Adeno-associated virus vector

Abstract

To provide recombinant viral vectors which are suitable for the delivery of therapeutic genes in vivo.SOLUTION: A recombinant adeno-associated virus (AAV) vector comprises: (a) a variant AAV3B capsid protein, where the variant AAV3B capsid protein comprises an amino acid substitution at position 312 in an AAV3B capsid protein sequence with respect to a wild type AAV3B capsid protein comprising a specific sequence; and (b) a heterologous nucleic acid comprising a nucleotide sequence encoding a gene product.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to the field of recombinant viral vectors. In particular, the present invention relates to recombinant viral vectors suitable for delivering therapeutic genes in vivo. [Background technology]

[0002] To date, adeno-associated viruses remain one of the most promising vectors for the delivery of therapeutic genes, and a substantial number of preclinical and clinical studies have firmly established that this approach is suitable for the development of gene-based drugs that can obtain marketing approval.

[0003] Since the development of AAV2 as a vector for gene therapy in the 1980s, significant progress has been made in optimizing this platform for various applications and target tissues. Perhaps the most significant development has been the discovery of a wide variety of serotypes, of which 10-12 serotypes are currently being widely explored. Among the most notable features of these various serotypes are their relative tissue tropism and, in some cases, their ability for retrograde transport in neuronal cells. Of these serotypes, AAV1-10 are widely used for preclinical and clinical purposes.

[0004] Newer platforms are being developed that involve processes that allow targeting and detargeting of specific tissue and cell subtypes in patients. The core technology of these approaches is based on trial-and-error evaluation of existing AAV variants (serotypes) and in vivo selection of randomly transduced AAV capsid mutants. Together, these two promising approaches result in dozens, if not hundreds, of vectors that may have different transduction behaviors.

[0005] The most interesting aspect of AAV serotypes is that they allow efficient transduction of specific tissues in animal models and humans. At present, a comprehensive molecular understanding of the underlying mechanisms of tissue tropism has not yet been presented, and therefore it is generally assumed that tissue-specific receptors available to each serotype play a central role in efficient transduction by the various serotypes. Summary of the Invention [Problem to be solved by the invention]

[0006] Thus, there remains a need for additional AAV vectors with improved properties in terms of transgene expression and tissue specificity in vivo, particularly as such vectors may offer significantly enhanced benefits in delivering genes to various target tissues in humans. [Means for solving the problem]

[0007] In one aspect, the invention provides a recombinant adeno-associated virus (AAV) vector comprising: (a) a variant AAV2 capsid protein comprising at least four amino acid substitutions present at positions 457, 492, 499 and 533 in the AAV2 capsid protein sequence compared to a wild-type AAV2 capsid protein; and (b) a heterologous nucleic acid comprising a nucleotide sequence encoding a gene product.

[0008] In one embodiment, the variant AAV capsid protein comprises a sequence of SEQ ID NO:2, or a sequence having at least 95% sequence identity thereto. The wild-type AAV capsid protein comprises the sequence of SEQ ID NO:1.

[0009] In one embodiment, the variant AAV2 capsid protein comprises one or more of the following residues: M457, A492, D499 and Y533. In a preferred embodiment, the variant AAV2 capsid protein comprises one or more of the following amino acid substitutions relative to the wild-type AAV2 capsid protein: Q457M, S492A, E499D and F533Y.

[0010] In one embodiment, the variant AAV2 capsid protein further comprises one or more amino acid substitutions at positions 125, 151, 162 and 205 in the AAV2 capsid protein sequence relative to the wild-type AAV capsid protein. In a preferred embodiment, the variant AAV2 capsid protein comprises one or more of the following residues: I125, A151, S162 and S205. In another preferred embodiment, the variant AAV2 capsid protein comprises one or more of the following amino acid substitutions relative to the wild-type AAV2 capsid protein: V125I, V151A, A162S and T205S.

[0011] In one embodiment, the variant AAV2 capsid protein further comprises one or more amino acid substitutions at positions 585 and 588 in the AAV2 capsid protein sequence relative to the wild-type AAV capsid protein. Preferably, the variant AAV2 capsid protein comprises one or more of the following residues: S585 and T588. More preferably, the variant AAV2 capsid protein comprises one or more of the following amino acid substitutions relative to the wild-type AAV2 capsid protein: R585S and R588T.

[0012] In one embodiment, the variant AAV2 capsid protein further comprises one or more amino acid substitutions at positions 546, 548 and 593 in the AAV2 capsid protein sequence relative to the wild-type AAV capsid protein. Preferably, the variant AAV2 capsid protein comprises one or more of the following residues: D546, G548 and S593. More preferably, the variant AAV2 capsid protein comprises one or more of the following amino acid substitutions relative to the wild-type AAV2 capsid protein: G546D, E548G and A593S.

[0013] In one embodiment, the variant AAV2 capsid protein comprises residue N312, i.e., the residue in the wild-type AAV2 capsid protein is at position 312. In this embodiment, the variant AAV2 capsid protein is not mutated at position 312 compared to the wild-type AAV2 capsid protein sequence.

[0014] In another aspect, the invention provides a recombinant adeno-associated virus (AAV) vector comprising: (a) a variant AAV8 capsid protein comprising an amino acid substitution at position 315 in the AAV8 capsid protein sequence compared to a wild-type AAV8 capsid protein; and (b) a heterologous nucleic acid comprising a nucleotide sequence encoding a gene product.

[0015] In one embodiment, the variant AAV capsid protein comprises a sequence having at least 95% sequence identity to SEQ ID NO: 6. In another embodiment, the wild-type AAV capsid protein comprises the sequence of SEQ ID NO: 6.

[0016] In one embodiment, the variant AAV8 capsid protein comprises the amino acid substitution S315N compared to the wild-type AAV8 capsid protein. and / or 596.

[0017] In preferred embodiments, the variant AAV8 capsid protein comprises one or more of the following amino acid substitutions relative to the wild-type AAV8 capsid protein: (a) V125I, Q151A, K163S, A206S, T460M, T495A, N502D, F536Y, N549D, A551G, Q588S and / or G596S, and / or (b) T591R.

[0018] In another aspect, the invention provides a recombinant adeno-associated virus (AAV) vector comprising: (a) a variant AAV3B capsid protein comprising an amino acid substitution at position 312 in the AAV3B capsid protein sequence compared to a wild-type AAV3B capsid protein; and (b) a heterologous nucleic acid comprising a nucleotide sequence encoding a gene product.

[0019] In one embodiment, the variant AAV3B capsid protein comprises a sequence having at least 95% sequence identity to SEQ ID NO: 11. In another embodiment, the wild-type AAV capsid protein comprises the sequence of SEQ ID NO:11.

[0020] In one embodiment, the variant AAV3B capsid protein comprises the amino acid substitution S312N compared to the wild-type AAV3B capsid protein.

[0021] In another aspect, the present invention provides a recombinant adeno-associated virus (AAV) vector comprising: (a) a variant AAV-LK03 capsid protein comprising an amino acid substitution at position 312 compared to the AAV-LK03 capsid protein sequence as defined in SEQ ID NO:12; and (b) a heterologous nucleic acid comprising a nucleotide sequence encoding a gene product.

[0022] In one embodiment, the variant AAV-LK03 capsid protein comprises a sequence having at least 95% sequence identity to SEQ ID NO:12.

[0023] In another aspect, the invention provides a recombinant adeno-associated virus (AAV) vector comprising: (a) a variant AAV capsid protein comprising at least one amino acid substitution at a position corresponding to one or more of positions 125, 151, 162, 205, 312, 457, 492, 499, 533, 546, 548, 585, 588 and / or 593 in the AAV2 capsid protein sequence relative to a wild-type AAV capsid protein; and (b) a heterologous nucleic acid comprising a nucleotide sequence encoding a gene product.

[0024] In one embodiment, at least one amino acid substitution is present at one or more of positions 125, 151, 162, 205, 312, 457, 492, 499, 533, 546, 548, 585, 588 and / or 593 in the AAV2 capsid protein sequence, or at one or more corresponding positions in an alternative AAV capsid protein sequence.

[0025] In one embodiment, the vector comprises a variant AAV2 capsid protein. In another embodiment, the variant AAV capsid protein comprises the sequence of SEQ ID NO:2 or a sequence having at least 95% sequence identity thereto. In another embodiment, the wild-type AAV capsid protein is derived from AAV2. In another embodiment, the wild-type AAV capsid protein comprises the sequence of SEQ ID NO:1.

[0026] In one embodiment, the variant AAV2 capsid protein comprises one or more of the following residues: I125, A151, S162, S205, S312, M457, A492, D In a preferred embodiment, the variant AAV2 capsid protein comprises one or more of the following amino acid substitutions relative to the wild-type AAV2 capsid protein: V125I, V151A, A162S, T205S, N312S, Q457M, S492A, E499D, F533Y, G546D, E548G, R585S, R588T and / or A593S.

[0027] In further embodiments, the variant AAV capsid protein is derived from AAV1, AAV5, AAV6, AAV8, AAV9 or AAV10.

[0028] In one embodiment, the vector comprises a variant AAV1 capsid protein. In another embodiment, the variant AAV capsid protein comprises a sequence having at least 95% sequence identity to SEQ ID NO:3. In another embodiment, the wild-type AAV capsid protein is derived from AAV1. In another embodiment, the wild-type AAV capsid protein comprises the sequence of SEQ ID NO:3.

[0029] In one embodiment, at least one amino acid substitution is present at one or more of positions 125, 151, 162, 205, 313, 458, 493, 500, 534, 547, 549, 586, 589 and / or 594 in the AAV1 capsid protein sequence. In a preferred embodiment, the variant AAV1 capsid protein comprises one or more of the following amino acid substitutions compared to the wild-type AAV1 capsid protein: V125I, Q151A, T162S, N313S, N458M, K493A, N500D, F534Y, S547D and / or G594S. In an alternative embodiment, the variant AAV1 capsid protein comprises one or more of the following amino acid substitutions compared to the wild-type AAV1 capsid protein: S205T, G549E, S586R and / or T589R.

[0030] In one embodiment, the vector comprises a variant AAV5 capsid protein. In another embodiment, the variant AAV capsid protein comprises a sequence having at least 95% sequence identity to SEQ ID NO:4. In another embodiment, the wild-type AAV capsid protein is derived from AAV5. In another embodiment, the wild-type AAV capsid protein comprises the sequence of SEQ ID NO:4.

[0031] In one embodiment, at least one amino acid substitution is present at one or more of positions 124, 150, 153, 195, 303, 444, 479, 486, 520, 533, 537, 575, 578 and / or 583 in the AAV5 capsid protein sequence. In a preferred embodiment, the variant AAV5 capsid protein comprises one or more of the following amino acid substitutions compared to the wild-type AAV5 capsid protein: V124I, K150A, K153S, A195S, R303S, T444M, S479A, V486D, T520Y, P533D and / or G583S. In an alternative embodiment, the variant AAV5 capsid protein comprises one or more of the following amino acid substitutions compared to the wild-type AAV5 capsid protein: G537E, S575R and / or T578R.

[0032] In one embodiment, the vector comprises a variant AAV6 capsid protein. In another embodiment, the variant AAV capsid protein comprises a sequence having at least 95% sequence identity to SEQ ID NO:5. In another embodiment, the wild-type AAV capsid protein is derived from AAV6. In another embodiment, the wild-type AAV capsid protein comprises the sequence of SEQ ID NO:5.

[0033] In one embodiment, at least one amino acid substitution is present at one or more of positions 125, 151, 162, 205, 313, 458, 493, 500, 534, 547, 549, 586, 589 and / or 594 in the AAV6 capsid protein sequence. In a preferred embodiment, the variant AAV6 capsid protein comprises one or more of the following amino acid substitutions compared to the wild-type AAV6 capsid protein: V125I, Q151A, T162S, N313S, N458M, K493A, N500D, F534Y, S547D and / or G594S. In an alternative embodiment, the variant AAV6 capsid protein comprises one or more of the following amino acid substitutions compared to the wild-type AAV6 capsid protein: S205T, G549E, S586R and / or T589R.

[0034] In one embodiment, the vector comprises a variant AAV8 capsid protein. In another embodiment, the variant AAV capsid protein comprises a sequence having at least 95% sequence identity to SEQ ID NO:6. In another embodiment, the wild-type AAV capsid protein is derived from AAV8. In another embodiment, the wild-type AAV capsid protein comprises the sequence of SEQ ID NO:6.

[0035] In one embodiment, at least one amino acid substitution is present at one or more of positions 125, 151, 163, 206, 315, 460, 495, 502, 536, 549, 551, 588, 591 and / or 596 in the AAV8 capsid protein sequence. In a preferred embodiment, the variant AAV8 capsid protein comprises one or more of the following amino acid substitutions compared to the wild-type AAV8 capsid protein: V125I, Q151A, K163S, A206S, T460M, T495A, N502D, F536Y, N549D, A551G, Q588S and / or G596S. In an alternative embodiment, the variant AAV8 capsid protein comprises one or more of the following amino acid substitutions compared to the wild-type AAV8 capsid protein: S315N and / or T591R.

[0036] In one embodiment, the vector comprises a variant AAV9 capsid protein. In another embodiment, the variant AAV capsid protein comprises a sequence having at least 95% sequence identity to SEQ ID NO:7. In another embodiment, the wild-type AAV capsid protein is derived from AAV9. In another embodiment, the wild-type AAV capsid protein comprises the sequence of SEQ ID NO:7.

[0037] In one embodiment, at least one amino acid substitution is present at one or more of positions 125, 151, 162, 205, 314, 458, 493, 500, 534, 547, 549, 586, 589 and / or 594 in the AAV9 capsid protein sequence. In a preferred embodiment, the variant AAV9 capsid protein comprises one or more of the following amino acid substitutions compared to the wild-type AAV9 capsid protein: L125I, Q151A, N314S, Q458M, V493A, E500D, F534Y, G547D, A589T and / or G594S. In an alternative embodiment, the variant AAV9 capsid protein comprises one or more of the following amino acid substitutions compared to the wild-type AAV9 capsid protein: S162A, S205T, G549E and / or S586R.

[0038] In one embodiment, the vector comprises a variant AAV10 capsid protein. In another embodiment, the variant AAV capsid protein comprises a sequence having at least 95% sequence identity to SEQ ID NO:8. In another embodiment, the wild-type AAV capsid protein is derived from AAV10. In another embodiment, the wild-type AAV capsid protein comprises the sequence of SEQ ID NO:8.

[0039] In one embodiment, at least one amino acid substitution is present at one or more of positions 125, 151, 163, 206, 315, 460, 495, 502, 536, 549, 551, 588, 591 and / or 596 in the AAV10 capsid protein sequence. In a preferred embodiment, the variant AAV10 capsid protein comprises one or more of the following amino acid substitutions compared to the wild-type AAV10 capsid protein: V125I, Q151A, K163S, A206S, N315S, T460M, L495A, N502D, F536Y, G549D, Q588S, A591T and / or G596S. In an alternative embodiment, the variant AAV10 capsid protein comprises the following amino acid substitution compared to the wild-type AAV10 capsid protein: G551E.

[0040] In one embodiment, the recombinant AAV vector exhibits increased transduction of neuronal or retinal tissue compared to an AAV vector comprising a corresponding wild-type AAV capsid protein.

[0041] In another embodiment, the recombinant AAV vector exhibits increased transduction of liver tissue compared to the corresponding wild-type AAV capsid protein.

[0042] In one embodiment, gene product comprises interference RNA or aptamer.In another embodiment, gene product comprises polypeptide.Preferably, gene product comprises neuroprotective polypeptide, antiangiogenic polypeptide, or polypeptide that enhances the function of neuronal or retinal cells.In a preferred embodiment, gene product comprises glial-derived neurotrophic factor, fibroblast growth factor, nerve growth factor, brain-derived neurotrophic factor, rhodopsin, retinoschisin, RPE65, or peripherin.

[0043] In another aspect, the present invention provides a pharmaceutical composition comprising (a) a recombinant AAV vector as defined above, and (b) a pharma- ceutically acceptable excipient.

[0044] In another aspect, the present invention provides a method for delivering a gene product to a tissue in a subject, the method comprising the step of administering to the subject a recombinant AAV vector or a pharmaceutical composition as defined above.

[0045] In some embodiments, the tissue is selected from blood, bone marrow, muscle tissue, neuronal tissue, retinal tissue, pancreatic tissue, liver tissue, kidney tissue, lung tissue, intestinal tissue or cardiac tissue. Preferably, the tissue is neuronal tissue, retinal tissue or liver tissue.

[0046] In another aspect, the present invention provides a method for treating a disorder in a subject, comprising administering to the subject a recombinant AAV vector or a pharmaceutical composition as defined above. In some embodiments, the disorder is a neurological disorder, an eye disorder or a liver disorder.

[0047] In another aspect, the present invention provides a recombinant AAV vector or pharmaceutical composition according to the above definition for use in treating a disorder in a subject.In some embodiments, the disorder is a neurological disorder, an eye disorder or a liver disorder.Preferably, the neurological disorder is a neurodegenerative disease.In an alternative embodiment, the eye disorder is glaucoma, retinitis pigmentosa, macular degeneration, retinoschisis or diabetic retinopathy.

[0048] In another aspect, the invention provides an isolated variant AAV capsid protein, which has a variant AAV capsid protein that is different from a wild-type AAV capsid protein at one or more of positions 125, 151, 162, 205, 312, 457, 492, 499, 533, 546, 548, 585, 588 and / or 593 in the AAV2 capsid protein sequence, or an alternative An isolated variant AAV capsid protein is provided that comprises at least one amino acid substitution present at one or more corresponding positions in the AAV capsid protein sequence.

[0049] In another aspect, the present invention provides an isolated nucleic acid comprising a nucleotide sequence encoding a variant AAV capsid protein according to the above definition.

[0050] In another aspect, the present invention provides an isolated host cell comprising a nucleic acid as defined above. That is, the present invention relates to the following. (1) A recombinant adeno-associated virus (AAV) vector comprising: (a) a variant AAV2 capsid protein comprising at least four amino acid substitutions at positions 457, 492, 499 and 533 in the AAV2 capsid protein sequence compared to a wild-type AAV2 capsid protein; and (b) a heterologous nucleic acid comprising a nucleotide sequence encoding a gene product; A recombinant AAV vector comprising: (2) A recombinant AAV vector as described in (1) above, wherein (i) the variant AAV capsid protein comprises the sequence of SEQ ID NO:2 or a sequence having at least 95% sequence identity thereto, and / or (ii) the wild-type AAV capsid protein comprises the sequence of SEQ ID NO:1. (3) The recombinant AAV vector described in (1) or (2) above, wherein the variant AAV2 capsid protein comprises one or more of the following residues: M457, A492, D499 and Y533. (4) The recombinant AAV vector according to (2) or (3) above, wherein the variant AAV2 capsid protein comprises one or more of the following amino acid substitutions compared to the wild-type AAV2 capsid protein: Q457M, S492A, E499D and F533Y. (5) A recombinant AAV vector described in any of (1) to (4) above, wherein the variant AAV2 capsid protein further comprises one or more amino acid substitutions at positions 125, 151, 162 and 205 in the AAV2 capsid protein sequence compared to the wild-type AAV capsid protein. (6) The recombinant AAV vector described in (5) above, wherein the variant AAV2 capsid protein comprises one or more of the following residues: I125, A151, S162 and S205. (7) The recombinant AAV vector according to (5) or (6) above, wherein the variant AAV2 capsid protein comprises one or more of the following amino acid substitutions compared to the wild-type AAV2 capsid protein: V125I, V151A, A162S and T205S. (8) A recombinant AAV vector according to any of (1) to (7) above, wherein the variant AAV2 capsid protein further comprises one or more amino acid substitutions at positions 585 and 588 in the AAV2 capsid protein sequence compared to the wild-type AAV capsid protein. (9) The recombinant AAV vector described in (8) above, wherein the variant AAV2 capsid protein comprises one or more of the following residues: S585 and T588. (10) The recombinant AAV vector according to (8) or (9) above, wherein the variant AAV2 capsid protein comprises one or more of the following amino acid substitutions compared to the wild-type AAV2 capsid protein: R585S and R588T. (11) A recombinant AAV vector according to any one of (1) to (10) above, wherein the variant AAV2 capsid protein further comprises one or more amino acid substitutions at positions 546, 548 and 593 in the AAV2 capsid protein sequence compared to the wild-type AAV capsid protein. (12) The recombinant AAV vector described in (11) above, wherein the variant AAV2 capsid protein comprises one or more of the following residues: D546, G548 and S593. (13) The recombinant AAV vector according to (11) or (12) above, wherein the variant AAV2 capsid protein comprises one or more of the following amino acid substitutions compared to the wild-type AAV2 capsid protein: G546D, E548G and A593S. (14) A recombinant AAV vector according to any one of (1) to (13) above, wherein the variant AAV2 capsid protein comprises residue N312. (15) A recombinant adeno-associated virus (AAV) vector comprising: (a) a variant AAV8 capsid protein comprising an amino acid substitution at position 315 in the AAV8 capsid protein sequence compared to a wild-type AAV8 capsid protein; and (b) a heterologous nucleic acid comprising a nucleotide sequence encoding a gene product; A recombinant AAV vector comprising: (16) A recombinant AAV vector as described in (15) above, wherein (i) the variant AAV capsid protein comprises a sequence having at least 95% sequence identity to SEQ ID NO:6, and / or (ii) the wild-type AAV capsid protein comprises the sequence of SEQ ID NO:6. (17) A recombinant AAV vector according to (15) or (16) above, wherein the variant AAV8 capsid protein comprises the amino acid substitution S315N compared to the wild-type AAV8 capsid protein. (18) The recombinant AAV vector according to any one of (15) to (17) above, further comprising one or more amino acid substitutions at one or more of positions 125, 151, 163, 206, 460, 495, 502, 536, 549, 551, 588, 591 and / or 596 in the AAV8 capsid protein sequence. (19) The variant AAV8 capsid protein has one or more of the following amino acid substitutions compared to the wild-type AAV8 capsid protein: (a) V125I, Q151A, K163S, A206S, T460M, T495A, N502D, F536Y, N549D, A551G, Q588S and / or G596S; and / or (b) T591R The recombinant AAV vector according to (18) above, (20) A recombinant adeno-associated virus (AAV) vector comprising: (a) a variant AAV3B capsid protein comprising an amino acid substitution at position 312 in the AAV3B capsid protein sequence compared to a wild-type AAV3B capsid protein; and (b) a heterologous nucleic acid comprising a nucleotide sequence encoding a gene product; A recombinant AAV vector comprising: (twenty one) A recombinant AAV vector as described in (20) above, wherein (i) the variant AAV3B capsid protein comprises a sequence having at least 95% sequence identity to SEQ ID NO:11, and / or (ii) the wild-type AAV capsid protein comprises the sequence of SEQ ID NO:11. (twenty two) A recombinant AAV vector according to (20) or (21) above, wherein the variant AAV3B capsid protein comprises the amino acid substitution S312N compared to the wild-type AAV3B capsid protein. (twenty three) A recombinant adeno-associated virus (AAV) vector comprising: (a) a variant AAV-LK03 capsid protein comprising an amino acid substitution at position 312 compared to the AAV-LK03 capsid protein sequence as defined in SEQ ID NO: 12; and (b) a heterologous nucleic acid comprising a nucleotide sequence encoding a gene product; A recombinant AAV vector comprising: (twenty four) The recombinant AAV vector described in (23) above, wherein the variant AAV-LK03 capsid protein comprises a sequence having at least 95% sequence identity to SEQ ID NO: 12. (twenty five) A recombinant AAV vector described in any of (1) to (14) above, wherein the recombinant AAV vector exhibits increased transduction of neuronal tissue or retinal tissue compared to an AAV vector containing a corresponding wild-type AAV capsid protein. (26) A recombinant AAV vector according to any one of (15) to (24) above, wherein the recombinant AAV vector exhibits increased transduction of liver tissue compared to the corresponding wild-type AAV capsid protein. (27) A recombinant AAV vector according to any one of (1) to (26) above, wherein the gene product comprises an interfering RNA or an aptamer. (28) A recombinant AAV vector according to any one of (1) to (27) above, wherein the gene product comprises a polypeptide. (29) The recombinant AAV vector described in (28) above, wherein the gene product comprises a neuroprotective polypeptide, an anti-angiogenic polypeptide, or a polypeptide that enhances the function of a neuronal or retinal cell. (30) The recombinant AAV vector described in (29) above, wherein the gene product comprises glial-derived neurotrophic factor, fibroblast growth factor, nerve growth factor, brain-derived neurotrophic factor, rhodopsin, retinoschisin, RPE65, or peripherin. (31) (a) a recombinant AAV vector according to any one of (1) to (30) above, and (b) Pharmaceutically acceptable excipients 23. A pharmaceutical composition comprising: (32) A method for delivering a gene product to a tissue in a subject, the method comprising the step of administering to the subject a recombinant AAV vector or pharmaceutical composition described in any one of (1) to (31) above. (33) The method according to (32) above, wherein the tissue is selected from blood, bone marrow, muscle tissue, neuronal tissue, retinal tissue, pancreatic tissue, liver tissue, kidney tissue, lung tissue, intestinal tissue or cardiac tissue. (34) The method according to (33) above, wherein the tissue is neuronal tissue, retinal tissue or liver tissue. (35) A method for treating a disorder in a subject, the method comprising the step of administering to the subject a recombinant AAV vector or pharmaceutical composition described in any one of (1) to (31) above. (36) A recombinant AAV vector or pharmaceutical composition according to any one of (1) to (31) above for use in treating a disorder in a subject. (37) The method, recombinant AAV vector or pharmaceutical composition for use according to (35) or (36) above, wherein the disorder is a neurological disorder, an eye disorder or a liver disorder. (38) The method, recombinant AAV vector or pharmaceutical composition for use according to (37) above, wherein the neurological disorder is a neurodegenerative disease. (39) The method, recombinant AAV vector or pharmaceutical composition for use according to (37) above, wherein the eye disorder is glaucoma, retinitis pigmentosa, macular degeneration, retinoschisis or diabetic retinopathy. [Brief description of the drawings]

[0051] [Figure 1] FIG. 1 shows the amino acid sequence of wild-type adeno-associated virus 2 capsid protein VP1 (SEQ ID NO:1; NCBI Reference Sequence: NC_001401), with the following residues highlighted: V125, V151, A162, T205, N312, Q457, S492, E499, F533, G546, E548, R585, R588, and A593. [Diagram 2] 1 shows the amino acid sequence of true adeno-associated virus 2 (ttAAV2) capsid protein VP1 (SEQ ID NO:2). The following residues are highlighted as different compared to VP1 of wild-type AAV2 (SEQ ID NO:1): I125, A151, S162, S205, S312, M457, A492, D499, Y533, D546, G548, S585, T588, S593. [Diagram 3]FIG. 1 shows the amino acid sequence of wild-type adeno-associated virus 1 capsid protein VP1 (SEQ ID NO:3; NCBI Reference Sequence: NC_002077). Highlighted residues: S205 (aligned with S205 in ttAAV2 (SEQ ID NO:2))-G549 (aligned with G548 in ttAAV2)-S586 (aligned with S585 in ttAAV2)-T589 (aligned with T588 in ttAAV2). [Figure 4] FIG. 1 shows the amino acid sequence of wild-type adeno-associated virus 5 capsid protein VP1 (SEQ ID NO:4; NCBI Reference Sequence: AF085716). Highlighted residues: G537 (aligns with G548 in ttAAV2)-S575 (aligns with S585 in ttAAV2)-T578 (aligns with T588 in ttAAV2). [Diagram 5] FIG. 1 shows the amino acid sequence of wild-type adeno-associated virus 6 capsid protein VP1 (SEQ ID NO:5; NCBI Reference Sequence: AF028704). Highlighted residues: S205 (aligned with S205 in ttAAV2)-G549 (aligned with G548 in ttAAV2)-S586 (aligned with S585 in ttAAV2)-T589 (aligned with T588 in ttAAV2). [Figure 6] FIG. 1 shows the amino acid sequence of wild-type adeno-associated virus 8 capsid protein VP1 (SEQ ID NO:6; NCBI Reference Sequence: NC_006261). Highlighted residues: S315 (aligned with S312 in ttAAV2)-T591 (aligned with T588 in ttAAV2). [Figure 7] FIG. 1 shows the amino acid sequence of wild-type adeno-associated virus 9 capsid protein VP1 (SEQ ID NO:7; NCBI Reference Sequence: AY530579). Highlighted residues: S162 (aligned with S162 in ttAAV2)-S205 (aligned with S205 in ttAAV2)-G549 (aligned with G548 in ttAAV2)-S586 (aligned with S585 in ttAAV2). [Figure 8]FIG. 8 shows the amino acid sequence of wild-type adeno-associated virus 10 capsid protein VP1 (SEQ ID NO:8). Highlighted residues: G551 (aligns with G548 in ttAAV2). [Figure 9] FIG. 1 shows an alignment of the amino acid sequence of AAV capsid protein VP1. [Figure 10] Figure 1 shows that the plasmids used to generate the AAV2 vectors were the packaging plasmid pDG. Top: pDG with wild-type AAV2 gene. Bottom: pDG-ttAAV2 with authentic AAV2 gene; two key mutations in the heparan-binding domain at positions 585 and 588 are highlighted. MMTV: promoter driving expression of AAV rep; E2a, E4ORF6 and VA are genes expressing adenoviral helper factors. [Figure 11A] Quantification of rAAV2 true type (TT) and wild type (WT) viral titers for in vivo injection. Krypton staining of proteins separated by SDS-PAGE was scanned using an infrared fluorescent scanner (Odyssey Imaging systems). 10 μl of AAV2 viral particles and 62.5 ng to 500 ng of BSA were separated on a 12% separation gel containing SDS and stained with Krypton Protein Stain. Images were converted to grayscale. Proteins of the capsid gene: VP1, VP2, VP3 are labeled on the left. [Figure 11B] Quantification of rAAV2 true type (TT) and wild type (WT) viral titers for in vivo injections. Table showing titers obtained from qPCR (vector genome [vg / ml]) and SDS-PAGE (capsid titer [capsid / ml]). [Figure 12A] Representative examples of rat brain sections stained with GFP-specific antibodies. Vectors were injected into the striatum as indicated by the arrow. [Figure 12B]FIG. 1 shows a representative example of an injection into the substantia nigra. [Figure 13A] Figure 1 shows GFP transduction of the eye using ttAAV2 and wtAAV2.Retinas in cross sections are shown after administration of ttAAV2 (top) and wtAAV2 (bottom) vectors. [Figure 13B] Figure 1 shows GFP transduction of the eye using ttAAV2 and wtAAV2. [Figure 14] Figure 1. Transduction of mouse brain following vector injection into neonates. iv: intravenous vector administration; ic: intracranial injection; AAV-2: wtAAV2; AAV-TT: ttAAV2. [Figure 15] Figure 1 shows a three-dimensional representation of the AAV2 capsid. Highlighted residues correspond to amino acid changes between ttAAV2 and wild-type particles and are colored according to their location. [Figure 16] FIG. 1 shows the three-fold spike on the AAV2 capsid. The highlighted residues correspond to the amino acid changes between authentic and wild-type particles. Residues in the heparin binding site are highlighted in green. [Figure 17] FIG. 1 shows the inside of the AAV2 capsid. The residues highlighted in light blue correspond to a single amino acid change in ttAAV2, which is located on the inside of the capsid. [Figure 18] 1 shows the 3-fold spike on the AAV2 capsid. The residues highlighted in beige correspond to two amino acid changes in the authentic vector that are spatially close and located in the groove between two adjacent 3-fold peaks on the AAV capsid. [Figure 19] FIG. 1 shows the three-fold spike on the AAV2 capsid. The residues highlighted in brown correspond to a single, isolated amino acid change (S593) in the authentic vector, which is located in the groove between adjacent three-fold peaks. [Figure 20]FIG. 1 shows the triply folded spike on the AAV2 capsid. The four amino acids highlighted in pink are involved in receptor binding and are in close proximity on the triply folded spike. [Figure 21] Figure 1 shows a three-dimensional alignment between the VP1 capsid monomer from AAV2 (light blue) and the VP1 monomer from AAV1 (orange). The highlighted residues in the center left of the figure correspond to G549 in AAV1 (orange sphere) and E548 in AAV2 (cyan sphere). The highlighted residues in the upper right of the figure correspond to S586 and T589 in AAV1 (orange sphere) and R585 and R588 in AAV2 (cyan sphere). [Figure 22] Figure 1 shows a three-dimensional alignment between VP1 capsid monomers from AAV2 (light blue) and AAV5 (purple). The highlighted residues in the center of the figure correspond to G537 in AAV5 (purple sphere) and E548 in AAV2 (cyan sphere). The highlighted residues in the upper right of the figure correspond to S575 and T578 in AAV5 (cyan sphere) and R585 and R588 in AAV2 (cyan sphere). [Diagram 23] A three-dimensional diagram of the alignment between the VP1 capsid monomer from AAV2 (light blue) and the VP1 monomer from AAV6 (yellow). The highlighted residues at the bottom of the diagram correspond to G549 in AAV6 (orange sphere) and E548 in AAV2 (cyan sphere). The highlighted residues at the top right of the diagram correspond to S586 and T589 in AAV6 (orange sphere) and R585 and R588 in AAV2 (cyan sphere). [Figure 24] Figure 1 shows a three-dimensional alignment between VP1 capsid monomers from AAV2 (light blue) and AAV8 (pink). The highlighted residues in the upper left of the figure correspond to S315 in AAV8 (red sphere) and N312 in AAV2 (cyan sphere). The highlighted residues in the lower right of the figure correspond to T591 in AAV8 (red sphere) and R588 in AAV2 (cyan sphere). [Diagram 25] Figure 1 shows a three-dimensional alignment between VP1 capsid monomers from AAV2 (light blue) and AAV9 (green). The highlighted residues in the center of the figure correspond to G549 in AAV9 (yellow sphere) and E548 in AAV2 (cyan sphere). The highlighted residues in the bottom left of the figure correspond to S586 in AAV9 (yellow sphere) and R585 in AAV2 (cyan sphere). [Figure 26A] Figure 26 shows the analysis of rAAV2TT and WT expression in the parafascicular nucleus after injection into the striatum in rat brain.Representative images of rat brain sections, showing the rostral aspect on the left and the caudal aspect on the right.Injection site in the striatum is shown; projection area in the hypothalamus observed in Figures 26B and C is shown (parafascicular nucleus: pf). [Figure 26B] 1 shows an analysis of rAAV2TT and WT expression in the parafascicular nucleus following striatal injection in the rat brain. High magnification images of GFP expression detected in the parafascicular nucleus (pf) following striatal injection of rAAV2WT. [Figure 26C] 1 shows analysis of rAAV2TT and WT expression in the parafascicular nucleus after striatal injection in rat brain. High magnification image of GFP expression detected in the parafascicular nucleus (pf) after striatal injection of TT. [Figure 27] Overview of intracranial injection of rAAV2TT and WT into neonatal mice. Representative examples of neonatal brain sections stained with GFP-specific antibody are shown. 5x1010vg of rAAV2TT (top) or rAAV2WT (middle) were injected into the lateral ventricle of neonatal mouse brain. Uninjected brains from neonatal mice were stained simultaneously and shown as negative controls (NT, no transduction). [Figure 28] High magnification photographs of neonatal mouse brain sections after intracranial injection of rAAV2TT or WT. Neonatal brain sections stained with GFP-specific antibody are shown. 5x1010vg of rAAV2TT (left panel) or rAAV2WT (right panel) were injected into the lateral ventricle of neonatal mouse brain. S1BF: barrel field primary somatosensory cortex. [Figure 29] Overview of brain transduction after systemic injection of rAAV2TT and WT into neonatal mice. Representative examples of neonatal brain sections stained with GFP-specific antibodies are shown. 2x10vg of rAAV2TT (top) or rAAV2WT (bottom) were injected into the jugular vein of neonatal mice. [Diagram 30] High magnification photographs of neonatal mouse brain sections after systemic injection of rAAV2TT or WT. Neonatal brain sections stained with GFP-specific antibody are shown. 2x10vg of rAAV2TT (left panel) or rAAV2WT (right panel) were injected into the jugular vein of neonatal mice. S1BF: barrel field primary somatosensory cortex. [Diagram 31] Figure 1 shows high magnification photographs of neonatal mouse tissue sections after systemic injection of rAAV2TT or WT. 2x10vg of rAAV2TT or rAAV2WT was injected into the jugular vein of neonatal mice. Uninjected mouse organs were used as negative controls. [Diagram 32] Figure 1 shows high magnification images of adult rat brain sections after injection of rAAV2TT, WT and HBnull into the striatum. Representative examples of rat brain sections stained with GFP-specific antibodies are shown. 3.5x109vg of rAAV2WT (left), TT (right) or AAV2-HBnull (middle) were injected into the striatum of adult rat brains, and representative pictures were taken in the thalamus or substantia nigra (SN). [Diagram 33] Figure 1 shows an overview of intracranial injection of intact AAV-TT compared to various TT mutants in neonatal mice. Representative examples of neonatal brain sections stained with GFP-specific antibodies are shown. 5x1010vg of rAAV2TT, TT-S312N, TT-S593A or TT-D546G / G548E (TT-DG) were injected into the lateral ventricle of neonatal mouse brain. Uninjected brains from neonatal mice were stained simultaneously and shown as negative controls (NT). [Diagram 34]Figure 1 shows high magnification pictures of neonatal mouse brain sections after intracranial injection of various TT mutant vectors. Neonatal brain sections stained with GFP-specific antibody are shown. 5x1010vg of vector was injected into the lateral ventricle of neonatal mouse brain. TT-DG: TT-D546G / G548E. [Diagram 35] Figure 1 shows an overview of intracranial injection of complete AAV-TT in neonatal mice compared to the TT-S312N mutant and the final potential TT vector containing 10 mutations. A representative example of a neonatal brain section stained with a GFP-specific antibody is shown. 5x1009vg of rAAV2TT, TT-S312N, TT or TT-S312N-D546G / G548E-S593A (TT-S312N-DG-S593A) was injected into the lateral ventricle of neonatal mouse brain. An uninjected brain from a neonatal mouse was stained in parallel and shown as a negative control (NT). [Diagram 36] Figure 1 shows high magnification pictures of neonatal mouse brain sections after intracranial injection of various TT mutant vectors. Sections of neonatal brain stained with GFP-specific antibody are shown. 5x1009 vg of vector was injected into the lateral ventricle of neonatal mouse brain. [Figure 37] Figure 1 shows ELISA quantification of GFP protein in neonatal mouse brains injected with full AAV-TT, TT-S312N mutant or TT-S312N-DG-S593A. 5x1009vg of vector was injected into the lateral ventricle of neonatal mouse brains, and total protein was extracted from whole brains collected. GFP-specific antibody was used to detect GFP expression in each brain sample, and standard GFP protein was used for quantification. N=5 animals per condition. Error bars indicate mean±SEM. [Figure 38] 1 shows the amino acid sequence of the VP1 capsid protein of AAV3B. Highlighted residues indicate residues that are identical to those in AAV-tt at the corresponding positions. The internal serine residue at position 312 is underlined. [Figure 39] FIG. 1 shows the amino acid sequence of the VP1 capsid protein of AAV-LK03. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0052] In one aspect, the present invention relates to recombinant adeno-associated virus (AAV) vectors.rAAV vectors typically comprise different variant capsid proteins compared to wild-type AAV capsid proteins.Advantageously, variant capsid proteins can enhance the infectivity of vectors in brain and / or eye, making vectors particularly suitable for delivering therapeutic agents by gene therapy into these tissues.

[0053] Recombinant AAV vectors The present disclosure provides recombinant adeno-associated virus (rAAV) vector. "AAV" is an abbreviation for adeno-associated virus, and can be used to refer to the virus itself or its derivatives. This term covers all subtypes, and both naturally occurring and recombinant forms, unless otherwise required. The abbreviation "rAAV" refers to recombinant adeno-associated virus, and recombinant adeno-associated virus is also called recombinant AAV vector (or "rAAV vector"). The term "AAV" includes, for example, AAV type 1 (AAV-1), AAV type 2 (AAV-2), AAV type 3 (AAV-3), AAV type 4 (AAV-4), AAV type 5 (AAV-5), AAV type 6 (AAV-6), AAV type 7 (AAV-7), AAV type 8 (AAV-8), AAV type 9 (AAV-9), AAV type 10 (AAV-10, including AAVrh10), AAV type 12 (AAV-12), 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 forth.

[0054] The genome sequences of various serotypes of AAV, as well as the sequences of native terminal repeats (TRs), Rep proteins and capsid subunits, are known in the art.Such sequences can be found in literature, or in public databases, for example, GenBank.See, for example, GenBank Accession Nos. NC-002077 (AAV-1), AF063497 (AAV-1), NC-001401 (AAV-2), AF043303 (AAV-2), NC-001729 (AAV-3), NC-001829 (AAV-4), U89790 (AAV-4), NC-006152 (AAV-5), AF513851 (AAV-7), AF513852 (AAV-8) and NC-006261 (AAV-8); the disclosures of which are incorporated herein by reference. See also, 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. (1999) J. Virology, 73:3994; Muramatsu et al. See also (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. (2004) Virology, 33:375-383; International Patent Publications WO00 / 28061, WO99 / 61601, WO98 / 11244; and U.S. Patent No. 6,156,303.

[0055] "rAAV vector" as used herein refers to an AAV vector that contains a sequence of a polynucleotide that is not of AAV origin (i.e., a polynucleotide that is heterologous to AAV), typically a sequence of interest for genetic transformation into a cell. In some embodiments, the heterologous polynucleotide may be flanked by at least one, and sometimes two, inverted terminal repeats (ITRs) of AAV. The term rAAV vector encompasses both rAAV vector particles and rAAV vector plasmids. rAAV vectors can be either single-stranded (ssAAV) or self-complementary (scAAV).

[0056] "AAV virus" or "AAV virus particle" or "rAAV vector particle" refers to a virus particle that is composed of at least one AAV capsid protein (typically all of the capsid proteins of wild-type AAV) and a polynucleotide rAAV vector that is enveloped in the capsid. When the particle contains a heterologous polynucleotide (i.e., a polynucleotide other than the wild-type AAV genome, for example, a transgene to be delivered to a mammalian cell), the particle is typically called "rAAV vector particle" or simply "rAAV vector". Thus, the production of rAAV particles necessarily includes the production of rAAV vectors, and thus the vector is contained inside the rAAV particle.

[0057] "Recombinant" as used herein means that the vector, polynucleotide, polypeptide, or cell is the product of various combinations of cloning, restriction, or ligation steps (e.g., steps involving the polynucleotide or polypeptide contained therein), and / or other procedures that result in a construct that is distinct from the product found in nature. A recombinant virus or recombinant vector is a viral particle that contains a recombinant polynucleotide. These terms include copies of the original polynucleotide construct and progeny copies of the original viral construct, respectively.

[0058] Variant AAV capsid proteins The rAAV vectors described herein include variant AAV capsid proteins. By "variant" it is meant that the AAV capsid protein is different from the corresponding wild-type AAV capsid protein of the same serotype. For example, the variant AAV capsid protein can contain one or more amino acid substitutions compared to the corresponding wild-type AAV capsid protein. In this context, "corresponding" refers to the capsid protein of the same serotype, in other words, the variant AAV1 capsid protein contains one or more amino acid substitutions compared to the corresponding wild-type AAV1 capsid protein, the variant AAV2 capsid protein contains one or more amino acid substitutions compared to the corresponding wild-type AAV2 capsid protein, etc.

[0059] A variant AAV capsid protein can include, for example, 1-50, 1-30, 1-20, or 1-15 amino acid substitutions compared to a wild-type AAV capsid protein. Preferably, the variant AAV capsid protein includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 amino acid substitutions compared to a corresponding wild-type AAV capsid protein. In preferred embodiments, the variant AAV capsid protein retains at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the wild-type capsid protein.

[0060] In embodiments of the invention, the variant AAV capsid protein has at least one of the following amino acids in the AAV2 capsid protein sequence compared to the wild-type AAV capsid protein: 125, 151, 162, 205, 312, 457, 492, 499, 533, 546, 548, 585, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880 The AAV2 capsid protein sequence includes at least one amino acid substitution at a position corresponding to one or more of positions 88 and / or 593. In this context, "corresponding" refers to a position in any AAV capsid protein sequence (e.g., in an AAV2 protein sequence or a non-AAV2 capsid protein sequence) that corresponds to one of the above positions in the AAV2 capsid protein. In one embodiment, the at least one amino acid substitution is present at one or more of positions 125, 151, 162, 205, 312, 457, 492, 499, 533, 546, 548, 585, 588 and / or 593 in the AAV2 capsid protein sequence, or at one or more corresponding positions in an alternative AAV capsid protein sequence.

[0061] Generally, AAV capsid proteins include VP1, VP2 and VP3. In a preferred embodiment, the capsid protein includes the AAV capsid protein VP1.

[0062] Nucleic acid and amino acid sequences and sequence identities The term "polynucleotide" refers to a polymeric form of nucleotides of any length, including deoxyribonucleotides or ribonucleotides, or their analogs. Polynucleotides can include modified nucleotides, such as methylated nucleotides and nucleotide analogs, and can be interposed with non-nucleotide components. Modifications to nucleotide structure, if present, can be introduced 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, in any embodiment of the invention described herein, polynucleotides encompass both double-stranded forms and each of the two complementary single-stranded forms that are known or predicted to constitute a double-stranded form.

[0063] The terms "polypeptide", "peptide" and "protein" are used interchangeably herein to refer to polymers of amino acids of any length. The terms also encompass polymers of amino acids that have been modified; modifications include, for example, disulfide bond formation, glycosylation, lipidation, phosphorylation, or conjugation with a labeling moiety. Polypeptides, such as antiangiogenic polypeptides, neuroprotective polypeptides, etc., when discussed in the context of delivery of gene products to mammalian subjects and compositions therefor, 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, references to nucleic acids encoding antiangiogenic polypeptides, neuroprotective polypeptides, and other such nucleic acids for use in delivering gene products to mammalian subjects (which may be referred to as "transgenes" to be delivered to recipient cells) also include polynucleotides that encode intact polypeptides or any fragments or genetically engineered derivatives that retain the desired biochemical function.

[0064] A polynucleotide or polypeptide has a certain percentage of "sequence identity" to another polynucleotide or polypeptide, meaning that when aligned and the two sequences are compared, the bases or amino acids are the same at that percentage. 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 on the world wide web at ncbi.nlm.nih.gov / BLAST / . Another alignment algorithm is described in Genetics and FASTA, available in the package of the Computer Computing Group (GCG) (Madison, Wisconsin, USA), GCG is a wholly owned subsidiary of Oxford Molecular Group, Inc. Other techniques for alignment are described in Methods in Enzymology, vol. 266: Computer Methods for Macromolecular Sequence Analysis (1996) edited by 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 aligning sequences. See Meth. Mol. Biol., 70:173-187 (1997). Sequences can also be aligned using the GAP program, which uses the alignment method of Needleman and Wunsch. See J. Mol. Biol., 48:443-453 (1970).

[0065] Of interest is the BestFit program, which uses the local homology algorithm of Smith and Waterman (Advances in Applied Mathematics 2:482-489 (1981)) to determine sequence identity. Gap creation penalties generally range from 1 to 5, usually 2 to 4, and in many embodiments are 3. Gap extension penalties generally range from about 0.01 to 0.20, and often are 0.10. This program has default parameters that are determined by the sequences that are input and to be compared. Preferably, the default parameters determined by the program are used to determine sequence identity. This program is also available from the Genetics Computing Group (GCG) (Madison, Wisconsin, USA) package.

[0066] Another interesting program is the FastDB algorithm. FastDB is a program that evaluates current methods in sequence comparison and Analysis, Macromolecule Sequencing and Synthesis, Selected Methods and Applications, pp. 127-149, 1988, Alan R. Liss, Inc. FastDB uses the following parameters: Mismatch penalty: 1.00; Gap penalty: 1.00; Gap size penalty: 0.33, and Linkage penalty: 30.0 The percent sequence identity is calculated based on the

[0067] Variant AAV2 capsid proteins In one embodiment, the vector comprises a variant AAV2 capsid protein, in which the variant AAV2 capsid protein comprises at least one amino acid substitution at one or more of positions 125, 151, 162, 205, 312, 457, 492, 499, 533, 546, 548, 585, 588, and / or 593 in the AAV2 capsid protein sequence.

[0068] The sequence of wild-type AAV2 capsid protein VP1 is known and is shown in Figure 1 (SEQ ID NO: 1). The wild-type AAV2 capsid protein sequence is also available in the databases under the accession numbers: NC-001401; UniProt P03135; NCBI reference sequence: YP_680426.1; GenBank: AAC03780.1.

[0069] Preferably, the variant AAV2 capsid protein has at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 1. In a preferred embodiment, the variant AAV2 capsid protein has at least 70%, at least 80%, at least 99% sequence identity to the sequence of SEQ ID NO: 2 or to that sequence. The present invention includes sequences having 0%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity.

[0070] In one embodiment, the variant AAV2 capsid protein comprises one or more of the following residues: I125, A151, S162, S205, S312, M457, A492, D499, Y533, D546, G548, S585, T588 and / or S593. In a preferred embodiment, the variant AAV2 capsid protein comprises one or more of the following amino acid substitutions compared to the wild-type AAV2 capsid protein: V125I, V151A, A162S, T205S, N312S, Q457M, S492A, E499D, F533Y, G546D, E548G, R585S, R588T and / or A593S.

[0071] Combinations of mutations in the AAV2 capsid protein The variant AAV2 capsid protein may comprise any combination of the above amino acid substitutions. Thus, in certain embodiments, the variant AAV2 capsid protein comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 amino acid substitutions selected from the above list. In one embodiment, the variant AAV2 capsid protein comprises all 14 amino acid substitutions disclosed above, for example, the variant AAV2 capsid protein comprises the sequence of SEQ ID NO: 2 (i.e., the sequence referred to herein as ttAAV2 or AAV2-TT).

[0072] In further embodiments, the variant AAV2 capsid protein can comprise a subset of the above 14 mutations. Without being bound by theory, in particular embodiments, the variant AAV2 capsid protein can comprise the following residues, which are divided into the following functional groups: 1) S585 and / or T588; these residues may be associated with reduced heparin binding and increased viral dissemination in brain tissue rich in heparan sulfate proteoglycans; 2) S312; this internal serine residue may play a role in capsid-DNA interactions; 3) D546 and / or G548; these residues may be involved in neutralizing antibody interactions and therefore contribute to transduction properties in vivo; 4) S593; this residue is located in a groove between adjacent 3-fold spikes; 5) M457, A492, D499 and / or Y533; these four amino acids may be involved in receptor binding and are closely spaced on the triplicate folded spike; 6) I125, A151, S162 and / or S205; these residues may be involved in PLA2 activity and / or trafficking of the entering virus.

[0073] It should be understood that the present specification also contemplates corresponding subgroups that include mutations corresponding to the above residues, if such mutations exist at corresponding positions in additional AAV serotypes (see below).

[0074] In a preferred embodiment, the variant AAV2 capsid protein comprises four or more mutations at the above-mentioned positions that may be associated with receptor binding, namely, residues 457, 492, 499 and 533. Thus, it is particularly preferred that the variant AAV2 capsid protein comprises the following residues: M457, A492, D499 and Y533.

[0075] In some preferred embodiments, the variant AAV2 capsid protein is not mutated at position 312 relative to the wild-type AAV2 capsid protein, e.g., the variant AAV2 capsid protein includes residue N312 (present in the wild-type AAV2 capsid protein). Thus, in some embodiments, the variant AAV2 capsid protein can include 1-13 of the specific mutations mentioned above, but does not include the mutation N312S.

[0076] Variant AAV capsid proteins from other serotypes In further embodiments, the variant AAV capsid protein is derived from an alternative AAV serotype, i.e., an AAV serotype other than AAV2. For example, the variant AAV capsid protein can be derived from the capsid protein of AAV1, AAV3B, AAV-LK03, AAV5, AAV6, AAV8, AAV9, or AAV10 (e.g., AAVrh10).

[0077] In these embodiments, the variant AAV capsid protein comprises at least one amino acid substitution at one or more positions corresponding to the positions set forth above for AAV2. In other words, the variant AAV capsid protein comprises at least one amino acid substitution at a position in an alternative (i.e., non-AAV2) AAV capsid protein sequence corresponding to positions 125, 151, 162, 205, 312, 457, 492, 499, 533, 546, 548, 585, 588, and / or 593 in the AAV2 capsid protein sequence.

[0078] It will be understood by those skilled in the art how to identify positions in capsid proteins from alternative AAV serotypes that correspond to positions 125, 151, 162, 205, 312, 457, 492, 499, 533, 546, 548, 585, 588 and / or 593 in the AAV2 capsid protein based on comparison of the amino acid sequences of the capsid proteins of various AAV serotypes. In particular, such positions can be readily identified by sequence alignments known in the art and described herein. For example, one such sequence alignment is provided in FIG. 9.

[0079] Particularly relevant in this context are positions in alternative AAV capsid protein sequences that correspond in three-dimensional space to positions 125, 151, 162, 205, 312, 457, 492, 499, 533, 546, 548, 585, 588 and / or 593 in the AAV2 capsid protein. Methods for three-dimensional modeling and alignment of protein structures are well known in the art and can be used to identify such corresponding positions in capsid protein sequences other than AAV2. Exemplary 3D alignments of the AAV2 capsid protein sequence with capsid protein sequences of alternative AAV serotypes (e.g., AAV1, AAV5, AAV6, AAV8 and AAV9) are shown in Figures 21-25 and discussed below. One skilled in the art can perform similar 3D alignments with capsid proteins from further serotypes, such as AAV2, AAV3, AAV7, AAV10 and AAV12, to identify positions in such sequences that correspond to the above-defined positions in AAV2.

[0080] Variant AAV1 capsid proteins In one embodiment, the vector comprises a variant AAV1 capsid protein. In this embodiment, the variant AAV1 capsid protein comprises at least one amino acid substitution at one or more of positions 125, 151, 162, 205, 313, 458, 493, 500, 534, 547, 549, 586, 589 and / or 594 in the AAV1 capsid protein sequence. These positions in the AAV1 capsid protein VP1 correspond to the positions disclosed above for AAV2.

[0081] The sequence of wild-type AAV1 capsid protein VP1 is known and is shown in Figure 3 (SEQ ID NO:3). The sequence of wild-type AAV1 capsid protein is also available from database accession number: NC-002077. Preferably, the variant AAV1 capsid protein has at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:3.

[0082] The wild-type AAV1 capsid protein VP1 already contains the following residues at positions that correspond to amino acid residues not present in wild-type AAV2 (SEQ ID NO: 1) but present in the variant AAV2 capsid protein disclosed above (SEQ ID NO: 2, ttAAV2): S205 (aligned with S205 in ttAAV2); G549 (aligned with G548 in ttAAV2); S586 (aligned with S585 in ttAAV2); and T589 (aligned with T588 in ttAAV2). Thus, in a preferred embodiment, the variant AAV1 capsid protein comprises one or more of the following amino acid substitutions compared to the wild-type AAV1 capsid protein: V125I, Q151A, T162S, N313S, N458M, K493A, N500D, F534Y, S547D and / or G594S. Typically, such variant AAV1 capsid proteins may share one or more functional properties with the variant AAV2 capsid protein (SEQ ID NO:2, ttAAV2), e.g., may increase infectivity and / or transduction of neuronal or retinal tissue compared to the wild-type AAV1 capsid protein.

[0083] In alternative embodiments, the variant AAV1 capsid protein comprises one or more amino acid substitutions that correspond to reversion of the mutations present in ttAAV2 back to the wild-type AAV2 sequence. For example, the variant AAV1 capsid protein can comprise one or more of the following substitutions: S205T, G549E, S586R and / or T589R. Typically, such variant AAV1 capsid proteins can share one or more functional properties with the wild-type AAV2 capsid protein (SEQ ID NO: 1), for example, can reduce infectivity and / or transduction of neuronal or retinal tissues compared to the wild-type AAV1 capsid protein.

[0084] Variant AAV5 capsid proteins In one embodiment, the vector comprises a variant AAV5 capsid protein. In this embodiment, the variant AAV5 capsid protein comprises at least one amino acid substitution at one or more of positions 124, 150, 153, 195, 303, 444, 479, 486, 520, 533, 537, 575, 578 and / or 583 in the AAV5 capsid protein sequence. These positions in the AAV5 capsid protein VP1 correspond to the positions disclosed above for AAV2.

[0085] The sequence of wild-type AAV5 capsid protein VP1 is known and is shown in Figure 4 (SEQ ID NO: 4). The sequence of wild-type AAV5 capsid protein is also available from database accession number: AF085716. Preferably, the variant AAV5 capsid protein has at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 4.

[0086] The wild-type AAV5 capsid protein VP1 contains the following residue at a position that corresponds to an amino acid residue that is not present in wild-type AAV2 (SEQ ID NO: 1) but is present in the variant AAV2 capsid protein disclosed above (SEQ ID NO: 2, ttAAV2): G537 (t tAAV2 already contains the following amino acid substitutions: S575 (aligned with G548 in tAAV2); S575 (aligned with S585 in ttAAV2); T578 (aligned with T588 in ttAAV2). Thus, in a preferred embodiment, the variant AAV5 capsid protein comprises one or more of the following amino acid substitutions compared to the wild-type AAV5 capsid protein: V124I, K150A, K153S, A195S, R303S, T444M, S479A, V486D, T520Y, P533D and / or G583S. Typically, such variant AAV5 capsid protein can share one or more functional properties with the variant AAV2 capsid protein (SEQ ID NO: 2, ttAAV2), for example, can increase infectivity and / or transduction of neuronal or retinal tissues compared to the wild-type AAV5 capsid protein.

[0087] In alternative embodiments, the variant AAV5 capsid protein comprises one or more amino acid substitutions that correspond to the reversion of the mutations present in ttAAV2 back to the wild-type AAV2 sequence. For example, the variant AAV5 capsid protein can comprise one or more of the following substitutions: G537E, S575R and / or T578R. Typically, such variant AAV5 capsid protein can share one or more functional properties with the wild-type AAV2 capsid protein (SEQ ID NO: 1), for example, can reduce the infectivity and / or transduction of neuronal or retinal tissues compared to the wild-type AAV5 capsid protein.

[0088] Variant AAV6 capsid proteins In one embodiment, the vector comprises a variant AAV6 capsid protein. In this embodiment, the variant AAV6 capsid protein comprises at least one amino acid substitution at one or more of the following positions in the AAV6 capsid protein sequence: 125, 151, 162, 205, 313, 458, 493, 500, 534, 547, 549, 586, 589 and / or 594. These positions in the AAV6 capsid protein VP1 correspond to the positions disclosed above for AAV2.

[0089] The sequence of wild-type AAV6 capsid protein VP1 is known and is shown in Figure 5 (SEQ ID NO:5). The sequence of wild-type AAV6 capsid protein is also available from database accession number: AF028704. Preferably, the variant AAV6 capsid protein has at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:5.

[0090] The wild-type AAV6 capsid protein VP1 already contains the following residues at positions that correspond to amino acid residues not present in wild-type AAV2 (SEQ ID NO: 1) but present in the variant AAV2 capsid protein disclosed above (SEQ ID NO: 2, ttAAV2): S205 (aligned with S205 in ttAAV2); G549 (aligned with G548 in ttAAV2); S586 (aligned with S585 in ttAAV2); T589 (aligned with T588 in ttAAV2). Thus, in a preferred embodiment, the variant AAV6 capsid protein comprises one or more of the following amino acid substitutions compared to the wild-type AAV6 capsid protein: V125I, Q151A, T162S, N313S, N458M, K493A, N500D, F534Y, S547D and / or G594S. Typically, such variant AAV6 capsid proteins may share one or more functional properties with the variant AAV2 capsid protein (SEQ ID NO:2, ttAAV2), e.g., may increase infectivity and / or transduction of neuronal or retinal tissue compared to the wild-type AAV6 capsid protein.

[0091] In alternative embodiments, the variant AAV6 capsid protein comprises one or more amino acid substitutions that correspond to the reversion of the mutations present in ttAAV2 back to the wild-type AAV2 sequence. For example, the variant AAV6 capsid protein can comprise one or more of the following substitutions: S205T, G549E, S586R and / or T589R. Typically, such variant AAV6 capsid proteins can share one or more functional properties with the wild-type AAV2 capsid protein (SEQ ID NO: 1), for example, can reduce the infectivity and / or transduction of neuronal or retinal tissues compared to the wild-type AAV6 capsid protein.

[0092] Variant AAV8 capsid proteins In one embodiment, the vector comprises a variant AAV8 capsid protein. In this embodiment, the variant AAV8 capsid protein comprises at least one amino acid substitution at one or more of positions 125, 151, 163, 206, 315, 460, 495, 502, 536, 549, 551, 588, 591 and / or 596 in the AAV8 capsid protein sequence. These positions in the AAV8 capsid protein VP1 correspond to the positions disclosed above for AAV2.

[0093] The sequence of wild-type AAV8 capsid protein VP1 is known and is shown in FIG. 6 (SEQ ID NO: 6). The sequence of wild-type AAV8 capsid protein is also available under database accession number NC _ 006261. Preferably, the variant AAV8 capsid protein has at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:6.

[0094] The wild-type AAV8 capsid protein VP1 already contains the following residues at positions that correspond to amino acid residues not present in wild-type AAV2 (SEQ ID NO: 1) but present in the variant AAV2 capsid protein disclosed above (SEQ ID NO: 2, ttAAV2): S315 (aligned with S312 in ttAAV2); T591 (aligned with T588 in ttAAV2). Thus, in a preferred embodiment, the variant AAV8 capsid protein comprises one or more of the following amino acid substitutions compared to the wild-type AAV8 capsid protein: V125I, Q151A, K163S, A206S, T460M, T495A, N502D, F536Y, N549D, A551G, Q588S and / or G596S. Typically, such variant AAV8 capsid proteins may share one or more functional properties with the variant AAV2 capsid protein (SEQ ID NO:2, ttAAV2), e.g., may increase infectivity and / or transduction of neuronal or retinal tissue compared to the wild-type AAV8 capsid protein.

[0095] In alternative embodiments, the variant AAV8 capsid protein comprises one or more amino acid substitutions that correspond to reversion of the mutations present in ttAAV2 back to the wild-type AAV2 sequence. For example, the variant AAV8 capsid protein can comprise one or more of the following substitutions: S315N and / or T591R. Typically, such variant AAV8 capsid proteins can share one or more functional properties with the wild-type AAV2 capsid protein (SEQ ID NO: 1), for example, reducing infectivity and / or transduction of neuronal or retinal tissues compared to the wild-type AAV8 capsid protein.

[0096] In one embodiment, the variant AAV8 capsid protein comprises an amino acid substitution at position 315 in the AAV8 capsid protein sequence compared to a wild-type AAV8 capsid protein. For example, the variant AAV8 capsid protein comprises residue N315. Thus, in one embodiment, the variant AAV8 capsid protein comprises the amino acid substitution S315N compared to the wild-type AAV8 capsid protein.

[0097] Variant AAV9 capsid proteins In one embodiment, the vector comprises a variant AAV9 capsid protein. In this embodiment, the variant AAV9 capsid protein comprises at least one amino acid substitution at one or more of positions 125, 151, 162, 205, 314, 458, 493, 500, 534, 547, 549, 586, 589 and / or 594 in the AAV9 capsid protein sequence. These positions in the AAV9 capsid protein VP1 correspond to the positions disclosed above for AAV2.

[0098] The sequence of wild-type AAV9 capsid protein VP1 is known and is shown in Figure 7 (SEQ ID NO: 7). The sequence of wild-type AAV9 capsid protein is also available from database accession number: AY530579. Preferably, the variant AAV9 capsid protein has at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 7.

[0099] The wild-type AAV9 capsid protein VP1 already contains the following residues at positions that correspond to amino acid residues not present in wild-type AAV2 (SEQ ID NO: 1) but present in the variant AAV2 capsid protein disclosed above (SEQ ID NO: 2, ttAAV2): S162 (aligned with S162 in ttAAV2); S205 (aligned with S205 in ttAAV2); G549 (aligned with G548 in ttAAV2); S586 (aligned with S585 in ttAAV2). Thus, in a preferred embodiment, the variant AAV9 capsid protein comprises one or more of the following amino acid substitutions compared to the wild-type AAV9 capsid protein: L125I, Q151A, N314S, Q458M, V493A, E500D, F534Y, G547D, A589T and / or G594S. Typically, such variant AAV9 capsid proteins may share one or more functional properties with the variant AAV2 capsid protein (SEQ ID NO:2, ttAAV2), e.g., may increase infectivity and / or transduction of neuronal or retinal tissue compared to the wild-type AAV9 capsid protein.

[0100] In alternative embodiments, the variant AAV9 capsid protein comprises one or more amino acid substitutions that correspond to reversion of the mutations present in ttAAV2 back to the wild-type AAV2 sequence. For example, the variant AAV9 capsid protein can comprise one or more of the following substitutions: S162A, S205T, G549E and / or S586R. Typically, such variant AAV9 capsid proteins can share one or more functional properties with the wild-type AAV2 capsid protein (SEQ ID NO: 1), for example, reducing infectivity and / or transduction of neuronal or retinal tissues compared to the wild-type AAV9 capsid protein.

[0101] Variant AAV10 capsid proteins In one embodiment, the vector comprises a variant AAV10 capsid protein. As used herein, "AAV10" includes AAVrh10. In this embodiment, the variant AAV10 (e.g., AAVrh10) capsid protein comprises at least one amino acid substitution at one or more of the following positions in the AAV10 capsid protein sequence: 125, 151, 163, 206, 315, 460, 495, 502, 536, 549, 551, 588, 591 and / or 596. These positions in the AAV10 capsid protein VP1 correspond to the positions disclosed above for AAV2.

[0102] The sequence of wild-type AAV10 capsid protein VP1 is known and is shown in Figure 8 (SEQ ID NO: 8). Preferably, the variant AAV10 capsid protein has at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:8.

[0103] The wild-type AAV10 capsid protein VP1 already contains the following residue at the position corresponding to the amino acid residue not present in wild-type AAV2 (SEQ ID NO: 1) but present in the variant AAV2 capsid protein disclosed above (SEQ ID NO: 2, ttAAV2): G551 (aligned with G548 in ttAAV2). Thus, in a preferred embodiment, the variant AAV10 capsid protein comprises one or more of the following amino acid substitutions compared to the wild-type AAV10 capsid protein: V125I, Q151A, K163S, A206S, N315S, T460M, L495A, N502D, F536Y, G549D, Q588S, A591T and / or G596S. Typically, such variant AAV10 capsid proteins may share one or more functional properties with the variant AAV2 capsid protein (SEQ ID NO:2, ttAAV2), e.g., may increase infectivity and / or transduction of neuronal or retinal tissue compared to the wild-type AAV10 capsid protein.

[0104] In an alternative embodiment, the variant AAV10 capsid protein comprises an amino acid substitution that reverts the mutation present in ttAAV2 back to the wild-type AAV2 sequence. For example, the variant AAV10 capsid protein can comprise the following substitution: G551E. Typically, such variant AAV10 capsid protein can share one or more functional properties with wild-type AAV2 capsid protein (SEQ ID NO: 1), for example, can reduce infectivity and / or transduction of neuronal or retinal tissues compared to wild-type AAV10 capsid protein.

[0105] Variant AAV3B capsid proteins In one embodiment, the vector comprises a variant AAV3B capsid protein. In this embodiment, the variant AAV3B capsid protein can comprise an amino acid substitution at position 312 compared to the wild-type AAV3B capsid protein. For example, the variant AAV3B capsid protein can comprise residue N312. Thus, in one embodiment, the variant AAV8 capsid protein comprises an amino acid substitution S312N compared to the wild-type AAV8 capsid protein. In a further embodiment, the variant AAV3B capsid protein can comprise one or more additional mutations at positions corresponding to those disclosed above for AAV2.

[0106] The sequence of wild-type AAV3B capsid protein VP1 is known and is shown in Figure 38 (SEQ ID NO: 11). The wild-type AAV3B capsid protein sequence is also available from the NCBI database under accession number: AF028705. Preferably, the variant AAV3B capsid protein has at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 11.

[0107] Variant AAV-LK03 capsid protein In one embodiment, the vector comprises a variant AAV-LK03 capsid protein. In this embodiment, the variant AAV-LK03 capsid protein can comprise an amino acid substitution at position 312 compared to the AAV-LK03 capsid protein sequence as defined in SEQ ID NO: 12. For example, the variant AAV-LK03 capsid protein can comprise The variant can comprise residue N312. Thus, in one embodiment, the variant AAV-LK03 capsid protein comprises the amino acid substitution S312N compared to the AAV-LK03 capsid protein sequence as defined in SEQ ID NO: 12. In a further embodiment, the variant AAV-LK03 capsid protein can comprise one or more additional mutations at positions corresponding to those disclosed above for AAV2.

[0108] The sequence of wild-type AAV-LK03 capsid protein VP1 is known and is shown in Figure 39 (SEQ ID NO: 12). The AAV-LK03 capsid protein sequence is also disclosed in WO2013 / 029030, where it is SEQ ID NO: 31. Preferably, the variant AAV-LK03 capsid protein has at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 12.

[0109] Gene product In one embodiment, rAAV further comprises a heterologous nucleic acid comprising a nucleotide sequence encoding a gene product. "Gene" refers to a polynucleotide that contains at least one open reading frame that can encode a specific protein after being transcribed and translated. "Gene product" is a molecule that results from the expression of a specific gene. Gene products include, for example, polypeptides, aptamers, interfering RNA, mRNA, etc.

[0110] "Heterologous" means that the genotype of the entity is derived from a distinct entity that is different from the genotype of the rest of the entity to which it is compared.For example, a polynucleotide introduced into a plasmid or vector derived from a different species by genetic engineering techniques is a heterologous polynucleotide.A promoter that is removed from a native coding sequence and is operably linked to a coding sequence that is not found in nature to be linked to such a promoter is a heterologous promoter.Thus, for example, a rAAV that comprises a heterologous nucleic acid that encodes a heterologous gene product is a rAAV that comprises 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.

[0111] In some embodiments, the gene product is an interfering RNA. In some embodiments, the gene product is an aptamer. In some embodiments, the gene product is a polypeptide.

[0112] Interfering RNA When gene product is interference RNA (RNAi), suitable RNAi includes RNAi that reduces the level of apoptotic or angiogenic factors in cells. For example, RNAi can be shRNA or siRNA that reduces the level of gene product that induces or promotes apoptosis in cells. Herein, genes whose gene products induce or promote apoptosis are referred to as "pro-apoptotic genes", and the products (mRNA; protein) of those genes are referred to as "pro-apoptotic gene products". Pro-apoptotic gene products include, for example, Bax, Bid, Bak and Bad gene products. For example, see U.S. Patent No. 7,846,730.

[0113] Interfering RNA can also be directed against angiogenic products, such as VEGF (e.g., Cand5; see, e.g., U.S. Patent Publication Nos. 2011 / 0143400; 2008 / 0188437; and Reich et al. (2003) Mol. Vis., 9:210), VEGFR1 (e.g., Sirna-027; see, e.g., Kaiser et al. (2010) Am. J. Ophthalmol., 150:33; and and Shen et al. (2006) Gene Ther., 13:225), or VEGFR2 (Kou et al. (2005) Biochem., 44:15064). See also U.S. Patent Nos. 6,649,596, 6,399,586, 5,661,135, 5,639,872, and 5,639,736; and U.S. Patent Nos. 7,947,659 and 7,919,473.

[0114] "Small interfering RNA" or siRNA is an RNA duplex nucleotide that is directed to a gene of interest ("target gene"). "RNA duplex" refers to a structure formed by complementary pairing between two regions of an RNA molecule. The nucleotide sequence of the duplex portion of the siRNA shows complementarity to the nucleotide sequence of the targeted gene, and thus the siRNA is "directed" to the gene. In some embodiments, the length of the siRNA duplex 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 long. In some embodiments, the duplex is 19-25 nucleotides long. The RNA duplex portion of the siRNA can be part of a hairpin structure. In addition to the duplex portion, the hairpin structure can contain a loop portion located between the two sequences that form the duplex. The length of the loop can vary. In some embodiments, the loop is 5, 6, 7, 8, 9, 10, 11, 12 or 13 nucleotides long. The hairpin structure can also contain a 3' or 5' overhang portion. In some embodiments, the overhang is a 0, 1, 2, 3, 4 or 5 nucleotide long 3' or 5' overhang.

[0115] "Small hairpin RNAs" or shRNAs are polynucleotide constructs that can be engineered to express an interfering RNA, eg, an siRNA.

[0116] Aptamers When gene product is aptamer, exemplary aptamer of interest includes aptamer for vascular endothelial growth factor (VEGF).See, for example, Ng et al. (2006) Nat.Rev.Drug Discovery, 5:123; and Lee et al. (2005) Proc.Natl.Acad.Sci.USA, 102:18902.Also suitable for use is aptamer specific for PDGF, such as E10030;See, for example, Ni and Hui (2009) Ophthalmologica, 223:401; and Akiyama et al. (2006) J.Cell Physiol., 207:407).

[0117] Polypeptides In one embodiment, the gene product is a therapeutic protein. A "therapeutic" peptide or protein is one that can alleviate or reduce symptoms resulting from the absence or deficiency of a protein in a cell or subject. Alternatively, a "therapeutic" peptide or protein is one that confers some other benefit to the subject, e.g., an anti-degenerative effect.

[0118] When the gene product is a polypeptide, the polypeptide is generally one that enhances the function of a cell, e.g., a cell present in neuronal tissue, retinal tissue or liver tissue, e.g., a hepatocyte, a neuron, a glial cell, a rod or cone photoreceptor cell, a retinal ganglion cell, a Muller cell, a bipolar cell, an amacrine cell, a horizontal cell, or a retinal pigment epithelial cell.

[0119] Exemplary polypeptides include neuroprotective polypeptides (e.g., GDNF, CNTF, NT4, NGF, and NTN); anti-angiogenic polypeptides (e.g., soluble vascular endothelial growth factor (VEGF) receptors; VEGF-binding antibodies; VEGF-binding antibody fragments (e.g., single chain anti-VEGF antibodies); endostatin; tumstatin; angiostatin; soluble Fit polypeptides (Lai et al. (2005) Mol. Ther. 12:659); Fc fusion proteins comprising soluble Fit polypeptides (e.g., Pechan et al. (2009) Gene Ther. 16:10); pigment epithelium-derived factor (PEDF); soluble Tie-2 receptor, etc.; tissue inhibitor of metalloproteinase-3 (TIMP-3); light-responsive opsins, e.g., rhodopsin; anti-apoptotic polypeptides (e.g., Bcl-2, Bcl-Xl); and the like. Suitable polypeptides include, but are not limited to, glial-derived neurotrophic factor (GDNF); fibroblast growth factor 2; neurturin (NTN); ciliary neurotrophic factor (CNTF); nerve growth factor (NGF); neurotrophin-4 (NT4); brain-derived neurotrophic factor (BDNF); epidermal growth factor; rhodopsin; X-linked inhibitor of apoptosis; and sonic hedgehog. Suitable polypeptides are disclosed, for example, in WO2012 / 145601.

[0120] Exemplary polypeptides for delivering genes to the liver include, for example, PBGD (porphobilinogen deaminase), IDUA (iduronidase), Fah (fumarylacetoacetate hydrolase), A1AT (alpha(1)-antitrypsin), 1A1 (hUGT1A1) (uridine diphosphate glucuronyltransferase), HCCS1 (hepatocellular carcinoma suppressor 1), CD (cytosine deaminase), SOCS3 (suppressor of cytokine signaling 3), TNF (tumor necrosis factor), thymidine kinase, IL-24 (interleukin-24), IL-12 (interleukin-12), and TRAIL (tumor necrosis factor-related apoptosis-inducing ligand).

[0121] Regulatory Sequences In some embodiments, the nucleotide sequence encoding the gene product of interest is operably linked to a constitutive promoter. In other embodiments, the nucleotide sequence encoding the gene product of interest is operably linked to an inducible promoter. In some cases, the nucleotide sequence encoding the gene product of interest is operably linked to a tissue-specific or cell type-specific regulatory element.

[0122] For example, in some cases, the nucleotide sequence encoding the gene product of interest is operably linked to a hepatocyte-specific, neuron-specific or photoreceptor-specific regulatory element (e.g., a photoreceptor-specific promoter), for example, a regulatory element that selectively expresses the gene that is operably linked in neuron or photoreceptor cells.Suitable photoreceptor-specific regulatory elements include, for example, the promoter of rhodopsin; the promoter of rhodopsin kinase (Young et al. (2003) Ophthalmol.Vis.Sci., 44:4076); the promoter of beta phosphodiesterase gene (Nicoud et al. (2007) J.Gene Med., 9:1015); the promoter of retinitis pigmentosa gene (Nicoud et al. (2007) supra); the enhancer of interphotoreceptor retinoid binding protein (IRBP) gene (Nicoud et al. (2007) supra); the promoter of IRBP gene (Yokoyama et al. (1992) Exp Eye Res., 55:225). Suitable neuron-specific promoters include, inter alia, the promoter of neuron-specific enolase (NSE), Andersen et al., Cell. Mol. Neurobiol., 13:503-15 (1993); the promoter of the neurofilament light chain gene, Piccioli et al., Proc. Natl. Acad. Sci. USA, 88:561 1-5 (1991); and the promoter of the neuron-specific vgf gene, Piccioli et al., Neuron, 15:373-84 (1995). Suitable hepatocyte-specific promoters include the promoter of albumin, (Heard et al., Mol Cell Biol, 1987;7:2425), or the alpha 1-antitrypsin promoter (Hafenrichter et al., Blood, 1994;84,3394-404).

[0123] "Regulatory elements" or "regulatory sequences" are nucleotide sequences involved in molecular interactions that contribute to the regulation of polynucleotide functions, including polynucleotide replication, duplication, transcription, splicing, translation or degradation. This regulation can affect the frequency, speed or specificity of the process and can be enhancing or inhibitory in nature. Regulatory elements known in the art include, for example, transcriptional regulatory sequences, such as promoters and enhancers. A promoter is a DNA region that, under certain conditions, can bind RNA polymerase and initiate transcription of the coding region that is usually located downstream (3' direction) from the promoter.

[0124] "Operably linked" or "operably linked" refers to the juxtaposition of genetic elements in such a relationship that they can function in an expected manner. For example, a promoter is operably linked to a coding region if it helps initiate transcription of the coding sequence. Intervening residues may be present between the promoter and the coding region so long as such a functional relationship is maintained.

[0125] The term "promoter" as used herein can refer to a DNA sequence located adjacent to a DNA sequence that codes for a recombinant product. A promoter is preferably operably linked to an adjacent DNA sequence. A promoter typically increases the amount of recombinant product expressed from a DNA sequence compared to the amount of recombinant product expressed in the absence of the promoter. A promoter from one organism can be utilized to enhance the expression of a recombinant product from a DNA sequence originating from another organism. For example, a vertebrate promoter can be used to express jellyfish GFP in a vertebrate. Furthermore, a single promoter element can also increase the amount of recombinant product expressed from multiple DNA sequences linked in parallel. Thus, a single promoter element can enhance the expression of one or more recombinant products. Multiple promoter elements are well known to those skilled in the art.

[0126] The term "enhancer" as used herein can refer to a DNA sequence located adjacent to a DNA sequence that codes for a recombinant product. Enhancer elements are typically located upstream of a promoter element, or may be located downstream or within a coding DNA sequence (e.g., a DNA sequence that is transcribed or translated into one or more recombinant products). Thus, enhancer elements can be located 100, 200, or 300 or more base pairs upstream or downstream from a DNA sequence that codes for a recombinant product. Enhancer elements can further increase the amount of recombinant product expressed from a DNA sequence in addition to the increased expression caused by a promoter element. A person skilled in the art can easily utilize multiple enhancer elements.

[0127] Pharmaceutical Compositions The present disclosure provides a pharmaceutical composition comprising: a) an rAAV vector as described above; and b) a pharma- ceutically acceptable carrier, diluent, excipient, or buffer. In some embodiments, the pharma- ceutically acceptable carrier, diluent, excipient, or buffer is suitable for use in humans.

[0128] Such excipients, carriers, diluents and buffers include any pharmaceutical agent that may be administered without undue toxicity. Pharmaceutically acceptable excipients include, but are not limited to, However, liquids include, for example, water, saline, glycerol, and ethanol.

[0129] Excipients can include pharma- ceutically acceptable salts, such as salts of mineral acids, e.g., hydrochlorides, hydrobromides, phosphates, sulfates, and the like; and salts of organic acids, e.g., acetates, propionates, malonates, benzoates, and the like. Additionally, auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, and the like, can also be present in such vehicles. A wide variety of pharma- ceutically acceptable excipients are known in the art and need not be discussed at length here. Pharmaceutically acceptable excipients are well described in a variety of publications, including, for example, A. Gennaro (2000) "Remington: The Science and Practice of Pharmacy", 20th ed., Lippincott, Williams, & Wilkins; Pharmaceutical Dosage Forms and Drug Delivery Systems (1999) eds. H. C. Ansel et al., 7th ed., Lippincott, Williams, & Wilkins; and Handbook of Pharmaceutical Excipients (2000) eds. A. H. Kibbe et al., 3rd ed., Amer. Pharmaceutical Assoc.

[0130] In certain embodiments, the present invention provides pharmaceutical compositions comprising the rAAV vectors described above in a pharma- ceutically acceptable carrier or other medicinal substances, pharmaceuticals, carriers, adjuvants, diluents, etc. For injection, the carrier is typically a liquid. For other methods of administration, the carrier may be either a solid or a liquid, for example, sterile, pyrogen-free water or sterile, pyrogen-free phosphate buffer solution. For inhalation administration, the carrier is respirable and preferably in the form of solid or liquid particles. As a vehicle for injection, it is preferred to use additives normally used for injection solutions, for example, water containing stabilizers, salts, or saline, and / or buffers.

[0131] By "pharmaceutical acceptable" is meant a substance that is biologically and otherwise desirable, e.g., that can be administered to a subject without causing any undesirable biological effects. Thus, such pharmaceutical compositions can be used, for example, in transfection of cells ex vivo or in administering viral particles or cells directly to a subject.

[0132] Methods for delivering gene products to tissues or cells (eg, hepatic, neuronal or retinal tissues or cells) and methods of treatment The method of the present invention provides a means for delivering heterologous nucleic acid sequences into host tissues or cells, including both dividing and non-dividing cells.In addition, the vectors and other reagents, methods and pharmaceutical preparations of the present invention are also useful in methods for administering proteins or peptides to subjects in need thereof, either as a therapeutic method or otherwise.Thus, in this manner, proteins or peptides can be produced in vivo in subjects.Subjects may require proteins or peptides because they have a deficiency in the protein or peptide, or because the production of the protein or peptide in the subject can provide some therapeutic effect, either as a therapeutic method or otherwise, as further described below.

[0133] As used herein, the terms "treatment," "treating," and the like refer to obtaining a desired pharmacological and / or physiological effect. The effect may be prophylactic, in the sense of completely or partially preventing a disease or its symptoms, and / or may be partial or complete relief of a disease and / or adverse effects resulting from the disease. It may be therapeutic in the sense of a complete cure. "Treatment", as used herein, encompasses any treatment of disease in a mammal, particularly a human, and includes (a) preventing the disease from developing in a subject who has not yet been diagnosed with the disease but who may be predisposed to or at risk of developing the disease, (b) inhibiting the disease, i.e., halting its progression, and (c) palliating the disease, i.e., causing the disease to recede.

[0134] In general, the present invention can be used to deliver any exogenous nucleic acid with biological effect to treat or ameliorate symptoms associated with any disorder associated with gene expression in any organ, tissue or cell, particularly symptoms associated with, for example, the liver, brain or eye. Exemplary disease states include, but are not limited to, lysosomal storage diseases, acute intermittent porphyria, ornithine transcarbamylase deficiency, alpha(1)-antitrypsin deficiency, acute liver failure, Pompe disease, tyrosinemia, Crigler-Najjar syndrome, hepatitis, cirrhosis, hepatocellular carcinoma, AIDS, Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, epilepsy, as well as other neurological disorders, cancers (e.g., brain tumors), retinal degenerative diseases and other eye diseases.

[0135] Gene transfer is likely to be useful in understanding and providing therapy for disease states. There are some genetic diseases for which the defective genes are known and have been cloned. In some cases, the functions of these cloned genes are known. In general, the disease states are divided into two classes: conditions that are usually recessively inherited, where an enzyme is usually deficient, and conditions that are dominantly inherited, where an imbalance at least temporarily involves a regulatory or structural protein. For diseases with a deficiency state, it is expected that gene transfer can be used to provide normal genes into affected tissues to perform replacement therapy, and animal models for the disease can be created using antisense mutations. For imbalance disease states, it is expected that gene transfer can be used to create disease states in model systems, which can then be used to attempt to correct the disease state. Thus, the methods of the present invention allow the treatment of genetic diseases. As used herein, a disease state is treated by partially or fully correcting the deficiency or imbalance that causes the disease or makes the disease more severe. Site-specific integration of nucleic acid sequences can also be used to create mutations or correct defects.

[0136] In one aspect, the invention provides a method of delivering a gene product to a tissue or cell (e.g., liver, neural or retinal tissue or cell) in a subject, comprising administering to the subject an rAAV vector as described above. The gene product can be, for example, a polypeptide or an interfering RNA (e.g., shRNA, siRNA, etc.), or an aptamer, as described above. The cell can be, for example, a blood cell, a stem cell, a bone marrow (e.g., hematopoietic) cell, a liver cell, a cancerous cell, a vascular cell, a pancreatic cell, a neural cell, a glial cell, an ocular or retinal cell, an epithelial or endothelial cell, a dendritic cell, a fibroblast, a lung cell, a muscle cell, a cardiac cell, an intestinal cell, or a kidney cell. Similarly, the tissue can be selected from, for example, blood, bone marrow, muscle tissue (e.g., skeletal muscle, cardiac muscle, or smooth muscle, including vascular smooth muscle), central or peripheral nervous system tissue (e.g., brain, neural tissue or retinal tissue), pancreatic tissue, liver tissue, kidney tissue, lung tissue, intestinal tissue, or heart tissue.

[0137] Gene products can be delivered to retinal cells to treat retinal diseases. Retinal cells can be photoreceptors, retinal ganglion cells, Muller cells, bipolar cells, amacrine cells, horizontal cells, or retinal pigment epithelial cells. In some cases, the retinal cells are photoreceptor cells, e.g., rod cells or cone cells. Similarly, gene products can be delivered to neural tissue or cells to treat neurological disorders. Gene products can be delivered to various cell types present in neural tissue, e.g., neurons or glial cells (e.g., astrocytes, olivine, and oocytes). The gene product can be delivered to liver cells, for example. Godendrocytes. Delivery of the gene product to the liver can provide treatment for liver damage. The gene product can be delivered to hepatocytes, for example.

[0138] The present disclosure provides a method of treating a disease (e.g., a liver disease, a neurological disease, or an eye disease) comprising administering to an individual in need thereof an effective amount of an rAAV vector as described above. The subject rAAV vectors can be administered by intracranial injection, intracerebral injection, intravitreal injection, retinal injection, intraocular injection by subretinal injection, intravenous injection, or any other convenient mode or route of administration.

[0139] Further exemplary modes of administration include oral administration, rectal administration, transmucosal administration, topical administration, transdermal administration, inhalation administration, parenteral administration (e.g., intravenous, subcutaneous, intradermal, intramuscular and intraarticular) administration, etc., as well as direct injection into tissue or organ, alternatively intrathecal injection, direct intramuscular injection, intracerebroventricular injection, intravenous injection, intraperitoneal injection, intranasal injection or intraocular injection. Injection can be prepared in a conventional dosage form, either as a liquid solution or suspension, a solid dosage form suitable for dissolving or suspending in liquid prior to injection, or as an emulsion. Alternatively, the virus can be administered locally, rather than systemically, for example, by forming a depot or sustained release.

[0140] The recombinant viral vector is preferably administered to a subject in an amount sufficient to cause infection (or transduction) and expression of the heterologous nucleic acid sequence in the subject's cells (e.g., hepatic, neuronal or retinal cells). Preferably, the target cell is a hepatic cell, a neuronal cell (including cells of the central and peripheral nervous system, particularly brain cells), or a retinal cell. In some cases, the retinal cell is a photoreceptor cell (e.g., rod and / or cone). In other cases, the retinal cell is an RGC cell. In other cases, the retinal cell is an RPE cell. In other cases, the retinal cell can include amacrine cells, bipolar cells, and horizontal cells.

[0141] Preferably, the vector is administered in a therapeutically effective amount. A "therapeutically effective" amount, as used herein, is an amount sufficient to alleviate (e.g., ameliorate, reduce, decrease) at least one symptom associated with a disease state. In other words, a "therapeutically effective" amount is an amount sufficient to bring about some improvement in the subject's condition. A "therapeutically effective amount" falls in a relatively broad range and can be determined by experimentation and / or clinical trials. For example, for in vivo injection, a therapeutically effective dose is approximately about 10 6 ~about 10 15 rAAV viral particles, e.g., about 10 8 ~10 12 For in vitro transduction, the effective amount of rAAV viral particles to be delivered to cells is approximately 10 8 ~about 10 13 Other effective doses can be readily established by those skilled in the art through routine trials to establish dose-response curves.

[0142] In some embodiments, two or more administrations (e.g., 2, 3, or 4 or more administrations) spaced at intervals of various lengths, e.g., daily, weekly, monthly, yearly, etc., can be administered to obtain a desired level of gene expression.

[0143] Neurological diseases that can be treated include any disease associated with the brain or CNS, including psychiatric diseases. Brain diseases are classified into two general categories: (a) pathological processes, such as infections, trauma, and neoplasms, and (b) diseases specific to the nervous system, including myelin diseases and neuronal degeneration. Diseases in either category can be treated. For example, neurological diseases include neurodegenerative diseases, such as Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis (ALS), spinal muscular atrophy, and cerebellar degeneration; schizophrenia; epilepsy; diseases related to ischemia and stroke; demyelinating diseases, such as multiple sclerosis, perivenous encephalitis, heterochromatin due to arylsulfatase A deficiency, and cerebellar degeneration. Leukodystrophies, such as Krabbe disease due to galactocerebroside beta-galactosidase deficiency, adrenoleukodystrophy and adrenomyeloneuropathy; post-viral diseases, such as progressive multifocal leukoencephalopathy, acute disseminated encephalomyelitis, acute necrotizing hemorrhagic leukoencephalitis; mitochondrial encephalomyopathy; neurological cancers, such as primary brain tumors, including gliomas, meningiomas, neurinomas, pituitary adenomas, medulloblastomas, craniopharyngiomas, hemangiomas, epidermoids, sarcomas, and intracranial metastases from other tumor sources; neurological infections, or neurological inflammatory conditions.

[0144] Ocular diseases that can be treated using the subject methods include, but are not limited to, acute macular neuroretinopathy; Behcet's disease; choroidal neovascularization; diabetic uveitis; histoplasmosis; macular degeneration, e.g., acute macular degeneration, nonexudative age-related macular degeneration, and exudative age-related macular degeneration; edema, e.g., macular edema, cystoid macular edema, and diabetic macular edema; multifocal choroiditis; ocular trauma affecting a site or location at the back of the eye; ocular tumors; retinal disorders, e.g., central retinal vein occlusion, diabetic retinopathy (including proliferative diabetic retinopathy), proliferative vitreoretinopathy (PVR), arterial occlusive disease of the retina, retinal detachment, uveoretinal disease; sympathetic ophthalmia; Vogt-Koyanagi-Harada (VKH) syndrome; doe's membrane exudation; conditions of the posterior part of the eye caused by ocular laser treatment or its effects; conditions of the posterior part of the eye caused by photodynamic therapy or its effects; photocoagulation, radiation retinopathy; epiretinal membrane disorder; branch retinal vein occlusion; anterior ischemic optic neuropathy; diabetic retinal dysfunction other than retinopathy; retinoschisis; retinitis pigmentosa; glaucoma; Usher syndrome, cone-rod dystrophy; Stargardt disease (fundus flammeus); hereditary macular degeneration; chorioretinal degeneration; Leber congenital amaurosis; congenital stationary night blindness; choroideremia; Bardet-Biedl syndrome; macular telangiectasia; Leber hereditary optic atrophy; retinopathy of prematurity; and color vision disorders, including achromatopsia, protanopia, deutazanomas, and tritanopia.

[0145] Liver diseases that can be treated include, for example, lysosomal storage diseases, such as acute intermittent porphyria, ornithine transcarbamylase deficiency, Wilson's disease, mucopolysaccharidoses (e.g., MPS type I or MPS type VI), Sly syndrome, Pompe disease, tyrosinemia, alpha(1)-antitrypsin deficiency, Crigler-Najjar syndrome; hepatitis A, hepatitis B, or hepatitis C; cirrhosis; liver cancer, such as hepatocellular carcinoma; or acute liver failure.

[0146] The present invention is useful in both veterinary and medical applications. Suitable subjects include both birds and mammals, with mammals being preferred. The term "birds" as used herein includes, but is not limited to, chickens, ducks, geese, quail, turkeys and pheasants. The term "mammals" as used herein includes, but is not limited to, humans, cows, sheep, goats, horses, cats, dogs, rabbits, and the like. Human subjects are most preferred. Human subjects include fetal, neonatal, infant, juvenile and adult subjects.

[0147] Transduction of tissues (e.g., liver tissue, neuronal tissue, or retinal tissue) In some embodiments, the rAAV vectors disclosed herein exhibit increased transduction of tissues (e.g., liver tissue, neuronal tissue and / or retinal tissue) when compared to a corresponding AAV vector (from the same serotype) that contains, for example, a wild-type AAV capsid protein. For example, the rAAV vectors can exhibit at least a 10%, 50%, 100%, 500% or 1000% increase in infectivity when compared to infectivity by AAV viral particles that contain the corresponding wild-type AAV capsid protein.

[0148] In further embodiments, the rAAV vectors disclosed herein can selectively or specifically infect a tissue (e.g., liver tissue, neuronal tissue, or retinal tissue), e.g. For example, the rAAV vector can exhibit increased transduction of hepatocytes, neurons, or retinal cells compared to other cell types. For example, the rAAV vector can exhibit at least a 10%, 50%, 100%, 500%, or 1000% increase in infectivity of a particular cell type (e.g., hepatocytes, neurons, or retinal cells) compared to another cell type (e.g., non-hepatic, non-neuronal, and / or non-retinal cells). For example, the rAAV vector can selectively infect hepatocytes, neurons, and / or photoreceptor cells compared to non-liver, brain, and / or eye cells.

[0149] Where a recombinant AAV vector exhibits increased transduction of neuronal or retinal tissue, for example when the vector is used to treat a neurological or ocular disorder, the vector preferably comprises a variant AAV2 capsid protein.

[0150] Where the recombinant AAV vector exhibits increased transduction of liver tissue, for example when the vector is used to treat liver damage, the vector preferably comprises a variant AAV3B, variant AAV-LK03 or variant AAV8 capsid protein.

[0151] Nucleic Acids and Host Cells The present disclosure provides an isolated nucleic acid comprising a nucleotide sequence encoding a variant adeno-associated virus (AAV) capsid protein as described above. The isolated nucleic acid can be included in an AAV vector, for example, a recombinant AAV vector.

[0152] Recombinant AAV vectors comprising such variant AAV capsid protein coding sequences can be used to generate recombinant AAV viral particles (i.e., recombinant AAV vector particles). Thus, the present disclosure provides recombinant AAV vectors that, when introduced into a suitable cell, can effect the generation of recombinant AAV viral particles.

[0153] The present invention further provides host cells, e.g., isolated (genetically modified) host cells, comprising a subject nucleic acid. A subject host cell can be an isolated cell, e.g., a cell cultured in vitro. As described below, a subject host cell is useful for producing a subject rAAV viral particle. When a subject host cell is used to produce a subject rAAV viral particle, the host cell is referred to as a "packaging cell." In some embodiments, a subject host cell is stably genetically modified with a subject nucleic acid. In other embodiments, a subject host cell is transiently genetically modified with a subject nucleic acid.

[0154] 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 generally further include a selectable marker, e.g., any of a number of well-known selectable markers, such as, for example, a neomycin resistance marker.

[0155] A subject host cell is generated by introducing a subject nucleic acid into any of a variety of cells, e.g., mammalian cells; mammalian cells include, for example, mouse cells and primate cells (e.g., human cells). Suitable mammalian cells include, but are not limited to, primary 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, and the like. Non-limiting examples of suitable host cells include, for example, HeLa cells (e.g., American Type Culture Collection (ATCC) No. CCL-2), CHO cells (e.g., ATCC Nos. CRL9618, CCL61, CRL9096), 293 cells (e.g., ATCC Nos. CRL9619, CRL9620, CRL9621, CRL9622, CRL9623, CRL9624, CRL9625, CRL9626, CRL9627, CRL9628, CRL9629 ... ATCC No.:CRL-1573), Vero cells, NIH3T3 cells (e.g., ATCC No.:CRL-1658), Huh-7 cells, BHK cells (e.g., ATCC No.:CCL10), PC12 cells (ATCC No.:CRL1721), COS cells, COS-7 cells (ATCC No.:CRL1651), RATI cells, mouse L cells (ATCC No.:CCLI.3), human embryonic kidney (HEK) cells (ATCC No.:CRL1573), HLHepG2 cells, and the like. Baculoviruses can also be used to infect insect cells, such as Sf9 cells, which produce AAV, to generate the subject host cells (see, e.g., U.S. Patent No. 7,271,002; U.S. Patent Application 12 / 297,958).

[0156] In some embodiments, the subject genetically modified host cell comprises a nucleic acid comprising a nucleotide sequence encoding one or more AAV rep proteins in addition to the nucleic acid comprising the nucleotide sequence encoding variant AAV capsid protein described above.In other embodiments, the subject host cell further comprises an rAAV vector.The subject host cell can be used to generate rAAV virus particles.Methods for generating rAAV virus particles are described, for example, in US Patent Publication No. 2005 / 0053922 and US Patent Publication No. 2009 / 0202490.

[0157] As used herein, "packaging" refers to a series of intracellular events that result in the assembly and encapsidation of AAV particles. AAV "rep" gene and AAV "cap" gene refer to polynucleotide sequences that code for the replication and encapsidation proteins of adeno-associated virus. Herein, AAV rep and AAV cap are referred to as the "packaging genes" of AAV. Assembly-associated proteins (AAPs) are products of open reading frames in the cap gene and may also be required for packaging.

[0158] "Helper virus" for AAV refers to a virus that allows AAV (e.g., wild-type AAV) to replicate and be packaged by mammalian cells. A variety of such helper viruses for AAV are known in the art, including adenovirus, herpesvirus, and poxvirus such as vaccinia. Adenoviruses include several different subgroups, but adenovirus type 5 of subgroup C is the most commonly used. Many adenoviruses of human, non-human mammalian and avian origin are known and available from depositories such as 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 depositories such as ATCC.

[0159] "Helper virus functions" refer to functions encoded in the genome of the helper virus that allow AAV replication and packaging (in conjunction with other functions required for replication and packaging as described herein). As described herein, "helper virus functions" can be provided in several ways, including providing a helper virus or, for example, providing a polynucleotide sequence encoding the required functions in trans to a producer cell. 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.

[0160] An "infectious" virus or virus particle is one that contains a competently assembled viral capsid and is capable of delivering a polynucleotide component into a cell for which the viral species is tropic. The term does not necessarily imply any replicative capacity of the virus. Infectious virus particles are counted. Assays for counting the infectious titer of TCID50 are described elsewhere in this disclosure and in the art. Virus 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 in the art. For example, see Grainger et al. (2005) Mol. Ther., 11:S337 (description of assay for TCID50 infectious titer); and Zolotukhin et al. (1999) Gene Ther., 6:973.

[0161] 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 competence generally requires the presence of functional AAV packaging genes. In general, the rAAV vectors described herein are not replication-competent in mammalian cells (especially human cells) due to the lack of one or more AAV packaging genes. Typically, such rAAV vectors lack any AAV packaging gene sequences to minimize the possibility of recombination of the AAV packaging genes with the incoming rAAV vector to generate replication-competent AAV. In many embodiments, the rAAV vector preparations described herein are preparations that contain little, if any, replication-competent AAV (rcAAV, also referred to as RCA) (e.g., 10 2 Approximately less than 1 rcAAV per 10 rAAV particles 4 Approximately less than 1 rcAAV per 10 rAAV particles 8 Approximately less than 1 rcAAV per 10 rAAV particles 12 less than about 1 rcAAV per rAAV particle, or no rcAAV).

[0162] An "isolated" nucleic acid, vector, virus, virus particle, host cell, or other material refers to a preparation of the material that is free of at least some of the other components that may also be present when the material or a similar material is naturally occurring or initially prepared. Thus, for example, an isolated material can be prepared by concentrating the material from a source mixture using purification techniques. Concentration may be measured as an absolute value, such as weight per volume of solution, or relative to potentially interfering materials present in a second, source mixture. Increasing the degree of concentration of the disclosed embodiments results in increasing degrees of isolation. The isolated nucleic acid, vector, virus, host cell, or other material is, in some embodiments, 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% or more purity.

[0163] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs.Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of this invention, the preferred methods and materials are described herein.All publications mentioned herein are incorporated by reference to disclose and describe the methods and / or materials, and publications related to such methods and / or materials are cited.

[0164] It is understood that certain features of the invention that are described, for clarity, in the context of separate embodiments, can also be provided in combination in a single embodiment. Conversely, various features of the invention that are described, for brevity, in the context of a single embodiment can also be provided separately or in any suitable combination of their parts. All combinations of the embodiments relating to the invention are specifically embraced by the invention and are disclosed herein as if each and every combination was individually and explicitly disclosed. Moreover, all subcombinations of the various embodiments and elements thereof are also specifically embraced by the invention and are disclosed herein as if each and every such subcombination was individually and explicitly disclosed. The subject matter is disclosed herein as if expressly disclosed herein.

[0165] The following examples are provided to illustrate certain embodiments of the present invention, but are not intended to limit the invention in any way. EXAMPLES

[0166] Example 1 In this example, we designed and constructed a novel AAV2 vector, named ttAAV2 (for authentic type). Furthermore, to evaluate whether ttAAV2 behaves differently compared with tissue culture-adapted (wild type) AAV2, we tested the novel vector in several animal models (rat, mouse and neonatal mouse). We demonstrated that ttAAV2 has advantages over AAV2 for gene delivery, and is particularly useful for in vivo transduction of brain tissue or eye tissue with heterologous sequences.

[0167] method 1. Cloning The capsid gene of wtAAV2 was obtained from our producer plasmid pDG (Figure 10). This plasmid contains the rep and cap genes of wtAAV2. Subfragments of the capsid gene (pDG nucleotides A: 3257-3759, B: 4025-4555, C: 4797-5287, and D: 5149-5425, respectively) were subcloned into pBS and subsequently subjected to mutagenesis. Four mutations were introduced into fragment A by site-directed mutagenesis, resulting in a construct encoding the following amino acid (AA) changes: V125I, V151A, A162S, and T205S. Fragment B was mutated to encode a single AA change: N312S. Fragment C was mutated to encode the following AA exchanges: Q457M, S492A, E499D, F533Y, G546D, E548G, R585S, R588T, and A593S. Upon confirmation of successful mutagenesis, fragments A-C were recloned into pDG, resulting in a producer plasmid (pDG-ttAAV2) that supports the generation of ttAAV2-encapsidated recombinant virus.

[0168] 2. Generation, purification and titer determination of ttAAV2-GFP viral vector Vector production was established according to standard protocols utilizing co-transfection of rAAV plasmids with pDG; pDG provides both the Ad helper functions and the AAV rep and cap genes. A variety of rAAV plasmids were used to generate recombinant plasmids.

[0169] pTR-UF11(CAG-GFP) was used as the rAAV plasmid. 8 × 10 per cell factory (CF10) 8293 cells were seeded. After 14-18 hours, cells were transfected with pDG or pDG-rrAAV2, and prAAV (e.g., pTR-UF11) using the CaPO4 co-precipitation method. After 72 hours, cells were harvested and resuspended in lysis buffer (20 mM Tris-HCl, pH 8, 150 mM NaCl, 0.5% deoxycholate). Cell pellets were lysed to release virus by four cycles of freezing and thawing, each cycle consisting of 30 min at -80°C followed by 30 min at 37°C. After the final thaw, the lysate was treated with benzonase at a concentration of 50 U / ml and incubated at 37°C for 30 min. Recombinant viruses were purified using gravity columns.

[0170] Purification. As a first step, the crude lysate was clarified by centrifugation at 4000 g for 15 min, to which a preformed iodixanol step gradient was applied. The viral fraction was then collected, rebuffered in lactated Ringer's solution, and purified using an Amicon centrifuge. Concentration was performed using a centrifuge filter.

[0171] The purity of the virus preparations was then assessed by SDS-polyacrylamide gel electrophoresis and the titers were determined using real-time PCR. 12 The collected viral fraction contained 1.5 x 10 particles. 12 At this point, this purification method recovered approximately 33% of the virus present in the crude extract.

[0172] 3. rAAV Vector Generation and Purification (Alternative Method) In an alternative embodiment at point 2 above, rAAV2 vectors are produced as follows: To produce rAAV2 viral particles, 5×10 8293T cells were seeded. After 14–18 h, the cells were double transfected with the GFP-containing vector PD10-pST2-CMV-GFP and either pDG or the pDG capsid mutant (pDG-ttAAV2) to generate AAV2-CMV-GFP wild-type or true-type vectors, respectively. Double transfection was achieved using PEI-max from Polysciences at a ratio of 3.5 ml of PEI per mg of DNA. After 72 h of incubation at 37°C, the cells were harvested by centrifuging the medium and cells at 2200 rpm for 10 min at 4°C. The supernatant was removed and saved for further processing, and the cell pellet was resuspended in lysis buffer (0.15 M NaCl, 50 mM Tris-HCl [pH 8.8]).

[0173] The cell pellet was then lysed to release the virus by four cycles of freezing and thawing, each cycle consisting of 30 minutes at -80°C followed by 30 minutes at 37°C. After the final thawing, the lysate was treated with benzonase at a concentration of 150U / ml and incubated at 37° for 30 minutes. The lysate was then spun at 2000 rpm for 20 minutes to clarify the lysate. The supernatant was filtered using a 0.22 μm cellulose acetate filter, and the recombinant AAV2 virus preparation was purified by FPLC using an AKTApurifier chromatography system (GE Healthcare) and an AVB Sepharose affinity column (bfr.A: PBS, pH8; bfr B: 0.5M glycine, pH2.7). The collected fractions were dialyzed overnight against PBS, and the titer of the virus preparation was then determined by SDS-polyacrylamide electrophoresis and real-time PCR.

[0174] result Transduction and propagation of ttAAV2 in vivo The ttAAV2 vector was tested in vivo to evaluate the bioactivity of the modified virus in such a situation. Samples of AAV2-CMV-GFP WT and AAV2-CMV-GFP TT viruses were prepared for injection into several in vivo models. For this purpose, the virus was concentrated, since only a small volume of vector can be injected in vivo. Then, qPCR and SDS-PAGE were performed to evaluate the new titer of the concentrated vector (Figure 11).

[0175] After qPCR and protein gel analysis, the new titers were: AAV2-CMV-GFP TT: 1.33 × 10 12 viral genomes / ml, and AAV2-CMV-GFP WT: 1.25 × 10 12 viral genomes / ml were obtained. The capsid titers were as follows: AAV2-CMV-GFP TT: 8.89 × 10 12 capsids / ml, and AAV2-CMV-GFP WT: 7.83 × 10 12 capsids / ml. The titer varies between genome copy number and capsid copy number, and at the same time, SDS-PAGE reveals that the genomic DNA fragments typically produced during the production of recombinant AAV vectors are not fully expressed. It was also shown that there are empty capsids forming the capsids, and therefore the capsid titers are higher than the viral genome titers obtained from qPCR.

[0176] Transduction into rat brain rAAV2 TT and WT viruses were injected into the substantia nigra or striatum of wild-type rats, with 2 × 10 9 vg or 3.5×10 9 vg. After 28 days, the brains were dissected and tissue sections were prepared for immunofluorescence analysis. Primary data are shown in Figures 12 and 26.

[0177] Both rAAV2 TT and WT viruses were able to transduce neurons and glial cells from their respective injection sites, although with different efficiencies. In comparison, it was observed that the TT vector transduced brain tissue more efficiently than the WT vector, and spread from the injection site was improved. In addition, after injection of rAAV2TT into the striatum, the presence of transduced neurons was also observed in one region of the hypothalamus, the parafascicular nucleus. This indicates that the TT vector was able to move from the transduced cell body at the injection site to the hypothalamus by active transport along the neuronal processes, revealing a strong ability for retrograde transport. This ability for retrograde transport was lost in the tissue culture-adapted WT rAAV2 vector; since no transduced cells could be observed in the same region (see FIG. 26).

[0178] Taken together, these results indicate a marked increase in transmission and transduction efficiency by ttAAV2 compared with titer-matched wtAAV2 in rat brain. Furthermore, AAV2TT also provides evidence for excellent retrograde transport capacity, which is lost in AAV2WT virus.

[0179] Transduction into mouse eye models ttAAV2 and wt-AAV2, obtained from the same batch used for the rat brain study, were injected intraocularly into adult mice at 2 × 10 per eye. 9vg dose. To avoid inter-animal variability, one eye of each mouse was injected with rAAV2TT and the contralateral eye with rAAV2WT. Three different routes of intraocular injection were analyzed: intracameral, intravitreal and subretinal. After 6 weeks, animals were harvested and GFP expression was assessed by immunofluorescence. The results are shown in FIG. 13. Taken together, these data indicate that transduction of photoreceptor cells by ttAAV2 after subretinal injection is significantly enhanced, both in terms of the level and number of transduced photoreceptor cells, when compared to wtAAV2 (used in the successful RPE65 clinical trial).

[0180] Transduction into a neonatal mouse model In summary, both ttAAV2GFP and wtAAV2GFP vectors were injected into neonatal mice. Two injection routes were tested: intravenous and intracranial injection. After 4 weeks, animals were sacrificed and all tissues were collected from all mice. Brains were collected and then analyzed, visualized by direct fluorescence of the organ on a fluorescent microscope. The results are shown in FIG. 14. Below, the results of intracranial and systemic injections are discussed in more detail.

[0181] Intracranial injection 5 × 10 of either vector 10 vg was injected into the lateral ventricle of P1 neonates. Four weeks after injection, the animals were sacrificed and the brains were dissected, sectioned, and stained using anti-GFP antibody. The results are shown in Figure 27.

[0182] Similar to what was observed in adult rat brain, these data indicate that AAV2TT results in enhanced transduction of mouse brain tissue compared to the AAV2WT vector following intracranial injection. The results show that the ttAAV2 vectors exhibited a higher spread of the ventricles and a higher rate of cell proliferation. When stained sections were observed at higher magnification, the difference in transduction efficiency between both vectors became even more evident: the TT vector performed better in terms of both the number of cells transduced and the expression level. AAV2TT and AAV2WT appeared to have the same cell type tropism, showing transduction of neuronal and glial cells, respectively, suggesting that the observed differences were not differences in cell type specificity but in efficiency (Figure 28). Taken together, these data show that ttAAV2 exhibits a significant enhancement of transduction of mouse brain tissue after ic injection compared to vectors based on wtAAV2. Furthermore, several lines of evidence suggest that transduction of the layer of ventricular epithelial cells that constitute the walls of the ventricles also occurs when using ttAAV2 vectors. This phenomenon is not observed with wtAAV2 vectors.

[0183] systemic injection 2 × 10 of either vector 11 vg was injected into the jugular vein in P1 neonates. Four weeks after injection, the animals were sacrificed and various organs (brain, liver, heart, muscle, lung, spleen and kidney) were harvested and evaluated for GFP transduction by immunohistochemistry using anti-GFP antibody. The results of brain staining are shown in Figure 29, and a higher magnification photograph is shown in Figure 30.

[0184] Good transgene expression in the CNS was observed after systemic injection of AAV2TT, whereas the AAV2WT vector, in comparison, showed only a few transduced neurons, which were poorly functional.

[0185] To assess the overall biodistribution of the AAV2TT vector, the level of transduction obtained in various tissues after systemic injection was evaluated. Harvested organs were fixed, paraffin embedded, sectioned, and stained for GFP expression (Figure 31).

[0186] These data indicate that the AAV-TT vector does not appear to have a strong tropism for other organs, but instead shows specificity primarily for neuronal tissues. This observation predicts that intravenous injection of AAV may prove beneficial for the treatment of neuronal genetic disorders, since this injection route ensures that the vector primarily transduces only the brain, and not non-target peripheral organs.

[0187] Taken together, our in vivo data suggest that ttAAV2 has exceptional transduction properties in ocular and brain tissues and exhibits almost exclusive specificity for neuronal tissues.

[0188] Example 2 Additional Considerations Without being bound by theory, it is believed that the mutations present in ttAAV2 compared to wtAAV2 comprise the following functional groups: 1) Heparin-binding residues (S585 and T588) located on the three-fold spike of the AAV2 capsid; these residues are thought to be involved in heparin binding in the wtAAV2 capsid. In ttAAV2, they are exchanged, which is speculated to support viral spread in brain tissue rich in heparan sulfate proteoglycans. 2) A single amino acid change in ttAAV2 (S312) located on the inside of the capsid; this internal serine residue is predicted to play a role in capsid-DNA interactions and thereby contribute to either viral stability, genome packaging or genome release during infection. 3) Located in the groove between three adjacent folds of the AAV capsid structure these residues are predicted to be involved in neutralizing antibody interactions and therefore may contribute to the transduction properties in vivo. 4) a single isolated amino acid change (S593) located in the groove between adjacent thrice folded spikes; 5) four closely spaced amino acids on the triplicate folded spike that are thought to be involved in receptor binding (M457, A492, D499, and Y533); 6) The four remaining amino acid changes (I125, A151, S162, and S205) present in the primary sequence of VP1 / VP2; these residues are within the capsid region that is not part of VP3. The VP1 / VP2 specific region within the viral capsid is known to contain PLA2 activity that may be involved in the transport of the entering virus. Changes in this region have been shown to affect viral infectivity.

[0189] The three-dimensional locations of these mutations in the AAV2 capsid protein are known and are shown in Figures 15-20. Corresponding locations can be identified in AAV capsid proteins from other serotypes (see below). To further characterize the role of individual amino acid changes involved in ttAAV2 and the improved ttAAV2 phenotype, the ttAAV2 capsid can be mutated to revert individual selected amino acids (or a series of amino acids based on, for example, groups 1-6 discussed above) back to their corresponding sequences in the wild-type AAV2 capsid. Each mutant vector can then be analyzed using the methods described in the Examples above.

[0190] For example, various mutant vectors expressing GFP can be first screened in animal model by intracranial (IC) injection into CD1 neonatal mice.Then, the GFP signal obtained from each mutant vector in the injected brain is observed by fluorescence microscopy, and compared with the signal obtained from the original wtAAV2 vector and ttAAV2 vector expressing GFP.If a new phenotype is identified (i.e., the strong GFP expression specific to ttAAV2 is abolished when a specific amino acid group is mutated), then the injected brain is further sectioned and analyzed by immunohistochemistry.

[0191] In parallel, these selected mutant vectors are subjected to a second screening by intracranial injection in adult rats. In addition, relevant mutation combinations are also analyzed by intravenous (IV) injection into neonatal mice to evaluate the biodistribution of the vectors. Selected organs (heart, lung, liver, spleen, kidney, muscle) are then processed for immunohistochemistry and evaluation of GFP expression.

[0192] Analysis of the contribution of each TT-specific residue to vector efficacy and biodistribution To further characterize AAV2TT and identify the amino acid changes responsible for the improved TT phenotype, the AAV2TT capsid was stepwise mutated to revert selected amino acids to their corresponding sequences in the wild-type AAV2 capsid. This strategy allowed us to more specifically identify the contribution of each of the 14 amino acid mutations to the AAV2TT phenotype and to define a minimal authentic vector containing only the necessary mutations.

[0193] As discussed above, the 14 TT-specific residues were divided into groups based on their location on the AAV capsid and their potential contribution to the transduction profile. To observe whether reversion of some TT-specific residues to their WT equivalents was associated with loss of phenotype, and thereby to identify key amino acid changes among the 14 TT residues, various AAVs were transfected by intracranial injection into neonatal mouse brain or adult rat brain. -TT mutations were screened.

[0194] Heparin-binding site (HBS) Residues 585 and 588 have been shown to be involved in heparin binding in AAV2WT capsid. In AAV-TT, they are exchanged, and it was speculated that this exchange supports viral spread in brain tissue rich in heparan sulfate proteoglycans. These two residues are located on three-fold adjacent spikes in the AAV2 capsid structure (see FIG. 16).

[0195] The heparin-binding ability of AAV2WT was abolished by engineering the R585S and R588T changes onto the WT capsid (AAV2-HBnull), i.e., mutating the residues to obtain the authentic equivalent. This first analysis was performed to confirm that AAV-TT is not simply the more broadly disseminating AAV2 without the heparin-binding site, but that some of the other 12 amino acid changes also play a role in improving the transduction profile of AAV-TT.

[0196] Intracranial injections into adult rats Titer-matched AAV2-TT, AAV2-WT and AAV2-HBnull vectors were delivered at 3.5 × 10 per injection into the substantia nigra or striatum of wild-type rats. 9 vg. After 28 days, the brains were dissected and tissue sections were prepared for immunohistochemical analysis of GFP expression (FIG. 32).

[0197] After injection of AAV-TT virus into the striatum, strong GFP expression in the thalamus and substantia nigra was observed, revealing the strong ability of the vector to retrograde transport. Meanwhile, AAV2-HBnull and AAV2WT showed much lower, if any, retrograde transport than AAV-TT; since transduction occurred in very few cell bodies in these regions after injection into the striatum. This observation indicated that AAV2-HBnull is different from AAV-TT, and that the absence of heparin-binding ability on the AAV2 capsid results in good spread of the authentic vector in the brain. However, this is not sufficient to explain the improved transduction profile of AAV-TT.

[0198] Residues: S312, D546-G548, and S593 The S312 residue is a TT-specific change, and the only change located on the inside of the AAV2 capsid. It was hypothesized that this internal residue may play a role in capsid-DNA interactions, thereby potentially contributing to either viral stability, genome packaging, or genome release during infection.

[0199] The D and G residues at positions 546 and 548, respectively, are located in a groove between adjacent 3-fold peaks.

[0200] A single lone serine, S593, resides in a groove between adjacent thrice folded spikes.

[0201] The locations of these TT-specific residues on the three-dimensional structure of the AAV2 capsid are shown in Figures 17 to 19. Given that the positions of these residues are not highly prominent in the structure, we hypothesized that these residues may not contribute to the transduction phenotype of ttAAV2.

[0202] Intracranial injection in neonatal mice The mutation S312N was engineered into the AAV2-TT capsid to produce TT-S31 The AAV-TT-DG mutant was generated. The AAV-TT-S593A mutation was engineered into the AAV2-TT capsid to generate the AAV-TT-S593A mutant. The AAV-TT-DG mutant was generated. The AAV-TT-S593A mutation was engineered into the AAV2-TT capsid to generate the AAV-TT-S593A mutant.

[0203] By reverting these selected TT amino acids to their corresponding sequences in the wild-type AAV2 capsid, we aimed to determine the contribution of these residues to the improved AAV-TT phenotype.

[0204] 5 × 10 of each mutant vector 10 vg was injected into the lateral ventricle of P1 neonates. Four weeks after injection, the animals were sacrificed and the brains were dissected, sectioned, and stained using anti-GFP antibody. The results are shown in Figure 33.

[0205] Interestingly, these data suggest that AAV TT-S312N exhibits enhanced transduction of mouse brain tissue compared to the complete AAV2TT vector. On the other hand, the amino acid changes S593A and D546E / G548D did not seem to affect the TT phenotype; since similar transduction profiles could be observed throughout the brain. When stained sections were observed at higher magnification, the difference in transduction efficiency became even more apparent (Figure 34).

[0206] From the high magnification, it could be observed that AAV TT-S312 seems to transduce neuronal tissues more efficiently than the original AAV-TT with 14 amino acid changes. In particular, stronger transgene expression, both in terms of the level and number of transduced cells, could be observed in the rostral brain (cortex, striatum, hippocampus) after injection of the TT-S312N vector. This observation was confirmed in all the animals analyzed, despite the high variability in the injected neonatal brain due to the difficulties associated with targeting the injection site. On the other hand, the reverted S593A and D546E / G548D did not seem to have much effect on the phenotype of transduction by the TT vector.

[0207] Example 3 Targets for amino acid mutations on the AAV2 true-type capsid selected from the results obtained using the mutation combinations in Example 2 Based on the results obtained from the mutation of a group of amino acids on the complete AAV TT capsid, we could determine that the mutation S312N appears to be beneficial to the TT phenotype and further increases its transduction efficiency in the brain. Furthermore, we observed that the reverted S593A and D546G / G548E did not appear to affect the AAV-TT phenotype in neuronal cells. Therefore, we hypothesized that the TT-specific residues: S593, D546 and G548 could be eliminated from the authentic capsid sequence, leaving instead the AAV2WT residues at these positions, resulting in a final TT vector with only 10 amino acid changes.

[0208] To confirm these hypotheses, we engineered the TT-S312N-D546G-G548D-S593A vector and tested its transduction efficiency by injection into neonatal mouse brains. Since the last neonatal intracranial injection seemed to saturate the detection signal of GFP expression, we decided to also inject the TT and TT-S312N vectors in parallel with this "pre-final" TT, using a dose 1 / 10 of the dose used previously. By using this lower dose, we aimed to avoid saturating the GFP staining level in the transduced brains and the difficulty of comparing transduction efficiencies between different mutants.

[0209] 5 × 10 of each mutant vector 09 vg was injected into the lateral ventricle of P1 neonates. Four weeks after injection, the animals were sacrificed and the brains were dissected, sectioned, and stained using anti-GFP antibody. The results are shown in Figure 35.

[0210] As previously observed, these data also suggest that AAV TT-S312N exhibits enhanced transduction of mouse brain tissue compared to the full AAV2TT vector. On the other hand, the minimal TT-S312N-D546G / G548E-S593A vector, despite also containing the S312N internal mutation, did not appear to reach these higher levels of transduction. This suggests that one or more of the amino acid changes play a role in the TT phenotype. Reverting these residues back to their AAV2WT equivalents lost some of the increased transduction capacity. Higher magnification of stained sections confirmed these observations (Figure 36).

[0211] We decided to further explore the transduction efficiency obtained with each of these vectors by quantifying the total amount of GFP expression obtained in the injected brains by enzyme-linked immunosorbent assay (ELISA) on protein extracts from whole brains. 09Four weeks after injection of the vg vector, the animals were sacrificed and the whole brains were collected, lysed, and total brain protein was extracted. Using GFP protein standards, we were then able to quantify the amount of GFP protein expressed in each injected brain (Figure 37).

[0212] It could be confirmed that the TT-S312N internal mutant transduced mouse brains more efficiently than the full TT vector; since TT-S312N resulted in a higher overall GFP expression in all injected brains. On the other hand, the minimal TT vector TT-S312N-D546G / G548E-S593A appeared to result in a lower level of transduction: on this graph, the average GFP expression per brain appears to be higher, but this was due to the extreme values ​​of GFP measured in one brain; this is indicated by the high error bar for this condition. With this minimal TT vector, the variability between animals was very high, with only one out of five animals performing better than animals transduced with TT-S312N. This high variability made us cautious in considering this provisional minimal TT vector; especially since immunohistochemical analysis also showed that the TT-S312N variant performed better than TT-S312N-DG-S593A.

[0213] Therefore, the TT-S312N variant was selected as the most preferred AAV TT vector, which consists of the following 13 amino acid mutations compared to wild-type AAV2: V125I, V151A, A162S, T205S, Q457M, S492A, E499D, F533Y, G546D, E548G, R585S, R588T and A593S.

[0214] Although the above studies suggest that TT-S312N is the most preferred AAV-TT vector, these studies also indicate that several TT-specific residues individually have and contribute functions. In particular, four amino acids in close position on the three-fold spike of the capsid are likely to be involved in receptor binding. In some embodiments, these residues are reverted in AAV-TT back to the corresponding residues in AAV2WT. For example, mutations M457Q, A492S, D499E and Y533F were engineered on the AAV-TT capsid and analyzed in this study to demonstrate the role of these residues. The mutated vector can be analyzed as described above. In a further embodiment, four amino acids present in the primary sequence of VP1 / VP2 that are likely involved in viral transport can be reverted in AAV-TT back to the corresponding residues in AAV2WT (I125V, A151V, S162A, S205T) and analyzed similarly.

[0215] Example 4 Construction of variant AAV vectors in other serotypes The function of the ttAAV2-specific amino acid changes in the context of other serotypes other than AAV2 can also be determined.

[0216] The amino acid sequences of the capsids of the major adeno-associated viruses (AAV), i.e., AAV1, AAV5, AAV6, AAV8, AAV9 and AAVrhlO, can be aligned with the sequence derived from ttAAV2, for example as shown in Figure 9. This allows identification of which ttAAV2-specific amino acids are already present at the same positions in other serotypes. The relevant residues in the various serotypes are then mutated to the corresponding residues in wtAAV2. These changes indicate the importance of the ttAAV2-specific residues for the efficiency and biodistribution of each serotype.

[0217] As discussed above, the various mutated vectors expressing GFP are then subjected to a first screening by intracranial (IC) injection into CD1 neonatal mice. The GFP signal obtained in the injected brain is then compared with that obtained from the appropriate GFP-expressing serotype control. The reduction or increase in GFP expression when the identified amino acids are mutated to their corresponding AAV2 residues indicates the importance of these specific residues at these specific positions. If appropriate, the injected brain is then further analyzed by sectioning and immunohistochemistry. In addition, selected mutant serotypes are also analyzed by intravenous (IV) injection into neonatal mice to evaluate the biodistribution of the vector and compare it with its original unmutated counterpart.

[0218] In further embodiments, the relevant amino acids identified in ttAAV2 are inserted as key mutations into the other major serotypes at the relevant positions. By inserting ttAAV2 specific residues into other AAV subtypes, it becomes possible to improve the transduction and biodistribution profile of each serotype.

[0219] Example 5 Alteration of ttAAV2-specific residues that are conserved in other AAV serotypes to the corresponding AAV2 residues Initially, comparative analysis of the amino acid sequences of the capsids of extant adeno-associated serotypes with sequences derived from ttAAV2 allowed the identification of ttAAV2-specific residues that are conserved in other serotypes (see FIG. 9). These residues consist of S162, S205, S312, G548, S585 and T588. Each serotype other than AAV2 contains one or a combination of these residues at the corresponding amino acid positions in its sequence.

[0220] In a specific embodiment, each of these residues in the various serotypes is converted to the corresponding wild-type AAV2 amino acid, and the transduction efficiency of these new mutants is tested.

[0221] 1) Modification of AAV1 serotype AAV1 contains residues: S205, G549, S586 and T589, which correspond to the following residues in ttAAV2: S205, G548, S585 and T588. A VP1 monomer from AAV1 is aligned in three dimensions with VP1 from AAV2. By performing the 3D structure analysis, it was possible to confirm that the corresponding residues in wtAAV2, i.e., T205, E548, R585 and R588, are located at the exact same positions in the 3D structure (Figure 21). Therefore, we concluded that it is significant that the conversion of each of the ttAAV2-specific residues in AAV1 to their corresponding wild-type AAV2 counterparts does not affect the three-dimensional structure of the protein. In a specific embodiment, the following mutations are made in the capsid sequence of AAV1: S205T, G549E, S586R, T589R.

[0222] 2) Modification of AAV5 serotype AAV5 contains residues G537, S575 and T578, which correspond to the following residues in ttAAV2: G548, S585 and T588. Residues R585 and R588 in AAV2 are in the same position as S575 and T578 in AAV5 on the 3D structure. According to the three-dimensional structure, residue E548 in AAV2 does not completely match residue G537 in AAV5 (Figure 22), but we still decided to include it in the study because both residues are relatively close in space. Therefore, in a specific embodiment, the following mutations are made in the capsid sequence of AAV5: G537E, S575R, T578R.

[0223] 3) AAV6 serotype modification AAV6 contains residues: S205, G549, S586, and T589, which correspond to the following residues in ttAAV2: S205, G548, S585, and T588. The corresponding residues in wtAAV2, i.e., T205, E548, R585, and R588, are in perfectly matching positions on the 3D structure of VP1 (Figure 23). Thus, in certain embodiments, the following mutations are made in the capsid sequence of AAV6: S205T, G549E, S586R, T589R.

[0224] 4) AAV8 serotype modification AAV8 contains residues: S315 and T591, which correspond in ttAAV2 to the following residues: S312 and T588. The corresponding residues in wtAAV2, i.e., N312 and R588, are at perfectly matching positions on the 3D structure of VP1 (Figure 24). Thus, in certain embodiments, the following mutations are made in the capsid sequence of AAV8: S315N, T591R.

[0225] In one embodiment, the improved transduction efficiency conferred by the S312N mutation in TT AAV2 can be transferred to the AAV8 serotype by applying the amino acid change S315N.

[0226] The capsid sequence of AAV8 was mutated by site-directed mutagenesis, thereby generating AAV8-S315N vector plasmid.This plasmid was used to generate recombinant AAV8-S315N vector expressing ITR-containing CMV-GFP transgene by double transfection of 293T cells.The vector was then purified from cell lysate and harvested culture supernatant by FPLC affinity chromatography using AVB Sepharose resin.The titer of capsid and the titer of vector genome were evaluated by SDS-PAGE and qPCR, respectively.

[0227] The mutant AAV8-S315N vectors expressing GFP are screened by systemic injection into CD1 neonatal mice. Titer-matched AAV8 vectors are used as controls. Then, 2×10 11 The expression of GFP obtained in different organs after intrajugular injection of vg is analyzed, focusing mainly on the liver, where AAV8 has already shown a rather strong transduction efficiency.

[0228] 5) AAV9 serotype modification AAV9 contains residues: S162, S205, G549 and S586, which correspond to the following residues in ttAAV2: S162, S205, G548 and S585. The corresponding residues in AAV2, i.e., A162, T205, E548 and R585, are in perfectly matched positions on the 3D structure of VP1 (Figure 25). Thus, in certain embodiments, the following mutations are made in the capsid sequence of AAV8: S162A, S205T, G549E and S586R.

[0229] 6) Modification of AAVrh10 serotype AAVrhlO contains residue G551, which corresponds to G548 in ttAAV2. Given that this residue and position appears to be conserved among various serotypes, it is speculated that G551 in AAVrhlO aligns three-dimensionally with E548 in wtAAV2. Thus, in one embodiment, the following mutation is made in the capsid sequence of AAVrhlO: G551E.

[0230] 7) Modification of AAV3B and AAV-LK03 serotypes Similar to the AAV8 serotype, the AAV3B serotype was also observed to contain an internal serine at position 312 when the sequence of the capsid protein VP1 was aligned with the sequence derived from AAV-TT (see the AAV3B capsid sequence in Figure 38).

[0231] The newly described LK03 AAV vector is a chimeric capsid composed of five different parental AAV capsids engineered by DNA shuffling by MAKay, which also contains residue S312 inside the capsid (Lisowski et al., Selection and evaluation of clinically relevant AAV variants in a xenograft). (See, Nature, 506, 382-386 (2014)). The capsid sequence of AAV-LK03 is disclosed in WO2013 / 029030 and is shown in FIG. 39.

[0232] Therefore, in a further embodiment, AAV3B vector and AAV-LK03 vector are mutated by applying amino acid change S312N in both vectors.These new mutants and their corresponding AAV control serotypes are also tested by intrajugular injection into newborn mice.Then, the expression of GFP obtained in various harvested organs is analyzed.

[0233] Example 6 Identification of ttAAV2-specific amino acids that are transferable between AAV serotypes. In a further embodiment, the key amino acids identified during the characterization of ttAAV2 are inserted into other major serotypes at relevant positions.Then, the newly engineered vectors are tested using unmutated appropriate serotypes as controls.This allows the validation of the importance of each amino acid at specific positions on AAV capsid, independent of serotype.

[0234] 1) Residues: S585, T588, S312, D546, G548 and S593 AAV1, AAV5 and AAV6 naturally contain the same amino acid residues in their capsid protein sequences at positions corresponding to G548, S585 and T588 in ttAAV2. Thus, in further embodiments, the capsid proteins in these serotypes are mutated at the corresponding positions to include the other residues present in ttAAV2: S312, D546 and S593. Similarly, AAV8 already contains the same amino acid residues as in ttAAV2, and these positions are not present in ttAAV2. AAV8 is also further mutated to contain residues corresponding to S585, D546, G548 and S593 in ttAAV2. AAV9 already contains residues corresponding to G548 and S585 in ttAAV2; AAV9 is mutated to include residues corresponding to T588, S312, D546 and S593 in ttAAV2. Finally, AAV10 already contains residues corresponding to G548; AAV10 is also further modified to contain residues corresponding to S585, T588, S312, G548 and S593.

[0235] 2) Residues: I125, A151, S162, S205, M457, A492, D499 and Y533 AAV1 and AAV6 naturally contain residue S205.Therefore, in further embodiments, these serotypes are mutated at positions corresponding to residues in ttAAV2: I125, A151, S162, M457, A492, D499 and Y533.Similarly, AAV9 already contains residues: S162 and S205; AAV9 is also further mutated to contain residues corresponding to residues in ttAAV2: I125, A151, M457, A492, D499 and Y533.Finally, AAV5, AAV8 and AAV10 are modified to have residues corresponding to residues in ttAAV2: I125, A151, S162, S205, M457, A492, D499 and Y533.

[0236] The positions of the mutations present in ttAAV2 and the corresponding residues present in the sequence of the wild-type capsid protein VP1 of other AAV serotypes are shown in Table 1 below. In general, vectors of variants other than AAV2 can be constructed by mutating any of the residues shown in Table 1 for these serotypes. The residues shown in italics are those that are already present in ttAAV2 at the corresponding positions. In a preferred embodiment, serotypes other than AAV2 are mutated at one or more residues shown in non-italic type. In this way, the advantageous properties exhibited by ttAAV2 can be transferred into alternative AAV serotypes.

[0237] [Table 1]

[0238] Further embodiments of the present invention The present invention also relates to further aspects defined in the sections summarized below. 1. A recombinant adeno-associated virus (AAV) vector comprising: (a) a variant AAV capsid protein comprising at least one amino acid substitution, relative to a wild-type AAV capsid protein, at a position corresponding to one or more of positions 125, 151, 162, 205, 312, 457, 492, 499, 533, 546, 548, 585, 588, and / or 593 in an AAV2 capsid protein sequence; and (b) a heterologous nucleic acid comprising a nucleotide sequence encoding a gene product; A recombinant AAV vector comprising: 2. The recombinant AAV vector of paragraph 1, wherein (i) the variant AAV2 capsid protein comprises a variant AAV2 capsid protein, (ii) the variant AAV capsid protein comprises a sequence of SEQ ID NO:2 or a sequence having at least 95% sequence identity thereto, (iii) the wild-type AAV capsid protein is derived from AAV2, and / or (iv) the wild-type AAV capsid protein comprises the sequence of SEQ ID NO:1. 3. The variant AAV2 capsid protein has one or more of the following residues: I125, A151, S162, S205, S312, M457, A492, D499, Y53 3, the recombinant AAV vector of paragraph 2, comprising D546, G548, S585, T588 and / or S593. 4. The recombinant AAV vector of paragraph 2 or 3, wherein the variant AAV2 capsid protein comprises one or more of the following amino acid substitutions compared to a wild-type AAV2 capsid protein: V125I, V151A, A162S, T205S, N312S, Q457M, S492A, E499D, F533Y, G546D, E548G, R585S, R588T and / or A593S. 5. The recombinant AAV vector of any of paragraphs 1 to 3, wherein the variant AAV capsid protein is derived from AAV1, AAV5, AAV6, AAV8, AAV9 or AAV10. 6. (i) comprises a variant AAV1 capsid protein; (ii) the variant AAV capsid protein comprises a sequence having at least 95% sequence identity to SEQ ID NO:3; (iii) the wild-type AAV capsid protein is derived from AAV1; and / or (iv) the wild-type AAV capsid protein comprises the sequence of SEQ ID NO:3; 6. The recombinant AAV vector of paragraph 5, wherein at least one amino acid substitution is present at one or more of positions 125, 151, 162, 205, 313, 458, 493, 500, 534, 547, 549, 586, 589 and / or 594 in the AAV1 capsid protein sequence. 7. The variant AAV1 capsid protein has one or more of the following amino acid substitutions compared to the wild-type AAV1 capsid protein: (a) V125I, Q151A, T162S, N313S, N458M, K493A, N500D, F534Y, S547D and / or G594S, and / or (b) S205T, G549E, S586R, and / or T589R 7. The recombinant AAV vector of claim 6, comprising: 8. (i) comprises a variant AAV5 capsid protein; (ii) the variant AAV capsid protein comprises a sequence having at least 95% sequence identity to SEQ ID NO:4; (iii) the wild-type AAV capsid protein is derived from AAV5; and / or (iv) the wild-type AAV capsid protein comprises the sequence of SEQ ID NO:4; 6. The recombinant AAV vector of paragraph 5, wherein at least one amino acid substitution is present at one or more of positions 124, 150, 153, 195, 303, 444, 479, 486, 520, 533, 537, 575, 578 and / or 583 in the AAV5 capsid protein sequence. 9. The variant AAV5 capsid protein has one or more of the following amino acid substitutions compared to the wild-type AAV5 capsid protein: (a) V124I, K150A, K153S, A195S, R303S, T444M, S479A, V486D, T520Y, P533D and / or G583S, and / or (b) G537E, S575R, and / or T578R 9. The recombinant AAV vector of claim 8, comprising: 10. (i) comprises a variant AAV6 capsid protein; (ii) the variant AAV capsid protein comprises a sequence having at least 95% sequence identity to SEQ ID NO:5; (iii) the wild-type AAV capsid protein is derived from AAV6; and / or (iv) the wild-type AAV capsid protein comprises the sequence of SEQ ID NO:5; The recombinant AAV vector of paragraph 5, wherein at least one amino acid substitution is present at one or more of positions 125, 151, 162, 205, 313, 458, 493, 500, 534, 547, 549, 586, 589 and / or 594 in the AAV6 capsid protein sequence. 11. The variant AAV6 capsid protein has one or more of the following amino acid substitutions compared to the wild-type AAV6 capsid protein: (a)V125I, Q151A, T162S, N313S, N458M, K493A, N500D, F534Y, S547D and / or G594S, and / or (b) S205T, G549E, S586R, and / or T589R The recombinant AAV vector of claim 10, comprising: 12. (i) comprises a variant AAV8 capsid protein; (ii) the variant AAV capsid protein comprises a sequence having at least 95% sequence identity to SEQ ID NO:6; (iii) the wild-type AAV capsid protein is derived from AAV8; and / or (iv) the wild-type AAV capsid protein comprises the sequence of SEQ ID NO:6; 6. The recombinant AAV vector of paragraph 5, wherein at least one amino acid substitution is present at one or more of positions 125, 151, 163, 206, 315, 460, 495, 502, 536, 549, 551, 588, 591 and / or 596 in the AAV8 capsid protein sequence. 13. The variant AAV8 capsid protein has one or more of the following amino acid substitutions compared to the wild-type AAV8 capsid protein: (a) V125I, Q151A, K163S, A206S, T460M, T495A, N502D, F536Y, N549D, A551G, Q588S and / or G596S, and / or (b) S315N and / or T591R 13. The recombinant AAV vector of claim 12, comprising: 14. (i) comprises a variant AAV9 capsid protein; (ii) the variant AAV capsid protein comprises a sequence having at least 95% sequence identity to SEQ ID NO:7; (iii) the wild-type AAV capsid protein is derived from AAV9; and / or (iv) the wild-type AAV capsid protein comprises the sequence of SEQ ID NO:7; 6. The recombinant AAV vector of paragraph 5, wherein at least one amino acid substitution is present at one or more of positions 125, 151, 162, 205, 314, 458, 493, 500, 534, 547, 549, 586, 589 and / or 594 in the AAV9 capsid protein sequence. 15. The variant AAV9 capsid protein has one or more of the following amino acid substitutions compared to the wild-type AAV9 capsid protein: (a) L125I, Q151A, N314S, Q458M, V493A, E500D, F534Y, G547D, A589T and / or G594S, and / or (b) S162A, S205T, G549E, and / or S586R 15. The recombinant AAV vector of claim 14, comprising: 16. (i) a variant AAVrhlO capsid protein; (ii) the variant AAV capsid protein comprises a sequence having at least 95% sequence identity to SEQ ID NO:8; (iii) the wild-type AAV capsid protein is derived from AAVrhlO; and / or (iv) the wild-type AAV capsid protein comprises the sequence of SEQ ID NO:8; The recombinant AAV vector of paragraph 5, wherein at least one amino acid substitution is present at one or more of positions 125, 151, 163, 206, 315, 460, 495, 502, 536, 549, 551, 588, 591 and / or 596 in the AAV10 capsid protein sequence. 17. The variant AAVrh10 capsid protein has one or more of the following amino acid substitutions compared to the wild-type AAVrh10 capsid protein: (a) V125I, Q151A, K163S, A206S, N315S, T460M, L495A, N502D, F536Y, G549D, Q588S, A591T and / or G596S, and / or (b) G551E 17. The recombinant AAV vector of claim 16, comprising: 18. The method of any of paragraphs 1 to 17, which exhibits increased transduction of neuronal or retinal tissue compared to an AAV vector containing a corresponding wild-type AAV capsid protein. Recombinant AAV vectors described. 19. The recombinant AAV vector of any of paragraphs 1 to 18, wherein the gene product comprises an interfering RNA or an aptamer. 20. The recombinant AAV vector of any of paragraphs 1 to 18, wherein the gene product comprises a polypeptide. 21. The recombinant AAV vector of paragraph 20, wherein the gene product comprises a neuroprotective polypeptide, an anti-angiogenic polypeptide, or a polypeptide that enhances the function of a neuronal or retinal cell. 22. The recombinant AAV vector of paragraph 21, wherein the gene product comprises glial-derived neurotrophic factor, fibroblast growth factor, nerve growth factor, brain-derived neurotrophic factor, rhodopsin, retinoschisin, RPE65, or peripherin. 23. (a) a recombinant AAV vector according to any one of paragraphs 1 to 22, and (b) Pharmaceutically acceptable excipients 23. A pharmaceutical composition comprising: 24. A method for delivering a gene product to neuronal or retinal tissue in a subject, comprising administering to the subject a recombinant AAV vector or pharmaceutical composition described in any of paragraphs 1 to 23. 25. A method for treating a neurological or eye disorder, comprising administering to a subject a recombinant AAV vector or pharmaceutical composition described in any of paragraphs 1 to 24. 26. A recombinant AAV vector or pharmaceutical composition according to any of paragraphs 1 to 23 for use in treating a neurological or eye disorder. 27. The method, recombinant AAV vector or pharmaceutical composition for use according to any of paragraphs 24 to 26, wherein the neurological disorder is a neurodegenerative disease. 28. The method, recombinant AAV vector or pharmaceutical composition for use according to any of paragraphs 24 to 26, wherein the eye disorder is glaucoma, retinitis pigmentosa, macular degeneration, retinoschisis or diabetic retinopathy. 29. An isolated variant AAV capsid protein, comprising at least one amino acid substitution relative to a wild-type AAV capsid protein at one or more of positions 125, 151, 162, 205, 312, 457, 492, 499, 533, 546, 548, 585, 588 and / or 593 in an AAV2 capsid protein sequence, or at one or more corresponding positions in an alternative AAV capsid protein sequence. 30. An isolated nucleic acid comprising a nucleotide sequence encoding a variant AAV capsid protein as defined in paragraph 29. 31. An isolated host cell comprising a nucleic acid as defined in paragraph 30. [Sequence List Free Text]

[0239] SEQ ID NO:1 is the amino acid sequence of wild-type adeno-associated virus 2 capsid protein VP1 (see FIG. 1). SEQ ID NO:2 is the amino acid sequence of the true adeno-associated virus 2 (ttAAV2) capsid protein (see FIG. 2). SEQ ID NO:3 is the amino acid sequence of wild-type adeno-associated virus 1 capsid protein VP1 (see FIG. 3). SEQ ID NO:4 is the amino acid sequence of wild-type adeno-associated virus 5 capsid protein VP1 (see FIG. 4). SEQ ID NO:5 is the amino acid sequence of wild-type adeno-associated virus 6 capsid protein VP1 (see FIG. 5). SEQ ID NO:6 is the amino acid sequence of wild-type adeno-associated virus 8 capsid protein VP1 (see FIG. 6). SEQ ID NO:7 is the amino acid sequence of wild-type adeno-associated virus 9 capsid protein VP1 (see FIG. 7). SEQ ID NO:8 is the amino acid sequence of wild-type adeno-associated virus 10Upenn capsid protein VP1 (see FIG. 8). SEQ ID NO:9 is the amino acid sequence of wild-type adeno-associated virus 10 Japanese capsid protein VP1 (see FIG. 9). SEQ ID NO:10 is the consensus amino acid sequence for the adeno-associated virus shown in FIG. SEQ ID NO:11 is the amino acid sequence of wild-type adeno-associated virus 3B capsid protein VP1 (see Figure 38). SEQ ID NO:12 is the amino acid sequence of the adeno-associated virus LK-03 capsid protein VP1 (see Figure 39).

Claims

1. 1. A recombinant adeno-associated virus (AAV) vector comprising: (a) a variant AAV3B capsid protein comprising an amino acid substitution at position 312 in the AAV3B capsid protein sequence compared to a wild-type AAV3B capsid protein comprising the sequence of SEQ ID NO:11; and (b) a heterologous nucleic acid comprising a nucleotide sequence encoding a gene product. Including, the amino acid substitution is S312N, the variant AAV3B capsid protein comprises a sequence having at least 90% sequence identity to SEQ ID NO:11, The recombinant AAV vector exhibits increased tissue transduction as compared to a corresponding AAV vector comprising the wild-type AAV3B capsid protein.

2. 2. The recombinant AAV vector of claim 1, wherein the variant AAV3B capsid protein comprises a sequence having at least 95% sequence identity to SEQ ID NO:

11.

3. 1. A recombinant adeno-associated virus (AAV) vector comprising: (a) a variant AAV-LK03 capsid protein comprising an amino acid substitution at position 312 compared to the wild-type AAV-LK03 capsid protein sequence as defined in SEQ ID NO: 12; and (b) a heterologous nucleic acid comprising a nucleotide sequence encoding a gene product. Including, the amino acid substitution is S312N, the variant AAV-LK03 capsid protein comprises a sequence having at least 90% sequence identity to SEQ ID NO: 12; The recombinant AAV vector exhibits increased tissue transduction as compared to a corresponding AAV vector comprising the wild-type AAV-LK03 capsid protein.

4. 4. The recombinant AAV vector of claim 3, wherein the variant AAV-LK03 capsid protein comprises a sequence having at least 95% sequence identity to SEQ ID NO:

12.

5. (a) a recombinant AAV vector according to any one of claims 1 to 4, and (b) a pharma- ceutically acceptable excipient 23. A pharmaceutical composition comprising:

6. A recombinant AAV vector or a pharmaceutical composition according to any one of claims 1 to 5 for use in the treatment of a disorder in a subject.

7. 7. The recombinant AAV vector or pharmaceutical composition for use according to claim 6, wherein the disorder is a neurological disorder, an eye disorder or a liver disorder, optionally wherein the neurological disorder is a neurodegenerative disease and / or the eye disorder is glaucoma, retinitis pigmentosa, macular degeneration, retinoschisis or diabetic retinopathy.

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