Variant AAV capsids for intravitreal delivery

Abstract

Provided herein are variant adeno-associated virus (AAV) capsid proteins, and recombinant AAV virions having one or more variant AAV capsid proteins. Also provided are compositions and methods for using the recombinant AAV virions, such as for treating or preventing diseases or disorders. Also provided herein are methods for providing a therapeutic gene product to the retina of a subject, comprising administering to the subject a recombinant AAV virion disclosed herein or a pharmaceutical composition disclosed herein by intravitreal injection.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 62 / 839,548, filed April 26, 2019, and U.S. Provisional Patent Application No. 62 / 923,924, filed October 21, 2019, the contents of each of which are incorporated by reference in their entirety herein.

[0002] Submission of sequence listing as an ASCII text file The contents of the following submissions, as ASCII text files, are incorporated herein by reference in their entirety: Sequence Listing in Computer Readable Form (CRF) (Filename: 627002001140SEQLIST.TXT, Recorded: April 21, 2020, Size: 49KB). [Background technology]

[0003] A promising approach to treat and prevent genetic and other diseases and disorders is the delivery of therapeutic gene products using gene therapy vectors such as virions.Examples of suitable virions for gene therapy include, but are not limited to, retrovirus virions, lentivirus virions, adenovirus virions, herpesvirus virions, alphavirus virions and adeno-associated virus (AAV) virions. AAV is a 4.7kb single-stranded DNA virus. Because wild-type AAV is non-pathogenic and has no etiological association with any known disease, recombinant virions (rAAV virions) based on AAV are associated with excellent clinical safety.In addition, AAV offers the ability for highly efficient gene delivery and sustained transgene expression in multiple tissues, including eye, muscle, lung and brain. Summary of the Invention [Means for solving the problem]

[0004] Certain remaining challenges in the design of virions for use in gene therapy include optimizing viral cell tropism, especially for ocular gene therapy, optimizing delivery to the retina.Therefore, there is a need for optimized virions for expressing genes in selected mammalian cells.The present invention addresses this need by providing modified AAV capsid proteins that are advantageous for the delivery of recombinant AAV virions to desired cells and tissues.

[0005] Provided herein is a recombinant AAV virion comprising: (a) a variant adeno-associated virus (AAV) capsid protein comprising a modified sequence that includes one or more amino acid substitutions within amino acid residues 570-579 relative to a parent AAV capsid protein, wherein the modified sequence comprises HKFKSGD (SEQ ID NO:1), where the numbering of the amino acid residues corresponds to that of the AAV5 VP1 capsid protein; and (b) a polynucleotide sequence encoding a therapeutic gene product.

[0006] In some embodiments, the parent AAV capsid protein is AAV5 capsid protein, or AAV5 and AAV2 hybrid capsid protein.In some embodiments, the parent AAV capsid protein is AAV2.5T capsid protein.In some embodiments, the parent AAV capsid protein is AAV2.5T VP1 capsid protein.

[0007] In some embodiments, the modified sequence comprises LAHKFKSGDA (SEQ ID NO: 3). In some embodiments, the variant AAV capsid protein comprises a capsid sequence having at least 85% homology to the amino acid sequence set forth in SEQ ID NO: 4 or SEQ ID NO: 5. In some embodiments, the variant AAV capsid protein comprises a capsid sequence set forth in SEQ ID NO: 6 or SEQ ID NO: 7.

[0008] In some embodiments, the rAAV virion is a variant AAV5, or a variant AAV2 and AAV5 hybrid virion. In some embodiments, the rAAV virion is a variant AAV2.5T virion.

[0009] In some embodiments, recombinant AAV virions can transduce cells of the retina when injected intravitreally into a mammal. In some embodiments, recombinant AAV virions can transduce one or more of photoreceptor cells, retinal ganglion cells, Muller cells, bipolar cells, amacrine cells, horizontal cells, and retinal pigment epithelial cells when injected intravitreally into a mammal. In some embodiments, recombinant AAV virions can transduce retinal pigment epithelial cells when injected intravitreally into a mammal.

[0010] In some embodiments, the therapeutic gene product is an siRNA, miRNA, or a protein. In some embodiments, the therapeutic gene product is an anti-vascular endothelial growth factor (anti-VEGF) gene product. In some embodiments, the therapeutic gene product is an opsin.

[0011] In some embodiments, the polynucleotide encoding therapeutic gene product is adjacent to one or more AAV ITR.In some embodiments, one or more AAV ITR is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, avian AAV, bovine AAV, canine AAV, equine AAV, primate AAV, non-primate AAV or ovine AAV ITR, or variant thereof.In some embodiments, one or more AAV ITR is AAV2 ITR or AAV5 ITR.

[0012] In some embodiments, the recombinant AAV virion has an altered cell tropism compared to AAV2.5T.

[0013] In some embodiments, the recombinant AAV virions disclosed herein are for use in a method of treating a retinal disease or disorder in a subject in need thereof, the method comprising administering to the subject a pharmaceutical composition comprising the recombinant AAV virion by intravitreal injection. In some embodiments, the recombinant AAV virions disclosed herein are for use in the preparation of a medicament for treating a retinal disease or disorder in a subject. In some embodiments, the disease or disorder is age-related macular degeneration (AMD), wet AMD, dry AMD, retinal neovascularization, choroidal neovascularization, diabetic retinopathy, proliferative diabetic retinopathy, retinal vein occlusion, central retinal vein occlusion, branch retinal vein occlusion, diabetic macular edema, diabetic retinal ischemia, ischemic retinopathy, or diabetic retinal edema.

[0014] Also provided herein are pharmaceutical compositions comprising the recombinant AAV virions disclosed herein.

[0015] Also provided herein is a method for producing rAAV virions, the method comprising: (a) culturing a host cell under conditions such that rAAV virions are produced, wherein the host cell comprises: (i) a polynucleotide encoding a variant AAV capsid protein comprising a modified sequence that includes one or more amino acid substitutions within amino acid residues 570-579 relative to the parent AAV capsid protein, the modified sequence comprising HKFKSGD (SEQ ID NO:1); (ii) a polynucleotide encoding a rep protein; (iii) a polynucleotide cassette comprising a sequence encoding a therapeutic gene product flanked by at least one AAV ITR; and (iv) AAV helper functions; and (b) recovering the rAAV virions produced by the host cell.

[0016] Also provided herein is a method of providing a therapeutic gene product to the retina of a subject, comprising administering to the subject a recombinant AAV virion as disclosed herein, or a pharmaceutical composition as disclosed herein, by intravitreal injection.

[0017] In some embodiments, the subject is diagnosed with acute macular neuroretinopathy, Behcet's disease, choroidal neovascularization, diabetic uveitis, histoplasmosis, macular degeneration, edema, multifocal choroiditis, ocular trauma affecting the posterior ocular site or area, ocular tumor, central retinal vein occlusion, diabetic retinopathy, proliferative vitreous retinopathy (PVR), retinal artery occlusive disease, retinal detachment, uveitic retinal disease, sympathetic ophthalmia, Vogt-Koyanagi-Harada (VKH) syndrome, uveal diffusion or is suspected of having one or more conditions selected from the group consisting of: ocular retinal diffusion, posterior ocular conditions caused or affected by ocular laser procedures, posterior ocular conditions caused or affected by photodynamic therapy, photocoagulation, radiation retinopathy, epiretinal membrane disorder, branch retinal vein occlusion, anterior ischemic optic neuropathy, diabetic retinal ischemia, ischemic retinopathy, non-retinopathy diabetic retinal dysfunction, retinoschisis, retinitis pigmentosa, glaucoma, Usher syndrome, cone-rod dystrophy, Stargardt disease, hereditary macular degeneration, chorioretinal degeneration, Leber congenital amaurosis, congenital stationary night blindness, total choroidal atrophy, Bardet-Biedl syndrome, macular telangiectasia, Leber hereditary optic neuropathy, retinopathy of prematurity, and disorders of color vision.

[0018] Also provided herein is a method of treating a retinal disease or disorder in a subject in need thereof, comprising administering to the subject a recombinant AAV virion as disclosed herein, or a pharmaceutical composition as disclosed herein, by intravitreal injection.

[0019] In some embodiments, the disease or disorder is acute macular neuroretinopathy, Behcet's disease, choroidal neovascularization, diabetic uveitis, histoplasmosis, macular degeneration, edema, multifocal choroiditis, ocular trauma affecting the posterior ocular site or area, ocular tumors, central retinal vein occlusion, diabetic retinopathy, proliferative vitreoretinopathy (PVR), retinal artery occlusive disease, retinal detachment, uveal retinal disease, sympathetic ophthalmia, Vogt-Koyanagi-Harada (VKH) syndrome, uveal diffusion, a posterior ocular condition caused or affected by ocular laser procedures, photodynamic therapy. posterior ocular conditions caused by or affected by, photocoagulation, radiation retinopathy, epiretinal membrane disorder, branch retinal vein occlusion, anterior ischemic optic neuropathy, diabetic retinal ischemia, ischemic retinopathy, non-retinopathy diabetic retinal dysfunction, retinoschisis, retinitis pigmentosa, glaucoma, Usher syndrome, cone-rod dystrophy, Stargardt disease, hereditary macular degeneration, chorioretinal degeneration, Leber congenital amaurosis, congenital stationary night blindness, total choroidal atrophy, Bardet-Biedl syndrome, macular telangiectasia, Leber hereditary optic neuropathy, retinopathy of prematurity, or disorders of color vision.

[0020] Also provided herein is a variant AAV capsid protein comprising a modified sequence that includes one or more amino acid substitutions within amino acid residues 570 to 579 relative to the parent AAV capsid protein, wherein the modified sequence comprises HKFKSGD (SEQ ID NO:1), and the numbering of the amino acid residues corresponds to that of the AAV5 VP1 capsid protein.

[0021] In some embodiments, the parent AAV capsid protein is AAV5 capsid protein, or AAV5 and AAV2 hybrid capsid protein.In some embodiments, the parent AAV capsid protein is AAV2.5T capsid protein.In some embodiments, the parent AAV capsid protein is AAV2.5T VP1 capsid protein.

[0022] In some embodiments, the modified AAV capsid protein comprises LAHKFKSGDA (SEQ ID NO:3) at amino acid residues 570-579 compared to the parent AAV capsid protein. In some embodiments, the variant AAV capsid protein comprises a capsid sequence having at least 85% homology to the amino acid sequence set forth in SEQ ID NO:4 or SEQ ID NO:5. In some embodiments, the variant AAV capsid protein comprises a capsid sequence set forth in SEQ ID NO:6 or SEQ ID NO:7.

[0023] Also provided herein are nucleic acids comprising a nucleic acid sequence encoding a variant AAV capsid protein disclosed herein.

[0024] Also provided herein is an expression vector comprising a nucleic acid disclosed herein, wherein a nucleic acid sequence encoding a variant AAV capsid protein is operably linked to a promoter sequence. In some embodiments, the expression vector further comprises a nucleic acid encoding a rep protein.

[0025] Also provided herein is a cell comprising the expression vector disclosed herein. In some embodiments, the cell further comprises a nucleic acid encoding a therapeutic gene product. In some embodiments, the therapeutic gene product is an siRNA, miRNA or protein. In some embodiments, the therapeutic gene product is an anti-vascular endothelial growth factor (anti-VEGF) gene product. In some embodiments, the therapeutic agent is an opsin.

[0026] In some embodiments, the polynucleotide encoding therapeutic gene product is adjacent to one or more AAV ITR.In some embodiments, one or more AAV ITR is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, avian AAV, bovine AAV, canine AAV, equine AAV, primate AAV, non-primate AAV or ovine AAV ITR, or variant thereof.In some embodiments, one or more AAV ITR is AAV2 ITR or AAV5 ITR.

[0027] All references cited herein, including patent applications and publications, are incorporated by reference in their entirety. The present invention provides, for example, the following items. (Item 1) (a) a variant adeno-associated virus (AAV) capsid protein comprising a modified sequence that includes one or more amino acid substitutions within amino acid residues 570-579 relative to a parent AAV capsid protein, the modified sequence comprising HKFKSGD (SEQ ID NO:1), wherein the numbering of the amino acid residues corresponds to that of the AAV5 VP1 capsid protein; and (b) a polynucleotide sequence encoding a therapeutic gene product. A recombinant AAV virion comprising: (Item 2) The recombinant AAV virion of item 1, wherein the parent AAV capsid protein is an AAV5 capsid protein or an AAV5 and AAV2 hybrid capsid protein. (Item 3) 3. The recombinant AAV virion of item 1 or 2, wherein the parent AAV capsid protein is an AAV2.5T capsid protein. (Item 4) 4. The recombinant AAV virion of any one of claims 1 to 3, wherein the parent AAV capsid protein is an AAV2.5T VP1 capsid protein. (Item 5) The recombinant AAV virion of any one of items 1 to 4, wherein the modified sequence comprises LAHKFKSGDA (sequence number 3). (Item 6) 6. The recombinant AAV virion of any one of items 1 to 5, wherein the variant AAV capsid protein comprises a capsid sequence having at least 85% homology to the amino acid sequence set forth in SEQ ID NO:4 or SEQ ID NO:5. (Item 7) 7. The recombinant AAV virion of any one of claims 1 to 6, wherein the variant AAV capsid protein comprises the capsid sequence set forth in SEQ ID NO:6 or SEQ ID NO:7. (Item 8) 8. The recombinant AAV virion of any one of items 1 to 7, wherein the rAAV virion is a variant AAV5, or a variant AAV2 and AAV5 hybrid virion. (Item 9) 9. The recombinant AAV virion of any one of claims 1 to 8, wherein the rAAV virion is a variant AAV2.5T virion. (Item 10) 10. The recombinant AAV virion of any one of items 1 to 9, which is capable of transducing cells of the retina when injected intravitreally into a mammal. (Item 11) The recombinant AAV virion of item 10, when injected intravitreally into a mammal, is capable of transducing one or more of photoreceptor cells, retinal ganglion cells, Muller cells, bipolar cells, amacrine cells, horizontal cells, and retinal pigment epithelial cells. (Item 12) 12. The recombinant AAV virion according to item 10 or 11, which is capable of transducing retinal pigment epithelial cells when injected intravitreally into a mammal. (Item 13) 13. The recombinant AAV virion of any one of items 1 to 12, wherein the therapeutic gene product is an siRNA, an miRNA or a protein. (Item 14) 14. The recombinant AAV virion of any one of items 1 to 13, wherein the therapeutic gene product is an anti-vascular endothelial growth factor (anti-VEGF) gene product. (Item 15) 14. The recombinant AAV virion of any one of items 1 to 13, wherein the therapeutic gene product is opsin. (Item 16) 16. The recombinant AAV virion of any one of items 1 to 15, wherein the polynucleotide encoding the therapeutic gene product is flanked by one or more AAV ITRs. (Item 17) The recombinant AAV virion of item 16, wherein the ITRs of the one or more AAVs are AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, avian AAV, bovine AAV, canine AAV, equine AAV, primate AAV, non-primate AAV or ovine AAV ITRs, or variants thereof. (Item 18) 18. The recombinant AAV virion of item 16 or 17, wherein the one or more AAV ITRs are AAV2 ITRs or AAV5 ITRs. (Item 19) 19. The recombinant AAV virion of any one of items 1 to 18, having an altered cell tropism compared to AAV2.5T. (Item 20) 20. A pharmaceutical composition comprising a recombinant AAV virion according to any one of items 1 to 19. (Item 21) 1. A method for producing rAAV virions, comprising: (a) culturing a host cell under conditions such that rAAV virions are produced, said host cell comprising: (i) a polynucleotide encoding a variant AAV capsid protein comprising a modified sequence that includes one or more amino acid substitutions within amino acid residues 570 to 579 relative to a parent AAV capsid protein, wherein the modified sequence comprises HKFKSGD (SEQ ID NO: 1); (ii) a polynucleotide encoding a rep protein; (iii) a polynucleotide cassette comprising a sequence encoding a therapeutic gene product flanked by at least one AAV ITR; and (iv) AAV helper function and (b) recovering the rAAV virions produced by the host cells. A method comprising: (Item 22) A method for providing a therapeutic gene product to the retina of a subject, comprising administering to the subject a recombinant AAV virion according to any one of items 1 to 19, or a pharmaceutical composition according to item 20, by intravitreal injection. (Item 23) The subject is diagnosed with acute macular neuroretinopathy, Behcet's disease, choroidal neovascularization, diabetic uveitis, histoplasmosis, macular degeneration, edema, multifocal choroiditis, ocular trauma affecting the posterior ocular region or area, ocular tumor, central retinal vein occlusion, diabetic retinopathy, proliferative vitreoretinopathy (PVR), retinal artery occlusive disease, retinal detachment, uveal retinal disease, sympathetic ophthalmia, Vogt-Koyanagi-Harada (VKH) syndrome, uveal diffusion, a posterior ocular condition caused or affected by ocular laser procedures, a posterior ocular condition caused or affected by photodynamic therapy, photocoagulation, radiation 23. The method of item 22, wherein the patient has been diagnosed with or is suspected of having one or more conditions selected from the group consisting of: retinal fibrosis, epiretinal membrane disorder, branch retinal vein occlusion, anterior ischemic optic neuropathy, diabetic retinal ischemia, ischemic retinopathy, non-retinopathy diabetic retinal dysfunction, retinoschisis, retinitis pigmentosa, glaucoma, Usher syndrome, cone-rod dystrophy, Stargardt disease, hereditary macular degeneration, chorioretinal degeneration, Leber congenital amaurosis, congenital stationary night blindness, total choroidal atrophy, Bardet-Biedl syndrome, macular telangiectasia, Leber hereditary optic neuropathy, retinopathy of prematurity, and disorders of color vision. (Item 24) 21. A method for treating a retinal disease or disorder in a subject in need of such treatment, comprising administering to the subject a recombinant AAV virion described in any one of items 1 to 19, or a pharmaceutical composition described in item 20, by intravitreal injection. (Item 25) The disease or disorder is selected from the group consisting of acute macular neuroretinopathy, Behcet's disease, choroidal neovascularization, diabetic uveitis, histoplasmosis, macular degeneration, edema, multifocal choroiditis, ocular trauma affecting the posterior ocular region or area, ocular tumors, central retinal vein occlusion, diabetic retinopathy, proliferative vitreoretinopathy (PVR), retinal artery occlusive disease, retinal detachment, uveal retinal disease, sympathetic ophthalmia, Vogt-Koyanagi-Harada (VKH) syndrome, uveal diffusion, a posterior ocular condition caused or affected by ocular laser procedures, and induced by photodynamic therapy. 25. The method of item 24, wherein the condition is a posterior ocular condition caused or affected by, photocoagulation, radiation retinopathy, epiretinal membrane disorder, branch retinal vein occlusion, anterior ischemic optic neuropathy, diabetic retinal ischemia, ischemic retinopathy, non-retinopathy diabetic retinal dysfunction, retinoschisis, retinitis pigmentosa, glaucoma, Usher syndrome, cone-rod dystrophy, Stargardt disease, hereditary macular degeneration, chorioretinal degeneration, Leber congenital amaurosis, congenital stationary night blindness, total choroidal atrophy, Bardet-Biedl syndrome, macular telangiectasia, Leber hereditary optic neuropathy, retinopathy of prematurity, or a disorder of color vision. (Item 26) 20. The recombinant AAV virion according to any one of items 1 to 19, for use in a method for treating a retinal disease or disorder in a subject in need of such treatment, the method comprising administering to the subject a pharmaceutical composition comprising the recombinant AAV virion by intravitreal injection. (Item 27) 20. The recombinant AAV virion of any one of items 1 to 19 for use in the preparation of a medicament for the treatment of a retinal disease or disorder in a subject. (Item 28) 28. The recombinant AAV virion of item 26 or 27, wherein the disease or disorder is age-related macular degeneration (AMD), wet AMD, dry AMD, retinal neovascularization, choroidal neovascularization, diabetic retinopathy, proliferative diabetic retinopathy, retinal vein occlusion, central retinal vein occlusion, branch retinal vein occlusion, diabetic macular edema, diabetic retinal ischemia, ischemic retinopathy or diabetic retinal edema. (Item 29) A variant AAV capsid protein comprising a modified sequence comprising one or more amino acid substitutions within amino acid residues 570 to 579 relative to a parent AAV capsid protein, wherein the modified sequence comprises HKFKSGD (SEQ ID NO: 1), and wherein the numbering of the amino acid residues corresponds to that of the AAV5 VP1 capsid protein. (Item 30) 30. The variant AAV capsid protein of item 29, wherein the parent AAV capsid protein is an AAV5 capsid protein or an AAV5 and AAV2 hybrid capsid protein. (Item 31) 31. The variant AAV capsid protein of claim 29 or 30, wherein the parent AAV capsid protein is an AAV2.5T capsid protein. (Item 32) 32. The variant AAV capsid protein of any one of items 29 to 31, wherein the parent AAV capsid protein is an AAV2.5T VP1 capsid protein. (Item 33) 33. The variant AAV capsid protein of any one of items 29 to 32, wherein the modified AAV capsid protein comprises LAHKFKSGDA (SEQ ID NO: 3) at amino acid residues 570 to 579 compared to the parent AAV capsid protein. (Item 34) 34. The variant AAV capsid protein of any one of items 29 to 33, comprising a capsid sequence having at least 85% homology to the amino acid sequence set forth in SEQ ID NO:4 or SEQ ID NO:5. (Item 35) 35. The variant AAV capsid protein of any one of items 29 to 34, comprising the capsid sequence set forth in SEQ ID NO:6 or SEQ ID NO:7. (Item 36) 36. A nucleic acid comprising a nucleic acid sequence encoding the variant AAV capsid protein of any one of items 29 to 35. (Item 37) 37. An expression vector comprising the nucleic acid of claim 36, wherein the nucleic acid sequence encoding the variant AAV capsid protein is operably linked to a promoter sequence. (Item 38) 38. The expression vector of item 37, further comprising a nucleic acid encoding a rep protein. (Item 39) A cell comprising the expression vector according to item 37 or 38. (Item 40) 40. The cell of item 39, further comprising a nucleic acid encoding a therapeutic gene product. (Item 41) 41. The cell according to item 40, wherein the therapeutic gene product is an siRNA, an miRNA or a protein. (Item 42) 42. The cell according to item 40 or 41, wherein the therapeutic gene product is an anti-vascular endothelial growth factor (anti-VEGF) gene product. (Item 43) 42. The cell of item 40 or 41, wherein the therapeutic agent is an opsin. (Item 44) 44. The cell of any one of items 40 to 43, wherein the polynucleotide encoding the therapeutic gene product is flanked by one or more AAV ITRs. (Item 45) 45. The cell of item 44, wherein the ITR of the one or more AAVs is an ITR of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, avian AAV, bovine AAV, canine AAV, equine AAV, primate AAV, non-primate AAV or ovine AAV, or a variant thereof. (Item 46) 46. ​​The cell of item 44 or 45, wherein the one or more AAV ITRs are AAV2 ITRs or AAV5 ITRs. [Brief description of the drawings]

[0028] [Figure 1-1] Figure 1A shows a schematic diagram of the retina and the perforation locations of the concentric specimens: Perforation 1 contains the macula (small circular depression); Perforation 2 contains the parafovea; the remaining tissue contains the peripheral retina including the blood arcade.

[0029] [Figure 1-2] Figures 1B-1E show the relative abundance of capsid variants at different stages of the selection process. Figure 1B shows the percentage of the top 100 variants in the original library; Figure 1C shows the percentage of the top 100 variants identified in parafoveal, macular or peripheral retinal regions from the second screening round. Figure 1D shows the percentage of the top 50 evolved variants successfully transduced into parafoveal and RPE cells from the round 2 library of parafoveal origin. Figure 1E shows the percentage of the top 50 evolved variants successfully transduced into macular + peripheral cells from the round 2 library of parafoveal origin.

[0030] [Diagram 2] Figure 2 shows the results of dot plots of heparin column fractions from AAV2.5T and variant capsid AAV2.5T.LSV1. Eluents E1-E10 have increasing concentrations of NaCl: 0.1 M (E1), 0.2 M (E2), 0.3 M (E3), 0.4 M (E4), 0.5 M (E5), 0.6 M (E6), 0.7 M (E7), 0.8 M (E8), 0.9 M (E9) and 1.0 M (E10).

[0031] [Diagram 3]Figure 3 shows live imaging of GFP expression in 6 mm porcine retinal explants at days 7 or 14 post-infection with AAV2.5T.LSV1 virions encoding 2E+10vg (i.e., 2E10vg) or 4E+10vg (i.e., 4E10vg) GFP.

[0032] [Figure 4-1] Figures 4A-4C provide OTC autofluorescence images of African green monkey retinas at days 14 (Figure 4A), 21 (Figure 4B), and 28 (Figure 4C) following intravitreal administration of AAV2.5T.LSV1 virions encoding GFP, demonstrating GFP expression within the fovea and vascular arcades.

[0033] [Figure 4-2] Figures 4D-4E provide Heidelberg Spectralis images of the left (Figure 4D) and right (Figure 4E) retinas of African green monkeys 28 days after intravitreal administration of AAV2.5T.LSV1 virions encoding GFP, demonstrating GFP expression within the fovea and vascular arcades.

[0034] [Figure 5-1] Figures 5A-5B provide fluorescent images of flat-mounted retinas of African green monkeys following intravitreal administration of AAV2.5T.LSV1 virions encoding GFP.

[0035] [Figure 5-2] Figures 5C-5D provide fluorescent images of flat-mounted retinas of African green monkeys following intravitreal administration of AAV2.5T.LSV1 virions encoding GFP for the left eye (Figure 5C) and right eye (Figure 5D).

[0036] Figure 5E provides GFP intensity profiles plotted over distance from the macula to the ciliary body for the eyes shown in Figures 5C-5D. The location of retinal regions was calculated from the average distance to the center of the fovea.

[0037] [Figure 6]Figures 6A-6C provide fluorescent images of the fovea to ciliary body of an African green monkey eye following intravitreal administration of AAV2.5T.LSV1 virions encoding GFP. Figure 6A shows GFP expression. Figure 6B shows DAPI staining of the nuclei. Figure 6C is a merged image of Figures 6A and 6B.

[0038] [Figure 7] FIG. 7 provides a fluorescent image of GFP expressed in the fovea of ​​an African green monkey eye following intravitreal administration of AAV2.5T.LSV1 virions encoding GFP.

[0039] [Figure 8] FIG. 8 provides a fluorescent image of GFP expressed in the fovea of ​​the African green monkey eye following intravitreal administration of AAV2.5T.LSV1 virions encoding GFP.

[0040] [Figure 9] Figures 9A-9D provide fluorescent images of the fovea of ​​an African green monkey eye following intravitreal administration of AAV2.5T.LSV1 virions encoding GFP. Figure 9A shows a merged image of Figures 9B-9D. Figure 9B shows DAPI staining of nuclei. Figure 9C shows GFP expression. Figure 9D shows rhodopsin antibody labeling of rod photoreceptor cells.

[0041] [Figure 10] Figures 10A-10D provide fluorescent images of the fovea of ​​an African green monkey eye following intravitreal administration of AAV2.5T.LSV1 virions encoding GFP. Figure 10A shows a merged image of Figures 10B-10D. Figure 10B shows DAPI staining of nuclei. Figure 10C shows GFP expression. Figure 10D shows glutamine synthetase antibody labeling of Müller cells.

[0042] [Figure 11] FIG. 11 provides a fluorescent image of GFP expressed in the retina of an African green monkey eye following intravitreal administration of AAV2.5T.LSV1 virions encoding GFP.

[0043] [Figure 12] Figures 12A-12C provide fluorescent images of the retina of an African green monkey eye following intravitreal administration of AAV2.5T.LSV1 virions encoding GFP. Figure 12A is a composite image of Figures 12B and 12C. Figure 12B shows DAPI staining of the nucleus. Figure 12C shows GFP expression.

[0044] [Figure 13] Figures 13A-13D provide fluorescent images of the retina of an African green monkey eye following intravitreal administration of AAV2.5T.LSV1 virions encoding GFP. Figure 13A shows a merged image of Figures 13B-13D. Figure 13B shows DAPI staining of nuclei. Figure 13C shows GFP expression. Figure 13D shows glutamine synthetase antibody labeling of Müller cells.

[0045] [Figure 14] Figures 14A-14D provide fluorescent images of the retina of an African green monkey eye following intravitreal administration of AAV2.5T.LSV1 virions encoding GFP. Figure 14A shows a merged image of Figures 14B-14D. Figure 14B shows DAPI staining of nuclei. Figure 14C shows GFP expression. Figure 14D shows glutamine synthetase antibody labeling of Müller cells.

[0046] [Figure 15] Figures 15A-15D provide fluorescent images of the retina of an African green monkey eye following intravitreal administration of AAV2.5T.LSV1 virions encoding GFP. Figure 15A shows a merged image of Figures 15B-15D. Figure 15B shows DAPI staining of nuclei. Figure 15C shows GFP expression. Figure 15D shows glutamine synthetase antibody labeling of Müller cells.

[0047] [Figure 16]Figures 16A-16E provide fluorescent images of the retina of an African green monkey eye following intravitreal administration of AAV2.5T.LSV1 virions encoding GFP. Figure 16A shows a merged image of Figures 16B-16E. Figure 16B shows DAPI staining of nuclei. Figure 16C shows GFP expression. Figure 16D shows PCK-alpha antibody labeling of bipolar cells. Figure 16E shows TUJ-1 antibody labeling of pathways of retinal ganglion axon terminals.

[0048] [Figure 17] Figures 17A-17D provide fluorescent images of the retina of an African green monkey eye following intravitreal administration of AAV2.5T.LSV1 virions encoding GFP. Figure 17A shows a merged image of Figures 17B-17D. Figure 17B shows DAPI staining of nuclei. Figure 17C shows GFP expression. Figure 17D shows PCK-alpha antibody labeling of bipolar cells.

[0049] [Figure 18] Figures 18A-18E provide fluorescent images of the retina of an African green monkey eye following intravitreal administration of AAV2.5T.LSV1 virions encoding GFP. Figure 18A shows a merged image of Figures 18B-18E. Figure 18B shows DAPI staining of nuclei. Figure 18C shows GFP expression. Figure 18D shows cone arrestin antibody labeling of cone photoreceptor cells. Figure 18E shows rhodopsin antibody labeling of rod photoreceptor cells.

[0050] [Figure 19] Figures 19A-19E provide fluorescent images of the retina of an African green monkey eye following intravitreal administration of AAV2.5T.LSV1 virions encoding GFP. Figure 19A shows a merged image of Figures 19B-19E. Figure 19B shows DAPI staining of nuclei. Figure 19C shows GFP expression. Figure 19D shows cone arrestin antibody labeling of cone photoreceptor cells. Figure 19E shows rhodopsin antibody labeling of rod photoreceptor cells.

[0051] [Figure 20]Figures 20A-20E provide fluorescent images of the retina of an African green monkey eye following intravitreal administration of AAV2.5T.LSV1 virions encoding GFP. Figure 20A shows a merged image of Figures 20B-20E. Figure 20B shows DAPI staining of nuclei. Figure 20C shows GFP expression. Figure 20D shows cone arrestin antibody labeling of cone photoreceptor cells. Figure 20E shows rhodopsin antibody labeling of rod photoreceptor cells.

[0052] [Figure 21] Figures 21A-21E provide fluorescent images of the retinal pigment epithelium (RPE) of African green monkey eyes following intravitreal administration of AAV2.5T.LSV1 virions encoding GFP. Figure 21A shows a merged image of Figures 21B-21E. Figure 21B shows DAPI staining of nuclei. Figure 21C shows native GFP expression without antibody labeling. Figure 21D shows RPE65 antibody labeling of cone photoreceptor cells. Figure 21E shows GFP expression at 647 nm.

[0053] [Figure 22] Figures 22A-22E provide fluorescent images of the retinal pigment epithelium (RPE) of African green monkey eyes following intravitreal administration of AAV2.5T.LSV1 virions encoding GFP. Figure 22A shows a merged image of Figures 22B-22E. Figure 22B shows DAPI staining of nuclei. Figure 22C shows native GFP expression without antibody labeling. Figure 22D shows RPE65 antibody labeling of cone photoreceptor cells. Figure 22E shows GFP expression at 647 nm.

[0054] [Figure 23] Figures 23A-23E provide planar fluorescence images of the retinal pigment epithelium (RPE) of an African green monkey eye following intravitreal administration of AAV2.5T.LSV1 virions encoding GFP. Figure 23A shows a composite image of Figures 23B-23E. Figure 23B shows DAPI staining of nuclei. Figure 23C shows native GFP expression without antibody labeling. Figure 23D shows RPE65 antibody labeling of cone photoreceptor cells. Figure 23E shows GFP expression at 647 nm.

[0055] [Figure 24] Figures 24A-24E provide planar fluorescence images of the retinal pigment epithelium (RPE) of an African green monkey eye following intravitreal administration of AAV2.5T.LSV1 virions encoding GFP. Figure 24A shows a composite image of Figures 24B-24E. Figure 24B shows DAPI staining of nuclei. Figure 24C shows native GFP expression without antibody labeling. Figure 24D shows RPE65 antibody labeling of cone photoreceptor cells. Figure 24E shows GFP expression at 647 nm.

[0056] [Diagram 25] Figures 25A-25E provide planar fluorescence images of the retinal pigment epithelium (RPE) of an African green monkey eye following intravitreal administration of AAV2.5T.LSV1 virions encoding GFP. Figure 25A shows a composite image of Figures 25B-25E. Figure 25B shows DAPI staining of nuclei. Figure 25C shows native GFP expression without antibody labeling. Figure 25D shows RPE65 antibody labeling of cone photoreceptor cells. Figure 25E shows GFP expression at 647 nm.

[0057] [Figure 26-1] Figures 26A-26C provide the average percentage of GFP-positive bipolar cells (Figure 26A), cone cells (Figure 26B), and RPE cells (Figure 26C) in various regions in the retina of African green monkey eyes following intravitreal administration of AAV2.5T.LSV1 virions encoding GFP. [Figure 26-2] Figures 26A-26C provide the average percentage of GFP-positive bipolar cells (Figure 26A), cone cells (Figure 26B), and RPE cells (Figure 26C) in various regions in the retina of African green monkey eyes following intravitreal administration of AAV2.5T.LSV1 virions encoding GFP. [Figure 26-3] Figures 26A-26C provide the average percentage of GFP-positive bipolar cells (Figure 26A), cone cells (Figure 26B), and RPE cells (Figure 26C) in various regions in the retina of African green monkey eyes following intravitreal administration of AAV2.5T.LSV1 virions encoding GFP.

[0058] [Figure 27] Figures 27A-27B provide neutralizing antibody (nAB) profiles against AAV2.5T.LSV1 and AAV2. Pooled human IgG antibodies (Gammagard IVIG) were combined with AAV2.5T.LSV1-CMV-GFP or AAV2-CMV-GFP. 293T cells were transduced with the mixture and incubated for 3 days before measuring GFP expression. Figure 27A shows the neutralizing antibody profile of IVIG against AAV2.5T.LSV1-CMV-GFP (N=3 for each dilution). Figure 27B shows the neutralizing antibody profile of IVIG against AAV2-CMV-GFP (N=2 or 3 for each dilution). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0059] The present disclosure provides variant AAV capsid proteins, and recombinant adeno-associated virus (AAV) virions having one or more variant AAV capsid proteins. In some embodiments, the recombinant AAV virions exhibit one or more of the following characteristics: 1) increased infectivity of retinal cells; 2) altered tropism; 3) increased binding to heparin and / or heparan sulfate proteoglycan and / or inner limiting membrane (ILM); and 4) increased ability to infect and / or cross ILM to deliver therapeutic gene products when administered intravitreally, compared to the corresponding viral vector that contains its native, wild-type and / or parent capsid protein instead of the modified capsid protein disclosed herein. Also provided are pharmaceutical compositions and methods for the use of any of the compositions disclosed herein to promote expression of therapeutic gene products in cells, such as retinal cells, in an individual, for example, for the treatment or prevention of a disease or disorder.

[0060] I. Definition Unless otherwise defined, all technical terms used herein have the same ordinary meaning as understood by one of ordinary skill in the art.

[0061] The terminology used herein is for the purpose of describing particular examples only and is not intended to be limiting. As used herein, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. Furthermore, to the extent the terms "including," "include," "having," "has," "with," or variations thereof are used in either the detailed description and / or claims, such terms are intended to be inclusive in a similar manner as the term "comprising." The term "comprising," as used herein, is synonymous with "including" or "containing," and is inclusive or open ended.

[0062] Any reference herein to "or" is intended to include "and / or" unless stated otherwise.

[0063] Reference herein to "about" a value or parameter includes (and describes) the variation that is directed to that value or parameter itself. For example, a reference to "about X" includes the description of "X."

[0064] In some embodiments of the invention, an "individual" or "subject" is a mammal. Mammals include, but are not limited to, domestic animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In certain embodiments, the individual or subject is a human.

[0065] As used herein, the terms "treat", "treating", "treatment", "ameliorate" or "ameliorating" and other grammatically equivalent terms refer to alleviating, attenuating or reversing an ocular disease or disorder or a symptom of an ocular disease or disorder, preventing additional symptoms of an ocular disease or disorder, reversing the underlying metabolic cause of the symptoms, inhibiting an ocular disease or disorder, e.g., arresting the development of an ocular disease or disorder, alleviating an ocular disease or disorder, causing regression of an ocular disease or disorder, or halting the symptoms of an ocular disease or disorder. The term further includes achieving a therapeutic benefit. The term "therapeutic benefit" refers to eradication or reversal of the ocular disease or disorder being treated. Therapeutic benefit may also be achieved by eradication or reversal of one or more physiological symptoms associated with an ocular disease or disorder, such that, in some embodiments, improvement is observed in a patient, subject or individual, even though the patient, subject or individual continues to be afflicted with an ocular disease or disorder.

[0066] In some embodiments, the methods of the invention provide a prophylactic benefit, e.g., the pharmaceutical compositions are administered to patients, subjects or individuals at risk of developing an ocular disease or disorder, or who report one or more physiological symptoms of an ocular disease or disorder, even in the absence of a diagnosis of the disease or disorder.

[0067] The terms "administer," "administering," "administration," and the like, as used herein, may refer to methods used to enable delivery of a therapeutic agent or pharmaceutical composition to a desired site of biological action. These methods include intravitreal or subretinal injection into the eye.

[0068] As used herein, the term "effective amount", "therapeutically effective amount" or "pharmaceutical effective amount" may refer to a sufficient amount of at least one pharmaceutical composition or compound administered to relieve to some extent one or more symptoms of the ocular disease or disorder being treated. An "effective amount", "therapeutically effective amount" or "pharmaceutical effective amount" of a pharmaceutical composition may be administered to a subject in need thereof in a unit dose (described in more detail elsewhere herein).

[0069] As used herein, the term "pharmaceutically acceptable" can refer to a substance, such as a carrier or diluent, that does not abrogate the biological activity or properties of the compounds disclosed herein and is relatively non-toxic (i.e., it does not cause undesirable biological effects when the substance is administered to an individual and does not interact in a deleterious manner with any of the components of the composition contained in the composition).

[0070] The term "pharmaceutical composition," as used herein, may refer to a biologically active compound optionally mixed with at least one pharma- ceutically acceptable chemical component, such as, for example, but not limited to, a carrier, stabilizer, diluent, dispersant, suspending agent, thickener, excipient, etc.

[0071] As used herein, "AAV vector" or "rAAV vector" refers to an adeno-associated virus (AAV) vector or recombinant AAV (rAAV) vector that contains a polynucleotide sequence that is not of AAV origin (e.g., a polynucleotide heterologous to AAV, such as a nucleic acid sequence encoding a therapeutic transgene (e.g., aflibercept)) for transduction into a target cell or tissue. Generally, the heterologous polynucleotide is flanked by at least one, and generally two, inverted terminal repeats (ITRs) of AAV. The term rAAV vector encompasses both rAAV vector particles and rAAV vector plasmids. rAAV vectors can be either single-stranded (ssAAV) or self-complementary (scAAV).

[0072] "AAV virus" or "AAV virus particle" or "rAAV vector particle" or "rAAV particle" or "recombinant AAV virion" refers to a virus particle that contains at least one capsid protein of AAV and a polynucleotide rAAV vector. When the particle contains a heterologous polynucleotide (e.g., a polynucleotide other than the genome of wild-type AAV, such as a transgene to be delivered to a target cell or tissue), it is typically referred to as a "rAAV vector particle" or "rAAV vector."

[0073] As used herein, the term "packaging" can refer to a series of intracellular events that can result in the assembly and encapsidation of rAAV particles.

[0074] The AAV "rep" and "cap" genes refer to polynucleotide sequences that encode the replication and encapsidation proteins of the adeno-associated virus. AAV rep and cap are referred to herein as AAV "packaging genes."

[0075] The term "polypeptide" or "protein" is used interchangeably herein to refer to a polymer of amino acids of any length. The polymer may be linear or branched, and may contain modified amino acids and may be interrupted by non-amino acids. The term also encompasses naturally or by intervention modified amino acid polymers, such as disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling moiety or toxin. Also included within this definition are polypeptides that contain, for example, one or more analogs of amino acids (including, for example, non-natural amino acids, etc.), and other modifications known in the art.

[0076] The term "polynucleotide" refers to a polymeric form of nucleotides of any length, including deoxyribonucleotides or ribonucleotides, or their analogs. A polynucleotide may contain modified nucleotides, such as methylated nucleotides and nucleotide analogs, and may be interrupted by non-nucleotide components. If present, modifications to the nucleotide structure may be imparted before or after assembly of the polymer. The term polynucleotide, as used herein, may refer to double-stranded and single-stranded molecules.

[0077] As used herein, "recombinant" may refer to a biological molecule, e.g., a gene or protein, that is (1) removed from its naturally occurring environment, (2) not associated with all or a portion of a polynucleotide with which the gene is found in nature, (3) operatively linked to a polynucleotide with which it is not naturally linked, or (4) not occurring in nature. The term "recombinant" may be used in reference to cloned DNA isolates, chemically synthesized polynucleotide analogs, or polynucleotide analogs biologically synthesized by heterologous systems, as well as proteins and / or mRNAs encoded by such nucleic acids. Thus, for example, a protein synthesized by a microorganism is recombinant if it is synthesized, for example, from an mRNA synthesized from a recombinant gene present in the cell.

[0078] The term "anti-VEGF gene product" includes any therapeutic agent, including a protein, polypeptide, peptide, fusion protein, multimeric protein, antibody, human monoclonal antibody, antibody fragment, aptamer, kinase inhibitor, receptor or receptor fragment, or nucleic acid molecule, that can reduce, interact with, interfere with, block, and / or inhibit the activity or function of endogenous VEGF and / or endogenous VEGF receptor (VEGFR), or VEGF-VEGFR interaction, or in vivo pathway. The anti-VEGF gene product can be any one of the known therapeutic gene products, e.g., ranibizumab, brolucizumab, or bevacizumab, that can reduce new blood vessel growth or formation and / or edema or swelling when delivered in vivo to a cell, tissue, or subject. In some embodiments, the anti-VEGF gene product can be naturally occurring, non-naturally occurring, or synthetic. In some embodiments, the anti-VEGF gene product can be derived from a naturally occurring molecule that is subsequently modified or mutated to confer anti-VEGF activity. In some embodiments, anti-VEGF gene product is fusion protein or chimeric protein.In such protein, functional domain or polypeptide is artificially fused to part or polypeptide to create fusion protein or chimeric protein that can sequester VEGF in vivo or function as VEGFR decoy.In some embodiments, anti-VEGF gene product is fusion protein or chimeric protein that blocks endogenous VEGFR from interacting with its ligand.

[0079] As used herein, unless otherwise required, "VEGF" may refer to any isoform of VEGF or VEGF family members, including, but not limited to, VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E, VEGF-F, placenta growth factor (PGF), or any combination thereof, or any functional fragment, or variant. As used herein, "VEGF receptor" or "VEGFR" or "VEGF-R" may be used to refer to any one of the receptors of VEGF, including, but not limited to, VEGFR-1 (or Flt-1), VEGFR-2 (or Flk-1 / KDR), and VEGFR-3 (or Flt-4). VEGFR may be a membrane-bound or soluble form, or a functional fragment, or a truncated form of the receptor.

[0080] As used herein, "sFlt-1 protein" refers to a polypeptide sequence or functional fragment thereof having at least 90% or greater homology with the naturally occurring human sFLT-1 sequence, unless expressly stated otherwise, such that the sFlt-1 protein or polypeptide binds to VEGF and / or VEGF receptors. Homology refers to the percent conservation of residues of alignment between two sequences. Naturally occurring human sFLT-1 proteins can include any suitable variant of sFLT-1, including, but not limited to, functional fragments, sequences containing insertions, deletions, substitutions, pseudofragments, pseudogenes, splice variants, or artificially optimized sequences.

[0081] "Operably linked" or "operably linked" or "coupled" can refer to the proximity of genetic elements, where the elements are in a relationship that allows them to operate in a predictable manner. For example, a promoter can be operably linked to a coding region if it serves to initiate transcription of the coding sequence. There can be intervening residues between the promoter and the coding region, so long as this functional relationship is maintained.

[0082] As used herein, "expression vector" includes vectors, such as plasmids, minicircles, viral vectors, liposomes, etc., discussed above or known in the art, that contain a polynucleotide encoding a gene product of interest and are used to effect expression of the gene product in the intended target cell. Expression vectors may also contain control elements operably linked to the coding region to facilitate expression of the gene product in the target. The combination of control elements, such as promoters, enhancers, UTRs, miRNA target sequences, etc., and one or more genes operably linked thereto for expression, may be referred to as "expression cassettes." Many such control elements are known and available in the art or can be readily constructed from components available in the art.

[0083] A "gene product" is a molecule resulting from the expression of a particular gene. Gene products include, for example, polypeptides, aptamers, interfering RNA, mRNA, etc. In certain embodiments, a "gene product" is a polypeptide, peptide, protein, or interfering RNA, including small interfering RNA (siRNA), miRNA, or small hairpin RNA (shRNA). In certain embodiments, the gene product is a therapeutic gene product, e.g., a therapeutic protein.

[0084] The term "heterologous" may refer to an entity that is genotypically different from the remainder of the entity to which it is being compared. For example, a polynucleotide introduced by genetic engineering techniques into a plasmid or vector derived from a different species may be a heterologous polynucleotide. A promoter that has been removed from its native coding sequence and operably linked to a coding sequence in a linkage not found in nature may be a heterologous promoter.

[0085] The methods, compositions and kits described herein may use conventional techniques and descriptions of molecular biology (including recombinant techniques), cell biology, biochemistry, immunochemistry and ophthalmology techniques that are within the skill of a person skilled in the art, unless otherwise indicated. Such conventional techniques include retina or vision observation and analysis in subjects, recombinant virus cloning and propagation, pharmaceutical composition formulation, and biochemical purification and immunochemistry methods. Specific examples of suitable techniques can be obtained by reference to the examples herein. However, it is understood that equivalent conventional procedures can also be used.Such conventional techniques and descriptions can be found in Green, et al., Eds., Genome Analysis: A Laboratory Manual Series (Vols. I-IV) (1999); Weiner, et al., Eds., Genetic Variation: A Laboratory Manual (2007); Dieffenbach, Dveksler, Eds., PCR Primer: A Laboratory Manual (2003); Bowtell and Sambrook, DNA Microarrays: A Molecular Cloning Manual (2003); Mount, Bioinformatics: Sequence and Genome Analysis (2004); Sambrook and Russell, Condensed Protocols from Molecular Cloning: A Laboratory Manual (2006); and Sambrook and Russell, Molecular Cloning: A Laboratory Manual (2002) (all from Cold Spring Harbor Laboratory Press); Stryer, L., Biochemistry (4th Ed.) WH Freeman, NY (1995); Gait, "Oligonucleotide Synthesis: A Practical Methods and techniques for determining the molecular chemistry of biological samples can be found in standard laboratory manuals, such as "Theoretical and Experimental Approach," IRL Press, London (1984); Nelson and Cox, Lehninger, Principles of Biochemistry, 3rd Ed., WH Freeman Pub., New York (2000); and Berg et al., Biochemistry, 5th Ed., WH Freeman Pub., New York (2002), all of which are incorporated herein by reference in their entireties for all purposes.

[0086] As used herein, a "variant AAV capsid protein" refers to an AAV capsid protein that contains at least one amino acid difference (e.g., an amino acid substitution, an amino acid insertion, an amino acid deletion) compared to a corresponding parent AAV capsid protein, and where the AAV capsid protein does not correspond to an amino acid sequence present in a naturally occurring AAV capsid protein. In some embodiments, the variant AAV capsid protein confers increased binding to heparin and / or heparan sulfate proteoglycans compared to binding by AAV virions containing the corresponding parent AAV capsid protein. In certain embodiments, the variant AAV capsid protein confers: a) increased infectivity of retinal cells compared to the infectivity of retinal cells by AAV virions containing the corresponding parent AAV capsid protein; b) altered cell tropism compared to the tropism of AAV virions containing the corresponding parent AAV capsid protein; and / or c) increased ability to bind to and / or pass through the ILM compared to AAV virions containing the corresponding parent AAV capsid protein.

[0087] As used herein, a "modified sequence" refers to a sequence that contains one or more substitutions, insertions and / or deletions compared to the corresponding sequence of a parent AAV or a parent AAV capsid protein.

[0088] "Helper virus" for AAV refers to a virus that allows AAV (e.g., wild-type AAV) to replicate and be packaged by mammalian cells. A variety of such helper viruses for AAV are known in the art, and include adenovirus, herpesvirus, and poxvirus such as vaccinia. Adenoviruses encompass several different subgroups, but adenovirus type 5 of subgroup C is the most commonly used. Numerous adenoviruses of human, non-human mammalian, and avian origin are known and available from repositories such as ATCC. Viruses of the herpes family include, for example, herpes simplex virus (HSV) and Epstein-Barr virus (EBV), as well as cytomegalovirus (CMV) and pseudorabies virus (PRV), which are also available from repositories such as ATCC.

[0089] "Helper functions" refers to functions encoded in the helper virus genome that allow AAV replication and packaging (along with other requirements for replication and packaging as described herein). As described herein, "helper functions" can be provided in several ways, including providing a helper virus or, for example, providing a polynucleotide sequence encoding the necessary functions to a producer cell in trans. For example, a plasmid or other expression vector containing a nucleotide sequence encoding one or more adenoviral proteins is transfected into a producer cell together with a rAAV vector.

[0090] An "infectious" virion or virus particle is one that contains a competently assembled viral capsid and can deliver polynucleotide components to cells for which the viral species is tropic. This term does not necessarily imply any replicative capacity of the virion. Assays for counting infectious virions are described elsewhere in this disclosure and in the art. The infectivity of a virus can be expressed as the ratio of infectious virions to total virions. Methods for determining the ratio of infectious virions to total virions are known in the art. See, for example, Grainger et al. (2005) Mol. Ther. 11:S337 (describing a TCID50infectious titer assay); and Zolotukhin et al. (1999) Gene Ther. 6:973.

[0091] II. Variant AAV Capsid Proteins Provided herein are variant adeno-associated virus (AAV) capsid proteins. In some embodiments, the variant AAV capsid proteins disclosed herein comprise a modified sequence comprising one or more amino acid substitutions within amino acid residues 570-579 relative to the parent AAV capsid protein, the modified sequence comprising HKFKSGD (SEQ ID NO: 1), where the numbering of the amino acid residues corresponds to that of the AAV5 VP1 capsid protein. In some embodiments, the variant AAV capsid proteins comprise a modified sequence comprising one or more amino acid substitutions within amino acid residues 570-579 relative to the parent AAV capsid protein, the modified sequence comprising X1X2HKFKSGDX3 (SEQ ID NO: 2), where the numbering of the amino acid residues corresponds to that of the AAV5 VP1 capsid protein. 1~3 may independently be any amino acid. 1~3 Each of X is independently selected from A, L, G, S, and T. 1~3are independently selected from A, L, G, S and T. In some embodiments, X1 is L. In some embodiments, X2 is A. In some embodiments, X3 is A. In some embodiments, the variant AAV capsid protein comprises a modified sequence comprising one or more amino acid substitutions within amino acid residues 570-579 relative to the parent AAV capsid protein, the modified sequence comprising LAHKFKSGDA (SEQ ID NO:3), a sequence having at least 80% or at least 90% homology to SEQ ID NO:3, a sequence having at least 80% or at least 90% sequence identity to SEQ ID NO:3, or a sequence having 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, or 9 or more consecutive amino acids within SEQ ID NO:3, where the numbering of the amino acid residues corresponds to the AAV5 VP1 capsid protein. In some embodiments, the modified sequence comprises LAHKFKSGDA (SEQ ID NO:3).

[0092] Herein, amino acid numbering corresponding to the AAV5 VP1 capsid protein is used to refer to amino acid modifications of the capsid protein (including specific amino acid substitutions and insertions), but it is understood that any of these amino acid modifications may also be introduced into the capsid protein of other serotypes of AAV, for example, at positions corresponding to those in AAV5 VP1. AAV protein sequences share considerable homology and similar amino acid numbering, and one of skill in the art can readily determine the amino acid residues in other AAV serotypes that correspond to those specifically described herein for AAV5 VP1.

[0093] In some embodiments, the parent AAV capsid protein is a wild-type AAV capsid protein, such as AAV type 1 (AAV1), AAV type 2 (AAV2), AAV type 3 (AAV3), AAV type 4 (AAV4), AAV type 5 (AAV5), AAV type 6 (AAV6), AAV type 7 (AAV7), AAV type 8 (AAV8), avian AAV, bovine AAV, canine AAV, equine AAV, primate AAV, non-primate AAV, or ovine AAV capsid protein. In some embodiments, the parent AAV capsid protein is an AAV5 capsid protein. In some embodiments, the parent AAV capsid protein is a VP1, VP2, or VP3 capsid protein. In some embodiments, the parent AAV capsid protein is an AAV5 VP1 capsid protein.

[0094] In some embodiments, the parent AAV capsid protein is a variant AAV capsid protein. In some embodiments, the parent AAV capsid protein is a variant of the capsid protein of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, avian AAV, bovine AAV, canine AAV, equine AAV, primate AAV, non-primate AAV, or ovine AAV. In some embodiments, the parent AAV capsid protein is a variant of the AAV5 VP1 capsid protein. In some embodiments, the parent AAV capsid protein is a variant of the AAV5 VP1 capsid protein that has at least 90%, at least 95%, at least 98%, or at least 99% homology with SEQ ID NO:4. In some embodiments, the parent AAV capsid protein is a variant of AAV5 VP1 capsid protein with at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with SEQ ID NO:4. In some embodiments, the parent AAV capsid protein is a hybrid capsid protein. In some embodiments, the parent AAV capsid protein is a hybrid of AAV2 capsid protein and AAV5 capsid protein. In some embodiments, the parent AAV capsid protein is AAV2.5T capsid protein or a variant thereof with at least 90%, at least 95%, at least 98%, or at least 99% homology with AAV2.5T capsid protein. In some embodiments, the parent AAV capsid protein is AAV2.5T capsid protein or its variant having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with AAV2.5T capsid protein.In some embodiments, the parent AAV capsid protein is AAV2.5T capsid protein.In some embodiments, the parent AAV capsid protein is VP1, VP2 or VP3 capsid protein.In some embodiments, the parent AAV capsid protein is an AAV2.5T VP1 capsid protein or a variant thereof having at least 90%, at least 95%, at least 98%, or at least 99% homology to the AAV2.5T VP1 capsid protein (SEQ ID NO: 5). In some embodiments, the parent AAV capsid protein is an AAV2.5T VP1 capsid protein or a variant thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to the AAV2.5T VP1 capsid protein (SEQ ID NO: 5).

[0095] In some embodiments, the variant AAV capsid protein comprises a capsid sequence having at least 85% homology to the amino acid sequence set forth in SEQ ID NO:4 or SEQ ID NO:5. In some embodiments, the variant AAV capsid protein comprises a capsid sequence having at least 85% sequence identity to the amino acid sequence set forth in SEQ ID NO:4 or SEQ ID NO:5. In some embodiments, the variant AAV capsid protein comprises a capsid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% homology to the amino acid sequence set forth in SEQ ID NO:6 or SEQ ID NO:7. In some embodiments, the variant AAV capsid protein comprises a capsid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO:6 or SEQ ID NO:7. In some embodiments, the variant AAV capsid protein comprises SEQ ID NO:6 or SEQ ID NO:7. In some embodiments, the variant AAV capsid protein comprises SEQ ID NO:7.

[0096] In certain embodiments, the variant AAV capsid protein, when present in a recombinant AAV virion, provides increased infectivity of retinal cells compared to the infectivity of retinal cells by recombinant AAV virions containing the corresponding parent AAV capsid protein. In certain embodiments, the variant AAV capsid protein, when present in a recombinant AAV virion, provides increased infectivity of retinal cells when administered by intravitreal injection compared to the infectivity of retinal cells by recombinant AAV virions containing the corresponding parent AAV capsid protein. In some embodiments, the retinal cell is one or more of a photoreceptor cell (e.g., rod; cone), a retinal ganglion cell (RGC), a retinal pigment epithelium (RPE) cell, a Müller cell, an amacrine cell, a bipolar cell, and a horizontal cell. In certain embodiments, the variant AAV capsid protein, when present in a recombinant AAV virion, confers an altered tropism for a recombinant AAV virion compared to the tropism of a recombinant AAV virion containing the corresponding parent AAV capsid protein. In certain embodiments, the variant AAV capsid protein, when present in a recombinant AAV virion, confers increased binding to heparin and / or heparan sulfate, and / or increased ability to bind to and cross the inner limiting membrane (ILM) following intravitreal injection compared to a recombinant AAV virion containing the corresponding parent AAV capsid protein.

[0097] III. Polynucleotides and Cells Also provided herein is a polynucleotide that encodes one or more variant AAV capsid proteins described herein. In certain embodiments, the polynucleotide is an expression vector. In some embodiments, the expression vector comprises a polynucleotide sequence that encodes a modified capsid protein described herein operably linked to a promoter sequence, for example, a promoter sequence that drives the expression of the polynucleotide in the cell. In certain embodiments, the cell is a host cell. The host cell can be used to produce virions that include modified capsid proteins. Exemplary host cells include mammalian cells (e.g., HEK293 cells), insect cells (e.g., SF9 cells), microorganisms, and yeast. In some embodiments, the cells contain an AAV rep gene, such as an AAV rep gene encoding an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, avian AAV, bovine AAV, canine AAV, equine AAV, primate AAV, non-primate AAV, or ovine AAV rep protein or a variant thereof. In some embodiments, the AAV rep gene is stably maintained in the cells.

[0098] Also provided herein is a cell comprising a polynucleotide or vector encoding the modified capsid protein described herein. In certain embodiments, the polynucleotide is an expression vector, and the expression vector comprises a polynucleotide sequence encoding the variant capsid described herein operably linked to a promoter sequence, e.g., a promoter sequence that drives the expression of the polynucleotide in the cell. In some embodiments, the polynucleotide or vector is stably maintained in the cell. In some embodiments, the cell further comprises an AAV rep gene. In some embodiments, the AAV rep gene is stably maintained in the cell. In certain embodiments, the polynucleotide or vector further comprises a sequence encoding a rep protein, e.g., the rep protein of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, avian AAV, bovine AAV, canine AAV, equine AAV, primate AAV, non-primate AAV, or ovine AAV, or a variant thereof.

[0099] In some embodiments, the cell further comprises a polynucleotide cassette comprising a sequence encoding a gene product, for example, a therapeutic gene product, such as the therapeutic gene product described herein.In certain embodiments, the polynucleotide cassette is flanked by one or more AAV inverted terminal repeats (ITRs).In certain embodiments, the polynucleotide cassette is flanked at the 5' and 3' ends by the ITRs of AAV.In some embodiments, the one or more ITRs are the ITRs of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, avian AAV, bovine AAV, canine AAV, equine AAV, primate AAV, non-primate AAV or ovine AAV, or variants thereof. In some embodiments, the one or more ITRs are AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, avian AAV, bovine AAV, canine AAV, equine AAV, primate AAV, non-primate AAV, or ovine AAV ITRs. In some embodiments, the one or more ITRs are AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, avian AAV, bovine AAV, canine AAV, equine AAV, primate AAV, non-primate AAV, or ovine AAV ITRs that comprise one or more insertions, deletions, and / or substitutions of nucleotides.

[0100] IV. Virions and Methods for Producing Them Also provided herein are virions, e.g., recombinant AAV virions, that comprise the variant AAV capsid proteins described herein.

[0101] In some embodiments, recombinant AAV virion further comprises a polynucleotide cassette comprising a sequence encoding a gene product, for example, a therapeutic gene product, for example, a therapeutic gene product as described herein.In certain embodiments, the polynucleotide cassette is adjacent to one or more AAV inverted terminal repeats (ITRs), for example, the ITRs of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, avian AAV, bovine AAV, canine AAV, equine AAV, primate AAV, non-primate AAV or ovine AAV, or variants thereof.In certain embodiments, the polynucleotide cassette is adjacent to the ITRs of AAV at the 5' and 3' ends. In some embodiments, the one or more ITRs are AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, avian, bovine, canine, equine, primate, non-primate, or ovine AAV ITRs, or variants thereof. In some embodiments, the one or more ITRs are AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, avian, bovine, canine, equine, primate, non-primate, or ovine AAV ITRs that include one or more nucleotide insertions, deletions, and / or substitutions. In some embodiments, one or more ITRs are an ITR of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, avian AAV, bovine AAV, canine AAV, equine AAV, primate AAV, non-primate AAV or ovine AAV.

[0102] In certain embodiments, the gene product inhibits angiogenesis in the retina of a subject, for example, choroidal neovascularization (CNV).Many cellular factors have been found to play an important role in regulating CNV generation, among which may include, but are not limited to, vascular endothelial growth factor (VEGF), VEGF receptor (VEGFR), platelet-derived growth factor (PDGF), hypoxia-inducible factor (HIF), angiopoietin (Ang) and other cytokines, mitogen-activated protein kinase (MAPK).In some embodiments, the gene product is one or more inhibitors of VEGF, VEGFR, PDGF, HIF, Ang and MAPK.

[0103] In certain embodiments, the gene product inhibits angiogenesis in the retina of a subject, for example, choroidal neovascularization (CNV).Many cellular factors have been found to play an important role in regulating CNV generation, among which may include, but are not limited to, vascular endothelial growth factor (VEGF), VEGF receptor (VEGFR), platelet-derived growth factor (PDGF), hypoxia-inducible factor (HIF), angiopoietin (Ang) and other cytokines, and mitogen-activated protein kinase (MAPK).In some embodiments, the gene product is one or more inhibitors of VEGF, VEGFR, PDGF, HIF, Ang and MAPK.

[0104] In some embodiments, the gene product is an interfering RNA, an aptamer, or a protein. In some embodiments, the therapeutic gene product is an interfering RNA, an aptamer, or a protein. In some embodiments, the gene product (or therapeutic gene product) is an siRNA, miRNA, or a protein. In some embodiments, the gene product (or therapeutic gene product) is a site-specific nuclease that provides site-specific knockdown of gene function. In some embodiments, the gene product (or therapeutic gene product) is an interfering RNA. In some embodiments, the gene product (or therapeutic gene product) is an siRNA or miRNA.

[0105] In some embodiments, the gene product (or therapeutic gene product) is an anti-VEGF gene product. In some embodiments, the gene product (or therapeutic gene product) is an anti-VEGF interfering RNA. In some embodiments, the gene product (or therapeutic gene product) is an anti-VEGF siRNA or an anti-VEGF miRNA.

[0106] In some embodiments, gene product (or therapeutic gene product) is aptamer.Exemplary aptamers of interest include aptamers for vascular endothelial growth factor (VEGF).See, for example, Ng et al. (2006) Nat. Rev. Drug Discovery 5:123; and Lee et al. (2005) Proc. Natl. Acad. Sci. USA 102:18902.PDGF-specific aptamers, such as E10030, are also suitable for use, see, for example, Niand Hui (2009) Ophthalmologica 223:401; and Akiyama et al. (2006) J. CellPhysiol. 207:407.

[0107] In some embodiments, the gene product (or therapeutic gene product) is a protein, generally a protein that enhances the function of a retinal cell, such as a rod or cone photoreceptor cell, a retinal ganglion cell, a Muller cell, a bipolar cell, an amacrine cell, a horizontal cell, or a retinal pigment epithelial cell. Exemplary proteins include neuroprotective polypeptides (e.g., GDNF, CNTF, NT4, NGF, and NTN); anti-angiogenic polypeptides (e.g., soluble vascular endothelial growth factor (VEGF) receptors, VEGF-binding antibodies, VEGF-binding antibody fragments (e.g., single chain anti-VEGF antibodies); endostatin; tumstatin; angiostatin; soluble Flt proteins (Lai et al. (2005) Mol. Ther. 12:659); Fc fusion proteins comprising soluble Flt proteins (see, e.g., Pechan et al. (2009) Gene Ther. 16:10); pigment epithelium-derived factor (PEDF); soluble Tie-2 receptor, and the like); tissue inhibitor of metalloproteinase-3 (TIMP-3); opsins, e.g., rhodopsin; anti-apoptotic polypeptides (e.g., Bcl-2, Bcl-Xl), and the like. Suitable polypeptides include, but are not limited to, glial-derived neurotrophic factor (GDNF); fibroblast growth factor 2; neurturin (NTN); ciliary neurotrophic factor (CNTF); nerve growth factor (NGF); neurotrophin-4 (NT4); brain-derived neurotrophic factor (BDNF); epidermal growth factor; rhodopsin; X-linked inhibitor of apoptosis; and sonic hedgehog.

[0108] Suitable opsins include, for example, light-responsive opsins described in U.S. Patent Application Publication No. 2007 / 0261127 (e.g., ChR2; Chop2); U.S. Patent Application Publication No. 2001 / 0086421; U.S. Patent Application Publication No. 2010 / 0015095; and Diester et al. (2011) Nat. Neurosci. 14:387.

[0109] Suitable proteins also include retinoschisin.Suitable polypeptides include, for example, retinitis pigmentosa GTPase regulator (RGPR) interacting protein-1 (see, for example, GenBank accession number Q96KN7, Q9EPQ2, and Q9GLM3); peripherin-2 (Prph2) (see, for example, GenBank accession number NP_000313; and Travis et al. (1991) Genomics 10:733); peripherin; retinal pigment epithelium specific protein (RPE65) (see, for example, GenBank AAC39660; and Morimura et al. (1998) Proc. Natl. Acad. Sci. USA 95:3088) and the like.

[0110] Suitable proteins also include CHM (Choroidal Atrophy (Rab Escort Protein 1)), a polypeptide that, when defective or absent, causes choroidal atrophy (see, e.g., Donnelly et al. (1994) Hum. Mol. Genet. 3:1017; and van Bokhoven et al. (1994) Hum. Mol. Genet. 3:1041); and Crumbs Homolog 1 (CRB1), a polypeptide that, when defective or absent, causes Leber's congenital amaurosis and retinitis pigmentosa (see, e.g., denHollander et al. (1999) Nat. Genet. 23:217; and GenBank Accession No. CAM23328).

[0111] Suitable proteins also include proteins that, when defective or absent, result in total color blindness, where such polypeptides include, for example, cone photoreceptor cGMP-gated channel subunit alpha (CNGA3) (see, e.g., GenBank Accession No. NP_001289; and Booij et al. (2011) Ophthalmology 118:160-167); cone photoreceptor cGMP-gated cation channel beta subunit (CNGB3) (see, e.g., Kohlet al. (2005) Eur J Hum Genet. 13(3):302); guanine nucleotide-binding proteins (G proteins); alpha transduction activity polypeptide 2 (GNAT2) (ACHM4); and ACHM5; as well as polypeptides that, when defective or absent, result in various forms of color blindness (e.g., L-opsin, M-opsin, and S-opsin). See Mancuso et al. (2009) Nature 461(7265):784-787.

[0112] In some embodiments, gene product (or therapeutic gene product) is anti-VEGF protein.In some embodiments, anti-VEGF protein is sFlt-1 protein or its fragment, or anti-VEGF antibody or its fragment.In some embodiments, anti-VEGF protein is anti-VEGF antibody or its fragment, such as aflibercept, ranibizumab and bevacizumab or their fragment.In some embodiments, anti-VEGF protein is sFlt-1 protein.

[0113] The soluble truncated form of the VEGF receptor FLT-1, sFLT-1, is the only known endogenous specific inhibitor of VEGF. In nature, it is generated by alternative mRNA splicing and lacks the membrane-proximal immunoglobulin-like domain, the transmembrane region and the intracellular tyrosine kinase domain. Structurally, both FLT-1 and sFLT-1 proteins can contain multiple functional domains. In some variants, FLT and sFLT proteins usually share six linking domains, three domains involved in protein dimerization and three domains involved in binding ligands such as VEGF. sFLT-1 is not restricted to the cell membrane. Unbound sFLT-1 can freely diffuse in the extracellular space or solution.

[0114] The interaction between sFLT-1 and VEGF receptor is specific and can compete with 100-fold excess of unlabeled VEGF. In some cases, the angiogenesis suppressing activity of sFLT-1 can result from two mechanisms: i) sequestration of VEGF, which binds with high affinity, and ii) inhibition of VEGF by forming inactive heterodimers with transmembrane isoforms of VEGF receptors FLTt-1 and FLK-1 / KDR. As known in the art, in vitro binding assays show that sFLT-1 binds to VEGF with high affinity and can also inhibit VEGF-driven proliferation of human umbilical vein endothelial cells. In animal models for cancer, sFLT-1 inhibits tumor growth. In some cases, sFLT-1 can function in a substoichiometric or dominant-negative manner, since excess VEGF in the extracellular space can prevent VEGF receptor binding and subsequent activation. These properties of sFLT-1 are described in Kendall and Thomas, 1993; Proc Natl Acad Sci. 90: 10705-10709, which is incorporated herein by reference in its entirety. Functional fragments of sFLT-1 can be used in place of the full-length protein. More specifically, the VEGF binding domain (domain 2) or alternatively domain 2 of sFLT-1 and domain 3 from sFLT1, KDR or another family member can be used to bind and inactivate VEGF. Such functional fragments are described in Wiesman et al., 1997; Cell, 91: 695-704, which is incorporated herein by reference in its entirety.

[0115] In some embodiments, sFlt-1 protein is the naturally occurring protein sFlt-1 described in the sequence disclosed by US Patent Publication No. 5,861,484 and SEQ ID NO: 109 of US Patent Application Publication No. 2013 / 0323302. An exemplary sFlt-1 amino acid sequence is disclosed herein as SEQ ID NO: 13. It also includes but is not limited to the sequence of sFlt-1 domain 2 or its functional fragments, including those described in SEQ ID NO: 121 of US Patent Application Publication No. 2013 / 0323302 and related constructs such as VEGF binding fusion proteins disclosed in US Patent Publication No. 7,635,474. An exemplary sFlt-1 functional fragment is disclosed herein as SEQ ID NO: 14. In some embodiments, the sFLT-1 protein is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99% or 100% homologous to SEQ ID NO: 13 or 14. In some embodiments, the sFLT-1 protein has at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99% or 100% sequence identity to SEQ ID NO: 13 or 14. The anti-VEGF protein may also include any of the sFLT-1 proteins, variants or fragments thereof described in U.S. Patent Application Publication No. 2013 / 0323302.

[0116] In some embodiments, the sFLT-1 protein is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99% or 100% homologous to the naturally occurring human sFLT-1 protein sequence. In some embodiments, the sFLT-1 protein is up to about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99% or 100% homologous to the naturally occurring human sFLT-1 protein sequence. In some embodiments, the sFLT-1 protein has at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99% or 100% sequence identity to a naturally occurring human sFLT-1 protein sequence. In some embodiments, the sFLT-1 protein has up to about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99% or 100% sequence identity to a naturally occurring human sFLT-1 protein sequence. In some embodiments, the sFLT-1 protein is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99% or 100% spatially homologous to the conformation of naturally occurring human sFLT-1 protein. In some cases, the sFLT-1 protein is up to about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99% or 100% spatially homologous to the conformation of naturally occurring human sFLT-1 protein.

[0117] In some embodiments, the anti-VEGF protein is an anti-VEGF antibody. In some embodiments, the anti-VEGF protein is ranibizumab (commercially available under the trademark Lucentis® (Genentech, San Francisco, CA), see Figure 1 of U.S. Patent No. 7,060,269 for the sequence of the heavy and light chain variable regions of ranibizumab); bevacizumab (commercially available under the trademark Avastin® (Genentech, San Francisco, CA), see Figure 1 of U.S. Patent No. 6,054,297 for the sequence of the heavy and light chain variable regions of bevacizumab); aflibercept (commercially available under the trademark Eylea® (Regeneron, Tarrytown, NY)); or brolucizumab, see U.S. Patent No. 10,035,850. In certain embodiments, bevacizumab is a VEGF antibody having the following heavy and light chain variable domain sequences: In certain embodiments, ranibizumab comprises the following heavy and light chain variable domain sequences: EVQLVESGGGLVQPGGSLRLSCAASGYTFTNYGMNWVRQAPGKGLEWVGWINTYTGEPTYAADFKRRFTFSLDTSKSTAYLQMNSLRAEDTAVYYCAKYPHYYGSSHWYFDVWGQGTL (SEQ ID NO: 8); and DIQMTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKVLIYFTSSLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSTVPWTFGQGTKVEIKRTV (SEQ ID NO: 9), respectively. In certain embodiments, aflibercept comprises the following amino acid sequence: EVQLVESGGGLVQPGGSLRLSCAASGYDFTHYGMNWVRQAPGKGLEWVGWINTYTGEPTYAADFKRRFTFSLDTSKSTAYLQMNSLRAEDTAVYYCAKYPYYYGTSHWYFDVWGQGTL (SEQ ID NO: 10); and DIQLTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKVLIYFTSSLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSTVPWTFGQGTKVEIKRTV (SEQ ID NO: 11), respectively. MVSYWDTGVLLCALLSCLLLTGSSSGSDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTN YLTHRQTNTIIDVVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHEKDKTH In certain embodiments, aflibercept comprises the following amino acid sequence: EIVMTQSPSTLSASVGDRVIITCQASEIIHSWLAWYQQKPGKAPKLLIYLASTLASGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCQNVYLASTNGANFGQGTKLTVLGGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSRLSCTASGFSLTDYYYMTWVRQAPGKGLEWVGFIDPDDDPYYATWAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAGGDHNSGWGLDIWGQGTLVTVSS (SEQ ID NO: 15).

[0118] These sequences can be expressed from DNA encoding such sequences using the genetic code, a standard technique understood by those of skill in the art. As can be understood by those of skill in the art, due to the degeneracy of the genetic code, anti-VEGF protein sequences can be readily expressed from several different DNA sequences.

[0119] In some cases, the gene product (or therapeutic gene product) is a site-specific endonuclease that provides site-specific knockdown of gene function, for example, where the endonuclease knocks out an allele associated with a retinal disease. For example, if a dominant allele encodes a defective copy of a gene that, when wild type, is a retinal structural protein and / or provides normal retinal function, the site-specific endonuclease can be targeted to the defective allele and knock out the defective allele.

[0120] In addition to knocking out defective alleles, site-specific nucleases can also be used to stimulate homologous recombination with donor DNA that codes for a functional copy of the protein encoded by the defective allele.Thus, for example, a viral vector can be used to deliver a site-specific endonuclease that knocks out the defective allele, and can be used to deliver a functional copy of the defective allele, which results in the repair of the defective allele, thereby providing the production of functional retinal proteins (e.g., functional retinoschisin, functional RPE65, functional peripherin, etc.).See, for example, Li et al. (2011) Nature 475:217.In some embodiments, the viral vector comprises a heterologous nucleotide sequence that codes for a site-specific endonuclease and a heterologous nucleotide sequence that codes for a functional copy of the defective allele, where the functional copy codes for a functional retinal protein. Functional retinal proteins include, for example, retinoschisin, RPE65, regulator of retinitis pigmentosa GTPase (RGPR)-interacting protein-1, peripherin, peripherin-2, and the like.

[0121] Suitable site-specific endonucleases for use include, for example, zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs), where such site-specific endonucleases are not naturally occurring and are modified to target specific genes.Such site-specific nucleases can be engineered to cleave specific positions in genomes, and then non-homologous end joining can repair the cleavage, inserting or deleting some nucleotides.Then, such insertion or deletion (also called "INDEL") takes the protein out of reading frame, effectively knocking out the gene.See, for example, US Patent Application Publication No. 2011 / 0301073.

[0122] In some embodiments, the sequence encoding the gene product is operably linked to a promoter. In some embodiments, the sequence encoding the gene product is operably linked to a constitutive promoter. In other embodiments, the nucleotide sequence encoding the gene product of interest is operably linked to an inducible promoter. In some examples, the sequence encoding the gene product is operably linked to a tissue-specific or cell type-specific regulatory element. For example, in some examples, the sequence encoding the gene product is operably linked to a photoreceptor cell-specific regulatory element (e.g., a photoreceptor cell-specific promoter), e.g., a regulatory element that confers selective expression of the operably linked gene in photoreceptor cells. Suitable photoreceptor cell-specific regulatory elements include, for example, the rhodopsin promoter; the rhodopsin kinase promoter (Young et al. (2003) Ophthalmol. Vis. Sci. 44:4076); the beta phosphodiesterase gene promoter (Nicoud et al. (2007) J. Gene Med. 9:1015); the retinitis pigmentosa gene promoter (Nicoud et al. (2007), supra); the interphotoreceptor retinoid binding protein (IRBP) gene enhancer (Nicoud et al. (2007), supra); the IRBP gene promoter (Yokoyama et al. (1992) Exp Eye Res. 55:225).

[0123] In certain embodiments, the recombinant AAV virions can bind to heparin and / or heparan sulfate proteoglycans (HSPGs) with at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, or at least 100-fold greater affinity than, for example, a recombinant AAV virion comprising the corresponding parent AAV capsid protein. In certain embodiments, the recombinant AAV virions can bind to heparin and / or heparan sulfate proteoglycans (HSPGs) with at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, or at least 100-fold greater affinity than, for example, an AAV5 virion. In certain embodiments, the recombinant AAV virion can bind to heparin and / or heparan sulfate proteoglycans (HSPGs) with at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, or at least 100-fold greater affinity than, for example, the AAV2.5T virion.

[0124] In certain embodiments, the recombinant AAV virion can bind to the inner limiting membrane (ILM) with at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, or at least 100-fold greater affinity than, for example, a recombinant AAV virion comprising a corresponding parent AAV capsid protein. In certain embodiments, the recombinant AAV virion can bind to the inner limiting membrane (ILM) with at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, or at least 100-fold greater affinity than, for example, an AAV5 virion. In certain embodiments, the recombinant AAV virion can bind to the inner limiting membrane (ILM) with at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, or at least 100-fold greater affinity than, for example, an AAV2.5T virion.

[0125] In certain embodiments, the recombinant AAV virion has an increased infectivity of retinal cells compared to the infectivity of retinal cells by recombinant AAV virions containing the corresponding parent AAV capsid proteins. In certain embodiments, the recombinant AAV virion can cross the ILM. In certain embodiments, the recombinant AAV virion has an increased infectivity of retinal cells compared to the infectivity of retinal cells by recombinant AAV virions containing the corresponding parent AAV capsid proteins and can cross the ILM. In certain embodiments, the recombinant AAV virion has an increased infectivity of retinal cells compared to the infectivity of retinal cells by AAV5 virions. In certain embodiments, the recombinant AAV virion has an increased infectivity of retinal cells compared to the infectivity of retinal cells by AAV2.5T virions. In certain embodiments, the recombinant AAV virion can transduce cells of the retina when injected intravitreally into a mammal. In certain embodiments, the recombinant AAV virion can cross the ILM. In certain embodiments, the recombinant AAV virion has an increased infectivity of retinal cells when administered by intravitreal injection, compared to the infectivity of retinal cells by recombinant AAV virions containing the corresponding parent AAV capsid protein when administered by intravitreal injection. In certain embodiments, the recombinant AAV virion has an increased infectivity of retinal cells when administered by intravitreal injection, compared to the infectivity of retinal cells by AAV5 virions when administered by intravitreal injection. In certain embodiments, the recombinant AAV virion has an increased infectivity of retinal cells when administered by intravitreal injection, compared to the infectivity of retinal cells by AAV2.5T virions when administered by intravitreal injection. In some embodiments, the retinal cells are one or more of photoreceptor cells (e.g., rods; cones), retinal ganglion cells (RGCs), retinal pigment epithelial (RPE) cells, Müller cells, amacrine cells, bipolar cells, and horizontal cells. In some embodiments, the retinal cells are Müller cells, cone cells, rod cells, bipolar cells and / or RPE cells. In some embodiments, the retinal cells are Müller cells. In some embodiments, the retinal cells are cone cells.In some embodiments, the retinal cells are bipolar cells. In some embodiments, the retinal cells are RPE cells.

[0126] In certain embodiments, recombinant AAV virions comprising the variant AAV capsid proteins described herein, when delivered via intravitreal injection, can deliver a gene product (such as a therapeutic gene product) to the retina, e.g., where the resulting expression is at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, or at least 100-fold higher than the corresponding recombinant AAV virion comprising the corresponding parent AAV capsid protein. In certain embodiments, recombinant AAV virions comprising the variant AAV capsid proteins described herein, when delivered via intravitreal injection, can deliver a gene product to the retina, e.g., where the resulting expression is at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, or at least 100-fold higher than the AAV5 and / or AAV2.5T vectors.

[0127] Numerous methods are known in the art for the production of recombinant AAV virions, 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. rAAV production cultures for the production of rAAV viral particles require 1) a suitable host cell, 2) suitable helper virus functions, 3) AAV rep and cap genes, 4) at least one AAV ITR sequence (e.g., an oversized rAAV vector genome) and flanking nucleic acid (such as a sequence encoding a gene product), and 5) appropriate media and media components that support rAAV production. In some embodiments, the suitable host cell is a primate host cell. In some embodiments, the suitable host cell is a human-derived cell line, such as HeLa, A549, 293, or Perc.6 cells.

[0128] In some embodiments, suitable helper functions are provided by wild-type or mutant adenovirus (such as temperature-sensitive adenovirus), herpesvirus (HSV), baculovirus, or a plasmid construct that provides helper functions. In some embodiments, AAV helper functions are provided by adenovirus or HSV. In some embodiments, AAV helper functions are provided by baculovirus, and the host cell is an insect cell (e.g., Spodoptera frugiperda (Sf9) cell).

[0129] In some embodiments, the AAV virions described herein are produced in insect cells. In some embodiments, the AAV virions described herein are produced in Spodoptera frugiperda (Sf9) cells. In some embodiments, the AAV virions described herein are produced in human-derived cell lines. In some embodiments, the AAV virions described herein are produced in HeLa, A549, 293 or Perc.6 cells. In some embodiments, the AAV virions described herein are produced in HEK-293 cells.

[0130] In some embodiments, the AAV rep gene can be from any AAV serotype. Generally, but not necessarily, the AAV rep gene is of the same serotype as the ITRs of the rAAV vector genome, so long as the rep protein encoded by the rep gene can function to replicate and package the rAAV genome.

[0131] Suitable media known in the art can be used for the production of rAAV vectors. These media include, but are not limited to, media made by Hyclone Laboratories and JRH, including modified Eagle's medium (MEM), Dulbecco's modified Eagle's medium (DMEM), custom formulations such as those described in U.S. Patent No. 6,566,118, each of which is incorporated herein by reference in its entirety, particularly with respect to custom media formulations for use in the production of recombinant AAV vectors, and Sf-900 II SFM medium described in U.S. Patent No. 6,723,551.

[0132] One method for producing rAAV virions is the triple transfection method.In brief, a plasmid containing a capsid gene, such as a rep gene and a polynucleotide encoding a variant AAV capsid protein disclosed herein, can be transfected (e.g., using calcium phosphate method) into a cell line (e.g., HEK-293 cells) together with a helper adenovirus plasmid, and virions can be collected and purified as necessary.Thus, in some embodiments, rAAV virions are produced by triple transfection of a vector genome, a nucleic acid encoding AAV rep and cap, such as a polynucleotide encoding a variant capsid protein disclosed herein, and a nucleic acid encoding AAV helper function into a host cell, where transfection of the nucleic acid into the host cell produces a host cell capable of producing rAAV virions.

[0133] In some embodiments, rAAV virions can be produced by a producer cell line method, such as the exemplary producer cell line method described in Martin et al., (2013) Human Gene Therapy Methods 24:253-269; US Patent Application Publication No. 2004 / 0224411; and Liu, XL et al. (1999) Gene Ther. 6:293-299. Briefly, a cell line (e.g., HeLa, 293, A549 or Perc.6 cell line) can be stably transfected with a plasmid containing a rep gene, a capsid gene, such as a polynucleotide encoding a variant capsid protein disclosed herein, and a vector genome. 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 a helper virus (e.g., adenovirus or HSV) to initiate rAAV production. The virions may then be harvested, the adenovirus may be inactivated (e.g., by heat) and / or removed, and the rAAV virions may be purified. Thus, in some embodiments, the rAAV virions have been produced by a producer cell line that includes one or more of a nucleic acid encoding a rAAV genome, a nucleic acid encoding AAV rep, a polynucleotide encoding a variant capsid protein disclosed herein, and a nucleic acid encoding AAV helper functions.

[0134] In some embodiments, the nucleic acid encoding the AAV rep gene, the AAV cap gene and / or the rAAV genome is stably maintained in the production cell line. In some embodiments, the nucleic acid encoding the AAV rep gene, the AAV cap gene and / or the rAAV genome is introduced into the cell line in one or more plasmids to generate the production cell line. In some embodiments, the AAV rep gene, the AAV cap gene and the rAAV genome are introduced into the cell in the same plasmid. In other embodiments, the AAV rep, the AAV cap and the rAAV genome are introduced into the cell in two or more different plasmids. In some embodiments, the cell line stably transfected with the plasmid maintains the plasmid over multiple cell line passages (e.g., 5, 10, 20, 30, 40, 50 or more cell line passages). For example, the plasmid may be replicated as a cell replicator or the plasmid may be integrated into the cell genome. A variety of sequences have been identified that allow plasmids to replicate autonomously in cells (e.g., human cells) (see, e.g., Krysan, PJ et al. (1989) Mol. Cell Biol. 9:1026-1033). In some embodiments, the plasmid may contain a selection marker (e.g., an antibiotic resistance marker) that allows for the selection of cells that maintain the plasmid. Commonly used selection markers in mammalian cells include, but are not limited to, blasticidin, G418, hygromycin B, zeocin, puromycin, and derivatives thereof.Methods for introducing nucleic acids into cells are known in the art and include, but are not limited to, viral transduction, cationic transfection (e.g., using cationic polymers such as DEAE-dextran, or cationic lipids such as lipofectamine), calcium phosphate transfection, microinjection, biolistic bombardment, electroporation, and nanoparticle transfection (for more details, see, e.g., Kim, TK and Eberwine, JH (2010) Anal. Bioanal. Chem. 397:3173-3178).

[0135] In some embodiments, the nucleic acid encoding the AAV rep gene, the AAV cap gene and / or the rAAV genome is stably integrated into the genome of the production cell line. In some embodiments, the nucleic acid encoding the AAV rep gene, the AAV cap gene and / or the rAAV genome is introduced into the cell line in one or more plasmids to generate the production cell line. In some embodiments, the AAV rep gene, the AAV cap gene and the rAAV genome are introduced into the cell on the same plasmid. In other embodiments, the AAV rep gene, the AAV cap gene and the rAAV genome are introduced into the cell on two or more different plasmids. In some embodiments, the plasmid may contain a selection marker (e.g., an antibiotic resistance marker) that allows for the selection of cells that maintain the plasmid. Methods for stable integration of nucleic acids into various host cell systems are known. For example, repeated selection (e.g., by using a selection marker) may be used to select for cells that have integrated the nucleic acid containing the selection marker (and the AAV cap gene, the AAV rep gene and / or the rAAV genome). In other embodiments, the nucleic acid can be integrated into a cell line in a site-specific manner to generate a production cell line. Several site-specific recombination systems are known in the art, such as FLP / FRT (see, e.g., O'Gorman, S. et al. (1991) Science 251:1351-1355), Cre / loxP (see, e.g., Sauer, B. and Henderson, N. (1988) Proc. Natl. Acad. Sci. 85:5166-5170), and phi C31-att (see, e.g., Groth, A. C. et al. (2000) Proc. Natl. Acad. Sci. 97:5995-6000).

[0136] In some embodiments, the producer cell line is derived from a primate cell line (e.g., a non-human primate cell line such as a Vero or FRhL-2 cell line). In some embodiments, the cell line is derived from a human cell line. In some embodiments, the producer cell line is derived from a HeLa, 293, A549, or PERC.6® (Crucell) cell. For example, the cell line is a HeLa, 293, A549, or PERC.6® (Crucell) cell line, or a derivative thereof, prior to the introduction and / or stable maintenance / integration of nucleic acids encoding the AAV rep gene, AAV cap gene, and / or rAAV genome into the cell line to generate the producer cell line.

[0137] In some embodiments, the production cell line is adapted to grow in suspension. As is known, anchorage-dependent cells typically cannot grow in suspension without a substrate such as microcarrier beads. Adapting a cell line to grow in suspension can include, for example, growing the cell line in agitated culture with agitation paddles, using culture medium lacking calcium and magnesium ions (and antifoaming agent if necessary) to prevent clumping, using culture vessels coated with siliconizing compounds, and selecting cells in the culture (not in large clumps or on the sides of the vessel) at each passage. For further explanation, see, for example, the ATCC Frequently Asked Questions document (available at www[dot]atcc[dot]org / Global / FAQs / 9 / 1 / Adapting%20a%20monolayer%20cell%20line%20to%20suspension-40[dot]aspx) and the references cited therein.

[0138] In some aspects, a method for producing rAAV virions disclosed herein is provided, comprising: (a) culturing a host cell under conditions in which rAAV virions are produced, the host cell comprising: (i) a polynucleotide encoding a variant AAV capsid protein disclosed herein; (ii) a polynucleotide encoding a rep protein; (iii) a polynucleotide cassette comprising a sequence encoding a gene product, such as a therapeutic gene product, flanked by at least one AAV ITR; and (iv) AAV helper functions; and (b) recovering the rAAV virions produced by the host cell. In some embodiments, the at least one AAV ITR is selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, avian AAV, bovine AAV, canine AAV, equine AAV, primate AAV, non-primate AAV, or ovine AAV serotype ITRs, etc. In some embodiments, the method further includes purifying the rAAV virions.

[0139] Suitable rAAV production culture media of the invention may be supplemented with serum or serum-derived recombinant proteins at levels of 0.5% to 20% (v / v or w / v). Alternatively, as known in the art, rAAV virions may be produced in serum-free conditions, which may also be referred to as animal-derived product-free media. One of skill in the art will appreciate that commercially available or custom media designed to support the production of rAAV virions may also be supplemented with one or more cell culture components known in the art, including, but not limited to, glucose, vitamins, amino acids and / or growth factors, to increase the titer of rAAV in the production culture.

[0140] rAAV production cultures can be grown under a variety of conditions (such as over a wide range of temperatures and for various lengths of time) appropriate for the particular host cell used. As is known in the art, rAAV production cultures include attachment-dependent cultures that can be cultured in suitable attachment-dependent vessels, such as roller bottles, hollow fiber filters, microcarriers, and packed bed or fluidized bed bioreactors. rAAV production cultures can also include suspension-adapted host cells, such as HeLa, 293, and SF-9 cells, that can be cultured in a variety of ways, including spinner flasks, stirred tank bioreactors, and disposable systems such as the Wave bag system.

[0141] V. Composition Also provided herein is a composition comprising a recombinant AAV virion disclosed herein that comprises a variant AAV capsid protein described herein. In some embodiments, the composition is a pharmaceutical composition comprising a recombinant AAV virion disclosed herein and one or more pharma- ceutically acceptable diluents, carriers, or excipients.

[0142] Recombinant AAV virions can be combined with pharma- ceutically acceptable carriers, diluents and reagents useful in the preparation of formulations, including excipients that are generally safe, non-toxic, desirable, and acceptable for use in primates. Such excipients can be solid, liquid, semi-solid, or, in the case of aerosol compositions, gaseous. Examples of such carriers or diluents include, but are not limited to, water, saline, Ringer's solution, dextrose solution, and 5% human serum albumin. Additional active compounds can also be incorporated into the formulation. Solutions or suspensions used for formulation can include sterile diluents such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerin, propylene glycol, or other synthetic solvents; antibacterial compounds such as benzyl alcohol or methylparabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating compounds such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetate buffer, citrate buffer, or phosphate buffer; detergents such as Tween® 20 to prevent aggregation; and compounds for adjusting tonicity such as sodium chloride or dextrose. The pH can be adjusted using an acid or base, such as hydrochloric acid or sodium hydroxide. In certain embodiments, the pharmaceutical composition is sterile. In examples where ocular cells are contacted in vivo, the recombinant AAV virion can be processed as necessary for delivery to the eye.

[0143] The pharmaceutical compositions may further include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include, but are not limited to, physiological saline, bacteriostatic water, or phosphate buffered saline (PBS). In some cases, the composition must be sterile and fluid to the extent that easy syringability exists. In certain embodiments, the pharmaceutical compositions are stable under the conditions of manufacture and storage and are preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier may be, for example, a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The appropriate fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In some embodiments, the pharmaceutical composition includes one or more isotonic agents, such as sugars, polyalcohols such as mannitol or sorbitol, and sodium chloride. In some embodiments, the pharmaceutical composition includes agents that delay absorption, such as aluminum monostearate and gelatin.

[0144] Sterile solution can be prepared by incorporating recombinant AAV virion in the required amount in suitable solvent with one or combination of the components listed above, and then optionally performing sterilization by filtration.Generally, dispersion is prepared by incorporating active compound into a sterile vehicle that contains a basic dispersion medium and other necessary components from those listed above.In the case of sterile powder for preparing sterile injectable solution, the method of preparation includes, but is not limited to, vacuum drying and freeze-drying, which produces a powder of active ingredient and any additional desired components from its solution that has been previously sterile-filtered.

[0145] In some embodiments, pharmaceutical compositions, such as controlled release formulations, including implants and microencapsulated delivery systems, are prepared with carriers that protect viral vectors from rapid excretion from the body.Biodegradable biocompatible polymers, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters and polylactic acid, can be used.Liposomal suspensions, including liposomes targeted to infected cells with monoclonal antibodies against viral antigens, can also be used as pharma- ceutically acceptable carriers, as described, for example, in U.S. Patent No. 4,522,811.

[0146] For ease of administration and uniformity of dosage, it may be advantageous to formulate oral, ophthalmic or parenteral compositions in dosage unit form.As used herein, dosage unit form may refer to physically discrete units suitable as unitary dosages for the subjects to be treated, each unit containing a predetermined amount of active compound calculated to produce the desired therapeutic effect.

[0147] Recombinant AAV virions can be prepared at any concentration suitable for efficient transduction of mammalian cells. For example, recombinant AAV virions can be prepared at approximately 10 per mL. 8 Vector genome or higher concentration, e.g., about 5 x 10 per mL 8 Vector genome, approximately 10 per mL 9 Vector genome, approximately 5 × 10 per mL 9 Vector genome, approximately 10 per mL 10 Vector genome, approximately 5 × 10 per mL 10 Vector genome, approximately 10 per mL 11 Vector genome, approximately 5 × 10 per mL 11 Vector genome, approximately 10 per mL 12 Vector genome, approximately 5 × 10 per mL 12 Vector genome, approximately 10 per mL 13 Vector genome, approximately 1.5 × 10 per mL 13 Vector genome, approximately 3 × 10 per mL 13Vector genome, approximately 5 × 10 per mL 13 Vector genome, approximately 7.5 × 10 per mL 13 Vector genome, approximately 9 × 10 per mL 13 Vector genome, approximately 1 × 10 per mL 14 Approximately 5 × 10 vector genomes per mL 14 The vector genome may be formulated at a concentration of about 1×10 per mL, or higher. In some embodiments, the viral particles are at a concentration of about 1×10 per mL. 15 It may be formulated at a concentration not exceeding the vector genome.

[0148] Recombinant AAV virions may include, but are not limited to, about 1×10 8 Vector genome or higher, e.g., about 1 x 10 9 , about 1×10 10 , about 1×10 11 , about 1×10 12 , or about 1×10 13 Vector genome or higher, or in certain instances, approximately 1 x 10 14 The vector genome may be formulated into any suitable unit dosage. In some embodiments, the recombinant AAV virion is about 4×10 15 The vector may be formulated in any suitable unit dosage not exceeding the vector genome. In some cases, the unit dosage is up to about 5×10 15 Vector genome, e.g., about 1 x 10 14 Vector genome or less, e.g., about 1×10 13 , about 1×10 12 , about 1×10 11 , about 1×10 10 , or approximately 1 × 10 9 Vector genome or less, or in certain instances, about 1×10 8 In some embodiments, the recombinant AAV virion is about 1×10 vector genome or less. 8 The vector genome may be formulated into any suitable unit dosage amount of about 1×10 or more. In some cases, the unit dosage amount is about 1×10 10 ~Approx. 1×10 11In some cases, the unit dosage is about 1×10 10 ~Approx. 3×10 12 In some cases, the unit dosage is about 1×10 9 ~Approx. 3×10 13 In some cases, the unit dosage is about 1×10 8 ~Approx. 3×10 14 The vector genome.

[0149] In some cases, the unit dosage of the pharmaceutical composition may be measured using the multiplicity of infection (MOI). MOI refers to the ratio or multiple of the number of cells to which the nucleic acid of the viral genome can be delivered. In some cases, the MOI is about 1×10 6 In some cases, the MOI can be about 1×10 5 ~Approx. 1×10 7 In some cases, the MOI can be about 1×10 4 ~Approx. 1×10 8 In some cases, the recombinant virions of the present disclosure may be at least about 1 x 10 1 , 1×10 2 , 1×10 3 , 1×10 4 , 1×10 5 , 1×10 6 , 1×10 7 , 1×10 8 , 1×10 9 , 1×10 10 , 1×10 11 , 1×10 12 , 1×10 13 , 1×10 14 , 1×10 15 , 1×10 16 , 1×10 17 and 1 × 10 18 In some cases, the recombinant AAV virions of the present disclosure are at an MOI of about 1 x 10 8 ~3×10 14 In some cases, the recombinant AAV virions of the present disclosure are at an MOI of up to about 1 x 10 1 , 1×10 2 , 1×10 3 , 1×104 , 1×10 5 , 1×10 6 , 1×10 7 , 1×10 8 , 1×10 9 , 1×10 10 , 1×10 11 , 1×10 12 , 1×10 13 , 1×10 14 , 1×10 15 , 1×10 16 , 1×10 17 and 1 × 10 18 The MOI is either

[0150] In some embodiments, the amount of the pharmaceutical composition is about 1×10 8 ~Approx. 1×10 15 Recombinant AAV virions, approximately 1 x 10 9 ~Approx. 1×10 14 Recombinant AAV virions, approximately 1 x 10 10 ~Approx. 1×10 13 of recombinant AAV virions, or approximately 1 x 10 11 ~Approx. 3×10 12 The recombinant AAV virion comprises

[0151] The pharmaceutical compositions can be included in a container, pack, or dispenser, such as a syringe, eg, a prefilled syringe, together with instructions for administration.

[0152] In some embodiments, the pharmaceutical composition comprises a pharma- ceutically acceptable salt.Various pharma- ceutically acceptable salts are known in the art and are described, for example, in "Remington's Pharmaceutical Sciences", 17th edition,Alfonso R. Gennaro (Ed.), Mark Publishing Company, Easton, PA, USA, 1985 (and its more recent editions), "Encyclopaedia of Pharmaceutical Technology", 3rd edition, James Swarbrick (Ed.), InformaHealthcare USA (Inc.), NY, USA, 2007, and J. Pharm. Sci. 66: 2 (1977).Also, for a general description of suitable salts, see Handbook of Pharmaceutical Salts: Properties, Selection, and Use by Stahl and Wermuth (Wiley-VCH, 2002).

[0153] Pharmaceutically acceptable base addition salts are formed with metals or amines, such as alkali and alkaline earth metals or organic amines. Metals used as cations include sodium, potassium, magnesium, calcium, and the like. Amines include N-N'-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, dicyclohexylamine, ethylenediamine, N-methylglucamine, and procaine (see, for example, Berge et al., "Pharmaceutical Salts," J. Pharma Sci., 1977, 66, 119). The base addition salts of the acidic compounds are prepared by contacting the free acid form with a sufficient amount of the desired base to produce a salt in a conventional manner. The free acid form can be regenerated by contacting the salt form with an acid and isolating the free acid in a conventional manner. The free acid forms differ somewhat from their respective salt forms in certain physical properties, such as solubility in polar solvents, but in other respects the salts are equivalent to their respective free acids for the purposes of this invention.

[0154] The pharmaceutical composition can be formulated for administration to a mammal, such as a primate, such as a human. In some embodiments, the pharmaceutical composition comprises an aqueous carrier, such as a non-toxic, inert, pharma- ceutically acceptable aqueous carrier. In some embodiments, the pH of the aqueous carrier is about 3 to about 8. In some embodiments, the pH of the aqueous carrier is about 6 to about 8.

[0155] In some embodiments, the pharmaceutical compositions provided herein comprise a therapeutically effective amount of the recombinant AAV virions disclosed herein in a mixture with a pharma- ceutically acceptable carrier and / or excipient, such as saline, phosphate buffered saline, phosphate, and amino acids, polymers, polyols, sugars, buffers, preservatives, and other proteins. Exemplary amino acids, polymers, sugars, and the like are octylphenoxypolyethoxyethanol compounds, polyethylene glycol monostearate compounds, polyoxyethylene sorbitan fatty acid esters, sucrose, fructose, dextrose, maltose, glucose, mannitol, dextran, sorbitol, inositol, galactitol, xylitol, lactose, trehalose, bovine or human serum albumin, citrate, acetate, Ringer's solution and Hank's solution, cysteine, arginine, carnitine, alanine, glycine, lysine, valine, leucine, polyvinylpyrrolidone, polyethylene, and glycol. In some embodiments, the pharmaceutical compositions are stable at about 4° C. for at least 6 months.

[0156] In some embodiments, the pharmaceutical compositions provided herein comprise a buffer such as phosphate buffered saline (PBS) or sodium phosphate / sodium sulfate, Tris buffer, glycine buffer, sterile water, and other buffers known to those of skill in the art such as those described in Good et al. (1966) Biochemistry 5:467.

[0157] Also provided herein is a composition comprising a recombinant AAV virion disclosed herein that comprises a variant AAV capsid protein described herein for use in the methods described herein. In some embodiments, the composition is a pharmaceutical composition comprising a recombinant AAV virion disclosed herein and one or more pharma- ceutically acceptable diluents, carriers, or excipients.

[0158] Also provided herein are compositions comprising the polynucleotides described herein that encode the variant AAV capsid proteins described herein. In some embodiments, the compositions are for use in the methods disclosed herein, such as the methods of producing recombinant AAV virions comprising the variant AAV capsid proteins.

[0159] VI. Methods and Uses As disclosed in the accompanying Examples, the modified capsid proteins described herein can confer enhanced or altered cell tropism to recombinant AAV virions comprising the variant AAV capsid protein. For example, certain variant AAV capsid proteins described herein are associated with increased infectivity of the retina or increased expression levels of a gene product in the retina, increased binding to the ILM, increased infectivity of retinal cells following intravitreal injection, and / or increased expression of a gene product in the retina following intravitreal injection.

[0160] Examples of retinal cellular components that can be transfected by the virions described herein include, but are not limited to, astrocytes, Muller cells, RGCs, RGC axons, photoreceptor cells, bipolar cells, amacrine cells, horizontal cells, and combinations thereof. In some examples, cellular markers are used to measure the transduction of nucleic acids encoding gene products into retinal cellular components. Cell markers include, but are not limited to, GFAP, vimentin, Fox, β-tubulin, rhodopsin, PKCa, parvalbumin, calbindin, and combinations thereof. Table 1 below shows exemplary cellular markers and corresponding retinal cellular components. [Table 1]

[0161] In some examples, the ability of rAAV virions to transduce gene products can be measured by the expression of receptor proteins, which in some embodiments can include GFP, YFP, Red Cherry, β-galactosidase, and other receptor proteins known in the art.

[0162] In some cases, the expression of receptor protein over time can be determined by capturing images of the eye at various time points. For example, the expression of GFP can be correlated to an increase in fluorescence. The amount of fluorescence at a given time point can be used to quantify the expression of the gene product.

[0163] In some embodiments, immunofluorescence can be used to determine the localization of the transduced gene in the retinal cellular components described herein. The retinal cellular components can be labeled by using an antibody specific for a cellular marker disclosed herein. In some examples, the antibody is coupled to a fluorophore. In some examples, a secondary antibody can be used to detect the binding of the first antibody. The transduced retina can be cryosectioned prior to incubation with an antibody against the cellular marker of interest, thereby allowing imaging of the bound antibody. The cryosections can then be labeled using an antibody to detect the transduction of the transgene, the presence of the cellular marker of interest, and the overlapping areas between the two.

[0164] Accordingly, provided herein is a method of enhancing or altering the tropism of a recombinant AAV virion, the method comprising incorporating a variant AAV capsid protein disclosed herein into the recombinant AAV virion. Also provided herein is a method of enhancing or altering the tropism of a recombinant AAV virion, the method comprising replacing amino acid residues 570-579 of a parent AAV capsid with an amino acid sequence comprising HKFKSGD (SEQ ID NO: 1) to generate a variant AAV capsid protein, and incorporating the variant AAV capsid protein into a recombinant AAV virion, the numbering of the amino acid residues corresponding to the AAV5 VP1 capsid protein. In some embodiments, the method comprises replacing amino acid residues 570-579 with an amino acid sequence comprising X1X2HKFKSGDX3 (SEQ ID NO: 2), the numbering of the amino acid residues corresponding to the AAV5 VP1 capsid protein. 1~3 may independently be any amino acid. 1~3 are each independently selected from A, L, G, S and T. In some embodiments, X1 is L. In some embodiments, X2 is A. In some embodiments, X3 is A. In some embodiments, the method comprises replacing amino acid residues 570-579 with an amino acid sequence comprising LAHKFKSGDA (SEQ ID NO:3), an amino acid sequence having at least 80% or at least 90% homology to SEQ ID NO:3, an amino acid sequence having at least 80% or at least 90% sequence identity to SEQ ID NO:3, or an amino acid sequence having 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, or 9 or more consecutive amino acids within SEQ ID NO:3. In some embodiments, the amino acid sequence comprises LAHKFKSGDA (SEQ ID NO:3).

[0165] In some embodiments, the parent AAV capsid protein is a wild-type AAV capsid protein, such as AAV type 1 (AAV1), AAV type 2 (AAV2), AAV type 3 (AAV3), AAV type 4 (AAV4), AAV type 5 (AAV5), AAV type 6 (AAV6), AAV type 7 (AAV7), AAV type 8 (AAV8), AAV type 9 (AAV9), AAV type 10 (AAV10), avian AAV, bovine AAV, canine AAV, equine AAV, primate AAV, non-primate AAV, or ovine AAV capsid protein. In some embodiments, the parent AAV capsid protein is an AAV5 capsid protein. In some embodiments, the parent AAV capsid protein is a VP1, VP2, or VP3 capsid protein. In some embodiments, the parent AAV capsid protein is an AAV5 VP1 capsid protein.

[0166] In some embodiments, the parent AAV capsid protein is a variant AAV capsid protein. In some embodiments, the parent AAV capsid protein is a variant of the capsid protein of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, avian AAV, bovine AAV, canine AAV, equine AAV, primate AAV, non-primate AAV, or ovine AAV. In some embodiments, the parent AAV capsid protein is a variant of the AAV5 VP1 capsid protein. In some embodiments, the parent AAV capsid protein is a variant of the AAV5 VP1 capsid protein that has at least 90%, at least 95%, at least 98%, or at least 99% homology with SEQ ID NO:4. In some embodiments, the parent AAV capsid protein is a variant of AAV5 VP1 capsid protein with at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with SEQ ID NO:4. In some embodiments, the parent AAV capsid protein is a hybrid. In some embodiments, the parent AAV capsid protein is a hybrid of AAV2 and AAV5. In some embodiments, the parent AAV capsid protein is AAV2.5T or a variant thereof with at least 90%, at least 95%, at least 98%, or at least 99% homology with AAV2.5T capsid protein. In some embodiments, the parent AAV capsid protein is AAV2.5T or a variant thereof with at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with AAV2.5T capsid protein. In some embodiments, the parent AAV capsid protein is AAV2.5T. In some embodiments, the parent AAV capsid protein is a VP1, VP2 or VP3 capsid protein.In some embodiments, the parent AAV capsid protein is an AAV2.5T VP1 capsid protein or a variant thereof having at least 90%, at least 95%, at least 98%, or at least 99% homology to the AAV2.5T VP1 capsid protein (SEQ ID NO: 5). In some embodiments, the parent AAV capsid protein is an AAV2.5T VP1 capsid protein or a variant thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to the AAV2.5T VP1 capsid protein (SEQ ID NO: 5).

[0167] The recombinant AAV virions described herein, including the variant AAV capsid proteins described herein, may be used in the delivery of gene products to cells, e.g., cells of an animal. For example, they may be used in research, e.g., to determine the effect that the gene products have on cell viability and / or function. As another example, they may be used in medicine, e.g., to treat disorders, e.g., by delivering therapeutic gene products to cells or tissues. Thus, in some embodiments, a method of expressing a gene product in a cell is provided herein, comprising contacting the cell with a composition comprising a recombinant AAV virion comprising a polynucleotide cassette comprising a sequence encoding the variant AAV capsid protein and gene product disclosed herein. In some embodiments, the contacting occurs in vitro. In some embodiments, the contacting occurs in vivo, i.e., the composition is administered to a subject. In certain embodiments, the composition is administered parenterally, e.g., intravenously, orally, or by injection. In certain embodiments, the composition is administered to the eye by injection, e.g., into the retina, subretina, or vitreous. In certain embodiments, the composition is administered by retinal, subretinal, or intravitreal injection. In certain embodiments, the composition is administered by intravitreal injection.

[0168] In certain embodiments in which mammalian cells are contacted in vitro with recombinant AAV virions comprising the variant AAV capsid proteins disclosed herein, the cells can be from any mammalian species, e.g., rodent (e.g., mouse, rat, gerbil, squirrel), rabbit, cat, dog, goat, sheep, pig, horse, cow, primate, human. The cells can be from an established cell line, e.g., WERI cells, 661W cells, or the cells can be primary cells.

[0169] In certain embodiments, to promote expression of the gene product, the cells are contacted with the recombinant AAV virion for about 30 minutes to about 24 hours or more, such as any one of about 1 hour, 1.5 hours, 2 hours, 2.5 hours, 3 hours, 3.5 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 12 hours, 16 hours, 18 hours, 20 hours, or 24 hours. The cells may be contacted with the recombinant AAV virion one or more times, such as one, two, three, or more than three times, and the cells are allowed to incubate with the recombinant AAV virion for some time after each contact event, such as about 16 to 24 hours. Contact with the cells may occur in any culture medium and under any culture conditions that promote cell survival. For example, the cells may be suspended in any convenient and suitable nutrient medium, such as Iscove's modified DMEM or RPMI 1640, supplemented with fetal bovine serum or heat-inactivated goat serum (about 5-10%), L-glutamine, thiols, particularly 2-mercaptoethanol, and antibiotics, such as penicillin and streptomycin. The culture may contain growth factors to which the cells are responsive. Growth factors, as defined herein, are molecules that can promote the survival, growth and / or differentiation of cells, either in culture or in intact tissues, by specific effects on transmembrane receptors. Growth factors include polypeptides, and non-polypeptide factors.

[0170] In certain embodiments, an effective amount of recombinant AAV virions comprising variant AAV capsid proteins is provided to cause expression of gene products in cells. In certain embodiments, the effective amount may be determined empirically, for example, by detecting the presence or level of gene products, by detecting effects on cell viability or function, etc. In certain embodiments, an effective amount of recombinant AAV virions promotes equal or higher expression of gene products in cells than the same amount of a reference viral vector, such as a recombinant AAV virion comprising parent AAV capsid proteins. In certain embodiments, expression is enhanced by about 2-fold or higher, for example, about 3-fold, 4-fold, or 5-fold or higher, and in some cases, about 10-fold, 20-fold, or 50-fold or higher, for example, 100-fold, compared to expression from a reference viral vector. In certain embodiments, enhanced expression occurs in a particular cell type, for example, any of the ocular cells described herein.

[0171] For the example of contacting a cell in vivo with a composition comprising a recombinant AAV virion comprising a polynucleotide cassette comprising a sequence encoding a variant AAV capsid protein and gene product disclosed herein, the subject may be any mammal, for example, a rodent (e.g., mouse, rat, gerbil), rabbit, cat, dog, goat, sheep, pig, horse, cow or primate (e.g., human or non-human primate). The disclosed methods and compositions find use in treating any condition that can be addressed, at least in part, by gene therapy of cells. Thus, the disclosed compositions and methods find use in treating individuals in need of cell therapy. Cells include, but are not limited to, blood, eye, liver, kidney, heart, muscle, stomach, intestine, pancreas and skin.

[0172] Also provided herein is a method for delivering gene products to the retina, comprising administering a recombinant AAV virion comprising a variant AAV capsid protein disclosed herein and a polynucleotide cassette comprising a sequence encoding a gene product by intravitreal injection. In some embodiments, provided herein is a method for delivering gene products through the ILM, comprising administering a recombinant AAV virion comprising a variant AAV capsid protein disclosed herein and a polynucleotide cassette comprising a sequence encoding a gene product by intravitreal injection. In certain embodiments, the present disclosure provides a method for providing gene products to the retina of a subject, comprising administering to the subject a pharmaceutical composition comprising a recombinant AAV virion as described herein by intravitreal injection, wherein the recombinant AAV virion comprises a variant AAV capsid as provided or disclosed herein and a polynucleotide sequence encoding a gene product.

[0173] In certain embodiments, the subject is a patient suffering from acute macular neuroretinopathy; Behcet's disease; choroidal neovascularization; diabetic uveitis; histoplasmosis; macular degeneration, such as acute macular degeneration, nonexudative age-related macular degeneration, and exudative age-related macular degeneration; edema, such as macular edema, cystoid macular edema, and diabetic macular edema; multifocal choroiditis; ocular trauma affecting the posterior ocular site or area; ocular tumors; retinal disorders, such as central retinal vein occlusion, diabetic retinopathy (including proliferative diabetic retinopathy), proliferative vitreous retinopathy (PVR), retinal artery occlusive disease, retinal detachment, uveal retinal disease; sympathetic ophthalmia; Vogt-Koyanagi-Harada (VKH) syndrome; uveal diffusion; posterior ocular conditions caused or affected by ocular laser procedures; photodynamic therapy. or have been diagnosed with or are suspected of having one or more diseases or disorders selected from the group consisting of: posterior ocular conditions caused or affected by retinal hypercalcaemia; photocoagulation, radiation retinopathy; epiretinal membrane disorder; branch retinal vein occlusion; anterior ischemic optic neuropathy; diabetic retinal ischemia; ischemic retinopathy, non-retinopathy; diabetic retinal dysfunction; retinoschisis; retinitis pigmentosa; glaucoma; Usher syndrome, cone-rod dystrophy; Stargardt disease (fundus flammeus); hereditary macular degeneration; chorioretinal degeneration; Leber's congenital amaurosis; congenital stationary night blindness; total choroidal atrophy; Bardet-Biedl syndrome, macular telangiectasia; Leber's hereditary optic neuropathy; retinopathy of prematurity; and disorders of color vision including total achromatopsia, protanopia, deutanopsia, and tritanopia.

[0174] In certain embodiments, the subject is a patient suffering from acute macular neuroretinopathy; Behcet's disease; choroidal neovascularization; diabetic uveitis; histoplasmosis; macular degeneration, such as acute macular degeneration, nonexudative age-related macular degeneration, and exudative age-related macular degeneration; edema, such as macular edema, cystoid macular edema, and diabetic macular edema; multifocal choroiditis; ocular trauma affecting the posterior ocular site or area; ocular tumors; disorders of the retina, such as central retinal vein occlusion, diabetic retinopathy (including proliferative diabetic retinopathy), proliferative vitreous retinopathy (PVR), retinal artery occlusive disease, retinal detachment, uveal retinal disease; sympathetic ophthalmia; Vogt-Koyanagi-Harada (VKH) syndrome; uveal diffusion; posterior ocular conditions caused or affected by ocular laser procedures; The patient is at risk of developing one or more diseases or disorders selected from the group consisting of: posterior ocular conditions caused or affected by photodynamic therapy; photocoagulation, radiation retinopathy; epiretinal membrane disorder; branch retinal vein occlusion; anterior ischemic optic neuropathy; diabetic retinal ischemia; ischemic retinopathy, non-retinopathy; diabetic retinal dysfunction; retinoschisis; retinitis pigmentosa; glaucoma; Usher syndrome, cone-rod dystrophy; Stargardt disease (fundus flammeus); hereditary macular degeneration; chorioretinal degeneration; Leber's congenital amaurosis; congenital stationary night blindness; total choroidal atrophy; Bardet-Biedl syndrome, macular telangiectasia; Leber's hereditary optic neuropathy; retinopathy of prematurity; and disorders of color vision including total achromatopsia, protanopia, deutanopsia, and tritanopia.

[0175] Also provided herein is a method for treating a disease or disorder of the retina in a subject in need thereof, comprising administering a recombinant AAV virion comprising a variant AAV capsid protein disclosed herein and a polynucleotide cassette comprising a sequence encoding a therapeutic gene product by intravitreal injection. Also provided herein is a method for prophylactically treating a disease or disorder of the retina in a subject in need thereof, comprising administering a recombinant AAV virion comprising a variant AAV capsid protein disclosed herein and a polynucleotide cassette comprising a sequence encoding a therapeutic gene product by intravitreal injection. Also provided herein is a method for treating an established or diagnosed disease or disorder of the retina in a subject in need thereof, comprising administering a recombinant AAV virion comprising a variant AAV capsid protein disclosed herein and a polynucleotide cassette comprising a sequence encoding a therapeutic gene product by intravitreal injection. In some embodiments, the methods include administering a pharmaceutical composition comprising a recombinant AAV virion comprising a polynucleotide cassette comprising a sequence encoding a variant AAV capsid protein and a therapeutic gene product as disclosed herein.

[0176] In some embodiments, the disease or disorder is acute macular neuroretinopathy; Behcet's disease; choroidal neovascularization; diabetic uveitis; histoplasmosis; macular degeneration, such as acute macular degeneration, nonexudative age-related macular degeneration, and exudative age-related macular degeneration; edema, such as macular edema, cystoid macular edema, and diabetic macular edema; multifocal choroiditis; ocular trauma affecting the posterior ocular site or area; ocular tumors; disorders of the retina, such as central retinal vein occlusion, diabetic retinopathy (including proliferative diabetic retinopathy), proliferative vitreous retinopathy (PVR), retinal artery occlusive disease, retinal detachment, uveal retinal disease; sympathetic ophthalmia; Vogt-Koyanagi-Harada (VKH) syndrome; uveal diffusion; or caused by or if caused by an ocular laser procedure. The present invention is directed to a method for treating or preventing the development of a retinal dysplasia, including but not limited to, posterior ocular conditions caused or affected by photodynamic therapy, posterior ocular conditions caused or affected by photodynamic therapy, photocoagulation, radiation retinopathy, epiretinal membrane disorder, branch retinal vein occlusion, anterior ischemic optic neuropathy, diabetic retinal ischemia, ischemic retinopathy, non-retinopathy, diabetic retinal dysfunction, retinoschisis, retinitis pigmentosa, glaucoma, Usher syndrome, cone-rod dystrophy, Stargardt disease (fundus flammeus), hereditary macular degeneration, chorioretinal degeneration, Leber's congenital amaurosis, congenital stationary night blindness, total choroidal atrophy, Bardet-Biedl syndrome, macular telangiectasia, Leber's hereditary optic neuropathy, retinopathy of prematurity, and disorders of color vision including total achromatopsia, protanopia, deutanopsia, and tritanopia.

[0177] In certain embodiments, the subject is a patient suffering from acute macular neuroretinopathy; Behcet's disease; choroidal neovascularization; diabetic uveitis; histoplasmosis; macular degeneration, such as acute macular degeneration, nonexudative age-related macular degeneration, and exudative age-related macular degeneration; edema, such as macular edema, cystoid macular edema, and diabetic macular edema; multifocal choroiditis; ocular trauma affecting the posterior ocular site or area; ocular tumors; disorders of the retina, such as central retinal vein occlusion, diabetic retinopathy (including proliferative diabetic retinopathy), proliferative vitreous retinopathy (PVR), retinal artery occlusive disease, retinal detachment, uveal retinal disease; sympathetic ophthalmia; Vogt-Koyanagi-Harada (VKH) syndrome; uveal diffusion; posterior ocular conditions caused or affected by ocular laser procedures; The patient is at risk of developing one or more diseases or disorders selected from the group consisting of: posterior ocular conditions caused or affected by photodynamic therapy; photocoagulation, radiation retinopathy; epiretinal membrane disorder; branch retinal vein occlusion; anterior ischemic optic neuropathy; diabetic retinal ischemia; ischemic retinopathy, non-retinopathy; diabetic retinal dysfunction; retinoschisis; retinitis pigmentosa; glaucoma; Usher syndrome, cone-rod dystrophy; Stargardt disease (fundus flammeus); hereditary macular degeneration; chorioretinal degeneration; Leber's congenital amaurosis; congenital stationary night blindness; total choroidal atrophy; Bardet-Biedl syndrome, macular telangiectasia; Leber's hereditary optic neuropathy; retinopathy of prematurity; and disorders of color vision including total achromatopsia, protanopia, deutanopsia, and tritanopia.

[0178] In certain embodiments, the gene product inhibits angiogenesis in the retina of a subject, for example, choroidal neovascularization (CNV).Many cellular factors have been found to play an important role in regulating CNV generation, among which may include, but are not limited to, vascular endothelial growth factor (VEGF), VEGF receptor (VEGFR), platelet-derived growth factor (PDGF), hypoxia-inducible factor (HIF), angiopoietin (Ang) and other cytokines, and mitogen-activated protein kinase (MAPK).In some embodiments, the gene product is one or more inhibitors of VEGF, VEGFR, PDGF, HIF, Ang and MAPK.

[0179] In some embodiments, the gene product is a gene product or a therapeutic gene product disclosed herein. In some embodiments, the gene product is an interfering RNA, an aptamer, or a protein. In some embodiments, the therapeutic gene product is an interfering RNA, an aptamer, or a protein. In some embodiments, the gene product (or therapeutic gene product) is an siRNA, miRNA, or a protein. In some embodiments, the gene product (or therapeutic gene product) is a site-specific nuclease that provides site-specific knockdown of gene function. In some embodiments, the gene product (or therapeutic gene product) is an interfering RNA. In some embodiments, the gene product (or therapeutic gene product) is an siRNA or miRNA.

[0180] In some embodiments, the method provides a therapeutic benefit, e.g., preventing the onset of a disease or disorder, halting the progression of a disease or disorder, reversing the progression of a disease or disorder, etc. In some embodiments, the method includes detecting that a therapeutic benefit has been achieved.

[0181] In some cases, the expression of the gene product, e.g., as detected by measuring the level of the gene product, by measuring therapeutic efficacy, etc., may be observed for 2 months or less after administration, e.g., about 4, 3, or 2 weeks or less after administration, e.g., 1 week after administration of the subject composition. The expression of the gene product is also expected to persist over time. Thus, in some cases, the expression of the gene product, e.g., as detected by measuring the level of the gene product, by measuring therapeutic efficacy, etc., may be observed for about 2 months or more after administration of the subject composition, e.g., about 4, 6, 8, or 10 months or more, and in some cases, about 1 year or more, e.g., 2, 3, 4, or 5 years, and in certain cases, more than 5 years.

[0182] In certain embodiments, subjects receive about 1×10 8Vector genome or more, in some cases, approximately 1 x 10 9 , 1×10 10 , 1×10 11 , 1×10 12 Or 1×10 13 Either vector genome or more, in one particular example, approximately 1 x 10 14 A vector genome or more is administered. In some examples, the amount of vector genome delivered is up to about 1×10 15 Vector genome, e.g., 1 x 10 14 Vector genome or less, e.g., about 1 x 10 13 , 1×10 12 , 1×10 11 , 1×10 10 Or 1×10 9 Vector genome or less, in one particular example, 1 x 10 8 Vector genome, sometimes 1×10 8 In some examples, the amount of vector genome delivered is about 1×10 10 ~Approx. 1×10 11 In some examples, the amount of vector genome delivered is about 1×10 10 ~Approx. 3×10 12 In some examples, the amount of vector genome delivered is about 1×10 9 ~Approx. 3×10 13 In some examples, the amount of vector genome delivered is about 1×10 8 ~Approx. 3×10 14 The vector genome.

[0183] In some cases, the amount of the pharmaceutical composition to be administered may be measured using the multiplicity of infection (MOI). In some cases, the MOI may refer to the ratio or multiple of the nucleic acid of the vector or virus genome to the cells to which it can be delivered. In some cases, the MOI is about 1×10 6 In some cases, the MOI can be about 1×10 5 ~Approx. 1×10 7 In some cases, the MOI can be about 1×104 ~Approx. 1×10 8 In some cases, the recombinant AAV virions of the present disclosure can be at least about 1 x 10 1 , 1×10 2 , 1×10 3 , 1×10 4 , 1×10 5 , 1×10 6 , 1×10 7 , 1×10 8 , 1×10 9 , 1×10 10 , 1×10 11 , 1×10 12 , 1×10 13 , 1×10 14 , 1×10 15 , 1×10 16 , 1×10 17 or 1×10 18 In some cases, the recombinant AAV virions of the present disclosure are at an MOI of about 1 x 10 8 ~Approx. 3×10 14 In some cases, the recombinant AAV virions of the present disclosure are at an MOI of up to about 1 x 10 1 , 1×10 2 , 1×10 3 , 1×10 4 , 1×10 5 , 1×10 6 , 1×10 7 , 1×10 8 , 1×10 9 , 1×10 10 , 1×10 11 , 1×10 12 , 1×10 13 , 1×10 14 , 1×10 15 , 1×10 16 , 1×10 17 or 1×10 18 The MOI is either

[0184] In some embodiments, the amount of the pharmaceutical composition is about 1×10 8 ~Approx. 1×10 15 Particles of recombinant AAV virions, approximately 1 x 10 9 ~Approx. 1×10 14Particles of recombinant AAV virions, approximately 1 x 10 10 ~Approx. 1×10 13 particles of recombinant AAV virions, or approximately 1 x 10 11 ~Approx. 3×10 12 The particles contain recombinant AAV virions.

[0185] In some embodiments, two or more administrations (e.g., 2, 3, 4 or more administrations) may be used over variously spaced periods, e.g., daily, weekly, monthly, yearly, etc., to achieve a desired level of gene expression.

[0186] In some embodiments, recombinant AAV virions are not detectable in tear, blood, saliva, or urine samples from a human subject about 7 days, about 14 days, about 21 days, or about 30 days after administration of the pharmaceutical composition. In some embodiments, the presence or absence of recombinant AAV virions is detected by qPCR or ELBA.

[0187] In some embodiments, a subject's best corrected visual acuity (BCVA) improves by about 1, 2, 3, 4, 5 or more lines following the methods of treatment described herein.

[0188] In some aspects, a reduction in angiogenesis as assessed by fluorescein angiography (FA) follows the administering step.

[0189] In some cases, retinal thickness may be measured to examine the effect of treatment.In some cases, the central retinal thickness of a human subject does not increase by more than about 50 microns, about 100 microns, or about 250 microns within about 12 months after treatment with the pharmaceutical composition of the present disclosure.In some cases, the central retinal thickness of a human subject decreases by at least about 50 microns, about 100 microns, about 200 microns, about 250 microns, about 300 microns, about 400 microns, about 500 microns, or about 600 microns within about 3 months, about 6 months, about 9 months, or about 12 months after treatment with the pharmaceutical composition of the present disclosure.The decrease in central retinal thickness of a human subject can be measured by comparing the central retinal thickness at one or more time points to a baseline measurement obtained at or within about 1 day, 3 days, 7 days, or 10 days after administration of the pharmaceutical composition of the present disclosure.

[0190] From the foregoing it will be appreciated that, although specific embodiments of the invention have been described herein for purposes of illustration, various modifications may be made without departing from the spirit and scope of the invention. Accordingly, the invention is not limited except as by the appended claims.

[0191] VII. Exemplary Embodiments Among the embodiments provided are the following: 1. (a) a variant adeno-associated virus (AAV) capsid protein comprising a modified sequence that includes one or more amino acid substitutions within amino acid residues 570-579 relative to a parent AAV capsid protein, the modified sequence comprising HKFKSGD (SEQ ID NO:1), wherein the numbering of the amino acid residues corresponds to that of the AAV5 VP1 capsid protein; and (b) a polynucleotide sequence encoding a therapeutic gene product. Recombinant AAV virions comprising: 2. The recombinant AAV virion of embodiment 1, wherein the parent AAV capsid protein is an AAV5 capsid protein, or an AAV5 and AAV2 hybrid capsid protein. 3. The recombinant AAV virion of embodiment 1 or 2, wherein the parent AAV capsid protein is an AAV2.5T capsid protein. 4. The recombinant AAV virion of any one of embodiments 1 to 3, wherein the parent AAV capsid protein is an AAV2.5T VP1 capsid protein. 5. The recombinant AAV virion of any one of embodiments 1 to 4, wherein the modified sequence comprises LAHKFKSGDA (sequence number 3). 6. The recombinant AAV virion of any one of embodiments 1 to 5, wherein the variant AAV capsid protein comprises a capsid sequence having at least 85% homology to the amino acid sequence set forth in SEQ ID NO:4 or SEQ ID NO:5. 7. The recombinant AAV virion of any one of embodiments 1 to 6, wherein the variant AAV capsid protein comprises the capsid sequence set forth in SEQ ID NO:6 or SEQ ID NO:7. 8. The recombinant AAV virion of any one of embodiments 1 to 7, wherein the rAAV virion is a variant AAV5, or a variant AAV2 and AAV5 hybrid virion. 9. The recombinant AAV virion of any one of embodiments 1 to 8, wherein the rAAV virion is a variant AAV2.5T virion. 10. A recombinant AAV virion according to any one of embodiments 1 to 9, which is capable of transducing cells of the retina when injected intravitreally into a mammal. 11. The recombinant AAV virion of embodiment 10, which is capable of transducing one or more of photoreceptor cells, retinal ganglion cells, Muller cells, bipolar cells, amacrine cells, horizontal cells, and retinal pigment epithelial cells when injected intravitreally into a mammal. 12. A recombinant AAV virion according to embodiment 10 or 11, which is capable of transducing retinal pigment epithelial cells when injected intravitreally into a mammal. 13. The recombinant AAV virion of any one of embodiments 1 to 12, wherein the therapeutic gene product is an siRNA, miRNA or a protein. 14. The recombinant AAV virion of any one of embodiments 1 to 13, wherein the therapeutic gene product is an anti-vascular endothelial growth factor (anti-VEGF) gene product. 15. The recombinant AAV virion of any one of embodiments 1 to 13, wherein the therapeutic gene product is opsin. 16. The recombinant AAV virion of any one of embodiments 1 to 15, wherein the polynucleotide encoding a therapeutic gene product is flanked by one or more AAV ITRs. 17. The recombinant AAV virion of embodiment 16, wherein the ITR of one or more AAVs is an ITR of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, avian AAV, bovine AAV, canine AAV, equine AAV, primate AAV, non-primate AAV or ovine AAV, or a variant thereof. 18. The recombinant AAV virion of embodiment 16 or 17, wherein the one or more AAV ITRs are AAV2 ITRs or AAV5 ITRs. 19. A recombinant AAV virion according to any one of embodiments 1 to 18, having an altered cell tropism compared to AAV2.5T. 20. A pharmaceutical composition comprising a recombinant AAV virion according to any one of embodiments 1 to 19. 21. A method for producing rAAV virions, comprising: (a) culturing a host cell under conditions such that rAAV virions are produced, the host cell comprising: (i) a polynucleotide encoding a variant AAV capsid protein comprising a modified sequence that includes one or more amino acid substitutions within amino acid residues 570 to 579 relative to a parent AAV capsid protein, wherein the modified sequence comprises HKFKSGD (SEQ ID NO:1); (ii) a polynucleotide encoding a rep protein; (iii) a polynucleotide cassette comprising a sequence encoding a therapeutic gene product flanked by at least one AAV ITR; and (iv) AAV helper function and (b) recovering the rAAV virions produced by the host cell. A method comprising: 22. A method for providing a therapeutic gene product to the retina of a subject, comprising administering to the subject a recombinant AAV virion described in any one of embodiments 1 to 19, or a pharmaceutical composition described in embodiment 20, by intravitreal injection. 23. The subject is diagnosed with acute macular neuroretinopathy, Behcet's disease, choroidal neovascularization, diabetic uveitis, histoplasmosis, macular degeneration, edema, multifocal choroiditis, ocular trauma affecting the posterior ocular site or area, ocular tumor, central retinal vein occlusion, diabetic retinopathy, proliferative vitreoretinopathy (PVR), retinal artery occlusive disease, retinal detachment, uveal retinal disease, sympathetic ophthalmia, Vogt-Koyanagi-Harada (VKH) syndrome, uveal diffusion, a posterior ocular condition caused or affected by ocular laser procedures, a posterior ocular condition caused or affected by photodynamic therapy, photocoagulation, radiation 23. The method of embodiment 22, wherein the patient has been diagnosed with or is suspected of having one or more conditions selected from the group consisting of: retinal fibrosis, epiretinal membrane disorder, branch retinal vein occlusion, anterior ischemic optic neuropathy, diabetic retinal ischemia, ischemic retinopathy, non-retinopathy diabetic retinal dysfunction, retinoschisis, retinitis pigmentosa, glaucoma, Usher syndrome, cone-rod dystrophy, Stargardt disease, hereditary macular degeneration, chorioretinal degeneration, Leber congenital amaurosis, congenital stationary night blindness, total choroidal atrophy, Bardet-Biedl syndrome, macular telangiectasia, Leber hereditary optic neuropathy, retinopathy of prematurity, and disorders of color vision. 24. A method for treating a retinal disease or disorder in a subject in need thereof, comprising administering to the subject a recombinant AAV virion described in any one of embodiments 1 to 19, or a pharmaceutical composition described in embodiment 20, by intravitreal injection. 25. The disease or disorder is acute macular neuroretinopathy, Behcet's disease, choroidal neovascularization, diabetic uveitis, histoplasmosis, macular degeneration, edema, multifocal choroiditis, ocular trauma affecting the posterior ocular region or area, ocular tumor, central retinal vein occlusion, diabetic retinopathy, proliferative vitreoretinopathy (PVR), retinal artery occlusive disease, retinal detachment, uveal retinal disease, sympathetic ophthalmia, Vogt-Koyanagi-Harada (VKH) syndrome, uveal diffusion, a posterior ocular condition caused by or affected by ocular laser procedures, or caused by photodynamic therapy 25. The method of embodiment 24, wherein the ocular condition is a posterior ocular condition caused or affected by, photocoagulation, radiation retinopathy, epiretinal membrane disorder, branch retinal vein occlusion, anterior ischemic optic neuropathy, diabetic retinal ischemia, ischemic retinopathy, non-retinopathy diabetic retinal dysfunction, retinoschisis, retinitis pigmentosa, glaucoma, Usher syndrome, cone-rod dystrophy, Stargardt disease, hereditary macular degeneration, chorioretinal degeneration, Leber congenital amaurosis, congenital stationary night blindness, total choroidal atrophy, Bardet-Biedl syndrome, macular telangiectasia, Leber hereditary optic neuropathy, retinopathy of prematurity, or a disorder of color vision. 26. The recombinant AAV virion of any one of embodiments 1 to 19 for use in a method for treating a retinal disease or disorder in a subject in need thereof, the method comprising administering to the subject a pharmaceutical composition comprising the recombinant AAV virion by intravitreal injection. 27. A recombinant AAV virion according to any one of embodiments 1 to 19 for use in the preparation of a medicament for the treatment of a disease or disorder of the retina in a subject. 28. The recombinant AAV virion of embodiment 26 or 27, wherein the disease or disorder is age-related macular degeneration (AMD), wet AMD, dry AMD, retinal neovascularization, choroidal neovascularization, diabetic retinopathy, proliferative diabetic retinopathy, retinal vein occlusion, central retinal vein occlusion, branch retinal vein occlusion, diabetic macular edema, diabetic retinal ischemia, ischemic retinopathy or diabetic retinal edema. 29. A variant AAV capsid protein comprising a modified sequence that includes one or more amino acid substitutions within amino acid residues 570-579 relative to the parent AAV capsid protein, wherein the modified sequence comprises HKFKSGD (SEQ ID NO:1), and the numbering of the amino acid residues corresponds to that of the AAV5 VP1 capsid protein. 30. The variant AAV capsid protein of embodiment 29, wherein the parent AAV capsid protein is an AAV5 capsid protein, or an AAV5 and AAV2 hybrid capsid protein. 31. The variant AAV capsid protein of embodiment 29 or 30, wherein the parent AAV capsid protein is an AAV2.5T capsid protein. 32. The variant AAV capsid protein of any one of embodiments 29 to 31, wherein the parent AAV capsid protein is an AAV2.5T VP1 capsid protein. 33. The variant AAV capsid protein of any one of embodiments 29 to 32, wherein the modified AAV capsid protein comprises LAHKFKSGDA (sequence number 3) at amino acid residues 570 to 579 compared to the parent AAV capsid protein. 34. The variant AAV capsid protein of any one of embodiments 29 to 33, comprising a capsid sequence having at least 85% homology to the amino acid sequence set forth in SEQ ID NO:4 or SEQ ID NO:5. 35. The variant AAV capsid protein of any one of embodiments 29 to 34, comprising the capsid sequence set forth in SEQ ID NO:6 or SEQ ID NO:7. 36. A nucleic acid comprising a nucleic acid sequence encoding a variant AAV capsid protein according to any of embodiments 29 to 35. 37. An expression vector comprising the nucleic acid of embodiment 36, wherein the nucleic acid sequence encoding the variant AAV capsid protein is operably linked to a promoter sequence. 38. An expression vector according to embodiment 37, further comprising a nucleic acid encoding a rep protein. 39. A cell comprising an expression vector according to embodiment 37 or 38. 40. The cell of embodiment 39, further comprising a nucleic acid encoding a therapeutic gene product. 41. The cell of embodiment 40, wherein the therapeutic gene product is an siRNA, miRNA or a protein. 42. The cell of embodiment 40 or 41, wherein the therapeutic gene product is an anti-vascular endothelial growth factor (anti-VEGF) gene product. 43. The cell of embodiment 40 or 41, wherein the therapeutic agent is an opsin. 44. A cell according to any one of embodiments 40 to 43, wherein the polynucleotide encoding a therapeutic gene product is flanked by one or more AAV ITRs. 45. The cell of embodiment 44, wherein the ITR of one or more AAVs is an ITR of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, avian AAV, bovine AAV, canine AAV, equine AAV, primate AAV, non-primate AAV or ovine AAV, or a variant thereof. 46. ​​The cell of embodiment 44 or 45, wherein the one or more AAV ITRs are AAV2 ITRs or AAV5 ITRs. EXAMPLES

[0192] Example 1: Construction of an AAV2.5T mutant library AAV2.5T is a hybrid capsid that contains regions from AAV2 and AAV5, as described in U.S. Patent No. 9,441,244, the disclosure of which is incorporated in its entirety. AAV2.5T can transduce the retina when delivered subretinal, but cannot transduce when injected intravitreally. The transduction of AAV2.5T can be blocked by the inner limiting membrane (ILM), which is rich in heparan sulfate proteoglycan (HSPG).

[0193] The surface-exposed domains of AAV2.5T are identical to those of AAV5 except for a single A to T substitution at aa582 of AAV2.5T (aa581 of AAV5), which appears to increase infectivity in mammalian cells without affecting the typical sialic acid receptor binding of AAV5. AAV5 and AAV2.5T have negligible heparan sulfate binding, whereas AAV2 has a high affinity for heparan sulfate.

[0194] To develop novel AAV variants with enhanced ability to cross the ILM and to transduce retinal cells upon delivery by intravitreal injection, an AAV mutant library with loop substitutions and insertions was generated from the AAV2.5T capsid in a region similar to the HSPG-binding region of AAV2 as determined by amino acid alignment.

[0195] Two mutant libraries were generated by synthesis of DNA fragments with random seven amino acid loops. Since there are 20 possible amino acids at each of the seven amino acid positions, the theoretical diversity of these fragment libraries is approximately 1×10 9 These mutant loops were flanked by amino acids LA at the 5' end and A at the 3' end.

[0196] Using these fragment libraries, a loop insertion library (LIL) was created by inserting a 10 amino acid loop at position 577 of the AAV2.5T capsid sequence in the receptor binding region.

[0197] A second library was created by replacing sequences in the surface-exposed region of the capsid with loop variant sequences between amino acids 571 and 580 (numbering from the first amino acid of VP1), and this library was designated the Loop Replacement Library (LSL).

[0198] These AAV variant capsids were cloned into plasmids to generate individual virus packaging cell libraries (by transient transfection with phiC31 integrase at a ratio of 1:50 (plasmid:phiC31) as described in WO2017112868A1). The resulting packaging cell libraries yield mutant AAV virions composed of vector genomes containing mutant Cap genes packaged within the capsid protein encoded by their genomes, with low levels of cross-packaging. To confirm the diversity of the cell libraries, gDNA was isolated from each library and NGS was performed. The diversity was approximately 725,000 unique variants in the LIL and 558,000 unique variants in the LSL.

[0199] Example 2: Screening an AAV library in NHP eyes To examine the ability of mutant AAV capsids to cross the ILM, a library of variant AAV virions was screened in vivo for their ability to cross the inner limiting membrane (ILM) and infect retinal cells in nonhuman primate (NHP) eyes.

[0200] The LIL and LSL mutant AAV capsid libraries were incubated at 6 × 10 9 ~9×10 9vg dose into the eyes of three African green monkeys (Chlorocebus sabaeus). Four to six weeks after injection, the animals were sacrificed and retinal tissue explants from each animal were isolated. The vitreous and retina along with the subchoroidal tissue were isolated, the globe was cut along the limbus, and the anterior part of the eye was discarded. The vitreous was then removed leaving the retina attached to the eyecup. The eyecup was transferred to CO2-dependent medium. The retina and RPE / choroid were cut to prepare a retinal flat mount. The entire flat mount was placed photoreceptor cell layer down on the polycarbonate insert membrane of a 10 cm transwell plate containing 10 mL of CO2-independent medium. Once the retina was settled flat on the insert, the medium was removed to expose the retina to air.

[0201] Retinas were transduced with human Ad5 virus (diluted in Neurobasal-A medium containing no antibiotics or antimycotics) at a multiplicity of infection (MOI) of 1000 by adding Ad5 solution on top of the tissue for 2 hours. Coinfection with Ad5 virus was used to increase the expression of Cap RNA from mutant AAV virions present in retinal tissue, thereby increasing the amount of mutant capsid RNA in infected cells for subsequent detection.

[0202] After the first incubation, Neurobasal-A+ (B27 supplement, 1x GlutaMAX, 1x antibiotic-antimycotic) medium was added to the bottom of the well so that it was in contact with the bottom of the insert but not covering the tissue (approximately 10 ml), and the tissue explants were incubated at 37°C with CO2 for 2 days. After incubation, the inserts were removed from the plate and placed on a clean flat surface, and three perforations were made as shown in Figure 1A. Briefly, a 5 mm perforation of the neural retina with RPE / choroid was obtained from the macula. A second concentric perforation was then made using a 10 mm perforation to isolate the area of ​​retina and RPE / choroid surrounding the macula within the vascular arcade, referred to as the parafoveal area. The remaining peripheral retina and RPE / choroid were then collected.

[0203] RNA was then extracted from each tissue punch and converted to cDNA by RT-PCR, followed by one round of PCR. The resulting PCR products were then cloned back into plasmids and sequenced to determine the sequence of mutant AAV capsids that were able to successfully cross the ILM and infect retinal cells in the monkey eyes.

[0204] For the second and third rounds of screening, the LIL and LSL variants extracted from each individual retinal location (perforation) were mixed to create one library of variants for each location. Pools of these libraries were then used to generate cell and AAV capsid libraries, which were separately injected into African green monkey (AGM) eyes and collected using the same retinal perforation strategy as above. Additionally, in one example, the retinal pigment epithelium was isolated and variants were recovered from the tissue.

[0205] Using NGS analysis, AAV2.5T.LSV1 was identified. AAV2.5T.LSV1 is a loop-substitution variant originating from an LSL library with the following amino acid loop sequence: LAHKFKSGDA (SEQ ID NO: 3). AAV2.5T.LSV1 was the 48th most abundant variant from the macular region in screening round 2. In screening round 3, AAV2.5T.LSV1 was the second most abundant variant found in the parafoveal and the most abundant variant found in the RPE region. The relative abundance of the top variants in each screening round is shown in Figures 1B-1E.

[0206] Example 3: Production of AAV2.5T.LSV1 in HEK293 cells AAV2.5T.LSV1 was produced in HEK293 cells for further characterization using a triple transfection method. The resulting titer of AAV2.5T.LSV1 was 1×10 13 vg / ml, demonstrating that AAV2.5T.LSV1 has good packaging capacity.

[0207] Example 4: Heparin-binding affinity of AAV2.5T.LSV1 The ability of AAV2.5T.LSV1 to bind HSPG was assessed by performing a heparin binding assay using a prepacked GE heparin column. Heparin binding of AAV2.5T.LSV1 was compared to AAV2.5T and two other variants produced in the screen described above. The vector was loaded onto the column, then washed and finally eluted with increasing concentrations of NaCl (100 mM to 1 M). Load, flow-through, wash and elution fractions were collected and analyzed by dot blot using the B1 antibody.

[0208] As shown in Figure 2, AAV2.5T does not bind to heparin. In contrast, AAV2.5T.LSV1 has the ability to bind to heparin.

[0209] Example 5: Expression and tropism in porcine retinal explants Ex vivo porcine retinal explants were maintained on transwells as described in PCT / US2017 / 030636, the disclosure of which is incorporated in its entirety, and were co-infected with 2×10 10 vg or 4×10 10 The porcine retina explants were transduced with a GFP reporter gene packaged in AAV2.5T.LSV1. Live fluorescent images were captured 1 and 2 weeks after transduction. Figure 3 shows live imaging of porcine retina explants. These images suggest that AAV2.5T.LSV1 can efficiently transduce at least some cells of the porcine retina in culture.

[0210] Example 6: Expression after intravitreal administration to the retina of non-human primates In vivo studies were performed using 5 × 10 cells carrying a GFP expression cassette under the control of a ubiquitous promoter. 11vg / eye of AAV2.5T.LSV1 was intravitreally injected in African green monkeys (Chlorocebus sabaeus). Expression in the eye was monitored weekly by fundus fluorescence and Heidelberg Spectralis imaging and is shown in Figures 4A-4E. Intravitreal delivery of AAV2.5T.LSV1-GFP resulted in GFP expression within the macula and vascular arcade. The vascular arcade region of the retina has the thickest ILM, which likely blocks transduction of retinal cells by most AAVs when injected intravitreally.

[0211] Example 7: Evaluation of expression after intravitreal administration The retinal tropism of AAV2.5T.LSV1 was assessed in non-human primates following IVT injection.

[0212] To assess the tropism of AAV2.5T.LSV1 in African green monkeys (Chlorocebus sabaeus), we used 5 × 10 vectors carrying a GFP expression cassette under the control of a ubiquitous promoter. 11 vg / eye of AAV2.5T.LSV1 was injected intravitreally (IVT).

[0213] Monkeys were sacrificed 35 days after IVT injection and eyes were enucleated. Eyes were fixed in 4% paraformaldehyde for 24 hours upon enucleation and then cryoprotected overnight with 30% sucrose / Sorenson's phosphate buffer. Eyes were dissected into quadrants and the retina, including the RPE and choroid, was removed from the sclera. Retinal quadrants were flat mounted onto slides and coverslipped under 30% sucrose / Sorenson's phosphate buffer. 5× tiled scans of native GFP were acquired using a Zeiss Axio.Z1 and merged using Zeiss Zen Blue software. Live fluorescent images obtained for AAV2.5T.LSV1-GFP are shown in Figures 5A-5D.

[0214] GFP intensity profiles were obtained from 5× scans of the flat-mounted retina sections shown in Figures 5C-5D. Measurements were taken from the edge of the macula closest to the optic nerve to the ciliary body. Approximately 20,000 measurement points, spanning a total length of 20 mm, were plotted over the distance from the macula to the ciliary body (Figure 5E).

[0215] Retinal flat mounts were embedded in OCT mounting medium and frozen on isopentane. Frozen sections were collected through the fovea, mid-periphery and periphery at 10 μm thickness and slides were stained for immunofluorescence labeling (IFL). Planar images of the RPE were made by peeling the RPE from the choroid of flat-mounted retinal sections and inverting the sections so that the RPE was placed face down on the slide, which was then stained for IFL. IFL was performed after 30 min of 0.1% Triton-x permeabilization followed by 1 h of serum protein blocking with 5% normal donkey serum / 6% bovine serum albumin in Sorenson's phosphate buffer. IFL was performed overnight at 4°C using the dyes and antibodies shown in Table 2. [Table 2]

[0216] Tissue sections were imaged and the resulting images are shown in Figures 6A-25E. A summary of the cell type AAV2.5T.LSV1 virions demonstrating tropism is shown in Table 3. [Table 3]

[0217] The percentage of GFP-positive cells was quantified by manual cell counting of colocalization with cell-specific markers. The numbers of GFP-positive bipolar, cone and RPE cells in different regions of the retina are shown in Figures 26A-26C, respectively, and summarized in Table 4. The number of GFP-positive cells varied with location in the retina. [Table 4]

[0218] Example 8: Determination of Neutralizing Antibody Profiles Neutralizing antibody (nAB) profiles are determined for AAV2.5T.LSV1 and AAV2.5T viruses by in vitro assays. Briefly, serial dilutions of pooled human IgG antibodies (Gammaguard IVIG) are combined with AAV2.5T.LSV1 or AAV2.5T vectors expressing GFP and used to transduce 293T cells. GFP expression is then measured and inhibition of GFP expression is used to determine IC 50 Calculate.

[0219] Example 9: Comparison of neutralizing antibody profiles of AAV2.5T.LSV1 and AAV2 Neutralizing antibody (nAB) profiles were determined for AAV2.5T.LSV1 and AAV2 viruses by in vitro assays. Briefly, a 3-fold dilution series of pooled human IgG antibodies (Gammaguard IVIG) was prepared and combined with AAV2.5T.LSV1 or AAV2 vectors expressing GFP (AAV2.5T.LSV1-CMV-GFP or AAV2-CMV-GFP, respectively). The mixture was then used to transduce 293T cells. The cells were incubated for 3 days, after which GFP expression was measured. Inhibition of GFP expression was used to determine IC 50 The IC 50 is reported as the reciprocal of the dilution of a pooled human IgG antibody sample at which GFP expression was reduced by 50% (e.g., IC 50 (A value of 100 indicates that IVIG diluted 1:100 reduced GFP expression by 50%). 50 Values ​​indicate increased inhibition of AAV transduction by antibodies present in pooled human IgG antibodies.

[0220] Inhibition curves for AAV2.5T.LSV1 and AAV2 are shown in Figures 27A-27B. AAV2 had a calculated IC 50The AAV2.5T.LSV1 has a calculated IC of 9.3. 50 had the following characteristics: array [Table 5-1] [Table 5-2] [Table 5-3] [Table 5-4]

Claims

1. (a) a variant adeno-associated virus (AAV) capsid protein comprising a modified sequence substituted at amino acid residues 571-580 relative to a parent AAV capsid protein, said modified sequence comprising LAHKFKSGDA (SEQ ID NO:3), said parent AAV capsid protein being an AAV2.5T VP1 capsid protein comprising the amino acid sequence of SEQ ID NO:5, and said variant AAV capsid protein comprising a VP1 capsid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:7; and (b) a polynucleotide comprising a sequence encoding a therapeutic gene product. wherein said recombinant AAV virion comprising said variant AAV capsid protein is capable of transducing cells of the retina when injected intravitreally into a mammal.

2. 2. The recombinant AAV virion of claim 1, wherein the variant AAV capsid protein comprises a VP1 capsid sequence having at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO:

7.

3. The recombinant AAV virion of claim 1 or 2, wherein the recombinant AAV virion is capable of transducing one or more of photoreceptor cells, retinal ganglion cells, Muller cells, bipolar cells, amacrine cells, horizontal cells, and / or retinal pigment epithelial cells when injected intravitreally into a mammal.

4. The recombinant AAV virion of claim 3, wherein the recombinant AAV virion is capable of transducing retinal pigment epithelial cells when injected intravitreally into a mammal.

5. The recombinant AAV virion of any one of claims 1 to 4, wherein the therapeutic gene product is an siRNA, an miRNA or a protein.

6. 6. The recombinant AAV virion of any one of claims 1 to 5, wherein the therapeutic gene product is an anti-vascular endothelial growth factor (anti-VEGF) gene product or an opsin.

7. 7. The recombinant AAV virion of any one of claims 1 to 6, wherein the polynucleotide encoding the therapeutic gene product is flanked by one or more AAV ITRs.

8. 8. The recombinant AAV virion of claim 7, wherein the ITRs of one or more AAVs are ITRs of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, avian AAV, bovine AAV, canine AAV, equine AAV, primate AAV, non-primate AAV or ovine AAV, or variants thereof.

9. 9. The recombinant AAV virion of claim 7 or 8, wherein the one or more AAV ITRs are AAV2 ITRs or AAV5 ITRs.

10. 10. The recombinant AAV virion of claim 1, wherein the recombinant AAV virion has an altered cell tropism compared to AAV2.5T.

11. A pharmaceutical composition comprising a recombinant AAV virion according to any one of claims 1 to 10.

12. 1. A method for producing recombinant AAV (rAAV) virions, the method comprising: (a) culturing a host cell under conditions in which rAAV virions are produced, said host cell comprising: (i) a polynucleotide encoding a variant AAV capsid protein comprising a modified sequence substituted at amino acid residues 571-580 relative to a parent AAV capsid protein, said modified sequence comprising LAHKFKSGDA (SEQ ID NO:3), said parent AAV capsid protein being an AAV2.5T VP1 capsid protein comprising the amino acid sequence of SEQ ID NO:5, and said variant AAV capsid protein comprising a VP1 capsid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:7; (ii) a polynucleotide encoding a rep protein; (iii) a polynucleotide cassette comprising a sequence encoding a therapeutic gene product flanked by at least one AAV ITR; and (iv) AAV helper function and (b) recovering the rAAV virions produced by the host cells. wherein said recombinant AAV virions comprising said variant AAV capsid protein are capable of transducing cells of the retina when injected intravitreally into a mammal.

13. A composition comprising a recombinant AAV virion described in any one of claims 1 to 10, or a pharmaceutical composition described in claim 11, for providing a therapeutic gene product to the retina of a subject, characterized in that the composition or pharmaceutical composition is administered to the subject by intravitreal injection.

14. The subject is diagnosed with acute macular neuroretinopathy, Behcet's disease, choroidal neovascularization, diabetic uveitis, histoplasmosis, macular degeneration, edema, multifocal choroiditis, ocular trauma affecting the posterior ocular site or area, ocular tumors, central retinal vein occlusion, diabetic retinopathy, proliferative vitreoretinopathy (PVR), retinal artery occlusive disease, retinal detachment, uveal retinal disease, sympathetic ophthalmia, Vogt-Koyanagi-Harada (VKH) syndrome, uveal diffusion, a posterior ocular condition caused or affected by ocular laser procedures, a posterior ocular condition caused or affected by photodynamic therapy, photocoagulation, radiation 14. The composition of claim 13, wherein the patient has been diagnosed with or is suspected of having one or more conditions selected from the group consisting of retinopathy, epiretinal membrane disorder, branch retinal vein occlusion, anterior ischemic optic neuropathy, diabetic retinal ischemia, ischemic retinopathy, non-retinopathy diabetic retinal dysfunction, retinoschisis, retinitis pigmentosa, glaucoma, Usher syndrome, cone-rod dystrophy, Stargardt disease, hereditary macular degeneration, chorioretinal degeneration, Leber congenital amaurosis, congenital stationary night blindness, total choroidal atrophy, Bardet-Biedl syndrome, macular telangiectasia, Leber hereditary optic neuropathy, retinopathy of prematurity, and disorders of color vision.

15. A composition comprising a recombinant AAV virion described in any one of claims 1 to 10, or a pharmaceutical composition described in claim 11, for treating a retinal disease or disorder in a subject in need of such treatment, wherein the composition or pharmaceutical composition is administered to the subject by intravitreal injection.

16. The disease or disorder is acute macular neuroretinopathy, Behcet's disease, choroidal neovascularization, diabetic uveitis, histoplasmosis, macular degeneration, edema, multifocal choroiditis, ocular trauma affecting the posterior ocular site or area, ocular tumor, central retinal vein occlusion, diabetic retinopathy, proliferative vitreoretinopathy (PVR), retinal artery occlusive disease, retinal detachment, uveal retinal disease, sympathetic ophthalmia, Vogt-Koyanagi-Harada (VKH) syndrome, uveal diffusion, a posterior ocular condition caused by or affected by ocular laser procedures, photodynamic therapy.

16. The composition of claim 15, wherein the condition is a posterior ocular condition in which the ocular condition is or is affected by, photocoagulation, radiation retinopathy, epiretinal membrane disorder, branch retinal vein occlusion, anterior ischemic optic neuropathy, diabetic retinal ischemia, ischemic retinopathy, non-retinopathy diabetic retinal dysfunction, retinoschisis, retinitis pigmentosa, glaucoma, Usher syndrome, cone-rod dystrophy, Stargardt disease, hereditary macular degeneration, chorioretinal degeneration, Leber congenital amaurosis, congenital stationary night blindness, total choroidal atrophy, Bardet-Biedl syndrome, macular telangiectasia, Leber hereditary optic neuropathy, retinopathy of prematurity, or a disorder of color vision.

17. 13. Use of a composition comprising a recombinant AAV virion according to any one of claims 1 to 10 for the manufacture of a medicament for the treatment of a retinal disease or disorder in a subject.

18. 18. The use of claim 17, wherein the disease or disorder is age-related macular degeneration (AMD), wet AMD, dry AMD, retinal neovascularization, choroidal neovascularization, diabetic retinopathy, proliferative diabetic retinopathy, retinal vein occlusion, central retinal vein occlusion, branch retinal vein occlusion, diabetic macular edema, diabetic retinal ischemia, ischemic retinopathy or diabetic retinal edema.

19. 1. A variant AAV capsid protein comprising a modified sequence substituted at amino acid residues 571-580 compared to a parent AAV capsid protein, said modified sequence comprising LAHKFKSGDA (SEQ ID NO:3), said parent AAV capsid protein being an AAV2.5T VP1 capsid protein comprising the amino acid sequence of SEQ ID NO:5, said variant AAV capsid protein comprising a VP1 capsid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:7, wherein a recombinant AAV virion comprising said variant AAV capsid protein is capable of transducing cells of the retina when injected intravitreally into a mammal.

20. 20. The variant AAV capsid protein of claim 19, comprising a VP1 capsid sequence having at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO:

7.

21. 21. A nucleic acid comprising a nucleic acid sequence encoding the variant AAV capsid protein of claim 19 or 20.

22. 22. An expression vector comprising the nucleic acid of claim 21 , wherein the nucleic acid sequence encoding the variant AAV capsid protein is operably linked to a promoter sequence.

23. 23. The expression vector of claim 22, further comprising a nucleic acid encoding a rep protein.

24. A cell comprising the expression vector of claim 22 or 23.

25. The cell of claim 24, further comprising a nucleic acid encoding a therapeutic gene product.

26. The cell of claim 25 , wherein the therapeutic gene product is an siRNA, miRNA or a protein.

27. 27. The cell of claim 25 or 26, wherein the therapeutic gene product is an anti-vascular endothelial growth factor (anti-VEGF) gene product or an opsin.

28. 28. The cell of any one of claims 25 to 27, wherein the nucleic acid encoding the therapeutic gene product is flanked by one or more AAV ITRs.

29. 29. The cell of claim 28, wherein the ITRs of one or more AAVs are ITRs of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, avian AAV, bovine AAV, canine AAV, equine AAV, primate AAV, non-primate AAV or ovine AAV, or variants thereof.

30. 30. The cell of claim 28 or 29, wherein the one or more AAV ITRs are AAV2 ITRs or AAV5 ITRs.

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