AAV Vectors for Retina and CNS Gene Therapy

Abstract

Recombinant adeno-associated viruses for enhanced gene therapy of ocular or CNS disorders are provided. [Solution] A method for delivering a heterologous nucleic acid to the eye of an individual, comprising administering recombinant adeno-associated virus (rAAV) particles subretinal to the individual, wherein the rAAV particles comprise: a) an rAAV capsid comprising an rAAV capsid protein containing one or more amino acid substitutions at one or more positions that interact with heparan sulfate proteoglycans or at one or more positions corresponding to amino acids 484, 487, 527, 532, 585, or 588, numbered based on the VP1 numbering of AAV2; and b) an rAAV vector comprising the heterologous nucleic acid and at least one AAV inverted terminal repeat sequence.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Patent Application No. 61 / 988,131, filed May 2, 2014, and U.S. Provisional Patent Application No. 62 / 114,575, filed February 10, 2015, each of which is incorporated herein by reference in its entirety.

[0002] Submitting a sequence listing as an ASCII text file The following submission in an ASCII text file is incorporated herein by reference in its entirety: Sequence Listing in Computer Readable Format (CRF) (Filename: 159792010440SEQLIST.txt, Data Recorded: April 29, 2015, Size: 85KB).

[0003] The present invention relates to mutant recombinant adeno-associated virus (rAAV) vectors for improved delivery to the eye and CNS; for example, for improved retinal gene therapy and improved CNS gene therapy. [Background technology]

[0004] Retinal degenerative diseases are promising targets for adeno-associated virus (AAV)-mediated gene therapy. AAV vectors can mediate long-term gene expression in the retina and can elicit minimal immune responses, making these vectors an attractive option for ocular gene delivery. The retina is a light-sensing tissue at the back of the eye composed of various cell types, including photoreceptor cells, retinal pigmented epithelial cells, and retinal ganglion cells. The target cell type and vector delivery route of AAV gene therapy vectors will depend on the disease manifestation. For example, a phase I clinical trial for age-related macular degeneration has utilized intravitreal delivery of vectors to achieve transduction of retinal ganglion cells, and a recent clinical trial for the treatment of patients with Leber congenital amaurosis type 2, a form of retinitis pigmentosa, has used subretinal delivery of the RPE65 gene to transduce retinal pigmented epithelial cells. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Kaplitt, MG et al. (2007) Lancet 369:2097-2105 [Non-patent document 2] Eberling, JL et al. (2008) Neurology 70:1980-1983 [Non-patent document 3] Fiandaca, MS et al. (2009) Neuroimage. 47 Suppl 2: T27-35 Summary of the Invention [Problem to be solved by the invention]

[0006] Given such utility, there is a need to develop new agents and methods to improve AAV delivery to the eye.

[0007] Adeno-associated virus (AAV)-based vectors have also become the vector system of choice for neurogene therapy, with excellent stability documented in several clinical trials (Non-Patent Document 1; Non-Patent Document 2; Non-Patent Document 3). However, effective treatment of neurological disorders has been largely hindered by problems associated with the delivery of AAV vectors to affected cell populations. This has been particularly problematic for disorders involving the central nervous system (CNS), and therefore there is a need to further improve AAV delivery to the CNS. [Means for solving the problem]

[0008] In some embodiments, the present invention provides a method for delivering a heterologous nucleic acid to the eye of an individual, comprising subretinal administration of recombinant adeno-associated virus (rAAV) particles to the individual, the rAAV particles comprising: (a) an rAAV capsid comprising an rAAV capsid protein containing one or more amino acid substitutions at one or more positions that interact with heparan sulfate proteoglycans, or at one or more positions corresponding to amino acids 484, 487, 527, 532, 585, or 588, numbered according to the VP1 numbering of AAV2; and (b) an rAAV vector comprising a heterologous nucleic acid and at least one AAV inverted terminal repeat. In some embodiments, the rAAV particles comprise an AAV serotype 2 (AAV2) capsid. In some embodiments, the one or more amino acid substitutions reduce binding of the rAAV particles to heparan sulfate proteoglycans. In some embodiments, the one or more amino acid substitutions reduce binding of the rAAV particle to heparan sulfate proteoglycans by at least about 10%, at least about 25%, at least about 50%, at least about 75%, or at least about 100%. In some embodiments, the one or more amino acid substitutions increase the transduction efficiency of the rAAV particle into cells of the eye or CNS. In some embodiments, the one or more amino acid substitutions increase the transduction efficiency of the rAAV particle into cells of the eye or CNS by at least about 10%, at least about 25%, at least about 50%, at least about 75%, or at least about 100%, for example, compared to a reference rAAV capsid comprising a wild-type AAV capsid protein. In some embodiments, the ocular cell is a retinal cell, photoreceptor cell, retinal pigmented epithelial cell, bipolar cell, horizontal cell, amacrine cell, Müller cell, and / or ganglion cell. In some embodiments, the cells of the CNS are oligodendrocytes, astrocytes, neurons, brain parenchymal cells, microglial cells, ependymal cells, and / or Purkinje cells.

[0009] In some embodiments, the AAV particles of the present invention comprise capsids having one or more amino acid substitutions at positions 484, 487, 527, 532, 585, or 588, based on the VP1 numbering system of AAV2. In some embodiments, the numbering is based on AAV2 VP1 comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, the one or more amino acid substitutions comprise substitution of a positively charged amino acid residue with an amino acid residue that does not bear a positive charge. In some embodiments, the positively charged amino acid residue is replaced with a hydrophobic amino acid residue. In further embodiments, the one or more amino acid substitutions comprise substitution of an arginine or lysine residue with an alanine residue. In some embodiments, the one or more amino acid substitutions comprise substitution of an arginine or lysine residue with an alanine residue. In some embodiments, the one or more amino acid substitutions comprise substitutions at positions R484, R487, K527, K532, R585, and / or R588, based on the VP1 numbering system of AAV2. In some embodiments, the rAAV particles comprise one or more rAAV capsid proteins having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity to SEQ ID NOs: 2, 4, and / or 6. In some embodiments, the one or more amino acid substitutions comprise a substitution at position R532. In some embodiments, the one or more amino acid substitutions comprise a substitution at positions R484 and R487 or at positions R585 and R588, numbered based on VP1 of AAV2. In further embodiments, one or more In some embodiments, the rAAV particles comprise an AAV1 capsid, an AAV2 capsid, an AAV3 capsid, an AAV6 capsid, an AAV8 capsid, an AAVrh8R capsid, an AAV9 capsid, or an AAVrh10 capsid.

[0010] In some embodiments, the AAV particles of the present invention comprise capsids with one or more amino acid substitutions at positions 485, 488, 528, 533, 586, or 589, as numbered based on the VP1 numbering of AAVrh8R. In some embodiments, the AAV particles comprise capsids with one or more amino acid substitutions at positions 485, 488, 528, or 533, as numbered based on the VP1 numbering of AAVrh8R. In some embodiments, the numbering is based on the VP1 of AAVrh8R comprising the amino acid sequence of SEQ ID NO: 9. In some embodiments, the one or more amino acid substitutions comprise substitution of a positively charged amino acid residue with an amino acid residue that does not bear a positive charge. In some embodiments, the positively charged amino acid residue is substituted with a hydrophobic amino acid residue. In further embodiments, the one or more amino acid substitutions comprise substitution of an arginine or lysine residue. In yet further embodiments, the one or more amino acid substitutions comprise substitution of an arginine or lysine residue with an alanine residue. In other embodiments, the one or more amino acid substitutions comprise substitution of an amino acid residue that does not bear a positive charge with an amino acid residue that does. In some embodiments, a hydrophobic amino acid residue is substituted with an amino acid residue that bears a positive charge. In further embodiments, the one or more amino acid substitutions comprise substitution of an alanine residue. In yet further embodiments, the one or more amino acid substitutions comprise substitution of an arginine or lysine residue with an alanine residue. In some embodiments, the amino acid substitution is at position 485, 488, 528, 533, or 589 based on the VP1 numbering of AAVrh8R. In some embodiments, the AAV particles of the present invention comprise capsids having one or more amino acid substitutions at positions 485, 488, 528, or 533 based on the VP1 numbering of AAVrh8R. In some embodiments, the numbering is based on VP1 of AAVrh8R comprising the amino acid sequence of SEQ ID NO:9.In some embodiments, the amino acid substitution comprises a substitution at position R485, R488, R533, or T589, numbered based on the VP1 numbering of AAVrh8R. In some embodiments, the rAAV particles comprise the rAAV capsid protein of SEQ ID NO: 11. In some embodiments, the rAAV particles comprise one or more rAAV capsid proteins having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity to SEQ ID NO: 11. In some embodiments, the single amino acid substitution comprises an R533A substitution, numbered based on VP1 of AAVrh8R.

[0011] In some embodiments, the rAAV particles comprise an AAV1, AAV6, or AAV9 capsid, and one or more amino acid substitutions are at positions 485, 488, 528, 533, 586, and / or 589, numbered based on the VP1 numbering of AAV1, AAV6, or AAV9; and / or the rAAV particles comprise an AAV8 or AAVrhlO capsid, and one or more amino acid substitutions are at positions 487, 490, 535, 588, and / or 591, numbered based on the VP1 numbering of AAV8 or AAVrhlO. .

[0012] In some embodiments, the AAV particles of the invention comprise a capsid comprising one or more amino acid substitutions that alter binding to HSPG (e.g., reduce or eliminate binding to HSPG), or one or more amino acid substitutions at one or more positions corresponding to amino acids 484, 487, 527, 532, 585, or 588, numbered based on the VP1 numbering of AAV2, and a rAAV vector comprising a heterologous nucleic acid encoding a therapeutic polypeptide or therapeutic nucleic acid. In some embodiments, the heterologous nucleic acid encodes a polypeptide selected from the group consisting of an antioxidant, a neurotrophic factor, an anti-apoptotic factor, an anti-angiogenic factor, and an anti-inflammatory factor. In further embodiments, the heterologous nucleic acid encodes a polypeptide selected from the group consisting of: Prph2, RPE65, AIPL1, GUCY2D, LCA5, CRX, CEP290, MYO 7a, Curarin, ABCA4, RDH12, IMPDH1, CRB1, LRAT, NMNAT1, TULP1, MERTK, RPGR, RP2, RPGRIP, CNGA3, CNGB3, GNAT2, GDNF, CNTF, FGF2, PEDF, EPO, BCL2, BCL-X, NFκB, endostatin, angiostatin, sFlt, sPDGF-R, IL10, anti-IL17, sIL17R, IL1-ra, anti-TGFβ, sTNF-RI, sTNF-RII, and IL4. In other embodiments, the heterologous nucleic acid encodes a therapeutic nucleic acid. In further embodiments, the therapeutic nucleic acid is an siRNA, shRNA, RNAi, miRNA, antisense RNA, ribozyme, or DNAzyme. In some embodiments, the rAAV vector is a self-complementary rAAV vector.

[0013] In some embodiments, the AAV particles of the present invention comprise a capsid containing one or more amino acid substitutions that alter HSPG binding (e.g., reduce or eliminate HSPG binding) or one or more amino acid substitutions at one or more positions corresponding to amino acids 484, 487, 527, 532, 585, or 588, numbered according to the VP1 numbering system of AAV2, and a heterologous nucleic acid encoding a therapeutic polypeptide or therapeutic nucleic acid, wherein the heterologous nucleic acid is under the control of a promoter sequence that is expressed in the retina. In some embodiments, the heterologous nucleic acid is operably linked to a promoter suitable for expression of the therapeutic polypeptide or therapeutic nucleic acid in one or more retinal cell types. In some embodiments, the retinal cells are photoreceptor cells, retinal pigmented epithelial cells, bipolar cells, horizontal cells, amacrine cells, Müller cells, and / or ganglion cells. In some embodiments, the promoter is a rhodopsin kinase (RK) promoter, an opsin promoter, a cytomegalovirus (CMV) promoter, or a chicken β-actin (CBA) promoter.

[0014] In some embodiments, the AAV particles of the invention comprise a capsid that includes one or more amino acid substitutions that alter binding to HSPG (e.g., reduce or eliminate binding to HSPG) or one or more amino acid substitutions at one or more positions corresponding to amino acids 484, 487, 527, 532, 585, or 588, numbered based on the VP1 numbering of AAV2, and a heterologous nucleic acid for delivery of the heterologous nucleic acid to the retina of an individual. In some embodiments, the individual is a human. In some embodiments, the heterologous nucleic acid is used to treat an ocular disorder selected from the group consisting of: autosomal recessive severe early-onset retinal degeneration (Leber's congenital amaurosis), congenital color blindness, Stargardt's disease, Best's disease, Doyne's disease, cone dystrophy, retinitis pigmentosa, X-linked retinoschisis, Usher syndrome, age-related macular degeneration, atrophic age-related macular degeneration, neovascular AMD, diabetic maculopathy, proliferative diabetic retinopathy (PDR), cystoid macular edema, central serous retinopathy, retinal detachment, intraocular inflammation, glaucoma, and posterior uveitis.

[0015] In some embodiments, the invention provides a method for improving rAAV transduction of cells following subretinal delivery of rAAV particles to the eye of an individual compared to transduction of cells with an rAAV comprising a wild-type capsid, comprising incorporating one or more amino acid substitutions into an AAV capsid protein at one or more positions that interact with heparan sulfate proteoglycans or at one or more positions corresponding to amino acids 484, 487, 527, 532, 585, or 588, numbered based on the VP1 numbering of AAV2; the rAAV particle comprises the rAAV capsid protein and an rAAV vector comprising a heterologous nucleic acid and at least one AAV terminal repeat sequence.

[0016] In another aspect, the invention provides a method for improving heterologous nucleic acid expression following subretinal delivery of rAAV particles to the eye of an individual, comprising incorporating one or more amino acid substitutions into an AAV capsid protein at one or more positions that interact with heparan sulfate proteoglycans or at one or more positions corresponding to amino acids 484, 487, 532, 585, or 588, numbered based on the VP1 numbering of AAV2; the rAAV particle comprises the rAAV capsid protein and an rAAV vector comprising a heterologous nucleic acid and at least one AAV terminal repeat sequence.

[0017] In some embodiments, the rAAV particles with improved transduction and / or improved expression of heterologous nucleic acids comprise an AAV serotype 2 (AAV2) capsid. In some embodiments, the one or more amino acid substitutions reduce binding of the rAAV particles to heparan sulfate proteoglycans. In some embodiments, the one or more amino acid substitutions reduce binding of the rAAV particles to heparan sulfate proteoglycans by at least about 10%, at least about 25%, at least about 50%, at least about 75%, or at least about 100%.

[0018] In some embodiments, rAAV particles with improved transduction and / or improved expression of heterologous nucleic acids comprise capsids with one or more amino acid substitutions at positions 484, 487, 527, 532, 585, or 588, as numbered based on the VP1 numbering of AAV2. In some embodiments, the numbering is based on AAV2 VP1 comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, the one or more amino acid substitutions comprise substitution of a positively charged amino acid residue with an amino acid residue that does not bear a positive charge. In some embodiments, the positively charged amino acid residue is substituted with a hydrophobic amino acid residue. In further embodiments, the one or more amino acid substitutions comprise substitution of an arginine or lysine residue with an alanine residue. In some embodiments, the one or more amino acid substitutions comprise substitutions at positions R484, R487, K527, K532, R585, and / or R588, numbered based on VP1 of AAV2. In some embodiments, the rAAV particles comprise one or more rAAV capsid proteins having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity to SEQ ID NOs: 2, 4, and / or 6. In some embodiments, the one or more amino acid substitutions comprise substitutions at positions R484 and R487 or at positions R585 and R588, numbered based on VP1 of AAV2. In a further embodiment, the one or more amino acid substitutions comprise R484A and R487A substitutions or R585A and R588A substitutions, numbered based on VP1 of AAV2. It contains the amino acid substitutions R585A and R588A, numbered based on VP1 of 2. In some embodiments, the rAAV particle comprises an AAV1 capsid, an AAV2 capsid, an AAV3 capsid, an AAV6 capsid, an AAV8 capsid, an AAVrh8R capsid, an AAV9 capsid, or an AAVrh10 capsid.

[0019] In some embodiments, rAAV particles with improved transduction and / or improved expression of heterologous nucleic acids comprise capsids with one or more amino acid substitutions at positions 485, 488, 528, 533, 586, or 589, as numbered based on the VP1 numbering of AAVrh8R. In some embodiments, AAV particles of the invention comprise capsids with one or more amino acid substitutions at positions 485, 488, 528, or 533, as numbered based on the VP1 numbering of AAVrh8R. In some embodiments, the numbering is based on the VP1 of AAVrh8R comprising the amino acid sequence of SEQ ID NO: 9. In some embodiments, the one or more amino acid substitutions comprise substitution of a positively charged amino acid residue with an amino acid residue that does not bear a positive charge. In some embodiments, the positively charged amino acid residue is substituted with a hydrophobic amino acid residue. In further embodiments, the one or more amino acid substitutions comprise substitution of an arginine or lysine residue. In still further embodiments, the one or more amino acid substitutions comprise substitutions of an arginine or lysine residue with an alanine residue. In other embodiments, the one or more amino acid substitutions comprise substitutions of an amino acid residue that does not bear a positive charge with an amino acid residue that bears a positive charge. In some embodiments, a hydrophobic amino acid residue is substituted with an amino acid residue that bears a positive charge. In further embodiments, the one or more amino acid substitutions comprise substitutions of an alanine residue. In still further embodiments, the one or more amino acid substitutions comprise substitutions of an arginine or lysine residue with an alanine residue. In some embodiments, the amino acid substitution is at position 485, 488, 528, 533, or 589, numbered based on the VP1 numbering of AAVrh8R. In some embodiments, the AAV particles of the present invention comprise capsids with one or more amino acid substitutions at positions 485, 488, 528, or 533, numbered based on the VP1 numbering of AAVrh8R.In some embodiments, the numbering is based on VP1 of AAVrh8R, which comprises the amino acid sequence of SEQ ID NO:9. In some embodiments, the amino acid substitution comprises a substitution at position R485, R488, R533, or T589, numbered based on the VP1 numbering of AAVrh8R. In some embodiments, the rAAV particles comprise the rAAV capsid protein of SEQ ID NO:11. In some embodiments, the rAAV particles comprise one or more rAAV capsid proteins having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity to SEQ ID NO:11. In some embodiments, one amino acid substitution comprises an R533A substitution, numbered based on VP1 of AAVrh8R.

[0020] In some embodiments, rAAV particles with improved transduction and / or improved expression of heterologous nucleic acids comprise a capsid containing one or more amino acid substitutions that alter HSPG binding (e.g., reduce or eliminate HSPG binding) or one or more amino acid substitutions at one or more positions corresponding to amino acids 484, 487, 527, 532, 585, or 588, numbered according to the VP1 numbering of AAV2, and a rAAV vector containing a heterologous nucleic acid encoding a therapeutic polypeptide or therapeutic nucleic acid. In some embodiments, the heterologous nucleic acid encodes a polypeptide selected from the group consisting of an antioxidant, a neurotrophic factor, an anti-apoptotic factor, an anti-angiogenic factor, and an anti-inflammatory factor. In further embodiments, the heterologous nucleic acid encodes a polypeptide selected from the group consisting of an antioxidant, a neurotrophic factor, an anti-apoptotic factor, an anti-angiogenic factor, and an anti-inflammatory factor. The nucleic acid encodes a polypeptide selected from the group consisting of: Prph2, RPE65, AIPL1, GUCY2D, LCA5, CRX, CEP290, MYO 7a, Curarin, ABCA4, RDH12, IMPDH1, CRB1, LRAT, NMNAT1, TULP1, MERTK, RPGR, RP2, RPGRIP, CNGA3, CNGB3, GNAT2, GDNF, CNTF, FGF2, PEDF, EPO, BCL2, BCL-X, NFκB, endostatin, angiostatin, sFlt, sPDGF-R, IL10, anti-IL17, sIL17R, IL1-ra, anti-TGFβ, sTNF-RI, sTNF-RII, and IL4. In other embodiments, the heterologous nucleic acid encodes a therapeutic nucleic acid. In further embodiments, the therapeutic nucleic acid is an siRNA, shRNA, RNAi, miRNA, antisense RNA, ribozyme, or DNAzyme. In some embodiments, the rAAV vector is a self-complementary rAAV vector.

[0021] In some embodiments, rAAV particles with improved transduction and / or improved expression of heterologous nucleic acids comprise a capsid containing one or more amino acid substitutions that alter HSPG binding (e.g., reduce or eliminate HSPG binding) or one or more amino acid substitutions at one or more positions corresponding to amino acids 484, 487, 532, 585, or 588, numbered according to the VP1 numbering system of AAV2, and a heterologous nucleic acid encoding a therapeutic polypeptide or therapeutic nucleic acid, wherein the heterologous nucleic acid is under the control of a promoter sequence that is expressed in the retina. In some embodiments, the heterologous nucleic acid is operably linked to a promoter suitable for expression of the therapeutic polypeptide or therapeutic nucleic acid in one or more retinal cell types. In some embodiments, the retinal cells are photoreceptor cells, retinal pigmented epithelial cells, and / or ganglion cells. In some embodiments, the promoter is a rhodopsin kinase (RK) promoter, an opsin promoter, a cytomegalovirus (CMV) promoter, or a chicken β-actin (CBA) promoter.

[0022] In some embodiments, rAAV particles with improved transduction and / or improved expression of a heterologous nucleic acid comprise a capsid that includes one or more amino acid substitutions that alter binding to HSPG (e.g., reduce or eliminate binding to HSPG) or one or more amino acid substitutions at one or more positions corresponding to amino acids 484, 487, 527, 532, 585, or 588, numbered based on the VP1 numbering of AAV2, and a heterologous nucleic acid for delivery of the heterologous nucleic acid to the retina of an individual. In some embodiments, the individual is a human. In some embodiments, the heterologous nucleic acid is used to treat an ocular disorder selected from the group consisting of: autosomal recessive severe early-onset retinal degeneration (Leber's congenital amaurosis), congenital color blindness, Stargardt's disease, Best's disease, Doyne's disease, cone dystrophy, retinitis pigmentosa, X-linked retinoschisis, Usher syndrome, age-related macular degeneration, atrophic age-related macular degeneration, neovascular AMD, diabetic maculopathy, proliferative diabetic retinopathy (PDR), cystoid macular edema, central serous retinopathy, retinal detachment, intraocular inflammation, glaucoma, and posterior uveitis.

[0023] In some embodiments, the present invention provides methods for treating an ocular disorder in an individual (e.g., a human), comprising delivering to the retina of the individual a composition comprising rAAV particles, the rAAV particles comprising: a) an rAAV capsid comprising an rAAV capsid protein comprising one or more amino acid substitutions at one or more positions that interact with heparan sulfate proteoglycans, or at one or more positions corresponding to amino acids 484, 487, 527, 532, 585, or 588, numbered according to the VP1 numbering of AAV2; and b) an rAAV vector comprising a heterologous nucleic acid and at least one AAV terminal repeat sequence. In some embodiments, the rAAV particles comprise an AAV serotype 2 (AAV2) capsid. In some embodiments, the one or more amino acid substitutions reduce binding of the rAAV particles to heparan sulfate proteoglycans. In some embodiments, the one or more amino acid substitutions reduce binding of the rAAV particle to heparan sulfate proteoglycans by at least about 10%, at least about 25%, at least about 50%, at least about 75%, or at least about 100%.

[0024] In some embodiments, the method includes subretinal delivery of rAAV particles comprising an rAAV vector encoding a heterologous nucleic acid used to treat an ocular disorder in an individual (e.g., a human), wherein the rAAV particles comprise a capsid having one or more amino acid substitutions at positions 484, 487, 527, 532, 585, or 588, as numbered based on the VP1 numbering of AAV2. In some embodiments, the numbering is based on the VP1 of AAV2 comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, the one or more amino acid substitutions comprise substitution of a positively charged amino acid residue with an amino acid residue that does not bear a positive charge. In some embodiments, the positively charged amino acid residue is substituted with a hydrophobic amino acid residue. In further embodiments, the one or more amino acid substitutions comprise substitution of an arginine or lysine residue. In yet further embodiments, the one or more amino acid substitutions comprise substitution of an arginine or lysine residue with an alanine residue. In some embodiments, the one or more amino acid substitutions comprise substitutions at positions R484, R487, K527, K532, R585, and / or R588, numbered based on VP1 of AAV2. In some embodiments, the rAAV particles comprise one or more rAAV capsid proteins having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity to SEQ ID NOs: 2, 4, and / or 6. In some embodiments, the one or more amino acid substitutions comprise substitutions at positions R484 and R487 or at positions R585 and R588, numbered based on VP1 of AAV2. In a further embodiment, the one or more amino acid substitutions comprise R484A and R487A substitutions or R585A and R588A substitutions, numbered based on VP1 of AAV2. In a further embodiment, the AAV capsid comprises amino acid substitutions R585A and R588A, numbered based on VP1 of AAV-2.In some embodiments, the rAAV particle comprises an AAV1 capsid, an AAV2 capsid, an AAV3 capsid, an AAV6 capsid, an AAV8 capsid, an AAVrh8R capsid, an AAV9 capsid, or an AAVrh10 capsid.

[0025] In some embodiments, the method comprises subretinal delivery of rAAV particles comprising an rAAV vector encoding a heterologous nucleic acid used to treat an ocular disorder in an individual (e.g., a human), wherein the rAAV particles comprise a capsid with one or more amino acid substitutions at positions 485, 488, 528, 533, 586, or 589, as numbered based on the VP1 numbering of AAVrh8R. In some embodiments, the AAV particles of the invention comprise a capsid with one or more amino acid substitutions at positions 485, 488, 528, or 533, as numbered based on the VP1 numbering of AAVrh8R. In some embodiments, the numbering is based on VP1 of AAVrh8R comprising the amino acid sequence of SEQ ID NO: 9. In some embodiments, the one or more amino acid substitutions comprise substitution of a positively charged amino acid residue with a non-positively charged amino acid residue. In some embodiments, the positively charged amino acid residue is substituted with a hydrophobic amino acid residue. In further embodiments, the one or more amino acid substitutions comprise substitutions of arginine or lysine residues. In yet further embodiments, the one or more amino acid substitutions comprise substitutions of arginine or lysine residues with alanine residues. In other embodiments, the one or more amino acid substitutions comprise substitutions of a non-positively charged amino acid residue with a positively charged amino acid residue. In some embodiments, the hydrophobic amino acid residue is substituted with a positively charged amino acid residue. In further embodiments, the one or more amino acid substitutions comprise substitutions of an alanine residue. In yet further embodiments, the one or more amino acid substitutions comprise substitutions of an arginine or lysine residue with an alanine residue. In some embodiments, the amino acid substitutions are at positions 485, 488, 528, 533, or 589, numbered based on the VP1 numbering of AAVrh8R. In some embodiments, the AAV particles of the present invention comprise capsids having one or more amino acid substitutions at positions 485, 488, 528, or 533, numbered based on the VP1 numbering of AAVrh8R. In some embodiments, the numbering is based on the VP1 of AAVrh8R comprising the amino acid sequence of SEQ ID NO:9. In some embodiments, the amino acid substitutions comprise substitutions at positions R485, R488, R533, or T589, numbered based on the VP1 numbering of AAVrh8R. In some embodiments, the rAAV particles comprise the rAAV capsid protein of SEQ ID NO: 11. In some embodiments, the rAAV particles comprise one or more rAAV capsid proteins having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity to SEQ ID NO: 11. In some embodiments, the single amino acid substitution comprises an R533A substitution, numbered based on VP1 of AAVrh8R.

[0026] In some embodiments, the methods include subretinal delivery of rAAV particles, wherein the rAAV particles comprise an rAAV vector encoding a heterologous nucleic acid used to treat an ocular disorder in an individual (e.g., a human), and a capsid comprising one or more amino acid substitutions that alter binding to HSPG (e.g., reduce or eliminate binding to HSPG), or one or more amino acid substitutions at one or more positions corresponding to amino acids 484, 487, 527, 532, 585, or 588, numbered based on the VP1 numbering of AAV2. In some embodiments, the heterologous nucleic acid encodes a polypeptide selected from the group consisting of an antioxidant, a neurotrophic factor, an anti-apoptotic factor, an anti-angiogenic factor, and an anti-inflammatory factor. In further embodiments, the heterologous nucleic acid encodes a polypeptide selected from the group consisting of: Prph2, RPE65, AIPL1, GUCY2D, LCA5, CRX, CEP290, MYO 7a, Curarin, ABCA4, RDH12, IMPDH1, CRB1, LRAT, NMNAT1, TULP1, MERTK, RPGR, RP2, RPGRIP, CNGA3, CNGB3, GNAT2, GDNF, CNTF, FGF2, PEDF, EPO, BCL2, BCL-X, NFκB, endostatin, angiostatin, sFlt, sPDGF-R, IL10, anti-IL17, sIL17R, IL1-ra, anti-TGFβ, sTNF-RI, sTNF-RII, and IL4. In other embodiments, the heterologous nucleic acid encodes a therapeutic nucleic acid. In further embodiments, the therapeutic nucleic acid is an siRNA, shRNA, RNAi, miRNA, antisense RNA, ribozyme, or DNAzyme. In some embodiments, the rAAV vector is a self-complementary rAAV vector.

[0027] In some embodiments, the method comprises subretinal delivery of rAAV particles, the rAAV particles comprising an rAAV vector encoding a heterologous nucleic acid for use in treating an ocular disorder in an individual (e.g., a human), and a capsid comprising one or more amino acid substitutions that alter binding to HSPG (e.g., reduce or eliminate binding to HSPG), or one or more amino acid substitutions at one or more positions corresponding to amino acids 484, 487, 527, 532, 585, or 588, numbered based on the VP1 numbering of AAV2, wherein the heterologous nucleic acid sequence is under the control of a promoter sequence that is expressed in the retina. In some embodiments, the heterologous nucleic acid is one or more The therapeutic polypeptide or nucleic acid is operably linked to a promoter suitable for expression in the above retinal cell types. In some embodiments, the retinal cells are photoreceptor cells, retinal pigmented epithelial cells, and / or ganglion cells. In some embodiments, the promoter is a rhodopsin kinase (RK) promoter, an opsin promoter, a cytomegalovirus (CMV) promoter, or a chicken beta-actin (CBA) promoter.

[0028] In some embodiments, the methods include subretinal delivery of rAAV particles, wherein the rAAV particles comprise an rAAV vector encoding a heterologous nucleic acid for use in treating an ocular disorder in an individual (e.g., a human), and one or more amino acid substitutions that alter binding to HSPG (e.g., reduce or eliminate binding to HSPG) or one or more amino acid substitutions at one or more positions corresponding to amino acids 484, 487, 527, 532, 585, or 588, numbered based on the VP1 numbering of AAV2. and a capsid containing one or more amino acid substitutions, wherein the ocular disorder is selected from the group consisting of autosomal recessive severe early-onset retinal degeneration (Leber's congenital amaurosis), congenital color blindness, Stargardt's disease, Best's disease, Doyne's disease, cone dystrophy, retinitis pigmentosa, X-linked retinoschisis, Usher syndrome, age-related macular degeneration, atrophic age-related macular degeneration, neovascular AMD, diabetic maculopathy, proliferative diabetic retinopathy (PDR), cystoid macular edema, central serous retinopathy, retinal detachment, intraocular inflammation, glaucoma, and posterior uveitis.

[0029] In some embodiments, the method comprises subretinal delivery of a composition comprising rAAV particles, the rAAV particles comprising an rAAV vector encoding a heterologous nucleic acid for use in treating an ocular disorder in an individual (e.g., a human), and a capsid comprising one or more amino acid substitutions that alter binding to HSPG (e.g., reduce or eliminate binding to HSPG), or one or more amino acid substitutions at one or more positions corresponding to amino acids 484, 487, 527, 532, 585, or 588, numbered based on the VP1 numbering of AAV2. In some embodiments, the concentration of particles in the composition is about 1 x 10 6 DRP / ml ~ approx. 1 x 10 14In some embodiments, the rAAV particle composition is effective in treating the visual function of an individual. In some embodiments, the visual function is assessed by microperimetry, scotopic perimetry, visual movement assessment, visual acuity, ERG, or reading assessment. In some embodiments, the method results in an improvement in the visual function of the individual. In some embodiments, the method results in the prevention or slowing of the progression of visual function decline in a person due to the progression of an eye disorder.

[0030] In some embodiments, the present invention provides a system for subretinal delivery of a vector to the eye of an individual, the system comprising: a) a composition comprising an effective amount of rAAV particles, wherein i) the capsid protein of the rAAV particles comprises one or more amino acid substitutions at one or more positions that interact with heparan sulfate proteoglycans or at one or more positions corresponding to amino acids 484, 487, 527, 532, 585, or 588, as numbered according to the VP1 numbering of AAV2; and ii) the vector comprises a heterologous nucleic acid encoding a therapeutic polypeptide or therapeutic RNA and at least one AAV terminal repeat sequence; and b) a device for retinal delivery of the rAAV. In some embodiments, the device comprises a thin cannula and a syringe, the thin cannula being 27-45 gauge. In some embodiments, the rAAV particle composition is contained within the syringe. In some embodiments, the cannula is attached to the syringe. In some embodiments, the concentration of particles in the composition is about 1 x 10 6 DRP / ml ~ approx. 1 x 10 14 DRP / ml.

[0031] In some embodiments, the rAAV particles of the system comprise an AAV2 capsid containing one or more amino acid substitutions that alter HSPG binding (e.g., reduce or eliminate binding). In some embodiments, the one or more amino acid substitutions reduce binding of the rAAV particles to heparan sulfate proteoglycans. In some embodiments, the one or more amino acid substitutions reduce binding of the rAAV particles to heparan sulfate proteoglycans by at least about 10%, at least about 25%, at least about 50%, at least about 75%, or at least about 100%. In some embodiments, the one or more amino acid substitutions increase the transduction efficiency of ocular or CNS cells by the rAAV particles. In some embodiments, the one or more amino acid substitutions increase the transduction efficiency of rAAV particles into cells of the eye or CNS by at least about 10%, at least about 25%, at least about 50%, at least about 75%, or at least about 100%, for example, compared to a reference rAAV capsid comprising a wild-type AAV capsid protein. In some embodiments, the cells of the eye are retinal cells, photoreceptor cells, retinal pigmented epithelial cells, bipolar cells, horizontal cells, amacrine cells, Müller cells, and / or ganglion cells. In some embodiments, the cells of the CNS are oligodendrocytes, astrocytes, neurons, brain parenchymal cells, microglial cells, ependymal cells, and / or Purkinje cells.

[0032] In some embodiments, the rAAV particles of the system comprise an AAV2 capsid comprising one or more amino acid substitutions that alter HSPG binding (e.g., reduce or eliminate binding). In some embodiments, the one or more amino acid substitutions are at positions 484, 487, 527, 532, 585, or 588, as numbered based on the AAV2 VP1 numbering system. In some embodiments, the numbering is based on the AAV2 VP1 comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, the one or more amino acid substitutions comprise a substitution of a positively charged amino acid residue with an amino acid residue that does not bear a positive charge. In some embodiments, the positively charged amino acid residue is substituted with a hydrophobic amino acid residue. In further embodiments, the one or more amino acid substitutions comprise a substitution of an arginine or lysine residue with an alanine residue. In some embodiments, the one or more amino acid substitutions comprise substitutions at positions R484, R487, K527, K532, R585, and / or R588, numbered based on VP1 of AAV2. In some embodiments, the rAAV particles comprise one or more rAAV capsid proteins having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity to SEQ ID NOs: 2, 4, and / or 6. In some embodiments, the one or more amino acid substitutions comprise substitutions at positions R484 and R487 or at positions R585 and R588, numbered based on VP1 of AAV2. In a further embodiment, the one or more amino acid substitutions comprise R484A and R487A substitutions or R585A and R588A substitutions, numbered based on VP1 of AAV2. In a further embodiment, the AAV capsid comprises amino acid substitutions R585A and R588A, numbered based on VP1 of AAV-2.In some embodiments, the rAAV particle comprises an AAV1 capsid, an AAV2 capsid, an AAV3 capsid, an AAV6 capsid, an AAV8 capsid, an AAVrh8R capsid, an AAV9 capsid, or an AAVrh10 capsid.

[0033] In some embodiments, the rAAV particles of the system comprise an AAV2 capsid comprising one or more amino acid substitutions that alter HSPG binding (e.g., reduce or eliminate binding). In some embodiments, the one or more amino acid substitutions are: The amino acid sequence is at positions 485, 488, 528, 533, 586, or 589 based on the VP1 numbering of AAVrh8R. In some embodiments, the numbering is based on VP1 of AAVrh8R comprising the amino acid sequence of SEQ ID NO:9. In some embodiments, the one or more amino acid substitutions comprise substitution of a positively charged amino acid residue with an amino acid residue that does not bear a positive charge. In some embodiments, the positively charged amino acid residue is substituted with a hydrophobic amino acid residue. In further embodiments, the one or more amino acid substitutions comprise substitution of an arginine or lysine residue with an alanine residue. In still further embodiments, the one or more amino acid substitutions comprise substitution of an arginine or lysine residue with an alanine residue. In other embodiments, the one or more amino acid substitutions comprise substitution of a non-positively charged amino acid residue with a positively charged amino acid residue. In some embodiments, the hydrophobic amino acid residue is substituted with a positively charged amino acid residue. In further embodiments, the one or more amino acid substitutions comprise substitution of an alanine residue. In still further embodiments, the one or more amino acid substitutions comprise substitutions of an arginine or lysine residue with an alanine residue. In some embodiments, the amino acid substitutions are at positions 485, 488, 528, 533, or 589, numbered based on the VP1 numbering of AAVrh8R. In some embodiments, the numbering is based on VP1 of AAVrh8R comprising the amino acid sequence of SEQ ID NO:9. In some embodiments, the amino acid substitutions comprise substitutions at positions R485, R488, R533, or T589, numbered based on the VP1 numbering of AAVrh8R. In some embodiments, the rAAV particles comprise the rAAV capsid protein of SEQ ID NO:11.In some embodiments, the rAAV particles comprise one or more rAAV capsid proteins having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity to SEQ ID NO: 11. In some embodiments, the single amino acid substitution comprises an R533A substitution, numbered based on VP1 of AAVrh8R.

[0034] In some embodiments, the rAAV particles of the system include an AAV capsid with one or more amino acid substitutions that alter HSPG binding (e.g., reduce or eliminate binding) or one or more amino acid substitutions at one or more positions corresponding to amino acids 484, 487, 527, 532, 585, or 588, numbered according to the VP1 numbering of AAV2, and a rAAV vector comprising a heterologous nucleic acid. In some embodiments, the heterologous nucleic acid encodes a therapeutic polypeptide or therapeutic nucleic acid. In some embodiments, the heterologous nucleic acid encodes a polypeptide selected from the group consisting of an antioxidant, a neurotrophic factor, an anti-apoptotic factor, an anti-angiogenic factor, and an anti-inflammatory factor. In further embodiments, the heterologous nucleic acid encodes a polypeptide selected from the group consisting of: Prph2, RPE65, AIPL1, GUCY2D, LCA5, CRX, CEP290, MYO 7a, Curarin, ABCA4, RDH12, IMPDH1, CRB1, LRAT, NMNAT1, TULP1, MERTK, RPGR, RP2, RPGRIP, CNGA3, CNGB3, GNAT2, GDNF, CNTF, FGF2, PEDF, EPO, BCL2, BCL-X, NFκB, endostatin, angiostatin, sFlt, sPDGF-R, IL10, anti-IL17, sIL17R, IL1-ra, anti-TGFβ, sTNF-RI, sTNF-RII, and IL4. In other embodiments, the heterologous nucleic acid encodes a therapeutic nucleic acid. In further embodiments, the therapeutic nucleic acid is an siRNA, RNAi, miRNA, antisense RNA, ribozyme, or DNAzyme. In some embodiments, the rAAV vector is a self-complementary rAAV vector.

[0035] In some embodiments, the rAAV particles of the system alter HSPG binding ( The rAAV vector includes an AAV capsid having one or more amino acid substitutions (e.g., that reduce or eliminate binding) or one or more amino acid substitutions at one or more positions corresponding to amino acids 484, 487, 527, 532, 585, or 588, numbered based on the VP1 numbering of AAV2, and a heterologous nucleic acid, wherein the heterologous nucleic acid is under the control of a promoter sequence that is expressed in the retina. In some embodiments, the heterologous nucleic acid is operably linked to a promoter suitable for expression of a therapeutic polypeptide or therapeutic nucleic acid in one or more retinal cell types. In some embodiments, the retinal cells are photoreceptor cells, retinal pigmented epithelial cells, and / or ganglion cells. In some embodiments, the promoter is a rhodopsin kinase (RK) promoter, an opsin promoter, a cytomegalovirus (CMV) promoter, or a chicken beta-actin (CBA) promoter.

[0036] In some embodiments, an rAAV particle of a system comprising an AAV capsid having one or more amino acid substitutions that alter HSPG binding (e.g., reduce or eliminate binding) or one or more amino acid substitutions at one or more positions corresponding to amino acids 484, 487, 527, 532, 585, or 588, numbered based on the VP1 numbering of AAV2, and an rAAV vector comprising a heterologous nucleic acid is used to deliver the heterologous nucleic acid to the retina of an individual. In some embodiments, the individual is human. In some embodiments, the heterologous nucleic acid is used to treat an ocular disorder selected from the group consisting of: autosomal recessive severe early-onset retinal degeneration (Leber's congenital amaurosis), congenital color blindness, Stargardt's disease, Best's disease, Doyne's disease, cone dystrophy, retinitis pigmentosa, X-linked retinoschisis, Usher syndrome, age-related macular degeneration, atrophic age-related macular degeneration, neovascular AMD, diabetic maculopathy, proliferative diabetic retinopathy (PDR), cystoid macular edema, central serous retinopathy, retinal detachment, intraocular inflammation, glaucoma, and posterior uveitis.

[0037] In some embodiments, the invention provides methods for delivering heterologous nucleic acid to the central nervous system (CNS) of an individual, the method comprising administering recombinant adeno-associated virus (rAAV) particles to the CNS of the individual, the rAAV particles comprising: a) an rAAV capsid comprising an rAAV capsid protein comprising one or more amino acid substitutions at one or more positions that interact with heparan sulfate proteoglycans or at one or more positions corresponding to amino acids 484, 487, 527, 532, 585, or 588, numbered based on the VP1 numbering of AAV2; and b) an rAAV vector comprising the heterologous nucleic acid and at least one AAV inverted terminal repeat. In some embodiments, the invention provides methods for improving rAAV transduction of cells of an individual's central nervous system (CNS) compared to transduction of cells with an rAAV comprising a wild-type capsid, comprising administering recombinant adeno-associated virus (rAAV) particles to the CNS of the individual, wherein the rAAV particles comprise: a) an rAAV capsid comprising an rAAV capsid protein comprising one or more amino acid substitutions at one or more positions that interact with heparan sulfate proteoglycans or at one or more positions corresponding to amino acids 484, 487, 527, 532, 585, or 588, numbered based on the VP1 numbering of AAV2; and b) an rAAV vector comprising a heterologous nucleic acid and at least one AAV inverted terminal repeat. In a further aspect, the invention provides a method for enhancing expression of a heterologous nucleic acid in the central nervous system (CNS) of an individual, comprising administering to the CNS of the individual recombinant adeno-associated virus (rAAV) particles comprising: a) an rAAV capsid comprising an rAAV capsid protein comprising one or more amino acid substitutions at one or more positions that interact with heparan sulfate proteoglycans, or at one or more positions corresponding to amino acids 484, 487, 527, 532, 585, or 588, numbered based on the VP1 numbering of AAV2; and b) a heterologous nucleic acid and at least one In yet a further aspect, the present invention provides a method for treating a disorder of the central nervous system (CNS) of an individual, comprising administering to the CNS of the individual an effective amount of a composition comprising rAAV particles, the rAAV particles comprising: a) an rAAV capsid comprising an rAAV capsid protein containing one or more amino acid substitutions at one or more positions that interact with heparan sulfate proteoglycans, or at one or more positions corresponding to amino acids 484, 487, 527, 532, 585, or 588, numbered according to the VP1 numbering of AAV2, and b) an rAAV vector comprising a heterologous nucleic acid and at least one AAV inverted terminal repeat.

[0038] In some embodiments of the above aspects and embodiments, the heterologous nucleic acid is expressed at an increased expression level compared to the expression level of the heterologous nucleic acid of an rAAV particle comprising a reference rAAV capsid. In some embodiments, nucleic acid expression is increased by at least about 10%, at least about 25%, at least about 50%, at least about 75%, or at least about 100%. In some embodiments, the rAAV particle results in reduced neuroinflammation compared to an rAAV particle comprising a reference rAAV capsid. In some embodiments, neuroinflammation is reduced by at least about 10%, at least about 25%, at least about 50%, at least about 75%, or at least about 100%. In some embodiments, the rAAV particle comprises an AAV serotype 2 (AAV2) capsid. In some embodiments, the one or more amino acid substitutions reduce binding of the rAAV particle to heparan sulfate proteoglycans. In some embodiments, the one or more amino acid substitutions reduce binding of the rAAV particles to heparan sulfate proteoglycans compared to binding of rAAV particles comprising a reference rAAV capsid to heparan sulfate proteoglycans. In some embodiments, the one or more amino acid substitutions reduce binding of the rAAV particles to heparan sulfate proteoglycans by at least about 10%, at least about 25%, at least about 50%, at least about 75%, or at least about 100%. In some embodiments, the one or more amino acid substitutions reduce binding of the rAAV particles to heparan sulfate proteoglycans by at least about 10%, at least about 25%, at least about 50%, at least about 75%, or at least about 100% compared to binding of rAAV particles comprising a reference capsid to heparan sulfate proteoglycans. In some embodiments, the reference rAAV capsid comprises a wild-type rAAV capsid or capsid protein. In some embodiments, the reference rAAV capsid comprises a rAAV capsid or capsid protein that lacks one or more amino acid substitutions at one or more positions that interact with heparan sulfate proteoglycans.In some embodiments, the one or more amino acid substitutions increase the transduction efficiency of rAAV particles into cells of the eye or CNS. In some embodiments, the one or more amino acid substitutions increase the transduction efficiency of rAAV particles into cells of the eye or CNS by at least about 10%, at least about 25%, at least about 50%, at least about 75%, or at least about 100%, for example, compared to a reference rAAV capsid comprising a wild-type AAV capsid protein. In some embodiments, the rAAV particles comprise an AAV1 capsid, an AAV2 capsid, an AAV3 capsid, an AAV6 capsid, an AAV8 capsid, an AAVrh8R capsid, an AAV9 capsid, or an AAVrh10 capsid.

[0039] In some embodiments of the above aspects and embodiments, the administration comprises direct spinal injection and / or intracerebral administration. In some embodiments, the intracerebral administration is to a site selected from the group consisting of the cerebrum, medulla, pons, cerebellum, intracranial cavity, meninges covering the brain, dura mater, arachnoid mater, pia mater, cerebrospinal fluid (CSF) in the subarachnoid space surrounding the brain, deep cerebellar nuclei of the cerebellum, ventricular system of the cerebrum, subarachnoid space, striatum, cortex, septum, thalamus, hypothalamus, and brain parenchyma. In some embodiments, the administration comprises intraventricular injection into at least one lateral cerebral ventricle. In some embodiments, administration is by intrathecal injection in the cervical, thoracic, and / or lumbar regions. In some embodiments, administration is by intrastriatal injection. In some embodiments, administration is by intrathalamic injection. In some embodiments, administration is by intraparenchymal injection. In some embodiments, administration includes direct spinal injection, intracranial and / or intracerebral administration. In some embodiments, the rAAV particles are administered to a single site.

[0040] In some embodiments of the above aspects and embodiments, the rAAV particles are delivered by stereotactic delivery. In some embodiments, the rAAV particles are delivered by convection-enhanced delivery. In some embodiments, the rAAV particles are administered using a CED delivery system. In some embodiments, the CED delivery system includes a cannula and / or a pump. In some embodiments, the cannula is an anti-reflux cannula or a stepped cannula. In some embodiments, the pump is a manual pump. In some embodiments, the pump is an osmotic pump. In some embodiments, the pump is an infusion pump.

[0041] In some of the above aspects and embodiments, the heparan sulfate proteoglycan is expressed on one or more cells of the CNS. In some embodiments, the one or more cells of the CNS are oligodendrocytes, astrocytes, neurons, brain parenchymal cells, microglial cells, ependymal cells, and / or Purkinje cells. In some embodiments, the heparan sulfate proteoglycan is expressed on neurons.

[0042] In some embodiments of the above aspects and embodiments, the heterologous nucleic acid is expressed in one or more cells of the CNS. In some embodiments, the one or more cells of the CNS are oligodendrocytes, astrocytes, neurons, brain parenchymal cells, microglial cells, ependymal cells, and / or Purkinje cells. In some embodiments, the heterologous nucleic acid is expressed in neurons. In some embodiments, the heterologous nucleic acid is exclusively expressed in neurons.

[0043] In some embodiments of the above aspects and embodiments, the one or more amino acid substitutions are at positions 448, 451, 484, 487, 527, 532, 585, and / or 588, numbered based on the AAV2 VP1 numbering system. In some embodiments, the one or more amino acid substitutions are at positions 484, 487, 527, 532, 585, or 588, numbered based on the AAV2 VP1 numbering system. In some embodiments, the numbering is based on an AAV2 VP1 comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, the one or more amino acid substitutions comprise a substitution of a positively charged amino acid residue with an amino acid residue that does not bear a positive charge. In some embodiments, the positively charged amino acid residue is substituted with a hydrophobic amino acid residue. In some embodiments, the one or more amino acid substitutions comprise a substitution of an arginine or lysine residue with an alanine residue. In some embodiments, the one or more amino acid substitutions comprise substitutions at positions R347, R350, K390, K395, R448, R451, R484, R487, K527, K532, R585, and / or R588, numbered based on VP1 of AAV2. In some embodiments, the one or more amino acid substitutions comprise substitutions at positions R484, R487, K527, K532, R585, and / or R588, numbered based on VP1 of AAV2. In some embodiments, the rAAV particles have an amino acid sequence that is at least about 90%, at least about 91%, at least about 92%, or at least about 93% identical to SEQ ID NOs: 2, 4, and / or 6. The rAAV capsid proteins may comprise one or more rAAV capsid proteins that share about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity with one another. In some embodiments, the one or more amino acid substitutions comprise R347A, R350A, K390A, K395A, R448A, R451A, R484A, R487A, K527A, K532A, R585A, and / or R588A substitutions, numbered based on VP1 of AAV2. In some embodiments, the one or more amino acid substitutions comprise substitutions at positions R484 and R487 or at positions R585 and R588, numbered based on VP1 of AAV2. In some embodiments, the one or more amino acid substitutions comprise R484A and R487A substitutions or R585A and R588A substitutions, numbered based on VP1 of AAV2. In some embodiments, the AAV capsid comprises amino acid substitutions R585A and R588A, numbered based on VP1 of AAV2. In some embodiments, the AAV capsid comprises amino acid substitution K532A, numbered based on VP1 of AAV2.

[0044] The one or more amino acid substitutions are at 485, 488, 528, 533, 586, or 589, based on the VP1 numbering of AAVrh8R. In some embodiments, the numbering is based on VP1 of AAVrh8R comprising the amino acid sequence of SEQ ID NO:9. In some embodiments, the one or more amino acid substitutions comprise substitution of a positively charged amino acid residue with an amino acid residue that does not bear a positive charge. In some embodiments, the positively charged amino acid residue is substituted with a hydrophobic amino acid residue. In further embodiments, the one or more amino acid substitutions comprise substitution of an arginine or lysine residue. In yet further embodiments, the one or more amino acid substitutions comprise substitution of an arginine or lysine residue with an alanine residue. In other embodiments, the one or more amino acid substitutions comprise substitution of a non-positively charged amino acid residue with a positively charged amino acid residue. In some embodiments, the hydrophobic amino acid residue is substituted with a positively charged amino acid residue. In further embodiments, the one or more amino acid substitutions comprise substitution of an alanine residue. In still further embodiments, the one or more amino acid substitutions comprise substitutions of an arginine or lysine residue with an alanine residue. In some embodiments, the amino acid substitutions are at positions 485, 488, 528, 533, or 589, numbered based on the VP1 numbering of AAVrh8R. In some embodiments, the numbering is based on VP1 of AAVrh8R comprising the amino acid sequence of SEQ ID NO:9. In some embodiments, the amino acid substitutions comprise substitutions at positions R485, R488, R533, or T589, numbered based on the VP numbering of AAVrh8R. In some embodiments, the rAAV particles comprise the rAAV capsid protein of SEQ ID NO:11.In some embodiments, the rAAV particles comprise one or more rAAV capsid proteins having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity to SEQ ID NO: 11. In some embodiments, the single amino acid substitution comprises an R533A substitution, numbered based on VP1 of AAVrh8R.

[0045] In some embodiments of the above aspects and embodiments, the heterologous nucleic acid encodes a therapeutic polypeptide or therapeutic nucleic acid. In some embodiments, the heterologous nucleic acid encodes a CNS-related gene. In some embodiments, the heterologous nucleic acid encodes an enzyme, a neurotrophic factor, a polypeptide deficient or mutated in individuals with CNS-related disorders, an antioxidant, an anti-apoptotic factor, an anti-angiogenic factor, and an anti-inflammatory factor, α-synuclein, acid β-glucosidase (GBA), β-galactosidase-1 (GLB1), iduronic acid 2- Sulfatase (IDS), galactosylceramidase (GALC), mannosidase, α-D-mannosidase (MAN2B1), β-mannosidase (MANBA), pseudoarylsulfatase A (ARSA), N-acetylglucosamine-1-phosphotransferase (GNPTAB), acid sphingomyelinase (ASM), Niemann-Pick C protein (NPC1), acid α-1,4-glucosidase (GAA), hexosaminidase β subunit, HEXB, N-sulfoglucosamine sulfohydrolase (MPS3A), N-α-acetylglucosaminidase (NAGLU), heparin acetyl-CoA, α-glucosaminidase N-acetyltransferase (MPS3C), N-acetylglucosamine-6-sulfotransferase (NEXB) The polypeptides encode polypeptides selected from the group consisting of: N-acetylgalactosamine-6-sulfatase (GNS), α-N-acetylgalactosaminidase (NAGA), β-glucuronidase (GUSB), hexosaminidase α subunit (HEXA), huntingtin (HTT), lysosomal acid lipase (LIPA), aspartylglucosaminidase, α-galactosidase A, palmitoyl protein thioesterase, tripentidyl peptidase, lysosomal transmembrane protein, cysteine ​​transporter, acid ceramidase, acid α-L-fucosidase, cathepsin A, α-L-iduronidase, arylsulfatase B, arylsulfatase A, N-acetylgalactosamine-6-sulfatase, acid β-galactosidase, and α-neuramidase. In some embodiments, the heterologous nucleic acid encodes a polypeptide selected from the group consisting of neuronal apoptosis inhibitory protein (NAIP), nerve growth factor (NGF), glial cell line-derived growth factor (GDNF), brain-derived growth factor (BDNF), ciliary neurotrophic factor (CNTF), tyrosine hydroxylase (TH), GTP-cyclohydrolase (GTPCH), amino acid decarboxylase (AADC), antioxidants, anti-angiogenic polypeptides, anti-inflammatory polypeptides, and aspartoacylase (ASPA). In some embodiments, the heterologous nucleic acid encodes a therapeutic nucleic acid.In some embodiments, the therapeutic nucleic acid is an siRNA, shRNA, RNAi, miRNA, antisense RNA, ribozyme, or DNAzyme. In some embodiments, the heterologous nucleic acid is under the control of a promoter sequence that is expressed in one or more cells of the CNS. In some embodiments, the heterologous nucleic acid is under the control of a promoter sequence selected from the group consisting of a cytomegalovirus (CMV) immediate early promoter, a RSV LTR, a MoMLV LTR, a phosphoglycerate kinase-1 (PGK) promoter, a simian virus 40 (SV40) promoter, a CK6 promoter, a transthyretin promoter (TTR), a TK promoter, a tetracycline-responsive promoter (TRE), an HBV promoter, a hAAT promoter, an LSP promoter, a chimeric liver-specific promoter (LSP), an E2F promoter, a telomerase (hTERT) promoter; a cytomegalovirus enhancer / chicken β-actin / rabbit β-globin promoter (CAG) promoter, an elongation factor 1-α promoter (EF1-α) promoter, a human β-glucuronidase promoter, a chicken β-actin (CBA) promoter, a retroviral Rous sarcoma virus (RSV) LTR promoter, a dihydrofolate reductase promoter, and a 13-actin promoter. In some embodiments, the heterologous nucleic acid is operably linked to a promoter suitable for expressing a therapeutic polypeptide or therapeutic nucleic acid in one or more cells of the CNS. In some embodiments, the one or more cells of the CNS include one or more cells of the brain. In some embodiments, the one or more cells of the CNS are oligodendrocytes, astrocytes, neurons, brain parenchymal cells, microglial cells, ependymal cells, and / or Purkinje cells. In some embodiments, the brain cells are neurons.

[0046] In some embodiments of the above aspects and embodiments, the rAAV vector is a self-complementary rAAV vector. In some embodiments, the vector comprises a first nucleic acid sequence encoding a heterologous nucleic acid and a second nucleic acid sequence encoding a complementary strand of the nucleic acid, wherein the first nucleic acid sequence shares interstrand base pairs with the second nucleic acid sequence over most or all of its length. In some embodiments, the first and second nucleic acid sequences are linked by a mutated AAV ITR, which comprises a deletion of the D region and a mutation of the truncation sequence.

[0047] In some of the above aspects and embodiments, the individual is a human.

[0048] In some of the above aspects and embodiments, the heterologous nucleic acid encodes a therapeutic polypeptide or therapeutic nucleic acid used to treat a CNS disorder. In some embodiments, the CNS disorder is lysosomal storage disease (LSD), Huntington's disease, epilepsy, Parkinson's disease, Alzheimer's disease, stroke, corticobasal degeneration (CBD), corticobasal ganglionic degeneration (CBGD), frontotemporal dementia (FTD), multiple system atrophy (MSA), progressive supranuclear palsy (PSP), or brain cancer. In some embodiments, the disorder is aspartylglucosaminuria, Fabry, childhood Batten disease (CNL1), classic late-onset childhood Batten disease (CNL2), juvenile Batten disease (CNL3), Batten CNL4, Batten CNL5, Batten CNL6, Batten CNL7, Batten CNL8, cystinosis, Farber, fucosidosis, galactosidosis, Gaucher disease type 1, Gaucher disease type 2, Gaucher disease type 3, GM1 gangliosidosis, Hunter disease, Krabbe disease, alpha-mannosidosis, In some embodiments, the CNS disorder is a lysosomal storage disease selected from the group consisting of leukemia, beta-mannosidosis, Maroteaux-Lamy, metachromatic leukodystrophy, Morquio A, Morquio B, mucolipidosis II / III, Niemann-Pick A, Niemann-Pick B, Niemann-Pick C, Pompe disease, Sandhoff disease, Sanfilippo A, Sanfilippo B, Sanfilippo C, Sanfilippo D, Schindler disease, Schindler-Kanzaki, sialidosis, Sly disease, Tay-Sachs disease, and Wolman disease. In some embodiments, the CNS disorder is Huntington's disease or Parkinson's disease.

[0049] In some embodiments, the present invention provides a method for treating Huntington's disease in an individual, comprising administering to the striatum of the individual an effective amount of a composition comprising recombinant adeno-associated virus (rAAV) particles, wherein the rAAV particles comprise: a) an rAAV capsid comprising an rAAV capsid protein containing one or more amino acid substitutions at one or more positions that interact with heparan sulfate proteoglycans or at one or more positions corresponding to amino acids 484, 487, 527, 532, 585, or 588, numbered according to the VP1 numbering of AAV2; and b) an rAAV vector comprising a heterologous nucleic acid and at least one AAV terminal repeat sequence. In some embodiments, the heterologous nucleic acid encodes a therapeutic polypeptide or therapeutic nucleic acid. In some embodiments, the therapeutic polypeptide is a huntingtin polypeptide or fragment thereof. In some embodiments, the huntingtin polypeptide or fragment thereof is a functional huntingtin polypeptide or functional fragment thereof. In some embodiments, the therapeutic nucleic acid comprises an RNAi against huntingtin. In some embodiments, the RNAi is a miRNA.

[0050] In some embodiments, the present invention provides a method of treating Parkinson's disease in an individual, comprising administering to the striatum of the individual an effective amount of a composition comprising recombinant adeno-associated virus (rAAV) particles, the rAAV particles comprising: a) an rAAV capsid comprising an rAAV capsid protein containing one or more amino acid substitutions at one or more positions that interact with heparan sulfate proteoglycans or at one or more positions corresponding to amino acids 484, 487, 527, 532, 585, or 588, numbered according to the VP1 numbering of AAV2; and b) an rAAV vector comprising a heterologous nucleic acid and at least one AAV terminal repeat sequence. In some embodiments, the heterologous nucleic acid encodes a therapeutic polypeptide or a therapeutic nucleic acid. In some embodiments, the therapeutic polypeptide is TH, GTPCII, GDNF, BDNF, and / or AADC; or a fragment thereof. In some embodiments, the therapeutic polypeptide is AADC or a fragment thereof.

[0051] In some embodiments of the above aspects and embodiments, the heterologous nucleic acid is expressed at an increased expression level compared to the expression level of the heterologous nucleic acid of an rAAV particle comprising a reference rAAV capsid. In some embodiments, the rAAV particle results in reduced neuroinflammation compared to an rAAV particle comprising a reference rAAV capsid. In some embodiments, the rAAV particle comprises an AAV serotype 2 (AAV2) capsid. In some embodiments, the one or more amino acid substitutions reduce binding of the rAAV particle to heparan sulfate proteoglycans. In some embodiments, the one or more amino acid substitutions reduce binding of the rAAV particle to heparan sulfate proteoglycans compared to binding of an rAAV particle comprising a reference rAAV capsid to heparan sulfate proteoglycans. In some embodiments, the one or more amino acid substitutions reduce binding of the rAAV particle to heparan sulfate proteoglycans by at least about 10%, at least about 25%, at least about 50%, at least about 75%, or at least about 100%. In some embodiments, the one or more amino acid substitutions reduce binding of the rAAV particle to heparan sulfate proteoglycans by at least about 10%, at least about 25%, at least about 50%, at least about 75%, or at least about 100% compared to binding of an rAAV particle comprising a reference capsid to heparan sulfate proteoglycans. In some embodiments, the reference rAAV capsid comprises a wild-type rAAV capsid or capsid protein. In some embodiments, the reference rAAV capsid comprises a rAAV capsid or capsid protein that lacks one or more amino acid substitutions at one or more positions that interact with heparan sulfate proteoglycans.

[0052] In some embodiments of the above aspects and embodiments, the rAAV particles are delivered by stereotactic delivery. In some embodiments, the rAAV particles are delivered by convection-enhanced delivery. In some embodiments, the rAAV particles are administered using a CED delivery system. In some embodiments, the cannula is an anti-reflux cannula or a stepped cannula. In some embodiments, the CED delivery system includes a cannula and / or a pump. In some embodiments, the rAAV particles are administered using a CED delivery system. In some embodiments, the pump is a manual pump. In some embodiments, the pump is an osmotic pump. In some embodiments, the pump is an infusion pump.

[0053] In some of the above aspects and embodiments, the heparan sulfate proteoglycan is expressed on one or more cells of the CNS. In some embodiments, the one or more cells of the CNS are oligodendrocytes, astrocytes, neurons, brain parenchymal cells, microglial cells, ependymal cells, and / or Purkinje cells. In some embodiments, the heparan sulfate proteoglycan is expressed on neurons.

[0054] In some embodiments of the above aspects and embodiments, the heterologous nucleic acid is expressed in one or more cells of the CNS. In some embodiments, the one or more cells of the CNS are oligodendrocytes, astrocytes, neurons, brain parenchymal cells, microglial cells, ependymal cells, and / or Purkinje cells. In some embodiments, the heterologous nucleic acid is expressed in neurons. In some embodiments, the heterologous nucleic acid is exclusively expressed in neurons.

[0055] One or more amino acid substitutions are numbered based on the VP1 numbering of AAV2. In some embodiments, the numbering is based on AAV2 VP1, which comprises the amino acid sequence of SEQ ID NO: 1. In some embodiments, the one or more amino acid substitutions comprise a substitution of a positively charged amino acid residue with an amino acid residue that does not bear a positive charge. In some embodiments, the positively charged amino acid residue is substituted with a hydrophobic amino acid residue. In some embodiments, the one or more amino acid substitutions comprise a substitution of an arginine or lysine residue with an alanine residue. In some embodiments, the one or more amino acid substitutions comprise a substitution at positions R347, R350, K390, K395, R448, R451, R484, R487, K527, K532, R585, and / or R588, numbered based on AAV2 VP1. In some embodiments, the one or more amino acid substitutions comprise substitutions at positions R484, R487, K527, K532, R585, and / or R588, numbered based on VP1 of AAV2. In some embodiments, the rAAV particles comprise one or more rAAV capsid proteins having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity to SEQ ID NOs: 2, 4, and / or 6. In some embodiments, the one or more amino acid substitutions comprise R347A, R350A, K390A, K395A, R448A, R451A, R484A, R487A, K527A, K532A, R585A, and / or R588A substitutions, numbered based on VP1 of AAV2. In some embodiments, the one or more amino acid substitutions comprise substitutions at positions R484 and R487 or at positions R585 and R588, numbered based on VP1 of AAV2.In some embodiments, the one or more amino acid substitutions comprise R484A and R487A substitutions or R585A and R588A substitutions, numbered based on VP1 of AAV2. In some embodiments, the AAV capsid comprises amino acid substitutions R585A and R588A, numbered based on VP1 of AAV2. In some embodiments, the AAV capsid comprises amino acid substitution K532A, numbered based on VP1 of AAV2.

[0056] In some embodiments of the above aspects and embodiments, the one or more amino acid substitutions are at positions 485, 488, 528, 533, 586, or 589, as numbered based on the VP1 numbering of AAVrh8R. In some embodiments, the AAV particles of the present invention comprise a capsid having one or more amino acid substitutions at positions 485, 488, 528, or 533, as numbered based on the VP1 numbering of AAVrh8R. In some embodiments, the numbering is based on the VP1 of AAVrh8R comprising the amino acid sequence of SEQ ID NO: 9. In some embodiments, the one or more amino acid substitutions comprise substitution of a positively charged amino acid residue with an amino acid residue that does not bear a positive charge. In some embodiments, the positively charged amino acid residue is substituted with a hydrophobic amino acid residue. In further embodiments, the one or more amino acid substitutions comprise substitution of an arginine or lysine residue. In yet further embodiments, the one or more amino acid substitutions comprise substitution of an arginine or lysine residue with an alanine residue. In other embodiments, the one or more amino acid substitutions comprise a substitution of an amino acid residue that does not bear a positive charge with an amino acid residue that does bear a positive charge. In some embodiments, a hydrophobic amino acid residue is substituted with an amino acid residue that bears a positive charge. In further embodiments, the one or more amino acid substitutions comprise a substitution of an alanine residue. In yet further embodiments, the one or more amino acid substitutions comprise a substitution of an arginine or lysine residue with an alanine residue. In some embodiments, the amino acid substitutions comprise a substitution of an arginine or lysine residue with an alanine residue. In some embodiments, the numbering is based on the VP1 of AAVrh8R, which comprises the amino acid sequence of SEQ ID NO:9. In some embodiments, the amino acid substitution comprises a substitution at position R485, R488, R533, or T589, numbered based on the VP1 numbering of AAVrh8R. In some embodiments, the numbering is based on the VP1 of AAVrh8R, which comprises the amino acid sequence of SEQ ID NO:9. In some embodiments, the amino acid substitution comprises a substitution at position R485, R488, R533, or T589, numbered based on the VP1 numbering of AAVrh8R. In some embodiments, the rAAV particles comprise an rAAV capsid protein of SEQ ID NO:11. In some embodiments, the rAAV particles comprise one or more rAAV capsid proteins having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity to SEQ ID NO:11. In some embodiments, the single amino acid substitution comprises an R533A substitution, numbered based on VP1 of AAVrh8R. In some embodiments, the rAAV particle comprises an AAV1 capsid, an AAV2 capsid, an AAV3 capsid, an AAV6 capsid, an AAV8 capsid, an AAVrh8R capsid, an AAV9 capsid, or an AAVrh10 capsid.

[0057] In some of the above aspects and embodiments, the heterologous nucleic acid is under the control of a promoter sequence that is expressed in one or more cells of the CNS. In some embodiments, the heterologous nucleic acid is under the control of a promoter sequence selected from the group consisting of a cytomegalovirus (CMV) immediate early promoter, a RSV LTR, a MoMLV LTR, a phosphoglycerate kinase-1 (PGK) promoter, a simian virus 40 (SV40) promoter, a CK6 promoter, a transthyretin promoter (TTR), a TK promoter, a tetracycline-responsive promoter (TRE), an HBV promoter, a hAAT promoter, an LSP promoter, a chimeric liver-specific promoter (LSP), an E2F promoter, a telomerase (hTERT) promoter; a cytomegalovirus enhancer / chicken β-actin / rabbit β-globin promoter (CAG) promoter, an elongation factor 1-α promoter (EF1-α) promoter, a human β-glucuronidase promoter, a chicken β-actin (CBA) promoter, a retroviral Rous sarcoma virus (RSV) LTR promoter, a dihydrofolate reductase promoter, and a 13-actin promoter. In some embodiments, the heterologous nucleic acid is operably linked to a promoter suitable for expressing a therapeutic polypeptide or therapeutic nucleic acid in one or more cells of the CNS. In some embodiments, the one or more cells of the CNS include one or more cells of the brain. In some embodiments, the one or more cells of the CNS are oligodendrocytes, astrocytes, neurons, brain parenchymal cells, microglial cells, ependymal cells, and / or Purkinje cells. In some embodiments, the brain cells are neurons.

[0058] In some embodiments of the above aspects and embodiments, the rAAV vector is a self-complementary rAAV vector. In some embodiments, the vector comprises a first nucleic acid sequence encoding a heterologous nucleic acid and a second nucleic acid sequence encoding a complementary strand of the nucleic acid, and the first nucleic acid sequence can form interstrand base pairs with the second nucleic acid sequence over most or all of its length. In some embodiments, the first nucleic acid sequence and the second nucleic acid sequence are linked by a mutant AAV ITR, and the mutant AAV ITR comprises a deletion of the D region and a mutation of the truncation sequence.

[0059] In some of the above aspects and embodiments, the individual is a human.

[0060] In some embodiments, the invention provides a kit for use in any of the above embodiments, comprising recombinant adeno-associated virus (rAAV) particles, the rAAV particles comprising: and b) an rAAV vector comprising a heterologous nucleic acid and at least one AAV inverted terminal repeat sequence. In some embodiments, the present invention provides kits for delivering heterologous nucleic acid to the central nervous system (CNS) of an individual, the kit comprising a) an rAAV capsid comprising an rAAV capsid protein comprising one or more amino acid substitutions at one or more positions that interact with heparan sulfate proteoglycans, or at one or more positions corresponding to amino acids 484, 487, 527, 532, 585, or 588, numbered based on the VP1 numbering of AAV2, and b) an rAAV vector comprising a heterologous nucleic acid and at least one AAV inverted terminal repeat sequence. In some embodiments, the present invention provides kits for delivering heterologous nucleic acid to the central nervous system (CNS) of an individual, the kit comprising a composition comprising recombinant adeno-associated virus (rAAV) particles, the rAAV particles comprising: a) an rAAV capsid comprising an rAAV capsid protein comprising one or more amino acid substitutions at one or more positions that interact with heparan sulfate proteoglycans, and b) an rAAV vector comprising a heterologous nucleic acid and at least one AAV inverted terminal repeat sequence. In some embodiments, the invention provides kits for treating a central nervous system (CNS) disorder in an individual, the kit comprising a composition comprising recombinant adeno-associated virus (rAAV) particles, the rAAV particles comprising: a) an rAAV capsid comprising an rAAV capsid protein comprising one or more amino acid substitutions at one or more positions that interact with heparan sulfate proteoglycan; and b) an rAAV vector comprising a heterologous nucleic acid for treating a CNS disorder and at least one AAV inverted terminal repeat.

[0061] In some embodiments of the above aspects and embodiments, the CNS disorder is Huntington's disease. In some embodiments, the heterologous nucleic acid encodes a therapeutic polypeptide or therapeutic nucleic acid. In some embodiments, the therapeutic polypeptide is a huntingtin polypeptide or a fragment thereof. In some embodiments, the huntingtin polypeptide or a fragment thereof is a functional huntingtin polypeptide or a functional fragment thereof. In some embodiments, the therapeutic nucleic acid comprises an RNAi against huntingtin. In some embodiments, the RNAi is an miRNA. In some embodiments, the CNS disorder is Parkinson's disease. In some embodiments, the heterologous nucleic acid encodes a therapeutic polypeptide or therapeutic nucleic acid. In some embodiments, the therapeutic polypeptide is TH, GTPCII, GDNF, BDNF and / or AADC; or a fragment thereof. In some embodiments, the therapeutic polypeptide is AADC or a fragment thereof.

[0062] In some aspects, the present invention provides recombinant adeno-associated virus (rAAV) particles for use in any of the above embodiments. In some aspects, the present invention provides recombinant adeno-associated virus (rAAV) particles for delivering a heterologous nucleic acid to the central nervous system (CNS) of an individual, the rAAV particles comprising: a) an rAAV capsid comprising an rAAV capsid protein containing one or more amino acid substitutions at one or more positions that interact with heparan sulfate proteoglycan, or at one or more positions corresponding to amino acids 484, 487, 527, 532, 585, or 588, numbered based on the VP1 numbering of AAV2; and b) an rAAV vector comprising a heterologous nucleic acid and at least one AAV inverted terminal repeat. In some embodiments, the present invention provides recombinant adeno-associated virus (rAAV) particles for treating a central nervous system (CNS) disorder in an individual, the rAAV particle comprising: a) an rAAV capsid comprising an rAAV capsid protein containing one or more amino acid substitutions at one or more positions that interact with heparan sulfate proteoglycans, or at one or more positions corresponding to amino acids 484, 487, 527, 532, 585, or 588, numbered based on the VP1 numbering of AAV2; and b) an rAAV vector comprising a heterologous nucleic acid and at least one AAV terminal repeat sequence. In some embodiments, the present invention provides recombinant adeno-associated virus (rAAV) particles for treating Huntington's disease in an individual, the rAAV particle comprising: a) an rAAV capsid protein containing one or more amino acid substitutions at one or more positions that interact with heparan sulfate proteoglycans, or at one or more positions corresponding to amino acids 484, 487, 527, 532, 585, or 588, numbered based on the VP1 numbering of AAV2. The present invention provides an rAAV particle comprising: a) an rAAV capsid comprising an rAAV capsid protein with one or more amino acid substitutions at one or more positions that interact with teoglycan, or at one or more positions corresponding to amino acids 484, 487, 527, 532, 585, or 588, numbered based on the VP1 numbering of AAV2; and b) an rAAV vector comprising a heterologous nucleic acid and at least one AAV terminal repeat sequence, wherein the rAAV particle is formulated for delivery to the striatum. In some embodiments, the present invention provides recombinant adeno-associated virus (rAAV) particles for treating Parkinson's disease in an individual, the rAAV particles comprising: a) an rAAV capsid comprising an rAAV capsid protein comprising one or more amino acid substitutions at one or more positions that interact with heparan sulfate proteoglycan, or at one or more positions corresponding to amino acids 484, 487, 527, 532, 585, or 588, numbered based on the VP1 numbering of AAV2; and b) an rAAV vector comprising a heterologous nucleic acid and at least one AAV terminal repeat, wherein the rAAV particles are formulated for delivery to the striatum. In some embodiments, the present invention provides recombinant adeno-associated virus (rAAV) particles for treating Huntington's disease in an individual, the rAAV particles comprising: a) an rAAV capsid comprising an rAAV capsid protein comprising one or more amino acid substitutions at one or more positions that interact with heparan sulfate proteoglycan, or at one or more positions corresponding to amino acids 484, 487, 527, 532, 585, or 588, numbered based on the VP1 numbering of AAV2; and b) an rAAV vector comprising a heterologous nucleic acid and at least one AAV terminal repeat sequence, wherein the rAAV particles are formulated for single-site delivery (e.g., to the CNS of an individual).In some embodiments, the present invention provides recombinant adeno-associated virus (rAAV) particles for treating Parkinson's disease in an individual, the rAAV particles comprising: a) an rAAV capsid comprising an rAAV capsid protein comprising one or more amino acid substitutions at one or more positions that interact with heparan sulfate proteoglycan, or at one or more positions corresponding to amino acids 484, 487, 527, 532, 585, or 588, numbered based on the VP1 numbering of AAV2; and b) an rAAV vector comprising a heterologous nucleic acid and at least one AAV terminal repeat sequence; and the rAAV particles are formulated for single-site delivery (e.g., to the CNS of an individual).

[0063] In some embodiments of the above aspects and embodiments, the heterologous nucleic acid is expressed at an increased expression level compared to the expression level of the heterologous nucleic acid of an rAAV particle comprising a reference rAAV capsid. In some embodiments, the rAAV particle results in reduced neuroinflammation compared to an rAAV particle comprising an rAAV capsid comprising the reference capsid. In some embodiments, the rAAV particle comprises an AAV serotype 2 (AAV2) capsid. In some embodiments, the one or more amino acid substitutions reduce binding of the rAAV particle to heparan sulfate proteoglycans. In some embodiments, the one or more amino acid substitutions reduce binding of the rAAV particle to heparan sulfate proteoglycans compared to binding of an rAAV particle comprising a reference rAAV capsid to heparan sulfate proteoglycans. In some embodiments, the one or more amino acid substitutions reduce binding of the rAAV particle to heparan sulfate proteoglycans by at least about 10%, at least about 25%, at least about 50%, at least about 75%, or at least about 100%. In some embodiments, the one or more amino acid substitutions reduce binding of the rAAV particle to heparan sulfate proteoglycans by at least about 10%, at least about 25%, at least about 50%, at least about 75%, or at least about 100% compared to binding of an rAAV particle comprising a reference capsid to heparan sulfate proteoglycans. In some embodiments, the reference rAAV capsid comprises a wild-type rAAV capsid or capsid protein. In some embodiments, the reference rAAV capsid comprises a rAAV capsid or capsid protein that lacks one or more amino acid substitutions at one or more positions that interact with heparan sulfate proteoglycans. .

[0064] In some of the above aspects and embodiments, the heparan sulfate proteoglycan is expressed on one or more cells of the CNS. In some embodiments, the one or more cells of the CNS are oligodendrocytes, astrocytes, neurons, brain parenchymal cells, microglial cells, ependymal cells, and / or Purkinje cells. In some embodiments, the heparan sulfate proteoglycan is expressed on neurons.

[0065] In some embodiments of the above aspects and embodiments, the heterologous nucleic acid is expressed in one or more cells of the CNS. In some embodiments, the one or more cells of the CNS are oligodendrocytes, astrocytes, neurons, brain parenchymal cells, microglial cells, ependymal cells, and / or Purkinje cells. In some embodiments, the heterologous nucleic acid is expressed in neurons. In some embodiments, the heterologous nucleic acid is exclusively expressed in neurons.

[0066] The one or more amino acid substitutions are at positions 484, 487, 527, 532, 585, or 588, numbered based on the VP1 numbering system of AAV2. In some embodiments, the one or more amino acid substitutions comprise a substitution of a positively charged amino acid residue with an amino acid residue that does not bear a positive charge. In some embodiments, the positively charged amino acid residue is substituted with a hydrophobic amino acid residue. In some embodiments, the one or more amino acid substitutions comprise a substitution of an arginine or lysine residue with an alanine residue. In some embodiments, the one or more amino acid substitutions comprise a substitution at positions R347, R350, K390, K395, R448, R451, R484, R487, K527, K532, R585, and / or R588, numbered based on the VP1 numbering system of AAV2. In some embodiments, the one or more amino acid substitutions comprise substitutions at positions R484, R487, K527, K532, R585, and / or R588, numbered based on VP1 of AAV2. In some embodiments, the rAAV particles comprise rAAV particles comprising one or more rAAV capsid proteins having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity to SEQ ID NOs: 2, 4, and / or 6. In some embodiments, the one or more amino acid substitutions comprise R347A, R350A, K390A, K395A, R448A, R451A, R484A, R487A, K527A, K532A, R585A, and / or R588A substitutions, numbered based on VP1 of AAV2. In some embodiments, the one or more amino acid substitutions comprise substitutions at positions R484 and R487 or at positions R585 and R588, numbered based on VP1 of AAV2.In some embodiments, the one or more amino acid substitutions comprise R484A and R487A substitutions or R585A and R588A substitutions, numbered based on VP1 of AAV2. In some embodiments, the AAV capsid comprises amino acid substitutions R585A and R588A, numbered based on VP1 of AAV2. In some embodiments, the AAV capsid comprises amino acid substitution K532A, numbered based on VP1 of AAV2. In some embodiments, the rAAV particle comprises an AAV1 capsid, an AAV2 capsid, an AAV3 capsid, an AAV6 capsid, an AAV8 capsid, an AAVrh8R capsid, an AAV9 capsid, or an AAVrh10 capsid.

[0067] In some of the above aspects and embodiments, the one or more amino acid substitutions are at positions 485, 488, 528, 533, 586, or 589, numbered according to the VP1 numbering system of AAVrh8R. In some embodiments, the numbering is based on VP1 of AAVrh8R comprising the amino acid sequence of SEQ ID NO:9. In some embodiments, the one or more amino acid substitutions comprise substitution of a positively charged amino acid residue with an amino acid residue that does not bear a positive charge. In some embodiments, the positively charged amino acid residue is substituted with a hydrophobic amino acid residue. In further embodiments, the one or more amino acid substitutions comprise substitution of an arginine or lysine residue with an alanine residue. In other embodiments, the one or more amino acid substitutions comprise substitution of a non-positively charged amino acid residue with a positively charged amino acid residue. In some embodiments, the hydrophobic amino acid residue is substituted with a positively charged amino acid residue. In further embodiments, the one or more amino acid substitutions comprise substitutions of alanine residues. In even further embodiments, the one or more amino acid substitutions comprise substitutions of arginine or lysine residues with alanine residues. In some embodiments, the amino acid substitutions are at positions 485, 488, 528, 533, or 589, numbered based on the VP1 numbering of AAVrh8R. In some embodiments, the numbering is based on the VP1 of AAVrh8R comprising the amino acid sequence of SEQ ID NO:9. In some embodiments, the amino acid substitutions comprise substitutions at positions R485, R488, R533, or T589, numbered based on the VP1 numbering of AAVrh8R. In some embodiments, the rAAV particles comprise the rAAV capsid protein of SEQ ID NO:11.In some embodiments, the rAAV particles comprise one or more rAAV capsid proteins having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity to SEQ ID NO: 11. In some embodiments, the single amino acid substitution comprises an R533A substitution, numbered based on VP1 of AAVrh8R.

[0068] In some of the above aspects and embodiments, the heterologous nucleic acid is under the control of a promoter sequence that is expressed in one or more cells of the CNS. In some embodiments, the heterologous nucleic acid is under the control of a promoter sequence selected from the group consisting of a cytomegalovirus (CMV) immediate early promoter, a RSV LTR, a MoMLV LTR, a phosphoglycerate kinase-1 (PGK) promoter, a simian virus 40 (SV40) promoter, a CK6 promoter, a transthyretin promoter (TTR), a TK promoter, a tetracycline-responsive promoter (TRE), an HBV promoter, a hAAT promoter, an LSP promoter, a chimeric liver-specific promoter (LSP), an E2F promoter, a telomerase (hTERT) promoter; a cytomegalovirus enhancer / chicken β-actin / rabbit β-globin promoter (CAG) promoter, an elongation factor 1-α promoter (EF1-α) promoter, a human β-glucuronidase promoter, a chicken β-actin (CBA) promoter, a retroviral Rous sarcoma virus (RSV) LTR promoter, a dihydrofolate reductase promoter, and a 13-actin promoter. In some embodiments, the heterologous nucleic acid is operably linked to a promoter suitable for expressing a therapeutic polypeptide or therapeutic nucleic acid in one or more cells of the CNS. In some embodiments, the one or more cells of the CNS include one or more cells of the brain. In some embodiments, the one or more cells of the CNS are oligodendrocytes, astrocytes, neurons, brain parenchymal cells, microglial cells, ependymal cells, and / or Purkinje cells.

[0069] In some embodiments of the above aspects and embodiments, the rAAV vector is a self-complementary rAAV vector. In some embodiments, the vector comprises a first nucleic acid sequence encoding a heterologous nucleic acid and a second nucleic acid sequence encoding a complementary strand of the nucleic acid, and the first nucleic acid sequence can form interstrand base pairs with the second nucleic acid sequence over most or all of its length. In some embodiments, the first nucleic acid sequence and the second nucleic acid sequence are linked by a mutant AAV ITR, and the mutant AAV ITR comprises a deletion of the D region and a mutation of the truncation sequence.

[0070] In some of the above aspects and embodiments, the individual is a human.

[0071] In some of the above aspects and embodiments, the rAAV particles are in a composition. In some embodiments, the composition includes a buffer and / or a pharmaceutically acceptable excipient. In some embodiments, the kit or rAAV particles further include instructions for delivery of the composition of rAAV particles to the CNS. In some embodiments, the kit or rAAV particles further include instructions for delivery of the composition of rAAV particles to the striatum.

[0072] In some aspects, the present invention provides rAAV particles comprising an AAVrh8R capsid protein, wherein the AAVrh8R capsid protein comprises one or more amino acid substitutions that increase binding of the rAAV particle to heparan sulfate proteoglycans compared to AAV particles comprising a wild-type AAVrh8R capsid protein, or the one or more amino acid substitutions are at one or more positions corresponding to amino acids 484, 487, 527, 532, 585, or 588, numbered according to the VP1 numbering of AAV2. In some embodiments, the one or more amino acid substitutions increase binding of the rAAV particle to heparan sulfate proteoglycans by at least about 10%, at least about 25%, at least about 50%, at least about 75%, or at least about 100%. In some embodiments, the amino acid substitution is at position 586, numbered according to the VP1 numbering of AAVrh8R. In some embodiments, the numbering is based on VP1 of AAVrh8R, which comprises the amino acid sequence of SEQ ID NO: 9. In some embodiments, the amino acid substitution comprises a substitution at position A586, numbered based on the VP1 numbering of AAVrh8R. In some embodiments, the amino acid substitution comprises an A586R or A586K substitution, numbered based on the VP1 numbering of AAVrh8R. In some embodiments, the rAAV particles comprise the rAAV capsid protein of SEQ ID NO: 10.

[0073] In some embodiments, the present invention provides methods for increasing heparan sulfate proteoglycan binding of rAAV particles comprising an AAVrh8R capsid protein, comprising introducing one or more amino acid substitutions into the capsid protein, wherein the one or more amino acid substitutions increase heparan sulfate proteoglycan binding of the rAAV particles compared to AAV particles comprising a wild-type AAVrh8R capsid protein. In some embodiments, the one or more amino acid substitutions increase heparan sulfate proteoglycan binding of the rAAV particles by at least about 10%, at least about 25%, at least about 50%, at least about 75%, or at least about 100%. In some embodiments, the amino acid substitution is at position 586 based on the VP1 numbering of AAVrh8R. In some embodiments, the numbering is based on the VP1 of AAVrh8R comprising the amino acid sequence of SEQ ID NO:9. In some embodiments, the amino acid substitution comprises a substitution at position A586, numbered based on VP1 of AAVrh8R. In some embodiments, the rAAV particles comprise the rAAV capsid protein of SEQ ID NO: 10.

[0074] In some embodiments, the invention provides methods for delivering a heterologous nucleic acid to the retina of an individual, the method comprising intravitreally administering to the individual recombinant adeno-associated virus (rAAV) particles, the rAAV particles comprising: a) an rAAV capsid comprising an rAAV capsid protein comprising one or more amino acid substitutions at one or more positions that interact with heparan sulfate proteoglycans or at one or more positions corresponding to amino acids 484, 487, 527, 532, 585, or 588, numbered based on the VP1 numbering of AAV2; and b) an rAAV vector comprising the heterologous nucleic acid and at least one AAV inverted terminal repeat. In some embodiments, the invention provides a method for improving rAAV transduction of cells following intravitreal delivery of rAAV particles to the eye of an individual compared to transduction of cells with an rAAV comprising a wild-type capsid, comprising incorporating one or more amino acid substitutions into an AAV capsid protein at one or more positions that interact with heparan sulfate proteoglycans or at one or more positions corresponding to amino acids 484, 487, 527, 532, 585, or 588, numbered based on the VP1 numbering of AAV2; the rAAV particle comprises the rAAV capsid protein and an rAAV vector comprising a heterologous nucleic acid and at least one AAV terminal repeat sequence. In some embodiments, the invention provides a method for improving heterologous nucleic acid expression following intravitreal delivery of rAAV particles to the eye of an individual, comprising incorporating one or more amino acid substitutions into an AAV capsid protein at one or more positions that interact with heparan sulfate proteoglycans or at one or more positions corresponding to amino acids 484, 487, 527, 532, 585, or 588, numbered based on the VP1 numbering of AAV2; the rAAV particle comprises the rAAV capsid protein and an rAAV vector comprising a heterologous nucleic acid and at least one AAV terminal repeat sequence.In some embodiments, the invention provides methods for treating an ocular disorder in an individual, the method comprising intravitreally delivering a composition comprising rAAV particles to the retina of the individual, the rAAV particles comprising: a) an rAAV capsid comprising an rAAV capsid protein comprising one or more amino acid substitutions at one or more positions that interact with heparan sulfate proteoglycans or at one or more positions corresponding to amino acids 484, 487, 527, 532, 585, or 588, numbered based on the VP1 numbering of AAV2; and b) an rAAV vector comprising a heterologous nucleic acid and at least one AAV long terminal repeat sequence. In some embodiments, the present invention provides a system for intravitreal delivery of a vector to the eye of an individual, the system comprising: (a) a composition comprising an effective amount of rAAV particles, wherein i) the capsid protein of the rAAV particles comprises one or more amino acid substitutions at one or more positions that interact with heparan sulfate proteoglycans or at one or more positions corresponding to amino acids 484, 487, 527, 532, 585, or 588, and ii) the vector comprises a heterologous nucleic acid encoding a therapeutic polypeptide or therapeutic RNA and at least one AAV terminal repeat sequence; and b) a device for intravitreal delivery of the rAAV. In some embodiments, the rAAV particles comprise an AAVrh8R, AAV1, AAV6, AAV8, AAV9, or AAVrh10 serotype capsid. In some embodiments, the invention provides a kit for treating an ocular disorder, comprising: a) a composition comprising rAAV particles, wherein the rAAV particles include: i) an rAAV capsid comprising an rAAV capsid protein comprising one or more amino acid substitutions at one or more positions that interact with heparan sulfate proteoglycans or at one or more positions corresponding to amino acids 484, 487, 527, 532, 585, or 588, numbered based on the VP1 numbering of AAV2; and and ii) an rAAV vector comprising a heterologous nucleic acid for treating an ocular disorder and at least one AAV inverted terminal repeat; and b) a pharmaceutical excipient suitable for intravitreal administration. In some embodiments, the rAAV particles comprise an AAVrh8R, AAV1, AAV6, AAV8, AAV9, or AAVrh10 capsid. In some aspects, the invention provides rAAV particles comprising an AAV1 capsid protein, wherein the AAV1 capsid protein comprises one or more amino acid substitutions that increase the transduction efficiency of the rAAV particles into ocular cells compared to AAV particles comprising a wild-type AAV1 capsid protein, or the one or more amino acid substitutions are at one or more positions corresponding to amino acids 484, 487, 527, 532, 585, or 588, as numbered based on the VP1 numbering of AAV2. In some embodiments, the present invention provides rAAV particles comprising an AAV6 capsid protein, wherein the AAV6 capsid protein comprises one or more amino acid substitutions that increase the transduction efficiency of the rAAV particle into ocular cells compared to AAV particles comprising wild-type AAV6 capsid protein, or wherein the one or more amino acid substitutions are at one or more positions corresponding to amino acids 484, 487, 527, 532, 585, or 588, as numbered based on the VP1 numbering of AAV2. In some embodiments, the present invention provides rAAV particles comprising an AAV8 capsid protein, wherein the AAV8 capsid protein comprises one or more amino acid substitutions that increase the transduction efficiency of the rAAV particle into ocular cells compared to AAV particles comprising wild-type AAV8 capsid protein, or wherein the one or more amino acid substitutions are at one or more positions corresponding to amino acids 484, 487, 527, 532, 585, or 588, numbered based on the VP1 numbering of AAV2.In some embodiments, the present invention provides rAAV particles comprising an AAV9 capsid protein, wherein the AAV9 capsid protein comprises one or more amino acid substitutions that increase the transduction efficiency of the rAAV particle into ocular cells compared to AAV particles comprising a wild-type AAV9 capsid protein, or wherein the one or more amino acid substitutions are at one or more positions corresponding to amino acids 484, 487, 527, 532, 585, or 588, as numbered based on the VP1 numbering of AAV2. In some embodiments, the invention provides rAAV particles comprising an AAVrhlO capsid protein, wherein the AAVrhlO capsid protein comprises one or more amino acid substitutions that increase the transduction efficiency of the rAAV particles into ocular cells compared to AAV particles comprising a wild-type AAVrhlO capsid protein, or wherein the one or more amino acid substitutions are at one or more positions corresponding to amino acids 484, 487, 527, 532, 585, or 588, numbered according to the AAV2 VP1 numbering. In some embodiments, the invention provides rAAV particles comprising an AAV3 capsid protein, wherein the AAV3 capsid comprises one or more amino acid substitutions at one or more positions corresponding to amino acids 484, 487, 527, 532, 585, or 588, numbered according to the AAV2 VP1 numbering. In some embodiments, the rAAV particles comprise an AAV1 capsid, an AAV2 capsid, an AAV3 capsid, an AAV6 capsid, an AAV8 capsid, an AAVrh8R capsid, an AAV9 capsid, or an AAVrh10 capsid. In some embodiments, the transduction efficiency is increased by at least about 10%, at least about 25%, at least about 50%, at least about 75%, or at least about 100%.

[0075] In some embodiments, one or more amino acid substitutions are made in the rAAV particle.Increased binding to heparan sulfate proteoglycans. In some embodiments, the one or more amino acid substitutions increase the binding of the rAAV particle to heparan sulfate proteoglycans by at least about 10%, at least about 25%, at least about 50%, at least about 75%, or at least about 100%. In some embodiments, the one or more amino acid substitutions increase the transduction efficiency of the rAAV particle into cells of the eye or central nervous system by at least about 10%, at least about 25%, at least about 50%, at least about 75%, or at least about 100% compared to AAV particles comprising a wild-type AAVrh8R capsid protein. In some embodiments, the ocular cells are retinal cells, photoreceptor cells, retinal pigmented epithelial cells, bipolar cells, horizontal cells, amacrine cells, Müller cells, and / or ganglion cells. In some embodiments, the one or more amino acid substitutions comprise the substitution of a non-positively charged amino acid residue with a positively charged amino acid residue. In some embodiments, a positively charged amino acid residue replaces a hydrophobic amino acid residue. In some embodiments, the one or more amino acid substitutions comprise substitutions with arginine or lysine residues. In some embodiments, the one or more amino acid substitutions comprise substitutions of alanine, serine, glutamine, or threonine residues with arginine or lysine residues. In some embodiments, the rAAV particles comprise an AAV serotype rh8R (AAVrh8R) capsid. In some embodiments, the one or more amino acid substitutions are at positions 586 and / or 589, numbered according to the VP1 numbering of AAVrh8R. In some embodiments, the numbering is based on the VP1 of AAVrh8R comprising the amino acid sequence of SEQ ID NO:9. In some embodiments, the one or more amino acid substitutions comprise substitutions at positions A586 and / or T589, numbered according to the VP1 numbering of AAVrh8R. In some embodiments, the one or more amino acid substitutions comprise an A586R or A586K substitution, numbered based on VP1 of AAVrh8R.In some embodiments, the one or more amino acid substitutions comprise a T589R or T589K substitution, numbered based on VP1 of AAVrh8R. In some embodiments, the rAAV particles comprise an AAV serotype 1 (AAV1) capsid. In some embodiments, the one or more amino acid substitutions are at positions 586 and / or 589, numbered based on VP1 numbering of AAV1. In some embodiments, VP1 of AAV1 comprises the amino acid sequence of SEQ ID NO: 12. In some embodiments, the one or more amino acid substitutions comprise a substitution at positions S586 and / or T589, numbered based on VP1 numbering of AAV1. In some embodiments, the one or more amino acid substitutions comprise a S586R or S586K substitution, numbered based on VP1 of AAV1. In some embodiments, the one or more amino acid substitutions comprise a T589R or T589K substitution, numbered based on VP1 of AAV1. In some embodiments, the rAAV particles comprise an AAV serotype 6 (AAV6) capsid. In some embodiments, the one or more amino acid substitutions are at positions 586 and / or 589, numbered based on the VP1 numbering of AAV6. In some embodiments, the numbering is based on an AAV6 VP1 comprising the amino acid sequence of SEQ ID NO: 13. In some embodiments, the one or more amino acid substitutions comprise substitutions at positions S586 and / or T589, numbered based on the VP1 numbering of AAV6. In some embodiments, the one or more amino acid substitutions comprise an S586R substitution, numbered based on the VP1 numbering of AAV6. In some embodiments, the one or more amino acid substitutions comprise a T589R or T589K substitution, numbered based on the VP1 numbering of AAV6. In some embodiments, the rAAV particles comprise an AAV serotype 8 (AAV8) capsid. In some embodiments, the one or more amino acid substitutions are at positions 588 and / or 591, numbered based on the VP1 numbering of AAV8.In some embodiments, VP1 of AAV8. The rAAV particles comprise an AAV serotype 9 (AAV9) capsid. In some embodiments, the one or more amino acid substitutions are at positions 586 and / or 589, numbered based on the VP1 numbering of AAV9. In some embodiments, the rAAV particles comprise an AAV serotype 9 (AAV9) capsid. In some embodiments, the one or more amino acid substitutions are at positions 586 and / or 589, numbered based on the VP1 numbering of AAV9. In some embodiments, the VP1 of AAV9 comprises the amino acid sequence of SEQ ID NO: 15. In some embodiments, the one or more amino acid substitutions are at positions S586 and / or S589, numbered based on the VP1 numbering of AAV9. In some embodiments, the one or more amino acid substitutions comprise a S586R or S586K substitution, numbered based on VP1 of AAV9. In some embodiments, the one or more amino acid substitutions comprise a A589R or A589K substitution, numbered based on VP1 of AAV9. In some embodiments, the rAAV particles comprise an AAV serotype rh10 (AAVrhlO) capsid. In some embodiments, the one or more amino acid substitutions are at positions 588 and / or 591, numbered based on VP1 numbering of AAVrhlO. In some embodiments, VP1 of AAVrhlO comprises the amino acid sequence of SEQ ID NO: 16. In some embodiments, the one or more amino acid substitutions comprise a substitution at positions Q588 and / or A591, numbered based on VP1 numbering of AAVrhlO. In some embodiments, the one or more amino acid substitutions comprise a Q588R or Q588K substitution, numbered based on VP1 of AAVrhlO.In some embodiments, the one or more amino acid substitutions comprise an A591R or A591K substitution, numbered based on VP1 of AAVrhlO. In some embodiments, the invention provides rAAV particles comprising an AAV3 capsid protein, wherein the AAV3 capsid comprises one or more amino acid substitutions at one or more positions corresponding to amino acids 484, 487, 527, 532, 585, or 588, numbered based on VP1 numbering of AAV2. In some embodiments, the one or more amino acid substitutions increase the transduction efficiency of the rAAV particles into cells of the eye or central nervous system by at least about 10%, at least about 25%, at least about 50%, at least about 75%, or at least about 100%, compared to AAV particles comprising a wild-type AAVrh8R capsid protein. In some embodiments, the rAAV particle comprises an AAV1 capsid, an AAV2 capsid, an AAV3 capsid, an AAV6 capsid, an AAV8 capsid, an AAVrh8R capsid, an AAV9 capsid, or an AAVrh10 capsid. In some embodiments, the heterologous nucleic acid encodes a therapeutic polypeptide or a therapeutic nucleic acid. In some embodiments, the heterologous nucleic acid encodes a polypeptide selected from the group consisting of an antioxidant, a neurotrophic factor, an anti-apoptotic factor, an anti-angiogenic factor, and an anti-inflammatory factor. In further embodiments, the heterologous nucleic acid encodes a polypeptide selected from the group consisting of: Prph2, RPE65, AIPL1, GUCY2D, LCA5, CRX, CEP290, MYO 7a, Curarin, ABCA4, RDH12, IMPDH1, CRB1, LRAT, NMNAT1, TULP1, MERTK, RPGR, RP2, RPGRIP, CNGA3, CNGB3, GNAT2, GDNF, CNTF, FGF2, PEDF, EPO, BCL2, BCL-X, NFκB, endostatin, angiostatin, sFlt, sPDGF-R, IL10, anti-IL17, sIL17R, IL1-ra, anti-TGFβ, sTNF-RI, sTNF-RII, and IL4. In other embodiments, the heterologous nucleic acid encodes a therapeutic nucleic acid. In a further embodiment, the therapeutic nucleic acid is an siRNA, shRNA, RNAi, miRNA, antisense RNA, ribozyme or It is a DNAzyme. In some embodiments, the rAAV vector is a self-complementary rAAV vector. In some embodiments, the AAV particles of the present invention comprise a capsid containing one or more amino acid substitutions that alter (e.g., reduce or eliminate) binding to HSPGs, or one or more amino acid substitutions at one or more positions corresponding to amino acids 484, 487, 527, 532, 585, or 588, numbered according to the VP1 numbering of AAV2, and a heterologous nucleic acid encoding a therapeutic polypeptide or therapeutic nucleic acid, wherein the heterologous nucleic acid is under the control of a promoter sequence that is expressed in the retina. In some embodiments, the heterologous nucleic acid is operably linked to a promoter suitable for expression of the therapeutic polypeptide or therapeutic nucleic acid in one or more retinal cell types. In some embodiments, the retinal cells are photoreceptor cells, retinal pigmented epithelial cells, bipolar cells, horizontal cells, amacrine cells, Müller cells, and / or ganglion cells. In some embodiments, the promoter is a rhodopsin kinase (RK) promoter, an opsin promoter, a cytomegalovirus (CMV) promoter, or a chicken beta-actin (CBA) promoter. In some embodiments, the individual is human. In some embodiments, the heterologous nucleic acid is used to treat an ocular disorder selected from the group consisting of: autosomal recessive severe early-onset retinal degeneration (Leber's congenital amaurosis), congenital color blindness, Stargardt's disease, Best's disease, Doyne's disease, cone dystrophy, retinitis pigmentosa, X-linked retinoschisis, Usher syndrome, age-related macular degeneration, atrophic age-related macular degeneration, neovascular AMD, diabetic maculopathy, proliferative diabetic retinopathy (PDR), cystoid macular edema, central serous retinopathy, retinal detachment, intraocular inflammation, glaucoma, and posterior uveitis. In some embodiments, the rAAV vector is a self-complementary rAAV vector. In some embodiments, the vector comprises a first nucleic acid sequence encoding a heterologous nucleic acid and a second nucleic acid sequence encoding the complementary strand of the nucleic acid, wherein the first nucleic acid sequence is capable of interstrand base pairing with the second nucleic acid sequence over most or all of its length.In some embodiments, the first and second nucleic acid sequences are linked by a mutant AAV ITR, wherein the mutant AAV ITR comprises a deletion of the D region and a mutation in the truncation sequence. In some embodiments, the individual is human. In some embodiments, the one or more amino acid substitutions increase the transduction efficiency of the rAAV particle into cells of the eye or central nervous system by at least about 10%, at least about 25%, at least about 50%, at least about 75%, or at least about 100%, compared to AAV particles comprising a wild-type AAVrh8R capsid protein.

[0076] All references cited herein, including patent applications and publications, are incorporated by reference in their entirety. [Brief explanation of the drawings]

[0077] [Figure 1]

[0023] Figure 1 shows capsid residues involved in heparan sulfate proteoglycan binding and the mutations introduced to generate the AAV2 HBKO mutants. Numbering is based on the VP1 amino acid sequence. [Figure 2] Figure 1 shows that reduced transduction of 293 cells in culture was observed with HBKO mutant AAV2 particles (AAV2 HBKO CBA-sFLT02) compared with wild-type AAV2 particles (AAV2 CBA-sFLT02). Transduction was assayed by measuring the amount of soluble Flt (sFLT) present in the culture medium 48 hours after injection of wild-type or HBKO mutant AAV2 particles carrying a vector using the CBA promoter to drive expression of Flt. [Figure 3]Figure 1 shows reduced transduction of 293 and HeLa cells in culture observed with HBKO mutant AAV2 particles (AAV2 HBKO CBA-GFP) compared to wild-type AAV2 particles (AAV2 CBA-GFP). Transduction was assayed by fluorescent imaging of cells 48 hours after injection with wild-type or HBKO mutant AAV2 particles carrying a vector using the CBA promoter to drive expression of EGFP. [Figure 4] Figures 4A and 4B show transduction observed after intravitreal (Figure 4A) or subretinal (Figure 4B) injection of wild-type AAV2 or HBKO mutant AAV2 particles. Transduction was assayed by expression of soluble Flt (sFLT) after transduction with a Flt-encoding vector. The number of injected vector genomes is indicated for each experiment (10 or 10 vg). [Figure 5] HBKO mutant AAV2 particles fail to transduce mouse eyes after intravitreal injection. Mice were injected intravitreally with wild-type (AAV2 CBA-GFP) or HBKO mutant (AAV2 HBKO CBA-GFP) AAV2 particles carrying a vector using the CBA promoter to drive EGFP expression, and sections were imaged by fluorescence microscopy. [Figure 6] Figure 1 shows that HBKO mutant AAV2 particles (AAV2 CBA HBKO) result in significantly increased transduction compared with wild-type particles (AAV2 CBA) after subretinal injection. Transduction was assayed by the expression of soluble Flt (sFLT) after injection of AAV2 particles carrying a vector using the CBA promoter to drive Flt expression. The number of injected vector genomes is indicated (10 or 10 vg). [Figure 7]HBKO mutant AAV2 particles (AAV2 HBKO CBA-GFP) result in significantly increased transduction of photoreceptor cells (as indicated) compared to wild-type particles (AAV2 CBA-GFP) after subretinal injection. Transduction was measured by fluorescent imaging of GFP expression after transduction with AAV2 particles carrying a vector using the CBA promoter to drive expression of EGFP. [Figure 8] This figure shows that HBKO mutant AAV2 particles (AAV2 RK HBKO) result in significantly increased transduction of photoreceptors compared with wild-type particles (AAV2 RK) after subretinal injection. Transduction was assayed by the expression of soluble Flt (sFLT) after injection of AAV2 particles carrying a vector that uses the rhodopsin kinase (RK) promoter to drive Flt expression. The number of vector genomes injected is indicated (10 or 10 vg). [Figure 9] Figures 9A and 9B show EGFP expression in mouse brain 30 days after intrastriatal injection of AAV2HBKO-EGFP (Figure 9A) compared to AAV2-EGFP (Figure 9B) in wild-type mice. Expression of EGFP in each panel was driven by the CBA promoter and visualized using fluorescence microscopy. [Figure 10] Figures 10A and 10B show GFP expression in the mouse brain 30 days after intrastriatal injection of AAV2HBKO-miRNA-Htt-GFP (Figure 10A) compared with AAV1-miRNA-Htt-GFP (Figure 10B) in YAC128HD mice. The miRNA-Htt-GFP vector refers to a construct expressing an artificial miRNA targeting human Htt and a GFP reporter. The expression of GFP in each panel was driven by the CBA promoter and visualized using fluorescence microscopy at three different magnifications (4X, 10X, and 20X, as indicated). [Figure 11A] FIG. 1 shows qPCR analysis of human HTT mRNA levels in striatal mouse brain punch sections 30 days after injection of AAV1-miRNA-Htt and AAV2HBKO-miRNA-Htt compared to untreated controls. [Figure 11B] FIG. 1 shows Western blot analysis of human Htt protein levels in cortical mouse brain punch sections 30 days after injection of AAV1-miRNA-Htt and AAV2HBKO-miRNA-Htt compared to untreated controls. [Figure 12] Figures 12A-12C show the expression of Iba1 in the striatum of YAC128 mice 30 days after injection of AAV2HBKO-miRNA-Htt-GFP (Figure 12B) or AAV1-miRNA-Htt-GFP (Figure 12C) compared to untreated controls (Figure 12A). [Figure 13] Figures 13A-13C show GFP expression in the striatum of YAC128 mice 30 days after injection of AAV2HBKO-miRNA-Htt-GFP (Figure 13B) or AAV1-miRNA-Htt-GFP (Figure 13C) compared to untreated controls (Figure 13A). [Figure 14] Figure 1 compares capsid residues involved in heparan sulfate proteoglycan binding between AAV2 and AAVrh8R capsids, with numbering based on the VP1 amino acid sequence. [Figure 15] 1 shows an amino acid alignment of AAV2 and AAVrh8R at residues involved in heparan binding in AAV2. The location of the AAVrh8R arginine capsid modification is circled. [Figure 16] Figure 16A shows improved in vitro transduction of HeLa cells by the AAVrh8R A586R mutant compared to wild-type AAVrh8R. Transduction was monitored by sFLT02 in the culture medium 48 hours after infection with AAVrh8R or AAVrh8R arginine-modified vectors. Figure 16B shows reduced in vitro transduction of HeLaRC32 cells by the AAVrh8R R533A mutant compared to wild-type AAVrh8R. Transduction was monitored by sFLT02 in the culture medium 48 hours after infection with AAVrh8R or AAVrh8R arginine-modified vectors. [Figure 17]Figures 17A-17D show the in vitro transduction levels exhibited by the AAVrh8R A586R and R533A mutants compared to wild-type AAVrh8R. The AAVrh8R A586R mutant (Figure 17B) exhibits increased in vitro transduction of NS1 cells compared to wild-type AAVrh8R (Figure 17A). The AAVrh8R R533A mutant (Figure 17D) exhibits decreased in vitro transduction of HeLa cells compared to wild-type AAVrh8R (Figure 17C). Transduction was monitored by EGFP expression in cells 48 hours after infection with AAVrh8R or AAVrh8R arginine-modified vectors. [Figure 18] Figures 18A and 18B show the levels of subretinal transduction in C57B16 mice demonstrated by the AAVrh8R A586R and R533A mutants. (Figure 18A) The AAVrh8R A586R mutant shows reduced subretinal transduction compared to wild-type AAVrh8R. AAV2 vectors were also tested. (Figure 18B) The AAVrh8R R533A mutant shows increased subretinal transduction compared to wild-type AAVrh8R and naive mice. Transduction was monitored by sFLT02 in retinal lysates of C57B16 mice 30 days after subretinal administration of AAVrh8R or AAVrh8R arginine-modified vectors. [Figure 19] FIG. 1 shows levels of sFLT02 in retinal lysates of C57B16 mice 30 days after intravitreal injection of AAV2, AAVrh8R, or AAVrh8R-A586R vectors. [Figure 20] FIG. 1 shows amino acid alignments of residues involved in heparan binding of AAVrh8R, AAV1, AAV6, AAV8, AAV9, and AAVrh10 with AAV2. DETAILED DESCRIPTION OF THE INVENTION

[0078] As described herein, the inventors have surprisingly discovered that modifications of rAAV particles corresponding to amino acids 484, 487, 532, 585, and / or 588, numbered based on the VP1 numbering of AAV2, demonstrate increased transduction of cells following administration to the eye or CNS of a subject. Without wishing to be bound by any theory, it is believed that these rAAV particles have reduced or eliminated binding to HSPGs or have modified capsid charge, such that administration of the rAAV particles results in increased transduction of cells in the eye or CNS of a subject. Accordingly, the present invention provides a method for delivering heterologous nucleic acid to the eye or CNS of an individual, comprising administering recombinant adeno-associated virus (rAAV) particles to the eye or CNS of the individual, wherein the rAAV particles: a) The methods include a) an rAAV capsid comprising an rAAV capsid protein comprising one or more amino acid substitutions at one or more positions that interact with heparan sulfate proteoglycans, and b) an rAAV vector comprising a heterologous nucleic acid and at least one AAV inverted terminal repeat sequence. In some embodiments, the amino acid substitutions result in reduced or eliminated binding to HSPGs.

[0079] In some embodiments, the invention provides methods for delivering a heterologous nucleic acid to the eye of an individual, comprising subretinal administration of recombinant adeno-associated virus (rAAV) particles to the individual, the rAAV particles comprising: a) an rAAV capsid comprising an rAAV capsid protein comprising one or more amino acid substitutions at one or more positions that interact with heparan sulfate proteoglycan; and b) an rAAV vector comprising a heterologous nucleic acid and at least one AAV inverted terminal repeat.

[0080] In some embodiments, the present invention provides a method for improving rAAV transduction of cells after subretinal delivery of rAAV particles into an individual's eye compared to transduction of cells with rAAV containing a wild-type capsid, comprising introducing one or more amino acid substitutions into an AAV capsid protein at one or more positions that interact with heparan sulfate proteoglycans; the rAAV particle comprises an rAAV capsid protein and an rAAV vector comprising a heterologous nucleic acid and at least one AAV terminal repeat sequence. In some embodiments, the amino acid substitutions reduce or eliminate binding to HSPG. In some embodiments, the rAAV particle comprises a capsid containing the R585A and R588A substitutions of rAAV2, numbered based on AAV2 VP1 (SEQ ID NO: 1). In some embodiments, the rAAV particles comprise capsids that include the A586R and / or R533A substitutions of AAVrh8R, numbered based on VP1 of AAVrh8R (SEQ ID NO: 9).

[0081] Provided is a method for improving expression of a heterologous nucleic acid after subretinal delivery of rAAV particles, comprising incorporating one or more amino acid substitutions into an AAV capsid protein at one or more positions that interact with heparan sulfate proteoglycans; the rAAV particle comprises an rAAV vector comprising the rAAV capsid protein, a heterologous nucleic acid, and at least one AAV terminal repeat sequence. In some embodiments, the amino acid substitutions result in reduced or eliminated binding to HSPG. In some embodiments, the rAAV particle comprises a capsid comprising the R585A and R588A substitutions of rAAV2, numbered based on VP1 of AAV2 (SEQ ID NO: 1). In some embodiments, the rAAV particle comprises a capsid comprising the A586R and / or R533A substitutions of AAVrh8R, numbered based on VP1 of AAVrh8R (SEQ ID NO: 9).

[0082] In some embodiments, the present invention provides a method for improving expression of a heterologous nucleic acid after subretinal delivery of rAAV particles into an individual's eye, comprising introducing one or more amino acid substitutions into an AAV capsid protein at one or more positions that interact with heparan sulfate proteoglycans; the rAAV particle comprises an rAAV vector comprising the rAAV capsid protein and a heterologous nucleic acid and at least one AAV terminal repeat sequence. The improved transduction is comparable to that of rAAV particles comprising wild-type capsids. In some embodiments, the amino acid substitutions reduce or eliminate binding to HSPGs. In some embodiments, the rAAV particle comprises a capsid comprising the R585A and R588A substitutions of the rAAV2 capsid, numbered based on AAV2 VP1 (SEQ ID NO: 1). In some embodiments, the rAAV particles comprise capsids that include the A586R and / or R533A substitutions of AAVrh8R, numbered based on VP1 of AAVrh8R (SEQ ID NO: 9).

[0083] In some embodiments, the present invention provides methods for treating an ocular disorder in an individual, comprising delivering to the individual's retina a composition comprising an effective amount of rAAV particles, the rAAV particles comprising: a) an rAAV capsid comprising an rAAV capsid protein comprising one or more amino acid substitutions at one or more positions that interact with heparan sulfate proteoglycans; and b) an rAAV vector comprising a heterologous nucleic acid and at least one AAV terminal repeat sequence. In some embodiments, the amino acid substitutions result in reduced or eliminated binding to HSPGs. In some embodiments, the rAAV particles comprise capsids comprising the R585A and R588A substitutions of rAAV2, numbered based on VP1 of AAV2 (SEQ ID NO: 1). In some embodiments, the rAAV particles comprise capsids comprising the A586R and / or R533A substitutions of AAVrh8R, numbered based on VP1 of AAVrh8R (SEQ ID NO: 9).

[0084] The present invention also provides a system for subretinal delivery of a vector to the eye of an individual, comprising: a) a composition comprising an effective amount of rAAV particles, wherein i) the capsid protein of the rAAV particles comprises one or more amino acid substitutions at one or more positions that interact with heparan sulfate proteoglycans; and ii) the vector comprises a heterologous nucleic acid encoding a therapeutic polypeptide or therapeutic RNA and at least one AAV terminal repeat; and b) a device for retinal delivery of the rAAV. In some embodiments, the amino acid substitutions reduce or eliminate binding to HSPGs. In some embodiments, the rAAV particles comprise capsids containing the R585A and R588A substitutions of rAAV2, numbered based on VP1 of AAV2 (SEQ ID NO: 1). In some embodiments, the rAAV particles comprise capsids containing the A586R and / or R533A substitutions of AAVrh8R, numbered based on VP1 of AAVrh8R (SEQ ID NO: 9).

[0085] In some embodiments, the present invention further provides methods for delivering heterologous nucleic acids to the central nervous system (CNS) of an individual, comprising administering recombinant adeno-associated virus (rAAV) particles to the CNS of the individual. The rAAV particles comprise (a) an rAAV capsid comprising an rAAV capsid protein containing one or more amino acid substitutions at one or more positions that interact with heparan sulfate proteoglycans, and (b) an rAAV vector comprising a heterologous nucleic acid and at least one AAV inverted terminal repeat sequence. These methods demonstrate improved cellular heterologous nucleic acid expression and / or rAAV transduction after delivery of the rAAV particles to the CNS of an individual, compared to transduction of cells with, for example, an rAAV comprising a wild-type capsid. Furthermore, the methods of the present invention can infect specific cells (e.g., neurons) while still achieving broad and robust transduction efficiency. Such rAAV particles and methods are suitable for treating CNS disorders, including, but not limited to, Huntington's disease. In some embodiments, the amino acid substitutions result in reduced or eliminated binding to HSPG. In some embodiments, the rAAV particles comprise capsids that include the R585A and R588A substitutions of rAAV2, numbered based on VP1 of AAV2 (SEQ ID NO: 1). In some embodiments, the rAAV particles comprise capsids that include the A586R and / or R533A substitutions of AAVrh8R, numbered based on VP1 of AAVrh8R (SEQ ID NO: 9).

[0086] The present invention also provides kits comprising rAAV particles or compositions containing rAAV particles, the rAAV particles having (a) an rAAV capsid comprising an rAAV capsid protein comprising one or more amino acid substitutions at one or more positions that interact with heparan sulfate proteoglycans, and (b) an rAAV vector comprising a heterologous nucleic acid and at least one AAV inverted terminal repeat. These kits are useful for delivery of heterologous nucleic acids to the eye or CNS of an individual, as well as for the administration of heterologous nucleic acids to the eye or CNS of an individual. They are also useful in treating ocular or CNS disorders (eg, treating retinopathy or Huntington's disease).

[0087] I. General technology The techniques and procedures described or referenced herein are generally well understood and can be readily understood by those of skill in the art, for example, in Molecular Cloning: A Laboratory Manual (Sambrook et al., 4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2012); Current Protocols in Molecular Biology (FMA Usubel et al., 2003); the series Methods in Enzymology (Academic Press, Inc.); PCR 2: A Practical Approach (MJ MacPherson, B.D. Hames, and G.R. Taylor, eds., 1995); Antibodies, A Laboratory Manual (Harlow and Lane, eds., 1988); Culture of Animal Cells: A Manual of Basic Technique and Specialized Applications (R.I. Freshney, 6th ed., J. Wiley and Sons, 2010); Oligonucleotide Synthesis (M.J. Gait, ed., 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Manual Notebook (ed. J.E.Cellis, Academic Press, 1998); Introduction to Cell and Tissue Culture (J.P. Mather and P.E. Roberts, Plenum Press, 1998); Cell and Tissue Culture: Laboratory Procedures (eds. A. Doyle, J.B. Griffiths, and D.G. Newell, J.W. Wiley and Sons, pp. 1993-1998); Handbook of Experimental Immunology (eds. D.M. Weir and C.C. Blackwell, 1996); Gene Transfer Vectors for Mammalian Cells (J.M.Miller and M.P. Calos, eds., 1987); PCR: The Polymerase Chain Reaction (Mullis et al., eds., 1994); Current Protocols in Immunology (J.E. Coligan et al., 1991); Short Protocols in Molecular Biology (Ausubel et al., eds., J. Wiley and Sons, 2002); Immunobiology (C.A. Janeway et al., 2004); Antibodies (P. Finch, 1997); Antibodies: A Practical Approach (D. Catty, ed., IRL Press, 1988-1989); Monoclonal Antibodies: A Practical Approach (P. Shepherd and C. Dean, eds., Oxford University Press, 2000); Using Antibodies: A Laboratory Manual (E. Harlow and D. Lane, Cold Spring Harbor Laboratory Press, 1999); The Antibodies (M. Zanetti and J.D. Capra, eds., Harwood Academic Press) Publishers, 1995); and Cancer: Principles and Practice of Oncology (V.T. DeVita et al., eds., J.B. Lippincott Company, 2011).

[0088] II. Definition As used herein, a "vector" refers to a vector, whether in vitro or in vivo. The term "transfected vector" refers to a recombinant plasmid or virus containing nucleic acid that is delivered to a host cell by either

[0089] The term "polynucleotide" or "nucleic acid" as used herein refers to any length of polymeric form of nucleotide, either ribonucleotide or deoxyribonucleotide. Thus, this term includes, but is not limited to, single-stranded, double-stranded, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrid, or polymers containing purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases. The backbone of a polynucleotide may contain sugar and phosphate groups (as typically found in RNA or DNA), or may contain modified or substituted sugar or phosphate groups. Alternatively, the backbone of a polynucleotide may comprise a polymer of synthetic subunits such as phosphoramidates, and thus may be an oligodeoxynucleoside phosphoramidate (P-NH2) or a mixed phosphoramidate-phosphodiester oligomer. Additionally, double-stranded polynucleotides can be obtained from a single-stranded polynucleotide product of chemical synthesis by synthesizing a complementary strand and annealing the strands under appropriate conditions, or by de novo synthesis of a complementary strand using a DNA polymerase and an appropriate primer.

[0090] The terms "polypeptide" and "protein" are used interchangeably to refer to polymers of amino acid residues and are not limited to a minimum length. Such polymers of amino acid residues may contain naturally occurring or non-naturally occurring amino acid residues, and include, but are not limited to, peptides, oligopeptides, dimers, trimers, and multimers of amino acid residues. Both full-length proteins and fragments thereof are encompassed by this definition. These terms also include post-expression modifications of the polypeptide, such as glycosylation, sialylation, acetylation, phosphorylation, and the like. Furthermore, for purposes of the present invention, "polypeptide" refers to proteins containing modifications (generally conservative in nature) to the native sequence, such as deletions, additions, and substitutions, provided that the protein maintains the desired activity. These modifications may be intentional, such as by site-directed mutagenesis, or may be accidental, such as errors resulting from mutations of hosts producing the protein or from PCR amplification.

[0091] "Recombinant viral vector" refers to a recombinant polynucleotide vector that contains one or more heterologous sequences (i.e., nucleic acid sequences not of viral origin). In the case of recombinant AAV vectors, the recombinant nucleic acid is flanked by at least one inverted terminal repeat (ITR). In some embodiments, the recombinant nucleic acid is flanked by two ITRs.

[0092] A "recombinant AAV vector (rAAV vector)" refers to a polynucleotide vector containing one or more heterologous sequences (i.e., nucleic acid sequences not derived from AAV) flanked by at least one AAV inverted terminal repeat (ITR). When present in a host cell infected with a suitable helper virus (or expressing suitable helper functions) and expressing AAV rep and cap gene products (i.e., AAV Rep and Cap proteins), such a rAAV vector is replicated and packaged into infectious viral particles. When the rAAV vector is integrated into a larger polynucleotide (e.g., in a chromosome or in another vector, such as a plasmid used for cloning or transfection), the rAAV vector is sometimes referred to as a "provector," which is "rescued" by replication and encapsidation in the presence of AAV packaging functions and suitable helper functions. rAAV vectors can be in any of a number of forms, including, but not limited to, plasmids, linear artificial chromosomes, can be complexed with lipids, can be encapsulated in liposomes, and can be viral. The rAAV vector can be packaged into an AAV viral capsid to generate a "recombinant adeno-associated viral particle (rAAV particle)."

[0093] "rAAV virus" or "rAAV viral particle" refers to a viral particle composed of at least one AAV capsid protein and an rAAV vector genome enclosed in the capsid.

[0094] "Heterologous" means derived from a genotypically distinct entity from that of the rest of the entity to which it is compared or introduced or incorporated. For example, a polynucleotide introduced into a different cell type by genetic engineering techniques is a heterologous polynucleotide (and can encode a heterologous polypeptide when expressed). Similarly, a cellular sequence (e.g., a gene or portion thereof) incorporated into a viral vector is a heterologous nucleotide sequence relative to that vector.

[0095] The term "transgene" refers to a polynucleotide that is introduced into a cell, and that can be transcribed into RNA and, optionally, translated and / or expressed under appropriate conditions.In several embodiments, the transgene confers desired characteristics to the cell into which the transgene is introduced, or otherwise produces a desired therapeutic or diagnostic outcome.In another embodiment, the transgene can be translated into a molecule that mediates RNA interference, such as miRNA, siRNA or shRNA.

[0096] The terms "genome particles (gp)," "genome equivalents," or "genome copies," when used with reference to viral titer, refer to the number of virions containing a recombinant AAV DNA genome, regardless of infectiousness or functionality. The number of genome particles in a particular vector preparation can be measured by procedures such as those described in the Examples herein or, for example, Clark et al. (1999) Hum. Gene Ther., 10:1031-1039; Veldwijk et al. (2002) Mol. Ther., 6:272-278.

[0097] The term "vector genome (vg)," as used herein, can refer to one or more polynucleotides comprising a set of polynucleotide sequences of a vector, e.g., a viral vector. A vector genome may be encapsulated in a viral particle capsid. Depending on the particular viral vector, the vector genome may comprise single-stranded DNA, double-stranded DNA, or single- or double-stranded RNA. A vector genome may comprise endogenous sequences associated with a particular viral vector and / or heterologous sequences inserted into a particular viral vector by recombinant techniques. For example, a recombinant AAV vector genome may comprise at least one ITR sequence flanked by a promoter, a stuffer, a sequence of interest (e.g., RNAi), and a polyadenylation sequence. A complete vector genome may comprise the complete set of polynucleotide sequences of a vector. In some embodiments, the nucleic acid titer of a viral vector may be measured in units of vg / mL. Suitable methods for measuring this titer are known in the art (e.g., quantitative PCR).

[0098] The terms "infectious unit (iu)," "infectious particle," or "replication unit," when used with respect to viral titer, refer to the number of infectious and replicative recombinant AAV vector particles, as measured by the infectious center assay, also known as the replication center assay, e.g., as described in McLaughlin et al. (1988) J. Virol., 62:1963-1973.

[0099] The term "transducing units (tu)" when used in reference to viral titer is used in the practice of this specification. It refers to the number of infectious recombinant AAV vector particles that result in the production of a functional transgene product, as measured by a functional assay such as those described in the Examples or, for example, those described in Xiao et al. (1997) Exp. Neurobiol., 144:113-124; or those described in Fisher et al. (1996) J. Virol., 70:520-532 (LFU assay).

[0100] "Inverted terminal repeat" or "ITR" sequences are a term well understood in the art and refer to relatively short sequences found at the ends of viral genomes that are in opposite orientation.

[0101] The term "AAV inverted terminal repeat (ITR)" is well understood in the art and refers to a sequence of approximately 145 nucleotides present at both ends of a native single-stranded AAV genome. The outermost 125 nucleotides of the ITR can exist in either of two alternative orientations, resulting in heterogeneity between different AAV genomes and between the two ends of a single AAV genome. The outermost 125 nucleotides also contain several shorter regions of self-complementarity (referred to as A, A', B, B', C, C', and D regions), which allow intrastrand base pairing to occur within this portion of the ITR.

[0102] "Truncation sequences" or "trs" are sequences within the D region of the AAV ITRs that are cleaved by the AAV rep protein during viral DNA replication. Mutant truncation sequences are less susceptible to cleavage by the AAV rep protein.

[0103] AAV "helper virus" refers to a virus that enables AAV (which is a defective parvovirus) to replicate and be packaged by the host cell. Many such helper viruses have been identified, including adenoviruses, herpesviruses, and poxviruses, such as vaccinia. Adenoviruses encompass many different subgroups, with adenovirus type 5 (Ad5) of subgroup C being the most commonly used. Numerous adenoviruses of human, nonhuman mammalian, and avian origin are known and available from depositories such as the American College of Cardiology (ATCC). Herpesviruses also available from depositories such as the ATCC include herpes simplex virus (HSV), Epstein-Barr virus (EBV), cytomegalovirus (CMV), and pseudorabies virus (PRV).

[0104] "Percent sequence identity (%)" to a reference polypeptide or nucleic acid sequence is defined as the percentage of amino acid residues or nucleotides in a candidate sequence that are identical to the amino acid residues or nucleotides in the reference polypeptide or nucleic acid sequence after aligning the sequences and, if necessary, introducing gaps to achieve the maximum percent sequence identity, not counting any conservative substitutions as part of the sequence identity. Alignment for determining percent amino acid or nucleic acid sequence identity can be achieved in a variety of ways within the skill of the art, for example, by using publicly available computer software programs, such as those described in Current Protocols in Molecular Biology (Ausubel et al., eds., 1987), Supplement 30, Section 7.7.18, Table 7.7.1, and including BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. An example of an alignment program is ALIGN Plus (Scientific and Educational Software, Pennsylvania). Those skilled in the art can determine the appropriate parameters for measuring alignment, including any algorithms required to achieve maximum alignment across the entire length of the sequences being compared. For purposes of this specification, a given amino acid sequence A having or containing a particular % amino acid sequence identity to, with, or against a given amino acid sequence B is referred to as a % amino acid identity of a given amino acid sequence A to, with, or against a given amino acid sequence B. The percent amino acid sequence identity of a given nucleic acid sequence C to, with, or to a given nucleic acid sequence D (alternatively, the percent nucleic acid identity of a given nucleic acid sequence C to, with, or to a given nucleic acid sequence D) is calculated as follows: 100 x fraction W / Z (where W is the number of nucleotides scored as identical matches by a sequence alignment program in an alignment of A and D in that program, and Z is the total number of nucleotides in D). It will be understood that if the length of amino acid sequence A is not identical to the length of amino acid sequence B, the percent amino acid sequence identity of A to B will not be the same as the percent amino acid sequence identity of B to A. For purposes herein, the percent nucleic acid identity of a given nucleic acid sequence C to, with, or to a given nucleic acid sequence D (alternatively, the percent nucleic acid identity of a given nucleic acid sequence C to, with, or to a given nucleic acid sequence D) is calculated as follows: 100 x fraction W / Z (where W is the number of nucleotides scored as identical matches by a sequence alignment program in an alignment of C and D in that program, and Z is the total number of nucleotides in D). It will be understood that if the length of nucleic acid sequence C is not identical to the length of nucleic acid sequence D, then the % nucleic acid sequence identity of C to D will not be the same as the % nucleic acid sequence identity of D to C.

[0105] An "isolated" molecule (eg, nucleic acid or protein) or cell means that it has been identified and separated and / or recovered from a component of its natural environment.

[0106] An "effective amount" is an amount sufficient to achieve beneficial or desired results, including clinical results (e.g., amelioration of symptoms, achievement of a clinical endpoint, etc.). An effective amount can be administered in one or more administrations. In terms of a disease state, an effective amount is an amount sufficient to ameliorate, stabilize, or delay the onset of a disease.

[0107] An "individual" or "subject" is a mammal. Mammals include, but are not limited to, domestic animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates, such as monkeys), rabbits, and rodents (e.g., mice and rats). In certain embodiments, the individual or subject is human.

[0108] As used herein, "treatment" refers to an approach to obtain beneficial or desired clinical results. For the purposes of the present invention, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, reduction in the extent of disease, stabilized (e.g., not worsening) disease state, prevention of disease spread (metastasis), delay or slowing of disease progression, improvement or palliation of disease state, and remission (whether partial or complete), whether detectable or undetectable. "Treatment" can also mean prolonging survival compared to the expected survival in the absence of treatment.

[0109] As used herein, the term "prophylactic treatment" refers to treatment when an individual is known or suspected to have or be at risk of having a disorder, but exhibits no or only mild symptoms of the disorder. Individuals receiving prophylactic treatment will be treated before the onset of symptoms.

[0110] As used herein, a "therapeutic" agent (e.g., a therapeutic polypeptide, nucleic acid, or transgene) is one that produces a beneficial or desired clinical result, such as the exemplary clinical results described above. Thus, a therapeutic agent can be used in the above treatments.

[0111] The term "central retina," as used herein, refers to the outer macula and / or inner macula and / or fovea. The term "central retinal cell type," as used herein, refers to cell types in the central retina, such as RPE and photoreceptor cells.

[0112] The term "macula" refers to the area with a high relative concentration of photoreceptor cells, particularly rods and cones, compared to the peripheral retina. The term "outer macula" refers to the central retinal region of a primate, containing the retina. As used herein, the term "outer macula" may also refer to the "peripheral macula." As used herein, the term "inner macula" may also refer to the "central macula."

[0113] The term "fovea" refers to a small region in the central retina of primates, approximately 0.5 mm in diameter or less, that contains a high relative concentration of photoreceptor cells, particularly cones, compared to the peripheral retina and macula.

[0114] The term "subretinal space," as used herein, refers to the location between photoreceptor cells and retinal pigment epithelial cells in the retina. The subretinal space may also be a potential space, such as before any subretinal injection of fluid. The subretinal space may also contain fluid that is injected into the potential space. In this case, the fluid is "in contact with the subretinal space." Cells "in contact with the subretinal space" include cells adjacent to the subretinal space, such as RPE and photoreceptor cells.

[0115] The term "bleb," as used herein, refers to a fluid cavity within the subretinal space of the eye. The blebs of the present invention may be formed by a single injection of fluid into a single cavity, by multiple injections of one or more fluids into the same cavity, by multiple injections into multiple cavities, or by multiple injections into multiple cavities that, when repositioned, create a total fluid cavity useful for achieving a therapeutic effect on a desired portion of the subretinal space.

[0116] "Rhodopsin kinase (RK) promoter" refers to a polynucleotide sequence derived from the rhodopsin kinase gene (e.g., human RK, represented by GenBank Entrez Gene ID 6011) that specifically drives expression in rod and cone photoreceptor cells and retinal cell lines, such as WERI Rb-1. As used herein, "rhodopsin kinase promoter" can also refer to the entire promoter sequence or a fragment of the promoter sequence sufficient to drive photoreceptor-specific expression, such as the sequences described in Khani, SC et al. (2007) Invest. Ophthalmol. Vis. Sci. 48(9):3954-61 and Young, JE et al. (2003) Invest. Ophthalmol. Vis. Sci. 44(9):4076-85. In some embodiments, the RK promoter spans from -112 to +180 relative to the transcription start site.

[0117] "Chicken β-actin (CBA) promoter" refers to a polynucleotide sequence derived from a chicken β-actin gene (e.g., chicken (Gallus gallus) β-actin represented by GenBank Entrez Gene ID 396526). As used herein, "chicken β-actin promoter" can also refer to a promoter containing a cytomegalovirus (CMV) early enhancer element and the promoter of the chicken β-actin gene, as well as the first exon and intron and splice acceptor of the rabbit β-globin gene, such as the sequence described in Miyazaki, J. et al. (1989) Gene 79(2):269-77. As used herein, the term "CAG promoter" can be used interchangeably. As used herein, the term "CMV early enhancer / chicken β-actin (CAG) promoter" can be used interchangeably.

[0118] Reference herein to "about" a value or parameter includes (and describes) embodiments that relate to that value or parameter per se. For example, a description that refers to "about X" includes a description of "X."

[0119] As used herein, the singular articles "a," "an," and "the" are used unless otherwise indicated. Unless otherwise indicated, plural references are included.

[0120] It will be understood that aspects and embodiments of the invention described herein encompass aspects and embodiments that "comprise," "consist," and / or "consist essentially of."

[0121] III. Virus particles It is known in the art that heparan sulfate proteoglycans (HSPGs) act as cellular receptors for AAV2 particles (Summerford, C. and Samulski, RJ (1998) J. Virol. 72(2):1438-45). Binding of AAV2 particles to HSPGs on the cell membrane helps attach the particles to the cell. Other cell surface proteins, such as fibroblast growth factor receptor and αvβ5 integrin, can also facilitate cell infection. After binding, AAV2 particles can enter cells by mechanisms including receptor-mediated endocytosis through clathrin-coated pits. AAV2 particles are released from intracellular vesicles by endosomal acidification. This allows AAV2 particles to migrate to the perinuclear region and then to the cell nucleus. It is also known that AAV3 particles bind to heparan (Rabinowitz, JE et al. (2002) J. Virol. 76(2):791-801).

[0122] Gene therapy protocols for ocular disorders require localized delivery of vectors to ocular cells (e.g., retinal cells). Cells targeted for treatment in these diseases can include, among others, one or more ocular cells (e.g., photoreceptors, ocular neurons, etc.). The methods and kits of the present invention are based, at least in part, on the discovery that certain rAAV capsids (e.g., those comprising rAAV capsid proteins containing one or more amino acid substitutions at one or more positions that interact with heparan sulfate proteoglycans) enable widespread vector distribution among ocular cells. Thus, these capsids may be particularly advantageous for delivering heterologous nucleic acids to an individual's eye, enhancing rAAV transduction of cells after delivery of rAAV particles to an individual's eye, enhancing expression of heterologous nucleic acids after delivery of rAAV particles to an individual's eye, and / or treating an individual's ocular disorder using rAAV particles.

[0123] Similarly, gene therapy protocols for CNS disorders require localized delivery of vectors to cells of the CNS. Cells targeted for treatment in these diseases can include, inter alia, one or more cells (e.g., neurons) of the brain. The methods and kits of the present invention are based, at least in part, on the discovery that certain rAAV capsids (e.g., those containing rAAV capsid proteins containing one or more amino acid substitutions at one or more positions that interact with heparan sulfate proteoglycans) enable widespread vector distribution among cells of the CNS. Thus, these capsids may be particularly advantageous for delivering heterologous nucleic acids to the central nervous system (CNS) of an individual, for enhancing rAAV transduction of cells after delivery of rAAV particles to the CNS of an individual, for enhancing expression of heterologous nucleic acids after delivery of rAAV particles to the CNS of an individual, and / or for treating disorders of the CNS of an individual using rAAV particles.

[0124] It is known that the capsid of AAV (e.g., AAV2, AAVrh8R, etc.) contains three capsid proteins: VP1, VP2, and VP3. These proteins contain a significant amount of overlapping amino acid sequence and unique N-terminal sequences. The AAV2 capsid contains 60 subunits arranged based on icosahedral symmetry (Xie, Q. et al. (2002) Proc. Natl. Acad. Sci. 99(16):10405-10). VP1, VP2, and VP3 are known to exist in a 1:1:10 ratio.

[0125] The binding of AAV2 capsid protein to HSPG is basic. These interactions are known to occur through electrostatic interactions between the capsid residues and negatively charged glycosaminoglycan residues (Opie, SR et al. (2003) J. Virol. 77:6995-7006; Kern, A et al. (2003) J. Virol. 77:11072-11081). Specific capsid residues involved in these interactions include R484, R487, K532, R585, and R588. Mutation of these residues has been shown to reduce AAV2 binding to HeLa cells and heparan itself (Opie, SR et al. (2003) J. Virol. 77:6995-7006; Kern, A et al. (2003) J. Virol. 77:11072-11081; WO2004 / 027019). A2, U.S. Patent No. 7,629,322). Furthermore, without wishing to be bound by theory, it is believed that amino acid substitutions at one or more of the residues corresponding to amino acids 484, 487, 532, 585, or 588, numbered based on the VP1 numbering of AAV2, may modify the transduction properties of AAV capsid types that do not bind HSPGs, or may modify the transduction properties of AAV capsid types independently of their ability to bind HSPGs.

[0126] Certain aspects of the present invention relate to delivery of heterologous nucleic acid to the eye or central nervous system (CNS) of an individual, comprising administering recombinant adeno-associated virus (rAAV) particles to the eye or CNS of the individual. In some embodiments, the rAAV particles comprise an rAAV capsid comprising an rAAV capsid protein containing one or more amino acid substitutions at one or more positions that interact with heparan sulfate proteoglycans. In some embodiments, the rAAV particles of the present invention comprise an AAV serotype 2 (AAV2) capsid. In some embodiments, the rAAV particles of the present invention comprise an AAV serotype rh8R (AAVrh8R) capsid.

[0127] As described herein, rAAV particles in which the capsid protein is mutated at residues that interact with HSPGs or at one or more corresponding residues at amino acids 484, 487, 532, 585, or 588, numbered based on the VP1 numbering of AAV2, exhibit advantageous properties, such as improved expression and / or reduced neuroinflammation. Thus, in some embodiments, upon delivery, a heterologous nucleic acid encoded by the rAAV vector is expressed at an increased expression level compared to the expression level of the heterologous nucleic acid of an rAAV particle comprising an rAAV capsid comprising a reference rAAV capsid protein (e.g., a wild-type rAAV capsid protein). In some embodiments, nucleic acid expression is increased by at least about 10%, at least about 25%, at least about 50%, at least about 75%, or at least about 100%. In some embodiments, when delivered, the rAAV particles result in reduced neuroinflammation compared to rAAV particles comprising a reference rAAV capsid protein (e.g., a wild-type rAAV capsid protein). In some embodiments, neuroinflammation is reduced by at least about 10%, at least about 25%, at least about 50%, at least about 75%, or at least about 100%. Suitable reference rAAV capsid proteins can include any capsid protein that lacks one or more amino acid substitutions at one or more positions that interact with heparan sulfate proteoglycans (thus, the reference capsid can have one or more "background" substitutions that do not alter binding to HSPGs).

[0128] In some embodiments, the invention provides methods for delivering a heterologous nucleic acid to the eye of an individual, comprising administering recombinant adeno-associated virus (rAAV) particles to the subretinal space of the individual, the rAAV particles comprising: a) an rAAV capsid comprising an rAAV capsid protein comprising one or more amino acid substitutions at one or more positions that interact with heparan sulfate proteoglycan; and b) an rAAV vector comprising a heterologous nucleic acid and at least one AAV inverted terminal repeat.

[0129] In some embodiments, the rAAV particles of the present invention comprise an AAV serotype 2 (AAV2) capsid. In some embodiments, the one or more amino acid substitutions are substitutions of amino acid residues in any one of VP1, VP2, and / or VP3 of AAV2, and the amino acid substitutions alter the interaction of the rAAV particle with HSPG (e.g., reduce or eliminate binding to HSPG). In some embodiments, the one or more amino acid substitutions are substitutions of amino acid residues in VP1 AAV2. In some embodiments, the one or more amino acid substitutions are substitutions of amino acid residues in VP2 AAV2. In some embodiments, the one or more amino acid substitutions are substitutions of amino acid residues in VP3 AAV2. In some embodiments, the one or more amino acid substitutions are substitutions of amino acid residues in a combination of VP1, VP2, and VP3 of AAV2. In some embodiments, the one or more amino acid substitutions are substitutions of amino acid residues in any one of VP1, VP2, and / or VP3 of AAV2. In some embodiments, the one or more amino acid substitutions are substitutions of amino acid residues in any one of the capsid proteins of SEQ ID NOs: 1, 3, and / or 5. In some embodiments, the rAAV particles of the invention comprise the capsid proteins of SEQ ID NOs: 2, 4, and / or 6.

[0130] In some embodiments, the rAAV particles of the present invention comprise an AAV serotype 3 (AAV3) capsid. In some embodiments, the one or more amino acid substitutions are substitutions of amino acid residues in any one of VP1, VP2, and / or VP3 of AAV3, and the amino acid substitutions alter the interaction of the rAAV particle with HSPG (e.g., reduce or eliminate binding to HSPG). In some embodiments, the one or more amino acid substitutions are substitutions of amino acid residues in VP1 AAV3. In some embodiments, the one or more amino acid substitutions are substitutions of amino acid residues in VP2 AAV3. In some embodiments, the one or more amino acid substitutions are substitutions of amino acid residues in VP3 AAV3. In some embodiments, the one or more amino acid substitutions are substitutions of amino acid residues in a combination of VP1, VP2, and VP3 of AAV3. In some embodiments, the one or more amino acid substitutions are substitutions of amino acid residues in any one of VP1, VP2, and / or VP3 of AAV3. In some embodiments, the one or more amino acid substitutions are at an amino acid residue corresponding to the capsid protein of SEQ ID NO:7.

[0131] In some embodiments, the rAAV particles of the invention comprise an AAV serotype rh8R (AAVrh8R) capsid, such as that described in U.S. Pre-Issue Application Publication No. 20090317417. In some embodiments, one or more amino acid substitutions are substitutions of amino acid residues in any one of VP1, VP2, and / or VP3 of AAVrh8R, and the amino acid substitutions alter the interaction of the rAAV particle with HSPG (e.g., reduce or eliminate binding to HSPG). In some embodiments, one or more amino acid substitutions are substitutions of amino acid residues in VP1 AAVrh8R. In some embodiments, one or more amino acid substitutions are substitutions of amino acid residues in VP2 AAVrh8R. In some embodiments, one or more amino acid substitutions are substitutions of amino acid residues in VP3 AAVrh8R. In some embodiments, one or more amino acid substitutions are substitutions of amino acid residues in a combination of VP1, VP2, and VP3 of AAVrh8R. In some embodiments, the one or more amino acid substitutions are substitutions of amino acid residues in any one of VP1, VP2, and / or VP3 of AAVrh8R. In some embodiments, the one or more amino acid substitutions are substitutions of amino acid residues in the capsid protein exemplified by SEQ ID NO: 9. In some embodiments, the rAAV particles of the invention comprise the capsid proteins of SEQ ID NOs: 10 and / or 11.

[0132] In some embodiments, the one or more amino acid substitutions reduce binding of the rAAV particle to heparan sulfate proteoglycans by at least about 10%, at least about 25%, at least about 50%, at least about 75%, or at least about 100%. In some embodiments, the one or more amino acid substitutions reduce binding of the rAAV particle to heparan sulfate proteoglycans (compared to binding of rAAV particles comprising wild-type capsids) by at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 100%. In some embodiments, the one or more amino acid substitutions reduce binding of rAAV particles to heparan sulfate proteoglycans by about 10% to about 100%, about 20% to about 100%, about 30% to about 100%, about 40% to about 100%, about 50% to about 100%, about 60% to about 100%, about 70% to about 100%, about 80% to about 100%, about 90% to about 100%, about 10% to about 90%, about 20% to about 90%, about 30% to about 90%, about 40% to about 90%, about 50% to about 90%, about 60% to about 90%, about 70% to about 90%, about 80% to about 90%, about 10% to about 80%, or about 20% to about 80%. , about 30% to about 80%, about 40% to about 80%, about 50% to about 80%, about 60% to about 80%, about 70% to about 80%, about 10% to about 70%, about 20% to about 70%, about 30% to about 70%, about 40% to about 70%, about 50% to about 70%, about 60% to about 70%, about 10% to about 60%, about 20% to about 60%, about 30% to about 60%, The reduction is by any one of about 40% to about 60%, about 50% to about 60%, about 10% to about 50%, about 20% to about 50%, about 30% to about 50%, about 40% to about 50%, about 10% to about 40%, about 20% to about 40%, about 30% to about 40%, about 10% to about 30%, about 20% to about 30%, or about 10% to about 20%.In some embodiments, one or more amino acid substitutions result in no detectable binding of the rAAV particle to heparan sulfate proteoglycans compared to the binding of wild-type rAAV particles. Means for measuring the binding of AAV particles to HSPGs are known in the art; for example, binding to heparan sulfate chromatography media or binding to cells known to express HSPGs on their surface. See, e.g., Opie, SR et al. (2003) J. Virol. 77:6995-7006 and Kern, A et al. (2003) J. Virol. 77:11072-11081.

[0133] In some embodiments, the invention provides rAAV particles for subretinal delivery of a therapeutic nucleic acid, comprising one or more amino acid substitutions in a capsid protein that reduce or eliminate binding of the rAAV particle to heparan sulfate proteoglycans, wherein the one or more amino acid substitutions are at positions 484, 487, 532, 585, or 588, numbered based on the VP1 numbering of AAV2. In some embodiments, the one or more amino acid substitutions are at positions 484, 487, 532, 585, or 588, numbered based on the VP1 numbering of AAV2. In some embodiments, the one or more amino acid substitutions are at positions 484, 487, 532, 585, or 588, numbered based on the VP1 numbering of AAV2. In some embodiments, the one or more amino acid substitutions are at positions 484, 487, 532, 585, or 588 of VP3 of AAV2, numbered based on VP1 of AAV2. In some embodiments, the one or more amino acid substitutions are at positions 484, 487, 532, 585, or 588 of VP1 of AAV2, VP2 of AAV2, and / or VP3 of AAV2, numbered based on VP1 of AAV2. In some embodiments, VP1 of rAAV2 comprises the amino acid sequence of SEQ ID NO:1.

[0134] In some embodiments, the one or more amino acid substitutions are at positions 484, 487, 532, 585, or 588 of AAV3 VP1, numbered based on AAV2 VP1. In some embodiments, the one or more amino acid substitutions are at positions 484, 487, 532, 585, or 588 of AAV3 VP2, numbered based on AAV2 VP1. In some embodiments, the one or more amino acid substitutions are at positions 484, 487, 532, 585, or 588 of AAV3 VP3, numbered based on AAV2 VP1. In some embodiments, the one or more amino acid substitutions are at positions 484, 487, 532, 585, or 588 of AAV3 VP1, AAV3 VP2, and / or AAV3 VP3, numbered based on rAAV2 VP1. In some embodiments, VP1 of rAAV2 comprises the amino acid sequence of SEQ ID NO:1.

[0135] In some embodiments, the AAV particles of the present invention comprise capsids having one or more amino acid substitutions at positions 485, 488, 528, 533, 586, or 589, as numbered based on the VP1 numbering of AAVrh8R. In some embodiments, the numbering is based on VP1 of AAVrh8R comprising the amino acid sequence of SEQ ID NO:9. In some embodiments, the one or more amino acid substitutions comprise substitution of a positively charged amino acid residue with an amino acid residue that does not bear a positive charge. In some embodiments, the positively charged amino acid residue is substituted with a hydrophobic amino acid residue. In further embodiments, the one or more amino acid substitutions comprise substitution of an arginine or lysine residue. In yet further embodiments, the one or more amino acid substitutions comprise substitution of an arginine or lysine residue with an alanine residue. In other embodiments, the one or more amino acid substitutions comprise substitution of a non-positively charged amino acid residue with a positively charged amino acid residue. In some embodiments, the hydrophobic amino acid residue is substituted with a positively charged amino acid residue. In further embodiments, the one or more amino acid substitutions comprise substitutions of alanine residues. In yet further embodiments, the one or more amino acid substitutions comprise substitutions of arginine or lysine residues with alanine residues. In some embodiments, the one or more amino acid substitutions comprise substitutions at positions R533 and / or A586, numbered based on VP1 of AAVrh8R. In further embodiments, the AAV capsid comprises amino acid substitutions A586R and R533A, numbered based on VP1 of AAVrh8R. In some embodiments, the rAAV particles comprise the rAAV capsid proteins of SEQ ID NOs: 10 and / or 11.

[0136] In some embodiments of the present invention, the one or more amino acid substitutions comprise substitution of a positively charged amino acid residue (e.g., an amino acid having a positively charged side chain) with an amino acid that does not have a positive charge (e.g., an amino acid that does not contain a positively charged side chain). Positively charged amino acids include arginine, histidine, and lysine. Examples of amino acid residues that do not have a positive charge include negatively charged amino acids (aspartic acid and glutamic acid), amino acids with uncharged polar side chains (serine, threonine, asparagine, and glutamine), amino acids with hydrophobic side chains (alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, and tryptophan), glycine, cysteine, and proline. In some embodiments, one or more positively charged amino acid residues of the AAV capsid are substituted with a hydrophobic amino acid residue. In some embodiments, the one or more amino acid substitutions comprise substitution of an arginine or lysine residue. In further embodiments, the one or more amino acid substitutions comprise substitution of an arginine or lysine residue with an alanine residue. In some embodiments, a hydrophobic amino acid residue is substituted with a positively charged amino acid residue. In further embodiments, one or more amino acid substitutions comprise substitution of an alanine residue. In yet further embodiments, one or more amino acid substitutions comprise substitution of an arginine or lysine residue with an alanine residue.

[0137] In some embodiments, the one or more amino acid substitutions comprise substitutions at positions R484, R487, K527, K532, R585, and / or R588 of VP1, VP2, and / or VP3, numbered based on VP1 of AAV2. In some embodiments, the one or more amino acid substitutions comprise substitutions at positions R484, R487, K527, K532, R585, and / or R588 of VP1, VP2, and / or VP3 of AAV2, numbered based on VP1 of AAV2. In some embodiments, the one or more amino acid substitutions comprise substitutions at positions R484, R487, K527, K532, and / or R588 of VP1, VP2, and / or VP3 of AAV2, numbered based on SEQ ID NO: 1. In some embodiments, the one or more amino acid substitutions comprise one or more of the substitutions R484A, R487A, R585A, and / or R588A in VP1, VP2, and / or VP3 of AAV2, numbered based on VP1 of AAV2. In some embodiments, the one or more amino acid substitutions comprise substitutions at positions R484, R487, K527, K532, R585, and / or R588 in VP1, VP2, and / or VP3 of AAV3, numbered based on VP1 of AAV2. In some embodiments, the one or more amino acid substitutions comprise one or more of the substitutions R484A, R487A, R585A, and / or R588A in VP1, VP2, and / or VP3 of AAV3, numbered based on VP1 of AAV2. In some embodiments, the rAAV particles comprise the rAAV capsid proteins of SEQ ID NOs: 2, 4, and / or 6.

[0138] In some embodiments, the one or more amino acid substitutions comprise substitutions at positions R485, R488, R533, A586, and / or T589 of VP1, VP2, and / or VP3, numbered based on VP1 of AAVrh8R. In some embodiments, the one or more amino acid substitutions comprise substitutions at positions R485, R488, R533, A586, and / or T589 of VP1, VP2, and / or VP3, numbered based on VP1 of AAVrh8R, numbered based on SEQ ID NO:9. In some embodiments, the one or more amino acid substitutions comprise one or more of the substitutions R533A and / or A586R in VP1, VP2, and / or VP3 of AAV2, numbered based on VP1 of AAVrh8R. In some embodiments, the rAAV particles comprise the rAAV capsid proteins of SEQ ID NOs: 10 and / or 11.

[0139] In some embodiments, the AAV capsid comprises one or more amino acid substitutions at one or more positions that interact with HSPG. In some embodiments, the AAV capsid comprises one or more amino acid substitutions at one or more positions that reduce or eliminate binding to HSPG. In some embodiments, the AAV capsid comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions that reduce or eliminate binding to HSPG. In some embodiments, the AAV capsid has 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions that reduce or eliminate binding to HSPG. In some embodiments, the AAV capsid comprises substitutions at positions R484 and R487, numbered based on VP1 of rAAV2. In some embodiments, the AAV capsid has substitutions at positions R484 and R487, numbered based on VP1 of rAAV2. In some embodiments, the AAV capsid comprises substitutions at positions R585 and R588, numbered based on VP1 of rAAV2. In some embodiments, the AAV capsid has substitutions at positions R585 and R588, numbered based on VP1 of rAAV2. In some embodiments, the AAV capsid comprises substitutions R484A and R487A, numbered based on VP1 of rAAV2. In some embodiments, the AAV capsid comprises substitutions R484A and R487A, numbered based on VP1 of rAAV2. In some embodiments, the AAV capsid comprises substitutions R585A and R588A, numbered based on VP1 of rAAV2. In some embodiments, the AAV capsid has substitutions R585A and R588A, numbered based on VP1 of rAAV2.

[0140] Heparan sulfate proteoglycans (HSPGs) are known to be expressed in many tissues throughout the body and to play important roles in the extracellular matrix, cell adhesion, and cell signaling. In some embodiments, heparan sulfate proteoglycans are expressed on one or more cells of the CNS. In certain embodiments, the one or more cells of the CNS are neurons.

[0141] In some embodiments, the present invention provides rAAV particles for CNS delivery of therapeutic nucleic acids, comprising one or more amino acid substitutions in a capsid protein that reduce or eliminate binding of the rAAV particle to heparan sulfate proteoglycans. In some embodiments, the one or more amino acid substitutions are at positions 347, 350, 390, 395, 448, 451, 484, 487, 527, 532, 585, and / or 588, numbered based on AAV2 VP1. As used herein, "numbering based on AAV2 VP1" refers to the recited amino acid in the capsid protein that corresponds to the recited amino acid in AAV2 VP1. For example, if the one or more amino acid substitutions are at positions 347, 350, 390, 395, 448, 451, 484, 487, 527, 532, 585, and / or 588, numbered based on VP1 of AAV2, then the one or more amino acid substitutions are at amino acids of the recited capsid protein corresponding to amino acids 347, 350, 390, 395, 448, 451, 484, 487, 527, 532, 585, and / or 588 of VP1 of AAV2. In some embodiments, the one or more amino acid substitutions are at positions R347, R350, K390, K395, R448, R451, R484, R487, K527, K532, R585, and / or R588, numbered based on VP1 of AAV2. In some embodiments, the one or more amino acid substitutions are at AAV2 position 484, 487, 532, 585, or 588. In some embodiments, the one or more amino acid substitutions are at AAV2 VP1, AAV2 VP2, and / or AAV2 VP3 position 484, 487, 532, 585, or 588, numbered based on AAV2 VP1. In some embodiments, VP1 of AAV2 (e.g., rAAV2) comprises the amino acid sequence of SEQ ID NO:1.

[0142] In some embodiments, the AAV capsid comprises substitutions at positions R484, R487, K527, K532, R585, and / or R588, numbered based on VP1 of rAAV2. In some embodiments, the rAAV particles of the invention comprise the capsid proteins of SEQ ID NOs: 2, 4, and / or 6. In some embodiments, the AAV capsid comprises substitutions at positions R484 and R487, or R585 and R588, numbered based on VP1 of rAAV2. In some embodiments, the AAV capsid comprises R484A and R487A substitutions or R585A and R588A substitutions, numbered based on VP1 of AAV2. In some embodiments, the AAV capsid comprises the amino acid substitutions R585A and R588A, numbered based on VP1 of AAV2. In some embodiments, the AAV capsid comprises the amino acid substitution K532A, numbered based on VP1 of AAV2. In some embodiments, the one or more amino acid substitutions comprise R347A, R350A, K390A, K395A, R448A, R451A, R484A, R487A, K527A, K532A, R585A, and / or R588A substitutions, numbered based on VP1 of AAV2.

[0143] In some embodiments, the present invention provides rAAV particles for CNS delivery of therapeutic nucleic acids, comprising one or more amino acid substitutions in a capsid protein that reduce or eliminate binding of the rAAV particles to heparan sulfate proteoglycans. In some embodiments, the rAAV particles of the present invention comprise an AAV serotype rh8R (AAVrh8R) capsid, such as that described in U.S. Pre-Issue Application Publication No. 20090317417. In some embodiments, the one or more amino acid substitutions are substitutions of amino acid residues in any one of VP1, VP2, and / or VP3 of AAVrh8R, and the amino acid substitutions alter the interaction of the rAAV particle with HSPG (e.g., reduce or eliminate binding to HSPG). In some embodiments, the one or more amino acid substitutions are substitutions of amino acid residues in VP1 AAVrh8R. In some embodiments, the one or more amino acid substitutions are substitutions of amino acid residues in VP2 AAVrh8R. In some embodiments, the one or more amino acid substitutions are substitutions of amino acid residues in VP3 AAVrh8R. In some embodiments, the one or more amino acid substitutions are substitutions of amino acid residues in a combination of VP1, VP2, and VP3 of AAVrh8R. In some embodiments, the one or more amino acid substitutions are substitutions of amino acid residues in any one of VP1, VP2, and / or VP3 of AAVrh8R. In some embodiments, the one or more amino acid substitutions are substitutions of amino acid residues in the capsid protein exemplified by SEQ ID NO:9. In some embodiments, the rAAV particles of the invention comprise capsid proteins of SEQ ID NOs:10 and / or 11.

[0144] In some embodiments, the rAAV particles comprise an AAV serotype rh8R (AAVrh8R) capsid. In some embodiments, the one or more amino acid substitutions are at positions 586 and / or 589, numbered based on the VP1 numbering of AAVrh8R. In some embodiments, the numbering is based on VP1 of AAVrh8R comprising the amino acid sequence of SEQ ID NO:9. In some embodiments, the one or more amino acid substitutions comprise substitutions at positions A586 and / or T589, numbered based on the VP1 numbering of AAVrh8R. In some embodiments, the one or more amino acid substitutions comprise an A586R substitution, numbered based on VP1 of AAVrh8R. In some embodiments, the one or more amino acid substitutions comprise a T589R or T589K substitution, numbered based on VP1 of AAVrh8R.

[0145] As discussed above, without wishing to be bound by theory, it is believed that one or more amino acid substitutions at residues corresponding to amino acids 484, 487, 532, 585, or 588, numbered according to the VP1 numbering of AAV2, may modify the transduction properties of AAV capsid types that do not bind HSPGs, or may modify the transduction properties of AAV capsid types independently of their ability to bind HSPGs. In some embodiments, the one or more amino acid substitutions include one or more amino acids corresponding to the amino acids shown in Figure 20. For example, in some embodiments, one or more amino acids at positions corresponding to amino acids 585 and / or 588 (numbered according to the VP1 numbering of AAV2) are replaced with arginine residues. or T589 for AAV1 or AAV6; S586 and / or A589 for AAV9; A586 and / or T589 for AAVrh8R; Q588 and / or T591 for AAV8; and Q588 and / or A591 for AAVrh10. These modified capsids can be used, inter alia, to improve intravitreal transduction targeted to the retina. In other embodiments, one or more amino acids (e.g., arginine or lysine) at positions corresponding to amino acids 484, 487, 527, and / or 532 (numbering based on VP1 of AAV2) are replaced with an amino acid that does not bear a positive charge, such as an alanine (e.g., R485, R488, K528, and / or K533 for AAV1 or AAV6; R485, R488, K528, and / or R533 for AAV9 or AAVrh8R; and R487, R490, K530, and / or R535 for AAV8 or AAVrh10). These modified capsids can be used, inter alia, for improved subretinal or CNS transduction.

[0146] In some embodiments, the rAAV particles comprise an AAV serotype 1 (AAV1) capsid. In some embodiments, the one or more amino acid substitutions are at positions 586 and / or 589, numbered based on the VP1 numbering of AAV1. In some embodiments, the VP1 of AAV1 comprises the amino acid sequence of SEQ ID NO: 12. In some embodiments, the one or more amino acid substitutions include substitutions at positions S586 and / or T589, numbered based on the VP1 numbering of AAV1. In some embodiments, the one or more amino acid substitutions include an S586R or S586K substitution, numbered based on the VP1 of AAV1. In some embodiments, the one or more amino acid substitutions include a T589R substitution, numbered based on the VP1 of AAV1. In some embodiments, the rAAV particles comprise an AAV serotype 6 (AAV6) capsid. In some embodiments, the one or more amino acid substitutions are at positions 586 and / or 589, numbered based on the VP1 numbering of AAV6. In some embodiments, the numbering is based on AAV6 VP1 comprising the amino acid sequence of SEQ ID NO: 13. In some embodiments, the one or more amino acid substitutions include substitutions at positions S586 and / or T589, numbered based on the VP1 numbering of AAV6. In some embodiments, the one or more amino acid substitutions include an S586R or S586K substitution, numbered based on the VP1 numbering of AAV6. In some embodiments, the one or more amino acid substitutions include a T589R substitution, numbered based on the VP1 numbering of AAV6. In some embodiments, the rAAV particles comprise an AAV serotype 8 (AAV8) capsid. In some embodiments, the one or more amino acid substitutions are at positions 588 and / or 591, numbered based on the VP1 numbering of AAV8. In some embodiments, the numbering is based on VP1 of AAV8, which comprises the amino acid sequence of SEQ ID NO:14.In some embodiments, the one or more amino acid substitutions comprise substitutions at positions Q588 and / or T591, numbered based on the VP1 numbering of AAV8. In some embodiments, the one or more amino acid substitutions comprise a Q588R or Q588K substitution, numbered based on the VP1 numbering of AAV8. In some embodiments, the one or more amino acid substitutions comprise a T591R substitution, numbered based on the VP1 numbering of AAV8. In some embodiments, the rAAV particles comprise an AAV serotype 9 (AAV9) capsid. In some embodiments, the one or more amino acid substitutions are at positions 586 and / or 589, numbered based on the VP1 numbering of AAV9. In some embodiments, the numbering is based on the VP1 of AAV9 comprising the amino acid sequence of SEQ ID NO: 15. In some embodiments, the one or more amino acid substitutions are based on the VP1 numbering of AAV9. In some embodiments, the rAAV particles comprise an AAV serotype rh10 (AAVrhlO) capsid. In some embodiments, the one or more amino acid substitutions are at positions 588 and / or 591, numbered based on the VP1 numbering of AAVrhlO. In some embodiments, the numbering is based on the VP1 of AAVrhlO, which comprises the amino acid sequence of SEQ ID NO: 16. In some embodiments, the one or more amino acid substitutions are at positions Q588 and / or A591, numbered based on the VP1 numbering of AAVrhlO. In some embodiments, the one or more amino acid substitutions comprise a Q588R or Q588K substitution, numbered based on VP1 of AAVrhlO. In some embodiments, the one or more amino acid substitutions comprise an A591R substitution, numbered based on VP1 of AAVrhlO.

[0147] IV. Treatment Methods Gene therapy protocols for retinal diseases, such as LCA, retinitis pigmentosa, and age-related macular degeneration, require localized delivery of vectors to retinal cells. The target cells for treatment in these diseases are photoreceptor cells of the retina or RPE cells beneath the neurosensory retina. Delivery of gene therapy vectors to these cells requires injection into the subretinal space between the retina and the RPE. In some embodiments, the present invention provides methods for delivering rAAV gene therapy vectors to retinal cells, wherein the rAAV vectors are packaged in AAV capsids containing substitutions of one or more amino acid residues that interact with HSPGs.

[0148] In some embodiments, the present invention provides a method for treating a CNS disorder in an individual, comprising delivering a composition comprising rAAV particles to the CNS of the individual, the rAAV particles comprising (a) an rAAV capsid comprising an rAAV capsid protein comprising one or more amino acid substitutions at one or more positions that interact with heparan sulfate proteoglycans, and (b) an rAAV vector comprising a heterologous nucleic acid and at least one AAV terminal repeat sequence. Furthermore, the methods for delivering heterologous nucleic acids to the central nervous system (CNS) of an individual, the methods for improving rAAV transduction of cells after delivery of rAAV particles to the CNS of an individual, and the methods for improving expression of heterologous nucleic acids after delivery of rAAV particles to the CNS of an individual described herein can be used to deliver heterologous nucleic acids, such as those encoding therapeutic polypeptides or therapeutic nucleic acids. These methods can be used, inter alia, to treat CNS disorders. In some embodiments, the individual is a human.

[0149] Therapeutic Vectors The present invention provides a method for gene therapy of ocular disorders, in which rAAV particles containing a therapeutic vector are delivered to the retina of an individual. Enhanced transduction of cells of the retina can be achieved by encapsulating the rAAV vector in an rAAV capsid (e.g., a particle such as rAAV2 or rAAVrh8R) in which one or more amino acids of the capsid that interact with HSPG have been substituted to reduce or eliminate binding of the rAAV particle to HSPG. The vector may contain heterologous nucleic acid encoding a polypeptide (e.g., a therapeutic or diagnostic polypeptide) and / or a therapeutic nucleic acid. Nucleic acids encoding therapeutic or diagnostic polypeptides and / or therapeutic nucleic acids can be synthesized using standard synthesis and recombinant methods known in the art. , can be generated. In some embodiments, the heterologous nucleic acid encodes a therapeutic polypeptide. In some embodiments, the heterologous nucleic acid encodes a diagnostic polypeptide. Non-limiting examples of nucleic acids encoding therapeutic polypeptides include: nucleic acids for replacement of defective or mutated genes known to cause retinal disease, e.g., Prph2, RPE65, MERTK, RPGR, RP2, RPGRIP, CNGA3, CNGB3, and GNAT2. Other non-limiting examples of nucleic acids encoding therapeutic polypeptides include those encoding neurotrophic factors (e.g., GDNF, CNTF, FGF2, PEDF, EPO), anti-apoptotic genes (e.g., BCL2, BCL-X, NFκB), anti-angiogenic factors (e.g., endostatin, angiostatin, sFlt), and anti-inflammatory factors (e.g., IL10, IL1-ra, TGFβ, IL4). Other therapeutic polypeptides for ocular disorders include, but are not limited to, Myo7a, ABCA4, REP1, GUCY2D, PDE6C, RS1, RPGRIP, Lpcat1, AIPL1, RDH12, CHM. In some embodiments, the encoded polypeptide is a human variant of the polypeptide.

[0150] The nucleic acid of the present invention may encode a polypeptide that is an intracellular protein, a polypeptide that is anchored in the cell membrane, a polypeptide that remains intracellular, or a polypeptide that is secreted by a cell transduced with the vector of the present invention. For a polypeptide that is secreted by a cell that has received the vector, the polypeptide can be soluble (i.e., not attached to the cell). For example, a soluble polypeptide lacks a transmembrane region and is secreted from the cell. Techniques for identifying and removing nucleic acid sequences that encode transmembrane domains are known in the art.

[0151] Vectors that can be administered according to the present invention also include vectors containing nucleic acids encoding RNA (e.g., RNAi, ribozymes, miRNA, siRNA, antisense RNA), which, when transcribed from the nucleic acid of the vector, can treat ocular disorders by interfering with the translation or transcription of abnormal or excessive proteins associated with the pathology of the present invention. For example, the nucleic acids of the present invention can encode RNA that treats diseases by highly specific removal or reduction of mRNAs encoding abnormal and / or excessive proteins. Therapeutic RNA sequences include RNAi, small inhibitory RNAs (siRNAs), microRNAs (miRNAs), and / or ribozymes (e.g., hammerhead ribozymes and hairpin ribozymes), which can treat diseases by highly specific removal or reduction of mRNAs encoding abnormal and / or excessive proteins, such as those occurring in various types of inherited retinal degeneration. Non-limiting examples of ocular disorders that can be treated by therapeutic RNA sequences include, for example, autosomal dominant retinitis pigmentosa (ADRP) and diabetic retinopathy. Examples of therapeutic RNA sequences and nucleic acids encoding these sequences that can be used in the present invention include those described in, for example, U.S. Patent No. 6,225,291, the disclosure of which is incorporated herein by reference in its entirety. In some embodiments, the therapeutic RNA sequence is miR-708. In some embodiments, miR-708 is used in combination with a nucleic acid encoding wild-type rhodopsin, either as part of the same rAAV vector or as part of a second rAAV vector. In some embodiments, the nucleic acid encoding wild-type rhodopsin does not have a miR-708 target sequence located in the 3' untranslated region of the rhodopsin gene. rAAV vectors encoding miR-708 and / or rhodopsin are provided in U.S. Provisional Patent Application No. 61 / 969,027, the entire contents of which are incorporated herein by reference.

[0152] Certain aspects of the invention relate to the use of rAAV particles (e.g., therapeutic vectors) that comprise: (a) an rAAV capsid protein that comprises one or more amino acid substitutions at one or more positions that interact with heparan sulfate proteoglycans; and (b) an rAAV capsid comprising a heterologous nucleic acid and at least one AAV terminal repeat sequence. In some embodiments, the heterologous nucleic acid encodes a therapeutic polypeptide or a therapeutic nucleic acid. As used herein, a therapeutic nucleic acid may express any therapeutic nucleic acid of the present disclosure, or may express any nucleic acid encoding a therapeutic polypeptide of the present disclosure. Therapeutic nucleic acids can be used, for example, to ameliorate symptoms of, prevent or delay the progression of, and / or treat a disorder (e.g., a disorder described herein).

[0153] Enhanced transduction of cells of the CNS can be achieved by packaging an rAAV vector into an rAAV capsid (e.g., rAAV2, rAAVrh8R, etc.) in which one or more amino acids of the capsid that interact with HSPG have been substituted to reduce or eliminate binding of the rAAV particle to HSPG. The vector may contain a heterologous nucleic acid encoding a polypeptide (e.g., a therapeutic or diagnostic polypeptide) and / or a therapeutic nucleic acid. Nucleic acids encoding therapeutic or diagnostic polypeptides and / or therapeutic nucleic acids can be generated using methods known in the art using standard synthetic and recombinant methods. In some embodiments, the heterologous nucleic acid encodes a therapeutic polypeptide. In some embodiments, the heterologous nucleic acid encodes a diagnostic polypeptide. In some embodiments, the heterologous nucleic acid encodes a CNS-related gene.

[0154] In some embodiments, the heterologous nucleic acid encodes a therapeutic nucleic acid. In some embodiments, therapeutic nucleic acids can include, but are not limited to, siRNA, shRNA, RNAi, miRNA, antisense RNA, ribozymes, or DNAzymes. Thus, a therapeutic nucleic acid can encode an RNA that, when transcribed from a nucleic acid in a vector, can treat a disorder of the invention (e.g., a CNS disorder) by interfering with the translation or transcription of aberrant or excessive proteins associated with the disorder. For example, a nucleic acid of the invention can encode an RNA that treats a disorder by highly specific removal or reduction of mRNA encoding aberrant and / or excessive proteins. Therapeutic RNA sequences include RNAi, small inhibitory RNA (siRNA), microRNA (miRNA), and / or ribozymes (e.g., hammerhead ribozymes and hairpin ribozymes), which can treat a disorder by highly specific removal or reduction of mRNA encoding aberrant and / or excessive proteins.

[0155] In some embodiments, the heterologous nucleic acid encodes a therapeutic polypeptide. A therapeutic polypeptide may, for example, provide a polypeptide and / or enzymatic activity that is absent or present at reduced levels in a cell or organism. Alternatively, a therapeutic polypeptide may provide a polypeptide and / or enzymatic activity that indirectly corrects an imbalance in a cell or organism. For example, a therapeutic polypeptide for a disorder associated with the accumulation of metabolites due to a deficiency in a metabolic enzyme or activity may provide a missing metabolic enzyme or activity, or may provide an alternative metabolic enzyme or activity that results in a reduction of the metabolite. A therapeutic polypeptide may also be used to reduce the activity of a polypeptide (e.g., one that is overexpressed, activated by a gain-of-function mutation, or whose activity is otherwise misregulated), for example, by acting as a dominant-negative polypeptide.

[0156] In some embodiments, the heterologous nucleic acid encodes a polypeptide selected from an enzyme, a neurotrophic factor, a polypeptide deficient or mutated in an individual with a CNS-related disorder, an antioxidant, an anti-apoptotic factor, an anti-angiogenic factor, and an anti-inflammatory factor. Such polypeptides can be used, for example, to provide polypeptides and / or enzyme activities that are reduced, absent, or misregulated during a CNS disorder, to treat a CNS disorder, or to treat a CNS disorder. CNS disorders can be treated by ameliorating the cause and / or symptoms and / or reducing damage to the CNS (e.g., apoptosis, inflammation, or other types of cell death) resulting from the CNS disorder. Non-limiting examples of nucleic acids encoding therapeutic polypeptides include nucleic acids for replacement of defective or mutated genes known to cause CNS disorders, such as Prph2, RPE65, MERTK, RPGR, RP2, RPGRIP, CNGA3, CNGB3, and GNAT2. Other non-limiting examples of nucleic acids encoding therapeutic polypeptides include those encoding neurotrophic factors (e.g., GDNF, CNTF, FGF2, PEDF, EPO), anti-apoptotic genes (e.g., BCL2, BCL-X, NFκB), anti-angiogenic factors (e.g., endostatin, angiostatin, sFlt), and anti-inflammatory factors (e.g., IL10, IL1-ra, TGFβ, IL4). Other therapeutic polypeptides for CNS disorders include, but are not limited to, Myo7a, ABCA4, REP1, GUCY2D, PDE6C, RS1, RPGRIP, Lpcat1, AIPL1, RDH12, and CHM. In some embodiments, the encoded polypeptide is a human variant of a polypeptide. In some embodiments, the heterologous nucleic acid encodes neuronal apoptosis inhibitory protein (NAIP), nerve growth factor (NGF), glial cell line-derived growth factor (GDNF), brain-derived growth factor (BDNF), ciliary neurotrophic factor (CNTF), tyrosine hydroxylase (TH), GTP-cyclohydrolase (GTPCH), amino acid decarboxylase (AADC), antioxidant, anti-angiogenic polypeptide, anti-inflammatory polypeptide, and / or aspartoacylase (ASPA). Examples of antioxidants include, but are not limited to, SOD1; SOD2; catalase; sirtuin 1, 3, 4, or 5; NRF2; PGC1a; GCL (catalytic subunit); GCL (modifier subunit); adiponectin; glutathione peroxidase 1; and neuroglobin.Examples of anti-angiogenic polypeptides include, but are not limited to, angiostatin, endostatin, PEDF, soluble VEGF receptors, and soluble PDGF receptors. Examples of anti-inflammatory polypeptides include, but are not limited to, IL-10, soluble IL17R, soluble TNF-R, TNF-R-Ig, IL-1 inhibitors, and IL18 inhibitors. Other exemplary polypeptides of these classes that can be used to treat CNS disorders are provided below.

[0157] The nucleic acid of the present invention may encode a polypeptide that is an intracellular protein, a polypeptide that is anchored in the cell membrane, a polypeptide that remains intracellular, or a polypeptide that is secreted by a cell transduced with the vector of the present invention. For a polypeptide that is secreted by a cell that has received the vector, the polypeptide can be soluble (i.e., not attached to the cell). For example, a soluble polypeptide lacks a transmembrane region and is secreted from the cell. Techniques for identifying and removing nucleic acid sequences that encode transmembrane domains are known in the art.

[0158] In some embodiments, the heterologous nucleic acid is operably linked to a promoter. Exemplary promoters include the cytomegalovirus (CMV) immediate early promoter, RSV LTR, MoMLV LTR, phosphoglycerate kinase-1 (PGK) promoter, simian virus 40 (SV40) promoter and CK6 promoter, transthyretin promoter (TTR), TK promoter, tetracycline-responsive promoter (TRE), HBV promoter, hAAT promoter, LSP promoter, chimeric liver-specific promoter (LSP), E2F promoter, telomerase (hTERT) promoter; the cytomegalovirus enhancer / chicken β-actin / rabbit β-globin promoter (CAG promoter; Niwa et al., Gene, 1991, 108(2):193-9), and the elongation factor 1-α promoter (EF1-α) promoter (Kim et al., Gene, 1990, 91(2):217-23 and Guo et al., Gene Ther., 1996, 3(9):802-10). Examples include, but are not limited to, the promoter. In some embodiments, the promoter comprises a cytomegalovirus enhancer linked to a human β-glucuronidase promoter or a chicken β-actin (CBA) promoter. The promoter can be a constitutive, inducible, or repressible promoter. In some embodiments, the present invention provides a recombinant vector comprising a nucleic acid encoding a heterologous transgene of the present disclosure operably linked to a CBA promoter. Exemplary promoters and descriptions can be found, for example, in U.S. Pre-Issue Application Publication No. 20140335054.

[0159] Examples of constitutive promoters include, but are not limited to, the retroviral Rous sarcoma virus (RSV) LTR promoter (optionally with the RSV enhancer), the cytomegalovirus (CMV) promoter (optionally with the CMV enhancer) [see, e.g., Boshart et al., Cell, 41:521-530 (1985)], the SV40 promoter, the dihydrofolate reductase promoter, the 13-actin promoter, the phosphoglycerol kinase (PGK) promoter, and the EF1a promoter [Invitrogen].

[0160] Inducible promoters allow for the regulation of gene expression and are regulated by exogenously supplied compounds, by environmental factors such as temperature, or by specific physiological conditions, e.g., acute phase, the presence of a particular differentiation state of cells, or only in replicating cells. Inducible promoters and inducible systems are available from a variety of commercial sources, including, but not limited to, Invitrogen, Clontech, and Ariad. Many other systems have been described, and those skilled in the art can readily select such systems. Examples of inducible promoters regulated by exogenously supplied promoters include the zinc-inducible sheep metallothionein (MT) promoter, the dexamethasone (Dex)-inducible mouse mammary tumor virus (MMTV) promoter, the T7 polymerase promoter system (WO 98 / 10088); the ecdysone insect promoter (No et al., Proc. Natl. Acad. Sci. USA, 93:3346-3351 (1996)), the tetracycline-repressible system (Gosse et al., J. Immunol. 2004, 10:101-102). n et al., Proc. Natl. Acad. Sci. USA, 89:5547-5551 (1992)), tetracycline-inducible system (Gossen et al., Science, 268:1766-1769 (1995); see also Harvey et al., Curr. Opin. Chem. Biol., 2:512-518 (1998)), RU486-inducible system (Wang et al., Nat. Biotech., 15:239-243 (1997) and Wang et al., Gene Ther., 4:432-441 (1997)), and rapamycin-inducible system (Magari et al., J. Clin. Invest., 100:2865-2872 (1997)). Still other types of inducible promoters that may be useful in this regard are those that are regulated by specific physiological conditions, such as temperature, acute phase, a particular differentiation state of the cell, or only in replicating cells.

[0161] In another embodiment, the native promoter of the transgene or a fragment thereof will be used. When it is desired that the expression of the transgene mimics native expression, the native promoter can be used. The native promoter may also be used when the expression of the transgene must be regulated temporally or developmentally, or in a tissue-specific manner, or in response to a specific transcriptional stimulus. In a further embodiment, other native expression control elements, such as enhancer elements, polyadenylation sites, or Kozak consensus sequences, may also be used to mimic native expression.

[0162] In some embodiments, the regulatory sequence confers tissue-specific gene expression capability. In some cases, the tissue-specific regulatory sequence binds to tissue-specific transcription factors that induce transcription in a tissue-specific manner. Such tissue-specific regulatory sequences (e.g., promoters, enhancers, etc.) can be used to express a gene in a tissue-specific manner. and the like) are well known in the art. Exemplary tissue-specific regulatory sequences include, but are not limited to, neuronal promoters, such as the neuron-specific enolase (NSE) promoter (Andersen et al., Cell. Mol. Neurobiol., 13:503-15 (1993)), the neurofilament light chain gene promoter (Piccioli et al., Proc. Natl. Acad. Sci. USA, 88:5611-5 (1991)), and the neuron-specific vgf gene promoter (Piccioli et al., Neuron, 15:373-84 (1995)). In some embodiments, the tissue-specific promoter is a promoter of a gene selected from the following: neuronal nucleus (NeuN), glial fibrillary acidic protein (GFAP), adenomatous polyposis coli (APC), and ionized calcium-binding adaptor molecule 1 (Iba-1). Other suitable tissue-specific promoters will be apparent to those skilled in the art. In some embodiments, the promoter is a chicken β-actin promoter.

[0163] In some embodiments, the heterologous nucleic acid is under the control of a promoter sequence that is expressed in one or more cells of the CNS. Many of the promoter sequences listed above (e.g., the CBA promoter) are known in the art to be expressed in one or more cells of the CNS. In some embodiments, the promoter sequence is ubiquitously expressed in an organism and can therefore be expressed in one or more cells of the CNS upon its delivery to the CNS. In other embodiments, a promoter sequence that is specifically expressed in the CNS or in one or more subsets of CNS cells may be used. In some embodiments, the heterologous nucleic acid is operably linked to a promoter suitable for expression of a therapeutic polypeptide or therapeutic nucleic acid in one or more cells of the CNS. Thus, in some embodiments, the therapeutic polypeptides or therapeutic nucleic acids of the present invention can be used to treat CNS disorders.

[0164] In some embodiments, the promoter expresses the heterologous nucleic acid in brain cells. Brain cells may refer to any brain cell known in the art, including, but not limited to, neurons (e.g., sensory neurons, motor neurons, interneurons, dopaminergic neurons, medium spiny neurons, cholinergic neurons, GABAergic neurons, pyramidal neurons, etc.), glial cells (e.g., microglia, macroglia, astrocytes, oligodendrocytes, ependymal cells, radial glia, etc.), brain parenchymal cells, microglial cells, ependymal cells, and / or Purkinje cells. In some embodiments, the promoter expresses the heterologous nucleic acid in neurons. In some embodiments, the heterologous nucleic acid is exclusively expressed in neurons (e.g., expressed in neurons and not expressed in other cells of the CNS, such as glial cells).

[0165] In some embodiments, the present invention provides rAAV vectors for use in methods for preventing or treating one or more genetic defects (e.g., inherited genetic defects, somatic genetic mutations, etc.) in a mammal, such as genetic defects that result in a polypeptide deficiency or excess in the subject, or for treating a deficiency or reducing the severity or extent of a deficiency in a subject manifesting a CNS-related disorder associated with a deficiency of such a polypeptide in cells and tissues. In some embodiments, the methods comprise administering to a subject an rAAV vector encoding one or more therapeutic peptides, polypeptides, functional RNAs, inhibitory nucleic acids, shRNAs, microRNAs, antisense nucleotides, etc., in a pharmaceutically acceptable carrier, in an amount and for a period of time sufficient to treat the CNS-related disorder in the subject having or suspected of having the CNS-related disorder.

[0166] The rAAV vector may contain a nucleic acid encoding a protein or functional RNA that regulates or treats a CNS-related disorder as a transgene. The following is a non-limiting list of genes associated with CNS-related disorders: neuronal apoptosis inhibitory protein (NAIP), nerve growth factor (NGF), glial cell line-derived growth factor (GDNF), brain-derived growth factor (BDNF), ciliary neurotrophic factor (CNTF), tyrosine hydroxylase (TH), GTP-cyclohydrolase (GTPCH), aspartoacylase (ASPA), superoxide dismutase (SOD1), antioxidants, anti-angiogenic polypeptides, anti-inflammatory polypeptides, and amino acid decarboxylase (AADC). For example, a transgene useful for treating Parkinson's disease encodes TH, the rate-limiting enzyme in dopamine synthesis. A transgene encoding GTPCII, which produces the TII cofactor tetrahydrobiopterin, may also be used to treat Parkinson's disease. Transgenes encoding GDNF or BDNF, or AADC, which promotes the conversion of L-dopa to DA, may also be used to treat Parkinson's disease. Transgenes useful for treating ALS may encode GDNF, BDNF, or CNTF. Transgenes useful for treating ALS may also encode functional RNAs, such as shRNAs and miRNAs, that inhibit SOD1 expression. Transgenes useful for treating ischemia may encode NAIP or NGF. Transgenes encoding β-glucuronidase (GUS) may be useful for treating certain lysosomal storage diseases (e.g., mucopolysaccharidosis type VII (MPS VII)). Transgenes encoding prodrug-activating genes, such as HSV-thymidine kinase, which converts ganciclovir into toxic nucleotides that disrupt DNA synthesis and cause cell death, may be useful for treating certain cancers, for example, when administered in combination with the prodrug. Transgenes encoding endogenous opioids, such as β-endorphin, may be useful for treating pain.Examples of antioxidants include, but are not limited to, SOD1; SOD2; catalase; sirtuin 1, 3, 4, or 5; NRF2; PGC1a; GCL (catalytic subunit); GCL (modifier subunit); adiponectin; glutathione peroxidase 1; and neuroglobin. Examples of anti-angiogenic polypeptides include, but are not limited to, angiostatin, endostatin, PEDF, soluble VEGF receptor, and soluble PDGF receptor. Examples of anti-inflammatory polypeptides include, but are not limited to, IL-10, soluble IL17R, soluble TNF-R, TNF-R-Ig, IL-1 inhibitor, and IL18 inhibitor. Other examples of transgenes that can be used in the rAAV vectors of the present invention will be apparent to those skilled in the art (see, for example, Costantini LC et al., Gene Therapy (2000) 7, 93-109).

[0167] In some embodiments, therapeutic polypeptides or therapeutic nucleic acids are used to treat CNS disorders. Without wishing to be bound by theory, it is believed that therapeutic polypeptides or therapeutic nucleic acids can be used to reduce or eliminate the expression and / or activity of a polypeptide whose gain of function is associated with a disorder, or to enhance the expression and / or activity of a polypeptide to complement a deficiency associated with a disorder (e.g., a mutation in a gene whose expression exhibits a similar or related activity). Non-limiting examples of inventive disorders that can be treated by the therapeutic polypeptides or therapeutic nucleic acids of the invention (exemplary genes targeted or delivered are provided in parentheses for each disorder) include stroke (e.g., caspase-3, Beclin 1, Ask 1, PAR 1, HIF 1 alpha, PUMA, and / or any of the genes described in Fukuda, AM and Badaut, J. (2013) Genes (Basel) 4:435-456), Huntington's disease (mutant HTT), epilepsy (e.g., SCN 1A, NMDAR, ADK, and / or any of the genes described in Boison, D. (2010) Epilepsia 51:1659-1668), Parkinson's disease (alpha-synuclein), Lou Gehrig's disease (also known as amyotrophic lateral sclerosis; SOD 1), Alzheimer's disease (tau, amyloid precursor protein), corticobasal leukemia (ALD) and leukemia (ALD). Corticobasal ganglionic degeneration or CBD (tau), corticobasal ganglionic degeneration or CBGD (tau), frontotemporal dementia or FTD (tau), progressive supranuclear palsy or PSP (tau), multiple system atrophy or MSA (alpha-synuclein), brain cancer (e.g., mutated or overexpressed oncogenes involved in brain cancer), and lysosomal storage diseases (LSD). Disorders of the present invention may involve large areas of the cortex, such as more than one functional area of ​​the cortex, more than one lobe of the cortex, and / or the entire cortex. Other non-limiting examples of disorders of the present invention that can be treated with therapeutic polypeptides or therapeutic nucleic acids of the present invention include traumatic brain injury, enzyme dysfunction disorders, psychiatric disorders (including post-traumatic stress syndrome), neurodegenerative diseases, and cognitive disorders (including dementia, autism, and depression). Enzyme dysfunction disorders include, but are not limited to, leukodystrophies (including Canavan disease) and any of the lysosomal storage diseases described below.

[0168] In some embodiments, therapeutic polypeptide or therapeutic nucleic acid is used to treat lysosomal storage disease.As is generally known in the art, lysosomal storage disease is a rare inherited metabolic disorder characterized by the defect of lysosomal function.Such disorders are often caused by the defect of the enzymes required for proper mucopolysaccharide, glycoprotein and / or lipid metabolism, which leads to the pathological accumulation of cellular material stored in lysosome. Non-limiting examples of lysosomal storage diseases of the invention that can be treated by a therapeutic polypeptide or therapeutic nucleic acid of the invention (exemplary genes targeted or delivered are provided in parentheses for each disorder) include Gaucher disease type 2 or 3 (acid β-glucosidase, GBA), GM1 gangliosidosis (β-galactosidase-1, GLB1), Hunter disease (iduronate 2-sulfatase, IDS), Krabbe disease (galactosylceramidase, GALC), mannosidosis (mannosidases, e.g., α-D-mannosidase, MAN2B1), β-mannosidosis (β-mannosidase, MANBA), metachromatic leukodystrophy (pseudoarylsulfatase A, ARSA), mucolipidosis type II / III (N-acetylglucosamine-1-phosphotransferase, GNPTAB), Niemann-Pick disease type A (acid sphingomyelinase, SNPTAB), and leukemia type B (acid sphingomyelinase, SNPTAB). Niemann-Pick disease type C (Niemann-Pick C protein, NPC1), Pompe disease (acid α-1,4-glucosidase, GAA), Sandhoff disease (hexosaminidase β subunit, HEXB), Sanfilippo disease type A (N-sulfoglucosamine sulfohydrolase, MPS3A), Sanfilippo disease type B (N-α-acetylglucosaminidase, NAGLU), Sanfilippo disease type C (heparin acetyl-C These include oA:α-glucosaminide N-acetyltransferase, MPS3C), Sanfilippo disease type D (N-acetylglucosamine-6-sulfatase, GNS), Schindler disease (α-N-acetylgalactosaminidase, NAGA), Sly disease (β-glucuronidase, GUSB), Tay-Sachs disease (hexosaminidase α subunit, HEXA), and Wolman disease (lysosomal acid lipase, LIPA).

[0169] Additional lysosomal storage diseases, as well as the defective enzymes associated with each disease, are listed below in Table 1. In some embodiments, the diseases listed in the table below are treated with therapeutic peptides or therapeutic nucleic acids of the invention that complement or otherwise compensate for the corresponding enzyme deficiency.

[0170] [Table 1]

[0171] [Table 2]

[0172] Huntington's disease One example of a disease in which the above-described vectors would be advantageous is Huntington's disease (HD), which is caused by a CAG repeat expansion mutation encoding an expanded polyglutamine (polyQ) repeat in mutant huntingtin protein (mHTT). HD is an autosomal dominant disease resulting from a single allele mutation, making it an attractive target for DNA- and RNA-based therapies. AAV vectors could provide an ideal delivery system for nucleic acid therapy, enabling long-term, continuous expression of these huntingtin-reducing molecules in the brain.

[0173] As described herein, intracranial administration of rAAV particles (e.g., therapeutic vectors) having rAAV capsid proteins containing one or more amino acid substitutions at one or more positions that interact with heparan sulfate proteoglycans results in widespread neuronal transduction. Accordingly, aspects of the present invention provide methods for delivering heterologous nucleic acids to the central nervous system using the recombinant viral particles described herein for the treatment of Huntington's disease. In some embodiments, the present invention provides methods and compositions for treating Huntington's disease in an individual, comprising administering to the mammal a pharmaceutical composition of the present disclosure (e.g., a pharmaceutical composition comprising a mutant viral particle of the present disclosure). In some aspects, the present invention provides methods and compositions for inhibiting the expression of htt in a mammal with Huntington's disease, comprising administering to the individual a pharmaceutical composition of the present disclosure (e.g., a pharmaceutical composition comprising a mutant viral particle of the present disclosure). In some embodiments, the present invention provides methods and compositions for inhibiting the accumulation of htt in cells of an individual with Huntington's disease, the methods and compositions comprising administering to the individual a pharmaceutical composition of the present disclosure (e.g., a pharmaceutical composition comprising a mutant viral particle of the present disclosure).

[0174] In some embodiments, the present invention provides methods and compositions for ameliorating symptoms of HD in an individual, comprising administering to the individual an effective amount of a recombinant viral particle comprising one or more amino acid substitutions at one or more positions that interact with heparan sulfate proteoglycans. The methods and compositions provide a method for treating HD, comprising administering to S, wherein the rAAV particles comprise a vector encoding an RNAi that inhibits the expression and / or accumulation of HTT in the individual. In some embodiments, symptoms of HD include, but are not limited to, chorea, rigidity, uncontrollable body movements, inability to control muscles, impaired coordination, restlessness, slow eye movements, abnormal posture, unsteadiness, ataxic gait, abnormal facial expressions, speech disorders, difficulty chewing and / or swallowing, sleep disorders, seizures, dementia, agnosia (e.g., impaired abilities related to planning, abstract thinking, flexibility, rule learning, interpersonal sensitivity, self-control, attention, learning, and memory), depression, anxiety, personality changes, aggression, compulsive behavior, obsessive-compulsive behavior, hypersexuality, psychosis, blunted affect, irritability, suicidal ideation, weight loss, muscle atrophy, heart failure, impaired glucose tolerance, testicular atrophy, and osteoporosis.

[0175] In some embodiments, the present invention provides a method for preventing or delaying the progression of HD. Autosomal dominant HD is a genetic disease that can be genotyped. For example, the number of CAG repeats of HTT can be determined by PCR-based repeat size measurement. This type of diagnosis can be made at any stage of life by direct testing of young people or adults (e.g., with the presentation of clinical symptoms), by parent screening or parent exclusion testing (e.g., by chorionic villus sampling or amniocentesis), or by preimplantation screening of fetuses. In addition, HD can be diagnosed by brain imaging to look for atrophy of the caudate nucleus and / or putamen and / or ventricular hypertrophy. These symptoms, combined with a family history of HD and / or clinical symptoms, will indicate HD.

[0176] The means for assessing the improvement of HD symptoms are known in the art. For example, the Unified Huntington's Disease Rating Scale (UHDRS) can be used to assess motor function, cognitive function, behavioral abnormalities, and functional ability (see, for example, Huntington Study Group (1996) Movement Disorders 11:136-42). This rating scale incorporates elements from tests such as the HD Movement and Daily Living Scale, Marsden and Quinn Chorea Severity Scale, Disability and Independence Scale, HD Motor Rating Scale (HDMRS), HD Functional Capacity Scale (HDFCS), and Quantitative Neurological Examination (QNE) to provide a unified and comprehensive test for multiple aspects of disease pathology. Other tests useful for assessing the improvement of HD symptoms include, but are not limited to, the Montreal Cognitive Assessment, brain imaging (e.g., MRI), category fluency test, trail making test, map search, Stroop word reading test, accelerated tapping task, and symbol-digit modality test.

[0177] In some embodiments of the present invention, the methods and compositions are used to treat people with HD.As described above, HD is inherited in an autosomal dominant manner and is caused by a CAG repeat expansion in the HTT gene.Juvenile-onset HD is most often inherited from the paternal side.Huntington's disease-like phenotypes have also been shown to be correlated with other genetic loci, such as HDL1, PRNP, HDL2, HDL3, and HDL4.Other genetic loci, including mutations in the GRIN2A, GRIN2B, MSX1, GRIK2, and APOE genes, may also alter the manifestation of HD symptoms.

[0178] rAAV composition In some embodiments, the present invention provides compositions comprising any of the rAAV particles described herein. Generally, compositions for use in the methods and systems of the present invention comprise an effective amount of rAAV particles, optionally in a pharmaceutically acceptable excipient, comprising a rAAV vector encoding a polypeptide and / or RNA. These viral particles comprise an AAV capsid (e.g., an AAV2 or AAVrh8R capsid) in which one or more amino acids that interact with HSPG have been substituted to reduce or eliminate rAAV particle binding to HSPG. As is well known in the art, pharmaceutically acceptable carriers can be used to deliver rAAV particles to a host cell. Acceptable excipients are relatively inert substances that facilitate the administration of pharmacologically active substances and can be supplied as solutions or suspensions, as emulsions, or as solid forms suitable for dissolving or suspending in liquid before use. For example, excipients can provide shape or consistency or act as diluents. Suitable excipients include, but are not limited to, stabilizers, wetting and emulsifying agents, salts for varying osmolality, encapsulating agents, pH buffering substances and buffers. Such excipients include any pharmaceutical suitable for direct delivery to the eye that can be administered without undue toxicity. Pharmaceutically acceptable excipients include, but are not limited to, sorbitol, any of various TWEEN compounds, and liquids such as water, saline, glycerol, and ethanol. Pharmaceutically acceptable salts can be included therein, such as inorganic acid salts, such as hydrochlorides, hydrobromides, phosphates, sulfates, and the like; and salts of organic acids, such as acetates, propionates, malonates, benzoates, and the like. A thorough discussion of pharmaceutically acceptable excipients is available in REMINGTON'S PHARMACEUTICAL SCIENCES (Mack Pub. Co., NJ 1991).

[0179] Generally, these compositions are formulated for administration by subretinal injection.Therefore, these compositions can be combined with a pharmaceutically acceptable vehicle, such as saline, Ringer's balanced salt solution (pH 7.4), etc.Although not required, in some cases, the compositions can be provided in a unit dosage form suitable for administration of a precise amount.

[0180] Subretinal delivery method of rAAV Subretinal delivery methods are known in the art.See, for example, WO2009 / 105690, which is incorporated herein by reference.In brief, the general method for delivering rAAV particles (such as particles such as rAAV2, rAAVrh8R) to the subretinal area of ​​the macula and fovea can be described by the following general outline.This example is intended only to illustrate certain features of the method and is not intended to be limiting in any way.

[0181] Generally, rAAV vectors can be delivered in the form of a composition that is injected intraocularly (subretinally) under direct observation using a surgical microscope. In some embodiments, the vector is encapsulated in the capsid of an rAAV particle, which comprises an rAAV capsid containing an rAAV capsid protein containing one or more amino acid substitutions at one or more positions that interact with heparan sulfate proteoglycans (e.g., inhibit or reduce HSPG binding), and an rAAV vector containing a heterologous nucleic acid and at least one AAV inverted terminal repeat sequence. This procedure involves a vitreous incision, followed by the injection of an rAAV vector suspension into the subretinal space using a thin cannula through one or more small retinal incisions.

[0182] Briefly, the injection cannula is sutured in place, allowing for the maintenance of normal ocular volume through infusion (e.g., saline solution) throughout the procedure. Vitrectomy is performed using a cannula of appropriate caliber (e.g., 20-27 gauge), and the volume of removed vitreous gel is replaced by infusion of saline or other isotonic fluid through the injection cannula. Vitrectomy is advantageous because (1) removal of the cortex (posterior hyaloid membrane) facilitates penetration of the retina by the cannula; (2) its removal and replacement with fluid (e.g., saline solution) creates a cavity suitable for intraocular injection of vector; and (3) its controlled removal reduces the likelihood of retinal tears and unplanned retinal detachment.

[0183] In some embodiments, a cannula of appropriate caliber (e.g., 27-45 gauge) is utilized to inject the rAAV composition directly into the subretinal space outside the central retina, thus forming a bleb in the subretinal space. In other embodiments, a small amount (e.g., 500 mg / mL) of rAAV is administered prior to retinal injection of the rAAV composition. A subretinal injection of about 0.1 to about 0.5 ml of an appropriate fluid (e.g., saline or Ringer's solution) is performed into the subretinal space outside the central retina. This initial injection into the subretinal space establishes an initial bleb within the subretinal space, thereby localizing retinal detachment to the location of the initial bleb. This initial fluid bleb can facilitate targeted delivery of the rAAV composition to the subretinal space (by defining the injection plane prior to rAAV delivery), minimize potential rAAV administration to the choroid, and minimize the possibility of rAAV injection or reflux into the vitreous cavity. In some embodiments, additional fluids containing one or more rAAV compositions and / or one or more additional therapeutic agents can be injected into the initial fluid bleb by administering these fluids directly into the initial fluid bleb using either the same or additional small-diameter cannula.

[0184] Intraocular administration of the rAAV composition and / or initial small volume of fluid can be performed using a thin cannula (e.g., 27-45 gauge) attached to a syringe. In some embodiments, the syringe plunger can be driven by a mechanized device, e.g., by depressing a foot pedal. The thin cannula is advanced through a sclerotomy, across the vitreous cavity, and to a predetermined site on each subject's retina (but outside the central retina) according to the region of the retina being targeted. Under direct visualization, the vector suspension is mechanically injected under the neurosensory retina, localizing the retinal detachment with a self-closing, non-enlarging retinotomy. As described above, the rAAV composition can be injected directly into the subretinal space to form a bleb outside the central retina, or the vector can be injected into an initial bleb outside the central retina to expand the bleb (and enlarge the area of ​​retinal detachment). In some embodiments, another fluid is injected into the bleb after injection of the rAAV composition.

[0185] Without wishing to be bound by theory, the speed and location of subretinal injection may result in local shear forces that may damage the macula, fovea, and / or underlying RPE cells. Subretinal injection may be performed at a speed that minimizes or avoids shear forces. In some embodiments, the rAAV composition is injected over a period of about 15-17 minutes. In some embodiments, the vector is injected over a period of about 17-20 minutes. In some embodiments, the rAAV composition is injected over a period of about 20-22 minutes. In some embodiments, the rAAV composition is injected at a rate of about 35 to about 65 μl / min. In some embodiments, the rAAV composition is injected at a rate of about 35 μl / min. In some embodiments, the rAAV composition is injected at a rate of about 40 μl / min. In some embodiments, the rAAV composition is injected at a rate of about 45 μl / min. In some embodiments, the rAAV composition is injected at a rate of about 50 μl / min. In some embodiments, the rAAV composition is injected at a rate of about 55 μl / min. In some embodiments, the rAAV composition is injected at a rate of about 60 μl / min. In some embodiments, the rAAV composition is injected at a rate of about 65 μl / min. Those skilled in the art will understand that the injection rate and time of the bleb may depend, for example, on the volume of rAAV composition or the size of the bleb required to create a retinal detachment sufficient to access cells of the central retina, the size of the cannula used to deliver the rAAV composition, and the ability to safely maintain the position of the cannula of the present invention.

[0186] In some embodiments of the invention, the volume of the composition injected into the subretinal space of the retina is greater than any one of about 1 μl, 2 μl, 3 μl, 4 μl, 5 μl, 6 μl, 7 μl, 8 μl, 9 μl, 10 μl, 15 μl, 20 μl, 25 μl, 50 μl, 75 μl, 100 μl, 200 μl, 300 μl, 400 μl, 500 μl, 600 μl, 700 μl, 800 μl, 900 μl or 1 mL, or any amount therebetween.

[0187] In some embodiments, the method comprises interacting with heparan sulfate proteoglycans. In some embodiments, the composition comprises administering to the eye (e.g., by subretinal and / or intravitreal administration) an effective amount of a recombinant viral particle comprising a vector encoding a heterologous nucleic acid, the recombinant viral particle comprising one or more amino acid substitutions at one or more positions corresponding to a viral titer of at least about 5 x 10 12 , 6×10 12 , 7×10 12 , 8×10 12 , 9×10 12 , 10×10 12 , 11×10 12 , 15×10 12 , 20×10 12 , 25×10 12 , 30×10 12 , or 50 × 10 12 In some embodiments, the viral titer of the composition is about 5 x 10 genome copies / mL. 12 ~6×10 12 , 6×10 12 ~7×10 12 , 7×10 12 ~8×10 12 , 8×10 12 ~9×10 12 , 9×10 12 ~10×10 12 , 10×10 12 ~11×10 12 , 11×10 12 ~15×10 12 , 15×10 12 ~20×10 12 , 20×10 12 ~25×10 12 , 25×10 12 ~30×10 12 , 30×10 12 ~50×10 12 , or 50 × 10 12 ~100×10 12 In some embodiments, the viral titer of the composition is about 5 x 10 genome copies / mL. 12 ~10×1012 , 10×10 12 ~25×10 12 , or 25 × 10 12 ~50×10 12 In some embodiments, the viral titer of the composition is at least about 5 x 10 genome copies / mL. 9 , 6×10 9 , 7×10 9 , 8×10 9 , 9×10 9 , 10×10 9 , 11×10 9 , 15×10 9 , 20×10 9 , 25×10 9 , 30×10 9 , or 50 × 10 9 In some embodiments, the viral titer of the composition is about 5 x 10 transducing units / mL. 9 ~6×10 9 , 6×10 9 ~7×10 9 , 7×10 9 ~8×10 9 , 8×10 9 ~9×10 9 , 9×10 9 ~10×10 9 , 10×10 9 ~11×10 9 , 11×10 9 ~15×10 9 , 15×10 9 ~20×10 9 , 20×10 9 ~25×10 9 , 25×10 9 ~30×10 9 , 30×10 9 ~50×10 9 , or 50 × 10 9 ~100×10 9 In some embodiments, the viral titer of the composition is about 5 x 10 transducing units / mL. 9 ~10×10 9 , 10×10 9 ~15×10 9 , 15×10 9~25×10 9 , or 25 × 10 9 ~50×10 9 In some embodiments, the viral titer of the composition is at least about 5 x 10 transducing units / mL. 10 , 6×10 10 , 7×10 10 , 8×10 10 , 9×10 10 , 10×10 10 , 11×10 10 , 15×10 10 , 20×10 10 , 25×10 10 , 30×10 10 , 40×10 10 , or 50 × 10 10 In some embodiments, the viral titer of the composition is at least about 5 x 10 infectious units / mL. 10 ~6×10 10 , 6×10 10 ~7×10 10 , 7×10 10 ~8×10 10 , 8×10 10 ~9×10 10 , 9×10 10 ~10×10 10 , 10×10 10 ~11×10 10 , 11×10 10 ~15×10 10 , 15×10 10 ~20×10 10 , 20×10 10 ~25×10 10 , 25×10 10 ~30×10 10 , 30×10 10 ~40×10 10 , 40×10 10 ~50×10 10 , or 50 × 10 10 ~100×10 10 In some embodiments, the viral titer of the composition is at least about 5 x 10 infectious units / mL. 10 ~10×10 10 , 10×10 10~15×10 10 , 15×10 10 ~25×10 10 , or 25 × 10 10 ~50×10 10 infectious units / mL.

[0188] In some embodiments, the methods involve administering to the eye of an individual (e.g., a human) (e.g., by subretinal and / or intravitreal administration) an effective amount of recombinant viral particles comprising one or more amino acid substitutions at one or more positions that interact with heparan sulfate proteoglycans. In some embodiments, the dose of viral particles administered to the individual is at least about 1 x 10 per kg of body weight. 8 ~Approx. 1×10 13 In some embodiments, the dose of viral particles administered to an individual is about 1 x 10 per kg of body weight. 8 ~Approx. 1×10 13 It is one of the genome copies.

[0189] One or more (e.g., two, three, or more) blebs can be formed. Generally, the total volume of blebs formed by the methods and systems of the present invention cannot exceed the fluid capacity of the eye, e.g., about 4 ml in a typical human subject. The total volume of each individual bleb can be at least about 0.3 ml, or at least about 5 ml, to promote a retinal detachment large enough to expose central retinal cell types and to generate blebs of sufficient amenability for optimal manipulation. Those skilled in the art will understand that when generating blebs according to the methods and systems of the present invention, adequate intraocular pressure must be maintained to avoid damage to ocular structures. The size of each individual bleb can be, for example, about 0.5 to about 1.2 ml, about 0.8 to about 1.2 ml, about 0.9 to about 1.2 ml, about 0.9 to about 1.0 ml, about 1.0 to about 2.0 ml, or about 1.0 to about 3.0 ml. Thus, in one example, three blebs of about 1 ml each can be constructed to inject a total of 3 ml of rAAV composition suspension. The total volume of all blebs taken together will be, for example, about 0.5 to about 3.0 ml, about 0.8 to about 3.0 ml, about 0.9 to about 3.0 ml, about 1.0 to about 3.0 ml, about 0.5 to about 1.5 ml, about 0.5 to about 1.2 ml, about 0.9 to about 3.0 ml, about 0.9 to about 2.0 ml, or about 0.9 to about 1.0 ml.

[0190] To safely and efficiently transduce target retinal (e.g., central) regions outside the bleb's original edge, the bleb may be manipulated to reposition the bleb to the target region for transduction. This manipulation of the bleb can be accomplished by repositioning the eye bearing the bleb, by realigning the human head with the eye or the eye bearing one or more blebs, depending on the volume of the bleb that creates dependency, and / or by using fluid-air exchange. This is particularly relevant for the central retina, as this region typically tolerates detachment from subretinal injections. In some embodiments, the bleb is repositioned using fluid-air exchange; fluid from the injection cannula is temporarily replaced with air, e.g., by blowing air onto the retinal surface. The large volume of air displaces vitreous fluid from the retinal surface, allowing the fluid within the vitreous cavity to flow out of the cannula. Due to the temporary lack of pressure from the vitreous fluid, the bleb moves and is drawn to the dependent portion of the eye. By properly positioning the eye, the subretinal rAAV composition bleb is manipulated to include adjacent areas (e.g., the macula and / or fovea). In some cases, the bleb is large enough to attract the bleb without the use of airflow replacement. The subject's head position may be changed to attract the bleb to the desired location within the eye, further encouraging movement of the bleb to the desired location. Once the desired configuration of the bleb is achieved, fluid is returned to the vitreous cavity. This fluid is an appropriate fluid, such as freshly prepared saline solution. Generally, the subretinal rAAV composition may be left in situ in vivo without retinal tear closure for the retinotomy and without intraocular tamponade, and the retina will spontaneously reattach within approximately 48 hours.

[0191] The methods of the present invention, in which a vector containing a therapeutic polypeptide or RNA sequence is safely and efficiently transduced into ocular cells (e.g., RPE and / or photoreceptor cells of the macula and / or fovea, e.g., RPE and / or photoreceptor cells) so that the transduced cells produce sufficient amounts of the therapeutic polypeptide or RNA sequence to treat an ocular disorder, can be used to treat individuals, e.g., humans, with ocular disorders. In some embodiments, transduction of ocular cells is improved by using rAAV particles (e.g., particles such as rAAV2 and rAAVrh8R) containing an AAV capsid protein containing one or more substitutions of amino acids that interact with HSPGs (e.g., that inhibit or eliminate binding to HSPGs). In some embodiments, the rAAV particles demonstrate reduced binding to HSPGs; for example, reduced binding of more than about 10%, 25%, 50%, 75%, 100%, or any number therebetween. In some embodiments, rAAV binding to HSPG is reduced by about 5% to about 100%, about 10% to about 50%, about 10% to about 30%, about 25% to about 75%, about 25% to about 50%, or about 30% to about 50%.

[0192] An effective amount of rAAV (in some embodiments, in the form of particles) is administered depending on the therapeutic objective. For example, if a desired therapeutic effect can be achieved with a low transduction percentage, the goal of treatment is generally to meet or exceed this transduction level. In some instances, this transduction level can be achieved by transducing only about 1-5% of target cells, in some embodiments, at least about 20%, in some embodiments, at least about 50%, in some embodiments, at least about 80%, in some embodiments, at least about 95%, and in some embodiments, at least about 99% of cells of the desired tissue type. As discussed above, substitution of one or more amino acids in the rAAV capsid that interact with HSPGs improves rAAV transduction. For reference, the number of particles administered per injection is generally about 1 x 10 particles. 6 ~1×10 14 Between the particles, about 1 × 10 7~1×10 13 Between the particles, about 1 × 10 9 ~1×10 12 or about 1 x 10 particles 11 The rAAV compositions may be administered by one or more subretinal injections during the same procedure or spaced apart by days, weeks, months, or years. In some embodiments, multiple vectors may be used to treat a person.

[0193] In some embodiments, administration of an effective amount of rAAV viral particles containing an rAAV capsid with one or more substitutions of HSPG-interacting amino acids to the retina results in transduction of photoreceptor cells at or near the site of administration. In some embodiments, greater than about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or 100% of the photoreceptor cells are transduced. In some embodiments, between about 5% and about 100%, 10% and about 50%, between about 10% and about 30%, between about 25% and about 75%, between about 25% and about 50%, or between about 30% and about 50% of the photoreceptor cells are transduced. Methods for identifying photoreceptor cells transduced by AAV viral particles containing rAAV capsids with one or more substitutions of amino acids that interact with HSPG are known in the art; for example, immunohistochemistry or the use of markers such as enhanced green fluorescent protein can be used to detect transduction of viral particles containing rAAV capsids with one or more substitutions of amino acids that interact with HSPG.

[0194] In some embodiments of the present invention, the method comprises administering an effective amount of AAV viral particles, which are viral particles comprising an rAAV capsid with one or more substitutions of amino acids that interact with HSPG, into the subretinal space (e.g., subretinal space) of a mammal to treat an individual with an ocular disorder, such as a human with an ocular disorder. In some embodiments, the composition is injected into one or more locations under the retina to express heterologous nucleic acid in photoreceptor cells. In some embodiments, the composition is injected into any one of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 locations under the retina.

[0195] In some embodiments, rAAV viral particles comprising rAAV capsids with one or more substitutions of HSPG-interacting amino acids are administered simultaneously or sequentially to more than one location, hi some embodiments, the multiple injections of rAAV viral particles are not separated by more than 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 9 hours, 12 hours, or 24 hours.

[0196] Intravitreal injection method The general method of intravitreal injection can be described by the following broad outline. This example is intended merely to illustrate certain features of the method and is in no way intended to be limiting. Intravitreal injection techniques are known in the art (see, e.g., Peyman, GA et al. (2009) Retina 29(7):875-912). See pages 114-117, and Fagan, XJ and Al-Qureshi, S. (2013) Clin. Experiment. Ophthalmol. 41(5):500-7).

[0197] Briefly, a subject undergoing intravitreal injection can be prepared for the procedure by dilating the pupil, disinfecting the eye, and administering an anesthetic. Any suitable mydriatic known in the art can be used to dilate the pupil. After sufficient pupil dilation is confirmed, treatment can be performed. Disinfection can be achieved by applying an ocular disinfectant, for example, an iodine-containing solution such as povidone-iodine (BETADINE®). Similar solutions can be used to cleanse the eyelids, eyelashes, and any other surrounding tissues (e.g., skin). Any suitable anesthetic, such as lidocaine or proparacaine, can be used in any suitable concentration. The anesthetic can be administered by any method known in the art, including, but not limited to, topical drops, gels or jellies, and subconjunctival application of the anesthetic.

[0198] Prior to injection, the area is cleared of eyelashes using a sterile eyelid speculum. The injection site may be marked with a syringe. The injection site may be selected based on the patient's lens. For example, the injection site may be 3-3.5 mm from the limbus for pseudophakic or aphakic patients and 3.5-4 mm from the limbus for phakic patients. The patient should be able to look away from the injection site.

[0199] In some embodiments, the method comprises administering to the eye (e.g., by subretinal and / or intravitreal administration) an effective amount of a recombinant viral particle comprising a vector encoding a heterologous nucleic acid, the recombinant viral particle comprising one or more amino acid substitutions at one or more positions that interact with heparan sulfate proteoglycans. In some embodiments, the viral titer of the composition is at least about 5 x 10 12 , 6×10 12 , 7×10 12 , 8×10 12 , 9×10 12 , 10×10 12 , 11×10 12 , 15×10 12 , 20×10 12 , 25×10 12 , 30×1012 , or 50 × 10 12 In some embodiments, the viral titer of the composition is about 5 x 10 genome copies / mL. 12 ~6×10 12 , 6×10 12 ~7×10 12 , 7×10 12 ~8×10 12 , 8×10 12 ~9×10 12 , 9×10 12 ~10×10 12 , 10×10 12 ~11×10 12 , 11×10 12 ~15×10 12 , 15×10 12 ~20×10 12 , 20×10 12 ~25×10 12 , 25×10 12 ~30×10 12 , 30×10 12 ~50×10 12 , or 50 × 10 12 ~100×10 12 In some embodiments, the viral titer of the composition is about 5 x 10 genome copies / mL. 12 ~10×10 12 , 10×10 12 ~25×10 12 , or 25 × 10 12 ~50×10 12 In some embodiments, the viral titer of the composition is at least about 5 x 10 genome copies / mL. 9 , 6×10 9 , 7×10 9 , 8×10 9 , 9×10 9 , 10×10 9 , 11×10 9 , 15×10 9 , 20×10 9 , 25×10 9 , 30×10 9 , or 50 × 10 9In some embodiments, the viral titer of the composition is about 5 x 10 transducing units / mL. 9 ~6×10 9 , 6×10 9 ~7×10 9 , 7×10 9 ~8×10 9 , 8×10 9 ~9×10 9 , 9×10 9 ~10×10 9 , 10×10 9 ~11×10 9 , 11×10 9 ~15×10 9 , 15×10 9 ~20×10 9 , 20×10 9 ~25×10 9 , 25×10 9 ~30×10 9 , 30×10 9 ~50×10 9 , or 50 × 10 9 ~100×10 9 In some embodiments, the viral titer of the composition is about 5 x 10 transducing units / mL. 9 ~10×10 9 , 10×10 9 ~15×10 9 , 15×10 9 ~25×10 9 , or 25 × 10 9 ~50×10 9 In some embodiments, the viral titer of the composition is at least about 5 x 10 transducing units / mL. 10 , 6×10 10 , 7×10 10 , 8×10 10 , 9×10 10 , 10×10 10 , 11×10 10 , 15×10 10 , 20×10 10 , 25×10 10 , 30×10 10 , 40×10 10 , or 50 × 10 10In some embodiments, the viral titer of the composition is at least about 5 x 10 infectious units / mL. 10 ~6×10 10 , 6×10 10 ~7×10 10 , 7×10 10 ~8×10 10 , 8×10 10 ~9×10 10 , 9×10 10 ~10×10 10 , 10×10 10 ~11×10 10 , 11×10 10 ~15×10 10 , 15×10 10 ~20×10 10 , 20×10 10 ~25×10 10 , 25×10 10 ~30×10 10 , 30×10 10 ~40×10 10 , 40×10 10 ~50×10 10 , or 50 × 10 10 ~100×10 10 In some embodiments, the viral titer of the composition is at least about 5 x 10 infectious units / mL. 10 ~10×10 10 , 10×10 10 ~15×10 10 , 15×10 10 ~25×10 10 , or 25 × 10 10 ~50×10 10 infectious units / mL.

[0200] In some embodiments, the methods involve administering to the eye of an individual (e.g., a human) (e.g., by subretinal and / or intravitreal administration) an effective amount of recombinant viral particles comprising one or more amino acid substitutions at one or more positions that interact with heparan sulfate proteoglycans. In some embodiments, the dose of viral particles administered to the individual is at least about 1 x 10 per kg of body weight. 8~Approx. 1×10 13 In some embodiments, the dose of viral particles administered to an individual is about 1 x 10 per kg of body weight. 8 ~Approx. 1×10 13 It is one of the genome copies.

[0201] During injection, the needle may be inserted perpendicular to the sclera and directed toward the center of the eye. The needle may be inserted so that the tip ends in the vitreous cavity rather than the subretinal space. Any suitable injection volume known in the art may be used. After injection, the eye may be treated with an antiseptic, such as an antibiotic. The eye may be rinsed to remove excess antiseptic.

[0202] Tools for determining retinal structure and efficacy of rAAV delivery The retina is known to have multiple layers. Retinal cell layers include the inner limiting membrane, nerve fibers, ganglion cells, inner plexiform layer, inner nuclear layer, outer plexiform layer, outer nuclear layer, outer limiting membrane, photoreceptors, and retinal pigment epithelium. The most proximal layer of the vitreous body is the inner limiting membrane. This layer can contain Müller cells, a type of glia. The nerve fiber layer can contain axons from ganglion cells that make up the optic nerve. The ganglion cell layer can include ganglion cells and amacrine cells. The inner plexiform layer can contain synapses between dendrites of ganglion and amacrine cells and axons of bipolar cells. The inner nuclear layer can contain nuclei of amacrine, bipolar, and horizontal cells. The outer plexiform layer can contain synapses between dendrites of horizontal cells and photoreceptor cell processes. The outer nuclear layer can contain photoreceptor cell bodies. The outer or external limiting membrane can contain cellular junctions, such as adherens junctions and desmosomes, between the apical processes of Müller cells and between these processes and the inner segments of photoreceptor cells. The photoreceptor layer, also known as the rod-cone layer and Jacob's membrane, can contain photoreceptor cells, including rods and cones. The most distal retinal layer to the vitreous is the retinal pigment epithelium (RPE), which can include a layer of hexagonal epithelial cells containing pigment granules.

[0203] It is also known that the retina contains many different cell types. Retinal neurons can include photoreceptor cells, bipolar cells, ganglion cells, amacrine cells, and horizontal cells. Photoreceptor cells are light sensitive. They can sense light and respond by transmitting signals to the optic nerve via bipolar cells and ganglion cells. Photoreceptor cells can include rod cells, which generally sense light in low light conditions, and cone cells, which generally sense color and perceive brighter light. Bipolar cells can receive input from photoreceptor cells and from synapsing onto amacrine or ganglion cells. Ganglion cells The retina can receive information from amacrine or horizontal cells and their axons from the optic nerve. Horizontal cells integrate input from multiple photoreceptors and can regulate light levels. Amacrine cells are interneurons that help regulate bipolar cells and provide input to ganglion cells. Glial cells in the retina include Müller cells, astroglia, and microglia.

[0204] The effectiveness of rAAV delivery by subretinal or intravitreal injection can be monitored by several criteria described herein.For example, after subject is treated using the method of the present invention, subject can be evaluated by one or more clinical parameters, including those described herein, for example, improvement and / or stabilization of one or more signs or symptoms of disease state and / or delay of progression.Examples of such tests are known in the art, and include objective as well as subjective (e.g., subject-reported) measurements. For example, to measure the effectiveness of a treatment on a subject's visual function, one or more of the following may be assessed: an improvement in the subject's subjective quality of vision or central visual function (e.g., an improvement in the subject's ability to read fluently and recognize faces), a subject's visual movements (e.g., a decrease in the time required to navigate a maze), visual acuity (e.g., an improvement in the subject's LogMAR score), microperimetry (e.g., an improvement in the subject's dB score), scotopic perimetry (e.g., an improvement in the subject's dB score), fine matrix mapping (e.g., an improvement in the subject's dB score), Goldmann perimetry (e.g., a reduction in the size of the scotoma area (i.e., blind area) and an improvement in the ability to resolve smaller targets), flicker sensitivity (e.g., an improvement in Hertz), autofluorescence, and electrophysiological measurements (e.g., an improvement in ERG). In some embodiments, visual function is measured by the subject's visual movements. In some embodiments, visual function is measured by the subject's visual acuity. In some embodiments, visual function is measured by microperimetry. In some embodiments, visual function is measured by scotopic perimetry. In some embodiments, visual function is measured by ERG. In some embodiments, visual function is measured by the subjective quality of vision of the subject.

[0205] In the case of diseases that result in progressively degenerative visual function, treating subjects at a young age can not only slow or halt disease progression, but can also improve or prevent the decline in visual function resulting from acquired amblyopia. Amblyopia may be of two types. In studies of nonhuman primates and kittens that are kept in total darkness from birth, even for a few months of age, the animals are functionally irreversibly blind, even when later exposed to light, despite having functional signals transmitted by the retina. This blindness occurs because neural connections and cortical "education" cease to develop from birth due to cessation of stimulation. It is unclear whether this function can ever be restored. In the case of retinal degenerative diseases, normal visual cortical circuitry was initially "learned," or developmentally appropriate, until degeneration produced significant functional impairment. In terms of signal transduction in the dysfunctional eye, reduced visual stimulation results in "acquired" or "learned" impairment ("acquired amblyopia"), in which the brain is unable to interpret signals or "use" the eye. In these cases of "acquired amblyopia," it is unclear whether there will be improved signaling from the retina as a result of gene therapy of the amblyopic eye, which could previously have resulted in a slowing of progression or stabilization of the disease as well as the gain of more normal function. In some embodiments, the human being treated is under 30 years of age. In some embodiments, the human being treated is under 20 years of age. In some embodiments, the human being treated is under 18 years of age. In some embodiments, the human being treated is under 15 years of age. In some embodiments, the human being treated is under 14 years of age. In some embodiments, the human being treated is under 13 years of age. In some embodiments, the human being treated is under 12 years of age. In some embodiments, the human being treated is under 10 years of age. In some embodiments, the human being treated is under 15 years of age. In some embodiments, the human being treated is under 16 years of age. is under the age of 8. In some embodiments, the human being treated is under the age of 6.

[0206] In some ocular disorders, there is a "nurse cell" phenomenon in which improving the function of one type of cell improves the function of another type of cell. For example, transduction of the RPE of the central retina with the rAAV of the present invention can improve rod function, and improved rod function can also lead to improved cone function. Thus, treatment of one type of cell can result in improved function for another type of cell.

[0207] The selection of a particular rAAV vector and composition depends on many different factors, including, but not limited to, the individual's medical history and the characteristics of the condition and individual being treated. The evaluation of such characteristics and the design of an appropriate treatment regimen are ultimately the responsibility of the prescribing physician.

[0208] In some embodiments, the human being treated has a genetic eye disorder but has not yet shown clinical signs or symptoms. In some embodiments, the human being treated has an eye disorder. In some embodiments, the human being treated shows one or more signs or symptoms of an eye disorder.

[0209] Non-limiting examples of eye disorders that can be treated by the systems and methods of the present invention include: autosomal recessive severe early-onset retinal degeneration (Leber's congenital amaurosis), congenital color blindness, Stargardt's disease, Best's disease, Doyne's disease, cone dystrophy, retinitis pigmentosa, X-linked retinoschisis, Usher syndrome, age-related macular degeneration, atrophic age-related macular degeneration, neovascular AMD, diabetic maculopathy, proliferative diabetic retinopathy (PDR), cystoid macular edema, central serous retinopathy, retinal detachment, intraocular inflammation, glaucoma, posterior uveitis, total choroidal atrophy, and Leber's hereditary optic neuropathy.

[0210] The compositions of the invention (e.g., AAV viral particles for subretinal or intravitreal delivery comprising an AAV capsid having one or more substitutions of amino acids that interact with HSPG or one or more substitutions at one or more positions corresponding to amino acids 484, 487, 532, 585, or 588) can be used alone or in combination with one or more additional therapeutic agents for treating ocular disorders. The interval between sequential administrations can be at least a few minutes, hours, or days (or, alternatively, less than a few minutes, hours, or days).

[0211] In some embodiments, one or more additional therapeutic agents may be administered subretinally or into the vitreous (e.g., by intravitreal administration). Non-limiting examples of additional therapeutic agents include polypeptide neurotrophic factors (e.g., GDNF, CNTF, BDNF, FGF2, PEDF, EPO), polypeptide anti-angiogenic factors (e.g., sFlt, angiostatin, endostatin), anti-angiogenic nucleic acids (e.g., siRNA, miRNA, ribozymes), e.g., anti-angiogenic nucleic acids against VEGF, anti-angiogenic morpholinos, e.g., anti-angiogenic morpholinos against VEGF, anti-angiogenic antibodies and / or antibody fragments (e.g., Fab fragments), e.g., anti-angiogenic antibodies and / or antibody fragments against VEGF.

[0212] Methods for CNS delivery In some embodiments, administration of an effective amount of a recombinant viral particle (e.g., an AAV2, AAVrh8R, etc. particle) comprising one or more amino acid substitutions at one or more positions that interact with heparan sulfate proteoglycans of the present disclosure results in the formation of a heparan sulfate proteoglycan into neurons (e.g., striatal neurons, e.g., spiny neurons) at or near the site of administration. Transduction occurs. In some embodiments, greater than about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or 100% of the neurons are transduced. In some embodiments, about 5% to about 100%, 10% to about 50%, about 10% to about 30%, about 25% to about 75%, about 25% to about 50%, or about 30% to about 50% of the neurons are transduced. Methods for identifying neurons transduced by recombinant viral particles expressing heterologous nucleic acids are known in the art; for example, expression can be detected using immunohistochemistry, RNA detection (e.g., qPCR, Northern blotting, RNA-seq, in situ hybridization, etc.), or the use of co-expressed markers such as enhanced green fluorescent protein.

[0213] In some embodiments of the present invention, the method comprises administering to the brain of a mammal, e.g., a human, an effective amount of a recombinant viral particle comprising one or more amino acid substitutions at one or more positions that interact with heparan sulfate proteoglycans of the present disclosure. In some embodiments, the composition is injected into one or more locations in the brain to express the heterologous nucleic acid of the present disclosure in at least one neuron. In some embodiments, the composition is injected into any one of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 locations in the brain. In some embodiments, the composition is injected into the striatum. In some embodiments, the composition is injected into the dorsal striatum. In some embodiments, the composition is injected into the putamen. In some embodiments, the composition is injected into the caudate nucleus. In some embodiments, the composition is injected into both the putamen and the caudate nucleus. In some embodiments, a recombinant viral particle comprising one or more amino acid substitutions at one or more positions that interact with heparan sulfate proteoglycans is administered to the CNS of an individual, e.g., into the striatum, by stereotaxic injection. In some embodiments, recombinant viral particles comprising one or more amino acid substitutions at one or more positions that interact with heparan sulfate proteoglycans are administered to the CNS of an individual by convection-enhanced delivery (CED); for example, CED to the striatum.

[0214] Administration of rAAV particles can be achieved by various routes. In some embodiments, administration includes direct spinal injection and / or intracerebral administration. In some embodiments, administration is to a site selected from the cerebrum, medulla, pons, cerebellum, intracranial cavity, meninges covering the brain, dura mater, arachnoid mater, pia mater, cerebrospinal fluid (CSF) in the subarachnoid space surrounding the brain, deep cerebellar nuclei of the cerebellum, the ventricular system of the cerebrum, the subarachnoid space, the striatum, cortex, septum, thalamus, hypothalamus, and brain parenchyma. In some embodiments, administration includes intraventricular injection into at least one lateral cerebral ventricle. In some embodiments, administration includes intrathecal injection in the cervical, thoracic, and / or lumbar regions. In some embodiments, administration includes intrastriatal injection. In some embodiments, administration includes intrathalamic injection. Various techniques and devices suitable for these administration routes, such as CED and / or stereotaxic injection, are described herein.

[0215] In some embodiments, recombinant viral particles containing one or more amino acid substitutions at one or more positions that interact with heparan sulfate proteoglycans are administered to one hemisphere of the brain, hi some embodiments, recombinant viral particles are administered to both hemispheres of the brain.

[0216] In some embodiments, recombinant viral particles comprising one or more amino acid substitutions at one or more positions that interact with heparan sulfate proteoglycans are administered simultaneously or sequentially to more than one location. In some embodiments, multiple injections of recombinant viral particles are administered at 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, or more. , not more than 6, 9, 12 or 24 hours apart.

[0217] In some embodiments, the present invention provides methods for treating a human having a CNS disorder by administering an effective amount of a pharmaceutical composition comprising a recombinant viral particle comprising one or more amino acid substitutions at one or more positions that interact with a heparan sulfate proteoglycan of the present disclosure to treat the CNS disorder. In some embodiments, the pharmaceutical composition comprises one or more pharmaceutically acceptable excipients.

[0218] Generally, about 1 μL to about 1 mL of a composition of the invention (e.g., about 100 μL to about 500 μL of composition) can be delivered. In some embodiments of the invention, the volume of recombinant viral particles containing one or more amino acid substitutions at one or more positions that interact with heparan sulfate proteoglycans injected into the striatum is any one or more of about 1 μL, 2 μL, 3 μL, 4 μL, 5 μL, 6 μL, 7 μL, 8 μL, 9 μL, 10 μL, 15 μL, 20 μL, 25 μL, 50 μL, 75 μL, 100 μL, 200 μL, 300 μL, 400 μL, 500 μL, 600 μL, 700 μL, 800 μL, 900 μL, or 1 mL, or any amount therebetween.

[0219] In some embodiments, a first volume of recombinant viral particles comprising one or more amino acid substitutions at one or more positions that interact with heparan sulfate proteoglycans is injected into a first region of the brain, and a second volume of the mutant rAAV particles is injected into a second region of the brain. For example, in some embodiments, a first amount of mutant rAAV particles is injected into the caudate nucleus, and a second volume of the composition is injected into the putamen. In some embodiments, a 1X volume of mutant rAAV particles is injected into the caudate nucleus, and a 1.5X, 2X, 2.5X, 3X, 3.5X, or 4X volume of mutant rAAV particles is injected into the putamen, where X is any one or more of about 1 μl, 2 μl, 3 μl, 4 μl, 5 μl, 6 μl, 7 μl, 8 μl, 9 μl, 10 μl, 15 μl, 20 μl, 25 μl, 50 μl, 75 μl, 100 μl, 200 μl, 300 μl, 400 μl, 500 μl, 600 μl, 700 μl, 800 μl, 900 μl, or 1 mL, or any amount therebetween.

[0220] Compositions of the invention (e.g., recombinant viral particles containing one or more amino acid substitutions at one or more positions that interact with heparan sulfate proteoglycans of the present disclosure) can be used alone or in combination with one or more additional therapeutic agents for treating CNS disorders (e.g., HD). The interval between sequential administrations can be at least a matter of minutes, hours, or days (or, alternatively, less than a matter of minutes, hours, or days).

[0221] In some embodiments, the method comprises administering to the CNS an effective amount of a recombinant viral particle comprising a vector encoding a heterologous nucleic acid, the recombinant viral particle comprising one or more amino acid substitutions at one or more positions that interact with heparan sulfate proteoglycans. In some embodiments, the viral titer of the composition is at least about 5 x 10 12 , 6×10 12 , 7×10 12 , 8×10 12 , 9×1012 , 10×10 12 , 11×10 12 , 15×10 12 , 20×10 12 , 25×10 12 , 30×10 12 , or 50 × 10 12 In some embodiments, the viral titer of the composition is about 5 x 10 genome copies / mL. 12 ~6×10 12 , 6×10 12 ~7×10 12 , 7×10 12 ~8×10 12 , 8×10 12 ~9×10 12 , 9×10 12 ~10×10 12 , 10×10 12 ~11×10 12 , 11×10 12 ~15×10 12 , 15×10 12 ~20×10 12 , 20×10 12 ~25×10 12 , 25×10 12 ~30×10 12 , 30×10 12 ~50×10 12 , or 50 × 10 12 ~100×10 12 genome copies / mL. In some embodiments, the viral titer of the composition is about 5 x 10 12 ~10×10 12 , 10×10 12 ~25×10 12 , or 25 × 10 12 ~50×10 12 In some embodiments, the viral titer of the composition is at least about 5 x 10 genome copies / mL. 9 , 6×10 9 , 7×10 9 , 8×10 9 , 9×10 9 , 10×10 9 , 11×10 9 , 15×109 , 20×10 9 , 25×10 9 , 30×10 9 , or 50 × 10 9 In some embodiments, the viral titer of the composition is about 5 x 10 transducing units / mL. 9 ~6×10 9 , 6×10 9 ~7×10 9 , 7×10 9 ~8×10 9 , 8×10 9 ~9×10 9 , 9×10 9 ~10×10 9 , 10×10 9 ~11×10 9 , 11×10 9 ~15×10 9 , 15×10 9 ~20×10 9 , 20×10 9 ~25×10 9 , 25×10 9 ~30×10 9 , 30×10 9 ~50×10 9 , or 50 × 10 9 ~100×10 9 In some embodiments, the viral titer of the composition is about 5 x 10 transducing units / mL. 9 ~10×10 9 , 10×10 9 ~15×10 9 , 15×10 9 ~25×10 9 , or 25 × 10 9 ~50×10 9 In some embodiments, the viral titer of the composition is at least about 5 x 10 transducing units / mL. 10 , 6×10 10 , 7×10 10 , 8×10 10 , 9×10 10 , 10×10 10 , 11×10 10 , 15×10 10 , 20×10 10, 25×10 10 , 30×10 10 , 40×10 10 , or 50 × 10 10 In some embodiments, the viral titer of the composition is at least about 5 x 10 infectious units / mL. 10 ~6×10 10 , 6×10 10 ~7×10 10 , 7×10 10 ~8×10 10 , 8×10 10 ~9×10 10 , 9×10 10 ~10×10 10 , 10×10 10 ~11×10 10 , 11×10 10 ~15×10 10 , 15×10 10 ~20×10 10 , 20×10 10 ~25×10 10 , 25×10 10 ~30×10 10 , 30×10 10 ~40×10 10 , 40×10 10 ~50×10 10 , or 50 × 10 10 ~100×10 10 In some embodiments, the viral titer of the composition is at least about 5 x 10 infectious units / mL. 10 ~10×10 10 , 10×10 10 ~15×10 10 , 15×10 10 ~25×10 10 , or 25 × 10 10 ~50×10 10 infectious units / mL.

[0222] In some embodiments, the methods involve administering to the CNS of an individual (e.g., a human) an effective amount of recombinant viral particles comprising one or more amino acid substitutions at one or more positions that interact with heparan sulfate proteoglycans. In some embodiments, the dose of viral particles administered to the individual is at least about 1 x 10 per kg of body weight. 8 ~Approx. 1×10 13 In some embodiments, the dose of viral particles administered to an individual is about 1 x 10 per kg of body weight. 8 ~Approx. 1×10 13 It is one of the genome copies.

[0223] In some embodiments, the methods involve administering to the CNS of an individual (e.g., a human) an effective amount of recombinant viral particles comprising one or more amino acid substitutions at one or more positions that interact with heparan sulfate proteoglycans to the individual. In some embodiments, the total amount of viral particles administered to the individual is at least about 1 x 10 9 ~Approx. 1×10 14 In some embodiments, the total amount of viral particles administered to an individual is about 1 x 10 9 ~Approx. 1×10 14 It is one of the genome copies.

[0224] V. Expression Constructs In some embodiments, the present invention provides methods for delivering heterologous nucleic acids to the eye by subretinal delivery of an rAAV vector comprising the heterologous nucleic acid, wherein the rAAV vector is packaged in an rAAV capsid (e.g., an rAAV2 capsid, an rAAVrh8R capsid, etc.) comprising one or more substitutions of amino acids that interact with HSPG. In some embodiments, the present invention provides methods for treating a CNS disorder in an individual, comprising administering to the individual a therapeutically effective amount of the ... The method includes delivering a composition comprising rAAV particles to the CNS of the body, the rAAV particles comprising (a) an rAAV capsid comprising an rAAV capsid protein comprising one or more amino acid substitutions at one or more positions that interact with heparan sulfate proteoglycans, and (b) an rAAV vector comprising a heterologous nucleic acid and at least one AAV terminal repeat sequence. In some embodiments, the heterologous nucleic acid (e.g., a transgene) is operably linked to a promoter. Exemplary promoters include the cytomegalovirus (CMV) immediate early promoter, RSV LTR, MoMLV LTR, phosphoglycerate kinase-1 (PGK) promoter, simian virus 40 (SV40) promoter and CK6 promoter, transthyretin promoter (TTR), TK promoter, tetracycline-responsive promoter (TRE), HBV promoter, hAAT promoter, LSP promoter, chimeric liver-specific promoter (LSP), E2F promoter, telomerase (hTERT) promoter; cytomegalovirus enhancer / chicken β-actin / rabbit β-globin promoter (CAG promoter; Niwa et al., Gene, 1991, 108(2):193-9), and elongation factor 1-α promoter (EF1-α) promoter (Kim et al., Gene, 1990, 91(2):217-23 and Guo et al., Gene, 1991, 108(2):217-23). Ther., 1996, 3(9):802-10). In some embodiments, the promoter comprises a cytomegalovirus enhancer linked to the human β-glucuronidase promoter or the chicken β-actin (CBA) promoter. The promoter can be a constitutive, inducible, or repressible promoter. In some embodiments, the promoter is capable of expressing a heterologous nucleic acid in ocular cells. In some embodiments, the promoter is capable of expressing a heterologous nucleic acid in photoreceptor cells or RPE. In embodiments, the promoter is a rhodopsin kinase (RK) promoter, e.g., a human RK promoter.In some embodiments, the promoter is an opsin promoter, for example, a human opsin promoter or a mouse opsin promoter.

[0225] The present invention contemplates the use of recombinant viral genomes to introduce one or more nucleic acid sequences encoding therapeutic polypeptides and / or nucleic acids for packaging into rAAV viral particles containing one or more substitutions of amino acids that interact with HSPGs. The recombinant viral genome may include any elements for establishing expression of the therapeutic polypeptides and / or nucleic acids, such as promoters, ITRs, ribosome binding elements, terminators, enhancers, selectable markers, introns, polyA signals, and / or origins of replication.

[0226] In some embodiments, the rAAV vector is a self-complementary rAAV vector, for example, one that comprises a recombinant self-complementary (sometimes referred to herein as "self-complementary") genome. AAV viral particles with self-complementary genomes and the method of using self-complementary AAV genomes are described in U.S. Patent Nos. 6,596,535, 7,125,717; 7,465,583; 7,785,888; 7,790,154; 7,846,729; 8,093,054; and 8,361,457; and Wang Z. et al. (2003) Gene Ther 10:2105-2111, each of which is incorporated herein by reference in its entirety. rAAVs containing self-complementary genomes will rapidly form double-stranded DNA molecules due to their partially complementary sequences (e.g., the complementary coding and non-coding strands of a transgene). In some embodiments, the vector comprises a first nucleic acid sequence encoding a heterologous nucleic acid and a second nucleic acid sequence encoding the complementary strand of the nucleic acid, wherein the first nucleic acid sequence can form interstrand base pairs with the second nucleic acid sequence over most or all of its length.

[0227] In some embodiments, the first heterologous nucleic acid sequence and the second heterologous nucleic acid sequence are linked by a mutated ITR (e.g., a right ITR). In some embodiments, the ITR comprises the polynucleotide sequence 5'-CACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCACGCCCGGGCTTTGCCCGGGCG-3' (SEQ ID NO: 8). The mutated ITR comprises a deletion of the D region, which includes a terminal cleavage sequence. As a result, during replication of the AAV viral genome, the rep protein will not cleave the viral genome at the mutated ITR, and thus a recombinant viral genome comprising, in 5' to 3' order, the following will be packaged into the viral capsid: the AAV ITR, the first heterologous polynucleotide sequence including the regulatory sequence, the mutated AAV ITR, the second polynucleotide sequence in the reverse orientation relative to the first heterologous polynucleotide, and the third AAV ITR.

[0228] VI. VIRUS PARTICLES AND METHODS FOR PRODUCING VIRUS PARTICLES rAAV viral particles In some embodiments, the present invention provides methods for delivering heterologous nucleic acids to the eye by subretinal delivery of an rAAV vector containing the heterologous nucleic acid, wherein the rAAV vector is packaged in an rAAV capsid (e.g., rAAV2, rAAVrh8R, etc.) that contains one or more substitutions of amino acids that interact with HSPG. In some embodiments, the present invention provides methods and kits for delivering rAAV particles to the CNS of an individual.

[0229] In some embodiments, the rAAV particle comprises an AAV vector. In some embodiments, the viral particle is a recombinant AAV particle comprising a nucleic acid comprising a heterologous transgene flanked by one or two AAV inverted terminal repeats (ITRs). The nucleic acid is encapsulated in the capsid of the AAV particle. The AAV particle also comprises capsid proteins. In some embodiments, the nucleic acid comprises a coding sequence of interest (e.g., a heterologous transgene) operably linked in the transcriptional direction, regulatory sequences including transcription start and termination sequences, thereby forming an expression cassette.

[0230] The expression cassette is flanked at its 5' and 3' ends by at least one functional AAV ITR sequence. "Functional AAV ITR sequence" means that the ITR sequence functions as intended for the rescue, replication, and packaging of AAV virions. See Davidson et al., PNAS, 2000, 97(7)3428-32; Passini et al., J. Virol., 2003, 77(12):7034-40; and Pechan et al., Gene Ther., 2009, 16:10-16. All of these references are incorporated herein by reference in their entirety. To implement some embodiments of the present invention, the recombinant vector contains at least all of the AAV sequences essential for encapsidation and the physical structure for infection by rAAV. AAV ITRs for use in the vectors of the invention need not have wild-type nucleotide sequences (e.g., as described in Kotin, Hum. Gene Ther., 1994, 5:793-801), but may be modified by nucleotide insertions, deletions, or substitutions, or may be derived from any of several AAV serotypes. More than 40 serotypes of AAV are now known, and new serotypes and variants of existing serotypes continue to be identified. See Gao et al., PNAS, 2002, 99(18):11854-6; Gao et al., PNAS, 2003, 100(10):6081-6; and Bossis et al., J. Virol., 2003, 77(12):6799-810.

[0231] The use of any AAV serotype is contemplated within the scope of the present invention. In some embodiments, the rAAV vector is selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV17, AAV18, AAV19, AAV The vector is derived from an AAV serotype, including, but not limited to, AAV ITRs such as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrh10, AAV11, AAV12, AAV2R471A, AAV DJ, caprine AAV, bovine AAV, or murine AAV ITRs. In some embodiments, the nucleic acid in the AAV comprises an IR of an AAV ITR such as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrh10, AAV11, AAV12, AAV2R471A, AAV DJ, caprine AAV, bovine AAV, or murine AAV. In a specific embodiment, the nucleic acid in the AAV comprises an AAV2 ITR. As explained above, the rAAV particles may further comprise a capsid comprising an rAAV capsid protein that includes one or more amino acid substitutions at one or more positions that interact with heparan sulfate proteoglycans.

[0232] In some embodiments, the vector may include a stuffer nucleic acid. In some embodiments, the stuffer nucleic acid may encode green fluorescent protein. In some embodiments, the stuffer nucleic acid may be located between the promoter and the nucleic acid encoding the RNAi.

[0233] Different AAV serotypes are used to optimize transduction into specific target cells or target specific cell types within specific target tissues (e.g., CNS tissues). rAAV particles can contain viral proteins and viral nucleic acids of the same serotype or mixed serotypes. For example, in some embodiments, rAAV particles can contain the AAV2 capsid protein of the present invention and at least one AAV2 ITR, or can contain the AAV2 capsid protein and at least one AAV1 ITR. All combinations of AAV serotypes for the production of rAAV particles are provided herein, as if each combination were expressly set forth herein. In some embodiments, the present invention provides rAAV particles comprising the AAV2 capsid of the present invention. In some embodiments, the present invention provides rAAV particles comprising the AAVrh8R capsid of the present invention.

[0234] Production of AAV particles Numerous methods are known in the art for producing rAAV vectors, including transfection, stable cell line production, and infectious hybrid virus production systems, including adenovirus-AAV hybrids, herpesvirus-AAV hybrids (Conway, J.E. et al. (1997) J. Virology 71(11):8780-8789), and baculovirus-AAV hybrids. All rAAV production cultures for the production of rAAV viral particles require: 1) a stable host cell, including, for example, a human-derived cell line, such as HeLa, A549, or 293 cells, or, in the case of a baculovirus production system, an insect-derived cell line, such as SF-9; 2) suitable helper virus functions, provided by wild-type or mutant adenovirus (e.g., temperature-sensitive adenovirus), herpesvirus, baculovirus, or a plasmid construct providing helper functions; 3) AAV rep and cap genes and gene products; 4) a transgene (e.g., a therapeutic transgene) flanked by at least one AAV ITR sequence; and 5) suitable media and media components that support rAAV production. Suitable media known in the art may be used for the production of rAAV vectors. These media include, but are not limited to, media produced by Hyclone Laboratories and JRH, including Modified Eagle's Medium (MEM), Dulbecco's Modified Eagle's Medium (DMEM), custom formulations such as those described in U.S. Pat. No. 6,566,118, and SF-900 II SFM medium as described in U.S. Pat. No. 6,723,551, each of which is incorporated herein by reference in its entirety, and specifically as set forth in U.S. Pat. No. 6,723,551. , which is incorporated herein by reference for custom media formulations for use in the production of recombinant AAV vectors.

[0235] rAAV particles can be produced using methods known in the art.See, for example, U.S. Patent Nos. 6,566,118; 6,989,264; and 6,995,006.When carrying out the present invention, host cells for producing rAAV particles include mammalian cells, insect cells, plant cells, microbial cells, and yeast.Host cells can also be packaging cells in which AAV rep and cap genes are stably maintained in the host cell, or producer cells in which AAV vector genomes are stably maintained.Exemplary packaging and producer cells are derived from 293, A549, or HeLa cells.AAV vectors are purified and formulated using standard techniques known in the art.

[0236] In some embodiments, rAAV particles may be produced by a triple transfection method, such as the exemplary triple transfection method provided below. Briefly, a plasmid containing the rep gene and the capsid gene can be transfected (e.g., using the calcium phosphate method) into a cell line (e.g., HEK-293 cells) along with a helper adenovirus plasmid, and the virus can be harvested and optionally purified.

[0237] In some embodiments, rAAV particles may be produced by a producer cell line method, such as the exemplary producer cell line method provided below (see also those referenced in Martin et al. (2013) Human Gene Therapy Methods 24:253-269). Briefly, a cell line (e.g., HeLa cells) can be stably transfected with a plasmid containing the rep gene, capsid gene, and promoter-transgene sequence. The cell line can be screened to select a lead clone for rAAV production, which can then be expanded into a production bioreactor and infected with adenovirus (e.g., wild-type adenovirus) as a helper to initiate rAAV production. The virus can then be harvested, the adenovirus can be inactivated (e.g., by heat) and / or removed, and the rAAV particles can be purified.

[0238] In some aspects, methods of producing any of the rAAV particles disclosed herein are provided, the methods comprising: (a) culturing host cells under conditions such that rAAV particles are produced, the host cells comprising: (i) one or more AAV packaging genes, each of which encodes an AAV replication and / or encapsidation protein; (ii) an rAAV provector comprising a nucleic acid encoding a therapeutic polypeptide and / or nucleic acid described herein, flanked by at least one AAV ITR; and (iii) AAV helper functions; and (b) recovering the rAAV particles produced by the host cells. In some embodiments, the at least one AAV ITR is selected from the group consisting of AAV ITRs, such as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrh10, AAV11, AAV12, AAV2R471A, AAV DJ, caprine AAV, bovine AAV, or murine AAV. In some embodiments, the capsid-encapsulating protein comprises one or more amino acid substitutions at one or more positions that interact with heparan sulfate proteoglycans. In some embodiments, the capsid-encapsulating protein is an AAV2 capsid-encapsulating protein. In some embodiments, the capsid-encapsulating protein is an AAVrh8R capsid-encapsulating protein.

[0239] Suitable rAAV production culture media of the present invention may be supplemented with serum or serum-derived recombinant proteins at levels of 0.5% to 20% (v / v or w / v). Alternatively, as is known in the art, rAAV vectors may be produced in serum-free conditions, sometimes referred to as animal-derived product-free media. Those skilled in the art will appreciate that commercial or custom media designed to support rAAV vector production may also be supplemented with one or more cell culture components known in the art, including, but not limited to, glucose, vitamins, amino acids, and / or growth factors, to increase the titer of rAAV in the production culture.

[0240] rAAV production cultures can be grown under a variety of conditions (such as over a wide temperature range and for various lengths of time) suitable for the particular host cells used. As is known in the art, rAAV production cultures include attachment-dependent cultures that can be cultured in suitable attachment-dependent vessels, such as roller bottles, hollow fiber filters, microcarriers, and packed-bed or fluidized-bed bioreactors. rAAV vector production cultures can also include suspension-adapted host cells, such as HeLa, 293, and SF-9 cells, that can be cultured in a variety of ways, including spinner flasks, stirred-tank bioreactors, and disposable systems (e.g., Wave bag systems).

[0241] The rAAV vector particles of the invention may be recovered from an rAAV production culture by lysis of the host cells of the production culture or by recovery of spent medium from the production culture, provided that the cells are cultured under conditions known in the art that result in the release of rAAV particles from intact cells into the medium, as more fully described in U.S. Patent No. 6,566,118. Suitable methods for lysing cells are also known in the art and include, for example, multiple freeze / thaw cycles, sonication, microfluidization, and treatment with chemicals, such as detergents / proteases.

[0242] In a further embodiment, the rAAV particles are purified. As used herein, the term "purified" includes preparations of rAAV particles that are free of at least some of the other components that may be present in the location in which the rAAV particles naturally occur or from which they were originally prepared. Thus, for example, isolated rAAV particles may be produced using a purification technique that enriches rAAV particles from a source mixture, such as a culture lysate or production culture supernatant. Enrichment can be measured in various ways, for example, by the proportion of DNase-resistant particles (DRP) or genome copies (gc) present in solution, or by infectivity, or can be measured with respect to secondary, potentially interfering substances present in the source mixture, such as impurities (including production culture impurities or in-process impurities, including helper viruses, media components, etc.).

[0243] In some embodiments, the rAAV production culture harvest is clarified to remove host cell debris. In some embodiments, the production culture harvest is clarified by filtration through a series of depth filters, including, for example, a grade DOHC Millipore Millistak+ HC Pod filter, a grade A1HC Millipore Millistak+ HC Pod filter, and a 0.2 μm filter Opticap XL10 Millipore Express SHC hydrophilic membrane filter. Clarification can also be achieved by a variety of other standard techniques known in the art, such as centrifugation or filtration through any cellulose acetate filter with a pore size of 0.2 μm or larger known in the art.

[0244] In some embodiments, the rAAV production culture harvest is further treated with Benzonase® to digest any high molecular weight DNA present in the production culture. In some embodiments, Benzonase® digestion is performed at a final concentration of, for example, 1-2 mL. The incubation is carried out under standard conditions known in the art, including 0.5 units / mL of Benzonase® at temperatures ranging from ambient to 37°C for periods ranging from 30 minutes to several hours.

[0245] rAAV particles may be isolated or purified using one or more of the following purification steps: equilibrium centrifugation; flow-through anion exchange filtration; tangential flow filtration (TFF) to concentrate rAAV particles; rAAV capture by apatite chromatography; heat inactivation of helper virus; rAAV capture by hydrophobic interaction chromatography; buffer exchange by size exclusion chromatography (SEC); nanofiltration; and rAAV capture by anion exchange chromatography, cation exchange chromatography, or affinity chromatography. These steps may be used alone, in various combinations, or in different orders. In some embodiments, the method includes all of the steps in the following order: Methods for purifying rAAV particles can be found, for example, in Xiao et al. (1998) Journal of Virology 72:2224-2232; U.S. Patent Nos. 6,989,264 and 8,137,948; and WO2010 / 148143.

[0246] Also provided herein is a pharmaceutical composition comprising an rAAV particle containing a heterologous nucleic acid encoding a therapeutic polypeptide and / or therapeutic nucleic acid, the rAAV particle comprising an rAAV capsid containing one or more substitutions of amino acids that interact with HSPG, and a pharmaceutically acceptable carrier. The pharmaceutical composition may be suitable for any of the administration modes described herein, such as subretinal administration.

[0247] In some embodiments, pharmaceutical compositions comprising the rAAV described herein and a pharmaceutically acceptable carrier are suitable for administration to humans. Such carriers are well known in the art (see, e.g., Remington's Pharmaceutical Sciences, 15th Edition, pp. 1035-1038 and 1570-1580). In some embodiments, pharmaceutical compositions comprising the rAAV described herein and a pharmaceutically acceptable carrier are suitable for ocular injection. Such pharmaceutically acceptable carriers can be sterile liquids, such as water and oils (including those of petroleum, animal, vegetable, or synthetic origin, e.g., peanut oil, soybean oil, mineral oil, etc.). Saline solutions and aqueous dextrose, polyethylene glycol (PEG), and glycerol solutions can also be used as liquid carriers, particularly for injectable solutions. Pharmaceutical compositions may further comprise additional components, such as preservatives, buffers, tonicity agents, antioxidants and stabilizers, non-ionic wetting or clarifying agents, thickeners, etc. The pharmaceutical compositions described herein may be packaged in single unit dosage form or in multidose form. The compositions are generally formulated as sterile, substantially isotonic solutions.

[0248] VII. Systems and Kits The rAAV compositions described herein may be included within a system designed for use in one of the inventive methods described herein.

[0249] subretinal delivery In some embodiments, the invention provides a system for subretinal delivery of a vector to the eye of an individual, the system comprising: a) a composition comprising an effective amount of rAAV particles, wherein i) the capsid protein of the rAAV particles comprises one or more amino acid substitutions at one or more positions that interact with heparan sulfate proteoglycans, and ii) the vector comprises a heterologous nucleic acid encoding a therapeutic polypeptide or therapeutic RNA and at least one AAV terminal repeat sequence; and b) a device for retinal delivery of the rAAV.

[0250] Generally, the system includes a small diameter cannula, such as 27-45 gauge, one or more (e.g., 1, 2, 3, 4 or more) syringes, and one or more (e.g., 1, 2, 3, 4 or more) fluids suitable for use in the methods of the invention.

[0251] A small diameter cannula is suitable for subretinal injection of vector suspensions and / or other fluids injected into the subretinal space. In some embodiments, the cannula is 27-45 gauge. In some embodiments, the small diameter cannula is 35-41 gauge. In some embodiments, the small diameter cannula is 40 or 41 gauge. In some embodiments, the small diameter cannula is 41 gauge. The cannula may be any suitable type of cannula, such as a de-Juan® cannula or an Eagle® cannula.

[0252] The syringe may be any suitable syringe, provided that it is capable of connection to a cannula for fluid delivery. In some embodiments, the syringe is an Accurus® system syringe. In some embodiments, the system has one syringe. In some embodiments, the system has two syringes. In some embodiments, the system has three syringes. In some embodiments, the system has four or more syringes.

[0253] The system may further include an automatic injection pump, which may be activated, for example, by a foot pedal.

[0254] Suitable fluids for use in the methods of the invention include those described herein, e.g., one or more fluids each comprising an effective amount of one or more vectors described herein, one or more fluids for forming the initial bleb (e.g., saline or other suitable fluids), and one or more fluids comprising one or more therapeutic agents.

[0255] Suitable fluids for use in the methods of the invention include those described herein, e.g., one or more fluids each containing an effective amount of one or more vectors described herein, one or more fluids for forming the initial bleb (e.g., saline or other suitable fluids), and one or more fluids containing one or more therapeutic agents.

[0256] In some embodiments, the volume of the fluid containing an effective amount of the vector is greater than about 0.8 ml. In some embodiments, the volume of the fluid containing an effective amount of the vector is at least about 0.9 ml. In some embodiments, the volume of the fluid containing an effective amount of the vector is at least about 1.0 ml. In some embodiments, the volume of the fluid containing an effective amount of the vector is at least about 1.5 ml. In some embodiments, the volume of the fluid containing an effective amount of the vector is at least about 2.0 ml. In some embodiments, the volume of the fluid containing an effective amount of the vector is greater than about 0.8 to about 3.0 ml. In some embodiments, the volume of the fluid containing an effective amount of the vector is greater than about 0.8 to about 2.5 ml. In some embodiments, the volume of the fluid containing an effective amount of the vector is greater than about 0.8 to about 2.0 ml. In some embodiments, the volume of the fluid containing an effective amount of the vector is greater than about 0.8 to about 1.5 ml. In some embodiments, the volume of the fluid containing an effective amount of the vector is greater than about 0.8 to about 1.0 ml. In some embodiments, the volume of the fluid containing the effective amount of the vector is about 0.9 to about 3.0 ml. In some embodiments, the volume of the fluid containing the effective amount of the vector is about 0.9 to about 2.5 ml. In some embodiments, the volume of the fluid containing the effective amount of the vector is about In some embodiments, the volume of the fluid containing an effective amount of the vector is about 0.9 to about 2.0 ml. In some embodiments, the volume of the fluid containing an effective amount of the vector is about 0.9 to about 1.5 ml. In some embodiments, the volume of the fluid containing an effective amount of the vector is about 0.9 to about 1.0 ml. In some embodiments, the volume of the fluid containing an effective amount of the vector is about 1.0 to about 3.0 ml. In some embodiments, the volume of the fluid containing an effective amount of the vector is about 1.0 to about 2.0 ml.

[0257] The fluid to form the initial bleb may be, for example, about 0.1 to about 0.5 ml. In some embodiments, the total volume of all fluids in the system is about 0.5 to about 3.0 ml.

[0258] In some embodiments, the system includes a single fluid (e.g., a fluid containing an effective amount of a vector). In some embodiments, the system includes two fluids. In some embodiments, the system includes three fluids. In some embodiments, the system includes four or more fluids.

[0259] The systems of the invention may be further packaged in a kit, which may further include instructions for use. In some embodiments, the kit further includes a device for subretinal delivery of a composition of rAAV particles. In some embodiments, the instructions include instructions according to one of the methods described herein. In some embodiments, the instructions include instructions for subretinal delivery of rAAV particles comprising capsids with one or more amino acid substitutions that alter, reduce, or eliminate binding of the rAAV particles to HSPGs.

[0260] CNS delivery The present invention provides a kit for delivering a heterologous nucleic acid to the CNS of an individual, the kit comprising a composition containing rAAV particles, the rAAV particles comprising (a) an rAAV capsid comprising an rAAV capsid protein comprising one or more amino acid substitutions at one or more positions that interact with heparan sulfate proteoglycans, and (b) an rAAV vector comprising a heterologous nucleic acid and at least one AAV inverted terminal repeat.Further provided herein is a kit for treating a CNS disorder in an individual, the kit comprising a composition containing rAAV particles, the rAAV particles comprising a) an rAAV capsid comprising an rAAV capsid protein comprising one or more amino acid substitutions at one or more positions that interact with heparan sulfate proteoglycans, and b) an rAAV vector comprising a heterologous nucleic acid for treating a CNS disorder and at least one AAV inverted terminal repeat.

[0261] The kit may include any rAAV particle or rAAV particle composition of the invention. For example, the kit may include an rAAV particle having an rAAV capsid containing an rAAV capsid protein containing one or more amino acid substitutions at one or more positions that interact with heparan sulfate proteoglycans (e.g., one or more amino acid substitutions that reduce binding of the rAAV particle to heparan sulfate proteoglycans, e.g., substitutions at R484, R487, K527, K532, R585, and / or R588, numbered based on VP1 of AAV2), and an rAAV vector containing a heterologous nucleic acid and at least one AAV inverted terminal repeat.

[0262] ...

Claims

1. 1. A composition comprising recombinant adeno-associated virus (rAAV) particles for use in delivering heterologous nucleic acid to the central nervous system (CNS) of an individual, comprising: wherein the rAAV particles are formulated for administration of recombinant adeno-associated virus (rAAV) particles to the CNS of the individual; (i) the rAAV particles are a) an AAV2 capsid comprising an AAV2 capsid protein that comprises an R585A substitution, an R588A substitution, or an R585A and an R588A substitution, numbered based on the VP1 numbering of AAV2; and b) a rAAV vector genome comprising said heterologous nucleic acid and at least one AAV inverted terminal repeat sequence. AAV serotype 2 (AAV2) particles comprising: The composition.

2. 1. A composition comprising recombinant adeno-associated virus (rAAV) particles for use in improving rAAV transduction of cells of the central nervous system (CNS) of an individual compared to transduction of the cells with a rAAV comprising a wild-type capsid, comprising: wherein the rAAV particles are formulated for administration of recombinant adeno-associated virus (rAAV) particles to the CNS of the individual; (i) the rAAV particles are a) an AAV2 capsid comprising an AAV2 capsid protein that comprises an R585A substitution, an R588A substitution, or an R585A and an R588A substitution, numbered based on the VP1 numbering of AAV2; and b) a rAAV vector genome comprising a heterologous nucleic acid and at least one AAV inverted terminal repeat sequence. AAV serotype 2 (AAV2) particles comprising: The composition.

3. For use in enhancing expression of a heterologous nucleic acid in the central nervous system (CNS) of an individual.

1. A composition comprising recombinant adeno-associated virus (rAAV) particles, wherein the rAAV particles are formulated for administration of recombinant adeno-associated virus (rAAV) particles to the CNS of the individual; (i) the rAAV particles are a) an AAV2 capsid comprising an AAV2 capsid protein that comprises an R585A substitution, an R588A substitution, or an R585A and an R588A substitution, numbered based on the VP1 numbering of AAV2; and b) a rAAV vector genome comprising a heterologous nucleic acid and at least one AAV inverted terminal repeat sequence. AAV serotype 2 (AAV2) particles comprising: The composition.

4. 1. A composition comprising recombinant adeno-associated virus (rAAV) particles for use in treating a disorder of the central nervous system (CNS) in an individual, comprising: wherein the rAAV particles are formulated for administration to the CNS of the individual of an effective amount of a composition comprising the rAAV particles; (i) the rAAV particles a) an AAV2 capsid comprising an AAV2 capsid protein that comprises an R585A substitution, an R588A substitution, or an R585A and an R588A substitution, numbered based on the VP1 numbering of AAV2; and b) a rAAV vector genome comprising a heterologous nucleic acid and at least one AAV inverted terminal repeat sequence. AAV serotype 2 (AAV2) particles comprising: The composition.

5. The composition of claim 1 , wherein administration comprises direct spinal injection, intracranial and / or intracerebral administration.

6. The composition of claim 1 , wherein the heterologous nucleic acid is expressed in one or more cells of the CNS.

7. The composition of claim 6, wherein the one or more cells of the CNS are oligodendrocytes, astrocytes, neurons, brain parenchymal cells, microglial cells, ependymal cells, and / or Purkinje cells.

8. The composition of claim 1 , wherein the heterologous nucleic acid is expressed in a neuron.

9. The composition of claim 1 , wherein the one or more amino acid substitutions comprise a substitution of a positively charged amino acid residue with a non-positively charged amino acid residue.

10. The composition of claim 9 , wherein a positively charged amino acid residue is replaced by a hydrophobic amino acid residue.

11. The composition of claim 1 , wherein the one or more amino acid substitutions comprise a substitution of an arginine or lysine residue.

12. 12. The composition of claim 11, wherein the one or more amino acid substitutions comprise substitution of an arginine or lysine residue with an alanine residue.

13. The rAAV particles are at least about 90% identical to SEQ ID NOs: 2, 4 and / or 6, 2. The composition of claim 1, comprising one or more rAAV capsid proteins having at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% sequence identity to said rAAV capsid protein.

14. The composition of claim 1 , wherein the one or more amino acid substitutions include R585A and R588A substitutions.

15. The composition of claim 1 , wherein the heterologous nucleic acid encodes a therapeutic polypeptide or a therapeutic nucleic acid.

16. The composition of claim 1 , wherein the heterologous nucleic acid encodes a CNS-related gene.

17. The heterologous nucleic acid may be an enzyme, a neurotrophic factor, a polypeptide deficient or mutated in an individual with a CNS-related disorder, an anti-apoptotic factor, an anti-angiogenic factor, and an anti-inflammatory factor, α-synuclein, acid β-glucosidase (GBA), β-galactosidase-1 (GLB1), iduronate 2-sulfatase (IDS), galactosylceramidase (GALC), mannosidase, α-D-mannosidase (MAN2B1), β-mannosidase (MAN2B2), β-mannosidase (MAN2B3), β-mannosidase (MAN2B4), β-mannosidase (MAN2B5), β-mannosidase (MAN2B6), β-mannosidase (MAN2B7), β-mannosidase (MAN2B8), β-mannosidase (MAN2B9), β-mannosidase (MAN2B10), β-mannosidase (MAN2B11), β-mannosidase (MAN2B12), β-mannosidase (MAN2B13), β-mannosidase (MAN2B14), β-mannosidase (MAN2B15), β-mannosidase (MAN2B16), β-mannosidase (MAN2B17), β-mannosidase (MAN2B18), β-mannosidase (MAN2B19), β-mannosidase (MAN2B19), β-mannosidase (MAN2B19), β-mannosidase (MAN2B19), β-mannosidase (MAN2B11), β-mannosidase (MAN2B12), β-mannosidase (MAN2B13), β-mannosidase (MAN2B14), β-mannosidase (MAN2B15), β-mannosidase (MAN2B16), β-mannosidase (MAN2B17), β-mannosidase (MAN2B18), β BA), pseudoarylsulfatase A (ARSA), N-acetylglucosamine 1-phosphotransferase (GNPTAB), acid sphingomyelinase (ASM), Niemann-Pick C protein (NPC1), acid α-1,4-glucosidase (GAA), hexosaminidase β subunit, HEXB, N-sulfoglucosamine sulfohydrolase (MPS3A), N-α-acetylglucosaminidase (NAGLU), heparin A Cetyl-CoA, α-glucosaminidase N-acetyltransferase (MPS3C), N-acetylglucosamine-6-sulfatase (GNS), α-N-acetylgalactosaminidase (NAGA), β-glucuronidase (GUSB), hexosaminidase α subunit (HEXA), huntingtin (HTT), lysosomal acid lipase (LIPA), aspartylglucosaminidase, α-galactosidase A, palmitoyl tan 2. The composition of claim 1, wherein the polypeptide encodes a polypeptide selected from the group consisting of protein thioesterase, tripentidyl peptidase, lysosomal transmembrane protein, cysteine ​​transporter, acid ceramidase, acid α-L-fucosidase, cathepsin A, α-L-iduronidase, arylsulfatase B, arylsulfatase A, N-acetylgalactosamine-6-sulfatase, acid β-galactosidase, or α-neuramidase.

18. 18. The composition of claim 17, wherein the heterologous nucleic acid encodes a polypeptide selected from the group consisting of neuronal apoptosis inhibitory protein (NAIP), nerve growth factor (NGF), glial cell line derived growth factor (GDNF), brain derived growth factor (BDNF), ciliary neurotrophic factor (CNTF), tyrosine hydroxylase (TH), GTP-cyclohydrolase (GTPCH), amino acid decarboxylase (AADC), anti-angiogenic polypeptides, anti-inflammatory polypeptides, and aspartoacylase (ASPA).

19. The composition of claim 1 , wherein the heterologous nucleic acid encodes a therapeutic nucleic acid.

20. The composition of claim 19, wherein the therapeutic nucleic acid is an siRNA, shRNA, RNAi, miRNA, antisense RNA, ribozyme or DNAzyme.

21. The composition of claim 1 , wherein the heterologous nucleic acid is under the control of a promoter sequence that is expressed in one or more cells of the CNS.

22. The heterologous nucleic acid may be a cytomegalovirus (CMV) immediate early promoter, an RSV LTR, or a MoMLV 2. The composition of claim 1, wherein the gene is under the control of a promoter selected from the group consisting of LTR, phosphoglycerate kinase-1 (PGK) promoter, simian virus 40 (SV40) promoter, CK6 promoter, transthyretin promoter (TTR), TK promoter, tetracycline responsive promoter (TRE), HBV promoter, hAAT promoter, LSP promoter, chimeric liver specific promoter (LSP), E2F promoter, telomerase (hTERT) promoter; cytomegalovirus enhancer / chicken β-actin / rabbit β-globin promoter (CAG) promoter, elongation factor 1-α promoter (EF1-α) promoter, human β-glucuronidase promoter, chicken β-actin (CBA) promoter, retroviral Rous sarcoma virus (RSV) LTR promoter, dihydrofolate reductase promoter, and β-actin promoter.

23. The composition of claim 21 , wherein the heterologous nucleic acid is operably linked to a promoter suitable for expression of a therapeutic polypeptide or therapeutic nucleic acid in one or more cells of the CNS.

24. 24. The composition of claim 23, wherein the one or more cells of the CNS are oligodendrocytes, astrocytes, neurons, brain parenchymal cells, microglial cells, ependymal cells, and / or Purkinje cells.

25. The composition of claim 23 , wherein the cell of the CNS is a neuron.

26. The composition of claim 1 , wherein the rAAV vector is a self-complementary rAAV vector.

27. The composition of claim 1 , wherein the individual is a human.

28. The composition of claim 1 , wherein the heterologous nucleic acid encodes a therapeutic polypeptide or therapeutic nucleic acid used to treat a disorder of the CNS.

29. 29. The composition of claim 28, wherein the CNS disorder is a lysosomal storage disease (LSD), Huntington's disease, epilepsy, Parkinson's disease, Alzheimer's disease, stroke, corticobasal degeneration (CBD), corticobasal ganglionic degeneration (CBGD), frontotemporal dementia (FTD), multiple system atrophy (MSA), progressive supranuclear palsy (PSP), or brain cancer.

30. Disorders include aspartylglucosaminuria, Fabry, childhood Batten disease (CNL1), classical late-onset childhood Batten disease (CNL2), juvenile Batten disease (CNL3), Batten CNL4, Batten CNL5, Batten CNL6, Batten CNL7, Batten CNL8, cystinosis, Farber, fucosidosis, galactosidic sialidosis, Gaucher disease type 1, Gaucher disease type 2, Gaucher disease type 3, GM1 gangliosidosis, Hunter disease, Krabbe disease, alpha-mannosidosis, beta-mannosidosis 30. The composition of claim 29, wherein the lysosomal storage disease is selected from the group consisting of leukodystrophy, Maroteaux-Lamy, metachromatic leukodystrophy, Morquio A, Morquio B, mucolipidosis II / III, Niemann-Pick disease A, Niemann-Pick disease B, Niemann-Pick disease C, Pompe disease, Sandhoff disease, Sanfilippo disease A, Sanfilippo disease B, Sanfilippo disease C, Sanfilippo disease D, Schindler disease, Schindler-Kanzaki, sialidosis, Sly disease, Tay-Sachs disease, and Wolman disease.

31. 1. A composition comprising recombinant adeno-associated virus (rAAV) particles for use in treating Huntington's disease in an individual, comprising: wherein the rAAV particles are formulated for administration to the striatum of the individual of an effective amount of a composition comprising recombinant adeno-associated virus (rAAV) particles; (i) the rAAV particles a) an AAV2 capsid comprising an AAV2 capsid protein that comprises an R585A substitution, an R588A substitution, or an R585A and an R588A substitution, numbered based on the VP1 numbering of AAV2; and b) a rAAV vector genome comprising a heterologous nucleic acid and at least one AAV inverted terminal repeat sequence. AAV serotype 2 (AAV2) particles comprising: The composition.

32. The heterologous nucleic acid is Therapeutic Polypeptides or Nucleic Acids The composition of claim 31 , wherein the composition encodes:

33. The composition of claim 32, wherein the therapeutic nucleic acid is a miRNA.

34. 1. A composition comprising a recombinant adeno-associated virus (rAAV) particle for use in treating Parkinson's disease in an individual, comprising: wherein the rAAV particles are formulated for administration to the striatum of the individual of an effective amount of a composition comprising recombinant adeno-associated virus (rAAV) particles; (i) the rAAV particles a) an AAV2 capsid comprising an AAV2 capsid protein that comprises an R585A substitution, an R588A substitution, or an R585A and an R588A substitution, numbered based on the VP1 numbering of AAV2; and b) a rAAV vector genome comprising a heterologous nucleic acid and at least one AAV inverted terminal repeat sequence. AAV serotype 2 (AAV2) particles comprising: The composition.

35. The composition of claim 34 , wherein the heterologous nucleic acid encodes a therapeutic polypeptide or a therapeutic nucleic acid.

36. 36. The composition of claim 35, wherein the therapeutic polypeptide is TH, GTPCII, GDNF, BDNF and / or AADC; or a fragment thereof.

37. The composition of claim 31 , wherein the rAAV particle comprises an AAV serotype 2 (AAV2) capsid.

38. The composition of claim 31 , wherein the heterologous nucleic acid is expressed in one or more cells of the CNS.

39. 39. The composition of claim 38, wherein the one or more cells of the CNS are oligodendrocytes, astrocytes, neurons, brain parenchymal cells, microglial cells, ependymal cells, and / or Purkinje cells.

40. The composition of claim 31 , wherein the heterologous nucleic acid is expressed in a neuron.

41. 32. The composition of claim 31, wherein the one or more amino acid substitutions comprise a substitution of a positively charged amino acid residue with a non-positively charged amino acid residue.

42. 42. The composition of claim 41, wherein a positively charged amino acid residue is replaced by a hydrophobic amino acid residue.

43. 32. The composition of claim 31, wherein the one or more amino acid substitutions comprise a substitution of an arginine or lysine residue.

44. 44. The composition of claim 43, wherein the one or more amino acid substitutions comprises substitution of an arginine or lysine residue with an alanine residue.

45. The composition of claim 31, wherein the rAAV particles comprise one or more rAAV capsid proteins having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% sequence identity to SEQ ID NOs: 2, 4 and / or 6.

46. The composition of claim 31 , wherein the heterologous nucleic acid is under the control of a promoter sequence that is expressed in one or more cells of the CNS.

47. The heterologous nucleic acid may be a cytomegalovirus (CMV) immediate early promoter, an RSV LTR, or a MoMLV 32. The composition of claim 31, wherein the nucleic acid sequence is under the control of a promoter sequence selected from the group consisting of LTR, phosphoglycerate kinase-1 (PGK) promoter, simian virus 40 (SV40) promoter, CK6 promoter, transthyretin promoter (TTR), TK promoter, tetracycline responsive promoter (TRE), HBV promoter, hAAT promoter, LSP promoter, chimeric liver specific promoter (LSP), E2F promoter, telomerase (hTERT) promoter; cytomegalovirus enhancer / chicken β-actin / rabbit β-globin promoter (CAG) promoter, elongation factor 1-α promoter (EF1-α) promoter, human β-glucuronidase promoter, chicken β-actin (CBA) promoter, retroviral Rous sarcoma virus (RSV) LTR promoter, dihydrofolate reductase promoter, and β13-actin promoter.

48. The composition of claim 46, wherein the heterologous nucleic acid is operably linked to a promoter suitable for expression of a therapeutic polypeptide or therapeutic nucleic acid in one or more cells of the CNS.

49. 49. The composition of claim 48, wherein the one or more cells of the CNS are oligodendrocytes, astrocytes, neurons, brain parenchymal cells, microglial cells, ependymal cells, and / or Purkinje cells.

50. The composition of claim 31 , wherein the rAAV vector is a self-complementary rAAV vector.

51. 51. The composition of claim 50, wherein the vector comprises a first nucleic acid sequence encoding a heterologous nucleic acid and a second nucleic acid sequence encoding a complementary strand of the nucleic acid, the first nucleic acid sequence being capable of interstrand base pairing with the second nucleic acid sequence over most or all of its length.

52. 52. The composition of claim 51 , wherein the first and second nucleic acid sequences are linked by a mutated AAV ITR, the mutated AAV ITR comprising a deletion of the D region and comprising a mutation in the truncation sequence.

53. The composition of claim 31 , wherein the individual is a human.

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