AAV vectors for retinal and CNS gene therapy

Abstract

To provide a method for delivering a heterologous nucleic acid to the eye of an individual, to provide a system for subretinal delivery of a vector to an eye of an individual, and to provide a kit for treating an ocular disorder.SOLUTION: Provided is a method for delivering a heterologous nucleic acid to the eye of an individual, comprising administering a recombinant adeno-associated virus (rAAV) particle to the subretina of the individual, the rAAV particle comprising: a) a rAAV capsid comprising rAAV capsid proteins comprising one or more amino acid substitutions at one or more positions that interacts with a heparan sulfate proteoglycan or at one or more positions corresponding to amino acids 484, 487, 527, 532, 585 or 588, numbering based on VP1 numbering of AAV2; and b) a rAAV vector comprising the heterologous nucleic acid and at least one AAV inverted terminal repeat.SELECTED DRAWING: None

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority to U.S. Provisional Patent Application No. 61 / 988,131, filed on 2 May 2014, and U.S. Provisional Patent Application No. 62 / 114,575, filed on 10 February 2015, each of which is incorporated herein by reference in its entirety.

[0002] Submission of sequence listings in ASCII text files. The following submission in ASCII text file is incorporated herein by reference in its entirety: a sequence listing in computer-readable format (CRF) (filename: 159792010440SEQLIST.txt, data recording date: April 29, 2015, size: 85KB).

[0003] The present invention relates to a mutant recombinant adeno-associated virus (rAAV) vector 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 a promising target for adeno-associated virus (AAV)-mediated gene therapy. AAV vectors are an attractive option for gene delivery to the eye because they can mediate long-term gene expression in the retina and evoke minimal immune responses. The retina is the light-sensing tissue at the back of the eyeball, composed of various cell types including photoreceptor cells, retinal pigment epithelial cells, and retinal ganglion cells. The target cell type and vector delivery pathway for AAV gene therapy vectors will depend on the manifestations of the disease. For example, in a Phase I trial for age-related macular degeneration, intravitreal delivery of the vector was used to achieve transduction into retinal ganglion cells, and in a recent trial treating patients with Leber congenital amaurosis type 2, a form of retinitis pigmentosa, subretinal delivery of the RPE65 gene was used to transduction into retinal pigment epithelial cells. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Kaplitt, MG et al. (2007) Lancet 369: pp. 2097-2105 [Non-Patent Document 2] Eberling, JL et al. (2008) Neurology 70: pp. 1980-1983 [Non-Patent Document 3] Fiandaca, MS et al. (2009) Neuroimage. 47 Supplement 2: pp. 27-35 [Overview of the project] [Problems that the invention aims to solve]

[0006] Given such usefulness, there is a need to develop novel drugs and methods to improve AAV delivery to the eye.

[0007] Adeno-associated virus (AAV)-based vectors are becoming a preferred vector system for neuronal gene therapy, and their excellent stability has been documented in multiple clinical trials (Non-Patent Literature 1; Non-Patent Literature 2; Non-Patent Literature 3). However, effective treatment of neurological disorders has been largely hindered by problems associated with the delivery of AAV vectors to affected cell populations. These delivery problems are Disorders involving the central nervous system (CNS) are a particular concern. Therefore, further improvements in AAV delivery to the CNS are necessary. [Means for solving the problem]

[0008] In some embodiments, the present invention provides a method for delivering heterologous nucleic acid to the eye of an individual, comprising administering recombinant adeno-associated virus (rAAV) particles subretinally to the individual, wherein the rAAV particles comprise an rAAV capsid containing an rAAV capsid protein with 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 an rAAV vector comprising heterologous nucleic acid and at least one AAV inverted terminal repeat sequence. In some embodiments, the rAAV particles comprise an AAV serotype 2 (AAV2) capsid. In some embodiments, one or more amino acid substitutions reduce the binding of the rAAV particles to heparan sulfate proteoglycans. In some embodiments, one or more amino acid substitutions reduce the binding of 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, one or more amino acid substitutions increase the transduction efficiency of rAAV particles to cells of the eye or CNS. In some embodiments, one or more amino acid substitutions increase the transduction efficiency of rAAV particles to 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% compared to a reference rAAV capsid containing, for example, wild-type AAV capsid protein. In some embodiments, cells of the eye are retinal cells, photoreceptor cells, retinal pigment 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, microglia, ependymal cells, and / or Purkinje cells.

[0009] In some embodiments, the AAV particles of the present invention include a capsid having one or more amino acid substitutions that are numbered based on the VP1 numbering of AAV2 and are at positions 484, 487, 527, 532, 585, or 588. In some embodiments, the numbering is based on the VP1 of AAV2, which includes the amino acid sequence of SEQ ID NO: 1. In some embodiments, one or more amino acid substitutions include the 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, one or more amino acid substitutions include the substitution of an arginine or lysine residue with an alanine residue. In some embodiments, one or more amino acid substitutions include substitutions at positions R484, R487, K527, K532, R585, and / or R588, which are numbered based on the VP1 of AAV2. In some embodiments, the rAAV particles contain 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, one or more amino acid substitutions include substitutions at position R532. In some embodiments, one or more amino acid substitutions include substitutions at positions R484 and R487 or R585 and R588, numbered based on VP1 of AAV2. In further embodiments, one Or further amino acid substitutions are numbered based on VP1 of AAV2 and include R484A and R487A substitutions or R585A and R588A substitutions. In a further embodiment, the AAV capsid is numbered based on VP1 of AAV-2 and includes amino acid substitutions R585A and R588A. In some embodiments, the rAAV particles include AAV1 capsid, AAV2 capsid, AAV3 capsid, AAV6 capsid, AAV8 capsid, AAVrh8R capsid, AAV9 capsid, or AAVrh10 capsid.

[0010] In some embodiments, the AAV particles of the present invention include a capsid having one or more amino acid substitutions that are numbered based on the VP1 numbering of AAVrh8R and are located at positions 485, 488, 528, 533, 586, or 589. In some embodiments, the AAV particles include a capsid having one or more amino acid substitutions that are numbered based on the VP1 numbering of AAVrh8R and are located at positions 485, 488, 528, or 533. In some embodiments, the numbering is based on the VP1 of AAVrh8R, which includes the amino acid sequence of SEQ ID NO: 9. In some embodiments, one or more amino acid substitutions include the 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, one or more amino acid substitutions include the substitution of an arginine or lysine residue with an alanine residue. In other embodiments, one or more amino acid substitutions include the substitution of a non-positively charged amino acid residue with a positively charged amino acid residue. In some embodiments, hydrophobic amino acid residues are substituted with positively charged amino acid residues. In further embodiments, one or more amino acid substitutions include the substitution of an alanine residue. In yet another embodiment, one or more amino acid substitutions include the substitution 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 include a capsid having one or more amino acid substitutions that are 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, which includes the amino acid sequence of SEQ ID NO: 9.In some embodiments, amino acid substitutions include substitutions at positions R485, R488, R533, or T589, numbered based on the VP1 numbering of AAVrh8R. In some embodiments, rAAV particles contain the rAAV capsid protein of SEQ ID NO: 11. In some embodiments, rAAV particles contain 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 with respect to SEQ ID NO: 11. In some embodiments, one amino acid substitution includes the R533A substitution, numbered based on the VP1 numbering of AAVrh8R.

[0011] In some embodiments, the rAAV particles comprise an AAV1, AAV6, or AAV9 capsid, with one or more amino acid substitutions 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 AAVrh10 capsid, with one or more amino acid substitutions at positions 487, 490, 535, 588, and / or 591, numbered based on the VP1 numbering of AAV8 or AAVrh10. .

[0012] In some embodiments, the AAV particles of the present invention include a capsid comprising one or more amino acid substitutions that modify the binding to HSPG (e.g., reduce or eliminate the 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 an rAAV vector comprising a heterogeneous nucleic acid encoding a therapeutic polypeptide or therapeutic nucleic acid. In some embodiments, the heterogeneous nucleic acid encodes a polypeptide selected from the group consisting of antioxidants, neurotrophic factors, anti-apoptotic factors, anti-angiogenic factors, and anti-inflammatory factors. 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, Clarine, 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-R I, sTNF-R II, and IL4. In other embodiments, the heterologous nucleic acid encodes a therapeutic nucleic acid. In further embodiments, the therapeutic nucleic acid is 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 include a capsid comprising 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, which modifies binding to HSPG (e.g., reducing or eliminating binding to HSPG), and a heterogeneous nucleic acid encoding a therapeutic polypeptide or therapeutic nucleic acid, the heterogeneous nucleic acid being under the control of a promoter sequence expressed in the retina. In some embodiments, the heterogeneous nucleic acid is operably linked to a promoter suitable for the 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 pigment 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 present invention include a capsid comprising one or more amino acid substitutions that modify the binding to HSPG (e.g., reduce or eliminate the 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 heterogeneous nucleic acids for delivery of heterogeneous nucleic acids to the retina of an individual. In some embodiments, the individual is a human. In some embodiments, heterologous nucleic acids are used to treat ocular disorders selected from the group consisting of: autosomal recessive severe early-onset retinal degeneration (Leber congenital amaurosis), congenital color blindness, Stargardt disease, Best's disease, Doin'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 present invention provides a method for improving rAAV transduction into cells after subretinal delivery of rAAV particles into the eye of an individual compared to transduction into cells with rAAV containing a wild-type capsid, comprising incorporating one or more amino acid substitutions into the 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 method provides that the rAAV particles comprise an rAAV capsid protein and an rAAV vector comprising heterogeneous nucleic acids and at least one AAV terminal repeat sequence.

[0016] In another embodiment, the present invention provides a method for improving heterologous nucleic acid expression after 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 method provides that the rAAV particles comprise an rAAV capsid protein and an rAAV vector comprising heterologous nucleic acid and at least one AAV terminal repeat sequence.

[0017] In some embodiments, rAAV particles with improved transduction and / or improved expression of heterologous nucleic acids contain an AAV serotype 2 (AAV2) capsid. In some embodiments, one or more amino acid substitutions reduce the binding of rAAV particles to heparan sulfate proteoglycans. In some embodiments, one or more amino acid substitutions reduce the binding of 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 include a capsid having one or more amino acid substitutions that are numbered based on the VP1 numbering of AAV2 and are at positions 484, 487, 527, 532, 585, or 588. In some embodiments, the numbering is based on the VP1 of AAV2, which includes the amino acid sequence of SEQ ID NO: 1. In some embodiments, one or more amino acid substitutions include the 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, one or more amino acid substitutions include the substitution of an arginine or lysine residue with an alanine residue. In some embodiments, one or more amino acid substitutions include substitutions at positions R484, R487, K527, K532, R585 and / or R588, numbered based on VP1 of AAV2. In some embodiments, the rAAV particles contain 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, one or more amino acid substitutions include substitutions at positions R484 and R487 or at positions R585 and R588, numbered based on VP1 of AAV2. In further embodiments, one or more amino acid substitutions are numbered based on VP1 of AAV2, including R484A and R487A substitutions or R585A and R588A substitutions. In further embodiments, the AAV capsid is AAV- Numbered based on VP1 of 2, the particles include amino acid substitutions R585A and R588A. In some embodiments, the rAAV particles include AAV1 capsid, AAV2 capsid, AAV3 capsid, AAV6 capsid, AAV8 capsid, AAVrh8R capsid, AAV9 capsid, or AAVrh10 capsid.

[0019] In some embodiments, rAAV particles with improved transduction and / or improved expression of heterologous nucleic acids include a capsid having one or more amino acid substitutions that are numbered based on the VP1 numbering of AAVrh8R and are located at positions 485, 488, 528, 533, 586, or 589. In some embodiments, the AAV particles of the present invention include a capsid having one or more amino acid substitutions that are numbered based on the VP1 numbering of AAVrh8R and are located at positions 485, 488, 528, or 533. In some embodiments, the numbering is based on the VP1 of AAVrh8R, which includes the amino acid sequence of SEQ ID NO: 9. In some embodiments, one or more amino acid substitutions include the 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, one or more amino acid substitutions include the substitution of an arginine or lysine residue. In further embodiments, one or more amino acid substitutions include the substitution of an arginine or lysine residue with an alanine residue. In other embodiments, one or more amino acid substitutions include the substitution of a non-positively charged amino acid residue with a positively charged amino acid residue. In some embodiments, hydrophobic amino acid residues are substituted with positively charged amino acid residues. In further embodiments, one or more amino acid substitutions include the substitution of an alanine residue. In further embodiments, one or more amino acid substitutions include the substitution 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 include a capsid having one or more amino acid substitutions that are 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 containing the amino acid sequence of SEQ ID NO: 9. In some embodiments, amino acid substitutions include substitutions at positions R485, R488, R533, or T589, numbered based on the VP1 numbering of AAVrh8R. In some embodiments, the rAAV particles contain the rAAV capsid protein of SEQ ID NO: 11. In some embodiments, the rAAV particles contain 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 with respect to SEQ ID NO: 11. In some embodiments, one amino acid substitution includes the R533A substitution, numbered based on VP1 of AAVrh8R.

[0020] In some embodiments, rAAV particles with enhanced transduction and / or enhanced expression of heterologous nucleic acids include an rAAV vector comprising a capsid containing 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 heterologous nucleic acids encoding a therapeutic polypeptide or therapeutic nucleic acid. In some embodiments, the heterologous nucleic acid encodes a polypeptide selected from the group consisting of antioxidants, neurotrophic factors, anti-apoptotic factors, anti-angiogenic factors, and anti-inflammatory factors. In further embodiments, heterologous nucleic acids The acid encodes a polypeptide selected from the group consisting of: Prph2, RPE65, AIPL1, GUCY2D, LCA5, CRX, CEP290, MYO 7a, Clarine, 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-R I, sTNF-R II, and IL4. In other embodiments, heterologous nucleic acids encode therapeutic nucleic acids. In further embodiments, the therapeutic nucleic acid is 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 enhanced transduction and / or enhanced heterologous nucleic acid expression include a capsid containing one or more amino acid substitutions that modify binding to HSPG (e.g., reducing or eliminating binding to HSPG) or one or more amino acid substitutions at one or more positions corresponding to amino acids 484, 487, 532, 585, or 588, numbered based on the VP1 numbering of AAV2, and a heterologous nucleic acid encoding a therapeutic polypeptide or therapeutic nucleic acid, the heterologous nucleic acid being under the control of a promoter sequence expressed in the retina. In some embodiments, the heterologous nucleic acid is operably linked to a promoter suitable for the 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 pigment 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, the transduction-enhanced and / or heterologous nucleic acid-enhanced rAAV particles include a capsid containing one or more amino acid substitutions that modify (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 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, heterologous nucleic acids are used to treat ocular disorders selected from the group consisting of: autosomal recessive severe early-onset retinal degeneration (Leber congenital amaurosis), congenital color blindness, Stargardt disease, Best's disease, Doin'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 a method for treating an eye disorder in an individual (e.g., a human), comprising delivery of a composition comprising rAAV particles to the retina of the individual, wherein 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, 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 rAAV particles comprise an AAV serotype 2 (AAV2) capsid. In some embodiments, one or more amino acid substitutions reduce the binding of the rAAV particles to heparan sulfate proteoglycans. Reduced. In some embodiments, one or more amino acid substitutions reduce the binding of 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%.

[0024] In some embodiments, the method comprises subretinal delivery of rAAV particles containing an rAAV vector encoding a heterologous nucleic acid used to treat an eye disorder in an individual (e.g., a human), wherein the rAAV particles contain a capsid having one or more amino acid substitutions that are numbered based on the VP1 numbering of AAV2 and are at positions 484, 487, 527, 532, 585, or 588. In some embodiments, the numbering is based on the VP1 of AAV2 containing the amino acid sequence of SEQ ID NO: 1. In some embodiments, one or more amino acid substitutions include the 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, one or more amino acid substitutions include the substitution of an arginine or lysine residue. In yet another embodiment, one or more amino acid substitutions include the substitution of an arginine or lysine residue with an alanine residue. In some embodiments, one or more amino acid substitutions include substitutions at positions R484, R487, K527, K532, R585 and / or R588, numbered based on VP1 of AAV2. In some embodiments, the rAAV particles contain 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, one or more amino acid substitutions include substitutions at positions R484 and R487 or at positions R585 and R588, numbered based on VP1 of AAV2. In a further embodiment, one or more amino acid substitutions include R484A and R487A substitutions or R585A and R588A substitutions, numbered based on VP1 of AAV2. In a further embodiment, the AAV capsid includes amino acid substitutions R585A and R588A, numbered based on VP1 of AAV-2.In some embodiments, the rAAV particles include AAV1 capsid, AAV2 capsid, AAV3 capsid, AAV6 capsid, AAV8 capsid, AAVrh8R capsid, AAV9 capsid, or AAVrh10 capsid.

[0025] In some embodiments, the method comprises subretinal delivery of rAAV particles containing an rAAV vector encoding a heterologous nucleic acid used to treat an eye disorder in an individual (e.g., a human), wherein the rAAV particles comprise a capsid having one or more amino acid substitutions that are at positions 485, 488, 528, 533, 586, or 589, 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 that are 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 containing the amino acid sequence of SEQ ID NO: 9. In some embodiments, one or more amino acid substitutions comprise the 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, one or more amino acid substitutions include substitution of an arginine or lysine residue. In yet another embodiment, one or more amino acid substitutions include substitution of an arginine or lysine residue with an alanine residue. In yet another embodiment, one or more amino acid substitutions include substitution of a non-positively charged amino acid residue with a positively charged amino acid residue. This includes substitutions by . In some embodiments, hydrophobic amino acid residues are substituted with positively charged amino acid residues. In further embodiments, one or more amino acid substitutions include substitutions of alanine residues. In yet further embodiments, one or more amino acid substitutions include 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 AAV particles of the present invention include a capsid having one or more amino acid substitutions that are 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 containing the amino acid sequence of SEQ ID NO: 9. In some embodiments, the amino acid substitutions include substitutions at positions R485, R488, R533, or T589, numbered based on the VP1 numbering of AAVrh8R. In some embodiments, the rAAV particles contain the rAAV capsid protein of SEQ ID NO: 11. In some embodiments, the rAAV particles contain 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 with respect to SEQ ID NO: 11. In some embodiments, one amino acid substitution includes the R533A substitution, numbered based on VP1 of AAVrh8R.

[0026] In some embodiments, the method comprises subretinal delivery of rAAV particles, the rAAV particles comprising an rAAV vector encoding a heterologous nucleic acid used to treat an eye disorder in an individual (e.g., a human), and a capsid comprising one or more amino acid substitutions that modify (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 antioxidants, neurotrophic factors, anti-apoptotic factors, anti-angiogenic factors, and anti-inflammatory factors. 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, Clarine, 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-R I, sTNF-R II, and IL4. In other embodiments, the heterologous nucleic acid encodes a therapeutic nucleic acid. In further embodiments, the therapeutic nucleic acid is 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 used to treat an eye disorder in an individual (e.g., a human), and a capsid comprising 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, which modifies (e.g., reduces or eliminates) binding to HSPG, and the heterologous nucleic acid sequence is under the control of a promoter sequence expressed in the retina. In some embodiments, the heterologous nucleic acid comprises one or more The above retinal cell types are operably linked to promoters suitable for the expression of therapeutic polypeptides or therapeutic nucleic acids. In some embodiments, the retinal cells are photoreceptor cells, retinal pigment epithelial cells, and / or ganglion cells. In some embodiments, the promoters are rhodopsin kinase (RK) promoters, opsin promoters, cytomegalovirus (CMV) promoters, and chicken β-actin (CBA) promoters.

[0028] In some embodiments, the method comprises subretinal delivery of rAAV particles, the rAAV particles comprising an rAAV vector encoding a heterologous nucleic acid used to treat eye disorders in an individual (e.g., a human), and one or more amino acid substitutions that modify (e.g., reduce or remove) the binding to HSPG or one or more amino acid substitutions at positions corresponding to amino acids 484, 487, 527, 532, 585 or 588, numbered based on the VP1 numbering of AAV2. The capsid comprises or contains further amino acid substitutions, and the eye disorder is selected from the group consisting of: autosomal recessive severe early-onset retinal degeneration (Leber congenital amaurosis), congenital color blindness, Stargardt disease, Best's disease, Doin'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 used to treat eye disorders in an individual (e.g., a human), and a capsid comprising one or more amino acid substitutions that modify (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 × 10⁻⁶ 6 DRP / ml ~ approximately 1 x 10 14The DRP / ml is effective in some embodiments for treating the visual function of an individual. In some embodiments, visual function is assessed by microperimeter measurement, dark-adapted perimetry, visual motion assessment, visual acuity, ERG, or reading assessment. In some embodiments, the method results in an improvement in the individual's visual function. In some embodiments, the method results in the prevention or slowing of the progression of visual impairment in a person due to the progression of eye damage.

[0030] In some embodiments, the present invention 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, 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 ii) the vector comprises a heteronucleotide 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 fine-diameter cannula and a syringe, the fine-diameter 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 × 10⁻⁶ 6 DRP / ml ~ approximately 1 x 10 14 It is DRP / ml.

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

[0032] In some embodiments, the rAAV particles of the system include an AAV2 capsid containing one or more amino acid substitutions that modify (e.g., reduce or eliminate) the HSPG bond. In some embodiments, one or more amino acid substitutions are at positions 484, 487, 527, 532, 585, or 588, numbered based on the VP1 numbering of AAV2. In some embodiments, the numbering is based on the VP1 of AAV2 containing the amino acid sequence of SEQ ID NO: 1. In some embodiments, one or more amino acid substitutions include the 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, one or more amino acid substitutions include the substitution of an arginine or lysine residue. In yet another embodiment, one or more amino acid substitutions include the substitution of an arginine or lysine residue with an alanine residue. In some embodiments, one or more amino acid substitutions include substitutions at positions R484, R487, K527, K532, R585 and / or R588, numbered based on VP1 of AAV2. In some embodiments, the rAAV particles contain 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, one or more amino acid substitutions include substitutions at positions R484 and R487 or at positions R585 and R588, numbered based on VP1 of AAV2. In a further embodiment, one or more amino acid substitutions include R484A and R487A substitutions or R585A and R588A substitutions, numbered based on VP1 of AAV2. In a further embodiment, the AAV capsid includes amino acid substitutions R585A and R588A, numbered based on VP1 of AAV-2.In some embodiments, the rAAV particles include AAV1 capsid, AAV2 capsid, AAV3 capsid, AAV6 capsid, AAV8 capsid, AAVrh8R capsid, AAV9 capsid, or AAVrh10 capsid.

[0033] In some embodiments, the rAAV particles of the system include an AAV2 capsid containing one or more amino acid substitutions that modify the HSPG bond (e.g., reduce or remove the bond). In some embodiments, the one or more amino acid substitutions are The numbering is based on the VP1 numbering of AAVrh8R and is at positions 485, 488, 528, 533, 586, or 589. In some embodiments, the numbering is based on the VP1 of AAVrh8R containing the amino acid sequence of SEQ ID NO: 9. In some embodiments, one or more amino acid substitutions include the 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, one or more amino acid substitutions include the substitution of an arginine or lysine residue. In yet another embodiment, one or more amino acid substitutions include the substitution of an arginine or lysine residue with an alanine residue. In yet another embodiment, one or more amino acid substitutions include the 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 yet another embodiment, one or more amino acid substitutions include the substitution of an alanine residue. In further embodiments, one or more amino acid substitutions include 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 containing the amino acid sequence of SEQ ID NO: 9. In some embodiments, the amino acid substitutions include substitutions at positions R485, R488, R533, or T589, numbered based on the VP1 numbering of AAVrh8R. In some embodiments, the rAAV particles contain the rAAV capsid protein of SEQ ID NO: 11.In some embodiments, the rAAV particles contain 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 with respect to SEQ ID NO: 11. In some embodiments, one amino acid substitution includes the R533A substitution, numbered based on VP1 of AAVrh8R.

[0034] In some embodiments, the rAAV particles of the system include an AAV capsid having 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 containing heterogeneous nucleic acids. In some embodiments, the heterogeneous nucleic acids encode therapeutic polypeptides or therapeutic nucleic acids. In some embodiments, the heterogeneous nucleic acids encode polypeptides selected from the group consisting of antioxidants, neurotrophic factors, anti-apoptotic factors, anti-angiogenic factors, and anti-inflammatory factors. 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, Clarine, 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-R I, sTNF-R II, and IL4. In other embodiments, the heterologous nucleic acid encodes a therapeutic nucleic acid. In further embodiments, the therapeutic nucleic acid is 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 modify the HSPG bond. For example, the rAAV vector comprises an AAV capsid having 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 heterogeneous nucleic acid, the heterogeneous nucleic acid being under the control of a promoter sequence expressed in the retina. In some embodiments, the heterogeneous nucleic acid is operably linked to a promoter suitable for the 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 pigment 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.

[0036] In some embodiments, rAAV particles of a system comprising an AAV capsid having one or more amino acid substitutions that modify the HSPG bond (e.g., reduce or remove the bond) 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 containing a heterologous nucleic acid, are used for delivery of the heterologous nucleic acid to the retina of an individual. In some embodiments, the individual is a human. In some embodiments, heterologous nucleic acids are used to treat ocular disorders selected from the group consisting of: autosomal recessive severe early-onset retinal degeneration (Leber congenital amaurosis), congenital color blindness, Stargardt disease, Best's disease, Doin'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 present invention provides a method 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, wherein the rAAV particles comprise a) an rAAV capsid containing 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 heterologous nucleic acids and at least one AAV inverted terminal repeat sequence. In some embodiments, the present invention provides a method for improving rAAV transduction into cells of an individual's central nervous system (CNS) compared to transduction into cells with rAAV comprising a wild-type capsid, the method comprising administering recombinant adeno-associated virus (rAAV) particles to the individual's CNS, the rAAV particles comprising a) 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 heterologous nucleic acid and at least one AAV inverted terminal repeat sequence. In a further embodiment, the present invention relates to a method for improving the expression of heterologous nucleic acids in the central nervous system (CNS) of an individual, comprising administering recombinant adeno-associated virus (rAAV) particles to the CNS of the individual, wherein the rAAV particles include: a) an rAAV capsid containing 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 The present invention provides a method comprising an rAAV vector comprising an AAV inverted terminal repeat sequence. In a further embodiment, the present invention provides a method for treating a disorder of the central nervous system (CNS) of an individual, comprising administering an effective amount of a composition comprising rAAV particles to the CNS of the individual, wherein the rAAV particles comprise a) 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 heterogeneous nucleic acid and at least one AAV terminal repeat sequence.

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

[0039] In some embodiments of the above aspects and embodiments, administration includes direct spinal injection and / or intracerebral administration. In some embodiments, intracerebral administration is directed 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) of 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, administration is directed to an intraventricular injection into at least one cerebral lateral ventricle. The administration method is injection. In some embodiments, the administration is intrathecal injection in the neck, chest, and / or lumbar region. In some embodiments, the administration is intrastriatal injection. In some embodiments, the administration is intrathalamic injection. In some embodiments, the administration is intraparenchymal injection. In some embodiments, the administration includes direct spinal injection, intracranial, and / or intracerebral administration. In some embodiments, rAAV particles are administered to a single site.

[0040] In some embodiments of the above aspects and embodiments, rAAV particles are delivered by stereotactic delivery. In some embodiments, rAAV particles are delivered by convection-enhanced delivery. In some embodiments, 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 a non-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 embodiments of the above aspects and embodiments, heparan sulfate proteoglycan is expressed on one or more cells of the CNS. In some embodiments, one or more cells of the CNS are oligodendrocytes, astrocytes, neurons, brain parenchymal cells, microglia, ependymal cells, and / or Purkinje cells. In some embodiments, heparan sulfate proteoglycan is expressed on neurons.

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

[0043] In some embodiments of the above aspects and embodiments, one or more amino acid substitutions are numbered based on the VP1 numbering of AAV2 at positions 448, 451, 484, 487, 527, 532, 585 and / or 588. In some embodiments, one or more amino acid substitutions are numbered based on the VP1 numbering of AAV2 at positions 484, 487, 527, 532, 585 or 588. In some embodiments, the numbering is based on the VP1 of AAV2 containing the amino acid sequence of SEQ ID NO: 1. In some embodiments, one or more amino acid substitutions include the 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 some embodiments, one or more amino acid substitutions include the substitution of an arginine or lysine residue. In some embodiments, one or more amino acid substitutions include the substitution of an arginine or lysine residue with an alanine residue. In some embodiments, one or more amino acid substitutions include 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, one or more amino acid substitutions include substitutions at positions R484, R487, K527, K532, R585 and / or R588, numbered based on VP1 of AAV2. In some embodiments, rAAV particles are at least about 90%, at least about 91%, at least about 92%, and less than 90% of SEQ ID NOs: 2, 4 and / or 6. Each comprises one or more rAAV capsid proteins having approximately 93%, at least approximately 94%, at least approximately 95%, at least approximately 96%, at least approximately 97%, at least approximately 98%, at least approximately 99%, or 100% sequence identity. In some embodiments, one or more amino acid substitutions, numbered based on VP1 of AAV2, include R347A, R350A, K390A, K395A, R448A, R451A, R484A, R487A, K527A, K532A, R585A, and / or R588A substitutions. In some embodiments, one or more amino acid substitutions, numbered based on VP1 of AAV2, include substitutions at positions R484 and R487 or at positions R585 and R588. In some embodiments, one or more amino acid substitutions include R484A and R487A substitutions or R585A and R588A substitutions, numbered based on VP1 of AAV2. In some embodiments, the AAV capsid includes amino acid substitutions R585A and R588A, numbered based on VP1 of AAV2. In some embodiments, the AAV capsid includes amino acid substitution K532A, numbered based on VP1 of AAV2.

[0044] One or more amino acid substitutions are numbered based on the VP1 numbering of AAVrh8R, specifically in 485, 488, 528, 533, 586, or 589. In some embodiments, the numbering is based on the VP1 of AAVrh8R containing the amino acid sequence of SEQ ID NO: 9. In some embodiments, one or more amino acid substitutions include the 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, one or more amino acid substitutions include the substitution of an arginine or lysine residue. In yet another embodiment, one or more amino acid substitutions include the substitution of an arginine or lysine residue with an alanine residue. In yet another embodiment, one or more amino acid substitutions include the 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 yet another embodiment, one or more amino acid substitutions include the substitution of an alanine residue. In further embodiments, one or more amino acid substitutions include 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 containing the amino acid sequence of SEQ ID NO: 9. In some embodiments, the amino acid substitutions include substitutions at positions R485, R488, R533, or T589, numbered based on the VP numbering of AAVrh8R. In some embodiments, the rAAV particles contain the rAAV capsid protein of SEQ ID NO: 11.In some embodiments, the rAAV particles contain 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 with respect to SEQ ID NO: 11. In some embodiments, one amino acid substitution includes the R533A substitution, numbered based on VP1 of AAVrh8R.

[0045] In some embodiments of the above aspects and embodiments, heterologous nucleic acids encode therapeutic polypeptides or therapeutic nucleic acids. In some embodiments, heterologous nucleic acids encode CNS-related genes. In some embodiments, heterologous nucleic acids encode enzymes, neurotrophic factors, deficient or mutant polypeptides in individuals with CNS-related disorders, antioxidants, anti-apoptotic factors, anti-angiogenic factors, and anti-inflammatory factors, 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), heparinacetyl-CoA, α-glucosaminidase N-acetyltransferase (MPS3C), N-acetylglucosamine-6-phosphotransferase The molecule encodes a polypeptide selected from the group consisting of ulphatase (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-idulonidase, arylsulfatase B, arylsulfatase A, N-acetylgalactosamine-6-sulfatase, acid β-galactosidase, or α-neuramidase. In some embodiments, heterologous nucleic acids encode polypeptides selected from the group consisting of neuronal apoptosis inhibitory proteins (NAIPs), nerve growth factors (NGFs), glial cell-derived growth factors (GDNFs), brain-derived growth factors (BDNFs), ciliary neurotrophic factor (CNTFs), tyrosine hydroxylase (TH), GTP-cyclohydrolase (GTPCH), amino acid decarboxylase (AADC), antioxidants, anti-angiogenic polypeptides, anti-inflammatory polypeptides, and aspartacylase (ASPA). In some embodiments, heterologous nucleic acids encode therapeutic nucleic acids.In some embodiments, the therapeutic nucleic acid is siRNA, shRNA, RNAi, miRNA, antisense RNA, ribozyme, or DNAzyme. In some embodiments, the heterologous nucleic acid is under the control of a promoter sequence expressed in one or more cells of the CNS. In some embodiments, heterologous nucleic acids are controlled by promoter sequences selected from the group consisting of the cytomegalovirus (CMV) initial promoter, RSV LTR, MoMLV LTR, phosphoglycerate kinase-1 (PGK) promoter, monkey 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, retrovirus Roussarcoma virus (RSV) LTR promoter, dihydrofolate reductase promoter, and 13-actin promoter. In some embodiments, heterologous nucleic acids are operably linked to a promoter suitable for the expression of a therapeutic polypeptide or therapeutic nucleic acid in one or more cells of the CNS. In some embodiments, one or more cells of the CNS include one or more cells of the brain. In some embodiments, one or more cells of the CNS are oligodendrocytes, astrocytes, neurons, brain parenchymal cells, microglia, ependymal cells, and / or Purkinje cells. In some embodiments, the cells of the brain 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 heterogeneous nucleic acid and a second nucleic acid sequence encoding a complementary strand of the nucleic acid, wherein the first nucleic acid sequence and the second nucleic acid sequence have inter-strand bases over most or all of its length. Pairs can be formed. In some embodiments, the first nucleic acid sequence and the second nucleic acid sequence are linked by a mutant AAV ITR, which includes a deletion in the D region and a mutation in the terminal cleavage sequence.

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

[0048] In some embodiments of the above aspects and embodiments, heterologous nucleic acids encode therapeutic polypeptides or therapeutic nucleic acids used to treat CNS disorders. In some embodiments, CNS disorders are lysosomal storage disorders (LSDs), Huntington's disease, epilepsy, Parkinson's disease, Alzheimer's disease, stroke, corticobasal degeneration (CBD), corticobasal ganglia degeneration (CBGD), frontotemporal dementia (FTD), multiple system atrophy (MSA), progressive supranuclear palsy (PSP), or brain cancer. In some embodiments, the disorder is aspartylglucosamineuria, Fabry, childhood Batten disease (CNL1), classic late-onset childhood Batten disease (CNL2), juvenile Batten disease (CNL3), CNL4 Batten, CNL5 Batten, CNL6 Batten, CNL7 Batten, CNL8 Batten, cystinosis, Faber, fucosidosis, galactosidosis, Gaucher disease type 1, Gaucher disease type 2, Gaucher disease type 3, GM1 gangliosidosis, Hunter's disease, Krabbe disease, α-mannosynosinus This is a lysosomal storage disorder selected from the group consisting of leukodystrophy, β-mannosidosis, Maloto-Lamy, metachromatic leukodystrophy, Morquio type A, Morquio type B, mucolipidosis type II / III, Niemann-Pick disease type A, Niemann-Pick disease type B, Niemann-Pick disease type C, Pompe disease, Sandhoff disease, Sanfilippo disease type A, Sanfilippo disease type B, Sanfilippo type C, Sanfilippo type D, Schindler disease, Schindler-Kanzaki disease, sialidosis, Sleigh disease, Tay-Sachs disease, and Wolmann 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 an effective amount of a composition comprising recombinant adeno-associated virus (rAAV) particles to the striatum of the individual, wherein 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, 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 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 RNAi against huntingtin. In some embodiments, RNAi is a miRNA.

[0050] In some embodiments, the present invention provides a method for treating Parkinson's disease in an individual, comprising administering an effective amount of a composition comprising recombinant adeno-associated virus (rAAV) particles to the striatum of the individual, wherein 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, 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 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.

[0051] In some embodiments of the above aspects and embodiments, heterologous nucleic acids are expressed at an increased expression level compared to the expression level of heterologous nucleic acids in rAAV particles containing a reference rAAV capsid. In some embodiments, rAAV particles result in reduced neuroinflammation compared to rAAV particles containing a reference rAAV capsid. In some embodiments, rAAV particles contain an AAV serotype 2 (AAV2) capsid. In some embodiments, one or more amino acid substitutions reduce the binding of rAAV particles to heparan sulfate proteoglycans. In some embodiments, one or more amino acid substitutions reduce the binding of rAAV particles to heparan sulfate proteoglycans compared to the binding of rAAV particles containing a reference rAAV capsid. In some embodiments, one or more amino acid substitutions reduce the binding of 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, one or more amino acid substitutions reduce the binding of 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 the binding of rAAV particles containing 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 an rAAV capsid or capsid protein that does not have 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, rAAV particles are delivered by stereotactic delivery. In some embodiments, rAAV particles are delivered by convection-enhanced delivery. In some embodiments, rAAV particles are administered using a CED delivery system. In some embodiments, the cannula is a non-reflux cannula or a stepped cannula. In some embodiments, the CED delivery system includes a cannula and / or a pump. In some embodiments, 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 embodiments of the above aspects and embodiments, heparan sulfate proteoglycan is expressed on one or more cells of the CNS. In some embodiments, one or more cells of the CNS are oligodendrocytes, astrocytes, neurons, brain parenchymal cells, microglia, ependymal cells, and / or Purkinje cells. In some embodiments, heparan sulfate proteoglycan is expressed on neurons.

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

[0055] One or more amino acid substitutions are numbered based on the VP1 numbering of AAV2. The numbering is based on the VP1 of AAV2 containing the amino acid sequence of SEQ ID NO: 1. In some embodiments, one or more amino acid substitutions include the 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 some embodiments, one or more amino acid substitutions include the substitution of an arginine or lysine residue. In some embodiments, one or more amino acid substitutions include the substitution of an arginine or lysine residue with an alanine residue. In some embodiments, one or more amino acid substitutions are numbered based on the VP1 of AAV2 and include substitutions at positions R347, R350, K390, K395, R448, R451, R484, R487, K527, K532, R585 and / or R588. In some embodiments, one or more amino acid substitutions are numbered based on VP1 of AAV2 and include substitutions at positions R484, R487, K527, K532, R585 and / or R588. In some embodiments, the rAAV particles contain 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, one or more amino acid substitutions, numbered based on VP1 of AAV2, include R347A, R350A, K390A, K395A, R448A, R451A, R484A, R487A, K527A, K532A, R585A and / or R588A substitutions. In some embodiments, one or more amino acid substitutions, numbered based on VP1 of AAV2, include substitutions at positions R484 and R487 or at positions R585 and R588.In some embodiments, one or more amino acid substitutions include R484A and R487A substitutions or R585A and R588A substitutions, numbered based on VP1 of AAV2. In some embodiments, the AAV capsid includes amino acid substitutions R585A and R588A, numbered based on VP1 of AAV2. In some embodiments, the AAV capsid includes amino acid substitution K532A, numbered based on VP1 of AAV2.

[0056] In some embodiments of the above aspects and embodiments, one or more amino acid substitutions are located at positions 485, 488, 528, 533, 586, or 589, numbered based on the VP1 numbering of AAVrh8R. In some embodiments, the AAV particles of the present invention include a capsid having one or more amino acid substitutions located 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, which includes the amino acid sequence of SEQ ID NO: 9. In some embodiments, one or more amino acid substitutions include the 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, one or more amino acid substitutions include the substitution of an arginine or lysine residue. In yet another embodiment, one or more amino acid substitutions include the substitution of an arginine or lysine residue with an alanine residue. In other embodiments, one or more amino acid substitutions include the substitution of a non-positively charged amino acid residue with a positively charged amino acid residue. In some embodiments, hydrophobic amino acid residues are substituted with positively charged amino acid residues. In further embodiments, one or more amino acid substitutions include the substitution of an alanine residue. In yet another embodiment, one or more amino acid substitutions include the substitution of an arginine or lysine residue with an alanine residue. In some embodiments, the amino acid substitution is AAVrh8 The numbering is based on the VP1 numbering of R and is at positions 485, 488, 528, 533, or 589. In some embodiments, the numbering is based on the VP1 of AAVrh8R containing the amino acid sequence of SEQ ID NO: 9. In some embodiments, the amino acid substitutions include substitutions at positions R485, R488, R533, or T589, numbered based on the VP1 numbering of AAVrh8R. In some embodiments, the rAAV particles contain the rAAV capsid protein of SEQ ID NO: 11. In some embodiments, the rAAV particles contain 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 with respect to SEQ ID NO: 11. In some embodiments, a single amino acid substitution includes the R533A substitution, numbered based on VP1 of AAVrh8R. In some embodiments, the rAAV particles include AAV1 capsid, AAV2 capsid, AAV3 capsid, AAV6 capsid, AAV8 capsid, AAVrh8R capsid, AAV9 capsid, or AAVrh10 capsid.

[0057] In some embodiments of the above aspects and embodiments, heterologous nucleic acids are under the control of promoter sequences expressed in one or more cells of the CNS. In some embodiments, heterologous nucleic acids are controlled by promoter sequences selected from the group consisting of the cytomegalovirus (CMV) initial promoter, RSV LTR, MoMLV LTR, phosphoglycerate kinase-1 (PGK) promoter, monkey 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, retrovirus Roussarcoma virus (RSV) LTR promoter, dihydrofolate reductase promoter, and 13-actin promoter. In some embodiments, heterologous nucleic acids are operably linked to a promoter suitable for the expression of a therapeutic polypeptide or therapeutic nucleic acid in one or more cells of the CNS. In some embodiments, one or more cells of the CNS include one or more cells of the brain. In some embodiments, one or more cells of the CNS are oligodendrocytes, astrocytes, neurons, brain parenchymal cells, microglia, ependymal cells, and / or Purkinje cells. In some embodiments, the cells of the brain 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 heterogeneous nucleic acid and a second nucleic acid sequence encoding a 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. In some embodiments, the first and second nucleic acid sequences are linked by a mutant AAV ITR, which includes a deletion in the D region and a mutation in the terminal cleavage sequence.

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

[0060] In some embodiments, the present invention is a kit for use in any of the above embodiments, comprising recombinant adeno-associated virus (rAAV) particles, the rAAV particles being a The present invention provides a kit comprising a) an rAAV capsid comprising an rAAV capsid protein having 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 heterogeneous nucleic acid and at least one AAV inverted terminal repeat sequence. In some embodiments, the present invention provides a kit for delivering heterogeneous nucleic acid to the central nervous system (CNS) of an individual, comprising a composition comprising recombinant adeno-associated virus (rAAV) particles, the rAAV particles comprising a) an rAAV capsid comprising an rAAV capsid protein having one or more amino acid substitutions at one or more positions that interact with heparan sulfate proteoglycans, and b) an rAAV vector comprising a heterogeneous nucleic acid and at least one AAV inverted terminal repeat sequence. In some embodiments, the present invention provides a kit for treating central nervous system (CNS) disorders in an individual, comprising a composition comprising recombinant adeno-associated virus (rAAV) particles, wherein the rAAV particles comprise a) an rAAV capsid comprising an rAAV capsid protein having one or more amino acid substitutions at one or more positions interacting with a heparan sulfate proteoglycan, and b) an rAAV vector comprising heterologous nucleic acid and at least one AAV inverted terminal repeat sequence for treating CNS disorders.

[0061] In some embodiments of the above aspects and embodiments, the CNS disorder is Huntington's disease. In some embodiments, the heterogeneous nucleic acid encodes a therapeutic polypeptide or therapeutic nucleic acid. In some embodiments, the therapeutic polypeptide is 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 includes RNAi for huntingtin. In some embodiments, the RNAi is miRNA. In some embodiments, the CNS disorder is Parkinson's disease. In some embodiments, the heterogeneous 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 embodiments, the present invention provides recombinant adeno-associated virus (rAAV) particles for use in any of the above embodiments. In some embodiments, the present invention provides recombinant adeno-associated virus (rAAV) particles for delivering heterologous nucleic acids to the central nervous system (CNS) of an individual, comprising: a) an rAAV capsid protein comprising one or more amino acid substitutions at one or more positions that interact with a 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 heterologous nucleic acid and at least one AAV inverted terminal repeat sequence. In some embodiments, the present invention provides recombinant adeno-associated virus (rAAV) particles for treating a disorder of the central nervous system (CNS) of an individual, comprising: a) an rAAV capsid comprising an rAAV capsid protein having 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 heterologous nucleic acids 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 of an individual, comprising: a) heparan sulfate pro The present invention provides an rAAV capsid comprising an rAAV capsid protein containing one or more amino acid substitutions at one or more positions that interact with theoglycans, 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 heterogeneous nucleic acid and at least one AAV terminal repeat sequence, which 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, 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 a 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, which is 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, comprising: a) an rAAV capsid comprising an rAAV capsid protein having one or more amino acid substitutions at one or more positions that interact with a 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 heterogeneous 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, 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 a 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).

[0063] In some embodiments of the above aspects and embodiments, heterologous nucleic acids are expressed at an increased expression level compared to the expression level of heterologous nucleic acids in rAAV particles containing a reference rAAV capsid. In some embodiments, rAAV particles result in reduced neuroinflammation compared to rAAV particles containing an rAAV capsid containing a reference capsid. In some embodiments, rAAV particles contain an AAV serotype 2 (AAV2) capsid. In some embodiments, one or more amino acid substitutions reduce the binding of rAAV particles to heparan sulfate proteoglycans. In some embodiments, one or more amino acid substitutions reduce the binding of rAAV particles to heparan sulfate proteoglycans compared to the binding of rAAV particles containing a reference rAAV capsid to heparan sulfate proteoglycans. In some embodiments, one or more amino acid substitutions reduce the binding of 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, one or more amino acid substitutions reduce the binding of 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 the binding of rAAV particles containing 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 an rAAV capsid or capsid protein that does not have one or more amino acid substitutions at one or more positions that interact with heparan sulfate proteoglycans. .

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

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

[0066] One or more amino acid substitutions are located at positions 484, 487, 527, 532, 585, or 588, numbered based on the VP1 numbering of AAV2. In some embodiments, one or more amino acid substitutions include the 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 some embodiments, one or more amino acid substitutions include the substitution of an arginine or lysine residue with an alanine residue. In some embodiments, one or more amino acid substitutions include substitutions at positions R347, R350, K390, K395, R448, R451, R484, R487, K527, K532, R585, and / or R588, numbered based on the VP1 numbering of AAV2. In some embodiments, one or more amino acid substitutions are numbered based on VP1 of AAV2 and include substitutions at positions R484, R487, K527, K532, R585 and / or R588. In some embodiments, the rAAV particles include rAAV particles containing 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 with respect to SEQ ID NOs. 2, 4 and / or 6. In some embodiments, one or more amino acid substitutions, numbered based on VP1 of AAV2, include R347A, R350A, K390A, K395A, R448A, R451A, R484A, R487A, K527A, K532A, R585A and / or R588A substitutions. In some embodiments, one or more amino acid substitutions, numbered based on VP1 of AAV2, include substitutions at positions R484 and R487 or at positions R585 and R588.In some embodiments, one or more amino acid substitutions include R484A and R487A substitutions or R585A and R588A substitutions, numbered based on VP1 of AAV2. In some embodiments, the AAV capsid includes amino acid substitutions R585A and R588A, numbered based on VP1 of AAV2. In some embodiments, the AAV capsid includes amino acid substitution K532A, numbered based on VP1 of AAV2. In some embodiments, the rAAV particles include AAV1 capsid, AAV2 capsid, AAV3 capsid, AAV6 capsid, AAV8 capsid, AAVrh8R capsid, AAV9 capsid, or AAVrh10 capsid.

[0067] In some embodiments of the above aspects and embodiments, one or more amino acid substitutions are numbered based on the VP1 numbering of AAVrh8R and are at positions 485, 488, 528, 533, 586, or 589. In some embodiments, the numbering is based on the VP1 of AAVrh8R containing the amino acid sequence of SEQ ID NO: 9. In some embodiments, one or more amino acid substitutions include the 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, one or more amino acid substitutions include the substitution of an arginine or lysine residue. In yet another embodiment, one or more amino acid substitutions include the substitution of an arginine or lysine residue with an alanine residue. In other embodiments, one or more amino acid substitutions include the 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, one or more amino acid substitutions include substitutions of alanine residues. In even further embodiments, one or more amino acid substitutions include substitutions of arginine or lysine residues with alanine residues. In some embodiments, the amino acid substitutions are numbered based on the VP1 numbering of AAVrh8R and are at positions 485, 488, 528, 533, or 589. In some embodiments, the numbering is based on the VP1 of AAVrh8R containing the amino acid sequence of SEQ ID NO: 9. In some embodiments, the amino acid substitutions are numbered based on the VP1 numbering of AAVrh8R and include substitutions at positions R485, R488, R533, or T589. In some embodiments, the rAAV particles contain the rAAV capsid protein of SEQ ID NO: 11.In some embodiments, the rAAV particles contain 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 with respect to SEQ ID NO: 11. In some embodiments, one amino acid substitution includes the R533A substitution, numbered based on VP1 of AAVrh8R.

[0068] In some embodiments of the above aspects and embodiments, heterologous nucleic acids are under the control of promoter sequences expressed in one or more cells of the CNS. In some embodiments, heterologous nucleic acids are controlled by promoter sequences selected from the group consisting of the cytomegalovirus (CMV) initial promoter, RSV LTR, MoMLV LTR, phosphoglycerate kinase-1 (PGK) promoter, monkey 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, retrovirus Roussarcoma virus (RSV) LTR promoter, dihydrofolate reductase promoter, and 13-actin promoter. In some embodiments, heterologous nucleic acids are operably linked to a promoter suitable for the expression of a therapeutic polypeptide or therapeutic nucleic acid in one or more cells of the CNS. In some embodiments, one or more cells of the CNS include one or more cells of the brain. In some embodiments, one or more cells of the CNS are oligodendrocytes, astrocytes, neurons, brain parenchymal cells, microglia, 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 heterogeneous nucleic acid and a second nucleic acid sequence encoding a 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. In some embodiments, the first and second nucleic acid sequences are linked by a mutant AAV ITR, which includes a deletion in the D region and a mutation in the terminal cleavage sequence.

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

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

[0072] In some embodiments, the present invention provides rAAV particles comprising an AAVrh8R capsid protein, wherein the AAVrh8R capsid protein comprises one or more amino acid substitutions, the one or more amino acid substitutions increasing the binding of the rAAV particles to heparan sulfate proteoglycans compared to AAV particles comprising 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 based on the VP1 numbering of AAV2. In some embodiments, the one or more amino acid substitutions increase the 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 amino acid substitution is at position 586, numbered based on the VP1 numbering of AAVrh8R. In some embodiments, the numbering is based on the VP1 of AAVrh8R containing the amino acid sequence of SEQ ID NO: 9. In some embodiments, the amino acid substitutions include substitutions at position A586, numbered based on the VP1 numbering of AAVrh8R. In some embodiments, the amino acid substitutions include A586R or A586K substitutions, numbered based on the VP1 numbering of AAVrh8R. In some embodiments, the rAAV particles contain the rAAV capsid protein of SEQ ID NO: 10.

[0073] In some embodiments, the present invention provides a method for increasing the binding of rAAV particles containing the AAVrh8R capsid protein to heparan sulfate proteoglycans, comprising introducing one or more amino acid substitutions into the capsid protein, wherein the one or more amino acid substitutions increase the binding of the rAAV particles to heparan sulfate proteoglycans compared to AAV particles containing the wild-type AAVrh8R capsid protein. In some embodiments, the one or more amino acid substitutions increase the 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 amino acid substitution is at position 586, numbered based on the VP1 numbering of AAVrh8R. In some embodiments, the numbering is based on the VP1 of AAVrh8R, which contains the amino acid sequence of SEQ ID NO: 9. In some embodiments, the amino acid substitutions include substitutions at position A586, numbered based on VP1 of AAVrh8R. The amino acid substitutions include A586R or A586K substitutions, numbered based on the VP1 numbering of AAVrh8R. In some embodiments, the rAAV particles contain the rAAV capsid protein of SEQ ID NO: 10.

[0074] In some embodiments, the present invention provides a method for delivering a heterologous nucleic acid to the retina of an individual, comprising intravitreal administration of recombinant adeno-associated virus (rAAV) particles to the individual, wherein the rAAV particles comprise a) 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 sequence. In some embodiments, the present invention provides a method for improving rAAV transduction into cells after intravitreous delivery of rAAV particles to the eye of an individual compared to transduction into cells with rAAV containing a wild-type capsid, comprising incorporating one or more amino acid substitutions into the 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 method provides that the rAAV particles comprise an rAAV capsid protein and an rAAV vector comprising heterogeneous nucleic acids and at least one AAV terminal repeat sequence. In some embodiments, the present invention provides a method for improving heterologous nucleic acid expression after intravitreous 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 method provides that the rAAV particles comprise an rAAV capsid protein and an rAAV vector comprising heterologous nucleic acids and at least one AAV terminal repeat sequence.In some embodiments, the present invention provides a method for treating an eye disorder in an individual, comprising intravitreal delivery of a composition comprising rAAV particles to the retina 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 a 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. In some embodiments, the present invention provides a system for intravitreous delivery of a vector into 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 interacting 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 heterologous nucleic acid encoding a therapeutic amount polypeptide or therapeutic RNA and at least one AAV terminal repeat sequence]; and b) a device for intravitreous delivery of the rAAV. In some embodiments, the rAAV particles comprise AAVrh8R, AAV1, AAV6, AAV8, AAV9 or AAVrh10 serotype capsids. In some embodiments, the present invention relates to a kit for treating an eye disorder, comprising a) a composition comprising rAAV particles [wherein the rAAV particles are 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 The present invention provides the kit comprising: (i) an rAAV vector comprising heterologous nucleic acid and at least one AAV inverted terminal repeat sequence for treating an eye disorder; and (ii) a pharmaceutical excipient suitable for intravitreal administration. In some embodiments, the rAAV particles comprise AAVrh8R, AAV1, AAV6, AAV8, AAV9, or AAVrh10 capsids. In some embodiments, the present invention provides rAAV particles comprising an AAV1 capsid protein, wherein the AAV1 capsid protein comprises one or more amino acid substitutions, the one or more amino acid substitutions increasing the transduction efficiency of the rAAV particles into eye cells compared to AAV particles comprising 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, 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, the one or more amino acid substitutions increasing the transduction efficiency of the rAAV particles into eye cells compared to AAV particles comprising wild-type AAV6 capsid protein, or the one or more amino acid substitutions are located 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 AAV8 capsid protein, wherein the AAV8 capsid protein comprises one or more amino acid substitutions, the one or more amino acid substitutions increasing the transduction efficiency of the rAAV particles into eye cells compared to AAV particles comprising wild-type AAV8 capsid protein, or the one or more amino acid substitutions are located 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, the one or more amino acid substitutions increasing the transduction efficiency of the rAAV particles into eye cells compared to AAV particles comprising wild-type AAV9 capsid protein, or the one or more amino acid substitutions are located 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 AAVrh10 capsid protein, wherein the AAVrh10 capsid protein comprises one or more amino acid substitutions, the one or more amino acid substitutions increasing the transduction efficiency of the rAAV particles into eye cells compared to AAV particles comprising wild-type AAVrh10 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 based on the VP1 numbering of AAV2. In some embodiments, the present 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 the VP1 numbering of AAV2. In some embodiments, the rAAV particles include AAV1 capsid, AAV2 capsid, AAV3 capsid, AAV6 capsid, AAV8 capsid, AAVrh8R capsid, AAV9 capsid, or 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 used in rAAV particles.The binding to heparan sulfate proteoglycans is increased. In some embodiments, one or more amino acid substitutions increase the binding of 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, one or more amino acid substitutions increase the transduction efficiency of rAAV particles to ocular or central nervous system cells 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 containing wild-type AAVrh8R capsid protein. In some embodiments, ocular cells are retinal cells, photoreceptor cells, retinal pigment epithelial cells, bipolar cells, horizontal cells, amacrine cells, Müller cells, and / or ganglion cells. In some embodiments, one or more amino acid substitutions include the substitution of a non-positively charged amino acid residue with a positively charged amino acid residue. In some embodiments, positively charged amino acid residues replace hydrophobic amino acid residues. In some embodiments, one or more amino acid substitutions include substitutions by arginine or lysine residues. In some embodiments, one or more amino acid substitutions include substitutions by arginine or lysine residues of alanine, serine, glutamine, or threonine residues. In some embodiments, rAAV particles contain an AAV serotype rh8R (AAVrh8R) capsid. In some embodiments, 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 the VP1 of AAVrh8R containing the amino acid sequence of SEQ ID NO: 9. In some embodiments, one or more amino acid substitutions include substitutions at positions A586 and / or T589, numbered based on the VP1 numbering of AAVrh8R. In some embodiments, one or more amino acid substitutions include A586R or A586K substitutions, numbered based on VP1 of AAVrh8R.In some embodiments, one or more amino acid substitutions include T589R or T589K substitutions, numbered based on VP1 of AAVrh8R. In some embodiments, the rAAV particle contains an AAV serotype 1 (AAV1) capsid. In some embodiments, 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 contains the amino acid sequence of SEQ ID NO: 12. In some embodiments, one or more amino acid substitutions include substitutions at positions S586 and / or T589, numbered based on VP1 numbering of AAV1. In some embodiments, one or more amino acid substitutions include S586R or S586K substitutions, numbered based on VP1 of AAV1. In some embodiments, one or more amino acid substitutions include T589R or T589K substitutions, numbered based on VP1 of AAV1. In some embodiments, the rAAV particles contain an AAV serotype 6 (AAV6) capsid. In some embodiments, one or more amino acid substitutions are located at positions 586 and / or 589, numbered based on the VP1 numbering of AAV6. In some embodiments, the numbering is based on the VP1 of AAV6 containing the amino acid sequence of SEQ ID NO: 13. In some embodiments, 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, one or more amino acid substitutions include an S586R substitution, numbered based on the VP1 of AAV6. In some embodiments, one or more amino acid substitutions include a T589R or T589K substitution, numbered based on the VP1 of AAV6. In some embodiments, the rAAV particles contain an AAV serotype 8 (AAV8) capsid. In some embodiments, one or more amino acid substitutions are located at positions 588 and / or 591, numbered based on the VP1 numbering of AAV8.In some embodiments, the VP1 of the AAV8 is... The amino acid sequence of SEQ ID NO: 14 is included. In some embodiments, one or more amino acid substitutions include substitutions at positions Q588 and / T591, numbered based on the VP1 numbering of AAV8. In some embodiments, one or more amino acid substitutions include Q588R or Q588K substitutions, numbered based on the VP1 numbering of AAV8. In some embodiments, one or more amino acid substitutions include T591R substitutions, numbered based on the VP1 numbering of AAV8. In some embodiments, the rAAV particle contains an AAV serotype 9 (AAV9) capsid. In some embodiments, 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 contains the amino acid sequence of SEQ ID NO: 15. In some embodiments, one or more amino acid substitutions include substitutions at positions S586 and / A589, numbered based on the VP1 numbering of AAV9. In some embodiments, one or more amino acid substitutions include S586R or S586K substitutions, numbered based on the VP1 of AAV9. In some embodiments, one or more amino acid substitutions include A589R or A589K substitutions, numbered based on the VP1 of AAV9. In some embodiments, the rAAV particle contains an AAV serotype rh10 (AAVrh10) capsid. In some embodiments, one or more amino acid substitutions are at positions 588 and / or 591, numbered based on the VP1 numbering of AAVrh10. In some embodiments, the VP1 of AAVrh10 contains the amino acid sequence of SEQ ID NO: 16. In some embodiments, one or more amino acid substitutions include substitutions at positions Q588 and / or A591, numbered based on the VP1 numbering of AAVrh10. In some embodiments, one or more amino acid substitutions include Q588R or Q588K substitutions, numbered based on the VP1 of AAVrh10.In some embodiments, one or more amino acid substitutions include A591R or A591K substitutions, numbered based on the VP1 of AAVrh10. In some embodiments, the present 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 the VP1 numbering of AAV2. In some embodiments, one or more amino acid substitutions increase the transduction efficiency of rAAV particles into eye or central nervous system cells 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 wild-type AAVrh8R capsid protein. In some embodiments, the rAAV particles include 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 therapeutic nucleic acid. In some embodiments, the heterologous nucleic acid encodes a polypeptide selected from the group consisting of antioxidants, neurotrophic factors, anti-apoptotic factors, anti-angiogenic factors, and anti-inflammatory factors. 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, Clarine, 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-R I, sTNF-R II, and IL4. In other embodiments, the heterologous nucleic acid encodes a therapeutic nucleic acid. In a further embodiment, the therapeutic nucleic acid is 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 include a capsid comprising 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, which modifies binding to HSPG (e.g., reducing or eliminating binding to HSPG), and a heterogeneous nucleic acid encoding a therapeutic polypeptide or therapeutic nucleic acid, the heterogeneous nucleic acid being under the control of a promoter sequence expressed in the retina. In some embodiments, the heterogeneous nucleic acid is operably linked to a promoter suitable for the 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 pigment 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. In some embodiments, the individual is a human. In some embodiments, the heterologous nucleic acid is used to treat an eye disorder selected from the group consisting of: autosomal recessive severe early-onset retinal degeneration (Leber congenital amaurosis), congenital color blindness, Stargardt disease, Best's disease, Doin'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 an autocomplementary rAAV vector. In some embodiments, the vector comprises a first nucleic acid sequence encoding a heterogeneous nucleic acid and a second nucleic acid sequence encoding a 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.In some embodiments, the first nucleic acid sequence and the second nucleic acid sequence are linked by a mutant AAV ITR, the mutant AAV ITR containing a deletion in the D region and a mutation in the terminal cleavage sequence. In some embodiments, the individual is human. In some embodiments, one or more amino acid substitutions increase the transduction efficiency of rAAV particles into eye or central nervous system cells 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 containing 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 drawing]

[0077] [Figure 1] This figure shows the capsid residues involved in heparan sulfate proteoglycan binding, and the mutations introduced to produce the AAV2 HBKO mutant. The numbering is based on the VP1 amino acid sequence. [Figure 2] This figure shows that transduction into 293 cells in culture was reduced with HBKO mutant AAV2 particles (AAV2 HBKO CBA-sFLT02) compared to 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 containing a vector that drives Flt expression using a CBA promoter. [Figure 3]This figure shows a reduction in transduction into heLa cells and 293 cells in culture observed with HBKO mutant AAV2 particles (AAV2 HBKO CBA-GFP) compared with wild-type AAV2 particles (AAV2 CBA-GFP). Transduction was assayed by fluorescence imaging of cells 48 hours after injection of wild-type or HBKO mutant AAV2 particles containing a vector driving EGFP expression using a CBA promoter. [Figure 4] Figures 4A and 4B show the transduction observed after intravitreous injection (Figure 4A) or subretinal injection (Figure 4B) of wild-type AAV2 or HBKO mutant AAV2 particles. Transduction was assayed by the expression of soluble Flt (sFLT) after transduction of the Flt-encoding vector. The number of injected vector genomes is shown for each experiment (10⁸ or 10⁹ vg). [Figure 5] This figure shows that HBKO mutant AAV2 particles could not be transduced into the mouse eye after intravitreous injection. Mice were intravitreously injected with wild-type (AAV2 CBA-GFP) or HBKO mutant (AAV2 HBKO CBA-GFP) AAV2 particles containing a vector that drives EGFP expression using a CBA promoter, and sections were imaged using fluorescence microscopy. [Figure 6] This figure shows that HBKO mutant AAV2 particles (AAV2 CBA HBKO) result in a significant increase in transduction compared to wild-type particles (AAV2 CBA) after subretinal injection. Transduction was assayed by the expression of soluble Flt (sFLT) after injection of AAV2 particles containing a vector that drives Flt expression using the CBA promoter. The number of injected vector genomes is shown (10⁸ or 10⁹ vg). [Figure 7]This figure shows that HBKO mutant AAV2 particles (AAV2 HBKO CBA-GFP) result in a significant increase in transduction into photoreceptor cells (as indicated) after subretinal injection compared to wild-type particles (AAV2 CBA-GFP). Transduction was measured by fluorescence imaging of GFP expression after transduction in AAV2 particles containing a vector that drives EGFP expression using a CBA promoter. [Figure 8] This figure shows that HBKO mutant AAV2 particles (AAV2 RK HBKO) result in a significant increase in photoreceptor transduction after subretinal injection compared to wild-type particles (AAV2 RK). Transduction was assayed by the expression of soluble Flt (sFLT) after injection of AAV2 particles containing a vector that drives Flt expression using a rhodopsin kinase (RK) promoter. The number of injected vector genomes is shown (10⁸ or 10⁹ vg). [Figure 9] Figures 9A and 9B show EGFP expression in the mouse brain 30 days after intrastriatal injection of AAV2HBKO-EGFP (Figure 9A) compared to AAV2-EGFP (Figure 9B) in wild-type mice. EGFP expression 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 that expresses an artificial miRNA targeting human Htt and a GFP reporter. GFP expression in each panel was driven by a CBA promoter and visualized using fluorescence microscopy at three different magnifications (as indicated, 4X, 10X, and 20X). [Figure 11A] This figure shows the qPCR analysis of human HTT mRNA levels in striatal mouse brain punch samples 30 days after injection of AAV1-miRNA-Htt and AAV2HBKO-miRNA-Htt, compared to an untreated control. [Figure 11B] This figure shows Western blot analysis of human Htt protein levels in cortical mouse brain punch pieces 30 days after injection of AAV1-miRNA-Htt and AAV2HBKO-miRNA-Htt, compared to an untreated control. [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 an untreated control (Figure 12A). [Figure 13] Figures 13A-13C show the expression of GFP 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 an untreated control (Figure 13A). [Figure 14] This figure compares the capsid residues involved in heparan sulfate proteoglycan binding between the AAV2 capsid and the AAVrh8R capsid. The numbering is based on the VP1 amino acid sequence. [Figure 15] This figure shows the amino acid alignment of AAV2 and AAVrh8R at the residues involved in heparan binding of AAV2. The location of the arginine capsid modification of AAVrh8R is circled. [Figure 16] Figure 16A shows the improved in vitro transduction into 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 the AAVrh8R arginine-modified vector. Figure 16B shows the decreased in vitro transduction into 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 the AAVrh8R arginine-modified vector. [Figure 17]Figures 17A–17D show the in vitro transduction levels demonstrated by AAVrh8R A586R and R533A mutants compared to wild-type AAVrh8R. The AAVrh8R A586R mutant (Figure 17B) shows increased in vitro transduction into NS1 cells compared to wild-type AAVrh8R (Figure 17A). The AAVrh8R R533A mutant (Figure 17D) shows decreased in vitro transduction into heLa cells compared to wild-type AAVrh8R (Figure 17C). Transduction was monitored by EGFP expression in cells 48 hours after infection with AAVrh8R or an AAVrh8R arginine-modified vector. [Figure 18] Figures 18A and 18B show the levels of subretinal transduction in C57Bl6 mice as demonstrated by AAVrh8R A586R and R533A mutants. (Figure 18A) The AAVrh8R A586R mutant shows decreased subretinal transduction compared to wild-type AAVrh8R. The AAV2 vector was 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 C57Bl6 mice 30 days after subretinal administration of AAVrh8R or the AAVrh8R arginine-modified vector. [Figure 19] This figure shows the level of sFLT02 in retinal lysis material of C57Bl6 mice 30 days after intravitreal injection of AAV2, AAVrh8R, or AAVrh8R-A586R vectors. [Figure 20] This figure shows the amino acid alignment of the residues involved in heparan binding in AAVrh8R, AAV1, AAV6, AAV8, AAV9, and AAVrh10 and AAV2. [Modes for carrying out the invention]

[0078] As described herein, the inventors have surprisingly discovered that modifications to rAAV particles corresponding to amino acids 484, 487, 532, 585 and / or 588, numbered based on the VP1 numbering of AAV2, demonstrate increased transduction into cells after administration to the eye or CNS of a subject. While we do not wish to be bound by any theory, it is thought that these rAAV particles have reduced or removed binding to HSPG, or that the capsid charge has been modified, resulting in increased transduction into cells of the eye or CNS of a subject upon administration of the rAAV particles. Accordingly, the present invention relates to 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 are a) The method is provided 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 a heparan sulfate proteoglycan, and b) an rAAV vector comprising a heterogeneous 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 present invention provides a method for delivering heterologous nucleic acid to the eye of an individual, comprising administering recombinant adeno-associated virus (rAAV) particles subretinate the individual, wherein the rAAV particles comprise a) an rAAV capsid comprising an rAAV capsid protein having one or more amino acid substitutions at one or more positions interacting with a heparan sulfate proteoglycan, and b) an rAAV vector comprising heterologous nucleic acid and at least one AAV inverted terminal repeat sequence.

[0080] In some embodiments, the present invention provides a method for improving rAAV transduction into cells after subretinal delivery of rAAV particles into an individual eye compared to transduction into cells with rAAV containing a wild-type capsid, comprising introducing one or more amino acid substitutions into the AAV capsid protein at one or more positions that interact with heparan sulfate proteoglycans; the method provides that the rAAV particles comprise 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 result in reduced or eliminated binding to HSPGs. In some embodiments, the rAAV particles are numbered based on VP1 (SEQ ID NO: 1) of AAV2 and comprise a capsid containing R585A and R588A substitutions of rAAV2. In some embodiments, the rAAV particles are numbered based on VP1 (SEQ ID NO: 9) of AAVrh8R and include a capsid containing A586R and / or R533A substitutions of AAVrh8R.

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

[0082] In some embodiments, the present invention provides a method for improving the expression of a heterologous nucleic acid after subretinal delivery of rAAV particles to the eye of an individual, 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 method provides that the rAAV particles comprise an rAAV capsid protein and an rAAV vector comprising a heterologous nucleic acid and at least one AAV terminal repeat sequence. The improved transduction is comparable to that of rAAV particles containing a wild-type capsid. In some embodiments, the amino acid substitutions result in reduced or eliminated binding to HSPGs. In some embodiments, the rAAV particles are numbered based on VP1 (SEQ ID NO: 1) of AAV2 and comprise a capsid containing R585A and R588A substitutions of the rAAV2 capsid. In some embodiments, the rAAV particles are numbered based on VP1 (SEQ ID NO: 9) of AAVrh8R and include a capsid containing A586R and / or R533A substitutions of AAVrh8R.

[0083] In some embodiments, the present invention provides a method for treating an eye disorder in an individual, comprising the delivery of a composition comprising an effective amount of rAAV particles to the retina 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 a heparan sulfate proteoglycan, and b) an rAAV vector comprising a heterogeneous 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 a capsid comprising R585A and R588A substitutions of rAAV2, numbered based on VP1 (SEQ ID NO: 1) of AAV2. In some embodiments, the rAAV particles comprise a capsid comprising A586R and / or R533A substitutions of AAVrh8R, numbered based on VP1 (SEQ ID NO: 9) of AAVrh8R.

[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 a heparan sulfate proteoglycan, and ii) the vector comprises a heterogeneous 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 amino acid substitutions result in reduced or eliminated binding with HSPG. In some embodiments, the rAAV particles comprise a capsid containing R585A and R588A substitutions of rAAV2, numbered based on VP1 (SEQ ID NO: 1) of AAV2. In some embodiments, the rAAV particles comprise a capsid containing A586R and / or R533A substitutions of AAVrh8R, numbered based on VP1 (SEQ ID NO: 9) of AAVrh8R.

[0085] In some embodiments, the present invention further provides a method 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 comprising one or more amino acid substitutions at one or more positions interacting with heparan sulfate proteoglycans, and (b) an rAAV vector comprising heterologous nucleic acids and at least one AAV inverted terminal repeat sequence. These methods exhibit improved heterologous nucleic acid expression in cells and / or rAAV transduction into cells after delivery of rAAV particles to the CNS of an individual, compared to transduction into cells with rAAV comprising, for example, a wild-type capsid. Furthermore, the methods of the present invention can infect specific cells (e.g., neurons), and moreover, broad and strong transduction efficiencies can still be achieved. Such rAAV particles and methods are suitable for treating CNS disorders, including (but not limited to) Huntington's disease. In some embodiments, amino acid substitutions result in reduced or removed binding to HSPG. In some embodiments, rAAV particles are numbered based on VP1 (SEQ ID NO: 1) of AAV2 and contain capsids with R585A and R588A substitutions of rAAV2. In some embodiments, rAAV particles are numbered based on VP1 (SEQ ID NO: 9) of AAVrh8R and contain capsids with A586R and / or R533A substitutions of AAVrh8R.

[0086] The present invention also provides a kit comprising rAAV particles or a composition comprising rAAV particles, 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 interacting with a heparan sulfate proteoglycan, and (b) an rAAV vector comprising a heterologous nucleic acid and at least one AAV inverted terminal repeat sequence. These kits are useful not only for the delivery of heterologous nucleic acids to the eye or CNS of an individual, but also for individual It is also useful in the treatment of ocular or CNS disorders (e.g., retinopathy or Huntington's disease).

[0087] I. General technology The techniques and procedures described or referenced herein are generally well understood by those skilled in the art, for example: Molecular Cloning: A Laboratory Manual (Sambrook et al., 4th edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2012); Current Protocols in Molecular Biology (FMAusubel 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 (RI Freshney, 6th edition, J. Wiley and Sons, 2010); Oligonucleotide Synthesis (MJ Gait, ed., 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (JECellis, ed., Academic Press, 1998); Introduction to Cell and Tissue Culture (JPMather and PERoberts, Plenum Press, 1998); Cell and Tissue Culture: Laboratory Procedures (A. Doyle, J. B. Griffiths, and D. G. Newell, eds., J. Wiley and Sons, 1993-8); Handbook of Experimental Immunology (DMWeir and C.C. Blackwell, eds., 1996); Gene Transfer Vectors for Mammalian Cells (JMMiller and MPCalos (eds.), 1987; PCR: The Polymerase Chain Reaction (Mullis et al., eds.), 1994; Current Protocols in Immunology (JEColigan et al., 1991); Short Protocols in Molecular Biology (Ausubel et al., J. Wiley and Sons, 2002); Immunobiology (CA 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 JDCapra, eds., Harwood Academic It is commonly used with conventional methodologies such as Publishers, 1995, and Cancer: Principles and Practice of Oncology (edited by V.T. DeVita et al., JBLippincott Company, 2011).

[0088] II. Definition As used herein, "vector" refers to an in vitro or in vivo vector. This refers to a recombinant plasmid or virus containing nucleic acids that are delivered to a host cell by either of the following means.

[0089] The terms “polynucleotide” or “nucleic acid,” as used herein, refer to any nucleotide, ribonucleotide, or deoxyribonucleotide in polymeric form of any length. Therefore, the term includes, but is not limited to, single-stranded, double-stranded, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or polymers containing purine and pyrimidine bases or other natural, chemically or biochemically modified, unnatural, or derivatized nucleotide bases. The backbone of a polynucleotide may contain sugar and phosphate ester groups (as commonly found in RNA or DNA), or modified or substituted sugar or phosphate ester groups. Alternatively, the backbone of a polynucleotide may contain polymers of synthetic subunits such as phosphoramides, and thus may be oligodeoxynucleoside phosphoramides (P-NH2) or mixed phosphoramide-phosphodiester oligomers. In addition, double-stranded polynucleotides can be obtained from chemically synthesized single-stranded polynucleotide products by synthesis of complementary strands and annealing of those strands under appropriate conditions, or by de novo synthesis of complementary strands using DNA polymerase and appropriate primers.

[0090] The terms “polypeptide” and “protein” are used synonymously to refer to polymers of amino acid residues, and are not limited to minimum length. Such polymers of amino acid residues may or may not contain native amino acid residues, and include, but are not limited to, peptides, oligopeptides, dimers, trimers, and polymers of amino acid residues. Both full-length proteins and their fragments are encompassed in this definition. These terms also include post-expression modifications of polypeptides, such as glycosylation, sialylation, acetylation, and phosphorylation. Furthermore, for the purposes of this invention, “polypeptide” refers to a protein that includes modifications to its native sequence, such as deletions, additions, and substitutions (generally, essentially conserved), provided that the protein maintains the desired activity. These modifications may be intentional, such as through site-directed mutagenesis, or accidental, such as mutations in the host producing the protein or errors resulting from PCR amplification.

[0091] A "recombinant viral vector" refers to a recombinant polynucleotide vector containing one or more heterogeneous sequences (i.e., non-viral nucleic acid sequences). In the case of a recombinant AAV vector, the recombinant nucleic acid has at least one inverted terminal repeat (ITR) adjacent to it. In some embodiments, the recombinant nucleic acid has two adjacent ITRs.

[0092] A "recombinant AAV vector (rAAV vector)" refers to a polynucleotide vector containing one or more heterologous sequences (i.e., non-AAV-derived nucleic acid sequences) adjacent to at least one AAV inverted terminal repeat (ITR). Such an rAAV vector, when present in a host cell infected with a suitable helper virus (or expressing suitable helper function) and expressing AAV rep and cap gene products (i.e., AAV Rep and Cap proteins), is replicated and packaged into infectious viral particles. If the rAAV vector is incorporated into a larger polynucleotide (e.g., in a chromosome or in another vector, e.g., in a plasmid used for cloning or transfection), the rAAV vector may also be called a "provector," and the provector is "rescued" by replication and capsid inclusion in the presence of AAV packaging function and suitable helper function. rAAV vectors may be any of many forms, including (but not limited to) plasmids and linear artificial chromosomes, and rAAV vectors can be complexed with lipids, encapsulated in liposomes, and are also viable. The rAAV vector can be encapsulated in the capsid of a rus particle, such as an AAV particle. By packaging an rAAV vector into an AAV virus capsid, "recombinant adeno-associated virus particles (rAAV particles)" can be produced.

[0093] The term "rAAV virus" or "rAAV virus particle" refers to a viral particle consisting of at least one AAV capsid protein and an rAAV vector genome encapsulated within the capsid.

[0094] The term "heterogeneous" means that it originates from an entity that is genotypeically different from the rest of the entity being compared, introduced, or incorporated. For example, a polynucleotide introduced into a different cell type by genetic engineering is a heterogeneous polynucleotide (and, when expressed, can encode a heterogeneous polypeptide). Similarly, a cell sequence (e.g., a gene or a portion thereof) incorporated into a viral vector is a heterogeneous nucleotide sequence relative to that vector.

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

[0096] When used in relation to viral titer, the terms "genomic particle (gp)," "genomic equivalent," or "genomic copy" refer to the number of virions containing recombinant AAV DNA genome, regardless of infectivity or functionality. The number of genomic particles in a particular vector formulation can be measured by the procedures 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 series of polynucleotide sequences of a vector, such as a viral vector. A vector genome may also be encapsulated within the capsid of a viral particle. Depending on the individual viral vector, a vector genome may contain single-stranded DNA, double-stranded DNA, or single-stranded or double-stranded RNA. A vector genome may contain endogenous sequences associated with a particular viral vector and / or heterologous sequences inserted into a particular viral vector by recombinant technology. For example, a recombinant AAV vector genome may contain a promoter, a stuffer, the sequence of interest (e.g., RNAi), and at least one ITR sequence adjacent to the polyadenylated sequence. A complete vector genome may contain the complete set of polynucleotide sequences of the 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] When used in relation to viral titer, the terms "infectious unit (iu)," "infectious particle," or "replication unit" refer to the number of infectious and replicable recombinant AAV vector particles measured by an infectious center assay, which is also known as a replication center assay, as described, for example, McLaughlin et al. (1988) J. Virol., 62: pp. 1963-1973.

[0099] The term "transduction unit (tu)" as used in relation to viral titer is defined in the context of the implementation of this specification. This refers to the number of infectious recombinant AAV vector particles that produce a functional transgene product, as measured by functional assays such as those described in the examples, or, for example, Xiao et al. (1997) Exp. Neurobiol., pp. 144:113-124; or Fisher et al. (1996) J. Virol., pp. 70:520-532 (LFU assay).

[0100] An "inverted terminal repeat" or "ITR" sequence is a well-understood term in the art, referring to a relatively short sequence found at the end of a viral genome in the opposite direction.

[0101] A term well understood in the art, the “AAV inverted terminal repeat (ITR)” sequence is a sequence of approximately 145 nucleotides present at both ends of a native single-stranded AAV genome. The outermost 125 nucleotides of an ITR can exist in one of two binary orientations, resulting in heterogeneity between different AAV genomes and between two ends of a single AAV genome. The outermost 125 nucleotides also contain several shorter, self-complementary regions (referred to as regions A, A', B, B', C, C', and D), which enable the occurrence of intra-strand base pairing within this portion of the ITR.

[0102] A "end-break sequence" or "trs" is a sequence within the D region of the AAV ITR that is cleaved by the AAV rep protein during viral DNA replication. Mutant end-break sequences are less susceptible to cleavage by the AAV rep protein.

[0103] A "helper virus" for AAV refers to a virus that enables the replication and packaging of AAV (a defective parvovirus) by host cells. Numerous such helper viruses have been identified, including adenoviruses, herpesviruses, and poxviruses, such as vaccinia. Adenoviruses encompass many different subgroups, but adenovirus type 5 (Ad5) of subgroup C is the most commonly used. A great many adenoviruses of human, non-human mammalian, and avian origin are publicly known and available from depositary organizations such as ATCC. Herpes viruses, also available from depositary organizations such as ATCC, include, for example, herpes simplex virus (HSV), Epstein-Barr virus (EBV), cytomegalovirus (CMV), and pseudorabies virus (PRV).

[0104] The “percentage of sequence identity (%)” for 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 an amino acid residue or nucleotide in the reference polypeptide or nucleic acid sequence, after the sequences have been aligned and gaps have been introduced as necessary to achieve the maximum percentage of sequence identity, excluding any conservative substitutions that are considered part of the sequence identity. Alignment for determining the percentage of amino acid or nucleic acid sequence identity can be achieved in various ways within the scope of the art, including, for example, using publicly available computer software programs, such as those described in Current Protocols in Molecular Biology (Ausubel et al., 1987), Appendix 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 appropriate parameters for measuring alignment, including any algorithm necessary to achieve the maximum alignment over the full length of the sequences being compared. For the purposes of this specification, a given amino acid sequence A has an amino acid identity % with respect to a given amino acid sequence B (or a given amino acid sequence B has or contains a specific amino acid sequence identity % with respect to a given amino acid sequence B). The amino acid sequence A (which can be expressed as amino acid sequence A) is calculated as follows: 100 × fraction X / Y (where X is the number of amino acid residues scored as identical matches in the alignment of A and B by the sequence alignment program, and Y is the total number of amino acid residues in B). It will be understood that if the length of amino acid sequence A is not the same as the length of amino acid sequence B, the amino acid sequence identity % of A with respect to B will not be the same as the amino acid sequence identity % of B with respect to A. For the purposes of this specification, the nucleic acid identity % of a given nucleic acid sequence C with respect to a given nucleic acid sequence D (or a given nuclear sequence C that has or contains a specific nucleic acid sequence identity % with respect to a given nucleic acid sequence D) is calculated as follows: 100 × fraction W / Z (where W is the number of nucleotides scored as identical matches in the alignment of C and D by the sequence alignment 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 the same as the length of nucleic acid sequence D, then the percentage of nucleic acid sequence identity of C with respect to D will not be the same as the percentage of nucleic acid sequence identity of D with respect to C.

[0105] The term "isolated" means that a molecule (e.g., nucleic acid or protein) or cell has been identified, separated, and / or recovered from its natural environment.

[0106] An "effective dose" is a sufficient amount to achieve a beneficial or desired outcome, including clinical outcomes (e.g., improvement of symptoms, achievement of clinical endpoints). An effective dose may be administered in one or more doses. From a disease perspective, an effective dose is a sufficient amount to improve, stabilize, or delay the onset of the disease.

[0107] The “individual” or “subject” is a mammal. Mammals include, but are not limited to, livestock (e.g., cattle, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates, e.g., monkeys), rabbits, and rodents (e.g., mice and rats). In certain embodiments, the individual or subject is a human.

[0108] As used herein, “treatment” is an approach to obtain a beneficial or desired clinical outcome. For the purposes of the present invention, beneficial or desired clinical outcomes include, but are not limited to, symptom relief, a reduction in the severity of the disease, a stabilized (e.g., non-worsening) condition, prevention of disease spread (metastasis), delay or slowing of disease progression, improvement or mitigation of the condition, and remission (whether partial or complete remission), whether detectable or undetectable. “Treatment” can also mean an extension of survival compared to the survival expected without treatment.

[0109] As used herein, the term “preventive treatment” refers to treatment given to an individual that is known or suspected to have a disability or be at risk of developing a disability, but is not exhibiting any symptoms of the disability, or only mild symptoms. Individuals receiving preventive treatment will be treated before symptoms develop.

[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 outcome, such as the exemplary clinical outcomes described above. Therefore, therapeutic agents can be used in the above-described procedures.

[0111] As used herein, the term “central retina” refers to the outer macula and / or inner macula and / or fovea. As used herein, the term “central retinal cell type” refers to the cell type of the central retina, such as RPE and photoreceptor cells.

[0112] The term "macula" refers to the area of ​​the retina with a high relative density of photoreceptor cells, particularly rods and cones. This refers to the central retinal region of primates, including the body. As used herein, the term "external macula" may also refer to the "peripheral macula." As used herein, the term "internal macula" may also refer to the "central macula."

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

[0114] As used herein, the term “subretinal space” refers to the space between photoreceptor cells and retinal pigment epithelial cells within the retina. The subretinal space may also be a latent space, such as before any subretinal injection of a fluid. The subretinal space may also contain the fluid to be injected into the latent 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] As used herein, the term "bleb" 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 rearranged, create a total fluid cavity useful for achieving a therapeutic effect on a desired portion of the subretinal space.

[0116] The term "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, as well as in retinal cell lines, such as WERI Rb-1. As used herein, the term "rhodopsin kinase promoter" may 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): pp. 3954-61 and Young, JE et al. (2003) Invest. Ophthalmol. Vis. Sci. 44(9): pp. 4076-85. In some embodiments, the RK promoter ranges from -112 to +180 relative to the transcription start site.

[0117] The term "chicken β-actin (CBA) promoter" refers to a polynucleotide sequence derived from the chicken β-actin gene (e.g., chicken (Gallus gallus) β-actin represented by GenBank Entrez Gene ID396526). As used herein, the term "chicken β-actin promoter" may also refer to a promoter containing the cytomegalovirus (CMV) early enhancer element and the chicken β-actin gene promoter, as well as the first exon and intron and the 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" may be used synonymously. As used herein, the term "CMV early enhancer / chicken β-actin (CAG) promoter" may be used synonymously.

[0118] References to values ​​or parameters “about” in this specification include (and describe) embodiments relating to the value or parameter itself. For example, a statement referring to “about X” includes a statement of “X”.

[0119] As used herein, the singular articles “a,” “an,” and “the” include plural referents unless otherwise indicated.

[0120] It is understood that aspects and embodiments of the invention described herein include aspects and embodiments that “comprise,” “consist of,” and / or “consist essentially of.”

[0121] III. Virus Particles It is known in the art that heparan sulfate proteoglycan (HSPG) acts as a cellular receptor for AAV2 particles (Summerford, C. and Samulski, R. J. (1998) J. Virol. 72(2):1438-1445). Binding of AAV2 particles to HSPG 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 the cell by mechanisms including receptor-dependent endocytosis by clathrin-coated pits. AAV2 particles are released from intracellular vesicles by endosomal acidification. This allows AAV2 particles to move to the perinuclear region and then into the cell nucleus. It is also known that AAV3 particles bind to heparan (Rabinowitz, J. E. et al. (2002) J. Virol. 76(2):79-801).

[0122] ​Gene therapy protocols for eye disorders require local delivery of vectors to eye cells (e.g., retinal cells). Cells targeted for treatment in these diseases include, among others, one or more cells of the eye (e.g., photoreceptors, ocular neurons, etc.). The methods and kits of the present invention are at least partly based on the discovery that broad distribution of vectors among eye cells is possible by specific rAAV capsids (e.g., rAAV capsid proteins comprising one or more amino acid substitutions at one or more positions interacting with heparan sulfate proteoglycans). Therefore, these capsids would be particularly advantageous for the delivery of heterologous nucleic acids to an individual's eye, for enhancing rAAV transduction into cells after delivery of rAAV particles to an individual's eye, for enhancing the expression of heterologous nucleic acids after delivery of rAAV particles to an individual's eye, and / or for the treatment of an individual's eye disorder using rAAV particles.

[0123] Similarly, gene therapy protocols for CNS disorders require local delivery of vectors to CNS cells. Cells targeted for treatment in these diseases include, among others, one or more cells of the brain (e.g., neurons). The methods and kits of the present invention are at least partly based on the discovery that broad distribution of vectors among CNS cells is possible by specific rAAV capsids (e.g., rAAV capsid proteins comprising one or more amino acid substitutions at one or more positions interacting with heparan sulfate proteoglycans). Therefore, these capsids would be particularly advantageous for the delivery of heterologous nucleic acids to the central nervous system (CNS) of an individual, for enhancing rAAV transduction into cells after delivery of rAAV particles to the CNS of an individual, for enhancing the expression of heterologous nucleic acids after delivery of rAAV particles to the CNS of an individual, and / or for treating CNS disorders in an individual using rAAV particles.

[0124] It is known that the capsids of AAVs (e.g., AAV2, AAVrh8R, etc.) contain the following three capsid proteins: VP1, VP2, and VP3. These proteins contain a significant amount of duplicate amino acid sequences 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 have been found to exist in a 1:1:10 ratio.

[0125] The binding of AAV2 capsid protein to HSPG is basic AAV2 capsid protein It is known that this occurs through electrostatic interactions between residues and negatively charged glycosaminoglycan residues (Opie, SR et al. (2003) J. Virol. 77: pp. 6995-7006; Kern, A et al. (2003) J. Virol. 77: pp. 11072-11081). Specific capsid residues involved in these interactions include R484, R487, K532, R585, and R588. Mutations in these residues have been shown to reduce AAV2 binding to heparan cells and heparan itself (Opie, SR et al. (2003) J. Virol. 77: pp. 6995-7006; Kern, A et al. (2003) J. Virol. 77: pp. 11072-11081; WO2004 / 027019). A2, U.S. Patent No. 7,629,322). Furthermore, although we do not wish to be bound by theory, based on the VP1 numbering of AAV2, it is conceivable that amino acid substitutions in one or more residues corresponding to amino acids 484, 487, 532, 585, or 588 may modify the transduction properties of AAV capsid types that do not bind to HSPGs, or modify the transduction properties of AAV capsid types independently of their ability to bind to HSPGs.

[0126] A particular aspect of the present invention relates to the delivery of a 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 having one or more amino acid substitutions at one or more positions that interact with a heparan sulfate proteoglycan. 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 HSPG, or at one or more corresponding residues of amino acids 484, 487, 532, 585, or 588, numbered based on the VP1 numbering of AAV2, exhibit advantageous properties, e.g., enhanced expression and / or reduced neuroinflammation. Thus, in some embodiments, when delivered, the 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 in rAAV particles containing an rAAV capsid containing a reference rAAV capsid protein (e.g., 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, upon delivery, rAAV particles result in reduced neuroinflammation compared to rAAV particles containing a reference rAAV capsid protein (e.g., 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 include any capsid protein that does not have one or more amino acid substitutions at one or more positions that interact with the heparan sulfate proteoglycan (therefore, the reference capsid may have one or more “background” substitutions that do not alter the binding with HSPG).

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

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

[0130] In some embodiments, the rAAV particles of the present invention contain an AAV serotype 3 (AAV3) capsid. In some embodiments, one or more amino acid substitutions are substitutions of any one amino acid residue of VP1, VP2, and / or VP3 of AAV3, the amino acid substitutions modify the interaction between the rAAV particles and HSPG (e.g., reducing or eliminating binding to HSPG). In some embodiments, one or more amino acid substitutions are substitutions of the VP1 amino acid residue of AAV3. In some embodiments, one or more amino acid substitutions are substitutions of the VP2 amino acid residue of AAV3. In some embodiments, one or more amino acid substitutions are substitutions of the VP3 amino acid residue of AAV3. In some embodiments, one or more amino acid substitutions are substitutions of amino acid residues of the combination of VP1, VP2, and VP3 of AAV3. In some embodiments, one or more amino acid substitutions are substitutions of any one amino acid residue of VP1, VP2, and / or VP3 of AAV3. In some embodiments, one or more amino acid substitutions are substitutions of amino acid residues corresponding to the capsid protein of SEQ ID NO: 7.

[0131] In some embodiments, the rAAV particles of the present invention comprise an AAV serotype rh8R (AAVrh8R) capsid, as described, for example, in U.S. Patent Preregistration Publication No. 20090317417. In some embodiments, one or more amino acid substitutions are substitutions of any one amino acid residue of VP1, VP2, and / or VP3 of AAVrh8R, the amino acid substitutions modify the interaction between the rAAV particles and HSPG (e.g., reducing or eliminating binding to HSPG). In some embodiments, one or more amino acid substitutions are substitutions of the VP1 amino acid residue of AAVrh8R. In some embodiments, one or more amino acid substitutions are substitutions of the VP2 amino acid residue of AAVrh8R. In some embodiments, one or more amino acid substitutions are substitutions of the VP3 amino acid residue of AAVrh8R. In some embodiments, one or more amino acid substitutions are substitutions of amino acid residues of a combination of VP1, VP2, and VP3 of AAVrh8R. In some embodiments, one or more amino acid substitutions are substitutions of any one amino acid residue of VP1, VP2, and / or VP3 of AAVrh8R. In some embodiments, one or more amino acid substitutions are substitutions of amino acid residues of the capsid protein exemplified by SEQ ID NO: 9. In some embodiments, the rAAV particles of the present invention contain the capsid protein of SEQ ID NO: 10 and / or 11.

[0132] In some embodiments, one or more amino acid substitutions reduce the binding of 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, one or more amino acid substitutions reduce the binding of rAAV particles to heparan sulfate proteoglycans (compared to the binding of rAAV particles containing 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, one or more amino acid substitutions increase the binding of rAAV particles to heparan sulfate proteoglycans (compared to the binding of rAAV particles containing wild-type capsids) by approximately 10% to 100%, 20% to 100%, 30% to 100%, 40% to 100%, 50% to 100%, 60% to 100%, 70% to 100%, 80% to 100%, 90% to 100%, 10% to 90%, 20% to 90%, 30% to 90%, 40% to 90%, 50% to 90%, 60% to 90%, 70% to 90%, 80% to 90%, 10% to 80%, and 20% to 80%. Approximately 30% to 80%, approximately 40% to 80%, approximately 50% to 80%, approximately 60% to 80%, approximately 70% to 80%, approximately 10% to 70%, approximately 20% to 70%, approximately 30% to 70%, approximately 40% to 70%, approximately 50% to 70%, approximately 60% to 70%, approximately 10% to 60%, approximately 20% to 60%, approximately 30% to 60%, Reduce by one of the following percentages: approximately 40% to 60%, 50% to 60%, 10% to 50%, 20% to 50%, 30% to 50%, 40% to 50%, 10% to 40%, 20% to 40%, 30% to 40%, 10% to 30%, 20% to 30%, or 10% to 20%.In some embodiments, one or more amino acid substitutions do not result in detectable binding of rAAV particles to heparan sulfate proteoglycans compared to binding of wild-type rAAV particles. Means for measuring the binding of AAV particles to HSPGs are known in the art; for example, means of binding to a heparan sulfate chromatography medium, or means of binding to cells on which HSPGs are known to express. See, for example, Opie, SR et al., (2003) J. Virol. 77: pp. 6995-7006 and Kern, A et al., (2003) J. Virol. 77: pp. 11072-11081.

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

[0134] In some embodiments, one or more amino acid substitutions are located at positions 484, 487, 532, 585, or 588 of VP1 of AAV3, numbered based on VP1 of AAV2. In some embodiments, one or more amino acid substitutions are located at positions 484, 487, 532, 585, or 588 of VP2 of AAV3, numbered based on VP1 of AAV2. In some embodiments, one or more amino acid substitutions are located at positions 484, 487, 532, 585, or 588 of VP3 of AAV3, numbered based on VP1 of AAV2. In some embodiments, one or more amino acid substitutions are located at positions 484, 487, 532, 585, or 588 of VP1 of AAV3, VP2 of AAV3, and / or VP3 of AVV3, numbered based on VP1 of rAAV2. In some embodiments, VP1 of rAAV2 contains the amino acid sequence of SEQ ID NO: 1.

[0135] In some embodiments, the AAV particles of the present invention include a capsid having one or more amino acid substitutions that are numbered based on the VP1 numbering of AAVrh8R and are at positions 485, 488, 528, 533, 586, or 589. In some embodiments, the numbering is based on the VP1 of AAVrh8R, which includes the amino acid sequence of SEQ ID NO: 9. In some embodiments, one or more amino acid substitutions include the 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, one or more amino acid substitutions include the substitution of an arginine or lysine residue. In yet another embodiment, one or more amino acid substitutions include the substitution of an arginine or lysine residue with an alanine residue. In other embodiments, one or more amino acid substitutions include the 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, one or more amino acid substitutions include substitutions of alanine residues. In even further embodiments, one or more amino acid substitutions include substitutions of arginine or lysine residues with alanine residues. In some embodiments, one or more amino acid substitutions include substitutions at positions R533 and / A586, numbered based on VP1 of AAVrh8R. In further embodiments, the AAV capsid includes amino acid substitutions A586R and R533A, numbered based on VP1 of AAVrh8R. In some embodiments, the rAAV particles include the rAAV capsid protein of SEQ ID NO: 10 and / or 11.

[0136] In some embodiments of the present invention, one or more amino acid substitutions include the 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). Examples of 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 polar side chains that do not have a charge (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 hydrophobic amino acid residues. In some embodiments, one or more amino acid substitutions include the substitution of an arginine or lysine residue. In further embodiments, one or more amino acid substitutions include substitution of an arginine or lysine residue with an alanine residue. In other embodiments, one or more amino acid substitutions have a positive charge. This includes substitution of a hydrophobic amino acid residue with a positively charged amino acid 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 include substitution of an alanine residue. In yet another embodiment, one or more amino acid substitutions include substitution of an arginine or lysine residue with an alanine residue.

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

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

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

[0140] It is known that heparan sulfate proteoglycan (HSPG) is expressed in many tissues in the body and plays an important role in the extracellular matrix, cell adhesion and cell signaling. In some embodiments, heparan sulfate proteoglycan is expressed on one or more cells of the CNS. In certain embodiments, 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 the binding of the rAAV particles to a heparan sulfate proteoglycan. In some embodiments, the one or more amino acid substitutions are numbered based on VP1 of AAV2, at positions 347, 350, 390, 395, 448, 451, 484, 487, 527, 532, 585 and / or 588. As used herein, “numbering based on VP1 of AAV2” refers to the listed amino acids of the listed capsid protein corresponding to the listed amino acids of VP1 of AAV2. For example, if one or more amino acid substitutions are located at positions 347, 350, 390, 395, 448, 451, 484, 487, 527, 532, 585 and / or 588, numbered based on VP1 of AAV2, then one or more amino acid substitutions are located at the listed amino acids of the 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, one or more amino acid substitutions are located 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, one or more amino acid substitutions are at positions 484, 487, 532, 585, or 588 of AAV2. In some embodiments, 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 AAV2 (e.g., rAAV2) contains the amino acid sequence of SEQ ID NO: 1.

[0142] In some embodiments, the AAV capsid includes 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 present invention include the capsid protein of SEQ ID NOs. 2, 4 and / or 6. In some embodiments, the AAV capsid includes substitutions at positions R484 and R487, or R585 and R588, numbered based on VP1 of rAAV2. In some embodiments, the AAV capsid includes substitutions at positions R484A and R487A or R585A and R588A, numbered based on VP1 of AAV2. In some embodiments, the AAV capsid includes amino acid substitutions R585A and R588A, numbered based on VP1 of AAV2. In some embodiments, the AAV capsid includes amino acid substitution K532A, numbered based on VP1 of AAV2. In some embodiments, one or more amino acid substitutions include 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 of a capsid protein that reduces or eliminates the 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, for example, in U.S. Patent Preregistration Publication No. 20090317417. In some embodiments, the one or more amino acid substitutions are substitutions of any one amino acid residue of VP1, VP2, and / or VP3 of AAVrh8R, and the amino acid substitutions modify the interaction between the rAAV particles and HSPGs (e.g., reducing or eliminating binding to HSPGs). In some embodiments, the one or more amino acid substitutions are substitutions of the VP1 amino acid residue of AAVrh8R. In some embodiments, the one or more amino acid substitutions are substitutions of the VP2 amino acid residue of 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, one or more amino acid substitutions are substitutions of any one amino acid residue in VP1, VP2, and / or VP3 of AAVrh8R. In some embodiments, 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 present invention contain the capsid protein of SEQ ID NO: 10 and / or 11.

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

[0145] As discussed above, while we do not wish to be bound by theory, it is conceivable that one or more amino acid substitutions at 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-type molecules that do not bind to HSPGs, or modify the transduction properties of AAV capsid-type molecules independently of their ability to bind to HSPGs. In some embodiments, 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 the positions corresponding to amino acids 585 and / or 588 (numbered based on the VP1 of AAV2) are arginine residues. Therefore, they are replaced (for example, S586 and / 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, among other things, to improve intravitreous transduction targeting 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 for AAV2) are replaced by an amino acid that does not have a positive charge, such as 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, among other things, to enhance subretinal or CNS transduction.

[0146] In some embodiments, the rAAV particles contain an AAV serotype 1 (AAV1) capsid. In some embodiments, 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 contains the amino acid sequence of SEQ ID NO: 12. In some embodiments, one or more amino acid substitutions include substitutions at positions S586 and / T589, numbered based on the VP1 numbering of AAV1. In some embodiments, one or more amino acid substitutions include S586R or S586K substitutions, numbered based on the VP1 of AAV1. In some embodiments, one or more amino acid substitutions include T589R substitutions, numbered based on the VP1 of AAV1. In some embodiments, the rAAV particles contain an AAV serotype 6 (AAV6) capsid. In some embodiments, one or more amino acid substitutions are located at positions 586 and / or 589, numbered based on the VP1 numbering of AAV6. In some embodiments, the numbering is based on the VP1 of AAV6 containing the amino acid sequence of SEQ ID NO: 13. In some embodiments, one or more amino acid substitutions include substitutions at positions S586 and / T589, numbered based on the VP1 numbering of AAV6. In some embodiments, one or more amino acid substitutions include S586R or S586K substitutions, numbered based on the VP1 of AAV6. In some embodiments, one or more amino acid substitutions include T589R substitutions, numbered based on the VP1 of AAV6. In some embodiments, the rAAV particle contains an AAV serotype 8 (AAV8) capsid. In some embodiments, one or more amino acid substitutions are located 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 containing the amino acid sequence of SEQ ID NO: 14.In some embodiments, one or more amino acid substitutions include substitutions at positions Q588 and / or T591, numbered based on the VP1 numbering of AAV8. In some embodiments, one or more amino acid substitutions include Q588R or Q588K substitutions, numbered based on the VP1 numbering of AAV8. In some embodiments, one or more amino acid substitutions include T591R substitutions, numbered based on the VP1 numbering of AAV8. In some embodiments, the rAAV particle contains an AAV serotype 9 (AAV9) capsid. In some embodiments, 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 numbering of AAV9, including the amino acid sequence of SEQ ID NO: 15. In some embodiments, one or more amino acid substitutions are based on the VP1 numbering of AAV9. Numbered based on the ring, the substitutions include those at positions S586 and / A589. In some embodiments, one or more amino acid substitutions include S586R or S586K substitutions, numbered based on the VP1 of AAV9. In some embodiments, one or more amino acid substitutions include A589R or A589K substitutions, numbered based on the VP1 of AAV9. In some embodiments, the rAAV particle contains an AAV serotype rh10 (AAVrh10) capsid. In some embodiments, one or more amino acid substitutions are at positions 588 and / or 591, numbered based on the VP1 numbering of AAVrh10. In some embodiments, the numbering is based on the VP1 of AAVrh10 containing the amino acid sequence of SEQ ID NO: 16. In some embodiments, one or more amino acid substitutions include substitutions at positions Q588 and / A591, numbered based on the VP1 numbering of AAVrh10. In some embodiments, one or more amino acid substitutions include Q588R or Q588K substitutions, numbered based on VP1 of AAVrh10. In some embodiments, one or more amino acid substitutions include A591R substitutions, numbered based on VP1 of AAVrh10.

[0147] IV. Treatment Methods Gene therapy protocols for retinal diseases, such as LCA, retinitis pigmentosa, and age-related macular degeneration, require local delivery of vectors to retinal cells. In these diseases, the target cells are either retinal photoreceptor cells or cells of the RPE located beneath the retinal neurosensory epithelium. 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 a method for delivering an rAAV gene therapy vector to retinal cells, wherein the rAAV vector is encapsulated in an AAV capsid comprising one or more amino acid residue substitutions that interact with HSPG.

[0148] In some embodiments, the present invention provides a method for treating a disorder of the central nervous system (CNS) of an individual, comprising the delivery of a composition comprising 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 interacting with a heparan sulfate proteoglycan, 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, methods for improving rAAV transduction into cells after delivery of rAAV particles to the CNS of an individual, and methods for improving the expression of heterologous nucleic acids after delivery of rAAV particles to the CNS of an individual, as described herein, can be used to deliver heterologous nucleic acids, such as those encoding therapeutic polypeptides or therapeutic nucleic acids. These methods can, among other things, be used to treat disorders of the CNS. In some embodiments, the individual is a human.

[0149] Therapeutic vectors The present invention provides a method for gene therapy of an eye disorder, wherein rAAV particles containing a therapeutic vector are delivered to the retina of an individual. The rAAV capsid (e.g., particles such as rAAV2, rAAVrh8R, etc.) is such that one or more amino acids of the capsid that interact with HSPG are substituted to reduce or eliminate the binding of the rAAV particle to HSPG. By encapsulating the rAAV vector in the rAAV capsid, improved transduction into retinal cells can be achieved. The vector may contain heterologous nucleic acids encoding polypeptides (e.g., therapeutic or diagnostic polypeptides) and / or therapeutic nucleic acids. The nucleic acids encoding therapeutic or diagnostic polypeptides and / or therapeutic nucleic acids are synthesized using methods known in the art, employing standard synthesis and recombination techniques. , can be produced. In some embodiments, heterologous nucleic acids encode therapeutic polypeptides. In some embodiments, heterologous nucleic acids encode diagnostic polypeptides. Non-limiting examples of nucleic acids encoding therapeutic polypeptides include: nucleic acids for substitution of deletion or mutation genes known to cause retinal diseases, 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 eye 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 the polypeptide.

[0150] The nucleic acids of the present invention may encode polypeptides that are intracellular proteins, polypeptides immobilized on the cell membrane, polypeptides that remain in the cell, or polypeptides secreted by cells transduced with the vector of the present invention. For polypeptides secreted by cells that have received the vector, the polypeptide may be soluble (i.e., not attached to the cell). For example, soluble polypeptides lack a transmembrane domain and are secreted from the cell. Techniques for identifying and removing nucleic acid sequences encoding 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) such that, when transcribed from the nucleic acid of the vector, the RNA can treat ocular disorders by interfering with the translation or transcription of abnormal or excessive proteins associated with the disease of the present invention. For example, the nucleic acids of the present invention may also encode RNA that treats the disease by highly specific removal or reduction of mRNA encoding abnormal and / or excessive proteins. Examples of therapeutic RNA sequences include RNAi, small inhibitory RNA (siRNA), microRNA (miRNA), and / or ribozymes (e.g., hammerhead ribozymes and hairpin ribozymes) that can treat the disease by highly specific removal or reduction of mRNA encoding abnormal and / or excessive proteins, such as those occurring in various types of hereditary retinal degeneration. Non-limiting examples of ocular disorders that can be treated with 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, for example, those described in 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 within the 3' untranslated region of the rhodopsin gene. An rAAV vector encoding miR-708 and / or rhodopsin is provided in U.S. Provisional Patent Application No. 61 / 969,027, which is incorporated herein by reference in its entirety.

[0152] A particular aspect of the present invention relates to the use of rAAV particles (e.g., therapeutic vectors), wherein the rAAV particles (a) an rAAV capsid protein comprising one or more amino acid substitutions at one or more positions that interact with a heparan sulfate proteoglycan (b) an rAAV capsid comprising (a) a heterogeneous nucleic acid and at least one AAV terminal repeat sequence comprising an rAAV vector. In some embodiments, the heterogeneous nucleic acid encodes a therapeutic polypeptide or a therapeutic nucleic acid. As used herein, the therapeutic nucleic acid may express any therapeutic nucleic acid of the disclosure or any nucleic acid encoding any therapeutic polypeptide of the disclosure. The therapeutic nucleic acid can be used, for example, to improve the symptoms of a disorder (e.g., a disorder described herein), to prevent or delay the progression of said disorder, and / or to treat said disorder.

[0153] Enhanced transduction of CNS cells can be achieved by encapsulating an rAAV vector in an rAAV capsid (e.g., rAAV2, rAAVrh8R, etc.) in which one or more amino acids of the capsid that interact with HSPG are substituted to reduce or eliminate the binding of the rAAV particle to HSPG. The vector may contain heterologous nucleic acids encoding polypeptides (e.g., therapeutic or diagnostic polypeptides) and / or therapeutic nucleic acids. Nucleic acids encoding therapeutic or diagnostic polypeptides and / or therapeutic nucleic acids can be produced using methods known in the art, employing standard synthesis and recombination 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, heterologous nucleic acids encode therapeutic nucleic acids. In some embodiments, therapeutic nucleic acids may include, but are not limited to, siRNA, shRNA, RNAi, miRNA, antisense RNA, ribozymes, or DNAzymes. Thus, therapeutic nucleic acids may also encode RNA that, when transcribed from the nucleic acid of a vector, can treat the disorders of the present invention (e.g., CNS disorders) by interfering with the translation or transcription of abnormal or excessive proteins associated with the disorders of the present invention. For example, the nucleic acids of the present invention may encode RNA that treats the disorder by highly specific removal or reduction of mRNA encoding abnormal and / or excessive proteins. Examples of therapeutic RNA sequences include RNAi, small inhibitory RNA (siRNA), microRNA (miRNA), and / or ribozymes (e.g., hammerhead ribozymes and hairpin ribozymes) that can treat the disorder by highly specific removal or reduction of mRNA encoding abnormal and / or excessive proteins.

[0155] In some embodiments, heterologous nucleic acids encode therapeutic polypeptides. Therapeutic polypeptides may, for example, supply polypeptide and / or enzyme activity that is absent or present at reduced levels within a cell or organism. Alternatively, therapeutic polypeptides may supply polypeptide and / or enzyme activity that indirectly resolve imbalances within a cell or organism. For example, therapeutic polypeptides for disorders associated with the accumulation of metabolites due to a deficiency of metabolic enzymes or activity may supply the deficient metabolic enzyme or activity, or supply an alternative metabolic enzyme or activity that results in a reduction of the metabolite. Therapeutic polypeptides may also be used to reduce the activity of polypeptides (e.g., those that are overexpressed, activated by gain-of-function mutations, or whose activity is otherwise misregulated) by acting, for example, as dominant-negative polypeptides.

[0156] In some embodiments, heterologous nucleic acids encode polypeptides selected from enzymes, neurotrophic factors, deficient or mutant polypeptides in individuals with CNS-related disorders, antioxidants, anti-apoptotic factors, anti-angiogenic factors, and anti-inflammatory factors. Such polypeptides are used, for example, to supply polypeptide and / or enzyme activity that is reduced, absent, or misregulated during CNS disorders. CNS disorders can be treated by improving the cause and / or symptoms, and / or by mitigating damage to the CNS resulting from the CNS disorder (e.g., apoptosis, inflammation, or other types of cell death). Non-limiting examples of nucleic acids encoding therapeutic polypeptides include: nucleic acids for substitution of deletion or mutational genes known to cause CNS disorders, 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 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-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 / or aspartacylase (ASPA). Examples of antioxidants, though not limited to them, include SOD1; SOD2; catalase; sirtuin 1, 3, 4 or 5; NRF2; PGC1a; GCL (catalytic subunit); GCL (modifier subunit); adiponectin; glutathione peroxidase 1; and neurorobin.Examples of anti-angiogenic polypeptides include, but are not limited to, angiostatins, endostatins, PEDF, soluble VEGF receptors, and soluble PDGF receptors. Examples of anti-inflammatory polypeptides include, but are not limited to, IL-10, soluble IL-17R, soluble TNF-R, TNF-R-Ig, IL-1 inhibitors, and IL-18 inhibitors. Other exemplary polypeptides of these classes that can be used to treat CNS disorders are provided below.

[0157] The nucleic acids of the present invention may encode polypeptides that are intracellular proteins, polypeptides immobilized on the cell membrane, polypeptides that remain in the cell, or polypeptides secreted by cells transduced with the vector of the present invention. For polypeptides secreted by cells that have received the vector, the polypeptide may be soluble (i.e., not attached to the cell). For example, soluble polypeptides lack a transmembrane domain and are secreted from the cell. Techniques for identifying and removing nucleic acid sequences encoding transmembrane domains are known in the art.

[0158] In some embodiments, heterologous nucleic acids are operably linked to a promoter. Examples of promoters include the cytomegalovirus (CMV) earliest promoter, RSV LTR, MoMLV LTR, phosphoglycerate kinase-1 (PGK) promoter, monkey 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): pp. 193-199) and elongation factor 1-α promoter (EF1-α) promoter (Kim et al., Gene, 1990, 91(2): pp. 217-23 and Guo et al., Gene Ther., 1996, 3(9): pp. 802-10), but The present invention is not limited to these. In some embodiments, the promoter includes a human β-glucuronidase promoter or a cytomegalovirus enhancer ligated to a chicken β-actin (CBA) promoter. The promoter may be constitutive, inductive, or repressive. In some embodiments, the present invention provides a recombinant vector comprising a nucleic acid encoding a heterologous transgene of the present disclosure operably ligated to a CBA promoter. Exemplary promoters and descriptions can be found, for example, in U.S. Pre-Registered Patent Application Publication No. 20140335054.

[0159] Examples of constitutive promoters include, but are not limited to, the retroviral Roussarcoma virus (RSV) LTR promoter (sometimes accompanied by an RSV enhancer), the cytomegalovirus (CMV) promoter (sometimes accompanied by a CMV enhancer) [see, for example, Boshart et al., Cell, 41: pp. 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 enable the regulation of gene expression, which is controlled by externally supplied compounds, by environmental factors such as temperature, or by specific physiological conditions, such as the acute phase, the presence of a specific differentiation state of the cell, 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 easily select them. Examples of inducible promoters controlled by externally supplied promoters include the zinc-inducible sheep metallothionein (MT) promoter, the dexamethasone (Dex)-inducible mouse mammary cancer virus (MMTV) promoter, the T7 polymerase promoter system (WO98 / 10088); the ecdysone insect promoter (No et al., Proc. Natl. Acad. Sci. USA, 93:3346-3351 (1996)); and the tetracycline inhibitory system (Gosse Examples include n et al., Proc. Natl. Acad. Sci. USA, 89: pp. 5547-5551 (1992), tetracycline-inducible systems (see also Gossen et al., Science, 268: pp. 1766-1769 (1995), and Harvey et al., Curr. Opin. Chem. Biol., 2: pp. 512-518 (1998)), RU486-inducible systems (Wang et al., Nat. Biotech., 15: pp. 239-243 (1997) and Wang et al., Gene Ther., 4: pp. 432-441 (1997)), and rapamycin-inducible systems (Magari et al., J. Clin. Invest., 100: pp. 2865-2872 (1997)). In this regard, other types of inductive promoters that may be useful are those that are regulated by specific physiological conditions, such as temperature, acute phase, a particular differentiation state of a cell, or only in replicating cells.

[0161] In another embodiment, the native promoter or a fragment thereof of the transgene may be used. A native promoter can be used when it is desired that the expression of the transgene mimics native expression. A native promoter may also be used when it is necessary to regulate the expression of the transgene temporally or developmentally, or in a tissue-specific manner, or in response to a specific transcriptional stimulus. In a further embodiment, other native expression regulatory 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 ability. In some cases, the tissue-specific regulatory sequence binds to a tissue-specific transcription factor that induces transcription in a tissue-specific manner. Such tissue-specific regulatory sequences (e.g., promoters, enhancers) Examples of such tissue-specific regulatory sequences are well known in the art. Exemplary tissue-specific promoters include, but are not limited to, the following: neuron promoters, e.g., neuron-specific enolase (NSE) promoter (Andersen et al., Cell. Mol. Neurobiol., 13:503-15 (1993)), neurofilament light chain gene promoter (Piccioli et al., Proc. Natl. Acad. Sci. USA, 88:5611-5 (1991)), and 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: neuronal nucleus (NeuN), glial fibrillary acidic protein (GFAP), adenomatous polyposis (APC), and ionized calcium-binding adapter molecule 1 (Iba-1). Other suitable tissue-specific promoters will be apparent to those skilled in the art. In some embodiments, the promoter is the chicken β-actin promoter.

[0163] In some embodiments, heterologous nucleic acids are under the control of a promoter sequence expressed in one or more cells of the CNS. It is known in the Art that many of the promoter sequences listed above (e.g., the CBA promoter) are expressed in one or more cells of the CNS. In some embodiments, the promoter sequence is ubiquitous in the organism and can therefore be expressed in one or more cells of the CNS by its delivery to the CNS. In other embodiments, promoter sequences that are specifically expressed in the CNS or in a subset of one or more CNS cells may be used. In some embodiments, heterologous nucleic acids are operably linked to a promoter suitable for the expression of a therapeutic polypeptide or therapeutic nucleic acid in one or more cells of the CNS. Therefore, in some embodiments, CNS disorders can be treated using the therapeutic polypeptide or therapeutic nucleic acid of the present invention.

[0164] In some embodiments, the promoter expresses heterologous nucleic acids in brain cells. Brain cells may refer to any brain cells 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.), parenchymal cells, microglial cells, ependymal cells, and / or Purkinje cells. In some embodiments, the promoter expresses heterologous nucleic acids in neurons. In some embodiments, the heterologous nucleic acids are expressed exclusively in neurons (e.g., expressed in neurons and not in other cells of the CNS, such as glial cells).

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

[0166] rAAV vectors may contain nucleic acids as transgenes that encode proteins or functional RNAs that modulate or treat CNS-related disorders. The following is a non-exclusive list of genes associated with CNS-related disorders: neuronal apoptosis inhibitory proteins (NAIPs), nerve growth factor (NGF), glial cell-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 decarboxylases (AADCs). For example, a transgene useful in 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 in treating Parkinson's disease. Transgenes encoding GDNF or BDNF, or AADC which facilitates 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 that inhibit SOD1 expression, such as shRNA and miRNA. Transgenes useful for treating ischemia may encode NAIP or NGF. Transgenes encoding β-glucuronidase (GUS) may be useful for treating certain lysosomal storage disorders (e.g., mucopolysaccharidosis type VII (MPS VII)). Transgenes encoding prodrug activators, such as HSV-thymidine kinase which converts ganciclovir into toxic nucleotides that disrupt DNA synthesis and lead to cell death, may be useful for treating certain cancers, for example, when administered in combination with the aforementioned 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 neurorobin. 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 inhibitors, and IL18 inhibitors. Other examples of transgenes that can be used in the rAAV vector of the present invention will be apparent to those skilled in the art (see, for example, Costantini LC et al., Gene Therapy (2000) 7, pp. 93-109).

[0167] In some embodiments, therapeutic polypeptides or therapeutic nucleic acids are used to treat CNS disorders. While we do not wish to be bound by theory, it is conceivable that therapeutic polypeptides or therapeutic nucleic acids can be used to reduce or eliminate the expression and / or activity of polypeptides whose gain of function is associated with the disorder, or to enhance the expression and / or activity of polypeptides to compensate for defects associated with the disorder (e.g., mutations in genes whose expression exhibits similar or related activity). Non-limiting examples of disorders of the invention that can be treated with the therapeutic polypeptides or therapeutic nucleic acids of the present invention (exemplary genes targeted or supplied are provided in parentheses for each disorder) include: stroke (e.g., caspase-3, Beclin1, Ask1, PAR1, HIF1α, PUMA, and / or any of the genes described in Fukuda, AM and Badaut, J. (2013) Genes (Basel) 4: pp. 435-456), Huntington's disease (mutant HTT), epilepsy (e.g., SCN1A, NMDAR, ADK, and / or any of the genes described in Boison, D. (2010) Epilepsy 51: pp. 1659-1668), Parkinson's disease (α-synuclein), Lou Gehrig's disease (also known as amyotrophic lateral sclerosis; SOD1), Alzheimer's disease (tau, amyloid precursor protein), and basal cortex Examples of disorders of the present invention include nuclear degeneration or CBD (tau), corticobasal ganglia ganglion 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., mutant or overexpressed oncogenes involved in brain cancer), and lysosomal storage disorders (LSD). Disorders of the present invention may include those involving large areas of the cortex, e.g., one or more functional areas of the cortex, one or more lobes of the cortex, and / or the entire cortex. Other non-limiting examples of disorders of the present invention that can be treated with the 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, leukodystrophy (including Canavan disease) and any of the lysosomal storage disorders described below.

[0168] In some embodiments, therapeutic polypeptides or therapeutic nucleic acids are used to treat lysosomal storage disorders. As is commonly known in the art, lysosomal storage disorders are rare hereditary metabolic disorders characterized by deficiencies in lysosomal function. Such disorders often result from deficiencies in enzymes necessary for proper mucopolysaccharide, glycoprotein, and / or lipid metabolism, leading to the pathological accumulation of cellular material stored in lysosomes. Non-limiting examples of lysosomal storage disorders of the present invention that can be treated with the therapeutic polypeptide or therapeutic nucleic acid of the present invention (exemplary genes targeted or supplied 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's disease (iduronate 2-sulfatase, IDS), Krabbe disease (galactosylceramidase, GALC), mannosidosis (mannosidase, 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), and Niemann-Pick disease type A (acid sphingomyelia Linase (ASM), 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 (heparinacetyl-C Examples include oA (α-glucosaminide N-acetyltransferase, MPS3C), Sanfilippo disease type D (N-acetylglucosamine-6-sulfatase, GNS), Schindler's disease (α-N-acetylgalactosaminidase, NAGA), Sleigh's disease (β-glucuronidase, GUSB), Tay-Sachs disease (hexosaminidase α subunit, HEXA), and Wolmann's disease (lysosomal acid lipase, LIPA).

[0169] Further lysosomal storage disorders, as well as the deficiency enzymes associated with each disease, are listed in Table 1 below. In some embodiments, the diseases listed in the table below are treated with therapeutic peptides or therapeutic nucleic acids of the present 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 vectors would be advantageous is Huntington's disease (HD), caused by a CAG repeat elongation mutation encoding an elongated polyglutamine (PolyQ) repeat sequence of mutant huntingtin protein (mHTT). Since HD is an autosomal dominant disorder resulting from a single allele mutation, it is an attractive target for DNA-based and RNA-based therapies. AAV vectors could provide an ideal delivery system for nucleic acid therapy, enabling long-term, sustained expression of these huntingtin-reducing molecules in the brain.

[0173] As described herein, intracranial administration of rAAV particles (e.g., therapeutic vectors) having an rAAV capsid protein 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 a method for delivering heterologous nucleic acids to the central nervous system using recombinant viral particles described herein for the treatment of Huntington's disease. In some embodiments, the present invention provides a method and composition for treating Huntington's disease in an individual, comprising administering the pharmaceutical composition of the present disclosure (e.g., a pharmaceutical composition comprising the mutant viral particles of the present disclosure) to a mammal. In some embodiments, the present invention provides a method and composition for inhibiting htt expression in a mammal having Huntington's disease, comprising administering the pharmaceutical composition of the present disclosure (e.g., a pharmaceutical composition comprising the mutant viral particles of the present disclosure) to an individual. In some embodiments, the present invention provides methods and compositions for inhibiting the accumulation of htt in the cells of an individual having Huntington's disease, comprising administering the pharmaceutical composition of the present disclosure (for example, a pharmaceutical composition comprising mutant virus particles of the present disclosure) to the individual.

[0174] In some embodiments, the present invention relates to a method and composition for improving the symptoms of HD in an individual, comprising an effective amount of recombinant virus particles having one or more amino acid substitutions at one or more positions that interact with heparan sulfate proteoglycans in the individual's CN The present invention provides a method and composition comprising administering to S, wherein the rAAV particles comprise a vector encoding 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, uncontrolled body movements, muscle incompatibility, dyscoordination, restlessness, slow eye movements, postural abnormalities, instability, ataxic gait, abnormal facial expressions, speech disorders, chewing and / or swallowing difficulties, sleep disorders, seizures, dementia, agnosia (e.g., impaired ability related to planning, abstract thinking, flexibility, rule-following, interpersonal sensitivity, self-control, attention, learning and memory), depression, anxiety, personality changes, aggression, compulsive behavior, obsessive-compulsive behavior, hypersexuality, psychosis, emotional blunting, irritability, suicidal ideation, weight loss, muscle atrophy, heart failure, impaired glucose tolerance, testicular atrophy and osteoporosis.

[0175] In some embodiments, the present invention provides methods for preventing or delaying the progression of HD. Autosomal dominant HD is a genetic disorder that can be determined by genotyping. For example, the number of CAG repeat sequences in HTT may be determined by PCR-based repeat sequence sizing. This type of diagnosis can be made at any stage of life by direct testing of young or adult individuals (e.g., along with the presentation of clinical symptoms), by parental screening or exclusion testing (e.g., by chorionic villus sampling or amniocentesis), or by preimplantation genetic screening of the fetus. In addition, HD may be diagnosed by imaging the brain and looking for atrophy of the caudate nucleus and / or putamen and / or ventricular hypertrophy. These symptoms, along with a family history of HD and / or clinical symptoms, would indicate HD.

[0176] Methods for determining improvement in HD symptoms are known in the art. For example, the Unified Huntington's Disease Rating Scale (UHDRS) may be used to assess motor function, cognitive function, behavioral abnormalities, and functional ability (see, e.g., Huntington Study Group (1996) Movement Disorders 11: pp. 136-142). This rating scale was developed to provide a unified and comprehensive test of multiple aspects of disease pathology by incorporating elements from tests such as the HD Motor and Daily Living Scale, the Marsden and Quinn Chorea Severity Scale, Physical Disability and Independent Scales, the HD Motor Rating Scale (HDMRS), the HD Functional Ability Scale (HDFCS), and the Quantified Neurological Examination (QNE). Other tests useful for determining HD symptom improvement include, but are not limited to, the Montreal Cognitive Assessment, brain imaging (e.g., MRI), categorical fluency tests, trail-making tests, map search, Stroop word reading tests, accelerated tapping tasks, and symbol-number modality tests.

[0177] In some embodiments of the present invention, the methods and compositions described above are used to treat humans having HD. As described above, HD is inherited in an autosomal dominant manner and is caused by the elongation of the CAG repeat sequence in the HTT gene. Juvenile-onset HD is almost always inherited paternally. Huntington's disease-like phenotypes have also been shown to correlate with other loci, such as HDL1, PRNP, HDL2, HDL3, and HDL4. Other loci, including mutations in the GRIN2A, GRIN2B, MSX1, GRIK2, and APOE genes, are also thought to 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 optionally comprise an effective amount of rAAV particles comprising a polypeptide and / or RNA-encoding rAAV vector in a pharmaceutically acceptable excipient. These viral particles comprise an AAV capsid (e.g., AAV2 or AAVrh8R capsid) in which one or more amino acids that interact with HSPG are substituted to reduce or eliminate rAAV particle binding to HSPG. As is well known in the art, pharmaceutically acceptable Acceptable excipients are relatively inert substances that facilitate the administration of pharmacologically effective substances and can be supplied as solutions or suspensions, emulsions, or in solid forms suitable for dissolving or suspending in liquid before use. For example, excipients can impart shape or consistency, or act as diluents. Suitable excipients include, but are not limited to, stabilizers, wetting and emulsifying agents, salts for altering molar osmotic pressure, mounting agents, pH buffers, and buffering agents. Such excipients include any pharmaceuticals suitable for direct delivery to the eye that can be administered without excessive toxicity. Medicinally acceptable excipients include, but are not limited to, sorbitol, any of the various TWEEN compounds, and liquids such as water, saline, glycerol, and ethanol. Medicinally acceptable salts, such as inorganic salts, such as hydrochloride, hydrobromide, phosphate, sulfate, etc., and salts of organic acids, such as acetate, propionate, malonate, benzoate, etc., may also be included. A detailed discussion of pharmaceutically acceptable excipients can be found 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 used in combination with pharmaceutically acceptable vehicles, such as saline solution or Ringer's equilibrium salt solution (pH 7.4). While not essential, the compositions may, in some cases, be supplied in unit dosage forms suitable for precise dose administration.

[0180] Subretinal delivery method of rAAV Subretinal delivery methods are known in the art. See, for example, WO2009 / 105690, incorporated herein by reference. Briefly, a general method for delivering rAAV particles (e.g., particles such as rAAV2 and rAAVrh8R) subretinally to the macula and fovea can be described by the following general outline. This example is intended to illustrate certain features of the method and not to limit it 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 a capsid of rAAV particles, the rAAV particles comprising an rAAV capsid protein containing one or more amino acid substitutions at one or more positions that interact with heparan sulfate proteoglycans (e.g., inhibiting or reducing HSPG binding), and an rAAV vector containing heterogeneous nucleic acids and at least one AAV inverted terminal repeat sequence. The procedure involves injecting the rAAV vector suspension using a thin cannula by vitrectomy, followed by one or more small retinal incisions into the subretinal space.

[0182] Simply put, the injection cannula is sutured in place, and normal ocular volume can be maintained throughout the surgery by injecting (e.g., saline solution). Vitrectomy is performed using a cannula of appropriate diameter (e.g., 20-27 gauge), and the volume of vitreous gel removed is replaced by the injection of saline solution or other isotonic solution through the injection cannula. Vitrectomy is advantageous because (1) the removal of its cortex (posterior vitreous membrane) facilitates retinal penetration by the cannula; (2) its removal and replacement with fluid (e.g., saline solution) creates a cavity suitable for intraocular injection of the vector; and (3) its controlled removal reduces the possibility of retinal tears and unplanned retinal detachment.

[0183] In some embodiments, the rAAV composition is directly injected into the subretinal space outside the central retina using a cannula of an appropriate diameter (e.g., 27-45 gauge), thus forming a bleb in the subretinal space. In other embodiments, a small amount (e.g.) is administered before the retinal injection of the rAAV composition. A suitable fluid (e.g., saline or Ringer's solution) of approximately 0.1 to 0.5 ml is injected subretinally into the subretinal space outside the central retina. This initial injection into the subretinal space establishes the first bleb within the subretinal space, thereby localizing the retinal detachment to the location of the first bleb. This first fluid bleb can facilitate targeted delivery of the rAAV composition into the subretinal space (by defining the injection surface before rAAV delivery), minimize possible rAAV administration to the choroid, and minimize the possibility of rAAV injection into or reflux of the vitreous cavity. In some embodiments, a fluid containing one or more rAAV compositions and / or one or more further therapeutic agents may be further injected into the first fluid bleb by direct administration of these fluids into the first fluid bleb using either the same or further small-diameter cannula.

[0184] Intraocular administration of the rAAV composition and / or the initial small amount of fluid can be performed using a small-diameter cannula (e.g., 27-45 gauge) attached to a syringe. In some embodiments, the plunger of this syringe can be driven by a mechanized device, for example, by pressing down a foot pedal. The small-diameter cannula is advanced by scleral incision across the vitreous cavity to a predetermined site on the retina of each target (but outside the central retina) according to the target retinal region. With direct visibility, the retinal detachment is localized with a non-expanding retinal incision that closes spontaneously, and the vector suspension is mechanically injected under the retinal neurosensory epithelium. 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 the first bleb outside the central retina to inflate that bleb (and enlarge the retinal detachment area). In some embodiments, another fluid is injected into the bleb after the injection of the rAAV composition.

[0185] While we do not wish to be bound by theory, the rate and location of subretinal injection may result in localized shear forces that could damage the macula, fovea, and / or underlying RPE cells. Subretinal injection may be performed at a rate that minimizes or avoids these shear forces. In some embodiments, the rAAV composition is injected over approximately 15–17 minutes. In some embodiments, the vector is injected over approximately 17–20 minutes. In some embodiments, the rAAV composition is injected over approximately 20–22 minutes. In some embodiments, the rAAV composition is injected at a rate of approximately 35–65 μl / min. In some embodiments, the rAAV composition is injected at a rate of approximately 35 μl / min. In some embodiments, the rAAV composition is injected at a rate of approximately 40 μl / min. In some embodiments, the rAAV composition is injected at a rate of approximately 45 μl / min. In some embodiments, the rAAV composition is injected at a rate of approximately 50 μl / min. In some embodiments, the rAAV composition is injected at a rate of approximately 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. It will be understood by those skilled in the art that the injection rate and time of the bleb may depend, for example, on the volume of the rAAV composition or the size of the bleb required to produce a retinal detachment sufficient to approach the 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 present invention, the volume of the composition injected into the subretinal space of the retina is greater than or any one of the following amounts: approximately 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.

[0187] In some embodiments, the method involves interacting with heparan sulfate proteoglycan a recombinant viral particle comprising one or more amino acid substitutions at one or more positions that interact with , the method comprising administering an effective amount of the recombinant viral particle comprising a vector encoding a heterologous nucleic acid to the eye (e.g., by subretinal and / or intravitreal administration). In some embodiments, the viral titer of the composition is at least about 5×10 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 genome copies / mL. In some embodiments, the viral titer of the composition is about 5×10 12 to 6×10 12 6×10 12 to 7×10 12 7×10 12 to 8×10 12 8×10 12 to 9×10 12 9×10 12 to 10×10 12 10×10 12 to 11×10 12 11×10 12 to 15×10 12 15×10 12 to 20×10 12 20×10 12 to 25×10 12 25×10 12 to 30×10 12 30×10 12 to 50×10 12 or 50×10 12 to 100×10 12 genome copies / mL. In some embodiments, the viral titer of the composition is about 5×10<00​12 , 10×10 12 ~25×10 12 , or 25×10 12 ~50×10 12 It is either genome copies / mL. In some embodiments, the viral titer of the composition is at least about 5 × 10⁶. 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 x 10 9 It is either transduction units / mL. In some embodiments, the viral titer of the composition is about 5 × 10⁻¹⁶. 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 x 10 9 ~100×10 9 It is either transduction units / mL. In some embodiments, the viral titer of the composition is about 5 × 10⁻¹⁶. 9 ~10×10 9 , 10×10 9 ~15×10 9 , 15×10 9~25×10 9 、 or 25×10 9 ~50×10 9 transfection units / mL. In some embodiments, the viral titer of the composition is at least about 5×10 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 infection units / mL. In some embodiments, the viral titer of the composition is at least about 5×10 10 ~6×10 10 、 6×10 10 ~7×10 10 、 7×10<z 10 ~8×10 10 、 8×10 10 ~9×10 10 、 9×10 10 ~10×10 10 [[ID=SO]]? 10×10 10 ~11×10 10 、 11×10 10 ~15×10 10 、 15×10 10 ~20×10 10 、 20×10 10 ~25×10 10 [[ID=GS]]、 25×10 10 ' ~30×10 10 、 30×10<a000124>~40×10 10 、 40×10 10 ~50×10 10 、 or 50×10 10 ~100×10 10 infection units / mL. In some embodiments, the viral titer of the composition is at least about 5×10 10 ~10×10 10 、 10×10 10 It should be noted that there may be some inaccuracies in the original text, such as the repeated use of some tags and the unclear context in some parts. The translation is done as accurately as possible based on the given rules.~15×10 10 , 15×10 10 ~25×10 10 , or 25×10 10 ~50×10 10 It is either infectious units / mL or one of the following:

[0188] In some embodiments, the method includes administering an effective amount of recombinant viral particles containing one or more amino acid substitutions at one or more positions that interact with heparan sulfate proteoglycans to the eye of an individual (e.g., a human) (e.g., by subretinal and / or intravitreal administration). In some embodiments, the dose of viral particles administered to the individual is at least about 1 × 10¹⁶ kg of body weight. 8 ~Approx. 1×10 13 It is one of the genome copies. In some embodiments, the dose of viral particles administered to an individual is about 1 × 10⁶ per kg of body weight. 8 ~Approx. 1×10 13 It is one of the genome copies.

[0189] One or more (for example, two, three, or more) blebs can be formed. Generally, the total volume of blebs formed by the method and system of the present invention cannot exceed the fluid volume of the eye, for example, 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, in order to promote a retinal detachment of sufficient size to expose the central retinal cell type and to produce a bleb of sufficient dependence for optimal operation. It will be understood by those skilled in the art that appropriate intraocular pressure must be maintained to avoid damage to the structure of the eye when producing blebs according to the method and system of the present invention. 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. In addition, the total volume of all the blebs would be, for example, approximately 0.5 to 3.0 ml, 0.8 to 3.0 ml, 0.9 to 3.0 ml, 1.0 to 3.0 ml, 0.5 to 1.5 ml, 0.5 to 1.2 ml, 0.9 to 3.0 ml, 0.9 to 2.0 ml, and 0.9 to 1.0 ml.

[0190] To safely and efficiently transduce a target retinal region (e.g., the central retina) outside the breb's situational edge, the breb may be manipulated to reposition it to the target region for transduction. This manipulation of the breb can be performed by realigning the human head and eye or an eye with one or more brebs, by changing the position of the breb on the eye due to the breb's dependency caused by its volume, and / or by using fluid-air displacement. This is particularly true for the central retina, as this region is usually resistant to detachment due to subretinal injection. In some embodiments, the breb is repositioned using fluid-air displacement; the fluid from the injection cannula is temporarily replaced with air, for example, by blowing air onto the retinal surface. As a large volume of air displaces the vitreous fluid from the retinal surface, the fluid within the vitreous cavity can flow out of the cannula. Due to the temporary lack of pressure from the vitreous fluid, the breb moves and is attracted to its dependent part in the eye. The bleb of the subretinal rAAV composition is manipulated to include adjacent regions (e.g., the macula and / or fovea) by appropriately positioning the eyeball. In some cases, the majority of the bleb is sufficient to attract it without the use of flowing air displacement. The movement of the bleb to the desired position may be further facilitated by changing the position of the subject's head to attract the bleb to the desired position within the eye. Once the desired stereochemical arrangement of the bleb is achieved, the fluid is returned to the vitreous cavity. This fluid is a suitable fluid, such as freshly prepared saline solution. In general, the subretinal rAAV composition may be left in vivo in situ without retinal tear closure for retinal incision and without intraocular tamponade, and the retina will spontaneously reattach within approximately 48 hours.

[0191] The present invention provides a method for safely and efficiently transducing a vector containing a therapeutic polypeptide or RNA sequence into ophthalmic cells (e.g., RPE and / or photoreceptor cells of the macula and / or fovea) to produce transduced cells that generate sufficient amounts of the therapeutic polypeptide or RNA sequence to treat an eye disorder. This method can be used to treat individuals with eye disorders; for example, humans. In some embodiments, transduction into ophthalmic cells is enhanced by using rAAV particles (e.g., particles such as rAAV2, rAAVrh8R, etc.) containing an AAV capsid protein with one or more amino acid substitutions that interact with HSPG (e.g., inhibit or remove binding to HSPG). In some embodiments, the rAAV particles exhibit such binding reduction; for example, reduced to about 10%, 25%, 50%, 75%, over 100%, or any number in between. In some embodiments, rAAV binding with HSPG is reduced by approximately 5% to approximately 100%, approximately 10% to approximately 50%, approximately 10% to approximately 30%, approximately 25% to approximately 75%, approximately 25% to approximately 50%, or approximately 30% to approximately 50%.

[0192] An effective amount of rAAV (in some embodiments, in the form of particles) is administered according to the therapeutic objective. For example, if the desired therapeutic effect can be achieved with a low transduction percentage, the objective of the treatment is generally to meet or exceed this transduction level. In some examples, this transduction level can be achieved by transducing only about 1-5% of target cells, at least about 20% of cells of the desired tissue type in some embodiments, at least about 50% in some embodiments, at least about 80% in some embodiments, at least about 95% in some embodiments, and at least about 99% of cells of the desired tissue type in some embodiments. As discussed above, substitution of one or more amino acids in the rAAV capsid that interacts with HSPG improves rAAV transduction. For reference, the number of particles administered in a single injection is generally about 1 × 10 particles. 6 ~1 × 10 14 During this time, the particles are approximately 1 × 10 7~1 × 10 13 During this time, the particles are approximately 1 × 10 9 ~1 × 10 12 Between, or particles approximately 1 × 10 11 The rAAV composition may be administered by subretinal injection, one or more times, during the same procedure or at intervals of several days, weeks, months, or years. In some embodiments, multiple vectors may be used to treat humans.

[0193] In some embodiments, administration of an effective amount of rAAV virus particles containing an rAAV capsid having one or more amino acid substitutions that interact with HSPG to the retina causes transduction into photoreceptor cells at or near the administration site. In some embodiments, more than about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or 100% of photoreceptor cells 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 photoreceptor cells are transduced. Methods for identifying photoreceptor cells transduced by AAV virus particles containing rAAV capsids having one or more amino acid substitutions that interact with HSPGs are known in the art; for example, transduction of viral particles containing rAAV capsids having one or more amino acid substitutions that interact with HSPGs can be detected using immunohistochemistry or the use of markers such as highly sensitive green fluorescent protein.

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

[0195] In some embodiments, rAAV virus particles containing an rAAV capsid having one or more amino acid substitutions that interact with HSPG are administered simultaneously or sequentially at more than one site. In some embodiments, multiple injections of rAAV virus particles are not spaced more than 1, 2, 3, 4, 5, 6, 9, 12, or 24 hours apart.

[0196] Method of intravitreal injection The general method of intravitreal injection can be described by the following general outline. This example is intended solely to illustrate certain characteristics of the method and is by no means intended to limit it. The technique of intravitreal injection is publicly known in the art (e.g., Peyman, GA et al. (2009) Retina 29(7):875~912). See also pages [page number], and Fagan, XJ and Al-Qureshi, S. (2013) Clin. Experiment. Ophthalmol. 41(5): pp. 500-57.

[0197] In short, preparation for intravitreal injection can be carried out by pupil dilation, disinfection of the eye, and administration of an anesthetic. Any suitable mydriatic agent known in the art may be used for pupil dilation. After confirming sufficient pupil dilation, the procedure can be performed. Disinfection can be carried out by applying an ocular disinfectant, such as an iodine-containing solution such as povidone-iodine (BETADINE®). Similar solutions may be used to cleanse the eyelids, eyelashes, and any other surrounding tissues (e.g., skin). Any suitable anesthetic, such as lidocaine or propalacaine, may be used at 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 the injection, remove the eyelashes from the area using a sterilized eyelid retractor. 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 edge in patients with pseudophakia or aphakia, and 3.5–4 mm from the edge in patients with phakia. The patient can look in the opposite direction from the injection site.

[0199] In some embodiments, the method comprises administering an effective amount of recombinant viral particles, comprising a vector encoding a heterologous nucleic acid, to the eye (e.g., by subretinal and / or intravitreal administration), wherein the recombinant viral particles include 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 × 10⁻¹⁶. 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 It is either genome copies / mL. In some embodiments, the viral titer of the composition is about 5 × 10⁶ 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 It is either genome copies / mL. In some embodiments, the viral titer of the composition is about 5 × 10⁶ 12 ~10×10 12 , 10×10 12 ~25×10 12 , or 25×10 12 ~50×10 12 It is either genome copies / mL. In some embodiments, the viral titer of the composition is at least about 5 × 10⁶. 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 9It is either transduction units / mL. In some embodiments, the viral titer of the composition is about 5 × 10⁻¹⁶. 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 It is either transduction units / mL. In some embodiments, the viral titer of the composition is about 5 × 10⁻¹⁶. 9 ~10×10 9 , 10×10 9 ~15×10 9 , 15×10 9 ~25×10 9 , or 25×10 9 ~50×10 9 It is either transduction units / mL. In some embodiments, the viral titer of the composition is at least about 5 × 10⁶ 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 10It is either infectious units / mL or one of the following. In some embodiments, the viral titer of the composition is at least about 5 × 10⁶ 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 It is either infectious units / mL or one of the following. In some embodiments, the viral titer of the composition is at least about 5 × 10⁶ 10 ~10×10 10 , 10×10 10 ~15×10 10 , 15×10 10 ~25×10 10 , or 25×10 10 ~50×10 10 It is either infectious units / mL or one of the following:

[0200] In some embodiments, the method includes administering an effective amount of recombinant viral particles containing one or more amino acid substitutions at one or more positions that interact with heparan sulfate proteoglycans to the eye of an individual (e.g., a human) (e.g., by subretinal and / or intravitreal administration). In some embodiments, the dose of viral particles administered to the individual is at least about 1 × 10¹⁶ kg of body weight. 8~Approx. 1×10 13 It is one of the genome copies. In some embodiments, the dose of viral particles administered to an individual is about 1 × 10⁶ per kg of body weight. 8 ~Approx. 1×10 13 It is one of the genome copies.

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

[0202] Means for determining the structure of the retina and the effectiveness of rAAV delivery It is well known that the retina has multiple layers. The cellular layers of the retina include the internal limiting membrane, nerve fibers, ganglion cells, internal plexiform layer, internal granular layer, external plexiform layer, external granular layer, external limiting membrane, photoreceptors, and retinal pigment epithelium. The layer closest to the vitreous humor is the internal limiting membrane. This layer may contain Müller cells, a type of glial cell. The nerve fiber layer may contain axons from ganglion cells that make up the optic nerve. The ganglion cell layer may contain ganglion cells and amacrine cells. The internal plexiform layer may contain synapses between the dendrites of ganglion and amacrine cells and the axons of bipolar cells. The internal granular layer may contain the cell nuclei of amacrine, bipolar, and horizontal cells. The external plexiform layer may contain synapses between the dendrites of horizontal cells and the processes of photoreceptor cells. The external granular layer may contain photoreceptor cell bodies. The outer or external boundary membrane may include cell junctions between Müller cell apical processes and between these processes and the internal segments of photoreceptor cells, such as adherent junctions and desmosomes. The photoreceptor layer, also known as the rod-cone layer and Jacob's membrane, may contain photoreceptor cells including rods and cones. The retinal layer at the most distal end of the vitreous humor is the retinal pigment epithelium (RPE), which may 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. These cells sense light and can respond by transmitting signals to the optic nerve via bipolar and ganglion cells. Photoreceptor cells may 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 synapses on amacrine or ganglion cells. Ganglion cells The retina can receive information from amacrine cells or horizontal cells, and from axons from the optic nerve. Horizontal cells can integrate input from multiple photoreceptors and regulate light levels. Amacrine cells are interneurons that help regulate bipolar cells and provide input to ganglion cells. Retinal glial cells can include Müller cells, astrocytes, and microglia.

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

[0205] In diseases that result in progressive degenerative vision, treating the subject at a young age can not only slow or halt the progression of the disease, but also improve or prevent the decline in visual function resulting from acquired amblyopia. Amblyopia may be of two types. In studies of non-human primates and kittens that are kept in complete darkness from birth, even up to 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 the "education" of neural connections and the cortex is halted from birth due to the cessation of stimulation. It is unclear whether this function can be restored. In the case of retinal degenerative diseases, the normal visual cortical network was developmentally appropriate until it was first "learned," or until the degeneration caused significant functional impairment. From the standpoint of signaling in the impaired eye, reduced visual stimulation results in "acquired" or "learned" functional impairment ("acquired amblyopia"), in which the brain is unable to interpret the signals or "use" the eye. In these cases of "acquired amblyopia," it is unclear whether there is improved signaling from the retina as a result of gene therapy for the amblyopic eye, which could have previously resulted in slower progression or stabilization of the condition, as well as the acquisition of more normal function. In some embodiments, the treated person is under 30 years of age. In some embodiments, the treated person is under 20 years of age. In some embodiments, the treated person is under 18 years of age. In some embodiments, the treated person is under 15 years of age. In some embodiments, the treated person is under 14 years of age. In some embodiments, the treated person is under 13 years of age. In some embodiments, the treated person is under 12 years of age. In some embodiments, the treated person is under 10 years of age. In some embodiments, the treated person is under 13 years of age. The subject is under 8 years of age. In some embodiments, the person being treated is under 6 years of age.

[0206] In some eye disorders, there is a "nurse cell" phenomenon where improving the function of one type of cell improves the function of another type of cell. For example, transduction of the central retina to the RPE with rAAV according to the present invention can improve rod function, and furthermore, improved rod function leads to improved cone function. Thus, treatment of one type of cell can lead to improved function of another type of cell.

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

[0208] In some embodiments, the person being treated has a hereditary eye disorder but does not yet show any clinical signs or symptoms. In some embodiments, the person being treated has an eye disorder. In some embodiments, the person 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 system and method of the present invention include: autosomal recessive severe early-onset retinal degeneration (Leber congenital amaurosis), congenital color blindness, Stargardt disease, Best's disease, Doin'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 hereditary optic nerve atrophy.

[0210] The composition of the present invention (for example, AAV virus particles for subretinal or intravitreous 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) may be used alone or in combination with one or more further therapeutic agents for treating ocular disorders. The interval between sequential doses may 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 further therapeutic agents may be administered subretinally or intravitreously (e.g., by intravitreal administration). Non-limiting examples of further 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 morpholino, e.g., anti-angiogenic morpholino 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 recombinant viral particles (e.g., particles such as AAV2, AAVrh8R) containing one or more amino acid substitutions at one or more positions that interact with the heparan sulfate proteoglycan of the present disclosure is shaped to neurons (e.g., striatal neurons, e.g., spinous neurons) at or near the administration site. Transduction occurs. In some embodiments, more than approximately 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or 100% of neurons are transduced. In some embodiments, approximately 5% to approximately 100%, 10% to approximately 50%, approximately 10% to approximately 30%, approximately 25% to approximately 75%, approximately 25% to approximately 50%, or approximately 30% to approximately 50% of 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, Insights hybridization, etc.), or the use of co-expression markers such as highly sensitive green fluorescent protein.

[0213] In some embodiments of the present invention, the method involves administering an effective amount of recombinant viral particles containing one or more amino acid substitutions at one or more sites interacting with heparan sulfate proteoglycans of the present disclosure into the brain of a mammal, for example, a human. In some embodiments, the composition is injected into one or more sites in the brain to express heterologous nucleic acids of the present disclosure in at least the neuron. In some embodiments, the composition is injected into one of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more sites 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 the putamen and the caudate nucleus. In some embodiments, recombinant viral particles containing one or more amino acid substitutions at one or more sites interacting with heparan sulfate proteoglycans are administered to the CNS of an individual, for example, the striatum, by stereotactic injection. 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 the CNS of an individual by convection-enhanced delivery (CED); for example, CED to the striatum.

[0214] rAAV particles can be administered via various routes. In some embodiments, administration includes direct spinal injection and / or intracerebral administration. In some embodiments, administration is to selected sites 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, ventricular system of the cerebrum, subarachnoid space, striatum, cortex, septum, thalamus, hypothalamus, and brain parenchyma. In some embodiments, administration includes intraventricular injection into at least one lateral ventricle of the cerebrum. In some embodiments, administration includes intraarachnoid injection in the cervical, thoracic, and / or lumbar region. In some embodiments, administration includes intrastriatal injection. In some embodiments, administration includes intrathalamic injection. Various techniques and devices suitable for these administration routes, e.g., CED and / or stereotactic 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. In some embodiments, recombinant viral particles are administered to both hemispheres of the brain.

[0216] In some embodiments, recombinant virus particles containing one or more amino acid substitutions at one or more sites that interact with heparan sulfate proteoglycans are administered simultaneously or sequentially at more than one site. In some embodiments, multiple injections of recombinant virus particles are administered at 1, 2, 3, 4, and 5 hours apart. Do not leave more than 6, 9, 12, or 24 hours between doses.

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

[0218] Generally, about 1 μL to about 1 mL (for example, about 100 μL to about 500 μL of the composition of the present invention) can be delivered. In some embodiments of the present invention, the volume of recombinant virus particles injected into the striatum, which include one or more amino acid substitutions at one or more positions that interact with heparan sulfate proteoglycans, is 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 in between.

[0219] In some embodiments, a first volume of recombinant viral particles containing one or more amino acid substitutions at one or more positions interacting 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 volume 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 one or more of approximately 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 in between.

[0220] The compositions of the present invention (for example, recombinant viral particles comprising one or more amino acid substitutions at one or more positions that interact with the heparan sulfate proteoglycan of this disclosure) may be used alone or in combination with one or more further therapeutic agents for treating CNS disorders (e.g., hemoglobin hemorrhage). The interval between sequential doses may be at least a few minutes, hours, or days (or alternatively, less than a few minutes, hours, or days).

[0221] In some embodiments, the method involves administering an effective amount of recombinant viral particles, comprising a vector encoding a heterologous nucleic acid, to the CNS, wherein the recombinant viral particles include 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 × 10⁻¹⁶. 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 It is either genome copies / mL. In some embodiments, the viral titer of the composition is about 5 × 10⁶ 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 It is either genome copies / mL or one of several implementations. In this state, the viral titer of the composition is approximately 5 × 10⁻⁶. 12 ~10×10 12 , 10×10 12 ~25×10 12 , or 25×10 12 ~50×10 12 It is either genome copies / mL. In some embodiments, the viral titer of the composition is at least about 5 × 10⁶. 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 It is either transduction units / mL. In some embodiments, the viral titer of the composition is about 5 × 10⁻¹⁶. 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 It is either transduction units / mL. In some embodiments, the viral titer of the composition is about 5 × 10⁻¹⁶. 9 ~10×10 9 , 10×10 9 ~15×10 9 , 15×10 9 ~25×10 9 , or 25×10 9 ~50×10 9 It is either transduction units / mL. In some embodiments, the viral titer of the composition is at least about 5 × 10⁶ 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 It is either infectious units / mL or one of the following. In some embodiments, the viral titer of the composition is at least about 5 × 10⁶ 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 It is either infectious units / mL or one of the following. In some embodiments, the viral titer of the composition is at least about 5 × 10⁶ 10 ~10×10 10 , 10×10 10 ~15×10 10 , 15×10 10 ~25×10 10 , or 25×10 10 ~50×10 10 It is either infectious units / mL or one of the following:

[0222] In some embodiments, the method involves administering an effective amount of recombinant viral particles containing one or more amino acid substitutions at one or more positions that interact with heparan sulfate proteoglycans to the CNS of an individual (e.g., a human). In some embodiments, the dose of viral particles administered to the individual is at least about 1 × 10¹⁶ per kg of body weight. 8 ~Approx. 1×10 13 It is one of the genome copies. In some embodiments, the dose of viral particles administered to an individual is about 1 × 10⁶ per kg of body weight. 8 ~Approx. 1×10 13 It is one of the genome copies.

[0223] In some embodiments, the method involves administering an effective amount of recombinant viral particles containing one or more amino acid substitutions at one or more positions that interact with heparan sulfate proteoglycans in the individual to the CNS of an individual (e.g., a human). In some embodiments, the total amount of viral particles administered to the individual is at least about 1 × 10⁻⁶ 9 ~Approx. 1×10 14 It is one of the genome copies. In some embodiments, the total amount of viral particles administered to an individual is about 1 × 10⁻⁶. 9 ~Approx. 1×10 14 It is one of the genome copies.

[0224] V. Expression constructs In some embodiments, the present invention provides a method for delivering heterologous nucleic acids to the eye by subretinal delivery of an rAAV vector containing heterologous nucleic acids, wherein the rAAV vector is encapsulated in an rAAV capsid (e.g., rAAV2 capsid, rAAVrh8R capsid, etc.) containing one or more amino acid substitutions that interact with HSPG. In some embodiments, the present invention provides a method for treating a CNS disorder in an individual, wherein the individual The method provides for the delivery of a composition comprising rAAV particles to the CNS of the body, wherein the rAAV particles comprise an rAAV capsid comprising an rAAV capsid protein comprising one or more amino acid substitutions at one or more positions interacting with a heparan sulfate proteoglycan, and (b) an rAAV vector comprising a heterogene and at least one AAV terminal repeat sequence. In some embodiments, the heterogene (e.g., a transgene) is operably ligated to a promoter. Exemplary promoters include the cytomegalovirus (CMV) earliest promoter, RSV LTR, MoMLV LTR, phosphoglycerate kinase-1 (PGK) promoter, monkey 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): pp. 193-199) and elongation factor 1-α promoter (EF1-α) promoter (Kim et al., Gene, 1990, 91(2): pp. 217-23 and Guo et al., Gene Examples include, but are not limited to, Ther., 1996, 3(9):802-810. In some embodiments, the promoter comprises a human β-glucuronidase promoter or a cytomegalovirus enhancer linked to a chicken β-actin (CBA) promoter. The promoter can be constitutive, inductive, or repressive. In some embodiments, the promoter has the ability to express heterologous nucleic acids in eye cells. In some embodiments, the promoter has the ability to express heterologous nucleic acids in photoreceptor cells or RPEs. In some embodiments, the promoter is a rhodopsin kinase (RK) promoter, for example, a human RK promoter.In some embodiments, the promoter is an opsin promoter, such as a human opsin promoter or a mouse opsin promoter.

[0225] The present invention envisions the use of recombinant viral genomes for introducing one or more nucleic acid sequences encoding therapeutic polypeptides and / or nucleic acids for packaging into rAAV viral particles containing one or more amino acid substitutions that interact with HSPGs. Recombinant viral genomes may include any elements for establishing the expression of therapeutic polypeptides and / or nucleic acids, such as promoters, ITRs, ribosome-binding elements, terminators, enhancers, selection markers, introns, poly(A) signals, and / or origins of replication.

[0226] In some embodiments, the rAAV vector includes a self-complementary rAAV vector, for example, a recombinant self-complementary genome (which may be used herein as synonymous with the term "self-complementary"). AAV virus particles having a self-complementary genome, and methods of using the self-complementary AAV genome are described in U.S. Patents 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 a self-complementary genome rapidly form a double-stranded DNA molecule through their partially complementary sequences (e.g., complementary coding and non-coding strands of the transgene). In some embodiments, the vector comprises a first nucleic acid sequence encoding a heterogeneous 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, a first heterologous nucleic acid sequence and a second heterologous nucleic acid sequence are linked by a mutant ITR (e.g., a right ITR). In some embodiments, the ITR contains the polynucleotide sequence 5'-CACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCACGCCCGGGCTTTGCCCGGGCG-3' (SEQ ID NO: 8). The mutant ITR contains a deletion in the D region, which includes a terminal cleavage sequence. As a result, during replication of the AAV viral genome, the rep protein does not cleave the viral genome at the mutant ITR, and therefore the recombinant viral genome, which includes the following in 5' to 3' order, is packaged into the viral capsid: AAV ITR, a first heterologous polynucleotide sequence containing a regulatory sequence, a mutant AAV ITR, a second polynucleotide sequence reversed relative to the first heterologous nucleotide, and a third AAV ITR.

[0228] VI. Virus particles and methods for producing virus particles rAAV virus particles In some embodiments, the present invention provides a method for delivering heterologous nucleic acids to the eye by subretinal delivery of an rAAV vector containing heterologous nucleic acids, wherein the rAAV vector is encapsulated in an rAAV capsid (e.g., rAAV2, rAAVrh8R, etc.) containing one or more amino acid substitutions that interact with HSPG. In some embodiments, the present invention provides methods and kits relating to the delivery of 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 containing a xenotransgene with one or two AAV inverted terminal repeat sequences (ITRs) adjacent to each other. The nucleic acid is encapsulated in the capsid of the AAV particle. The AAV particle also comprises a capsid protein. In some embodiments, the nucleic acid comprises a coding sequence of interest (e.g., a xenotransgene), a transcription start sequence and a transcription termination sequence, and a regulatory sequence, the components of which are operably linked in the transcription direction, thereby forming an expression cassette.

[0230] The expression cassette has at least one functional AAV ITR sequence adjacent to its 5' and 3' ends. “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) pp. 3428-32; Passini et al., J. Virol., 2003, 77(12):70 pp. 34-40; and Pechan et al., Gene Ther., 2009, 16:10-16. All of these references are incorporated herein by reference in their entirety. To carry out some aspects of the present invention, the recombinant vector includes at least all of the AAV sequences essential for capsid inclusion and the physical structure for infection by rAAV. The AAV ITR for use in the vector of the present invention does not need to have a wild-type nucleotide sequence (as described, for example, Kotin, Hum. Gene Ther., 1994, 5:793-801), may be modified by nucleotide insertions, deletions, or substitutions, or may be derived from any of several AAV serotypes. More than 40 AAV serotypes are now publicly known, and new serotypes and variants of existing serotypes continue to be identified. See Gao et al., PNAS, 2002, 99(18):11854-11866; Gao et al., PNAS, 2003, 100(10):6081-11866; and Bossis et al., J. Virol., 2003, 77(12):6799-110.

[0231] The use of any AAV serotype is conceivable within the scope of the present invention. In some embodiments, the rAAV vector is AAV1, AAV2, AAV3, AAV4, AAV5, AAV 6. A vector derived from AAV serotypes, including, but not limited to, AAV ITRs such as AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrh10, AAV11, AAV12, AAV2R471A, AAV DJ, goat AAV, bovine AAV, or mouse AAV ITR. In some embodiments, the nucleic acids in the AAV include IRs of AAV ITRs such as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrh10, AAV11, AAV12, AAV2R471A, AAV DJ, goat AAV, bovine AAV, or mouse AAV. In certain embodiments, the nucleic acids in the AAV include AAV2 ITR. As described above, rAAV particles may further contain a capsid containing an rAAV capsid protein that includes one or more amino acid substitutions at one or more positions that interact with the heparan sulfate proteoglycan.

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

[0233] Different AAV serotypes are used to optimize transduction to specific target cells or to target specific cell types within specific target tissues (e.g., CNS tissue). rAAV particles may contain viral proteins and viral nucleic acids of the same or mixed serotypes. For example, in some embodiments, rAAV particles may contain the AAV2 capsid protein of the present invention and at least one AAV2 ITR, or may contain the AAV2 capsid protein and at least one AAV1 ITR. Any combination of AAV serotypes for the production of rAAV particles is provided herein, as each combination is clearly stated herein. In some embodiments, the present invention provides rAAV particles containing the AAV2 capsid of the present invention. In some embodiments, the present invention provides rAAV particles containing the AAVrh8R capsid of the present invention.

[0234] Production of AAV particles A great many methods for producing rAAV vectors are known in the art, including transfection, stable cell line production, and infectious hybrid virus production systems, including adenovirus-AAV hybrids, herpesvirus-AAV hybrids (Conway, JE et al. (1997) J. Virology 71(11):8780-8789) and baculovirus-AAV hybrids. All rAAV production cultures for the production of rAAV virus particles require: 1) stable host cells, including, for example, human cell lines such as HeLa, A549, or 293 cells, or, in the case of baculovirus production systems, insect cell lines such as SF-9; 2) suitable helper virus functions provided by wild-type or mutant adenoviruses (e.g., temperature-sensitive adenoviruses), herpesviruses, baculoviruses, or plasmid constructs that provide helper functions; 3) AAV rep and cap genes and gene products; 4) transgenes (e.g., therapeutic transgenes) adjacent to 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, denatured Eagle medium (MEM), Dulbecco's denatured Aigle medium (DMEM), custom formulations, such as those described in U.S. Patent No. 6,566,118, and SF-900 II SFM medium as described in U.S. Patent No. 6,723,551, and media produced by Hyclone Laboratories and JRH, each of the aforementioned patents being described in whole, in particular Custom culture medium formulations for use in the production of recombinant AAV vectors are incorporated herein by reference.

[0235] rAAV particles can be produced using methods known in the art. See, for example, U.S. Patents 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. The host cell may be a packaging cell in which the AAV rep and cap genes are stably maintained within the host cell, or a producer cell in which the AAV vector genome is stably maintained. Exemplary packaging and producer cells are derived from 293, A549, or heLa cells. The AAV vector is purified and formulated using standard techniques known in the art.

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

[0237] In some embodiments, rAAV particles may also be produced by producer cell line methods, such as the exemplary producer cell line method provided below (see also Martin et al. (2013) Human Gene Therapy Methods 24: pp. 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 an adenovirus (e.g., wild-type adenovirus) as a helper to initiate rAAV production. The virus can then be recovered, the adenovirus can be inactivated (e.g., by heat), and / or removed, and the rAAV particles can be purified.

[0238] In some embodiments, a method is provided for producing any rAAV particles disclosed herein, the method comprising: (a) culturing a host cell under conditions under which rAAV particles are produced, wherein the host cell comprises (i) one or more AAV packaging genes [wherein each of the AAV packaging genes encodes an AAV replication and / or capsid inclusion protein]; (ii) an rAAV provector comprising a nucleic acid encoding a therapeutic polypeptide and / or nucleic acid as described herein, adjacent to at least one AAV ITR; and (iii) an AAV helper function; and (b) recovering the rAAV particles produced by the host cell. 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, goat AAV, bovine AAV, or mouse AAV. In some embodiments, the capsid-encapsulating protein contains one or more amino acid substitutions at one or more positions that interact with the heparan sulfate proteoglycan. In some embodiments, the capsid-encapsulating protein is the AAV2 capsid-encapsulating protein. In some embodiments, the capsid-encapsulating protein is the AAVrh8R capsid-encapsulating protein.

[0239] The preferred rAAV production culture medium of the present invention may be supplemented with serum or serum-derived recombinant protein at a level of 0.5% to 20% (v / v or w / v). Alternatively, as is well known in the art, the rAAV vector may be produced under serum-free conditions, sometimes referred to as a medium free of animal-derived products. It will be understood by those skilled in the art that the titer of rAAV in the production culture can also be increased by supplementing a commercial or custom medium designed to support the production of rAAV vectors with one or more cell culture components known in the art, including but not limited to glucose, vitamins, amino acids, and / or growth factors.

[0240] rAAV production cultures can be grown under a variety of conditions suitable for the specific host cells used (e.g., over a wide temperature range, over various time periods). As is well known in the art, rAAV production cultures include adhesion-dependent cultures that can be cultured in suitable adhesion-dependent containers such as roller bottles, hollow fiber filters, microcarriers, and packed or fluidized bed bioreactors. rAAV vector production cultures may 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., WaveBag systems).

[0241] The rAAV vector particles of the present invention may be recovered from an rAAV production culture by lysing the host cells of the production culture, or by recovering the used culture medium from the production culture, provided that the cells are cultured under conditions known in the art for releasing rAAV particles into the culture medium from intact cells, as better 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, micro-solution, and treatment with chemicals, such as surfactants / proteases.

[0242] In further embodiments, rAAV particles are purified. The term “purified,” as used herein, includes a product of rAAV particles that is free from at least some of the other components that may be present in the location from which the rAAV particles were originally prepared or that are naturally present in the location from which the rAAV particles were originally prepared. Thus, isolated rAAV particles may be produced by purification techniques that concentrate rAAV particles from a source mixture, e.g., a culture lysate or production culture supernatant. Concentration can be measured in various ways, e.g., by the ratio of DNase-resistant particles (DRPs) or genome copies (gc) present in the solution, or by infectivity, or with respect to a second, potentially interfering substance present in the source mixture, e.g., impurities [including production culture impurities or process impurities (including helper viruses, culture medium components, etc.)].

[0243] In some embodiments, the rAAV production culture recovery is clarified to remove host cell fragments. In some embodiments, the production culture recovery is clarified by filtration through a series of depth filters, including, for example, Grade DOHC Millipore Millistak+ HC Pod filters, Grade A1HC Millipore Millistak+ HC Pod filters, and 0.2 μm filter Opticap XL10 Millipore Express SHC hydrophilic membrane filters. Clarification can also be achieved by various 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 recovery is further treated with Benzonase® to digest any high molecular weight DNA present in the production culture. In some embodiments, Benzonase® digestion is performed, for example, to a final concentration of 1-2 This is carried out under standard conditions known in the art, including a period of 30 minutes to several hours, with 0.5 units / mL of Benzonase® at a temperature in the range of ambient temperature ~37°C.

[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) for concentration of rAAV particles; rAAV capture by apatite chromatography; thermal inactivation of helper viruses; 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 individually, in various combinations, or in different orders. In some embodiments, the method includes all 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. Patents 6,989,264 and 8,137,948; and WO2010 / 148143.

[0246] Also provided herein are rAAV particles comprising heterologous nucleic acids encoding therapeutic polypeptides and / or therapeutic nucleic acids, wherein the rAAV particles comprise an rAAV capsid comprising one or more amino acid substitutions that interact with HSPG, and a pharmaceutical composition comprising a pharmaceutically acceptable carrier. The pharmaceutical composition would be suitable for any administration method described herein; for example, by subretinal administration.

[0247] In some embodiments, pharmaceutical compositions comprising rAAV as described herein and a pharmaceutically acceptable carrier are suitable for administration to humans. Such carriers are well known in the art (see, for example, Remington's Pharmaceutical Sciences, 15th edition, pp. 1035-1038 and 1570-1580). In some embodiments, pharmaceutical compositions comprising rAAV as described herein and a pharmaceutically acceptable carrier are suitable for ocular injection. Such pharmaceutically acceptable carriers may be sterile solutions, such as water and oil (of petroleum, animal, plant, or synthetic origin, including peanut oil, soybean oil, mineral oil, etc.). Salt solutions and aqueous solutions of dextrose, polyethylene glycol (PEG), and glycerol can also be used as liquid carriers, particularly for injectable solutions. Pharmaceutical compositions may further contain, for example, preservatives, buffers, isotonic agents, antioxidants and stabilizers, nonionic wetting or clarifying agents, thickeners, and the like. The pharmaceutical compositions described herein may be packaged in single-unit dosage forms or in multiple-dose forms. The compositions are generally formulated as sterile and substantially isotonic solutions.

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

[0249] subretinal delivery In some embodiments, the present invention 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 interacting with a heparan sulfate proteoglycan, and ii) the vector comprises a heterogeneous 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 comprises a small-diameter cannula of 27-45 gauge, one or more syringes (e.g., 1, 2, 3, 4 or more), and one or more fluids (e.g., 1, 2, 3, 4 or more) suitable for use in the method of the present invention.

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

[0252] The syringe may be any suitable syringe, provided that it is capable of connecting 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 can be operated, for example, by a foot pedal.

[0254] Suitable fluids for use in the method of the present invention include those described herein, for example, 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 solution or other suitable fluid), and one or more fluids containing one or more therapeutic agents.

[0255] Suitable fluids for use in the method of the present invention include those described herein, for example, 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 solution or other suitable fluid), and one or more fluids containing one or more therapeutic agents.

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

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

[0258] In some embodiments, the system includes a single fluid (e.g., a fluid containing an effective amount of the 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 system of the present 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 for use include instructions relating to one of the methods described herein. In some embodiments, the instructions for use include instructions for subretinal delivery of rAAV particles comprising a capsid having one or more amino acid substitutions that modify, reduce or remove the binding of rAAV particles to HSPG.

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

[0261] The kit may comprise any of the rAAV particles or rAAV particle compositions of the present invention. For example, the kit may comprise rAAV particles comprising an rAAV capsid protein containing an rAAV capsid protein with 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 the binding of rAAV particles 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 comprising heterogeneous nucleic acids and at least one AAV inverted terminal repeat sequence.

[0262] In some embodiments, the kit further includes a device for CNS delivery of a composition of rAAV particles. Devices for CNS delivery (e.g., for delivery of a composition containing rAAV particles) are known in the art and include pumps (e.g., osmotic and / or infusion pumps, as described below) and injection devi...

Claims

1. A composition comprising recombinant adeno-associated virus (rAAV) particles for use in delivering heterologous nucleic acids to the central nervous system (CNS) of an individual, Here, the rAAV particles are formulated to administer recombinant adeno-associated virus (rAAV) particles to the CNS of the individual. The rAAV particle is, a) AAVrh8R capsid protein with amino acid substitutions, wherein the amino acid substitution is numbered R533A based on the VP1 numbering of AAVrh8R, and b) an rAAV vector genome comprising the heterogeneous nucleic acid and at least one AAV inverted terminal repeat sequence. including, The aforementioned composition.

2. A composition comprising recombinant adeno-associated virus (rAAV) particles for use in improving rAAV transduction into cells of an individual's central nervous system (CNS) compared to transduction into cells with rAAV containing a wild-type capsid, Here, the rAAV particles are formulated to administer recombinant adeno-associated virus (rAAV) particles to the CNS of the individual. The rAAV particle is, a) an AAVrh8R capsid protein with amino acid substitutions, wherein the amino acid substitution is numbered R533A based on the VP1 numbering of AAVrh8R, and b) An rAAV vector genome comprising heterologous nucleic acids and at least one AAV inverted terminal repeat sequence. including, The aforementioned composition.

3. A composition comprising recombinant adeno-associated virus (rAAV) particles for use in improving the expression of heterologous nucleic acids in the central nervous system (CNS) of an individual, Here, the rAAV particles are formulated to administer recombinant adeno-associated virus (rAAV) particles to the CNS of the individual. The rAAV particle is, a) an AAVrh8R capsid protein with amino acid substitutions, wherein the amino acid substitution is numbered R533A based on the VP1 numbering of AAVrh8R, and b) An rAAV vector genome comprising heterologous nucleic acids and at least one AAV inverted terminal repeat sequence. including, The aforementioned composition.

4. A composition comprising recombinant adeno-associated virus (rAAV) particles for use in treating central nervous system (CNS) disorders in an individual, Here, the rAAV particles are formulated to administer an effective amount of the composition containing the rAAV particles to the CNS of the individual. The rAAV particle is, a) an AAVrh8R capsid protein with amino acid substitutions, wherein the amino acid substitution is numbered R533A based on the VP1 numbering of AAVrh8R, and b) An rAAV vector genome comprising heterologous nucleic acids and at least one AAV inverted terminal repeat sequence. including, The aforementioned composition.

5. The composition according to claim 1, wherein administration includes direct spinal injection, intracranial and / or intracerebral administration.

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

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

8. The composition according to claim 1, wherein the heterologous nucleic acid is expressed in neurons.

9. Heterogeneous nucleic acids include enzymes, neurotrophic factors, deficient or mutant polypeptides in individuals with CNS-related disorders, anti-apoptotic factors, anti-angiogenic factors, and anti-inflammatory factors, α-synuclein, acid β-glucosidase (GBA), β-galactosidase-1 (GLB1), iduronate 2-sulfatase (IDS), galactosylceramidase (GALC), mannosidase, α-D-mannosidase (MAN2B1), β-mannosidase (MANBA), pseudoarylsulfatase A (ARSA), N-acetylglucosamine-1-phosphotransferase (GNPTAB), and 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), heparinacetyl-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 lipoprotein The composition according to claim 1, encoding a polypeptide selected from the group consisting of ze (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-idulonidase, arylsulfatase B, arylsulfatase A, N-acetylgalactosamine-6-sulfatase, acid β-galactosidase, or α-neuramidase.

10. The composition according to claim 1, wherein the heterogeneous nucleic acid encodes a polypeptide selected from the group consisting of neuronal apoptosis inhibitory protein (NAIP), nerve growth factor (NGF), glial cell-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 polypeptide, anti-inflammatory polypeptide, and aspartoacylase (ASPA).

11. The composition according to claim 1, wherein the heterogeneous nucleic acid encodes a therapeutic nucleic acid.

12. The composition according to claim 11, wherein the therapeutic nucleic acid is siRNA, shRNA, RNAi, miRNA, antisense RNA, ribozyme, or DNAzyme.

13. The heterologous nucleic acids include the cytomegalovirus (CMV) initial promoter, RSV LTR, and MoMLV. The composition according to claim 1, which is under the control of a promoter selected from the group consisting of LTR, phosphoglycerate kinase-1 (PGK) promoter, monkey 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, retrovirus Roussarcoma virus (RSV) LTR promoter, dihydrofolate reductase promoter, and β-actin promoter.

14. The composition according to claim 1, wherein the rAAV vector is a self-complementary rAAV vector.

15. The composition according to claim 1, wherein the individual is a human.

16. The composition according to claim 1, wherein the heterogeneous nucleic acid encodes a therapeutic polypeptide or therapeutic nucleic acid used to treat a CNS disorder.

17. The composition according to claim 16, wherein the CNS disorder is lysosomal storage disorder (LSD), Huntington's disease, epilepsy, Parkinson's disease, Alzheimer's disease, stroke, corticobasal degeneration (CBD), corticobasal ganglia degeneration (CBGD), frontotemporal dementia (FTD), multiple system atrophy (MSA), progressive supranuclear palsy (PSP), or brain cancer.

18. A composition comprising recombinant adeno-associated virus (rAAV) particles for use in treating Huntington's disease in individuals, Here, the rAAV particles are formulated to administer an effective amount of a composition containing recombinant adeno-associated virus (rAAV) particles to the striatum of the individual. The rAAV particle is, a) an AAVrh8R capsid protein with amino acid substitutions, wherein the amino acid substitution is numbered R533A based on the VP1 numbering of AAVrh8R, and b) An rAAV vector genome comprising heterologous nucleic acids and at least one AAV inverted terminal repeat sequence. including, The aforementioned composition.

19. The composition according to claim 18, wherein the heterogeneous nucleic acid encodes a therapeutic polypeptide or a therapeutic nucleic acid.

20. The composition according to claim 19, wherein the therapeutic nucleic acid is miRNA.

21. A composition comprising recombinant adeno-associated virus (rAAV) particles for use in treating Parkinson's disease in individuals, Here, the rAAV particles are formulated to administer an effective amount of a composition containing recombinant adeno-associated virus (rAAV) particles to the striatum of the individual. The rAAV particle is, a) an AAVrh8R capsid protein with amino acid substitutions, wherein the amino acid substitution is numbered R533A based on the VP1 numbering of AAVrh8R, and b) An rAAV vector genome comprising heterologous nucleic acids and at least one AAV inverted terminal repeat sequence. including, The aforementioned composition.

22. The composition according to claim 21, wherein the heterogeneous nucleic acid encodes a therapeutic polypeptide or a therapeutic nucleic acid.

23. The composition according to claim 22, wherein the therapeutic polypeptide is TH, GTPCII, GDNF, BDNF and / or AADC; or a fragment thereof.

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