala Pro Gly Lys
Lys Arg 130 135 140Pro Val Asp Gln Ser Pro Gln Glu Pro Asp Ser Ser
Ser Gly Val Gly145 150 155 160Lys Ser Gly Lys Gln Pro Ala Arg Lys
Arg Leu Asn Phe Gly Gln Thr 165 170 175Gly Asp Ser Glu Ser Val Pro
Asp Pro Gln Pro Leu Gly Glu Pro Pro 180 185 190Ala Ala Pro Thr Ser
Leu Gly Ser Asn Thr Met Ala Ser Gly Gly Gly 195 200 205Ala Pro Met
Ala Asp Asn Asn Glu Gly Ala Asp Gly Val Gly Asn Ser 210 215 220Ser
Gly Asn Trp His Cys Asp Ser Gln Trp Leu Gly Asp Arg Val Ile225 230
235 240Thr Thr Ser Thr Arg Thr Trp Ala Leu Pro Thr Tyr Asn Asn His
Leu 245 250 255Tyr Lys Gln Ile Ser Ser Gln Ser Gly Ala Ser Asn Asp
Asn His Tyr 260 265 270Phe Gly Tyr Ser Thr Pro Trp Gly Tyr Phe Asp
Phe Asn Arg Phe His 275 280 285Cys His Phe Ser Pro Arg Asp Trp Gln
Arg Leu Ile Asn Asn Asn Trp 290 295 300Gly Phe Arg Pro Lys Lys Leu
Ser Phe Lys Leu Phe Asn Ile Gln Val305 310 315 320Lys Glu Val Thr
Gln Asn Asp Gly Thr Thr Thr Ile Ala Asn Asn Leu 325 330 335Thr Ser
Thr Val Gln Val Phe Thr Asp Ser Glu Tyr Gln Leu Pro Tyr 340 345
350Val Leu Gly Ser Ala His Gln Gly Cys Leu Pro Pro Phe Pro Ala Asp
355 360 365Val Phe Met Val Pro Gln Tyr Gly Tyr Leu Thr Leu Asn Asn
Gly Ser 370 375 380Gln Ala Val Gly Arg Ser Ser Phe Tyr Cys Leu Glu
Tyr Phe Pro Ser385 390 395 400Gln Met Leu Arg Thr Gly Asn Asn Phe
Gln Phe Ser Tyr Thr Phe Glu 405 410 415Asp Val Pro Phe His Ser Ser
Tyr Ala His Ser Gln Ser Leu Asp Arg 420 425 430Leu Met Asn Pro Leu
Ile Asp Gln Tyr Leu Tyr Tyr Leu Asn Arg Thr 435 440 445Gln Gly Thr
Thr Ser Gly Thr Thr Asn Gln Ser Arg Leu Leu Phe Ser 450 455 460Gln
Ala Gly Pro Gln Ser Met Ser Leu Gln Ala Arg Asn Trp Leu Pro465 470
475 480Gly Pro Cys Tyr Arg Gln Gln Arg Leu Ser Lys Thr Ala Asn Asp
Asn 485 490 495Asn Asn Ser Asn Phe Pro Trp Thr Ala Ala Ser Lys Tyr
His Leu Asn 500 505 510Gly Arg Asp Ser Leu Val Asn Pro Gly Pro Ala
Met Ala Ser His Lys 515 520 525Asp Asp Glu Glu Lys Phe Phe Pro Met
His Gly Asn Leu Ile Phe Gly 530 535 540Lys Glu Gly Thr Thr Ala Ser
Asn Ala Glu Leu Asp Asn Val Met Ile545 550 555 560Thr Asp Glu Glu
Glu Ile Arg Thr Thr Asn Pro Val Ala Thr Glu Gln 565 570 575Tyr Gly
Thr Val Ala Asn Asn Leu Gln Ser Ser Asn Thr Ala Pro Thr 580 585
590Thr Arg Thr Val Asn Asp Gln Gly Ala Leu Pro Gly Met Val Trp Gln
595 600 605Asp Arg Asp Val Tyr Leu Gln Gly Pro Ile Trp Ala Lys Ile
Pro His 610 615 620Thr Asp Gly His Phe His Pro Ser Pro Leu Met Gly
Gly Phe Gly Leu625 630 635 640Lys His Pro Pro Pro Gln Ile Met Ile
Lys Asn Thr Pro Val Pro Ala 645 650 655Asn Pro Pro Thr Thr Phe Ser
Pro Ala Lys Phe Ala Ser Phe Ile Thr 660 665 670Gln Tyr Ser Thr Gly
Gln Val Ser Val Glu Ile Glu Trp Glu Leu Gln 675 680 685Lys Glu Asn
Ser Lys Arg Trp Asn Pro Glu Ile Gln Tyr Thr Ser Asn 690 695 700Tyr
Asn Lys Ser Val Asn Val Asp Phe Thr Val Asp Thr Asn Gly Val705 710
715 720Tyr Ser Glu Pro Arg Pro Ile Gly Thr Arg Tyr Leu Thr Arg Pro
Leu 725 730 735
* * * * *