Compositions and methods for treating overt optic atrophy and X-linked retinoschisis

Abstract

Provided herein are recombinant AAV vectors, AAV viral vectors, capsid proteins, and administration methods for improved gene therapy, as well as methods for their production and use. These AAV vectors can be used to treat retinoschisis (e.g., X-linked retinoschisis) or manifest optic atrophy (DOA). In one aspect, the present disclosure provides a method of treating retinoschisis in a subject in need thereof, comprising administering an AAV viral vector to the subject via the juxtaretinal or subretinal route.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 63 / 332,015, filed April 18, 2022, the contents of which are incorporated by reference in their entirety.

[0002] Electronic Sequence Listing Reference 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 their entirety. [Background technology]

[0003] X-linked retinoschisis (XLRS) is a rare monogenic disorder that results in severe visual impairment. While female carriers are asymptomatic, affected males usually begin to show symptoms of the disease within the first decade, sometimes in infancy. The disease results from mutations in the RS1 gene, which is expressed in photoreceptors and retinal bipolar cells. In individuals with XLRS, adhesion of adjacent retinal layers is disrupted, resulting in discontinuities in the retinal circuitry, degeneration of photoreceptors, and cavities that result in impaired vision. The current standard of care for XLRS patients is palliative and includes correction of refractive errors, low vision aids, and genetic counseling. Complications such as retinal detachment (up to 22% of patients) and vitreous hemorrhage (up to 40% of patients) occur most frequently in the later stages of the disease and can be treated surgically. Early intervention with gene therapy has significant potential to reverse or stabilize disease progression at an early stage of the disease, preventing significant vision loss as well as the development of these more severe complications.

[0004] Autosomal dominant optic atrophy (ADOA) results in vision loss in the first to second decades of life. ADOA subjects typically have retinal degeneration, neurological defects, and musculoskeletal complications over time. ADOA is caused by mutation(s) in Opa1. Opa1 has been shown to be involved in mitochondrial cristae structure, mitochondrial fusion, and inner mitochondrial membrane remodeling.

[0005] New gene therapies for treating XLRS or ADOA are needed. Summary of the Invention

[0006] The present disclosure relates generally to the field of gene therapy, and specifically to recombinant adeno-associated virus (AAV) vector particles (also known as AAV viral vectors) containing novel expression cassettes, and their uses, for the delivery of transgenes to treat or prevent diseases or disorders, such as XLRS or ADOA.

[0007] In one aspect, the disclosure provides a method of treating retinoschisis in a subject in need thereof, comprising juxtaretinal or subretinal administration of an AAV viral vector to the subject. 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 beta 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, 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. In an embodiment, the method comprises juxtaretinal administration of an AAV viral vector to the subject. In an embodiment, the subject is a human. In an embodiment, the AAV viral vector is administered to a subject in a manner that is at least about 10 minutes after administration of the AAV viral vector. 10 ~about 10 12 In an embodiment, the retinoschisis is X-linked retinoschisis. In an embodiment, the transgene is RS1. In an embodiment, 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. In an embodiment, 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.

[0008] In one aspect, the disclosure provides a method of treating an ocular disease or disorder in a subject in need of such treatment comprising administering to the subject an AAV viral vector, wherein the AAV viral vector comprises an AAV vector genome comprising, in a 5' to 3' orientation, (a) a first AAV inverted terminal repeat, (b) a promoter, (c) a heterologous nucleic acid encoding Opa1, (d) a polyadenylation signal, and (e) a second AAV inverted terminal repeat.

[0009] In embodiments, the promoter is a CBh 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: 154. In embodiments, the promoter is a MeCP2 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: 156.

[0010] 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: 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 in between.

[0011] In embodiments, the heterologous nucleic acid encoding Opal comprises a 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 NOs: 175, 182, and 184. In embodiments, the heterologous nucleic acid encodes an Opal protein comprising an amino acid 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 NOs: 180, 183, and 185. In embodiments, the heterologous nucleic acid encodes an Opal protein comprising 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: 180.

[0012] In embodiments, 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.

[0013] In embodiments, the AAV vector genome does not contain any telomeric repeat sequences.

[0014] In an embodiment, the AAV vector genome comprises a first telomeric repeat sequence located between the polyadenylation signal and the second AAV inverted terminal repeat. In an embodiment, 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. In an embodiment, the AAV vector genome comprises a second telomeric repeat sequence located between the first AAV inverted terminal repeat and the promoter. In an embodiment, the second 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: 203.

[0015] In embodiments, the first AAV inverted terminal repeat comprises or consists of 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: 253. In embodiments, the second AAV inverted terminal repeat comprises or consists of 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: 254.

[0016] In an embodiment, the AAV vector genome comprises, in a 5' to 3' orientation, (a) a first AAV inverted terminal repeat, (b) a 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: 154, (c) a heterologous nucleic acid encoding Opal, (d) a polyadenylation signal 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: 201, and (e) a second AAV inverted terminal repeat. In an embodiment, 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. In an embodiment, the intron sequence is located between the promoter and the heterologous nucleic acid encoding Opal. In embodiments, the AAV vector genome 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: 228. In embodiments, the Opal protein comprises 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: 180.

[0017] In embodiments, the AAV vector genome 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 NOs: 230-239.

[0018] In embodiments, the eye disease or disorder is autosomal dominant optic atrophy.

[0019] In one aspect, the disclosure provides a method of treating an ocular disease or disorder in a subject in need of such treatment comprising administering to the subject an AAV viral vector, wherein the AAV viral vector comprises an AAV vector genome comprising, in a 5' to 3' orientation, (a) a first AAV inverted terminal repeat, (b) a promoter, (c) a heterologous nucleic acid encoding RS1, (d) a polyadenylation signal, and (e) a second AAV inverted terminal repeat.

[0020] 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 a 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 an embodiment, the promoter is a PDE 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:198.

[0021] In embodiments, the promoter is a CBh 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:154.

[0022] In embodiments, the AAV vector genome comprises an IRBP enhancer sequence upstream of the promoter. In embodiments, the IRBP enhancer sequence has at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 199. In embodiments, the IRBP enhancer sequence is located immediately upstream of the promoter without any additional nucleotides in between.

[0023] In embodiments, the AAV vector genome comprises a CVA-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.

[0024] 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: 200 or 222. In embodiments, the intron is located between the promoter and the heterologous nucleic acid encoding RS1.

[0025] In embodiments, the heterologous nucleic acid encoding RS1 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: 117. In embodiments, the heterologous nucleic acid encodes an RS1 protein comprising 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.

[0026] In embodiments, 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.

[0027] In embodiments, the AAV vector genome does not contain any telomeric repeat sequences.

[0028] In embodiments, the AAV vector genome comprises a first telomeric repeat sequence located between the polyadenylation signal and the second AAV inverted terminal repeat. In embodiments, 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:203.

[0029] In an embodiment, the AAV vector genome comprises a human beta-globin scaffold / matrix attachment region (βGlo_s / MAR) sequence. In an embodiment, the βGlo_s / MAR sequence is located between the polyadenylation signal and the second AAV inverted terminal repeat. In an embodiment, the βGlo_s / MAR sequence is located between the polyadenylation signal and the first telomeric repeat sequence. In an embodiment, the βGlo_s / MAR sequence has at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO:221.

[0030] In embodiments, the first AAV inverted terminal repeat comprises or consists of 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: 255. In embodiments, the second AAV inverted terminal repeat comprises or consists of 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: 256.

[0031] In embodiments, the AAV vector genome comprises, in the 5' to 3' orientation, (a) a first AAV inverted terminal repeat, (b) an IRBP enhancer sequence having at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, at least 99%, or 100% identity to SEQ ID NO: 199, (c) 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, (d) a heterologous nucleic acid encoding RS1, (e) a polyadenylation signal 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: 225, and (f) a second AAV inverted terminal repeat.

[0032] 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 a CBA sequence of SEQ ID NO: 229, or a sequence having up to 5, up to 4, up to 3, up to 2, or up to 1 mutation thereto. In embodiments, the CBA sequence is located immediately upstream of the intron sequence without any additional nucleotides in between. 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.

[0033] In embodiments, the AAV vector genome 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:224.

[0034] In embodiments, the AAV vector genome 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 NOs: 224 and 240-252.

[0035] In an embodiment, the eye disease or disorder is X-linked retinoschisis.

[0036] In embodiments, 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. In embodiments, 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. In embodiments, 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.

[0037] In embodiments, the administration is pararetinal administration, hi embodiments, pararetinal administration includes injection at a distance of 0-13 millimeters (mm), 0-10 mm, 0-5 mm, or 0-3 mm from the surface of the retina in the posterior vitreous cavity of the eye.

[0038] In an embodiment, the subject is a human.

[0039] In one aspect, the disclosure provides a nucleic acid comprising, in 5' to 3' orientation: (a) a first AAV inverted terminal repeat, (b) a promoter, (c) a heterologous nucleic acid encoding Opa1, (d) a polyadenylation signal, and (e) a second AAV inverted terminal repeat.

[0040] In embodiments, the promoter is a CBh 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: 154. In embodiments, the promoter is a MeCP2 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: 156.

[0041] 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: 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 in between.

[0042] In embodiments, the heterologous nucleic acid encoding Opal comprises a 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 NOs: 175, 182, and 184. In embodiments, the heterologous nucleic acid encodes an Opal protein comprising an amino acid 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 NOs: 180, 183, and 185. In embodiments, the heterologous nucleic acid encodes an Opal protein comprising 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: 180.

[0043] In embodiments, 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.

[0044] In embodiments, the AAV vector genome does not contain any telomeric repeat sequences.

[0045] In an embodiment, the AAV vector genome comprises a first telomeric repeat sequence located between the polyadenylation signal and the second AAV inverted terminal repeat. In an embodiment, 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. In an embodiment, the AAV vector genome comprises a second telomeric repeat sequence located between the first AAV inverted terminal repeat and the promoter. In an embodiment, the second 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: 203.

[0046] In embodiments, the first AAV inverted terminal repeat comprises or consists of 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: 253. In embodiments, the second AAV inverted terminal repeat comprises or consists of 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: 254.

[0047] In embodiments, the AAV vector genome comprises, in a 5' to 3' orientation, (a) a first AAV inverted terminal repeat, (b) a 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: 154, (c) a heterologous nucleic acid encoding Opa1, (d) a polyadenylation signal 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: 201, and (e) a second AAV inverted terminal repeat.

[0048] In embodiments, a nucleic acid 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. In embodiments, the intron sequence is located between the promoter and the heterologous nucleic acid encoding Opa1.

[0049] In embodiments, the Opal protein comprises 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:180.

[0050] In embodiments, the nucleic acid 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: 228. In embodiments, the nucleic acid 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 NOs: 230-239.

[0051] In one aspect, the disclosure provides a nucleic acid comprising, in 5' to 3' orientation, (a) a first AAV inverted terminal repeat, (b) a promoter, (c) a heterologous nucleic acid encoding RS1, (d) a polyadenylation signal, and (e) a second AAV inverted terminal repeat.

[0052] In embodiments, the promoter is a photoreceptor-specific promoter, hi 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.

[0053] In embodiments, the promoter is a CBh 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:154.

[0054] In embodiments, the promoter is a 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 embodiments, 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 having at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 198.

[0055] In embodiments, the nucleic acid comprises an IRBP enhancer sequence upstream of the promoter. In embodiments, the IRBP enhancer sequence has at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 199. In embodiments, the IRBP enhancer sequence is located immediately upstream of the promoter without any additional nucleotides in between.

[0056] In embodiments, the AAV vector genome comprises a CVA-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.

[0057] 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: 200 or 222. In embodiments, the intron is located between the promoter and the heterologous nucleic acid encoding RS1.

[0058] In embodiments, the heterologous nucleic acid encoding RS1 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: 117. In embodiments, the heterologous nucleic acid encodes an RS1 protein comprising 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.

[0059] In embodiments, 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.

[0060] In embodiments, the AAV vector genome does not contain any telomeric repeat sequences.

[0061] In embodiments, the AAV vector genome comprises a first telomeric repeat sequence located between the polyadenylation signal and the second AAV inverted terminal repeat. In embodiments, 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:203.

[0062] In an embodiment, the AAV vector genome comprises a human beta-globin scaffold / matrix attachment region (βGlo_s / MAR) sequence. In an embodiment, the βGlo_s / MAR sequence is located between the polyadenylation signal and the second AAV inverted terminal repeat. In an embodiment, the βGlo_s / MAR sequence is located between the polyadenylation signal and the first telomeric repeat sequence. In an embodiment, the βGlo_s / MAR sequence has at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO:221.

[0063] In embodiments, the first AAV inverted terminal repeat comprises or consists of 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: 255. In embodiments, the second AAV inverted terminal repeat comprises or consists of 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: 256.

[0064] In embodiments, the AAV vector genome comprises, in the 5' to 3' orientation, (a) a first AAV inverted terminal repeat, (b) an IRBP enhancer sequence having at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, at least 99%, or 100% identity to SEQ ID NO: 199, (c) 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, (d) a heterologous nucleic acid encoding RS1, (e) a polyadenylation signal 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: 225, and (f) a second AAV inverted terminal repeat.

[0065] In embodiments, the nucleic acid 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.

[0066] In embodiments, the nucleic acid comprises a CBA sequence of SEQ ID NO: 229, or a sequence having up to 5, up to 4, up to 3, up to 2, or up to 1 mutation thereto. In embodiments, the CBA sequence is located immediately upstream of an intron sequence without any additional nucleotides in between. 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.

[0067] In embodiments, the nucleic acid 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:224.

[0068] In embodiments, the nucleic acid 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 NOs: 224 and 240-252.

[0069] In one aspect, the disclosure provides a nucleic acid comprising, in 5' to 3' orientation, (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 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 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.

[0070] In embodiments, the nucleic acid 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. In embodiments, the intron is located between the promoter and the heterologous nucleic acid encoding the transgene.

[0071] In an embodiment, the nucleic acid comprises a first ITR located 5' to the promoter and a second ITR located 3' to the polyadenylation signal.

[0072] In an embodiment, the nucleic acid does not include any telomeric repeat sequences.

[0073] In an embodiment, the nucleic acid comprises a first telomeric repeat sequence located between the polyadenylation signal and the second AAV ITR. In an embodiment, 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. In an embodiment, the nucleic acid comprises a second telomeric repeat sequence located between the first AAV inverted terminal repeat and the promoter. In an embodiment, the second 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:203.

[0074] In one aspect, the disclosure provides a vector comprising a nucleic acid of the disclosure.

[0075] In one aspect, the disclosure provides an AAV vector genome comprising a nucleic acid of the disclosure.

[0076] In one aspect, the disclosure provides an AAV viral vector comprising an AAV vector genome of the disclosure. In embodiments, 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.

[0077] 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.

[0078] In one aspect, the present disclosure provides a method for treating a disease or disorder, comprising administering to a subject the AAV viral vector of the present disclosure.In an embodiment, the AAV viral vector is administered to a subject intraocularly, periocularly, intravitreally, juxtaretinal, or subretinal.In an embodiment, the disease or disorder is macular degeneration, retinitis pigmentosa, autosomal overt optic atrophy, retinoschisis, Stargardt disease, Vietti crystalline dystrophy, or BEST vitelliform macular dystrophy.In an embodiment, the disease or disorder is X-linked retinoschisis.

[0079] This 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. [Brief description of the drawings]

[0080] [Figure 1] Illustrative of different modes of intraocular administration (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). [Figure 2A]Figure 2 shows AAV viral vector-mediated GFP expression in the eye of a non-human primate animal model via intravitreal or pararetinal administration. Scanning laser ophthalmoscopy (SLO) imaging was performed 26 days after injection of the indicated AAV viral vector. Figure 2A shows the spread of transduction mediated by intravitreal injection of AAV viral vectors containing the AAV204 capsid protein. [Figure 2B] Figure 2A shows AAV viral vector-mediated GFP expression in the eye of a non-human primate animal model via intravitreal or pararetinal administration. Scanning laser ophthalmoscopy (SLO) imaging was performed 26 days after injection of the indicated AAV viral vector. Figure 2B shows the spread of transduction mediated by pararetinal injection of AAV viral vectors containing the AAV204 capsid protein. [Figure 2C] Figure 2 shows AAV viral vector-mediated GFP expression in the eye of a non-human primate animal model via intravitreal or pararetinal administration. Scanning laser ophthalmoscopy (SLO) imaging was performed 26 days after injection of the indicated AAV viral vector. Figure 2C shows the spread of transduction mediated by pararetinal injection of AAV viral vectors containing the AAV8 capsid protein. [Figure 2D] Figure 2 shows AAV viral vector-mediated GFP expression in the eye of a non-human primate animal model via intravitreal or pararetinal administration. Scanning laser ophthalmoscopy (SLO) imaging was performed 26 days after injection of the indicated AAV viral vector. Figure 2D shows the spread of transduction mediated by pararetinal injection of AAV viral vectors containing the AAV214 capsid protein. [Figure 2E] Figure 2 shows AAV viral vector-mediated GFP expression in the eye of a non-human primate animal model via intravitreal or pararetinal administration. Scanning laser ophthalmoscopy (SLO) imaging was performed 26 days after injection of the indicated AAV viral vector. Figure 2E shows the spread of transduction mediated by pararetinal injection of AAV viral vectors containing the AAV214-D5 capsid protein. [Figure 3A]Figure 3 shows image analysis of the retina after AAV administration. Figure 3A shows a merged image of the retina after intravitreal administration of AAV204. [Figure 3B] Image analysis of the retina after AAV administration. Figure 3B shows the merged (top left), rhodopsin (top right), and zoomed merged (bottom) images of the retina after pararetinal administration of AAV204. [Figure 3C] Image analysis of the retina after AAV administration. Figure 3C shows the merged (top left), rhodopsin (top right), and zoomed merged (bottom) images of the retina after pararetinal administration of AAV204. [Figure 3D] Figure 3D shows image analysis of the retina after AAV administration, and Figure 3D shows immunohistochemical analysis of rhodopsin and GFP expression one month after pararetinal injection of AAV204 or AAV8 viral vectors. [Figure 3E] Figure 3E shows image analysis of the retina after AAV administration, and Figure 3F shows rhodopsin and GFP expression in the fovea after pararetinal injection of the AAV204 viral vector. [Figure 3F] Figure 3F shows image analysis of the retina after AAV administration, and shows rhodopsin and GFP expression along the papillomacular bundle after pararetinal injection of AAV204 viral vector. [Figure 4A] Figure 4 shows AAV viral vector-mediated GFP expression in the eye of a non-human primate animal model via subretinal administration. SLO imaging was performed 27 days after injection of the indicated AAV viral vector. Figure 4A shows the spread of transduction mediated by subretinal injection of AAV viral vectors containing the AAV8 capsid protein. [Figure 4B] Figure 4A shows AAV viral vector-mediated GFP expression in the eye of a non-human primate animal model via subretinal administration. SLO imaging was performed 27 days after injection of the indicated AAV viral vector. Figure 4B shows the spread of transduction mediated by subretinal injection of AAV viral vectors containing the AAV214 capsid protein. [Figure 4C]Figure 4A shows AAV viral vector-mediated GFP expression in the eye of a non-human primate animal model via subretinal administration. SLO imaging was performed 27 days after injection of the indicated AAV viral vector. Figure 4B shows the spread of transduction mediated by subretinal injection of an AAV viral vector containing the AAV214-D5 capsid protein. [Figure 4D] FIG. 4D shows a merged image of the retina (top left), rhodopsin (top right), and zoomed merged (bottom) images following subretinal administration of AAV8. [Figure 4E] FIG. 4E shows a merged image of the retina (top left), rhodopsin (top right), and zoomed merged (bottom) images following subretinal administration of AAV214. [Figure 4F] Figure 4F shows overlay images of the retina (top left), rhodopsin (top right), and zoomed overlay (bottom) images after subretinal administration of AAV214-D5. [Diagram 5] A diagram of the VP1, VP2, and VP3 portions of the capsid protein is shown. The VP1 and VP2 specific portions are shown along with the VP3 portion that is identical to the produced VP3 protein. The amino acid sequence of AAV214 VP3 (SEQ ID NO:41) is shown, showing variable regions I-IX. The complete VP1 protein amino acid sequence for AAV214 is provided as SEQ ID NO:3. [Figure 6-1] 1 shows an alignment of the VP1 protein amino acid sequences of AAV214 (sequence number 3) and AAV214-D5 (sequence number 164). [Figure 6-2] Same as above. [Figure 7] 1 shows the design of various Opa1-encoding AAV vector genomes. [Figure 8A] Expression of Opa1 in 293 cells transfected with each of the indicated vectors is shown. [Figure 8B] 1 is a chart showing viral production yields for each of the indicated vectors. [Figure 9A] 1 shows the expression of Opa1 in a viral potency assay. [Figure 9B] 1 shows the expression of Opa1 in a viral potency assay. [Figure 9C] Protein staining results of cells transfected with each of the indicated vectors are shown. [Figure 10A] FIG. 1 shows a schematic of a proof-of-concept study evaluating AAV204 viral vectors encoding Opa1. [Figure 10B] Western analysis of Opa1 and FLAG tag expression in heterozygous treated mice. [Figure 10C] Western analysis of the expression of Opa1, FLAG tag, Brn3a and Rho is shown. [Figure 10D] RT-PCR analysis of human Opal, mouse Opal, and FLAG tag RNA transcript levels in wild-type or Opal heterozygous, untreated or treated animals at 2 months post-injection. [Figure 11A] FIG. 1 shows a schematic of a proof-of-concept (POC) study to evaluate AAV204 viral vectors encoding Opa1. [Figure 11B] FIG. 1 shows a schematic of a proof-of-concept (POC) study to evaluate AAV204 viral vectors encoding Opa1. [Figure 12A] A summary table of AAV vector genomes encoding various RS1s is provided. [Figure 12B] 1 shows the design of AAV vector genomes encoding various RS1s. [Figure 13] Western analysis of RS1 protein expression is shown. [Figure 14A] 1 is a chart showing expression of secreted RS1 protein in Lec2 cells transduced with each of the indicated AAV viral vectors. [Figure 14B] 1 is a chart showing expression of targeted transgene mRNA in Lec2 cells transduced with each of the indicated AAV viral vectors. [Figure 14C] Western analysis of secreted RS1 protein in Lec2 cells transduced with each of the indicated AAV viral vectors is shown. [Figure 14D]Western analysis comparing the molecular weights of myc-tagged RS1 and wild-type RS1 is shown. [Figure 15A] FIG. 1 shows a diagram of a proof-of-concept (POC) study to evaluate AAV204 viral vectors encoding RS1. [Figure 15B] 1 is a chart showing expression of RS1 protein in wild-type mice transduced with the indicated AAVs. [Figure 16A] Western analysis of RS1 expression in mice transduced with the indicated AAVs is shown. [Figure 16B] Western analysis of RS1 expression in mice transduced with the indicated AAVs is shown. [Figure 17A] FIG. 1 shows a schematic of a proof-of-concept (POC) study to evaluate AAV204 viral vectors encoding RS1. [Figure 17B] FIG. 1 shows a schematic of a proof-of-concept (POC) study to evaluate AAV204 viral vectors encoding RS1. [Figure 18A] 1 is a table showing the various treatment groups for a mouse study of RS1 expression. [Figure 18B] Expression of endogenous mouse RS1 (mRs1) measured by qPCR in groups 5-8 at 2 months post treatment (mpt) is shown. [Figure 18C] Expression of RS1 from the virally delivered transgene at 2 mpt is shown. Data have been log transformed to facilitate visualization of large differential comparisons. nd: not detectable. [Figure 18D] Results of simultaneous detection of endogenous Rs1 and virus-derived myc-RS1 using the highly specific RS1 antibody are shown in Figure 18D and quantified in Figure 18E. Due to the presence of the myc tag, the RS1-specific bands in groups 5 and 6 are larger than the endogenous protein in group 8. A myc-RS1 positive control derived from transduced tissue culture cells is included in the last lane. nd: not detected. [Figure 18E]Results of simultaneous detection of endogenous Rs1 and virus-derived myc-RS1 using the highly specific RS1 antibody are shown in Figure 18D and quantified in Figure 18E. Due to the presence of the myc tag, the RS1-specific bands in groups 5 and 6 are larger than the endogenous protein in group 8. A myc-RS1 positive control derived from transduced tissue culture cells is included in the last lane. nd: not detected. [Figure 19A] IHC staining of wild type (WT) retina at 2 mpt is shown. [Figure 19B] IHC staining of untreated mutant retinas at 2 mpt is shown. [Figure 19C] IHC staining of mutant retinas transduced with RS1_46 at 2 mpt is shown. [Figure 19D] IHC staining of mutant retina transduced with RS1_46 in the right eye of animal #123 at 2 mpt is shown. [Figure 19E] IHC staining of mutant retinas transduced with RS1_48 at 2 mpt is shown. [Figure 19F] 13 is a chart showing cone density analysis at p90, 2 mpt. [Figure 19G] Staining of retinal samples is shown. Top panels A-C show retinal samples transduced with AAV204.RK:RS1_28. RS1 is stained red and PNA is stained green to indicate the extent of cone degeneration. Bottom panels are higher magnification images showing cone density in transduced and non-transduced areas (sections E and F, respectively) of similarly prepared WT retinas (section D) and treated mutant retinas. [Figure 20A] Western analysis of RS1 protein expression is shown. The positive control in the last lane is recombinant RS1 from transfected tissue culture cells. [Figure 20B] 1 is a chart showing quantification of band intensity. nd = not detected. [Figure 20C]IHC of WT retinas at 6 mpt is shown. Expression of RS1 (red) in WT eyes (group 3) is uniform across the retina and enriched in photoreceptors. The right panel shows a higher magnification view of the boxed area. [Figure 20D] IHC of mutant retinas at 6 mpt. Staining for RS1 (red) is absent in mutant retinas (group 1). The right panel shows a higher magnification view of the boxed area. In the absence of RS1, the retinal vasculature is labeled with the secondary antibody (arrow). [Figure 20E] IHC of mutant retina treated with RS1_28 at 6 mpt is shown. Expression of RS1 (red) is seen 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. [Figure 20F] IHC of mutant retinas treated with RS1_26 at 6 mpt is shown. None of the eyes in this group had detectable expression of RS1. [Figure 21] 1 is a chart showing quantification of cone density at 6 mpt. Cone density was measured from sections stained with peanut agglutinin (PNA). Measurements from group 2 are separated by expression of RS1. Within group 2, individual data points of the same color reflect RS1-positive and RS1-negative areas of the same section. In four of the five eyes with detectable RS1 expression, cone density was slightly higher in the adjacent area with RS1 expression. Eyes that did not have detectable RS1 expression are represented by black dots. [Figure 22A] OCT imaging of representative eyes at 6 mpt is shown. The yellow bar in each image indicates the ONL thickness. The arrow indicates an example of retinal separation that was present in one eye. [Figure 22B] 1 is a chart showing quantitative analysis of ONL measurements across all groups. [Figure 23A] IHC of WT retina at 6 mpt is shown. [Figure 23B] IHC of untreated mutant retinas at 6 mpt is shown. [Figure 23C] IHC of mutant retinas treated with AAV204.CBh:RS1_16 at 6 mpt is shown. Most eyes treated with AAV204.CBh:RS1_16 showed extensive degeneration in the dorsal retina where the bleb would have been. Visible RS1 expression was rare and absent in this eye. The magnified boxed area in the right panel shows improved cone density (PNA staining) despite the absence of detectable RS1 staining. [Figure 23D] IHC of mutant retinas treated with AAV204.CBh:RS1_16 at 6 mpt in one particular eye (with injection injury). The asterisk on the left indicates a treated retina with severe injection-related injury, showing expression of RS1 (red) throughout the inner retina. The right shows a deeper section of the same eye, showing RS1 expression in the inner retina (B') and expression within photoreceptors in the area adjacent to the lesion (B''). In all panels, PNA (grey) labels cone outer segments and Iba1 (green) labels inflammatory cells. [Figure 23E] IHC of mutant retinas treated with AAV204.CBh:RS1_18 at 6 mpt is shown. In this example, strong RS1 expression was observed in the dorsal photoreceptors and spread to the inner retina (box a). In contrast, RS1 staining was absent in the ventral retina (box b). Most of the dorsal retina was devoid of cones, despite being otherwise structurally intact. However, the region just dorsal to box b was both RS1 positive and cone-enriched, compared with the adjacent RS1-negative region. [Figure 23F] Figure 13 is a chart summarizing the cone density analysis at 6 mpt. Cone density was significantly improved in all treated eyes, even in areas where RS1 immunostaining was undetectable. For this analysis, areas of severe degeneration and cone depletion were omitted. [Figure 24A]24B and 24C are charts summarizing the mean ONL thickness across all groups. For treated eyes, separate measurements were made for RS1+ and RS1- regions of the retina, with similarly colored data points within each group reflecting measurements obtained from the same section. Severely degenerated regions were not included. Data from representative eyes of groups 10 and 11 are shown in FIG. 24B and FIG. 24C, where star-shaped labels indicate areas of treated retina with RS1 expression. [Figure 24B] Same as above. [Figure 24C] Same as above. [Figure 25A] Western analysis of RS1 protein expression is shown. The positive control in the last lane is recombinant RS1 from transfected tissue culture cells. [Figure 25B] 1 is a chart showing quantification of band intensity. nd = not detected. [Figure 26A] 1 shows OCT imaging of an untreated mutant eye. [Figure 26B] 1 shows OCT imaging of a wild type eye. [Figure 26C] OCT imaging of the postoperative bleb to confirm successful injection is shown. [Figure 26D] Shown is OCT imaging of a treated eye 6 months after injection. [Figure 26E] Shown is OCT imaging of a treated eye 6 months post-injection, where the bleb margin was captured. [Figure 26F] 1 shows OCT imaging of treated eyes 6 months after injection. The yellow line in these figures indicates the thickness of the ONL. [Figure 26G] 1 is a chart showing quantification of ONL thickness. [Fig. 26H] FIG. 13 is a paired t-test estimate plot showing increased ONL thickness in treated dorsal retina (11D) compared to untreated ventral retina (11V) in the same eye. [Figure 27] A representative flicker ERG is shown at 6 mpt. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0081] Some embodiments according to the present disclosure are described more fully below. However, aspects of the present disclosure may be embodied in different forms and should not be construed as being limited to the embodiments described herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the present invention to those skilled in the art. The terms used in the description of this specification are for the purpose of describing specific embodiments only, and are not intended to be limiting.

[0082] Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by those skilled in the art to which the present invention belongs. 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 related art, and should not be interpreted in an idealized or overly formal sense unless expressly defined herein.

[0083] Unless the context indicates otherwise, it is specifically intended that the various features of the invention described herein can be used in any combination. Moreover, the present disclosure also contemplates that in embodiments, any feature or combination of features described herein can be excluded or omitted. To illustrate, if the present specification describes a complex as comprising components A, B, and C, it is specifically intended that any of A, B, or C, or combinations thereof, can be omitted and discarded, either alone or in any combination.

[0084] Unless expressly stated otherwise, all specific embodiments, features, and terms are intended to include both the recited embodiment, feature, or term and biological equivalents thereof.

[0085] Incorporation by Reference All references, articles, publications, patents, patent publications, and patent applications cited herein are incorporated by reference in their entirety for all purposes. However, mention of any references, articles, publications, patents, patent publications, and patent applications cited herein is not, and should not be considered as, an acknowledgment or any form of suggestion that they constitute valid prior art or form part of the general knowledge anywhere in the world.

[0086] definition The practice of the present technology employs, unless otherwise indicated, conventional techniques of organic chemistry, pharmacology, immunology, molecular biology, microbiology, cell biology, and recombinant DNA within the skill of the art. See, e.g., Sambrook, Fritsch and Maniatis, Molecular Cloning: A Laboratory Manual, 2nd edition (1989), Current Protocols in Molecular Biology (FMA Usubel, et al. eds., (1987)), the series Methods in Enzymology (Academic Press, Inc.): PCR 2: A Practical Approach (MJ MacPherson, BD Hames and GR Taylor eds. (1995)), Harlow and Lane, eds. (1988) Antibodies, a Laboratory Manual, and Animal Cell Culture (RI Freshney, ed. (1987)).

[0087] Additionally, although not always explicitly stated, it is understood that the reagents described herein are merely exemplary and that equivalents of such are known in the art.

[0088] As used herein when referring to measurable values ​​such as amounts or concentrations and the like, the term "about" is meant to encompass a 10% variation of the specified amount.

[0089] When used to describe the selection of any component, range, dosage form, etc. disclosed herein, the terms "acceptable," "effective," or "sufficient" intend that the component, range, dosage form, etc. is suitable for the purposes of the disclosure.

[0090] Unless specifically recited, 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 monkey, bovine, porcine, murine, rat, avian, reptilian, and human, e.g., HEK293 cells and 293T cells.

[0091] As used herein, the term "isolated" refers to a molecule or biological or cellular material that is substantially free of other substances.

[0092] 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, the term includes, but is not limited to, single-, double-, or multistranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or polymers that contain, consist essentially of, or consist of purine and pyrimidine bases, or other naturally occurring, chemically or biochemically modified, non-natural or derivatized nucleotide bases.

[0093] "Gene" refers to a polynucleotide containing at least one open reading frame (ORF) capable of encoding a particular polypeptide or protein. "Gene product," or alternatively, "gene expression product," refers to the amino acid sequence (e.g., peptide or polypeptide) produced when a gene is transcribed and translated.

[0094] As used herein, "expression" refers to the two-step process by which a polynucleotide is transcribed into mRNA and / or the transcribed mRNA is subsequently translated into a peptide, polypeptide, or protein. If the polynucleotide is derived from genomic DNA, expression may also include splicing of the mRNA in eukaryotic cells.

[0095] "Under transcriptional control" is a term well understood in the art and indicates that transcription of a polynucleotide sequence, usually a DNA sequence, is dependent on being operably linked to elements that contribute to or facilitate the initiation of transcription. "Operably linked" refers to being positioned in a manner that allows the polynucleotide to function within a cell. In one aspect, the invention provides a promoter operably linked to a downstream sequence.

[0096] The term "encodes," as applied to a polynucleotide, refers to a polynucleotide that is said to "encode" a polypeptide if, in its natural state, or when manipulated by methods well known to those of skill in the art, it can be transcribed to produce mRNA for the polypeptide and / or fragments thereof. The antisense strand is the complement of such a nucleic acid, and the coding sequence can be deduced therefrom.

[0097] As used herein, the term "promoter" refers to a control sequence, which is a region of a polynucleotide sequence at which the initiation and rate of transcription of a coding sequence, such as a gene or transgene, is controlled. A promoter may be, for example, constitutive, inducible, repressible, or tissue-specific. A promoter may contain genetic elements to which regulatory proteins and molecules, such as RNA polymerase and transcription factors, may bind. Non-limiting exemplary promoters include the Rous sarcoma virus (RSV) LTR promoter (optionally with the RSV enhancer), the cytomegalovirus (CMV) promoter, the SV40 promoter, the dihydrofolate reductase promoter, the β-actin promoter, the phosphoglycerol kinase (PGK) promoter, the U6 promoter, the H1 promoter, the ubiquitous chicken β-actin hybrid (CBh) promoter, the small nuclear RNA (U1a or U1b) promoter, the MeCP2 promoter, the MeP418 promoter, the MeP426 promoter, the minimal MeCP2 promoter, the VMD2 promoter, the mRho promoter, or the EF1 promoter.

[0098] Additional non-limiting exemplary promoters provided herein include, but are not limited to, EFla, Ubc, human β-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 sequences of such promoters 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 TATA box of 7SK, U6 and H1 promoters to abolish RNA polymerase III transcription and stimulate RNA polymerase II-dependent mRNA transcription). Synthetic derived promoters can be used for ubiquitous or tissue-specific expression. Additionally, viral derived promoters, some of which are described above, such as CMV, HIV, adenovirus, and AAV promoters, can be useful in the methods disclosed herein. In embodiments, the promoter is used in conjunction 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.

[0099] Enhancers are regulatory elements that increase the expression of a target sequence. A "promoter / enhancer" is a polynucleotide that contains a sequence capable of providing both promoter and enhancer functions. For example, retroviral long terminal repeats contain both promoter and enhancer functions. Enhancers / promoters can be "endogenous", "exogenous", or "heterologous". An "endogenous" enhancer / promoter is one that is naturally linked to a given gene in a genome. An "exogenous" or "heterologous" enhancer / promoter is juxtaposed to a gene by genetic engineering (i.e., molecular biology techniques) such that transcription of that gene is directed by the linked enhancer / promoter. Non-limiting examples of linked enhancers / promoters for use in the methods, compositions, and constructs provided herein include PDE promoter + IRBP enhancer or CMV enhancer + U1a promoter. It is understood in the art that an enhancer can act from a distance and regardless of its orientation relative to the location of the endogenous or heterologous promoter. Thus, an enhancer that acts at a distance from a promoter is therefore further understood to be "operably linked" to that promoter, regardless of its location within the vector, or its orientation relative to the location of the promoter.

[0100] The terms "protein", "peptide" and "polypeptide" are used interchangeably and in their broadest sense to refer to a compound of two or more subunits of amino acids, amino acid analogs, or peptidomimetics. The subunits may be linked by peptide bonds. In alternative embodiments, the subunits may be linked by other bonds, such as esters, ethers, and the like. A protein or peptide must contain at least two amino acids, and there is no limit to the maximum number of amino acids that may comprise, consist essentially of, or consist of a protein or peptide sequence. As used herein, the term "amino acid" refers to either natural and / or unnatural or synthetic amino acids, including glycine and both D and L optical isomers, amino acid analogs, and peptidomimetics.

[0101] As used herein, the term "signal peptide" or "signal polypeptide" refers to an amino acid sequence that is usually present at the N-terminus of a newly synthesized secretory or membrane polypeptide or protein. It acts to direct the polypeptide to a specific cellular location, for example, through the cell membrane, to the cell membrane, or to the nucleus. In an embodiment, the signal peptide is removed after localization. Examples of signal peptides are well known in the art. Non-limiting examples are those described in U.S. Pat. Nos. 8,853,381, 5,958,736, and 8,795,965. In an embodiment, the signal peptide may be an IDUA signal peptide.

[0102] The terms "equivalent" or "biological equivalent" are used interchangeably when referring to a particular molecule, biological material, or cellular material and are intended to have minimal homology while still maintaining a desired structure or functionality. 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 to a reference polypeptide (e.g., a wild-type polypeptide), or polypeptides encoded by a polynucleotide 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 to a reference polynucleotide (e.g., a wild-type polynucleotide).

[0103] "Homology" or "identity" or "similarity" refers to the sequence similarity between two peptides or two nucleic acid molecules. The percent identity can be determined by comparing positions in each sequence that may be aligned for comparison purposes. If a position in the compared sequences is occupied by the same base or amino acid, 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. An "unrelated" or "non-homologous" sequence shares less than 40% identity, less than 25% identity with one of the sequences of the present disclosure. Alignments and percent sequence identity for nucleic acid or amino acid sequences provided herein can be determined by importing the nucleic acid or amino acid sequence into and using ClustalW (available at genome.jp / tools-bin / clustalw / ) and the Gonnet (for proteins) weight matrix. In embodiments, the ClustalW parameters used to perform nucleic acid sequence alignments using the nucleic acid sequences found herein are generated using the ClustalW (for DNA) weight matrix.

[0104] As used herein, an amino acid modification can be a substitution, deletion, or insertion. An amino acid substitution can be a conservative amino acid substitution or a non-conservative amino acid substitution. A conservative substitution (also called a conservative mutation, conservative substitution, or conservative variation) is a replacement of an amino acid in a protein that changes a given amino acid to a different amino acid with similar biochemical properties (e.g., charge, hydrophobicity, or size). As used herein, a "conservative variation" refers to the replacement of an amino acid residue with another biologically similar residue. Examples of conservative variations include the replacement of one hydrophobic residue, such as isoleucine, valine, leucine, or methionine, with another, or the replacement of one charged or polar residue with another, such as the replacement of arginine with lysine, glutamic acid with aspartic acid, glutamine with asparagine, and the like. Other illustrative examples of conservative substitutions include 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.

[0105] As used herein, the term "vector" refers to a nucleic acid that comprises, consists essentially of, or consists of an intact replicon such that the vector can be replicated when placed into a cell, for example, by a process of transfection, infection, or transformation. It is understood in the art that once inside a cell, the vector may replicate as an extrachromosomal (episomal) element or may integrate into a host cell chromosome. A vector may comprise a nucleic acid derived from a retrovirus, adenovirus, herpes virus, baculovirus, modified baculovirus, papovavirus, or modified naturally occurring virus. Exemplary non-viral vectors for delivering nucleic acids include the use of naked DNA; DNA complexed with cationic lipids, alone or in combination with cationic polymers; anionic and cationic liposomes; DNA-protein complexes and particles comprising, consisting essentially of, or consisting of DNA condensed with cationic polymers such as heterologous polylysine, defined length oligopeptides, and polyethyleneimine, optionally contained in liposomes; and ternary complexes comprising, consisting essentially of, or consisting of viruses and polylysine-DNA.

[0106] For general recombinant techniques, vectors containing both a promoter and a cloning site to which a polynucleotide can be operably linked are well known in the art. Such vectors have the ability to transcribe RNA in vitro or in vivo and are commercially available from sources such as Agilent Technologies (Santa Clara, Calif) and Promega Biotech (Madison, Wis.). To optimize expression and / or in vitro transcription, it may be necessary to remove, add, or modify the 5' and / or 3' untranslated portions of the cloned transgene to remove excess, potentially inappropriate alternative translation initiation codons or other sequences that may prevent or reduce expression, either at the transcription or translation level. Alternatively, a consensus ribosome binding site can be inserted immediately at the 5' initiation codon to enhance expression.

[0107] "Viral vector" is defined as a recombinant virus or virus particle that contains a polynucleotide that is delivered to a host cell either in vivo, ex vivo, or in vitro. Examples of viral vectors include retroviral vectors, AAV viral vectors, lentiviral vectors, adenoviral vectors, alphaviral vectors, and the like. Alphaviral vectors, such as Semliki Forest virus-based vectors and Sindbis virus-based vectors, have also been developed for use in 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.

[0108] As used herein, the term "recombinant expression system" or "recombinant vector" refers to a genetic construct or constructs for the expression of certain genetic material formed by recombinant means.

[0109] A "gene delivery vehicle" is defined as any molecule capable of carrying an inserted polynucleotide into a host cell. Examples of gene delivery vehicles are liposomes, micelles, biocompatible polymers, including natural and synthetic polymers; lipoproteins; polypeptides; polysaccharides; lipopolysaccharides; artificial viral envelopes; metal particles; bacteria; viruses, such as baculoviruses, adenoviruses, and retroviruses; bacteriophages, cosmids, plasmids, and fungal vectors; and other recombinant vehicles commonly used in the art that have been described for expression in a variety of eukaryotic and prokaryotic hosts and can be used for gene therapy and simple protein expression. Also, liposomes that comprise, consist essentially of, or consist of targeting antibodies or fragments thereof can be used in the methods disclosed herein. In addition to delivery of polynucleotides to cells or cell populations, direct introduction of the proteins described herein into cells or cell populations can be performed by non-limiting techniques of protein transfection, alternatively fostering conditions that can enhance expression and / or promote activity of the proteins disclosed herein are other non-limiting techniques.

[0110] The polynucleotides disclosed herein can be delivered to cells or tissues using gene delivery vehicles. "Gene delivery", "gene transfer", "transduction" and the like, as used herein, are terms that refer to the introduction of an exogenous polynucleotide into a host cell (sometimes also referred to as a "transgene"), regardless of the method used for the transfer. Such methods include a variety of well-known techniques, such as vector-mediated gene transfer (e.g., by viral infection / transfection, or various other protein- or lipid-based gene delivery complexes), as well as techniques that facilitate the delivery of "naked" polynucleotides (electroporation, "gene gun" delivery, and various other techniques used for the transfer of polynucleotides). The introduced polynucleotide may be stably or transiently maintained in the host cell. Stable maintenance typically requires that the introduced polynucleotide contains an origin of replication compatible with the host cell, or is integrated into a host cell replicon, such as an extrachromosomal replicon (e.g., a plasmid) or a nuclear or mitochondrial chromosome. A number of vectors are known in the art and are known to have the ability to mediate the transfer of genes into mammalian cells, as described herein.

[0111] A "plasmid" is a DNA molecule that is usually separate from chromosomal DNA and has the ability to replicate independently of chromosomal DNA. It is often circular and double-stranded. Plasmids provide a mechanism for horizontal gene transfer within a microbial population and typically provide a selective advantage under a given environmental condition. Plasmids may carry genes that provide resistance to antibiotics that occur naturally in competing environmental niches, or alternatively, the proteins produced may act as toxins under similar circumstances. Although plasmid vectors often exist as extrachromosomal circular DNA molecules, it is known in the art that plasmid vectors may be designed to stably integrate into host chromosomes in either a random or targeted manner, and such integration may be achieved using either circular plasmids or plasmids that are linearized prior to introduction into the host cell.

[0112] "Plasmids" used in genetic engineering are called "plasmid vectors". Many plasmids are commercially available for such use. The gene to be replicated is inserted into a copy of the plasmid, which contains a gene that makes the cell resistant to a particular antibiotic, and into a multiple cloning site (MCS, or polylinker), a short region that contains several commonly used restriction sites and allows easy insertion of DNA fragments at this location. Another major use of plasmids is to make large amounts of proteins. In this case, researchers grow bacteria or eukaryotic cells containing a plasmid carrying the gene of interest and can induce them to produce large amounts of the protein from the inserted gene.

[0113] In aspects in which gene transfer is mediated by a DNA viral vector, such as adenovirus (Ad) or adeno-associated virus (AAV), vector construct refers to a polynucleotide that comprises, consists essentially of, or consists of the viral genome or a portion thereof and the transgene.

[0114] The term "adeno-associated virus" or "AAV" as used herein refers to a member of the class of viruses associated with this name and belonging to the genus Dependoparvovirus, family Parvoviridae. Adeno-associated viruses are single-stranded DNA viruses that grow only in cells in which certain functions are provided by a coinfecting helper virus. General information and reviews of AAV can be found, for example, in Carter, 1989, Handbook of Parvoviruses, Vol. 1, pp. 169-228, and Berns, 1990, Virology, pp. 1743-1764, Raven Press, (New York). Since it is well known that the various serotypes are very closely related, both structurally and functionally, even at the genetic level, it is fully expected that the same principles described in these reviews will be applicable to additional AAV serotypes that are characterized after the publication date of the reviews. (See, e.g., Blacklowe, 1988, pp. 165-174, in Parvoviruses and Human Disease, J.R. Pattison, ed., and Rose, Comprehensive Virology 3:1-61 (1974).) For example, all AAV serotypes apparently exhibit very similar replication properties mediated by homologous rep genes, and they all have three related capsid proteins, such as those expressed in AAV2. The degree of relatedness is further suggested by extensive cross-hybridization between serotypes along the length of the genome, and heteroduplex analysis revealing the presence of similar self-annealing segments at the ends corresponding to "inverted terminal repeats" (ITRs). Similar infectivity patterns also suggest that the replication functions in each serotype are under similar regulatory control. Multiple serotypes of this virus are known to be suitable for gene delivery, and all known serotypes can infect cells from a variety of tissue types. At least eleven consecutive AAV serotypes are known in the art.Non-limiting exemplary serotypes useful in the methods disclosed herein include any of eleven 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, AAV refers to serotypes AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVPHP.B, or AAVrh74.

[0115] As used herein, "AAV vector" refers to a vector that includes one or more heterologous nucleic acid (HNA) sequences and one or more AAV inverted terminal repeats (ITRs). Such AAV vectors, when present in a host cell that provides the functionality of the rep and cap gene products, can replicate, allowing the ITRs and the nucleic acid between the ITRs to be packaged into infectious viral particles. In embodiments, the AAV vector includes a promoter, at least one nucleic acid that can code for at least one protein or RNA, and / or an enhancer and / or terminator in the adjacent ITRs that is packaged into the infectious AAV particle. The nucleic acid between the ITRs and the ITRs can be encapsidated into an AAV capsid, and this encapsidated portion of the nucleic acid can be referred to as the "AAV vector genome." In addition to the encapsidated portion, the AAV vector can contain antibiotic resistance genes or other elements known in the art that are included in a plasmid for manufacturing purposes, but are not packaged into the AAV particle.

[0116] As used herein, the term "viral capsid" or "capsid" refers to the proteinaceous shell or coat of a viral particle. The capsid functions to encapsidate, protect, transport, and release the viral genome into the host cell. Capsids are generally composed of oligomeric structural subunits of proteins ("capsid proteins"). As used herein, the term "encapsidated" means enclosed within the 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 dodecahedron 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.

[0117] "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.

[0118] As used herein, the term "helper" with respect to a virus or plasmid refers to a virus or plasmid used to provide additional components necessary for the replication and packaging of any one of the AAV vector genomes disclosed herein. The components encoded by the helper virus can include any gene required for virion assembly, encapsidation, genome replication, and / or packaging. For example, the helper virus or plasmid can encode an enzyme required 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 herpesvirus (virus). In an embodiment, the pHELP plasmid can be a pHELPK plasmid, in which the ampicillin expression cassette is replaced with a kanamycin expression cassette, and pHELPK has the sequence shown in SEQ ID NO:92.

[0119] As used herein, packaging cells (or helper cells) are cells used to produce viral vectors. To produce recombinant AAV viral vectors, Rep and Cap proteins provided in trans, as well as gene sequences from adenovirus that help AAV replication, are required. In some embodiments, the packaging / helper cells containing the plasmid are stably integrated into the genome of the cell. In other embodiments, the packaging cells can be transiently transfected. Typically, the packaging cells are eukaryotic cells, such as mammalian or insect cells.

[0120] As used herein, a reporter protein is a detectable protein operably linked to a promoter to assay the expression (e.g., tissue specificity and / or strength) of the promoter. In embodiments, a reporter protein may be operably linked to a polypeptide. In embodiments, reporter proteins may be used in DNA delivery methods, functional identification and characterization of promoter and enhancer elements, translational and transcriptional regulation, mRNA processing, and monitoring protein:protein interactions. Non-limiting examples of reporter proteins are β-galactosidase, fluorescent proteins such as green fluorescent protein (GFP) or red fluorescent protein (RFP), luciferase, glutathione-S-transferase, and maltose binding protein.

[0121] A "pharmaceutical composition" is intended to comprise a combination of an active ingredient, such as a polypeptide, polynucleotide, antibody, or viral vector, with an inert or active carrier, such as a solid support, making the composition suitable for diagnostic or therapeutic use in vitro, in vivo or ex vivo.

[0122] As used herein, the term "pharmaceutical acceptable carrier" includes standard pharmaceutical carriers such as phosphate buffered saline, water, and emulsions such as oil / water or water / oil emulsions, as well as any of various types of wetting agents. The composition may also include stabilizers and preservatives. For examples of carriers, stabilizers, and adjuvants, see Martin (1975) Remington's Pharm.Sci., 15th Ed. (Mack Publ.Co., Easton).

[0123] A "subject" of diagnosis or treatment may be a cell, or an animal, such as a mammal, or a human. A subject is not limited to a particular species, and includes non-human animals that are the subject of diagnosis or treatment, and non-human animals that are the subject of infection or animal models, including, but not limited to, monkey, murine, rat, dog, or rabbit species, as well as other farm animals, sport animals, or pets. In an embodiment, the subject is a human.

[0124] The term "tissue" is used herein to refer to tissue of a living or dead organism, or any tissue derived from or designed to mimic a living or dead organism. Tissues may be healthy, diseased, or genetically altered. Biological tissues may include any single tissue (e.g., a collection of cells that may be interconnected), or a group of tissues making up an organ or part or region of the body of an organism. Tissues may include, consist of, or consist of homogeneous cellular material, or may be composite structures such as found in regions of the body including the thorax, which may include, for example, lung tissue, skeletal tissue, and / or muscle tissue. Exemplary tissues include, but are not limited to, those derived from the liver, lung, thyroid, skin, pancreas, blood vessels, bladder, kidney, brain, biliary tree, duodenum, abdominal aorta, iliac vein, heart, and intestine, including any combination thereof.

[0125] As used herein, "treating" or "treatment" of a disease in a subject refers to (1) preventing a symptom or disease from occurring in a subject who is predisposed to the disease or who does not yet exhibit symptoms thereof, (2) inhibiting or arresting the development of a disease, or (3) ameliorating or causing regression of a disease or symptoms of a disease. As understood in the art, "treatment" is an approach to obtain beneficial or desired results, including clinical results. For purposes of the present technology, beneficial or desired results may include, but are not limited to, one or more of the following: alleviation or amelioration of one or more symptoms, whether detectable or undetectable, reduction in the extent of a condition (including a disease), a stable (i.e., non-worsening) stage of a condition (including a disease), delay or slowing of a condition (including a disease), progression, amelioration, or alleviation, stage, and remission (partial or complete) of a condition (including a disease).

[0126] As used herein, the term "effective amount" is intended to mean an amount sufficient to achieve a desired effect. In the context of therapeutic or prophylactic applications, the effective amount will depend on the type and severity of the condition in question, as well as the characteristics of the individual subject, such as general health, age, sex, weight, and tolerance to the pharmaceutical composition. In the context of gene therapy, in an embodiment, the effective amount is an amount sufficient to cause partial or full function reacquisition of a missing gene in a subject. In an embodiment, an effective amount of AAV viral particles is an amount sufficient to cause expression of a gene in a subject. Those skilled in the art will be able to determine the appropriate amount depending on these and other factors.

[0127] In embodiments, the effective amount will depend on the size and nature of the application in question. It will also depend on the nature and sensitivity of the target subject, and the method of use. Those skilled in the art will be able to determine the effective amount based on these and other considerations. The effective amount may comprise, consist essentially of, or consist of one or more doses of the composition, depending on the embodiment.

[0128] 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 human. Administration may occur in one dose, continuously or intermittently throughout the course of treatment. Methods of determining the most effective means and dosage of administration will vary with the composition used in the therapy, the purpose of the therapy, and the age, health, or sex of the subject being treated. Single or multiple administrations may be carried out with the dose level and pattern selected by the treating physician, or in the case of pets and other animals, by the treating veterinarian.

[0129] Structure and function of AAV AAV is a replication-deficient parvovirus whose single-stranded DNA genome is approximately 4.7 kb in length, containing two approximately 145 nucleotide inverted terminal repeats (ITRs). There are multiple serotypes of AAV. The nucleotide sequences of the genomes of the 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 in 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, 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, and the AAV-9 genome is described in Gao et al. 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. Pat. No. 9,434,928, which is incorporated herein by reference in its entirety. U.S. Pat. No. 9,434,928 also provides the sequences of the capsid proteins and the self-complementary genome. In one embodiment, the genome is a self-complementary genome. Cis-acting sequences that direct viral DNA replication (rep), encapsidation / packaging, and host cell chromosomal integration are contained within the AAV ITRs. Three AAV promoters (designated p5, p19, and p40 for their relative map locations) drive expression of two AAV internal open reading frames encoding the rep and cap genes.Two rep promoters (p5 and p19) coupled with differential splicing of a single AAV intron (at nucleotides 2107 and 2227) result in the production of four rep proteins (rep 78, rep 68, rep 52, and rep 40) from the rep gene. The rep proteins have multiple enzymatic properties that are ultimately responsible for the replication of the viral genome.

[0130] The cap gene is expressed from the p40 promoter and encodes three capsid proteins, VP1, VP2, and VP3. Alternative splicing and non-consensus translation start sites are responsible for the production of the three related capsid proteins. More specifically, after a single mRNA into which each of the VP1, VP2, and VP3 proteins is translated is transcribed, it can be spliced ​​in two different ways, i.e., either a longer or a shorter intron can be excised, resulting in the formation of two pools of mRNAs (2.3 kb and 2.6 kb long mRNA pools). The longer intron is often preferred, and therefore the 2.3 kb long mRNA can be referred to as the major splice variant. This form lacks the first AUG codon, from which the synthesis of the VP1 protein is initiated, resulting in a reduced overall level of VP1 protein synthesis. The first AUG codon remaining in the major splice variant is the start codon for the VP3 protein. However, upstream of that codon in the same open reading frame is an ACG sequence (encoding threonine) surrounded by an optimal Kozak (translation start) context.This contributes to the low level synthesis of the VP2 protein, which, like VP1, is actually a VP3 protein that contains additional N-terminal residues, each of which is incorporated herein by reference, Becerra SP et al., (December 1985). "Direct mapping of adeno-associated viral 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 viral 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 codon". "Alternate mRNA splicing is required for synthesis of adeno-associated viral VP1 capsid protein". Journal of Virology. 62(9):3356-63; and Trempe JP, Carter BJ (September 1988). "Alternate mRNA splicing is required for synthesis of adeno-associated viral VP1 capsid protein". Journal of Virology. 62(9):3356-63. A single consensus polyadenylation signal is located at map position 95 of the AAV genome.The life cycle and genetics of AAV are reviewed in Muzyczka, Current Topics in Microbiology and Immunology, 158:97-129 (1992).

[0131] Each VP1 protein contains a VP1 portion, a VP2 portion, and a VP3 portion. The VP1 portion is an N-terminal portion of the VP1 protein that is unique to the VP1 protein, corresponding to amino acids 1-137 of SEQ ID NO: 164. The VP2 portion is an amino acid sequence present in the VP1 protein that is also found in the N-terminal portion of the VP2 protein, corresponding to amino acids 138-202 of SEQ ID NO: 164. The VP3 portion and the VP3 protein have the same sequence. The VP3 portion is a C-terminal portion of the VP1 protein that is shared by the VP1 and VP2 proteins, corresponding to amino acids 203-737 of SEQ ID NO: 164. See FIG. 5.

[0132] The VP3 protein can be further divided into separate variable surface regions I to IX (VR-I to IX). As described in DiMatta et al., "Structural Insight into the Unique Properties of Adeno-Associated Virus Serotype 9," J. Virol., Vol. 86(12):6947-6958, June 2012, the contents of which are incorporated herein by reference, each of the variable surface regions (VRs) can comprise or contain specific amino acid sequences that, either alone or in combination with specific amino acid sequences of each of the other VRs, can confer a unique infection phenotype (e.g., reduced antigenicity, improved transduction, and / or tissue-specific tropism compared to other AAV serotypes) to the particular serotype.

[0133] AAV has unique characteristics that make it attractive as a viral vector for delivering foreign DNA to cells, for example in gene therapy. AAV infection of cells in culture is non-cytopathic, and natural infection of humans and other animals is silent and asymptomatic. Furthermore, AAV can infect many mammalian cells, allowing the possibility of targeting many different tissues in vivo. Furthermore, AAV can transduce slowly dividing and non-dividing cells and essentially persist for the lifetime of those cells as transcriptionally active nuclear episomes (extrachromosomal elements). The AAV proviral genome is inserted as cloned DNA into a plasmid, making the construction of recombinant genomes feasible. Furthermore, signals directing AAV replication and genome encapsidation are contained within the ITRs of the AAV genome, so that part or all of the internal approximately 4.3 kb of the genome (encoding replication and structural capsid proteins, rep-cap) can be replaced with foreign DNA to generate an AAV vector genome. The rep and cap proteins may be provided in trans. Another important feature of AAV is that it is an extremely stable and robust virus. It easily withstands the conditions used to inactivate adenovirus (56°C to 65°C for several hours), making cryopreservation of AAV less hazardous. AAV can even be lyophilized. Finally, AAV-infected cells are not resistant to superinfection.

[0134] Several studies have demonstrated long-term (>1.5 years) recombinant AAV-mediated protein expression in muscle. 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). Furthermore, as described by 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), muscle is highly vascularized, so recombinant AAV transduction results in the appearance of the transgene product in the systemic circulation after intramuscular injection. Furthermore, Lewis et al., J Virol, 76:8769-8775 (2002) demonstrated that skeletal muscle fibers possess the cellular factors necessary for correct antibody glycosylation, folding, and secretion, indicating that muscle has the capacity for stable expression of 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 may be from any AAV serotype capable of deriving a recombinant virus, 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 rAAV is disclosed, for example, in WO2001 / 083692. Other types of rAAV variants are also contemplated, for example, rAAVs with capsid mutations.See, e.g., 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.

[0135] AAV vector particles, capsid proteins, and AAV vectors Provided herein are AAV vector particles, AAV vectors, and capsid proteins that have desired tissue specificity and find use in delivering a variety of therapeutic payloads, including nucleic acids and proteins, useful for the treatment of diseases.

[0136] AAV Capsid Protein The present disclosure provides AAV particles with the characteristics of high gene transfer efficiency and increased tissue tropism.Currently, AAV viral vector delivery relies on the use of serotype selection for tissue targeting based on the natural tropism of the virus or by direct injection into target tissue.However, many currently available AAV viral vectors are not optimal for delivering genes to specific target sites.

[0137] The present disclosure provides AAV capsid protein sequences that confer high gene transfer efficiency and increased tissue specificity to AAV particles comprising the AAV capsid protein sequences. In embodiments, AAV particles comprising such AAV capsid proteins are administered via specific delivery routes to achieve optimal delivery to specific target sites.

[0138] 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 acids mutated, deleted, or added 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 may 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 relative to any one of the nucleic acid sequences listed in Table 1. [Table 1]

[0139] 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 that differ from the amino acid sequence of 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 relative to the sequence of SEQ ID NO: 15, 18-23, 47, 82, 98, or 167.

[0140] 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).

[0141] 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).

[0142] Exemplary VP2 and VP3 protein sequences are provided in Tables 2 and 3. Given the VP2 and VP3 sequences, the VP1 portion may be determined by alignment with the complete VP1 protein sequence. [Table 2]

[0143] 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 that has up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 different amino acids from SEQ ID NO: 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 that has up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 different amino acids from SEQ ID NO: 165.

[0144] Also provided are polynucleotides encoding these VP2 proteins. In embodiments, the polynucleotides encoding the VP2 proteins comprise, consist essentially of, or consist of the sequence of SEQ ID NO: 47, or a sequence having up to 5, up to 10, or up to 30 nucleotide changes relative to SEQ ID NO: 47. In embodiments, the polynucleotides encoding the VP2 proteins comprise, consist essentially of, or consist of the sequence of SEQ ID NO: 168, or a sequence having up to 5, up to 10, or up to 30 nucleotide changes relative to SEQ ID NO: 168.

[0145] Exemplary nucleic acids for other capsid VP2 portions may be derived from the corresponding portions of the VP1 capsid protein nucleic acid. [Table 3]

[0146] 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.

[0147] In embodiments, the AAV VP3 protein comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 17, 41-46, 51, 86, or 166, or a sequence that has up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 different amino acids from SEQ ID NO: 17, 41-46, 51, 86, or 166. In embodiments, the AAV VP3 protein comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 166, or a sequence that has up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 different amino acids from SEQ ID NO: 166.

[0148] Also provided are polynucleotides encoding these VP3 proteins. In embodiments, a polynucleotide encodes a protein comprising, consisting essentially of, or consisting 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 relative to SEQ ID NO: 16, 24-29, 48, 83, or 169. In embodiments, a polynucleotide encodes a protein comprising, consisting essentially of, or consisting of a sequence of SEQ ID NO: 169, or a sequence having up to 5, up to 10, or up to 30 nucleotide changes relative to SEQ ID NO: 169.

[0149] 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 may be replaced with a VP1, VP2, or VP3 portion from a different AAV capsid protein disclosed herein.

[0150] In embodiments, provided herein is an AAV capsid protein comprising a leucine residue at amino acid position 129, an asparagine residue at amino acid position 586, and a glutamic acid residue at amino acid position 723, where 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 may be introduced into other capsid proteins.

[0151] In embodiments, 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 position 129; the VP2 portion comprises a threonine (T) or asparagine (N) residue at amino acid position 157, a lysine (K) or serine (S) residue at amino acid position 162; the VP3 portion comprises an asparagine (N) residue at amino acid position 223, an alanine (A) residue at amino acid position 224, a histidine (H) residue at amino acid position 272, a threonine (T) residue at amino acid position 410, a histidine (H) residue at amino acid position 724, and a proline (P) residue at amino acid position 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).

[0152] In embodiments, the VP1 portion further comprises an aspartic acid (D) or alanine (A) residue at amino acid position 24, where 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 embodiments, the VP2 portion further comprises one or more of: (i) a proline (P) residue at amino acid position 148, (ii) an arginine (R) residue inserted at amino acid position 152, (iii) an arginine (R) residue at amino acid position 168, (iv) an isoleucine (I) residue at amino acid position 189, and (v) a serine (S) residue at amino acid position 200, where 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.

[0153] In embodiments, one or more of the variable regions I-IX in the capsid protein of the disclosed VP3 portion (see FIG. 5) may be removed and replaced with alternative regions. Suitable alternatives are identified in Table 6 below. The positions for these, and the identities of additional alternatives, may be identified by alignment to SEQ ID NO: 41, as shown in FIG. 5. In embodiments, one or more of the VRs may have an insertion of one, two, or three amino acids. In embodiments, one or more of the VRs may have a deletion of one, two, or three amino acids. [Table 6-1] [Table 6-2]

[0154] The present disclosure provides a nucleic acid encoding any one of the AAV capsid proteins disclosed herein. The present disclosure also provides a vector comprising any one of the nucleic acids disclosed herein.

[0155] In an embodiment, the AAV is an AAV9 serotype. Alternative serotypes or modified capsid viruses can be used to optimize neurotropism. Alternative vectors include AAV9 serotype vectors modified for higher neurotropism than standard AAV9, such as PHP.B, which uses the Cre-lox recombination system to specify a neurotargeting vector. Alternatively, AAV9 PHP.B has a modified amino acid 498 of VP1 from asparagine to lysine to reduce liver tropism. Further variants of AAVrh74 with several mutated amino acids can be used for very broad tissue tropism, including the brain.

[0156] AAV vectors The AAV vector also provides the nucleic acid to be encapsidated into the AAV vector particle, which contains the element(s) responsible for controlling the expression of the nucleic acid in the subject, and the ITRs to facilitate encapsidation. In an embodiment, the AAV vector disclosed herein comprises at least one heterologous nucleic acid (HNA) sequence, which is effective for treating a disease or disorder when expressed in the 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.

[0157] In embodiments, the control of transgene expression in a host cell may be regulated by regulatory elements contained within the AAV vector, including a promoter sequence and a polyadenylation signal. In embodiments, the AAV vector may also encode a signal peptide. In embodiments, the AAV vector has a 5' inverted terminal repeat (ITR) and a 3' inverted terminal repeat (ITR). The 5' ITR is located upstream of the promoter, which is also upstream of the transgene. In embodiments, the 5' ITR and the 3' ITR have the same sequence. In embodiments, they have different sequences. In embodiments, the AAV vector of the present disclosure may include, in a 5' to 3' orientation, a first (5') ITR, a promoter, a transgene, a polyadenylation signal, and a second (3') ITR.

[0158] 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: 253. 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: 254. In embodiments, the corresponding AAV vector is for expression of the Opal transgene.

[0159] 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 for expression of an RS1 transgene.

[0160] Further description of ITRs can be found, for example, in McCarty et al., Gene Ther. 2003 Dec;10(26):2112-8, the contents of which are incorporated herein by reference in their entirety.

[0161] In embodiments, the HNA (eg, an HNA comprising a transgene) is operably linked to a promoter.

[0162] In an embodiment, 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 an embodiment, 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, beta actin promoter, phosphoglycerol kinase (PGK) promoter, U6 promoter, H1 promoter, hybrid chicken beta actin promoter, MeCP2 promoter, H1 promoter, U1a promoter, mMeP418 promoter, mMeP426 promoter, minimal MeCP2 promoter, CAG promoter, or EF1 promoter. It is known in the art that the nucleotide sequence of such promoters can be modified to increase or decrease the efficiency of mRNA transcription. See, e.g., Gao et al. (2018) Mol. Ther.: Nucleic Acids 12:135-145 (modifying 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, a lung-specific promoter, a skin-specific promoter, a muscle-specific promoter, a liver-specific promoter, or an eye-specific promoter (e.g., a VMD2 or mRho promoter).

[0163] In embodiments, the promoter may comprise, consist essentially of, or consist of a polynucleotide having the 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 to 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 to any one of the nucleic acid sequences listed in Table 5.

[0164] 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.

[0165] 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.

[0166] 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.

[0167] In embodiments, the promoter is a PDE promoter. In embodiments, the PDE 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:198. [Table 5-1] [Table 5-2]

[0168] 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.

[0169] In embodiments, the enhancer is an interphotoreceptor retinoid binding protein (IRBP) enhancer. In embodiments, the IRBP enhancer 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:199.

[0170] In an embodiment, the HNA sequence is operably linked to additional regulatory elements. The additional regulatory elements can be woodchuck hepatitis virus post-transcriptional regulatory elements (WPRE). In an embodiment, the AAV vector can include regulatory components suitable for vector growth and culture in bacterial hosts for vector production purposes. For example, the vector can include genes for antibiotic resistance and plasmid maintenance in bacteria, as well as related regulatory elements for controlling protein expression in bacteria.

[0171] 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 MeCP2 polyadenylation signal, retinol dehydrogenase 1 (RDH1) polyadenylation signal, bovine growth hormone (BGH) polyadenylation signal, SV40 polyadenylation signal, SPA49 polyadenylation signal, sNRP-TK65 polyadenylation signal, sNRP polyadenylation signal, or TK65 polyadenylation signal.Exemplary SPA49 polyadenylation signals are described in Ostedgaard et al., Proc. Nat'l Acad. Sci. USA (Feb. 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 having at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 201. In embodiments, the polyadenylation signal 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: 225.

[0172] In an embodiment, the intron is inserted between the promoter and the HNA. In an embodiment, 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:200. In an embodiment, 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:222. In an embodiment, 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:227. In embodiments, the AAV vector genome comprises a CBA sequence located immediately upstream of an intron sequence without any additional nucleotides in between, wherein the CBA sequence comprises, consists essentially of, or consists of the nucleic acid sequence SEQ ID NO: 229, or a sequence having up to 10, up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2, or up to 1 mutation thereto. 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".

[0173] In an embodiment, the first telomeric repeat is inserted between the polyadenylation signal and the 3'ITR. In an embodiment, the telomeric repeat comprises a repeat unit of CCCTAA (SEQ ID NO: 217). In an embodiment, the telomeric repeat comprises an intermediate repeat comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more consecutive copies of a repeat unit of CCCTAA (SEQ ID NO: 217). In an embodiment, the telomeric repeat comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more copies of an intermediate repeat. In an embodiment, the copies of the intermediate repeat are separated by a spacer comprising TTTTT (SEQ ID NO: 218). In an embodiment, the first telomeric repeat 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.

[0174] In an embodiment, the first telomeric repeat is inserted between the polyadenylation signal and the 3'ITR. In an embodiment, the telomeric repeat comprises a repeat unit of TTAGGG (SEQ ID NO: 219). In an embodiment, the telomeric repeat comprises an intermediate repeat comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more consecutive copies of a repeat unit of TTAGGG (SEQ ID NO: 219). In an embodiment, the telomeric repeat comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more copies of an intermediate repeat. In an embodiment, the copies of the intermediate repeat are separated by a spacer comprising AAAAA (SEQ ID NO: 220). In an embodiment, the first telomeric repeat 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.

[0175] In an embodiment, the second telomeric repeat is inserted between the 5'ITR and the promoter. In an embodiment, the telomeric repeat comprises a repeat unit of TTAGGG (SEQ ID NO: 219). In an embodiment, the telomeric repeat comprises an intermediate repeat comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more consecutive copies of a repeat unit of TTAGGG (SEQ ID NO: 219). In an embodiment, the telomeric repeat comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more copies of an intermediate repeat. In an embodiment, the copies of the intermediate repeat are separated by a spacer comprising AAAAA (SEQ ID NO: 220). In an embodiment, the second telomeric repeat 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.

[0176] In embodiments, the human beta-globin scaffold / matrix attachment region (βGlo_s / MAR) sequence is inserted between the polyadenylation signal and the downstream telomeric repeats 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.

[0177] Heterologous nucleic acid (HNA) The AAV viral vectors disclosed herein infect and deliver one or more heterologous nucleic acids (HNA) to a target tissue. In embodiments, the HNA sequences are transcribed and, optionally, translated within the cells of the target tissue.

[0178] In some cases, the HNA encodes an antisense RNA, a microRNA, an siRNA, or a 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 efficiently delivered to target tissues and cells to mediate gene repair via the CRISPR system, and current CRISPR / Cas9 gene correction protocols suffer from several shortcomings. Long-term expression of Cas9 may induce a host immune response. Additional guide RNAs may be delivered via a separate vector due to packaging constraints. In an embodiment, the HNA encodes a Cas9 protein or its equivalent.

[0179] In embodiments, HNA comprises a transgene encoding a protein that can be expressed in a subject's cells to treat a disease or disorder resulting from the reduction or elimination of the activity of a native protein. Thus, in embodiments, the transgene is selected from the group consisting of cystic fibrosis transmembrane conductance regulator (CFTR), N-acetylglucosaminidase (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 beta (GBA1), iduronidase alpha-L- (IDUA), methyl-CpG binding protein 2 (MeCP2), ceroid lipofuscinosis, neuron, 1 (CLN1), rhodopsin (Rho), cytochrome P450 family 4 subfamily V member 2 (CYP4V2), retinoschisin 1 (RS1), phosphodiesterase 6B (PDE6B), ATP-binding cassette subfamily A member 4 (ABCA4), bestrophin-1 (BEST1), OPA1 mitochondrial dynamin-like GTPase (OPA1), and optic atrophy 3 (OPA3).

[0180] In an embodiment, the transgene encodes a cytochrome P450 family 4 subfamily V member 2 (CYP4V2). In an embodiment, CYP4V2 comprises a mutated, codon-optimized, and / or truncated sequence of CYP4V2. In an embodiment, 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 an embodiment, 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 disclosure encodes CYP4V2 and is for treating Vietti crystalline dystrophy.

[0181] In embodiments, the transgene encodes retinoschisin 1 (RS1). In embodiments, the RS1 transgene comprises a mutated, codon-optimized, and / or 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, consists essentially of, or consists 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 for treating retinoschisis.

[0182] In an embodiment, the transgene encodes phosphodiesterase 6B (PDE6B). In an embodiment, the PDE6B transgene comprises a mutated, codon-optimized, and / or truncated sequence of PDE6B. In an embodiment, 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 an embodiment, 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 for treating retinitis pigmentosa.

[0183] In an embodiment, the transgene encodes ATP-binding cassette subfamily A member 4 (ABCA4). In an embodiment, the ABCA4 transgene comprises a mutated, codon-optimized, and / or truncated sequence of ABCA4. In an embodiment, 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 an embodiment, 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 disclosure encodes ABCA4 and is for treating Stargardt's disease.

[0184] In an embodiment, the transgene encodes bestrophin 1 (BEST1). In an embodiment, the BEST1 transgene comprises a mutated, codon-optimized, and / or truncated sequence of BEST1. In an embodiment, 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 an embodiment, 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 embodiments, the AAV vector or AAV vector genome of the disclosure encodes BEST1 and is for treating BEST vitelliform macular dystrophy.

[0185] In embodiments, the transgene encodes the OPA1 mitochondrial dynamin-like GTPase (OPA1). In embodiments, the OPA1 transgene comprises a mutated, codon-optimized, and / or 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 DeltaS1 (ΔS1) isoform that comprises, consists essentially of, or consists of a nucleic acid having a sequence of SEQ ID NO: 182, 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: 182. In embodiments, the OPA1 transgene is a DeltaS1 isoform that encodes a protein that comprises, consists essentially of, or consists of an amino acid sequence of SEQ ID NO: 183, 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: 183. In embodiments, the OPA1 transgene is an E5b isoform comprising, consisting essentially of, or consisting of a nucleic acid having the 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 a transgenic E5b isoform encoding a protein comprising, consisting essentially of, or consisting of the 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, an AAV vector or AAV vector genome of the disclosure encodes OPA1 and is for treating manifest optic atrophy.

[0186] In embodiments, the transgene encodes Optic Atrophy 3 (OPA3). In embodiments, the OPA3 transgene comprises a mutated, codon-optimized, and / or 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 disclosure encodes OPA3 and is for treating manifest optic atrophy.

[0187] 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 relative to 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 different amino acids from any one of the amino acid sequences listed in Table 4 (SEQ ID NOs: 142-144 and 177-118). [Table 4-1] [Table 4-2]

[0188] In embodiments, the transgene comprises a nucleic acid sequence 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 relative to any one of SEQ ID NOs: 99-133 and 172-176. In embodiments, the transgene encodes an amino acid sequence 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 any one of the amino acid sequences SEQ ID NOs: 134-151 and 177-181.

[0189] In embodiments, the heterologous nucleic acid encodes a reporter protein, for example a fluorescent protein.

[0190] Methods for Producing AAV Viral Vectors Various approaches can be used to produce AAV viral vectors. In an embodiment, packaging is achieved by using helper virus or helper plasmid and cell line. Helper virus or helper plasmid contains elements and sequences that facilitate viral vector production. In another aspect, helper plasmid is stably integrated into the genome of packaging cell line, so that packaging cell line does not need additional transfection with helper plasmid.

[0191] In embodiments, the cells are packaging or helper cell lines. In embodiments, the helper cell lines are eukaryotic cells, such as HEK293 cells or 293T cells. In embodiments, the helper cells are yeast cells or insect cells.

[0192] In embodiments, the cell comprises a nucleic acid encoding a tetracycline activator protein and a promoter that regulates expression of the tetracycline activator protein. In embodiments, the promoter that regulates expression of the tetracycline activator protein is a constitutive promoter. In embodiments, the promoter is a phosphoglycerate kinase promoter (PGK) or a CMV promoter.

[0193] The helper plasmid can contain at least one viral helper DNA sequence derived from a replication-incompetent viral genome, for example, to encode in trans all virion proteins required for packaging replication-incompetent AAV without producing replication-incompetent AAV and to produce virion proteins capable of packaging replication-incompetent AAV at high titers.

[0194] Helper plasmids for packaging AAV are known in the art, see, for example, US Patent Publication No. 2004 / 0235174A1, which is incorporated herein by reference. As described herein, AAV helper plasmids may contain, as non-limiting examples, the Ad5 genes E2A, E4, and VA controlled by their respective native or heterologous promoters as helper virus DNA sequences. AAV helper plasmids may additionally contain expression cassettes for the expression of marker proteins, such as fluorescent proteins, to allow simple detection of transfection of desired target cells.

[0195] The present disclosure provides a method for producing AAV particles, comprising transfecting a packaging cell line with any one of the AAV helper plasmids disclosed herein and any one of the AAV vectors 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, for example, a human embryonic kidney (HEK) 293 cell line. The present disclosure provides a cell comprising any one of the AAV vectors and / or AAV particles disclosed herein.

[0196] Pharmaceutical Compositions The present disclosure provides pharmaceutical compositions comprising any one of the AAV vectors, AAV capsids, and / or AAV particles described herein. Typically, the AAV particles are administered for therapeutic purposes.

[0197] Pharmaceutical compositions as described herein may be formulated by any method known or developed in the art of pharmacology, including, but not limited to, contacting an active ingredient (e.g., a viral particle or an AAV vector) with excipients or other accessory ingredients and dividing or packaging the product into dosage units. The viral particles of the present disclosure may be formulated to have desirable characteristics, such as increased stability, increased cell transfection, sustained or delayed release, biodistribution or disposition, regulated or enhanced translation of the encoded protein in vivo, and release profile of the encoded protein in vivo.

[0198] Thus, the pharmaceutical composition may further comprise saline, lipidoid, liposome, lipid nanoparticle, polymer, lipoplex, core-shell nanoparticle, peptide, protein, cell transfected with a viral vector or transduced with an AAV viral particle (e.g., for implantation into a subject), nanoparticle mimic, or combinations thereof. In an embodiment, the pharmaceutical composition is formulated as a nanoparticle. In an embodiment, the nanoparticle is a self-assembled nucleic acid nanoparticle.

[0199] Pharmaceutical compositions according to the present disclosure may be prepared, packaged, and / or sold in bulk, as single unit doses, and / or as multiple single unit doses. The amount of active ingredient is generally equal to the dosage of active ingredient to be administered to a subject, and / or a convenient fraction of such a dosage (e.g., 1 / 2 or 1 / 3 of such a dosage, etc.). The formulations of the present invention may include one or more excipients, each in an amount that together increases the stability of the viral vector, increases the transfection or transduction of cells by the viral vector, increases the expression of the protein encoded by the viral vector, and / or modifies the release profile of the protein encoded by the viral vector. In an embodiment, the pharmaceutical composition includes an excipient. Non-limiting examples of excipients include a solvent, dispersion medium, diluent, or other liquid vehicle, a dispersing or suspending aid, a surfactant, an isotonic agent, a thickening or emulsifying agent, a preservative, or a combination thereof.

[0200] In an embodiment, the pharmaceutical composition comprises a cryoprotectant. The term "cryoprotectant" refers to an agent capable of reducing or eliminating damage to a substance during freezing. Non-limiting examples of cryoprotectants include sucrose, trehalose, lactose, glycerol, dextrose, raffinose, and / or mannitol.

[0201] Treatment method The present disclosure provides a method of preventing or treating a disorder comprising, consisting essentially of, or consisting of administering to a subject a therapeutically effective amount of any one of the pharmaceutical compositions disclosed herein.

[0202] In embodiments, the disorder is a CNS disorder, a skin disorder, a lung disorder, a muscle disorder, a liver disorder, or an eye disease (or a retinal disease). In embodiments, the disorder is cystic fibrosis. In embodiments, the disorder is an eye disease. In embodiments, the disorder is a retinal disease.

[0203] In embodiments, the disorder is hypophosphatasia, amyotrophic lateral sclerosis (ALS), spinal muscular atrophy (SMA), recessive dystrophic epidermolysis bullosa (RDEB), lysosomal storage disorders (including Duchenne muscular dystrophy and Becker muscular dystrophy), juvenile Batten disease, infantile Batten disease, autosomal overt disorders, muscular dystrophies, Vietti crystalline dystrophy, retinoschisis (e.g., degenerative, hereditary , traction, 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, muscle wasting, heart disease, intimal hyperplasia, Rett syndrome, epilepsy, Huntington's disease, Parkinson's disease, Alzheimer's disease, autoimmune diseases, 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.

[0204] In an embodiment, the disorder is X-linked retinoschisis (XLRS), a rare monogenic disease resulting in severe visual impairment. While female carriers are asymptomatic, affected males usually begin to show symptoms of the disease within the first decade, sometimes in infancy. The disease is due to mutations in the RS1 gene, which is expressed in photoreceptors and retinal bipolar cells. The gene product is a secreted protein that is localized primarily in the inner segments of photoreceptor cells and more diffusely in the remainder of the neural retina. RS1 forms homooctameric complexes and is thought to mediate cell-cell adhesion through interactions with extracellular epitopes of membrane proteins. In individuals with XLRS, cavities develop where adhesion of adjacent retinal layers is disrupted, usually in the outer plexiform layer where photoreceptors synapse with inner retinal neurons. This results in discontinuities in the retinal circuitry, degeneration of photoreceptors, and vision impairment. Many of the RS1 mutations reported in the literature are point mutations predicted to disrupt protein secretion and are therefore functionally equivalent to null alleles. The current standard of care for XLRS patients is palliative and includes correction of refractive errors, low vision assistance, and genetic counseling. Complications such as retinal detachment (up to 22% of patients) and vitreous hemorrhage (up to 40% of patients) occur most frequently in the later stages of the disease and can be treated surgically. Early intervention with gene therapy has the significant potential to reverse or stabilize disease progression at an early stage of the disease, preventing significant vision loss as well as the development of these more severe complications.

[0205] In an embodiment, the disorder is autosomal overt optic atrophy (ADOA). ADOA is caused by mutations in Opa1, resulting in vision loss in the first to second decades of life. In an embodiment, Opa1 homozygous mutants are embryonic lethal and therefore do not survive past E9-12. Heterozygous (HT) animals survive to that stage, but over time have retinal degeneration, neurological defects, and musculoskeletal complications. Mice exhibit optic nerve atrophy on fundus and scanning laser ophthalmoscopy (SLO) examination, as well as reduced electroretinogram (ERG) amplitude, and fibrosis in 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 inner mitochondrial membrane remodeling.

[0206] In an embodiment, the present disclosure provides a method for 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.

[0207] In embodiments, the target cells of the present disclosure include retinal cells. In embodiments, the retinal cells include photoreceptors, bipolar cells, retinal ganglion cells, horizontal cells, or amacrine cells. In embodiments, the retinal cells include retinal ganglion cells. In embodiments, the retinal cells include bipolar cells. In embodiments, the retinal cells include horizontal cells. In embodiments, the retinal cells include amacrine cells. In embodiments, the retinal cells include photoreceptors. In embodiments, the photoreceptors include rod cells and / or cone cells.

[0208] In embodiments, the target cells of the present disclosure comprise, consist essentially of, or consist of photoreceptor cells, hi embodiments, the transgenes of the present disclosure are operably linked to an RK promoter for selective expression in photoreceptor cells.

[0209] In addition to the specific transgenes disclosed herein, sequences of known active enzymes may be used as transgenes to deliver functional enzyme activity.

[0210] 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 genetic mutation in the CLN3 gene. CLN3 disease is a progressive neurodegenerative disorder that predominantly affects the central nervous system (CNS), resulting in behavioral problems, vision loss, and other cognitive impairments.

[0211] In an embodiment, the disorder is Fabry disease. Fabry disease is an X-linked lysosomal storage disorder caused by a deficiency in alpha-galactosidase A (GLA) activity, which leads to the accumulation of glycolipid products, globotriaosylceramide (Gb3) and lyso-Gb3, in lysosomes. The manifestations of the disease are highly heterogeneous, but usually include frequent attacks of peripheral neurotrophic pain, angiokeratoma, decreased sweating, corneal dystrophy, and gastrointestinal complications. As the disease progresses, patients suffer from cardiomyopathy, renal failure, and cerebrovascular disease, all of which are the main causes of shortened life span in Fabry patients. Men are the most severely affected population of patients with mutations in the GLA gene, but it is becoming more evident that female patients are also frequently symptomatic, but are often misdiagnosed. Enzyme replacement therapy (ERT) is currently the only FDA-approved therapy for treating Fabry, and requires biweekly injections of relatively large amounts of recombinant protein. ERT reduces the accumulation of Gb3 in the heart, kidneys, and vasculature, but does not completely treat all symptoms of Fabry, primarily due to its inability to efficiently enter the CNS. Gene therapy strategies are being investigated, and many show great promise in correcting glycolipid accumulation, but most are unable to efficiently enter the CNS and suffer from the immune response often seen during GLA supplementation.

[0212] In embodiments, the AAV viral vectors disclosed herein are used to treat Fabry disease in patients who do not respond to ERT or when ERT does not address all symptoms. In embodiments, the AAV viral vectors disclosed herein are used to treat Fabry disease in patients who are already receiving ERT.

[0213] In an embodiment, the disorder is Pompe disease. Pompe disease is a lysosomal storage disorder caused by a deficiency in acid alpha-glucosidase (GAA) activity, which results in the accumulation of glycogen within lysosomes. The disease primarily affects both smooth and striated muscle tissues, as well as the central nervous system (CNS), and is presented as a form of muscular dystrophy with early death. Enzyme replacement therapy (ERT) is currently the only FDA-approved therapy for treating Pompe, and requires biweekly injections of relatively large amounts of recombinant protein. Although ERT significantly reduces the mortality rate of infant Pompe patients, who typically die by age 2 without treatment, it cannot completely alleviate all symptoms of Pompe, primarily due to its inability to efficiently enter the CNS and the resulting immune response against the GAA protein. Gene therapy strategies are being investigated, and many have shown great promise in correcting the accumulation of glycogen and other symptoms of Pompe. Most suffer from the severe immune response seen during GAA replacement. Previous studies have demonstrated that liver-specific expression can render animals tolerant to GAA protein and significantly reduce the humoral response.

[0214] In embodiments, the AAV viral vectors disclosed herein are used to treat Pompe disease in patients who have already been administered ERT, e.g., patients who do not respond to ERT or where ERT does not address all of their symptoms.

[0215] In embodiments, the AAV viral vectors disclosed herein are used to treat cancer. In embodiments, the cancer is a solid cancer, such as 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.

[0216] In embodiments, the disorder is an ocular disease. The eye is an immune privileged tissue. Very low numbers of the virus are required for therapeutic benefit. In embodiments, the ocular disease affects photoreceptors and RPE cells. In embodiments, the ocular disease is retinitis pigmentosa (e.g., autosomal recessive (SPATA7 gene, LRAT gene, TULP1 gene), autosomal dominant (AIPL1 gene), and X-linked (RPGR gene)), ocular disorders associated with mutations in the bestrophin-1 (BEST-1 or BEST1) gene (e.g., vitelliform macular dystrophy, age-related macular degeneration, autosomal dominant vitreoretinochoroidopathy, glaucoma, cataract), Leber's congenital amaurosis (LCA, aryl-hydrocarbon interacting protein-like 1 (AIPL1) gene), cone-rod dystrophy (CRD, ABCA4 gene). , Stargardt (ABCA4 gene), congenital choroideremia (CHM gene), Usher syndrome (MYO7A gene, CDH23 gene, USH2A gene, CLRN1 gene), overt optic atrophy (e.g., autosomal (OPA1 gene, OPA3 gene)), retinitis pigmentosa (PDE6B gene), retinoschisis (RS1 gene), Vietti crystalline dystrophy (CYP4V2 gene), or color vision deficiency (CNGA3 gene, CNGB3 gene, GNAT2 gene, PDE6C gene, or PDE6H gene).

[0217] In an embodiment, the present disclosure provides a method for 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.

[0218] In embodiments, the target cells of the present disclosure include retinal cells. In embodiments, the retinal cells include photoreceptors, bipolar cells, retinal ganglion cells, horizontal cells, or amacrine cells. In embodiments, the retinal cells include retinal ganglion cells. In embodiments, the retinal cells include bipolar cells. In embodiments, the retinal cells include horizontal cells. In embodiments, the retinal cells include amacrine cells. In embodiments, the retinal cells include photoreceptors. In embodiments, the photoreceptors include rod cells and / or cone cells.

[0219] In embodiments, the target cells of the present disclosure comprise, consist essentially of, or consist of photoreceptor cells.

[0220] In embodiments, the subject is a mammal, e.g., a human. In certain aspects, the human is an infant human, e.g., under 3 years of age, under 2 years of age, or under 1 year of age.

[0221] The methods of treatment and prevention disclosed herein may be combined with appropriate diagnostic techniques to identify and select patients for treatment or prevention. For example, the methods of treating or preventing a disorder disclosed herein may further comprise a step of performing a genetic test to identify a genetic mutation or deletion associated with the disorder in the subject. In embodiments, the methods of treating or preventing a disorder comprise administering to a subject previously identified as having a mutation associated with the disorder or at high risk of developing the disorder (e.g., based on genetic factors).

[0222] The present disclosure provides a method for increasing the level of a protein in a host cell, comprising contacting the host cell with any one of the AAV particles disclosed herein, wherein the AAV particle comprises any one of the AAV vector genomes disclosed herein, comprising an HNA sequence encoding the 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 disorder that results in a decrease in the level and / or functionality of the protein compared to the level and / or functionality of the protein in a normal subject.

[0223] In embodiments, the level of protein is about 1×10 -7 ng, approximately 3 × 10 -7 ng, approximately 5 × 10 -7 ng, approximately 7 × 10 -7 ng, approximately 9 × 10 -7 ng, approximately 1 × 10 -6 ng, approximately 2 × 10 -6 ng, approximately 3 × 10 -6 ng, approximately 4 × 10 -6 ng, approximately 6 × 10 -6 ng, approximately 7 × 10 -6 ng, approximately 8 × 10 -6 ng, approximately 9 × 10 -6 ng, approximately 10 × 10 -6 ng, approximately 12 × 10 -6 ng, approx. 14x10 -6 ng, about 16x10 -6 ng, approx. 18x10 -6 ng, about 20x10 -6 ng, about 25x10 -6 ng, about 30x10 -6 ng, approx. 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 approximately 90x10 -5 increases to ng levels.

[0224] The present disclosure provides a method of introducing a gene of interest into a cell of a subject, comprising contacting the cell with an effective amount of any one of the AAV viral particles disclosed herein, wherein the AAV viral particle contains any one of the AAV vector genomes disclosed herein that includes the gene of interest.

[0225] Dosage and Administration Methods for determining the most effective means and dosage administration are known to those skilled in the art and will vary depending on the composition used for treatment, the purpose of the treatment, and the subject being treated. Single or multiple administrations can be performed with the dosage level and pattern selected by the treating physician. Note that this can be influenced by the route of administration. Suitable dosage forms and methods of administration of drugs are known in the art. A non-limiting example of such a suitable dosage is 10 mg / dose per administration. 9 From as few as 10 17 The vector genome can be as many as 1 or more.

[0226] In embodiments, the disclosure provides a method of treating a disease or disorder in a subject in need thereof, comprising administering to the subject an effective amount of a therapeutic agent (e.g., an AAV viral vector).

[0227] In embodiments of the methods described herein, the number of viral particles (e.g., AAV) administered to a subject is about 10 9 ~about 10 17 In an embodiment, the range is about 10 10 ~about 10 12 , about 10 11 ~about 10 13 , about 10 11 ~about 10 12 , about 10 11 ~about 10 14 , about 5×l0 11 ~Approx. 5×10 12 , or about 10 12 ~about 10 13 In embodiments, the amount of viral genome (vg) administered to the subject is about 10 9 ~about 10 17 In embodiments, the range is about 10 10 , 2x10 10 , 3x10 10 , 4x10 10 , 5x10 10 , 6x10 10 , 7x10 10 , 8x10 10 , 9x10 10 , 10 11 , 2x10 11 , 3x10 11 , 4x10 11 , 5x10 11 , 6x10 11 , 7x10 11 , 8x10 11 , 9x10 11 , or 10 12 In embodiments, about 10 viral genomes (vg) are administered to the subject. 10 ~about 10 11 , about 10 11 ~about 10 12 , about 10 12 ~about 10 13 vg, about 5×l0 9 ~Approx. 5×10 10 , about 5×l0 10 ~Approx. 5×10 11 , about 5×l0 11 ~Approx. 5×10 12 , about 10 10~about 10 12 , about 10 11 ~about 10 13 , about 10 10 ~about 10 13 , or about 10 11 ~about 10 14 of viral genome (vg) is administered to the subject. For administration to the human eye, approximately 1 × 10 10 A total dose of 5×10 vg / eye may be used for mouse eyes. 9 A total dose of vg / eye may be used. 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, and fluorescein angiography.

[0228] In embodiments, the AAV particles repair a genetic defect in a subject. In embodiments, the ratio of repaired to unrepaired target polynucleotides or polypeptides in a successfully treated cell, tissue, organ, or subject 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 repaired target polynucleotides or polypeptides may be determined by any method known in the art, including, but not limited to, Western blot, Northern blot, Southern blot, PCR, sequencing, mass spectrometry, flow cytometry, immunohistochemistry (IHC), immunofluorescence, fluorescent in situ hybridization, next generation sequencing, immunoblot, and ELISA.

[0229] In embodiments, the viral particles are introduced into the subject intravenously, intrathecally, intracerebral, intraventricular, intranasal, intratracheal, intraaural, intraocular or periocular, orally, rectally, transmucosally, inhalationally, transdermal, parenterally, subcutaneously, intradermal, intramuscularly, intrapleurally, topically, intralymphatically, intravesically, and such introduction may also be intraarterially, intracardiacly, intraventricularly, subventricularly, epidurally, intracerebrally, intraventricularly, subretinally, pararetinally, intravitreally, intraarticularly, intraperitoneally, intrauterinely, or any combination thereof. In embodiments, the viral particles are delivered to the desired target tissue, such as, for example, the lung, eye, or CNS, as non-limiting examples. In embodiments, the delivery of the viral particles is systemic. The intravesical administration route involves administering the drug directly into the cerebrospinal fluid of the ventricles. This can be done by direct injection into the cisterna magna or via a tube that is permanently placed.

[0230] To treat eye diseases (or eye disorders) intraocularly, there are several 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, intracavitary administration (anterior chamber), intravitreal administration, subretinal administration, pararetinal administration, systemic administration, or combinations thereof. 80% of genetic eye disorders occur within the photoreceptors. Intravitreal delivery of small amounts of gene therapy can be performed in an outpatient clinic.

[0231] In an embodiment, the mode of administration is pararetinal administration. As used herein, "pararetinal administration" refers to a form of intravitreal administration in which a therapeutic agent (e.g., an AAV viral vector) is injected (i.e., targeted delivery) into the vitreous cavity in close proximity to a desired area of ​​the retina. In an embodiment, the desired area of ​​the retina is near the foveal region of the retina. In contrast to routine intravitreal administration, which is performed using a short needle designed to deposit the product in the middle vitreous cavity and does not require direct visualization, pararetinal injection is performed under direct visualization of a long needle that has the ability to deliver the product to the posterior vitreous cavity close to the retina. In an embodiment, the therapeutic agent is deposited in the vitreous cavity at a distance of 0 mm to 13 mm from the surface of the retina, at a distance of 0 mm to 10 mm from the surface of the retina, at a distance of 0 mm to 5 mm from the surface of the retina, or at a distance of 0 mm to 3 mm from the surface of the retina. In embodiments, the therapeutic agent is deposited within the vitreous cavity at a distance of 0-13 mm, 0-12 mm, 0-11 mm, 0-10 mm, 0-9 mm, 0-8 mm, 0-7 mm, 0-6 mm, 0-5 mm, 0-4 mm, 0-3 mm, 0-2 mm, or 0-1 mm from the surface of the retina.

[0232] In embodiments, pararetinal administration is used in situations where subretinal injection is not appropriate. In embodiments, pararetinal administration is used for targeted transduction of the optic nerve. In embodiments, pararetinal administration is used to treat a disease or disorder associated with dysfunction of the optic nerve. In embodiments, pararetinal administration is used to treat overt optic atrophy or retinoschisis.

[0233] In embodiments, pararetinal administration involves the use of a small gauge needle (30 gauge or equivalent) with sufficient length (25 mm or equivalent) to reach the posterior pole of the human eye, external or internal illumination and visualization with a microscope, and the use of a corneal contact lens that allows focusing on the posterior vitreous cavity and retina. This is usually done after appropriate analgesia and disinfection, at which point the corneal contact lens is fitted to the eye and the microscope is placed to view the posterior retina. The needle is inserted through the ocular wall in the pars plana region and its tip is visualized. Under direct visualization, the tip of the needle is advanced to the desired location close to the retinal surface. The syringe plunger is advanced to slowly deposit the viral vector (which may be contained in any suitable composition or formulation). The needle is withdrawn and the eye is inspected. The port may be closed with a suture, but with a sufficiently small gauge needle (such as 30 gauge), no suture is required to close the needle track. Ointments and eye shields may be applied, and if necessary, the subject may be kept in a supine position for a period of time after surgery to further promote high pararetinal concentrations of the product. Variations in the delivery device include the creation of a sclerotomy with or without the use of a vitrectomy port to allow for the use of a blunt cannula, and / or cannula designs with tapered and / or flexible extendable tips or side ports to optimize access to and safety on the retinal surface, and / or the use of pneumatic systems instead of simple syringe plungers. Additional descriptions of pararetinal administration are disclosed, for example, in WO2020 / 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.

[0234] In an embodiment, the mode of administration is subretinal administration, in which the substance is injected into the subretinal space between the retinal pigment epithelium (RPE) cells and the photoreceptors. In the subretinal space, the injected substance is in direct contact with the cell membrane of the photoreceptors, and the RPE cells, and the subretinal blebs. In an embodiment, the AAV used for subretinal administration comprises the capsid protein of AAV214 or AAV214-D5. In an embodiment, the subretinal administration is for treating ADOA, XLRS, Stargardt's disease, Vietti crystalline dystrophy, or BEST vitelliform macular dystrophy. Additional description of subretinal administration is disclosed, for example, in Peng et.al., Ophthalmic Res 2017;58:217-226, and Hartman et al., J Ocul Pharmacol Ther.2018 Mar 1;34(1-2):141-153, the contents of each of which are incorporated herein by reference in their entirety for all purposes.

[0235] Administration of the AAV viral particles or compositions of the present disclosure can be performed in one dose, continuously or intermittently throughout the course of treatment, hi embodiments, the AAV viral particles or compositions of the present disclosure are administered parenterally by injection, infusion, or implantation.

[0236] In embodiments, the AAV particles of the present disclosure exhibit enhanced tropism for the brain and cervical spine. In embodiments, the viral particles of the present disclosure can cross the blood-brain barrier (BBB). In embodiments, the AAV particles of the present disclosure exhibit high retinal tropism by pararetinal, subretinal, and / or intravitreal injection. In embodiments, the AAV particles of the present disclosure target multiple ocular cell types, such as, for example, cones, rods, and retinal pigment epithelium (RPE). In embodiments, the AAV particles of the present disclosure evade neutralizing antibodies against native serotypes, allowing for the possibility of re-administration. In further aspects, the AAV particles and compositions of the present disclosure may be administered in combination with other known therapies for the disorder being treated.

[0237] kit The agents, viral vectors, or compositions described herein may, in embodiments, be combined into pharmaceutical or diagnostic or research kits to facilitate their use in therapeutic, diagnostic, or research applications. In embodiments, the kits of the present disclosure include any one of the modified AAV capsid proteins, AAV vectors, AAV viral particles, host cells, isolated tissues, compositions, or pharmaceutical compositions as described herein.

[0238] In an embodiment, the kit further includes instructions for use. Specifically, such a kit may include one or more of the agents described herein, together with instructions describing the intended use and proper use of these agents. By way of example, in an embodiment, the kit may include instructions for mixing one or more components of the kit, and / or for isolating and mixing samples and applying to a subject. In an embodiment, the agents in the kit are in pharmaceutical formulations and dosages suitable for a particular use and method of administering the agents. Kits for research purposes may contain components in appropriate concentrations or amounts for carrying out various experiments.

[0239] The kits may be designed to facilitate the use of the methods described herein and may take many forms. Each of the compositions of the kit may be provided in liquid form (e.g., solution) or solid form (e.g., dry powder), as applicable. In certain cases, some of the compositions may be configurable or otherwise processable (e.g., into an active form), for example, by the addition of a suitable solvent or other species (e.g., water or cell culture medium), which may or may not be provided with the kit. In embodiments, the compositions may be provided in a preservation solution (e.g., a cryopreservation solution). Non-limiting examples of preservation solutions include DMSO, paraformaldehyde, and CryoStor® (Stem Cell Technologies, Vancouver, Canada). In embodiments, the preservation solution contains an amount of a metalloprotease inhibitor.

[0240] In an embodiment, the kit contains any one or more of the components described herein in one or more containers. Thus, in an embodiment, the kit may include a container that contains the agent described herein. The agent may be in liquid, gel, or solid (powder) form. The agent may be sterilely prepared, packaged in a syringe, and shipped refrigerated. Alternatively, they may be housed in a vial or other container for storage. A second container may have another agent that is sterilely prepared. Alternatively, the kit may contain an active agent that is premixed and shipped in a syringe, vial, tube, or other container. The kit may have one or more or all of the components required to administer the agent to a subject, such as a syringe, topical application device, or IV needle tube and bag.

[0241] While the invention has been described in conjunction with the above embodiments, it should be understood that the foregoing description and examples are intended to illustrate, but not to limit, the scope of the invention. Other aspects, advantages, and modifications within the scope of the invention will be apparent to those skilled in the art to which the invention pertains.

[0242] In addition, where features or aspects of the invention are described in terms of a Markush group, those skilled in the art will recognize that the invention is thereby also described in terms of any individual members or subgroups of members of the Markush group. EXAMPLES

[0243] Example 1: Characterization of pararetinal and subretinal administration in non-human primates using various AAV viral vectors The transduction efficiency of AAV viral vectors containing AAV204, AAV214, AAV214-D5, or AAV8 capsids via multiple ocular administration modes was evaluated as described below.

[0244] All AAV viral vectors used in this example contain a recombinant nucleic acid encoding an enhanced green fluorescent protein (hereinafter "EGFP" or "GFP") reporter transgene operably linked to the CBh promoter.

[0245] To test the transduction efficiency of AAV viral vectors in vivo, non-human primates (NHPs) Macaca fascicularis were administered the indicated AAV viral vectors via juxtaretinal, subretinal, or intravitreal administration. The dose / volume for each administration mode was as follows: Pararetinal administration - 1.0E+11vg in an injection volume of 100μL Subretinal administration - 2.5E+10vg in 100μL injection volume Intravitreal administration - 1.5E+12vg in an injection volume of 150μL Pararetinal administration was performed by layering the virus on top of the retina between the vitreous and the internal limiting membrane, therefore not resulting in subretinal detachment. GFP expression was monitored using scanning laser ophthalmoscopy (SLO). SLO images were acquired for samples harvested 26-27 days after injection. At 28 days after injection, eyes were harvested, processed, and analyzed by immunohistochemistry.

[0246] Figures 2B-2E show the SLO results of pararetinal injection of AAV viral vectors containing the capsid proteins of AAV204 (Figure 2B), AAV8 (Figure 2C), AAV214 (Figure 2D), or AAV214-D5 (Figure 2E). Among the capsid proteins tested, AAV viral vectors containing the AAV204 capsid show robust transduction in the macula, papillomacular bundle, and retinal nerve fibers via pararetinal injection, with much higher transduction efficiency compared to the other capsids tested.

[0247] In addition, pararetinal administration of AAV viral vectors containing the AAV204 capsid (Figure 2B) also demonstrates much higher local transduction efficiency (especially for optic nerve transduction) compared to conventional intravitreal administration of the same AAV viral vector (Figure 2A).

[0248] To further compare the transduction efficiency of these two administration routes, retinas were processed for image analysis (Figures 3A-3C). Again, retinas receiving pararetinal administration of AAV viral vectors containing the AAV204 capsid (Figures 3B-3C) show much more robust macular and optic nerve transduction than retinas receiving intravitreal administration of the same AAV viral vectors (Figure 3A).

[0249] Further immunohistochemical analysis of rhodopsin and GFP one 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 entering the optic nerve. In comparison, pararetinal injection of AAV8 viral vector (Figure 3D) showed much lower GFP expression in RGCs. As shown in Figures 3E-3F, pararetinal administration of AAV204 viral vector also resulted in robust GFP expression in the NHP fovea and along the papillomacular bundle between the macula and the optic nerve. These results were consistent with the SLO analysis. Hence, pararetinal administration of AAV204 viral vector results in efficient transduction of target cells in the macula and foveal pit as well as retinal ganglion cells and associated retinal nerve fibers extending to the optic nerve at a dose at least 10-fold lower compared to intravitreal AAV injections commonly used in the art.

[0250] We also evaluated the transduction efficiency of AAV viral vectors containing the capsid proteins of AAV8 (Figures 4A, 4D), AAV214 (Figures 4B, 4E), and AAV214-D5 (Figures 4C, 4F). The results show that these three capsids show similar transduction efficiency when administered subretinally.

[0251] Conclusions: These results demonstrate that pararetinal injection of AAV vectors containing the AAV204 capsid can efficiently deliver payload to the macula or optic nerve / retinal ganglion cell layer.

[0252] Example 2: In vitro characterization of AAV vector genomes containing a human Opa1 expression cassette According to FIG. 7, multiple AAV vector genome expression vectors were constructed for expression of human Opal (hOpa1), including: - pA-Opa1_1 (comprising SEQ ID NO: 230 and generated from a DNA template vector comprising SEQ ID NO: 186), - pA-Opa1_3 (comprising SEQ ID NO: 231 and generated from a DNA template vector comprising SEQ ID NO: 187), - pA-Opa1_5 (comprising SEQ ID NO: 232 and generated from a DNA template vector comprising SEQ ID NO: 188), - pA-Opa1_11 (comprising SEQ ID NO: 233 and generated from a DNA template vector comprising SEQ ID NO: 189), - pA-Opa1_13 (comprising SEQ ID NO: 234 and generated from a DNA template vector comprising SEQ ID NO: 190), - pA-Opa1_15 (comprising SEQ ID NO: 235 and generated from a DNA template vector comprising SEQ ID NO: 191), - pA-Opa1_17 (comprising SEQ ID NO: 236 and generated from a DNA template vector comprising SEQ ID NO: 192), - pA-Opa1_21 (comprising SEQ ID NO: 237 and generated from a DNA template vector comprising SEQ ID NO: 193), - pA-Opa1_25 (containing SEQ ID NO: 238 and generated from a DNA template vector containing SEQ ID NO: 194), and - pA-Opa1_27 (comprising SEQ ID NO: 239 and generated from a DNA template vector comprising SEQ ID NO: 195). Each vector genome construct contains, 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'ITR and 3'ITR. In addition, most constructs contained telomeric repeats (TRs) downstream of the BGH polyA signal and, optionally, upstream of the promoter. Specifically, pA-Opa1_1 does not contain a TR, pA-Opa1-3 and pA-Opa1-5 both contain 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 contain 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. The Opal transgene is operably linked to the CBh promoter in most AAV vector genomes, with the exception that in pA-Opa1_5 and pA-Opa1_13, the Opal transgene is operably linked to the MeCP2 promoter. In addition, the transgene in pA-Opa1_15 and pA-Opa1_25 is the human Opal ΔS1 isoform, and the transgene in pA-Opa1_17 and pA-Opa1_27 is the human Opal E5b isoform. The pA-Opa1_2x construct further comprises a 3xFLAG tag fused to the 3' end of the human Opal open reading frame.

[0253] After preparation of plasmid DNA for each AAV vector genome, 293 cells were transfected with 1 μg of plasmid DNA and incubated at 37 °C, 5% CO2 for 48 h before collecting protein lysates. Expression of Opa1 was examined using Western analysis (Figure 8A). In addition, the AAV capsid packaging ability of these AAV vector genomes was analyzed by measuring the virus production yield (Figure 8B, X-axis shows relative units corresponding to the number of DNase-resistant virus genomes in a given volume of virus production medium). The ability of AAV viral vectors to infect cells and express Opa1 protein was evaluated for the indicated vector genomes. Expression of Opa1 by each vector genome was detected by anti-Opa1 antibody (Figure 9A, upper panel and Figure 9B, lanes 5-13) or by anti-FLAG antibody (Figure 9A, lower panel and Figure 9B, lanes 2-4). In addition, Opa1 expression was also analyzed by protein staining in transfected Opa1(- / -) cell lines (Fig. 9C).

[0254] The results showed that pA-Opa1_3 resulted in approximately 4-fold lower expression of Opa1 compared to pA-Opa1_1 in 293 cells, and expression from pA-Opa1_5 was even lower than pA-Opa1_3. Neither pA-Opa1_3 nor pA-Opa1_5 had the ability to efficiently package into AAV capsids. On the other hand, pA-Opa1_11 resulted in better expression of Opa1 than pA-Opa1_1, and pA-Opa1_13 also showed better expression of Opa1 compared to pA-Opa1_5. In addition, both pA-Opa1_11 and pA-Opa1_13 had the ability to efficiently package into AAV capsids. Therefore, introducing telomeric repeats flanking both the 5' and 3' sides of the Opa1 expression cassette improves the expression of Opa1 as well as the packaging of the AAV vector genome.

[0255] Example 3: In vivo study of treatment of overt optic atrophy using an AAV viral vector encoding OPA1 To investigate Opa1 expression in vivo, animal studies were performed according to Figure 10A. Briefly, three treatment groups of Opa1- / + mice were injected at 1 month of age with AAV204 viral vectors containing pA-Opa1_21 (high or low dose), or pA-Opa1_27, by intravitreal injection into the vitreoretinal space using a 33-34G needle and syringe, according to Table 7 below. The left eye was penetrated temporally and the right eye nasal to the pupil. Optical coherence tomography (OCT) was performed after injection to assess postoperative damage, and either buprenorphine (0.01-0.05 mg / ml) or meloxicam (5 mg / ml) was administered to aid recovery from anesthesia. Either ofloxacin or neiomycin polymyxin B gramicidin was administered after the procedure to prevent infection. Mice were also administered atipamezole (0.1-1.0 mg / kg) to reverse adverse effects from the metabolism of xylazine. Mice were then allowed to recover on a heating pad until fully conscious. Mice were monitored daily for 2 months for morbidity and mortality after injection. Six mice were used for each of the three treatment groups and two control untreated groups (30 mice total). They were sacrificed at 3 months of age, mice were euthanized by CO2 exposure, and eyes were first removed with blunt scissors to dissect the eyelids, followed by separation of the optic nerve with curved forceps and 1 mm surgical scissors. Corneas were cut and the vitreous removed to create "eye cup" specimens for subsequent analysis. [Table 7]

[0256] When the expression of Opa1-Flag was assayed, the expression of Opa1-21 protein was observed in all treated animals with the correct size, the correct isoform in two bands (Figure 10B). The pattern of isoforms is identical to the endogenous isoforms in untreated and treated wild-type eyes (although the wild-type band was only visible upon overexposure). Opa1-27 protein was expressed in the eye samples, but not in the expected isoform size as in cell culture. Opa1-27 protein expression appeared to be limited to the uncleaved long / S1 isoform(s) without the short isoform(s) that may regulate mitochondrial fusion (as described in Wang et al., Mol Biol Cell. 2021 Jan 15;32(2):157-168). The amount of Brn3a and rhodopsin (Rho) in the samples was also analyzed using equivalent protein loading (Figure 10C). Brn3a, a marker for retinal ganglion cells, was more consistent across samples.

[0257] These results show that compared to wild-type (WT) animals, all three treatment groups showed a significant increase in the expression of heterologous Opa1, with the high dose group of construct pA-Opa1-21 achieving the most significant expression of Opa1. The Opa1 expression data correlated with the levels of expression of the FLAG tag from all three groups, with a clear absence of FLAG expression from untreated samples.

[0258] In addition to Western analysis, RT-PCR was used to analyze the expression levels of RNA transcripts. As shown in Figure 10D, significant expression of RNA transcripts of both high and low doses of pA-Opa1_21 (isoform 1), as well as pA-Opa1_27 (isoform 7) was observed in all treated HT animals, but as expected, no transcripts encoding human Opal or FLAG tags were detected in untreated animals. Interestingly, pA-Opa1_27 appeared to be expressed at a higher level at the mRNA level, but the protein level was much lower than that of the high dose of pA-Opa1_21.

[0259] On the other hand, construct pA-Opa1_25 did not result in detectable expression of Opa1 at either the mRNA or protein level (data not shown).

[0260] In another proposed study (Figure 11A and Figure 11B), Opa1- / + mice in each treatment group are administered AAV204 viral vectors containing the indicated vector genome. The two control groups are untreated Opa1- / + mice and untreated Opa1+ / + mice, respectively. For the treatment group delivered with the Opa1 transgene without the 3xFLAG tag, the expression level of Opa1 and treatment efficacy are evaluated by RT-qPCR and counting retinal ganglion cells (RGCs) (Figure 11A). Visual acuity (VA), optical coherence tomography (OCT), scotopic threshold response (STR), and photopic negative response (PhNR) are measured 10 months after injection. In addition, for the treatment group delivered with the Opa1 transgene containing the 3xFLAG tag, the expression of Opa1 is evaluated using RT-qPCR and IHC 4 months after injection (Figure 11B).

[0261] Example 4: In vitro characterization of AAV vector genomes containing human RS1 expression cassettes AAV vector genomes were constructed for the expression of RS1 protein according to FIG. 12A, and the overall design is shown in FIG. 12B. Each construct contains, from 5' to 3', a promoter, either a CBh-MVM (SEQ ID NO: 200) or MVM (SEQ ID NO: 222) intron, an RS1 open reading frame, a BGH polyA site (SEQ ID NO: 201 or 225), and a telomeric repeat (SEQ ID NO: 203), flanked by 5'ITR and 3'ITR. For the promoter, each construct ending with "6" contains a CBh promoter (SEQ ID NO: 154), each construct ending with "8" contains a RK promoter (SEQ ID NO: 196), each construct ending with "0" contains a Rho promoter (SEQ ID NO: 197), and each construct ending with "2" contains a PDE promoter (SEQ ID NO: 198). Some constructs also contain a beta Glo_s / MAR sequence (SEQ ID NO: 221) between the BGH polyA site and the telomeric repeat.

[0262] The AAV vector genomes used in this study include: - pA-RS1_8 (comprising SEQ ID NO: 240 and generated from a DNA template vector comprising SEQ ID NO: 204), - pA-RS1_16 (comprising SEQ ID NO: 241 and generated from a DNA template vector comprising SEQ ID NO: 205), - pA-RS1_18 (comprising SEQ ID NO: 242 and generated from a DNA template vector comprising SEQ ID NO: 206), - pA-RS1_20 (comprising SEQ ID NO: 243 and generated from a DNA template vector comprising SEQ ID NO: 207), - pA-RS1_22 (comprising SEQ ID NO: 244 and generated from a DNA template vector comprising SEQ ID NO: 208), - pA-RS1_26 (comprising SEQ ID NO: 245 and generated from a DNA template vector comprising SEQ ID NO: 209), - pA-RS1_28 (comprising SEQ ID NO: 246 and generated from a DNA template vector comprising SEQ ID NO: 210), - pA-RS1_30 (comprising SEQ ID NO: 247 and generated from a DNA template vector comprising SEQ ID NO: 211), - pA-RS1_32 (comprising SEQ ID NO: 248 and generated from a DNA template vector comprising SEQ ID NO: 212), - pA-RS1_36 (comprising SEQ ID NO: 249 and generated from a DNA template vector comprising SEQ ID NO: 213), - pA-RS1_38 (comprising SEQ ID NO: 250 and generated from a DNA template vector comprising SEQ ID NO: 214), - pA-RS1_46 (comprising SEQ ID NO: 251 and generated from a DNA template vector comprising SEQ ID NO: 215), - pA-RS1_48 (containing SEQ ID NO: 252 and generated from a DNA template vector containing SEQ ID NO: 216), and - pA-RS1_58 (containing SEQ ID NO: 224 and generated from a DNA template vector containing SEQ ID NO: 223).

[0263] The expression levels of RS1 protein of three AAV vector genomes (pA-RS1_8, pA-RS1_18, and pA-RS1_28) were assessed using Western analysis ( FIG. 13 ). Both pA-RS1_18 and pA-RS1_28 yielded a higher percentage of secreted RS1 protein compared to pA-RS1_8, although the total protein yield was comparable between these constructs.

[0264] The efficacy of AAV204 viral vectors containing the indicated RS1 expression vector genomes was also evaluated in Lec2 cell cultures. As shown in Figures 14A-14C, myc-tagged RS1 was expressed at similar levels as untagged RS1 in transduced Lec2 cells and was properly secreted. The CBh promoter also allowed higher expression of RS1 compared to the RK promoter. Secreted myc-RS1 is approximately 4 kD larger than native RS1 (Figure 14D).

[0265] Example 5: In vivo study of X-linked retinoschisis treatment using an AAV viral vector encoding RS1 In one study, wild-type mice were administered an AAV204 viral vector containing an RS1 expression cassette via intravitreal injection. Expression of RS1 protein in each group of animals was assessed 1 month after injection by RT-qPCR (mRNA copies / ng of total RNA) and Western analysis, and protein distribution by IHC (Figure 15A).

[0266] As shown in Figure 15B, expression of mouse RS1 (mRS1) protein was detected in each mouse tested, and the mean values ​​of mRS1 expression were not statistically different between the test groups. Mice administered AAV204.pA-RS1_26 exhibited robust expression of myc-tagged human RS1 (hRS1) at levels comparable to native mRS1 expression. Meanwhile, there was little detectable expression of hRS1 from the RK promoter in mice at this time point (with one exception).

[0267] In the next study, wild-type mice were administered AAV204 viral vectors containing either pA-RS1_36 or pA-RS1_38 vector genomes at a dose of 3e+9vg / eye via intravitreal injection into the eye cup. Thirty days after injection, samples were collected and total protein extracts were examined by Western analysis using an anti-myc antibody (Figure 16A). Myc-hRS1 protein expression was detected in all eyes administered with AAV204.pA-RS1_36 (containing the CBh promoter). In contrast, myc-RS1 protein was not detected in the treatment group administered with AAV204.pA-RS1_38 (containing the RK promoter) or in untreated controls. This result was confirmed using an anti-human RS1 (hRS1) antibody (Figure 16B). This anti-hRS1 antibody actually detected both human and mouse RS1 proteins, 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.

[0268] To assess ocular expression (FIG. 17A), RS1(y / -) mice are administered an AAV204 viral vector containing the pA-RS1_26 vector genome into the eyecup. The efficacy of the treatment group is compared to untreated control groups (either RS1(y / -) or wild-type RS1(y / +) mice) at 6 months using optical coherence tomography (OCT) and electroretinograms (ERG), and RS1 expression levels are assessed at 7 months using IHC.

[0269] In another proof-of-concept study (Figure 17B), three groups of mice are administered AAV204 viral vectors containing pA-RS1_28, pA-RS1_46, or pA-RS1_48 vector genomes, respectively, and compared to untreated control groups. Expression levels of RS1 are assessed at 3 months using Western analysis or RT-qPCR, or at 8 months using IHC. Treatment efficacy is assessed at 8 months using OCT and ERG.

[0270] In another proof-of-concept study, male RS1(- / Y) mice and wild-type male siblings were enrolled at 21±3 days of age and acclimated to the study for at least 3 days prior to dosing. Treatment groups were designed according to FIG. 18A. Briefly, - Groups 5 to 9 were used to demonstrate the in vivo infectivity of the intravitreally delivered vector and to analyze the distribution of virus-derived proteins in the retina at 2 months post-treatment (mpt). To facilitate protein detection, constructs with N-terminal myc-tag ends were used (pA-RS1_46, pA-RS1_48). Groups 1 to 4 were used to determine the longevity of expression and to analyze the effect of transgene expression on disease progression at 6 months after intravitreal injection. The RS1 transgene used in these vectors did not have a myc tag. - Groups 10-11 were used to evaluate the effect of subretinal injections as an alternative drug delivery procedure, 6 months after intravitreal injection, and directly compared with IVT injections performed in parallel. Subretinal injections for the treatment of retinoschisis may carry higher risks, since it is a more invasive technique that may lead to an increased rate of retinal detachment, given the structurally compromised state of the pathological retina. However, this has not been directly tested. The AAV viral constructs used in these two groups did not contain sequences encoding myc tags and S / MARs. An "eye cup" was prepared and the neural retina was then separated from the pigmented retinal pigment epithelium (RPE) to generate a dissociated retinal preparation.

[0271] Analysis of retinal samples 2 months after intravitreal delivery Using primers directed against mouse RS1 (mRs1), expression of the endogenous gene was measured across all groups at the 2 mpt endpoint. Expression of mRs1 was significantly reduced in all mutant animals compared to WT, regardless of treatment (Figure 18B). This is most likely a result of the degenerative state of the mutant retina, specifically the loss of photoreceptors where mRs1 is highly expressed. In the untreated mutant group (group 9), the mutant retina similarly reduced expression of mRs1, suggesting that treatment does not affect endogenous mRs1 expression.

[0272] The viral-derived RS1 transcripts were then detected and distinguished from endogenous transcripts using primers targeting the mycRS1 transgene (FIG. 18C). mycRS1 was not detectable in untreated retinas (groups 8, 9), but was readily detected in all treated retinas (groups 5, 6). The promoter used did not significantly affect the total amount of viral-derived transcripts. Expression of transgenic myc-RS1 was approximately 2 log units lower than that of endogenous Rs1 in mutant animals.

[0273] Both endogenous and virally derived proteins were simultaneously detected by Western analysis using RS1-specific monoclonal antibody 3R10 to measure global RS1 protein expression levels (Figures 18D and 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 that driven by the RK promoter (group 6) was 7% of WT. The difference in size of the RS1 protein between the treated and WT groups was due to the presence of the myc tag. In untreated mutant animals (group 9), RS1 was not detectable.

[0274] At 2 mpt, additional immunohistochemical staining of eyes was performed using 3R10 antibody to confirm transgene expression and evaluate protein localization. In addition, lectin PNA was used to label cone photoreceptor outer segments and Iba1 was used to label retinal microglia. Low magnification images of whole retinas show the extent of transduction, with higher magnification images of boxed regions to show PNA and Iba1 staining as well as distribution of RS1 across retinal layers. In some cases, multiple boxed regions were included to allow direct comparison of transduced and non-transduced areas of the same section.

[0275] In WT animals from group 8, Rs1 was prominently localized in the inner segments of photoreceptors and more diffuse in the synaptic layers of the retina. Cone photoreceptors were regularly spaced throughout the length of the retina (12.2±1.5 cones per 100 μm) and Iba1 staining was minimal (FIG. 19A). In contrast, untreated mutant retinas (group 9) had no detectable staining for RS1, cone density was reduced (7.8±0.9 cones per 100 μm), and Iba1 staining was more prominent, indicating a hyperinflammatory state that is common in retinal diseases (FIG. 19B). Small bright spots that appeared with RS1 staining in retinas that did not express RS1 were IgG-containing retinal blood vessels that became labeled with anti-mouse IgG secondary antibody in the absence of RS1 expression. Some examples of these are indicated by arrows in FIG. 19B.

[0276] RS1 labeling in mutant retinas treated with AAV204.CBh:RS1_46 (group 5) was generally restricted to the inner nuclear layer (INL) with small, associated patches of labeled photoreceptor inner segments. There were no examples of labeled photoreceptors without adjacent inner retinal labeling. Cone density and Iba1 labeling were similar to those of untreated retinas, regardless of RS1 expression. A representative retina from this treatment group is shown in Figure 19C. Arrows indicate small areas of transgene expression in photoreceptors. The right eye from one of the animals (#123) was exceptional in that transgene expression was observed along 58% of the retina, including the entire dorsal half of the retina, in both the inner retina and photoreceptors (Figure 19D). However, there was still no obvious change in cone density or Iba1 labeling in this eye sample.

[0277] In contrast, none of the animals treated with AAV204.RK:RS1_48 (Group 6) had observable immunolabeling of RS1 (FIG. 19E). Cone density and Iba1 labeling in these animals were indistinguishable from untreated mutants. This was unexpected, given that viral expression was detected by RT-PCR and Western analysis from other animals in this group.

[0278] Cone density was then quantified from IHC images of right eyes from WT and untreated mutant animals, as well as animal #123, which had high levels of transgene expression in photoreceptors. Cone density in mutant eyes was reduced to 63% of that in WT animals, as expected (Figure 19F). Cone density was also measured in clearly defined RS1-positive and RS1-negative areas of eye #123OD (group 5). The entire eye had lower cone density than the WT average, but there was a slight improvement in the RS1-positive areas of that eye when compared to the RS1-negative areas from the same eye.

[0279] Analysis of retinal samples 6 months after intravitreal delivery To show 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 restricted to a well-defined region in the periphery, covering approximately 10% of the entire retina. The extent of transduction was similar in the fellow eye. Cone density in the non-transduced portion of the retina was approximately 19% of that in similarly prepared WT controls. In the transduced portion of the retina, cone density improved to 46% of WT. The results showed that expression of the RS1 transgene was more restricted than expected and was concentrated near the injection site in the dorsal retina.

[0280] Western analysis was performed on retinas collected at 6 mpt (Figures 20A and 20B). Using the RS1-specific antibody 3R10, expression of RS1 was detected in mutant eyes treated with either AAV204.CBh:RS1_28 (group 2) or AAV204.RK:RS1_26 (group 4). Among all treated animals, the levels of RS1 expression ranged from 1.5% to 5.5% of that detected in WT retinas. Unexpectedly, expression from the CBh promoter-controlled transgene was not significantly higher than that from the RK promoter-controlled transgene. In untreated mutant animals, no expression was detected.

[0281] Immunohistochemistry (IHC) was then used to examine the distribution of RS1 expression in treated eyes at this time point and to determine the effect of RS1 expression on disease phenotype. Similar to the previous 2 mpt time point, expression of RS1 in WT animals was concentrated in the photoreceptor inner segments, with more diffuse expression in the inner retina (Figure 20C). In contrast, no expression was seen in untreated mutant animals (Figure 20D). Labeling of blood vessels with an anti-mouse secondary antibody was again seen in mutant retinas (Figure 20D, example indicated by arrows). At this time point, cone density was similarly reduced in mutants, and Iba1 labeling was more prominent.

[0282] In animals from Group 2 treated with AAV204.RK:RS1_28 that had detectable RS1 expression, it ranged from 14% to 59% of sections. This should not be interpreted as a percentage of the entire retina, as only sections with maximal expression were imaged. A representative example from this group is shown in Figure 20E. RS1 labeling was not primarily restricted to the inner retina, although most transduced areas contained significant photoreceptor expression as well. In contrast, none of the Group 4 eyes treated with AAV204.CBh:RS1_26 had detectable expression (Figure 20F).

[0283] Notably, the localization of recombinant RS1 in the retina of group 2 animals was identical to that of the endogenous protein, with a significant amount of staining in the photoreceptor inner segments and more diffuse staining throughout the inner retina, even in animals treated with the photoreceptor-specific promoter, indicating that secreted RS1 has the ability to diffuse radially through the retina to reach receptors in adjacent layers.

[0284] Cone density was quantified in all animals at 6 months (Figure 21). In mutants treated with AAV204.RK:RS1_28 (group 2), RS1-positive and RS1-negative regions were treated as separate data points. Cone density in untreated mutants was 31±6% of wild type, and the average density in group 2 was unchanged, regardless of RS1 expression. However, in all but one of the animals in group 2, cone density was higher in RS1-positive regions compared to adjacent RS1-negative regions of the same sections. This improvement ranged from 15% to 71%. Strikingly, animals in group 4 treated with AAV204CBh:RS1_26 had a mean cone density 63% higher than the untreated group, despite having no observable expression of RS1 (p=0.0217). Thus, in the case of RK promoter-controlled RS1, cone density improved after treatment with either viral construct, even though such improvement did not correlate with observable RS1 immunoreactivity.

[0285] Optical coherence tomography (OCT) was then used to analyze the retinal status. OCT is a non-invasive imaging technique that produces cross-sectional images of the retina and is routinely used to aid in the diagnosis of retinal diseases. This is a particularly useful technique for retinoschisis, as the large retinal cavities that are characteristic of the disease are easily observed. However, by 7 months of age, most separations are 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. Thus, interpretation of these images must include the assumption that transgene expression is widespread, or at least that it overlaps with the OCT field.

[0286] Analysis of the OCT data from this study revealed that, regardless of treatment, the separation phenotype was evident in only about one-third of the mutant eyes imaged, consistent with disease features that begin to resolve around this time point in mutant mice. No separation was observed in WT eyes.

[0287] Representative OCT images are shown in FIG. 22A, including two treated eyes from group 2 where maximal RS1 expression was observed by either IHC or Western analysis. Each image is of the dorsal retina in the mid-periphery. At 10 OD, this is the approximate area where strong RS1 expression was observed by IHC. Untreated mutant eyes from group 1 had a significantly thinner outer nuclear layer (ONL) than WT eyes from group 3 (26.9±1.9 μm vs. 52.8±2.6 μm). Eyes from both treatment groups had a thicker ONL than untreated animals, but they remained thinner than WT eyes (FIG. 22B).

[0288] The current study was completed using isolated retinas instead of whole eyes. Isolating the retina has the effect of enriching the target tissue within the sample and visualizing limited amounts of expression from the RK promoter. Furthermore, using isolated retinas eliminates extraretinal expression occurring with the ubiquitous CBh promoter and provides a clear indication of RS1 expression levels within the target tissue. 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 2 months after treatment was approximately 1.5 log units lower than endogenous RS1 expression.

[0289] Analysis of retinal samples 6 months after subretinal delivery In addition to intravitreal injection, selected AAV viral vectors were also delivered by subretinal injection (Groups 10 and 11 in FIG. 18A). Corresponding samples were analyzed at 6 mpt along with samples from control Groups 1 and 3 (untreated mutant and wild type, respectively).

[0290] The RS1-specific monoclonal antibody 3R10 was used to confirm transgene expression and to assess protein localization. In addition, the lectin PNA was used to label cone photoreceptor outer segments and Iba1 was used to label retinal microglia. Low magnification images of whole retinas are shown to show the extent of transduction, with higher magnification images of boxed regions to show PNA and Iba1 staining as well as the distribution of RS1 across retinal layers. In some cases, multiple boxed regions were included to allow direct comparison of transduced and non-transduced areas of the same section.

[0291] In WT animals from group 3, endogenous Rs1 is prominently localized to the inner segments of photoreceptors and more diffuse in the synaptic layers of the retina. Cone photoreceptors are regularly spaced throughout the length of the retina (10.2±1.6 cones per 100 μm), with minimal Iba1 staining (FIG. 23A).

[0292] In contrast, untreated mutant retinas (group 1) had no detectable RS1 staining, cone density was reduced (3.2±0.6 cones per 100 μm), and Iba1 staining was more prominent, indicating a hyperinflammatory state common in retinal disease (FIG. 23B). The small bright spots that appear with RS1 staining in retinas that do not express RS1 are IgG-containing retinal vessels that become labeled with anti-mouse IgG secondary antibodies in the absence of RS1 expression. Some examples of these are indicated by arrows in FIG. 23B.

[0293] Mutant animals treated with AAV204.CBh:RS1_16 (group 10) exhibited severe retinal degeneration associated with the injection site, with no retinal cells remaining (Figure 23C). In some cases, there were adjacent sections of retinas with expression of RS1, but cone density was not improved in these RS1+ regions. However, the average cone density in the RS1- regions of these retinas that did not suffer from injection-related degeneration was approximately twice that of untreated animals (6.2±1.4 vs. 3.2±0.6 cones per 100 μm, respectively), suggesting that expression of RS1 below the level of IHC detectability may extend beyond the bleb and still provide some therapeutic benefit. No changes were seen in Iba1 staining of treated animals. The right eye from one animal (#167) was unique in that it had a large lesion in the ventral retina, most likely mechanical damage sustained during the injection. Nonetheless, uniform RS1 staining was seen throughout the inner retina, as well as in photoreceptors adjacent to the lesion (Figure 23D), which may impair the physical barrier that normally inhibits diffusion of viral particles through the retina and allows more widespread expression.

[0294] In contrast, seven of eight eyes treated with AAV204.RK:RS1_18 (group 11, FIG. 23E) had highly significant RS1 expression in photoreceptors, with only one of these seven animals having the severe degeneration that was common in animals treated with CBh:RS1. Expression was generally localized to the dorsal retina at the bleb. Although the RK promoter is rod specific, 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 diffuse radially. In four of the seven eyes, the cone-depleted region was associated with the central bleb, while the remainder of the retina was intact. Outside this cone-depleted region was an RS1-positive margin where cone density was actually increased compared to the RS1-negative portion of the same section. In fact, this margin region had cone density comparable to wild-type eyes. Taken together, these results suggest that extreme overexpression of RS1 in mutant animals can be severely retinotoxic, and that moderate overexpression is detrimental to cones but may have therapeutic outcomes with appropriate dosing.

[0295] The chart in Figure 23F summarizes the cone density measurements. Areas of severe degeneration were omitted. For Group 11 eyes treated with RK:RS1, the cone-depleted RS1-positive zone was omitted. Thus, RS1+ in Group 11 is representative of the cone-enriched area surrounding the depleted zone. All treated eyes had higher cone density than untreated control eyes, even in areas lacking visible RS1 expression. Furthermore, the cone-enriched RS1-positive areas in Group 11 are indistinguishable from WT.

[0296] The thickness of the outer nuclear layer (ONL) was also measured from frozen sections stained with DAPI (Figures 24A-24C). For this analysis, areas of severe degeneration were omitted. In group 10 animals treated with CBh:RS1, the thickness of the ONL was equivalent to that of untreated mutant animals. In all group 11 animals treated with RK:RS1, the ONL was specifically thickened in areas of the retina with expression of RS1. In those eye regions that did not express RS1, the thickness of the ONL was approximately equivalent to that of untreated mutants.

[0297] Protein analysis: Western analysis was performed on frozen tissues harvested 6 months after treatment (Figures 25A and 25B). RS1-specific antibody 3E10 was used to detect expression of RS1 in mutant eyes treated with either AAV204.CBh:RS1_16 (group 10) or AAV204.RK:RS1_18 (group 11). Expression of recombinant RS1 in both groups was statistically equivalent to the expression of endogenous RS1 in WT animals (group 3).

[0298] Optical coherence tomography (OCT): As mentioned above, interpretation of OCT images assumes that transgene expression is widespread, or at least that it overlaps with the OCT field. This was reasonable in this study since the injections were subretinal, and examination of IHC images from animals treated with RK:RS1_18 (group 11) suggests that expression spread through the dorsal retina and was excluded from the ventral side.

[0299] OCT data are shown in Figures 26A-26F. The ONL in untreated mutant eyes was nearly one-third that of wild-type eyes (17.9 ± 1.9 vs. 48.7 ± 1.9 μm, Figures 26A-26B). All treated eyes were imaged immediately after injection to confirm the presence of a bleb (Figure 26C). All 13 eyes successfully injected with CBh:RS1_16 showed severe degeneration (Figure 26D). In some cases, some retina overlapped with the margin of the remaining bleb, but the thickness of the ONL there, as well as that of the ventral, untreated retina, was not significantly different from uninjected mutant animals.

[0300] In contrast, among the RK:RS1_18 treated eyes (Group 11), only 7 of 13 eyes with interpretable images had signs of severe injection-related degeneration, although 4 of those 7 still had measurable areas at the margins of the bleb (Figures 26E-26F).Of the 6 non-degenerated eyes and 4 degenerated eyes with measurable margins, all of them showed improvement in ONL thickness compared to the corresponding ventral retina as well as the untreated mutant eyes from Group 1 (Figures 26G-26H).

[0301] Electroretinogram (ERG): Electroretinogram is a non-invasive technique that measures retinal function in response to flashes of light and can be modified to elicit either rod-based or cone-based responses. The intensity of the flash stimulus can also be varied, with the amplitudes of the ERG components responding in parallel. Dark-adapted (DA) ERGs elicit rod-dominated responses and are composed of two main components: the a-wave, which is an early negative amplitude peak and reflects rod photoreceptor activity; and the b-wave, which is a large positive amplitude peak and reflects activity primarily of ON bipolar cells. Light-adapted (LA) ERGs use a dim background light to desensitize rod photoreceptors, allowing the cone response to be elicited. Cone responses can also be measured using a flicker paradigm, in which the frequency of stimulation is faster than the recovery time of the rod photoreceptors. A more detailed description 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.

[0302] Because the ERG protocol used in this study utilizes whole-field stimulation, recordings reflect the net response across the entire visual field, and therefore the ability to resolve the effects of rAAV-based treatments in the ERG depends on the extent of transgene expression across the retina.

[0303] In the early stages of retinoschisis disease, the synapses between the photoreceptors and the inner retinal neurons are destroyed. In the DA ERG, this is initially manifested as a decrease in b-wave amplitude. As the disease progresses, photoreceptors are lost, resulting in a decrease in a-wave amplitude. In severe cases, the ERG response may be undetectable.

[0304] A summary of the ERG results is presented in Table 8. Flicker ERGs showed significant improvement in cone responses in Group 11 animals treated with RK:RS1_18, although no improvement was seen in treated animals following a single flash stimulus. Representative Flicker ERG traces are shown in FIG. 27. [Table 8]

[0305] Summary of Results The results of subretinal injections indicate that RS1 expression from AAV204.CBh:RS1_16 at the doses used in this study may result in certain toxicities, such as possible retinal degeneration at the injection site. CBh is a strong and ubiquitous promoter. Overexpression of RS1 in cells that do not normally express RS1 may be causative of degeneration, suggesting that lower doses may be used to deliver RS1 transgenes operably linked to strong and ubiquitous promoters. In contrast, only about half of the eyes treated with AAV204.RK:RS1_18 had similar areas of degeneration. RK is a photoreceptor-specific promoter that is the primary site of RS1 expression in the WT retina. Western analysis showed that the two promoters yielded equal amounts of recombinant protein, but elimination of RS1 expression in non-photoreceptors was clearly beneficial. Even with RK-driven photoreceptor-specific expression, secreted RS1 was observed in the inner retina adjacent to RS1-positive photoreceptors, indicating that it had the ability to diffuse radially through the retina. This expression pattern is consistent with that of endogenous RS1 expression in WT animals. Notably, Western analysis showed that the expression of RS1 in the treated eyes was equivalent to that of endogenous expression in WT mice, even though only a portion of the retina was treated, indicating that (i) the RS1 protein load in the treated area was likely significantly above physiological levels, and (ii) the degeneration associated with the RK promoter may be resolved by using a lower dose.

[0306] Because mutations in RS1 cause severe structural damage to the retina, this study was initially designed for intravitreal injection, which is less invasive than subretinal, with the intention of minimizing the risk of causing additional damage to the already damaged retina. However, animals treated intravitreally with AAV204.CBh:RS1_26 and AAV204.RK:RS1_28 had minimal expression of recombinant protein, which prompted studies with subretinal treatment. With subretinal delivery, the vector is restricted to a much smaller space directly adjacent to the target layer, thereby increasing transduction rates and promoting transgene expression. Indeed, a comparison of these two studies demonstrates that subretinal injection results in better expression than intravitreal.

[0307] Efficacy in this study was assessed via three different measurements: cone density, ONL thickness, and ERG, all of which were reduced in untreated mutant mice. Expression of RS1 from the CBh promoter caused fatal retinal degeneration in all treated animals. The RK promoter, with an expression profile targeting the desired cell type, was much better tolerated, with corresponding treated eyes having more cones, thicker ONL, and improved ERG cone response compared to untreated animals. Degenerated areas were still observed in some RK-treated eyes, but these areas were still directly related to the injection site. Margins of the degenerated areas, where the effective dose was presumably 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 treated eyes where no immunoreactivity of RS1 was detected. This was true for both CBh and RK treated eyes, supporting the hypothesis that low doses, below the threshold of immunodetectability, can still be cone protective.

[0308] Given the promising results from subretinal delivery, juxtaretinal administration may be the preferred vector delivery method for treating XLRS. Similar to intravitreal injections, and unlike subretinal injections, juxtaretinal injections do not penetrate the retina and are therefore less likely to cause mechanical damage to the retina (which is usually fragile in XLRS cases) than subretinal injections. On the other hand, juxtaretinal injections are superior to intravitreal injections by localizing the injection bolus directly adjacent to the retina. Previous studies in NHP models using AAV204.CBh:GFP vectors have demonstrated that this technique allows for efficient transduction of all retinal layers. Although mouse eyes are too small to achieve juxtaretinal delivery, the results of subretinal injections in the current mouse study are nevertheless a model for the efficacy of juxtaretinal injections in nonhuman primates (NHPs) and humans.

[0309] Further Numbered Embodiments Further numbered embodiments of the present disclosure are provided as follows:

[0310] Embodiment 1. A method of treating retinoschisis in a subject in need thereof, comprising pararetinal or subretinal administration of an AAV viral vector to the subject.

[0311] 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.

[0312] Embodiment 3. The method of embodiment 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.

[0313] Embodiment 4. The method of embodiment 2, wherein the photoreceptor-specific promoter is a rhodopsin kinase (RK) promoter.

[0314] Embodiment 5. The method of embodiment 4, wherein the RK promoter comprises, consists essentially of, or consists of a nucleic acid having at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO:196.

[0315] Embodiment 6. The method of any one of embodiments 1 to 5, comprising pararetinal administration of an AAV viral vector to a subject.

[0316] Embodiment 7. The method of any one of embodiments 1 to 6, wherein the subject is a human and the AAV viral vector is administered at a dose of about 10 10 to about 10 12 viral genomes (vg).

[0317] Embodiment 8. The method of any one of embodiments 1 to 6, wherein the retinoschisis is X-linked retinoschisis.

[0318] Embodiment 9. The method of any one of embodiments 2 to 8, wherein the transgene is RS1.

[0319] Embodiment 10. The method of embodiment 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.

[0320] Embodiment 11. A method of treating an ocular disease or disorder in a subject in need of treatment thereof, comprising administering to the subject an AAV viral vector, wherein the AAV viral vector comprises an AAV vector genome, the AAV vector genome comprising, in a 5' to 3' orientation: (a) the first AAV inverted terminal repeat, (b) a promoter; (c) a heterologous nucleic acid encoding Opa1; (d) a polyadenylation signal, and (e) a second AAV inverted terminal repeat.

[0321] Embodiment 12. The method of embodiment 11, wherein the promoter is a CBh 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: 154.

[0322] Embodiment 13. The method of embodiment 11, wherein the promoter is a MeCP2 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: 156.

[0323] Embodiment 14. The method of any one of embodiments 11 to 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 the intron sequence is located between the promoter and the heterologous nucleic acid encoding Opa1.

[0324] Embodiment 15. The method of embodiment 14, wherein the intron sequence is located immediately downstream of the promoter without any additional nucleotides in between.

[0325] Embodiment 16. The method of any one of embodiments 11 to 15, wherein the heterologous nucleic acid encoding Opal comprises a 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 NOs: 175, 182, and 184.

[0326] Embodiment 17. The method of any one of embodiments 11 to 16, wherein the heterologous nucleic acid encodes an Opa1 protein comprising an amino acid 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 NOs: 180, 183, and 185.

[0327] Embodiment 18. The method of any one of embodiments 11 to 16, wherein the heterologous nucleic acid encodes an Opa1 protein comprising 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: 180.

[0328] Embodiment 19. The method of any one of embodiments 11 to 18, 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.

[0329] Embodiment 20. The method of any one of embodiments 11 to 19, wherein the AAV vector genome does not contain any telomeric repeat sequences.

[0330] Embodiment 21. The method of any one of embodiments 11 to 19, wherein the AAV vector genome comprises a first telomeric repeat sequence located between the polyadenylation signal and the second AAV inverted terminal repeat, and 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.

[0331] Embodiment 22. The method of any one of embodiments 11 to 21, wherein the AAV vector genome comprises a second telomeric repeat sequence located between the first AAV inverted terminal repeat and the promoter, and the second 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: 203.

[0332] Embodiment 23. The method of any one of embodiments 11 to 22, wherein the first AAV inverted terminal repeat comprises or consists of 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: 253, and / or the second AAV inverted terminal repeat comprises or consists of 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: 254.

[0333] Embodiment 24. The AAV vector genome, in a 5' to 3' orientation, (a) the first AAV inverted terminal repeat, (b) a 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: 154; (c) a heterologous nucleic acid encoding Opa1; (d) a polyadenylation signal 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: 201, and (e) a second AAV inverted terminal repeat.

[0334] Embodiment 25. The method of embodiment 24, 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, and the intron sequence is located between the promoter and the heterologous nucleic acid encoding Opa1.

[0335] Embodiment 26. The method of embodiment 24 or 25, wherein the AAV vector genome 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: 228.

[0336] Embodiment 27. The method of any one of embodiments 24 to 26, wherein the Opal protein comprises 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: 180.

[0337] Embodiment 28. The method of any one of embodiments 11 to 27, wherein the AAV vector genome 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 NOs: 230 to 239.

[0338] Embodiment 29. The method of any one of embodiments 11 to 28, wherein the eye disease or disorder is autosomal overt optic atrophy.

[0339] Embodiment 30. A method of treating an ocular disease or disorder in a subject in need of treatment thereof, comprising administering to the subject an AAV viral vector, wherein the AAV viral vector comprises an AAV vector genome, the AAV vector genome comprising, in a 5' to 3' orientation: (a) the first AAV inverted terminal repeat, (b) a promoter; (c) a heterologous nucleic acid encoding RS1; (d) a polyadenylation signal, and (e) a second AAV inverted terminal repeat.

[0340] Embodiment 31 The method of embodiment 30, wherein the promoter is a photoreceptor-specific promoter.

[0341] Embodiment 32. The method of 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.

[0342] Embodiment 33. The method of embodiment 30, wherein the promoter is a CBh 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: 154.

[0343] Embodiment 34. The method of embodiment 30, wherein the promoter is a 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.

[0344] Embodiment 35. The method of embodiment 30, wherein 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.

[0345] Embodiment 36. The method of embodiment 30, wherein the promoter is a PDE 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: 198.

[0346] Embodiment 37. The method of any one of embodiments 30 to 36, wherein the AAV vector genome comprises an IRBP enhancer sequence upstream of the promoter, and the IRBP enhancer sequence has at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 199.

[0347] Embodiment 38 The method of embodiment 37, wherein the IRBP enhancer sequence is located immediately upstream of the promoter without any additional nucleotides in between.

[0348] Embodiment 39. The method of any one of embodiments 30 to 38, wherein the AAV vector genome comprises a CVA-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, and the CVA-MVM intron sequence is located between the promoter and the heterologous nucleic acid encoding RS1.

[0349] Embodiment 40. The method of any one of embodiments 30 to 38, 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 222, and the intron is located between the promoter and the heterologous nucleic acid encoding RS1.

[0350] Embodiment 41. The method of any one of embodiments 30 to 40, wherein the heterologous nucleic acid encoding RS1 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: 117.

[0351] Embodiment 42. The method of any one of embodiments 30 to 41, wherein the heterologous nucleic acid encodes an RS1 protein comprising 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.

[0352] Embodiment 43. The method of any one of embodiments 30 to 42, 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.

[0353] Embodiment 44. The method of any one of embodiments 30 to 43, wherein the AAV vector genome does not contain any telomeric repeat sequences.

[0354] Embodiment 45. The method of any one of embodiments 30 to 43, wherein the AAV vector genome comprises a first telomeric repeat sequence located between the polyadenylation signal and the second AAV inverted terminal repeat, and 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: 203.

[0355] Embodiment 46. The method of any one of embodiments 30 to 45, wherein the AAV vector genome comprises a human beta-globin scaffold / matrix attachment region (βGlo_s / MAR) sequence located between the polyadenylation signal and the second AAV inverted terminal repeat or between the polyadenylation signal and the first telomeric repeat sequence, and βGlo_s / MAR has at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 221.

[0356] Embodiment 47. The method of any one of embodiments 30 to 46, wherein the first AAV inverted terminal repeat comprises or consists of 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: 255, and / or the second AAV inverted terminal repeat comprises or consists of 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: 256.

[0357] Embodiment 48. The AAV vector genome comprises, in a 5' to 3' orientation: (a) the first AAV inverted terminal repeat, (b) an 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) 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; (d) a heterologous nucleic acid encoding RS1; (e) a polyadenylation signal 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: 225, and (f) a second AAV inverted terminal repeat.

[0358] Embodiment 49. The method of any one of embodiments 30 to 48, 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: 222, and the intron sequence is located between the promoter and the heterologous nucleic acid encoding RS1.

[0359] Embodiment 50. The method of embodiment 49, wherein the AAV vector genome comprises a CBA sequence of SEQ ID NO: 229, or a sequence having up to 5, up to 4, up to 3, up to 2, or up to 1 mutation therein, and the CBA sequence is located immediately upstream of the intron sequence without any additional nucleotides in between.

[0360] Embodiment 51. The method of embodiment 49 or 50, wherein 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, and the CVA-MVM intron sequence is located between the promoter and the heterologous nucleic acid encoding RS1.

[0361] Embodiment 52. The method of any one of embodiments 48 to 51, wherein the AAV vector genome 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: 224.

[0362] Embodiment 53. The method of any one of embodiments 30 to 52, wherein the AAV vector genome 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 NOs: 224 and 240 to 252.

[0363] Embodiment 54. The method of any one of embodiments 30 to 53, wherein the ocular disease or disorder is X-linked retinoschisis.

[0364] Embodiment 55. The method of any one of embodiments 1 to 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.

[0365] Embodiment 56. The method of embodiment 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.

[0366] Embodiment 57. The method of embodiment 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.

[0367] Embodiment 58. The method of any one of embodiments 11 to 57, wherein the administration is pararetinal administration.

[0368] Embodiment 59. The method of any one of embodiments 1-10 and 58, wherein the pararetinal administration comprises injecting at a distance of 0-13 millimeters (mm), 0-10 mm, 0-5 mm, or 0-3 mm from the surface of the retina in the posterior vitreous cavity of the eye.

[0369] Embodiment 60. The method of any one of embodiments 1 to 59, wherein the AAV viral vector is administered at a dose of about 10 10 to about 10 12 viral genomes (vg).

[0370] Embodiment 61. The method of any one of embodiments 1 to 60, wherein the subject is a human.

[0371] In the 5' to 3' orientation of embodiment 62, (a) the first AAV inverted terminal repeat, (b) a promoter; (c) a heterologous nucleic acid encoding Opa1; (d) a polyadenylation signal, and (e) a second AAV inverted terminal repeat,

[0372] Embodiment 63. The nucleic acid of embodiment 62, wherein the promoter is a CBh 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: 154.

[0373] Embodiment 64. The nucleic acid of embodiment 62, wherein the promoter is a MeCP2 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: 156.

[0374] Embodiment 65. The nucleic acid described in any one of embodiments 62 to 64, 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 the intron sequence is located between the promoter and the heterologous nucleic acid encoding Opa1.

[0375] Embodiment 66. The nucleic acid of any one of embodiments 62 to 65, wherein the intron sequence is located immediately downstream of the promoter without any additional nucleotides in between.

[0376] Embodiment 67. A nucleic acid described in any one of embodiments 62 to 66, wherein the heterologous nucleic acid encoding Opal comprises a 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 NOs: 175, 182, and 184.

[0377] Embodiment 68. A nucleic acid described in any one of embodiments 62 to 67, wherein the heterologous nucleic acid encodes an Opa1 protein comprising an amino acid 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 NOs: 180, 183, and 185.

[0378] Embodiment 69. The nucleic acid of any one of embodiments 62 to 67, wherein the heterologous nucleic acid encodes an Opa1 protein comprising 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: 180.

[0379] Embodiment 70. The nucleic acid according to any one of embodiments 62 to 69, 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.

[0380] Embodiment 71. The nucleic acid of any one of embodiments 62 to 70, wherein the AAV vector genome does not contain any telomeric repeat sequences.

[0381] Embodiment 72. The nucleic acid of any one of embodiments 62 to 70, wherein the AAV vector genome comprises a first telomeric repeat sequence located between the polyadenylation signal and the second AAV inverted terminal repeat, and 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.

[0382] Embodiment 73. The nucleic acid of any one of embodiments 62 to 72, wherein the AAV vector genome comprises a second telomeric repeat sequence located between the first AAV inverted terminal repeat and the promoter, and the second 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: 203.

[0383] Embodiment 74. The nucleic acid according to any one of embodiments 62 to 73, wherein the first AAV inverted terminal repeat comprises or consists of 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: 253, and / or the second AAV inverted terminal repeat comprises or consists of 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: 254.

[0384] Embodiment 75. The AAV vector genome comprises, in a 5' to 3' orientation: (a) the first AAV inverted terminal repeat, (b) a 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: 154; (c) a heterologous nucleic acid encoding Opa1; (d) a polyadenylation signal 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: 201, and (e) a second AAV inverted terminal repeat.

[0385] Embodiment 76. The nucleic acid of 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, wherein the intron sequence is located between the promoter and the heterologous nucleic acid encoding Opa1.

[0386] Embodiment 77. The nucleic acid of embodiment 75 or 76, wherein the Opal protein comprises 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: 180.

[0387] Embodiment 78. A nucleic acid described in any one of embodiments 75 to 77, 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: 228.

[0388] Embodiment 79. A nucleic acid described in any one of embodiments 62 to 78, comprising 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 NOs: 230 to 239.

[0389] In an embodiment 80.5' to 3' orientation, (a) the first AAV inverted terminal repeat, (b) a promoter; (c) a heterologous nucleic acid encoding RS1; (d) a polyadenylation signal, and (e) a second AAV inverted terminal repeat,

[0390] Embodiment 81. The nucleic acid of embodiment 80, wherein the promoter is a photoreceptor-specific promoter.

[0391] Embodiment 82. The nucleic acid of 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.

[0392] Embodiment 83. The nucleic acid of embodiment 80, wherein the promoter is a CBh 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: 154.

[0393] Embodiment 84. The nucleic acid of embodiment 80, wherein the promoter is a 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.

[0394] Embodiment 85. The nucleic acid of embodiment 80, wherein 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.

[0395] Embodiment 86. The nucleic acid of embodiment 80, wherein the promoter is a PDE 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: 198.

[0396] Embodiment 87. A nucleic acid described in any one of embodiments 80 to 86, comprising an IRBP enhancer sequence upstream of the promoter, wherein the IRBP enhancer sequence has at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 199.

[0397] 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.

[0398] Embodiment 89. The nucleic acid described in any one of embodiments 80 to 88, wherein the AAV vector genome comprises a CVA-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, and the CVA-MVM intron sequence is located between the promoter and the heterologous nucleic acid encoding RS1.

[0399] Embodiment 90. The nucleic acid of any one of embodiments 80 to 88, 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 222, and the intron is located between the promoter and the heterologous nucleic acid encoding RS1.

[0400] Embodiment 91. The nucleic acid described in any one of embodiments 80 to 88, wherein the heterologous nucleic acid encoding RS1 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: 117.

[0401] Embodiment 92. The nucleic acid of any one of embodiments 80 to 91, wherein the heterologous nucleic acid encodes an RS1 protein comprising 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.

[0402] Embodiment 93. The nucleic acid according to any one of embodiments 80 to 92, 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.

[0403] Embodiment 94. The nucleic acid of any one of embodiments 80 to 93, wherein the AAV vector genome does not contain any telomeric repeat sequences.

[0404] Embodiment 95. The nucleic acid of any one of embodiments 80 to 93, wherein the AAV vector genome comprises a first telomeric repeat sequence located between the polyadenylation signal and the second AAV inverted terminal repeat, and 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: 203.

[0405] Embodiment 96. The nucleic acid of any one of embodiments 80 to 95, wherein the AAV vector genome comprises a human beta-globin scaffold / matrix attachment region (βGlo_s / MAR) sequence located between the polyadenylation signal and the second AAV inverted terminal repeat or between the polyadenylation signal and the first telomeric repeat sequence, and the βGlo_s / MAR sequence has at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 221.

[0406] Embodiment 97. The nucleic acid according to any one of embodiments 80 to 96, wherein the first AAV inverted terminal repeat comprises or consists of 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: 255, and / or the second AAV inverted terminal repeat comprises or consists of 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: 256.

[0407] Embodiment 98. The AAV vector genome comprises, in a 5' to 3' orientation: (a) the first AAV inverted terminal repeat, (b) an 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) 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; (d) a heterologous nucleic acid encoding RS1; (e) a polyadenylation signal 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: 225, and (f) a second AAV inverted terminal repeat.

[0408] Embodiment 99. A nucleic acid described in any one of embodiments 80 to 98, 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: 222, wherein the intron sequence is located between the promoter and the heterologous nucleic acid encoding RS1.

[0409] Embodiment 100. The nucleic acid of embodiment 99, comprising a CBA sequence of SEQ ID NO: 229 or a sequence having up to 5, up to 4, up to 3, up to 2, or up to 1 mutation therein, wherein the CBA sequence is located immediately upstream of the intron sequence without any additional nucleotides in between.

[0410] Embodiment 101. The nucleic acid described in embodiment 99 or 100, comprising 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, wherein the CBA-MVM intron sequence is located between the promoter and the heterologous nucleic acid encoding RS1.

[0411] Embodiment 102. A nucleic acid according to any one of embodiments 98 to 101, 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: 224.

[0412] Embodiment 103. A nucleic acid according to any one of embodiments 80 to 102, comprising 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 NOs: 224 and 240 to 252.

[0413] In the 5' to 3' orientation of embodiment 104, (a) a promoter; (b) a heterologous nucleic acid encoding a transgene, and (c) a polyadenylation signal, A nucleic acid wherein the promoter is a CBh 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: 154, and 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.

[0414] Embodiment 105. The nucleic acid of embodiment 104, 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, 222, 226, or 227, wherein the intron is located between the promoter and the heterologous nucleic acid encoding the transgene.

[0415] Embodiment 106. The nucleic acid according to any one of embodiments 104 to 105, comprising a first ITR located 5' of the promoter and a second ITR located 3' of the polyadenylation signal.

[0416] Embodiment 107. The nucleic acid of any one of embodiments 104 to 106, which does not contain any telomeric repeat sequences.

[0417] Embodiment 108. The nucleic acid according to any one of embodiments 104 to 106, comprising a first telomeric repeat sequence located between the polyadenylation signal and the second AAV ITR, 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.

[0418] Embodiment 109. The nucleic acid of any one of embodiments 104 to 108, comprising a second telomeric repeat sequence located between the first AAV inverted terminal repeat and the promoter, wherein the second 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: 203.

[0419] Embodiment 110. A vector comprising a nucleic acid according to any one of embodiments 62 to 109.

[0420] Embodiment 111. An AAV vector genome comprising a nucleic acid according to any one of embodiments 62 to 109.

[0421] Embodiment 112. An AAV viral vector comprising the AAV vector genome described in embodiment 111.

[0422] Embodiment 113. An AAV viral vector described in embodiment 112, comprising 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 to 3, 30 to 34, 49, 84, and 164.

[0423] Embodiment 114. A method for expressing a transgene in a retinal cell, comprising delivering to the retinal cell a nucleic acid described in any one of embodiments 62 to 109, or transducing the retinal cell with an AAV viral vector described in any one of embodiments 112 to 113.

[0424] Embodiment 115. The method of embodiment 114, wherein the retinal cells are retinal ganglion cells.

[0425] Embodiment 116. A method for treating a disease or disorder, comprising administering to a subject an AAV viral vector described in any one of embodiments 112 to 113.

[0426] Embodiment 117. The method of embodiment 116, wherein the AAV viral vector is administered to the subject intraocularly, periocularly, intravitreally, pararetinal, or subretinal.

[0427] Embodiment 118. The method of embodiment 116 or 117, wherein the disease or disorder is macular degeneration, retinitis pigmentosa, autosomal dominant optic atrophy, retinoschisis, Stargardt disease, Vietti crystalline dystrophy or BEST vitelliform macular dystrophy.

[0428] Embodiment 119. The method of embodiment 116 or 117, wherein the disease or disorder is X-linked retinoschisis (XLRS).

Claims

1. A method for treating retinoschisis in a subject in need thereof, comprising pararetinal or subretinal administration of an AAV viral vector to the subject.

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

196.

6. The method of any one of claims 1 to 5, comprising pararetinal administration of the AAV viral vector to the subject.

7. The subject is a human, and the AAV viral vector is １０ ~about 10 １２ The method of any one of claims 1 to 6, wherein the vaccine is administered at a dose of 0.1 mg / kg of viral genome (vg).

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

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

10. 10. The method of claim 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.

11. 1. A method of treating an ocular disease or disorder in a subject in need of such treatment, comprising administering to the subject an AAV viral vector, the AAV viral vector comprising an AAV vector genome, the AAV vector genome comprising, in a 5' to 3' orientation: (a) a first AAV inverted terminal repeat, (b) a promoter, (c) a heterologous nucleic acid encoding Opa1; (d) a polyadenylation signal, and (e) a second AAV inverted terminal repeat.

12. 12. The method of claim 11, wherein the promoter is a CBh 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:

154.

13. 12. The method of claim 11, wherein the promoter is a MeCP2 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:

156.

14. 14. The method of any one of claims 11 to 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 the intron sequence is located between the promoter and the heterologous nucleic acid encoding Opa1.

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

16. The method of any one of claims 11 to 15, wherein the heterologous nucleic acid encoding Opa1 comprises a 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 NOs: 175, 182, and 184.

17. The method of any one of claims 11 to 16, wherein the heterologous nucleic acid encodes an Opa1 protein comprising an amino acid 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 NOs: 180, 183, and 185.

18. The method of any one of claims 11 to 16, wherein the heterologous nucleic acid encodes an Opa1 protein comprising 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:

180.

19. 19. The method of any one of claims 11 to 18, 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.

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

21. 20. The method of any one of claims 11 to 19, wherein the AAV vector genome comprises a first telomeric repeat sequence located between the polyadenylation signal and the second AAV inverted terminal repeat, 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.

22. 22. The method of any one of claims 11 to 21, wherein the AAV vector genome comprises a second telomeric repeat sequence located between the first AAV inverted terminal repeat and the promoter, and the second 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:

203.

23. 23. The method of any one of claims 11 to 22, wherein the first AAV inverted terminal repeat comprises or consists of 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:253 and / or the second AAV inverted terminal repeat comprises or consists of 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:

254.

24. the AAV vector genome, in a 5' to 3' orientation, (a) the first AAV inverted terminal repeat, (b) the 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: 154; (c) the heterologous nucleic acid encoding Opa1; (d) the polyadenylation signal 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: 201; and (e) the second AAV inverted terminal repeat.

25. 25. The method of claim 24, 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, and the intron sequence is located between the promoter and the heterologous nucleic acid encoding Opa1.

26. 26. The method of claim 24 or 25, wherein the AAV vector genome 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:

228.

27. The method of any one of claims 24 to 26, wherein the Opa1 protein comprises 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:

180.

28. 28. The method of any one of claims 11-27, wherein the AAV vector genome 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 NOs:230-239.

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

30. 1. A method of treating an ocular disease or disorder in a subject in need of such treatment, comprising administering to the subject an AAV viral vector, the AAV viral vector comprising an AAV vector genome, the AAV vector genome comprising, in a 5' to 3' orientation: (a) a first AAV inverted terminal repeat, (b) a promoter, (c) a heterologous nucleic acid encoding RS1; (d) a polyadenylation signal, and (e) a second AAV inverted terminal repeat.

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

32. 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. 31. The method of claim 30, wherein the promoter is a CBh 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:

154.

34. 31. The method of claim 30, wherein 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.

35. 31. The method of claim 30, wherein 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.

36. 31. The method of claim 30, wherein the promoter is a PDE 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:

198.

37. 37. The method of any one of claims 30-36, wherein the AAV vector genome comprises an IRBP enhancer sequence upstream of the promoter, and the IRBP enhancer sequence has at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO:

199.

38. 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. 39. The method of any one of claims 30-38, wherein the AAV vector genome comprises a CVA-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, and the CVA-MVM intron sequence is located between the promoter and the heterologous nucleic acid encoding RS1.

40. 39. The method of any one of claims 30-38, 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 222, and the intron is located between the promoter and the heterologous nucleic acid encoding RS1.

41. 41. The method of any one of claims 30 to 40, wherein the heterologous nucleic acid encoding RS1 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:

117.

42. 42. The method of any one of claims 30-41, wherein the heterologous nucleic acid encodes an RS1 protein comprising 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.

43. 43. The method of any one of claims 30 to 42, 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.

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

45. 44. 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, 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:

203.

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

221.

47. 47. The method of any one of claims 30 to 46, wherein the first AAV inverted terminal repeat comprises or consists of 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:255 and / or the second AAV inverted terminal repeat comprises or consists of 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:

256.

48. the AAV vector genome, in a 5' to 3' orientation, (a) the first AAV inverted terminal repeat, (b) the 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) the 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; (d) the heterologous nucleic acid encoding RS1; (e) the polyadenylation signal 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: 225; and (f) the second AAV inverted terminal repeat.

49. 49. The method of any one of claims 30-48, 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:222, and the intron sequence is located between the promoter and the heterologous nucleic acid encoding RS1.

50. 50. The method of claim 49, wherein the AAV vector genome comprises a CBA sequence of SEQ ID NO: 229, or a sequence having up to 5, up to 4, up to 3, up to 2, or up to 1 mutation therein, and wherein the CBA sequence is located immediately upstream of the intron sequence without any additional nucleotides in between.

51. 51. The method of claim 49 or 50, wherein the AAV vector genome comprises a CVA-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, and the CVA-MVM intron sequence is located between the promoter and the heterologous nucleic acid encoding RS1.

52. 52. The method of any one of claims 48 to 51, wherein the AAV vector genome 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:

224.

53. 53. The method of any one of claims 30-52, wherein the AAV vector genome 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 NOs: 224 and 240-252.

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

55. 55. The method of any one of claims 1 to 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. 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. 56. The method of claim 55, wherein the AAV viral vector comprises an AAV capsid protein that comprises, or consists 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 to 57, wherein the administration is pararetinal administration.

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

60. The AAV viral vector is about 10 １０ ~about 10 １２ 60. The method of any one of claims 1 to 59, wherein the vaccine is administered at a dose of 0.1 mg / kg of viral genome (vg).

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

62. In a 5' to 3' orientation, (a) a first AAV inverted terminal repeat, (b) a promoter, (c) a heterologous nucleic acid encoding Opa1; (d) a polyadenylation signal, and (e) a second AAV inverted terminal repeat.

63. 63. The nucleic acid of claim 62, wherein the promoter is a CBh 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:

154.

64. 63. The nucleic acid of claim 62, wherein the promoter is a MeCP2 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:

156.

65. 65. The nucleic acid of any one of claims 62-64, 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 the intron sequence is located between the promoter and the heterologous nucleic acid encoding Opa1.

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

67. 67. The nucleic acid of any one of claims 62 to 66, wherein the heterologous nucleic acid encoding Opa1 comprises a 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 NOs: 175, 182, and 184.

68. The nucleic acid of any one of claims 62 to 67, wherein the heterologous nucleic acid encodes an Opa1 protein comprising an amino acid 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 NOs: 180, 183, and 185.

69. The nucleic acid of any one of claims 62 to 67, wherein the heterologous nucleic acid encodes an Opa1 protein comprising 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:

180.

70. 70. The nucleic acid of any one of claims 62 to 69, 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.

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

72. 71. The nucleic acid of 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, 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.

73. 73. The nucleic acid of 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 and the promoter, and the second 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:

203.

74. 74. The nucleic acid of any one of claims 62 to 73, wherein the first AAV inverted terminal repeat comprises or consists of 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:253 and / or the second AAV inverted terminal repeat comprises or consists of 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:

254.

75. the AAV vector genome, in a 5' to 3' orientation, (a) the first AAV inverted terminal repeat, (b) the 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: 154; (c) the heterologous nucleic acid encoding Opa1; (d) the polyadenylation signal 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: 201; and (e) the second AAV inverted terminal repeat.

76. 76. The nucleic acid of claim 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, wherein the intron sequence is located between the promoter and the heterologous nucleic acid encoding Opa1.

77. The nucleic acid of claim 75 or 76, wherein the Opa1 protein comprises 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:

180.

78. 78. The nucleic acid of any one of claims 75 to 77, 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:

228.

79. 79. The nucleic acid of any one of claims 62 to 78, comprising 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 NOs: 230 to 239.

80. In a 5' to 3' orientation, (a) a first AAV inverted terminal repeat, (b) a promoter, (c) a heterologous nucleic acid encoding RS1; (d) a polyadenylation signal, and (e) a second AAV inverted terminal repeat.

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

82. 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. 81. The nucleic acid of claim 80, wherein the promoter is a CBh 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:

154.

84. 81. The nucleic acid of claim 80, wherein 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.

85. 81. The nucleic acid of claim 80, wherein 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.

86. 81. The nucleic acid of claim 80, wherein the promoter is a PDE 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:

198.

87. 87. The nucleic acid of any one of claims 80 to 86, comprising an IRBP enhancer sequence upstream of the promoter, the 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.

88. 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. 89. The nucleic acid of any one of claims 80-88, wherein the AAV vector genome comprises a CVA-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, and the CVA-MVM intron sequence is located between the promoter and the heterologous nucleic acid encoding RS1.

90. 89. The nucleic acid of any one of claims 80-88, 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 222, and the intron is located between the promoter and the heterologous nucleic acid encoding RS1.

91. 89. The nucleic acid of any one of claims 80 to 88, wherein the heterologous nucleic acid encoding RS1 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:

117.

92. 92. The nucleic acid of any one of claims 80 to 91, wherein the heterologous nucleic acid encodes an RS1 protein comprising 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.

93. 93. The nucleic acid of any one of claims 80 to 92, 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.

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

95. 94. 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, and 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:

203.

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

221.

97. 97. The nucleic acid of any one of claims 80 to 96, wherein the first AAV inverted terminal repeat comprises or consists of 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:255 and / or the second AAV inverted terminal repeat comprises or consists of 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:

256.

98. the AAV vector genome, in a 5' to 3' orientation, (a) the first AAV inverted terminal repeat, (b) the 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) the 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; (d) the heterologous nucleic acid encoding RS1; (e) the polyadenylation signal 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: 225; and (f) the second AAV inverted terminal repeat.

99. 99. The nucleic acid of any one of claims 80-98, 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:222, wherein the intron sequence is located between the promoter and the heterologous nucleic acid encoding RS1.

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

101. 101. The nucleic acid of claim 99 or 100, comprising a CVA-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, wherein the CVA-MVM intron sequence is located between the promoter and the heterologous nucleic acid encoding RS1.

102. 102. The nucleic acid of any one of claims 98 to 101, 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:

224.

103. 103. The nucleic acid of any one of claims 80 to 102, comprising 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 NOs: 224 and 240 to 252.

104. In a 5' to 3' orientation, (a) a promoter, (b) a heterologous nucleic acid encoding a transgene, and (c) a polyadenylation signal, A nucleic acid wherein the promoter is a CBh 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: 154, and 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.

105. 105. The nucleic acid of claim 104, 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, 222, 226, or 227, wherein the intron is located between the promoter and the heterologous nucleic acid encoding the transgene.

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

107. 107. The nucleic acid of any one of claims 104 to 106, which does not contain any telomeric repeat sequences.

108. 107. The nucleic acid of any one of claims 104 to 106, comprising a first telomeric repeat sequence located between the polyadenylation signal and the second AAV ITR, 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.

109. 109. The nucleic acid of any one of claims 104 to 108, comprising a second telomeric repeat sequence located between the first AAV inverted terminal repeat and the promoter, wherein the second 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:

203.

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

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

112. An AAV viral vector comprising the AAV vector genome described in claim 111.

113. 113. The AAV viral vector of claim 112, comprising 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, the method comprising delivering to said retinal cell a nucleic acid according to any one of claims 62 to 109, or transducing said retinal cell with an AAV viral vector according to any one of claims 112 to 113.

115. 115. The method of claim 114, wherein the retinal cell is a retinal ganglion cell.

116. A method for treating a disease or disorder, comprising administering to a subject an AAV viral vector described in any one of claims 112 to 113.

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

118. The method of claim 116 or 117, wherein the disease or disorder is macular degeneration, retinitis pigmentosa, autosomal dominant optic atrophy, retinoschisis, Stargardt disease, Vietti crystalline dystrophy, or BEST vitelliform macular dystrophy.

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

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