RU2846967C2 - Adeno-associated virus vector - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: described is a recombinant adeno-associated virus (AAV) vector for delivering a gene product into a subject's tissue, comprising: (a) a variant AAV8 capsid protein, wherein the variant AAV8 capsid protein comprises an amino acid substitution compared to the non-mutant type AAV8 capsid protein at position 315 in the AAV8 capsid protein sequence, where the non-mutant type AAV8 capsid protein contains the sequence SEQ ID NO: 6; and (b) a heterologous nucleic acid containing a nucleotide sequence coding a gene product. Also disclosed is a recombinant adeno-associated virus (AAV) vector for delivering a gene product into a subject's tissue, comprising: (a) a variant AAV3B capsid protein, wherein the variant AAV3B capsid protein comprises an amino acid substitution compared to the non-mutant type AAV3B capsid protein at position 312 in the AAV3B capsid protein sequence, wherein the non-mutant type AAV3B capsid protein comprises the sequence SEQ ID NO: 11; and (b) a heterologous nucleic acid containing a nucleotide sequence coding a gene product. Also disclosed is a recombinant adeno-associated virus (AAV) vector for delivering a gene product into a subject's tissue, comprising: (a) a variant AAV-LK03 capsid protein, wherein the variant AAV-LK03 capsid protein comprises 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 containing a nucleotide sequence coding a gene product.

EFFECT: invention extends the range of means for delivering a gene product into a subject’s tissue.

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Description

FIELD OF INVENTION

The present invention relates to the field of recombinant viral vectors. In particular, the present invention concerns recombinant viral vectors that are suitable for the delivery of therapeutic genes in vivo .

PREREQUISITES FOR THE CREATION OF THE INVENTION

To date, the adeno-associated virus remains one of the most promising vectors for delivering therapeutic genes. A significant number of preclinical and clinical studies have firmly established that this approach is suitable for developing gene-based drugs with the potential to achieve market approval.

Since the early development of AAV2 as a gene therapy vector in the 1980s, significant progress has been made in optimizing this platform for various applications and target tissues. Perhaps the most significant of these developments is the discovery of a wide range of serotypes, of which ten of the twelve are currently under extensive investigation. Key characteristics of these different serotypes include their respective tissue tropisms and, in some cases, the ability to retrogradely transport neurons. Of these serotypes, AAV1-10 is widely used for preclinical and clinical purposes.

A newer platform has been developed that incorporates processes that allow for targeting and non-targeting of specific tissues and cell subtypes in patients. The core technology of these approaches relies on trial-and-error evaluation of existing AAV variants (serotypes) and in vivo selection of randomly introduced AAV capsid mutants. Together, these two promising approaches provide dozens, if not hundreds, of potential vectors with diverse transduction behaviors.

The most intriguing aspect of AAV serotypes is their ability to efficiently transduce specific tissues in animal models and humans. To date, a comprehensive molecular understanding of the underlying mechanisms of tissue tropism has not been developed, and thus, it is generally assumed that commonly available tissue-specific receptors for each serotype play a central role in the efficient transduction of different serotypes.

Accordingly, there remains a need for additional AAV vectors with improved properties in terms of in vivo transgene expression and tissue specificity. Specifically, such vectors have the efficiency to provide significantly enhanced gene delivery to various target tissues in the human body.

SUMMARY OF THE INVENTION

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

In one 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 non-mutant AAV capsid protein comprises the sequence of SEQ ID NO: 1.

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 compared to the wild-type AAV2 capsid protein: Q457M, S492A, E499D, and F533Y.

In one embodiment, the variant AAV2 capsid protein further comprises one or more amino acid substitutions compared to the wild-type AAV capsid protein at the following positions in the AAV2 capsid protein sequence: 125, 151, 162, and 205. In a preferred embodiment, the variant AAV2 capsid protein comprises one or more of 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 compared to the wild-type AAV2 capsid protein: V125I, V151A, A162S, and T205S.

In one embodiment, the variant AAV2 capsid protein further comprises one or more amino acid substitutions compared to the wild-type AAV capsid protein at the following positions in the AAV2 capsid protein sequence: 585 and 588. Preferably, the variant AAV2 capsid protein comprises one or more of 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 compared to the wild-type AAV2 capsid protein: R585S and R588T.

In one embodiment, the variant AAV2 capsid protein further comprises one or more amino acid substitutions compared to the wild-type AAV capsid protein at the following positions in the AAV2 capsid protein sequence: 546, 548, and 593. Preferably, the variant AAV2 capsid protein comprises one or more of 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 compared to the wild-type AAV2 capsid protein: G546D, E548G, and A593S.

In one embodiment, the variant AAV2 capsid protein comprises residue N312, i.e., the residue that is present in the non-mutated type of AAV2 capsid protein at position 312. In this embodiment, the variant AAV2 capsid protein is non-mutated at position 312 compared to the non-mutated type of AAV2 capsid protein sequence.

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

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.

In one embodiment, the variant AAV8 capsid protein comprises an amino acid substitution S315N compared to the wild-type AAV8 capsid protein. Preferably, the AAV8 capsid protein sequence comprises one or more amino acid substitutions present at one or more of the following positions: 125, 151, 163, 206, 460, 495, 502, 536, 549, 551, 588, 591 and/or 596.

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: (a) V125I, Q151A, K163S, A206S, T460M, T495A, N502D, F536Y, N549D, A551G, Q588S and/or G596S; and/or (b) T591R.

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

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.

In one embodiment, the variant AAV3B capsid protein comprises an S312N amino acid substitution compared to a non-mutant type AAV3B capsid protein.

In another aspect, the present invention provides a recombinant adeno-associated virus (AAV) vector comprising (a) a variant AAV-LK03 capsid protein, wherein the variant AAV-LK03 capsid protein comprises 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 the gene product.

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

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

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

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 from AAV2. In another embodiment, the wild-type AAV capsid protein comprises the sequence of SEQ ID NO: 1.

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.

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

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 from AAV1. In another embodiment, the wild-type AAV capsid protein comprises the sequence of SEQ ID NO:3.

In one embodiment, at least one amino acid substitution is present at one or more of the following positions in the AAV1 capsid protein sequence: 125, 151, 162, 205, 313, 458, 493, 500, 534, 547, 549, 586, 589 and/or 594. 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.

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 from AAV5. In another embodiment, the wild-type AAV capsid protein comprises the sequence of SEQ ID NO:4.

In one embodiment, at least one amino acid substitution is present at one or more of the following positions in the AAV5 capsid protein sequence: 124, 150, 153, 195, 303, 444, 479, 486, 520, 533, 537, 575, 578 and/or 583. 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.

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 from AAV6. In another embodiment, the wild-type AAV capsid protein comprises the sequence of SEQ ID NO:5.

In one embodiment, at least one amino acid substitution is present 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. 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.

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 from AAV8. In another embodiment, the wild-type AAV capsid protein comprises the sequence of SEQ ID NO:6.

In one embodiment, at least one amino acid substitution is present at one or more of the following positions in the AAV8 capsid protein sequence: 125, 151, 163, 206, 315, 460, 495, 502, 536, 549, 551, 588, 591 and/or 596. 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.

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 from AAV9. In another embodiment, the wild-type AAV capsid protein comprises the sequence of SEQ ID NO:7.

In one embodiment, at least one amino acid substitution is present at one or more of the following positions in the AAV9 capsid protein sequence: 125, 151, 162, 205, 314, 458, 493, 500, 534, 547, 549, 586, 589 and/or 594. 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.

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 from AAV10. In another embodiment, the wild-type AAV capsid protein comprises the sequence of SEQ ID NO: 8.

In one embodiment, at least one amino acid substitution is present 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. 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.

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

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

In one embodiment, the gene product comprises interfering RNA or an aptamer. In another embodiment, the gene product comprises a polypeptide. Preferably, the gene product comprises a neuroprotective polypeptide, an antiangiogenic polypeptide, or a polypeptide that improves neuronal or retinal cell function. In preferred embodiments, the gene product comprises glial cell-derived neurotrophic factor, fibroblast growth factor, nerve growth factor, brain-derived neurotrophic factor, rhodopsin, retinoschisin, RPE65, or peripherin.

In another aspect, the present invention provides a pharmaceutical composition comprising: (a) a recombinant AAV vector as defined above; and (b) a pharmaceutically acceptable excipient.

In another aspect, the present invention provides a method of delivering a gene product to tissue in a subject, wherein the method comprises administering to the subject a recombinant AAV vector or pharmaceutical composition as defined above.

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, retinal, or liver tissue.

In another aspect, the present invention provides a method for treating a disorder in a subject, wherein the method comprises administering to the subject a recombinant AAV vector or pharmaceutical composition as defined above. In some embodiments, the disorder is a neurological disorder, an ocular disorder, or a liver disease.

In another aspect, the present invention provides a recombinant AAV vector or pharmaceutical composition, as defined above, for use in treating a disorder in a subject. In some embodiments, the disorder is a neurological, ocular, or liver disorder. Preferably, the neurological disorder is a neurodegenerative disease. In an alternative embodiment, the ocular disorder is glaucoma, retinal pigmentosa, macular degeneration, retinoschisis, or diabetic retinopathy.

In another aspect, the present invention provides an isolated variant AAV capsid protein, wherein the variant AAV capsid protein comprises at least one amino acid substitution compared to a wild-type AAV capsid protein; wherein the at least one amino acid substitution is present at one or more of the following positions in the AAV2 capsid protein sequence: 125, 151, 162, 205, 312, 457, 492, 499, 533, 546, 548, 585, 588 and/or 593; or at one or more of the corresponding positions in an alternative AAV capsid protein sequence.

In another aspect, the present invention provides an isolated nucleic acid comprising a nucleotide sequence that encodes a variant AAV capsid protein as defined above.

In another aspect, the present invention provides an isolated host cell comprising a nucleic acid as defined above.

BRIEF DESCRIPTION OF DRAWINGS

Figure 1 shows the amino acid sequence of the VP1 capsid protein of the wild-type adeno-associated virus 2 (SEQ ID NO:1; NCBI reference sequence: NC_001401). Residues V125, V151, A162, T205, N312, Q457, S492, E499, F533, G546, E548, R585, R588, and A593 are highlighted.

Figure 2 shows the amino acid sequence of the VP1 capsid protein of the true type AAV2 adeno-associated virus 2 (ttAAV2) (SEQ ID NO:2). Residues I125, A151, S162, S205, S312, M457, A492, D499, Y533, D546, G548, S585, T588, S593 differ compared to the wild-type AAV2 VP1 (SEQ ID NO:1) and are highlighted.

Figure 3 shows the amino acid sequence of the VP1 capsid protein of wild-type adeno-associated virus 1 (SEQ ID NO:3; NCBI reference sequence: NC_002077). Highlighted residues: S205 (matches S205 in ttAAV2 (SEQ ID NO:2)) – G549 (matches G548 in ttAAV2) – S586 (matches S585 in ttAAV2) – T589 (matches T588 in ttAAV2).

Figure 4 shows the amino acid sequence of the VP1 capsid protein of wild-type adeno-associated virus 5 (SEQ ID NO:4; NCBI reference sequence: AF085716). Highlighted residues: G537 (matches G548 in ttAAV2) – S575 (matches S585 in ttAAV2) – T578 (matches T588 in ttAAV2).

Figure 5 shows the amino acid sequence of the VP1 capsid protein of wild-type adeno-associated virus 6 (SEQ ID NO:5; NCBI reference sequence: AF028704). Highlighted residues: S205 (matches S205 in ttAAV2) – G549 (matches G548 in ttAAV2) – S586 (matches S585 in ttAAV2) – T589 (matches T588 in ttAAV2).

Figure 6 shows the amino acid sequence of the VP1 capsid protein of the wild-type adeno-associated virus 8 (SEQ ID NO:6; NCBI reference sequence: NC_006261). Highlighted residues: S315 (matches S312 in ttAAV2) – T591 (matches T588 in ttAAV2).

Figure 7 shows the amino acid sequence of the capsid protein VP1 of the wild-type adeno-associated virus 9 (SEQ ID NO:7; NCBI reference sequence: AY530579). Highlighted residues: S162 (matches S162 in ttAAV2) – S205 (matches S205 in ttAAV2) – G549 (matches G548 in ttAAV2) – S586 (matches S585 in ttAAV2).

Figure 8 shows the amino acid sequence of the VP1 capsid protein of the wild-type adeno-associated virus 10 (SEQ ID NO:8). Highlighted residue: G551 (matches G548 in ttAAV2).

Figure 9 shows a comparative analysis of the primary structure of the amino acid sequences of the AAV capsid protein VP1.

Figure 10: The plasmid used to generate the AAV2 vectors was the packaging plasmid pDG. Above: pDG with the wild-type AAV2 genes. Below: pDG-ttAAV2 with the true AAV2 genes, highlighting two key mutations in the heparan-binding domains at positions 585 and 588. MMTV: the promoter driving the expression of AAV rep, E2a, E4ORF6, and VA are genes expressing adenoviral helper factors.

Figure 11: Quantification of viral titers of true-type (TT) and wild-type (WT) rAAV2 in vivo by SDS-PAGE showing krypton staining for separated proteins and scanned using an infrared fluorescence scanner (Odyssey Imaging Systems). A: 10 μl of AAV2 viral particles and 62.5 ng – 500 ng of BSA were separated on a 12% separating gel containing SDS and stained with krypton dye for protein. The image was converted to grayscale. Capsid proteins of the VP1, VP2, VP3 gene are labeled on the left side. B: Table showing titers from qPCR (vector genome [vg/ml]) and SDS-Page (capsid titer [capsid/ml]).

Figure 12 A: Representative examples of rat brain regions stained with a GFP-specific antibody are shown. The vector was injected into the striatum, as indicated by the arrow. B: A representative example of an injection into the substantia nigra is shown.

Figure 13: GFP transduction of the eye using ttAAV2 and wtAAV2 is shown. A. The retina is shown in cross-section after administration of the ttAAV2 (top) and wtAAV2 (bottom) vectors. B. The concentrations of the dotted boxes in A are shown.

Figure 14: Mouse brain transduction after neonatal vector injection: i.v., intravenous vector administration; i.c., intracranial injection; AAV-2, wtAAV2; AAV-TT, ttAAV2.

Figure 15: Three-dimensional representation of the AAV2 capsid. Highlighted residues correspond to amino acid changes between ttAAV2 and wild-type particles, grouped by color based on their position.

Figure 16: Representation of the triple spike structure on the AAV2 capsid. The residues correspond to amino acid changes between the authentic and non-mutant particles. Heparin-binding site residues are highlighted in green.

Figure 17: Representation of the interior of the AAV2 capsid. The residues highlighted in light blue correspond to a single amino acid change in ttAAV2, which is located on the interior of the capsid.

Figure 18: Representation of the triple spike structure on the AAV2 capsid. The residues highlighted in beige correspond to two amino acid changes in the authentic-type vector that are spatially close and located in the groove between the two triply proximal peaks on the AAV capsid.

Figure 19: Representation of the triple spike on the AAV2 capsid. The residue highlighted in brown corresponds to one highlighted amino acid change (S593) in the authentic-type vector, which is located in the groove between the tripartite proximal peaks.

Figure 20: Representation of the triple spike on the AAV2 capsid. The four amino acids highlighted in pink are involved in receptor binding and are closely spaced in the triple spike.

Figure 21: Three-dimensional representation of the primary structure comparison between the VP1 capsid monomer from AAV2 (light blue) and the VP1 monomer from AAV1 (orange). The highlighted residues in the middle left of the image correspond to G549 in AAV1 (orange spheres) and E548 in AAV2 (green-blue sphere). The highlighted residues in the upper right corner of the image correspond to S586 and T589 in AAV1 (orange spheres) and R585 and R588 in AAV2 (green-blue sphere).

Figure 22: Three-dimensional representation of the primary structure comparison between the VP1 capsid monomer from AAV2 (light blue) and the VP1 monomer from AAV5 (purple). The highlighted residues in the middle of the image correspond to G537 in AAV5 (purple spheres) and E548 in AAV2 (green-blue sphere). The highlighted residues in the upper right corner of the image correspond to S575 and T578 in AAV5 (purple spheres) and R585 and R588 in AAV2 (green-blue sphere).

Figure 23: Three-dimensional representation of the primary structure comparison between the VP1 capsid monomer from AAV2 (light blue) and the VP1 monomer from AAV6 (yellow). The highlighted residues at the bottom of the image correspond to G549 in AAV6 (orange spheres) and E548 in AAV2 (green-blue sphere). The highlighted residues in the upper right corner of the image correspond to S586 and T589 in AAV6 (orange spheres) and R585 and R588 in AAV2 (green-blue sphere).

Figure 24: Three-dimensional representation of the primary structure comparison between the VP1 capsid monomer from AAV2 (light blue) and the VP1 monomer from AAV8 (pink). The highlighted residues in the upper left corner of the image correspond to S315 in AAV8 (red spheres) and N312 in AAV2 (green-blue sphere). The highlighted residues in the lower right corner of the image correspond to T591 in AAV8 (red spheres) and R588 in AAV2 (green-blue sphere).

Figure 25: Three-dimensional representation of the primary structure comparison between the VP1 capsid monomer from AAV2 (light blue) and the VP1 monomer from AAV9 (green). The highlighted residues in the middle of the image correspond to G549 in AAV9 (yellow spheres) and E548 in AAV2 (green-blue sphere). The highlighted residues in the lower left corner of the image correspond to S586 in AAV9 (yellow spheres) and R585 in AAV2 (green-blue sphere).

Figure 26: Expression analysis of rAAV2 TT and WT in the parafascicular nucleus following intrastriatal injection in rat brains. A: Representative images of rat brain sections are shown, showing the rostral side on the left and the caudal side on the right. The injection site in the striatum is indicated, and the projection area in the hypothalamus is observed in B and C (parafascicular nucleus, pf). B and C: High magnification image of GFP expression detected in the parafascicular nucleus (pf) following intrastriatal injection of rAAV2 WT (B) or TT (C).

Figure 27: Overview of intracranial injections of rAAV2 TT and WT in neonatal mice. Representative examples of neonatal brain sections stained with a GFP-specific antibody are shown. 5 × 10 ¹⁰ g of rAAV2 TT (top) or rAAV2 WT (middle) were injected into the lateral ventricle of neonatal mouse brains. Uninjected brains from neonatal mice, stained simultaneously, are shown as a negative control (NT, non-transduced).

Figure 28: High-magnification images of neonatal mouse brain sections after intracranial injections of rAAV2 TT or WT. Neonatal brain sections stained with a GFP-specific antibody are shown. 5×10 ¹⁰ g rAAV2 TT (left panels) or rAAV2 WT (right panels) were injected into the lateral ventricle of neonatal mouse brains. S1BF: brainstem area of the primary somatosensory cortex.

Figure 29: Overview of brain transduction following systemic injection of rAAV2 TT and WT in neonatal mice. Representative examples of neonatal brain sections stained with a GFP-specific antibody are shown. 2× ¹⁰¹¹ g of rAAV2 TT (top) or rAAV2 WT (bottom) were injected into the jugular veins of neonatal mice.

Figure 30: High-magnification images of neonatal mouse brain sections after systemic injections of rAAV2 TT or WT. Neonatal brain sections stained with a GFP-specific antibody are shown. 2× ¹⁰¹¹ vg rAAV2 TT (left panels) or rAAV2 WT (right panels) were injected into the jugular veins of neonatal mice. S1BF: brainstem area of the primary somatosensory cortex.

Figure 31: High-magnification images of neonatal mouse tissue sections after systemic injections of rAAV2 TT or WT. 2× ¹⁰¹¹ vg of rAAV2 TT or rAAV2 WT were injected into the jugular veins of neonatal mice. Uninjected mouse organs were used as negative controls.

Figure 32: High-magnification images of adult rat brain sections after intrastriatal injections of rAAV2 TT, WT, and HBnull. Representative examples of rat brain sections stained with a GFP-specific antibody are shown. 3.5× ¹⁰⁹ g of rAAV2 WT (left), TT (right), or AAV2-HBnull (middle) were injected into the striatum of adult rat brains, and representative images were obtained for the thalamus or substantia nigra (SN).

Figure 33: Overview of intracranial injections of whole AAV-TT versus different TT mutants in neonatal mice. Representative examples of neonatal brain sections stained with a GFP-specific antibody are shown. 5×10 ¹⁰ ug rAAV2 TT, TT-S312N, TT-S593A, or TT-D546G/G548E (TT-DG) were injected into the lateral ventricle of neonatal mouse brains. Uninjected brain from a neonatal mouse, stained simultaneously, is shown as a negative control (NT).

Figure 34: High-magnification images of neonatal mouse brain sections after intracranial injections of various TT mutant vectors. Neonatal brain sections stained with a GFP-specific antibody are shown. 5×10 ¹⁰ vg of vectors were injected into the lateral ventricle of neonatal mouse brains. TT-DG: TT-D546G/G548E.

Figure 35: Overview of intracranial injections of full-length AAV-TT compared with the TT-S312N mutant and a potential final TT vector containing 10 mutations in neonatal mice. Representative examples of neonatal brain sections stained with a GFP-specific antibody are shown. 5× ¹⁰⁹ vg of rAAV2 TT, TT-S312N, TT, or TT-S312N-D546G/G548E-S593A (TT-S312N-DG-S593A) were injected into the lateral ventricle of neonatal mouse brains. Uninjected brain from a neonatal mouse, stained simultaneously, is shown as a negative control (NT).

Figure 36: High-magnification images of neonatal mouse brain sections after intracranial injections of various TT mutant vectors. Neonatal brain sections stained with a GFP-specific antibody are shown. 5×10 ⁹ vg of vectors were injected into the lateral ventricle of neonatal mouse brains.

Figure 37: ELISA quantification of GFP protein in the brains of neonatal mice injected with full AAV-TT, TT-S312N mutant, or TT-S312N-DG-S593A. 5 × 10 ⁹ vg of vectors were injected into the lateral ventricle of neonatal mice brains, and total proteins were extracted from the whole harvested brains. A 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 represent the mean ± SEM.

Figure 38: Amino acid sequence of the AAV3B capsid protein VP1. Highlighted residues represent residues identical to those in AAV-tt at the corresponding positions. The internal serine residue at position 312 is underlined.

Figure 39: Amino acid sequence of VP1 capsid protein of AAV-LK03.

SEQUENCE LIST

SEQ ID NO:1 is the amino acid sequence of the capsid protein of the wild-type adeno-associated virus 2 VP1 (see Figure 1).

SEQ ID NO:2 is the amino acid sequence of the capsid protein of true type A adeno-associated virus 2 (ttAAV2) (see Figure 2).

SEQ ID NO:3 is the amino acid sequence of the capsid protein of the wild-type adeno-associated virus 1 VP1 (see Figure 3).

SEQ ID NO:4 is the amino acid sequence of the capsid protein of the wild-type adeno-associated virus 5 VP1 (see Figure 4).

SEQ ID NO:5 is the amino acid sequence of the capsid protein of the wild-type adeno-associated virus 6 VP1 (see Figure 5).

SEQ ID NO:6 is the amino acid sequence of the capsid protein of the wild-type adeno-associated virus 8 VP1 (see Figure 6).

SEQ ID NO:7 is the amino acid sequence of the capsid protein of the wild-type adeno-associated virus 9 VP1 (see Figure 7).

SEQ ID NO:8 is the amino acid sequence of the capsid protein Upenn of the wild-type adeno-associated virus 10 VP1 (see Figure 8).

SEQ ID NO:9 is the amino acid sequence of Japanese capsid protein of the wild-type adeno-associated virus 10 VP1 (see Figure 9).

SEQ ID NO:10 is the consensus amino acid sequence for the adeno-associated viruses shown in Figure 9.

SEQ ID NO:11 is the amino acid sequence of the capsid protein of the wild-type adeno-associated virus 3B VP1 (see Figure 38).

SEQ ID NO:12 is the amino acid sequence of the capsid protein of adeno-associated virus LK-03 VP1 (see Figure 39).

DETAILED DESCRIPTION OF THE INVENTION

In one aspect, the present invention concerns a recombinant adeno-associated virus (AAV) vector. The rAAV vector typically contains a variant capsid protein that differs from the capsid protein of the wild-type AAV. The variant capsid protein can advantageously confer increased infectivity to the vector in the brain and/or eye, making the vector particularly suitable for delivering therapeutic agents via gene therapy to these tissues.

Recombinant AAV vector

The present disclosure provides a 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 a derivative thereof. The term encompasses all subtypes and both naturally occurring and recombinant forms, except where otherwise specified. The abbreviation "rAAV" refers to a recombinant adeno-associated virus, also referred to as a 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 AAVs that infect primates, "non-primate AAV" refers to AAVs that infect non-primate mammals, "bovine AAV" refers to AAVs that infect mammals, cattle, and so on.

The genomic sequences of the various AAV serotypes, as well as the sequences of the native terminal repeats (TR), Rep proteins, and capsid subunits, are known in the art. Such sequences can be found in the literature or in publicly available databases such as GenBank. See, for example, GenBank accession numbers 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, e.g., 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. (1996) Virology 221:208; Shade et al. (1986) J. Virol. 58:921; Gao et al. (2002) Proc. Nat. Acad. Sci. USA 99: 11854; Moris et al. (2004) Virology 33:375-383; International Patent Publications WO 00/28061, WO 99/61601, WO 98/11244; and U.S. Patent No. 6,156,303.

An "rAAV vector," as used herein, refers to an AAV vector comprising a polynucleotide sequence of non-AAV origin (i.e., a polynucleotide heterologous to AAV), typically a sequence of interest for genetic transformation of a cell. In some embodiments, the heterologous polynucleotide may be flanked by at least one, and sometimes two, AAV inverted terminal repeat (ITR) sequences. The term rAAV vector encompasses both rAAV vector particles and rAAV vector plasmids. An rAAV vector may be either single-stranded (ssAAV) or self-complementary (scAAV).

An "AAV virus," or "AAV viral particle," or "rAAV vector particle" refers to a viral particle consisting of at least one AAV capsid protein (typically all capsid proteins of a wild-type AAV) and a capsid-enclosed rAAV vector polynucleotide. If the particle contains a heterologous polynucleotide (i.e., a polynucleotide not related to the wild-type AAV genome, such as a transgene for delivery to a mammalian cell), it is typically referred to as an "rAAV vector particle" or simply an "rAAV vector." Thus, production of an rAAV particle necessarily includes production of an rAAV vector, since such a vector is contained within the rAAV particle.

"Recombinant," as used herein, means that a vector, polynucleotide, polypeptide, or cell is the product of various combinations of cloning, restriction, or ligation steps (e.g., related to the polynucleotide or polypeptide contained therein), and/or other procedures that result in a construct that differs from the product found in nature. A recombinant virus or vector is a viral particle containing a recombinant polynucleotide. The terms respectively include replications of the original polynucleotide construct and progeny of the original viral construct.

Variant AAV Capsid Proteins

The rAAV vectors described herein contain a variant AAV capsid protein. By "variant" is meant that the AAV capsid protein differs from the corresponding wild-type AAV capsid protein of the same serotype. For example, a variant AAV capsid protein may 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, i.e., a variant AAV1 capsid protein contains one or more amino acid substitutions compared to the corresponding wild-type AAV1 capsid protein, a variant AAV2 capsid protein contains one or more amino acid substitutions compared to the corresponding wild-type AAV2 capsid protein, and so on.

The variant AAV capsid protein may comprise, for example, 1 to 50, 1 to 30, 1 to 20, or 1 to 15 amino acid substitutions compared to the wild-type AAV capsid protein. Preferably, the variant AAV capsid protein comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 amino acid substitutions compared to the 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.

In embodiments of the present invention, the variant AAV capsid protein comprises at least one amino acid substitution compared to the wild-type AAV capsid protein at a position corresponding to one or more of the following positions in the AAV2 capsid protein sequence: 125, 151, 162, 205, 312, 457, 492, 499, 533, 546, 548, 585, 588 and/or 593. As used herein, “corresponding to” 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-mentioned positions in the AAV2 capsid protein. In one embodiment, at least one amino acid substitution is present at one or more of the following positions in the AAV2 capsid protein sequence: 125, 151, 162, 205, 312, 457, 492, 499, 533, 546, 548, 585, 588 and/or 593; or at one or more corresponding positions in an alternative AAV capsid protein sequence.

In general, AAV capsid proteins include VP1, VP2, and VP3. In a preferred embodiment, the capsid protein comprises AAV capsid protein VP1.

NUCLEIC ACID SEQUENCES AND AMINO ACID SEQUENCES AND SEQUENCE IDENTITY

The term "polynucleotide" refers to a polymeric form of nucleotides of any length, including deoxyribonucleotides or ribonucleotides, or analogs thereof. A polynucleotide may contain modified nucleotides, such as methylated nucleotides and nucleotide analogs, and may be interrupted by non-nucleotide components. If present, modifications to the nucleotide structure may be provided before or after assembly of the polymer. The term "polynucleotide," as used herein, refers interchangeably to double-stranded and single-stranded molecules. Unless otherwise stated or required, any embodiment of the invention described herein that is a polynucleotide encompasses both the double-stranded form and each of the two complementary single-stranded forms known or predicted to form the double-stranded form.

The terms "polypeptide," "peptide," and "protein," as used interchangeably herein, refer to polymers of amino acids of any length. The terms also encompass an amino acid polymer that has been modified, for example, by disulfide bond formation, glycosylation, lipidation, phosphorylation, or conjugation with a labeling component. Polypeptides, such as antiangiogenic polypeptides, neuroprotective polypeptides, and the like, when considered in the context of delivering a gene product to a mammalian subject, and compositions thereof, refer to the corresponding intact polypeptide, or any fragment or derivative thereof obtained by genetic engineering methods that retains the desired biochemical properties of the intact protein. Similarly, references to nucleic acids encoding antiangiogenic polypeptides, nucleic acids encoding neuroprotective polypeptides, and other such nucleic acids for use in delivering a gene product to a mammalian subject (which may be referred to as "transgenes" that are delivered to a recipient cell) include polynucleotides encoding the intact polypeptide or any fragment or derivative produced by genetic engineering that has the desired biochemical function.

A polynucleotide or polypeptide has a certain percentage of "sequence identity" to another polynucleotide or polypeptide, meaning that when aligned, the percentage of bases or amino acids is the same as when the two sequences are compared. Sequence similarity can be determined in a number of different ways. To determine sequence identity, sequences can be aligned using methods and computer programs including BLAST, available online at ncbi.nlm.nih.gov/BLAST/. Another alignment algorithm is FASTA, available from the Genetics Computing Group (GCG), Madison, Wisconsin, USA, a wholly owned subsidiary of Oxford Molecular Group, Inc. Other alignment methods are described in Methods in Enzymology, vol. 266: Computer Methods for Macromolecular Sequence Analysis (1996), ed. Doolittle, Academic Press, Inc., a division of Harcourt Brace & Co., San Diego, California, USA. Of particular interest are alignment programs that allow gaps in sequence. The Smith-Waterman algorithm is one type of algorithm that allows gaps in sequence alignments. See Meth. Mol. Biol. 70: 173–187 (1997). Additionally, the GAP program using the Nildman-Wunsch alignment method can be used to align sequences. See J. Mol. Biol. 48: 443–453 (1970).

Of interest is the BestFit program, which uses the Smith-Waterman local homology algorithm (Advances in Applied Mathematics 2: 482–489 (1981)) to determine sequence identity. The gap generation penalty will typically range from 1 to 5, typically from 2 to 4, and in many embodiments will be 3. The gap extension penalty will typically range from approximately 0.01 to 0.20, and in many cases will be 0.10. The program has default parameters determined by the sequences entered for comparison. Preferably, sequence identity is determined using the default parameters determined by the program. This program is also available from the Genetics Computing Group (GCG) package, Madison, Wisconsin, USA.

Another program of interest is the FastDB algorithm. FastDB is described in Current Methods in Sequence Comparison and Analysis, Macromolecule Sequencing and Synthesis, Selected Methods and Applications, pp. 127–149, 1988, Alan R. Liss, Inc. Percent sequence identity is calculated using FastDB based on the following parameters:

Penalty for mismatch: 1.00;

Penalty for missing a sequence: 1.00;

Penalty for the size of the pass: 0.33; And

Merger penalty: 30.0.

Variant AAV2 Capsid Protein

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

The sequence of the AAV2 wild-type capsid protein VP1 is known and is shown in Figure 1 (SEQ ID NO:1). The sequences of the AAV2 wild-type capsid protein are also available from the database with accession numbers: NC-001401; UniProt P03135; NCBI reference sequence: YP_680426.1; GenBank: AAC03780.1.

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 comprises the sequence of SEQ ID NO: 2, or a sequence having 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 thereto.

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.

COMBINATIONS OF MUTATIONS IN THE AAV2 CAPSID PROTEIN

The variant AAV2 capsid protein may comprise any combination of the amino acid substitutions described above. Therefore, in specific 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 list above. 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., ttAAV2 or AAV2-TT as referred to herein).

In further embodiments, the variant AAV2 capsid protein may contain a subset of the 14 mutations noted above. Without wishing to be bound by theory, in certain embodiments, the variant AAV2 capsid protein may contain the following residues, which are categorized below into functional groups:

1) S585 and/or T588; these residues may be associated with reduced heparin binding and increased viral dissemination in heparin sulfate proteoglycan-rich brain tissue;

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 interactions with neutralizing antibodies and thus contribute to in vivo transduction properties;

4) S593; this residue is located in the depression between the triple-procymal spinous processes;

5) M457, A492, D499 and/or Y533; these four amino acids may be involved in receptor binding and are closely located on the triple spines;

6) I125, A151, S162 and/or S205; these residues may be associated with PLA2 activity and/or directional migration of the incoming virus.

It should be noted that also considered in this document are the corresponding subgroups containing mutations corresponding to the above residues when present at the corresponding positions in further AAV serotypes (see below).

In preferred embodiments, the variant AAV2 capsid protein comprises four or more mutations at the positions mentioned above that may be associated with receptor binding, i.e., 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.

In some preferred embodiments, the variant AAV2 capsid protein is unmutated compared to the wild-type AAV2 capsid protein at position 312, for example, the variant AAV2 capsid protein comprises residue N312 (which is present in the wild-type AAV2 capsid protein). Thus, in some embodiments, the variant AAV2 capsid protein may contain from 1 to 13 specific mutations noted above, but not the N312S mutation.

Variant AAV Capsid Proteins from Other Serotypes

In further embodiments, the variant AAV capsid protein is 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 AAV1, AAV3B, AAV-LK03, AAV5, AAV6, AAV8, AAV9, or AAV10 (e.g., AAVrh10) capsid protein.

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

Those skilled in the art know, based on comparison of the amino acid sequences of capsid proteins of different AAV serotypes, 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. In particular, such positions can be readily identified using sequence alignments, as is known in the art and described herein. For example, one such sequence alignment is shown in Figure 9.

Of particular importance in this context are positions in the 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 non-AAV2 capsid protein sequences. Illustrative 3D alignments of the primary structures of AAV2 capsid protein sequences with the capsid protein sequences of alternative AAV serotypes (e.g., AAV1, AAV5, AAV6, AAV8, and AAV9) are shown in Figures 21 through 25 and are discussed below. A skilled artisan can perform similar 3D primary structure comparisons with capsid proteins from subsequent serotypes (e.g., AAV2, AAV3, AAV7, AAV10, and AAV12) and identify positions in those sequences that correspond to the positions identified above in AAV2.

Variant AAV1 Capsid Protein

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 the following positions in the AAV1 capsid protein sequence: 125, 151, 162, 205, 313, 458, 493, 500, 534, 547, 549, 586, 589 and/or 594. These positions in the AAV1 capsid protein VP1 correspond to those disclosed above with respect to AAV2.

The sequence of the AAV1 capsid protein of the wild-type VP1 is known and is shown in Figure 3 (SEQ ID NO:3). Sequences of the AAV1 capsid protein of the wild-type are also available from the database with the 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.

The wild-type AAV1 capsid protein VP1 already contains the following residues at positions that correspond to the amino acid residues present in the variant AAV2 capsid protein disclosed above (SEQ ID NO:2, ttAAV2), but not in wild-type AAV2 (SEQ ID NO:1): S205 (matches S205 in ttAAV2); G549 (matches G548 in ttAAV2); S586 (matches S585 in ttAAV2); and T589 (matches T588 in ttAAV2). Accordingly, 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 a variant AAV1 capsid protein may share one or more functional properties with the variant AAV2 capsid protein (SEQ ID NO:2, ttAAV2), for example, it may provide increased infectivity and/or transduction of retinal neurons compared to the wild-type AAV1 capsid protein.

In alternative embodiments, the variant AAV1 capsid protein comprises one or more amino acid substitutions that correspond to the reverse permutations of the mutations present in ttAAV2 in the sequence of the wild-type AAV2. For example, the variant AAV1 capsid protein may comprise one or more of the following substitutions: S205T, G549E, S586R, and/or T589R. Typically, such a variant AAV1 capsid protein may share one or more functional properties with the wild-type AAV2 capsid protein (SEQ ID NO:1), for example, it may provide reduced infectivity and/or transduction of retinal neuron tissue compared to the wild-type AAV1 capsid protein.

Variant AAV5 Capsid Protein

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 the following positions in the AAV5 capsid protein sequence: 124, 150, 153, 195, 303, 444, 479, 486, 520, 533, 537, 575, 578 and/or 583. These positions in the AAV5 capsid protein VP1 correspond to those disclosed above with respect to AAV2.

The sequence of the AAV5 capsid protein of the wild-type VP1 is known and is shown in Figure 4 (SEQ ID NO:4). Sequences of the AAV5 capsid protein of the wild-type are also available from the database with the 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.

The AAV5 wild-type capsid protein VP1 already contains the following residues in positions that correspond to amino acid residues that are present in the variant AAV2 capsid protein disclosed above (SEQ ID NO:2, ttAAV2), but not in wild-type AAV2 (SEQ ID NO:1): G537 (matches G548 in ttAAV2); S575 (matches S585 in ttAAV2); T578 (matches T588 in ttAAV2). Accordingly, 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 a variant AAV5 capsid protein may share one or more functional properties with the variant AAV2 capsid protein (SEQ ID NO:2, ttAAV2), for example, it may provide increased infectivity and/or transduction of retinal neuron compared to the wild-type AAV5 capsid protein.

In alternative embodiments, the variant AAV5 capsid protein comprises one or more amino acid substitutions that correspond to the permutations of mutations present in the ttAAV2 reverse sequence of the wild-type AAV2. For example, the variant AAV5 capsid protein may comprise one or more of the following substitutions: G537E, S575R, and/or T578R. Typically, such a variant AAV5 capsid protein may share one or more functional properties with the wild-type AAV2 capsid protein (SEQ ID NO: 1), for example, it may provide reduced infectivity and/or transduction of retinal neuron tissue compared to the wild-type AAV5 capsid protein.

Variant AAV6 Capsid Protein

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 those disclosed above with respect to AAV2.

The sequence of the AAV6 capsid protein of the wild-type VP1 is known and is shown in Figure 5 (SEQ ID NO:5). Sequences of the AAV6 capsid protein of the wild-type are also available from the database with the 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.

The AAV6 wild-type capsid protein VP1 already contains the following residues in positions that correspond to amino acid residues present in the variant AAV2 capsid protein disclosed above (SEQ ID NO:2, ttAAV2), but not in wild-type AAV2 (SEQ ID NO:1): S205 (matches S205 in ttAAV2); G549 (matches G548 in ttAAV2); S586 (matches S585 in ttAAV2); T589 (matches T588 in ttAAV2). Accordingly, 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 a variant AAV6 capsid protein may share one or more functional properties with the variant AAV2 capsid protein (SEQ ID NO:2, ttAAV2), for example, it may provide increased infectivity and/or transduction of retinal neuron compared to the wild-type AAV6 capsid protein.

In alternative embodiments, the variant AAV6 capsid protein comprises one or more amino acid substitutions that correspond to the permutations of mutations present in ttAAV2 inversely compared to the wild-type AAV2 sequence. For example, the variant AAV6 capsid protein may comprise one or more of the following substitutions: S205T, G549E, S586R, and/or T589R. Typically, such a variant AAV6 capsid protein may share one or more functional properties with the wild-type AAV2 capsid protein (SEQ ID NO:1), for example, it may provide reduced infectivity and/or transduction of retinal neuron tissue compared to the wild-type AAV6 capsid protein.

Variant AAV8 Capsid Protein

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 the following positions in the AAV8 capsid protein sequence: 125, 151, 163, 206, 315, 460, 495, 502, 536, 549, 551, 588, 591 and/or 596. These positions in the AAV8 capsid protein VP1 correspond to those disclosed above with respect to AAV2.

The sequence of the AAV8 capsid protein of the wild-type VP1 is known and is shown in Figure 6 (SEQ ID NO:6). Sequences of the AAV8 capsid protein of the wild-type are also available from the database with the 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.

The wild-type AAV8 capsid protein VP1 already comprises the following residues at positions that correspond to the amino acid residues present in the variant AAV2 capsid protein disclosed above (SEQ ID NO:2, ttAAV2), but not in the wild-type AAV2 (SEQ ID NO:1): S315 (matches S312 in ttAAV2); T591 (matches T588 in ttAAV2). Accordingly, 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 a variant AAV8 capsid protein may share one or more functional properties with a variant AAV2 capsid protein (SEQ ID NO:2, ttAAV2), such as providing increased infectivity and/or retinal neuronal transduction compared to a wild-type AAV8 capsid protein.

In alternative embodiments, the variant AAV8 capsid protein comprises one or more amino acid substitutions that correspond to the permutations of mutations present in the ttAAV2 reverse sequence of the wild-type AAV2. For example, a variant AAV8 capsid protein may comprise one or more of the following substitutions: S315N and/or T591R. Typically, such a variant AAV8 capsid protein may share one or more functional properties with the wild-type AAV2 capsid protein (SEQ ID NO: 1), for example, it may provide reduced infectivity and/or transduction of retinal neuron tissue compared to the wild-type AAV8 capsid protein.

In one embodiment, the variant AAV8 capsid protein comprises an amino acid substitution compared to the wild-type AAV8 capsid protein at position 315 in the AAV8 capsid protein sequence. For example, the variant AAV8 capsid protein may comprise 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.

AAV9 VARIANT CAPSID PROTEIN

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 the following positions in the AAV9 capsid protein sequence: 125, 151, 162, 205, 314, 458, 493, 500, 534, 547, 549, 586, 589 and/or 594. These positions in the AAV9 capsid protein VP1 correspond to those disclosed above with respect to AAV2.

The sequence of the AAV9 capsid protein of the wild-type VP1 is known and is shown in Figure 7 (SEQ ID NO:7). Sequences of the AAV9 capsid protein of the wild-type are also available from the database with the 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.

The AAV9 wild-type capsid protein VP1 already contains the following residues in positions that correspond to amino acid residues that are present in the variant AAV2 capsid protein disclosed above (SEQ ID NO:2, ttAAV2), but not in the wild-type AAV2 (SEQ ID NO:1): S162 (matches S162 in ttAAV2); S205 (matches S205 in ttAAV2); G549 (matches G548 in ttAAV2); S586 (matches S585 in ttAAV2). Accordingly, 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 a variant AAV9 capsid protein may share one or more functional properties with the variant AAV2 capsid protein (SEQ ID NO:2, ttAAV2), for example, it may provide increased infectivity and/or transduction of retinal neuron compared to the wild-type AAV9 capsid protein.

In alternative embodiments, the variant AAV9 capsid protein comprises one or more amino acid substitutions that correspond to the reverse permutations of mutations present in ttAAV2 of the wild-type AAV2 sequence. For example, a variant AAV9 capsid protein may comprise one or more of the following substitutions: S162A, S205T, G549E, and/or S586R. Typically, such a variant AAV9 capsid protein may share one or more functional properties with the wild-type AAV2 capsid protein (SEQ ID NO:1), for example, it may provide reduced infectivity and/or transduction of retinal neuron tissue compared to the wild-type AAV9 capsid protein.

AAV10 VARIANT CAPSID PROTEIN

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 those disclosed above with respect to AAV2.

The sequence of the AAV10 capsid protein of the non-mutant type 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.

The wild-type AAV10 capsid protein VP1 already contains the following residue at a position that corresponds to an amino acid residue that is present in the variant AAV2 capsid protein disclosed above (SEQ ID NO:2, ttAAV2), but not in the wild-type AAV2 (SEQ ID NO:1): G551 (matches G548 in ttAAV2). Accordingly, in a preferred embodiment, the variant AAV10 capsid protein contains 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 a variant AAV10 capsid protein may share one or more functional properties with a variant AAV2 capsid protein (SEQ ID NO:2, ttAAV2), such as may provide increased infectivity and/or retinal neuronal transduction compared to a wild-type AAV10 capsid protein.

In alternative embodiments, the variant AAV10 capsid protein comprises an amino acid substitution that corresponds to a reversal of the mutations present in ttAAV2 to the wild-type AAV2 sequence. For example, a variant AAV10 capsid protein may comprise the following substitution: G551E. Typically, such a variant AAV10 capsid protein may share one or more functional properties with the wild-type AAV2 capsid protein (SEQ ID NO: 1), for example, it may provide reduced infectivity and/or transduction of retinal neurons compared to the wild-type AAV10 capsid protein.

Variant AAV3B Capsid Protein

In one embodiment, the vector comprises a variant AAV3B capsid protein. In this embodiment, the variant AAV3B capsid protein may comprise an amino acid substitution at position 312 compared to the wild-type AAV3B capsid protein. For example, the variant AAV3B capsid protein may 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 further embodiments, the variant AAV3B capsid protein may comprise one or more additional mutations at positions that correspond to those described above with respect to AAV2.

The sequence of the AAV3B capsid protein of the wild-type VP1 is known and is shown in Figure 38 (SEQ ID NO:11). The sequence of the AAV3B capsid protein of the wild-type 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.

VARIANT AAV-LK03 CAPSIDS PROTEIN

In one embodiment, the vector comprises a variant AAV-LK03 capsid protein. In this embodiment, the variant AAV-LK03 capsid protein may 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 may comprise residue N312. Thus, in one embodiment, the variant AAV-LK03 capsid protein comprises an amino acid substitution S312N compared to the AAV-LK03 capsid protein sequence as defined in SEQ ID NO: 12. In further embodiments, the variant AAV-LK03 capsid protein may comprise one or more additional mutations at positions that correspond to those disclosed above with respect to AAV2.

The sequence of the AAV-LK03 capsid protein of the non-mutant type VP1 is known and is shown in Figure 39 (SEQ ID NO:12). The sequence of the AAV-LK03 capsid protein is also disclosed in WO2013/029030, as sequence number 31 therein. 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.

GENE PRODUCTS

In one embodiment, the rAAV further comprises a heterologous nucleic acid comprising a nucleotide sequence encoding a gene product. A "gene" refers to a polynucleotide containing at least one open reading frame that is capable of encoding a specific protein after being transcribed and translated. A "gene product" is a molecule resulting from the expression of a specific gene. Gene products include, for example, a polypeptide, an aptamer, interfering RNA, mRNA, and the like.

"Heterologous" means derived from a genotypically distinct entity from what remains of the entity to which it is being compared. For example, a polynucleotide genetically engineered into a plasmid or vector derived from a different species is a heterologous polynucleotide. A linked promoter, removed from its native coding sequence and operably linked to a coding sequence with which it does not occur in nature, is a heterologous promoter. Thus, for example, an rAAV that includes a heterologous nucleic acid encoding a heterologous gene product is an rAAV that includes a nucleic acid not normally included in a non-mutant type of AAV that does not occur in nature, and the encoded heterologous gene product is a gene product that is not normally encoded by a non-mutant type of AAV that does not occur in nature.

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.

Interfering RNA

When the gene product is interfering RNA (RNAi), suitable RNAi includes RNAi that reduces the level of an apoptotic or angiogenic factor in a cell. For example, RNAi can be shRNA or siRNA that reduces the level of a gene product that induces or promotes apoptosis in a cell. Genes whose gene products induce or promote apoptosis are referred to herein as "pro-apoptotic genes," and the products of these genes (mRNA; protein) are referred to as "pro-apoptotic gene products." Pro-apoptotic gene products include, for example, the Bax, Bid, Bak, and Bad gene products. See, for example, U.S. Patent No. 7,846,730.

RNAi may also be against an angiogenic product, such as VEGF (e.g., Cand5; see, e.g., U.S. Patent Publication No. 2011/0143400; U.S. Patent Publication No. 2008/0188437; and Reich et al. (2003) Mol. Vis. 9:210), VEGFR1 (e.g., miRNA-027; see, e.g., Kaiser et al. (2010) Am. J. Ophthalmol. 150:33; 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.

Small interfering RNA, or short interfering RNA, or siRNA, is a duplex of RNA nucleotides that targets a gene of interest (the "target gene"). "Duplex RNA" refers to the structure formed by a complementary pair between two regions of an RNA molecule. SiRNA is "targeted" for a gene in that the nucleotide sequence of the duplex portion of the siRNA is complementary to the nucleotide sequence of the target gene. In some embodiments, the length of the siRNA duplex is less than 30 nucleotides. In some embodiments, the duplex may be 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, or 10 nucleotides in length. In some embodiments, the duplex is 19-25 nucleotides in length. The duplex RNA portion of the siRNA may be part of a hairpin structure. In addition to the duplex portion, the hairpin structure may comprise a portion of a loop located between the two sequences that form the duplex. The loop may vary in length. In some embodiments, the loop is 5, 6, 7, 8, 9, 10, 11, 12, or 13 nucleotides in length. The hairpin structure may also comprise 3' or 5' overhangs. In some embodiments, the overhang is a 3' or 5' overhang of 0, 1, 2, 3, 4, or 5 nucleotides in length.

A "short hairpin RNA," or shRNA, is a polynucleotide construct that can be made to express interfering RNA, such as siRNA.

Aptamers

When the gene product is an aptamer, exemplary aptamers of interest include an aptamer against vascular endothelial growth factor (VEGF). See, e.g., 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 a PDGF-specific aptamer, such as E10030; see, e.g., Ni and Hui (2009) Ophthalmologica 223:401; and Akiyama et al. (2006) J. Cell Physiol. 207:407).

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 a protein deficiency or defect in a cell or subject. Alternatively, a "therapeutic" peptide or protein is one that otherwise provides a benefit to the subject, such as anti-degenerative effects.

When the gene product is a polypeptide, the polypeptide is typically a polypeptide that improves the function of a cell, such as a cell present in neuronal, retinal, or liver tissue, such as a hepatocyte, neuron, glial cell, rod or cone photoreceptor cell, retinal ganglion cell, Müller cell, bipolar cell, amacrine cell, horizontal cell, or retinal pigmented epithelial cell.

Exemplary polypeptides include neuroprotective polypeptides (e.g., GDNF, CNTF, NT4, NGF, and NTN); antiangiogenic polypeptides (e.g., soluble vascular endothelial growth factor (VEGF) receptor; VEGF-binding antibody; VEGF-binding antibody fragment (e.g., single-chain anti-VEGF antibody); endostatin; tumstatin; angiostatin; soluble Fit polypeptide (Lai et al. (2005) Mol. Ther. 12:659); an Fc fusion protein comprising soluble Fit polypeptide (see, e.g., Pechan et al. (2009) Gene Ther. 16:10); pigment epithelial growth factor (PEDF); soluble Tie-2 receptor; etc.); tissue inhibitor of metalloproteinase-3 (TIMP-3); a light-sensitive opsin, such as rhodopsin; anti-apoptotic polypeptides (e.g., Bcl-2, Bcl-Xl); and the like. Suitable polypeptides include, but are not limited to, glial cell line derived neurotrophic factor (GDNF); fibroblast growth factor 2; neuroturin (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 WO 2012/145601.

Exemplary polypeptides for gene delivery to the liver include, for example, PBGD (porphobilinogen deaminase), IDUA (iduronidase), Fah (fumaryl acetoacetate hydrolase), A1AT (alpha(1)-antitrypsin), 1A1(hUGT1A1) (uridine diphosphate glucuronyl transferase), 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).

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-specific regulatory element.

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), e.g., a regulatory element that provides for selective expression of the operably linked gene in a neuronal or photoreceptor cell. Suitable photoreceptor-specific regulatory elements include, for example, the rhodopsin promoter; the rhodopsin kinase promoter (Young et al. (2003) Ophthalmol. Vis. Sci. 44:4076); the beta-phosphodiesterase gene promoter (Nicoud et al. (2007) J. Gene Med. 9: 1015); the retinal pigmentosa gene promoter (Nicoud et al. (2007) supra); interphotoreceptor retinoid-binding protein (IRBP) gene enhancer (Nicoud et al. (2007) supra); IRBP gene promoter (Yokoyama et al. (1992) Exp Eye Res. 55:225). Acceptable neuron-specific promoters include the neuron-specific enolase (NSE) promoter, Andersen et al. Cell. Mol. Neurobiol., 13:503–15 (1993; neurofilament light chain gene promoter, Piccioli et al., Proc. Natl. Acad. Sci. USA, 88:561 1–5 (1991); and neuron-specific vgf gene promoter, Piccioli et al., Neuron, 15:373–84 (1995)], among others. Suitable hepatocyte-specific promoters include the albumin promoter (Heard et al., Mol Cell Biol 1987; 7: 2425) or the alpha 1-antitrypsin promoter (Hafenrichter et al. Blood 1994; 84, 3394–404).

A "control element" or "control sequence" is a nucleotide sequence involved in the interaction of molecules that facilitates the functional regulation of a polynucleotide, including replication, duplication, transcription, splicing, translation, or degradation of the polynucleotide. Regulation can affect the frequency, rate, or specificity of a process and can be either upregulating or inhibitory in nature. Control elements known in the art include, for example, transcriptional regulatory sequences such as promoters and enhancers. A promoter is a region of DNA capable, under certain conditions, of binding RNA polymerase and initiating transcription of a coding region, typically located downstream (in the 3' direction) of the promoter.

"Functionally linked" or "functionally linked" refers to adjacent genetic elements in which the elements are in a relationship that allows them to act in the expected order. For example, a promoter is functionally linked to a coding region if the promoter helps initiate transcription of the coding sequence. Intervening residues may exist between the promoter and the coding region, provided this functional relationship is maintained.

The term "promoters" or "promoter," as used herein, may refer to a DNA sequence that is adjacent to a DNA sequence that encodes a recombinant product. A promoter is preferably operably linked to the 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 when no promoter exists. A promoter from one organism can be used to increase 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 vertebrates. Furthermore, a single promoter element can increase the amount of recombinant products expressed for multiple DNA sequences attached in tandem. Therefore, 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 from the prior art.

The term "enhancers" or "enhancer," as used herein, may refer to a DNA sequence that is located adjacent to a DNA sequence that encodes a recombinant product. Enhancer elements are typically located in the 3'-5' direction of a promoter element, or may be located in the 5'-3' direction or within the coding DNA sequence (e.g., the DNA sequence transcribed or translated into a recombinant product or products). Accordingly, an enhancer element may be located 100 base pairs, 200 base pairs, or 300 or more base pairs in the 3'-5' direction or 5'-3' direction of the DNA sequence that encodes the recombinant product. Enhancer elements can increase the amount of recombinant product expressed from the DNA sequence specified above, where the increased expression is mediated by the promoter element. Multiple enhancer elements are readily available to those skilled in the art from the state of the art.

PHARMACEUTICAL COMPOSITIONS

The present disclosure provides a pharmaceutical composition comprising: a) an rAAV vector as described above; and b) a pharmaceutically acceptable carrier, diluent, excipient, or buffer. In some embodiments, the pharmaceutically acceptable carrier, diluent, excipient, or buffer is suitable for human use.

Such excipients, carriers, diluents, and buffers include any pharmaceutical agent that can be administered without undue toxicity. Pharmaceutically acceptable excipients include, but are not limited to, liquids such as water, saline, glycerol, and ethanol.

Pharmaceutically acceptable salts may be included therein, for example, mineral acid salts such as hydrochlorides, hydrobromides, phosphates, sulfates, and the like; and organic acid salts such as acetates, propionates, malonates, benzoates, and the like. In addition, auxiliary substances such as wetting or emulsifying agents, pH buffering substances, and the like may be present in such vehicles. A wide variety of pharmaceutically acceptable excipients are known in the art and need not be discussed in detail herein. Pharmaceutically acceptable excipients are well described in various publications, including, for example, A. Gennaro (2000) "Remington: The Science and Practice of Pharmacy," 20th edition, Lippincott, Williams, &Wilkins; Pharmaceutical Dosage Forms and Drug Delivery Systems (1999) H. C. Ansel et al., eds., 7(th) ed., Lippincott, Williams, &Wilkins; and Handbook of Pharmaceutical Excipients (2000) A.H. Kibbe et al., eds., 3rd ed. Amer. Pharmaceutical Assoc.

In specific embodiments, the present invention provides a pharmaceutical composition comprising an rAAV vector as described above in a pharmaceutically acceptable carrier or other medicinal agents, pharmaceutical agents, excipients, adjuvants, diluents, etc. For injection, the carrier will typically be a liquid. For other modes of administration, the carrier may be either solid or liquid, such as sterile, pyrogen-free water or sterile, pyrogen-free, phosphate-buffered saline. For inhalation administration, the carrier will be respirable and will preferably be in the form of solid or liquid particles. Water containing additives common to injection solutions, such as stabilizing agents, salts or saline, and/or buffers, is preferably used as an injection medium.

The term "pharmaceutically acceptable" refers to a material that is not biologically or otherwise undesirable, such as a material that can be administered to a subject without causing any undesirable biological effects. Thus, such a pharmaceutical composition can be used, for example, in ex vivo cell transfection or by directly administering a viral particle or cell to a subject.

Methods of delivering a gene product to a tissue or cell (e.g., liver tissue or cells, neuronal or retinal tissues or cells) and methods of treatment

The methods of the present invention provide methods for delivering heterologous nucleic acid sequences to a host tissue or cell, including both dividing and non-dividing cells. The vectors and other reagents, methods, and pharmaceuticals of the present invention are also useful for administering a protein or peptide to a subject in need thereof, as a treatment or otherwise. The protein or peptide can thus be produced in vivo in the subject. The subject may need the protein or peptide because the subject has a deficiency of the protein or peptide, or because production of the protein or peptide in the subject can provide some therapeutic effect, as a treatment or otherwise, and as described further below.

As used herein, the terms "treatment," "treating," and the like refer to the production of a desired pharmacological and/or physiological effect. The effect may be prophylactic in terms of complete or partial prevention of the disease or symptoms thereof and/or may be therapeutic in terms of partial or complete relief from the disease and/or adverse effects attributable to the disease. "Treatment," as used herein, encompasses any treatment of a disease in a mammal, particularly a human, and includes: (a) preventing the onset of the disease in a subject who may be predisposed to the disease or at risk of contracting the disease but who has not yet been diagnosed as having it; (b) containing the disease, i.e., suppressing its progression; and (c) alleviating the disease, i.e., causing an alleviation of the symptoms of the disease.

In general, the present invention can be used to deliver any foreign nucleic acid with a biological effect for the treatment or alleviation of symptoms associated with any disorder associated with gene expression in any organ, tissue or cell, especially those associated with, for example, the liver, brain or eye. Illustrative disease states include, but are not limited to: lysosomal storage disease, acute intermittent porphyria, ornithine transcarbamylase deficiency, alpha(1)-antitrypsin deficiency, acute liver failure, Pompe disease, tyrosinemia, Crigler-Najjar syndrome, hepatitis, liver cirrhosis, hepatocellular carcinoma, AIDS, Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, epilepsy, and other neurological disorders, cancer (e.g., brain cancer), degenerative retinal diseases, and other eye diseases.

Gene transfer has significant potential for use in understanding and providing therapy for disease states. There are a number of inherited diseases in which the defective genes are known and have been cloned. In some cases, the function of these cloned genes is known. Generally, these disease states are divided into two classes: deficiency states, typically of enzymes, which are usually inherited recessively, and unbalanced states, which at least sometimes involve regulatory or structural proteins, which are inherited dominantly. For deficiency states, gene transfer can be used to introduce a normal gene into affected tissues during replacement therapy, as well as to create animal models of the disease using antisense mutations. For unbalanced disease states, gene transfer can be used to create the disease state in a model system, which can then be used to counteract the disease state. Thus, the methods according to the present invention enable the treatment of genetic diseases. As used herein, a disease condition is treated partially or completely by correcting the deficiency or imbalance that causes the disease or makes it more severe. Site-specific integration of nucleic acid sequences to induce mutations or correct defects is also possible.

In one aspect, the present invention provides a method for delivering a gene product to tissue or a cell (e.g., liver tissue or cell, neuronal or retinal tissue or cell) of a subject, wherein the method comprises administering to the subject an rAAV vector as described above. The gene product can be a polypeptide or an interfering RNA (e.g., shRNA, siRNA, etc.), or an aptamer, for example, as described above. The cell, for example, can be a blood cell, a stem cell, a bone marrow cell (e.g., a hematopoietic cell), a liver cell, a cancer cell, a vascular cell, a pancreatic cell, a nerve 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 may, for example, be selected from 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 tissue, neuronal tissue, or retinal tissue), pancreatic tissue, liver tissue, kidney tissue, lung tissue, intestinal tissue, or cardiac tissue.

Delivery of a gene product to a retinal cell may provide treatment for a retinal disease. The retinal cell may be a photoreceptor, retinal ganglion cell, Müller cell, bipolar cell, amacrine cell, horizontal cell, or retinal pigmented epithelial cell. In some cases, the retinal cell is a photoreceptor cell, such as a rod or cone cell. Similarly, delivery of a gene product to neuronal tissue or cell may provide treatment for a neurological disorder. The gene product may be delivered to various cell types present in neuronal tissue, such as neurons or glial cells (e.g., astrocytes, oligodendrocytes, etc.). Delivery of a gene product to the liver may provide treatment for liver diseases. The gene product may be delivered to, for example, hepatocytes.

The present disclosure provides a method for treating a disease (e.g., a liver disease, a neurological disease, or an ocular disease), wherein the method comprises administering to a subject in need thereof an effective amount of an rAAV vector, as described above. The rAAV vector may be administered to the subject by intracranial injection, intracerebral injection, intraocular injection, intravitreal injection, retinal injection, subretinal injection, intravenous injection, or any other convenient route or route of administration.

Other exemplary routes of administration include oral, rectal, transmucosal, topical, transdermal, inhalational, parenteral (e.g., intravenous, subcutaneous, intradermal, intramuscular, and intra-articular) administration, and the like, as well as direct administration into tissue or organ, intrathecal, direct intramuscular, intraventricular, intravenous, intraperitoneal, intranasal, or intraocular injections. Injections can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution or suspension in liquid prior to injection, or as emulsions. Alternatively, the virus can be administered locally rather than systemically, for example, as a depot or sustained-release composition.

Recombinant viral vectors are preferably administered to a subject in an amount sufficient to result in infection (or transduction) and expression of a heterologous nucleic acid sequence in cells (e.g., liver cells, neuronal cells, or retinal cells) of the subject. Preferably, the target cells are hepatocytes, neural cells (including cells of the central and peripheral nervous systems, particularly brain cells), or retinal cells. In some cases, the retinal cell is a photoreceptor cell (e.g., rods and/or spines). In other cases, the retinal cell is an RGC cell. In other cases, the retinal cell is an RPE cell. In other cases, retinal cells may include amacrine cells, bipolar cells, and horizontal cells.

Preferably, the vector is administered in a therapeutically effective amount. A "therapeutically effective" amount, as used herein, is an amount that is sufficient to alleviate (e.g., mitigate, reduce, decrease) at least one of the symptoms associated with a disease state. Alternatively, a "therapeutically effective" amount is said to be an amount that is sufficient to provide some improvement in the subject's condition. A "therapeutically effective amount" will fall within a relatively wide range that can be determined experimentally and/or during clinical trials. For example, for in vivo injections, a therapeutically effective dose will be on the order of about 10 ⁶ to about 10 ¹⁵ rAAV virions, such as from about 10 ⁸ to 10 ¹² rAAV virions. For in vitro transduction, the effective amount of rAAV virions to be delivered to cells will be on the order of about 10 ⁸ to about 10 ¹³ rAAV virions. Other effective dosages can be readily established by one skilled in the art by routine tests establishing dose-response curves.

In some embodiments, more than one administration (e.g., two, three, four, or more administrations) may be used to achieve the desired level of gene expression over a period of various intervals, such as daily, weekly, monthly, annually, etc.

Treatable neurological diseases include any disease involving the brain or central nervous system, including psychiatric diseases. Brain diseases are divided into two main categories: (a) pathological processes, such as infections, trauma, and neoplasms; and (b) diseases unique to the nervous system, which include myelin diseases and neuronal degeneration. Diseases from either category may be treatable. For example, a neurological disease may be selected from neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis (ALS), spinal muscular atrophy, and cerebellar degeneration; schizophrenia; epilepsy; ischemia-related diseases and stroke; Demyelinating diseases such as multiple sclerosis, perivenous encephalitis, leukodystrophies such as metachromatic leukodystrophy due to arylsulfatase A deficiency, 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 encephalomyopathies; neurological cancers such as primary brain tumors including glioma, meningioma, neurinoma, pituitary adenoma, medulloblastoma, craniopharyngioma, hemangioma, epidermoid, sarcoma and intracranial metastasis from other tumor sources; neurological infections or neurological inflammatory conditions.

Eye diseases that can be treated in a subject using the method include, but are not limited to, acute macular neuroretinopathy; Behcet's disease; choroidal neovascularization; diabetic uveitis; histoplasmosis; macular degeneration such as acute macular degeneration, non-exudative age-related macular degeneration, and exudative age-related macular degeneration; edema such as macular edema, cystoid macular edema, and diabetic macular edema; multifocal choroiditis; multifocal choroiditis; ocular trauma that affects the ocular posterior site or location; ocular tumors; Retinal disorders such as central retinal vein occlusion, diabetic retinopathy (including proliferative diabetic retinopathy), proliferative vitreoretinopathy (PVR), retinal artery occlusive disease, retinal detachment, uveal retinal disease; sympathetic ophthalmia; Vogt-Koyanagi-Harada syndrome (VKH); uveal diffusion; posterior eye condition caused or influenced by laser eye treatment; posterior eye condition caused or influenced by photodynamic therapy; photocoagulation, radiation retinopathy; epiretinal membrane disorders; branch retinal vein occlusion; anterior ischemic optic neuropathy; non-retinopathy diabetic retinal dysfunction; retinoschisis; pigmentary retinal degeneration; glaucoma; Usher syndrome, cone-rod dystrophy; Stargardt disease (yellow macular retinal abiotrophy); hereditary macular degeneration; chorioretinal degeneration; Leber's amaurosis; congenital stationary night blindness; choroideremia; Bardet-Biedl syndrome; macular telangiectasia; Leber's hereditary optic neuropathy; retinopathy of prematurity; and color vision defects including achromatopsia, protanopia, deuteranopia, and tritanopia.

Treatable liver diseases include, for example, lysosomal storage diseases such as acute intermittent porphyria, ornithine transcarbamylase deficiency, Wilson's disease, mucopolysaccharidoses (eg, MPS type I or MPS type VI), Sly syndrome, Pompe disease, tyrosinemia, alpha(1)-antitrypsin deficiency, Crigler-Najjar syndrome; hepatitis A, B, or C; cirrhosis of the liver; liver cancer such as hepatocellular carcinoma; or acute liver failure.

The present invention finds application in both the veterinary and medical fields. 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 "mammal," as used herein, includes, but is not limited to, humans, bovines, ovine, caprine, equine, feline, canine, lagomorph, etc. Human subjects are most preferred. Human subjects include fetal, neonatal, infant, juvenile, and adult subjects.

TISSUE TRANSDUCTION (E.G., LIVER, NEURONAL, OR RETINAL TISSUE)

In some embodiments, the rAAV vectors disclosed herein exhibit increased tissue transduction (e.g., liver tissue, neuronal and/or retinal tissue), for example, compared to a corresponding AAV vector (from the same serotype) containing a wild-type AAV capsid protein. For example, an rAAV vector can exhibit at least 10%, 50%, 100%, 500%, or 1000% increased infectivity compared to the infectivity of an AAV virion containing a corresponding wild-type AAV capsid protein.

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

When a recombinant AAV vector exhibits enhanced transduction of neuronal or retinal tissue, such as when the vector is used to treat a neurological or ocular disorder, the vector preferably contains a variant AAV2 capsid protein.

When a recombinant AAV vector exhibits enhanced transduction of liver tissue, such as when the vector is used to treat liver diseases, the vector preferably comprises a variant AAV3B, AAV-LK03, or AAV8 capsid protein.

Nucleic Acids and Host Cells

The present disclosure provides an isolated nucleic acid comprising a nucleotide sequence encoding a variant capsid protein of an adeno-associated virus (AAV), as described above. The isolated nucleic acid may be contained in an AAV vector, such as a recombinant AAV vector.

A recombinant AAV vector containing such a variant AAV capsid protein coding sequence can be used to produce a recombinant AAV virion (i.e., a recombinant AAV vector particle). Thus, the present disclosure provides a recombinant AAV vector that, when introduced into a suitable cell, can provide for the production of a recombinant AAV virion.

The present invention also provides host cells, such as isolated (genetically modified) host cells, containing the subject's nucleic acid. The subject's host cell may be an isolated cell, such as a cell in vitro culture. The subject's host cell is useful for producing the subject's rAAV virion, as described below. When the subject's host cell is used to produce the subject's rAAV virion, it is referred to as a "packaging cell." In some embodiments, the subject's host cell is stably genetically modified with the subject's nucleic acid. In other embodiments, the subject's host cell is transiently genetically modified with the subject's nucleic acid.

The subject's nucleic acid is introduced stably or transiently into the host cell using established methods, including, but not limited to, electroporation, calcium phosphate precipitation, liposome-mediated transfection, and the like. For stable transformation, the subject's nucleic acid will typically additionally include a selectable marker, such as one of several well-known selectable markers, such as neomycin resistance.

A host cell of a subject is generated by introducing a nucleic acid of the subject into any of a plurality of cells, for example, a plurality of cells including, 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, wherein 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 No. CRL-1573), Vero cells, NIH 3T3 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. The subject's host cell can also be engineered using baculovirus to infect insect cells, such as Sf9 cells, which produce AAV (see, e.g., U.S. Patent No. 7,271,002; U.S. Patent Application No. 12/297,958).

In some embodiments, the genetically modified host cell of the subject comprises, in addition to a nucleic acid comprising a nucleotide sequence encoding a variant AAV capsid protein, as described above, wherein the nucleic acid comprises a nucleotide sequence encoding one or more AAV rep proteins. In other embodiments, the host cell of the subject further comprises an anrAAV vector. The anrAAV virion can be produced using the host cell of the subject. Methods for producing an anrAAV virion are described in, for example, U.S. Patent Publication No. 2005/0053922 and U.S. Patent Publication No. 2009/0202490.

As used in this document, "packaging" refers to a series of intracellular events that ultimately lead to the assembly and encapsidation of an AAV particle. The AAV "rep" and "cap" genes refer to polynucleotide sequences encoding the replication and encapsidation proteins of the adeno-associated virus. AAV rep and cap are referred to herein as AAV "packaging genes." The assembly-associated protein (AAP) is the product of an open reading frame within the cap gene and may also require packaging.

A "helper virus" for AAV refers to a virus that enables AAV (e.g., a wild-type AAV) to replicate and be packaged by a mammalian cell. A variety of such AAV helper viruses are known in the art, including adenoviruses, herpesviruses, and poxviruses such as vaccinia virus. Adenoviruses encompass a number of different subgroups, although adenovirus type 5 subgroup C is the most commonly used. Numerous adenoviruses of human, non-human mammalian, and avian origin are known and available from depositories such as the ATCC. Viruses of the herpes family include, for example, herpes simplex virus (HSV) and Epstein-Barr virus (EBV), as well as cytomegalovirus (CMV) and pseudorabies virus (PRV); these are also available from depositories such as the ATCC.

"Helper virus function(s)" refers to the function(s) encoded in the helper virus genome that enables AAV replication and packaging (in conjunction with other replication and packaging requirements described herein). As described herein, "helper virus function" can be achieved in several ways, including providing the helper virus or providing, for example, polynucleotide sequences encoding the desired function(s) to the producer cell in trans. For example, a plasmid or other expression vector containing nucleotide sequences encoding one or more adenoviral proteins is transfected into the producer cell along with the anrAAV vector.

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 virus species is tropic. The term does not necessarily imply any ability for the virus to replicate. Assays for enumerating infectious virus particles are described elsewhere in this disclosure and in the prior art. Viral infectivity can be expressed as the ratio of infectious virus particles to total virus particles. Methods for determining the ratio of infectious virus particle to total virus particle are known in the art. See, for example, Grainger et al. (2005) Mol. Ther. 11: S337 (describing a TCID50 infectious titer assay); and Zolotukhinet al. (1999) Gene Ther. 6:973.

A "replication-competent" virus (e.g., replication-competent AAV) refers to a phenotypic virus of the wild-type that is infectious and also capable of replication in an infected cell (i.e., in the presence of a helper virus or helper virus functions). In the case of AAV, replication competence typically requires the presence of functional AAV packaging genes. In general, rAAV vectors, as described herein, are replication-incompetent 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 that replication-competent AAV is generated by recombination between AAV packaging genes and the incoming rAAV vector. In many embodiments, rAAV vector preparations as described herein are those that contain little, if any, replication-competent AAV (rcAAV, also referred to as RCA) (e.g., less than about 1 rcAAV per ¹⁰² rAAV particles, less than about 1 rcAAV per ¹⁰⁴ rAAV particles, less than about 1 rcAAV per ¹⁰⁸ rAAV particles, less than about 1 rcAAV per ¹⁰¹² rAAV particles, or no rcAAV).

An "isolated" nucleic acid, vector, virus, virion, host cell, or other substance refers to a preparation of the substance devoid of at least some other components that may also be present when the substance or a similar substance occurs in nature or was originally obtained from one. Thus, for example, an isolated substance may be obtained using purification technology to enrich it from an initial mixture. Enrichment may be measured on an absolute basis, such as mass per volume of solution, or it may be measured relative to a second, potentially interfering substance present in the initial mixture. Increasing enrichments according to embodiments of this disclosure are increasingly isolated. The isolated nucleic acid, vector, virus, host cell, or other substance in some embodiments is purified, such as from about 80% to about 90% pure, at least about 90% pure, at least about 95% pure, at least about 98% pure, or at least about 99% pure, or more.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, the preferred methods and materials are described below. All publications mentioned herein are hereby incorporated by reference for the disclosure and description of the methods and/or materials in connection with which these publications are cited.

It should be understood that certain features of the invention, which for clarity are described in the context of separate embodiments, may also be presented in combination in a single embodiment. Conversely, various features of the invention, which for brevity are described in the context of a single embodiment, may also be presented separately or in any suitable subcombination. All combinations of embodiments related to the present invention are specifically encompassed by the present invention and are disclosed herein as if each combination were individually and expressly disclosed. Furthermore, all sub-combinations of various embodiments and their elements are also specifically encompassed by the present invention and are disclosed herein as if each such sub-combination were individually and expressly disclosed herein.

The following example is presented to illustrate certain embodiments of the present invention. It is in no way intended to limit the invention.

EXAMPLES

Example 1

In this example, the inventors designed and constructed a new AAV2 vector, designated ttAAV2 (as in the true type). Furthermore, the new vector was tested in a number of animal models (rats, mice, and neonatal mice) to assess how ttAAV2 behaved differently from tissue-culture-adapted (non-mutant type) AAV2. The inventors demonstrated that ttAAV2 has gene delivery advantages for AAV2 and is particularly useful for in vivo transduction of brain or eye tissues with heterologous sequences.

Methods

1. Cloning: The wtAAV2 capsid gene was taken from our pDG producer plasmid (Figure 10). This plasmid contains the wtAAV2 rep and cap genes. 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 for subsequent mutagenesis. Four mutations were introduced into fragment A using site-directed mutagenesis, resulting in a construct encoding the amino acid (AA) changes V125I, V151A, A162S, and T205S. Fragment B was mutated to encode one AA change, N312S. Fragment C was mutated to encode the AA changes Q457M, S492A, E499D, F533Y, G546D, E548G, R585S, R588T, and A593S. After confirmation of successful mutagenesis, fragments A-C were recloned into pDG, resulting in a producer plasmid (pDG-ttAAV2) that will support the production of recombinant virus encapsidated by the ttAAV2 capsid.

2. Purification and titration of ttAAV2-GFP viral vector production

Vector production was based on standard protocols using cotransfections of rAAV plasmids with pDG, which provides both Ad helper functions and the AAV rep and cap genes. A variety of rAAV plasmids was used to generate recombinant plasmids.

pTR-UF11 (CAG-GFP) was used as the rAAV plasmid. 8 × 10 ⁸ 293 cells were seeded in a cell factory (CF10). After 14–18 h, the cells were transfected with pDG or pDG-rrAAV2 and prAAV (e.g., pTR-UF11) using the CaPO ₄ coprecipitation method. After 72 h, the cells were harvested and resuspended in lysis buffer (20 mM Tris-HCl, pH 8, 150 mM NaCl, 0.5% deoxycholate). Cell pellets were lysed using four freeze-thaw cycles to release virus, where each cycle consists of 30 min at -80°C followed by 30 min at 37°C. After the final thawing, the lysate was treated with benzonase at a concentration of 50 U/ml and incubated for 30 minutes at 37°C. The recombinant virus was purified using a gravity column.

Purification. As a first step, the crude lysate was clarified by centrifugation at 4000g for 15 minutes and applied to a preformed iodixanol step gradient. The viral fraction was then collected, rebuffered in lactated Ringer's solution, and concentrated using Amicon centrifugation filters.

The purity of the viral preparations was then assessed using SDS-polyacrylamide electrophoresis and titrated using real-time PCR methods. The crude extract contained 4.5 x 10 ¹² particles; the collected viral fractions contained 1.5 x 10 ¹² particles. At this point, the purification method recovered approximately 33% of the virus present in the crude extract.

3. Production and purification of rAAV vector (alternative method)

In an alternative embodiment to that described in point 2 above, the rAAV2 vector was prepared as follows. To produce rAAV2 virions, 5 × 10 ⁸ 293T cells were seeded in a cell factory (CF10). After 14–18 hours, the cells were transfected twice with the GFP-containing vector PD10-pST2-CMV-GFP and either pDG or the pDG capsid mutant (pDG-ttAAV2) to produce wild-type or true-type AAV2-CMV-GFP vectors, respectively. Double transfections were performed using PEI-max from Polysciences at a ratio of 3.5 ml PEI per mg DNA. Cells were harvested after 72 h of incubation at 37°C by centrifuging the srcd and cells at 2200 rpm for 10 min at 4°C. The supernatant was removed and saved for further processing, and the cell pellets were resuspended in lysis buffer (0.15 M NaCl, 50 mM Tris-HCl [pH 8.8]).

Cell pellets were lysed using four freeze-thaw cycles to release virus, where each cycle consisted 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 150 U/ml and incubated for 30 min at 37°C. The lysate was then spun at 2000 rpm for 20 min to clarify the lysate. The supernatant was filtered using a 0.22 μm cellulose acetate filter, and recombinant AAV2 virus preparations were purified by FPLC using an ÄKTA Purification Chromatography System (GE Healthcare) and an AVB-Sepharose affinity column (buffer A: PBS, pH 8; buffer B: 0.5 M glycine, pH 2.7). The collected fractions were dialyzed against PBS overnight, and the viral preparations were then titrated using SDS-polyacrylamide electrophoresis and real-time PCR methods.

Results

In vivo transduction and spread of ttAAV2

The ttAAV2 vector was studied in vivo to assess the biological activity of the modified virus in this context. Samples of AAV2-CMV-GFP WT and TT viruses were prepared for injection into a number of in vivo models. For this purpose, we concentrated the viruses, as only limited volumes of vectors can be injected in vivo . We then performed quantitative PCR (qPCR) and SDS-PAGE to assess new titers of the concentrated vectors (Figure 11).

After qPCR and protein–protein gel analysis, we obtained the following new titers: AAV2-CMV-GFP TT – 1.33 x ¹⁰¹² viral genomes/ml and AAV2-CMV-GFP WT – 1.25 x ¹⁰¹² viral genomes/ml. Capsid titers were as follows: AAV2-CMV-GFP TT – 8.89 x ¹⁰¹² capsids/ml – AAV2-CMV-GFP WT at 7.83 x ¹⁰¹² capsids/ml. Titers between genomic copies and capsid copies differ because SDS-PAGE also shows empty capsids, which are typically generated during the production of recombinant AAV vectors; therefore, the capsid titer is higher than the viral genome titer obtained by qPCR.

Transduction in the rat brain

The rAAV2 TT and WT viruses were injected into the substantia nigra or striatum of wild-type rats, with three rats per condition injected at a dose of 2× ¹⁰⁹ vg or 3.5× ¹⁰⁹ vg per injection. After 28 days, the brains were dissected, and tissue sections were prepared for immunofluorescence analysis. The primary data are shown in Figure 12 and Figure 26.

Both rAAV2 TT and WT viruses were able to transduce neuronal and glial cells from each injection site, albeit with different efficiencies. In comparison, we observed that the TT vector transduced brain tissue more efficiently and disseminated more from the injection site than the WT vector. Furthermore, we observed the presence of transduced neurons in the parafascicular nucleus, a region of the hypothalamus, following intrastriatal injection of rAAV2 TT. This indicates that the TT vector was able to migrate from transduced cell bodies at the injection site to the hypothalamus via active transport along neuronal projections, highlighting a strong retrograde transport capacity. This retrograde transport capacity was lost in tissue culture of the adapted WT rAAV2 vector, as no transduced cells could be observed in the same region (see Figure 26).

Taken together, these results in rat brain indicate significantly higher spread and transduction efficiency from ttAAV2 compared to titer-matched wtAAV2. Furthermore, AAV2 TT demonstrates evidence of excellent retrograde transport capacity, which has been lost in AAV2 WT virus.

Transduction in the mouse eye model

ttAAV2 and wt-AAV2 from the same lot as those used for our rat brain studies were injected into the eyes of adult mice at a dose of 2 × 10 ⁹ vg/eye. To avoid interanimal variability, each mouse received an injection of rAAV2 TT into one eye and an injection of rAAV2 WT into the contralateral eye. Three different intraocular injection routes were analyzed: intracameral, intravitreal, and subretinal. Animals were collected, and GFP expression was assessed by immunofluorescence after 6 weeks. The results are shown in Figure 13. Collectively, these data indicate a marked increase in photoreceptor cell transduction after subretinal injection of ttAAV2, in terms of both the level and the number of transduced photoreceptor cells, compared with wtAAV2 (which was used in the successful RPE65 clinical trial).

Transduction in a neonatal mouse model

Thus, both ttAAV2 and wtAAV2 GFP vectors were injected into neonatal mice. Two injection routes were tested: intravenous and intracranial. After 4 weeks, the animals were sacrificed, and all tissues were collected from all mice. We analyzed the brains, which were visualized after collection by direct fluorescence microscopy. The results are shown in Figure 14. The results of intracranial and systemic injections are discussed in more detail below.

Intracranial injections

5 × 10 ¹⁰ vg of any vector were injected into the lateral ventricle of P1 neonates. The animals were sacrificed 4 weeks after injection, and the brains were dissected, sectioned, and stained using an anti-GFP antibody. The results are shown in Figure 27.

As observed in adult rat brain, these data indicate that AAV2 TT exhibits enhanced transduction of mouse brain tissue and greater dissemination following intracranial injection compared to the AAV2 WT vector. When viewing stained tissue sections at higher magnification, differences in transduction efficiency between the two vectors were further highlighted, with the TT vector performing better in terms of both expression level and the number of transduced cells. AAV2 TT and WT appeared to have the same cell-type affinity, each showing transduction of neuronal as well as glial cells, suggesting that the observed differences represent differences in efficiency rather than cell-type specificity (Figure 28). Taken together, these data indicate that ttAAV2 exhibits significantly greater transduction of mouse brain tissue following intradermal (i.c.) injection compared to wtAAV2-based vectors. Furthermore, some evidence suggests transduction of the ependymal cell layer lining the ventricles when ttAAV2 vectors are used. This phenomenon was not observed with the wtAAV2 vector.

Systemic injections

Intrajugular injections of 2 × 10 ¹¹ vg of either vector were administered at P1 of neonates. Animals were sacrificed 4 weeks post-injection, and various organs (brain, liver, heart, muscle, lung, spleen, and kidney) were collected and assessed for GFP transduction using immunohistochemistry with an anti-GFP antibody. Brain staining results are shown in Figure 29, and high-magnification images are presented in Figure 30.

We observed good transgene expression in the central nervous system after systemic injection of AAV2 TT. The AAV2 WT vector performed poorly compared to the few transduced neurons observed.

To assess the overall biodistribution of the AAV2 TT vector, we evaluated the transduction levels achieved in various tissues following systemic injection. Harvested organs were fixed, paraffin-embedded, and stained for GFP expression (Figure 31).

These data indicate that the AAV-TT vector does not appear to have a strong affinity for other organs, but instead exhibits specificity primarily for neuronal tissues. This observation may prove useful for the treatment of neuronal genetic disorders via intravenous injection of AAV, as it ensures that the vector will not transduce non-target peripheral organs, but primarily the brain, with this injection route.

Taken together, our in vivo data suggest that ttAAV2 has exceptional transduction characteristics in ocular and brain tissues, demonstrating almost exclusive specificity for neuronal tissues.

Example 2

Additional considerations

Without wishing to be bound by theory, it is believed that the mutations present in ttAAV2 compared to wtAAV2 contain the following functional groups:

1) heparin-binding residues located on the triple spikes of the AAV2 capsid (S585 and T588); these residues are believed to be responsible for heparin binding of the wtAAV2 capsid. In ttAAV2, this is replaced, and we hypothesize that this substitution supports viral spread to brain tissue rich in heparan sulfate proteoglycan.

2) a single amino acid change in ttAAV2 located on the internal face of the capsid (S312); this internal serine residue may play a role in capsid-DNA interactions, thereby potentially contributing to either virus stability, genome packaging, or genome release during infection.

3) two spatially adjacent amino acids (D546 and G548) located in the recess between the triple proximal spikes on the AAV capsid structure; these residues may be involved in interactions with neutralizing antibodies and thus contribute to in vivo transduction properties.

4) one isolated amino acid change (S593) located in the cleft between the triple proximal spines;

5) four amino acids are thought to be involved in receptor binding and are closely located on the triple spines (M457, A492, D499, and Y533);

6) four remaining amino acid changes located in the VP1/VP2 primary sequence (I125, A151, S162, and S205); these residues are within the capsid region and are not part of VP3. VP1/VP2-specific regions within the viral capsid are known to have PLA2 activity, which may be involved in the directional migration of incoming virus. Changes in this region have been shown to affect viral infectivity.

The three-dimensional positions of these mutations in the AAV2 capsid protein are known and are shown in Figures 15–20. Corresponding positions can be identified in AAV capsid proteins from other serotypes (see below). To further characterize ttAAV2 and the role of individual amino acid changes that are responsible for the improved phenotype of ttAAV2, the ttAAV2 capsid can be mutated to completely change individual selected amino acids (or groups of amino acids, e.g., based on groups 1–6 discussed above) to their corresponding sequence in the capsid of the wild-type AAV2. Each mutant vector can then be analyzed using methods as described in the examples above.

For example, various mutant GFP-expressing vectors could be submitted for initial screening in an animal model via intracranial (IC) injection into CD1 neonatal mice. The GFP signal obtained in the injected brain from each mutant vector was then observed using fluorescence microscopy and compared with that obtained from the original GFP-expressing wtAAV2 and ttAAV2 vectors. After identifying a new phenotype (i.e., abolition of ttAAV2-specific strong GFP expression when a specific amino acid group is mutated), the injected brains were further sectioned and examined using immunohistochemistry.

In parallel, the selected mutant vectors are submitted for a second screening by intracranial injection into adult rats. Furthermore, the corresponding mutant combinations are analyzed using intravenous (IV) injections into neonatal mice to assess vector biodistribution. Individual organs (heart, lungs, liver, spleen, kidneys, and muscle) are then processed for immunohistochemistry and assessment of GFP expression.

Analysis of the contribution of each TT-specific residue to the vector efficiency and biodistribution

To further characterize AAV2 TT and identify the amino acid changes responsible for the enhanced TT phenotype, the AAV2 TT capsid was mutated step by step to completely alter selected amino acids to their corresponding sequences in the wild-type AAV2 capsid. This strategy allowed us to more specifically distinguish the contribution of each of the 14 amino acid mutations to the AAV2 TT phenotype and determine a minimal true-type vector containing only the necessary mutations.

As discussed above, we grouped the 14 TT-specific residues into groups based on their positions on the AAV capsid and their associated potential contributions to the transduction profile. Various AAV-TT mutants were screened using intracranial injections into the brains of neonatal mice or adult rats to observe whether rearrangement of some TT-specific residues to WT equivalents was associated with a loss of phenotype, thereby identifying important amino acid changes among the 14 TT residues.

Heparin binding site (HBS)

Residues 585 and 588 were shown to be responsible for heparin binding by the AAV2 WT capsid. In AAV-TT, these residues are substituted, and we hypothesized that this substitution supports viral spread in brain tissue enriched in heparin sulfate proteoglycan. These two residues are located on the triple proximal spikes of the AAV2 capsid structure (see Figure 16).

We abolished the heparin-binding ability of WT AAV2 by engineering the R585S and R588T substitutions into the WT capsid (AAV2-HBnull), mutating these residues to their true-type equivalents. This initial analysis was conducted to ensure that AAV-TT was not simply AAV2 lacking the heparin-binding site, capable of greater transduction, but not in the same way, as several of the other 12 amino acid substitutions also play a role in AAV-TT's improved transduction profile.

Intracranial injections into an adult rat

AAV2-TT, AAV2-WT, and AAV2-HBnull vectors were injected with the appropriate titer into the substantia nigra or striatum of wild-type rats at a dose of 3.5× ¹⁰⁹ vg per injection. After 28 days, the brains were dissected, and tissue sections were prepared for immunohistochemical analysis of GFP expression (Figure 32).

We observed strong GFP expression in the thalamus and substantia nigra after intrastriatal injection of the AAV-TT virus, highlighting the vector's robust retrograde transport capacity. In comparison, AAV2-HBnull and AAV2 WT demonstrated much less retrograde transport—if any at all—than AAV-TT, as very few cell bodies were transduced to these regions after intrastriatal injection. This observation indicates that AAV2-HBnull is distinct from AAV-TT and that the lack of heparin binding on the AAV2 capsid facilitates the robust spread of the true-type vector in the brain. However, this is insufficient to explain its enhanced transduction profile.

Residues S312, D546-G548 and S593

The S312 residue is the only TT-specific change located on the internal side of the AAV2 capsid. We hypothesized that this internal residue may play a role in capsid-DNA interactions, potentially contributing to either viral stability, genome packaging, or genome release during infection.

The D and G residues at positions 546 and 548, respectively, are located in the depression between the proximal 3-fold peaks.

One isolated serine, S593, is located in the depression between the triple procymal spines.

The positions of these TT-specific residues in the three-dimensional structure of the AAV2 capsid are shown in Figures 17–19. Given their less prominent position in the structure, we hypothesized that these residues could not contribute to the transduction of the ttAAV2 phenotype.

Intracranial injections into neonatal mice

The S312N mutation was engineered into the AAV2-TT capsid to create the TT-S312N mutant. The D546G and G548E mutations were engineered into the AAV2-TT capsid to create the AAV-TT-DG mutant. The S593A mutation was engineered into the AAV2-TT capsid to create the AAV-TT-S593A mutant.

By reversing these selected TT amino acids to their corresponding sequence in the wild-type AAV2 capsid, we sought to determine the contribution of these residues to the improved AAV-TT phenotype.

5 × 10 ¹⁰ vg of each mutant vector were injected into the lateral ventricle of P1 neonates. Animals were sacrificed 4 weeks after injection, and the brains were dissected, sectioned, and stained using an anti-GFP antibody. The results are shown in Figure 33.

Interestingly, these data indicate that the AAV TT-S312N demonstrates enhanced transduction of mouse brain tissue compared to the full AAV2 TT vector. On the other hand, the amino acid changes S593A and D546E/G548D did not appear to affect the TT phenotype, as similar transduction profiles were observed throughout the brain. When examining the stained areas at higher magnification, differences in transduction efficiency were further highlighted (Figure 34).

At high magnification, we observed that AAV TT-S312 transduced neuronal tissues with higher efficiency than the original AAV-TT with 14 amino acid changes. Specifically, we observed stronger transgene expression in the rostral brain (cerebral cortex, striatum, hippocampus) following injection of the TT-S312N vector, in terms of both the level and number of transduced cells. Despite high variability in the injected neonatal brain due to difficulties in targeting the injection site, this observation was confirmed in all animals analyzed. On the other hand, the S593A or D546E/G548D rearrangements did not appear to have a significant impact on the TT vector's transduction phenotype.

Example 3

Targeted amino acid mutations on the authentic type AAV2 capsid selected from the results obtained with the mutant combinations in Example 2

Based on the results of amino acid group mutations on the complete AAV TT capsid, we determined that the S312N mutation appears to be beneficial for the TT phenotype, further enhancing its transduction efficiency in the brain. Furthermore, we observed that the S593A and D546G/G548E rearrangements appeared to have no effect on the neuronal phenotype of AAV-TT. We therefore hypothesized that the TT-specific residues S593, D546, and G548 could be excluded from the true-type capsid sequence, leaving the WT residues at these positions in place of AAV2, yielding a final TT vector with only 10 amino acid changes.

To test these hypotheses, we constructed the TT-S312N-D546G-G548D-S593A vector and examined its transduction efficiency using intracerebral injections into neonatal mice. Because intracranial injections into neonates were found to result in a saturated signal of detectable GFP expression, we decided to additionally administer the TT and TT-S312N vectors along with this "pre-final" TT, using a dose 10-fold lower than previously used. By using this lower dose, we aimed to avoid reaching saturated levels of GFP staining in the transduced brain and to avoid difficulties in comparing transduction efficiencies between different mutants.

5×10 ⁹ vg of each mutant vector were injected into the lateral ventricle of P1 neonates. Animals were sacrificed 4 weeks after injection, and the brains were dissected, sectioned, and stained with an anti-GFP antibody. The results are shown in Figure 35.

As observed previously, these data suggest that AAV TT-S312N exhibits enhanced transduction of mouse brain tissue compared to the full AAV2 TT vector. On the other hand, the minimal TT-S312N-D546G/G548E-S593A vector did not achieve these higher transduction levels, even though it also contained the internal S312N mutation. This suggests that one or more amino acid substitutions play a role in the TT phenotype. By completely changing these residues back to their AAV2 WT equivalents, we lost some of the enhanced transduction capacity. These observations were confirmed when the stained areas were examined at higher magnification (Figure 36).

We further investigated the transduction efficiency of each of these vectors by quantifying total GFP expression in injected brains using enzyme-linked immunosorbent assay (ELISA) on total brain protein extracts. Briefly, 4 weeks after injection of 5 × 10 ^g of vectors, animals were sacrificed, whole brains were collected and lysed, and total brain protein was extracted. Using a GFP protein standard, we were then able to quantify the amounts of GFP protein expressed in each injected brain (Figure 37).

We could confirm that the internal mutant TT-S312N transduces mouse brains with greater efficiency than the full TT vector, as it results in higher overall GFP expression throughout the injected brain. On the other hand, the minimal TT vector, TT-S312N-D546G/G548E-S593A, appeared to result in lower transduction levels: although the average amount of GFP expressed per brain appears higher in this graph, this was due to the extreme GFP values measured in a single brain, as illustrated by the high error bar for this condition. With this minimal TT vector, interanimal variability was very high, with only one animal out of five performing better than the animals transduced with TT-S312N. With this minimal TT vector, interanimal variability This high variability led us to consider this temporary minimal TT vector with caution, especially because immunohistochemical analyses also showed that the TT-S312N variant performed better than TT-S312N-DG-S593A.

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

Although the above studies suggest TT-S312N as the preferred AAV-TT vector, these studies illustrate the distinct functions and contributions of several TT-specific residues. In particular, four amino acids closely located on the capsid's triple spike are likely involved in receptor binding. In some embodiments, these residues revert AAV-TT to the corresponding residues of WT AAV2. For example, the M457Q, A492S, D499E, and Y533F mutations can be constructed on the AAV-TT capsid; this mutant vector was analyzed as described previously to illustrate the role of these residues. In further embodiments, four amino acids located in the VP1/VP2 primary sequence that are likely to be involved in viral homing may be reverted in AAV-TT back to the corresponding residues of AAV2 WT (I125V, A151V, S162A, S205T) and analyzed in a similar manner.

Example 4

Construction of AAV vector variants in other serotypes

The function of ttAAV2-specific amino acid changes in the context of other, non-AAV2 serotypes can also be determined.

The capsid amino acid sequences of the major adeno-associated viruses (AAVs), namely AAV1, 5, 6, 8, 9, and rh10, can be aligned with one of the ttAAV2 serotypes, for example, as shown in Figure 9. This identification allowed us to identify ttAAV2-specific amino acids already present at the same positions in other serotypes. The corresponding residues in the various serotypes were then mutated to the corresponding residues in wtAAV2. These changes demonstrate the importance of ttAAV2-specific residues for the efficacy and biodistribution of each serotype.

As discussed above, various mutant GFP-expressing vectors were then submitted to an initial screening by intracranial (IC) injection of CD1 into neonatal mice. The GFP signal obtained in the injected brains was then compared with that obtained from controls of the corresponding GFP-expressing serotype. The decrease or increase in GFP expression upon mutating the identified amino acids to their corresponding AAV2 residues demonstrates the importance of these particular residues at these specific positions. Where applicable, injected brains were then further sectioned and examined by immunohistochemistry. Additionally, selected mutant serotypes were analyzed by intravenous (IV) injections into neonatal mice to assess the biodistribution of the vectors and compare it with the original, unmutated counterparts.

In further embodiments, the corresponding amino acids identified in ttAAV2 are inserted as key mutations into other known serotypes at relevant positions. Inserting ttAAV2-specific residues into other AAV subtypes allows us to improve the transduction and biodistribution profiles of each serotype.

Example 5

Modification of ttAAV2-specific residues conserved in other AAV serotypes to corresponding AAV2 residues

Comparative analysis of the capsid amino acid sequences of existing adeno-associated serotypes with one of the ttAAV2 serotypes allowed us for the first time to identify ttAAV2-specific residues that are conserved in other serotypes (see Figure 9). These residues consist of S162, S205, S312, G548, S585, and T588. Each non-AAV2 serotype contains one or a combination of several of these residues at the corresponding amino acid position in its sequence.

In specific embodiments, each of these residues in the different serotypes is converted to the corresponding amino acid(s) of the non-mutant type of AAV2, and the transduction efficiency of these new mutants is examined.

1) Modification of the AAV1 serotype

AAV1 contains residues S205, G549, S586, and T589, which correspond to the following residues in ttAAV2: S205, G548, S585, and T588. When the VP1 monomer from AAV1 was three-dimensionally aligned with VP1 from AAV2, we could verify that the corresponding residues in wtAAV2, namely T205, E548, R585, and R588, are predominantly at the corresponding positions in the 3D structure (Figure 21). Thus, we concluded that it would be essential to convert each of the ttAAV2-specific residues in AAV1 to the corresponding equivalents of the wild-type AAV2 without affecting the three-dimensional structure of the protein. In specific embodiments, the following mutations were made in the AAV1 capsid sequence: S205T, G549E, S586R, T589R.

2) Modification of the 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 corresponding positions with S575 and T578 in AAV5 in the 3D structure. Although residue E548 in AAV2 did not correspond substantially to residue G537 in AAV5 according to the 3D structure (Figure 22), we nevertheless decided to include it in the study because both residues are relatively close in space. Therefore, in specific embodiments, the following mutations were made in the AAV5 capsid sequence: G537E, S575R, T578R.

3) Modification of the AAV6 serotype

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, namely T205, E548, R585, and R588 (Figure 23), are predominantly in the corresponding positions on the VP1 3D structures. Therefore, in specific embodiments, the following mutations were made in the AAV6 capsid sequence: S205T, G549E, S586R, T589R.

4) Modification of the AAV8 serotype

AAV8 contains residues S315 and T591, which correspond to the following residues in ttAAV2: S312 and T588. The corresponding residues in wtAAV2, namely N312 and R588, are predominantly located at the corresponding positions on the VP1 3D structures (Figure 24). Therefore, in specific embodiments, the following mutations were made in the AAV8 capsid sequence: S315N, T591R.

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

We mutated the AAV8 capsid sequence using site-directed mutagenesis, thereby constructing the AAV8-S315N vector plasmid. This plasmid was used to produce recombinant AAV8-S315N vectors expressing the ITR-containing CMV-GFP transgene by double transfection of 293T cells. The vector was then purified from cell lysates and harvested culture supernatants by FPLC affinity chromatography using AVB Sepharose resin. Capsid titer and vector genome titer were assessed using SDS-PAGE and qPCR, respectively.

The mutant AAV8-S315N vector expressing GFP was screened by systemic injection of CD1 into neonatal mice. The titer of the corresponding AAV8 vector served as a control. GFP expression was then analyzed in various organs following intrajugular vein injection of 2 × 10 ¹¹ vg, focusing primarily on the liver, where AAV8 had previously demonstrated some strong transduction efficiency.

5) Modification of AAV9 serotype

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, namely A162, T205, E548, and R585, are predominantly in the corresponding positions on the VP1 3D structures (Figure 25). Therefore, in specific embodiments, the following mutations were made in the AAV8 capsid sequence: S162A, S205T, G549E, and S586R.

6) Modification of AAVrh10 serotype

AAVrh10 contains residue G551, which corresponds to G548 in ttAAV2. Given the conservation of this residue and its position across different serotypes, we hypothesize that G551 in AAVrh10 will align three-dimensionally with E548 in wtAAV2. Therefore, in one embodiment, the following mutation is made in the AAVrh10 capsid sequence: G551E.

7) Modification of AAV3B and AAV-LK03 serotypes

Similar to the AAV8 serotype, we observed that the AAV3B serotype also contains an internal serine at position 312 after alignment of the VP1 capsid protein sequence with one of the AAV-TTs (see AAV3B capsid sequence in Figure 38).

The recently described LK03 AAV vector, a chimeric capsid composed of five different parental AAV capsids constructed by M.A. Kay using DNA shuffling, also contains residue S312 in the capsid interior (see Lisowski et al., Selection and evaluation of clically relevant AAV variants in a xenograft liver model, Nature 506, 382–386 (2014)). The AAV-LK03 capsid sequence is disclosed in WO2013/029030 and is shown in Figure 39.

Therefore, in the following embodiments, the AAV3B and AAV-LK03 vectors are mutated by introducing the S312N amino acid change in both vectors. These new mutants, along with their corresponding AAV control serotypes, are also tested by intrajugular injection into neonatal mice. GFP expression obtained in various harvested organs is then analyzed.

Example 6

Identification of ttAAV2-specific amino acids that are transferable between AAV serotypes

In further embodiments, key amino acids identified during characterization of ttAAV2 are inserted into other known serotypes at corresponding positions. The newly constructed vectors are then tested using the corresponding unmutated serotypes as controls. This confirms the importance of individual amino acids at specific positions on AAV capsids, regardless of serotype.

1) Residues S585, T588, S312, D546, G548 and S593

AAV1, AAV5, and AAV6 naturally contain the same amino acid residue at positions in their capsid protein sequences corresponding to G548, S585, and T588 in ttAAV2. Therefore, in further embodiments, the capsid proteins in these serotypes are mutated at the corresponding positions to include other residues S312, D546, and S593 present in ttAAV2. Similarly, AAV8, which already contains the same amino acid residue as ttAAV2 at positions corresponding to S312 and T588 in ttAAV2, is further mutated to contain residues corresponding to S585, D546, G548, and S593 in ttAAV2. AAV9, which already contains the corresponding residues G548 and S585 in ttAAV2, is mutated to include residues corresponding to T588, S312, D546, and S593 in ttAAV2. Finally, AAV10, which already contains a residue corresponding to G548, is further modified to also contain residues corresponding to S585, T588, S312, G548, and S593.

2) Residues I125, A151, S162, S205, M457, A492, D499 and Y533

AAV1 and AAV6 already naturally contain the S205 residue. Therefore, in the following embodiments, these serotypes are mutated at positions corresponding to residues I125, A151, S162, M457, A492, D499, and Y533 in ttAAV2. The analogous AAV9, which already contains residues S162 and S205, is further mutated to contain residues corresponding to I125, A151, M457, A492, D499, and Y533 in ttAAV2. Finally, AAV5, 8, and 10 are modified to exhibit residues corresponding to I125, A151, S162, S205, M457, A492, D499, and Y533 in ttAAV2.

The positions of the mutations present in ttAAV2 and the corresponding residues present in the wild-type VP1 capsid protein sequences of other AAV serotypes are shown in Table 1 below. In general, non-AAV2 vector variants can be constructed by mutating any of the residues shown in Table 1 for these serotypes. Residues shown in italics represent residues already present in ttAAV2 at the corresponding position. In a preferred embodiment, non-AAV2 serotypes are mutated at one or more residues shown in non-italic font. In this sense, the preferred properties exhibited by ttAAV2 can be transferred to alternative AAV serotypes.

Table 1

ttAAV2 AAV1 AAV5 AAV6 AAV8 AAV9 AAV10 I125 V125 V124 V125 V125 L125 V125 A151 Q151 K150 Q151 Q151 Q151 Q151 S162 T162 K153 T162 K163 S162 K163 S205 S205 A195 S205 A206 S205 A206 S312 N313 R303 N313 S315 N314 N315 M457 N458 T444 N458 T460 Q458 T460 A492 K493 S479 K493 T495 V493 L495 D499 N500 V486 N500 N502 E500 N502 Y533 F534 T520 F534 F536 F534 F536 D546 S547 P533 S547 N549 G547 G549 G548 G549 G537 G549 A551 G549 G551 S585 S586 S575 S586 Q588 S586 Q588 T588 T589 T578 T589 T591 A589 A591 S593 G594 G583 G594 G596 G594 G596

Additional embodiments of the invention

The invention further relates to further aspects as defined in the following brief paragraphs:

1. Recombinant adeno-associated virus (AAV) vector containing:

(a) a variant AAV capsid protein, wherein the variant AAV capsid protein comprises at least one amino acid substitution compared to a wild-type AAV capsid protein; wherein the at least one amino acid substitution is present at a position corresponding to one or more of the following positions in the AAV2 capsid protein sequence: 125, 151, 162, 205, 312, 457, 492, 499, 533, 546, 548, 585, 588 and/or 593; and

(b) a heterologous nucleic acid containing a nucleotide sequence encoding a gene product.

2. A recombinant AAV vector according to paragraph 1, wherein (i) the vector comprises a variant AAV2 capsid protein; (ii) the variant AAV capsid protein comprises the sequence of SEQ ID NO:2, or a sequence having at least 95% sequence identity thereto; (iii) the wild-type AAV capsid protein is from AAV2; and/or (iv) the wild-type AAV capsid protein comprises the sequence of SEQ ID NO:1.

3. The recombinant AAV vector of paragraph 2, wherein 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.

4. The recombinant AAV vector according to paragraph 2 or paragraph 3, wherein the variant AAV2 capsid protein comprises one or more of the following amino acid substitutions compared to the capsid protein of the wild-type AAV2: V125I, V151A, A162S, T205S, N312S, Q457M, S492A, E499D, F533Y, G546D, E548G, R585S, R588T and/or A593S.

5. A recombinant AAV vector according to any of paragraphs 1-3, wherein the variant AAV capsid protein is from AAV1, AAV5, AAV6, AAV8, AAV9 or AAV10.

6. A recombinant AAV vector according to paragraph 5, wherein (i) the vector 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 from AAV1; and/or (iv) the wild-type AAV capsid protein comprises the sequence of SEQ ID NO:3;

and wherein at least one amino acid substitution is present at one or more of the following positions in the AAV1 capsid protein sequence: 125, 151, 162, 205, 313, 458, 493, 500, 534, 547, 549, 586, 589 and/or 594.

7. A recombinant AAV vector according to claim 6, wherein the variant AAV1 capsid protein comprises one or more of the following amino acid substitutions compared to the capsid protein of the wild-type AAV1:

(a) V125I, Q151A, T162S, N313S, N458M, K493A, N500D, F534Y, S547D, and/or G594S; and/or

(b) S205T, G549E, S586R and/or T589R.

8. A recombinant AAV vector according to paragraph 5, wherein (i) the vector 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 from AAV5; and/or (iv) the wild-type AAV capsid protein comprises the sequence of SEQ ID NO:4;

and wherein at least one amino acid substitution is present at one or more of the following positions in the AAV5 capsid protein sequence: 124, 150, 153, 195, 303, 444, 479, 486, 520, 533, 537, 575, 578 and/or 583.

9. A recombinant AAV vector according to claim 8, wherein the variant AAV5 capsid protein comprises one or more of the following amino acid substitutions compared to the non-mutant 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.

10. The recombinant AAV vector of claim 5, wherein (i) the vector 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 from AAV6; and/or (iv) the wild-type AAV capsid protein comprises the sequence of SEQ ID NO:5;

and wherein at least one amino acid substitution is present 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.

11. A recombinant AAV vector according to claim 10, wherein the variant AAV6 capsid protein comprises one or more of the following amino acid substitutions compared to the non-mutant 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.

12. The recombinant AAV vector of claim 5, wherein (i) the vector 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 from AAV8; and/or (iv) the wild-type AAV capsid protein comprises the sequence of SEQ ID NO:6;

and wherein at least one amino acid substitution is present at one or more of the following positions in the AAV8 capsid protein sequence: 125, 151, 163, 206, 315, 460, 495, 502, 536, 549, 551, 588, 591 and/or 596.

13. A recombinant AAV vector according to claim 12, wherein the variant AAV8 capsid protein comprises one or more of the following amino acid substitutions compared to the non-mutant 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.

14. The recombinant AAV vector of claim 5, wherein (i) the vector 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 from AAV9; and/or (iv) the wild-type AAV capsid protein comprises the sequence of SEQ ID NO:7;

and wherein at least one amino acid substitution is present at one or more of the following positions in the AAV9 capsid protein sequence: 125, 151, 162, 205, 314, 458, 493, 500, 534, 547, 549, 586, 589 and/or 594.

15. A recombinant AAV vector according to claim 14, wherein the variant AAV9 capsid protein comprises one or more of the following amino acid substitutions compared to the non-mutant 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.

16. The recombinant AAV vector of paragraph 5, wherein (i) the vector comprises a variant AAVrh10 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 from AAVrh10; and/or (iv) the wild-type AAV capsid protein comprises the sequence of SEQ ID NO:8;

and wherein at least one amino acid substitution is present 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.

17. A recombinant AAV vector according to claim 16, wherein the variant AAVrh10 capsid protein comprises one or more of the following amino acid substitutions compared to the non-mutant type AAVrh10 capsid protein:

(a) V125I, Q151A, K163S, A206S, N315S, T460M, L495A, N502D, F536Y, G549D, Q588S, A591T and/or G596S; and/or

(b) G551E.

18. A recombinant AAV vector according to any of the preceding paragraphs, wherein the recombinant AAV vector exhibits increased transduction of neuronal or retinal tissue compared to an AAV vector containing the corresponding wild-type AAV capsid protein.

19. A recombinant AAV vector according to any of the preceding paragraphs, wherein the gene product comprises an interfering RNA or an aptamer.

20. A recombinant AAV vector according to any of paragraphs 1-18, wherein the gene product comprises a polypeptide.

21. A recombinant AAV vector according to claim 20, wherein the gene product comprises a neuroprotective polypeptide, an antiangiogenic polypeptide, or a polypeptide that improves neuronal or retinal cell function.

22. A recombinant AAV vector according to paragraph 21, wherein the gene product comprises glial cell line-derived neurotrophic factor, fibroblast growth factor, nerve growth factor, brain-derived neurotrophic factor, rhodopsin, retinoschisin, RPE65, or peripherin.

23. A pharmaceutical composition containing:

(a) a recombinant AAV vector according to any of the preceding paragraphs; and

(b) a pharmaceutically acceptable excipient.

24. A method of delivering a gene product to neuronal or retinal tissue in a subject, wherein the method comprises administering to the subject a recombinant AAV vector or a pharmaceutical composition according to any of the preceding paragraphs.

25. A method of treating a neurological or ocular disorder, wherein the method comprises administering to the subject a recombinant AAV vector or a pharmaceutical composition according to any of the preceding paragraphs.

26. A recombinant AAV vector or pharmaceutical composition according to any of paragraphs 1-23, for use in the treatment of a neurological or ocular disorder.

27. The method, recombinant AAV vector or pharmaceutical composition for use according to any of paragraphs 24-26, wherein the neurological disorder is a neurodegenerative disease.

28. The method, recombinant AAV vector, or pharmaceutical composition for use according to any of paragraphs 24-26, wherein the ocular disorder is glaucoma, retinitis pigmentosa, macular degeneration, retinoschisis, or diabetic retinopathy.

29. An isolated variant AAV capsid protein, wherein the variant AAV capsid protein comprises at least one amino acid substitution compared to a wild-type AAV capsid protein; wherein the at least one amino acid substitution is present at one or more of the following positions in the AAV2 capsid protein sequence: 125, 151, 162, 205, 312, 457, 492, 499, 533, 546, 548, 585, 588 and/or 593; or at one or more corresponding positions in an alternative AAV capsid protein sequence.

30. An isolated nucleic acid comprising a nucleotide sequence that encodes a variant AAV capsid protein as defined in paragraph 29.

31. An isolated host cell containing a nucleic acid as defined in paragraph 30.

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SEQUENCE LIST

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AIKEN SCHOOL OF MEDICINE AT MOUNT SINAI

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Lys Ser Gly Gln Gln Pro Ala Arg Lys Arg Leu Asn Phe Gly Gln Thr

165 170 175

Gly Asp Ala Asp Ser Val Pro Asp Pro Gln Pro Leu Gly Gln Pro Pro

180 185 190

Ala Ala Pro Ser Gly Leu Gly Thr Asn Thr Met Ala Ser Gly Ser Gly

195 200 205

Ala Pro Met Ala Asp Asn Asn Glu Gly Ala Asp Gly Val Gly Asn Ser

210 215 220

Ser Gly Asn Trp His Cys Asp Ser Thr Trp Met Gly Asp Arg Val Ile

225 230 235 240

Thr Thr Ser Thr Arg Thr Trp Ala Leu Pro Thr Tyr Asn Asn His Leu

245 250 255

Tyr Lys Gln Ile Ser Ser Gln Ser Gly Ala Ser Asn Asp Asn His Tyr

260 265 270

Phe Gly Tyr Ser Thr Pro Trp Gly Tyr Phe Asp Phe Asn Arg Phe His

275 280 285

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

290 295 300

Gly Phe Arg Pro Lys Arg Leu Ser Phe Lys Leu Phe Asn Ile Gln Val

305 310 315 320

Lys Glu Val Thr Gln Asn Asp Gly Thr Thr Thr Ile Ala Asn Asn Leu

325 330 335

Thr Ser Thr Val Gln Val Phe Thr Asp Ser Glu Tyr Gln Leu Pro Tyr

340 345 350

Val Leu Gly Ser Ala His Gln Gly Cys Leu Pro Pro Phe Pro Ala Asp

355 360 365

Val Phe Met Val Pro Gln Tyr Gly Tyr Leu Thr Leu Asn Asn Gly Ser

370 375 380

Gln Ala Val Gly Arg Ser Ser Phe Tyr Cys Leu Glu Tyr Phe Pro Ser

385 390 395 400

Gln Met Leu Arg Thr Gly Asn Asn Phe Thr Phe Ser Tyr Thr Phe Glu

405 410 415

Asp Val Pro Phe His Ser Ser Tyr Ala His Ser Gln Ser Leu Asp Arg

420 425 430

Leu Met Asn Pro Leu Ile Asp Gln Tyr Leu Tyr Tyr Leu Ser Arg Thr

435 440 445

Asn Thr Pro Ser Gly Thr Thr Thr Met Ser Arg Leu Gln Phe Ser Gln

450 455 460

Ala Gly Ala Ser Asp Ile Arg Asp Gln Ser Arg Asn Trp Leu Pro Gly

465 470 475 480

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

485 490 495

Asn Ser Asp Tyr Ser Trp Thr Gly Ala Thr Lys Tyr His Leu Asn Gly

500 505 510

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

515 520 525

Asp Glu Glu Lys Tyr Phe Pro Gln Ser Gly Val Leu Ile Phe Gly Lys

530 535 540

Gln Asp Ser Gly Lys Thr Asn Val Asp Ile Glu Lys Val Met Ile Thr

545 550 555 560

Asp Glu Glu Glu Ile Arg Thr Thr Asn Pro Val Ala Thr Glu Gln Tyr

565 570 575

Gly Ser Val Ser Thr Asn Leu Gln Ser Gly Asn Thr Gln Ala Ala Thr

580 585 590

Ser Asp Val Asn Thr Gln Gly Val Leu Pro Gly Met Val Trp Gln Asp

595 600 605

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

610 615 620

Asp Gly His Phe His Pro Ser Pro Leu Met Gly Gly Phe Gly Leu Lys

625 630 635 640

His Pro Pro Pro Gln Ile Leu Ile Lys Asn Thr Pro Val Pro Ala Asn

645 650 655

Pro Ser Thr Thr Phe Ser Ala Ala Lys Phe Ala Ser Phe Ile Thr Gln

660 665 670

Tyr Ser Thr Gly Gln Val Ser Val Glu Ile Glu Trp Glu Leu Gln Lys

675 680 685

Glu Asn Ser Lys Arg Trp Asn Pro Glu Ile Gln Tyr Thr Ser Asn Tyr

690 695 700

Asn Lys Ser Val Asn Val Asp Phe Thr Val Asp Thr Asn Gly Val Tyr

705 710 715 720

Ser Glu Pro Arg Pro Ile Gly Thr Arg Tyr Leu Thr Arg Asn Leu

725 730 735

<210> 3

<211> 736

<212> PRT

<213> Adeno-associated virus-1

<400> 3

Met Ala Ala Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Asn Leu Ser

1 5 10 15

Glu Gly Ile Arg Glu Trp Trp Asp Leu Lys Pro Gly Ala Pro Lys Pro

20 25 30

Lys Ala Asn Gln Gln Lys Gln Asp Asp Gly Arg Gly Leu Val Leu Pro

35 40 45

Gly Tyr Lys Tyr Leu Gly Pro Phe Asn Gly Leu Asp Lys Gly Glu Pro

50 55 60

Val Asn Ala Ala Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp

65 70 75 80

Gln Gln Leu Lys Ala Gly Asp Asn Pro Tyr Leu Arg Tyr Asn His Ala

85 90 95

Asp Ala Glu Phe Gln Glu Arg Leu Gln Glu Asp Thr Ser Phe Gly Gly

100 105 110

Asn Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg Val Leu Glu Pro

115 120 125

Leu Gly Leu Val Glu Glu Gly Ala Lys Thr Ala Pro Gly Lys Lys Arg

130 135 140

Pro Val Glu Gln Ser Pro Gln Glu Pro Asp Ser Ser Ser Gly Ile Gly

145 150 155 160

Lys Thr Gly Gln Gln Pro Ala Lys Lys Arg Leu Asn Phe Gly Gln Thr

165 170 175

Gly Asp Ser Glu Ser Val Pro Asp Pro Gln Pro Leu Gly Glu Pro Pro

180 185 190

Ala Thr Pro Ala Ala Val Gly Pro Thr Thr Met Ala Ser Gly Gly Gly

195 200 205

Ala Pro Met Ala Asp Asn Asn Glu Gly Ala Asp Gly Val Gly Asn Ala

210 215 220

Ser Gly Asn Trp His Cys Asp Ser Thr Trp Leu Gly Asp Arg Val Ile

225 230 235 240

Thr Thr Ser Thr Arg Thr Trp Ala Leu Pro Thr Tyr Asn Asn His Leu

245 250 255

Tyr Lys Gln Ile Ser Ser Ala Ser Thr Gly Ala Ser Asn Asp Asn His

260 265 270

Tyr Phe Gly Tyr Ser Thr Pro Trp Gly Tyr Phe Asp Phe Asn Arg Phe

275 280 285

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

290 295 300

Trp Gly Phe Arg Pro Lys Arg Leu Asn Phe Lys Leu Phe Asn Ile Gln

305 310 315 320

Val Lys Glu Val Thr Thr Asn Asp Gly Val Thr Thr Ile Ala Asn Asn

325 330 335

Leu Thr Ser Thr Val Gln Val Phe Ser Asp Ser Glu Tyr Gln Leu Pro

340 345 350

Tyr Val Leu Gly Ser Ala His Gln Gly Cys Leu Pro Pro Phe Pro Ala

355 360 365

Asp Val Phe Met Ile Pro Gln Tyr Gly Tyr Leu Thr Leu Asn Asn Gly

370 375 380

Ser Gln Ala Val Gly Arg Ser Ser Phe Tyr Cys Leu Glu Tyr Phe Pro

385 390 395 400

Ser Gln Met Leu Arg Thr Gly Asn Asn Phe Thr Phe Ser Tyr Thr Phe

405 410 415

Glu Glu Val Pro Phe His Ser Ser Tyr Ala His Ser Gln Ser Leu Asp

420 425 430

Arg Leu Met Asn Pro Leu Ile Asp Gln Tyr Leu Tyr Tyr Leu Asn Arg

435 440 445

Thr Gln Asn Gln Ser Gly Ser Ala Gln Asn Lys Asp Leu Leu Phe Ser

450 455 460

Arg Gly Ser Pro Ala Gly Met Ser Val Gln Pro Lys Asn Trp Leu Pro

465 470 475 480

Gly Pro Cys Tyr Arg Gln Gln Arg Val Ser Lys Thr Lys Thr Asp Asn

485 490 495

Asn Asn Ser Asn Phe Thr Trp Thr Gly Ala Ser Lys Tyr Asn Leu Asn

500 505 510

Gly Arg Glu Ser Ile Ile Asn Pro Gly Thr Ala Met Ala Ser His Lys

515 520 525

Asp Asp Glu Asp Lys Phe Phe Pro Met Ser Gly Val Met Ile Phe Gly

530 535 540

Lys Glu Ser Ala Gly Ala Ser Asn Thr Ala Leu Asp Asn Val Met Ile

545 550 555 560

Thr Asp Glu Glu Glu Ile Lys Ala Thr Asn Pro Val Ala Thr Glu Arg

565 570 575

Phe Gly Thr Val Ala Val Asn Phe Gln Ser Ser Ser Thr Asp Pro Ala

580 585 590

Thr Gly Asp Val His Ala Met Gly Ala Leu Pro Gly Met Val Trp Gln

595 600 605

Asp Arg Asp Val Tyr Leu Gln Gly Pro Ile Trp Ala Lys Ile Pro His

610 615 620

Thr Asp Gly His Phe His Pro Ser Pro Leu Met Gly Gly Phe Gly Leu

625 630 635 640

Lys Asn Pro Pro Pro Gln Ile Leu Ile Lys Asn Thr Pro Val Pro Ala

645 650 655

Asn Pro Pro Ala Glu Phe Ser Ala Thr Lys Phe Ala Ser Phe Ile Thr

660 665 670

Gln Tyr Ser Thr Gly Gln Val Ser Val Glu Ile Glu Trp Glu Leu Gln

675 680 685

Lys Glu Asn Ser Lys Arg Trp Asn Pro Glu Val Gln Tyr Thr Ser Asn

690 695 700

Tyr Ala Lys Ser Ala Asn Val Asp Phe Thr Val Asp Asn Asn Gly Leu

705 710 715 720

Tyr Thr Glu Pro Arg Pro Ile Gly Thr Arg Tyr Leu Thr Arg Pro Leu

725 730 735

<210> 4

<211> 724

<212> PRT

<213> Adeno-associated virus-5

<400> 4

Met Ser Phe Val Asp His Pro Pro Asp Trp Leu Glu Glu Val Gly Glu

1 5 10 15

Gly Leu Arg Glu Phe Leu Gly Leu Glu Ala Gly Pro Pro Lys Pro Lys

20 25 30

Pro Asn Gln Gln His Gln Asp Gln Ala Arg Gly Leu Val Leu Pro Gly

35 40 45

Tyr Asn Tyr Leu Gly Pro Gly Asn Gly Leu Asp Arg Gly Glu Pro Val

50 55 60

Asn Arg Ala Asp Glu Val Ala Arg Glu His Asp Ile Ser Tyr Asn Glu

65 70 75 80

Gln Leu Glu Ala Gly Asp Asn Pro Tyr Leu Lys Tyr Asn His Ala Asp

85 90 95

Ala Glu Phe Gln Glu Lys Leu Ala Asp Asp Thr Ser Phe Gly Gly Asn

100 105 110

Leu Gly Lys Ala Val Phe Gln Ala Lys Lys Arg Val Leu Glu Pro Phe

115 120 125

Gly Leu Val Glu Glu Gly Ala Lys Thr Ala Pro Thr Gly Lys Arg Ile

130 135 140

Asp Asp His Phe Pro Lys Arg Lys Lys Ala Arg Thr Glu Glu Asp Ser

145 150 155 160

Lys Pro Ser Thr Ser Ser Asp Ala Glu Ala Gly Pro Ser Gly Ser Gln

165 170 175

Gln Leu Gln Ile Pro Ala Gln Pro Ala Ser Ser Leu Gly Ala Asp Thr

180 185 190

Met Ser Ala Gly Gly Gly Gly Pro Leu Gly Asp Asn Asn Gln Gly Ala

195 200 205

Asp Gly Val Gly Asn Ala Ser Gly Asp Trp His Cys Asp Ser Thr Trp

210 215 220

Met Gly Asp Arg Val Val Thr Lys Ser Thr Arg Thr Trp Val Leu Pro

225 230 235 240

Ser Tyr Asn Asn His Gln Tyr Arg Glu Ile Lys Ser Gly Ser Val Asp

245 250 255

Gly Ser Asn Ala Asn Ala Tyr Phe Gly Tyr Ser Thr Pro Trp Gly Tyr

260 265 270

Phe Asp Phe Asn Arg Phe His Ser His Trp Ser Pro Arg Asp Trp Gln

275 280 285

Arg Leu Ile Asn Asn Tyr Trp Gly Phe Arg Pro Arg Ser Leu Arg Val

290 295 300

Lys Ile Phe Asn Ile Gln Val Lys Glu Val Thr Val Gln Asp Ser Thr

305 310 315 320

Thr Thr Ile Ala Asn Asn Leu Thr Ser Thr Val Gln Val Phe Thr Asp

325 330 335

Asp Asp Tyr Gln Leu Pro Tyr Val Val Gly Asn Gly Thr Glu Gly Cys

340 345 350

Leu Pro Ala Phe Pro Pro Gln Val Phe Thr Leu Pro Gln Tyr Gly Tyr

355 360 365

Ala Thr Leu Asn Arg Asp Asn Thr Glu Asn Pro Thr Glu Arg Ser Ser

370 375 380

Phe Phe Cys Leu Glu Tyr Phe Pro Ser Lys Met Leu Arg Thr Gly Asn

385 390 395 400

Asn Phe Glu Phe Thr Tyr Asn Phe Glu Glu Val Pro Phe His Ser Ser

405 410 415

Phe Ala Pro Ser Gln Asn Leu Phe Lys Leu Ala Asn Pro Leu Val Asp

420 425 430

Gln Tyr Leu Tyr Arg Phe Val Ser Thr Asn Asn Thr Gly Gly Val Gln

435 440 445

Phe Asn Lys Asn Leu Ala Gly Arg Tyr Ala Asn Thr Tyr Lys Asn Trp

450 455 460

Phe Pro Gly Pro Met Gly Arg Thr Gln Gly Trp Asn Leu Gly Ser Gly

465 470 475 480

Val Asn Arg Ala Ser Val Ser Ala Phe Ala Thr Thr Asn Arg Met Glu

485 490 495

Leu Glu Gly Ala Ser Tyr Gln Val Pro Pro Gln Pro Asn Gly Met Thr

500 505 510

Asn Asn Leu Gln Gly Ser Asn Thr Tyr Ala Leu Glu Asn Thr Met Ile

515 520 525

Phe Asn Ser Gln Pro Ala Asn Pro Gly Thr Thr Ala Thr Tyr Leu Glu

530 535 540

Gly Asn Met Leu Ile Thr Ser Glu Ser Glu Thr Gln Pro Val Asn Arg

545 550 555 560

Val Ala Tyr Asn Val Gly Gly Gln Met Ala Thr Asn Asn Gln Ser Ser

565 570 575

Thr Thr Ala Pro Ala Thr Gly Thr Tyr Asn Leu Gln Glu Ile Val Pro

580 585 590

Gly Ser Val Trp Met Glu Arg Asp Val Tyr Leu Gln Gly Pro Ile Trp

595 600 605

Ala Lys Ile Pro Glu Thr Gly Ala His Phe His Pro Ser Pro Ala Met

610 615 620

Gly Gly Phe Gly Leu Lys His Pro Pro Pro Met Met Leu Ile Lys Asn

625 630 635 640

Thr Pro Val Pro Gly Asn Ile Thr Ser Phe Ser Asp Val Pro Val Ser

645 650 655

Ser Phe Ile Thr Gln Tyr Ser Thr Gly Gln Val Thr Val Glu Met Glu

660 665 670

Trp Glu Leu Lys Lys Glu Asn Ser Lys Arg Trp Asn Pro Glu Ile Gln

675 680 685

Tyr Thr Asn Asn Tyr Asn Asp Pro Gln Phe Val Asp Phe Ala Pro Asp

690 695 700

Ser Thr Gly Glu Tyr Arg Thr Thr Arg Pro Ile Gly Thr Arg Tyr Leu

705 710 715 720

Thr Arg Pro Leu

<210> 5

<211> 736

<212> PRT

<213> Adeno-associated virus-6

<400> 5

Met Ala Ala Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Asn Leu Ser

1 5 10 15

Glu Gly Ile Arg Glu Trp Trp Asp Leu Lys Pro Gly Ala Pro Lys Pro

20 25 30

Lys Ala Asn Gln Gln Lys Gln Asp Asp Gly Arg Gly Leu Val Leu Pro

35 40 45

Gly Tyr Lys Tyr Leu Gly Pro Phe Asn Gly Leu Asp Lys Gly Glu Pro

50 55 60

Val Asn Ala Ala Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp

65 70 75 80

Gln Gln Leu Lys Ala Gly Asp Asn Pro Tyr Leu Arg Tyr Asn His Ala

85 90 95

Asp Ala Glu Phe Gln Glu Arg Leu Gln Glu Asp Thr Ser Phe Gly Gly

100 105 110

Asn Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg Val Leu Glu Pro

115 120 125

Phe Gly Leu Val Glu Glu Gly Ala Lys Thr Ala Pro Gly Lys Lys Arg

130 135 140

Pro Val Glu Gln Ser Pro Gln Glu Pro Asp Ser Ser Ser Gly Ile Gly

145 150 155 160

Lys Thr Gly Gln Gln Pro Ala Lys Lys Arg Leu Asn Phe Gly Gln Thr

165 170 175

Gly Asp Ser Glu Ser Val Pro Asp Pro Gln Pro Leu Gly Glu Pro Pro

180 185 190

Ala Thr Pro Ala Ala Val Gly Pro Thr Thr Met Ala Ser Gly Gly Gly

195 200 205

Ala Pro Met Ala Asp Asn Asn Glu Gly Ala Asp Gly Val Gly Asn Ala

210 215 220

Ser Gly Asn Trp His Cys Asp Ser Thr Trp Leu Gly Asp Arg Val Ile

225 230 235 240

Thr Thr Ser Thr Arg Thr Trp Ala Leu Pro Thr Tyr Asn Asn His Leu

245 250 255

Tyr Lys Gln Ile Ser Ser Ala Ser Thr Gly Ala Ser Asn Asp Asn His

260 265 270

Tyr Phe Gly Tyr Ser Thr Pro Trp Gly Tyr Phe Asp Phe Asn Arg Phe

275 280 285

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

290 295 300

Trp Gly Phe Arg Pro Lys Arg Leu Asn Phe Lys Leu Phe Asn Ile Gln

305 310 315 320

Val Lys Glu Val Thr Thr Asn Asp Gly Val Thr Thr Ile Ala Asn Asn

325 330 335

Leu Thr Ser Thr Val Gln Val Phe Ser Asp Ser Glu Tyr Gln Leu Pro

340 345 350

Tyr Val Leu Gly Ser Ala His Gln Gly Cys Leu Pro Pro Phe Pro Ala

355 360 365

Asp Val Phe Met Ile Pro Gln Tyr Gly Tyr Leu Thr Leu Asn Asn Gly

370 375 380

Ser Gln Ala Val Gly Arg Ser Ser Phe Tyr Cys Leu Glu Tyr Phe Pro

385 390 395 400

Ser Gln Met Leu Arg Thr Gly Asn Asn Phe Thr Phe Ser Tyr Thr Phe

405 410 415

Glu Asp Val Pro Phe His Ser Ser Tyr Ala His Ser Gln Ser Leu Asp

420 425 430

Arg Leu Met Asn Pro Leu Ile Asp Gln Tyr Leu Tyr Tyr Leu Asn Arg

435 440 445

Thr Gln Asn Gln Ser Gly Ser Ala Gln Asn Lys Asp Leu Leu Phe Ser

450 455 460

Arg Gly Ser Pro Ala Gly Met Ser Val Gln Pro Lys Asn Trp Leu Pro

465 470 475 480

Gly Pro Cys Tyr Arg Gln Gln Arg Val Ser Lys Thr Lys Thr Asp Asn

485 490 495

Asn Asn Ser Asn Phe Thr Trp Thr Gly Ala Ser Lys Tyr Asn Leu Asn

500 505 510

Gly Arg Glu Ser Ile Ile Asn Pro Gly Thr Ala Met Ala Ser His Lys

515 520 525

Asp Asp Lys Asp Lys Phe Phe Pro Met Ser Gly Val Met Ile Phe Gly

530 535 540

Lys Glu Ser Ala Gly Ala Ser Asn Thr Ala Leu Asp Asn Val Met Ile

545 550 555 560

Thr Asp Glu Glu Glu Ile Lys Ala Thr Asn Pro Val Ala Thr Glu Arg

565 570 575

Phe Gly Thr Val Ala Val Asn Leu Gln Ser Ser Ser Thr Asp Pro Ala

580 585 590

Thr Gly Asp Val His Val Met Gly Ala Leu Pro Gly Met Val Trp Gln

595 600 605

Asp Arg Asp Val Tyr Leu Gln Gly Pro Ile Trp Ala Lys Ile Pro His

610 615 620

Thr Asp Gly His Phe His Pro Ser Pro Leu Met Gly Gly Phe Gly Leu

625 630 635 640

Lys His Pro Pro Pro Gln Ile Leu Ile Lys Asn Thr Pro Val Pro Ala

645 650 655

Asn Pro Pro Ala Glu Phe Ser Ala Thr Lys Phe Ala Ser Phe Ile Thr

660 665 670

Gln Tyr Ser Thr Gly Gln Val Ser Val Glu Ile Glu Trp Glu Leu Gln

675 680 685

Lys Glu Asn Ser Lys Arg Trp Asn Pro Glu Val Gln Tyr Thr Ser Asn

690 695 700

Tyr Ala Lys Ser Ala Asn Val Asp Phe Thr Val Asp Asn Asn Gly Leu

705 710 715 720

Tyr Thr Glu Pro Arg Pro Ile Gly Thr Arg Tyr Leu Thr Arg Pro Leu

725 730 735

<210> 6

<211> 738

<212> PRT

<213> Adeno-associated virus-8

<400> 6

Met Ala Ala Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Asn Leu Ser

1 5 10 15

Glu Gly Ile Arg Glu Trp Trp Ala Leu Lys Pro Gly Ala Pro Lys Pro

20 25 30

Lys Ala Asn Gln Gln Lys Gln Asp Asp Gly Arg Gly Leu Val Leu Pro

35 40 45

Gly Tyr Lys Tyr Leu Gly Pro Phe Asn Gly Leu Asp Lys Gly Glu Pro

50 55 60

Val Asn Ala Ala Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp

65 70 75 80

Gln Gln Leu Gln Ala Gly Asp Asn Pro Tyr Leu Arg Tyr Asn His Ala

85 90 95

Asp Ala Glu Phe Gln Glu Arg Leu Gln Glu Asp Thr Ser Phe Gly Gly

100 105 110

Asn Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg Val Leu Glu Pro

115 120 125

Leu Gly Leu Val Glu Glu Gly Ala Lys Thr Ala Pro Gly Lys Lys Arg

130 135 140

Pro Val Glu Pro Ser Pro Gln Arg Ser Pro Asp Ser Ser Thr Gly Ile

145 150 155 160

Gly Lys Lys Gly Gln Gln Pro Ala Arg Lys Arg Leu Asn Phe Gly Gln

165 170 175

Thr Gly Asp Ser Glu Ser Val Pro Asp Pro Gln Pro Leu Gly Glu Pro

180 185 190

Pro Ala Ala Pro Ser Gly Val Gly Pro Asn Thr Met Ala Ala Gly Gly

195 200 205

Gly Ala Pro Met Ala Asp Asn Asn Glu Gly Ala Asp Gly Val Gly Ser

210 215 220

Ser Ser Gly Asn Trp His Cys Asp Ser Thr Trp Leu Gly Asp Arg Val

225 230 235 240

Ile Thr Thr Ser Thr Arg Thr Trp Ala Leu Pro Thr Tyr Asn Asn His

245 250 255

Leu Tyr Lys Gln Ile Ser Asn Gly Thr Ser Gly Gly Ala Thr Asn Asp

260 265 270

Asn Thr Tyr Phe Gly Tyr Ser Thr Pro Trp Gly Tyr Phe Asp Phe Asn

275 280 285

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

290 295 300

Asn Asn Trp Gly Phe Arg Pro Lys Arg Leu Ser Phe Lys Leu Phe Asn

305 310 315 320

Ile Gln Val Lys Glu Val Thr Gln Asn Glu Gly Thr Lys Thr Ile Ala

325 330 335

Asn Asn Leu Thr Ser Thr Ile Gln Val Phe Thr Asp Ser Glu Tyr Gln

340 345 350

Leu Pro Tyr Val Leu Gly Ser Ala His Gln Gly Cys Leu Pro Pro Phe

355 360 365

Pro Ala Asp Val Phe Met Ile Pro Gln Tyr Gly Tyr Leu Thr Leu Asn

370 375 380

Asn Gly Ser Gln Ala Val Gly Arg Ser Ser Phe Tyr Cys Leu Glu Tyr

385 390 395 400

Phe Pro Ser Gln Met Leu Arg Thr Gly Asn Asn Phe Gln Phe Thr Tyr

405 410 415

Thr Phe Glu Asp Val Pro Phe His Ser Ser Tyr Ala His Ser Gln Ser

420 425 430

Leu Asp Arg Leu Met Asn Pro Leu Ile Asp Gln Tyr Leu Tyr Tyr Leu

435 440 445

Ser Arg Thr Gln Thr Thr Gly Gly Thr Ala Asn Thr Gln Thr Leu Gly

450 455 460

Phe Ser Gln Gly Gly Pro Asn Thr Met Ala Asn Gln Ala Lys Asn Trp

465 470 475 480

Leu Pro Gly Pro Cys Tyr Arg Gln Gln Arg Val Ser Thr Thr Thr Gly

485 490 495

Gln Asn Asn Asn Ser Asn Phe Ala Trp Thr Ala Gly Thr Lys Tyr His

500 505 510

Leu Asn Gly Arg Asn Ser Leu Ala Asn Pro Gly Ile Ala Met Ala Thr

515 520 525

His Lys Asp Asp Glu Glu Arg Phe Phe Pro Ser Asn Gly Ile Leu Ile

530 535 540

Phe Gly Lys Gln Asn Ala Ala Arg Asp Asn Ala Asp Tyr Ser Asp Val

545 550 555 560

Met Leu Thr Ser Glu Glu Glu Ile Lys Thr Thr Asn Pro Val Ala Thr

565 570 575

Glu Glu Tyr Gly Ile Val Ala Asp Asn Leu Gln Gln Gln Asn Thr Ala

580 585 590

Pro Gln Ile Gly Thr Val Asn Ser Gln Gly Ala Leu Pro Gly Met Val

595 600 605

Trp Gln Asn Arg Asp Val Tyr Leu Gln Gly Pro Ile Trp Ala Lys Ile

610 615 620

Pro His Thr Asp Gly Asn Phe His Pro Ser Pro Leu Met Gly Gly Phe

625 630 635 640

Gly Leu Lys His Pro Pro Pro Gln Ile Leu Ile Lys Asn Thr Pro Val

645 650 655

Pro Ala Asp Pro Pro Thr Thr Phe Asn Gln Ser Lys Leu Asn Ser Phe

660 665 670

Ile Thr Gln Tyr Ser Thr Gly Gln Val Ser Val Glu Ile Glu Trp Glu

675 680 685

Leu Gln Lys Glu Asn Ser Lys Arg Trp Asn Pro Glu Ile Gln Tyr Thr

690 695 700

Ser Asn Tyr Tyr Lys Ser Thr Ser Val Asp Phe Ala Val Asn Thr Glu

705 710 715 720

Gly Val Tyr Ser Glu Pro Arg Pro Ile Gly Thr Arg Tyr Leu Thr Arg

725 730 735

Asn Leu

<210> 7

<211> 736

<212> PRT

<213> Adeno-associated virus-9

<400> 7

Met Ala Ala Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Asn Leu Ser

1 5 10 15

Glu Gly Ile Arg Glu Trp Trp Ala Leu Lys Pro Gly Ala Pro Gln Pro

20 25 30

Lys Ala Asn Gln Gln His Gln Asp Asn Ala Arg Gly Leu Val Leu Pro

35 40 45

Gly Tyr Lys Tyr Leu Gly Pro Gly Asn Gly Leu Asp Lys Gly Glu Pro

50 55 60

Val Asn Ala Ala Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp

65 70 75 80

Gln Gln Leu Lys Ala Gly Asp Asn Pro Tyr Leu Lys Tyr Asn His Ala

85 90 95

Asp Ala Glu Phe Gln Glu Arg Leu Lys Glu Asp Thr Ser Phe Gly Gly

100 105 110

Asn Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg Leu Leu Glu Pro

115 120 125

Leu Gly Leu Val Glu Glu Ala Ala Lys Thr Ala Pro Gly Lys Lys Arg

130 135 140

Pro Val Glu Gln Ser Pro Gln Glu Pro Asp Ser Ser Ala Gly Ile Gly

145 150 155 160

Lys Ser Gly Ala Gln Pro Ala Lys Lys Arg Leu Asn Phe Gly Gln Thr

165 170 175

Gly Asp Thr Glu Ser Val Pro Asp Pro Gln Pro Ile Gly Glu Pro Pro

180 185 190

Ala Ala Pro Ser Gly Val Gly Ser Leu Thr Met Ala Ser Gly Gly Gly

195 200 205

Ala Pro Val Ala Asp Asn Asn Glu Gly Ala Asp Gly Val Gly Ser Ser

210 215 220

Ser Gly Asn Trp His Cys Asp Ser Gln Trp Leu Gly Asp Arg Val Ile

225 230 235 240

Thr Thr Ser Thr Arg Thr Trp Ala Leu Pro Thr Tyr Asn Asn His Leu

245 250 255

Tyr Lys Gln Ile Ser Asn Ser Thr Ser Gly Gly Ser Ser Asn Asp Asn

260 265 270

Ala Tyr Phe Gly Tyr Ser Thr Pro Trp Gly Tyr Phe Asp Phe Asn Arg

275 280 285

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

290 295 300

Asn Trp Gly Phe Arg Pro Lys Arg Leu Asn Phe Lys Leu Phe Asn Ile

305 310 315 320

Gln Val Lys Glu Val Thr Asp Asn Asn Gly Val Lys Thr Ile Ala Asn

325 330 335

Asn Leu Thr Ser Thr Val Gln Val Phe Thr Asp Ser Asp Tyr Gln Leu

340 345 350

Pro Tyr Val Leu Gly Ser Ala His Glu Gly Cys Leu Pro Pro Phe Pro

355 360 365

Ala Asp Val Phe Met Ile Pro Gln Tyr Gly Tyr Leu Thr Leu Asn Asp

370 375 380

Gly Ser Gln Ala Val Gly Arg Ser Ser Phe Tyr Cys Leu Glu Tyr Phe

385 390 395 400

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

405 410 415

Phe Glu Asn Val Pro Phe His Ser Ser Tyr Ala His Ser Gln Ser Leu

420 425 430

Asp Arg Leu Met Asn Pro Leu Ile Asp Gln Tyr Leu Tyr Tyr Leu Ser

435 440 445

Lys Thr Ile Asn Gly Ser Gly Gln Asn Gln Gln Thr Leu Lys Phe Ser

450 455 460

Val Ala Gly Pro Ser Asn Met Ala Val Gln Gly Arg Asn Tyr Ile Pro

465 470 475 480

Gly Pro Ser Tyr Arg Gln Gln Arg Val Ser Thr Thr Val Thr Gln Asn

485 490 495

Asn Asn Ser Glu Phe Ala Trp Pro Gly Ala Ser Ser Trp Ala Leu Asn

500 505 510

Gly Arg Asn Ser Leu Met Asn Pro Gly Pro Ala Met Ala Ser His Lys

515 520 525

Glu Gly Glu Asp Arg Phe Phe Pro Leu Ser Gly Ser Leu Ile Phe Gly

530 535 540

Lys Gln Gly Thr Gly Arg Asp Asn Val Asp Ala Asp Lys Val Met Ile

545 550 555 560

Thr Asn Glu Glu Glu Ile Lys Thr Thr Asn Pro Val Ala Thr Glu Ser

565 570 575

Tyr Gly Gln Val Ala Thr Asn His Gln Ser Ala Gln Ala Gln Ala Gln

580 585 590

Thr Gly Trp Val Gln Asn Gln Gly Ile Leu Pro Gly Met Val Trp Gln

595 600 605

Asp Arg Asp Val Tyr Leu Gln Gly Pro Ile Trp Ala Lys Ile Pro His

610 615 620

Thr Asp Gly Asn Phe His Pro Ser Pro Leu Met Gly Gly Phe Gly Met

625 630 635 640

Lys His Pro Pro Pro Gln Ile Leu Ile Lys Asn Thr Pro Val Pro Ala

645 650 655

Asp Pro Pro Thr Ala Phe Asn Lys Asp Lys Leu Asn Ser Phe Ile Thr

660 665 670

Gln Tyr Ser Thr Gly Gln Val Ser Val Glu Ile Glu Trp Glu Leu Gln

675 680 685

Lys Glu Asn Ser Lys Arg Trp Asn Pro Glu Ile Gln Tyr Thr Ser Asn

690 695 700

Tyr Tyr Lys Ser Asn Asn Val Glu Phe Ala Val Asn Thr Glu Gly Val

705 710 715 720

Tyr Ser Glu Pro Arg Pro Ile Gly Thr Arg Tyr Leu Thr Arg Asn Leu

725 730 735

<210> 8

<211> 738

<212> PRT

<213> Adeno-associated virus-10

<400> 8

Met Ala Ala Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Asn Leu Ser

1 5 10 15

Glu Gly Ile Arg Glu Trp Trp Asp Leu Lys Pro Gly Ala Pro Lys Pro

20 25 30

Lys Ala Asn Gln Gln Lys Gln Asp Asp Gly Arg Gly Leu Val Leu Pro

35 40 45

Gly Tyr Lys Tyr Leu Gly Pro Phe Asn Gly Leu Asp Lys Gly Glu Pro

50 55 60

Val Asn Ala Ala Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp

65 70 75 80

Gln Gln Leu Lys Ala Gly Asp Asn Pro Tyr Leu Arg Tyr Asn His Ala

85 90 95

Asp Ala Glu Phe Gln Glu Arg Leu Gln Glu Asp Thr Ser Phe Gly Gly

100 105 110

Asn Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg Val Leu Glu Pro

115 120 125

Leu Gly Leu Val Glu Glu Gly Ala Lys Thr Ala Pro Gly Lys Lys Arg

130 135 140

Pro Val Glu Pro Ser Pro Gln Arg Ser Pro Asp Ser Ser Thr Gly Ile

145 150 155 160

Gly Lys Lys Gly Gln Gln Pro Ala Lys Lys Arg Leu Asn Phe Gly Gln

165 170 175

Thr Gly Asp Ser Glu Ser Val Pro Asp Pro Gln Pro Ile Gly Glu Pro

180 185 190

Pro Ala Gly Pro Ser Gly Leu Gly Ser Gly Thr Met Ala Ala Gly Gly

195 200 205

Gly Ala Pro Met Ala Asp Asn Asn Glu Gly Ala Asp Gly Val Gly Ser

210 215 220

Ser Ser Gly Asn Trp His Cys Asp Ser Thr Trp Leu Gly Asp Arg Val

225 230 235 240

Ile Thr Thr Ser Thr Arg Thr Trp Ala Leu Pro Thr Tyr Asn Asn His

245 250 255

Leu Tyr Lys Gln Ile Ser Asn Gly Thr Ser Gly Gly Ser Thr Asn Asp

260 265 270

Asn Thr Tyr Phe Gly Tyr Ser Thr Pro Trp Gly Tyr Phe Asp Phe Asn

275 280 285

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

290 295 300

Asn Asn Trp Gly Phe Arg Pro Lys Arg Leu Asn Phe Lys Leu Phe Asn

305 310 315 320

Ile Gln Val Lys Glu Val Thr Gln Asn Glu Gly Thr Lys Thr Ile Ala

325 330 335

Asn Asn Leu Thr Ser Thr Ile Gln Val Phe Thr Asp Ser Glu Tyr Gln

340 345 350

Leu Pro Tyr Val Leu Gly Ser Ala His Gln Gly Cys Leu Pro Pro Phe

355 360 365

Pro Ala Asp Val Phe Met Ile Pro Gln Tyr Gly Tyr Leu Thr Leu Asn

370 375 380

Asn Gly Ser Gln Ala Val Gly Arg Ser Ser Phe Tyr Cys Leu Glu Tyr

385 390 395 400

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

405 410 415

Gln Phe Glu Asp Val Pro Phe His Ser Ser Tyr Ala His Ser Gln Ser

420 425 430

Leu Asp Arg Leu Met Asn Pro Leu Ile Asp Gln Tyr Leu Tyr Tyr Leu

435 440 445

Ser Arg Thr Gln Ser Thr Gly Gly Thr Ala Gly Thr Gln Gln Leu Leu

450 455 460

Phe Ser Gln Ala Gly Pro Asn Asn Met Ser Ala Gln Ala Lys Asn Trp

465 470 475 480

Leu Pro Gly Pro Cys Tyr Arg Gln Gln Arg Val Ser Thr Thr Leu Ser

485 490 495

Gln Asn Asn Asn Ser Asn Phe Ala Trp Thr Gly Ala Thr Lys Tyr His

500 505 510

Leu Asn Gly Arg Asp Ser Leu Val Asn Pro Gly Val Ala Met Ala Thr

515 520 525

His Lys Asp Asp Glu Glu Arg Phe Phe Pro Ser Ser Gly Val Leu Met

530 535 540

Phe Gly Lys Gln Gly Ala Gly Lys Asp Asn Val Asp Tyr Ser Ser Val

545 550 555 560

Met Leu Thr Ser Glu Glu Glu Ile Lys Thr Thr Asn Pro Val Ala Thr

565 570 575

Glu Gln Tyr Gly Val Val Ala Asp Asn Leu Gln Gln Gln Asn Ala Ala

580 585 590

Pro Ile Val Gly Ala Val Asn Ser Gln Gly Ala Leu Pro Gly Met Val

595 600 605

Trp Gln Asn Arg Asp Val Tyr Leu Gln Gly Pro Ile Trp Ala Lys Ile

610 615 620

Pro His Thr Asp Gly Asn Phe His Pro Ser Pro Leu Met Gly Gly Phe

625 630 635 640

Gly Leu Lys His Pro Pro Pro Gln Ile Leu Ile Lys Asn Thr Pro Val

645 650 655

Pro Ala Asp Pro Pro Thr Thr Phe Ser Gln Ala Lys Leu Ala Ser Phe

660 665 670

Ile Thr Gln Tyr Ser Thr Gly Gln Val Ser Val Glu Ile Glu Trp Glu

675 680 685

Leu Gln Lys Glu Asn Ser Lys Arg Trp Asn Pro Glu Ile Gln Tyr Thr

690 695 700

Ser Asn Tyr Tyr Lys Ser Thr Asn Val Asp Phe Ala Val Asn Thr Asp

705 710 715 720

Gly Thr Tyr Ser Glu Pro Arg Pro Ile Gly Thr Arg Tyr Leu Thr Arg

725 730 735

Asn Leu

<210> 9

<211> 738

<212> PRT

<213> Adeno-associated virus 10

<400> 9

Met Ala Ala Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Asn Leu Ser

1 5 10 15

Glu Gly Ile Arg Glu Trp Trp Asp Leu Lys Pro Gly Ala Pro Lys Pro

20 25 30

Lys Ala Asn Gln Gln Lys Gln Asp Asp Gly Arg Gly Leu Val Leu Pro

35 40 45

Gly Tyr Lys Tyr Leu Gly Pro Phe Asn Gly Leu Asp Lys Gly Glu Pro

50 55 60

Val Asn Ala Ala Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp

65 70 75 80

Gln Gln Leu Lys Ala Gly Asp Asn Pro Tyr Leu Arg Tyr Asn His Ala

85 90 95

Asp Ala Glu Phe Gln Glu Arg Leu Gln Glu Asp Thr Ser Phe Gly Gly

100 105 110

Asn Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg Val Leu Glu Pro

115 120 125

Leu Gly Leu Val Glu Glu Ala Ala Lys Thr Ala Pro Gly Lys Lys Arg

130 135 140

Pro Val Glu Pro Ser Pro Gln Arg Ser Pro Asp Ser Ser Thr Gly Ile

145 150 155 160

Gly Lys Lys Gly Gln Gln Pro Ala Lys Lys Arg Leu Asn Phe Gly Gln

165 170 175

Thr Gly Glu Ser Glu Ser Val Pro Asp Pro Gln Pro Ile Gly Glu Pro

180 185 190

Pro Ala Gly Pro Ser Gly Leu Gly Ser Gly Thr Met Ala Ala Gly Gly

195 200 205

Gly Ala Pro Met Ala Asp Asn Asn Glu Gly Ala Asp Gly Val Gly Ser

210 215 220

Ser Ser Gly Asn Trp His Cys Asp Ser Thr Trp Leu Gly Asp Arg Val

225 230 235 240

Ile Thr Thr Ser Thr Arg Thr Trp Ala Leu Pro Thr Tyr Asn Asn His

245 250 255

Leu Tyr Lys Gln Ile Ser Asn Gly Thr Ser Gly Gly Ser Thr Asn Asp

260 265 270

Asn Thr Tyr Phe Gly Tyr Ser Thr Pro Trp Gly Tyr Phe Asp Phe Asn

275 280 285

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

290 295 300

Asn Asn Trp Gly Phe Arg Pro Lys Arg Leu Ser Phe Lys Leu Phe Asn

305 310 315 320

Ile Gln Val Lys Glu Val Thr Gln Asn Glu Gly Thr Lys Thr Ile Ala

325 330 335

Asn Asn Leu Thr Ser Thr Ile Gln Val Phe Thr Asp Ser Glu Tyr Gln

340 345 350

Leu Pro Tyr Val Leu Gly Ser Ala His Gln Gly Cys Leu Pro Pro Phe

355 360 365

Pro Ala Asp Val Phe Met Ile Pro Gln Tyr Gly Tyr Leu Thr Leu Asn

370 375 380

Asn Gly Ser Gln Ala Val Gly Arg Ser Ser Phe Tyr Cys Leu Glu Tyr

385 390 395 400

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

405 410 415

Thr Phe Glu Asp Val Pro Phe His Ser Ser Tyr Ala His Ser Gln Ser

420 425 430

Leu Asp Arg Leu Met Asn Pro Leu Ile Asp Gln Tyr Leu Tyr Tyr Leu

435 440 445

Ser Arg Thr Gln Ser Thr Gly Gly Thr Gln Gly Thr Gln Gln Leu Leu

450 455 460

Phe Ser Gln Ala Gly Pro Ala Asn Met Ser Ala Gln Ala Lys Asn Trp

465 470 475 480

Leu Pro Gly Pro Cys Tyr Arg Gln Gln Arg Val Ser Thr Thr Leu Ser

485 490 495

Gln Asn Asn Asn Ser Asn Phe Ala Trp Thr Gly Ala Thr Lys Tyr His

500 505 510

Leu Asn Gly Arg Asp Ser Leu Val Asn Pro Gly Val Ala Met Ala Thr

515 520 525

His Lys Asp Asp Glu Glu Arg Phe Phe Pro Ser Ser Gly Val Leu Met

530 535 540

Phe Gly Lys Gln Gly Ala Gly Arg Asp Asn Val Asp Tyr Ser Ser Val

545 550 555 560

Met Leu Thr Ser Glu Glu Glu Ile Lys Thr Thr Asn Pro Val Ala Thr

565 570 575

Glu Gln Tyr Gly Val Val Ala Asp Asn Leu Gln Gln Ala Asn Thr Gly

580 585 590

Pro Ile Val Gly Asn Val Asn Ser Gln Gly Ala Leu Pro Gly Met Val

595 600 605

Trp Gln Asn Arg Asp Val Tyr Leu Gln Gly Pro Ile Trp Ala Lys Ile

610 615 620

Pro His Thr Asp Gly Asn Phe His Pro Ser Pro Leu Met Gly Gly Phe

625 630 635 640

Gly Leu Lys His Pro Pro Pro Gln Ile Leu Ile Lys Asn Thr Pro Val

645 650 655

Pro Ala Asp Pro Pro Thr Thr Phe Ser Gln Ala Lys Leu Ala Ser Phe

660 665 670

Ile Thr Gln Tyr Ser Thr Gly Gln Val Ser Val Glu Ile Glu Trp Glu

675 680 685

Leu Gln Lys Glu Asn Ser Lys Arg Trp Asn Pro Glu Ile Gln Tyr Thr

690 695 700

Ser Asn Tyr Tyr Lys Ser Thr Asn Val Asp Phe Ala Val Asn Thr Glu

705 710 715 720

Gly Thr Tyr Ser Glu Pro Arg Pro Ile Gly Thr Arg Tyr Leu Thr Arg

725 730 735

Asn Leu

<210> 10

<211> 744

<212> PRT

<213> Artificial sequence

<220>

<223> Consensus sequence

<220>

<221> SITE

<222> (148)..(148)

<223> Xaa does not indicate general consensus

<220>

<221> SITE

<222> (202)..(202)

<223> Xaa does not indicate general consensus

<220>

<221> SITE

<222> (207)..(207)

<223> Xaa does not indicate general consensus

<220>

<221> SITE

<222> (270)..(270)

<223> Xaa does not indicate general consensus

<220>

<221> SITE

<222> (458)..(458)

<223> Xaa does not indicate general consensus

<220>

<221> SITE

<222> (462)..(462)

<223> Xaa does not indicate general consensus

<220>

<221> SITE

<222> (465)..(465)

<223> Xaa does not indicate general consensus

<220>

<221> SITE

<222> (474)..(474)

<223> Xaa does not indicate general consensus

<220>

<221> SITE

<222> (496)..(496)

<223> Xaa does not indicate general consensus

<220>

<221> SITE

<222> (498)..(498)

<223> Xaa does not indicate general consensus

<220>

<221> SITE

<222> (500) .. (500)

<223> Xaa does not indicate general consensus

<220>

<221> SITE

<222> (538)..(538)

<223> Xaa does not indicate general consensus

<220>

<221> SITE

<222> (557) ..(557)

<223> Xaa does not indicate general consensus

<220>

<221> SITE

<222> (562)..(563)

<223> Xaa does not indicate general consensus

<220>

<221> SITE

<222> (565) .. (565)

<223> Xaa does not indicate general consensus

<220>

<221> SITE

<222> (570) .. (570)

<223> Xaa does not indicate general consensus

<220>

<221> SITE

<222> (587) .. (587)

<223> Xaa does not indicate general consensus

<220>

<221> SITE

<222> (598) ..(598)

<223> Xaa does not indicate general consensus

<220>

<221> SITE

<222> (675) .. (675)

<223> Xaa does not indicate general consensus

<400> 10

Met Ala Ala Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Asn Leu Ser

1 5 10 15

Glu Gly Ile Arg Glu Trp Trp Asp Leu Lys Pro Gly Ala Pro Lys Pro

20 25 30

Lys Ala Asn Gln Gln Lys Gln Asp Asp Gly Arg Gly Leu Val Leu Pro

35 40 45

Gly Tyr Lys Tyr Leu Gly Pro Phe Asn Gly Leu Asp Lys Gly Glu Pro

50 55 60

Val Asn Ala Ala Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp

65 70 75 80

Gln Gln Leu Lys Ala Gly Asp Asn Pro Tyr Leu Arg Tyr Asn His Ala

85 90 95

Asp Ala Glu Phe Gln Glu Arg Leu Gln Glu Asp Thr Ser Phe Gly Gly

100 105 110

Asn Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg Val Leu Glu Pro

115 120 125

Leu Gly Leu Val Glu Glu Gly Ala Lys Thr Ala Pro Gly Lys Lys Arg

130 135 140

Pro Val Glu Xaa Ser Pro Gln Arg Glu Pro Asp Ser Ser Ser Gly Ile

145 150 155 160

Gly Lys Lys Gly Gln Gln Pro Ala Lys Lys Arg Leu Asn Phe Gly Gln

165 170 175

Thr Gly Asp Ser Glu Ser Val Pro Asp Gly Pro Gln Pro Leu Gly Glu

180 185 190

Pro Pro Ala Ala Pro Ser Gly Leu Gly Xaa Asn Thr Met Ala Xaa Gly

195 200 205

Gly Gly Ala Pro Met Ala Asp Asn Asn Glu Gly Ala Asp Gly Val Gly

210 215 220

Asn Ser Ser Gly Asn Trp His Cys Asp Ser Thr Trp Leu Gly Asp Arg

225 230 235 240

Val Ile Thr Thr Ser Thr Arg Thr Trp Ala Leu Pro Thr Tyr Asn Asn

245 250 255

His Leu Tyr Lys Gln Ile Ser Ser Gly Ser Ser Gly Gly Xaa Ser Asn

260 265 270

Asp Asn His Tyr Phe Gly Tyr Ser Thr Pro Trp Gly Tyr Phe Asp Phe

275 280 285

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

290 295 300

Asn Asn Asn Trp Gly Phe Arg Pro Lys Arg Leu Asn Phe Lys Leu Phe

305 310 315 320

Asn Ile Gln Val Lys Glu Val Thr Gln Asn Asp Gly Thr Thr Thr Ile

325 330 335

Ala Asn Asn Leu Thr Ser Thr Val Gln Val Phe Thr Asp Ser Glu Tyr

340 345 350

Gln Leu Pro Tyr Val Leu Gly Ser Ala His Gln Gly Cys Leu Pro Pro

355 360 365

Phe Pro Ala Asp Val Phe Met Ile Pro Gln Tyr Gly Tyr Leu Thr Leu

370 375 380

Asn Arg Asp Asn Gly Ser Gln Ala Val Gly Arg Ser Ser Phe Tyr Cys

385 390 395 400

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

405 410 415

Phe Ser Tyr Thr Phe Glu Asp Val Pro Phe His Ser Ser Tyr Ala His

420 425 430

Ser Gln Ser Leu Asp Arg Leu Met Asn Pro Leu Ile Asp Gln Tyr Leu

435 440 445

Tyr Tyr Leu Ser Arg Thr Gln Asn Thr Xaa Gly Thr Ala Xaa Thr Gln

450 455 460

Xaa Leu Leu Phe Ser Gln Ala Gly Pro Xaa Asn Met Ser Val Gln Ala

465 470 475 480

Lys Asn Trp Leu Pro Gly Pro Cys Tyr Arg Gln Gln Arg Val Ser Xaa

485 490 495

Thr Xaa Thr Xaa Asn Asn Asn Ser Asn Phe Ala Trp Thr Gly Ala Thr

500 505 510

Lys Tyr His Leu Asn Gly Arg Asp Ser Leu Val Asn Pro Gly Pro Ala

515 520 525

Met Ala Ser His Lys Asp Asp Glu Glu Xaa Phe Phe Pro Ser Ser Gly

530 535 540

Val Leu Ile Phe Gly Lys Gln Gly Ala Asn Pro Gly Xaa Asp Asn Val

545 550 555 560

Asp Xaa Xaa Gly Xaa Val Met Ile Thr Xaa Glu Glu Glu Ile Lys Thr

565 570 575

Thr Asn Pro Val Ala Thr Glu Gln Tyr Gly Xaa Val Ala Thr Asn Leu

580 585 590

Gln Ser Ser Asn Thr Xaa Pro Ala Thr Gly Asp Val Asn Ser Gln Gly

595 600 605

Ala Leu Pro Gly Met Val Trp Gln Asp Arg Asp Val Tyr Leu Gln Gly

610 615 620

Pro Ile Trp Ala Lys Ile Pro His Thr Asp Gly His Phe His Pro Ser

625 630 635 640

Pro Leu Met Gly Gly Phe Gly Leu Lys His Pro Pro Pro Gln Ile Leu

645 650 655

Ile Lys Asn Thr Pro Val Pro Ala Asn Pro Pro Thr Thr Phe Ser Ala

660 665 670

Ala Lys Xaa Ala Ser Phe Ile Thr Gln Tyr Ser Thr Gly Gln Val Ser

675 680 685

Val Glu Ile Glu Trp Glu Leu Gln Lys Glu Asn Ser Lys Arg Trp Asn

690 695 700

Pro Glu Ile Gln Tyr Thr Ser Asn Tyr Tyr Lys Ser Thr Asn Val Asp

705 710 715 720

Phe Ala Val Asp Thr Asn Gly Val Tyr Ser Glu Pro Arg Pro Ile Gly

725 730 735

Thr Arg Tyr Leu Thr Arg Asn Leu

740

<210> 11

<211> 736

<212> PRT

<213> Adeno-associated virus 3B

<400> 11

Met Ala Ala Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Asn Leu Ser

1 5 10 15

Glu Gly Ile Arg Glu Trp Trp Ala Leu Lys Pro Gly Val Pro Gln Pro

20 25 30

Lys Ala Asn Gln Gln His Gln Asp Asn Arg Arg Gly Leu Val Leu Pro

35 40 45

Gly Tyr Lys Tyr Leu Gly Pro Gly Asn Gly Leu Asp Lys Gly Glu Pro

50 55 60

Val Asn Glu Ala Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp

65 70 75 80

Gln Gln Leu Lys Ala Gly Asp Asn Pro Tyr Leu Lys Tyr Asn His Ala

85 90 95

Asp Ala Glu Phe Gln Glu Arg Leu Gln Glu Asp Thr Ser Phe Gly Gly

100 105 110

Asn Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg Ile Leu Glu Pro

115 120 125

Leu Gly Leu Val Glu Glu Ala Ala Lys Thr Ala Pro Gly Lys Lys Arg

130 135 140

Pro Val Asp Gln Ser Pro Gln Glu Pro Asp Ser Ser Ser Gly Val Gly

145 150 155 160

Lys Ser Gly Lys Gln Pro Ala Arg Lys Arg Leu Asn Phe Gly Gln Thr

165 170 175

Gly Asp Ser Glu Ser Val Pro Asp Pro Gln Pro Leu Gly Glu Pro Pro

180 185 190

Ala Ala Pro Thr Ser Leu Gly Ser Asn Thr Met Ala Ser Gly Gly Gly

195 200 205

Ala Pro Met Ala Asp Asn Asn Glu Gly Ala Asp Gly Val Gly Asn Ser

210 215 220

Ser Gly Asn Trp His Cys Asp Ser Gln Trp Leu Gly Asp Arg Val Ile

225 230 235 240

Thr Thr Ser Thr Arg Thr Trp Ala Leu Pro Thr Tyr Asn Asn His Leu

245 250 255

Tyr Lys Gln Ile Ser Ser Gln Ser Gly Ala Ser Asn Asp Asn His Tyr

260 265 270

Phe Gly Tyr Ser Thr Pro Trp Gly Tyr Phe Asp Phe Asn Arg Phe His

275 280 285

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

290 295 300

Gly Phe Arg Pro Lys Lys Leu Ser Phe Lys Leu Phe Asn Ile Gln Val

305 310 315 320

Lys Glu Val Thr Gln Asn Asp Gly Thr Thr Thr Ile Ala Asn Asn Leu

325 330 335

Thr Ser Thr Val Gln Val Phe Thr Asp Ser Glu Tyr Gln Leu Pro Tyr

340 345 350

Val Leu Gly Ser Ala His Gln Gly Cys Leu Pro Pro Phe Pro Ala Asp

355 360 365

Val Phe Met Val Pro Gln Tyr Gly Tyr Leu Thr Leu Asn Asn Gly Ser

370 375 380

Gln Ala Val Gly Arg Ser Ser Phe Tyr Cys Leu Glu Tyr Phe Pro Ser

385 390 395 400

Gln Met Leu Arg Thr Gly Asn Asn Phe Gln Phe Ser Tyr Thr Phe Glu

405 410 415

Asp Val Pro Phe His Ser Ser Tyr Ala His Ser Gln Ser Leu Asp Arg

420 425 430

Leu Met Asn Pro Leu Ile Asp Gln Tyr Leu Tyr Tyr Leu Asn Arg Thr

435 440 445

Gln Gly Thr Thr Ser Gly Thr Thr Asn Gln Ser Arg Leu Leu Phe Ser

450 455 460

Gln Ala Gly Pro Gln Ser Met Ser Leu Gln Ala Arg Asn Trp Leu Pro

465 470 475 480

Gly Pro Cys Tyr Arg Gln Gln Arg Leu Ser Lys Thr Ala Asn Asp Asn

485 490 495

Asn Asn Ser Asn Phe Pro Trp Thr Ala Ala Ser Lys Tyr His Leu Asn

500 505 510

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

515 520 525

Asp Asp Glu Glu Lys Phe Phe Pro Met His Gly Asn Leu Ile Phe Gly

530 535 540

Lys Glu Gly Thr Thr Ala Ser Asn Ala Glu Leu Asp Asn Val Met Ile

545 550 555 560

Thr Asp Glu Glu Glu Ile Arg Thr Thr Asn Pro Val Ala Thr Glu Gln

565 570 575

Tyr Gly Thr Val Ala Asn Asn Leu Gln Ser Ser Asn Thr Ala Pro Thr

580 585 590

Thr Arg Thr Val Asn Asp Gln Gly Ala Leu Pro Gly Met Val Trp Gln

595 600 605

Asp Arg Asp Val Tyr Leu Gln Gly Pro Ile Trp Ala Lys Ile Pro His

610 615 620

Thr Asp Gly His Phe His Pro Ser Pro Leu Met Gly Gly Phe Gly Leu

625 630 635 640

Lys His Pro Pro Pro Gln Ile Met Ile Lys Asn Thr Pro Val Pro Ala

645 650 655

Asn Pro Pro Thr Thr Phe Ser Pro Ala Lys Phe Ala Ser Phe Ile Thr

660 665 670

Gln Tyr Ser Thr Gly Gln Val Ser Val Glu Ile Glu Trp Glu Leu Gln

675 680 685

Lys Glu Asn Ser Lys Arg Trp Asn Pro Glu Ile Gln Tyr Thr Ser Asn

690 695 700

Tyr Asn Lys Ser Val Asn Val Asp Phe Thr Val Asp Thr Asn Gly Val

705 710 715 720

Tyr Ser Glu Pro Arg Pro Ile Gly Thr Arg Tyr Leu Thr Arg Asn Leu

725 730 735

<210> 12

<211> 736

<212> PRT

<213> Artificial sequence

<220>

<223> Recombinant sequence

<400> 12

Met Ala Ala Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Asn Leu Ser

1 5 10 15

Glu Gly Ile Arg Glu Trp Trp Ala Leu Gln Pro Gly Ala Pro Lys Pro

20 25 30

Lys Ala Asn Gln Gln His Gln Asp Asn Ala Arg Gly Leu Val Leu Pro

35 40 45

Gly Tyr Lys Tyr Leu Gly Pro Gly Asn Gly Leu Asp Lys Gly Glu Pro

50 55 60

Val Asn Ala Ala Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp

65 70 75 80

Gln Gln Leu Lys Ala Gly Asp Asn Pro Tyr Leu Lys Tyr Asn His Ala

85 90 95

Asp Ala Glu Phe Gln Glu Arg Leu Lys Glu Asp Thr Ser Phe Gly Gly

100 105 110

Asn Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg Leu Leu Glu Pro

115 120 125

Leu Gly Leu Val Glu Glu Ala Ala Lys Thr Ala Pro Gly Lys Lys Arg

130 135 140

Pro Val Asp Gln Ser Pro Gln Glu Pro Asp Ser Ser Ser Gly Val Gly

145 150 155 160

Lys Ser Gly Lys Gln Pro Ala Arg Lys Arg Leu Asn Phe Gly Gln Thr

165 170 175

Gly Asp Ser Glu Ser Val Pro Asp Pro Gln Pro Leu Gly Glu Pro Pro

180 185 190

Ala Ala Pro Thr Ser Leu Gly Ser Asn Thr Met Ala Ser Gly Gly Gly

195 200 205

Ala Pro Met Ala Asp Asn Asn Glu Gly Ala Asp Gly Val Gly Asn Ser

210 215 220

Ser Gly Asn Trp His Cys Asp Ser Gln Trp Leu Gly Asp Arg Val Ile

225 230 235 240

Thr Thr Ser Thr Arg Thr Trp Ala Leu Pro Thr Tyr Asn Asn His Leu

245 250 255

Tyr Lys Gln Ile Ser Ser Gln Ser Gly Ala Ser Asn Asp Asn His Tyr

260 265 270

Phe Gly Tyr Ser Thr Pro Trp Gly Tyr Phe Asp Phe Asn Arg Phe His

275 280 285

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

290 295 300

Gly Phe Arg Pro Lys Lys Leu Ser Phe Lys Leu Phe Asn Ile Gln Val

305 310 315 320

Lys Glu Val Thr Gln Asn Asp Gly Thr Thr Thr Ile Ala Asn Asn Leu

325 330 335

Thr Ser Thr Val Gln Val Phe Thr Asp Ser Glu Tyr Gln Leu Pro Tyr

340 345 350

Val Leu Gly Ser Ala His Gln Gly Cys Leu Pro Pro Phe Pro Ala Asp

355 360 365

Val Phe Met Val Pro Gln Tyr Gly Tyr Leu Thr Leu Asn Asn Gly Ser

370 375 380

Gln Ala Val Gly Arg Ser Ser Phe Tyr Cys Leu Glu Tyr Phe Pro Ser

385 390 395 400

Gln Met Leu Arg Thr Gly Asn Asn Phe Gln Phe Ser Tyr Thr Phe Glu

405 410 415

Asp Val Pro Phe His Ser Ser Tyr Ala His Ser Gln Ser Leu Asp Arg

420 425 430

Leu Met Asn Pro Leu Ile Asp Gln Tyr Leu Tyr Tyr Leu Asn Arg Thr

435 440 445

Gln Gly Thr Thr Ser Gly Thr Thr Asn Gln Ser Arg Leu Leu Phe Ser

450 455 460

Gln Ala Gly Pro Gln Ser Met Ser Leu Gln Ala Arg Asn Trp Leu Pro

465 470 475 480

Gly Pro Cys Tyr Arg Gln Gln Arg Leu Ser Lys Thr Ala Asn Asp Asn

485 490 495

Asn Asn Ser Asn Phe Pro Trp Thr Ala Ala Ser Lys Tyr His Leu Asn

500 505 510

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

515 520 525

Asp Asp Glu Glu Lys Phe Phe Pro Met His Gly Asn Leu Ile Phe Gly

530 535 540

Lys Glu Gly Thr Thr Ala Ser Asn Ala Glu Leu Asp Asn Val Met Ile

545 550 555 560

Thr Asp Glu Glu Glu Ile Arg Thr Thr Asn Pro Val Ala Thr Glu Gln

565 570 575

Tyr Gly Thr Val Ala Asn Asn Leu Gln Ser Ser Asn Thr Ala Pro Thr

580 585 590

Thr Arg Thr Val Asn Asp Gln Gly Ala Leu Pro Gly Met Val Trp Gln

595 600 605

Asp Arg Asp Val Tyr Leu Gln Gly Pro Ile Trp Ala Lys Ile Pro His

610 615 620

Thr Asp Gly His Phe His Pro Ser Pro Leu Met Gly Gly Phe Gly Leu

625 630 635 640

Lys His Pro Pro Pro Gln Ile Met Ile Lys Asn Thr Pro Val Pro Ala

645 650 655

Asn Pro Pro Thr Thr Phe Ser Pro Ala Lys Phe Ala Ser Phe Ile Thr

660 665 670

Gln Tyr Ser Thr Gly Gln Val Ser Val Glu Ile Glu Trp Glu Leu Gln

675 680 685

Lys Glu Asn Ser Lys Arg Trp Asn Pro Glu Ile Gln Tyr Thr Ser Asn

690 695 700

Tyr Asn Lys Ser Val Asn Val Asp Phe Thr Val Asp Thr Asn Gly Val

705 710 715 720

Tyr Ser Glu Pro Arg Pro Ile Gly Thr Arg Tyr Leu Thr Arg Pro Leu

725 730 735

<---

Claims (15)

1. A recombinant adeno-associated virus (AAV) vector for delivery of a gene product into a subject's tissue, comprising: (a) a variant AAV8 capsid protein, wherein the variant AAV8 capsid protein comprises an amino acid substitution compared to a wild-type AAV8 capsid protein at position 315 in the sequence of the AAV8 capsid protein, wherein the wild-type AAV8 capsid protein comprises the sequence of SEQ ID NO:6; and (b) a heterologous nucleic acid containing a nucleotide sequence encoding a gene product. 2. The recombinant AAV vector of claim 1, wherein the variant AAV capsid protein comprises a sequence having at least 95% sequence identity to SEQ ID NO:6. 3. The recombinant AAV vector of claim 1 or 2, further comprising one or more amino acid substitutions present at one or more of the following positions in the AAV8 capsid protein sequence: 125, 151, 163, 206, 460, 495, 502, 536, 549, 551, 588, 591 and/or 596. 4. A recombinant adeno-associated virus (AAV) vector for delivery of a gene product into a subject's tissue, comprising: (a) a variant AAV3B capsid protein, wherein the variant AAV3B capsid protein comprises an amino acid substitution compared to a wild-type AAV3B capsid protein at position 312 in the sequence of the AAV3B capsid protein, wherein the wild-type AAV3B capsid protein comprises the sequence of SEQ ID NO:11; and (b) a heterologous nucleic acid containing a nucleotide sequence encoding a gene product. 5. The recombinant AAV vector of claim 4, wherein (i) the variant AAV3B capsid protein comprises a sequence having at least 95% sequence identity to SEQ ID NO:11. 6. A recombinant adeno-associated virus (AAV) vector for delivery of a gene product into a subject's tissue, comprising: (a) a variant AAV-LK03 capsid protein, wherein the variant AAV-LK03 capsid protein comprises an amino acid substitution at position 312 compared to the sequence of the AAV-LK03 capsid protein as defined in SEQ ID NO:12; and (b) a heterologous nucleic acid containing a nucleotide sequence encoding a gene product. 7. The recombinant AAV vector of claim 6, wherein the variant AAV-LK03 capsid protein comprises a sequence having at least 95% sequence identity to SEQ ID NO:12. 8. A recombinant AAV vector according to any one of claims 1-7 for use in treating a disorder in a subject. 9. A recombinant AAV vector for use according to claim 8, wherein the disorder is a neurological, ocular disorder or liver disease, optionally wherein the neurological disorder is a neurodegenerative disease and/or the ocular disorder is glaucoma, retinal pigmentary degeneration, macular degeneration, retinoschisis or diabetic retinopathy.