U.S. patent application number 11/893781 was filed with the patent office on 2008-02-28 for adeno-associated virus serotype i nucleic acid sequences, vectors and host cell containing same.
This patent application is currently assigned to The Trustees of the University of Pennsylvania. Invention is credited to James M. Wilson, Weidong Xiao.
Application Number | 20080050343 11/893781 |
Document ID | / |
Family ID | 31996504 |
Filed Date | 2008-02-28 |
United States Patent
Application |
20080050343 |
Kind Code |
A1 |
Wilson; James M. ; et
al. |
February 28, 2008 |
Adeno-associated virus serotype I nucleic acid sequences, vectors
and host cell containing same
Abstract
The nucleic acid sequences of adeno-associated virus (AAV)
serotype 1 are provided, as are vectors and host cells containing
these sequences and functional fragments thereof. Also provided are
methods of delivering genes via AAV-1 derived vectors.
Inventors: |
Wilson; James M.; (Gladwyne,
PA) ; Xiao; Weidong; (Jenkintown, PA) |
Correspondence
Address: |
HOWSON AND HOWSON
SUITE 210
501 OFFICE CENTER DRIVE
FT WASHINGTON
PA
19034
US
|
Assignee: |
The Trustees of the University of
Pennsylvania
Philadelphia
PA
|
Family ID: |
31996504 |
Appl. No.: |
11/893781 |
Filed: |
August 17, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11708785 |
Feb 20, 2007 |
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11893781 |
Aug 17, 2007 |
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10696900 |
Oct 30, 2003 |
7186552 |
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11708785 |
Feb 20, 2007 |
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09807802 |
Nov 29, 2001 |
6759237 |
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PCT/US99/25694 |
Nov 2, 1999 |
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10696900 |
Oct 30, 2003 |
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11430226 |
May 8, 2006 |
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11893781 |
Aug 17, 2007 |
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10696282 |
Oct 29, 2003 |
7105345 |
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11430226 |
May 8, 2006 |
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60107114 |
Nov 5, 1998 |
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Current U.S.
Class: |
424/93.2 ;
435/235.1; 435/252.3; 435/254.11; 435/258.1; 435/320.1; 435/325;
435/419; 435/456; 536/23.1 |
Current CPC
Class: |
A61K 48/00 20130101;
C07K 14/505 20130101; C07K 14/8125 20130101; C12N 2799/025
20130101; C12N 7/00 20130101; C12N 2750/14042 20130101; C12N
2750/14122 20130101; C12N 2750/14143 20130101; C12N 2750/14121
20130101; C12N 15/86 20130101; C07K 14/005 20130101 |
Class at
Publication: |
424/093.2 ;
435/235.1; 435/252.3; 435/254.11; 435/258.1; 435/320.1; 435/325;
435/419; 435/456; 536/023.1 |
International
Class: |
A61K 48/00 20060101
A61K048/00; C12N 1/15 20060101 C12N001/15; C12N 1/21 20060101
C12N001/21; C12N 15/11 20060101 C12N015/11; C12N 15/63 20060101
C12N015/63; C12N 15/86 20060101 C12N015/86; C12N 5/10 20060101
C12N005/10; C12N 7/00 20060101 C12N007/00; C12N 7/01 20060101
C12N007/01 |
Goverment Interests
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
[0003] This work was supported by the National Institutes of
Health, grant no. P30 DK47757-06 and PO1 HD32649-04. The US
government may have certain rights in this invention.
Claims
1. An isolated AAV-1 nucleic acid molecule comprising a sequence
selected from the group consisting of: (a) SEQ ID NO: 1; (b) a DNA
sequence complementary to SEQ ID NO: 1; (c) cDNA complementary to
(a) or (b); and (d) RNA complementary to any of (a) to (c).
2. A nucleic acid molecule comprising an AAV-1 inverted terminal
repeat (ITR) sequence selected from the group consisting of: (a) nt
1 to 143 of SEQ ID NO: 1; (b) nt 4576 to 4718 of SEQ ID NO: 1; (c)
a nucleic acid sequence complementary to (a) or (b); and (d) a
functional fragment of (a), (b), or (c).
3. A recombinant vector comprising a 5' AAV-1 inverted terminal
repeat (ITR) and a selected transgene, wherein said ITR has the
sequence selected from the group consisting of: (a) nt 1 to 143 of
SEQ ID NO: 1; (b) a nucleic acid sequence complementary to (a); and
(c) a functional fragment of (a) or (b).
4. The recombinant vector according to claim 3, wherein said vector
further comprises a 3' AAV-1 ITR.
5. A recombinant vector comprising a 3' AAV-1 inverted terminal
repeat (ITR) and a selected transgene, wherein said ITR has the
sequence selected from the group consisting of: (a) nt 4576 to 4718
of SEQ ID NO: 1; (b) a nucleic acid sequence complementary to (a);
and (c) a functional fragment of (a) or (b).
6. The recombinant vector according to claim 5, wherein said vector
further comprises a 5' AAV-1 ITR.
7. A pharmaceutical composition comprising a carrier and a virus
comprising the vector according to claim 5.
8. A method for producing a selected gene product comprising the
steps of transfecting a mammalian cell with the molecule according
to claim 1 or a functional fragment thereof and culturing said cell
under conditions suitable to express said gene product.
9. The recombinant vector according to claim 3, wherein said vector
further comprises AAV-1 capsid proteins having the sequence of SEQ
ID NO: 13, 15 or 17 or functional fragments thereof.
10. The recombinant vector according to claim 3, wherein said
vector further comprises adenovirus sequences.
11. The host cell transduced with a recombinant viral vector
according to claim 3.
12. The host cell transduced with a nucleic acid molecule according
to claim 1.
13. The host cell transduced with a nucleic acid molecule according
to claim 2.
14. The pharmaceutical composition comprising a carrier and a virus
comprising the vector according to claim 3.
15. The method for delivery of a transgene comprising the step of
delivering to a host cell a recombinant virus comprising a
recombinant vector according to claim 3.
16. A recombinant host cell transformed with a nucleic acid
sequence expressing one or more AAV-1 rep proteins selected from
among rep78 having the amino acid sequence of SEQ ID NO: 7, rep 68
having the amino acid sequence of SEQ ID NO: 7, rep 52 having the
amino acid sequence of SEQ ID NO: 9, and rep 40 having the amino
acid sequence of SEQ ID NO: 11.
17. A composition comprising a recombinant virus having an AAV-1
capsid comprising an AAV-1 protein selected from among AAV-1 vp1
having the amino acid sequence of SEQ ID No: 13; AAV-1 vp2 having
the amino acid sequence of SEQ ID NO: 15 and AAV-1 vp3 having the
amino acid sequence of SEQ ID NO: 17 and a heterologous molecule
which comprises an AAV 5' inverted terminal repeat sequence (ITR),
a transgene, and an AAV 3' ITR.
18. The composition of claim 17 wherein the AAV-1 protein vp1 is
encoded by a nucleic acid having at least about 98% identity to
nucleotides 2223-4431 of SEQ ID NO: 1, as measured by MacVector
6.0.
19. The composition of claim 17 wherein the AAV-1 protein vp2 is
encoded by a nucleic acid having at least about 98% identity to
nucleotides 2634-4432 of SEQ ID NO: 1, as measured by MacVector
6.0.
20. The composition of claim 17 wherein the AAV-1 protein vp3 is
encoded by a nucleic acid having at least about 98% identity to
nucleotides 2829-4432 of SEQ ID NO: 1, as measured by
MacVector6.0.
21. The composition of claim 17 wherein the AAV 5' ITR and 3' ITR
are of AAV serotype 2.
22. The composition of claim 21 wherein the recombinant virus
further comprises a regulatable promoter which directs expression
of the transgene.
23. A recombinant vector comprising an AAV-1 P5 promoter having the
sequence of nt 236 to 299 of SEQ ID NO: 1 or a functional fragment
thereof.
24. A nucleic acid molecule encoding AAV-1 helper functions, said
molecule comprising an AAV rep coding region and an AAV cap coding
region, wherein said cap coding region comprises at least one
member is selected from the group consisting of: (a) vp1, nt 2223
to 4431 of SEQ ID NO: 1; (b) vp2, nt 2634 to 4432 of SEQ ID NO: 1;
and (c) vp3, nt 2829 to 4432 of SEQ ID NO: 1.
25. A host cell stably transduced with an AAV-1 P5 promoter having
the sequence of nt 236 to 299 of SEQ ID NO: 1.
26. A pharmaceutical composition comprising a carrier and a virus
comprising the vector according to claim 25.
27. A method for AAV-mediated delivery of a transgene comprising
the step of delivering to a host cell an AAV virion which
comprises: (a) a capsid comprising at least one capsid protein
encoded by an AAV-1 cap gene; and (b) a DNA molecule comprising a
transgene under the control of regulatory sequences directing its
expression.
28. A method for AAV-mediated delivery of a transgene to a host
comprising the steps of: (a) assaying a sample from the host to
determine the presence of neutralizing antibodies specific against
any serotype of AAV; and (b) delivering to the host an AAV virion
which comprises: (i) a capsid comprising at least one capsid
protein encoded by a cap gene of an AAV serotype against which the
host has no antibodies as determined in step (a); and (ii) a DNA
molecule comprising a transgene under the control of regulatory
sequences directing its expression.
29. The method according to claim 28, comprising the additional
step of repeating steps (a) and (b).
30. The host cell transduced with a nucleic acid molecule according
to claim 24.
31. A method for transducing a muscle cell, said method comprising
the step of infecting the cell with a recombinant AAV vector
comprising an AAV1 capsid.
32. A method for transducing a liver cell, said method comprising
the step of infecting the cell with a recombinant AAV vector
comprising an AAV1 capsid.
33. A method of delivering a heterologous nucleic acid to at least
one muscle cell in a mammalian subject, comprising: (a) providing
at least one recombinant adeno-associated virus (rAAV) virion, said
rAAV virion comprising an AAV-1 capsid and a heterologous nucleic
acid operably linked to expression control elements; and (b)
administering said rAAV virions to said muscle cell, whereby
expression of said heterologous nucleic acid provides for a
therapeutic effect.
34. The method of claim 33, wherein said heterologous nucleic acid
is a gene encoding a protein.
35. The method of claim 33, wherein said heterologous nucleic acid
is an antisense RNA.
36. The method of claim 33, wherein said protein is a secreted
protein.
37. The method of claim 35, wherein said secreted protein is
selected from the group consisting of cytokines, growth factors,
and differentiation factors.
38. The method of claim 34, wherein said protein is
.alpha.1-antitrypsin or erthryopoietin.
39. The method of claim 33, wherein said administering of said rAAV
virions is by way of direct injection to said muscle cell of said
mammalian subject.
40. The method of claim 39, wherein said muscle cell is a skeletal
muscle cell.
41. The method of claim 33, wherein said administering of said rAAV
virions is by way of administration to a vascular conduit of said
mammalian subject.
42. The method of claim 41, wherein said vascular conduit is a
vein.
43. A chimeric virus particle comprising: (a) a chimeric
adeno-associated virus serotype-2 (AAV2) capsid comprising a
chimeric capsid protein comprising at least one capsid region from
AAV1; and (b) a nucleic acid comprising 5' and 3' AAV inverted
terminal repeats and at least one heterologous nucleotide sequence,
wherein said nucleic acid is packaged within the chimeric
parvovirus capsid.
44. The chimeric virus particle of claim 43, wherein said at least
one heterologous nucleotide sequence encodes a protein or
peptide.
45. The chimeric virus particle of claim 44, wherein said protein
or peptide is a therapeutic protein or peptide.
46. The chimeric virus particle of claim 45, wherein said protein
or peptide is dystrophin or a mini-dystrophin.
47. The chimeric virus particle of claim 44, wherein said protein
or peptide is an immunogenic protein or peptide.
48. The chimeric virus particle of claim 43, wherein said at least
one heterologous nucleotide sequence encodes an untranslated RNA
sequence.
49. The chimeric virus particle of claim 43, wherein said AAV1
capsid region is inserted into the chimeric capsid protein but does
not replace a region of said chimeric capsid protein.
50. The chimeric virus particle of claim 43, wherein said at least
one AAV1 capsid region replaces a region within said chimeric
capsid region.
51. The chimeric virus particle of claim 43, wherein said at least
one AAV1 capsid region is a loop region of the major vp1 capsid
subunit.
52. The chimeric virus particle of claim 51, wherein said loop
region replaces a loop region in the major Vp1 capsid subunit.
53. The chimeric virus particle of claim 43, wherein said 5' and 3'
AAV inverted terminal repeats are AAV2 inverted terminal
repeats.
54. The chimeric virus particle of claim 43, wherein said nucleic
acid does not comprise the AAV cap genes or the AAV rep genes.
55. The chimeric virus particle of claim 43, wherein an antigenic
property of said chimeric AAV2 capsid is reduced as compared with
the wild-type AAV2 capsid.
56. A chimeric virus particle comprising: (a) a chimeric
adeno-associated virus serotype-2 (AAV2) capsid comprising at least
one AAV1 capsid region; and (b) a nucleic acid comprising 5' and 3'
AAV inverted terminal repeats and at least one heterologous
nucleotide sequence, wherein said nucleic acid is packaged within
the chimeric AAV2 capsid.
57. The chimeric virus particle of claim 56, wherein said at least
one heterologous nucleotide sequence encodes a protein or
peptide.
58. The chimeric virus particle of claim 57, wherein said protein
or peptide is a therapeutic protein or peptide.
59. The chimeric virus particle of claim 58, wherein said protein
or peptide is dystrophin or a mini-dystrophin.
60. The chimeric virus particle of claim 57, wherein said protein
or peptide is an immunogenic protein or peptide.
61. The chimeric virus particle of claim 57, wherein said at least
one heterologous nucleotide sequence encodes an untranslated RNA
sequence.
62. The chimeric virus particle of claim 57, wherein said AAV1
capsid region is inserted into the chimeric capsid protein but does
not replace a region of said chimeric capsid protein.
63. The chimeric virus particle of claim 57, wherein said at least
one AAV1 capsid region replaces a region within said chimeric
capsid region.
64. The chimeric virus particle of claim 57, wherein said at least
one AAV1 capsid region is a loop region of the major Vp1 capsid
subunit.
65. The chimeric virus particle of claim 64, wherein said loop
region replaces a loop region in the major Vp1 capsid subunit.
66. The chimeric virus particle of claim 56, wherein said 5' and 3'
AAV inverted terminal repeats are AAV2 inverted terminal
repeats.
67. The chimeric virus particle of claim 56, wherein said nucleic
acid does not comprise the AAV cap genes or the AAV rep genes.
68. The chimeric virus particle of claim 56, wherein an antigenic
property of said chimeric AAV2 capsid is reduced as compared with
the wild-type AAV2 capsid.
69. The chimeric virus particle of claim 56, wherein said at least
one AAV1 capsid region replaces a capsid subunit in said AAV2
capsid.
70. A composition comprising the chimeric virus particle of claim
43.
71. A composition comprising the chimeric virus particle of claim
57.
72. An isolated nucleic acid encoding the adeno-associated virus
serotype-2 (AAV2) cap genes and AAV rep genes, wherein the AAV2 cap
genes encode a chimeric AAV2 capsid comprising at least one AAV1
capsid region.
73. A vector comprising the isolated nucleic acid of claim 43.
74. A cell comprising the vector of claim 57.
75. A cell comprising the isolated nucleic acid of claim 74 stably
integrated into the genome of the cell.
76. A method of producing a chimeric virus particle, the method
comprising: providing a cell with chimeric adeno-associated virus
serotype-2 (AAV2) cap genes, AAV rep genes, a nucleic acid
comprising 5' and 3' AAV inverted terminal repeats and at least one
heterologous nucleotide sequence, and helper functions for
generating a productive AAV infection, wherein the chimeric AAV cap
genes comprise at least one nucleic acid sequence from AAV1 cap
genes such that the chimeric AAV2 cap genes encode a chimeric AAV2
capsid comprising at least one AAV1 capsid region; and allowing
assembly of the chimeric virus particles.
77. The method of claim 76, further comprising collecting the
chimeric virus particles.
78. The method of claim 76, wherein the chimeric AAV2 cap genes and
AAV rep genes are provided by one or more transcomplementing
packaging vectors.
79. The method of claim 76, wherein the chimeric AAV2 cap genes and
AAV rep genes are provided by a plasmid.
80. The method of claim 76, wherein the chimeric AAV2 cap genes and
AAV rep genes are stably integrated into the genome of the
cell.
81. The method of claim 76, wherein the AAV rep genes are AAV2 rep
genes.
82. A chimeric virus particle produced by the method of claim
78.
83. A method of delivering a nucleotide sequence to a cell, the
method comprising introducing into a cell the chimeric virus
particle of claim 57.
84. A method of administering a nucleotide sequence to a subject,
the method comprising administering to a subject the chimeric virus
particle of claim 57.
85. A pharmaceutical formulation comprising the chimeric virus
particle of claim 57 in a pharmaceutically-acceptable carrier.
86. A hybrid virus particle comprising: a parvovirus capsid; and a
nucleic acid comprising at least one adeno-associated virus (AAV)
serotype 5 inverted terminal repeat packaged within said parvovirus
capsid, subject to the proviso that said parvovirus capsid is not
an AAV serotype 5 capsid.
87. The hybrid virus particle of claim 86, wherein said nucleic
acid comprises at least one heterologous nucleotide sequence.
88. The hybrid virus particle of claim 86, wherein said parvovirus
capsid is an autonomous parvovirus capsid.
89. The hybrid virus particle of claim 86, wherein said parvovirus
capsid is a B 19 capsid.
90. The hybrid virus particle of claim 86, wherein said parvovirus
capsid is an AAV capsid.
91. The hybrid virus particle of claim 90, wherein said AAV capsid
is of a serotype selected from the group consisting of AAV
serotypes 1, 2, 3, 4 and 6.
92. The hybrid virus particle of claim 91 selected from the group
consisting of: (a) a hybrid virus particle comprising an AAV
serotype-1 capsid and at least one AAV serotype-5 inverted terminal
repeat, (b) a hybrid virus particle comprising an AAV serotype-2
capsid and at least one AAV serotype-5 inverted terminal repeat,
and (c) a hybrid virus particle comprising an AAV serotype-6 capsid
and at least one AAV serotype-5 inverted terminal repeat.
93. The hybrid virus particle of claim 86, wherein said nucleic
acid does not comprise the AAV cap genes or the AAV rep genes.
94. The hybrid virus particle of claim 86 comprising two AAV
inverted terminal repeats that flank said at least one heterologous
nucleotide sequence.
95. The hybrid virus particle of claim 87, wherein said at least
one heterologous nucleotide sequence encodes a protein or
peptide.
96. The hybrid virus-particle of claim 95, wherein said protein or
peptide is a therapeutic protein or peptide.
97. The hybrid virus particle of claim 95, wherein said protein or
peptide is an immunogenic protein or peptide.
98. The hybrid virus particle of claim 95, wherein said at least
one heterologous nucleotide sequence encodes dystrophin, a
mini-dystrophin, a clotting factor, .beta.-glucocerebrosidase,
erythropoietin, cystic fibrosis transmembrane regulator protein, a
cytokine, .beta.-globin, a hormone, .alpha.-globin or a growth
factor.
99. The hybrid virus particle of claim 87, wherein said at least
one heterologous nucleotide sequence encodes an untranslated
RNA.
100. A pharmaceutical formulation comprising the hybrid virus
particle of claim 86 in a pharmaceutically-acceptable carrier.
101. An isolated nucleic acid for producing the hybrid virus
particle of claim 86, wherein said isolated nucleic acid comprises
parvovirus cap genes and adeno-associated virus (AAV) rep genes,
subject to the proviso that said parvovirus cap genes are not AAV
serotype 5 cap genes.
102. The isolated nucleic acid of claim 101, wherein said
parvovirus cap genes are operably associated with an authentic
parvovirus promoter.
103. The isolated nucleic acid of claim 101, wherein said
parvovirus cap genes are B19 cap genes.
104. The isolated nucleic acid of claim 103, wherein said AAV rep
genes are AAV serotype-5 rep genes.
105. The isolated nucleic acid of claim 103, wherein said AAV cap
genes are of a serotype selected from the group consisting of AAV
serotypes 1, 2, 3, 4 and 6.
106. The isolated nucleic acid of claim 105 selected from the group
consisting of: (a) an isolated nucleic acid comprising AAV
serotype-1 cap genes and AAV serotype-5 rep genes, (b) an isolated
nucleic acid comprising AAV serotype-2 cap genes and AAV serotype-5
rep genes, (c) an isolated nucleic acid comprising AAV serotype-3
cap genes and AAV serotype-5 rep genes, (d) an isolated nucleic
acid comprising AAV serotype-4 cap genes and AAV serotype-5 rep
genes, and (e) an isolated nucleic acid comprising AAV serotype-6
cap genes and AAV serotype-5 rep genes.
107. The isolated nucleic acid of claim 106, wherein said cap genes
are AAV cap genes.
108. The isolated nucleic acid of claim 107, wherein said AAV cap
genes are operably associated with an authentic AAV promoter.
109. The isolated nucleic acid of claim 108, wherein said authentic
AAV promoter is an AAV p40 promoter.
110. A vector comprising the isolated nucleic acid of claim
101.
111. The vector of claim 110, wherein said vector is selected from
the group consisting of plasmids, naked DNA vectors, bacterial
artificial chromosomes, yeast artificial chromosomes, and viral
vectors.
112. The vector of claim 110, wherein said vector is a plasmid.
113. A cell comprising the vector of claim 110.
114. The cell of claim 113, wherein said cell is selected from the
group consisting of bacterial, protozoan, yeast, fungus, plant, and
animal cells.
115. A cell comprising a vector, the vector comprising: parvovirus
cap genes, adeno-associated virus (AAV) rep genes, and a nucleic
acid comprising at least one AAV inverted serotype 5 terminal
repeat, subject to the proviso that said parvovirus cap genes are
not AAV serotype 5 cap genes.
116. The cell of claim 115, wherein said cell is a mammalian
cell.
117. A cell comprising parvovirus cap genes and adeno-associated
virus (AAV) rep genes stably integrated into the genome of the
cell, subject to the proviso that if said parvovirus cap genes are
not AAV serotype 2 cap genes, the serotypes of said AAV cap genes
and said AAV rep genes are different.
118. The cell of claim 117 further comprising a nucleic acid
comprising at least one AAV serotype 5 inverted terminal repeat,
subject to the proviso that said parvovirus cap genes are not AAV
serotype 5 cap genes.
119. A method of producing a hybrid virus particle, the method
comprising: providing a cell with adeno-associated virus (AAV) rep
genes, parvovirus cap genes, a nucleic acid comprising at least one
AAV serotype 5 inverted terminal repeat, and helper functions for
generating a productive AAV infection; subject to the proviso that
the parvovirus cap genes are not AAV serotype 5 cap genes, and
allowing assembly of the hybrid virus particles.
120. The method of claim 119, further comprising collecting the
hybrid virus particles.
121. The method of claim 119, wherein the nucleic acid comprises at
least one heterologous nucleotide sequence.
122. The method of claim 119, wherein the parvovirus cap genes and
AAV rep genes are provided by one or more transcomplementing
packaging vectors.
123. The method of claim 119, wherein the parvovirus cap genes and
AAV rep genes are provided by a plasmid.
124. The method of claim 119, wherein the parvovirus cap genes and
AAV rep genes are provided by an adenovirus vector.
125. The method of claim 119, wherein the parvovirus cap genes and
AAV rep genes are stably integrated into the genome of the
cell.
126. The method of claim 119, wherein the parvovirus cap genes are
AAV cap genes.
127. A hybrid virus particle produced by the method of claim
119.
128. A method of delivering a nucleotide sequence to a cell,
comprising introducing into a cell the hybrid virus particle
according to claim 87.
129. A method of administering a nucleotide sequence to a subject,
comprising administering the cell of claim 113 to a subject.
130. A method of administering a nucleotide sequence to a subject,
comprising administering to a subject the hybrid virus particle
according to claim 127.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This is a continuation of U.S. patent application Ser. No.
11/708,785, filed Feb. 20, 2007, which is a continuation of U.S.
patent application Ser. No. 10/696,900, filed Oct. 30, 2003, now
U.S. Pat. No. 7,186,552, which is a continuation of U.S. patent
application Ser. No. 09/807,802, filed Nov. 29, 2001, now U.S. Pat.
No. 6,759,237, which is a national stage under 35 USC 371 of
PCT/US99/25694, filed Nov. 2, 1999, which claims the benefit under
35 USC 119(e) of the priority of U.S. Patent Application No.
60/107,114, filed Nov. 5, 1998.
[0002] This is also a continuation of U.S. patent application Ser.
No. 11/430,226, filed May 8, 2006, which is a divisional of U.S.
patent application Ser. No. 10/696,282, filed Oct. 29, 2003, now
U.S. Pat. No. 7,105,345, which is a divisional of U.S. patent
application Ser. No. 09/807,802, filed Nov. 29, 2001, now U.S. Pat.
No. 6,759,237, which is a national stage under 35 USC 371 of
PCT/US99/25694, filed Nov. 2, 1999, which claims the benefit of the
priority of U.S. Patent Application No. 60/107,114, filed Nov. 5,
1998.
BACKGROUND OF THE INVENTION
[0004] This invention relates generally to viral vector, and more
particularly, to recombinant viral vectors useful for gene
delivery.
[0005] Adeno-associated viruses are small, single-stranded DNA
viruses which require helper virus to facilitate efficient
replication [K. I. Berns, Parvoviridae: the viruses and their
replication, p. 1007-1041, in F. N. Fields et al., Fundamental
virology, 3rd ed., vol. 2, (Lippencott-Raven Publishers,
Philadelphia, Pa.) (1995)]. The 4.7 kb genome of AAV is
characterized by two inverted terminal repeats (ITR) and two open
reading frames which encode the Rep proteins and Cap proteins,
respectively. The Rep reading frame encodes four proteins of
molecular weight 78 kD, 68 kD, 52 kD and 40 kD. These proteins
function mainly in regulating AAV replication and integration of
the AAV into a host cell's chromosomes. The Cap reading frame
encodes three structural proteins in molecular weight 85 kD (VP 1),
72 kD (VP2) and 61 kD (VP3) [Berns, cited above]. More than 80% of
total proteins in AAV virion comprise VP3. The two ITRs are the
only cis elements essential for AAV replication, packaging and
integration. There are two conformations of AAV ITRs called "flip"
and "flop". These differences in conformation originated from the
replication model of adeno-associated virus which use the ITR to
initiate and reinitiate the replication [R. O. Snyder et al., J.
Virol., 67:6096-6104 (1993); K. I. Berns, Microbiological Reviews,
54:316-329 (1990)].
[0006] AAVs have been found in many animal species, including
primates, canine, fowl and human [F. A. Murphy et al., "The
Classification and Nomenclature of Viruses: Sixth Report of the
International Committee on Taxonomy of Viruses", Archives of
Virology, (Springer-Verlag, Vienna) (1995)]. In addition to five
known primate AAVs (AAV-1 to AAV-5), AAV-6, another serotype
closely related to AAV-2 and AAV-1 has also been isolated [E. A.
Rutledge et al., J. Virol., 72:309-319 (1998)]. Among all known AAV
serotypes, AAV-2 is perhaps the most well-characterized serotype,
because its infectious clone was the first made [R. J. Samulski et
al., Proc. Natl. Acad. Sci. USA, 79:2077-2081 (1982)].
Subsequently, the full sequences for AAV-3A, AAV-3B, AAV-4 and
AAV-6 have also been determined [Rutledge, cited above; J. A.
Chiorini et al., J. Virol., 71:6823-6833 (1997); S. Muramatsu et
al., Virol., 221:208-217 (1996)]. Generally, all AAVs share more
than 80% homology in nucleotide sequence.
[0007] A number of unique properties make AAV a promising vector
for human gene therapy [Muzyczka, Current Topics in Microbiology
and Immunology, 158:97-129 (1992)]. Unlike other viral vectors,
AAVs have not been shown to be associated with any known human
disease and are generally not considered pathogenic. Wild type AAV
is capable of integrating into host chromosomes in a site specific
manner [R. M. Kotin et al., Proc. Natl. Acad. Sci. USA,
87:2211-2215 (1990); R. J. Samulski, EMBO J., 10(12):3941-3950
(1991)]. Recombinant AAV vectors can integrate into tissue cultured
cells in chromosome 19 if the rep proteins are supplied in trans
[C. Balague et al., J. Virol., 71:3299-3306 (1997); R. T. Surosky
et al., J. Virol., 71:7951-7959 (1997)]. The integrated genomes of
AAV have been shown to allow long term gene expression in a number
of tissues, including, muscle, liver, and brain [K. J. Fisher,
Nature Med., 3(3):306-312 (1997); R. O. Snyder et al., Nature
Genetics, 16:270-276 (1997); X. Xiao et al., Experimental
Neurology, 144:113-124 (1997); Xiao, J. Virol., 70(11):8098-8108
(1996)].
[0008] AAV-2 has been shown to be present in about 80-90% of the
human population. Earlier studies showed that neutralizing
antibodies for AAV-2 are prevalent [W. P. Parks et al., J. Virol.,
2:716-722 (1970)]. The presence of such antibodies may
significantly decrease the usefulness of AAV vectors based on AAV-2
despite its other merits. What are needed in the art are vectors
characterized by the advantages of AAV-2, including those described
above, without the disadvantages, including the presence of
neutralizing antibodies.
SUMMARY OF THE INVENTION
[0009] In one aspect, the invention provides an isolated AAV-1
nucleic acid molecule which is selected from among SEQ ID NO: 1,
the strand complementary to SEQ ID NO: 1, and cDNA and RNA
sequences complementary to SEQ ID NO: 1 and its complementary
strand.
[0010] In another aspect, the present invention provides AAV ITR
sequences, which include the 5' ITR sequences, nt 1 to 143 of SEQ
ID NO: 1; the 3' ITR sequences, nt 4576 to 4718 of SEQ ID NO: 1,
and fragments thereof.
[0011] In yet another aspect, the present invention provides a
recombinant vector comprising an AAV-1 ITR and a selected
transgene. Preferably, the vector comprises both the 5' and 3'
AAV-1 ITRs between which the selected transgene is located.
[0012] In still another aspect, the invention provides a
recombinant vector comprising an AAV-1 P5 promoter having the
sequence of nt 236 to 299 of SEQ ID NO: 1 or a functional fragment
thereof.
[0013] In a further aspect, the present invention provides a
nucleic acid molecule encoding an AAV-1 rep coding region and an
AAV-1 cap coding region. In still another aspect, the present
invention provides a host cell transduced with a recombinant viral
vector of the invention. The invention further provides a host cell
stably transduced with an AAV-1 P5 promoter of the invention.
[0014] In still a further aspect, the present invention provides a
pharmaceutical composition comprising a carrier and a vector of the
invention.
[0015] In yet another aspect, the present invention provides a
method for AAV mediated delivery of a transgene to a host involving
the step of delivering to a selected host a recombinant viral
vector comprising a selected transgene under the control of
sequences which direct expression thereof and an adeno-associated
virus 1 (AAV-1) virion.
[0016] In another aspect, the invention provides a method for in
vitro production of a selected gene product using a vector of the
invention.
[0017] Other aspects and advantages of the invention will be
readily apparent to one of skill in the art from the detailed
description of the invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0018] FIGS. 1A-1F illustrate the alignment of nucleotides of AAV-1
[SEQ ID NO: 1], AAV-2 [SEQ ID NO: 18] and AAV-6 [SEQ ID NO: 19].
The alignment was done with MacVector 6.0. The full sequences of
AAV-1 are shown in the top line. Nucleotides in AAV-2 and AAV-6
identical to AAV-1 are symbolized by "." and gaps by "-". Some of
the conserved features among AAVs are marked in this figure. Note
the 3' ITRs of AAV-1 and AAV-6 are shown in different
orientations.
[0019] FIG. 2 illustrates the predicted secondary structure of
AAV-1 ITR (nt 1-146 of SEQ ID NO:1). The nucleotides in AAV-2 (nt
1-144 of SEQ ID NO:18) and AAV-6 (nt 1-136 of SEQ ID NO:19) are
shown in italic and bold respectively.
[0020] FIG. 3A illustrates a hypothesis of how AAV-6 arose from the
homologous recombination between AAV-1 and AAV-2. The major
elements of AAV-1 are indicated in the graph. A region that is
shared between AAV-1, AAV-2 and AAV-6 is shown in box with waved
lines.
[0021] FIG. 3B is a detailed illustration of a 71 bp homologous
region among AAV-1 (438-531 of SEQ ID NO:1), AAV-2 (424-513 of SEQ
ID NO:18) and AAV-6 (423-512 of SEQ ID NO:19). Nucleotides that
differ among these serotypes are indicated by arrows.
[0022] FIG. 4A is a bar chart illustrating expression levels of
human alpha 1 anti-trypsin (.alpha.1AT) in serum following delivery
of hAAT via recombinant AAV-1 and recombinant AAV-2 viruses.
[0023] FIG. 4B is a bar chart illustrating expression levels of
erythropoietin (epo) in serum following delivery of the epo gene
via recombinant AAV-1 and recombinant AAV-2 viruses.
[0024] FIG. 5A is a bar chart illustrating expression levels of
.alpha.1AT in liver following delivery of .alpha.1AT as described
in Example 7.
[0025] FIG. 5B is a bar chart demonstrating expression levels of
epo in liver following delivery of epo as described in Example
7.
[0026] FIG. 5C is a bar chart demonstrating neutralizing antibodies
(NAB) directed to AAV-1 following delivery of .alpha.1AT or epo to
liver as described in Example 7.
[0027] FIG. 5D is a bar chart demonstrating neutralizing antibodies
(NAB) directed to AAV-2 following delivery of .alpha.1AT or epo to
liver as described in Example 7.
[0028] FIG. 6A is a bar chart illustrating expression levels of
.alpha.1AT in muscle following delivery of .alpha.1AT as described
in Example 7.
[0029] FIG. 6B is a bar chart demonstrating expression levels of
epo in muscle following delivery of epo as described in Example
7.
[0030] FIG. 6C is a bar chart demonstrating neutralizing antibodies
(NAB) directed to AAV-1 following delivery of .alpha.1AT or epo to
muscle as described in Example 7.
[0031] FIG. 6D is a bar chart demonstrating neutralizing antibodies
(NAB) directed to AAV-2 following delivery of .alpha.1AT or epo to
muscle as described in Example 7.
DETAILED DESCRIPTION OF THE INVENTION
[0032] The present invention provides novel nucleic acid sequences
for an adeno-associated virus of serotype 1 (AAV-1). Also provided
are fragments of these AAV-1 sequences. Among particularly
desirable AAV-1 fragments are the inverted terminal repeat
sequences (ITRs), rep and cap. Each of these fragments may be
readily utilized, e.g., as a cassette, in a variety of vector
systems and host cells. Such fragments may be used alone, in
combination with other AAV-1 sequences or fragments, or in
combination with elements from other AAV or non-AAV viral
sequences. In one particularly desirable embodiment, a cassette may
contain the AAV-1 ITRs of the invention flanking a selected
transgene. In another desirable embodiment, a cassette may contain
the AAV-1 rep and/or cap proteins, e.g., for use in producing
recombinant (rAAV) virus.
[0033] Thus, the AAV-1 sequences and fragments thereof are useful
in production of rAAV, and are also useful as antisense delivery
vectors, gene therapy vectors, or vaccine vectors. The invention
further provides nucleic acid molecules, gene delivery vectors, and
host cells which contain the AAV-1 sequences of the invention. Also
provided a novel methods of gene delivery using AAV vectors.
[0034] As described herein, the vectors of the invention containing
the AAV-1 capsid proteins of the invention are particularly well
suited for use in applications in which the neutralizing antibodies
diminish the effectiveness of other AAV serotype based vectors, as
well as other viral vectors. The rAAV vectors of the invention are
particularly advantageous in rAAV readministration and repeat gene
therapy.
[0035] These and other embodiments and advantages of the invention
are described in more detail below. As used throughout this
specification and the claims, the term "comprising" is inclusive of
other components, elements, integers, steps and the like.
I. AAV1 Nucleic Acid and Protein Sequences
[0036] The AAV-1 nucleic acid sequences of the invention include
the DNA sequences of SEQ ID NO: 1 (FIGS. 1A-1F), which consists of
4718 nucleotides. The AAV-1 nucleic acid sequences of the invention
further encompass the strand which is complementary to SEQ ID NO:
1, as well as the RNA and cDNA sequences corresponding to SEQ ID
NO: 1 and its complementary strand. Also included in the nucleic
acid sequences of the invention are natural variants and engineered
modifications of SEQ ID NO: 1 and its complementary strand. Such
modifications include, for example, labels which are known in the
art, methylation, and substitution of one or more of the naturally
occurring nucleotides with an analog.
[0037] Further included in this invention are nucleic acid
sequences which are greater than 85%, preferably at least about
90%, more preferably at least about 95%, and most preferably at
least about 98-99% identical or homologous to SEQ ID NO:1.
[0038] The term "percent sequence identity" or "identical" in the
context of nucleic acid sequences refers to the residues in the two
sequences which are the same when aligned for maximum
correspondence. The length of sequence identity comparison may be
over the full-length sequence, or a fragment at least about nine
nucleotides, usually at least about 20-24 nucleotides, at least
about 28-32 nucleotides, and preferably at least about 36 or more
nucleotides. There are a number of different algorithms known in
the art which can be used to measure nucleotide sequence identity.
For instance, polynucleotide sequences can be compared using Fasta,
a program in GCG Version 6.1. Fasta provides alignments and percent
sequence identity of the regions of the best overlap between the
query and search sequences (Pearson, 1990, herein incorporated by
reference). For instance, percent sequence identity between nucleic
acid sequences can be determined using Fasta with its default
parameters (a word size of 6 and the NOPAM factor for the scoring
matrix) as provided in GCG Version 6.1, herein incorporated by
reference.
[0039] The term "substantial homology" or "substantial similarity,"
when referring to a nucleic acid or fragment thereof, indicates
that, when optimally aligned with appropriate nucleotide insertions
or deletions with another nucleic acid (or its complementary
strand), there is nucleotide sequence identity in at least about
95-99% of the sequence.
[0040] Also included within the invention are fragments of SEQ ID
NO: 1, its complementary strand, cDNA and RNA complementary
thereto. Suitable fragments are at least 15 nucleotides in length,
and encompass functional fragments which are of biological
interest. Certain of these fragments may be identified by reference
to FIGS. 1A-1F. Examples of particularly desirable functional
fragments include the AAV-1 inverted terminal repeat (ITR)
sequences of the invention. In contrast to the 145 nt ITRs of
AAV-2, AAV-3, and AAV-4, the AAV-1 ITRs have been found to consist
of only 143 nucleotides, yet advantageously are characterized by
the T-shaped hairpin structure which is believed to be responsible
for the ability of the AAV-2 ITRs to direct site-specific
integration. In addition, AAV-1 is unique among other AAV
serotypes, in that the 5' and 3' ITRs are identical. The
full-length 5' ITR sequences of AAV-1 are provided at nucleotides
1-143 of SEQ ID NO: 1 (FIG. 1A) and the full-length 3' ITR
sequences of AAV-1 are provided at nt 4576-4718 of SEQ ID NO: 1
(FIG. 1F). One of skill in the art can readily utilize less than
the full-length 5' and/or 3' ITR sequences for various purposes and
may construct modified ITRs using conventional techniques, e.g., as
described for AAV-2 ITRs in Samulski et al, Cell, 33:135-143
(1983).
[0041] Another desirable functional fragment of the AAV-1 genome is
the P5 promoter of AAV-1 which has sequences unique among AAV P5
promoters, while maintaining critical regulatory elements and
functions. This promoter is located within nt 236-299 of SEQ ID NO:
1 (FIG. 1A). Other examples of functional fragments of interest
include the sequences at the junction of the rep/cap, e.g., the
sequences spanning nt 2306-2223, as well as larger fragments which
encompass this junction which may comprise 50 nucleotides on either
side of this junction. Still other examples of functional fragments
include the sequences encoding the rep proteins. Rep 78 is located
in the region of nt 334-2306 of SEQ ID NO: 1; Rep 68 is located in
the region of nt 334-2272, and contains an intron spanning nt
1924-2220 of SEQ ID NO: 1. Rep 52 is located in the region of nt
1007-2304 of SEQ ID NO: 1; rep 40 is located in the region of nt
1007-2272, and contains an intron spanning nt 1924-2246 of SEQ ID
NO: 1. Also of interest are the sequences encoding the capsid
proteins, VP 1 [nt 2223-4431 of SEQ ID NO: 1], VP2 [nt 2634-4432 of
SEQ ID NO: 1] and VP3 [nt 2829-4432 of SEQ ID NO: 1]. Other
fragments of interest may include the AAV-1 P19 sequences, AAV-1
P40 sequences, the rep binding site, and the terminal resolute site
(TRS).
[0042] The invention further provides the proteins and fragments
thereof which are encoded by the AAV-1 nucleic acids of the
invention. Particularly desirable proteins include the rep and cap
proteins, which are encoded by the nucleotide sequences identified
above. These proteins include rep 78 [SEQ ID NO:5], rep 68 [SEQ ID
NO:7], rep 52 [SEQ ID NO:9], rep 40 [SEQ ID NO: 11], vp1 [SEQ ID
NO: 13], vp2 [SEQ ID NO: 15], and vp3 [SEQ ID NO: 17] and
functional fragments thereof while the sequences of the rep and cap
proteins have been found to be closely related to those of AAV-6,
there are differences in the amino acid sequences (see Table 1
below), as well as differences in the recognition of these proteins
by the immune system. However, one of skill in the art may readily
select other suitable proteins or protein fragments of biological
interest. Suitably, such fragments are at least 8 amino acids in
length. However, fragments of other desired lengths may be readily
utilized. Such fragments may be produced recombinantly or by other
suitable means, e.g., chemical synthesis.
[0043] The sequences, proteins, and fragments of the invention may
be produced by any suitable means, including recombinant
production, chemical synthesis, or other synthetic means. Such
production methods are within the knowledge of those of skill in
the art and are not a limitation of the present invention.
II. Viral Vectors
[0044] In another aspect, the present invention provides vectors
which utilize the AAV-1 sequences of the invention, including
fragments thereof, for delivery of a heterologous gene or other
nucleic acid sequences to a target cell. Suitably, these
heterologous sequences (i.e., a transgene) encode a protein or gene
product which is capable of being expressed in the target cell.
Such a transgene may be constructed in the form of a "minigene".
Such a "minigene" includes selected heterologous gene sequences and
the other regulatory elements necessary to transcribe the gene and
express the gene product in a host cell. Thus, the gene sequences
are operatively linked to regulatory components in a manner which
permit their transcription. Such components include conventional
regulatory elements necessary to drive expression of the transgene
in a cell containing the viral vector. The minigene may also
contain a selected promoter which is linked to the transgene and
located, with other regulatory elements, within the selected viral
sequences of the recombinant vector.
[0045] Selection of the promoter is a routine matter and is not a
limitation of this invention. Useful promoters may be constitutive
promoters or regulated (inducible) promoters, which will enable
control of the timing and amount of the transgene to be expressed.
For example, desirable promoters include the cytomegalovirus (CMV)
immediate early promoter/enhancer [see, e.g., Boshart et al, Cell,
41:521-530 (1985)], the Rous sarcoma virus LTR promoter/enhancer,
and the chicken cytoplasmic .beta.-actin promoter [T. A. Kost et
al, Nucl. Acids Res., 11(23):8287 (1983)]. Still other desirable
promoters are the albumin promoter and an AAV P5 promoter.
Optionally, the selected promoter is used in conjunction with a
heterologous enhancer, e.g., the .beta.-actin promoter may be used
in conjunction with the CMV enhancer. Yet other suitable or
desirable promoters and enhancers may be selected by one of skill
in the art.
[0046] The minigene may also desirably contain nucleic acid
sequences heterologous to the viral vector sequences including
sequences providing signals required for efficient polyadenylation
of the transcript (poly-A or pA) and introns with functional splice
donor and acceptor sites. A common poly-A sequence which is
employed in the exemplary vectors of this invention is that derived
from the papovavirus SV-40. The poly-A sequence generally is
inserted in the minigene downstream of the transgene sequences and
upstream of the viral vector sequences. A common intron sequence is
also derived from SV-40, and is referred to as the SV40 T intron
sequence. A minigene of the present invention may also contain such
an intron, desirably located between the promoter/enhancer sequence
and the transgene. Selection of these and other common vector
elements are conventional [see, e.g., Sambrook et al, "Molecular
Cloning. A Laboratory Manual", 2d edit., Cold Spring Harbor
Laboratory, New York (1989) and references cited therein] and many
such sequences are available from commercial and industrial sources
as well as from Genbank.
[0047] The selection of the transgene is not a limitation of the
present invention. Suitable transgenes may be readily selected from
among desirable reporter genes, therapeutic genes, and optionally,
genes encoding immunogenic polypeptides. Examples of suitable
reporter genes include .beta.-galactosidase (.beta.-gal), an
alkaline phosphatase gene, and green fluorescent protein (GFP).
Examples of therapeutic genes include, cytokines, growth factors,
hormones, and differentiation factors, among others. The transgene
may be readily selected by one of skill in the art. See, e.g., WO
98/09657, which identifies other suitable transgenes.
[0048] Suitably, the vectors of the invention contain, at a
minimum, cassettes which consist of fragments of the AAV-1
sequences and proteins. In one embodiment, a vector of the
invention comprises a selected transgene, which is flanked by a 5'
ITR and a 3' ITR, at least one of which is an AAV-1 ITR of the
invention. Suitably, vectors of the invention may contain a AAV-1
P5 promoter of the invention. In yet another embodiment, a plasmid
or vector of the invention contains AAV-1 rep sequences. In still
another embodiment, a plasmid or vector of the invention contains
at least one of the AAV-1 cap proteins of the invention. Most
suitably, these AAV-1-derived vectors are assembled into viral
vectors, as described herein.
[0049] A. AAV Viral Vectors
[0050] In one aspect, the present invention provides a recombinant
AAV-1 viral vector produced using the AAV-1 capsid proteins of the
invention. The packaged rAAV-1 virions of the invention may
contain, in addition to a selected minigene, other AAV-1 sequences,
or may contain sequences from other AAV serotypes.
[0051] Methods of generating rAAV virions are well known and the
selection of a suitable method is not a limitation on the present
invention. See, e.g., K. Fisher et al, J. Virol., 70:520-532 (1993)
and U.S. Pat. No. 5,478,745. In one suitable method, a selected
host cell is provided with the AAV sequence encoding a rep protein,
the gene encoding the AAV cap protein and with the sequences for
packaging and subsequent delivery. Desirably, the method utilizes
the sequences encoding the AAV-1 rep and/or cap proteins of the
invention.
[0052] In one embodiment, the rep/cap genes and the sequences for
delivery are supplied by co-transfection of vectors carrying these
genes and sequences. In one currently preferred embodiment, a cis
(vector) plasmid, a trans plasmid containing the rep and cap genes,
and a plasmid containing the adenovirus helper genes are
co-transfected into a suitable cell line, e.g., 293. Alternatively,
one or more of these functions may be provided in trans via
separate vectors, or may be found in a suitably engineered
packaging cell line.
[0053] An exemplary cis plasmid will contain, in 5' to 3' order,
AAV 5' ITR, the selected transgene, and AAV 3' ITR. In one
desirable embodiment, at least one of the AAV ITRs is a 143 nt
AAV-1 ITR. However, other AAV serotype ITRs may be readily
selected. Suitably, the full-length ITRs are utilized. However, one
of skill in the art can readily prepare modified AAV ITRs using
conventional techniques. Similarly, methods for construction of
such plasmids is well known to those of skill in the art.
[0054] A trans plasmid for use in the production of the rAAV-1
virion particle may be prepared according to known techniques. In
one desired embodiment, this plasmid contains the rep and cap
proteins of AAV-1, or functional fragments thereof. Alternatively,
the rep sequences may be from another selected AAV serotype.
[0055] The cis and trans plasmid may then be co-transfected with a
wild-type helper virus (e.g., Ad2, Ad5, or a herpesvirus), or more
desirably, a replication defective adenovirus, into a selected host
cell. Alternatively, the cis and trans plasmid may be
co-transfected into a selected host cell together with a
transfected plasmid which provides the necessary helper functions.
Selection of a suitable host cell is well within the skill of those
in the art and include such mammalian cells as 293 cells, HeLa
cells, among others.
[0056] Alternatively, the cis plasmid and, optionally the trans
plasmid, may be transfected into a packaging cell line which
provides the remaining helper functions necessary for production of
a rAAV containing the desired AAV-1 sequences of the invention. An
example of a suitable packaging cell line, where an AAV-2 capsid is
desired, is B-50, which stably expresses AAV-2 rep and cap genes
under the control of a homologous P5 promoter. This cell line is
characterized by integration into the cellular chromosome of
multiple copies (at least 5 copies) of P5-rep-cap gene cassettes in
a concatomer form. This B-50 cell line was deposited with the
American Type Culture Collection, 10801 University Boulevard,
Manassas, Va. 20110-2209, on Sep. 18, 1997 under Accession No.
CRL-12401 pursuant to the provisions of the Budapest Treaty.
However, the present invention is not limited as to the selection
of the packaging cell line.
[0057] Exemplary transducing vectors based on AAV-1 capsid proteins
have been tested both in vivo and in vitro, as described in more
detail in Example 4. In these studies, it was demonstrated that
recombinant AAV vector with an AAV-1 virion can transduce both
mouse liver and muscle. These, and other AAV-1 based gene therapy
vectors which may be generated by one of skill in the art are
beneficial for gene delivery to selected host cells and gene
therapy patients since the neutralization antibodies of AAV-1
present in much of the human population exhibit different patterns
from other AAV serotypes and therefore do not neutralize the AAV-1
virions. One of skill in the art may readily prepare other rAAV
viral vectors containing the AAV-1 capsid proteins provided herein
using a variety of techniques known to those of skill in the art.
One may similarly prepare still other rAAV viral vectors containing
AAV-1 sequence and AAV capsids of another serotype.
[0058] B. Other Viral Vectors
[0059] One of skill in the art will readily understand that the
AAV-1 sequences of the invention can be readily adapted for use in
these and other viral vector systems for in vitro, ex vivo or in
vivo gene delivery. Particularly well suited for use in such viral
vector systems are the AAV-1 ITR sequences, the AAV-1 rep, the
AAV-1 cap, and the AAV-1 P5 promoter sequences.
[0060] For example, in one desirable embodiment, the AAV-1 ITR
sequences of the invention may be used in an expression cassette
which includes AAV-1 5' ITR, a non-AAV DNA sequences of interest
(e.g., a minigene), and 3' ITR and which lacks functional rep/cap.
Such a cassette containing an AAV-1 ITR may be located on a plasmid
for subsequent transfection into a desired host cell, such as the
cis plasmid described above. This expression cassette may further
be provided with an AAV capsid of a selected serotype to permit
infection of a cell or stably transfected into a desired host cell
for packaging of rAAV virions. Such an expression cassette may be
readily adapted for use in other viral systems, including
adenovirus systems and lentivirus systems. Methods of producing
Ad/AAV vectors are well known to those of skill in the art. One
desirable method is described in PCT/US95/14018. However, the
present invention is not limited to any particular method.
[0061] Another aspect of the present invention is the novel AAV-1
P5 promoter sequences which are located in the region spanning nt
236-299 of SEQ ID NO: 1. This promoter is useful in a variety of
viral vectors for driving expression of a desired transgene.
[0062] Similarly, one of skill in the art can readily select other
fragments of the AAV-1 genome of the invention for use in a variety
of vector systems. Such vectors systems may include, e.g.,
lentiviruses, retroviruses, poxviruses, vaccinia viruses, and
adenoviral systems, among others. Selection of these vector systems
is not a limitation of the present invention.
[0063] C. Host Cells and Packaging Cell Lines
[0064] In yet another aspect, the present invention provides host
cells which may be transiently transfected with AAV-1 nucleic acid
sequences of the invention to permit expression of a desired
transgene or production of a rAAV particle. For example, a selected
host cell may be transfected with the AAV-1 P5 promoter sequences
and/or the AAV-1 5' ITR sequences using conventional techniques.
Providing AAV helper functions to the transfected cell lines of the
invention results in packaging of the rAAV as infectious rAAV
particles. Such cell lines may be produced in accordance with known
techniques [see, e.g, U.S. Pat. No. 5,658,785], making use of the
AAV-1 sequences of the invention.
[0065] Alternatively, host cells of the invention may be stably
transfected with a rAAV expression cassette of the invention, and
with copies of AAV-1 rep and cap genes. Suitable parental cell
lines include mammalian cell lines and it may be desirable to
select host cells from among non-simian mammalian cells. Examples
of suitable parental cell lines include, without limitation, HeLa
[ATCC CCL 2], A549 [ATCC Accession No. CCL 185], KB [CCL 17],
Detroit [e.g., Detroit 510, CCL 72] and WI38 [CCL 75] cells. These
cell lines are all available from the American Type Culture
Collection, 10801 University Boulevard, Manassas, Va. 20110-2209
USA. Other suitable parent cell lines may be obtained from other
sources and may be used to construct stable cell lines containing
the P5 and/or AAV rep and cap sequences of the invention.
[0066] Recombinant vectors generated as described above are useful
for delivery of the DNA of interest to cells.
III. Methods of Delivering Genes Via AAV-1 Derived Vectors
[0067] In another aspect, the present invention provides a method
for delivery of a transgene to a host which involves transfecting
or infecting a selected host cell with a recombinant viral vector
generated with the AAV-1 sequences (or functional fragments
thereof) of the invention. Methods for delivery are well known to
those of skill in the art and are not a limitation of the present
invention.
[0068] In one desirable embodiment, the invention provides a method
for AAV-mediated delivery of a transgene to a host. This method
involves transfecting or infecting a selected host cell with a
recombinant viral vector containing a selected transgene under the
control of sequences which direct expression thereof and AAV-1
capsid proteins.
[0069] Optionally, a sample from the host may be first assayed for
the presence of antibodies to a selected AAV serotype. A variety of
assay formats for detecting neutralizing antibodies are well known
to those of skill in the art. The selection of such an assay is not
a limitation of the present invention. See, e.g., Fisher et al,
Nature Med., 3(3):306-312 (March 1997) and W. C. Manning et al,
Human Gene Therapy, 9:477-485 (Mar. 1, 1998). The results of this
assay may be used to determine which AAV vector containing capsid
proteins of a particular serotype are preferred for delivery, e.g.,
by the absence of neutralizing antibodies specific for that capsid
serotype.
[0070] In one aspect of this method, the delivery of vector with
AAV-1 capsid proteins may precede or follow delivery of a gene via
a vector with a different serotype AAV capsid protein. Thus, gene
delivery via rAAV vectors may be used for repeat gene delivery to a
selected host cell. Desirably, subsequently administered rAAV
vectors carry the same transgene as the first rAAV vector, but the
subsequently administered vectors contain capsid proteins of
serotypes which differ from the first vector. For example, if a
first vector has AAV-2 capsid proteins, subsequently administered
vectors may have capsid proteins selected from among the other
serotypes, including AAV-1, AAV-3A, AAV-3B, AAV-4 and AAV-6.
[0071] Thus, a rAAV-1-derived recombinant viral vector of the
invention provides an efficient gene transfer vehicle which can
deliver a selected transgene to a selected host cell in vivo or ex
vivo even where the organism has neutralizing antibodies to one or
more AAV serotypes. These compositions are particularly well suited
to gene delivery for therapeutic purposes. However, the
compositions of the invention may also be useful in immunization.
Further, the compositions of the invention may also be used for
production of a desired gene product in vitro.
[0072] The above-described recombinant vectors may be delivered to
host cells according to published methods. An AAV viral vector
bearing the selected transgene may be administered to a patient,
preferably suspended in a biologically compatible solution or
pharmaceutically acceptable delivery vehicle. A suitable vehicle
includes sterile saline. Other aqueous and non-aqueous isotonic
sterile injection solutions and aqueous and non-aqueous sterile
suspensions known to be pharmaceutically acceptable carriers and
well known to those of skill in the art may be employed for this
purpose.
[0073] The viral vectors are administered in sufficient amounts to
transfect the cells and to provide sufficient levels of gene
transfer and expression to provide a therapeutic benefit without
undue adverse effects, or with medically acceptable physiological
effects, which can be determined by those skilled in the medical
arts. Conventional and pharmaceutically acceptable routes of
administration include, but are not limited to, direct delivery to
the liver, oral, intranasal, intravenous, intramuscular,
subcutaneous, intradermal, and other parental routes of
administration. Routes of administration may be combined, if
desired.
[0074] Dosages of the viral vector will depend primarily on factors
such as the condition being treated, the age, weight and health of
the patient, and may thus vary among patients. For example, a
therapeutically effective human dosage of the viral vector is
generally in the range of from about 1 ml to about 100 ml of
solution containing concentrations of from about 1.times.10.sup.9
to 1.times.10.sup.16 genomes virus vector. A preferred human dosage
may be about 1.times.10.sup.13 to 1.times.10.sup.16 AAV genomes.
The dosage will be adjusted to balance the therapeutic benefit
against any side effects and such dosages may vary depending upon
the therapeutic application for which the recombinant vector is
employed. The levels of expression of the transgene can be
monitored to determine the frequency of dosage resulting in viral
vectors, preferably AAV vectors containing the minigene.
Optionally, dosage regimens similar to those described for
therapeutic purposes may be utilized for immunization using the
compositions of the invention. For in vitro production, a desired
protein may be obtained from a desired culture following
transfection of host cells with a rAAV containing the gene encoding
the desired protein and culturing the cell culture under conditions
which permits expression. The expressed protein may then be
purified and isolated, as desired. Suitable techniques for
transfection, cell culturing, purification, and isolation are known
to those of skill in the art.
[0075] The following examples illustrate several aspects and
embodiments of the invention.
EXAMPLE 1
Generation of Infectious Clone of AAV-1
[0076] The replicated form DNA of AAV-1 was extracted from 293
cells that were infected by AAV-1 and wild type adenovirus type
5.
[0077] A. Cell Culture and Virus
[0078] AAV-free 293 cells and 84-31 cells were provided by the
human application laboratory of the University of Pennsylvania.
These cells were cultured in Dulbecco's Modified Eagle Medium with
10% fetal bovine serum (Hyclone), penicillin (100 U/ml) and
streptomycin at 37EC in a moisturized environment supplied with 5%
C0.sub.2. The 84-31 cell line constitutively expresses adenovirus
genes E1a, E1b, E4/ORF6, and has been described previously [K. J.
Fisher, J. Virol., 70:520-532 (1996)]. AAV-1 (ATCC VR-645) seed
stock was purchased from American Type Culture Collection (ATCC,
Manassas, Va.). AAV viruses were propagated in 293 cells with wild
type Ad5 as a helper virus.
[0079] B. Recombinant AAV Generation
[0080] The recombinant AAV viruses were generated by transfection
using an adenovirus free method. Briefly, the cis plasmid (with AAV
ITR), trans plasmid (with AAV rep gene and cap gene) and helper
plasmid (pF.DELTA.13, with essential regions from the adenovirus
genome) were simultaneously co-transfected into 293 cells in a
ratio of 1:1:2 by calcium phosphate precipitation. The pF.DELTA.13
helper plasmid has an 8 kb deletion in the adenovirus E2B region
and has deletions in most of the late genes. This helper plasmid
was generated by deleting the RsrII fragment from pFG140 (Microbix,
Canada). Typically, 50 .mu.g of DNA (cis:trans:PF.DELTA.13 at
ratios of 1:1:2, respectively) was transfected onto a 15 cm tissue
culture dish. The cells were harvested 96 hours post-transfection,
sonicated and treated with 0.5% sodium deoxycholate (37.degree. C.
for 10 min). Cell lysates were then subjected to two rounds of a
CsCl gradient. Peak fractions containing AAV vector were collected,
pooled, and dialyzed against PBS before injecting into animals. To
make rAAV virus with AAV-1 virion, the pAV1H or p5E18 (2/1) was
used as the trans plasmid to provide rep and cap function.
[0081] For the generation of rAAV based on AAV-2, p5E 18 was used
as the trans plasmid since it greatly improved the rAAV yield. This
plasmid, p5E18(2/2), expresses AAV-2 Rep and Cap and contains a P5
promoter relocated to a position 3' to the Cap gene, thereby
minimizing expression of Rep78 and Rep68. The strategy was
initially described by Li et al, J. Virol., 71:5236-5243 (1997).
P5E18(2/2) was constructed in the following way. The previously
described pMMTV-trans vector (i.e., the mouse mammary tumor virus
promoter substituted for the P5 promoter in an AAV-2-based vector)
was digested with SmaI and ClaI, filled in with the Klenow enzyme,
and then recircularized with DNA ligase. The resulting construct
was digested with XbaI, filled in, and ligated to the blunt-ended
BamHI-XbaI fragment from pCR-p5, constructed in the following way.
The P5 promoter of AAV was amplified by PCR and the amplified
fragment was subsequently cloned into pCR2.1 (Invitrogen) to yield
pCR-P5. The helper plasmid pAV1H was constructed by cloning the
BfaI fragment of pAAV-2 into pBluescript II-SK(+) at the BcorV and
SmaI sites. The 3.0-kb XbaI-KpnI fragment from p5E18(2/2), the
2.3-kb XbaI-KpnI fragment from pAV1H, and the 1.7-kb KpnI fragment
from p5E18(2/2) were incorporated into a separate plasmid
P5E18(2/1), which contains AAV-2 Rep, AAV-1 Cap, and the AAV-2 P5
promoter located 3' to the Cap gene. Plasmid p5E18(2/1) produced
10- to 20-fold higher quantities of the vector than pAV1H (i.e.,
10.sup.12 genomes/50 15-cm.sup.2 plates).
[0082] C. DNA Techniques
[0083] Hirt DNA extraction was performed as described in the art
with minor modification [R. J. Samulski et al., Cell, 33:135-143
(1983)]. More particularly, Hirst solution without SDS was used
instead of using original Hirt solution containing SDS. The amount
of SDS present in the original Hirst solution was added after the
cells had been fully suspended. To construct AAV-1 infectious
clone, the Hirt DNA from AAV-1 infected 293 cells was repaired with
Klenow enzyme (New England Biolabs) to ensure the ends were blunt.
The treated AAV-1 Hirt DNA was then digested with BamHI and cloned
into three vectors, respectively. The internal BamHI was cloned
into pBlueScript II-SK+ cut with BamHI to get pAV1-BM. The left and
right fragments were cloned into pBlueScript II-SK+ cut with
BamHI+EcoRV to obtain pAV1-BL and pAV1-BR, respectively. The AAV
sequence in these three plasmids were subsequently assembled into
the same vector to get AAV-1 infectious clone pAAV-1. The helper
plasmid for recombinant AAV-1 virus generation was constructed by
cloning the BfaI fragment of pAAV-1 into pBlueScript II-SK+ at the
EcoRV site.
[0084] Analysis of the Hirt DNA revealed three bands, a dimer at
9.4 kb, a monomer at 4.7 kb and single-stranded DNA at 1.7 kb,
which correlated to different replication forms of AAV-1. The
monomer band was excised from the gel and then digested with BamHI.
This resulted in three fragments of 1.1 kb, 0.8 kb and 2.8 kb. This
pattern is in accordance with the description by Bantel-schaal and
zur Hausen, Virol., 134(1):52-63 (1984). The 1.1 kb and 2.8 kb
BamHI fragments were cloned into pBlueScript-KS(+) at BamHI and
EcoRV site. The internal 0.8 kb fragment was cloned into BamHI site
of pBlueScript-KS(+).
[0085] These three fragments were then subcloned into the same
construct to obtain a plasmid (pAAV-1) that contained the full
sequence of AAV-1. The pAAV-1 was then tested for its ability to
rescue from the plasmid backbone and package infectious virus. The
pAAV-1 was then transfected to 293 cells and supplied with
adenovirus type as helper at MOI 10. The virus supernatant was used
to reinfect 293 cells.
[0086] For Southern blot analysis, Hirt DNA was digested with DpnI
to remove bacteria-borne plasmid and probed with internal BamHI
fragment of AAV-1. The membrane was then washed at high stringency
conditions, which included: twice 30 minutes with 2.times.SSC, 0.1%
SDS at 65EC and twice 30 minutes with 0.1.times.SSC, 0.1% SDS at
65EC. The membrane was then analyzed by both phosphor image and
X-ray autoradiography. The results confirmed that pAAV-1 is indeed
an infectious clone of AAV serotype 1.
EXAMPLE 2
Sequencing Analysis of AAV-1
[0087] The entire AAV-1 genome was then determined by automatic
sequencing and was found to be 4718 nucleotides in length (FIGS.
1A-1F). For sequencing, an ABI 373 automatic sequencer as used to
determine the sequences for all plasmids and PCR fragments related
to this study using the FS dye chemistry. All sequences were
confirmed by sequencing both plus and minus strands. These
sequences were also confirmed by sequencing two independent clones
of pAV-BM, pAV-BL and pAV-BR. Since the replicated form of AAV-1
DNA served as the template for sequence determination, these
sequences were also confirmed by sequencing a series of PCR
products using original AAV-1 seed stock as a template.
[0088] The length of AAV-1 was found to be within the range of the
other serotypes: AAV-3 (4726 nucleotides), AAV-4 (4774
nucleotides), AAV-2 (4681 nucleotides), and AAV-6 (4683
nucleotides).
[0089] The AAV-1 genome exhibited similarities to other serotypes
of adeno-associated viruses. Overall, it shares more than 80%
identity with other known AAV viruses as determined by the computer
program Megalign using default settings [DNASTAR, Madison, Wis.].
The key features in AAV-2 can also be found in AAV-1. First, AAV-1
has the same type of inverted terminal repeat which is capable of
forming T-shaped hairpin structures, despite the differences at the
nucleotide level (FIGS. 2 and 3). The sequences of right ITRs and
left ITRs of AAV-1 are identical. The AAV TR sequence is subdivided
into A, A', B, B', C, C', D and D' [Bern, cited above].
[0090] These AAV ITR sequences are also virtually the same as those
found in AAV-6 right ITR, there being one nucleotide difference in
each of A and A' sequence, and the last nucleotide of the D
sequence. Second, the AAV-2 rep binding motif [GCTCGCTCGCTCGCTG
(SEQ ID NO: 20)] is well conserved. Such motif can also be found in
the human chromosome 19 AAV-2 pre-integration region. Finally,
non-structural and structural coding regions, and regulatory
elements similar to those of other AAV serotypes also exist in
AAV-1 genome.
[0091] Although the overall features of AAV terminal repeats are
very much conserved, the total length of the AAV terminal repeat
exhibits divergence. The terminal repeat of AAV-1 consists of 143
nucleotides while those of AAV-2, AAV-3, and AAV-4 are about 145 or
146 nucleotides. The loop region of AAV-1 ITR most closely
resembles that of AAV-4 in that it also uses TCT instead of the TTT
found in AAV-2 and AAV-3. The possibility of sequencing error was
eliminated using restriction enzyme digestion, since these three
nucleotides are part of the SacI site (gagctc; nt 69-74 of SEQ ID
NO: 1). The p5 promoter region of AAV-1 shows more variations in
nucleotide sequences with other AAV serotypes. However, it still
maintains the critical regulatory elements. The two copies of YY1
[See, FIGS. 1A-1F] sites seemed to be preserved in all known AAV
serotypes, which have been shown to be involved in regulating AAV
gene expression. In AAV-4, there are 56 additional nucleotides
inserted between YY1 and E-box/USF site, while in AAV-1, there are
26 additional nucleotides inserted before the E-box/USF site. The
p19 promoter, p40 promoter and polyA can also be identified from
the AAV-1 genome by analogy to known AAV serotypes, which are also
highly conserved.
[0092] Thus, the analysis of AAV terminal repeats of various
serotypes showed that the A and A' sequence is very much conserved.
One of the reasons may be the Rep binding motif (GCTC).sub.3GCTG
[SEQ ID NO: 20]. These sequences appear to be essential for AAV DNA
replication and site-specific integration. The same sequence has
also been shown to be preserved in a monkey genome [Samulski,
personal communication]. The first 8 nucleotides of the D sequence
are also identical in all known AAV serotypes. This is in
accordance with the observation of the Srivastava group that only
the first 10 nucleotides are essential for AAV packaging [X. S.
Wang et al, J. Virol., 71:3077-3082 (1997); X. S. Wang et al, J.
Virol., 71:1140-1146 (1997)]. The function of the rest of the D
sequences still remain unclear. They may be somehow related to
their tissue specificities. The variation of nucleotide in B and C
sequence may also suggest that the secondary structure of the ITRs
is more critical for its biological function, which has been
demonstrated in many previous publications.
EXAMPLE 3
Comparison of AAV-1 Sequences
[0093] The nucleotide sequences of AAV-1, obtained as described
above, were compared with known AAV sequences, including AAV-2,
AAV-4 and AAV-6 using DNA Star Megalign. This comparison revealed a
stretch of 71 identical nucleotides shared by AAV-1, AAV-2 and
AAV-6. See, FIGS. 1A-1F.
[0094] This comparison further suggested that AAV-6 is a hybrid
formed by homologous recombination of AAV-1 and AAV-2. See, FIGS.
3A and 3B. These nucleotides divide the AAV-6 genome into two
regions. The 5' half of AAV-6 of 522 nucleotides is identical to
that of AAV-2 except in 2 positions. The 3' half of AAV-6 including
the majority of the rep gene, complete cap gene and 3' ITR is 98%
identical to AAV-1.
[0095] Biologically, such recombination may enable AAV-1 to acquire
the ability to transmit through the human population. It is also
interesting to note that the ITRs of AAV-6 comprise one AAV-1 ITR
and one AAV-2 ITR. The replication model of defective parvovirus
can maintain this special arrangement. Studies on AAV integration
have shown that a majority of AAV integrants carries deletions in
at least one of the terminal repeats. These deletions have been
shown to be able to be repaired through gene conversion using the
other intact terminal repeat as a template. Therefore, it would be
very difficult to maintain AAV-6 as a homogenous population when an
integrated copy of AAV-6 is rescued from host cells with helper
virus infection. The AAV-6 with two identical AAV-2 ITRs or two
identical AAV-1 ITRs should be the dominant variants. The AAV-6
with two AAV-1 ITRs has been observed by Russell's group [Rutledge,
cited above (1998)]. So far there is no report on AAV-6 with two
AAV-2 ITRs. Acquirement of AAV-2 P5 promoter by AAV-6 may have
explained that AAV-6 have been isolated from human origin while
AAV-1 with the same virion has not. The regulation of P5 promoter
between different species of AAV may be different in vivo. This
observation suggests the capsid proteins of AAV were not the only
determinants for tissue specificity.
[0096] Although it is clear that AAV-6 is a hybrid of AAV-1 and
AAV-2, AAV-6 has already exhibited divergence from either AAV-1 or
AAV-2. There are two nucleotide differences between AAV-6 and AAV-2
in their first 450 nucleotides. There are about 1% differences
between AAV-6 and AAV-1 in nucleotide levels from nucleotides 522
to the 3' end. There also exists a quite divergent region
(nucleotide 4486-4593) between AAV-6 and AAV-1 (FIGS. 1A-1F). This
region does not encode any known proteins for AAVs. These
differences in nucleotide sequences may suggest that AAV-6 and
AAV-1 have gone through some evolution since the recombination took
place. Another possible explanation is that there exists another
variant of AAV-1 which has yet to be identified. So far, there is
no evidence to rule out either possibility. It is still unknown if
other hybrids (AAV-2 to AAV-4, etc.) existed in nature.
[0097] The coding region of AAV-1 was deduced by comparison with
other known AAV serotypes. Table 1 illustrates the coding region
differences between AAV-1 and AAV-6. The amino acid residues are
deduced according to AAV-2.
[0098] With reference to the amino acid position of AAV-1, Table 1
lists the amino acids of AAV-1 which have been changed to the
corresponding ones of AAV-6. The amino acids of AAV-1 are shown to
the left of the arrow. Reference may be made to SEQ ID NO: 5 of the
amino acid sequence of AAV-1 Rep 78 and to SEQ ID NO: 13 for the
amino acid sequence of AAV-1 VP1. TABLE-US-00001 TABLE 1 Coding
region variations between AAV-1 and AAV-6 Rep protein Cap protein
(Rep78) (VP1) Position(s) Amino acids Position(s) Amino acids 28
S6N 129 L6F 191 Q6H 418 E6D 192 H6D 531 E6K 308 E6D 584 F6L 598 A6V
642 N6H
[0099] It was surprising to see that the sequence of the AAV-1
coding region is almost identical to that of AAV-6 from position
452 to the end of coding region (99%). The first 508 nucleotides of
AAV-6 have been shown to be identical to those of AAV-2 [Rutledge,
cited above (1998)]. Since the components of AAV-6 genome seemed to
be AAV-2 left ITR-AAV-2 p5 promoter-AAV-1 coding region-AAV-1 right
ITR, it was concluded that AAV-6 is a naturally occurred hybrid
between AAV-1 and AAV-2.
EXAMPLE 4
Gene Therapy Vector Based on AAV-1
[0100] Recombinant gene transfer vectors based on AAV-1 viruses
were constructed by the methods described in Example 1. To produce
a hybrid recombinant virus with AAV-1 virion and AAV-2 ITR, the
AAV-1 trans plasmid (pAV1H) and the AAV-2 cis-lacZ plasmid (with
AAV-2 ITR) were used. The AAV-2 ITR was used in this vector in view
of its known ability to direct site-specific integration. Also
constructed for use in this experiment was an AAV-1 vector carrying
the green fluorescent protein (GFP) marker gene under the control
of the immediate early promoter of CMV using pAV1H as the trans
plasmid.
[0101] A. rAAV-1 Viruses Transfect Host Cells in Vitro
[0102] 84-31 cells, which are subclones of 293 cells (which express
adenovirus E1a, E1b) which stably express E4/ORF5, were infected
with rAAV-1 GFP or rAAV-lacZ. High levels of expression of GFP and
lacZ was detected in the cultured 84-31 cells. This suggested that
rAAV-1 based vector was very similar to AAV-2 based vectors in
ability to infect and expression levels.
[0103] B. rAAV-1 Viruses Transfect Cells in Vivo
[0104] The performance of AAV-1 based vectors was also tested in
vivo. The rAAV-1 CMV-.alpha.1AT virus was constructed as follows.
The EcoRI fragment of pAT85 (ATCC) containing human
.alpha.1-antitrypsin (.alpha.1AT) cDNA fragment was blunted and
cloned into PCR (Promega) at a SmaI site to obtain PCR-.alpha.1AT.
The CMV promoter was cloned into PCR-.alpha.1AT at the XbaI site.
The Alb-.alpha.1AT expression cassette was removed by XhoI and ClaI
and cloned into pAV1H at the XbaI site. This vector plasmid was
used to generate AAV-1-CMV-.alpha.1AT virus used in the experiment
described below.
[0105] For screening human antibodies against AAV, purified AAV
virus is lysed with Ripa buffer (10 mM Tris pH 8.2, 1% Triton
X-100, 1% SDS, 0.15 M NaCl) and separated in 10% SDS-PAGE gel. The
heat inactivated human serum was used at a 1 to 1000 dilution in
this assay. The rAAV-1 CMV-.alpha.1AT viruses were injected into
Rag-1 mice through tail vein injection at different dosages. The
concentration of human .alpha.1-antitrypsin in mouse serum was
measured using ELISA. The coating antibody is rabbit anti-human
human .alpha.1-antitrypsin (Sigma). The goat-antihuman
.alpha.1-antitrypsin (Sigma) was used as the primary detection
antibodies. The sensitivity of this assay is around 0.3 ng/ml to 30
ng/ml. The expression of human .alpha.-antitrypsin in mouse blood
can be detected in a very encouraging level. This result is shown
in Table 2. TABLE-US-00002 TABLE 2 Human Antitrypsin Expressed in
Mouse Liver Amount of virus injected Week 2 (ng/ml) Week 4 (ng/ml)
2 .times. 10.sup.10 genomes 214.2 171.4 1 .times. 10.sup.10 genomes
117.8 109.8 5 .times. 10.sup.10 genomes 64.5 67.8 2.5 .times.
10.sup.10 genomes 30.9 58.4
[0106] rAAV-1 CMV-lacZ viruses were also injected into the muscle
of C57BL6 mice and similar results were obtained. Collectively,
these results suggested that AAV-1 based vector would be
appropriate for both liver and muscle gene delivery.
EXAMPLE 5
Neutralizing Antibodies Against AAV-1
[0107] Simple and quantitative assays for neutralizing antibodies
(NAB) to AAV-1 and AAV-2 were developed with recombinant vectors. A
total of 33 rhesus monkeys and 77 normal human subjects were
screened.
[0108] 1. Nonhuman Primates
[0109] Wild-caught juvenile rhesus monkeys were purchased from
Covance (Alice, Tex.) and LABS of Virginia (Yemassee, S.C.) and
kept in full quarantine. The monkeys weighed approximately 3 to 4
kg. The nonhuman primates used in the Institute for Human Gene
Therapy research program are purposefully bred in the United States
from specific-pathogen-free closed colonies. All vendors are US
Department of Agriculture class A dealers. The rhesus macaques are
therefore not infected with important simian pathogens, including
the tuberculosis agent, major simian lentiviruses (simian
immunodeficiency virus and simian retroviruses), and cercopithecine
herpesvirus. The animals are also free of internal and external
parasites. The excellent health status of these premium animals
minimized the potential for extraneous variables. For this study,
serum was obtained from monkeys prior to initiation of any
protocol.
[0110] NAB titers were analyzed by assessing the ability of serum
antibody to inhibit the transduction of reporter virus expressing
green fluorescent protein (GFP) (AAV1-GFP or AAV2-GFP) into 84-31
cells. Various dilutions of antibodies preincubated with reporter
virus for 1 hour at 37.degree. C. were added to 90% confluent cell
cultures. Cells were incubated for 48 hours and the expression of
green fluorescent protein was measured by FluoroImaging (Molecular
Dynamics). NAB titers were calculated as the highest dilution at
which 50% of the cells stained green.
[0111] Analysis of NAB in rhesus monkeys showed that 61% of animals
tested positive for AAV-1; a minority (24%) has NAB to AAV-2. Over
one-third of animals had antibodies to AAV-1 but not AAV-2 (i.e.,
were monospecific for AAV-1), whereas no animals were positive for
AAV-2 without reacting to AAV-1. These data support the hypothesis
that AAV-1 is endemic in rhesus monkeys. The presence of true AAV-2
infections in this group of nonhuman primates is less clear, since
cross-neutralizing activity of an AAV-1 response to AAV-2 can not
be ruled out. It is interesting that there is a linear relationship
between AAV-2 NAB and AAV-1 NAB in animals that had both.
[0112] 2. Humans
[0113] For these neutralization antibody assays, human serum
samples were incubated at 56.degree. C. for 30 min to inactivate
complement and then diluted in DMEM. The virus (rAAV or rAd with
either lacZ or GFP) was then mixed with each serum dilution
(20.times., 400.times., 2000.times., 4000.times., etc.) and
incubated for 1 hour at 37.degree. C. before applied to 90%
confluent cultures of 84-31 cells (for AAV) or Hela cells (for
adenovirus) in 96-well plates. After 60 minutes of incubation at
culture condition, 100 .mu.l additional media containing 20% FCS
was added to make final culture media containing 10% FCS.
[0114] The results are summarized in Table 3. TABLE-US-00003 TABLE
3 Adenovirus AAV-1 AAV-2 # of samples Percentage - - - 41 53.2% + -
- 16 20.8% - + - 0 0.0% - - + 2 2.6% - + + 2 2.6% + - + 3 3.9% + +
- 0 0.0% + + + 13 16.9% Total 77 100%
[0115] The human neutralizing antibodies against these three
viruses seemed to be unrelated since the existence of neutralizing
antibodies against AAV are not indications for antibodies against
adenovirus. However, AAV requires adenovirus as helper virus, in
most of the cases, the neutralizing antibodies against AAV
correlated with the existence of neutralizing antibodies to
adenovirus. Among the 77 human serum samples screened, 41% of the
samples can neutralize the infectivity of recombinant adenovirus
based on Ad5. 15/77 (19%) of serum samples can neutralize the
transduction of rAAV-1 while 20/77 (20%) of the samples inhibit
rAAV-2 transduction at 1 to 80 dilutions or higher. All serum
samples positive in neutralizing antibodies for AAV-1 in are also
positive for AAV-2. However, there are five (6%) rAAV-2 positive
samples that failed to neutralize rAAV-1. In samples that are
positive for neutralizing antibodies, the titer of antibodies also
varied in the positive ones. The results from screening human sera
for antibodies against AAVs supported the conclusion that AAV-1
presents the same epitome as that of AAV-2 to interact with
cellular receptors since AAV-1 neutralizing human serums can also
decrease the infectivity of AAV-2. However, the profile of
neutralizing antibodies for these AAVs is not identical, there are
additional specific receptors for each AAV serotype.
EXAMPLE 6
Recombinant AAV Viruses Exhibit Tissue Tropism
[0116] The recombinant AAV-1 vectors of the invention and the
recombinant AAV-2 vectors [containing the gene encoding human
.alpha.1-antitrypsin (.alpha.1AT) or murine erythropoietin (Epo)
from a cytomegalovirus-enhanced .beta.-actin promoter (CB)] were
evaluated in a direct comparison to equivalent copies of AAV-2
vectors containing the same vector genes.
[0117] Recombinant viruses with AAV-1 capsids were constructed
using the techniques in Example 1. To make rAAV with AAV-1 virions,
pAV1H or p5E18 (2/1) was used as the trans plasmid to provide Rep
and Cap functions. For the generation of the rAAV based on AAV-2,
p5E18(2/2) was used as the trans plasmid, since it greatly improved
the rAAV yield. [Early experiments indicated similar in vivo
performances of AAV-1 vectors produced with pAV1H and p5E19 (2/1).
All subsequent studies used AAV-1 vectors derived from p5E18(2/1)
because of the increased yield.]
[0118] Equivalent stocks of the AAV-1 and AAV-2 vectors were
injected intramuscularly (5.times.10.sup.10 genomes) or liver via
the portal circulation (1.times.10.sup.11 genomes) into
immunodeficient mice, and the animals (four groups) were analyzed
on day 30 for expression of transgene. See, FIGS. 4A and 4B.
[0119] AAV-2 vectors consistently produced 10- to 50-fold more
serum erythropoietin or .alpha.1-antitrypsin when injected into
liver compared to muscle. (However, the AAV-1-delivered genes did
achieve acceptable expression levels in the liver.) This result was
very different from that for AAV-1 vectors, with which muscle
expression was equivalent to or greater than liver expression. In
fact, AAV-1 outperformed AAV-2 in muscle when equivalent titers
based on genomes were administered.
EXAMPLE 7
Gene Delivery Via rAAV-1
[0120] C57BL/6 mice (6- to 8-week old males, Jackson Laboratories)
were analyzed for AAV mediated gene transfer to liver following
intrasplenic injection of vector (i.e., targeted to liver). A total
of 10.sup.11 genome equivalents of rAAV-1 or rAAV-2 vector were
injected into the circulation in 100 .mu.l buffered saline. The
first vector contained either an AAV-1 capsid or an AAV-2 capsid
and expressed .alpha.1AT under the control of the chicken
.beta.-actin (CB) promoter. Day 28 sera were analyzed for
antibodies against AAV-1 or AAV-2 and serum .alpha.1AT levels were
checked. Animals were then injected with an AAV-1 or AAV-2
construct expressing erythropoietin (Epo, also under the control of
the CB promoter). One month later sera was analyzed for serum
levels of Epo. The following groups were analyzed (FIGS.
5A-5D).
[0121] In Group 1, vector 1 was AAV-2 expressing .alpha.1AT and
vector 2 was AAV-2 expressing Epo. Animals generated antibodies
against AAV-2 following the first vector administration which
prevented the readministration of the AAV-2 based vector. There was
no evidence for cross-neutralizing the antibody to AAV-1.
[0122] In Group 2, vector 1 was AAV-1 expressing .alpha.1AT while
vector 2 was AAV-1 expressing Epo. The first vector administration
did result in significant .alpha.1AT expression at one month
associated with antibodies to neutralizing antibodies to AAV-1. The
animals were not successfully readministered with the AAV-1 Epo
expressing construct.
[0123] In Group 3, the effectiveness of an AAV-2 vector expressing
Epo injected into a naive animal was measured. The animals were
injected with PBS and injected with AAV-2 Epo vector at day 28 and
analyzed for Epo expression one month later. The neutralizing
antibodies were evaluated at day 28 so we did not expect to see
anything since they received PBS with the first vector injection.
This shows that in naive animals AAV-2 is very efficient at
transferring the Epo gene as demonstrated by high level of serum
Epo one month later.
[0124] Group 4 was an experiment similar to Group 3 in which the
animals originally received PBS for vector 1 and then the AAV-1
expressing Epo construct 28 days later. At the time of vector
injection, there obviously were no antibodies to either AAV-1 or
AAV-2. The AAV-1 based vector was capable of generating significant
expression of Epo when measured one month later.
[0125] Group 5 is a cross-over experiment where the initial vector
is AAV-2 expressing .alpha.1AT followed by the AAV-1 construct
expressing Epo. The animals, as expected, were efficiently infected
with the AAV-2 vector expressing .alpha.1AT as shown by high levels
of the protein in blood at 28 days. This was associated with
significant neutralizing antibodies to AAV-2. Importantly, the
animals were successfully administered AAV-1 following the AAV-2
vector as shown by the presence of Epo in serum 28 days following
the second vector administration. At the time of this vector
administration, there was high level AAV-2 neutralizing antibodies
and very low cross-reaction to AAV-1. The level of Epo was slightly
diminished possibly due to a small amount of cross-reactivity.
Group 6 was the opposite cross-over experiment in which the initial
vector was AAV-1 based, whereas the second experiment was AAV-2
based. The AAV-1 vector did lead to significant gene expression of
.alpha.1AT, which also resulted in high level AAV-1 neutralizing
antibody. The animals were very efficiently administered AAV-2
following the initial AAV-1 vector as evidenced by high level
Epo.
[0126] A substantially identical experiment was performed in muscle
in which 5.times.10.sup.10 genomes were injected into the tibialis
anterior of C57BL/6 mice as a model for muscle directed gene
therapy. The results are illustrated in FIGS. 6A-6D and are
essentially the same as for liver.
[0127] In summary, this experiment demonstrates the utility of
using an AAV-1 vector in patients who have pre-existing antibodies
to AAV-2 or who had initially received an AAV-2 vector and need
readministration.
EXAMPLE 8
Construction of Recombinant Viruses Containing AAV-1 ITRs
[0128] This example illustrates the construction of recombinant AAV
vectors which contain AAV-1 ITRs of the invention.
[0129] An AAV-1 cis plasmid is constructed as follows. A 160 bp
Xho-NruI AAV-1 fragment containing the AAV-1 5' ITR is obtained
from pAV1-BL. pAV1-BL was generated as described in Example 1. The
Xho-NruI fragment is then cloned into a second pAV1-BL plasmid at
an XbaI site to provide the plasmid with two AAV-1 ITRs. The
desired transgene is then cloned into the modified pAV-1BL at the
NruI and BamHI site, which is located between the AAV-1 ITR
sequences. The resulting AAV-1 cis plasmid contains AAV-1 ITRs
flanking the transgene and lacks functional AAV-1 rep and cap.
[0130] Recombinant AAV is produced by simultaneously transfecting
three plasmids into 293 cells. These include the AAV-1 cis plasmid
described above; a trans plasmid which provides AAV rep/cap
functions and lacks AAV ITRs; and a plasmid providing adenovirus
helper functions. The rep and/or cap functions may be provided in
trans by AAV-1 or another AAV serotype, depending on the immunity
profile of the intended recipient. Alternatively, the rep or cap
functions may be provided in cis by AAV-1 or another serotype,
again depending on the patient's immunity profile.
[0131] In a typical cotransfection, 50 .mu.g of DNA
(cis:trans:helper at ratios of 1:1:2, respectively) is transfected
onto a 15 cm tissue culture dish. Cells are harvested 96 hours post
transfection, sonicated and treated with 0.5% sodium deoxycholate
(37E for 10 min). Cell lysates are then subjected to 2-3 rounds of
ultracentrifugation in a cesium gradient. Peak fractions containing
rAAV are collected, pooled and dialyzed against PBS. A typical
yield is 1.times.10.sup.13 genomes/10.sup.9 cells.
[0132] Using this method, one recombinant virus construct is
prepared which contains the AAV-1 ITRs flanking the transgene, with
an AAV-1 capsid. Another recombinant virus construct is prepared
with contains the AAV-1 ITRs flanking the transgene, with an AAV-2
capsid.
[0133] All publications cited in this specification are
incorporated herein by reference. While the invention has been
described with reference to a particularly preferred embodiments,
it will be appreciated that modifications can be made without
departing from the spirit of the invention. Such modifications are
intended to fall within the scope of the claims.
Sequence CWU 1
1
20 1 4718 DNA AAV-1 CDS (335)..(2206) CDS (2223)..(4430) 1
ttgcccactc cctctctgcg cgctcgctcg ctcggtgggg cctgcggacc aaaggtccgc
60 agacggcaga gctctgctct gccggcccca ccgagcgagc gagcgcgcag
agagggagtg 120 ggcaactcca tcactagggg taatcgcgaa gcgcctccca
cgctgccgcg tcagcgctga 180 cgtaaattac gtcatagggg agtggtcctg
tattagctgt cacgtgagtg cttttgcgac 240 attttgcgac accacgtggc
catttagggt atatatggcc gagtgagcga gcaggatctc 300 cattttgacc
gcgaaatttg aacgagcagc agcc atg ccg ggc ttc tac gag atc 355 Met Pro
Gly Phe Tyr Glu Ile 1 5 gtg atc aag gtg ccg agc gac ctg gac gag cac
ctg ccg ggc att tct 403 Val Ile Lys Val Pro Ser Asp Leu Asp Glu His
Leu Pro Gly Ile Ser 10 15 20 gac tcg ttt gtg agc tgg gtg gcc gag
aag gaa tgg gag ctg ccc ccg 451 Asp Ser Phe Val Ser Trp Val Ala Glu
Lys Glu Trp Glu Leu Pro Pro 25 30 35 gat tct gac atg gat ctg aat
ctg att gag cag gca ccc ctg acc gtg 499 Asp Ser Asp Met Asp Leu Asn
Leu Ile Glu Gln Ala Pro Leu Thr Val 40 45 50 55 gcc gag aag ctg cag
cgc gac ttc ctg gtc caa tgg cgc cgc gtg agt 547 Ala Glu Lys Leu Gln
Arg Asp Phe Leu Val Gln Trp Arg Arg Val Ser 60 65 70 aag gcc ccg
gag gcc ctc ttc ttt gtt cag ttc gag aag ggc gag tcc 595 Lys Ala Pro
Glu Ala Leu Phe Phe Val Gln Phe Glu Lys Gly Glu Ser 75 80 85 tac
ttc cac ctc cat att ctg gtg gag acc acg ggg gtc aaa tcc atg 643 Tyr
Phe His Leu His Ile Leu Val Glu Thr Thr Gly Val Lys Ser Met 90 95
100 gtg ctg ggc cgc ttc ctg agt cag att agg gac aag ctg gtg cag acc
691 Val Leu Gly Arg Phe Leu Ser Gln Ile Arg Asp Lys Leu Val Gln Thr
105 110 115 atc tac cgc ggg atc gag ccg acc ctg ccc aac tgg ttc gcg
gtg acc 739 Ile Tyr Arg Gly Ile Glu Pro Thr Leu Pro Asn Trp Phe Ala
Val Thr 120 125 130 135 aag acg cgt aat ggc gcc gga ggg ggg aac aag
gtg gtg gac gag tgc 787 Lys Thr Arg Asn Gly Ala Gly Gly Gly Asn Lys
Val Val Asp Glu Cys 140 145 150 tac atc ccc aac tac ctc ctg ccc aag
act cag ccc gag ctg cag tgg 835 Tyr Ile Pro Asn Tyr Leu Leu Pro Lys
Thr Gln Pro Glu Leu Gln Trp 155 160 165 gcg tgg act aac atg gag gag
tat ata agc gcc tgt ttg aac ctg gcc 883 Ala Trp Thr Asn Met Glu Glu
Tyr Ile Ser Ala Cys Leu Asn Leu Ala 170 175 180 gag cgc aaa cgg ctc
gtg gcg cag cac ctg acc cac gtc agc cag acc 931 Glu Arg Lys Arg Leu
Val Ala Gln His Leu Thr His Val Ser Gln Thr 185 190 195 cag gag cag
aac aag gag aat ctg aac ccc aat tct gac gcg cct gtc 979 Gln Glu Gln
Asn Lys Glu Asn Leu Asn Pro Asn Ser Asp Ala Pro Val 200 205 210 215
atc cgg tca aaa acc tcc gcg cgc tac atg gag ctg gtc ggg tgg ctg
1027 Ile Arg Ser Lys Thr Ser Ala Arg Tyr Met Glu Leu Val Gly Trp
Leu 220 225 230 gtg gac cgg ggc atc acc tcc gag aag cag tgg atc cag
gag gac cag 1075 Val Asp Arg Gly Ile Thr Ser Glu Lys Gln Trp Ile
Gln Glu Asp Gln 235 240 245 gcc tcg tac atc tcc ttc aac gcc gct tcc
aac tcg cgg tcc cag atc 1123 Ala Ser Tyr Ile Ser Phe Asn Ala Ala
Ser Asn Ser Arg Ser Gln Ile 250 255 260 aag gcc gct ctg gac aat gcc
ggc aag atc atg gcg ctg acc aaa tcc 1171 Lys Ala Ala Leu Asp Asn
Ala Gly Lys Ile Met Ala Leu Thr Lys Ser 265 270 275 gcg ccc gac tac
ctg gta ggc ccc gct ccg ccc gcg gac att aaa acc 1219 Ala Pro Asp
Tyr Leu Val Gly Pro Ala Pro Pro Ala Asp Ile Lys Thr 280 285 290 295
aac cgc atc tac cgc atc ctg gag ctg aac ggc tac gaa cct gcc tac
1267 Asn Arg Ile Tyr Arg Ile Leu Glu Leu Asn Gly Tyr Glu Pro Ala
Tyr 300 305 310 gcc ggc tcc gtc ttt ctc ggc tgg gcc cag aaa agg ttc
ggg aag cgc 1315 Ala Gly Ser Val Phe Leu Gly Trp Ala Gln Lys Arg
Phe Gly Lys Arg 315 320 325 aac acc atc tgg ctg ttt ggg ccg gcc acc
acg ggc aag acc aac atc 1363 Asn Thr Ile Trp Leu Phe Gly Pro Ala
Thr Thr Gly Lys Thr Asn Ile 330 335 340 gcg gaa gcc atc gcc cac gcc
gtg ccc ttc tac ggc tgc gtc aac tgg 1411 Ala Glu Ala Ile Ala His
Ala Val Pro Phe Tyr Gly Cys Val Asn Trp 345 350 355 acc aat gag aac
ttt ccc ttc aat gat tgc gtc gac aag atg gtg atc 1459 Thr Asn Glu
Asn Phe Pro Phe Asn Asp Cys Val Asp Lys Met Val Ile 360 365 370 375
tgg tgg gag gag ggc aag atg acg gcc aag gtc gtg gag tcc gcc aag
1507 Trp Trp Glu Glu Gly Lys Met Thr Ala Lys Val Val Glu Ser Ala
Lys 380 385 390 gcc att ctc ggc ggc agc aag gtg cgc gtg gac caa aag
tgc aag tcg 1555 Ala Ile Leu Gly Gly Ser Lys Val Arg Val Asp Gln
Lys Cys Lys Ser 395 400 405 tcc gcc cag atc gac ccc acc ccc gtg atc
gtc acc tcc aac acc aac 1603 Ser Ala Gln Ile Asp Pro Thr Pro Val
Ile Val Thr Ser Asn Thr Asn 410 415 420 atg tgc gcc gtg att gac ggg
aac agc acc acc ttc gag cac cag cag 1651 Met Cys Ala Val Ile Asp
Gly Asn Ser Thr Thr Phe Glu His Gln Gln 425 430 435 ccg ttg cag gac
cgg atg ttc aaa ttt gaa ctc acc cgc cgt ctg gag 1699 Pro Leu Gln
Asp Arg Met Phe Lys Phe Glu Leu Thr Arg Arg Leu Glu 440 445 450 455
cat gac ttt ggc aag gtg aca aag cag gaa gtc aaa gag ttc ttc cgc
1747 His Asp Phe Gly Lys Val Thr Lys Gln Glu Val Lys Glu Phe Phe
Arg 460 465 470 tgg gcg cag gat cac gtg acc gag gtg gcg cat gag ttc
tac gtc aga 1795 Trp Ala Gln Asp His Val Thr Glu Val Ala His Glu
Phe Tyr Val Arg 475 480 485 aag ggt gga gcc aac aaa aga ccc gcc ccc
gat gac gcg gat aaa agc 1843 Lys Gly Gly Ala Asn Lys Arg Pro Ala
Pro Asp Asp Ala Asp Lys Ser 490 495 500 gag ccc aag cgg gcc tgc ccc
tca gtc gcg gat cca tcg acg tca gac 1891 Glu Pro Lys Arg Ala Cys
Pro Ser Val Ala Asp Pro Ser Thr Ser Asp 505 510 515 gcg gaa gga gct
ccg gtg gac ttt gcc gac agg tac caa aac aaa tgt 1939 Ala Glu Gly
Ala Pro Val Asp Phe Ala Asp Arg Tyr Gln Asn Lys Cys 520 525 530 535
tct cgt cac gcg ggc atg ctt cag atg ctg ttt ccc tgc aag aca tgc
1987 Ser Arg His Ala Gly Met Leu Gln Met Leu Phe Pro Cys Lys Thr
Cys 540 545 550 gag aga atg aat cag aat ttc aac att tgc ttc acg cac
ggg acg aga 2035 Glu Arg Met Asn Gln Asn Phe Asn Ile Cys Phe Thr
His Gly Thr Arg 555 560 565 gac tgt tca gag tgc ttc ccc ggc gtg tca
gaa tct caa ccg gtc gtc 2083 Asp Cys Ser Glu Cys Phe Pro Gly Val
Ser Glu Ser Gln Pro Val Val 570 575 580 aga aag agg acg tat cgg aaa
ctc tgt gcc att cat cat ctg ctg ggg 2131 Arg Lys Arg Thr Tyr Arg
Lys Leu Cys Ala Ile His His Leu Leu Gly 585 590 595 cgg gct ccc gag
att gct tgc tcg gcc tgc gat ctg gtc aac gtg gac 2179 Arg Ala Pro
Glu Ile Ala Cys Ser Ala Cys Asp Leu Val Asn Val Asp 600 605 610 615
ctg gat gac tgt gtt tct gag caa taa atgacttaaa ccaggt atg gct gcc
2231 Leu Asp Asp Cys Val Ser Glu Gln Met Ala Ala 620 625 gat ggt
tat ctt cca gat tgg ctc gag gac aac ctc tct gag ggc att 2279 Asp
Gly Tyr Leu Pro Asp Trp Leu Glu Asp Asn Leu Ser Glu Gly Ile 630 635
640 cgc gag tgg tgg gac ttg aaa cct gga gcc ccg aag ccc aaa gcc aac
2327 Arg Glu Trp Trp Asp Leu Lys Pro Gly Ala Pro Lys Pro Lys Ala
Asn 645 650 655 cag caa aag cag gac gac ggc cgg ggt ctg gtg ctt cct
ggc tac aag 2375 Gln Gln Lys Gln Asp Asp Gly Arg Gly Leu Val Leu
Pro Gly Tyr Lys 660 665 670 tac ctc gga ccc ttc aac gga ctc gac aag
ggg gag ccc gtc aac gcg 2423 Tyr Leu Gly Pro Phe Asn Gly Leu Asp
Lys Gly Glu Pro Val Asn Ala 675 680 685 690 gcg gac gca gcg gcc ctc
gag cac gac aag gcc tac gac cag cag ctc 2471 Ala Asp Ala Ala Ala
Leu Glu His Asp Lys Ala Tyr Asp Gln Gln Leu 695 700 705 aaa gcg ggt
gac aat ccg tac ctg cgg tat aac cac gcc gac gcc gag 2519 Lys Ala
Gly Asp Asn Pro Tyr Leu Arg Tyr Asn His Ala Asp Ala Glu 710 715 720
ttt cag gag cgt ctg caa gaa gat acg tct ttt ggg ggc aac ctc ggg
2567 Phe Gln Glu Arg Leu Gln Glu Asp Thr Ser Phe Gly Gly Asn Leu
Gly 725 730 735 cga gca gtc ttc cag gcc aag aag cgg gtt ctc gaa cct
ctc ggt ctg 2615 Arg Ala Val Phe Gln Ala Lys Lys Arg Val Leu Glu
Pro Leu Gly Leu 740 745 750 gtt gag gaa ggc gct aag acg gct cct gga
aag aaa cgt ccg gta gag 2663 Val Glu Glu Gly Ala Lys Thr Ala Pro
Gly Lys Lys Arg Pro Val Glu 755 760 765 770 cag tcg cca caa gag cca
gac tcc tcc tcg ggc atc ggc aag aca ggc 2711 Gln Ser Pro Gln Glu
Pro Asp Ser Ser Ser Gly Ile Gly Lys Thr Gly 775 780 785 cag cag ccc
gct aaa aag aga ctc aat ttt ggt cag act ggc gac tca 2759 Gln Gln
Pro Ala Lys Lys Arg Leu Asn Phe Gly Gln Thr Gly Asp Ser 790 795 800
gag tca gtc ccc gat cca caa cct ctc gga gaa cct cca gca acc ccc
2807 Glu Ser Val Pro Asp Pro Gln Pro Leu Gly Glu Pro Pro Ala Thr
Pro 805 810 815 gct gct gtg gga cct act aca atg gct tca ggc ggt ggc
gca cca atg 2855 Ala Ala Val Gly Pro Thr Thr Met Ala Ser Gly Gly
Gly Ala Pro Met 820 825 830 gca gac aat aac gaa ggc gcc gac gga gtg
ggt aat gcc tca gga aat 2903 Ala Asp Asn Asn Glu Gly Ala Asp Gly
Val Gly Asn Ala Ser Gly Asn 835 840 845 850 tgg cat tgc gat tcc aca
tgg ctg ggc gac aga gtc atc acc acc agc 2951 Trp His Cys Asp Ser
Thr Trp Leu Gly Asp Arg Val Ile Thr Thr Ser 855 860 865 acc cgc acc
tgg gcc ttg ccc acc tac aat aac cac ctc tac aag caa 2999 Thr Arg
Thr Trp Ala Leu Pro Thr Tyr Asn Asn His Leu Tyr Lys Gln 870 875 880
atc tcc agt gct tca acg ggg gcc agc aac gac aac cac tac ttc ggc
3047 Ile Ser Ser Ala Ser Thr Gly Ala Ser Asn Asp Asn His Tyr Phe
Gly 885 890 895 tac agc acc ccc tgg ggg tat ttt gat ttc aac aga ttc
cac tgc cac 3095 Tyr Ser Thr Pro Trp Gly Tyr Phe Asp Phe Asn Arg
Phe His Cys His 900 905 910 ttt tca cca cgt gac tgg cag cga ctc atc
aac aac aat tgg gga ttc 3143 Phe Ser Pro Arg Asp Trp Gln Arg Leu
Ile Asn Asn Asn Trp Gly Phe 915 920 925 930 cgg ccc aag aga ctc aac
ttc aaa ctc ttc aac atc caa gtc aag gag 3191 Arg Pro Lys Arg Leu
Asn Phe Lys Leu Phe Asn Ile Gln Val Lys Glu 935 940 945 gtc acg acg
aat gat ggc gtc aca acc atc gct aat aac ctt acc agc 3239 Val Thr
Thr Asn Asp Gly Val Thr Thr Ile Ala Asn Asn Leu Thr Ser 950 955 960
acg gtt caa gtc ttc tcg gac tcg gag tac cag ctt ccg tac gtc ctc
3287 Thr Val Gln Val Phe Ser Asp Ser Glu Tyr Gln Leu Pro Tyr Val
Leu 965 970 975 ggc tct gcg cac cag ggc tgc ctc cct ccg ttc ccg gcg
gac gtg ttc 3335 Gly Ser Ala His Gln Gly Cys Leu Pro Pro Phe Pro
Ala Asp Val Phe 980 985 990 atg att ccg caa tac ggc tac ctg acg ctc
aac aat ggc agc caa 3380 Met Ile Pro Gln Tyr Gly Tyr Leu Thr Leu
Asn Asn Gly Ser Gln 995 1000 1005 gcc gtg gga cgt tca tcc ttt tac
tgc ctg gaa tat ttc cct tct 3425 Ala Val Gly Arg Ser Ser Phe Tyr
Cys Leu Glu Tyr Phe Pro Ser 1010 1015 1020 cag atg ctg aga acg ggc
aac aac ttt acc ttc agc tac acc ttt 3470 Gln Met Leu Arg Thr Gly
Asn Asn Phe Thr Phe Ser Tyr Thr Phe 1025 1030 1035 gag gaa gtg cct
ttc cac agc agc tac gcg cac agc cag agc ctg 3515 Glu Glu Val Pro
Phe His Ser Ser Tyr Ala His Ser Gln Ser Leu 1040 1045 1050 gac cgg
ctg atg aat cct ctc atc gac caa tac ctg tat tac ctg 3560 Asp Arg
Leu Met Asn Pro Leu Ile Asp Gln Tyr Leu Tyr Tyr Leu 1055 1060 1065
aac aga act caa aat cag tcc gga agt gcc caa aac aag gac ttg 3605
Asn Arg Thr Gln Asn Gln Ser Gly Ser Ala Gln Asn Lys Asp Leu 1070
1075 1080 ctg ttt agc cgt ggg tct cca gct ggc atg tct gtt cag ccc
aaa 3650 Leu Phe Ser Arg Gly Ser Pro Ala Gly Met Ser Val Gln Pro
Lys 1085 1090 1095 aac tgg cta cct gga ccc tgt tat cgg cag cag cgc
gtt tct aaa 3695 Asn Trp Leu Pro Gly Pro Cys Tyr Arg Gln Gln Arg
Val Ser Lys 1100 1105 1110 aca aaa aca gac aac aac aac agc aat ttt
acc tgg act ggt gct 3740 Thr Lys Thr Asp Asn Asn Asn Ser Asn Phe
Thr Trp Thr Gly Ala 1115 1120 1125 tca aaa tat aac ctc aat ggg cgt
gaa tcc atc atc aac cct ggc 3785 Ser Lys Tyr Asn Leu Asn Gly Arg
Glu Ser Ile Ile Asn Pro Gly 1130 1135 1140 act gct atg gcc tca cac
aaa gac gac gaa gac aag ttc ttt ccc 3830 Thr Ala Met Ala Ser His
Lys Asp Asp Glu Asp Lys Phe Phe Pro 1145 1150 1155 atg agc ggt gtc
atg att ttt gga aaa gag agc gcc gga gct tca 3875 Met Ser Gly Val
Met Ile Phe Gly Lys Glu Ser Ala Gly Ala Ser 1160 1165 1170 aac act
gca ttg gac aat gtc atg att aca gac gaa gag gaa att 3920 Asn Thr
Ala Leu Asp Asn Val Met Ile Thr Asp Glu Glu Glu Ile 1175 1180 1185
aaa gcc act aac cct gtg gcc acc gaa aga ttt ggg acc gtg gca 3965
Lys Ala Thr Asn Pro Val Ala Thr Glu Arg Phe Gly Thr Val Ala 1190
1195 1200 gtc aat ttc cag agc agc agc aca gac cct gcg acc gga gat
gtg 4010 Val Asn Phe Gln Ser Ser Ser Thr Asp Pro Ala Thr Gly Asp
Val 1205 1210 1215 cat gct atg gga gca tta cct ggc atg gtg tgg caa
gat aga gac 4055 His Ala Met Gly Ala Leu Pro Gly Met Val Trp Gln
Asp Arg Asp 1220 1225 1230 gtg tac ctg cag ggt ccc att tgg gcc aaa
att cct cac aca gat 4100 Val Tyr Leu Gln Gly Pro Ile Trp Ala Lys
Ile Pro His Thr Asp 1235 1240 1245 gga cac ttt cac ccg tct cct ctt
atg ggc ggc ttt gga ctc aag 4145 Gly His Phe His Pro Ser Pro Leu
Met Gly Gly Phe Gly Leu Lys 1250 1255 1260 aac ccg cct cct cag atc
ctc atc aaa aac acg cct gtt cct gcg 4190 Asn Pro Pro Pro Gln Ile
Leu Ile Lys Asn Thr Pro Val Pro Ala 1265 1270 1275 aat cct ccg gcg
gag ttt tca gct aca aag ttt gct tca ttc atc 4235 Asn Pro Pro Ala
Glu Phe Ser Ala Thr Lys Phe Ala Ser Phe Ile 1280 1285 1290 acc caa
tac tcc aca gga caa gtg agt gtg gaa att gaa tgg gag 4280 Thr Gln
Tyr Ser Thr Gly Gln Val Ser Val Glu Ile Glu Trp Glu 1295 1300 1305
ctg cag aaa gaa aac agc aag cgc tgg aat ccc gaa gtg cag tac 4325
Leu Gln Lys Glu Asn Ser Lys Arg Trp Asn Pro Glu Val Gln Tyr 1310
1315 1320 aca tcc aat tat gca aaa tct gcc aac gtt gat ttt act gtg
gac 4370 Thr Ser Asn Tyr Ala Lys Ser Ala Asn Val Asp Phe Thr Val
Asp 1325 1330 1335 aac aat gga ctt tat act gag cct cgc ccc att ggc
acc cgt tac 4415 Asn Asn Gly Leu Tyr Thr Glu Pro Arg Pro Ile Gly
Thr Arg Tyr 1340 1345 1350 ctt acc cgt ccc ctg taattacgtg
ttaatcaata aaccggttga ttcgtttcag 4470 Leu Thr Arg Pro Leu 1355
ttgaactttg gtctcctgtc cttcttatct tatcggttac catggttata gcttacacat
4530 taactgcttg gttgcgcttc gcgataaaag acttacgtca tcgggttacc
cctagtgatg 4590 gagttgccca ctccctctct gcgcgctcgc tcgctcggtg
gggcctgcgg accaaaggtc 4650 cgcagacggc agagctctgc tctgccggcc
ccaccgagcg agcgagcgcg cagagaggga 4710 gtgggcaa 4718 2 623 PRT AAV-1
2 Met Pro Gly Phe Tyr Glu Ile Val Ile Lys Val Pro Ser Asp Leu Asp 1
5 10 15 Glu His Leu Pro Gly Ile Ser Asp Ser Phe Val Ser Trp Val Ala
Glu 20 25 30 Lys Glu Trp Glu Leu Pro Pro Asp Ser Asp Met Asp Leu
Asn Leu Ile 35 40 45 Glu Gln Ala Pro Leu Thr Val Ala Glu Lys Leu
Gln Arg Asp Phe Leu 50 55 60 Val Gln Trp Arg Arg Val Ser Lys Ala
Pro Glu Ala Leu Phe Phe Val 65 70 75 80 Gln Phe Glu Lys Gly Glu Ser
Tyr Phe His Leu His Ile Leu Val Glu 85 90 95 Thr Thr Gly Val Lys
Ser Met Val Leu Gly Arg Phe Leu Ser Gln Ile 100 105 110 Arg Asp Lys
Leu Val Gln Thr Ile Tyr Arg Gly Ile Glu Pro Thr Leu
115 120 125 Pro Asn Trp Phe Ala Val Thr Lys Thr Arg Asn Gly Ala Gly
Gly Gly 130 135 140 Asn Lys Val Val Asp Glu Cys Tyr Ile Pro Asn Tyr
Leu Leu Pro Lys 145 150 155 160 Thr Gln Pro Glu Leu Gln Trp Ala Trp
Thr Asn Met Glu Glu Tyr Ile 165 170 175 Ser Ala Cys Leu Asn Leu Ala
Glu Arg Lys Arg Leu Val Ala Gln His 180 185 190 Leu Thr His Val Ser
Gln Thr Gln Glu Gln Asn Lys Glu Asn Leu Asn 195 200 205 Pro Asn Ser
Asp Ala Pro Val Ile Arg Ser Lys Thr Ser Ala Arg Tyr 210 215 220 Met
Glu Leu Val Gly Trp Leu Val Asp Arg Gly Ile Thr Ser Glu Lys 225 230
235 240 Gln Trp Ile Gln Glu Asp Gln Ala Ser Tyr Ile Ser Phe Asn Ala
Ala 245 250 255 Ser Asn Ser Arg Ser Gln Ile Lys Ala Ala Leu Asp Asn
Ala Gly Lys 260 265 270 Ile Met Ala Leu Thr Lys Ser Ala Pro Asp Tyr
Leu Val Gly Pro Ala 275 280 285 Pro Pro Ala Asp Ile Lys Thr Asn Arg
Ile Tyr Arg Ile Leu Glu Leu 290 295 300 Asn Gly Tyr Glu Pro Ala Tyr
Ala Gly Ser Val Phe Leu Gly Trp Ala 305 310 315 320 Gln Lys Arg Phe
Gly Lys Arg Asn Thr Ile Trp Leu Phe Gly Pro Ala 325 330 335 Thr Thr
Gly Lys Thr Asn Ile Ala Glu Ala Ile Ala His Ala Val Pro 340 345 350
Phe Tyr Gly Cys Val Asn Trp Thr Asn Glu Asn Phe Pro Phe Asn Asp 355
360 365 Cys Val Asp Lys Met Val Ile Trp Trp Glu Glu Gly Lys Met Thr
Ala 370 375 380 Lys Val Val Glu Ser Ala Lys Ala Ile Leu Gly Gly Ser
Lys Val Arg 385 390 395 400 Val Asp Gln Lys Cys Lys Ser Ser Ala Gln
Ile Asp Pro Thr Pro Val 405 410 415 Ile Val Thr Ser Asn Thr Asn Met
Cys Ala Val Ile Asp Gly Asn Ser 420 425 430 Thr Thr Phe Glu His Gln
Gln Pro Leu Gln Asp Arg Met Phe Lys Phe 435 440 445 Glu Leu Thr Arg
Arg Leu Glu His Asp Phe Gly Lys Val Thr Lys Gln 450 455 460 Glu Val
Lys Glu Phe Phe Arg Trp Ala Gln Asp His Val Thr Glu Val 465 470 475
480 Ala His Glu Phe Tyr Val Arg Lys Gly Gly Ala Asn Lys Arg Pro Ala
485 490 495 Pro Asp Asp Ala Asp Lys Ser Glu Pro Lys Arg Ala Cys Pro
Ser Val 500 505 510 Ala Asp Pro Ser Thr Ser Asp Ala Glu Gly Ala Pro
Val Asp Phe Ala 515 520 525 Asp Arg Tyr Gln Asn Lys Cys Ser Arg His
Ala Gly Met Leu Gln Met 530 535 540 Leu Phe Pro Cys Lys Thr Cys Glu
Arg Met Asn Gln Asn Phe Asn Ile 545 550 555 560 Cys Phe Thr His Gly
Thr Arg Asp Cys Ser Glu Cys Phe Pro Gly Val 565 570 575 Ser Glu Ser
Gln Pro Val Val Arg Lys Arg Thr Tyr Arg Lys Leu Cys 580 585 590 Ala
Ile His His Leu Leu Gly Arg Ala Pro Glu Ile Ala Cys Ser Ala 595 600
605 Cys Asp Leu Val Asn Val Asp Leu Asp Asp Cys Val Ser Glu Gln 610
615 620 3 736 PRT AAV-1 3 Met Ala Ala Asp Gly Tyr Leu Pro Asp Trp
Leu Glu Asp Asn Leu Ser 1 5 10 15 Glu Gly Ile Arg Glu Trp Trp Asp
Leu Lys Pro Gly Ala Pro Lys Pro 20 25 30 Lys Ala Asn Gln Gln Lys
Gln Asp Asp Gly Arg Gly Leu Val Leu Pro 35 40 45 Gly Tyr Lys Tyr
Leu Gly Pro Phe Asn Gly Leu Asp Lys Gly Glu Pro 50 55 60 Val Asn
Ala Ala Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp 65 70 75 80
Gln Gln Leu Lys Ala Gly Asp Asn Pro Tyr Leu Arg Tyr Asn His Ala 85
90 95 Asp Ala Glu Phe Gln Glu Arg Leu Gln Glu Asp Thr Ser Phe Gly
Gly 100 105 110 Asn Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg Val
Leu Glu Pro 115 120 125 Leu Gly Leu Val Glu Glu Gly Ala Lys Thr Ala
Pro Gly Lys Lys Arg 130 135 140 Pro Val Glu Gln Ser Pro Gln Glu Pro
Asp Ser Ser Ser Gly Ile Gly 145 150 155 160 Lys Thr Gly Gln Gln Pro
Ala Lys Lys Arg Leu Asn Phe Gly Gln Thr 165 170 175 Gly Asp Ser Glu
Ser Val Pro Asp Pro Gln Pro Leu Gly Glu Pro Pro 180 185 190 Ala Thr
Pro Ala Ala Val Gly Pro Thr Thr Met Ala Ser Gly Gly Gly 195 200 205
Ala Pro Met Ala Asp Asn Asn Glu Gly Ala Asp Gly Val Gly Asn Ala 210
215 220 Ser Gly Asn Trp His Cys Asp Ser Thr Trp Leu Gly Asp Arg Val
Ile 225 230 235 240 Thr Thr Ser Thr Arg Thr Trp Ala Leu Pro Thr Tyr
Asn Asn His Leu 245 250 255 Tyr Lys Gln Ile Ser Ser Ala Ser Thr Gly
Ala Ser Asn Asp Asn His 260 265 270 Tyr Phe Gly Tyr Ser Thr Pro Trp
Gly Tyr Phe Asp Phe Asn Arg Phe 275 280 285 His Cys His Phe Ser Pro
Arg Asp Trp Gln Arg Leu Ile Asn Asn Asn 290 295 300 Trp Gly Phe Arg
Pro Lys Arg Leu Asn Phe Lys Leu Phe Asn Ile Gln 305 310 315 320 Val
Lys Glu Val Thr Thr Asn Asp Gly Val Thr Thr Ile Ala Asn Asn 325 330
335 Leu Thr Ser Thr Val Gln Val Phe Ser Asp Ser Glu Tyr Gln Leu Pro
340 345 350 Tyr Val Leu Gly Ser Ala His Gln Gly Cys Leu Pro Pro Phe
Pro Ala 355 360 365 Asp Val Phe Met Ile Pro Gln Tyr Gly Tyr Leu Thr
Leu Asn Asn Gly 370 375 380 Ser Gln Ala Val Gly Arg Ser Ser Phe Tyr
Cys Leu Glu Tyr Phe Pro 385 390 395 400 Ser Gln Met Leu Arg Thr Gly
Asn Asn Phe Thr Phe Ser Tyr Thr Phe 405 410 415 Glu Glu Val Pro Phe
His Ser Ser Tyr Ala His Ser Gln Ser Leu Asp 420 425 430 Arg Leu Met
Asn Pro Leu Ile Asp Gln Tyr Leu Tyr Tyr Leu Asn Arg 435 440 445 Thr
Gln Asn Gln Ser Gly Ser Ala Gln Asn Lys Asp Leu Leu Phe Ser 450 455
460 Arg Gly Ser Pro Ala Gly Met Ser Val Gln Pro Lys Asn Trp Leu Pro
465 470 475 480 Gly Pro Cys Tyr Arg Gln Gln Arg Val Ser Lys Thr Lys
Thr Asp Asn 485 490 495 Asn Asn Ser Asn Phe Thr Trp Thr Gly Ala Ser
Lys Tyr Asn Leu Asn 500 505 510 Gly Arg Glu Ser Ile Ile Asn Pro Gly
Thr Ala Met Ala Ser His Lys 515 520 525 Asp Asp Glu Asp Lys Phe Phe
Pro Met Ser Gly Val Met Ile Phe Gly 530 535 540 Lys Glu Ser Ala Gly
Ala Ser Asn Thr Ala Leu Asp Asn Val Met Ile 545 550 555 560 Thr Asp
Glu Glu Glu Ile Lys Ala Thr Asn Pro Val Ala Thr Glu Arg 565 570 575
Phe Gly Thr Val Ala Val Asn Phe Gln Ser Ser Ser Thr Asp Pro Ala 580
585 590 Thr Gly Asp Val His Ala Met Gly Ala Leu Pro Gly Met Val Trp
Gln 595 600 605 Asp Arg Asp Val Tyr Leu Gln Gly Pro Ile Trp Ala Lys
Ile Pro His 610 615 620 Thr Asp Gly His Phe His Pro Ser Pro Leu Met
Gly Gly Phe Gly Leu 625 630 635 640 Lys Asn Pro Pro Pro Gln Ile Leu
Ile Lys Asn Thr Pro Val Pro Ala 645 650 655 Asn Pro Pro Ala Glu Phe
Ser Ala Thr Lys Phe Ala Ser Phe Ile Thr 660 665 670 Gln Tyr Ser Thr
Gly Gln Val Ser Val Glu Ile Glu Trp Glu Leu Gln 675 680 685 Lys Glu
Asn Ser Lys Arg Trp Asn Pro Glu Val Gln Tyr Thr Ser Asn 690 695 700
Tyr Ala Lys Ser Ala Asn Val Asp Phe Thr Val Asp Asn Asn Gly Leu 705
710 715 720 Tyr Thr Glu Pro Arg Pro Ile Gly Thr Arg Tyr Leu Thr Arg
Pro Leu 725 730 735 4 1872 DNA AAV-1 CDS (1)..(1869) 4 atg ccg ggc
ttc tac gag atc gtg atc aag gtg ccg agc gac ctg gac 48 Met Pro Gly
Phe Tyr Glu Ile Val Ile Lys Val Pro Ser Asp Leu Asp 1 5 10 15 gag
cac ctg ccg ggc att tct gac tcg ttt gtg agc tgg gtg gcc gag 96 Glu
His Leu Pro Gly Ile Ser Asp Ser Phe Val Ser Trp Val Ala Glu 20 25
30 aag gaa tgg gag ctg ccc ccg gat tct gac atg gat ctg aat ctg att
144 Lys Glu Trp Glu Leu Pro Pro Asp Ser Asp Met Asp Leu Asn Leu Ile
35 40 45 gag cag gca ccc ctg acc gtg gcc gag aag ctg cag cgc gac
ttc ctg 192 Glu Gln Ala Pro Leu Thr Val Ala Glu Lys Leu Gln Arg Asp
Phe Leu 50 55 60 gtc caa tgg cgc cgc gtg agt aag gcc ccg gag gcc
ctc ttc ttt gtt 240 Val Gln Trp Arg Arg Val Ser Lys Ala Pro Glu Ala
Leu Phe Phe Val 65 70 75 80 cag ttc gag aag ggc gag tcc tac ttc cac
ctc cat att ctg gtg gag 288 Gln Phe Glu Lys Gly Glu Ser Tyr Phe His
Leu His Ile Leu Val Glu 85 90 95 acc acg ggg gtc aaa tcc atg gtg
ctg ggc cgc ttc ctg agt cag att 336 Thr Thr Gly Val Lys Ser Met Val
Leu Gly Arg Phe Leu Ser Gln Ile 100 105 110 agg gac aag ctg gtg cag
acc atc tac cgc ggg atc gag ccg acc ctg 384 Arg Asp Lys Leu Val Gln
Thr Ile Tyr Arg Gly Ile Glu Pro Thr Leu 115 120 125 ccc aac tgg ttc
gcg gtg acc aag acg cgt aat ggc gcc gga ggg ggg 432 Pro Asn Trp Phe
Ala Val Thr Lys Thr Arg Asn Gly Ala Gly Gly Gly 130 135 140 aac aag
gtg gtg gac gag tgc tac atc ccc aac tac ctc ctg ccc aag 480 Asn Lys
Val Val Asp Glu Cys Tyr Ile Pro Asn Tyr Leu Leu Pro Lys 145 150 155
160 act cag ccc gag ctg cag tgg gcg tgg act aac atg gag gag tat ata
528 Thr Gln Pro Glu Leu Gln Trp Ala Trp Thr Asn Met Glu Glu Tyr Ile
165 170 175 agc gcc tgt ttg aac ctg gcc gag cgc aaa cgg ctc gtg gcg
cag cac 576 Ser Ala Cys Leu Asn Leu Ala Glu Arg Lys Arg Leu Val Ala
Gln His 180 185 190 ctg acc cac gtc agc cag acc cag gag cag aac aag
gag aat ctg aac 624 Leu Thr His Val Ser Gln Thr Gln Glu Gln Asn Lys
Glu Asn Leu Asn 195 200 205 ccc aat tct gac gcg cct gtc atc cgg tca
aaa acc tcc gcg cgc tac 672 Pro Asn Ser Asp Ala Pro Val Ile Arg Ser
Lys Thr Ser Ala Arg Tyr 210 215 220 atg gag ctg gtc ggg tgg ctg gtg
gac cgg ggc atc acc tcc gag aag 720 Met Glu Leu Val Gly Trp Leu Val
Asp Arg Gly Ile Thr Ser Glu Lys 225 230 235 240 cag tgg atc cag gag
gac cag gcc tcg tac atc tcc ttc aac gcc gct 768 Gln Trp Ile Gln Glu
Asp Gln Ala Ser Tyr Ile Ser Phe Asn Ala Ala 245 250 255 tcc aac tcg
cgg tcc cag atc aag gcc gct ctg gac aat gcc ggc aag 816 Ser Asn Ser
Arg Ser Gln Ile Lys Ala Ala Leu Asp Asn Ala Gly Lys 260 265 270 atc
atg gcg ctg acc aaa tcc gcg ccc gac tac ctg gta ggc ccc gct 864 Ile
Met Ala Leu Thr Lys Ser Ala Pro Asp Tyr Leu Val Gly Pro Ala 275 280
285 ccg ccc gcg gac att aaa acc aac cgc atc tac cgc atc ctg gag ctg
912 Pro Pro Ala Asp Ile Lys Thr Asn Arg Ile Tyr Arg Ile Leu Glu Leu
290 295 300 aac ggc tac gaa cct gcc tac gcc ggc tcc gtc ttt ctc ggc
tgg gcc 960 Asn Gly Tyr Glu Pro Ala Tyr Ala Gly Ser Val Phe Leu Gly
Trp Ala 305 310 315 320 cag aaa agg ttc ggg aag cgc aac acc atc tgg
ctg ttt ggg ccg gcc 1008 Gln Lys Arg Phe Gly Lys Arg Asn Thr Ile
Trp Leu Phe Gly Pro Ala 325 330 335 acc acg ggc aag acc aac atc gcg
gaa gcc atc gcc cac gcc gtg ccc 1056 Thr Thr Gly Lys Thr Asn Ile
Ala Glu Ala Ile Ala His Ala Val Pro 340 345 350 ttc tac ggc tgc gtc
aac tgg acc aat gag aac ttt ccc ttc aat gat 1104 Phe Tyr Gly Cys
Val Asn Trp Thr Asn Glu Asn Phe Pro Phe Asn Asp 355 360 365 tgc gtc
gac aag atg gtg atc tgg tgg gag gag ggc aag atg acg gcc 1152 Cys
Val Asp Lys Met Val Ile Trp Trp Glu Glu Gly Lys Met Thr Ala 370 375
380 aag gtc gtg gag tcc gcc aag gcc att ctc ggc ggc agc aag gtg cgc
1200 Lys Val Val Glu Ser Ala Lys Ala Ile Leu Gly Gly Ser Lys Val
Arg 385 390 395 400 gtg gac caa aag tgc aag tcg tcc gcc cag atc gac
ccc acc ccc gtg 1248 Val Asp Gln Lys Cys Lys Ser Ser Ala Gln Ile
Asp Pro Thr Pro Val 405 410 415 atc gtc acc tcc aac acc aac atg tgc
gcc gtg att gac ggg aac agc 1296 Ile Val Thr Ser Asn Thr Asn Met
Cys Ala Val Ile Asp Gly Asn Ser 420 425 430 acc acc ttc gag cac cag
cag ccg ttg cag gac cgg atg ttc aaa ttt 1344 Thr Thr Phe Glu His
Gln Gln Pro Leu Gln Asp Arg Met Phe Lys Phe 435 440 445 gaa ctc acc
cgc cgt ctg gag cat gac ttt ggc aag gtg aca aag cag 1392 Glu Leu
Thr Arg Arg Leu Glu His Asp Phe Gly Lys Val Thr Lys Gln 450 455 460
gaa gtc aaa gag ttc ttc cgc tgg gcg cag gat cac gtg acc gag gtg
1440 Glu Val Lys Glu Phe Phe Arg Trp Ala Gln Asp His Val Thr Glu
Val 465 470 475 480 gcg cat gag ttc tac gtc aga aag ggt gga gcc aac
aaa aga ccc gcc 1488 Ala His Glu Phe Tyr Val Arg Lys Gly Gly Ala
Asn Lys Arg Pro Ala 485 490 495 ccc gat gac gcg gat aaa agc gag ccc
aag cgg gcc tgc ccc tca gtc 1536 Pro Asp Asp Ala Asp Lys Ser Glu
Pro Lys Arg Ala Cys Pro Ser Val 500 505 510 gcg gat cca tcg acg tca
gac gcg gaa gga gct ccg gtg gac ttt gcc 1584 Ala Asp Pro Ser Thr
Ser Asp Ala Glu Gly Ala Pro Val Asp Phe Ala 515 520 525 gac agg tac
caa aac aaa tgt tct cgt cac gcg ggc atg ctt cag atg 1632 Asp Arg
Tyr Gln Asn Lys Cys Ser Arg His Ala Gly Met Leu Gln Met 530 535 540
ctg ttt ccc tgc aag aca tgc gag aga atg aat cag aat ttc aac att
1680 Leu Phe Pro Cys Lys Thr Cys Glu Arg Met Asn Gln Asn Phe Asn
Ile 545 550 555 560 tgc ttc acg cac ggg acg aga gac tgt tca gag tgc
ttc ccc ggc gtg 1728 Cys Phe Thr His Gly Thr Arg Asp Cys Ser Glu
Cys Phe Pro Gly Val 565 570 575 tca gaa tct caa ccg gtc gtc aga aag
agg acg tat cgg aaa ctc tgt 1776 Ser Glu Ser Gln Pro Val Val Arg
Lys Arg Thr Tyr Arg Lys Leu Cys 580 585 590 gcc att cat cat ctg ctg
ggg cgg gct ccc gag att gct tgc tcg gcc 1824 Ala Ile His His Leu
Leu Gly Arg Ala Pro Glu Ile Ala Cys Ser Ala 595 600 605 tgc gat ctg
gtc aac gtg gac ctg gat gac tgt gtt tct gag caa taa 1872 Cys Asp
Leu Val Asn Val Asp Leu Asp Asp Cys Val Ser Glu Gln 610 615 620 5
623 PRT AAV-1 5 Met Pro Gly Phe Tyr Glu Ile Val Ile Lys Val Pro Ser
Asp Leu Asp 1 5 10 15 Glu His Leu Pro Gly Ile Ser Asp Ser Phe Val
Ser Trp Val Ala Glu 20 25 30 Lys Glu Trp Glu Leu Pro Pro Asp Ser
Asp Met Asp Leu Asn Leu Ile 35 40 45 Glu Gln Ala Pro Leu Thr Val
Ala Glu Lys Leu Gln Arg Asp Phe Leu 50 55 60 Val Gln Trp Arg Arg
Val Ser Lys Ala Pro Glu Ala Leu Phe Phe Val 65 70 75 80 Gln Phe Glu
Lys Gly Glu Ser Tyr Phe His Leu His Ile Leu Val Glu 85 90 95 Thr
Thr Gly Val Lys Ser Met Val Leu Gly Arg Phe Leu Ser Gln Ile 100 105
110 Arg Asp Lys Leu Val Gln Thr Ile Tyr Arg Gly Ile Glu Pro Thr Leu
115 120 125 Pro Asn Trp Phe Ala Val Thr Lys Thr Arg Asn Gly Ala Gly
Gly Gly 130 135 140 Asn Lys Val Val Asp Glu Cys Tyr Ile Pro Asn Tyr
Leu Leu Pro Lys 145 150 155 160 Thr Gln Pro Glu Leu Gln Trp Ala Trp
Thr Asn Met Glu Glu Tyr Ile 165 170 175 Ser Ala Cys Leu Asn Leu Ala
Glu Arg Lys Arg Leu Val Ala Gln His 180 185 190 Leu Thr His Val Ser
Gln Thr Gln Glu Gln Asn
Lys Glu Asn Leu Asn 195 200 205 Pro Asn Ser Asp Ala Pro Val Ile Arg
Ser Lys Thr Ser Ala Arg Tyr 210 215 220 Met Glu Leu Val Gly Trp Leu
Val Asp Arg Gly Ile Thr Ser Glu Lys 225 230 235 240 Gln Trp Ile Gln
Glu Asp Gln Ala Ser Tyr Ile Ser Phe Asn Ala Ala 245 250 255 Ser Asn
Ser Arg Ser Gln Ile Lys Ala Ala Leu Asp Asn Ala Gly Lys 260 265 270
Ile Met Ala Leu Thr Lys Ser Ala Pro Asp Tyr Leu Val Gly Pro Ala 275
280 285 Pro Pro Ala Asp Ile Lys Thr Asn Arg Ile Tyr Arg Ile Leu Glu
Leu 290 295 300 Asn Gly Tyr Glu Pro Ala Tyr Ala Gly Ser Val Phe Leu
Gly Trp Ala 305 310 315 320 Gln Lys Arg Phe Gly Lys Arg Asn Thr Ile
Trp Leu Phe Gly Pro Ala 325 330 335 Thr Thr Gly Lys Thr Asn Ile Ala
Glu Ala Ile Ala His Ala Val Pro 340 345 350 Phe Tyr Gly Cys Val Asn
Trp Thr Asn Glu Asn Phe Pro Phe Asn Asp 355 360 365 Cys Val Asp Lys
Met Val Ile Trp Trp Glu Glu Gly Lys Met Thr Ala 370 375 380 Lys Val
Val Glu Ser Ala Lys Ala Ile Leu Gly Gly Ser Lys Val Arg 385 390 395
400 Val Asp Gln Lys Cys Lys Ser Ser Ala Gln Ile Asp Pro Thr Pro Val
405 410 415 Ile Val Thr Ser Asn Thr Asn Met Cys Ala Val Ile Asp Gly
Asn Ser 420 425 430 Thr Thr Phe Glu His Gln Gln Pro Leu Gln Asp Arg
Met Phe Lys Phe 435 440 445 Glu Leu Thr Arg Arg Leu Glu His Asp Phe
Gly Lys Val Thr Lys Gln 450 455 460 Glu Val Lys Glu Phe Phe Arg Trp
Ala Gln Asp His Val Thr Glu Val 465 470 475 480 Ala His Glu Phe Tyr
Val Arg Lys Gly Gly Ala Asn Lys Arg Pro Ala 485 490 495 Pro Asp Asp
Ala Asp Lys Ser Glu Pro Lys Arg Ala Cys Pro Ser Val 500 505 510 Ala
Asp Pro Ser Thr Ser Asp Ala Glu Gly Ala Pro Val Asp Phe Ala 515 520
525 Asp Arg Tyr Gln Asn Lys Cys Ser Arg His Ala Gly Met Leu Gln Met
530 535 540 Leu Phe Pro Cys Lys Thr Cys Glu Arg Met Asn Gln Asn Phe
Asn Ile 545 550 555 560 Cys Phe Thr His Gly Thr Arg Asp Cys Ser Glu
Cys Phe Pro Gly Val 565 570 575 Ser Glu Ser Gln Pro Val Val Arg Lys
Arg Thr Tyr Arg Lys Leu Cys 580 585 590 Ala Ile His His Leu Leu Gly
Arg Ala Pro Glu Ile Ala Cys Ser Ala 595 600 605 Cys Asp Leu Val Asn
Val Asp Leu Asp Asp Cys Val Ser Glu Gln 610 615 620 6 1641 DNA
AAV-1 CDS (1)..(1638) 6 atg ccg ggc ttc tac gag atc gtg atc aag gtg
ccg agc gac ctg gac 48 Met Pro Gly Phe Tyr Glu Ile Val Ile Lys Val
Pro Ser Asp Leu Asp 1 5 10 15 gag cac ctg ccg ggc att tct gac tcg
ttt gtg agc tgg gtg gcc gag 96 Glu His Leu Pro Gly Ile Ser Asp Ser
Phe Val Ser Trp Val Ala Glu 20 25 30 aag gaa tgg gag ctg ccc ccg
gat tct gac atg gat ctg aat ctg att 144 Lys Glu Trp Glu Leu Pro Pro
Asp Ser Asp Met Asp Leu Asn Leu Ile 35 40 45 gag cag gca ccc ctg
acc gtg gcc gag aag ctg cag cgc gac ttc ctg 192 Glu Gln Ala Pro Leu
Thr Val Ala Glu Lys Leu Gln Arg Asp Phe Leu 50 55 60 gtc caa tgg
cgc cgc gtg agt aag gcc ccg gag gcc ctc ttc ttt gtt 240 Val Gln Trp
Arg Arg Val Ser Lys Ala Pro Glu Ala Leu Phe Phe Val 65 70 75 80 cag
ttc gag aag ggc gag tcc tac ttc cac ctc cat att ctg gtg gag 288 Gln
Phe Glu Lys Gly Glu Ser Tyr Phe His Leu His Ile Leu Val Glu 85 90
95 acc acg ggg gtc aaa tcc atg gtg ctg ggc cgc ttc ctg agt cag att
336 Thr Thr Gly Val Lys Ser Met Val Leu Gly Arg Phe Leu Ser Gln Ile
100 105 110 agg gac aag ctg gtg cag acc atc tac cgc ggg atc gag ccg
acc ctg 384 Arg Asp Lys Leu Val Gln Thr Ile Tyr Arg Gly Ile Glu Pro
Thr Leu 115 120 125 ccc aac tgg ttc gcg gtg acc aag acg cgt aat ggc
gcc gga ggg ggg 432 Pro Asn Trp Phe Ala Val Thr Lys Thr Arg Asn Gly
Ala Gly Gly Gly 130 135 140 aac aag gtg gtg gac gag tgc tac atc ccc
aac tac ctc ctg ccc aag 480 Asn Lys Val Val Asp Glu Cys Tyr Ile Pro
Asn Tyr Leu Leu Pro Lys 145 150 155 160 act cag ccc gag ctg cag tgg
gcg tgg act aac atg gag gag tat ata 528 Thr Gln Pro Glu Leu Gln Trp
Ala Trp Thr Asn Met Glu Glu Tyr Ile 165 170 175 agc gcc tgt ttg aac
ctg gcc gag cgc aaa cgg ctc gtg gcg cag cac 576 Ser Ala Cys Leu Asn
Leu Ala Glu Arg Lys Arg Leu Val Ala Gln His 180 185 190 ctg acc cac
gtc agc cag acc cag gag cag aac aag gag aat ctg aac 624 Leu Thr His
Val Ser Gln Thr Gln Glu Gln Asn Lys Glu Asn Leu Asn 195 200 205 ccc
aat tct gac gcg cct gtc atc cgg tca aaa acc tcc gcg cgc tac 672 Pro
Asn Ser Asp Ala Pro Val Ile Arg Ser Lys Thr Ser Ala Arg Tyr 210 215
220 atg gag ctg gtc ggg tgg ctg gtg gac cgg ggc atc acc tcc gag aag
720 Met Glu Leu Val Gly Trp Leu Val Asp Arg Gly Ile Thr Ser Glu Lys
225 230 235 240 cag tgg atc cag gag gac cag gcc tcg tac atc tcc ttc
aac gcc gct 768 Gln Trp Ile Gln Glu Asp Gln Ala Ser Tyr Ile Ser Phe
Asn Ala Ala 245 250 255 tcc aac tcg cgg tcc cag atc aag gcc gct ctg
gac aat gcc ggc aag 816 Ser Asn Ser Arg Ser Gln Ile Lys Ala Ala Leu
Asp Asn Ala Gly Lys 260 265 270 atc atg gcg ctg acc aaa tcc gcg ccc
gac tac ctg gta ggc ccc gct 864 Ile Met Ala Leu Thr Lys Ser Ala Pro
Asp Tyr Leu Val Gly Pro Ala 275 280 285 ccg ccc gcg gac att aaa acc
aac cgc atc tac cgc atc ctg gag ctg 912 Pro Pro Ala Asp Ile Lys Thr
Asn Arg Ile Tyr Arg Ile Leu Glu Leu 290 295 300 aac ggc tac gaa cct
gcc tac gcc ggc tcc gtc ttt ctc ggc tgg gcc 960 Asn Gly Tyr Glu Pro
Ala Tyr Ala Gly Ser Val Phe Leu Gly Trp Ala 305 310 315 320 cag aaa
agg ttc ggg aag cgc aac acc atc tgg ctg ttt ggg ccg gcc 1008 Gln
Lys Arg Phe Gly Lys Arg Asn Thr Ile Trp Leu Phe Gly Pro Ala 325 330
335 acc acg ggc aag acc aac atc gcg gaa gcc atc gcc cac gcc gtg ccc
1056 Thr Thr Gly Lys Thr Asn Ile Ala Glu Ala Ile Ala His Ala Val
Pro 340 345 350 ttc tac ggc tgc gtc aac tgg acc aat gag aac ttt ccc
ttc aat gat 1104 Phe Tyr Gly Cys Val Asn Trp Thr Asn Glu Asn Phe
Pro Phe Asn Asp 355 360 365 tgc gtc gac aag atg gtg atc tgg tgg gag
gag ggc aag atg acg gcc 1152 Cys Val Asp Lys Met Val Ile Trp Trp
Glu Glu Gly Lys Met Thr Ala 370 375 380 aag gtc gtg gag tcc gcc aag
gcc att ctc ggc ggc agc aag gtg cgc 1200 Lys Val Val Glu Ser Ala
Lys Ala Ile Leu Gly Gly Ser Lys Val Arg 385 390 395 400 gtg gac caa
aag tgc aag tcg tcc gcc cag atc gac ccc acc ccc gtg 1248 Val Asp
Gln Lys Cys Lys Ser Ser Ala Gln Ile Asp Pro Thr Pro Val 405 410 415
atc gtc acc tcc aac acc aac atg tgc gcc gtg att gac ggg aac agc
1296 Ile Val Thr Ser Asn Thr Asn Met Cys Ala Val Ile Asp Gly Asn
Ser 420 425 430 acc acc ttc gag cac cag cag ccg ttg cag gac cgg atg
ttc aaa ttt 1344 Thr Thr Phe Glu His Gln Gln Pro Leu Gln Asp Arg
Met Phe Lys Phe 435 440 445 gaa ctc acc cgc cgt ctg gag cat gac ttt
ggc aag gtg aca aag cag 1392 Glu Leu Thr Arg Arg Leu Glu His Asp
Phe Gly Lys Val Thr Lys Gln 450 455 460 gaa gtc aaa gag ttc ttc cgc
tgg gcg cag gat cac gtg acc gag gtg 1440 Glu Val Lys Glu Phe Phe
Arg Trp Ala Gln Asp His Val Thr Glu Val 465 470 475 480 gcg cat gag
ttc tac gtc aga aag ggt gga gcc aac aaa aga ccc gcc 1488 Ala His
Glu Phe Tyr Val Arg Lys Gly Gly Ala Asn Lys Arg Pro Ala 485 490 495
ccc gat gac gcg gat aaa agc gag ccc aag cgg gcc tgc ccc tca gtc
1536 Pro Asp Asp Ala Asp Lys Ser Glu Pro Lys Arg Ala Cys Pro Ser
Val 500 505 510 gcg gat cca tcg acg tca gac gcg gaa gga gct ccg gtg
gac ttt gcc 1584 Ala Asp Pro Ser Thr Ser Asp Ala Glu Gly Ala Pro
Val Asp Phe Ala 515 520 525 gac agg tat ggc tgc cga tgg tta tct tcc
aga ttg gct cga gga caa 1632 Asp Arg Tyr Gly Cys Arg Trp Leu Ser
Ser Arg Leu Ala Arg Gly Gln 530 535 540 cct ctc tga 1641 Pro Leu
545 7 546 PRT AAV-1 7 Met Pro Gly Phe Tyr Glu Ile Val Ile Lys Val
Pro Ser Asp Leu Asp 1 5 10 15 Glu His Leu Pro Gly Ile Ser Asp Ser
Phe Val Ser Trp Val Ala Glu 20 25 30 Lys Glu Trp Glu Leu Pro Pro
Asp Ser Asp Met Asp Leu Asn Leu Ile 35 40 45 Glu Gln Ala Pro Leu
Thr Val Ala Glu Lys Leu Gln Arg Asp Phe Leu 50 55 60 Val Gln Trp
Arg Arg Val Ser Lys Ala Pro Glu Ala Leu Phe Phe Val 65 70 75 80 Gln
Phe Glu Lys Gly Glu Ser Tyr Phe His Leu His Ile Leu Val Glu 85 90
95 Thr Thr Gly Val Lys Ser Met Val Leu Gly Arg Phe Leu Ser Gln Ile
100 105 110 Arg Asp Lys Leu Val Gln Thr Ile Tyr Arg Gly Ile Glu Pro
Thr Leu 115 120 125 Pro Asn Trp Phe Ala Val Thr Lys Thr Arg Asn Gly
Ala Gly Gly Gly 130 135 140 Asn Lys Val Val Asp Glu Cys Tyr Ile Pro
Asn Tyr Leu Leu Pro Lys 145 150 155 160 Thr Gln Pro Glu Leu Gln Trp
Ala Trp Thr Asn Met Glu Glu Tyr Ile 165 170 175 Ser Ala Cys Leu Asn
Leu Ala Glu Arg Lys Arg Leu Val Ala Gln His 180 185 190 Leu Thr His
Val Ser Gln Thr Gln Glu Gln Asn Lys Glu Asn Leu Asn 195 200 205 Pro
Asn Ser Asp Ala Pro Val Ile Arg Ser Lys Thr Ser Ala Arg Tyr 210 215
220 Met Glu Leu Val Gly Trp Leu Val Asp Arg Gly Ile Thr Ser Glu Lys
225 230 235 240 Gln Trp Ile Gln Glu Asp Gln Ala Ser Tyr Ile Ser Phe
Asn Ala Ala 245 250 255 Ser Asn Ser Arg Ser Gln Ile Lys Ala Ala Leu
Asp Asn Ala Gly Lys 260 265 270 Ile Met Ala Leu Thr Lys Ser Ala Pro
Asp Tyr Leu Val Gly Pro Ala 275 280 285 Pro Pro Ala Asp Ile Lys Thr
Asn Arg Ile Tyr Arg Ile Leu Glu Leu 290 295 300 Asn Gly Tyr Glu Pro
Ala Tyr Ala Gly Ser Val Phe Leu Gly Trp Ala 305 310 315 320 Gln Lys
Arg Phe Gly Lys Arg Asn Thr Ile Trp Leu Phe Gly Pro Ala 325 330 335
Thr Thr Gly Lys Thr Asn Ile Ala Glu Ala Ile Ala His Ala Val Pro 340
345 350 Phe Tyr Gly Cys Val Asn Trp Thr Asn Glu Asn Phe Pro Phe Asn
Asp 355 360 365 Cys Val Asp Lys Met Val Ile Trp Trp Glu Glu Gly Lys
Met Thr Ala 370 375 380 Lys Val Val Glu Ser Ala Lys Ala Ile Leu Gly
Gly Ser Lys Val Arg 385 390 395 400 Val Asp Gln Lys Cys Lys Ser Ser
Ala Gln Ile Asp Pro Thr Pro Val 405 410 415 Ile Val Thr Ser Asn Thr
Asn Met Cys Ala Val Ile Asp Gly Asn Ser 420 425 430 Thr Thr Phe Glu
His Gln Gln Pro Leu Gln Asp Arg Met Phe Lys Phe 435 440 445 Glu Leu
Thr Arg Arg Leu Glu His Asp Phe Gly Lys Val Thr Lys Gln 450 455 460
Glu Val Lys Glu Phe Phe Arg Trp Ala Gln Asp His Val Thr Glu Val 465
470 475 480 Ala His Glu Phe Tyr Val Arg Lys Gly Gly Ala Asn Lys Arg
Pro Ala 485 490 495 Pro Asp Asp Ala Asp Lys Ser Glu Pro Lys Arg Ala
Cys Pro Ser Val 500 505 510 Ala Asp Pro Ser Thr Ser Asp Ala Glu Gly
Ala Pro Val Asp Phe Ala 515 520 525 Asp Arg Tyr Gly Cys Arg Trp Leu
Ser Ser Arg Leu Ala Arg Gly Gln 530 535 540 Pro Leu 545 8 1200 DNA
AAV-1 CDS (1)..(1197) 8 atg gag ctg gtc ggg tgg ctg gtg gac cgg ggc
atc acc tcc gag aag 48 Met Glu Leu Val Gly Trp Leu Val Asp Arg Gly
Ile Thr Ser Glu Lys 1 5 10 15 cag tgg atc cag gag gac cag gcc tcg
tac atc tcc ttc aac gcc gct 96 Gln Trp Ile Gln Glu Asp Gln Ala Ser
Tyr Ile Ser Phe Asn Ala Ala 20 25 30 tcc aac tcg cgg tcc cag atc
aag gcc gct ctg gac aat gcc ggc aag 144 Ser Asn Ser Arg Ser Gln Ile
Lys Ala Ala Leu Asp Asn Ala Gly Lys 35 40 45 atc atg gcg ctg acc
aaa tcc gcg ccc gac tac ctg gta ggc ccc gct 192 Ile Met Ala Leu Thr
Lys Ser Ala Pro Asp Tyr Leu Val Gly Pro Ala 50 55 60 ccg ccc gcg
gac att aaa acc aac cgc atc tac cgc atc ctg gag ctg 240 Pro Pro Ala
Asp Ile Lys Thr Asn Arg Ile Tyr Arg Ile Leu Glu Leu 65 70 75 80 aac
ggc tac gaa cct gcc tac gcc ggc tcc gtc ttt ctc ggc tgg gcc 288 Asn
Gly Tyr Glu Pro Ala Tyr Ala Gly Ser Val Phe Leu Gly Trp Ala 85 90
95 cag aaa agg ttc ggg aag cgc aac acc atc tgg ctg ttt ggg ccg gcc
336 Gln Lys Arg Phe Gly Lys Arg Asn Thr Ile Trp Leu Phe Gly Pro Ala
100 105 110 acc acg ggc aag acc aac atc gcg gaa gcc atc gcc cac gcc
gtg ccc 384 Thr Thr Gly Lys Thr Asn Ile Ala Glu Ala Ile Ala His Ala
Val Pro 115 120 125 ttc tac ggc tgc gtc aac tgg acc aat gag aac ttt
ccc ttc aat gat 432 Phe Tyr Gly Cys Val Asn Trp Thr Asn Glu Asn Phe
Pro Phe Asn Asp 130 135 140 tgc gtc gac aag atg gtg atc tgg tgg gag
gag ggc aag atg acg gcc 480 Cys Val Asp Lys Met Val Ile Trp Trp Glu
Glu Gly Lys Met Thr Ala 145 150 155 160 aag gtc gtg gag tcc gcc aag
gcc att ctc ggc ggc agc aag gtg cgc 528 Lys Val Val Glu Ser Ala Lys
Ala Ile Leu Gly Gly Ser Lys Val Arg 165 170 175 gtg gac caa aag tgc
aag tcg tcc gcc cag atc gac ccc acc ccc gtg 576 Val Asp Gln Lys Cys
Lys Ser Ser Ala Gln Ile Asp Pro Thr Pro Val 180 185 190 atc gtc acc
tcc aac acc aac atg tgc gcc gtg att gac ggg aac agc 624 Ile Val Thr
Ser Asn Thr Asn Met Cys Ala Val Ile Asp Gly Asn Ser 195 200 205 acc
acc ttc gag cac cag cag ccg ttg cag gac cgg atg ttc aaa ttt 672 Thr
Thr Phe Glu His Gln Gln Pro Leu Gln Asp Arg Met Phe Lys Phe 210 215
220 gaa ctc acc cgc cgt ctg gag cat gac ttt ggc aag gtg aca aag cag
720 Glu Leu Thr Arg Arg Leu Glu His Asp Phe Gly Lys Val Thr Lys Gln
225 230 235 240 gaa gtc aaa gag ttc ttc cgc tgg gcg cag gat cac gtg
acc gag gtg 768 Glu Val Lys Glu Phe Phe Arg Trp Ala Gln Asp His Val
Thr Glu Val 245 250 255 gcg cat gag ttc tac gtc aga aag ggt gga gcc
aac aaa aga ccc gcc 816 Ala His Glu Phe Tyr Val Arg Lys Gly Gly Ala
Asn Lys Arg Pro Ala 260 265 270 ccc gat gac gcg gat aaa agc gag ccc
aag cgg gcc tgc ccc tca gtc 864 Pro Asp Asp Ala Asp Lys Ser Glu Pro
Lys Arg Ala Cys Pro Ser Val 275 280 285 gcg gat cca tcg acg tca gac
gcg gaa gga gct ccg gtg gac ttt gcc 912 Ala Asp Pro Ser Thr Ser Asp
Ala Glu Gly Ala Pro Val Asp Phe Ala 290 295 300 gac agg tac caa aac
aaa tgt tct cgt cac gcg ggc atg ctt cag atg 960 Asp Arg Tyr Gln Asn
Lys Cys Ser Arg His Ala Gly Met Leu Gln Met 305 310 315 320 ctg ttt
ccc tgc aag aca tgc gag aga atg aat cag aat ttc aac att 1008 Leu
Phe Pro Cys Lys Thr Cys Glu Arg Met Asn Gln Asn Phe Asn Ile 325 330
335 tgc ttc acg cac ggg acg aga gac tgt tca gag tgc ttc ccc ggc gtg
1056 Cys Phe Thr His Gly Thr Arg Asp Cys Ser Glu Cys Phe Pro Gly
Val 340 345 350 tca gaa tct caa ccg gtc gtc aga aag agg acg tat cgg
aaa ctc tgt 1104 Ser Glu Ser Gln Pro Val Val Arg Lys Arg Thr Tyr
Arg Lys Leu Cys
355 360 365 gcc att cat cat ctg ctg ggg cgg gct ccc gag att gct tgc
tcg gcc 1152 Ala Ile His His Leu Leu Gly Arg Ala Pro Glu Ile Ala
Cys Ser Ala 370 375 380 tgc gat ctg gtc aac gtg gac ctg gat gac tgt
gtt tct gag caa taa 1200 Cys Asp Leu Val Asn Val Asp Leu Asp Asp
Cys Val Ser Glu Gln 385 390 395 9 399 PRT AAV-1 9 Met Glu Leu Val
Gly Trp Leu Val Asp Arg Gly Ile Thr Ser Glu Lys 1 5 10 15 Gln Trp
Ile Gln Glu Asp Gln Ala Ser Tyr Ile Ser Phe Asn Ala Ala 20 25 30
Ser Asn Ser Arg Ser Gln Ile Lys Ala Ala Leu Asp Asn Ala Gly Lys 35
40 45 Ile Met Ala Leu Thr Lys Ser Ala Pro Asp Tyr Leu Val Gly Pro
Ala 50 55 60 Pro Pro Ala Asp Ile Lys Thr Asn Arg Ile Tyr Arg Ile
Leu Glu Leu 65 70 75 80 Asn Gly Tyr Glu Pro Ala Tyr Ala Gly Ser Val
Phe Leu Gly Trp Ala 85 90 95 Gln Lys Arg Phe Gly Lys Arg Asn Thr
Ile Trp Leu Phe Gly Pro Ala 100 105 110 Thr Thr Gly Lys Thr Asn Ile
Ala Glu Ala Ile Ala His Ala Val Pro 115 120 125 Phe Tyr Gly Cys Val
Asn Trp Thr Asn Glu Asn Phe Pro Phe Asn Asp 130 135 140 Cys Val Asp
Lys Met Val Ile Trp Trp Glu Glu Gly Lys Met Thr Ala 145 150 155 160
Lys Val Val Glu Ser Ala Lys Ala Ile Leu Gly Gly Ser Lys Val Arg 165
170 175 Val Asp Gln Lys Cys Lys Ser Ser Ala Gln Ile Asp Pro Thr Pro
Val 180 185 190 Ile Val Thr Ser Asn Thr Asn Met Cys Ala Val Ile Asp
Gly Asn Ser 195 200 205 Thr Thr Phe Glu His Gln Gln Pro Leu Gln Asp
Arg Met Phe Lys Phe 210 215 220 Glu Leu Thr Arg Arg Leu Glu His Asp
Phe Gly Lys Val Thr Lys Gln 225 230 235 240 Glu Val Lys Glu Phe Phe
Arg Trp Ala Gln Asp His Val Thr Glu Val 245 250 255 Ala His Glu Phe
Tyr Val Arg Lys Gly Gly Ala Asn Lys Arg Pro Ala 260 265 270 Pro Asp
Asp Ala Asp Lys Ser Glu Pro Lys Arg Ala Cys Pro Ser Val 275 280 285
Ala Asp Pro Ser Thr Ser Asp Ala Glu Gly Ala Pro Val Asp Phe Ala 290
295 300 Asp Arg Tyr Gln Asn Lys Cys Ser Arg His Ala Gly Met Leu Gln
Met 305 310 315 320 Leu Phe Pro Cys Lys Thr Cys Glu Arg Met Asn Gln
Asn Phe Asn Ile 325 330 335 Cys Phe Thr His Gly Thr Arg Asp Cys Ser
Glu Cys Phe Pro Gly Val 340 345 350 Ser Glu Ser Gln Pro Val Val Arg
Lys Arg Thr Tyr Arg Lys Leu Cys 355 360 365 Ala Ile His His Leu Leu
Gly Arg Ala Pro Glu Ile Ala Cys Ser Ala 370 375 380 Cys Asp Leu Val
Asn Val Asp Leu Asp Asp Cys Val Ser Glu Gln 385 390 395 10 969 DNA
AAV-1 CDS (1)..(966) misc_feature (943)..(944) minor splice site 10
atg gag ctg gtc ggg tgg ctg gtg gac cgg ggc atc acc tcc gag aag 48
Met Glu Leu Val Gly Trp Leu Val Asp Arg Gly Ile Thr Ser Glu Lys 1 5
10 15 cag tgg atc cag gag gac cag gcc tcg tac atc tcc ttc aac gcc
gct 96 Gln Trp Ile Gln Glu Asp Gln Ala Ser Tyr Ile Ser Phe Asn Ala
Ala 20 25 30 tcc aac tcg cgg tcc cag atc aag gcc gct ctg gac aat
gcc ggc aag 144 Ser Asn Ser Arg Ser Gln Ile Lys Ala Ala Leu Asp Asn
Ala Gly Lys 35 40 45 atc atg gcg ctg acc aaa tcc gcg ccc gac tac
ctg gta ggc ccc gct 192 Ile Met Ala Leu Thr Lys Ser Ala Pro Asp Tyr
Leu Val Gly Pro Ala 50 55 60 ccg ccc gcg gac att aaa acc aac cgc
atc tac cgc atc ctg gag ctg 240 Pro Pro Ala Asp Ile Lys Thr Asn Arg
Ile Tyr Arg Ile Leu Glu Leu 65 70 75 80 aac ggc tac gaa cct gcc tac
gcc ggc tcc gtc ttt ctc ggc tgg gcc 288 Asn Gly Tyr Glu Pro Ala Tyr
Ala Gly Ser Val Phe Leu Gly Trp Ala 85 90 95 cag aaa agg ttc ggg
aag cgc aac acc atc tgg ctg ttt ggg ccg gcc 336 Gln Lys Arg Phe Gly
Lys Arg Asn Thr Ile Trp Leu Phe Gly Pro Ala 100 105 110 acc acg ggc
aag acc aac atc gcg gaa gcc atc gcc cac gcc gtg ccc 384 Thr Thr Gly
Lys Thr Asn Ile Ala Glu Ala Ile Ala His Ala Val Pro 115 120 125 ttc
tac ggc tgc gtc aac tgg acc aat gag aac ttt ccc ttc aat gat 432 Phe
Tyr Gly Cys Val Asn Trp Thr Asn Glu Asn Phe Pro Phe Asn Asp 130 135
140 tgc gtc gac aag atg gtg atc tgg tgg gag gag ggc aag atg acg gcc
480 Cys Val Asp Lys Met Val Ile Trp Trp Glu Glu Gly Lys Met Thr Ala
145 150 155 160 aag gtc gtg gag tcc gcc aag gcc att ctc ggc ggc agc
aag gtg cgc 528 Lys Val Val Glu Ser Ala Lys Ala Ile Leu Gly Gly Ser
Lys Val Arg 165 170 175 gtg gac caa aag tgc aag tcg tcc gcc cag atc
gac ccc acc ccc gtg 576 Val Asp Gln Lys Cys Lys Ser Ser Ala Gln Ile
Asp Pro Thr Pro Val 180 185 190 atc gtc acc tcc aac acc aac atg tgc
gcc gtg att gac ggg aac agc 624 Ile Val Thr Ser Asn Thr Asn Met Cys
Ala Val Ile Asp Gly Asn Ser 195 200 205 acc acc ttc gag cac cag cag
ccg ttg cag gac cgg atg ttc aaa ttt 672 Thr Thr Phe Glu His Gln Gln
Pro Leu Gln Asp Arg Met Phe Lys Phe 210 215 220 gaa ctc acc cgc cgt
ctg gag cat gac ttt ggc aag gtg aca aag cag 720 Glu Leu Thr Arg Arg
Leu Glu His Asp Phe Gly Lys Val Thr Lys Gln 225 230 235 240 gaa gtc
aaa gag ttc ttc cgc tgg gcg cag gat cac gtg acc gag gtg 768 Glu Val
Lys Glu Phe Phe Arg Trp Ala Gln Asp His Val Thr Glu Val 245 250 255
gcg cat gag ttc tac gtc aga aag ggt gga gcc aac aaa aga ccc gcc 816
Ala His Glu Phe Tyr Val Arg Lys Gly Gly Ala Asn Lys Arg Pro Ala 260
265 270 ccc gat gac gcg gat aaa agc gag ccc aag cgg gcc tgc ccc tca
gtc 864 Pro Asp Asp Ala Asp Lys Ser Glu Pro Lys Arg Ala Cys Pro Ser
Val 275 280 285 gcg gat cca tcg acg tca gac gcg gaa gga gct ccg gtg
gac ttt gcc 912 Ala Asp Pro Ser Thr Ser Asp Ala Glu Gly Ala Pro Val
Asp Phe Ala 290 295 300 gac agg tat ggc tgc cga tgg tta tct tcc aga
ttg gct cga gga caa 960 Asp Arg Tyr Gly Cys Arg Trp Leu Ser Ser Arg
Leu Ala Arg Gly Gln 305 310 315 320 cct ctc tga 969 Pro Leu 11 322
PRT AAV-1 misc_feature (943)..(944) minor splice site 11 Met Glu
Leu Val Gly Trp Leu Val Asp Arg Gly Ile Thr Ser Glu Lys 1 5 10 15
Gln Trp Ile Gln Glu Asp Gln Ala Ser Tyr Ile Ser Phe Asn Ala Ala 20
25 30 Ser Asn Ser Arg Ser Gln Ile Lys Ala Ala Leu Asp Asn Ala Gly
Lys 35 40 45 Ile Met Ala Leu Thr Lys Ser Ala Pro Asp Tyr Leu Val
Gly Pro Ala 50 55 60 Pro Pro Ala Asp Ile Lys Thr Asn Arg Ile Tyr
Arg Ile Leu Glu Leu 65 70 75 80 Asn Gly Tyr Glu Pro Ala Tyr Ala Gly
Ser Val Phe Leu Gly Trp Ala 85 90 95 Gln Lys Arg Phe Gly Lys Arg
Asn Thr Ile Trp Leu Phe Gly Pro Ala 100 105 110 Thr Thr Gly Lys Thr
Asn Ile Ala Glu Ala Ile Ala His Ala Val Pro 115 120 125 Phe Tyr Gly
Cys Val Asn Trp Thr Asn Glu Asn Phe Pro Phe Asn Asp 130 135 140 Cys
Val Asp Lys Met Val Ile Trp Trp Glu Glu Gly Lys Met Thr Ala 145 150
155 160 Lys Val Val Glu Ser Ala Lys Ala Ile Leu Gly Gly Ser Lys Val
Arg 165 170 175 Val Asp Gln Lys Cys Lys Ser Ser Ala Gln Ile Asp Pro
Thr Pro Val 180 185 190 Ile Val Thr Ser Asn Thr Asn Met Cys Ala Val
Ile Asp Gly Asn Ser 195 200 205 Thr Thr Phe Glu His Gln Gln Pro Leu
Gln Asp Arg Met Phe Lys Phe 210 215 220 Glu Leu Thr Arg Arg Leu Glu
His Asp Phe Gly Lys Val Thr Lys Gln 225 230 235 240 Glu Val Lys Glu
Phe Phe Arg Trp Ala Gln Asp His Val Thr Glu Val 245 250 255 Ala His
Glu Phe Tyr Val Arg Lys Gly Gly Ala Asn Lys Arg Pro Ala 260 265 270
Pro Asp Asp Ala Asp Lys Ser Glu Pro Lys Arg Ala Cys Pro Ser Val 275
280 285 Ala Asp Pro Ser Thr Ser Asp Ala Glu Gly Ala Pro Val Asp Phe
Ala 290 295 300 Asp Arg Tyr Gly Cys Arg Trp Leu Ser Ser Arg Leu Ala
Arg Gly Gln 305 310 315 320 Pro Leu 12 2211 DNA AAV-1 CDS
(1)..(2208) 12 atg gct gcc gat ggt tat ctt cca gat tgg ctc gag gac
aac ctc tct 48 Met Ala Ala Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp
Asn Leu Ser 1 5 10 15 gag ggc att cgc gag tgg tgg gac ttg aaa cct
gga gcc ccg aag ccc 96 Glu Gly Ile Arg Glu Trp Trp Asp Leu Lys Pro
Gly Ala Pro Lys Pro 20 25 30 aaa gcc aac cag caa aag cag gac gac
ggc cgg ggt ctg gtg ctt cct 144 Lys Ala Asn Gln Gln Lys Gln Asp Asp
Gly Arg Gly Leu Val Leu Pro 35 40 45 ggc tac aag tac ctc gga ccc
ttc aac gga ctc gac aag ggg gag ccc 192 Gly Tyr Lys Tyr Leu Gly Pro
Phe Asn Gly Leu Asp Lys Gly Glu Pro 50 55 60 gtc aac gcg gcg gac
gca gcg gcc ctc gag cac gac aag gcc tac gac 240 Val Asn Ala Ala Asp
Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp 65 70 75 80 cag cag ctc
aaa gcg ggt gac aat ccg tac ctg cgg tat aac cac gcc 288 Gln Gln Leu
Lys Ala Gly Asp Asn Pro Tyr Leu Arg Tyr Asn His Ala 85 90 95 gac
gcc gag ttt cag gag cgt ctg caa gaa gat acg tct ttt ggg ggc 336 Asp
Ala Glu Phe Gln Glu Arg Leu Gln Glu Asp Thr Ser Phe Gly Gly 100 105
110 aac ctc ggg cga gca gtc ttc cag gcc aag aag cgg gtt ctc gaa cct
384 Asn Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg Val Leu Glu Pro
115 120 125 ctc ggt ctg gtt gag gaa ggc gct aag acg gct cct gga aag
aaa cgt 432 Leu Gly Leu Val Glu Glu Gly Ala Lys Thr Ala Pro Gly Lys
Lys Arg 130 135 140 ccg gta gag cag tcg cca caa gag cca gac tcc tcc
tcg ggc atc ggc 480 Pro Val Glu Gln Ser Pro Gln Glu Pro Asp Ser Ser
Ser Gly Ile Gly 145 150 155 160 aag aca ggc cag cag ccc gct aaa aag
aga ctc aat ttt ggt cag act 528 Lys Thr Gly Gln Gln Pro Ala Lys Lys
Arg Leu Asn Phe Gly Gln Thr 165 170 175 ggc gac tca gag tca gtc ccc
gat cca caa cct ctc gga gaa cct cca 576 Gly Asp Ser Glu Ser Val Pro
Asp Pro Gln Pro Leu Gly Glu Pro Pro 180 185 190 gca acc ccc gct gct
gtg gga cct act aca atg gct tca ggc ggt ggc 624 Ala Thr Pro Ala Ala
Val Gly Pro Thr Thr Met Ala Ser Gly Gly Gly 195 200 205 gca cca atg
gca gac aat aac gaa ggc gcc gac gga gtg ggt aat gcc 672 Ala Pro Met
Ala Asp Asn Asn Glu Gly Ala Asp Gly Val Gly Asn Ala 210 215 220 tca
gga aat tgg cat tgc gat tcc aca tgg ctg ggc gac aga gtc atc 720 Ser
Gly Asn Trp His Cys Asp Ser Thr Trp Leu Gly Asp Arg Val Ile 225 230
235 240 acc acc agc acc cgc acc tgg gcc ttg ccc acc tac aat aac cac
ctc 768 Thr Thr Ser Thr Arg Thr Trp Ala Leu Pro Thr Tyr Asn Asn His
Leu 245 250 255 tac aag caa atc tcc agt gct tca acg ggg gcc agc aac
gac aac cac 816 Tyr Lys Gln Ile Ser Ser Ala Ser Thr Gly Ala Ser Asn
Asp Asn His 260 265 270 tac ttc ggc tac agc acc ccc tgg ggg tat ttt
gat ttc aac aga ttc 864 Tyr Phe Gly Tyr Ser Thr Pro Trp Gly Tyr Phe
Asp Phe Asn Arg Phe 275 280 285 cac tgc cac ttt tca cca cgt gac tgg
cag cga ctc atc aac aac aat 912 His Cys His Phe Ser Pro Arg Asp Trp
Gln Arg Leu Ile Asn Asn Asn 290 295 300 tgg gga ttc cgg ccc aag aga
ctc aac ttc aaa ctc ttc aac atc caa 960 Trp Gly Phe Arg Pro Lys Arg
Leu Asn Phe Lys Leu Phe Asn Ile Gln 305 310 315 320 gtc aag gag gtc
acg acg aat gat ggc gtc aca acc atc gct aat aac 1008 Val Lys Glu
Val Thr Thr Asn Asp Gly Val Thr Thr Ile Ala Asn Asn 325 330 335 ctt
acc agc acg gtt caa gtc ttc tcg gac tcg gag tac cag ctt ccg 1056
Leu Thr Ser Thr Val Gln Val Phe Ser Asp Ser Glu Tyr Gln Leu Pro 340
345 350 tac gtc ctc ggc tct gcg cac cag ggc tgc ctc cct ccg ttc ccg
gcg 1104 Tyr Val Leu Gly Ser Ala His Gln Gly Cys Leu Pro Pro Phe
Pro Ala 355 360 365 gac gtg ttc atg att ccg caa tac ggc tac ctg acg
ctc aac aat ggc 1152 Asp Val Phe Met Ile Pro Gln Tyr Gly Tyr Leu
Thr Leu Asn Asn Gly 370 375 380 agc caa gcc gtg gga cgt tca tcc ttt
tac tgc ctg gaa tat ttc cct 1200 Ser Gln Ala Val Gly Arg Ser Ser
Phe Tyr Cys Leu Glu Tyr Phe Pro 385 390 395 400 tct cag atg ctg aga
acg ggc aac aac ttt acc ttc agc tac acc ttt 1248 Ser Gln Met Leu
Arg Thr Gly Asn Asn Phe Thr Phe Ser Tyr Thr Phe 405 410 415 gag gaa
gtg cct ttc cac agc agc tac gcg cac agc cag agc ctg gac 1296 Glu
Glu Val Pro Phe His Ser Ser Tyr Ala His Ser Gln Ser Leu Asp 420 425
430 cgg ctg atg aat cct ctc atc gac caa tac ctg tat tac ctg aac aga
1344 Arg Leu Met Asn Pro Leu Ile Asp Gln Tyr Leu Tyr Tyr Leu Asn
Arg 435 440 445 act caa aat cag tcc gga agt gcc caa aac aag gac ttg
ctg ttt agc 1392 Thr Gln Asn Gln Ser Gly Ser Ala Gln Asn Lys Asp
Leu Leu Phe Ser 450 455 460 cgt ggg tct cca gct ggc atg tct gtt cag
ccc aaa aac tgg cta cct 1440 Arg Gly Ser Pro Ala Gly Met Ser Val
Gln Pro Lys Asn Trp Leu Pro 465 470 475 480 gga ccc tgt tat cgg cag
cag cgc gtt tct aaa aca aaa aca gac aac 1488 Gly Pro Cys Tyr Arg
Gln Gln Arg Val Ser Lys Thr Lys Thr Asp Asn 485 490 495 aac aac agc
aat ttt acc tgg act ggt gct tca aaa tat aac ctc aat 1536 Asn Asn
Ser Asn Phe Thr Trp Thr Gly Ala Ser Lys Tyr Asn Leu Asn 500 505 510
ggg cgt gaa tcc atc atc aac cct ggc act gct atg gcc tca cac aaa
1584 Gly Arg Glu Ser Ile Ile Asn Pro Gly Thr Ala Met Ala Ser His
Lys 515 520 525 gac gac gaa gac aag ttc ttt ccc atg agc ggt gtc atg
att ttt gga 1632 Asp Asp Glu Asp Lys Phe Phe Pro Met Ser Gly Val
Met Ile Phe Gly 530 535 540 aaa gag agc gcc gga gct tca aac act gca
ttg gac aat gtc atg att 1680 Lys Glu Ser Ala Gly Ala Ser Asn Thr
Ala Leu Asp Asn Val Met Ile 545 550 555 560 aca gac gaa gag gaa att
aaa gcc act aac cct gtg gcc acc gaa aga 1728 Thr Asp Glu Glu Glu
Ile Lys Ala Thr Asn Pro Val Ala Thr Glu Arg 565 570 575 ttt ggg acc
gtg gca gtc aat ttc cag agc agc agc aca gac cct gcg 1776 Phe Gly
Thr Val Ala Val Asn Phe Gln Ser Ser Ser Thr Asp Pro Ala 580 585 590
acc gga gat gtg cat gct atg gga gca tta cct ggc atg gtg tgg caa
1824 Thr Gly Asp Val His Ala Met Gly Ala Leu Pro Gly Met Val Trp
Gln 595 600 605 gat aga gac gtg tac ctg cag ggt ccc att tgg gcc aaa
att cct cac 1872 Asp Arg Asp Val Tyr Leu Gln Gly Pro Ile Trp Ala
Lys Ile Pro His 610 615 620 aca gat gga cac ttt cac ccg tct cct ctt
atg ggc ggc ttt gga ctc 1920 Thr Asp Gly His Phe His Pro Ser Pro
Leu Met Gly Gly Phe Gly Leu 625 630 635 640 aag aac ccg cct cct cag
atc ctc atc aaa aac acg cct gtt cct gcg 1968 Lys Asn Pro Pro Pro
Gln Ile Leu Ile Lys Asn Thr Pro Val Pro Ala 645 650 655 aat cct ccg
gcg gag ttt tca gct aca aag ttt gct tca ttc atc acc 2016 Asn Pro
Pro Ala Glu Phe Ser Ala Thr Lys Phe Ala Ser Phe Ile Thr 660 665 670
caa tac tcc aca gga caa gtg agt gtg gaa att gaa tgg gag ctg cag
2064 Gln Tyr Ser Thr Gly Gln Val Ser Val Glu Ile Glu Trp Glu Leu
Gln 675 680 685 aaa gaa aac agc aag cgc tgg aat ccc gaa gtg cag tac
aca tcc aat 2112 Lys Glu Asn Ser Lys Arg Trp Asn Pro Glu Val Gln
Tyr Thr Ser
Asn 690 695 700 tat gca aaa tct gcc aac gtt gat ttt act gtg gac aac
aat gga ctt 2160 Tyr Ala Lys Ser Ala Asn Val Asp Phe Thr Val Asp
Asn Asn Gly Leu 705 710 715 720 tat act gag cct cgc ccc att ggc acc
cgt tac ctt acc cgt ccc ctg 2208 Tyr Thr Glu Pro Arg Pro Ile Gly
Thr Arg Tyr Leu Thr Arg Pro Leu 725 730 735 taa 2211 13 736 PRT
AAV-1 13 Met Ala Ala Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Asn
Leu Ser 1 5 10 15 Glu Gly Ile Arg Glu Trp Trp Asp Leu Lys Pro Gly
Ala Pro Lys Pro 20 25 30 Lys Ala Asn Gln Gln Lys Gln Asp Asp Gly
Arg Gly Leu Val Leu Pro 35 40 45 Gly Tyr Lys Tyr Leu Gly Pro Phe
Asn Gly Leu Asp Lys Gly Glu Pro 50 55 60 Val Asn Ala Ala Asp Ala
Ala Ala Leu Glu His Asp Lys Ala Tyr Asp 65 70 75 80 Gln Gln Leu Lys
Ala Gly Asp Asn Pro Tyr Leu Arg Tyr Asn His Ala 85 90 95 Asp Ala
Glu Phe Gln Glu Arg Leu Gln Glu Asp Thr Ser Phe Gly Gly 100 105 110
Asn Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg Val Leu Glu Pro 115
120 125 Leu Gly Leu Val Glu Glu Gly Ala Lys Thr Ala Pro Gly Lys Lys
Arg 130 135 140 Pro Val Glu Gln Ser Pro Gln Glu Pro Asp Ser Ser Ser
Gly Ile Gly 145 150 155 160 Lys Thr Gly Gln Gln Pro Ala Lys Lys Arg
Leu Asn Phe Gly Gln Thr 165 170 175 Gly Asp Ser Glu Ser Val Pro Asp
Pro Gln Pro Leu Gly Glu Pro Pro 180 185 190 Ala Thr Pro Ala Ala Val
Gly Pro Thr Thr Met Ala Ser Gly Gly Gly 195 200 205 Ala Pro Met Ala
Asp Asn Asn Glu Gly Ala Asp Gly Val Gly Asn Ala 210 215 220 Ser Gly
Asn Trp His Cys Asp Ser Thr Trp Leu Gly Asp Arg Val Ile 225 230 235
240 Thr Thr Ser Thr Arg Thr Trp Ala Leu Pro Thr Tyr Asn Asn His Leu
245 250 255 Tyr Lys Gln Ile Ser Ser Ala Ser Thr Gly Ala Ser Asn Asp
Asn His 260 265 270 Tyr Phe Gly Tyr Ser Thr Pro Trp Gly Tyr Phe Asp
Phe Asn Arg Phe 275 280 285 His Cys His Phe Ser Pro Arg Asp Trp Gln
Arg Leu Ile Asn Asn Asn 290 295 300 Trp Gly Phe Arg Pro Lys Arg Leu
Asn Phe Lys Leu Phe Asn Ile Gln 305 310 315 320 Val Lys Glu Val Thr
Thr Asn Asp Gly Val Thr Thr Ile Ala Asn Asn 325 330 335 Leu Thr Ser
Thr Val Gln Val Phe Ser Asp Ser Glu Tyr Gln Leu Pro 340 345 350 Tyr
Val Leu Gly Ser Ala His Gln Gly Cys Leu Pro Pro Phe Pro Ala 355 360
365 Asp Val Phe Met Ile Pro Gln Tyr Gly Tyr Leu Thr Leu Asn Asn Gly
370 375 380 Ser Gln Ala Val Gly Arg Ser Ser Phe Tyr Cys Leu Glu Tyr
Phe Pro 385 390 395 400 Ser Gln Met Leu Arg Thr Gly Asn Asn Phe Thr
Phe Ser Tyr Thr Phe 405 410 415 Glu Glu Val Pro Phe His Ser Ser Tyr
Ala His Ser Gln Ser Leu Asp 420 425 430 Arg Leu Met Asn Pro Leu Ile
Asp Gln Tyr Leu Tyr Tyr Leu Asn Arg 435 440 445 Thr Gln Asn Gln Ser
Gly Ser Ala Gln Asn Lys Asp Leu Leu Phe Ser 450 455 460 Arg Gly Ser
Pro Ala Gly Met Ser Val Gln Pro Lys Asn Trp Leu Pro 465 470 475 480
Gly Pro Cys Tyr Arg Gln Gln Arg Val Ser Lys Thr Lys Thr Asp Asn 485
490 495 Asn Asn Ser Asn Phe Thr Trp Thr Gly Ala Ser Lys Tyr Asn Leu
Asn 500 505 510 Gly Arg Glu Ser Ile Ile Asn Pro Gly Thr Ala Met Ala
Ser His Lys 515 520 525 Asp Asp Glu Asp Lys Phe Phe Pro Met Ser Gly
Val Met Ile Phe Gly 530 535 540 Lys Glu Ser Ala Gly Ala Ser Asn Thr
Ala Leu Asp Asn Val Met Ile 545 550 555 560 Thr Asp Glu Glu Glu Ile
Lys Ala Thr Asn Pro Val Ala Thr Glu Arg 565 570 575 Phe Gly Thr Val
Ala Val Asn Phe Gln Ser Ser Ser Thr Asp Pro Ala 580 585 590 Thr Gly
Asp Val His Ala Met Gly Ala Leu Pro Gly Met Val Trp Gln 595 600 605
Asp Arg Asp Val Tyr Leu Gln Gly Pro Ile Trp Ala Lys Ile Pro His 610
615 620 Thr Asp Gly His Phe His Pro Ser Pro Leu Met Gly Gly Phe Gly
Leu 625 630 635 640 Lys Asn Pro Pro Pro Gln Ile Leu Ile Lys Asn Thr
Pro Val Pro Ala 645 650 655 Asn Pro Pro Ala Glu Phe Ser Ala Thr Lys
Phe Ala Ser Phe Ile Thr 660 665 670 Gln Tyr Ser Thr Gly Gln Val Ser
Val Glu Ile Glu Trp Glu Leu Gln 675 680 685 Lys Glu Asn Ser Lys Arg
Trp Asn Pro Glu Val Gln Tyr Thr Ser Asn 690 695 700 Tyr Ala Lys Ser
Ala Asn Val Asp Phe Thr Val Asp Asn Asn Gly Leu 705 710 715 720 Tyr
Thr Glu Pro Arg Pro Ile Gly Thr Arg Tyr Leu Thr Arg Pro Leu 725 730
735 14 1800 DNA AAV-1 CDS (1)..(1797) 14 acg gct cct gga aag aaa
cgt ccg gta gag cag tcg cca caa gag cca 48 Thr Ala Pro Gly Lys Lys
Arg Pro Val Glu Gln Ser Pro Gln Glu Pro 1 5 10 15 gac tcc tcc tcg
ggc atc ggc aag aca ggc cag cag ccc gct aaa aag 96 Asp Ser Ser Ser
Gly Ile Gly Lys Thr Gly Gln Gln Pro Ala Lys Lys 20 25 30 aga ctc
aat ttt ggt cag act ggc gac tca gag tca gtc ccc gat cca 144 Arg Leu
Asn Phe Gly Gln Thr Gly Asp Ser Glu Ser Val Pro Asp Pro 35 40 45
caa cct ctc gga gaa cct cca gca acc ccc gct gct gtg gga cct act 192
Gln Pro Leu Gly Glu Pro Pro Ala Thr Pro Ala Ala Val Gly Pro Thr 50
55 60 aca atg gct tca ggc ggt ggc gca cca atg gca gac aat aac gaa
ggc 240 Thr Met Ala Ser Gly Gly Gly Ala Pro Met Ala Asp Asn Asn Glu
Gly 65 70 75 80 gcc gac gga gtg ggt aat gcc tca gga aat tgg cat tgc
gat tcc aca 288 Ala Asp Gly Val Gly Asn Ala Ser Gly Asn Trp His Cys
Asp Ser Thr 85 90 95 tgg ctg ggc gac aga gtc atc acc acc agc acc
cgc acc tgg gcc ttg 336 Trp Leu Gly Asp Arg Val Ile Thr Thr Ser Thr
Arg Thr Trp Ala Leu 100 105 110 ccc acc tac aat aac cac ctc tac aag
caa atc tcc agt gct tca acg 384 Pro Thr Tyr Asn Asn His Leu Tyr Lys
Gln Ile Ser Ser Ala Ser Thr 115 120 125 ggg gcc agc aac gac aac cac
tac ttc ggc tac agc acc ccc tgg ggg 432 Gly Ala Ser Asn Asp Asn His
Tyr Phe Gly Tyr Ser Thr Pro Trp Gly 130 135 140 tat ttt gat ttc aac
aga ttc cac tgc cac ttt tca cca cgt gac tgg 480 Tyr Phe Asp Phe Asn
Arg Phe His Cys His Phe Ser Pro Arg Asp Trp 145 150 155 160 cag cga
ctc atc aac aac aat tgg gga ttc cgg ccc aag aga ctc aac 528 Gln Arg
Leu Ile Asn Asn Asn Trp Gly Phe Arg Pro Lys Arg Leu Asn 165 170 175
ttc aaa ctc ttc aac atc caa gtc aag gag gtc acg acg aat gat ggc 576
Phe Lys Leu Phe Asn Ile Gln Val Lys Glu Val Thr Thr Asn Asp Gly 180
185 190 gtc aca acc atc gct aat aac ctt acc agc acg gtt caa gtc ttc
tcg 624 Val Thr Thr Ile Ala Asn Asn Leu Thr Ser Thr Val Gln Val Phe
Ser 195 200 205 gac tcg gag tac cag ctt ccg tac gtc ctc ggc tct gcg
cac cag ggc 672 Asp Ser Glu Tyr Gln Leu Pro Tyr Val Leu Gly Ser Ala
His Gln Gly 210 215 220 tgc ctc cct ccg ttc ccg gcg gac gtg ttc atg
att ccg caa tac ggc 720 Cys Leu Pro Pro Phe Pro Ala Asp Val Phe Met
Ile Pro Gln Tyr Gly 225 230 235 240 tac ctg acg ctc aac aat ggc agc
caa gcc gtg gga cgt tca tcc ttt 768 Tyr Leu Thr Leu Asn Asn Gly Ser
Gln Ala Val Gly Arg Ser Ser Phe 245 250 255 tac tgc ctg gaa tat ttc
cct tct cag atg ctg aga acg ggc aac aac 816 Tyr Cys Leu Glu Tyr Phe
Pro Ser Gln Met Leu Arg Thr Gly Asn Asn 260 265 270 ttt acc ttc agc
tac acc ttt gag gaa gtg cct ttc cac agc agc tac 864 Phe Thr Phe Ser
Tyr Thr Phe Glu Glu Val Pro Phe His Ser Ser Tyr 275 280 285 gcg cac
agc cag agc ctg gac cgg ctg atg aat cct ctc atc gac caa 912 Ala His
Ser Gln Ser Leu Asp Arg Leu Met Asn Pro Leu Ile Asp Gln 290 295 300
tac ctg tat tac ctg aac aga act caa aat cag tcc gga agt gcc caa 960
Tyr Leu Tyr Tyr Leu Asn Arg Thr Gln Asn Gln Ser Gly Ser Ala Gln 305
310 315 320 aac aag gac ttg ctg ttt agc cgt ggg tct cca gct ggc atg
tct gtt 1008 Asn Lys Asp Leu Leu Phe Ser Arg Gly Ser Pro Ala Gly
Met Ser Val 325 330 335 cag ccc aaa aac tgg cta cct gga ccc tgt tat
cgg cag cag cgc gtt 1056 Gln Pro Lys Asn Trp Leu Pro Gly Pro Cys
Tyr Arg Gln Gln Arg Val 340 345 350 tct aaa aca aaa aca gac aac aac
aac agc aat ttt acc tgg act ggt 1104 Ser Lys Thr Lys Thr Asp Asn
Asn Asn Ser Asn Phe Thr Trp Thr Gly 355 360 365 gct tca aaa tat aac
ctc aat ggg cgt gaa tcc atc atc aac cct ggc 1152 Ala Ser Lys Tyr
Asn Leu Asn Gly Arg Glu Ser Ile Ile Asn Pro Gly 370 375 380 act gct
atg gcc tca cac aaa gac gac gaa gac aag ttc ttt ccc atg 1200 Thr
Ala Met Ala Ser His Lys Asp Asp Glu Asp Lys Phe Phe Pro Met 385 390
395 400 agc ggt gtc atg att ttt gga aaa gag agc gcc gga gct tca aac
act 1248 Ser Gly Val Met Ile Phe Gly Lys Glu Ser Ala Gly Ala Ser
Asn Thr 405 410 415 gca ttg gac aat gtc atg att aca gac gaa gag gaa
att aaa gcc act 1296 Ala Leu Asp Asn Val Met Ile Thr Asp Glu Glu
Glu Ile Lys Ala Thr 420 425 430 aac cct gtg gcc acc gaa aga ttt ggg
acc gtg gca gtc aat ttc cag 1344 Asn Pro Val Ala Thr Glu Arg Phe
Gly Thr Val Ala Val Asn Phe Gln 435 440 445 agc agc agc aca gac cct
gcg acc gga gat gtg cat gct atg gga gca 1392 Ser Ser Ser Thr Asp
Pro Ala Thr Gly Asp Val His Ala Met Gly Ala 450 455 460 tta cct ggc
atg gtg tgg caa gat aga gac gtg tac ctg cag ggt ccc 1440 Leu Pro
Gly Met Val Trp Gln Asp Arg Asp Val Tyr Leu Gln Gly Pro 465 470 475
480 att tgg gcc aaa att cct cac aca gat gga cac ttt cac ccg tct cct
1488 Ile Trp Ala Lys Ile Pro His Thr Asp Gly His Phe His Pro Ser
Pro 485 490 495 ctt atg ggc ggc ttt gga ctc aag aac ccg cct cct cag
atc ctc atc 1536 Leu Met Gly Gly Phe Gly Leu Lys Asn Pro Pro Pro
Gln Ile Leu Ile 500 505 510 aaa aac acg cct gtt cct gcg aat cct ccg
gcg gag ttt tca gct aca 1584 Lys Asn Thr Pro Val Pro Ala Asn Pro
Pro Ala Glu Phe Ser Ala Thr 515 520 525 aag ttt gct tca ttc atc acc
caa tac tcc aca gga caa gtg agt gtg 1632 Lys Phe Ala Ser Phe Ile
Thr Gln Tyr Ser Thr Gly Gln Val Ser Val 530 535 540 gaa att gaa tgg
gag ctg cag aaa gaa aac agc aag cgc tgg aat ccc 1680 Glu Ile Glu
Trp Glu Leu Gln Lys Glu Asn Ser Lys Arg Trp Asn Pro 545 550 555 560
gaa gtg cag tac aca tcc aat tat gca aaa tct gcc aac gtt gat ttt
1728 Glu Val Gln Tyr Thr Ser Asn Tyr Ala Lys Ser Ala Asn Val Asp
Phe 565 570 575 act gtg gac aac aat gga ctt tat act gag cct cgc ccc
att ggc acc 1776 Thr Val Asp Asn Asn Gly Leu Tyr Thr Glu Pro Arg
Pro Ile Gly Thr 580 585 590 cgt tac ctt acc cgt ccc ctg taa 1800
Arg Tyr Leu Thr Arg Pro Leu 595 15 599 PRT AAV-1 15 Thr Ala Pro Gly
Lys Lys Arg Pro Val Glu Gln Ser Pro Gln Glu Pro 1 5 10 15 Asp Ser
Ser Ser Gly Ile Gly Lys Thr Gly Gln Gln Pro Ala Lys Lys 20 25 30
Arg Leu Asn Phe Gly Gln Thr Gly Asp Ser Glu Ser Val Pro Asp Pro 35
40 45 Gln Pro Leu Gly Glu Pro Pro Ala Thr Pro Ala Ala Val Gly Pro
Thr 50 55 60 Thr Met Ala Ser Gly Gly Gly Ala Pro Met Ala Asp Asn
Asn Glu Gly 65 70 75 80 Ala Asp Gly Val Gly Asn Ala Ser Gly Asn Trp
His Cys Asp Ser Thr 85 90 95 Trp Leu Gly Asp Arg Val Ile Thr Thr
Ser Thr Arg Thr Trp Ala Leu 100 105 110 Pro Thr Tyr Asn Asn His Leu
Tyr Lys Gln Ile Ser Ser Ala Ser Thr 115 120 125 Gly Ala Ser Asn Asp
Asn His Tyr Phe Gly Tyr Ser Thr Pro Trp Gly 130 135 140 Tyr Phe Asp
Phe Asn Arg Phe His Cys His Phe Ser Pro Arg Asp Trp 145 150 155 160
Gln Arg Leu Ile Asn Asn Asn Trp Gly Phe Arg Pro Lys Arg Leu Asn 165
170 175 Phe Lys Leu Phe Asn Ile Gln Val Lys Glu Val Thr Thr Asn Asp
Gly 180 185 190 Val Thr Thr Ile Ala Asn Asn Leu Thr Ser Thr Val Gln
Val Phe Ser 195 200 205 Asp Ser Glu Tyr Gln Leu Pro Tyr Val Leu Gly
Ser Ala His Gln Gly 210 215 220 Cys Leu Pro Pro Phe Pro Ala Asp Val
Phe Met Ile Pro Gln Tyr Gly 225 230 235 240 Tyr Leu Thr Leu Asn Asn
Gly Ser Gln Ala Val Gly Arg Ser Ser Phe 245 250 255 Tyr Cys Leu Glu
Tyr Phe Pro Ser Gln Met Leu Arg Thr Gly Asn Asn 260 265 270 Phe Thr
Phe Ser Tyr Thr Phe Glu Glu Val Pro Phe His Ser Ser Tyr 275 280 285
Ala His Ser Gln Ser Leu Asp Arg Leu Met Asn Pro Leu Ile Asp Gln 290
295 300 Tyr Leu Tyr Tyr Leu Asn Arg Thr Gln Asn Gln Ser Gly Ser Ala
Gln 305 310 315 320 Asn Lys Asp Leu Leu Phe Ser Arg Gly Ser Pro Ala
Gly Met Ser Val 325 330 335 Gln Pro Lys Asn Trp Leu Pro Gly Pro Cys
Tyr Arg Gln Gln Arg Val 340 345 350 Ser Lys Thr Lys Thr Asp Asn Asn
Asn Ser Asn Phe Thr Trp Thr Gly 355 360 365 Ala Ser Lys Tyr Asn Leu
Asn Gly Arg Glu Ser Ile Ile Asn Pro Gly 370 375 380 Thr Ala Met Ala
Ser His Lys Asp Asp Glu Asp Lys Phe Phe Pro Met 385 390 395 400 Ser
Gly Val Met Ile Phe Gly Lys Glu Ser Ala Gly Ala Ser Asn Thr 405 410
415 Ala Leu Asp Asn Val Met Ile Thr Asp Glu Glu Glu Ile Lys Ala Thr
420 425 430 Asn Pro Val Ala Thr Glu Arg Phe Gly Thr Val Ala Val Asn
Phe Gln 435 440 445 Ser Ser Ser Thr Asp Pro Ala Thr Gly Asp Val His
Ala Met Gly Ala 450 455 460 Leu Pro Gly Met Val Trp Gln Asp Arg Asp
Val Tyr Leu Gln Gly Pro 465 470 475 480 Ile Trp Ala Lys Ile Pro His
Thr Asp Gly His Phe His Pro Ser Pro 485 490 495 Leu Met Gly Gly Phe
Gly Leu Lys Asn Pro Pro Pro Gln Ile Leu Ile 500 505 510 Lys Asn Thr
Pro Val Pro Ala Asn Pro Pro Ala Glu Phe Ser Ala Thr 515 520 525 Lys
Phe Ala Ser Phe Ile Thr Gln Tyr Ser Thr Gly Gln Val Ser Val 530 535
540 Glu Ile Glu Trp Glu Leu Gln Lys Glu Asn Ser Lys Arg Trp Asn Pro
545 550 555 560 Glu Val Gln Tyr Thr Ser Asn Tyr Ala Lys Ser Ala Asn
Val Asp Phe 565 570 575 Thr Val Asp Asn Asn Gly Leu Tyr Thr Glu Pro
Arg Pro Ile Gly Thr 580 585 590 Arg Tyr Leu Thr Arg Pro Leu 595 16
1605 DNA AAV-1 CDS (1)..(1602) 16 atg gct tca ggc ggt ggc gca cca
atg gca gac aat aac gaa ggc gcc 48 Met Ala Ser Gly Gly Gly Ala Pro
Met Ala Asp Asn Asn Glu Gly Ala 1 5 10 15 gac gga gtg ggt aat gcc
tca gga aat tgg cat tgc gat tcc aca tgg 96 Asp Gly Val Gly Asn Ala
Ser Gly Asn Trp His Cys Asp Ser Thr Trp 20 25 30 ctg ggc gac aga
gtc atc acc acc agc acc cgc acc tgg gcc ttg ccc 144 Leu Gly Asp
Arg Val Ile Thr Thr Ser Thr Arg Thr Trp Ala Leu Pro 35 40 45 acc
tac aat aac cac ctc tac aag caa atc tcc agt gct tca acg ggg 192 Thr
Tyr Asn Asn His Leu Tyr Lys Gln Ile Ser Ser Ala Ser Thr Gly 50 55
60 gcc agc aac gac aac cac tac ttc ggc tac agc acc ccc tgg ggg tat
240 Ala Ser Asn Asp Asn His Tyr Phe Gly Tyr Ser Thr Pro Trp Gly Tyr
65 70 75 80 ttt gat ttc aac aga ttc cac tgc cac ttt tca cca cgt gac
tgg cag 288 Phe Asp Phe Asn Arg Phe His Cys His Phe Ser Pro Arg Asp
Trp Gln 85 90 95 cga ctc atc aac aac aat tgg gga ttc cgg ccc aag
aga ctc aac ttc 336 Arg Leu Ile Asn Asn Asn Trp Gly Phe Arg Pro Lys
Arg Leu Asn Phe 100 105 110 aaa ctc ttc aac atc caa gtc aag gag gtc
acg acg aat gat ggc gtc 384 Lys Leu Phe Asn Ile Gln Val Lys Glu Val
Thr Thr Asn Asp Gly Val 115 120 125 aca acc atc gct aat aac ctt acc
agc acg gtt caa gtc ttc tcg gac 432 Thr Thr Ile Ala Asn Asn Leu Thr
Ser Thr Val Gln Val Phe Ser Asp 130 135 140 tcg gag tac cag ctt ccg
tac gtc ctc ggc tct gcg cac cag ggc tgc 480 Ser Glu Tyr Gln Leu Pro
Tyr Val Leu Gly Ser Ala His Gln Gly Cys 145 150 155 160 ctc cct ccg
ttc ccg gcg gac gtg ttc atg att ccg caa tac ggc tac 528 Leu Pro Pro
Phe Pro Ala Asp Val Phe Met Ile Pro Gln Tyr Gly Tyr 165 170 175 ctg
acg ctc aac aat ggc agc caa gcc gtg gga cgt tca tcc ttt tac 576 Leu
Thr Leu Asn Asn Gly Ser Gln Ala Val Gly Arg Ser Ser Phe Tyr 180 185
190 tgc ctg gaa tat ttc cct tct cag atg ctg aga acg ggc aac aac ttt
624 Cys Leu Glu Tyr Phe Pro Ser Gln Met Leu Arg Thr Gly Asn Asn Phe
195 200 205 acc ttc agc tac acc ttt gag gaa gtg cct ttc cac agc agc
tac gcg 672 Thr Phe Ser Tyr Thr Phe Glu Glu Val Pro Phe His Ser Ser
Tyr Ala 210 215 220 cac agc cag agc ctg gac cgg ctg atg aat cct ctc
atc gac caa tac 720 His Ser Gln Ser Leu Asp Arg Leu Met Asn Pro Leu
Ile Asp Gln Tyr 225 230 235 240 ctg tat tac ctg aac aga act caa aat
cag tcc gga agt gcc caa aac 768 Leu Tyr Tyr Leu Asn Arg Thr Gln Asn
Gln Ser Gly Ser Ala Gln Asn 245 250 255 aag gac ttg ctg ttt agc cgt
ggg tct cca gct ggc atg tct gtt cag 816 Lys Asp Leu Leu Phe Ser Arg
Gly Ser Pro Ala Gly Met Ser Val Gln 260 265 270 ccc aaa aac tgg cta
cct gga ccc tgt tat cgg cag cag cgc gtt tct 864 Pro Lys Asn Trp Leu
Pro Gly Pro Cys Tyr Arg Gln Gln Arg Val Ser 275 280 285 aaa aca aaa
aca gac aac aac aac agc aat ttt acc tgg act ggt gct 912 Lys Thr Lys
Thr Asp Asn Asn Asn Ser Asn Phe Thr Trp Thr Gly Ala 290 295 300 tca
aaa tat aac ctc aat ggg cgt gaa tcc atc atc aac cct ggc act 960 Ser
Lys Tyr Asn Leu Asn Gly Arg Glu Ser Ile Ile Asn Pro Gly Thr 305 310
315 320 gct atg gcc tca cac aaa gac gac gaa gac aag ttc ttt ccc atg
agc 1008 Ala Met Ala Ser His Lys Asp Asp Glu Asp Lys Phe Phe Pro
Met Ser 325 330 335 ggt gtc atg att ttt gga aaa gag agc gcc gga gct
tca aac act gca 1056 Gly Val Met Ile Phe Gly Lys Glu Ser Ala Gly
Ala Ser Asn Thr Ala 340 345 350 ttg gac aat gtc atg att aca gac gaa
gag gaa att aaa gcc act aac 1104 Leu Asp Asn Val Met Ile Thr Asp
Glu Glu Glu Ile Lys Ala Thr Asn 355 360 365 cct gtg gcc acc gaa aga
ttt ggg acc gtg gca gtc aat ttc cag agc 1152 Pro Val Ala Thr Glu
Arg Phe Gly Thr Val Ala Val Asn Phe Gln Ser 370 375 380 agc agc aca
gac cct gcg acc gga gat gtg cat gct atg gga gca tta 1200 Ser Ser
Thr Asp Pro Ala Thr Gly Asp Val His Ala Met Gly Ala Leu 385 390 395
400 cct ggc atg gtg tgg caa gat aga gac gtg tac ctg cag ggt ccc att
1248 Pro Gly Met Val Trp Gln Asp Arg Asp Val Tyr Leu Gln Gly Pro
Ile 405 410 415 tgg gcc aaa att cct cac aca gat gga cac ttt cac ccg
tct cct ctt 1296 Trp Ala Lys Ile Pro His Thr Asp Gly His Phe His
Pro Ser Pro Leu 420 425 430 atg ggc ggc ttt gga ctc aag aac ccg cct
cct cag atc ctc atc aaa 1344 Met Gly Gly Phe Gly Leu Lys Asn Pro
Pro Pro Gln Ile Leu Ile Lys 435 440 445 aac acg cct gtt cct gcg aat
cct ccg gcg gag ttt tca gct aca aag 1392 Asn Thr Pro Val Pro Ala
Asn Pro Pro Ala Glu Phe Ser Ala Thr Lys 450 455 460 ttt gct tca ttc
atc acc caa tac tcc aca gga caa gtg agt gtg gaa 1440 Phe Ala Ser
Phe Ile Thr Gln Tyr Ser Thr Gly Gln Val Ser Val Glu 465 470 475 480
att gaa tgg gag ctg cag aaa gaa aac agc aag cgc tgg aat ccc gaa
1488 Ile Glu Trp Glu Leu Gln Lys Glu Asn Ser Lys Arg Trp Asn Pro
Glu 485 490 495 gtg cag tac aca tcc aat tat gca aaa tct gcc aac gtt
gat ttt act 1536 Val Gln Tyr Thr Ser Asn Tyr Ala Lys Ser Ala Asn
Val Asp Phe Thr 500 505 510 gtg gac aac aat gga ctt tat act gag cct
cgc ccc att ggc acc cgt 1584 Val Asp Asn Asn Gly Leu Tyr Thr Glu
Pro Arg Pro Ile Gly Thr Arg 515 520 525 tac ctt acc cgt ccc ctg taa
1605 Tyr Leu Thr Arg Pro Leu 530 17 534 PRT AAV-1 17 Met Ala Ser
Gly Gly Gly Ala Pro Met Ala Asp Asn Asn Glu Gly Ala 1 5 10 15 Asp
Gly Val Gly Asn Ala Ser Gly Asn Trp His Cys Asp Ser Thr Trp 20 25
30 Leu Gly Asp Arg Val Ile Thr Thr Ser Thr Arg Thr Trp Ala Leu Pro
35 40 45 Thr Tyr Asn Asn His Leu Tyr Lys Gln Ile Ser Ser Ala Ser
Thr Gly 50 55 60 Ala Ser Asn Asp Asn His Tyr Phe Gly Tyr Ser Thr
Pro Trp Gly Tyr 65 70 75 80 Phe Asp Phe Asn Arg Phe His Cys His Phe
Ser Pro Arg Asp Trp Gln 85 90 95 Arg Leu Ile Asn Asn Asn Trp Gly
Phe Arg Pro Lys Arg Leu Asn Phe 100 105 110 Lys Leu Phe Asn Ile Gln
Val Lys Glu Val Thr Thr Asn Asp Gly Val 115 120 125 Thr Thr Ile Ala
Asn Asn Leu Thr Ser Thr Val Gln Val Phe Ser Asp 130 135 140 Ser Glu
Tyr Gln Leu Pro Tyr Val Leu Gly Ser Ala His Gln Gly Cys 145 150 155
160 Leu Pro Pro Phe Pro Ala Asp Val Phe Met Ile Pro Gln Tyr Gly Tyr
165 170 175 Leu Thr Leu Asn Asn Gly Ser Gln Ala Val Gly Arg Ser Ser
Phe Tyr 180 185 190 Cys Leu Glu Tyr Phe Pro Ser Gln Met Leu Arg Thr
Gly Asn Asn Phe 195 200 205 Thr Phe Ser Tyr Thr Phe Glu Glu Val Pro
Phe His Ser Ser Tyr Ala 210 215 220 His Ser Gln Ser Leu Asp Arg Leu
Met Asn Pro Leu Ile Asp Gln Tyr 225 230 235 240 Leu Tyr Tyr Leu Asn
Arg Thr Gln Asn Gln Ser Gly Ser Ala Gln Asn 245 250 255 Lys Asp Leu
Leu Phe Ser Arg Gly Ser Pro Ala Gly Met Ser Val Gln 260 265 270 Pro
Lys Asn Trp Leu Pro Gly Pro Cys Tyr Arg Gln Gln Arg Val Ser 275 280
285 Lys Thr Lys Thr Asp Asn Asn Asn Ser Asn Phe Thr Trp Thr Gly Ala
290 295 300 Ser Lys Tyr Asn Leu Asn Gly Arg Glu Ser Ile Ile Asn Pro
Gly Thr 305 310 315 320 Ala Met Ala Ser His Lys Asp Asp Glu Asp Lys
Phe Phe Pro Met Ser 325 330 335 Gly Val Met Ile Phe Gly Lys Glu Ser
Ala Gly Ala Ser Asn Thr Ala 340 345 350 Leu Asp Asn Val Met Ile Thr
Asp Glu Glu Glu Ile Lys Ala Thr Asn 355 360 365 Pro Val Ala Thr Glu
Arg Phe Gly Thr Val Ala Val Asn Phe Gln Ser 370 375 380 Ser Ser Thr
Asp Pro Ala Thr Gly Asp Val His Ala Met Gly Ala Leu 385 390 395 400
Pro Gly Met Val Trp Gln Asp Arg Asp Val Tyr Leu Gln Gly Pro Ile 405
410 415 Trp Ala Lys Ile Pro His Thr Asp Gly His Phe His Pro Ser Pro
Leu 420 425 430 Met Gly Gly Phe Gly Leu Lys Asn Pro Pro Pro Gln Ile
Leu Ile Lys 435 440 445 Asn Thr Pro Val Pro Ala Asn Pro Pro Ala Glu
Phe Ser Ala Thr Lys 450 455 460 Phe Ala Ser Phe Ile Thr Gln Tyr Ser
Thr Gly Gln Val Ser Val Glu 465 470 475 480 Ile Glu Trp Glu Leu Gln
Lys Glu Asn Ser Lys Arg Trp Asn Pro Glu 485 490 495 Val Gln Tyr Thr
Ser Asn Tyr Ala Lys Ser Ala Asn Val Asp Phe Thr 500 505 510 Val Asp
Asn Asn Gly Leu Tyr Thr Glu Pro Arg Pro Ile Gly Thr Arg 515 520 525
Tyr Leu Thr Arg Pro Leu 530 18 4681 DNA AAV-2 18 ttggccactc
cctctctgcg cgctcgctcg ctcactgagg ccgggcgacc aaaggtcgcc 60
cgacgcccgg gctttgcccg ggcggcctca gtgagcgagc gagcgcgcag agagggagtg
120 gccaactcca tcactagggg ttcctggagg ggtggagtcg tgacgtgaat
tacgtcatag 180 ggttagggag gtcctgtatt agaggtcacg tgagtgtttt
gcgacatttt gcgacaccat 240 gtggtcacgc tgggtattta agcccgagtg
agcacgcagg gtctccattt tgaagcggga 300 ggtttgaacg cgcagccgcc
atgccggggt tttacgagat tgtgattaag gtccccagcg 360 accttgacgg
gcatctgccc ggcatttctg acagctttgt gaactgggtg gccgagaagg 420
aatgggagtt gccgccagat tctgacatgg atctgaatct gattgagcag gcacccctga
480 ccgtggccga gaagctgcag cgcgactttc tgacggaatg gcgccgtgtg
agtaaggccc 540 cggaggccct tttctttgtg caatttgaga agggagagag
ctacttccac atgcacgtgc 600 tcgtggaaac caccggggtg aaatccatgg
ttttgggacg tttcctgagt cagattcgcg 660 aaaaactgat tcagagaatt
taccgcggga tcgagccgac tttgccaaac tggttcgcgg 720 tcacaaagac
cagaaatggc gccggaggcg ggaacaaggt ggtggatgag tgctacatcc 780
ccaattactt gctccccaaa acccagcctg agctccagtg ggcgtggact aatatggaac
840 agtatttaag cgcctgtttg aatctcacgg agcgtaaacg gttggtggcg
cagcatctga 900 cgcacgtgtc gcagacgcag gagcagaaca aagagaatca
gaatcccaat tctgatgcgc 960 cggtgatcag atcaaaaact tcagccaggt
acatggagct ggtcgggtgg ctcgtggaca 1020 aggggattac ctcggagaag
cagtggatcc aggaggacca ggcctcatac atctccttca 1080 atgcggcctc
caactcgcgg tcccaaatca aggctgcctt ggacaatgcg ggaaagatta 1140
tgagcctgac taaaaccgcc cccgactacc tggtgggcca gcagcccgtg gaggacattt
1200 ccagcaatcg gatttataaa attttggaac taaacgggta cgatccccaa
tatgcggctt 1260 ccgtctttct gggatgggcc acgaaaaagt tcggcaagag
gaacaccatc tggctgtttg 1320 ggcctgcaac taccgggaag accaacatcg
cggaggccat agcccacact gtgcccttct 1380 acgggtgcgt aaactggacc
aatgagaact ttcccttcaa cgactgtgtc gacaagatgg 1440 tgatctggtg
ggaggagggg aagatgaccg ccaaggtcgt ggagtcggcc aaagccattc 1500
tcggaggaag caaggtgcgc gtggaccaga aatgcaagtc ctcggcccag atagacccga
1560 ctcccgtgat cgtcacctcc aacaccaaca tgtgcgccgt gattgacggg
aactcaacga 1620 ccttcgaaca ccagcagccg ttgcaagacc ggatgttcaa
atttgaactc acccgccgtc 1680 tggatcatga ctttgggaag gtcaccaagc
aggaagtcaa agactttttc cggtgggcaa 1740 aggatcacgt ggttgaggtg
gagcatgaat tctacgtcaa aaagggtgga gccaagaaaa 1800 gacccgcccc
cagtgacgca gatataagtg agcccaaacg ggtgcgcgag tcagttgcgc 1860
agccatcgac gtcagacgcg gaagcttcga tcaactacgc agacaggtac caaaacaaat
1920 gttctcgtca cgtgggcatg aatctgatgc tgtttccctg cagacaatgc
gagagaatga 1980 atcagaattc aaatatctgc ttcactcacg gacagaaaga
ctgtttagag tgctttcccg 2040 tgtcagaatc tcaaccggtt tctgtcgtca
aaaaggcgta tcagaaactg tgctacattc 2100 atcatatcat gggaaaggtg
ccagacgctt gcactgcctg cgatctggtc aatgtggatt 2160 tggatgactg
catctttgaa caataaatga tttaaatcag gtatggctgc cgatggttat 2220
cttccagatt ggctcgagga cactctctct gaaggaataa gacagtggtg gaagctcaaa
2280 cctggcccac caccaccaaa gcccgcagag cggcataagg acgacagcag
gggtcttgtg 2340 cttcctgggt acaagtacct cggacccttc aacggactcg
acaagggaga gccggtcaac 2400 gaggcagacg ccgcggccct cgagcacgac
aaagcctacg accggcagct cgacagcgga 2460 gacaacccgt acctcaagta
caaccacgcc gacgcggagt ttcaggagcg ccttaaagaa 2520 gatacgtctt
ttgggggcaa cctcggacga gcagtcttcc aggcgaaaaa gagggttctt 2580
gaacctctcg gcctggttga ggaacctgtt aagacggctc cgggaaaaaa gaggccggta
2640 gagcactctc ctgtggagcc agactcctcc tcgggaaccg gaaagccggg
ccagcagcct 2700 gcaagaaaaa gattgaattt tggtcagact ggagacgcag
actcagtacc tgacccccag 2760 cctctcggac agccaccagc agccccctct
ggtctgggaa ctaatacgat ggctacaggc 2820 agtggcgcac caatggcaga
caataacgag ggcgccgacg gagtgggtaa ttcctccgga 2880 aattggcatt
gcgattccac atggatgggc gacagagtca tcaccaccag cacccgaacc 2940
tgggccctgc ccacctacaa caaccacctc tacaaacaaa tttccagcca atcaggagcc
3000 tcgaacgaca atcactactt tggctacagc accccttggg ggtattttga
cttcaacaga 3060 ttccactgcc acttttcacc acgtgactgg caaagactca
tcaacaacaa ctggggattc 3120 cgacccaaga gactcaactt caacctcttt
aacattcaag tcaaagaggt cacgcagaat 3180 gacggtacga cgacgattgc
caataacctt accagcacgg ttcaggtgtt tactgactcg 3240 gagtaccagc
tcccgtacgt cctcggctcg gcgcatcaag gatgcctccc gccgttccca 3300
gcagacgtct tcatggtgcc acagtatgga tacctcaccc tgaacaacgg gagtcaggca
3360 gtaggacgct cttcatttta ctgcctggag tactttcctt ctcagatgct
gcgtaccgga 3420 aacaacttta ccttcagcta cacttttgag gacgttcctt
tccacagcag ctacgctcac 3480 agccagagtc tggaccgtct catgaatcct
ctcatcgacc agtacctgta ttacttgagc 3540 agaacaaaca ctccaagtgg
aaccaccacg cagtcaaggc ttcagttttc tcaggcccca 3600 gccagtgaca
ttcgggacca gtctaggaac tggcttcctg gaccctgtta ccgccagcag 3660
cgagtatgaa agacatctgc ggataacaac aacagtgaat actcgtggac tggagctacc
3720 aagtaccacc tcaatggcag agactctctg gtgaatccgg ggcccgccat
ggcaagccac 3780 aaggacgatg aagaaaagtt ttttcctcag agcggggttc
tcatctttgg gaagcaaggc 3840 tcagagaaaa caaatgtgaa cattgaaaag
gtcatgatta cagacgaaga ggaaatccca 3900 acaaccaatc ccgtggctac
ggagcagtat ggttctgtat ctaccaacct ccagagaggc 3960 aacagacaag
cagctaccgc agatgtcaac acacaaggcg ttcttccagg catggtctgg 4020
caggacagag atgtgtacct tcaggggccc atctgggcaa agattccaca cacggacgga
4080 cattttcacc cctctcccct catgggtgga ttcggactta aacaccctcc
tccacagatt 4140 ctcatcaaga acaccccggt acctgcgaat ccttcgacca
ccttcagtgc ggcaaagttt 4200 gcttccttca tcacacagta ctccacggga
cacggtcagc gtggagatcg agtgggagct 4260 gcagaacgaa aacagcaaac
gctggaatcc cgaaattcag tacacttcca actacaacaa 4320 gtctgttaat
cgtggacttt accgtggata ctaatggcgt gtattcagag cctcgcccca 4380
ttggcaccag atacctgact cgtaatctgt aattgcttgt taatcaataa accgtttaat
4440 tcgtttcagt tgaactttgg tctctgcgta tttctttctt atctagtttc
catggctacg 4500 tagataagta gcatggcggg ttaatcatta actacaagga
acccctagtg atggagttgg 4560 ccactccctc tctgcgcgct cgctcgctca
ctgaggccgg gcgaccaaag gtcgcccgac 4620 gcccgggctt tgccccggcg
gcctcagtga gcgagcgagc gcgcagagag ggagtgggca 4680 a 4681 19 4683 DNA
AAV-6 19 ttggccactc cctctctgcg cgctcgctcg ctcactgagg ccgggcgacc
aaaggtcgcc 60 cgacgcccgg gctttgcccg ggcggcctca gtgagcgagc
gagcgcgcag agagggagtg 120 gccaactcca tcactagggg ttcctggagg
ggtggagtcg tgacgtgaat tacgtcatag 180 ggttagggag gtcctgtatt
agaggtcacg tgagtgtttt gcgacatttt gcgacaccat 240 gtggtcacgc
tgggtattta agcccgagtg agcacgcagg gtctccattt tgaagcggga 300
ggtttgaacg cgcagcgcca tgccggggtt ttacgagatt gtgattaagg tccccagcga
360 ccttgacgag catctgcccg gcatttctga cagctttgtg aactgggtgg
ccgagaagga 420 atgggagttg ccgccagatt ctgacatgga tctgaatctg
attgagcagg cacccctgac 480 cgtggccgag aagctgcagc gcgacttcct
ggtccactgg cgccgcgtga gtaaggcccc 540 ggaggccctc ttctttgttc
agttcgagaa gggcgagtcc tacttccacc tccatattct 600 ggtggagacc
acgggggtca aatccatggt gctgggccgc ttcctgagtc agattagcga 660
caagctggtg cagaccatct accgcgggat cgagccgacc ctgcccaact ggttcgcggt
720 gaccaagacg cgtaatggcg ccggaggggg gaacaaggtg gtggacgagt
gctacatccc 780 caactacctc ctgcccaaga ctcagcccga gctgcagtgg
gcgtggacta acatggagga 840 gtatataagc gcgtgtttaa acctggccga
gcgcaaacgg ctcgtggcgc acgacctgac 900 ccacgtcagc cagacccagg
agcagaacaa ggagaatctg aaccccaatt ctgacgcgcc 960 tgtcatccgg
tcaaaaacct ccgcacgcta catggagctg gtcgggtggc tggtggaccg 1020
gggcatcacc tccgagaagc agtggatcca ggaggaccag gcctcgtaca tctccttcaa
1080 cgccgcctcc aactcgcggt cccagatcaa ggccgctctg gacaatgccg
gcaagatcat 1140 ggcgctgacc aaatccgcgc ccgactacct ggtaggcccc
gctccgcccg ccgacattaa 1200 aaccaaccgc atttaccgca tcctggagct
gaacggctac gaccctgcct acgccggctc 1260 cgtctttctc ggctgggccc
agaaaaggtt cggaaaacgc aacaccatct ggctgtttgg 1320 gccggccacc
acgggcaaga ccaacatcgc ggaagccatc gcccacgccg tgcccttcta 1380
cggctgcgtc aactggacca atgagaactt tcccttcaac gattgcgtcg acaagatggt
1440 gatctggtgg gaggagggca agatgacggc caaggtcgtg gagtccgcca
aggccattct 1500 cggcggcagc aaggtgcgcg tggaccaaaa gtgcaagtcg
tccgcccaga tcgatcccac 1560 ccccgtgatc gtcacctcca acaccaacat
gtgcgccgtg attgacggga acagcaccac 1620 cttcgagcac cagcagccgt
tgcaggaccg gatgttcaaa tttgaactca cccgccgtct 1680 ggagcatgac
tttggcaagg tgacaaagca ggaagtcaaa gagttcttcc gctgggcgca 1740
ggatcacgtg accgaggtgg cgcatgagtt ctacgtcaga aagggtggag ccaacaacag
1800 acccgccccc gatgacgcgg ataaaagcga gcccaagcgg gcctgcccct
cagtcgcgga 1860 tccatcgacg tcagacgcgg aaggagctcc ggtggacttt
gccgacaggt accaaaacaa 1920 atgttctcgt cacgcgggca tgcttcagat
gctgtttccc tgcaaaacat gcgagagaat 1980 gaatcagaat ttcaacattt
gcttcacgca cgggaccaga gactgttcag aatgtttccc 2040 cggcgtgtca
gaatctcaac cggtcgtcag
aaagaggacg tatcggaaac tctgtgccat 2100 tcatcatctg ctggggcggg
ctcccgagat tgcttgctcg gcctgcgatc tggtcaacgt 2160 ggatctggat
gactgtgttt ctgagcaata aatgacttaa accaggtatg gctgccgatg 2220
gttatcttcc agattggctc gaggacaacc tctctgaggg cattcggcag tggtgggact
2280 tgaaacctgg agccccgaaa cccaaagcca accagcaaaa gcaggacgac
ggccggggtc 2340 tggtgcttcc tggctacaag tacctcggac ccttcaacgg
actcgacaag ggggagcccg 2400 tcaacgcggc ggatgcagcg gccctcgagc
acgacaaggc ctacgaccag cagctcaaag 2460 cgggtgacaa tccgtacctg
cggtataacc acgccgacgc cgagtttcag gagcgtctgc 2520 aagaagatac
gtcttttggg ggcaacctcg ggcgagcagt cttccaggcc aagaagaggg 2580
ttctcgaacc ttttggtctg gttgaggaag gtgctaagac ggctcctgga aagaaacgtc
2640 cggtagagca gtcgccacaa gagccagact cctcctcggg cattggcaag
acaggccagc 2700 agcccgctaa aaagagactc aattttggtc agactggcga
ctcagagtca gtccccgacc 2760 cacaacctct cggagaacct ccagcaaccc
ccgctgctgt gggacctact acaatggctt 2820 caggcggtgg cgcaccaatg
gcagacaata acgaaggcgc cgacggagtg ggtaatgcct 2880 caggaaattg
gcattgcgat tccacatggc tgggcgacag agtcatcacc accagcaccc 2940
gaacatgggc cttgcccacc tataacaacc acctctacaa gcaaatctcc agtgcttcaa
3000 cgggggccag caacgacaac cactacttcg gctacagcac cccctggggg
tattttgatt 3060 tcaacagatt ccactgccat ttctcaccac gtgactggca
gcgactcatc aacaacaatt 3120 ggggattccg gcccaagaga ctcaacttca
agctcttcaa catccaagtc aaggaggtca 3180 cgacgaatga tggcgtcacg
accatcgcta ataaccttac cagcacggtt caagtcttgt 3240 cggactcgga
gtaccagttc ccgtacgtcc tcggctctgc gcaccagggc tgcctccctc 3300
cgttcccggc ggacgtgttc atgattccgc agtacggcta cctaacgctc aacaatggca
3360 gccaggcagt gggacgctca tccttttact gcctggaata tttcccatcg
cagatgctga 3420 gaacgggcaa taactttacc ttcagctaca ccttcgagga
cgtgcctttc cacagcagct 3480 acgcgcacag ccagagcctg gaccggctga
tgaatcctct catcgaccag tacctgtatt 3540 acctgaacag aactcacaat
cagtccggaa gtgcccaaaa caaggacttg ctgtttagcc 3600 gtgggtctcc
agctggcatg tctgttcagc ccaaaaactg gctacctgga ccctgttacc 3660
ggcagcagcg cgtttctaaa acaaaaacag acaacaacaa cagcaacttt acctggactg
3720 gtgcttcaaa atataacctt aatgggcgtg aatctataat caaccctggc
actgctatgg 3780 cctcacacaa agacgacaaa gacaagttct ttcccatgag
cggtgtcatg atttttggaa 3840 aggagagcgc cggagcttca aacactgcat
tggacaatgt catgatcaca gacgaagagg 3900 aaatcaaagc cactaacccc
gtggccaccg aaagatttgg gactgtggca gtcaatctcc 3960 agagcagcag
cacagaccct gcgaccggag atgtgcatgt tatgggagcc ttacctggaa 4020
tggtgtggca agacagagac gtatacctgc agggtcctat ttgggccaaa attcctcaca
4080 cggatggaca ctttcacccg tctcctctca tgggcggctt tggacttaag
cacccgcctc 4140 ctcagatcct catcaaaaac acgcctgttc ctgcgaatcc
tccggcagag ttttcggcta 4200 caaagtttgc ttcattcatc acccagtatt
ccacaggaca agtgagcgtg gagattgaat 4260 gggagctgca gaaagaaaac
agcaaacgct ggaatcccga agtgcagtat acatctaact 4320 atgcaaaatc
tgccaacgtt gatttcactg tggacaacaa tggactttat actgagcctc 4380
gccccattgg cacccgttac ctcacccgtc ccctgtaatt gtgtgttaat caataaaccg
4440 gttaattcgt gtcagttgaa ctttggtctc atgtccttat tatcttatct
ggtcaccata 4500 gcaaccggtt acacattaac tgcttagttg cgcttcgcga
atacccctag tgatggagtt 4560 gcccactccc tctatgcgcg ctcgctcgct
cggtggggcc ggcagagcag agctctgccg 4620 tctgcggacc tttggtccgc
aggccccacc gagcgagcga gcgcgcatag agggagtggc 4680 caa 4683 20 16 DNA
rep binding motif 20 gctcgctcgc tcgctg 16
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