Adeno-associated virus mutants and methods of use thereof

Abstract

The present invention provides an infectious recombinant adeno-associated virus (rAAV) virion containing heterologous nucleic acid that has improved resistance to human AAV neutralizing antibodies. The present invention also provides a method for delivering heterologous nucleic acid-containing rAAV virions to target cells. [Solution] An infectious recombinant adeno-associated virus virion comprising (a) a mutant adeno-associated virus capsid protein comprising an amino acid sequence having at least about 90% amino acid sequence identity to a specific amino acid sequence, and (b) a heterologous nucleic acid.

Description

[Technical Field]

[0001] cross reference This application claims the benefit of U.S. Provisional Patent Application No. 61 / 829,735, filed May 31, 2013, which is incorporated herein by reference in its entirety.

[0002] Federally Sponsored Research Statement This invention was made with government support under Grant No. HL081527 awarded by the National Institutes of Health. The government has certain rights in this invention.

[0003] Incorporation by reference of sequence listings provided as text files The Sequence Listing is provided herewith as a text file entitled "BERK-216WO_ST25.txt" having a size of 169 KB, created on May 28, 2014. The contents of this text file are incorporated herein by reference in their entirety. [Background technology]

[0004] introduction Gene delivery vectors based on adeno-associated viruses (AAVs) have demonstrated promise in both preclinical disease models and, recently, human clinical trials for several disease targets. Because wild-type AAVs are nonpathogenic and have no etiological association with any known disease, AAV-based vectors are maximally safe. Furthermore, AAVs offer the capability for highly efficient gene delivery and sustained transgene expression in many tissues, including the liver, muscle, lung, retina, and brain.

[0005] AAV is a single-stranded DNA virus containing two open reading frames, rep and cap. The first gene encodes four proteins (Rep78, Rep68, Rep52, and Rep40) required for genome replication, while the second gene expresses three structural proteins (VP1-VP3) that assemble to form the viral capsid. As its name implies, AAV depends on the presence of a helper virus, such as adenovirus or herpesvirus, for active replication. In the absence of a helper, AAV establishes a latent state in which its genome is maintained episomally or integrated into the host chromosome. Several homologous primate AAV serotypes and numerous nonhuman primate types have been identified. AAV2 is the best characterized gene delivery vehicle.

[0006] As of 2010, there were 75 ongoing clinical trials using AAV as a gene delivery vehicle. However, the high prevalence of anti-capsid neutralizing antibodies due to widespread exposure to numerous AAV variants and serotypes within the human population reduces the efficacy of AAV gene therapy. This pre-existing immunity, as well as subsequent immune development due to vector administration, may hinder the wider implementation of AAV gene therapy. For example, to date, AAV has been most successful in clinical studies involving delivery to immune-privileged regions.

[0007] Recent analyses have shown that the prevalence of anti-AAV IgG antibodies in humans is highest for AAV2 (72%) and AAV1 (67%), but AAV9 (47%), AAV6 (46%), AAV5 (40%), and AAV8 (38%) antibodies were also present in the majority of the population studied. Some studies have found that humoral immunity to AAV capsids during gene therapy can be prevented by lowering the amount of rAAV particles delivered. Unfortunately, administration of low vector doses results in low transduction and therefore low therapeutic gene expression.

[0008] There is a need in the art for the development of novel AAV variants that are resistant to neutralization by anti-AAV antibodies. [Advanced Technology Documents]

Non-licensed literature

[0009] [Non-licensed document 1] Asuri et al.,Mol Ther.2012 Feb;20(2):329-38 [Non-licensed document 2] Bainbridge et al.,N Engl J Med.2008 May 22;358(21):2231-9 [Non-licensed document 3] Excoffon et al.,Proc Natl Acad Sci US A.2009Mar10;106(10):3865-70

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[0010] [Patent Document 1] WO2012145601 No. [Patent Document 2] U.S. Patent Publication No. US20050053922 Summary of the Invention

[0011] overview The present disclosure provides infectious recombinant adeno-associated virus (rAAV) virions comprising a mutant capsid protein and a heterologous nucleic acid. The present disclosure further provides mutant adeno-associated virus (AAV) capsid proteins (and / or nucleic acids encoding the mutant AAV capsid proteins) that confer increased resistance to human AAV neutralizing antibodies on infectious rAAV virions. The present disclosure further provides host cells comprising infectious rAAV virions and / or nucleic acids encoding the subject mutant AAV capsid proteins. The present disclosure further provides libraries of the above-described virions, capsid proteins, nucleic acids, and / or host cells, wherein the mutant AAV capsid protein of at least one member of the library comprises an amino acid sequence having at least one amino acid substitution compared to the amino acid sequence set forth in one of SEQ ID NOs: 10-13 and 26-33.

[0012] The present disclosure further provides methods for delivering heterologous nucleic acids to target cells, wherein the target cells are contacted with a subject infectious rAAV virion. The present disclosure further provides methods for delivering gene products to an individual, generally comprising administering an effective amount of a subject rAAV virion to an individual in need thereof. Compositions and kits for practicing the subject methods are also provided herein.

[0013] Features Features of the present disclosure include infectious recombinant adeno-associated virus (rAAV) virions comprising (a) a mutant adeno-associated virus (AAV) capsid protein comprising an amino acid sequence having at least about 90% amino acid sequence identity to an amino acid sequence set forth in one of SEQ ID NOs: 11-13 and 26-33, and (b) a heterologous nucleic acid. In some cases, the mutant AAV capsid protein comprises an amino acid sequence having at least about 95% amino acid sequence identity to an amino acid sequence set forth in one of SEQ ID NOs: 11-13 and 26-33. In some cases, the mutant AAV capsid protein comprises an amino acid sequence set forth in one of SEQ ID NOs: 11-13 and 26-33.

[0014] Features of the present disclosure include infectious recombinant adeno-associated virus (rAAV) virions comprising: (a) a mutant adeno-associated virus (AAV) capsid protein comprising an amino acid sequence having at least about 95% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 10, and including the amino acid substitutions N312K, N449D, D472N, N551S, I698V, and L735Q relative to SEQ ID NO: 2; and (b) a heterologous nucleic acid. In some cases, the mutant AAV capsid protein comprises the amino acid sequence set forth in SEQ ID NO: 10. In some cases, the rAAV exhibits increased resistance to human AAV neutralizing antibodies compared to the resistance exhibited by AAV2 (wild-type AAV serotype 2). In some cases, the rAAV exhibits at least about 1.5-fold (e.g., at least about 3-fold, at least about 5-fold, at least about 10-fold, at least about 30-fold, etc.) greater resistance to human AAV neutralizing antibodies than the resistance exhibited by AAV2. In some cases, the rAAV exhibits increased transduction of mammalian cells in the presence of human AAV neutralizing antibodies compared to transduction of mammalian cells exhibited by wild-type AAV serotype 2 (AAV2). In some cases, the mammalian cell is a liver cell, a pancreatic cell, a skeletal muscle cell, a cardiac muscle cell, a fibroblast, a retinal cell, a synovial joint cell, a lung cell, a T cell, a neuron, a glial cell, a stem cell (e.g., a hematopoietic stem cell, a hematopoietic progenitor cell, a neural stem cell, a neural progenitor cell, a neural crest stem cell, an embryonic stem cell, an induced pluripotent stem cell (iPS cell), a mesenchymal stem cell, a mesodermal stem cell, a liver stem cell, a pancreatic stem cell, a pancreatic progenitor cell, a muscle stem cell, a retinal stem cell, etc.), an endothelial cell, or a cancer cell. In some cases, the heterologous nucleic acid comprises an RNA interference agent. In some cases, the heterologous nucleic acid comprises a nucleotide sequence encoding a polypeptide.

[0015] Features of the present disclosure include isolated nucleic acids comprising a nucleotide sequence encoding a mutant adeno-associated virus (AAV) capsid protein comprising an amino acid sequence having at least about 90% amino acid sequence identity to the amino acid sequence set forth in one of SEQ ID NOs: 11-13 and 26-33. In some cases, the encoded mutant AAV capsid protein comprises an amino acid sequence having at least about 95% amino acid sequence identity to the amino acid sequence set forth in one of SEQ ID NOs: 11-13 and 26-33. In some cases, the encoded mutant AAV capsid protein comprises the amino acid sequence set forth in one of SEQ ID NOs: 11-13 and 26-33.

[0016] A feature of the disclosure includes an isolated nucleic acid comprising a nucleotide sequence encoding a mutant adeno-associated virus (AAV) capsid protein comprising an amino acid sequence having at least about 95% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO:10, and comprising the amino acid substitutions N312K, N449D, D472N, N551S, I698V, and L735Q relative to SEQ ID NO:2.

[0017] In some cases, the encoded mutant AAV capsid protein (encoded by the isolated nucleic acid) confers increased resistance to human AAV neutralizing antibodies to infectious recombinant adeno-associated virus (rAAV) virions compared to the resistance exhibited by AAV2 (wild-type AAV serotype 2). In some cases, the increased resistance is at least about 1.5-fold (e.g., at least about 3-fold, at least about 5-fold, at least about 10-fold, at least about 30-fold, etc.) greater than the resistance exhibited by AAV2. In some cases, the encoded mutant AAV capsid protein (encoded by the isolated nucleic acid) confers increased transduction of mammalian cells in the presence of human AAV neutralizing antibodies to infectious recombinant adeno-associated virus (rAAV) virions compared to the transduction exhibited by AAV2.

[0018] A feature of the present disclosure includes an isolated host cell comprising the subject nucleic acid described above. In some cases, the host cell is stably transfected with the nucleic acid. In some cases, the host cell further comprises a nucleic acid comprising a nucleotide sequence encoding an AAVrep protein. In some cases, the host cell further comprises a recombinant AAV vector.

[0019] A feature of the present disclosure includes a method of delivering a heterologous nucleic acid to a target cell, comprising contacting the target cell with a subject virion (described above). In some cases, the target cell is a liver cell, a pancreatic cell, a skeletal muscle cell, a cardiac muscle cell, a fibroblast, a retinal cell, a synovial joint cell, a lung cell, a T cell, a neuron, a glial cell, a stem cell (e.g., a hematopoietic stem cell, a hematopoietic progenitor cell, a neural stem cell, a neural progenitor cell, a neural crest stem cell, an embryonic stem cell, an induced pluripotent stem cell (iPS cell), a mesenchymal stem cell, a mesodermal stem cell, a liver stem cell, a pancreatic stem cell, a pancreatic progenitor cell, a muscle stem cell, or a retinal stem cell, etc.), an endothelial cell, or a cancer cell. In some cases, the target cell is in vitro. In some cases, the target cell is in vivo.

[0020] A feature of the present disclosure includes a method for delivering a gene product to an individual in need thereof, the method comprising administering to the individual an effective amount of a subject infectious recombinant adeno-associated virus (rAAV) virion (described above). In some cases, the heterologous nucleic acid of the rAAV virion comprises an RNA interference agent. In some cases, the heterologous nucleic acid of the rAAV virion comprises a nucleotide sequence encoding a polypeptide. In some cases, the administering step comprises indirect delivery of the infectious rAAV virion. In some cases, the administering step comprises direct delivery of the infectious rAAV virion.

[0021] Features of the present disclosure include mutant adeno-associated virus (AAV) capsid proteins comprising an amino acid sequence having at least about 90% amino acid sequence identity to an amino acid sequence set forth in one of SEQ ID NOs: 11-13 and 26-33. In some cases, the AAV capsid protein comprises an amino acid sequence having at least about 95% amino acid sequence identity to an amino acid sequence set forth in one of SEQ ID NOs: 11-13 and 26-33. In some cases, the AAV capsid protein comprises an amino acid sequence set forth in one of SEQ ID NOs: 11-13 and 26-33.

[0022] A feature of the present disclosure includes a mutant adeno-associated virus (AAV) capsid protein comprising an amino acid sequence having at least about 95% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 10, and comprising the amino acid substitutions N312K, N449D, D472N, N551S, I698V, and L735Q relative to SEQ ID NO: 2. In some cases, the mutant AAV capsid protein comprises the amino acid sequence set forth in SEQ ID NO: 10. In some cases, the mutant AAV capsid protein confers increased resistance to human AAV neutralizing antibodies to infectious recombinant adeno-associated virus (rAAV) virions compared to the resistance exhibited by AAV2. In some cases, the increased resistance is at least about 1.5-fold (e.g., at least about 3-fold, at least about 5-fold, at least about 10-fold, at least about 30-fold, etc.) greater than the resistance exhibited by AAV2. In some cases, mutant AAV capsid proteins confer increased transduction of infectious recombinant adeno-associated virus (rAAV) virions into mammalian cells in the presence of human AAV neutralizing antibodies compared to the transduction exhibited by AAV2.

[0023] A feature of the present disclosure includes a library comprising at least one of: (i) two or more infectious rAAV virions, each comprising a mutant adeno-associated virus (AAV) capsid protein and a heterologous nucleic acid; (ii) two or more isolated nucleic acids, each comprising a nucleotide sequence encoding a mutant AAV capsid protein; (iii) two or more host cells, each comprising a nucleic acid comprising a nucleotide sequence encoding a mutant AAV capsid protein; and (iv) two or more mutant AAV capsid proteins, wherein the mutant AAV capsid protein of at least one member of the library comprises an amino acid sequence having at least one amino acid substitution compared to the amino acid sequence set forth in one of SEQ ID NOs: 10-13 and 26-33.

[0024] A feature of the present disclosure includes a method for generating and identifying modified infectious rAAV virions that exhibit altered infectious properties relative to a starter (parent) virion comprising a starter capsid protein, the method comprising: (a) generating mutant adeno-associated virus (AAV) capsid proteins from a starter capsid protein, wherein the starter capsid proteins comprise an amino acid sequence set forth in one of SEQ ID NOs: 10-13 and 26-33, and each mutant AAV capsid protein comprises at least one amino acid substitution relative to the starter capsid protein; (b) generating mutant AAV virions, each comprising the mutant capsid AAV protein generated in step (a); and (c) assaying the mutant AAV virions generated in step (b) for altered infectious properties to identify the modified infectious rAAV virions. In some cases, generating the library of mutant AAV capsid proteins comprises a mutagenesis method selected from the group consisting of polymerase chain reaction mutagenesis, oligonucleotide-directed mutagenesis, saturation mutagenesis, loop-swap mutagenesis, fragment-shuffling mutagenesis, and combinations thereof. In some cases, the altered infection characteristic is increased resistance to human neutralizing AAV antibodies compared to the resistance exhibited by the starter virion. In some cases, the altered infection characteristic is increased transduction of mammalian cells in the presence of human AAV neutralizing antibodies compared to the transduction exhibited by the starter virion. In some cases, the modified infectious rAAV virions comprise a modified AAV capsid protein comprising an amino acid sequence having at least about 90% amino acid sequence identity with a starter capsid protein.

[0025] A feature of the disclosure includes a method of generating a mutant AAV capsid protein from a starter capsid protein, the method comprising subjecting a nucleic acid comprising a nucleotide sequence encoding a starter capsid protein to a type of mutagenesis selected from the group consisting of polymerase chain reaction mutagenesis, oligonucleotide-directed mutagenesis, saturation mutagenesis, loop swap mutagenesis, fragment shuffling mutagenesis, and combinations thereof, wherein the starter capsid protein comprises an amino acid sequence set forth in one of SEQ ID NOs: 10-13 and 26-33. [The present invention 1001] (a) a mutant adeno-associated virus (AAV) capsid protein comprising an amino acid sequence having at least about 90% amino acid sequence identity to amino acids 203 to 736 of the amino acid sequence set forth in one of SEQ ID NOs: 11 to 13 and 26 to 33; and (b) Heterologous nucleic acid Infectious recombinant adeno-associated virus (rAAV) virions comprising: [The present invention 1002] The infectious rAAV of the present invention 1001, wherein the mutant AAV capsid protein comprises an amino acid sequence having at least about 95% amino acid sequence identity with amino acids 203 to 736 of the amino acid sequence set forth in one of SEQ ID NOs: 11 to 13 and 26 to 33. [The present invention 1003] 1001. The infectious rAAV of the present invention, wherein the mutant AAV capsid protein comprises an amino acid sequence set forth in one of SEQ ID NOs: 11-13 and 26-33. [The present invention 1004] (a) a mutant adeno-associated virus (AAV) capsid protein comprising an amino acid sequence having at least about 95% amino acid sequence identity to amino acids 203 to 736 of the amino acid sequence set forth in SEQ ID NO: 10, and comprising the amino acid substitutions N312K, N449D, D472N, N551S, I698V, and L735Q compared to SEQ ID NO: 2; and (b) Heterologous nucleic acid Infectious recombinant adeno-associated virus (rAAV) virions comprising: [The present invention 1005] 1004. The infectious rAAV of the present invention, wherein the mutant AAV capsid protein comprises the amino acid sequence set forth in SEQ ID NO:10. [The present invention 1006] 1001 or 1004, wherein the infectious rAAV exhibits increased resistance to human AAV neutralizing antibodies compared to the resistance exhibited by AAV2 (wild-type AAV serotype 2). [The present invention 1007] 1006. The infectious rAAV of the present invention, wherein the rAAV virions exhibit at least about 1.5-fold greater resistance to human AAV neutralizing antibodies than the resistance exhibited by AAV2. [The present invention 1008] 1006. The infectious rAAV of the present invention, wherein the rAAV virions exhibit at least about three-fold greater resistance to human AAV neutralizing antibodies than the resistance exhibited by AAV2. [The present invention 1009] 1006. The infectious rAAV of the present invention, wherein the rAAV virions exhibit at least about 5-fold greater resistance to human AAV neutralizing antibodies than the resistance exhibited by AAV2. [The present invention 1010] 1006. The infectious rAAV of the present invention, wherein the rAAV virions exhibit at least about 10-fold greater resistance to human AAV neutralizing antibodies than the resistance exhibited by AAV2. [The present invention 1011] 1006. The infectious rAAV of the present invention, wherein said rAAV virions exhibit at least about 30-fold greater resistance to human AAV neutralizing antibodies than the resistance exhibited by AAV2. [The present invention 1012] 1001 or 1004, wherein the infectious rAAV of the present invention exhibits increased transduction of mammalian cells in the presence of human AAV neutralizing antibodies, as compared to the transduction of mammalian cells exhibited by wild-type AAV serotype 2 (AAV2). [The present invention 1013] The infectious rAAV of the present invention, wherein the mammalian cell is a liver cell, a pancreatic cell, a skeletal muscle cell, a cardiac muscle cell, a fibroblast, a retinal cell, a synovial joint cell, a lung cell, a T cell, a neuron, a glial cell, a stem cell, an endothelial cell, or a cancer cell. [The present invention 1014] The infectious rAAV of the present invention, wherein the stem cell is a hematopoietic stem cell, a hematopoietic progenitor cell, a neural stem cell, a neural progenitor cell, a neural crest stem cell, an embryonic stem cell, an induced pluripotent stem cell (iPS cell), a mesenchymal stem cell, a mesodermal stem cell, a liver stem cell, a pancreatic stem cell, a pancreatic progenitor cell, a muscle stem cell, or a retinal stem cell. [The present invention 1015] 1001 or 1004, wherein the heterologous nucleic acid comprises an RNA interfering agent. [The present invention 1016] 1005. The infectious rAAV of claim 1001 or claim 1004, wherein said heterologous nucleic acid comprises a nucleotide sequence encoding a polypeptide. [The present invention 1017] An isolated nucleic acid comprising a nucleotide sequence encoding a mutant adeno-associated virus (AAV) capsid protein comprising an amino acid sequence having at least about 90% amino acid sequence identity to amino acids 203 to 736 of the amino acid sequence set forth in one of SEQ ID NOs: 11 to 13 and 26 to 33. [The present invention 1018] 1017. The isolated nucleic acid of claim 1017, wherein the encoded mutant AAV capsid protein comprises an amino acid sequence having at least about 95% amino acid sequence identity to amino acids 203 to 736 of the amino acid sequence set forth in one of SEQ ID NOs: 11 to 13 and 26 to 33. [The present invention 1019] 1017. The isolated nucleic acid of claim 1017, wherein the encoded mutant AAV capsid protein comprises an amino acid sequence set forth in one of SEQ ID NOs: 11-13 and 26-33. [The present invention 1020] A mutant adeno-associated virus (AAV) capsid protein comprising an amino acid sequence having at least about 95% amino acid sequence identity with amino acids 203 to 736 of the amino acid sequence set forth in SEQ ID NO: 10, and comprising the amino acid substitutions N312K, N449D, D472N, N551S, I698V, and L735Q compared to SEQ ID NO: 2. An isolated nucleic acid comprising a nucleotide sequence encoding [The present invention 1021] 1020. The isolated nucleic acid of claim 1017, wherein the encoded mutant AAV capsid protein confers increased resistance to human AAV neutralizing antibodies to infectious recombinant adeno-associated virus (rAAV) virions compared to the resistance exhibited by AAV2 (wild-type AAV serotype 2). [The present invention 1022] 1021. The isolated nucleic acid of claim 1021, wherein said increased resistance is at least about 1.5-fold higher than the resistance exhibited by AAV2. [The present invention 1023] 1021. The isolated nucleic acid of claim 1021, wherein said increased resistance is at least about three times higher than the resistance exhibited by AAV2. [The present invention 1024] 1021. The isolated nucleic acid of claim 1021, wherein said increased resistance is at least about 5-fold greater than the resistance exhibited by AAV2. [The present invention 1025] 1021. The isolated nucleic acid of claim 1021, wherein said increased resistance is at least about 10-fold greater than the resistance exhibited by AAV2. [The present invention 1026] 1021. The isolated nucleic acid of claim 1021, wherein said increased resistance is at least about 30-fold greater than the resistance exhibited by AAV2. [The present invention 1027] 1020. The isolated nucleic acid of claim 1017 or 1020, wherein the mutant AAV capsid protein confers increased transduction of infectious recombinant adeno-associated virus (rAAV) virions into mammalian cells in the presence of human AAV neutralizing antibodies compared to the transduction exhibited by AAV2 (wild-type AAV serotype 2). [The present invention 1028] An isolated host cell comprising the nucleic acid of the present invention. [The present invention 1029] An isolated host cell comprising said nucleic acid of the present invention 1020. [The present invention 1030] 1028 or 1029, wherein the isolated host cell is stably transfected with the nucleic acid. [The present invention 1031] 1029. The isolated host cell of claim 1028 or claim 1029, further comprising a nucleic acid comprising a nucleotide sequence encoding an AAVrep protein. [The present invention 1032] 1028. The isolated host cell of claim 1029, further comprising a recombinant AAV vector. [The present invention 1033] A method of delivering heterologous nucleic acid to a target cell, comprising contacting said target cell with a virion of invention 1001 or invention 1004. [The present invention 1034] 103. The method of claim 1033, wherein the target cell is a liver cell, a pancreatic cell, a skeletal muscle cell, a cardiac muscle cell, a fibroblast, a retinal cell, a synovial joint cell, a lung cell, a T cell, a neuron, a glial cell, a stem cell, an endothelial cell, or a cancer cell. [This invention 1035] 103. The method of claim 1034, wherein the stem cells are hematopoietic stem cells, hematopoietic progenitor cells, neural stem cells, neural progenitor cells, neural crest stem cells, embryonic stem cells, induced pluripotent stem cells (iPS cells), mesenchymal stem cells, mesodermal stem cells, liver stem cells, pancreatic stem cells, pancreatic progenitor cells, muscle stem cells, or retinal stem cells. [The present invention 1036] 1034. The method of claim 1033, wherein said target cell is in vitro. [This invention 1037] The method of claim 1033, wherein the target cell is in vivo. [The present invention 1038] 10. A method of delivering a gene product to an individual in need thereof, comprising administering to said individual an effective amount of an infectious recombinant adeno-associated virus (rAAV) virion of invention 1001 or invention 1004. [This invention 1039] 1038. The method of claim 1038, wherein the heterologous nucleic acid comprises an RNA interfering agent. [The present invention 1040] 1038. The method of claim 1038, wherein said heterologous nucleic acid comprises a nucleotide sequence encoding a polypeptide. [This invention 1041] 1038. The method of claim 1038, wherein said administering step comprises indirect delivery of said infectious rAAV virions. [The present invention 1042] 1038. The method of claim 1038, wherein said administering step comprises direct delivery of said infectious rAAV virions. [This invention 1043] A mutant adeno-associated virus (AAV) capsid protein comprising an amino acid sequence having at least about 90% amino acid sequence identity with amino acids 203 to 736 of the amino acid sequence set forth in one of SEQ ID NOs: 11 to 13 and 26 to 33. [This invention 1044] 1043. The mutant AAV capsid protein of the present invention, comprising an amino acid sequence having at least about 95% amino acid sequence identity with amino acids 203 to 736 of the amino acid sequence set forth in one of SEQ ID NOs: 11 to 13 and 26 to 33. [This invention 1045] 1043. The mutant AAV capsid protein of the present invention, comprising an amino acid sequence set forth in one of SEQ ID NOs: 11 to 13 and 26 to 33. [The present invention 1046] A mutant adeno-associated virus (AAV) capsid protein comprising an amino acid sequence having at least about 95% amino acid sequence identity with amino acids 203 to 736 of the amino acid sequence set forth in SEQ ID NO: 10, and comprising the amino acid substitutions N312K, N449D, D472N, N551S, I698V, and L735Q compared to SEQ ID NO: 2. [This invention 1047] 1046. The mutant AAV capsid protein of the present invention, comprising the amino acid sequence set forth in SEQ ID NO: 10. [This invention 1048] The mutant AAV capsid protein of invention 1043 or invention 1046, wherein the mutant AAV capsid protein confers increased resistance to human AAV neutralizing antibodies to infectious recombinant adeno-associated virus (rAAV) virions compared to the resistance exhibited by AAV2 (wild-type AAV serotype 2). [This invention 1049] 1048. The mutant AAV capsid protein of the present invention, wherein said increased resistance is at least about 1.5-fold higher than the resistance exhibited by AAV2. [The present invention 1050] 1048. The mutant AAV capsid protein of the present invention, wherein said increased resistance is at least about three times higher than the resistance exhibited by AAV2. [This invention 1051] 1048. The mutant AAV capsid protein of the present invention, wherein said increased resistance is at least about 5-fold higher than the resistance exhibited by AAV2. [This invention 1052] 1048. The mutant AAV capsid protein of the present invention, wherein said increased resistance is at least about 10-fold higher than the resistance exhibited by AAV2. [This invention 1053] 1048. The mutant AAV capsid protein of the present invention, wherein said increased resistance is at least about 30-fold higher than the resistance exhibited by AAV2. [This invention 1054] The mutant AAV capsid protein of invention 1043 or invention 1046, which confers increased transduction of infectious recombinant adeno-associated virus (rAAV) virions into mammalian cells in the presence of human AAV neutralizing antibodies, compared to the transduction exhibited by AAV2 (wild-type AAV serotype 2). [This invention 1055] (i) two or more infectious rAAV virions, each comprising a mutant adeno-associated virus (AAV) capsid protein and a heterologous nucleic acid; (ii) two or more isolated nucleic acids, each comprising a nucleotide sequence encoding a mutant AAV capsid protein; (iii) two or more host cells, each containing a nucleic acid comprising a nucleotide sequence encoding a mutant AAV capsid protein; and (iv) two or more mutant AAV capsid proteins A library comprising at least one of: The library, wherein the mutant AAV capsid protein of at least one member of the library comprises an amino acid sequence having at least one amino acid substitution compared to the amino acid sequence set forth in one of SEQ ID NOs: 10 to 13 and 26 to 33. [The present invention 1056] 1. A method for generating and identifying modified infectious recombinant adeno-associated virus (rAAV) virions that exhibit altered infectious properties compared to starter virions containing a starter capsid protein, comprising: (a) generating mutant adeno-associated virus (AAV) capsid proteins from the starter capsid protein, wherein the starter capsid protein comprises an amino acid sequence set forth in one of SEQ ID NOs: 10-13 and 26-33, and each mutant AAV capsid protein comprises at least one amino acid substitution compared to the starter capsid protein; (b) generating mutant AAV virions, each comprising the mutant capsid AAV proteins produced in step (a); (c) assaying the mutant AAV virions produced in step (b) for altered infectious properties to identify said modified infectious rAAV virions; The method comprising: [This invention 1057] 1056. The method of claim 1056, wherein generating said library of mutant AAV capsid proteins comprises a mutagenesis method selected from the group consisting of polymerase chain reaction mutagenesis, oligonucleotide-directed mutagenesis, saturation mutagenesis, loop swap mutagenesis, fragment shuffling mutagenesis, and combinations thereof. [This invention 1058] 1056. The method of claim 1056, wherein said altered infectivity characteristics is increased resistance to human neutralizing AAV antibodies compared to the resistance exhibited by said starter virions. [This invention 1059] 1056. The method of claim 1056, wherein said altered infection characteristic is increased transduction of mammalian cells in the presence of human AAV neutralizing antibodies compared to the transduction exhibited by said starter virions. [The present invention 1060] 1056. The method of claim 1056, wherein the modified infectious rAAV virion comprises a modified AAV capsid protein comprising an amino acid sequence having at least about 90% amino acid sequence identity with said starter capsid protein. [Brief explanation of the drawings]

[0026] [Figure 1] Figures 1A-B show the directed evolution of AAV for enhanced antibody evasion. [Figure 2A] 1 shows the neutralization profile of antibody escape mutants using human IVIG. [Figure 2B] 1 shows the neutralization profile of antibody escape mutants using human IVIG. [Figure 3A] 1 shows the neutralization profile of antibody escape mutants using human serum obtained from an individual who was excluded from a hemophilia B clinical trial due to the presence of high neutralizing antibody titers to AAV. [Figure 3B] 1 shows the neutralization profile of antibody escape mutants using human serum obtained from an individual who was excluded from a hemophilia B clinical trial due to the presence of high neutralizing antibody titers to AAV. [Figure 3C] 1 shows the neutralization profile of antibody escape mutants using human serum obtained from an individual who was excluded from a hemophilia B clinical trial due to the presence of high neutralizing antibody titers to AAV. [Figure 4] Figure 4A-B shows the amino acid sequences of the loop swap / shuffle and saturation mutagenesis clones. [Figure 5] Demonstrate the in vitro tropism of AAV mutants. [Figure 6] 6A-B show the in vivo localization and neutralization of novel AAV mutants. [Figure 7] 7A-D demonstrate the generation of human antibody escapees. [Figure 8A] The capsid protein sequence of shuffle 100-1 (SEQ ID NO: 11) aligned with the wild-type capsid protein sequences of AAV1 to 9 (SEQ ID NOs: 1 to 9) is shown. [Figure 8B] The capsid protein sequence of shuffle 100-1 (SEQ ID NO: 11) aligned with the wild-type capsid protein sequences of AAV1 to 9 (SEQ ID NOs: 1 to 9) is shown. [Figure 8C] The capsid protein sequence of shuffle 100-1 (SEQ ID NO: 11) aligned with the wild-type capsid protein sequences of AAV1 to 9 (SEQ ID NOs: 1 to 9) is shown. [Figure 8D] The capsid protein sequence of shuffle 100-1 (SEQ ID NO: 11) aligned with the wild-type capsid protein sequences of AAV1 to 9 (SEQ ID NOs: 1 to 9) is shown. [Figure 8E] The capsid protein sequence of shuffle 100-1 (SEQ ID NO: 11) aligned with the wild-type capsid protein sequences of AAV1 to 9 (SEQ ID NOs: 1 to 9) is shown. [Figure 8F] The capsid protein sequence of shuffle 100-1 (SEQ ID NO: 11) aligned with the wild-type capsid protein sequences of AAV1 to 9 (SEQ ID NOs: 1 to 9) is shown. [Figure 8G] The capsid protein sequence of shuffle 100-1 (SEQ ID NO: 11) aligned with the wild-type capsid protein sequences of AAV1 to 9 (SEQ ID NOs: 1 to 9) is shown. [Figure 8H] The capsid protein sequence of shuffle 100-1 (SEQ ID NO: 11) aligned with the wild-type capsid protein sequences of AAV1 to 9 (SEQ ID NOs: 1 to 9) is shown. [Figure 8I] The capsid protein sequence of shuffle 100-1 (SEQ ID NO: 11) aligned with the wild-type capsid protein sequences of AAV1 to 9 (SEQ ID NOs: 1 to 9) is shown. [Figure 9A]The capsid protein sequence of shuffle 100-3 (SEQ ID NO: 12) aligned with the wild-type capsid protein sequences of AAV1 to 9 (SEQ ID NOs: 1 to 9) is shown. [Figure 9B] The capsid protein sequence of shuffle 100-3 (SEQ ID NO: 12) aligned with the wild-type capsid protein sequences of AAV1 to 9 (SEQ ID NOs: 1 to 9) is shown. [Figure 9C] The capsid protein sequence of shuffle 100-3 (SEQ ID NO: 12) aligned with the wild-type capsid protein sequences of AAV1 to 9 (SEQ ID NOs: 1 to 9) is shown. [Figure 9D] The capsid protein sequence of shuffle 100-3 (SEQ ID NO: 12) aligned with the wild-type capsid protein sequences of AAV1 to 9 (SEQ ID NOs: 1 to 9) is shown. [Figure 9E] The capsid protein sequence of shuffle 100-3 (SEQ ID NO: 12) aligned with the wild-type capsid protein sequences of AAV1 to 9 (SEQ ID NOs: 1 to 9) is shown. [Figure 9F] The capsid protein sequence of shuffle 100-3 (SEQ ID NO: 12) aligned with the wild-type capsid protein sequences of AAV1 to 9 (SEQ ID NOs: 1 to 9) is shown. [Figure 9G] The capsid protein sequence of shuffle 100-3 (SEQ ID NO: 12) aligned with the wild-type capsid protein sequences of AAV1 to 9 (SEQ ID NOs: 1 to 9) is shown. [Figure 9H] The capsid protein sequence of shuffle 100-3 (SEQ ID NO: 12) aligned with the wild-type capsid protein sequences of AAV1 to 9 (SEQ ID NOs: 1 to 9) is shown. [Figure 9I] The capsid protein sequence of shuffle 100-3 (SEQ ID NO: 12) aligned with the wild-type capsid protein sequences of AAV1 to 9 (SEQ ID NOs: 1 to 9) is shown. [Figure 10A] The capsid protein sequence of shuffle 100-7 (SEQ ID NO: 13) aligned with the wild-type capsid protein sequences of AAV1 to 9 (SEQ ID NOs: 1 to 9) is shown. [Figure 10B]The capsid protein sequence of shuffle 100-7 (SEQ ID NO: 13) aligned with the wild-type capsid protein sequences of AAV1 to 9 (SEQ ID NOs: 1 to 9) is shown. [Figure 10C] The capsid protein sequence of shuffle 100-7 (SEQ ID NO: 13) aligned with the wild-type capsid protein sequences of AAV1 to 9 (SEQ ID NOs: 1 to 9) is shown. [Figure 10D] The capsid protein sequence of shuffle 100-7 (SEQ ID NO: 13) aligned with the wild-type capsid protein sequences of AAV1 to 9 (SEQ ID NOs: 1 to 9) is shown. [Figure 10E] The capsid protein sequence of shuffle 100-7 (SEQ ID NO: 13) aligned with the wild-type capsid protein sequences of AAV1 to 9 (SEQ ID NOs: 1 to 9) is shown. [Figure 10F] The capsid protein sequence of shuffle 100-7 (SEQ ID NO: 13) aligned with the wild-type capsid protein sequences of AAV1 to 9 (SEQ ID NOs: 1 to 9) is shown. [Figure 10G] The capsid protein sequence of shuffle 100-7 (SEQ ID NO: 13) aligned with the wild-type capsid protein sequences of AAV1 to 9 (SEQ ID NOs: 1 to 9) is shown. [Figure 10H] The capsid protein sequence of shuffle 100-7 (SEQ ID NO: 13) aligned with the wild-type capsid protein sequences of AAV1 to 9 (SEQ ID NOs: 1 to 9) is shown. [Figure 10I] The capsid protein sequence of shuffle 100-7 (SEQ ID NO: 13) aligned with the wild-type capsid protein sequences of AAV1 to 9 (SEQ ID NOs: 1 to 9) is shown. [Figure 11] Neutralizing antibody titers of library clones and parent serotypes in immunized mouse sera are shown. DETAILED DESCRIPTION OF THE INVENTION

[0027] definition Adeno-associated virus is a nonpathogenic parvovirus composed of a 4.7 kb single-stranded DNA genome within a nonenveloped icosahedral capsid. "AAV" is short for adeno-associated virus and can be used to refer to the virus itself or its derivatives. The genome contains three open reading frames (ORFs) flanked by inverted terminal repeats (ITRs) that function as viral origins of replication and packaging signals. The rep ORF encodes four nonstructural proteins that play roles in viral replication, transcriptional regulation, site-specific integration, and virion assembly. The cap ORF encodes three structural proteins (VP1-VP3) that assemble to form the 60-mer viral capsid. Finally, an ORF present as an alternative reading frame within the cap gene generates the assembly activating protein (AAP), a viral protein that localizes AAV capsid proteins to the nucleolus and functions in the capsid assembly process.

[0028] There are several naturally occurring serotypes and over 100 variants of AAV, each of which differs in amino acid sequence, particularly within the hypervariable regions of the capsid protein, and thus in its gene delivery properties. None of the AAVs have been associated with human disease, making recombinant AAVs attractive for clinical applications.

[0029] As used herein, the term "AAV" encompasses all subtypes and both naturally occurring and recombinant forms, unless otherwise required. The term "AAV" includes AAV type 1 (AAV-1 or AAV1), AAV type 2 (AAV-2 or AAV2), AAV type 3 (AAV-3 or AAV3), AAV type 4 (AAV-4 or AAV4), AAV type 5 (AAV-5 or AAV5), AAV type 6 (AAV-6 or AAV6), AAV type 7 (AAV-7 or AAV7), AAV type 8 (AAV-8 or AAV8), AAV type 9 (AAV-9 or AAV9), avian AAV, bovine AAV, canine AAV, equine AAV, primate AAV, non-primate AAV, and ovine AAV. "Primate AAV" refers to AAV that infects primates, "non-primate AAV" refers to AAV that infects non-primate mammals, "bovine AAV" refers to AAV that infects bovine mammals, and so on.

[0030] The genome sequences of various AAV serotypes, as well as the sequences of native terminal sequences (TRs), Rep proteins, and capsid subunits, are known to those skilled in the art. Such sequences can be found in the literature or in public databases such as GenBank. For example, GenBank accession numbers NC_002077.1 (AAV-1), AF063497.1 (AAV-1), NC_001401.2 (AAV-2), AF043303.1 (AAV-2), J01901.1 (AAV-2), U48704.1 (AAV-3), NC_001729.1 (AAV-3), NC_001829.1 (AAV-4), U89790.1 (AAV-4), NC_006152.1 (AAV-5), NC_006152.1 (AAV-6), NC_006152.1 (AAV-7), NC_006152.1 (AAV-8), NC_006152.1 (AAV-9), NC_006152.1 (AAV-10), NC_006152.1 (AAV-11), NC_006152.1 (AAV-12), NC_006152.1 (AAV-13), NC_006152.1 (AAV-14), NC_006152.1 (AAV-15), NC_006152.1 (AAV-16), NC_006152.1 (AAV-17), NC_006152.1 (AAV-18), NC_006152.1 (AAV-19), NC_006152.1 (AAV-19), NC_006152.1 (A See AAV_5), AF085716.1 (AAV-5), AF028704.1 (AAV-6), NC_006260.1 (AAV-7), AF513851.1 (AAV-7), AF513852.1 (AAV-8), NC_006261.1 (AAV-8), and AY530579.1 (AAV-9), the disclosures of which are incorporated herein by reference for their teaching of AAV nucleic acid and amino acid sequences. For example, Srivistava et al. (1983) J. Virology 45:555, Chiorini et al. (1998) J. Virology 71: 6823, Chiorini et al. (1999) J. Virology 73: 1309, Bantel-Schaal et al. (1999) J. Virology 73: 939, Xiao et al. al. (1999) J. Virology 73:3994, Muramatsu et al. (1996) Virology 221: 208, Shade et al., (1986) J. Virol. 58: 921, Gao et al. (2002) Proc. Nat. Acad. Sci. USA 99: 11854, Morris et al. See also, al. (2004) Virology 33:375-383, International Patent Publication Nos. WO 00 / 28061, WO 99 / 61601, WO 98 / 11244, and U.S. Patent No. 6,156,303.

[0031] The sequences of naturally occurring cap (capsid) proteins associated with AAV serotypes are known to those of skill in the art and include AAV1 (SEQ ID NO: 1), AAV2 (SEQ ID NO: 2), AAV3 (SEQ ID NO: 3), AAV4 (SEQ ID NO: 4), AAV5 (SEQ ID NO: 5), AAV6 (SEQ ID NO: 6), AAV7 (SEQ ID NO: 7), AAV8 (SEQ ID NO: 8), and AAV9 (SEQ ID NO: 9). The term "mutant AAV capsid protein" refers to an AAV capsid protein comprising an amino acid sequence that contains at least one substitution (including deletion, insertion, etc.) compared to one of the naturally occurring AAV capsid protein sequences set forth in SEQ ID NOs: 1-9.

[0032] An "AAV virion" or "AAV viral particle" refers to a viral particle composed of at least one AAV capsid protein and an encapsidated AAV polynucleotide.

[0033] "Recombinant," when 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 differs from polynucleotides found in nature. A recombinant virus is a viral particle that includes a recombinant polynucleotide. These terms include copies of the original polynucleotide construct and progeny of the original viral construct, respectively.

[0034] When an AAV virion contains a heterologous polynucleotide (i.e., a polynucleotide other than the wild-type AAV genome, such as a transgene delivered to a target cell, an RNAi agent or a CRISPR agent delivered to a target cell, etc.), it is typically referred to as a "recombinant AAV (rAAV) virion" or "rAAV virus particle." Generally, the heterologous polynucleotide is flanked by at least one, and generally two, AAV inverted terminal repeat (ITR) sequences.

[0035] The term "rAAV vector" by definition encompasses rAAV virions (i.e., rAAV virus particles) that contain rAAV polynucleotides (e.g., infectious rAAV virions), and also encompasses polynucleotides encoding rAAV (e.g., single-stranded polynucleotides encoding rAAV (ss-rAAV), double-stranded polynucleotides encoding rAAV (ds-rAAV), plasmids encoding rAAV, etc.).

[0036] "Packaging" refers to the series of intracellular events that lead to assembly and encapsidation of AAV particles.

[0037] AAV "rep" and "cap" genes refer to polynucleotide sequences that encode the replication and encapsidation proteins of adeno-associated virus. AAV rep and cap are referred to herein as AAV "packaging genes."

[0038] A "helper virus" for AAV refers to a virus that enables AAV (e.g., wild-type AAV) to be replicated and packaged by mammalian cells. Various such helper viruses for AAV are known to those skilled in the art and include adenoviruses, herpesviruses, and poxviruses such as vaccinia. Although adenovirus type 5 of subgroup C is most commonly used, adenoviruses encompass many different subgroups. Numerous adenoviruses derived from humans, non-human mammals, and birds are known and available from repositories such as the ATCC. Viruses of the herpes family include, for example, herpes simplex virus (HSV) and Epstein-Barr virus (EBV), as well as cytomegalovirus (CMV) and pseudorabies virus (PRV), which are also available from repositories such as the ATCC.

[0039] "Helper virus function(s)" refers to the function(s) encoded within the helper virus genome that enable AAV replication and packaging (in conjunction with other requirements for replication and packaging described herein). As described herein, "helper virus functions" can be provided in a number of ways, including providing a helper virus to the producer cell in trans, or, for example, by providing a polynucleotide sequence encoding the necessary function(s). For example, a plasmid or other expression vector containing a nucleotide sequence encoding one or more adenoviral proteins is transfected into the producer cell along with the rAAV vector.

[0040] An "infectious" virus or virus particle is one that contains a competently assembled viral capsid and is capable of delivering polynucleotide components to cells for which the viral species is tropic. This term does not necessarily imply any replication capability of the virus. Assays for counting infectious virus particles are described elsewhere in this disclosure and in the art. Viral infectivity can be expressed as the ratio of infectious virus particles to total virus particles. Methods for determining the ratio of infectious virus particles to total virus particles are known to those skilled in the art. See, for example, Grainger et al. (2005) Mol. Ther. 11:S337 (describing a TCID50 infectious titer assay) and Zolotukhin et al. (1999) Gene Ther. 6:973. See also the Examples.

[0041] The term "tropism," as used herein, refers to the preferential targeting of a specific host species or specific cell type within a host species by a virus (e.g., AAV). For example, a virus that can infect cells of the heart, lung, liver, and muscle has a broader (i.e., increased) tropism than a virus that can infect only lung and muscle cells. Tropism can also include the dependency of a virus on specific types of host cell surface molecules. For example, some viruses can infect only cells with surface glycosaminoglycans, while other viruses can infect only cells with sialic acid (such dependency can be tested using various cell lines deficient in specific classes of molecules as potential host cells for viral infection). In some cases, viral tropism describes the relative preference of the virus. For example, a first virus may be able to infect all cell types but is much more successful at infecting cells with surface glycosaminoglycans. If a second virus also favors the same characteristic (e.g., the second virus is also more successful at infecting cells with surface glycosaminoglycans), the second virus can be considered to have a similar (or identical) tropism to the first virus, even if the absolute transduction efficiency is not similar. For example, a second virus may be more efficient at infecting every given cell tested than the first virus, but if the relative preferences are similar (or identical), the second virus can still be considered to have a similar (or identical) tropism to the first virus. In some embodiments, the tropism of virions comprising a subject mutant AAV capsid protein is not altered compared to naturally occurring virions. In some embodiments, the tropism of virions comprising a subject mutant AAV capsid protein is expanded (i.e., enhanced) compared to naturally occurring virions. In some embodiments, the tropism of virions comprising a subject mutant AAV capsid protein is reduced compared to naturally occurring virions.

[0042] A "replication-competent" virus (e.g., replication-competent AAV) refers to a phenotypically wild-type virus that is infectious and also capable of replicating in infected cells (i.e., in the presence of a helper virus or helper virus functions). In the case of AAV, replication ability generally requires the presence of functional AAV packaging genes. Generally, the rAAV vectors described herein are replication-incompetent in mammalian cells (particularly human cells) due to the lack of one or more AAV packaging genes. Typically, such rAAV vectors lack any AAV packaging gene sequence to minimize the possibility that replication-competent AAV is generated by recombination between the AAV packaging gene and the incoming rAAV vector. In many embodiments, the rAAV vector preparations described herein contain, in some cases, only a small amount of replication-competent AAV (rcAAV, also referred to as RCA) (e.g., 10 2 Approximately 1 rcAAV per rAAV particle, less than 10 4 Approximately 1 rcAAV per rAAV particle, less than 10 8 Approximately 1 rcAAV per rAAV particle, less than 10 12 There is less than about 1 rcAAV per rAAV particle, or no rcAAV.

[0043] The term "polynucleotide" refers to a polymeric form of nucleotides of any length, containing deoxyribonucleotides or ribonucleotides, or their analogs. Polynucleotides may contain modified nucleotides, such as methylated nucleotides and nucleotide analogs, and may be separated 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-stranded and single-stranded molecules. Unless otherwise specified or required, any embodiment herein containing a polynucleotide encompasses both the double-stranded form and each of the two complementary single-stranded forms known or predicted to comprise the duplex.

[0044] A polynucleotide or polypeptide has a certain percentage of "sequence identity" with 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 several different ways. To determine sequence identity, sequences can be aligned using methods and computer programs, including BLAST, available via the World Wide Web at ncbi.nlm.nih.gov / BLAST / . Another alignment algorithm is FASTA, available in the Genetics Computing Group (GCG) package, a wholly owned subsidiary of Oxford Molecular Group, Inc., Madison, Wisconsin, USA. Other alignment techniques 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 & Co., San Diego, California, USA. Of particular interest are alignment programs that permit gaps in sequences. Smith-Waterman is one type of algorithm that permits gaps in sequence alignments. See Meth. Mol. Biol. 70:173-187 (1997). The GAP program, which uses the Needleman and Wunsch alignment method, can also be utilized to align sequences. See J. Mol. Biol. 48:443-453 (1970).

[0045] "Gene" refers to a polynucleotide that performs some type of function within a cell. For example, a gene can contain an open reading frame that can encode a specific protein after being transcribed and translated. Alternatively, a gene can encode a functional RNA product that is not translated (e.g., an aptamer, interfering RNA, ribosomal RNA (rRNA), transfer RNA (tRNA), etc.).

[0046] A "gene expression product" or "gene product" is a molecule resulting from the expression of a particular gene, as defined above. Gene expression products include, for example, polypeptides, aptamers, interfering RNA, messenger RNA (mRNA), rRNA, tRNA, non-coding RNA (ncRNA), etc.

[0047] "RNA interference agent" or "RNAi agent" encompasses any agent (or polynucleotide encoding such an agent) that can be used to alter the expression of a gene (as defined above). Examples of RNAi agents known to those skilled in the art include, but are not limited to, (i) siRNA agents, (ii) antisense RNA, (iii) CRISPR agents, (iv) zinc finger nuclease agents, and (v) transcription activator-like effector nuclease (TALEN) agents.

[0048] (i) siRNA agents ("small interfering" or "short interfering RNA" (or siRNA)) are RNA duplexes of nucleotides targeted to a genetic target ("target gene"). "RNA duplex" refers to a structure formed by complementary pairing between two regions of an RNA molecule, forming a region of double-stranded RNA (dsRNA). An siRNA is "targeted" to a gene in that the nucleotide sequence of its duplex is complementary to the nucleotide sequence of the target gene. In some embodiments, the length of the duplex of an siRNA 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. The RNA duplex portion of an siRNA can be part of a hairpin structure. An siRNA agent containing a hairpin may also be referred to as an "shRNA (short hairpin RNA) agent." In addition to the double-stranded portion, the hairpin structure may contain a loop portion located between the two sequences forming the duplex. The loop may 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 may also contain a 3' or 5' overhang portion. In some embodiments, the overhang is 0, 1, 2, 3, 4, or 5 nucleotides in length. Generally, the level of an expression product (e.g., mRNA, polypeptide, etc.) of a target gene is reduced by an siRNA agent (e.g., siRNA, shRNA, etc.) containing a specific double-stranded nucleotide sequence 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, ORF, or 3'UT region. In some embodiments, the short interfering RNA is approximately 19-25 nt in length.For example, for a description of siRNA technology, see PCT Publication Nos. WO0 / 44895, WO99 / 32619, WO01 / 75164, WO01 / 92513, WO01 / 29058, WO01 / 89304, WO02 / 16620, and WO02 / 29858, and U.S. Patent Publication No. 20040023390. The siRNA and / or shRNA 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 polyadenylation signal.

[0049] (ii) Antisense RNA is RNA that is complementary to a gene expression product. For example, antisense RNA targeted to a specific mRNA is an RNA-based agent (or can be a modified RNA) that is complementary to the mRNA, and the hybridization of the antisense RNA to the mRNA changes the expression of the mRNA (for example, by changing the stability of the RNA, changing the translation of the RNA, etc.). Also included within "antisense RNA" is a nucleic acid that encodes the antisense RNA.

[0050] (iii) CRISPR Agents. The CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) / CRISPR-associated (Cas) system provides bacteria and archaea with adaptive immunity against viruses and plasmids by using CRISPR RNA (crRNA) to induce silencing of invading nucleic acids. The Cas9 protein (or its functional equivalents and / or variants, i.e., Cas9-like proteins) naturally contains DNA endonuclease activity that depends on the association of the protein with two naturally occurring or synthetic RNA molecules, termed crRNA and tracrRNA (also called guide RNAs). In some cases, the two molecules are covalently linked to form a single molecule (also called single guide RNA ("sgRNA")). Thus, the Cas9 or Cas9-like protein associates with a DNA-targeting RNA (this term encompasses both bimolecular and single-molecule guide RNA configurations), which activates and guides the Cas9 or Cas9-like protein to the target nucleic acid sequence. When Cas9 or Cas9-like proteins retain their native enzymatic function, they cleave target DNA to create double-strand breaks, which can result in genome alterations (i.e., edits such as deletions, insertions (if a donor polynucleotide is present), exchanges, etc.), thereby altering gene expression. Some variants of Cas9 (which are encompassed by the term Cas9-like) have been altered to have reduced DNA cleavage activity (in some cases, they cleave one strand instead of both strands of the target DNA, while in other cases, they have severely reduced DNA cleavage activity to the point of abolishing it). Cas9-like proteins with reduced (and even no) DNA cleavage activity can still be directed to target DNA and inhibit RNA polymerase activity. Thus, enzymatically inactive Cas9-like proteins can be targeted to specific locations within target DNA by DNA-targeting RNA to inhibit transcription of the target DNA.Detailed information regarding CRISPR agents can be found, for example, in (a) Jinek et al., Science. 2012 Aug 17;337(6096):816-21: "A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity," (b) Qi et al., Cell. 2013 Feb 28;152(5):1173-83: "Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression," and (c) U.S. Patent Application No. 13 / 842,859 and PCT Application No. PCT / US13 / 32589, all of which are incorporated by reference in their entireties. Thus, the term "CRISPR agent," as used herein, encompasses any agent (nucleic acid encoding such an agent) comprising naturally occurring and / or synthetic sequences that can be used within a Cas9-based system (e.g., a Cas9 or Cas9-like protein; any component of a DNA-targeting RNA, e.g., a crRNA-like RNA, a tracrRNA-like RNA, a single guide RNA, etc.; a donor polynucleotide, etc.).

[0051] (iv) Zinc finger nuclease (ZFN) agents. Zinc finger nucleases (ZFNs) are artificial DNA endonucleases generated by fusing a zinc finger DNA binding domain to a DNA cleavage domain. ZFNs can be engineered to target a desired DNA sequence, allowing the zinc finger nuclease to cleave a unique target sequence. When introduced into a cell, ZFNs can be used to edit target DNA within the cell (e.g., the cell's genome) by causing a double-strand break. For more information on the use of ZFNs, see, e.g., Asuri et al., Mol Ther. 2012 Feb;20(2):329-38; Bibikova et al., Science. 2003 May 2;300(5620):764; Wood et al., Science. 2011 Jul 15;333(6040):307; Ochiai et al., Genes Cells. 2010 Aug;15(8):875-85; Takasu et al., Insect Biochem Mol Biol. 2010 Oct;40(10):759-65; Ekker et al., Zebrafish 2008 Summer;5(2):121-3; Young et al., Proc Natl Acad Sci U S A. 2011 Apr 26;108(17):7052-7; Goldberg et al., Cell. 2010 Mar 5;140(5):678-91, Geurts et al, Science. 2009 Jul 24;325(5939):433, Flisikowska et al, PLoS One. 2011;6(6):e21045. doi:10.1371 / journal.pone.0021045. Epub 2011 Jun 13, Hauschild et al, Proc Natl Acad Sci US A. 2011 Jul 19;108(29):12013-7, and Yu et al, Cell Res. 2011 Nov;21(11):1638-40, all of which are incorporated by reference herein for their teachings related to ZFNs.The term "ZFN agent" encompasses zinc finger nucleases and / or polynucleotides comprising a nucleotide sequence encoding a zinc finger nuclease.

[0052] (v) Transcription activator-like effector nuclease (TALEN) agents. Transcription activator-like effector nucleases (TALENs) are artificial DNA endonucleases generated by fusing a TAL (transcription activator-like) effector DNA binding domain to a DNA cleavage domain. TALENs can be rapidly engineered to bind to virtually any desired DNA sequence, and when introduced into cells, TALENs can be used to edit target DNA (e.g., the cell's genome) within the cell by causing double-strand breaks. For more information regarding the use of TALENs, see, for example, Hockemeyer et al. Nat Biotechnol. 2011 Jul 7; 29(8): 731-4, Wood et al. Science. 2011 Jul 15; 333(6040): 307, Tesson et al. Nat Biotechnol. 2011 Aug 5; 29(8): 695-6, and Huang et al., Nat Biotechnol. 2011 Aug 5; 29(8): 699-700, all of which are incorporated herein by reference for their teachings related to TALENs. The term "TALEN agent" encompasses TALENs and / or polynucleotides comprising a nucleotide sequence encoding a TALEN.

[0053] A "control element" or "control sequence" is a nucleotide sequence involved in a molecular interaction that contributes to the functional control of a polynucleotide, including replication, duplication, transcription, splicing, translation, or degradation of the polynucleotide. Control can affect the frequency, rate, or specificity of the process and can be enhancing or repressive in nature. Control elements known to those skilled in the art include, for example, transcription control sequences, such as promoters and enhancers. A promoter is a DNA region that, under certain conditions, is capable of binding RNA polymerase and initiating translation of a coding region, usually located downstream (3' direction) from the promoter.

[0054] "Operably linked" or "operably linked" refers to the juxtaposition of genetic elements, wherein the elements are in a relationship permitting them to function in the expected manner. For example, a promoter is operably linked to a coding region if the promoter helps initiate transcription of the coding sequence. There can be intervening residues between the promoter and the coding region, so long as this functional relationship is maintained.

[0055] An "expression vector" is a vector containing a region encoding a polypeptide of interest and is used to effect expression of the protein in an intended target cell. Expression vectors also contain control elements operably linked to the coding region to facilitate expression of the protein in the target. The combination of control elements and the gene or genes to which they are operably linked for expression is sometimes referred to as an "expression cassette," many of which are known and available to those of skill in the art or can be readily constructed from components available to those of skill in the art.

[0056] "Heterologous" means that the genotype is derived from a different entity from the other entity to which it is compared.For example, a polynucleotide introduced into a plasmid or vector derived from a different species by genetic engineering technology is a heterologous polynucleotide.A promoter that is removed from its native coding sequence and operably linked to a coding sequence that is not naturally found to be linked to is a heterologous promoter.Thus, for example, an rAAV that contains a heterologous nucleic acid encoding a heterologous gene product is an rAAV that contains a nucleic acid that is not normally contained in naturally occurring wild-type AAV, and the encoded heterologous gene product is a gene product that is not normally encoded by naturally occurring wild-type AAV.

[0057] The terms "genetic alteration" and "genetic modification" (and grammatical variations thereof) are used interchangeably herein and refer to a process by which a genetic element (e.g., a polynucleotide) is introduced into a cell by other than mitosis or meiosis. The element may be heterologous to the cell, or may be an additional copy or improved version of an element already present in the cell. Genetic alteration may be effected by transfecting the cell with a recombinant plasmid or other polynucleotide through any process known to those of skill in the art, such as, for example, electroporation, calcium phosphate precipitation, or contacting with a polynucleotide-liposome complex. Genetic alteration may also be effected by, for example, transduction or infection with a DNA or RNA virus or viral vector. Generally, the genetic element is introduced into a chromosome or microchromosome within the cell, but any alteration that alters the phenotype and / or genotype of the cell and its progeny is included in the term.

[0058] A cell has been "genetically modified" or "transformed" or "transfected" by exogenous DNA when the exogenous DNA has been introduced inside the cell (e.g., via a recombinant virus). The presence of the exogenous DNA results in a permanent or transient genetic change. The transforming DNA may or may not be integrated (covalently linked) into the genome of the cell. A "clone" is a collection of cells derived from a single cell or common ancestor by mitosis. A "cell line" is a clone of a primary cell that is capable of stable in vitro growth for many generations.

[0059] A cell is said to be "stably" altered, transduced, genetically modified, or transformed with a gene sequence if the gene sequence is available to perform its function during extended culture of the cell in vitro and / or over long periods in vivo. Generally, such cells are "genetically" altered (genetically modified) in that a genetic change is introduced that is also heritable by the progeny of the altered cell.

[0060] The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to polymers of amino acids of any length. The terms also encompass modified amino acid polymers, such as those that undergo disulfide bond formation, glycosylation, lipidation, phosphorylation, or conjugation with a labeling component. When discussed in the context of delivering gene products to a mammalian subject, polypeptides such as anti-angiogenic polypeptides, neuroprotective polypeptides, etc., and compositions thereof refer to the respective intact polypeptides or any fragments or genetically engineered derivatives thereof that retain the desired biochemical function of the intact protein. Similarly, reference to nucleic acids encoding anti-angiogenic polypeptides, nucleic acids encoding neuroprotective polypeptides, and other such nucleic acids for use in delivering gene products to a mammalian subject (which may be referred to as "transgenes" delivered to recipient cells) includes polynucleotides encoding the intact polypeptides or any fragments or genetically engineered derivatives that possess the desired biochemical function.

[0061] An "isolated" plasmid, nucleic acid, vector, virus, virion, host cell, protein, or other substance refers to a preparation of that substance that lacks at least some of the other components that may be present when the substance naturally occurs or is originally prepared from that substance. Thus, for example, an isolated substance can be prepared by using purification techniques to enrich it from a source mixture. Enrichment can be measured in absolute terms, such as weight per volume of solution, or relative to a second, potentially interfering substance present in the source mixture. Increasing enrichment in embodiments of the present disclosure translates to greater isolation. In some embodiments, an isolated plasmid, nucleic acid, vector, virus, host cell, or other substance is purified to, for example, about 80% to about 90% purity, at least about 90% purity, at least about 95% purity, at least about 98% purity, or at least about 99% purity, or greater purity.

[0062] As used herein, the terms "treatment," "treating," and the like refer to obtaining a desired pharmacological and / or physiological effect. The effect can be preventative, in that a disease or its symptoms are completely or partially prevented, and / or therapeutic, in that a disease and / or adverse effects attributable to the disease are partially or completely cured. "Treatment," as used herein, encompasses any treatment of a disease in a mammal, particularly a human, and includes (a) preventing the disease (and / or symptoms caused by the disease) in a subject who may be predisposed to or at risk of developing the disease, but who has not yet been diagnosed with it; (b) inhibiting the disease (and / or symptoms caused by the disease), i.e., arresting its progression; and (c) alleviating the disease (and / or symptoms caused by the disease), i.e., causing regression of the disease (and / or symptoms caused by the disease).

[0063] The terms "individual," "host," "subject," and "patient" are used interchangeably herein and refer to mammals, including, but not limited to, humans, non-human primates, including monkeys, mammalian sport animals (e.g., horses), mammalian livestock animals (e.g., sheep, goats, etc.), mammalian pets (dogs, cats, etc.), and rodents (e.g., mice, rats, etc.).

[0064] In some embodiments, the individual is a human who has previously been naturally exposed to AAV and consequently harbors anti-AAV antibodies (i.e., AAV neutralizing antibodies). In some embodiments, the individual is a human who has been administered an AAV vector (and may consequently harbor anti-AAV antibodies) and requires readministration of the vector for treatment of a different condition or for further treatment of the same condition. There are many such therapeutic applications / disease targets, for example, based on positive results in clinical trials involving AAV gene delivery to the liver, muscle, and retina (all tissues affected by neutralizing antibodies to this vehicle).

[0065] The term "effective amount," as used herein, is an amount sufficient to produce a beneficial or desired clinical result. An effective amount can be administered in one or more administrations. For purposes of this disclosure, an effective amount of a compound (e.g., infectious rAAV virions) is an amount sufficient to palliate, ameliorate, stabilize, reverse, prevent, slow, or delay the progression of (and / or symptoms associated with) a particular disease state (e.g., cancer). Thus, an effective amount of infectious rAAV virions is an amount of infectious rAAV virions that evades the neutralizing activity of an individual's anti-AAV antibodies and can thereby effectively deliver heterologous nucleic acid to a target cell(s) in the individual.

[0066] Before further describing the present invention, it is to be understood that this invention is not limited to the specific 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 specific embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

[0067] Where a range of values ​​is provided, each intervening value, to the tenth of the unit of the lower limit, between the upper and lower limit of that range, unless the context clearly dictates otherwise, 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 be independently included in the smaller ranges and are encompassed within the invention, but 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.

[0068] Unless otherwise defined, 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 and testing of the present invention, the preferred methods and materials are now described. All publications mentioned herein are incorporated by reference to disclose and describe the methods and / or materials in the context in which the publications are described.

[0069] As used herein and in the appended claims, the singular forms "a," "an," and "the" should also be understood to include plural referents unless the context clearly dictates otherwise. Thus, for example, a reference to "an infectious recombinant adeno-associated virus (rAAV) virion" includes a plurality of such virions; a reference to "the infectious recombinant adeno-associated virus (rAAV) virion" includes one or more such virions and equivalents thereof known to those skilled in the art, and so forth. It is further noted that the claims may be drafted to exclude any additional elements. Accordingly, this statement is intended to serve as a warning against the use of exclusionary language such as "solely," "only," or the use of "negative" limitations in connection with the recitation of claim elements.

[0070] It is understood that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination. All combinations of the embodiments related to the present invention are specifically embraced by the present invention and are disclosed herein as if each and every combination were individually and expressly disclosed. Furthermore, all subcombinations of the various embodiments and elements thereof are also specifically embraced by the present invention and are disclosed herein as if each and every such subcombination were individually and expressly disclosed herein.

[0071] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein should 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.

[0072] Detailed Description The present disclosure provides infectious recombinant adeno-associated virus (rAAV) virions comprising a mutant capsid protein and a heterologous nucleic acid. The present disclosure further provides mutant adeno-associated virus (AAV) capsid proteins (and / or nucleic acids encoding the mutant AAV capsid proteins) that confer increased resistance to human AAV neutralizing antibodies on infectious rAAV virions. The present disclosure further provides host cells comprising infectious rAAV virions and / or nucleic acids encoding the subject mutant AAV capsid proteins. The present disclosure further provides libraries of the above-described virions, capsid proteins, nucleic acids, and / or host cells, wherein the mutant AAV capsid protein of at least one member of the library comprises an amino acid sequence having at least one amino acid substitution compared to the amino acid sequence set forth in one of SEQ ID NOs: 10-13 and 26-33.

[0073] The present disclosure further provides methods for delivering heterologous nucleic acids to target cells, wherein the target cells are contacted with a subject infectious rAAV virion. The present disclosure further provides methods for delivering gene products to an individual, generally comprising administering an effective amount of a subject rAAV virion to an individual in need thereof. Also provided herein are compositions and kits for practicing the subject methods. In many embodiments, a subject infectious rAAV virion, a subject nucleic acid, a subject mutant AAV capsid protein, a subject host cell, or the like, is isolated.

[0074] Mutant AAV capsid polypeptides A subject mutant AAV capsid polypeptide (or mutant AAV capsid protein encoded by a subject nucleic acid) confers increased resistance to human AAV neutralizing antibodies to infectious rAAV virions comprising the mutant AAV capsid polypeptide, compared to the resistance exhibited by wild-type AAV (e.g., AAV2 (wild-type AAV serotype 2)) or AAV comprising the wild-type capsid protein. In some embodiments, the increased resistance is at least about 1.5-fold (e.g., at least about 1.5-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 7.5-fold, at least about 10-fold, at least about 12-fold, at least about 15-fold, at least about 17-fold, at least about 20-fold, at least about 25-fold, at least about 30-fold, at least about 40-fold, at least about 50-fold, at least about 75-fold, at least about 100-fold, at least about 150-fold, at least about 200-fold, at least about 250-fold, at least about 300-fold, etc.) greater than the resistance exhibited by wild-type AAV (e.g., AAV2 (wild-type AAV serotype 2)) or an AAV comprising a wild-type capsid protein.

[0075] A subject mutant AAV capsid protein (or a mutant AAV capsid protein encoded by a subject nucleic acid) can be said to confer on infectious rAAV virions increased transduction of mammalian cells in the presence of human AAV neutralizing antibodies, compared to the transduction exhibited by wild-type AAV (e.g., AAV2 (wild-type AAV serotype 2)) or an AAV comprising the wild-type capsid protein. In some embodiments, the increased transduction is at least about 1.5-fold (e.g., at least about 1.5-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 7.5-fold, at least about 10-fold, at least about 12-fold, at least about 15-fold, at least about 17-fold, at least about 20-fold, at least about 25-fold, at least about 30-fold, at least about 40-fold, at least about 50-fold, at least about 75-fold, at least about 100-fold, at least about 150-fold, at least about 200-fold, at least about 250-fold, at least about 300-fold, etc.) greater than the transduction exhibited by wild-type AAV (e.g., AAV2 (wild-type AAV serotype 2)) or an AAV comprising a wild-type capsid protein.

[0076] In some embodiments, a subject mutant AAV capsid protein (or a mutant AAV capsid protein encoded by a subject nucleic acid) exhibits reduced binding to neutralizing antibodies that bind to wild-type AAV capsid protein. For example, a subject mutant AAV capsid protein may exhibit at least about 1.5-fold (e.g., at least about 1.5-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 7.5-fold, at least about 10-fold, at least about 12-fold, at least about 15-fold, at least about 17-fold, at least about 20-fold, at least about 25-fold, at least about 30-fold, at least about 40-fold, at least about 50-fold, at least about 75-fold, at least about 100-fold, at least about 150-fold, at least about 200-fold, at least about 250-fold, at least about 300-fold, etc.) reduced binding (e.g., reduced affinity) to a neutralizing antibody that binds to a wild-type AAV capsid protein, compared to the binding affinity of the antibody to the wild-type AAV capsid protein.

[0077] In some embodiments, the anti-AAV neutralizing antibody is about 10 -7 Less than M, approximately 5 x 10 -6 Less than M, about 10 -6 Less than M, approximately 5 x 10 -5 Less than M, about 10 -5 Less than M, about 10 -4 binds to a subject mutant AAV capsid protein (or a mutant AAV capsid protein encoded by a subject nucleic acid) with less than M, or with lower affinity.

[0078] The term "mutant capsid protein" does not encompass wild-type AAV capsid proteins. A "mutant AAV capsid protein" does not include an amino acid sequence present in a naturally occurring AAV capsid protein. For example, a subject mutant capsid protein does not include an amino acid sequence having 100% sequence identity with any of the sequences set forth in SEQ ID NOs: 1-9. In other words, a subject mutant capsid protein does not include an amino acid sequence set forth in any of SEQ ID NOs: 1-9. A mutant capsid protein can differ in amino acid sequence from a "starter" or "parent" AAV capsid protein, which can be a wild-type AAV capsid protein or a non-wild-type AAV capsid protein.

[0079] In some embodiments, a subject mutant AAV capsid protein (or a mutant AAV capsid protein encoded by a subject nucleic acid) comprises an amino acid sequence having at least about 90% (e.g., at least about 92%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5%, or 100%) amino acid sequence identity to amino acids 203-736 of the amino acid sequence set forth in one of SEQ ID NOs: 10-13 and 26-33.

[0080] In some embodiments, a subject mutant AAV capsid protein (or a mutant AAV capsid protein encoded by a subject nucleic acid) comprises an amino acid sequence having at least about 90% (e.g., at least about 92%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5%, or 100%) amino acid sequence identity to an amino acid sequence set forth in one of SEQ ID NOs: 10-13 and 26-33.

[0081] In some embodiments, a subject mutant AAV capsid protein (or a mutant AAV capsid protein encoded by a subject nucleic acid) comprises an amino acid sequence having at least about 95% (e.g., at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5%, or 100%) amino acid sequence identity to amino acids 203-736 of the amino acid sequence set forth in SEQ ID NO: 10, and comprises the amino acid substitutions N312K, N449D, D472N, N551S, I698V, and L735Q, or the corresponding positions in another AAV parent serotype, relative to the AAV capsid protein of AAV2 (e.g., SEQ ID NO: 2).

[0082] In some embodiments, a subject mutant AAV capsid protein (or a mutant AAV capsid protein encoded by a subject nucleic acid) comprises an amino acid sequence having at least about 95% (e.g., at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5%, or 100%) amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 10, and includes the amino acid substitutions N312K, N449D, D472N, N551S, I698V, and L735Q relative to the AAV capsid protein of AAV2 (e.g., SEQ ID NO: 2), or the corresponding positions in another AAV parent serotype.

[0083] In some embodiments, a subject mutant AAV capsid protein (or a mutant AAV capsid protein encoded by a subject nucleic acid) comprises an amino acid sequence having at least about 95% (e.g., at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5%, or 100%) amino acid sequence identity to amino acids 203-736 of the amino acid sequence set forth in SEQ ID NO:31, and comprises the amino acid substitutions N312K, N449D, N551S, and I698V relative to the AAV capsid protein of AAV2 (e.g., SEQ ID NO:2), or the corresponding positions in another AAV parent serotype.

[0084] In some embodiments, a subject mutant AAV capsid protein (or a mutant AAV capsid protein encoded by a subject nucleic acid) comprises an amino acid sequence having at least about 95% (e.g., at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5%, or 100%) amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO:31, and includes the amino acid substitutions N312K, N449D, N551S, and I698V relative to the AAV capsid protein of AAV2 (e.g., SEQ ID NO:2), or the corresponding positions in another AAV parent serotype.

[0085] In some embodiments, a subject mutant AAV capsid protein (a mutant AAV capsid protein encoded by a subject nucleic acid) comprises an amino acid sequence having at least about 95% (e.g., at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5%, or 100%) amino acid sequence identity to amino acids 203-736 of the amino acid sequence set forth in SEQ ID NO: 32, and comprises the amino acid substitutions D180N, N312K, Q385R, N449D, N551S, I698V, and S721T relative to the AAV capsid protein of AAV2 (e.g., SEQ ID NO: 2), or the corresponding positions in another AAV parent serotype.

[0086] In some embodiments, a subject mutant AAV capsid protein (or a mutant AAV capsid protein encoded by a subject nucleic acid) comprises an amino acid sequence having at least about 95% (e.g., at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5%, or 100%) amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 32, and includes the amino acid substitutions D180N, N312K, Q385R, N449D, N551S, I698V, and S721T relative to the AAV capsid protein of AAV2 (e.g., SEQ ID NO: 2), or the corresponding positions in another AAV parent serotype.

[0087] In some embodiments, a subject mutant AAV capsid protein (or a mutant AAV capsid protein encoded by a subject nucleic acid) comprises an amino acid sequence having at least about 95% (e.g., at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5%, or 100%) amino acid sequence identity to amino acids 203-736 of the amino acid sequence set forth in SEQ ID NO:33, and includes the amino acid substitutions N312K, N449D, T450A, N551S, and I698V relative to the AAV capsid protein of AAV2 (e.g., SEQ ID NO:2), or the corresponding positions in another AAV parent serotype.

[0088] In some embodiments, a subject mutant AAV capsid protein (or a mutant AAV capsid protein encoded by a subject nucleic acid) comprises an amino acid sequence having at least about 95% (e.g., at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5%, or 100%) amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 33, and includes the amino acid substitutions N312K, N449D, T450A, N551S, and I698V relative to the AAV capsid protein of AAV2 (e.g., SEQ ID NO: 2), or the corresponding positions in another AAV parent serotype.

[0089] Exemplary mutant AAV capsid proteins include, but are not limited to, the following (see Figures 8-10 for selected exemplary sequence alignments):

[0090] SM10-2 (amino acid sequence) TIFF0007813169000001.tif108152

[0091] SM10-2 (nucleotide sequence) TIFF0007813169000002.tif202153

[0092] Shuffle 100-1 (amino acid sequence) TIFF0007813169000003.tif108153

[0093] Shuffle 100-1 (nucleotide sequence) TIFF0007813169000004.tif203153

[0094] Shuffle 100-3 (amino acid sequence) TIFF0007813169000005.tif108152

[0095] Shuffle 100-3 (nucleotide sequence) TIFF0007813169000006.tif203153

[0096] Shuffle 100-7 (amino acid sequence) TIFF0007813169000007.tif108152

[0097] Shuffle 100-7 (nucleotide sequence) TIFF0007813169000008.tif204153

[0098] Shuffle 10-2 (amino acid sequence) TIFF0007813169000009.tif108153

[0099] Shuffle 10-2 (nucleotide sequence) TIFF0007813169000010.tif203153

[0100] Shuffle 10-6 (amino acid sequence) TIFF0007813169000011.tif107152

[0101] Shuffle 10-6 (nucleotide sequence) TIFF0007813169000012.tif204153

[0102] Shuffle 10-8 (amino acid sequence) TIFF0007813169000013.tif108153

[0103] Shuffle 10-8 (nucleotide sequence) TIFF0007813169000014.tif203153

[0104] Shuffle 100-2 (amino acid sequence) TIFF0007813169000015.tif108153

[0105] Shuffle 100-2 (nucleotide sequence) TIFF0007813169000016.tif203153

[0106] SM10-1 (amino acid sequence) TIFF0007813169000017.tif108153

[0107] SM10-1 (nucleotide sequence) TIFF0007813169000018.tif203153

[0108] SM10-8 (amino acid sequence) TIFF0007813169000019.tif108152

[0109] SM10-8 (nucleotide sequence) TIFF0007813169000020.tif203153

[0110] SM100-3 (amino acid sequence) TIFF0007813169000021.tif107153

[0111] SM100-3 (nucleotide sequence) TIFF0007813169000022.tif203153

[0112] SM100-10 (amino acid sequence) TIFF0007813169000023.tif107152

[0113] SM100-10 (nucleotide sequence) TIFF0007813169000024.tif203153

[0114] Nucleic Acids and Host Cells The present disclosure provides nucleic acids comprising nucleotide sequences encoding mutant AAV capsid proteins (as described above), as well as host cells comprising the subject nucleic acids. The nucleic acids and host cells are useful for producing rAAV virions (as described below).

[0115] The present disclosure provides host cells, e.g., isolated host cells, comprising a subject nucleic acid. A subject host cell may be referred to as a "genetically modified host cell" and 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. When 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 (i.e., stably transfected) with a subject nucleic acid. In other embodiments, a subject host cell is transiently genetically modified (i.e., transiently transfected) with a subject nucleic acid.

[0116] The subject nucleic acids are stably or transiently introduced into host cells using established techniques, including, but not limited to, electroporation, calcium phosphate precipitation, liposome-mediated transfection, etc. For stable transformation, the subject nucleic acids will generally further include a selectable marker, e.g., any of several known selectable markers such as neomycin resistance.

[0117] A subject host cell is produced by introducing a subject nucleic acid into any of a variety of cells, e.g., mammalian cells, including, for example, mouse cells and primate cells (e.g., human cells). Suitable mammalian cells include, but are not limited to, primate cells and cell lines, and 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, etc.

[0118] In some embodiments, a subject host cell comprises a nucleic acid comprising a nucleotide sequence encoding one or more AAV rep proteins in addition to a nucleic acid comprising a nucleotide sequence encoding a mutant capsid protein. In other embodiments, a subject host cell further comprises an rAAV vector, as described below. rAAV virions are produced using a subject host cell, as described in more detail below.

[0119] Infectious rAAV virions The subject infectious rAAV virions comprise a mutant AAV capsid protein and a heterologous nucleic acid (described in more detail below) and exhibit increased resistance to human AAV neutralizing antibodies compared to the resistance exhibited by wild-type AAV (e.g., AAV2 (wild-type AAV serotype 2)) or an AAV comprising a wild-type capsid protein. "Increased resistance" means that the subject infectious rAAV virions exhibit increased infectivity in the presence of human anti-AAV antibodies. As described above, viral infectivity can be expressed as the ratio of infectious viral particles to total viral particles. Thus, increased infectivity refers to an increased ratio of infectious viral particles to total viral particles. To determine the resistance of AAV to human anti-AAV antibodies, the infectivity of the AAV is measured in the presence of various concentrations of human anti-AAV antibodies to obtain the antibody concentration (e.g., serum concentration, IVIG concentration, etc.) (mg / mL) required to reduce gene delivery efficiency (i.e., infectivity) to 50% of that in the absence of human anti-AAV antibodies. A virus that requires a higher antibody concentration to reduce gene delivery efficiency to 50% of that in the absence of human anti-AAV antibodies is said to have increased resistance to antibody neutralization. Thus, a two-fold increase in resistance means a two-fold increase in the antibody concentration required to reduce gene delivery efficiency to 50% of that in the absence of human anti-AAV antibodies. In some embodiments, a subject infectious rAAV virion exhibits at least about 1.5-fold (e.g., at least about 1.5-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 7.5-fold, at least about 10-fold, at least about 12-fold, at least about 15-fold, at least about 17-fold, at least about 20-fold, at least about 25-fold, at least about 30-fold, at least about 40-fold, at least about 50-fold, at least about 75-fold, at least about 100-fold, at least about 150-fold, at least about 200-fold, at least about 250-fold, at least about 300-fold, etc.) greater resistance to human AAV neutralizing antibodies than the resistance exhibited by wild-type AAV (e.g., AAV2 (wild-type AAV serotype 2)) or an AAV comprising a wild-type capsid protein.

[0120] A subject infectious rAAV virion can be said to exhibit increased transduction of mammalian cells in the presence of human AAV neutralizing antibodies. In some embodiments, a subject infectious rAAV virion exhibits at least about 1.5-fold (e.g., at least about 1.5-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 7.5-fold, at least about 10-fold, at least about 12-fold, at least about 15-fold, at least about 17-fold, at least about 20-fold, at least about 25-fold, at least about 30-fold, at least about 40-fold, at least about 50-fold, at least about 75-fold, at least about 100-fold, at least about 150-fold, at least about 200-fold, at least about 250-fold, at least about 300-fold, etc.) greater transduction of mammalian cells in the presence of human AAV neutralizing antibodies than the transduction exhibited by wild-type AAV (e.g., AAV2 (wild-type AAV serotype 2)) or an AAV comprising a wild-type capsid protein.

[0121] In some embodiments, a subject infectious rAAV virion exhibits reduced binding to a neutralizing antibody that binds to a wild-type AAV capsid protein. For example, a subject infectious rAAV virion may exhibit reduced binding (e.g., reduced affinity) to a neutralizing antibody that binds to a wild-type AAV capsid protein by at least about 1.5-fold (e.g., at least about 1.5-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 7.5-fold, at least about 10-fold, at least about 12-fold, at least about 15-fold, at least about 17-fold, at least about 20-fold, at least about 25-fold, at least about 30-fold, at least about 40-fold, at least about 50-fold, at least about 75-fold, at least about 100-fold, at least about 150-fold, at least about 200-fold, at least about 250-fold, at least about 300-fold, etc.) compared to the antibody's binding affinity to the wild-type AAV capsid protein.

[0122] In some embodiments, the anti-AAV neutralizing antibody is about 10 -7 Less than M, approximately 5 x 10 -6 Less than M, about 10 -6 Less than M, approximately 5 x 10 -5 Less than M, about 10 -5Less than M, about 10 -4 binds to the subject infectious rAAV virions with less than or equal to M.

[0123] In some embodiments, a subject infectious rAAV virion exhibits an increased in vivo residence time compared to wild-type AAV, e.g., a subject infectious rAAV virion 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 wild-type AAV.

[0124] Whether infectious rAAV virions of a given subject exhibit reduced binding to and / or increased resistance to neutralizing antibodies can be determined using any convenient assay known to those of skill in the art.

[0125] In some embodiments, a subject infectious rAAV virion comprises wild-type Rep78, Rep68, Rep52, and Rep40 proteins, hi other embodiments, a subject infectious rAAV virion comprises one or more mutant capsid proteins, as well as one or more mutations in one or more of the Rep78, Rep68, Rep52, and Rep40 proteins.

[0126] heterologous nucleic acid Heterologous DNA molecules (also referred to herein as "heterologous nucleic acids") suitable for use in a subject rAAV vector (e.g., a subject infectious rAAV virion) can be any heterologous nucleic acid. In some embodiments, the heterologous nucleic acid comprises a nucleotide sequence encoding a polypeptide (e.g., a protein that confers some desired characteristic to the target cell, such as a fluorescent protein that allows cell tracking, an enzyme that provides a missing or altered activity in the target cell, etc.). In some embodiments, the heterologous nucleic acid comprises an RNA interference agent (as defined above).

[0127] The subject heterologous nucleic acids will generally be less than about 5 kilobases (kb) in size and will include, for example, a gene (nucleotide sequence) encoding a protein that is deficient or missing from a recipient individual or target cell, a gene encoding a protein having a desired biological or therapeutic effect (e.g., antibacterial, antiviral, or antitumor / anticancer function), a nucleotide sequence encoding an RNA that inhibits or reduces the production of a harmful or undesirable protein (e.g., a nucleotide sequence encoding an RNA interfering agent as defined above), and / or a nucleotide sequence encoding an antigenic protein.

[0128] Preferred heterologous nucleic acids are those used to treat acquired immune deficiency syndrome (AIDS), cancer, hypercholestemia; activator deficiency / GM2 gangliosidosis, alpha-mannosidosis, aspartylglucosaminuria, cholesterol ester storage disease, chronic hexosaminidase A deficiency, cystinosis, Danon disease, Fabry disease, Farber disease, fucosidosis, galactosialidosis, Gaucher disease, GM1 gangliosidosis, I-cell disease / mucolipidosis II, infantile free sialic acid storage disease / ISSD, juvenile hexosaminidase A deficiency, Krabbe disease, lysosomalic acid lipase deficiency, metachromatic leukodystrophy, mucopolysaccharidosis diseases (pseudo-Hurler polydystrophy / mucolipidosis IIIA, MPS I-Hurler syndrome, MPS I-Scheie syndrome, MPS I-Hurler-Scheie syndrome, MPS II-Hunter syndrome, Sanfilippo syndrome type A / MPS MPS III A, Sanfilippo syndrome type B / MPS III B, Sanfilippo syndrome type C / MPS III C, Sanfilippo syndrome type D / MPS III D, Morquio A / MPS IV A, Morquio B / MPS IV B, MPS IX hyaluronidase deficiency, MPS VI maroteolamy, MPS VII Sly syndrome (including mucolipidosis I / sialidosis, mucolipidosis IIIC, and mucolipidosis IV), multiple sulfatase deficiency, Niemann-Pick disease, neuronal ceroid lipofuscinosis, Pompe disease / glycogenosis type II, pyknodysostosis, Sandhoff disease / adult-onset / GM2 gangliosidosis, Sandhoff disease / GM2 gangliosidosis-infantile, Sandhoff disease / GM2 gangliosidosis-juvenile, Schindler disease, Salla disease / sialic acid storage disease, Tay-Sachs / GM2 gangliosidosis These include, but are not limited to, those encoding proteins used to treat endocrine, metabolic, hematological, cardiovascular, neurological, musculoskeletal, urinary, pulmonary, and immune disorders, including disorders such as inflammatory, autoimmune, chronic, and infectious diseases, such as lysosomal storage diseases such as leukemia, leukemia, and Wolman disease, insulin disorders such as diabetes mellitus, growth disorders, various blood disorders including various anemias, thalassemias, and hemophilia; genetic defects such as cystic fibrosis, Gaucher disease, Hurler disease, adenosine deaminase (ADA) deficiency, emphysema, and the like.

[0129] Suitable heterologous nucleic acids include interferons (e.g., IFN-γ, IFN-α, IFN-β, IFN-ω; IFN-τ); insulin (e.g., Novolin, Humulin, Humalog, Lantus, Ultralente, etc.); erythropoietin ("EPO"; e.g., Procrit®, Eprex®, or Epogen® (epoetin-α); Aranesp® (darbepoetin-α); NeoRecormon®, Epogin® (epoetin-β), etc.); monoclonal antibodies (e.g., IFN-γ, IFN-α, IFN-β, IFN-τ); insulin (e.g., Novolin, Humulin, Humalog, Lantus, Ultralente, etc.); erythropoietin ("EPO"; e.g., Procrit®, Eprex®, or Epogen® (epoetin-α); Aranesp® (darbepoetin-α); NeoRecormon®, Epogin® (epoetin-β), etc.); monoclonal antibodies (e.g., IFN-γ, IFN-α, IFN-τ); Antibodies (e.g., monoclonal antibodies) (e.g., Rituxan® (rituximab), Remicade® (infliximab), Herceptin® (trastuzumab), Humira™ (adalimumab), Xolair® (omalizumab), Bexxar® (tositumomab), Raptiva™ (efalizumab), Erbitux™ (cetuximab), Avaconazole™ (available from avian venom ... stin® (bevacizumab), etc.); blood factors (e.g., Activase® (alteplase) tissue plasminogen activator, NovoSeven® (recombinant human Factor VIIa), Factor VIIa, Factor VIII (e.g., Kogenate®), Factor IX, β-globin, hemoglobin, etc.); colony-stimulating factors (e.g., Neupogen® (filgrastim; G-CSF), Neulasta (pegfilgrastim), granulocyte colony-stimulating factor (G-CSF), granulocyte-monocyte colony-stimulating factor (G-CSF), knee-stimulating factor, macrophage colony-stimulating factor, megakaryocyte colony-stimulating factor, etc.; growth hormones (e.g., somatropins, e.g., Genotropin®, Nutropin®, Norditropin®, Saizen®, Serostim®, Humatrope®, etc., human growth hormone, etc.); interleukins (e.g., IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, etc., including e.g., Proleukin®);Growth factors (e.g., Regranex® (beclapermin; PDGF), Fiblast® (trafermin; bFGF), Stemgen® (ansestim; stem cell factor), keratinocyte growth factor, acidic fibroblast growth factor, stem cell factor, basic fibroblast growth factor, hepatocyte growth factor, etc.); soluble receptors (e.g., TNF-α binding soluble receptors such as Enbrel® (etanercept), soluble VEGF receptors, soluble interleukin receptors, soluble gamma / delta receptors, etc. T cell receptors, etc.; enzymes (e.g., α-glucosidase; Cerazyme® (imiglucarase; β-glucocerebrosidase, Ceredase® (alglucerase); enzyme activators (e.g., tissue plasminogen activator); chemokines (e.g., IP-10; Mig; Groα / IL-8, RANTES; MIP-1α; MIP-1β; MCP-1; PF-4, etc.); angiogenic agents (e.g., vascular endothelial growth factor (VEGF)); anti-angiogenic agents (e.g., soluble VEGF receptors); protein vaccines; bradykinin, cholecystokinin, gastin, secretin, oxytocin, gonadotropin-releasing hormone, beta-endorphin, enkephalin, substance P, somatostatin, prolactin, galanin, growth hormone-releasing hormone, bombesin, dynorphin, neurotensin, mochi Neuroactive peptides such as phospholipid, thyroid-stimulating hormone, neuropeptide Y, progesterone, calcitonin, insulin, glucagon, vasopressin, angiotensin II, thyrotropin-releasing hormone, vasoactive intestinal peptide, sleep peptide; other proteins such as thrombolytic agents, atrial natriuretic peptide, bone morphogenetic protein, thrombopoietin, relaxin, glial fibrillary acidic protein, follicle-stimulating hormone, human alpha-1 antitrypsin, leukemia inhibitory factor, transforming growth factor, insulin-like growth factor, luteinizing hormone, macrophage-activating factor, tumor necrosis factor, neutrophil chemotactic factor, nerve growth factor, tissue inhibitor of metalloproteinases; vasoactive intestinal peptide, angiogenin, angiotropin, fibrin; hirudin; leukemia inhibitory factor; IL-1 receptor antagonists (e.g., Kineret® (anakinra));Suitable nucleic acids include, but are not limited to, those encoding any of a variety of proteins, including, but not limited to, ion channels such as the cystic fibrosis transmembrane conductance regulator (CFTR); dystrophin; utrophin, a tumor suppressor; the lysosomal enzyme acid alpha-glucosidase (GAA); etc. Suitable nucleic acids also include those encoding functional fragments of any of the foregoing proteins, and nucleic acids encoding functional variants of any of the foregoing proteins;

[0130] Suitable heterologous nucleic acids also include those encoding antigenic proteins. The subject rAAV vectors comprising heterologous nucleic acids encoding antigenic proteins are suitable for stimulating an immune response to antigenic proteins in mammalian hosts. The antigenic proteins are derived from autoantigens, allergens, tumor / cancer-associated antigens, pathogenic viruses, pathogenic bacteria, pathogenic protozoa, pathogenic helminths, or any other pathogenic microorganisms that infect mammalian hosts. As used herein, the term "nucleic acid encoding an antigenic protein derived from" includes nucleic acids encoding wild-type antigenic proteins, such as nucleic acids isolated from a pathogenic virus encoding a viral protein; synthetic nucleic acids generated in a laboratory that encode an antigenic protein having an amino acid sequence identical to that of a naturally occurring antigenic protein; synthetic nucleic acids generated in a laboratory that encode an antigenic protein having an amino acid sequence that differs from that of a naturally occurring antigenic protein (e.g., from 1 amino acid to about 15 amino acids) but that still elicit an immune response to the corresponding naturally occurring antigenic protein; and synthetic nucleic acids generated in a laboratory that encode fragments of an antigenic protein (e.g., a fragment of about 5 amino acids to about 50 amino acids, which fragment contains one or more antigenic epitopes), which elicit an immune response to the corresponding naturally occurring antigenic protein.

[0131] Similarly, antigenic proteins "derived from" autoantigens, allergens, tumor / cancer-associated antigens, pathogenic viruses, pathogenic bacteria, pathogenic protozoa, pathogenic helminths, or any other pathogenic microorganism that infects a mammalian host include proteins that have the same amino acid sequence as the naturally occurring antigenic protein, and proteins that have an amino acid sequence that differs from the naturally occurring antigenic protein (e.g., by 1 amino acid to about 15 amino acids) but that still elicit an immune response to the corresponding naturally occurring antigenic protein, as well as fragments of the antigenic protein (e.g., a fragment of about 5 amino acids to about 100 amino acids, e.g., about 5 to about 50 amino acids, which fragment contains one or more antigenic epitopes) that elicit an immune response to the corresponding naturally occurring antigenic protein.

[0132] In some embodiments, an immune response to an antigenic protein encoded by a subject rAAV vector will stimulate a protective immune response in a mammalian host against a pathogenic microorganism that presents the antigenic protein or antigenic epitope (or a protein or epitope cross-reactive with the rAAV-encoded antigenic protein or antigenic epitope). In some embodiments, a cytotoxic T lymphocyte (CTL) response to the rAAV-encoded antigenic protein will be raised in the mammalian host. In other embodiments, a humoral response to the rAAV-encoded antigenic protein will be raised in mammalian host cells such that antibodies specific for the antigenic protein are generated. In many embodiments, a TH1 immune response to the rAAV-encoded antigenic protein will be raised in the mammalian host. Suitable antigenic proteins include tumor / cancer-associated antigens, viral antigens, bacterial antigens, and protozoan antigens, as well as antigenic fragments thereof. In some embodiments, the antigenic protein is derived from an intracellular pathogen. In other embodiments, the antigenic protein is an autoantigen. In still other embodiments, the antigenic protein is an allergen.

[0133] Tumor / cancer-specific antigens include, but are not limited to, any of the various MAGEs (melanoma-associated antigen E), including MAGE1 (e.g., GenBank accession number M77481), MAGE2 (e.g., GenBank accession number U03735), MAGE3, MAGE4, ​​etc.; any of the various tyrosinases; mutant ras; mutant p53 (e.g., GenBank accession numbers X54156 and AA494311); and p97 melanoma antigen (e.g., GenBank accession number M12154). Other tumor / cancer-specific antigens include Ras and p53 peptides associated with advanced cancers, HPV16 / 18 and E6 / E7 antigens associated with cervical cancer, MUCI1-KLH associated with breast cancer (e.g., GenBank accession number J03651), CEA (carcinoembryonic antigen) associated with colorectal cancer (e.g., GenBank accession number X98311), gp100 (e.g., GenBank accession number S73003), or MART1 antigen associated with melanoma, and PSA antigen associated with prostate cancer (e.g., GenBank accession number X14810). The p53 gene sequence is known (see, e.g., Harris et al. (1986) Mol. Cell. Biol., 6:4650-4656) and is deposited in GenBank under accession number M14694. Thus, the subject proteins, nucleic acids, and / or virions may be used as immunotherapies for cancers including, but not limited to, cervical cancer, breast cancer, colorectal cancer, prostate cancer, lung cancer, and melanoma.

[0134] Viral antigens are derived from known pathogens that cause diseases, including, but not limited to, measles, mumps, rubella, polio, 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 fever (e.g., GenBank Accession No. M24444), hantavirus, and human immunodeficiency virus (e.g., GenBank Accession No. U18552).

[0135] Suitable bacterial and parasitic antigens include those against diphtheria, pertussis (e.g., GenBank Accession No. M35274), tetanus (e.g., GenBank Accession No. M64353), tuberculosis, bacterial and fungal pneumonia (e.g., Haemophilus influenzae, Pneumocystis carinii, etc.), cholera, typhoid fever, plague, shigellosis, salmonellosis (e.g., GenBank Accession No. L03833), Legionnaires' disease, Lyme disease (e.g., GenBank Accession No. U59487), malaria (e.g., GenBank Accession No. X 53832), hookworms, onchocerciasis (e.g., GenBank Accession No. M27807), schistosomiasis (e.g., GenBank Accession No. L08198), trypanosomiasis, leshmaniasis, giardiasis (e.g., GenBank Accession No. M33641), amebiasis, filariasis (e.g., GenBank Accession No. J03266), borreliosis, and trichinellosis.

[0136] Suitable heterologous nucleic acids encoding heterologous gene products include untranslated RNAs such as RNAi agents (as described in more detail above) (e.g., antisense RNA, siRNA, shRNA, double-stranded RNA (dsRNA), CRISPR agents, such as Cas9 or Cas9-like proteins, crRNA-like RNA, tracrRNA-like RNA, single guide RNA, and / or donor polynucleotides), ribozymes, and the like. RNAi agents can be used to silence gene expression. Some RNAi agents provide tools that can then be used to silence gene expression (e.g., CRISPR agents such as cas9 or cas9-like proteins).

[0137] Target genes include any gene that encodes a target gene product (RNA or protein) that is harmful (e.g., pathological), e.g., a dysfunctional target gene product (e.g., due to a mutation in the coding protein sequence, due to a mutation in a non-coding sequence that controls the steady-state level of the gene product, etc.) Target gene products include, but are not limited to, huntingtin, hepatitis C virus, human immunodeficiency virus, amyloid precursor protein, tau, proteins containing polyglutamine repeats, herpes viruses (e.g., varicella zoster), any pathological virus, etc.

[0138] Thus, the subject rAAVs comprising heterologous nucleic acids encoding RNAi agents are useful for treating a variety of disorders and conditions, including, but not limited to, neurodegenerative diseases, e.g., trinucleotide repeat diseases, such as diseases associated with polyglutamine repeat sequences, e.g., Huntington's disease, spinocerebellar ataxia, spinal-bulbar muscular atrophy (SBMA), dentatorubral-pallidoluysian atrophy (DRPLA), and other diseases associated with polyglutamine repeat sequences; acquired pathologies (e.g., diseases or syndromes manifested by abnormal physiological, biochemical, cellular, structural, or molecular biology) such as viral infections, e.g., hepatitis that occurs or may occur as a result of HCV infection, acquired immune deficiency syndrome that occurs as a result of HIV infection; cancer, and the like.

[0139] In many embodiments, the heterologous nucleic acid encoding RNAi agent is operably linked to promoter.Suitable promoters are known to those skilled in the art, and include the promoter of any protein-coding gene, for example, the promoter of endogenously regulated gene or constitutively expressed gene.For example, the promoter of the gene that is regulated by cell physiological events, for example, hypoxia, for example, heat shock, oxygen level, and / or carbon monoxide level, can be operably linked to siRNA-encoding nucleic acid.

[0140] A selected heterologous nucleotide sequence, such as an EPO-encoding or nucleic acid of interest, is operably linked to regulatory elements that direct its transcription or expression in vivo. Such regulatory elements may include regulatory sequences normally associated with the selected gene (e.g., endogenous cellular regulatory elements). Alternatively, heterologous regulatory sequences may be used. Useful heterologous regulatory sequences generally include those derived from sequences encoding mammalian or viral genes. Examples include, but are not limited to, the SV40 early promoter, the mouse mammary tumor virus long terminal repeat (LTR) promoter, the adenovirus major late promoter (Ad MLP), herpes simplex virus (HSV) promoter, endogenous cellular promoters heterologous to the gene of interest, cytomegalovirus (CMV) promoters such as the CMV immediate early promoter region (CMVIE), the Rous sarcoma virus (RSV) promoter, synthetic promoters, hybrid promoters, and the like. Additionally, sequences derived from non-viral genes, such as the mouse metallothionein gene, may also be useful herein. Such promoter sequences are commercially available, for example, from Stratagene (San Diego, CA).

[0141] In some embodiments, a cell-type- or tissue-specific promoter may be operably linked to a heterologous nucleic acid encoding a heterologous gene product such that the gene product is selectively or preferentially produced in a particular cell type(s) or tissue(s). In some embodiments, an inducible promoter may be operably linked to a heterologous nucleic acid.

[0142] For example, muscle-specific and inducible promoters, enhancers, etc. are useful for delivery of gene products to muscle cells. Such regulatory elements include, but are not limited to, those from the actin and myosin gene family, such as from the myoD gene family, the myocyte-specific enhancer-binding factor MEF-2, regulatory elements from the human skeletal actin gene and cardiac actin gene, muscle creatine kinase sequence elements and mouse creatine kinase enhancer (mCK) elements, regulatory elements 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 factor, steroid-inducible elements and promoters such as glucocorticoid response elements (GREs), fusion consensus elements for RU486 induction, and elements providing tetracycline-regulated gene expression.

[0143] AAV expression vectors harboring a DNA molecule of interest (heterologous DNA) bounded by AAV ITRs can be constructed by directly inserting a selected sequence(s) into an AAV genome from which the major AAV open reading frame ("ORF") has been excised. Other portions of the AAV genome can also be deleted, so long as a sufficient portion of the ITRs remains capable of replication and packaging functions. Such constructs can be designed using techniques known to those skilled in the art. See, for example, U.S. Pat. Nos. 5,173,414 and 5,139,941, International Publication Nos. WO92 / 01070 (published January 23, 1992) and WO93 / 03769 (published March 4, 1993), Lebkowski et al. (1988) Molec. Cell. Biol. 8:3988-3996, Vincent et al. (1990) Vaccines 90 (Cold Spring Harbor Laboratory Press), Carter, BJ (1992) Current Opinion in Biotechnology 3:533-539, Muzyczka, N. (1992) Current Topics in Microbiol. and Immunol. 158:97-129, Kotin, RM (1994) Human Gene Therapy 5:793-801, Shelling and See Smith (1994) Gene Therapy 1:165-169, and Zhou et al. (1994) J. Exp. Med. 179:1867-1875.

[0144] Alternatively, AAV ITRs can be excised from the viral genome or from an AAV vector containing the same fused 5' and 3' ITRs of a selected nucleic acid construct present in another vector using any convenient method known to those of skill in the art. For example, one suitable approach uses standard ligation techniques, such as those described in Sambrook et al., supra. For example, ligation can be accomplished in 20 mM Tris-Cl pH 7.5, 10 mM MgCl2, 10 mM DTT, 33 μg / ml BSA, 10 mM-50 mM NaCl, and 40 μM ATP at 0°C-16°C with either 0.01-0.02 (Weiss) units T4 DNA ligase ("sticky end" ligation), or 1 mM ATP at 14°C with 0.3-0.6 (Weiss) units T4 DNA ligase ("blunt end" ligation). Intramolecular "sticky end" ligations are often performed at a total DNA concentration of 30-100 μg / ml (total final concentration of 5-100 nM). AAV vectors containing ITRs are described, for example, in U.S. Patent No. 5,139,941. In particular, that document describes several AAV vectors available from the American Type Culture Collection ("ATCC") under accession numbers 53222, 53223, 53224, 53225, and 53226.

[0145] Additionally, chimeric genes can be synthetically generated to include AAV ITR sequences positioned 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.

[0146] Generation of subject infectious rAAV virions Typically, host or "producer" cells are used for introduction for rAAV vector replication and packaging. Such producer cells (usually mammalian host cells) generally contain, or are modified to contain, several different types of components for rAAV production. The first component is a recombinant adeno-associated viral (rAAV) vector genome (or "rAAV provector") that can be replicated and packaged into vector particles by a host packaging cell. The rAAV provector typically contains a heterologous polynucleotide (or "transgene") that is desirable for genetically altering another cell in the context of gene therapy (since packaging of such a transgene into an rAAV vector 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 contain sequences recognized during AAV vector excision, replication, and packaging, as well as during vector integration into the host cell genome.

[0147] The second component is a helper virus, which provides helper functions for AAV replication. While adenovirus is commonly used, other helper viruses can also be used, as known to those skilled in the art. Alternatively, the necessary helper virus functions can be genetically isolated from the helper virus, and the encoding genes can be used to provide the 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.

[0148] The final components within the producer somatic cell to provide for AAV production are the "AAV packaging genes," such as the AAV rep and cap genes, which provide the replication and encapsidation proteins, respectively. Several different versions of the AAV packaging genes can be provided, including the rep-cap cassette and separate rep and / or cap cassettes, in which the rep and / or cap genes can remain under the control of their native promoters or can be operably linked to heterologous promoters. Such AAV packaging genes can be transiently or stably introduced into the host packaging cell, as known to those of skill in the art and described in more detail below.

[0149] 1. rAAV vector A subject rAAV virion containing a heterologous DNA of interest ("heterologous DNA of interest" is also referred to herein as "heterologous nucleic acid") can be produced using standard methods known to those of skill in the art. The methods generally include the steps of: (1) introducing a subject rAAV vector into a host cell, (2) introducing an AAV helper construct into the host cell, the helper construct comprising an AAV coding region capable of being expressed in the host cell to compensate for AAV helper functions lacking from the AAV vector, (3) introducing one or more helper viruses and / or accessory function vectors into the host cell, the helper viruses and / or accessory function vectors providing 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 helper virus or accessory function vector(s) can be introduced into host cells either simultaneously or sequentially using standard transfection techniques.

[0150] AAV expression vectors are constructed using known techniques to provide at least the operably linked components in the direction of transcription, regulatory elements including a transcription initiation region, the DNA of interest, and a transcription termination region. The regulatory elements are selected to be functional in mammalian muscle cells. The resulting construct containing the operably linked components is bounded (5' and 3') by functional AAV ITR sequences.

[0151] The nucleotide sequences of the AAV ITR regions are known. For example, for the AAV-2 sequence, see Kotin, RM (1994) Human Gene Therapy 5:793-801; Berns, KI "Parvoviridae and Their Replication" in Fundamental Virology, 2nd Edition, (BN Fields and DMK Knipe, eds.). The AAV ITRs used in the vectors of the present invention do not need to have wild-type nucleotide sequences and can be altered, for example, by nucleotide insertion, deletion, or substitution. Furthermore, the AAV ITRs can be derived from any of several AAV serotypes, including, but not limited to, AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-7, etc. Furthermore, the 5' and 3' ITRs flanking a selected nucleotide sequence in an AAV expression vector need not be identical or derived from the same AAV serotype or isolate, so long as they function as intended, i.e., allow for excision and rescue of the desired sequence from the host cell genome or vector, and allow for integration of the DNA molecule into the recipient cell genome when the AAV Rep gene products are present in the cell. The ITRs allow replication of the vector sequences in the presence of the appropriate mixture of Rep proteins. The ITRs also allow for incorporation of the vector sequences into the capsid to generate AAV particles.

[0152] To produce rAAV virions, the AAV expression vector is introduced into a suitable host cell using known techniques, such as transfection. Numerous transfection techniques are generally known to those skilled in the art. See, for example, 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 coprecipitation (Graham et al. (1973) Virol. 52:456-467), direct microinjection into cultured cells (Capecchi, MR (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).

[0153] For purposes of this disclosure, host cells suitable for producing rAAV virions include bacteria, yeast cells, insect cells, and mammalian cells that can be or have been used as recipients of heterologous DNA molecules. This term includes the progeny of the original transfected cell. Thus, a "host cell" for producing rAAV virions generally refers to a cell transfected with an exogenous DNA sequence. Cells from the stable human cell line, 293 (readily available, for example, through the American Type Culture Collection under accession number ATCC CRL1573), are used in many embodiments. In particular, human cell line 293 is a human embryonic kidney cell line 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 easily transfected and provides a particularly convenient platform for producing rAAV virions.

[0154] 2.AAV helper function To replicate and encapsidate the nucleotide sequences flanked by the AAV ITRs to produce rAAV virions, host cells containing the above-described AAV expression vectors must be capable of providing AAV helper functions. AAV helper functions are generally AAV-derived coding sequences that 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 compensate for essential AAV functions missing from an AAV expression vector. Thus, AAV helper functions include one or both of the major AAV ORFs, i.e., the rep and cap coding regions, or functional homologs thereof. In the context of the present disclosure, the cap function includes one or more mutant capsid proteins, at least one of which contains at least one mutation as described above.

[0155] By "AAV rep coding region" is meant the art-recognized region of the AAV genome that encodes the replication proteins Rep78, Rep68, Rep52, and Rep40. 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 regulation of transcription from AAV (or other heterologous) promoters. The Rep expression products are collectively required for replication of 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, RM (1994) Human Gene Therapy 5:793-801. Suitable homologs 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).

[0156] AAV cap proteins include VP1, VP2, and VP3, at least one of which includes at least one mutation as described above.

[0157] AAV helper functions are introduced into host cells by transfecting them with an AAV helper construct, either before or simultaneously with transfection of the AAV expression vector. AAV helper constructs are therefore used to provide at least transient expression of the AAV rep and / or cap genes to compensate for missing AAV functions essential for productive AAV infection. AAV helper constructs lack AAV ITRs and are unable to replicate or package themselves. These constructs can be in the form of plasmids, phages, transposons, cosmids, viruses, or virions. Numerous 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, for example, Samulski et al. (1989) J. Virol. 63:3822-3828 and McCarty et al. (1991) J. Virol. 65:2936-2945. Many other vectors encoding Rep and / or Cap expression products have been described, see, e.g., U.S. Patent No. 5,139,941.

[0158] Both AAV expression vectors and AAV helper constructs can be constructed to contain one or more optional selectable markers. Suitable markers include genes that confer antibiotic resistance or sensitivity to cells transfected with a nucleic acid construct containing the selectable marker, confer color to the cells, or alter the antigenic characteristics of the cells when the cells are grown in an appropriate selective medium. Some selectable markers useful in practicing the disclosed methods include the hygromycin B resistance gene (encoding aminoglycoside phosphotransferase (APH)), which confers resistance to hygromycin, thereby allowing selection in mammalian cells, and the neomycin phosphotransferase gene (encoding neomycin phosphotransferase), which confers resistance to G418, thereby allowing selection in mammalian cells. Other suitable markers are known to those of skill in the art.

[0159] 3. AAV accessory functions To produce rAAV virions, the host cell (or packaging cell) must also be adapted to provide non-AAV-derived functions or "accessory functions." Accessory functions are non-AAV-derived viral and / or cellular functions on which AAV is dependent for its replication. Thus, accessory functions include at least those non-AAV proteins and RNAs required for 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. The accessory functions of viral systems can be derived from any of the known helper viruses.

[0160] In particular, accessory functions can be introduced into and expressed in host cells using methods known to those skilled in the art. Typically, accessory functions are provided by infection of host cells with an unrelated helper virus. Many suitable helper viruses are known, including adenoviruses, herpesviruses such as herpes simplex virus types 1 and 2, and vaccinia viruses. Non-viral accessory functions, such as those provided by cell synchronization using any of a variety of known agents, can also be useful herein. See, for example, 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.

[0161] Alternatively, accessory functions can be provided using accessory function vectors. Accessory function vectors contain nucleotide sequences that provide one or more accessory functions. The accessory function vectors can be introduced into suitable host cells to support efficient AAV virion production in the host cells. 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 that, when associated with the appropriate control elements and enzymes, can be transcribed or expressed in a host cell to provide the accessory functions.

[0162] Nucleic acid sequences providing accessory functions can be obtained from natural sources, such as from the genome of an adenovirus particle, or can be constructed using recombinant or synthetic methods known to those skilled in the art. In this regard, accessory functions from adenoviruses have been extensively studied, and numerous adenoviral genes involved in accessory functions have been identified and partially characterized. See, for example, Carter, BJ (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. In particular, the early adenoviral gene regions E1a, E2a, E4, VAI RNA, and possibly E1b are thought to be involved in the accessory process. Janik et al. (1981) Proc. Natl. Acad. Sci. USA 78:1925-1929. Accessory functions from herpes viruses have been described. See, e.g., Young et al. (1979) Prog. Med. Virol. 25:113. Accessory functions from vaccinia viruses have also been described. See, e.g., Carter, BJ (1990), supra; Schlehofer et al. (1986) Virology 152:110-117.

[0163] Infection of host cells with a helper virus or transfection of host cells with an accessory function vector results in the expression of accessory functions that transcriptionally activate the AAV helper construct to produce AAV Rep and / or Cap proteins. The Rep expression products excise recombinant DNA (containing the DNA of interest, e.g., heterologous nucleic acid) from the AAV expression vector. The Rep proteins also serve to replicate the AAV genome. The expressed Cap proteins assemble into capsids, and the recombinant AAV genome is packaged into the capsids. Productive AAV replication thus ensues, and DNA is packaged into rAAV virions.

[0164] After recombinant AAV replication, rAAV virions can be purified from host cells using a variety of conventional purification methods, such as CsCl gradients. Furthermore, if infection is used to express accessory functions, residual helper virus can be inactivated using known methods. For example, adenovirus can be inactivated by heating to a temperature of approximately 60° C. for, e.g., 20 minutes or more. Because AAV is highly heat-stable, while helper adenovirus is heat-labile, this treatment effectively inactivates only the helper virus.

[0165] The resulting rAAV virions are ready to be used for DNA delivery, such as in gene therapy applications, or delivery of gene products to a mammalian host.

[0166] Delivery of heterologous nucleic acids The present disclosure further provides methods for delivering heterologous nucleic acids to target cells and / or individuals in need thereof. In some embodiments, the individuals in need thereof are humans who have previously been naturally exposed to AAV and consequently possess anti-AAV antibodies (i.e., AAV neutralizing antibodies). For example, there are many such therapeutic applications / disease targets, based on positive results in clinical trials involving AAV gene delivery to liver, muscle, and retina (all tissues affected by neutralizing antibodies to the vehicle).

[0167] The subject methods generally involve (i) administering an effective amount of a subject rAAV virion to an individual, and / or (ii) contacting target cells with a subject virion. Generally, the rAAV virion is administered to a subject using either in vivo ("direct") or in vitro ("indirect") transduction techniques. When transduced in vitro ("indirectly"), the desired recipient cells (i.e., "target cells") can be removed from the individual, transduced with the rAAV virion, and reintroduced into the individual. Alternatively, syngeneic or xenogeneic cells can be used if they would not generate an inappropriate immune response in the individual.

[0168] Suitable methods for delivery and introduction of transduced target cells into an individual have been described. For example, cells can be transduced in vitro, e.g., by combining recombinant AAV virions with the cells in an appropriate medium and screening for cells harboring the DNA of interest using conventional techniques such as Southern blot and / or PCR, or by using a selectable marker. The transduced cells can then be formulated into pharmaceutical compositions, described more fully below, which can be introduced into a subject by a variety of techniques, such as by intramuscular, intravenous, subcutaneous, and intraperitoneal injection.

[0169] For in vivo (i.e., "direct") delivery, the rAAV virions can be formulated into a pharmaceutical composition and administered parenterally (e.g., via intramuscular, subcutaneous, intratumoral, transdermal, intrathecal, intravenous routes, etc.).

[0170] Pharmaceutical compositions may contain sufficient genetic material to produce a therapeutically effective amount of the desired gene expression product, i.e., an amount sufficient to reduce or ameliorate the symptoms of the disease state in question, or an amount sufficient to provide the desired benefit. Pharmaceutical compositions may 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 may be included therein, for example, mineral acid salts such as hydrochloride, hydrobromide, phosphate, sulfate, and the like, and salts of organic acids such as acetate, propionate, malonate, benzoate, 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 to those skilled in the art and need not be discussed in detail herein. Pharmaceutically acceptable excipients are described, for example, in A. Gennaro (2000) "Remington: The Science and Practice of Pharmacy," 20th edition, Lippincott, Williams, & Wilkins; Pharmaceutical Dosage Forms and Drug Delivery Systems (1999) H.C. Cansel et al., eds., 7 th ed., Lippincott, Williams, & Wilkins, and Handbook of Pharmaceutical Excipients (2000) AHKibbe et al., eds.,3 rd It is described in detail in various publications, including ed. Amer. Pharmaceutical Assoc.

[0171] The appropriate dosage may depend, among other factors, on the mammal being treated (e.g., a human or non-human primate, or other mammal), the age and general condition of the individual being treated, the severity of the condition being treated, the particular therapeutic protein in question, and its mode of administration. An appropriate effective amount can be readily determined by one of ordinary skill in the art.

[0172] Thus, a "therapeutically effective amount" will fall in a relatively broad range that can be determined by clinical trials. For example, for in vivo injection, i.e., injection directly into skeletal or cardiac muscle, a therapeutically effective dose may be about 10 6 ~about 10 15 rAAV virions, e.g., approximately 10 8 ~10 12 For in vitro transduction, the effective amount of rAAV virions delivered to cells will be approximately 10 8 ~about 10 13 Other effective doses can be readily established by one skilled in the art through routine testing to establish dose-response curves.

[0173] Dosage treatment can be a single dose schedule or a multiple dose schedule. Furthermore, a subject can be administered as many doses as appropriate. Those skilled in the art can easily determine the appropriate number of doses.

[0174] The cells of interest (i.e., "target cells") are typically mammalian, a term that refers to any animal classified as a mammal, including humans, domestic and livestock animals, and zoo, laboratory, sport, or pet animals, such as dogs, horses, cats, cows, mice, rats, rabbits, etc. In some embodiments, the target cells are human cells.

[0175] Target cells of interest include any cell susceptible to infection by the subject rAAV virions. In some cases, for example, when the method is a method of delivering heterologous nucleic acid to a target cell, the target cell may be a cell removed from an individual (e.g., a "primary" cell), or the target cell may be a tissue culture cell (e.g., from an established cell line).

[0176] Exemplary target cells include, but are not limited to, liver cells, pancreatic cells (e.g., islet cells: alpha cells, beta cells, delta cells, gamma cells, and / or epsilon cells), skeletal muscle cells, cardiomyocytes, fibroblasts, retinal cells, synovial joint cells, lung cells, T cells, neurons, glial cells, stem cells, hematopoietic progenitor cells, neural progenitor cells, endothelial cells, and cancer cells. Exemplary stem cell target cells include, but are not limited to, hematopoietic stem cells, neural stem cells, neural crest stem cells, embryonic stem cells, induced pluripotent stem cells (iPS cells), mesenchymal stem cells, mesodermal stem cells, liver stem cells, pancreatic stem cells, muscle stem cells, and retinal stem cells.

[0177] The term "stem cell" is used herein to refer to a mammalian cell capable of both self-renewal and generating differentiated progeny (see, e.g., Morrison et al. (1997) Cell 88:287-298). Stem cells generally possess one or more of the following characteristics: the ability to undergo asynchronous or symmetric replication, where the two daughter cells after division may have different phenotypes; extensive self-renewal; the ability to exist in a mitotically quiescent form; and the ability to clonal regeneration of all tissues in which they reside, e.g., hematopoietic stem cells reconstitute all hematopoietic lineages. As will be understood by those skilled in the art, "progenitor cells" differ from stem cells in that they typically do not possess extensive self-renewal capacity and can generate a more restricted subset of lineages in the tissue from which they originate, e.g., only lymphocytes, or, in a hematopoietic environment, the erythroid lineage. As used herein, the term "stem cell" encompasses both "stem cells" and "progenitor cells" as defined above.

[0178] Stem cells can be characterized both by the presence of markers associated with specific epitopes identified by antibodies, and by the absence of certain markers identified by the lack of binding of specific antibodies. Stem cells can also be identified by functional assays, both in vitro and in vivo, particularly assays related to the ability of stem cells to give rise to multiple differentiated progeny.

[0179] Suitable stem cells of interest include, but are not limited to, hematopoietic stem cells and progenitor cells derived therefrom (U.S. Patent No. 5,061,620); neural crest stem cells (see Morrison et al. (1999) Cell 96:737-749); neural stem cells and neural progenitor cells; embryonic stem cells; mesenchymal stem cells; mesodermal stem cells; liver stem cells, muscle stem cells, retinal stem cells, induced pluripotent stem cells (iPS cells), etc. Other hematopoietic "progenitor" cells of interest include cells committed to the lymphoid lineage, such as immature T cell and B cell populations.

[0180] Purified populations of stem or progenitor cells can be used. For example, human hematopoietic stem cells can be positively selected using antibodies specific for CD34, thy-1, or negatively selected using lineage-specific markers, T cell-specific markers, tumor / cancer-specific markers, etc., which may include glycophorin A, CD3, CD24, CD16, CD14, CD38, CD45RA, CD36, CD2, CD19, CD56, CD66a, and CD66b. Markers useful for isolating mesodermal stem cells include FcγRII, FcγRIII, Thy-1, CD44, VLA-4α, LFA-1β, HSA, ICAM-1, CD45, Aa4.1, Sca-1, etc. Neural crest stem cells can be positively selected with antibodies specific for the low-affinity nerve growth factor receptor (LNGFR) and the markers sulfatide, glial fibrillary acidic protein (GFAP), myelin protein P, etc. o Human mesenchymal stem cells can be positively isolated using the markers SH2, SH3, and SH4.

[0181] The target cells used may be fresh, frozen, or pre-cultured. They may be fetal, neonatal, or 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 stem cells are separated from other cells of hematopoietic or other lineages is not critical to the present disclosure. As described above, a substantially homogeneous population of stem or progenitor cells may be obtained by selective isolation of cells that exhibit epitope characteristics associated with stem cells but lack markers associated with differentiated cells.

[0182] Nucleic acids that can be delivered to an individual include any of the heterologous nucleic acids defined above. Proteins that can be delivered using the subject methods also include functional fragments of any of the aforementioned proteins and functional variants of any of the aforementioned proteins.

[0183] In some embodiments, a therapeutically effective amount of protein is produced in a mammalian host. Whether a therapeutically effective amount of a particular protein is produced in a mammalian host using the subject method can be easily determined using an assay appropriate for the particular protein. For example, if the protein is EPO, hematocrit is measured.

[0184] When the rAAV encodes an antigenic protein, suitable antigenic proteins that can be delivered to an individual using the subject method include, but are not limited to, tumor / cancer-associated antigens, autoantigens ("self" antigens), viral antigens, bacterial antigens, protozoan antigens, and allergens, as well as antigenic fragments thereof. In some embodiments, a cytotoxic T lymphocyte (CTL) response to the rAAV-encoded antigenic protein will be elicited in the mammalian host. In other embodiments, a humoral response to the rAAV-encoded antigenic protein will be elicited 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 elicited in the mammalian host. Whether an immune response to the antigenic protein has been generated is easily determined using well-established methods. For example, enzyme-linked immunosorbent assays can be used to determine whether antibodies to the antigenic protein have been generated. Methods for detecting antigen-specific CTLs are known to those of skill in the art. For example, detectably labeled target cells expressing the antigenic protein on their surface are used to assay for the presence of antigen-specific CTLs in a blood sample.

[0185] Whether a therapeutically effective amount of a heterologous nucleic acid (e.g., a nucleic acid encoding a polypeptide, an RNAi agent, etc.) has been delivered to a mammalian host using the subject methods is readily determined using any suitable assay, for example, if the gene product is an RNAi agent that inhibits HIV, viral load can be measured.

[0186] Methods for generating and identifying modified rAAV virions The present disclosure provides methods for generating and identifying modified infectious recombinant adeno-associated virus (rAAV) virions comprising a mutant capsid protein comprising an amino acid sequence having at least one amino acid substitution (including deletion, insertion, etc.) compared to a starter AAV capsid protein, the starter AAV capsid protein comprising the amino acid sequence set forth in one of SEQ ID NOs: 10-13 and 26-33.

[0187] The methods generally involve generating a mutant rAAV virion library and selecting the library for modified rAAV virions that have altered properties relative to a starter rAAV virion. The starter rAAV virion comprises a mutant AAV capsid protein comprising an amino acid sequence set forth in one of SEQ ID NOs: 10-13 and 26-33. The disclosure further provides libraries and compositions comprising the libraries.

[0188] In some embodiments, a given selection step is repeated two, three, four or more times to enrich the subject AAV library for altered virion properties, hi some embodiments, after selection of the AAV library, individual clones are isolated and sequenced.

[0189] Generation of mutant AAV libraries A mutant AAV library is generated containing one or more mutations relative to the starter AAV cap gene. The starter cap gene is a cap containing a nucleotide sequence encoding a mutant AAV capsid protein containing the amino acid sequence set forth in one of SEQ ID NOS: 10-13 and 26-33. Mutations in the rAAV cap gene are generated using any known method. Suitable methods for mutagenesis of the starter AAV cap gene include, but are not limited to, polymerase chain reaction (PCR)-based methods, oligonucleotide-directed mutagenesis, saturation mutagenesis, loop-swap mutagenesis, fragment-shuffling mutagenesis (i.e., DNA shuffling), and the like. Methods for generating mutations are well described in the art. See, e.g., Zhao et al. Nat Biotechnol. 1998 Mar;16(3):234-5; Koerber et al., Mol Ther. 2008 Oct;16(10):1703-9; Koerber et al., Mol Ther. 2009 Dec;17(12):2088-95; U.S. Patent No. 6,579,678; U.S. Patent No. 6,573,098; and U.S. Patent No. 6,582,914, all of which are incorporated by reference herein for their teachings regarding mutagenesis.

[0190] In some embodiments, mutant AAV libraries containing mutations in the cap gene can be generated using an annealing-extension process. The annealing-extension process involves amplification of the cap gene using a PCR-based method. A 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, growing fragments anneal to different templates based on sequence complementarity and are further extended. Cycles of denaturation, annealing, and extension are repeated until full-length sequences are formed. The resulting full-length sequences contain at least one mutation in the cap gene compared to the wild-type AAV cap gene.

[0191] The PCR product containing the AAV cap sequence containing one or more mutations is inserted into a plasmid containing the wild-type AAV genome. The result is a library of AAV cap mutants. Thus, the present disclosure provides a library of AAV cap mutants, ranging from about 10 to about 10 10 Mutant AAV cap gene libraries are provided, each containing a member and comprising a mutation in the AAV cap gene. A given member of the library has from about 1 to about 50 mutations in the AAV cap gene. The subject libraries may contain from 10 to about 10 9 Each of these comprises distinct members of the AAV cap gene, each with a different mutation(s) in the AAV cap gene.

[0192] Once the cap mutation library is generated, viral particles are generated that can then be selected based on their altered capsid characteristics. The library plasmid DNA is transfected into suitable host cells (e.g., 293 cells), followed by introduction of a helper virus into the cells. The viral particles (rAAV library particles) produced by the transfected host cells are harvested.

[0193] Library Selection Once the library is generated, it is selected for specific virion properties (i.e., altered infection properties). Virus particles are generated as described above (thus generating a library of modified rAAV virions) and undergo one or more selection steps to identify modified rAAV virions with altered infection properties (relative to infectious rAAV virions containing a mutant capsid protein comprising an amino acid sequence set forth in one of SEQ ID NOS: 10-13 and 26-33). The infection properties selected for may include, but are not limited to, 1) altered binding (e.g., reduced binding) to AAV neutralizing antibodies, 2) increased evasion of AAV neutralizing antibodies, 3) increased infectivity of cells resistant to infection with AAV, and 4) altered heparin binding.

[0194] 1. Selection for reduced binding to AAV neutralizing antibodies In some embodiments, the subject AAV libraries are selected for altered (e.g., reduced) binding to neutralizing antibodies that bind to and neutralize wild-type AAV virions, compared to the binding of the neutralizing antibodies to wild-type AAV virions and neutralization of wild-type AAV virions (or compared to infectious rAAV virions comprising a mutant capsid protein comprising an amino acid sequence set forth in one of SEQ ID NOS: 10-13 and 26-33). AAV library particles (AAV library virions) are contacted with the neutralizing antibodies, and the ability of the AAV library particles to infect permissive host cells is tested. Typically, the AAV library particles are contacted with various concentrations of the neutralizing antibodies. The higher the concentration of neutralizing antibody required to reduce the infectivity of the AAV library particles, the more resistant the AAV particles are to neutralization. Any convenient assay known to those of skill in the art can be used to directly measure the binding of AAV library virions to neutralizing anti-AAV antibodies (e.g., to measure binding affinity).

[0195] 2. Selection for elevated escape from AAV neutralizing antibodies In some embodiments, the subject AAV libraries are selected for increased escape from neutralizing antibodies (i.e., increased resistance to human neutralizing AAV antibodies) relative to infectious rAAV virions comprising a variant capsid protein comprising an amino acid sequence set forth in one of SEQ ID NOS: 10-13 and 26-33. The AAV library particles are contacted with target cells in the presence of a neutralizing AAV antibody (usually a human neutralizing anti-AAV antibody). After a suitable length of time to allow infection of the cells with the AAV library particles, helper virus is added, and AAV library particles that successfully infect the cell(s) are recovered. In some embodiments, infectivity is measured for virions that exhibit successful infection (e.g., as described above). In some embodiments, the cycle of infection, addition of helper virus, and recovery of AAV particles is repeated one, two, three, or more times. Selection can occur using different amounts (concentrations) of neutralizing AAV antibody to select for different degrees of escape (e.g., each repeated round can utilize an increased concentration of antibody compared to the previous round).

[0196] 3. Selection for increased infectivity of non-permissive cells In some embodiments, the subject AAV libraries are selected for increased infectivity of non-permissive cells (relative to infectious rAAV virions comprising a mutant capsid protein comprising an amino acid sequence set forth in one of SEQ ID NOS: 10-13 and 26-33). The AAV library particles are contacted with non-permissive cells (e.g., a population of non-permissive cells). After a suitable length of time to allow infection of the cells with the AAV library particles, helper virus is added, and AAV library particles that successfully infect the non-permissive cell(s) are recovered. In some embodiments, the cycle of infection, addition of helper virus, and recovery of AAV particles is repeated one, two, three, or more times.

[0197] 4. Selection for Altered Heparin Binding In some embodiments, the subject libraries are selected for altered heparin binding, including increased and decreased heparin binding, relative to wild-type AAV virion heparin binding (or relative to infectious rAAV virions comprising a mutant capsid protein comprising an amino acid sequence set forth in one of SEQ ID NOS: 10-13 and 26-33). The AAV library particles are contacted with a heparin affinity matrix. For example, the AAV library particles are placed on a heparin affinity column under conditions that allow binding of the AAV library particles to heparin. Exemplary conditions include equilibrating the column with 0.15 M NaCl and 50 mM Tris at pH 7.5. After allowing the AAV library particles to bind to the heparin affinity matrix, the AAV library particle / heparin affinity matrix complexes are washed with a volume of buffer containing increasing concentrations of NaCl, and eluted AAV library particles are collected at each NaCl concentration. For example, after binding, the AAV library particle / heparin affinity matrix complexes are washed with 50 mM Tris buffer, pH 7.5, containing 200 mM NaCl, and the eluted AAV library particles are collected. The elution step is repeated with 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.

[0198] AAV library particles eluting at NaCl concentrations below about 450 mM NaCl exhibit reduced heparin binding characteristics compared to wild-type AAV, and AAV library particles eluting at NaCl concentrations above about 550 mM NaCl exhibit increased heparin binding characteristics compared to wild-type AAV.

[0199] In some embodiments, the eluted AAV library particles are amplified by co-infection of permissive cells with a helper virus and re-fractionated on a heparin affinity matrix, a step that can be repeated multiple times to enrich for AAV library particles with altered heparin-binding characteristics.

[0200] In this method, one or more selection steps can follow the generation of AAV library particles. For example, in some embodiments, the method involves selecting for increased heparin binding, followed by selecting for reduced binding to neutralizing antibodies. In other embodiments, the method involves selecting for reduced binding to neutralizing antibodies, followed by selecting for increased heparin binding. In other embodiments, the method involves selecting for reduced binding to neutralizing antibodies, followed by selecting for reduced heparin binding. In other embodiments, the method involves selecting for reduced binding to neutralizing antibodies, followed by selecting for reduced heparin binding. In other embodiments, the method involves selecting for reduced binding to neutralizing antibodies, followed by selecting for increased infectivity of stem cells. In other embodiments, the method involves selecting for reduced binding to neutralizing antibodies, followed by selecting for increased evasion of neutralizing antibodies. In other embodiments, the method involves selecting for increased evasion of neutralizing antibodies, followed by selecting for reduced binding to neutralizing antibodies.

[0201] Accordingly, the present disclosure provides an adeno-associated virus (AAV) library comprising a plurality of nucleic acids, each nucleic acid comprising a nucleotide sequence encoding a mutant AAV capsid protein. The encoded mutant AAV capsid protein comprises at least one amino acid substitution compared to the sequence set forth in one of SEQ ID NOs: 10-13 and 26-33. The present disclosure provides a library of mutant adeno-associated virus (AAV) particles, comprising a plurality of AAV particles, each comprising an AAV capsid protein comprising at least one amino acid substitution compared to the sequence set forth in one of SEQ ID NOs: 10-13 and 26-33. The nucleic acids encoding the mutant AAV capsid proteins are described above, as are the characteristics of the encoded mutant AAV capsid proteins.

[0202] The present disclosure further provides (i) two or more infectious rAAV virions, each comprising a mutant adeno-associated virus (AAV) capsid protein and a heterologous nucleic acid; (ii) two or more isolated nucleic acids, each comprising a nucleotide sequence encoding a mutant AAV capsid protein; (iii) two or more host cells, each comprising a nucleic acid comprising a nucleotide sequence encoding a mutant AAV capsid protein; and (iv) a library comprising at least one of the two or more mutant AAV capsid proteins, wherein the mutant AAV capsid protein of at least one member of the library comprises an amino acid sequence having at least one amino acid substitution compared to the amino acid sequence set forth in one of SEQ ID NOs: 10-13 and 26-33.

[0203] Compositions and Kits Compositions and kits for use in the methods of the present disclosure are also provided. The subject compositions and kits include at least one of a subject infectious rAAV virion, a subject rAAV vector, a subject nucleotide acid comprising a nucleotide sequence encoding a subject mutant AAV capsid protein, an isolated host cell comprising a subject nucleic acid (i.e., a subject genetically modified host cell comprising a nucleic acid comprising a nucleotide sequence encoding a subject mutant AAV capsid protein), a subject library (e.g., any of the libraries described above), and a subject mutant AAV capsid protein. The composition or kit may include any convenient combination of the above. The composition or kit may also include a helper virus and / or a nucleic acid comprising a nucleotide sequence encoding the helper virus. The kit may also include reagents for generating nucleic acids encoding modified mutant AAV capsid proteins (i.e., "mutant" nucleic acids).

[0204] In addition to the above compositions, the subject kits (in certain embodiments) may further comprise instructions for practicing the subject methods.These instructions may be present in the subject kits in various forms, one or more of which may be present in the kit.One form in which these instructions may be present is information printed on a suitable medium or substrate, such as in the kit packaging, in a package insert, etc., for example, paper on which the information is printed.Another form in which these instructions may be present is a computer-readable medium on which the information is recorded, such as a floppy disk, a compact disk (CD), a flash drive, etc.Another form in which these instructions may be present is a website address that can be used via the Internet to access the information at a remote site.

[0205] Now that the present invention is fully described, it will be apparent to those skilled in the art that various changes and modifications can be made therein without departing from the spirit or scope of the invention. [Example]

[0206] The following examples are set forth 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 described below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should be accounted for. Unless otherwise indicated, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric. Standard abbreviations may be used, such as bp, base pair(s); kb, kilobase(s); ml, milliliter(s), μl, microliter(s), nl, nanoliter(s), pl, picoliter(s), s or sec, second(s), min, minute(s), h or hr, hour(s), aa, amino acid(s), kb, kilobase(s), bp, base pair(s), nt, nucleotide(s), im, intramuscular, ip, intraperitoneal, sc, subcutaneous, iv, intravenous, etc.

[0207] Example 1: Adeno-associated virus (AAV) gene therapy vectors have demonstrated considerable promise in several clinical trials to date. However, circulating anti-AAV antibodies resulting from early childhood exposure or prior administration of AAV vectors have prevented the implementation of AAV gene therapy for many potential patients. The present inventors have isolated novel AAV variants capable of enhanced anti-AAV antibody evasion both in vitro and in vivo. Stringent pressure resulting from selection using low- and high-potency human serum pools and human IVIG has led to the evolution of AAV variants capable of evading antibody neutralization from individual human sera, human IVIG, and mouse sera, making them the most broadly evasive variants to date.

[0208] Materials and Methods cell line Cell lines were cultured at 37°C in 5% CO2 and were obtained from the American Type Culture Collection (Manassas, VA) unless otherwise indicated. HEK293T, HeLa, and HT1080 cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (Gibco, Carlsbad, CA) and 1% penicillin / streptomycin (Invitrogen, Carlsbad, CA). CHO K1 and CHO pgsA cells were cultured in F-12K medium (ATCC) supplemented with 10% fetal bovine serum (Gibco) and 1% penicillin / streptomycin (Invitrogen). Pro5 and Lec1 cells were cultured in MEM-alpha medium (Gibco) supplemented with 10% fetal bovine serum (Gibco) and 1% penicillin / streptomycin (Invitrogen).

[0209] Human serum pool for selection Eighteen individual human serum samples were obtained from Innovative Research, Inc. (Southfield, MI), and neutralizing antibody titers against wild-type AAV2 were determined for each sample (Table 2). Because individual samples were likely to have variation in both antibody affinity and epitope specificity, three effective serum pools (α = A + F + G, β = B + H + M, and γ = I + J + N) were generated by mixing equal amounts of the individual serum samples. Selection in the presence of these antibody variations should result in a general enhancement of resistance to many pre-existing human antibodies. To select for resistance to an even broader range of antibodies, subsequent selections were performed in the presence of Gamimune N, 10% human IVIG (Bayer, Elkhart, IN).

[0210] Table 2: Neutralizing antibody titers of individual human serum samples The neutralizing antibody (NAb) titer for each sample is reported as the reciprocal of the volume fraction of serum required to reduce infectivity to 37% of that measured in the absence of serum. Three serum pools (α = A + F + G, β = B + H + M, and γ = I + J + N) were then generated by mixing equal volumes of the three individual serum samples. (Table 2) TIFF0007813169000025.tif53128

[0211] Library generation and virus production then by error-prone PCR to generate a saturation mutagenesis library An AAV2 cap library was generated by the tethered extension process described by Zhao et al. using TIFF0007813169000026.tif19149 as the forward and reverse primers, respectively. Selection using pooled individual human serum revealed a variant containing four point mutations (listed in the Results section) that served as the basis for a saturation mutagenesis library. The cap gene for this variant underwent further mutagenesis by changing amino acids at specific sites. Primers TIFF0007813169000027.tif5128 and the corresponding reverse complement primer were used to mutate the R471 amino acid site. TIFF0007813169000028.tif14158 and the corresponding reverse complement primers were used to mutate the K532 and E548 amino acid sites. TIFF0007813169000029.tif5128 and the corresponding reverse complement primer were used to mutate the N587 amino acid site. TIFF0007813169000030.tif5147 and the corresponding reverse complement primer were used to mutagenize the V708 and T716 amino acid sites. A library consisting of AAV2 containing randomized cap loop regions and a library containing shuffled DNA from wild-type AAV1, AAV2, AAV4, AAV5, AAV6, AAV8, and AAV9 cap genes were packaged and pooled for the initial selection step (Koerber et al.; Mol Ther. 2008 Oct;16(10):1703-9, and Koerber et al.; Mol Ther. 2009 Dec;17(12):2088-95, both of which are incorporated herein by reference in their entireties).

[0212] For the second and third rounds of evolution, the cap genes from the loop swap / shuffle library and the saturation mutagenesis library were used as previously described. Random mutagenesis libraries were generated by error-prone PCR using TIFF0007813169000031.tif12128 as forward and reverse primers, respectively. Replication-competent AAV libraries and recombinant AAV vectors expressing GFP under the control of the CMV promoter were packaged in HEK293T cells (ATCC) using the calcium phosphate transfection method, and the viruses were purified by iodixonal gradient centrifugation. For in vivo use, recombinant AAV vectors expressing GFP or luciferase under the control of the CMV promoter were further purified by Amicon filtration. DNase resistance genome titers were determined by quantitative PCR (Excoffon et al., Proc Natl Acad Sci US A. 2009 Mar 10;106(10):3865-70, and Maheshri et al., Nat Biotechnol. 2006 Feb;24(2):198-204, both of which are incorporated herein by reference in their entireties).

[0213] Library selection and evolution A round of selection was defined as infection of HEK293T cells with the AAV starting library (30 minutes at room temperature for pooled individual human sera or 1 hour at 37°C with heat-inactivated IVIG prior to infection), followed by adenovirus rescue and recovery of successful mutants. Each round of evolution consisted of mutagenesis of the cap gene to generate the starting library and three rounds of selection. Three rounds of evolution were performed for each library, with clonal analysis performed between each round of evolution. The starting library for each round of evolution was generated as described above. After the third round of selection, the AAV cap gene was isolated from the pool of successful AAV mutants and amplified by PCR. The cap gene was inserted into the pXX2 recombinant AAV packaging plasmid using NotI and HindIII. The cap gene was then sequenced at the University of California, Berkeley DNA Sequencing Facility and analyzed using Geneious software (Biomatters, Auckland, New Zealand). A three-dimensional model of the AAV2 capsid (Protein Databank accession number 1LP3) was rendered in Pymol (DeLano Scientific, San Carlos, CA).

[0214] In vitro transduction analysis of antibody escape mutants HEK293T cells were infected with 3 × 10 4Cells were seeded at a density of 1000 cells / well. Mutants were incubated with heat-inactivated IVIG, individual human serum, or individual mouse serum at 37°C for 1 hour before infection, and cells were then infected with rAAV-GFP at a genomic MOI of 2000. The percentage of GFP-positive cells was assessed 48 hours postinfection using the ImageXpress Micro Cellular Imaging and Analysis System (Molecular Devices, Sunnyvale, CA) and MetaXpress Image Analysis Software, version 3.1.0, Multi Wavelength Cell Scoring Application Module (Molecular Devices).

[0215] In vitro transduction analysis To determine relative transduction efficiencies, selected mutants were compared to the parental wild-type AAV serotype, HEK293T, CHO K1, CHO pgsA (lacking all surface glycosaminoglycans), CHO Pro5 (parental line for several glycosylation mutants, including Lec1 cells), CHO Lec1 (glycosylation-deficient), HeLa, and HT1080 cells (human fibrosarcoma cell line) at 2.5 × 10 per well 24 hours prior to infection. 4 Cells were seeded at a density of 100x100 / mL. Cells were infected with rAAV1-GFP, rAAV2-GFP, rAAV6-GFP, shuffle100.1-GFP, shuffle100.3-GFP, SM10.2-GFP, or shuffle100.7-GFP at MOIs ranging from 100 to 1000. The percentage of GFP-positive cells was assessed 48 hours postinfection using a Beckman-Coulter Cytomics FC500 flow cytometer (Beckman-Coulter, Brea, CA).

[0216] In vivo analysis of antibody escape mutants For analysis of gene expression in vivo, 8-week-old female Balb / c mice were primed with 4 mg of IVIG or phosphate-buffered saline (control mice) per mouse via tail vein injection 24 hours before administration of recombinant shuffle 100-3 (see SEQ ID NO: 12), SM10-2 (see SEQ ID NO: 10), or AAV2 vectors. Mice received 10 mg of a recombinant AAV vector encoding luciferase under the control of a CMV promoter via tail vein injection. 11 Mice were infected with the viral genome. For bioluminescence imaging, mice were anesthetized with 2% isofluorane and oxygen. D-luciferin substrate (GOLD Biotechnology, St. Louis, MO) was injected intraperitoneally at a dose of 500 μg / g of body weight. Images were generated using a VivoVision IVIS Lumina imaging system (Xenogen, Alameda, CA). Ventral images were taken for each mouse 7–10 min after substrate injection every week for 4 weeks. Five weeks after injection, serum was collected via cardiac puncture, and the mice were then perfused with 0.9% saline solution. Heart, liver, lungs, kidneys, spleen, brain, spinal cord, and hindlimb muscles were harvested and frozen. Frozen tissue samples were homogenized and resuspended in Reporter Lysis Buffer (Promega, Mannheim, Germany) for in vitro luciferase analysis. The luciferase-containing lysate was clarified by centrifugation at 10,000 g for 10 minutes. To assay the samples, 20 μL of lysate was added to 100 μL of luciferase assay buffer, mixed, incubated for 5 minutes, and placed in a luminometer. Signals were integrated for 30 seconds with a 2-second delay and reported in relative light units (RLU) as detected by a TD20 / 20 luminometer (Turner Designs, Sunnyvale, CA). Luciferase signals were normalized to total protein content as determined by the bicinchoninic acid assay (Pierce).

[0217] result Our results demonstrated that AAV can evolve to significantly overcome neutralization by anti-AAV antibodies both in vitro and in vivo. We isolated novel AAV mutants that required 2- to 35-fold higher neutralizing antibody titers (using human IVIG) than wild-type AAV in vitro. The antibody neutralizing properties also translated into enhanced in vivo transduction in the presence of neutralizing antibodies. The isolation of such novel clones resistant to anti-AAV antibodies will enable broader implementation of AAV-based therapies as nucleic acid delivery vectors, including in individuals with high antibody titers who are currently ineligible for AAV gene therapy.

[0218] AAV library generation and selection by directed evolution Figure 1a shows a schematic diagram of the directed evolution approach used to isolate novel AAV variants capable of evading human antibody neutralization. A viral library was generated using the DNA mutagenesis technique described in the following paragraphs (Figure 1a, steps 1 and 2). During the first selection, a viral library pool generated by error-prone PCR mutations in the AAV2 cap gene was incubated with various dilutions of a low-potency α-human serum pool at room temperature for 30 minutes before infection of HEK293T cells (step 3). After three rounds of selection against the low-potency α-human serum pool (Figure 1a, steps 4 and 5), several mutants with enhanced resistance to this neutralizing serum pool were obtained (Figure 1a, step 6, Figure 7a). Mutant 1.45 contained two point mutations (N312K, N449D), which conferred more than 10-fold higher resistance to neutralization by the α-pool compared to wild-type AAV2.

[0219] The cap gene from mutant 1.45 was subjected to additional random mutagenesis, and the resulting library was selected for three more rounds of selection against the β and γ pools in parallel. Because only slight improvements in antibody evasion were observed (data not shown), the recovered cap genes were pooled and subjected to additional diversification by DNA shuffling and EP PCR. Three more rounds of selection against increasing amounts of serum from both the β and γ pools resulted in substantial enrichment in the amount of virus recovered from the viral library compared to wild-type AAV2 (Fig. 7b, c). Sequencing of successful cap genes from both pools revealed several low-frequency mutants and a single dominant mutant, mutant γ4.3, which contained four point mutations (N312K, N449D, N551S, and I698V) present in both libraries. In the presence of human IVIG, mutant 1.45 demonstrated a modest 1.2-fold increased resistance to neutralization, while γ4.3 demonstrated a 3.1-fold increased resistance to neutralization ( FIG. 7d ). This observation supports the hypothesis that pools of individual human sera can be used to isolate AAV mutants capable of enhanced evasion of antibodies present in the general human population.

[0220] The moderate success of mutant γ4.3 in resisting neutralization by anti-AAV antibodies prompted the development of a library based on the γ4.3 cap gene. Amino acid sites R471, K532, E548, N587, V708, and T716, previously determined to be immunogenic sites on the AAV2 capsid, were subjected to saturation mutagenesis in an attempt to discover amino acid mutations that might improve the antibody resistance of γ4.3. This "saturation mutagenesis" library was subjected to three additional rounds of selection, along with a "shuffle" library consisting of random cap chimeras of seven parental AAV serotypes and a "loop swap" library consisting of AAV2 caps with permuted loop regions. In this selection, the viral library pool was incubated with various dilutions of human IVIG at 37°C for 1 hour before infection of HEK293T cells. After infection with the AAV library and amplification of infectious AAV variants by adenovirus superinfection, the number of viral genomes, or viral titer, from each library condition was quantified and compared to the titer of wild-type AAV2 as a way to determine the success of selection (Figure 1b). For each round of selection using saturation mutagenesis and loop-swap / shuffle libraries, viral pools from the 1:10 and 1:100 IVIG dilution conditions that produced viral titers higher than wild-type AAV2 were used as starting points for subsequent rounds of selection. After three rounds of selection, successful viral cap genes were isolated and individually tested to determine the viruses with the most efficient gene delivery. Furthermore, cap genes isolated from the third round of selection underwent an additional round of error-prone PCR mutagenesis, and this process was repeated to iteratively increase viral fitness.

[0221] Figure 1 depicts the directed evolution of AAV for enhanced antibody evasion. (a) Schematic of directed evolution. 1) A viral library is generated by genetically diversifying the cap gene using several complementary approaches. 2) Virus is packaged into HEK293T cells using plasmid transfection, followed by recovery and purification. 3) The viral library is incubated with several concentrations of human IVIG and introduced into HEK293T cells in vitro. 4) Successful viruses are amplified and recovered via adenovirus superinfection. 5) Successful clones are enriched by repeated selection at lower MOIs. 6) Isolated viral DNA reveals successful cap genes. 7) Successful cap genes are remutated to serve as new starting points for selection. (b) Selection of antibody evasion mutants from the loop swap / shuffle and saturation mutagenesis library. HEK293T cells were infected with the viral library for 24 hours. Viral particles that productively infected cells were amplified by adenovirus infection, and rescued AAV was quantified by qPCR. A 1:10 dilution of IVIG corresponds to a concentration of 10 mg IVIG / mL. Error bars indicate standard deviation (n=3).

[0222] Figure 7 demonstrates the generation of AAV2-based human antibody escapees. (a) Four viral clones selected after three rounds of selection against the low-stringency α pool demonstrate enhanced resistance to 1 μL of α serum at an MOI of 1. Two additional rounds of diversification (i.e., mutagenesis and DNA shuffling) and selection (three rounds of increasing serum volume) resulted in significantly improved virus recovery in the presence of large amounts of the highly potent (b) β and (c) γ pools. (d) Furthermore, two viral clones (1.45 and γ4.3) demonstrated 1.23- and 3.10-fold enhanced resistance to a highly diverse pool of pre-existing antibodies present with pooled human intravenous immunoglobulin (IVIg) from approximately 100,000 individuals compared to wild-type AAV2.

[0223] Enhanced antibody evasion of newly evolved AAV variants in vitro Of the 12 clones selected and packaged for individual analysis from the saturation mutagenesis and loop-swap / shuffle library after nine rounds of screening against human IVIG, all 12 required higher neutralizing antibody titers than both wild-type AAV1 and AAV2 (Fig. 2a and Table 1). The mutant shuffle 100-3 (see SEQ ID NO: 12), which required a 35-fold higher in vitro IVIG concentration than wild-type AAV2 for neutralization, was still able to transduce approximately 10% of cells in the presence of 1 mg / mL IVIG (Fig. 2b). Furthermore, the mutant SM10-2 from the AAV2 saturation mutagenesis library required a 7.5-fold higher in vitro IVIG concentration than wild-type AAV2 for neutralization. Furthermore, mutant shuffle 100-3 and SM10-2 (see SEQ ID NO: 10) showed enhanced transduction in the presence of serum samples from individual patients who were excluded from hemophilia B clinical trials (Figure 3) (Nathwani et al., N Engl J Med. 2011 Dec 22;365(25):2357-65).

[0224] Figure 2 shows the neutralization profiles of antibody escape mutants. The cap genes of antibody escape mutants isolated after three rounds of evolution were used to package recombinant AAV encoding GFP and incubated with human IVIG before infection of HEK293T cells. The percentage of remaining infectious particles was determined using high-content fluorescence imaging and normalized to the infectious titer in the absence of IVIG. Two clones from each library that were resistant to IVIG are shown. Data for other clones analyzed are shown in Table 1. (a) Neutralization curves. Error bars indicate standard deviation (n = 3). (b) Representative fluorescence images from several IVIG dilutions show that the mutants are capable of transducing HEK293T cells in the presence of high concentrations of neutralizing antibodies.

[0225] Figure 3 shows the neutralization profiles of antibody escape mutants. Human serum was obtained from an individual who was excluded from a hemophilia B clinical trial due to the presence of high neutralizing antibody titers to AAV. Recombinant AAV encoding GFP was incubated with individual human serum samples prior to infection of HEK293T cells. The percentage of remaining infectious particles was determined using fluorescence microscopy and normalized to the infectious titer in the absence of human serum. Error bars indicate standard deviation (n = 3).

[0226] Sequence analysis of the 12 clones revealed that the two mutants with the highest neutralizing antibody resistance, shuffle100-3 (see SEQ ID NO: 12) and shuffle100-1 (see SEQ ID NO: 11), were nearly identical shuffled capsids containing fragments of AAV1-4, AAV6, and AAV9 (Figure 4). The differences at amino acids 469 (AAV6 residue vs. AAV7 residue) and 598 (AAV6 residue vs. AAV1 residue) between the two mutants translated into a nearly three-fold increase in neutralizing antibody titer for shuffle100-3 (see SEQ ID NO: 12) (Table 1). Mutant shuffle100-7 (see SEQ ID NO: 13), which had the fourth highest neutralizing antibody resistance (Table 1), was also a shuffled capsid containing fragments of AAV1, AAV6, and AAV8 (Figure 4), which is in good agreement with reported data showing that wild-type AAV1 and AAV8 are effective in evading anti-AAV2 antibodies. Interestingly, mutant SM10-2 (see SEQ ID NO: 10) retained the point mutation obtained by mutation γ4.3 and also retained the wild-type residue at the saturation mutagenesis site. Mutant SM10-2 (see SEQ ID NO: 10) gained additional point mutations at surface residue D472N and internal residue L735Q. Figure 4 shows the amino acid sequences of the loop-swap / shuffle and saturation mutagenesis clones. (a) Schematic diagrams of the capsid proteins are shown for two clones from each library with the highest neutralizing IVIG concentrations. Each region is shaded according to the parent serotype from which it was derived. Black arrows indicate the initiation codons of the VP1, VP2, and VP3 capsid proteins (from left to right). Gray arrows indicate surface loop regions I, II, III, IV, and V based on the AAV2 capsid (from left to right). (b) Molecular models of the complete AAV2 capsid based on the lytic structure are shown for two clones from each library with the highest neutralizing IVIG concentrations. Each region is shaded according to the parent serotype from which it is derived. For mutant shuffle 100-3 (see SEQ ID NO: 12), black arrows indicate differences from mutant shuffle 100-1 (see SEQ ID NO: 11). For mutant SM10-2 (see SEQ ID NO: 10), mutations N449D, D472N, N551S, and I698V are surface mutations (black).

[0227] Table 1: IVIG neutralizing antibody titers of library clones and parent serotypes Human IVIG was used to neutralize recombinant AAV-GFP vectors bearing capsids from wild-type AAV1, AAV2, AAV8, and mutants recovered from loop-swap / shuffle and saturation mutagenesis libraries. The IVIG concentrations (mg / mL) required to reduce gene delivery efficiency to 50% of that in the absence of IVIG are shown and compared to the concentration required to reduce AAV2 delivery. All mutants analyzed required higher concentrations of IVIG than wild-type AAV1 and AAV2. Neutralizing antibody titers were determined by exponential fitting of the curves in Figure 2. SEQ ID NOs are listed as "amino acid, nucleotide." (Table 1) TIFF0007813169000032.tif135156

[0228] Mutants shuffle100-3 (see SEQ ID NO: 12), shuffle100-1 (see SEQ ID NO: 11), and shuffle100-7 (see SEQ ID NO: 13) have transduction profiles that mimic those of the parent serotypes AAV1 and AAV6 (Figure 5). Furthermore, mutations in SM10-2 (see SEQ ID NO: 10) do not prevent heparin dependence (as found in the parent serotype AAV2), resulting in a profile similar to AAV2 (Figure 5).

[0229] Figure 5 demonstrates the in vitro tropism of the novel AAV variants. Recombinant AAV vectors expressing green fluorescent protein were used to transduce a panel of cell lines: CHO, pgsA (lacking all surface glycosaminoglycans), Pro5, Lec1 (lacking sialic acid), HEK293T, HeLa, and HT1080 (a human fibrosarcoma cell line), to profile the transduction characteristics of the novel AAV variants. Error bars indicate standard deviation (n = 3).

[0230] Enhanced antibody evasion of newly evolved AAV variants in vivo To determine the localization patterns of the shuffle100-3 and shuffle100-7 mutants, luciferase enzyme activity was examined in various tissues of naive mice injected with AAV2, shuffle100-3, or shuffle100-7 (Fig. 6a). The shuffle100-7 mutant showed similar in vivo tropism to AAV2, except for a 7-fold higher transduction of the heart, a 5-fold higher transduction of the lung, and a 4.5-fold lower transduction of the liver. The shuffle100-3 mutant showed over 4-fold higher transduction of the brain, over 3-fold higher transduction of the lung, and 27-fold higher transduction of the muscle than AAV2. Analysis of sera from these mice showed that mutant shuffle100-3 required equal or greater in vitro serum concentrations for neutralization than AAV1 and AAV8 for sera from mice receiving AAV1, AAV2, AAV8, or shuffle100-3 gene delivery vectors (Figure 11). Shuffle100-7 required equal or greater in vitro serum concentrations for neutralization than AAV1 for sera from mice receiving AAV1, AAV2, AAV8, shuffle100-3, or SM10-2 gene delivery vectors (Figure 11). Furthermore, both mutants were less neutralized by sera from mice receiving the AAV2 gene delivery vector than all wild-type AAV serotypes tested. Interestingly, mutant shuffle100-3 was also less neutralized by sera from mice immunized against it than any of the other serotypes or mutants tested (Figure 11). This data indicates that these mutants may be used in combination with wild-type AAV serotypes or other mutants in applications requiring multiple vector administrations.

[0231] Figure 11 shows the neutralizing antibody titers of library clones and parent serotypes in immunized mouse sera. Serum from mice administered library clones or wild-type AAV was used to neutralize recombinant AAV-GFP vectors carrying capsids from wild-type AAV1, AAV2, and AAV8, as well as mutants recovered from loop-swap / shuffle and saturation mutagenesis libraries. The serum dilution required to reduce gene delivery efficiency to 50% of the efficiency in the absence of serum is shown.

[0232] To determine the ability of shuffle100-7 and shuffle100-3 mutants to evade antibody neutralization in vivo, mice were passively immunized with human IVIG before AAV injection. The shuffle100-7 mutant had significantly higher transduction in the heart, liver, and muscle than AAV2, as measured by luciferase enzyme activity (Fig. 6b). The shuffle100-3 mutant had significantly higher transduction in the heart and muscle compared to AAV2 (Fig. 6b).

[0233] Figure 6 shows the in vivo localization and neutralization of novel AAV mutants. (a) A recombinant AAV vector encoding luciferase was administered to female BALB / c mice via tail vein injection. Five weeks later, the level of luciferase activity was determined and normalized to total protein for each sample analyzed. (b) A recombinant AAV vector expressing luciferase was administered to female BALB / c mice via tail vein injection 24 hours after tail vein injection of 4 mg of human IVIG. Five weeks later, the level of luciferase expression was normalized to total protein for each sample analyzed. Error bars indicate standard deviation (n = 3); * = p < 0.05. RLU, relative luciferase units.

[0234] Mutant γ4.3, isolated from an AAV2-based error-prone library selected against a pool of individual human sera, contained four point mutations (N312K, N449D, N551S, and I698V). Interestingly, two of these positions (N449 and N551) had previously been identified as immunogenic residues using other pools of human sera, demonstrating that antigenic epitopes encompassing these sites are targeted by many different neutralizing antibodies. Therefore, these sites represent interesting and valuable targets for mutation. Combining directed evolution and rational design within a saturation mutagenesis library led to the isolation of mutant SM10-2, which was capable of greater antibody resistance than both AAV1 and AAV2 in vitro. Mutant SM10-2 incorporates two additional point mutations (D472N and L735Q) relative to those found in mutant γ4.3. The D472N mutation was previously shown to increase the level of capsid synthesis in HEK293 cells. Similarly, the exchange of the positively charged lysine side chain at amino acid position 735 with an uncharged glutamine side chain, which is also present in mutant shuffle100-7 despite being located within the interior of the assembled capsid, may function to stabilize the capsid (Figure 4).

[0235] The creation of chimeric AAV capsids allows for the generation of viral variants that can combine desirable characteristics from multiple AAV serotypes. AAV8 and AAV9 have also been shown to be much more resistant to neutralization by IVIG than AAV2, but amino acids specific to these capsids were present only within a small span on the surface of the shuffled variants isolated during our selection (Figure 4). Shuffled 100-3, a variant that exhibited more efficient evasion of antibody neutralization in vitro, exhibited similar in vitro tropism to its parent serotypes, AAV1 and AAV6, but was more infectious than either wild-type serotype. The differences in amino acids 469 and 598 between shuffled 100-1 and shuffled 100-3 translated into a nearly threefold increase in neutralizing antibody titers for shuffled 100-3. A study by Lochrie et al. reported that the immunogenic residues recognized by human serum and IVIG differ, suggesting that different individuals can generate different neutralizing antibodies to different sets of epitopes on the AAV capsid and that complete escape from neutralization is not easy (Lochrie et al., J Virol. 2006 Jan;80(2):821-34). Our study demonstrates that the use of multiple rounds of directed evolution using several different serum pools containing anti-AAV antibodies of varying amounts and potencies will lead to the isolation of novel AAV variants capable of enhanced cell transduction both in vitro and in vivo in the presence of multiple anti-AAV antibody pools.

[0236] Adaptive immune responses to AAV vector components in animals and humans often prevent re-administration of AAV vectors of the same serotype, complicating gene delivery applications requiring multiple vector administrations. In vitro neutralization assays using serum from mice used in biodistribution studies demonstrated that the mutants were less neutralized by these sera than wild-type AAV (Figure 11), which may be useful for gene therapy strategies requiring vector re-administration. For example, shuffle 100-3 was not neutralized by serum from mice injected with AAV2, and AAV2 was not neutralized by serum from mice injected with shuffle 100-3, suggesting that this mutant could be used in combination with wild-type AAV serotypes or in applications requiring multiple vector administrations. In conclusion, we used directed evolution to isolate novel AAV mutants capable of reduced neutralization by anti-AAV antibodies derived from individual human patients, pooled human serum, and mouse serum, both in vitro and in vivo.

[0237] While the present invention has been described with reference to specific embodiments thereof, it will 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(s), 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.

Claims

1. (a) a mutant capsid protein comprising an amino acid sequence having at least 95% amino acid sequence identity with amino acids 203 to 736 of the amino acid sequence set forth in SEQ ID NO: 12 and 100% amino acid sequence identity with amino acids 469 and 598 of the amino acid sequence set forth in SEQ ID NO: 12; and (b) Heterologous nucleic acid and exhibiting at least 5-fold greater resistance to human neutralizing antibodies compared to the resistance exhibited by wild-type AAV serotype 2 (AAV2).

2. The rAAV of claim 1, wherein the amino acid sequence has at least 96% amino acid sequence identity with amino acids 203 to 736 of the amino acid sequence set forth in SEQ ID NO:

12.

3. The rAAV of claim 1, wherein the amino acid sequence has at least 97% amino acid sequence identity with amino acids 203 to 736 of the amino acid sequence set forth in SEQ ID NO:

12.

4. The rAAV of claim 1, wherein the amino acid sequence has at least 98% amino acid sequence identity with amino acids 203 to 736 of the amino acid sequence set forth in SEQ ID NO:

12.

5. The rAAV of claim 1, wherein the amino acid sequence has at least 99% amino acid sequence identity with amino acids 203 to 736 of the amino acid sequence set forth in SEQ ID NO:

12.

6. 6. The rAAV of any one of claims 1 to 5, wherein the rAAV exhibits resistance to human neutralizing antibodies that is at least 10-fold greater than the resistance exhibited by AAV2.

7. 6. The rAAV of any one of claims 1 to 5, wherein the rAAV exhibits at least 15-fold greater resistance to human neutralizing antibodies than the resistance exhibited by AAV2.

8. 6. The rAAV of any one of claims 1 to 5, wherein the rAAV exhibits resistance to human neutralizing antibodies that is at least 20-fold greater than the resistance exhibited by AAV2.

9. 6. The rAAV of any one of claims 1 to 5, wherein the rAAV exhibits resistance to human neutralizing antibodies that is at least 25-fold greater than the resistance exhibited by AAV2.

10. 6. The rAAV of any one of claims 1 to 5, wherein the rAAV exhibits resistance to human neutralizing antibodies that is at least 30-fold greater than the resistance exhibited by AAV2.

11. 6. The rAAV of any one of claims 1 to 5, wherein the rAAV exhibits at least 35-fold greater resistance to human neutralizing antibodies than the resistance exhibited by AAV2.

12. An rAAV described in any one of claims 1 to 11, which exhibits increased transduction of mammalian cells in the presence of human neutralizing antibodies compared to the transduction of mammalian cells exhibited by AAV2.

13. The rAAV of claim 12, wherein the mammalian cell is a liver cell, a pancreatic cell, a skeletal muscle cell, a cardiac muscle cell, a fibroblast, a retinal cell, a synovial joint cell, a lung cell, a T cell, a neuron, a glial cell, a stem cell, an endothelial cell, or a cancer cell.

14. The rAAV of any one of claims 1 to 13, wherein the heterologous nucleic acid comprises an interfering RNA.

15. The rAAV of any one of claims 1 to 13, wherein the heterologous nucleic acid comprises a nucleotide sequence encoding a polypeptide.

16. An isolated host cell comprising the rAAV of any one of claims 1 to 15.

17. An isolated host cell comprising nucleic acid encoding the rAAV of any one of claims 1 to 15.

18. A gene product delivery agent comprising the recombinant adeno-associated virus (rAAV) of any one of claims 1 to 15.

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