Patent Publication Number: US-2019169237-A1

Title: Mutant adeno-associated virus virions and methods of use thereof

Description:
CROSS-REFERENCE 
     This application claims the benefit of U.S. Provisional Patent Application No. 60/484,111 filed Jun. 30, 2003, which application is incorporated herein by reference in its entirety. 
    
    
     FIELD OF THE INVENTION 
     The present invention is in the field of recombinant adeno-associated virus vectors. 
     BACKGROUND OF THE INVENTION 
     Adeno-associated virus (AAV) is a 4.7 kb, single stranded DNA virus that contains two open reading frames, rep and cap. The first gene encodes four proteins necessary for genome replication (Rep78, Rep68, Rep52, and Rep40), and the second expresses three structural proteins (VP1-3) that assemble to form the viral capsid. As its name implies, AAV is dependent upon the presence of a helper virus, such as an adenovirus or herpesvirus, for active replication. In the absence of a helper it establishes a latent state in which its genome is maintained episomally or integrated into the host chromosome. To date, eight homologous primate AAV serotypes and numerous non-human primate types have been identified, although AAV2 is the best characterized as a gene delivery vehicle. 
     In 1989 a recombinant AAV2 (rAAV) gene delivery vector system was first generated, and vectors based on AAV have subsequently been shown to offer numerous major advantages. First, vectors based on AAV are extremely safe, since wild-type AAV is nonpathogenic and has no etiologic association with any known diseases. In addition, AAV offers the capability for highly efficient gene delivery and sustained transgene expression in numerous tissues, including muscle, lung, and brain. Furthermore, AAV has enjoyed success in human clinical trials. 
     Despite this success, vector design problems remain. One major concern is the fact that much of the human population has already been exposed to various AAV serotypes, and as a result a significant fraction of any future patient population harbors neutralizing antibodies (NABs) that block gene delivery. Additional problems with rAAV vectors include limited tissue dispersion for serotypes that employ heparan sulfate as a receptor (AAV2 and 3), poor infection of non-permissive cell types such as stem cells, challenges with high efficiency targeting of gene delivery to selected cell populations, and a finite transgene carrying capacity. 
     There is a need in the art for improved AAV vectors that evade neutralization by serum antibodies to AAV. The present invention addresses this need. 
     LITERATURE 
     Halbert et al. (2000)  J Virol  74, 1524-32; Blacklow et al. (1971)  Am J Epidemiol  94, 359-66. (1971); Erles et al. (1999)  J Med Virol  59, 406-11; Moskalenko et al. (2000)  J Virol  74, 1761-6; Wobus et al. (2000)  J Virol  74, 9281-93; Sun et al. (2003)  Gene Ther  10, 964-76; Nguyen (2001)  Neuroreport  12, 1961-4; Davidson et al. (2000)  Proc Natl Acad Sci USA  97, 3428-32; Rabinowitz et al. (1999)  Virology  265, 274-85; Opie et al. (2003)  J Virol  77, 6995-7006; U.S. Pat. Nos. 6,596,539; 6,733,757; 6,710,036; 6,703,237. 
     SUMMARY OF THE INVENTION 
     The present invention provides mutant adeno-associated virus (AAV) that exhibit altered capsid properties, e.g., reduced binding to neutralizing antibodies in serum and/or altered heparin binding and/or altered infectivity of particular cell types. The present invention further provides libraries of mutant AAV comprising one or more mutations in a capsid gene. The present invention further provides methods of generating the mutant AAV and mutant AAV libraries, and compositions comprising the mutant AAV. The present invention further provides recombinant AAV (rAAV) virions that comprise a mutant capsid protein. The present invention further provides nucleic acids comprising nucleotide sequences that encode mutant capsid proteins, and host cells comprising the nucleic acids. The present invention further provides methods of delivering a gene product to an individual, the methods generally involving administering an effective amount of a subject rAAV virion to an individual in need thereof. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIGS. 1 a  and 1 b    depict heparin binding characteristics of wild type AAV versus the viral library. 
         FIGS. 2 a  and 2 b    depict generation of antibody neutralization escape mutants.  FIG. 2 a    depicts the fraction rescued (normalized with respect to zero stringency) at various stringencies (given as the ratio of neutralizing antibody titer (reciprocal dilution) divided by the actual dilution).  FIG. 2 b    depicts the percent knockdown (reduction) in infection by rabbit antisera for wild type AAV, AAV library, and individual AAV escape mutants. 
         FIGS. 3  A-C depict the nucleotide sequence of wild-type AAV cap (SEQ ID NO:1) aligned with nucleotide sequences of cap of the neutralizing antibody escape mutants AbE2 (SEQ ID NO:2) and AbE L (SEQ ID NO:3). Boxes indicate changes in nucleotide sequence compared to wild-type. 
         FIGS. 4A-G  depict an alignment of VP-1-encoding nucleotide sequences of wild-type AAV-2 VP1 (SEQ ID NO:4), and exemplary neutralizing antibody evasion mutants. 
         FIGS. 5A-C  depict an alignment of VP-1-encoding amino acid sequences of wild-type AAV-2 VP1 (SEQ ID NO5), and exemplary neutralizing antibody evasion mutants. 
         FIGS. 6A-J  depict an alignment of VP-1-encoding nucleotide sequences of wild-type AAV-2 VP1, and exemplary neutralizing antibody evasion mutants. 
         FIGS. 7A-D  depict an alignment of VP-1-encoding amino acid sequences of wild-type AAV-2 VP1, and exemplary neutralizing antibody evasion mutants. 
         FIGS. 8A-G  depict an alignment of VP-1-encoding nucleotide sequences of wild-type AAV-2 VP1, and exemplary heparin binding mutants. 
         FIGS. 9A-C  depict an alignment of VP-1-encoding amino acid sequences of wild-type AAV-2 VP1, and exemplary heparin binding mutants. 
     
    
    
     DEFINITIONS 
     A “vector” as used herein refers to a macromolecule or association of macromolecules that comprises or associates with a polynucleotide and which can be used to mediate delivery of the polynucleotide to a cell. Illustrative vectors include, for example, plasmids, viral vectors, liposomes, and other gene delivery vehicles. 
     “AAV” is an abbreviation for adeno-associated virus, and may be used to refer to the virus itself or derivatives thereof. The term covers all subtypes and both naturally occurring and recombinant forms, except where required otherwise. The abbreviation “rAAV” refers to recombinant adeno-associated virus, also referred to as a recombinant AAV vector (or “rAAV vector”). The term “AAV” includes AAV type 1 (AAV-1), AAV type 2 (AAV-2), AAV type 3 (AAV-3), AAV type 4 (AAV-4), AAV type 5 (AAV-5), AAV type 6 (AAV-6), AAV type 7 (AAV-7), AAV type 8 (AAV-8), avian AAV, bovine AAV, canine AAV, equine AAV, primate AAV, non-primate AAV, and ovine AAV. “Primate AAV” refers to AAV that infect primates, “non-primate AAV” refers to AAV that infect non-primate mammals, “bovine AAV” refers to AAV that infect bovine mammals, etc. 
     An “rAAV vector” as used herein refers to an AAV vector comprising a polynucleotide sequence not of AAV origin (i.e., a polynucleotide heterologous to AAV), typically a sequence of interest for the genetic transformation of a cell. In general, the heterologous polynucleotide is flanked by at least one, and generally by two AAV inverted terminal repeat sequences (ITRs). The term rAAV vector encompasses both rAAV vector particles and rAAV vector plasmids. 
     An “AAV virus” or “AAV viral particle” or “rAAV vector particle” refers to a viral particle composed of at least one AAV capsid protein (typically by all of the capsid proteins of a wild-type AAV) and an encapsidated polynucleotide rAAV vector. If the particle comprises a heterologous polynucleotide (i.e. a polynucleotide other than a wild-type AAV genome such as a transgene to be delivered to a mammalian cell), it is typically referred to as an “rAAV vector particle” or simply an “rAAV vector”. Thus, production of rAAV particle necessarily includes production of rAAV vector, as such a vector is contained within an rAAV particle. 
     “Packaging” refers to a series of intracellular events that result in the assembly and encapsidation of an AAV particle. 
     AAV “rep” and “cap” genes refer to polynucleotide sequences encoding replication and encapsidation proteins of adeno-associated virus. AAV rep and cap are referred to herein as AAV “packaging genes.” 
     A “helper virus” for AAV refers to a virus that allows AAV (e.g. wild-type AAV) to be replicated and packaged by a mammalian cell. A variety of such helper viruses for AAV are known in the art, including adenoviruses, herpesviruses and poxviruses such as vaccinia. The adenoviruses encompass a number of different subgroups, although Adenovirus type 5 of subgroup C is most commonly used. Numerous adenoviruses of human, non-human mammalian and avian origin are known and available from depositories such as the ATCC. Viruses of the herpes family include, for example, herpes simplex viruses (HSV) and Epstein-Barr viruses (EBV), as well as cytomegaloviruses (CMV) and pseudorabies viruses (PRV); which are also available from depositories such as ATCC. 
     “Helper virus function(s)” refers to function(s) encoded in a helper virus genome which allow AAV replication and packaging (in conjunction with other requirements for replication and packaging described herein). As described herein, “helper virus function” may be provided in a number of ways, including by providing helper virus or providing, for example, polynucleotide sequences encoding the requisite function(s) to a producer cell in trans. 
     An “infectious” virus or viral particle is one that comprises a polynucleotide component which it is capable of delivering into a cell for which the viral species is trophic. The term does not necessarily imply any replication capacity of the virus. Assays for counting infectious viral particles are described elsewhere in this disclosure and in the art. Viral infectivity can be expressed as the P:I ratio, or the ratio of total viral particles to infective viral particles. 
     A “replication-competent” virus (e.g. a replication-competent AAV) refers to a phenotypically wild-type virus that is infectious, and is also capable of being replicated in an infected cell (i.e. in the presence of a helper virus or helper virus functions). In the case of AAV, replication competence generally requires the presence of functional AAV packaging genes. In general, rAAV vectors as described herein are replication-incompetent in mammalian cells (especially in human cells) by virtue of the lack of one or more AAV packaging genes. Typically, such rAAV vectors lack any AAV packaging gene sequences in order to minimize the possibility that replication competent AAV are generated by recombination between AAV packaging genes and an incoming rAAV vector. In many embodiments, rAAV vector preparations as described herein are those which contain few if any replication competent AAV (rcAAV, also referred to as RCA) (e.g., less than about 1 rcAAV per 10 2  rAAV particles, less than about 1 rcAAV per 10 4  rAAV particles, less than about 1 rcAAV per 10 8  rAAV particles, less than about 1 rcAAV per 10 12  rAAV particles, or no rcAAV). 
     The term “polynucleotide” refers to a polymeric form of nucleotides of any length, including deoxyribonucleotides or ribonucleotides, or analogs thereof. A polynucleotide may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs, and may be interrupted by non-nucleotide components. If present, modifications to the nucleotide structure may be imparted before or after assembly of the polymer. The term polynucleotide, as used herein, refers interchangeably to double- and single-stranded molecules. Unless otherwise specified or required, any embodiment of the invention described herein that is a polynucleotide encompasses both the double-stranded form and each of two complementary single-stranded forms known or predicted to make up the double-stranded form. 
     Nucleic acid hybridization reactions can be performed under conditions of different “stringency”. Conditions that increase stringency of a hybridization reaction of widely known and published in the art. See, e.g., Sambrook et al.  Molecular Cloning, A Laboratory Manual,  2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989, herein incorporated by reference. For example, see page 7.52 of Sambrook et al. Examples of relevant conditions include (in order of increasing stringency): incubation temperatures of 25° C., 37° C., 50° C. and 68° C.; buffer concentrations of 10×SSC, 6×SSC, 1×SSC, 0.1×SSC (where 1×SSC is 0.15 M NaCl and 15 mM citrate buffer) and their equivalents using other buffer systems; formamide concentrations of 0%, 25%, 50%, and 75%; incubation times from 5 minutes to 24 hours; 1, 2, or more washing steps; wash incubation times of 1, 2, or 15 minutes; and wash solutions of 6×SSC, 1×SSC, 0.1×SSC, or deionized water. An example of stringent hybridization conditions is hybridization at 50° C. or higher and 0.1×SSC (15 mM sodium chloride/1.5 mM sodium citrate). Another example of stringent hybridization conditions is overnight incubation at 42° C. in a solution: 50% formamide, 1×SSC (150 mM NaCl, 15 mM sodium citrate), 50 mM sodium phosphate (pH 7.6), 5×Denhardt&#39;s solution, 10% dextran sulfate, and 20 μg/ml denatured, sheared salmon sperm DNA, followed by washing the filters in 0.1×SSC at about 65° C. As another example, stringent hybridization conditions comprise: prehybridization for 8 hours to overnight at 65° C. in a solution comprising 6× single strength citrate (SSC) (1×SSC is 0.15 M NaCl, 0.015 M Na citrate; pH 7.0), 5×Denhardt&#39;s solution, 0.05% sodium pyrophosphate and 100 μg/ml herring sperm DNA; hybridization for 18-20 hours at 65° C. in a solution containing 6×SSC, 1×Denhardt&#39;s solution, 100 μg/ml yeast tRNA and 0.05% sodium pyrophosphate; and washing of filters at 65° C. for 1 h in a solution containing 0.2×SSC and 0.1% SDS (sodium dodecyl sulfate). 
     Stringent hybridization conditions are hybridization conditions that are at least as stringent as the above representative conditions. Other stringent hybridization conditions are known in the art and may also be employed to identify nucleic acids of this particular embodiment of the invention. 
     “T m ” is the temperature in degrees Celsius at which 50% of a polynucleotide duplex made of complementary strands hydrogen bonded in anti-parallel direction by Watson-Crick base pairing dissociates into single strands under conditions of the experiment. T m  may be predicted according to a standard formula, such as: 
         T   m =81.5+16.6 log[ X   + ]+0.41(%  G/C )−0.61(%  F )−600/ L  
 
     where [X + ] is the cation concentration (usually sodium ion, Na + ) in mol/L; 
     (% G/C) is the number of G and C residues as a percentage of total residues in the duplex; (% F) is the percent formamide in solution (wt/vol); and L is the number of nucleotides in each strand of the duplex. 
     A polynucleotide or polypeptide has a certain percent “sequence identity” to another polynucleotide or polypeptide, meaning that, when aligned, that percentage of bases or amino acids are the same when comparing the two sequences. Sequence similarity can be determined in a number of different manners. To determine sequence identity, sequences can be aligned using the methods and computer programs, including BLAST, available over the world wide web at ncbi.nlm.nih.gov/BLAST/. Another alignment algorithm is FASTA, available in the Genetics Computing Group (GCG) package, from Madison, Wis., USA, a wholly owned subsidiary of Oxford Molecular Group, Inc. Other techniques for alignment are described in Methods in Enzymology, vol. 266: Computer Methods for Macromolecular Sequence Analysis (1996), ed. Doolittle, Academic Press, Inc., a division of Harcourt Brace &amp; Co., San Diego, Calif., USA. Of particular interest are alignment programs that permit gaps in the sequence. The Smith-Waterman is one type of algorithm that permits gaps in sequence alignments. See  Meth. Mol. Biol.  70: 173-187 (1997). Also, the GAP program using the Needleman and Wunsch alignment method can be utilized to align sequences. See  J. Mol. Biol.  48: 443-453 (1970) 
     Of interest is the BestFit program using the local homology algorithm of Smith Waterman (Advances in Applied Mathematics 2: 482-489 (1981) to determine sequence identity. The gap generation penalty will generally range from 1 to 5, usually 2 to 4 and in many embodiments will be 3. The gap extension penalty will generally range from about 0.01 to 0.20 and in many instances will be 0.10. The program has default parameters determined by the sequences inputted to be compared. Preferably, the sequence identity is determined using the default parameters determined by the program. This program is available also from Genetics Computing Group (GCG) package, from Madison, Wis., USA. 
     Another program of interest is the FastDB algorithm. FastDB is described in Current Methods in Sequence Comparison and Analysis, Macromolecule Sequencing and Synthesis, Selected Methods and Applications, pp. 127-149, 1988, Alan R. Liss, Inc. Percent sequence identity is calculated by FastDB based upon the following parameters: 
     Mismatch Penalty: 1.00; 
     Gap Penalty: 1.00; 
     Gap Size Penalty: 0.33; and 
     Joining Penalty: 30.0. 
     A “gene” refers to a polynucleotide containing at least one open reading frame that is capable of encoding a particular protein after being transcribed and translated. 
     A “small interfering” or “short interfering RNA” or siRNA is a RNA duplex of nucleotides that is targeted to a gene interest (a “target gene”). An “RNA duplex” refers to the structure formed by the complementary pairing between two regions of a RNA molecule. siRNA is “targeted” to a gene in that the nucleotide sequence of the duplex portion of the siRNA is complementary to a nucleotide sequence of the targeted gene. In some embodiments, the length of the duplex of siRNAs is less than 30 nucleotides. In some embodiments, the duplex can be 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11 or 10 nucleotides in length. In some embodiments, the length of the duplex is 19-25 nucleotides in length. The RNA duplex portion of the siRNA can be part of a hairpin structure. In addition to the duplex portion, the hairpin structure may contain a loop portion positioned between the two sequences that form the duplex. The loop can vary in length. In some embodiments the loop is 5, 6, 7, 8, 9, 10, 11, 12 or 13 nucleotides in length. The hairpin structure can also contain 3′ or 5′ overhang portions. In some embodiments, the overhang is a 3′ or a 5′ overhang 0, 1, 2, 3, 4 or 5 nucleotides in length. 
     “Recombinant,” as applied to a polynucleotide means that the polynucleotide is the product of various combinations of cloning, restriction or ligation steps, and other procedures that result in a construct that is distinct from a polynucleotide found in nature. A recombinant virus is a viral particle comprising a recombinant polynucleotide. The terms respectively include replicates of the original polynucleotide construct and progeny of the original virus construct. 
     A “control element” or “control sequence” is a nucleotide sequence involved in an interaction of molecules that contributes to the functional regulation of a polynucleotide, including replication, duplication, transcription, splicing, translation, or degradation of the polynucleotide. The regulation may affect the frequency, speed, or specificity of the process, and may be enhancing or inhibitory in nature. Control elements known in the art include, for example, transcriptional regulatory sequences such as promoters and enhancers. A promoter is a DNA region capable under certain conditions of binding RNA polymerase and initiating transcription of a coding region usually located downstream (in the 3′ direction) from the promoter. 
     “Operatively linked” or “operably linked” refers to a juxtaposition of genetic elements, wherein the elements are in a relationship permitting them to operate in the expected manner. For instance, a promoter is operatively linked to a coding region if the promoter helps initiate transcription of the coding sequence. There may be intervening residues between the promoter and coding region so long as this functional relationship is maintained. 
     An “expression vector” is a vector comp rising a region which encodes a polypeptide of interest, and is used for effecting the expression of the protein in an intended target cell. An expression vector also comprises control elements operatively linked to the encoding region to facilitate expression of the protein in the target. The combination of control elements and a gene or genes to which they are operably linked for expression is sometimes referred to as an “expression cassette,” a large number of which are known and available in the art or can be readily constructed from components that are available in the art. 
     “Heterologous” means derived from a genotypically distinct entity from that of the rest of the entity to which it is being compared. For example, a polynucleotide introduced by genetic engineering techniques into a plasmid or vector derived from a different species is a heterologous polynucleotide. A promoter removed from its native coding sequence and operatively linked to a coding sequence with which it is not naturally found linked is a heterologous promoter. Thus, for example, an rAAV that includes a heterologous nucleic acid encoding a heterologous gene product is an rAAV that includes a nucleic acid not normally included in a naturally-occurring, wild-type AAV, and the encoded heterologous gene product is a gene product not normally encoded by a naturally-occurring, wild-type AAV. 
     The terms “genetic alteration” and “genetic modification” (and grammatical variants thereof), are used interchangeably herein to refer to a process wherein a genetic element (e.g., a polynucleotide) is introduced into a cell other than by mitosis or meiosis. The element may be heterologous to the cell, or it may be an additional copy or improved version of an element already present in the cell. Genetic alteration may be effected, for example, by transfecting a cell with a recombinant plasmid or other polynucleotide through any process known in the art, such as electroporation, calcium phosphate precipitation, or contacting with a polynucleotide-liposome complex. Genetic alteration may also be effected, for example, by transduction or infection with a DNA or RNA virus or viral vector. Generally, the genetic element is introduced into a chromosome or mini-chromosome in the cell; but any alteration that changes the phenotype and/or genotype of the cell and its progeny is included in this term. 
     A cell is said to be “stably” altered, transduced, genetically modified, or transformed with a genetic sequence if the sequence is available to perform its function during extended culture of the cell in vitro. Generally, such a cell is “heritably” altered (genetically modified) in that a genetic alteration is introduced which is also inheritable by progeny of the altered cell. 
     The terms “polypeptide,” “peptide,” and “protein” are used interchangeably herein to refer to polymers of amino acids of any length. The terms also encompass an amino acid polymer that has been modified; for example, disulfide bond formation, glycosylation, lipidation, phosphorylation, or conjugation with a labeling component. Polypeptides such as “CFTR,” “p53,” “EPO” and the like, when discussed in the context of delivering a gene product to a mammalian subject, and compositions therefor, refer to the respective intact polypeptide, or any fragment or genetically engineered derivative thereof, that retains the desired biochemical function of the intact protein. Similarly, references to CFTR, p53, EPO genes, and other such genes for use in delivery of a gene product to a mammalian subject (which may be referred to as “transgenes” to be delivered to a recipient cell), include polynucleotides encoding the intact polypeptide or any fragment or genetically engineered derivative possessing the desired biochemical function. 
     An “isolated” plasmid, nucleic acid, vector, virus, host cell, or other substance refers to a preparation of the substance devoid of at least some of the other components that may also be present where the substance or a similar substance naturally occurs or is initially prepared from. Thus, for example, an isolated substance may be prepared by using a purification technique to enrich it from a source mixture. Enrichment can be measured on an absolute basis, such as weight per volume of solution, or it can be measured in relation to a second, potentially interfering substance present in the source mixture. Increasing enrichments of the embodiments of this invention are increasingly more isolated. An isolated plasmid, nucleic acid, vector, virus, host cell, or other substance is in some embodiments purified, e.g., from about 80% to about 90% pure, at least about 90% pure, at least about 95% pure, at least about 98% pure, or at least about 99%, or more, pure. 
     As used herein, the terms “treatment,” “treating,” and the like, refer to obtaining a desired pharmacologic and/or physiologic effect. The effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and/or may be therapeutic in terms of a partial or complete cure for a disease and/or adverse affect attributable to the disease. “Treatment,” as used herein, covers any treatment of a disease in a mammal, particularly in a human, and includes: (a) preventing the disease from occurring in a subject which may be predisposed to the disease or at risk of acquiring the disease but has not yet been diagnosed as having it; (b) inhibiting the disease, i.e., arresting its development; and (c) relieving the disease, i.e., causing regression of the disease. 
     The terms “individual,” “host,” “subject,” and “patient” are used interchangeably herein, and refer to a mammal, including, but not limited to, human and non-human primates, including simians and humans; mammalian sport animals (e.g., horses); mammalian farm animals (e.g., sheep, goats, etc.); mammalian pets (dogs, cats, etc.); and rodents (e.g., mice, rats, etc.). 
     Before the present invention is further described, it is to be understood that this invention is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims. 
     Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention. 
     Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited. 
     It must be noted that as used herein and in the appended claims, the singular forms “a”, “and”, and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “an rAAV vector” includes a plurality of such vectors and reference to “the mutant AAV capsid protein” includes reference to one or more mutant AAV capsid proteins and equivalents thereof known to those skilled in the art, and so forth. 
     The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed. 
     DETAILED DESCRIPTION OF THE INVENTION 
     The present invention provides mutant adeno-associated virus (AAV) that exhibit altered capsid properties, e.g., reduced binding to neutralizing antibodies in serum and/or altered heparin binding and/or altered infectivity of particular cell types. The present invention further provides libraries of mutant AAV comprising one or more mutations in a capsid gene. The present invention further provides methods of generating the mutant AAV, mutant AAV libraries, and compositions comprising the mutant AAV or mutant AAV libraries. The present invention further provides recombinant AAV (rAAV) virions that comprise a mutant capsid protein. The present invention further provides nucleic acids comprising nucleotide sequences that encode mutant capsid proteins, and host cells comprising the nucleic acids. The present invention further provides methods of delivering a gene product to an individual, the methods generally involving administering an effective amount of a subject rAAV virion to an individual in need thereof. In many embodiments, a subject mutant AAV virion, a subject nucleic acid, etc., is isolated. 
     A subject mutant AAV virion or a subject rAAV virion exhibits one or more of the following properties: 1) increased heparan sulfate binding affinity relative to wild-type AAV; 2) decreased heparan sulfate binding affinity relative to wild-type AAV; 3) increased infectivity of a cell that is resistant to infection with AAV, or that is less permissive to infection with AAV than a prototypical permissive cell; 4) increased evasion of neutralizing antibodies; and 5) increased ability to cross an endothelial cell layer. 
     A subject nucleic acid encoding a mutant AAV capsid protein is useful for generating recombinant AAV virions that exhibit altered properties such as increased heparan sulfate binding, decreased heparan sulfate binding, increased infectivity of a cell that is resistant to infection with AAV or that is less permissive to infection with AAV, increased evasion of neutralizing antibodies, increased ability to cross an endothelial cell layer, and the like. A subject rAAV virion is useful for delivering a gene product to an individual. 
     Cell membrane-associated heparan sulfate proteoglycan is a primary cell surface receptor for AAV, e.g., AAV-2. Increased heparan sulfate binding (e.g., increased heparin affinity) is advantageous where, e.g., the rAAV particle is being delivered in a localized manner, e.g., where diffusion of the rAAV particle away from the site of delivery is not desired. Many cell types produce heparan sulfate, which remains on the surface of the cell or in the immediate environment of the cell. Thus, an rAAV virion with increased heparan sulfate binding affinity would remain relatively close to the site of administration. For example, localized delivery of a subject rAAV virion is advantageous for delivery of a gene product to a tumor that is localized to a particular anatomical site (but not, e.g., to surrounding non-cancerous tissue), for delivery of a gene product to a diseased cardiac vessel (but not to the surrounding healthy heart tissue), etc. 
     In many embodiments of the present invention, AAV-2, and mutants of AAV-2, are exemplified. However, the exemplification of AAV-2 herein is in no way meant to be limiting. Those skilled in the art can readily adapt the methods as discussed herein to generate capsid mutants of other AAV, including, e.g., AAV-3, AAV-4, AAV-5, etc. Thus, e.g., where an AAV binds to the ß 5  subunit of integrin α v ß 5 , the present invention contemplates mutant AAV that exhibit increased or decreased binding to the ß 5  subunit of integrin α v ß 5 , compared to the corresponding wild-type AAV. As another example, where an AAV (e.g., AAV-4) binds to O-linked sialic acid, the present invention contemplates mutant AAV that exhibit increased or decreased binding to O-linked sialic acid. As another example, where an AAV (e.g., AAV-5) binds to N-linked sialic acid or to a platelet-derived growth factor receptor (PDGFR), the present invention contemplates mutant AAV that exhibit increased or decreased binding to N-linked sialic acid or PDGFR. See, e.g., Kaludov et al. ((2001)  J. Virol.  75:6884); Pasquale et al. ((2003)  Nat. Med.  9:1306); and Walters et al. ((2001)  J. Biol. Chem.  276:20610) for descriptions of AAV receptors. 
     Increased heparin affinity is also advantageous in that it confers increased infectivity of cell types that are typically refractory to infection with AAV, e.g., non-permissive cell types and “less-permissive” cell types (e.g., cells that are less permissive than a prototypical permissive cell). Such cell types include those with relatively low amounts of heparan sulfate on their surface. Increased heparan sulfate binding affinity allows an increased level of binding to cells that have relatively low levels of surface heparan sulfate, and therefore leads to increased infectivity of these cells. An example of cells that are refractory to infection with AAV is a stem cell. Thus, a subject rAAV virion is advantageous because it can infect stem cells and can deliver gene products to stem cells. Other examples of cells that are non-permissive or less permissive to infection with AAV include lung epithelial cells and hepatocytes. 
     Decreased heparan sulfate binding (e.g., decreased heparin affinity) is advantageous for therapeutic strategies in which more widespread, or systemic delivery of a subject rAAV virion is desired. Such rAAV virions diffuse away from the site of administration, and thus infect a greater number of cells than rAAV virions with wild-type capsid protein(s). 
     Decreased binding to neutralizing antibodies is advantageous. Neutralizing antibodies bind to wild-type capsid proteins. Binding of neutralizing antibodies to wild-type capsid proteins may have several effects, including limiting the residence time of an rAAV virions that comprises wild-type capsid proteins in the viral particle, preventing the virus from binding to the cell surface, aggregating the virus, induction of structural alterations in the capsid, and prevention of viral disassembly and uncoating (a step necessary to release the DNA). An rAAV particle that has decreased binding to neutralizing antibodies thus has increased capacity to infect cells, and increased residence time in the body of an individual administered with the rAAV virion Thus, the effective duration of delivery of gene product is increased. 
     Increased ability to cross an endothelial cell layer allows the rAAV virion to gain access to tissues and cells that are separated from the site of administration by an endothelial cell layer. For example, the blood-brain barrier, the tumor vasculature, and the cardiovascular system all present endothelial cell layers that form a barrier to access of a particular anatomical site. A subject rAAV virion thus may exhibit one or more of the following properties: 1) increased ability to cross the blood-brain barrier; 2) increased ability to cross the tumor vasculature and infect tumor cells; and 3) increased ability to cross the endothelial layer within the heart. 
     Mutant Adeno-Associated Virus Virions 
     The present invention provides mutant adeno-associated virus comprising mutant capsid proteins that exhibit altered capsid properties. By virtue of comprising one or more mutant capsid proteins, a subject mutant AAV exhibits one or more of the following properties: 1) increased heparin binding affinity relative to wild-type AAV; 2) decreased heparin binding affinity relative to wild-type AAV; 3) increased infectivity of a cell that is resistant to infection with AAV; 4) increased evasion of neutralizing antibodies; and 5) increased ability to cross an endothelial cell layer. The properties of a subject mutant AAV are compared to a corresponding parental, wild-type AAV. Thus, e.g., where the parental, wild-type AAV is AAV-2, and the subject mutant AAV is a mutant of wild-type AAV-2, the properties of the subject mutant is compared to that same property of wild-type AAV-2. 
     Mutants with Increased Heparin Affinity 
     In some embodiments, a capsid protein encoded by a subject mutant AAV exhibits increased binding affinity to heparan sulfate relative to wild-type AAV. In these embodiments, a capsid protein encoded by a subject mutant AAV exhibits at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 2-fold, at least 2.5-fold, at least about 5-fold, at least about 10-fold, at least about 15-fold, at least about 50 fold, at least about 75-fold, or at least about 100-fold or more, higher affinity for heparan sulfate than wild-type AAV capsid. Because heparin is a molecule that is structurally similar to heparan sulfate, heparin is frequently used to determine experimentally whether a capsid protein has altered binding to heparan sulfate. Thus, the terms “heparin binding affinity,” and “heparan sulfate binding affinity,” and similar terms, are used interchangeably herein. 
     For example, whereas the binding affinity of AAV-2 to heparin has a K(d) value of approximately 2.0 nM, a subject mutant AAV has a binding affinity to heparin that is at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 2-fold, at least 2.5-fold, at least about 5-fold, at least about 10-fold, at least about 15-fold, at least about 50 fold, at least about 75-fold, or at least about 100-fold or more, higher than the affinity of wild-type AAV-2 to heparin. 
     Typically, wild-type AAV elutes from a heparin affinity chromatography medium with a NaCl concentration in a range of from about 450 mM to about 550 mM. In some embodiments, a subject mutant AAV elutes from a heparin affinity chromatography medium with a NaCl concentration of greater than about 550 mM, e.g., from about 575 mM NaCl to about 600 mM NaCl, from about 600 mM NaCl to about 625 mM NaCl, from about 625 mM NaCl to about 650 mM NaCl, from about 650 mM NaCl to about 675 mM NaCl, from about 675 mM NaCl to about 700 mM NaCl, from about 700 mM NaCl to about 725 mM NaCl, from about 725 mM NaCl to about 750 mM NaCl, from about 750 mM NaCl to about 775 mM NaCl, or from about 775 mM NaCl to about 800 mM NaCl, or higher. 
     Mutants with Decreased Heparin Affinity 
     In other embodiments, a subject mutant AAV exhibits a lower affinity for heparan sulfate than wild-type AAV. In these embodiments, a subject mutant AAV, when packaged in a viral particle, has at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, or at least about 85% lower affinity for heparin than wild-type AAV (e.g., wild-type AAV-2). In some embodiments, a subject mutant AAV, when packaged into a viral particle, elutes from a heparin affinity chromatography medium with concentration of NaCl in the range of from about 440 mM NaCl to about 400 mM NaCl, from about 400 mM NaCl to about 375 mM NaCl, from about 375 mM NaCl to about 350 mM NaCl, from about 350 mM NaCl to about 325 mM NaCl, from about 325 mM NaCl to about 300 mM NaCl, from about 300 mM NaCl to about 275 mM NaCl, from about 275 mM NaCl to about 250 mM NaCl, from about 250 mM NaCl to about 225 mM NaCl, from about 225 mM NaCl to about 200 mM NaCl or lower. 
     Heparin binding affinity can be determined using any known assay. For example, affinity of variant capsids for heparan sulfate can be measured by binding viral particles to immobilized heparin. See, e.g., Qui et al. (2000)  Virology  269:137-147. 
     Neutralizing Antibody-Evading Mutants 
     In some embodiments, a subject mutant AAV exhibits increased resistance to neutralizing antibodies compared to wild-type AAV (“wt AAV”) or AAV comprising a wild-type capsid protein. In these embodiments, a subject mutant AAV has from about 10-fold to about 10,000-fold greater resistance to neutralizing antibodies than wt AAV, e.g., a subject mutant AAV has from about 10-fold to about 25-fold, from about 25-fold to about 50-fold, from about 50-fold to about 75-fold, from about 75-fold to about 100-fold, from about 100-fold to about 150-fold, from about 150-fold to about 200-fold, from about 200-fold to about 250-fold, from about 250-fold to about 300-fold, at least about 350-fold, at least about 400-fold, from about 400-fold to about 450-fold, from about 450-fold to about 500-fold, from about 500-fold to about 550-fold, from about 550-fold to about 600-fold, from about 600-fold to about 700-fold, from about 700-fold to about 800-fold, from about 800-fold to about 900-fold, from about 900-fold to about 1000-fold, from about 1,000-fold to about 2,000-fold, from about 2,000-fold to about 3,000-fold, from about 3,000-fold to about 4,000-fold, from about 4,000-fold to about 5,000-fold, from about 5,000-fold to about 6,000-fold, from about 6,000-fold to about 7,000-fold, from about 7,000-fold to about 8,000-fold, from about 8,000-fold to about 9,000-fold, or from about 9,000-fold to about 10,000-fold greater resistance to neutralizing antibodies than a wild-type AAV or an AAV comprising a wild-type capsid protein. 
     In some embodiments, a subject mutant AAV exhibits decreased binding to a neutralizing antibody that binds a wild-type AAV capsid protein. For example, a subject mutant AAV exhibits from about 10-fold to about 10,000-fold reduced binding to a neutralizing antibody that binds a wild-type AAV capsid protein. For example, a subject mutant AAV exhibits from about 10-fold to about 25-fold, from about 25-fold to about 50-fold, from about 50-fold to about 75-fold, from about 75-fold to about 100-fold, from about 100-fold to about 150-fold, from about 150-fold to about 200-fold, from about 200-fold to about 250-fold, from about 250-fold to about 300-fold, at least about 350-fold, at least about 400-fold, from about 400-fold to about 450-fold, from about 450-fold to about 500-fold, from about 500-fold to about 550-fold, from about 550-fold to about 600-fold, from about 600-fold to about 700-fold, from about 700-fold to about 800-fold, from about 800-fold to about 900-fold, from about 900-fold to about 1000-fold, from about 1,000-fold to about 2,000-fold, from about 2,000-fold to about 3,000-fold, from about 3,000-fold to about 4,000-fold, from about 4,000-fold to about 5,000-fold, from about 5,000-fold to about 6,000-fold, from about 6,000-fold to about 7,000-fold, from about 7,000-fold to about 8,000-fold, from about 8,000-fold to about 9,000-fold, or from about 9,000-fold to about 10,000-fold reduced binding (e.g., reduced affinity) to a neutralizing antibody that binds a wild-type capsid AAV protein, compared to the binding affinity of the antibody to wild-type AAV capsid protein. 
     In some embodiments, an anti-AAV neutralizing antibody binds to a subject neutralizing antibody escape mutant AAV with an affinity of less than about 10 −7  M, less than about 5×10 −6  M, less than about 10 −6  M, less than about 5×10 −5  M, less than about 10 −5  M, less than about 10 −4  M, or lower. 
     In some embodiments, a subject mutant AAV exhibits increased in vivo residence time compared to a wild-type AAV. For example, a subject mutant AAV exhibits a residence time that is at least about 10%, at least about 25%, at least about 50%, at least about 100%, at least about 3-fold, at least about 5-fold, at least about 10-fold, at least about 25-fold, at least about 50-fold, at least about 100-fold, or more, longer than the residence time of a wild-type AAV. 
     Whether a given mutant AAV exhibits reduced binding to a neutralizing antibody and/or increased resistance to neutralizing antibody can be determined using any known assay, including the assay described in Example 1. For example, mutant AAV is contacted with a permissive cell type, e.g., 293 cells, in the presence of neutralizing antibody. A control sample contains the cells, mutant AAV, and no neutralizing antibody. After a suitable time, the cells are contacted with adenovirus, and AAV particles are detected. The level of AAV particles is compared to the amount of AAV particles that are generated in the absence of neutralizing antibody. 
     Mutants with Increased Infectivity 
     In some embodiments, a subject mutant AAV that exhibits increased infectivity of cells that are non-permissive to infection with AAV, and cells that are less permissive to infection with AAV. Cells that are non-permissive to infection with AAV, and cells that are less permissive to infection with AAV, are collectively referred to herein as “non-permissive cells.” When a population of permissive cells is contacted in vitro or in vivo with AAV at a multiplicity of infection (moi) of 5, from about 70% to about 100% of the cell population becomes infected with the AAV. When a population of non-permissive cells is contacted in vitro or in vivo with AAV at an moi of 5, less than about 70% of the population becomes infected with AAV, e.g., no greater than from about 60% to about 69%, from about 50% to about 60%, from about 40% to about 50%, from about 30% to about 40%, from about 20% to about 30%, from about 10% to about 20%, or from about 1% to about 10%, of the population becomes infected with AAV, and in some cell types, essentially none of the cells becomes infected with AAV. 
     Whether a cell is permissive or non-permissive to infection with AAV can be readily determined by contacting in vitro or in vivo a population of a particular cell type with an rAAV construct that comprises a nucleotide sequence encoding a protein that provides a detectable signal (e.g., a fluorescent protein such as a green fluorescent protein), at an moi of 5. The proportion of cells that become positive for the detectable protein is an indication of the percentage of cells that became infected with the rAAV. Where from about 0% to about 69% of the cells become infected with the rAAV, the cells are said to be non-permissive to infection with AAV. Where from about 70% to about 100% of the cells become infected with the rAAV, the cells are said to be permissive to infection with AAV. Infectivity can be expressed relative to infectivity of 293 cells. In some embodiments, a non-permissive cell exhibits reduced infectivity with AAV compared to 293 cells, e.g., a non-permissive cell exhibits less than about 70% of the infectivity of 293 cells to AAV, e.g., a non-permissive cells exhibits less than about 70%, less than about 60%, less than about 50%, less than about 40%, less than about 30%, less than about 20%, less than about 10%, or less, of the infectivity of 293 cells to AAV. 
     In some embodiments, a subject mutant AAV exhibits increased ability to infect a cell that is relatively refractory to AAV infection (e.g., a non-permissive cell). In these embodiments, a subject mutant AAV exhibits at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 2-fold, at least about 4-fold, at least about 5-fold, at least about 10-fold, at least about 25-fold, or more, greater infectivity of a non-permissive cell than a wild-type AAV. 
     Examples of cells that are relatively refractory to AAV infection include stem cells. Further examples of non-permissive cell types include, but are not limited to, lung epithelial cells, and hepatocytes. 
     The term “stem cell” is used herein to refer to a mammalian cell that has the ability both to self-renew, and to generate differentiated progeny (see, e.g., Morrison et al. (1997)  Cell  88:287-298). Generally, stem cells also have one or more of the following properties: an ability to undergo asynchronous, or symmetric replication, that is where the two daughter cells after division can have different phenotypes; extensive self-renewal capacity; capacity for existence in a mitotically quiescent form; and clonal regeneration of all the tissue in which they exist, for example the ability of hematopoietic stem cells to reconstitute all hematopoietic lineages. “Progenitor cells” differ from stem cells in that they typically do not have the extensive self-renewal capacity, and often can only regenerate a subset of the lineages in the tissue from which they derive, for example only lymphoid, or erythroid lineages in a hematopoietic setting. 
     Stem cells may be characterized by both the presence of markers associated with specific epitopes identified by antibodies and the absence of certain markers as identified by the lack of binding of specific antibodies. Stem cells may also be identified by functional assays both in vitro and in vivo, particularly assays relating to the ability of stem cells to give rise to multiple differentiated progeny. 
     Stem cells of interest include hematopoietic stem cells and progenitor cells derived therefrom (U.S. Pat. No. 5,061,620); neural crest stem cells (see Morrison et al. (1999) Cell 96:737-749); adult neural stem cells and neural progenitor cells; embryonic stem cells; mesenchymal stem cells; mesodermal stem cells; etc. Other hematopoietic “progenitor” cells of interest include cells dedicated to lymphoid lineages, e.g. immature T cell and B cell populations. 
     Structural Features 
     A subject mutant AAV virion comprises a mutation in at least one capsid protein (e.g., at least one of VP1, VP2, and VP3). Thus, at least one of VP1, VP2, and VP3 has at least one amino acid substitution compared to wild-type AAV capsid protein. In some embodiments, at least one of VP1, VP2, and VP3 has from one to about 25 amino acid substitutions compared to wild-type AAV VP1, VP2, and VP3, e.g., from about one to about 5, from about 5 to about 10, from about 10 to about 15, from about 15 to about 20, or from about 20 to about 25 amino acid substitutions compared to wild-type AAV VP1, VP2, and VP3. Alternatively, a subject mutant AAV virion comprises one or more amino acid deletions and/or insertions in at least one capsid protein relative to wild-type capsid protein. In some embodiments, a subject mutant AAV virion comprises one or more amino acid substitutions and/or deletions and/or insertions in a capsid protein relative to a wild-type capsid protein. 
     In some embodiments, a subject mutant AAV virion exhibits reduced binding to neutralizing antibody compared to wild-type AAV, and comprises a VP1 that has an amino acid sequence that has at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99%, or greater, amino acid sequence identity to an amino acid sequence as set forth in one of SEQ ID NOs:7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, and 33, or as set forth in  FIGS. 5A-C  or  FIGS. 7A-D . In some embodiments, a subject mutant AAV virion exhibits reduced binding to neutralizing antibody compared to wild-type AAV, and comprises a VP1 that has an amino acid sequence that has from 1-5, from 5-10, or from 10-20 amino acid differences from an amino acid sequence as set forth in one of SEQ ID NOs:7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, and 33 or as set forth in  FIGS. 5A-C  or  FIGS. 7A-D . In some embodiments, a subject mutant AAV virion exhibits reduced binding to neutralizing antibody compared to wild-type AAV, and comprises a VP1 that has an amino acid sequence as set forth in one of SEQ ID NOs:7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, and 33 or as set forth in  FIGS. 5A-C  or  FIGS. 7A-D . 
     In some embodiments, a subject mutant AAV virion exhibits increased heparan sulfate affinity compared to wild-type AAV, and comprises a VP1 that has an amino acid sequence that has at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99%, or greater, amino acid sequence identity to an amino acid sequence as set forth in SEQ ID NO:35 or SEQ ID NO:37, or as set forth in  FIGS. 9A-C (D14H1 or D14L3). In some embodiments, a subject mutant AAV virion exhibits increased heparan sulfate affinity compared to wild-type AAV, and comprises a VP1 that has an amino acid sequence that has from 1-5, from 5-10, or from 10-20 amino acid differences from an amino acid sequence as set forth in SEQ ID NO:35 or SEQ ID NO:37, or as set forth in  FIGS. 9A-C (D14H1 or D14L3). In some embodiments, a subject mutant AAV virion exhibits increased heparan sulfate affinity compared to wild-type AAV, and comprises a VP1 that has an amino acid sequence as set forth in SEQ ID NO:35 or SEQ ID NO:37, or as set forth in  FIGS. 9A-C (D14H1 or D14L3). 
     In some embodiments, a subject mutant AAV virion exhibits reduced heparan sulfate affinity compared to wild-type AAV, and comprises a VP1 that has an amino acid sequence that has at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99%, or greater, amino acid sequence identity to an amino acid sequence as set forth in SEQ ID NO:39 or SEQ ID NO41, or as set forth in  FIGS. 9A-C (P1BH1 or P1BH2). In some embodiments, a subject mutant AAV virion exhibits reduced heparan sulfate affinity compared to wild-type AAV, and comprises a VP1 that has an amino acid sequence that has from 1-5, from 5-10, or from 10-20 amino acid differences from an amino acid sequence as set forth in SEQ ID NO:39 or SEQ ID NO:41, or as set forth in  FIGS. 9A-C (P1BH1 or P1BH2). In some embodiments, a subject mutant AAV virion exhibits decreased heparan sulfate affinity compared to wild-type AAV, and comprises a VP1 that has an amino acid sequence as set forth in SEQ ID NO:39 or SEQ ID NO:41, or as set forth in  FIGS. 9A-C (P1BH1 or P1BH2). 
     In some embodiments, a subject mutant AAV virion comprises wild-type Rep78, Rep68, Rep52, and Rep40 proteins. In other embodiments, a subject mutant AAV comprises, in addition to one or more mutant capsid proteins, one or more mutations in one or more of Rep78, Rep68, Rep52, and Rep40 proteins. 
     Nucleic Acids and Host Cells 
     The present invention provides nucleic acids comprising nucleotide sequences encoding a mutant AAV capsid protein, as well as host cells comprising a subject nucleic acid. The nucleic acids and host cells are useful for generating rAAV virions, as described below. A subject nucleic acid encodes one or more of VP1, VP2, and VP3 comprising one or more amino acid substitutions. A subject nucleic acid comprises a nucleotide sequence encoding at least one of VP1, VP2, and VP3, wherein the encoded capsid protein comprises from one to about 15 amino acid substitutions compared to a wild-type AAV capsid protein, e.g., from about one to about 5, from about 5 to about 10, from about 10 to about 15, from about 15 to about 20, or from about 20 to about 25 amino acid substitutions compared to a wild-type AAV capsid protein. The encoded capsid protein may, alternatively or in addition, comprise one or more amino acid deletions and/or insertions relative to a wild-type AAV capsid protein. 
     In some embodiments, a subject nucleic acid comprises a nucleotide sequence that comprises from about 1 to about 30 nucleotide differences (e.g., from about 1 to about 5, from about 5 to about 10, from about 10 to about 20, or from about 20 to about 30 nucleotide differences) from a nucleotide sequence as set forth in any one of SEQ ID NOs:2, 3, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, and 40, or any one of the nucleotide sequences set forth in  FIGS. 4A-G ,  FIGS. 6A-J , or  FIGS. 8A-G . In some embodiments, a subject nucleic acid comprises a nucleotide sequence that hybridizes under stringent hybridization conditions to a nucleic acid having a nucleotide sequence as set forth in any one of SEQ ID NOs:2, 3, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, and 40, or any one of the nucleotide sequences set forth in  FIGS. 4A-G ,  FIGS. 6A-J , or  FIGS. 8A-G . In some embodiments, a subject nucleic acid comprises a nucleotide sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or more, identical to a nucleotide sequence as set forth in any one of SEQ ID NOs:2, 3, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, and 40, or any one of the nucleotide sequences set forth in  FIGS. 4A-G ,  FIGS. 6A-J , or  FIGS. 8A-G . In some embodiments, a subject nucleic acid comprises a nucleic acid having a nucleotide sequence as set forth in any one of SEQ ID NOs:2, 3, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, and 40, or any one of the nucleotide sequences set forth in  FIGS. 4A-G ,  FIGS. 6A-J , or  FIGS. 8A-G . 
     The present invention further provides host cells, e.g., isolated host cells, comprising a subject nucleic acid. A subject host cell is typically an isolated cell, e.g., a cell in in vitro culture. A subject host cell is useful for producing a subject rAAV virion, as described below. Where a subject host cell is used to produce a subject rAAV virion, it is referred to as a “packaging cell.” In some embodiments, a subject host cell is stably genetically modified with a subject nucleic acid. In other embodiments, a subject host cell is transiently genetically modified with a subject nucleic acid. 
     A subject nucleic acid is introduced stably or transiently into a host cell, using established techniques, including, but not limited to, electroporation, calcium phosphate precipitation, liposome-mediated transfection, and the like. For stable transformation, a subject nucleic acid will generally further include a selectable marker, e.g., any of several well-known selectable markers such as neomycin resistance, and the like. 
     A subject host cell is generated by introducing a subject nucleic acid into any of a variety of cells, e.g., mammalian cells, including, e.g., murine cells, and primate cells (e.g., human cells). Suitable mammalian cells include, but are not limited to, primary cells and cell lines, where suitable cell lines include, but are not limited to, 293 cells, COS cells, HeLa cells, Vero cells, 3T3 mouse fibroblasts, C3H10T1/2 fibroblasts, CHO cells, and the like. 
     In some embodiments, a subject host cell includes, in addition to a nucleic acid comprising a nucleotide sequence encoding a mutant capsid protein, a nucleic acid that comprises a nucleotide sequence encoding one or more AAV rep proteins. In other embodiments, a subject host cell further comprises an rAAV vector, as described below. As described in more detail below, an rAAV virion is generated using a subject host cell. 
     rAAV Virions 
     A mutant capsid protein may be incorporated into an AAV that comprises a heterologous nucleic acid that provides for production of a heterologous gene product (e.g., a heterologous nucleic acid or a heterologous protein). A subject recombinant AAV virion (“rAAV virion”) comprises a mutant capsid protein, and includes a heterologous nucleic acid that encodes a heterologous gene product. Thus, the present invention provides rAAV virions that comprise a mutant capsid protein, as described above; and a heterologous nucleic acid. A subject rAAV virion is useful for introducing a gene product into an individual 
     A subject rAAV virion comprises a mutant capsid protein, as described above. By virtue of comprising a mutant capsid protein, a subject rAAV virion exhibits one or more of the following properties: 1) increased heparin binding affinity relative to wild-type AAV; 2) decreased heparin binding affinity relative to wild-type AAV; 3) increased infectivity of a cell that is non-permissive to infection with AAV; 4) increased evasion of neutralizing antibodies; and 5) increased ability to cross an endothelial cell layer. 
     In some embodiments, a subject rAAV virion exhibits increased binding affinity to heparin relative to a wild-type AAV virion. In these embodiments, a capsid protein encoded by a subject rAAV virion exhibits at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 2-fold, at least 2.5-fold, at least about 5-fold, at least about 10-fold, at least about 15-fold, at least about 50 fold, at least about 75-fold, or at least about 100-fold or more, higher affinity for heparin than wild-type AAV capsid. Typically, wild-type AAV elutes from a heparin affinity chromatography medium with a NaCl concentration in a range of from about 450 mM to about 550 mM. In some embodiments, a subject rAAV virion elutes from a heparin affinity chromatography medium with a NaCl concentration of greater than about 550 mM, e.g., from about 575 mM NaCl to about 600 mM NaCl, from about 600 mM NaCl to about 625 mM NaCl, from about 625 mM NaCl to about 650 mM NaCl, from about 650 mM NaCl to about 675 mM NaCl, from about 675 mM NaCl to about 700 mM NaCl, from about 700 mM NaCl to about 725 mM NaCl, from about 725 mM NaCl to about 750 mM NaCl, from about 750 mM NaCl to about 775 mM NaCl, or from about 775 mM NaCl to about 800 mM NaCl, or higher. 
     In other embodiments, a subject rAAV virion exhibits a lower affinity for heparin than wild-type AAV. In these embodiments, a subject rAAV virion has at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, or at least about 85% lower affinity for heparin than wild-type AAV. In some embodiments, a subject rAAV virion elutes from a heparin affinity chromatography medium with concentration of NaCl in the range of from about 440 mM NaCl to about 400 mM NaCl, from about 400 mM NaCl to about 375 mM NaCl, from about 375 mM NaCl to about 350 mM NaCl, from about 350 mM NaCl to about 325 mM NaCl, from about 325 mM NaCl to about 300 mM NaCl, from about 300 mM NaCl to about 275 mM NaCl, from about 275 mM NaCl to about 250 mM NaCl, from about 250 mM NaCl to about 225 mM NaCl, from about 225 mM NaCl to about 200 mM NaCl or lower. 
     Heparin binding affinity can be determined using any known assay. For example, affinity of variant capsids for heparan sulfate can be measured by binding viral particles to immobilized heparin. See, e.g., Qui et al. (2000)  Virology  269:137-147. 
     In some embodiments, a subject rAAV virion exhibits increased resistance to neutralizing antibodies compared to wild-type AAV or AAV comprising a wild-typo capsid protein. In these embodiments, a subject rAAV virion has from about 10-fold to about 10,000-fold greater resistance to neutralizing antibodies than wt AAV, e.g., a subject rAAV virion has from about 10-fold to about 25-fold, from about 25-fold to about 50-fold, from about 50-fold to about 75-fold, from about 75-fold to about 100-fold, from about 100-fold to about 150-fold, from about 150-fold to about 200-fold, from about 200-fold to about 250-fold, from about 250-fold to about 300-fold, at least about 350-fold, at least about 400-fold, from about 400-fold to about 450-fold, from about 450-fold to about 500-fold, from about 500-fold to about 550-fold, from about 550-fold to about 600-fold, from about 600-fold to about 700-fold, from about 700-fold to about 800-fold, from about 800-fold to about 900-fold, from about 900-fold to about 1000-fold, from about 1,000-fold to about 2,000-fold, from about 2,000-fold to about 3,000-fold, from about 3,000-fold to about 4,000-fold, from about 4,000-fold to about 5,000-fold, from about 5,000-fold to about 6,000-fold, from about 6,000-fold to about 7,000-fold, from about 7,000-fold to about 8,000-fold, from about 8,000-fold to about 9,000-fold, or from about 9,000-fold to about 10,000-fold greater resistance to neutralizing antibodies than a wild-type AAV or an AAV comprising a wild-type capsid protein. 
     In some embodiments, a subject rAAV virion exhibits decreased binding to a neutralizing antibody that binds a wild-type AAV capsid protein. For example, a subject mutant rAAV virion exhibits from about 10-fold to about 10,000-fold reduced binding to a neutralizing antibody that binds a wild-type AAV capsid protein. For example, a subject mutant rAAV virion exhibits from about 10-fold to about 25-fold, from about 25-fold to about 50-fold, from about 50-fold to about 75-fold, from about 75-fold to about 100-fold, from about 100-fold to about 150-fold, from about 150-fold to about 200-fold, from about 200-fold to about 250-fold, from about 250-fold to about 300-fold, at least about 350-fold, at least about 400-fold, from about 400-fold to about 450-fold, from about 450-fold to about 500-fold, from about 500-fold to about 550-fold, from about 550-fold to about 600-fold, from about 600-fold to about 700-fold, from about 700-fold to about 800-fold, from about 800-fold to about 900-fold, from about 900-fold to about 1000-fold, from about 1,000-fold to about 2,000-fold, from about 2,000-fold to about 3,000-fold, from about 3,000-fold to about 4,000-fold, from about 4,000-fold to about 5,000-fold, from about 5,000-fold to about 6,000-fold, from about 6,000-fold to about 7,000-fold, from about 7,000-fold to about 8,000-fold, from about 8,000-fold to about 9,000-fold, or from about 9,000-fold to about 10,000-fold reduced binding to a neutralizing antibody that binds a wild-type capsid AAV protein, compared to the binding affinity of the antibody to wild-type AAV capsid protein. 
     In some embodiments, an anti-AAV neutralizing antibody binds to a subject rAAV virion with an affinity of less than about 10 −7  M, less than about 5×10 −6  M, less than about 10 −6  M, less than about 5×10 −5  M, less than about 10 −5  M, less than about 10 −4  M, or lower. 
     A subject rAAV virion that exhibits reduced binding to neutralizing antibodies has increased residence time in the body, compared to the residence time of an AAV virion comprising wild-type capsid proteins. Thus, e.g., a subject rAAV virion has at least about 25%, at least about 50%, at least about 75%, at least about 2-fold, at least about 2.5-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 50-fold, or more, increased residence time in vivo compared to the residence time of an AAV virion comprising wild-type capsid proteins. 
     Whether a given mutant rAAV virion exhibits reduced binding to a neutralizing antibody and/or increased resistance to neutralizing antibody can be determined using any known assay, including the assay described in the Example. For example, mutant rAAV virion is contacted with a permissive cell type, e.g., 293 cells, in the presence of neutralizing antibody. A control sample contains the cells, mutant rAAV virion, and no neutralizing antibody. After a suitable time, the cells are contacted with adenovirus, and rAAV particles are detected. The level of rAAV particles is compared to the amount of rAAV particles that are generated in the absence of neutralizing antibody. 
     In some embodiments, a subject rAAV virion exhibits increased ability to infect a cell that is relatively refractory to AAV infection (e.g., a non-permissive cell). In these embodiments, a subject mutant AAV exhibits at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 2-fold, at least about 4-fold, at least about 10-fold, at least about 20-fold, or at least about 50-fold, or more, greater infectivity of a non-permissive cell than a wild-type AAV or an rAAV virion comprising wild-type capsid protein. 
     Examples of cells that are relatively refractory to AAV infection include, but are not limited to stem cells, hepatocytes, and lung epithelial cells. 
     The term “stem cell” is used herein to refer to a mammalian cell that has the ability both to self-renew, and to generate differentiated progeny (see, e.g., Morrison et al. (1997)  Cell  88:287-298). Generally, stem cells also have one or more of the following properties: an ability to undergo asynchronous, or symmetric replication, that is where the two daughter cells after division can have different phenotypes; extensive self-renewal capacity; capacity for existence in a mitotically quiescent form; and clonal regeneration of all the tissue in which they exist, for example the ability of hematopoietic stem cells to reconstitute all hematopoietic lineages. “Progenitor cells” differ from stem cells in that they typically do not have the extensive self-renewal capacity, and often can only regenerate a subset of the lineages in the tissue from which they derive, for example only lymphoid, or erythroid lineages in a hematopoietic setting. 
     Stem cells may be characterized by both the presence of markers associated with specific epitopes identified by antibodies and the absence of certain markers as identified by the lack of binding of specific antibodies. Stem cells may also be identified by functional assays both in vitro and in vivo, particularly assays relating to the ability of stem cells to give rise to multiple differentiated progeny. 
     Stem cells of interest include hematopoietic stem cells and progenitor cells derived therefrom (U.S. Pat. No. 5,061,620); neural crest stem cells (see Morrison et al. (1999) Cell 96:737-749); adult neural stem cells and neural progenitor cells; embryonic stem cells; mesenchymal stem cells; mesodermal stem cells; etc. Other hematopoietic “progenitor” cells of interest include cells dedicated to lymphoid lineages, e.g. immature T cell and B cell populations. 
     In some embodiments, a subject rAAV virion exhibits increased ability to cross an endothelial cell layer. For example, in these embodiments, a subject rAAV virion exhibits at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 2-fold, at least about 5-fold, at least about 10-fold, at least about 25-fold, or at least about 50-fold increase in ability to cross an endothelial cell layer. 
     Whether a given rAAV virion exhibits an increased ability to cross an endothelial cell layer can be determined experimentally using well-known systems. 
     A subject rAAV virion comprises a mutation in at least one capsid protein (e.g., at least one of VP1, VP2, and VP3). Thus, at least one of VP1, VP2, and VP3 has at least one amino acid substitution compared to wild-type AAV capsid protein. In some embodiments, at least one of VP1, VP2, and VP3 has from one to about 25 amino acid substitutions compared to wild-type AAV VP1, VP2, and VP3, e.g., from about one to about 5, from about 5 to about 10, from about 10 to about 15, from about 15 to about 20, or from about 20 to about 25 amino acid substitutions compared to wild-type AAV VP1, VP2, and VP3. Alternatively, a subject rAAV virion comprises one or more amino acid deletions and/or insertions in at least one capsid protein relative to wild-type capsid protein. In some embodiments, a subject rAAV virion comprises one or more amino acid substitutions and/or deletions and/or insertions in a capsid protein relative to a wild-type capsid protein. 
     In some embodiments, a subject rAAV virion exhibits reduced binding to neutralizing antibody compared to wild-type AAV, and comprises a VP1 that has an amino acid sequence that has at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99%, or greater, amino acid sequence identity to an amino acid sequence as set forth in one of SEQ ID NOs:7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, and 33, or as set forth in  FIGS. 5A-C  or  FIGS. 7A-D . In some embodiments, a subject rAAV virion exhibits reduced binding to neutralizing antibody compared to wild-type AAV, and comprises a VP1 that has an amino acid sequence that has from 1-5, from 5-10, or from 10-20 amino acid differences from an amino acid sequence as set forth in one of SEQ ID NOs: 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, and 33, or as set forth in  FIGS. 5A-C  or  FIGS. 7A-D . In some embodiments, a subject rAAV virion exhibits reduced binding to neutralizing antibody compared to wild-type AAV, and comprises a VP1 that has an amino acid sequence as set forth in one of SEQ ID NOs: 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, and 33, or as set forth in  FIGS. 5A-C  or  FIGS. 7A-D . 
     In some embodiments, a subject rAAV virion exhibits increased heparan sulfate affinity compared to wild-type AAV, and comprises a VP1 that has an amino acid sequence that has at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99%, or greater, amino acid sequence identity to an amino acid sequence as set forth in SEQ ID NO35 or SEQ ID NO37, or as set forth in  FIGS. 9A-C (D14H1 or D14L3). In some embodiments, a subject rAAV virion exhibits increased heparan sulfate affinity compared to wild-type AAV, and comprises a VP1 that has an amino acid sequence that has from 1-5, from 5-10, or from 10-20 amino acid differences from an amino acid sequence as set forth in SEQ ID NO35 or SEQ ID NO:37, or as set forth in  FIGS. 9A-C (D14H1 or D14L3). In some embodiments, a subject rAAV virion exhibits increased heparan sulfate affinity compared to wild-type AAV, and comprises a VP1 that has an amino acid sequence as set forth in SEQ ID NO:35 or SEQ ID NO:37, or as set forth in  FIGS. 9A-C (D14H1 or D14L3). 
     In some embodiments, a subject rAAV virion exhibits reduced heparan sulfate affinity compared to wild-type AAV, and comprises a VP1 that has an amino acid sequence that has at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99%, or greater, amino acid sequence identity to an amino acid sequence as set forth in SEQ ID NO39 or SEQ ID NO:41, or as set forth in  FIGS. 9A-C (P1BH1 or P1BH2). In some embodiments, a subject rAAV virion exhibits reduced heparan sulfate affinity compared to wild-type AAV, and comprises a VP1 that has an amino acid sequence that has from 1-5, from 5-10, or from 10-20 amino acid differences from an amino acid sequence as set forth in SEQ ID NO:39 or SEQ ID NO:41, or as set forth in  FIGS. 9A-C (P1BH1 or P1BH2). In some embodiments, a subject rAAV virion exhibits decreased heparan sulfate affinity compared to wild-type AAV, and comprises a VP1 that has an amino acid sequence as set forth in SEQ ID NO:39 or SEQ ID NO:41, or as set forth in  FIGS. 9A-C (P1BH1 or P1BH2). 
     Generation of Subject rAAV Virions 
     By way of introduction, it is typical to employ a host or “producer” cell for rAAV vector replication and packaging. Such a producer cell (usually a mammalian host cell) generally comprises or is modified to comprise several different types of components for rAAV production. The first component is a recombinant adeno-associated viral (rAAV) vector genome (or “rAAV pro-vector”) that can be replicated and packaged into vector particles by the host packaging cell. The rAAV pro-vector will normally comprise a heterologous polynucleotide (or “transgene”), with which it is desired to genetically alter another cell in the context of gene therapy (since the packaging of such a transgene into rAAV vector particles can be effectively used to deliver the transgene to a variety of mammalian cells). The transgene is generally flanked by two AAV inverted terminal repeats (ITRs) which comprise sequences that are recognized during excision, replication and packaging of the AAV vector, as well as during integration of the vector into a host cell genome. 
     A second component is a helper virus that can provide helper functions for AAV replication. Although adenovirus is commonly employed, other helper viruses can also be used as is known in the art. Alternatively, the requisite helper virus functions can be isolated genetically from a helper virus and the encoding genes can be used to provide helper virus functions in trans. The AAV vector elements and the helper virus (or helper virus functions) can be introduced into the host cell either simultaneously or sequentially in any order. 
     The final components for AAV production to be provided in the producer cell are “AAV packaging genes” such as AAV rep and cap genes that provide replication and encapsidation proteins, respectively. Several different versions of AAV packaging genes can be provided (including rep-cap cassettes and separate rep and/or cap cassettes in which the rep and/or cap genes can be left under the control of the native promoters or operably linked to heterologous promoters. Such AAV packaging genes can be introduced either transiently or stably into the host packaging cell, as is known in the art and described in more detail below. 
     1. rAAV Vector 
     A subject rAAV virion, including the heterologous DNA of interest (where “heterologous DNA of interest” is also referred to herein as “heterologous nucleic acid”), can be produced using standard methodology, known to those of skill in the art. The methods generally involve the steps of (1) introducing a subject rAAV vector into a host cell; (2) introducing an AAV helper construct into the host cell, where the helper construct includes AAV coding regions capable of being expressed in the host cell to complement AAV helper functions missing from the AAV vector; (3) introducing one or more helper viruses and/or accessory function vectors into the host cell, wherein the helper virus and/or accessory function vectors provide accessory functions capable of supporting efficient recombinant AAV (“rAAV”) virion production in the host cell; and (4) culturing the host cell to produce rAAV virions. The AAV expression vector, AAV helper construct and the helper virus or accessory function vector(s) can be introduced into the host cell, either simultaneously or serially, using standard transfection techniques. 
     AAV expression vectors are constructed using known techniques to at least provide as operatively linked components in the direction of transcription, control elements including a transcriptional initiation region, the DNA of interest and a transcriptional termination region. The control elements are selected to be functional in a mammalian muscle cell. The resulting construct which contains the operatively linked components is bounded (5′ and 3′) with functional AAV ITR sequences. 
     The nucleotide sequences of AAV ITR regions are known. See, e.g., Kotin, R. M. (1994) Human Gene Therapy 5:793-801; Berns, K. I. “Parvoviridae and their Replication” in Fundamental Virology, 2nd Edition, (B. N. Fields and D. M. Knipe, eds.) for the AAV-2 sequence. AAV ITRs used in the vectors of the invention need not have a wild-type nucleotide sequence, and may be altered, e.g., by the insertion, deletion or substitution of nucleotides. Additionally, AAV ITRs may be derived from any of several AAV serotypes, including without limitation, AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-7, etc. Furthermore, 5′ and 3′ ITRs which flank a selected nucleotide sequence in an AAV expression vector need not necessarily be identical or derived from the same AAV serotype or isolate, so long as they function as intended, i.e., to allow for excision and rescue of the sequence of interest from a host cell genome or vector, and to allow integration of the DNA molecule into the recipient cell genome when AAV Rep gene products are present in the cell. ITRs allow replication of the vector sequence in the presence of an appropriate mixture of Rep proteins. ITRs also allow for the incorporation of the vector sequence into the capsid to generate an AAV particle. 
     A suitable heterologous DNA molecule (also referred to herein as a “heterologous nucleic acid”) for use in a subject rAAV vector will generally be less than about 5 kilobases (kb) in size and will include, for example, a gene (a nucleotide sequence) that encodes a protein that is defective or missing from a recipient subject; a gene that encodes a protein having a desired biological or therapeutic effect (e.g., an antibacterial, antiviral or antitumor function); a nucleotide sequence that encodes an RNA that inhibits or reduces production of a deleterious or otherwise undesired protein; a nucleotide sequence that encodes an antigenic protein; or a nucleotide sequence that encodes an RNA that inhibits or reduces production of a protein. 
     Suitable heterologous nucleic acids include, but are not limited to, those encoding proteins used for the treatment of endocrine, metabolic, hematologic, cardiovascular, neurologic, musculoskeletal, urologic, pulmonary and immune disorders, including such disorders as inflammatory diseases, autoimmune, chronic and infectious diseases, such as acquired immunodeficiency syndrome (AIDS), cancer, hypercholestemia, insulin disorders such as diabetes, growth disorders, various blood disorders including various anemias, thalassemias and hemophilia; genetic defects such as cystic fibrosis, Gaucher&#39;s Disease, Hurler&#39;s Disease, adenosine deaminase (ADA) deficiency, emphysema, or the like. 
     Suitable heterologous nucleic acids include, but are not limited to, those encoding any of a variety of proteins, including, but not limited to: an interferon (e.g., IFN-?, IFN-α, IFN-ß, IFN-?; IFN-t); an insulin (e.g., Novolin, Humulin, Humalog, Lantus, Ultralente, etc.); an erythropoietin (“EPO”; e.g., Procrit®, Eprex®, or Epogen® (epoetin-a); Aranesp® (darbepoietin-a); NeoRecormon®, Epogin® (epoetin-ß); and the like); an antibody (e.g., a monoclonal antibody) (e.g., Rituxan® (rituximab); Remicade® (infliximab); Herceptin® (trastuzumab); Humira™ (adalimumab); Xolair® (omalizumab); Bexxar® (tositumomab); Raptiva™ (efalizumab); Erbitux™ (cetuximab); and the like), including an antigen-binding fragment of a monoclonal antibody; a blood factor (e.g., Activase® (alteplase) tissue plasminogen activator; NovoSeven® (recombinant human factor VIIa); Factor VIIa; Factor VIII (e.g., Kogenate®); Factor IX; ß-globin; hemoglobin; and the like); a colony stimulating factor (e.g., Neupogen® (filgrastim; GCSF); Neulasta (pegfilgrastim); granulocyte colony stimulating factor (G-CSF), granulocyte-monocyte colony stimulating factor, macrophage colony stimulating factor, megakaryocyte colony stimulating factor; and the like); a growth hormone (e.g., a somatotropin, e.g., Genotropin®, Nutropin®, Norditropin®, Saizen®, Serostim®, Humatrope®, etc.; a human growth hormone; and the like); an interleukin (e.g., IL-1; IL-2, including, e.g., Proleukin®; IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9; etc.); a growth factor (e.g., Regranex® (beclapermin; PDGF); Fiblast® (trafermin; bFGF); Stemgen® (ancestim; stem cell factor); keratinocyte growth factor; an acidic fibroblast growth factor, a stem cell factor, a basic fibroblast growth factor, a hepatocyte growth factor; and the like); a soluble receptor (e.g., a TNF-α-binding soluble receptor such as Enbrel® (etanercept); a soluble VEGF receptor; a soluble interleukin receptor; a soluble ?/d T cell receptor; and the like); an enzyme (e.g., a-glucosidase; Cerazyme® (imiglucarase; ß-glucocerebrosidase, Ceredase® (alglucerase); an enzyme activator (e.g., tissue plasminogen activator); a chemokine (e.g., IP-10; Mig; Groa/IL-8, RANTES; MIP-1a; MIP-1ß; MCP-1; PF-4; and the like); an angiogenic agent (e.g., vascular endothelial growth factor (VEGF); an anti-angiogenic agent (e.g., a soluble VEGF receptor); a protein vaccine; a neuroactive peptide such as bradykinin, cholecystokinin, gastin, secretin, oxytocin, gonadotropin-releasing hormone, beta-endorphin, enkephalin, substance P, somatostatin, prolactin, galanin, growth hormone-releasing hormone, bombesin, dynorphin, neurotensin, motilin, thyrotropin, neuropeptide Y, luteinizing hormone, calcitonin, insulin, glucagon, vasopressin, angiotensin II, thyrotropin-releasing hormone, vasoactive intestinal peptide, a sleep peptide, etc.; other proteins such as a thrombolytic agent, an atrial natriuretic peptide, bone morphogenic protein, thrombopoietin, relaxin, glial fibrillary acidic protein, follicle stimulating hormone, a human alpha-1 antitrypsin, a leukemia inhibitory factor, a transforming growth factor, an insulin-like growth factor, a luteinizing hormone, a macrophage activating factor, tumor necrosis factor, a neutrophil chemotactic factor, a nerve growth factor a tissue inhibitor of metalloproteinases; a vasoactive intestinal peptide, angiogenin, angiotropin, fibrin; hirudin; a leukemia inhibitory factor; an IL-1 receptor antagonist (e.g., Kineret® (anakinra)); an ion channel, e.g., cystic fibrosis transmembrane conductance regulator (CFTR); dystrophin; utrophin, a tumor suppressor; lysosomal enzyme acid a-glucosidase (GAA); and the like. Suitable nucleic acids also include those that encode a functional fragment of any of the aforementioned proteins; and nucleic acids that encode functional variants of any of the aforementioned proteins. 
     Suitable heterologous nucleic acids also include those that encode antigenic proteins. A subject rAAV that comprises a heterologous nucleic acid that encodes an antigenic protein is suitable for stimulating an immune response to the antigenic protein in a mammalian host. The antigenic protein is derived from an autoantigen, an allergen, a tumor-associated antigen, a pathogenic virus, a pathogenic bacterium, a pathogenic protozoan, a pathogenic helminth, or any other pathogenic organism that infects a mammalian host. As used herein, the term “a nucleic acid encoding an antigenic protein derived from” includes nucleic acids encoding wild-type antigenic proteins, e.g., a nucleic acid isolated from a pathogenic virus that encodes a viral protein; synthetic nucleic acids generated in the laboratory that encode antigenic proteins that are identical in amino acid sequence to a naturally-occurring antigenic protein; synthetic nucleic acids generated in the laboratory that encode antigenic proteins that differ in amino acid sequence (e.g., by from one amino acid to about 15 amino acids) from a naturally-occurring antigenic protein, but that nonetheless induce an immune response to the corresponding naturally-occurring antigenic protein; synthetic nucleic acids generated in the laboratory that encode fragments of antigenic proteins (e.g., fragments of from about 5 amino acids to about 50 amino acids, which fragments comprises one or more antigenic epitopes), which fragments induce an immune response to the corresponding naturally-occurring antigenic protein; etc. 
     Similarly, an antigenic protein “derived from” an autoantigen, an allergen, a tumor-associated antigen, a pathogenic virus, a pathogenic bacterium, a pathogenic protozoan, a pathogenic helminth, or any other pathogenic organism that infects a mammalian host, includes proteins that are identical in amino acid sequence to a naturally-occurring antigenic protein, and proteins that differ in amino acid sequence (e.g., by from one amino acid to about 15 amino acids) from a naturally-occurring antigenic protein, but that nonetheless induce an immune response to the corresponding naturally-occurring antigenic protein; and fragments of antigenic proteins (e.g., fragments of from about 5 amino acids to about 50 amino acids, which fragments comprises one or more antigenic epitopes), which fragments induce an immune response to the corresponding naturally-occurring antigenic protein. 
     In some embodiments, an immune response to an antigenic protein encoded by a subject rAAV will stimulate a protective immune response to a pathogenic organism that displays the antigenic protein or antigenic epitope (or a protein or an epitope that is cross-reactive with the rAAV-encoded antigenic protein or antigenic epitopes) in the mammalian host. In some embodiments, a cytotoxic T lymphocyte (CTL) response to the rAAV-encoded antigenic protein will be induced in the mammalian host. In other embodiments, a humoral response to the rAAV-encoded antigenic protein will be induced in the mammalian host, such that antibodies specific to the antigenic protein are generated. In many embodiments, a TH1 immune response to the rAAV-encoded antigenic protein will be induced in the mammalian host. Suitable antigenic proteins include tumor-associated antigens, viral antigens, bacterial antigens, and protozoal antigens; and antigenic fragments thereof. In some embodiments, the antigenic protein is derived from an intracellular pathogen. In other embodiments, the antigenic protein is a self-antigen. In yet other embodiments, the antigenic protein is an allergen 
     Tumor-specific antigens include, but are not limited to, any of the various MAGEs (Melanoma-Associated Antigen E), including MAGE 1 (e.g., GenBank Accession No. M77481), MAGE 2 (e.g., GenBank Accession No. U03735), MAGE 3, MAGE 4, etc.; any of the various tyrosinases; mutant ras; mutant p53 (e.g., GenBank Accession No. X54156 and AA494311); and p97 melanoma antigen (e.g., GenBank Accession No. M12154). Other tumor-specific antigens include the Ras peptide and p53 peptide associated with advanced cancers, the HPV 16/18 and E6/E7 antigens associated with cervical cancers, MUCI1-KLH antigen associated with breast carcinoma (e.g., GenBank Accession No. J03651), CEA (carcinoembryonic antigen) associated with colorectal cancer (e.g., GenBank Accession No. X98311), gp100 (e.g., GenBank Accession No. 573003) or MART1 antigens associated with melanoma, and the PSA antigen associated with prostate cancer (e.g., GenBank Accession No. X14810). The p53 gene sequence is known (See e.g., Harris et al. (1986) Mol. Cell. Biol., 6:4650-4656) and is deposited with GenBank under Accession No. M14694. Thus, the present invention can be used as immunotherapeutics for cancers including, but not limited to, cervical, breast, colorectal, prostate, lung cancers, and for melanomas. 
     Viral antigens are derived from known causative agents responsible for diseases including, but not limited to, measles, mumps, rubella, poliomyelitis, hepatitis A, B (e.g., GenBank Accession No. E02707), and C (e.g., GenBank Accession No. E06890), as well as other hepatitis viruses, influenza, adenovirus (e.g., types 4 and 7), rabies (e.g., GenBank Accession No. M34678), yellow fever, Japanese encephalitis (e.g., GenBank Accession No. E07883), dengue (e.g., GenBank Accession No. M24444), hantavirus, and human immunodeficiency virus (e.g., GenBank Accession No. U18552). 
     Suitable bacterial and parasitic antigens include those derived from known causative agents responsible for diseases including, but not limited to, diphtheria, pertussis (e.g., GenBank Accession No. M35274), tetanus (e.g., GenBank Accession No. M64353), tuberculosis, bacterial and fungal pneumonias (e.g.,  Haemophilus influenzae, Pneumocystis carinii , etc.), cholera, typhoid, plague, shigellosis,  salmonellosis  (e.g., GenBank Accession No. L03833), Legionnaire&#39;s Disease, Lyme disease (e.g, GenBank Accession No. U59487), malaria (e.g., GenBank Accession No. X53832), hookworm, onchocerciasis (e.g., GenBank Accession No. M27807), schistosomiasis (e.g., GenBank Accession No. L08198), trypanosomiasis, leshmaniasis, giardiasis (e.g., GenBank Accession No. M33641), amoebiasis, filariasis (e.g., GenBank Accession No. J03266), borreliosis, and trichinosis. 
     Suitable heterologous nucleic acids that encode heterologous gene products include non-translated RNAs, such as an antisense RNA, a ribozyme, an RNAi and an siRNA. Interfering RNA (RNAi) fragments, particularly double-stranded (ds) RNAi, can be used to inhibit gene expression One approach well known in the art for inhibiting gene expression is short interfering RNA (siRNA) mediated gene silencing, where the level of expression product of a target gene is reduced by specific double stranded siRNA nucleotide sequences that are complementary to at least a 19-25 nucleotide long segment (e.g., a 20-21 nucleotide sequence) of the target gene transcript, including the 5′ untranslated (UT) region, the ORF, or the 3′ UT region. In some embodiments, short interfering RNAs are about 19-25 nt in length. See, e.g., PCT applications WO0/44895, WO99/32619, WO01/75164, WO01/92513, WO01/29058, WO01/89304, WO02/16620, and WO02/29858; and U.S. Patent Publication No. 20040023390 for descriptions of siRNA technology. The siRNA can be encoded by a nucleic acid sequence, and the nucleic acid sequence can also include a promoter. The nucleic acid sequence can also include a polyadenylation signal. In some embodiments, the polyadenylation signal is a synthetic minimal polyadelylation signal. 
     Target genes include any gene encoding a target gene product (RNA or protein) that is deleterious (e.g., pathological); a target gene product that is malfunctioning; a target gene product. Target gene products include, but are not limited to, huntingtin; hepatitis C virus; human immunodeficiency virus; amyloid precursor protein; tau; a protein that includes a polyglutamine repeat; a herpes virus (e.g., varicella zoster); any pathological virus; and the like. 
     As such a subject rAAV that includes a heterologous nucleic acid encoding an siRNA is useful for treating a variety of disorders and conditions, including, but not limited to, neurodegenerative diseases, e.g., a trinucleotide-repeat disease, such as a disease associated with polyglutamine repeats, e.g., Huntington&#39;s disease, spinocerebellar ataxia, spinal and bulbar muscular atrophy (SBMA), dentatorubropallidoluysian atrophy (DRPLA), etc.; an acquired pathology (e.g., a disease or syndrome manifested by an abnormal physiological, biochemical, cellular, structural, or molecular biological state) such as a viral infection, e.g., hepatitis that occurs or may occur as a result of an HCV infection, acquired immunodeficiency syndrome, which occurs as a result of an HIV infection; and the like. 
     In many embodiments, a heterologous nucleic acid encoding an siRNA is operably linked to a promoter. Suitable promoters are known those skilled in the art and include the promoter of any protein-encoding gene, e.g., an endogenously regulated gene or a constitutively expressed gene. For example, the promoters of genes regulated by cellular physiological events, e.g., heat shock, oxygen levels and/or carbon monoxide levels, e.g., in hypoxia, may be operably linked to an siRNA-encoding nucleic acid. 
     The selected heterologous nucleotide sequence, such as EPO-encoding or nucleic acid of interest, is operably linked to control elements that direct the transcription or expression thereof in the nucleotide sequence in vivo. Such control elements can comprise control sequences normally associated with the selected gene (e.g., endogenous cellular control elements). Alternatively, heterologous control sequences can be employed. Useful heterologous control sequences generally include those derived from sequences encoding mammalian or viral genes. Examples include, but are not limited to, the SV40 early promoter, mouse mammary tumor virus long terminal repeat (LTR) promoter; adenovirus major late promoter (Ad MLP); a herpes simplex virus (HSV) promoter, an endogenous cellular promoter that is heterologous to the gene of interest, a cytomegalovirus (CMV) promoter such as the CMV immediate early promoter region (CMVIE), a rous sarcoma virus (RSV) promoter, synthetic promoters, hybrid promoters, and the like. In addition, sequences derived from nonviral genes, such as the murine metallothionein gene, will also find use herein. Such promoter sequences are commercially available from, e.g., Stratagene (San Diego, Calif.). 
     In some embodiments, cell type-specific or tissue-specific promoter will be operably linked to the heterologous nucleic acid encoding the heterologous gene product, such that the gene product is produced selectively or preferentially in a particular cell type(s) or tissue(s). In some embodiments, an inducible promoter will be operably linked to the heterologous nucleic acid. 
     For example, muscle-specific and inducible promoters, enhancers and the like, are useful for delivery of a gene product to a muscle cell. Such control elements include, but are not limited to, those derived from the actin and myosin gene families, such as from the myoD gene family; the myocyte-specific enhancer binding factor MEF-2; control elements derived from the human skeletal actin gene and the cardiac actin gene; muscle creatine kinase sequence elements and the murine creatine kinase enhancer (mCK) element; control elements derived from the skeletal fast-twitch troponin C gene, the slow-twitch cardiac troponin C gene and the slow-twitch troponin I gene; hypoxia-inducible nuclear factors; steroid-inducible elements and promoters, such as the glucocorticoid response element (GRE); the fusion consensus element for RU486 induction; and elements that provide for tetracycline regulated gene expression. 
     The AAV expression vector which harbors the DNA molecule of interest (the heterologous DNA) bounded by AAV ITRs, can be constructed by directly inserting the selected sequence(s) into an AAV genome which has had the major AAV open reading frames (“ORFs”) excised therefrom. Other portions of the AAV genome can also be deleted, so long as a sufficient portion of the ITRs remain to allow for replication and packaging functions. Such constructs can be designed using techniques well known in the art. See, e.g., U.S. Pat. Nos. 5,173,414 and 5,139,941; International Publication Nos. WO 92/01070 (published Jan. 23, 1992) and WO 93/03769 (published Mar. 4, 1993); Lebkowski et al. (1988) Molec. Cell. Biol. 8:3988-3996; Vincent et al. (1990) Vaccines 90 (Cold Spring Harbor Laboratory Press); Carter, B. J. (1992) Current Opinion in Biotechnology 3:533-539; Muzyczka, N. (1992) Current Topics in Microbiol. and Immunol. 158:97-129; Kotin, R. M. (1994) Human Gene Therapy 5:793-801; Shelling and Smith (1994) Gene Therapy 1:165-169; and Zhou et al. (1994) J. Exp. Med. 179:1867-1875. 
     Alternatively, AAV ITRs can be excised from the viral genome or from an AAV vector containing the same and fused 5′ and 3′ of a selected nucleic acid construct that is present in another vector using standard ligation techniques, such as those described in Sambrook et al., supra. For example, ligations can be accomplished in 20 mM Tris-Cl pH 7.5, 10 mM MgCl 2 , 10 mM DTT, 33 μg/ml BSA, 10 mM-50 mM NaCl, and either 40 μM ATP, 0.01-0.02 (Weiss) units T4 DNA ligase at 0° C. to 16° C. (for “sticky end” ligation) or 1 mM ATP, 0.3-0.6 (Weiss) units T4 DNA ligase at 14° C. (for “blunt end” ligation). Intermolecular “sticky end” ligations are usually performed at 30-100 μg/ml total DNA concentrations (5-100 nM total end concentration). AAV vectors which contain ITRs have been described in, e.g., U.S. Pat. No. 5,139,941. In particular, several AAV vectors are described therein which are available from the American Type Culture Collection (“ATCC”) under Accession Numbers 53222, 53223, 53224, 53225 and 53226. 
     Additionally, chimeric genes can be produced synthetically to include AAV ITR sequences arranged 5′ and 3′ of one or more selected nucleic acid sequences. Preferred codons for expression of the chimeric gene sequence in mammalian muscle cells can be used. The complete chimeric sequence is assembled from overlapping oligonucleotides prepared by standard methods. See, e.g., Edge, Nature (1981) 292:756; Nambair et al. Science (1984) 223:1299; Jay et al. J. Biol. Chem. (1984) 259:6311. 
     In order to produce rAAV virions, an AAV expression vector is introduced into a suitable host cell using known techniques, such as by transfection. A number of transfection techniques are generally known in the art. See, e.g., Graham et al. (1973) Virology, 52:456, Sambrook et al. (1989) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratories, New York, Davis et al. (1986) Basic Methods in Molecular Biology, Elsevier, and Chu et al. (1981) Gene 13:197. Particularly suitable transfection methods include calcium phosphate co-precipitation (Graham et al. (1973) Virol. 52:456-467), direct micro-injection into cultured cells (Capecchi, M. R. (1980) Cell 22:479-488), electroporation (Shigekawa et al. (1988) BioTechnigues 6:742-751), liposome mediated gene transfer (Mannino et al. (1988) BioTechniques 6:682-690), lipid-mediated transduction (Felgner et al. (1987) Proc. Natl. Acad. Sci. USA 84:7413-7417), and nucleic acid delivery using high-velocity microprojectiles (Klein et al. (1987) Nature 327:70-73). 
     For the purposes of the invention, suitable host cells for producing rAAV virions include microorganisms, yeast cells, insect cells, and mammalian cells, that can be, or have been, used as recipients of a heterologous DNA molecule. The term includes the progeny of the original cell which has been transfected. Thus, a “host cell” as used herein generally refers to a cell which has been transfected with an exogenous DNA sequence. Cells from the stable human cell line, 293 (readily available through, e.g., the American Type Culture Collection under Accession Number ATCC CRL1573) are used in many embodiments. Particularly, the human cell line 293 is a human embryonic kidney cell line that has been transformed with adenovirus type-5 DNA fragments (Graham et al. (1977) J. Gen. Virol. 36:59), and expresses the adenoviral E1a and E1b genes (Aiello et al. (1979) Virology 94:460). The 293 cell line is readily transfected, and provides a particularly convenient platform in which to produce rAAV virions. 
     2. AAV Helper Functions 
     Host cells containing the above-described AAV expression vectors must be rendered capable of providing AAV helper functions in order to replicate and encapsidate the nucleotide sequences flanked by the AAV ITRs to produce rAAV virions. AAV helper functions are generally AAV-derived coding sequences which can be expressed to provide AAV gene products that, in turn, function in trans for productive AAV replication. AAV helper functions are used herein to complement necessary AAV functions that are missing from the AAV expression vectors. Thus, AAV helper functions include one, or both of the major AAV ORFs, namely the rep and cap coding regions, or functional homologues thereof. In the context of the instant invention, the cap functions include one or more mutant capsid proteins, wherein at least one capsid protein comprises at least one mutation, as described above. 
     By “AAV rep coding region” is meant the art-recognized region of the AAV genome which encodes the replication proteins Rep 78, Rep 68, Rep 52 and Rep 40. These Rep expression products have been shown to possess many functions, including recognition, binding and nicking of the AAV origin of DNA replication, DNA helicase activity and modulation of transcription from AAV (or other heterologous) promoters. The Rep expression products are collectively required for replicating the AAV genome. For a description of the AAV rep coding region, see, e.g., Muzyczka, N. (1992) Current Topics in Microbiol. and Immunol. 158:97-129; and Kotin, R. M. (1994) Human Gene Therapy 5:793-801. Suitable homologues of the AAV rep coding region include the human herpesvirus 6 (HHV-6) rep gene which is also known to mediate AAV-2 DNA replication (Thomson et al. (1994) Virology 204:304-311). 
     AAV cap proteins include VP1, VP2, and VP3, wherein at least one of VP1, VP2, and VP3 comprises at least one mutation, as described above. 
     AAV helper functions are introduced into the host cell by transfecting the host cell with an AAV helper construct either prior to, or concurrently with, the transfection of the AAV expression vector. AAV helper constructs are thus used to provide at least transient expression of AAV rep and/or cap genes to complement missing AAV functions that are necessary for productive AAV infection. AAV helper constructs lack AAV ITRs and can neither replicate nor package themselves. These constructs can be in the form of a plasmid, phage, transposon, cosmid, virus, or virion. A number of AAV helper constructs have been described, such as the commonly used plasmids pAAV/Ad and pIM29+45 which encode both Rep and Cap expression products. See, e.g., Samulski et al. (1989) J. Virol. 63:3822-3828; and McCarty et al. (1991) J. Virol. 65:2936-2945. A number of other vectors have been described which encode Rep and/or Cap expression products. See, e.g., U.S. Pat. No. 5,139,941. 
     Both AAV expression vectors and AAV helper constructs can be constructed to contain one or more optional selectable markers. Suitable markers include genes which confer antibiotic resistance or sensitivity to, impart color to, or change the antigenic characteristics of those cells which have been transfected with a nucleic acid construct containing the selectable marker when the cells are grown in an appropriate selective medium. Several selectable marker genes that are useful in the practice of the invention include the hygromycin B resistance gene (encoding Aminoglycoside phosphotranferase (APH)) that allows selection in mammalian cells by conferring resistance to hygromycin; the neomycin phosphotranferase gene (encoding neomycin phosphotransferase) that allows selection in mammalian cells by conferring resistance to G418; and the like. Other suitable markers are known to those of skill in the art. 
     3. AAV Accessory Functions 
     The host cell (or packaging cell) must also be rendered capable of providing non AAV derived functions, or “accessory functions,” in order to produce rAAV virions. Accessory functions are non AAV derived viral and/or cellular functions upon which AAV is dependent for its replication. Thus, accessory functions include at least those non AAV proteins and RNAs that are required in AAV replication, including those involved in activation of AAV gene transcription, stage specific AAV mRNA splicing, AAV DNA replication, synthesis of Cap expression products and AAV capsid assembly. Viral-based accessory functions can be derived from any of the known helper viruses. 
     Particularly, accessory functions can be introduced into and then expressed in host cells using methods known to those of skill in the art. Commonly, accessory functions are provided by infection of the host cells with an unrelated helper virus. A number of suitable helper viruses are known, including adenoviruses; herpesviruses such as herpes simplex virus types 1 and 2; and vaccinia viruses. Nonviral accessory functions will also find use herein, such as those provided by cell synchronization using any of various known agents. See, e.g., Buller et al. (1981) J. Virol. 40:241-247; McPherson et al. (1985) Virology 147:217-222; Schlehofer et al. (1986) Virology 152:110-117. 
     Alternatively, accessory functions can be provided using an accessory function vector. Accessory function vectors include nucleotide sequences that provide one or more accessory functions. An accessory function vector is capable of being introduced into a suitable host cell in order to support efficient AAV virion production in the host cell. Accessory function vectors can be in the form of a plasmid, phage, transposon, cosmid, or another virus. Accessory vectors can also be in the form of one or more linearized DNA or RNA fragments which, when associated with the appropriate control elements and enzymes, can be transcribed or expressed in a host cell to provide accessory functions. 
     Nucleic acid sequences providing the accessory functions can be obtained from natural sources, such as from the genome of an adenovirus particle, or constructed using recombinant or synthetic methods known in the art. In this regard, adenovirus-derived accessory functions have been widely studied, and a number of adenovirus genes involved in accessory functions have been identified and partially characterized. See, e.g., Carter, B. J. (1990) “Adeno-Associated Virus Helper Functions,” in CRC Handbook of Parvoviruses, vol. I (P. Tijssen, ed.), and Muzyczka, N. (1992) Curr. Topics. Microbiol. and Immun. 158:97-129. Specifically, early adenoviral gene regions E1a, E2a, E4, VAI RNA and, possibly, E1b are thought to participate in the accessory process. Janik et al. (1981) Proc. Natl. Acad. Sci. USA 78:1925-1929. Herpesvirus-derived accessory functions have been described. See, e.g., Young et al. (1979) Prog. Med. Virol. 25:113. Vaccinia virus-derived accessory functions have also been described. See, e.g., Carter, B. J. (1990), supra., Schlehofer et al. (1986) Virology 152:110-117. 
     As a consequence of the infection of the host cell with a helper virus, or transfection of the host cell with an accessory function vector, accessory functions are expressed which transactivate the AAV helper construct to produce AAV Rep and/or Cap proteins. The Rep expression products excise the recombinant DNA (including the DNA of interest, e.g., the heterologous nucleic acid) from the AAV expression vector. The Rep proteins also serve to duplicate the AAV genome. The expressed Cap proteins assemble into capsids, and the recombinant AAV genome is packaged into the capsids. Thus, productive AAV replication ensues, and the DNA is packaged into rAAV virions. 
     Following recombinant AAV replication, rAAV virions can be purified from the host cell using a variety of conventional purification methods, such as CsCl gradients. Further, if infection is employed to express the accessory functions, residual helper virus can be inactivated, using known methods. For example, adenovirus can be inactivated by heating to temperatures of approximately 60° C. for, e.g., 20 minutes or more. This treatment effectively inactivates only the helper virus since AAV is extremely heat stable while the helper adenovirus is heat labile. 
     The resulting rAAV virions are then ready for use for DNA delivery, such as in gene therapy applications, or for the delivery of a gene product to a mammalian host. 
     Delivery of a Gene Product 
     The present invention further provides methods of delivering a gene product to an individual in need thereof. The methods generally involve introducing a subject rAAV virion into an individual. 
     Generally, rAAV virions are introduced into a cell using either in vivo or in vitro transduction techniques. If transduced in vitro, the desired recipient cell will be removed from the subject, transduced with rAAV virions and reintroduced into the subject. Alternatively, syngeneic or xenogeneic cells can be used where those cells will not generate an inappropriate immune response in the subject. 
     Suitable methods for the delivery and introduction of transduced cells into a subject have been described. For example, cells can be transduced in vitro by combining recombinant AAV virions with cells e.g., in appropriate media, and screening for those cells harboring the DNA of interest using conventional techniques such as Southern blots and/or PCR, or by using selectable markers. Transduced cells can then be formulated into pharmaceutical compositions, described more fully below, and the composition introduced into the subject by various techniques, such as by intramuscular, intravenous, subcutaneous and intraperitoneal injection. 
     For in vivo delivery, the rAAV virions will be formulated into pharmaceutical compositions and will generally be administered parenterally (e.g., administered via an intramuscular, subcutaneous, intratumoral, transdermal, intrathecal, etc., route of administration. 
     Pharmaceutical compositions will comprise sufficient genetic material to produce a therapeutically effective amount of the gene product of interest, i.e., an amount sufficient to reduce or ameliorate symptoms of the disease state in question or an amount sufficient to confer the desired benefit. The pharmaceutical compositions will also contain a pharmaceutically acceptable excipient. Such excipients include any pharmaceutical agent that does not itself induce the production of antibodies harmful to the individual receiving the composition, and which may be administered without undue toxicity. Pharmaceutically acceptable excipients include, but are not limited to, liquids such as water, saline, glycerol and ethanol. Pharmaceutically acceptable salts can be included therein, for example, mineral acid salts such as hydrochlorides, hydrobromides, phosphates, sulfates, and the like; and the salts of organic acids such as acetates, propionates, malonates, benzoates, and the like. Additionally, auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, and the like, may be present in such vehicles. A wide variety of pharmaceutically acceptable excipients are known in the art and need not be discussed in detail herein. Pharmaceutically acceptable excipients have been amply described in a variety of publications, including, for example, A. Gennaro (2000) “Remington: The Science and Practice of Pharmacy,” 20th edition, Lippincott, Williams, &amp; Wilkins; Pharmaceutical Dosage Forms and Drug Delivery Systems (1999) H. C. Ansel et al., eds., 7 th  ed., Lippincott, Williams, &amp; Wilkins; and Handbook of Pharmaceutical Excipients (2000) A. H. Kibbe et al., eds., 3 rd  ed. Amer. Pharmaceutical Assoc. 
     Appropriate doses will depend on the mammal being treated (e.g., human or nonhuman primate or other mammal), age and general condition of the subject to be treated, the severity of the condition being treated, the particular therapeutic protein in question, its mode of administration, among other factors. An appropriate effective amount can be readily determined by one of skill in the art. 
     Thus, a “therapeutically effective amount” will fall in a relatively broad range that can be determined through clinical trials. For example, for in vivo injection, i.e., injection directly to skeletal or cardiac muscle, a therapeutically effective dose will be on the order of from about 10 6  to about 10 15  of the rAAV virions, e.g., from about 10 8  to 10 12  rAAV virions. For in vitro transduction, an effective amount of rAAV virions to be delivered to cells will be on the order of from about 10 8  to about 10 13  of the rAAV virions. Other effective dosages can be readily established by one of ordinary skill in the art through routine trials establishing dose response curves. 
     Dosage treatment may be a single dose schedule or a multiple dose schedule. Moreover, the subject may be administered as many doses as appropriate. One of skill in the art can readily determine an appropriate number of doses. 
     In some embodiments, the present invention provides methods of delivering a gene product to a stem cell. In these embodiments, a subject rAAV virion is introduced into a stem cell, either in vitro or in vivo. Stem cells of interest include hematopoietic stem cells and progenitor cells derived therefrom (U.S. Pat. No. 5,061,620); neural crest stem cells (see Morrison et al. (1999) Cell 96:737-749); adult neural stem cells or neural progenitor cells; embryonic stem cells; mesenchymal stem cells; mesodermal stem cells; etc. Other hematopoietic “progenitor” cells of interest include cells dedicated to lymphoid lineages, e.g. immature T cell and B cell populations. 
     Purified populations of stem or progenitor cells may be used. For example, human hematopoietic stem cells may be positively selected using antibodies specific for CD34, thy-1; or negatively selected using lineage specific markers which may include glycophorin A, CD3, CD24, CD16, CD14, CD38, CD45RA, CD36, CD2, CD19, CD56, CD66a, and CD66b; T cell specific markers, tumor specific markers, etc. Markers useful for the separation of mesodermal stem cells include Fc?RII, Fc?RIII, Thy-1, CD44, VLA-4a, LFA-1ß, HSA, ICAM-1, CD45, Aa4.1, Sca-1, etc. Neural crest stem cells may be positively selected with antibodies specific for low-affinity nerve growth factor receptor (LNGFR), and negatively selected for the markers sulfatide, glial fibrillary acidic protein (GFAP), myelin protein P o , peripherin and neurofilament. Human mesenchymal stem cells may be positively separated using the markers SH2, SH3 and SH4. 
     The cells of interest are typically mammalian, where the term refers to any animal classified as a mammal, including humans, domestic and farm animals, and zoo, laboratory, sports, or pet animals, such as dogs, horses, cats, cows, mice, rats, rabbits, etc. In some embodiments, the stem cell is a human stem cell. 
     The cells which are employed may be fresh, frozen, or have been subject to prior culture. They may be fetal, neonate, adult. Hematopoietic cells may be obtained from fetal liver, bone marrow, blood, particularly G-CSF or GM-CSF mobilized peripheral blood, or any other conventional source. The manner in which the stem cells are separated from other cells of the hematopoietic or other lineage is not critical to this invention. As described above, a substantially homogeneous population of stem or progenitor cells may be obtained by selective isolation of cells free of markers associated with differentiated cells, while displaying epitopic characteristics associated with the stem cells. 
     Any of a variety of proteins can be delivered to an individual using a subject method. Suitable proteins include, but are not limited to, an interferon (e.g., IFN-?, IFN-a, IFN-ß, IFN-?; IFN-t); an insulin (e.g., Novolin, Humulin, Humalog, Lantus, Ultralente, etc.); an erythropoietin (“EPO”; e.g., Procrit®, Eprex®, or Epogen® (epoetin-a); Aranesp® (darbepoietin-a); NeoRecormon®, Epogin® (epoetin-ß); and the like); an antibody (e.g., a monoclonal antibody) (e.g., Rituxan® (rituximab); Remicade® (infliximab); Herceptin® (trastuzumab); Humira™ (adalimumab); Xolair® (omalizumab); Bexxar® (tositumomab); Raptiva™ (efalizumab); Erbitux™ (cetuximab); and the like), including an antigen-binding fragment of a monoclonal antibody; a blood factor (e.g., Activase® (alteplase) tissue plasminogen activator; NovoSeven® (recombinant human factor VIIa); Factor VIIa; Factor VIII (e.g., Kogenate®); Factor IX; ß-globin; hemoglobin; and the like); a colony stimulating factor (e.g., Neupogen® (filgrastim; GCSF); Neulasta (pegfilgrastim); granulocyte colony stimulating factor (G-CSF), granulocyte-monocyte colony stimulating factor, macrophage colony stimulating factor, megakaryocyte colony stimulating factor; and the like); a growth hormone (e.g., a somatotropin, e.g., Genotropin®, Nutropin®, Norditropin®, Saizen®, Serostim®, Humatrope®, etc.; a human growth hormone; and the like); an interleukin (e.g., IL-1; IL-2, including, e.g., Proleukin®; IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9; etc.); a growth factor (e.g., Regranex® (beclapermin; PDGF); Fiblast® (trafermin; bFGF); Stemgen® (ancestim; stem cell factor); keratinocyte growth factor; an acidic fibroblast growth factor, a stem cell factor, a basic fibroblast growth factor, a hepatocyte growth factor; and the like); a soluble receptor (e.g., a TNF-α-binding soluble receptor such as Enbrel® (etanercept); a soluble VEGF receptor; a soluble interleukin receptor; a soluble ?/d T cell receptor; and the like); an enzyme (e.g., a-glucosidase; Cerazyme® (imiglucarase; ß-glucocerebrosidase, Ceredase® (alglucerase); an enzyme activator (e.g., tissue plasminogen activator); a chemokine (e.g., IP-10; Mig; Groa/IL-8, RANTES; MIP-1a; MIP-1ß; MCP-1; PF-4; and the like); an angiogenic agent (e.g., vascular endothelial growth factor (VEGF); an anti-angiogenic agent (e.g., a soluble VEGF receptor); a protein vaccine; a neuroactive peptide such as bradykinin, cholecystokinin, gastin, secretin, oxytocin, gonadotropin-releasing hormone, beta-endorphin, enkephalin, substance P, somatostatin, prolactin, galanin, growth hormone-releasing hormone, bombesin, dynorphin, neurotensin, motilin, thyrotropin, neuropeptide Y, luteinizing hormone, calcitonin, insulin, glucagon, vasopressin, angiotensin II, thyrotropin-releasing hormone, vasoactive intestinal peptide, a sleep peptide, etc.; other proteins such as a thrombolytic agent, an atrial natriuretic peptide, bone morphogenic protein, thrombopoietin, relaxin, glial fibrillary acidic protein, follicle stimulating hormone, a human alpha-1 antitrypsin, a leukemia inhibitory factor, a transforming growth factor, an insulin-like growth factor, a luteinizing hormone, a macrophage activating factor, tumor necrosis factor, a neutrophil chemotactic factor, a nerve growth factor a tissue inhibitor of metalloproteinases; a vasoactive intestinal peptide, angiogenin, angiotropin, fibrin; hirudin; a leukemia inhibitory factor; an IL-1 receptor antagonist (e.g., Kineret® (anakinra)); an ion channel, e.g., cystic fibrosis transmembrane conductance regulator (CFTR); dystrophin; utrophin, a tumor suppressor; lysosomal enzyme acid a-glucosidase (GAA); and the like. Proteins that can be delivered using a subject method also include a functional fragment of any of the aforementioned proteins; and functional variants of any of the aforementioned proteins. 
     In some embodiments, a therapeutically effective amount of a protein is produced in the mammalian host. Whether a therapeutically effective amount of a particular protein is produced in the mammalian host using a subject method is readily determined using assays appropriate to the particular protein. For example, where the protein is EPO, hematocrit is measured. 
     Where the rAAV encodes an antigenic protein, suitable antigenic proteins that can be delivered to an individual using a subject method include, but are not limited to, tumor-associated antigens, autoantigens (“self” antigens), viral antigens, bacterial antigens, protozoal antigens, and allergens; and antigenic fragments thereof. In some embodiments, a cytotoxic T lymphocyte (CTL) response to the rAAV-encoded antigenic protein will be induced in the mammalian host. In other embodiments, a humoral response to the rAAV-encoded antigenic protein will be induced in the mammalian host, such that antibodies specific to the antigenic protein are generated. In many embodiments, a TH1 immune response to the rAAV-encoded antigenic protein will be induced in the mammalian host. Whether an immune response to the antigenic protein has been generated is readily determined using well-established methods. For example, an enzyme-linked immunosorbent assay can be used to determine whether antibody to an antigenic protein has been generated. Methods of detecting antigen-specific CTL are well known in the art. For example, a detectably labeled target cell expressing the antigenic protein on its surface is used to assay for the presence of antigen-specific CTL in a blood sample. 
     Nucleic acids that can be delivered to an individual using a subject method include non-translated RNAs, such as an antisense RNA, a ribozyme, an RNAi and an siRNA. In some embodiments, a therapeutically effective amount of the non-translated RNA is produced in the mammalian host. Whether a therapeutically effective amount of a non-translated RNA has been delivered to a mammalian host using a subject method is readily determined using any appropriate assay. For example, where the gene product is an siRNA that inhibits HIV, viral load can be measured. 
     Methods of Generating Mutant AAV Virions 
     The present invention provides a method of generating a mutant AAV virion comprising one or more mutations in one or more of VP1, VP2, and VP3. The method generally involves generating a mutant AAV library; and selecting the library for capsid mutants with altered capsid properties. The present invention further provides mutant AAV libraries, and compositions comprising the mutant AAV libraries. 
     In some embodiments, a given selection step is repeated two, three, four, or more times to enrich a subject AAV library for altered capsid properties. In some embodiments, following selection of an AAV library, individual clones are isolated and sequenced. 
     Generation of Mutant AAV Library 
     A mutant AAV library is generated that comprises one or more mutations in an AAV cap gene. Mutations in the AAV cap gene are generated using any known method. Suitable methods for mutagenesis of an AAV cap gene include, but are not limited to, a polymerase chain reaction (PCR)-based method, oligonucleotide-directed mutagenesis, and the like. Methods for generating mutations are well described in the art. See, e.g., Zhao et al. (1998)  Nat. Biotechnol.  16:234-235; U.S. Pat. Nos. 6,579,678; 6,573,098; and 6,582,914. 
     In some embodiments, a mutant AAV library comprising mutations in the cap gene will be generated using a staggered extension process. The staggered extension process involves amplification of the cap gene using a PCR-based method. The template cap gene is primed using specific PCR primers, followed by repeated cycles of denaturation and very short annealing/polymerase-catalyzed extension. In each cycle, the growing fragments anneal to different templates based on sequence complementarity and extend further. The cycles of denaturation, annealing, and extension are repeated until full-length sequences form. The resulting full-length sequences include at least one mutation in the cap gene compared to a wild-type AAV cap gene. 
     The PCR products comprising AAV cap sequences that include one or more mutations are inserted into a plasmid containing a wild-type AAV genome. The result is a library of AAV cap mutants. Thus, the present invention provides a mutant AAV cap gene library comprising from about 10 to about 10 10  members, and comprising mutations in the AAV cap gene. A given member of the library has from about one to about 50 mutations in the AAV cap gene. A subject library comprises from 10 to about 10 9  distinct members, each having a different mutation(s) in the AAV cap gene. 
     Once a cap mutant library is generated, viral particles are produced that can then be selected on the basis of altered capsid properties. Library plasmid DNA is transfected into a suitable host cell (e.g., 293 cells), followed by introduction into the cell of helper virus. Viral particles produced by the transfected host cells (“AAV library particles) are collected. 
     Library Selection 
     Once a library is generated, it is selected for a particular capsid property. Viral particles are generated as discussed above, and subjected to one or more selection steps. Capsid properties that are selected for include, but are not limited to: 1) increased heparin binding affinity relative to wild-type AAV; 2) decreased heparin binding affinity relative to wild-type AAV; 3) increased infectivity of a cell that is resistant to infection with AAV; 4) increased evasion of neutralizing antibodies; and 5) increased ability to cross an endothelial cell layer. 
     1. Selection for Altered Heparin Binding 
     In some embodiments, a subject library is selected for altered heparin binding, including increased heparin binding and decreased heparin binding relative to wild-type AAV virion heparin binding. AAV library particles are contacted with a heparin affinity matrix. For example, AAV library particles are loaded onto a heparin affinity column under conditions that permit binding of the AAV library particles to the heparin. Exemplary conditions include equilibration of the column with 0.15 M NaCl and 50 mM Tris at pH 7.5. After allowing the AAV library particle to bind to the heparin affinity matrix, the AAV library particle/heparin affinity matrix complex is washed with volumes of buffer containing progressively increasing concentrations of NaCl, and at each NaCl concentration, eluted AAV library particles are collected. For example, after binding the AAV library particle/heparin affinity matrix complex is washed with a volume of 50 mM Tris buffer, pH 7.5, containing 200 mM NaCl, and eluted AAV library particles are collected. The elution step is repeated with a 50 mM Tris buffer, pH 7.5, containing about 250 mM NaCl, about 300 mM NaCl, about 350 mM, about 400 mM NaCl, about 450 mM NaCl, about 500 mM NaCl, about 550 mM NaCl, about 600 mM NaCl, about 650 mM NaCl, about 700 mM NaCl, or about 750 mM NaCl 
     AAV library particles that elute at NaCl concentrations lower than about 450 mM NaCl exhibit decreased heparin binding properties relative to wild-type AAV. AAV library particles that elute at NaCl concentrations higher than about 550 mM NaCl exhibit increased heparin binding properties relative to wild-type AAV. 
     In some embodiments, eluted AAV library particles are amplified by co-infection of permissive cells with a helper virus, and are re-fractionated on heparin affinity matrix. This step can be repeated a number of times to enrich for AAV library particles with altered heparin binding properties. 
     2. Selection for Reduced Binding to Neutralizing Antibodies 
     In some embodiments, a subject AAV library is selected for reducing binding to neutralizing antibodies that bind to an neutralize wild-type AAV virions, compared to the binding of such antibodies to wild-type AAV virions and neutralization of wild-type AAV virions. AAV library particles are contacted with neutralizing antibodies and the ability of the AAV library particles to infect a permissive host cell is tested. Typically, AAV library particles are contacted with various concentrations of neutralizing antibodies. The higher the concentration of neutralizing antibodies that is required to reduce infectivity of the AAV library particles, the more resistant the AAV particles are to neutralization. 
     3. Selection for Increased Infectivity of Non-Permissive Cells 
     In some embodiments, a subject AAV library is selected for increased infectivity of non-permissive cells. AAV library particles are contacted with a non-permissive cell (e.g., a population of non-permissive cells). After a suitable amount of time to allow for infection of the cells with AAV library particles, helper virus is added, and AAV library particles that successfully infected the non-permissive cell(s) are harvested. In some embodiments, the cycle of infection, addition of helper virus, and harvesting of AAV particles is repeated one, two, three, or more times. 
     In the present methods, one or more selection steps may follow generation of AAV library particles. For example, in some embodiments, the method comprises selecting for increased heparin binding, followed by selecting for decreased binding to neutralizing antibodies. In other embodiments, the method comprises selecting for decreased binding to neutralizing antibodies, followed by selecting for increased heparin binding. In other embodiments, the method comprises selecting for decreased heparin binding, followed by selecting for decreased binding to neutralizing antibodies. In other embodiments, the method comprises selecting for decreased binding to neutralizing antibodies, followed by selecting for decreased heparin binding. In other embodiments, the method comprises selecting for decreased binding to neutralizing antibodies, followed by selecting for increased infectivity of a stem cell. 
     Thus, the present invention provides an adeno-associated virus (AAV) library, that includes a plurality of nucleic acids, each of which nucleic acids include a nucleotide sequence that encodes a mutant AAV capsid protein. The encoded mutant AAV capsid protein includes at least one amino acid substitution relative to a wild-type AAV capsid protein. The present invention provides a library of mutant adeno-associated virus (AAV) particles, including a plurality of AAV particles each of which includes an AAV capsid protein that includes at least one amino acid substitution relative to a wild-type AAV capsid protein. Nucleic acids encoding mutant AAV capsid proteins are described above, as are the properties of the encoded mutant AAV capsid proteins. 
     EXAMPLES 
     The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g. amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Centigrade, and pressure is at or near atmospheric. Standard abbreviations may be used, e.g., bp, base pair(s); kb, kilobase(s); pl, picoliter(s); s, second(s); min, minute(s); hr, hour(s); and the like. 
     Example 1: Generation and Characterization of AAV Capsid Variants 
     Methods 
     Library Generation and Vector Packaging 
     An AAV2 cap ORF genetic library was generated using the staggered extension process described by Zhao at al. ((1998)  Nat. Biotechnol.  16:258-261), and the resulting cap product was inserted into a plasmid containing the wild type AAV2 genome. The result was transformed into  E. coli  for large scale plasmid production and purification. AAV was then produced and purified by CsCl centrifugation essentially as previously described (Kaspar et al. (2002)  Proc. Natl. Acad. Sci. USA  99:2320-2325; and Lai et al. (2003)  Nat. Neurosci.  6:21-27). Briefly, the library plasmid DNA was transfected into 293 human embryonic kidney cells (ATCC) using the calcium phosphate method, followed by addition of serotype 5 adenovirus (Ad5) at a multiplicity of infection (MOI) of 3. Virus was purified using CsCl density centrifugation. For all experiments, the AAV genomic titer was determined by extracting vector DNA as previously described (Kaspar et al. supra; and Lai et al., supra) followed by quantification using real time PCR with SYBR-Green dye (Molecular Probes) and a Biorad iCycler. 
     Heparin Column Chromatography 
     Approximately 10 12  AAV library particles were loaded onto a 1 mL HiTrap heparin column (Amersham) previously equilibrated with 0.15 M NaCl and 50 mM Tris at pH 7.5. Washes were performed using 0.75 ml volumes of the same buffer with increasing increments of 50 mM NaCl up to 750 mM, followed by a 1 M wash. As a control, rAAV-GFP was also subjected to heparin affinity chromatography. To isolate individual viral clones from library fractions that eluted at different salt concentrations, viral DNA was extracted from the fractions, amplified by PCR, and inserted into a rAAV packaging plasmid based on pAd8. rAAV-GFP was then packaged in order to analyze the ability of these mutant capsids to package rAAV vector. Capsid variants were sequenced at the U.C. Berkeley DNA sequencing facility. Finally, the affinity of the variant capsids for heparin was quantified by using the method of Qiu et al. ((2000)  Virol.  269:137-147), except that virus bound to the immobilized heparin was quantified by real time PCR. 
     Antisera Generation and Antibody Neutralization Screen 
     Polyclonal sera containing neutralizing antibodies (NABs) against AAV2 were generated in two New Zealand White rabbits in accordance with the U.C. Berkeley Animal Care and Use Committee and NIH standards for laboratory animal care. Briefly, 5×10 10  CsCl-purified rAAV2 particles were mixed with 0.5 mL TitreMax adjuvant (CytRx) and injected into the anterior hindlimb muscle. Two boosts were performed at 3-week intervals using the same AAV dosage, followed by antiserum collection. 
     Both wild type and a mutant AAV library were incubated with varying amounts of serum (0-6.25 μl) in 75 μl of phosphate-buffered saline (PBS) (pH 7.4) for 30 minutes at room temperature, followed by addition to 2.5×10 5  293 cells in a 6-well format. After 48 hours, AAV was rescued from infected cells by addition of Ad5, and cells were harvested 24 hours later. 
     Individual viral clones from the library fraction that successfully infected cells even in the presence of NAB were inserted into the rAAV packaging plasmid, and rAAV-GFP was produced as above. rAAV with mutant capsids were then incubated in 5 μl polyclonal sera as above, followed by addition to 1×10 5  293 cells. At 72 hours post-infection, the fraction of green cells was quantified by flow cytometry at the U.C. Berkeley Cancer Center (Beckman-Coulter EPICS). 
     Results 
     Library Generation 
     The staggered extension process (Zhao et al., supra), a polymerase chain reaction (PCR)-based method that generates diverse genetic libraries in a manner similar to that of DNA shuffling, was used to generate a library of cap mutants with point mutations randomly distributed throughout VP1-3. This product was inserted into a plasmid containing the complete wild type AAV genome to yield a viral library with approximately 10 6  independent clones, as determined by quantifying the number of colonies following bacterial transformation. To assess its degree of sequence diversity, the plasmid library was sequenced. The plasmid was then packaged into virus by transient transfection into 293 cells followed by Ad5 infection to yield a viral particle library. This large AAV library is selected for viral variants with any variety of new properties or functions, and the capsid structure conferring these new functions are readily recovered by DNA sequence analysis of the AAV genome encapsidated in the particles. 
     Heparin Binding Mutants 
     As an initial gauge of how the library&#39;s sequence diversity translated into capsid functional diversity, CsCl-purified library particles were subjected to heparin affinity chromatography with steps of increasing NaCl concentration. As previously reported (Zolotukhin et al. (1999)  Gene Ther.  6:973-985) wild type AAV elutes from heparin between 450 and 550 mM NaCl ( FIG. 1 ). In stark contrast, the AAV mutant library elutes at a wide range of salt concentrations, from the 150 mM load to the final 750 mM fraction. This result demonstrates that the library encompasses significant sequence and functional diversity. Since its affinity for heparan sulfate limits the spread of rAAV2 vectors upon injection in vivo, lower affinity mutants may be desirable as gene delivery vectors when wide dissemination through a large tissue or region is needed. In contrast, a higher affinity mutant may be advantageous for regionally pinpointed, high level gene expression. 
       FIGS. 1 a  and 1 b   . Heparin binding characteristics of wild type AAV vs. the viral library. a) The heparin affinity column chromatogram of elution of wild type AAV (hatched bars) and the mutant library (open bars) is shown. Virus gradually elutes from the column as the NaCl concentration is increased. b) Chromatograms of pools from the mutant library selected for lower (hatched bars) and higher (open bars) heparin affinities. 
     To isolate mutants with both low and high heparin affinity, the 150 mM and 700 mM NaCl fractions from the initial library were separately amplified by co-infection of 293 cells with Ad5, and refractionated on the heparin column. After three rounds of enrichment, the majority of the two resulting viral pools eluted from the column at 150 mM and 750 mM ( FIG. 1 b   ). Importantly, since each round of enrichment involved 293 cell infection, these pools are still composed of highly infectious virus. Next, individual capsid clones were isolated from each of these salt fractions and sequenced. 
     The fact that these mutants eluted from the heparin column at different salt concentrations indicates that they had different affinities for heparin. To accurately measure these affinities, however, the method of Qiu et al supra can be used. By performing Scatchard binding analysis of virus to heparin immobilized to microtiter plates, the difference in the affinity of the mutants for heparin, compared to the affinity of wild-type AAV for heparin, is determined. 
     Neutralizing Antiserum Escape Mutants 
     The isolation of functionally diverse heparin binding mutants from our AAV mutant library demonstrates the utility of this approach for creating vectors with novel properties. This approach was applied to a much more significant problem: vector elimination by neutralizing antibodies. Rabbit anti-AAV2 neutralizing antiserum was generated, and a 1:1500 serum dilution was sufficient to inhibit rAAV-GFP gene delivery to 293 cells by 50%, comparable to NAB titers in human serum. 
       FIGS. 2 a  and 2 b   . Generation of antibody neutralization escape mutants. a) Virus was incubated with antiserum before addition to 293 cells. The level of virus that successfully evaded antibody neutralization as compared to control infection in the absence of antiserum was measured by addition of Ad5 and titering of rescued AAV by quantitative real time PCR on AAV genomes. b) The data on individual variants is presented. 
     Antibody escape mutants were isolated from the viral library. During successive rounds of selection, the library was subjected to increasing stringencies, where stringency is defined as the ratio of the dilution necessary to reduce rAAV2 with wild type capsid by 50% (a 1500-fold for the rabbit antiserum) to the dilution actually used. Therefore, the more antiserum added to the virus, the higher the stringency. After three rounds of selection, followed by co-infection of 293 cells with Ad5 for amplification, the resulting viral pool was less susceptible to antibody neutralization as compared to wild type virus. Next, five individual capsid clones from this pool were analyzed by using them to package rAAV-GFP. The results demonstrated that the resulting recombinant vector is between 100 and 1000-fold more resistant to neutralizing antibodies. This is a therapeutically relevant result. 
     Neutralizing antibody inhibition of rAAV gene delivery can be observed as follows. rAAV2 with wild type capsid is added to 293 cells and visualized after 24 hours. Virus is visualized in green (Alexa 488), microtubules in red (Alexa 546), and the nucleus in blue (TO-PRO-3). After preincubation in neutralizing antiserum, the intracellular transport of rAAV2 with wild type capsid is expected to be significantly inhibited. 
     In  FIGS. 3  A-C, the cap nucleotide sequences of two neutralizing antibody escape mutants is shown, compared to wild-type cap sequence. 
     Example 2: Characterization of Further AAV Capsid Variants 
     AAV mutants—including antibody evaders, mutants with reduced heparin binding, and mutants with increased heparin binding—were generated as follows. 
     Library Generation: 
     A combination of error prone PCR and the staggered extension process (StEP) was used to generate a library of mutant DNA fragments encoding the capsid protein. This library was then inserted into a plasmid in order to package it into virus, by transfection of these plasmids plus an adenoviral helper plasmid into 293 cells. The library was then purified by cesium chloride density centrifugation. 
     Selection: 
     The library was then passed through screening steps. For example, for the antibody evasion mutant, a sample of the library was mixed together with polyclonal anti-AAV antiserum, then added to 293 cells. Addition of adenovirus amplifies the variants that are able to successfully evade the antiserum. 
     After amplification of the ‘successful’ variants (e.g., variants that infected the 293 cells in the presence of anti-AAV antibody) in 293 cells, the variants were purified, mixed again with antiserum, and allowed to infect 293 cells, and finally reamplified with adenovirus to further enrich for variants. After several rounds of enrichment, the viral DNA was purified, cloned, and sequenced to determine which mutations resulted in successful variants. 
     Generating Vector: 
     At this point, only the viral capsid DNA had been used to package replication competent virus, i.e. viruses with a genome that contained rep and the mutant cap genes and were therefore capable of replicating in the presence of adenovirus. However, to use the variants in a gene therapy setting, it was important to use the mutant cap gene to package recombinant particles containing a therapeutic gene. To demonstrate that the mutant cap genes described herein are capable of packaging recombinant virus, the cap gene was moved into a packaging/helper plasmid. Addition of this helper along with an adenoviral helper plasmid and a vector plasmid containing a promoter driving the expression of a reporter gene (GFP) to cells results in the generation of recombinant particles. 
     Antibody Evasion Mutants 
     Two sources of antiserum were used to generate antibody evasion mutants. One is rabbit serum that was generated by injecting rabbits with wild type AAV particles (Anti-AAV Rabbit Serum produced from New Zealand White rabbits). The second is human serum pooled from a number of individuals (Sigma Product #14-1388; Lot #122K0424; Origin: 40 North American donors). 
     Heparin Binding Mutants 
     To generate heparin binding mutants, CsCl-purified library viral particles were subjected to heparin affinity chromatography with steps of increasing NaCl concentration, as described in Example 1. Mutants with reduced heparin binding were eluted with the same NaCl concentration used to load the viral particles (e.g., 0.15 M NaCl). Mutants with reduced heparin binding were eluted at between 650-700 mM NaCl. 
     Characterization 
     Tables 1-4, below, provide the location of the amino acid differences in VP1 from wild-type AAV VP1 for the various mutants. All amino acid differences are relative to the VP1 amino acid sequence shown in  FIGS. 5A-C (SEQ ID NO5). VP-1-encoding nucleotide sequence of the various mutants are provided in  FIGS. 4A-G ,  FIGS. 6A-J , and  FIGS. 8A-G . In  FIGS. 4A-G ,  FIGS. 6A-J , and  FIGS. 8A-G , nucleotide differences from wild-type AAV-2 VP1 are shown in bold. VP-1 amino acid sequences of the various mutants are provided in  FIGS. 5A-C ,  FIGS. 7A-D , and  FIGS. 9A-C . In  FIGS. 5A-C ,  FIGS. 7A-D , and  FIGS. 9A-C , conservative amino acid changes are indicated with a box; and non-conservative amino acid changes are indicated in bold. 
     Table 1 provides the clone number and amino acid changes for various AAV neutralizing antibody escape mutants. 
     
       
         
           
               
             
               
                 TABLE 1 
               
             
            
               
                   
               
               
                 Mutations in 5 rabbit antisera evader clones 
               
            
           
           
               
               
               
               
            
               
                   
                 Clone 
                 Mutation 
                 Region 
               
               
                   
                   
               
               
                   
                 rAbE1 
                 T713R 
                 2-fold dimple* 
               
               
                   
                   
                 T716A 
                 2-fold dimple* 
               
               
                   
                 rAbE2 
                 V418L 
                 Antigenic peptide 53* 
               
               
                   
                   
                 T713R 
                 2-fold dimple* 
               
               
                   
                   
                 T716A 
                 2-fold dimple* 
               
               
                   
                 rAbE3 
                 T716A 
                 2-fold dimple* 
               
               
                   
                 rAbE4 
                 D180N 
                 A69 linear epitope †   
               
               
                   
                   
                 T716A 
                 2-fold dimple* 
               
               
                   
                 rAbE5 
                 A493G 
                 C37-B conformational 
               
               
                   
                   
                   
                 epitope †   
               
               
                   
                   
                 T716A 
                 2-fold dimple* 
               
               
                   
                   
               
               
                   
                 Moskalenko et al. (2000)  J. Virol . 74: 1761-1766 
               
               
                   
                   † Wobus et al. (2000)  J. Virol . 74: 9281-9293 
               
               
                   
                   ‡ Xie et al. (2002)  Proc. Natl. Acad. Sci. USA  99: 10405-10410 
               
            
           
         
       
     
     Table 2 provides the clone number and amino acid changes for various AAV neutralizing antibody escape mutants. 
     
       
         
           
               
             
               
                 TABLE 2 
               
             
            
               
                   
               
               
                 Mutations in 9 human antisera evader clones 
               
            
           
           
               
               
               
            
               
                 Clone 
                 Mutation 
                 Region 
               
               
                   
               
               
                 S1CM5 
                 K169R 
                 Peptide 22-23* &amp; Bordering A69 linear epitope †   
               
               
                   
                 S181P 
                 A69 linear epitope †   
               
               
                   
                 A333V 
                 5-fold cylinder* 
               
               
                   
                 P363T 
                 Between 5-fold cylinder and A20 
               
               
                   
                   
                 conformational epitope 
               
               
                   
                 A493E 
                 C37-B conformational epitope †   
               
               
                 S2CM5 
                 I19V 
                 “Lip” insertion (Peptide 4-5)* 
               
               
                   
                 V369A 
                 A20 conformational epitope †   
               
               
                   
                 A593E 
                 Accessible surface region in Loop 4 on β-GH13 ‡   
               
               
                 S2CM2 
                 G189E 
                 6 aa proximity to A69 linear epitope †   
               
               
                   
                 N215I 
                 Near N-terminus VP3 
               
               
                   
                 A367E 
                 Between 5-fold cylinder and A20 conformational 
               
               
                   
                   
                 epitope, 2 aa proximity to A20 
               
               
                   
                 S429G 
                 α-GH1 ‡   
               
               
                   
                 A493E 
                 C37-B conformational epitope †   
               
               
                   
                 S580P 
                 End of b-GH12, 1 aa proximity to accessible 
               
               
                   
                   
                 surface region loop 4, β-GH13 ‡   
               
               
                   
                 P643L 
                 Proline residue between β-GH16 and β-H in 
               
               
                   
                   
                 β barrel core ‡   
               
               
                   
                 E685V 
               
               
                 S3CM2 
                 R20S 
                 “Lip” insertion (Peptide 4-5)* 
               
               
                   
                 N57S 
               
               
                   
                 E347V 
                 5-fold cylinder* 
               
               
                   
                 A493E 
                 C37-B conformational epitope †   
               
               
                   
                 N551D 
               
               
                   
                 D594E 
                 Accessible surface region in Loop 4 on β-GH13 ‡   
               
               
                 A1CM5 
                 K26R 
                 “Lip” insertion (Peptide 4-5)* 
               
               
                   
                 N215D 
               
               
                   
                 G355S 
                 5-fold cylinder* 
               
               
                   
                 A593E 
                 Accessible surface region in Loop 4 on β-GH13 ‡   
               
               
                 A3CM5 
                 G49E 
               
               
                   
                 S196P 
               
               
                   
                 T337P 
                 5-fold cylinder* 
               
               
                 A1CM2 
                 K24E 
                 “Lip” insertion (Peptide 4-5)* 
               
               
                   
                 L91P 
               
               
                   
                 K137T 
               
               
                   
                 Q186L 
                 3 aa proximity A69 linear epitope* 
               
               
                   
                 T251A 
                 Minor Peptide 33, Canyon Epitope* 
               
               
                   
                 H290L 
               
               
                   
                 F306L 
                 End of 5-fold cylinder* 
               
               
                   
                 S390T 
               
               
                   
                 A493E 
                 C37-B conformational epitope †   
               
               
                   
                 A505V 
                 1 aa proximity to C37-B conformational epitope †   
               
               
                   
                 V557I 
               
               
                   
                 T651A 
               
               
                 A2CM2 
                 V46A 
               
               
                   
                 S196P 
                 Between A20 and C37 minor Conformational 
               
               
                   
                   
                 Epitope †   
               
               
                   
                 A593E 
                 Accessible surface region in Loop 4 on β-GH13 ‡   
               
               
                 A3CM2 
                 P31L 
               
               
                   
                 F100S 
               
               
                   
                 I260T 
                 Minor Peptide 33, Canyon Epitope* 
               
               
                   
                 R459G 
                 3-fold spike in Loop 3 (peptide 58)* 
               
               
                   
                 A522T 
               
               
                   
                 A663V 
               
               
                   
                 E681G 
               
               
                   
                 W694R 
               
               
                   
               
            
           
         
       
     
     Table 3 provides the clone number and amino acid changes for various AAV mutants with increased heparin binding. 
     
       
         
           
               
             
               
                 TABLE 3 
               
             
            
               
                   
               
               
                 Mutations of Clones with Increased Heparin Affinity 
               
            
           
           
               
               
               
            
               
                   
                 Clone 
                 Mutation 
               
               
                   
                   
               
               
                   
                 D14H1 
                 W23L 
               
               
                   
                   
                 D231G 
               
               
                   
                   
                 S261F 
               
               
                   
                   
                 V323F 
               
               
                   
                   
                 Q349P 
               
               
                   
                   
                 G406E 
               
               
                   
                   
                 N408D 
               
               
                   
                 D14L3 
                 W23L 
               
               
                   
                   
                 S196T 
               
               
                   
                   
                 D231G 
               
               
                   
                   
                 S261F 
               
               
                   
                   
                 Q349P 
               
               
                   
                   
                 G406E 
               
               
                   
                   
                 N408D 
               
               
                   
                   
                 N569D 
               
               
                   
                   
                 N596D 
               
               
                   
                   
               
            
           
         
       
     
     Table 4 provides the clone number and amino acid changes for various AAV mutants with reduced heparin binding. 
     
       
         
           
               
             
               
                 TABLE 4 
               
             
            
               
                   
               
               
                 Mutations of Clones with Reduced Heparin Affinity 
               
            
           
           
               
               
               
            
               
                   
                 Clone 
                 Mutation 
               
               
                   
                   
               
               
                   
                 P1BH1 
                 M235V 
               
               
                   
                   
                 Q401R 
               
               
                   
                   
                 L437H 
               
               
                   
                   
                 N582D 
               
               
                   
                   
                 T660A 
               
               
                   
                 P1BH2 
                 G111R 
               
               
                   
                   
                 K258E 
               
               
                   
                   
                 L315P 
               
               
                   
                   
                 E322G 
               
               
                   
                   
                 N551D 
               
               
                   
                   
                 V605I 
               
               
                   
                   
               
            
           
         
       
     
     While the present invention has been described with reference to the specific embodiments thereof, it should be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the true spirit and scope of the invention. In addition, many modifications may be made to adapt a particular situation, material, composition of matter, process, process step or steps, to the objective, spirit and scope of the present invention. All such modifications are intended to be within the scope of the claims appended hereto.