Methods of treating ocular neovascular diseases using AAV2 variants encoding aflibercept

Abstract

To provide therapeutic methods for ocular neovascular diseases such as wAMD, DR, or DME that are effective, reduce the risk of adverse effects, and are amenable to high long-term patient compliance.SOLUTION: A method of treating an ocular neovascular disease in an individual is provided, the method comprising administering a unit dose of recombinant adeno-associated virus (rAAV) particles to an eye of the individual, wherein the rAAV particles comprise: a) a nucleic acid encoding a polypeptide comprising an amino acid sequence having at least about 95% identity to a specific amino acid sequence and flanked by AAV2 inverted terminal repeats (ITRs), and b) an AAV2 capsid protein comprising an amino acid sequence LGETTRP inserted between positions 587 and 588 of the capsid protein, wherein the amino acid residue numbering corresponds to an AAV2 VP1 capsid protein.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application is a continuation of U.S. Provisional Patent Application No. 62 / 899,070, filed September 11, 2019; U.S. Provisional Patent Application No. 62 / 913,648, filed October 10, 2019; International Application No. PCT / US2019 / 062066, filed November 18, 2019; and U.S. Provisional Patent Application No. PCT / US2019 / 062066, filed January 10, 2020, the disclosures of each of which are hereby incorporated by reference in their entirety. This application claims the benefit of U.S. Provisional Patent Application No. 62 / 959,784, filed February 7, 2020, U.S. Provisional Patent Application No. 62 / 971,835, filed May 1, 2020, U.S. Provisional Patent Application No. 63 / 019,190, filed May 27, 2020, U.S. Provisional Patent Application No. 63 / 030,819, filed May 27, 2020, and U.S. Provisional Patent Application No. 63 / 063,203, filed August 7, 2020.

[0002] Field The present disclosure relates to methods of treating intraocular neovascular diseases and disorders in an individual, the methods comprising administering to the eye of the individual a single unit dose of recombinant adeno-associated virus (rAAV) particles encoding an anti-VEGF agent (e.g., aflibercept).

[0003] Submitting a sequence listing as an ASCII text file The contents of the following submission in an ASCII text file are incorporated herein by reference in their entirety: Sequence Listing in Computer Readable Form (CRF) (Filename: 627002001241SEQLIST.TXT, Recorded: September 9, 2020, Size: 41KB).

[0004] background Age-related macular degeneration (AMD) is a degenerative eye disease that affects the macula, a small, light-sensitive area in the center of the retina responsible for reading and fine-grained vision. Conditions affecting the macula reduce central vision, while peripheral vision remains unaffected. In severe cases, the disease can result in central blindness. AMD is a significant cause of vision loss in people aged 65 and older in the US population, with the estimated prevalence of any AMD in people over the age of 40 being approximately 6.5% (Klein et al., (2011) Arch Ophthalmol, 129(1):75-80). Neovascular or wet or exudative AMD (nAMD, wAMD, or nwAMD) is an advanced form of AMD. A typical feature of wAMD is choroidal neovascularization (CNV), which is the infiltration of abnormal blood vessels from the underlying choroid layer into the retina, resulting in retinal cell damage and central blindness. This abnormal angiogenesis process is modulated by growth factors, particularly vascular endothelial growth factor (VEGF). The standard treatment for wAMD is a class of molecules that bind to and sequester VEGF, such as ranibizumab (Lucentis) and aflibercept (Eylea).

[0005] Diabetic retinopathy (DR) is a major complication of diabetes and a leading cause of blindness in the working-age population. DR can be nonproliferative (NPDR), which is associated with no new blood vessel growth, or proliferative (PDR), which involves new, abnormal blood vessel growth within the retina or choroid. Diabetic macular edema (DME), a complication of DR, is another example of an eye disease that affects the macula. DME affects up to 10% of people with diabetes and is caused by fluid accumulation in the macula. DME is the most frequent cause of vision loss in people with DR. Available treatments for DME include lasers and anti-vascular endothelial growth factor (anti-VEGF) drugs, such as aflibercept.

[0006] Aflibercept is a recombinant fusion protein that acts as a decoy receptor for vascular endothelial growth factor subtypes A and B (VEGF-A and VEGF-B) and placental growth factor (PGF). By binding to these ligands, aflibercept prevents their binding to vascular endothelial growth factor receptors (VEGFR), VEGFR-1 and VEGFR-2, inhibiting angiogenesis and reducing vascular permeability. Aflibercept consists of domain 2 of VEGFR-1 and domain 3 of VEGFR-2 fused to the Fc fragment of IgG1.

[0007] Current standard-of-care anti-VEGF agents, such as aflibercept, must be re-administered every 4–8 weeks via intravitreal (IVT) injection to achieve optimal treatment outcomes and maintain vision. Compliance with such regimens is burdensome for patients, their caregivers, and healthcare systems, and most patients fall out of optimal regimen compliance over time, which correlates with vision loss (Khanani AM, et al.). Additionally, complications exist, including endophthalmitis, retinal detachment, traumatic cataract, and elevated intraocular pressure (IOP), and the risk of these complications may increase with repeated IVT injections (Falavarjani et al., (2013) Eye (Lond), 27(7):787-794). [Prior art documents] [Non-patent literature]

[0008] [Non-Patent Document 1] Klein et al., (2011) Arch Ophthalmol, 129(1):75-80 [Non-patent document 2] Falavarjani et al., (2013) Eye (Lond), 27(7):787-794 Summary of the Invention [Problem to be solved by the invention]

[0009] Thus, there is a need in the art for treatments for intraocular neovascular diseases such as wAMD, DR, or DME that are effective, have a reduced risk of adverse effects, and are amenable to high long-term patient compliance. [Means for solving the problem]

[0010] Summary of the Disclosure In one aspect, provided herein is a method for treating an intraocular neovascular disease in an individual, comprising administering about 6×10 recombinant adeno-associated virus (rAAV) particles to the individual. 11 Methods are provided, comprising administering a unit dose of a vector genome (vg) or less to one eye of an individual, wherein the individual is a human, and the rAAV particles comprise: (a) a nucleic acid encoding a polypeptide comprising an amino acid sequence having at least about 95% identity to the amino acid sequence of SEQ ID NO: 35, and flanked by AAV2 inverted terminal repeats (ITRs), and (b) an AAV2 capsid protein comprising, or consisting of, the amino acid sequence LGETTRP (SEQ ID NO: 14) inserted between positions 587 and 588 of the capsid protein, wherein the amino acid residue numbering corresponds to that of the AAV2 VP1 capsid protein. In some embodiments, the method comprises reducing retinal fluid in the eye of the individual.

[0011] In another aspect, provided herein is a method for reducing retinal fluid in an eye of an individual with an intraocular neovascular disease, comprising administering a unit dose of rAAV particles to one eye of the individual, wherein the individual is human, and the rAAV particles comprise: (a) a nucleic acid encoding a polypeptide comprising an amino acid sequence having at least about 95% identity to the amino acid sequence of SEQ ID NO: 35, flanked by AAV2 inverted terminal repeats (ITRs); and (b) an AAV2 capsid protein comprising the amino acid sequence LGETTRP (SEQ ID NO: 14) inserted between positions 587 and 588 of the capsid protein, wherein the amino acid residue numbering corresponds to that of the AAV2 VP1 capsid protein. In some embodiments, the individual has received at least one treatment with an anti-VEGF agent within approximately the last 12 weeks prior to administration of the unit dose of rAAV particles. In some embodiments, the amount or presence of retinal fluid in one eye of the individual is refractory to previous treatment with an anti-VEGF agent. In some embodiments, the anti-VEGF agent is aflibercept. In some embodiments, the retinal fluid in one eye is reduced by at least about 60%. In some embodiments, the retinal fluid in one eye is reduced by about 80% compared to the retinal fluid level in one eye of the individual before administering the rAAV to the individual. In some embodiments, the retinal fluid is subretinal fluid (SRF) or intraretinal fluid (IRF). In some embodiments, the unit dose of rAAV particles is about 6 x 10 11 vector genome / eye (vg / eye) or less.

[0012] In another aspect, a method for treating an intraocular neovascular disease in an individual includes the steps of: (a) administering an anti-VEGF agent to one eye of the individual; and (b) administering about 6×10 recombinant adeno-associated virus (rAAV) particles to one eye of the individual after administration of the anti-VEGF agent. 11and administering a unit dose of a vector genome (vg) or less to one eye of the individual, wherein the individual is a human, and the rAAV particles comprise (i) a nucleic acid encoding a polypeptide comprising an amino acid sequence having at least about 95% identity to the amino acid sequence of SEQ ID NO: 35 and flanked by AAV2 inverted terminal repeats (ITRs), and (ii) an AAV2 capsid protein comprising the amino acid sequence LGETTRP (SEQ ID NO: 14) inserted between positions 587 and 588 of the capsid protein, wherein the amino acid residue numbering corresponds to that of the AAV2 VP1 capsid protein. In some embodiments, the method comprises administering the unit dose of the rAAV particles to one eye of the individual about 1 week or about 7 days after administration of the anti-VEGF agent. In some embodiments, the method comprises administering the unit dose of the rAAV particles to one eye of the individual about 1 week to about 2 weeks after administration of the anti-VEGF agent. In some embodiments, the method comprises administering a unit dose of rAAV particles to one eye of the individual about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, about 14 days, or about 15 days after administration of the anti-VEGF agent. In some embodiments, the method comprises administering an anti-VEGF agent to one eye of the individual on day 1 and administering a unit dose of rAAV particles to one eye of the individual on day 8. In some embodiments, the anti-VEGF agent comprises aflibercept. In some embodiments, the aflibercept is administered by intravitreal injection at a dose of about 2 mg. In some embodiments, the method further comprises administering topical steroid treatment. In some embodiments, the topical steroid treatment is difluprednate treatment. In some embodiments, the topical steroid treatment comprises administering the topical steroid about four times per day for about four weeks, followed by administering the topical steroid about three times per day for about one week, followed by administering the topical steroid about two times per day for about one week, and then administering the topical steroid about once per day for about one week, timing starting with and following administration of the anti-VEGF agent.In some embodiments, the topical steroid treatment comprises administering the topical steroid about four times per day for about one month, followed by administering the topical steroid about three times per day for about one month, followed by administering the topical steroid about twice per day for about one month, and then administering the topical steroid about once per day for about one month, timing starting with and following administration of the anti-VEGF agent. In some embodiments, the topical steroid treatment comprises administering the topical steroid about four times per day for about one month, followed by administering the topical steroid about three times per day for about one month, followed by administering the topical steroid about twice per day for about one month, and then administering the topical steroid about once per day for about one month, timing starting with and following administration of the rAAV particles. In some embodiments, the topical steroid comprises an about 1 μg to about 3 μg dose of 0.05% difluprednate. In some embodiments, the topical steroid comprises an about 2.5 μg dose of 0.05% difluprednate.

[0013] In some embodiments, which may be combined with any of the preceding embodiments, the unit dose of rAAV particles is about 6×10 10 ~about 2×10 11 In some embodiments, the unit dose of rAAV particles is about 2 x 10 11 or about 6 x 10 10 In some embodiments, which may be combined with any of the preceding embodiments, the unit dose of rAAV particles is about 6×10 10 ~Approx. 6×10 11 In some embodiments, which may be combined with any of the preceding embodiments, the unit dose of rAAV particles is about 6×10 10 ~about 2×10 11 In some embodiments, which may be combined with any of the preceding embodiments, the unit dose of rAAV particles is about 2×10 11 ~Approx. 6×10 11In some embodiments, the unit dose of rAAV particles is about 2 x 10 11 or about 6 x 10 10 In some embodiments, the unit dose of rAAV particles is about 2 x 10 11 vg / eye. In some embodiments, the unit dose of rAAV particles is about 6 x 10 11 vg / eye.

[0014] In some embodiments, which may be in combination with any of the preceding embodiments, the individual has one or more symptoms of intraocular neovascular disease in the contralateral eye.

[0015] In some embodiments, which may be combined with any of the preceding embodiments, the methods provided herein further include administering a unit dose of rAAV particles to the contralateral eye of the individual. In some embodiments, administering the unit dose of rAAV particles to the contralateral eye is up to about two weeks after administering the unit dose of rAAV particles to one eye. In some embodiments, administering the unit dose of rAAV particles to the contralateral eye is on the same day as administering the unit dose of rAAV particles to one eye, or administering the unit dose of rAAV particles to the contralateral eye is between about one day and about 14 days after administering the unit dose of rAAV particles to one eye. In some embodiments, the unit dose of rAAV particles administered to the contralateral eye of the individual contains the same or fewer vector genomes / eye (vg / eye) as the unit dose of rAAV particles administered to one eye of the individual. In some embodiments, the step of administering the unit dose of rAAV particles to the contralateral eye occurs at least about two weeks after the step of administering the unit dose of rAAV particles to one eye. In some embodiments, the unit dose of rAAV particles administered to the contralateral eye of the individual contains more vector genomes / eye (vg / eye) than the unit dose of rAAV particles administered to one eye of the individual.

[0016] In some embodiments, which may be in combination with any of the preceding embodiments, the nucleic acid comprises the nucleic acid sequence of SEQ ID NO: 40, or a sequence having at least 85% identity thereto.

[0017] In some embodiments that may be combined with any of the preceding embodiments, the polypeptide comprises the amino acid sequence of SEQ ID NO: 35. In some embodiments that may be combined with any of the preceding embodiments, the polypeptide comprises the amino acid sequence of SEQ ID NO: 41. In some embodiments, the polypeptide is aflibercept.

[0018] In some embodiments, which may be combined with any of the preceding embodiments, the nucleic acid further comprises a first enhancer region, a promoter region, a 5'UTR region, a second enhancer region, and a polyadenylation site. In some embodiments, the nucleic acid comprises, in 5' to 3' order: (a) a first enhancer region; (b) a promoter region; (c) a 5'UTR region; (d) a nucleic acid encoding a polypeptide comprising an amino acid sequence having at least about 95% identity to the amino acid sequence of SEQ ID NO: 35; (e) a second enhancer region; and (f) a polyadenylation site, and is flanked by AAV2 inverted terminal repeats (ITRs). In some embodiments, the first enhancer region comprises a CMV sequence comprising the sequence of SEQ ID NO: 22 or a sequence having at least 85% identity thereto. In some embodiments, the promoter region comprises a CMV sequence comprising the sequence of SEQ ID NO: 23 or a sequence having at least 85% identity thereto. In some embodiments, the nucleic acid encoding the polypeptide comprises the nucleic acid sequence of SEQ ID NO: 40 or a sequence having at least 85% identity thereto. In some embodiments, the polypeptide comprises the amino acid sequence of SEQ ID NO: 35 or a sequence having at least 85% identity thereto. In some embodiments, the polypeptide comprises the amino acid sequence of SEQ ID NO: 41 or a sequence having at least 85% identity thereto. In some embodiments, the polypeptide is aflibercept. In some embodiments, the 5'UTR region comprises, in 5' to 3' order, a TPL sequence comprising the sequence of SEQ ID NO: 24 or a sequence having at least 85% identity thereto, and an eMLP sequence comprising the sequence of SEQ ID NO: 25 or a sequence having at least 85% identity thereto. In some embodiments, the second enhancer region comprises a full-length EES sequence comprising the sequence of SEQ ID NO: 26 or a sequence having at least 85% identity thereto. In some embodiments, the polyadenylation site comprises an HGH polyadenylation site comprising the sequence of SEQ ID NO: 27 or a sequence having at least 85% identity thereto.In some embodiments, the nucleic acid further comprises: (a) a first enhancer region comprising a CMV sequence comprising the sequence of SEQ ID NO: 22 or a sequence having at least 85% identity thereto; (b) a promoter region comprising a CMV sequence comprising the sequence of SEQ ID NO: 23 or a sequence having at least 85% identity thereto; (c) a 5'UTR region comprising, in 5' to 3' order, a TPL sequence comprising the sequence of SEQ ID NO: 24 or a sequence having at least 85% identity thereto, and an eMLP sequence comprising the sequence of SEQ ID NO: 25 or a sequence having at least 85% identity thereto; (d) a second enhancer region comprising a full-length EES sequence comprising the sequence of SEQ ID NO: 26 or a sequence having at least 85% identity thereto; and (e) an HGH polyadenylation site comprising the sequence of SEQ ID NO: 27 or a sequence having at least 85% identity thereto. In some embodiments, the nucleic acid comprises AAV ITRs on both ends of the element.

[0019] In some embodiments, which may be in combination with any of the preceding embodiments, the nucleic acid comprises the sequence of SEQ ID NO: 39, or a sequence having at least 85% identity thereto.

[0020] In some embodiments, which may be in combination with any of the preceding embodiments, the rAAV particles comprise an AAV2 VP1 capsid protein comprising a GH loop comprising the amino acid sequence of SEQ ID NO: 38, or an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 38. In some embodiments, the rAAV particles comprise an AAV2 VP1 capsid protein comprising a GH loop comprising an amino acid sequence having any of at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 38.

[0021] In some embodiments, which may be in combination with any of the preceding embodiments, the rAAV particles comprise an AAV2 VP1 capsid protein comprising the amino acid sequence of SEQ ID NO: 37, or an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 37. In some embodiments, the rAAV particles comprise an AAV2 VP1 capsid protein comprising an amino acid sequence having any of at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 37.

[0022] In some embodiments, which may be in combination with any of the preceding embodiments, the AAV2 capsid protein comprises or consists of the amino acid sequence LGETTRP (SEQ ID NO: 14) inserted between positions 587 and 588 of AAV2 VP1 comprising the sequence of SEQ ID NO: 13. In some embodiments, the AAV2 capsid protein comprises the amino acid sequence LALGETTRPA (SEQ ID NO: 1) inserted between positions 587 and 588 of the capsid protein, where the amino acid residue numbering corresponds to that of the AAV2 VP1 capsid protein. In some embodiments, the AAV2 capsid protein comprises the amino acid sequence LALGETTRPA (SEQ ID NO: 1) inserted between positions 587 and 588 of AAV2 VP1 comprising the sequence of SEQ ID NO: 13. In some embodiments, the AAV2 capsid protein comprises the amino acid sequence LALGETTRPA (SEQ ID NO: 1) inserted between positions 587 and 588 of the capsid protein, where the amino acid residue numbering corresponds to that of the AAV2 VP1 capsid protein. In some embodiments, the AAV2 capsid protein comprises the amino acid sequence LALGETTRPA (SEQ ID NO: 1) inserted between positions 587 and 588 of AAV2 VP1 comprising the sequence of SEQ ID NO: 13.

[0023] In some embodiments, which may be combined with any of the preceding embodiments, administration of the unit dose of rAAV particles to one eye and / or the contralateral eye is by intravitreal administration.

[0024] In some embodiments, which may be combined with any of the preceding embodiments, the unit dose of rAAV particles is in a pharmaceutical formulation. In some embodiments, the pharmaceutical formulation comprises rAAV particles, sodium chloride, sodium phosphate, and a surfactant. In some embodiments, the pharmaceutical formulation comprises about 150 to about 200 mM sodium chloride, about 1 to about 10 mM sodium dihydrogen phosphate, about 1 to about 10 mM sodium dihydrogen phosphate, about 0.0005% (wt / vol) to about 0.005% (wt / vol) poloxamer 188, and about 6 x 10 13 ~Approx. 6×10 10 In some embodiments, the pharmaceutical formulation comprises about 180 mM sodium chloride, about 5 mM sodium dihydrogen phosphate, about 5 mM sodium dihydrogen phosphate, about 6 x 10 12 vg / mL rAAV particles, and about 0.001% (weight / volume) poloxamer 188, and the pharmaceutical formulation has a pH of about 7.3. In some embodiments, the pharmaceutical formulation comprises about 180 mM sodium chloride, about 5 mM sodium dihydrogen phosphate, about 5 mM sodium dihydrogen phosphate, about 2 x 10 12 vg / mL rAAV particles, and about 0.001% (weight / volume) poloxamer 188, and the pharmaceutical formulation has a pH of about 7.3. In some embodiments, the pharmaceutical formulation comprises about 180 mM sodium chloride, about 5 mM sodium dihydrogen phosphate, about 5 mM sodium dihydrogen phosphate, about 6 x 10 11 vg / mL rAAV particles, and about 0.001% (weight / volume) poloxamer 188, and the pharmaceutical formulation has a pH of about 7.3.

[0025] In some embodiments that may be combined with any of the preceding embodiments, the unit dose of rAAV particles comprises a volume of about 25 μL to about 250 μL. In some embodiments, the unit dose of rAAV particles comprises a volume of about 100 μL. In some embodiments, the unit dose of rAAV particles comprises a volume of about 30 μL. In some embodiments that may be combined with any of the preceding embodiments, the unit dose of rAAV particles administered to one eye and / or the contralateral eye comprises a volume of about 25 μL to about 250 μL. In some embodiments, the unit dose of rAAV particles administered to one eye and / or the contralateral eye comprises a volume of about 100 μL. In some embodiments, the unit dose of rAAV particles administered to one eye and / or the contralateral eye comprises a volume of about 30 μL.

[0026] In some embodiments, which may be combined with any of the above-mentioned embodiments, the individual has previously been treated with an anti-VEGF agent for intraocular neovascular disease.In some embodiments, the individual has previously been injected with an anti-VEGF agent into one eye and / or the other eye before administering rAAV particles to one eye and / or the other eye.In some embodiments, the individual has not previously been treated with an anti-VEGF agent for intraocular neovascular disease.In some embodiments, the anti-VEGF agent is aflibercept.

[0027] In some embodiments, which may be in combination with any of the preceding embodiments, the intraocular neovascular disease is exudative age-related macular degeneration (AMD), retinal neovascularization, choroidal neovascularization, diabetic retinopathy, proliferative diabetic retinopathy, retinal vein occlusion, central retinal vein occlusion, branch retinal vein occlusion, diabetic macular edema, diabetic retinal ischemia, ischemic retinopathy, diabetic retinal edema, or any combination thereof.

[0028] In some embodiments, which may be combined with any of the preceding embodiments, the unit dose of rAAV particles is administered in combination with steroid treatment. In some embodiments, the steroid treatment is corticosteroid treatment. In some embodiments, the steroid treatment is systemic steroid treatment. In some embodiments, the steroid treatment is oral steroid treatment. In some embodiments, the steroid treatment is prednisone treatment. In some embodiments, the oral prednisone treatment includes, starting three days before administration of the unit dose of rAAV particles to one eye and / or the contralateral eye, administering prednisone at a dose of about 60 mg per day for a total of six days, followed by administering prednisone at a dose of about 40 mg per day for a total of three days, followed by administering prednisone at a dose of about 20 mg per day for a total of two days, followed by administering prednisone at a dose of about 10 mg per day for a total of two days. In some embodiments, the steroid treatment is topical steroid treatment. In some embodiments, the steroid treatment is difluprednate treatment. In some embodiments, the steroid is administered before, during, and / or after administration of a unit dose of rAAV particles, hi some embodiments, the steroid is administered before, during, and / or after administration of a unit dose of rAAV particles to one eye and / or the contralateral eye.

[0029] In some embodiments, which may be combined with any of the preceding embodiments, the steroid treatment is topical steroid treatment, and the topical steroid treatment is daily steroid treatment for up to about 4 weeks, up to about 6 weeks, or up to about 8 weeks after administration of a unit dose of rAAV particles. In some embodiments, the topical steroid treatment comprises about four administrations of topical steroid at about week 1, about three administrations of topical steroid at about week 2, about two administrations of topical steroid at about week 3, and about one administration of topical steroid at about week 4, timing starting with and following administration of a unit dose of rAAV particles. In some embodiments, the topical steroid treatment comprises about four administrations of topical steroid per day for about three weeks after administration of a unit dose of rAAV particles, followed by about three administrations of topical steroid per day for about one week thereafter, about two administrations of topical steroid per day for about one week thereafter, and about one administration of topical steroid per day for about one week thereafter. In some embodiments, the topical steroid treatment comprises administering the topical steroid about four times per day for about four weeks, followed by administering the topical steroid about three times per day for about one week, followed by administering the topical steroid about twice per day for about one week, and then administering the topical steroid about once per day for about one week, timing starting about one week before administration of the unit dose of rAAV particles. In some embodiments, the topical steroid treatment comprises administering the topical steroid about four times per day for about one month, followed by administering the topical steroid about three times per day for about one month, followed by administering the topical steroid about twice per day for about one month, and then administering the topical steroid about once per day for about one month, timing starting about the time of administration of the unit dose of rAAV particles. In some embodiments, the topical steroid treatment comprises administering the topical steroid about four times per day for about one month, followed by administering the topical steroid about three times per day for about one month, followed by administering the topical steroid about two times per day for about one month, and then administering the topical steroid about once per day for about one month, starting about one week before administration of the unit dose of rAAV particles.In some embodiments, the topical steroid comprises about a 1 μg to about a 3 μg dose of 0.05% difluprednate, hi some embodiments, the topical steroid comprises about a 2.5 μg dose of 0.05% difluprednate.

[0030] In some embodiments, the intraocular neovascular disease is wet age-related macular degeneration (wAMD).

[0031] In some embodiments, which may be combined with any of the preceding embodiments, administering a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual results in a maintenance or reduction in retinal thickness compared to the retinal thickness before administration of the unit dose of rAAV particles. In some embodiments, the maintenance or reduction in retinal thickness compared to the retinal thickness before administration of the unit dose of rAAV particles exists for about 30 weeks, about 34 weeks, about 44 weeks, about 6 months, about 1 year, about 1.5 years, about 2 years, about 3 years, about 5 years, about 10 years, or longer after administration of the unit dose of rAAV particles to one eye and / or the contralateral eye of the individual. In some embodiments, administering a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual results in a reduction in retinal thickness compared to the retinal thickness before administration of the unit dose of rAAV particles. In some embodiments, the reduction in retinal thickness is at least about 10% compared to the retinal thickness before administration of the unit dose of rAAV particles. In some embodiments, the retinal thickness is central zone retinal thickness (CST) or central foveal retinal thickness (CRT).

[0032] In some embodiments, which may be combined with any of the preceding embodiments, administering a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual results in a maintenance or reduction in macular volume compared to the macular volume before administration of the unit dose of rAAV particles. In some embodiments, the maintenance or reduction in macular volume compared to the macular volume before administration of the unit dose of rAAV particles exists for about 30 weeks, about 34 weeks, about 44 weeks, about 6 months, about 1 year, about 1.5 years, about 2 years, about 3 years, about 5 years, about 10 years, or longer after administration of the unit dose of rAAV particles to one eye and / or the contralateral eye of the individual. In some embodiments, administering a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual results in a reduction in macular volume compared to the macular volume before administration of the unit dose of rAAV particles. In some embodiments, the reduction in macular volume is at least about 10% compared to the macular volume before administration of the unit dose of rAAV particles.

[0033] In some embodiments, which may be combined with any of the preceding embodiments, administering a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual results in the maintenance or improvement of visual acuity compared to the visual acuity before administration of the unit dose of rAAV particles. In some embodiments, the maintenance or improvement of visual acuity compared to the visual acuity before administration of the unit dose of rAAV particles exists for about 30 weeks, about 34 weeks, about 44 weeks, about 6 months, about 1 year, about 1.5 years, about 2 years, about 3 years, about 5 years, about 10 years, or longer after administration of the unit dose of rAAV particles to one eye and / or the contralateral eye of the individual. In some embodiments, administering a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual results in the improvement of visual acuity compared to the visual acuity before administration of the unit dose of rAAV particles. In some embodiments, the visual acuity is best-corrected visual acuity (BCVA).

[0034] In some embodiments, which may be combined with any of the preceding embodiments, administration of a single unit dose of rAAV particles to one eye and / or the contralateral eye of an individual results in a therapeutic benefit (e.g., treatment of an intraocular neovascular disease, reduction of retinal fluid, maintenance or reduction of retinal thickness, maintenance or reduction of macular volume, and / or maintenance or improvement of vision). In some embodiments, administration of a single unit dose of rAAV particles to one eye and / or the contralateral eye of an individual results in a therapeutic benefit that exists for about 30 weeks or longer, about 34 weeks or longer, about 44 weeks or longer, about 6 months or longer, about 1 year or longer, about 1.5 years or longer, or about 2 years, about 3 years, about 5 years, about 10 years, or longer, after administration of the unit dose of rAAV particles to the one eye and / or the contralateral eye of the individual.

[0035] In some embodiments, which may be combined with any of the preceding embodiments, administration of a unit dose of rAAV particles to one eye and / or contralateral eye of a plurality of individuals results in at least about 50% of the individuals in the plurality not requiring anti-VEGF rescue treatment, hi some embodiments, at least about 50% of the individuals in the plurality not requiring anti-VEGF rescue treatment for at least about 20 weeks, at least about 36 weeks, at least about 52 weeks, at least about 56 weeks, or longer after administration of the unit dose of rAAV particles.

[0036] In some embodiments, which may be in combination with any of the preceding embodiments, administration of a single unit dose of rAAV particles to one eye and / or contralateral eye of a plurality of individuals results in at least about 67% of the individuals in the plurality not requiring anti-VEGF rescue treatment, hi some embodiments, at least about 67% of the individuals in the plurality not requiring anti-VEGF rescue treatment for at least about 20 weeks, at least about 36 weeks, at least about 52 weeks, at least about 60 weeks, at least about 64 weeks, or at least about 66 weeks after administration of the unit dose of rAAV particles.

[0037] In some embodiments, which may be in combination with any of the preceding embodiments, administration of a unit dose of rAAV particles to one eye and / or contralateral eye of a plurality of individuals results in at least about 78% of the individuals in the plurality not requiring anti-VEGF rescue treatment, hi some embodiments, at least about 78% of the individuals in the plurality not requiring anti-VEGF rescue treatment for at least about 20 weeks, at least about 36 weeks, or longer after administration of the unit dose of rAAV particles.

[0038] In some embodiments, which may be combined with any of the preceding embodiments, administration of a unit dose of rAAV particles to one eye and / or contralateral eye of a plurality of individuals results in 100% of the individuals in the plurality not requiring anti-VEGF rescue treatment, hi some embodiments, 100% of the individuals in the plurality not requiring anti-VEGF rescue treatment for at least about 64 weeks, at least about 68 weeks, at least about 72 weeks, at least about 76 weeks, at least about 80 weeks, at least about 84 weeks, or longer after administration of the unit dose of rAAV particles.

[0039] In some embodiments, which may be in combination with any of the preceding embodiments, administration of a unit dose of rAAV particles to one eye and / or contralateral eyes of multiple individuals results in a reduction in the annual anti-VEGF injection rate of at least about 80%, at least about 85%, at least about 87%, at least about 90%, at least about 95%, at least about 99%, or 100% compared to the annual anti-VEGF injection rate before administration of the unit dose of rAAV particles.

[0040] In some embodiments, the intraocular neovascular disease is diabetic macular edema (DME). In some embodiments, administering a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual results in a two- or three-level improvement in the Diabetic Retinopathy Severity Scale (DRSS). In some embodiments, administering a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual results in a two-level improvement in the Diabetic Retinopathy Severity Scale (DRSS). In some embodiments, administering a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual results in a three-level improvement in the Diabetic Retinopathy Severity Scale (DRSS). Incorporation by Reference

[0041] All references cited herein, including patent applications and publications, are hereby incorporated by reference in their entirety.

[0042] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description and accompanying drawings that set forth illustrative embodiments, in which the principles of the invention are utilized. [Brief explanation of the drawings]

[0043] [Figure 1A] Figures 1A-1B present a schematic diagram of the investigational drug and the Phase I study described in Examples 1 and 2. Figure 1A is a schematic diagram of AAV2.7m8-aflibercept. AAV2.7m8-aflibercept is a recombinant replication-deficient adeno-associated virus (rAAV) vector containing an AAV2.7m8 protein capsid and a vector genome containing an expression cassette (C11) of a codon-optimized version of the aflibercept cDNA under the control of a ubiquitous chimeric promoter. The AAV2.7m8-aflibercept vector genome also contains two AAV2 inverted terminal repeats (ITRs) on either side of the aflibercept cDNA expression cassette. Figure 1B is a diagram summarizing the study design of the Phase I study described in Examples 1 and 2. [Figure 1B] Same as above.

[0044] [Figure 1C] FIG. 1C presents a diagram of the AAV2.7m8-aflibercept vector genome (SEQ ID NO: 39). The vector genome contains an expression cassette composed of two AAV serotype 2 inverted terminal repeats (ITRs) (positions 1-145 and 3772-3916 of SEQ ID NO:39), a CMV promoter (positions 180-693 of SEQ ID NO:39), a 5' untranslated region (UTR) composed of an adenoviral tripartite leader sequence and a synthetic intron (positions 694-1314 of SEQ ID NO:39), a Kozak sequence (positions 1329-1340 of SEQ ID NO:39), codon-optimized aflibercept cDNA (positions 1338-2714 of SEQ ID NO:39), a 3' UTR composed of a human scaffold attachment region (positions 2717-3527 of SEQ ID NO:39), and a human growth hormone polyadenylation / transcription termination signal (positions 3546-3748 of SEQ ID NO:39). AAV = adeno-associated virus; CMV = cytomegalovirus; GH = growth hormone; ITR = inverted terminal repeat; polyA = polyadenylation; SAR = scaffold attachment region; TPL = tripartite leader.

[0045] [Figure 2A]Figures 2A-2L show optical coherence tomography (OCT) images and retinal thickness maps derived from the OCT images obtained from subjects in Cohort 1 of the study described in Example 1. OCT images were obtained at the indicated time points before and after administration of AAV2.7m8-aflibercept (Day 1). Anti-VEGF IVT treatment intervals are shown for all subjects. Figure 2A presents OCT images and retinal thickness maps derived from the OCT images obtained from Subject 1 at five visits at the indicated time points before the screening aflibercept injection. The OCT images were obtained immediately before treatment with aflibercept standard therapy. Subject 1 required aflibercept IVT every 5 to 7 weeks and exhibited refractory subretinal fluid and pigment epithelial detachment (PED) despite treatment with aflibercept standard therapy. Figure 2B presents OCT images and retinal thickness maps derived from OCT images acquired from subject 1 at the time of aflibercept injection at screening (day -7), AAV2.7m8-aflibercept injection (day 1), and follow-up visits at the indicated time points. Subject 1 did not require any rescue injections after AAV2.7m8-aflibercept injection. Subject 1 demonstrated resolution of subretinal fluid beginning at week 4, with subretinal and intraretinal fluid remaining absent (dry). Figure 2C presents OCT images and retinal thickness maps derived from OCT images acquired from subject 2 at five visits at the indicated time points prior to aflibercept injection at screening. OCT images were acquired immediately prior to treatment with aflibercept standard of care. Subject 2 required six aflibercept IVT treatments during the 8 months prior to treatment with AAV2.7m8-aflibercept to maintain retinal anatomy. Figure 2D presents OCT images and retinal thickness maps derived from OCT images acquired from subject 2 at the time of aflibercept injection at screening (day -7), AAV2.7m8-aflibercept injection (day 1), and follow-up visits at the indicated time points. Subject 2 did not require any rescue injections after AAV2.7m8-aflibercept injection. Subject 2 demonstrated stable retinal anatomy through week 24, with no subretinal or intraretinal fluid present.Figure 2E presents OCT images and retinal thickness maps derived from OCT images from subject 3 acquired at four visits at the indicated time points before the screening aflibercept injection. An OCT image acquired at week -27 is not shown. An OCT image was acquired immediately prior to treatment with aflibercept standard therapy. Subject 3 exhibited subretinal fluid, which increased as the interval between aflibercept IVTs increased from 5 to 7 weeks. Figure 2F presents OCT images and retinal thickness maps derived from OCT images acquired at the time of the screening aflibercept injection (day -7), the AAV2.7m8-aflibercept injection (day 1), and the follow-up visits at the indicated time points. Subject 3 did not require any rescue injections after the AAV2.7m8-aflibercept injection. Subject 3 demonstrated resolution of refractory subretinal fluid by week 8 and stable retinal anatomy through week 24. Figure 2G presents OCT images and retinal thickness maps derived from OCT images acquired from subject 4 at five visits at the indicated time points prior to the screening aflibercept injection. The OCT images were acquired immediately prior to treatment with ranibizumab 0.5 mg IVT standard of care. Subject 4 demonstrated subretinal fluid refractory to ranibizumab IVT injection. Figure 2H presents OCT images and retinal thickness maps derived from OCT images acquired from subject 4 at the time of the screening aflibercept injection (day -14), the AAV2.7m8-aflibercept injection (day 1), and the follow-up visits at the indicated time points. Subject 4 did not require any rescue injections after the AAV2.7m8-aflibercept injection. Subject 4 demonstrated resolution of refractory subretinal fluid by week 8 and stable retinal anatomy through week 24. Figure 2I presents OCT images and retinal thickness maps derived from the OCT images acquired at visit 5 at the indicated time points prior to aflibercept injection at screening from subject 5. The OCT images were acquired immediately prior to treatment with aflibercept standard of care.Figure 2J presents OCT images and retinal thickness maps derived from OCT images from subject 5 acquired at the time of aflibercept injection at screening (day -14), AAV2.7m8-aflibercept injection (day 1), and follow-up visits at the indicated times. Subject 5 did not require any rescue injections after AAV2.7m8-aflibercept injection. Subretinal fluid and PED were present during treatment with AAV2.7m8-aflibercept and resolved over time. The retinal anatomy remained stable through week 24, with no subretinal or intraretinal fluid present. Figure 2K presents OCT images and retinal thickness maps derived from OCT images acquired at five visits at the indicated times before aflibercept injection at screening from subject 6. OCT images were acquired immediately before treatment with either bevacizumab 1.5 mg IVT or ranibizumab 0.5 mg IVT, as indicated. The retinal appearance of Subject 6 was consistent with polypoidal choroidal vasculopathy (PCV). Figure 2L presents OCT images and retinal thickness maps derived from the OCT images acquired from Subject 6 at the time of aflibercept injection at screening (day -10), AAV2.7m8-aflibercept injection (day 1), and follow-up visits at the indicated time points. Subject 6 did not require any rescue injections after AAV2.7m8-aflibercept injection. Subject 6 did not demonstrate any increase in subretinal fluid through week 24, and some anatomical improvement was achieved. Subject 6's contralateral eye received standard-of-care aflibercept injections every 4 weeks over the course of the study and demonstrated similar retinal morphology to the AAV2.7m8-aflibercept-treated eye. [Figure 2B] Same as above. [Figure 2C] Same as above. [Figure 2D] Same as above. [Figure 2E] Same as above. [Figure 2F] Same as above. [Figure 2G] Same as above. [Figure 2H] Same as above. [Figure 2I] Same as above. [Figure 2J] Same as above. [Figure 2K] Same as above. [Figure 2L] Same as above.

[0046] [Figure 3] Figure 3 shows the mean change in central retinal thickness (CST) at the indicated time points for subjects in Cohort 1 of the study described in Example 1. Error bars represent 90% confidence intervals calculated using a T-distribution. Baseline (BL) indicates measurements obtained prior to the aflibercept injection at screening, 7 to 15 days (e.g., 7 to 14 days) prior to treatment with AAV2.7m8-aflibercept on Day 1. At 24 weeks after treatment with AAV2.7m8-aflibercept, subjects demonstrated a mean change in CST of -52.7 μm (90% CI -86.5, -18.8). BL = baseline; D = day; W = week. The Day 1 visit occurred 7 to 14 days after the baseline visit.

[0047] [Figure 4] Figure 4 shows mean best-corrected visual acuity (BCVA) measurements based on the Early Treatment Diabetic Retinopathy Study (ETDRS) letter rating at the indicated time points for subjects in Cohort 1 of the study described in Example 1. Error bars indicate 90% confidence intervals calculated using a T-distribution. Baseline (BL) indicates measurements obtained prior to the aflibercept injection at screening, 7 to 15 days (e.g., 7 to 14 days) prior to treatment with AAV2.7m8-aflibercept on Day 1. At 24 weeks after treatment with AAV2.7m8-aflibercept, subjects demonstrated a mean change in BCVA of -2 letters (90% CI -9.1, 5.1). BL = baseline; D = day; W = week. The Day 1 visit occurred 7 to 14 days after the baseline visit.

[0048] [Figure 5] FIG. 5 presents the nucleic acid sequence of aflibercept (SEQ ID NO: 36).

[0049] [Figure 6] Figure 6 shows plots of anterior chamber and vitreous cell counts after treatment with AAV2.7m8-aflibercept for subjects 1-6 from the study described in Example 1. The steroid treatment administered to each patient is indicated below each plot. Aqueous humor cell count categories were based on the Standardization of Uveitis Nomenclature (SUN) criteria (Jabs, DA et al., J Ophthalmol. 2005; 140: 509-516). Vitreous cell count categories were based on the National Institutes of Health (NIH) guidelines. For aqueous humor cells, a cell count of 0.5+ indicates 1-5 cells, a cell count of 1+ indicates 6-15 cells, a cell count of 2+ indicates 16-25 cells, a cell count of 3+ indicates 26-50 cells, and a cell count of 4+ indicates >50 cells. For vitreous cells, a cell count of 0.5+ indicates 1-10 cells, a cell count of 1+ indicates 11-20 cells, a cell count of 2+ indicates 21-30 cells, a cell count of 3+ indicates 31-100 cells, and a cell count of 4+ indicates >100 cells. For the analysis shown in this figure, rare cells were captured as 0.5+.

[0050] [Figure 7A]Figures 7A-7B show optical coherence tomography (OCT) images and retinal thickness maps derived from OCT images acquired at a median follow-up time of 34 weeks for subjects 1-6 of cohort 1 of the study described in Example 1. Additionally, the change from baseline in BCVA, the number of anti-VEGF IVT injections during the 8 months prior to AAV2.7m8-aflibercept administration, and the number of rescue anti-VEGF IVT injections administered during the study are also presented for each of subjects 1-6. The actual week in which the OCT images and retinal thickness maps were obtained for each subject is indicated (subject 1 = week 44; subject 2 = week 40; subject 3 = week 36; subject 4 = week 32; subject 5 = week 28; and subject 6 = week 28). No subjects required rescue anti-VEGF IVT injections during the study, and no retreatment criteria were met at any time during the maximum 44-week follow-up period. No subjects showed signs of disease reactivation on OCT imaging. [Figure 7B] Same as above.

[0051] [Figure 8]Figure 8 shows plots of aqueous humor and vitreous cell counts for subjects 1-6 in Cohort 1 of the study described in Example 1 after treatment with AAV2.7m8-aflibercept up to a median follow-up time of 44 weeks (range, 40-52 weeks). The steroid treatment administered to each subject is indicated below each plot. Aqueous humor cell grade categories were based on the Standardization of Uveitis Nomenclature (SUN) criteria (Jabs, DA et al. J Ophthalmol. 2005;140:509-516). Vitreous cell grade categories were based on the National Institutes of Health (NIH) guidelines. Regarding aqueous humor cells, a cell grade of 0.5+ indicates 1-5 cells, a cell grade of 1+ indicates 6-15 cells, a cell grade of 2+ indicates 16-25 cells, a cell grade of 3+ indicates 26-50 cells, and a cell grade of 4+ indicates >50 cells. Regarding vitreous cells, cell grade 0.5+ indicates 1-10 cells, cell grade 1+ indicates 11-20 cells, cell grade 2+ indicates 21-30 cells, cell grade 3+ indicates 31-100 cells, and cell grade 4+ indicates >100 cells. Rare cells were captured as cell grade 0.5+.

[0052] [Figure 9A] Figures 9A-9B show optical coherence tomography (OCT) images and retinal thickness maps derived from the OCT images acquired at a median follow-up time of 44 weeks for subjects 1-6 of cohort 1 of the study described in Example 1. Changes from baseline in BCVA, ETDRS letter size, and CST are also presented. The actual week in which the OCT image and retinal thickness map were obtained for each subject is indicated. An asterisk indicates that subject 4 underwent retinal detachment repair using a gas bubble injection to repair spontaneous pseudophakic macular detachment rhegmatogenous retinal detachment (RRD) (Figure 9B). Subject 4 is still under follow-up; the OCT image and BCVA value for subject 4 correspond to the last observation before retinal detachment. [Figure 9B] Same as above.

[0053] [Figure 10] Figure 10 shows mean best-corrected visual acuity (BCVA) measurements based on the Early Treatment Diabetic Retinopathy Study (ETDRS) letter rating at the indicated time points for subjects in Cohort 2 of the study described in Examples 2 and 3. Error bars indicate 90% confidence intervals for the mean absolute BCVA calculated using a T-distribution. Baseline (BL) indicates measurements obtained prior to the aflibercept injection at screening, 7 to 15 days (e.g., 7 to 14 days) prior to treatment with AAV2.7m8-aflibercept on Day 1. At 24 weeks after treatment with AAV2.7m8-aflibercept, subjects demonstrated a mean change in BCVA of -4.8 letters. BL = baseline; D = day; W = week. The Day 1 visit occurred 7 to 15 days after the baseline visit.

[0054] [Figure 11] Figure 11 shows the mean central retinal thickness (CST) at the indicated time points for subjects in Cohort 2 of the study described in Examples 2 and 3. Error bars represent the 90% confidence intervals of the mean absolute CST calculated using the T-distribution. Baseline (BL) indicates measurements obtained prior to aflibercept injection at screening, 7 to 15 days (e.g., 7 to 14 days) prior to treatment with AAV2.7m8-aflibercept on Day 1. At 24 weeks after treatment with AAV2.7m8-aflibercept, subjects demonstrated a mean change in CST of -27.8 μm. BL = baseline; D = day; W = week. The Day 1 visit occurred 7 to 15 days after the baseline visit.

[0055] [Figure 12]Figure 12 shows plots of aqueous humor cell counts and vitreous cell counts after treatment with AAV2.7m8-aflibercept for subjects in cohort 2 of the study described in Examples 2 and 3 up to a 24-week follow-up period. The steroid treatment administered to each subject is shown below each plot; the frequency of topical steroid (difluprednate) eye drops administration is shown (e.g., 1x = once per day; 2x = twice per day; 3x = three times per day; 4x = four times per day). Aqueous humor cell grade categories were based on the Standardization of Uveitis Nomenclature (SUN) criteria (Jabs, DA et al. J Ophthalmol. 2005; 140: 509-516). Vitreous cell grade categories were based on the National Institutes of Health (NIH) guidelines. For aqueous humor cells, a cell grade of 0.5+ indicates 1-5 cells, a cell grade of 1+ indicates 6-15 cells, a cell grade of 2+ indicates 16-25 cells, a cell grade of 3+ indicates 26-50 cells, and a cell grade of 4+ indicates >50 cells. For vitreous cells, a cell grade of 0.5+ indicates 1-10 cells, a cell grade of 1+ indicates 11-20 cells, a cell grade of 2+ indicates 21-30 cells, a cell grade of 3+ indicates 31-100 cells, and a cell grade of 4+ indicates >100 cells. For this analysis, rare cells were captured as a cell grade of 0.5+.

[0056] [Figure 13A]Figures 13A-13B show optical coherence tomography (OCT) images and retinal thickness maps derived from the OCT images obtained from subjects 1-6 of cohort 2 of the study described in Examples 2 and 3, up to a 24-week follow-up period. The change from baseline in BCVA, change from baseline in CST, and the number of rescue anti-VEGF injections administered during the 24-week follow-up period are also presented. In Figure 13A, the asterisk indicates that subject 3 received three rescue anti-VEGF injections, including at week 24, due to a ≥10-letter decrease in BCVA from baseline, all of which were attributed to intraretinal or subretinal fluid. In Figure 13B, the asterisk indicates that subject 5 received three rescue anti-VEGF injections due to an increase in central retinal thickness of >75 μm from baseline, with the final injection administered at week 20. [Figure 13B] Same as above.

[0057] [Figure 14] Figure 14 is a Swimmer's Lane Plot showing the number of anti-VEGF injections for subjects in Cohorts 1 and 2 of the study described in Examples 1-3. The x-axis represents time points in weeks relative to the time point at which AAV2.7m8-aflibercept was administered. The y-axis represents individual subjects in Cohorts 1 and 2. Circles represent anti-VEGF IVT injections administered before and after treatment with AAV2.7m8-aflibercept. The asterisk indicates that Subject 6 in Cohort 2 was diagnosed with nAMD 6.4 months before administration of AAV2.7m8-aflibercept. The vertical line bisecting the plot indicates Day 1, the time at which AAV2.7m8-aflibercept was administered. To the right of the bisecting vertical line are each of the 61 subsequent study visits.

[0058] [Figure 15A]Figures 15A-15C show plots of aqueous humor and vitreous cell counts measured by slit-lamp examination after treatment with AAV2.7m8-aflibercept for subjects in Cohorts 1, 2, and 3 of the study described in Examples 1-5. Figure 15A shows plots of aqueous humor and vitreous cell counts for subjects in Cohort 1 up to a 64-week follow-up time (median = 60 weeks; range = 52-64 weeks). Figure 15B shows plots of aqueous humor and vitreous cell counts for subjects in Cohort 2 up to a 40-week follow-up time (median = 36 weeks; range = 32-40 weeks). Figure 15C shows plots of aqueous humor and vitreous cell counts for subjects in Cohort 3 up to a 20-week follow-up time. In Figures 15A-15C, the steroid treatment administered to each subject is shown below each plot; aqueous humor cell grade categories were based on the Standardization of Uveitis Nomenclature (SUN) criteria: Jabs DA, et al. J Ophthalmol 2005; 140: 509-516; vitreous cell grade categories were based on the National Institutes of Health (NIH) guidelines; aqueous humor cells: 0.5+ = 1-5 cells; 1+ = 6-15 cells; 2+ = 16-25 cells; 3+ = 26-50 cells; 4+ = > 50 cells; vitreous cells: 0.5+ = 1-10 cells; 1+ = 11-20 cells; 2+ = 21-30 cells; 3+ = 31-100 cells; 4+ = > 100 cells; for this analysis, rare cells were captured as 0.5+. QID = 4 times per day; TID = 3 times per day; BID = 2 times per day; QD = 1 time per day; QOD = 1 time per 2 days; "QD>>" indicates that the subject is continuing to receive topical steroids once per day, "BID>>" indicates that the subject is continuing to receive topical steroids twice per day, and "QID>>" indicates that the subject is continuing to receive topical steroids 4 times per day. [Figure 15B] Same as above. [Figure 15C] Same as above.

[0059] [Figure 16A] Figures 16A-16B present the mean BCVA and mean CST from baseline to week 52 for subjects (n=6) in Cohort 1 of the study described in Examples 1-5. Figure 16A shows the mean BCVA (ETDRS letters) from baseline to week 52. The asterisk indicates one subject had low BCVA scores at weeks 44 and 48 due to retinal detachment. Figure 16B shows the mean CST (μm) from baseline to week 52. The asterisk indicates one subject had no CST data at weeks 44 and 48 due to retinal detachment. In Figures 16A-16B, error bars indicate 90% confidence intervals for mean absolute BCVA and CST values using a T-distribution; BL = baseline; D = day; W = week. [Figure 16B] Same as above.

[0060] [Figure 17A] Figures 17A-17B present the mean BCVA and mean CST from baseline to week 36 for subjects (n=6) in Cohort 2 of the study described in Examples 1-5. Figure 17A shows the mean BCVA (ETDRS letters) from baseline to week 36. Figure 17B shows the mean CST (μm) from baseline to week 36. In Figures 17A-17B, error bars indicate 90% confidence intervals for mean absolute BCVA and CST values using a T-distribution; BL=baseline; D=day; W=week; one subject missed the week 36 visit. [Figure 17B] Same as above.

[0061] [Figure 18A]Figures 18A-18B present the mean BCVA and mean CST from baseline to week 20 for five subjects in Cohort 3 of the study described in Examples 1-5. Figure 18A shows the mean BCVA (ETDRS letters) from baseline to week 20. Figure 18B shows the mean CST (μm) from baseline to week 20. In Figures 18A-18B, error bars indicate 90% confidence intervals for mean absolute BCVA and CST values using a T-distribution; BL = baseline; D = day; W = week; one subject missed the week 36 visit. [Figure 18B] Same as above.

[0062] [Figure 19] 19 is a Swimmer's Lane Plot showing the number of anti-VEGF injections administered to subjects in Cohorts 1, 2, and 3 of the study described in Examples 1-5. The x-axis represents time points in weeks relative to the time points at which AAV2.7m8-aflibercept was administered. The y-axis represents individual subjects in Cohorts 1-3. Circles represent anti-VEGF IVT injections administered before and after treatment with AAV2.7m8-aflibercept. The vertical line bisecting the plot indicates Day 1, the time at which AAV2.7m8-aflibercept was administered. To the right of the bisecting vertical line are each of the subsequent study visits.

[0063] [Figure 20A]Figures 20A-20B show optical coherence tomography (OCT) images and retinal thickness maps derived from the OCT images obtained from subject 4 of cohort 1 of the study described in Examples 1-5 at the indicated time points before and after administration of AAV2.7m8-aflibercept. Figure 20A shows OCT images and retinal thickness maps for subject 4 of cohort 1 at the indicated time points (weeks) before administration of AAV2.7m8-aflibercept while the subject was receiving an anti-VEGF IVT injection (ranibizumab). Figure 20B shows OCT images and retinal thickness maps for subject 4 of cohort 1 at the indicated time points (weeks) before and after administration of AAV2.7m8-aflibercept. The times of administration of the anti-VEGF IVT injection at screening ("anti-VEGF IVT at screening") and administration of AAV2.7m8-aflibercept ("AAV2.7m8-aflibercept") are shown. BCVA (ETDRS letter) and CST (μm) are shown at the indicated times before and after administration of AAV2.7m8-aflibercept. An asterisk indicates that the subject had a retinal detachment event unrelated to AAV2.7m8-aflibercept. [Figure 20B] Same as above.

[0064] [Figure 21A]Figures 21A-21B show optical coherence tomography (OCT) images and retinal thickness maps derived from the OCT images obtained from subject 5 of cohort 3 of the study described in Examples 1-5 at the indicated time points before and after administration of AAV2.7m8-aflibercept. Figure 21A shows OCT images and retinal thickness maps for subject 5 of cohort 3 at the indicated time points (weeks) before administration of AAV2.7m8-aflibercept while the subject was receiving an anti-VEGF IVT injection (aflibercept). Figure 21B shows OCT images and retinal thickness maps for subject 5 of cohort 3 at the indicated time points (weeks) before and after administration of AAV2.7m8-aflibercept. The times of administration of the anti-VEGF IVT injection at screening ("anti-VEGF IVT at screening") and administration of AAV2.7m8-aflibercept ("AAV2.7m8-aflibercept") are shown. BCVA (ETDRS letters) and CST (μm) are presented at each indicated time point before and after administration of AAV2.7m8-aflibercept. [Figure 21B] Same as above.

[0065] [Figure 22A]Figures 22A-22D show plots of aqueous humor cell counts and vitreous cell counts measured by slit-lamp examination for subjects in cohorts 1, 2, 3, and 4 of the study described in Examples 1-5 and 7 after treatment with AAV2.7m8-aflibercept. Figure 22A shows plots of aqueous humor cell counts and vitreous cell counts for subjects in cohort 1 up to 80 weeks of follow-up. Figure 22B shows plots of aqueous humor cell counts and vitreous cell counts for subjects in cohort 2 up to 56 weeks of follow-up. Figure 22C shows plots of aqueous humor cell counts and vitreous cell counts for subjects in cohort 3 up to 36 weeks of follow-up. Figure 22D shows plots of aqueous humor cell counts and vitreous cell counts for subjects in cohort 4 up to 8 weeks of follow-up. In Figures 22A-22D, the steroid treatment administered to each subject is indicated below each plot; aqueous humor cell grade categories were based on the Standardization of Uveitis Nomenclature (SUN) criteria: Jabs DA, et al. J Ophthalmol 2005; 140: 509-516; vitreous cell grade categories were based on the National Institutes of Health (NIH) guidelines; aqueous humor cells: 0.5+ = 1-5 cells; 1+ = 6-15 cells; 2+ = 16-25 cells; 3+ = 26-50 cells; 4+ = > 50 cells; vitreous cells: 0.5+ = 1-10 cells; 1+ = 11-20 cells; 2+ = 21-30 cells; 3+ = 31-100 cells; 4+ = > 100 cells; for this analysis, rare cells were captured as 0.5+. QID = 4 times per day; TID = 3 times per day; BID = 2 times per day; QD = 1 time per day; QOD = 1 time per 2 days; "QD>>" indicates that the subject is continuing to receive topical steroids once per day, "BID>>" indicates that the subject is continuing to receive topical steroids twice per day, "TID>>" indicates that the subject is continuing to receive topical steroids 3 times per day, and "QID>>" indicates that the subject is continuing to receive topical steroids 4 times per day. [Figure 22B] Same as above. [Figure 22C] Same as above. [Figure 22D] Same as above.

[0066] [Figure 23A] Figures 23A-23B present the mean BCVA and mean CST from baseline to week 72 for subjects (n=6) in Cohort 1 of the study described in Examples 1-5 and 7. Figure 23A shows the mean BCVA (ETDRS letters) from baseline to week 72. One subject had low BCVA scores at weeks 44 and 48 due to retinal detachment. *n=5 from weeks 56 to 72. Figure 23B shows the mean CST (μm) from baseline to week 72. One subject did not have CST data at weeks 44 and 48 due to retinal detachment. *n=5 from weeks 56 to 72. In Figures 23A-23B, error bars indicate 90% confidence intervals for mean absolute BCVA and CST values using a T-distribution; BL=baseline; D=day; W=week. [Figure 23B] Same as above.

[0067] [Figure 24A] Figures 24A-24B present the mean BCVA and mean CST from baseline to week 52 for subjects (n=6) in Cohort 2 of the study described in Examples 1-5 and 7. Figure 24A shows the mean BCVA (ETDRS letters) from baseline to week 52. *n=5 for weeks 36 and 40. Figure 24B shows the mean CST (μm) from baseline to week 52. *n=5 for weeks 36 and 40. In Figures 24A-24B, error bars indicate 90% confidence intervals for mean absolute BCVA and CST values using the T-distribution; BL=baseline; D=day; W=week. [Figure 24B] Same as above.

[0068] [Figure 25A]Figures 25A-25B present the mean BCVA and mean CST from baseline to week 20 for subjects (n=9) in Cohort 3 of the study described in Examples 1-5 and 7. Figure 25A shows the mean BCVA (ETDRS letters) from baseline to week 20. *n=8 for weeks 4, 16, and 20. Figure 25B shows the mean CST (μm) from baseline to week 20. *n=8 for weeks 4, 16, and 20. In Figures 25A-25B, error bars indicate 90% confidence intervals for mean absolute BCVA and CST values using a T-distribution; BL=baseline; D=day; W=week. [Figure 25B] Same as above.

[0069] [Figure 26] Figure 26 is a Swimmer's Lane Plot showing the number of anti-VEGF injections administered to subjects in Cohorts 1, 2, 3, and 4 of the study described in Examples 1-5 and 7. The x-axis represents time point in weeks relative to the time of AAV2.7m8-aflibercept administration. The y-axis represents individual subjects in Cohorts 1-4. Circles represent anti-VEGF IVT injections administered before and after treatment with AAV2.7m8-aflibercept. The vertical line bisecting the plot indicates Day 1, when AAV2.7m8-aflibercept was administered. To the right of the bisecting vertical line are each of the subsequent study visits. Five subjects were diagnosed <1 year prior to the AAV2.7m8-aflibercept injection (one subject each in Cohorts 2 and 3, and three subjects in Cohort 4). In cohort 4, data prior to day 1 are incomplete for subject 2 due to emigration and for subject 5 due to participation in a clinical trial with an unknown drug after diagnosis.

[0070] [Figure 27A]Figures 27A-27B show the mean annual anti-VEGF injection rates for subjects in Cohorts 1-3 of the study described in Examples 1-5 and 7. Figure 27A presents a comparison of the mean annual anti-VEGF injection rates for subjects in Cohort 1 (received the "high dose" of 6x10vg / eye; n=6) and for subjects in Cohorts 2 and 3 (received the "low dose" of 2x10vg / eye; n=15). Figure 27B presents a comparison of the mean annual anti-VEGF injection rates for subjects in Cohorts 1 (n=6), 2 (n=6), and 3 (n=9), respectively. In Figures 27A-27B, annualized rate (before) = (number of anti-VEGF IVT injections in the 12 months prior to AAV2.7m8-aflibercept) / (number of days from first anti-VEGF IVT injection in the past 12 months to time of AAV2.7m8-aflibercept injection / 365.25); and annualized rate (after) = (number of anti-VEGF IVT injections from AAV2.7m8-aflibercept) / (number of days from AAV2.7m8-aflibercept to last study follow-up / 365.25). [Figure 27B] Same as above.

[0071] [Figure 28A] Figures 28A-28B show optical coherence tomography (OCT) images and retinal thickness maps derived from the OCT images obtained at the indicated time points before and after administration of AAV2.7m8-aflibercept from subject 5 of cohort 3 of the study described in Examples 1-5 and 7. Figure 28A shows OCT images and retinal thickness maps for subject 5 of cohort 3 at the indicated time points (weeks) before administration of AAV2.7m8-aflibercept while the subject was receiving an anti-VEGF IVT injection (aflibercept). Figure 28B shows OCT images and retinal thickness maps for subject 5 of cohort 3 at the indicated time points (weeks) before and after administration of AAV2.7m8-aflibercept. The number of administrations of anti-VEGF IVT injections ("aflibercept IVT") and AAV2.7m8-aflibercept at screening is shown. BCVA (ETDRS letters) and CST (μm) are presented at each indicated time point before and after administration of AAV2.7m8-aflibercept. [Figure 28B] Same as above.

[0072] [Figure 29] Figure 29 shows a diagram of the study design described in Example 6 evaluating the durability of a single intravitreal (IVT) injection of AAV2.7m8-aflibercept in subjects with diabetic macular edema (DME). DRSS = Diabetic Retinopathy Severity Score; OCT = Optical Coherence Tomography; CST = Central Space Retinal Thickness. *All subjects received a 7-week course of difluprednate eye drops, starting QID and tapering to QD. **PE = Primary Endpoint Evaluation. ***EOS = End of Study Evaluation. DETAILED DESCRIPTION OF THE INVENTION

[0073] Detailed Description Some aspects are described below with reference to example applications for illustration. It should be understood that numerous specific details, relationships, and methods are described to provide a thorough understanding of the features described herein. However, those skilled in the relevant art will readily recognize that the features described herein can be practiced without one or more of the specific details or by other methods. The features described herein are not limited to the illustrated order of acts or events, as some acts may occur in a different order and / or simultaneously with other acts or events. Furthermore, not all illustrated acts or events are necessarily required to implement a methodology in accordance with the features described herein. definition

[0074] Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0075] The terms used herein are for the purpose of describing particular examples only and are not intended to be limiting. As used herein, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly dictates otherwise. Furthermore, when the terms "including," "includes," "having," "has," "with," or variations thereof are used in either the detailed description and / or claims, such terms are intended to be inclusive in a manner similar to the term "comprising." The term "comprising," as used herein, is synonymous with "including" or "containing" and is inclusive or open-ended.

[0076] As used herein, the reference to "or" is intended to encompass "and / or" unless otherwise indicated. As used herein, the term "about" a number refers to that number plus or minus 10%. Reference to "about" a value or parameter herein includes (and describes) embodiments that are directed to that value or parameter itself.

[0077] The terms "subject," "patient," or "individual" refer to primates, e.g., humans and non-human primates, e.g., African green monkeys and rhesus monkeys. In some embodiments, the subject is a human.

[0078] The terms "treat," "treating," "treatment," "improve," or "improving," and other grammatical equivalents, as used herein, refer to alleviating, attenuating, or improving an intraocular neovascular disease or disorder, or the symptoms of an intraocular neovascular disease or disorder, preventing additional symptoms of an intraocular neovascular disease or disorder, improving or preventing the underlying metabolic cause of the symptoms, inhibiting an intraocular neovascular disease or disorder, e.g., halting the development of an intraocular neovascular disease or disorder, alleviating an intraocular neovascular disease or disorder, causing regression of an intraocular neovascular disease or disorder, or halting the symptoms of an intraocular neovascular disease or disorder, and are intended to include prevention. The term further includes achieving a therapeutic benefit and / or a prophylactic benefit. The term "therapeutic benefit" refers to the eradication or amelioration of the intraocular neovascular disease or disorder being treated. The term "therapeutic benefit" refers to the eradication or amelioration of the intraocular neovascular disease or disorder being treated. Similarly, a therapeutic benefit can also be achieved in some embodiments by eradicating or ameliorating one or more physiological symptoms associated with an intraocular neovascular disease or disorder, such that an improvement is observed in a subject even though the subject still suffers from the intraocular neovascular disease or disorder. For a preventative benefit, the pharmaceutical composition is administered to a subject at risk of developing an intraocular neovascular disease or disorder, or to a subject who reports one or more physiological symptoms of an intraocular neovascular disease or disorder, even if the disease or disorder has not been diagnosed.

[0079] The terms "administer," "administering," "administration," and the like, as used herein, may refer to methods used to enable delivery of a therapeutic agent or pharmaceutical composition to a desired site of biological effect. These methods include intravitreal or subretinal injection into the eye.

[0080] The terms "effective amount," "therapeutically effective amount," or "pharmaceutically effective amount," as used herein, may refer to a sufficient quantity of at least one pharmaceutical composition or compound being administered to relieve to some extent one or more symptoms of the ocular disease or disorder being treated. An "effective amount," "therapeutically effective amount," or "pharmaceutically effective amount" of a pharmaceutical composition may be administered as a unit dose to a subject in need thereof (described in more detail elsewhere herein).

[0081] The term "pharmaceutically acceptable," as used herein, can refer to a material, such as a carrier or diluent, that does not eliminate the biological activity or properties of the compounds disclosed herein and that is relatively non-toxic (i.e., does not cause undesired biological effects when the material is administered to an individual and does not adversely interact with any of the components of the composition in which it is contained).

[0082] The term "pharmaceutical composition" or simply "composition," as used herein, may refer to a biologically active compound optionally mixed with at least one pharmaceutically acceptable chemical component such as, but not limited to, a carrier, stabilizer, diluent, dispersant, suspending agent, thickener, excipient, and the like.

[0083] As used herein, "AAV vector" or "rAAV vector" refers to an adeno-associated virus (AAV) vector or recombinant AAV (rAAV) vector that contains a polynucleotide sequence that is not of AAV origin (e.g., a polynucleotide heterologous to AAV, such as a nucleic acid sequence encoding a therapeutic transgene, e.g., aflibercept) for transduction into target cells or target tissues. Generally, the heterologous polynucleotide is flanked by at least one AAV inverted terminal repeat (ITR), and generally has two AAV inverted terminal repeat (ITR) on both ends. The term rAAV vector encompasses both rAAV vector particles and rAAV vector plasmids. rAAV vectors can be either single-stranded (ssAAV) or self-complementary (scAAV).

[0084] "AAV virus" or "AAV virus particle" or "rAAV vector particle" or "rAAV particle" refers to a virus particle that contains at least one AAV capsid protein and a polynucleotide rAAV vector.In some cases, at least one AAV capsid protein is derived from wild-type AAV or is a variant AAV capsid protein (for example, an AAV capsid protein with an insertion, such as the insertion of the 7m8 amino acid sequence described below).When a particle contains a heterologous polynucleotide (for example, a polynucleotide other than the wild-type AAV genome, such as a transgene that is delivered to target cells or target tissues), it is referred to as "rAAV particle," "rAAV vector particle," or "rAAV vector."In this way, the production of rAAV particles necessarily includes the production of rAAV vectors, since such vectors are contained within rAAV particles.

[0085] The term "packaging," as used herein, may refer to a series of intracellular events that may result in the assembly and encapsidation of rAAV particles.

[0086] AAV "rep" and "cap" genes refer to polynucleotide sequences that encode the replication and encapsidation proteins of adeno-associated virus. AAV rep and cap are referred to herein as AAV "packaging genes."

[0087] The term "polypeptide" can encompass both naturally occurring and non-naturally occurring proteins (e.g., fusion proteins), peptides, fragments, mutants, derivatives, and analogs thereof. A polypeptide can be a monomer, dimer, trimer, or polymer. Furthermore, a polypeptide can contain several different domains, each of which has one or more distinct activities. For the avoidance of doubt, a "polypeptide" can be of any length greater than two amino acids.

[0088] As used herein, "polypeptide variant" or simply "variant" refers to a polypeptide whose sequence contains amino acid modifications. In some embodiments, the modification is an insertion, duplication, deletion, rearrangement, or substitution of one or more amino acids compared to the amino acid sequence of a reference protein or polypeptide, such as a native or wild-type protein. A variant may have one or more amino acid point substitutions, where a single amino acid at a position is changed to another amino acid, one or more insertions and / or deletions, where one or more amino acids are inserted or deleted, respectively, in the sequence of the reference protein, and / or truncation of the amino acid sequence at either or both the amino or carboxy terminus. A variant may have the same or different biological activity compared to a reference protein or an unmodified protein.

[0089] In some embodiments, a variant may have, for example, at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% overall sequence homology with its counterpart reference protein. In some embodiments, a variant may have at least about 90% overall sequence homology with the wild-type protein. In some embodiments, a variant exhibits at least about 95%, at least about 98%, at least about 99%, at least about 99.5%, or at least about 99.9% overall sequence identity.

[0090] As used herein, "recombinant" can refer to a biological molecule, e.g., a gene or protein, that (1) has been removed from its naturally occurring environment, (2) is not associated with all or part of a polynucleotide in which it is found in nature, (3) is operably linked to a polynucleotide to which it is not linked in nature, or (4) is not naturally occurring. The term "recombinant" can be used in reference to cloned DNA isolates, chemically synthesized polynucleotide analogs, or polynucleotide analogs biologically synthesized by heterologous systems, as well as proteins and / or mRNAs encoded by such nucleic acids. Thus, for example, a protein synthesized by a microorganism is recombinant if, for example, it is synthesized from mRNA synthesized from a recombinant gene present in the cell.

[0091] The term "anti-VEGF agent" includes any therapeutic agent, including a protein, polypeptide, peptide, fusion protein, multimeric protein, gene product, antibody, human monoclonal antibody, antibody fragment, aptamer, small molecule, kinase inhibitor, receptor or receptor fragment, or nucleic acid molecule, that can reduce, interfere with, disrupt, block, and / or inhibit the activity or function of endogenous VEGF and / or endogenous VEGF receptor (VEGFR), or VEGF-VEGFR interaction or pathway in vivo. The anti-VEGF agent can be any one of known therapeutic agents that can reduce the growth or formation of new blood vessels and / or edema or swelling when delivered to a cell, tissue, or subject in vivo, such as ranibizumab, brolucizumab, or bevacizumab. In some embodiments, the anti-VEGF agent can be naturally occurring, non-naturally occurring, or synthetic. In some embodiments, the anti-VEGF agent can be derived from a naturally occurring molecule that has subsequently been modified or mutated to confer anti-VEGF activity. In some embodiments, the anti-VEGF agent is a fusion protein or chimeric protein. In such proteins, functional domains or polypeptides are artificially fused to moieties or polypeptides to create fusion or chimeric proteins that can sequester VEGF in vivo or function as VEGFR decoys. In some embodiments, the anti-VEGF agent is a fusion or chimeric protein that blocks endogenous VEGFR from interacting with its ligands.

[0092] As used herein, "VEGF," unless otherwise required, may refer to any isoform of VEGF, including, but not limited to, VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E, VEGF-F, or any combination thereof, or any functional fragment or variant thereof. Unless otherwise required, "VEGF" may refer to any member of the VEGF family, including the members: VEGF-A, placental growth factor (PGF), VEGF-B, VEGF-C, and VEGF-D, or any combination, functional fragment, or variant thereof. As used herein, "VEGF receptor" or "VEGFR" or "VEGF-R" may be used to refer to any one of the receptors for VEGF, including, but not limited to, VEGFR-1 (or Flt-1), VEGFR-2 (or Flk-1 / KDR), and VEGFR-3 (or Flt-4). VEGFRs may be membrane-bound or soluble, or functional fragments or truncated forms of the receptor. Examples of anti-VEGF agents include, but are not limited to, ranibizumab, bevacizumab, brolucizumab, or any combination, variant, or functional fragment thereof.

[0093] "Operably linked" or "operably linked" or "coupled" can refer to the juxtaposition of genetic elements in a relationship permitting them to operate in their expected manner. For example, a promoter can be operably linked to a coding region if the promoter helps initiate transcription of the coding sequence. Intervening residues can be present between the promoter and the coding region so long as this functional relationship is maintained.

[0094] The terms "expression vector" or "expression construct" or "cassette" or "plasmid" or simply "vector" can include any type of gene product, including AAV or rAAV vectors containing nucleic acids or polynucleotides encoding gene products in which some or all of the nucleic acid encoding sequence is transcribable and compatible with gene therapy. The transcript can be translated into protein. In some embodiments, the transcript is partially translated or not translated. In certain aspects, expression includes both transcription of the gene and translation of mRNA into a gene product. In other aspects, expression alone includes transcription of the nucleic acid encoding the gene of interest. The expression vector can also include control elements operably linked to the coding region to facilitate expression of the protein in the target cell. The combination of control elements and the gene or genes to which they are operably linked for expression can sometimes be referred to as an "expression cassette," many of which are known and available in the art or can be readily constructed from components available in the art.

[0095] The term "heterologous" can refer to an entity that differs in genotype from the rest of the entity to which it is compared. For example, a polynucleotide introduced into a plasmid or vector from a different species by genetic engineering techniques can be a heterologous polynucleotide. A promoter removed from its native coding sequence and operably linked to a coding sequence with which it is not naturally found can be a heterologous promoter.

[0096] As used herein, "7m8" refers to the amino acid sequence LALGETTRPA (SEQ ID NO: 1).

[0097] "7m8 variant" refers to an rAAV, which may be of any serotype, in which the amino acid sequence LALGETTRPA (SEQ ID NO: 1) is inserted into the solvent-exposed GH loop of the capsid protein.

[0098] When 7m8 is inserted into rAAV2 (also referred to as AAV2.7m8), the amino acid sequence LALGETTRPA (SEQ ID NO: 1) is inserted into the GH loop within amino acids 570-611 of the AAV2 capsid protein, for example, between positions 587 and 588 of the AAV2 capsid protein VP1. In some examples, when 7m8 is inserted into rAAV2 (also referred to as AAV2.7m8), the amino acid sequence LALGETTRPA (SEQ ID NO: 1) is inserted into the GH loop of the AAV2 capsid protein, for example, between positions 587 and 588 of the AAV2 VP1 comprising the sequence of SEQ ID NO: 13. When 7m8 is inserted into rAAV1 (also referred to as AAV1.7m8), the amino acid sequence LALGETTRPA (SEQ ID NO: 1) is inserted into the GH loop within amino acids 571-612 of the AAV1 capsid protein, for example, between amino acids 590 and 591 of the AAV1 capsid protein. When 7m8 is inserted into rAAV5 (also referred to as AAV5.7m8), the amino acid sequence LALGETTRPA (SEQ ID NO: 1) is inserted into the GH loop within amino acids 560-601 of the AAV5 capsid protein, e.g., between amino acids 575 and 576 of the AAV5 capsid protein. When 7m8 is inserted into rAAV6 (also referred to as AAV6.7m8), the amino acid sequence LALGETTRPA (SEQ ID NO: 1) is inserted into the GH loop within amino acids 571-612 of the AAV6 capsid protein, e.g., between amino acids 590 and 591 of the AAV6 capsid protein. When 7m8 is inserted into rAAV7 (also referred to as AAV7.7m8), the amino acid sequence LALGETTRPA (SEQ ID NO: 1) is inserted into the GH loop within amino acids 572-613 of the AAV7 capsid protein, e.g., between amino acids 589 and 590 of the AAV7 capsid protein. When 7m8 is inserted into rAAV8 (also referred to as AAV8.7m8), the amino acid sequence LALGETTRPA (SEQ ID NO: 1) is inserted into the GH loop within amino acids 573 to 614 of the AAV8 capsid protein, for example, between amino acids 590 and 591 of the AAV8 capsid protein.When 7m8 is inserted into rAAV9 (also referred to as AAV9.7m8), the amino acid sequence LALGETTRPA (SEQ ID NO: 1) is inserted into the GH loop of the AAV9 capsid protein, for example, between amino acids 588 and 589 of the AAV9 capsid protein. When 7m8 is inserted into rAAV10 (also referred to as AAV10.7m8), the amino acid sequence LALGETTRPA (SEQ ID NO: 1) is inserted into the GH loop within amino acids 573-614 of the AAV10 capsid protein, for example, between amino acids 589 and 590 of the AAV10 capsid protein. overview

[0099] Current treatments for intraocular neovascular diseases, such as wAMD (e.g., aflibercept recombinant protein, ranibizumab recombinant protein), require lifelong IVT administration approximately every 4–8 weeks. This can increase the risk of inflammation, infection, and other adverse effects in some patients. Furthermore, current treatments have compliance issues due to repeated and / or frequent trips to the clinic for treatment, especially in elderly patients, who are most commonly affected by wAMD. Reduced frequency of administration is associated with vision loss and worsening of ocular diseases or conditions. The ability of AAV vectors to efficiently transduce target retinal cells after IVT injection has been successfully utilized to transfer therapeutic genes to photoreceptors, retinal pigment epithelium, and the inner retina to treat various retinal diseases. Thus, administration of rAAV particles encoding anti-VEGF agents (e.g., aflibercept) can provide long-term and / or sustained release of the anti-VEGF agent in vivo.

[0100] Unexpectedly, 6 × 10 of aflibercept-encoding rAAV particles 11 Administration of a single low dose of vector genome (vg) / eye to the eyes of individuals with intraocular neovascular disease resulted in disease stabilization and robust anatomical responses in all treated individuals (see Example 1). In addition, administration of 6 x 10 rAAV particles encoding aflibercept 11After administration of a single low-dose of 1000 mg / eye, vision stabilized in all treated individuals, and none required rescue anti-VEGF treatment (e.g., IVT injection of aflibercept). Moreover, administration of a single unit dose of rAAV particles encoding aflibercept to the eyes of individuals with intraocular neovascular disease unexpectedly resulted in a reduction (e.g., resolution) of symptoms, including intraretinal and subretinal fluid, that were refractory to previous anti-VEGF treatment (e.g., chronic IVT injection of aflibercept, ranibizumab, or bevacizumab).

[0101] Thus, the present disclosure provides a method for the production of 6×10 rAAV particles encoding an anti-VEGF agent (e.g., aflibercept). 11 The present disclosure provides a method for treating intraocular neovascular disease in an individual by administering a single unit dose of 1000 mg / eye or less.In addition, the present disclosure provides a method for reducing retinal fluid in the eye of an individual with intraocular neovascular disease by administering a single unit dose of rAAV particles encoding an anti-VEGF agent (e.g., aflibercept).The method disclosed herein reduces or eliminates the need for repeated IVT injections, providing long-term efficacy while addressing the problems of non-compliance and non-adherence.In addition, the method provided herein reduces the adverse effects associated with multiple IVT injections. Treatment method

[0102] Provided herein are methods for treating an intraocular neovascular disease in an individual, the method comprising administering a unit dose of recombinant adeno-associated virus (rAAV) particles to the eye of the individual.

[0103] Also provided herein is a method for reducing retinal fluid in the eye of an individual having an intraocular neovascular disease, the method comprising administering a unit dose of rAAV particles to the eye of the individual.

[0104] Also provided herein is a method for treating an intraocular neovascular disease in an individual, the method comprising administering an anti-VEGF agent (e.g., aflibercept) to the eye of the individual, and administering a unit dose of recombinant adeno-associated virus (rAAV) particles to the eye of the individual after administration of the anti-VEGF agent.

[0105] In some embodiments, the intraocular neovascular disease is exudative age-related macular degeneration (wAMD), retinal neovascularization, choroidal neovascularization, diabetic retinopathy, proliferative diabetic retinopathy, retinal vein occlusion, central retinal vein occlusion, branch retinal vein occlusion, diabetic macular edema, diabetic retinal ischemia, ischemic retinopathy, diabetic retinal edema, or any combination thereof.

[0106] In some embodiments, the term intraocular neovascular disease also encompasses VEGF-driven pre-neovascular diseases that progress to neovascular forms if left untreated, hi some embodiments, the intraocular neovascular disease is the pre-neovascular disease, non-proliferative diabetic retinopathy.

[0107] In some embodiments, the individual is human. In some embodiments, the individual has received at least one previous treatment (e.g., at least one, at least two, at least three, at least four, at least five, or more treatments) of an intraocular neovascular disease with an anti-VEGF agent (e.g., bevacizumab, brolucizumab, ranibizumab, faricimab, abicipar pegol, conbercept, OPT-302, KSI-301, injectable sunitinib maleate (GB-102), PAN-90806 (PanOptica), and / or aflibercept) within about the last 8 weeks, about the last 9 weeks, about the last 10 weeks, about the last 11 weeks, about the last 12 weeks, about the last 13 weeks, about the last 14 weeks, about the last 15 weeks, or about the last 16 weeks prior to administration of the unit dose of rAAV particles. In some embodiments, the individual has had a meaningful response to previous treatment with an anti-VEGF agent (e.g., bevacizumab, brolucizumab, ranibizumab, faricimab, abicipar pegol, conbercept, OPT-302, KSI-301, injectable sunitinib maleate (GB-102), PAN-90806 (PanOptica), and / or aflibercept). In some embodiments, the anti-VEGF agent is aflibercept, a functional variant thereof, or a functional fragment thereof. In some embodiments, the anti-VEGF agent comprises a polypeptide comprising an amino acid sequence having at least about 95% identity to the amino acid sequence of SEQ ID NO: 35. In some embodiments, the retinal fluid of the individual's eye is intraretinal fluid (IRF) and / or subretinal fluid (SRF). In some embodiments, the amount or presence of retinal fluid in the individual's eye is refractory to previous treatment with an anti-VEGF agent (e.g., bevacizumab, brolucizumab, ranibizumab, faricimab, abicipar pegol, conbercept, OPT-302, KSI-301, injectable sunitinib maleate (GB-102), PAN-90806 (PanOptica), and / or aflibercept). In some embodiments, the anti-VEGF agent is aflibercept, a functional variant thereof, or a functional fragment thereof.In some embodiments, the anti-VEGF agent comprises a polypeptide comprising an amino acid sequence having at least about 95% identity to the amino acid sequence of SEQ ID NO:35.

[0108] In some embodiments, the intraocular neovascular disease is diabetic macular edema (DME). In some embodiments, the individual is human. In some embodiments, the individual has type 1 or type 2 diabetes. In some embodiments, the individual has visual impairment due to diabetic macular edema involving the fovea. In some embodiments, the individual has a visual acuity (BCVA) of about 78 to 50 ETDRS characters (e.g., any of 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, or 78 ETDRS characters) for the eye receiving the rAAV particles prior to administration of the unit dose of rAAV particles. In some embodiments, the individual has a visual acuity (Snellen equivalent) of about 20 / 32 to about 20 / 100 for the eye receiving the rAAV particles prior to administration of the unit dose of rAAV particles. In some embodiments, the individual has a central retinal thickness (CST) of ≥ 325 μm using a Heidelberg Spectralis® for the eye receiving the rAAV particles prior to administration of the unit dose of rAAV particles, with an IRF spanning the fovea (1 mm central to the fovea). In some embodiments, the individual has a decrease in vision primarily due to diabetic macular edema for the eye receiving the rAAV particles prior to administration of the unit dose of rAAV particles. In some embodiments, the individual has been diagnosed with diabetic macular edema for about 6 months or less prior to administration of the unit dose of rAAV particles, e.g., about 6 months, about 5 months, about 4 months, about 3 months, about 2 months, about 1 month, or less prior to administration of the unit dose of rAAV particles. In some embodiments, the individual has received zero, one, or two previous treatments for DME, e.g., zero, one, or two intravitreal injections of an anti-VEGF agent, e.g., aflibercept, for the eye receiving the rAAV particles prior to administration of the unit dose of rAAV particles.In some embodiments, the individual has received previous treatment with an anti-VEGF agent for at least about 60 days (i.e., about 2 months) prior to administration of the unit dose of rAAV particles, with respect to the eye receiving the rAAV particles. In some embodiments, the eye receiving the rAAV particles has shown a meaningful response in terms of central zone retinal thickness to previous treatment with an anti-VEGF agent, e.g., at least a 10% reduction in central zone retinal thickness, prior to administration of the unit dose of rAAV particles. In some embodiments, the individual has not experienced an adverse reaction to previous treatment with an anti-VEGF agent prior to administration of the unit dose of rAAV particles. In some embodiments, the individual does not have neutralizing antibodies against AAV2.7m8 prior to administration of the unit dose of rAAV particles. In some embodiments, the individual does not have an anti-AAV2.7m8 neutralizing antibody titer of greater than 1:125 prior to administration of the unit dose of rAAV particles, e.g., within about 6 months prior to administration of the unit dose of rAAV particles. In some embodiments, the individual has no history of allergies to aflibercept, corticosteroids, or fluorescein dyes or sodium fluorescein (e.g., used in angiography) prior to administration of the unit dose of rAAV particles. In some embodiments, the individual has a history of mild allergies to aflibercept, corticosteroids, or fluorescein dyes or sodium fluorescein (e.g., used in angiography) prior to administration of the unit dose of rAAV particles, where the allergy is suitable for treatment. In some embodiments, the individual does not have uncontrolled diabetes, e.g., an HbA1c greater than 10%, prior to administration of the unit dose of rAAV particles. In some embodiments, the individual does not have a history of diabetic ketoacidosis within about three months prior to administration of the unit dose of rAAV particles. In some embodiments, the individual has not initiated intensive insulin treatment, e.g., using an insulin pump or multiple daily insulin injections, prior to administration of the unit dose of rAAV particles. In some embodiments, the individual does not plan to start intensive insulin treatment, for example, using an insulin pump or multiple daily insulin injections, within about three months after administration of the unit dose of rAAV particles.In some embodiments, the individual does not have a history of systemic autoimmune disease requiring systemic steroids or immunosuppressive treatment, such as methotrexate or adalimumab, prior to administration of the unit dose of rAAV particles. In some embodiments, the individual does not receive systemic drugs known to cause macular edema, such as fingolimod, tamoxifen, chloroquine, or hydroxychloroquine, prior to administration of the unit dose of rAAV particles. In some embodiments, the individual does not receive systemic anti-VEGF treatment prior to administration of the unit dose of rAAV particles. In some embodiments, the individual does not have a high risk of proliferative diabetic retinopathy (PDR) in the eye receiving the rAAV particles prior to administration of the unit dose of rAAV particles. In some embodiments, PDR is defined as any vitreous or preretinal hemorrhage, neovascularization elsewhere in >½ the papillary area within an area equivalent to a standard ETDRS 7 visual field on clinical examination, or neovascularization of the papillary area >⅓ the papillary area on clinical examination. In some embodiments, the individual has not undergone focal or grid laser photocoagulation with respect to the eye receiving the rAAV particles prior to administration of the unit dose of rAAV particles. In some embodiments, the individual has not undergone any previous panretinal photocoagulation (PRP) with respect to the eye receiving the rAAV particles prior to administration of the unit dose of rAAV particles. In some embodiments, the individual has not undergone anti-VEGF treatment (e.g., an IVT injection of aflibercept) with respect to the eye receiving the rAAV particles prior to administration of the unit dose of rAAV particles. In some embodiments, the individual has not undergone anti-VEGF treatment (e.g., an IVT injection of aflibercept) with respect to the eye receiving the rAAV particles for at least 60 days prior to administration of the unit dose of rAAV particles. In some embodiments, the individual has not undergone more than two anti-VEGF treatments (e.g., an IVT injection of aflibercept) with respect to the eye receiving the rAAV particles prior to administration of the unit dose of rAAV particles.In some embodiments, the individual does not have a history of anterior segment neovascularization (e.g., neovascularization of the iris [NVI] or neovascular glaucoma [NVG]), significant vitreous hemorrhage, fibrovascular tissue proliferation, or tractional retinal detachment in the eye receiving the rAAV particles prior to administration of the unit dose of rAAV particles. In some embodiments, the individual does not have a foveal structural abnormality (e.g., thick, hard exudate, pigmentary abnormalities, foveal atrophy, vitreomacular traction, or epiretinal membrane) that contributes to macular edema or visual impairment in the eye receiving the rAAV particles prior to administration of the unit dose of rAAV particles. In some embodiments, the foveal structural abnormality is assessed by clinical examination or OCT. In some embodiments, the individual has no history of retinal disease other than diabetic retinopathy (e.g., age-related macular degeneration (in either eye), retinal vein occlusion, retinal artery occlusion, or pathological myopia) with respect to the eye receiving the rAAV particles prior to administration of the unit dose of rAAV particles. In some embodiments, the individual has no history of ocular disease other than diabetic macular edema, e.g., a history of significant cataract or macular traction, or evidence of posterior subcapsular cataract, with respect to the eye receiving the rAAV particles prior to administration of the unit dose of rAAV particles. In some embodiments, the individual has no history of cataract extraction or yttrium aluminum garnet (YAG) capsulotomy with respect to the eye receiving the rAAV particles within at least about 3 months prior to administration of the unit dose of rAAV particles. In some embodiments, the individual has no history of retinal detachment (with or without repair) with respect to the eye receiving the rAAV particles prior to administration of the unit dose of rAAV particles. In some embodiments, the individual has no history of trabeculectomy, glaucoma shunting, or minimally invasive glaucoma surgery (MIGS) with respect to the eye receiving the rAAV particles prior to administration of the unit dose of rAAV particles. In some embodiments, the individual has no history of vitrectomy or other filtration surgery with respect to the eye receiving the rAAV particles prior to administration of the unit dose of rAAV particles. In some embodiments, the individual has no aphakia or the presence of an anterior chamber intraocular lens with respect to the eye receiving the rAAV particles prior to administration of the unit dose of rAAV particles.In some embodiments, the individual does not have uncontrolled ocular hypertension or glaucoma, e.g., an IOP > 22 mmHg despite treatment with an anti-glaucoma medication or current use of an IOP-lowering medication > 2, with respect to the eye receiving the rAAV particles prior to administration of the unit dose of rAAV particles. In some embodiments, the individual does not have a history of intraocular or periocular steroid treatment (e.g., IVT Triesence, Iluvien, or Ozurdex) for any ocular condition with respect to the eye receiving the rAAV particles prior to administration of the unit dose of rAAV particles. In some embodiments, the individual has not undergone refractive surgery with respect to the eye receiving the rAAV particles within at least about 90 days prior to administration of the unit dose of rAAV particles. In some embodiments, the individual does not have previous penetrating keratoplasty, endothelial keratoplasty, or ocular radiation with respect to the eye receiving the rAAV particles prior to administration of the unit dose of rAAV particles. In some embodiments, the individual has not undergone any previous vitreoretinal surgery with respect to the eye receiving the rAAV particles prior to administration of the unit dose of rAAV particles. In some embodiments, the individual has no history of uveitis or intraocular inflammation, e.g., evidence of or exceeding the expected grade of mild, resolved post-operative inflammation, prior to administration of the unit dose of rAAV particles. In some embodiments, the individual has no history of IOP elevation associated with topical steroid administration prior to administration of the unit dose of rAAV particles. In some embodiments, the individual has no history of ocular herpes simplex virus (HSV), varicella-zoster virus (VZV), or cytomegalovirus (CMV), including viral uveitis, retinitis, or keratitis, prior to administration of the unit dose of rAAV particles. In some embodiments, the individual has no evidence of any external ocular infection, including conjunctivitis, chalazion, or significant blepharitis, prior to administration of the unit dose of rAAV particles. In some embodiments, the individual has no history of ocular toxoplasmosis prior to administration of the unit dose of rAAV particles.

[0109] In some embodiments, the unit dose is expressed as the number of vector genomes (vg). In some embodiments, the unit dose is about 6 x 10 of rAAV particles.11 In some embodiments, the unit dose is expressed as vector genomes (vg) / eye (vg / eye). In some embodiments, the unit dose is about 6 x 10 of rAAV particles. 11 In some embodiments, the unit dose of rAAV particles is about 6 x 10 vg / eye or less. 10 ~Approx. 2×10 11 In some embodiments, the unit dose of rAAV particles is about 2 x 10 vg / eye. 11 or about 6 x 10 10 vg / eye.

[0110] In some embodiments, a unit dose of rAAV particles is administered to one eye of an individual. In some embodiments, the one eye of the individual is the right eye or the left eye. In some embodiments, the one eye of the individual is the right eye. In some embodiments, the one eye of the individual is the left eye. In some embodiments, the methods provided herein further comprise administering a unit dose of rAAV particles to the other eye of the individual. In some embodiments, the one eye of the individual is the right eye and the other eye is the left eye. In some embodiments, the one eye of the individual is the left eye and the other eye is the right eye.

[0111] In some embodiments, the step of administering a unit dose of rAAV particles to the contralateral eye of the individual occurs up to about two weeks (e.g., about day 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14) after the step of administering a unit dose of rAAV particles to one eye. In some embodiments, the unit dose of rAAV particles administered to the contralateral eye of the individual is about the same (e.g., less than 1% higher or lower, less than 5% higher or lower, less than 10% higher or lower, or less than 20% higher or lower) or lower (e.g., about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, or about 90% lower) as the unit dose of rAAV particles administered to one eye of the individual.

[0112] In some embodiments, the step of administering a unit dose of rAAV particles to the contralateral eye occurs at least about 2 weeks (e.g., at least about 2 weeks, at least about 3 weeks, at least about 4 weeks, at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, at least about 6 months, at least about 7 months, at least about 8 months, at least about 9 months, at least about 10 months, at least about 11 months, at least about 12 months, at least about 1 year, at least about 2 years, at least about 3 years, at least about 4 years, at least about 5 years, or longer) after the step of administering a unit dose of rAAV particles to one eye. In some embodiments, the unit dose of rAAV particles administered to the contralateral eye of an individual is higher than the unit dose of rAAV particles administered to one eye of the individual (e.g., about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 125%, about 150%, about 175%, about 200%, about 225%, about 250%, about 275%, about 300% or more higher).

[0113] In some embodiments, the rAAV particles comprise: a) a nucleic acid encoding a polypeptide comprising an amino acid sequence having at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.99%, or 100% identity to the amino acid sequence of SEQ ID NO:35, flanked by AAV2 inverted terminal repeats (ITRs), and b) an AAV2 capsid protein comprising the amino acid sequence LGETTRP (SEQ ID NO:14) inserted between positions 587 and 588 of the capsid protein, wherein the amino acid residue numbering corresponds to that of the AAV2 VP1 capsid protein. The sequence of SEQ ID NO:35 is provided below: SDTGRPFVEMYSEIPEIIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVLSPS HGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHEKDKTHTCPPCPAPEL LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 35)

[0114] In some embodiments, the rAAV particles comprise: a) a nucleic acid encoding a polypeptide comprising an amino acid sequence having at least about 95% identity to the amino acid sequence of SEQ ID NO: 35, flanked by AAV2 inverted terminal repeats (ITRs); and b) an AAV2 capsid protein comprising the amino acid sequence LGETTRP (SEQ ID NO: 14) inserted between positions 587 and 588 of the capsid protein, wherein the amino acid residue numbering corresponds to that of the AAV2 VP1 capsid protein.

[0115] In some embodiments, the rAAV particles comprise a nucleic acid encoding a polypeptide comprising an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.99%, or 100% identity to the amino acid sequence of SEQ ID NO: 35, and flanked by AAV2 inverted repeat repeats (ITRs). In some embodiments, the rAAV particles comprise a nucleic acid encoding a polypeptide comprising an amino acid sequence having at least about 95% identity to the amino acid sequence of SEQ ID NO: 35, and flanked by AAV2 inverted repeat repeats (ITRs). In some embodiments, the polypeptide comprises the amino acid sequence of SEQ ID NO: 35. In some embodiments, the polypeptide is aflibercept, or a functional variant or functional fragment thereof.

[0116] In some embodiments, the rAAV particles comprise a nucleic acid that includes a codon-optimized sequence encoding an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.99%, or 100% identity to the amino acid sequence of SEQ ID NO: 35, and is flanked by AAV2 inverted terminal repeats (ITRs). In some embodiments, the rAAV particles comprise a nucleic acid that includes a codon-optimized sequence encoding an amino acid sequence having at least about 95% identity to the amino acid sequence of SEQ ID NO: 35, and is flanked by AAV2 inverted terminal repeats (ITRs). In some embodiments, the rAAV particles comprise a nucleic acid that includes a codon-optimized sequence encoding an amino acid sequence having 100% identity to the amino acid sequence of SEQ ID NO: 35, and is flanked by AAV2 inverted terminal repeats (ITRs).

[0117] In some embodiments, the rAAV particles comprise a nucleic acid comprising the cDNA sequence of aflibercept, or a functional variant thereof, or a functional fragment thereof, and flanked by AAV2 inverted repeat repeats (ITRs). In some embodiments, the rAAV particles comprise a nucleic acid comprising the codon-optimized cDNA sequence of aflibercept, or a functional variant thereof, or a functional fragment thereof, and flanked by AAV2 inverted repeat repeats (ITRs). In some embodiments, the rAAV particles comprise a nucleic acid comprising the nucleic acid sequence of SEQ ID NO: 36.

[0118] In some embodiments, the nucleic acid further comprises (a) a first enhancer region comprising a CMV sequence; (b) a promoter region comprising a CMV sequence; (c) a 5'UTR region comprising, in 5' to 3' order, a TPL sequence and an eMLP sequence; (d) a second enhancer region comprising the full-length EES sequence; and (e) an HGH polyadenylation site. In some embodiments, the enhancer region comprising the CMV sequence comprises the sequence of SEQ ID NO: 22. In some embodiments, the promoter region comprising the CMV sequence comprises the sequence of SEQ ID NO: 23. In some embodiments, the TPL sequence comprises the sequence of SEQ ID NO: 24. In some embodiments, the eMLP sequence comprises the sequence of SEQ ID NO: 25. In some embodiments, the second enhancer region comprising the full-length EES sequence comprises the sequence of SEQ ID NO: 26. In some embodiments, the HGH polyadenylation site comprises the sequence of SEQ ID NO: 27.

[0119] In some embodiments, the rAAV particles comprise an AAV2 capsid protein comprising the amino acid sequence LGETTRP (SEQ ID NO: 14) inserted between positions 587 and 588 of AAV2 VP1 comprising the sequence of SEQ ID NO: 13. The sequence of SEQ ID NO: 13 is provided below: MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPGYKYLGPFNGLD KGEPVNEADAAALEHDKAYDRQLDSGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQ AKKRVLEPLGLVEEPVKTAPGKKRPVEHSPVEPDSSSGTGKAGQQPARKRLNFGQTGDAD SVPDPQPLGQPPAAPSGLGTNTMATGSGAPMADNNEGADGVGNSSGNWHCDSTWMGDRVI TTSTRTWALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLI NNNWGFRPKRLNFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQG CLPPFPADVFMVPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEDVPF HSSYAHSQSLDRLMNPLIDQYLYYLSRTNTPSGTTTQSRLQFSQAGASDIRDQSRNWLPG PCYRQQRVSKTSADNNNSEYSWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVL IFGKQGSEKTNVDIEKVMITDEEIRTTNPVATEQYGSVSTNLQRGNRQAATADVNTQGV LPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPSTT FSAAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSVNVDFTVDTNGVY SEPRPIGTRYLTRNL (SEQ ID NO: 13)

[0120] In some embodiments, the rAAV particles comprise an AAV2 capsid protein comprising the amino acid sequence LALGETTRPA (SEQ ID NO: 1) inserted between positions 587 and 588 of the capsid protein, where the amino acid residue numbering corresponds to that of the AAV2 VP1 capsid protein. In some embodiments, the rAAV particles comprise an AAV2 capsid protein comprising the amino acid sequence LALGETTRPA (SEQ ID NO: 1) inserted between positions 587 and 588 of AAV2 VP1, which comprises the sequence of SEQ ID NO: 13.

[0121] In some embodiments, the rAAV particles contain the following amino acid sequences inserted between positions 587 and 588 of the capsid protein: LALGETTRPA (SEQ ID NO: 1); LANETITRPA (SEQ ID NO: 2), LAKAGQANNA (SEQ ID NO: 3), LAKDPKTTNA (SEQ ID NO: 4), KDTDTTR (SEQ ID NO: 5), RAGGSVG (SEQ ID NO: 6), AVDTTKF (SEQ ID NO: 7), STGKVPN (SEQ ID NO: 8), LAKDTDTTRA (SEQ ID NO: 9), LARAGGSVGA (SEQ ID NO: 10), LAGAGGSVGA (SEQ ID NO: 11), LAGAGGSVGA (SEQ ID NO: 12), LAGAGGSVGA (SEQ ID NO: 13), LAGAGGSVGA (SEQ ID NO: 14), LAGAGGSVGA (SEQ ID NO: 15), LAGAGGSVGA (SEQ ID NO: 16), LAGAGGSVGA (SEQ ID NO: 17), LAGAGGSVGA (SEQ ID NO: 18), LAGAGGSVGA (SEQ ID NO: 19), LAGAGGSVGA (SEQ ID NO: 20), LAGAGGSVGA (SEQ ID NO: 21), LAGAGGSVGA (SEQ ID NO: 22), LAGAGGSVGA (SEQ ID NO: 23), LAGAGGSVGA (SEQ ID NO: 24), LAGAGGSVGA (SEQ ID NO: 25), LAGAGGSVGA (SEQ ID NO: 26), LAGAGGSVGA (SEQ ID NO: 27), LAGAGGSVGA (SEQ ID NO: 28), LAGAGGSVGA (SEQ ID NO: 29), LAGAGGSVGA (SEQ ID NO: 30), LAGAGGSVGA (SEQ ID NO: 3 In some embodiments, the rAAV particles comprise an AAV2 capsid protein comprising any of the following: LAAVDTTKFA (SEQ ID NO: 11), LASTGKVPNA (SEQ ID NO: 12), LGETTRP (SEQ ID NO: 14), NETITRP (SEQ ID NO: 15), KAGQANN (SEQ ID NO: 16), KDPKTTN (SEQ ID NO: 17), KDTDTTR (SEQ ID NO: 18), RAGGSVG (SEQ ID NO: 19), AVDTTKF (SEQ ID NO: 20), and STGKVPN (SEQ ID NO: 21), where the amino acid residue numbering corresponds to that of the AAV2 VP1 capsid protein. In some embodiments, the rAAV particles comprise an AAV2 capsid protein comprising the sequence of SEQ ID NO: 13. The AAV2 capsid protein contains any of the following amino acid sequences inserted between positions 587 and 588 of VP1: LALGETTRPA (SEQ ID NO: 1); LANETITRPA (SEQ ID NO: 2), LAKAGQANNA (SEQ ID NO: 3), LAKDPKTTNA (SEQ ID NO: 4), KDTDTTR (SEQ ID NO: 5), RAGGSVG (SEQ ID NO: 6), AVDTTKF (SEQ ID NO: 7), STGKVPN (SEQ ID NO: 8), LAKDTDTTRA (SEQ ID NO: 9), LARAGGSVGA (SEQ ID NO: 10), LAAVDTTKFA (SEQ ID NO: 11), LASTGKVPNA (SEQ ID NO: 12), LGETTRP (SEQ ID NO: 14), NETITRP (SEQ ID NO: 15), KAGQANN (SEQ ID NO: 16), KDPKTTN (SEQ ID NO: 17), KDTDTTR (SEQ ID NO: 18), RAGGSVG (SEQ ID NO: 19), AVDTTKF (SEQ ID NO: 20), and STGKVPN (SEQ ID NO: 21).

[0122] In some embodiments, the unit dose of rAAV particles is administered to one eye and / or the contralateral eye of an individual by intravitreal (IVT) injection, intraocular administration, or intraretinal injection. In some embodiments, the unit dose of rAAV particles is administered to one eye and / or the contralateral eye of an individual by intravitreal (IVT) injection.

[0123] In some embodiments, the unit dose of rAAV particles is in a pharmaceutical formulation. In some embodiments, the pharmaceutical formulation comprises rAAV particles, one or more osmolality or ionic strength adjusters, one or more buffering agents, one or more surfactants, and one or more solvents. In some embodiments, the osmolality or ionic strength adjuster is sodium chloride. In some embodiments, the one or more buffering agents are sodium dihydrogen phosphate and / or sodium hydrogen phosphate. In some embodiments, the surfactant is poloxamer 188. In some embodiments, the solvent is water. In some embodiments, the pharmaceutical formulation comprises rAAV particles, sodium chloride, sodium phosphate, and a surfactant. In some embodiments, the pharmaceutical formulation comprises about 1 x 10 10 vg / mL ~ approx. 1×10 13 In some embodiments, the pharmaceutical formulation comprises about 6 x 10 vg / mL of rAAV particles. 11 vg / mL ~ approx. 6×10 12vg / mL of rAAV particles. In some embodiments, the pharmaceutical formulation comprises about 150 mM to about 200 mM sodium chloride (e.g., about 150 mM, about 160 mM, about 170 mM, about 180 mM, about 190 mM, or about 200 mM). In some embodiments, the pharmaceutical formulation comprises about 1 mM to about 10 mM sodium dihydrogen phosphate (e.g., about 1 mM, about 2 mM, about 3 mM, about 4 mM, about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, or about 10 mM). In some embodiments, the pharmaceutical formulation comprises about 1 mM to about 10 mM sodium dihydrogen phosphate (e.g., about 1 mM, about 2 mM, about 3 mM, about 4 mM, about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, or about 10 mM). In some embodiments, the pharmaceutical formulation comprises about 0.0005% (wt / vol) to about 0.005% (wt / vol) poloxamer 188 (e.g., about 0.0005% (wt / vol), 0.0006% (wt / vol), 0.0007% (wt / vol), 0.0008% (wt / vol), 0.0009% (wt / vol), 0.001% (wt / vol), 0.002% (wt / vol), 0.003% (wt / vol), 0.004% (wt / vol), or about 0.005% (wt / vol)). In some embodiments, the pharmaceutical formulation has a pH of about 7.0 to about 7.5 (e.g., about 7.0, about 7.1, about 7.2, about 7.3, about 7.4, or about 7.5). In some embodiments, the pharmaceutical formulation comprises about 6×10 12 vg / mL rAAV particles, about 180 mM sodium chloride, about 5 mM sodium dihydrogen phosphate, about 5 mM sodium dihydrogen phosphate, and about 0.001% (weight / volume) poloxamer 188, and the pharmaceutical formulation has a pH of about 7.3. In some embodiments, the pharmaceutical formulation contains about 6 x 10 11 vg / mL rAAV particles, about 180 mM sodium chloride, about 5 mM sodium dihydrogen phosphate, about 5 mM sodium hydrogen phosphate, and about 0.001% (weight / volume) poloxamer 188, and the pharmaceutical formulation has a pH of about 7.3.

[0124] In some embodiments, a unit dose of rAAV particles comprises a volume of between about 25 μL and about 250 μL (e.g., any of about 25 μL, about 30 μL, about 40 μL, about 50 μL, about 60 μL, about 70 μL, about 80 μL, about 90 μL, about 100 μL, about 110 μL, about 120 μL, about 130 μL, about 140 μL, about 150 μL, about 160 μL, about 170 μL, about 180 μL, about 190 μL, about 200 μL, about 210 μL, about 220 μL, about 230 μL, about 240 μL, or about 250 μL). In some embodiments, the concentration of rAAV particles in the pharmaceutical formulation is adjusted so that the volume of a unit dose of rAAV particles administered to the eye of an individual is between about 25 μL and about 250 μL. In some embodiments, a unit dose of rAAV particles comprises a volume of about 100 μL, hi some embodiments, a unit dose of rAAV particles comprises a volume of about 30 μL.

[0125] In some embodiments, the unit dose of rAAV particles is administered in combination with a steroid treatment. In some embodiments, the steroid treatment is a corticosteroid treatment. In some embodiments, the steroid treatment is a systemic steroid treatment. In some embodiments, the steroid treatment is an oral steroid treatment. In some embodiments, the steroid treatment is a prednisone treatment. In some embodiments, the steroid treatment is an ophthalmic steroid treatment. In some embodiments, the ophthalmic steroid treatment is a topical steroid treatment (e.g., eye drops), a periocular steroid treatment (e.g., subtenon, subconjunctival), an intravitreal steroid treatment, or a suprachoroidal steroid treatment. In some embodiments, the topical steroid treatment is a difluprednate treatment, a medrysone treatment, a loteprednol treatment, a prednisolone treatment, a fluocinolone treatment, a triamcinolone treatment, a rimexolone treatment, a dexamethasone treatment, a fluorometholone treatment, a fluocinolone treatment, a rimexolone treatment, or a prednisone treatment. In some embodiments, the topical steroid treatment is a difluprednate treatment. In some embodiments, steroid treatment is administered before, during, and / or after administration of a unit dose of rAAV particles. In some embodiments, steroid treatment is administered before administration of a unit dose of rAAV particles. In some embodiments, steroid treatment is administered during administration of a unit dose of rAAV particles. In some embodiments, steroid treatment is administered after administration of a unit dose of rAAV particles. In some embodiments, steroid treatment is administered before and during administration of a unit dose of rAAV particles. In some embodiments, steroid treatment is administered before and after administration of a unit dose of rAAV particles. In some embodiments, steroid treatment is administered during and after administration of a unit dose of rAAV particles. In some embodiments, steroid treatment is administered before, during, and after administration of a unit dose of rAAV particles.

[0126] In some embodiments, the steroid treatment is an ophthalmic steroid treatment (e.g., difluprednate). In some embodiments, the ophthalmic steroid treatment (e.g., difluprednate) is daily steroid treatment for up to about 4 weeks, about 6 weeks, or about 8 weeks from administration of a unit dose of rAAV particles. In some embodiments, the ophthalmic steroid treatment comprises about four administrations of ophthalmic steroid at about week 1, about three administrations of ophthalmic steroid at about week 2, about two administrations of ophthalmic steroid at about week 3, and about one administration of ophthalmic steroid at about week 4, timing starting from and following administration of a unit dose of rAAV particles. In some embodiments, the ophthalmic steroid is about 0.005% to about 0.5% difluprednate. In some embodiments, the ophthalmic steroid is about 0.005%, about 0.006%, about 0.007%, about 0.008%, about 0.009%, about 0.01%, about 0.02%, about 0.03%, about 0.4%, about 0.05%, about 0.06%, about 0.07%, about 0.08%, about 0.09%, or about 0.1% difluprednate. In some embodiments, the ophthalmic steroid is 0.05% difluprednate. In some embodiments, the dose of 0.05% difluprednate is one drop of ophthalmic solution. In some embodiments, one drop is about 50 μl (e.g., about 25 μl to about 50 μl, about 50 μl to about 100 μl). In some embodiments, the dose of difluprednate comprises about 1 μg to about 5 μg, or about 2 μg to about 3 μg, or about 2.5 μg difluprednate. In some embodiments, the dose of difluprednate comprises about 2.5 μg difluprednate.

[0127] In some embodiments, the steroid treatment is an ophthalmic steroid treatment (e.g., difluprednate). In some embodiments, the ophthalmic steroid treatment (e.g., difluprednate) is daily topical steroid treatment for up to about 4 weeks, about 6 weeks, or about 8 weeks from administration of a unit dose of rAAV particles. In some embodiments, the topical steroid treatment comprises about 4 administrations of topical steroid at about week 1, about 3 administrations of topical steroid at about week 2, about 2 administrations of topical steroid at about week 3, and about 1 administration of topical steroid at about week 4, timing starting from and following administration of a unit dose of rAAV particles. In some embodiments, the topical steroid treatment comprises, after administration of a unit dose of rAAV particles, administering the topical steroid about four times per day (i.e., QID) for about three weeks, followed by administering the topical steroid about three times per day (i.e., TID) for about one week, followed by administering the topical steroid about twice per day (i.e., BID) for about one week, and then administering the topical steroid about once per day (i.e., QD) for about one week. In some embodiments, the topical steroid comprises about 1 μg to about 3 μg of 0.05% difluprednate. In some embodiments, the topical steroid comprises about 2.5 μg of 0.05% difluprednate. In some embodiments, the topical steroid comprises about 0.005% to about 0.5% difluprednate. In some embodiments, the topical steroid is about 0.005%, about 0.006%, about 0.007%, about 0.008%, about 0.009%, about 0.01%, about 0.02%, about 0.03%, about 0.04%, about 0.05%, about 0.06%, about 0.07%, about 0.08%, about 0.09%, or about 0.1% difluprednate. In some embodiments, the topical steroid is 0.05% difluprednate. In some embodiments, the dose of 0.05% difluprednate is one drop of ophthalmic solution. In some embodiments, one drop is about 50 μl (e.g., about 25 μl to about 50 μl, about 50 μl to about 100 μl). In some embodiments, the dose of difluprednate comprises about 1 μg to about 5 μg, or about 2 μg to about 3 μg, or about 2.5 μg difluprednate.In some embodiments, the dose of difluprednate comprises about 2.5 μg difluprednate.

[0128] In some embodiments, the retinal fluid (e.g., SRF and / or IRF) in the individual's eye is reduced by more than about 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% after administration of a unit dose of rAAV particles to one eye and / or the contralateral eye of the individual. In some embodiments, the retinal fluid (e.g., SRF and / or IRF) in the individual's eye is reduced by more than about 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% after administration of a unit dose of rAAV particles to one eye and / or the contralateral eye of the individual, compared to the level of retinal fluid in the individual's eye before administration of the unit dose of rAAV particles. In some embodiments, the retinal fluid (e.g., SRF and / or IRF) in the individual's eye is reduced by about 100% after administration of a unit dose of rAAV particles to one eye and / or the contralateral eye of the individual, compared to the level of retinal fluid in the individual's eye before administration of the unit dose of rAAV particles.

[0129] In some embodiments, the methods provided herein further comprise monitoring the level of retinal fluid (e.g., SRF and / or IRF) in one eye and / or the contralateral eye of the individual after administration of a unit dose of rAAV particles. In some embodiments, a reduction in retinal fluid (e.g., SRF and / or IRF) in the eye is first observed after about 1 day, about 3 days, about 8 days, about 2 weeks, about 4 weeks, about 6 weeks, about 8 weeks, about 12 weeks, about 16 weeks, about 20 weeks, about 24 weeks, about 28 weeks, about 32 weeks, about 36 weeks, about 40 weeks, about 44 weeks, about 48 weeks, about 52 weeks, about 56 weeks, about 60 weeks, about 64 weeks, about 68 weeks, about 72 weeks, about 76 weeks, about 80 weeks, about 84 weeks, about 88 weeks, about 92 weeks, about 96 weeks, about 100 weeks, about 104 weeks, or longer, after administration of the unit dose of rAAV particles. In some embodiments, the reduction in retinal fluid (e.g., SRF and / or IRF) in the eye persists or is maintained for at least 1 week, at least 2 weeks, at least 4 weeks, at least 6 weeks, at least 8 weeks, at least 12 weeks, at least 16 weeks, at least 20 weeks, at least 24 weeks, at least 28 weeks, at least 32 weeks, at least 36 weeks, at least 40 weeks, at least 44 weeks, at least 48 weeks, at least 52 weeks, at least 56 weeks, at least 60 weeks, at least 64 weeks, at least 68 weeks, at least 72 weeks, at least 76 weeks, at least 80 weeks, at least 84 weeks, at least 88 weeks, at least 92 weeks, at least 96 weeks, at least 100 weeks, at least 104 weeks, or longer, after administration of a unit dose of the rAAV particles.

[0130] In some embodiments, the reduction in retinal fluid of the eye (e.g., SRF and / or IRF) is determined by any method known in the art. In some embodiments, the reduction in retinal fluid of the eye (e.g., SRF and / or IRF) is determined by optical coherence tomography (OCT), spectral domain OCT (SD-OCT), OCT angiography, fluorescein angiography, or direct retinal observation. In some embodiments, the reduction in retinal fluid of the eye (e.g., SRF and / or IRF) is determined by optical coherence tomography (OCT). In some embodiments, the reduction in retinal fluid of the eye (e.g., SRF and / or IRF) is determined by spectral domain OCT (SD-OCT). In some embodiments, the reduction in retinal fluid of the eye (e.g., SRF and / or IRF) is determined by OCT angiography. In some embodiments, the reduction in retinal fluid of the eye (e.g., SRF and / or IRF) is determined by fluorescein angiography. In some embodiments, the reduction in retinal fluid (eg, SRF and / or IRF) in the eye is determined by direct retinal observation.

[0131] In some embodiments, whether an intraocular neovascular disease is treated in an individual after administration of a unit dose of rAAV particles to one eye and / or the contralateral eye is assessed based on the level of retinal fluid (e.g., intraretinal fluid (IRF) and / or subretinal fluid (SRF)) compared to the level of retinal fluid (e.g., intraretinal fluid (IRF) and / or subretinal fluid (SRF)) before administration of a unit dose of AAV particles to one eye and / or the contralateral eye (e.g., as described above). In some embodiments, the retinal fluid is subretinal fluid (SRF) or intraretinal fluid (IRF). In some embodiments, the retinal fluid is subretinal fluid (SRF). In some embodiments, the retinal fluid is intraretinal fluid (IRF). In some embodiments, if a reduction in retinal fluid (e.g., IRF and / or SRF) is observed after administration of a unit dose of rAAV particles to one eye and / or the other eye compared to the level of retinal fluid (e.g., IRF and / or SRF) before administration of the unit dose of rAAV particles to one eye and / or the other eye, it is determined (e.g., as described above) that the intraocular neovascular disease has been treated in the individual after administration of the unit dose of rAAV particles to one eye and / or the other eye. In some embodiments, the intraocular neovascular disease is AMD.

[0132] In some embodiments, administering a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual results in a maintenance or reduction in retinal thickness compared to the retinal thickness before administration of the unit dose of rAAV particles. In some embodiments, administering a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual results in a reduction in retinal thickness compared to the retinal thickness before administration of the unit dose of rAAV particles. In some embodiments, the retinal thickness is central sclera retinal thickness (CST) or central foveal retinal thickness (CRT). In some embodiments, administering a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual results in a reduction in retinal thickness of greater than any of about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or about 100% compared to the retinal thickness before administration of the unit dose of rAAV particles. In some embodiments, the retinal thickness (e.g., CST or CRT) is determined by OCT or SD-OCT.

[0133] In some embodiments, administering a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual results in a decrease in central retinal thickness (CRT) or central zone retinal thickness (CST) of about 10 μm to about 100 μm (e.g., greater than or equal to any of about 10 μm, about 15 μm, about 20 μm, about 25 μm, about 30 μm, about 35 μm, about 40 μm, about 45 μm, about 50 μm, about 55 μm, about 60 μm, about 65 μm, about 70 μm, about 75 μm, about 80 μm, about 85 μm, about 90 μm, about 95 μm, or about 100 μm). In some embodiments, administering a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual results in a decrease in central retinal thickness (CRT) or central regional retinal thickness (CST) of about 5 μm to about 50 μm (e.g., about 5 μm, about 10 μm, about 15 μm, about 20 μm, about 25 μm, about 30 μm, about 35 μm, about 40 μm, about 45 μm, or about 50 μm). In some embodiments, administering a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual results in a decrease in central retinal thickness (CRT) or central regional retinal thickness (CST) of about 5 μm to about 40 μm (e.g., about 5 μm, about 10 μm, about 15 μm, about 20 μm, about 25 μm, about 30 μm, about 35 μm, or about 40 μm). In some embodiments, administering a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual results in a decrease in central retinal thickness (CRT) or central zone retinal thickness (CST) of about 5 μm to about 30 μm (e.g., any of about 5 μm, about 6 μm, about 7 μm, about 8 μm, about 9 μm, about 10 μm, about 11 μm, about 12 μm, about 13 μm, about 14 μm, about 15 μm, about 16 μm, about 17 μm, about 18 μm, about 19 μm, about 20 μm, about 21 μm, about 22 μm, about 23 μm, about 24 μm, about 25 μm, about 26 μm, about 27 μm, about 28 μm, about 29 μm, or about 30 μm).In some embodiments, administering a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual results in a decrease in central retinal thickness (CRT) or central zone retinal thickness (CST) of about 5 μm to about 25 μm (e.g., any of about 5 μm, about 6 μm, about 7 μm, about 8 μm, about 9 μm, about 10 μm, about 11 μm, about 12 μm, about 13 μm, about 14 μm, about 15 μm, about 16 μm, about 17 μm, about 18 μm, about 19 μm, about 20 μm, about 21 μm, about 22 μm, about 23 μm, about 24 μm, or about 25 μm). In some embodiments, administering a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual results in a decrease in central retinal thickness (CRT) or central regional retinal thickness (CST) of about 5 μm to about 20 μm (e.g., about 5 μm, about 6 μm, about 7 μm, about 8 μm, about 9 μm, about 10 μm, about 11 μm, about 12 μm, about 13 μm, about 14 μm, about 15 μm, about 16 μm, about 17 μm, about 18 μm, about 19 μm, or about 20 μm). In some embodiments, administering a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual results in a decrease in central retinal thickness (CRT) or central regional retinal thickness (CST) of about 18 μm to about 75 μm. In some embodiments, administering a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual results in a decrease of about 18.5 μm in central retinal thickness (CRT) or central regional retinal thickness (CST). In some embodiments, administering a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual results in a decrease of about 21.0 μm in central retinal thickness (CRT) or central regional retinal thickness (CST). In some embodiments, administering a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual results in a decrease of about 8.3 μm in central retinal thickness (CRT) or central regional retinal thickness (CST). In some embodiments, administering a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual results in a decrease of about 25.5 μm in central retinal thickness (CRT) or central regional retinal thickness (CST). In some embodiments, administering a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual results in a decrease of central retinal thickness (CRT) or central zone retinal thickness (CST) of about 24.8 μm.In some embodiments, administering a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual results in a decrease of central retinal thickness (CRT) or central regional retinal thickness (CST) of about 75 μm. In some embodiments, administering a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual results in a decrease of central retinal thickness (CRT) or central regional retinal thickness (CST) of about 100 μm or more (e.g., any of about 100 μm or more, about 110 μm or more, about 120 μm or more, about 130 μm or more, about 140 μm or more, about 150 μm or more, about 160 μm or more, about 170 μm or more, about 180 μm or more, about 190 μm or more, or about 200 μm or more). In some embodiments, administering a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual results in a decrease in central retinal thickness (CRT) or central zone retinal thickness (CST) of about 10 μm to about 200 μm (e.g., any of about 10 μm, about 20 μm, about 30 μm, about 40 μm, about 50 μm, about 60 μm, about 70 μm, about 80 μm, about 90 μm, about 100 μm, about 110 μm, about 120 μm, about 130 μm, about 140 μm, about 150 μm, about 160 μm, about 170 μm, about 180 μm, about 190 μm, or about 200 μm). In some embodiments, administering a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual results in a decrease in central retinal thickness (CRT) or central zone retinal thickness (CST) of about 50 μm to about 200 μm (e.g., any of about 50 μm, about 60 μm, about 70 μm, about 80 μm, about 90 μm, about 100 μm, about 110 μm, about 120 μm, about 130 μm, about 140 μm, about 150 μm, about 160 μm, about 170 μm, about 180 μm, about 190 μm, or about 200 μm).In some embodiments, administering a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual results in a decrease in central retinal thickness (CRT) or central zone retinal thickness (CST) of about 60 μm to about 200 μm (e.g., any of about 60 μm, about 70 μm, about 80 μm, about 90 μm, about 100 μm, about 110 μm, about 120 μm, about 130 μm, about 140 μm, about 150 μm, about 160 μm, about 170 μm, about 180 μm, about 190 μm, or about 200 μm). In some embodiments, administering a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual results in a decrease in central retinal thickness (CRT) or central zone retinal thickness (CST) of about 70 μm to about 200 μm (e.g., any of about 70 μm, about 80 μm, about 90 μm, about 100 μm, about 110 μm, about 120 μm, about 130 μm, about 140 μm, about 150 μm, about 160 μm, about 170 μm, about 180 μm, about 190 μm, or about 200 μm). In some embodiments, administering a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual results in a decrease in central retinal thickness (CRT) or central zone retinal thickness (CST) of about 80 μm to about 200 μm (e.g., any of about 80 μm, about 90 μm, about 100 μm, about 110 μm, about 120 μm, about 130 μm, about 140 μm, about 150 μm, about 160 μm, about 170 μm, about 180 μm, about 190 μm, or about 200 μm). In some embodiments, administering a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual results in a decrease in central retinal thickness (CRT) or central zone retinal thickness (CST) of about 90 μm to about 200 μm (e.g., any of about 90 μm, about 100 μm, about 110 μm, about 120 μm, about 130 μm, about 140 μm, about 150 μm, about 160 μm, about 170 μm, about 180 μm, about 190 μm, or about 200 μm). In some embodiments, administering a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual results in a decrease in central retinal thickness (CRT) or central zone retinal thickness (CST) of about 100 μm to about 200 μm (e.g., any of about 100 μm, about 110 μm, about 120 μm, about 130 μm, about 140 μm, about 150 μm, about 160 μm, about 170 μm, about 180 μm, about 190 μm, or about 200 μm).In some embodiments, administering a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual results in a decrease in central retinal thickness (CRT) or central zone retinal thickness (CST) of about 110 μm to about 200 μm (e.g., any of about 110 μm, about 120 μm, about 130 μm, about 140 μm, about 150 μm, about 160 μm, about 170 μm, about 180 μm, about 190 μm, or about 200 μm). In some embodiments, administering a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual results in a decrease in central retinal thickness (CRT) or central zone retinal thickness (CST) of about 115 μm to about 200 μm (e.g., any of about 115 μm, about 120 μm, about 125 μm, about 130 μm, about 135 μm, about 140 μm, about 145 μm, about 150 μm, about 155 μm, about 160 μm, about 165 μm, about 170 μm, about 175 μm, about 180 μm, about 185 μm, about 190 μm, about 195 μm, or about 200 μm). In some embodiments, administering a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual results in a decrease in central retinal thickness (CRT) or central zone retinal thickness (CST) of about 120 μm to about 200 μm (e.g., any of about 120 μm, about 125 μm, about 130 μm, about 135 μm, about 140 μm, about 145 μm, about 150 μm, about 155 μm, about 160 μm, about 165 μm, about 170 μm, about 175 μm, about 180 μm, about 185 μm, about 190 μm, about 195 μm, or about 200 μm). In some embodiments, administering a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual results in a decrease in central retinal thickness (CRT) or central zone retinal thickness (CST) of about 125 μm to about 200 μm (e.g., any of about 125 μm, about 130 μm, about 135 μm, about 140 μm, about 145 μm, about 150 μm, about 155 μm, about 160 μm, about 165 μm, about 170 μm, about 175 μm, about 180 μm, about 185 μm, about 190 μm, about 195 μm, or about 200 μm).In some embodiments, administering a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual results in a decrease in central retinal thickness (CRT) or central zone retinal thickness (CST) of about 130 μm to about 200 μm (e.g., any of about 130 μm, about 135 μm, about 140 μm, about 145 μm, about 150 μm, about 155 μm, about 160 μm, about 165 μm, about 170 μm, about 175 μm, about 180 μm, about 185 μm, about 190 μm, about 195 μm, or about 200 μm). In some embodiments, administering a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual results in a decrease in central retinal thickness (CRT) or central zone retinal thickness (CST) of about 135 μm to about 200 μm (e.g., any of about 135 μm, about 140 μm, about 145 μm, about 150 μm, about 155 μm, about 160 μm, about 165 μm, about 170 μm, about 175 μm, about 180 μm, about 185 μm, about 190 μm, about 195 μm, or about 200 μm). In some embodiments, administering a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual results in a central foveal retinal thickness (CRT) or central zone retinal thickness (CST) of about 140 μm to about 200 μm (e.g., about 140 μm, about 145 μm, about 150 μm, about 155 μm, about 160 μm, about 165 μm, about 170 μm, about 175 μm, about 176 μm, about 177 μm, about 178 μm, about 179 μm, about 180 μm, about 181 μm, about 182 μm, about 183 μm, about 184 μm, about 185 μm, about 186 μm, about 187 μm, about 188 μm, about 189 μm, about In some embodiments, administering a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual results in a decrease in central retinal thickness (CRT) or central zone retinal thickness (CST) of about 145 μm to about 200 μm (e.g., about 145 μm, about 150 μm, about 155 μm, about 160 μm, about 165 μm, about 170 μm, about 175 μm, about 180 μm, about 185 μm, about 190 μm, about 195 μm, or about 200 μm). In some embodiments, administering a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual results in a decrease in central retinal thickness (CRT) or central zone retinal thickness (CST) of about 150 μm to about 200 μm (e.g., any of about 150 μm, about 155 μm, about 160 μm, about 165 μm, about 170 μm, about 175 μm, about 180 μm, about 185 μm, about 190 μm, about 195 μm, or about 200 μm). In some embodiments, administering a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual results in a decrease in central retinal thickness (CRT) or central zone retinal thickness (CST) of about 155 μm to about 200 μm (e.g., any of about 155 μm, about 160 μm, about 165 μm, about 170 μm, about 175 μm, about 180 μm, about 185 μm, about 190 μm, about 195 μm, or about 200 μm). In some embodiments, administering a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual results in a decrease in central retinal thickness (CRT) or central subretinal thickness (CST) of about 160 μm to about 200 μm (e.g., any of about 160 μm, about 165 μm, about 170 μm, about 175 μm, about 180 μm, about 185 μm, about 190 μm, about 195 μm, or about 200 μm). In some embodiments, administering a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual results in a decrease in central retinal thickness (CRT) or central subretinal thickness (CST) of about 165 μm to about 200 μm (e.g., any of about 165 μm, about 170 μm, about 175 μm, about 180 μm, about 185 μm, about 190 μm, about 195 μm, or about 200 μm).In some embodiments, administering a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual results in a decrease in central foveal thickness (CRT) or central regional retinal thickness (CST) of about 170 μm to about 200 μm (e.g., about 170 μm, about 175 μm, about 180 μm, about 185 μm, about 190 μm, about 195 μm, or about 200 μm). In some embodiments, administering a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual results in a decrease in central foveal thickness (CRT) or central regional retinal thickness (CST) of about 175 μm to about 200 μm (e.g., about 175 μm, about 180 μm, about 185 μm, about 190 μm, about 195 μm, or about 200 μm). In some embodiments, administering a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual results in a decrease in central retinal thickness (CRT) or central regional retinal thickness (CST) of about 180 μm to about 200 μm (e.g., about 180 μm, about 185 μm, about 190 μm, about 195 μm, or about 200 μm). In some embodiments, administering a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual results in a decrease in central retinal thickness (CRT) or central regional retinal thickness (CST) of about 21.0 μm compared to the central retinal thickness (CRT) or central regional retinal thickness (CST) before administration of the unit dose of rAAV particles. In some embodiments, administering a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual results in a decrease in central retinal thickness (CRT) or central regional retinal thickness (CST) of about 8.3 μm compared to the central retinal thickness (CRT) or central regional retinal thickness (CST) before administration of the unit dose of rAAV particles. In some embodiments, administering a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual results in a decrease in central retinal thickness (CRT) or central regional retinal thickness (CST) of about 26.2 μm compared to the central retinal thickness (CRT) or central regional retinal thickness (CST) before administration of the unit dose of rAAV particles.In some embodiments, administering a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual results in a decrease in central retinal thickness (CRT) or central regional retinal thickness (CST) of about 24.8 μm compared to the central retinal thickness (CRT) or central regional retinal thickness (CST) before administration of the unit dose of rAAV particles. In some embodiments, administering a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual results in a decrease in central retinal thickness (CRT) or central regional retinal thickness (CST) of about 40.8 μm compared to the central retinal thickness (CRT) or central regional retinal thickness (CST) before administration of the unit dose of rAAV particles. In some embodiments, administering a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual results in a decrease in central retinal thickness (CRT) or central regional retinal thickness (CST) of about 30.0 μm compared to the central retinal thickness (CRT) or central regional retinal thickness (CST) before administration of the unit dose of rAAV particles. In some embodiments, administering a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual results in a decrease in central retinal thickness (CRT) or central regional retinal thickness (CST) of about 118.6 μm compared to the central retinal thickness (CRT) or central regional retinal thickness (CST) before administration of the unit dose of rAAV particles. In some embodiments, administering a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual results in a decrease in central retinal thickness (CRT) or central regional retinal thickness (CST) of about 119.0 μm compared to the central retinal thickness (CRT) or central regional retinal thickness (CST) before administration of the unit dose of rAAV particles. In some embodiments, administering a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual results in a decrease in central retinal thickness (CRT) or central regional retinal thickness (CST) of about 137.8 μm compared to the central retinal thickness (CRT) or central regional retinal thickness (CST) before administration of the unit dose of rAAV particles. In some embodiments, administering a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual results in a decrease in central retinal thickness (CRT) or central regional retinal thickness (CST) of about 152.7 μm compared to the central retinal thickness (CRT) or central regional retinal thickness (CST) before administration of the unit dose of rAAV particles.In some embodiments, administering a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual results in a decrease in central retinal thickness (CRT) or central regional retinal thickness (CST) of about 153.3 μm compared to the central retinal thickness (CRT) or central regional retinal thickness (CST) before administration of the unit dose of rAAV particles. In some embodiments, administering a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual results in a decrease in central retinal thickness (CRT) or central regional retinal thickness (CST) of about 149.8 μm compared to the central retinal thickness (CRT) or central regional retinal thickness (CST) before administration of the unit dose of rAAV particles.

[0134] In some embodiments, administering a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual results in a change in central retinal thickness (CRT) or central zone retinal thickness (CST) of about -200 μm to about +40 μm (e.g., any of about -200 μm, about -180 μm, about -160 μm, about -140 μm, about -120 μm, about -100 μm, about -80 μm, about -60 μm, about -40 μm, about -20 μm, about 0 μm, about +5 μm, about +10 μm, about +15 μm, about +20 μm, about +25 μm, about +30 μm, about +35 μm, or about +40 μm) compared to the retinal thickness before administration of the unit dose of rAAV particles. In some embodiments, administering a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual results in a decrease in central retinal thickness (CRT) or central regional retinal thickness (CST) of about 8 μm, about 11 μm, about 16 μm, about 29 μm, about 33 μm, about 38 μm, about 55 μm, about 61 μm, or about 117 μm. In some embodiments, administering a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual results in a decrease in central retinal thickness (CRT) or central regional retinal thickness (CST) of about 27.8 μm or about 30.8 μm. In some embodiments, administering a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual results in an increase in central retinal thickness (CRT) or central regional retinal thickness (CST) of about 4 μm, about 12 μm, or about 32 μm. In some embodiments, administering a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual results in a change in central retinal thickness (CRT) or central regional retinal thickness (CST) of about -21.0 μm compared to the central retinal thickness (CRT) or central regional retinal thickness (CST) before administration of the unit dose of rAAV particles. In some embodiments, administering a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual results in a change in central retinal thickness (CRT) or central regional retinal thickness (CST) of about -8.3 μm compared to the central retinal thickness (CRT) or central regional retinal thickness (CST) before administration of the unit dose of rAAV particles.In some embodiments, administering a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual results in a change in central retinal thickness (CRT) or central regional retinal thickness (CST) of about -26.2 μm compared to the central retinal thickness (CRT) or central regional retinal thickness (CST) before administration of the unit dose of rAAV particles. In some embodiments, administering a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual results in a change in central retinal thickness (CRT) or central regional retinal thickness (CST) of about -24.8 μm compared to the central retinal thickness (CRT) or central regional retinal thickness (CST) before administration of the unit dose of rAAV particles. In some embodiments, administering a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual results in a change in central retinal thickness (CRT) or central regional retinal thickness (CST) of about -40.8 μm compared to the central retinal thickness (CRT) or central regional retinal thickness (CST) before administration of the unit dose of rAAV particles. In some embodiments, administering a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual results in a change in central retinal thickness (CRT) or central regional retinal thickness (CST) of about -30.0 μm compared to the central retinal thickness (CRT) or central regional retinal thickness (CST) before administration of the unit dose of rAAV particles. In some embodiments, administering a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual results in a change in central retinal thickness (CRT) or central regional retinal thickness (CST) of about -118.6 μm compared to the central retinal thickness (CRT) or central regional retinal thickness (CST) before administration of the unit dose of rAAV particles. In some embodiments, administering a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual results in a change in central retinal thickness (CRT) or central regional retinal thickness (CST) of about -119.0 μm compared to the central retinal thickness (CRT) or central regional retinal thickness (CST) before administration of the unit dose of rAAV particles. In some embodiments, administering a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual results in a change in central retinal thickness (CRT) or central regional retinal thickness (CST) of approximately -137.8 μm compared to the central retinal thickness (CRT) or central regional retinal thickness (CST) before administration of the unit dose of rAAV particles.In some embodiments, administering a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual results in a change in central retinal thickness (CRT) or central regional retinal thickness (CST) of about -152.7 μm compared to the central retinal thickness (CRT) or central regional retinal thickness (CST) before administration of the unit dose of rAAV particles. In some embodiments, administering a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual results in a change in central retinal thickness (CRT) or central regional retinal thickness (CST) of about -153.3 μm compared to the central retinal thickness (CRT) or central regional retinal thickness (CST) before administration of the unit dose of rAAV particles. In some embodiments, administering a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual results in a change in central retinal thickness (CRT) or central regional retinal thickness (CST) of approximately -149.8 μm compared to the central retinal thickness (CRT) or central regional retinal thickness (CST) before administration of the unit dose of rAAV particles.

[0135] In some embodiments, the change (e.g., decrease) in central foveal thickness (CRT) or central regional retinal thickness (CST) compared to the CRT or CST before administration of the unit dose of rAAV particles is present at any time point about 1 day, about 1 week, about 2 weeks, about 4 weeks, about 8 weeks, about 16 weeks, about 24 weeks, about 30 weeks, about 32 weeks, about 34 weeks, about 40 weeks, about 44 weeks, about 48 weeks, about 52 weeks, or more after administration of the unit dose of rAAV particles to one eye and / or the contralateral eye of the individual. In some embodiments, a change (e.g., a decrease) in central foveal thickness (CRT) or central regional retinal thickness (CST) compared to the CRT or CST before administration of the unit dose of rAAV particles is observed at about 1 day, about 1 week, about 2 weeks, about 4 weeks, about 8 weeks, about 12 weeks, about 16 weeks, about 20 weeks after administration of the unit dose of rAAV particles to one eye and / or the contralateral eye of the individual. , about 24 weeks, about 28 weeks, about 32 weeks, about 36 weeks, about 40 weeks, about 44 weeks, about 48 weeks, about 52 weeks, about 56 weeks, about 60 weeks, about 64 weeks, about 68 weeks, about 72 weeks, about 76 weeks, about 80 weeks, about 84 weeks, about 88 weeks, about 92 weeks, about 96 weeks, about 100 weeks, about 104 weeks, about 108 weeks, or more.

[0136] In some embodiments, administering a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual results in a reduction of central retinal thickness (CRT) or central regional retinal thickness (CST) by more than about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% compared to the retinal thickness before administration of the unit dose of rAAV particles. In some embodiments, administering a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual results in a reduction of central retinal thickness (CRT) or central regional retinal thickness (CST) by at least about 10% compared to the retinal thickness before administration of the unit dose of rAAV particles. In some embodiments, administering a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual results in a decrease in central retinal thickness (CRT) or central zone retinal thickness (CST) of about 15% or more compared to the retinal thickness before administration of the unit dose of rAAV particles.

[0137] In some embodiments, administering a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual results in an increase in central retinal thickness (CRT) or central regional retinal thickness (CST) of less than about 40 μm (e.g., less than about 40 μm, less than about 35 μm, less than about 30 μm, less than about 25 μm, less than about 20 μm, less than about 15 μm, less than about 10 μm, less than about 5 μm, or less than about 1 μm) compared to the retinal thickness before administration of the unit dose of rAAV particles. In some embodiments, administering a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual results in an increase in central retinal thickness (CRT) or central regional retinal thickness (CST) of about 32 μm or less compared to the retinal thickness before administration of the unit dose of rAAV particles.

[0138] In some embodiments, the maintenance, reduction, or increase in retinal thickness compared to the retinal thickness before administration of the unit dose of rAAV particles is present at about 30 weeks or more after administration of the unit dose of rAAV particles to one eye and / or the contralateral eye of the individual. In some embodiments, the maintenance, reduction, or increase in retinal thickness compared to the retinal thickness before administration of the unit dose of rAAV particles is present at any time point about 30 weeks, about 34 weeks, about 44 weeks, about 6 months, about 1 year, about 1.5 years, about 2 years, about 3 years, about 5 years, about 10 years, or more after administration of the unit dose of rAAV particles to one eye and / or the contralateral eye of the individual.

[0139] In some embodiments, administering a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual results in a maintenance or reduction in macular volume compared to the macular volume before administration of the unit dose of rAAV particles. In some embodiments, administering a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual results in a reduction in macular volume compared to the macular volume before administration of the unit dose of rAAV particles. In some embodiments, administering a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual results in a reduction in macular volume of greater than any of about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, or about 50% compared to the macular volume before administration of the unit dose of rAAV particles. In some embodiments, administering a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual results in a decrease in macular volume of at least about 10% compared to the macular volume before administration of the unit dose of rAAV particles. In some embodiments, the macular volume is determined by OCT or SD-OCT. In some embodiments, administering a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual results in a decrease in macular volume of at least about 10% compared to the macular volume before administration of the unit dose of rAAV particles. In some embodiments, administering a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual results in a decrease in macular volume of about 15% or more compared to the macular volume before administration of the unit dose of rAAV particles. In some embodiments, the macular volume is determined by OCT or SD-OCT.

[0140] In some embodiments, the maintenance or reduction in macular volume compared to the macular volume before administration of the unit dose of rAAV particles is present at about 30 weeks or more after administration of the unit dose of rAAV particles to one eye and / or the contralateral eye of the individual. In some embodiments, the maintenance or reduction in macular volume compared to the macular volume before administration of the unit dose of rAAV particles is present at any time point about 30 weeks, about 34 weeks, about 44 weeks, about 6 months, about 1 year, about 1.5 years, about 2 years, about 3 years, about 5 years, about 10 years, or more after administration of the unit dose of rAAV particles to one eye and / or the contralateral eye of the individual.

[0141] In some embodiments, administering a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual results in maintained or improved vision compared to the vision before administration of the unit dose of rAAV particles. In some embodiments, administering a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual results in improved vision compared to the vision before administration of the unit dose of rAAV particles. In some embodiments, administering a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual results in an improvement in vision of greater than about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 125%, about 150%, about 175%, about 200%, about 225%, about 250%, about 275%, about 300%, or more, compared to the vision before administration of the unit dose of rAAV particles. In some embodiments, the visual acuity is best-corrected visual acuity (BCVA). In some embodiments, administering a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual results in an improvement in BCVA compared to the BCVA before administration of the unit dose of rAAV particles. In some embodiments, BCVA is expressed as an ETDRS score, which corresponds to the number of letters read correctly (Vitale et al., (2016) JAMA Opthalmol 134(9):1041:1047).

[0142] In some embodiments, administering a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual results in an improvement in BCVA of at least 15 letters (Vitale et al., (2016) JAMA Opthalmol 134 (9): 1041: 1047) (e.g., at least about 15 letters, at least about 20 letters) on the ETDRS compared to the BCVA before administration of the unit dose of rAAV particles. In some embodiments, administering a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual results in an improvement in BCVA of about 1 to about 15 letters (e.g., about 1 letter, about 2 letters, about 3 letters, about 4 letters, about 5 letters, about 6 letters, about 7 letters, about 8 letters, about 9 letters, about 10 letters, about 11 letters, about 12 letters, about 13 letters, about 14 letters, or about 15 letters) on the ETDRS compared to the BCVA before administration of the unit dose of rAAV particles. In some embodiments, administering a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual results in an improvement in BCVA of about 5 letters on the ETDRS compared to the BCVA before administration of the unit dose of rAAV particles. In some embodiments, administering a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual results in an improvement in BCVA of about 1 letter, about 2 letters, about 3 letters, about 4 letters, about 5 letters, about 6 letters, or about 7 letters on the ETDRS compared to the BCVA before administration of the unit dose of rAAV particles. In some embodiments, administering a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual results in an improvement in BCVA of about 3 letters or more on the ETDRS compared to the BCVA before administration of the unit dose of rAAV particles. In some embodiments, administering a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual results in an improvement in BCVA of about 4 letters or more on the ETDRS compared to the BCVA before administration of the unit dose of rAAV particles. In some embodiments, administering a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual results in an improvement in BCVA of about 5.1 letters or greater on the ETDRS compared to the BCVA before administration of the unit dose of rAAV particles. In some embodiments, administering a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual results in an improvement in BCVA of about 6.4 letters or greater on the ETDRS compared to the BCVA before administration of the unit dose of rAAV particles.In some embodiments, administering a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual results in an improvement in BCVA of about 6.8 letters on the ETDRS compared to the BCVA before administration of the unit dose of rAAV particles. In some embodiments, administering a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual results in an improvement in BCVA of about 8.8 letters on the ETDRS compared to the BCVA before administration of the unit dose of rAAV particles. In some embodiments, administering a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual results in an improvement in BCVA of about 2.3 letters on the ETDRS compared to the BCVA before administration of the unit dose of rAAV particles.

[0143] In some embodiments, administering a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual results in maintenance of BCVA compared to the BCVA before administration of the unit dose of rAAV particles, wherein the individual experiences a loss of less than 15 ETDRS letters (Vitale et al., 2004). al., (2016) JAMA Opthalmol 134 (9): 1041: 1047) (e.g., 15 characters or or less, 14 or less, 13 or less, 12 or less, 11 or less, 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, 2 or less, 1 letter, or 0 letters). In some embodiments, administering a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual results in maintained BCVA compared to the BCVA before administration of the unit dose of rAAV particles, wherein the individual's letter reduction is about 2 letters. In some embodiments, administering a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual results in maintained BCVA compared to the BCVA before administration of the unit dose of rAAV particles, and wherein the individual's ETDRS letter reduction is about 1 letter, about 2 letters, about 3 letters, about 4 letters, about 5 letters, about 6 letters, about 7 letters, about 8 letters, or about 9 letters. In some embodiments, administering a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual results in maintained BCVA compared to the BCVA before administration of the unit dose of rAAV particles, and wherein the individual's letter reduction is 0 letters. In some embodiments, administering a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual results in maintained BCVA compared to the BCVA before administration of the unit dose of rAAV particles, and wherein the individual's letter reduction is about 1 letter. In some embodiments, administering a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual results in maintained BCVA compared to the BCVA before administration of the unit dose of rAAV particles, wherein the individual has a letter reduction of about 2.7 letters. In some embodiments, administering a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual results in maintained BCVA compared to the BCVA before administration of the unit dose of rAAV particles, wherein the individual has a letter reduction of about 2.8 letters.In some embodiments, administering a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual results in maintained BCVA compared to the BCVA before administration of the unit dose of rAAV particles, wherein the individual experiences a letter reduction of about 2 or less. In some embodiments, administering a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual results in maintained BCVA compared to the BCVA before administration of the unit dose of rAAV particles, wherein the individual experiences a letter reduction of about 3.2 or less. In some embodiments, administering a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual results in maintenance of BCVA compared to the BCVA before administration of the unit dose of rAAV particles, wherein the individual experiences a letter loss of between about 15 and about 0 letters (e.g., about 15 letters, about 14 letters, about 13 letters, about 12 letters, about 11 letters, about 10 letters, about 9 letters, about 8 letters, about 7 letters, about 6 letters, about 5 letters, about 4 letters, about 3 letters, about 2 letters, about 1 letter, or 0 letters). In some embodiments, administering a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual results in a maintained BCVA compared to the BCVA before administration of the unit dose of rAAV particles, wherein the individual experiences a letter loss of about 10 to about 0 letters (e.g., about 10 letters, about 9 letters, about 8 letters, about 7 letters, about 6 letters, about 5 letters, about 4 letters, about 3 letters, about 2 letters, about 1 letter, or 0 letters). In some embodiments, administering a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual results in a maintained BCVA compared to the BCVA before administration of the unit dose of rAAV particles, wherein the individual experiences a letter loss of about 5 to about 0 letters (e.g., about 5 letters, about 4 letters, about 3 letters, about 2 letters, about 1 letter, or 0 letters). In some embodiments, administering a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual results in maintenance of BCVA compared to the BCVA before administration of the unit dose of rAAV particles, wherein the individual experiences a letter loss of between about 4 and about 0 letters (e.g., about 4 letters, about 3 letters, about 2 letters, about 1 letter, or 0 letters).In some embodiments, administering a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual results in maintained BCVA compared to the BCVA before administration of the unit dose of rAAV particles, wherein the individual experiences a letter reduction of about 3 to about 0 letters (e.g., about 3 letters, about 2 letters, about 1 letter, or 0 letters). In some embodiments, administering a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual results in maintained BCVA compared to the BCVA before administration of the unit dose of rAAV particles, wherein the individual experiences a letter reduction of about 2 to about 0 letters (e.g., about 2 letters, about 1 letter, or 0 letters). In some embodiments, administering a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual results in maintained BCVA compared to the BCVA before administration of the unit dose of rAAV particles, wherein the individual experiences a letter reduction of about 1 to about 0 letters.

[0144] In some embodiments, administering a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual results in a change in BCVA of about -20 to +7 letters or more on the ETDRS compared to the BCVA before administration of the unit dose of rAAV particles (e.g., any of -20, -19, -18, -17, -16, -15, -14, -13, -12, -11, -10, -9, -8, -7, -6, -5, -4, -3, -2, -1, 0, +1, +2, +3, +4, +5, +6, +7, +8, +9, +10, +11, +12, +13, +14, +15, +16, +17, +18, +19, +20, or more). In some embodiments, administering a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual results in an increase in BCVA of about 16 letters, 7 letters, or 5 letters on the ETDRS compared to the BCVA before administration of the unit dose of rAAV particles. In some embodiments, administering a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual results in an increase in BCVA of about 19 letters, 14 letters, 7 letters, 6 letters, 5 letters, 4 letters, 3 letters, 2 letters, or 1 letter on the ETDRS compared to the BCVA before administration of the unit dose of rAAV particles. In some embodiments, administering a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual results in an increase in BCVA of about 4.8 letters or about 0.8 letters on the ETDRS compared to the BCVA before administration of the unit dose of rAAV particles. In some embodiments, administering a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual results in a decrease in BCVA of about 2 letters or less on the ETDRS compared to the BCVA before administration of the unit dose of rAAV particles. In some embodiments, administering a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual results in a decrease in BCVA of about 3.2 letters or less on the ETDRS compared to the BCVA before administration of the unit dose of rAAV particles.

[0145] In some embodiments, administering a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual results in a change in BCVA of about -15 to +7 letters or more on the ETDRS compared to the BCVA before administration of the unit dose of rAAV particles (e.g., any of -15 letters, -14 letters, -13 letters, -12 letters, -11 letters, -10 letters, -9 letters, -8 letters, -7 letters, -6 letters, -5 letters, -4 letters, -3 letters, -2 letters, -1 letter, 0 letters, +1 letter, +2 letters, +3 letters, +4 letters, +5 letters, +6 letters, +7 letters, +8 letters, +9 letters, +10 letters, +11 letters, +12 letters, +13 letters, +14 letters, +15 letters, +16 letters, +17 letters, +18 letters, +19 letters, +20 letters, or more). In some embodiments, administering a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual results in a change in BCVA of about -10 to +7 letters or more on the ETDRS compared to the BCVA before administration of the unit dose of rAAV particles (e.g., any of -10 letters, -9 letters, -8 letters, -7 letters, -6 letters, -5 letters, -4 letters, -3 letters, -2 letters, -1 letter, 0 letters, +1 letter, +2 letters, +3 letters, +4 letters, +5 letters, +6 letters, +7 letters, +8 letters, +9 letters, +10 letters, +11 letters, +12 letters, +13 letters, +14 letters, +15 letters, +16 letters, +17 letters, +18 letters, +19 letters, +20 letters, or more). In some embodiments, administering a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual results in a change in BCVA of about -5 to +7 letters or more on the ETDRS compared to the BCVA before administration of the unit dose of rAAV particles (e.g., any of -5 letters, -4 letters, -3 letters, -2 letters, -1 letter, 0 letters, +1 letter, +2 letters, +3 letters, +4 letters, +5 letters, +6 letters, +7 letters, +8 letters, +9 letters, +10 letters, +11 letters, +12 letters, +13 letters, +14 letters, +15 letters, +16 letters, +17 letters, +18 letters, +19 letters, +20 letters, or more).In some embodiments, administering a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual results in a change in BCVA of about -4 to +7 letters or more on the ETDRS compared to the BCVA before administration of the unit dose of rAAV particles (e.g., any of -4 letters, -3 letters, -2 letters, -1 letter, 0 letters, +1 letter, +2 letters, +3 letters, +4 letters, +5 letters, +6 letters, +7 letters, +8 letters, +9 letters, +10 letters, +11 letters, +12 letters, +13 letters, +14 letters, +15 letters, +16 letters, +17 letters, +18 letters, +19 letters, +20 letters, or more). In some embodiments, administering a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual results in a change in BCVA of about -3 to +7 letters or more on the ETDRS compared to the BCVA before administration of the unit dose of rAAV particles (e.g., any of -3 letters, -2 letters, -1 letter, 0 letters, +1 letter, +2 letters, +3 letters, +4 letters, +5 letters, +6 letters, +7 letters, +8 letters, +9 letters, +10 letters, +11 letters, +12 letters, +13 letters, +14 letters, +15 letters, +16 letters, +17 letters, +18 letters, +19 letters, +20 letters, or more). In some embodiments, administering a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual results in a change in BCVA of about -2 to +7 letters or more on the ETDRS compared to the BCVA before administration of the unit dose of rAAV particles (e.g., any of -2 letters, -1 letter, 0 letters, +1 letter, +2 letters, +3 letters, +4 letters, +5 letters, +6 letters, +7 letters, +8 letters, +9 letters, +10 letters, +11 letters, +12 letters, +13 letters, +14 letters, +15 letters, +16 letters, +17 letters, +18 letters, +19 letters, +20 letters, or more).In some embodiments, administering a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual results in a change in BCVA of about -1 to +7 letters or more on the ETDRS compared to the BCVA before administration of the unit dose of rAAV particles (e.g., any of -1 letter, 0 letter, +1 letter, +2 letter, +3 letter, +4 letter, +5 letter, +6 letter, +7 letter, +8 letter, +9 letter, +10 letter, +11 letter, +12 letter, +13 letter, +14 letter, +15 letter, +16 letter, +17 letter, +18 letter, +19 letter, +20 letter, or more). In some embodiments, administering a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual results in a change in BCVA of about 0 to +7 letters or more (e.g., any of 0 letters, +1 letter, +2 letters, +3 letters, +4 letters, +5 letters, +6 letters, +7 letters, +8 letters, +9 letters, +10 letters, +11 letters, +12 letters, +13 letters, +14 letters, +15 letters, +16 letters, +17 letters, +18 letters, +19 letters, +20 letters, or more) on the ETDRS compared to the BCVA before administration of the unit dose of rAAV particles. In some embodiments, administering a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual results in a change in BCVA of about +1 to +7 letters or more on the ETDRS compared to the BCVA before administration of the unit dose of rAAV particles (e.g., any of +1 letter, +2 letters, +3 letters, +4 letters, +5 letters, +6 letters, +7 letters, +8 letters, +9 letters, +10 letters, +11 letters, +12 letters, +13 letters, +14 letters, +15 letters, +16 letters, +17 letters, +18 letters, +19 letters, +20 letters, or more). In some embodiments, administering a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual results in a change in BCVA of about +2 to +7 letters or more on the ETDRS compared to the BCVA before administration of the unit dose of rAAV particles (e.g., any of +2 letters, +3 letters, +4 letters, +5 letters, +6 letters, +7 letters, +8 letters, +9 letters, +10 letters, +11 letters, +12 letters, +13 letters, +14 letters, +15 letters, +16 letters, +17 letters, +18 letters, +19 letters, +20 letters, or more).In some embodiments, administering a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual results in a change in BCVA of about +3 to +7 letters or more (e.g., +3 letters, +4 letters, +5 letters, +6 letters, +7 letters, +8 letters, +9 letters, +10 letters, +11 letters, +12 letters, +13 letters, +14 letters, +15 letters, +16 letters, +17 letters, +18 letters, +19 letters, +20 letters, or more) on the ETDRS compared to the BCVA before administration of the unit dose of rAAV particles. In some embodiments, administering a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual results in a change in BCVA of about +4 to +7 letters or more (e.g., any of +4 letters, +5 letters, +6 letters, +7 letters, +8 letters, +9 letters, +10 letters, +11 letters, +12 letters, +13 letters, +14 letters, +15 letters, +16 letters, +17 letters, +18 letters, +19 letters, +20 letters, or more) on the ETDRS compared to the BCVA before administration of the unit dose of rAAV particles. In some embodiments, administering a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual results in a change in BCVA of about +5 to +7 letters or more (e.g., +5 letters, +6 letters, +7 letters, +8 letters, +9 letters, +10 letters, +11 letters, +12 letters, +13 letters, +14 letters, +15 letters, +16 letters, +17 letters, +18 letters, +19 letters, +20 letters, or more) on the ETDRS compared to the BCVA before administration of the unit dose of rAAV particles. In some embodiments, administering a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual results in a change in BCVA of about +6 letters or about +7 letters on the ETDRS compared to the BCVA before administration of the unit dose of rAAV particles.

[0146] In some embodiments, administration of a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual results in transient inflammation (e.g., inflammation driven by aqueous humor cells and / or vitreous cells, aqueous flare, posterior synechiae, hypodilated pupils). In some embodiments, administration of a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual results in inflammation (e.g., inflammation driven by aqueous humor cells and / or vitreous cells, aqueous flare, posterior synechiae, hypodilated pupils) that improves after administration of oral and / or topical steroid treatment and / or mydriatic medication. In some embodiments, administration of a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual results in inflammation (e.g., inflammation driven by aqueous humor cells and / or vitreous cells) that resolves after administration of oral and / or topical steroid treatment. Inflammation (e.g., aqueous humor and / or vitreous cell-driven inflammation, aqueous humor flare, posterior synechiae, hypodilation) can be measured using any method known in the art, such as slit lamp examination.

[0147] In some embodiments, the maintenance or improvement in visual acuity (e.g., BCVA) compared to the visual acuity before administration of the unit dose of rAAV particles is present at any time point about 1 day, about 1 week, about 2 weeks, about 4 weeks, about 8 weeks, about 12 weeks, about 16 weeks, about 20 weeks, about 24 weeks, about 28 weeks, about 32 weeks, about 36 weeks, about 40 weeks, about 44 weeks, about 48 weeks, about 52 weeks, about 56 weeks, about 60 weeks, about 64 weeks, about 68 weeks, about 72 weeks, about 76 weeks, about 80 weeks, about 84 weeks, about 88 weeks, about 92 weeks, about 96 weeks, about 100 weeks, about 104 weeks, about 108 weeks, or more after administration of the unit dose of rAAV particles to one eye and / or the contralateral eye of the individual. In some embodiments, the maintenance or improvement in visual acuity (e.g., BCVA) compared to the visual acuity before administration of the unit dose of rAAV particles is present at about 30 weeks or more after administration of the unit dose of rAAV particles to one eye and / or the contralateral eye of the individual. In some embodiments, the maintenance or improvement in visual acuity (e.g., BCVA) compared to the visual acuity before administration of the unit dose of rAAV particles is present at any time point about 30 weeks, about 34 weeks, about 44 weeks, about 6 months, about 1 year, about 1.5 years, about 2 years, about 3 years, about 5 years, about 10 years, or more after administration of the unit dose of rAAV particles to one eye and / or the contralateral eye of the individual.

[0148] In some embodiments, treatment of an ocular neovascular disease in an individual after administration of a unit dose of rAAV particles to one eye and / or the other eye is assessed based on best corrected visual acuity (BCVA) in one eye and / or the other eye. In some embodiments, BCVA is expressed as an ETDRS score, which corresponds to the number of letters read correctly (Vitale et al., (2016) JAMA Opthalmol 134(9):1041:1047). In some embodiments, an individual is determined to have preserved vision and / or visual acuity if they have a decrease in ETDRS score of less than 15 letters (e.g., 15 or less, 14 or less, 13 or less, 12 or less, 11 or less, 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, 2 or less, 1 letter, or 0 letters) compared to before administration of a unit dose of rAAV particles to one eye and / or the contralateral eye. In some embodiments, an individual is determined to have improved vision and / or visual acuity if they have an increase of at least 15 letters (e.g., at least about 15, at least about 20, at least about 30, at least about 40, at least about 50, at least about 60, or at least about 70 letters) compared to before administration of a unit dose of rAAV particles to one eye and / or the contralateral eye.

[0149] In some embodiments, whether an intraocular neovascular disease has been treated in an individual after administration of a unit dose of rAAV particles to one eye and / or the contralateral eye is assessed based on the central retinal thickness (CST) or central retinal thickness (CRT) in one eye and / or the contralateral eye. In some embodiments, the CST or CRT is determined by SD-OCT. In some embodiments, whether an intraocular neovascular disease has been treated in an individual after administration of a unit dose of rAAV particles to one eye and / or the contralateral eye is determined if the CST or CRT, as assessed by SD-OCT, is reduced after administration of a unit dose of rAAV particles to one eye and / or the contralateral eye compared to before administration of the unit dose of rAAV particles to the one eye and / or the contralateral eye. In some embodiments, it is determined that an intraocular neovascular disease has been treated in an individual after administration of a unit dose of rAAV particles to one eye and / or the contralateral eye if the CST or CRT, as assessed by SD-OCT, is maintained after administration of a unit dose of rAAV particles to one eye and / or the contralateral eye compared to before administration of the unit dose of rAAV particles to one eye and / or the contralateral eye.

[0150] In some embodiments, whether an intraocular neovascular disease has been treated in an individual after administration of a unit dose of rAAV particles to one eye and / or the contralateral eye is assessed based on the macular volume in one eye and / or the contralateral eye. In some embodiments, the macular volume is determined by SD-OCT. In some embodiments, whether an intraocular neovascular disease has been treated in an individual after administration of a unit dose of rAAV particles to one eye and / or the contralateral eye is determined if the macular volume assessed by SD-OCT decreases after administration of a unit dose of rAAV particles to one eye and / or the contralateral eye compared to before administration of the unit dose of rAAV particles to the one eye and / or the contralateral eye. In some embodiments, an intraocular neovascular disease is determined to be treated in an individual after administration of a unit dose of rAAV particles to one eye and / or the contralateral eye if the macular volume assessed by SD-OCT is maintained after administration of a unit dose of rAAV particles to one eye and / or the contralateral eye compared to before administration of the unit dose of rAAV particles to one eye and / or the contralateral eye.

[0151] In some embodiments, whether an intraocular neovascular disease has been treated in an individual after administration of a unit dose of rAAV particles to one eye and / or the contralateral eye is assessed based on the retinal thickness (e.g., central retinal thickness (CRT) or central zone retinal thickness (CST)) and macular volume in one eye and / or the contralateral eye. In some embodiments, the CST and macular volume are determined by SD-OCT. In some embodiments, whether an intraocular neovascular disease has been treated in an individual after administration of a unit dose of rAAV particles to one eye and / or the contralateral eye is determined if the CST and macular volume assessed by SD-OCT are reduced after administration of a unit dose of rAAV particles to one eye and / or the contralateral eye compared to before administration of the unit dose of rAAV particles to the one eye and / or the contralateral eye. In some embodiments, an intraocular neovascular disease is determined to be treated in an individual after administration of a unit dose of rAAV particles to one eye and / or the contralateral eye if the CST and macular volume assessed by SD-OCT are maintained after administration of a unit dose of rAAV particles to one eye and / or the contralateral eye compared to before administration of the unit dose of rAAV particles to one eye and / or the contralateral eye.

[0152] In some embodiments, whether an intraocular neovascular disease has been treated in an individual after administration of a unit dose of rAAV particles to one eye and / or the other eye is assessed based on the number of rescue treatments (e.g., aflibercept injections) required by the individual after administration of a unit dose of rAAV particles to one eye and / or the other eye. In some embodiments, if the individual requires less than one rescue treatment (e.g., aflibercept injection) every 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, or more after administration of a unit dose of rAAV particles to one eye and / or the other eye, it is determined that an intraocular neovascular disease has been treated in an individual after administration of a unit dose of rAAV particles to one eye and / or the other eye.

[0153] In some embodiments, an ocular neovascular disease is determined to be treated in an individual after administration of a unit dose of rAAV particles to one eye and / or the contralateral eye if the individual does not require any rescue therapeutic treatment (e.g., an aflibercept injection) for any of at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, at least 7 weeks, at least 8 weeks, at least 9 weeks, at least 10 weeks, at least 15 weeks, at least 20 weeks, at least 30 weeks, at least 40 weeks, at least 50 weeks, at least 60 weeks, at least 70 weeks, at least 80 weeks, at least 90 weeks, at least 100 weeks, at least 110 weeks, or longer, after administration of the unit dose of rAAV particles to one eye and / or the contralateral eye.

[0154] In some embodiments, the individual does not require any rescue therapy treatment (e.g., aflibercept injections) for any of at least about 24 months, at least about 23 months, at least about 22 months, at least about 21 months, at least about 20 months, at least about 19 months, at least about 18 months, at least about 17 months, at least about 16 months, at least about 15 months, at least about 14 months, at least about 13 months, at least about 12 months, at least about 11 months, at least about 10 months, at least about 9 months, at least about 8 months, at least about 7 months, at least about 6 months, at least about 5 months, at least about 4 months, at least about 3 months, at least about 2 months, at least about 1 month, at least about 3 weeks, at least about 2 weeks, or at least about 1 week after administration of a unit dose of rAAV particles to one eye and / or the contralateral eye. In some embodiments, the individual does not require any rescue treatment (e.g., aflibercept injection) for at least about 12 months after administration of a unit dose of rAAV particles to one eye and / or the other eye. In some embodiments, the individual does not require any rescue treatment (e.g., aflibercept injection) for at least about 10 months after administration of a unit dose of rAAV particles to one eye and / or the other eye. In some embodiments, the individual does not require any rescue treatment (e.g., aflibercept injection) for at least about 7 months after administration of a unit dose of rAAV particles to one eye and / or the other eye. In some embodiments, the individual does not require any rescue treatment (e.g., aflibercept injection) for at least about 6 months after administration of a unit dose of rAAV particles to one eye and / or the other eye. In some embodiments, the individual does not require any rescue therapy (e.g., aflibercept injections) for at least about two months after administration of a unit dose of rAAV particles to one eye and / or the contralateral eye. In some embodiments, the individual does not require any rescue therapy (e.g., aflibercept injections) for at least about one month after administration of a unit dose of rAAV particles to one eye and / or the contralateral eye.

[0155] In some embodiments, administration of a single unit dose of rAAV particles to one eye and / or contralateral eye of a plurality of individuals results in at least about 50% (e.g., any of at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, or 100%) of the individuals within the plurality not requiring anti-VEGF rescue treatment (e.g., aflibercept injections). In some embodiments, administration of a single unit dose of rAAV particles to one eye and / or the contralateral eye of a plurality of individuals results in at least about 67% (e.g., at least about 67%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, or 100%) of the individuals within the plurality not requiring anti-VEGF rescue treatment (e.g., an aflibercept injection). In some embodiments, administration of a single unit dose of rAAV particles to one eye and / or the contralateral eye of a plurality of individuals results in at least about 50% of the individuals within the plurality not requiring anti-VEGF rescue treatment (e.g., an aflibercept injection). In some embodiments, administration of a single unit dose of rAAV particles to one eye and / or the contralateral eye of a plurality of individuals results in at least about 78% of the individuals within the plurality not requiring anti-VEGF rescue treatment (e.g., an aflibercept injection). In some embodiments, administration of a single unit dose of rAAV particles to one eye and / or the contralateral eye of a plurality of individuals results in at least about 80% of the individuals in the plurality not requiring anti-VEGF rescue treatment (e.g., aflibercept injections). In some embodiments, administration of a single unit dose of rAAV particles to one eye and / or the contralateral eye of a plurality of individuals results in at least about 82% of the individuals in the plurality not requiring anti-VEGF rescue treatment (e.g., aflibercept injections). In some embodiments, administration of a single unit dose of rAAV particles to one eye and / or the contralateral eye of a plurality of individuals results in 100% of the individuals in the plurality not requiring anti-VEGF rescue treatment (e.g., aflibercept injections).

[0156] In some embodiments, administration of a single unit dose of rAAV particles to one eye and / or contralateral eye of a plurality of individuals results in at least about 50% (e.g., at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, or 100%) of the individuals within the plurality experiencing a cytopenia at least about 4 weeks after administration of the rAAV particles, e.g., at least about 4 weeks, at least about 8 weeks, at least about 12 weeks, at least about 16 weeks, at least about 20 weeks, at least about 24 weeks, at least about 30 weeks, at least about 32 weeks, at least about 34 weeks, at least about 36 weeks, at least about 38 weeks, at least about 39 weeks, at least about 40 weeks, at least about 41 weeks, at least about 42 weeks, at least about 43 weeks, at least about 44 weeks, at least about 45 weeks, at least about 46 weeks, at least about 47 weeks, at least about 48 weeks, at least about 49 weeks, at least about 50 weeks, at least about 51 weeks, at least about 52 weeks, at least about 53 weeks, at least about 54 weeks, at least about 55 weeks, at least about 56 weeks, at least about 57 weeks, at least about 58 weeks, at least about 59 weeks, at least about 60 weeks, at least about 61 weeks, at least about 62 weeks, at least about 63 weeks, at least about 64 weeks, at least about 65 weeks, at least about 66 weeks, at least about 67 weeks, at least about 68 weeks, at least about 69 weeks, at least about 70 weeks, at least about 71 Anti-VEGF rescue treatment (e.g., aflibercept injections) is not required for any of 4 weeks, at least about 28 weeks, at least about 32 weeks, at least about 36 weeks, at least about 40 weeks, at least about 44 weeks, at least about 48 weeks, at least about 52 weeks, at least about 56 weeks, at least about 60 weeks, at least about 64 weeks, at least about 68 weeks, at least about 72 weeks, at least about 76 weeks, at least about 80 weeks, at least about 84 weeks, at least about 88 weeks, at least about 92 weeks, at least about 96 weeks, at least about 100 weeks, at least about 104 weeks, at least about 108 weeks, or longer. In some embodiments, administration of a single unit dose of rAAV particles to one eye and / or the contralateral eye of a plurality of individuals results in at least about 50% of the individuals within the plurality not requiring anti-VEGF rescue treatment (e.g., aflibercept injections) for about 52 weeks or longer, or about 56 weeks or longer after administration of the rAAV particles.In some embodiments, administration of a single unit dose of rAAV particles to one eye and / or contralateral eye of a plurality of individuals results in at least about 67% (e.g., any of at least about 67%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, or 100%) of the individuals within the plurality not requiring anti-VEGF rescue treatment (e.g., aflibercept injections) for at least about 20 weeks after administration of the rAAV particles, e.g., any of at least about 20 weeks, at least about 24 weeks, at least about 28 weeks, at least about 32 weeks, at least about 36 weeks, at least about 40 weeks, at least about 44 weeks, at least about 48 weeks, at least about 52 weeks, at least about 56 weeks, at least about 60 weeks, at least about 64 weeks, at least about 66 weeks, or longer after administration of the rAAV particles. In some embodiments, administration of a single unit dose of rAAV particles to one eye and / or contralateral eye of a plurality of individuals results in at least about 78% of the individuals in the plurality experiencing a response at least about 4 weeks, e.g., at least about 4 weeks, at least about 8 weeks, at least about 12 weeks, at least about 16 weeks, at least about 20 weeks, at least about 24 weeks, at least about 28 weeks, at least about 32 weeks, at least about 36 weeks, at least about 40 weeks, at least about 44 weeks, at least about 50 weeks, at least about 52 weeks, at least about 54 weeks, at least about 56 weeks, at least about 58 weeks, at least about 59 weeks, at least about 60 weeks, at least about 61 weeks, at least about 62 weeks, at least about 63 weeks, at least about 64 weeks, at least about 65 weeks, at least about 66 weeks, at least about 67 weeks, at least about 68 weeks, at least about 69 weeks, at least about 70 weeks, at least about 71 weeks, at least about 72 weeks, at least about 73 weeks, at least about 74 weeks, at least about 75 weeks, at least about 76 weeks, at least about 77 weeks, at least about 78 weeks, at least about 79 weeks, at least about 80 weeks, at least about 81 weeks, at least about 82 weeks, at least about 83 weeks, at least about 84 weeks, at least about 85 weeks, at least about 86 weeks, at least about 87 weeks, at least about 88 weeks, at least about 89 weeks, at least about 90 weeks, at least about 91 weeks, at least about 92 weeks, at least about 93 weeks, at least about 94 weeks, at least about 95 weeks, at least about 96 weeks, at least about 97 weeks, at least about 98 weeks, at least about Anti-VEGF rescue treatment (e.g., aflibercept injections) is not required for any of about 48 weeks, at least about 52 weeks, at least about 56 weeks, at least about 60 weeks, at least about 64 weeks, at least about 68 weeks, at least about 72 weeks, at least about 76 weeks, at least about 80 weeks, at least about 84 weeks, at least about 88 weeks, at least about 92 weeks, at least about 96 weeks, at least about 100 weeks, at least about 104 weeks, at least about 108 weeks, or longer.In some embodiments, administration of a single unit dose of rAAV particles to one eye and / or contralateral eye of a plurality of individuals results in at least about 78% of the individuals within the plurality not requiring anti-VEGF rescue treatment (e.g., aflibercept injections) for about 20 weeks or longer, or about 36 weeks or longer after administration of the rAAV particles. In some embodiments, administration of a single unit dose of rAAV particles to one eye and / or contralateral eye of a plurality of individuals results in at least about 80% of the individuals in the plurality not requiring anti-VEGF rescue treatment (e.g., aflibercept injections) for at least about 20 weeks after administration of the rAAV particles, e.g., any of at least about 20 weeks, at least about 24 weeks, at least about 28 weeks, at least about 32 weeks, at least about 36 weeks, at least about 40 weeks, at least about 44 weeks, at least about 48 weeks, at least about 52 weeks, at least about 56 weeks, at least about 60 weeks, at least about 64 weeks, at least about 66 weeks, or longer after administration of the rAAV particles. In some embodiments, administration of a single unit dose of rAAV particles to one eye and / or contralateral eye of a plurality of individuals results in at least about 82% of the individuals within the plurality not requiring anti-VEGF rescue treatment (e.g., aflibercept injections) for at least about 20 weeks after administration of the rAAV particles, e.g., any of at least about 20 weeks, at least about 24 weeks, at least about 28 weeks, at least about 32 weeks, at least about 36 weeks, at least about 40 weeks, at least about 44 weeks, at least about 48 weeks, at least about 52 weeks, at least about 56 weeks, at least about 60 weeks, at least about 64 weeks, at least about 66 weeks, or longer after administration of the rAAV particles.In some embodiments, administration of a single unit dose of rAAV particles to one eye and / or contralateral eye of a plurality of individuals results in 100% of the individuals in the plurality not requiring anti-VEGF rescue treatment (e.g., aflibercept injections) for at least about 20 weeks after administration of the rAAV particles, e.g., any of at least about 20 weeks, at least about 24 weeks, at least about 28 weeks, at least about 32 weeks, at least about 36 weeks, at least about 40 weeks, at least about 44 weeks, at least about 48 weeks, at least about 52 weeks, at least about 56 weeks, at least about 60 weeks, at least about 64 weeks, at least about 66 weeks, or longer after administration of the rAAV particles. In some embodiments, administration of a single unit dose of rAAV particles to one eye and / or contralateral eye of a plurality of individuals results in 100% of the individuals in the plurality remaining healthy for at least about 4 weeks after administration of the rAAV particles, e.g., at least about 4 weeks, at least about 8 weeks, at least about 12 weeks, at least about 16 weeks, at least about 20 weeks, at least about 24 weeks, at least about 28 weeks, at least about 32 weeks, at least about 36 weeks, at least about 40 weeks, at least about 44 weeks, at least about 48 weeks, at least about 50 weeks, at least about 52 weeks, at least about 54 weeks, at least about 56 weeks, at least about 58 weeks, at least about 59 weeks, at least about 60 weeks, at least about 61 weeks, at least about 62 weeks, at least about 63 weeks, at least about 64 weeks, at least about 65 weeks, at least about 66 weeks, at least about 67 weeks, at least about 68 weeks, at least about 69 weeks, at least about 70 weeks, at least about 71 weeks, at least about 72 weeks, at least about 73 weeks, at least about 74 weeks, at least about 75 weeks, at least about 76 weeks, at least about 77 weeks, at least about 78 weeks, at least about 79 weeks, at least about 80 weeks, at least about 81 weeks, at least about 82 weeks, at least about 83 weeks, at least about 84 weeks, at least about 85 weeks, at least about 86 weeks, at least about 87 weeks, at least about 88 weeks, at least about 89 weeks, at least about 90 weeks, at least about 91 weeks, at least about 92 weeks, at least about 93 weeks, at least about 94 weeks, at least about 95 weeks, at least about 96 weeks, at least about 9 Anti-VEGF rescue treatment (e.g., aflibercept injections) is not required for any of 8 weeks, at least about 52 weeks, at least about 56 weeks, at least about 60 weeks, at least about 64 weeks, at least about 68 weeks, at least about 72 weeks, at least about 76 weeks, at least about 80 weeks, at least about 84 weeks, at least about 88 weeks, at least about 92 weeks, at least about 96 weeks, at least about 100 weeks, at least about 104 weeks, at least about 108 weeks, or longer. In some embodiments, administration of a single unit dose of rAAV particles to one eye and / or the contralateral eye of a plurality of individuals results in 100% of the individuals within the plurality not requiring anti-VEGF rescue treatment (e.g., aflibercept injections) for any of about 64 weeks or longer, 72 weeks or longer, or 84 weeks or longer after administration of the rAAV particles.

[0157] In some embodiments, administration of a unit dose of rAAV particles to one eye and / or the contralateral eye of a plurality of individuals results in about 78% or less of the individuals within the plurality (e.g., any of about 78% or less, about 75% or less, about 70% or less, about 65% or less, about 60% or less, about 55% or less, about 50% or less, about 45% or less, about 40% or less, about 35% or less, about 30% or less, about 25% or less, about 20% or less, about 15% or less, about 10% or less, about 5% or less, about 2.5% or less, about 1% or less, or about 0.5% or less) requiring rescue treatment (e.g., aflibercept injection) for the one eye and / or the contralateral eye. In some embodiments, administration of a unit dose of rAAV particles to one eye and / or the contralateral eye of a plurality of individuals results in about 50% or less (e.g., any of about 50% or less, about 45% or less, about 40% or less, about 35% or less, about 30% or less, about 25% or less, about 20% or less, about 15% or less, about 10% or less, about 5% or less, about 2.5% or less, about 1% or less, or about 0.5% or less) of the individuals within the plurality requiring some kind of rescue treatment (e.g., an aflibercept injection) to the one eye and / or the contralateral eye. In some embodiments, administration of a unit dose of rAAV particles to one eye and / or the contralateral eye of a plurality of individuals results in less than about 30% of the individuals (e.g., less than any of about 30%, about 25%, about 20%, about 15%, about 10%, about 5%, about 2.5%, about 1%, or about 0.5%) requiring rescue treatment (e.g., aflibercept injection) for the one eye and / or the contralateral eye. In some embodiments, administration of a unit dose of rAAV particles to one eye and / or the contralateral eye of a plurality of individuals results in less than about 30% of the individuals in the plurality requiring rescue treatment (e.g., aflibercept injection) for the one eye and / or the contralateral eye.In some embodiments, administration of a unit dose of rAAV particles to one eye and / or the contralateral eye of a plurality of individuals results in less than about 20% of the individuals in the plurality requiring rescue treatment (e.g., aflibercept injection) for the one eye and / or the contralateral eye. In some embodiments, administration of a unit dose of rAAV particles to one eye and / or the contralateral eye of a plurality of individuals results in 0% of the individuals in the plurality requiring rescue treatment (e.g., aflibercept injection) for the one eye and / or the contralateral eye.

[0158] In some embodiments, administration of a unit dose of rAAV particles to one eye and / or contralateral eye of a plurality of individuals results in about 78% or less of the individuals within the plurality (e.g., any of about 78% or less, about 75% or less, about 70% or less, about 65% or less, about 60% or less, about 55% or less, about 50% or less, about 45% or less, about 40% or less, about 35% or less, about 30% or less, about 25% or less, about 20% or less, about 15% or less, about 10% or less, about 5% or less, about 2.5% or less, about 1% or less, or about 0.5% or less) experiencing rAAV phenotype at least about 4 weeks after administration of the rAAV particles, e.g., rAAV phenotype. Rescue treatment (e.g., aflibercept injection) is required for at least about 4 weeks, at least about 8 weeks, at least about 12 weeks, at least about 16 weeks, at least about 20 weeks, at least about 24 weeks, at least about 28 weeks, at least about 32 weeks, at least about 36 weeks, at least about 40 weeks, at least about 44 weeks, at least about 48 weeks, at least about 52 weeks, at least about 56 weeks, at least about 60 weeks, at least about 64 weeks, at least about 68 weeks, at least about 72 weeks, at least about 76 weeks, at least about 80 weeks, at least about 84 weeks, at least about 88 weeks, at least about 92 weeks, at least about 96 weeks, at least about 100 weeks, at least about 104 weeks, at least about 108 weeks, or longer, after administration of the V particles.In some embodiments, administration of a unit dose of rAAV particles to one eye and / or contralateral eye of a plurality of individuals results in less than about 30% (e.g., less than any of about 30%, about 25%, about 20%, about 15%, about 10%, about 5%, about 2.5%, about 1%, or about 0.5%) of the individuals within the plurality requiring rescue treatment (e.g., aflibercept injection) for at least about 20 weeks after administration of the rAAV particles, e.g., at least about 20 weeks, at least about 24 weeks, at least about 28 weeks, at least about 32 weeks, at least about 36 weeks, at least about 40 weeks, at least about 44 weeks, at least about 48 weeks, at least about 52 weeks, at least about 56 weeks, at least about 60 weeks, at least about 64 weeks, at least about 66 weeks, or longer after administration of the rAAV particles. In some embodiments, administration of a unit dose of rAAV particles to one eye and / or the contralateral eye of a plurality of individuals results in less than about 30% of the individuals in the plurality requiring rescue treatment (e.g., an aflibercept injection) for one eye and / or the contralateral eye for at least about 20 weeks after administration of the rAAV particles, e.g., any of at least about 20 weeks, at least about 24 weeks, at least about 28 weeks, at least about 32 weeks, at least about 36 weeks, at least about 40 weeks, at least about 44 weeks, at least about 48 weeks, at least about 52 weeks, at least about 56 weeks, at least about 60 weeks, at least about 64 weeks, at least about 66 weeks, or longer after administration of the rAAV particles. In some embodiments, administration of a unit dose of rAAV particles to one eye and / or the contralateral eye of a plurality of individuals results in less than about 20% of the individuals in the plurality requiring rescue treatment (e.g., an aflibercept injection) for one eye and / or the contralateral eye for at least about 20 weeks after administration of the rAAV particles, e.g., at least about 20 weeks, at least about 24 weeks, at least about 28 weeks, at least about 32 weeks, at least about 36 weeks, at least about 40 weeks, at least about 44 weeks, at least about 48 weeks, at least about 52 weeks, at least about 56 weeks, at least about 60 weeks, at least about 64 weeks, at least about 66 weeks, or longer, after administration of the rAAV particles.In some embodiments, administration of a unit dose of rAAV particles to one eye and / or the contralateral eye of a plurality of individuals results in 0.1% of the individuals in the plurality requiring rescue treatment (e.g., an aflibercept injection) for one eye and / or the contralateral eye for at least about 20 weeks after administration of the rAAV particles, e.g., at least about 20 weeks, at least about 24 weeks, at least about 28 weeks, at least about 32 weeks, at least about 36 weeks, at least about 40 weeks, at least about 44 weeks, at least about 48 weeks, at least about 52 weeks, at least about 56 weeks, at least about 60 weeks, at least about 64 weeks, at least about 66 weeks, or longer, after administration of the rAAV particles. In some embodiments, administration of a unit dose of rAAV particles to one eye and / or contralateral eye of a plurality of individuals results in 0.1% of the individuals in the plurality experiencing a response at least about 4 weeks after administration of the rAAV particles, e.g., at least about 4 weeks, at least about 8 weeks, at least about 12 weeks, at least about 16 weeks, at least about 20 weeks, at least about 24 weeks, at least about 28 weeks, at least about 32 weeks, at least about 36 weeks, at least about 40 weeks, at least about 44 weeks, at least about 48 weeks, at least about 50 weeks, at least about 51 weeks, at least about 52 weeks, at least about 53 weeks, at least about 54 weeks, at least about 55 weeks, at least about 56 weeks, at least about 57 weeks, at least about 58 weeks, at least about 59 weeks, at least about 60 weeks, at least about 61 weeks, at least about 62 weeks, at least about 63 weeks, at least about 64 weeks, at least about 65 weeks, at least about 66 weeks, at least about 67 weeks, at least about 68 weeks, at least about 69 weeks, at least about 70 weeks, at least about 71 weeks, at least about 72 weeks, at least about 73 weeks, at least about 74 weeks, at least about 75 weeks, at least about 76 weeks, at least about 77 weeks, at least about 78 weeks, at least about 79 weeks, at least about 80 weeks, at least about 81 weeks, at least about 82 weeks, at least about 83 weeks, at least about 84 weeks, at least about 85 weeks, at least about 86 weeks, at least about 87 weeks, at least about 88 weeks, at least about 89 weeks, at least about 90 weeks, at least about 91 weeks, at least about 92 weeks, at least about 93 Requires some rescue treatment (e.g., aflibercept injection) in one eye and / or the contralateral eye for any of 2 weeks, at least about 56 weeks, at least about 60 weeks, at least about 64 weeks, at least about 68 weeks, at least about 72 weeks, at least about 76 weeks, at least about 80 weeks, at least about 84 weeks, at least about 88 weeks, at least about 92 weeks, at least about 96 weeks, at least about 100 weeks, at least about 104 weeks, at least about 108 weeks, or longer.

[0159] In some embodiments, administration of a unit dose of rAAV particles to one eye and / or the contralateral eye of a plurality of individuals results in a reduction in the average annual anti-VEGF injection rate by at least about 80%, at least about 85%, at least about 87%, at least about 90%, at least about 95%, at least about 99%, or 100% compared to the average annual anti-VEGF injection rate before administration of the unit dose of rAAV particles. In some embodiments, administration of a unit dose of rAAV particles to one eye and / or the contralateral eye of a plurality of individuals results in a reduction in the average annual anti-VEGF injection rate by about 87% or more compared to the average annual anti-VEGF injection rate before administration of the unit dose of rAAV particles. In some embodiments, administration of a unit dose of rAAV particles to one eye and / or the contralateral eye of a plurality of individuals results in a reduction in the average annual anti-VEGF injection rate by 100% compared to the average annual anti-VEGF injection rate before administration of the unit dose of rAAV particles.

[0160] In some embodiments, the average annual anti-VEGF injection rate prior to administration of a unit dose of rAAV particles is calculated according to the following formula: Annual rate before administration of a unit dose of rAAV particles = (number of anti-VEGF injections during the 12 months before administration of a unit dose of rAAV particles) / (number of days from the first anti-VEGF injection during the 12 months before administration of a unit dose of rAAV particles to administration of a unit dose of rAAV particles / 365.25)

[0161] In some embodiments, the average annual anti-VEGF injection rate after administration of a unit dose of rAAV particles is calculated according to the following formula: Annual rate after administration of a unit dose of rAAV particles = (number of anti-VEGF injections after administration of a unit dose of rAAV particles) / (number of days after administration of a unit dose of rAAV particles / 365.25).

[0162] In some embodiments, an individual is determined to require rescue treatment (e.g., an intravitreal anti-VEGF injection, such as an aflibercept injection) after administration of rAAV particles if the individual exhibits a 10-letter or greater decrease in BCVA (e.g., using an ETDRS protocol) due to intraretinal or subretinal fluid (e.g., as determined by SD-OCT) for one eye and / or the contralateral eye receiving the rAAV particles compared to the BCVA for the one eye and / or the contralateral eye receiving the rAAV particles prior to administration of the rAAV particles. In some embodiments, if the central retinal thickness (e.g., CST or CRT) of one eye and / or the contralateral eye receiving the rAAV particles is increased by, for example, SD-OCT, by more than 75 μm compared to the central retinal thickness of the one eye and / or the contralateral eye receiving the rAAV particles before administration of the rAAV particles, the individual is determined to require rescue treatment (e.g., intravitreal anti-VEGF injection, such as an aflibercept injection) after administration of the rAAV particles. In some embodiments, if the individual exhibits sight-threatening hemorrhage due to AMD in the one eye and / or the contralateral eye receiving the rAAV particles, the individual is determined to require rescue treatment (e.g., intravitreal anti-VEGF injection, such as an aflibercept injection) after administration of the rAAV particles.

[0163] In some embodiments, the rescue treatment comprises administering standard anti-VEGF treatment.Such standard anti-VEGF treatment comprises one or more anti-VEGF treatments (for example, anti-VEGF intravitreal injections).In some embodiments, the rescue treatment comprises one or more IVT injections of aflibercept.In some embodiments, the rescue treatment comprises one or more IVT injections of aflibercept, including about 2 mg of aflibercept.

[0164] In some embodiments, whether an intraocular neovascular disease has been treated in an individual after administration of a unit dose of rAAV particles to one eye and / or the other eye is assessed based on the level of retinal fluid compared to the level of retinal fluid before administration of a unit dose of rAAV particles to one eye and / or the other eye. In some embodiments, if a reduction in retinal fluid is observed after administration of a unit dose of rAAV particles to one eye and / or the other eye compared to the level of retinal fluid before administration of a unit dose of rAAV particles to one eye and / or the other eye, it is determined that an intraocular neovascular disease has been treated in an individual after administration of a unit dose of rAAV particles to one eye and / or the other eye. In some embodiments, the intraocular neovascular disease is AMD.

[0165] In some embodiments, whether an intraocular neovascular disease has been treated in an individual after administration of a unit dose of rAAV particles to one eye and / or the other eye is assessed based on the resolution of pigment epithelial detachment (PED) compared to the PED before administration of a unit dose of rAAV particles to one eye and / or the other eye. In some embodiments, if resolution of PED is observed after administration of a unit dose of rAAV particles to one eye and / or the other eye compared to the PED before administration of a unit dose of rAAV particles to one eye and / or the other eye, it is determined that an intraocular neovascular disease has been treated in an individual after administration of a unit dose of rAAV particles to one eye and / or the other eye. In some embodiments, the intraocular neovascular disease is wAMD.

[0166] In some embodiments, whether an intraocular neovascular disease has been treated in an individual after administration of a unit dose of rAAV particles to one eye and / or the contralateral eye is assessed based on the growth of choroidal neovascularization (CNV) lesions as determined by fluorescein angiography. In some embodiments, whether an intraocular neovascular disease has been treated in an individual after administration of a unit dose of rAAV particles to one eye and / or the contralateral eye is determined if the CNV lesions shrink (e.g., by more than about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, or 100%) after administration of a unit dose of rAAV particles to one eye and / or the contralateral eye compared to the CNV lesions present before administration of the unit dose of rAAV particles to the one eye and / or the contralateral eye. In some embodiments, it is determined that an intraocular neovascular disease has been treated in an individual after administration of a unit dose of rAAV particles to one eye and / or the other eye if the CNV lesions do not grow (e.g., grow by less than about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 15%, or about 20%) after administration of a unit dose of rAAV particles to one eye and / or the other eye compared to the CNV lesions present before administration of the unit dose of rAAV particles to the one eye and / or the other eye. In some embodiments, the intraocular neovascular disease is AMD.

[0167] In some embodiments, whether an intraocular neovascular disease has been treated in an individual after administration of a unit dose of rAAV particles to one eye and / or the contralateral eye is assessed based on anatomical features of the one eye and / or the contralateral eye using any method known in the art (e.g., SD-OCT, OCT, fluorescein angiography, digital color fundus photography, etc.). In some embodiments, if an improvement in the anatomical features of the one eye and / or the contralateral eye is observed after administration of a unit dose of rAAV particles to the one eye and / or the contralateral eye, it is determined that an intraocular neovascular disease has been treated in an individual after administration of a unit dose of rAAV particles to the one eye and / or the contralateral eye. In some embodiments, the intraocular neovascular disease is wAMD.

[0168] In some embodiments, whether an intraocular neovascular disease is treated in an individual after administration of a unit dose of rAAV particles to one eye and / or the contralateral eye is determined by ophthalmologic examination, intraocular pressure (e.g., using a Goldmann applanation tonometer, or a Tono-pen), indirect ophthalmoscopy, examination of one eye and / or the contralateral eye and ocular adnexa, eyelid and / or pupil responsiveness, ptosis, abnormal pupil shape, anisocoria, abnormal response to light, Assessment is based on afferent pupillary defect, slit lamp examination (including examination of the eyelid, conjunctiva, cornea, lens, iris, and anterior chamber), abnormalities of the vitreous body, optic nerve, peripheral retina, and posterior segment of the retinal vasculature, SD-OCT, fluorescein angiography, digital color fundus photography (including images of the retina, optic disc, and / or macula), aqueous humor sampling, vitreous humor sampling, OCT-angiography (OCT-A), refraction, and / or visual acuity (BCVA). In some embodiments, SD-OCT is performed to evaluate retinal thickness (e.g., central retinal thickness or central area retinal thickness), macular volume, and / or the presence of fluid (e.g., subretinal fluid or intraretinal fluid). In some embodiments, the intraocular neovascular disease is wAMD.

[0169] A unit dose of rAAV particles can be administered to one eye and / or the other eye of an individual by any method known in the art. For example, the unit dose of rAAV particles can be administered to one eye and / or the other eye of an individual by intraocular or intravitreal injection. In some embodiments, the administration of a unit dose of rAAV particles to one eye and / or the other eye of an individual is intraocular. In some embodiments, the administration of a unit dose of rAAV particles to one eye and / or the other eye of an individual is by intravitreal injection (IVT) or subretinal injection. In some embodiments, the administration of a unit dose of rAAV particles to one eye and / or the other eye of an individual is by IVT injection. In some embodiments, the unit dose of rAAV particles is administered by intravitreal injection using aseptic technique. In some embodiments, the unit dose of rAAV particles is administered by intravitreal injection using aseptic technique with povidone iodine. It is administered by injection.

[0170] In some embodiments, the individual has not received previous treatment for intraocular neovascular disease. In some embodiments, the individual has not received previous treatment for intraocular neovascular disease in one eye and / or the other eye. In some embodiments, the individual has not received previous treatment with an anti-VEGF agent (e.g., bevacizumab, brolucizumab, ranibizumab, faricimab, abicipar pegol, conbercept, OPT-302, KSI-301, injectable sunitinib maleate (GB-102), PAN-90806 (PanOptica), and / or aflibercept). In some embodiments, the individual has not received prior treatment with an anti-VEGF agent (e.g., bevacizumab, brolucizumab, ranibizumab, faricimab, abicipar pegol, conbercept, OPT-302, KSI-301, injectable sunitinib maleate (GB-102), PAN-90806 (PanOptica), and / or aflibercept) in one eye and / or the other eye. In some embodiments, the individual has not received prior aflibercept treatment. In some embodiments, the individual has not received prior aflibercept treatment in one eye and / or the other eye. steroid treatment

[0171] In some embodiments, the unit dose of rAAV particles is administered in combination with steroid treatment, hi some embodiments, the steroid treatment is corticosteroid treatment. Exemplary corticosteroids include aclometasone, amcinomide, beclomethasone, betamethasone, budesonide, ciclesonide, clobetasol, clobetasone, clocortolone, cloprednol, cortivazol, deflazacort, deoxycorticosterone, desonide, desoximetasone, dexamethasone, diflorasone, diflucortolone, difluprednate, fluchloron, fludrocortisone, fludroxycortide, flumethasone, flunisolide, fluocinolone acetonide, fluocinonide, fluocortin, fluocortolone, fluorometholone, fluperolone, fluticasone, fuprednidene, formocortal, halcinonide, halcinonide, halcinonide, halcinolone ... Steroid treatments include, but are not limited to, lometasone, hydrocortisone aceponate, hydrocortisone buteprate, hydrocortisone butyrate, loteprednol, medrysone, meprednisone, methylprednisolone, methylprednisolone aceponate, mometasone furoate, paramethasone, prednicarbate, prednisone, prednisolone, prednylidene, remexolone, tixocortol, triamcinolone, and urobetasol. In some embodiments, the steroid treatment is a systemic steroid treatment. In some embodiments, the steroid treatment is an oral steroid treatment. In some embodiments, the steroid treatment is an ophthalmic steroid treatment. In some embodiments, the ophthalmic steroid treatment is a topical steroid treatment (e.g., eye drops), a periocular steroid treatment (e.g., subtenon, subconjunctival), an intravitreal steroid treatment, or a suprachoroidal steroid treatment. In some embodiments, the topical steroid treatment is difluprednate treatment, medrysone treatment, loteprednol treatment, prednisolone treatment, fluocinolone treatment, triamcinolone treatment, rimexolone treatment, dexamethasone treatment, fluorometholone treatment, fluocinolone treatment, rimexolone treatment, or prednisone treatment. In some embodiments, the ophthalmic steroid treatment is difluprednate treatment. In some embodiments, the steroid treatment is prednisone treatment. In some embodiments, the steroid treatment is difluprednate treatment.

[0172] In some embodiments, the steroid treatment includes systemic steroid treatment and topical steroid treatment. In some embodiments, the systemic steroid treatment is oral steroid treatment. In some embodiments, the systemic steroid treatment is prednisone treatment. In some embodiments, the topical steroid treatment is difluprednate treatment. In some embodiments, the systemic steroid treatment and topical steroid treatment are administered simultaneously (e.g., on the same day). In some embodiments, the systemic steroid treatment and topical steroid treatment are administered separately (e.g., on different days).

[0173] In some embodiments, the steroid is administered before, during, and / or after administration of a unit dose of rAAV particles. In some embodiments, the steroid is administered before, during, and after administration of a unit dose of rAAV particles. In some embodiments, the steroid is administered during and after administration of a unit dose of rAAV particles. In some embodiments, the steroid is administered before administration of a unit dose of rAAV particles. In some embodiments, the steroid is administered during administration of a unit dose of rAAV particles. In some embodiments, the steroid is administered before and during administration of a unit dose of rAAV particles. In some embodiments, the steroid is administered after administration of a unit dose of rAAV particles. In some embodiments, the steroid is administered during and after administration of a unit dose of rAAV particles. In some embodiments, the steroid is administered before and / or after administration of a unit dose of rAAV particles. In some embodiments, the steroid is administered before and after administration of a unit dose of rAAV particles.

[0174] In some embodiments, the steroid treatment is systemic steroid treatment. In some embodiments, the systemic steroid treatment is oral steroid treatment.

[0175] In some embodiments, the steroid treatment is oral prednisone treatment. In some embodiments, oral prednisone treatment is initiated before administration of a unit dose of rAAV particles. In some embodiments, the initial oral prednisone treatment is administered at a dose of about 40 mg, about 45 mg, about 50 mg, about 55 mg, about 60 mg, about 65 mg, or about 70 mg / day of prednisone about 7 days, about 6 days, about 5 days, about 4 days, about 3 days, about 2 days, about 1 day, or 0 days before administration of a unit dose of rAAV particles, and lasts for about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, or about 10 days, or longer. In some embodiments, the initial oral prednisone treatment is administered at a dose of about 60 mg / day of prednisone about 3 days before administration of a unit dose of rAAV and lasts for about 3 days.

[0176] In some embodiments, after the initial oral prednisone treatment, the oral prednisone treatment is tapered. In some embodiments, the oral prednisone treatment is tapered at a dose of about 20 mg, about 25 mg, about 30 mg, about 35 mg, about 40 mg, about 45 mg, or about 50 mg per day of prednisone for a total of about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, or about 7 days, followed by about 10 mg, about 15 mg, about 20 mg, or about 25 mg per day of prednisone for about 1 day, about 2 days, about 3 days, or about 4 days, followed by about 5 mg, about 10 mg, or about 15 mg per day of prednisone for about 1 day, about 2 days, about 3 days, or about 4 days. In some embodiments, the tapering of the prednisone dose is administered at a dose of about 40 mg / day of prednisone for three days, followed by a dose of about 20 mg / day of prednisone for two days, followed by a dose of about 10 mg / day of prednisone for two days.

[0177] In some embodiments, the initial oral prednisone treatment is initiated 3 days prior to administration of the unit dose of rAAV particles at a dose of 60 mg / day of prednisone for a total of 6 days, followed by a dose of 40 mg / day of prednisone for a total of 3 days, followed by a dose of 20 mg / day of prednisone for 2 days, followed by a dose of 10 mg / day of prednisone for 2 days.

[0178] In some embodiments, the steroid treatment is an ophthalmic steroid treatment. In some embodiments, the ophthalmic steroid treatment is difluprednate treatment. In some embodiments, the steroid treatment is administered before, during, and / or after administration of a unit dose of rAAV particles. In some embodiments, the steroid treatment is administered before administration of a unit dose of rAAV particles. In some embodiments, the steroid treatment is administered during administration of a unit dose of rAAV particles. In some embodiments, the steroid treatment is administered after administration of a unit dose of rAAV particles. In some embodiments, the steroid treatment is administered before and during administration of a unit dose of rAAV particles. In some embodiments, the steroid treatment is administered before and after administration of a unit dose of rAAV particles. In some embodiments, the steroid treatment is administered during and after administration of a unit dose of rAAV particles. In some embodiments, the steroid treatment is administered before, during, and after administration of a unit dose of rAAV particles.

[0179] In some embodiments, the steroid treatment is an ophthalmic steroid treatment, e.g., a topical steroid treatment. In some embodiments, the ophthalmic steroid treatment, e.g., a topical steroid treatment, is a daily steroid treatment for up to 4 weeks, up to 6 weeks, up to 8 weeks, up to 3 months, up to 4 months, up to 5 months, or up to 6 months after administration of a unit dose of rAAV particles. In some embodiments, the topical steroid treatment comprises about four administrations of topical steroid at about week 1, about three administrations of topical steroid at about week 2, about two administrations of topical steroid at about week 3, and about one administration of topical steroid at about week 4, timing starting with and following administration of a unit dose of rAAV particles. In some embodiments, the topical steroid treatment comprises administering the topical steroid about four times per day (i.e., QID) for about three weeks after administration of the unit dose of rAAV particles, followed by administering the topical steroid about three times per day (i.e., TID) for about one week, followed by administering the topical steroid about two times per day (i.e., BID) for about one week, and then administering the topical steroid about once per day (i.e., QD) for about one week. In some embodiments, the ophthalmic steroid treatment is extended at the discretion of the treating physician.

[0180] In some embodiments, the ophthalmic steroid is about 0.005% to about 0.5% difluprednate. In some embodiments, the ophthalmic steroid is about 0.005%, about 0.006%, about 0.007%, about 0.008%, about 0.009%, about 0.01%, about 0.02%, about 0.03%, about 0.4%, about 0.05%, about 0.06%, about 0.07%, about 0.08%, about 0.09%, or about 0.1% difluprednate. In some embodiments, the ophthalmic steroid is 0.05% difluprednate. In some embodiments, the dose of 0.05% difluprednate is one drop of ophthalmic solution. In some embodiments, one drop is about 50 μl (e.g., about 25 μl to about 50 μl, or about 50 μl to about 100 μl). In some embodiments, the dose of difluprednate comprises about 1 μg to about 5 μg, or about 2 μg to about 3 μg, or about 2.5 μg difluprednate. In some embodiments, the dose of difluprednate comprises about 2.5 μg difluprednate.

[0181] In some embodiments, the topical steroid treatment comprises 7 weeks of topical steroid treatment, e.g., 0.05% difluprednate. In some embodiments, the topical steroid treatment comprises about 4 doses per day (i.e., QID) for about 4 weeks, followed by about 3 doses per day (i.e., TID) for about 1 week, followed by about 2 doses per day (i.e., BID) for about 1 week, and then about 1 dose per day (i.e., QD) for about 1 week, starting about 1 week before administration of the unit dose of rAAV particles. In some embodiments, the topical steroid treatment comprises about four administrations of topical steroid per day (i.e., QID) for about 28 days, followed by about three administrations of topical steroid per day (i.e., TID) for about 7 days, followed by about two administrations of topical steroid per day (i.e., BID) for about 7 days, and then about one administration of topical steroid per day (i.e., QD) for about 7 days, starting about 7 days before administration of the unit dose of rAAV particles. In some embodiments, the topical steroid treatment comprises about four administrations of topical steroid per day (i.e., QID) from day 1 to about day 28, followed by about three administrations of topical steroid per day (i.e., TID) from about day 29 to about day 35, followed by about two administrations of topical steroid per day (i.e., BID) from about day 36 to about day 42, and then about one administration of topical steroid per day (i.e., QD) from about day 43 to about day 49, starting on day 1. In some embodiments, if inflammation is present, the topical steroid treatment is continued.

[0182] In some embodiments, the treatment methods provided herein include administering an anti-VEGF agent (e.g., an IVT injection of aflibercept) to one eye of an individual before administering a unit dose of rAAV particles to one eye of the individual. In some embodiments, the anti-VEGF agent is administered about 7 days or about 1 week before administration of the unit dose of rAAV particles. In some embodiments, the anti-VEGF agent is administered on about day 1, and the unit dose of rAAV particles is administered on about day 8. In some embodiments, the topical steroid treatment includes 7 weeks of topical steroid treatment, for example, 0.05% difluprednate. In some embodiments, the topical steroid treatment comprises about four administrations of topical steroid per day (i.e., QID) for about four weeks, followed by about three administrations of topical steroid per day (i.e., TID) for about one week, followed by about two administrations of topical steroid per day (i.e., BID) for about one week, and about one administration of topical steroid per day (i.e., QD) for about one week, timing starting with and following administration of the anti-VEGF agent. In some embodiments, the topical steroid treatment comprises about four administrations of topical steroid per day (i.e., QID) for about 28 days, followed by about three administrations of topical steroid per day (i.e., TID) for about seven days, followed by about two administrations of topical steroid per day (i.e., BID) for about seven days, and about one administration of topical steroid per day (i.e., QD) for about seven days, timing starting with and following administration of the anti-VEGF agent. In some embodiments, the topical steroid treatment comprises about four administrations of topical steroid per day (i.e., QID) from day 1 to about day 28, followed by about three administrations of topical steroid per day (i.e., TID) from about day 29 to about day 35, followed by about two administrations of topical steroid per day (i.e., BID) from about day 36 to about day 42, and then about one administration of topical steroid per day (i.e., QD) from about day 43 to about day 49, starting on day 1. In some embodiments, if inflammation is present, the topical steroid treatment is continued.

[0183] In some embodiments, the topical steroid treatment comprises four months of topical steroid treatment, e.g., 0.05% difluprednate. In some embodiments, the topical steroid treatment comprises about four administrations of topical steroid per day (i.e., QID) for about one month, followed by about three administrations of topical steroid per day (i.e., TID) for about one month, followed by about two administrations of topical steroid per day (i.e., BID) for about one month, and then about one administration of topical steroid per day (i.e., QD) for about one month, starting about one week before administration of the unit dose of rAAV particles. In some embodiments, the topical steroid treatment comprises about four administrations of topical steroid per day (i.e., QID) for about 30 days, followed by about three administrations of topical steroid per day (i.e., TID) for about 30 days, followed by about two administrations of topical steroid per day (i.e., BID) for about 30 days, and then about one administration of topical steroid per day (i.e., QD) for about 30 days, starting about 7 days before administration of the unit dose of rAAV particles. In some embodiments, the topical steroid treatment comprises about four administrations of topical steroid per day (i.e., QID) from day 1 to about day 30, followed by about three administrations of topical steroid per day (i.e., TID) from about day 31 to about day 60, followed by about two administrations of topical steroid per day (i.e., BID) from about day 61 to about day 90, and then about one administration of topical steroid per day (i.e., QD) from about day 91 to about day 120, starting on day 1. In some embodiments, if inflammation is present, the topical steroid treatment is continued.

[0184] In some embodiments, the treatment methods provided herein include administering an anti-VEGF agent (e.g., an IVT injection of aflibercept) to one eye of an individual before administering a unit dose of rAAV particles to one eye of the individual. In some embodiments, the anti-VEGF agent is administered about 7 days or about 1 week before administration of the unit dose of rAAV particles. In some embodiments, the anti-VEGF agent is administered on about day 1, and the unit dose of rAAV particles is administered on about day 8. In some embodiments, the topical steroid treatment includes 4 months of topical steroid treatment, for example, 0.05% difluprednate. In some embodiments, the topical steroid treatment comprises about four administrations of topical steroid per day (i.e., QID) for about one month, followed by about three administrations of topical steroid per day (i.e., TID) for about one month, followed by about two administrations of topical steroid per day (i.e., BID) for about one month, and then about one administration of topical steroid per day (i.e., QD) for about one month, timing starting with and following administration of the anti-VEGF agent. In some embodiments, the topical steroid treatment comprises about four administrations of topical steroid per day (i.e., QID) for about 30 days, followed by about three administrations of topical steroid per day (i.e., TID) for about 30 days, followed by about two administrations of topical steroid per day (i.e., BID) for about 30 days, and then about one administration of topical steroid per day (i.e., QD) for about 30 days, timing starting with and following administration of the anti-VEGF agent. In some embodiments, the topical steroid treatment comprises about four administrations of topical steroid per day (i.e., QID) from day 1 to about day 30, followed by about three administrations of topical steroid per day (i.e., TID) from about day 31 to about day 60, followed by about two administrations of topical steroid per day (i.e., BID) from about day 61 to about day 90, and then about one administration of topical steroid per day (i.e., QD) from about day 91 to about day 120, starting on day 1. In some embodiments, if inflammation is present, the topical steroid treatment is continued. Vectors for delivering transgenes to target cells

[0185] In some embodiments, the recombinant adeno-associated virus (rAAV) particle comprises a recombinant viral vector derived from an adeno-associated virus (AAV) that has been modified to be replication-deficient in a subject (e.g., a human or non-human primate). In some embodiments, the adeno-associated virus (AAV) is a recombinant AAV (rAAV).

[0186] AAV or rAAV is a small, non-enveloped, single-stranded DNA virus. rAAV is a non-pathogenic human parvovirus, and can be made to depend on helper viruses for replication, including adenovirus, herpes simplex virus, vaccinia virus, and CMV.

[0187] Since exposure to wild-type (wt) AAV is known not to be associated with or cause any human pathology and is common in the general population, AAV or rAAV is a suitable delivery system for gene therapy. The AAV and rAAV used for gene therapy to deliver anti-VEGF agents, such as aflibercept, can be of any serotype. In some embodiments, the method of the present disclosure provides for the use of any suitable AAV serotype, including AAV1, AAV2, AAV2.5, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, rh10, AAV-DJ, and any hybrid or chimeric AAV thereof. In some embodiments, the serotype used is based on the viral tropism or infectivity of the intended target cell. In some embodiments, several AAV vectors can be generated to allow the selection of the most optimal serotype for use with anti-VEGF agent transgenes (e.g., aflibercept transgenes).

[0188] In some embodiments, the methods of the present disclosure provide for the use of pseudotyped AAV. Pseudotyped AAV particles contain the AAV genome inverted terminal repeats (ITRs) of one AAV serotype encapsidated by the AAV capsid of another AAV serotype. Typically, pseudotyped AAVs are designated as "AAV# / #", where the first "#" indicates the AAV ITR serotype and the second "#" indicates the capsid serotype. For example, an AAV particle containing AAV2 ITRs and an AAV1 capsid is designated as "AAV2 / 1".

[0189] In some embodiments, the rAAV particle comprises a nucleic acid, e.g., a heterologous nucleic acid. In some embodiments, the nucleic acid encodes a transgene, e.g., an anti-VEGF agent (e.g., aflibercept). In some embodiments, the encoded transgene, e.g., an anti-VEGF agent, is under the transcriptional control of a promoter that initiates transcription of the nucleic acid. In some embodiments, the promoter is a "ubiquitous" promoter. In some embodiments, the promoter is a "strong" or constitutively active promoter, such as a cytomegalovirus (CMV) promoter, an elongation factor 1 alpha (EFla) promoter, a glyceraldehyde 3-phosphate dehydrogenase (GAPDH) promoter, or a connexin 36 (or "Cx36") promoter. In some embodiments, the promoter is a tissue-specific promoter that is activated in specific tissues or cells, e.g., retinal cells, to reduce potential toxicity or undesirable effects on non-targeted cells. In some aspects, several AAV vectors can be generated to allow for the selection of the most optimal serotype and promoter for use with the anti-VEGF agent transgene (e.g., an aflibercept transgene). In some embodiments, the nucleic acid is flanked by AAV inverted terminal repeats (ITRs). In some embodiments, the nucleic acid is flanked by AAV2 ITRs.

[0190] In some embodiments, the AAV vector comprises a polynucleotide cassette for enhancing expression of a transgene (e.g., an anti-VEGF agent such as aflibercept) in target cells (e.g., retinal cells). In some embodiments, the polynucleotide cassette comprises, in 5' to 3' order: (a) a first enhancer region comprising a CMV sequence (SEQ ID NO: 22); (b) a promoter region comprising a CMV sequence (SEQ ID NO: 23); (c) a 5' UTR region comprising, in 5' to 3' order, TPL and eMLP sequences (SEQ ID NO: 24 and SEQ ID NO: 25, respectively); (d) a coding sequence encoding a peptide or polypeptide (e.g., an anti-VEGF agent such as aflibercept); (e) a second enhancer region comprising the full-length EES sequence (SEQ ID NO: 26); and (f) an HGH polyadenylation site (SEQ ID NO: 27). In certain of these embodiments, the polynucleotide cassette comprises one or more sequences selected from SEQ ID NOs: 28-32, or sequences having at least 85% identity thereto. In certain of these embodiments, the 5' arm of the polynucleotide cassette comprises or consists of SEQ ID NO: 33 or a sequence having at least 85% identity thereto. In certain of these embodiments, the 3' arm of the polynucleotide cassette comprises or consists of SEQ ID NO: 34 or a sequence having at least 85% identity thereto. The nucleic acid sequences of SEQ ID NOs: 22-34 are provided below: ACTTACGGTA AATGGCCCGC CTGGCTGACC GCCCAACGAC CCCCGCCCAT TGACGTCAAT AATGACGTAT GTTCCCATAG TAACGCCAAT AGGGACTTTC CATTGACGTC AATGGGTGGA GTATTTACGG TAAACTGCCC ACTTGGCAGT ACATCAAGTG TATCATATGC CAAGTCCGCC CCCTATTGAC GTCAATGACG GTAAATGGCC CGCCTGGCAT TATGCCCAGT ACATGACCTT ACGGGACTTT CCTACTTGGC AGTACATCTA CGTATTAGTC ATCGCTATTA CCA(SEQ ID NO: 22) TGCTGATGCG GTTTTGGCAG TACACCAATG GGCGTGGATA GCGGTTTGAC TCACGGGGAT TTCCAAGTCT CCACCCCATT GACGTCAATG GGAGTTTGTT TTGGCACCAA AATCAACGGG ACTTTCCAAA ATGTCGTAAT AACCCCGCCC CGTTGACGCA AATGGGCGGT AGGCGTGTAC GGTGGGAGGT CTATATAAGC AGAGCTCGTT TAGTGAACCG(SEQ ID NO: 23) CTCACTCTCT TCCGCATCGC TGTCTGCGAG GGCCAGCTGT TGGGCTCGCG GTTGAGGACA AACTCTTCGC GGTCTTTCCA GTACTCTTGG ATCGGAAACC CGTCGGCCTC CGAACGGTAC TCCGCCACCG AGGGACCTGA GCGAGTCCGC ATCGACCGGA TCGGAAAACC TCTCGAGAAA GGCGTCTAAC CAGTCACAGT CGCAAGGTAG GCTGAGCACC GTGGCGGGCG GCAGCGGGTG GCGGTCGGGG TTGTTTCTGG CGGAGGTGCT GCTGATGATG TAATTAAAGT AGGCGGTCTT GAGACGGCGG ATGGTCGA(SEQ ID NO: 24) CCAGCTGTTG GGGTGAGTAC TCCCTCTCAA AAGCGGGCAT TACTTCTGCG CTAAGATTGT CAGTTTCCAA AAACGAGGAG GATTTGATAT TCACCTGGCC CG(SEQ ID NO: 25) CTGTTCTCAT CACATCATAT CAAGGTTATA TACCATCAAT ATTGCCACAG ATGTTACTTA GCCTTTTAAT ATTTCTCTAA TTTAGTGTAT ATGCAATGAT AGTTCTCTGA TTTCTGAGAT TGAGTTTCTC ATGTGTAATG ATTATTTAGA GTTTCTCTTT CATCTGTTCA AATTTTTGTC TAGTTTTATT TTTTACTGAT TTGTAAGACT TCTTTTTATA ATCTGCATAT TACAATTCTC TTTACTGGGG TGTTGCAAAT ATTTTCTGTC ATTCTATGGC CTGACTTTTC TTAATGGTTT TTTAATTTTA AAAATAAGTC TTAATTTCA TGCAATCTAA TTAACAATCT TTTCTTTGTG GTTAGGACTT TGAGTCATAA GAAATTTTTC TCTACACTGA AGTCATGATG GCATGCTTCT ATATTATTTT CTAAAAGATT TAAAGTTTTG CCTTCTCCAT TTAGACTAT AATTCACTGG AATTTTTTTG TGTGTATGGT ATGACATATG GGTTCCCTTT TATTTTTTAC ATATAAATAT ATTTCCCTGT TTTTCTAAAA AAGAAAAAGA TCATCATTTT CCCATTGTAA AATGCCATAT TTTTTTCATA GGTCACTTAC ATATATCAAT GGGTCTGTTT CTGAGCTCTA CTCTATTTTA TCAGCCTCAC TGTCTATCCC CACACATCTC ATGCTTTGCT CTAAATCTTG ATATTTAGTG GAACATTCTT TCCCATTTTG TTCTACAAGA ATATTTTTGT TATTGTCTTT GGGCTTTCTA TATACATTTT GAAATGAGGT TGACAAGTTA (SEQ ID NO: 26) CTGCCCGGGT GGCATCCCTG TGACCCCTCC CCAGTGCCTC TCCTGGCCCT GGAAGTTGCC ACTCCAGTGC CCACCAGCCT TGTCCTAATA AAATTAAGTT GCATCATTTT GTCTGACTAG GTGTCCTTCT ATAATATTAT GGGGTGGAGG GGGGTGGTAT GGAGCAAGGG GCCCAAGTTG GGAAGAAACC TGTAGGGCCT GC (SEQ ID NO: 27) AGGCGGTCTT GAGACGGCGG ATGGTCGAGG TGAGGTGTGG CAGGCTTGAG ATCCAGCTGT TGGGGTGA (SEQ ID NO: 28) CGCTGTTTTG ACCTCCATAG TGGACACCGG GACCGATCCA GCCTCCGCGT CTCAGGGGAG ATCTCGTTTA GTGAACCGTC AGATCCTCAC TCTCTTCCGC ATCGCTGTCT GCGAGGGCCA GCTGTTGGG (SEQ ID NO: 29) TTGATATTCA CCTGGCCCGA TCTGGCCATA CACTTG (SEQ ID NO: 30) CCCAGGTCCA AGTTTAAACG CC (SEQ ID NO: 31) TCTTTGGGCT TTCTATATAC ATTTTGAAAT GAGGTTGACA AGTTACCTAG GAAAACTGTC TTCCTGCCCG GGTGGCA (SEQ ID NO: 32) CTCTGGAGAC GACTTACGGT AAATGGCCCG CCTGGCTGAC CGCCCAACGA CCCCCGCCCA TTGACGTCAA TAATGACGTA TGTTCCCATA GTAACGCCAA TAGGGACTTT CCATTGACGT CAATGGGTGG AGTATTTACG GTAAACTGCC CACTTGGCAG TACATCAAGT GTATCATATG CCAAGTCCGC CCCCTATTGA CGTCAATGAC GGTAAATGGC CCGCCTGGCA TTATGCCCAG TACATGACCT TACGGGACTT TCCTACTTGG CAGTACATCT ACGTATTAGT CATCGCTATT ACCATGCTGA TGCGGTTTTG GCAGTACACC AATGGGCGTG GATAGCGGTT TGACTCACGG GGATTTCCAA GTCTCCACCC CATTGACGTC AATGGGAGTT TGTTTTGGCA CCAAAATCAA CGGGACTTTC CAAAATGTCG TAATAACCCC GCCCCGTTGA CGCAAATGGG CGGTAGGCGT GTACGGTGGG AGGTCTATAT AAGCAGAGCT CGTTTAGTGA ACCGTCAGAT CGCCTGGAGA GGCCATCCAC GCTGTTTTGA CCTCCATAGT GGACACCGGG ACCGATCCAG CCTCCGCGTC TCAGGGGAGA TCTCGTTTAG TGAACCGTCA GATCCTCACT CTCTTCCGCA TCGCTGTCTG CGAGGGCCAG CTGTTGGGCT CGCGGTTGAG GACAAACTCT TCGCGGTCTT TCCAGTACTC TTGGATCGGA AACCCGTCGG CCTCCGAACG GTACTCCGCC ACCGAGGGAC CTGAGCGAGT CCGCATCGAC CGGATCGGAA AACCTCTCGA GAAAGGCGTC TAACCAGTCA CAGTCGCAAG GTAGGCTGAG CACCGTGGCG GGCGGCAGCG GGTGGCGGTC GGGGTTGTTT CTGGCGGAGG TGCTGCTGAT GATGTAATTA AAGTAGGCGG TCTTGAGACG GCGGATGGTC GAGGTGAGGT GTGGCAGGCT TGAGATCCAG CTGTTGGGGT GAGTACTCCC TCTCAAAAGC GGGCATTACT TCTGCGCTAA GATTGTCAGT TTCCAAAAAC GAGGAGGATT TGATATTCAC CTGGCCCGAT CTGGCCATAC ACTTGAGTGA CAATGACATC CACTTTGCCT TTCTCTCCAC AGGTGTCCAC TCCCAGGTCC AAGTTTAAAC GCCGCCACCA TG(SEQ ID NO: 33) ACTGTTCTCA TCACATCATA TCAAGGTTAT ATACCATCAA TATTGCCACA GATGTTACTT AGCCTTTTAA TATTTCTCTA ATTTAGTGTA TATGCAATGA TAGTTCTCTG ATTTCTGAGA TTGAGTTTCT CATGTGTAAT GATTATTTAG AGTTTCTCTT TCATCTGTTC AAATTTTTGT CTAGTTTTAT TTTTTACTGA TTTGTAAGAC TTCTTTTTAT AATCTGCATA TTACAATTCT CTTTACTGGG GTGTTGCAAA TATTTTCTGT CATTCTATGG CCTGACTTTT CTTAATGGTT TTTTAATTTT AAAAATAAGT CTTAATATTC ATGCAATCTA ATTAACAATC TTTTCTTTGT GGTTAGGACT TTGAGTCATA AGAAATTTTT CTCTACACTG AAGTCATGAT GGCATGCTTC TATATTATTT TCTAAAAGAT TTAAAGTTTT GCCTTCTCCA TTTAGACTTA TAATTCACTG GAATTTTTT GTGTGTATGG TATGACATAT GGGTTCCCTT TTATTTTTTA CATATAAATA TATTTCCCTG TTTTTCTAAA AAAGAAAAAG ATCATCATTT TCCCATTGTA AAATGCCATA TTTTTTTCAT AGGTCACTTA CATATATCAA TGGGTCTGTT TCTGAGCTCT ACTCTATTTT ATCAGCCTCA CTGTCTATCC CCACACATCT CATGCTTTGC TCTAAATCTT GATATTTAGT GGAACATTCT TTCCCATTTT GTTCTACAAG AATATTTTTG TTATTGTCTT TGGGCTTTCT ATATACATTT TGAAATGAGG TTGACAAGTT ACCTAGGAAA ACTGTCTTCC TGCCCGGGTG GCATCCCTGT GACCCCTCCC CAGTGCCTCT CCTGGCCCTG GAAGTTGCCA CTCCAGTGCC CACCAGCCTT GTCCTAATAA AATTAAGTTG CATCATTTTG TCTGACTAGG TGTCCTTCTA TAATATTATG GGGTGGAGGG GGGTGGTATG GAGCAAGGGG CCCAAGTTGG GAAGAAACCT GTAGGGCCTG CGAAGACAGT CAG (SEQ ID NO: 34)

[0191] In some embodiments, the polynucleotide cassette comprises or consists of SEQ ID NO: 39, or a sequence having at least 85% identity thereto. gcgcgctcgctcgctcactgaggccgcccgggcaaagcccgggcgtcgggcgacctttggtcgcccggcctcagtgagcgagcgagcgcgcagagagggagtggccaactccatcactaggggttccttgtagttaatgattaacccgccatgctacttatctacgtactctggagacgacttacggtaaatggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaataatgacgtatgttcccatagtaacgccaatagggactttccattgacgtcaatgggtggagtatttacggtaaactgcccacttggcagtacatcaagtgtatcatatgccaagtccgccccctattgacgtcaatgacggtaaatggcccgcctggcattatgcccagtacatgaccttacgggactttcctacttggcagtacatctacgtattagtcatcgctattaccatgctgatgcggttttggcagtacaccaatgggcgtggatagcggtttgactcacggggatttccaagtctccaccccattgacgtcaatgggagtttgttttggcaccaaaatcaacgggactttccaaaatgtcgtaataaccccgccccgttgacgcaaatgggcggtaggcgtgtacggtgggaggtctatataagcagagctcgtttagtgaaccgtcagatcgcctggagaggccatccacgctgttttgacctccatagtggacaccgggaccgatccagcctccgcgtctcaggggagatctcgtttagtgaaccgtcagatcctcactctcttccgcatcgctgtctgcgagggccagctgttgggctcgcggttgaggacaaactcttcgcggtctttccagtactcttggatcggaaacccgtcggcctccgaacggtactccgccaccgagggacctgagcgagtccgcatcgaccg

[0192] SEQ ID NO:39, as set forth above, includes, from 5' to 3', an AAV serotype 2 inverted terminal repeat (ITR) comprising nucleotides 1-145 of SEQ ID NO:39; a CMV promoter comprising nucleotides 180-693 of SEQ ID NO:39; a 5' untranslated region (UTR) comprising an adenovirus tripartite leader sequence and a synthetic intron, comprising nucleotides 694-1314 of SEQ ID NO:39; a Kozak sequence comprising nucleotides 1329-1340 of SEQ ID NO:39; a codon-optimized aflibercept cDNA sequence comprising nucleotides 1338-2714 of SEQ ID NO:39; a 3' UTR comprising a human scaffold attachment region, comprising nucleotides 2717-3527 of SEQ ID NO:39; a human growth hormone polyadenylation / transcription termination signal comprising nucleotides 3546-3748 of SEQ ID NO:39; and an AAV serotype 2 inverted terminal repeat (ITR) comprising nucleotides 3772-3916 of SEQ ID NO:39. See, for example, Figure 1C.

[0193] Additional polynucleotide cassettes for enhancing expression of a transgene (e.g., a transgene encoding an anti-VEGF agent such as aflibercept) in a target cell (e.g., a retinal cell) are disclosed in WO2018 / 170473, the contents of which relating to polynucleotide cassettes for enhancing expression of a transgene in a target cell are incorporated herein by reference.

[0194] In some embodiments, the rAAV particles comprise a variant capsid protein with increased infectivity of target cells, such as retinal cells, and are used to increase transduction of retinal cells or to increase targeting of gene delivery to retinal cells in an individual. In some embodiments, the rAAV particles comprise an amino acid modification in the capsid protein GH loop / loop IV of the AAV capsid protein. In some embodiments, the modification site is a solvent-accessible portion of the GH loop / loop IV of the AAV capsid protein. For descriptions of the GH loop / loop IV of the AAV capsid, see, for example, van Vliet et al. (2006) Mol. Ther. 14:809; Padron et al. (2005) J. Virol. 79:5047; and Shen See, e.g., S. et al. (2007) Mol. Ther. 15:1955. Several AAV capsid variants are known. In some embodiments, the rAAV particles comprise a variant AAV capsid protein that comprises an insertion of between 5 and 11 amino acids, e.g., a 7 amino acid sequence, in the GH loop of the capsid protein compared to the corresponding parent AAV capsid protein, and the variant capsid protein confers increased infectivity of retinal cells compared to the infectivity of retinal cells by AAV particles comprising the corresponding parent or unmodified AAV capsid protein. In some embodiments, any one of the following amino acid sequences may be inserted into the GH loop of the capsid protein: LALGETTRPA (SEQ ID NO: 1); LANETITRPA (SEQ ID NO: 2), LAKAGQANNA (SEQ ID NO: 3), LAKDPKTTNA (SEQ ID NO: 4), KDTDTTR (SEQ ID NO: 5), RAGGSVG (SEQ ID NO: 6), AVDTTKF (SEQ ID NO: 7), STGKVPN (SEQ ID NO: 8), LAKDTDTTRA (SEQ ID NO: 9), LARAGGSVGA (SEQ ID NO: 10), LAAVDTTKFA (SEQ ID NO: 11), and LASTGKVPNA (SEQ ID NO: 12), LGETTRP (SEQ ID NO: 14), NETITRP (SEQ ID NO: 15), KAGQANN (SEQ ID NO: 16), KDPKTTN (SEQ ID NO: 17), KDTDTTR (SEQ ID NO: 18), RAGGSVG (SEQ ID NO: 19), AVDTTKF (SEQ ID NO: 20), and STGKVPN (SEQ ID NO: 21). In some embodiments, any one of the amino acid sequences set forth in SEQ ID NOS: 1-12 and 14-21 is inserted into the solvent-exposed GH loop of the VP1 capsid protein in rAAV. Additional details regarding amino acid sequences that can be inserted into the GH loop of a capsid protein to facilitate transduction of a nucleic acid of interest into retinal cells, for example, after IVT injection, are provided in WO2012145601, US9587282, US10202657, and US10214785, the contents of which relating to amino acid sequences that can be inserted into the GH loop of a capsid protein are incorporated herein by reference.

[0195] In some embodiments, the rAAV particles contain one of the following amino acid sequences inserted between positions 587 and 588 of the AAV2 capsid protein; between amino acids 590 and 591 of the AAV1 capsid protein; between amino acids 575 and 576 of the AAV5 capsid protein; between amino acids 590 and 591 of the AAV6 capsid protein; between amino acids 589 and 590 of the AAV7 capsid protein; between amino acids 590 and 591 of the AAV8 capsid protein; between amino acids 588 and 589 of the AAV9 capsid protein; or between amino acids 589 and 590 of the AAV10 capsid protein: LALGETTRPA (SEQ ID NO: 1); LANETITRPA (SEQ ID NO: 2), LAKAGQANNA (SEQ ID NO: 3), LAKDPK TTNA (SEQ ID NO: 4), KDTDTTR (SEQ ID NO: 5), RAGGSVG (SEQ ID NO: 6), AVDTTKF (SEQ ID NO: 7), STGKVPN (SEQ ID NO: 8), LAKDTDTTRA (SEQ ID NO: 9), LARAGGSVGA (SEQ ID NO: 10), LAAVDTTKFA (SEQ ID NO: 11), and LASTGKVPNA (SEQ ID NO: 12), LGETTRP (SEQ ID NO: 14), NETITRP (SEQ ID NO: 15), KAGQANN (SEQ ID NO: 16), KDPKTTN (SEQ ID NO: 17), KDTDTTR (SEQ ID NO: 18), RAGGSVG (SEQ ID NO: 19), AVDTTKF (SEQ ID NO: 20), and STGKVPN (SEQ ID NO: 21). In some embodiments, the rAAV particles comprise an AAV2 capsid protein comprising the amino acid sequence LALGETTRPA (SEQ ID NO: 1) inserted between positions 587 and 588 of the capsid protein, where the amino acid residue numbering corresponds to that of the AAV2 VP1 capsid protein. In some embodiments, the rAAV particles comprise an AAV2 capsid protein comprising the amino acid sequence LALGETTRPA (SEQ ID NO: 1) inserted between positions 587 and 588 of AAV2 VP1, which comprises the sequence of SEQ ID NO: 13.

[0196] In some embodiments, the rAAV particles comprise a 7m8 variant capsid protein from AAV2 that includes the amino acid sequence LALGETTRPA (SEQ ID NO: 1) inserted within the GH loop of the AAV2 VP1 protein, between positions 587 and 588 of AAV2 VP1. In some embodiments, the rAAV particles comprise an AAV2 GH loop that includes the amino acid sequence of SEQ ID NO: 38 or an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 38. In some embodiments, the rAAV particles comprise an AAV2 VP1 capsid protein that includes a GH loop comprising an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:38. FSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTNTPSGTTTQSRLQFSQAGASDI RDQSRNWLPGPCYRQQRVSKTSADNNNSEYSWTGATKYHLNGRDSLVNPGPAMASHKDDE EKFFPQSGVLIFGKQGSEKTNVDIEKVMITDEEIRTTNPVATEQYGSVSTNLQRGNLAL GETTRPARQAATADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLK HPPPQILIKN (SEQ ID NO: 38)

[0197] In some embodiments, the rAAV particles comprise a 7m8 variant capsid protein from AAV2 that includes the amino acid sequence LALGETTRPA (SEQ ID NO: 1) inserted between positions 587 and 588 of AAV2 VP1. The sequence of the 7m8 variant capsid protein from AAV2 that includes the amino acid sequence LALGETTRPA (SEQ ID NO: 1) inserted between positions 587 and 588 of AAV2 VP1 is provided below: (SEQ ID NO: 37)

[0198] In some embodiments, the rAAV particles comprise a capsid protein VP1 comprising the amino acid sequence LGETTRP (SEQ ID NO: 14) inserted between positions 587 and 588 of the capsid protein, where the amino acid residue numbering corresponds to that of the AAV2 VP1 capsid protein. In some embodiments, the rAAV particles comprise a capsid protein VP2 comprising the amino acid sequence LGETTRP (SEQ ID NO: 14) inserted between positions 587 and 588 of the capsid protein, where the amino acid residue numbering corresponds to that of the AAV2 VP1 capsid protein. In some embodiments, the rAAV particles comprise a capsid protein VP3 comprising the amino acid sequence LGETTRP (SEQ ID NO: 14) inserted between positions 587 and 588 of the capsid protein, where the amino acid residue numbering corresponds to that of the AAV2 VP1 capsid protein. In some embodiments, the rAAV particles comprise capsid proteins VP1, VP2, and VP3, each of which comprises the amino acid sequence LGETTRP (SEQ ID NO: 14) inserted between positions 587 and 588 of the capsid protein, with amino acid residue numbering corresponding to that of the AAV2 VP1 capsid protein.

[0199] In some embodiments, the rAAV particles comprise a capsid protein VP1 comprising the amino acid sequence LALGETTRPA (SEQ ID NO: 1) inserted between positions 587 and 588 of the capsid protein, where the amino acid residue numbering corresponds to that of the AAV2 VP1 capsid protein. In some embodiments, the rAAV particles comprise a capsid protein VP2 comprising the amino acid sequence LALGETTRPA (SEQ ID NO: 1) inserted between positions 587 and 588 of the capsid protein, where the amino acid residue numbering corresponds to that of the AAV2 VP1 capsid protein. In some embodiments, the rAAV particles comprise a capsid protein VP3 comprising the amino acid sequence LALGETTRPA (SEQ ID NO: 1) inserted between positions 587 and 588 of the capsid protein, where the amino acid residue numbering corresponds to that of the AAV2 VP1 capsid protein. In some embodiments, the rAAV particles comprise capsid proteins VP1, VP2, and VP3, each of which comprises the amino acid sequence LALGETTRPA (SEQ ID NO: 1) inserted between positions 587 and 588 of the capsid protein, with amino acid residue numbering corresponding to that of the AAV2 VP1 capsid protein.

[0200] In some embodiments, the recombinant virus and / or plasmid used to generate the rAAV virus contains other transcriptional or regulatory elements, such as a polyA (polyadenylation) sequence, an untranslated region (UTR), a 3'UTR, or a termination sequence. In some embodiments, more than one gene is expressed from a vector or plasmid using an internal ribosome entry site (IRES) or similar element that allows for simultaneous expression of two or more proteins or creates a multigene or polycistronic mRNA.

[0201] In some embodiments, the rAAV and / or the plasmid used to generate the rAAV comprises one or more of the following nucleic acid elements: first ITR sequence; promoter sequence; intron sequence; first UTR sequence; heterologous nucleic acid encoding anti-VEGF agent (e.g., aflibercept); second UTR sequence; polyA sequence; and second ITR sequence.In some embodiments, linker sequence(s) are inserted between two or more of the nucleic acid elements.In some embodiments, the heterologous nucleic acid encoding therapeutic polypeptide encodes, for example, aflibercept (or its functional fragment or functional variant).

[0202] In some embodiments, the vector is a targeted vector, particularly a targeted rAAV (e.g., AAV2.7m8) that exhibits high infectivity of specific cells, such as retinal cells (e.g., photoreceptors, retinal ganglion cells, Müller cells, bipolar cells, amacrine cells, horizontal cells, or retinal pigment epithelial cells). Viral vectors for use in the present disclosure may include vectors that exhibit low toxicity and / or low immunogenicity in individuals and express a therapeutically effective amount of an anti-VEGF agent (e.g., aflibercept) in individuals, such as humans. Any suitable method known in the art can be used for biochemical purification of recombinant viruses (e.g., rAAV) for preparing, for example, pharmaceutical compositions described elsewhere herein. Recombinant AAV viruses can be harvested directly from cells or from culture media containing cells. Viruses can be purified using various biochemical means, such as gel filtration, filtration, chromatography, affinity purification, density gradient ultracentrifugation, or size exclusion. In some embodiments, the virus is lyophilized.

[0203] In some embodiments, the rAAV particles comprise a 7m8 variant capsid protein, e.g., rAAV2.7m8, and a nucleic acid sequence encoding an anti-VEGF agent (e.g., aflibercept, or a functional fragment or variant thereof). In some embodiments, the rAAV particles (e.g., 7m8 variants) have an increase in retinal cell infectivity of at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 100% compared to AAV particles comprising the corresponding parent or unmodified AAV capsid protein. In some embodiments, the increase in infectivity of retinal cells is between 5% and 100%, between 5% and 95%, between 5% and 90%, between 5% and 85%, between 5% and 80%, between 5% and 75%, between 5% and 70%, between 5% and 65%, between 5% and 60%, between 5% and 55%, between 5% and 50%, between 5% and 45%, between 5% and 40%, between 5% and 35%, between 5% and 30%, between 5% and 25%, between 5% and 20%, between 5% and 15%, or between 5% and 10%, compared to AAV particles containing the corresponding parent or unmodified AAV capsid protein.

[0204] In some embodiments, the increase in retinal cell infectivity of an rAAV variant, such as rAAV2.7m8, is at least 1-fold, at least 1.1-fold, at least 1.2-fold, at least 1.3-fold, at least 1.4-fold, at least 1.5-fold, at least 1.6-fold, at least 1.7-fold, at least 1.8-fold, at least 1.9-fold, or at least 2-fold compared to AAV particles comprising the corresponding parental or unmodified AAV capsid proteins. In some embodiments, the increase in infectivity is at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, or at least 10-fold compared to AAV particles comprising the corresponding parental AAV capsid proteins. In some embodiments, the increase in infectivity is at least 15-fold, at least 20-fold, at least 25-fold, at least 30-fold, at least 35-fold, at least 40-fold, at least 45-fold, at least 50-fold, at least 55-fold, at least 60-fold, at least 65-fold, at least 70-fold, at least 75-fold, at least 80-fold, at least 85-fold, at least 90-fold, or at least 100-fold compared to AAV particles comprising the corresponding parent or unmodified AAV capsid proteins.

[0205] In some embodiments, the increase in retinal cell infectivity of the rAAV variant, e.g., rAAV2.7m8, is between 10-fold and 100-fold, between 10-fold and 95-fold, between 10-fold and 90-fold, between 10-fold and 85-fold, between 10-fold and 80-fold, between 10-fold and 75-fold, between 10-fold and 70-fold, between 10-fold and 65-fold, between 10-fold and 60-fold, between 10-fold and 55-fold, between 10-fold and 50-fold, between 10-fold and 45-fold, between 10-fold and 40-fold, between 10-fold and 35-fold, between 10-fold and 30-fold, between 10-fold and 25-fold, between 10-fold and 20-fold, or between 10-fold and 15-fold, compared to AAV particles comprising the corresponding parental or unmodified AAV capsid protein.

[0206] In some embodiments, the increase in retinal cell infectivity is between 2-fold and 20-fold, between 2-fold and 19-fold, between 2-fold and 18-fold, between 2-fold and 17-fold, between 2-fold and 16-fold, between 2-fold and 15-fold, between 2-fold and 14-fold, between 2-fold and 13-fold, between 2-fold and 12-fold, between 2-fold and 11-fold, between 2-fold and 10-fold, between 2-fold and 9-fold, between 2-fold and 8-fold, between 2-fold and 7-fold, between 2-fold and 6-fold, between 2-fold and 5-fold, between 2-fold and 4-fold, or between 2-fold and 3-fold compared to AAV particles comprising the corresponding parental or unmodified AAV capsid proteins.

[0207] In some embodiments, the amino acid modifications of the capsid proteins described herein may confer an increased ability to penetrate the inner limiting membrane (ILM) of an individual's, e.g., a human's, eye, compared to the ability of AAV particles containing the corresponding parent or unmodified AAV capsid protein to penetrate the ILM of the subject's eye. In some embodiments, the increased ability of the rAAV variant, e.g., rAAV2.7m8, to penetrate the ILM is at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 100% compared to AAV particles containing the corresponding parent or unmodified AAV capsid protein. In some embodiments, the increase in ability to cross the ILM is between 5% and 100%, between 5% and 95%, between 5% and 90%, between 5% and 85%, between 5% and 80%, between 5% and 75%, between 5% and 70%, between 5% and 65%, between 5% and 60%, between 5% and 55%, between 5% and 50%, between 5% and 45%, between 5% and 40%, between 5% and 35%, between 5% and 30%, between 5% and 25%, between 5% and 20%, between 5% and 15%, or between 5% and 10% increase compared to the parent or unmodified AAV capsid protein.

[0208] In some embodiments, the increased ability of the rAAV variant, e.g., rAAV2.7m8, to cross the ILM is at least 1-fold, at least 1.1-fold, at least 1.2-fold, at least 1.3-fold, at least 1.4-fold, at least 1.5-fold, at least 1.6-fold, at least 1.7-fold, at least 1.8-fold, at least 1.9-fold, or at least 2-fold compared to AAV particles comprising the corresponding parental AAV capsid proteins. In some embodiments, the increased ability to cross the ILM is at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, or at least 10-fold compared to AAV particles comprising the corresponding parental AAV capsid proteins. In some embodiments, the increase in ability to cross the ILM is at least 15-fold, at least 20-fold, at least 25-fold, at least 30-fold, at least 35-fold, at least 40-fold, at least 45-fold, at least 50-fold, at least 55-fold, at least 60-fold, at least 65-fold, at least 70-fold, at least 75-fold, at least 80-fold, at least 85-fold, at least 90-fold, or at least 100-fold compared to AAV particles comprising the corresponding parent or unmodified AAV capsid proteins.

[0209] In some embodiments, the increase in the ability of the rAAV variant, e.g., rAAV2.7m8, to cross the ILM is between 10-fold and 100-fold, between 10-fold and 95-fold, between 10-fold and 90-fold, between 10-fold and 85-fold, between 10-fold and 80-fold, between 10-fold and 75-fold, between 10-fold and 70-fold, between 10-fold and 65-fold, between 10-fold and 60-fold, between 10-fold and 55-fold, between 10-fold and 50-fold, between 10-fold and 45-fold, between 10-fold and 40-fold, between 10-fold and 35-fold, between 10-fold and 30-fold, between 10-fold and 25-fold, between 10-fold and 20-fold, or between 10-fold and 15-fold, compared to AAV particles comprising the corresponding parental or unmodified AAV capsid proteins.

[0210] In some embodiments, the increase in the ability of the rAAV variant, e.g., rAAV2.7m8, to cross the ILM is between 2-fold and 20-fold, between 2-fold and 19-fold, between 2-fold and 18-fold, between 2-fold and 17-fold, between 2-fold and 16-fold, between 2-fold and 15-fold, between 2-fold and 14-fold, between 2-fold and 13-fold, between 2-fold and 12-fold, between 2-fold and 11-fold, between 2-fold and 10-fold, between 2-fold and 9-fold, between 2-fold and 8-fold, between 2-fold and 7-fold, between 2-fold and 6-fold, between 2-fold and 5-fold, between 2-fold and 4-fold, or between 2-fold and 3-fold, compared to AAV particles comprising the corresponding parental or unmodified AAV capsid proteins.

[0211] In some embodiments, rAAV.7m8 containing aflibercept-encoding nucleic acid is used for gene therapy.In some embodiments, AAV2 or rAAV2 is used to deliver the nucleic acid sequence encoding an anti-VEGF agent (such as aflibercept) to the eye or retinal cells of a subject via intravitreal or subretinal injection.In some embodiments, AAV2 or rAAV2 is used to deliver the nucleic acid sequence encoding an anti-VEGF agent (such as aflibercept) to the eye or retinal cells of a subject via intravitreal injection.In some embodiments, rAAV2.7m8 is used to deliver the nucleic acid sequence of an anti-VEGF agent (such as aflibercept) to the retinal cells of a subject.In some embodiments, heterologous nucleic acid (such as the nucleic acid encoding an anti-VEGF agent such as aflibercept) is integrated into the target cell genome (such as the retinal cell genome), resulting in the long-term expression of, for example, an anti-VEGF agent (such as aflibercept) in the target cell. In some embodiments, the viral vector delivers a plasmid or other extrachromosomal genetic element containing a heterologous nucleic acid (e.g., a nucleic acid encoding an anti-VEGF agent such as aflibercept) to a target cell (e.g., a retinal cell).

[0212] In some embodiments, the rAAV particles comprise a nucleic acid encoding a polypeptide comprising an amino acid sequence having at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% identity to the amino acid sequence of SEQ ID NO: 35, and flanked by AAV2 inverted repeat units (ITRs). In some embodiments, the rAAV particles comprise a nucleic acid encoding a polypeptide comprising an amino acid sequence having at least about 95% identity to the amino acid sequence of SEQ ID NO: 35, and flanked by AAV2 inverted repeat units (ITRs). In some embodiments, the rAAV particles comprise a nucleic acid encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 35, and flanked by AAV2 inverted repeat units (ITRs). In some embodiments, the rAAV particles comprise a nucleic acid encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 35. In some embodiments, the rAAV particles comprise a nucleic acid encoding aflibercept, and flanked by AAV2 inverted repeat units (ITRs). The sequence of SEQ ID NO: 35 is provided below: SDTGRPFVEMYSEIPEIIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVLSPS HGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHEKDKTHTCPPCPAPEL LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 35)

[0213] In some embodiments, the rAAV particles comprise a nucleic acid having at least about 75%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.9%, or at least about 100% sequence identity to the nucleic acid sequence of SEQ ID NO: 36, and flanked by AAV2 inverted terminal repeats (ITRs). The sequence of SEQ ID NO: 36 is provided in Figure 5. In some embodiments, the rAAV particles contain a nucleic acid having at least about 75%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.9%, or at least about 100% sequence identity with the nucleic acid sequence of aflibercept (e.g., SEQ ID NO: 36), and the nucleic acid is flanked by AAV2 inverted terminal repeats (ITRs). In some embodiments, the nucleic acid sequence of aflibercept is derived from its amino acid sequence. In some embodiments, the nucleic acid sequence of aflibercept is codon-optimized to improve its expression in a subject.In some embodiments, rAAV particles comprise a nucleic acid having any of at least about 75%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.9%, or 100% sequence identity to the nucleic acid sequence of SEQ ID NO: 40, wherein the nucleic acid is flanked by AAV2 inverted terminal repeats (ITRs). In some embodiments, rAAV particles comprise a nucleic acid comprising the nucleic acid sequence of SEQ ID NO: 40. In some embodiments, rAAV particles comprise a nucleic acid comprising the nucleic acid sequence of SEQ ID NO: 40, wherein the nucleic acid is flanked by AAV2 inverted terminal repeats (ITRs). atggtgtcatactgggatactggagtcttgctttgtgccctgctgtcctgcctcctcctgactggctccagctcgggctcagataccggtcgccccttcgtggagatgtactccgagatcccggaaattatccacatgactgaggggcgcgaacttgtgatcccctgccgggtcaccagcccgaacattactgtgactttgaagaagttccccctggacaccctgattccggatgggaagagaattatctgggattcacggaagggattcatcatcagcaacgcgacctacaaggaaattggcctcctcacttgcgaagccactgtgaacggacacttgtacaagaccaactacctgacccaccgccagaccaacaccatcatcgacgtcgtcctgtccccttcgcacgggatcgagctctcggtgggagagaagttggtgcttaactgcaccgcccggacggaactgaatgtgggaatcgacttcaactgggaatacccgtccagcaagcatcagcataagaagctggtgaaccgggacctcaagactcagtccggcagcgaaatgaagaagttcctgtcgaccctcactattgacggagtgaccagatccgaccagggcctctacacttgcgccgcttccagcggactcatgaccaagaagaacagcactttcgtgagggtgcatgagaaggacaagacccacacgtgtccgccgtgcccagccccagagctgctgggaggcccttccgtgttcctgtttccgcccaagccaaaggat accctgatgatctcaaggacccctgaggtcacatgcgtcgtggtggatgtgtcgcacgaggaccctgaagtcaaattcaattggtatgtggacggagtggaagtccacaacgcgaaaaccaagccgagagaagaacagtacaattccacctaccgggtggtgtcggtgctgactgtgctgcaccaggactggctcaacggaaaggagtacaagtgcaaggtgtccaacaaggctctgcccgcacctattgaaaagaccatctccaaggccaagggtcaacctcgcgagcctcaggtgtacactctgcctccaagccgggacgaactgactaagaaccaagtctctctgacctgtttggtgaagggcttctacccgtcagacatcgcagtggagtgggagtcaaacggtcagccggagaacaactacaaaacaaccccccccgtgctggactccgacggctccttcttcctgtactccaagcttaccgtggataagagccgctggcaacagggcaacgtgttttcctgctccgtcatgcacgaagccctgcacaaccattatacccagaagtccctgtcgctgtcccccgggaaatag(SEQ ID NO: 40)

[0214] In some embodiments, the rAAV particles comprise a nucleic acid encoding a polypeptide comprising an amino acid sequence having at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identity to the amino acid sequence of SEQ ID NO:41, and flanked by AAV2 inverted repeat units (ITRs). In some embodiments, the rAAV particles comprise a nucleic acid encoding a polypeptide comprising an amino acid sequence having at least about 95% identity to the amino acid sequence of SEQ ID NO:41, and flanked by AAV2 inverted repeat units (ITRs). In some embodiments, the rAAV particles comprise a nucleic acid encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:41, and flanked by AAV2 inverted repeat units (ITRs). In some embodiments, the rAAV particles comprise a nucleic acid encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:41. MVSYWDTGVLLCALLSCLLLTGSSSGSDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKT NYLTHRQTNTIIDVVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHEKDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 41)

[0215] In some embodiments, the nucleic acid sequence of aflibercept is codon-optimized for expression in a primate or human subject. Construction of a synthetic gene corresponding to the aflibercept amino acid sequence is described in the literature, e.g., Kanda A, Noda K, Saito W, Ishida S. Aflibercept Traps Galectin-1, an Angiogenic Factor Associated with Diabetic Retinopathy. Scientific Reports 5:17946 (2015) ("VEGF-Trap"). R1R2 (a (2003) describes that aflibercept cDNA was generated as a synthetic gene by IDT (Coralville, IA). Given the available amino acid sequence of aflibercept, any method known in the art can be used to generate aflibercept cDNA for use in gene therapy or rAAV as described herein.

[0216] Codon optimization can be achieved by any method known in the art.Codon optimization refers to the process of modifying nucleic acid sequence by replacing at least one codon (for example, about 1 or more, 2, 3, 4, 5, 10, 15, 20, 25, 50, 100 or more codons) of native sequence with more frequently used or most frequently used codon in host cell, while maintaining native amino acid sequence, in order to enhance the expression of gene in target or host cell of interest, for example, human retinal cell.For example, GenScript Codon Usage Frequency Table Tool at www(dot)genscript(dot)com / tools / codon-frequency-table;Codon Usage Database at www(dot)kazusa(dot)or(dot)jp / codon / ; and Nakamura, Y., et al. "Codon usage tabulated from the international DNA sequence databases: status for the year 2000" Nucl. Acids Res. 28:292 (2000). Codon usage tables including the following are readily available.

[0217] Homology refers to the percent conservation of residues in an alignment between two sequences, including but not limited to functional fragments, sequences containing insertions, deletions, substitutions, pseudofragments, pseudogenes, splice variants, or artificially optimized sequences.

[0218] In some embodiments, the rAAV particles comprise a nucleic acid encoding aflibercept. In some embodiments, the polypeptide is aflibercept.

[0219] As used herein, "aflibercept" refers to a polypeptide or protein sequence, or a functional fragment, variant, or mutant thereof, having at least 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher, or 100% homology to the aflibercept amino acid sequence (SEQ ID NO: 35) identified above. Homology refers to the percent conservation of residues in the alignment between two sequences, including, but not limited to, functional fragments, sequences containing insertions, deletions, substitutions, pseudofragments, pseudogenes, splice variants, or artificially optimized sequences.

[0220] In some embodiments, the amino acid sequence of aflibercept is at least 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% homologous to the aflibercept amino acid sequence of SEQ ID NO: 35. In some embodiments, the nucleic acid sequences encoding aflibercept disclosed herein exhibit at least any of 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% sequence identity with the nucleic acid sequence of aflibercept (e.g., SEQ ID NO: 36) when compared to the corresponding cDNA sequence of the aflibercept amino acid sequence identified above. In some embodiments, aflibercept is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9% or 100% spatially homologous to aflibercept (e.g., in terms of its secondary structure, tertiary structure, and quaternary structure or conformation).In some embodiments, aflibercept is at most 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9% or 100% spatially homologous to aflibercept used in standard treatment (e.g., in terms of its secondary structure, tertiary structure, and quaternary structure or conformation).

[0221] In some embodiments, the aflibercept gene product or aflibercept transgene included in rAAV-based gene therapy comprises a capsid variant (e.g., 7m8 variant) disclosed herein and encodes a protein, fusion protein, or polypeptide having at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% homology with the above amino acid sequence of SEQ ID NO: 35 or the corresponding cDNA sequence of aflibercept (e.g., the cDNA of the aflibercept sequence used in gene therapy compared with SEQ ID NO: 36). In some embodiments, the methods and compositions disclosed herein comprise a functional fragment of aflibercept, or a variant or mutant thereof. In some embodiments, the nucleic acid sequence of aflibercept is modified or codon-optimized to enhance its activity, expression, stability, and / or solubility in vivo.

[0222] Aflibercept is a 115 kDa fusion protein that can be glycosylated. It contains an IgG backbone fused to the extracellular VEGF receptor sequences of human VEGFR-1 and VEGFR-2, and functions as a soluble decoy receptor by binding to VEGF-A with higher affinity than its native or endogenous receptor. See, e.g., Stewart MW. Aflibercept (VEGF Trap-eye): the newest anti-VEGF drug. Br. J. Ophthalmol. 2012 Sep;96(9):1157-8. Its high affinity for VEGF prevents or destroys the subsequent binding and activation of native or endogenous VEGF receptors. Reduction of VEGF activity can result in reduced angiogenesis and vascular permeability. The inhibition of placental growth factor PIGF and VEGF-B by aflibercept can also contribute to the treatment of eye diseases or disorders characterized by abnormal (e.g., excessive) angiogenesis and / or neovascularization. PIGF is associated with certain eye diseases or disorders, such as angiogenesis and exudative AMD, which may be associated with elevated levels of PIGF. Overexpression of VEGF-B can be associated with the breakdown of the blood-retinal barrier and retinal angiogenesis. Thus, the inhibition of VEGF-A, VEGF-B, and PIGF can all contribute to the effectiveness of aflibercept. Method for preparing a vector for delivering a transgene to a target cell

[0223] In some embodiments, rAAV particles are produced using any method known in the art. In some embodiments, rAAV particles are produced using a baculovirus expression vector system in Sf9 cells. Sf9 cells are an insect cell culture cell system that is commonly used for recombinant protein production using baculovirus. In some embodiments, rAAV particles are produced using two baculoviruses in Sf9 cells. In some embodiments, rAAV particles are produced using two baculoviruses in Sf9 cells, where the first baculovirus encodes the genes for AAV2 Rep and AAV2.7m8 Cap protein, and the second baculovirus encodes an anti-VEGF agent. In some embodiments, rAAV particles are produced using two baculoviruses in Sf9 cells, where the first baculovirus encodes the genes for AAV2 Rep and AAV2.7m8 Cap protein, and the second baculovirus encodes an aflibercept (for example, human aflibercept) cDNA expression cassette. In some embodiments, the rAAV particles are produced in Sf9 cells using two baculoviruses, where the first baculovirus encodes genes for the AAV2 Rep and AAV2.7m8 Cap proteins, and the second baculovirus comprises a nucleic acid encoding a polypeptide comprising an amino acid sequence having at least about 95% identity to the amino acid sequence of SEQ ID NO: 35 and flanked by AAV2 inverted terminal repeats (ITRs). In some embodiments, the polypeptide comprises the amino acid sequence of SEQ ID NO: 35. In some embodiments, the polypeptide is aflibercept. dose

[0224] In some embodiments, a unit dose of rAAV particles is administered to one eye of an individual. In some embodiments, the one eye of the individual is the right eye or the left eye. In some embodiments, the one eye of the individual is the right eye. In some embodiments, the one eye of the individual is the left eye. In some embodiments, the methods provided herein further comprise administering a unit dose of rAAV particles to the other eye of the individual. In some embodiments, the one eye of the individual is the right eye and the other eye is the left eye. In some embodiments, the one eye of the individual is the left eye and the other eye is the right eye.

[0225] In some embodiments, the step of administering a unit dose of rAAV particles to the contralateral eye occurs at least about 2 weeks (e.g., at least about 2 weeks, at least about 3 weeks, at least about 4 weeks, at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, at least about 6 months, at least about 7 months, at least about 8 months, at least about 9 months, at least about 10 months, at least about 11 months, at least about 12 months, at least about 1 year, at least about 2 years, at least about 3 years, at ...

Claims

[Claim 1] The invention described in this specification.

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