AU9721001A

AU9721001A - AAV4 vector and uses thereof

Info

Publication number: AU9721001A
Application number: AU97210/01A
Authority: AU
Inventors: John A. Chiorini; Robert M. Kotin; Brian Safer
Original assignee: US Department of Health and Human Services; US Government
Current assignee: US Department of Health and Human Services
Priority date: 1996-09-11
Filing date: 2001-12-12
Publication date: 2002-02-07
Anticipated expiration: 2017-09-11
Also published as: AU771545B2

Description

AUSTRALIA

Patents Act 1990 The Government of the United States of America, represented by The Secretary of the Department of Health and Human Services COMPLETE SPECIFICATION STANDARD PATENT Invention Title: AAV4 vector and uses thereof The following statement is a full description of this invention including the best method of performing it known to us:- AAV4 VECTOR AND USES THEREOF BACKGROUND OF THE INVENTION Field of the Invention The present invention provides adeno-associated virus 4 (AAV4) and vectors derived therefrom. Thus, the present invention relates to AAV4 vectors for and methods of delivering nucleic acids to cells of subjects.

Background Art Adeno associated virus (AAV) is a small nonpathogenic virus of the parvoviridae 15 family (for review see 28). AAV is distinct from the other members of this family by its dependence upon a helper virus for replication. In the absence of a helper virus, AAV may integrate in a locus specific manner into the q arm of chromosome 19 The approximately 5 kb genome of AAV consists of one segment of single stranded DNA of .either plus or minus polarity. The ends of the genome are short inverted terminal S. 20 repeats which can fold into hairpin structures and serve as the origin of viral DNA replication. Physically, the parvovirus virion is non-enveloped and its icosohedral capsid is approximately 20 nm in diameter.

To date 7 serologically distinct AAVs have been identified and 5 have been isolated from humans or primates and are referred to as AAV types 1-5 The most extensively studied of these isolates is AAV type 2 (AAV2). The genome of AAV2 is 4680 nucleotides in length and contains two open reading frames (ORFs). The left ORF encodes the non-structural Rep proteins, Rep40, Rep 52, Rep68 and Rep 78, which are involved in regulation of replication and transcription in addition to the production of single-stranded progeny genomes 11, 12, 15, 17, 19, 21-23, 25, 34, 37-40).

Furthermore, two of the Rep proteins have been associated with the preferential integration of AAV genomes into a region of the q arm of human chromosome 19.

Rep68/78 have also been shown to possess NTP binding activity as well as DNA and RNA helicase activities. The Rep proteins possess a nuclear localization signal as well as several potential phosphorylation sites. Mutation of one of these kinase sites resulted in a loss of replication activity.

The ends of the genome are short inverted terminal repeats which have the potential to fold into T-shaped hairpin structures that serve as the origin of viral DNA replication. Within the ITR region two elements have been described which are central to the function of the ITR, a GAGC repeat motif and the terminal resolution site (trs).

The repeat motif has been shown to bind Rep when the ITR is in either a linear or hairpin conformation 8, 26). This binding serves to position Rep68/78 for cleavage at the trs which occurs in a site- and strand-specific manner. In addition to their role in replication, these two elements appear to be central to viral integration. Contained within the chromosome 19 integration locus is a Rep binding site with an adjacent trs.

15 These elements have been shown to be functional and necessary for locus specific integration.

The AAV2 virion is a non-enveloped, icosohedral particle approximately 25 nm in diameter, consisting of three related proteins referred to as VPI,2 and 3. The right ORF encodes the capsid proteins, VP1, VP2, and VP3. These proteins are found in a 20 ratio of 1:1:10 respectively and are all derived from the right-hand ORF. The capsid proteins differ from each other by the use of alternative splicing and an unusual start codon. Deletion analysis has shown that removal or alteration of VPI which is translated from an alternatively spliced message results in a reduced yield of infections particles 16, 38). Mutations within the VP3 coding region result in the failure to produce any single-stranded progeny DNA or infectious particles (15, 16, 38).

The following features of AAV have made it an attractive vector for gene transfer AAV vectors have been shown in vitro to stably integrate into the cellular genome; possess a broad host range; transduce both dividing and non dividing cells in vitro and in vivo (13, 20, 30, 32) and maintain high levels of expression of the transduced genes Viral particles are heat stable, resistant to solvents, detergents, changes in pH, temperature, and can be concentrated on CsC1 gradients Integration ofAAV provirus is not associated with any long term negative effects on cell growth or differentiation The ITRs have been shown to be the only cis elements required for replication, packaging and integration (35) and may contain some promoter activities (14).

Initial data indicate that AAV4 is a unique member of this family. DNA hybridization data indicated a similar level of homology for AAV1-4 However, in contrast to the other AAVs only one ORF corresponding to the capsid proteins was identified in AAV4 and no ORF was detected for the Rep proteins (27).

AAV2 was originally thought to infect a wide variety of cell types provided the appropriate helper virus was present. Recent work has shown that some cell lines are transduced very poorly by AAV2 While the receptor has not been completely "characterized, binding studies have indicated that it is poorly expressed on erythroid cells Recombinant AAV2 transduction of CD34', bone marrow pluripotent cells, requires a multiplicity of infection (MOI) of 104 particles per cell W. Nienhuis unpublished results). This suggests that transduction is occurring by a non-specific S...mechanism or that the density of receptors displayed on.the cell surface is low compared to other cell types.

S" The present invention provides a vector comprising the AAV4 virus as well as AAV4 viral particles. While AAV4 is similar to AAV2, the two viruses are found herein to be physically and genetically distinct. These differences endow AAV4 with some unique advantages which better suit it as a vector for gene therapy. For example, the wt AAV4 genome is larger than AAV2, allowing for efficient encapsidation of a larger recombinant genome. Furthermore, wt AAV4-particles have a greater buoyant density than AAV2 particles and therefore are more easily separated from contaminating helper virus and empty AAV particles than AAV2-based particles. Additionally, in contrast to AAV1, 2, and 3, AAV4, is able to hemagglutinate human, guinea pig, and sheep erythrocytes (18).

Furthermore, as shown herein, AAV4 capsid protein, again surprisingly, is distinct from AAV2 capsid protein and exhibits different tissue tropism. AAV2 and AAV4 have been shown to be serologically distinct and thus, in a gene therapy application, AAV4 would allow for transduction of a patient who already possess neutralizing antibodies to AAV2 either as a result of natural immunological defense or from prior exposure to AAV2 vectors. Thus, the present invention, by providing these new recombinant vectors and particles based on AAV4 provides a new and highly useful series of vectors.

S.e e SUMMARY OF THE INVENTION The present invention provides a nucleic acid vector comprising a pair of adenoassociated virus 4 (AAV4) inverted terminal repeats and a promoter between the inverted terminal repeats.

The present invention further provides an AAV4 particle containing a vector comprising a pair of AAV2 inverted terminal repeats.

Additionally, the instant invention provides an isolated nucleic acid comprising the nucleotide sequence set forth in SEQ ID NO:1 [AAV4 genome]. Furthermore, the present invention provides an isolated nucleic acid consisting essentially of the nucleotide sequence set forth in SEQ ID NO:1 [AAV4 genome].

The present invention provides an isolated nucleic acid encoding an adenoassociated virus 4 Rep protein. Additionally provided is an isolated AAV4 Rep protein having the amino acid sequence set forth in SEQ ID NO:2, or a unique fragment thereof.

Additionally provided is an isolated AAV4 Rep protein having the amino acid sequence set forth in SEQ ID NO:8, or a unique fragment thereof. Additionally provided is an isolated AAV4 Rep protein having the amino acid sequence set forth in SEQ ID NO:9, or a unique fragment thereof. Additionally provided is an isolated AAV4 Rep protein having the amino acid sequence set forth in SEQ ID NO: 10, or a unique fragment thereof. Additionally provided is an isolated AAV4 Rep protein having the amino acid sequence set forth in SEQ ID NO: 11, or a unique fragment thereof.

The present invention further provides an isolated AAV4 capsid protein having the amino acid sequence set forth in SEQ ID NO:4. Additionally provided is an isolated AAV4 capsid protein having the amino acid sequence set forth in SEQ ID NO:16. Also provided is an isolated AAV4 capsid protein having the amino acid sequence set forth in SEQ ID NO:18.

The present invention additionally provides an isolated nucleic acid encoding adeno-associated virus 4 capsid protein.

The present invention further provides an AAV4 particle comprising a capsid protein consisting essentially of the amino acid sequence set forth in SEQ ID NO:4.

Additionally provided by the present invention is an isolated nucleic acid comprising an AAV4 p5 promoter.

The instant invention provides a method of screening a cell for infectivity by AAV4 comprising contacting the cell with AAV4 and detecting the presence of AAV4 in the cells.

0**000 0 The present invention further provides a method of delivering a nucleic acid to a cell comprising administering to the cell an AAV4 particle containing a-vector comprising the nucleic acid inserted between a pair ofAAV inverted terminal repeats, thereby delivering the nucleic acid to the cell.

The present invention also provides a method of delivering a nucleic acid to a 20 subject comprising administering to a cell from the subject an AAV4 particle comprising the nucleic acid inserted between a pair of AAV inverted terminal repeats, and returning the cell to the subject, thereby delivering the nucleic acid to the subject.

0 The present invention further provides a method of delivering a nucleic acid to a subject comprising administering to a cell from the subject an AAV4 particle comprising the nucleic acid inserted between a pair of AAV inverted terminal repeats, and returning the cell to the subject, thereby delivering the nucleic acid to the subject.

The present invention also provides a method of delivering a nucleic acid to a cell in a subject comprising administering to the subject an AAV4 particle comprising the nucleic acid inserted between a pair of AAV inverted terminal repeats, thereby delivering the nucleic acid to a cell in the subject.

The instant invention furither provides a method of delivering a nucleic acid to a cell in a subject having antibodies to AAV2 comprising administering to the subject an AAV4 particle comprising the nucleic acid, thereby delivering the nucleic acid to a cell in the subject.

*oe 0 S O 0*000: BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 shows a schematic outline of AAV 4. Promoters are indicated by horizontal arrows with their corresponding map positions indicated above. The polyadenylation site is indicated by a vertical arrow and the two open reading frames are indicated by black boxes. The splice region is indicated by a shaded box.

Fig. 2 shows AAV4 ITR.The sequence of the ITR (SEQ ID NO: 20) is shown in the hairpin conformation. The putative Rep binding site is boxed. The cleavage site in the trs is indicated by an arrow. Bases which differ from the ITR of AAV2 are outlined.

Fig. 3 shows cotransduction of rAAV2 and rAAV4. Cos cells were transduced with a constant amount of rAAV2 or rAAV4 expressing beta galactosidase and increasing amounts of rAAV2 expressing human factor IX (rAAV2FIX) For the competition the *o 15 number of positive cells detected in the cotransduced wells was divided by the number Sof positive cells in the control wells (cells transduced with only rAAV2LacZ or rAAV4LacZ) and expressed as a percent of the control. This value was plotted against the number of particles of rAAV2FIX.

20 Fig. 4 shows effect of trypsin treatment on cos cell transduction. Cos cell monolayers were trypsinized and diluted in complete media. Cells were incubated with virus at an MOI of 260 and following cell attachment the virus was removed. As a control an equal number of cos cells were plated and allowed to attach overnight before transduction with virus for the same amount of time. The number of positive cells was determined by staining 50 hrs post transduction. The data is presented as a ratio of the number of positive cells seen with the trypsinized group and the control group.

DETAILED DESCRIPTION OF THE INVENTION As used in the specification and in the claims, can mean one or more, depending upon the context in which it is used.

The present invention provides the nucleotide sequence of the adeno-associated virus 4 (AAV4) genome and vectors and particles derived therefrom. Specifically, the present invention provides a nucleic acid vector comprising a pair of AAV4 inverted terminal repeats (ITRs) and a promoter between the inverted terminal repeats. The AAV4 ITRs are exemplified by the nucleotide sequence set forth in SEQ ID NO:6 and SEQ ID NO:20; however, these sequences can have minor modifications and still be contemplated to constitute AAV4 ITRs. The nucleic acid listed in SEQ ID NO:6 depicts the ITR in the "flip" orientation of the ITR. The nucleic acid listed in SEQ ID 15 NO:20 depicts the ITR in the "flop" orientation of the ITR. Minor modifications in an ITR of either orientation are those that will not interfere with the hairpin structure formed by the AAV4 ITR as described herein and known in the art. Furthermore, to be considered within the term "AAV4 ITRs" the nucleotide sequence must retain the Rep binding site described herein and exemplified in SEQ ID NO:6 and SEQ ID NO:20, i.e., 20 it must retain one or both features described herein that distinguish the AAV4 ITR from the AAV2 ITR: four (rather than three as in AAV2) "GAGC" repeats and in the AAV4 ITR Rep binding site the fourth nucleotide in the first two "GAGC" repeats is a T 0 rather than a C.

The promoter can be any desired promoter, selected by known considerations, such as the level of expression of a nucleic acid functionally linked to the promoter and the cell type in which the vector is to be used. Promoters can be an exogenous or an endogenous promoter. Promoters can include, for example, known strong promoters such as SV40 or the inducible metallothionein promoter, or an AAV promoter, such as an AAV p5 promoter. Additional examples of promoters include promoters derived from actin genes, immunoglobulin genes, cytomegalovirus (CMV), adenovirus, bovine papilloma virus, adenoviral promoters, such as the adenoviral major late promoter, an inducible heat shock promoter, respiratory syncytial virus, Rous sarcomas virus (RSV), etc. Specifically, the promoter can be AAV2 p 5 promoter or AAV4 p5 promoter.

More specifically, the AAV4 p 5 promoter can be about nucleotides 130 to 291 of SEQ ID NO: 1. Additionally, the p5 promoter may be enhanced by nucleotides 1-130.

Furthermore, smaller fragments of p5 promoter that retain promoter activity can readily be determined by standard procedures including, for example, constructing a series of deletions in the p5 promoter, linking the deletion to a reporter gene, and determining whether the reporter gene is expressed, transcribed and/or translated.

It should be recognized that the nucleotide and amino acid sequences set forth S herein may contain minor sequencing errors. Such errors in the nucleotide sequences can be corrected, for example, by using the hybridization procedure described above with various probes derived from the described sequences such that the coding sequence oo o 15 can be reisolated and resequenced. The corresponding amino acid sequence can then be corrected accordingly.

*oo The AAV4 vector can further comprise an exogenous nucleic acid functionally 20 linked to the promoter. By "heterologous nucleic acid" is meant that any heterologous Sor exogenous nucleic acid can be inserted into the vector for transfer into a cell, tissue or organism. The nucleic acid can encode a polypeptide or protein or an antisense RNA, for example. By "functionally linked" is meant such that the promoter can promote expression of the heterologous nucleic acid, as is known in the art, such as appropriate orientation of the promoter relative to the heterologous nucleic acid.

Furthermore, the heterologous nucleic acid preferably has all appropriate sequences for expression of the nucleic acid, as known in the art, to functionally encode, allow the nucleic acid to be expressed. The nucleic acid can include, for example, expression control sequences, such as an enhancer, and necessary information processing sites, such as ribosome binding sites, RNA splice sites, polyadenylation sites, and transcriptional terminator sequences.

The heterologous nucleic acid can encode beneficial proteins that replace missing or defective proteins required by the subject into which the vector in transferred or can encode a cytotoxic polypeptide that can be directed, to cancer cells or other cells whose death would be beneficial to the subject. The heterologous nucleic acid can also encode antisense RNAs that can bind to, and thereby inactivate, mRNAs made by the subject that encode harmful proteins. In one embodiment, antisense polynucleotides can be produced from a heterologous expression cassette in an AAV4 viral construct where the expression cassette contains a sequence that promotes cell-type specific expression (Wirak et al., EMBO 10:289 (1991)). For general methods relating to antisense polynucleotides, see Antisense RNA and DNA, D. A. Melton, Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1988).

Examples of heterologous nucleic acids which can be administered to a cell or subject as part of the present AAV4 vector can include, but are not limited to the 15 following: nucleic acids encoding therapeutic agents, such as tumor necrosis factors (TNF), such as TNF-a; interferons, such as interferon-a, interferon-P, and interferon-y; interleukins, such as IL-1, IL-1 P, and ILs -2 through -14; GM-CSF; adenosine deaminase; cellular growth factors, such as lymphokines; soluble CD4; Factor VIII; Factor IX; T-cell receptors; LDL receptor; ApoE; ApoC; alpha-1 antitrypsin; ornithine e• transcarbamylase (OTC); cystic fibrosis transmembrane receptor (CFTR); insulin; Fc receptors for antigen binding domains of antibodies, such as immunoglobulins; and antisense sequences which inhibit viral replication, such as antisense sequences which inhibit replication of hepatitis B or hepatitis non-A, non-B virus. The nucleic acid is chosen considering several factors, including the cell to be transfected. Where the target cell is a blood cell, for example, particularly useful nucleic acids to use are those which allow the blood cells to exert a therapeutic effect, such as a gene encoding a clotting factor for use in treatment of hemophilia. Furthermore, the nucleic acid can encode more than one gene product, limited only, if the nucleic acid is to be packaged in a capsid, by the size of nucleic acid that can be packaged.

12 Furthermore, suitable nucleic acids can include those that, when transferred into a primary cell, such as a blood cell, cause the transferred cell to target a site in the body where that cell's presence would be beneficial. For example, blood cells such as TIL cells can be modified, such as by transfer into the cell of a Fab portion of a monoclonal antibody, to recognize a selected antigen. Another example would be to introduce a nucleic acid that would target a therapeutic blood cell to tumor cells. Nucleic acids useful in treating cancer cells include those encoding chemotactic factors which cause an inflammatory response at a specific site, thereby having a therapeutic effect.

Cells, particularly blood cells, having such nucleic acids transferred into them can be useful in a variety of diseases, syndromes and conditions. For example, suitable nucleic acids include nucleic acids encoding soluble CD4, used in the treatment of AIDS and a-antitrypsin, used in the treatment of emphysema caused by a-antitrypsin S.deficiency. Other diseases, syndromes and conditions in which such cells can be useful 15 include, for example, adenosine deaminase deficiency, sickle cell deficiency, brain disorders such as Alzheimer's disease, thalassemia, hemophilia, diabetes, phenylketonuria, growth disorders and heart diseases, such as those caused by alterations in cholesterol metabolism, and defects of the immune system.

20 As another example, hepatocytes can be transfected with the present vectors having useful nucleic acids to treat liver disease. For example, a nucleic acid encoding OTC can be used to transfect hepatocytes (ex vivo and returned to the liver or in vivo) to treat congenital hyperammonemia, caused by an inherited deficiency in OTC.

Another example is to use a nucleic acid encoding LDL to target hepatocytes ex vivo or in vivo to treat inherited LDL receptor deficiency. Such transfected hepatocytes can also be used to treat acquired infectious diseases, such as diseases resulting from a viral infection. For example, transduced hepatocyte precursors can be used to treat viral hepatitis, such as hepatitis B and non-A, non-B hepatitis, for example by transducing the hepatocyte precursor with a nucleic acid encoding an antisense RNA that inhibits viral replication. Another example includes transferring a vector of the present invention having a nucleic acid encoding a protein, such as a-interferon, which can confer resistance to the hepatitis virus.

For a procedure using transfected hepatocytes or hepatocyte precursors, hepatocyte precursors having a vector of the present invention transferred in can be grown in tissue culture, removed form the tissue culture vessel, and introduced to the body, such as by a surgical method. In this example, the tissue would be placed directly into the liver, or into the body cavity in proximity to the liver, as in a transplant or graft.

Alternatively, the cells can simply be directly injected into the liver, into the portal circulatory system, or into the spleen, from which the cells can be transported to the liver via the circulatory system. Furthermore, the cells can be attached to a support, oo such as microcarrier beads, which can then be introduced, such as by injection, into the peritoneal cavity. Once the cells are in the liver, by whatever means, the cells can then express the nucleic acid and/or differentiate into mature hepatocytes which can express 15 the nucleic acid.

The present invention also contemplates any unique fragment of these AAV4 nucleic acids, including the AAV4 nucleic acids set forth in SEQ ID NOs: 1, 3, 5, 6, 7, 12-15, 17 and 19. To be unique, the fragment must be of sufficient size to distinguish it 20 from other known sequences, most readily determined by comparing any nucleic acid fragment to the nucleotide sequences of nucleic acids in computer databases, such as GenBank. Such comparative searches are standard in the art. Typically, a unique fragment useful as a primer or probe will be at least about 8 or 10 to about 20 or nucleotides in length, depending upon the specific nucleotide content of the sequence.

Additionally, fragments can be, for example, at least about 30, 40, 50, 75, 100, 200 or 500 nucleotides in length. The nucleic acid can be single or double stranded, depending upon the purpose for which it is intended.

The present invention further provides an AAV4 capsid protein. In particular, the present invention provides not only a polypeptide comprising all three AAV4 coat proteins, VP1, VP2 and VP3, but also a polypeptide comprising each AAV4 coat 14 protein individually. Thus an AAV4 particle comprising an AAV4 capsid protein comprises at least one AAV4 coat protein VP1, VP2 or VP3. An AAV4 particle comprising an AAV4 capsid protein can be utilized to deliver a nucleic acid vector to a cell, tissue or subject. For example, the herein described AAV4 vectors can be encapsulated in an AAV4 particle and utilized in a gene delivery method. Furthermore, other viral nucleic acids can be encapsidated in the AAV4 particle and utilized in such delivery methods. For example, an AAV2 vector can be encapsidated in an AAV4 particle and administered. Furthermore, a chimeric capsid protein incorporating both AAV2 and AAV4 sequences can be generated, by standard cloning methods, selecting regions from each protein as desired. For example, particularly antigenic regions of the AAV2 capsid protein can be replaced with the corresponding region of the AAV4 capsid protein.

The herein described AAV4 nucleic acid vector can be encapsidated in an AAV *oo 15 particle. In particular, it can be encapsidated in an AAV1 particle, an AAV2 particle, an AAV3 particle, an AAV4 particle, or an AAV5 particle by standard methods using the appropriate capsid proteins in the encapsidation process, as long as the nucleic acid vector fits within the size limitation of the particle utilized. The encapsidation process itself is standard in the art.

An AAV4 particle is a viral particle comprising an AAV4 capsid protein. An AAV4 capsid polypeptide encoding the entire VP1, VP2, and VP3 polypeptide can overall have at least about 63% homology to the polypeptide having the amino acid sequence encoded by nucleotides 2260-4464 set forth in SEQ ID NO: 1 (AAV4 capsid protein). The capsid protein can have about 70% homology, about 75% homology, homology, 85% homology, 90% homology, 95% homology, 98% homology, 99% homology, or even 100% homology to the protein having the amino acid sequence encoded by nucleotides 2260-4464 set forth in SEQ ID NO: 1. The particle can be a particle comprising both AAV4 and AAV2 capsid protein, a chimeric protein.

Variations in the amino acid sequence of the AAV4 capsid protein are contemplated herein, as long as the resulting viral particle comprising the AAV4 capsid remains antigenically or immunologically distinct from AAV2, as can be routinely determined by standard methods. Specifically, for example, ELISA and Western blots can be used to determine whether a viral particle is antigenically or immunologically distinct from AAV2. Furthermore, the AAV4 viral particle preferably retains tissue tropism distinction from AAV2, such as that exemplified in the examples herein, though an AAV4 chimeric particle comprising at least one AAV4 coat protein may have a different tissue tropism from that of an AAV4 particle consisting only of AAV4 coat proteins.

The invention further provides an AAV4 particle containing, encapsidating, a vector comprising a pair of AAV2 inverted terminal repeats. The nucleotide sequence of AAV2 ITRs is known in the art. Furthermore, the particle can be a particle comprising both AAV4 and AAV2 capsid protein, a chimeric protein. The vector encapsidated in the particle can further comprise an exogenous nucleic acid inserted 15 between the inverted terminal repeats.

The present invention further provides an isolated nucleic acid comprising the nucleotide sequence set forth in SEQ ID NO:1 (AAV4 genome). This nucleic acid, or 2 portions thereof, can be inserted into other vectors, such as plasmids, yeast artificial 20 chromosomes, or other viral vectors, if desired, by standard cloning methods. The present invention also provides an isolated nucleic acid consisting essentially of the .nucleotide sequence set forth in SEQ ID NO:1. The nucleotides of SEQ ID NO:1 can •have minor modifications and still be contemplated by the present invention. For example, modifications that do not alter the amino acid encoded by any given codon (such as by modification of the third, "wobble," position in a codon) can readily be made, and such alterations are known in the art. Furthermore, modifications that cause a resulting neutral amino acid substitution of a similar amino acid can be made in a coding region of the genome. Additionally, modifications as described herein for the AAV4 components, such as the ITRs, the p5 promoter, etc. are contemplated in this invention.

The present invention additionally provides an isolated nucleic acid that selectively hybridizes with an isolated nucleic acid consisting essentially of the nucleotide sequence set forth in SEQ ID NO: 1 (AAV4 genome). The present invention further provides an isolated nucleic acid that selectively hybridizes with an isolated nucleic acid comprising the nucleotide sequence set forth in SEQ ID NO: 1 (AAV4 genome). By "selectively hybridizes" as used in the claims is meant a nucleic acid that specifically hybridizes to the particular target nucleic acid under sufficient stringency conditions to selectively hybridize to the target nucleic acid without significant background hybridization to a nucleic acid encoding an unrelated protein, and particularly, without detectably hybridizing to AAV2. Thus, a nucleic acid that selectively hybridizes with a nucleic acid of the present invention will not selectively hybridize under stringent conditions with a nucleic acid encoding a different protein, and vice versa. Therefore, nucleic acids for use, for example, as primers and probes to detect or amplify the target nucleic acids are contemplated herein. Nucleic acid 15 fragments that selectively hybridize to any given nucleic acid can be used, .as primers and or probes for further hybridization or for amplification methods polymerase chain reaction (PCR), ligase chain reaction Additionally, for example, a primer or probe can be designed that selectively hybridizes with both AAV4 and a gene of interest carried within the AAV4 vector a chimeric nucleic acid).

Stringency of hybridization is controlled by both temperature and salt concentration of either or both of the hybridization and washing steps. Typically, the stringency of hybridization to achieve selective hybridization involves hybridization in high ionic strength solution (6X SSC or 6X SSPE) at a temperature that is about 12- 25C below the Tm (the melting temperature at which half of the molecules dissociate from its partner) followed by washing at a combination of temperature and salt concentration chosen so that the washing temperature is about 5°C to 20°C below the The temperature and salt conditions are readily determined empirically in preliminary experiments in which samples of reference DNA immobilized on filters are hybridized to a labeled nucleic acid of interest and then washed under conditions of different stringencies. Hybridization temperatures are typically higher for DNA-RNA and RNA- RNA hybridizations. The washing temperatures can be used as described above to achieve selective stringency, as is known in the art. (Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1989; Kunkel et al. Methods Enzymol. 1987:154:367, 1987). A preferable stringent hybridization condition for a DNA:DNA hybridization can be at about 68 C (in aqueous solution) in 6X SSC or 6X SSPE followed by washing at 68 C.

Stringency of hybridization and washing, if desired, can be reduced accordingly as homology desired is decreased, and further, depending upon the G-C or A-T richness of any area wherein variability is searched for. Likewise, stringency of hybridization and washing, if desired, can be increased accordingly as homology desired is increased, and further, depending upon the G-C or A-T richness of any area wherein high homology is desired, all as known in the art.

A nucleic acid that selectively hybridizes to any portion of the AAV4 genome is 15 contemplated herein. Therefore, a nucleic acid that selectively hybridizes to AAV4 can be of longer length than the AAV4 genome, it can be about the same length as the AAV4 genome or it can be shorter than the AAV4 genome. The length of the nucleic ev* acid is limited on the shorter end of the size range only by its specificity for hybridization to AAV4, once it is too short, typically less than about 5 to 7 nucleotides in length, 20 it will no longer bind specifically to AAV4, but rather will hybridize to numerous background nucleic acids. Additionally contemplated by this invention is a nucleic acid that has a portion that specifically hybridizes to AAV4 and a portion that specifically O* hybridizes to a gene of interest inserted within AAV4.

The present invention further provides an isolated nucleic acid encoding an adeno-associated virus 4 Rep protein. The AAV4 Rep proteins are encoded by open reading frame (ORF) 1 of the AAV4 genome. The AAV4 Rep genes are exemplified by the nucleic acid set forth in SEQ ID NO:3 (AAV4 ORF1), and include a nucleic acid consisting essentially of the nucleotide sequence set forth in SEQ ID NO:3 and a nucleic acid comprising the nucleotide sequence set forth in SEQ ID NO:3. The present invention also includes a nucleic acid encoding the amino acid sequence set forth in SEQ 18 ID NO: 2 (polypeptide encoded by AAV4 ORF1). However, the present invention includes that the Rep genes nucleic acid can include any one, two, three, or four of the four Rep proteins, in any order, in such a nucleic acid. Furthermore, minor modifications are contemplated in the nucleic acid, such as silent mutations in the coding sequences, mutations that make neutral or conservative changes in the encoded amino acid sequence, and mutations in regulatory regions that do not disrupt the expression of the gene. Examples of other minor modifications are known in the art. Further modifications can be made in the nucleic acid, such as to disrupt or alter expression of one or more of the Rep proteins in order to, for example, determine the effect of such a disruption; such as to mutate one or more of the Rep proteins to determine the resulting effect, etc. However, in general, a modified nucleic acid encoding all four Rep proteins will have at least about 90%, about 93%, about 95%, about 98% or 100% homology to the sequence set forth in SEQ ID NO:3, and the Rep polypeptide encoded therein will have overall about 93%, about 95%, about 98%, about 99% or 100% homology with the amino acid sequence set forth in SEQ ID NO:2.

The present invention also provides an isolated nucleic acid that selectively hybridizes with a nucleic acid consisting essentially of the nucleotide sequence set forth in SEQ ID NO:3 and an isolated nucleic acid that selectively hybridizes with a nucleic acid comprising the nucleotide sequence set forth in SEQ ID NO:3. "Selectively hybridizing" is defined elsewhere herein.

The present invention also provides each individual AAV4 Rep protein and the nucleic acid encoding each. Thus the present invention provides the nucleic acid encoding a Rep 40 protein, and in particular an isolated nucleic acid comprising the nucleotide sequence set forth in SEQ ID NO: 12, an isolated nucleic acid consisting essentially of the nucleotide sequence set forth in SEQ ID NO:12, and a nucleic acid encoding the adeno-associated virus 4 Rep protein having the amino acid sequence set forth in SEQ ID NO:8. The present invention also provides the nucleic acid encoding a Rep 52 protein, and in particular an isolated nucleic acid comprising the nucleotide sequence set forth in SEQ ID NO: 13, an isolated nucleic acid consisting essentially of 19 the nucleotide sequence set forth in SEQ ID NO: 13, and a nucleic acid encoding the adeno-associated virus 4 Rep protein having the amino acid sequence set forth in SEQ ID NO:9. The present invention further provides the nucleic acid encoding a Rep 68 protein, and in particular an isolated nucleic acid comprising the nucleotide sequence set forth in SEQ ID NO: 14, an isolated nucleic acid consisting essentially of the nucleotide sequence set forth in SEQ ID NO:14, and a nucleic acid encoding the adeno-associated virus 4 Rep protein having the amino acid sequence set forth in SEQ ID NO: 10. And, further, the present invention provides the nucleic acid encoding a Rep 78 protein, and in particular an isolated nucleic acid comprising the nucleotide sequence set forth in SEQ ID NO:15, an isolated nucleic acid consisting essentially of the nucleotide sequence set forth in SEQ ID NO:15, and a nucleic acid encoding the adeno-associated virus 4 Rep protein having the amino acid sequence set forth in SEQ ID NO:11. As described elsewhere herein, these nucleic acids can have minor modifications, including silent nucleotide substitutions, mutations causing neutral amino acid substitutions in the encoded proteins, and mutations in control regions that do not or minimally affect the encoded amino acid sequence.

The present invention further provides a nucleic acid encoding the entire AAV4 Capsid polypeptide. Specifically, the present invention provides a nucleic acid having the nucleotide sequence set for the nucleotides 2260-4464 of SEQ ID NO:1.

Furthermore, the present invention provides a nucleic acid encoding each of the three Ooeoo AAV4 coat proteins, VP 1, VP2, and VP3. Thus, the present invention provides a nucleic acid encoding AAV4 VP1, a nucleic acid encoding AAV4 VP2, and a nucleic acid encoding AAV4 VP3. Thus, the present invention provides a nucleic acid encoding the amino acid sequence set forth in SEQ ID NO:4 (VP1); a nucleic acid encoding the amino acid sequence set forth in SEQ ID NO:16 (VP2), and a nucleic acid encoding the amino acid sequence set forth in SEQ ID NO: 18 (VP3). The present invention also specifically provides a nucleic acid comprising SEQ ID NO:5 (VP1 gene); a nucleic acid comprising SEQ ID NO:17 (VP2 gene); and a nucleic acid comprising SEQ ID NO: 19 (VP3 gene). The present invention also specifically provides a nucleic acid consisting essentially of SEQ ID NO:5 (VP1 gene), a nucleic acid consisting essentially of SEQ ID NO: 17 (VP2 gene), and a nucleic acid consisting essentially of SEQ ID NO:19 (VP3 gene). Furthermore, a nucleic acid encoding an AAV4 capsid protein VP1 is set forth as nucleotides 2157-4361 of SEQ ID NO:1; a nucleic acid encoding an AAV4 capsid protein VP2 is set forth as nucleotides 2565-4361 of SEQ ID NO:1; and a nucleic acid encoding an AAV4 capsid protein VP3 is set forth as nucleotides 2745-4361 of SEQ ID NO:1. Minor modifications in the nucleotide sequences encoding the capsid, or coat, proteins are contemplated, as described above for other AAV4 nucleic acids.

The present invention also provides a cell containing one or more of the herein described nucleic acids, such as the AAV4 genome, AAV4 ORF1 and ORF2, each AAV4 Rep protein gene, and each AAV4 capsid protein gene. Such a cell can be any desired cell and can be selected based upon the use intended. For example, cells can include human HeLa cells, cos cells, other human and mammalian cells and cell lines.

Primary cultures as well as established cultures and cell lines can be used. Nucleic acids of the present invention can be delivered into cells by any selected means, in particular depending upon the target cells. Many delivery means are well-known in the art. For example, electroporation, calcium phosphate precipitation, microinjection, cationic or anionic liposomes, and liposomes in combination with a nuclear localization signal peptide for delivery to the nucleus can be utilized, as is known in the art. Additionally, if in a viral particle, the cells can simply be transfected with the particle by standard means known in the art for AAV transfection.

The term "polypeptide" as used herein refers to a polymer of amino acids and includes full-length proteins and fragments thereof. Thus, "protein," polypeptide," and "peptide" are often used interchangeably herein. Substitutions can be selected by known parameters to be neutral (see, Robinson WE Jr, and Mitchell WM., AIDS 4:S151-S162 (1990)). As will be appreciated by those skilled in the art, the invention also includes those polypeptides having slight variations in amino acid sequences or other properties. Such variations may arise naturally as allelic variations due to genetic polymorphism) or may be produced by human intervention by mutagenesis of cloned DNA sequences), such as induced point, deletion, insertion and substitution 21 mutants. Minor changes in amino acid sequence are generally preferred, such as conservative amino acid replacements, small internal deletions or insertions, and additions or deletions at the ends of the molecules. Substitutions may be designed based on, for example, the model of Dayhoff, et al. (in Atlas of Protein Sequence and Structure 1978, Nat'l Biomed. Res. Found., Washington, These modifications can result in changes in the amino acid sequence, provide silent mutations, modify a restriction site, or provide other specific mutations.

A polypeptide of the present invention can be readily obtained by any of several means. For example, polypeptide of interest can be synthesized mechanically by standard methods. Additionally, the coding regions of the genes can be expressed and the resulting polypeptide isolated by standard methods. Furthermore, an antibody *o specific for the resulting polypeptide can be raised by standard methods (see, e.g., Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1988), and the protein can be isolated from a cell i expressing the nucleic acid encoding the polypeptide by selective hybridization with the antibody. This protein can be purified to the extent desired by standard methods of protein purification (see, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1989).

'Typically, to be unique, a polypeptide fragment of the present invention will be at least about 5 amino acids in length; however, unique fragments can be 6, 7, 8, 9, 30, 40, 50, 60, 70, 80, 90, 100 or more amino acids in length. A unique polypeptide will typically comprise such a unique fragment; however, a unique polypeptide can also be determined by its overall homology. A unique polypeptide can be 6, 7, 8, 9, 10, 40, 50, 60, 70, 80, 90, 100 or more amino acids in length. Uniqueness of a polypeptide fragment can readily be determined by standard methods such as searches of computer databases of known peptide or nucleic acid sequences or by hybridization studies to the nucleic acid encoding the protein or to the protein itself, as known in the art.

22 The present invention provides an isolated AAV4 Rep protein. AAV4 Rep polypeptide is encoded by ORFI of AAV4 Specifically, the present invention provides an AAV4 Rep polypeptide comprising the amino acid sequence set forth in SEQ ID NO:2, or a unique fragment thereof. The present invention also provides an AAV4 Rep polypeptide consisting essentially of the amino acid sequence set forth in SEQ ID NO:2, or a unique fragment thereof. Additionally, nucleotides 291-2306 of the AAV4 genome, which genome is set forth in SEQ ID NO: 1, encode the AAV4 Rep polypeptide. The present invention also provides each AAV4 Rep protein. Thus the present invention provides AAV4 Rep 40, or a unique fragment thereof The present invention particularly provides Rep 40 having the amino acid sequence set forth in SEQ ID NO:8.

The present invention provides AAV4 Rep 52, or a unique fragment thereof. The present invention particularly provides Rep 52 having the amino acid sequence set forth in SEQ ID NO:9. The present invention provides AAV4 Rep 68, or a unique fragment thereof The present invention particularly provides Rep 68 having the amino acid sequence set forth in SEQ ID NO: 10. The present invention provides AAV4 Rep 78, or a unique fragment thereof The present invention particularly provides Rep 78 having S"the amino acid sequence set forth in SEQ ID NO: 11. By "unique fragment thereof' is meant any smaller polypeptide fragment encoded by AAV rep gene that is of sufficient length to be unique to the Rep polypeptide. Substitutions and modifications of the amino acid sequence can be made as described above and, further, can include protein processing modifications, such as glycosylation, to the polypeptide. However, a polypeptide including all four Rep proteins will encode a polypeptide having at least about 91% overall homology to the sequence set forth in SEQ ID NO:2, and it can have about 93%, about 95%, about 98%, about 99% or 100% homology with the amino acid sequence set forth in SEQ ID NO:2.

The present invention further provides an AAV4 Capsid polypeptide or a unique fragment thereof. AAV4 capsid polypeptide is encoded by ORF 2 of AAV4.

Specifically, the present invention provides an AAV4 Capsid protein comprising the amino acid sequence encoded by nucleotides 2260-4464 of the nucleotide sequence set forth in SEQ ID NO:1, or a unique fragment of such protein. The present invention also provides an AAV4 Capsid protein consisting essentially of the amino acid sequence encoded by nucleotides 2260-4464 of the nucleotide sequence set forth in SEQ ID NO:1, or a unique fragment of such protein. The present invention further provides the individual AAV4 coat proteins, VP1, VP2 and VP3. Thus, the present invention provides an isolated polypeptide having the amino acid sequence set forth in SEQ ID NO:4 (VP1). The present invention additionally provides an isolated polypeptide having the amino acid sequence set forth in SEQ ID NO:16 (VP2). The present invention also provides an isolated polypeptide having the amino acid sequence set forth in SEQ ID NO:18 (VP3). By "unique fragment thereof' is meant any smaller S. polypeptide fragment encoded by any AAV4 capsid gene that is of sufficient length to be unique to the AAV4 Capsid protein. Substitutions and modifications of the amino acid sequence can be made as described above and, further, can include protein processing modifications, such as glycosylation, to the polypeptide. However, an AAV4 Capsid S• ~polypeptide including all three coat proteins will have at least about 63% overall homology to the polypeptide encoded by nucleotides 2260-4464 of the sequence set forth in SEQ ID NO: 1. The protein can have about 65%, about 70%, about about 80%, about 85%, about 90%, about 95% or even 100% homology to the amino acid sequence encoded by the nucleotides 2260-4464 of the sequence set forth in SEQ ID NO:4. An AAV4 VP2 polypeptide can have at least about 58%, about 60%, about 70%, about 80%, about 90% about 95% or about 100% homology to the amino acid sequence set forth in SEQ ID NO:16. An AAV4 VP3 polypeptide can have at least about 60%, about 70%, about 80%, about 90% about 95% or about 100% homology to the amino acid sequence set forth in SEQ ID NO:18.

The present invention further provides an isolated antibody that specifically binds AAV4 Rep protein. Also provided is an isolated antibody that specifically binds the AAV4 Rep protein having the amino acid sequence set forth in SEQ ID NO:2, or that specifically binds a unique fragment thereof. Clearly, any given antibody can recognize and bind one of a number of possible epitopes present in the polypeptide; thus only a 24 unique portion of a polypeptide (having the epitope) may need to be present in an assay to determine if the antibody specifically binds the polypeptide.

The present invention additionally provides an isolated antibody that specifically binds any adeno-associated virus 4 Capsid protein or the polypeptide comprising all three AAV4 coat proteins. Also provided is an isolated antibody that specifically binds the AAV4 Capsid protein having the amino acid sequence set forth in SEQ ID NO:4, or that specifically binds a unique fragment thereof. The present invention further provides an isolated antibody that specifically binds the AAV4 Capsid protein having the amino acid sequence set forth in SEQ ID NO: 16, or that specifically binds a unique fragment thereof. The invention additionally provides an isolated antibody that specifically binds the AAV4 Capsid protein having the amino acid sequence set forth in SEQ ID NO:18, or that specifically binds a unique fragment thereof. Again, any given antibody can recognize and bind one of a number of possible epitopes present in the polypeptide; thus only a unique portion of a polypeptide (having the epitope) may need to be present in an assay to determine if the antibody specifically binds the polypeptide.

The antibody can be a component of a composition that comprises an antibody that specifically binds the AAV4 protein. The composition can further comprise, e.g., serum, serum-free medium, or a pharmaceutically acceptable carrier such as physiological saline, etc..

By "an antibody that specifically binds" an AAV4 polypeptide or protein is meant an antibody that selectively binds to an epitope on any portion of the AAV4 peptide such that the antibody selectively binds to the AAV4 polypeptide, such that the antibody binds specifically to the corresponding AAV4 polypeptide without significant background. Specific binding by an antibody further means that the antibody can be used to selectively remove the target polypeptide from a sample comprising the polypeptide or and can readily be determined by radioimmuno assay (RIA), bioassay, or enzyme-linked immunosorbant (ELISA) technology. An ELISA method effective for the detection of the specific antibody-antigen binding can, for example, be as follows: bind the antibody to a substrate; contact the bound antibody with a sample containing the antigen; contact the above with a secondary antibody bound to a detectable moiety horseradish peroxidase enzyme or alkaline phosphatase enzyme); contact the above with the substrate for the enzyme; contact the above with a color reagent; observe the color change.

An antibody can include antibody fragments such as Fab fragments which retain the binding activity. Antibodies can be made as described in, Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring 10 Harbor, New York (1988). Briefly, purified antigen can be injected into an animal in an oooo amount and in intervals sufficient to elicit an immune response. Antibodies can either be purified directly, or spleen cells can be obtained from the animal. The cells are then fused with an immortal cell line and screened for antibody secretion. Individual hybridomas are then propagated as individual clones serving as a source for a particular monoclonal antibody.

The present invention additionally provides a method of screening a cell for infectivity by AAV4 comprising contacting the cell with AAV4 and detecting the presence ofAAV4 in the cells. AAV4 particles can be detected using any standard physical or biochemical methods. For example, physical methods that can be used for this detection include 1) polymerase chain reaction (PCR) for viral DNA or RNA, 2) direct hybridization with labeled probes, 3) antibody directed against the viral structural or non- structural proteins. Catalytic methods of viral detection include, but are not limited to, detection of site and strand specific DNA nicking activity of Rep proteins or replication of an AAV origin- containing substrate. Additional detection methods are outlined in Fields, Virology, Raven Press, New York, New York. 1996.

For screening a cell for infectivity by AAV4 wherein the presence of AAV4 in the cells is determined by nucleic acid hybridization methods, a nucleic acid probe for such detection can comprise, for example, a unique fragment of any of the AAV4 nucleic acids provided herein. The uniqueness of any nucleic acid probe can readily be 26 determined as described herein for unique nucleic acids. The nucleic acid can be, for example, the nucleic acid whose nucleotide sequence is set forth in SEQ ID NO: 1, 3, 6, 7, 12, 13, 14, 15, 17 or 19, or a unique fragment thereof.

The present invention includes a method of determining the suitability of an AAV4 vector for administration to a subject comprising administering to an antibodycontaining sample from the subject an antigenic fragment of an isolated AAV4 capsid protein, and detecting an antibody-antigen reaction in the sample, the presence of a reaction indicating the AAV4 vector to be unsuitable for use in the subject. The AAV4 capsid protein from which an antigenic fragment is selected can have the amino acid sequence set forth in SEQ ID NO:4. An immunogenic fragment of an isolated AAV4 capsid protein can also be used in these methods. The AAV4 capsid protein from which .i an antigenic fragment is selected can have the amino acid sequence set forth in SEQ ID NO: 17. The AAV4 capsid protein from which an antigenic fragment is selected can have the amino acid sequence set forth in SEQ ID NO: 19.

Alternatively, or additionally, an antigenic fragment of an isolated AAV4 Rep protein can be utilized in this determination method. An immunogenic fragment of an isolated AAV4 Rep protein can also be used in these methods. Thus the present invention further provides a method of determining the suitability of an AAV4 vector for administration to a subject comprising administering to an antibody-containing sample from the subject an antigenic fragment of an AAV4 Rep protein and detecting an antibody-antigen reaction in the sample, the presence of a reaction indicating the AAV4 vector to be unsuitable for use in the subject. The AAV4 Rep protein from which an antigenic fragment is selected can have the amino acid sequence set forth in SEQ ID NO:2. The AAV4 Rep protein from which an antigenic fragment is selected can have the amino acid sequence set forth in SEQ ID NO:8. The AAV4 Rep protein from which an antigenic fragment is selected can have the amino acid sequence set forth in SEQ ID NO:9. The AAV4 Rep protein from which an antigenic fragment is selected can have the amino acid sequence set forth in SEQ ID NO:10. The AAV4 Rep protein from which an antigenic fragment is selected can have the amino acid sequence set forth in SEQ ID NO:11.

An antigenic or immunoreactive fragment is typically an amino acid sequence of at least about 5 consecutive amino acids, and it can be derived from the AAV4 polypeptide amino acid sequence. An antigenic fragment is any fragment unique to the AAV4 protein, as described herein, against which an AAV4-specific antibody can be raised, by standard methods. Thus, the resulting antibody-antigen reaction should be specific for AAV4.

The AAV4 polypeptide fragments can be analyzed to determine their antigenicity, immunogenicity and/or specificity. Briefly, various concentrations of a putative immunogenically specific fragment are prepared and administered to a subject and the immunological response the production of antibodies or cell mediated immunity) of an animal to each concentration is determined. The amounts of antigen administered depend on the subject, e.g. a human, rabbit or a guinea pig, the condition of the subject, the size of the subject, etc. Thereafter an animal so inoculated with the antigen can be exposed to the AAV4 viral particle or AAV4 protein to test the immunoreactivity or the antigenicity of the specific immunogenic fragment. The specificity of a putative antigenic or immunogenic fragment can be ascertained by testing sera, other fluids or lymphocytes from the inoculated animal for cross reactivity with other closely related viruses, such as AAV1, AAV2, AAV3 and As will be recognized by those skilled in the art, numerous types of immunoassays are available for use in the present invention to detect binding between an antibody and an AAV4 polypeptide of this invention. For instance, direct and indirect binding assays, competitive assays, sandwich assays, and the like, as are generally described in, U.S. Pat. Nos. 4,642,285;-4,376,110; 4,016,043; 3,879,262; 3,852,157; 3,850,752; 3,839,153; 3,791,932; and Harlow and Lane, Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, N.Y. (1988). For example, enzyme immunoassays such as immunofluorescence assays (IFA), enzyme linked immunosorbent assays (ELISA) and immunoblotting can be readily adapted to accomplish the detection of the antibody. An ELISA method effective for the detection of the antibody bound to the antigen can, for example, be as follows: bind the antigen to a substrate; contact the bound antigen with a fluid or tissue sample containing the antibody; contact the above with a secondary antibody specific for the antigen and bound to a detectable moiety horseradish peroxidase enzyme or alkaline phosphatase enzyme); contact the above with the substrate for the enzyme; contact the above with a color reagent; observe color change.

The antibody-containing sample of this method can comprise any biological sample which would contain the antibody or a cell containing the antibody, such as blood, plasma, serum, bone marrow, saliva and urine.

By the "suitability of an AAV4 vector for administration to a subject" is meant a determination of whether the AAV4 vector will elicit a neutralizing immune response upon administration to a particular subject. A vector that does not elicit a significant immune response is a potentially suitable vector, whereas a vector that elicits a significant, neutralizing immune response is thus indicated to be unsuitable for use in S" that subject. Significance of any detectable immune response is a standard parameter ooooo 20 understood by the skilled artisan in the field. For example, one can incubate the subject's serum with the virus, then determine whether that virus retains its ability to ~transduce cells in culture. If such virus cannot transduce cells in culture, the vector likely has elicited a significant immune response.

The present method further provides a method of delivering a nucleic acid to a cell comprising administering to the cell an AAV4 particle containing a vector comprising the nucleic acid inserted between a pair of AAV inverted terminal repeats, thereby delivering the nucleic acid to the cell. Administration to the cell can be accomplished by any means, including simply contacting the particle, optionally contained in a desired liquid such as tissue culture medium, or a buffered saline solution, with the cells. The particle can be allowed to remain in contact with the cells for any desired length of time, and typically the particle is administered and allowed to remain indefinitely. For such in vitro methods, the virus can be administered to the cell by standard viral transduction methods, as known in the art and as exemplified herein.

Titers of virus to administer can vary, particularly depending upon the cell type, but will be typical of that used for AAV transduction in general. Additionally the titers used to transduce the particular cells in the present examples can be utilized. The cells can include any desired cell, such as the following cells and cells derived from the following tissues, in humans as well as other mammals, such as primates, horse, sheep, goat, pig, dog, rat, and mouse: Adipocytes, Adenocyte, Adrenal cortex, Amnion, Aorta, Ascites, 10 Astrocyte, Bladder, Bone, Bone marrow, Brain, Breast, Bronchus, Cardiac muscle, Cecum, Cervix, Chorion, Colon, Conjunctiva, Connective tissue, Cornea, Dermis, Duodenum, Endometrium, Endothelium, Epithelial tissue, Epidermis, Esophagus, Eye, Fascia, Fibroblasts, Foreskin, Gastric, Glial cells, Glioblast, Gonad, Hepatic cells, Histocyte, Ileum, Intestine, small Intestine, Jejunum, Keratinocytes, Kidney, Larynx, Leukocytes, Lipocyte, Liver, Lung, Lymph node, Lymphoblast, Lymphocytes, Macrophages, Mammary alveolar nodule, Mammary gland, Mastocyte, Maxilla, Melanocytes, Monocytes, Mouth, Myelin, Nervous tissue, Neuroblast, Neurons, :Neuroglia, Osteoblasts, Osteogenic cells, Ovary, Palate, Pancreas, Papilloma, Peritoneum, Pituicytes, Pharynx, Placenta, Plasma cells, Pleura, Prostate, Rectum, Salivary gland, Skeletal muscle, Skin, Smooth muscle, Somatic, Spleen, Squamous, Stomach, Submandibular gland, Submaxillary gland, Synoviocytes, Testis, Thymus, Thyroid, Trabeculae, Trachea, Turbinate, Umbilical cord, Ureter, and Uterus.

The AAV inverted terminal repeats in the vector for the herein described delivery methods can be AAV4 inverted terminal repeats. Specifically, they can comprise the nucleic acid whose nucleotide sequence is set forth in SEQ ID NO:6 or the nucleic acid whose nucleotide sequence is set forth in SEQ ID NO:20, or any fragment thereof demonstrated to have ITR functioning. The ITRs can also consist essentially of the nucleic acid whose nucleotide sequence is set forth in SEQ ID NO:6 or the nucleic acid whose nucleotide sequence is set forth in SEQ ID NO:20. Furthermore, the AAV inverted terminal repeats in the vector for the herein described nucleic acid delivery methods can also comprise AAV2 inverted terminal repeats. Additionally, the AAV inverted terminal repeats in the vector for this delivery method can also consist essentially of AAV2 inverted terminal repeats.

The present invention also includes a method of delivering a nucleic acid to a subject comprising administering to a cell from the subject an AAV4 particle comprising the nucleic acid inserted between a pair of AAV inverted terminal repeats, and returning the cell to the subject, thereby delivering the nucleic acid to the subject. The AAV ITRs can be any AAV ITRs, including AAV4 ITRs and AAV2 ITRs. For such an ex vivo administration, cells are isolated from a subject by standard means according to the cell type and placed in appropriate culture medium, again according to cell type (see, e.g., ATCC catalog). Viral particles are then contacted with the cells as described above, and the virus is allowed to transfect the cells. Cells can then be transplanted back into the subject's body, again by means standard for the cell type and tissue in general, 15 U.S. Patent No. 5,399,346; for neural cells, Dunnett, S.B. and Bjorklund, eds., Transplantation: Neural Transplantation-A Practical Approach, Oxford University Press, Oxford (1992)). If desired, prior to transplantation, the cells can be studied for degree of transfection by the virus, by known detection means and as described herein.

Cells for ex vivo transfection followed by transplantation into a subject can be selected 20 from those listed above, or can be any other selected cell. Preferably, a selected cell type is examined for its capability to be transfected by AAV4. Preferably, the selected *e cell will be a cell readily transduced with AAV4 particles; however, depending upon the application, even cells with relatively low transduction efficiencies can be useful, particularly if the cell is from a tissue or organ in which even production of a small amount of the protein or antisense RNA encoded by the vector will be beneficial to the subject.

The present invention further provides a method of delivering a nucleic acid to a cell in a subject comprising administering to the subject an AAV4 particle comprising the nucleic acid inserted between a pair of AAV inverted terminal repeats, thereby delivering the nucleic acid to a cell in the subject. Administration can be an ex vivo administration directly to a cell removed from a subject, such as any of the cells listed above, followed by replacement of the cell back into the subject, or administration can be in vivo administration to a cell in the subject. For ex vivo administration, cells are isolated from a subject by standard means according to the cell type and placed in appropriate culture medium, again according to cell type (see, ATCC catalog).

Viral particles are then contacted with the cells as described above, and the virus is allowed to transfect the cells. Cells can then be transplanted back into the subject's body, again by means standard for the cell type and tissue for neural cells, Dunnett, S.B. and Bjorklund, eds., Transplantation: Neural Transplantation-A PracticalApproach, Oxford University Press, Oxford (1992)). If desired, prior to transplantation, the cells can be studied for degree oftransfection by the virus, by known detection means and as described herein.

In vivo administration to a human subject or an animal model can be by any of 15 many standard means for administering viruses, depending upon the target organ, tissue or cell. Virus particles can be administered orally, parenterally intravenously), by intramuscular injection, by direct tissue or organ injection, by intraperitoneal injection, topically, transdermally, or the like. Viral nucleic acids (non-encapsidated) can be administered, as a complex with cationic liposomes, or encapsulated in anionic 20 liposomes. Compositions can include various amounts of the selected viral particle or non-encapsidated viral nucleic acid in combination with a pharmaceutically acceptable carrier and, in addition, if desired, may include other medicinal agents, pharmaceutical agents, carriers, adjuvants, diluents, etc. Parental administration, if used, is generally characterized by injection. Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution or suspension in liquid prior to injection, or as emulsions. Dosages will depend upon the mode of administration, the disease or condition to be treated, and the individual subject's condition, but will be that dosage typical for and used in administration of other AAV vectors, such as AAV2 vectors. Often a single dose can be sufficient; however, the dose can be repeated if desirable.

32 The present invention further provides a method of delivering a nucleic acid to a cell in a subject having antibodies to AAV2 comprising administering to the subject an AAV4 particle comprising the nucleic acid, thereby delivering the nucleic acid to a cell in the subject. A subject that has antibodies to AAV2 can readily be determined by any of several known means, such as contacting AAV2 protein(s) with an antibodycontaining sample, such as blood, from a subject and detecting an antigen-antibody reaction in the sample. Delivery of the AAV4 particle can be by either ex vivo or in vivo administration as herein described. Thus, a subject who might have an adverse immunogenic reaction to a vector administered in an AAV2 viral particle can have a desired nucleic acid delivered using an AAV4 particle. This delivery system can be particularly useful for subjects who have received therapy utilizing AAV2 particles in the past and have developed antibodies to AAV2. An AAV4 regimen can now be substituted to deliver the desired nucleic acid.

Ce STATEMENT OF UTILITY The present invention provides recombinant vectors based on AAV4. Such vectors may be useful for transducing erythroid progenitor cells which is very inefficient with AAV2 based vectors. In addition to transduction of other cell types, transduction of erythroid cells would be useful for the treatment of cancer and genetic diseases which can be corrected by bone marrow transplants using matched donors. Some examples of this type of treatment include, but are not limited to, the introduction of a therapeutic gene such as genes encoding interferons, interleukins, tumor necrosis factors, adenosine deaminase, cellular growth factors such as lymphokines, blood coagulation factors such as factor VIII and IX, cholesterol metabolism uptake and transport protein such as EpoE and LDL receptor, and antisense sequences to inhibit viral replication of, for S. example, hepatitis or HIV.

The present invention provides a vector comprising the AAV4 virus as well as AAV4 viral particles. While AAV4 is similar to AAV2, the two viruses are found herein to be physically and genetically distinct. These differences endow AAV4 with some unique advantages which better suit it as a vector for gene therapy. For example, the wt S. 20 AAV4 genome is larger than AAV2, allowing for efficient encapsidation of a larger recombinant genome. Furthermore, wt AAV4 particles have a greater buoyant density than AAV2 particles and therefore are more easily separated from contaminating helper virus and empty AAV particles than AAV2-based particles.

Furthermore, as shown herein, AAV4 capsid protein is distinct from AAV2 capsid protein and exhibits different tissue tropism. AAV2 and AAV4 are shown herein to utilize distinct cellular receptors. AAV2 and AAV4 have been shown to be serologically distinct and thus, in a gene therapy application, AAV4 would allow for transduction of a patient who already possess neutralizing antibodies to AAV2 either as a result of natural immunological defense or from prior exposure to AAV2 vectors.

34 The present invention is more particularly described in the following examples which are intended as illustrative only since numerous modifications and variations therein will be apparent to those skilled in the art.

EXAMPLES

To understand the nature of AAV4 virus and to determine its usefulness as a vector for gene transfer, it was cloned and sequenced.

Cell culture and virus propagation Cos and HeLa cells were maintained as monolayer cultures in D10 medium (Dulbecco's modified Eagle's medium containing 10% fetal calf serum, loo ug/ml i penicillin, 100 units/ml streptomycin and IX Fungizone as recommended by the S- 15 manufacturer, (GIBCO, Gaithersburg, MD, USA) All other cell types were grown under standard conditions which have been previously reported. AAV4 stocks were obtained from American Type Culture Collection VR- 64 6.

Virus was produced as previously described for AAV2 using the Beta galactosidase vector plasmid and a helper plasmid containing the AAV4 Rep and Cap genes The helper plasmid was constructed in such a way as not to allow any homologous sequence between the helper and vector plasmids. This step was taken to minimize the potential for wild-type (wt) particle formation by homologous recombination.

Virus was isolated from 5x10 7 cos cells by CsCI banding and the distribution of Beta galactosidase genomes across the genome was determined by DNA dot blots of aliquots of gradient fractions. The majority of packaged genomes were found in fractions with a density of 1.43 which is similar to that reported for wt AAV4. This preparation of virus yielded 2.5 X10" particles or 5000 particles/producer cell. In comparison AAV2 isolated and CsCI banded from 8X10 7 cells yielded 1.2 particles or 1500 particles/producer cell. Thus, typical yields of rAAV4 particles/producer cell were 3-5 fold greater than that of rAAV2 particles.

DNA Cloning and Sequencing and Analysis In order to clone the genome of AAV4, viral lysate was amplified in cos cells and then HeLa cells with the resulting viral particles isolated by CsCI banding. DNA dot blots of aliquots of the gradient fractions indicated that peak genomes were contained in fractions with a density of 1.41-1.45. This is very similar to the buoyant density previously reported for AAV4 Analysis of annealed DNA obtained from these fractions indicated a major species of 4.8kb in length which upon restriction analysis gave bands similar in size to those previously reported. Additional restriction analysis indicated the presence ofBssHII restriction sites near the ends of the DNA. Digestion with BssHII yielded a 4.5kb fragment which was then cloned into Bluescript SKII+ and two independent clones were sequenced.

The viral sequence is now available through Genebank, accession number 15 U89790. DNA sequence was determined using an ABI 373A automated sequencer and the FS dye terminator chemistry. Both strands of the plasmids were sequenced and confirmed by sequencing of a second clone. As further confirmation of the authenticity of the sequence, bases 91-600 were PCR amlified from the original seed material and directly sequenced. The sequence of this region, which contains a 56 base insertion 20 compared to AAV2 and 3, was found to be identical to that derived from the cloned material. The ITR was cloned using Deep Vent Polymerase (New England Biolabs) according to the manufactures instructions using the following primers, primer 1: 5'TCTAGTCTAGACTTGGCCACTCCCTCTCTGCGCGC(SEQ ID NO:21); primer 2: *•*oo 51 AGGCCTTAAGAGCAGTCGTCCACCACCTTGTTCC (SEQ ID NO:22).

Cycling conditions were 97°C 20 sec, 65"C 30 sec, 75 C 1 min for 35 rounds.

Following the PCR reaction, the mixture was treated with XbaI and EcoRI endonucleases and the amplified band purified by agarose gel electrophoresis. The recovered DNA fragment was ligated into Bluescript SKII+ (Stratagene) and transformed into competent Sure strain bacteria (Stratagene). The helper plasmid (pSV40oriAAV 4 2 used for the production of recombinant virus, which contains the rep and cap genes of AAV4, was produced by PCR with Pfii polymerase (Stratagene) according to the manufactures instructions. The amplified sequence, nt 216-4440, was ligated into a plasmid that contains the SV40 origin of replication previously described 10). Cycling conditions were 95°C 30 sec, 55°C 30 sec, 72 0 C 3 min for 20 rounds.

The final clone was confirmed by sequencing. The Pgal reporter vector has been described previously Sequencing of this fragment revealed two open reading frames (ORF) instead of only one as previously suggested. In addition to the previously identified Capsid ORF in the right-hand side of the genome, an additional ORF is present on the left-hand side.

Computer analysis indicated that the left-hand ORF has a high degree ofhomology to the Rep gene of AAV2. At the amino acid level the ORF is 90% identical to that of AAV2 with only 5% of the changes being non-conserved (SEQ ID NO:2). In contrast, the right ORF is only 62% identical at the amino acid level when compared to the corrected AAV2 sequence. While the internal start site of VP2 appears to be conserved, the start site for VP3 is in the middle of one of the two blocks of divergent sequence.

15 The second divergent block is in the middle of VP3. By using three dimensional structure analysis of the canine parvovirus and computer aided sequence comparisons, regions of AAV2 which might be exposed on the surface of the virus have been identified. Comparison of the AAV2 and AAV4 sequences indicates that these regions are not well conserved between the two viruses and suggests altered tissue tropism for the two viruses.

Comparison of the p5 promoter region of the two viruses shows a high degree of conservation of known functional elements (SEQ ID NO:7). Initial work by Chang et al. identified two YY1 binding sites at -60 and +1 and a TATA Box at -30 which are all conserved between AAV2 and AAV4 A binding site for the Rep has been identified in the p5 promoter at -17 and is also conserved The only divergence between the two viruses in this region appears to be in the sequence surrounding these elements.

AAV4 also contains an additional 56 bases in this region between the p5 promoter and the TRS (nt 209-269). Based on its positioning in the viral genome and efficient use of the limited genome space, this sequence may possess some promoter activity or be involved in rescue, replication or packaging of the virus.

The inverted terminal repeats were cloned by PCR using a probe derived from the terminal resolution site (TRS)ofthe BssHII fragment and a primer in the Rep ORF.

The TRS is a sequence at the end of the stem of the ITR and the reverse compliment of TRS sequence was contained within the BssHII fragment. The resulting fragments were cloned and found to contain a number of sequence changes compared to AAV2.

However, these changes were found to be complementary and did not affect the ability of this region to fold into a hairpin structure (Fig While the TRS site was conserved between AAV2 and AAV4 the Rep binding site contained two alterations which expand the binding site from 3 GAGC repeats to 4. The first two repeats in AAV4 both contain a T in the fourth position instead of a C. This type of repeat is present in the promoter and is present in the consensus sequence that has been proposed for Rep binding (10) and its expansion may affect its affinity for Rep. Methylation interference data has suggested the importance of the CTTTG motif found at the tip of one palindrome in Rep binding with the underlined T residues clearly affecting Rep binding 15 to both the flip and flop forms. While most of this motif is conserved in AAV4 the middle T residue is changed to a C (33).

Hemagglutination assays Hemagglutination was measured essentially as described previously Serial 20 two fold dilutions of virus in Veronal-buffered saline were mixed with an equal volume of 0.4% human erythrocytes (type 0) in plastic U bottom 96 well plates. The reaction was complete after a 2 hr incubation at 8 C. HA units (HAU) are defined as the reciprocal of the dilution causing 50% hemagglutination.

The results show that both the wild type and recombinant AAV4 viruses can hemagglutinate human red blood cells (RBCS) with HA titers of approximately 1024 HAU/gl and 512 HAU/pl respectively. No HA activity was detected with AAV type 3 or recombinant AAV type 2 as well as the helper adenovirus. If the temperature was raised to 22 0 C, HA activity decreased 32-fold. Comparison of the viral particle number per RBC at the end point dilution indicated that approximately 1-10 particles per RBC were required for hemagglutination. This value is similar to that previously reported (18).

38 Tissue tropism analysis The sequence divergence in the capsid proteins ORF which are predicted to be exposed on the surface of the virus may result in an altered binding specificity for AAV4 compared to AAV2. Very little is known about the tissue tropism of any dependovirus.

While it had been shown to hemagglutinate human, guinea pig, and sheep erythrocytes, it is thought to be exclusively a simian virus Therefore, to examine AAV4 tissue tropism and its species specificity, recombinant AAV4 particles which contained the gene for nuclear localized Beta galactosidase were constructed. Because of the similarity in genetic organization of AAV4 and AAV2, it was determined whether AAV4 particles could be constructed containing a recombinant genome. Furthermore, because of the structural similarities of the AAV type 2 and type 4 ITRs, a genome containing AAV2 ITRs which had been previously described was used.

Tissue tropism analysis 1. To study AAV transduction, a variety of cell lines 15 were transduced with 5 fold serial dilutions of either recombinant AAV2 or AAV4 particles expressing the gene for nuclear localized Beta galactosidase activity (Table 1).

Approximately 4 X10 4 cells were exposed to virus in 0.5ml serum free media for 1 hour and then 1 ml of the appropriate complete media was added and the cells were incubated for 48-60 hours. The cells were then fixed and stained for P-galactosidase activity with 5-Bromo-4-Chloro-3-Indolyl-P-D-galactopyranoside (Xgal) (ICN Biomedicals) (36).

Biological titers were determined by counting the number of positive cells in the different dilutions using a calibrated microscope ocular (3.1mm 2 then multiplying by the area of the well and the dilution of the virus. Typically dilutions which gave 1-10 positive cells per field (100-1000 positive cells per 2cm well) were used for titer determination. Titers were determined by the average number of cells in a minimum of fields/well.

To examine difference in tissue tropism, a number of cell lines were transduced with serial dilutions of either AAV4 or AAV2 and the biological titers determined. As shown in Table 1, when Cos cells were transduced with a similar number of viral particles, a similar level of transduction was observed with AAV2 and AAV4.

However, other cell lines exhibited differential transducibility by AAV2 or AAV4.

Transduction of the human colon adenocarcinoma cell line SW480 with AAV2 was over 100 times higher than that obtained with AAV4. Furthermore, both vectors transduced SW1116, SW1463 and NIH3T3 cells relatively poorly.

TABLE 1 Cell type AAV2 AAV4 Cos 4.5 X10 7 1.9 X10 7 SW480 3.8 X10 6 2.8 X10 4 SW 1116 5.2 X10 4 8 X10 3 SW1463 8.8 X10 4 8 X10 3 SW620 8.8 X10 4

ND

NIH 3T3 2 X10 4 8X10 3 Tissue tropism analysis 2.

S. A. Transduction of cells. Exponentially growing cells (2 X 104) were plated in each well of a 12 well plate and transduced with serial dilutions of virus in 200 pl of medium 20 for I hr. After this period, 800 pl of additional medium was added and incubated for 48 hrs. The cells were then fixed and stained for P-galactosidase activity overnight with 5-bromo-4-chloro-3-indolyl-P-D-galactopyranoside (Xgal) (ICN Biomedicals) No endogenous P-galactosidase activity was visible after 24 hr incubation in Xgal solution.

Infectious titers were determined by counting the number of positive cells in the different dilutions using a calibrated microscope ocular diameter 3.1 mm 2 then multiplying by the area of the well and the dilution of the virus. Titers were determined by the average number of cells in a minimum of 10 fields/well.

As shown in Table 2, cos cells transduced with equivalent amounts of rAAV2 and rAAV4particles resulted in similar transduction levels. However, other cell lines exhibited differential transducibility. Transduction of the human colon adenocarcinoma cell line, SW480, with rAAV2 was 60 times higher than that obtained with rAAV4. Hela and SW620 cells were also transduced more efficiently with rAAV2 than rAAV4. In contrast, transduction of primary rat brain cultures exhibited a greater transduction of glial and neuronal cells with rAAV4 compared to rAAV2. Because of the heterogeneous nature of the cell population in the rat brain cultures, only relative transduction efficiencies are reported (Table 2).

As a control for adenovirus contamination of the viral preparations cos and Hela cells were coinfected with RAAV and adenovirus then stained after 24 hr. While the titer of rAAV2 increased in the presence of Ad in both cos and Hela, adenovirus only increased the titer in the cos cells transduced with rAAV4 and not the HeLa cells, suggesting the difference in transduction efficiencies is not the result of adenovirus contamination. Furthermore, both vectors transduced SW1116, SW1463, NIH3T3 and monkey fibroblasts FL2 cells very poorly. Thus AAV4 may utilize a cellular receptor distinct from that of AAV2.

15 TABLE 2 CELL TYPE AAV2 AAV4 Primary Rat Brain 1 4.3+ 0.7 cos 4.2X10 7 '4.6X10 6 2.2X10 7 2.5X10 6 20 SW 480 7.75X10 6 ±1.7X10 6 1.3X10 5 6.8X10 4 Hela 2.1X10 7 d:X10 6 1.3X10 6 SW620 1.2X10 5 '3.9X10 4 4X10 4 KLEB 1.2X10s 5 3.5X10 4 9X10 4 ±1.4X10 4 HB 5.6X10S'2X10 5 3.8X10 4 1l.8X0 4 SW1116 5.2 X 10 4 8 X 10 3 SW1463 8.8 X 10 4 8 X 10 3 NIH 3T3 3 X 10 3 2 X 10 3 B. Competition assay. Cos cells were plated at 2x 104 /well in 12 well plates 12-24 hrs prior to transduction. Cells were transduced with 0.5x 107 particles of rAAV2 or rAAV4 (containing the LacZ gene) in 200 pl of DMEM and increasing amounts of rAAV2 containing the gene for the human coagulation factor IX. Prior to transduction the CsCI was removed from the virus by dialysis against isotonic saline. After lhr incubation with the recombinant virus the culture medium was supplemented with complete medium and allowed to incubate for 48-60 hrs. The cells were then stained and counted as described above.

AAV4 utilization of a cellular receptor distinct from that of AAV2 was further examined by cotransduction experiments with rAAV2 and rAAV4. Cos cells were transduced with an equal number of rAAV2 or rAAV4 particles containing the LacZ gene and increasing amounts of rAAV2 particles containing the human 'coagulation factor IX gene (rAAV2FIX) At a 72:1 ratio of rAAV2FIX:rAAV4LacZ only a two-fold effect on the level of rAAV4LacZ 15 transduction was obtained (Fig However this same ratio of .rAAV2FIX:rAAV2LacZ reduced the transduction efficiency of rAAV2LacZ approximately 10 fold. Comparison of the 50% inhibition points for the two viruses indicated a 7 fold difference in sensitivity.

C. Trypsinization of cells. An 80% confluent monolayer of cos cells (lx 107) was treated with 0.05% trypsin/0.02% versene solution (Biofluids) for 3-5 min at 37C. Following detachment the trypsin was inactivated by the addition of an equal volume of media containing 10% fetal calf serum. The cells were then further diluted to a final concentration of lx 10 4 /ml. One ml of cells was plated in a 12 well dish and incubated with virus at a multiplicity of infection (MOI) of 260 for 1-2 hrs.

Following attachment of the cells the media containing the virus was removed, the cells washed and fresh media was added. Control cells were plated at the same time but were not transduced until the next day. Transduction conditions were done as described above for the trypsinized cell group. The number of transduced cells was determined by staining 48-60 hrs post transduction and counted as described above.

Previous research had shown that binding and infection of AAV2 is inhibited by trypsin treatment of cells Transduction of cos cells with rAAV21acZ gene was also inhibited by trypsin treatment prior to transduction (Fig In contrast trypsin treatment had a minimal effect on rAAV4 lacZ transduction. This result and the previous competition experiment are both consistent with the utilization of distinct cellular receptors for AAV2 and AAV4.

AAV4 is a distinct virus based on sequence analysis, physical properties of the virion, hemagglutination activity, and tissue tropism. The sequence data indicates that AAV4 is a distinct virus from that of AAV2. In contrast to original reports, AAV4 contains two open reading frames which code for either Rep proteins or Capsid proteins. AAV4 contains additional sequence upstream of the p5 promoter which may affect promoter activity, packaging or particle stability.

o oo :Furthermore, AAV4 contains an expanded Rep binding site in its ITR which could alter its activity as an origin of replication or promoter. The majority of the differences in the Capsid proteins lies in regions which have been proposed to be on the exterior surface of the parvovirus. These changes are most likely responsible for the lack of cross reacting antibodies, hemagglutinate activity, and the altered tissue tropism compared to AAV2. Furthermore, in contrast to previous reports AAV4 is able to transduce human as well as monkey cells.

Throughout this application, various publications are referenced. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this invention pertains.

Although the present process has been described with reference to specific details of certain embodiments thereof, it is not intended that such details should be regarded as limitations upon the scope of the invention except as and to the extent that they are included in the accompanying claims.

43 References: 1. Arella, S. Garzon, J. Bergeron, and P. Tijssen. Handbook of Parvoviruses. Vol. 1. ed. P. Tijssen. Boca Raton, Florida, CRC Press, 1990.

2. Bachmann, M.D. Hoggan, E. Kurstak, J.L. Melnick, H.G.

Pereira, P. Tattersall, and C. Vago. 1979. Interverology 11: 248-254.

3. Bantel-Schaal, U. and M. Stohr. 1992. J. Virol. 66: 773-779.

4. Chang, Y. Shi, and T. Shenk. 1989. J1 Virol. 63: 3479-88.

Chejanovsky, N. and B.J. Carter. 1989. Virology 173: 120-128.

O* 6. Chejanovsky, N. and B.J. Carter. 1989. Virology 171: 239-247.

Osee 7. Chiorini, S.M. Wiener, R.M. Kotin, R.A. Owens, SRM Ky6stiii, and B. Safer. 1994. 1. Virol. 68: 7448-7457.

fee.0 15 8. Chiorini, M.D. Weitzman, R.A. Owens, E. Urcelay, B. Safer, and 000 R.M. Kotin. 1994. 1. Virol. 68: 797-804.

9. Chiorini, C.M. Wendtner, E. Urcelay, B. Safer, M. Hallek, and R.M. Kotin. 1995. Human Gene Therapy 6: 1531-1541.

Chiorini, L. Yang, B. Safer, and R.M. Kotin. 1995. J1 Virol. 69: 7334-7338.

11. Dixit, M.S. Webb, W.C. Smart, and S. Ohi. 1991. Gene 104: 253- 7.

S12. Fisher, R.E. and H.D. Mayor. 1991. J Theor Biol 149: 429-39.

13. Flotte, S.A. Af ione, C. Conrad, S.A. McG rath, R. Solow, H. Oka, P.L. Zeitlin, W.B. Guggino, and B.J. Carter. 1993. Proc. Natl. Acad.

Sci. 90: 10613-10617.

14. Flotte, S.A. Aione, R. Solow, M.L. Drumm, D. Markakis, W.B.

Guggino, P.L. Zeitlin, and B.J. Carter. 1993. J Biol Chem 268: 378 1- 15. Hermonat, M.A. Labow, R. Wright, K.I. B~erns, and N.

Muzyczka. 1984. J. Virol. 51: 329-339.

44 16. Hermonat, F.L. and N. Muzyczka. 1984. Proc Nati Acad Sci USA 81: 6466-70.

17. Hunter, L.A. and R.J. Samulski. 1992. J. Virol. 66: 3 17-24.

18. Ito, M. and H.D. Mayor. 1968. J. Immuno. 100: 61-68.

19. Janik, M.M. Huston, K. Cho, and J.A. Rose. 1989. Virology 168: 320-9.

Kaplitt, P. Leone, R.J. Samulski, X. Xiao, D.W. Pfaff, K.L.

O'Malley, and J.M. During. 1994. Nature Genetics 8: 148-154.

21. Kotin, M. Siniscalco, R.J. Samuiski, X. Zhu, L. Hunter, C.A.

Laughlin, S. McLaughlin, N. Muzyczka, M. Rocchi, -and K.T. Berns.

1990. Proc. Natl. Acad. Sci. (USA) 87: 2211-2215.

Laughlin, N. Jones, and B.J. Carter. 1982. J. Virol. 41: 868-76.

23. Laughlin, M.W. Myers, D.L. Risin, B.J. Carter. 1979. Virology 94: 162-74.

24. McCarty, J. Pereira, I. Zolotukhin, X. Zhou, J.H. Ryan, and N.

Muzyczka. 1994. J. Virol. 68: 4988-4997.

Mendelson, J.P. Trempe, and B.J. Carter. 1986. J. Virol. 60: 823- :*:832.

26. Mizukami, N.S. Young, and K.E. Brown. 1996. Virology 217: 124- 130.

.*00 027. Muster, Y.S. Lee, J.E. Newbold, and J. Leis, 1980. J. Virol. 653-6 1.

28. iM'uzyczka, N. 1992. Curr Top Microbiol Immunol 158: 97-129.

29. Parks, J.L. Melnick, R. Rongey, and H.D. Mayor. 1967. J. Virol.

1: 171-180.

PodsakofT, K.K. Jr Wong, and S. Chatterjee. 1994. J. Virol. 68: 5656-5666.

31. Rose, M.D. Hoggan, F. Koczot, and A.J. Shatkin. 1968. J. Virol.

2: 999-1005.

32. Russell, A.D. Miller, and I.E. Alexander. 1994. Proc. Natl. Acad.

Sci. USA 91: 8915-8919.

33. Ryan, S. Zolotukhin, and N. Muzyczka. 1996. 1. Viral. 70: 1542- 1553.

34. Samulski, K.I. Berns, M. Tan, and N. Muzyczka. 1982. Proc Nati Acad Sci USA 79: 2077-8 1.

Samulski, L.S. Chang, and T. Shenk. 1989. 1. Virol. 63: 3 822-8.

36. Sanes, J.L.R Rubenstein, and J.F. Nicocas. 1986. EMBO 3133-3142.

37. Senapathy, J.D. Tratschin, and B.J. Carter. 1984. J Mol Biol 179: 1-20.

:38. Tratscbin, I.L. Miller, and B.J. Carter. 1984. 1. Viral. 51: 611- 619.

39. Trempe, J.P. and B.J. Carter. 1988. J. Viral. 62: 68-74.

40. Trempe, E. Mendelson, and B.J. Carter. 1987. Virology 161: 18- 28.

41. Walsh, J.M. Liu, X. Xiao, N.S. Young, A.W. Nienhuis, and R.J.

Saniulski. 1992. Proc Nati Acad Sci USA 89: 7257-6 1.

.42. Winocour, M.F. Callaham, and E. Huberman. 1988. Virology 167: 393-9.

S

S.

S

SEQUENCE LISTING GENERAL INFORMATION: APPLICANT: Chiorini, John A.

Kotin, Robert M.

Safer, Brian (ii) TITLE OF INVENTION: AAV4 VECTOR AND USES THEREOF (iii) NUMBER OF SEQUENCES: 22 (iv) CORRESPONDENCE ADDRESS: ADDRESSEE: Needle Rosenberg STREET: 127 Peachtree CITY: Atlanta STATE: Georgia COUNTRY: USA ZIP: 30303 COMPUTER READABLE FORM: MEDIUM TYPE: Floppy disk COMPUTER: IBM PC compatible OPERATING SYSTEM: PC-DOS/MS-DOS SOFTWARE: PatentIn Release Version #1.30 (vi) CURRENT APPLICATION DATA: APPLICATION NUMBER: FILING DATE:

CLASSIFICATION:

(viii) ATTORNEY/AGENT INFORMATION: NAME: Selby, Elizabeth REGISTRATION NUMBER: 38,298 REFERENCE/DOCKET NUMBER: 14014.0252 INFORMATION FCR SEQ ID NO:1: SEQUENCE CHARACTERISTICS: LENGTH: 4768 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (ix) OT-ER INFO: AAV4 genome (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:

TTGGCCACTC

AGACTGCCGG

GCCAACTCCA

GTCCCTGTAT

AAGCTGCCAC

GAGGGTATAT

CCTCTATGCG

CCTCTGGCCG

ATATCTAGGT

TAGCAGTCAC

GTCACAGCCA

AACCGCGAGT

CGCTCGCTCA

GCAGGGCCGA

TTGCCCACTG

GTGAGTGTCG

CGTGGTCCGT

GAGCCAGCGA

CTCACTCGGC

GTGAGTGAGC

ACGTCAATGT

TATTTCGCGG

TTGCGACAGT

GGAGCTCCAT

CCTGGAGACC

GAGCGCGCAT

GACGTCCTAG

AGCGTAGCGG

TTGCGACACC

TTTGCCCGCG

AAAGGTCTCC

AGAGGGAGTG

GGTTAGGGAG

AGCGCATACC

ATGTGGTCAG

AATTTTGAAC

0

GAGCAGCAGC

AGCACCTGCC

TGCCGCCGGA

AAAAGCTGCA

TCTTCTTTGT

CCGTGGGCGT

TGACCCGCAT

CGCGTAATGG

TGCTCCCCAA

GCGCCTGTTT

CGCAGACGCA

GGTCAAAAAC

CGTCAGAAAA

CCAACTCGCG

CAAAGACGGC

GCATCTACCG

TGGGCTGGGC

CGACGGGTAA

TGAACTGGAC

GGGAGGAGGG

GCAAGGTGCG

TCGTCACCTC

ACCAACAACC

ACTTTGGCAA

TGACCGAGGT

CCAATGACGC

CGTCAGACGC

ACGTGGGTAT

TGGACATTTG

CTCAACCCGT

TCATGGGGAG

CATGCCGGGG

CGGCATTTCT

TTCTGACATG

ACGCGAGTTC

CCAGTTCGAG

CAAATCCATG

CTACCGCGGG

CGCCGGAGGC

GACCCAGCCC

GAATCTCGCG

GGAGCAGAAC

CTCCGCCAGG

GCAATGGATC

GTCACAAATC

TCCGGACTAC

AATCCTCGAG

GCAAAAGAAG

AACCAACATC

CAATGAGAAC

CAAGATGACG

CGTGGACCAA

CAACACCAAC

ACTCCAGGAC

GGTCACCAAG

GACTCACGAG

AGATATAAGT

GGAAGCTCCG

GAATCTGATG

CTTCACGCAC

GTCTGTCGTC

GGCGCCCGAG

TTCTACGAGA

GACTCTTTTG

GACTTGAATC

CTGGTCGAGT

AAGGGGGACA

GTGGTGGGCC

GTCGAGCCGC

GGGAACAAGG

GAGCTCCAGT

GAGCGTAAAC

AAGGAAAACC

TACATGGAGC

CAGGAGGACC

AAGGCCGCGC

CTGGTGGGCC

ATGAACIGGGT

TTCGGGAAGA

GCGGAAGCCA

TTTCCGTTCA

GCCAAGGTCG

AAGTGCAAGT

ATGTGCGCGG

CGGATGTTCA

CAG-GAAGTCA

TTTTACGTCA

GAGCCCAAGC

GTGGACTACG

CTTTTTCCCT

GGGGTCATGG

AGAAAGCGGA

GTGGCCTGCT

TCGTGCTGAA

TGAGCTGGGT

TGATTGAGCA

GGCGCCGC GT

GCTACTTCCA

GCTACGTGAG

AGCTTCCGAA

TGGTGGACGA

GGGCGTGGAC

GGCTGGTGGC

AGAACCCCAA

TGGTCGGGTG

AGGCGTCCTA

TGGACAATGC

AGAACCCGCC

ACGATCCGCA

GGAACACCAT

TCGCCCACGC

ACGATTGCGT

TAGAGAGCGC

CATCGGCCCA

TCATCGACGG

AGTTCGAGCT

AAGACTTTTT

GAAAGGGTGG

GGGCCTGTCC

CGGACAGGTA

GCCGGCAATG

ACTGTGCCGA

CGTATCAGA

CGGCCTGCGA

GGTGCCCAGC

GGCCGAGAAG

GGCACCCCTG

GAGTAAGGCC

CCTGCACATC

CCAGATTAAA

CTGGTTCGCG

CTGCTACATC

TAACATGGAC

GCAGCATCTG

TTCTGACGCG

GCTGGTGGAC

CATCTCCTTC

CTCCAAAATC

GGAGGACATT

GTACGCGGCC

CTGGCTCTTT

CGTGCCCTTC

CGACAAGATG

CAAGGCCATC

GATCGACCCA

AAACTCGACC

CACCAAGCGC

CCGGTGGGCG

AGCTAGAAAG

GTCAGTTGCG

CCAAAACAAA

CGAGAGAATG

GTGCTTCCCC

ACTGTGTCCG

ACTGGCCAAT

TGACTGACGG

GACCTGGACG

GAATGGGAGC

ACCGTGGCCG

CCGGAGGCCC

CTGGTGGAG-A

GAGAAGCTGG

GTGACCAAGA

CCCAACTACC

CAGTATATAA

ACGCACGTGT

CCGGTCATCA

CGCGGGATCA

AACGCCGCCT

ATGAGCCTGA

TCCAGCAACC

TCCGTCTTCC

GGGCCGGCCA

TACGGCTGCG

GTGATCTGGT

CTGGGCGGAA

ACTCCCGTGA

ACCTTCGAGC

CTGGAGCACG

TCAGATCACG

AGGCCCGCCC

CAGCCATCGA

TGTTCTCGTC

AATCAGAATG

GTGTCAGAAT

ATTCATCACA

GTGGACTTGG

TTACCTTCCA

420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 1920 1980 2040 2100 2160 2220 2280 ATGACTGTGA CATGGAACAA TAAATGACTC AAACCAGATA GATTGGCTAG AGGACAACCT CTCTGAAGGC

GCCCCTAAAC

GGTTACAAAT

GACGCGGCAG

CCCTACCTCA

CCGTTTGGGG

CTTGGTCTGG

TCCCCCCAGC

AAGAAGCTCG

TCCGGAGCCA

GGSGGACAAG

TGGTCTGAGG

AACCACCTN'T

ACCCCCTGGG

CAGCGACTCA

AACATCCAGG

ACCAGCACGG

GGTCAAGAGG

TACTGTGGAC

TGCCTGGAGT

AGTTTTGAGA

ATGAACCCTC

CTGAATGCCG

TTTAAAAAGA

AATCAAAACT

AGCACTCTGG

CCTGCGGACA

AACACGGCCA

AACGCCACCG

CTGCCGACCG

AGAGACATTT

CACCCCTCAC

CCAAGGCAAA

ACCTCGGACC

CCCTCGAGCA

AGTACAACCA

GCAACCTCGG

TTGAGCAAGC

AGCCCGACTC

TTTTCGAAGA

TGTCTGATGA

GTGCCGATGG

GCCACGTCAC

ACAAGCGACT

GATACTTTGA

TCAACAACAA

TCAAGGAGGT

TTCAGATCTT

GCAGCCTGCC

TGGTGACCC-G

ACTTTCCTTC

AGGTGCCTTT

TCATCGACCA

GGACTGCCAC

ACTGGCTGCC

ACAAGATCCC

ACGGAAGATG

GCAAGTTCAG

CCGTACCCGG

ATACGGACAT

TGGACAGACT

ACTACCAGGG

CGCTGATTGG

TCAACAACAT

CGGCAACGGA

CGACAAGGCC

CGCCGACGCG

CAGAGCAGTC

GGGTGAGACG

CTCCACGGGT

CGAAACTGGA

CAGTGAGATG

AGTGGGTAAT

GACCACCAGC

CGGAGAGAGC

CTTCAACCGC

CTGGGGCATG

CACGACGTCG

TGCGGACTCG

TCCTTTTCCC

CAACACTTCG

GCAGATGCTG

CCACTCGATG

GTACCTGTGG

CACCAACTTT

CGGGCCTTCA

TGCCACCGGG

GAGTGCCCTG

CAACAGCCAG

GACTCTGATC

GTGGGGCAAC

GACAGCCTTG

TCCCATTTGG

TGGGTTTGGG

GTTCGAGAGT

CAGGACAACG

CTCGACAAGG

TACGACCAGC

GAGTTCCAGC

TTCCAGGCCA

GCTCCTGGAA

ATCGGCAAAA

GCAGGCGACG

CGTGCAGCAG

GCCTCGGGTG

ACCAGAACCT

CTGCAGTCCA

TTCCACTGCC

CGACCCAAAG

AACGGCGAGA

TCGTACGAAC

AACGACGTCT

CAGCAACAGA

CGGACTGGCA

TACGCGCACA

GGACTGCAAT

ACCAAGCTGC

ATCAAGCAGC

TCAGACAGTC

ACCCCCGGAC

CTCATCTTTG

TTCACCTCTG

CTACCTGGCG

GGAGCCGTGC

GCCAAGATTC

CTGAAACACC

GGTGGGCGCT

CTCGGGGTCT

GGGAACCCGT

AGCTCAAGGC

AGCGGCTTCA

AAAAGAGGGT

AGAAGAGACC

AAGGCAAGCA

GACCCCCTGA

CTGGCGGAGC

ATTGGCATTG

GGGTCTTGCC

ACACCTACAA

ACTTCTCACC

CCATGCGGGT

CAACGGTGGC

TGCCGTACGT

TTATGGTGCC

CTGACAGAAA

ACAACTTTGA

GCCAGAGCCT

CGACCACCAC

GGCCTACCAA

AGGGCTTCTC

TCATCAAATA

CTCCAATGGC

CGGGGCCTAA

AGGAGGAGCT

GTGACCAGAG

CTGGAATGGT

CTCATACCGA

CGCCTCCTCA

GCAACCTGGA

TGTGCTTCCG

CAACGCAGCG

CGGTGACAAC

GGGCGACACA

TCTTGAACCT

GTTGATTGAA

GCCGGCTAAA

GGGATCAACT

TGCAGTCGAG

CGATTCCACC

CACCTACAAC

CGGATTCTCC

ACGTGACTGG

CAAAATCTTC

TAATAACCTT

GATGGATGCG

CCAGTACGGC

TGCCTTCTAC

AATTACGTAC

GGACCGGCTG

CGGAACCACC

CTTTTCCAAC

AAAGACTGCC

CGAGACGCAC

CACGGCTGGA

ACAGAACGGC

GGCAGCCACC

CAACAGCAAC

CTGGCAAAAC

TGGACACTTT

AATTTTTATC

2340 2400 2460 2520 2580 2640 2700 2760 2820 2880 2940 3000 3060 3120 3180 3240 3300 3360 3420 3480 3540 3600 3660 3720 3780 3840 3900 3960 4020 4080 -4140 4200

AAGAACACCC

TTCATTACTC

GAGCGGTCCA

TCTCTGTTGT

CGCTACCTCA

GTTGP.ACTTT

TAAGCAGCGG

TCTGGCAAAC

GGCCCTGGAG

AGCGAGCGCG

CGGTACCTGC

AGTACAGCAC

AACGCTGGAA

GGGCTCCCGA

CCCACCACCT

GGTCTCCGTG

CCTGCGGCGC

CATGATGATG

ACCAAAGGTC

CATAGAGGGA

GAATCCTGCA

TGGCCAGGTG

CCCCGAGGTC

TGCGGCTGGG

GTAATAACCT

TCCTTCTTAT

TTGCGCTTCG

GAGTTGGCCA

TCCAGACTGC

GTGGCCAA

ACGACCTTCA

TCGGTGCAGA

CAGTTTACCT

AAATACACTG

GTTAATCAAT

CTTATCTCGT

CGGTTTACAA

CTCCCTCTAT

CGGCCTCTGG

GCTCTACTCC

TTGACTGGGA

CCAACTACGG

AGCCTAGGGC

AAACCGGTTT

TTCCATGGCT

CTGCCGGTTA

GCGCGCTCGC

CCGGCAGGGC

GGTAAACTCC

GATCCAGAAG

ACAGCAAAAC

TATCGGTACC

ATTCGTTTCA.

ACTGCGTACA

ATCAGTAACT

TCACTCACTC

CGAGTGAGTG

4260 4320 4380 4440 4500 4560 4620 4680 4740 4768 INFORMATION FOR SEQ ID NO:2: Wi SEQUENCE CHARACTERISTICS: LENGTH: 624 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (ix) OTHER INFO: AAV4 Rep protein (full length) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: Met Pro Gly Phe Tyr Glu Ile Val Leu Lys 1 Glu.

Lys His Leu Pro Glu Trp, Glu.

5 Gly Ile Leu Pro Val Pro Val Ser Ser Asp Leu Asp Ser Asp Ser 25 Pro Asp Ser Trp Asp Met Asp 35 Glu Gin Ala Leu Arg Val. Ala Giu Asn Leu Ile Glu Phe Leu Pro Leu Thr Glu Lys Leu so Val Giu.

Gin Ala Gin Trp Arg Arg Val 70 Glu Lys Gly Asp Lys Ala Pro Leu Phe Phe Val1 Phe Ser Tyr Phe His Ile Leu Val Giu Thr Val Gly Lys Glu Lys 115 Pro Asn Trp Lys Ser Met Vai Val 105 Tyr Arg Tyr Val Val Thr Arg Arg Gly Val Ser Gln Ile 110 Pro Gin Leu Gly Giy Gly Phe Ala Val 130 Asn Lys 145 Val Val Asp ASP 150 Thr 135 Cys Lys Thr Arg Asn Gly 140 Tyr Tyr Ile Pro Asn 155 Leu Leu Pro Lys 160 0 0..

.0 0 Thr Ser Leu Pro Met 225 Gin Ser Ile Pro As n 305 Gin Thr Phe Cys Lys 385 Val Ile Thr Giu Glu 465 Thr Gin Aila Thr Asn 210 Giu Trp Asn Met Pro 290 Giy Lys Thr Tyr Val1 370 Vai Asp Vai Thr Leu 450 Vali His Pro Cys His 195 Ser Leu Ile Ser Ser 275 Giu Tyr Lys Gly Gly 355 Asp Vai Gin Thr Phe 435 Thr Lys Giu Giu Leu 180 Vai Asp Val Gin Arg 260 Leu Asp Asp Phe Lys 340 Cys Lys Giu Lys Ser 420 Giu Lys Asp Phe Leu 165 Asn Ser Aila Gly Giu 245 Ser Thr Ile Pro Gly 325 Thr Vai Met Ser Cys 405 As n His Arg Phe Tyr 485 Gin Leu Gin Pro Trp 230 Asp Gin Lys Ser Gin 310 Lys Asn Asn Vai Al a 390 Lys Thr C-in Leu Phe 470 Val Trp Aia Thr Vai 215 Leu Gin Ile Thr Ser 295 Tyr Arg Ile Trp Ile 375 Lys Ser Asn Gin Giu 455 Arg Arg Aia Giu Gin 260 Ile Val Aila Lys Ala 280 Asn Aila Asn Ala Thr 360 Trp Ala Ser Met Pro 440 His Trp Lys Trp Arg 185 Giu Arg Asp Ser Ala 265 Pro Arg Ala Thr Giu 345 Asn Trp Ile Ala Cys 425 Leu Asp Aia Gly Thr 170 Lys Gin Ser Arg Tyr 250 Al a Asp Ile Ser Ile 330 Ala Giu Giu Leu Gin 410 Al a Gin Phe Ser Giy 490 As n Arg As n Lys Giy 235 Ile Leu Tyr Tyr Val 315 Trp Ile Asn Glu Giy 395 Ile Vai Asp Gly Asp 475 Ala Met Leu Lys Thr 220 Ile Ser Asp Leu Arg 300 Phe Leu Ala Phe Giy 380 Giy Asp Ile Arg Lys 460 His Arg Asp Val Giu 205 Ser Thr Phe Asn Val 285 Ile Leu Phe His Pro 365 Lys Ser Pro Asp Met 445 Val Val Lys Ile His Asn Tyr Lys 240 Al a Lys Asn Met Al a 320 Ala Pro Asp Ala Arg 400 Val Ser Phe Gin Val 480 Ala Pro Ala Arg Phe 545 Phe Ser Pro Cys Asn Gin Tyr 530 Pro Thr Gin Ile Glu 610 Ala 500 Ser Asn Arg Gly Val 580 His Ala Asp lie Ser Ser Cys Cys 550 Met Val Met Val Asp Ser 535 Glu Asp Val Gly Asp 615 Glu Ala 520 Arg Arg Cys Arg Arg 600 Leu 51 Pro 505 Glu His Met Ala Lys 585 Ala Asp Lys Ala Val Asn Glu 570 Arg Pro Asp Arg Pro Gly Gin 555 Cys Thr Glu Cys Cys Asp 525 Asn Val Pro Gin Ala 605 Met

C

C. C

C

INFORMATION FOR SEQ ID NO:3: SEQUENCE CHARACTERISTICS: LENGTH: 1872 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (ix) OTHER INFO: AAV4 Rep gene (full length) (ix) FEATURE: NAME/KEY: CDS LOCATION: 1..1872 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: ATG CCG GGG TTC TAC GAG ATC GTG CTG AAG GTG CCC AGC GAC Met Pro Gly Phe Tyr Glu Ile Val Leu Lys Val Pro Ser Asp 1 5 10 GAG CAC CTG CCC GGC ATT TCT GAC TCT TTT GTG AGC TGG GTG Glu His Leu Pro Gly Ile Ser Asp Ser Phe Val Ser Trp Val 25 AAG GAA TGG GAG CTG CCG CCG GAT TCT GAC ATG GAC TTG AAT Lys Glu Trp Glu Leu Pro Pro Asp Ser Asp Met Asp Leu Asn 40 GAG CAG GCA CCC CTG ACC GTG GCC GAA AAG CTG CAA CGC GAG Glu Gin Ala Pro Leu Thr Val Ala Glu Lys Leu Gin Arg Glu 55 Ser Val Ala Asp Met Leu Ile Cys 560 Ser Glu 575 Leu Cys Ser Ala Gin CTG GAC Leu Asp GCC GAG Ala Glu CTG ATT Leu Ile TTC CTG Phe Leu 52 GTC GAG TGG CGC CGC GTG AGT AAG GCC CCG GAG GCC CTC TTC TTT GTC Val Giu Trp, Arg Arg Val Ser Lys Ala Pro Glu Ala Leu Phe Phe Val 70

C

C. C C

C.

CAG

Gin

ACC

Thr

AAA

Lys

CCG

Pro

AAC

Asn 145

ACC

Thr

AGC

Ser

CTG

Leu

CCC

Pro

ATG

Met 225

CAA

Gin

TCC

Ser

ATC

Ile

CCG

Pro

TTC

Phe

GTG

Vai

GAG

Giu

AAC

Asn 130

AAG

Lys

CAG

Gin

GCC

Ala

ACG

Thr

AAT

As n 210

GAG

Giu

TGG

Trp

AAC

As n

ATG

Met

CCG

Pro 290

GAG

Glu

GGC

Gly

AAG

Lys 115

TGG

Trp

GTG

Vai

CCC

Pro

TGT

Cys

CAC

His 195

TCT

Ser

CTG

Leu

ATC

Ile

TCG

Ser

AGC

Ser 275

GAG

Glu

AAG

Lys

GTC

Vai 100

CTG

Leu

TTC

Phe

GTG

Vai

GAG

Giu

TTG

Leu 180

GTG

Val

GAC

Asp

GTC

Val1

CAG

Gin

CGG

Arg 260

CTG

Leu

GAC

Asp

GGG

Gly

AAA

Lys

GTG

Val

GCG

Ala

GAC

Asp

CTC

Leu 165

AAT

As n

TCG

Ser

GCG

Al a

GGG

Gly

GAG

Giu 245

TCA

Ser

ACA

Thr

ATT

Ile

GAC

Asp

TCC

Ser

ACC

Thr

GTG

Val1

GAC

Asp 150

CAG

Gin

CTC

Leu

CAG

Gin

CCG

Pro

TGG

Trp 230

GAC

Asp

CAA

Gin

AAG

Lys

TCC

Ser

AGC

Ser

ATG

Met

CGC

Arg

ACC

Thr 135

TGC

Cys

TGG

Trp

GCG

Ala

ACG

Thr

GTC

Val 215

CTG

Leu

CAG

Gin

ATC

Ile

ACG

Thr

AGC

Ser 295

TAC

TTC

Phe

GTG

Val1 105

TAC

Tyr

ACG

Thr

ATC

Ile

TGG

Trp

CGT

Arg 185

GAG

AGG

Arg

GAC

Asp

TCC

Ser

GCC

Al a 265

CCG

Pro

CGC

Arg

GCC

CAC

His 90

GGC

Gly

CGC

Arg

CGT

Arg

CCC

Pro

ACT

Thr 170

AAA

Lys

CAG

Gin

TCA

Ser

CGC

Arg

TAC

Tyr 250

GCG

Ala

GAC

Asp

ATC

Ile

TCC

75

CTG

Leu

CGC

Arg

GGG

Gly

AAT

Asn

AAC

Asn 155

AAC

Asn

CGG

Arg

AAC

Asn

AAA

Lys

GGG

Gly 235

ATC

Ilie

CTG

Leu

TAC

Tyr

TAC

Tyr

GTC

Val1 315

CAC

His

TAC

Tyr

GTC

Val

GGC

Gly 140

TAC

Tyr

ATG

Met

CTG

Leu

AAG

Lys

ACC

Thr 220

ATC

Ile

TCC

Ser

GAC

Asp

CTG

Leu

CGA

Arg 300

TTC

Phe

ATC

Ile

GTG

Val

GAG

Glu 125

GCC

Ala

CTG

Leu

GAC

Asp

GTG

Val1

GAA

Giu 205

TCC

Ser

ACG

Thr

TTC

Phe

AAT

Asn

GTG

Val 285

ATC

Ile

CTG

Leu

GAG

Giu

ATT

Ile

CTT

Leu

GGG

Gly

AAG

Lys 160

ATA

Ile

CAT

His

AAC

As n

TAC

Tyr

AAG

Lys 240

GCC

Ala

AAA

Lys

AAC

Asn

ATG

Met

GCG

Ala 320 AAC GGG TAC GAT CCG CAG Asn 305 Gly Tyr Asp Pro Gin Tyr Ala Ala Ser 310 53 AAG AAG TTC GGG AAG AGG AAC ACC ATC TGG CTC TTT GGG CCG GCC CAA Gin Lys Lys Phe

ACG

Thr

TTC

Phe

TGC

Cys

AAG

Lys 385

GTG

Val1

ATC

Ile

ACC

Thr

GAG

Glu

GAA

Glu 465

ACT

Thr

CCC

Pro

GCG

Al a

AGG

Arg

TTT

Phe 545

TTC

Phe

ACG

Thr

TAC

Tyr

GTC

Val 370

GTC

Val

GAC

Asp

GTC

Val

ACC

Thr

CTC

Leu 450

GTC

Val

CAC

His

AAT

As n

CAG

Gin

TAC

Tyr 530

CCC

Pro

ACG

Thr

GGT

Giy

GGC

Giy 355

GAC

Asp

GTA

Val

CAA

Gin

ACC

Thr

TTC

Phe 435

ACC

Thr

AAA

Lys

GAG

Giu

GAC

Asp

CCA

Pro i5

CAA

Gin

TGC

Cys

CAC

His

AAA

Lys 340

TGC

Cys

AAG

Lys

GAG

Glu

AAG

Lys

TCC

Ser 420

GAG

Giu

AAG

Lys

GAC

Asp

TTT

Phe

GCA

Ala 500

TCG

Ser

AAC

Asn

CGG

Arg

GGG

Giy Gly 325

ACC

Thr

GTG

Vai

ATG

Met

AGC

Ser

TGC

Cys 405

AAC

Asn

CAC

His

CGC

Arg

TTT

Phe

TAC

Tyr 485

GAT

Asp

ACG

Thr

AAA

Lys

CAA

Gin

GTC

Vai 565

AAC

Asn

AAC

Asn

GTG

Vali

GCC

Al a 390

AAG

Lys

ACC

Thr

CAA

Gin

CTG

Leu

TTC

Phe 470

GTC

Val

ATA

Ile

TCA

Ser

TGT

Cys

TGC

Cys 550

ATG

Met

GCG

Ala

ACC

Thr 360

TGG

Trp

GCC

Ala

TCG.

Ser

ATG

Met

CCA

Pro 440

CAC

His

TGG

Trp

AAG

Lys

GAG

Giu

GCG

Al a 520

CGT

Arg

AGA

Arg

TGT

Cys

GAA

Giu 345

AAT

Asn

TGG

Trp

ATC

Ile

GCC

Al a

TGC

Cys 425

CTC

Leu

GAC

Asp

GCG

Al a

GGT

Gly

CCC

Pro 505

GAA

Glu

CAC

His

ATG

Met

GCC

Ala Lys Arg Asn Thr Ile 330

GCC

Al a

GAG

Glu

GAG

Glu

CTG

Leu

CAG

Gln 410

GCG

Al a

CAG

Gin

TTT

Phe

TCA

Ser

GGA

Gly 490

AAG

Lys

GCT

Al a

GTG

Val

AAT

As n

GAG

Giu 570 Trp

ATC

Ile

AAC

Asn

GAG

Giu

GGC

Gly 395

ATC

Ile

GTC

Val

GAC

Asp

GGC

Gly

GAT

Asp 475

GCT

Al a

CGG

Arg

CCG

Pro

GGT

Gly

CAG

Gin 555

TGC

Cys Leu

GCC

Al a

TTT

Phe

GGC

Gly 380

GGA

Gly

GAC

Asp

ATC

Ile

CGG

Arg

AAG

Lys 460

CAC

His

AGA

Arg

GCC

Ala

GTG

Val1

ATG

Met 540

AAT

Asn

TTC

Phe Phe

CAC

His

CCG

Pro 365

AG

Lys

AGC

Ser

CCA

Pro

GAC

Asp

ATG

Met 445

GTC

Val

GTG

Val

AAG

Lys

TGT

Cys

GAC

Asp 525

AAT

Asn

GTG

Val

CCC

Pro Gly

GCC

Al a 350

TTC

Phe

ATG

Met

AAG

Lys

ACT

Thr

GGA

Gly 430

TTC

Phe

ACC

Thr

ACC

Thr

AGG

Arg

CCG

Pro 510

TAC

Tyr

CTG

Leu

GAC

Asp

GTG

Val Pro 335

GTG

Val1

AAC

Asn

ACG

Thr

GTG

Val1

CCC

Pro 415

AAC

Asn

AAG

Lys

AAG

Lys

GAG

Giu

CCC

Pro 495

TCA

Ser

GCG

Al a

ATG

Met

ATT

Ile

TCA

Ser 575 Al a

CCC

Pro

GAT

Asp

GCC

Al a

CGC

Arg 400

GTG

Val

TCG

Ser

TTC

Phe

CAG

Gln

GTG

Val 480

GCC

Al a

GTT

Val

GAC

Asp

CTT

Leu

TGC

Cys 560

GAA

Glu 1008 1056 1104 1152 1200 1248 1296 1344 1392 1440 1488 1536 1584 1632 1680 -1728 TCT CAA CCC Ser Gin Pro CCG ATT CAT Pro Ile His 595

GTG

Val1 580

C.AC

His TCT GTC GTC AGA Ser Vai Vai Arg

AAG

Lys 585

GCG

Ala CGG ACG TAT CAG Arg Thr Tyr Gin ATC ATG GGG Ile Met Gly CCC GAG GTG Pro Giu Val AAA CTG TGT Lys Leu Cys 590 TGC TCG GCC Cys Ser Ala GAA CAA TAA Giu Gin 1776 1824 1872 TGC GAA Cys Giu 610 CTG GCC AAT GTG Leu Ala Asn Vai

GAC

Asp 615 GAT GAC TGT Asp Asp Cys

GAC

Asp 620 INFORMATION FOR SEQ ID NO:4: SEQUErNCE CHARACTERISTICS: LENGTH: 734 amino acids TYPE: amino acid (ii) MOLECULE TYPE: DESCRIPTION: protein (ix) OTHER INFO: AAV4 capsid protein VP1 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: Met Thr Asp Giy Gly Val Ala Asn Tyr Lys 50 Asn Ala 65 Gin Leu Ala Glu Leu Giy Gly Leu 130 Leu Ile 145 Lys Gly Gly Ala Asp Asp Gly Gin 210 Asp Ser 225 Trp Val Ser Leu Arg Gin Tyr Al a Lys Phe Arg 115 Val1 Glu Lys Gly Ser 195 Gly Thr Leu Gin Glu 20 Gin Leu As'o Al a Gin 100 Al a Glu Ser Gin

ASD

Giu Al a Trp Pro Ser 260 Tvr Fis Gly Ala Gly Gin Val Gin -=ro 1.65 Gly Met P s Ser Thr 245 s n Leu T rp Gin Pro Ala 70 Asp Arg P:he Al a Gin I50 Al a Pro Arg Giy Glu 230 Tyr Pro Ala Asp Gly Al a Asn Leu Gin Gly 135 Gin Lys Pro Ala Val1 215 Giy Asn Asp Leu Asn 40 Asn Leu Pro Gin Ala 120 Giu Pro Lys Glu Al a 200 Gly His Asn Trp Gin 25 Al a Gly Glu Tyr Gly 105 Lys Thr Asp Lys Gly 185 Al a Asn Val His Gly 265 Leu 10 Pro Arg Leu His Leu 90 Asp Lys Ala Ser Leu 170 Ser Gly Ala Thr Leu 250 Giu Gly Gly Asp Asp 75 Lys Thr Arg Pro Ser 155 Val Thr Gly Ser Thr 235 Tyr Asp Al a Leu Lys Lys Tyr Ser Val Gly 140 Thr Phe Ser Al a Gly 220 Thr Lys Asn Pro Val1 Gly Al a Asn Phe Leu 125 Lys Gly Glu Gly Ala 205 Asp Ser Arg Leu Ser Lys Pro Leu Pro Glu Pro Tyr Asp His Ala Gly Gly 110 Glu Pro Lys Arg Ile Gly Asp Glu 175 Ala Met 190 Val Glu Trp His Thr Arg Leu Gly 255 Trp Gly 270 Glu Lys Gly Val1 Gin Asp Asn Leu Pro Lys 160 Thr Ser Gly Cys Thr 240 Glu Tyr Thr Tyr Asn Phe Ser Thr Pro Phe Asp Phe Asn Arg Phe His Cys His Phe Ser Pro Arg Asp Trp Gin 275 280 285 Arg Leu Ile Asn Asn Asn Trp Gly Met Arg Pro Lys Ala Met Arg Val 290 295 3.00 Lys Ile Phe Asn Ile Gin Val Lys Glu Val Thr Thr Ser Asn Gly Glu 305 310 315 320 Thr Thr Val Ala Asn Asn Leu Thr Ser Thr Val Gin Ile Phe Ala Asp 325 330 335 Ser Ser Tyr Glu Leu Pro Tyr Val Met Asp Ala Gly Gin Glu Gly Ser 340 345 350 Leu Pro Pro Phe Pro Asn Asp Val Phe Met Val Pro Gln Tyr Gly Tyr 355 360 365 Cys Gly Leu Val Thr Gly Asn Thr Ser Gin Gin Gin Thr Asp Arg Asn 370 375 380 Ala Phe Tyr Cys Leu Glu Tyr Phe Pro Ser Gin Met Leu Arg Thr Gly 385 390 395 400 Asn Asn Phe Glu Ile Thr Tyr Ser Phe Glu Lys Val Pro Phe His Ser 405 410 415 Met Tyr Ala His Ser Gin Ser Leu Asp Arg Leu Met Asn Pro Leu Ile 420 425 430 Asp Gin Tyr Leu Trp Gly Leu Gin Ser Thr Thr Thr Gly Thr Thr Leu 435 440 445 Asn Ala Gly Thr Ala Thr Thr Asn Phe Thr Lys Leu Arg Pro Thr Asn 450 455 460 .Phe Ser Asn Phe Lys Lys Asn Trp Leu Pro Gly Pro Ser Ile Lys Gin 465 470 475 480 Gin Gly Phe Ser Lys Thr Ala Asn Gin Asn Tyr Lys Ile Pro Ala Thr 485 490 495 Gly Ser Asp Ser Leu Ile Lys Tyr Glu Thr His Ser Thr Leu Asp Gly 500 505 510 Arg Trp Ser Ala Leu Thr Pro Gly Pro Pro Met Ala Thr Ala Gly Pro 515 520 525 Ala Asp Ser Lys Phe Ser Asn Ser Gin Leu Ile Phe Ala Gly Pro Lys 530 535 540 Gin Asn Gly Asn Thr Ala Thr Val Pro Gly Thr Leu Ile Phe Thr Ser 545 550 555 560 Glu Glu Glu Leu Ala Ala Thr Asn Ala Thr Asp Thr Asp Met Trp Gly 565 570 575 *Asn Leu Pro Gly Gly Asp Gin Ser Asn Ser Asn Leu Pro Thr Val Asp 580 585 590 Arg Leu Thr Ala Leu Gly Ala Val Pro Gly Met Val Trp Gin Asn Arg 595 600 605 SAsp Ile Tyr Tyr Gin Gly Pro Ile Trp Ala Lys Ile Pro His Thr Asp 610 615 620 Gly His Phe His Pro Ser Pro Leu Ile Gly Gly Phe Gly Leu Lys His 625 630 635 640 Pro Pro Pro Gin Ile Phe Ile Lys Asn Thr Pro Val Pro Ala Asn Pro 645 650 655 Ala Thr Thr Phe Ser Ser Thr Pro Val Asn Ser Phe Ile Thr Gin Tyr 660 665 670 Ser Thr Gly Gin Val Ser Val Gin Ile Asp Trp Glu Ile Gin Lys Glu 675 680 685 Arg Ser Lys Arg Trp Asn Pro Glu Val Gin Phe Thr Ser Asn Tyr Gly 690 695 700 Gin Gin Asn Ser Leu Leu Trp Ala Pro Asp Ala Ala Gly Lys Tyr Thr 705 710 715 720 Glu Pro Arg Ala Ile Gly Thr Arg Tyr Leu Thr His His Leu 725 730 INFORMATION FOR SEQ ID Wi SEQUENCE CHARACTERISTICS: LENGTH: 2208 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ix) OTHER INFO: AAV4 capsid protein VPl gene (xi) SEQUENCE DESCRIPTION: SEQ ID 55.4.0

S

ATGACTGACG

TGGTGGGCGC

GCTCGGGGTC

GGGGAACCCG

CAGCTCAAGG

CAGCGGCTTC

AAAAAGAGGG

AAGAAGAGAC

AAAGGCAAGC

GGACCCCCTG

GCTGGCGGAG

GATTGGCATT

TGGGTCTTGC

AACACCTACA

CACTTCTCAC

GCCATGCGGG

ACAACGGTGG

CTGCCGTACG

TTTATGGTGC

ACTGACAGAA

AACAACTTTG

AGCCAGAGCC

TCGACCACCA

CGGCCTACCA

CAGGGCTTCT

CTCATCAAAT

CCTCCAATGG

GCGGGGCCTA

GAC-GAGGAGC

GGTGACCAGA

CCTGGAATGG

CCTCATACCG

CCGCCTCCTC

AGCTCTACTC

ATTGACTGGG

TCCAACTACG

GAGCCTAGGG

GTTACCTTCC

TGCAACCTGG

TTGTGCTTCC

TCAACGCAGC

CCGGTGACAA

AGGGCGACAC

TTCTTGAACC

CGTTGATTGA

AGCCGGCTAA

AGGGATCAAC

CTGCAGTCGA

GCGATTCCAC

CCACCTACAA

ACGGATTCTC

CACGTGACTG

TCAAAATCTT

CTAATAACCT

TGATGGATCC

CCCAGTACG

ATGCCTTCTA

AAATTACGTA

TGGACCGGC'T

CCGGAACC.AC

ACTTTCC.:A

CAAAGACTCC

ACGAGACGCA

CC.ACGGCTCG

AACAGA-ACCG

TGGCAGCCAC

GCAACAGCAA

TCTC-GCAAAA

ATGGACAC-T

AAATTTTTAT

CGGTAAACTC

AGATCCAGAA

GACAGCAAAA

CTATCGGTAC

AGATTGGCTA

AGCCCCTAAA

GGGTTACAAA

GGACGCGGCA

CCCCTACCTC

ATCGTTTGGG

TCTTGGTCTG

ATCCCCCCAG

AAAGAAGCTC

TTCCGGAGCC

GGGSGGACAA

CTGGTCTGAG

CAACCACCTN

CACCCCCTGG

GCAGCGACTC

CAACATCCAG

TACCAGCACG

GGGTCAAGAG

CTACTGTGGA

CTGCCTGGAG

CAGTTTTGAG

GATGAACCCT

CCTGAATGCC

CTTTAAAAAG

CAATCAAAAC

CAGCACTCTG

ACCTGCGGAC

CAACACGGCC

CAACGCCACC

CCTGCCGACC

CAGAGACATT

TCACCCCTCA

CAAGAACACC

CTTCATTACT

GGAGCGGTCC

CTCTCTGTTG

CCGCTACCTC

GAGGACAACC

CCCAAGGCAA

TACCTCGGAC

GCCCTCGAGC

AAGTACAACC

GGCAACCTCG

GTTGAGCAAG

CAGCCCGACT

GTTTTCG.AAG

ATGTCTGATG

GGTGCCGATG

GGCCACGTCA

TACAAGCGAC

GGATACTTTG

ATCAACAACA

GTCAAGGAGG

GTTCAGATCT

GGCAGCCTGC

CTGGTGACCG

TACTTTCCTT

AAGGTGCCTT

CTCATCGACC

GGGACTGCCA

AACTGGCTGC

TACAAGATCC

GACGGAAGAT

AGCAAGTTCA

ACCGTACCCG

GATACGGACA

GTGGACAGAC

TACTACCAGG

CCGCTGATTG

CCGGTACCTG

CAGTACAGCA

AAACGCTGGA

TGGGCTCCCG

ACCCACCACC

TCTCTGAAGG

ATCAACAACA

CCGGCAACGG

ACGACAAGGC

ACGCCGACGC

GCAGAGCAGT

CGGGTGAGAC

CCTCCACGGG

ACGAAACTGG

ACAGTGAGAT

GAGTGGGTAA

CGACCACCAG

TCGGAGAGAG

ACTTCAACCG

ACTGGGGCAT

TCACGACGTC

TTGCGGACTC

CTCCTTTTCC

GCAACACTTC

CGCAGATGCT

TCCACTCGAT

AGTACCTGTG

CCACCAACTT

CCGGGCCTTC

CTGCCACCGG

GGAGTGCCCT

GCAACAGCCA

.GGACTCTGAT

TGTGGGGCAA

TGACAGCCTT

GTCCCATTTG

GTGGGTTTGG

CGAATCCTGC

CTGGCCAGGT

ACCCCGAGGT

ATGCGGCTGG

TGTAATAA

CGTTCGAGAG

TCAGGACAAC

ACTCGACAAG

CTACGACCAG

GGAGTTCCAG

CTTCCAGGCC

GGCTCCTGGA

TATCGGCAAA

AGCAGGCGAC

GCGTGCAGCA

TGCCTCGGGT

CACCAGAACC

CCTGCAGTCC

CTTCCACTGC

GCGACCCAAA

GAACGGCGAG

GTCGTACGAA

CAACGACGTC

GCAGCAACAG

GCGGACTGGC

GTACGCGCAC

GGGACTGCAA

TACCAAGCTG

AATCAAGCAG

GTCAGACAGT

GACCCCCGGA

GCTCATCTTT

CTTCACCTCT

CCTACCTGGC

GGGAGCCGTG

GGCCAAGATT

GCTGAAACAC

AACGACCTTC

GTCGGTGCAG

CCAGTTTACC

GAAATACACT

120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 1920 1980 2040 2100 2160 2208 INFORMATION FOR SEQ ID NO:6: SEQUENCE CHARACTERISTICS: LENGTH: 125 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ix) OTHER INFO: AAV4 ITR "flip" orientation (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: TTGGCCACTC CCTCTATGCG CGCTCGCTCA CTCACTCGGC CCTGGAGACC AAAGGTCTCC AGACTGCCGG CCTCTGGCCG GCAGGGCCGA GTGAGTGAGC GAGCGCGCAT AGAGGGAGTG 120 GCCAA 125 INFORMATION FOR SEQ ID NO:7: SEQUENCE CHARACTERISTICS: LENGTH: 245 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ix) OTHER INFO: AAV4 p5 promoter (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: CTCCATCATC TAGGTTTGCC CACTGACGTC AATGTGACGT CCTAGGGTTA GGGAGGTCCC TGTATTAGCA GTCACGTGAG TGTCGTATTT CGCGGAGCGT AGCGGAGCGC ATACCAAGCT 120 GCCACGTCAC AGCCACGTGG TCCGTTTGCG ACAGTTTGCG ACACCATGTG GTCAGGAGGG 180 TATATAACCG CGAGTGAGCC AGCGAGGAGC TCCATTTTGC CCGCGAATTT TGAACGAGCA 240 GCAGC 245 INFORMATION FOR SEQ ID NO:8: SEQUENCE CHARACTERISTICS: LENGTH: 313 amino acids TYPE: amino acid STRANDEDNESS: not relevant TOPOLOGY: not relevant oeo (ii) MOLECULE TYPE: DESCRIPTION: protein (ix) OTHER INFO: AAV4 Rep protein *SSS°e (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: Met Glu Leu Val Gly Trp Leu Val Asp Arg Gly Ile Thr Ser Glu Lys S1 5 10 Gin Trp Ile Gin Glu Asp Gin Ala Ser Tyr Ile Ser Phe Asn Ala Ala 25 Ser Asn Ser Arg Ser Gin Ile Lys Ala Ala Leu Asp Asn Ala Ser Lys 40 Ile Met Ser Leu Thr Lys Thr Ala Pro Asp Tyr Leu Val Gly Gin Asn 55 Pro Pro Glu Asp Ile Ser Ser Asn Arg Ile Tyr Arg Ile Leu Glu Met 70 75 Asn Gly Tyr Asp Pro Gin Tyr Ala Ala Ser Val Phe Leu Gly Trp Ala 90 Gin Lys Lys Phe Gly Lys Arg Asn Thr Ile Trp Leu Phe Gly Pro Ala 100 105 110 Thr Thr Gly Lys Thr Asn Ile Ala Glu Ala Ile Ala His Ala Val Pro 115 120 125 Phe Tyr Gly Cys Val Asn Trp Thr Asn Glu Asn Phe Pro Phe Asn Asp 130 135 140 Cys Val Asp Lys Met Val Ile Trp Trp Glu Glu Gly Lys Met Thr Ala 145 150 155 160 Lys Val Val Glu Ser Ala Lys Ala Ile Leu Gly Gly Ser Lys Val Arg 165 170 175 Val Ile Thr Glu 225 Giu Thr Pro Ala Arg 305 Asp Val Thr 210 Leu Val His Asn Gin 290 Leu Gin Thr 195 Phe Thr Lys Giu Asp 275 Pro Al a Cys Asn His Arg Phe 245 Tyr Asp Thr Gly Lys Thr Gin Leu 230 Phe Val Ile Ser Gin 310 Ser Met 200 Pro His Trp Lys Giu 280 Al a Leu Gin Ala Gin Phe Ser 250 Gly Lys Ala Ile Val1 Asp Gly 235 Asp Aia Arg Pro Asp Ile Arg 220 Lys His Arg Ala Val 300 Pro Asp 205 Met Val Val Lys Cys 285 Asp Thr 190 Giy Phe Thr Thr Arg 270 Pro Tyr Val Ser Phe Gin 240 Val Al a Val1 Asp 0@@e 0*@S 0 @00.6.

0 *0 S 0@ @0 SeeS 0 0@*0 @5 *5 0 INFORMATION FOR SEQ ID NO:9: SEQUENCE CHARACTERISTICS: LENGTH: 399 amino acids TYPE: amino acid STRAN7DEDNESS: not relevant TOPOLOGY: not relevant (ii) MOLECULE TYPE: DESCRIPTION: protein (ix) OTHER INFO: AAV4 Rep protein 52 (xi) S7EQU---CE DESCRIPTION: SEQ ID NO:9: Met Glu Leu Val Gly 00 0@ 05 *00505 0 5* 0 05 00

S

005550 0 Gin Ser Ile Pro 65 Asn Gin Thr Phe Cys 145 Lys Vai Ile T rp As n Met 50 Pro Gly Lys Thr Tyr 130 Val Vai AspD Val1 Ile Ser Ser Giu Tyr Lys Gly Gly Asp, Vali Gin Thr Gln 20 Arc Leu Asp Asp) Phe 100 Lvs Cvs Lvs Glu Lvs iso Ser Glu Ser -hr Ilie Pro Gly Thr Val1 Met Ser Cys As n Trm Asp Gin Lys Ser 70 Gin Lys As n As n Val1 150 Al a Lys Thr Leu Gin Ile Thr Ser Tyr Arg Ile Trp 135 Ile Lys Ser Asn Val Al a Lys 40 Ala Asn Al a Asn Al a 120 Thr Trp Ala Ser Met Asp Ser 25 Ala Pro Arg Al a Thr 105 Giu Asn Trp Ile Al a 185 Cys Arg 10 Tyr Al a Asp Ile Ser 90 Ile Al a Glu Glu Leu 170 Gin Al a Gly Ile Leu Tyr Tyr 75 Val1 Trp Ile Asn Glu 155 Gly Ile Val Ile Ser Asp Leu Arg Phe Leu Al a Phe 140 Gly Giy Asp Ile Arg 220 Thr Phe As n Val Ile Leu Phe His 125 Pro Lys Ser Pro Asp 205 Ser Asn Ala Gly Leu Gly Giy 110 Aia Phe Met Lys Thr 190 Gly Glu Al a Ser Gin Giu Trp Pro Vai Asn Thr Val 175 Pro Asn Lys Al a Lys Asn Met Al a Ala Pro Asp Al a 160 Arg Val Ser 195 200 Thr Thr 210 Phe Giu His Gin Gin 215 Pro Leu Gin Asp Met Phe Lys Phe Glu Leu Thr Lys Arg Leu Glu His Asp Phe Gly Lys Val Thr Lys Gin 225 230 235 240 Glu Val Lys Asp Phe Phe Arg Trp Ala Ser Asp His Val Thr Glu Val 245 250 255 Thr His Glu Phe Tyr Val Arg Lys Gly Gly Ala Arg Lys Arg Pro Ala 260 265 270 Pro Asn Asp Ala Asp Ile Ser Glu Pro Lys Arg Ala Cys Pro Ser Val 275 280 285 Ala Gln Pro Ser Thr Ser Asp Ala Glu Ala Pro Val Asp Tyr Ala Asp 290 295 300 Arg Tyr Gin Asn Lys Cys Ser Arg His Val Gly Met Asn Leu Met Leu 305 310 315 320 Phe Pro Cys Arg Gin Cys Glu Arg Met Asn Gin Asn Val Asp Ile Cys 325 330 335 Phe Thr His Gly Val Met Asp Cys Ala Glu Cys Phe Pro Val Ser Glu 340 345 350 Ser Gin Pro Val Ser Val Val Arg Lys Arg Thr Tyr Gin Lys Leu Cys 355 360 365 Pro Ile His His Ile Met Gly Arg Ala Pro Glu Val Ala Cys Ser Ala 370 375 380 Cys Glu Leu Ala Asn Val Asp Leu Asp Asp Cys Asp Met Glu Gln S385 390 395 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 537 amino acids TYPE: amino acid STRANDEDNESS: not relevant TOPOLOGY: not relevant (ii) MOLECULE TYPE: DESCRIPTION: protein (ix) OTHER INFO: AAV4 Rep protein 68 S(xi) SEQUENCE DESCRIPTION: SEQ ID Met Pro Gly Phe Tyr Glu Ile Val Leu Lys Val Pro Ser Asp Leu Asp 1 5 10 Glu His Leu Pro Gly Ile Ser Asp Ser Phe Val Ser Trp Val Ala Glu 25 Lys Glu Trp Glu Leu Pro Pro Asp Ser Asp Met Asp Leu Asn Leu Ile 35 40 Glu Gin Ala Pro Leu Thr Val Ala Glu Lys Leu Gin Arg Glu Phe Leu 55 Val Glu Trp Arg Arg Val Ser Lys Ala Pro Glu Ala Leu Phe Phe Val 70 75 Gin Phe Glu Lys Gly Asp Ser Tyr Phe His Leu His Ile Leu Val Glu 90 Thr Val Gly Val Lys Ser Met Val Val Gly Arg Tyr Val Ser Gin Ile 100 105 110 Lys Glu Lys Leu Val Thr Arg Ile Tyr Arg Gly Val Glu Pro Gin Leu 115 120 125 Pro Asn Trp Phe Ala Val Thr Lys Thr Arg Asn Gly Ala Gly Gly Gly 130 135 140 Asn Lys Val Val Asp Asp Cys Tyr Ile Pro Asn Tyr Leu Leu Pro Lys 145 150 155 160 Thr Gin Pro Glu Leu Gin Trp Ala Trp Thr Asn Met Asp Gin Tyr Ile 165 170 175 Ser Ala Cys Leu Asn Leu Ala Glu Arg Lys Arg Leu Val Ala Gin His 180 185 190 Leu Thr His Val Ser Gin Thr Gin Glu Gin Asn Lys Glu Asn Gin Asn 195 200 205 Pro Asn Ser Asp Ala Pro Val Ile Arg Ser Lys Thr Ser Ala Arg Tyr 210 215 220 Met Glu Leu Val Gly Trp Leu Val Asp Arg Gly Ile Thr Ser Glu Lys 225 230 235 240 Gin Trp Ile Gin Glu Asp Gin Ala Ser Tyr Ile Ser Phe Asn Ala Ala 245 250 255 Ser Asn Ser Arg Ser Gin Ile Lys Ala Ala Leu Asp Asn Ala Ser Lys 260 265 270 Ile Met Ser Leu Thr Lys Thr Ala Pro Asp Tyr Leu Val Gly Gin Asn 275 280 285 Pro Pro Glu Asp Ile Ser Ser Asn Arg Ile Tyr Arg Ile Leu Glu Met 290 295 300 Asn Gly Tyr Asp Pro Gin Tyr Ala Ala Ser Val Phe Leu Gly Trp Ala 305 310 315 320 Gin Lys Lys Phe Gly Lys Arg Asn Thr Ile Trp Leu Phe Gly Pro Ala 325 330 335 Thr Thr Gly Lys Thr Asn Ile Ala Glu Ala Ile Ala His Ala Val Pro 340 345 350 Phe Tyr Gly Cys Val Asn Trp Thr Asn Glu Asn Phe Pro Phe Asn Asp 355 360 365 Cys Val Asp Lys Met Val Ile Trp Trp Glu Glu Gly Lys Met Thr Ala 370 375 380 Lys Val Val Glu Ser Ala Lys Ala Ile Leu Gly Gly Ser Lys Val Arg .385 390 395 400 Val Asp Gin Lys Cys Lys Ser Ser Ala Gin Ile Asp Pro Thr Pro Val 405 410 415 Ile Val Thr Ser Asn Thr Asn Met Cys Ala Val Ile Asp Gly Asn Ser 420 425 430 Thr Thr Phe Glu His Gin Gin Pro Leu Gin Asp Arg Met Phe Lys Phe 435 440 445 Glu Leu Thr Lys Arg Leu Glu His Asp Phe Gly Lys Val Thr Lys Gin 450 455 460 Glu Val Lys Asp Phe Phe Arg Trp Ala Ser Asp His Val Thr Glu Val 465 470 475 480 Thr His Glu Phe Tyr Val Arg Lys Gly Gly Ala Arg Lys Arg Pro Ala 0 485 490 495 Pro Asn Asp Ala Asp Ile Ser Glu Pro Lys Arg Ala Cys Pro Ser Val 500 505 510 Ala Gin Pro Ser Thr Ser Asp Ala Glu Ala Pro Val Asp Tyr Ala Asp 515 520 525 Arg Leu Ala Arg Gly Gin Pro Leu Xaa 530 535 INFORMATION FOR SEQ ID NO:11: SEQUENCE CHARACTERISTICS: LENGTH: 623 amino acids TYPE: amino acid STRANDEDNESS: not relevant TOPOLOGY: not relevant (ii) MOLECULE TYPE: DESCRIPTION: protein (ix) OTHER INFO: AAV4 Rep protein 78 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: Met Pro Gly Phe Tyr Glu Ile Val Leu Lys Val Pro Ser Asp Leu Asp 1 5 10 Glu Lys Glu Val Gin Thr Lys Pro Asn 145 Thr Ser Leu Pro Met 225 Gin Ser Ile Pro Asn 305 Gin Thr Phe Cys Lys 385 Val Ile Thr Glu Glu 465 Thr Pro His Glu Gin Glu Phe Val Glu Asn 130 Lys Gin Ala Thr Asn 210 Glu Trp Asn Met Pro 290 Gly Lys Thr Tyr Val 370 Val Asp Val Thr Leu 450 Val His Asn Leu Trp Ala Trp Glu Gly Lys 115 Trp Val Pro Cys His 195 Ser Leu Ile Ser Ser 275 Glu Tyr Lys Gly Gly 355 Asp Val Gin Thr Phe 435 Thr Lys Glu Asp Pro Glu Pro Arg Lys Val 100 Leu Phe Val Glu Leu 180 Val Asp Val Gin Arg 260 Leu Asp Asp Phe Lys 340 Cys Lys Glu Lys Ser 420 Glu Lys Asp Phe Ala 500 Gly Leu Leu Arg Gly Lys Val Ala Asp Leu 165 Asn Ser Ala Gly Glu 245 Ser Thr Ile Pro Gly 325 Thr Val Met Ser Cys 405 Asn His Arg Phe Tyr 485 Asp Ile Pro Thr Val 70 Asp Ser Thr Val Asp 150 Gin Leu Gin Pro Trp 230 Asp Gin Lys Ser Gin 310 Lys Asn Asn Val Ala 390 Lys Thr Gin Leu Phe 470 Val Ile Ser Pro Val 55 Ser Ser Met Arg Thr 135 Cys Trp Ala Thr Val 215 Leu Gin Ile Thr Ser 295 Tyr Arg Ile Trp Ile 375 Lys Ser Asn Gin Glu 455 Arg Arg Ser

I

Ser 25 Ser Glu Ala Phe Val 105 Tyr Thr Ile Trp Arg 185 Glu Arg Asp Ser Ala 265 Pro Arg Ala Thr Glu 345 Asn Trp Ile Ala Cys 425 Leu Asp Ala Gly Pro 505 Phe Asp Lys Pro His 90 Gly Arg Arg Pro Thr 170 Lys Gin Ser Arg Tyr 250 Ala Asp Ile Ser Ile 330 Ala Glu Glu Leu Gin 410 Ala Gin Phe Ser Gly 490 Lys Val Met Leu Glu 75 Leu Arg Gly Asn Asn 155 Asn Arg Asn Lys Gly 235 Ile Leu Tyr Tyr Val 315 Trp Ile Asn Glu Gly 395 Ile Val Asp Gly Asp 475 Ala Arg Ser Asp Gin Ala His Tyr Val Gly 140 Tyr Met Leu Lys Thr 220 Ile Ser Asp Leu Arg 300 Phe Leu Ala Phe Gly 380 Gly Asp Ile Arg Lys 460 His Arg Ala Val Asn Glu Phe Leu Ser 110 Pro Gly Leu Gin Ala 190 Asn Ala Ser Asn Ala 270 Gly Leu Gly Gly Ala 350 Phe Met Lys Thr Gly 430 Phe Thr Thr Arg Pro 510 Ala Leu Phe Phe Val Gin Gin Gly Pro Tyr 175 Gin Gin Arg Glu Ala 255 Ser Gin Glu Trp Pro 335 Val Asn Thr Val Pro 415 Asn Lys Lys Glu Pro 495 Ser Glu Ile Leu Val Glu Ile Leu Gly Lys 160 Ile His Asn Tyr Lys 240 Ala Lys Asn Met Ala 320 Ala Pro Asp Ala Arg 400 Val Ser Phe Gin Val 480 Ala Val Ala Gin Pro 515 Arg Tyr Gin 530 Phe Pro Cys 545 Phe Thr His Ser Gin Pro Pro Ile His 595 Ser Thr Ser Asp Asn Lys Cys Arg Gly Val 580 His Gin Val1 Ser Arg 535 Glu Arg Asp Cys Glu Ala Pro Vai His Val Gly Met 540 Met Asn Gin Asn 555 Ala Giu Cys Phe Tyr Ala Asp Leu Met Leu Val Asp Ile Cys 560 Glu Pro Val Ser Val Vai Arg Ile Met Gly Arg 600 Lys 570 Arg Thr Tyr Gin Lys 590 Pro Glu Val Ala Cys Leu Cys Ser Ala 605 Met Glu Gin Cys Giu Leu Ala Asn Val Asp Leu 610 615 Asp Asp Cys INFORMATION FOR SEQ ID NO:12: Ci) SEQUENCE CHARACTERISTICS: LENGTH: 939 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLCGY: linear (ix) OTHER INFO: AAV4 Rep 40 gene (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:

ATGGAGCTGG

GAGGACCAGG

GCCGCGCTGG

GTGGGCCAGA

AACGGGTACG

GGGAAGAGGA

GAAGCCATCG

CCGTTCAACG

AAGGTCGTAG

TGCAAGTCAT

TGCGCGGTCA

ATGTTCAAGT

GAAGTCAAAG

TACGTCAGAA

CCCAAGCGGG

GACTACGCGG

TCGC-GTGGCT

CGTCCTACAT

ACAATGCCTC

ACCCGCCGGA

ATCCGCAGTA

ACACCACTG

CCCACGCCGT

ATTGCG:-CGA

AGAGCGCCAA

CGG C CCAGAT

TCGACGGAAA

TCGAGCTCAC

ACTTTTT-CCG

AGGGTGGAGC

CCTGTCC-GTC

ACAGATTGGC

GGTGGACCGC

CTCCTTCAAC

CAAAATCATG

GGACATTTCC

CGCGGCCTCC

GCTCTTTGGG

GCCCTTCTAC

CAAGATGGTG

GGCCATCCTG

CGACCCAACT

CTCGACCACC

CAAGCGCCTG

GTGGGCGTCA

TAGAAAGAGG

AGTTGCGCAG

TAGAGGACAA

GGGATCACGT

GCCGCCTCCA

AGCCTGACAA

AGCAACCGCA

GTCTTCCTGG

CCGGCCACGA

GGCTGCGTGA

ATCTGGTGGG

GGCGGAAGCA

CCCGTGATCG

TTCGAGCACC

GAGCACGACT

GATCACGTGA

CCCGCCCCCA

CCATCGACGT

CCTCTCTGA

CAGAAAAGCA

ACTCGCGGTC

AGACGGCTCC

TCTACCGAAT

GCTGGGCGCA

CGGGTAAAAC

ACTGGACCAA

AGGAGGGCAA

AGGTGCGCGT

TCACCTCCAA

AACAACCACT

TTGGCAAGGT

CCGAGGTGAC

ATGACGCAGA

CAGACGCGGA

ATGGATCCAG

ACAAATCAAG

GGACTACCTG

CCTCGAGATG

AAAGAAGTTC

CAACATCGCG

TGAGAACTTT

GATGACGGCC

GGACCAAAAG

CACCAACATG

CCAGGACCGG

CACCAAGCAG

TCACGAGTTT

TATAAGTGAG

AGCTCCGGTG

120 180 240 300 360 420 480 540 600 660 720 780 840 900 939 INFORMATI-ON FOR SEQ ID NO:i3: Ci) SEQUENCE CHARACTERISTICS: CA) LENGTH: 1:-97 base pairs TYPE: nucleic acid STRANDEDNESS: double CD) TOPOLOGY: linear (ix) OTHER INFO: AAV4 Rep 52 gene (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13: ATGGAGCTGG TCGC-GTGGCT GGTGGACCGC GGGATCACGT CAGAAAAGCA GAGGACCAGG CGTCCTACAT CTCCTTCAAC! GCCGCCTCCA ACTCGCGGTC GCCGCGCTGG ACAATC-CCTC CAAAATCATG AGCCTGACAA AGACGGCTCC GTGGGCCAGA ACCCGCCGGA GGACATTTCC AGCAACCGCA TCTACCGAAT

ATGGATCCAG

ACAAATCAAG

GGACTACCTG

CCTCGAGATG

AACGGGTACG

GGGAAGAGGA

GAAGCCATCG

CCGTTCA.ACG

AAGGTCGTAG

TGCAAGTCAT

TGCGCGGTCAk

ATGTTCAAGT

GAAGTCAAAG

TACGTCAGAA

CCCAAGCGGG

GACTACGCGG

TTTCCCTGCC

GTCATGGACT

AAGCGGACGT

GCCTGCTCGG

ATCCGCAGTA

ACACCATCTG

CCCACGCCGT

ATTGCGTCGA

AGAGCGCCAA

CGGCCCAGAT

TCGACGGAAA

TCGAGCTCAC

ACTTTTTCCG

AGGGTGGAGC

CCTGTCCGTC

ACAGGTACCA

GGCAATGCG.A

GTGCCGAGTG

ATCAGAAACT

CCTGCGAACT

CGCGGCCTCC

GCTCTTTGGG

GCCCTTCTAC

C.AAGATGGTG

GGCCATCCTG

CGACCCAACT

CTCGACCACC

CAAGCGCCTG

GTGGGCGTCA

TAGAAAGAGG

AGTTGCGCAG

AAACAA.ATGT

GAGAATGAAT

CTTCCCCGTG

GTGTCCGATT

GGCCAATGTG

GTCTTCCTGG

CCGGCCACGA

GGCTGCGTGA

ATCTGGTGGG

GGCGGAAGCA

CCCGTGATCG

TTCGAGCACC

GAGCACGACT

GATCACGTGA

CC CGCCC CCA

CCATCGACGT

TCTCGTCACG

CAGAATGTGG

TCAGAATCTC

CATCACATCA

GACTTGGATG

GCTGGGCGCA.

CGGGTAAAAC

ACTGGACCAA

AGGAGGGCAA

AGGTGCGCGT

TCACCTCCAA

AACAACCACT

TTGGCAAGGT

CCGAGGTGAC

ATGACGCAGA

CAGACGCGGA

TGGGTATGAA

ACATTTGCTT

AACCCGTGTC

TGGGGAGGGC

ACTGTGACAT

AAAGAAGTTC

CAACATCGCG

TGAGAACTTT

GATGACGGCC

GGACCAAAAG.

CACCAACATG

CCAGGACCGG

CACCAAGCAG

TCACGAGTTT

TATAAGTGAG

AGCTCCGGTG

TCTGATGCTT

CACGCACGGG

TGTCGTCAGA.

GCCCGAGGTG

GGAACAA

300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1197

S.

S

INFORMATION FOR SEQ ID NO:14: SEQUENCE CHARACTERISTICS: LENGTH: 1611 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ix) OTHER INFO: AAV4 Rep 68 gene (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:

ATGCCGGGGT

GGCATTTCTG

TCTGACATGG

CGCGAGTTCC

CAGTTCGAGA

AAATCCATGG

TACCGCGGGG

GCCGGAGGCG

ACCCAGCCCG

AATCTCGCGG

GAGCAGAACA

TCCGCCAGGT

CAATGGATCC

TCACAAATCA

CCGGACTACC

ATCCTCGAGA

CAAAAGAAGT

ACCAACATCG

AATGAGAACT

AAGATGACGG

GTGGACCAAA

AACACCAACA

CTCCAGGACC

GTCACCAAGC

ACTCACGAGT

GATATA.AGTG

GAAGCTCCGG

TCTACGAGAT

ACTCTTTTGT

ACTTGAATCT

TGGTCGAGTG

AGGGGGACAG

TGGTGGGCCG

TCGAGCCGCA

GGAACAAGGT

AGCTCCAGTG

AGCGTAAACG

AGGAAAACCA

ACATGGAGCT

AGGAGGACCA

AGO CCGCGCT

TGGTGGGCC.A

TGAACGGGTA

TCGGGAAGAG

CGGAAGCCAT

TTCCGTTCAA

CCAAGGTCGT

AGTGCA.AGTC

TGTGCGCGGT

GGATGTTCAA

AGGAAGTCAA

TTTACGTCAG

AGCCCAAGCG

TGGACTACGC

CGTGCTGAAG

GAGCTGGGTG

GATTGAGCAG

GCGCCGCGTG

CTACTTCCAC

CTACGTGAGC

GCTTCCGAAC

GGTGGACGAC

GCTGGACT

GCTGGTGGCG

GAACCCCAAT

GGTCGGGTGG

GGCGTCCTAC

GGACAATGCC

GAACCCGCCG

CGATCCGCAG

GAACACCATC

CGCCCACGCC

CGATTGCGTC

AGAGAGCGCC

ATCGGCCCAG

CATCGACGGA

GTTCGAGCTC

AGACTTTTTC

AAAGGGTGGA

GGCCTGTCCG

GGACAGATTG

GTGCCCAGCG

GCCGAGAAGG

GCACCCCTGA

AGTAAGGCCC

CTGCACATCC

CAGATTAAAG

TGGTTCGCGG

TGCTACATCC

AACATGGACC

CAGCATCTGA

TCTGACGCGC

CTGGTGGACC

ATCTCCTTCA

TCCAAAATCA

GAGGACATTT

TACGCGGCCT

TGGCTCTTTG

GTGCCCTTCT

GACAAGATGG

AAGGCCATCC

ATCGACCCAA

AACTCGACCA

ACCAAGCGCC

CGGTGGGCGT

GCTAGAAAGA

TCAGTTGCGC

GCTAGAGGAC

ACCTGGACGA

AATGGGAGCT

CCGTGGCCGA

CGGAGGCCCT

TGGTGGAGAC!

AGA.AGCTGGT

TGACCAAGAC

CCAACTACCT

AGTATATAAG

CGCACGTGTC

CGGTCATCAG

GCGGGATCAC

ACGCCGCCTC

TGAGCCTGAC

CCAGCAACCG

CCGTCTTCCT

GGCCGGCCAC

ACGGCTGCGT

TGATCTGGTG

TGGGCGGAAG

CTCCCGTGAT

CCTTCGAGCA

TGGAGCACGA

CAGATCACGT

GGCCCGCCCC

AGCCATCGAC

AA.CCTCTCTG

GCACCTGCCC

GCCGCCGGAT

AAAGCTGCAA

CTTCTTTGTC

CGTGGGCGTC

GACCCGCATC

GCGTAATGGC

GCTCCCCAAG

CGCCTGTTTG

GCAGACGCAG

GTCAAAAACC

GTCAGAAAAG

CAACTCGCGG

AAAGACGGCT

CATCTACCGA

GGGCTGGGCG

GACGGGTAAA

GAACTGGACC

GGAGGAGGGC

CAAGGTGCGC

CGTCACCTCC

CCAACAACCA

CTTTGGCAAG

GACCGAGGTG

CAATGACGCA

GTCAGACGCG

120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1611 INFORMATION FOR SEQ ID Ci SEQUENCE CHARACTERISTICS: LENGTH: 1872 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ix) OTHER INFO: AAV4 Rep 78 gene (xi) SE-QENCE DESCRIPTION: SEQ ID

ATGCCGGGGT

GGCATTTCTG,

TCTGACATGG

CGCGAGTTCC

CAGTTCGAGA

AAATCCATGG

TACCGCGGGG

GCCGGAGGCG

ACCCAGCCCG

AATCTCGCGG

GAGCAGAACA

TCCGCCAGGT

CAATGGATCC

TCACAAATCA

CCGGACTACC

ATCCTCGAGA

CAAAAGAAGT

ACCAACATCG

AATGAGAACT

AAGATGACGG

GTGGACCAAA

AACACCAACA

CTCCAGGACC

GTCACCAAGC

ACTCACGAGT

GATATAAGTG

GAAGCTCCGG

AATCTGATGC

TTCACGCACG

TCTGTCGTCA

GCGCCCGAGG

ATGGAACAAT

TCTACGAGAT

ACTCTTTTGT

ACTTGAATCT

TGGTCGAGTG

AGGGGACAG

TGGTGC-GCCG

TCGAGCCGCA

GGAAC.A AGGT

AGCTCCAGTG

AGCGTAAACG

AGGAAAACCA

ACATGGAGCT

AGGAGGACCA,

AC-GCCGCGCT

TGGTGGGCCA

TGAACGGGTA

TCGGGAAGAG

CGGAAGCCAT

TTCCGTTCAA

CCAAC-GTCGT

AGTGCAAGTC

TGTGCGCGGT

C-GATG7!TCAA

AGGAAGTCAA

TTTACGTCAG

AOCCCAAGCG

T-2GACTACGC

TT-TTTCCCTG

C-CGTCATGGA

GAAAGCGGAC

TGGCCTGCTC

AA

CGTGCTGAAG

GAGCTGGGTG

GATTGAGCAG

GCGCCGCGTG,

CTACTTCCAC

CTACGTGAGC

GCTTCCGAAC

GGTGGACGAC

GGCGTGGACT

GCTGGTGGCG

GAACCCCAAT

GGTCGGGTGG

GGCGTCCTAC

GGACAATGCC

GAACCCGCCG

CGATCCGCAG

GAACACCATC

CGCCCACGCC

CGATTGCGTC

AGAGAGCGCC

ATCGGCCCAG

CATCGACGGA

GTTCGAGCTC

AGACTTTTTC

AAAGGGTGGA

GGCCTGTCCG

GGACAGGTAC

CCGGCAATGC

CTGTGCCGAG

GTATCAGAAA

GGCCTGCGAA

GTGCCCAGCG

GCCGAGAAGG

GCACCCCTGA

AGTAAGGCCC

CTGCACATCC

CAGATTAAAG

TGGTTCGCGG

TGCTACATCC

AACATGGACC

CAGCATCTGA

TCTGACGCGC

CTGGTGGACC

ATCTCCTTCA

TCCAAAATCA

GAGGACATTT

TACGCGGCCT

TGGCTCTTTG

GTGCCCTTCT

GACAAGATGG

AAGGCCATCC

ATCGACCCAA

AACTCGACCA

ACCAAGCGCC

CGGTGGGCGT

GCTAGAAAGA

TCAGTTGCGC

CAAAACAAAT

GAGAGAATGA

TGCTTCCCCG

CTGTGTCCGA

CTGGCCAATG

ACCTGGACGA

AATGGGAGCT

CCGTGGCCGA

CGGAGGCCCT

TGGTGGAGAC

AGAAGCTGGT

TGACCAAGAC

CCAACTACCT

AGTATATAAG

CGCACGTGTC

CGGTCATCAG

GCGGGATCAC

ACGCCGCCTC

TGAGCCTGAC

CCAGCAACCG

CCGTCTTCCT

GGCCGGCCAC

ACGGCTGCGT

TGATCTGGTG

TGGGCGGAAG

CTCCCGTGAT

CCTTCGAGCA

TGGAGCACGA

CAGATCACGT

GGCCCGCCCC

AGCCATCGAC

GTTCTCGTCA

ATCAGAATGT

TGTCAGAATC

TTCATCACAT

TGGACTTGGA

GCACCTGCCC

GCCGCCGGAT

AAAGCTGCAA

CTTCTTTGTC

CGTGGGCGTC

GACCCGCATC

GCGTAATGGC

GCTCCCCAAG

CGCCTGTTTG

GCAGACGCAG

GTCAAAAACC

GTCAGAAAAG

CAACTCGCGG

AAAGACGGCT

CATCTACCGA

GGGCTGGGCG

GACGGGTAAA

GAACTGGACC

GGAGGAGGGC

CAAGGTGCGC

CGTCACCTCC

CCAACAACCA

CTTTGGCAAG

GACCGAGGTG

CAATGACGCA

GTCAGACGCG

CGTGGGTATG

GGACATTTGC

TCAACCCGTG

CATGGGGAGG

TGACTGTGAC

120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 1872 INFORMATION FOR SEQ ID NO:16: Wi SEQUENCE CHARACTERISTICS: LENGTH: 598 amino acids TYPE: amino acid STRANDEDNESS: not relevant TOPOLOGY: not relevant (ii) MOLECULE TYPE: DESCRIPTION: protein (ix) OTH4ER INFO: AAV4 capsid protein VP2 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16: Thr Ala Pro Gly Lys Lys Arg Pro Leu Ile Glu Ser Pro Gin Gin Pro 10 1s Asp Ser Ser Thr Gly lie Gly Lys Lys Gly Lys Gin Pro Ala Lys Lys 25 Lys Gly Al a Asn Val1 His Gly His 145 Met Giu Ser Met Phe 225 Ser Pro Phe Asp Ser 305 Phe Leu Gin Giu Pro 385 Gin Pro Al a Asn Pro 465 Trp Ile Asn Leu Ser s0 Giy Al a Thr Leu Phe 130 Phe Arg Vai Thr Asp 210 Met Gin Ser Giu Arg 290 Thr Thr Pro As n Thr 370 Pro Leu Gly Thr Ser 450 Gly Ala Giy Thr Val Thr Gly Ser Thr Tyr 115 Ser Ser Pro Thr Val 195 Al a Val1 Gin Gin Lys 275 Leu Thr Lys Gly Tyr 355 His Met Ile Thr Asp 435 As n Met Lys Gly Pro 515 Phe Ser Al a Gly Thr 100 Lys Thr Pro Lys Thr 180 Gin Gly Pro Gin Met 260 Val Met Thr Leu Pro 340 Lys Ser Aila Phe Leu 420 Thr Leu Val Ile Phe S00 Val1 Giu Gly Al a Asp Ser Arg Pro Arg Ala 165 Ser Ile Gin Gin Thr 245 Leu Pro Asn Gly Arg 325 Ser Ile Thr Thr Ala 405 Ile Asp Pro Trp Pro 485 Gly Pro Asp Al a Val Trp Thr Leu Trp Asp 150 Met Asn Phe Giu Tyr 230 Asp Arg Phe Pro Thr 310 Pro Ile Pro Leu Ala 390 Gly Phe Met Thr Gin 470 His Leu Al a Giu Met 55 Glu His Arg Gly Gly 135 Trp Arg Giy Al a Gly 215 Gly Arg Thr His Leu 295 Thr Thr Lys Ala Asp 375 Gly Pro Thr Trp Val 455 Asn Thr Lys Asn Thr 40 Ser Giy Cys Thr Giu 120 Tyr Gin Val1 Glu Asp 200 Ser Tyr Asn Gly Ser 280 Ile Leu Asn Gin Thr 360 Gly Pro Lys Ser Gly 440 Asp Arg Asp His Pro 520 Gly Asp Giy Asp Trp 105 Ser Phe Arg Lys Thr 185 Ser Leu Cys Ala Asn 265 Met Asp Asn Phe Gin 345 Gly Arg Ala Gin Giu 425 Asn Arg Asp Gly Pro 505 Ala Al a Asp Gin Ser Val Leu Asp Leu Ile 170 Thr Ser Pro Gly Phe 250 Asn Tyr Gin Al a Ser 330 Gly Ser Trp Asp Asn 410 Glu Leu Leu Ile His 490 Pro Thr Gly Ser Gly 75 Thr Leu Gin Phe Ile 155 Phe Val1 Tyr Pro Leu 235 Tyr Phe Al a Tyr Gly 315 As n Phe Asp Ser Ser 395 Gly Giu Pro Thr Tyr 475 Phe Pro Thr Asp Giu Al a Trp Pro Ser Asn 140 Asn Asn Al a Giu Phe 220 Val Cys Giu His Leu 300 Thr Phe Ser Ser Al a 380 Lys Asn Leu Gly Ala 460 Tyr His Gin Phe Gly Met Asp Ser Thr Asn 125 Arg Asn Ile Asn Leu 205 Pro Thr Leu Ile Ser 285 Trp Ala Lys Lys Leu 365 Leu Phe Thr Al a Gly 445 Leu Gin Pro Ile Ser 525 Pro Arg Gly Giu Tyr 110 Thr Phe As n Gin As n 190 Pro As n Gly Giu Thr 270 Gin Gly Thr Lys Thr 350 Ile Thr Ser Al a Ala 430 Asp Gly Gly Ser Phe 510 Ser Giu Al a Gly His As n Asn Cys Gly 160 Lys Thr Val Val Thr 240 Phe Ser Leu Gin As n 320 Trp As n Tyr Giy Ser 400 Val Asn Ser Val Ile 480 Leu Lys Pro Val Asn Ser Phe Ile Thr Gin Tyr Ser Thr 530 535 Ile Asp Trp Giu Ile Gin Lys Giu Arg Ser Gly Gin Val Ser Val Gin 540 Lys Arg Trp Asn Pro Giu 555 560 Asn Ser Leu Leu Trp Ala Val Pro Tyr 550 Gin Phe Thr Ser Asn Tyr Giy Gin 565 Asp Ala Ala Gly Lys Tyr Thr Giu 580 585 Leu Thr His His Leu 595 Gin 570 Pro 575 Thr Arg Arg Ala Ile INFORMATION FOR SEQ ID NO:17: Wi SEQUENCE CHARACTERISTICS: LENGTH: 1800 base pairs TYPE: nucieic acid STRANDEDNESS: double TOPOLOGY: linear (ix) OTHER INFO: AAV4 capsid protein VP2 gene (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:

ACGGCTCCTG

GGTATCGGCA

GGAGCAGGCG

ATGCGTGCAG

AATGCCTCGG

AGCACCAGAA

AGCCTGCAGT

CGCTTCCACT

ATGCGACCCA

TCGAACGGCG

TCGTCGTACG

CCCAACGACG

TCGCAGCAAC

CTGCGGACTG

ATGTACGCGC

TGGGGACTGC

TTTACCAAGC

TCAATCAAGC

GGGTCAGACA

CTGACCCCCG

CAGCTCATCT

ATCTTCACCT

AACCTACCTG

TTGGGAGCCG

TGGGCCAAGA

GGGCTGAAAC

GCAACGACCT

GTGTCGGTGC

GTCCAGTTTA

GGGAAATACA

GAAAGAAGAG

AAAAAGGCAA.

ACGGACCCCC

CAGCTGGCGG

GTGATTGGCA

CCTGGGTCTT

CCAACACCTA

GCCACTTCTC

AAGCCATGCG

AGACAACGGT

AACTGCCGTA

TCTTTATGGT

AGACTGACAG

GCAACAACTT

ACAGCCAGAG

AATCGACCAC

TGCGGCCTAC

AGCAGGGCTT

GTCTCATCAA

GACCTCCAAT

TTGCGGGGCC

CTGAGGAGGA

GCGGTGACCA

TGCCTGGAAT

TTCCTCATAC

ACCCGCCTCC

TCAGCTCTAC

AGATTGACTG

CCTCCAACTA

CTGAGCCTAG

ACCGTTGATT

GCAGCCGGCT

TGAGGGATCA

AGCTGCAGTC

TTGCGATTCC

GCCCACCTAC

CAACGGATTC

ACCACGTGAC

GGTCAAAATC

GGCTAATAAC

CGTGATGGAT

GCCCCAGTAC

AAATGCCTTC

TGAAATTACG

CCTGGACCGG

CACCGGAACC

CAACTTTTCC

CTCAAAGACT

ATACGAGACG

GGCCACGGCT

TAAACAGAAC

GCTGGCAGCC

GAGCAACAGC

GGTCTGGCAA

CGATGGACAC

TCAAATTTTT

TCCGGTAAAC

GGAGATCCAG

CGGACAGCAA

GGCTATCGGT

GAATC CC CC C

AAAAAGAAGC

ACTTCCGGAG

GAGGGSGGAC

ACCTGGTCTG

AACAACCACC

TCCACCCCCT

TGGCAGCGAC

TTCAACATCC

CTTACCAGCA

GCGGGTCAAG

GGCTACTGTG

TACTGCCTGG

TACAGTTTTG

CTGATGAACC

ACCCTGAATG

AACTTTAAAA

GCCAATCAAA

CACAGCACTC

GGACCTGCGG

GGCAACACGG

ACCAACGCCA

AACCTGCCGA

AACAGAGACA

TTTCACCCCT

ATCAAGAACA

TCCTTCATTA

AAGGAGCGGT

AACTCTCTGT

ACCCGCTACC

AGCAGCCCGA

TCGTTTTCGA

CCA.TGTCTGA

AAGGTGCCGA

AGGGCCACGT

TNTACAAGCG

GGGGATACTT

TCATCAACAA

AGGTCAAGGA

CGGTTCAGAT

AGGGCAGCCT

GACTGGTGAC

AGTACTTTCC

AGAAGGTGCC

CTCTCATCGA

CCGGGACTGC

AGAACTGGCT

ACTACAAGAT

TGGACGGAAG

ACAGCAAGTT

CCACCGTACC

CCGATACGGA

CCGTGGACAG

TTTACTACCA

CACCGCTGAT

CCCCGGTACC

CTCAGTACAG

CCA.AACGCTG

TGTGGGCTCC

TCACCCACCA

CTCCTCCACG

AGACGAAACT

TGACAGTGAG

TGGAGTGGGT

CACGACCACC

ACTCGGAGAG

TGACTTCAAC

CAACTGGGGC

GGTCACGACG

CTTTGCGGAC

GCCTCCTTTT

CGGCAACACT

TTCGCAGATG

TTTCCACTCG

CCAGTACCTG

CACCACCAAC

GCCCGGGCCT

CCCTGCCACC

ATGGAGTGCC

CAGCAACAGC

CGGGACTCTG

CATGTGGGGC

ACTGACAGCC

GGGTCCCATT

TGGTGGGTTT

TGCGAATCCT

CACTGGCCAG

GAACCCCGAG

CGATGCGGCT

CCTGTAATAA

120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 INFORMATION FOR SEQ ID NO:18: SEQUENCE CHARACTERISTICS: LENGTH: 544 amino acids TYPE: amino acid STRANDEDNESS: not relevant TOPOLOGY: not relevant (ii) MOLECULE TYPE: DESCRIPTION: protein (ix) OTHER INFO: AAV4 capsid protein VP3 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18: Met 1 Glu His Arg Gly Gly Trp Arg Gly Al a 145 Gly Giy Arg Thr His 225 Leu Thr Thr Lys Ala 305 Asp Gly Pro Thr Trp 385 Val Ser Giy Cys Thr Giu Tyr Gin Val1 Giu 130 Asp Ser Tyr As n Gly 210 Ser Ile Leu Asn Gin 290 Thr Gly Pro Lys Ser 370 Gly Asp Asp Gly Asp Trp Ser Phe Arg Lys 115 Thr Ser Leu Cys Al a 195 As n Met Asp As n Phe 275 Gin Gly Arg Al a Gin 355 Giu Asn Arg Asp Ser Glu 5 Gin Gly Ala Ser Thr Trp Val Leu Pro Leu Gin Ser 70 Asp Phe Asn 85 Leu Ile Asn 100 Ile Phe Asn Thr Val Ala Ser Tyr Giu iso Pro Pro Phe 165 Giy Leu Vai 180 Phe Tyr Cys Asn Phe Giu Tyr Ala His 230 Gin Tyr Leu 245 Ala Giy Thr 260 Ser Asn Phe Giy Phe Ser Ser Asp Ser 310 Trp Ser Ala 325 Asp Ser Lys 340 Asn Gly Asn Glu Giu Leu Leu Pro Gly 390 Leu Thr Aia 405 Met Asp Ser Thr As n Arg Asn Ile Asn 135 Leu Pro Thr Leu Ile 215 Ser Trp Ala Lys Lys 295 Leu Leu Phe Thr Al a 375 Gly Arg Giy Giu 40 Tyr Thr Phe Asn Gln 120 Asn Pro Asn Gly Giu 200 Thr Gin Gly Thr Lys 280 Thr Ile Thr Ser Ala 360 Ala Asp Al a Vai 25 Giy Asn Tyr His Trp 105 Vali Leu Tyr Asp Asn 185 Tyr Tyr Ser Leu Thr 265 Asn Ala Lys Pro Asn 345 Thr Thr Gin Ala Ala Giy Asn His Vai Asn His Asn Gly Cys His 90 Gly Met Lys Giu Thr Ser Val Met 2.55 Val Phe 170 Thr Ser Phe Pro Ser Plie Leu Asp 235 Gin Ser 250 Asn Phe Trp Leu Asn Gin Tyr Glu 315 Gly Pro 330 Ser Gin Val Pro Asn Ala Ser Asn 395 Gly Al a Thr Leu Phe Phe Arg Val1 Thr 140 Asp Met Gin Ser Giu 220 Arg Thr Thr Pro Asn 300 Thr Pro Leu Gly Thr 380 Ser Gly Ser Thr Tyr Ser Ser Pro Thr 125 Val Ala Val Gin Gin 205 Lys Leu Thr Lys Gly 285 Tyr His Met Ile Thr 365 Asp Asn Al a Giy Thr Lys Thr Pro Lys 110 Thr Gin Gly Pro Gln 190 Met Val1 Met Thr Leu 270 Pro Lys Ser Ala Phe 350 Leu Thr Leu Ala Asp Ser Arg Pro Arg Ala Ser Ile Gin Gin 175 Thr Leu Pro As n Gly 255 Arg Ser Ile Thr Thr 335 Al a Ile Asp Pro Val Trp Thr Leu Trp Asp Met Asn Phe Giu 160 Tyr Asp Arg Phe Pro 240 Thr Pro Ile Pro Leu 320 Al a Giy Phe Met Thr 400

C

Leu Giy Ala Val Pro Gly Met Val 410 Trp Gin 415 Asn Arg Asp Ile Tyr Tyr Gin Gly Pro Ile Trp 425 Pro Leu Ile Thr Asp Gly 435 Lys His Pro 450 Asn Pro Ala Phe His Pro Ser Ala Lys Ile Pro His 430 Gly Giy Phe Giy Leu 445 Thr Pro Vai Pro Ala Pro Pro Gin Thr Thr Phe 470 Thr Gly Gin Ile 455 S er Phe Ile Lys Asn 460 As n Ser Thr Pro Ser Phe Ile 465 Gin Thr 480 Tyr Ser Val Ser Val 485 Lys Gin 490 Giu Asp Trp Giu Ile Gin 495 Ser Asn Lys Giu Arg Tyr Gly Gin Tyr Thr Glu 530 Ser 500 Gin Arg Trp Asn Pro 505 Trp Val Gin Phe Thr Asn Ser Leu Leu 520 Gly Ala Pro Asp Al a 525 Thr Ala Giy Lys His His Leu Pro Arg Ala Ile 535 Thr Arg Tyr Leu 540 a. a.

a a a a a. a a a.

a.

a INFORMATION FOR SEQ ID NO:i9: i)SEQUENCE CHARACTERISTICS: LENGTH: i6i7 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ix) OTHER INFO: AAV4 capsid protein VP3 gene (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:

ATGCGTGCAG

AATGCCTCGG

AGCACCAGAA

AGCCTGCAGT

CGCTTCCACT

ATGCGACCCA

TCGAACGGCG

TCGTCGTACG

CCCAACGACG

TCGCAGCAAC

CTGCGGACTG

ATGTACGCGC

TGGGGACTGC

TTTACCAAGC

TCAATCAAGC

GGGTCAGACA

CTGACCCCCG

CAGCTCATCT

ATCTTCACCT

AACCTACCTG

TTGGGAGCCG

TGGGCCAAGA

GGGCTGAAAC

GCAACGACCT

GTGTCGGTGC

GTCCAGTTTA

GGGAAATACA

CAGCTGGCGG

GTGATTGGCA

CCTGC-GTCTT

CCAACACCTA

GCCACTTCTC

AAGCCATGCG

AGACAACGGT

AACTGC-CGTA

TCTTTATGGT

AGACTGACAG

GCAACAACTT

ACAGCCAGAG

AATCGACCAC

TGCGGCCTAC

AGCAGGGCTT

GTCTCATCAA

GACCTCCAAT

TTGCGGC-GCC

CTGAC-GAGGA

GCC-GTGACCA

TGCCTGCGAAT

TTCCTCATAC

ACCCGCCTCC

TCAGCTCTAC

AGATTGACTG

CCTCCAACTA

CTGAGCCTAG

AGCTGCAGTC

TTGCGATTCC

GCCCACCTAC

CAACGGATTC

ACCACGTGAC

GGTCAAAATC

GGCTAATAAC

CGTGATGGAT

GCCCCAGTAC

AAATGCCTTC

TGAAATTACG

CCTGGACCGG

CACCGGAACC

CAACTTTTCC

CTCAAAGACT

ATACGAGACG

GGCCACGGCT

TAAACAGAAC

GCTGGCAGCC

GAGCAACAGC

GGTCTGGCAA

CGATGGACAC

TCAAATTTTT

TCCGGTAAAC

GGAGATCCAG

CGGACAGCAA

GGCTATCGGT

GAGGGSGGAC

ACCTGGTCTG

AACAACCACC

TCCACCCCCT

TGGCAGCGAC

TTCAACATCC

CTTACCAGCA

GCGGGTCAAG

GGCTACTGTG

TACTGCCTGG

TACAGTTTTG

CTGATGAACC

ACCCTGAATG

AACTTTAAAA

GCCAATCAAA

CACAGCACTC

GGACCTGCGG

GGCAACACGG

ACCAACGCCA

AACCTGCCGA

AACAGAGACA

TTTCACCCCT

ATCAAGAACA.

TCCTTCATTA

AAGGAGCGGT

AACTCTCTGT

ACCCGCTACC

AAGGTGCCGA

AGGGCCACGT

TNTACAAGCG

GGGGATACTT

TCATCAACAA

AGGTCAAGGA

CGGTTCAGAT

AGGGCAGCCT

GACTGGTGAC

AGTACTTTCC

AGAAGGTGCC

CTCTCATCGA

CCGGGACTGC

AGAACTGGCT

ACTACAAGAT

TGGACGGAAG

ACAGCAAGTT

CCACCGTACC

CCGATACGGA

CCGTGGACAG

TTTACTACCA

CACCGCTGAT

CCCCGGTACC

CTCAGTACAG

CCAAACGCTG

TGTGGGCTCC

TCACCCACCA

TGGAGTGGGT

CACGACCACC

ACTCGGAGAG

TGACTTCAAC

CAACTGGGGC

GGTCACGACG

CTTTGCGGAC

GCCTCCTTTT

CGGCAACACT

TTCGCAGATG

TTTCCACTCG

CCAGTACCTG

CACCACCAAC

GCCCGGGCCT

CCCTGCCACC

ATGGAGTGCC

CAGCAACAGC

CGGGACTCTG

CATGTGGGGC

ACTGACAGCC

GGGTCCCATT

TGGTGGGTTT

TGCGAATCCT

CACTGGCCAG

GAACCCCGAG

CGATGCGGCT

CCTGTAA

120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1617 INFOPUNATION FOR SEQ ID Ci) SEQUENCE CHARACTERISTICS: LENGTH: 129 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ix) OTHER INFO: AAV4 ITR "flop" orientation (xi) SEQUENCE DESCRIPTION: SEQ ID TTGGCCACTC CCTCTATGCG CGCTCGCTCA CTCACTCGGC CCTGCGGCCA GAGGCCGGCA GTCTGGAGAC CTTTGGTGTC CAGGGCAGGG CCGAGTGAGT GAGCGAGCGC GCATAGAGGG 120 AGTGGCCAA 129 INFORMATION FOR SEQ ID NO:21: SEQUENCE CHARACTERISTICS: LENGTH: 35 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21: TCTAGTCTAG ACTTGGCCAC TCCCTCTCTG CGCGC INFORMATION FOR SEQ ID NO:22: SEQUENCE CHARACTERISTICS: LENGTH: 34 base pairs TYPE: nucleic acid STRANDEDNESS: single S(D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22: AGGCCTTAAG AGCAGTCGTC CACCACCTTG TTCC 34

S

1* 0

Claims

2. A nucleic acid vector including a pair of adeno-associated virus 4 (AAV4) inverted terminal repeats and a promoter between the inverted terminal repeats, wherein the AAV4 inverted terminal repeats include the nucleotide sequence set forth in SEQ ID NO: 6.
3. A nucleic acid vector including a pair of adeno-associated virus 4 (AAV4) inverted terminal repeats and a promoter between the inverted terminal repeats, wherein the AAV4 inverted terminal repeats include the nucleotide sequence set forth in SEQ ID NO:
4. A vector according to claim 2, wherein the promoter is an AAV promoter p 5
5. A vector according to claim 4, wherein the p5 promoter is AAV4 20 promoter. "i 6. A vector according to any one of claims 1 to 5, further including an exogenous nucleic acid functionally linked to the promoter. 25 7. A vector according to any one of claims 1 to 6 encapsidated in an adeno- associated virus particle.
8. A particle of claim 7, wherein the particle is an AAV4 particle, including a capsid protein including an amino acid sequence defined by amino acids 438- 601 shown in SEQ ID NO:4.
9. A particle of claim 7, wherein the particle is an AAV1 particle, an AAV2 particle, an AAV3 particle or an AAV5 particle.
10. An AAV4 particle, including a capsid protein including an amino acid sequence defined by amino acids 438-601 shown in SEQ ID NO:4.
11. A particle of claim 10, wherein the vector further includes an exogenous nucleic acid inserted between the inverted terminal repeats.
12. An isolated nucleic acid including the nucleotide sequence set forth in SEQ ID NO:1.
13. An isolated nucleic acid consisting essentially of the nucleotide sequence set forth in SEQ ID NO: 1.
14. An isolated nucleic acid that selectively hybridizes with the nucleic acid of claim 13.
15. An isolated nucleic acid encoding an adeno-associated virus 4 Rep 15 protein.
16. A nucleic acid according to claim 15, wherein the adeno-associated virus 4 Rep protein has the amino acid sequence set forth in SEQ ID NO:2.
17. A nucleic acid according to claim 15, wherein the adeno-associated virus 4 Rep protein has the amino acid sequence set forth in SEQ ID NO:8.
18. A nucleic acid according to claim 15, wherein the adeno-associated virus 4 Rep protein has the amino acid sequence set forth in SEQ ID NO:9.
19. A nucleic acid according to claim 15, wherein the adeno-associated virus 4 Rep protein has the amino acid sequence set forth in SEQ ID A nucleic acid according to claim 15, wherein the adeno-associated virus 4 Rep protein has the amino acid sequence set forth in SEQ ID NO:11.
21. A nucleic acid according to claim 15, wherein the nucleic acid includes the nucleotide sequence set forth in SEQ ID NO:3.
22. A nucleic acid according to claim 15, wherein the nucleic acid consists essentially of the nucleotide sequence set forth in SEQ ID NO:3.
23. An isolated nucleic acid that selectively hybridizes with the nucleic acid of claim 22.
24. A nucleic acid according to claim 15, wherein the nucleic acid includes the nucleotide sequence set forth in SEQ ID NO:12. A nucleic acid according to claim 15, wherein the nucleic acid includes the nucleotide sequence set forth in SEQ ID NO:13.
26. The nucleic acid of claim 15, wherein the nucleic acid includes the nucleotide sequence set forth in SEQ ID NO:14.
27. A nucleic acid according to claim 15, wherein the nucleic acid includes 15 the nucleotide sequence set forth in SEQ ID
28. An isolated AAV4 Rep protein having the amino acid sequence set forth in SEQ ID NO:2, or a unique fragment thereof.
29. An isolated AAV4 Rep protein having the amino acid sequence set forth in SEQ ID NO:8, or a unique fragment thereof. An isolated AAV4 Rep protein having the amino acid sequence set forth in SEQ ID NO:9, or a unique fragment thereof.
31. An isolated AAV4 Rep protein having the amino acid sequence set forth in SEQ ID NO:10, or a unique fragment thereof.
32. An isolated AAV4 Rep protein having the amino acid sequence set forth in SEQ ID NO:11, or a unique fragment thereof.
33. An isolated antibody that specifically binds the protein of claim 28.
34. An isolated AAV4 capsid protein having the amino acid sequence set forth in SEQ ID NO:4. 73 An isolated antibody that specifically binds the protein of claim 34.
36. An isolated AAV4 capsid protein having the amino acid sequence set forth in SEQ ID NO:16.
37. An isolated antibody that specifically binds the protein of claim 36.
38. An isolated AAV4 capsid protein having the amino acid sequence set forth in SEQ ID NO:18.
39. An isolated antibody that specifically binds the protein of claim 38.
40. An isolated nucleic acid encoding the adeno-associated virus 4 capsid protein of SEQ ID NO:16.
41. An isolated nucleic acid encoding the adeno-associated virus 4 capsid protein of SEQ ID NO:4.
42. A nucleic acid according to claim 41, wherein the nucleic acid includes the nucleic acid sequence set forth in SEQ ID A nucleic acid according to claim 41, wherein the nucleic acid consists essentially of the nucleic acid sequence set forth in SEQ ID 25 44. An isolated nucleic acid that selectively hybridizes with the nucleic acid of claim 39. An isolated nucleic acid that selectively hybridizes with the nucleic acid of SEQ ID NO:4.
46. An isolated nucleic acid including the AAV4 p5 promoter including nucleotides 130-291 of SEQ ID NO:1.
47. A method of screening a cell for infectivity by AAV4 including contacting the cell with an AAV4 particle including a capsid protein including an amino acid sequence defined by amino acids 438-601 shown in SEQ ID NO:4 and detecting the presence of the AAV4 particle in the cells.
48. A method of screening a cell for infectivity by AAV4 including contacting the cell with an AAV4 vector including an AAV4 particle including a capsid protein including an amino acid sequence defined by amino acids 438-601 shown in SEQ ID NO:4 and including a known nucleic acid, wherein the presence of the AAV4 vector is detected in the cells by detecting the presence of the known nucleic acid.
49. A method of determining, the suitability of an AAV4 vector for administration to a subject including administering to an antibody-containing i sample from the subject an antigenic protein including an amino acid sequence selected from the group consisting of SEQ ID NO:4, SEQ ID NO:16, SEQ ID 15 NO:18, SEQ ID NO:2, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11 and residues 438-601 of SEQ ID NO:2 and detecting an antibody-antigen reaction in the sample, the presence of a reaction indicating the AAV4 vector to be unsuitable for use in the subject.
50. A method of determining the presence in a subject of an AAV4-specific antibody including administering to an antibody-containing sample from the subject an antigenic protein including an amino acid sequence selected from the group consisting of SEQ ID NO:4, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:2, i: SEQ ID NO:8, SEQ IDNO:9, SEQ ID NO:10, SEQ ID NO:11 and residues 438-601 25 shown in SEQ ID NO:4 and detecting an antibody-antigen reaction in the sample, the presence of a reaction indicating the presence of an AAV4-specific antibody in the subject.
51. A method of delivering a nucleic acid to a cell including administering to the cell an AAV4 particle, including a capsid protein including an amino acid sequence defined by amino acids 438-601 shown in SEQ ID NO:4, containing a vector including the nucleic acid inserted between a pair of AAV inverted terminal repeats, thereby delivering the nucleic acid to the cell.
52. A method according to claim 52, wherein the AAV inverted terminal repeats are AAV4 inverted terminal repeats.
53. A method according to claim 52, wherein the AAV inverted terminal repeats are AAV2 inverted terminal repeats.
54. A method of delivering a nucleic acid to a subject including administering to a cell from the subject an AAV4 particle, including a capsid protein including an amino acid sequence defined by amino acids 438-601 shown in SEQ ID NO:4, including the nucleic acid inserted between a pair of AAV inverted terminal repeats, and returning the cell to the subject, thereby delivering the nucleic acid to the subject.
55. A method of delivering a nucleic acid to a cell in a subject including administering to the subject an AAV4 particle, including a capsid protein including an amino acid sequence defined by amino acids 438-601 shown in 15 SEQ ID NO:4, including the nucleic acid inserted between a pair of AAV inverted terminal repeats, thereby delivering the nucleic acid to a cell in the subject.
56. A method of delivering a nucleic acid to a cell in a subject having antibodies to AAV2 including administering to the subject an AAV4 particle, including a capsid protein including an amino acid sequence defined by amino acids 438-601 shown in SEQ ID NO:4, including the nucleic acid, thereby delivering the nucleic acid to a cell in the subject. 25 57. A vector according to claim 3, wherein the promoter is an AAV promoter
58. A vector according to claim 3, wherein the p5 promoter is AAV4 promoter.
59. A vector according to claim 3, further including an exogenous nucleic acid functionally linked to the promoter. A vector according to claim 3, encapsidated in an adeno-associated virus particle.
61. A particle of claim 61, wherein the particle is an AAV4 particle, including a capsid protein including an amino acid sequence defined by amino acids 438- 601 shown in SEQ ID NO:4.
62. A particle of claim 61, wherein the particle is an AAV1 particle, an AAV2 particle, an AAV3 particle or an AAV5 particle.
63. An isolated nucleic acid encoding the adeno-associated virus 4 capsid protein of SEQ ID NO:18.
64. A particle of claim 7, wherein the particle is an AAV4 particle, including a capsid protein including an amino acid sequence selected from the group consisting of SEQ ID NO:4, SEQ ID NO:16 and SEQ ID NO:18. 15 65. An AAV4 particle, including a capsid protein consisting essentially of an amino acid sequence selected from the group consisting of SEQ ID NO:4, SEQ ID NO:16 and SEQ ID NO:18, containing a vector including a pair of AAV2 inverted terminal repeats.
66. A method of delivering a nucleic acid to a cell including administering to S.i: the cell an AAV4 particle, including a capsid protein consisting essentially of an amino acid sequence selected from the group consisting of SEQ ID NO:4, SEQ ID NO:16 and SEQ ID NO:18, containing a vector including the nucleic acid inserted between a pair of AAV inverted terminal repeats, thereby delivering the 25 nucleic acid to the cell.
67. A method of delivering a nucleic acid to a subject including administering to a cell from the subject an AAV4 particle, including a capsid protein consisting essentially of an amino acid sequence selected from the group consisting of SEQ ID NO:4, SEQ ID NO:16 and SEQ ID NO:18, including the nucleic acid inserted between a pair of AAV inverted terminal repeats, and returning the cell to the subject, thereby delivering the nucleic acid to the subject.
68. A method of delivering a nucleic acid to a cell in a subject including administering to the subject an AAV4 particle, including a capsid protein consisting essentially of an amino acid sequence selected from the group 77 consisting of SEQ ID NO:4, SEQ ID NO:16 and SEQ ID NO:18, including the nucleic acid inserted between a pair of AAV inverted terminal repeats, thereby delivering the nucleic acid to a cell in the subject.
69. A method of delivering a nucleic acid to a cell in a subject having antibodies to AAV2 including administering to the subject an AAV4 particle, including a capsid protein consisting essentially of an amino acid sequence selected from the group consisting of SEQ ID NO:4, SEQ ID NO:16 and SEQ ID NO:18, including the nucleic acid, thereby delivering the nucleic acid to a cell in the subject.
70. An AAV4 vector, including a nucleic acid selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:17, SEQ ID 15 NO:19, SEQ ID NO:20, SEQ ID NO:21 and SEQ ID NO:22.
71. An isolated adeno-associated virus 4 Rep protein.
72. A nucleic acid vector according to claim 1 substantially as hereinbefore described with reference to any of the Examples or Figures. e 9*