Patent Abstract:
The invention relates to Adeno-associated virus vectors. In particular, it relates to Adeno-associated virus vectors with modified capsid proteins and materials and methods for their preparation and use.

Full Description:
RELATED APPLICATIONS  
       [0001]    The present application claims priority benefit of U.S. Provisional Application No. 60/260,124 filed Jan. 5, 2001 which is herein incorporated by reference in its entirety. 
     
    
     
       FIELD OF THE INVENTION  
         [0002]    The invention relates to Adeno-associated virus vectors. In particular, it relates to Adeno-associated virus vectors with modified capsid proteins and materials and methods for their preparation and use.  
         BACKGROUND  
         [0003]    Adeno-associated virus (AAV) is a replication-deficient parvovirus, the single-stranded DNA genome of which is about 4.7 kb in length including 145 nucleotide inverted terminal repeat (ITRs). The nucleotide sequence of the AAV serotype 2 (AAV2) genome is presented in Srivastava et al.,  J. Virol.,  45: 555-564 (1983) as corrected by Ruffing et al.,  J. Gen. Virol.,  75: 3385-3392 (1994) Cis-acting sequences directing viral DNA replication (rep), encapsidation/packaging and host cell chromosome integration are contained within the ITRs. Three AAV promoters, p5, p19, and p40 (named for their relative map locations), drive the expression of the two AAV internal open reading frames encoding rep and cap genes. The two rep promoters (p5 and p19), coupled with the differential splicing of the single AAV intron (at nucleotides 2107 and 2227), result in the production of four rep proteins (rep 78, rep 68, rep 52, and rep 40)from the rep gene. Rep proteins possess multiple enzymatic properties which are ultimately responsible for replicating the viral genome. The cap gene is expressed from the p40 promoter and it encodes the three capsid proteins VP1, VP2, and VP3. Alternative splicing and non-consensus translational start sites are responsible for the production of the three related capsid proteins. A single consensus polyadenylation site is located at map position 95 of the AAV genome. The life cycle and genetics of AAV are reviewed in Muzyczka,  Current Topics in Microbiology and Immunology,  158: 97-129 (1992).  
           [0004]    When AAV infects a human cell, the viral genome can integrate into chromosome 19 resulting in latent infection of the cell. Production of infectious virus does not occur unless the cell is infected with a helper virus (for example, adenovirus or herpesvirus). In the case of adenovirus, genes E1A, E1B, E2A, E4 and VA provide helper functions. Upon infection with a helper virus, the AAV provirus is rescued and amplified, and both AAV and adenovirus are produced.  
           [0005]    AAV possesses unique features that make it attractive as a vaccine vector for expressing immunogenic peptides/polypeptides and as a vector for delivering foreign DNA to cells, for example, in gene therapy. AAV infection of cells in culture is noncytopathic, and natural infection of humans and other animals is silent and asymptomatic Moreover, AAV infects many mammalian cells allowing the possibility of targeting many different tissues in vivo. Replication of the viral DNA is not required for integration, and thus helper virus is not required for this process. The AAV proviral genome is infectious as cloned DNA in plasmids which makes construction of recombinant genomes feasible. Furthermore, because the signals directing AAV replication, genome encapsidation and integration are contained within the ITRs of the AAV genome, some or all of the internal approximately 4.3 kb of the genome (encoding replication and structural capsid proteins, rep-cap) may be replaced with foreign DNA such as a gene cassette containing a promoter, a DNA of interest and a polyadenylation signal. The rep and cap proteins may be provided in trans. Another significant feature of AAV is that it is an extremely stable and hearty virus. It easily withstands the conditions used to inactivate adenovirus (56° to 65° C. for several hours), making cold preservation of rAAV-vectors less critical. AAV may even be lyophilized. Finally, AAV-infected cells are not resistant to superinfection.  
           [0006]    Recent research on AAV has therefore involved attempts to modify the viral genome. As the range of cells that AAV will infect is so broad, some researches have focused on modifying the virus so that it targets specific types of cells for infection. The cellular range or tropism of the virus is determined by the binding of AAV capsid protein(s) to receptor and/or coreceptor proteins expressed on the surface of target cells. Heparin-sulfate proteoglycan (HSPG) is the primary cellular attachment receptor for AAV2. In attempts to enable AAV to bind other cellular receptors, mutagenesis of the AAV capsid-encoding DNA to encode heterologous targeting peptides as part of a capsid protein has produced varying results. For example, Girod et al. ( Nature Medicine,  5: 1052-1056, 1999) describes AAV2 insertional mutants generated to target L14-specific integrin receptors. These mutant AAV2 vectors expressed capsid proteins which had a fourteen amino acid peptide comprising the RGD domain of the laminin fragment P1 inserted at six different sites. Rabinowitz et al. ( Virology,  265: 274-285, 1999) attempted to identify capsid domains and positions which were capable of tolerating insertions without loss of function. Related PCT application WO 00/28004 describes the modified capsid proteins containing insertions such as melanocyte stimulating hormone, poly-histidine tracts, poly-lysine tracts, an RGD domain and bradykinin. Only a few of the modified capsid proteins could be incorporated into functional viral particles and titers of the viruses were drastically lower than wild-type virus.  
         SUMMARY OF THE INVENTION  
         [0007]    The present inventors recognized a need in the art for identification of sites in the AAV capsid protein(s) from which peptides/polypeptides of interest may be presented in a desired conformation to allow the development of AAV vectors that deliver DNA to specific target cells and the development of AAV vectors that present/display on their surface immunogenic peptides/polypeptides. Their invention is based on the elucidation of sites/regions in the AAV2 capsid protein that are amenable to insertion of heterologous peptides, the development of scaffolding sequences required for proper conformation of peptides, and the construction of AAV2 vectors with altered tropism. The full length nucleotide sequence of the wild type AAV2 vector is set out as SEQ ID NO: 12. The amino acid sequence of VP1capsid protein (SEQ ID NO: 13) is encoded by the nucleotides 2203-4410 of SEQ ID NO: 12, the amino acid sequence of VP2 capsid protein (SEQ ID NO: 14) is encoded by nucleotides 2614-4410 of SEQ ID NO: 12 and the amino acid sequence of VP3 capsid protein (SEQ ID NO: 15) is encoded by nucleotides 2809-4410 of SEQ ID NO: 12.  
           [0008]    The present invention provides AAV vectors (viral particles) encoding capsid proteins that comprise insertions of amino acids of interest (i.e., peptides or polypeptides). Preferably, the AAV vectors are AAV2 vectors. Also preferably, DNA encoding the insertions follows the cap gene DNA encoding amino acid position 139 and/or position 161 in the VP1/VP2 capsid region, and/or amino acid position 459, 584, 588 and/or 657 in the VP3 region. While the capsid sites/regions amenable to insertions have been described herein with respect to AAV2, those skilled in the art will understand that corresponding sites in other parvoviruses, both autonomously-replicating parvoviruses and other AAV dependent viruses, are also sites/regions amenable to insertions in those viruses. The amino acids of interest may impart a different binding/targeting ability to the vector or may themselves be immunogenic. As a result, the vectors of the invention exhibit altered characteristics in comparison to wild type AAV, including but not limited to, altered cellular tropism and/or antigenic properties. The invention also contemplates cells, plasmids and viruses which comprise polynucleotides encoding the capsid proteins of the invention.  
           [0009]    It is contemplated that in addition to amino acids of interest, amino acids serving as linker/scaffolding sequences as described herein may be included in the AAV vector capsid insert to maintain the functional conformation of the capsid. The linker/scaffolding sequences are short sequences which flank the insertion of interest in the mutated capsid protein. For example, the insertion may have the amino acids TG at its amino terminus and the tripeptide ALS, GLS or LLA at its carboxy terminus.  
           [0010]    Techniques to produce AAV vectors, in which a AAV genome to be packaged, rep and cap genes, and helper virus functions are provided to a cell are standard in the art. Production of AAV vectors requires that the following components are present within a single cell (denoted herein as a packaging cell): a rAAV construct consisting of a DNA of interest flanked by AAV inverted terminal repeats, an AAV helper construct containing the capsid gene (which may or may not be comprise an insert) and the rep gene, and an adenovirus helper plasmid or infected with an adenovirus. The rAAV construct may be delivered to a packaging cell by transfection in a plasmid, infection by a viral genome or may be integrated into the packaging cell genome. The AAV helper construct may be delivered to a packaging cell by transfection of a plasmid or integrated into the packaging cell genome. The adenovirus helper plasmid or adenovirus may be delivered to the packaging cell by transfection/infection. The term “helper virus functions” refers to the functions carried out by the addition of an adenovirus helper plasmid or infection of adenovirus to support production of AAV viral particles.  
           [0011]    One method generating a packaging cell with all the necessary components for AAV production is the triple transfection method. In this method a cell such as a 293 cell is transfected with the rAAV construct, the AAV helper construct and a adenovirus helper plasmid or infected with adenovirus. The advantages of the triple transfection method are that it is easily adaptable and straightforward. Generally, this method is used for small scale vector preparations.  
           [0012]    Another method of generating a packaging cell is to create a cell line which stably expresses all the necessary components for AAV vector production. For example, a plasmid expressing the rAAV construct, a helper construct expressing the rep and cap proteins (modified or wild type) and a selectable marker, such as Neo, are integrated into the genome of a cell. The packaging cell line is then infected with a helper virus such as adenovirus. The advantages of this method are that the cells are selectable and are suitable for large-scale production of the vector.  
           [0013]    In another aspect the invention provides AAV helper constructs encoding a AAV cap gene comprising DNA encoding an insertion of one or more amino acids in the encoded capsid protein(s). The insertion is at a position of the encoded capsid protein(s) that is exposed on the surface of an AAV vector comprising the capsid protein(s) and that does not disrupt conformation of the capsid protein(s) in a manner that prevents assembly of the vector or infectivity of the vector. Limited by these criteria, the size of the insert may vary from as short as two amino acids to as long as amino acids encoding an entire protein. Also provided are cells that stably or transiently produce AAV vectors of the invention. Methods of producing AAV vectors using such cells are contemplated by the invention.  
           [0014]    In one embodiment, the AAV vectors of the invention comprising capsid proteins with binding/targeting amino acids inserted are useful for the therapeutic delivery and/or transfer of nucleic acids to animal (including human) cells both in vitro and in vivo. Nucleic acids of interest include nucleic acids encoding peptides and polypeptides, such as therapeutic (e.g., for medical or veterinary uses) peptides or polypeptides. A therapeutic peptide or polypeptide is one that may prevent or reduce symptoms that result from an absence or defect in a protein in a cell or person. Alternatively, a therapeutic peptide or polypeptide is one that otherwise confers a benefit to a subject, e.g., anti-cancer effects. As a further alternative, the nucleic acid may encode a reporter peptide or protein (e.g., an enzyme). In yet still another alternative, the nucleic acid of interest may be an antisense nucleic acid or a ribozyme.  
           [0015]    In another embodiment, the AAV vectors are useful as vaccines. The use of parvoviruses as vaccines is known in the art. Immunogenic amino acids (peptides or polypeptides) may be presented as inserts in the AAV vector capsid. Alternatively, immunogenic amino acids may be expressed from a heterologous nucleic acid introduced into a recombinant AAV genome and carried by the AAV vector. If the immunogenic amino acids are expressed from a recombinant AAV genome, the AAV vector of the invention preferably exhibits an altered cellular tropism and comprises a capsid protein with an insertion of targeting amino acids that are different from those of wild type AAV. Immunogenic amino acids may be from any source (e.g., bacterial, viral or tumor antigens).  
           [0016]    AAV vectors of the invention that exhibit an altered cellular tropism may differ from wild type in that the natural tropism of AAV may be reduced or abolished by insertion or substitution of amino acids of interest in a capsid protein of the vector. Alternatively, the insertion or substitution of the amino acids may target the vector to a particular cell type(s) perhaps not targeted by wild type AAV. Cell types of interest contemplated by the invention include, for example, glial cells, airway epithelium cells, hematopoietic progenitors cells and tumor cells. In preferred embodiments, capsid amino acids are modified to remove wild type tropism and to introduce a new tropism. The inserted or substituted amino acid may comprise targeting peptides and polypeptides that are ligands and other peptides that bind to cell surface receptors and glycoproteins as well as fragments thereof that retain the ability to target vectors to cells. The targeting peptide or polypeptide may be any type of antibody or antigen-binding fragment thereof that recognizes, e.g., a cell-surface epitope. The binding domain from a toxin can be used to target the AAV vector to particular target cells of interest. It is also contemplated that AAV vectors of the invention may be targeted to a cell using a “nonclassical” import/export signal peptide (e.g., fibroblast growth factor-1 and -2, interleukin 1, HIV-1 Tat protein, herpes virus VP22 protein, and the like).  
           [0017]    Also contemplated as targeting peptides are peptides that direct uptake of the AAV vector by specific cells. For example, a FVFLP peptide (SEQ ID NO: 18) triggers uptake by liver cells. Another peptide contemplated to direct uptake by cancer cells is the RGD peptide, e.g., 4C-RGD. The RGD domain is known to mediate interactions between extracelluar matrix proteins and integrin receptors located on the surface of cancer cells. It is contemplated that the insertion of an RGD peptide into the capsid of the AAV vector will act as a cell entry mechanism specific to cancer cells. The receptor-binding peptide from luteinizing hormone is also contemplated as a peptide which when inserted into the capsid of an AAV vector will direct entry into ovarian cells since ovarian cells express luteinizing hormone receptors.  
           [0018]    Other targeting peptide contemplated influence cellular trafficking of viral particles. Phage display techniques, as well as other techniques known in the art, may be used to identify peptides that recognize, preferably specifically, a cell type of interest. Alternatively, the targeting sequence comprises amino acids that may be used for chemical coupling (e.g., through amino acid side groups of arginine or lysine residues) of the capsid to another molecule that directs entry of the AAV vector into a cell.  
           [0019]    The present invention also encompasses modified AAV vectors, the capsid protein(s) of which are biotinylated in vivo. For example, the invention contemplates AAV capsids engineered to include the biotin acceptor peptide (BAP). Expression of the  E. coli  enzyme biotin protein ligase during AAV vector biosynthesis in the presence of biotin results in biotinylation of the AAV capsid proteins as they are made and assembled into viral particles.  
           [0020]    In order to biotinylate the AAV viral particles, a system for expressing the biotin ligase enzyme in packaging cell lines is contemplated by the present invention. The invention provides for plasmids, such as the pCMV plasmid, which direct expression of the biotin ligase gene within the packaging cell line. For production of the biotinylated AAV vector the following components need to be transfected into a packaging cell: a rAAV vector comprising DNA of interest flanked by AAV inverted terminal repeats, an AAV helper construct containing a capsid gene with a BAP insert and the rep gene, adenovirus helper plasmid or infected with adenovirus, and the biotin ligase gene (BirA). In this system, the biotin ligase gene may be expressed by a plasmid including the BirA gene (such as pCMV-BirA) infection with an adenovirus which expresses the BirA gene or by using a packaging cell line that is stably transfected with the BirA gene.  
           [0021]    It is contemplated that the biotinylated AAV viral particles will serve as substrates for conjugation of targeting motifs (e.g., monoclonal antibodies, growth factors, cytokines) to the surface of vector particles through utilizing avidin/strepavidin-biotin chemistry. In addition, the biotinylated AAV viral particles are contemplated to be useful for visualizing the biodistribution of the viral particles both in vivo and in vitro. The biotinylated viral particles can be visualized with fluorescence or enzymatically with labeled strepavidin compounds. Biotinylation is also useful for conjugating epitope shielding moieties, such as polyethylene glycol, to the AAV vector. The conjugation of shielding moieties allows the vector to evade immune recognition. Biotinylation of the AAV vector is also contemplated to enhance intracellular trafficking of viral particles through conjugation of proteins or peptides such as nuclear transport proteins. Biotinylation may also be used to conjugate proteins or peptides which affect the processing of AAV vector genomes such as increasing the efficiency of integration. In addition, biotinylation may also be used to conjugate proteins or peptides that affect the target cells, e.g., proteins that make a target cell more susceptible to infection or proteins that activate a target cell thereby making it a better target for the expression of a therapeutic or antigenic peptide.  
           [0022]    The present invention also provides compositions comprising an AAV vector of the invention in a pharmaceutically acceptable carrier. The compositions may also comprise other ingredients such as diluents and adjuvants. Acceptable carriers, diluents and adjuvants are nontoxic to recipients and are preferably inert at the dosages and concentrations employed, and include buffers such as phosphate, citrate, or other organic acids; antioxidants such as ascorbic acid; low molecular weight polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and/or nonionic surfactants such as Tween, pluronics or polyethylene glycol (PEG).  
           [0023]    Methods of eliciting an immune response to amino acids of interest are contemplated by the invention. The methods comprise a step of administering an immunogenic dose of a composition comprising a AAV vector of the invention to a animal (including a human person) in need thereof. In the methods, the immunogenic amino acids may be inserted in the AAV vector capsid protein(s) or may be encoded by a recombinant genome encapsidated as the AAV vector. An immunogenic dose of a composition of the invention is one that generates, after administration, a detectable humoral and/or cellular immune response in comparison to the immune response detectable before administration or in comparison to a standard immune response before administration. The invention contemplates that the immune response resulting from the methods may be protective and/or therapeutic.  
           [0024]    Therapeutic methods of delivering and/or transferring nucleic acids of interest to a host cell are also contemplated by the invention. The methods comprise the step of administering a therapeutically effective dose of a composition comprising a AAV vector of the invention to an animal (including a human person) in need thereof. A therapeutically effective dose is a dose sufficient to alleviate (eliminate or reduce) at least one symptom associated with the disease state being treated. Administration of the therapeutically effective dose of the compositions may be by routes standard in the art, for example, parenteral, intravenous, oral, buccal, nasal, pulmonary, rectal, or vaginal.  
           [0025]    Titers of AAV vector to be administered in methods of the invention will vary depending, for example, on the particular virus vector, the mode of administration, the treatment goal, the individual, and the cell type(s) being targeted, and may be determined by methods standard in the art. 
       
    
    
     DETAILED DESCRIPTION  
       [0026]    The present invention is illustrated by the following examples that are not intended to limit the invention. Example I describes construction of AAV packaging plasmids encoding altered capsid proteins and analysis of the ability of the altered capsid proteins to be assembled into infectious AAV vectors. Example 2 presents assays for the surface expression of epitopes inserted in the altered capsid proteins. Example 3 describes experiments testing whether the AAV vectors retained HSPG-binding ability. Example 4 describes construction and characterization of a mutant AAV vector containing a double insertion within the capsid protein. Example 5 includes analysis of the effect of linker and scaffold sequences on the altered capsid proteins. Example 6 presents the results of experiments in which AAV vectors encoding capsid proteins with an insertion of an luteinizing hormone receptor binding peptide were able to transduce OVCAR-3 cells. Example 6 also discusses various indications amenable to use of AAV vectors of the invention. Example 7 and 8 describe fourteen additional modified AAV vectors, wherein the RGD-4C peptide motif was inserted into the capsid proteins. The experiments described in Example 9 demonstrate that the AAV-RGD vectors attach to and enter cells via integrin receptors. Example 10 demonstrates that the AAV-RGD vectors were capable of mediating gene delivery via integrin receptors. Example demonstrates that the AAV-RGD vectors transferred genes to ovarian adenocarcinoma cell lines. Example 12 describes AAV mediated eGFP gene delivery to human ovarian tumor xenografts established in SCID mice. Example 13 describes construction of mutant AAV vectors which are biotinylated in vivo through an insertion of the biotin acceptor peptide in the capsid protein. Finally, Example 14 describes a packaging system for biotinylated AAV vectors.  
       EXAMPLE 1  
       [0027]    In order to identify sites within the AAV2 capsid that could tolerate insertion of targeting epitopes, an extensive site-specific mutagenesis strategy was designed. Regions of the AAV2 capsid DNA to be modified were chosen by analyzing data from a number of sources to predict which ones encoded capsid amino acids that were exposed on the surface of the virion and which encoded amino acids that could be replaced with other amino acids without significantly altering the conformation of the rest of the capsid protein(s). One source of data was a comparison of structural information from five related autonomous parvoviruses. The five parvoviruses had solved virion structures and included canine parvovirus (CPV) (Tsao et al.,  Science,  251: 1456-1464 and Wu et al.,  J. Mol. Biol,  233: 231-244), feline panleukopenia virus (FPV)(Agbandje et al.,  Proteins,  16: 155-171), minute virus of mice (MVM)(Agbandje-McKenna et al.,  Structure,  6: 1369-1381 and Llamas-Saiz et al.,  Acta Crystallogr. Sect. D. Biol. Crystallogr.,  53: 93-102), parvovirus B19 (B19)(Chipman et al.,  Proc. Natl. Acad. Sci. USA,  93: 7502-7506) and Aleutian mink disease parvovirus (ADV)(McKenna et al.,  J. Virol.,  73: 6882-6891). This information was compared to a computer-predicted secondary structure of the AAV2 capsid based on its known primary amino acid sequence. Other sources of data were previous reports of immunogenic regions of the AAV2 capsid and previous reports of effects of random capsid mutations. Finally, the AAV2 capsid primary amino acid sequence was compared with that of other AAV and other parvoviridae for regions of defined secondary structure to create a model of the AAV2 capsid. From the model sites for insertion of small peptides two to fifteen amino acids in length were chosen. A series of thirty-eight virus mutants containing peptide insertions at twenty-five unique sites within the AAV2 capsid protein was generated. Most of the insertions were within the VP1 capsid protein (19/25), four were within the VP1 unique region and two were within the VP1/VP2 unique region. Epitopes inserted within the VP3 protein are expected to be displayed on every capsid monomer within the AAV virion (60/virion). Insertions within the VP1 or VP1/VP2 unique regions would be expected to be displayed three and six times, respectively, per virion.  
         [0028]    Site-directed mutagenesis was performed on plasmid pUC-Cap (a subclone of the AAV2 Rep and Cap open reading frames (ORF)). Mutagenesis was confirmed by restriction endonuclease digestion. The altered Cap genes were then substituted for the wild-type AAV2 sequences in plasmid pACG2 to generate the series of mutant helper plasmids described in Table 1 below, wherein epitope AgeI is the amino acids encoded by an AgeI restriction site, epitope NgoMI is the amino acids encoded by an NgoMI restriction site, epitope 4C-RGD is a cyclic RGD-based peptide (CDCRGDCFC; SEQ ID NO: 10) that has been shown to bind a number of integrins, including α v β 3 , α v β 5 , α 5 β 1 , α 5 β 1 , α 3 β 1 , α 2 β 1  and α 6 β 1 , present on the surface of mammalian cells that is useful for targeting to tumor endothelium and other cell types, epitope BPV is a peptide from bovine papilloma virus (TPPYLK; SEQ ID NO: 16), and epitope LH is a receptor-binding peptide from luteinizing hormone (HCSTCYYHKS; SEQ ID NO: 17). Plasmid nomenclature in the Table  1  can be understood by reference to plasmid pACG-A139 wherein pACG refers to the starting plasmid in which mutant cap sequences were inserted and A139 refers to insertion of an AgeI restriction site after position 139 of the capsid, and by reference to plasmid pACG-A139BPV/GLS wherein BPV indicates the peptide of interest that is inserted and /GLS indicates inclusion of linker amino acids at the carboxy terminus of the inserted epitope.  
                             TABLE 1                           Mutant AAV Packaging Plasmids            Mutant Plasmid               Designation   Location   Insertion (epitope)               pACG-A26   VP1   TG (Age I)       pACG-A46   VP1   TG (Age I)       pACG-A115-4C-   VP1   TGCDCRGDCFCGLS (SEQ ID       RGD/GLS       NO: 1) (4C-RGD)       pACG-A120   VP1   TG (Age I)       pACG-A139   VP2   TG (Age I)       pACG-A139BPV/GLS   VP2   TGTPFYLKGLS               (SEQ ID NO: 2) (BPV)       pACG-A139LH/GLS   VP2   TGHCSTCYYHKSGLS (SEQ ID               NO: 3) (LH)       pACG-A161BPV/ALS   VP2   TGTPFYLKALS (SEQ ID NO: 4)               (BPV)       pACG-A161BPV/LLA   VP2   TGTPFYLKLLA (SEQ ID NO: 5)               (BPV)       pACG-A161BPV/GLS   VP2   TGTPFYLKGLS (SEQ ID NO: 2)               (BPV)       pACG-A161LH/GLS   VP2   TGHCSTCYYHKSGLS (SEQ ID               NO: 3) (LH)       pACG-A312   VP3   TG (Age I)       pACG-N319   VP3   AG (NgoMI)       pACG-A323-4C-   VP3   TGCDCRGDCFCGLS (SEQ ID       RGD/GLS       NO: 1) (4C-RGD)       pACG-A339BPV   VP3   TGTPFYLK (SEQ ID NO: 6)               (BPV       pACG-A375BPV   VP3   TGTPFYLK (SEQ ID NO: 6)               (BPV)       pACG-A441   VP3   TG (Age I)       pACG-A459   VP3   TG (Age I)       pACG-A459BPV/GLS   VP3   TGTPFYLKGLS (SEQ ID NO: 2)               (BPV)       pACG-A459LH/GLS   VP3   TGHCSTCYYHKSGLS (SEQ IS               NO: 3) (LH)       pACG-A466   VP3   TG (Age I)       pACG-A480-4C-   VP3   TGCDCRGDCFCGLS (SEQ ID       RGD/GLS       NO: 1) (4C-RGD)       pACG-N496   VP3   AG (NgoMI)       pACG-A520LH/GLS   VP3   TGHCSTCYYHKSGLS (SEQ ID               NO: 3) (LH)       pACG-A520BPV/LLA   VP3   TGTPFYLKLLA (SEQ ID NO: 5)               (BPV)       pACG-A540   VP3   TG (Age I)       pACG-N549   VP3   AG (NgoMI)       pACG-N584   VP3   AG (NgoMI)       pACG-A584BPV/ALS   VP3   TGTPFYLKALS (SEQ ID NO: 4)               (BPV)       pACG-A584BPV/LLA   VP3   TGTPFYLKLLA (SEQ ID NO: 5)               (BPV)       pACG-A584BPV/GLS   VP3   TGTPFYLKGLS (SEQ ID NO: 2)               (BPV)       pACG-N472   VP3   AG (NgoMI)       pACG-A587BPV/ALS   VP3   TGTPFYLKALS (SEQ ID NO: 4)               (BPV)       pACG-A587BPV/LLA   VP3   TGTPFYLKLLA (SEQ ID NO: 5)               (BPV)       pACG-A587BPV/GLS   VP3   TGTPFYLKGLS (SEQ ID NO: 2)               (BPV)       pACG-A595-4C-   VP3   TGCDCRGDCFCGLS (SEQ ID       RGD/GLS       NO: 1) (4C-RGD)       pACG-A597-4C-   VP3   TGCDCRGDCFCGLS (SEQ ID       RGD/GLS       NO: 1) (4C-RGD)       pACG-A657   VP3   TG (Age I)                  
 
         [0029]    The mutant AAV packaging plasmids were tested for their ability to generate AAV vectors with altered capsids by triple transfection with plasmid pAAV-LacZ (a plasmid containing LacZ flanked by AAV ITRs) and pXX6-80 (a plasmid containing Adenovirus helper DNA) according to established procedures. AAV vector preparations were assessed for particle formation and vector infectivity. Particles were identified by ELISA using A20 monoclonal antibody, whereas DNA-containing particles were identified by dot-blot and/or PCR. Vector particles were tested for infectivity by cellular transduction assay on Adenovirus-infected C12 cells. Capsid mutants were grouped into three types. Capsid mutants that did not give rise to any viral particles were classified as Type I (7/38). Mutants that produced non-infectious particles were classified as Type II ( 11/38) and mutants that produced fully infectious viral particles were classified as Type III (20/38). See Table 2 below wherein the actual titers are listed as values for comparison with the wild type titer unless the titer (−) is four orders of magnitude or more less than wild type vector and a titer (+) is below the sensitivity of DNA dot blot but detectable by PCR.  
                                     TABLE 2                           Mutant AAV Vector Characterization       Particle titer            Mutant Vector           Infections   Mutant       Designation   Dot-blot   A20 ELISA   titer   Type               AAV-A26   (+)   7.5 × 10 7     —   II       AAV-A46   9.2 × 10 7     8.0 × 10 7     1.2 × 10 3     III       AAV-A115-4C-   5.6 × 10 7     7.5 × 10 7     1.2 × 10 2     III       RGD/GLS       AAV-A120   3.4 × 10 7     8.0 × 10 7     1.0 × 10 3     III       AAV-A139   2.0 × 10 7     9.0 × 10 7     5.0 × 10 5     III       AAV-A139BPV/GLS   1.4 × 10 8     9.0 × 10 7     6.8 × 10 5     III       AAV-A139LH/GLS   1.2 × 10 8     8.0 × 10 7     3.3 × 10 5     III       AAV-A161BPV/ALS   4.0 × 10 7     8.0 × 10 7     1.2 × 10 5     III       AAV-A161BPV/LLA   1.4 × 10 6     7.5 × 10 5     5.9 × 10 2     III       AAV-A161BPV/GLS   1.2 × 10 7     7.5 × 10 6     8.7 × 10 4     III       AAV-A161LH/GLS   4.0 × 10 6     8.0 × 10 7     3.4 × 10 4     III       AAV-A312   1.8 × 10 6     —   5.3 × 10 2     III       AAV-N319   2.4 × 10 7     4.5 × 10 5     0.6 × 10 3     III       AAV-A323-4C-   (+)   —   —   I       RGD/GLS       AAV-A339BPV   (+)   —   —   II       AAV-A375BPV   —   —   —   I       AAV-A441   —   —   —   I       AAV-A459   7.2 × 10 6     8.0 × 10 7     6.5 × 10 4     III       AAV-A459BPV/GLS   5.6 × 10 7     4.5 × 10 6     2.2 × 10 5     III       AAV-A459LH/GLS   3.2 × 10 6     4.5 × 10 5     —   II       AAV-A466   (+)   7.5 × 10 7     —   II       AAV-N472   —   —   —   I       AAV-A480-4C-   —   —   —   I       RGD/GLS       AAV-N496   22 × 10 6     —   1.1 × 10 2     III       AAV-A520LH/GLS   (+)   7.5 × 10 7     —   II       AAV-A520BPV/LLA   (+)   7.5 × 10 7     —   II       AAV-N540   (+)   8.0 × 10 7     —   II       AAV-N549   (+)   4.5 × 10 6     —   II       AAV-N584   1.1 × 10 8     8.0 × 10 7     4.0 × 10 5     III       AAV-A584BPV/ALS   3.0 × 10 7     8.0 × 10 7     6.5 × 10 2     III       AAV-A584BPV/LLA   1.3 × 10 7     9.0 × 10 6     —   II       AAV-A584BPV/GLS   (+)   7.5 × 10 5     —   II       AAV-A587BPV/ALS   1.8 × 10 7     8.0 × 10 6     5.0 ×10 1     III       AAV-A587BPV/LLA   7.2 × 10 5     9.0 × 10 5     —   II       AAV-A587BPV/GLS   3.5 × 10 7     9.0 × 10 7     2.7 × 10 2     III       AAV-A595-4C-   —   2.5 × 10 4     —   I       RGD/GLS       AAV-A597-4C-   —   2.5 × 10 4     —   I       RGD/GLS       AAV-A657   1.8 × 10 7     7.5 × 10 7     5.2 × 10 4     III       AAV (wild-type)   4.8 × 10 7     9.0 × 10 7     6.2 × 10 5     N/A                  
 
         [0030]    Of the sites chosen for linker insertion, 20 (80%) tolerated this manipulation as assessed by particle formation. Infectious virus could be produced containing linker insertions at twelve of the sites that were tolerated for viral assembly (12/20; 60%). This represents 48% of the sites originally selected for mutagenesis.  
         [0031]    Although twelve sites within the AAV2 capsid protein(s) could be altered, and the mutant capsid monomers still assemble, package viral genomes, and infect cells, the infectious titers of these viruses varied greatly. These ranged from essentially wild-type levels to greater than four orders of magnitude less infectious than wild-type. Significantly, several sites could tolerate a wide range of genetic insertions without effects on virus titer. Both of the sites within the VP1/VP2 unique region of the capsid ORF proved able to tolerate genetic insertions without a loss in viral titer. See results for mutant vectors with insertions after amino acid positions A139 and A161. However, insertion after position A161 showed some dependence on surrounding sequence elements. See Example 5 below. Within the VP3 region of the capsid ORF, results were more variable. Although many insertions were tolerated with essentially no loss in vector titer (for example, after positions R459 and Q584), there was a greater dependence on linker sequences (compare AAV-N584BPV/ALS to AAV-N584BPV/LLA; also see Example 5, below) and the primary sequence of the epitope being inserted (compare AAV-A459BPV/GLS to AAV-A459LH/GLS).  
       EXAMPLE 2  
       [0032]    The surface accessibility of inserted BPV epitopes in the mutant AAV vectors described in Example 1 was examined by immunoprecipitation.  
         [0033]    Iodixanol grandient-purified vectors were precipitated with anti-BPV monoclonal antibody using protein-G Sepharose, subjected to SDS-PAGE, blotted to nylon membranes and probed with anti-AAV B1 monoclonal antibody. A summary of epitope display for each BPV insertion mutant is shown in Table 3 below.  
                             TABLE 3                           Surface Display of Inserted BPV Epitopes                Mutant Vector Designation   Epitope Display                       AAV-A139BPV/GLS   +           AAV-A161BPV/ALS   +           AAV-A161BPV/LLA   +           AAV-A161BPV/GLS   +           AAV-A339BPV   −           AAV-A459BPV/GLS   +           AAV-A520BPV/LLA   +           AAV-A584BPV/ALS   +           AAV-A584BPV/LLA   +           AAV-A584BPV/GLS   −           AAV-A587BPV/ALS   +           AAV-A587BPV/LLA   −           AAV-A587BPV/GLS   +                      
 
         [0034]    Inserted peptide epitopes could be displayed efficiently on the surface of viral particles at each site tested which were all sites that insertion gave rise to infectious vectors. However, display was often dependent on inclusion of appropriate linker/scaffolding sequences.  
       EXAMPLE 3  
       [0035]    The mutant AAV vectors of Example 1 were also tested for retention of the ability to bind HSPG.  
         [0036]    The ability of the AAV vectors to bind HSPG was assessed by purifying the AAV preparations on an iodixanol gradient. The 40% iodixanol layer was collected and diluted in PBS-MK containing heparin sulfate affinity resin. The mixtures were incubated for two hours with gentle shaking at 4° C. followed by centrifugation. The viral bound resin was washed three times with PBS-MK for ten minutes at room temperature and resuspended in loading buffer. The samples were then boiled and analyzed by Western blotting with monoclonal antibody B1 directed against the AAV2 VP3 capsid protein.. A summary of the HS-binding characteristic for all of the mutant is presented in Table 4 below.  
                             TABLE 4                           HSPG Binding                Mutant Vector Designation   HSPG Binding                       AAV-A26   −           AAV-A46   +           AAV-A115-4C-RGD/GLS   +           AAV-A139   +           AAV-A139BPV/GLS   +           AAV-A139LH/GLS   +           AAV-A161BPV/ALS   +           AAV-A161LH/GLS   +           AAV-A312   −           AAV-A323-4C-RGD/GLS   −           AAV-A375BPV   +           AAV-A459   +           AAV-A459LH/GLS   +           AAV-A466   +           AAV-N472   +           AAV-A480-4C-RGD/GLS   +           AAV-A520LH/GLS   −           AAV-A520BPV/LLA   −           AAV-A540   +           AAV-N549   −           AAV-A584BPV/ALS   +           AAV-A584BPV/LLA   +           AAV-A584BPV/GLS   −           AAV-A587BPV/ALS   +           AAV-A587BPV/LLA   +           AAV-A587BPV/GLS   +           AAV-A595-4C-RGD/GLS   +           AAV-A597-4C-RGD/GLS   +           AAV (wild-type)   +                      
 
         [0037]    Some of the Type II mutants may have been non-infectious because they no longer bound HSPG (see the A26 or A520 mutants). These mutants are valuable because the endogenous tropism of the virus has been ablated and any binding capability added to the virus would be exclusive. In situations in which loss of receptor-binding ability as a result of introducing mutations at a specific capsid site is not desirable, the foregoing data demonstrates that binding can often be rescued by inclusion of appropriate flexible linker sequences.  
       EXAMPLE 4  
       [0038]    A mutant AAV2 vectors containing a peptide insertion at two different sites within the capsid protein was generated using the methods described herein. The 4C-RGD peptide (SEQ ID NO: 10) was inserted using site directed mutagenesis as described in Example I after amino acid position 520 and position 588 within the VP3 capsid protein. The double mutant AAV2 vector (denoted herein as A520RGD4C588RGD4C) was assessed for particle formation and vector infectivity. Particles were identified by ELISA using A20 monoclonal antibody, whereas DNA-containing particles were identified by dot-blot. Vector particles were tested for infectivity by cellular tranduction assay on Adenovirus-infected C12 cells. The double mutant was able to infect cells and produce viral particles at a similar rate as other mutant and wild-type vectors. In Table 5, infectivity is presented as the percentage of target cells expressing the vector-encoded transgene and particle titer is presented as particles/μl.  
                                                 TABLE 5                                   HS   Particle Titer            Capsid   Infectivity   Binding   A20 ELISA   DNA Dot Blot               A520RGD4C   −   −   7.5 × 10 4     −       A588RGD4C   52.1%   +     7 × 10 5     8 × 10 4         A520RGD4C588RGD4C   45.8%   −     2 × 10 5     5 × 10 4         ACG   49.9%   +     1 × 10 6     2 × 10 5                    
 
         [0039]    The ability of the double mutant AAV capsids to bind HSPG was assessed as describe in Example 3. The double mutant was unable to bind to HSPG like the A520RGD4C vector, but retained the ability to infect the target cells similar to A5884RGD4C. See Table 9 above. Thus, the double mutant, A520RGD4C588RGD4C, is a receptor-targeted mutant that was produced at a reasonable titer and is defective in binding the AAV2 endogenous receptor HSPG.  
       EXAMPLE 5  
       [0040]    It was envisioned that insertion of larger peptide epitopes might disrupt the AAV capsid by conformationally straining neighboring sequences. To circumvent this problem, two different approaches were employed in generating various mutant AAV packaging plasmids described in Example 1. First, in some altered capsids the structure of neighboring capsid regions was maintained by the introduction of a disulfide bond, and second, in other altered capsids flexible linker sequences were included to minimize conformational stress. See Table 6 below, wherein linker sequence TG-ALS indicates that linker amino acids TG were included at the amino terminus of the inserted epitope and amino acids ALS were included at the carboxy terminus of the inserted epitope.  
                                             TABLE 6                           Dependence on Appropriate Linker/Scaffolding Sequences            Mutant Vector   Linker   Particle   Infectious   HSPG   Epitope           Designation   Sequence   Titer   Titer   Binding   Display   Type               AAV-   TG-ALS   ++++   ++++   +   +   III       A161BPV/ALS   (SEQ ID           NO: 7)       AAV-   TG-LLA   ++   ++   +   +   III       A161BPV/LLA   (SEQ ID           NO: 8)       AAV-   TG-GLS   +++   ++++   +   +   III       A161BPV/GLS   (SEQ ID           NO: 9)       AAV-   TG-ALS   ++++   ++++   +   +   III       N584BPV/ALS   (SEQ ID           NO: 7)       AAV-   TG-LLA   +++   −   +   +   II       N584BPV/LLA   (SEQ ID           NO: 8)       AAV-   TG-GLS   +   −   −   −   II       N584BPV/GLS   (SEQ ID           NO: 9)       AAV-   TG-ALS   +++   +++   +   +   III       A587BPV/ALS   (SEQ ID           NO: 7)       AAV-   TG-LLA   ++   −   +   −   II       A587BPV/LLA   (SEQ ID           NO: 8)       AAV-   TG-GLS   ++++   ++   +   +   III       A587BPV/GLS   (SEQ ID           NO: 9)                  
 
         [0041]    Through the choice of appropriate linkers, infectious virus was rescued from previously dead mutants. In other instances, titers were influenced over several orders of magnitude. From this analysis it is clear that incorporation of flexible linkers containing small uncharged amino acids (such as alanine or serine) is extremely important for rescuing virus structure, infectivity, and for efficient epitope display.  
       EXAMPLE 6  
       [0042]    The ability of vector AAV-A139LH (containing the LH receptor binding peptide) to target the human ovarian cancer cell line OVCAR-3 was tested. Expression of the LH receptor is upregulated on these cells. Because OVCAR-3 cells also express HSPG control experiments were performed to demonstrate that the AAV vector indeed exhibited an altered tropism.  
         [0043]    Briefly, equal numbers of AAV-A139LH vector particles or vector particles with BPV inserts instead of LH inserts were applied to the surface of OVCAR-3 cells for 2 hours at 4° C. HeLa cells which express HSPG but not the LH receptor were used as a control cell line. Experiments were performed either in the presence or absence of 500 μg/ml soluble heparin sulfate (HS) which competes with binding between AAV and HSPG and in the presence or absence of progesterone which increases expression of the LH receptor. The cells were then washed of unbound vector, shifted to 37° C. and maintained for 48 hours at which time gene transfer was assessed.  
         [0044]    In the experiments, AAV-A139LH transduced both HeLa and OVCAR-3 cells in the absence of HS. In the presence of HS, transduction of OVCAR-3 cells was reduced more than 10-fold and transduction of Hela cells was reduced more than 100-fold. Addition of progesterone restored transduction of ovarian cells that was lost in the presence of HS. The addition of progesterone increased transduction of OVCAR-3 cells by AAV-A139LH but not by AAV-A139BPV.  
         [0045]    These results demonstrate that AAV-A139LH has acquired tropism for cells expressing the LH receptor.  
         [0046]    As demonstrated by the foregoing data, AAV vectors of the invention may therefore be used for targeted DNA delivery. Some indications include: cancer gene therapy (e.g., for toxin or “suicide” gene delivery) and therapeutic gene transfer to cell and/or tissue types that have been refractive to gene transfer with conventional AAV vectors (e.g., airway epithelium for the treatment of cystic fibrosis, glia for the treatment of primary brain cancers, and hematopoietic progenitors cells for the treatment of any number of other disorders). For therapeutic gene delivery, AAV vectors of the invention may be targeted to non-antigen presenting cells in order to avoid an immune response to a gene or protein of interest and/or may incorporate epitope shielding moieties and/or mutations of immunodominant epitopes.  
         [0047]    Alternatively, AAV vectors may be used as vaccines. Viral particles containing foreign epitopes may be used directly as immunogns. AAV vectors displaying such epitopes may also contain DNA that would lead to the expression of the same or related sequences within target cells. Such a dual immunization approach is contemplated to generate a more robust and wider range response. For vaccine use, targeted AAV vectors may specifically transduce APC (while avoiding other cells).  
         [0048]    Finally, AAV vectors of the invention may be used as non-therapeutic reagents such as imaging reagents for the determination of vector pharmokinetics and biodistribution, for example, through the attachment of radio tracer elements and real-time scintography.  
       EXAMPLE 7  
       [0049]    Fourteen additional AAV capsid mutants were generated in the non-infectious AAV plasmid, pACG, by PCR-based site-directed mutagenesis as described in Example 1. In all thirteen, the 4C-RGD peptide (CDCRGDCFC; SEQ ID NO: 10) was inserted into the AAV capsid monomer.  
         [0050]    4C-RGD encoding oligonucleotide were inserted into seven different sites within the AAV capsid gene. One site was within the VP1 unique region of the AAV2 capsid protein gene, three were within the VP1/VP2 unique region, and the three remaining sites were located within the VP3 region of the capsid ORF. DNA encoding the 4C-RGD peptide epitope was either inserted alone or flanked by one of two different five amino acid connecting peptide linkers, as described in Example 5. See Table 7 below. Producer cell lines based on 293 cells were used to generate modified AAV vectors comprising the altered capsids. These modified vectors are denoted as “AAV-RGD” collectively herein.  
                               TABLE 7                               Inserted                       Peptide       Particle       Vector   Upstream   (SEQ ID NO:   Downstream   Titer       Designation   Linker   10)   Linker   (ELISA)                   A46-RGD4C   TG   CDCRGDCFC   —   8.5 × 10 7         A46-RGD4CGLS   TG   CDCRGDCFC   GLS   4.5 × 10 6         A115-RGD4C   TG   CDCRGDCFC   —   4.5 × 10 6         A115-   TG   CDCRGDCFC   GLS   6.0 × 10 7         RGD4CGLS       A139-RGD4C   TG   CDCRGDCFC   —   8.5 × 10 7         A139-   TG   CDCRGDCFC   GLS   9.0 × 10 7         RGD4CGLS       A161-RGD4C   TG   CDCRGDCFC   —   4.5 × 10 6         A161-   TG   CDCRGDCFC   ALS   5.0 × 10 6         RGD4CALS       A459-RGD4C   TG   CDCRGDCFC   —   4.5 × 10 6         A459-   TG   CDCRGDCFC   GLS   4.5 × 10 6         RGD4CGLS       A584-RGD4C   TG   CDCRGDCFC   —   8.5 × 10 7         A584-   TG   CDCRGDCFC   ALS   9.0 × 10 7         RGD4CALS       A588-RGD4C   TG   CDCRGDCFC   —   9.0 × 10 7         A588-   TG   CDCRGDCFC   GLS   9.0 × 10 7         RGD4CGLS       Wild-type   —   —   —   7.5 × 10 7                    
 
         [0051]    All the mutant capsid proteins were efficiently assembled and packaged. Furthermore, all of the modified AAV vectors generated were infectious, although there were significant differences in their efficiency of mediating gene transduction. See Table 8 below.  
                                             TABLE 8                                       Percent eGFP Positive Cells                            rAVVeGFP                   (+500 μg/ml           Capsid   rAVVeGFP (alone)   Heparin Sulfate)                       A46-RGD4C    2.5%     1%           A46-RGD4CGLS    3.%    0.5%           A115-RGD4C     5%     1%           A115-RGD4CGLS    7.5%     1%           A139-RGD4C     35%    2.5%           A139-RGD4CGLS     40%     2%           A161-RGD4C     4%    0.5%           A161-RGD4CALS     5%     1%           A459-RGD4C    3.5%     1%           A459-RGD4CGLS     3%   0.25%           A584-RGD4C     49%     30%           A584-RGD4CALS     51%     37%           A588-RGD4C     40%     32%           A588-RGD4CGLS     46%     38%           Wild-type   47.5%     1%                      
 
         [0052]    The differences in gene transduction among the AAV-RGD vectors were related to both the site of peptide insertion and the presence, or absence, of linker sequences flanking the inserted 4C-RGD peptide. Insertion of the RGD epitope following AAV VP1 amino acids at positions 46, 115, 161 or 459 severely diminished infectious titer. However, insertions following the AAV amino acids at positions 139, 584 and 588 were well tolerated and did not affect titer appreciably.  
         [0053]    For all the AAV-RGD vectors, inclusion of linker/scaffolding sequences resulted in slightly more efficient infection and maintenance of titer. To determine if the inserted 4C-RGD peptide had imparted to the modified vectors HSPG-independence, gene transduction assays were performed in the presence of heparin sulfate as described in Example 5. Although, AAV vectors containing unmodified capsids were unable to transduce cells in the presence of heparin sulfate, AAV-RGD vectors containing the 4C-RGD epitope following amino acids 584 and 588 transduced all types of cells tested in the presence of heparin sulfate. These results strongly suggest that AAV-RGD vectors set out in Table 6 are infecting cells via a HSPG-independent mechanism..  
       EXAMPLE 8  
       [0054]    To assess if the AAV-RGD viral particles bind integrin receptors, a solid-phase ELISA assay using purified α v β 3  integrin was carried out as follows.  
         [0055]    Neutravidin-coated plates (Pierce, Rockford, Ill.) were incubated with 1 μg/well of biotinylated heparin in PBST (0.05% Tween 20, 0.2% BSA) overnight at 4° C. The wells were then washed five times with wash buffer (PBS containing 0.05% Tween-20 and 0.1 % BSA) and AAV particles were bound at room temperature for two hours with gentle shaking. Subsequently, the plate was washed five times with wash buffer and purified integrin α v β 3  (Chemicon, Temecula, Calif.) in binding buffer (20 mM Tris-HCl , 150 mM NaCl , 2 mM CaCl 2 . 1 mM MgCl 2 . 1 mM MnCl 2  and 0.1% BSA, pH 7.5) was added to each well at a concentration of 1 μg/ml. The plates were incubated overnight at 4° C, washed three times with wash buffer and incubated with VNR139 monoclonal antibody (anti-α v  subunit, GIBCO-BRL; Gaithersburg, Md.) in binding buffer for 2 hours at room temperature. The plates are then washed five times and incubated with secondary antibody (HRP-conjugated anti-mouse IgG) for 1 hour at room temperature. Following a final wash the ELISA plate was developed with ABTS substrate solution and the VECTASTAIN kit (Vector Laboratories, Burlingame, Calif.) as recommended by the manufacturer. Color development was stopped by the addition of 1N H 2 SO 4 , and plates were read in a plate reader set at 405 nM.  
         [0056]    This analysis clearly indicated that the AAV-RGD viral particles bound α v β 3  integrin. The unmodified viral particles bound only at background level at all concentrations tested.  
       EXAMPLE 9  
       [0057]    The insertion of the RGD peptide in the capsid protein of AAV-RGD vectors modified the cellular tropism of these vectors. The cell entry pathway of the AAV RGD vectors was investigated by measuring gene transfer to cell lines expressing various levels of HSPG as well as intergrins α v β 3  and α v β 5 . The following cell lines were tested: Hela cells, K562 human chronic myelogenous leukemia cells and Raji human lymphoblast-like cells.  
         [0058]    First, flow cytometry was used to analyze the integrin and HSPG expression profile of these cell lines. Briefly, the cells were resuspended in SM buffer (HEPES-buffered saline containing 1% bovine serum albumin) at 2×10 6  cell/ml. The cells were incubated briefly at 37° C. to allow regeneration of surface integrins, then incubated with FITC-labeled LM609 antibody or FITC-labeled PIF6 antibody (1:200 dilution, Chemicon, Temeula, Calif.) for two hours at 4° C. HSPG expression in these cells was analyzed with anti-HSPG monoclonal antibody, HepSS-1 (1:200 dilution) for two hours at 4° C. Subsequently the cells were washed five times with SM buffer and incubated with FITC labeled goat anti-mouse IgM serum (1:800 dilution) for one hour at 4° C, the cells were washed with SM buffer and analyzed by flow cytometry. This analysis demonstrated that Hela cells expressed high levels of HSPG and α v β 5  integrin and low levels of α v β 3  integrin. K562 cells expressed low levels of HSPG, but α v β 5  integrin was expressed at high levels. Raji cells were negative for HSPG expression and expressed high levels of α v β 3  and α v β 5  integrins. Subsequently, the ability of the wild-type AAV-eGFP and the modified vectors (A584-RGD4C-eGFP, A584-RGD4CALS-eGFP, A588-RGD4C-eGFP, A588-RGD4CGLS) to transfer the eGFP gene to Hela, Raji and K562 cells was analyzed. The cells were seeded in a 24-well plates the day prior to infection in order to reach 75% confluence or about 5×10 5  cell/ml on the following day. Serial dilutions of the vectors were added to the cells in the presence of Ad5 at the MOI of 3iu/cell. The cells and viruses were incubated at 37° C. for 48 hours, after which the media was removed and the cells washed two time with PBS. The cells were then fixed and analyzed for GFP transduction by FACS analysis using an anti-GFP antibody.  
         [0059]    Due to the low expression of HSPG, K562 and Raji cells were poorly transduced by AAVeGFP vectors containing unmodified AAV capsid protein, but these cells were efficiently transduced by the same vector packaged into A5884C-RGD capsids. The efficiency of eGFP gene transduction by the A5884C-RGD vector was similar to that observed by the unmodified AAV vector in Hela cells. Furthermore, gene transfer mediated by the RGD-containing particles was 4-fold higher in the K562 cells and 13-fold higher in the Raji cells as compared to transduction by vectors comprising unmodified capsids. These experiments clearly demonstrate that incorporation of the 4C-RGD epitope into the VP3 monomer of AAV2 vectors resulted in dramatic changes in the initial steps of virus-cell interaction, presumably by creating an alternative cell attachment and entry pathway.  
         [0060]    Experiments were also carried out to compare the binding profiles of the wild type AAV2 vector and that containing the 4C-RGD capsid protein using soluble heparin sulfate to compete for binding, and anti-AAV monoclonal antibody A20 and FACS analysis to detect binding. In these experiments, wild type AAV2 vector did not bind to Hela cells in the presence of heparin sulfate. However, vectors containing A5884C-RGD capsid protein bound to Hela cells in the presence of soluble heparin sulfate. Binding of modified AAV viral particles to Hela cells was blocked by treatment with synthetic RGD peptide. Since the RGD peptides could efficiently block binding, these data further suggest that AAV-RGD capsids use cellular integrins as receptors during the cell attachment process.  
       EXAMPLE 10  
       [0061]    Experiments were carried out to determine if the AAV-RGD vectors were capable of mediating gene delivery via integrin receptors.  
         [0062]    Competitive inhibition assays using soluble heparin sulfate to inhibit AAV-mediated gene delivery were carried out as follows. AAV-RGD vectors or control vector AAVeGFP and modified vectors A584-RGD4C-eGFP, A584-RGD4CALS-eGFP, A588-RGD4C-eGFP, A588-RGD4CGLS were first incubated with 1500 μg/ml soluble heparin sulfate for two hours at 37° C. and then incubated with the Hela cells at 4° C. in the presence of 500 μg/ml heparin sulfate for an additional four hours. The cells were subsequently washed three times with fresh medium to remove unbound vector and incubated for 48 hours at 37° C., after which the cells were washed two times with PBS, fixed and analyzed for GFP gene transduction by FACS analysis in Hela cells.  
         [0063]    When infected with the control virus, AAVeGFP comprising the unmodified capsid, GFP gene expression in Hela cells was efficiently blocked by soluble heparin sulfate. The same concentrations of heparin sulfate only blocked about 20% of A5884C-RGD capsid-mediated GFP expression in Hela cells. These experiments further demonstrated that the A5884-RGD capsids were capable of using an alternative HSPG-independent cell entry pathway.  
         [0064]    To assess the specificity of the alternate cell entry pathway through integrin receptor, synthetic RGD peptide (200 μg/ml) or anti-integrin antibody VNR139 was used to determine if AAV-RGD mediated gene-transduction was inhibited in the presence of soluble heparin sulfate. The addition of the RGD specific inhibitor in combination with heparin sulfate completely inhibited A5884C-RGD-mediated gene expression. This experiment demonstrated that the HSPG-independent interaction was due to interaction with RGD-binding integrins expressed on the Hela cells.  
       EXAMPLE 11  
       [0065]    The ability of unmodified AAV vector (wild type) to mediate GFP gene transductlon was tested in various ovarian adenocarcinoma cell lines. Transduction of the eGFP gene was measured by FACS. Unmodified AAV vector mediated gene transfer and expression in the human ovarian adenocarcinoma cell lines PA-1, OVCAR-3, OVCAR-3N and OV4. Unmodified AAV vector did not transduce the ovarian adenocarcinoma cell lines Hey, SKOV-3 and OV3. The unmodified AAV vector transfers the eGFP gene via the HSPG receptor. HSPG expression in ovarian cancer cells was determined by FACS analysis using an anti-HSGP antibody (Seikagaku America, Falmouth, Mass.). The unmodified AAV vector was unable to transduce the Hey and OV3 cell line since these cell lines were negative for HSPG expression. See Table 8 .  
         [0066]    Since some human ovarian adenocarcinoma cell lines do not express HSPG, it was of interest to determine if ovarian tumor antigens (e.g., integrin) would facilitate AAV-mediated gene transfer in ovarian cancer cells. Integrin expression was analyzed by FACS analysis using an anti-α v  antibody and the data is displayed in Table 9. All ovarian cancer cells tested expressed a member of the α v  integrin family.  
                                 TABLE 9                           Integrin and HSPG Expression on Human Ovarian Adenocarcinoma                Ovarian                   Adenocarcinoma   HSPG Expression   α, Integrin Expression                       PA-1   +   +           Hey   −   +           OVCAR-3   +   +           OVCAR-3N   +   +           OV4   +   +           SKOV-3ip   −   +           OV3   −   +                      
 
         [0067]    The AAV-RGD vectors A588-RGD4C-eGFP and A588-RGD4CGLS were tested for their ability to target gene transfer to the ovarian cell lines as described in Example 9. These AAV-RGD vectors were able to transduce all ovarian cancer cell lines tested. The AAV-RGD vectors were able to more efficiently direct gene transfer in the ovarian cell lines PA-1, Hey, OVCAR-3, OVCAR-3N, OV4, SKOV-3ip and OV3 in comparison compared to wild-type AAV vector containing unmodified capsid.  
         [0068]    AAV-RGD mediated gene transfer was demonstrated to be independent of HSPG interaction. Competitive gene transfer experiments in the OVCAR-3 cell line were carried out with soluble heparin sulfate as described in Example 10. A5884C-RGD vector efficiently directed gene transfer in the presence of soluble heparin sulfate in OVCAR-3 cells. However, gene transfer was completely blocked by the addition of RGD peptide or anti-integrin antibody in the presence of soluble heparin sulfate. The A5884C-RGD mediated gene transfer proceeded through integrin receptors.  
       EXAMPLE 12  
       [0069]    Side-by-side comparison of the effectiveness of the unmodi fied AAV2 vector and the RGD-AAV vector for gene transfer to ovarian tumors was carried out in vivo. Human SKOV-3 cells were delivered intraperitoneally into SKID mice and developed tumors in the peritoneal cavity five days after implantation. The tumors were allowed to develop for five-seven days. Subsequently, matched doses of AAV-RGD vector or unmodified AAV vectors engineered to express the eGFP gene were administered intraperitoneally to the mice at 5×10 8  particles/mouse. At 15, 25, and 35 days post vector administration, the mice were sacrificed and the tumors were analyzed for the extent of gene delivery and expression. eGFP expression was detected in paraffin sections of tumor tissue using an anti-GFP antibody. In Table 10, GFP gene expression is indicated as a percent of tumor tissue expressing the gene, AAV-RGD indicates tumor tissue harvested from mice treated with AAV-RGD vector and ACG indicates tumor tissue harvested from mice treated with wild type vector.  
                                         TABLE 10                                       GFP Expression                Day   AAV-RGD   ACG               15   15%   3%       25   60%   7%       35   95%   7%                  
 
         [0070]    It is generally accepted that for an anti-tumor gene therapy to be effective a genetic vector must be able to deliver and express a gene in as much of the tumor as possible. In studies with other transgenes, (e.g., HSV-TK) it has been established that at least 10-15% of the tumor needs to be transduced in order to be effective. This experiment suggest that the unmodified AAV2-vectors would not be effective anti-tumor agents since the transduction rate in vivo was low. In contrast, the modified RGD-AAV vector had a high rate of gene transduction and therefore may an excellent candidate for anti-tumor therapy. The fact that the eGFP expression comes on slowly (increasing over a 5 week period) is not unexpected and is a characteristic of rAAV.  
       EXAMPLE 13  
       [0071]    In addition to inserting peptide ligands into the AAV2 vector to modify viral tropism, peptide insertions in the AAV2 vector can also be used as substrates for an enzymatic reaction covalently linking a biotin molecule in a site-specific manner to the AAV capsid. AAV capsids have been engineered to include a unique fifteen amino acid long biotin acceptor (BAP) peptide that is recognized by an  E. coli  enzyme, biotin protein ligase. In the presence of ATP, the ligase specifically attaches biotin to the lysine residue in this sequence. When the bacterial enzyme was expressed in a packaging cell line where AAV vector biosynthesis was occurring, vector capsid proteins were biotinylated as they were made and assembled into viral particles. The result of such a packaging scheme was in vivo biotinylated AAV particles. The advantages to labeling the AAV vector by biotinylation is that the reaction is enzymatic and therefore the conditions are gentle and the labeling is highly specific.  
         [0072]    The AAV-BAP vectors were generated by methods similar to those described for the AAV-BPV, AAV-LH and AAV-RGD vectors in Example 1. Six AAV-mutants were generated and the packaging plasmids encoding these mutants are designated herein as pAB139BAP/ALS, pAB139BAP/GLS, pAB161BAP/ALS, pAB161BAP/GLS, pAB584BAP/GLS, and pAB584BAP/ALS. These mutants contain BAP insertions of the peptide sequence (GLNDIFEAQKIEWHE; SEQ ID NO: 11) flanked by either TG-ALS, or TG-GLS linker sequence (SEQ ID NO: 7 and 9, respectively). BAP insertions within the AAV vector following amino acids at positions 139 and 161 (regardless of the linker sequence) produced infectious mutant AAV vector particles at a level similar to wild-type. Insertion of the BAP peptide following amino acid 584 with the GLS linker causes a slight, but insignificant (less than 10-fold), decease in particle titer. Insertion of the BAP peptide at the same site within the AAV vector with the ALS linker caused a significant (&gt;10,00 fold) decrease particle titer. All of the insertion sites within the AAV vector contemplated by the present invention (positions 139 and 161 in the VP1/VP2 region and positions 459, 584, 588 and 657) are candidate sites for the BAP insertion.  
       EXAMPLE 14  
       [0073]    In order to label the AAV particles containing the BAP insert with biotin, a system for expressing the biotin ligase (BirA) enzyme in a packaging cell line was developed to create an in vivo biotinylated AAV vector. The BirA gene was inserted into the pCMV plasmid and is designated herein as pCMV-BirA. This plasmid was used to direct BirA gene expression in 283 cells and used with the AAV-BAP vector to produce in vivo biotinylated AAV vector. Briefly, 293 cells were transfected with the pCMV-BirA plasmid with a selectable maker gene (Neo). The resulting packaging cell was stably transfected with a rAAV comprising a DNA of interest flanked by AAV inverted terminal repeats, an AAV helper construct containing cap gene with a mutant BAP insertion (Example 12), an adenovirus helper plasmid or infected with adenovirus. Alternatively, 293 cells (which are standard AAV vector packaging cells) stably transfected with pCMV-BirA may be used as the packaging cell line. In addition, 293 cells infected with the adenovirus engineered to express the BirA gene may be used as the packaging cell line. AAV particles containing capsids with BAP insertions can also be labeled in vitro (post-purification) using purified BirA enzyme (available commercially).  
         [0074]    Alternatively, a recombinant replication-competent adenovirus that expresses BirA was also developed for biotinylated AAV vector synthesis, eliminating the need for a separate BirA expression plasmid. This system allowed for large-scale AAV vector production of the biotinylated AAV utilizing packaging cell lines that have integrated copies of both AAV vector and AAV helper sequences. The Ad-based BirA expression system also was able to drive the expression of much larger amounts of the BirA gene product. The adenovirus expressed a BirA-eGFP fusion protein from a CMV promoter in the Ad E3 region, which allowed for monitoring BirA expression via GFP fluorescence.  
         [0075]    A sensitive ELISA assay was used to quantitate the extent and efficiency of in vivo (and/or in vitro) biotinylation. AAV containing the 584BAP/GLS insertion was shown to be efficiently biotinylated in vivo (and in vitro) using either the plasmid based or Ad-based BirA expression systems. The biotinylated AAV vectors when conjugated to biotinylated ligands (e.g., monoclonal antibodies) via strepavidin can be specifically targeted to cell surface receptors of interest.  
         [0076]    The advantages of using the biotinylation reaction to label the AAV viral particles is that it is an enzymatic reaction and therefore the conditions are gentle while the labeling is highly specific. In addition, the in vivo biotinylation reaction described herein has a much higher biotinylation efficiency than chemical biotinylation utilizing cross-linking reagents.  
         [0077]    The biotinylated AAV viral particles are contemplated to serve as substrates for conjugation of targeting motifs(e.g., monoclonal antibodies, growth factors, cytokines) to the surface of vector particles through utilizing avidin/strepavidin-biotin chemistry. In addition, the biotinylated AAV viral particles are contemplated to be useful for visualizing the biodistribution of the viral particles both in vivo and in vitro. The biotinylated viral particles can be visualized with fluorescence or enzymatically with labeled strepavidin compounds. Biotinylation may also be useful for conjugating epitope shielding moieties, such as polyethylene glycol, to the AAV vector. The conjugation of shielding moieties will allow the vector to evade immune recognition. Biotinylation of the AAV vector is also contemplated to enhance intracellular trafficking of viral particles through conjugation of proteins or peptides such as nuclear transport proteins. Biotinylation may also be use to conjugate proteins or peptides which effect the processing of AAV vector genomes such as increasing the efficiency of integration. In addition, biotinylation may also be used to conjugate proteins or peptides that effect the target cells, e.g., proteins that make a target cell more susceptible to infection or proteins that activate a target cell thereby making it a better target for the expression of a therapeutic or antigenic peptide.  
         [0078]    While the present invention has been described in terms of preferred embodiments, it understood that variations and improvements will occur to those skilled in the art. Therefore, only such limitations as appear in the claims should be placed on the invention.  
     
       
       
         1 
         
           
             18  
           
           
             1  
             14  
             PRT  
             Artificial Sequence  
             
               RGD Peptide  
             
           
            1 

Thr Gly Cys Asp Cys Arg Gly Asp Cys Phe Cys Gly Leu Ser 
1               5                   10 

 
           
             2  
             11  
             PRT  
             Artificial Sequence  
             
               Bovine Papilloma Virus Peptide  
             
           
            2 

Thr Gly Thr Pro Phe Tyr Leu Lys Gly Leu Ser 
1               5                   10 

 
           
             3  
             15  
             PRT  
             Artificial Sequence  
             
               Luteinizing hormone Peptide  
             
           
            3 

Thr Gly His Cys Ser Thr Cys Tyr Tyr His Lys Ser Gly Leu Ser 
1               5                   10                  15 

 
           
             4  
             11  
             PRT  
             Artificial Sequence  
             
               Bovine Papilloma Virus Peptide  
             
           
            4 

Thr Gly Thr Pro Phe Tyr Leu Lys Ala Leu Ser 
1               5                   10 

 
           
             5  
             11  
             PRT  
             Artificial Sequence  
             
               Bovine Papilloma Virus Peptide  
             
           
            5 

Thr Gly Thr Pro Phe Tyr Leu Lys Leu Leu Ala 
1               5                   10 

 
           
             6  
             8  
             PRT  
             Artificial Sequence  
             
               Bovine Papilloma Virus Peptide  
             
           
            6 

Thr Gly Thr Pro Phe Tyr Leu Lys 
1               5 

 
           
             7  
             5  
             PRT  
             Artificial Sequence  
             
               Synthetic Linker Peptide  
             
           
            7 

Thr Gly Ala Leu Ser 
1               5 

 
           
             8  
             5  
             PRT  
             Artificial Sequence  
             
               Synthetic Linker Peptide  
             
           
            8 

Thr Gly Leu Leu Ala 
1               5 

 
           
             9  
             5  
             PRT  
             Artificial Sequence  
             
               Synthetic Linker Peptide  
             
           
            9 

Thr Gly Gly Leu Ser 
1               5 

 
           
             10  
             9  
             PRT  
             Artificial Sequence  
             
               4C-RGD Peptide  
             
           
            10 

Cys Asp Cys Arg Gly Asp Cys Phe Cys 
1               5 

 
           
             11  
             15  
             PRT  
             Artificial Sequence  
             
               Biotin acceptor peptide  
             
           
            11 

Gly Leu Asn Asp Ile Phe Glu Ala Gln Lys Ile Glu Trp His Glu 
1               5                   10                  15 

 
           
             12  
             4679  
             DNA  
             adeno-associated virus 2  
           
            12 

ttggccactc cctctctgcg cgctcgctcg ctcactgagg ccgggcgacc aaaggtcgcc     60 

cgacgcccgg gctttgcccg ggcggcctca gtgagcgagc gagcgcgcag agagggagtg    120 

gccaactcca tcactagggg ttcctggagg ggtggagtcg tgacgtgaat tacgtcatag    180 

ggttagggag gtcctgtatt agaggtcacg tgagtgtttt gcgacatttt gcgacaccat    240 

gtggtcacgc tgggtattta agcccgagtg agcacgcagg gtctccattt tgaagcggga    300 

ggtttgaacg cgcagccgcc atgccggggt tttacgagat tgtgattaag gtccccagcg    360 

accttgacga gcatctgccc ggcatttctg acagctttgt gaactgggtg gccgagaagg    420 

aatgggagtt gccgccagat tctgacatgg atctgaatct gattgagcag gcacccctga    480 

ccgtggccga gaagctgcag cgcgactttc tgacggaatg gcgccgtgtg agtaaggccc    540 

cggaggccct tttctttgtg caatttgaga agggagagag ctacttccac atgcacgtgc    600 

tcgtggaaac caccggggtg aaatccatgg ttttgggacg tttcctgagt cagattcgcg    660 

aaaaactgat tcagagaatt taccgcggga tcgagccgac tttgccaaac tggttcgcgg    720 

tcacaaagac cagaaatggc gccggaggcg ggaacaaggt ggtggatgag tgctacatcc    780 

ccaattactt gctccccaaa acccagcctg agctccagtg ggcgtggact aatatggaac    840 

agtatttaag cgcctgtttg aatctcacgg agcgtaaacg gttggtggcg cagcatctga    900 

cgcacgtgtc gcagacgcag gagcagaaca aagagaatca gaatcccaat tctgatgcgc    960 

cggtgatcag atcaaaaact tcagccaggt acatggagct ggtcgggtgg ctcgtggaca   1020 

aggggattac ctcggagaag cagtggatcc aggaggacca ggcctcatac atctccttca   1080 

atgcggcctc caactcgcgg tcccaaatca aggctgcctt ggacaatgcg ggaaagatta   1140 

tgagcctgac taaaaccgcc cccgactacc tggtgggcca gcagcccgtg gaggacattt   1200 

ccagcaatcg gatttataaa attttggaac taaacgggta cgatccccaa tatgcggctt   1260 

ccgtctttct gggatgggcc acgaaaaagt tcggcaagag gaacaccatc tggctgtttg   1320 

ggcctgcaac taccgggaag accaacatcg cggaggccat agcccacact gtgcccttct   1380 

acgggtgcgt aaactggacc aatgagaact ttcccttcaa cgactgtgtc gacaagatgg   1440 

tgatctggtg ggaggagggg aagatgaccg ccaaggtcgt ggagtcggcc aaagccattc   1500 

tcggaggaag caaggtgcgc gtggaccaga aatgcaagtc ctcggcccag atagacccga   1560 

ctcccgtgat cgtcacctcc aacaccaaca tgtgcgccgt gattgacggg aactcaacga   1620 

ccttcgaaca ccagcagccg ttgcaagacc ggatgttcaa atttgaactc acccgccgtc   1680 

tggatcatga ctttgggaag gtcaccaagc aggaagtcaa agactttttc cggtgggcaa   1740 

aggatcacgt ggttgaggtg gagcatgaat tctacgtcaa aaagggtgga gccaagaaaa   1800 

gacccgcccc cagtgacgca gatataagtg agcccaaacg ggtgcgcgag tcagttgcgc   1860 

agccatcgac gtcagacgcg gaagcttcga tcaactacgc agacaggtac caaaacaaat   1920 

gttctcgtca cgtgggcatg aatctgatgc tgtttccctg cagacaatgc gagagaatga   1980 

atcagaattc aaatatctgc ttcactcacg gacagaaaga ctgtttagag tgctttcccg   2040 

tgtcagaatc tcaacccgtt tctgtcgtca aaaaggcgta tcagaaactg tgctacattc   2100 

atcatatcat gggaaaggtg ccagacgctt gcactgcctg cgatctggtc aatgtggatt   2160 

tggatgactg catctttgaa caataaatga tttaaatcag gtatggctgc cgatggttat   2220 

cttccagatt ggctcgagga cactctctct gaaggaataa gacagtggtg gaagctcaaa   2280 

cctggcccac caccaccaaa gcccgcagag cggcataagg acgacagcag gggtcttgtg   2340 

cttcctgggt acaagtacct cggacccttc aacggactcg acaagggaga gccggtcaac   2400 

gaggcagacg ccgcggccct cgagcacgac aaagcctacg accggcagct cgacagcgga   2460 

gacaacccgt acctcaagta caaccacgcc gacgcggagt ttcaggagcg ccttaaagaa   2520 

gatacgtctt ttgggggcaa cctcggacga gcagtcttcc aggcgaaaaa gagggttctt   2580 

gaacctctgg gcctggttga ggaacctgtt aagacggctc cgggaaaaaa gaggccggta   2640 

gagcactctc ctgtggagcc agactcctcc tcgggaaccg gaaaggcggg ccagcagcct   2700 

gcaagaaaaa gattgaattt tggtcagact ggagacgcag actcagtacc tgacccccag   2760 

cctctcggac agccaccagc agccccctct ggtctgggaa ctaatacgat ggctacaggc   2820 

agtggcgcac caatggcaga caataacgag ggcgccgacg gagtgggtaa ttcctcggga   2880 

aattggcatt gcgattccac atggatgggc gacagagtca tcaccaccag cacccgaacc   2940 

tgggccctgc ccacctacaa caaccacctc tacaaacaaa tttccagcca atcaggagcc   3000 

tcgaacgaca atcactactt tggctacagc accccttggg ggtattttga cttcaacaga   3060 

ttccactgcc acttttcacc acgtgactgg caaagactca tcaacaacaa ctggggattc   3120 

cgacccaaga gactcaactt caagctcttt aacattcaag tcaaagaggt cacgcagaat   3180 

gacggtacga cgacgattgc caataacctt accagcacgg ttcaggtgtt tactgactcg   3240 

gagtaccagc tcccgtacgt cctcggctcg gcgcatcaag gatgcctccc gccgttccca   3300 

gcagacgtct tcatggtgcc acagtatgga tacctcaccc tgaacaacgg gagtcaggca   3360 

gtaggacgct cttcatttta ctgcctggag tactttcctt ctcagatgct gcgtaccgga   3420 

aacaacttta ccttcagcta cacttttgag gacgttcctt tccacagcag ctacgctcac   3480 

agccagagtc tggaccgtct catgaatcct ctcatcgacc agtacctgta ttacttgagc   3540 

agaacaaaca ctccaagtgg aaccaccacg cagtcaaggc ttcagttttc tcaggccgga   3600 

gcgagtgaca ttcgggacca gtctaggaac tggcttcctg gaccctgtta ccgccagcag   3660 

cgagtatcaa agacatctgc ggataacaac aacagtgaat actcgtggac tggagctacc   3720 

aagtaccacc tcaatggcag agactctctg gtgaatccgg gcccggccat ggcaagccac   3780 

aaggacgatg aagaaaagtt ttttcctcag agcggggttc tcatctttgg gaagcaaggc   3840 

tcagagaaaa caaatgtgga cattgaaaag gtcatgatta cagacgaaga ggaaatcagg   3900 

acaaccaatc ccgtggctac ggagcagtat ggttctgtat ctaccaacct ccagagaggc   3960 

aacagacaag cagctaccgc agatgtcaac acacaaggcg ttcttccagg catggtctgg   4020 

caggacagag atgtgtacct tcaggggccc atctgggcaa agattccaca cacggacgga   4080 

cattttcacc cctctcccct catgggtgga ttcggactta aacaccctcc tccacagatt   4140 

ctcatcaaga acaccccggt acctgcgaat ccttcgacca ccttcagtgc ggcaaagttt   4200 

gcttccttca tcacacagta ctccacggga caggtcagcg tggagatcga gtgggagctg   4260 

cagaaggaaa acagcaaacg ctggaatccc gaaattcagt acacttccaa ctacaacaag   4320 

tctgttaatg tggactttac tgtggacact aatggcgtgt attcagagcc tcgccccatt   4380 

ggcaccagat acctgactcg taatctgtaa ttgcttgtta atcaataaac cgtttaattc   4440 

gtttcagttg aactttggtc tctgcgtatt tctttcttat ctagtttcca tggctacgta   4500 

gataagtagc atggcgggtt aatcattaac tacaaggaac ccctagtgat ggagttggcc   4560 

actccctctc tgcgcgctcg ctcgctcact gaggccgggc gaccaaaggt cgcccgacgc   4620 

ccgggctttg cccgggcggc ctcagtgagc gagcgagcgc gcagagaggg agtggccaa    4679 

 
           
             13  
             735  
             PRT  
             adeno-associated virus 2 VP1 caspid protien  
           
            13 

Met Ala Ala Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Thr Leu Ser 
1               5                   10                  15 

Glu Gly Ile Arg Gln Trp Trp Lys Leu Lys Pro Gly Pro Pro Pro Pro 
            20                  25                  30 

Lys Pro Ala Glu Arg His Lys Asp Asp Ser Arg Gly Leu Val Leu Pro 
        35                  40                  45 

Gly Tyr Lys Tyr Leu Gly Pro Phe Asn Gly Leu Asp Lys Gly Glu Pro 
    50                  55                  60 

Val Asn Glu Ala Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp 
65                  70                  75                  80 

Arg Gln Leu Asp Ser Gly Asp Asn Pro Tyr Leu Lys Tyr Asn His Ala 
                85                  90                  95 

Asp Ala Glu Phe Gln Glu Arg Leu Lys Glu Asp Thr Ser Phe Gly Gly 
            100                 105                 110 

Asn Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg Val Leu Glu Pro 
        115                 120                 125 

Leu Gly Leu Val Glu Glu Pro Val Lys Thr Ala Pro Gly Lys Lys Arg 
    130                 135                 140 

Pro Val Glu His Ser Pro Val Glu Pro Asp Ser Ser Ser Gly Thr Gly 
145                 150                 155                 160 

Lys Ala Gly Gln Gln Pro Ala Arg Lys Arg Leu Asn Phe Gly Gln Thr 
                165                 170                 175 

Gly Asp Ala Asp Ser Val Pro Asp Pro Gln Pro Leu Gly Gln Pro Pro 
            180                 185                 190 

Ala Ala Pro Ser Gly Leu Gly Thr Asn Thr Met Ala Thr Gly Ser Gly 
        195                 200                 205 

Ala Pro Met Ala Asp Asn Asn Glu Gly Ala Asp Gly Val Gly Asn Ser 
    210                 215                 220 

Ser Gly Asn Trp His Cys Asp Ser Thr Trp Met Gly Asp Arg Val Ile 
225                 230                 235                 240 

Thr Thr Ser Thr Arg Thr Trp Ala Leu Pro Thr Tyr Asn Asn His Leu 
                245                 250                 255 

Tyr Lys Gln Ile Ser Ser Gln Ser Gly Ala Ser Asn Asp Asn His Tyr 
            260                 265                 270 

Phe Gly Tyr Ser Thr Pro Trp Gly Tyr Phe Asp Phe Asn Arg Phe His 
        275                 280                 285 

Cys His Phe Ser Pro Arg Asp Trp Gln Arg Leu Ile Asn Asn Asn Trp 
    290                 295                 300 

Gly Phe Arg Pro Lys Arg Leu Asn Phe Lys Leu Phe Asn Ile Gln Val 
305                 310                 315                 320 

Lys Glu Val Thr Gln Asn Asp Gly Thr Thr Thr Ile Ala Asn Asn Leu 
                325                 330                 335 

Thr Ser Thr Val Gln Val Phe Thr Asp Ser Glu Tyr Gln Leu Pro Tyr 
            340                 345                 350 

Val Leu Gly Ser Ala His Gln Gly Cys Leu Pro Pro Phe Pro Ala Asp 
        355                 360                 365 

Val Phe Met Val Pro Gln Tyr Gly Tyr Leu Thr Leu Asn Asn Gly Ser 
    370                 375                 380 

Gln Ala Val Gly Arg Ser Ser Phe Tyr Cys Leu Glu Tyr Phe Pro Ser 
385                 390                 395                 400 

Gln Met Leu Arg Thr Gly Asn Asn Phe Thr Phe Ser Tyr Thr Phe Glu 
                405                 410                 415 

Asp Val Pro Phe His Ser Ser Tyr Ala His Ser Gln Ser Leu Asp Arg 
            420                 425                 430 

Leu Met Asn Pro Leu Ile Asp Gln Tyr Leu Tyr Tyr Leu Ser Arg Thr 
        435                 440                 445 

Asn Thr Pro Ser Gly Thr Thr Thr Gln Ser Arg Leu Gln Phe Ser Gln 
    450                 455                 460 

Ala Gly Ala Ser Asp Ile Arg Asp Gln Ser Arg Asn Trp Leu Pro Gly 
465                 470                 475                 480 

Pro Cys Tyr Arg Gln Gln Arg Val Ser Lys Thr Ser Ala Asp Asn Asn 
                485                 490                 495 

Asn Ser Glu Tyr Ser Trp Thr Gly Ala Thr Lys Tyr His Leu Asn Gly 
            500                 505                 510 

Arg Asp Ser Leu Val Asn Pro Gly Pro Ala Met Ala Ser His Lys Asp 
        515                 520                 525 

Asp Glu Glu Lys Phe Phe Pro Gln Ser Gly Val Leu Ile Phe Gly Lys 
    530                 535                 540 

Gln Gly Ser Glu Lys Thr Asn Val Asp Ile Glu Lys Val Met Ile Thr 
545                 550                 555                 560 

Asp Glu Glu Glu Ile Arg Thr Thr Asn Pro Val Ala Thr Glu Gln Tyr 
                565                 570                 575 

Gly Ser Val Ser Thr Asn Leu Gln Arg Gly Asn Arg Gln Ala Ala Thr 
            580                 585                 590 

Ala Asp Val Asn Thr Gln Gly Val Leu Pro Gly Met Val Trp Gln Asp 
        595                 600                 605 

Arg Asp Val Tyr Leu Gln Gly Pro Ile Trp Ala Lys Ile Pro His Thr 
    610                 615                 620 

Asp Gly His Phe His Pro Ser Pro Leu Met Gly Gly Phe Gly Leu Lys 
625                 630                 635                 640 

His Pro Pro Pro Gln Ile Leu Ile Lys Asn Thr Pro Val Pro Ala Asn 
                645                 650                 655 

Pro Ser Thr Thr Phe Ser Ala Ala Lys Phe Ala Ser Phe Ile Thr Gln 
            660                 665                 670 

Tyr Ser Thr Gly Gln Val Ser Val Glu Ile Glu Trp Glu Leu Gln Lys 
        675                 680                 685 

Glu Asn Ser Lys Arg Trp Asn Pro Glu Ile Gln Tyr Thr Ser Asn Tyr 
    690                 695                 700 

Asn Lys Ser Val Asn Val Asp Phe Thr Val Asp Thr Asn Gly Val Tyr 
705                 710                 715                 720 

Ser Glu Pro Arg Pro Ile Gly Thr Arg Tyr Leu Thr Arg Asn Leu 
                725                 730                 735 

 
           
             14  
             598  
             PRT  
             adeno-associated virus 2 VP2 capsid protien  
           
            14 

Met Ala Pro Gly Lys Lys Arg Pro Val Glu His Ser Pro Val Glu Pro 
1               5                   10                  15 

Asp Ser Ser Ser Gly Thr Gly Lys Ala Gly Gln Gln Pro Ala Arg Lys 
            20                  25                  30 

Arg Leu Asn Phe Gly Gln Thr Gly Asp Ala Asp Ser Val Pro Asp Pro 
        35                  40                  45 

Gln Pro Leu Gly Gln Pro Pro Ala Ala Pro Ser Gly Leu Gly Thr Asn 
    50                  55                  60 

Thr Met Ala Thr Gly Ser Gly Ala Pro Met Ala Asp Asn Asn Glu Gly 
65                  70                  75                  80 

Ala Asp Gly Val Gly Asn Ser Ser Gly Asn Trp His Cys Asp Ser Thr 
                85                  90                  95 

Trp Met Gly Asp Arg Val Ile Thr Thr Ser Thr Arg Thr Trp Ala Leu 
            100                 105                 110 

Pro Thr Tyr Asn Asn His Leu Tyr Lys Gln Ile Ser Ser Gln Ser Gly 
        115                 120                 125 

Ala Ser Asn Asp Asn His Tyr Phe Gly Tyr Ser Thr Pro Trp Gly Tyr 
    130                 135                 140 

Phe Asp Phe Asn Arg Phe His Cys His Phe Ser Pro Arg Asp Trp Gln 
145                 150                 155                 160 

Arg Leu Ile Asn Asn Asn Trp Gly Phe Arg Pro Lys Arg Leu Asn Phe 
                165                 170                 175 

Lys Leu Phe Asn Ile Gln Val Lys Glu Val Thr Gln Asn Asp Gly Thr 
            180                 185                 190 

Thr Thr Ile Ala Asn Asn Leu Thr Ser Thr Val Gln Val Phe Thr Asp 
        195                 200                 205 

Ser Glu Tyr Gln Leu Pro Tyr Val Leu Gly Ser Ala His Gln Gly Cys 
    210                 215                 220 

Leu Pro Pro Phe Pro Ala Asp Val Phe Met Val Pro Gln Tyr Gly Tyr 
225                 230                 235                 240 

Leu Thr Leu Asn Asn Gly Ser Gln Ala Val Gly Arg Ser Ser Phe Tyr 
                245                 250                 255 

Cys Leu Glu Tyr Phe Pro Ser Gln Met Leu Arg Thr Gly Asn Asn Phe 
            260                 265                 270 

Thr Phe Ser Tyr Thr Phe Glu Asp Val Pro Phe His Ser Ser Tyr Ala 
        275                 280                 285 

His Ser Gln Ser Leu Asp Arg Leu Met Asn Pro Leu Ile Asp Gln Tyr 
    290                 295                 300 

Leu Tyr Tyr Leu Ser Arg Thr Asn Thr Pro Ser Gly Thr Thr Thr Gln 
305                 310                 315                 320 

Ser Arg Leu Gln Phe Ser Gln Ala Gly Ala Ser Asp Ile Arg Asp Gln 
                325                 330                 335 

Ser Arg Asn Trp Leu Pro Gly Pro Cys Tyr Arg Gln Gln Arg Val Ser 
            340                 345                 350 

Lys Thr Ser Ala Asp Asn Asn Asn Ser Glu Tyr Ser Trp Thr Gly Ala 
        355                 360                 365 

Thr Lys Tyr His Leu Asn Gly Arg Asp Ser Leu Val Asn Pro Gly Pro 
    370                 375                 380 

Ala Met Ala Ser His Lys Asp Asp Glu Glu Lys Phe Phe Pro Gln Ser 
385                 390                 395                 400 

Gly Val Leu Ile Phe Gly Lys Gln Gly Ser Glu Lys Thr Asn Val Asp 
                405                 410                 415 

Ile Glu Lys Val Met Ile Thr Asp Glu Glu Glu Ile Arg Thr Thr Asn 
            420                 425                 430 

Pro Val Ala Thr Glu Gln Tyr Gly Ser Val Ser Thr Asn Leu Gln Arg 
        435                 440                 445 

Gly Asn Arg Gln Ala Ala Thr Ala Asp Val Asn Thr Gln Gly Val Leu 
    450                 455                 460 

Pro Gly Met Val Trp Gln Asp Arg Asp Val Tyr Leu Gln Gly Pro Ile 
465                 470                 475                 480 

Trp Ala Lys Ile Pro His Thr Asp Gly His Phe His Pro Ser Pro Leu 
                485                 490                 495 

Met Gly Gly Phe Gly Leu Lys His Pro Pro Pro Gln Ile Leu Ile Lys 
            500                 505                 510 

Asn Thr Pro Val Pro Ala Asn Pro Ser Thr Thr Phe Ser Ala Ala Lys 
        515                 520                 525 

Phe Ala Ser Phe Ile Thr Gln Tyr Ser Thr Gly Gln Val Ser Val Glu 
    530                 535                 540 

Ile Glu Trp Glu Leu Gln Lys Glu Asn Ser Lys Arg Trp Asn Pro Glu 
545                 550                 555                 560 

Ile Gln Tyr Thr Ser Asn Tyr Asn Lys Ser Val Asn Val Asp Phe Thr 
                565                 570                 575 

Val Asp Thr Asn Gly Val Tyr Ser Glu Pro Arg Pro Ile Gly Thr Arg 
            580                 585                 590 

Tyr Leu Thr Arg Asn Leu 
        595 

 
           
             15  
             533  
             PRT  
             adeno-associated virus 2 VP3 capsid protien  
           
            15 

Met Ala Thr Gly Ser Gly Ala Pro Met Ala Asp Asn Asn Glu Gly Ala 
1               5                   10                  15 

Asp Gly Val Gly Asn Ser Ser Gly Asn Trp His Cys Asp Ser Thr Trp 
            20                  25                  30 

Met Gly Asp Arg Val Ile Thr Thr Ser Thr Arg Thr Trp Ala Leu Pro 
        35                  40                  45 

Thr Tyr Asn Asn His Leu Tyr Lys Gln Ile Ser Ser Gln Ser Gly Ala 
    50                  55                  60 

Ser Asn Asp Asn His Tyr Phe Gly Tyr Ser Thr Pro Trp Gly Tyr Phe 
65                  70                  75                  80 

Asp Phe Asn Arg Phe His Cys His Phe Ser Pro Arg Asp Trp Gln Arg 
                85                  90                  95 

Leu Ile Asn Asn Asn Trp Gly Phe Arg Pro Lys Arg Leu Asn Phe Lys 
            100                 105                 110 

Leu Phe Asn Ile Gln Val Lys Glu Val Thr Gln Asn Asp Gly Thr Thr 
        115                 120                 125 

Thr Ile Ala Asn Asn Leu Thr Ser Thr Val Gln Val Phe Thr Asp Ser 
    130                 135                 140 

Glu Tyr Gln Leu Pro Tyr Val Leu Gly Ser Ala His Gln Gly Cys Leu 
145                 150                 155                 160 

Pro Pro Phe Pro Ala Asp Val Phe Met Val Pro Gln Tyr Gly Tyr Leu 
                165                 170                 175 

Thr Leu Asn Asn Gly Ser Gln Ala Val Gly Arg Ser Ser Phe Tyr Cys 
            180                 185                 190 

Leu Glu Tyr Phe Pro Ser Gln Met Leu Arg Thr Gly Asn Asn Phe Thr 
        195                 200                 205 

Phe Ser Tyr Thr Phe Glu Asp Val Pro Phe His Ser Ser Tyr Ala His 
    210                 215                 220 

Ser Gln Ser Leu Asp Arg Leu Met Asn Pro Leu Ile Asp Gln Tyr Leu 
225                 230                 235                 240 

Tyr Tyr Leu Ser Arg Thr Asn Thr Pro Ser Gly Thr Thr Thr Gln Ser 
                245                 250                 255 

Arg Leu Gln Phe Ser Gln Ala Gly Ala Ser Asp Ile Arg Asp Gln Ser 
            260                 265                 270 

Arg Asn Trp Leu Pro Gly Pro Cys Tyr Arg Gln Gln Arg Val Ser Lys 
        275                 280                 285 

Thr Ser Ala Asp Asn Asn Asn Ser Glu Tyr Ser Trp Thr Gly Ala Thr 
    290                 295                 300 

Lys Tyr His Leu Asn Gly Arg Asp Ser Leu Val Asn Pro Gly Pro Ala 
305                 310                 315                 320 

Met Ala Ser His Lys Asp Asp Glu Glu Lys Phe Phe Pro Gln Ser Gly 
                325                 330                 335 

Val Leu Ile Phe Gly Lys Gln Gly Ser Glu Lys Thr Asn Val Asp Ile 
            340                 345                 350 

Glu Lys Val Met Ile Thr Asp Glu Glu Glu Ile Arg Thr Thr Asn Pro 
        355                 360                 365 

Val Ala Thr Glu Gln Tyr Gly Ser Val Ser Thr Asn Leu Gln Arg Gly 
    370                 375                 380 

Asn Arg Gln Ala Ala Thr Ala Asp Val Asn Thr Gln Gly Val Leu Pro 
385                 390                 395                 400 

Gly Met Val Trp Gln Asp Arg Asp Val Tyr Leu Gln Gly Pro Ile Trp 
                405                 410                 415 

Ala Lys Ile Pro His Thr Asp Gly His Phe His Pro Ser Pro Leu Met 
            420                 425                 430 

Gly Gly Phe Gly Leu Lys His Pro Pro Pro Gln Ile Leu Ile Lys Asn 
        435                 440                 445 

Thr Pro Val Pro Ala Asn Pro Ser Thr Thr Phe Ser Ala Ala Lys Phe 
    450                 455                 460 

Ala Ser Phe Ile Thr Gln Tyr Ser Thr Gly Gln Val Ser Val Glu Ile 
465                 470                 475                 480 

Glu Trp Glu Leu Gln Lys Glu Asn Ser Lys Arg Trp Asn Pro Glu Ile 
                485                 490                 495 

Gln Tyr Thr Ser Asn Tyr Asn Lys Ser Val Asn Val Asp Phe Thr Val 
            500                 505                 510 

Asp Thr Asn Gly Val Tyr Ser Glu Pro Arg Pro Ile Gly Thr Arg Tyr 
        515                 520                 525 

Leu Thr Arg Asn Leu 
    530 

 
           
             16  
             6  
             PRT  
             Artificial Sequence  
             
               Bovine Papilloma Virus peptide  
             
           
            16 

Thr Pro Phe Tyr Leu Lys 
1               5 

 
           
             17  
             10  
             PRT  
             Artificial Sequence  
             
               Luteinizing Hormone peptide  
             
           
            17 

His Cys Ser Thr Cys Tyr Tyr His Lys Ser 
1               5                   10 

 
           
             18  
             5  
             PRT  
             Artificial Sequence  
             
               synthetic targeting peptide  
             
           
            18 

Phe Val Phe Lys Pro 
1               5

Technology Classification (CPC): 2