Compositions and methods for treating dominant optic atrophy and X-linked retinal cleavage

Abstract

Provided herein are recombinant AAV vectors, AAV viral vectors, capsid proteins and methods of administration for improved gene therapy, as well as methods of making and using the same. These AAV vectors may be used to treat retinal cleavage (e.g., X-linked retinal cleavage) or dominant optic atrophy (DOA).

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 332,015, filed on April 18, 2022, the contents of which are incorporated by reference in their entirety.

[0003] References to Electronic Sequence Listings

[0004] The contents of the electronic Sequence Listing (ABEO_009_01WO_SeqList_ST26.xml; size: 732,965 bytes; and creation date: April 13, 2023) are incorporated herein by reference in its entirety. Background Art

[0005] X-linked retinoschisis (XLRS) is a rare monogenic disease that causes severe visual impairment. Although female carriers are asymptomatic, affected males usually begin to show symptoms of the disease within the first decade, and occasionally in infancy. The disease is caused by mutations in the RS1 gene, which is expressed in photoreceptors and retinal bipolar cells. In individuals with XLRS, holes appear where the adhesion of adjacent retinal layers is destroyed, resulting in discontinuity within the retinal circuit, photoreceptor degeneration, and impaired visual acuity. The current standard of care for XLRS patients is palliative and involves correction of refractive error, low vision assistance, and genetic counseling. Complications such as retinal detachment (up to 22% of patients) and vitreous hemorrhage (up to 40% of patients) are most common in the late stage of the disease and can be treated surgically. Early intervention via gene therapy has great potential to reverse or stabilize disease progression and prevent severe vision loss and the occurrence of these more serious complications in the early stages of the disease.

[0006] Autosomal dominant optic atrophy (ADOA) causes vision loss in the second to third decade of life. Over time, ADOA subjects typically have retinal degeneration, neurological deficits, and musculoskeletal complications. ADOA is caused by mutations in Opa1. Opa1 has been shown to be involved in mitochondrial christaestructure, mitochondrial fusion, and mitochondrial inner membrane remodeling.

[0007] New gene therapies are needed to treat XLRS or ADOA. Summary of the invention

[0008] The present disclosure relates generally to the field of gene therapy, and in particular to recombinant adeno-associated virus (AAV) vector particles (also referred to as AAV viral vectors) with novel expression cassettes, and their use for delivering transgenes to treat or prevent diseases or disorders such as XLRS or ADOA.

[0009] In one aspect, the present disclosure provides a method for treating retinoschisis in a subject in need thereof, comprising administering an AAV viral vector to the subject's retina or subretina. In an embodiment, the AAV viral vector comprises a photoreceptor-specific promoter operably linked to a transgene encoded by a heterologous nucleic acid. In an embodiment, the photoreceptor-specific promoter is selected from the group consisting of a rhodopsin kinase (RK) promoter, a rhodopsin (RHO) promoter, a β phosphodiesterase (PDE) promoter, and a retinitis pigmentosa (RP1) promoter. In an embodiment, the photoreceptor-specific promoter is a rhodopsin kinase (RK) promoter. In an embodiment, the RK promoter comprises a nucleic acid sequence having at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to SEQ ID NO: 196, is essentially composed of or consists of it. In an embodiment, the method comprises administering an AAV viral vector to the subject's retina. In an embodiment, the subject is a human. In an embodiment, the AAV viral vector is administered at about 10 10 to about 10 12 In some embodiments, the transgene is an RS1. In some embodiments, the transgene comprises a nucleic acid sequence that is at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 117. In some embodiments, the transgene encodes an amino acid sequence that is at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 143.

[0010] In one aspect, the present disclosure provides a method of treating an ocular disease or condition in a subject in need thereof, comprising administering to the subject an AAV viral vector, wherein the AAV viral vector comprises an AAV vector genome, wherein the AAV vector genome comprises in the 5' to 3' direction: (a) a first AAV inverted terminal repeat sequence, (b) a promoter, (c) a heterologous nucleic acid encoding Opa1, (d) a polyadenylation signal, and (e) a second AAV inverted terminal repeat sequence.

[0011] In embodiments, the promoter is a CBh promoter comprising a sequence at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 154. In embodiments, the promoter is a MeCP2 promoter comprising a sequence at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 156.

[0012] In embodiments, the AAV vector genome comprises an intron sequence that is at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 200 or 227. In embodiments, the intron sequence is located between the promoter and the heterologous nucleic acid encoding Opal. In embodiments, the intron sequence is located immediately downstream of the promoter without any additional nucleotides therebetween.

[0013] In embodiments, the heterologous nucleic acid encoding Opal comprises a sequence at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 175, 182, and 184. In embodiments, the heterologous nucleic acid encodes an Opal protein comprising an amino acid sequence at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 180, 183, and 185. In embodiments, the heterologous nucleic acid encodes an Opal protein comprising an amino acid sequence at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 180.

[0014] In embodiments, the polyadenylation signal comprises a sequence that is at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 201 or 225.

[0015] In embodiments, the AAV vector genome does not comprise any telomeric repeat sequences.

[0016] In embodiments, the AAV vector genome comprises a first telomeric repeat sequence located between a polyadenylation signal and a second AAV inverted terminal repeat sequence. In embodiments, the first telomeric repeat sequence is at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 202. In embodiments, the AAV vector genome comprises a second telomeric repeat sequence located between the first AAV inverted terminal repeat sequence and a promoter. In embodiments, the second telomeric repeat sequence is at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 203.

[0017] In embodiments, the first AAV inverted terminal repeat comprises or consists of a sequence that is at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 253. In embodiments, the second AAV inverted terminal repeat comprises or consists of a sequence that is at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 254.

[0018] In embodiments, the AAV vector genome comprises, in the 5' to 3' direction: (a) a first AAV inverted terminal repeat sequence, (b) a promoter comprising a sequence at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 154, (c) a heterologous nucleic acid encoding Opa1, (d) a polyadenylation signal comprising a sequence at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 201, and (e) a second AAV inverted terminal repeat sequence. In embodiments, the AAV vector genome comprises an intron sequence at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 200. In embodiments, the intron sequence is located between the promoter and the heterologous nucleic acid encoding Opa1. In embodiments, the AAV vector genome comprises a sequence at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 228. In embodiments, the Opal protein comprises an amino acid sequence at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 180.

[0019] In embodiments, the AAV vector genome comprises a polynucleotide sequence that is at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 230-239.

[0020] In embodiments, the ocular disease or disorder is autosomal dominant optic atrophy.

[0021] In one aspect, the present disclosure provides a method of treating an ocular disease or condition in a subject in need thereof, comprising administering to the subject an AAV viral vector, wherein the AAV viral vector comprises an AAV vector genome, wherein the AAV vector genome comprises in the 5'' to 3' direction: (a) a first AAV inverted terminal repeat sequence, (b) a promoter, (c) a heterologous nucleic acid encoding RS1, (d) a polyadenylation signal, and (e) a second AAV inverted terminal repeat sequence.

[0022] In an embodiment, the promoter is a photoreceptor-specific promoter. In an embodiment, the photoreceptor-specific promoter is selected from the group consisting of a rhodopsin kinase (RK) promoter, a rhodopsin (Rho) promoter, a beta phosphodiesterase (PDE) promoter, and a retinitis pigmentosa (RP1) promoter. In an embodiment, the promoter is an RK promoter comprising a sequence having at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to SEQ ID NO: 196. In an embodiment, the promoter is a Rho promoter comprising a sequence having at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to SEQ ID NO: 197. In embodiments, the promoter is a PDE promoter comprising a sequence at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:198.

[0023] In embodiments, the promoter is a CBh promoter comprising a sequence at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:154.

[0024] In embodiments, the AAV vector genome comprises an IRBP enhancer sequence upstream of the promoter. In embodiments, the IRBP enhancer sequence is at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 199. In embodiments, the IRBP enhancer sequence is located immediately upstream of the promoter without any additional nucleotides therebetween.

[0025] In embodiments, the AAV vector genome comprises a CVA-MVM intron sequence that is at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 226. In embodiments, the CVA-MVM intron sequence is located between the promoter and the heterologous nucleic acid encoding RS1.

[0026] In embodiments, the AAV vector genome comprises an intron sequence that is at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 200 or 222. In embodiments, the intron is located between the promoter and the heterologous nucleic acid encoding RS1.

[0027] In embodiments, the heterologous nucleic acid encoding RS1 comprises a sequence that is at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 117. In embodiments, the heterologous nucleic acid encodes a RS1 protein that comprises an amino acid sequence that is at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 143.

[0028] In embodiments, the polyadenylation signal comprises a sequence that is at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 201 or 225.

[0029] In embodiments, the AAV vector genome does not comprise any telomeric repeat sequences.

[0030] In embodiments, the AAV vector genome comprises a first telomeric repeat sequence located between a polyadenylation signal and a second AAV inverted terminal repeat sequence. In embodiments, the first telomeric repeat sequence is at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 203.

[0031] In embodiments, the AAV vector genome comprises a human β-globin scaffold / matrix attachment region (βGlo_s / MAR) sequence. In embodiments, the βGlo_s / MAR sequence is located between the polyadenylation signal and the second AAV inverted terminal repeat sequence. In embodiments, the βGlo_s / MAR sequence is located between the polyadenylation signal and the first telomeric repeat sequence. In embodiments, the βGlo_s / MAR sequence is at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 221.

[0032] In embodiments, the first AAV inverted terminal repeat comprises or consists of a sequence that is at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 255. In embodiments, the second AAV inverted terminal repeat comprises or consists of a sequence that is at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 256.

[0033] In an embodiment, the AAV vector genome comprises in the 5' to 3' direction: (a) a first AAV inverted terminal repeat sequence, (b) an IRBP enhancer sequence that is at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 199, (c) an RK promoter comprising a sequence that is at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 196, (d) a heterologous nucleic acid encoding RS1, (e) a polyadenylation signal comprising a sequence that is at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 225, and (f) a second AAV inverted terminal repeat sequence.

[0034] In embodiments, the AAV vector genome comprises an intron sequence having at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to SEQ ID NO: 222. In embodiments, the intron sequence is located between the promoter and the heterologous nucleic acid encoding RS1. In embodiments, the AAV vector genome comprises the CBA sequence of SEQ ID NO: 229, or a sequence having at most 5, at most 4, at most 3, at most 2 or at most 1 mutations thereto. In embodiments, the CBA sequence is located immediately upstream of the intron sequence without any additional nucleotides therebetween. In embodiments, the AAV vector genome comprises a CBA-MVM intron sequence having at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to SEQ ID NO: 226. In embodiments, the CVA-MVM intron sequence is located between the promoter and the heterologous nucleic acid encoding RS1.

[0035] In embodiments, the AAV vector genome comprises a sequence that is at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:224.

[0036] In embodiments, the AAV vector genome comprises a polynucleotide sequence that is at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 224 and 240-252.

[0037] In embodiments, the ocular disease or condition is X-linked retinoschisis.

[0038] In embodiments, the AAV viral vector comprises an AAV capsid protein comprising an amino acid sequence at least 95% identical to SEQ ID NO: 1-3, 30-34, 49, 67, 84, or 164. In embodiments, the AAV viral vector comprises an AAV capsid protein comprising an amino acid sequence at least 98%, at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 1-3, 30-34, 49, 67, 84, or 164. In embodiments, the AAV viral vector comprises an AAV capsid protein comprising, or consisting of, an amino acid sequence at least 95%, at least 98%, at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 2.

[0039] In embodiments, administration is juxtaretinal administration. In embodiments, juxtaretinal administration comprises injection at a distance between 0 and 13 millimeters (mm), between 0 and 10 mm, between 0 and 5 mm, or between 0 and 3 mm from the surface of the retina in the posterior vitreous cavity of the eye.

[0040] In embodiments, the subject is a human.

[0041] In one aspect, the disclosure provides a nucleic acid comprising, in the 5' to 3' direction: (a) a first AAV inverted terminal repeat sequence, (b) a promoter, (c) a heterologous nucleic acid encoding Opa1, (d) a polyadenylation signal, and (e) a second AAV inverted terminal repeat sequence.

[0042] In embodiments, the promoter is a CBh promoter comprising a sequence at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 154. In embodiments, the promoter is a MeCP2 promoter comprising a sequence at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 156.

[0043] In embodiments, the AAV vector genome comprises an intron sequence that is at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 200 or 227. In embodiments, the intron sequence is located between the promoter and the heterologous nucleic acid encoding Opal. In embodiments, the intron sequence is located immediately downstream of the promoter without any additional nucleotides therebetween.

[0044] In embodiments, the heterologous nucleic acid encoding Opal comprises a sequence at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 175, 182, and 184. In embodiments, the heterologous nucleic acid encodes an Opal protein comprising an amino acid sequence at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 180, 183, and 185. In embodiments, the heterologous nucleic acid encodes an Opal protein comprising an amino acid sequence at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 180.

[0045] In embodiments, the polyadenylation signal comprises a sequence that is at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 201 or 225.

[0046] In embodiments, the AAV vector genome does not comprise any telomeric repeat sequences.

[0047] In embodiments, the AAV vector genome comprises a first telomeric repeat sequence located between a polyadenylation signal and a second AAV inverted terminal repeat sequence. In embodiments, the first telomeric repeat sequence is at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 202. In embodiments, the AAV vector genome comprises a second telomeric repeat sequence located between the first AAV inverted terminal repeat sequence and a promoter. In embodiments, the second telomeric repeat sequence is at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 203.

[0048] In embodiments, the first AAV inverted terminal repeat comprises or consists of a sequence that is at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 253. In embodiments, the second AAV inverted terminal repeat comprises or consists of a sequence that is at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 254.

[0049] In an embodiment, the AAV vector genome comprises, in the 5' to 3' direction: (a) a first AAV inverted terminal repeat, (b) a promoter comprising a sequence at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 154, (c) a heterologous nucleic acid encoding Opa1, (d) a polyadenylation signal comprising a sequence at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 201, and (e) a second AAV inverted terminal repeat.

[0050] In embodiments, the nucleic acid comprises an intron sequence that is at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 200. In embodiments, the intron sequence is located between the promoter and the heterologous nucleic acid encoding Opal.

[0051] In embodiments, the Opal protein comprises an amino acid sequence that is at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:180.

[0052] In embodiments, the nucleic acid comprises a sequence that is at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 228. In embodiments, the nucleic acid comprises a polynucleotide sequence that is at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 230-239.

[0053] In one aspect, the present disclosure provides a nucleic acid comprising, in the 5' to 3' direction: (a) a first AAV inverted terminal repeat sequence, (b) a promoter, (c) a heterologous nucleic acid encoding RS1, (d) a polyadenylation signal, and (e) a second AAV inverted terminal repeat sequence.

[0054] In embodiments, the promoter is a photoreceptor-specific promoter. In embodiments, the photoreceptor-specific promoter is selected from the group consisting of a rhodopsin kinase (RK) promoter, a rhodopsin (Rho) promoter, a beta phosphodiesterase (PDE) promoter, and a retinitis pigmentosa (RP1) promoter.

[0055] In embodiments, the promoter is a CBh promoter comprising a sequence at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:154.

[0056] In embodiments, the promoter is an RK promoter comprising a sequence at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 196. In embodiments, the promoter is a Rho promoter comprising a sequence at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 197. In embodiments, the promoter is a PDE promoter comprising a sequence at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 198.

[0057] In embodiments, the nucleic acid comprises an IRBP enhancer sequence upstream of the promoter. In embodiments, the IRBP enhancer sequence is at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 199. In embodiments, the IRBP enhancer sequence is located immediately upstream of the promoter without any additional nucleotides therebetween.

[0058] In embodiments, the AAV vector genome comprises a CVA-MVM intron sequence that is at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 226. In embodiments, the CVA-MVM intron sequence is located between the promoter and the heterologous nucleic acid encoding RS1.

[0059] In embodiments, the AAV vector genome comprises an intron sequence that is at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 200 or 222. In embodiments, the intron is located between the promoter and the heterologous nucleic acid encoding RS1.

[0060] In embodiments, the heterologous nucleic acid encoding RS1 comprises a sequence that is at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 117. In embodiments, the heterologous nucleic acid encodes a RS1 protein that comprises an amino acid sequence that is at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 143.

[0061] In embodiments, the polyadenylation signal comprises a sequence that is at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 201 or 225.

[0062] In embodiments, the AAV vector genome does not comprise any telomeric repeat sequences.

[0063] In embodiments, the AAV vector genome comprises a first telomeric repeat sequence located between a polyadenylation signal and a second AAV inverted terminal repeat sequence. In embodiments, the first telomeric repeat sequence is at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 203.

[0064] In embodiments, the AAV vector genome comprises a human β-globin scaffold / matrix attachment region (βGlo_s / MAR) sequence. In embodiments, the βGlo_s / MAR sequence is located between the polyadenylation signal and the second AAV inverted terminal repeat sequence. In embodiments, the βGlo_s / MAR sequence is located between the polyadenylation signal and the first telomeric repeat sequence. In embodiments, the βGlo_s / MAR sequence is at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 221.

[0065] In embodiments, the first AAV inverted terminal repeat comprises or consists of a sequence that is at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 255. In embodiments, the second AAV inverted terminal repeat comprises or consists of a sequence that is at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 256.

[0066] In an embodiment, the AAV vector genome comprises in the 5' to 3' direction: (a) a first AAV inverted terminal repeat sequence, (b) an IRBP enhancer sequence that is at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 199, (c) an RK promoter comprising a sequence that is at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 196, (d) a heterologous nucleic acid encoding RS1, (e) a polyadenylation signal comprising a sequence that is at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 225, and (f) a second AAV inverted terminal repeat sequence.

[0067] In embodiments, the nucleic acid comprises an intron sequence that is at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 222. In embodiments, the intron sequence is located between the promoter and the heterologous nucleic acid encoding RS1.

[0068] In embodiments, the nucleic acid comprises the CBA sequence of SEQ ID NO: 229, or a sequence having at most 5, at most 4, at most 3, at most 2, or at most 1 mutations therefrom. In embodiments, the CBA sequence is located immediately upstream of the intron sequence without any additional nucleotides therebetween. In embodiments, the nucleic acid comprises a CBA-MVM intron sequence having at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 226. In embodiments, the CVA-MVM intron sequence is located between the promoter and the heterologous nucleic acid encoding RS1.

[0069] In embodiments, the nucleic acid comprises a sequence that is at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:224.

[0070] In embodiments, the nucleic acid comprises a polynucleotide sequence at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identical to any one of SEQ ID NOs: 224 and 240-252.

[0071] In one aspect, the disclosure provides a nucleic acid comprising, in the 5' to 3' direction: (a) a promoter, (b) a heterologous nucleic acid encoding a transgene, and (c) a polyadenylation signal, wherein the promoter is a CBh promoter comprising a sequence at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 154, and wherein the polyadenylation signal comprises a sequence at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 201 or 225.

[0072] In embodiments, the nucleic acid comprises an intron sequence that is at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 200, 222, 226, or 227. In embodiments, the intron is located between the promoter and the heterologous nucleic acid encoding the transgene.

[0073] In embodiments, the nucleic acid comprises a first ITR located 5' to the promoter and a second ITR located 3' to the polyadenylation signal.

[0074] In embodiments, the nucleic acid does not comprise any telomeric repeat sequences.

[0075] In embodiments, the nucleic acid comprises a first telomeric repeat sequence located between a polyadenylation signal and a second AAV ITR. In embodiments, the first telomeric repeat sequence is at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 202. In embodiments, the nucleic acid comprises a second telomeric repeat sequence located between a first AAV inverted terminal repeat sequence and a promoter. In embodiments, the second telomeric repeat sequence is at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 203.

[0076] In one aspect, the disclosure provides a vector comprising a nucleic acid of the disclosure.

[0077] In one aspect, the disclosure provides an AAV vector genome comprising a nucleic acid of the disclosure.

[0078] In one aspect, the present disclosure provides an AAV viral vector comprising an AAV vector genome of the present disclosure. In an embodiment, the AAV viral vector comprises an AAV capsid protein comprising an amino acid sequence that is at least 95%, at least 98%, at least 99%, at least 99.5% or 100% identical to any one of SEQ ID NOs: 1-3, 30-34, 49, 84 and 164.

[0079] In one aspect, the present disclosure provides a method of expressing a transgene in a retinal cell, comprising delivering a nucleic acid of the present disclosure to the retinal cell, or transducing the retinal cell with an AAV viral vector of the present disclosure. In an embodiment, the retinal cell is a retinal ganglion cell.

[0080] In one aspect, the present disclosure provides a method for treating a disease or illness, the method comprising administering an AAV viral vector of the present disclosure to a subject. In an embodiment, an AAV viral vector is administered to a subject intraocularly, periocularly, intravitreally, next to the retina, or subretinaly. In an embodiment, the disease or illness is macular degeneration, retinitis pigmentosa, autosomal dominant optic atrophy, retinoschisis, Stargardt disease, Bietti's Crystalline Dystrophy, or Best's yolk-shaped macular dystrophy. In an embodiment, the disease or illness is X-linked retinoschisis. BRIEF DESCRIPTION OF THE DRAWINGS

[0081] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0082] Figure 1 is a schematic representation of different intraocular administration modes (adapted from Yiu et al., Mol Ther Methods Clin Dev. 2020 Jan 21;16:179-191, the contents of which are incorporated herein by reference in their entirety).

[0083] Figure 2A-2E AAV viral vector-mediated GFP expression in the eyes of non-human primate models via intravitreal or pararetinal administration is shown. Scanning laser ophthalmoscopy (SLO) imaging was performed 26 days after injection of the indicated AAV viral vectors. Figure 2A Shown is the spread of transduction mediated by intravitreal injection of an AAV viral vector containing the AAV204 capsid protein. Figure 2B Shown is the spread of transduction mediated by pararetinal injection of an AAV viral vector containing the AAV204 capsid protein. Figure 2C Shown is the spread of transduction mediated by pararetinal injection of an AAV viral vector containing the AAV8 capsid protein. Figure 2D Shown is the spread of transduction mediated by pararetinal injection of an AAV viral vector containing the AAV214 capsid protein. Figure 2E Shown is the spread of transduction mediated by pararetinal injection of an AAV viral vector containing the AAV214-D5 capsid protein.

[0084] Figure 3A-Figure 3F Shown are imaging analyses of the retina following AAV administration. Figure 3A Shown are composite images of the retina following intravitreal administration of AAV204. Figure 3B Shown are composite images of the retina (upper left), rhodopsin (upper right), and a magnified composite image (lower) after juxtaretinal administration of AAV204. Figure 3C Shown are composite images of the retina (upper left), rhodopsin (upper right), and a magnified composite image (lower) after juxtaretinal administration of AAV204. Figure 3D Shown are immunohistochemical analyses of rhodopsin and GFP expression 1 month after pararetinal injection of AAV204 or AAV8 viral vectors. Figure 3E Shown is rhodopsin and GFP expression in the fovea following pararetinal injection of AAV204 viral vectors. Figure 3F Shown is the expression of rhodopsin and GFP along the papillomacular bundle following pararetinal injection of AAV204 viral vector.

[0085] Figure 4A-4C AAV viral vector-mediated GFP expression in the eye of a non-human primate model via subretinal administration is shown. SLO imaging was performed on day 27 after injection of the indicated AAV viral vectors. Figure 4A Shown is the spread of transduction mediated by subretinal injection of an AAV viral vector containing the AAV8 capsid protein. Figure 4B Shown is the spread of transduction mediated by subretinal injection of an AAV viral vector containing the AAV214 capsid protein. Figure 4C Shown is the spread of transduction mediated by subretinal injection of an AAV viral vector containing the AAV214-D5 capsid protein. Figure 4D Shown are a composite image of the retina (upper left), rhodopsin (upper right), and a magnified composite image (lower) after subretinal administration of AAV8. Figure 4E Shown are a composite image of the retina (upper left), rhodopsin (upper right), and a magnified composite image (lower) after subretinal administration of AAV214. Figure 4F Shown are composite images of the retina (upper left), rhodopsin (upper right), and a magnified composite image (lower) after subretinal administration of AAV214-D5.

[0086] Figure 5A diagram of the VP1, VP2 and VP3 portions of the capsid protein is shown. The VP1-specific portion and the VP2-specific portion are indicated together with the VP3 portion, which is identical to the VP3 protein produced. The amino acid sequence of AAV214 VP3 is shown (SEQ ID NO:41) and variable regions I-IX are indicated. The complete VP1 protein amino acid sequence of AAV214 is provided as SEQ ID NO:3.

[0087] Figure 6 An alignment of the amino acid sequences of the VP1 proteins of AAV214 (SEQ ID NO: 3) and AAV214-D5 (SEQ ID NO: 164) is shown.

[0088] Figure 7 The designs of various AAV vector genomes encoding Opa1 are shown.

[0089] Fig. 8A Shown is the expression of Opal in 293 cells transfected with each of the indicated vectors. Figure 8B is a graph showing the viral yield of each indicated vector.

[0090] Fig.9A and Fig. 9B Opal expression in viral potency testing is shown. Fig. 9C Shown are protein staining results of cells transfected with each indicated vector.

[0091] Fig. 10A A schematic diagram of a proof-of-concept study evaluating AAV204 viral vector encoding Opa1 is shown. Fig. 10B Western analysis of Opa1 and FLAG tag expression in heterozygous treated mice is shown. Fig. 10C Western analysis of Opal, FLAG-tag, Brn3a and Rho expression is shown. Fig. 10D Shown are RT-PCR analyses of human Opal, mouse Opal and FLAG-tagged RNA transcript levels in wild-type or Opal heterozygous, untreated or treated animals at 2 months post-injection.

[0092] Fig.11A and Fig. 11B A schematic diagram of a proof-of-concept (POC) study evaluating AAV204 viral vector encoding Opa1 is shown.

[0093] Fig. 12A A tabular summary of various AAV vector genomes encoding RS1 is shown. Fig. 12B Designs of various RS1-encoding AAV vector genomes are shown.

[0094] Fig.13 Western analysis of RS1 protein expression is shown.

[0095] Fig.14A is a graph showing the expression of secreted RS1 protein in Lec2 cells transduced with each of the indicated AAV viral vectors. Fig. 14B is a graph showing mRNA expression of target transgenes in Lec2 cells transduced with each of the indicated AAV viral vectors. Fig. 14C Shown are Western analyses of secreted RS1 proteins in Lec2 cells transduced with each of the indicated AAV viral vectors. Fig.14D Western analysis comparing the molecular weight of myc-tagged RS1 and wild-type RS1 is shown.

[0096] Fig.15A Shown is a diagram of a proof-of-concept (POC) study evaluating AAV204 viral vector encoding RS1. Fig. 15B is a graph showing RS1 protein expression in wild-type mice transduced with the indicated AAVs.

[0097] Fig.16A and Fig. 16B Shown are Western analyses of RS1 expression in mice transduced with the indicated AAVs.

[0098] Fig.17A and Fig. 17B A schematic diagram of a proof-of-concept (POC) study evaluating AAV204 viral vector encoding RS1 is shown.

[0099] Fig.18A is a table showing the different treatment groups for the mouse study of RS1 expression. Fig.18B Shown are endogenous mouse RS1 (mRs1) expression as measured by qPCR for Groups 5-8 at the 2 months post-treatment (mpt) time point. Fig. 18C RS1 expression from virally delivered transgenes at the 2 mpt time point is shown. Data were log transformed to facilitate visualization of comparisons with larger differences. nd: not detected. Fig.18D and Fig.18E The results of simultaneous detection of endogenous RS1 and viral-derived myc-RS1 using a highly specific RS1 antibody are shown. Western analysis was performed on Fig.18D is shown in and Fig.18E Quantification was performed in . The RS1-specific bands in groups 5 and 6 were larger than the endogenous protein in group 8 due to the presence of the myc tag. A myc-RS1 positive control derived from transduced tissue culture cells was included in the last lane. nd: not detected.

[0100] Fig.19A Shown is IHC staining of wild-type (WT) retina at the 2 mpt time point. Fig.19B Shown is IHC staining of untreated mutant retinas at the 2 mpt time point. Fig.19C Shown is IHC staining of mutant retinas transduced with RS1_46 at the 2 mpt time point. Fig.19D Shown is IHC staining of mutant retinas transduced with RS1_46 in the right eye of animal #123 at the 2 mpt time point. Fig.19E Shown is IHC staining of mutant retinas transduced with RS1_48 at the 2 mpt time point. Fig.19F It is a graph showing the analysis of cone density at p90, 2 mpt time points. Figure 19G Staining of retinal samples is shown. Top panels AC show retinal samples transduced with AAV204.RK: RS1_28: RS1 is stained red and PNA is stained green to demonstrate the extent of cone degeneration. Bottom panels are higher magnification images showing cone density in transduced and untransduced areas of similarly prepared WT retinas (section D) and treated mutant retinas (sections E and F, respectively).

[0101] Fig. 20A Western analysis of RS1 protein expression is shown. The positive control in the last lane is recombinant RS1 from transfected tissue culture cells. Fig. 20B is a graph showing quantification of band intensity. nd = not detected. Fig. 20C IHC of WT retina at 6 mpt time point is shown. RS1 expression (red) in WT eyes (Group 3) is uniform throughout the retina and concentrated in photoreceptors. The figure on the right shows a higher magnification view of the boxed area. Fig.20D IHC of mutant retinas at 6 mpt time points is shown. RS1 staining (red) is absent in mutant retinas (Group 1). The figure on the right shows a higher magnification view of the boxed area. In the absence of RS1, the retinal vasculature is labeled with a secondary antibody (arrow). Fig.20E IHC of mutant retinas treated with RS1_28 at the 6 mpt time point is shown. RS1 expression (red) is visible in the dorsal retina of this eye from Group 2. Higher magnification views of transduced (a) and non-transduced (b) areas are shown on the right. Fig.20F IHC of mutant retinas treated with RS1_26 at the 6 mpt time point is shown. None of the eyes in this group had detectable RS1 expression.

[0102] Fig.21is a graph showing quantification of cone density at the 6 mpt time point. Cone density was measured from sections stained with peanut agglutinin (PNA). Measurements from Group 2 were separated by RS1 expression. In Group 2, each data point of the same color reflects RS1 positive and negative areas of the same section. In four of the five eyes with detectable RS1 expression, cone density was slightly higher in areas with adjacent RS1 expression. Eyes without detectable RS1 expression are represented by black dots.

[0103] Fig.22A OCT imaging of a representative eye at the 6 mpt time point is shown. The yellow bar in each image indicates the thickness of the ONL. The arrows show examples of retinal schises present in one eye. Fig. 22B is a graph showing quantitative analysis of ONL measurements in all groups.

[0104] Fig.23A IHC of WT retina at 6 mpt time point is shown. Fig. 23B Shown is IHC of untreated mutant retinas at the 6 mpt time point. Fig.23C IHC of mutant retinas treated with AAV204.CBh:RS1_16 at the 6 mpt time point is shown. In most eyes treated with AAV204.CBh:RS1_16, extensive degeneration was observed in large areas in the dorsal retina where vesicles should have appeared. Visible RS1 expression is rare and absent in this eye. Although there is no detectable RS1 staining, the boxed area enlarged in the figure on the right shows improved cone density (PNA staining). Fig.23D IHC of mutant retinas treated with AAV204.CBh:RS1_16 at the 6 mpt time point in one particular eye (with injection injury) is shown. On the left, asterisks show treated retinas with severe injection-related injury and RS1 expression (red) throughout the inner retina. Deeper sections of the same eye are shown on the right, showing RS1 expression in the inner retina (B') and expression in photoreceptors in areas adjacent to the lesion (B"). In all figures, PNA (grey) marks the outer segments of cones, and Iba1 (green) marks inflammatory cells. Fig.23EIHC of mutant retinas treated with AAV204.CBh:RS1_18 at the 6 mpt time point is shown. In this example, strong RS1 expression is observed in dorsal photoreceptors and extends into the inner retina (frame a). In contrast, RS1 staining is absent in the ventral retina (frame b). Although otherwise structurally intact, most of the dorsal retina lacks cones. However, the area immediately behind frame b is RS1 positive and is also rich in cones compared to the adjacent RS1 negative area. Fig.23F is a graph summarizing the analysis of cone density at the 6 mpt time point. Cone density was significantly improved in all treated eyes, even in areas where RS1 immunostaining could not be detected. For this analysis, areas of severe degeneration and areas of cone depletion were omitted.

[0105] Fig.24A is a graph summarizing the mean ONL thickness in all groups. For treated eyes, separate measurements were obtained for RS1+ and RS1- areas of the retina, and data points of similar color within each group reflect measurements taken from the same section. Areas of severe degeneration are not included. Data from representative eyes in Groups 10 and 11 are shown in Fig. 24B and Fig.24C Shown in , where star labels indicate areas of treated retina with RS1 expression.

[0106] Fig.25A Western analysis of RS1 protein expression is shown. The positive control in the last lane is recombinant RS1 from transfected tissue culture cells. Fig.25B is a graph showing quantification of band intensity. nd = not detected.

[0107] Fig.26A OCT imaging of an untreated mutant eye is shown. Fig.26B OCT imaging of wild-type eyes is shown. Fig.26C Postoperative OCT imaging of the vesicle is shown to confirm successful injection. Fig.26D Shown are OCT images of treated eyes 6 months after injection. Fig.26E Shown is an OCT image of the treated eye 6 months after injection; the edge of the vesicle is captured here. Fig.26F OCT images of treated eyes 6 months after injection are shown. The yellow line in these figures indicates the thickness of the ONL. Figure 26G is a graph showing quantification of ONL thickness. Fig.26H is a paired t-test estimate showing increased ONL thickness in the treated dorsal retina (11D) compared to the untreated ventral retina (11V) in the same eye.

[0108] Fig. 27 A representative flicker ERG at the 6 mpt time point is shown. DETAILED DESCRIPTION

[0109] The following will be more fully described according to certain embodiments of the present disclosure. However, aspects of the present disclosure can be embodied in many different forms and should not be construed as being limited to the embodiments set forth herein. In fact, these embodiments are provided to make the present disclosure thorough and complete, and to fully convey the scope of the present invention to those skilled in the art. The terms used in this specification are only used for the purpose of describing specific embodiments and are not intended to be restrictive.

[0110] Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as those commonly understood by those skilled in the art to which the present invention belongs. It should be further understood that terms such as those defined in commonly used dictionaries should be interpreted as having a meaning consistent with their meaning in the context of this application and the relevant art, and unless explicitly defined as such herein, should not be interpreted in an idealized or overly formal sense.

[0111] Unless the context indicates otherwise, it is expressly intended that the various features of the invention described herein may be used in any combination. In addition, the present disclosure also contemplates that, in embodiments, any feature or combination of features set forth herein may be excluded or omitted. For example, if the specification states that a composite comprises components A, B, and C, it is expressly intended that any one or combination of A, B, or C may be omitted and abandoned, either alone or in any combination.

[0112] Unless expressly stated otherwise, all specified embodiments, features, and terms are intended to include the stated embodiments, features, or terms and their biological equivalents.

[0113] Incorporated by Reference

[0114] All references, articles, publications, patents, patent publications and patent applications cited herein are incorporated by reference in their entirety for all purposes. However, the mention of any reference, article, publication, patent, patent publication and patent application cited herein is not and should not be taken as an admission or any form of suggestion that they constitute valid prior art or form part of the common general knowledge in any country in the world.

[0115] definition

[0116] Unless otherwise indicated, the practice of the present technology will employ conventional techniques of organic chemistry, pharmacology, immunology, molecular biology, microbiology, cell biology, and recombinant DNA, which are within the skill of the art. See, e.g., Sambrook, Fritsch, and Maniatis, Molecular Cloning: A Laboratory Manual, Second Edition (1989); Current Protocols in Molecular Biology (FM Ausubel et al., ed., (1987)); these series Methods in Enzymology (Academic Press, Inc.): PCR 2: A Practical Approach (MJ MacPherson, BD Hames, and GR Taylor, ed. (1995)), Harlow and Lane, ed. (1988) Antibodies, a Laboratory Manual, and Animal Cell Culture (RI. Freshney, ed. (1987)).

[0117] It is also to be understood, although not always explicitly stated, that the reagents described herein are exemplary only and that equivalents thereof are known in the art.

[0118] As used herein, the term "about" when referring to a measurable value such as an amount or concentration, is intended to encompass variations of 10% of the specified amount.

[0119] The terms "acceptable," "effective," or "sufficient" when used to describe the selection of any component, range, dosage form, etc. disclosed herein, mean that the component, range, dosage form, etc. is suitable for the disclosed purpose.

[0120] Unless otherwise specified, the term "host cell" includes eukaryotic host cells, including, for example, fungal cells, yeast cells, higher plant cells, insect cells and mammalian cells. Non-limiting examples of eukaryotic host cells include apes, cows, pigs, mice, rats, birds, reptiles and humans, such as HEK293 cells and 293T cells.

[0121] As used herein, the term "isolated" refers to molecular or biological or cellular material that is substantially free of other material.

[0122] As used herein, the terms "nucleic acid sequence" and "polynucleotide" are used interchangeably to refer to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. Thus, this term includes, but is not limited to, single-stranded, double-stranded, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or polymers comprising, consisting essentially of, or contributed to by purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural or derived nucleotide bases.

[0123] "Gene" refers to a polynucleotide containing at least one open reading frame (ORF) capable of encoding a specific polypeptide or protein. "Gene product" or alternatively "gene expression product" refers to an amino acid sequence (eg, a peptide or polypeptide) produced when a gene is transcribed and translated.

[0124] As used herein, "expression" refers to the two-step process in which a polynucleotide is transcribed into mRNA and / or the process in which the transcribed mRNA is subsequently translated into a peptide, polypeptide or protein. If the polynucleotide is derived from genomic DNA, expression may include splicing of mRNA in a eukaryotic cell.

[0125] "Under transcriptional control" is a term well known in the art, indicating that transcription of a polynucleotide sequence (usually a DNA sequence) is dependent upon its operably linked to an element that aids in the initiation of transcription or promotes transcription. "Operably linked" means that the polynucleotides are arranged in a manner that allows them to function in a cell. In one aspect, the invention provides a promoter that is operably linked to a downstream sequence.

[0126] The term "encoding" as it applies to a polynucleotide refers to a polynucleotide that, if in its native state or when manipulated by methods well known to those skilled in the art, can be transcribed to produce mRNA for a polypeptide and / or fragment thereof, the polynucleotide is said to "encode" the polypeptide. The antisense strand is the complement of such nucleic acid, and the coding sequence can be deduced therefrom.

[0127] As used herein, the term "promoter" means a control sequence, which is a region of a polynucleotide sequence that controls the initiation and rate of transcription of a coding sequence such as a gene or transgene in this region. For example, a promoter can be constitutive, inducible, repressive or tissue-specific. A promoter may contain genetic elements that can bind regulatory proteins and molecules (such as RNA polymerases and transcription factors). Non-limiting exemplary promoters include Rous sarcoma virus (RSV) LTR promoters (optionally with RSV enhancers), cytomegalovirus (CMV) promoters, SV40 promoters, dihydrofolate reductase promoters, β-actin promoters, phosphoglycerol kinase (PGK) promoters, U6 promoters, H1 promoters, ubiquitous chicken β-actin hybrid (CBh) promoters, small nuclear RNA (U1a or U1b) promoters, MeCP2 promoters, MeP418 promoters, MeP426 promoters, minimal MeCP2 promoters, VMD2 promoters, mRho promoters or EFI promoters.

[0128] Other non-limiting exemplary promoters provided herein include, but are not limited to, EF1a, Ubc, human beta-actin, CAG, TRE, Ac5, polyhedrin, CaMKIIa, Gal1, TEF1, GDS, ADH1, Ubi, and alpha-1-antitrypsin (hAAT). It is known in the art that the nucleotide sequence of such promoters can be modified to increase or reduce the efficiency of mRNA transcription. See, for example, Gao et al. (2018) Mol. Ther.: Nucleic Acids 12: 135-145 (modification of the TATA box of 7SK, U6, and H1 promoters to eliminate RNA polymerase III transcription and stimulate RNA polymerase II dependent mRNA transcription). Synthetic derived promoters can be used for ubiquitous or tissue-specific expression. Further, viral derived promoters (some of which have been noted above) can be used for methods disclosed herein, such as CMV, HIV, adenovirus, and AAV promoters. In an embodiment, a promoter is used together with an enhancer to increase transcription efficiency. Non-limiting examples of enhancers include the interstitial retinoid binding protein (IRBP) enhancer, the RSV enhancer, or the CMV enhancer.

[0129] Enhancers are regulatory elements that increase the expression of a target sequence. "Promoters / enhancers" are polynucleotides containing sequences that can provide promoter and enhancer functions. For example, the long terminal repeats of retroviruses contain promoter and enhancer functions. Enhancers / promoters can be "endogenous" or "exogenous" or "heterologous". "Endogenous" enhancers / promoters are enhancers / promoters that are naturally connected to a given gene in a genome. "Exogenous" or "heterologous" enhancers / promoters are enhancers / promoters that are juxtaposed to a gene by genetic manipulation (i.e., molecular biology techniques) so that transcription of the gene is guided by the connected enhancer / promoter. Non-limiting examples of enhancers / promoters connected for the methods, compositions, and constructs provided herein include PDE promoters plus IRBP enhancers or CMV enhancers plus U1a promoters. It will be appreciated in the art that enhancers can be operated remotely and regardless of the direction of their position relative to endogenous or heterologous promoters. Thus, it is further understood that an enhancer that operates at a distance from a promoter is therefore "operably linked" to the promoter, regardless of its position in the vector or its orientation relative to the promoter's location.

[0130] The terms "protein", "peptide" and "polypeptide" are used interchangeably and in their broadest sense refer to a compound of two or more subunit amino acids, amino acid analogs or peptide mimetics. The subunits may be linked by peptide bonds. On the other hand, the subunits may be linked by other bonds, such as esters, ethers, etc. A protein or peptide must contain at least two amino acids and there is no limit on the maximum number of amino acids, which may comprise, consist essentially of, or consist of the sequence of a protein or peptide. As used herein, the term "amino acid" refers to natural and / or non-natural or synthetic amino acids, including glycine and both D and L optical isomers, amino acid analogs and peptide mimetics.

[0131] As used herein, the term "signal peptide" or "signal polypeptide" means an amino acid sequence that is usually present at the N-terminus of a newly synthesized secretory or membrane polypeptide or protein. Its function is to guide the polypeptide to a specific cell location, for example, through a cell membrane, into a cell membrane, or into a nucleus. In an embodiment, the signal peptide is removed after positioning. Examples of signal peptides are well known in the art. Non-limiting examples are those described in U.S. Patent Nos. 8,853,381, 5,958,736, and 8,795,965. In an embodiment, the signal peptide can be an IDUA signal peptide.

[0132] When referring to a specific molecule, biological material or cell material, the term "equivalent" or "biological equivalent" is used interchangeably and is intended to have minimal homology while still maintaining those of the desired structure or function. Non-limiting examples of equivalent polypeptides include polypeptides having at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95% identity or at least about 99% identity with a reference polypeptide (e.g., a wild-type polypeptide); or polypeptides encoded by polynucleotides having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95% identity, at least about 97% sequence identity or at least about 99% sequence identity with a reference polynucleotide (e.g., a wild-type polynucleotide).

[0133] The term "homology" or "identity" or "similarity" refers to the sequence similarity between two peptides or between two nucleic acid molecules. Percent identity can be determined by comparing the positions in each sequence that can be aligned for comparison purposes. When a position in the compared sequences is occupied by the same base or amino acid, then the molecules are identical at that position. The degree of identity between sequences is a function of the number of matching positions shared by the sequences. "Unrelated" or "non-homologous" sequences share less than 40% identity, less than 25% identity with one of the sequences disclosed herein. The percent identity can be determined by importing the nucleic acid or amino acid sequences provided herein and using ClustalW (available in genome.jp / tools-bin / clustalw / The comparison and percentage sequence identity of the nucleic acid or amino acid sequence are determined using the ClustalW parameters for nucleic acid sequence alignment using the nucleic acid sequences found herein (obtained) and Gonnet (for proteins) weight matrices. In an embodiment, the ClustalW parameters for nucleic acid sequence alignment using the nucleic acid sequences found herein are generated using the ClustalW (for DNA) weight matrix.

[0134] As used herein, amino acid modifications can be substitutions, deletions or insertions. Amino acid substitutions can be conservative amino acid substitutions or non-conservative amino acid substitutions. Conservative substitutions (also referred to as conservative mutations, conservative substitutions or conservative variations) are amino acid substitutions in proteins that change a given amino acid into a different amino acid with similar biochemical properties (e.g., charge, hydrophobicity, or size). As used herein, "conservative variation" refers to the replacement of an amino acid residue by another biologically similar residue. Examples of conservative variations include replacing another hydrophobic residue with a hydrophobic residue such as isoleucine, valine, leucine, or methionine; or replacing another residue with a charged residue or polar residue, such as replacing lysine with arginine, replacing aspartic acid with glutamic acid, replacing asparagine with glutamine, etc. Other illustrative examples of conservative substitutions include the following changes: alanine to serine; asparagine to glutamine or histidine; aspartic acid to glutamic acid; cysteine ​​to serine; glycine to proline; histidine to asparagine or glutamine; lysine to arginine, glutamine or glutamic acid; phenylalanine to tyrosine, serine to threonine; threonine to serine; tryptophan to tyrosine; tyrosine to tryptophan or phenylalanine; and the like.

[0135] As used herein, the term "vector" refers to a nucleic acid that contains, consists essentially of, or consists of a complete replicon, so that the vector can be replicated when placed in a cell, for example, by transfection, infection, or transformation. It is understood in the art that once inside a cell, the vector can be replicated as an extrachromosomal (episomal) element or can be integrated into a host cell chromosome. The vector may include nucleic acids derived from retroviruses, adenoviruses, herpes viruses, baculoviruses, modified baculoviruses, papovaviruses, or otherwise modified naturally occurring viruses. Exemplary non-viral vectors for delivering nucleic acids include naked DNA; DNA complexed with cationic lipids alone or in combination with cationic polymers; anionic liposomes and cationic liposomes; DNA-protein complexes and particles that contain DNA condensed with cationic polymers, consisting essentially of or consisting of the DNA, the cationic polymers such as heterologous polylysine, oligopeptides of defined length, and polyethyleneimine, in some cases contained in liposomes; and the use of ternary complexes that contain viruses and polylysine-DNA, consisting essentially of or consisting of it.

[0136] In general, recombinant techniques are well known in the art, containing promoters and cloning sites that can be operably connected to polynucleotides. Such vectors can transcribe RNA in vitro or in vivo, and can be commercially available from sources such as Agilent Technologies (Santa Clara, Calif) and Promega Biotech (Madison, Wis.). In order to optimize expression and / or in vitro transcription, it may be necessary to remove, add or change the 5' and / or 3' non-translated portion of the cloned transgenic, to increase or reduce expression at the transcription or translation level to eliminate additional, potentially inappropriate alternative translation initiation codons or other sequences that may interfere. Alternatively, a common ribosome binding site can be directly inserted into the 5' of the start codon to enhance expression.

[0137] "Viral vector" is defined as a recombinantly produced virus or virus particle containing a polynucleotide to be delivered to a host cell in vivo, in vitro or in vitro. Examples of viral vectors include retroviral vectors, AAV viral vectors, lentiviral vectors, adenoviral vectors, alphaviral vectors, etc. Alphaviral vectors, such as vectors based on Semliki Forest virus and vectors based on Sindbis virus, have also been developed for gene therapy and immunotherapy. See, for example, Schlesinger and Dubensky (1999) Curr. Opin. Biotechnol. 5: 434-439, and Ying, et al. (1999) Nat. Med. 5 (7): 823-827.

[0138] As used herein, the term "recombinant expression system" or "recombinant vector" refers to one or more genetic constructs formed by recombination and used to express certain genetic materials.

[0139] "Gene delivery vector" is defined as any molecule that can carry an inserted polynucleotide into a host cell. Examples of gene delivery vectors are liposomes, micelles, biocompatible polymers, including natural polymers and synthetic polymers; lipoproteins; polypeptides; polysaccharides; lipopolysaccharides; artificial viral envelopes; metal particles; bacteria; viruses, such as baculovirus, adenovirus and retrovirus; phage, cosmids, plasmids and fungal vectors; and other recombinant vectors commonly used in the art, which have been described for expression in a variety of eukaryotic hosts and prokaryotic hosts, and can be used for gene therapy as well as simple protein expression. Liposomes that also contain, consist essentially of or consist of targeting antibodies or fragments thereof can be used in the methods disclosed herein. In addition to delivering polynucleotides to cells or cell populations, the proteins described herein can be directly introduced into cells or cell populations by non-limiting techniques of protein transfection, alternatively, culture conditions that can enhance the expression of proteins disclosed herein and / or promote their activity are other non-limiting techniques.

[0140] Gene delivery vectors can be used to deliver the polynucleotides disclosed herein to cells or tissues. "Gene delivery", "gene transfer", "transduction", etc. as used herein refer to the term of introducing exogenous polynucleotides (sometimes referred to as "transgenic") into host cells, regardless of the method used for introduction. Such methods include a variety of well-known techniques, such as vector-mediated gene transfer (by, for example, viral infection / transfection, or various other protein-based or lipid-based gene delivery complexes) and technologies (such as electroporation, "gene gun" delivery and various other technologies for introducing polynucleotides) that promote delivery of "naked" polynucleotides. The introduced polynucleotides can be stably or transiently maintained in the host cell. Stable maintenance generally requires that the introduced polynucleotides contain a replication origin compatible with the host cell or are integrated into the replicon of the host cell, such as an extrachromosomal replicon (e.g., plasmid) or a nuclear or mitochondrial chromosome. Many known vectors are capable of mediating the transfer of genes to mammalian cells, as known in the art and described herein.

[0141] "Plasmid" is a DNA molecule that is usually separated from chromosomal DNA and can replicate independently of chromosomal DNA. In many cases, it is circular and double-stranded. Plasmid provides a mechanism for horizontal gene transfer within a microbial population, and generally provides a selection advantage under a given environmental state. Plasmids may carry genes that provide resistance to naturally occurring antibiotics in competitive environmental niches, or alternatively, the protein produced may act as a toxin under similar circumstances. It is known in the art that although plasmid vectors are generally present as extrachromosomal circular DNA molecules, plasmid vectors can also be designed to be stably integrated into the host chromosome randomly or in a targeted manner, and such integration can use circular plasmids or plasmids that have been linearized before being introduced into the host cell.

[0142] "Plasmids" used in genetic engineering are called "plasmid vectors." Many plasmids are commercially available for such uses. The gene to be replicated is inserted into a copy of the plasmid that contains a gene that makes the cell resistant to a specific antibiotic, as well as a multiple cloning site (MCS, or polylinker), which is a short region containing several common restriction sites that allow easy insertion of DNA fragments at this location. Another major use of plasmids is to make large amounts of proteins. In this case, researchers culture bacterial or eukaryotic cells containing a plasmid carrying the gene of interest, which can induce the plasmid to produce large amounts of protein from the inserted gene.

[0143] In aspects where gene transfer is mediated by a DNA viral vector such as adenovirus (Ad) or adeno-associated virus (AAV), vector construct refers to a polynucleotide comprising, consisting essentially of, or consisting of: the viral genome or a portion thereof and a transgene.

[0144] The term "adeno-associated virus" or "AAV" as used herein refers to a member of the virus class associated with the name and belonging to the Parvoviridae family that depends on the genus Parvovirus. Adeno-associated virus is a single-stranded DNA virus that grows only in cells, where some functions are provided by co-infected helper viruses. General information and reviews of AAV can be found in, for example, Carter, 1989, Handbook of Parvoviruses, Vol. 1, pp. 169-228 and Berns, 1990, Virology, pp. 1743-1764, Raven Press, (New York). It is fully expected that the same principles described in these reviews will apply to other AAV serotypes after the publication date of the review, because it is well known that various serotypes are very closely related in structure and function, even at the genetic level. (See, for example, Blacklowe, 1988, Pages 165-174 of Parvoviruses and Human Disease, edited by JR Pattison; and Rose, Comprehensive Virology 3: 1-61 (1974)). For example, all AAV serotypes apparently exhibit very similar replication properties mediated by homologous replication genes; and all carry three related capsid proteins, such as those expressed in AAV2. The extent of the correlation is further indicated by heteroduplex analysis, which reveals extensive cross-hybridization between serotypes along the length of the genome; and the presence of similar self-annealing segments at the ends, which correspond to "inverted terminal repeat sequences" (ITRs). Similar infectivity patterns also indicate that the replication function of each serotype is under similar regulatory control. It is known that multiple serotypes of the virus are suitable for gene delivery; all known serotypes can infect cells from various tissue types. At least 11 consecutively numbered AAV serotypes are known in the art. Non-limiting exemplary serotypes that can be used for the methods disclosed herein include any of the 11 serotypes, such as AAV2, AAV8, AAV9, or variant serotypes, such as AAV-DJ and AAV PHP.B. AAV particles comprise, consist essentially of, or consist of three major viral proteins: VP1, VP2, and VP3. In embodiments, the AAV refers to serotype AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVPHP.B, or AAVrh74.

[0145] As used herein, "AAV vector" refers to a vector comprising one or more heterologous nucleic acid (HNA) sequences and one or more AAV reverse terminal repeats (ITRs). Such AAV vectors can be replicated when present in a functional host cell providing rep and cap gene products, and allow the nucleic acid between ITR and ITR to be packaged into infectious viral particles. In an embodiment, the AAV vector comprises a promoter, at least one nucleic acid that can encode at least one protein or RNA and / or an enhancer and / or terminator in the flanking ITR packaged in infectious AAV particles. The nucleic acid between ITR and ITR can be encapsidated into the AAV capsid, and the encapsidated portion of the nucleic acid can be referred to as an "AAV vector genome". The AAV vector can also include elements, such as antibiotic resistance genes or other elements known in the art, in addition to the encapsidated portion, which are contained in a plasmid for manufacturing purposes but are not packaged into AAV particles.

[0146] As used herein, the term "viral capsid" or "capsid" refers to the protein shell or outer shell of the virus particle. The function of the capsid is to encapsulate, protect, transport the viral genome and release it into the host cell. The capsid is usually composed of oligomeric structural subunits of proteins ("capsid proteins"). As used herein, the term "encapsidation" refers to being enclosed in a viral capsid. The viral capsid of AAV is composed of a mixture of three viral capsid proteins: VP1, VP2 and VP3. The mixture of VP1, VP2 and VP3 contains 60 monomers arranged in a T=1 icosahedral symmetry in a ratio of 1:1:10 (VP1:VP2:VP3) or 1:1:20 (VP1:VP2:VP3), as described in Sonntag F et al. (June 2010), “A viral assembly factor promotes AAV2 capsid formation in the nucleolus”. Proceedings of the National Academy of Sciences of the United States of America. 107(22):10220–5, and Rabinowitz JE, Samulski RJ (December 2000), “Building a better vector: the manipulation of AAV virions”. Virology. 278(2):301–8, each of which is incorporated herein by reference in its entirety.

[0147] "AAV virion" or "AAV viral particle" or "AAV viral vector" or "AAV vector particle" or "AAV particle" refers to a viral particle composed of at least one AAV capsid protein and an encapsidated AAV vector genome.

[0148] As used herein, the term "helper" referring to a virus or plasmid refers to a virus or plasmid for providing any of the AAV vector genomes disclosed herein for replication and packaging of the additional components necessary. The components encoded by the helper virus may include any gene required for virion assembly, encapsidation, genome replication and / or packaging. For example, a helper virus or plasmid may encode an enzyme for replication of the viral genome. Non-limiting examples of helper viruses and plasmids suitable for use with AAV constructs include pHELP (plasmid), adenovirus (virus) or herpes virus (virus). In an embodiment, the pHELP plasmid may be a pHELPK plasmid, in which an ampicillin expression cassette is exchanged with a kanamycin expression cassette; pHELPK has the sequence shown in SEQ ID NO:92.

[0149] As used herein, packaging cells (or helper cells) are cells used to produce viral vectors. Rep and Cap proteins provided in trans and adenovirus gene sequences from AAV replication are required to produce recombinant AAV viral vectors. In some aspects, packaging / helper cells contain plasmids stably incorporated into the genome of the cell. In other aspects, packaging cells can be transiently transfected. Typically, packaging cells are eukaryotic cells, such as mammalian cells or insect cells.

[0150] As used herein, reporter protein is a detectable protein that is operably connected to a promoter to measure the expression (e.g., tissue specificity and / or intensity) of a promoter. In some aspects, a reporter protein can be operably connected to a polypeptide. In some aspects, a reporter protein can be used to monitor the functional identification and characterization of DNA delivery methods, promoters and enhancer elements, translation and transcriptional regulation, mRNA processing and protein: protein interactions. Non-limiting examples of reporter proteins are beta-galactosidase; fluorescent proteins, such as green fluorescent protein (GFP) or red fluorescent protein (RFP); luciferase; glutathione S-transferase; and maltose binding protein.

[0151] A "pharmaceutical composition" is intended to comprise an active ingredient (such as a polypeptide, polynucleotide, antibody or viral vector) in combination with an inert or active carrier (such as a solid support) such that the composition is suitable for diagnostic or therapeutic use in vitro, in vivo or ex vivo.

[0152] As used herein, the term "pharmaceutically acceptable carrier" encompasses any standard pharmaceutical carrier, such as phosphate buffered saline solution, water and emulsions, such as oil / water or water / oil emulsions, and various types of wetting agents. The composition may also contain stabilizers and preservatives. For examples of carriers, stabilizers and adjuvants, see Martin (1975) Remington's Pharm. Sci., 15th Edition, (Mack Publ. Co., Easton).

[0153] A "subject" of diagnosis or treatment is a cell or an animal such as a mammal or a human. The subject is not limited to a particular species and includes non-human animals receiving diagnosis or treatment and those infected or animal models, including but not limited to apes, mice, rats, dogs, or rabbits, and other livestock, sports animals, or pets. In an embodiment, the subject is a human.

[0154] The term "tissue" as used herein refers to the tissue of a living or dead organism or any tissue derived from or designed to mimic a living or dead organism. The tissue may be healthy, sick and / or have a genetic mutation. Biological tissue may include any single tissue (e.g., a collection of cells that can be interconnected), or a group of tissues that constitute an organ or part or region of an organism. Tissue may include, be substantially composed of, or be composed of homogenous cell material, or it may be a composite structure, such as a composite structure found in a body region including a chest, which may include, for example, lung tissue, bone tissue, and / or muscle tissue. Exemplary tissues include, but are not limited to, tissues derived from liver, lung, thyroid, skin, pancreas, blood vessel, bladder, kidney, brain, bile duct tree, duodenum, abdominal aorta, iliac vein, heart, and intestine, including any combination thereof.

[0155] As used herein, "treating" or "treatment" of a disease in a subject refers to (1) preventing the occurrence of symptoms of a disease in a subject susceptible to or not yet showing symptoms of the disease; (2) inhibiting a disease or preventing its development; or (3) ameliorating or causing regression of a disease or symptoms of a disease. As understood in the art, "treatment" is a method for obtaining a beneficial or desired result (including a clinical result). For purposes of the present technology, a beneficial or desired result may include, but is not limited to, one or more relief or improvement of one or more symptoms, a reduction in the extent of a condition (including a disease), a stable (i.e., non-worsening) state of a condition (including a disease), a delay or mitigation of a condition (including a disease), progression, improvement or alleviation of a condition (including a disease), a state and relief (whether partial or complete), whether detectable or undetectable.

[0156] As used herein, the term "effective amount" is intended to mean an amount sufficient to achieve the desired effect. In the context of therapeutic or preventive applications, the effective amount will depend on the type and severity of the condition in question and the characteristics of the individual subject, such as overall health, age, sex, weight, and tolerance to the pharmaceutical composition. In the context of gene therapy, in an embodiment, an effective amount can be an amount sufficient to cause restoration of some or all of the function of a defective gene in a subject. In an embodiment, the effective amount of AAV viral particles is an amount sufficient to cause expression of a gene in a subject. A skilled technician will be able to determine an appropriate amount based on these and other factors.

[0157] In embodiments, the effective amount will depend on the size and nature of the application in question. It also depends on the nature and sensitivity of the target subject and the method used. A skilled artisan will be able to determine the effective amount based on these and other considerations. Depending on the embodiment, the effective amount may comprise, consist essentially of, or consist of one or more administrations of the composition.

[0158] As used herein, the term "administer" or "administration" is intended to mean the delivery of a substance to a subject such as an animal or a human. Administration can be performed continuously or intermittently in one dose throughout the course of treatment. The method of determining the most effective means of administration and dosage will vary with the composition used for therapy, the purpose of therapy, and the age, health status, or sex of the subject being treated. Single or multiple administrations may be performed, with dosage levels and patterns selected by the treating physician, or in the case of pets and other animals, by the treating veterinarian.

[0159] AAV structure and function

[0160] AAV is a replication-deficient parvovirus with a single-stranded DNA genome of about 4.7 kb in length, including two inverted terminal repeats (ITRs) of about 145 nucleotides. AAV has multiple serotypes. The nucleotide sequences of the genomes of AAV serotypes are known. For example, the complete genome of AAV-1 is provided in GenBank Accession No. NC_002077; the complete genome of AAV-2 is provided in GenBank Accession No. NC_001401 and Srivastava et al., J. Virol., 45:555-564 (1983); the complete genome of AAV-3 is provided in GenBank Accession No. NC_1829; the complete genome of AAV-4 is provided in GenBank Accession No. NC_001829; the AAV-5 genome is provided in GenBank Accession No. AF085716; AAV- The complete genome of AAV-6 is provided in GenBank Accession No. NC_001862; at least portions of the AAV-7 and AAV-8 genomes are provided in GenBank Accession Nos. AX753246 and AX753249, respectively; the AAV-9 genome is provided in Gao et al., J. Virol., 78:6381-6388 (2004); the AAV-10 genome is provided in Mol. Ther., 13(1):67-76 (2006); and the AAV-11 genome is provided in Virology, 330(2):375-383 (2004). The sequence of the AAV rh.74 genome is provided in U.S. Patent No. 9,434,928, which is incorporated herein by reference in its entirety. U.S. Patent No. 9,434,928 also provides the sequences of the capsid protein and the self-complementary genome. In one aspect, the genome is a self-complementary genome. Contained within the AAV ITR are cis-acting sequences that direct viral DNA replication (rep), encapsidation / packaging, and host cell chromosomal integration. Three AAV promoters (named p5, p19, and p40 for their relative map positions) drive expression of two AAV internal open reading frames encoding the rep and cap genes. The two rep promoters (p5 and p19) are combined with differential splicing of a single AAV intron (at nucleotides 2107 and 2227) to produce four rep proteins (rep 78, rep 68, rep 52, and rep 40) from the rep gene. Rep proteins have multiple enzymatic properties and are ultimately responsible for replicating the viral genome.

[0161] The Cap gene is expressed from the p40 promoter and encodes three capsid proteins, VP1, VP2 and VP3. Alternative splicing and non-shared translation start sites are responsible for the production of three related capsid proteins. More specifically, after the single mRNA that translates VP1, VP2 and VP3 proteins is transcribed, splicing can be performed in two different ways: longer or shorter introns can be excised, thereby forming two pools of mRNA: 2.3kb and 2.6kb long mRNA pools. Longer introns are generally preferred, and therefore the 2.3kb long mRNA can be called the major splice variant. This form lacks the first AUG codon, and the synthesis of VP1 protein starts from this codon, resulting in a decrease in the overall level of VP1 protein synthesis. The first AUG codon retained in the major splice variant is the start codon of the VP3 protein. However, there is an ACG sequence (encoding threonine) upstream of this codon in the same open reading frame, and the ACG sequence is surrounded by the optimal Kozak (translation start) context. This results in lower levels of synthesis of the VP2 protein, which is actually the VP3 protein with an additional N-terminal residue, like VP1, as described in Becerra SP et al. (December 1985). "Direct mapping of adeno-associated virus capsid proteins B and C: a possible ACG initiation codon". Proceedings of the National Academy of Sciences of the United States of America. 82(23): 7919–23, Cassinotti P et al., (November 1988), "Organization of the adeno-associated virus (AAV) capsid gene: mapping of a minor spliced ​​mRNA coding for virus capsid protein 1". Virology. 167(1): 176–84, Muralidhar S et al., (January 1994)."Site-directed mutagenesis of adeno-associated virus type 2 structural protein initiation codons: effects on regulation of synthesis and biological activity". Journal of Virology. 68(1):170–6, and Trempe JP, Carter BJ (September 1988). "Alternate mRNA splicing is required for synthesis of adeno-associated virus VP1 capsid protein". Journal of Virology. 62(9):3356–63, each of which is hereby incorporated by reference. A single common polyadenylation signal is located at map position 95 of the AAV genome. Muzyczka, Current Topics in Microbiology and Immunology, 158:97-129 (1992) reviewed the life cycle and genetics of AAV.

[0162] Each VP1 protein contains a VP1 portion, a VP2 portion, and a VP3 portion. The VP1 portion is the N-terminal portion of the VP1 protein, which is unique to the VP1 protein and corresponds to amino acids 1-137 of SEQ ID NO:164. The VP2 portion is the amino acid sequence present in the VP1 protein, which is also present in the N-terminal portion of the VP2 protein and corresponds to amino acids 138-202 of SEQ ID NO:164. The VP3 portion has the same sequence as the VP3 protein. The VP3 portion is the C-terminal portion of the VP1 protein, which is shared with the VP1 and VP2 proteins and corresponds to amino acids 203-737 of SEQ ID NO:164. See Figure 5 .

[0163] The VP3 protein can be further divided into discrete variable surface regions I-IX (VR-I-IX). Each variable surface region (VR) can comprise or contain a specific amino acid sequence, which alone or in combination with the specific amino acid sequence of each other VR can confer a unique infection phenotype (e.g., reduced antigenicity, improved transduction and / or tissue-specific tropism relative to other AAV serotypes) to a specific serotype, as described in DiMatta et al., "Structural Insight into the Unique Properties of Adeno-Associated Virus Serotype 9", J. Virol., Volume 86(12): 6947-6958, June 2012, the contents of which are incorporated herein by reference.

[0164] AAV has unique characteristics that make it attractive as a viral vector for delivering foreign DNA to cells, for example in gene therapy. Infection of cultured cells by AAV is non-cytopathic, while natural infection in humans and other animals is silent and asymptomatic. In addition, AAV infects many mammalian cells, allowing it to potentially target many different tissues in the body. In addition, AAV transduces slowly dividing and non-dividing cells and can persist essentially throughout the life of these cells as transcriptionally active nuclear episomes (extrachromosomal elements). The AAV proviral genome is inserted into a plasmid as cloned DNA, which makes the construction of recombinant genomes feasible. In addition, since the signals directing AAV replication and genome encapsidation are contained within the ITRs of the AAV genome, part or all of the internal approximately 4.3 kb of the genome (encoding replication and structural capsid proteins, rep-cap) may be replaced by foreign DNA to generate AAV vector genomes. The rep and cap proteins can be provided in trans. Another notable feature of AAV is that it is an extremely stable and robust virus. It can easily withstand the conditions used to inactivate adenovirus (56°C to 65°C for several hours), making refrigeration of AAV less critical. AAV can even be freeze-dried. Finally, AAV-infected cells are not resistant to repeated infection.

[0165] Several studies have demonstrated that recombinant AAV-mediated proteins can be expressed in muscle for a long time (>1.5 years). See Clark et al., Hum Gene Ther, 8:659-669 (1997); Kessler et al., Proc Nat. Acad Sc. USA, 93:14082-14087 (1996); and Xiao et al., J Virol, 70:8098-8108 (1996). See also Chao et al., Mol Ther, 2:619-623 (2000) and Chao et al., Mol Ther, 4:217-222 (2001). In addition, because muscle is highly vascularized, recombinant AAV transduction results in the appearance of transgene products in the systemic circulation after intramuscular injection, as described in Herzog et al., Proc Natl Acad Sci USA, 94:5804-5809 (1997) and Murphy et al., Proc Natl Acad Sci USA, 94:13921-13926 (1997). In addition, Lewis et al., J Virol, 76:8769-8775 (2002) demonstrated that skeletal muscle fibers have cytokines necessary for proper antibody glycosylation, folding and secretion, indicating that muscle is capable of stably expressing secreted protein therapeutics. The recombinant AAV (rAAV) genome of the present invention comprises, consists essentially of, or consists of a nucleic acid molecule encoding a therapeutic protein (e.g., CYP4V2, RS1, PDE6B, ABCA4, BEST1, OPA1 or OPA3) and one or more AAV ITRs flanking the nucleic acid molecule. The AAV DNA in the rAAV genome can be from any AAV serotype from which recombinant viruses can be derived, including but not limited to AAV serotypes AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10, AAV-11, AAV-12, AAV-13, AAV PHP.B, and AAV rh74. The production of pseudotyped rAAVs is disclosed, for example, in WO2001083692. Other types of rAAV variants, such as rAAVs with capsid mutations, are also contemplated. See, for example, Marsic et al., Molecular Therapy, 22(11):1900-1909(2014). The nucleotide sequences of the genomes of various AAV serotypes are known in the art.

[0166] AAV vector particles, capsid protein and AAV vector

[0167] Provided herein are AAV vector particles, AAV vectors, and capsid proteins that have desired tissue specificity and can be used to deliver a variety of therapeutic payloads, including nucleic acids and proteins that can be used to treat disease.

[0168] AAV capsid protein

[0169] The present disclosure provides AAV particles with the properties of high gene transfer efficiency and increased tissue tropism. AAV viral vector delivery currently relies on tissue targeting using serotype selection based on the natural tropism of the virus or by direct injection into the target tissue. However, many currently available AAV viral vectors are suboptimal for delivering genes to specific target sites.

[0170] The present disclosure provides AAV capsid protein sequences that confer high gene transfer efficiency and increased tissue specificity to AAV particles containing them. In embodiments, AAV particles containing such AAV capsid proteins are administered via a specific delivery route to achieve optimal delivery to a specific target site.

[0171] In embodiments, the VP1 capsid protein comprises any one of the amino acid sequences listed in Table 1, or a sequence having up to 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid mutations, deletions or additions compared to any one of the amino acid sequences listed in Table 1. In embodiments, up to 15 amino acids, up to 20 amino acids, up to 30 amino acids or up to 40 amino acids can be mutated, deleted or added compared to these sequences. In embodiments, the VP1 capsid protein is encoded by any one of the nucleic acid sequences listed in Table 1, or a sequence having up to 5, up to 10, up to 30 or up to 60 nucleotide changes compared to any one of the nucleic acid sequences listed in Table 1.

[0172] Table 1: VP1 capsid protein

[0173] Amino acid SEQ ID NO: NA SEQ ID NO: AAV capsid name 1 98 AAV 110 2 15 AAV 204 3 18 AAV 214 30 19 AAV 214A 31 20 AAV 214e 32 21 AAV 214e8 33 22 AAV 214e9 34 23 AAV 214e10 49 47 AAV ITB102_45 84 82 AAV 214AB 164 167 AAV 214-D5

[0174] In embodiments, the AAV VP1 protein comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 1-3, 30-34, 49, 84, or 164, or a sequence having up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids different from SEQ ID NO: 1-3, 30-34, 49, 84, or 164. Polynucleotides encoding these VP1 proteins are also provided. In embodiments, the polynucleotide encoding the VP1 protein comprises, consists essentially of, or consists of the sequence of SEQ ID NO: 15, 18-23, 47, 82, 98, or 167, or a sequence having up to 5, up to 10, or up to 30 nucleotide changes from SEQ ID NO: 15, 18-23, 47, 82, 98, or 167.

[0175] In an embodiment, the AAV capsid sequence is AAV-110 capsid protein (SEQ ID NO: 1), AAV204 capsid protein (SEQ ID NO: 2), AAV214 capsid protein (SEQ ID NO: 3) or AAV ITB102_45 capsid protein (SEQ ID NO: 49). In an embodiment, the AAV capsid protein is a variant of the AAV214 capsid protein. In an embodiment, the AAV capsid protein is AAV214A (SEQ ID NO: 30), AAV-214-AB (SEQ ID NO: 84), AAV214e (SEQ ID NO: 31), AAV214e8 (SEQ ID NO: 32), AAV214e9 (SEQ ID NO: 33), AAV214e10 (SEQ ID NO: 34) or AAV214-D5 (SEQ ID NO: 164). In an embodiment, the AAV capsid protein is AAV214-D5 (SEQ ID NO: 164).

[0176] In embodiments, the AAV capsid sequence is an AAV204 capsid protein (SEQ ID NO: 2), an AAV214 capsid protein (SEQ ID NO: 3), an AAV214-D5 capsid protein (SEQ ID NO: 164), or an AAV8 capsid protein (SEQ ID NO: 67).

[0177] Sequences of exemplary VP2 and VP3 proteins are provided in Tables 2 and 3. Given the VP2 and VP3 sequences, the VP1 portion can be determined by alignment with the complete VP1 protein sequence.

[0178] Table 2: VP2 capsid protein

[0179] Amino acid SEQ ID NO: name 35 214 36 214A 37 214e 38 214e8 39 214e9 40 214e10 85 214AB 50 ITB102_45 165 214-D5

[0180] In embodiments, the AAV VP2 protein comprises, consists essentially of, or consists of the amino acid sequence of any one of SEQ ID NOs: 35-40, 50, 85, and 165, or a sequence having up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids different from SEQ ID NOs: 35-40, 50, 85, or 165. In embodiments, the AAV VP2 protein comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 165, or a sequence having up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids different from SEQ ID NO: 165.

[0181] Polynucleotides encoding these VP2 proteins are also provided. In embodiments, the polynucleotide encoding the VP2 protein comprises, consists essentially of, or consists of the sequence of SEQ ID NO:47, or a sequence having up to 5, up to 10, or up to 30 nucleotide changes from SEQ ID NO:47. In embodiments, the polynucleotide encoding the VP2 protein comprises, consists essentially of, or consists of the sequence of SEQ ID NO:168, or a sequence having up to 5, up to 10, or up to 30 nucleotide changes from SEQ ID NO:168.

[0182] Exemplary nucleic acids for other capsid VP2 portions can be derived from the corresponding portion of the VP1 capsid protein nucleic acid.

[0183] Table 3: VP3 capsid protein

[0184] Amino acid SEQ ID NO: NA SEQ ID NO: AAV capsid name 17 16 204 41 24 214 42 25 214A 43 26 214e 44 27 214e8 45 28 214e9 46 29 214e10 86 83 214AB 51 48 ITB102_45 166 169 214-D5

[0185] The VP3 proteins of AAV214, AAV214e, AAV214e8, AAV214e9, and AAV214e10 have the same amino acid (SEQ ID NO:41) and nucleic acid (SEQ ID NO:24) sequences.

[0186] In embodiments, the AAV VP3 protein comprises, consists essentially of, or consists of an amino acid sequence of SEQ ID NO: 17, 41-46, 51, 86, or 166, or a sequence having up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids different from SEQ ID NO: 17, 41-46, 51, 86, or 166. In embodiments, the AAV VP3 protein comprises, consists essentially of, or consists of an amino acid sequence of SEQ ID NO: 166, or a sequence having up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids different from SEQ ID NO: 166.

[0187] Polynucleotides encoding these VP3 proteins are also provided. In embodiments, the polynucleotide encoding the protein comprises, consists essentially of, or consists of a sequence of SEQ ID NO: 16, 24-29, 48, 83, or 169, or a sequence having up to 5, up to 10, or up to 30 nucleotide changes from SEQ ID NO: 16, 24-29, 48, 83, or 169. In embodiments, the polynucleotide encoding the protein comprises, consists essentially of, or consists of a sequence of SEQ ID NO: 169, or a sequence having up to 5, up to 10, or up to 30 nucleotide changes from SEQ ID NO: 169.

[0188] In embodiments, the AAV capsid protein is a chimeric protein. In embodiments, the VP1, VP2, or VP3 portion of an AAV capsid protein disclosed herein can be replaced with a VP1, VP2, or VP3 portion from a different AAV capsid protein disclosed herein.

[0189] In an embodiment, an AAV capsid protein is provided herein, comprising a leucine residue at amino acid 129, an asparagine residue at amino acid 586, and a glutamic acid residue at amino acid 723, wherein the amino acid positions in the AAV capsid protein are numbered relative to the amino acid positions in the amino acid sequence of SEQ ID NO: 2. In some cases, the protein comprises the amino acid sequence of SEQ ID NO: 2. In other cases, these amino acids can be introduced into other capsid proteins.

[0190] In an embodiment, provided herein is an AAV VP1 capsid protein comprising a VP1 portion, a VP2 portion, and a VP3 portion, wherein the VP1 portion comprises a leucine (L) residue at amino acid 129, wherein the VP2 portion comprises a threonine (T) or asparagine (N) residue at amino acid 157 and a lysine (K) or serine (S) residue at amino acid 162, and wherein the VP3 portion comprises an asparagine (N) residue at amino acid 223, an alanine (A) residue at amino acid 224, a histidine (H) residue at amino acid 272, a threonine (T) residue at amino acid 410, a histidine (H) residue at amino acid 724, and a proline (P) residue at amino acid 734, wherein the amino acid positions in the AAV capsid protein are numbered relative to the amino acid positions in the amino acid sequence of SEQ ID NO: 3 (i.e., VP1 capsid subunit numbering).

[0191] In an embodiment, the VP1 portion further comprises an aspartic acid (D) or an alanine (A) residue at amino acid 24, wherein the amino acid positions in the AAV capsid protein are numbered relative to the amino acid positions in the amino acid sequence of SEQ ID NO: 3. In an embodiment, the VP2 portion further comprises one or more of the following: (i) a proline (P) residue at amino acid 148; (ii) an inserted arginine (R) residue at amino acid 152; (iii) an arginine (R) residue at amino acid 168; (iv) an isoleucine (I) residue at amino acid 189; (v) a serine (S) residue at amino acid 200, wherein the amino acid positions in the AAV capsid protein are numbered relative to the amino acid positions in the amino acid sequence of SEQ ID NO: 3.

[0192] In the embodiments, one or more variable regions I to IX (see Figure 5 ) can be removed and replaced with alternative regions. Suitable alternatives are identified in Table 6 below. The positions of these and the properties of additional alternatives can be identified by comparison with SEQ ID NO: 41, such as Figure 5 In an embodiment, one or more VRs may have an insertion of 1, 2, or 3 amino acids. In an embodiment, one or more VRs may have a deletion of 1, 2, or 3 amino acids.

[0193] Table 6: Variable Regions

[0194]

[0195] The present disclosure provides nucleic acids encoding any one of the AAV capsid proteins disclosed herein. The present disclosure also provides vectors comprising any one of the nucleic acids disclosed herein.

[0196] In an embodiment, the AAV is an AAV9 serotype. Alternative serotypes or modified capsid viruses can be used to optimize neuronal tropism. Alternative vectors include: modified AAV9 serotype vectors for higher neuronal tropism than standard AAV9, such as PHP.B, which uses the Cre-lox recombination system to identify neural targeting vectors. Alternatively, AAV9PHP.B has a modified amino acid 498 of VP1 from asparagine to lysine to reduce liver tropism. Additional variants of AAVrh74 that have mutated several amino acids can be used for very wide tissue tropism, including the brain.

[0197] AAV vectors

[0198] AAV vectors provide nucleic acids that become encapsidated into AAV vector particles, including elements involved in controlling the expression of nucleic acids in subjects, and ITRs that promote encapsidation. In an embodiment, the AAV vector disclosed herein comprises at least one heterologous nucleic acid (HNA) sequence that can effectively treat a disease or disorder when expressed in a subject's cells. In an embodiment, the HNA sequence comprises a transgene. In an embodiment, the AAV vector comprises at least one ITR sequence and at least one transgene. In an embodiment, the transgene encodes a therapeutic protein or a therapeutic RNA.

[0199] In an embodiment, the control of transgenic expression in a host cell can be regulated by regulatory elements contained in an AAV vector, including a promoter sequence and a polyadenylation signal. In an embodiment, the AAV vector can also encode a signal peptide. In an embodiment, the AAV vector has 5' and 3' inverted terminal repeats (ITRs). The 5' ITR is located upstream of the promoter, and the promoter is located upstream of the transgenic. In an embodiment, the 5' and 3' ITRs have the same sequence. In an embodiment, they have different sequences. In an embodiment, the AAV vector of the present disclosure may include a first (5') ITR, a promoter, a transgenic, a polyadenylation signal, and a second (3') ITR in the 5' to 3' direction.

[0200] In embodiments, the 5'ITR comprises, consists essentially of, or consists of a nucleic acid sequence at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 253. In embodiments, the 3'ITR comprises, consists essentially of, or consists of a nucleic acid sequence at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 254. In embodiments, the corresponding AAV vector is used to express an Opa1 transgene.

[0201] In embodiments, the 5’ ITR comprises, consists essentially of, or consists of a nucleic acid sequence having at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 255. In embodiments, the 3’ ITR comprises, consists essentially of, or consists of a nucleic acid sequence having at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 256. In embodiments, the corresponding AAV vector is used to express the RS1 transgene.

[0202] Additional descriptions of ITRs can be found, for example, in McCarty et al., Gene Ther. December 2003;10(26):2112-8, the content of which is incorporated herein by reference in its entirety.

[0203] In embodiments, the HNA (e.g., HNA comprising a transgene) is operably linked to a promoter.

[0204] In embodiments, the HNA is operably linked to a constitutive promoter. The constitutive promoter can be any constitutive promoter known in the art and / or provided herein. In embodiments, the constitutive promoter comprises, consists essentially of, or consists of: Rous sarcoma virus (RSV) LTR promoter (optionally with RSV enhancer), cytomegalovirus (CMV) promoter, SV40 promoter, dihydrofolate reductase promoter, β-actin promoter, phosphoglycerate kinase (PGK) promoter, U6 promoter, H1 promoter, hybrid chicken β-actin promoter, MeCP2 promoter, H1 promoter, U1a promoter, mMeP418 promoter, mMeP426 promoter, minimal MeCP2 promoter, CAG promoter, or EF1 promoter. The nucleotide sequences of such promoters known in the art can be modified to increase or decrease the efficiency of mRNA transcription. See, for example, Gao et al. (2018) Mol. Ther.: Nucleic Acids 12:135-145 (modifying the TATA box of 7SK, U6, and H1 promoters to abolish RNA polymerase III transcription and stimulate RNA polymerase II-dependent mRNA transcription). In embodiments, the HNA sequence is operably linked to a tissue-specific control promoter or an inducible promoter. In embodiments, the tissue-specific control promoter is a central nervous system (CNS) cell-specific promoter, lung-specific promoter, skin-specific promoter, muscle-specific promoter, liver-specific promoter, eye-specific promoter (e.g., VMD2 or mRho promoter).

[0205] In embodiments, the promoter may comprise, consist essentially of, or consist of a polynucleotide having a sequence of SEQ ID NO:96 (mouse U1 promoter) or SEQ ID NO:97 (H1 promoter). In embodiments, the promoter is a U1a or U1b promoter, an EF1 promoter, or CBA (chicken beta-actin). In embodiments, the promoter may comprise, consist essentially of, or consist of any one of the nucleic acid sequences listed in Table 5, or a sequence having up to 5, up to 10, up to 20, or up to 30 nucleotide changes with any one of the nucleic acid sequences listed in Table 5. In embodiments, the promoter may comprise, consist essentially of, or consist of a nucleic acid sequence having at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identity with any one of the nucleic acid sequences listed in Table 5.

[0206] In embodiments, the promoter is a chicken beta-actin hybrid (CBh) promoter. In embodiments, the CBh promoter comprises, consists essentially of, or consists of a nucleic acid sequence having at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to SEQ ID NO: 154.

[0207] In embodiments, the promoter is a rhodopsin kinase (RK) promoter. In embodiments, the RK promoter comprises, consists essentially of, or consists of a nucleic acid sequence having at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to SEQ ID NO: 196.

[0208] In embodiments, the promoter is a Rho promoter.In embodiments, the Rho promoter comprises, consists essentially of, or consists of a nucleic acid sequence having at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to SEQ ID NO: 197.

[0209] In embodiments, the promoter is a PDE promoter.In embodiments, the PDE promoter comprises, consists essentially of, or consists of a nucleic acid sequence that is at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 198.

[0210] Table 5: Non-limiting examples of promoters

[0211]

[0212]

[0213] In an embodiment, the AAV vector comprises an enhancer. In an embodiment, the enhancer is operably linked to the HNA sequence. In an embodiment, the enhancer is located upstream of the promoter. In an embodiment, the enhancer is located immediately upstream of the promoter without any additional nucleotides in between.

[0214] In embodiments, the enhancer is an inter-photoreceptor retinoic acid binding protein (IRBP) enhancer. In embodiments, the IRBP enhancer comprises, consists essentially of, or consists of a nucleic acid sequence that is at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 199.

[0215] In an embodiment, the HNA sequence is operably linked to an additional regulatory element. The additional regulatory element can be a woodchuck hepatitis virus post-transcriptional regulatory element ("WPRE"). In an embodiment, the AAV vector can include regulatory components suitable for growth and cultivation of the vector in a bacterial host for the purpose of vector production. For example, the vector can include genes for antibiotic resistance and genes for maintenance of plasmids in bacteria, as well as related regulatory elements for controlling protein expression in bacteria.

[0216] In an embodiment, the HNA sequence is operably linked to a polyadenylation signal. In an embodiment, the polyadenylation signal comprises, consists essentially of, or consists of a MeCP2 polyadenylation signal, a retinol dehydrogenase 1 (RDH1) polyadenylation signal, a bovine growth hormone (BGH) polyadenylation signal, an SV40 polyadenylation signal, a SPA49 polyadenylation signal, a sNRP-TK65 polyadenylation signal, a sNRP polyadenylation signal, or a TK65 polyadenylation signal. Exemplary SPA49 polyadenylation signals are described in Ostedgaard et al., Proc. Nat'l Acad. Sci. USA (February 22, 2005) 102: 2952-2957, which is incorporated herein by reference. In embodiments, the polyadenylation signal sequence comprises, consists essentially of, or consists of a nucleic acid sequence that is at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 201. In embodiments, the polyadenylation signal sequence comprises, consists essentially of, or consists of a nucleic acid sequence that is at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 225.

[0217] In an embodiment, an intron is inserted between a promoter and an HNA. In an embodiment, an intron comprises, consists essentially of, or consists of a nucleic acid sequence having at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to SEQ ID NO: 200. In an embodiment, an intron comprises, consists essentially of, or consists of a nucleic acid sequence having at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to SEQ ID NO: 222. In an embodiment, an intron comprises, consists essentially of, or consists of a nucleic acid sequence having at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to SEQ ID NO: 227. In embodiments, the AAV vector genome comprises a CBA sequence located immediately upstream of an intron sequence without any additional nucleotides therebetween, and wherein the CBA sequence comprises, consists essentially of, or consists of the nucleic acid sequence SEQ ID NO: 229, or a sequence thereof having at most 10, at most 9, at most 8, at most 7, at most 6, at most 5, at most 4, at most 3, at most 2, or at most 1 mutation. In embodiments, the intron comprises, consists essentially of, or consists of a nucleic acid sequence having at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 226. In embodiments, the intron does not comprise the polynucleotide sequence "ATG".

[0218] In embodiments, the first telomeric repeat sequence is inserted between the polyadenylation signal and the 3'ITR. In embodiments, the telomeric repeat sequence comprises a repeating unit of CCCTAA (SEQ ID NO: 217). In embodiments, the telomeric repeat sequence comprises an intermediate repeat sequence comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more consecutive copies of a repeating unit of CCCTAA (SEQ ID NO: 217). In embodiments, the telomeric repeat sequence comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more copies of the intermediate repeat sequence. In embodiments, the copies of the intermediate repeat sequence are separated by a spacer comprising TTTTT (SEQ ID NO: 218). In embodiments, the first telomeric repeat sequence comprises, consists essentially of, or consists of a nucleic acid sequence having at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to SEQ ID NO: 202.

[0219] In embodiments, the first telomeric repeat sequence is inserted between the polyadenylation signal and the 3'ITR. In embodiments, the telomeric repeat sequence comprises a repeating unit of TTAGGG (SEQ ID NO: 219). In embodiments, the telomeric repeat sequence comprises an intermediate repeat sequence comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more consecutive copies of a repeating unit of TTAGGG (SEQ ID NO: 219). In embodiments, the telomeric repeat sequence comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more copies of the intermediate repeat sequence. In embodiments, the copies of the intermediate repeat sequence are separated by a spacer comprising AAAAA (SEQ ID NO: 220). In embodiments, the first telomeric repeat sequence comprises, consists essentially of, or consists of a nucleic acid sequence having at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to SEQ ID NO: 203.

[0220] In an embodiment, the second telomeric repeat sequence is inserted between the 5'ITR and the promoter. In an embodiment, the telomeric repeat sequence comprises a repeating unit of TTAGGG (SEQ ID NO: 219). In an embodiment, the telomeric repeat sequence comprises an intermediate repeat sequence, which comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more consecutive copies of a repeating unit of TTAGGG (SEQ ID NO: 219). In an embodiment, the telomeric repeat sequence comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more copies of the intermediate repeat sequence. In an embodiment, the copies of the intermediate repeat sequence are separated by a spacer comprising AAAAA (SEQ ID NO: 220). In an embodiment, the second telomeric repeat sequence comprises, consists essentially of, or consists of a nucleic acid sequence having at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to SEQ ID NO: 203.

[0221] In embodiments, a human β-globin scaffold / matrix attachment region (βGlo_s / MAR) sequence is inserted between the polyadenylation signal and the downstream telomeric repeat sequence, or between the polyadenylation signal and the downstream 3'ITR. In embodiments, the βGlo_s / MAR sequence comprises, consists essentially of, or consists of a nucleic acid sequence having at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to SEQ ID NO: 221.

[0222] Heterologous Nucleic Acid (HNA)

[0223] The AAV viral vectors disclosed herein are infected with one or more heterologous nucleic acids (HNA) and delivered to a target tissue. In embodiments, the HNA sequence is transcribed and optionally translated in cells of the target tissue.

[0224] In some cases, HNA encodes antisense RNA, microRNA, siRNA or guide RNA (gRNA). CRISPR technology has been used to target the genome of living cells for modification. Cas9 protein is a large enzyme that must be effectively delivered to target tissues and cells to mediate gene repair by the CRISPR system, and there are many shortcomings in the current CRISPR / Cas9 gene correction scheme. The long-term expression of Cas9 can trigger a host immune response. Due to packaging restrictions, additional guide RNA may be delivered via a separate vector. In an embodiment, HNA encodes Cas9 protein or its equivalent.

[0225] In an embodiment, the HNA comprises a transgene encoding a protein that can be expressed in a subject's cells to treat a disease or condition caused by reduced or eliminated activity of a native protein. Thus, in an embodiment, the transgene can encode a protein selected from the group consisting of cystic fibrosis transmembrane conductance regulator (CFTR), N-acetyl-α-glucosaminidase (NAGLU), N-sulfoglucosamine sulfohydrolase (SGSH), palmitoyl-protein thioesterase 1 (PPT1), survival of motor neuron 1, telomere (SMN1), alkaline phosphatase, biomineralization-related (ALPL, also known as TNALP), glial cell line-derived neurotrophic factor (GDNF), glucosylceramidase β (GBA1), iduronidase α-L- (IDUA), methyl-CpG binding protein 2 (MeCP2), ceroid lipofuscinosis (ceroid lipofuscinosis), CLN1, rhodopsin (Rho), cytochrome P450 family 4 subfamily V member 2 (CYP4V2), retinol 1 (RS1), phosphodiesterase 6B (PDE6B), ATP-binding cassette subfamily A member 4 (ABCA4), benzygote-like maculopathy protein-1 (BEST1), OPA1 mitochondrial dynamin-like GTPase (OPA1), and optic atrophy 3 (OPA3).

[0226] In embodiments, the transgene encodes cytochrome P450 family 4 subfamily V member 2 (CYP4V2). In embodiments, CYP4V2 comprises a mutant sequence, a codon optimized sequence and / or a truncated sequence of CYP4V2. In embodiments, CYP4V2 comprises, consists essentially of, or consists of a nucleic acid having a sequence of SEQ ID NO: 116, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or at least 99.5% identity to SEQ ID NO: 116. In embodiments, CYP4V2 encodes a protein comprising, consists essentially of, or consists of an amino acid sequence of SEQ ID NO: 142, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or at least 99.5% identity to SEQ ID NO: 142. In embodiments, the AAV vector or AAV vector genome of the present disclosure encodes CYP4V2 and is used to treat Bietti crystal dystrophy.

[0227] In embodiments, the transgene encodes retinoschisis 1 (RS1). In embodiments, the RS1 transgene comprises a mutant sequence, a codon-optimized sequence, and / or a truncated sequence of RS1. In embodiments, RS1 comprises, consists essentially of, or consists of a nucleic acid having a sequence of SEQ ID NO: 117, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or at least 99.5% identity to SEQ ID NO: 117. In embodiments, RS1 encodes a protein comprising, consisting essentially of, or consisting of an amino acid sequence of SEQ ID NO: 143, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or at least 99.5% identity to SEQ ID NO: 143. In embodiments, the AAV vector or AAV vector genome of the present disclosure encodes RS1 and is used to treat retinoschisis.

[0228] In embodiments, the transgene encodes phosphodiesterase 6B (PDE6B). In embodiments, the PDE6B transgene comprises a mutant sequence, a codon optimized sequence and / or a truncated sequence of PDE6B. In embodiments, RS1 comprises, consists essentially of, or consists of a nucleic acid having a sequence of SEQ ID NO: 118, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or at least 99.5% identity to SEQ ID NO: 118. In embodiments, PDE6B encodes a protein comprising, consists essentially of, or consists of an amino acid sequence of SEQ ID NO: 144, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or at least 99.5% identity to SEQ ID NO: 144. In embodiments, the AAV vector or AAV vector genome of the present disclosure encodes PDE6B and is used to treat retinitis pigmentosa.

[0229] In embodiments, the transgene encodes ATP-binding cassette subfamily A member 4 (ABCA4). In embodiments, the ABCA4 transgene comprises a mutant sequence, a codon-optimized sequence, and / or a truncated sequence of ABCA4. In embodiments, ABCA4 comprises, consists essentially of, or consists of a nucleic acid having a sequence of SEQ ID NO: 172, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or at least 99.5% identity to SEQ ID NO: 172. In embodiments, ABCA4 encodes a protein comprising, consists essentially of, or consists of an amino acid sequence of SEQ ID NO: 177, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or at least 99.5% identity to SEQ ID NO: 177. In embodiments, the AAV vector or AAV vector genome of the present disclosure encodes ABCA4 and is used to treat Stargardt's disease.

[0230] In embodiments, the transgene encodes BEST1 (BEST1). In embodiments, the BEST1 transgene comprises a mutant sequence, a codon-optimized sequence, and / or a truncated sequence of BEST1. In embodiments, BEST1 comprises, consists essentially of, or consists of a nucleic acid having a sequence of SEQ ID NO: 173 or 174, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or at least 99.5% identity to SEQ ID NO: 173 or 174. In embodiments, BEST1 encodes a protein comprising, consists essentially of, or consists of an amino acid sequence of SEQ ID NO: 178 or 179, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or at least 99.5% identity to SEQ ID NO: 178 or 179. In an embodiment, the AAV vector or AAV vector genome of the present disclosure encodes BEST1 and is used to treat Best's vitelliform macular dystrophy.

[0231] In embodiments, the transgene encodes an OPA1 mitochondrial dynamin-like GTPase (OPA1). In embodiments, the OPA1 transgene comprises a mutant sequence, a codon-optimized sequence, and / or a truncated sequence of OPA1. In embodiments, the OPA1 transgene comprises, consists essentially of, or consists of a nucleic acid having a sequence of SEQ ID NO: 175, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or at least 99.5% identity to SEQ ID NO: 175. In embodiments, the OPA1 transgene encodes a protein comprising, consists essentially of, or consists of an amino acid sequence of SEQ ID NO: 180, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or at least 99.5% identity to SEQ ID NO: 180. In embodiments, the OPA1 transgene is a ΔS1 (ΔS1) isoform comprising, consisting essentially of, or consisting of a nucleic acid having a sequence of SEQ ID NO: 182, or a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to SEQ ID NO: 182. In embodiments, the OPA1 transgene is a ΔS1 isoform encoding a protein comprising, consisting essentially of, or consisting of an amino acid sequence of SEQ ID NO: 183, or a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to SEQ ID NO: 183. In embodiments, the OPA1 transgene is an E5b isoform comprising, consisting essentially of, or consisting of a nucleic acid having a sequence of SEQ ID NO: 184, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or at least 99.5% identity to SEQ ID NO: 184. In embodiments, OPA1 is an E5b isoform of the transgene encoding a protein comprising, consisting essentially of, or consisting of an amino acid sequence of SEQ ID NO: 185, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or at least 99.5% identity to SEQ ID NO: 185. In embodiments, the AAV vector or AAV vector genome of the present disclosure encodes OPA1 and is used to treat dominant optic atrophy.

[0232] In embodiments, the transgene encodes optic atrophy 3 (OPA3). In embodiments, the OPA3 transgene comprises a mutant sequence, a codon-optimized sequence, and / or a truncated sequence of OPA3. In embodiments, OPA3 comprises, consists essentially of, or consists of a nucleic acid having a sequence of SEQ ID NO: 176, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or at least 99.5% identity to SEQ ID NO: 176. In embodiments, OPA3 encodes a protein comprising, consists essentially of, or consists of an amino acid sequence of SEQ ID NO: 181, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or at least 99.5% identity to SEQ ID NO: 181. In embodiments, the AAV vector or AAV vector genome of the present disclosure encodes OPA3 and is used to treat dominant optic atrophy.

[0233] In embodiments, the transgene comprises any one of the nucleic acid sequences listed in Table 4, or a sequence having up to 5, up to 10, or up to 30 nucleotide changes from any one of the DNA sequences in Table 4 (SEQ ID NOs: 116-118 and 172-176). In embodiments, the transgene encodes any one of the amino acid sequences listed in Table 4, or a sequence having up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids that differ from any one of the amino acid sequences listed in Table 4 (SEQ ID NOs: 142-144 and 177-118).

[0234] Table 4: Non-limiting examples of transgenics

[0235]

[0236] In embodiments, the transgene comprises a nucleic acid sequence as set forth in any one of SEQ ID NOs: 99-133 and 172-176, or a sequence having up to 5, up to 10, or up to 30 nucleotide changes from SEQ ID NOs: 99-133 and 172-176. In embodiments, the transgene encodes an amino acid sequence as set forth in any one of SEQ ID NOs: 134-151 and 177-181, or a sequence having up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids that differ from the amino acid sequence of any one of SEQ ID NOs: 134-151 and 177-181.

[0237] In embodiments, the heterologous nucleic acid encodes a reporter protein; for example, a fluorescent protein.

[0238] Methods for producing AAV viral vectors

[0239] AAV viral vectors can be produced using a variety of methods. In an embodiment, packaging is achieved by using a helper virus or a helper plasmid and a cell line. The helper virus or helper plasmid contains elements and sequences that promote the production of viral vectors. In another aspect, the helper plasmid is stably incorporated into the genome of the packaging cell line so that the packaging cell line does not need to be additionally transfected with a helper plasmid.

[0240] In an embodiment, the cell is a packaging cell line or a helper cell line. In an embodiment, the helper cell line is a eukaryotic cell; for example, a HEK 293 cell or a 293T cell. In an embodiment, the helper cell is a yeast cell or an insect cell.

[0241] In an embodiment, the cell comprises a nucleic acid encoding a tetracycline activator protein; and a promoter that regulates the expression of the tetracycline activator protein. In an embodiment, the promoter that regulates the expression of the tetracycline activator protein is a constitutive promoter. In an embodiment, the promoter is a phosphoglycerate kinase promoter (PGK) or a CMV promoter.

[0242] The helper plasmid can comprise, for example, at least one viral helper DNA sequence derived from a replication-incompetent viral genome, which trans-encodes all virion proteins required for packaging replication-incompetent AAV and is used to produce virion proteins capable of packaging replication-incompetent AAV at high titers, but does not produce replication-competent AAV.

[0243] Helper plasmids for packaging AAV are known in the art, see, e.g., U.S. Patent Publication No. 2004 / 0235174A1, which is incorporated herein by reference. As described therein, the AAV helper plasmid may contain, as a non-limiting example, the Ad5 genes E2A, E4, and VA as helper viral DNA sequences, controlled by their respective native promoters or heterologous promoters. The AAV helper plasmid may also contain an expression cassette for the expression of a marker protein (such as a fluorescent protein) to allow simple detection of transfection of desired target cells.

[0244] The present disclosure provides a method for producing AAV particles, comprising transfecting a packaging cell line with any AAV helper plasmid disclosed herein; and any AAV vector disclosed herein. In an embodiment, the AAV helper plasmid and the AAV vector are co-transfected into the packaging cell line. In an embodiment, the cell line is a mammalian cell line, such as a human embryonic kidney (HEK) 293 cell line. The present disclosure provides a cell comprising any of the AAV vectors and / or AAV particles disclosed herein.

[0245] Pharmaceutical composition

[0246] The present disclosure provides pharmaceutical compositions that comprise any one of the AAV vectors, AAV capsids, and / or AAV particles described herein. Typically, AAV particles are administered for therapy.

[0247] The pharmaceutical compositions described herein can be formulated by any method known or developed in the art of pharmacology, including but not limited to contacting the active ingredient (e.g., viral particles or AAV vectors) with excipients or other auxiliary components, and dividing or packaging the product into dosage units. The viral particles of the present disclosure can be formulated to have desired characteristics, such as increased stability, increased cell transfection, sustained or delayed release, biodistribution or tropism, regulation or enhancement of translation in vivo of the encoded protein, and release profile of the encoded protein in vivo.

[0248] Accordingly, the pharmaceutical compositions can further comprise saline, lipids, liposomes, lipid nanoparticles, polymers, lipid complexes, core-shell nanoparticles, peptides, proteins, cells transfected with an AAV vector or transduced with AAV viral particles (e.g., for transplantation into a subject), nanoparticle mimics, or combinations thereof. In embodiments, the pharmaceutical compositions are formulated as nanoparticles. In embodiments, the nanoparticles are self-assembling nucleic acid nanoparticles.

[0249] The pharmaceutical compositions according to the present disclosure can be prepared, packaged, and / or sold as a single unit dose and / or as multiple single unit doses in bulk. The amount of the active ingredient generally equals the dose of the active ingredient administered to a subject and / or a convenient fraction of this dose, such as, for example, one-half or one-third of this dose. The formulations of the present invention can comprise one or more excipients, each in an amount that together increases the stability of the viral vector, increases cell transfection or transduction by the viral vector, increases the expression of the protein encoded by the viral vector, and / or alters the release profile of the protein encoded by the viral vector. In embodiments, the pharmaceutical composition comprises an excipient. Non-limiting examples of excipients include solvents, dispersion media, diluents, or other liquid vehicles, dispersing or suspending aids, surfactants, isotonic agents, thickening or emulsifying agents, preservatives, or combinations thereof.

[0250] In embodiments, the pharmaceutical composition comprises a cryoprotectant. The term "cryoprotectant" refers to an agent that can reduce or eliminate damage to a substance during freezing. Non-limiting examples of cryoprotectants include sucrose, trehalose, lactose, glycerol, dextran, raffinose, and / or mannitol.

[0251] Methods of Treatment

[0252] The present disclosure provides methods of preventing or treating a disorder, comprising administering to a subject a therapeutically effective amount of any one of the pharmaceutical compositions disclosed herein, consisting essentially of, or consisting of the same.

[0253] In embodiments, the disorder is a CNS disorder, a skin disorder, a lung disorder, a muscle disorder, a liver disorder, or an ophthalmic disease (or a retinal disease). In embodiments, the disorder is cystic fibrosis. In embodiments, the disorder is an ophthalmic disease. In embodiments, the disorder is a retinal disease.

[0254] In embodiments, the disorder is hypophosphatasia, amyotrophic lateral sclerosis (ALS), spinal muscular atrophy (SMA), recessive dystrophic epidermolysis bullosa (RDEB), lysosomal storage diseases (including Duchenne muscular dystrophy and Becker muscular dystrophy), juvenile Batten disease, infantile Batten disease, autosomal dominant genetic diseases, muscular dystrophy, Bietti crystal dystrophy, retinoschisis (e.g., degenerative, hereditary, tractional, exudative), hemophilia A, hemophilia B, multiple sclerosis, diabetes, Fabry disease, Pompe disease, neuronal ceroid lipofuscinosis 1 (CLN1), CLN3 disease (or juvenile neuronal ceroid lipofuscinosis), Gaucher disease, cancer, arthritis, muscular dystrophy, heart disease, intimal hyperplasia, Rett syndrome, epilepsy, Huntington disease, Parkinson disease, Alzheimer disease, autoimmune disease, cystic fibrosis, thalassemia, Hurler syndrome (MPS IH), Sly syndrome, Scheie syndrome, Hurler-Scheie syndrome, Hunter syndrome, Sanfilippo syndrome A (mucopolysaccharidosis IIIA or MPS IIIA), Sanfilippo syndrome B (mucopolysaccharidosis IIIB or MPS IIIB), Sanfilippo syndrome C, Sanfilippo syndrome D, Morquio syndrome, Maroteaux-Lamy syndrome, Krabbe disease, phenylketonuria, spinal cerebral ataxia, LDL receptor deficiency, hyperammonemia, anemia, arthritis, or adenosine deaminase deficiency.

[0255] In an embodiment, the disease is X-linked retinoschisis (XLRS), a rare monogenic disease causing severe visual impairment. Although female carriers are asymptomatic, affected males usually begin to show disease symptoms within the first decade, and occasionally show disease symptoms in infancy. The disease is caused by a mutation in the RS1 gene, which is expressed in photoreceptors and retinal bipolar cells. The gene product is a secreted protein that is mainly located in the inner segment of photoreceptors and more widely located in the rest of the neural retina. RS1 forms a homologous octamer complex, and is considered to mediate cell-to-cell adhesion via interactions with the extracellular epitopes of membrane proteins. In individuals suffering from XLRS, holes are formed where the adhesion of adjacent retinal layers is destroyed, typically in the outer plexiform layer of the synapses of photoreceptors and inner retinal neurons. This can cause discontinuity, photoreceptor degeneration and impaired visual acuity in the retinal circuit. Many RS1 mutations reported in the literature are point mutations, which are expected to destroy the secretion of protein, and are therefore functionally equivalent to null alleles. The current standard of care for XLRS patients is palliative and involves correction of refractive error, low vision assistance, and genetic counseling. Complications such as retinal detachment (up to 22% of patients) and vitreous hemorrhage (up to 40% of patients) are most common in the late stages of the disease and can be treated surgically. Early intervention via gene therapy has great potential to reverse or stabilize disease progression in the early stages of the disease and prevent severe vision loss and the occurrence of these more serious complications.

[0256] In an embodiment, the disease is autosomal dominant optic atrophy (ADOA). ADOA is caused by Opa1 mutations, leading to vision loss in the second to third decade of life. In an embodiment, Opa1 homozygous mutants are embryonic lethal and therefore cannot survive beyond E9-12. Heterozygous (HT) animals can survive to term, but have retinal degeneration, neurological defects and musculoskeletal complications over time. Mice show optic atrophy in fundus and scanning laser ophthalmoscopy (SLO) examinations, as well as reduced electroretinogram (ERG) amplitude, and fibrosis of the inner limiting membrane (ILM) and retinal nerve fiber layer (RNFL). Opa1 has been shown to be involved in mitochondrial cristae structure, mitochondrial fusion and mitochondrial inner membrane remodeling.

[0257] In an embodiment, the present disclosure provides a method of expressing a transgene in a retinal cell. In an embodiment, the method comprises delivering a nucleic acid of the present disclosure to a retinal cell. In an embodiment, the method comprises transducing a retinal cell with an AAV viral vector of the present disclosure.

[0258] In embodiments, target cells of the present disclosure include retinal cells. In embodiments, retinal cells include photoreceptors, bipolar cells, retinal ganglion cells, horizontal cells, or amacrine cells. In embodiments, retinal cells include retinal ganglion cells. In embodiments, retinal cells include bipolar cells. In embodiments, retinal cells include horizontal cells. In embodiments, retinal cells include amacrine cells. In embodiments, retinal cells include photoreceptors. In embodiments, photoreceptors include rods and / or cones.

[0259] In embodiments, the target cells of the present disclosure comprise, consist essentially of, or consist of photoreceptor cells. In embodiments, the transgenes of the present disclosure are operably linked to an RK promoter for selective expression in photoreceptor cells.

[0260] In addition to the specific transgenes disclosed herein, known active enzyme sequences can be used as transgenes to deliver functional enzyme activity.

[0261] In an embodiment, the disorder is CLN3 disease. CLN3 disease or juvenile neuronal ceroid lipofuscinosis is a lysosomal storage disease caused by an autosomal recessive mutation in the CLN3 gene. CLN3 disease is a progressive neurodegenerative disorder in which the central nervous system (CNS) is greatly affected, leading to behavioral problems, vision loss, and other cognitive impairments.

[0262] In an embodiment, the disorder is Fabry disease. Fabry disease is an X-linked lysosomal storage disease caused by a defect in the activity of alpha galactosidase A (GLA) that results in the accumulation of glycolipid products, triacylceramide (globotriaosylceramide) (Gb3) and lyso-Gb3 in lysosomes. The disease manifestations are highly heterogeneous, but typically include frequent episodes of peripheral neurotrophic pain, angiokeratoma, reduced sweating, corneal dystrophy and gastrointestinal complications. As the disease progresses, patients will experience cardiomyopathy, renal insufficiency and cerebrovascular disease, all of which are the main causes of shortened life spans in patients with Fabry disease. Although men are the most severely affected group among patients with mutations in the GLA gene, it is becoming increasingly apparent that female patients also often experience symptoms, but are often misdiagnosed. Enzyme replacement therapy (ERT) is the only FDA-approved therapy for Fabry disease, and requires injection of relatively large amounts of recombinant protein every two weeks. Although ERT reduces the accumulation of Gb3 in the heart, kidneys and vasculature, it cannot completely treat all symptoms of Fabry, primarily because it cannot effectively enter the CNS. Gene therapy strategies have been investigated, and while many show great promise in correcting glycolipid accumulation, most fail to effectively enter the CNS and also suffer from the immune responses commonly seen during GLA replacement.

[0263] In embodiments, the AAV viral vectors disclosed herein are used to treat Fabry disease in patients who are unresponsive to ERT or when ERT fails to resolve all symptoms. In embodiments, the AAV viral vectors disclosed herein are used to treat Fabry disease in patients who have been administered ERT.

[0264] In an embodiment, the disease is Pompe disease. Pompe disease is a lysosomal storage disease caused by a defect in the activity of acid alpha glucosidase (GAA) that causes glycogen to accumulate in lysosomes. The disease manifests itself as a form of muscular dystrophy that primarily affects smooth and striated muscle tissues and the central nervous system (CNS), and leads to early death. Enzyme replacement therapy (ERT) is the only therapy currently approved by the FDA for the treatment of Pompe disease, and requires a relatively large amount of recombinant protein to be injected every two weeks. Although ERT significantly reduces the mortality rate of infant Pompe disease patients (these patients usually die by the age of two without therapy), it cannot completely improve all symptoms of Pompe disease, mainly because it cannot effectively enter the CNS and cause an immune response to GAA protein. Gene therapy strategies have been studied, and although many strategies have shown great promise in correcting glycogen accumulation and other symptoms of Pompe disease. Most people have suffered from severe immune responses observed during GAA replacement. Previous work has demonstrated that liver-specific expression can make animals tolerant to GAA protein and significantly reduce humoral responses.

[0265] In embodiments, the AAV viral vectors disclosed herein are used to treat Pompe disease in patients who have been administered ERT; for example, those patients who are unresponsive to ERT, or when ERT has failed to resolve all of their symptoms.

[0266] In an embodiment, the AAV viral vector disclosed herein is used to treat cancer. In an embodiment, the cancer is a solid cancer; for example, bladder cancer, breast cancer, cervical cancer, colon cancer, rectal cancer, endometrial cancer, kidney cancer, lip cancer, oral cancer, liver cancer, melanoma, mesothelioma, non-small cell lung cancer, non-melanoma skin cancer, ovarian cancer, pancreatic cancer, prostate cancer, sarcoma, small cell lung tumor or thyroid cancer.

[0267] In an embodiment, the disease is an ophthalmic disease. The eye is an immune privileged tissue. Only a very small amount of virus is needed for therapeutic benefit. In an embodiment, the ophthalmic disease affects photoreceptor cells and RPE cells. In an embodiment, the ophthalmic disease includes, is essentially composed of or is composed of: retinitis pigmentosa (e.g., autosomal recessive inheritance (SPATA7 gene; LRAT gene; TULP1 gene), autosomal dominant inheritance (AIPL1 gene) and X-linked inheritance (RPGR gene)), the eye disease associated with the mutation of yolk-like maculopathy protein-1 (BEST-1 or BEST1) gene (e.g., yolk-like macular dystrophy, age-related macular degeneration, autosomal dominant vitreoretinal choroidopathy, glaucoma, cataract), Leber congenital amaurosis (LCA; aryl hydrocarbon interacting protein-like 1 (AIPL1) gene ), cone-rod dystrophy (CRD; ABCA4 gene), Stargardt disease (ABCA4 gene), choroideremia (CHM gene), Usher syndrome (MYO7A gene; CDH23 gene; USH2A gene; CLRN1 gene), dominant optic atrophy (e.g., autosomal (OPA1 gene; OPA3 gene)), retinitis pigmentosa (PDE6B gene), retinoschisis (RS1 gene), Bietti crystal dystrophy (CYP4V2 gene), or achromatopsia (CNGA3 gene, CNGB3 gene, GNAT2 gene, PDE6C gene, or PDE6H gene).

[0268] In an embodiment, the present disclosure provides a method of expressing a transgene in a retinal cell. In an embodiment, the method comprises delivering a nucleic acid of the present disclosure to a retinal cell. In an embodiment, the method comprises transducing a retinal cell with an AAV viral vector of the present disclosure.

[0269] In embodiments, target cells of the present disclosure include retinal cells. In embodiments, retinal cells include photoreceptors, bipolar cells, retinal ganglion cells, horizontal cells, or amacrine cells. In embodiments, retinal cells include retinal ganglion cells. In embodiments, retinal cells include bipolar cells. In embodiments, retinal cells include horizontal cells. In embodiments, retinal cells include amacrine cells. In embodiments, retinal cells include photoreceptors. In embodiments, photoreceptors include rods and / or cones.

[0270] In embodiments, the target cells of the present disclosure comprise, consist essentially of, or consist of photoreceptor cells.

[0271] In an embodiment, the subject is a mammal; for example, a human. In a specific aspect, the human is an infant human; for example, under 3 years old, under 2 years old, or under 1 year old.

[0272] The method for treating and preventing disclosed herein can be combined with appropriate diagnostic techniques to identify and select patients for treatment or prevention. For example, the method for treating or preventing a disease disclosed herein can further include performing a gene test to identify a genetic mutation or a missing step associated with the disease in a subject. In an embodiment, the method for treating or preventing a disease includes administering to a subject who has previously been identified as carrying a mutation associated with the disease or who has been identified as being at a high risk (e.g., based on genetic factors) of developing a disease.

[0273] The present disclosure provides a method for increasing the level of a protein in a host cell, comprising contacting a host cell with any AAV particle disclosed herein, wherein the AAV particle comprises any AAV vector genome disclosed herein, and the AAV vector genome comprises an HNA sequence encoding a protein. In an embodiment, the protein is a therapeutic protein. In an embodiment, the host cell is in vitro, in vivo or ex vivo. In an embodiment, the host cell is derived from a subject. In an embodiment, the subject suffers from a disease that causes a level and / or functionality of a protein reduced compared to the level and / or functionality of a protein in a normal subject.

[0274] In an embodiment, the level of protein in the host cell is increased to about 1×10 -7 ng, about 3x10 -7 ng, about 5x10 -7 ng, about 7x10 -7 ng, about 9x10 -7 ng, about 1x10 -6 ng, about 2x10 -6 ng, about 3x10 -6 ng, about 4x10 -6ng, about 6x10 -6 ng, about 7x10 -6 ng, about 8x10 -6 ng, about 9x10 -6 ng, about 10x10 -6 ng, about 12x10 -6 ng, about 14x10 -6 ng, about 16x10 -6 ng, about 18x10 -6 ng, about 20x10 -6 ng, about 25x10 -6 ng, about 30x10 -6 ng, about 35x10 -6 ng, about 40x10 -6 ng, about 45x10 -6 ng, about 50x10 -6 ng, about 55x10 -6 ng, about 60x10 -6 ng, about 65x10 -6 ng, about 70x10 -6 ng, about 75x10 -6 ng, about 80x10 -6 ng, about 85x10 -6 ng, about 90x10 -6 ng, about 95x10 -6 ng, about 10x10 -5 ng, about 20x10 -5 ng, about 30x10 -5 ng, about 40x10 -5 ng, about 50x10 -5 ng, about 60x10 -5 ng, about 70x10 -5 ng, about 80x10 -5 ng or about 90x10 -5 ng level.

[0275] The present disclosure provides a method for introducing a gene of interest into a cell in a subject, comprising contacting the cell with an effective amount of any one of the AAV viral particles disclosed herein, wherein the AAV viral particles comprise any one of the AAV vector genomes disclosed herein, the AAV vector genome comprising the gene of interest.

[0276] Dosage and Administration

[0277] Methods for determining the most effective mode of administration and dosage are known to those skilled in the art and will vary with the composition used for treatment, the purpose of the treatment, and the subject being treated. Single or multiple administrations may be performed with the dosage level and pattern selected by the treating physician. It is worth noting that dosage may be affected by the route of administration. Suitable dosage formulations and methods of administering the agents are known in the art. Non-limiting examples of such suitable dosages may be as low as 10 mg / kg per administration. 9 vector genomes up to 10 17 vector genome.

[0278] In embodiments, the present disclosure provides methods of treating a disease or disorder in a subject in need thereof, comprising administering to the subject an effective amount of a therapeutic agent (eg, an AAV viral vector).

[0279] In embodiments of the methods described herein, the number of viral particles (e.g., AAV) administered to a subject is about 10 9 to about 10 17 In an embodiment, about 10 10 to about 10 12 , about 10 11 to about 10 13 , about 10 11 to about 10 12 , about 10 11 to about 10 14 , about 5x 10 11 About 5x 10 12 or about 10 12 to about 10 13 In an embodiment, the amount of viral genome (vg) administered to a subject is about 10 9 to about 10 17 In an embodiment, about 10 10 , 2x 10 10 , 3x 10 10 , 4x 10 10 , 5x 10 10 , 6x 10 10 , 7x 10 10 , 8x 10 10 , 9x 10 10 , 10 11 , 2x 10 11 , 3x 10 11 , 4x 10 11 , 5x 10 11 , 6x 10 11 , 7x10 11 , 8x 1011 , 9x 10 11 or 10 12 In an embodiment, about 10 10 to about 10 11 , about 10 11 to about 10 12 , about 10 12 to about 10 13 vg, about 5x 10 9 About 5x10 10 , about 5x 10 10 About 5x 10 11 , about 5x 10 11 About 5x 10 12 , about 10 10 to about 10 12 , about 10 11 to about 10 13 , about 10 10 to about 10 13 or about 10 11 to about 10 14 Viral genome (vg). For administration to the human eye, approximately 1 x 10 10 vg / eye, and for mouse eyes, approximately 5 x 10 9 Total dose of vg / eye. Non-invasive in vivo imaging techniques can be used to monitor efficacy / safety in animals, including but not limited to scanning laser ophthalmoscopy (SLO), optical coherence tomography (OCT), multiphoton microscopy, fluorescein angiography.

[0280] In an embodiment, the AAV particles repair the gene defect in the subject. In an embodiment, the ratio of the target polynucleotide or polypeptide repaired to the unrepaired target polynucleotide or polypeptide in the successfully treated cells, tissues, organs or subjects is at least about 1.5: 1, about 2: 1, about 3: 1, about 4: 1, about 5: 1, about 6: 1, about 7: 1, about 8: 1, about 9: 1, about 10: 1, about 20: 1, about 50: 1, about 100: 1, about 1000: 1, about 10,000: 1, about 100,000: 1 or about 1,000,000: 1. The amount or ratio of the target polynucleotide or polypeptide repaired can be determined by any method known in the art, including but not limited to Western analysis, Northern analysis, Southern analysis, PCR, sequencing, mass spectrometry, flow cytometry, immunohistochemistry (IHC), immunofluorescence, fluorescence in situ hybridization, next generation sequencing, immunoblotting and ELISA.

[0281] In an embodiment, the viral particles are introduced into the subject intravenously, intrathecally, intracerebrally, intraventricularly, intranasally, intratracheally, intraauricularly, intraocularly or periocularly, orally, rectally, transmucosally, by inhalation, transdermally, parenterally, subcutaneously, intradermally, intramuscularly, intrapleurally, topically, intralymphatically, intracisternally; such introduction may also be intraarterially, intracardiacly, subventricularly, epidurally, intracerebrally, intraventricularly, subretinally, pararetinally, intravitreally, intraarticularly, intraperitoneally, intrauterinely, or any combination thereof. In an embodiment, the viral particles are delivered to a desired target tissue, for example, as a non-limiting example, delivered to the lungs, eyes, or CNS. In an embodiment, the delivery of the viral particles is systemic. The route of intracisternal administration involves administering the drug directly into the cerebrospinal fluid of the ventricles. It can be performed by direct injection into the cerebellar cisterna or via a permanently positioned tube.

[0282] For intraocular treatment of ophthalmic diseases (or eye disorders), there are a variety of modes of administration known to those skilled in the art, including but not limited to: lacrimal gland (LG) administration, topical eye drops, intrastromal administration to the cornea, intracameral administration (anterior chamber), intravitreal administration, subretinal administration, juxtaretinal administration, systemic administration, or a combination thereof. 80% of inherited eye disorders occur in the photoreceptors. Intravitreal delivery of small volume gene therapy can be performed in an out-patient clinic.

[0283] In an embodiment, the mode of administration is juxta-retinal administration. As used herein, the term "juxta-retinal administration" refers to a form of intravitreal administration, which injects therapeutic agents (e.g., AAV viral vectors) into the vitreous cavity of the desired area next to the retina (i.e., targeted delivery). In an embodiment, the desired area of ​​the retina is close to the foveal region of the retina. Compared with conventional intravitreal administration, conventional intravitreal administration is completed using a short needle designed to deposit the product in the middle of the vitreous cavity and does not require direct visualization, and juxta-retinal injection is completed under the direct visualization of the longer needle in the rear vitreous cavity near the retina. In an embodiment, therapeutic agents are deposited at a distance of 0mm to 13mm from the surface of the retina, a distance of 0mm to 10mm from the surface of the retina, a distance of 0mm to 5mm from the surface of the retina, or a distance of 0mm to 3mm from the surface of the retina. In embodiments, the therapeutic agent is deposited in the vitreous cavity at a distance of between 0-13 mm, between 0-12 mm, between 0-11 mm, between 0-10 mm, between 0-9 mm, between 0-8 mm, between 0-7 mm, between 0-6 mm, between 0-5 mm, between 0-4 mm, between 0-3 mm, between 0-2 mm, or between 0-1 mm from the surface of the retina.

[0284] In embodiments, juxtaretinal administration is used for situations where subretinal injection is not suitable. In embodiments, juxtaretinal administration is used for targeted transduction of the optic nerve. In embodiments, juxtaretinal administration is used to treat diseases or disorders associated with dysfunction of the optic nerve. In embodiments, juxtaretinal administration is used to treat dominant optic atrophy or retinoschisis.

[0285] In an embodiment, juxtaretinal administration involves the use of a small gauge needle (30 gauge or the like) long enough to reach the posterior pole (25 mm or the like) of the human eye, external or internal illumination and visualization using a microscope, and the use of a contact lens to allow focusing on the posterior vitreous cavity and retina. This is usually done after adequate analgesia and disinfection, at which time the contact lens is coupled to the eye and the microscope is positioned to observe the posterior retina. The needle is inserted through the eye wall in the pars plana region, and its tip is visualized. Under direct visualization, the needle tip is advanced to the desired position close to the retinal surface. The syringe plunger advances to slowly deposit the viral vector (which can be included in any suitable composition or formulation). Pull out the needle and check the eye. The port can be closed with sutures, but for sufficiently small-caliber needles (such as 30 gauge), sutures are not required to close the needle tract. Ointments and eye masks can be used, and if necessary, the subject can be kept in a supine position for a period of time after surgery to further promote high concentrations of the product next to the retina. Variations of the delivery instrument may include creating a sclerotomy with or without a vitrectomy port to allow for the use of a blunt cannula, and / or a cannula design with a tapered and / or flexible extendable tip or side port to optimize access and safety to the retinal surface, and / or using a pneumatic system instead of a simple syringe plunger. Additional descriptions of juxtaretinal administration are disclosed in, for example, WO 2020 / 018766 and Zeng et al., Mol Ther Methods Clin Dev. 2020 Sep 11; 18:422–427; the contents of each of which are incorporated herein by reference in their entirety for all purposes.

[0286] In an embodiment, the mode of administration is subretinal administration, which injects the material into the subretinal space between the retinal pigment epithelium (RPE) cells and the photoreceptors. In the subretinal space, the injected material is in direct contact with the plasma membrane of the photoreceptors and the RPE cells and subretinal vesicles. In an embodiment, the AAV for subretinal administration includes the capsid protein of AAV214 or AAV214-D5. In an embodiment, subretinal administration is used to treat ADOA, XLRS, Stargardt's disease, Bietti's crystal dystrophy or Best's yolk-shaped macular dystrophy. Additional descriptions of subretinal administration are disclosed in, for example, Peng et al., Ophthalmic Res 2017; 58: 217-226; and Hartman et al., J Ocul Pharmacol Ther. March 1, 2018; 34 (1-2): 141–153; The contents of each of them are incorporated herein by reference in their entirety for all purposes.

[0287] Administration of the AAV viral particles or compositions of the present disclosure can be achieved in a single dose, continuously or intermittently throughout the course of treatment. In embodiments, the AAV viral particles or compositions of the present disclosure are administered parenterally by injection, infusion, or implantation.

[0288] In an embodiment, the AAV particles of the present disclosure show enhanced tropism for the brain and cervical spine. In an embodiment, the viral particles of the present disclosure can cross the blood-brain barrier (BBB). In an embodiment, the AAV particles of the present disclosure show high retinal tropism by pararetinal, subretinal and / or intravitreal injection. In an embodiment, the AAV particles of the present disclosure target a variety of eye cell types, such as, for example, cones, rods and retinal pigment epithelium (RPE). In an embodiment, the AAV particles of the present disclosure escape neutralizing antibodies against natural serotypes, and therefore can achieve potential re-administration. In another aspect, the AAV particles and compositions of the present disclosure can be administered in combination with other known treatments for the illness being treated.

[0289] Reagent test kit

[0290] In embodiments, the agents, viral vectors or compositions described herein can be assembled into pharmaceutical or diagnostic or research kits to facilitate their use in therapeutic applications, diagnostic applications or research applications. In embodiments, the kits disclosed herein include any of the modified AAV capsid proteins, AAV vectors, AAV viral particles, host cells, isolated tissues, compositions or pharmaceutical compositions as described herein.

[0291] In an embodiment, the kit further includes instructions for use. Specifically, such kits may include one or more medicaments as described herein, and instructions for describing the intended application and proper use of these reagents. As an example, in an embodiment, the kit may include instructions for mixing one or more components of the kit and / or separating and mixing samples and applying to a subject. In an embodiment, the medicament in the kit is a pharmaceutical formulation and dosage suitable for a method of administration of a specific application and medicament. Kits for research purposes may be used to carry out various experiments containing components of appropriate concentration or quantity.

[0292] Kits can be designed to facilitate the use of the methods described herein and can take a variety of forms. Where applicable, each composition in the kit can be provided in liquid form (e.g., in solution) or in solid form (e.g., dry powder). In some cases, some compositions can be composed or processable (e.g., processed into an active form), for example, by adding a suitable solvent or other substance (e.g., water or cell culture medium), which may or may not be provided with the kit. In an embodiment, the composition can be provided in a preservation solution (e.g., a cryopreservation solution). Non-limiting examples of preservation solutions include DMSO, paraformaldehyde and (Stem Cell Technologies, Vancouver, Canada) In embodiments, the storage solution comprises an amount of a metalloproteinase inhibitor.

[0293] In an embodiment, the kit includes any one or more of the components described herein in one or more containers. Therefore, in an embodiment, the kit may include a container for holding a medicament described herein. The medicament may be in the form of a liquid, a colloid, or a solid (powder). The medicament may be aseptically prepared, packaged in a syringe, and transported refrigerated. Alternatively, they may be contained in a vial or used to store other containers. The second container may have other reagents prepared aseptically. Alternatively, the kit may include an active agent premixed and transported in a syringe, a vial, a tube, or other containers. The kit may have one or more or all of the components required for administering the medicament to the subject, such as a syringe, a local application device, or an IV needle and bag.

[0294] It should be understood that although the present invention has been described in conjunction with the above embodiments, the foregoing description and examples are intended to illustrate rather than limit the scope of the present invention. Other aspects, advantages and modifications within the scope of the present invention will be apparent to those skilled in the art to which the present invention belongs.

[0295] In addition, where features or aspects of the invention are described in terms of Markush groups, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group.

[0296] Examples

[0297] Example 1: Characterization of pararetinal and subretinal drug delivery in non-human primates using various AAV viral vectors

[0298] The transduction efficiency of AAV viral vectors comprising AAV204, AAV214, AAV214-D5, or AAV8 capsids via various modes of ocular administration was evaluated as described below.

[0299] All AAV viral vectors used in this example comprised a recombinant nucleic acid encoding an enhanced green fluorescent protein (hereinafter "EGFP" or "GFP") reporter gene transgene operably linked to a CBh promoter.

[0300] To test the in vivo transduction efficiency of AAV viral vectors, non-human primate (NHP) cynomolgus monkeys were dosed with the indicated AAV viral vectors by pararetinal, subretinal or intravitreal administration. The dose / volume for each mode of administration was:

[0301] Peritoneal administration – 1.0E+11 vg in 100 μL injection volume

[0302] Subretinal administration – 2.5E+10 vg in 100 μL injection volume

[0303] Intravitreal administration – 1.5E+12 vg in 150 μL injection volume

[0304] Juxtaretinal administration was performed by layering the virus on top of the retina between the vitreous and the inner limiting membrane, so no subretinal detachment would occur. GFP expression was monitored using a scanning laser ophthalmoscope (SLO). SLO images were taken on samples collected 26-27 days after injection. At 28 days after injection, eyes were collected, processed, and analyzed by immunohistochemistry.

[0305] Figure 2B-2E The pararetinal injection of AAV204 ( Figure 2B )、AAV8( Figure 2C )、AAV214( Figure 2D ) or AAV214-D5( Figure 2E) capsid protein. Among the capsid proteins tested, the AAV viral vector containing the AAV204 capsid showed robust transduction in the macula, papillomacular bundle, and retinal nerve fibers via pararetinal injection, with much higher transduction efficiency than the other tested capsids.

[0306] In addition, the same AAV viral vector ( Figure 2A ) compared to pararetinal administration of AAV viral vectors containing AAV204 capsids ( Figure 2B ) also showed much higher local transduction efficiency ((especially for optic nerve transduction).

[0307] To further compare the transduction efficiency of these two administration routes, imaging analysis of the retina was performed ( Figure 3A-3C ). Again, the retina received pararetinal administration of an AAV viral vector containing an AAV204 capsid ( Figure 3B-Figure 3C ) than the retina that received intravitreal administration of the same AAV viral vector ( Figure 3A ) showed more robust macular and optic nerve transduction.

[0308] Further immunohistochemical analysis of rhodopsin and GFP 1 month after pararetinal injection of AAV204 viral vector ( Figure 3D ) showed high GFP expression in retinal ganglion cells (RGCs) throughout the retina, and nerve fibers with high GFP expression were observed along the retina and into the optic nerve. In contrast, pararetinal injection of AAV8 viral vector ( Figure 3D ) shows much lower GFP expression in RGCs. Figure 3E-Figure 3F As shown in , pararetinal administration of AAV204 viral vectors also resulted in robust GFP expression in the NHP fovea and along the papilla-macular bundle between the macula and the optic nerve. These results are consistent with the SLO analysis. Thus, at a dose at least 10-fold lower than that of intravitreal AAV injections typically used in the art, pararetinal administration of AAV204 viral vectors resulted in efficient transduction of target cells in the macula and fovea, as well as retinal ganglion cells and associated retinal nerve fibers extending to the optic nerve.

[0309] The inclusion of AAV8 ( Figure 4A , Figure 4D )、AAV214( Figure 4B , Figure 4E ) and AAV214-D5( Figure 4C , Figure 4F The results showed that these three capsids showed similar transduction efficiencies when administered subretinaly.

[0310] Conclusions: These results suggest that pararetinal injection of AAV vectors containing the AAV204 capsid can effectively deliver payloads to the macula or optic nerve / retinal ganglion cell layer.

[0311] Example 2: In vitro characterization of AAV vector genomes containing the human Opa1 expression cassette

[0312] according to Figure 7 , construct multiple AAV vector genome expression vectors for expressing human Opa1 (hOpa1), including:

[0313] -pA-Opa1_1 (comprising SEQ ID NO: 230; generated from a DNA template vector comprising SEQ ID NO: 186;

[0314] -pA-Opa1_3 (comprising SEQ ID NO: 231; generated from a DNA template vector comprising SEQ ID NO: 187;

[0315] -pA-Opa1_5 (comprising SEQ ID NO: 232; generated from a DNA template vector comprising SEQ ID NO: 188;

[0316] -pA-Opa1_11 (comprising SEQ ID NO: 233; generated from a DNA template vector comprising SEQ ID NO: 189;

[0317] -pA-Opa1_13 (comprising SEQ ID NO: 234; generated from a DNA template vector comprising SEQ ID NO: 190;

[0318] -pA-Opa1_15 (comprising SEQ ID NO: 235; generated from a DNA template vector comprising SEQ ID NO: 191;

[0319] -pA-Opa1_17 (comprising SEQ ID NO: 236; generated from a DNA template vector comprising SEQ ID NO: 192;

[0320] -pA-Opa1_21 (comprising SEQ ID NO: 237; generated from a DNA template vector comprising SEQ ID NO: 193;

[0321] -pA-Opa1_25 (comprising SEQ ID NO: 238; generated from a DNA template vector comprising SEQ ID NO: 194; and

[0322] - pA-Opa1_27 (comprising SEQ ID NO: 239; generated from a DNA template vector comprising SEQ ID NO: 195).

[0323] Each vector genome construct comprises, from 5' to 3', a promoter (CBh promoter (SEQ ID NO: 154) or MeCP2 promoter (SEQ ID NO: 156)), an intron (SEQ ID NO: 200), an hOpa1 open reading frame, and a BGH polyA signal (SEQ ID NO: 201 or 225), flanked by 5' and 3' ITRs. In addition, most constructs comprise telomeric repeats (TRs) downstream of the BGH polyA signal and optionally upstream of the promoter. Specifically, pA-Opa1-1 does not comprise a TR; both pA-Opa1-3 and pA-Opa1-5 comprise a first TR (SEQ ID NO: 202) and a second TR (SEQ ID NO: 203) downstream of the BGH polyA signal; and the other AAV vector genomes comprise both a first TR (SEQ ID NO: 202) downstream of the BGH polyA signal and a second TR (SEQ ID NO: 203) upstream of the promoter. In most AAV vector genomes, the Opa1 transgene is operably linked to the CBh promoter, except in pA-Opa1_5 and pA-Opa1_13, where the Opa1 transgene is operably linked to the MeCP2 promoter. In addition, the transgenes in pA-Opa1_15 and pA-Opa1_25 are human Opa1ΔS1 isoforms, and the transgenes in pA-Opa1_17 and pA-Opa1_27 are human Opa1 E5b isoforms. The pA-Opa1_2x construct further comprises a 3xFLAG tag fused to the 3' end of the human Opa1 open reading frame.

[0324] After preparing plasmid DNA of each AAV vector genome, 293 cells were transfected with 1 μg of plasmid DNA and incubated at 37°C, 5% CO 2 The cells were incubated for 48 hours at 4 °C and protein lysates were collected. Western analysis was used to examine Opa1 expression ( Fig. 8A In addition, the AAV capsid packaging capacity of these AAV vector genomes was analyzed by measuring viral yield ( Figure 8B The ability of AAV viral vectors to infect cells and express Opa1 protein was evaluated for the indicated vector genomes. Fig.9A , the above figure and Fig. 9B , lanes 5-13) or by anti-FLAG antibody ( Fig.9A , the following figure and Fig. 9B , lanes 2-4) to detect Opa1 expression by each vector genome. In addition, Opa1 expression was also analyzed by protein staining in the transfected Opa1(- / -) cell line ( Fig. 9C ).

[0325] The results showed that in 293 cells, pA-Opa1_3 produced about 4-fold lower Opa1 expression than pA-Opa1_1, and the expression from pA-Opa1_5 was even lower than pA-Opa1_3. Both pA-Opa1_3 and pA-Opa1_5 were not efficiently packaged into AAV capsids. On the other hand, pA-Opa1_11 produced better Opa1 expression than pA-Opa1_1, and pA-Opa1_13 also showed better Opa1 expression compared to pA-Opa1_5. In addition, both pA-Opa1_11 and pA-Opa1_13 were able to be efficiently packaged into AAV capsids. Therefore, the introduction of telomeric repeat sequences flanking both the 5' and 3' sides of the Opa1 expression cassette improved Opa1 expression as well as the packaging of the AAV vector genome.

[0326] Example 3: In vivo study of dominant optic atrophy treatment using AAV viral vector encoding OPA1

[0327] To study the in vivo expression of Opa1, Fig. 10A Animal studies were performed. In brief, according to Table 7 below, Opa1- / + mice in three treatment groups were injected with AAV204 viral vectors containing pA-Opa1_21 (high or low dose) or pA-Opa1_27 at one month old via intravitreal injection into the retinal vitreous space with a 33-34G needle and syringe. The left eye was penetrated on the temporal side, and the right eye was penetrated on the nasal side of the pupil. Optical coherence tomography (OCT) was performed after injection to assess any postoperative damage, and buprenorphine (0.01-0.05mg / ml) or meloxicam (5mg / ml) was administered to help recover from anesthesia. Ofloxacin or neomycin polymyxin B gramicidin was administered postoperatively to prevent infection. Atipamezole (0.1-1.0mg / kg) was also administered to mice to reverse the side effects caused by the metabolism of xylazine. The mice were then allowed to recover on a heating pad until fully awake. The morbidity and mortality of mice were monitored every day after injection for two months. Six mice (30 mice in total) were used for each of the three treatment groups and two control untreated groups. They were killed at three months old and euthanized via CO2 exposure, and the eyes were first removed with blunt scissors to remove the eyelids, followed by curved forceps and 1 mm surgical scissors to separate the optic nerve. The cornea was cut and the lens was removed to make an "eye cup" sample for subsequent analysis.

[0328] Table 7: Mouse Cohorts Used for Intravitreal Injections

[0329]

[0330] After measuring Opa1-Flag expression, expression of Opa1-21 protein was observed in all treated animals at the correct size and in the correct isoform of two bands ( Fig. 10B ). The isoform pattern was identical to the endogenous isoform in untreated and treated wild-type eyes (although the wild-type band was only visible upon overexposure). Opa1-27 protein was expressed in eye samples, but not at the predicted isoform size as in cell culture. Expression of Opa1-27 protein appears to be restricted to the uncleaved long / S1 isoform, without the short isoform that may regulate mitochondrial fusion (as described in Wang et al., Mol Biol Cell. 2021 Jan 15;32(2):157-168). The amounts of Brn3a and rhodopsin (Rho) in the samples were also analyzed using equivalent protein loading ( Fig. 10C ). Brn3a (a marker for retinal ganglion cells) was more consistent between samples.

[0331] These results show that all three treatment groups showed significantly increased expression of heterologous Opa1 compared to wild-type (WT) animals, with the most significant Opa1 expression achieved in the high-dose group with construct pA-Opa1-21. The Opa1 expression data correlated with the level of FLAG tag expression from all three groups, with untreated samples clearly lacking FLAG expression.

[0332] In addition to Western analysis, RT-PCR was used to analyze the expression levels of RNA transcripts. Fig. 10D As shown, significant expression of RNA transcripts for both high and low doses of pA-Opa1_21 (isoform 1) and pA-Opa1_27 (isoform 7) was observed in all treated HT animals, whereas, as expected, no transcripts encoding human Opa1 or FLAG tag were detected in untreated animals. Interestingly, although pA-Opa1_27 appeared to be expressed at a higher level at the mRNA level, the protein level was much lower than that of the high dose of pA-Opa1_21.

[0333] On the other hand, construct pA-Opal_25 did not result in detectable expression of Opal at either the mRNA or protein level (data not shown).

[0334] In another proposed study ( Fig.11A and Fig. 11B), Opa1- / + mice in each treatment group were administered AAV204 viral vectors containing the indicated vector genomes. The two control groups were untreated Opa1- / + mice and untreated Opa1+ / + mice. For the treatment groups delivered with Opa1 transgene without 3xFLAG tag, Opa1 expression levels and therapeutic efficacy were evaluated by RT-qPCR and counting retinal ganglion cells (RGCs) ( Fig.11A ). Visual acuity (VA), optical coherence tomography (OCT), scotopic threshold response (STR), and photopic negative response (PhNR) were measured at 10 months after injection. In addition, and for the treatment group delivered with the Opa1 transgene containing the 3xFLAG tag, Opa1 expression was assessed using RT-qPCR and IHC at 4 months after injection ( Fig. 11B Example 4: In vitro characterization of AAV vector genomes containing human RS1 expression cassettes

[0335] according to Fig. 12A , multiple AAV vector genomes for expressing RS1 protein were constructed, with the overall design being Fig. 12B .Each construct comprises a promoter, an intron of CBh-MVM (SEQ ID NO:200) or MVM (SEQ ID NO:222), an RS1 open reading frame, a BGH polyA site (SEQ ID NO:201 or 225) and a telomeric repeat sequence (SEQ ID NO:203) from 5' to 3', and is flanked by 5' and 3' ITRs.For promoters, each construct ending with "6" comprises a CBh promoter (SEQ ID NO:154), each construct ending with "8" comprises an RK promoter (SEQ ID NO:196), each construct ending with "0" comprises a Rho promoter (SEQ ID NO:197), and each construct ending with "2" comprises a PDE promoter (SEQ ID NO:198).Some constructs also comprise a βGlo_s / MAR sequence (SEQ ID NO:221) between the BGH polyA site and the telomeric repeat sequence.

[0336] The AAV vector genomes used in this study included:

[0337] -pA-RS1_8 (comprising SEQ ID NO: 240; generated from a DNA template vector comprising SEQ ID NO: 204;

[0338] -pA-RS1_16 (comprising SEQ ID NO:241; generated from a DNA template vector comprising SEQ ID NO:205);

[0339] -pA-RS1_18 (comprising SEQ ID NO: 242; generated from a DNA template vector comprising SEQ ID NO: 206;

[0340] -pA-RS1_20 (comprising SEQ ID NO: 243; generated from a DNA template vector comprising SEQ ID NO: 207;

[0341] -pA-RS1_22 (comprising SEQ ID NO: 244; generated from a DNA template vector comprising SEQ ID NO: 208;

[0342] -pA-RS1_26 (comprising SEQ ID NO: 245; generated from a DNA template vector comprising SEQ ID NO: 209;

[0343] -pA-RS1_28 (comprising SEQ ID NO: 246; generated from a DNA template vector comprising SEQ ID NO: 210;

[0344] -pA-RS1_30 (comprising SEQ ID NO: 247; generated from a DNA template vector comprising SEQ ID NO: 211;

[0345] -pA-RS1_32 (comprising SEQ ID NO: 248; generated from a DNA template vector comprising SEQ ID NO: 212;

[0346] -pA-RS1_36 (comprising SEQ ID NO: 249; generated from a DNA template vector comprising SEQ ID NO: 213;

[0347] -pA-RS1_38 (comprising SEQ ID NO: 250; generated from a DNA template vector comprising SEQ ID NO: 214;

[0348] -pA-RS1_46 (comprising SEQ ID NO: 251; generated from a DNA template vector comprising SEQ ID NO: 215;

[0349] -pA-RS1_48 (comprising SEQ ID NO: 252; generated from a DNA template vector comprising SEQ ID NO: 216; and

[0350] -pA-RS1_58 (comprising SEQ ID NO: 224; generated from a DNA template vector comprising SEQ ID NO: 223).

[0351] Western analysis was used to evaluate the RS1 protein expression levels of three AAV vector genomes (pA-RS1_8, pA-RS1_18, and pA-RS1_28). Fig.13 ). Both pA-RS1_18 and pA-RS1_28 produced a higher proportion of secreted RS1 protein compared to pA-RS1_8, although total protein production was comparable in these constructs.

[0352] The efficacy of AAV204 viral vectors containing the indicated RS1-expressing vector genomes was also evaluated in Lec2 cell cultures. Figure 14A-Figure 14C As shown, myc-tagged RS1 was expressed at similar levels to untagged RS1 in transduced Lec2 cells and was properly secreted. Moreover, the CBh promoter was able to achieve higher RS1 expression compared to the RK promoter. Secreted myc-RS1 was approximately 4 kD larger than native RS1 ( Fig.14D ).

[0353] Example 5: In vivo study of treatment of X-linked retinoschisis using AAV viral vector encoding RS1

[0354] In one study, wild-type mice were administered an AAV204 viral vector containing an RS1 expression cassette via intravitreal injection. At the 1-month post-injection time point, the expression of RS1 protein in each animal group was assessed by RT-qPCR (mRNA copies / ng total RNA) and Western analysis, and the protein distribution was assessed by IHC ( Fig.15A ).

[0355] like Fig. 15B As shown, expression of mouse RS1 (mRS1) protein was detected in each mouse tested, and there was no statistical difference in the mean mRS1 expression between the test groups. Mice administered AAV204.pA-RS1_26 showed robust expression of myc-tagged human RS1 (hRS1), with levels comparable to native mRS1 expression. On the other hand, there was almost no detectable hRS1 expression from the RK promoter in mice at this time (with one exception).

[0356] In the next study, AAV204 viral vectors containing pA-RS1_36 or pA-RS1_38 vector genomes were administered to the eye cups of wild-type mice via intravitreal injection at a dose of 3e+9 vg / eye. At 30 days after injection, samples were collected and total protein extracts were examined by Western analysis using an anti-myc antibody ( Fig.16A). Myc-hRS1 protein expression was detected in all eyes administered AAV204.pA-RS1_36 (containing the CBh promoter). In contrast, no myc-RS1 protein was detected in the treatment group administered AAV204.pA-RS1_38 (containing the RK promoter) or in the untreated controls. This result was confirmed using anti-human RS1 (hRS1) antibodies ( Fig. 16B ). The anti-hRS1 antibody actually detected both human RS1 protein and mouse RS1 protein, and the expression pattern of recombinant human RS1 (hRS1) based on the anti-hRS1 antibody was consistent with the expression pattern based on the anti-myc antibody.

[0357] To assess ocular expression ( Fig.17A ), the AAV204 viral vector containing the pA-RS1_26 vector genome was administered to the eye cup of RS1(y / -) mice. The efficacy of the treatment group was compared with that of the untreated control group (RS1(y / -) mice or wild-type RS1(y / +) mice) at the 6-month time point using optical coherence tomography (OCT) and electroretinography (ERG), and the expression level of RS1 was evaluated at the 7-month time point using IHC.

[0358] In another proof-of-concept study ( Fig. 17B ), AAV204 viral vectors containing pA-RS1_28, pA-RS1_46 or pA-RS1_48 vector genomes were administered to three mouse groups, respectively, and compared with unadministered control groups. The expression level of RS1 was assessed at a 3-month time point using Western analysis or RT-qPCR, or at an 8-month time point using IHC. The efficacy of treatment was assessed at an 8-month time point using OCT and ERG.

[0359] In another proof-of-concept study, male RS1(- / Y) mice and wild-type male littermates were recruited at 21 ± 3 days of age and acclimated to the study for at least 3 days prior to dosing. Fig.18A To design the treatment groups.

[0360] - Groups 5-9 were used to demonstrate the in vivo infectivity of the intravitreal delivered vectors and to analyze the distribution of virus-derived proteins in the retina at the 2 month post-treatment (mpt) time point. To facilitate protein detection, constructs with N-terminal myc tag ends were used (pA-RS1_46, pA-RS1_48).

[0361] - Groups 1-4 were used to confirm the longevity of expression at the 6-month time point after intravitreal injection and to analyze the effect of transgene expression on disease progression. The RS1 transgene used in these vectors does not have a myc tag.

[0362] - Groups 10-11 were used to evaluate the effect of subretinal injections as an alternative drug delivery procedure at a time point of 6 months after intravitreal injections and to compare directly with IVT injections performed in parallel. Subretinal injections for the treatment of retinoschisis may carry a greater risk as it is a more invasive technique that may result in increased rates of retinal detachment given the structurally compromised state of the diseased retina. However, this has not been directly tested. The AAV viral constructs used in these two groups did not contain a myc tag encoding sequence or S / MAR.

[0363] An "eye cup" was prepared, and the neural retina was then separated from the pigmented retinal pigment epithelium (RPE) to create a retinal dissection sample.

[0364] Analysis of retinal samples 2 months after intravitreal delivery

[0365] Endogenous gene expression was measured in all groups at the 2 mpt endpoint using primers against mouse RS1 (mRs1). mRs1 expression was significantly reduced in all mutant animals, regardless of treatment, compared to WT ( Fig.18B This is likely a result of the degenerative state of the mutant retina, and specifically the loss of photoreceptors, where mRs1 is highly expressed. In the untreated mutant group (Group 9), the mutant retina had similarly reduced mRs1 expression, indicating that the treatment did not affect endogenous mRs1 expression.

[0366] Primers targeting the mycRS1 transgene were then used to detect virus-derived RS1 transcripts and distinguish them from endogenous transcripts ( Fig. 18C ). Although mycRS1 was undetectable in untreated retinas (Groups 8, 9), it was readily detected in all treated retinas (Groups 5, 6). The promoter used had no significant effect on the total amount of virus-derived transcripts. Expression of transgenic myc-RS1 was nearly 2 log units lower than that of endogenous Rs1 in mutant animals.

[0367] The total RS1 protein expression level was measured by Western analysis using RS1-specific monoclonal antibody 3R10 to simultaneously detect endogenous and virus-derived proteins ( Fig.18D and Fig.18E ). Although expression varied between individual retinas, the average RS1 protein expression driven by the CBh promoter (Group 5) was 30% of the RS1 produced in WT animals (Group 8), and the average RS1 protein expression driven by the RK promoter (Group 6) was 7% of WT. The difference in the size of RS1 protein between the treated and WT groups is due to the presence of the myc tag. RS1 was not detected in untreated mutant animals (Group 9).

[0368] Additional eyes were immunohistochemically stained at the 2 mpt time point using the 3R10 antibody to confirm transgene expression and assess protein localization. In addition, the lectin PNA was used to label the outer segments of cone photoreceptors and Iba1 was used to label retinal microglia. Low magnification images of the entire retina demonstrate the extent of transduction, with higher magnification images of the boxed areas demonstrating PNA and Iba1 staining and the distribution of RS1 across the retinal layers. In some cases, multiple boxed areas were included to allow direct comparison of transduced and non-transduced areas of the same section.

[0369] In WT animals from group 8, Rs1 was clearly localized to the inner segments of photoreceptors and more broadly to the synaptic layer of the retina. Cone photoreceptors were regularly spaced throughout the length of the retina (12.2 ± 1.5 cones / 100 μm), and Iba1 staining was minimal ( Fig.19A In contrast, untreated mutant retinas (group 9) had no detectable RS1 staining, decreased cone density (7.8 ± 0.9 cones / 100 μm), and more prominent Iba1 staining, suggesting a hyperinflammatory state commonly seen in retinal diseases ( Fig.19B ). In retinas that do not express RS1, small bright foci that appear by RS1 staining are retinal vessels containing IgG, which in the absence of RS1 expression become labeled by the anti-mouse IgG secondary antibody. Several examples of these are given in Fig.19B Indicated by arrows.

[0370] RS1 labeling in mutant retinas treated with AAV204.CBh:RS1_46 (Group 5) was generally confined to the inner nuclear layer (INL) and was accompanied by small spots of labeled photoreceptor inner segments. There were no instances of labeled photoreceptors without adjacent inner retinal labeling. Cone density and Iba1 labeling were similar to those of untreated retinas regardless of RS1 expression. Representative retinas from this treatment group were shown in Figure 5. Fig.19C Arrows indicate small areas of transgene expression in photoreceptors. The right eye from one animal (#123) was an exception, as transgene expression was observed in the inner retina as well as in photoreceptors along 58% of the retina (including the entire dorsal half) ( Fig.19D ); however, there were still no apparent changes in cone density or Iba1 labeling in this eye sample.

[0371] In contrast, none of the animals treated with AAV204.RK:RS1_48 (Group 6) had observable RS1 immunolabeling ( Fig.19E). Cone density and Iba1 labeling in these animals were indistinguishable from untreated mutants. This was unexpected, as viral expression was detected in other animals in this group by RT-PCR and Western analysis.

[0372] Cone density was then quantified from IHC images of the right eye of WT and untreated mutant animals, as well as animal #123, which has high levels of transgene expression in photoreceptors. As expected, cone density in the mutant eye was reduced to 63% of that in WT animals ( Fig.19F ). Cone density was also measured in well-defined RS1 positive and RS1 negative areas of the eye #123OD (Group 5). This eye overall had lower cone density than the WT average, but there was a modest improvement in the RS1 positive areas of this eye when compared to the RS1 negative areas from the same eye.

[0373] Analysis of retinal samples 6 months after intravitreal delivery

[0374] To demonstrate the extent of transduction in IVT-injected eyes, retinas from one animal (Group 2) treated with AAV204.RK:RS1_28 were stained as flat mounts ( Figure 19G ). Transgene expression was limited to a well-defined area in the periphery, covering approximately 10% of the entire retina. The extent of transduction was similar in the other eye. Cone density in the non-transduced portion of the retina was approximately 19% of that in a similarly prepared WT control. In the transduced portion of the retina, cone density was increased to 46% of WT. The results indicate that expression of the RS1 transgene is more limited than expected and is concentrated near the injection site in the dorsal retina.

[0375] Western analysis was performed on retinas harvested at 6 mpt ( Fig. 20A and Fig. 20B ). Using the RS1-specific antibody 3R10, RS1 expression was detected in mutant eyes treated with AAV204.CBh:RS1_28 (Group 2) or AAV204.RK:RS1_26 (Group 4). In all treated animals, the levels of RS1 expression ranged from 1.5% to 5.5% of the levels detected in the WT retina. Unexpectedly, expression from the transgene controlled by the CBh promoter was not significantly higher than expression from the transgene controlled by the RK promoter. No expression was detected in untreated mutant animals.

[0376] Immunohistochemistry (IHC) was then used to examine the distribution of RS1 expression in treated eyes at this time point and to determine the impact of RS1 expression on disease phenotype. Similar to the previous 2mpt time point, RS1 expression in WT animals was concentrated in the photoreceptor inner segments, with more diffuse expression in the inner retina ( Fig. 20C In contrast, no expression was observed in untreated mutant animals ( Fig.20D ). Blood vessels were again observed in the mutant retina with anti-mouse secondary antibody ( Fig.20D , examples indicated with arrows). Cone density was also reduced in the mutant at this time point, and Iba1 labeling was more prominent.

[0377] In animals from group 2 treated with AAV204.RK:RS1_28, RS1 expression was detectable, ranging from 14% to 59% of the sections. This should not be interpreted as a percentage of the entire retina, as only sections with maximal expression were imaged. Representative examples from this group are shown in Fig.20E RS1 labeling was not primarily restricted to the inner retina, but most transduced areas also included considerable photoreceptor expression. In contrast, none of the eyes in Group 4 treated with AAV204.CBh:RS1_26 had detectable expression ( Fig.20F ).

[0378] Notably, the localization of recombinant RS1 in the retina of group 2 animals was identical to that of the endogenous protein, with abundant staining in photoreceptor inner segments and more diffuse staining throughout the inner retina, even in animals treated with the photoreceptor-specific promoter. This suggests that secreted RS1 is able to diffuse radially through the retina to reach receptors in adjacent layers.

[0379] Cone density was quantified in all animals at the 6-month time point ( Fig.21). In mutants treated with AAV204.RK:RS1_28 (Group 2), RS1-positive and RS1-negative areas were considered separate data points. Cone density in untreated mutants was 31±6% of wild type, and the average density in Group 2 did not change regardless of RS1 expression. However, in all animals in Group 2, except for one animal, cone density was higher in RS1-positive areas than in adjacent RS1-negative areas in the same section. This improvement ranged from 15% to 71%. Surprisingly, despite the absence of observable RS1 expression, animals in Group 4 treated with AAV204CBh:RS1_26 had an average cone density that was 63% higher than that of the untreated group (p=0.0217). Therefore, cone density was improved after treatment with either viral construct, although in the case of RS1 controlled by the RK promoter, such improvement was not associated with observable RS1 immunoreactivity.

[0380] Optical coherence tomography (OCT) was then used to analyze the status of the retina. OCT is a non-invasive imaging technique that produces cross-sectional images of the retina and is often used to help diagnose retinal diseases. This is a particularly useful technique for retinoschisis because the large retinal cavities that are characteristic of the disease are easily observed. However, by 7 months of age, most of the schisis is resolved in RS1 mutant mice. OCT images are similar to histological sections, but are more limited in that they cannot be directly correlated with expression data. Therefore, the interpretation of these images must include the assumption that expression of the transgene is widespread, or at least that it overlaps with the OCT field.

[0381] Analysis of the OCT data from this study showed that, regardless of treatment, the cleavage phenotype only appeared in about one-third of the mutant eyes imaged. This is consistent with the fact that the disease begins to regress around this time point in the mutant mice. No cleavage was observed in WT eyes.

[0382] Representative OCT images of Fig.22A Shown are two treated eyes from Group 2 where maximal RS1 expression was observed by IHC or Western analysis. Each image is of the dorsal retina in the mid-periphery. At 10 OD, this is roughly the area where strong RS1 expression was observed by IHC. The untreated mutant eyes from Group 1 had a significantly thinner outer nuclear layer (ONL) than the WT eyes from Group 3 (26.9 ± 1.9 μm vs. 52.8 ± 2.6 μm). Eyes from both treated groups had thicker ONLs than untreated animals, but they were still thinner than WT eyes ( Fig. 22B ).

[0383] The current studies were done using isolated retinas rather than whole eyes. Isolating the retina has the effect of concentrating the target tissue in the sample and making a limited amount of expression from the RK promoter visible. In addition, the use of isolated retinas excludes extraretinal expression that occurs in the presence of the ubiquitous CBh promoter, providing a clear indication of RS1 expression levels in target tissues. The results show that (i) RS1 expression from either promoter was readily detectable in treated mutant eyes, regardless of the promoter used; and (ii) unexpectedly, CBh-driven RS1 expression in the retina was nearly 1.5 log units lower than endogenous RS1 expression 2 months after treatment.

[0384] Analysis of retinal samples 6 months after subretinal delivery

[0385] In addition to intravitreal injections, selected AAV viral vectors were delivered by subretinal injection ( Fig.18A At the 6 mpt time point, the corresponding samples were analyzed together with samples from control groups 1 and 3 (untreated mutant and wild type, respectively).

[0386] RS1-specific monoclonal antibody 3R10 was used to confirm transgene expression and assess protein localization. In addition, lectin PNA was used to label the outer segments of cone photoreceptors, and Iba1 was used to label retinal microglia. Low magnification images of the entire retina are shown to demonstrate the extent of transduction, with higher magnification images of the boxed areas showing PNA and Iba1 staining and the distribution of RS1 across the retinal layers. In some cases, multiple boxed areas were included to allow direct comparison of transduced and non-transduced areas of the same section.

[0387] In WT animals from group 3, endogenous Rs1 was clearly localized to the inner segments of photoreceptors and more broadly to the synaptic layer of the retina. Cone photoreceptors were regularly spaced throughout the length of the retina (10.2 ± 1.6 cones / 100 μm), and Iba1 staining was minimal ( Fig.23A ).

[0388] In contrast, untreated mutant retinas (Group 1) had no detectable RS1 staining, decreased cone density (3.2 ± 0.6 cones / 100 μm), and more prominent Iba1 staining, suggesting a hyperinflammatory state commonly seen in retinal diseases ( Fig. 23B ). The small bright foci that appear by RS1 staining in the retina that does not express RS1 are retinal vessels containing IgG, which become labeled by the anti-mouse IgG secondary antibody in the absence of RS1 expression. Several examples of these are given in Fig. 23BIndicated by arrows.

[0389] Mutant animals treated with AAV204.CBh:RS1_16 (Group 10) exhibited severe retinal degeneration associated with the injection site and no remaining retinal cells ( Fig.23C ). In some cases, adjacent sections of the retina had RS1 expression, despite no improvement in cone density in these RS1+ areas. However, the mean cone density in RS1- areas of these retinas (which did not suffer injection-related degeneration) was approximately twice that of untreated animals (6.2±1.4 vs. 3.2±0.6 cones / 100μm, respectively), suggesting that RS1 expression below IHC detectable levels may extend outside the vesicle but still provide some therapeutic benefit. No changes were observed in Iba1 staining in treated animals. The right eye from one animal (#167) was unique in that it had severe damage in the ventral retina, most likely from mechanical damage sustained during injection. Despite this, uniform RS1 staining was observed throughout the inner retina and in photoreceptors adjacent to the lesion ( Fig.23D ). It is likely that this lesion compromises the physical barrier that normally inhibits the spread of viral particles through the retina, allowing for more widespread expression.

[0390] In contrast, seven of eight eyes treated with AAV204.RK:RS1_18 (Group 11, Fig.23E ) had very significant RS1 expression in photoreceptors, and only one of the seven animals had the severe degeneration common in animals treated with CBh:RS1. Expression was typically localized to the dorsal retina at the vesicle. The RK promoter is rod-specific, but labeling was also observed in the inner retina adjacent to RS1+ photoreceptors in all seven eyes, indicating that RS1 produced in the outer retina can spread radially. In four of the seven eyes, areas of cone depletion were associated with central vesicles, despite the rest of the retina being intact. Outside this cone-depleted area, there was an RS1-positive edge in which cone density was actually increased compared to the RS1-negative portion of the same section. In fact, this edge region had a cone density equivalent to that of wild-type eyes. Taken together, these results suggest that extreme overexpression of RS1 in mutant animals can be severely retinotoxic, while moderate overexpression is harmful to cones, but appropriate administration can produce a therapeutic effect.

[0391] Fig.23FThe graphs in summarize the cone density measurements. Areas of severe degeneration were omitted. For Group 11 eyes treated with RK:RS1, RS1-positive areas with cone depletion were omitted. Therefore, Group 11 RS1+ represents cone-enriched areas surrounding depleted areas. All treated eyes had higher cone density than untreated control eyes, even in areas lacking visible RS1 expression. Furthermore, cone-enriched RS1-positive areas in Group 11 were indistinguishable from WT.

[0392] The thickness of the outer nuclear layer (ONL) was also measured from DAPI-stained cryosections ( Figure 24A-Figure 24C ). For this analysis, areas of severe degeneration were omitted. In group 10 animals treated with CBh:RS1, ONL thickness was equal to that of untreated mutant animals. In all group 11 animals treated with RK:RS1, ONL was thicker, particularly in areas of the retina with RS1 expression. In those areas of the eye that did not express RS1, ONL thickness was roughly equal to that of untreated mutants.

[0393] Protein analysis: Western analysis was performed on frozen tissues harvested 6 months after treatment ( Fig.25A and Fig.25B ). Using the RS1-specific antibody 3E10, RS1 expression was detected in mutant eyes treated with AAV204.CBh:RS1_16 (Group 10) or AAV204.RK:RS1_18 (Group 11). The expression of recombinant RS1 in both groups was statistically equivalent to the expression of endogenous RS1 in WT animals (Group 3).

[0394] Optical coherence tomography (OCT): As mentioned above, the interpretation of OCT images assumes that expression of the transgene is widespread, or at least that it overlaps with the OCT field. This is reasonable in the present study, as injections were performed subretinal, and investigation of IHC images from animals treated with RK:RS1_18 (Group 11) showed that expression was widespread throughout the dorsal retina, excluding the ventral side.

[0395] OCT data Figure 26A-26F The ONL of untreated mutant eyes was almost one-third of that of wild-type eyes (17.9 ± 1.9 vs. 48.7 ± 1.9 μm, Figure 26A-26B All treated eyes were imaged immediately after injection to confirm the presence of vesicles ( Fig.26C All 13 eyes successfully injected with CBh:RS1_16 showed severe degeneration ( Fig.26D). Several instances overlapped with the edge of the vesicle, where some retina was preserved, but the thickness of the ONL there, as well as the thickness of the ventral, untreated retina, was not significantly different from that of uninjected mutant animals.

[0396] In contrast, in eyes treated with RK:RS1_18 (Group 11), only 7 of 13 eyes with interpretable images had signs of severe injection-related degeneration, but 4 of 7 eyes still had measurable areas in the margins of the vesicles ( Figure 26E-26F In six non-degenerative eyes and four degenerative eyes with measurable margins, all showed improvement in ONL thickness when compared to the corresponding ventral retina as well as untreated mutant eyes from Group 1 ( Figure 26G-26H ).

[0397] Electroretinography (ERG): Electroretinography is a non-invasive technique that measures the function of the retina's response to flashes and can be modified to elicit either rod- or cone-based responses. The intensity of the flash stimulus can also be varied, and the amplitude of the ERG components will respond in parallel. The dark-adapted (DA) ERG elicits a rod-dominated response and consists of two main components: an a-wave, which is an initial negative amplitude peak and reflects rod photoreceptor activity; and a b-wave, which is a large positive amplitude peak and primarily reflects the activity of ON-bipolar cells. In the light-adapted (LA) ERG, a dim background light is used to reduce the sensitivity of the rod photoreceptors, allowing the cone response to be isolated. Cone responses can also be measured using a flicker paradigm, where the frequency of the stimulus is faster than the recovery time of the rod photoreceptors. More descriptions of the ERG can be found, for example, in Georgiou, Anne L. et al., Current eye research 39.5 (2014): 472-486, the contents of which are incorporated herein by reference in their entirety.

[0398] The ERG protocol used in this study employed full-field stimulation, so recordings reflect the net response across the entire visual field. Therefore, the ability to resolve the effects of rAAV-based treatments on ERGs depends on the extent of transgene expression in the retina.

[0399] In the early stages of retinoschisis, the synapses between the photoreceptors and the inner retinal neurons are destroyed. This initially manifests as a reduction in the b-wave amplitude in the DA ERG. As the disease progresses, the photoreceptors disappear and, as a result, the a-wave amplitude also decreases. In severe cases, the ERG response may not be detectable.

[0400] A summary of the ERG results is presented in Table 8. Although no improvements were seen in treated animals after a single flash stimulus, the flicker ERGs showed a significant improvement in cone responses in Group 11 animals treated with RK:RS1_18. Representative flicker ERG traces are shown in Table 8. Fig. 27 Shown in.

[0401] Table 8: ERG results matrix

[0402]

[0403]

[0404] Summary of ERG results at the 6-month time point. All values ​​are mean ± SD. IT = implicit time; T-

[0405] P = valley to peak.

[0406] Summary of results

[0407] The results of subretinal injections show that at the dose used in this study, RS1 expression from AAV204.CBh:RS1_16 may cause certain toxicity, such as possible retinal degeneration at the injection site.CBh is a strong, ubiquitous promoter. Overexpression of RS1 in cells that do not normally express RS1 may be the cause of degeneration, which suggests that lower doses can be used to deliver RS1 transgenes that are operably linked to a strong, ubiquitous promoter. In contrast, only about half of the eyes treated with AAV204.RK:RS1_18 have similar degenerative areas. RK is a photoreceptor-specific promoter, which is the main site of RS1 expression in the WT retina. Although Western analysis shows that the two promoters produce equal amounts of recombinant proteins, it is obviously beneficial to exclude RS1 expression in non-photoreceptors. Even with RK-driven photoreceptor-specific expression, secreted RS1 is observed in the inner retina adjacent to RS1-positive photoreceptors, indicating that it can diffuse radially through the retina. This expression pattern is very compatible with the endogenous RS1 expression pattern in WT animals. Notably, Western analysis showed that RS1 expression in treated eyes was equal to endogenous expression in WT mice, even though only a small portion of the retina was treated, suggesting that (i) the load of RS1 protein in the treated area may be significantly higher than physiological levels and (ii) RK promoter-related degeneration may be addressed by using a lower dose.

[0408] Because RS1 mutations cause severe structural damage to the retina, this study was initially designed for intravitreal injections, a less invasive technique than subretinal injections and with the goal of minimizing the risk of additional damage to an already compromised retina. However, animals treated intravitreally with AAV204.CBh:RS1_26 and AAV204.RK:RS1_28 had minimal expression of recombinant protein, prompting studies in the context of subretinal treatment. With subretinal delivery, the vector is confined to a smaller space immediately adjacent to the target layer, thereby increasing transduction efficiency and promoting transgene expression. Indeed, a comparison of the two studies showed that subretinal injections produced better expression than intravitreal injections.

[0409] The efficacy in this study was evaluated via three different measurements: cone density, ONL thickness, and ERG, all of which were reduced in untreated mutant mice. RS1 expression from the CBh promoter caused catastrophic retinal degeneration in all treated animals. The RK promoter, which has an expression profile targeting the desired cell type, is better tolerated, and the corresponding treated eyes have more cones, thicker ONL, and improved ERG cone responses compared to untreated animals. Areas of degeneration were still observed in some RK-treated eyes, but these areas were still directly related to the injection site. The edges of the degenerated areas, where the effective dose may be lower, showed improvements in both cone density and ONL thickness. Surprisingly, IHC analysis showed that cone density was slightly but significantly improved in areas of the treated eyes where RS1 immunoreactivity was not detected. This is true for both CBh-treated eyes and RK-treated eyes, and supports the hypothesis that low doses, even below the threshold of immunodetection, can still have a cone-protective effect.

[0410] In view of the encouraging results from subretinal delivery, pararetinal administration may be a preferred vector delivery method for treating XLRS. Similar to intravitreal injection and different from subretinal injection, pararetinal injection does not penetrate the retina, and is therefore less likely to cause mechanical damage to the retina than subretinal injection, and in XLRS cases, the retina is usually fragile. On the other hand, by positioning the injection bolus directly near the retina, pararetinal injection is superior to intravitreal injection. Previous studies in the NHP model using AAV204.CBh: GFP vectors have shown that this technology allows effective transduction of all retinal layers. Although mouse eyes are too small to achieve pararetinal delivery, the results of subretinal injections in current mouse studies still simulate the efficacy of pararetinal injections in non-human primates (NHPs) and humans.

[0411] Additional numbered embodiments

[0412] Additional numbered embodiments of the present disclosure are provided below:

[0413] Example 1. A method of treating retinoschisis in a subject in need thereof, comprising administering an AAV viral vector to the subject adjacent to the retina or subretina.

[0414] Embodiment 2. The method of embodiment 1, wherein the AAV viral vector comprises a photoreceptor-specific promoter operably linked to a transgene encoded by a heterologous nucleic acid.

[0415] Embodiment 3. The method according to embodiment 2, wherein the photoreceptor-specific promoter is selected from the group consisting of rhodopsin kinase (RK) promoter, rhodopsin (RHO) promoter, beta phosphodiesterase (PDE) promoter and retinitis pigmentosa (RP1) promoter.

[0416] Embodiment 4. The method of embodiment 2, wherein the photoreceptor-specific promoter is a rhodopsin kinase (RK) promoter.

[0417] Embodiment 5. A method according to embodiment 4, wherein the RK promoter comprises, consists essentially of, or consists of a nucleic acid sequence that is at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO:196.

[0418] Embodiment 6. The method according to any one of embodiments 1-5, comprising administering the AAV viral vector to the subject's retina.

[0419] Embodiment 7. The method of any one of embodiments 1-6, wherein the subject is a human, and wherein the AAV viral vector is administered at a dose of about 1010 to about 1012 viral genomes (vg).

[0420] Embodiment 8. The method of any one of embodiments 1-6, wherein the retinoschisis is X-linked retinoschisis.

[0421] Embodiment 9. The method of any one of embodiments 2-8, wherein the transgene is RS1.

[0422] Example 10. The method according to Example 9, wherein the transgene comprises a nucleic acid sequence having at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to SEQ ID NO: 117, or wherein the transgene encodes an amino acid sequence having at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to SEQ ID NO: 143.

[0423] Example 11. A method of treating an eye disease or disorder in a subject in need thereof, comprising administering to the subject an AAV viral vector, wherein the AAV viral vector comprises an AAV vector genome, and wherein the AAV vector genome comprises, in the 5' to 3' direction:

[0424] (a) a first AAV inverted terminal repeat,

[0425] (b) a promoter,

[0426] (c) a heterologous nucleic acid encoding Opa1,

[0427] (d) a polyadenylation signal, and

[0428] (e) a second AAV inverted terminal repeat.

[0429] Example 12. The method according to Example 11, wherein the promoter is the CBh promoter, and the CBh promoter comprises a sequence having at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to SEQ ID NO: 154.

[0430] Example 13. The method according to Example 11, wherein the promoter is the MeCP2 promoter, and the MeCP2 promoter comprises a sequence having at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to SEQ ID NO: 156.

[0431] Example 14. The method according to any one of Examples 11-13, wherein the AAV vector genome comprises an intron sequence having at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to SEQ ID NO: 200 or 227, and wherein the intron sequence is located between the promoter and the heterologous nucleic acid encoding Opa1.

[0432] Example 15. The method according to Example 14, wherein the intron sequence is located immediately downstream of the promoter, with no additional nucleotides therebetween.

[0433] Embodiment 16. The method of any one of embodiments 11-15, wherein the heterologous nucleic acid encoding Opal comprises a sequence that is at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identical to any one of SEQ ID NOs: 175, 182 and 184.

[0434] Embodiment 17. The method of any one of embodiments 11-16, wherein the heterologous nucleic acid encodes an Opa1 protein comprising an amino acid sequence that is at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identical to any one of SEQ ID NOs: 180, 183 and 185.

[0435] Embodiment 18. The method of any one of embodiments 11-16, wherein the heterologous nucleic acid encodes an Opa1 protein comprising an amino acid sequence that is at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 180.

[0436] Embodiment 19. A method according to any one of embodiments 11-18, wherein the polyadenylation signal comprises a sequence that is at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 201 or 225.

[0437] Embodiment 20. A method according to any one of embodiments 11-19, wherein the AAV vector genome does not contain any telomeric repeat sequences.

[0438] Embodiment 21. The method of any one of Embodiments 11-19, wherein the AAV vector genome comprises a first telomeric repeat sequence located between the polyadenylation signal and the second AAV inverted terminal repeat sequence, and wherein the first telomeric repeat sequence is at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO:202.

[0439] Embodiment 22. A method according to any one of embodiments 11-21, wherein the AAV vector genome comprises a second telomeric repeat sequence located between the first AAV inverted terminal repeat sequence and the promoter, and wherein the second telomeric repeat sequence is at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO:203.

[0440] Embodiment 23. A method according to any one of Embodiments 11-22, wherein the first AAV inverted terminal repeat sequence comprises, or consists of, a sequence that is at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 253, and / or wherein the second AAV inverted terminal repeat sequence comprises, or consists of, a sequence that is at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 254.

[0441] Embodiment 24. The method of any one of embodiments 11-23, wherein the AAV vector genome comprises in the 5' to 3' direction:

[0442] (a) said first AAV inverted terminal repeat sequence,

[0443] (b) said promoter comprising a sequence that is at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 154,

[0444] (c) a heterologous nucleic acid encoding Opa1,

[0445] (d) said polyadenylation signal comprising a sequence that is at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 201, and

[0446] (e) the second AAV inverted terminal repeat sequence.

[0447] Embodiment 25. A method according to embodiment 24, wherein the AAV vector genome comprises an intron sequence that is at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 200, and wherein the intron sequence is located between the promoter and the heterologous nucleic acid encoding Opa1.

[0448] Embodiment 26. A method according to embodiment 24 or 25, wherein the AAV vector genome comprises a sequence that is at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 228.

[0449] Embodiment 27. The method of any one of embodiments 24-26, wherein the Opal protein comprises an amino acid sequence that is at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO:180.

[0450] Embodiment 28. A method according to any one of embodiments 11-27, wherein the AAV vector genome comprises a polynucleotide sequence that is at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identical to any one of SEQ ID NOs: 230-239.

[0451] Embodiment 29. The method of any one of embodiments 11-28, wherein the ocular disease or disorder is autosomal dominant optic atrophy.

[0452] Embodiment 30. A method of treating an ocular disease or disorder in a subject in need thereof, comprising administering to the subject an AAV viral vector, wherein the AAV viral vector comprises an AAV vector genome, wherein the AAV vector genome comprises in the 5' to 3' direction:

[0453] (a) the first AAV inverted terminal repeat sequence,

[0454] (b) a promoter,

[0455] (c) a heterologous nucleic acid encoding RS1,

[0456] (d) a polyadenylation signal, and

[0457] (e) Second AAV inverted terminal repeat sequence.

[0458] Embodiment 31. The method of embodiment 30, wherein the promoter is a photoreceptor-specific promoter.

[0459] Embodiment 32. The method according to embodiment 31, wherein the photoreceptor-specific promoter is selected from the group consisting of a rhodopsin kinase (RK) promoter, a rhodopsin (Rho) promoter, a beta phosphodiesterase (PDE) promoter, and a retinitis pigmentosa (RP1) promoter.

[0460] Embodiment 33. A method according to embodiment 30, wherein the promoter is a CBh promoter, and the CBh promoter comprises a sequence that is at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO:154.

[0461] Embodiment 34. A method according to embodiment 30, wherein the promoter is an RK promoter, and the RK promoter comprises a sequence that is at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO:196.

[0462] Embodiment 35. A method according to embodiment 30, wherein the promoter is a Rho promoter, and the Rho promoter comprises a sequence that is at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 197.

[0463] Embodiment 36. A method according to embodiment 30, wherein the promoter is a PDE promoter, and the PDE promoter comprises a sequence that is at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 198.

[0464] Embodiment 37. A method according to any one of embodiments 30-36, wherein the AAV vector genome comprises an IRBP enhancer sequence upstream of the promoter, wherein the IRBP enhancer sequence is at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO:199.

[0465] Embodiment 38. The method according to embodiment 37, wherein the IRBP enhancer sequence is located immediately upstream of the promoter without any additional nucleotides in between.

[0466] Embodiment 39. A method according to any one of embodiments 30-38, wherein the AAV vector genome comprises a CVA-MVM intron sequence that is at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 226, and wherein the CVA-MVM intron sequence is located between the promoter and the heterologous nucleic acid encoding RS1.

[0467] Embodiment 40. A method according to any one of embodiments 30-38, wherein the AAV vector genome comprises an intron sequence that is at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 200 or 222, and wherein the intron is located between the promoter and the heterologous nucleic acid encoding RS1.

[0468] Embodiment 41. A method according to any one of embodiments 30-40, wherein the heterologous nucleic acid encoding RS1 comprises a sequence that is at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO:117.

[0469] Embodiment 42. A method according to any one of embodiments 30-41, wherein the heterologous nucleic acid encodes an RS1 protein, and the RS1 protein comprises an amino acid sequence that is at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 143.

[0470] Embodiment 43. A method according to any one of embodiments 30-42, wherein the polyadenylation signal comprises a sequence that is at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 201 or 225.

[0471] Embodiment 44. A method according to any one of embodiments 30-43, wherein the AAV vector genome does not contain any telomeric repeat sequences.

[0472] Embodiment 45. The method of any one of Embodiments 30-43, wherein the AAV vector genome comprises a first telomeric repeat sequence located between the polyadenylation signal and the second AAV inverted terminal repeat sequence, and wherein the first telomeric repeat sequence is at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO:203.

[0473] Embodiment 46. A method according to any one of embodiments 30-45, wherein the AAV vector genome comprises a human β-globin scaffold / matrix attachment region (βGlo_s / MAR) sequence located between the polyadenylation signal and the second AAV inverted terminal repeat sequence or between the polyadenylation signal and the first telomeric repeat sequence, wherein βGlo_s / MAR has at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity with SEQ ID NO:221.

[0474] Embodiment 47. A method according to any one of Embodiments 30-46, wherein the first AAV inverted terminal repeat sequence comprises, or consists of, a sequence that is at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 255, and / or wherein the second AAV inverted terminal repeat sequence comprises, or consists of, a sequence that is at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 256.

[0475] Embodiment 48. The method of any one of embodiments 30-47, wherein the AAV vector genome comprises in the 5' to 3' direction:

[0476] (a) said first AAV inverted terminal repeat sequence,

[0477] (b) said IRBP enhancer sequence having at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to SEQ ID NO: 199,

[0478] (c) said RK promoter comprising a sequence that is at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 196,

[0479] (d) the heterologous nucleic acid encoding RS1,

[0480] (e) said polyadenylation signal comprising a sequence that is at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 225, and

[0481] (f) the second AAV inverted terminal repeat sequence.

[0482] Embodiment 49. A method according to any one of embodiments 30-48, wherein the AAV vector genome comprises an intron sequence that is at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 222, and wherein the intron sequence is located between the promoter and the heterologous nucleic acid encoding RS1.

[0483] Embodiment 50. A method according to embodiment 49, wherein the AAV vector genome comprises a CBA sequence of SEQ ID NO: 229, or a sequence having at most 5, at most 4, at most 3, at most 2 or at most 1 mutations thereto, and wherein the CBA sequence is located immediately upstream of the intron sequence without any additional nucleotides in between.

[0484] Example 51. A method according to Example 49 or 50, wherein the AAV vector genome comprises a CBA-MVM intron sequence that is at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 226, and wherein the CVA-MVM intron sequence is located between the promoter and the heterologous nucleic acid encoding RS1.

[0485] Embodiment 52. A method according to any one of embodiments 48-51, wherein the AAV vector genome comprises a sequence that is at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 224.

[0486] Embodiment 53. A method according to any one of embodiments 30-52, wherein the AAV vector genome comprises a polynucleotide sequence that is at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identical to any one of SEQ ID NOs: 224 and 240-252.

[0487] Embodiment 54. The method of any one of embodiments 30-53, wherein the ocular disease or disorder is X-linked retinoschisis.

[0488] Embodiment 55. A method according to any one of embodiments 1-54, wherein the AAV viral vector comprises an AAV capsid protein, and the AAV capsid protein comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 1-3, 30-34, 49, 67, 84 or 164.

[0489] Example 56. A method according to Example 55, wherein the AAV viral vector comprises an AAV capsid protein, and the AAV capsid protein comprises an amino acid sequence that is at least 98%, at least 99%, at least 99.5% or 100% identical to SEQ ID NO: 1-3, 30-34, 49, 67, 84 or 164.

[0490] Embodiment 57. A method according to embodiment 55, wherein the AAV viral vector comprises an AAV capsid protein, and the AAV capsid protein comprises an amino acid sequence that is at least 95%, at least 98%, at least 99%, at least 99.5% or 100% identical to SEQ ID NO:2, or is composed thereof.

[0491] Embodiment 58. The method of any one of embodiments 11-57, wherein said administration is pararetinal administration.

[0492] Example 59. The method of any one of Examples 1-10 and 58, wherein the pararetinal administration comprises injection at a distance between 0 and 13 millimeters (mm), between 0 and 10 mm, between 0 and 5 mm, or between 0 and 3 mm from the surface of the retina in the posterior vitreous cavity of the eye.

[0493] Embodiment 60. The method of any one of embodiments 1-59, wherein the AAV viral vector is administered at a dose of about 1010 to about 1012 viral genomes (vg).

[0494] Embodiment 61. A method according to any one of embodiments 1-60, wherein the subject is human.

[0495] Embodiment 62. A nucleic acid comprising, in the 5' to 3' direction:

[0496] (a) the first AAV inverted terminal repeat sequence,

[0497] (b) a promoter,

[0498] (c) a heterologous nucleic acid encoding Opa1,

[0499] (d) a polyadenylation signal, and

[0500] (e) Second AAV inverted terminal repeat sequence.

[0501] Embodiment 63. The nucleic acid of embodiment 62, wherein the promoter is a CBh promoter comprising a sequence that is at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 154.

[0502] Embodiment 64. The nucleic acid of embodiment 62, wherein the promoter is a MeCP2 promoter, comprising a sequence that is at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 156.

[0503] Embodiment 65. A nucleic acid according to any one of embodiments 62-64, wherein the AAV vector genome comprises an intron sequence that is at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 200 or 227, and wherein the intron sequence is located between the promoter and the heterologous nucleic acid encoding Opa1.

[0504] Embodiment 66. A nucleic acid according to any one of embodiments 62-65, wherein the intron sequence is located immediately downstream of the promoter without any additional nucleotides in between.

[0505] Embodiment 67. The nucleic acid of any one of embodiments 62-66, wherein the heterologous nucleic acid encoding Opa1 comprises a sequence that is at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identical to any one of SEQ ID NOs: 175, 182, and 184.

[0506] Embodiment 68. The nucleic acid of any one of embodiments 62-67, wherein the heterologous nucleic acid encodes an Opa1 protein comprising an amino acid sequence that is at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identical to any one of SEQ ID NOs: 180, 183, and 185.

[0507] Embodiment 69. The nucleic acid of any one of embodiments 62-67, wherein the heterologous nucleic acid encodes an Opal protein comprising an amino acid sequence that is at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 180.

[0508] Embodiment 70. A nucleic acid according to any one of embodiments 62-69, wherein the polyadenylation signal comprises a sequence that is at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 201 or 225.

[0509] Embodiment 71. A nucleic acid according to any one of embodiments 62-70, wherein the AAV vector genome does not contain any telomeric repeat sequences.

[0510] Embodiment 72. A nucleic acid according to any one of embodiments 62-70, wherein the AAV vector genome comprises a first telomeric repeat sequence located between the polyadenylation signal and the second AAV inverted terminal repeat sequence, and wherein the first telomeric repeat sequence is at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO:202.

[0511] Embodiment 73. A nucleic acid according to any one of embodiments 62-72, wherein the AAV vector genome comprises a second telomeric repeat sequence located between the first AAV inverted terminal repeat sequence and the promoter, and wherein the second telomeric repeat sequence is at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO:203.

[0512] Embodiment 74. A nucleic acid according to any one of embodiments 62-73, wherein the first AAV inverted terminal repeat sequence comprises, or consists of, a sequence that is at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 253, and / or wherein the second AAV inverted terminal repeat sequence comprises, or consists of, a sequence that is at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 254.

[0513] Embodiment 75. The nucleic acid of any one of embodiments 62-74, wherein the AAV vector genome comprises in the 5' to 3' direction:

[0514] (a) said first AAV inverted terminal repeat sequence,

[0515] (b) said promoter comprising a sequence that is at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 154,

[0516] (c) a heterologous nucleic acid encoding Opa1,

[0517] (d) said polyadenylation signal comprising a sequence that is at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 201, and

[0518] (e) the second AAV inverted terminal repeat sequence.

[0519] Embodiment 76. A nucleic acid according to embodiment 75, comprising an intron sequence having at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to SEQ ID NO: 200, and wherein the intron sequence is located between the promoter and the heterologous nucleic acid encoding Opa1.

[0520] Embodiment 77. The nucleic acid of embodiment 75 or 76, wherein the Opal protein comprises an amino acid sequence that is at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO:180.

[0521] Embodiment 78. A nucleic acid according to any one of embodiments 75-77, comprising a sequence that is at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 228.

[0522] Embodiment 79. A nucleic acid according to any one of embodiments 62-78, comprising a polynucleotide sequence that is at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identical to any one of SEQ ID NOs: 230-239.

[0523] Embodiment 80. A nucleic acid comprising, in the 5' to 3' direction:

[0524] (a) the first AAV inverted terminal repeat sequence,

[0525] (b) a promoter,

[0526] (c) a heterologous nucleic acid encoding RS1,

[0527] (d) a polyadenylation signal, and

[0528] (e) Second AAV inverted terminal repeat sequence.

[0529] Embodiment 81. The nucleic acid of embodiment 80, wherein the promoter is a photoreceptor-specific promoter.

[0530] Embodiment 82. A nucleic acid according to embodiment 81, wherein the photoreceptor-specific promoter is selected from the group consisting of a rhodopsin kinase (RK) promoter, a rhodopsin (RHO) promoter, a beta phosphodiesterase (PDE) promoter, and a retinitis pigmentosa (RP1) promoter.

[0531] Embodiment 83. A nucleic acid according to embodiment 80, wherein the promoter is a CBh promoter, and the CBh promoter comprises a sequence that is at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 154.

[0532] Embodiment 84. A nucleic acid according to embodiment 80, wherein the promoter is an RK promoter, and the RK promoter comprises a sequence that is at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO:196.

[0533] Embodiment 85. A nucleic acid according to embodiment 80, wherein the promoter is a Rho promoter, and the Rho promoter comprises a sequence that is at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 197.

[0534] Embodiment 86. A nucleic acid according to embodiment 80, wherein the promoter is a PDE promoter, and the PDE promoter comprises a sequence that is at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 198.

[0535] Embodiment 87. A nucleic acid according to any one of embodiments 80-86, comprising an IRBP enhancer sequence upstream of the promoter, wherein the IRBP enhancer sequence is at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO:199.

[0536] Embodiment 88. The nucleic acid of embodiment 87, wherein the IRBP enhancer sequence is located immediately upstream of the promoter without any additional nucleotides in between.

[0537] Embodiment 89. A nucleic acid according to any one of embodiments 80-88, wherein the AAV vector genome comprises a CVA-MVM intron sequence that is at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 226, and wherein the CVA-MVM intron sequence is located between the promoter and the heterologous nucleic acid encoding RS1.

[0538] Embodiment 90. A nucleic acid according to any one of embodiments 80-88, wherein the AAV vector genome comprises an intron sequence that is at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 200 or 222, and wherein the intron is located between the promoter and the heterologous nucleic acid encoding RS1.

[0539] Embodiment 91. A nucleic acid according to any one of embodiments 80-88, wherein the heterologous nucleic acid encoding RS1 comprises a sequence that is at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 117.

[0540] Embodiment 92. A nucleic acid according to any one of embodiments 80-91, wherein the heterologous nucleic acid encodes an RS1 protein, wherein the RS1 protein comprises an amino acid sequence that is at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 143.

[0541] Embodiment 93. A nucleic acid according to any one of embodiments 80-92, wherein the polyadenylation signal comprises a sequence that is at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 201 or 225.

[0542] Embodiment 94. A nucleic acid according to any one of embodiments 80-93, wherein the AAV vector genome does not contain any telomeric repeat sequences.

[0543] Embodiment 95. A nucleic acid according to any one of embodiments 80-93, wherein the AAV vector genome comprises a first telomeric repeat sequence located between the polyadenylation signal and the second AAV inverted terminal repeat sequence, and wherein the first telomeric repeat sequence is at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO:203.

[0544] Embodiment 96. A nucleic acid according to any one of embodiments 80-95, wherein the AAV vector genome comprises a human β-globin scaffold / matrix attachment region (βGlo_s / MAR) sequence located between the polyadenylation signal and the second AAV inverted terminal repeat sequence or between the polyadenylation signal and the first telomeric repeat sequence, wherein the βGlo_s / MAR sequence is at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO:221.

[0545] Embodiment 97. A nucleic acid according to any one of embodiments 80-96, wherein the first AAV inverted terminal repeat sequence comprises, or consists of, a sequence that is at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 255, and / or wherein the second AAV inverted terminal repeat sequence comprises, or consists of, a sequence that is at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 256.

[0546] Embodiment 98. The nucleic acid of any one of embodiments 80-97, wherein the AAV vector genome comprises in the 5' to 3' direction:

[0547] (a) said first AAV inverted terminal repeat sequence,

[0548] (b) said IRBP enhancer sequence having at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to SEQ ID NO: 199,

[0549] (c) said RK promoter comprising a sequence that is at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 196,

[0550] (d) the heterologous nucleic acid encoding RS1,

[0551] (e) said polyadenylation signal comprising a sequence that is at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 225, and

[0552] (f) the second AAV inverted terminal repeat sequence.

[0553] Embodiment 99. A nucleic acid according to any one of embodiments 80-98, comprising an intron sequence that is at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 222, and wherein the intron sequence is located between the promoter and the heterologous nucleic acid encoding RS1.

[0554] Embodiment 100. A nucleic acid according to embodiment 99, comprising a CBA sequence of SEQ ID NO: 229, or a sequence thereof having at most 5, at most 4, at most 3, at most 2, or at most 1 mutation, and wherein the CBA sequence is located immediately upstream of the intron sequence without any additional nucleotides in between.

[0555] Embodiment 101. A nucleic acid according to embodiment 99 or 100, comprising a CBA-MVM intron sequence that is at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 226, and wherein the CVA-MVM intron sequence is located between the promoter and the heterologous nucleic acid encoding RS1.

[0556] Embodiment 102. A nucleic acid according to any one of embodiments 98-101, comprising a sequence that is at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 224.

[0557] Example 103. The nucleic acid according to any one of Examples 80-102, which comprises a polynucleotide sequence having at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to any one of SEQ ID NO: 224 and 240-252.

[0558] Example 104. A nucleic acid, which comprises, in the 5' to 3' direction:

[0559] (a) a promoter,

[0560] (b) a heterologous nucleic acid encoding a transgene, and

[0561] (c) a polyadenylation signal,

[0562] wherein the promoter is the CBh promoter, the CBh promoter comprises a sequence having at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to SEQ ID NO: 154, and wherein the polyadenylation signal comprises a sequence having at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to SEQ ID NO: 201 or 225.

[0563] Example 105. The nucleic acid according to Example 104, which comprises an intron sequence having at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to SEQ ID NO: 200, 222, 226 or 227, and wherein the intron is located between the promoter and the heterologous nucleic acid encoding the transgene.

[0564] Example 106. The nucleic acid according to any one of Examples 104-105, which comprises a first ITR located 5' of the promoter and a second ITR located 3' of the polyadenylation signal.

[0565] Example 107. The nucleic acid according to any one of Examples 104-106, wherein the nucleic acid does not comprise any telomeric repeat sequences.

[0566] Example 108. The nucleic acid according to any one of Examples 104-106, which comprises a first telomeric repeat sequence located between the polyadenylation signal and the second AAV ITR, and wherein the first telomeric repeat sequence has at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to SEQ ID NO: 202.

[0567] Embodiment 109. A nucleic acid according to any one of embodiments 104-108, comprising a second telomeric repeat sequence located between the first AAV inverted terminal repeat sequence and the promoter, and wherein the second telomeric repeat sequence is at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO:203.

[0568] Embodiment 110. A vector comprising the nucleic acid of any one of embodiments 62-109.

[0569] Embodiment 111. An AAV vector genome comprising the nucleic acid of any one of embodiments 62-109.

[0570] Embodiment 112. An AAV viral vector comprising the AAV vector genome according to embodiment 111.

[0571] Embodiment 113. An AAV viral vector according to Embodiment 112, wherein the AAV viral vector comprises an AAV capsid protein, and the AAV capsid protein comprises an amino acid sequence that is at least 95%, at least 98%, at least 99%, at least 99.5% or 100% identical to any one of SEQ ID NOs: 1-3, 30-34, 49, 84 and 164.

[0572] Embodiment 114. A method of expressing a transgene in a retinal cell, comprising delivering a nucleic acid according to any one of embodiments 62-109 to the retinal cell, or transducing the retinal cell with an AAV viral vector according to any one of embodiments 112-113.

[0573] Embodiment 115. The method of Embodiment 114, wherein the retinal cells are retinal ganglion cells.

[0574] Embodiment 116. A method of treating a disease or disorder comprising administering to a subject the AAV viral vector of any one of Embodiments 112-113.

[0575] Embodiment 117. The method of Embodiment 116, wherein the AAV viral vector is administered intraocularly, periocularly, intravitreally, adjacent to the retina, or subretinaly to the subject.

[0576] Embodiment 118. The method of embodiment 116 or 117, wherein the disease or condition is macular degeneration, retinitis pigmentosa, autosomal dominant optic atrophy, retinoschisis, Stargardt's disease, Bietti's crystalline dystrophy, or Best's vitelliform macular dystrophy.

[0577] Embodiment 119. The method of embodiment 116 or 117, wherein the disease or disorder is X-linked retinoschisis (XLRS).

Claims

1. A method of treating retinoschisis in a subject in need thereof, comprising administering an AAV viral vector to the subject adjacent to the retina or subretina.

2. The method of claim 1, wherein the AAV viral vector comprises a photoreceptor-specific promoter operably linked to a transgene encoded by a heterologous nucleic acid.

3. The method of claim 2, wherein the photoreceptor-specific promoter is selected from the group consisting of a rhodopsin kinase (RK) promoter, a rhodopsin (RHO) promoter, a beta phosphodiesterase (PDE) promoter, and a retinitis pigmentosa (RP1) promoter.

4. The method of claim 2, wherein the photoreceptor-specific promoter is a rhodopsin kinase (RK) promoter.

5. The method of claim 4, wherein the RK promoter comprises, consists essentially of, or consists of a nucleic acid sequence that is at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO:

196.

6. The method of any one of claims 1-5, comprising administering the AAV viral vector adjacent to the retina of the subject.

7. The method according to any one of claims 1-6, wherein the subject is a human, and wherein the AAV viral vector is expressed in an amount of about 10 10 to about 10 12 Dosage administration of viral genome (vg).

8. The method of any one of claims 1-6, wherein the retinoschisis is X-linked retinoschisis.

9. The method of any one of claims 2-8, wherein the transgene is RS1.

10. The method of claim 9, wherein the transgene comprises a nucleic acid sequence that is at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 117, or wherein the transgene encodes an amino acid sequence that is at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:

143.

11. A method of treating an ocular disease or disorder in a subject in need thereof, comprising administering to the subject an AAV viral vector, wherein the AAV viral vector comprises an AAV vector genome, wherein the AAV vector genome comprises in the 5' to 3' direction: (a) the first AAV inverted terminal repeat sequence, (b) a promoter, (c) a heterologous nucleic acid encoding Opa1, (d) a polyadenylation signal, and (e) Second AAV inverted terminal repeat sequence.

12. The method of claim 11, wherein the promoter is a CBh promoter comprising a sequence that is at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO:

154.

13. The method of claim 11, wherein the promoter is a MeCP2 promoter comprising a sequence that is at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO:

156.

14. The method of any one of claims 11-13, wherein the AAV vector genome comprises an intron sequence that is at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 200 or 227, and wherein the intron sequence is located between the promoter and the heterologous nucleic acid encoding Opa1.

15. The method according to claim 14, wherein the intron sequence is located immediately downstream of the promoter without any additional nucleotides therebetween.

16. The method of any one of claims 11-15, wherein the heterologous nucleic acid encoding Opal comprises a sequence at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identical to any one of SEQ ID NOs: 175, 182 and 184.

17. The method of any one of claims 11-16, wherein the heterologous nucleic acid encodes an Opal protein comprising an amino acid sequence at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identical to any one of SEQ ID NOs: 180, 183 and 185.

18. The method of any one of claims 11-16, wherein the heterologous nucleic acid encodes an Opal protein comprising an amino acid sequence at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO:

180.

19. The method of any one of claims 11-18, wherein the polyadenylation signal comprises a sequence that is at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 201 or 225.

20. The method of any one of claims 11-19, wherein the AAV vector genome does not contain any telomeric repeat sequences.

21. The method of any one of claims 11-19, wherein the AAV vector genome comprises a first telomeric repeat sequence located between the polyadenylation signal and the second AAV inverted terminal repeat sequence, and wherein the first telomeric repeat sequence is at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO:

202.

22. The method of any one of claims 11-21, wherein the AAV vector genome comprises a second telomeric repeat sequence located between the first AAV inverted terminal repeat sequence and the promoter, and wherein the second telomeric repeat sequence is at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO:

203.

23. The method of any one of claims 11-22, wherein the first AAV inverted terminal repeat comprises, or consists of, a sequence that is at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 253, and / or wherein the second AAV inverted terminal repeat comprises, or consists of, a sequence that is at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO:

254.

24. The method of any one of claims 11-23, wherein the AAV vector genome comprises in the 5' to 3' direction: (a) said first AAV inverted terminal repeat sequence, (b) said promoter comprising a sequence that is at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 154, (c) the heterologous nucleic acid encoding Opa1, (d) said polyadenylation signal comprising a sequence that is at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 201, and (e) the second AAV inverted terminal repeat sequence.

25. The method of claim 24, wherein the AAV vector genome comprises an intron sequence that is at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 200, and wherein the intron sequence is located between the promoter and the heterologous nucleic acid encoding Opa1.

26. The method of claim 24 or 25, wherein the AAV vector genome comprises a sequence that is at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO:

228.

27. The method of any one of claims 24-26, wherein the Opal protein comprises an amino acid sequence at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO:

180.

28. The method of any one of claims 11-27, wherein the AAV vector genome comprises a polynucleotide sequence that is at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identical to any one of SEQ ID NOs: 230-239.

29. The method of any one of claims 11-28, wherein the ocular disease or disorder is autosomal dominant optic atrophy.

30. A method of treating an ocular disease or disorder in a subject in need thereof, comprising administering to the subject an AAV viral vector, wherein the AAV viral vector comprises an AAV vector genome, wherein the AAV vector genome comprises in the 5' to 3' direction: (a) the first AAV inverted terminal repeat sequence, (b) a promoter, (c) a heterologous nucleic acid encoding RS1, (d) a polyadenylation signal, and (e) Second AAV inverted terminal repeat sequence.

31. The method of claim 30, wherein the promoter is a photoreceptor-specific promoter.

32. The method of claim 31, wherein the photoreceptor-specific promoter is selected from the group consisting of a rhodopsin kinase (RK) promoter, a rhodopsin (Rho) promoter, a beta phosphodiesterase (PDE) promoter, and a retinitis pigmentosa (RP1) promoter.

33. The method of claim 30, wherein the promoter is a CBh promoter comprising a sequence at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO:

154.

34. The method of claim 30, wherein the promoter is a RK promoter comprising a sequence that is at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO:

196.

35. The method of claim 30, wherein the promoter is a Rho promoter comprising a sequence that is at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO:

197.

36. The method of claim 30, wherein the promoter is a PDE promoter comprising a sequence that is at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO:

198.

37. A method according to any one of claims 30-36, wherein the AAV vector genome contains an IRBP enhancer sequence upstream of the promoter, wherein the IRBP enhancer sequence is at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO:

199.

38. The method of claim 37, wherein the IRBP enhancer sequence is located immediately upstream of the promoter without any additional nucleotides in between.

39. The method of any one of claims 30-38, wherein the AAV vector genome comprises a CVA-MVM intron sequence that is at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 226, and wherein the CVA-MVM intron sequence is located between the promoter and the heterologous nucleic acid encoding RS1.

40. The method of any one of claims 30-38, wherein the AAV vector genome comprises an intron sequence that is at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 200 or 222, and wherein the intron is located between the promoter and the heterologous nucleic acid encoding RS1.

41. The method of any one of claims 30-40, wherein the heterologous nucleic acid encoding RS1 comprises a sequence that is at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO:

117.

42. The method of any one of claims 30-41, wherein the heterologous nucleic acid encodes an RS1 protein comprising an amino acid sequence that is at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO:

143.

43. The method of any one of claims 30-42, wherein the polyadenylation signal comprises a sequence that is at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 201 or 225.

44. The method of any one of claims 30-43, wherein the AAV vector genome does not comprise any telomeric repeat sequences.

45. The method of any one of claims 30-43, wherein the AAV vector genome comprises a first telomeric repeat sequence located between the polyadenylation signal and the second AAV inverted terminal repeat sequence, and wherein the first telomeric repeat sequence is at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO:

203.

46. ​​A method according to any one of claims 30-45, wherein the AAV vector genome comprises a human β-globin scaffold / matrix attachment region (βGlo_s / MAR) sequence located between the polyadenylation signal and the second AAV inverted terminal repeat sequence or between the polyadenylation signal and the first telomeric repeat sequence, wherein βGlo_s / MAR has at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity with SEQ ID NO:

221.

47. The method of any one of claims 30-46, wherein the first AAV inverted terminal repeat comprises, or consists of, a sequence that is at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 255, and / or wherein the second AAV inverted terminal repeat comprises, or consists of, a sequence that is at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO:

256.

48. The method of any one of claims 30-47, wherein the AAV vector genome comprises in the 5' to 3'' direction: (a) said first AAV inverted terminal repeat sequence, (b) said IRBP enhancer sequence having at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to SEQ ID NO: 199, (c) said RK promoter comprising a sequence that is at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 196, (d) the heterologous nucleic acid encoding RS1, (e) said polyadenylation signal comprising a sequence that is at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 225, and (f) the second AAV inverted terminal repeat sequence.

49. The method of any one of claims 30-48, wherein the AAV vector genome comprises an intron sequence that is at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 222, and wherein the intron sequence is located between the promoter and the heterologous nucleic acid encoding RS1.

50. The method of claim 49, wherein the AAV vector genome comprises a CBA sequence of SEQ ID NO: 229, or a sequence having at most 5, at most 4, at most 3, at most 2 or at most 1 mutations thereto, and wherein the CBA sequence is located immediately upstream of the intron sequence without any additional nucleotides therebetween.

51. A method according to claim 49 or 50, wherein the AAV vector genome comprises a CBA-MVM intron sequence that is at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO:226, and wherein the CVA-MVM intron sequence is located between the promoter and the heterologous nucleic acid encoding RS1.

52. The method of any one of claims 48-51, wherein the AAV vector genome comprises a sequence that is at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO:

224.

53. The method of any one of claims 30-52, wherein the AAV vector genome comprises a polynucleotide sequence that is at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identical to any one of SEQ ID NOs: 224 and 240-252.

54. The method of any one of claims 30-53, wherein the ocular disease or disorder is X-linked retinoschisis.

55. The method of any one of claims 1-54, wherein the AAV viral vector comprises an AAV capsid protein comprising an amino acid sequence that is at least 95% identical to SEQ ID NO: 1-3, 30-34, 49, 67, 84 or 164.

56. The method of claim 55, wherein the AAV viral vector comprises an AAV capsid protein comprising an amino acid sequence that is at least 98%, at least 99%, at least 99.5% or 100% identical to SEQ ID NO: 1-3, 30-34, 49, 67, 84 or 164.

57. The method of claim 55, wherein the AAV viral vector comprises an AAV capsid protein comprising, or consisting of, an amino acid sequence that is at least 95%, at least 98%, at least 99%, at least 99.5% or 100% identical to SEQ ID NO:

2.

58. The method of any one of claims 11-57, wherein the administration is pararetinal administration.

59. The method of any one of claims 1-10 and 58, wherein the pararetinal administration comprises injecting at a distance between 0 and 13 millimeters (mm), between 0 and 10 mm, between 0 and 5 mm, or between 0 and 3 mm from the surface of the retina in the posterior vitreous cavity of the eye.

60. The method of any one of claims 1-59, wherein the AAV viral vector is present in an amount of about 10 10 to about 10 12 Dosage administration of viral genome (vg).

61. The method of any one of claims 1-60, wherein the subject is a human.

62. A nucleic acid comprising, in the 5' to 3' direction: (a) the first AAV inverted terminal repeat sequence, (b) a promoter, (c) a heterologous nucleic acid encoding Opa1, (d) a polyadenylation signal, and (e) Second AAV inverted terminal repeat sequence.

63. The nucleic acid of claim 62, wherein the promoter is a CBh promoter comprising a sequence that is at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO:

154.

64. The nucleic acid of claim 62, wherein the promoter is a MeCP2 promoter comprising a sequence that is at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO:

156.

65. The nucleic acid of any one of claims 62-64, wherein the AAV vector genome comprises an intron sequence that is at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 200 or 227, and wherein the intron sequence is located between the promoter and the heterologous nucleic acid encoding Opa1.

66. The nucleic acid according to any one of claims 62-65, wherein the intron sequence is located immediately downstream of the promoter without any additional nucleotides in between.

67. The nucleic acid of any one of claims 62-66, wherein the heterologous nucleic acid encoding Opal comprises a sequence at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identical to any one of SEQ ID NOs: 175, 182 and 184.

68. The nucleic acid of any one of claims 62-67, wherein the heterologous nucleic acid encodes an Opal protein comprising an amino acid sequence at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identical to any one of SEQ ID NOs: 180, 183 and 185.

69. The nucleic acid of any one of claims 62-67, wherein the heterologous nucleic acid encodes an Opal protein comprising an amino acid sequence at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO:

180.

70. The nucleic acid of any one of claims 62-69, wherein the polyadenylation signal comprises a sequence that is at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 201 or 225.

71. The nucleic acid of any one of claims 62-70, wherein the AAV vector genome does not comprise any telomeric repeat sequences.

72. A nucleic acid according to any one of claims 62-70, wherein the AAV vector genome comprises a first telomeric repeat sequence located between the polyadenylation signal and the second AAV inverted terminal repeat sequence, and wherein the first telomeric repeat sequence is at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO:

202.

73. A nucleic acid according to any one of claims 62-72, wherein the AAV vector genome comprises a second telomeric repeat sequence located between the first AAV inverted terminal repeat sequence and the promoter, and wherein the second telomeric repeat sequence is at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO:

203.

74. The nucleic acid of any one of claims 62-73, wherein the first AAV inverted terminal repeat comprises, or consists of, a sequence that is at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 253, and / or wherein the second AAV inverted terminal repeat comprises, or consists of, a sequence that is at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO:

254.

75. The nucleic acid of any one of claims 62-74, wherein the AAV vector genome comprises in the 5' to 3' direction: (a) said first AAV inverted terminal repeat sequence, (b) said promoter comprising a sequence that is at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 154, (c) the heterologous nucleic acid encoding Opa1, (d) said polyadenylation signal comprising a sequence that is at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 201, and (e) the second AAV inverted terminal repeat sequence.

76. The nucleic acid of claim 75, comprising an intron sequence that is at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO:200, and wherein the intron sequence is located between the promoter and the heterologous nucleic acid encoding Opal.

77. The nucleic acid of claim 75 or 76, wherein the Opal protein comprises an amino acid sequence that is at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO:

180.

78. The nucleic acid of any one of claims 75-77, comprising a sequence that is at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO:

228.

79. The nucleic acid of any one of claims 62-78, comprising a polynucleotide sequence that is at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identical to any one of SEQ ID NOs: 230-239.

80. A nucleic acid comprising, in the 5' to 3' direction: (a) the first AAV inverted terminal repeat sequence, (b) a promoter, (c) a heterologous nucleic acid encoding RS1, (d) a polyadenylation signal, and (e) Second AAV inverted terminal repeat sequence.

81. The nucleic acid of claim 80, wherein the promoter is a photoreceptor-specific promoter.

82. The nucleic acid of claim 81, wherein the photoreceptor-specific promoter is selected from the group consisting of a rhodopsin kinase (RK) promoter, a rhodopsin (RHO) promoter, a beta phosphodiesterase (PDE) promoter, and a retinitis pigmentosa (RP1) promoter.

83. The nucleic acid of claim 80, wherein the promoter is a CBh promoter comprising a sequence that is at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO:

154.

84. The nucleic acid of claim 80, wherein the promoter is an RK promoter comprising a sequence that is at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO:

196.

85. The nucleic acid of claim 80, wherein the promoter is a Rho promoter comprising a sequence that is at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO:

197.

86. The nucleic acid of claim 80, wherein the promoter is a PDE promoter comprising a sequence that is at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO:

198.

87. A nucleic acid according to any one of claims 80-86, comprising an IRBP enhancer sequence upstream of the promoter, wherein the IRBP enhancer sequence is at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO:

199.

88. The nucleic acid of claim 87, wherein the IRBP enhancer sequence is located immediately upstream of the promoter without any additional nucleotides in between.

89. A nucleic acid according to any one of claims 80-88, wherein the AAV vector genome comprises a CVA-MVM intron sequence that is at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ IDNO:226, and wherein the CVA-MVM intron sequence is located between the promoter and the heterologous nucleic acid encoding RS1.

90. A nucleic acid according to any one of claims 80-88, wherein the AAV vector genome comprises an intron sequence that is at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO:200 or 222, and wherein the intron is located between the promoter and the heterologous nucleic acid encoding RS1.

91. The nucleic acid of any one of claims 80-88, wherein the heterologous nucleic acid encoding RS1 comprises a sequence that is at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO:

117.

92. A nucleic acid according to any one of claims 80-91, wherein the heterologous nucleic acid encodes an RS1 protein, which comprises an amino acid sequence that is at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO:

143.

93. The nucleic acid of any one of claims 80-92, wherein the polyadenylation signal comprises a sequence that is at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 201 or 225.

94. The nucleic acid of any one of claims 80-93, wherein the AAV vector genome does not comprise any telomeric repeat sequences.

95. The nucleic acid of any one of claims 80-93, wherein the AAV vector genome comprises a first telomeric repeat sequence located between the polyadenylation signal and the second AAV inverted terminal repeat sequence, and wherein the first telomeric repeat sequence is at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO:

203.

96. The nucleic acid of any one of claims 80-95, wherein the AAV vector genome comprises a human β-globin scaffold / matrix attachment region (βGlo_s / MAR) sequence located between the polyadenylation signal and the second AAV inverted terminal repeat sequence or between the polyadenylation signal and the first telomeric repeat sequence, wherein the βGlo_s / MAR sequence is at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO:

221.

97. The nucleic acid of any one of claims 80-96, wherein the first AAV inverted terminal repeat comprises, or consists of, a sequence that is at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO:255, and / or wherein the second AAV inverted terminal repeat comprises, or consists of, a sequence that is at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO:

256.

98. The nucleic acid of any one of claims 80-97, wherein the AAV vector genome comprises in the 5' to 3' direction: (a) said first AAV inverted terminal repeat sequence, (b) said IRBP enhancer sequence having at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to SEQ ID NO: 199, (c) said RK promoter comprising a sequence that is at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 196, (d) the heterologous nucleic acid encoding RS1, (e) said polyadenylation signal comprising a sequence that is at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 225, and (f) the second AAV inverted terminal repeat sequence.

99. A nucleic acid according to any one of claims 80-98, comprising an intron sequence that is at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO:222, and wherein the intron sequence is located between the promoter and the heterologous nucleic acid encoding RS1.

100. The nucleic acid of claim 99, comprising the CBA sequence of SEQ ID NO: 229, or a sequence having at most 5, at most 4, at most 3, at most 2 or at most 1 mutations therein, and wherein the CBA sequence is located immediately upstream of the intron sequence without any additional nucleotides in between.

101. A nucleic acid according to claim 99 or 100, which comprises a CBA-MVM intron sequence that is at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO:226, and wherein the CVA-MVM intron sequence is located between the promoter and the heterologous nucleic acid encoding RS1.

102. The nucleic acid of any one of claims 98-101, comprising a sequence that is at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO:

224.

103. The nucleic acid of any one of claims 80-102, comprising a polynucleotide sequence that is at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identical to any one of SEQ ID NOs: 224 and 240-252.

104. A nucleic acid comprising, in the 5' to 3' direction: (a) a promoter, (b) a heterologous nucleic acid encoding a transgene, and (c) polyadenylation signal, wherein the promoter is a CBh promoter comprising a sequence that is at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 154, and wherein the polyadenylation signal comprises a sequence that is at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 201 or 225.

105. The nucleic acid of claim 104, comprising an intron sequence that is at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 200, 222, 226 or 227, and wherein the intron is located between the promoter and the heterologous nucleic acid encoding the transgene.

106. The nucleic acid according to any one of claims 104-105, comprising a first ITR located 5' to the promoter and a second ITR located 3' to the polyadenylation signal.

107. The nucleic acid of any one of claims 104-106, wherein the nucleic acid does not comprise any telomeric repeat sequences.

108. The nucleic acid of any one of claims 104-106, comprising a first telomeric repeat sequence located between the polyadenylation signal and a second AAV ITR, and wherein the first telomeric repeat sequence is at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO:

202.

109. The nucleic acid of any one of claims 104-108, comprising a second telomeric repeat sequence located between the first AAV inverted terminal repeat sequence and the promoter, and wherein the second telomeric repeat sequence is at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO:

203.

110. A vector comprising the nucleic acid of any one of claims 62-109.

111. An AAV vector genome comprising the nucleic acid of any one of claims 62-109.

112. An AAV viral vector comprising the AAV vector genome according to claim 111.

113. The AAV viral vector of claim 112, wherein the AAV viral vector comprises an AAV capsid protein comprising an amino acid sequence that is at least 95%, at least 98%, at least 99%, at least 99.5% or 100% identical to any one of SEQ ID NOs: 1-3, 30-34, 49, 84 and 164.

114. A method for expressing a transgene in a retinal cell, comprising delivering a nucleic acid according to any one of claims 62-109 to the retinal cell, or transducing the retinal cell with an AAV viral vector according to any one of claims 112-113.

115. The method of claim 114, wherein the retinal cells are retinal ganglion cells.

116. A method of treating a disease or condition comprising administering to a subject an AAV viral vector according to any one of claims 112-113.

117. The method of claim 116, wherein the AAV viral vector is administered intraocularly, periocularly, intravitreally, pararetinaly, or subretinaly to the subject.

118. The method of claim 116 or 117, wherein the disease or condition is macular degeneration, retinitis pigmentosa, autosomal dominant optic atrophy, retinoschisis, Stargardt's disease, Bietti's crystalline dystrophy, or Best's vitelliform macular dystrophy.

119. The method of claim 116 or 117, wherein the disease or condition is X-linked retinoschisis (XLRS).

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