Methods for treating xlrs

Abstract

Provided herein are compositions and methods for the treatment of XLRS.

Description

WSGR Docket No. 58774-736.601METHODS FOR TREATING XLRSCROSS-REFERENCE

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 709,355, filed October 18, 2024, and U.S. Provisional Application No. 63 / 721,318, filed November 15,2024, which applications are incorporated herein by reference in its entirety.INCORPORATION BY REFERENCE OF SEQUENCE LISTING

[0002] The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled 58774-736.601.xml, created October 16,2025, which is 158 kilobytes in size. The information in the electronic format of the Sequence Listing is incorporated by reference in its entirety.BACKGROUND

[0003] Retinoschisis is a genetic condition characterized by the splitting of the retina into two layers. There are two forms of this disorder. The more common form, known commonly as senile retinoschisis, typically develops in middle age or beyond and can affect both men and women. The rarer form, X-linked retinoschisis (XLRS) is present at birth and affects boys and young men. The main symptom of XLRS is impaired vision that cannot be improved with eyeglasses. While some people with XLRS may experience progressive vision loss throughout their life, other people may have relatively stable vision throughout their lifetime. XLRS is one type of a broader disorder called macular degeneration, as it primarily affects the macula. XLRS affects about 30,000 men in the United States and European Union. There are no treatments available for patients with XLRS. However, the use of topical dorzolamide and oral acetazolamide in reducing cystic spaces and foveal thickness with a concomitant increase in visual acuity has been reported.SUMMARY

[0004] XLRS is caused by mutations in a gene on the X chromosome called RSI which encodes a protein called retinoschisin. Retinoschisin is a structural protein expressed and secreted by photoreceptor and bipolar cells that binds strongly and specifically to the surfaces of many cells in the retina. This protein serves as an adhesive to maintain the structural integrity of the layers of the retina. Without normal retinoschisin protein, the layers of the retina split, inter-cellWSGR Docket No. 58774-736.601 communication is disrupted, and retinal cells and ultimately vision is lost. XLRS patients typically present with a diminished b-wave in electroretinogram (ERG) measurements of their retina.Patients with nonsense mutations generally have more severe disease than those with missense mutations.

[0005] The present disclosure provides for rAAV therapies for the treatment of XLRS. These therapies are designed for administration to the eyes of subjects, such as human subjects, including humans diagnosed with or suffering from XLRS. The disclosed rAAV vectors and particles may provide for improved retinal structure and function after administration to subjects. The disclosed vectors may provide for amelioration or reversal, e.g., a partial or complete reversal, of the symptoms of retinoschisis. Particular doses for administration of AAV particles include about 1 .5 x E10 vg per eye to about 5.0 x E10 vg per eye, or about 3.0 x E10 vg per eye.

[0006] Example disclosed AAV vectors comprise the human RSI transgene (or heterologous nucleic acid) lacking the 5’ and 3 ’ untranslated regions, or UTRs. In some embodiments, the transgene contains mutations relative to the wild-type sequence that do not result in any amino acid change (i.e., are silent mutations). The present disclosure also provides for the use of photoreceptor-specific promoters, such as the human rhodopsin promoter, and AAV capsids that display improved retinal transduction efficiency and that exhibit enhanced lateral spread after subretinal injection, e.g., at about 1.5 x 1010vg per eye.

[0007] In some embodiments, the disclosed AAV particles comprise an AAV capsid AAV44.9(E531D), which is capable of highly efficient transduction of rods, cones, and retinal pigment epithelium (“RPE”) following subretinal injection. In addition, AAV44.9(E531D) exhibits increased lateral spread, transducing photoreceptors, and retinal pigment epithelium outside the subretinal injection bleb.

[0008] In certain aspects, disclosed herein is a method of treating X-linked retinoschisis (XLRS) in a human subject in need thereof, the method comprising administering to an eye of the human subject about 1.5 x 1010vg to about 5.0 x 1010vg of an AAV particle. In some embodiments, the about 1.5 x 1010vgto about 5.0 x 1010vg of the AAV particle is administered to each eye of the human subject. In some embodiments, about 1.5 x 1010vg of the AAV particle is administered to the eye or to each eye of the human subject. In some embodiments, about 3. O x 1010vg of the AAV particle is administered to the eye or to each eye of the human subject. In some embodiments, the AAV particle comprises a polynucleotide comprising a heterologous nucleic acid encoding a retinoschisin (RS) protein. In some embodiments, the RS protein is a human RS protein. In some embodiments, the RS protein has the amino acid sequence of SEQ ID NO: 12. In some embodiments, the RS protein is encodedby a nucleic acid sequence at least about 80%, 81%, 82%,WSGR Docket No. 58774-736.60183%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the nucleic acid sequence of SEQ ID NO: 8. In some embodiments, the polynucleotide comprises a human rhodopsin kinase promoter. In some embodiments, the human rhodopsin kinase promoter comprises a sequence at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 7. In some embodiments, the polynucleotide comprises a sequence at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 33 (pTR-X002-3pSR). In some embodiments, the AAV particle comprises a capsid. In some embodiments, the capsid comprises a VP1 amino acid sequence at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1. In some embodiments, the capsid comprises a VP2 amino acid sequence at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 2. In some embodiments, the capsid comprises a VP3 amino acid sequence at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to: SEQ ID NO: 3. In some embodiments, the human subject has a mutation in the RSI gene. In some embodiments, the human subject has an increased risk of retinal detachment as compared to a human subject that does not have the mutation in the RSI gene. In some embodiments, the human subject has a reduced risk of retinal detachment after the administering. In some embodiments, the administering comprises subretinal administration to a fovea of one or both eyes of the mammal. In some embodiments, detachment of the fovea is minimized. In some embodiments, the human subject has a significant improvement as measured by patient global impression of change (PGIC) as compared to before the administering. In some embodiments, the human subject has loss of vision prior to the administering. In some embodiments, the human subject regains vision after the administering. In some embodiments, regaining vision comprises an improvement in visual acuity or an improvement in the visual field. In some embodiments, the human subject expresses native RSI after the administering, optionally wherein the native RSI is SEQ ID NO: 12. In some embodiments, the human subject expresses native RSI in photoreceptors after the administering. In some embodiments, the human subject expresses native RSI in bipolar cells after the administering. In some embodiments, the human subject is 6 years of age or older. In some embodiments, the human subject is 18 years old or older. In some embodiments, the human subject is between 6 years of age and 65 years of age. In some embodiments, the human subject has XLRS caused by a pathogenic mutation in RSI. In some embodiments, prior to the administering the human subject has foveal schisis in the eye. In someWSGR Docket No. 58774-736.601 embodiments, prior to the administering the human subject has parafoveal / perifoveal schisis in the eye. In some embodiments, before and / or after the administering, the method comprises performing optical coherence tomography on the human subject. In some embodiments, before and / or after the administering, the method comprises performing microperimetry on the human subject. In some embodiments, the AAV particle is administered via a subretinal injection. In some embodiments, the AAV particle is administered in a volume of about 150 microliters. In some embodiments, the AAV particle is administered in 2 blebs. In some embodiments, the AAV particle is administered in a manner that avoids foveal detachment. In some embodiments, the corticosteroid comprises prednisone, triamcinolone acetonide, methylprednisolone, prenidsolone acetate, or a combination thereof. In some embodiments, the corticosteroid comprises prednisone. In some embodiments, the corticosteroid is administered starting about 7 weeks prior to the administering of the AAV particle. In some embodiments, the corticosteroid is administered at about 1 mg / kg of the human subject per day. In some embodiments, the triamcinolone acetonide is administered starting about 7 weeks prior to the administering of the AAV particle. In some embodiments, about 20 mg of the triamcinolone acetonide is administered. In some embodiments, the triamcinolone acetonide is administered in a periocular injection. In some embodiments, the methylprednisolone is administered starting about 7 weeks prior to the administering of the AAV particle. In some embodiments, about 250 mg of the methylprednisolone is administered. In some embodiments, the methylprednisolone is administered via IV. In some embodiments, the prenidsolone acetate is administered starting about 28 days prior to the administering of the AAV particle. In some embodiments, about 1% of the prenidsolone acetate is administered. In some embodiments, the prenidsolone acetate is administered topically. In some embodiments, the administering comprises administering prednisone at a dose of Img / kg / day for seven weeks. In some embodiments, the administering comprises administering 20 mg of triamcinalone acetonide by periocular injection. In some embodiments, the administering comprises administering 250 mg of methylprednisolone intravenously. In some embodiments, the administering comprises administering 1% prenidsolone acetate topically for 28 days. In some embodiments, after the administering, the human subject has a significant improvement in PGIC. In some embodiments, after the administering the human subject has closure of a foveal schisis. In some embodiments, after the administering the human subject has a reduction in central retinal thickness. In some embodiments, afterthe administering the human subject has improvement in retinal sensitivity. In some embodiments, the administering a) preserves one or more photoreceptor cells, b) restores laminar retinal structure, c) restores one or more rod- and / or cone-mediated functions, d) restores completely or partially visual behavior in one or both eyes, or e) any combination thereof. In someWSGR Docket No. 58774-736.601 embodiments, the administering restores laminar retinal structure. In some embodiments, the human subject is sensitive to inflammation. In some embodiments, the dose of the AAV particle reduces inflammation in the human subject as compared to a higher dose of the AAV particle.

[0009] In certain aspects, disclosed herein is a method of improving visual acuity in an eye in a human subject with X-linked retinoschisis (XLRS), the method comprising administering to the eye of the human subject an AAV particle, wherein the visual acuity is improved by at least 5 ETDRS letters at least 6 months after administering. In some embodiments, the visual acuity comprises Low Light Visual Acuity (LLVA). In some embodiments, the visual acuity comprises Best Corrected Visual Acuity (BCVA). In some embodiments, the visual acuity is improved at least 12 months, at least 18 months, at least 24 months, at least 36 months, at least 48 months, or at least 60 months after administering. In some embodiments, the AAV particle comprises a polynucleotide comprising a heterologous nucleic acid encoding a retinoschisin (RS) protein. In some embodiments, the RS protein is a human RS protein. In some embodiments, the RS protein has the amino acid sequence of SEQ ID NO: 12. In some embodiments, the RS protein is encoded by a nucleic acid sequence at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the nucleic acid sequence of SEQ ID NO: 8. In some embodiments, the polynucleotide comprises a human rhodopsin kinase promoter. In some embodiments, the human rhodopsin kinase promoter comprises a sequence at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 7. In some embodiments, the polynucleotide comprises a sequence at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 33 (pTR-X002-3pSR). In some embodiments, the AAV particle comprises a capsid. In some embodiments, the capsid comprises a VP1 amino acid sequence at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1. In some embodiments, the capsid comprises a VP2 amino acid sequence at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 2. In some embodiments, the capsid comprises a VP3 amino acid sequence at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to: SEQ ID NO: 3. In some embodiments, the human subject has a mutation in the RSI gene. In some embodiments, the human subject has an increased risk of retinal detachment as compared to a human subject that does not have the mutation in the RSI gene. In some embodiments, the human subject has a reduced risk of retinal detachment after the administering. In some embodiments, theWSGR Docket No. 58774-736.601 administering comprises subretinal administration to a fovea of one or both eyes of the mammal. In some embodiments, detachment of the fovea is minimized. In some embodiments, the human subject has a significant improvement as measured by patient global impression of change (PGIC) as compared to before the administering. In some embodiments, the human subject has loss of vision prior to the administering. In some embodiments, the human subject regains vision after the administering. In some embodiments, regaining vision comprises an improvement in visual acuity or an improvement in the visual field. In some embodiments, the human subject expresses native RSI after the administering, optionally wherein the native RSI is SEQ ID NO: 12. In some embodiments, the human subject expresses native RSI in photoreceptors after the administering. In some embodiments, the human subject expresses native RSI in bipolar cells after the administering In some embodiments, the human subject is 6 years of age or older. In some embodiments, the human subject is 18 years old or older. In some embodiments, the human subject is between 6 years of age and 65 years of age. In some embodiments, the human subject has XLRS caused by a pathogenic mutation in RSI . In some embodiments, prior to the administering the human subject has foveal schisis in the eye. In some embodiments, prior to the administering the human subject has parafoveal / perifoveal schisis in the eye. In some embodiments, before and / or after the administering, the method comprises performing optical coherence tomography on the human subject. In some embodiments, before and / or after the administering, the method comprises performing microperimetry on the human subject. In some embodiments, the AAV particle is administered via a subretinal injection. In some embodiments, the AAV particle is administered in a volume of about 150 microliters. In some embodiments, the AAV particle is administered in 2 blebs. In some embodiments, the AAV particle is administered in a manner that avoids foveal detachment. In some embodiments, the corticosteroid comprises prednisone, triamcinolone acetonide, methylprednisolone, prenidsolone acetate, or a combination thereof. In some embodiments, the corticosteroid comprises prednisone. In some embodiments, the corticosteroid is administered starting about 7 weeks prior to the administering of the AAV particle. In some embodiments, the corticosteroid is administered at about 1 mg / kg of the human subject per day. In some embodiments, the triamcinolone acetonide is administered starting about 7 weeks prior to the administering of the AAV particle. In some embodiments, about 20 mg of the triamcinolone acetonide is administered. In some embodiments, the triamcinolone acetonide is administered in a periocular injection. In some embodiments, the methylprednisolone is administered starting about 7 weeks prior to the administering of the AAV particle. In some embodiments, about 250 mg of the methylprednisolone is administered. In some embodiments, the methylprednisolone is administered via IV. In some embodiments, the prenidsolone acetate is administered starting about 28 days priorWSGR Docket No. 58774-736.601 to the administering of the AAV particle. In some embodiments, about 1% of the prenidsolone acetate is administered. In some embodiments, the prenidsolone acetate is administered topically. In some embodiments, the administering comprises administering prednisone at a dose of Img / kg / day for seven weeks. In some embodiments, the administering comprises administering 20 mg of triamcinalone acetonide by periocular injection. In some embodiments, the administering comprises administering 250 mg of methylprednisolone intravenously. In some embodiments, the administering comprises administering 1% prenidsolone acetate topically for 28 days. In some embodiments, after the administering, the human subject has a significant improvement in PGIC. In some embodiments, after the administering the human subject has closure of a foveal schisis. In some embodiments, after the administering the human subject has a reduction in central retinal thickness. In some embodiments, after the administering the human subject has improvement in retinal sensitivity. In some embodiments, the administering a) preserves one or more photoreceptor cells, b) restores laminar retinal structure, c) restores one or more rod - and / or cone-mediated functions, d) restores completely or partially visual behavior in one or both eyes, or e) any combination thereof. In some embodiments, the administering restores laminar retinal structure. In some embodiments, the human subject is sensitive to inflammation. In some embodiments, the dose of the AAV particle reduces inflammation in the human subject as compared to a higher dose of the AAV particle.

[0010] In certain aspects, disclosed herein is a method of improving vision in an eye in a human subject with X-linked retinoschisis (XLRS), the method comprising administering to the eye of the human subject an AAV particle, wherein at least one schisis cavity in the eye is reduced in size for at least one month compared to prior to administering the AAV particle. In some embodiments, the at least one schisis cavity is reduced in size by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%. In some embodiments, the at least one schisis cavity is reduced in size at least 6 months, 12 months, at least 18 months, at least 24 months, at least 36 months, at least 48 months, or at least 60 months after administering. In some embodiments, there is a complete closure of the at least one schisis cavity. In some embodiments, the eye comprises at least two schisis cavities before administering, and there is complete closure of all schisis cavities in the eye. In some embodiments, the size of the at least one schisis cavity is measured using optical coherence tomography. In some embodiments, the AAV particle is at administered in a subretinal bleb and the at least one schisis cavity that is reduced in size is located in an area of the retina outside of the subretinal bleb. In some embodiments, the AAV particle comprises a polynucleotide comprising a heterologous nucleic acid encoding a retinoschisin (RS) protein. In some embodiments, the RS protein is a human RS protein. In some embodiments, the RS protein has the amino acid sequenceWSGR Docket No. 58774-736.601 of SEQ ID NO: 12. In some embodiments, the RS protein is encodedby a nucleic acid sequence at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the nucleic acid sequence of SEQ ID NO: 8. In some embodiments, the polynucleotide comprises a human rhodopsin kinase promoter. In some embodiments, the human rhodopsin kinase promoter comprises a sequence at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 7. In some embodiments, the polynucleotide comprises a sequence atleast about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 33 (pTR-X002-3pSR). In some embodiments, the AAV particle comprises a capsid. In some embodiments, the capsid comprises a VP1 amino acid sequence atleast about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1. In some embodiments, the capsid comprises a VP2 amino acid sequence at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 2. In some embodiments, the capsid comprises a VP3 amino acid sequence at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to: SEQ ID NO: 3. In some embodiments, the human subject has a mutation in the RSI gene. In some embodiments, the human subject has an increased risk of retinal detachment as compared to a human subject that does not have the mutation in the RSI gene. In some embodiments, the human subject has a reduced risk of retinal detachment after the administering. In some embodiments, the administering comprises subretinal administration to a fovea of one or both eyes of the mammal. In some embodiments, detachment of the fovea is minimized. In some embodiments, the human subject has a significant improvement as measured by patient global impression of change (PGIC) as compared to before the administering. In some embodiments, the human subject has loss of vision prior to the administering. In some embodiments, the human subject regains vision after the administering. In some embodiments, regaining vision comprises an improvement in visual acuity or an improvement in the visual field. In some embodiments, the human subject expresses native RSI after the administering, optionally wherein the native RSI is SEQ ID NO: 12. In some embodiments, the human subject expresses native RSI in photoreceptors after the administering. In some embodiments, the human subject expresses native RSI in bipolar cells after the administering. In some embodiments, the human subject is 6 years of age or older. In some embodiments, the human subject is 18 years old or older. In some embodiments, the human subject is between 6 years of age and 65 years of age. In some embodiments, the human subject has XLRS caused by a pathogenic mutation in RSI. In someWSGR Docket No. 58774-736.601 embodiments, prior to the administering the human subject has foveal schisis in the eye. In some embodiments, prior to the administering the human subject has parafoveal / perifoveal schisis in the eye. In some embodiments, before and / or after the administering, the method comprises performing optical coherence tomography on the human subject. In some embodiments, before and / or after the administering, the method comprises performing microperimetry on the human subject. In some embodiments, the AAV particle is administered via a subretinal injection. In some embodiments, the AAV particle is administered in a volume of about 150 microliters. In some embodiments, the AAV particle is administered in 2 blebs. In some embodiments, the AAV particle is administered in a manner that avoids foveal detachment. In some embodiments, the corticosteroid comprises prednisone, triamcinolone acetonide, methylprednisolone, prenidsolone acetate, or a combination thereof. In some embodiments, the corticosteroid comprises prednisone. In some embodiments, the corticosteroid is administered starting about 7 weeks prior to the administering of the AAV particle. In some embodiments, the corticosteroid is administered at about 1 mg / kgof the human subject per day. In some embodiments, the triamcinolone acetonide is administered starting about 7 weeks prior to the administering of the AAV particle. In some embodiments, about 20 mg of the triamcinolone acetonide is administered. In some embodiments, the triamcinolone acetonide is administered in a periocular injection. In some embodiments, the methylprednisolone is administered starting about 7 weeks prior to the administering of the AAV particle. In some embodiments, about 250 mg of the methylprednisolone is administered. In some embodiments, the methylprednisolone is administered via IV. In some embodiments, the prenidsolone acetate is administered starting about 28 days prior to the administering of the AAV particle. In some embodiments, about 1% of the prenidsolone acetate is administered. In some embodiments, the prenidsolone acetate is administered topically. In some embodiments, the administering comprises administering prednisone at a dose of Img / kg / day for seven weeks. In some embodiments, the administering comprises administering 20 mg of triamcinalone acetonide by periocular injection. In some embodiments, the administering comprises administering 250 mg of methylprednisolone intravenously. In some embodiments, the administering comprises administering 1% prenidsolone acetate topically for 28 days. In some embodiments, after the administering, the human subject has a significant improvement in PGIC. In some embodiments, after the administering the human subject has closure of a foveal schisis. In some embodiments, after the administering the human subject has a reduction in central retinal thickness. In some embodiments, after the administering the human subject has improvement in retinal sensitivity. In some embodiments, the administering a) preserves one or more photoreceptor cells, b) restores laminar retinal structure, c) restores one or more rod- and / or cone-mediated functions, d) restoresWSGR Docket No. 58774-736.601 completely or partially visual behavior in one or both eyes, or e) any combination thereof. In some embodiments, the administering restores laminar retinal structure. In some embodiments, the human subject is sensitive to inflammation. In some embodiments, the dose of the AAV particle reduces inflammation in the human subject as compared to a higher dose of the AAV particle.

[0011] In certain aspects, disclosed herein is a method of improving vision in an eye in a human subject with X-linked retinoschisis (XLRS), the method comprising administering to the eye of the human subject an AAV particle, wherein the retinal thickness of the eye is reduced in size compared to prior to administering the AAV particle. In some embodiments, the retinal thickness is reduced in size at least 6 months, 12 months, at least 18 months, at least 24 months, at least 36 months, at least 48 months, or at least 60 months after administering. In some embodiments, the reduction in retinal thickness is specific to the central retina or the macula of the eye. In some embodiments, the AAV particle comprises a polynucleotide comprising a heterologous nucleic acid encoding a retinoschisin (RS) protein. In some embodiments, the RS protein is a human RS protein. In some embodiments, the RS protein has the amino acid sequence of SEQ ID NO: 12. In some embodiments, the RS protein is encoded by a nucleic acid sequence at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the nucleic acid sequence of SEQ ID NO: 8. In some embodiments, the polynucleotide comprises a human rhodopsin kinase promoter. In some embodiments, the human rhodopsin kinase promoter comprises a sequence at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 7. In some embodiments, the polynucleotide comprises a sequence at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 33 (pTR-X002-3pSR). In some embodiments, the AAV particle comprises a capsid. In some embodiments, the capsid comprises a VP1 amino acid sequence at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1. In some embodiments, the capsid comprises a VP2 amino acid sequence at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 2. In some embodiments, the capsid comprises a VP3 amino acid sequence at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to: SEQ ID NO: 3. In some embodiments, the human subject has a mutation in the RSI gene. In some embodiments, the human subject has an increased risk of retinal detachment as compared to a human subject that does not have the mutation in the RSI gene. In some embodiments, the human subject has a reduced risk of retinal detachment after theWSGR Docket No. 58774-736.601 administering. In some embodiments, the administering comprises subretinal administration to a fovea of one or both eyes of the mammal. In some embodiments, detachment of the fovea is minimized. In some embodiments, the human subject has a significant improvement as measured by patient global impression of change (PGIC) as compared to before the administering. In some embodiments, the human subject has loss of vision prior to the administering. In some embodiments, the human subject regains vision after the administering. In some embodiments, regaining vision comprises an improvement in visual acuity or an improvement in the visual field. In some embodiments, the human subject expresses native RSI after the administering, optionally wherein the native RSI is SEQ ID NO: 12. In some embodiments, the human subject expresses native RSI in photoreceptors after the administering. In some embodiments, the human subject expresses native RSI in bipolar cells after the administering. In some embodiments, the human subject is 6 years of age or older. In some embodiments, the human subject is 18 years old or older. In some embodiments, the human subject is between 6 years of age and 65 years of age. In some embodiments, the human subject has XLRS caused by a pathogenic mutation in RSI. In some embodiments, prior to the administering the human subject has foveal schisis in the eye. In some embodiments, prior to the administering the human subject has parafoveal / perifoveal schisis in the eye. In some embodiments, before and / or after the administering, the method comprises performing optical coherence tomography on the human subject. In some embodiments, before and / or after the administering, the method comprises performing microperimetry on the human subject. In some embodiments, the AAV particle is administered via a subretinal injection. In some embodiments, the AAV particle is administered in a volume of about 150 microliters. In some embodiments, the AAV particle is administered in 2 blebs. In some embodiments, the AAV particle is administered in a manner that avoids foveal detachment. In some embodiments, the corticosteroid comprises prednisone, triamcinolone acetonide, methylprednisolone, prenidsolone acetate, or a combination thereof. In some embodiments, the corticosteroid comprises prednisone. In some embodiments, the corticosteroid is administered starting about 7 weeks prior to the administering of the AAV particle. In some embodiments, the corticosteroid is administered at about 1 mg / kg of the human subject per day. In some embodiments, the triamcinolone acetonide is administered starting about 7 weeks prior to the administering of the AAV particle. In some embodiments, about 20 mg of the triamcinolone acetonide is administered. In some embodiments, the triamcinolone acetonide is administered in a periocular injection. In some embodiments, the methylprednisolone is administered starting about 7 weeks prior to the administering of the AAV particle. In some embodiments, about 250 mg of the methylprednisolone is administered. In some embodiments, the methylprednisolone is administered via IV. In some embodiments, theWSGR Docket No. 58774-736.601 prenidsolone acetate is administered starting about 28 days prior to the administering of the AAV particle. In some embodiments, about 1% of the prenidsolone acetate is administered. In some embodiments, the prenidsolone acetate is administered topically. In some embodiments, the administering comprises administering prednisone at a dose of Img / kg / day for seven weeks. In some embodiments, the administering comprises administering 20 mg of triamcinalone acetonide by periocular injection. In some embodiments, the administering comprises administering 250 mg of methylprednisolone intravenously. In some embodiments, the administering comprises administering 1% prenidsolone acetate topically for 28 days. In some embodiments, after the administering, the human subject has a significant improvement in PGIC. In some embodiments, after the administering the human subject has closure of a foveal schisis. In some embodiments, after the administering the human subject has a reduction in central retinal thickness. In some embodiments, after the administering the human subject has improvement in retinal sensitivity. In some embodiments, the administering a) preserves one or more photoreceptor cells, b) restores laminar retinal structure, c) restores one or more rod- and / or cone-mediated functions, d) restores completely or partially visual behavior in one or both eyes, or e) any combination thereof. In some embodiments, the administering restores laminar retinal structure. In some embodiments, the human subject is sensitive to inflammation. In some embodiments, the dose of the AAV particle reduces inflammation in the human subject as compared to a higher dose of the AAV particle.

[0012] In certain aspects, disclosed herein is a method of visual field improvement in a human subject with X-linked retinoschisis (XLRS), the method comprising administering to an eye of the human subject an AAV particle, wherein the visual field improvement is at least 4 dB, as compared to before administering. In some embodiments, the visual field is measured at at least 4 fixed loci, 5 fixed loci, 6 fixed loci, 7 fixed loci, 8 fixed loci, 9 fixed loci, or 10 fixed loci. In some embodiments, the visual field improvement is at least 5dB, 6 dB, 7dB, 8 dB, 9 dB, or 10 dB. In some embodiments, the visual improvement is at least 7dB averaged across at least 5 fixed loci. In some embodiments, the fixed loci are pre-selected. In some embodiments, the visual field improvement is maintained at least 6 months, 12 months, at least 18 months, at least 24 months, at least 36 months, at least 48 months, or at least 60 months after administering. In some embodiments, the visual field is measured using microperimetry. In some embodiments, the visual field is measured using static automatic perimetry, optionally wherein the static automatic perimetry comprises a Humphrey Field Analyzer. In some embodiments, the AAV particle comprises a polynucleotide comprising a heterologous nucleic acid encoding a retinoschisin (RS) protein. In some embodiments, the RS protein is a human RS protein. In some embodiments, the RS protein has the amino acid sequence of SEQ ID NO: 12. In some embodiments, the RS proteinWSGR Docket No. 58774-736.601 is encoded by a nucleic acid sequence at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the nucleic acid sequence of SEQ ID NO: 8. In some embodiments, the polynucleotide comprises a human rhodopsin kinase promoter. In some embodiments, the human rhodopsin kinase promoter comprises a sequence at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 7. In some embodiments, the polynucleotide comprises a sequence at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 33 (pTR-X002-3pSR). In some embodiments, the AAV particle comprises a capsid. In some embodiments, the capsid comprises a VP1 amino acid sequence at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1. In some embodiments, the capsid comprises a VP2 amino acid sequence at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 2. In some embodiments, the capsid comprises a VP3 amino acid sequence at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to: SEQ ID NO: 3. In some embodiments, the human subject has a mutation in the RSI gene. In some embodiments, the human subject has an increased risk of retinal detachment as compared to a human subject that does not have the mutation in the RSI gene. In some embodiments, the human subject has a reduced risk of retinal detachment after the administering. In some embodiments, the administering comprises subretinal administration to a fovea of one or both eyes of the mammal. In some embodiments, detachment of the fovea is minimized. In some embodiments, the human subject has a significant improvement as measured by patient global impression of change (PGIC) as compared to before the administering. In some embodiments, the human subject has loss of vision prior to the administering. In some embodiments, the human subject regains vision after the administering. In some embodiments, regaining vision comprises an improvement in visual acuity or an improvement in the visual field. In some embodiments, the human subject expresses native RSI after the administering, optionally wherein the native RSI is SEQ ID NO: 12. In some embodiments, the human subject expresses native RSI in photoreceptors after the administering. In some embodiments, the human subject expresses native RSI in bipolar cells after the administering In some embodiments, the human subject is 6 years of age or older. In some embodiments, the human subject is 18 years old or older. In some embodiments, the human subject is between 6 years of age and 65 years of age. In some embodiments, the human subject has XLRS caused by a pathogenic mutation in RSI . In some embodiments, prior to the administering the human subjectWSGR Docket No. 58774-736.601 has foveal schisis in the eye. In some embodiments, prior to the administering the human subject has parafoveal / perifoveal schisis in the eye. In some embodiments, before and / or after the administering, the method comprises performing optical coherence tomography on the human subject. In some embodiments, before and / or after the administering, the method comprises performing microperimetry on the human subject. In some embodiments, the AAV particle is administered via a subretinal injection. In some embodiments, the AAV particle is administered in a volume of about 150 microliters. In some embodiments, the AAV particle is administered in 2 blebs. In some embodiments, the AAV particle is administered in a manner that avoids foveal detachment. In some embodiments, the corticosteroid comprises prednisone, triamcinolone acetonide, methylprednisolone, prenidsolone acetate, or a combination thereof. In some embodiments, the corticosteroid comprises prednisone. In some embodiments, the corticosteroid is administered starting about 7 weeks prior to the administering of the AAV particle. In some embodiments, the corticosteroid is administered at about 1 mg / kg of the human subject per day. In some embodiments, the triamcinolone acetonide is administered starting about 7 weeks prior to the administering of the AAV particle. In some embodiments, about 20 mg of the triamcinolone acetonide is administered. In some embodiments, the triamcinolone acetonide is administered in a periocular injection. In some embodiments, the methylprednisolone is administered starting about 7 weeks prior to the administering of the AAV particle. In some embodiments, about 250 mg of the methylprednisolone is administered. In some embodiments, the methylprednisolone is administered via IV. In some embodiments, the prenidsolone acetate is administered starting about 28 days prior to the administering of the AAV particle. In some embodiments, about 1% of the prenidsolone acetate is administered. In some embodiments, the prenidsolone acetate is administered topically. In some embodiments, the administering comprises administering prednisone at a dose of Img / kg / day for seven weeks. In some embodiments, the administering comprises administering 20 mg of triamcinalone acetonide by periocular injection. In some embodiments, the administering comprises administering 250 mg of methylprednisolone intravenously. In some embodiments, the administering comprises administering 1% prenidsolone acetate topically for 28 days. In some embodiments, after the administering, the human subject has a significant improvement in PGIC. In some embodiments, after the administering the human subject has closure of a foveal schisis. In some embodiments, after the administering the human subject has a reduction in central retinal thickness. In some embodiments, after the administering the human subject has improvement in retinal sensitivity. In some embodiments, the administering a) preserves one or more photoreceptor cells, b) restores laminar retinal structure, c) restores one or more rod- and / or cone-mediated functions, d) restores completely or partially visual behavior in one or both eyes, or e) anyWSGR Docket No. 58774-736.601 combination thereof. In some embodiments, the administering restores laminar retinal structure. In some embodiments, the human subject is sensitive to inflammation. In some embodiments, the dose of the AAV particle reduces inflammation in the human subject as compared to a higher dose of the AAV particle.

[0013] In certain aspects, disclosed herein is a method of treating X-linked retinoschisis (XLRS) in a human subject in need thereof, the method comprising administering to an eye of the human subject an AAV particle, wherein the human subject has a Best Correct Visual Acuity (BCVA) between 34 and 73 letters in the eye before administering. In some embodiments, the BCVA is improved by at least 5 ETDRS letters at least 6 months after administering. In some embodiments, the BCVA is improved at least 12 months, at least 18 months, at least 24 months, at least 36 months, at least 48 months, or at least 60 months after administering. In some embodiments, the AAV particle comprises a polynucleotide comprising a heterologous nucleic acid encoding a retinoschisin (RS) protein. In some embodiments, the RS protein is a human RS protein. In some embodiments, the RS protein has the amino acid sequence of SEQ ID NO: 12. In some embodiments, the RS protein is encodedby a nucleic acid sequence at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the nucleic acid sequence of SEQ ID NO: 8. In some embodiments, the polynucleotide comprises a human rhodopsin kinase promoter. In some embodiments, the human rhodopsin kinase promoter comprises a sequence at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 7. In some embodiments, the polynucleotide comprises a sequence at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 33 (pTR-X002-3pSR). In some embodiments, the AAV particle comprises a capsid. In some embodiments, the capsid comprises a VP1 amino acid sequence at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1. In some embodiments, the capsid comprises a VP2 amino acid sequence at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 2. In some embodiments, the capsid comprises a VP3 amino acid sequence at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to: SEQ ID NO: 3. In some embodiments, the human subject has a mutation in the RSI gene. In some embodiments, the human subject has an increased risk of retinal detachment as compared to a human subject that does not have the mutation in the RSI gene. In some embodiments, the human subject has a reduced risk of retinal detachment after theWSGR Docket No. 58774-736.601 administering. In some embodiments, the administering comprises subretinal administration to a fovea of one or both eyes of the mammal. In some embodiments, detachment of the fovea is minimized. In some embodiments, the human subject has a significant improvement as measured by patient global impression of change (PGIC) as compared to before the administering. In some embodiments, the human subject has loss of vision prior to the administering. In some embodiments, the human subject regains vision after the administering. In some embodiments, regaining vision comprises an improvement in visual acuity or an improvement in the visual field. In some embodiments, the human subject expresses native RSI after the administering, optionally wherein the native RSI is SEQ ID NO: 12. In some embodiments, the human subject expresses native RSI in photoreceptors after the administering. In some embodiments, the human subject expresses native RSI in bipolar cells after the administering. In some embodiments, the human subject is 6 years of age or older. In some embodiments, the human subject is 18 years old or older. In some embodiments, the human subject is between 6 years of age and 65 years of age. In some embodiments, the human subject has XLRS caused by a pathogenic mutation in RSI. In some embodiments, prior to the administering the human subject has foveal schisis in the eye. In some embodiments, prior to the administering the human subject has parafoveal / perifoveal schisis in the eye. In some embodiments, before and / or after the administering, the method comprises performing optical coherence tomography on the human subject. In some embodiments, before and / or after the administering, the method comprises performing microperimetry on the human subject. In some embodiments, the AAV particle is administered via a subretinal injection. In some embodiments, the AAV particle is administered in a volume of about 150 microliters. In some embodiments, the AAV particle is administered in 2 blebs. In some embodiments, the AAV particle is administered in a manner that avoids foveal detachment. In some embodiments, the corticosteroid comprises prednisone, triamcinolone acetonide, methylprednisolone, prenidsolone acetate, or a combination thereof. In some embodiments, the corticosteroid comprises prednisone. In some embodiments, the corticosteroid is administered starting about 7 weeks prior to the administering of the AAV particle. In some embodiments, the corticosteroid is administered at about 1 mg / kg of the human subject per day. In some embodiments, the triamcinolone acetonide is administered starting about 7 weeks prior to the administering of the AAV particle. In some embodiments, about 20 mg of the triamcinolone acetonide is administered. In some embodiments, the triamcinolone acetonide is administered in a periocular injection. In some embodiments, the methylprednisolone is administered starting about 7 weeks prior to the administering of the AAV particle. In some embodiments, about 250 mg of the methylprednisolone is administered. In some embodiments, the methylprednisolone is administered via IV. In some embodiments, theWSGR Docket No. 58774-736.601 prenidsolone acetate is administered starting about 28 days prior to the administering of the AAV particle. In some embodiments, about 1% of the prenidsolone acetate is administered. In some embodiments, the prenidsolone acetate is administered topically. In some embodiments, the administering comprises administering prednisone at a dose of Img / kg / day for seven weeks. In some embodiments, the administering comprises administering 20 mg of triamcinalone acetonide by periocular injection. In some embodiments, the administering comprises administering 250 mg of methylprednisolone intravenously. In some embodiments, the administering comprises administering 1% prenidsolone acetate topically for 28 days. In some embodiments, after the administering, the human subject has a significant improvement in PGIC. In some embodiments, after the administering the human subject has closure of a foveal schisis. In some embodiments, after the administering the human subject has a reduction in central retinal thickness. In some embodiments, after the administering the human subject has improvement in retinal sensitivity. In some embodiments, the administering a) preserves one or more photoreceptor cells, b) restores laminar retinal structure, c) restores one or more rod- and / or cone-mediated functions, d) restores completely or partially visual behavior in one or both eyes, or e) any combination thereof. In some embodiments, the administering restores laminar retinal structure. In some embodiments, the human subject is sensitive to inflammation. In some embodiments, the dose of the AAV particle reduces inflammation in the human subject as compared to a higher dose of the AAV particle.

[0014] In certain aspects, disclosed herein is a method of treating X-linked retinoschisis (XLRS) in a human subject in need thereof, the method comprising administering to an eye of the human subject about 150 pL of a solution comprising an AAV particle. In some embodiments, the AAV particle comprises a polynucleotide comprising a heterologous nucleic acid encoding a retinoschisin (RS) protein. In some embodiments, the RS protein is a human RS protein. In some embodiments, the RS protein has the amino acid sequence of SEQ ID NO: 12. In some embodiments, the RS protein is encodedby a nucleic acid sequence at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the nucleic acid sequence of SEQ ID NO: 8. In some embodiments, the polynucleotide comprises a human rhodopsin kinase promoter. In some embodiments, the human rhodopsin kinase promoter comprises a sequence at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 7. In some embodiments, the polynucleotide comprises a sequence at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 33 (pTR-X002-3pSR). In some embodiments, the AAV particle comprises a capsid. In some embodiments, the capsid comprises a VP1 amino acidWSGR Docket No. 58774-736.601 sequence at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1. In some embodiments, the capsid comprises a VP2 amino acid sequence at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 2. In some embodiments, the capsid comprises a VP3 amino acid sequence at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to: SEQ ID NO: 3. In some embodiments, the human subject has a mutation in the RSI gene. In some embodiments, the human subject has an increased risk of retinal detachment as compared to a human subject that does not have the mutation in the RSI gene. In some embodiments, the human subject has a reduced risk of retinal detachment after the administering. In some embodiments, the administering comprises subretinal administration to a fovea of one or both eyes of the mammal. In some embodiments, detachment of the fovea is minimized. In some embodiments, the human subject has a significant improvement as measured by patient global impression of change (PGIC) as compared to before the administering. In some embodiments, the human subject has loss of vision prior to the administering. In some embodiments, the human subject regains vision after the administering. In some embodiments, regaining vision comprises an improvement in visual acuity or an improvement in the visual field. In some embodiments, the human subject expresses native RSI after the administering, optionally wherein the native RSI is SEQ ID NO: 12. In some embodiments, the human subject expresses native RSI in photoreceptors after the administering. In some embodiments, the human subject expresses native RSI in bipolar cells after the administering. In some embodiments, the human subject is 6 years of age or older. In some embodiments, the human subject is 18 years old or older. In some embodiments, the human subject is between 6 years of age and 65 years of age. In some embodiments, the human subject has XLRS caused by a pathogenic mutation in RSI. In some embodiments, prior to the administering the human subject has foveal schisis in the eye. In some embodiments, prior to the administering the human subject has parafoveal / perifoveal schisis in the eye. In some embodiments, before and / or after the administering, the method comprises performing optical coherence tomography on the human subject. In some embodiments, before and / or after the administering, the method comprises performing microperimetry on the human subject. In some embodiments, the AAV particle is administered via a subretinal injection. In some embodiments, the AAV particle is administered in a volume of about 150 microliters. In some embodiments, the AAV particle is administered in 2 blebs. In some embodiments, the AAV particle is administered in a manner that avoids foveal detachment. In some embodiments, the corticosteroid comprises prednisone, triamcinolone acetonide, methylprednisolone, prenidsoloneWSGR Docket No. 58774-736.601 acetate, or a combination thereof. In some embodiments, the corticosteroid comprises prednisone. In some embodiments, the corticosteroid is administered starting about 7 weeks prior to the administering of the AAV particle. In some embodiments, the corticosteroid is administered at about 1 mg / kg of the human subject per day. In some embodiments, the triamcinolone acetonide is administered starting about 7 weeks prior to the administering of the AAV particle. In some embodiments, about 20 mg of the triamcinolone acetonide is administered. In some embodiments, the triamcinolone acetonide is administered in a periocular injection. In some embodiments, the methylprednisolone is administered starting about 7 weeks prior to the administering of the AAV particle. In some embodiments, about 250 mg of the methylprednisolone is administered. In some embodiments, the methylprednisolone is administered via IV. In some embodiments, the prenidsolone acetate is administered starting about 28 days prior to the administering of the AAV particle. In some embodiments, about 1% of the prenidsolone acetate is administered. In some embodiments, the prenidsolone acetate is administered topically. In some embodiments, the administering comprises administering prednisone at a dose of Img / kg / day for seven weeks. In some embodiments, the administering comprises administering 20 mg of triamcinalone acetonide by periocular injection. In some embodiments, the administering comprises administering 250 mg of methylprednisolone intravenously. In some embodiments, the administering comprises administering 1% prenidsolone acetate topically for 28 days. In some embodiments, after the administering, the human subject has a significant improvement in PGIC. In some embodiments, after the administering the human subject has closure of a foveal schisis. In some embodiments, after the administering the human subject has a reduction in central retinal thickness. In some embodiments, after the administering the human subject has improvement in retinal sensitivity. In some embodiments, the administering a) preserves one or more photoreceptor cells, b) restores laminar retinal structure, c) restores one or more rod- and / or cone-mediated functions, d) restores completely or partially visual behavior in one or both eyes, ore) any combination thereof. In some embodiments, the administering restores laminar retinal structure. In some embodiments, the human subject is sensitive to inflammation. In some embodiments, the dose of the AAV particle reduces inflammation in the human subject as compared to a higher dose of the AAV particle.

[0015] In certain aspects, disclosed herein is a method of treating X-linked retinoschisis (XLRS) in a human subject in need thereof, the method comprising administering to an eye of the human subject an AAV particle and a corticosteroid. In some embodiments, the AAV particle comprises a polynucleotide comprising a heterologous nucleic acid encoding a retinoschisin (RS) protein. In some embodiments, the RS protein is a human RS protein. In some embodiments, the RS protein has the amino acid sequence of SEQ ID NO: 12. In some embodiments, the RS proteinWSGR Docket No. 58774-736.601 is encoded by a nucleic acid sequence at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the nucleic acid sequence of SEQ ID NO: 8. In some embodiments, the polynucleotide comprises a human rhodopsin kinase promoter. In some embodiments, the human rhodopsin kinase promoter comprises a sequence at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 7. In some embodiments, the polynucleotide comprises a sequence at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 33 (pTR-X002-3pSR). In some embodiments, the AAV particle comprises a capsid. In some embodiments, the capsid comprises a VP1 amino acid sequence at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1. In some embodiments, the capsid comprises a VP2 amino acid sequence at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 2. In some embodiments, the capsid comprises a VP3 amino acid sequence at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to: SEQ ID NO: 3. In some embodiments, the human subject has a mutation in the RSI gene. In some embodiments, the human subject has an increased risk of retinal detachment as compared to a human subject that does not have the mutation in the RSI gene. In some embodiments, the human subject has a reduced risk of retinal detachment after the administering. In some embodiments, the administering comprises subretinal administration to a fovea of one or both eyes of the mammal. In some embodiments, detachment of the fovea is minimized. In some embodiments, the human subject has a significant improvement as measured by patient global impression of change (PGIC) as compared to before the administering. In some embodiments, the human subject has loss of vision prior to the administering. In some embodiments, the human subject regains vision after the administering. In some embodiments, regaining vision comprises an improvement in visual acuity or an improvement in the visual field. In some embodiments, the human subject expresses native RSI after the administering, optionally wherein the native RSI is SEQ ID NO: 12. In some embodiments, the human subject expresses native RSI in photoreceptors after the administering. In some embodiments, the human subject expresses native RSI in bipolar cells after the administering In some embodiments, the human subject is 6 years of age or older. In some embodiments, the human subject is 18 years old or older. In some embodiments, the human subject is between 6 years of age and 65 years of age. In some embodiments, the human subject has XLRS caused by a pathogenic mutation in RSI . In some embodiments, prior to the administering the human subjectWSGR Docket No. 58774-736.601 has foveal schisis in the eye. In some embodiments, prior to the administering the human subject has parafoveal / perifoveal schisis in the eye. In some embodiments, before and / or after the administering, the method comprises performing optical coherence tomography on the human subject. In some embodiments, before and / or after the administering, the method comprises performing microperimetry on the human subject. In some embodiments, the AAV particle is administered via a subretinal injection. In some embodiments, the AAV particle is administered in a volume of about 150 microliters. In some embodiments, the AAV particle is administered in 2 blebs. In some embodiments, the AAV particle is administered in a manner that avoids foveal detachment. In some embodiments, the corticosteroid comprises prednisone, triamcinolone acetonide, methylprednisolone, prenidsolone acetate, or a combination thereof. In some embodiments, the corticosteroid comprises prednisone. In some embodiments, the corticosteroid is administered starting about 7 weeks prior to the administering of the AAV particle. In some embodiments, the corticosteroid is administered at about 1 mg / kg of the human subject per day. In some embodiments, the triamcinolone acetonide is administered starting about 7 weeks prior to the administering of the AAV particle. In some embodiments, about 20 mg of the triamcinolone acetonide is administered. In some embodiments, the triamcinolone acetonide is administered in a periocular injection. In some embodiments, the methylprednisolone is administered starting about 7 weeks prior to the administering of the AAV particle. In some embodiments, about 250 mg of the methylprednisolone is administered. In some embodiments, the methylprednisolone is administered via IV. In some embodiments, the prenidsolone acetate is administered starting about 28 days prior to the administering of the AAV particle. In some embodiments, about 1% of the prenidsolone acetate is administered. In some embodiments, the prenidsolone acetate is administered topically. In some embodiments, the administering comprises administering prednisone at a dose of Img / kg / day for seven weeks. In some embodiments, the administering comprises administering 20 mg of triamcinalone acetonide by periocular injection. In some embodiments, the administering comprises administering 250 mg of methylprednisolone intravenously. In some embodiments, the administering comprises administering 1% prenidsolone acetate topically for 28 days. In some embodiments, after the administering, the human subject has a significant improvement in PGIC. In some embodiments, after the administering the human subject has closure of a foveal schisis. In some embodiments, after the administering the human subject has a reduction in central retinal thickness. In some embodiments, after the administering the human subject has improvement in retinal sensitivity. In some embodiments, the administering a) preserves one or more photoreceptor cells, b) restores laminar retinal structure, c) restores one or more rod- and / or cone-mediated functions, d) restores completely or partially visual behavior in one or both eyes, or e) anyWSGR Docket No. 58774-736.601 combination thereof. In some embodiments, the administering restores laminar retinal structure. In some embodiments, the human subject is sensitive to inflammation. In some embodiments, the dose of the AAV particle reduces inflammation in the human subject as compared to a higher dose of the AAV particle.BRIEF DESCRIPTION OF THE DRAWINGS

[0016] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:

[0017] FIG. 1 shows AAV44.9-hGRKl-GFP (SEQ ID NO: 56) and AAV44.9(E531D)- hGRKl-GFP (SEQ ID NO: 56) exhibit enhanced lateral spread and potency in subretinally injected macaques. Vector delivered at l ><1012vg / mL. Initial boundaries of blebs on day of dosing and borders of resulting GFP expression are outlined in white dotted line. Identical vasculature is highlighted in thickened dark lines for reference.

[0018] FIG. 2 shows optical coherence tomography (OCT) scans of three subretinal injection blebs created (see negative contrast fundus image on the left) totaling 90 pL of vector volume, following extrafoveal subretinal injection of AAV44.9-hGRKl-GFP (SEQ ID NO: 56) (1 *1012vg / mL) in macaque. OCT allows for longitudinal, in vivo assessments of retinal structure, such as schisis lesions.

[0019] FIG. 3 shows OCT images of macaque retinas following extrafoveal subretinal injection of AAV44.9-hGRKl-GFP (SEQ ID NO: 56). Arrows in SLO image (top left) indicate the locations of retinal sections shown in the scans in the lower part of the figure. Sections were stained for cone arrestin and DAPI. The percentage of rods / cones expressing GFP is plotted in each zone. ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer. Staining for native GFP is indicated with circles, staining for cone arrestin is indicated with triangles, and staining for DAP is indicated with squares, in this figure and throughout the drawings.

[0020] FIG. 4 shows OCT images of macaque retinas following extrafoveal subretinal injection of AAV44.9(E531D)-hGRKl-GFP (SEQ ID NO: 56), three blebs totaling 90 pL of vector volume. Arrows in SLO image (top left) indicate the locations of retinal sections shown in the scans in the lower part of the figure. Sections were stained for cone arrestin and DAPI. The percentage of rods / cones expressing GFP is plotted in each zone.WSGR Docket No. 58774-736.601

[0021] FIGS. 5A-5D show representative OCT images of parafoveal regions of macaque retinas following extrafoveal subretinal injection of AAV44.9(E531D)-hGRKl-GFP (SEQ ID NO: 56) and AAV44.9-hGRKl-GFP (SEQ ID NO: 56). Scale bars are in A=40 microns, B=20 microns.

[0022] FIGS. 6A and 6B show representative OCT images of perifoveal regions of macaque retinas following injections of AAV44.9(E53 lD)-hGRKl-GFP (SEQ ID NO: 56) and AAV44.9- hGRKl-GFP (SEQ ID NO: 56).

[0023] FIG. 7 shows a schematic of a subretinal injection.

[0024] FIGS. 8A-8C show the transduction profile of sc-smCBA-mCherry (SEQ ID NO. 57) packaged into AAV44.9, AAV44.9(Y73 IF), AAV44.9(E53 ID), AAV5 and AAV8(Y733F) - following subretinal injection (SRI) in Nrl-GFP mice. (FIG. 8A) AAV-mediated mCherry expression in representative fluorescent fundus images (FIG. 8A, top row) and retinal cross sections (FIG. 8A, middle and bottom row) taken at 4 weeks post-injection with 2*1012vg / mL. Transduction profile of AAV44.9(E531D)-IRBP / GNAT2-GFP (SEQ ID NO: 58) following subretinal injection (SRI) in C57BL6J mice. (FIG. 8B) Six weeks post-injection with 2*1012vg / mL (2*109vg delivered), retinal cross sections were stained with an antibody directed against cone arrestin and counterstained with DAPI. (FIG. 8C) * Kruskal -Wallis tests were performed, followed by a posthoc Dunn’s test to make pairwise comparisons between groups. For the non-rod values: H=44.912, df=6, P<0.001. For the rod values: H=85.546, df=6, P<0.001. Scale bars in A, B=50 microns. RPE — retinal pigment epithelium, ONL — outer nuclear layer, INL — inner nuclear layer, GCL-ganglion cell layer.

[0025] FIGS. 9A-9E show natural history electroretinogram measurements (ERGs) at various months of wild-type, heterozygous and homozygous XLRS knockout mice. FIG. 9A shows natural history ERG at 1 month. FIG. 9B shows natural history ERG at 2 months. FIG. 9C shows natural history ERG at 3 months. FIG. 9D shows natural history ERG at 4 months. FIG. 9E shows natural history ERG at 5 months.

[0026] FIG. 10 shows a 1-5 month summary of the natural history ERG of RSI wildtype and knockout mice.

[0027] FIG. 11 shows the quantification of schisis lesions in Rsl KO mice. The extent of retinoschisis cavities was analyzed using Bioptigen OCT and facilitated by the Diver software. Eight (8) spots are designated by a 3 *3 grid. The center spot is the optical nerve (ON) where cavities are not present. The presence, abundance, and magnitude of schisis cavities within a 0.5 mm (500 m) segment were used to generate a score. Examples of scans and corresponding scores as well as descriptions of observations used to factor scores are shown in FIG. 12.WSGR Docket No. 58774-736.601

[0028] FIG. 12 shows the grading system for severity of schisis cavities (0-4), with 0 equaling no schisis cavities and 4 being the highest cavity count.

[0029] FIG. 13 shows a graph of retinoschisis cavity count. Cavity score peaks at 2 to 3 months and then improves with time.

[0030] FIG. 14 shows the OCT measurements and schisis activity scores associated with the natural history immunohistochemistry (IHC) assay. 1 month old mouse retinal sections stained with monoclonal antibody to RSI. RSI expression localized to the inner / outer segment junction of photoreceptors in the female heterozygous and wildtype male mice and is absent in male and female RSI KO mice.

[0031] FIGS. 15A-15F show schematics for six cassettes for exemplary vectors of the disclosure. Exemplary vectors include AAV-hGRKl-hRSlsyn, whichhas a length of 1723 bpfrom the 5' end of the first ITR to the 3' end of the second ITR(FIG. 15A, SEQ ID NO: 16), AAV-CBA- hRSlsyn, which has a length of 2977 bp from the 5' end of the first ITR to the 3' end of the second ITR (FIG. 15B, SEQ ID NO: 59), AAV-hGRKl -hRSl syn-WPREsf, which has a length of 2311 bp from the 5' end of the first ITR to the 3' end of the second ITR (FIG. 15C, SEQ ID NO: 17), pTR- X001-3p, which has a length of 4534 bp from the 5' end of the first ITR to the 3' end of the second ITR (FIG. 15D, SEQ ID NO: 31), pTR-X001-5p, which has a length of 4528 bp from the 5' end of the first ITR to the 3 ' end of the second ITR (FIG. 15E, SEQ ID NO: 32), and pTR-X002-3p, which has a length of 4549 bp from the 5' end of the first ITR to the 3' end of the second ITR (FIG. 15F, SEQ ID NO: 33). pTR-GRKl -hRSl syn was packaged in AAV5 and AAV44.9(E531D). pTR- CBA-hRSl syn vector plasmid (pTR-UFl 1 backbone) was packaged in AAV5 to serve as a control.

[0032] FIG. 16 shows OCT measurements (at 1 month post injection of AAV44.9(E531D)- hGRKlsyn (SEQ ID NO: 16), AAV5 -hGRKl -hRSl syn (SEQ ID NO: 16), and AAV5-CBA- hRSlsyn).

[0033] FIG. 17A shows data for the pilot AAV-RS1 study with AAV44.9(E53 lD)-hGRKl- hRSl (SEQ ID NO: 16), AAV5-GRK1-RS1 (SEQ ID NO: 16) and AAV5-CBA-RS1 (SEQ ID NO. 59). Test Injection: Retinoschisis cavity score (1 month post injection). Schisis cavities were completely resolved in all treated eyes. FIG. 17B shows data for the pilot AAV -RSI study with AAV44.9(E531D)-hGRKl-hRSl (SEQ ID NO: 16), AAV5-GRK1-RS1 (SEQ ID NO: 16) and AAV5-CBA-RS1 (SEQ ID NO. 59). ERG shown at 1 month post subretinal AAV-hRSl injection. Preliminary Conclusions: hGRKl promoter drove therapeutic RSI expression, and AAV44.9(E531D) vectored construct was therapeutic.

[0034] FIGS. 18A-18C show OCT monthly data after administration of rAAV44.9(E531D)- hGRKl-hRSlsyn (SEQ ID NO: 16) or rAAV5 -hGRKl -hRSl syn (SEQ ID NO: 16). FIG. 18AWSGR Docket No. 58774-736.601 shows OCT monthly data at 1 month and 2 months. FIG. 18B shows OCT monthly data at 3 months and 4 months. FIG. 18C shows OCT monthly data at 5 months and 6 months.

[0035] FIG. 19 shows OCT data of the left eye versus the right eye after administration of rAAV44.9(E531D)-hGRKl -hRSlsyn (SEQ ID NO: 16) or rAAV5-hGRKl -hRSlsyn (SEQ ID NO: 16).

[0036] FIG. 20 shows ERG measurements at one month after administration of rAAV44.9(E531D)-hGRKl -hRSlsyn (SEQ ID NO: 16) or rAAV5-hGRKl -hRSlsyn (SEQ ID NO: 16).

[0037] FIG. 21 shows ERG at two months after administration of rAAV44.9(E53 lD)-hGRKl - hRSlsyn (SEQ ID NO: 16) or rAAV5-hGRKl -hRSlsyn (SEQ ID NO: 16).

[0038] FIG. 22 shows ERG at three months after administration of rAAV44.9(E531D)- hGRKl -hRSlsyn (SEQ ID NO: 16) or rAAV5-hGRKl -hRSlsyn (SEQ ID NO: 16).

[0039] FIG. 23 shows ERG at four months after administration of rAAV44.9(E53 lD)-hGRKl - hRSlsyn (SEQ ID NO: 16) or rAAV5-hGRKl -hRSlsyn (SEQ ID NO: 16).

[0040] FIG. 24 shows ERG at five months after administration of rAAV44.9(E531D)-hGRKl- hRSlsyn (SEQ ID NO: 16) or rAAV5-hGRKl -hRSlsyn (SEQ ID NO: 16).

[0041] FIG. 25 shows ERG at six months after administration of rAAV44.9(E53 lD)-hGRKl- hRSlsyn (SEQ ID NO: 16) or rAAV5-hGRKl -hRSlsyn (SEQ ID NO: 16).

[0042] FIG. 26 shows a statistic analysis of b waves of the injected virus after administration of rAAV44.9(E53 lD)-hGRKl -hRSlsyn (SEQ ID NO: 16) or rAAV5-hGRKl -hRSlsyn (SEQ ID NO: 16).

[0043] FIG. 27 shows a statistic analysis of b waves of the injected eyes between 1 and 6 months after administration of rAAV44.9(E53 lD)-hGRKl -hRSlsyn (SEQ ID NO: 16) or rAAV5- hGRKl -hRSlsyn (SEQ ID NO: 16).

[0044] FIG. 28 shows a statistic analysis of b waves of AAV44.9 (E53 ID) injected eyes after administration of rAAV44.9(E53 lD)-hGRKl -hRSlsyn (SEQ ID NO: 16).

[0045] FIG. 29 shows a statistic analysis of b waves of the AAV5 injected eyes with rAAV5- hGRKl -hRSlsyn (SEQ ID NO: 16).

[0046] FIG. 30 shows a statistic analysis of scotopic a waves of the injected virus (rAAV44.9(E53 lD)-hGRKl -hRSlsyn (SEQ ID NO: 16)) relative to the empty vehicle.

[0047] FIG. 31 shows IHC measurements at 4 months post injection with rAAV44.9(E531D)- hGRKl -hRSlsyn (SEQ ID NO: 16).

[0048] FIG. 32 shows restoration of retinal structure in RS1KO mice treated with rAAV44.9(E53 ID) containing stuffed cassettes. Quantification of schisis cavity scores in RS1KOWSGR Docket No. 58774-736.601 mice treated with either vehicle or rAAV44.9(E53 ID) vectors containing the following cassettes: X001 (SEQ ID NO. 34), X001-3p (SEQ ID NO. 31), X001-5p (SEQ ID NO. 32), orX002-3p (SEQ ID NOS: 33). All vectors improved retinoschisis scores at both timepoints, except for X001-5p at 1- month post-injection. Nominal descriptive statistical significance was determined with a two-way ANOVA with a Tukey’s post-test on the treated eyes.

[0049] FIGS. 33A-33B show restoration of retinal function in RS1KO mice treated with rAAV44.9(E53 ID) containing stuffed cassettes. Average maximum scotopic (left) and photopic (right) b-wave amplitudes in RS1KO mice measured 1 - and 2-months after subretinal injection in one eye with either vehicle or rAAV44.9(E53 ID) containing the following cassettes: X001 (SEQ ID NO. 34), X001-3p (SEQ ID NO. 31), X001-5p (SEQ ID NO. 32), orX002-3p (SEQ ID NO. 33). Vector was dosed at either 1 *108vg(FIG. 33A) or 5*108vg (FIG. 33B). Retinal function in eyes treated with all vectors was improved over untreated control eyes. Nominal descriptive statistical significance was determined with two-way ANOVA with Tukey’s post-test on each individual data set. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0050] FIGS. 34A-34B shows RSI expression in retinas of RS1KO mice treated with rAAV44.9(E53 lD)-X002-3p (SEQ ID NO: 33). Representative retinal cross sections from RS1KO mice treated (top) in one eye only either vehicle or rAAV44.9(E53 ID) containing the X002-3p cassette (SEQ ID NO: 33). Vector was dosed at either l ><108vg (FIG. 34A) or 5 * 108vg (FIG. 34B) Contralateral untreated eyes are shown in the bottom row. All retinas stained with an antibody raised against RSI (triangles) and counterstained with DAPI (squares).

[0051] FIG. 35 shows a summary of the baseline characteristics for inclusion in an XLRS Phase 1 / 2 Clinical Trial for the administration of pTR-X002-3pSR Gene Therapy (SEQ ID NO. 33) in male subjects. The first cohort was administered low dose(1.5E10vg / eye) pTR-X002-3pSR. The second cohort was administered low dose (5.0E10vg / eye) pTR-X002-3pSR. The third cohort was administered 3.0 x 1O10vg / eye. The fourth cohort was administered a high volume (2.3 x 1O10vg / eye, 225 pL) or a low volume (1.5 x 1010vg / eye, 150 pL).

[0052] FIG. 36 shows OCT data for closure of foveal schisis in treated eyes, collected from the XLRS Phase 1 / 2 Clinical Trial in both cohorts.

[0053] FIG. 37 shows OCT data for closure of foveal schisis in untreated eyes, collected from the XLRS Phase 1 / 2 Clinical Trial in both cohorts.

[0054] FIG. 38 shows the CSF thickness of eyes treated with pTR-X002-3pSR. Data were collected from the XLRS Phase 1 / 2 Clinical Trial in both cohorts.WSGR Docket No. 58774-736.601

[0055] FIG. 39 shows OCT data for closure of foveal schisis in treated eyes corresponds with administration of pTR-X002-3pSR Gene Therapy as demonstrated by GFP expression. Data were collected from the XLRS Phase 1 / 2 Clinical Trial in both cohorts.

[0056] FIG. 40 shows data demonstrating visual acuity in subjects treated with pTR-X002- 3pSR. Data were collected from the XLRS Phase 1 / 2 Clinical Trial in both cohorts.

[0057] FIGs. 41A-41B show AAV44.9(E53 ID) exhibits enhanced lateral spread and potency in subretinally injected macaques. AAV44.9(E53 ID) containing either myc-tagged human RSI [AAV44.9(E53 ID)-RSl(myc)] (SEQ ID NO. 60) or green fluorescent protein (AAV44.9(E531D)- GFP) (SEQ ID NO. 56), both driven by a photoreceptor specific promoter were co -delivered at a concentration of 6.6E11 vg / mL. AAV5 vectors containing identical constructs were used as a control. Initial boundaries of blebs on day of dosing and borders of resulting GFP expression are outlined in dotted line. Foveal transduction was achieved following placement of either one or two injection blebs. In contrast, AAV5 -mediated GFP remained confined to margins of the original bleb, and no foveal transduction was achieved. Identical vasculature is highlighted in thickened dark lines for reference.

[0058] FIGs. 42A-42D show representative immunohistochemistry images of perifoveal regions of macaque retinas following the injections described in FIGs. 41A-41B. FIGs. 42A-42B show retinal transduction by AAV44.9(E53 lD)-hGRKl-GFP (SEQ ID NOS: 56) and AAV44.9(E53 lD)-hGRKl-mycRSl (SEQ ID NOS: 60) vectors delivered in two 50 mL blebs (FIG. 42A), or one 100 mLbleb (FIG. 42B). Two 50 pL blebs containing both vectors (3.3 x 1010vg each) (FIG. 42A), or a single 100 pL bleb containing both vectors (3.3 x 1010vg each) (FIG. 42B) were placed subretinally outside the macula. In life cSLO images (inset) reveal location of original injection blebs at 1-week post-injection and the full extent of spread at 7-weeks postinjection. Widefield image (top) from the central retinal block (containing the fovea) stained with antibodies raised against myc and RSI and counterstained with DAPI revealed GFP and myc expression beyond the margins of the original subretinal injection bleb. High magnification images of the fovea reveal that the majority of foveal cones were GFP positive, that AAV44.9(E531D)- mediated myc expression (red) was found in the fovea and that it localized to the same locations as endogenous RSI expression (yellow). IS / OS = inner segments / outer segments; ONL = outer nuclear layer; INL = inner nuclear layer; GC = ganglion cells . FIG. 42C shows retinal transduction by AAV5-hGRKl-GFP and AAV5-hGRKl-mycRSl vectors. Two 50 pL blebs containing both vectors (3.3 x 1010vgeach) were placed subretinally outside the macula. In life cSLO images are included (inset, top) to indicate location of original blebs at 1 -week post-injection and the extent of GFP expression at 7-weeks post-injection. No AAV5-mediated transgene expression was observedWSGR Docket No. 58774-736.601 outside the original injection blebs. Widefield images from the central retinal block (left) and high magnification foveal images (right) stained with antibodies against myc and RSI and counterstained with DAPI revealed an absence of AAV5-mediated GFP or myc expression Red (myc) and green (GFP) channels were brightened to emphasize this absence of expression. OS = outer segments; IS = inner segments; ONL = outer nuclear layer; INL = inner nuclear layer; GC = ganglion cells. FIG. 42D shows a graph demonstrating the lateral spread of AAV44.9(E53 ID) vs. AAV5 injected eyes. GFP and mycRSl expression in retinal cross sections located equal distances away from subretinal injection bleb margins in macaques injected with AAV5 (left) or AAV.SPR (right) vectors. The percent of photoreceptors transduced in each location by AAV -GFP is quantified on the bottom left. GFP and mycRSl expression are seen at the bleb margins in both AAV5 (albeit at lower levels) and AAV.SPR injected animals. No AAV5 -mediated GFP or mycRSl is observed beyond the bleb margins whereas AAV. SPR-mediated expression of both transgenes can be seen multiple millimeters away. MycRSl expression is not observed in areas devoid of GFP expression. IS = inner segments; ONL = outer nuclear layer; INL = inner nuclear layer; GC = ganglion cells.

[0059] FIG. 43 shows electroretinogram measurements (ERGs) at month 1, month 2, month 3, month 4, month 5 and month 6 post-injection and a graph of retinoschisis score of AAV44.9(E53 lD)-hGRKl-RSl (SEQ IDNO: 16) injected eyes at low 3.0 x 108vg / eye, mid 1.0 x 109vg / eye, and high 3.0 x 109vg / eye doses in RSI KO mice.

[0060] FIG. 44 shows electroretinogram measurements (ERGs) at month 1 and month 3 postinjection and a graph of retinoschisis score of AAV44.9(E53 lD)-hGRKl-RSl (SEQ ID NO: 16) injected eyes at low (3.0 x 107vg / eye (n=20)), middle (1.3 x 108vg / eye (n=20)), or high (High 5.3 x 108vg / eye (n=20)) or max (2.1 x 109vg / eye (n=20)) dose in RSI KO mice.

[0061] FIGs. 45A-45D shows evaluation of retinal structure and function, and RSI expression following subretinal injection of AAV vectors rAAV44.9(E53 lD)-X001 (SEQ ID NO: 34) delivering human RSI to RS1KO mice. FIG. 45A depicts scotopic b-wave amplitude changes (Mean ± SE) and FIG. 45B depicts photopic b-wave amplitude changes (Mean ± SE) over time per group. Data were collected for both the injected and uninjected eyes. Change was computed as the observed value from the injected eye minus the observed value from the uninjected eye (injected - uninjected) per individual mouse. FIG. 45C depicts retinoschisis score changes (Mean ± SE) over time per group. Data were collected for both the injected and uninjected eyes. Retinoschisis score change was computed as the observed value from the injected eye minus the observed value from the uninjected eye (injected - uninjected) per individual mouse. FIG. 45D depicts representative retinal cross sections from RS1KO mice unilaterally injectedWSGR Docket No. 58774-736.601 rAAV44.9(E531D)-XOO1 (SEQ ID NO: 34) at a low (3.0 x 108vg / eye), mid 1.0 x 109vg / eye), or high dose (3.0 x 109vg / eye), or with vehicle. Contralateral untreated eyes are shown in the right row. All retinas were stained with an antibody raised against RSI (red) and counterstained with DAPI (blue). INL = inner nuclear layer, IS = inner segments, ONL= outer nuclear layer.DETAILED DESCRIPTION

[0062] The present disclosure provides rAAV vectors, compositions, methods, for administration of rAAV particles for treatment of XLRS. These vectors are designed for delivery of a therapeutic agent comprising a synthetic retinoschisin gene to mammalian subjects, such as human subjects. Advantageously, the methods of rAAV particle administration disclosed herein have improved efficiency in transducing the retina of the mammalian eye, and in particular, in transducing the photoreceptor (PR) and retinal pigment epithelial (RPE) cells in vivo. Specifically, the disclosed rAAV vectors and compositions are capable of lateral spread beyond the site of vector injection, which will lead to efficient transduction of both intact as well as schisis (split) retina while utilizing relatively small volumes or doses of AAV vector. Accordingly, the disclosed methods may reduce risk of additional damage occurring due to surgical intervention near a schisis lesion, while ensuring that highly efficient photoreceptor transduction is achieved. The disclosed methods may thus provide for safe and efficient delivery of the retinoschisin (RSI) gene to photoreceptors. The disclosure also provides cells, such as host cells, containing any of the disclosed rAAV vectors.

[0063] In particular embodiments, delivery of this therapeutic agent a) preserves one or more photoreceptor (PR) cells, b) restores laminar retinal structure, c) restores one or more rod - and / or cone-mediated functions, d) restores completely or partially visual behavior in one or both eyes, or e) any combination thereof. In some embodiments, delivery of this therapeutic agent results in the closure of at least one schisis cavity. In some embodiments, production of the therapeutic agent persists in the one or more photoreceptor cells or the one or more RPE cells substantially for a period of at least six months following an initial administration of the rAAV particle into the one or both eyes of the mammal. In some embodiments, production of the therapeutic agent persists in the one or more PR cells or the one or more RPE cells for a period of at least 12 months, at least 18 months, or at least 24 months following an initial administration of the rAAV particle.

[0064] In some embodiments, a polynucleotide comprising a heterologous nucleic acid that is encapsidated into an rAAV particle is provided. In particular embodiments, the heterologous nucleic acid is administered to the subject to provide a functional protein, e.g., human retinoschisinWSGR Docket No. 58774-736.601(RSI), to restore, e.g., completely, or partially, photoreceptor function to a subject (e.g., a human) as well as restore laminar retinal structure, i.e. reduce schisis lesions. In some embodiments, one or both alleles of a target coding sequence of the subject are silenced by administering an rAAV particle comprising a heterologous nucleic acid disclosed herein to the subject (e.g., to a human suffering from XLRS).

[0065] In some aspects, the disclosure provides compositions comprising a rAAV particle and a pharmaceutically acceptable carrier, excipient, diluent and / or buffer. In some aspects, the disclosure provides a method of transducing RPE and photoreceptor cells to modulate expression of the heterologous nucleic acid (or transgene) in a subject, the method comprising administering to the subject, such as a human subject, a composition comprising an rAAV particle as described herein and a pharmaceutically acceptable carrier, excipient, diluent, buffer, and any combination thereof. In some aspects, the disclosure provides a method of treating XLRS in a subject, the method comprising administering a composition to the eye of a subject.

[0066] In some aspects, the disclosure provides a composition for use in treating retinal disease and a composition for use in the manufacture of a medicament to treat retinal disease. In some aspects, the disclosure provides a composition comprising an rAAV particle as described herein for use in treatment by subretinally or intravitreally administering to one or both eyes of the mammal.

[0067] In some aspects, administration of the disclosed rAAV vectors is performed in a manner and / or dose that reduces or prevents inflammation . In a non-limiting example, the administration is subretinal (see FIG. 7). Some research suggests administration of high doses of AAV particles causes inflammation. Systemic administration of high doses of AAV particles that target the liver, neurons and / or muscles have led to deaths of subjects in a handful of clinical trials following excessive inflammation and onset of liver disease. Despite strong proof of concept studies in mouse models for subretinally delivered AAV -RSI vectors, the first clinical trials utilized intravitreally (IVT) delivered AAVs because the risk:benefit ratio precluded use of subretinal injection f or the treatment of XLRS. With the advent of intra-operative optical coherence tomography (OCT), vector placement can be tightly controlled by surgeons. In particular, the vector can be injected to areas beyond the schisis lesions. In certain embodiments, AAV vectors herein exhibit lateral spread beyond the injection site for efficient transduction of both intact as well as schisis retinal areas. The dosing may be about 1.5 x 1010vgto about 5.0 x 1010vg per eye. The dosing may be 3.0 x 1010vg per eye. The dosing may be 1.5 x 1010vg per eye.

[0068] The disclosed rAAV vectors utilize capsid variants that exhibit increased lateral spread and high transduction efficiencies, such as AAV44.9(E53 ID) and other AAV44.9 variants. The high efficiency and lateral spread mediated by AAV44.9(E531D) allows for smaller injection blebWSGR Docket No. 58774-736.601 volumes, thereby further reducing risk by limiting the area of detachment. Accordingly, the use of any of the disclosed rAAV vectors may facilitate reduction of bleb volumes and / or doses for ocular administration necessary to achieve a therapeutic effect in subjects, such as human subjects. Methods of administration of the disclosed rAAV vectors may enable targeting of subretinal injections to areas of the retina (e.g., the retina of an XLRS patient) that do not contain lesions or are relatively intact. These methods may be facilitated by the use of intra-operative OCT to guide the vitreoretinal surgeon’s placement of blebs.

[0069] Accordingly, the disclosed rAAV particles may be administered in injection bleb volumes of less than 250 pL, less than 200 pL, less than 175 pL, less than 150 pL, less than 125 pL, less than 100 pL, less than 90 pL, less than 75 pL, less than 50 pL, or less than 45 pL, wherein the minimal injection volume is 10 pL. In some embodiments, 0.0001 mL to 10 mL (e.g., 0.0001 mL, 0.001 mL, 0.01 mL, 0.1 mL, 1 mL, 10 mLs) are delivered to the retina of a subject in a dose. In some embodiments, the disclosed rAAV particles are administered in an inject bleb volume of about 150 pL. In some embodiments, a dose of between 5 ^1010to 1 xio12vector genomes (vgs) / mL is administered to the retina. In some embodiments, a dose of about 1.5 x io10vg / eye is delivered to the eye. In the non -human primate experiments of Examples provided herein, a total of 90 pL (3 individual injection blebs of 30 pL each) was administered subretinally to primate retina (see Example 1). These volumes are substantially smaller than the volumes used in standard clinical application of AAV gene therapy. For example, Luxturna(voretigeneneparvovec-rzyl), an rAAV- RPE65 vector, is delivered in a 300 pL subretinal injection. Accordingly, in some embodiments, a single subretinal injection of any of the disclosed vectors may achieve therapeutic effects. In some embodiments, no more than 3-5 subretinal injections may achieve therapeutic effects. In some embodiments, subretinal injections of disclosed rAAV particles or compositions thereof ranging in total volumes of between 30 pL and 300 pL, or more preferably between 30 pL and 150 pL may be administered. In particular embodiments, 3 subretinal injections ranging in bleb volumes of between 10 pL and 10 pL each may be administered. In some embodiments, the volume is 150 uL.Synthetic RSI Sequences

[0070] In some embodiments, the heterologous nucleic acid of any of the polynucleotides of the disclosure has a sequence that has at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98%, at least 99% identity, at least 99.5% identity, or 100% identity to a nucleotide sequence set forth as SEQ ID NO: 8. This synthetic RSI transgene lacks 5' and 3 ' untranslated regions relative to the cDNA of wild-type RSI . This sequence is referred to herein is “hRSl syn.” The length of SEQ ID NO: 8 is 684 nucleotides (nt). In some embodiments, the heterologousWSGR Docket No. 58774-736.601 nucleic acid differs by 5, 10, 15, 20, 25, or more than 25 nucleotides from the nucleotide sequence of SEQ ID NO: 8.

[0071] In various embodiments, the heterologous nucleic acid of any of the polynucleotides of the disclosure does not comprise the 5' untranslated region (UTR) of the (wild-type) gene (or cDNA) encoding human retinoschisin (SEQ ID NO: 39). In some embodiments, the heterologous nucleic acid does not comprise the 3' UTR of the human retinoschisin gene (SEQ ID NO: 40). In various embodiments, the heterologous nucleic acid does not comprise the 3 ' UTR or the 5' UTR of the human retinoschisin gene. In various embodiments, the heterologous nucleic acid does not comprise truncated versions of the 3 ' UTR or the 5' UTR of the human retinoschisin gene. In some embodiments, the heterologous nucleic acid does not comprise a wild -type RSI cDNA. In some embodiments, the heterologous nucleic acid consists of an RSI coding region.

[0072] In some embodiments, the heterologous nucleic acid comprises the nucleic acid sequence of any one of SEQ ID NOs: 8, 9 or 10. In some embodiments, the heterologous nucleic acid consists of the nucleic acid sequence of any one of SEQ ID NOs: 8, 9 or 10.

[0073] A nucleotide sequence encoding a synthetic human retinoschisin 1 (RSI) transgene (SEQ ID NO: 8) are shown below. The start codon is underlined; and the stop codon is bolded.

[0074] hRSlsyn: (SEQ ID NO: 8) ATGTCACGCAAGATAGAAGGCTTTTTGTTATTACTTCTCTTTGGCTATGAAGCCACATTGGGATTATCGTCTACCGAGGATGAAGGCGAGGACCCATGGTATCAAAAA GCCTGCAAGTGCGATTGCCAAGGAGGACCCAATGCTCTGTGGTCTGCAGGTGCCACCT CCTTGGACTGTATACCAGAATGCCCATATCACAAGCCTCTGGGTTTCGAGTCAGGGGA GGTCACACCGGACCAGATCACCTGCTCTAACCCGGAGCAGTATGTGGGCTGGTATTCT TCGTGGACTGCAAACAAGGCCCGGCTCAACAGTCAAGGCTTTGGGTGTGCCTGGCTCT CCAAGTTCCAGGACAGTAGCCAGTGGTTACAGATAGATCTGAAGGAGATCAAAGTGAT TTCAGGCATCCTCACCCAGGGGCGCTGTGACATCGATGAGTGGATGACCAAGTACAGC GTGCAGTACAGGACCGATGAGCGCCTGAACTGGATTTACTACAAGGACCAGACTGGA AACAACCGGGTCTTCTATGGCAACTCGGACCGCACCTCCACGGTTCAGAACCTGCTGC GGCCCCCCATCATCTCCCGCTTCATCCGCCTCATCCCGCTGGGCTGGCACGTCCGCATT GCCATCCGGATGGAGCTGCTGGAGTGCGTCAGCAAGTGTGCCTGA.

[0075] By a nucleic acid molecule (e.g., a heterologous nucleic acid, or transgene) comprising a nucleotide sequence having at least, for example, 95% “identity” to a query nucleic acid sequence, it is intended that the nucleotide sequence of the subject nucleic acid molecule is identical to the query sequence except that the subject nucleic acid molecule sequence may include up to five nucleotide alterations per each 100 nucleotides of the query sequence. In other words, to obtain aWSGR Docket No. 58774-736.601 promoter having a nucleotide sequence at least 95% identical to a reference (query) sequence, up to 5% of the nucleotides in the subject sequence maybe inserted, deleted, or substituted with another nucleotide. These alterations of the reference sequence may occur at the 5' or 3' ends of the reference sequence or anywhere between those positions, interspersed either individually among nucleotides in the reference sequence or in one or more contiguous groups within the reference sequence.

[0076] As a practical matter, whether any particular nucleic acid molecule is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to, for instance, the nucleotide sequence of a synthetic RSI cDNA, can be determined conventionally using known computer programs.Preferred methods for determining the best overall match between a query sequence (a sequence of the present disclosure) and a subject sequence, also referred to as a global sequence alignment, can be determined using the FASTA program analysis described by Pearson and Lipman (1988) and FASTDB and blastn computer programs based on the algorithm of Brutlag et al. (Comp. App. Biosci. 6:237-245 (1990)). In a sequence alignment the query and subject sequences are either both nucleotide sequences or both amino acid sequences. The result of said global sequence alignment is expressed as percent identity. Preferred parameters used in a FASTDB amino acid alignment are: Matrix=PAM 0, k-tuple=2, Mismatch Penalty=l, Joining Penalty=20, Randomization Group Length =0, Cutoff Score=l, Window Size=sequence length, Gap Penalty =5, Gap Size Penalty=0.05, Window Size=500 or the length of the subject amino acid sequence, whichever is shorter.

[0077] Whether a nucleotide is matched / aligned is determined by results of the FASTDB sequence alignment. This percentage is then subtracted from the percent identity, calculated by the above FASTDB program using the specified parameters, to arrive at a final percent identity score. This final percent identity score is what is used for the purposes of the present disclosure. For subject sequences truncated at the 5' and / or 3 ' ends, relative to the query sequence, the percent identity is corrected by calculating the number of nucleotides of the query sequence that are positioned 5 ' to or 3' to the query sequence, which are not matched / aligned with a corresponding subject nucleotide, as a percent of the total bases of the query sequence.

[0078] In some embodiments, vectors containing any of the polynucleotides as described herein (such as the polynucleotides of SEQ ID NOs: 8-10) may comprise a Kozak sequence immediately 5' of the start codon sequence (i.e., ATG). In some embodiments, this Kozak sequence comprises the nucleic acid sequence of GCCGCCACC (SEQ ID NO: 55). In some embodiments, the Kozak sequences of any of the disclosed vectors (e.g., those set forth in SEQ ID NOs: 31 -35) contain a Kozak sequence having 1, 2, or 3 nucleotides that differ relative to SEQ ID NO: 55. In some embodiments, any of these Kozak sequences constitutes a ribosomal entry site.WSGR Docket No. 58774-736.601

[0079] In some embodiments, the polynucleotides as described herein may comprise a nucleic acid sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 nucleotides that differ relative to the sequence as set forth in any one of SEQ ID NOs: 7-10 and 15-17, e.g., SEQ ID NOs: 8-10, 16 and 17. In some embodiments, the polynucleotides as described herein may comprise a nucleic acid sequence having 1 -10 (e.g., 5 or 9) nucleotides that differ relative to the sequence of any one of SEQ ID NOs: 8, 9 and 10. These differences may comprise nucleotides that have been inserted, deleted, or substituted relative to the sequence of any one of SEQ ID NOs: 7-10 and 15-17. In some embodiments, the polynucleotides comprise truncations at the 5' or 3' end relative to any one of SEQ ID NOs: 7-10 and 15-17. In some embodiments, the disclosed polynucleotides contain stretches of about 50, about 75, about 100, about 125, about 150, about 175, or about 180 nucleotides in common with the sequence of any one of SEQ ID NOs: 7-10 and 15-17. In some embodiments, the disclosed polynucleotides contain stretches of about 50, about 75, about 100, about 125, about 150, about 175, about 180, about200, about 300, about 400, about 500, about 600, about 700, about 800, about 900, about 1000, or more than about 1000 nucleotides in common with the sequence of any one of SEQ ID NOs: 16 and 17.

[0080] In some embodiments, the disclosed polynucleotides contain stretches of about 50, 75 or more nucleotides in common with any one of SEQ ID NOs: 8-10, 16 and 17 in regions of the sequence in which CpG islands are absent. The polynucleotides of the disclosure may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more than 12 silent mutations that do not result in mutations in the encoded RSI protein sequence. In some embodiments, the disclosed polynucleotides may comprise between 30 and 40 silent mutations relative to the wild-type RSI sequence. In some embodiments, a heterologous nucleic acid that varies in identity of up to 30% relative to (i.e., has at least 70% identity to) any of the sequences of SEQ ID NOs: 8-10, 16 and 17 encodes a polypeptide that has at least 90% amino acid sequence identity to a wild-type RSI protein. In some embodiments, a heterologous nucleic acid that varies in identity of up to 30% relative to any of the sequences of SEQ ID NOs: 8-10, 16 and 17 encodes a polypeptide that has at least 90% amino acid sequence identity to any of the sequences of SEQ ID NOs: 12-14. In some embodiments, a heterologous nucleic acid comprises at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98%, at least 99% identity, at least 99.5% identity, or 100% identity to the sequences of SEQ ID NOs: 8 -10, 16 and 17.

[0081] In some embodiments, the heterologous nucleic acid of any of the rAAV vectors of the disclosure has a sequence that has at least 75% identity, at least 80% identity, at least 85% identity,WSGR Docket No. 58774-736.601 at least 90% identity, at least 91 % identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98%, at least 99% identity, at least 99.5% identity, or 100% identity to the nucleotide sequence set forth as SEQ ID NO: 9. The heterologous nucleic acid may comprise SEQ ID NO: 9. A nucleotide sequence encoding a synthetic human retinoschisin 1 (RSI) transgene that has been codon -optimized for human expression and wherein CpG islands have been eliminated (SEQ ID NO: 9) is shown below. The start codon is underlined; and the stop codon is bolded.

[0082] (SEQ ID NO: 9)ATGTCAAGAAAGATAGAAGGCTTTTTGTTATTACTTCTCTTTGGCTATGAAGCCACATT GGGATTATCGTCTACAGAGGATGAAGGAGAGGACCCATGGTATCAAAAAGCCTGCAA GTGTGATTGCCAAGGAGGACCCAATGCTCTGTGGTCTGCAGGTGCCACCTCCTTGGAC TGTATACCAGAATGCCCATATCACAAGCCTCTGGGTTTTGAGTCAGGGGAGGTCACAC CTGACCAGATCACCTGCTCTAACCCTGAGCAGTATGTGGGCTGGTATTCTTCTTGGACT GCAAACAAGGCCAGACTCAACAGTCAAGGCTTTGGGTGTGCCTGGCTCTCCAAGTTCC AGGACAGTAGCCAGTGGTTACAGATAGATCTGAAGGAGATCAAAGTGATTTCAGGGA TCCTCACCCAGGGGAGATGTGACATTGATGAGTGGATGACCAAGTACTCTGTGCAGTA CAGGACAGATGAGCGCCTGAACTGGATTTACTACAAGGACCAGACTGGAAACAACAG AGTCTTCTATGGCAACTCTGACAGAACCTCCACAGTTCAGAACCTGCTGAGACCCCCC ATCATCTCCAGATTCATCAGACTCATCCCACTGGGCTGGCATGTCAGAATTGCCATCAG GATGGAGCTGCTGGAGTGTGTCAGCAAGTGTGCCTGA.

[0083] CpG islands are regions of the genome that contain a large number of cytosine -guanine dinucleotide repeats in the 5 '— 3 ' direction. In mammalian genomes, CpG islands usually extend for 300-3000base pairs. Cytosines in CpG dinucleotides may be, and are often, methylated through cellular epigenetic mechanisms to form 5 -methylcytosines. Methylation of these cytosines reduces expression of coding regions of any genes in which these islands appear (e.g., the RSI gene). In some embodiments, any of the disclosed polynucleotides comprise mutations (e.g., silent mutations) that interrupt thirty-six instances of CpG dinucleotides.

[0084] In some embodiments, the polynucleotide of the rAAV vector comprises a myc-tagged polynucleotide. In some embodiments, the heterologous nucleic acid of any of the rAAV nucleic acid vectors of the disclosure has a sequence that has at least 80% identity, at least 85% identity, at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98%, at least 99% identity, at least 99.5% identity, or 100% identity to the nucleotide sequence set forth as SEQ ID NO: 10. The heterologous nucleic acid may comprise SEQ ID NO: 10. A nucleotide sequenceWSGR Docket No. 58774-736.601 encoding a synthetic myc tagged-human retinoschisin 1 (RSI) transgene (SEQ ID NO: 10) is shown below. The start codon is underlined; and the stop codon is bolded.

[0085] (SEQ ID NO: 10)ATGTCACGCAAGATAGAAGGCTTTTTGTTATTACTTCTCTTTGGCTATGAAGCCACATT GGGATTATCGGAGCAGAAATTAATCAGTGAGGAAGATCTGTCTACCGAGGATGAAGG CGAGGACCCATGGTATCAAAAAGCCTGCAAGTGCGATTGCCAAGGAGGACCCAATGC TCTGTGGTCTGCAGGTGCCACCTCCTTGGACTGTATACCAGAATGCCCATATCACAAGC CTCTGGGTTTCGAGTCAGGGGAGGTCACACCGGACCAGATCACCTGCTCTAACCCGGA GCAGTATGTGGGCTGGTATTCTTCGTGGACTGCAAACAAGGCCCGGCTCAACAGTCAA GGCTTTGGGTGTGCCTGGCTCTCCAAGTTCCAGGACAGTAGCCAGTGGTTACAGATAG ATCTGAAGGAGATCAAAGTGATTTCAGGCATCCTCACCCAGGGGCGCTGTGACATCGA TGAGTGGATGACCAAGTACAGCGTGCAGTACAGGACCGATGAGCGCCTGAACTGGATT TACTACAAGGACCAGACTGGAAACAACCGGGTCTTCTATGGCAACTCGGACCGCACCT CCACGGTTCAGAACCTGCTGCGGCCCCCCATCATCTCCCGCTTCATCCGCCTCATCCCG CTGGGCTGGCACGTCCGCATTGCCATCCGGATGGAGCTGCTGGAGTGCGTCAGCAAGT GTGCCTGA.

[0086] In various embodiments, the rAAV vectors comprising a heterologous nucleic acid encode human retinoschisin 1 (RSI) protein, or a variant thereof. The amino acid sequences of the RSI protein variants encoded by each of the nucleotide sequences of SEQ ID NOs : 8-10, respectively, are shown below as SEQ ID NOs: 12-14. In some embodiments, the encoded RSI protein is a human retinoschisin protein defined by the amino acid sequence of SEQ ID NO: 12. In some embodiments, the encoded RSI protein may comprise a sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 amino acids that differ relative to the sequence of any one of SEQ ID NOs: 12-14. In some embodiments, the encoded RSI protein may comprise a sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 amino acids that differ relative to SEQ ID NO: 12. These differences may comprise amino acids that have been inserted, deleted, or substituted relative to the sequence of any one of SEQ ID NOs: 12-14. In some embodiments, the disclosed rAAV vectors encode a protein having an amino acid sequence having at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, atleast 98%, atleast 99% identity, at least 99.5% identity, or 100% identity to any of the amino acid sequences of SEQ ID NOs: 12-14.

[0087] Human synthetic RSI (SEQ ID NO: 12)MSRKIEGFLLLLLFGYEATLGLSSTEDEGEDPWYQKACKCDCQGGPNALWSAGATSLDCIWSGR Docket No. 58774-736.601PECPYHKPLGFESGEVTPDQITCSNPEQYVGWYSSWTANKARLNSQGFGCAWLSKFQDSS QWLQIDLKEIKVISGILTQGRCDIDEWMTKYSVQYRTDERLNWIYYKDQTGNNRVFYGNS DRTSTVQNLLRPPIISRFIRLIPLGWHVRIAIRMELLECVSKCA.

[0088] Human synthetic RSI, CpG islands eliminated (SEQ ID NO: 13) MSRKIEGFLLLLLFGYEATLGLSSTEDEGEDPWYQKACKCDCQGGPNALWSAGATSLDCI PECPYHKPLGFESGEVTPDQITCSNPEQYVGWYSSWTANKARLNSQGFGCAWLSKFQDSS QWLQIDLKEIKVISGILTQGRCDIDEWMTKYSVQYRTDERLNWIYYKDQTGNNRVFYGNS DRTSTVQNLLRPPIISRFIRLIPLGWHVRIAIRMELLECVSKCA.

[0089] Human synthetic myc tagged-RSl; C-myc tag underlined (SEQ ID NO: 14)MSRKIEGFLLLLLFGYEATLGLSEQKLISEEDLSTEDEGEDPWYOKACKCDCOGGPNALWS AGATSLDCIPECPYHKPLGFESGEVTPDQITCSNPEQYVGWYSSWTANKARLNSQGFGCA WLSKFQDSSQWLQIDLKEIKVISGILTQGRCDIDEWMTKYSVQYRTDERLNWIYYKDQTG NNRVFYGNSDRTSTVQNLLRPPIISRFIRLIPLGWHVRIAIRMELLECVSKCA.

[0090] In some embodiments, the nucleic acid vector comprising the heterologous nucleic acid (e.g., RSI) has a length of between 1700 nucleotides (nt, or base pairs (bp)) and about 5000, about 4550, about4549, about 4535, about4534, about 4528, about4525, about4500, about 3200, about 3000, about 2977, about 2900, about 2800, about 2311, about 2300, about 1725, about 1723, or about 1700 nucleotides from the 5 'beginning of a first ITR to the 3' end of a second ITR. In some embodiments, the nucleic acid vector has a length of about 1700 to about 1800 (e.g., about 1723) nucleotides from the 5 'beginning of a first ITR to the 3' end of a second ITR (e.g., FIG. 15A). In some embodiments, the nucleic acid vector has a length of about 2900 to about 3100 (e.g., about 2977) nucleotides from the 5' beginning of a first ITR to the 3' end of a second ITR (e.g., FIG.15B) In some embodiments, the nucleic acid vector has a length of about 2200 to about 2400 (e.g., about 2311) nucleotides from the 5' beginning of a first ITR to the 3' end of a second ITR (e.g., FIG. 15C). In some embodiments, the nucleic acid vector has a length of about 4400 to about 4600 (e.g., about 4534) nucleotides from the 5' beginning of a first ITR to the 3' end of a second ITR (e.g., FIG. 15D). In some embodiments, the nucleic acid vector has a length of about 4400 to about 4600 (e.g., about 4528) nucleotides from the 5' beginning of a first ITR to the 3' end of a second ITR (e.g., FIG. 15E). In some embodiments, the nucleic acid vector has a length of about 4400 to about 4700 (e.g., about 4549) nucleotides from the 5' beginning of a first ITR to the 3' end of a second ITR (e.g., FIG. 15F). In some embodiments, the nucleic acid vector comprising the heterologous nucleic acid comprises a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to anyWSGR Docket No. 58774-736.601 one of SEQ ID NOs: 16-17 and 31 -35. In some embodiments, the nucleic acid vector comprising the heterologous nucleic acid comprises the sequence of any one of SEQ ID NOs: 16-17 and 31-35.

[0091] In some embodiments, the polynucleotides as described herein may comprise a nucleic acid sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 nucleotides that differ relative to the sequence as set forth in any one of SEQ ID NOs: 31-35. These differences may comprise nucleotides that have been inserted, deleted, or substituted relative to the sequence of any one of SEQ ID NOs: 31-35. In some embodiments, the polynucleotides comprise truncations at the 5' or 3' end relative to any one of SEQ ID NOs: 31 -35. In some embodiments, the disclosed polynucleotides contain stretches of about 50, about 75, about 100, about 125, about 150, about 175, or about 180 nucleotides in common with the sequence of any one of SEQ ID NOs: 31 -35. In some embodiments, the disclosed polynucleotides contain stretches of about 50, about 75, about 100, about 125, about 150, about 175, about 180, about200, about 300, about400, about 500, about 600, about 700, about 800, about 900, about 1000, or more than about 1000 nucleotides in common with the sequence of any one of SEQ ID NOs: 31 -35.Splice Donor, Splice Acceptor Regions, and Introns

[0092] In some embodiments, the nucleic acid vector comprises an intron. In some embodiments, the nucleic acid vector comprises a splice donor and splice acceptor regions independent of an intron.

[0093] In some embodiments, the nucleic acid vector comprises an SV40 intron. In some embodiments, the SV40 intron comprises an SV40 splice donor region. In some embodiments, the SV40 intron comprises an SV40 splice acceptor region. In some embodiments, the SV40 intron comprises SV40 splice donor and splice acceptor regions (SV40 SD / SA) (e.g., see FIGS. 15A-15B and 15D-15F).

[0094] The length of the SV40 intron is 99 nucleotides. It was first reported by Ostedgaard et al. that the presence of the SV40 intron between the promoter and the transgene in an AAV expression cassette provided a two-fold increase of transgene expression in lung carcinoma cells, while under the control of a CMV promoter and enhancer (PNAS 2005; 102(8): 2952 -2957). Recently, it was shown that positioning an SV40 intron downstream of the expression cassette in a non-viral vector resulted in highest levels of expression of a reporter transgene (in Chinese hamster ovary cells). See Xu et al., J. Cell. Mol. Med. Vol 22, No 4 (2018): 2231 -2239. In some embodiments of the disclosed rAAV vectors, the SV40 intron is positioned downstream (3') of the heterologous nucleic acid. In some embodiments, the SV40 intron is positioned upstream (5') of the heterologous nucleic acid.WSGR Docket No. 58774-736.601

[0095] In some embodiments, the SV40 intron comprises a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 20. In some embodiments, the SV40 intron comprises a sequence having at least 90% or 95% identity to SEQ ID NO: 20. In some embodiments, the SV40 intron comprises SEQ ID NO: 20: TCTAGAGGATCCGGTACTCGAGGAACTGAAAAACCAGAAAGTTAACTGGTAAGTTTAG TCTTTTTGTCTTTTATTTCAGGTCCCGGATCCGGTGGTGGTGCAAATCAAAGAACTGCT CCTCAGTGGATGTTGCCTTTACTTCTAGGCCTGTACGGAAGTGTTAC.

[0096] In some embodiments, the SV40 intron contains stretches of about 50, about 75, about 80, about 85, about 95, or about 99 nucleotides in common with the sequence of SEQ ID NO: 20.

[0097] In some embodiments, the nucleic acid vector comprises a minute virus of mice (MVM) intron. For instance, the MVM intron having sequence AAGAGGTAAGGGTTTAAGGGATGGTTGGTTGGTGGGGTATTAATGTTTAATTACC TGGAGCACCTGCCTGAAATCACTTTTTTTCAGGTTGG (SEQ ID NO: 21), or a sequence about or at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 21.

[0098] In some embodiments, the nucleic acid vector comprises an exon 1 splice site. In some embodiments, the nucleic acid vector comprises an intron 1 splice site.

[0099] In some embodiments, the nucleic acid vector comprising the heterologous nucleic acid and an intron, splice acceptor site, and / or splice donor site (e.g., SV40 intron) has a length of between 1700 nucleotides (nt, or base pairs (bp)) and about 5000, about 4550, about 4549, about 4535, about 4534, about 4528, about 4525, about 4500, about 3200, about 3000, about 2977, about 2900, about2800, about 2311, about2300, about 1725, about 1723, or about 1700 nucleotides from the 5' beginning of a first ITRto the 3' end of a second ITR. In some embodiments, the nucleic acid vector has a length of about 1700 to about 1800 (e.g., about 1723) nucleotides from the 5' beginning of a first ITR to the 3 ' end of a second ITR (e.g., FIG. 15A). In some embodiments, the nucleic acid vector has a length of about 2900 to about 3100 (e.g., about 2977) nucleotides from the 5 ' beginning of a first ITR to the 3' end of a second ITR (e.g., FIG. 15B). In some embodiments, the nucleic acid vector has a length of about 2200 to about 2400 (e.g., about 2311) nucleotides from the 5' beginning of a first ITR to the 3 ' end of a second ITR (e.g., FIG. 15C). In some embodiments, the nucleic acid vector has a length of about 4400 to about 4600 (e.g., about 4534) nucleotides from the 5 ' beginning of a first ITR to the 3 ' end of a second ITR (e.g., FIG. 15D). In some embodiments, the nucleic acid vector has a length of about 4400 to about 4600 (e.g., about 4528) nucleotides from the 5' beginning of a first ITR to the 3' end of a second ITR (e.g., FIG.WSGR Docket No. 58774-736.60115E). In some embodiments, the nucleic acid vector has a length of about 4400 to about 4700 (e.g., about 4549) nucleotides from the 5' beginning of a first ITR to the 3' end of a second ITR (e.g., FIG. 15F). In some embodiments, the nucleic acid vector comprising the heterologous nucleic acid and an intron, splice acceptor site, and / or splice donor site (e.g., SV40 intron) comprises a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to any one of SEQ ID NO s: 16-17 and 31-35. In some embodiments, the nucleic acid vector comprising the heterologous nucleic acid and an intron, splice acceptor site, and / or splice donor site (e.g., SV40 intron) comprises a sequence of any one of SEQ ID NOs: 16-17 and 31 -35.Promoters

[0100] In some embodiments, the polynucleotide within the rAAV particle comprises regulatory sequences, such as transcription and translation initiation and termination codons, which are specific to the type of host (e.g., bacterium, fungus, plant, or animal) into which the rAAV particle is to be introduced. Preferably, the nucleic acid molecule within the rAAV particle comprises regulatory sequences that are specific to the genus of the host. Most preferably, the molecule comprises regulatory sequences that are specific to the species of the host. The polynucleotide within the rAAV particle may comprise expression control sequences, such as promoters, enhancers, polyadenylation signals, transcription terminators, internal ribosome entry sites (IRES), and the like, that provide for the expression of the heterologous nucleic acid(s) in a host cell. Exemplary expression control sequences are known in the art. In some embodiments, the heterologous nucleic acid is operably linked to one or more regulatory sequences which direct expression of the heterologous nucleic acid in a photoreceptor cell or retinal pigment epithelium cell.

[0101] In some embodiments, the polynucleotide of any of the disclosed rAAV vectors comprises a promoter that is capable of expressing the nucleic acid sequence in one or more photoreceptors (PR) or retinal pigment epithelial (RPE) cells of a mammalian eye. In particular embodiments, the disclosure provides a PR- or RPE-cell-specific promoter operably linked to at least a first hetereologous nucleic acid sequence that encodes a therapeutic agent. Exemplary PR- or RPE-cell-specific promoters may comprise a) photoreceptor-specific promoters (active in rod and cone cells), e.g., IRBP promoter (hIRPB, IRBP, IRBP241), rhodopsin kinase promoter (hGRKl, GRK1, GRK, RK), and / or chimeric human Retinoschisin -IRBP enhancer (RS / IRPB); cone-specific promoters, e.g., red / green cone opsin promoter (which may comprise the 2.1 kb (PR2.1) version or 1.7 kb (PR1.7) version, see U.S. Patent Publication No. 2018 / 0112231, the sequences of which are herein incorporated by reference), Cone Arrestin promoter (hCAR, CAR), chimeric IRBPWSGR Docket No. 58774-736.601 enhancer-cone transducin promoter (IRBP / GNAT2, IRBPe-GNAT2); rod-specific promoters, e.g., human rhodopsin promoter (RHO, RHOP, etc.), human NRL promoter (NRL); or RPE-specific promoters such as RPE65 or Bestrophin / VMD2 (BEST1, BEST, VMD2). In some embodiments, the promoter is a photoreceptor-specific promoter such as an IRBP promoter (hIRPB, IRBP, IRBP241). In some embodiments, the promoter is a rod-specific promoter such as a human rhodopsin promoter (RHO, RHOP).

[0102] In exemplary embodiments, the polynucleotide comprises an hGRKl promoter. In some embodiments, the polynucleotide comprises a CBA promoter. In some embodiments, the polynucleotide comprises a truncated chimeric CBA-CMV promoter (smCBA) promoter, which contains a CMV enhancer and a truncated CBA promoter.

[0103] Exemplary vectors of the disclosure that comprise hGRKl promoters include AAV- hGRKl-hRSlsyn (FIG. 15A), AAV-hGRKl-hRSl syn-WPREsf (FIG. 15C), pTR-X001-3p (FIG.15D), pTR-X001-5p (FIG. 15E), and pTR-X002-3p (FIG. 15F). An exemplary vector of the disclosure that comprise an smCBA promoter is AAV-CBA-hRSl syn (FIG. 15B).

[0104] In some embodiments, the promoter of any of the disclosed rAAV vectors comprises a nucleotide sequence that has at least 90% identity, at least 92.5% identity, at least 95% identity, at least 98%, at least 99% identity, or 100% identity to the sequence of the hGRKl promoter as set forth in SEQ ID NO: 7: (SEQ ID NO: 7) GGGCCCCAGAAGCCTGGTGGTTGTTTGTCCTTCTCAGGGGAAAAGTGAGGCGGCCCCT TGGAGGAAGGGGCCGGGCAGAATGATCTAATCGGATTCCAAGCAGCTCAGGGGATTG TCTTTTTCTAGCACCTTCTTGCCACTCCTAAGCGTCCTCCGTGACCCCGGCTGGGATTTAGCCTGGTGCTGTGTCAGCCCCGGTCTCCCAGGGGCTTCCCAGTGGTCCCCAGGAACCCT CGACAGGGCCCGGTCTCTCTCGTCCAGCAAGGGCAGGGACGGGCCACAGGCCAAGGG C.

[0105] In some embodiments, the disclosure provides constitutive promoters operably linked to at least a first polynucleotide that may comprise rhodopsin, synl (synapsin), CMV, CBA, CB, smCBA, CBh, or EFl -alpha promoter.

[0106] In some embodiments, the nucleic acid vector comprising the heterologous nucleic acid and promoter has a length of between 1700 nucleotides (nt, or base pairs (bp)) and about 5000, about 4550, about 4549, about4535, about 4534, about4528, about 4525, about 4500, about 3200, about 3000, about 2977, about 2900, about 2800, about 2311, about 2300, about 1725, about 1723, or about 1700 nucleotides from the 5' beginning of a first ITR to the 3 ' end of a second ITR. In some embodiments, the nucleic acid vector has a length of about 1700 to about 1800 (e.g., about 1723) nucleotides from the 5' beginning of a first ITR to the 3' end of a second ITR (e.g., FIG.WSGR Docket No. 58774-736.60115A). In some embodiments, the nucleic acid vector has a length of about2900to about 3100 (e.g., about 2977) nucleotides from the 5' beginning of a first ITR to the 3' end of a second ITR (e.g., FIG. 15B) In some embodiments, the nucleic acid vector has a length of about 2200 to about 2400 (e.g., about 2311) nucleotides from the 5' beginning of a first ITR to the 3 ' end of a second ITR (e.g., FIG. 15C). In some embodiments, the nucleic acid vector has a length of about 4400 to about 4600 (e.g., about4534) nucleotides from the 5' beginning of a first ITR to the 3' end of a second ITR (e.g., FIG. 15D). In some embodiments, the nucleic acid vector has a length of about 4400 to about 4600 (e.g., about 4528) nucleotides from the 5' beginning of a first ITR to the 3' end of a second ITR (e.g., FIG. 15E). In some embodiments, the nucleic acid vector has a length of about 4400 to about 4700 (e.g., about 4549) nucleotides from the 5 ' beginning of a first ITR to the 3 ' end of a second ITR (e.g., FIG. 15F). In some embodiments, the nucleic acid vector comprising the heterologous nucleic acid and promoter comprises a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to any one of SEQ ID NOs: 16-17, 31-35. In some embodiments, the nucleic acid vector comprising the heterologous nucleic acid and promoter comprises a sequence of any one of SEQ ID NOs: 16-17, 31-35.Post-Transcription Regulatory Elements

[0107] In some embodiments, the nucleic acid vector comprises a post -transcriptional regulatory sequence. In some embodiments, the nucleic acid vector comprises a woodchuck hepatitis virus post-transcription regulatory element (WPRE). The polynucleotide may comprises a WPRE element, such as a WPRE elementthat comprises the nucleotide sequence of SEQ ID NO: 15. In some embodiments, the WPRE element is positioned 3 ' of the heterologous nucleic acid. In some embodiments, the WPRE element is positioned 5' of the heterologous nucleic acid.

[0108] In some embodiments, the nucleic acid vector comprises a WPRE. In some cases, the WPRE is a WPREsf sequence, where the “sf” suffix denotes safe for administration. In some embodiments, the polynucleotide of any of the disclosed rAAV vectors comprises a WPRE element having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity y to the nucleotide sequence of SEQ ID NO: 15. In some embodiments, the polynucleotide comprises the sequence of SEQ ID NO: 15.

[0109] WPREsf sequence: (SEQ ID NO:15)TCGACCTCTGGATTACAAAATTTGTGAAAGATTGACTGGTATTCTTAACTATGTTGC TCCTTTTACGCTATGTGGATACGCTGCTTTAATGCCTTTGTATCATGCTATTGCTTCCCG TATGGCTTTCATTTTCTCCTCCTTGTATAAATCCTGGTTGCTGTCTCTTTATGAGGAGTT GTGGCCCGTTGTCAGGCAACGTGGCGTGGTGTGCACTGTGTTTGCTGACGCAACCCCCWSGR Docket No. 58774-736.601ACTGGTTGGGGCATTGCCACCACCTGTCAGCTCCTTTCCGGGACTTTCGCTTTCCCCCT CCCTATTGCCACGGCGGAACTCATCGCCGCCTGCCTTGCCCGCTGCTGGACAGGGGCT CGGCTGTTGGGCACTGACAATTCCGTGGTGTTGTCGGGGAAATCATCGTCCTTTCCTTG GCTGCTCGCCTGTGTTGCCACCTGGATTCTGCGCGGGACGTCCTTCTGCTACGTCCCTT CGGCCCTCAATCCAGCGGACCTTCCTTCCCGCGGCCTGCTGCCGGCTCTGCGGCCTCTT CCGCGTCTTCGCCTTCGCCCTCAGACGAGTCGGATCTCCCTTTGGGCCGCCTCCCCGC.

[0110] In some embodiments, the nucleic acid vector comprising the heterologous nucleic acid and post-transcription regulatory element has a length of between 1700 nucleotides (nt, or base pairs (bp)) and about 5000, about 4550, about 4549, about 4535, about 4534 , about 4528, about 4525, about4500, about 3200, about 3000, about 2977, about2900, about2800, about 2311, about 2300, about 1725, about 1723, or about 1700 nucleotides from the 5' beginning of a first ITR to the 3 ' end of a second ITR. In some embodiments, the nucleic acid vector has a length of about 1700 to about 1800 (e.g., about 1723) nucleotides from the 5' beginning of a first ITR to the 3 ' end of a second ITR (e.g., FIG. 15A). In some embodiments, the nucleic acid vector has a length of about 2900 to about 3100 (e.g., about 2977) nucleotides from the 5 ' beginning of a first ITR to the 3 ' end of a second ITR (e.g., FIG. 15B). In some embodiments, the nucleic acid vector has a length of about 2200 to about 2400 (e.g., about 2311) nucleotides from the 5' beginning of a first ITR to the 3 ' end of a second ITR (e.g., FIG. 15C). In some embodiments, the nucleic acid vector has a length of about 4400 to about 4600 (e.g., about 4534) nucleotides from the 5' beginning of a first ITR to the 3' end of a second ITR (e.g., FIG. 15D). In some embodiments, the nucleic acid vector has a length of about 4400 to about 4600 (e.g., about 4528) nucleotides from the 5' beginning of a first ITR to the 3' end of a second ITR (e.g., FIG. 15E). In some embodiments, the nucleic acid vector has a length of about 4400 to about 4700 (e.g., about 4549) nucleotides from the 5' beginning of a first ITR to the 3 ' end of a second ITR (e.g., FIG. 15F). In some embodiments, the nucleic acid vector comprising the heterologous nucleic acid and post -transcription regulatory element comprises a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to any one of SEQ ID NOs: 16-17 and 31 -35. In some embodiments, the nucleic acid vector comprising the heterologous nucleic acid and post-transcription regulatory element comprises a sequence of any one of SEQ ID NOs: 16-17 and 31-35.Polyadenylation (poly A) Signal Sequence

[0111] In some embodiments, the nucleic acid vector comprises a polyadenylation (poly A) signal sequence. In some embodiments, the polyadenylation signal is selected from a bovine growth factor hormone (bGH) polyadenylation signal, an SV40 polyadenylation signal, a human growthWSGR Docket No. 58774-736.601 factor hormone (hGH) polyadenylation signal, and a rabbit beta -globin (rbGlob) polyadenylation signal.

[0112] In some embodiments, the vector comprises a bGH polyA signal. In some embodiments, the vector comprises an SV40 polyA signal.

[0113] In some embodiments, the vector comprises a bGH polyA signal having a nucleic acid sequence having atleast 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 19. In some embodiments, the polyA signal of any of the disclosed vectors comprises the nucleic acid sequence of SEQ ID NO: 19.

[0114] bGH polyA signal: (SEQ ID NO: 19)TCGACTAGAGCTCGCTGATCAGCCTCGACTGTGCCTTCTAGTTGCCAGCCATCTGTTGT TTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTA ATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGT GGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCAT.

[0115] In some embodiments, the nucleic acid vector comprising the heterologous nucleic acid and polyA signal has a length of between 1700 nucleotides (nt, or base pairs (bp)) and about 5000, about 4550, about 4549, about4535, about 4534, about4528, about 4525, about4500, about 3200, about 3000, about 2977, about 2900, about 2800, about 2311, about 2300, about 1725, about 1723, or about 1700 nucleotides from the 5' beginning of a first ITR to the 3 ' end of a second ITR. In some embodiments, the nucleic acid vector has a length of about 1700 to about 1800 (e.g., about 1723) nucleotides from the 5' beginning of a first ITR to the 3' end of a second ITR (e.g., FIG.15A). In some embodiments, the nucleic acid vector has a length of about2900 to about 3100 (e.g., about 2977) nucleotides from the 5' beginning of a first ITR to the 3' end of a second ITR (e.g., FIG. 15B) In some embodiments, the nucleic acid vector has a length of about 2200 to about 2400 (e.g., about 2311) nucleotides from the 5' beginning of a first ITR to the 3' end of a second ITR (e.g., FIG. 15C). In some embodiments, the nucleic acid vector has a length of about 4400 to about 4600 (e.g., about 4534) nucleotides from the 5' beginning of a first ITR to the 3' end of a second ITR (e.g., FIG. 15D). In some embodiments, the nucleic acid vector has a length of about 4400 to about 4600 (e.g., about 4528) nucleotides from the 5' beginning of a first ITR to the 3 ' end of a second ITR (e.g., FIG. 15E). In some embodiments, the nucleic acid vector has a length of about 4400 to about 4700 (e.g., about 4549) nucleotides from the 5 ' beginning of a first ITR to the 3 ' end of a second ITR (e.g., FIG. 15F). In some embodiments, the nucleic acid vector comprising the heterologous nucleic acid and polyA signal comprises a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, orWSGR Docket No. 58774-736.60199% identity to any one of SEQ ID NOs: 16-17, 31-35. In some embodiments, the nucleic acid vector comprising the heterologous nucleic acid and poly A signal comprises a sequence of any one of SEQ ID NOs: 16-17, 31-35.Terminal Repeats

[0116] In some embodiments, the nucleic acid vector comprises one or more terminal repeats.In some embodiments, the nucleic acid vector comprises a first inverted terminal repeat (ITR) and a second inverted terminal repeat (ITR). In some embodiments, an inverted terminal repeat comprises a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 22 or 23. In some embodiments, an inverted terminal repeat comprises SEQ ID NO: 22 or 23. In some embodiments, an inverted terminal repeat comprises a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 24 or 25. In some embodiments, an inverted terminal repeat comprises SEQ ID NO: 24 or 25.

[0117] Inverted terminal repeat (e.g., first ITR) (SEQ ID NO: 22) GGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGG CGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGA GTGGCCAACTCCATCACTAGGGGTTCCT

[0118] Inverted terminal repeat (e.g., first ITR) (SEQ ID NO: 23) TTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCG GGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGG GAGTGGCCAACTCCATCACTAGGGGTTCCT.

[0119] Inverted terminal repeat (e.g., second ITR) (SEQ ID NO: 24) AGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAG GCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCG AGCGAGCGCGC AGAGAGGGAGTGGCC .

[0120] Inverted terminal repeat (e.g., second ITR) (SEQ ID NO: 25) AGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAG GCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCG AGCGAGCGCGCAGAGAGGGAGTGGCCAA.

[0121] In some embodiments, the nucleic acid vector comprising the heterologous nucleic acid and terminal repeat has a length of between 1700 nucleotides (nt, or base pairs (bp)) and about 5000, about 4550, about 4549, about 4535, about 4534, about4528, about4525, about 4500, about 3200, about 3000, about 2977, about 2900, about 2800, about2311, about2300, about 1725, aboutWSGR Docket No. 58774-736.6011723, or about 1700 nucleotides from the 5 ' beginning of a first ITR to the 3 ' end of a second ITR. In some embodiments, the nucleic acid vector has a length of about 1700 to about 1800 (e.g., about 1723) nucleotides from the 5' beginning of a first ITR to the 3 ' end of a second ITR (e.g., FIG.15A). In some embodiments, the nucleic acid vector has a length of about2900 to about 3100 (e.g., about 2977) nucleotides from the 5' beginning of a first ITR to the 3' end of a second ITR (e.g., FIG. 15B) In some embodiments, the nucleic acid vector has a length of about 2200 to about 2400 (e.g., about 2311) nucleotides from the 5' beginning of a first ITR to the 3 ' end of a second ITR (e.g., FIG. 15C). In some embodiments, the nucleic acid vector has a length of about 4400 to about 4600 (e.g., about 4534) nucleotides from the 5' beginning of a first ITR to the 3' end of a second ITR (e.g., FIG. 15D). In some embodiments, the nucleic acid vector has a length of about 4400 to about 4600 (e.g., about 4528) nucleotides from the 5' beginning of a first ITR to the 3 ' end of a second ITR (e.g., FIG. 15E). In some embodiments, the nucleic acid vector has a length of about 4400 to about 4700 (e.g., about 4549) nucleotides from the 5 ' beginning of a first ITR to the 3 ' end of a second ITR (e.g., FIG. 15F). In some embodiments, the nucleic acid vector comprising the heterologous nucleic acid and terminal repeat comprises a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to any one of SEQ ID NOs: 16, 17, and 31 -35. In some embodiments, the nucleic acid vector comprising the heterologous nucleic acid and terminal repeat comprises a sequence of any one of SEQ ID NOs: 16, 17, and 31 -35.St ffer Sequences

[0122] In some embodiments, the rAAV vectors of the disclosure contain one or more stuffer sequences. In some embodiments, a “stuffer” sequence refers to a generic, inert, non-coding sequence that increases the length of the rAAV vectors. In some embodiments, the stuffer sequence increases the length of any of the disclosed rAAV vectors such that the cassette is close to wtAAV genome size (e.g., ~4.8 kb from ITR to ITR).

[0123] In some embodiments, the nucleic acid stuffer sequence has a length of about 100 to about 5000 nucleobases. In some embodiments, the nucleic acid stuffer sequence has a length of about 100 to about 5000, about 100 to about 4000, about 100 to about 3000, or about 100 to about 2000 nucleobases. In some embodiments, the nucleic acid stuffer sequence has a length of about 100 to about 5000, about 500 to about 5000, about 1000 to about 5000, about 2000 to about 5000, or about 3000 to about 5000 nucleobases. In some embodiments, the nucleic acid stuffer sequence has a length of about 1000 to about 5000, about 1050 to about 4500, or about 2000 to about 3000 nucleobases. In some embodiments, the nucleic acid stuffer sequence has a length of about 2822 nucleobases. In some embodiments, the nucleic acid stuffer sequence has a length of about 2820WSGR Docket No. 58774-736.601 nucleobases. In some embodiments, the nucleic acid stuffer sequence has a length of about 2249 nucleobases. In some embodiments, the nucleic acid stuffer sequence has a length of about 2235 nucleobases. In some embodiments, the nucleic acid stuffer sequence has a length of about 2219 nucleobases.

[0124] In some embodiments, a cassette (from the beginning of a first ITR to the end of a second ITR) comprising the nucleic acid stuffer sequence has a length of about 2000 to about 5000 nucleobases. In some embodiments, the cassette comprising the nucleic acid stuffer sequence has a length of about 3000 to about 5000, about 3500 to about 5000, about 4000 to about 5000, about 4100 to about 5000, about 4200 to about 5000, about 4300 to about 5000, about 4400 to about 5000, about4500 to about 5000, about4000to about4900, about 4100 to about 4900, about 4200 to about 4900, about 4300 to about 4900, about 4400 to about 4900, about 4000 to about 4800, about 4100 to about 4800, about 4200 to about 4800, about 4300 to about 4800, about 4400 to about 4800, about 4000 to about 4700, about 4100 to about 5000, about 4200 to about 4700, about 4300 to about 4700, about 4400 to about 4700, about 4000 to about 4600, about 4100 to about 4600, about 4200 to about 4600, about 4300 to about 4600, about 4400 to about 4600, about 4000 to about 4500, about 4100 to about4500, about 4200 to about 4500, about 4300 to about 4500, or about 4400 to about 4500 nucleobases. In some embodiments, the cassette comprising the nucleic acid stuffer sequence has a length of about 4500, 4510, 4520, 4530, 4540, 4550, 4560, 4570, 4580, 4590, or 5000 nucleobases. In some embodiments, the cassette comprising the nucleic acid stuffer sequence has a length of about 4534 nucleobases. In some embodiments, the cassette comprising the nucleic acid stuffer sequence has a length of about 4528 nucleobases. In some embodiments, the cassette comprising the nucleic acid stuffer sequence has a length of about 4549 nucleobases.

[0125] In some embodiments, the nucleic acid stuffer sequence comprises a sequence about or at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOS: 26-30 or 41 -43. In some embodiments, the nucleic acid stuffer sequence comprises a sequence about or at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOS: 26-30 or 41-43. In some embodiments, the nucleic acid stuffer sequence comprises a sequence about or at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOS: 26-30 or 41-43.

[0126] In some embodiments, the nucleic acid stuffer sequence comprises a sequence about or at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to bases 1 -100, 2-101, 3-102, 4-103, 5-104, 6-105, 7-WSGR Docket No. 58774-736.601106. 8-107. 9-108. 10-109. 11-110. 12-111. 13-112. 14-113. 15-114. 16-115. 17-116. 18-117. 19118, 20-119, 21-120, 22-121, 23-122, 24-123, 25 -124, 26-125, 27-126, 28-127, 29-128, 30-129, 31- 130, 32-131, 33-132, 34-133, 35-134, 36-135, 37 -136, 38-137, 39-138, 40-139, 41-140, 42-141, 43- 142, 44-143, 45-144, 46-145, 47-146, 48-147, 49 -148, 50-149, 51-150, 52-151, 53-152, 54-153, 55- 154, 56-155, 57-156, 58-157, 59-158, 60-159, 61 -160, 62-161, 63-162, 64-163, 65-164, 66-165, 67- 166, 68-167, 69-168, 70-169, 71-170, 72-171, 73 -172, 74-173, 75-174, 76-175, 77-176, 78-177, 79- 178, 80-179, 81-180, 82-181, 83-182, 84-183, 85 -184, 86-185, 87-186, 88-187, 89-188, 90-189, 91- 190, 92-191, 93-192, 94-193, 95-194, 96-195, 97 -196, 98-197, 99-198, 100-199, 101-200, 102-201, 103-202, 104-203, 105-204, 106-205, 107-206, 108-207, 109-208, 110-209, 111-210, 112-211, 113-212, 114-213, 115-214, 116-215, 117-216, 118-217, 119-218, 120-219, 121-220, 122-221, 123-222, 124-223, 125-224, 126-225, 127-226, 128-227, 129-228, 130-229, 131-230, 132-231, 133-232, 134-233, 135-234, 136-235, 137-236, 138-237, 139-238, 140-239, 141-240, 142-241, 143-242, 144-243, 145-244, 146-245, 147-246, 148-247, 149-248, 150-249, 151-250, 152-251, 153-252, 154-253, 155-254, 156-255, 157-256, 158-257, 159-258, 160-259, 161-260, 162-261, 163-262, 164-263, 165-264, 166-265, 167-266, 168-267, 169-268, 170-269, 171-270, 172-271, 173-272, 174-273, 175-274, 176-275, 177-276, 178-277, 179-278, 180-279, 181-280, 182-281, 183-282, 184-283, 185-284, 186-285, 187-286, 188-287, 189-288, 190-289, 191-290, 192-291, 193-292, 194-293, 195-294, 196-295, 197-296, 198-297, 199-298, 200-299, 201-300, 202-301, 203-302, 204-303, 205-304, 206-305, 207-306, 208-307, 209-308, 210-309, 211-310, 212-311, 213-312, 214-313, 215-314, 216-315, 217-316, 218-317, 219-318, 220-319, 221-320, 222-321, 223-322, 224-323, 225-324, 226-325, 227-326, 228-327, 229-328, 230-329, 231-330, 232-331, 233-332, 234-333, 235-334, 236-335, 237-336, 238-337, 239-338, 240-339, 241-340, 242-341, 243-342, 244-343, 245-344, 246-345, 247-346, 248-347, 249-348, 250-349, 251-350, 252-351, 253-352, 254-353, 255-354, 256-355, 257-356, 258-357, 259-358, 260-359, 261-360, 262-361, 263-362, 264-363, 265-364, 266-365, 267-366, 268-367, 269-368, 270-369, 271-370, 272-371, 273-372, 274-373, 275-374, 276-375, 277-376, 278-377, 279-378, 280-379, 281-380, 282-381, 283-382, 284-383, 285-384, 286-385, 287-386, 288-387, 289-388, 290-389, 291-390, 292-391, 293-392, 294-393, 295-394, 296-395, 297-396, 298-397, 299-398, 300-399, 301-400, 302-401, 303-402, 304-403, 305-404, 306-405, 307-406, 308-407, 309-408, 310-409, 311-410, 312-411, 313-412, 314-413, 315-414, 316-415, 317-416, 318-417, 319-418, 320-419, 321-420, 322-421, 323-422, 324-423, 325-424, 326-425, 327-426, 328-427, 329-428, 330-429, 331-430, 332-431, 333-432, 334-433, 335-434, 336-435, 337-436, 338-437, 339-438, 340-439, 341-440, 342-441, 343-442, 344-443, 345-444, 346-445, 347-446, 348-447, 349-448, 350-449, 351-450, 352-451, 353-452, 354-453, 355-454, 356-455, 357-456, 358-457, 359-458, 360-459, 361-460, 362-461,WSGR Docket No. 58774-736.601363-462, 364-463, 365-464, 366-465, 367-466, 368 -467, 369-468, 370-469, 371-470, 372-471, 373-472, 374-473, 375-474, 376-475, 377-476, 378 -477, 379-478, 380-479, 381-480, 382-481, 383-482, 384-483, 385-484, 386-485, 387-486, 388 -487, 389-488, 390-489, 391-490, 392-491, 393-492, 394-493, 395-494, 396-495, 397-496, 398 -497, 399-498, 400-499, 401-500, 402-501, 403-502, 404-503, 405-504, 406-505, 407-506, 408 -507, 409-508, 410-509, 411-510, 412-511, 413-512, 414-513, 415-514, 416-515, 417-516, 418 -517, 419-518, 420-519, 421-520, 422-521, 423-522, 424-523, 425-524, 426-525, 427-526, 428 -527, 429-528, 430-529, 431-530, 432-531, 433-532, 434-533, 435-534, 436-535, 437-536, 438 -537, 439-538, 440-539, 441-540, 442-541, 443-542, 444-543, 445-544, 446-545, 447-546, 448 -547, 449-548, 450-549, 451-550, 452-551, 453-552, 454-553, 455-554, 456-555, 457-556, 458 -557, 459-558, 460-559, 461-560, 462-561, 463-562, 464-563, 465-564, 466-565, 467-566, 468 -567, 469-568, 470-569, 471-570, 472-571, 473-572, 474-573, 475-574, 476-575, 477-576, 478 -577, 479-578, 480-579, 481-580, 482-581, 483-582, 484-583, 485-584, 486-585, 487-586, 488 -587, 489-588, 490-589, 491-590, 492-591, 493-592, 494-593, 495-594, 496-595, 497-596, 498 -597, 499-598, 500-599, 501-600, 502-601, 503-602, 504-603, 505-604, 506-605, 507-606, 508 -607, 509-608, 510-609, 511-610, 512-611, 513-612, 514-613, 515-614, 516-615, 517-616, 518 -617, 519-618, 520-619, 521-620, 522-621, 523-622, 524-623, 525-624, 526-625, 527-626, 528 -627, 529-628, 530-629, 531-630, 532-631, 533-632, 534-633, 535-634, 536-635, 537-636, 538 -637, 539-638, 540-639, 541-640, 542-641, 543-642, 544-643, 545-644, 546-645, 547-646, 548 -647, 549-648, 550-649, 551-650, 552-651, 553-652, 554-653, 555-654, 556-655, 557-656, 558 -657, 559-658, 560-659, 561-660, 562-661, 563-662, 564-663, 565-664, 566-665, 567-666, 568 -667, 569-668, 570-669, 571-670, 572-671, 573-672, 574-673, 575-674, 576-675, 577-676, 578 -677, 579-678, 580-679, 581-680, 582-681, 583-682, 584-683, 585-684, 586-685, 587-686, 588 -687, 589-688, 590-689, 591-690, 592-691, 593-692, 594-693, 595-694, 596-695, 597-696, 598 -697, 599-698, 600-699, 601-700, 602-701, 603-702, 604-703, 605-704, 606-705, 607-706, 608 -707, 609-708, 610-709, 611-710, 612-711, 613-712, 614-713, 615-714, 616-715, 617-716, 618 -717, 619-718, 620-719, 621-720, 622-721, 623-722, 624-723, 625-724, 626-725, 627-726, 628 -727, 629-728, 630-729, 631-730, 632-731, 633-732, 634-733, 635-734, 636-735, 637-736, 638 -737, 639-738, 640-739, 641-740, 642-741, 643-742, 644-743, 645-744, 646-745, 647-746, 648 -747, 649-748, 650-749, 651-750, 652-751, 653-752, 654-753, 655-754, 656-755, 657-756, 658 -757, 659-758, 660-759, 661-760, 662-761, 663-762, 664-763, 665-764, 666-765, 667-766, 668 -767, 669-768, 670-769, 671-770, 672-771, 673-772, 674-773, 675-774, 676-775, 677-776, 678 -777, 679-778, 680-779, 681-780, 682-781, 683-782, 684-783, 685-784, 686-785, 687-786, 688 -787, 689-788, 690-789, 691-790, 692-791, 693-792, 694-793, 695-794, 696-795, 697-796, 698 -797, 699-798, 700-799, 701-800, 702-801,WSGR Docket No. 58774-736.601703-802, 704-803, 705-804, 706-805, 707-806, 708-807, 709-808, 710-809, 711-810, 712-811, 713-812, 714-813, 715-814, 716-815, 717-816, 718-817, 719-818, 720-819, 721-820, 722-821, 723-822, 724-823, 725-824, 726-825, 727-826, 728-827, 729-828, 730-829, 731-830, 732-831, 733-832, 734-833, 735-834, 736-835, 737-836, 738-837, 739-838, 740-839, 741-840, 742-841, 743-842, 744-843, 745-844, 746-845, 747-846, 748-847, 749-848, 750-849, 751-850, 752-851, 753-852, 754-853, 755-854, 756-855, 757-856, 758-857, 759-858, 760-859, 761-860, 762-861, 763-862, 764-863, 765-864, 766-865, 767-866, 768-867, 769-868, 770-869, 771-870, 772-871, 773-872, 774-873, 775-874, 776-875, 777-876, 778-877, 779-878, 780-879, 781-880, 782-881, 783-882, 784-883, 785-884, 786-885, 787-886, 788-887, 789-888, 790-889, 791-890, 792-891, 793-892, 794-893, 795-894, 796-895, 797-896, 798-897, 799-898, 800-899, 801-900, 802-901, 803-902, 804-903, 805-904, 806-905, 807-906, 808-907, 809-908, 810-909, 811-910, 812-911, 813-912, 814-913, 815-914, 816-915, 817-916, 818-917, 819-918, 820-919, 821-920, 822-921, 823-922, 824-923, 825-924, 826-925, 827-926, 828-927, 829-928, 830-929, 831-930, 832-931, 833-932, 834-933, 835-934, 836-935, 837-936, 838-937, 839-938, 840-939, 841-940, 842-941, 843-942, 844-943, 845-944, 846-945, 847-946, 848-947, 849-948, 850-949, 851-950, 852-951, 853-952, 854-953, 855-954, 856-955, 857-956, 858-957, 859-958, 860-959, 861-960, 862-961, 863-962, 864-963, 865-964, 866-965, 867-966, 868-967, 869-968, 870-969, 871-970, 872-971, 873-972, 874-973, 875-974, 876-975, 877-976, 878-977, 879-978, 880-979, 881-980, 882-981, 883-982, 884-983, 885-984, 886-985, 887-986, 888-987, 889-988, 890-989, 891-990, 892-991, 893-992, 894-993, 895-994, 896-995, 897-996, 898-997, 899-998, 900-999, 901-1000, 902-1001, 903-1002, 904-1003, 905-1004, 906-1005, 907-1006, 908-1007, 909-1008, 910-1009, 911-1010, 912-1011, 913-1012, 914-1013, 915-1014, 916-1015, 917-1016, 918-1017, 919-1018, 920-1019, 921-1020, 922-1021, 923-1022, 924-1023, 925-1024, 926-1025, 927-1026, 928-1027, 929-1028, 930-1029, 931-1030, 932-1031, 933-1032, 934-1033, 935-1034, 936-1035, 937-1036, 938-1037, 939-1038, 940-1039, 941-1040, 942-1041, 943-1042, 944-1043, 945-1044, 946-1045, 947-1046, 948-1047, 949-1048, 950-1049, 951-1050, 952-1051, 953-1052, 954-1053, 955-1054, 956-1055, 957-1056, 958-1057, 959-1058, 960-1059, 961-1060, 962-1061, 963-1062, 964-1063, 965-1064, 966-1065, 967-1066, 968-1067, 969-1068, 970-1069, 971-1070, 972-1071, 973-1072, 974-1073, 975-1074, 976-1075, 977-1076, 978-1077, 979-1078, 980-1079, 981-1080, 982-1081, 983-1082, 984-1083, 985-1084, 986-1085, 987-1086, 988-1087, 989-1088, 990-1089, 991-1090, 992-1091, 993-1092, 994-1093, 995-1094, 996-1095, 997-1096, 998-1097, 999-1098, 1000-1099, 1000-1100, 1001-1101, 1002-1102, 1003-1103, 1004-1104, 1005-1105, 1006-1106, 1007-1107, 1008-1108, 1009-1109, 1010-1110, 1011-1111, 1012-1112, 1013-1113, 1014-1114, 1015-1115, 1016-1116, 1017-1117, 1018-1118, 1019-1119, 1020-1120, 1021-1121, 1022-1122, 1023-1123, 1024-1124,WSGR Docket No. 58774-736.6011025-1125, 1026-1126, 1027-1127, 1028-1128, 1029-1129, 1030-1130, 1031-1131, 1032-1132, 1033-1133, 1034-1134, 1035-1135, 1036-1136, 1037-1137, 1038-1138, 1039-1139, 1040-1140, 1041-1141, 1042-1142, 1043-1143, 1044-1144, 1045-1145, 1046-1146, 1047-1147, 1048-1148, 1049-1149, 1050-1150, 1051-1151, 1052-1152, 1053-1153, 1054-1154, 1055-1155, 1056-1156, 1057-1157, 1058-1158, 1059-1159, 1060-1160, 1061-1161, 1062-1162, 1063-1163, 1064-1164, 1065-1165, 1066-1166, 1067-1167, 1068-1168, 1069-1169, 1070-1170, 1071-1171, 1072-1172, 1073-1173, 1074-1174, 1075-1175, 1076-1176, 1077-1177, 1078-1178, 1079-1179, 1080-1180, 1081-1181, 1082-1182, 1083-1183, 1084-1184, 1085-1185, 1086-1186, 1087-1187, 1088-1188, 1089-1189, 1090-1190, 1091-1191, 1092-1192, 1093-1193, 1094-1194, 1095-1195, 1096-1196, 1097-1197, 1098-1198, 1099-1199, 1100-1200, 1101-1201, 1102-1202, 1103-1203, 1104-1204, 1105-1205, 1106-1206, 1107-1207, 1108-1208, 1109-1209, 1110-1210, 1111-1211, 1112-1212, 1113-1213, 1114-1214, 1115-1215, 1116-1216, 1117-1217, 1118-1218, 1119-1219, 1120-1220, 1121-1221, 1122-1222, 1123-1223, 1124-1224, 1125-1225, 1126-1226, 1127-1227, 1128-1228, 1129-1229, 1130-1230, 1131-1231, 1132-1232, 1133-1233, 1134-1234, 1135-1235, 1136-1236, 1137-1237, 1138-1238, 1139-1239, 1140-1240, 1141-1241, 1142-1242, 1143-1243, 1144-1244, 1145-1245, 1146-1246, 1147-1247, 1148-1248, 1149-1249, 1150-1250, 1151-1251, 1152-1252, 1153-1253, 1154-1254, 1155-1255, 1156-1256, 1157-1257, 1158-1258, 1159-1259, 1160-1260, 1161-1261, 1162-1262, 1163-1263, 1164-1264, 1165-1265, 1166-1266, 1167-1267, 1168-1268, 1169-1269, 1170-1270, 1171-1271, 1172-1272, 1173-1273, 1174-1274, 1175-1275, 1176-1276, 1177-1277, 1178-1278, 1179-1279, 1180-1280, 1181-1281, 1182-1282, 1183-1283, 1184-1284, 1185-1285, 1186-1286, 1187-1287, 1188-1288, 1189-1289, 1190-1290, 1191-1291, 1192-1292, 1193-1293, 1194-1294, 1195-1295, 1196-1296, 1197-1297, 1198-1298, 1199-1299, 1200-1300, 1201-1301, 1202-1302, 1203-1303, 1204-1304, 1205-1305, 1206-1306, 1207-1307, 1208-1308, 1209-1309, 1210-1310, 1211-1311, 1212-1312, 1213-1313, 1214-1314, 1215-1315, 1216-1316, 1217-1317, 1218-1318, 1219-1319, 1220-1320, 1221-1321, 1222-1322, 1223-1323, 1224-1324, 1225-1325, 1226-1326, 1227-1327, 1228-1328, 1229-1329, 1230-1330, 1231-1331, 1232-1332, 1233-1333, 1234-1334, 1235-1335, 1236-1336, 1237-1337, 1238-1338, 1239-1339, 1240-1340, 1241-1341, 1242-1342, 1243-1343, 1244-1344, 1245-1345, 1246-1346, 1247-1347, 1248-1348, 1249-1349, 1250-1350, 1251-1351, 1252-1352, 1253-1353, 1254-1354, 1255-1355, 1256-1356, 1257-1357, 1258-1358, 1259-1359, 1260-1360, 1261-1361, 1262-1362, 1263-1363, 1264-1364, 1265-1365, 1266-1366, 1267-1367, 1268-1368, 1269-1369, 1270-1370, 1271-1371, 1272-1372, 1273-1373, 1274-1374, 1275-1375, 1276-1376, 1277-1377, 1278-1378, 1279-1379, 1280-1380, 1281-1381, 1282-1382, 1283-1383, 1284-1384, 1285-1385, 1286-1386, 1287-1387, 1288-1388, 1289-1389, 1290-1390, 1291-1391, 1292-1392, 1293-1393, 1294-1394, 1295-1395, 1296-1396,WSGR Docket No. 58774-736.6011297-1397, 1298-1398, 1299-1399, 1300-1400, 1301-1401, 1302-1402, 1303-1403, 1304-1404 1305-1405, 1306-1406, 1307-1407, 1308-1408, 1309-1409, 1310-1410, 1311-1411, 1312-1412 1313-1413, 1314-1414, 1315-1415, 1316-1416, 1317-1417, 1318-1418, 1319-1419, 1320-1420 1321-1421, 1322-1422, 1323-1423, 1324-1424, 1325-1425, 1326-1426, 1327-1427, 1328-1428 1329-1429, 1330-1430, 1331-1431, 1332-1432, 1333-1433, 1334-1434, 1335-1435, 1336-1436 1337-1437, 1338-1438, 1339-1439, 1340-1440, 1341-1441, 1342-1442, 1343-1443, 1344-1444 1345-1445, 1346-1446, 1347-1447, 1348-1448, 1349-1449, 1350-1450, 1351-1451, 1352-1452 1353-1453, 1354-1454, 1355-1455, 1356-1456, 1357-1457, 1358-1458, 1359-1459, 1360-1460 1361-1461, 1362-1462, 1363-1463, 1364-1464, 1365-1465, 1366-1466, 1367-1467, 1368-1468 1369-1469, 1370-1470, 1371-1471, 1372-1472, 1373-1473, 1374-1474, 1375-1475, 1376-1476 1377-1477, 1378-1478, 1379-1479, 1380-1480, 1381-1481, 1382-1482, 1383-1483, 1384-1484 1385-1485, 1386-1486, 1387-1487, 1388-1488, 1389-1489, 1390-1490, 1391-1491, 1392-1492 1393-1493, 1394-1494, 1395-1495, 1396-1496, 1397-1497, 1398-1498, 1399-1499, 1400-1500 1401-1501, 1402-1502, 1403-1503, 1404-1504, 1405-1505, 1406-1506, 1407-1507, 1408-1508 1409-1509, 1410-1510, 1411-1511, 1412-1512, 1413-1513, 1414-1514, 1415-1515, 1416-1516 1417-1517, 1418-1518, 1419-1519, 1420-1520, 1421-1521, 1422-1522, 1423-1523, 1424-1524 1425-1525, 1426-1526, 1427-1527, 1428-1528, 1429-1529, 1430-1530, 1431-1531, 1432-1532 1433-1533, 1434-1534, 1435-1535, 1436-1536, 1437-1537, 1438-1538, 1439-1539, 1440-1540 1441-1541, 1442-1542, 1443-1543, 1444-1544, 1445-1545, 1446-1546, 1447-1547, 1448-1548 1449-1549, 1450-1550, 1451-1551, 1452-1552, 1453-1553, 1454-1554, 1455-1555, 1456-1556 1457-1557, 1458-1558, 1459-1559, 1460-1560, 1461-1561, 1462-1562, 1463-1563, 1464-1564 1465-1565, 1466-1566, 1467-1567, 1468-1568, 1469-1569, 1470-1570, 1471-1571, 1472-1572 1473-1573, 1474-1574, 1475-1575, 1476-1576, 1477-1577, 1478-1578, 1479-1579, 1480-1580 1481-1581, 1482-1582, 1483-1583, 1484-1584, 1485-1585, 1486-1586, 1487-1587, 1488-1588 1489-1589, 1490-1590, 1491-1591, 1492-1592, 1493-1593, 1494-1594, 1495-1595, 1496-1596 1497-1597, 1498-1598, 1499-1599, 1500-1600, 1501-1601, 1502-1602, 1503-1603, 1504-1604 1505-1605, 1506-1606, 1507-1607, 1508-1608, 1509-1609, 1510-1610, 1511-1611, 1512-1612 1513-1613, 1514-1614, 1515-1615, 1516-1616, 1517-1617, 1518-1618, 1519-1619, 1520-1620 1521-1621, 1522-1622, 1523-1623, 1524-1624, 1525-1625, 1526-1626, 1527-1627, 1528-1628 1529-1629, 1530-1630, 1531-1631, 1532-1632, 1533-1633, 1534-1634, 1535-1635, 1536-1636 1537-1637, 1538-1638, 1539-1639, 1540-1640, 1541-1641, 1542-1642, 1543-1643, 1544-1644 1545-1645, 1546-1646, 1547-1647, 1548-1648, 1549-1649, 1550-1650, 1551-1651, 1552-1652 1553-1653, 1554-1654, 1555-1655, 1556-1656, 1557-1657, 1558-1658, 1559-1659, 1560-1660 1561-1661, 1562-1662, 1563-1663, 1564-1664, 1565-1665, 1566-1666, 1567-1667, 1568-1668WSGR Docket No. 58774-736.6011569-1669, 1570-1670, 1571-1671, 1572-1672, 1573-1673, 1574-1674, 1575-1675, 1576-1676 1577-1677, 1578-1678, 1579-1679, 1580-1680, 1581-1681, 1582-1682, 1583-1683, 1584-1684 1585-1685, 1586-1686, 1587-1687, 1588-1688, 1589-1689, 1590-1690, 1591-1691, 1592-1692 1593-1693, 1594-1694, 1595-1695, 1596-1696, 1597-1697, 1598-1698, 1599-1699, 1600-1700 1601-1701, 1602-1702, 1603-1703, 1604-1704, 1605-1705, 1606-1706, 1607-1707, 1608-1708 1609-1709, 1610-1710, 1611-1711, 1612-1712, 1613-1713, 1614-1714, 1615-1715, 1616-1716 1617-1717, 1618-1718, 1619-1719, 1620-1720, 1621-1721, 1622-1722, 1623-1723, 1624-1724 1625-1725, 1626-1726, 1627-1727, 1628-1728, 1629-1729, 1630-1730, 1631-1731, 1632-1732 1633-1733, 1634-1734, 1635-1735, 1636-1736, 1637-1737, 1638-1738, 1639-1739, 1640-1740 1641-1741, 1642-1742, 1643-1743, 1644-1744, 1645-1745, 1646-1746, 1647-1747, 1648-1748 1649-1749, 1650-1750, 1651-1751, 1652-1752, 1653-1753, 1654-1754, 1655-1755, 1656-1756 1657-1757, 1658-1758, 1659-1759, 1660-1760, 1661-1761, 1662-1762, 1663-1763, 1664-1764 1665-1765, 1666-1766, 1667-1767, 1668-1768, 1669-1769, 1670-1770, 1671-1771, 1672-1772 1673-1773, 1674-1774, 1675-1775, 1676-1776, 1677-1777, 1678-1778, 1679-1779, 1680-1780 1681-1781, 1682-1782, 1683-1783, 1684-1784, 1685-1785, 1686-1786, 1687-1787, 1688-1788 1689-1789, 1690-1790, 1691-1791, 1692-1792, 1693-1793, 1694-1794, 1695-1795, 1696-1796 1697-1797, 1698-1798, 1699-1799, 1700-1800, 1701-1801, 1702-1802, 1703-1803, 1704-1804 1705-1805, 1706-1806, 1707-1807, 1708-1808, 1709-1809, 1710-1810, 1711-1811, 1712-1812 1713-1813, 1714-1814, 1715-1815, 1716-1816, 1717-1817, 1718-1818, 1719-1819, 1720-1820 1721-1821, 1722-1822, 1723-1823, 1724-1824, 1725-1825, 1726-1826, 1727-1827, 1728-1828 1729-1829, 1730-1830, 1731-1831, 1732-1832, 1733-1833, 1734-1834, 1735-1835, 1736-1836 1737-1837, 1738-1838, 1739-1839, 1740-1840, 1741-1841, 1742-1842, 1743-1843, 1744-1844 1745-1845, 1746-1846, 1747-1847, 1748-1848, 1749-1849, 1750-1850, 1751-1851, 1752-1852 1753-1853, 1754-1854, 1755-1855, 1756-1856, 1757-1857, 1758-1858, 1759-1859, 1760-1860 1761-1861, 1762-1862, 1763-1863, 1764-1864, 1765-1865, 1766-1866, 1767-1867, 1768-1868 1769-1869, 1770-1870, 1771-1871, 1772-1872, 1773-1873, 1774-1874, 1775-1875, 1776-1876 1777-1877, 1778-1878, 1779-1879, 1780-1880, 1781-1881, 1782-1882, 1783-1883, 1784-1884 1785-1885, 1786-1886, 1787-1887, 1788-1888, 1789-1889, 1790-1890, 1791-1891, 1792-1892 1793-1893, 1794-1894, 1795-1895, 1796-1896, 1797-1897, 1798-1898, 1799-1899, 1800-1900 1801-1901, 1802-1902, 1803-1903, 1804-1904, 1805-1905, 1806-1906, 1807-1907, 1808-1908 1809-1909, 1810-1910, 1811-1911, 1812-1912, 1813-1913, 1814-1914, 1815-1915, 1816-1916 1817-1917, 1818-1918, 1819-1919, 1820-1920, 1821-1921, 1822-1922, 1823-1923, 1824-1924 1825-1925, 1826-1926, 1827-1927, 1828-1928, 1829-1929, 1830-1930, 1831-1931, 1832-1932 1833-1933, 1834-1934, 1835-1935, 1836-1936, 1837-1937, 1838-1938, 1839-1939, 1840-1940WSGR Docket No. 58774-736.6011841-1941, 1842-1942, 1843-1943, 1844-1944, 1845-1945, 1846-1946, 1847-1947, 1848-1948, 1849-1949, 1850-1950, 1851-1951, 1852-1952, 1853-1953, 1854-1954, 1855-1955, 1856-1956, 1857-1957, 1858-1958, 1859-1959, 1860-1960, 1861-1961, 1862-1962, 1863-1963, 1864-1964, 1865-1965, 1866-1966, 1867-1967, 1868-1968, 1869-1969, 1870-1970, 1871-1971, 1872-1972, 1873-1973, 1874-1974, 1875-1975, 1876-1976, 1877-1977, 1878-1978, 1879-1979, 1880-1980, 1881-1981, 1882-1982, 1883-1983, 1884-1984, 1885-1985, 1886-1986, 1887-1987, 1888-1988, 1889-1989, 1890-1990, 1891-1991, 1892-1992, 1893-1993, 1894-1994, 1895-1995, 1896-1996, 1897-1997, 1898-1998, 1899-1999, 1900-2000, 1901-2001, 1902-2002, 1903-2003, 1904-2004, 1905-2005, 1906-2006, 1907-2007, 1908-2008, 1909-2009, 1910-2010, 1911-2011, 1912-2012, 1913-2013, 1914-2014, 1915-2015, 1916-2016, 1917-2017, 1918-2018, 1919-2019, 1920-2020, 1921-2021, 1922-2022, 1923-2023, 1924-2024, 1925-2025, 1926-2026, 1927-2027, 1928-2028, 1929-2029, 1930-2030, 1931-2031, 1932-2032, 1933-2033, 1934-2034, 1935-2035, 1936-2036, 1937-2037, 1938-2038, 1939-2039, 1940-2040, 1941-2041, 1942-2042, 1943-2043, 1944-2044, 1945-2045, 1946-2046, 1947-2047, 1948-2048, 1949-2049, 1950-2050, 1951-2051, 1952-2052, 1953-2053, 1954-2054, 1955-2055, 1956-2056, 1957-2057, 1958-2058, 1959-2059, 1960-2060, 1961-2061, 1962-2062, 1963-2063, 1964-2064, 1965-2065, 1966-2066, 1967-2067, 1968-2068, 1969-2069, 1970-2070, 1971-2071, 1972-2072, 1973-2073, 1974-2074, 1975-2075, 1976-2076, 1977-2077, 1978-2078, 1979-2079, 1980-2080, 1981-2081, 1982-2082, 1983-2083, 1984-2084, 1985-2085, 1986-2086, 1987-2087, 1988-2088, 1989-2089, 1990-2090, 1991-2091, 1992-2092, 1993-2093, 1994-2094, 1995-2095, 1996-2096, 1997-2097, 1998-2098, 1999-2099, 2000-2100, 2001-2101, 2002-2102, 2003-2103, 2004-2104, 2005-2105, 2006-2106, 2007-2107, 2008-2108, 2009-2109, 2010-2110, 2011-2111, 2012-2112, 2013-2113, 2014-2114, 2015-2115, 2016-2116, 2017-2117, 2018-2118, 2019-2119, 2020-2120, 2021-2121, 2022-2122, 2023-2123, 2024-2124, 2025-2125, 2026-2126, 2027-2127, 2028-2128, 2029-2129, 2030-2130, 2031-2131, 2032-2132, 2033-2133, 2034-2134, 2035-2135, 2036-2136, 2037-2137, 2038-2138, 2039-2139, 2040-2140, 2041-2141, 2042-2142, 2043-2143, 2044-2144, 2045-2145, 2046-2146, 2047-2147, 2048-2148, 2049-2149, 2050-2150, 2051-2151, 2052-2152, 2053-2153, 2054-2154, 2055-2155, 2056-2156, 2057-2157, 2058-2158, 2059-2159, 2060-2160, 2061-2161, 2062-2162, 2063-2163, 2064-2164, 2065-2165, 2066-2166, 2067-2167, 2068-2168, 2069-2169, 2070-2170, 2071-2171, 2072-2172, 2073-2173, 2074-2174, 2075-2175, 2076-2176, 2077-2177, 2078-2178, 2079-2179, 2080-2180, 2081-2181, 2082-2182, 2083-2183, 2084-2184, 2085-2185, 2086-2186, 2087-2187, 2088-2188, 2089-2189, 2090-2190, 2091-2191, 2092-2192, 2093-2193, 2094-2194, 2095-2195, 2096-2196, 2097-2197, 2098-2198, 2099-2199, 2100-2200, 2101-2201, 2102-2202, 2103-2203, 2104-2204, 2105-2205, 2106-2206, 2107-2207, 2108-2208, 2109-2209, 2110-2210, 2111-2211, 2112-2212,WSGR Docket No. 58774-736.6012113-2213, 2114-2214, 2115-2215, 2116-2216, 2117-2217, 2118-2218, 2119-2219, 2120-2220, 2121-2221, 2122-2222, 2123-2223, 2124-2224, 2125-2225, 2126-2226, 2127-2227, 2128-2228, 2129-2229, 2130-2230, 2131-2231, 2132-2232, 2133-2233, 2134-2234, or 2135-2235 of any one of SEQ ID NOS: 26-30 or 41-43.

[0127] Accordingly, provided herein are rAAV vectors comprising a polynucleotide comprising a nucleic acid encoding a human retinoschisin protein and a stuffer sequence. In some embodiments, the stuffer sequence has a length of between about 1000 nucleotides and about 4000 nucleotides. In some embodiments, the stuffer sequence has a length of between about 2000 nucleotides and about 3500, about 3200, about 3000, about 2900, or about 2800 nucleotides. In some embodiments, the stuffer sequence has a length of between about 2500 nucleotides and about 3000 nucleotides. In some embodiments, the stuffer sequence has a length of about 2800 nucleotides.

[0128] In some embodiments, the nucleic acid vector comprising the heterologous nucleic acid and stuff er sequence comprises a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to any one of SEQ ID NOs: 16-17, 31-35. In some embodiments, the nucleic acid vector comprising the heterologous nucleic acid and stuffer sequence comprises a sequence of any one of SEQ ID NOs: 31-34.

[0129] In some embodiments, the AAV vector comprises a first ITR and a second ITR and the length of the AAV vector between the first ITR and the second ITR (i.e., “the length between the ITRs”) is between about 2000 nucleotides and about 6000 nucleotides. In some embodiments, such an AAV vector comprises a stuffer sequence. In some embodiments, the length of the AAV vector between the first ITR and the second ITR is between about 3000 nucleotides and about 5000 nucleotides. In some embodiments, the length of the AAV vector between the first ITR and the second ITR is between about 4000 nucleotides and about 5000 nucleotides. This length between the ITRs may be about 4500 nucleotides.

[0130] In some embodiments, the total length of the heterologous nucleic acid and the stuffer sequence is between about 2000 nucleotides and about 5000 nucleotides. In some embodiments, this total length is between about 2500 nucleotides and about 4500, about 4200, about 4000, about 3800, about 3700, about 3600, or about 3500 nucleotides. In some embodiments, the stuffer sequence has a length of about 3500 nucleotides.

[0131] In some embodiments, the stuffer sequence is positioned downstream (3') of the heterologous nucleic acid encoding human retinoschisin protein. In some embodiments, the stuffer sequence is positioned upstream (5') of the poly A sequence. In some embodiments, the stufferWSGR Docket No. 58774-736.601 sequence is positioned 3' of the polyA sequence. In some embodiments, the stuffer sequence is positioned between two AAV ITR sequences.Table 1: Stuffer SequencesWSGR Docket No. 58774-736.601WSGR Docket No. 58774-736.601WSGR Docket No. 58774-736.601Example AAV Vectors

[0132] In some embodiments, provided herein are nucleic acid vectors comprising a heterologous nucleic acid (e.g., encoding a retinoschisin protein) and one or more additional elements. In some embodiments, the additional element comprises an intron. In some embodiments, the additional element comprises a splice donor region. In some embodiments, the additional element comprises a splice acceptor region. In some embodiments, the additional element comprises a promoter. In some embodiments, the additional element comprises a polyA signal. In some embodiments, the additional element comprises one or more terminal repeats. In some embodiments, the additional element comprises a WPRE element. In some embodiments, the additional element comprises two or more of the elements described above.

[0133] Accordingly, exemplary rAAV vectors described in the disclosure may comprise any one of the following structures: AAV-hGRKl-hRSl syn, AAV-hGRKl-hRSlsyn-WPREsf, AAV- hGRKl-GFP, AAV-pTR-X001-3p, AAV-pTR-X001-5p, AAV-pTR-X002-3p, AAV-pTR-X002- 3pSR, AAV-pTR-XOOl, and AAV-pTR-X002. Exemplary vectors encoding an RSI protein may comprise any of the following: AAV44.9(E53 lD)-hGRKl-hRSlsyn, AAV44.9(E53 lD)-hGRKl- hRS 1 sy n-WPREsf , AAV44.9(Y446F+T492 V+E531 D)-hGRK 1 -hRS 1 sy n, AAV44.9(Y446F+T492V+E531D)-hGRKl -hRSl syn-WPREsf, AAV44.9(Y446F+E531D)- hGRKl -hRSl syn, AAV44.9(Y446F+E53 lD)-hGRKl-hRSl syn-WPREsf, AAV44.9(T492V+E53 lD)-hGRKl-hRSl syn, AAV44.9(T492V+E53 lD)-hGRKl-hRSlsyn- WPREsf, AAV44.9-hGRKl-hRSl syn, AAV44.9-hGRKl-hRSl syn-WPREsf, AAV44.9(Y731F)- hGRK 1 -hRS 1 sy n, AAV44.9(Y731 F)-hGRK 1 -hRS 1 sy n-WPREsf , AAV5 -hGRK 1 -hRS 1 syn, AAV5-hGRKl -hRSl syn-WPREsf, AAV2(4pMut)AHS-hGRKl -hRSl syn, AAV2(4pMut)AHS- hGRKl -hRS Isy n-WPREsf, AAV8(Y447F+Y733F+T494V)-hGRKl -hRSl syn, or AAV8(Y447F+Y733F+T494V)-hGRKl -hRSl syn-WPREsf. In certain embodiments, exemplary vectors comprise AAV44.9(E53 lD)-hGRKl -hRSl syn, AAV44.9(E53 lD)-hGRKl -hRSl syn- WPREsf, AAV44.9(Y446F+T492V+E531D)-hGRKl-hRSlsyn, orAAV44 .9(Y446F+T492 V+E531 D)-hGRK 1 -hRS 1 syn-WPREsf.WSGR Docket No. 58774-736.601

[0134] In certain embodiments, the vector comprises AAV44.9-pTR-X001-3p. In certain embodiments, the vector comprises AAV44.9-pTR-X001-5p. In certain embodiments, the vector comprises AAV44.9-pTR-X002-3p. In certain embodiments, the vector comprises AAV44.9-pTR- X002-3pSR. In certain embodiments, the vector comprises AAV44.9-pTR-X001. In certain embodiments, the vector comprises AAV44.9-pTR-X002.

[0135] In certain embodiments, the vector comprises AAV44.9(E53 lD)-pTR-X001-3p. In certain embodiments, the vector comprises AAV-44.9(E53 lD)-pTR-X001-5p. In certain embodiments, the vector comprises AAV44.9(E531D)-pTR-X002-3p. In certain embodiments, the vector comprises AAV44.9(E53 lD)-pTR-X002-3pSR. In certain embodiments, the vector comprises AAV44.9(E53 lD)-pTR-X001. In certain embodiments, the vector comprises AAV44.9(E53 lD)-pTR-X002.

[0136] In certain embodiments, the vector comprises AAV2-pTR-X001-3p. In certain embodiments, the vector comprises AAV2-pTR-X001-5p. In certain embodiments, the vector comprises AAV2-pTR-X002-3p. In certain embodiments, the vector comprises AAV2-pTR-X002- 3pSR. In certain embodiments, the vector comprises AAV2-pTR-X001. In certain embodiments, the vector comprises AAV2-pTR-X002.

[0137] In certain embodiments, the vector comprises AAV2(4pMut)AHS-pTR-X001-3p. In certain embodiments, the vector comprises AAV2(4pMut)AHS-pTR-X001-5p. In certain embodiments, the vector comprises AAV2(4pMut)AHS-pTR-X002-3p. In certain embodiments, the vector comprises AAV2(4pMut)AHS-pTR-X002-3pSR. In certain embodiments, the vector comprises AAV2(4pMut)AHS-pTR-X001. In certain embodiments, the vector comprises AAV2(4pMut)AHS-pTR-X002.

[0138] Further exemplary vectors containing a reporter transgene (e.g., GFP) may comprise any of the following: AAV44.9(Y73 lF)-hGRKl-GFP, AAV44.9(E53 lD)-IRBP / GNAT2-hGFP, AAV44.9(Y73 lF)-IRBP / GNAT2-hGFP.

[0139] In some embodiments, the AAV vector comprises a sequence having about or at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 31. For example, the AAV vector comprises SEQ ID NO: 31.

[0140] In some embodiments, the AAV vector comprises a sequence having about or at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 32. For example, the AAV vector comprises SEQ ID NO: 32.WSGR Docket No. 58774-736.601

[0141] In some embodiments, the AAV vector comprises a sequence having about or at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 33. For example, the AAV vector comprises SEQ ID NO: 33.

[0142] In some embodiments, the AAV vector comprises a sequence having about or at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 34. For example, the AAV vector comprises SEQ ID NO: 34.

[0143] In some embodiments, the AAV vector comprises a sequence having about or at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 35. For example, the AAV vector comprises SEQ ID NO: 35.

[0144] In some embodiments, the AAV vector comprises a sequence having about or at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 33. For example, the AAV vector comprises SEQ ID NO: 33.

[0145] In some embodiments, the AAV vector has the following architecture: 5' ITR — promoter — intron and / or splice donor and / or splice acceptor — hRSl coding sequence — poly A — stuffer — 3' ITR. The promoter may be an hGRKl promoter. The promoter may be a CBA or smCBA promoter. The polyA sequence may be a bGH polyA sequence. The intron and / or splice donor and / or splice acceptor may be a SV40 intron.

[0146] In some embodiments, the AAV vector has the following architecture: 5' ITR — stuffer — promoter — intron and / or splice donor and / or splice acceptor — hRSl coding sequence — polyA — 3' ITR. The promoter may be an hGRKl promoter. The promoter may be a CBA or smCBA promoter. The polyA sequence may be a bGH polyA sequence. The intron and / or splice donor and / or splice acceptor may be a SV40 intron.

[0147] In some embodiments, the AAV vector has the following architecture: 5' ITR — promoter — intron and / or splice donor and / or splice acceptor — hRSl coding sequence — posttranscription regulatory element — polyA — stuffer — 3' ITR. The promoter may be an hGRKl promoter. The promoter may be a CBA or smCBA promoter. The polyA sequence may be a bGH polyA sequence. The intron and / or splice donor and / or splice acceptor may be a SV40 intron. The post-transcription regulatory element may be a WPRE element.

[0148] In some embodiments, the AAV vector has the following architecture: 5' ITR — promoter — intron and / or splice donor and / or splice acceptor — hRSl coding sequence — polyA — 3 'WSGR Docket No. 58774-736.601ITR. The promoter may be an hGRKl promoter. The promoter may be a CBA or smCBA promoter. The polyA sequence may be a bGH polyA sequence. The intron and / or splice donor and / or splice acceptor may be a SV40 intron.

[0149] In some embodiments, the AAV vector has the following architecture: 5' ITR — promoter — intron and / or splice donor and / or splice acceptor — hRSl coding sequence — posttranscription regulatory element — polyA — 3' ITR. The promoter may be an hGRKl promoter. The promoter may be a CBA or smCBA promoter. The polyA sequence may be a bGH polyA sequence. The intron and / or splice donor and / or splice acceptor may be a SV40 intron. The post -transcription regulatory element may be a WPRE element.

[0150] The disclosed rAAV vectors may comprise a nucleotide sequence having at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, atleast97% identity, atleast98%, atleast99% identity, at least 99.5% identity, or 100% identity to the nucleotide sequence of SEQ ID NO: 16 or 17. In some embodiments, the vector comprises the nucleotide sequence of SEQ ID NO: 16 or 17. In particular embodiments, the disclosure provides rAAV vectors comprising the AAV-hGRKl-hRSlsyn structure that comprises the nucleotide sequence of SEQ ID NO: 16, provided below. The length of SEQ ID NO: 16 is 1740 nucleotides (nt). In particular embodiments, the disclosure provides rAAV vectors comprising the AAV-hGRKl-hRSlsyn-WPREsf structure that comprises the nucleotide sequence of SEQ ID NO: 17, provided below. The length of SEQ ID NO: 17 is 2320 nucleotides (nt).

[0151] In some embodiments, exemplary rAAV vectors of the disclosure contain the following architecture: 5' ITR — promoter — SV40 intron — hRSl coding sequence — polyA — stuffer — 3' ITR. In some embodiments, exemplary rAAV vectors of the disclosure contain the following architecture: 5' ITR — promoter — SV40 intron — hRSl coding sequence — polyA — 3' ITR. In some embodiments, exemplary rAAV vectors of the disclosure contain the following architecture: 5' ITR — promoter — SV40 intron — hRSl coding sequence — WPREsf — polyA — stuffer — 3' ITR. In some embodiments, exemplary rAAV vectors of the disclosure contain the following architecture: 5' ITR — promoter — SV40 intron — hRSl coding sequence WPREsf — polyA — 3' ITR. The promoter of a vector in accordance with any of these architectures may be an hGRKl promoter. The promoter of a vector in accordance with any of these architectures may be a CBA or smCBA promoter. The polyA sequence of a vector in accordance with any of these architectures may be a bGH polyA sequence. In the below constructs, oneor more modified Kozak sequences is indicated in italics.Table 2: AAV Vector sequencesWSGR Docket No. 58774-736.601WSGR Docket No. 58774-736.601WSGR Docket No. 58774-736.601WSGR Docket No. 58774-736.601WSGR Docket No. 58774-736.601WSGR Docket No. 58774-736.601WSGR Docket No. 58774-736.601WSGR Docket No. 58774-736.601WSGR Docket No. 58774-736.601WSGR Docket No. 58774-736.601Capsid Variants

[0152] In some embodiments, the disclosure provides improved rAAV particles that have been derived from a number of different serotypes, including but not limited to AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10 and combinations thereof. In particular embodiments, the capsid comprises AAV5 or AAV44.9(E53 ID). In some embodiments, the capsid may comprise a variant of AAV2, a variant of AAV5, a variant of AAV7, a variant of AAV8, or a variant of AAV9. In some embodiments, the capsid comprises AAV44.9(E53 ID), AAV2(4pMut)AHS, AAV44.9, AAVrh.8, AAVrh.8R, AAVrh.10, AAVrh.74, AAV2TT, AAV2HBKO, AAV8(Y447F+Y733F+T494V), or AAVAnc80. In some embodiments, the capsid comprises AAV2, AAV6, or a capsid variant derived from AAV2 or AAV6. Accordingly, in some embodiments, the capsid comprises AAV2(7m8), AAV-DJ, AAV2 / 2-MAX, AAVSHhlO, AAVSHhlOY, AAV3, AAV3b, AAVLK03, AAV7BP2, AAV1(E531K), AAV6(D532N), AAV6- 3pmut, AAV2G9 or elements thereof.

[0153] In particular embodiments, the present disclosure provides rAAV particles comprising capsid variants of the capsid serotype AAV44.9. In some embodiments, the disclosed particles comprise the capsid protein AAV44.9(E531D). As described in the Examples herein, it was found that AAV44.9(E53 ID) mediates improved retinal transduction relative to unmodified AAV44.9 and AAVrh.8, and significantly higher transduction than benchmark capsids (e.g., AAV5 - and AAV8-based vectors) in both species. Accordingly, the disclosure provides rAAV particles comprising a capsid protein of the AAV44.9(E53 ID) serotype, and related compositions and methods. In some embodiments, the rAAV particle comprises a heterologous nucleic acid, e.g., encoding a therapeutic or diagnostic agent. The heterologous nucleic acid may be in the form of a single-stranded (ss) or self-complementary (sc) AAV nucleic acid vector, such as single -stranded or self-complementary recombinant viral genome.

[0154] The disclosure further provides rAAV particles having AAV44.9 capsids that comprise the E53 ID substitution and one or more additional substitutions, such as a Y-F mutation at residue 446, a T-V mutation at residue 492, or both. Accordingly, the disclosure provides rAAV particles comprising an AAV44.9(T492V+E53 ID) capsid, an AAV44.9(Y446F+E53 ID) capsid, or an AAV44.9(Y446F+T492V+E531D) capsid, and related compositions and methods.

[0155] In some embodiments, the disclosure provides rAAV particles comprising an AAV44.9 capsid. This capsid may be highly suitable for mediating improved transduction of retinal tissues by subretinal injection. The inventors have discovered that intravitreal injection of AAV44.9 particles may not effectively transduce retinal cells.WSGR Docket No. 58774-736.601

[0156] In some embodiments, the disclosure provides rAAV particles comprising an AAV2(4pMut)AHS capsid. The AAV2(4pMut)AHS capsid was shown to mediate enhanced lateral spread in primate retina and display efficient photoreceptor transduction. The disclosure also provides particles comprising AAV8(Y733F) and AAV8(Y447F+Y733F+T494V) capsid variants.

[0157] Aspects of this disclosure relate to vectors comprising an AAV44.9(E531D) capsid that exhibits enhanced lateral spread after subretinal injection to the fovea, wherein detachment of the fovea (e.g., a temporary bullous detachment) is minimized. In some embodiments, the disclosure provides a capsid protein, e.g., a VP1, VP2 or VP3 capsid protein, comprising the amino acid sequence of SEQ ID NO: 1, 2, and / or 3.

[0158] In some embodiments, the disclosure provides an rAAV particle comprising a capsid comprising a VP1, VP2, and / or VP3 protein, wherein the rAAV particle further comprises a polynucleotide comprising a heterologous nucleic acid. In some embodiments, the rAAV particle comprises a capsid comprising a VP1, VP2, and / or VP3 protein, wherein the VP1 protein comprises the amino acid sequence of SEQ ID NO: 1, the VP2 protein comprises the amino acid sequence of SEQ ID NO: 2, and / or the VP3 protein comprises the amino acid sequence of SEQ ID NO: 3, and wherein the AAV further comprises a polynucleotide comprising a heterologous nucleic acid. The polynucleotide may be flanked by one or more inverted terminal repeat (ITR) sequences.

[0159] In some embodiments, the disclosure provides a capsid protein comprising an amino acid sequence having at least 80% identity, at least 85% identity, at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98%, at least 99% identity, at least 99.5% identity, or 100% identity to any of SEQ ID NOs: 1, 2, or 3. In some embodiments, the disclosure provides a capsid protein comprising the amino acid sequence of SEQ ID NO: 1, 2, and / or 3. In particular embodiments, capsids comprising the amino acid sequence set forth as SEQ ID NO: 1 are provided (an AAV44.9(E53 ID) capsid VP1). In some embodiments, the disclosure provides a capsid protein comprising an amino acid sequence having at least 80% identity, at least 85% identity, at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98%, at least 99% identity, at least 99.5% identity, or 100% identity to SEQ ID NO: 18. In particular embodiments, capsids comprising the amino acid sequence set forth as SEQ ID NO: 18 are provided (an AAV44.9(Y446F+T492V+E53 ID) capsid VP1). In some embodiments, the disclosed capsids may comprise a sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 amino acids that differ relative to the sequence of any one of SEQ ID NOs: 1 -3, 18 and 44-53. These differencesWSGR Docket No. 58774-736.601 may comprise amino acidsthathave been inserted, deleted, or substituted relative to the sequence of any one of SEQ ID NOs: 1-3, 18 and 44-53.Table 3: Capsid sequencesWSGR Docket No. 58774-736.601WSGR Docket No. 58774-736.601WSGR Docket No. 58774-736.601WSGR Docket No. 58774-736.601WSGR Docket No. 58774-736.601

[0160] In some embodiments, the disclosure provides a nucleic acid, e.g., a plasmid or viral vector, comprisingthe nucleic acid sequence of SEQ ID NO: 4 (which encodes AAV44.9(E53 ID) VP1). In some embodiments, the disclosure provides a nucleic acid, e.g., a plasmid or viral vector, comprisingthe nucleic acid sequence of SEQ ID NO: 5 (which encodes AAV44.9(E53 ID) VP2). In some embodiments, the disclosure provides a nucleic acid, e.g., a plasmid or viral vector, comprisingthe nucleic acid sequence of SEQ ID NO: 6 (which encodes AAV44.9(E53 ID) VP3). In some embodiments, the viral vector is a recombinant adeno -associated viral (rAAV) vector. In some embodiments, the rAAV vector is self -complementary. In some embodiments, the nucleic acid is comprised within a cell, e.g., a mammalian or insect cell.

[0161] All substitutions in the AAV44.9 capsid protein as described herein are based on the VP1 amino acid sequence set forth in SEQ ID NO: 1. As would be appreciated by one of skill in the art, all substitutions described herein in the VP1 sequence are equally applicable to the sequences of the VP2 and VP3 proteins.

[0162] In some embodiments, an AAV-DJ capsid is used in conjunction with the rAAV vectors of the disclosure. AAV-DJ comprises the insertion of 7 amino acids into the HSPG binding domain of the AAV2 capsid and has high expression efficiency in Muller cells following intravitreal injection. In some embodiments, an AAV2(7m8) is used in conjunction with the rAAV vectors of the disclosure. The AAV2(7m8) capsid is closely related to AAV-DJ. In some embodiments, theWSGR Docket No. 58774-736.601AAV2 / 2-MAX capsid comprises five point mutations, Y272F, Y444F, Y500F, Y730F, T491V. In some embodiments, the AAVSHhlO and AAV6(D532N) capsids are derivatives of AAV6. In some embodiments, the AAV6-3pmut is (also known as AAV6(TM6) and AAV6(Y705+Y731F+T492V)).

[0163] In some embodiments, the capsid used in conjunction with the disclosed rAAV vectors is a capsid comprising non-native amino acid substitutions at amino acid residues of a wild-type AAV2 capsid. In some embodiments, the non-native amino acid substitutions comprise one or more of Y272F, Y444F, T491V, Y500F, Y700F, Y704F, Y730F or a combination thereof. In some embodiments, the capsids comprise non-native amino acid substitutions at amino acid residues of a wild-type AAV6 capsid as set forth in SEQ ID NO: 6. In some embodiments, the non-native amino acid substitutions comprise one or more of Y445F, Y705F, Y73 IF, T492V, S663 V or a combination thereof. In some embodiments, the capsid comprises AAV2G9, a variant of AAV2.

[0164] In some embodiments, the capsid comprises a non-native amino acid substitution at amino acid residue 533 of a wild-type AAV8 capsid. In some embodiments, the non-native amino acid substitution is E533K, Y733F, or a combination thereof. In some embodiments, the capsid comprises AAV7BP2, a variant of AAV8.

[0165] In some embodiments, the capsid comprises non-native amino acid substitutions of a wild-type AAV2 capsid. In some embodiments, the capsid comprises one or more of:

[0166] (a) Y444F;

[0167] (b) Y444F+Y500F+Y730F;

[0168] (c) Y272F+Y444F+Y500F+Y730F;

[0169] (d) Y444F+Y500F+Y730F+T491 V; or

[0170] (e) Y272F+Y444F+Y500F+Y730F+T491 V, or at equivalent amino acid positions corresponding thereto in any one of the wild-type AAV1, AAV3, AAV4, AAV5, AAV7, AAV9, or AAV10 capsid proteins.

[0171] In some embodiments, the capsid comprises non-native amino acid substitutions of a wild-type AAV6 capsid. In some embodiments, the capsid comprises one or more of:

[0172] (a) Y445F;

[0173] (b) Y705F+Y731F;

[0174] (c) T492V;

[0175] (d) Y705F+Y731F+T492V;

[0176] (e) S663V; or

[0177] (f) S663V+T492V.WSGR Docket No. 58774-736.601

[0178] In various embodiments, the rAAV particles comprise one of the following capsids, i.e., capsid variants of AAV2: DGE-DF (also known as ‘V1V4 VR-V’), P2-V2, P2-V3, and ME-B(Y- F+T-V). The DGE-DF capsid variant contains aspartic acid, glycine, glutamic acid, aspartic acid, and phenylalanine at amino acid positions 492, 493, 494, 499, and 500 of wild-type AAV2 VP1. The P2-V2 capsid variant contains alanine, threonine, proline, aspartic acid, phenylalanine, and aspartic acid at positions 263, 490, 492, 499, 500, and 530 of AAV2 VP1. The P2-V3 capsid variant contains asparagine, alanine, phenylalanine, alanine, asparagine, valine, threonine, arginine, aspartic acid, and aspartic acid atpositions 263, 264, 444, 451, 454, 455, 459, 527, 530, and 531 of AAV2 VP1. The ME-B(Y-F+T-V) capsid variant contains aspartic acid, glycine, glutamic acid, aspartic acid, and phenylalanine at positions 492, 493, 494, 499, and 500 of AAV2 VP1, respectively, SAAGADXAXDS at positions 546-556 of AAV2 VP1, and the following substitutions: Y272F, Y444F, and T491 V. In some embodiments, the rAAV particles comprise a capsid selected from AAV6(3pMut), AAV2(quadYF+T-V), or AAV2(trpYF). In some embodiments, the rAAV particles comprise any of the capsid variants described in International Patent Publication No. WO 2018 / 156654, which is incorporated by reference herein.

[0179] In some embodiments, the AAV particles comprise one or more capsids from AAV2, e.g., AAV2(4pMut)AHS capsid.

[0180] As shown in FIGS. 8A-8C, the transduction profile of subretinally injected rAAV vectors in Nrl-GFP mice retina at 4 and 6 weeks post-injection was evaluated. Flow cytometry was used to determine transduction efficiencies. As shown in FIG. 8C, vector-mediated GFP expression and cone arrestin colocalized, indicating that the evaluated AAV44.9, AAV44.9(Y731F), AAV44.9(E531D), AAVrh.8 and AAV8(Y733F) capsids efficiently transduces cone photoreceptors.

[0181] OCT measurements and schisis activity scores associated with the natural history study were evaluated. 1 month old mouse retinal sections stained with monoclonal antibody to RSI (shaded), as shown in FIG. 14 . RSI expression localized to the inner / outer segment junction of photoreceptors in the female heterozygous and wildtype male mice and is absent in male and female RSI KO mice. Schisis cavities were observed in the inner retina.Vectors

[0182] Recombinant AAV vectors are described herein. In some embodiments, the vector described herein is a self-complementary rAAV (scAAV) vector. In some embodiments, the vector is a single-stranded (ss) vector. In some embodiments, the vector is provided to the one or both eyes by one or more administrations of an infectious adeno-associated viral particle, an rAAV virion, orWSGR Docket No. 58774-736.601 a plurality of infectious rAAV particles in an amount and for a time sufficient to treat or ameliorate one or more symptoms of the disease or condition being treated.

[0183] In some aspects, a method for providing a mammal in need thereof with a therapeutically-effective amount of a selected therapeutic agent (e.g., a synthetic human RSI) is described herein. In some embodiments, the therapeutic agent is encoded in a heterologous nucleic acid, or transgene, that is inserted into a recombinant AAV nucleic acid vector. In some embodiments, the nucleic acid vector comprises one or more heterologous nucleic acids comprising a sequence encoding a protein or polypeptide of interest operably linked to a promoter (e.g., an hGRKl promoter), wherein the one or more transgenes are flanked on each side with an ITR sequence. The disclosed nucleic acid vectors may comprise AAV inverted terminal repeats flanking a polynucleotide comprising the RSI heterologous nucleic acid (transgene) and other regulatory elements. In some embodiments, the disclosed nucleic acid vectors comprise AAV ITRs flanking a polynucleotide comprising the RSI heterologous nucleic acid, SV40 intron, WPREsf element, and polyA signal sequence. In some embodiments, the vectors comprise AAV ITRs flanking a polynucleotide comprising the RSI heterologous nucleic acid, SV40 intron, WPREsf element, stuff sequence, and polyA sequence.

[0184] The ITR sequences canbe derived from any AAV serotype (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) or can be derived from more than one serotype. In some embodiments, the ITR sequences of the first serotype are derived from AAV2, AAV5, AAV7 AAV8, orAAV9. In some embodiments, the ITR sequences are derived from AAV2 or AAV5. In some embodiments, the ITR sequences are the same serotype as the capsid (e.g., AAV5 ITR sequences and AAV5 capsid, etc.).

[0185] ITR sequences and plasmids containing ITR sequences are known in the art and commercially available (see, e.g., products and services available from Vector Biolabs, Philadelphia, Pa.; Cellbiolabs, San Diego, Calif.; Agilent Technologies, Santa Clara, Ca; and Addgene, Cambridge, Mass.; and Gene delivery to skeletal muscle results in sustained expression and systemic delivery of a therapeutic protein. In some embodiments, the nucleic acid vector comprises a pTR-UF-11 plasmid backbone, which is a plasmid that contains AAV2 ITRs. This plasmid is commercially available from the American Type Culture Collection (ATCC MBA-331).

[0186] In some aspects, the rAAV vectors described herein may comprise multiple (two, three, four, five, six, seven, eight, nine, or ten) heterologous nucleic acids. In certain embodiments, the multiple heterologous nucleic acids are comprised on a single polynucleotide molecule. Multiple heterologous nucleic acids may be used, for example, to correct or ameliorate a gene defect caused by a multi-subunit protein. In various embodiments, a different heterologous nucleic acid may be used to encode each subunit of a protein, or to encode different peptides or proteins. This isWSGR Docket No. 58774-736.601 desirable when the size of the nucleic acid encoding the protein subunit is large, e.g., for an immunoglobulin, the platelet-derived growth factor, or a dystrophin protein. In order for the cell to produce the multi-subunit protein, a cell is infected with the rAAV particle containing each of the different subunits. Alternatively, different subunits of a protein may be encoded by the same nucleic acid sequence. In various embodiments, a single heterologous nucleic acid includes the nucleic acid encoding each of the subunits, with the nucleic acid for each subunit separated by an internal ribozyme entry site (IRES). This is desirable when the size of the nucleic acid encoding each of the subunits is small, e.g., the total size of the nucleic acid encoding the subunits and the IRES is less than five kilobases.

[0187] As an alternative to an IRES, the nucleic acid may be separated by sequences encoding a 2 A peptide, which self-cleaves in a post-translational event. This 2 A peptide is significantly smaller than an IRES, making it well suited for use when space is a limiting factor. More often, when the heterologous nucleic acid is large, consists of multi -subunits, or two heterologous nucleic acids are co-delivered, or rAAV particle carrying the desired heterologous nucleic acid(s) or subunits are co-administered to allow them to concatamerize in vivo to form a single vector genome. In such an embodiment, a first rAAV particle may carry an expression cassette which expresses a single heterologous nucleic acid and a second rAAV particle may carry an expression cassette which expresses a different heterologous nucleic acid for co -expression in the host cell. However, the selected heterologous nucleic acid may encode any biologically active product or other product, e.g., a product desirable for study.

[0188] In some aspects, the rAAV vectors may be codon-optimized for human expression. In some embodiments, the rAAV vectors may have modified Kozak nucleic acid sequences that provide for enhanced transduction or fitness in the target cell, e.g., a PR cell. Kozak sequences include the translation initiation codon (ATG) and a stretch of nucleotides positioned 5' of the initiation codon.Methods of Treatment and Formulations

[0189] In some embodiments, methods are provided involving providing a mammal in need thereof with a therapeutically effective amount of a selected therapeutic agent, the method comprising administering to one or both eyes of the mammal, an amount of the rAAV particles described herein; and for a time effective to provide the mammal with a therapeutically -effective amount of the selected therapeutic agent.

[0190] In certain embodiments, the mammal is suspected of having, is at risk for developing, or has been diagnosed with X-linked retinoschisis. In some embodiments, the mammal is a human. In some embodiments, the human is atleast 1, 2, 3, 4, 5, or 6 years of age. In some embodiments theWSGR Docket No. 58774-736.601 human is between 6 years of age and 18 years of age. In some embodiments, the human is between 6 years of age and 65 years of age. In some embodiments, the human is at least 18 years of age.

[0191] In some embodiments, methods are provided for transducing a mammalian photoreceptor cell or retinal pigment epithelium cell, the method comprising administering to one or both eyes of a mammal the rAAV particles described herein. In particular embodiments, methods are provided for expressing a polynucleotide in one or more photoreceptor cells of a mammal, the method comprising subretinally or intravitreally administering to one or both eyes of the mammal the rAAV particles described herein, or compositions thereof, wherein the rAAV particle comprises a polynucleotide comprising at least a first polynucleotide that comprises a PR- or an RPE-cell-specific promoter operably linked to at least a first heterologous nucleic acid sequence that encodes a therapeutic agent, for a time effective to produce the therapeutic agent in the one or more PR or RPE cells of the mammal.

[0192] In particular embodiments, a replacement coding sequence is administered to the subject to provide a functional protein, e.g., RSI protein, to restore, e.g., completely, or partially, photoreceptor function to a subject (e.g., a human). In some embodiments, one or both alleles of a target coding sequence of the subject are silenced by administering an rAAV particle comprising a heterologous nucleic acid disclosed herein to the subject (e.g., to a human having dominant conerod dystrophy). In particular embodiments, the endogenous mutant alleles of one or more target coding sequences are silenced or suppressed by administering an rAAV particle disclosed herein.

[0193] In some embodiments, the mammal is a human subject. In some embodiments, the mammal is a non-human primate subject. Non-limiting examples of non-human primate subjects include macaques (e.g., cynomolgus or rhesus macaques), marmosets, tamarins, spider monkeys, owl monkeys, vervet monkeys, squirrel monkeys, baboons, gorillas, chimpanzees, and orangutans. Other exemplary subjects include domesticated animals such as dogs and cats; livestock such as horses, cattle, pigs, sheep, goats, and chickens; and other animals such as mice, rats, guinea pigs, and hamsters.

[0194] In certain embodiments, methods are provided for subretinally administering to a fovea (e.g., foveal cone cells) of the mammal the rAAV particles described herein or compositions thereof. In particular embodiments, detachment of the fovea is minimized during and / or after subretinal administration. In particular embodiments, subretinal administration of the rAAV particle is performed in the absence of any detachment of the fovea.

[0195] In some embodiments, the dose of rAAV particles administered to a cell or a subject may be on the order ranging from 106to 1014vgs / mL (or particles / mL) or 103to 1015vgs / mL, or any values there between for either range, such as for example, about 106, 107, 108, 109, 1010, 1011,WSGR Docket No. 58774-736.6011012, 1013, or 1014vgs / mL. In particular embodiments, rAAV particles in an amount of 5 x1010vgs / mL are be administered. In some embodiments, rAAV particles in an amount of 1 x1011vgs / mL are be administered. In some embodiments, rAAV particles in an amount of 1 x 1012vgs / mL are be administered. In some embodiments, the dose of rAAV particles administered to a subject may be on the order ranging from 106to 1014vgs / mL, or 103to 1015vgs / mL, or any values therebetween for either range, such as for example, about 106, 107, 108, 109, 1010, 1011, 1012, 1013, or 1014vgs / mL. In some embodiments, rAAV particles of higher than 1013vgs / mL are be administered. The rAAV particles can be administered as a single dose, or divided into two or more administrations as may be required to achieve therapy of the particular disease, disorder or condition being treated.

[0196] In particular embodiments, rAAV particle titers range from 5 xl010-l xlO12vg / ml. In some embodiments, rAAV particle titers can be 1x1010, 2.5x1010, 5 xl010, 1 xlO11, 2.5 xlO11, 5 xl0n, I xlO12, 2.5xl012, 5 xl012, 1 X1013, 2.5 X1013, or 5xl013vg / mL. In some embodiments, particle titers are less than 5 xl010vg / mL. In some embodiments, rAAV particles are administered via methods described herein (e.g., subretinally, or intravitreally). In particular embodiments, particles are administered subretinally. In some embodiments, about 1.5xl010vg / eye is administered to the subject.

[0197] The rAAV particles can be administered as a single dose, or divided into two or more administrations (e.g., up to three subretinal injection blebs) as may be required to achieve therapy of the particular disease, disorder or condition being treated. In some embodiments, from 1 to 500 microliters of a composition (e.g., comprising an rAAV particle) described in this application is administered to one orboth eyesof a subject. For example, in some embodiments, about 10, about 30, about45, about 50, about 75, about 90, about 100, about 200 microliters can be administered to each eye. In particular embodiments, 3 subretinal injections ranging in bleb volumes of 10 pL to 100 pL each may be administered. In some embodiments, about 150 microliters is administered to each eye.

[0198] In some embodiments, the disclosure provides formulations of one or more rAAV -based compositions disclosed herein in pharmaceutically acceptable solutions for administration to a cell or an animal, either alone or in combination with one or more other modalities of therapy, and in particular, for therapy of human cells, tissues, and diseases affecting man.

[0199] The rAAV particles described herein may be administered in combination with other agents as well, such as, e.g., proteins or polypeptides or various pharmaceutically -active agents, including one or more systemic or topical administrations of therapeutic polypeptides, biologically active fragments, or variants thereof. In particular embodiments, the described rAAV particles may be administered in combination with one or more carbonic anhydrase inhibitors (CAIs). In someWSGR Docket No. 58774-736.601 embodiments, they may be co-administered with any of the CAIs acetazolamide, dichlorphenamide (also known as diclofenamide), methazolamide, dorzolamide, brinzolamide, ethoxzolamide, and zonisamide. There is virtually no limit to other components that may also be included, given that the additional agents do not cause a significant adverse effect upon contact with the target cells or host tissues. The rAAV particles may thus be delivered along with various other agents as required in the particular instance. Such compositions may be purified from host cells or other biological sources, or alternatively may be chemically synthesized as described herein.

[0200] In some embodiments, described herein are uses of the described rAAV vectors, viral particles, compositions, and (host) cells described herein in the preparation of medicaments for diagnosing, preventing, treating, or ameliorating at least one or more symptoms of XLRS. In some embodiments, the methods comprise direct administration to the vitreous of one or both eyes of a mammal in need thereof, one or more of the described vectors, viral particles, cells, compositi ons, or pluralities thereof, in an amount and for a time sufficient to diagnose, prevent, treat, or lessen one or more symptoms of such a disease, dysfunction, disorder, abnormal condition, deficiency, injury, or trauma in one or both eyes of the affected mammal. In various embodiments, the mammal is a human subject.

[0201] In some embodiments, described herein are compositions useful in treating XLRS in the preparation of medicaments to treat XLRS, comprising one or more of the described rAAV vectors, particles, compositions, and host cells. In some embodiments, the compositions comprise at least a first pharmaceutically -acceptable excipient for use in the manufacture of medicaments and methods involving therapeutic administration of such rAAV particles or vectors. In some embodiments, pharmaceutical formulations are suitable for intravitreal administration into one or both eyes of a human or other mammal.

[0202] In some embodiments, described herein are methods and uses of the described rAAV vectors and compositions for treating or ameliorating the symptoms of XLRS in human photoreceptors or RPE cells. In some embodiments, the disclosed methods and uses comprise intravitreal or subretinal administration to one or both eyes of a subj ect in need thereof, one or more of the described particles vectors, particles, host cells, or compositions, in an amount andfor a time sufficient to treat or ameliorate the symptoms of such a deficiency in the affected mammal. In some embodiments, the methods comprise prophylactic treatment of an animals suspected of having such conditions, or administration of such compositions to those animals at risk for developing such conditions either following diagnosis, or prior to the onset of symptoms.

[0203] In some embodiments, administration of the rAAV vectors and compositions described herein to the eye improves vision as compared to before administration. In some embodiments,WSGR Docket No. 58774-736.601 administration improves visual acuity. In some embodiments, visual acuity in the eye is measured based on Best-Corrected Visual Acuity (BVCA). In some embodiments, visual acuity in the eye is measured based on Low Luminence Visual Acuity (LLVA).

[0204] In some embodiments, the BVCA before administration is between 34 to 73 ETDRS letters. In some embodiments, the BVCA before administration is less than 30 ETDR letters, 35 ETDRS letters, 40 ETDRS letters, 45 ETDRS letters, 50 ETDRS letters, 55 ETDRS letters, 60 ETDRS letters, 65 ETDRS letters, 60 ETDRS letters, 75 ETDRS letters, or 80 ETDRS letters. In some embodiments, the BVCA before administration is between 20 / 200 to 20 / 40. In some embodiments, visual acuity is improved atleast 5%, 10%, 15%, 20%, 25%, 30%, 35% or more as measured by BCVA. In some embodiments, the BCVA is improved by at least 3 ETDRS letters, at least 4, ETDRS letters, at least 5 ETDRS letters, at least 6 ETDRS letters, at least 7 ETDRS letters, at least 8 E ETDRS TDRs letters, at least 9 ETDRS letters, or at least 10 ETDRS letters as compared to before administration of the rAAV vectors and compositions. In some embodiments, the improvement is maintained at least 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 12 months, at least 18 months, at least 24 months, at least 36 months, at least 48 months, or at least 60 months after administration.

[0205] In some embodiments, the LLVA before administration is 28 and 64 letters, is less than 30 ETDR letters, 35 ETDRS letters, 40 ETDRS letters, 45 ETDRS letters, 50 ETDRS letters, 55 ETDRS letters, 60 ETDRS letters, 65 ETDRS letters, 60 ETDRS letters, 75 ETDRS letters, or 80 ETDRS letters. In some embodiments, visual acuity is improved at least 5%, 10%, 15%, 20%, 25%, 30%, 35% or more as measured by LLVA. In some embodiments, the LLVA is improved by at least 3 ETDRS letters, at least 4, ETDRS letters, at least 5 ETDRS letters, at least 6 ETDRS letters, at least 7 ETDRS letters, at least 8 ETDRS letters, at least 9 ETDRS letters, or at least 10 ETDRS letters as compared to before administration of the rAAV vectors and compositions. In some embodiments, the improvement is maintained at least 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 12 months, atleast 18 months, at least 24 months, atleast 36 months, at least 48 months, or at least 60 months after administration.

[0206] In some embodiments, there is a reduction in size of at least one schisis cavity in the eye compared to prior to administering the AAV particle. The schisis cavity may be measured using optical coherency tomography (OCT). In some embodiments, there is at least a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% reduction in the size of at least one schisis cavity. In some embodiments, there is a complete closure of the at least one schisis cavity. In some embodiments, there is at least a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% reduction in the size of all one schisis cavities. In some embodiments, there is complete closure of all schisis cavities. In someWSGR Docket No. 58774-736.601 embodiments, the improvement is maintained at least 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 12 months, at least 18 months, at least 24 months, at least 36 months, at least 48 months, or at least 60 months after administration. In some embodiments, the at least one schisis cavity that is reduced in size is located in an area of the retina outside of the subretinal bleb.

[0207] In some embodiments, there is a reduction in retinal thickness compared to prior to administering the AAV particle. The retinal thickness may be measured using optical coherency tomography (OCT). In some embodiments, there is at least a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% reduction in the size of retinal thickness. In some embodiments, the reduction in retinal thickness is specific to the central retina or the macula of the eye.

[0208] In some embodiments, the improvement is maintained at least 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 12 months, at least 18 months, at least 24 months, at least 36 months, at least 48 months, or at least 60 months after administration.

[0209] In some embodiments, there is an improvement in the visual field of the subject. In some embodiments, the visual field is measured by microperimetry. In some embodiments, the visual field is measured using static automatic perimetry. In some embodiments, the static automatic perimetry comprises a Humphrey Field Analyzer. In some embodiments, measurements are taken at at least 4 fixed loci, 5 fixed loci, 6 fixed loci, 7 fixed loci, 8 fixed loci, 9 fixed loci, or 10 fixed loci. In some embodiments, the fixed loci are pre-selected. In some embodiments, the visual field is improved by at least 3 dB, 4 dB, 5dB, 6 dB, 7dB, 8 dB, 9 dB, or 10 dB. In some embodiments, the improvement is an improvement of at least 4 dB averaged over at least 9 loci. In some embodiments, the improvement is an improvement of at least 7 dB averaged over at least 5 loci. In some embodiments, the improvement is maintained at least 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 12 months, at least 18 months, at least 24 months, at least 36 months, at least 48 months, or at least 60 months after administration.

[0210] In some embodiments, administration of the rAAV vectors and compositions described herein to the eye improves vision as compared to before administration in a measurement based on patient feedback. In some embodiments, the measurement is a patient global impression of change (PGIC). The PGIC may ask the subject or patient to rate their overall change following administration. In some embodiments, the PGIC comprises the subject or patient rating the overall change in at least one of activity limitations, symptoms, emotions, vision, and overall quality of life on a scale from 1 to 7. In some embodiments, the PGIC comprises the subject or patient rating the overall change in at least one of activity limitations, symptoms, emotions, vision, and overall quality of life on a scale from 1 to 10. In some embodiments, the PGIC comprises the subject or patient rating the overall change in at least one of activity limitations, symptoms, emotions, vision,WSGR Docket No. 58774-736.601 and overall quality of life on a scale from no change to a significant change. In some embodiments, the measurement is a patient global impression of severity (PGIS). The PGIS may ask the subject or patientto rate their overall impression of severity on a scale from 1 to 7. In some embodiments, the PGIS may comprise the subject or patient rating their overall impression of severity from least severe to most severe. In some embodiments, the PGIS, the PGIC, or a combination thereof correlates with a clinical endpoint such as a measurement of visual acuity, visual field, or a physical measurement of the retina (such as retinal thickness or area of at least one schisis cavity). In some embodiments, there is a significant improvement in the measurement as compared to before the administration. In some embodiments, the improvement is maintained at least 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 12 months, at least 18 months, at least 24 months, at least 36 months, at least 48 months, or at least 60 months after administration.Pharmaceutical Compositions and Cells

[0211] In some aspects, the disclosure contemplates host cells that comprise a particle that incorporates an AAV44.9(E53 ID) capsid, a nucleic acid encoding a AAV44.9(E53 ID) capsid or an rAAV particle as described herein. Such host cells include mammalian host cells, with human host cells being preferred, and may be isolated, e.g., in cell or tissue culture. In some embodiments, the host cell is a cell of the eye.

[0212] In some embodiments, a composition is provided which comprises an rAAV particle as described herein (e.g., comprising a AAV44.9(E53 ID) capsid) and optionally a pharmaceutically acceptable carrier, excipient, diluent and / or buffer. In some embodiments, the compositions described herein can be administered to a mammal (or subject) in need of treatment. In some embodiments, the subject has or is suspected of having retinoschisis. In some embodiments, the subject has one or more endogenous mutant alleles (e.g., associated with or that cause a disease, disorder or condition of the eye or retina, such as retinoschisis).

[0213] Formulation of pharmaceutically -acceptable excipients and carrier solutions is well- known to those of skill in the art, as is the development of suitable dosing and treatment regimens for using the particular compositions described herein in a variety of treatment regimens, including e.g., subretinal, intravitreal, parenteral, intravenous, intranasal, intra -articular, and intramuscular administration and formulation.

[0214] Typically, these formulations may contain at least about 0.1% of the therapeutic agent (e.g., rAAV particle) or more, although the percentage of the active ingredient(s) may, of course, be varied and may conveniently be between about 1 or 2% and about 70% or 80% or more of the weight or volume of the total formulation. Naturally, the amount of therapeutic agent(s) (e.g., rAAV particle) in each therapeutically -useful composition may be prepared in such a way that aWSGR Docket No. 58774-736.601 suitable dosage will be obtained in any given unit dose of the compound. Factors such as solubility, bioavailability, biological half-life, route of administration, product shelf life, as well as other pharmacological considerations will be contemplated by one skilled in the art of preparing such pharmaceutical formulations, and as such, a variety of dosages and treatment regimens may be desirable.

[0215] In certain circumstances it will be desirable to deliver an rAAV particle as described herein (e.g., comprising a AAV44.9(E53 ID) capsid) in suitably formulated pharmaceutical compositions disclosed herein either subretinally, intraocularly, intravitreally, parenterally, subcutaneously, intravenously, intracerebro-ventricularly, intramuscularly, intrathecally, orally, intraperitoneally, by oral or nasal inhalation, or by direct injection to one or more cells, tissues, or organs by direct injection.

[0216] The pharmaceutical forms of compositions (e.g., comprising an rAAV particle as described herein) suitable for injectable use include sterile aqueous solutions or dispersions. In some embodiments, the form is sterile and fluid to the extent that easy syringability exists. In some embodiments, the form is stable under the conditions of manufacture and storage and is preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, saline, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and / or vegetable oils. Proper fluidity may 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 dispersion and by the use of surfactants.

[0217] The term “carrier” refers to a diluent, adjuvant, excipient, or vehicle with which the rAAV particle as described herein is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum oil such as mineral oil, vegetable oil such as peanut oil, soybean oil, and sesame oil, animal oil, or oil of synthetic origin. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers.

[0218] The compositions of the present disclosure can be delivered to the eye through a variety of routes. They may be delivered intraocularly, by topical application to the eye or by intraocular injection into, for example the vitreous (intravitreal injection) or subretinal (subretinal injection) inter-photoreceptor space. In some embodiments, they are delivered to rod photoreceptor cells. Alternatively, they may be delivered locally by insertion or injection into the tissue surrounding the eye. They may be delivered systemically through an oral route or by subcutaneous, intravenous, or intramuscular injection. Alternatively, they may be delivered by means of a catheter or by means of an implant, wherein such an implant is made of a porous, non -porous or gelatinous material,WSGR Docket No. 58774-736.601 including membranes such as silastic membranes or fibers, biodegradable polymers, or proteinaceous material. They can be administered prior to the onset of the condition, to prevent its occurrence, for example, during surgery on the eye, or immediately after the onset of the pathological condition or during the occurrence of an acute or protracted condition.

[0219] For administration of an injectable aqueous solution, for example, the solution may be suitably buffered, if necessary, and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, intravitreal, subcutaneous, and intraperitoneal administration. In this connection, a sterile aqueous medium that can be employed will be known to those of skill in the art in light of the present disclosure. For example, one dosage may be dissolvedin 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion, (see for example, “Remington's Pharmaceutical Sciences” 15thEdition, pages 1035-1038 and 1570-1580). Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject. Moreover, for human administration, preparations should meet sterility, pyrogenicity, and the general safety and purity standards as required by, e.g., FDA Office of Biologies standards.

[0220] Sterile injectable solutions may be prepared by incorporating an rAAV particle as described herein in the required amount in the appropriate solvent with several of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile -filtered solution thereof.

[0221] The amount of composition (e.g., comprising an rAAV particle as described herein) and time of administration of such composition will be within the purview of the skilled artisan having benefit of the present teachings. It is likely, however, that the administration of therapeutically - effective amounts of the disclosed compositions may be achieved by a single administration, such as for example, a single injection of sufficient numbers of rAAV particles to provide therapeutic benefit to the patient undergoing such treatment. Alternatively, in some circumstances, it may be desirable to provide multiple, or successive administrations of the composition, either over aWSGR Docket No. 58774-736.601 relatively short, or a relatively prolonged period of time, as may be determined by the medical practitioner overseeing the administration of such compositions.

[0222] In some embodiments, visual acuity can be maintained or restored (e.g., partially, or completely) after administering one or more compositions described in this application. In some embodiments, one or more photoreceptor cells or one or more RPE cells may be preserved, partially or completely, and / or one or more rod- and / or cone-mediated functions may be restored, partially or completely, after administering one or more compositions described in this application.

[0223] To “treat” a disease as the term is used herein, means to reduce the frequency or severity of at least one sign or symptom of a disease, disorder or condition experienced by a subject (e.g., cone-rod dystrophy). The compositions described above are typically administered to a subject in an effective amount, that is, an amount capable of producing a desirable result. The desirable result will depend upon the active agent being administered. For example, an effective amount of a rAAV particle may be an amount of the particle that is capable of transferring a heterologous nucleic acid to a host organ, tissue, or cell.

[0224] Toxicity and efficacy of the compositions utilized in methods of the disclosure can be determined by standard pharmaceutical procedures, using either cells in culture or experimental animals to determine the LD50 (the dose lethal to 50% of the population). The dose ratio between toxicity and efficacy the therapeutic index and it can be expressed as the ratio LD50 / ED50. Those compositions that exhibit large therapeutic indices are preferred. While those that exhibit toxic side effects may be used, care should be taken to design a delivery system that minimizes the potential damage of such side effects. The dosage of compositions as described herein lies generally within a range that includes an ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized.Methods of Producing rAAV Particles

[0225] In some embodiments, a plasmid containing the heterologous nucleic acid may be combined with one or more helper plasmids, e.g., that contain a rep gene (e.g., encoding Rep78, Rep68, Rep52 and Rep40) and a cap gene (encoding VP1, VP2, and VP3, including a modified VP3 region as described herein), and transfected or permanently integrated into a producer cell line such that the rAAV particle may be packaged and subsequently purified.

[0226] In some embodiments, the one or more helper plasmids include a first helper plasmid comprising a rep gene and a cap gene (e.g., encoding a rAAV capsid protein as described herein) and a second helper plasmid comprising a Ela gene, a Elb gene, a E4 gene, a E2a gene, and a VA gene. In some embodiments, the rep gene is a rep gene derived from AAV2 and the cap gene is derived from AAV44.9 and may include modifications to the gene in order to produce the modifiedWSGR Docket No. 58774-736.601 capsid protein described herein. Helper plasmids, and methods of making such plasmids, are known in the art and commercially available (see, e.g., pDM, pDG, pDPlrs, pDP2rs, pDP3rs, pDP4rs, pDP5rs, pDP6rs, pDG(R484E / R585E), and pDP8.ape plasmids from PlasmidFactory, Bielefeld, Germany; other products and services available from Vector Biolabs, Philadelphia, Pa.;Cellbiolabs, San Diego, Calif.; Agilent Technologies, Santa Clara, Ca; and Addgene, Cambridge, Mass.; pxx6; Grimm et al. (1998), Novel Tools for Production and Purification of Recombinant Adenoassociated Virus Vectors, Human Gene Therapy, Vol. 9, 2745 -2760; Kern, A. et al. (2003), Identification of a Heparin -Binding Motif on Adeno- Associated Virus Type 2 Capsids, Journal of Virology, Vol. 77, 11072-11081.; Grimm et al. (2003), Helper Virus-Free, Optically Controllable, and Two-Plasmid-Based Production of Adeno-associated Virus Vectors of Serotypes 1 to 6, Molecular Therapy, Vol. 7, 839-850; Kronenberg et al. (2005), A Conformational Change in the Adeno-Associated Virus Type 2 Capsid Leads to the Exposure of Hidden VP1 N Termini, Journal of Virology, Vol. 79, 5296-5303; and Moullier, P. and Snyder, R. O. (2008), International efforts for recombinant adeno-associated viral vector reference standards, Molecular Therapy, Vol. 16, 1185-1188).

[0227] An exemplary, non-limiting, rAAV particle production method is described next. One or more helper plasmids are produced or obtained, which comprise rep and cap ORFs for the desired AAV serotype and the adenoviral VA, E2A (DBP), and E4 genes under the transcriptional control of their native promoters. The cap ORF may also comprise one or more modifications to produce a modified capsid protein as described herein. HEK293 cells (available from ATCC®) are transfected via CaPO4 -mediated transfection, lipids, or polymeric molecules such as Polyethylenimine (PEI) with the helper plasmid(s) and a plasmid containing a nucleic acid vector described herein. The HEK293 cells are then incubated for at least 60 hours to allow for rAAV particle production. Alternatively, in another example Sf9-based producer stable cell lines are infected with a single recombinant baculovirus containing the heterologous nucleic acid sequence. As a further alternative, in another example HEK293 or BHK cell lines are infected with a HSV containing the heterologous nucleic acid sequence and optionally one or more helper HSVs containing rep and cap ORFs as described herein and the adenoviral VA, E2A (DBP), and E4 genes under the transcriptional control of their native promoters. The HEK293, BHK, or Sf9 cells may then incubated for at least 60 hours to allow for rAAV particle production. The rAAV particles can then be purified using any method known the art or described herein, e.g., by iodixanol step gradient, CsCl gradient, chromatography, polyethylene glycol (PEG) precipitation, and / or affinity capture.WSGR Docket No. 58774-736.601

[0228] In various embodiments, an iodixanol step gradient purification method is used. Vectors may be packaged into mammalian cells (e.g., HEK293T cells) and purified by iodixanol gradient centrifugation, followed by buffer exchange and concentration into BSS / Tween buffer. An affinity capture step may be added.Numbered Embodiments

[0229] The following numbered embodiments are intended to be exemplary: Embodiment 1 comprises an adeno-associated virus (AAV) vector comprising a polynucleotide that comprises a heterologous nucleic acid encoding a retinoschisin protein and an SV40 intron. Embodiment 2 comprises the AAV vector of embodiment 1, wherein the SV40 intron comprises a splice donor region and / or a splice acceptor region. Embodiment s comprises the AAV vector of embodiment 1 or 2, wherein the SV40 intron comprises a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the nucleic acid sequence of SEQ ID NO: 20. Embodiment 4 comprises the AAV vector of embodiment 1 or 2, wherein the SV40 intron comprises the nucleic acid sequence of SEQ ID NO: 20. Embodiment 5 comprises the AAV vector of any one of embodiments 1 -4, wherein the retinoschisin protein is a human retinoschisin protein. Embodiment 6 comprises the AAV vector of any one of embodiments 1 -5, wherein the heterologous nucleic acid does not comprise the 5' untranslated region of human retinoschisin. Embodiment 7 comprises the AAV vector of embodiment 6, wherein the 5 ' untranslated region comprises the nucleic acid sequence of SEQ ID NO: 39. Embodiment 8 comprises the AAV vector of any one of embodiments 1 -7, wherein the nucleic acid does not comprise the 3 ' untranslated region of human retinoschisin. Embodiment 9 comprises the AAV vector of embodiment 8, wherein the 3' untranslated region comprises the nucleic acid sequence of SEQ ID NO: 40. Embodiment 10 comprises a recombinant AAV (rAAV) vector comprising a polynucleotide that comprises a heterologous nucleic acid comprising a sequence having atleast 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 92.5%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the nucleic acid sequence of SEQ ID NO: 8. Embodiment 11 comprises the rAAV vector of any one of embodiments 1 -10, wherein the heterologous nucleic acid comprises the nucleic acid sequence set forth as SEQ ID NO: 8, 9 or 10. Embodiment 12 comprises the rAAV vector of any one of embodiments 1 -11, wherein the heterologous nucleic acid is operably linked to one or more regulatory elements that direct expression of the heterologous nucleic acid in a photoreceptor cell or retinal pigment epithelium cell. Embodiment 13 comprises the rAAV vector of any one of embodiments 1 -12, wherein the retinoschisin protein comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity toWSGR Docket No. 58774-736.601 the amino acid sequence of SEQ ID NO: 12. Embodiment 14 comprises the rAAV vector of any one of embodiments 1-13, wherein the retinoschisin protein comprises the amino acid sequence of SEQ ID NO: 12. Embodiment 15 comprises the rAAV vector of any one of embodiments 1 -14, wherein the polynucleotide comprises a promoter. Embodiment 16 comprises the rAAV vector of embodiment 15, wherein the promoter is selected from a rhodopsin kinase promoter, a rhodopsin promoter, an IRBP promoter, a chimeric human Retinoschisin -IRBP enhancer (RS / IRPB); a red / green cone opsin promoter, a Cone Arrestin promoter, a chimeric IRBP enhancer-cone transducin promoter, a chicken beta actin promoter, and a truncated chimeric chicken beta actin (smCBA) promoter. Embodiment 17 comprisesthe rAAV vector of embodiment 15 or 16, wherein the promoter is a rhodopsin kinase promoter. Embodiment 18 comprises the rAAV vector of any one of embodiments 15-17, wherein the promoter is a human rhodopsin kinase (hGRKl) promoter. Embodiment 19 comprises the rAAV vector of any one of embodiments 15-18, wherein the promoter comprises a nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 7. Embodiment 20 comprisesthe rAAV vector of any one of embodiments 15-18, wherein the promoter comprises the nucleic acid sequence of SEQ ID NO: 7. Embodiment 21 comprises the rAAV vector of any one of embodiments 1 -20, wherein the polynucleotide comprises a WPRE element. Embodiment 22 comprises the rAAV vector of embodiment 21, wherein the WPRE element comprises a nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 15, or comprises the nucleotide sequence of SEQ ID NO: 15. Embodiment 23 comprises the rAAV vector of embodiment 21 or 22, wherein the WPRE element is positioned 3 ' of the heterologous nucleic acid. Embodiment 24 comprises the rAAV vector of any one of embodiments 1-23, wherein the vector is self-complementary. Embodiment 25 comprisesthe rAAV vector of any one of embodiments 1-24, wherein the polynucleotide is flanked by one or more inverted terminal repeats (ITRs). Embodiment 26 comprises the rAAV vector of embodiment 25, wherein the one or more ITR sequences comprises a first ITR sequence and a second ITR sequence. Embodiment 27 comprises the rAAV vector of embodiment 26, wherein the first ITR sequence comprises a nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to any one of SEQ ID NOs: 22-23. Embodiment 28 comprises the rAAV vector of embodiment 26 or 27, wherein the first ITR sequence comprises the nucleic acid sequence of any one of SEQ ID NOs: 22-23. Embodiment 29 comprises the rAAV vector of any one of embodiments 26-28, wherein the second ITR sequence comprises a nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,WSGR Docket No. 58774-736.60188%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to any one of SEQ ID NOs: 24-25. Embodiment 30 comprises the rAAV vector of any one of embodiments 26-29, wherein the second ITR sequence comprises the nucleic acid sequence of any one of SEQ ID NOs: 24-25. Embodiment 31 comprises the rAAV vector of any one of embodiments 1-30, wherein the polynucleotide further comprises a polyadenylation signal. Embodiment 32 comprises the rAAV vector of embodiment 31, wherein the polyadenylation signal is selected from a bovine growth factor hormone polyadenylation signal, an SV40 polyadenylation signal, a human growth factor hormone polyadenylation signal, andan rbGlob polyadenylation signal. Embodiment 33 comprises the rAAV vector of embodiment 32, wherein the polyadenylation signal comprises a bovine growth factor polyadenylation signal. Embodiment 34 comprises the rAAV vector of any one of embodiments 31-33, wherein the poly adenylation signal comprises a sequence having least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 19. Embodiment 35 comprises the rAAV vector of any one of embodiments 31-34, wherein the polyadenylation signal comprises SEQ ID NO: 19. Embodiment 36 comprises the rAAV vector of any one of embodiments 1 -35, wherein the vector comprises a nucleotide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the nucleotide sequence of any one of SEQ ID NOS: 16, 17, and 31-35. Embodiment 37 comprises the rAAV vector of any one of embodiments 1-36, wherein the vector comprises any one of the nucleotide sequences of any one of SEQ ID NOS: 16, 17, and 31-35. Embodiment 38 comprises the rAAV vector of any one of embodiments 1-37, wherein the SV40 intron is positioned 5' of the heterologous nucleic acid. Embodiment 39 comprises the rAAV vector of any one of embodiments 1-38, wherein the SV40 intron is positioned 3' of the promoter. Embodiment 40 comprises the rAAV vector of any one of embodiments 1 -39, wherein the SV40 intron is positioned between the promoter and heterologous nucleic acid. Embodiment 41 comprises the rAAV vector of any one of embodiments 1 -40, wherein the SV40 intron is positioned 3' of the heterologous nucleic acid. Embodiment 42 comprises an AAV vector comprising a polynucleotide comprising a heterologous nucleic acid encoding a retinoschisin protein operably linked to a rhodopsin kinase promoter. Embodiment 43 comprises the rAAV vector of embodiment 42, wherein the promoter is a human rhodopsin kinase (hGRKl) promoter. Embodiment 44 comprises the AAV vector of embodiment 42 or embodiment 43, wherein the promoter comprises a nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 7. Embodiment 45 comprises the AAV vector of any one of embodiments 42-44, wherein the promoter comprises the nucleic acid sequence of SEQ ID NO: 7. Embodiment 46 comprises theWSGR Docket No. 58774-736.601AAV vector of any one of embodiments 42-45, comprising a polyadenylation signal. Embodiment 47 comprises the AAV vector of embodiment 46, wherein the polyadenylation signal is selected from a bovine growth factor hormone polyadenylation signal, an SV40 polyadenylation signal, a human growth factor hormone polyadenylation signal, and an rbGlob polyadenylation signal. Embodiment 48 comprises the AAV vector of embodiment 47, wherein the polyadenylation signal comprises a bovine growth factor polyadenylation signal. Embodiment 49 comprises the AAV vector of any one of embodiments 46-48, wherein the polyadenylation signal comprises a sequence having least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 19. Embodiment 50 comprises the AAV vector of any one of embodiments 46-49, wherein the polyadenylation signal comprises SEQ ID NO: 19. Embodiment 51 comprises the AAV vector of any one of embodiments, 42-50, comprising one or more ITRs. Embodiment 52 comprises the AAV vector of embodiment 51, wherein the one or more ITRs comprises a first ITR sequence and a second ITR sequence. Embodiment 53 comprises the AAV vector of embodiment 52, wherein the first ITR sequence comprises a nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NOs: 22-23. Embodiment 54 comprises the AAV vector of embodiment 52 or 53, wherein the first ITR sequence comprises the nucleic acid sequence of SEQ ID NOs: 22-23. Embodiment 55 comprises the AAV vector of any one of embodiments 52-54, wherein the second ITR sequence comprises a nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NOs: 24-25. Embodiment 56 comprises the AAV vector of any one of embodiments 52-55, wherein the second ITR sequence comprises the nucleic acid sequence of SEQ ID NOs: 24-25. Embodiment 57 comprises the rAAV vector of any one of embodiments 42-56, comprising a WPRE element. Embodiment 58 comprises the rAAV vector of embodiment 57, wherein the WPRE element comprises a nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 15. Embodiment 59 comprises the rAAV vector of embodiment 58, wherein the WPRE element comprises the nucleotide sequence of SEQ ID NO: 15. Embodiment 60 comprises the rAAV vector of any one of embodiments 57-59, wherein the WPRE element is positioned 3' of the heterologous nucleic acid. Embodiment 61 comprises an AAV vector comprising a polynucleotide comprising a heterologous nucleic acid encoding a retinoschisin protein and a polyadenylation signal. Embodiment 62 comprises the AAV vector of embodiment 61, wherein the polyadenylation signal is selected from a bovine growth factor hormone polyadenylation signal, an SV40 polyadenylation signal, a human growth factor hormoneWSGR Docket No. 58774-736.601 polyadenylation signal, and an rbGlob polyadenylation signal. Embodiment 63 comprises the AAV vector of embodiment 62, wherein the polyadenylation signal comprises a bovine growth factor polyadenylation signal. Embodiment 64 comprises the AAV vector of any one of embodiments 61 - 63, wherein the polyadenylation signal comprises a sequence having least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 19. Embodiment 65 comprises the AAV vector of any one of embodiments 61-64, wherein the polyadenylation signal comprises SEQ ID NO: 19. Embodiment 66 comprises the AAV vector of any one of embodiments, 61 -65, comprising one or morelTRs. Embodiment 67 comprises the AAV vector of embodiment 66, wherein the one or more ITRs comprises a first ITR sequence and a second ITR sequence. Embodiment 68 comprises the AAV vector of embodiment 67, wherein the first ITR sequence comprises a nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NOs: 22-23. Embodiment 69 comprises the AAV vector of embodiment 67 or 68, wherein the first ITR sequence comprises the nucleic acid sequence of SEQ ID NOs: 22 - 23. Embodiment 70 comprises the AAV vector of any one of embodiments 67-69, wherein the second ITR sequence comprises a nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NOs: 24-25. Embodiment 71 comprises the AAV vector of any one of embodiments 67-70, wherein the second ITR sequence comprises the nucleic acid sequence of SEQ ID NOs: 24-25. Embodiment 72 comprises the rAAV vector of any one of embodiments 61 -71, comprising a WPRE element. Embodiment 73 comprises the rAAV vector of embodiment 72, wherein the WPRE element comprises a nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 15. Embodiment 74 comprises the rAAV vector of embodiment 73, wherein the WPRE element comprises the nucleotide sequence of SEQ ID NO: 15. Embodiment 75 comprises the rAAV vector of any one of embodiments 72-74, wherein the WPRE element is positioned 3' of the heterologous nucleic acid. Embodiment 76 comprises the rAAV vector of any one of embodiments 61 -75, wherein the polynucleotide comprises a promoter. Embodiment 77 comprises the rAAV vector of embodiment 76, wherein the promoter is selected from a rhodopsin kinase promoter, a rhodopsin promoter, an IRBP promoter, a RS / IRPB promoter; a red / green cone opsin promoter, a Cone Arrestin promoter, a chimeric IRBP enhancer-cone transducin promoter, a chicken beta actin promoter, and a smCBA promoter. Embodiment 78 comprises the AAV vector of embodiment 76, wherein the promoter is a rhodopsin kinase promoter. Embodiment 79 comprises the rAAV vector of embodiment 78, wherein the promoter is a human rhodopsin kinaseWSGR Docket No. 58774-736.601(hGRKl) promoter. Embodiment 80 comprises the AAV vector of any one of embodiments 76-79, wherein the promoter comprises a nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 7. Embodiment 81 comprises the AAV vector of any one of embodiments 76-80, wherein the promoter comprises the nucleic acid sequence of SEQ ID NO: 7. Embodiment 82 comprises an AAV vector comprising i) a polynucleotide comprising a heterologous nucleic acid encoding a retinoschisin protein andii) one or more ITRs. Embodiment 83 comprises the AAV vector of embodiment 82, wherein the one or more ITRs comprises a first ITR sequence and a second ITR sequence. Embodiment 84 comprises the AAV vector of embodiment 83, wherein the first ITR sequence comprises a nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NOs: 22-23. Embodiment 85 comprises the AAV vector of embodiment 83 or 84, wherein the first ITR sequence comprisesthe nucleic acid sequence of SEQ ID NOs: 22-23. Embodiment 86 comprises the AAV vector of any one of embodiments 83 -85, wherein the second ITR sequence comprises a nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NOs: 24-25. Embodiment 87 comprises the AAV vector of any one of embodiments 83-86, wherein the second ITR sequence comprises the nucleic acid sequence of SEQ ID NOs: 24-25. Embodiment 88 comprises the AAV vector of any one of embodiments 82-87, comprising a polyadenylation signal. Embodiment 89 comprises the AAV vector of embodiment 88, wherein the polyadenylation signal is selected from a bovine growth factor hormone polyadenylation signal, an SV40 polyadenylation signal, a human growth factor hormone polyadenylation signal, and an rbGlob polyadenylation signal. Embodiment 90 comprises the AAV vector of embodiment 89, wherein the poly adenylation signal comprises a bovine growth factor polyadenylation signal. Embodiment 91 comprises the AAV vector of any one of embodiments 88-90, wherein the polyadenylation signal comprises a sequence having least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 19. Embodiment 92 comprises the AAV vector of any one of embodiments 88-91, wherein the polyadenylation signal comprises SEQ ID NO: 19. Embodiment 93 comprises the rAAV vector of any one of embodiments 82-92, comprising a WPRE element. Embodiment 94 comprises the rAAV vector of embodiment 93, wherein the WPRE element comprises a nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 15. Embodiment 95 comprises the rAAV vector of embodiment 94, wherein the WPRE element comprises the nucleotide sequence of SEQ ID NO: 15. Embodiment 96WSGR Docket No. 58774-736.601 comprises the rAAV vector of any one of embodiments 93 -95, wherein the WPRE element is positioned 3' of the heterologous nucleic acid. Embodiment 97 comprises the rAAV vector of any one of embodiments 82-96, wherein the polynucleotide comprises a promoter. Embodiment 98 comprises the rAAV vector of embodiment 97, wherein the promoter is selected from a rhodopsin kinase promoter, a rhodopsin promoter, an IRBP promoter, a RS / IRPB promoter; a red / green cone opsin promoter, a Cone Arrestin promoter, a chimeric IRBP enhancer-cone transducin promoter, a chicken beta actin promoter, and a smCBA promoter. Embodiment 99 comprises the AAV vector of embodiment 97, wherein the promoter is a rhodopsin kinase promoter. Embodiment 100 comprises the rAAV vector of embodiment 99, wherein the promoter is a human rhodopsin kinase (hGRKl) promoter. Embodiment 101 comprises the AAV vector of any one of embodiments 97- 100, wherein the promoter comprises a nucleic acid sequence having at least 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 7. Embodiment 102 comprises the AAV vector of any one of embodiments 97-101, wherein the promoter comprises the nucleic acid sequence of SEQ ID NO: 7. Embodiment 103 comprises an AAV vector comprising i) a polynucleotide comprising a heterologous nucleic acid encoding a retinoschisin protein and ii) a WPRE element. Embodiment 104 comprises the rAAV vector of embodiment 103, wherein the WPRE element comprises a nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 15. Embodiment 105 comprises the rAAV vector of embodiment 104, wherein the WPRE element comprises the nucleotide sequence of SEQ ID NO: 15. Embodiment 106 comprises the rAAV vector of any one of embodiments 103 - 105, wherein the WPRE element is positioned 3' of the heterologous nucleic acid. Embodiment 107 comprises the AAV vector of any one of embodiments, 103 -106, comprising one or more ITRs. Embodiment 108 comprises the AAV vector of embodiment 107, wherein the one or more ITRs comprises a first ITR sequence and a second ITR sequence. Embodiment 109 comprises the AAV vector of embodiment 108, wherein the first ITR sequence comprises a nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NOs: 22-23. Embodiment 110 comprises the AAV vector of embodiment 108 or 109, wherein the first ITR sequence comprises the nucleic acid sequence of SEQ ID NOs: 22-23. Embodiment 111 comprises the AAV vector of any one of embodiments 108-110, wherein the second ITR sequence comprises a nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NOs: 24-25. Embodiment 112 comprises the AAV vector of any one of embodiments 108-111, wherein the second ITR sequence comprises theWSGR Docket No. 58774-736.601 nucleic acid sequence of SEQ ID NOs: 24-25. Embodiment 113 comprises the AAV vector of any one of embodiments 103-112, comprising a polyadenylation signal. Embodiment 114 comprises the AAV vector of embodiment 113, wherein the polyadenylation signal is selected from a bovine growth factor hormone polyadenylation signal, an SV40 polyadenylation signal, a human growth factor hormone polyadenylation signal, and an rbGlob polyadenylation signal. Embodiment 115 comprises the AAV vector of embodiment 114, wherein the polyadenylation signal comprises a bovine growth factor polyadenylation signal. Embodiment 116 comprises the AAV vector of any one of embodiments 113-115, wherein the polyadenylation signal comprises a sequence having least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 19. Embodiment 117 comprises the AAV vector of any one of embodiments 113-116, wherein the polyadenylation signal comprises SEQ ID NO: 19. Embodiment 118 comprises the rAAV vector of any one of embodiments 103-117, wherein the polynucleotide comprises a promoter. Embodiment 119 comprises the rAAV vector of embodiment 118, wherein the promoter is selected from a rhodopsin kinase promoter, a rhodopsin promoter, an IRBP promoter, a RS / IRPB promoter; a red / green cone opsin promoter, a Cone Arrestin promoter, a chimeric IRBP enhancer-cone transducin promoter, a chicken beta actin promoter, and a smCBA promoter. Embodiment 120 comprises the AAV vector of embodiment 118, wherein the promoter is a rhodopsin kinase promoter. Embodiment 121 comprises the rAAV vector of any one of embodiments 118-120, wherein the promoter is a human rhodopsin kinase (hGRKl) promoter. Embodiment 122 comprises the AAV vector of any one of embodiments 118-121, wherein the promoter comprises a nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 7. Embodiment 123 comprises the AAV vector of any one of embodiments 118-122, wherein the promoter comprises the nucleic acid sequence of SEQ ID NO: 7. Embodiment 124 comprises the AAV vector of any one of embodiments 42-123, wherein the polynucleotide further comprises an SV40 intron. Embodiment 125 comprises the AAV vector of embodiment 124, wherein the SV40 intron comprises a splice donor region and / or a splice acceptor region. Embodiment 126 comprises the AAV vector of embodiment 124 or 125, wherein the SV40 intron comprises a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the nucleic acid sequence of SEQ ID NO: 20. Embodiment 127 comprises the AAV vector of embodiment 126, wherein the SV40 intron comprises the nucleic acid sequence of SEQ ID NO: 20. Embodiment 128 comprises the AAV vector of any one of embodiments 42-127, wherein the retinoschisin protein is a human retinoschisin protein. Embodiment 129 comprises the AAV vector of any one of embodiments 42-WSGR Docket No. 58774-736.601128, wherein the heterologous nucleic acid does not comprise the 5 ' untranslated region of human retinoschisin. Embodiment 130 comprises the AAV vector of embodiment 129, wherein the 5' untranslated region comprises the nucleic acid sequence of SEQ ID NO: 39. Embodiment 131 comprises the AAV vector of any one of embodiments 42-130, wherein the nucleic acid does not comprise the 3 ' untranslated region of human retinoschisin. Embodiment 132 comprises the AAV vector of embodiment 131, wherein the 3 ' untranslated region comprises the nucleic acid sequence of SEQ ID NO: 40. Embodiment 133 comprises the rAAV particle of any one of embodiments 42- 132, wherein the heterologous nucleic acid comprises the nucleic acid sequence set forth as SEQ ID NO: 8, 9 or 10. Embodiment 134 comprises the AAV vector of any one of embodiments 42-133, wherein the retinoschisin protein comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of SEQ ID NO: 12. Embodiment 135 comprises the AAV vector of any one of embodiments 42-134, wherein the retinoschisin protein comprises the amino acid sequence of SEQ ID NO: 12. Embodiment 136 comprises the AAV vector of any one of embodiments 42-135, wherein the polynucleotide comprises a splice donor region and / or a splice acceptor region. Embodiment 137 comprises the AAV vector of any one of embodiments 1 -136, wherein the AAV vector comprises a first ITR and a second ITR and the length of the AAV vector between the first ITR and the second ITR is between about 2000 nucleotides and about 6000 nucleotides. Embodiment 138 comprises the AAV vector of embodiment 137, wherein the length of the AAV vectorbetween the first ITR and the second ITR is between about 3000 nucleotides and about 5000 nucleotides. Embodiment 139 comprises the AAV vector of embodiment 138, wherein the length of the AAV vector between the first ITR and the second ITR is between about 4000 nucleotides and about 5000 nucleotides. Embodiment 140 comprises the AAV vector of embodiment 139, wherein the length of the AAV vectorbetween the first ITR and the second ITR is about 4500 nucleotides. Embodiment 141 comprises the AAV vector of any one of embodiments 1-140, wherein the AAV vector comprises a stuffer sequence. Embodiment 142 comprises an AAV vector comprising a polynucleotide comprising a nucleic acid encoding human retinoschisin protein and a stuffer sequence. Embodiment 143 comprises the AAV vector of embodiment 141 or 142, wherein the stuffer sequence has a length of between about 1000 nucleotides and about 4000 nucleotides. Embodiment 144 comprises the AAV vector of any one of embodiments 141 -143, wherein the stuffer sequence has a length of between 2000 nucleotides and about 3000 nucleotides. Embodiment 145 comprises the AAV vector of any one of embodiments 141 -144, wherein the stuffer sequence has a length of about 2800 nucleotides. Embodiment 146 comprises the AAV vector of any one of embodiments 141 -144, wherein the stuffer sequence has a total length of aboutWSGR Docket No. 58774-736.6012200 nucleotides. Embodiment 147 comprises the AAV vector of any one of embodiments 141 - 146, wherein the stuffer sequence comprises a sequence about or at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to at least 100 contiguous bases of any one of SEQ ID NOS: 26-30. Embodiment 148 comprises the AAV vector of any one of embodiments 141 -147, wherein the stuffer sequence is positioned 3' of the heterologous nucleic acid. Embodiment 149 comprises the AAV vector of any one of embodiments 141-147, wherein the stuffer sequence is positioned 5' of the heterologous nucleic acid. Embodiment 150 comprises the AAV vector of any one of embodiments 141 -149, wherein the stuffer sequence is positioned between ITR sequences. Embodiment 151 comprises a nucleic acid stuffer sequence comprising a sequence about or at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to at least a 100 contiguous bases of any one of SEQ ID NOS: 26-30. Embodiment 152 comprises an AAV vector comprising the nucleic acid stuffer sequence of embodiment 151. Embodiment 153 comprises an AAV particle that comprises the AAV vector of any one of embodiments 1-152 and a capsid. Embodiment 154 comprises the AAV particle of embodiment 153, wherein the capsid is selected from AAV44.9(E531D), AAV44.9(T492V+E53 ID), AAV44.9(Y446F+E53 ID), and AAV44.9(Y446F+T492V+E53 ID), AAVS and variants thereof, AAV7 and variants thereof, AAV8 and variants thereof, AAVS and variants thereof, AAV2(4pMut)AHS, AAV44.9, AAV8(Y447F+Y733F+T494V), AAVrh.8, AAVrh.8R, AAVrh.10, AAVrh.74, AAV2TT, AAV2HBKO, AAVAnc80, DGE-DF, P2-V2, P2-V3, and ME-B(Y-F+T-V). Embodiment 155 comprises the AAV particle of embodiment 153, wherein the AAV capsid is selected from AAV44.9(E53 ID), AAV44.9(T492V+E53 ID), AAV44.9(Y446F+E53 ID), and AAV44.9(Y446F+T492V+E53 ID). Embodiment 156 comprises the AAV particle of embodiment 153, wherein the AAV capsid is AAV44.9(E531D). Embodiment 157 comprises the AAV particle of embodiment 153, wherein the AAV capsid is selected from DGE-DF, P2-V2, P2-V3, and ME- B(Y-F+T-V). Embodiment 158 comprisesthe AAV particle of embodiment 153, wherein the AAV capsid comprises an AAVS capsid protein, or a variant thereof . Embodiment 159 comprises a composition comprising the AAV particle of any one of embodiments 153 -158 and a pharmaceutically acceptable carrier, buffer, diluent, or excipient, or any combination thereof. Embodiment 160 comprises a cell comprising the AAV vector of any one of embodiments 1 -152, the AAV particle of any one of embodiments 153 -158, or the composition of embodiment 159. Embodiment 161 comprises a method for transducing a cell, the method comprising administering to the cell the AAV vector of any one of embodiments 1 -152, the AAV particle of any one of embodiments 153-158, or the composition of embodiment 159. Embodiment 162 comprises theWSGR Docket No. 58774-736.601 method of embodiment 160 or 161, wherein the cell is a photoreceptor cell. Embodiment 163 comprises the method of embodiment 160 or 161, wherein the cell is a retinal pigment epithelial (RPE) cell. Embodiment 164 comprises a method for treating X-linked retinoschisis (XLRS) in a mammal, the method comprising administering to the mammal the AAV vector of any one of embodiments 1 -152, the AAV particle of any one of embodiments 153 -158, the composition of embodiment 159, or the cell of embodiment 160. Embodiment 165 comprises the method of embodiment 164, wherein the administration is to one or both eyes of the mammal. Embodiment 166 comprises the method of embodiment 164 or 165, wherein the step of administering comprises intravitreal administration or subretinal administration to one or both eyes of the mammal. Embodiment 167 comprises the method of any one of embodiments 164-166, wherein a volume of between 30 pL and 150 pL of the vector, particle orthe composition is administered. Embodiment 168 comprises the method of embodiment 167, wherein a volume of about 90 pL of the vector, particle orthe composition is administered. Embodiment 169 comprises the method of any one of embodiments 164-168, wherein the particle is administered in an amount of between 5 * 1010and 1 *1012vector genomes (vgs) / mL. Embodiment 170 comprises the method of any one of embodiments 164-169, wherein about 1 .4E10 to 3.2E10vg of the particle is administered to one of both eyes of the human. Embodiment 171 comprises the method of any one of embodiments 164- 170, wherein about 1.4E10 to 1.6E10vg of the particle is administered to one of both eyes of the human. Embodiment 172 comprises the method of any one of embodiments 164-171, wherein about 1.5 El 0 vg of the particle is administered to one of both eyes of the human. Embodiment 173 comprises the method of any one of embodiments 164-172, wherein about 2.8E10 to 3.2E10 vg of the particle is administered to one of both eyes of the human. Embodiment 174 comprises the method of any one of embodiments 164-173, wherein about 3.0E10 vg of the particle is administered to one of both eyes of the human. Embodiment 175 comprises the method of any one of embodiments 164-174, wherein step of administering a) preserves one or more photoreceptor cells, b) restores laminar retinal structure, c) restores one or more rod - and / or cone-mediated functions, d) restores completely or partially visual behavior in one or both eyes, or e) any combination thereof. Embodiment 176 comprises the method of any one of embodiments 164-175, wherein the mammal is human. Embodiment 177 comprises the method of any one of embodiments 164-176 further comprising administering a corticosteroid to the subject. Embodiment 178 comprises the method of embodiment 177, wherein the corticosteroid comprises prednisone, triamcinolone acetonide, methylprednisolone, prenidsolone acetate, or a combination thereof.Embodiment 179 comprises the method of embodiment 177, wherein the administering comprises administering prednisone at a dose of Img / kg / day for seven weeks. Embodiment 180 comprisesWSGR Docket No. 58774-736.601 the method of embodiment 177, wherein the administering comprises administering 20 mg of triamcinolone acetonide by periocular injection. Embodiment 181 comprises the method of embodiment 177, wherein the administering comprises administering 250 mg of methylprednisolone intravenously. Embodiment 182 comprises the method of embodiment 177, wherein the administering comprises administering 1% prenidsolone acetate topically for 28 days. Embodiment 183 comprises the method of treating X-linked retinoschisis (XLRS) in a subject in need thereof, the method comprising administering to an eye of the subject about 1.5 x 1010vg to about 5.0 x 1010vg of an AAV particle. Embodiment 184 comprises the method of embodiment183, wherein the about 1.5 x 1010vgto about 5.0 x 1010vg of the AAV particle is administered to each eye of the subject. Embodiment 185 comprisesthe method of embodiment 183 or embodiment184, wherein about 3.0 x 1010vg of the AAV particle is administered to the eye or to each eye of the subject. Embodiment 186 comprisesthe method of any one of embodiments 183 -185, wherein the AAV particle comprises a polynucleotide comprising a heterologous nucleic acid encoding a retinoschisin (RS) protein. Embodiment 187 comprises the method of embodiment 186, wherein the RS protein is a human RS protein. Embodiment 188 comprises the method of embodiment 186 or embodiment 187, wherein the RS protein has the amino acid sequence of SEQ ID NO: 12. Embodiment 189 comprises the method of any one of embodiments 186-188, wherein the RS protein is encoded by a nucleic acid sequence at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the nucleic acid sequence of SEQ ID NO: 8. Embodiment 190 comprises the method of any one of embodiments 186-189, wherein the polynucleotide comprises a human rhodopsin kinase promoter. Embodiment 191 comprises the method of embodiment 190, wherein the human rhodopsin kinase promoter has a sequence at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 7. Embodiment 192 comprises the method of any one of embodiments 186-191, wherein the polynucleotide comprises a sequence at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 33 (pTR-X002-3pSR) . Embodiment 193 comprises the method of any one of embodiments 183 -192, wherein the AAV particle comprises a capsid, such as an AAV44.9(E53 ID) capsid. Embodiment 194 comprises the method of embodiment 193, wherein the capsid comprises a VP1 amino acid sequence atleast about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to: SEQ ID NO: 1. Embodiment 195 comprises the method of embodiment 193 or embodiment 194, wherein the capsid comprises a VP2 amino acid sequence at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,WSGR Docket No. 58774-736.60192%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to: SEQ ID NO: 2. Embodiment 196 comprises the method of any one of embodiments 193 -195, wherein the capsid comprises a VP3 amino acid sequence atleast about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to: SEQ ID NO: 3. Embodiment 197 comprises the method of any one of embodiments 183-196, wherein the subject has a mutation in the RSI gene. Embodiment 198 comprises the method of embodiment 197, wherein the subject has an increased risk of retinal detachment as compared to a subject that does not have the mutation in the RSI gene. Embodiment 199 comprises the method of any one of embodiments 183 -198, wherein the subject has a reduced risk of retinal detachment after the administering. Embodiment 200 comprises the method of any one of embodiments 183 -199, wherein the subject has loss of vision prior to the administering. Embodiment 201 comprises the method of embodiment 200, wherein the subject regains vision after the administering. Embodiment 202 comprises the method of any one of any one of embodiments 183 -201, wherein the subject expresses native RSI (e.g., SEQ ID NO: 12) after the administering. Embodiment203 comprises the method of 202, wherein the subject expresses native RSI in photoreceptors after the administering. Embodiment 204 comprises the method of embodiment 202 or embodiment 203, wherein the subject expresses native RSI in bipolar cells after the administering. Embodiment 205 comprises the method of any one of embodiments 183-204, wherein the subject is 18 years old or older. Embodiment206 comprises the method of any one of embodiments 183-205, wherein the subject has XLRS caused by a pathogenic mutation in RSI . Embodiment 207 comprises the method of any one of embodiments 183 -206, wherein prior to the administering the subject has a BVCA of 34 to 73 ETDRS letters (20 / 200 to 20 / 40). Embodiment 208 comprises the method of any one of embodiments 183 -207, wherein prior to the administering the subject has the presence of foveal schisis in the eye. Embodiment 209 comprises the method of any one of embodiments 183 -208, wherein prior to the administering the subject has parafoveal / perifoveal schisis in the eye. Embodiment 210 comprises the method of any one of embodiments 183-209, wherein before and / or after the administering, the method comprises performing optical coherence tomography on the subject. Embodiment 211 comprises the method of any one of embodiments 183 -210, wherein before and / or after the administering, the method comprises performing microperimetry on the subject. Embodiment 212 comprises the method of any one of embodiments 183-211, wherein the AAV particle is administered via a subretinal injection. Embodiment 213 comprises the method of any one of embodiments 183-212, wherein the AAV particle is administered in a volume of about 150 microliters. Embodiment 214 comprises the method of any one of embodiments 183-213, wherein the AAV particle is administered in 2 blebs. Embodiment 215 comprises the method of any one of embodiments 183 -214, wherein the AAVWSGR Docket No. 58774-736.601 particle is administered in a manner that avoids foveal detachment. Embodiment 216 comprises the method of any one of embodiments 183 -215, comprising administering a corticosteroid to the subject. Embodiment 217 comprises the method of embodiment 216, wherein the corticosteroid comprises prednisone. Embodiment 218 comprises the method of embodiment 216 or embodiment 217, wherein the corticosteroid is administered starting about 7 weeks prior to the administering of the AAV particle. Embodiment 219 comprises the method of any one of embodiments 216-218 wherein the corticosteroid is administered at about 1 mg / kg of the subject per day. Embodiment 220 comprises the method of any one of embodiments 183 -219, comprising administering to the subject triamcinolone acetonide. Embodiment 221 comprises the method of embodiment 220, wherein the triamcinolone acetonide is administered starting about 7 weeks prior to the administering of the AAV particle. Embodiment 222 comprises the method of embodiment 220 or embodiment 221, wherein about 20 mg of the triamcinolone acetonide is administered.Embodiment 223 comprises the method of any one of embodiments 220-223, wherein the triamcinolone acetonide is administered in a periocular injection. Embodiment 224 comprises the method of any one of embodiments 183 -223, comprising administering to the subject methylprednisolone. Embodiment 225 comprises the method of embodiment 224, wherein the methylprednisolone is administered starting about 7 weeks prior to the administering of the AAV particle. Embodiment 226 comprises the method of embodiment 224 or embodiment 225, wherein about 250 mg of the methylprednisolone is administered. Embodiment 227 comprises the method of any one of embodiments 224-226, wherein the methylprednisolone is administered via IV. Embodiment 228 comprises the method of any one of embodiments 183 -227, comprising administering to the subject prenidsolone acetate. Embodiment 229 comprises the method of embodiment 228, wherein the prenidsolone acetate is administered starting about 28 days prior to the administering of the AAV particle. Embodiment 230 comprises the method of embodiment 228 or embodiment 229, wherein about 1% of the prenidsolone acetate is administered. Embodiment 231 comprises the method of any one of embodiments 228-230, wherein the prenidsolone acetate is administered topically. Embodiment 232 comprises the method of any one of embodiments 183 - 231, wherein after the administering the subject has closure of a foveal schisis. Embodiment 233 comprises the method of any one of embodiments 183 -232, wherein after the administering the subject has a reduction in central retinal thickness. Embodiment 234 comprises the method of any one of embodiments 183-233, wherein after the administering the subject has improvement in retinal sensitivity. Embodiment 235 comprises the method of any one of embodiments 183 -234, wherein about 1 .4E10 to 3.2E10 vg of the particle is administered to one of both eyes of the human. Embodiment 236 comprises the method of any one of embodiments 183-235, wherein about 1 .4E10WSGR Docket No. 58774-736.601 to 1.6E10 vg of the particle is administered to one of both eyes of the human. Embodiment 237 comprises the method of any one of embodiments 183 -236, wherein about 1.5 E10 vg of the particle is administered to one of both eyes of the human. Embodiment 238 comprises the method of any one of embodiments 183-237, wherein about 2.8E10 to 3.2E10 vg of the particle is administered to one of both eyesof the human. Embodiment239 comprises the method of any one of embodiments 183-238, wherein about 3.0E10 vg of the particle is administered to one of both eyes of the human. Embodiment 240 comprisesthe method of any one of embodiments 183 -239, wherein the administering a) preserves one or more photoreceptor cells, b) restores laminar retinal structure, c) restores one or more rod- and / or cone-mediated functions, d) restores completely or partially visual behavior in one or both eyes, or e) any combination thereof. Embodiment 241 comprises the method of any one of embodiments 183 -240, wherein the administering restores laminar retinal structure. Embodiment 242 comprises the method of any one of embodiments 183 - 241, wherein the administering comprises subretinal administration to a fovea of one or both eyes of the mammal. Embodiment 243 comprises the method of embodiment 242, wherein detachment of the fovea is minimized. Embodiment 244 comprises the method of any one of embodiments 183- 243, further comprising administering a corticosteroid to the subject. Embodiment 245 comprises the method of embodiment 244, wherein the corticosteroid comprises prednisone, triamcinolone acetonide, methylprednisolone, prenidsolone acetate, or a combination thereof. Embodiment 246 comprises the method of embodiment 244, wherein the administering comprises administering prednisone at a dose of Img / kg / day for seven weeks. Embodiment 247 comprises the method of embodiment 244, wherein the administering comprises administering 20 mg of triamcinalone acetonide by periocular injection. Embodiment 248 comprises the method of embodiment 244, wherein the administering comprises administering 250 mg of methylprednisolone intravenously. Embodiment 249 comprises the method of embodiment 244, wherein the administering comprises administering 1% prenidsolone acetate topically for 28 days. Embodiment 250 comprises the method of any one of embodiments 173-250, wherein the capsid enhances potency of the AAV particle as compared to an AAV particle having a different AAV capsid. Embodiment 251 comprises the method of any one of embodiments 183 -250, wherein the subject is sensitive to inflammation. Embodiment 252 comprises the method of any one of embodiments 183 -251, wherein the dose of the AAV particle reduces inflammation in the subject as compared to a higher dose of the AAV particle.WSGR Docket No. 58774-736.601EXAMPLES

[0230] The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples that follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus may be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes may be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.Example 1 — Enhanced Lateral Spread and Foveal Transduction Following Subretinal Administration of an AAV Vector Encoding a Reporter Gene in Macaque

[0231] An evaluation of the performance of vectors and particles incorporating the AAV44.9(E531D) capsid variant in subretinally injected mice and macaques relative to benchmark vectors, the closely related AAVrh.8, and unmodified AAV44.9, was pursued. It was determined that the human rhodopsin kinase (hGRKl) promoter has exclusive activity in non -human primate rods and cones. As such, the hGRKl promoter was evaluated for its ability to drive green fluorescent protein (GFP) reporter expression in macaque eyes in the improved AAV44.9(E53 ID) vector. The degree of lateral spread from the initial bleb boundaries was also evaluated.

[0232] The parafoveal region is the zone of the eye that circumscribes the fovea, approximately 4 degrees eccentricity from the central fixation point. The parafovea has the highest density of rods, while still also containing a large number of cones. It is a transitional zone between cone- and roddominant retina and is important in the context of diseases where degeneration proceeds from the outer to inner retina, such as retinitis pigmentosa (RP). The perifoveal region is the zone that circumscribes the parafovea, and represents the outermost band of the macula. Like the parafovea, the periovea has an important role in progression of diseases like RP, where retinal degeration starts in the periphery and progresses to the central retina. The perifovea is the first zone of the macula to undergo degeneration in RP.

[0233] Two rAAV vectors— AAV44.9-hGRKl -GFP and AAV44.9(E53 lD)-hGRKl-GFP— were subretinally administered to macaque eyes. Vectors were delivered at concentrations of 1 *1012vg / mL. A control vector, AAV5 -hGRKl -GFP, was also administered to the eyes.

[0234] Particles incorporating both modified and unmodified AAV44.9 vectors exhibited enhanced lateral spread and potency in subretinally injected macaque subjects (see FIG. 1). Initial boundaries of the bleb and boundaries of resulting GFP expression are outlined in white dotted line in FIG. 1. Identical vasculature is highlighted in thickened dark line for reference. GFP expressionWSGR Docket No. 58774-736.601 mediated by AAV44.9(E53 ID) was visible at 1 week post injection. Both AAV44.9 and AAV44.9(E53 ID) were well tolerated in the primate retina at the 1 *1012vg / mL dose. The control vector, AAV5, mediated GFP restriction that remained sequestered within the original injection bleb.

[0235] An extrafoveal subretinal injection of AAV44.9-hGRKl-GFP (concentration of 1 *1012vg / mL) was performed in macaque subjects. Optical coherence tomography (OCT) scans revealed thatthe fovea was not detached duringthe injection (FIG. 2). This extrafoveal subretinal injection transduced 98% of foveal cones even and 100% of central rods, even though the fovea did not detach (see top right of FIG. 3). The capsid exhibited enhanced lateral spread, as bleb boundaries were expanded relative to the initial boundaries.

[0236] Images were also captured from macaque eyes injected with AAV44.9(E531D)- hGRKl-GFP. A qualitative analysis in a single eye revealed ~50% of foveal cone transduction mediated by AAV44.9(E53 ID).

[0237] Three subretinal injections totaling a volume of 90 pL (30 pL each) of AAV44.9(E53 lD)-hGRKl-GFP were performed in the superior, temporal, and inferior retina outside the fovea of macaque eyes. Retinal sections were stained with an antibody directed against cone arrestin and three blinded observers counted the number of GFP positive cones and rods in 5 retinal regions across a single plane traversing the foveal pit. Results of this administration are shown in FIG. 4. OCT scans revealed that the fovea was not detached during the injection (see right panel of FIG. 4).

[0238] These results indicate that extrafoveal subretinal injection in macaque of AAV44.9(E53 lD)-hGRKl -GFP exhibited remarkable transduction of central cone and rod cells in the absence of foveal detachment. Peripheral rods and cones were also transduced very efficiently. Accordingly, extrafoveal subretinal injection resulted in highly efficient transduction across the foveal region.

[0239] As shown in FIGS. 5A-5D, an examination of the parafovea following this injection revealed thatthe AAV44.9(E53 ID) particles transduced parafoveal cones located both nasal and temporal to the foveal pit. Notably, however, parafoveal cone transduction was not achieved with unmodified AAV44.9. This finding is of interest at least because i) parafoveal cones are refractory to transduction by a variety of AAV capsid variants, and ii) the earliest loss of structure and function due to aging and inherited retinal disease often occurs in the parafoveal region. Despite the dissimilarity in their ability to transduce cones in this region, the modified and unmodified AAV44.9 vectors efficiently transduced parafoveal rods to a substantially equal degree.WSGR Docket No. 58774-736.601

[0240] As shown in FIGS. 6A and 6B, an examination of the perifovea revealed a similar pattern: particles that incorporated the AAV44.9(E531D) capsid transduced perifoveal cones, but unmodified AAV44.9 did not. Perifoveal rods were efficiently transduced by both capsids in this region. The perifovea circumscribes the parafovea.

[0241] These results demonstrated that the enhanced lateral spread of transduction provided by the improved AAV44.9(E53 ID) capsid variant vectors may allow subretinal injection in the parafoveal region to produce transduction of the foveal cells while circumventing the deleterious effects of inducing a foveal detachment in human subjects. They further demonstrate that enhanced lateral spread and transduction maybe achieved with a total injection volume of AAV particles of as low as 90 pL (3 injection blebs of 30 pL each).

[0242] Materials and Methods

[0243] Vector Production

[0244] A self-complementary AAV construct containing the truncated chimeric CMV-Chicken Beta Actin (smCBA) promoter driving mCherry (sc-smCBA-mCherry) (SEQ ID NO. 57) was packaged into AAV44.9, AAV44.9(Y731F), AAV44.9(E53 ID), AAV5 and AAV8(Y733F) using a triple transfection-plasmid based system in adherent HEK293T seeded in double -stack cell factories (1,272 cm2 cell growth area). Cells were harvested and lysed by successive freeze thaw cycles. Virus within the lysate was purified by iodixanol density gradient and was buffer exchanged into Alcon BSS supplemented with Tween 20 (0.014%). Virus was quantified by qPCR relative to a standard and stored at -80 C. Addition of Y73 IF and E53 ID substitutions to the AAV44.9 capsid were accomplished by site-directed mutagenesis of the AAV2rep-44.9cap plasmid and confirmed by Sanger sequencing. An additional construct containing the cone-specific, IRBPe-GNAT2 chimeric promoter driving green fluorescent protein (GFP) (SEQ ID NO: 58) was packaged in the AAV44.9 and AAV44.9 variant vectors.

[0245] In-Vitro Transduction Assay

[0246] ARPE-19 (human retinal pigment epithelial cell line) and 661W (mouse cone cell line) cells were seeded in 96 well plates at a concentration of 1.0x104 cells / well. The following day, cells were infected at 10,000 p / cell. Three days post -infection, fluorescent microscopy at a fixed exposure was performed, cells were detached and flow-cytometry was used to quantify reporter protein expression (mCherry) via fluorescence. mCherry expression was calculated by multiplying the mean mCherry fluorescence times the number of positive cells. Graphs represent expression levels minus the level of cells only.

[0247] Injection- I l l -WSGR Docket No. 58774-736.601

[0248] 2 *109vg in 1 pL of vector containing solution was delivered either intravitreally or subretinally to macaque retinas. A minimum of 6 eyes receiving successful injections were analyzed in each experiment.

[0249] Fundoscopy

[0250] At 4 weeks post-injection, fundoscopy was performed using a Micron III camera (Phoenix Research Laboratories, Pleasanton, Calif.). Bright field and red fluorescent images were taken to visualize retinal health and mCherry expression, respectively. Exposure settings were constant between experiments and are indicated in the figure legends.

[0251] Measurement of Retinal Transduction Via Flow-Cytometry

[0252] Neural retinas (with RPE manually stripped from retina) from between 4 to 6 Nrl-GFP eyes per cohort were harvested and dissociated with papain. Flow -cytometry was performed on treated, dissociated retinas and untreated controls to quantify the percentage of cells that were positive for GFP (rod photoreceptors), mCherry (non-rod retinal neurons transduced by rAAV), or both (rod photoreceptors transduced by rAAV). The percentage of rods and non-rod neural retinal cells transduced by each vector were separately averaged.

[0253] Tissue Preparation and Immunostaining

[0254] Four weeks post-injection, the eyes were enucleated, fixed overnight at 4° C. in freshly prepared 4% paraformaldehyde (PF A) in phosphate-buffered saline (PBS). Cornea and lens were removed, and the eye cup was incubated in 30% sucrose solution overnight at 4° C. Eyes were embedded in cryostat compound and frozen at -80° C. Sections (12 pm thick) were cut using a cryostat (Leica Microsystem, Buffalo Grove, Ill.) and transferred to glass slides. Retinal cryosections were rinsed with 1 x phosphate-buffered saline (PBS), blocked with 0.5% Triton-XlOO and 1% bovine serum albumin (BSA) for 1 hour each and then incubated overnight at 4° C. with mouse monoclonal anti cone arrestin antibody (1 :1000, generously provided by Dr Clay Smith). The following day slides were rinsed with 1 PBS and then incubated at room temperature for 1 hour with Alexa Fluor donkey-anti-mouse secondary antibody (1 : 500) in 1 PBS and counterstained with DAPI. Images were acquired using confocal laser scanning microscope (Leica TCS SP8) and Fluorescence microscope (EVOS).Example 2 — Enhanced Lateral Spread and Foveal Transduction Following Subretinal Administration of an AAV Vector Encoding an RSI Gene in Macaque

[0255] An evaluation of the performance of vectors and particles incorporating the AAV44.9(E53 ID) capsid variant in subretinally injected relative to control vectors was pursued. It was determined that the human rhodopsin kinase (hGRKl) promoter has exclusive activity in nonhuman primate rods and cones. As such, the hGRKl promoter was evaluated for its ability to driveWSGR Docket No. 58774-736.601 retinoschisinl (RSI) expression in macaque eyes in the improved AAV44.9(E53 ID) vector. The degree of lateral spread from the initial bleb boundaries was also evaluated.

[0256] Two rAAV vectors — AAV44.9(E53 lD)-hGRKl-mycRSl (mycRSl sequence comprises SEQ ID NO. 10) and AAV5-hGRKl-RSl — were subretinally administered to macaque eyes. Vectors were co-delivered after being blended at 3.3 xlO11vg / mL / vector for a total concentration of 6.6xlOnvg / mL in either a single 100 pL extrafoveal injection or two 50 pL extrafoveal injections.

[0257] Particles incorporating AAV44.9(E531D)-hGRKl -RSI vectors exhibited enhanced lateral spread and potency in subretinally injected macaque subjects (see FIGs. 41A and 41B). Initial boundaries of the bleb and boundaries of resulting GFP expression are outlined in dotted line in FIG. 41 A and 4 IB. Identical vasculature is highlighted in thickened dark line for reference. GFP expression mediated by AAV44.9(E531D) was visible at 1 week post injection. The control vector, AAV5, mediated GFP restriction that remained sequestered within the original injection bleb.

[0258] As shown in FIGs. 42A-42D, an immunohistological examination of the perifovea revealed that particles that from the AAV44.9(E53 ID) capsid had spread from the injection site leading to transduction of RSI and GFP within foveal cones.

[0259] These results are summarized in Table 4.Table 4 - Systemic Biodistribution of AAV44.9(E531D) and AAV5 vectors in NHP (7 weeks- post-subretinal injection).WSGR Docket No. 58774-736.601gDNA); ID = Identification number; L = Left; LGN = Lateral geniculate nucleus; LN = Lymph node; OR = Right eye; OS = Left eye; R = Right; Vg = Vector genomes

[0260] These results demonstrated that the enhanced lateral spread of transduction provided by the improved AAV44.9(E53 ID) capsid variant vectors may allow subretinal injection in the parafoveal region to produce transduction of the foveal cells while circumventing the deleterious effects of inducing a foveal detachment in human subjects. They further demonstrated that enhanced lateral spread and transduction may be achieved with a total injection volume of AAV particles of as low as 100 pL.Example 3 — Natural History Study of Retinoschisis Mice

[0261] A 5 -month natural history study of RSI knockout (KO) mice was conducted to confirm that mice display the characteristic phenotypes of XLRS. Abnormal retinal function of the mice was observed by ERG, and schisis formations in retinal structure were measured by OCT. Measurements were taken at the end of each month for five months. See FIGS. 9A-9E and 10-13,. Scores were averaged to generate an overall cavity score across individual retinas. Mice were grouped into the following cohorts:

[0262] Male Rsl hemizygous (Rsl- / y) KO (diseased)

[0263] Female Rsl homozygous KO (diseased)

[0264] Male Rsl+ / y wild type (normal)

[0265] Female Rsl+ / - heterozygous (normal)

[0266] A single cohort of mice were sacrificed at 1 month of age, and their retinas were evaluated for expression of retinoschisin (RSI) by immunohistochemistry. Results are shown in FIG. 14.

[0267] To interpret the OCT results, a grading system for severity of schisis cavities (0-4), with 0 equaling no schisis cavities and 4 being the highest cavity count. OCT measurements and schisis cavity scoring charts are shown in FIG. 13 for each of 5 months of the study. The grading system is defined as follows:WSGR Docket No. 58774-736.601

[0268] 0 : No schisis cavities; 1 : Isolated schisis cavities; 2: Multiple small schisis cavities; 3:Multiple medium schisis cavities with some communication; 4: Large and numerous schisis cavities with some communication

[0269] The natural history study showed that the cavity score of RSI KO mice peaked at 2 to 3 months, and then improved with time. H4C measurements showed that RSI expression localized to the inner / outer segment junction of photoreceptors in the female heterozygous and wildtype male mice and is absent in male and female Rsl KO mice.Example 4 — Evaluation of Therapeutic Efficacy of AAV Capsids in Subretinal Delivery of Synthetic hRSl to Retinoschisis Mice

[0270] The biodistribution of various AAV capsids, including AAV44.9(E53 ID), in retina andRPE relative to AAVS capsids across multiple doses of rAAV particles were evaluated over a five- month study. rAAV particles containing one of the evaluated capsids were administered to Rsl knockout mice.

[0271] Particles were administered to four mice in each of four cohorts in a dose of 1 pl of 5 *1012vector genomes (vgs) / mL. Accordingly, a total of 5*109vector genomes were delivered to each animal. Particles were administered by subretinal injectioninto the OD eye, and contralateral eyes were not treated.

[0272] It was evaluated whether subretinal injections of rAAV vectors expressing a synthetic human a synthetic human RSI transgene could provide therapeutic efficacy to Rsl knockout (Rsl - KO) mice. This synthetic transgene was truncated, lacking 5' and 3' untranslated regions (UTRs), such that it contained only the coding region of RSI. The nucleotide sequence of this transgene is provided above as SEQ ID NO: 8. In some experiments, the transgene was myc-tagged (SEQ ID NO: 10).

[0273] In other experiments, the synthetic transgene also had four CpG islands have been removed from the coding sequence and was codon -optimized for human expression (SEQ ID NO: 10).

[0274] Several different capsids were evaluated for inducing strongest biodistribution in PRs. rAAV44.9(E53 lD)-hGRKl-hRSl and rAAV5-hGRKl-hRSl vectors were administered to Rsl KO mice restored retinal function and structure. Both of these capsids have the hGRKl promoter operably controlling the hRSl transgene. Results were compared to those in Rsl KO mice treated with benchmark vector rAAV5-CBA-hRSl, and those of contralateral (OS) untreated eyes (in all groups).

[0275] Viral particles comprising the AAV44.9(E53 ID) capsid carrying hGRKl -hRSl successfully transduced photoreceptor cells and provided therapeutic levels of human retinoschisinWSGR Docket No. 58774-736.601 in these cells. Surprisingly, retinoschisis cavities were completely resolved in all treated eyes as early as a single month after rAAV injection. Likewise, retinal function was substantially improved in all treated eyes as early as a single month after rAAV injection. Table 5 (below) shows 6-month ERG and OCT data from mice in Groups 1 -4, and 4-month ERG and OCT data in mice from Groups 6 and 7. “vg”=vector genomes; “ — N*” signifies the number (N) of animals dead or sacrificed for IHC.

[0276] The human rhodopsin kinase promoter (hGRKl) was confirmed as the strongest evaluated promoter for mediating expression of therapeutic levels of human RSI in PRs of treated eyes.

[0277] In addition, a 1-month proof-of-concept (POC) study evaluating the impact of subretinally delivered AAV5 or AAV44.9(E53 ID), containing hGRKl or the ubiquitious CBA promoter driving the hRSlsyn gene, on retinal structure and function was performed in RSI -KO deficient mice (hemizygous Rsl- / y males). A single dose (2.3 ><1012vg / mL; 2.3><109vg / eye) of rAAV44.9(E53 lD)-hGRKl -hRSlsyn (SEQ ID NO: 16), rAAV5 -hGRKl -hRSlsyn (SEQ ID NO: 16), or AAV5-CBA-hRSlsyn (SEQ ID NO. 59) was administered a postnatal day 25 (P25). The P25 time point was chosen because it corresponds to an immature adult (early teens), a time point where it is expected that XLRS patients will pursue treatment. The contralateral eye remained un- injected. At 1-month post-injection, retinal structure and function were assessed via optical coherence tomography (OCT) and electroretinogram (ERG), respectively. Mice were euthanized immediately thereafter and both eyes from mice in all groups were collected, fixed, cry o -protected and evaluated with immunohistochemistry. FIG. 16 shows restoration of retinal structure in rAAV- treated RSI -KO mice at 1 month post-injection by OCT analysis (complete resolution of schisis cavities in RSI -KO mice treated with all three vectors). Further, rod- and cone-mediated retinal function was improved in RSI -KO mouse eyes treated with all vectors relative to un-injected controls. Immunohistochemical analysis revealed the presence of RSI expression in photoreceptor inner segments in cross sections of treated retinas. RSI expression was absent from contralateral untreated eyes. These results suggested that both capsids rAAV44.9(E53 lD)-hGRKl -hRSlsyn, rAAV5-hGRKl -hRSlsyn, were efficacious, and outcomes were similar following treatment with vectors containing the photoreceptor-specific hGRKl promoter and the ubiquitous CBA promoter. Further studies were performed using hGRKl for limited expression to the intended target (photoreceptors), thus providing an additional safety feature by avoiding off -target expression. Retinoschisis cavity scores at 1 month are shown in FIG. 17 A. Associated ERG measurements at 1 month are shown in FIG. 17B. A comparison of OCT data of the left eye and right eye are shown in FIG. 19. A comparison of ERG data of the left eye and right eye are shown in FIG. 20.WSGR Docket No. 58774-736.601

[0278] Following the POC study, a six -month, pharmacology study was conducted to evaluate dose responsive improvements in retinal structure and function following a single subretinal administration of an AAV5 or AAV44.9(E53 ID) capsid containing the hGRKl promoter driving the hRSl syn gene (rAAV5 -hGRKl -hRSl syn (SEQ ID NO. 16) or rAAV44.9(E53 lD)-hGRKl- hRSl syn (SEQ ID NO. 16), respectively) in RSI -KO mice. Mice were subretinally injected between P24 and P26 in one eye with either vehicle, AAV5 or AAV44.9(E53 ID) at low (1.4x1011vg / ml; 1.4 xlO8vg / eye), middle (4.7x1011vg / ml; 4.7 xlO8vg / ml), or high (1.4x1012vg / ml; 1 ,4xl09vg / eye) dose. The contralateral eyes remained un -injected. Retinal structure and function were assessed via OCT and ERG, respectively, monthly over the course of 6 months. Following euthanasia at 6 months post-injection, retinas were cryosectioned and evaluated for RSI expression via immunohistochemistry.

[0279] OCT analysis revealed stable resolution of schisis cavities in RSI -KO mice treated with both vectors at all doses starting at one-month post-injection. An apparent dose-response was observed, as lower retinoschisis scores were observed in the two higher dose cohorts than in the low dose group. Significant improvements in both rod- and cone-mediated ERG function were also observed following treatment with both vectors at all doses, including the low dose. For scotopic b - wave amplitudes, the number of timepoints at which a significant difference between rAAV44.9(E53 lD)-hGRKl -hRSl syn-treated and vehicle-treated animals was observed, increased from 3 timepoints (2, 5, 6 months) at the low dose, 4 timepoints (2, 3, 5, 6 months) at the mid dose, to all 6 timepoints in the high dose-treated animals. A similar observation was made for photopic b- wave amplitudes. These data suggest a dose response of rAAV44.9(E53 ID) -hGRKl -hRSl syn in correcting retinal function in RSI -KO mice. Similar observations were seen in animals injected with rA A V5 -hGRKl -hRSlsyn. Immunohistochemical analysis revealed the presence of RSI expression in photoreceptor inner segments in cross sections of retinas from treated eyes of RSI - KO mice. RSI expression was absent from contralateral untreated eyes and those treated with vehicle alone.

[0280] The ERG measurement results at each of months 1 -6 are shown in FIGS. 20-25, respectively. Statistical analyses ofb waves of the injected virus versus vehicle, as determined by ERG measurements of Group 6 and 7 are shown in FIGS. 26-30. H4C measurements at the 4-month timepoint after injection are shown in FIG. 31.

[0281] In conclusion, these study results show that 1) subretinal injections of rAAV5 and rAAV44.9(E53 ID) vectors have comparable potency across a range of doses in the RSI -KO mice, 2) a dose response relationship was observed between both rAAV44.9(E53 ID) -hGRKl -hRSlsyn and rAAV5 -hGRKl -hRSlsyn and correction of the structural and functional deficits in RSI -KOWSGR Docket No. 58774-736.601 mice. A minimal effective dose (MED) of approximately 1.4x1011vg / ml (1.4 xlO8vg / eye) was identified. Further, 3) the photoreceptor-specific hGRKl promoter drives therapeutic levels of RSI expression in treated mice.

[0282] Additional studies evaluated dose ranging transduction and biodistribution of rAAV particles having optimal capsids in non-human primates with and without induced retinoschisis. Additional studies will involve administration of Cloning AAV-hGRKl-hRSl vectors having a woodchuck hepatitis virus post-transcription regulatory element (WPRE), wherein the WPRE is safe for administration (“WPREsf ’). It is envisioned that this construct will lead to higher expression of RSI allowing for reduction of vector dose to further improve the safety profile of these ocular gene therapies.Table 5. ERG / OCT data in MiceExample 5 - Efficacy and Tolerability was Evaluated in a Hybrid Toxicology / Pharmacology Study in RS1KO mice

[0283] A three-month, hybrid toxicology / pharmacology study was conducted to evaluate dose responsive improvements in retinal structure and function following a single subretinal administration of an AAV44.9(E53 ID) capsid containing the hGRKl promoter driving the hRSlsyn gene (rAAV44.9(E531D)-hGRKl-hRSlsyn) in RSI -KO mice. Mice were subretinally injected between P24 and P26 in one eye with either vehicle or AAV44.9(E531D) at low (3.0 x 107vg / eye (n=20)), middle (1.3 x 108vg / eye (n=20)), or high (High 5.3 x 108vg / eye (n=20)) or max (2.1 x 109vg / eye (n=20)) dose. The contralateral eyes remained un -injected. Retinal structure and function were assessed via ERG and retinoschisis score, respectively, over the course of 3 months. The results are shown in FIG. 44 and significant dose dependent improvements in retinal structure and function were observed. The study design and results are summarized in Table 6.Table 6WSGR Docket No. 58774-736.601Example 6 - Durable Correction of Retinal Structure and Function Mediated by rAAV44.9(E531D) -hRSl in RS1KO Mice

[0284] A 6- month pharmacology study was performed evaluating rAAV44.9(E53 ID) and AAV5- vectors containing a hGRKl -hRSl syn-XOOl transgene (SEQ ID NO: 34) for their ability to rescue retinal structure and function in subretinally injected RS1KO mice. The results are shown in FIGs. 43 and 45A-45D. The study design is summarized in Table 7.Table 7WSGR Docket No. 58774-736.601Example 7 — Evaluation of hRSl -Containing rAAV Vectors with Stuffer Sequences

[0285] This study was conducted to evaluate optimized hRSl -containing AAV44.9(E53 ID) vectors with genome sizes conducive to efficient packaging. The goal was to identify a construct that was at least as effective as rAAV44.9(E531D)-XOO1 (SEQ ID NO: 34).

[0286] Due to the small packaging size of pTR-XOOl (1723 bp), and the possibility for heterologous genome packaging, several new constructs were designed with cassette sizes approaching the natural carrying capacity of AAV (~4.7 Kb ITR to ITR cassette). This was accomplished by addition of an inert stuffer sequence inserted within the vector cassette either 5 ' to (X001-5p), or 3' to (X001-3p) hGRKl -hRSl syn-bGH poly A (pTR-XOOl -5p, SEQ ID NO. 31 and pTR-XOOl -3p, SEQ ID NO. 32, respectively). The stuffer DNA was de novo synthesized (Genscript, NJ). Additionally, a version was created incorporating the woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) positioned between hRSlsyn and bGH polyA (pTR-X002-3p, SEQ ID NO. 33). The mutant version of WPRE used has previously been incorporated in AAV vectors used in other ocular gene therapy clinical trials. All constructs contained the same hGRKl promoter, SV40 SD / SA and bGH polyA signal sequence. rAAV44.9(E53 ID) vectors were produced by packaging these expression cassettes, and the vector genomes ranged in size from 4534-4549 nucleotides. The multiple constructs evaluated are represented in FIGS. 15D-15F.

[0287] A 3 -month nonclinical study using two vector doses was performed to test restoration of retinal structure / function in RSI -KO mice. The ‘unstuffed’ vector pTR-XOO 1 (rAAV44.9(E531D)- X001), which rescued retinal structure / function in the earlier described RSI -KO mouse studies, was included as the comparator control.

[0288] A summary of the cassette selection study design is presentedin Table 8. RSI -KO mice were subretinally injected in one eye with either vehicle (Group 1), rAAV44.9(E531D)-XOO1 (Groups 2 and 3), rAAV44.9(E53 lD)-X001-3p (Groups 4 and 5), rAAV44.9(E53 lD)-X001-5p (Groups 6 and 7), or rAAV44.9(E53 lD)-X002-3p (Groups 8 and 9) vectors. Vectors were delivered at either 1.OxlO11vg / mL; 1.OxlO8vg / eye ( Groups 2, 4, 6, 8) or 5.0X1011vg / mL; 5. OxlO8vg / eye (WSGR Docket No. 58774-736.601Groups 3, 5, 7, 9). The contralateral eyes remained un -injected. Retinal structure and function were assessed via OCT and ERG, respectively, at approximately 1 -, and 2-months post-injection.Animals were euthanized at approximately 3 months post-injection. Following euthanasia, retinas were cryosectioned and evaluated for RSI expression via immunohistochemistry.Table 8 Study Design

[0289] OCT analysis revealed resolution of schisis cavities in vector -treated eyes (FIG. 32). With the exception of eyes treated with the low dose of rAAV44.9(E531D)-X001-5p, and evaluated at 1 -month post-injection, there was a statically significant difference in the retinoschisis score between vehicle and vector-treated eyes at both timepoints. At a concentration of l ><1011vg / mL (1.OxlO8vg / eye), rAAV44.9(E531D)-X002-3p led to significant improvements in rod- and cone- mediated function relative to eyes injected with vehicle alone (FIG. 33A). At the higher concentration of 5xl0nvg / mL (5. OxlO8vg / eye), all three stuffer constructs conferred significant improvements in retinal function relative to vehicle -injected controls (FIG. 33B). No significant differences in ERG amplitudesbetweeneyesinjected with rAAV44.9(E53 ID) carrying stuffed vs. unstuffed constructs were observed at this higher dose.

[0290] Immunohistochemical analysis revealed the presence of R...

Claims

WSGR Docket No. 58774-736.601CLAIMSWhat is claimed is:

1. A method of treating X-linked retinoschisis (XLRS) in a human subject in need thereof, the method comprising administering to an eye of the human subject about 1.5 x 1010vg to about 5.0 x 1010vg of an AAV particle.

2. The method of claim 1, wherein the about 1 .5 x 1010vg to about 5.0 x 1010vg of the AAV particle is administered to each eye of the human subject.

3. The method of claim 1 or claim 2, wherein about 1.5 x 1010vg of the AAV particle is administered to the eye or to each eye of the human subject.

4. The method of claim 1 or claim 2, wherein about 3.0 x 1010vg of the AAV particle is administered to the eye or to each eye of the human subject.

5. A method of improving visual acuity in an eye in a human subject with X-linked retinoschisis (XLRS), the method comprising administering to the eye of the human subject an AAV particle, wherein the visual acuity is improved by at least 5 ETDRS letters at least 6 months after administering.

6. The method of claim 5, wherein the visual acuity comprises Low Light Visual Acuity (LLVA).

7. The method of claim 5, wherein the visual acuity comprises Best Corrected Visual Acuity (BCVA).

8. The method of any one of claims 5-7, wherein the visual acuity is improved at least 12 months, at least 18 months, at least 24 months, at least 36 months, at least 48 months, or at least 60 months after administering.

9. A method of improving vision in an eye in a human subject with X-linked retinoschisis (XLRS), the method comprising administering to the eye of the human subject an AAV particle, wherein at least one schisis cavity in the eye is reduced in size for at least one month compared to prior to administering the AAV particle.

10. The method of claim 9, wherein the at least one schisis cavity is reduced in size by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%.

11. The method of claim 9 or 10, wherein the at least one schisis cavity is reduced in size at least 6 months, 12 months, at least 18 months, at least 24 months, at least 36 months, at least 48 months, or at least 60 months after administering.

12. The method of any one of claims 9-11, wherein there is a complete closure of the at least one schisis cavity.WSGR Docket No. 58774-736.60113. The method of claim 12, wherein the eye comprises at least two schisis cavities before administering, and there is complete closure of all schisis cavities in the eye.

14. The method of any one of claims 9- 13, wherein the size of the at least one schisis cavity is measured using optical coherence tomography.

15. The method of any one of claims 9-14, wherein the AAV particle is at administered in a subretinal bleb and the at least one schisis cavity that is reduced in size is located in an area of the retina outside of the subretinal bleb.

16. A method of improving vision in an eye in a human subject with X-linked retinoschisis (XLRS), the method comprising administering to the eye of the human subject an AAV particle, wherein the retinal thickness of the eye is reduced in size compared to prior to administering the AAV particle.

17. The method of claim 16, wherein the retinal thickness is reduced in size at least 6 months, 12 months, at least 18 months, at least 24 months, at least 36 months, at least 48 months, or at least 60 months after administering.

18. The method of claim 16 or 17, wherein the reduction in retinal thickness is specific to the central retina or the macula of the eye.

19. A method of visual field improvement in a human subject with X-linked retinoschisis (XLRS), the method comprising administering to an eye of the human subject an AAV particle, wherein the visual field improvement is at least 4 dB, as compared to before administering.

20. The method of claim 19, wherein the visual field is measured at at least 4 fixed loci, 5 fixed loci, 6 fixed loci, 7 fixed loci, 8 fixed loci, 9 fixed loci, or 10 fixed loci.

21. The method of claim 19 or 20, wherein the visual field improvement is at least 5dB, 6 dB, 7dB, 8 dB, 9 dB, or 10 dB.

22. The method of claim 21, wherein the visual improvement is at least 7dB averaged across at least 5 fixed loci.

23. The method of any one of claims 19-22, wherein the fixed loci are pre-selected.

24. The method of any one of claims 19-23, wherein the visual field improvementis maintained at least 6 months, 12 months, at least 18 months, at least 24 months, at least 36 months, at least 48 months, or at least 60 months after administering.

25. The method of any one of claims 19-24, wherein the visual field is measured using microperimetry.WSGR Docket No. 58774-736.60126. The method of any one of claims 19-24, wherein the visual field is measured using static automatic perimetry, optionally wherein the static automatic perimetry comprises a Humphrey Field Analyzer.

27. A method of treating X-linked retinoschisis (XLRS) in a human subject in need thereof, the method comprising administering to an eye of the human subject an AAV particle, wherein the human subject has a Best Correct Visual Acuity (BCVA) between 34 and 73 letters in the eye before administering.

28. The method of claim 27, wherein the BCVA is improved by at least 5 ETDRS letters at least 6 months after administering.

29. The method of claim 27 or 28, wherein the BCVA is improved at least 12 months, at least 18 months, at least 24 months, at least 36 months, at least 48 months, or at least 60 months after administering.

30. The method of any one of the preceding claims, wherein the AAV particle comprises a polynucleotide comprising a heterologous nucleic acid encoding a retinoschisin (RS) protein.

31. The method of claim 30, wherein the RS protein is a human RS protein.

32. The method of claim 30 or claim 31 , wherein the RS protein has the amino acid sequence of SEQ ID NO: 12.

33. The method of any one of claims 30-32, wherein the RS protein is encoded by a nucleic acid sequence at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the nucleic acid sequence of SEQ ID NO: 8.

34. The method of any one of claims 30-33, wherein the polynucleotide comprises a human rhodopsin kinase promoter.

35. The method of claim 34, wherein the human rhodopsin kinase promoter comprises a sequence at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 7.

36. The method of any one of claims 30-35, wherein the polynucleotide comprises a sequence at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 33 (pTR-X002-3pSR).

37. The method of any one of the preceding claims, wherein the AAV particle comprises a capsid.WSGR Docket No. 58774-736.60138. The method of claim 37, wherein the capsid comprises a VP1 amino acid sequence at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1.

39. The method of claim 37 or claim 38, wherein the capsid comprises a VP2 amino acid sequence at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 2.

40. The method of any one of claims 37-38, wherein the capsid comprises a VP3 amino acid sequence at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to: SEQ ID NO: 3.

41. The method of any one of the preceding claims, wherein the human subject has a mutation in the RSI gene.

42. The method of claim 41, wherein the human subject has an increased risk of retinal detachment as compared to a human subject that does not have the mutation in the RSI gene.

43. The method of any one of the preceding claims, wherein the human subject has a reduced risk of retinal detachment after the administering.

44. The method of any one of the preceding claims, wherein the administering comprises subretinal administration to a fovea of one or both eyes of the mammal.

45. The method of claim 44, wherein detachment of the fovea is minimized.

46. The method of any one of the preceding claims, wherein the human subject has a significant improvement as measured by patient global impression of change (PGIC) as compared to before the administering.

47. The method of any one of the preceding claims, wherein the human subject has loss of vision prior to the administering.

48. The method of claim 47, wherein the human subject regains vision after the administering.

49. The method of claim 48, wherein regaining vision comprises an improvement in visual acuity or an improvement in the visual field.

50. The method of any one of any one of the preceding claims, wherein the human subject expresses native RSI after the administering, optionally wherein the native RSI is SEQ ID NO: 12.

51. The method of claim 50, wherein the human subject expresses nativeRSl in photoreceptors after the administering.

52. The method of claim 50 or claim 51, wherein the human subject expresses native RSI in bipolar cells after the administering.WSGR Docket No. 58774-736.60153. The method of any one of the preceding claims, wherein the human subject is 6 years of age or older.

54. The method of any one of the preceding claims, wherein the human subject is 18 years old or older.

55. The method of claim 53, wherein the human subject is between 6 years of age and 65 years of age.

56. The method of any one of the preceding claims, wherein the human subject has XLRS caused by a pathogenic mutation in RSI .

57. The method of any one of the preceding claims, wherein prior to the administering the human subject has foveal schisis in the eye.

58. The method of any one of the preceding claims, wherein prior to the administering the human subject has parafoveal / perifoveal schisis in the eye.

59. The method of any one of the preceding claims, wherein before and / or after the administering, the method comprises performing optical coherence tomography on the human subject.

60. The method of any one of the preceding claims, wherein before and / or after the administering, the method comprises performing microperimetry on the human subject.

61. The method of any one of the preceding claims, wherein the AAV particle is administered via a subretinal injection.

62. A method of treating X-linked retinoschisis (XLRS) in a human subject in need thereof, the method comprising administering to an eye of the human subject about 150 pL of a solution comprising an AAV particle.

63. The method of any one of claims 1 -61, wherein the AAV particle is administered in a volume of about 150 microliters.

64. The method of any one of the preceding claims, wherein the AAV particle is administered in 2 blebs.

65. The method of any one of the preceding claims, wherein the AAV particle is administered in a manner that avoids foveal detachment.

66. A method of treating X-linked retinoschisis (XLRS) in a human subject in need thereof, the method comprising administering to an eye of the human subject an AAV particle and a corticosteroid.

67. The method of any one of claims 1-65, comprising administering a corticosteroid to the human subject.WSGR Docket No. 58774-736.60168. The method of claim 66 or claim 67, wherein the corticosteroid comprises prednisone, triamcinolone acetonide, methylprednisolone, prenidsolone acetate, or a combination thereof.

69. The method of claim 68, wherein the corticosteroid comprises prednisone.

70. The method of any one of claims 66-69, wherein the corticosteroid is administered starting about 7 weeks prior to the administering of the AAV particle.

71. The method of any one of claims 66-70, wherein the corticosteroid is administered at about 1 mg / kg of the human subject per day.

72. The method of any one of claims 66-71, comprising administering to the human subject triamcinolone acetonide.

73. The method of claim 72, wherein the triamcinolone acetonide is administered starting about 7 weeks prior to the administering of the AAV particle.

74. The method of claim 72 or claim 73, wherein about 20 mg of the triamcinolone acetonide is administered.

75. The method of any one of claims 72-74, wherein the triamcinolone acetonide is administered in a periocular injection.

76. The method of any one of claims 66-75, comprising administering to the human subject methylprednisolone.

77. The method of claim 76 wherein the methylprednisolone is administered starting about 7 weeks prior to the administering of the AAV particle.

78. The method of claim 76 or claim 77, wherein about 250 mg of the methylprednisolone is administered.

79. The method of any one of claims 76 -78, wherein the methylprednisolone is administered via IV.

80. The method of any one of claims 66-79, comprising administering to the human subject prenidsolone acetate.

81. The method of claim 80, wherein the prenidsolone acetate is administered starting about 28 days prior to the administering of the AAV particle.

82. The method of claim 80 or claim 81, wherein about 1% of the prenidsolone acetate is administered.

83. The method of any one of claims 80-82, wherein the prenidsolone acetate is administered topically.

84. The method of claim 83, wherein the administering comprises administering prednisone at a dose of Img / kg / day for seven weeks.WSGR Docket No. 58774-736.60185. The method of claim 83, wherein the administering comprises administering 20 mg of triamcinalone acetonide by periocular injection.

86. The method of claim 83, wherein the administering comprises administering 250 mg of methylprednisolone intravenously.

87. The method of claim 83, wherein the administering comprises administering 1% prenidsolone acetate topically for 28 days.

88. The method of any one of the preceding claims, wherein after the administering, the human subject has a significant improvement in PGIC.

89. The method of any one of the preceding claims, wherein after the administering the human subject has closure of a foveal schisis.

90. The method of any one of the preceding claims, wherein after the administering the human subject has a reduction in central retinal thickness.

91. The method of any one of the preceding claims, wherein after the administering the human subject has improvement in retinal sensitivity.

92. The method of any one of the preceding claims, wherein the administering a) preserves one or more photoreceptor cells, b) restores laminar retinal structure, c) restores one or more rod- and / or cone-mediated functions, d) restores completely or partially visual behavior in one or both eyes, or e) any combination thereof.

93. The method of any one of the preceding claims, wherein the administering restores laminar retinal structure.

94. The method of any one of the preceding claims, wherein the human subject is sensitive to inflammation.

95. The method of any one of the preceding claims, wherein the dose of the AAV particle reduces inflammation in the human subject as compared to a higher dose of the AAV particle.

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