U.S. patent application number 16/776850 was filed with the patent office on 2020-05-21 for adeno-associated virus (aav) clades, sequences, vectors containing same, and uses therefor.
The applicant listed for this patent is The Trustees of the University of Pennsylvania. Invention is credited to Mauricio R. Alvira, Guangping Gao, Luc H. Vandenberghe, James M. Wilson.
Application Number | 20200155704 16/776850 |
Document ID | / |
Family ID | 34426044 |
Filed Date | 2020-05-21 |
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United States Patent
Application |
20200155704 |
Kind Code |
A1 |
Wilson; James M. ; et
al. |
May 21, 2020 |
ADENO-ASSOCIATED VIRUS (AAV) CLADES, SEQUENCES, VECTORS CONTAINING
SAME, AND USES THEREFOR
Abstract
Sequences of novel adeno-associated virus capsids and vectors
and host cells containing these sequences are provided. Also
described are methods of using such host cells and vectors in
production of rAAV particles. AAV-mediated delivery of therapeutic
and immunogenic genes using the vectors of the invention is also
provided.
Inventors: |
Wilson; James M.;
(Philadelphia, PA) ; Gao; Guangping; (Westborough,
MA) ; Alvira; Mauricio R.; (Philadelphia, PA)
; Vandenberghe; Luc H.; (Weston, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
The Trustees of the University of Pennsylvania |
Philadelphia |
PA |
US |
|
|
Family ID: |
34426044 |
Appl. No.: |
16/776850 |
Filed: |
January 30, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16045043 |
Jul 25, 2018 |
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16776850 |
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15227418 |
Aug 3, 2016 |
10265417 |
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16045043 |
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13023918 |
Feb 9, 2011 |
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15227418 |
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10573600 |
Mar 24, 2006 |
7906111 |
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PCT/US2004/028817 |
Sep 30, 2004 |
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13023918 |
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60566546 |
Apr 29, 2004 |
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60508226 |
Sep 30, 2003 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 14/005 20130101;
C12N 2750/14142 20130101; A61P 31/12 20180101; A61P 35/00 20180101;
C12Y 304/21022 20130101; C12N 2830/48 20130101; A61P 43/00
20180101; C12N 2750/14122 20130101; C12N 2750/14121 20130101; C12N
2830/85 20130101; A61P 11/06 20180101; A61K 48/00 20130101; A61P
37/02 20180101; C12N 9/644 20130101; A61P 7/00 20180101; C12N 7/00
20130101; C12N 2830/008 20130101; A61P 31/20 20180101; A61P 31/04
20180101; C12N 2750/14143 20130101; C12N 2750/14151 20130101; A61P
7/04 20180101; C07K 14/705 20130101; A61P 11/00 20180101; A61P
31/10 20180101; C12N 15/86 20130101 |
International
Class: |
A61K 48/00 20060101
A61K048/00; C12N 9/64 20060101 C12N009/64; C07K 14/705 20060101
C07K014/705; C12N 7/00 20060101 C12N007/00; C12N 15/86 20060101
C12N015/86; C07K 14/005 20060101 C07K014/005 |
Claims
1. A method of generating a recombinant adeno-associated virus
(AAV) comprising culturing a host cell containing: (a) a molecule
encoding the AAV vp1 capsid protein having a sequence of amino
acids 1 to 736 of SEQ ID NO: 123, or a sequence which is at least
95% identical to the full length of amino acids 1 to 736 of SEQ ID
NO: 123; (b) a functional rep gene; (c) a nucleic acid molecule
comprising at least one AAV inverted terminal repeat (ITR) and a
non-AAV nucleic acid sequence encoding a gene product operably
linked to sequences which direct expression of the gene product in
a host cell; and (d) sufficient helper functions to permit
packaging of the minigene into the AAV capsid protein under
conditions which permit packaging of the minigene into the AAV
capsid.
2. The method of claim 1, wherein the sequence of the vp1 protein
is at least 97% identical to the full length of amino acids 1 to
736 of SEQ ID NO: 123.
3. The method of claim 1, wherein the sequence of the vp1 protein
is at least 99% identical to the full-length of amino acids 1 to
736 of SEQ ID NO: 123.
4. The method of claim 1, wherein the sequence of the vp1 protein
is the full-length of amino acids 1 to 736 of SEQ ID NO: 123.
5. The method of claim 1, wherein the gene product is a low density
lipoprotein (LDL) receptor, a high density lipoprotein (HDL)
receptor, a very low density lipoprotein (VLDL) receptor, Factor
VIII, Factor IX, erythropoietin, ornithine transcarbamylase (OTC),
vascular endothelial growth factor (VEGF), glucose-6-phosphatase,
alpha-1 antitrypsin, or an antibody.
6. The method of claim 1, wherein the molecule is a nucleic acid
molecule comprising nucleotides 1 to 2208 of SEQ ID NO: 3, or a
nucleotide sequence at least 99% identical to nucleotides 1 to 2208
of SEQ ID NO: 3.
7. The method of claim 1, wherein the rep gene is from AAV2.
8. A method of generating a recombinant adeno-associated virus
(AAV) comprising culturing a host cell containing: (a) a molecule
encoding the AAV vp2 capsid protein having a sequence of amino
acids 138 to 736 of SEQ ID NO: 123, or a sequence which is at least
95% identical to the full length of amino acids 138 to 736 of SEQ
ID NO: 123; (b) a functional rep gene; (c) a nucleic acid molecule
comprising at least one AAV inverted terminal repeat (ITR) and a
non-AAV nucleic acid sequence encoding a gene product operably
linked to sequences which direct expression of the gene product in
a host cell; and (d) sufficient helper functions to permit
packaging of the minigene into the AAV capsid protein under
conditions which permit packaging of the minigene into the AAV
capsid.
9. The method of claim 8, wherein the sequence of the vp2 protein
is at least 97% identical to the full length of amino acids 138 to
736 of SEQ ID NO: 123.
10. The method of claim 8, wherein the sequence of the vp2 protein
is at least 99% identical to the full-length of amino acids 138 to
736 of SEQ ID NO: 123.
11. The method of claim 8, wherein the sequence of the vp2 protein
is the full-length of amino acids 138 to 736 of SEQ ID NO: 123.
12. The method of claim 8, wherein the gene product is a low
density lipoprotein (LDL) receptor, a high density lipoprotein
(HDL) receptor, a very low density lipoprotein (VLDL) receptor,
Factor VIII, Factor IX, erythropoietin, ornithine transcarbamylase
(OTC), vascular endothelial growth factor (VEGF),
glucose-6-phosphatase, alpha-1 antitrypsin, or an antibody.
13. The method of claim 8, wherein the molecule is a nucleic acid
molecule comprising nucleotides 412 to 2208 of SEQ ID NO: 3, or a
nucleotide sequence at least 99% identical to nucleotides 412 to
2208 of SEQ ID NO: 3.
14. The method of claim 8, wherein the rep gene is from AAV2.
15. A method of generating a recombinant adeno-associated virus
(AAV) comprising culturing a host cell containing: (a) a molecule
encoding the AAV vp3 capsid protein having a sequence of amino
acids 203 to 736 of SEQ ID NO: 123, or a sequence which is at least
95% identical to the full length of amino acids 203 to 736 of SEQ
ID NO: 123; (b) a functional rep gene; (c) a nucleic acid molecule
comprising at least one AAV inverted terminal repeat (ITR) and a
non-AAV nucleic acid sequence encoding a gene product operably
linked to sequences which direct expression of the gene product in
a host cell; and (d) sufficient helper functions to permit
packaging of the minigene into the AAV capsid protein under
conditions which permit packaging of the minigene into the AAV
capsid.
16. The method of claim 15, wherein the sequence of the vp3 protein
is at least 97% identical to the full length of amino acids 203 to
736 of SEQ ID NO: 123.
17. The method of claim 15, wherein the sequence of the vp3 protein
is at least 99% identical to the full-length of amino acids 203 to
736 of SEQ ID NO: 123.
18. The method of claim 15, wherein the sequence of the vp3 protein
is the full-length of amino acids 203 to 736 of SEQ ID NO: 123.
19. The method of claim 15, wherein the gene product is a low
density lipoprotein (LDL) receptor, a high density lipoprotein
(HDL) receptor, a very low density lipoprotein (VLDL) receptor,
Factor VIII, Factor IX, erythropoietin, ornithine transcarbamylase
(OTC), vascular endothelial growth factor (VEGF),
glucose-6-phosphatase, alpha-1 antitrypsin, or an antibody
20. The method of claim 15, wherein the molecule is a nucleic acid
molecule comprising nucleotides 607 to 2208 of SEQ ID NO: 3, or a
nucleotide sequence at least 99% identical to nucleotides 607 to
2208 of SEQ ID NO: 3.
21. The method of claim 15, wherein the rep gene is from AAV2.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 16/045,043, filed Jul. 25, 2018, which is
continuation of U.S. patent application Ser. No. 15/227,418, filed
Aug. 3, 2016, now U.S. Pat. No. 10,265,417, issued Apr. 23, 2019,
which is a continuation of U.S. patent application Ser. No.
13/023,918, filed Feb. 9, 2011, which is a continuation of U.S.
patent application Ser. No. 10/573,600, filed Mar. 24, 2006, now
U.S. Pat. No. 7,906,111, issued Mar. 15, 2011, which is a national
stage application under 35 USC .sctn. 371 of PCT/US04/028817, filed
Sep. 30, 2004, which claims the benefit under 35 USC .sctn. 119(e)
of the priority of U.S. Patent Application No. 60/508,226, filed
Sep. 30, 2003, now expired, and U.S. Patent Application No.
60/566,546, filed Apr. 29, 2004, now expired. Each of these
applications is hereby incorporated by reference in their
entirety.
BACKGROUND OF THE INVENTION
[0002] Adeno-associated virus (AAV), a member of the Parvovirus
family, is a small nonenveloped, icosahedral virus with
single-stranded linear DNA genomes of 4.7 kilobases (kb) to 6 kb.
AAV is assigned to the genus, Dependovirus, because the virus was
discovered as a contaminant in purified adenovirus stocks. AAV's
life cycle includes a latent phase at which AAV genomes, after
infection, are site specifically integrated into host chromosomes
and an infectious phase in which, following either adenovirus or
herpes simplex virus infection, the integrated genomes are
subsequently rescued, replicated, and packaged into infectious
viruses. The properties of non-pathogenicity, broad host range of
infectivity, including non-dividing cells, and potential
site-specific chromosomal integration make AAV an attractive tool
for gene transfer.
[0003] Recent studies suggest that AAV vectors may be the preferred
vehicle for gene delivery. To date, there have been several
different well-characterized AAVs isolated from human or non-human
primates (NHP).
[0004] It has been found that AAVs of different serotypes exhibit
different transfection efficiencies, and also exhibit tropism for
different cells or tissues. However, the relationship between these
different serotypes has not previously been explored.
[0005] What is desirable are AAV-based constructs for delivery of
heterologous molecules.
SUMMARY OF THE INVENTION
[0006] The present invention provides "superfamilies" or "clades"
of AAV of phylogenetically related sequences. These AAV clades
provide a source of AAV sequences useful for targeting and/or
delivering molecules to desired target cells or tissues.
[0007] In one aspect, the invention provides an AAV clade having at
least three AAV members which are phylogenetically related as
determined using a Neighbor-Joining heuristic by a bootstrap value
of at least 75% (based on at least 1000 replicates) and a Poisson
correction distance measurement of no more than 0.05, based on
alignment of the AAV vp1 amino acid sequence. Suitably, the AAV
clade is composed of AAV sequences useful in generating
vectors.
[0008] The present invention further provides a human AAV serotype
previously unknown, designated herein as clone 28.4/hu.14, or
alternatively, AAV serotype 9. Thus, in another aspect, the
invention provides an AAV of serotype 9 composed of AAV capsid
which is serologically related to a capsid of the sequence of amino
acids 1 to 736 of SEQ ID NO: 123 and serologically distinct from a
capsid protein of any of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7
or AAV8.
[0009] Vectors constructed with capsid of this huAAV9 have
exhibited gene transfer efficacies similar to AAV8 in liver,
superior to AAV1 in muscle and 200 fold higher than AAV 5 in lung.
Further, this novel human AAV serotype shares less than 85%
sequence identity to previously described AAV1 through AAV8 and is
not cross-neutralized by any of these AAVs.
[0010] The present invention also provides other novel AAV
sequences, compositions containing these sequences, and uses
therefor. Advantageously, these compositions are particularly well
suited for use in compositions requiring re-administration of AAV
vectors for therapeutic or prophylactic purposes.
[0011] These and other aspects of the invention will be readily
apparent from the following detailed description of the
invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0012] FIG. 1 is a tree showing the phylogenic relationship
constructed using the Neighbor-Joining heuristic with Poisson
correction distance measurement. The relationship was determined
based on the isolated AAV vp1 capsid protein, with the isolated AAV
grouped in clades. Groups of individual capsid clones are
classified in clades based on their common ancestry. Clade
nomenclature goes from A through F; subtypes are represented by the
clade letter followed by a number.
[0013] FIGS. 2A-2AE are an alignment of the amino acid sequences of
AAV vp1 capsid proteins of the invention, with the numbering of the
individual sequences reported, and previously published AAV1 [SEQ
ID NO: 219]; AAV2 [SEQ ID NO: 221]; AAV3-3 [SEQ ID NO: 217]; AAV4-4
[SEQ ID NO: 218]; AAV5 [SEQ ID NO: 216]; AAV6 [SEQ ID NO: 220];
AAV7 [SEQ ID NO: 222]; AAV8 [SEQ ID NO: 223], and; rh. 25/42-15;
29.3/bb.1; cy.2; 29.5/bb.2; rh.32, rh.33, rh.34, rh.10; rh.24;
rh14, rh.16, rh.17, rh.12, rh.18, rh.21 (formerly termed 41.10);
rh.25 (formerly termed 41.15); rh2; rh.31; cy.3; cy.5; rh.13; cy.4;
cy.6; rh.22; rh.19; rh.35; rh.37; rh.36; rh.23; rh.8; and ch.5
Published Patent Application No. 2003/0138772 A1 (Jul. 24, 2003)].
The sequences of the invention include hu.14/AAV9 [SEQ ID NO:123];
hu.17 [SEQ ID NO: 83], hu. 6 [SEQ ID NO: 84], hu.42 [SEQ ID NO:
85], rh.38 [SEQ ID NO: 86], hu.40 [SEQ ID NO: 87], hu.37 [SEQ ID
NO: 88], rh.40 [SEQ ID NO: 92], rh.52 [SEQ ID NO: 96]; rh.53 [SEQ
ID NO: 97]; rh.49 [SEQ ID NO: 103]; rh.51 [SEQ ID NO: 104]; rh.57
[SEQ ID NO: 105]; rh.58 [SEQ ID NO: 106], rh.61 [SEQ ID NO: 107];
rh.50 [SEQ ID NO: 108]; rh.43 [SEQ ID NO: 163]; rh.62 [SEQ ID NO:
114]; rh.48 [SEQ ID NO: 115]; 4-9/rh.54 (SEQ ID No: 116); and
4-19/rh.55 (SEQ ID Nos: 117); hu.31 [SEQ ID NO:121]; hu.32 [SEQ ID
NO:122]; hu.34 [SEQ ID NO: 125]; hu.45 [SEQ ID NO: 127]; hu.47 [SEQ
ID NO: 128]; hu.13 [SEQ ID NO:129]; hu.28 [SEQ ID NO:130]; hu.29
[SEQ ID NO:132]; hu.19 [SEQ ID NO: 133]; hu.20 [SEQ ID NO: 134];
hu.21 [SEQ ID NO:135]; hu.23.2 [SEQ ID NO:137]; hu.22 [SEQ ID NO:
138]; hu.27 [SEQ ID NO: 140]; hu.4 [SEQ ID NO: 141]; hu.2 [SEQ ID
NO: 143]; hu.1 [SEQ ID NO: 144]; hu.3 [SEQ ID NO: 145]; hu.25 [SEQ
ID NO: 146]; hu.15 [SEQ ID NO: 147]; hu.16 [SEQ ID NO: 148]; hu.18
[SEQ ID NO: 149]; hu.7 [SEQ ID NO: 150]; hu.11 [SEQ ID NO: 153];
hu.9 [SEQ ID NO: 155]; hu.10 [SEQ ID NO: 156]; hu.48 [SEQ ID NO:
157]; hu.44 [SEQ ID NO: 158]; hu.46 [SEQ ID NO: 159]; hu.43 [SEQ ID
NO: 160]; hu.35 [SEQ ID NO: 164]; hu.24 [SEQ ID NO: 136]; rh.64
[SEQ ID NO: 99]; hu.41 [SEQ ID NO: 91]; hu.39 [SEQ ID NO: 102];
hu.67 [SEQ ID NO: 198]; hu.66 [SEQ ID NO: 197]; hu.51 [SEQ ID NO:
190]; hu.52 [SEQ ID NO: 191]; hu.49 [SEQ ID NO: 189]; hu.56 [SEQ ID
NO: 192]; hu.57 [SEQ ID NO: 193]; hu.58 [SEQ ID NO: 194]; hu.63
[SEQ ID NO: 195]; hu.64 [SEQ ID NO: 196]; hu.60 [SEQ ID NO: 184];
hu.61 [SEQ ID NO: 185]; hu.53 [SEQ ID NO: 186]; hu.55 [SEQ ID NO:
187]; hu.54 [SEQ ID NO: 188]; hu.6 [SEQ ID NO: 84]; and rh.56 [SEQ
ID NO: 152]. These capsid sequences are also reproduced in the
Sequence Listing, which is incorporated by reference herein.
[0014] FIGS. 3A-3CN are an alignment of the nucleic acid sequences
of AAV vp1 capsid proteins of the invention, with the numbering of
the individual sequences reported, and previously published AAV5
(SEQ ID NO: 199); AAV3-3 (SEQ ID NO: 200); AAV4-4 (SEQ ID NO: 201);
AAV1 (SEQ ID NO: 202); AAV6 (SEQ ID NO: 203); AAV2(SEQ ID NO: 211);
AAV7 (SEQ ID NO: 213) and AAV8 (SEQ ID NO: 214); rh. 25/42-15;
29.3/bb.1; cy.2; 29.5/bb.2; rh.32, rh.33, rh.34, rh.10; rh.24;
rh14, rh.16, rh.17, rh.12, rh.18, rh.21 (formerly termed 41.10);
rh.25 (formerly termed 41.15; GenBank accession AY530557); rh2;
rh.31; cy.3; cy.5; rh.13; cy.4; cy.6; rh.22; rh.19; rh.35; rh.37;
rh.36; rh.23; rh.8; and ch.5 [US Published Patent Application No.
2003/0138772 A1 (Jul. 24, 2003)]. The nucleic acid sequences of the
invention include, hu.14/AAV9 (SEQ ID No: 3); LG-4/rh.38 (SEQ ID
No: 7); LG-10/rh.40 (SEQ ID No: 14); N721-8/rh.43 (SEQ ID No: 43);
1-8/rh.49 (SEQ ID NO: 25); 2-4/rh.50 (SEQ ID No: 23); 2-5/rh.51
(SEQ ID No: 22); 3-9/rh.52 (SEQ ID No: 18); 3-11/rh.53 (SEQ ID NO:
17); 5-3/rh.57 (SEQ ID No: 26); 5-22/rh.58 (SEQ ID No: 27);
2-3/rh.61 (SEQ ID NO: 21); 4-8/rh.64 (SEQ ID No: 15); 3.1/hu.6 (SEQ
ID NO: 5); 33.12/hu.17 (SEQ ID NO:4); 106.1/hu.37 (SEQ ID No: 10);
LG-9/hu.39 (SEQ ID No: 24); 114.3/hu.40 (SEQ ID No: 11);
127.2/hu.41 (SEQ ID NO:6); 127.5/hu.42 (SEQ ID No: 8); and hu.66
(SEQ ID NO: 173); 2-15/rh.62 (SEQ ID NO: 33); 1-7/rh.48 (SEQ ID NO:
32); 4-9/rh.54 (SEQ ID No: 40); 4-19/rh.55 (SEQ ID NO: 37);
52/hu.19 (SEQ ID NO: 62), 52.1/hu.20 (SEQ ID NO: 63), 54.5/hu.23
(SEQ ID No: 60), 54.2/hu.22 (SEQ ID No: 67), 54.7/hu.24 (SEQ ID No:
66), 54.1/hu.21 (SEQ ID No: 65), 54.4R/hu.27 (SEQ ID No: 64);
46.2/hu.28 (SEQ ID No: 68); 46.6/hu.29 (SEQ ID No: 69); 128.1/hu.43
(SEQ ID No: 80); 128.3/hu.44 (SEQ ID No: 81) and 130.4/hu.48 (SEQ
ID NO: 78); 3.1/hu.9 (SEQ ID No: 58); 16.8/hu.10 (SEQ ID No: 56);
16.12/hu.11 (SEQ ID No: 57); 145.1/hu.53 (SEQ ID No: 176);
145.6/hu.55 (SEQ ID No: 178); 145.5/hu.54 (SEQ ID No: 177);
7.3/hu.7 (SEQ ID No: 55); 52/hu.19 (SEQ ID No: 62); 33.4/hu.15 (SEQ
ID No: 50); 33.8/hu.16 (SEQ ID No: 51); 58.2/hu.25 (SEQ ID No: 49);
161.10/hu.60 (SEQ ID No: 170); H-5/hu.3 (SEQ ID No: 44); H-1/hu.1
(SEQ ID No: 46); 161.6/hu.61 (SEQ ID No: 174); hu.31 (SEQ ID No:
1); hu.32 (SEQ ID No: 2); hu.46 (SEQ ID NO: 82); hu.34 (SEQ ID NO:
72); hu.47 (SEQ ID NO: 77); hu.63 (SEQ ID NO: 204); hu.56 (SEQ ID
NO: 205); hu.45 (SEQ ID NO: 76); hu.57 (SEQ ID NO: 206); hu.35 (SEQ
ID NO: 73); hu.58 (SEQ ID NO: 207); hu.51(SEQ ID NO: 208); hu.49
(SEQ ID NO: 209); hu.52 (SEQ ID NO: 210); hu.13 (SEQ ID NO: 71);
hu.64 (SEQ ID NO: 212); rh.56 (SEQ ID NO: 54); hu.2 (SEQ ID NO:
48); hu.18 (SEQ ID NO: 52); hu.4 (SEQ ID NO: 47); and hu.67 (SEQ ID
NO: 215). These sequences are also reproduced in the Sequence
Listing, which is incorporated by reference herein.
[0015] FIGS. 4A-4D provide an evaluation of gene transfer
efficiency of novel primate AAV-based vectors in vitro and in vivo.
AAV vectors were pseudotyped as described [Gao et al, Proc Natl
Acad Sci USA, 99:11854-11859 (Sep. 3, 2002)] with capsids of AAVs
1, 2, 5, 7, 8 and 6 and ch.5, rh.34, cy.5, rh.20, rh.8 and AAV9.
For in vitro study, FIG. 4A, 84-32 cells (293 cells expressing E4
of adenovirus serotypes) seeded in a 95 well plate were infected
with pseudotyped AAVCMVEGFP vectors at an MOI of 1.times.10.sup.4GC
per cell. Relative EGFP transduction efficiency was estimated as
percentage of green cells using a UV microscope at 48 hours
post-infection and shown on the Y axis. For in vivo study, the
vectors expressing the secreted reporter gene A 1AT were
administered to the liver (FIG. 4B), lung (FIG. 4C) and muscle
(FIG. 4D) of NCR nude mice (4-6 weeks old) at a dose of
1.times.10.sup.11 GC per animal by intraportal (FIG. 4B),
intratracheal (FIG. 4C) and intramuscular injections (FIG. 4D),
respectively. Serum A1AT levels (ng/mL) were compared at day 28
post gene transfer and presented on the Y axis. The X axis
indicates the AAVs analyzed and the clades to which they
belong.
DETAILED DESCRIPTION OF THE INVENTION
[0016] In any arsenal of vectors useful in therapy or prophylaxis,
a variety of distinct vectors capable of carrying a macromolecule
to a target cell is desirable, in order to permit selection of a
vector source for a desired application. To date, one of the
concerns regarding the use of AAV as vectors was the lack of a
variety of different virus sources. One way in which the present
invention overcomes this problem is by providing clades of AAV,
which are useful for selecting phylogenetically related, or where
desired for a selected regimen, phylogenetically distinct, AAV and
for predicting function. The invention further provides novel AAV
viruses.
[0017] 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
to 99% of the aligned sequences. Preferably, the homology is over
full-length sequence, or an open reading frame thereof, or another
suitable fragment which is at least 15 nucleotides in length.
Examples of suitable fragments are described herein.
[0018] The terms "sequence identity" "percent sequence identity" or
"percent 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 of the genome, the
full-length of a gene coding sequence, or a fragment of at least
about 500 to 5000 nucleotides, is desired. However, identity among
smaller fragments, e.g. of at least about nine nucleotides, usually
at least about 20 to 24 nucleotides, at least about 28 to 32
nucleotides, at least about 36 or more nucleotides, may also be
desired. Similarly, "percent sequence identity" may be readily
determined for amino acid sequences, over the full-length of a
protein, or a fragment thereof. Suitably, a fragment is at least
about 8 amino acids in length, and may be up to about 700 amino
acids. Examples of suitable fragments are described herein.
[0019] The term "substantial homology" or "substantial similarity,"
when referring to amino acids or fragments thereof, indicates that,
when optimally aligned with appropriate amino acid insertions or
deletions with another amino acid (or its complementary strand),
there is amino acid sequence identity in at least about 95 to 99%
of the aligned sequences. Preferably, the homology is over
full-length sequence, or a protein thereof, e.g., a cap protein, a
rep protein, or a fragment thereof which is at least 8 amino acids,
or more desirably, at least 15 amino acids in length. Examples of
suitable fragments are described herein.
[0020] By the term "highly conserved" is meant at least 80%
identity, preferably at least 90% identity, and more preferably,
over 97% identity. Identity is readily determined by one of skill
in the art by resort to algorithms and computer programs known by
those of skill in the art.
[0021] Generally, when referring to "identity", "homology", or
"similarity" between two different adeno-associated viruses,
"identity", "homology" or "similarity" is determined in reference
to "aligned" sequences. "Aligned" sequences or "alignments" refer
to multiple nucleic acid sequences or protein (amino acids)
sequences, often containing corrections for missing or additional
bases or amino acids as compared to a reference sequence. In the
examples, AAV alignments are performed using the published AAV2 or
AAV1 sequences as a reference point. However, one of skill in the
art can readily select another AAV sequence as a reference.
[0022] Alignments are performed using any of a variety of publicly
or commercially available Multiple Sequence Alignment Programs.
Examples of such programs include, "Clustal W", "CAP Sequence
Assembly", "MAP", and "MEME", which are accessible through Web
Servers on the internet. Other sources for such programs are known
to those of skill in the art. Alternatively, Vector NTI utilities
are also used. There are also a number of algorithms known in the
art that can be used to measure nucleotide sequence identity,
including those contained in the programs described above. As
another example, polynucleotide sequences can be compared using
Fasta.TM., a program in GCG Version 6.1. Fasta.TM. provides
alignments and percent sequence identity of the regions of the best
overlap between the query and search sequences. For instance,
percent sequence identity between nucleic acid sequences can be
determined using Fasta.TM. 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. Multiple
sequence alignment programs are also available for amino acid
sequences, e.g., the "Clustal X", "MAP", "PIMA", "MSA",
"BLOCKMAKER", "MEME", and "Match-Box" programs. Generally, any of
these programs are used at default settings, although one of skill
in the art can alter these settings as needed. Alternatively, one
of skill in the art can utilize another algorithm or computer
program which provides at least the level of identity or alignment
as that provided by the referenced algorithms and programs. See,
e.g., J. D. Thomson et al, Nucl. Acids. Res., "A comprehensive
comparison of multiple sequence alignments", 27(13):2682-2690
(1999).
[0023] The term "serotype" is a distinction with respect to an AAV
having a capsid which is serologically distinct from other AAV
serotypes. Serologic distinctiveness is determined on the basis of
the lack of cross-reactivity between antibodies to the AAV as
compared to other AAV.
[0024] Cross-reactivity is typically measured in a neutralizing
antibody assay. For this assay polyclonal serum is generated
against a specific AAV in a rabbit or other suitable animal model
using the adeno-associated viruses. In this assay, the serum
generated against a specific AAV is then tested in its ability to
neutralize either the same (homologous) or a heterologous AAV. The
dilution that achieves 50% neutralization is considered the
neutralizing antibody titer. If for two AAVs the quotient of the
heterologous titer divided by the homologous titer is lower than 16
in a reciprocal manner, those two vectors are considered as the
same serotype. Conversely, if the ratio of the heterologous titer
over the homologous titer is 16 or more in a reciprocal manner the
two AAVs are considered distinct serotypes.
[0025] As defined herein, to form serotype 9, antibodies generated
to a selected AAV capsid must not be cross-reactive with any of AAV
1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7 or AAV8. In one embodiment,
the present invention provides an AAV capsid of a novel serotype,
identified herein, as human AAV serotype 9.
[0026] As used throughout this specification and the claims, the
terms "comprising" and "including" are inclusive of other
components, elements, integers, steps and the like. Conversely, the
term "consisting" and its variants are exclusive of other
components, elements, integers, steps and the like.
I. Clades
[0027] In one aspect, the invention provides clades of AAV. A clade
is a group of AAV which are phylogenetically related to one another
as determined using a Neighbor-Joining algorithm by a bootstrap
value of at least 75% (of at least 1000 replicates) and a Poisson
correction distance measurement of no more than 0.05, based on
alignment of the AAV vp1 amino acid sequence.
[0028] The Neighbor-Joining algorithm has been described
extensively in the literature. See, e.g., M. Nei and S. Kumar,
Molecular Evolution and Phylogenetics (Oxford University Press, New
York (2000). Computer programs are available that can be used to
implement this algorithm. For example, the MEGA v2.1 program
implements the modified Nei-Gojobori method. Using these techniques
and computer programs, and the sequence of an AAV vp1 capsid
protein, one of skill in the art can readily determine whether a
selected AAV is contained in one of the clades identified herein,
in another clade, or is outside these clades.
[0029] While the clades defined herein are based primarily upon
naturally occurring AAV vp1 capsids, the clades are not limited to
naturally occurring AAV. The clades can encompass non-naturally
occurring AAV, including, without limitation, recombinant, modified
or altered, chimeric, hybrid, synthetic, artificial, etc., AAV
which are phylogenetic ally related as determined using a
Neighbor-Joining algorithm at least 75% (of at least 1000
replicates) and a Poisson correction distance measurement of no
more than 0.05, based on alignment of the AAV vp1 amino acid
sequence.
[0030] The clades described herein include Clade A (represented by
AAV1 and AAV6), Clade B (represented by AAV2) and Clade C
(represented by the AAV2-AAV3 hybrid), Clade D (represented by
AAV7), Clade E (represented by AAV8), and Clade F (represented by
human AAV9). These clades are represented by a member of the clade
that is a previously described AAV serotype. Previously described
AAV1 and AAV6 are members of a single clade (Clade A) in which 4
isolates were recovered from 3 humans. Previously described AAV3
and AAV5 serotypes are clearly distinct from one another, but were
not detected in the screen described herein, and have not been
included in any of these clades.
[0031] Clade B (AAV2) and Clade C (the AAV2-AAV3 hybrid) are the
most abundant of those found in humans (22 isolates from 12
individuals for AAV2 and 17 isolates from 8 individuals for Clade
C).
[0032] There are a large number of sequences grouped in either
Clade D (AAV7) or Clade E (AAV8). Interestingly, both of these
clades are prevalent in different species. Clade D is unique to
rhesus and cynomologus macaques with 15 members being isolated from
10 different animals. Clade E is interesting because it is found in
both human and nonhuman primates: 9 isolates were recovered from 7
humans and 21 isolates were obtained in 9 different nonhuman
primates including rhesus macaques, a baboon and a pigtail
monkey.
[0033] In two other animals the hybrid nature of certain sequences
was proven, although all sequences in this case seem to have
originated through individual and different recombinations of two
co-infecting viruses (in both animals a Clade D with a Clade E
virus). None of these recombinants were identified in other animals
or subjects.
[0034] Since Clade C (the AAV2-AAV3 hybrid) clade was identified in
6 different human subjects, the recombination event resulted in a
fit progeny. In the case of the AAV7-AAV8 hybrids on the other
hand, only few conclusions can be drawn as to the implication of
recombination in AAV evolution. These recombination events show
that AAV is capable of recombining, thereby creating in-frame genes
and in some cases packagable and/or infectious capsid structures.
Clade C (the AAV2-AAV3 hybrid clade) on the other hand is a group
of viruses that has acquired a selective advantage through
recombination that made them sustain certain environmental
pressures.
[0035] A. Clade A (represented by AAV1 and AAV6):
[0036] In another aspect, the invention provides Clade A, which is
characterized by containing the previously published AAV1 and AAV6.
See, e.g., International Publication No. WO 00/28061, 18 May 2000;
Rutledge et al, J Virol, 72(1):309-319 (January 1998). In addition,
this clade contains novel AAV including, without limitation,
128.1/hu. 43 [SEQ ID NOs: 80 and 160]; 128.3/hu. 44 [SEQ ID Nos: 81
and 158]; 130.4/hu.48 [SEQ ID NO: 78 and 157]; and hu.46 [SEQ ID
NOs: 82 and 159]. The invention further provides a modified hu. 43
capsid [SEQ ID NO:236] and a modified hu. 46 capsid [SEQ ID
NO:224].
[0037] In one embodiment, one or more of the members of this clade
has a capsid with an amino acid identity of at least 85% identity,
at least 90% identity, at least 95% identity, or at least 97%
identity over the full-length of the vp1, the vp2, or the vp3 of
the AAV1 and/or AAV6 capsid.
[0038] In another embodiment, the invention provides novel AAV of
Clade A, provided that none of the novel AAV comprises a capsid of
any of AAV1 or AAV6. These AAV may include, without limitation, an
AAV having a capsid derived from one or more of 128.1/hu. 43 [SEQ
ID Nos: 80 and 160]; modified hu.43 [SEQ ID NO:236] 128.3/hu. 44
[SEQ ID Nos: 81 and 158]; hu.46 [SEQ ID NOs: 82 and 159]; modified
hu. 46 [SEQ ID NO:224]; and 130.4/hu.48 [SEQ ID NO: 78 and
157].
[0039] B. Clade B (AAV2 Clade):
[0040] In another embodiment, the invention provides a Clade B.
[0041] This clade is characterized by containing, at a minimum, the
previously described AAV2 and novel AAV of the invention including,
52/hu.19 [SEQ ID NOs: 62 and 133], 52.1/hu.20 [SEQ ID NOs: 63 and
134], 54.5/hu.23 [SEQ ID Nos: 60 and 137], 54.2/hu.22 [SEQ ID Nos:
67 and 138], 54.7/hu.24 [SEQ ID Nos: 66 and 136], 54.1/hu.21 [SEQ
ID Nos: 65 and 135], 54.4R/hu.27 [SEQ ID Nos: 64 and 140];
46.2/hu.28 [SEQ ID Nos: 68 and 130]; 46.6/hu.29 [SEQ ID Nos: 69 and
132]; modified hu. 29 [SEQ ID NO: 225]; 172.1/hu.63 [SEQ ID NO: 171
and 195; GenBank Accession No. AY530624]; 172.2/hu. 64 [SEQ ID NO:
172 and 196; GenBank Accession No. AY530625]; 24.5/hu.13 [SEQ ID
NO: 71 and 129; GenBank Accession No. AY530578]; 145.6/hu.56 [SEQ
ID NO: 168 and 192]; hu.57 [SEQ ID Nos: 169 and 193]; 136.1/hu.49
[SEQ ID NO: 165 and 189]; 156.1/hu.58 [SEQ ID NO: 179 and 194];
72.2/hu.34 [SEQ ID NO: 72 and 125; GenBank Accession No. AY530598];
72.3/hu.35 [SEQ ID NO: 73 and 164; GenBank Accession No. AY530599];
130.1/hu.47 [SEQ ID NO: 77 and 128]; 129.1/hu.45 (SEQ ID NO: 76 and
127; GenBank Accession No. AY530608); 140.1/hu.51 [SEQ ID NO: 161
and 190; GenBank Accession No. AY530613]; and 140.2/hu.52 [SEQ ID
NO: 167 and 191; GenBank Accession No. AY530614].
[0042] In one embodiment, one or more of the members of this clade
has a capsid with an amino acid identity of at least 85% identity,
at least 90% identity, at least 95% identity, or at least 97%
identity over the full-length of the vp1, the vp2, or the vp3 of
the AAV2 capsid.
[0043] In another embodiment, the invention provides novel AAV of
Clade B, provided that none of the AAV has an AAV2 capsid. These
AAV may include, without limitation, an AAV having a capsid derived
from one or more of the following: 52/hu.19 [SEQ ID NOs: 62 and
133], 52.1/hu.20 [SEQ ID NOs: 63 and 134], 54.5/hu.23 [SEQ ID Nos:
60 and 137], 54.2/hu.22 [SEQ ID Nos: 67 and 138], 54.7/hu.24 [SEQ
ID Nos: 66 and 136], 54.1/hu.21 [SEQ ID Nos: 65 and 135],
54.4R/hu.27 [SEQ ID Nos: 64 and 140]; 46.2/hu.28 [SEQ ID Nos: 68
and 130]; 46.6/hu.29 [SEQ ID Nos: 69 and 132]; modified hu. 29 [SEQ
ID NO: 225]; 172.1/hu.63 [SEQ ID NO: 171 and 195]; 172.2/hu. 64
[SEQ ID NO: 172 and 196]; 24.5/hu.13 [SEQ ID NO: 71 and 129];
145.6/hu.56 [SEQ ID NO: 168 and 192; GenBank Accession No.
AY530618]; hu.57 [SEQ ID Nos: 169 and 193; GenBank Accession No.
AY530619]; 136.1/hu.49 [SEQ ID NO: 165 and 189; GenBank Accession
No. AY530612]; 156.1/hu.58 [SEQ ID NO: 179 and 194; GenBank
Accession No. AY530620]; 72.2/hu.34 [SEQ ID NO: 72 and 125];
72.3/hu.35 [SEQ ID NO: 73 and 164]; 129.1/hu.45 [SEQ ID NO: 76 and
127]; 130.1/hu.47 [SEQ ID NO:77 and 128; GenBank Accession No.
AY530610]; 140.1/hu.51 [SEQ ID NO: 161 and 190; GenBank Accession
No. AY530613]; and 140.2/hu.52 [SEQ ID NO: 167 and 191; GenBank
Accession No. AY530614].
[0044] C. Clade C (AAV2-AAV3 Hybrid Clade)
[0045] In another aspect, the invention provides Clade C, which is
characterized by containing AAV that are hybrids of the previously
published AAV2 and AAV3 such as H-6/hu.4; H-2/hu.2 [US Patent
Application 2003/0138772 (Jun. 24, 2003). In addition, this clade
contains novel AAV including, without limitation, 3.1/hu.9 [SEQ ID
Nos: 58 and 155]; 16.8/hu.10 [SEQ ID Nos: 56 and 156]; 16.12/hu.11
[SEQ ID Nos: 57 and 153]; 145.1/hu.53 [SEQ ID Nos: 176 and 186];
145.6/hu.55 [SEQ ID Nos: 178 and 187]; 145.5/hu.54 [SEQ ID Nos: 177
and 188]; 7.3/hu.7 [SEQ ID Nos: 55 and 150; now deposited as
GenBank Accession No. AY530628]; modified hu. 7 [SEQ ID NO: 226];
33.4/hu.15 [SEQ ID Nos: 50 and 147]; 33.8/hu.16 [SEQ ID Nos: 51 and
148]; hu.18 [SEQ ID NOs: 52 and 149]; 58.2/hu.25 [SEQ ID Nos: 49
and 146]; 161.10/hu.60 [SEQ ID Nos: 170 and 184]; H-5/hu.3 [SEQ ID
Nos: 44 and 145]; H-1/hu.1 [SEQ ID Nos: 46 and 144]; and
161.6/hu.61 [SEQ ID Nos: 174 and 185].
[0046] In one embodiment, one or more of the members of this clade
has a capsid with an amino acid identity of at least 85% identity,
at least 90% identity, at least 95% identity, or at least 97%
identity over the full-length of the vp1, the vp2, or the vp3 of
the hu.4 and/or hu.2 capsid.
[0047] In another embodiment, the invention provides novel AAV of
Clade C (the AAV2-AAV3 hybrid clade), provided that none of the
novel AAV comprises a capsid of hu.2 or hu.4. These AAV may
include, without limitation, an AAV having a capsid derived from
one or more of 3.1/hu.9 [SEQ ID Nos: 58 and 155]; 16.8/hu.10 [SEQ
ID Nos: 56 and 156]; 16.12/hu.11 [SEQ ID Nos: 57 and 153];
145.1/hu.53 [SEQ ID Nos: 176 and 186]; 145.6/hu.55 [SEQ ID Nos: 178
and 187]; 145.5/hu.54 [SEQ ID Nos: 177 and 188]; 7.3/hu.7 [SEQ ID
Nos: 55 and 150]; modified hu.7 [SEQ ID NO:226]; 33.4/hu.15 [SEQ ID
Nos: 50 and 147]; 33.8/hu.16 [SEQ ID Nos: 51 and 148]; 58.2/hu.25
[SEQ ID Nos: 49 and 146]; 161.10/hu.60 [SEQ ID Nos: 170 and 184];
H-5/hu.3 [SEQ ID Nos: 44 and 145]; H-1/hu.1 [SEQ ID Nos: 46 and
144]; and 161.6/hu.61 [SEQ ID Nos: 174 and 185].
[0048] D. Clade D (AAV7 Clade)
[0049] In another embodiment, the invention provides Clade D. This
clade is characterized by containing the previously described AAV7
[G. Gao et al, Proc. Natl Acad. Sci USA, 99:11854-9 (Sep. 3, 2002).
The nucleic acid sequences encoding the AAV7 capsid are reproduced
in SEQ ID NO: 184; the amino acid sequences of the AAV7 capsid are
reproduced in SEQ ID NO: 185. In addition, the clade contains a
number of previously described AAV sequences, including: cy.2;
cy.3; cy.4; cy.5; cy.6; rh.13; rh.37; rh. 36; and rh.35 [US
Published Patent Application No. US 2003/0138772 A1 (Jul. 24,
2003)]. Additionally, the AAV7 clade contains novel AAV sequences,
including, without limitation, 2-15/rh.62 [SEQ ID Nos: 33 and 114];
1-7/rh.48 [SEQ ID Nos: 32 and 115]; 4-9/rh.54 [SEQ ID Nos: 40 and
116]; and 4-19/rh.55 [SEQ ID Nos: 37 and 117]. The invention
further includes modified cy. 5 [SEQ ID NO: 227]; modified rh.13
[SEQ ID NO: 228]; and modified rh. 37 [SEQ ID NO: 229].
[0050] In one embodiment, one or more of the members of this clade
has a capsid with an amino acid identity of at least 85% identity,
at least 90% identity, at least 95% identity, or at least 97%
identity over the full-length of the vp1, the vp2, or the vp3 of
the AAV7 capsid, SEQ ID NO: 184 and 185.
[0051] In another embodiment, the invention provides novel AAV of
Clade D, provided that none of the novel AAV comprises a capsid of
any of cy.2; cy.3; cy.4; cy.5; cy.6; rh.13; rh.37; rh. 36; and
rh.35. These AAV may include, without limitation, an AAV having a
capsid derived from one or more of the following 2-15/rh.62 [SEQ ID
Nos: 33 and 114]; 1-7/rh.48 [SEQ ID Nos: 32 and 115]; 4-9/rh.54
[SEQ ID Nos: 40 and 116]; and 4-19/rh.55 [SEQ ID Nos: 37 and
117].
[0052] E. Clade E (AAV8 Clade)
[0053] In one aspect, the invention provides Clade E. This clade is
characterized by containing the previously described AAV8 [G. Gao
et al, Proc. Natl Acad. Sci USA, 99:11854-9 (Sep. 3, 2002)],
43.1/rh.2; 44.2/rh.10; rh. 25; 29.3/bb.1; and 29.5/bb.2 [US
Published Patent Application No. US 2003/0138772 A1 (Jul. 24,
2003)].
[0054] Further, the clade novel AAV sequences, including, without
limitation, including, e.g., 30.10/pi.1 [SEQ ID NOs: 28 and 93],
30.12/pi.2 [SEQ ID NOs: 30 and 95, 30.19/pi.3 [SEQ ID NOs: 29 and
94], LG-4/rh.38 [SEQ ID Nos: 7 and 86]; LG-10/rh.40 [SEQ ID Nos: 14
and 92]; N721-8/rh.43 [SEQ ID Nos: 43 and 163]; 1-8/rh.49 [SEQ ID
NOs: 25 and 103]; 2-4/rh.50 [SEQ ID Nos: 23 and 108]; 2-5/rh.51
[SEQ ID Nos: 22 and 104]; 3-9/rh.52 [SEQ ID Nos: 18 and 96];
3-11/rh.53 [SEQ ID NOs: 17 and 97]; 5-3/rh.57 [SEQ ID Nos: 26 and
105]; 5-22/rh.58 [SEQ ID Nos: 27 and 58]; 2-3/rh.61 [SEQ ID NOs: 21
and 107]; 4-8/rh.64 [SEQ ID Nos: 15 and 99]; 3.1/hu.6 [SEQ ID NO: 5
and 84]; 33.12/hu.17 [SEQ ID NO:4 and 83]; 106.1/hu.37 [SEQ ID Nos:
10 and 88]; LG-9/hu.39 [SEQ ID Nos: 24 and 102]; 114.3/hu. 40 [SEQ
ID Nos: 11 and 87]; 127.2/hu.41 [SEQ ID NO:6 and 91]; 127.5/hu.42
[SEQ ID Nos: 8 and 85]; hu. 66 [SEQ ID NOs: 173 and 197]; and hu.67
[SEQ ID NOs: 174 and 198]. This clade further includes modified rh.
2 [SEQ ID NO: 231]; modified rh. 58 [SEQ ID NO: 232]; modified rh.
64 [SEQ ID NO: 233].
[0055] In one embodiment, one or more of the members of this clade
has a capsid with an amino acid identity of at least 85% identity,
at least 90% identity, at least 95% identity, or at least 97%
identity over the full-length of the vp1, the vp2, or the vp3 of
the AAV8 capsid. The nucleic acid sequences encoding the AAV8
capsid are reproduced in SEQ ID NO: 186 and the amino acid
sequences of the capsid are reproduced in SEQ ID NO:187.
[0056] In another embodiment, the invention provides novel AAV of
Clade E, provided that none of the novel AAV comprises a capsid of
any of AAV8, rh.8; 44.2/rh.10; rh. 25; 29.3/bb.1; and 29.5/bb.2 [US
Published Patent Application No. US 2003/0138772 A1 (Jul. 24,
2003)]. These AAV may include, without limitation, an AAV having a
capsid derived from one or more of the following: 30.10/pi.1 [SEQ
ID NOs:28 and 93], 30.12/pi.2 [SEQ ID NOs:30 and 95, 30.19/pi.3
[SEQ ID NOs:29 and 94], LG-4/rh.38 [SEQ ID Nos: 7 and 86];
LG-10/rh.40 [SEQ ID Nos: 14 and 92]; N721-8/rh.43 [SEQ ID Nos: 43
and 163]; 1-8/rh.49 [SEQ ID NOs: 25 and 103]; 2-4/rh.50 [SEQ ID
Nos: 23 and 108]; 2-5/rh.51 [SEQ ID Nos: 22 and 104]; 3-9/rh.52
[SEQ ID Nos: 18 and 96]; 3-11/rh.53 [SEQ ID NOs: 17 and 97];
5-3/rh.57 [SEQ ID Nos: 26 and 105]; 5-22/rh.58 [SEQ ID Nos: 27 and
58]; modified rh. 58 [SEQ ID NO: 232]; 2-3/rh.61 [SEQ ID NOs: 21
and 107]; 4-8/rh.64 [SEQ ID Nos: 15 and 99]; modified rh. 64[SEQ ID
NO: 233]; 3.1/hu.6 [SEQ ID NO: 5 and 84]; 33.12/hu.17 [SEQ ID NO:4
and 83]; 106.1/hu.37 [SEQ ID Nos: 10 and 88]; LG-9/hu.39 [SEQ ID
Nos: 24 and 102]; 114.3/hu. 40 [SEQ ID Nos: 11 and 87]; 127.2/hu.41
[SEQ ID NO:6 and 91]; 127.5/hu.42 [SEQ ID Nos: 8 and 85]; hu. 66
[SEQ ID NOs: 173 and 197]; and hu.67 [SEQ ID NOs: 174 and 198].
[0057] F. Clade F (AAV 9 Clade)
[0058] This clade is identified by the name of a novel AAV serotype
identified herein as hu.14/AAV9 [SEQ ID Nos: 3 and 123]. In
addition, this clade contains other novel sequences including,
hu.31 [SEQ ID NOs:1 and 121]; and hu.32 [SEQ ID Nos: 2 and
122].
[0059] In one embodiment, one or more of the members of this clade
has a capsid with an amino acid identity of at least 85% identity,
at least 90% identity, at least 95% identity, or at least 97%
identity over the full-length of the vp1, the vp2, or the vp3 of
the AAV9 capsid, SEQ ID NO: 3 and 123.
[0060] In another embodiment, the invention provides novel AAV of
Clade F, which include, without limitation, an AAV having a capsid
derived from one or more of hu.14/AAV9 [SEQ ID Nos: 3 and 123],
hu.31 [SEQ ID NOs:1 and 121] and hu.32 [SEQ ID Nos: 1 and 122].
[0061] The AAV clades of the invention are useful for a variety of
purposes, including providing ready collections of related AAV for
generating viral vectors, and for generating targeting molecules.
These clades may also be used as tools for a variety of purposes
that will be readily apparent to one of skill in the art.
II. Novel AAV Sequences
[0062] The invention provides the nucleic acid sequences and amino
acids of a novel AAV serotype, which is termed interchangeably
herein as clone hu.14/28.4 and huAAV9. These sequences are useful
for constructing vectors that are highly efficient in transduction
of liver, muscle and lung. This novel AAV and its sequences are
also useful for a variety of other purposes. These sequences are
being submitted with GenBank and have been assigned the accession
numbers identified herein.
[0063] The invention further provides the nucleic acid sequences
and amino acid sequences of a number of novel AAV. Many of these
sequence include those described above as members of a clade, as
summarized below.
[0064] 128.1/hu. 43 [SEQ ID Nos: 80 and 160 GenBank Accession No.
AY530606]; modified hu. 43 [SEQ ID NO:236]; 128.3/hu. 44 [SEQ ID
Nos: 81 and 158; GenBank Accession No. AY530607] and 130.4/hu.48
[SEQ ID NO: 78 and 157; GenBank Accession No. AY530611]; from the
Clade A;
[0065] 52/hu.19 [SEQ ID NOs: 62 and 133; GenBank Accession No.
AY530584], 52.1/hu.20 [SEQ ID NOs: 63 and 134; GenBank Accession
No. AY530586], 54.5/hu.23 [SEQ ID Nos: 60 and 137; GenBank
Accession No. AY530589], 54.2/hu.22 [SEQ ID Nos: 67 and 138;
GenBank Accession No. AY530588], 54.7/hu.24 [SEQ ID Nos: 66 and
136; GenBank Accession No. AY530590], 54.1/hu.21 [SEQ ID Nos: 65
and 135; GenBank Accession No. AY530587], 54.4R/hu.27 [SEQ ID Nos:
64 and 140; GenBank Accession No. AY530592]; 46.2/hu.28 [SEQ ID
Nos: 68 and 130; GenBank Accession No. AY530593]; 46.6/hu.29 [SEQ
ID Nos: 69 and 132; GenBank Accession No. AY530594]; modified hu.
29 [SEQ ID NO: 225]; 172.1/hu.63 [SEQ ID NO: 171 and 195]; and
140.2/hu.52 (SEQ ID NO: 167 and 191; from Clade B;
[0066] 3.1/hu.9 [SEQ ID Nos: 58 and 155; GenBank Accession No.
AY530626]; 16.8/hu.10 [SEQ ID Nos: 56 and 156; GenBank Accession
No. AY530576]; 16.12/hu.11 [SEQ ID Nos: 57 and 153; GenBank
Accession No. AY530577]; 145.1/hu.53 [SEQ ID Nos: 176 and 186;
GenBank Accession No. AY530615]; 145.6/hu.55 [SEQ ID Nos: 178 and
187; GenBank Accession No. AY530617]; 145.5/hu.54 [SEQ ID Nos: 177
and 188; GenBank Accession No. AY530616]; 7.3/hu.7 [SEQ ID Nos: 55
and 150; GenBank Accession No. AY530628]; modified hu. 7 [SEQ ID
NO: 226]; hu.18 [SEQ ID Nos: 52 and 149; GenBank Accession No.
AY530583]; 33.4/hu.15 [SEQ ID Nos: 50 and 147; GenBank Accession
No. AY530580]; 33.8/hu.16 [SEQ ID Nos: 51 and 148; GenBank
Accession No. AY530581]; 58.2/hu.25 [SEQ ID Nos: 49 and 146;
GenBank Accession No. AY530591]; 161.10/hu.60 [SEQ ID Nos: 170 and
184; GenBank Accession No. AY530622]; H-5/hu.3 [SEQ ID Nos: 44 and
145; GenBank Accession No. AY530595]; H-1/hu.1 [SEQ ID Nos: 46 and
144; GenBank Accession No. AY530575]; and 161.6/hu.61 [SEQ ID Nos:
174 and 185; GenBank Accession No. AY530623] from Clade C;
[0067] 2-15/rh.62 [SEQ ID Nos: 33 and 114; GenBank Accession No.
AY530573]; 1-7/rh.48 [SEQ ID Nos: 32 and 115; GenBank Accession No.
AY530561]; 4-9/rh.54 [SEQ ID Nos: 40 and 116; GenBank Accession No.
AY530567]; and 4-19/rh.55 [SEQ ID Nos: 37 and 117; GenBank
Accession No. AY530568]; modified cy. 5 [SEQ ID NO: 227]; modified
rh.13 [SEQ ID NO: 228]; and modified rh. 37 [SEQ ID NO: 229] from
the Clade D;
[0068] 30.10/pi.1 [SEQ ID NOs:28 and 93; GenBank Accession No.
AY53055], 30.12/pi.2 [SEQ ID NOs:30 and 95; GenBank Accession No.
AY 530554], 30.19/pi.3 [SEQ ID NOs:29 and 94; GenBank Accession No.
AY530555], LG-4/rh.38 [SEQ ID Nos: 7 and 86; GenBank Accession No.
AY 530558]; LG-10/rh.40 [SEQ ID Nos: 14 and 92; GenBank Accession
No. AY530559]; N721-8/rh.43 [SEQ ID Nos: 43 and 163; GenBank
Accession No. AY530560]; 1-8/rh.49 [SEQ ID NOs: 25 and 103; GenBank
Accession No. AY530561]; 2-4/rh.50 [SEQ ID Nos: 23 and 108; GenBank
Accession No. AY530563]; 2-5/rh.51 [SEQ ID Nos: 22 and 104; GenBank
Accession No. 530564]; 3-9/rh.52 [SEQ ID Nos: 18 and 96; GenBank
Accession No. AY530565]; 3-11/rh.53 [SEQ ID Nos: 17 and 97; GenBank
Accession No. AY530566]; 5-3/rh.57 [SEQ ID Nos: 26 and 105; GenBank
Accession No. AY530569]; 5-22/rh.58 [SEQ ID Nos: 27 and 58; GenBank
Accession No. 530570]; modified rh. 58 [SEQ ID NO: 232]; 2-3/rh.61
[SEQ ID Nos: 21 and 107; GenBank Accession No. AY530572]; 4-8/rh.64
[SEQ ID Nos: 15 and 99; GenBank Accession No. AY530574]; modified
rh. 64[SEQ ID NO: 233]; 3.1/hu.6 [SEQ ID NO: 5 and 84; GenBank
Accession No. AY530621]; 33.12/hu.17 [SEQ ID NO:4 and 83; GenBank
Accession No. AY530582]; 106.1/hu.37 [SEQ ID Nos: 10 and 88;
GenBank Accession No. AY530600]; LG-9/hu.39 [SEQ ID Nos: 24 and
102; GenBank Accession No. AY530601]; 114.3/hu. 40 [SEQ ID Nos: 11
and 87; GenBank Accession No. AY530603]; 127.2/hu.41 [SEQ ID NO:6
and 91; GenBank Accession No. AY530604]; 127.5/hu.42 [SEQ ID Nos: 8
and 85; GenBank Accession No. AY530605]; and hu. 66 [SEQ ID NOs:
173 and 197; GenBank Accession No. AY530626]; and hu.67 [SEQ ID
NOs: 174 and 198; GenBank Accession No. AY530627]; and modified
rh.2 [SEQ ID NO:231]; from Clade E;
[0069] hu.14/AAV9 [SEQ ID Nos: 3 and 123; GenBank Accession No.
AY530579], hu.31 [SEQ ID NOs:1 and 121; AY530596] and hu.32 [SEQ ID
Nos: 1 and 122; GenBank Accession No. AY530597] from Clade F.
[0070] In addition, the present invention provides AAV sequences,
including, rh.59 [SEQ ID NO: 49 and 110]; rh.60 [SEQ ID NO: 31 and
120; GenBank Accession No. AY530571], modified ch.5 [SEQ ID NO:
234]; and modified rh. 8 [SEQ ID NO: 235], which are outside the
definition of the clades described above.
[0071] Also provided are fragments of the AAV sequences of the
invention. Each of these fragments may be readily utilized in a
variety of vector systems and host cells. Among desirable AAV
fragments are the cap proteins, including the vp1, vp2, vp3 and
hypervariable regions. Where desired, the methodology described in
published US Patent Publication No. US 2003/0138772 A1 (Jul. 24,
2003)] can be used to obtain the rep sequences for the AAV clones
identified above. Such rep sequences include, e.g., rep 78, rep 68,
rep 52, and rep 40, and the sequences encoding these proteins.
Similarly, other fragments of these clones may be obtained using
the techniques described in the referenced patent publication,
including the AAV inverted terminal repeat (ITRs), AAV P19
sequences, AAV P40 sequences, the rep binding site, and the
terminal resolute site (TRS). Still other suitable fragments will
be readily apparent to those of skill in the art.
[0072] The capsid and other fragments of the invention can be
readily utilized in a variety of vector systems and host cells.
Such fragments may be used alone, in combination with other AAV
sequences or fragments, or in combination with elements from other
AAV or non-AAV viral sequences. In one particularly desirable
embodiment, a vector contains the AAV cap and/or rep sequences of
the invention.
[0073] The AAV 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 sequences of the invention.
[0074] Suitable fragments can be determined using the information
provided herein.
[0075] As described herein, the vectors of the invention containing
the AAV 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.
[0076] These and other embodiments and advantages of the invention
are described in more detail below.
[0077] A. AAV Serotype 9/Hu14 Sequences
[0078] The invention provides the nucleic acid sequences and amino
acids of a novel AAV, which is termed interchangeable herein as
clone hu.14 (formerly termed 28.4) and huAAV9. As defined herein,
novel serotype AAV9 refers to AAV having a capsid which generates
antibodies which cross-react serologically with the capsid having
the sequence of hu. 14 [SEQ ID NO: 123] and which antibodies do not
cross-react serologically with antibodies generated to the capsids
of any of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7 or AAV8.
[0079] 1. Nucleic Acid Sequences
[0080] The AAV9 nucleic acid sequences of the invention include the
DNA sequences of SEQ ID NO: 3, which consists of 2211
nucleotides.
[0081] The nucleic acid sequences of the invention further
encompass the strand which is complementary to SEQ ID NO: 3, as
well as the RNA and cDNA sequences corresponding to SEQ ID NO: 3,
and its complementary strand. Also included in the nucleic acid
sequences of the invention are natural variants and engineered
modifications of SEQ ID NO: 3 and its complementary strand. Such
modifications include, for example, labels that are known in the
art, methylation, and substitution of one or more of the naturally
occurring nucleotides with a degenerate nucleotide.
[0082] Further included in this invention are nucleic acid
sequences which are greater than about 90%, more preferably at
least about 95%, and most preferably at least about 98 to 99%,
identical or homologous to SEQ ID NO: 3.
[0083] Also included within the invention are fragments of SEQ ID
NO: 3, its complementary strand, and cDNA and RNA complementary
thereto. Suitable fragments are at least 15 nucleotides in length,
and encompass functional fragments, i.e., fragments which are of
biological interest. Such fragments include the sequences encoding
the three variable proteins (vp) of the AAV9/HU.14 capsid which are
alternative splice variants: vp1 [nt 1 to 2211 of SEQ ID NO:3]; vp2
[about nt 411 to 2211 of SEQ ID NO:3]; and vp 3 [about nt 609 to
2211 of SEQ ID NO:3]. Other suitable fragments of SEQ ID NO: 3,
include the fragment which contains the start codon for the
AAV9/HU.14 capsid protein, and the fragments encoding the
hypervariable regions of the vp1 capsid protein, which are
described herein.
[0084] In addition to including the nucleic acid sequences provided
in the figures and Sequence Listing, the present invention includes
nucleic acid molecules and sequences which are designed to express
the amino acid sequences, proteins and peptides of the AAV
serotypes of the invention. Thus, the invention includes nucleic
acid sequences which encode the following novel AAV amino acid
sequences and artificial AAV serotypes generated using these
sequences and/or unique fragments thereof.
[0085] As used herein, artificial AAV serotypes include, without
limitation, AAVs with a non-naturally occurring capsid protein.
Such an artificial capsid may be generated by any suitable
technique, using a novel AAV sequence of the invention (e.g., a
fragment of a vp1 capsid protein) in combination with heterologous
sequences which may be obtained from another AAV serotype (known or
novel), non-contiguous portions of the same AAV serotype, from a
non-AAV viral source, or from a non-viral source. An artificial AAV
serotype may be, without limitation, a chimeric AAV capsid, a
recombinant AAV capsid, or a "humanized" AAV capsid.
[0086] 2. HU.14/AAV9 Amino Acid Sequences, Proteins and
Peptides
[0087] The invention further provides proteins and fragments
thereof which are encoded by the hu.14/AAV9 nucleic acids of the
invention, and hu.14/AAV9 proteins and fragments which are
generated by other methods. As used herein, these proteins include
the assembled capsid. The invention further encompasses AAV
serotypes generated using sequences of the novel AAV serotype of
the invention, which are generated using synthetic, recombinant or
other techniques known to those of skill in the art. The invention
is not limited to novel AAV amino acid sequences, peptides and
proteins expressed from the novel AAV nucleic acid sequences of the
invention, but encompasses amino acid sequences, peptides and
proteins generated by other methods known in the art, including,
e.g., by chemical synthesis, by other synthetic techniques, or by
other methods. The sequences of any of the AAV capsids provided
herein can be readily generated using a variety of techniques.
[0088] Suitable production techniques are well known to those of
skill in the art. See, e.g., Sambrook et al, Molecular Cloning: A
Laboratory Manual, Cold Spring Harbor Press (Cold Spring Harbor,
N.Y.). Alternatively, peptides can also be synthesized by the
well-known solid phase peptide synthesis methods (Merrifield, J.
Am. Chem. Soc., 85:2149 (1962); Stewart and Young, Solid Phase
Peptide Synthesis (Freeman, San Francisco, 1969) pp. 27-62). These
and other suitable production methods are within the knowledge of
those of skill in the art and are not a limitation of the present
invention.
[0089] Particularly desirable proteins include the AAV capsid
proteins, which are encoded by the nucleotide sequences identified
above. The AAV capsid is composed of three proteins, vp1, vp2 and
vp3, which are alternative splice variants. The full-length
sequence provided in FIG. 2 is that of vp1. The AAV9/HU.14 capsid
proteins include vp1 [amino acids (aa) 1 to 736 of SEQ ID NO: 123],
vp2 [about aa 138 to 736 of SEQ ID NO: 123], vp3 [about aa 203 to
736 of SEQ ID NO: 123], and functional fragments thereof. Other
desirable fragments of the capsid protein include the constant and
variable regions, located between hypervariable regions (HVR).
Other desirable fragments of the capsid protein include the HVR
themselves.
[0090] An algorithm developed to determine areas of sequence
divergence in AAV2 has yielded 12 hypervariable regions (HVR) of
which 5 overlap or are part of the four previously described
variable regions. [Chiorini et al, J. Virol, 73:1309-19 (1999);
Rutledge et al, J. Virol., 72:309-319] Using this algorithm and/or
the alignment techniques described herein, the HVR of the novel AAV
serotypes are determined. For example, the HVR are located as
follows: HVR1, aa 146-152; HVR2, aa 182-186; HVR3, aa 262-264;
HVR4, aa 381-383; HVR5, aa 450-474; HVR6, aa 490-495; HVR7, aa
500-504; HVR8, aa 514-522; HVR9, aa 534-555; HVR10, aa 581-594;
HVR11, aa 658-667; and HVR12, aa 705-719 the numbering system is
based on an alignment which uses the AAV2 vp1 as a point of
reference]. Using the alignment provided herein performed using the
Clustal X program at default settings, or using other commercially
or publicly available alignment programs at default settings such
as are described herein, one of skill in the art can readily
determine corresponding fragments of the novel AAV capsids of the
invention.
[0091] Still other desirable fragments of the AAV9/HU.14 capsid
protein include amino acids 1 to 184 of SEQ ID NO: 123, amino acids
199 to 259; amino acids 274 to 446; amino acids 603 to 659; amino
acids 670 to 706; amino acids 724 to 736 of SEQ ID NO: 123; aa
185-198; aa 260-273; aa447-477; aa495-602; aa660-669; and
aa707-723. Additionally, examples of other suitable fragments of
AAV capsids include, with respect to the numbering of AAV9 [SEQ ID
NO: 123], aa 24-42, aa 25-28; aa 81-85; aa133-165; aa 134-165; aa
137-143; aa 154-156; aa 194-208; aa 261-274; aa 262-274; aa
171-173; aa 413-417; aa 449-478; aa 494-525; aa 534-571; aa
581-601; aa 660-671; aa 709-723. Using the alignment provided
herein performed using the Clustal X program at default settings,
or using other commercially or publicly available alignment
programs at default settings, one of skill in the art can readily
determine corresponding fragments of the novel AAV capsids of the
invention.
[0092] Still other desirable AAV9/HU.14 proteins include the rep
proteins include rep68/78 and rep40/52.
[0093] Suitably, 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.
[0094] The invention further provides other AAV9/HU.14 sequences
which are identified using the sequence information provided
herein. For example, given the AAV9/HU.14 sequences provided
herein, infectious AAV9/HU.14 may be isolated using genome walking
technology (Siebert et al., 1995, Nucleic Acid Research,
23:1087-1088, Friezner-Degen et al., 1986, J. Biol. Chem.
261:6972-6985, BD Biosciences Clontech, Palo Alto, Calif.). Genome
walking is particularly well suited for identifying and isolating
the sequences adjacent to the novel sequences identified according
to the method of the invention. This technique is also useful for
isolating inverted terminal repeat (ITRs) of the novel AAV9/HU.14
serotype, based upon the novel AAV capsid and rep sequences
provided herein.
[0095] 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.
[0096] III. Production of rAAV with Novel AAV Capsids
[0097] The invention encompasses novel AAV capsid sequences of
which are free of DNA and/or cellular material with these viruses
are associated in nature. To avoid repeating all of the novel AAV
capsids provided herein, reference is made throughout this and the
following sections to the hu.14/AAV9 capsid. However, it should be
appreciated that the other novel AAV capsid sequences of the
invention can be used in a similar manner.
[0098] In another aspect, the present invention provides molecules
that utilize the novel AAV sequences of the invention, including
fragments thereof, for production of molecules useful in delivery
of a heterologous gene or other nucleic acid sequences to a target
cell.
[0099] In another aspect, the present invention provides molecules
that utilize the AAV sequences of the invention, including
fragments thereof, for production of viral vectors useful in
delivery of a heterologous gene or other nucleic acid sequences to
a target cell.
[0100] The molecules of the invention which contain AAV sequences
include any genetic element (vector) which may be delivered to a
host cell, e.g., naked DNA, a plasmid, phage, transposon, cosmid,
episome, a protein in a non-viral delivery vehicle (e.g., a
lipid-based carrier), virus, etc., which transfers the sequences
carried thereon. The selected vector may be delivered by any
suitable method, including transfection, electroporation, liposome
delivery, membrane fusion techniques, high velocity DNA-coated
pellets, viral infection and protoplast fusion. The methods used to
construct any embodiment of this invention are known to those with
skill in nucleic acid manipulation and include genetic engineering,
recombinant engineering, and synthetic techniques. See, e.g.,
Sambrook et al, Molecular Cloning: A Laboratory Manual, Cold Spring
Harbor Press, Cold Spring Harbor, N.Y.
[0101] In one embodiment, the vectors of the invention contain,
inter alia, sequences encoding an AAV capsid of the invention or a
fragment thereof. In another embodiment, the vectors of the
invention contain, at a minimum, sequences encoding an AAV rep
protein or a fragment thereof. Optionally, vectors of the invention
may contain both AAV cap and rep proteins. In vectors in which both
AAV rep and cap are provided, the AAV rep and AAV cap sequences can
originate from an AAV of the same clade. Alternatively, the present
invention provides vectors in which the rep sequences are from an
AAV source which differs from that which is providing the cap
sequences. In one embodiment, the rep and cap sequences are
expressed from separate sources (e.g., separate vectors, or a host
cell and a vector). In another embodiment, these rep sequences are
fused in frame to cap sequences of a different AAV source to form a
chimeric AAV vector. Optionally, the vectors of the invention are
vectors packaged in an AAV capsid of the invention. These vectors
and other vectors described herein can further contain a minigene
comprising a selected transgene which is flanked by AAV 5' ITR and
AAV 3' ITR.
[0102] Thus, in one embodiment, the vectors described herein
contain nucleic acid sequences encoding an intact AAV capsid which
may be from a single AAV sequence (e.g., AAV9/HU.14). Such a capsid
may comprise amino acids 1 to 736 of SEQ ID NO:123. Alternatively,
these vectors contain sequences encoding artificial capsids which
contain one or more fragments of the AAV9/HU.14 capsid fused to
heterologous AAV or non-AAV capsid proteins (or fragments thereof).
These artificial capsid proteins are selected from non-contiguous
portions of the AAV9/HU.14 capsid or from capsids of other AAVs.
For example, a rAAV may have a capsid protein comprising one or
more of the AAV9/HU.14 capsid regions selected from the vp2 and/or
vp3, or from vp 1, or fragments thereof selected from amino acids 1
to 184, amino acids 199 to 259; amino acids 274 to 446; amino acids
603 to 659; amino acids 670 to 706; amino acids 724 to 738 of the
AAV9/HU.14 capsid, SEQ ID NO: 123. In another example, it may be
desirable to alter the start codon of the vp3 protein to GTG.
Alternatively, the rAAV may contain one or more of the AAV serotype
9 capsid protein hypervariable regions which are identified herein,
or other fragment including, without limitation, aa 185-198; aa
260-273; aa447-477; aa495-602; aa660-669; and aa707-723 of the
AAV9/HU.14 capsid. See, SEQ ID NO: 123. These modifications may be
to increase expression, yield, and/or to improve purification in
the selected expression systems, or for another desired purpose
(e.g., to change tropism or alter neutralizing antibody
epitopes).
[0103] The vectors described herein, e.g., a plasmid, are useful
for a variety of purposes, but are particularly well suited for use
in production of a rAAV containing a capsid comprising AAV
sequences or a fragment thereof. These vectors, including rAAV,
their elements, construction, and uses are described in detail
herein.
[0104] In one aspect, the invention provides a method of generating
a recombinant adeno-associated virus (AAV) having an AAV serotype 9
capsid, or a portion thereof. Such a method involves culturing a
host cell which contains a nucleic acid sequence encoding an AAV
serotype 9 capsid protein, or fragment thereof, as defined herein;
a functional rep gene; a minigene composed of, at a minimum, AAV
inverted terminal repeats (ITRs) and a transgene; and sufficient
helper functions to permit packaging of the minigene into the
AAV9/HU.14 capsid protein.
[0105] The components required to be cultured in the host cell to
package an AAV minigene in an AAV capsid may be provided to the
host cell in trans. Alternatively, any one or more of the required
components (e.g., minigene, rep sequences, cap sequences, and/or
helper functions) may be provided by a stable host cell which has
been engineered to contain one or more of the required components
using methods known to those of skill in the art. Most suitably,
such a stable host cell will contain the required component(s)
under the control of an inducible promoter. However, the required
component(s) may be under the control of a constitutive promoter.
Examples of suitable inducible and constitutive promoters are
provided herein, in the discussion of regulatory elements suitable
for use with the transgene. In still another alternative, a
selected stable host cell may contain selected component(s) under
the control of a constitutive promoter and other selected
component(s) under the control of one or more inducible promoters.
For example, a stable host cell may be generated which is derived
from 293 cells (which contain E1 helper functions under the control
of a constitutive promoter), but which contains the rep and/or cap
proteins under the control of inducible promoters. Still other
stable host cells may be generated by one of skill in the art.
[0106] The minigene, rep sequences, cap sequences, and helper
functions required for producing the rAAV of the invention may be
delivered to the packaging host cell in the form of any genetic
element which transfer the sequences carried thereon. The selected
genetic element may be delivered by any suitable method, including
those described herein. The methods used to construct any
embodiment of this invention are known to those with skill in
nucleic acid manipulation and include genetic engineering,
recombinant engineering, and synthetic techniques. See, e.g.,
Sambrook et al, Molecular Cloning: A Laboratory Manual, Cold Spring
Harbor Press, Cold Spring Harbor, N.Y. Similarly, methods of
generating rAAV virions are well known and the selection of a
suitable method is not a limitation on the present invention. See,
e.g., K. Fisher et al, J Virol., 70:520-532 (1993) and U.S. Pat.
No. 5,478,745.
[0107] Unless otherwise specified, the AAV ITRs, and other selected
AAV components described herein, may be readily selected from among
any AAV, including, without limitation, AAV1, AAV2, AAV3, AAV4,
AAV5, AAV6, AAV7, AAV9 and one of the other novel AAV sequences of
the invention. These ITRs or other AAV components may be readily
isolated using techniques available to those of skill in the art
from an AAV sequence. Such AAV may be isolated or obtained from
academic, commercial, or public sources (e.g., the American Type
Culture Collection, Manassas, Va.). Alternatively, the AAV
sequences may be obtained through synthetic or other suitable means
by reference to published sequences such as are available in the
literature or in databases such as, e.g., GenBank.RTM.,
PubMed.RTM., or the like.
[0108] A. The Minigene
[0109] The minigene is composed of, at a minimum, a transgene and
its regulatory sequences, and 5' and 3' AAV inverted terminal
repeats (ITRs). In one desirable embodiment, the ITRs of AAV
serotype 2 are used. However, ITRs from other suitable sources may
be selected. It is this minigene that is packaged into a capsid
protein and delivered to a selected host cell.
[0110] 1. The Transgene
[0111] The transgene is a nucleic acid sequence, heterologous to
the vector sequences flanking the transgene, which encodes a
polypeptide, protein, or other product, of interest. The nucleic
acid coding sequence is operatively linked to regulatory components
in a manner which permits transgene transcription, translation,
and/or expression in a host cell.
[0112] The composition of the transgene sequence will depend upon
the use to which the resulting vector will be put. For example, one
type of transgene sequence includes a reporter sequence, which upon
expression produces a detectable signal. Such reporter sequences
include, without limitation, DNA sequences encoding
.beta.-lactamase, .beta.-galactosidase (LacZ), alkaline
phosphatase, thymidine kinase, green fluorescent protein (GFP),
enhanced GFP (EGFP), chloramphenicol acetyltransferase (CAT),
luciferase, membrane bound proteins including, for example, CD2,
CD4, CD8, the influenza hemagglutinin protein, and others well
known in the art, to which high affinity antibodies directed
thereto exist or can be produced by conventional means, and fusion
proteins comprising a membrane bound protein appropriately fused to
an antigen tag domain from, among others, hemagglutinin or Myc.
[0113] These coding sequences, when associated with regulatory
elements which drive their expression, provide signals detectable
by conventional means, including enzymatic, radiographic,
colorimetric, fluorescence or other spectrographic assays,
fluorescent activating cell sorting assays and immunological
assays, including enzyme linked immunosorbent assay (ELISA),
radioimmunoassay (RIA) and immunohistochemistry. For example, where
the marker sequence is the LacZ gene, the presence of the vector
carrying the signal is detected by assays for beta-galactosidase
activity. Where the transgene is green fluorescent protein or
luciferase, the vector carrying the signal may be measured visually
by color or light production in a luminometer.
[0114] However, desirably, the transgene is a non-marker sequence
encoding a product which is useful in biology and medicine, such as
proteins, peptides, RNA, enzymes, dominant negative mutants, or
catalytic RNAs. Desirable RNA molecules include tRNA, dsRNA,
ribosomal RNA, catalytic RNAs, siRNA, small hairpin RNA,
trans-splicing RNA, and antisense RNAs. One example of a useful RNA
sequence is a sequence which inhibits or extinguishes expression of
a targeted nucleic acid sequence in the treated animal Typically,
suitable target sequences include oncologic targets and viral
diseases. See, for examples of such targets the oncologic targets
and viruses identified below in the section relating to
immunogens.
[0115] The transgene may be used to correct or ameliorate gene
deficiencies, which may include deficiencies in which normal genes
are expressed at less than normal levels or deficiencies in which
the functional gene product is not expressed. Alternatively, the
transgene may provide a product to a cell which is not natively
expressed in the cell type or in the host. A preferred type of
transgene sequence encodes a therapeutic protein or polypeptide
which is expressed in a host cell. The invention further includes
using multiple transgenes. In certain situations, a different
transgene may be used to encode each subunit of a protein, or to
encode different peptides or proteins. This is desirable when the
size of the DNA encoding the protein subunit is large, e.g., for an
immunoglobulin, the platelet-derived growth factor, or a dystrophin
protein. In order for the cell to produce the multi-subunit
protein, a cell is infected with the recombinant virus containing
each of the different subunits. Alternatively, different subunits
of a protein may be encoded by the same transgene. In this case, a
single transgene includes the DNA encoding each of the subunits,
with the DNA for each subunit separated by an internal ribozyme
entry site (IRES). This is desirable when the size of the DNA
encoding each of the subunits is small, e.g., the total size of the
DNA encoding the subunits and the IRES is less than five kilobases.
As an alternative to an IRES, the DNA may be separated by sequences
encoding a 2A peptide, which self-cleaves in a post-translational
event. See, e.g., M. L. Donnelly, et al, J. Gen. Viral., 78(Pt
1):13-21 (January 1997); Furler, S., et al, Gene Ther.,
8(11):864-873 (June 2001); Klump H., et al., Gene Ther.,
8(10):811-817 (May 2001). This 2A peptide is significantly smaller
than an IRES, making it well suited for use when space is a
limiting factor. More often, when the transgene is large, consists
of multi-subunits, or two transgenes are co-delivered, rAAV
carrying the desired transgene(s) or subunits are co-administered
to allow them to concatamerize in vivo to form a single vector
genome. In such an embodiment, a first AAV may carry an expression
cassette which expresses a single transgene and a second AAV may
carry an expression cassette which expresses a different transgene
for co-expression in the host cell. However, the selected transgene
may encode any biologically active product or other product, e.g.,
a product desirable for study.
[0116] Suitable transgenes may be readily selected by one of skill
in the art. The selection of the transgene is not considered to be
a limitation of this invention.
[0117] 2. Regulatory Elements
[0118] In addition to the major elements identified above for the
minigene, the vector also includes conventional control elements
which are operably linked to the transgene in a manner which
permits its transcription, translation and/or expression in a cell
transfected with the plasmid vector or infected with the virus
produced by the invention. As used herein, "operably linked"
sequences include both expression control sequences that are
contiguous with the gene of interest and expression control
sequences that act in trans or at a distance to control the gene of
interest.
[0119] Expression control sequences include appropriate
transcription initiation, termination, promoter and enhancer
sequences; efficient RNA processing signals such as splicing and
polyadenylation (polyA) signals; sequences that stabilize
cytoplasmic mRNA; sequences that enhance translation efficiency
(i.e., Kozak consensus sequence); sequences that enhance protein
stability; and when desired, sequences that enhance secretion of
the encoded product. A great number of expression control
sequences, including promoters which are native, constitutive,
inducible and/or tissue-specific, are known in the art and may be
utilized.
[0120] Examples of constitutive promoters include, without
limitation, the retroviral Rous sarcoma virus (RSV) LTR promoter
(optionally with the RSV enhancer), the cytomegalovirus (CMV)
promoter (optionally with the CMV enhancer) [see, e.g., Boshart et
al, Cell, 41:521-530 (1985)], the SV40 promoter, the dihydrofolate
reductase promoter, the .beta.-actin promoter, the phosphoglycerol
kinase (PGK) promoter, and the EF1 promoter [Invitrogen]. Inducible
promoters allow regulation of gene expression and can be regulated
by exogenously supplied compounds, environmental factors such as
temperature, or the presence of a specific physiological state,
e.g., acute phase, a particular differentiation state of the cell,
or in replicating cells only. Inducible promoters and inducible
systems are available from a variety of commercial sources,
including, without limitation, Invitrogen, Clontech and Ariad. Many
other systems have been described and can be readily selected by
one of skill in the art. Examples of inducible promoters regulated
by exogenously supplied compounds, include, the zinc-inducible
sheep metallothionine (MT) promoter, the dexamethasone
(Dex)-inducible mouse mammary tumor virus (MMTV) promoter, the T7
polymerase promoter system [International Patent Publication No. WO
98/10088]; the ecdysone insect promoter [No et al, Proc. Natl.
Acad. Sci. USA, 93:3346-3351 (1996)], the tetracycline-repressible
system [Gossen et al, Proc. Natl. Acad. Sci. USA, 89:5547-5551
(1992)], the tetracycline-inducible system [Gossen et al, Science,
268:1766-1769 (1995), see also Harvey et al, Curr. Opin. Chem.
Biol., 2:512-518 (1998)], the RU486-inducible system [Wang et al,
Nat. Biotech., 15:239-243 (1997) and Wang et al, Gene Ther.,
4:432-441 (1997)] and the rapamycin-inducible system [Magari et al,
J. Clin. Invest., 100:2865-2872 (1997)]. Other types of inducible
promoters which may be useful in this context are those which are
regulated by a specific physiological state, e.g., temperature,
acute phase, a particular differentiation state of the cell, or in
replicating cells only.
[0121] In another embodiment, the native promoter for the transgene
will be used. The native promoter may be preferred when it is
desired that expression of the transgene should mimic the native
expression. The native promoter may be used when expression of the
transgene must be regulated temporally or developmentally, or in a
tissue-specific manner, or in response to specific transcriptional
stimuli. In a further embodiment, other native expression control
elements, such as enhancer elements, polyadenylation sites or Kozak
consensus sequences may also be used to mimic the native
expression.
[0122] Another embodiment of the transgene includes a gene operably
linked to a tissue-specific promoter. For instance, if expression
in skeletal muscle is desired, a promoter active in muscle should
be used. These include the promoters from genes encoding skeletal
.beta.-actin, myosin light chain 2A, dystrophin, muscle creatine
kinase, as well as synthetic muscle promoters with activities
higher than naturally-occurring promoters (see Li et al., Nat.
Biotech., 17:241-245 (1999)). Examples of promoters that are
tissue-specific are known for liver (albumin, Miyatake et al., J.
Vivol., 71:5124-32 (1997); hepatitis B virus core promoter, Sandig
et al., Gene Ther., 3:1002-9 (1996); alpha-fetoprotein (AFP),
Arbuthnot et al., Hum. Gene Ther., 7:1503-14 (1996)), bone
osteocalcin (Stein et al., Mol. Biol. Rep., 24:185-96 (1997)); bone
sialoprotein (Chen et al., J. Bone Miner. Res., 11:654-64 (1996)),
lymphocytes (CD2, Hansal et al., J. Immunol., 161:1063-8 (1998);
immunoglobulin heavy chain; T cell receptor chain), neuronal such
as neuron-specific enolase (NSE) promoter (Andersen et al., Cell.
Mol. Neurobiol., 13:503-15 (1993)), neurofilament light-chain gene
(Piccioli et al., Proc. Natl. Acad. Sci. USA, 88:5611-5 (1991)),
and the neuron-specific vgf gene (Piccioli et al., Neuron,
15:373-84 (1995)), among others.
[0123] Optionally, plasmids carrying therapeutically useful
transgenes may also include selectable markers or reporter genes
may include sequences encoding geneticin, hygromicin or purimycin
resistance, among others. Such selectable reporters or marker genes
(preferably located outside the viral genome to be rescued by the
method of the invention) can be used to signal the presence of the
plasmids in bacterial cells, such as ampicillin resistance. Other
components of the plasmid may include an origin of replication.
Selection of these and other promoters and vector elements are
conventional and many such sequences are available [see, e.g.,
Sambrook et al, and references cited therein].
[0124] The combination of the transgene, promoter/enhancer, and 5'
and 3' AAV ITRs is referred to as a "minigene" for ease of
reference herein. Provided with the teachings of this invention,
the design of such a minigene can be made by resort to conventional
techniques.
[0125] 3. Delivery of the Minigene to a Packaging Host Cell
[0126] The minigene can be carried on any suitable vector, e.g., a
plasmid, which is delivered to a host cell. The plasmids useful in
this invention may be engineered such that they are suitable for
replication and, optionally, integration in prokaryotic cells,
mammalian cells, or both. These plasmids (or other vectors carrying
the 5' AAV ITR-heterologous molecule-3' AAV ITR) contain sequences
permitting replication of the minigene in eukaryotes and/or
prokaryotes and selection markers for these systems. Selectable
markers or reporter genes may include sequences encoding geneticin,
hygromicin or purimycin resistance, among others. The plasmids may
also contain certain selectable reporters or marker genes that can
be used to signal the presence of the vector in bacterial cells,
such as ampicillin resistance. Other components of the plasmid may
include an origin of replication and an amplicon, such as the
amplicon system employing the Epstein Barr virus nuclear antigen.
This amplicon system, or other similar amplicon components permit
high copy episomal replication in the cells. Preferably, the
molecule carrying the minigene is transfected into the cell, where
it may exist transiently. Alternatively, the minigene (carrying the
5' AAV ITR-heterologous molecule-3' ITR) may be stably integrated
into the genome of the host cell, either chromosomally or as an
episome. In certain embodiments, the minigene may be present in
multiple copies, optionally in head-to-head, head-to-tail, or
tail-to-tail concatamers. Suitable transfection techniques are
known and may readily be utilized to deliver the minigene to the
host cell.
[0127] Generally, when delivering the vector comprising the
minigene by transfection, the vector is delivered in an amount from
about 5 .mu.g to about 100 .mu.g DNA, about 10 .mu.g to about 50
.mu.g DNA to about 1.times.10.sup.4 cells to about
1.times.10.sup.13 cells, or about 1.times.10.sup.5 cells. However,
the relative amounts of vector DNA to host cells may be adjusted,
taking into consideration such factors as the selected vector, the
delivery method and the host cells selected.
[0128] B. Rep and Cap Sequences
[0129] In addition to the minigene, the host cell contains the
sequences which drive expression of a novel AAV capsid protein of
the invention (or a capsid protein comprising a fragment thereof)
in the host cell and rep sequences of the same source as the source
of the AAV ITRs found in the minigene, or a cross-complementing
source. The AAV cap and rep sequences may be independently obtained
from an AAV source as described above and may be introduced into
the host cell in any manner known to one in the art as described
above. Additionally, when pseudotyping an AAV vector in (e.g., an
AAV9/HU.14 capsid), the sequences encoding each of the essential
rep proteins may be supplied by different AAV sources (e.g., AAV1,
AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8). For example, the
rep78/68 sequences may be from AAV2, whereas the rep52/40 sequences
may be from AAV8.
[0130] In one embodiment, the host cell stably contains the capsid
protein under the control of a suitable promoter, such as those
described above. Most desirably, in this embodiment, the capsid
protein is expressed under the control of an inducible promoter. In
another embodiment, the capsid protein is supplied to the host cell
in trans. When delivered to the host cell in trans, the capsid
protein may be delivered via a plasmid which contains the sequences
necessary to direct expression of the selected capsid protein in
the host cell. Most desirably, when delivered to the host cell in
trans, the plasmid carrying the capsid protein also carries other
sequences required for packaging the rAAV, e.g., the rep
sequences.
[0131] In another embodiment, the host cell stably contains the rep
sequences under the control of a suitable promoter, such as those
described above. Most desirably, in this embodiment, the essential
rep proteins are expressed under the control of an inducible
promoter. In another embodiment, the rep proteins are supplied to
the host cell in trans. When delivered to the host cell in trans,
the rep proteins may be delivered via a plasmid which contains the
sequences necessary to direct expression of the selected rep
proteins in the host cell. Most desirably, when delivered to the
host cell in trans, the plasmid carrying the capsid protein also
carries other sequences required for packaging the rAAV, e.g., the
rep and cap sequences.
[0132] Thus, in one embodiment, the rep and cap sequences may be
transfected into the host cell on a single nucleic acid molecule
and exist stably in the cell as an episome. In another embodiment,
the rep and cap sequences are stably integrated into the chromosome
of the cell. Another embodiment has the rep and cap sequences
transiently expressed in the host cell. For example, a useful
nucleic acid molecule for such transfection comprises, from 5' to
3', a promoter, an optional spacer interposed between the promoter
and the start site of the rep gene sequence, an AAV rep gene
sequence, and an AAV cap gene sequence.
[0133] Optionally, the rep and/or cap sequences may be supplied on
a vector that contains other DNA sequences that are to be
introduced into the host cells. For instance, the vector may
contain the rAAV construct comprising the minigene. The vector may
comprise one or more of the genes encoding the helper functions,
e.g., the adenoviral proteins E1, E2a, and E4 ORF6, and the gene
for VAI RNA.
[0134] Preferably, the promoter used in this construct may be any
of the constitutive, inducible or native promoters known to one of
skill in the art or as discussed above. In one embodiment, an AAV
P5 promoter sequence is employed. The selection of the AAV to
provide any of these sequences does not limit the invention.
[0135] In another preferred embodiment, the promoter for rep is an
inducible promoter, such as are discussed above in connection with
the transgene regulatory elements. One preferred promoter for rep
expression is the T7 promoter. The vector comprising the rep gene
regulated by the T7 promoter and the cap gene, is transfected or
transformed into a cell which either constitutively or inducibly
expresses the T7 polymerase. See International Patent Publication
No. WO 98/10088, published Mar. 12, 1998.
[0136] The spacer is an optional element in the design of the
vector. The spacer is a DNA sequence interposed between the
promoter and the rep gene ATG start site. The spacer may have any
desired design; that is, it may be a random sequence of
nucleotides, or alternatively, it may encode a gene product, such
as a marker gene. The spacer may contain genes which typically
incorporate start/stop and polyA sites. The spacer may be a
non-coding DNA sequence from a prokaryote or eukaryote, a
repetitive non-coding sequence, a coding sequence without
transcriptional controls or a coding sequence with transcriptional
controls. Two exemplary sources of spacer sequences are the phage
ladder sequences or yeast ladder sequences, which are available
commercially, e.g., from Gibco or Invitrogen, among others. The
spacer may be of any size sufficient to reduce expression of the
rep78 and rep68 gene products, leaving the rep52, rep40 and cap
gene products expressed at normal levels. The length of the spacer
may therefore range from about 10 bp to about 10.0 kbp, preferably
in the range of about 100 bp to about 8.0 kbp. To reduce the
possibility of recombination, the spacer is preferably less than 2
kbp in length; however, the invention is not so limited.
[0137] Although the molecule(s) providing rep and cap may exist in
the host cell transiently (i.e., through transfection), it is
preferred that one or both of the rep and cap proteins and the
promoter(s) controlling their expression be stably expressed in the
host cell, e.g., as an episome or by integration into the
chromosome of the host cell. The methods employed for constructing
embodiments of this invention are conventional genetic engineering
or recombinant engineering techniques such as those described in
the references above. While this specification provides
illustrative examples of specific constructs, using the information
provided herein, one of skill in the art may select and design
other suitable constructs, using a choice of spacers, P5 promoters,
and other elements, including at least one translational start and
stop signal, and the optional addition of polyadenylation
sites.
[0138] In another embodiment of this invention, the rep or cap
protein may be provided stably by a host cell.
[0139] C. The Helper Functions
[0140] The packaging host cell also requires helper functions in
order to package the rAAV of the invention. Optionally, these
functions may be supplied by a herpesvirus. Most desirably, the
necessary helper functions are each provided from a human or
non-human primate adenovirus source, such as those described above
and/or are available from a variety of sources, including the
American Type Culture Collection (ATCC), Manassas, Va. (US). In one
currently preferred embodiment, the host cell is provided with
and/or contains an E1a gene product, an E1b gene product, an E2a
gene product, and/or an E4 ORF6 gene product. The host cell may
contain other adenoviral genes such as VAI RNA, but these genes are
not required. In a preferred embodiment, no other adenovirus genes
or gene functions are present in the host cell.
[0141] By "adenoviral DNA which expresses the E1a gene product", it
is meant any adenovirus sequence encoding E1a or any functional E1a
portion. Adenoviral DNA which expresses the E2a gene product and
adenoviral DNA which expresses the E4 ORF6 gene products are
defined similarly. Also included are any alleles or other
modifications of the adenoviral gene or functional portion thereof.
Such modifications may be deliberately introduced by resort to
conventional genetic engineering or mutagenic techniques to enhance
the adenoviral function in some manner, as well as naturally
occurring allelic variants thereof. Such modifications and methods
for manipulating DNA to achieve these adenovirus gene functions are
known to those of skill in the art.
[0142] The adenovirus E1a, E1b, E2a, and/or E4ORF6 gene products,
as well as any other desired helper functions, can be provided
using any means that allows their expression in a cell. Each of the
sequences encoding these products may be on a separate vector, or
one or more genes may be on the same vector. The vector may be any
vector known in the art or disclosed above, including plasmids,
cosmids and viruses. Introduction into the host cell of the vector
may be achieved by any means known in the art or as disclosed
above, including transfection, infection, electroporation, liposome
delivery, membrane fusion techniques, high velocity DNA-coated
pellets, viral infection and protoplast fusion, among others. One
or more of the adenoviral genes may be stably integrated into the
genome of the host cell, stably expressed as episomes, or expressed
transiently. The gene products may all be expressed transiently, on
an episome or stably integrated, or some of the gene products may
be expressed stably while others are expressed transiently.
Furthermore, the promoters for each of the adenoviral genes may be
selected independently from a constitutive promoter, an inducible
promoter or a native adenoviral promoter. The promoters may be
regulated by a specific physiological state of the organism or cell
(i.e., by the differentiation state or in replicating or quiescent
cells) or by exogenously added factors, for example.
[0143] D. Host Cells And Packaging Cell Lines
[0144] The host cell itself may be selected from any biological
organism, including prokaryotic (e.g., bacterial) cells, and
eukaryotic cells, including, insect cells, yeast cells and
mammalian cells. Particularly desirable host cells are selected
from among any mammalian species, including, without limitation,
cells such as A549, WEHI, 3T3, 10T1/2, BHK, MDCK, COS 1, COS 7, BSC
1, BSC 40, BMT 10, VERO, WI38, HeLa, 293 cells (which express
functional adenoviral E1), Saos, C2C12, L cells, HT1080, HepG2 and
primary fibroblast, hepatocyte and myoblast cells derived from
mammals including human, monkey, mouse, rat, rabbit, and hamster.
The selection of the mammalian species providing the cells is not a
limitation of this invention; nor is the type of mammalian cell,
i.e., fibroblast, hepatocyte, tumor cell, etc. The requirements for
the cell used is that it not carry any adenovirus gene other than
E1, E2a and/or E4 ORF6; it not contain any other virus gene which
could result in homologous recombination of a contaminating virus
during the production of rAAV; and it is capable of infection or
transfection of DNA and expression of the transfected DNA. In a
preferred embodiment, the host cell is one that has rep and cap
stably transfected in the cell.
[0145] One host cell useful in the present invention is a host cell
stably transformed with the sequences encoding rep and cap, and
which is transfected with the adenovirus E1, E2a, and E4ORF6 DNA
and a construct carrying the minigene as described above. Stable
rep and/or cap expressing cell lines, such as B-50 (International
Patent Application Publication No. WO 99/15685), or those described
in U.S. Pat. No. 5,658,785, may also be similarly employed. Another
desirable host cell contains the minimum adenoviral DNA which is
sufficient to express E4 ORF6. Yet other cell lines can be
constructed using the novel AAV9 cap sequences of the
invention.
[0146] The preparation of a host cell according to this invention
involves techniques such as assembly of selected DNA sequences.
This assembly may be accomplished utilizing conventional
techniques. Such techniques include cDNA and genomic cloning, which
are well known and are described in Sambrook et al., cited above,
use of overlapping oligonucleotide sequences of the adenovirus and
AAV genomes, combined with polymerase chain reaction, synthetic
methods, and any other suitable methods which provide the desired
nucleotide sequence.
[0147] Introduction of the molecules (as plasmids or viruses) into
the host cell may also be accomplished using techniques known to
the skilled artisan and as discussed throughout the specification.
In preferred embodiment, standard transfection techniques are used,
e.g., CaPO.sub.4 transfection or electroporation, and/or infection
by hybrid adenovirus/AAV vectors into cell lines such as the human
embryonic kidney cell line HEK 293 (a human kidney cell line
containing functional adenovirus E1 genes which provides
trans-acting E1 proteins).
[0148] The AAV9/HU.14 based vectors which are generated by one of
skill in the art are beneficial for gene delivery to selected host
cells and gene therapy patients since no neutralization antibodies
to AAV9/HU.14 have been found in the human population. One of skill
in the art may readily prepare other rAAV viral vectors containing
the AAV9/HU.14 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 AAV9/HU.14
sequence and AAV capsids from another source.
[0149] One of skill in the art will readily understand that the
novel AAV 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. Similarly, one of skill in the art can readily
select other fragments of the AAV genome of the invention for use
in a variety of rAAV and non-rAAV 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.
[0150] Thus, the invention further provides vectors generated using
the nucleic acid and amino acid sequences of the novel AAV of the
invention. Such vectors are useful for a variety of purposes,
including for delivery of therapeutic molecules and for use in
vaccine regimens. Particularly desirable for delivery of
therapeutic molecules are recombinant AAV containing capsids of the
novel AAV of the invention. These, or other vector constructs
containing novel AAV sequences of the invention may be used in
vaccine regimens, e.g., for co-delivery of a cytokine, or for
delivery of the immunogen itself.
IV. Recombinant Viruses And Uses Therefor
[0151] Using the techniques described herein, one of skill in the
art can generate a rAAV having a capsid of an AAV of the invention
or having a capsid containing one or more fragments of an AAV of
the invention. In one embodiment, a full-length capsid from a
single AAV, e.g., hu.14/AAV9 [SEQ ID NO: 123] can be utilized. In
another embodiment, a full-length capsid may be generated which
contains one or more fragments of the novel AAV capsid of the
invention fused in frame with sequences from another selected AAV,
or from heterologous (i.e., non-contiguous) portions of the same
AAV. For example, a rAAV may contain one or more of the novel
hypervariable region sequences of AAV9/HU.14. Alternatively, the
unique AAV sequences of the invention may be used in constructs
containing other viral or non-viral sequences. Optionally, a
recombinant virus may carry AAV rep sequences encoding one or more
of the AAV rep proteins.
[0152] A. Delivery of Viruses
[0153] 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 AAV9/HU.14 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.
[0154] 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 that direct expression thereof and AAV9 capsid
proteins.
[0155] Optionally, a sample from the host may be first assayed for
the presence of antibodies to a selected AAV source (e.g., a
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 source
are preferred for delivery, e.g., by the absence of neutralizing
antibodies specific for that capsid source.
[0156] In one aspect of this method, the delivery of vector with
AAV capsid proteins of the invention may precede or follow delivery
of a gene via a vector with a different 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
sources (and preferably, different serotypes) which differ from the
first vector. For example, if a first vector has AAV9/HU.14 capsid
proteins, subsequently administered vectors may have capsid
proteins selected from among the other AAV, optionally, from
another serotype or from another clade.
[0157] Optionally, multiple rAAV vectors can be used to deliver
large transgenes or multiple transgenes by co-administration of
rAAV vectors concatamerize in vivo to form a single vector genome.
In such an embodiment, a first AAV may carry an expression cassette
which expresses a single transgene (or a subunit thereof) and a
second AAV may carry an expression cassette which expresses a
second transgene (or a different subunit) for co-expression in the
host cell. A first AAV may carry an expression cassette which is a
first piece of a polycistronic construct (e.g., a promoter and
transgene, or subunit) and a second AAV may carry an expression
cassette which is a second piece of a polycistronic construct
(e.g., transgene or subunit and a polyA sequence). These two pieces
of a polycistronic construct concatamerize in vivo to form a single
vector genome that co-expresses the transgenes delivered by the
first and second AAV. In such embodiments, the rAAV vector carrying
the first expression cassette and the rAAV vector carrying the
second expression cassette can be delivered in a single
pharmaceutical composition. In other embodiments, the two or more
rAAV vectors are delivered as separate pharmaceutical compositions
which can be administered substantially simultaneously, or shortly
before or after one another.
[0158] The above-described recombinant vectors may be delivered to
host cells according to published methods. The rAAV, preferably
suspended in a physiologically compatible carrier, may be
administered to a human or non-human mammalian patient. Suitable
carriers may be readily selected by one of skill in the art in view
of the indication for which the transfer virus is directed. For
example, one suitable carrier includes saline, which may be
formulated with a variety of buffering solutions (e.g., phosphate
buffered saline). Other exemplary carriers include sterile saline,
lactose, sucrose, calcium phosphate, gelatin, dextran, agar,
pectin, peanut oil, sesame oil, and water. The selection of the
carrier is not a limitation of the present invention.
[0159] Optionally, the compositions of the invention may contain,
in addition to the rAAV and carrier(s), other conventional
pharmaceutical ingredients, such as preservatives, or chemical
stabilizers. Suitable exemplary preservatives include
chlorobutanol, potassium sorbate, sorbic acid, sulfur dioxide,
propyl gallate, the parabens, ethyl vanillin, glycerin, phenol, and
parachlorophenol. Suitable chemical stabilizers include gelatin and
albumin
[0160] The 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
a desired organ (e.g., the liver (optionally via the hepatic
artery) or lung), oral, inhalation, intranasal, intratracheal,
intraarterial, intraocular, intravenous, intramuscular,
subcutaneous, intradermal, and other parental routes of
administration. Routes of administration may be combined, if
desired.
[0161] 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 0.1 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
for delivery to large organs (e.g., liver, muscle, heart and lung)
may be about 5.times.10.sup.10 to 5.times.10.sup.13 AAV genomes per
1 kg, at a volume of about 1 to 100 mL. A preferred dosage for
delivery to eye is about 5.times.10.sup.9 to 5.times.10.sup.12
genome copies, at a volume of about 0.1 mL to 1 mL. 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.
[0162] Examples of therapeutic products and immunogenic products
for delivery by the AAV-containing vectors of the invention are
provided below. These vectors may be used for a variety of
therapeutic or vaccinal regimens, as described herein.
Additionally, these vectors may be delivered in combination with
one or more other vectors or active ingredients in a desired
therapeutic and/or vaccinal regimen.
[0163] B. Therapeutic Transgenes
[0164] Useful therapeutic products encoded by the transgene include
hormones and growth and differentiation factors including, without
limitation, insulin, glucagon, growth hormone (GH), parathyroid
hormone (PTH), growth hormone releasing factor (GRF), follicle
stimulating hormone (FSH), luteinizing hormone (LH), human
chorionic gonadotropin (hCG), vascular endothelial growth factor
(VEGF), angiopoietins, angiostatin, granulocyte colony stimulating
factor (GCSF), erythropoietin (EPO), connective tissue growth
factor (CTGF), basic fibroblast growth factor (bFGF), acidic
fibroblast growth factor (aFGF), epidermal growth factor (EGF),
platelet-derived growth factor (PDGF), insulin growth factors I and
II (IGF-I and IGF-II), any one of the transforming growth factor
.alpha. superfamily, including TGF.alpha., activins, inhibins, or
any of the bone morphogenic proteins (BMP) BMPs 1-15, any one of
the heregluin/neuregulin/ARIA/neu differentiation factor (NDF)
family of growth factors, nerve growth factor (NGF), brain-derived
neurotrophic factor (BDNF), neurotrophins NT-3 and NT-4/5, ciliary
neurotrophic factor (CNTF), glial cell line derived neurotrophic
factor (GDNF), neurturin, agrin, any one of the family of
semaphorins/collapsins, netrin-1 and netrin-2, hepatocyte growth
factor (HGF), ephrins, noggin, sonic hedgehog and tyrosine
hydroxylase.
[0165] Other useful transgene products include proteins that
regulate the immune system including, without limitation, cytokines
and lymphokines such as thrombopoietin (TPO), interleukins (IL)
IL-1 through IL-25 (including, e.g., IL-2, IL-4, IL-12 and IL-18),
monocyte chemoattractant protein, leukemia inhibitory factor,
granulocyte-macrophage colony stimulating factor, Fas ligand, tumor
necrosis factors .alpha. and .beta., interferons .alpha., .beta.,
and .gamma., stem cell factor, flk-2/flt3 ligand. Gene products
produced by the immune system are also useful in the invention.
These include, without limitations, immunoglobulins IgG, IgM, IgA,
IgD and IgE, chimeric immunoglobulins, humanized antibodies, single
chain antibodies, T cell receptors, chimeric T cell receptors,
single chain T cell receptors, class I and class II MHC molecules,
as well as engineered immunoglobulins and MHC molecules. Useful
gene products also include complement regulatory proteins such as
complement regulatory proteins, membrane cofactor protein (MCP),
decay accelerating factor (DAF), CR1, CF2 and CD59.
[0166] Still other useful gene products include any one of the
receptors for the hormones, growth factors, cytokines, lymphokines,
regulatory proteins and immune system proteins. The invention
encompasses receptors for cholesterol regulation and/or lipid
modulation, including the low density lipoprotein (LDL) receptor,
high density lipoprotein (HDL) receptor, the very low density
lipoprotein (VLDL) receptor, and scavenger receptors. The invention
also encompasses gene products such as members of the steroid
hormone receptor superfamily including glucocorticoid receptors and
estrogen receptors, Vitamin D receptors and other nuclear
receptors. In addition, useful gene products include transcription
factors such as jun, fos, max, mad, serum response factor (SRF),
AP-1, AP2, myb, MyoD and myogenin, ETS-box containing proteins,
TFE3, E2F, ATF1, ATF2, ATF3, ATF4, ZF5, NFAT, CREB, HNF-4, C/EBP,
SP1, CCAAT-box binding proteins, interferon regulation factor
(IRF-1), Wilms tumor protein, ETS-binding protein, STAT, GATA-box
binding proteins, e.g., GATA-3, and the forkhead family of winged
helix proteins.
[0167] Other useful gene products include, carbamoyl synthetase I,
ornithine transcarbamylase, arginosuccinate synthetase,
arginosuccinate lyase, arginase, fumarylacetacetate hydrolase,
phenylalanine hydroxylase, alpha-1 antitrypsin,
glucose-6-phosphatase, porphobilinogen deaminase, cystathione
beta-synthase, branched chain ketoacid decarboxylase, albumin,
isovaleryl-coA dehydrogenase, propionyl CoA carboxylase, methyl
malonyl CoA mutase, glutaryl CoA dehydrogenase, insulin,
beta-glucosidase, pyruvate carboxylate, hepatic phosphorylase,
phosphorylase kinase, glycine decarboxylase, H-protein, T-protein,
a cystic fibrosis transmembrane regulator (CFTR) sequence, and a
dystrophin gene product [e.g., a mini- or micro-dystrophin]. Still
other useful gene products include enzymes such as may be useful in
enzyme replacement therapy, which is useful in a variety of
conditions resulting from deficient activity of enzyme. For
example, enzymes that contain mannose-6-phosphate may be utilized
in therapies for lysosomal storage diseases (e.g., a suitable gene
includes that encoding .beta.-glucuronidase (GUSB)).
[0168] Still other useful gene products include those used for
treatment of hemophilia, including hemophilia B (including Factor
IX) and hemophilia A (including Factor VIII and its variants, such
as the light chain and heavy chain of the heterodimer and the
B-deleted domain; U.S. Pat. Nos. 6,200,560 and 6,221,349). The
Factor VIII gene codes for 2351 amino acids and the protein has six
domains, designated from the amino to the terminal carboxy terminus
as A1-A2-B-A3-C1-C2 [Wood et al, Nature, 312:330 (1984); Vehar et
al., Nature 312:337 (1984); and Toole et al, Nature, 342:337
(1984)]. Human Factor VIII is processed within the cell to yield a
heterodimer primarily comprising a heavy chain containing the A1,
A2 and B domains and a light chain containing the A3, C1 and C2
domains. Both the single chain polypeptide and the heterodimer
circulate in the plasma as inactive precursors, until activated by
thrombin cleavage between the A2 and B domains, which releases the
B domain and results in a heavy chain consisting of the A 1 and A2
domains. The B domain is deleted in the activated procoagulant form
of the protein. Additionally, in the native protein, two
polypeptide chains ("a" and "b"), flanking the B domain, are bound
to a divalent calcium cation.
[0169] In some embodiments, the minigene comprises first 57 base
pairs of the Factor VIII heavy chain which encodes the 10 amino
acid signal sequence, as well as the human growth hormone (hGH)
polyadenylation sequence. In alternative embodiments, the minigene
further comprises the A1 and A2 domains, as well as 5 amino acids
from the N-terminus of the B domain, and/or 85 amino acids of the
C-terminus of the B domain, as well as the A3, C1 and C2 domains.
In yet other embodiments, the nucleic acids encoding Factor VIII
heavy chain and light chain are provided in a single minigene
separated by 42 nucleic acids coding for 14 amino acids of the B
domain [U.S. Pat. No. 6,200,560].
[0170] As used herein, a therapeutically effective amount is an
amount of AAV vector that produces sufficient amounts of Factor
VIII to decrease the time it takes for a subject's blood to clot.
Generally, severe hemophiliacs having less than 1% of normal levels
of Factor VIII have a whole blood clotting time of greater than 60
minutes as compared to approximately 10 minutes for
non-hemophiliacs.
[0171] The present invention is not limited to any specific Factor
VIII sequence. Many natural and recombinant forms of Factor VIII
have been isolated and generated. Examples of naturally occurring
and recombinant forms of Factor VII can be found in the patent and
scientific literature including, U.S. Pat. Nos. 5,563,045,
5,451,521, 5,422,260, 5,004,803, 4,757,006, 5,661,008, 5,789,203,
5,681,746, 5,595,886, 5,045,455, 5,668,108, 5,633,150, 5,693,499,
5,587,310, 5,171,844, 5,149,637, 5,112,950, 4,886,876;
International Patent Publication Nos. WO 94/11503, WO 87/07144, WO
92/16557, WO 91/09122, WO 97/03195, WO 96/21035, and WO 91/07490;
European Patent Application Nos. EP 0 672 138, EP 0 270 618, EP 0
182 448, EP 0 162 067, EP 0 786 474, EP 0 533 862, EP 0 506 757, EP
0 874 057, EP 0 795 021, EP 0 670 332, EP 0 500 734, EP 0 232 112,
and EP 0 160 457; Sanberg et al., XXth Int. Congress of the World
Fed. Of Hemophilia (1992), and Lind et al., Eur. J. Biochem.,
232:19 (1995).
[0172] Nucleic acids sequences coding for the above-described
Factor VIII can be obtained using recombinant methods or by
deriving the sequence from a vector known to include the same.
Furthermore, the desired sequence can be isolated directly from
cells and tissues containing the same, using standard techniques,
such as phenol extraction and PCR of cDNA or genomic DNA [See,
e.g., Sambrook et al]. Nucleotide sequences can also be produced
synthetically, rather than cloned. The complete sequence can be
assembled from overlapping oligonucleotides prepared by standard
methods and assembled into a complete coding sequence [See, e.g.,
Edge, Nature 292:757 (1981); Nambari et al, Science, 223:1299
(1984); and Jay et al, J. Biol. Chem. 259:6311 (1984).
[0173] Furthermore, the invention is not limited to human Factor
VIII. Indeed, it is intended that the present invention encompass
Factor VIII from animals other than humans, including but not
limited to companion animals (e.g., canine, felines, and equines),
livestock (e.g., bovines, caprines and ovines), laboratory animals,
marine mammals, large cats, etc.
[0174] The AAV vectors may contain a nucleic acid coding for
fragments of Factor VIII which is itself not biologically active,
yet when administered into the subject improves or restores the
blood clotting time. For example, as discussed above, the Factor
VIII protein comprises two polypeptide chains: a heavy chain and a
light chain separated by a B-domain which is cleaved during
processing. As demonstrated by the present invention, co-tranducing
recipient cells with the Factor VIII heavy and light chains leads
to the expression of biologically active Factor VIII. Because most
hemophiliacs contain a mutation or deletion in only one of the
chains (e.g., heavy or light chain), it may be possible to
administer only the chain defective in the patient to supply the
other chain
[0175] Other useful gene products include non-naturally occurring
polypeptides, such as chimeric or hybrid polypeptides having a
non-naturally occurring amino acid sequence containing insertions,
deletions or amino acid substitutions. For example, single-chain
engineered immunoglobulins could be useful in certain
immunocompromised patients. Other types of non-naturally occurring
gene sequences include antisense molecules and catalytic nucleic
acids, such as ribozymes, which could be used to reduce
overexpression of a target.
[0176] Reduction and/or modulation of expression of a gene is
particularly desirable for treatment of hyperproliferative
conditions characterized by hyperproliferating cells, as are
cancers and psoriasis. Target polypeptides include those
polypeptides which are produced exclusively or at higher levels in
hyperproliferative cells as compared to normal cells. Target
antigens include polypeptides encoded by oncogenes such as myb,
myc, fyn, and the translocation gene bcr/abl, ras, src, P53, neu,
trk and EGRF. In addition to oncogene products as target antigens,
target polypeptides for anti-cancer treatments and protective
regimens include variable regions of antibodies made by B cell
lymphomas and variable regions of T cell receptors of T cell
lymphomas which, in some embodiments, are also used as target
antigens for autoimmune disease. Other tumor-associated
polypeptides can be used as target polypeptides such as
polypeptides which are found at higher levels in tumor cells
including the polypeptide recognized by monoclonal antibody 17-1A
and folate binding polypeptides.
[0177] Other suitable therapeutic polypeptides and proteins include
those which may be useful for treating individuals suffering from
autoimmune diseases and disorders by conferring a broad based
protective immune response against targets that are associated with
autoimmunity including cell receptors and cells which produce
"self"-directed antibodies. T cell mediated autoimmune diseases
include Rheumatoid arthritis (RA), multiple sclerosis (MS),
Sjogren's syndrome, sarcoidosis, insulin dependent diabetes
mellitus (IDDM), autoimmune thyroiditis, reactive arthritis,
ankylosing spondylitis, scleroderma, polymyositis, dermatomyositis,
psoriasis, vasculitis, Wegener's granulomatosis, Crohn's disease
and ulcerative colitis. Each of these diseases is characterized by
T cell receptors (TCRs) that bind to endogenous antigens and
initiate the inflammatory cascade associated with autoimmune
diseases.
[0178] C. Immunogenic Transgenes
[0179] Suitably, the AAV vectors of the invention avoid the
generation of immune responses to the AAV sequences contained
within the vector. However, these vectors may nonetheless be
formulated in a manner that permits the expression of a transgene
carried by the vectors to induce an immune response to a selected
antigen. For example, in order to promote an immune response, the
transgene may be expressed from a constitutive promoter, the vector
can be adjuvanted as described herein, and/or the vector can be put
into degenerating tissue.
[0180] Examples of suitable immunogenic transgenes include those
selected from a variety of viral families. Examples of desirable
viral families against which an immune response would be desirable
include, the picornavirus family, which includes the genera
rhinoviruses, which are responsible for about 50% of cases of the
common cold; the genera enteroviruses, which include polioviruses,
coxsackieviruses, echoviruses, and human enteroviruses such as
hepatitis A virus; and the genera apthoviruses, which are
responsible for foot and mouth diseases, primarily in non-human
animals. Within the picornavirus family of viruses, target antigens
include the VP1, VP2, VP3, VP4, and VPG. Other viral families
include the astroviruses and the calcivirus family. The calcivirus
family encompasses the Norwalk group of viruses, which are an
important causative agent of epidemic gastroenteritis. Still
another viral family desirable for use in targeting antigens for
inducing immune responses in humans and non-human animals is the
togavirus family, which includes the genera alphavirus, which
include Sindbis viruses, RossRiver virus, and Venezuelan, Eastern
& Western Equine encephalitis, and rubivirus, including Rubella
virus. The flaviviridae family includes dengue, yellow fever,
Japanese encephalitis, St. Louis encephalitis and tick borne
encephalitis viruses. Other target antigens may be generated from
the Hepatitis C or the coronavirus family, which includes a number
of non-human viruses such as infectious bronchitis virus (poultry),
porcine transmissible gastroenteric virus (pig), porcine
hemagglutinatin encephalomyelitis virus (pig), feline infectious
peritonitis virus (cat), feline enteric coronavirus (cat), canine
coronavirus (dog), and human respiratory coronaviruses, which may
cause the common cold and/or non-A, B or C hepatitis, and which
include the putative cause of sudden acute respiratory syndrome
(SARS). Within the coronavirus family, target antigens include the
E1 (also called M or matrix protein), E2 (also called S or Spike
protein), E3 (also called HE or hemagglutin-elterose) glycoprotein
(not present in all coronaviruses), or N (nucleocapsid). Still
other antigens may be targeted against the arterivirus family and
the rhabdovirus family. The rhabdovirus family includes the genera
vesiculovirus (e.g., Vesicular Stomatitis Virus), and the general
lyssavirus (e.g., rabies). Within the rhabdovirus family, suitable
antigens may be derived from the G protein or the N protein. The
family filoviridae, which includes hemorrhagic fever viruses such
as Marburg and Ebola virus may be a suitable source of antigens.
The paramyxovirus family includes parainfluenza Virus Type 1,
parainfluenza Virus Type 3, bovine parainfluenza Virus Type 3,
rubulavirus (mumps virus, parainfluenza Virus Type 2, parainfluenza
virus Type 4, Newcastle disease virus (chickens), rinderpest,
morbillivirus, which includes measles and canine distemper, and
pneumovirus, which includes respiratory syncytial virus. The
influenza virus is classified within the family orthomyxovirus and
is a suitable source of antigen (e.g., the HA protein, the N1
protein). The bunyavirus family includes the genera bunyavirus
(California encephalitis, La Crosse), phlebovirus (Rift Valley
Fever), hantavirus (puremala is a hemahagin fever virus),
nairovirus (Nairobi sheep disease) and various unassigned
bungaviruses. The arenavirus family provides a source of antigens
against LCM and Lassa fever virus. Another source of antigens is
the bornavirus family. The reovirus family includes the genera
reovirus, rotavirus (which causes acute gastroenteritis in
children), orbiviruses, and cultivirus (Colorado Tick fever,
Lebombo (humans), equine encephalosis, blue tongue). The retrovirus
family includes the sub-family oncorivirinal which encompasses such
human and veterinary diseases as feline leukemia virus, HTLVI and
HTLVII, lentivirinal (which includes HIV, simian immunodeficiency
virus, feline immunodeficiency virus, equine infectious anemia
virus, and spumavirinal). The papovavirus family includes the
sub-family polyomaviruses (BKU and JCU viruses) and the sub-family
papillomavirus (associated with cancers or malignant progression of
papilloma). The adenovirus family includes viruses (EX, AD7, ARD,
O.B.) which cause respiratory disease and/or enteritis. The
parvovirus family includes feline parvovirus (feline enteritis),
feline panleucopeniavirus, canine parvovirus, and porcine
parvovirus. The herpesvirus family includes the sub-family
alphaherpesvirinae, which encompasses the genera simplexvirus
(HSVI, HSVII), varicellovirus (pseudorabies, varicella zoster) and
the sub-family betaherpesvirinae, which includes the genera
cytomegalovirus (HCMV, muromegalovirus) and the sub-family
gammaherpesvirinae, which includes the genera lymphocryptovirus,
EBV (Burkitts lymphoma), human herpesviruses 6A, 6B and 7, Kaposi's
sarcoma-associated herpesvirus and cercopithecine herpesvirus (B
virus), infectious rhinotracheitis, Marek's disease virus, and
rhadinovirus. The poxvirus family includes the sub-family
chordopoxvirinae, which encompasses the genera orthopoxvirus
(Variola major (Smallpox) and Vaccinia (Cowpox)), parapoxvirus,
avipoxvirus, capripoxvirus, leporipoxvirus, suipoxvirus, and the
sub-family entomopoxvirinae. The hepadnavirus family includes the
Hepatitis B virus. One unclassified virus which may be suitable
source of antigens is the Hepatitis delta virus, Hepatitis E virus,
and prions. Another virus which is a source of antigens is Nipan
Virus. Still other viral sources may include avian infectious
bursal disease virus and porcine respiratory and reproductive
syndrome virus. The alphavirus family includes equine arteritis
virus and various Encephalitis viruses.
[0181] The present invention may also encompass immunogens which
are useful to immunize a human or non-human animal against other
pathogens including bacteria, fungi, parasitic microorganisms or
multicellular parasites which infect human and non-human
vertebrates, or from a cancer cell or tumor cell. Examples of
bacterial pathogens include pathogenic gram-positive cocci include
pneumococci; staphylococci (and the toxins produced thereby, e.g.,
enterotoxin B); and streptococci. Pathogenic gram-negative cocci
include meningococcus; gonococcus. Pathogenic enteric gram-negative
bacilli include enterobacteriaceae; pseudomonas, acinetobacteria
and eikenella; melioidosis; salmonella; shigella; haemophilus;
moraxella; H ducreyi (which causes chancroid); brucella species
(brucellosis); Francisella tularensis (which causes tularemia);
Yersinia pestis (plague) and other yersinia (pasteurella);
streptobacillus moniliformis and spirillum; Gram-positive bacilli
include Listeria monocytogenes; erysipelothrix rhusiopathiae;
Corynebacterium diphtheria (diphtheria); cholera; B. anthracia
(anthrax); donovanosis (granuloma inguinale); and bartonellosis.
Diseases caused by pathogenic anaerobic bacteria include tetanus;
botulism (Clostridum botulinum and its toxin); Clostridium
perfringens and its epsilon toxin; other clostridia; tuberculosis;
leprosy; and other mycobacteria. Pathogenic spirochetal diseases
include syphilis; treponematoses: yaws, pinta and endemic syphilis;
and leptospirosis. Other infections caused by higher pathogen
bacteria and pathogenic fungi include glanders (Burkholderia
mallei); actinomycosis; nocardiosis; cryptococcosis, blastomycosis,
histoplasmosis and coccidioidomycosis; candidiasis, aspergillosis,
and mucormycosis; sporotrichosis; paracoccidiodomycosis,
petriellidiosis, torulopsosis, mycetoma and chromomycosis; and
dermatophytosis. Rickettsial infections include Typhus fever, Rocky
Mountain spotted fever, Q fever (Coxiella burnetti), and
Rickettsialpox. Examples of mycoplasma and chlamydial infections
include: Mycoplasma pneumoniae; lymphogranuloma venereum;
psittacosis; and perinatal chlamydial infections. Pathogenic
eukaryotes encompass pathogenic protozoans and helminths and
infections produced thereby include: amebiasis; malaria;
leishmaniasis; trypanosomiasis; toxoplasmosis; Pneumocystis
carinii; Trichans; Toxoplasma gondii; babesiosis; giardiasis;
trichinosis; filariasis; schistosomiasis; nematodes; trematodes or
flukes; and cestode (tapeworm) infections.
[0182] Many of these organisms and/or the toxins produced thereby
have been identified by the Centers for Disease Control [(CDC),
Department of Heath and Human Services, USA], as agents which have
potential for use in biological attacks. For example, some of these
biological agents, include, Bacillus anthracia (anthrax),
Clostridium botulinum and its toxin (botulism), Yersinia pestis
(plague), variola major (smallpox), Francisella tularensis
(tularemia), and viral hemorrhagic fevers [filoviruses (e.g.,
Ebola, Marburg], and arenaviruses [e.g., Lassa, Machupo]), all of
which are currently classified as Category A agents; Coxiella
burnetti (Q fever); Brucella species (brucellosis), Burkholderia
mallei (glanders), Burkholderia pseudomallei (meloidosis), Ricinus
communis and its toxin (ricin toxin), Clostridium perfringens and
its toxin (epsilon toxin), Staphylococcus species and their toxins
(enterotoxin B), Chlamydia psittaci (psittacosis), water safety
threats (e.g., Vibrio cholerae, Crytosporidium parvum), Typhus
fever (Richettsia powazekii), and viral encephalitis (alphaviruses,
e.g., Venezuelan equine encephalitis; eastern equine encephalitis;
western equine encephalitis); all of which are currently classified
as Category B agents; and Nipan virus and hantaviruses, which are
currently classified as Category C agents. In addition, other
organisms, which are so classified or differently classified, may
be identified and/or used for such a purpose in the future. It will
be readily understood that the viral vectors and other constructs
described herein are useful to deliver antigens from these
organisms, viruses, their toxins or other by-products, which will
prevent and/or treat infection or other adverse reactions with
these biological agents.
[0183] Administration of the vectors of the invention to deliver
immunogens against the variable region of the T cells elicit an
immune response including CTLs to eliminate those T cells. In
rheumatoid arthritis (RA), several specific variable regions of
TCRs which are involved in the disease have been characterized.
These TCRs include V-3, V-14, V-17 and V-17. Thus, delivery of a
nucleic acid sequence that encodes at least one of these
polypeptides will elicit an immune response that will target T
cells involved in RA. In multiple sclerosis (MS), several specific
variable regions of TCRs which are involved in the disease have
been characterized. These TCRs include V-7 and V-10. Thus, delivery
of a nucleic acid sequence that encodes at least one of these
polypeptides will elicit an immune response that will target T
cells involved in MS. In scleroderma, several specific variable
regions of TCRs which are involved in the disease have been
characterized. These TCRs include V-6, V-8, V-14 and V-16, V-3C,
V-7, V-14, V-15, V-16, V-28 and V-12. Thus, delivery of a nucleic
acid molecule that encodes at least one of these polypeptides will
elicit an immune response that will target T cells involved in
scleroderma.
[0184] Thus, a rAAV-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 sources. In one embodiment, the rAAV and the cells are
mixed ex vivo; the infected cells are cultured using conventional
methodologies; and the transduced cells are re-infused into the
patient.
[0185] These compositions are particularly well suited to gene
delivery for therapeutic purposes and for immunization, including
inducing protective immunity. Further, the compositions of the
invention may also be used for production of a desired gene product
in vitro. For in vitro production, a desired product (e.g., a
protein) may be obtained from a desired culture following
transfection of host cells with a rAAV containing the molecule
encoding the desired product and culturing the cell culture under
conditions which permit expression. The expressed product 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.
[0186] The following examples illustrate several aspects and
embodiments of the invention.
Example 1--Computational Analysis of Primate AAV Sequences
[0187] A. Collection of Primate Tissues
[0188] Sources of nonhuman primate tissues were described
previously [N. Muzyczka, K. I. Berns, in Fields Virology D. M.
Knipe, P. M. Howley, Eds. (Lippincott Williams & Wilkins,
Philadelphia, 2001), vol. 2, pp. 2327-2359]. Human tissues were
collected from either surgical procedures or postmortem examination
or organ donors through two major national human tissue providers,
Cooperative Human Tissue Network (CHTN) and National Disease
Research Interchange (NDRI). Human tissues used for this study were
comprised of 18 different tissue types that included colon, liver,
lung, spleen, kidney, brain, small bowel, bone marrow, heart, lymph
nodes, skeletal muscle, ovary, pancreas, stomach, esophagus,
cervix, testis and prostate. The tissue samples came from a diverse
group of individuals of different gender, races (Caucasian,
African-American, Asian and Hispanic) and ages (23-83 years). Among
259 samples from 250 individuals analyzed, approximately 28% of
tissues were associated with pathology.
[0189] B. Detection and Isolation of AAV Sequences
[0190] Total cellular DNAs were extracted from human and nonhuman
primate tissues as described previously [R. W. Atchison, et al.,
Science 194, 754-756 (1965)]. Molecular prevalence and tissue
distribution of AAVs in humans were determined by either signature
or full-length cap PCR using the primers and conditions that were
similar to those used for the nonhuman primate analysis. The same
PCR cloning strategy used for the isolation and characterization of
an expanded family of AAVs in nonhuman primates was deployed in the
isolation of AAVs from selected human tissues. Briefly, a 3.1 kb
fragment containing a part of rep and full length cap sequence was
amplified from tissue DNAs by PCR and Topo-cloned (Invitrogen). The
human AAV clones were initially analyzed by restriction mapping to
help identify diversity of AAV sequences, which were subsequently
subjected to full sequence analysis by SeqWright (SeqWright,
Houston, Tex.) with an accuracy of 99.9%. A total of 67 capsid
clones isolated from human tissues were characterized (hu.1-hu.67).
From nonhuman primate tissues, 86 cap clones were sequenced, among
which 70 clones were from rhesus macaques, 6 clones from
cynomologus macaques, 3 clones from pigtailed macaques, 2 clones
from a baboon and 5 clones from a chimpanzee.
[0191] C. Analysis of AAV Sequences
[0192] From all contiguous sequences, AAV capsid viral protein
(vp1) open reading frames (ORFs) were analyzed. The AAV capsid VP1
protein sequences were aligned with the ClustalX1.81.TM. program
[H. D. Mayor, J. L. Melnick, Nature 210, 331-332 (1966)] and an
in-frame DNA alignment was produced with the BioEdit.TM. [U.
Bantel-Schaal, H. Zur Hausen, Virology 134, 52-63 (1984)] software
package. Phylogenies were inferred with the MEGA.TM. v2.1 and the
TreePuzzle.TM. package. Neighbor-Joining, Maximum Parsimony, and
Maximum Likelihood [M. Nei, S. Kumar, Molecular Evolution and
Phylogenetics (Oxford University Press, New York, 2000); H. A.
Schmidt, K. Strimmer, M. Vingron, A. von Haeseler, Bioinformatics
18, 502-4 (March, 2002); N. Saitou, M. Nei, Mol Biol Evol 4, 406-25
(July, 1987)] algorithms were used to confirm similar clustering of
sequences in monophylic groups.
[0193] Clades were then defined from a Neighbor-Joining
phylogenetic tree of all protein sequences. The amino-acid
distances were estimated by making use of Poisson-correction.
Bootstrap analysis was performed with a 1000 replicates. Sequences
were considered monophylic when they had a connecting node within a
0.05 genetic distance. A group of sequences originating from 3 or
more sources was considered a clade. The phylogeny of AAV was
further evaluated for evidence of recombination through a
sequential analysis. Homoplasy was screened for by implementation
of the Split Decomposition algorithm [H. J. Bandelt, A. W. Dress,
Mol Phylogenet Evol 1, 242-52 (September 1992)]. Splits that were
picked up in this manner were then further analyzed for
recombination making use of the Bootscan algorithm in the Simplot
software [M. Nei and S. Kumar, Molecular Evolution and
Phylogenetics (Oxford University Press, New York, 2000)]. A sliding
window of 400 nt (10 nt/step) was used to obtain 100 bootstrap
replicate neighbor joining trees. Subsequently, Split Decomposition
and Neighbor-Joining phylogenies were inferred from the putative
recombination fragments. Significant improvement of bootstrap
values, reduction of splits and regrouping of the hybrid sequences
with their parental sources were considered the criterion for
recombination.
[0194] A number of different cap sequences amplified from 8
different human subjects showed phylogenetic relationships to AAV2
(5') and AAV3 (3') around a common breakpoint at position 1400 of
the Cap DNA sequence, consistent with recombination and the
formation of a hybrid virus. This is the general region of the cap
gene where recombination was detected from isolates from a
mesenteric lymph node of a rhesus macaque [Gao et al., Proc Natl
Acad Sci USA 100, 6081-6086 (May 13, 2002)]. An overall codon based
Z-test for selection was performed implementing the Neib-Gojobori
method [R. M. Kotin, Hum Gene Ther 5, 793-801 (July, 1994)].
[0195] The phylogenetic analyses were repeated excluding the clones
that were positively identified as hybrids. In this analysis, goose
and avian AAVs were included as outgroups [(I. Bossis, J. A.
Chiorini, J Virol 77, 6799-810 (June 2003)]. FIG. 1 is a neighbor
joining tree; similar relationships were obtained using maximum
parsimony and maximum likelihood analyses.
[0196] This analysis demonstrated 11 phylogenetic groups, which are
summarized in Table 1. The species origin of the 6 AAV clades and 5
individual AAV clones (or sets of clones) is represented by the
number or sources from which the sequences were retrieved in the
sampling. The total number of sequences gathered per species and
per grouping is shown in between brackets. References for
previously described sequences per clade are in the right column
Rhesus--rhesus macaques; cyno--cynomologus macaques;
chimp--chimpanzees; pigtail--pigtail macaques.
TABLE-US-00001 TABLE 1 Classification of the number of sources
(sequences) per species and per clade or clone Ba- Pig- Human
Rhesus Cyno boon Chimp tail Clade/ representative A/AAV1 (AAV6) 3
(4) B/AAV2 12 (22) C/AAV2-AAV3 8 (17) hybrid D/AAV7 5 (10) 5 (5)
E/AAV8 7 (9) 7 (16) 1 (2) 1 (3) F/AAV9 3 (3) Clones AAV3 AAV4 1 (3)
AAV5 Ch. 5 1 (1) Rh. 8 2 (2)
[0197] Since, as noted above, recombination is not implemented in
the standard phylogenetic algorithms used, in order to build a
proper phylogenetic tree, those sequences were excluded from the
analysis, of which their recombinative ancestry was established. A
neighbor-joining analysis of all non-recombined sequences is
represented side by side with the clades that did evolve making use
of recombination. A similar output was generated with the different
algorithm used and with the nucleotide sequence as input.
[0198] Additional experiments were performed to evaluate the
relationship of phylogenetic relatedness to function as measured by
serologic activity and tropism, as described in the following
examples.
Example 2--Serological Analysis of Novel Human AAVs
[0199] The last clade obtained as described in the preceding
example was derived from isolates of 3 humans and did not contain a
previously described serotype. Polyclonal antisera were generated
against a representative member of this clade and a comprehensive
study of serologic cross reactivity between the previously
described serotypes was performed. This showed that the new human
clade is serologically distinct from the other known serotypes and
therefore is called Clade F (represented by AAV9).
[0200] Rabbit polyclonal antibodies against AAV serotypes 1-9 were
generated by intramuscularly inoculating the animals with
1.times.10.sup.13 genome copies each of AAV vectors together with
an equal volume of incomplete Freud's adjuvant. The injections were
repeated at day 34 to boost antibody titers. Serological cross
reactivity between AAV 1-9 was determined by assessing the
inhibitory effect of rabbit antisera on transduction of 293 cells
by vectors carrying a reporter gene (AAVCMVEGFP, which carries
enhanced green fluorescent protein) pseudotyped with capsids
derived from different AAV sources. Transduction of 84-31 cells by
AAVCMVEGFP vectors was assessed under a UV microscope. In assessing
serologic relationships between two AAVs, the ability of both
heterologous and homologous sera to neutralize vectors from each
AAV were tested. If neutralization by the serum was at least
16-fold lower against heterologous vectors than homologous vectors
in a reciprocal manner, the two AAVs are considered distinct
serotypes. Neutralization titers were defined as described
previously [(G. P. Gao et al., Proc Natl Acad Sci USA 99, 11854-9
(Sep. 3, 2002)].
TABLE-US-00002 TABLE 2 Serologic evaluation of novel AAV vectors
Vector pseudotypes used in the neutralization assay from rabbit
immunized with AAV2/1 AAV2/2 AAV2/3 AAV2/4 AAV2/5 AAV2/6 AAV2/7
AAV2/8 AAV2/9 AAV2/1 1/163,840 No NAB No NAB No NAB 1/40,960
1/40,960 1/40 No NAB No NAB AAV2/2 1/80 1/81,920 1/5,120 1/20 No
NAB 1/80 1/40 1/40 No NAB AAV2/3 1/1,280 1/2,560 1/40,960 1/20 1/40
1/2,560 1/1,280 1/1,280 No NAB AAV2/4 1/20 No NAB No NAB 1/1,280
1/40 No NAB No NAB No NAB 1/40 AAV2/5 1/20,480 No NAB 1/80 No NAB
1/163,840 1/5,120 1/40 No NAB No NAB AAV2/6 1/81,920 No NAB
1/640.sup. 1/40 1/40 1/327,680 1/40 No NAB 1/40 AAV2/7 1/1,280
1/640.sup. 1/1,280 1/20 No NAB 1/1,280 1/163,840 1/5,120 1/80
AAV2/8 1/20 1/1,280 1/1,280 No NAB 1/20 No NAB 1/640 1/327,680
1/2,560 AAV2/9 No NAB No NAB No NAB No NAB No NAB No NAB 1/20
1/640.sup. 1/20,480
[0201] These data confirm the phylogenetic groupings of the
different clones and clades except for unanticipated serological
reactivity of the structurally distinct AAV5 and AAV1 serotypes
(i.e., ratio of heterologous/homologous titer were 1/4 and 1/8 in
reciprocal titrations).
[0202] The result further indicated that AAVhu.14 had a distinct
serological property and did not have significant cross reactivity
with antisera generated from any known AAV serotypes. The
serological distinctiveness of AAVhu.14 was further supported by
its uniqueness in the capsid structure which shared less than 85%
amino acid sequence identity with all other AAV serotypes compared
in this study. Those findings provided the basis for us to name
AAVhu.14 as a new serotype, AAV9.
Example 3--Evaluation of Primate AAVs as Gene Transfer Vectors
[0203] The biological tropisms of AAVs were studied by generating
vector pseudotyped in which recombinant AAV2 genomes expressing
either GFP or the secreted reporter gene .alpha.-1 antitrypsin
(A1AT) were packaged with capsids derived from various clones and
one representative member from each primate AAV clade for
comparison. For instance, the data obtained from AAV1 was used to
represent Clade A, followed by AAV2 for Clade B, Rh.34 for AAV4,
AAV7 for Clade D, AAV8 for Clade E, and AAVHu.14 for Clade F. AAV5,
AAVCh.5 and AAVRh.8 stand as single AAV genotypes for the
comparison.
[0204] The vectors were evaluated for transduction efficiency in
vitro, based on GFP transduction, and transduction efficiency in
vivo in liver, muscle or lung (FIG. 4).
[0205] A. In Vitro
[0206] Vectors expressing enhanced green fluorescent protein (EGFP)
were used to examine their in vitro transduction efficiency in
84-31 cells and to study their serological properties. For
functional analysis, in vitro transduction of different AAVCMVEGFP
vectors was measured in 84-31 cells that were seeded in a 96 well
plate and infected with pseudotyped AAVCMVEGFP vectors at an MOI of
1.times.10.sup.4 GC per cell. AAV vectors were pseudotyped with
capsids of AAVs 1, 2, 5, 7, 8 and 6 other novel AAVs (Ch.5, Rh.34,
Cy5, rh.20, Rh.8 and AAV9) using the technique described in G. Gao
et al., Proc Natl Acad Sci USA 99, 11854-9 (Sep. 3, 2002). Relative
EGFP transduction efficiency was scored as 0, 1, 2 and 3
corresponding to 0-10%, 10-30%, 30-70% and 70-100% of green cells
estimated using a UV microscope at 48 hours post infection.
[0207] B. In Vivo
[0208] For in vivo studies, human .alpha.-antitrypsin (A1AT) was
selected as a sensitive and quantitative reporter gene in the
vectors and expressed under the control of CMV-enhanced chicken
.beta.-actin promoter. Employment of the CB promoter enables high
levels of tissue non-specific and constitutive A1AT gene transfer
to be achieved and also permits use of the same vector preparation
for gene transfer studies in any tissue of interest. Four to six
week old NCR nude mice were treated with novel AAV vectors
(AAVCBhA1AT) at a dose of 1.times.10.sup.11 genome copies per
animal through intraportal, intratracheal and intramuscular
injections for liver, lung and muscle directed gene transfer,
respectively. Serum samples were collected at different time points
post gene transfer and A1AT concentrations were determined by an
ELISA-based assay and scored as 0, 1, 2 and 3 relative to different
serum A1AT levels at day 28 post gene transfer, depending on the
route of vector administration (Liver: 0=A1AT<400 ng/ml, 1=A1AT
400-1000 ng/ml, 2=A1AT 1000-10,000 ng/ml, 3=A1AT>10,000 ng/ml;
Lung: 0=A1AT<200 ng/ml, 1=A1AT 200-1000 ng/ml, 2=A1AT
1000-10,000 ng/ml, 3=A1AT>10,000 ng/ml; Muscle: 0=A1AT<100
ng/ml, 1=A1AT 100-1000 ng/ml, 2=A1AT 1000-10,000 ng/ml,
3=A1AT>10,000 ng/ml).
[0209] A human AAV, clone 28.4/hu.14 (now named AAV9), has the
ability to transduce liver at a efficiency similar to AAV8, lung 2
logs better than AAV5 and muscle superior to AAV1, whereas the
performance of two other human clones, 24.5 and 16.12 (hu.12 and
hu.13) was marginal in all 3 target tissues. Clone N721.8
(AAVrh.43) is also a high performer in all three tissues.
[0210] To further analyze gene transfer efficiency of AAV9 and rh
43 in comparison with that of bench markers for liver (AAV8), lung
(AAV5) and muscle (AAV1), a dose response experiment was carried
out. Both new vectors demonstrated at least 10 fold more gene
transfer than AAV1 in muscle, similar performance to AAV8 in liver
and 2 logs more efficient than AAV5 in lung.
[0211] A group of AAVs demonstrated efficient gene transfer in all
3 tissues that was similar or superior to the performance of their
bench marker in each tissue has emerged. To date, 3 novel AAVs have
fallen into this category, two from rhesus (rh10 and 43) and one
from human (hu.14 or AAV9). A direct comparison of relative gene
transfer efficiency of those 3 AAVs to their bench markers in the
murine liver, lung and muscle suggests that some primate AAVs with
the best fitness might have evolved from rigorous biological
selection and evolution as "super" viruses. These are particularly
well suited for gene transfer applications.
[0212] C. Profiles of Biological Activity
[0213] Unique profiles of biological activity, in terms of
efficiency of gene transfer, were demonstrated for the different
AAVs with substantial concordance within members of a set of clones
or clade. However, in vitro transduction did not predict the
efficiency of gene transfer in vivo. An algorithm for comparing the
biological activity between two different AAV pseudotypes was
developed based on relative scoring of the level of transgene
expression and a cumulative analysis of differences.
[0214] Cumulative differences of the gene transfer scores in vitro
and in vivo between pairs of AAVs were calculated and presented in
the table (ND=not determined) according to the following formula.
Cumulative functional difference in terms of scores between vectors
A and B=in vitro (A-B)+lung (A-B)+liver (A-B)+muscle (A-B). The
smaller the number, the more similar in function the AAVs. In the
grey shaded area, the percentage difference in sequence is
represented in bold italic. The percentage difference in cap
structure was determined by dividing the number of amino-acid
differences after a pairwise deletion of gaps by 750, the length of
the VP1 protein sequence alignment.
TABLE-US-00003 AAV1 AAV2 AAV3 Ch. 5 AAV4 AAV5 AAV7 AAV8 Rh. 8 AAV9
AAV1 0 5 ND 4 4 4 2 4 5 4 AAV2 0 ND 3 2 4 7 7 6 9 AAV3 0 ND ND ND
ND ND ND ND Ch. 5 0 2 4 6 6 5 8 AAV4 0 2 7 6 5 8 AAV5 0 4 4 3 6
AAV7 0 2 3 2 AAV8 0 1 2 Rh. 8 0 3 AAV9 0
[0215] These studies point out a number of issues relevant to the
study of parvoviruses in humans. The prevalence of endogenous AAV
sequences in a wide array of human tissues suggests that natural
infections with this group of viruses are quite common. The wide
tissue distribution of viral sequences and the frequent detection
in liver, spleen and gut indicate that transmission occurs via the
gastrointestinal track and that viremia may be a feature of the
infection.
[0216] The tremendous diversity of sequence present in both human
and nonhuman primates has functional correlates in terms of tropism
and serology, suggesting it is driven by real biological pressures
such as immune escape. Clearly, recombination contributes to this
diversity as evidenced by the second most common human clade, which
is a hybrid of two previously described AAVs.
[0217] Inspection of the topology of the phylogenetic analysis
reveals insight into the relationship between the evolution of the
virus and its host restriction. The entire genus of dependoviruses
appears to be derived from avian AAV consistent with Lukashov and
Goudsmit RV. V. Lukashov, J. Goudsmit, J Vivol 75, 2729-40 (March,
2001)]. The AAV4 and AAV5 isolates diverged early from the
subsequent development of the other AAVs. The next important node
divides the species into two major monophilic groups. The first
group contains clones isolated solely from humans and includes
Clade B, AAV3 clone, Clade C and Clade A; the only exception to the
species restriction of this group is the single clone from
chimpanzees, called ch.5. The other monophilic group, representing
the remaining members of the genus, is derived from both human and
nonhuman primates. This group includes Clade D and the rh.8 clone,
which were isolated exclusively from macaques, and the Clade F,
which is human specific. The remaining clade within this group
(i.e., Clade E) has members from both humans and a number of
nonhuman primate species suggesting transmission of this clade
across species barriers. It is interesting that the capsid
structures of Clade E members isolated from some humans are
essentially identical to some from nonhuman primates, indicating
that very little host adaptation has occurred. Analysis of the
biology of AAV8 derived vectors demonstrated a broad range of
tissue tropism with high levels of gene transfer, which is
consistent with a more promiscuous range of infectivity, and may
explain its apparent zoonosis. An even greater range and efficiency
of gene transfer was noted for the Clade F, highlighting the
potential for cross species transmission, which to date has not
been detected.
[0218] The presence of latent AAVs widely disseminated throughout
human and nonhuman primates and their apparent predisposition to
recombine and to cross species barriers raises important issues.
This combination of events has the potential to lead to the
emergence of new infectious agents with modified virulence.
Assessing this potential is confounded by the fact that the
clinical sequalae of AAV infections in primates has yet to be
defined. In addition, the high prevalence of AAV sequences in liver
may contribute to dissemination of the virus in the human
population in the setting of allogeneic and xenogenic liver
transplantation. Finally, the finding of endogenous AAVs in humans
has implications in the use of AAV for human gene therapy. The fact
that wild type AAV is so prevalent in primates without ever being
associated with a malignancy suggests it is not particularly
oncogenic. In fact, expression of AAV rep genes has been shown to
suppress transformation P. L. Hermonat, Virology 172, 253-61
(September, 1989)].
Example 4--AAV 2/9 Vector for the Treatment of Cystic Fibrosis
Airway Disease
[0219] To date, CFTR gene transfer to the lung for the treatment of
CF airway disease has been limited by poor vector performance
combined with the significant barriers that the airway epithelium
poses to effective gene transfer. The AAV2 genome packaged in the
AAV9 capsid (AAV2/9) was compared to AAV2/5 in various airway model
systems.
[0220] A 50 .mu.l single dose of 1.times.10.sup.11 genome copies
(gc) of AAV2/9 expressing either the nuclear targeted
.beta.-galactosidase (nLacZ) gene or the green fluorescence protein
(GFP) gene under the transcriptional control of the chicken
.beta.-actin promoter was instilled intranasally into nude and also
C57Bl/6 mice. Twenty-one days later, the lung and nose were
processed for gene expression. In control animals transduced with
AAV2/9-GFP, no LacZ positive cells were seen. AAV2/9-nLacZ
successfully transduced mainly airways, whereas AAV2/5-nLacZ
transduced mainly alveoli and few airways. Across the nasal airway
epithelium, both AAV2/5 and AAV2/9 transduced ciliated and
non-ciliated epithelial cells.
[0221] Epithelial cell specific promoters are currently being
evaluated to improve targeting to the airway cells in vivo. Based
on the in vivo findings, the gene transfer efficiency of AAV2/9 to
human airway epithelial cells was tested next. Airway epithelial
cells were isolated from human trachea and bronchi and grown at
air-liquid-interface (ALI) on collagen coated membrane supports.
Once the cells polarized and differentiated, they were transduced
with AAV2/9 or AAV2/5 expressing GFP from the apical as well as the
basolateral side. Both AAV2/5 and AAV2/9 were successful at
transducing epithelial cells from the basolateral surface. However,
when applied onto the apical surface AAV2/9 resulted in a 10-fold
increase in the number of transduced cells compared to AAV2/5.
Currently, the gene transfer performance of AAV2/9 in the lungs and
nasal airways of nonhuman primates is being evaluated.
[0222] This experiment demonstrates that AAV2/9 can efficiently
transduce the airways of murine lung and well-differentiated human
airway epithelial cells grown at ALI.
Example 5--Comparison of Direct Injection of AAV1(2/1) and
AAV9(2/9) in Adult Rat Hearts
[0223] Two adult (3 month old) rats received a single injection of
5.times.10.sup.11 particles of AAV2/1 or AAV2/9 in the left
ventricle
[0224] The results were spectacular, with significantly more
expression observed in the adult rat heart with AAV2/9 vectors as
compared to AAV2/1, as assessed by lacZ histochemistry. AAV2/9 also
shows superior gene transfer in neonatal mouse heart.
Example 6--AAV2/9 Vector for Hemophilia B Gene Therapy
[0225] In this study, AAV 2/9 vectors are shown to be more
efficient and less immunogenic vectors for both liver and
muscle-directed gene therapy for hemophilia B than the traditional
AAV sources.
[0226] For a liver-directed approach, evaluation of the AAV2/9
pseudotyped vector was performed in mouse and dog hemophilic
models. In immunocompetent hemophilia B mice (in C57BL/6
background), long-term superphysiological levels of canine Factor
IX (cFIX, 41-70 .mu.g/ml) and shortened activated partial
thromboplastin time (aPTT) have been achieved following intraportal
injection of 1.times.10.sup.11 genome copies (GC)/mouse of AAV2/7,
2/8, and 2/9 vectors in which the cFIX is expressed under a liver
specific promoter (LSP) and woodchuck hepatitis B
post-transcriptional responsive element (WPRE). A 10-fold lower
dose (1.times.10.sup.10 GC/mouse) of AAV2/8 vector generated normal
level of cFIX and aPTT time. In University of North Caroline (UNC)
hemophilia B dogs, it was previously demonstrated that
administration of an AAV2/8 vector into a dog previously treated
with an AAV2 vector was successful; cFIX expression peaked at 10
.mu.g/ml day 6 after the r.sup.d intraportal injection
(dose=5.times.10.sup.12 GC/kg), then gradually decreased and
stabilized around 700 ng/ml (16% of the normal level) throughout
the study (11/2 years). This level was about 3-fold higher than
that from a hemophilia B dog that received a single injection of
AAV2-cFIX at the similar dose. Recently, two naive hemophilia B
dogs were injected with AAV2/8 vectors intraportally at the dose of
5.25.times.10.sup.12 GC/kg. cFIX levels in one dog (male) reached
30% of normal level (1.5 .mu.g/ml) ten weeks after injection and
has sustained at 1.3-1.5 .mu.g/ml, while the second dog (female)
maintained cFIX expression at about 10% of normal level. Whole
blood clotting time (WBCT) and aPTT were both shortened after the
injection, suggesting the antigen was biologically active. Liver
enzymes (aspartate amino transferase (SGOT), alanine amino
transferase (SGPT) in both dogs remained in the normal range after
surgery. These AAV were also evaluated for muscle-targeted gene
therapy of hemophilia B. AAV-CMV-cFIX-WPRE [an AAV carrying cFIX
under the control of a CMV promoter and containing the WPRE]
packaged with six different AAV sources were compared in
immunocompetent hemophilia B mice (in C57BL/6 background) after
intramuscular injection at the dose of 1.times.10.sup.11 GC/mouse.
cFIX gene expression and antibody formation were monitored. Highest
expression was detected in the plasma of the mice injected with
AAV2/8 vectors (1460+392 ng/ml at day 42), followed by AAV2/9
(773+171 ng/ml at day 42) and AAV2/7 (500+311 ng/ml at day 42).
Levels were maintained for 5 months. Surprisingly, cFIX expression
by AAV2/1 ranged from 0-253 ng/ml (average: 66+82 ng/ml). Anti-cFIX
inhibitor (IgG) was detected in some of the AAV2/1-injected mice.
cFIX expression levels in these mice correlated well with inhibitor
levels. Further screening of inhibitor formation was performed on
day 28 samples for all AAV. Hemophilia B mice showed highest
inhibitor formation against AAV2/2, followed by AAV2/5, and AAV2/1.
Only sporadic and low level inhibitors were detected in animals
injected with AAV2/7, AAV2/8 and AAV2/9. Thus, the advantages of
the new AAV serotype 2/9 vectors for muscle-directed gene therapy
for hemophilia B as more efficient and safe vectors without
eliciting any significant anti-FIX antibody formation are
shown.
Example 7--Novel Rh.43 Vectors of Invention
[0227] A. Comparison of AAVrh.43 Based A1AT Expression Vector with
AAV8 and AAV9 in Mouse Liver Directed Gene Transfer
[0228] Novel AAVrh.43, which belongs to Clade E by phylogenetic
analysis vector was compared to AAV8 and novel AAV9 for hA1AT
levels after intraportal infusion to the mouse liver. More
particularly, pseudotyped AAVrh.43, AAV2/8 and AAV2/9 vectors were
compared in mouse liver-directed gene transfer. Pseudotyped vectors
at doses of 1.times.10.sup.11GC, 3.times.10.sup.10 GC and
1.times.10.sup.10 GC per animal were administrated to 4-6 week old
C57BL/6 mouse intramuscularly. Serum samples were collected from
animals at day 28 post vector infusion for the human alpha 1
anti-trypsin (hA1AT) assay.
[0229] The data indicated that the novel AAVrh.43 vector had indeed
a performance similar to that of AAV9 in the mouse model.
[0230] B. Nuclear Target LacZ Gene Transfer to Mouse Liver and
Muscle Mediated by Pseudotyped AAV Vectors.
[0231] Novel AAV9 and AAVrh.43 based vectors of the invention were
compared to AAV1 and AAV2-based vector. The vectors were injected
at a dose of 1.times.10.sup.11 GC per mouse either intraportally to
target liver or intramuscularly to the right anterior tibialis
muscle of C57BL/6 mice intramuscularly. The animals were sacrificed
at day 28 post gene transfer and tissues of interest harvested for
X-gal histochemical staining.
[0232] The AAVrh.43 vector demonstrated gene transfer efficiency
that was close to AAV9 but at least 5 fold higher than AAV1. The
property of AAVrh.43 was further analyzed in both liver and muscle
using nuclear targeted LacZ gene as a reporter to visualize extend
of gene transfer histochemically.
[0233] C. Comparison of AAVrh.43 Based A1AT Expression Vector with
AAV5 in Mouse Lung Directed Gene Transfer
[0234] A novel rh.43-based vector of the invention also
demonstrated superb gene transfer potency in lung tissue. Different
doses (1.times.10.sup.10, 3.times.10.sup.10 and 1.times.10.sup.11
GC per animal) of pseudotyped v