Methods of Treating Intraocular Neovascular Disease Using AAV2 Variants Encoding Aflibercept

Abstract

Provided is a method for treating an intraocular neovascular disease in an individual, comprising administering to an eye of the individual a unit dose of recombinant adeno-associated virus (rAAV) particles, wherein 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.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority under 35 U.S.C. §119(e) to U.S. Provisional Application No. 63 / 435,103, filed December 23, 2022, U.S. Provisional Application No. 63 / 339,795, filed May 9, 2022, U.S. Provisional Application No. 63 / 336,789, filed April 29, 2022, U.S. Provisional Application No. 63 / 336,191, filed April 28, 2022, and U.S. Provisional Application No. 63 / 305,838, filed February 2, 2022, each of which is incorporated by reference in its entirety.

[0002] SEQUENCE LISTING STATEMENT A public use copy of the sequence listing will be submitted contemporaneously with the specification as an XML file in WIPO standard ST.26 format with the filename "17234-036WO1_SequenceListing.xml", a creation date of January 31, 2023, and a size of 50,228 bytes. The sequence listing submitted through the USPTO Patent Division is a part of the specification and is incorporated herein by reference in its entirety.

[0003] The present disclosure relates to methods of treating intraocular neovascular diseases and disorders in an individual, the method 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). [Background technology]

[0004] Age-related macular degeneration (AMD) is a degenerative disease of the eye that affects the macula, a small light-sensitive area in the center of the retina responsible for reading and fine 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. The 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 angiogenic process is modulated by growth factors, particularly vascular endothelial growth factor (VEGF). The standard treatment for wAMD is a class of molecules that bind and sequester VEGF, such as ranibizumab (Lucentis) and aflibercept (Eylea).

[0005] Diabetic retinopathy (DR) is a major complication of diabetes and the leading cause of blindness in the working age population. DR can be non-proliferative (NPDR) without new blood vessel growth, or proliferative (PDR) with new abnormal blood vessel growth in the retina or choroid. Diabetic macular edema (DME) is a complication of DR and is another example of an eye disease that affects the macula. DME affects up to 10% of people with diabetes and is caused by the accumulation of fluid in the macula. DME is the most frequent cause of vision loss in people with DR. Available treatments to treat 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 can prevent them from 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] The 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 preserve vision. Compliance with such regimens is burdensome for patients, their caregivers, and the health system, and most patients fall out of optimal regimen compliance over time, which correlates with vision loss (Khanani AM, et al.). In addition, complications exist, including endophthalmitis, retinal detachment, traumatic cataract, and elevated intraocular pressure (IOP), and the risk of these complications may be increased with repeated IVT injections (Falavarjani et al., (2013) Eye (Lond), 27 (7): 787-794). Thus, there is a need in the art for a treatment for intraocular neovascular diseases, such as wAMD, DR, or DME, that is effective, has a reduced risk of adverse effects, and is amenable to high long-term patient compliance. [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

[0009] 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 that include 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, 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, where the amino acid residue numbering corresponds to that of the AAV2 VP1 capsid protein. In some embodiments, the method includes reducing retinal fluid in the eye of the individual.

[0010] In another aspect, provided herein is a method for reducing retinal fluid in an eye of an individual having an intraocular neovascular disease, comprising administering a unit dose of rAAV particles to one eye of the 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, 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, the amino acid residue numbering corresponding to that of the AAV2 VP1 capsid protein. In some embodiments, the individual has received at least one treatment with an anti-VEGF agent in about the last 12 weeks prior to administration of the unit dose of the 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 level of retinal fluid 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 6×10 11 vector genome / eye (vg / eye) or less.

[0011] In another aspect, a method for treating an intraocular neovascular disease in an individual, comprising: (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. 11Provided herein are methods comprising 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 particle comprises (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, 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, the amino acid residue numbering corresponding to that of the AAV2 VP1 capsid protein. In some embodiments, the method comprises administering a unit dose of the rAAV particle 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 a unit dose of the rAAV particle 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 a 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 about 4 doses of topical steroid per day for about 1 month, followed by about 3 doses of topical steroid per day for about 1 month, followed by about 2 doses of topical steroid per day for about 1 month, and followed by about 1 dose of topical steroid per day for about 1 month, timing starting from and after administration of the anti-VEGF agent. In some embodiments, the topical steroid treatment comprises about 4 doses of topical steroid per day for about 1 month, followed by about 3 doses of topical steroid per day for about 1 month, followed by about 2 doses of topical steroid per day for about 1 month, and followed by about 1 dose of topical steroid per day for about 1 month, timing starting from and after administration of the rAAV particles. In some embodiments, the topical steroid comprises about 1 μg to about 3 μg dose of 0.05% difluprednate. In some embodiments, the topical steroid comprises about 2.5 μg dose of 0.05% difluprednate.

[0012] In some embodiments, which may be combined with any of the preceding embodiments, the unit dose of the rAAV particles is about 6×10 10 ~Approx. 2×10 11 vector genome / eye (vg / eye). In some embodiments, the unit dose of the rAAV particles is about 2×10 11 or about 6 x 10 10 In some embodiments, which may be combined with any of the preceding embodiments, the unit dose of the rAAV particles is about 6×10 10 ~about 6×10 11 In some embodiments, which may be combined with any of the preceding embodiments, the unit dose of the rAAV particles is about 6×10 10 ~Approx. 2×10 11 In some embodiments, which may be combined with any of the preceding embodiments, the unit dose of the rAAV particles is about 2×10 11 ~about 6×10 11vector genome / eye (vg / eye). In some embodiments, the unit dose of the rAAV particles is about 2×10 11 or about 6 x 10 10 vector genome / eye (vg / eye). In some embodiments, the unit dose of the rAAV particles is about 2×10 11 vg / eye. In some embodiments, the unit dose of the rAAV particles is about 6×10 11 vg / eye.

[0013] In some embodiments, which may be in combination with any of the preceding embodiments, the individual has one or more symptoms of an intraocular neovascular disease in the contralateral eye.

[0014] 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 a unit dose of rAAV particles to the contralateral eye is up to about 2 weeks after administering a unit dose of rAAV particles to one eye. In some embodiments, administering a unit dose of rAAV particles to the contralateral eye is on the same day as administering a unit dose of rAAV particles to one eye, or administering a unit dose of rAAV particles to the contralateral eye is between about 1 day and about 14 days after 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 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 a unit dose of rAAV particles to the contralateral eye is at least about 2 weeks 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 contains more vector genomes / eye (vg / eye) than the unit dose of rAAV particles administered to one eye of the individual.

[0015] 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.

[0016] 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.

[0017] In some embodiments, which may be combined with any of the above-mentioned 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 order from 5' to 3': (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 with 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 a 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 a 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 a nucleic acid sequence of SEQ ID NO: 40 or a sequence having at least 85% identity thereto. In some embodiments, the polypeptide comprises an amino acid sequence of SEQ ID NO: 35 or a sequence having at least 85% identity thereto. In some embodiments, the polypeptide comprises an 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 order from 5' to 3', a TPL sequence comprising a sequence of SEQ ID NO: 24 or a sequence having at least 85% identity thereto, and an eMLP sequence comprising a 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 a 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 a 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 a sequence of SEQ ID NO: 22 or a sequence having at least 85% identity thereto; (b) a promoter region comprising a CMV sequence comprising a sequence of SEQ ID NO: 23 or a sequence having at least 85% identity thereto; (c) a 5'UTR region comprising, in order from 5' to 3', a TPL sequence comprising a sequence of SEQ ID NO: 24 or a sequence having at least 85% identity thereto, and an eMLP sequence comprising a 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 a sequence of SEQ ID NO: 26 or a sequence having at least 85% identity thereto; and (e) an HGH polyadenylation site comprising a sequence of SEQ ID NO: 27 or a sequence having at least 85% identity thereto. In some embodiments, the nucleic acid comprises an AAV ITR on both ends of the element.

[0018] 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.

[0019] In some embodiments, which may be in combination with any of the preceding embodiments, the rAAV particles comprise an AAV2 VP1 capsid protein that includes a GH loop that includes an 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 an amino acid sequence that has 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.

[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 an 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.

[0021] 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 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 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.

[0022] 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.

[0023] In some embodiments, which may be combined with any of the preceding embodiments, the unit dose of the rAAV particles is in a pharmaceutical formulation. In some embodiments, the pharmaceutical formulation comprises the 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% (weight / volume) to about 0.005% (weight / volume) poloxamer 188, and about 6×10 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 6×10 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 2×10 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 6×10 11 vg / mL rAAV particles, and about 0.001% (weight / volume) poloxamer 188, and the pharmaceutical formulation has a pH of about 7.3.

[0024] In some embodiments, which 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, which 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 50 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.

[0025] In some embodiments, which may be combined with any of the above-mentioned embodiments, the individual has been previously treated with an anti-VEGF agent for intraocular neovascular disease.In some embodiments, the individual has been previously injected with an anti-VEGF agent into one eye and / or the other eye before administration of rAAV particles into one eye and / or the other eye.In some embodiments, the individual has not been previously treated with an anti-VEGF agent for intraocular neovascular disease.In some embodiments, the anti-VEGF agent is aflibercept.

[0026] 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.

[0027] In some embodiments, which may be combined with any of the preceding 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 oral prednisone treatment includes administering prednisone at a dose of about 60 mg per day for a total of 6 days, followed by administering prednisone at a dose of about 40 mg per day for a total of 3 days, followed by administering prednisone at a dose of about 20 mg per day for a total of 2 days, followed by administering prednisone at a dose of about 10 mg per day for a total of 2 days, beginning 3 days prior to administration of the unit dose of rAAV particles to one eye and / or the contralateral eye. In some embodiments, the steroid treatment is a topical steroid treatment. In some embodiments, the steroid treatment is a 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.

[0028] In some embodiments, which may be in combination with any of the preceding embodiments, the steroid treatment is a topical steroid treatment, and the topical steroid treatment is a daily steroid treatment for up to about 4 weeks, up to about 6 weeks, or up to about 8 weeks from administration of a unit dose of rAAV particles. In some embodiments, the topical steroid treatment includes about 4 doses of topical steroid at about week 1, about 3 doses of topical steroid at about week 2, about 2 doses of topical steroid at about week 3, and about 1 dose 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 includes about 4 doses of topical steroid per day for about 3 weeks after administration of a unit dose of rAAV particles, about 3 doses of topical steroid per day for about 1 week thereafter, about 2 doses of topical steroid per day for about 1 week thereafter, and about 1 dose of topical steroid per day for about 1 week thereafter. In some embodiments, the topical steroid treatment comprises about four administrations of topical steroid per day for about four weeks, followed by about three administrations of topical steroid per day for about one week, followed by about two administrations of topical steroid per day for about one week, and about one administration of topical steroid per day for about one week thereafter, timing starting about one week prior to administration of the unit dose of rAAV particles. In some embodiments, the topical steroid treatment comprises about four administrations of topical steroid per day for about one month, followed by about three administrations of topical steroid per day for about one month, followed by about two administrations of topical steroid per day for about one month, and about one administration of topical steroid per day for about one month thereafter, timing starting about at the time of administration of the unit dose of rAAV particles. In some embodiments, the topical steroid treatment comprises about four administrations of topical steroid per day for about one month, followed by about three administrations of topical steroid per day for about one month, followed by about two administrations of topical steroid per day for about one month, and then about one administration of topical steroid per day for about one month, starting about one week prior to 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.

[0029] In some embodiments, the intraocular neovascular disease is wet age-related macular degeneration (wAMD).

[0030] In some embodiments, which may be in combination 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 an 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).

[0031] In some embodiments, which may be in combination 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 an 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.

[0032] In some embodiments, which may be in combination 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 a unit dose of rAAV particles to one eye and / or the contralateral eye of an 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).

[0033] 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 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 is present for about 30 weeks or more, about 34 weeks or more, about 44 weeks or more, about 6 months or more, about 1 year or more, about 1.5 years or more, or about 2 years, about 3 years, about 5 years, about 10 years, or more after administration of the unit dose of rAAV particles to the one eye and / or the contralateral eye of the individual.

[0034] 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 50% of the individuals in the plurality not requiring anti-VEGF rescue treatment, in 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.

[0035] 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. In some embodiments, at least about 67% of the individuals in the plurality do not require 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.

[0036] 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. In some embodiments, at least about 78% of the individuals in the plurality do not require 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.

[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 100% of the individuals in the plurality not requiring anti-VEGF rescue treatment, in 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, following administration of the unit dose of rAAV particles.

[0038] 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 the contralateral eye of a plurality of 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 prior to administration of the unit dose of rAAV particles.

[0039] 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 opposite eye of an individual results in a two or three step improvement in the Diabetic Retinopathy Severity Scale (DRSS). In some embodiments, administering a unit dose of rAAV particles to one eye and / or the opposite eye of an individual results in a two step improvement in the Diabetic Retinopathy Severity Scale (DRSS). In some embodiments, administering a unit dose of rAAV particles to one eye and / or the opposite eye of an individual results in a three step improvement in the Diabetic Retinopathy Severity Scale (DRSS).

[0040] In one embodiment, a method of treating an intraocular neovascular disease in an individual is provided. The method comprises administering to an individual a dose of about 2×10 11 The method includes administering a unit dose of a recombinant adeno-associated virus (rAAV) particle having a vector genome (vg) or less to one eye of an individual, the individual being a human, the rAAV particle comprising: (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, the amino acid residue numbering corresponding to that of the AAV2 VP1 capsid protein. In various embodiments of the method, retinal fluid in the eye of an individual having an intraocular neovascular disease is reduced.

[0041] In one embodiment, a method is provided for reducing retinal fluid in an eye of an individual having an intraocular neovascular disease. The method includes administering a unit dose of rAAV particles to one eye of the individual, the individual being a human, the rAAV particles comprising (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, the amino acid residue numbering corresponding to that of the AAV2 VP1 capsid protein. In an aspect of the method, the individual has received at least one treatment with an anti-VEGF agent within about 12 weeks prior to administration of the unit dose of the rAAV particles. In various method aspects, the amount or presence of retinal fluid in one eye of the individual is refractory to a prior treatment with an anti-VEGF agent. In a further embodiment of the method, the anti-VEGF agent is aflibercept.In any embodiment of the above method, the retinal fluid of one eye is reduced by about 80% compared to the retinal fluid level of one eye of the individual before administering rAAV to the individual.In any embodiment of the above method, the retinal fluid is subretinal fluid (SRF) or intraretinal fluid (IRF).

[0042] Methods for treating intraocular neovascular disease in an individual are provided, comprising: (a) administering an anti-VEGF agent to one eye of the individual; and (b) administering about 6×10 vector genomes (vg) or less of a unit dose of recombinant adeno-associated virus (rAAV) particles to one eye of the individual after administration of the anti-VEGF agent, 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, flanked by AAV2 inverted terminal repeats (ITRs), and (ii) an AAV capsid protein 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. An embodiment of the method includes administering a unit dose of rAAV particles to one eye of the individual about one week or about seven days after administration of the anti-VEGF agent. An embodiment of the method includes 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 an embodiment of the method, the anti-VEGF agent includes aflibercept. In an embodiment of the method, the aflibercept is administered by intravitreal injection at a dose of about 2 mg.

[0043] In an embodiment of the above method, the unit dose of rAAV particles is about 6×10 10 ~Approx. 6×10 11 In an embodiment of the above method, the unit dose of rAAV particles is about 6×10 10 ~Approx. 2×10 11 In some embodiments of the above methods, the unit dose of the rAAV particles is about 2×10 11 In another embodiment of the above method, the unit dose of the rAAV particles is about 6×10 11 Vector genome / eye (vg / eye).

[0044] In any of the above methods, the individual has one or more symptoms of intraocular neovascular disease in the opposite eye. Various embodiments of the above methods further include administering a unit dose of rAAV particles to the opposite eye of the individual. In some embodiments, the unit dose of rAAV particles is administered to the opposite eye up to about 2 weeks after the unit dose of rAAV particles is administered to one eye. In some embodiments, the unit dose of rAAV particles is administered to the opposite eye on the same day as the unit dose of rAAV particles is administered to one eye, or the unit dose of rAAV particles is administered to the opposite eye between about 1 day and about 14 days after the unit dose of rAAV particles is administered to one eye. In some embodiments, the unit dose of rAAV particles administered to the opposite eye of the individual contains the same or less vector genomes / eye (vg / eye) compared to the unit dose of rAAV particles administered to one eye of the individual. In some embodiments, the unit dose of rAAV particles is administered to the contralateral eye at least about 2 weeks after the unit dose of rAAV particles is administered to one eye. In some embodiments of the above methods, the unit dose of rAAV particles administered to the contralateral eye of the individual comprises more vector genomes / eye (vg / eye) compared to the unit dose of rAAV particles administered to one eye of the individual.

[0045] In some embodiments of the above methods, the nucleic acid comprises a nucleic acid sequence of SEQ ID NO: 40, or a sequence having at least 85% identity thereto. In some embodiments of the above methods, the polypeptide comprises an amino acid sequence of SEQ ID NO: 35. In some embodiments of the above methods, the polypeptide comprises an amino acid sequence of SEQ ID NO: 41. In some embodiments of the methods, the polypeptide is aflibercept. In some embodiments of the methods, 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 of the methods, the nucleic acid comprises, in order from 5' to 3': (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, flanked by AAV2 inverted terminal repeats (ITRs). In some aspects, the first enhancer region comprises a CMV sequence comprising a sequence of SEQ ID NO:22 or a sequence having at least 85% identity thereto. In some aspects, the promoter region comprises a CMV sequence comprising a sequence of SEQ ID NO:23 or a sequence having at least 85% identity thereto. In some aspects of the method, the nucleic acid encoding the polypeptide comprises a nucleic acid sequence of SEQ ID NO:40 or a sequence having at least 85% identity thereto. In various aspects, the polypeptide comprises an amino acid sequence of SEQ ID NO:35 or a sequence having at least 95% identity thereto. In various aspects, the polypeptide comprises an amino acid sequence of SEQ ID NO:41 or a sequence having at least 95% identity thereto. In some aspects, the polypeptide is aflibercept. In various aspects, the 5'UTR region comprises, from 5' to 3', a TPL sequence comprising a sequence of SEQ ID NO:24 or a sequence having at least 85% identity thereto, and an eMLP sequence comprising a sequence of SEQ ID NO:25 or a sequence having at least 85% identity thereto. In some embodiments of the method, 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 certain embodiments of the method, 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.

[0046] In one embodiment of the method, the nucleic acid further comprises: (a) a first enhancer region comprising a CMV sequence comprising the sequence set forth in 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 order from 5' to 3', 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.

[0047] In any embodiment of the method, the nucleic acid comprises the sequence of SEQ ID NO: 39, or a sequence having at least 85% identity thereto.

[0048] In embodiments of any of the above methods, the AAV2 capsid protein comprises the amino acid sequence LGETTRPA (SEQ ID NO:1) inserted between positions 587 and 588 of the capsid protein, where the amino acid residue numbering corresponds to the AAV2 VP1 capsid protein comprising the sequence of SEQ ID NO: 13. In embodiments of any of the above methods, 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 the AAV2 VP1 capsid protein comprising the sequence of SEQ ID NO: 13.

[0049] In embodiments of any of the above methods, the rAAV particles comprise an AAV2 VP1 capsid protein that includes 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.

[0050] In embodiments of any of the above methods, administration of a unit dose of rAAV particles to one eye and / or the contralateral eye is by intravitreal administration.

[0051] In any of the above methods, the unit dose of the rAAV particles is in a pharmaceutical formulation. In some embodiments, the pharmaceutical formulation comprises the rAAV particles, sodium chloride, sodium phosphate, and a surfactant. In one embodiment of the above methods, the pharmaceutical composition 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% (weight / volume) to about 0.0005% (weight / volume) poloxamer 188, and about 6×10 13 ~Approx. 6×10 10 In some embodiments, the pharmaceutical composition comprises about 180 mM sodium chloride, about 5 mM sodium dihydrogen phosphate, about 5 mM sodium dihydrogen phosphate, about 6×10 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 composition comprises about 180 mM sodium chloride, about 5 mM sodium dihydrogen phosphate, about 5 mM sodium dihydrogen phosphate, about 6×10 11 vg / mL rAAV particles, and about 0.001% (weight / volume) poloxamer 188, and the pharmaceutical formulation has a pH of about 7.3.

[0052] In any of the above methods, the unit dose of rAAV particles administered to the one eye and / or the contralateral eye is a volume of about 25 μl to about 250 μl. In some embodiments, the unit dose of rAAV particles administered to the 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 the one eye and / or the contralateral eye comprises a volume of about 30 μl.

[0053] In embodiments of the above methods, the individual has had prior treatment for an intraocular neovascular disease with an anti-VEGF agent, hi some embodiments, the individual has had one or two injections of the anti-VEGF agent in one eye and / or the contralateral eye prior to administration of the rAAV particles to the one eye and / or the contralateral eye.

[0054] In some aspects of the above methods, the individual has not had prior treatment for the intraocular neovascular disease with an anti-VEGF agent.

[0055] In an embodiment of any one of the above methods, the anti-VEGF agent is aflibercept.

[0056] In one embodiment of any of the above methods, 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.

[0057] In embodiments of the above methods, the unit dose of rAAV particles is administered in combination with steroid treatment. In certain embodiments, the steroid treatment is a corticosteroid treatment. In some embodiments, the steroid treatment is a systemic steroid treatment. In various embodiments, the steroid treatment is an oral steroid treatment. In some embodiments, the steroid treatment is a prednisone treatment. In certain embodiments of the methods, the steroid treatment is a topical steroid treatment. In embodiments of the methods, the steroid treatment is a difluprednate treatment. In embodiments of the above methods, the steroid is administered before, during, and / or after administration of the unit dose of rAAV particles to one eye and / or the contralateral eye.

[0058] In some method embodiments, the steroid treatment is a topical steroid treatment, and the topical steroid treatment is a daily steroid treatment for up to about 4 weeks, up to about 6 weeks, or up to about 8 weeks from administration of a unit dose of rAAV particles. In some embodiments of the above methods, the topical steroid treatment comprises about 4 administrations of topical steroid per day in about the first week, about 3 administrations of topical steroid per day in about the second week, about 2 administrations of topical steroid per day in about the third week, and about 1 administration of topical steroid per day in about the fourth week, with the time period measured beginning at the time of administration of the unit dose of rAAV particles. In some method embodiments, the topical steroid treatment comprises about 4 administrations of topical steroid per day for about 3 weeks after administration of the unit dose of rAAV particles, about 3 administrations of topical steroid per day for about one week thereafter, about 2 administrations of topical steroid per day for about one week thereafter, and about 1 administration of topical steroid per day for about one week thereafter. In some embodiments of the method, 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, followed by administering the topical steroid about once per day for about one week, the time period being measured beginning about one week prior to administration of the unit dose of rAAV particles.

[0059] In some embodiments of the method, the method further comprises administering a topical steroid treatment. In some embodiments of the method, the topical steroid treatment is difluprednate treatment. In some embodiments of the method, the topical steroid treatment comprises about 4 doses of topical steroid per day for about 4 weeks, followed by about 3 doses of topical steroid per day for about 1 week, followed by about 2 doses of topical steroid per day for about 1 week, followed by about 1 dose of topical steroid per day for about 1 week, with the time period measured starting from the time of administration of the anti-VEGF agent. In some embodiments of the method, the topical steroid comprises about 1 μg to about 3 μg of 0.05% difluprednate. In some embodiments of the method, the topical steroid comprises about 2.5 μg of 0.05% difluprednate.

[0060] In any of the above method embodiments, administering a unit dose of rAAV particles to one eye and / or the contralateral eye of the 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 the individual results in a reduction in retinal thickness compared to the retinal thickness before administration of the unit dose of rAAV particles. In certain method 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 certain method embodiments, the retinal thickness is central zone retinal thickness (CST) or central foveal retinal thickness (CRT).

[0061] In any of the above methods, administering a unit dose of rAAV particles to one eye and / or the contralateral eye of the 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 one method embodiment, administering a unit dose of rAAV particles to one eye and / or the contralateral eye of the individual results in a reduction in macular volume compared to the macular volume before administration of the unit dose of rAAV particles. In method 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.

[0062] In any of the above methods, administering a unit dose of rAAV particles to one eye and / or the contralateral eye of the individual results in maintained or improved visual acuity compared to the visual acuity before administration of the unit dose of rAAV particles. In some methods, administering a unit dose of rAAV particles to one eye and / or the contralateral eye of the individual results in improved visual acuity compared to the visual acuity before administration of the unit dose of rAAV particles. In methods, the visual acuity is best corrected visual acuity (BCVA).

[0063] In any of the above methods, administration of a single unit dose of the rAAV particles to one eye and / or contralateral eye of the plurality of individuals results in at least about 50% of the plurality of individuals not requiring anti-VEGF rescue treatment. In some embodiments of the methods, at least about 50% of the plurality of individuals do not require 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 the rAAV particles.

[0064] In some embodiments of the methods, administration of the unit dose of the rAAV particles to one eye and / or the contralateral eye of the plurality of individuals results in at least about 67% of the plurality of individuals not requiring anti-VEGF rescue treatment. In some embodiments of the methods, at least about 67% of the plurality of individuals are 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 the rAAV particles.

[0065] In embodiments of the above methods, 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 plurality of individuals not requiring anti-VEGF rescue treatment, hi various embodiments, at least about 78% of the plurality of individuals 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.

[0066] In embodiments of the above methods, 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 plurality of individuals not requiring anti-VEGF rescue treatment. In embodiments of the methods, 100% of the plurality of individuals are 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, following administration of the unit dose of rAAV particles.

[0067] Any embodiment of the above methods provides for administration of a unit dose of rAAV particles to one eye and / or contralateral eye of a plurality of individuals that 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 prior to administration of the unit dose of rAAV particles.

[0068] In various method embodiments, the intraocular neovascular disease is exudative age-related macular degeneration (AMD).

[0069] In various method embodiments, the intraocular neovascular disease is diabetic macular edema (DME). In various 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 step improvement in the Diabetic Retinopathy Severity Scale (DRSS).

[0070] An embodiment of the present application provides a unit dose of about 6 x 1011 vector genomes (vg) or less of recombinant adeno-associated virus (rAAV) particles for use in a method of treating an intraocular neovascular disease in an individual, the method comprising administering the unit dose to one eye of the individual, the individual being a human, the rAAV particles comprising: (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 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.

[0071] One embodiment of the present application provides a unit dose of rAAV particles for use in a method of reducing retinal fluid in an eye of an individual having an intraocular neovascular disease, the method comprising administering the unit dose to one eye of the individual, the individual being a human, the rAAV particles comprising (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, where the amino acid residue numbering corresponds to that of the AAV2 VP1 capsid protein. The above unit dose aspects for use in the indicated methods, wherein the method further comprises administering an anti-VEGF agent to one eye of the individual prior to administration of the unit dose.

[0072] Incorporation by Reference All references cited herein, including patent applications and publications, are hereby incorporated by reference in their entirety. [Brief description of the drawings]

[0073] [Figure 1]Provides a summary of harvested eyes used for in-life fluid collection in aqueous humor (AH) or vitreous humor (VH) at the indicated time points (30, 60 and 98 days). The eye or eyes from which collection was performed are indicated. "OD" stands for right eye, or right eye. "OS" stands for left eye, or left eye. "OU" stands for both eyes, or both eyes. Subject identifiers and group numbers are also shown.

[0074] [Diagram 2] A table is provided summarizing aflibercept levels obtained in the vitreous fluid in the indicated eye or eyes of subjects at the indicated time points. BLOQ stands for "below the level of quantification." Dosages are expressed in vg / eye. Eyes were treated with either 3E10 or 1E11 viral genomes (vg) / treated eye. Subjects are identified.

[0075] [Diagram 3] Provides a table summarizing aflibercept levels obtained in the aqueous humor in the indicated eye or eyes of subjects at the indicated time points. BLOQ stands for "below the level of quantification." Dosage is expressed in vg / eye. Eyes were treated with either 3E10 or 1E11 viral genome (vg) / treated eye. Subjects are identified.

[0076] [Figure 4] A chart summarizing aqueous aflibercept concentrations in either the aqueous humor or the vitreous humor is provided. Following a dose of 3.00E10 vg / mL, peak aqueous aflibercept values ​​exceeded or approached 500 ng / mL. Animals were treated with doses of 3.00E10 (gray) or 1.00E11 (black) vg / mL. Aqueous humor and vitreous humor were collected 30, 60, and 90 days after dosing. Aflibercept levels were measured using an aflibercept ELISA. Peak aflibercept values ​​detected in the aqueous humor and vitreous humor for each animal are represented here. Black horizontal bars represent group means, black vertical bars represent standard error, and asterisks represent outlier samples hypothesized to be the result of anti-drug antibodies.

[0077] [Diagram 5] We provide charts summarizing historical aqueous humor data showing a flat dose response across three logarithmic dosage levels. The chart on the right is displayed on a logarithmic scale.

[0078] [Figure 6]

[0023] Figure 1 provides a graph summarizing the distribution of ADVM-022 in tissues from non-human primates (NHPs) dosed with 3x1010 (left) and 1x1011 vg / eye (right) of ADVM-022 vector genome (vg). Tissues were harvested 97 days after dosing. "*" represents pooled data from right (R) and left (L) organs, LN represents lymph node, and LGN represents lateral geniculate body. Horizontal bars correspond to the geometric mean. Dosing affects ADVM-022 distribution to a greater extent in some tissues than others.

[0079] [Figure 7] Graphs are provided summarizing the distribution of ADVM-022 expressed mRNA in tissues from non-human primates (NHPs) dosed with 3x1010 (left) and 1x1011 vg / eye (right) of ADVM-022. Tissues were harvested 97 days after dosing. "*" represents pooled data from right (R) and left (L) organs, LN represents lymph node, and LGN represents lateral geniculate body. Horizontal bars correspond to the geometric mean. Dose modification affects ADVM-022 mRNA expression in certain tissues.

[0080] [Figure 8]

[0023] Figure 1 provides a graph summarizing the distribution of ADVM-022 vector genomes (vg) in whole blood from non-human primates (NHPs) dosed with ADVM-022 at 3x1010 (left) and 1x1011 vg / eye (right). Time of whole blood collection is indicated on the x-axis. Whole blood was collected at BL (baseline), 8 hours (0.33 days), 48 hours (2 days), 144 hours (6 days), 336 hours (14 days), 43 days, and 87 days after dosing.

[0081] [Figure 9]1 provides a visual representation of ADVM-022 vector genome biodistribution in NHP eyes dosed with 3E10vg / eye. In photographs of samples taken from the indicated areas, arrowheads point to some of the ADVM-022 DNA positive cells and extracellular particles. In the macula (area A), intracellular vector DNA is detected in the macular retinal ganglion cells and in the nucleated cell layer. In the central retina (area B), no intracellular vector DNA is detected, and vector DNA is detected as particles associated with the internal limiting membrane. In the peripheral retina (area C), intracellular vector DNA is detected in the retinal ganglion cell layer and in the nucleated cell layer. In the ciliary body (area D), intracellular vector DNA is detected in some non-pigmented epithelial cells of the ciliary processes.

[0082] [Figure 10] 1 provides a visual representation of the biodistribution of ADVM-022 transgene mRNA in NHP eyes dosed with 3E10vg / eye. In photographs of samples taken from the indicated areas, arrowheads point to some of the ADVM-022 positive cells. In the macula (area a), transgene mRNA is detected in the macular retinal ganglion cells and in the nucleated cell layer. In the peripheral retina (area b), transgene mRNA is detected in the retinal ganglion cell layer and in the nucleated cell layer. In the ciliary body (area c), transgene mRNA is detected in some non-pigmented epithelial cells of the ciliary processes and in the area of ​​the ciliary muscle.

[0083] [Figure 11]FIG. 1 depicts the design of a Phase 2 study of AAV2.7m8-aflibercept in anti-VEGF treatment-experienced patients with neovascular (exudative) age-related macular degeneration (nAMD) as described in Example 1 herein. The study includes eight study arms (arms 1A, 1B, 2A, 2B, 3A, 3B, 4A, and 4B), with participants within each arm receiving a single dose of AAV2.7m8-aflibercept at a unit dose of 2×10 vg / eye or 6×10 vg / eye, accompanied by a prophylactic corticosteroid treatment regimen (topical, IVT, topical + oral, or IVT + oral). The study includes a screening period (14-21 days) and a 50-week study period following dosing with AAV2.7m8-aflibercept. IVT=intravitreal, nAb=neutralizing antibody, AFL=aflibercept, D=day, W=week, q4W=every 4 weeks, CST=central field retinal thickness, SD-OCT=spectral domain optical coherence tomography, CRC=central reading center, SOC=standard of care, mos=month, QID=4 times daily, TID=3 times daily, BID=2 times daily, QD=once daily, QOD=alternate day, wk=week, PI=principal investigator. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0084] Some embodiments 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 full 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, since some acts may occur in different orders and / or simultaneously with other acts or events. Moreover, not all illustrated acts or events are necessary to implement the methodology according to the features described herein.

[0085] definition Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0086] The terms used herein are merely for the purpose of describing particular examples 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 indicates 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.

[0087] References herein to "or" are intended to encompass "and / or" unless otherwise indicated. As used herein, the term "about" a number refers to that number plus or minus 10%. The term "about" a range refers to a range minus 10% of its minimum value and plus 10% of its maximum value. References herein to "about" a value or parameter include (and describe) embodiments that are directed to that value or parameter per se.

[0088] The term "subject," "patient," or "individual" refers to primates, including humans and non-human primates, such as African green monkeys and rhesus monkeys. In some embodiments, the subject is a human.

[0089] 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 a symptom 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 symptom, inhibiting an intraocular neovascular disease or disorder, such as 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 a symptom of an intraocular neovascular disease or disorder, and are intended to include prevention. The terms further include achieving a therapeutic benefit and / or a prophylactic benefit. The term "therapeutic benefit" refers to the eradication or improvement of the intraocular neovascular disease or disorder being treated. The term "therapeutic benefit" refers to the eradication or improvement of the intraocular neovascular disease or disorder being treated. Similarly, therapeutic benefit is also achieved in some embodiments by eradicating or improving one or more physiological symptoms associated with intraocular neovascular disease or disorder, so that improvement is observed in the subject, even though the subject still suffers from intraocular neovascular disease or disorder.For preventive benefit, the pharmaceutical composition is administered to a subject who is at risk of developing intraocular neovascular disease or disorder, or who reports one or more physiological symptoms of intraocular neovascular disease or disorder, even if the disease or disorder has not been diagnosed.

[0090] The terms "administer," "administering," "administration," and the like, as used herein, may refer to methods used to enable delivery of a therapeutic agent or pharmaceutical composition to a desired site of biological action. These methods include intravitreal or subretinal injection into the eye.

[0091] The terms "effective amount", "therapeutically effective amount", or "pharmaceutical effective amount", as used herein, may refer to a sufficient amount of at least one pharmaceutical composition or compound being administered that relieves to some extent one or more symptoms of the ocular disease or disorder being treated. An "effective amount", "therapeutically effective amount", or "pharmaceutical effective amount" of a pharmaceutical composition may be administered as a unit dose to a subject in need thereof (described in more detail elsewhere herein).

[0092] The term "pharmacologically 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 is relatively non-toxic (i.e., does not elicit undesirable 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).

[0093] The term "pharmaceutical composition" or simply "composition," as used herein, may refer to a biologically active compound optionally mixed with at least one pharma- ceutically acceptable chemical component, such as, but not limited to, a carrier, stabilizer, diluent, dispersant, suspending agent, thickener, excipient, and the like.

[0094] "AAV vector" or "rAAV vector" as used herein refers to an adeno-associated virus (AAV) vector or recombinant AAV (rAAV) vector that contains a polynucleotide sequence that is not of AAV origin (e.g., a polynucleotide heterologous to AAV, such as a nucleic acid sequence encoding a therapeutic transgene, e.g., aflibercept) for transduction into a target cell or into a target tissue. Generally, the heterologous polynucleotide is flanked by at least one AAV inverted repeat repeat (ITR), and generally has two AAV inverted repeat repeats (ITR) at 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).

[0095] "AAV virus" or "AAV virus particle" or "rAAV vector particle" or "rAAV particle" refers to a virus particle that comprises at least one AAV capsid protein and a polynucleotide rAAV vector. In some examples, at least one AAV capsid protein is derived from wild-type AAV or is a variant AAV capsid protein (e.g., an AAV capsid protein with an insertion, such as the insertion of the 7m8 amino acid sequence described below). When the particle comprises a heterologous polynucleotide (e.g., a polynucleotide other than the wild-type AAV genome, such as a transgene that is delivered to a target cell or target tissue), it is called "rAAV particle", "rAAV vector particle", or "rAAV vector". Thus, the production of rAAV particles necessarily includes the production of rAAV vectors, since such vectors are contained within the rAAV particles.

[0096] 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.

[0097] AAV "rep" and "cap" genes refer to polynucleotide sequences that encode the replication and encapsidation proteins of the adeno-associated virus. AAV rep and cap are referred to herein as AAV "packaging genes."

[0098] The term "polypeptide" may encompass both naturally occurring and non-naturally occurring proteins (e.g., fusion proteins), peptides, fragments, mutants, derivatives and analogs thereof. A polypeptide may be a monomer, dimer, trimer, or polymer. Furthermore, a polypeptide may contain several different domains, each of which has one or more distinct activities. For the avoidance of doubt, a "polypeptide" may be any length greater than two amino acids.

[0099] As used herein, "polypeptide variant" or simply "variant" refers to a polypeptide whose sequence contains amino acid modification. 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 of the amino or carboxy termini. A variant may have the same or different biological activity compared to the reference protein or unmodified protein.

[0100] 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.

[0101] As used herein, "recombinant" may refer to a biological molecule, e.g., a gene or protein, that is (1) removed from its naturally occurring environment, (2) not associated with all or a portion of a polynucleotide with which the gene is found in nature, (3) operably linked to a polynucleotide with which it is not linked in nature, or (4) not occurring in nature. The term "recombinant" may be used in reference to cloned DNA isolates, chemically synthesized polynucleotide analogs, or polynucleotide analogs biologically synthesized by heterologous systems, as well as proteins and / or mRNAs encoded by such nucleic acids. Thus, for example, a protein synthesized by a microorganism is recombinant if, for example, it is synthesized from an mRNA synthesized from a recombinant gene present in the cell.

[0102] 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 the 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 then been modified or mutated to confer anti-VEGF activity. In some embodiments, the anti-VEGF agent is a fusion protein or a 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.

[0103] As used herein, "VEGF" 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. 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 of VEGF, including but not limited to VEGFR-1 (or Flt-1), VEGFR-2 (or Flk-1 / KDR), and VEGFR-3 (or Flt-4). VEGFR may be membrane-bound or soluble, or a functional fragment or truncated form 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.

[0104] "Operably linked" or "operably linked" or "coupled" can refer to the juxtaposition of genetic elements in a relationship that allows them to operate in an expected manner. For example, a promoter can be operably linked to a coding region if the promoter helps initiate transcription of the coding sequence. There can be intervening residues between the promoter and the coding region so long as this functional relationship is maintained.

[0105] The term "expression vector" or "expression construct" or "cassette" or "plasmid" or simply "vector" may 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 may be translated into a 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 the mRNA into a gene product. In other aspects, expression only includes transcription of the nucleic acid encoding the gene of interest. The expression vector may also include control elements operably linked to the coding region to facilitate expression of the protein in the target cell. The combination of the 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.

[0106] The term "heterologous" may refer to an entity that is genotypically different 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 may be a heterologous polynucleotide. A promoter that has been removed from its native coding sequence and operably linked to a coding sequence with which it is not naturally found may be a heterologous promoter.

[0107] As used herein, "7m8" refers to the amino acid sequence LALGETTRPA (SEQ ID NO:1).

[0108] "7m8 variant" refers to a 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.

[0109] 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, e.g., 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, e.g., between amino acids 589 and 590 of the AAV10 capsid protein.

[0110] overview 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 may increase the risk of inflammation, infection, and other adverse effects in some patients. In addition, current treatments have compliance issues due to repeated and / or frequent trips to the clinic to administer treatment, especially in elderly patients who are most affected by wAMD. Reduction in the number of administrations 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 utilized to successfully transfer therapeutic genes into photoreceptors, retinal pigment epithelium, and inner retina to treat a variety of retinal diseases. Thus, administration of rAAV particles encoding an anti-VEGF agent (e.g., aflibercept) can provide long-term and / or sustained release of the anti-VEGF agent in vivo.

[0111] Unexpectedly, 6 × 10 of aflibercept-encoding rAAV particles 11 Administration of a single low dose of vector genome (vg) / eye to the eye of individuals with intraocular neovascular disease resulted in disease stabilization and robust anatomical responses in all treated individuals (see Example 1). In addition, 6×10 rAAV particles encoding aflibercept were administered to 11After administration of a single low dose of 10000 vg / eye, vision was stabilized in all treated individuals, and none of them 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 eye of an individual with intraocular neovascular disease unexpectedly caused 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).

[0112] Thus, the present disclosure provides a method for the production of 6×10 rAAV particles encoding an anti-VEGF agent (e.g., aflibercept). 11 Provided herein is a method for treating intraocular neovascular disease in an individual by administering a single unit dose of 10 ...

[0113] Treatment Method Provided herein is a method for treating an intraocular neovascular disease in an individual, the method comprising administering a unit dose of recombinant adeno-associated virus (rAAV) particles to an eye of the individual.

[0114] Also provided herein is a method for reducing retinal fluid in the eye of an individual having an ocular neovascular disease, the method comprising administering a unit dose of rAAV particles to the eye of the individual.

[0115] 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 an 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.

[0116] 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.

[0117] In some embodiments, the term intraocular neovascular disease also encompasses VEGF-driven pre-neovascular diseases that progress to neovascular morphology if left untreated. In some embodiments, the intraocular neovascular disease is the pre-neovascular disease, non-proliferative diabetic retinopathy.

[0118] In some embodiments, the individual is a human. In some embodiments, the individual has received at least one prior 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) during 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 a 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.

[0119] In some embodiments, the intraocular neovascular disease is diabetic macular edema (DME). In some embodiments, the individual is a 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 the 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 the rAAV particles. In some embodiments, the individual has a central zone retinal thickness (CST) of ≥ 325 μm using Heidelberg Spectralis® for the eye receiving the rAAV particles prior to administration of the unit dose of the 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 the rAAV particles. In some embodiments, the individual has a diagnosis of diabetic macular edema for the eye receiving the rAAV particles for about 6 months or less prior to administration of the unit dose of the rAAV particles, e.g., any of 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 the rAAV particles. In some embodiments, the individual has received 0, 1, or 2 prior treatments for DME, e.g., 0, 1, or 2 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 the rAAV particles.In some embodiments, the individual has undergone a 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 the rAAV particles, for the eye receiving the rAAV particles. In some embodiments, the individual has demonstrated a meaningful response in terms of central zone retinal thickness, e.g., at least a 10% reduction in central zone retinal thickness, to a previous treatment with an anti-VEGF agent prior to administration of the unit dose of the rAAV particles, for the eye receiving the rAAV particles. In some embodiments, the individual has not experienced an adverse reaction to a previous treatment with an anti-VEGF agent prior to administration of the unit dose of the rAAV particles. In some embodiments, the individual does not have neutralizing antibodies against AAV2.7m8 prior to administration of the unit dose of the 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 the rAAV particles, e.g., within about 6 months prior to administration of the unit dose of the 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 a 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 a unit dose of rAAV particles, where the allergy is suitable for treatment. In some embodiments, the individual does not have uncontrolled diabetes, e.g., HbA1c greater than 10%, prior to administration of a unit dose of rAAV particles. In some embodiments, the individual does not have a history of diabetic ketoacidosis within about 3 months prior to administration of a unit dose of rAAV particles. In some embodiments, the individual has not begun intensive insulin treatment, e.g., using an insulin pump or multiple daily insulin injections, prior to administration of a unit dose of rAAV particles. In some embodiments, the individual does not plan to begin intensive insulin treatment, e.g., 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 the 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 the rAAV particles. In some embodiments, the individual does not receive systemic anti-VEGF treatment prior to administration of the unit dose of the rAAV particles. In some embodiments, the individual does not have a high risk of proliferative diabetic retinopathy (PDR) for the eye receiving the rAAV particles prior to administration of the unit dose of the rAAV particles. In some embodiments, PDR is defined as any vitreous or preretinal hemorrhage, neovascularization elsewhere in >1 / 2 the papilla area within an area equivalent to a standard ETDRS 7 field on clinical examination, or papilla neovascularization >1 / 3 the papilla area on clinical examination. In some embodiments, the individual has not undergone focal or grid laser photocoagulation for the eye receiving the rAAV particle before administration of the unit dose of the rAAV particle. In some embodiments, the individual has not undergone any previous panretinal photocoagulation (PRP) for the eye receiving the rAAV particle before administration of the unit dose of the rAAV particle. In some embodiments, the individual has not undergone anti-VEGF treatment (e.g., an IVT injection of aflibercept) for the eye receiving the rAAV particle before administration of the unit dose of the rAAV particle. In some embodiments, the individual has not undergone anti-VEGF treatment (e.g., an IVT injection of aflibercept) for at least 60 days before administration of the unit dose of the rAAV particle for the eye receiving the rAAV particle. In some embodiments, the individual has not undergone more than two anti-VEGF treatments (e.g., an IVT injection of aflibercept) for the eye receiving the rAAV particle before administration of the unit dose of the rAAV particle.In some embodiments, the individual does not have a history of anterior ocular neovascularization (e.g., neovascularization of the iris [NVI] or neovascular glaucoma [NVG]), significant vitreous hemorrhage, fibrovascular proliferation, or tractional retinal detachment in the eye receiving the rAAV particles prior to administration of the unit dose of the rAAV particles. In some embodiments, the individual does not have a structural abnormality of the fovea that contributes to macular edema or vision impairment (e.g., thick, hard exudate, pigmentary abnormalities, foveal atrophy, vitreomacular traction, or epiretinal membrane) in the eye receiving the rAAV particles prior to administration of the unit dose of the rAAV particles. In some embodiments, the structural abnormality of the fovea 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 the 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 the 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 the 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 the rAAV particles. In some embodiments, the individual does not have a 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 the rAAV particles.In some embodiments, the individual does not have a history of vitrectomy or other filtration surgery with respect to the eye receiving the rAAV particles prior to administration of the unit dose of the rAAV particles.In some embodiments, the individual does not have 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 the rAAV particles.In some embodiments, the individual does not have uncontrolled ocular hypertension or glaucoma, e.g., IOP>22 mmHg despite treatment with anti-glaucoma medication or current use of IOP-lowering medications >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 for any ocular condition (e.g., IVT Triesence, Iluvien, or Ozurdex) 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 had 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 the rAAV particles. In some embodiments, the individual has no history of uveitis or intraocular inflammation, e.g., any grade other than a minor expected postoperative inflammation that has resolved or exceeded, prior to administration of the unit dose of the 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 the 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 the 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 the rAAV particles. In some embodiments, the individual has no history of ocular toxoplasmosis prior to administration of the unit dose of rAAV particles.

[0120] In some embodiments, the unit dose is expressed as vector genomes (vg). In some embodiments, the unit dose is about 6×10 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 6×10 of rAAV particles. 11 vg / eye or less, e.g., 1×10 11 In some embodiments, the unit dose of the rAAV particles is about 6×10 10 ~Approx. 2×10 11 vg / eye. In some embodiments, the unit dose is 3×10 11 In some embodiments, the unit dose of the rAAV particles is about 2×10 11 or about 6 x 10 10 vg / eye.

[0121] In some embodiments, the unit dose of the rAAV particles is administered to one eye of the 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 method provided herein further comprises administering the unit dose of the 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.

[0122] In some embodiments, the step of administering a unit dose of rAAV particles to the contralateral eye of the individual is up to about 2 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) than the unit dose of rAAV particles administered to one eye of the individual.

[0123] In some embodiments, the step of administering a unit dose of rAAV particles to the contralateral eye is 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 greater, higher).

[0124] 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, where 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)

[0125] 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, where the amino acid residue numbering corresponds to that of the AAV2 VP1 capsid protein.

[0126] 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, flanked by AAV2 inverted terminal 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, 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 or a functional variant or functional fragment thereof.

[0127] In some embodiments, the rAAV particles comprise a nucleic acid that includes a codon-optimized sequence that encodes 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 that encodes 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 that encodes 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).

[0128] In some embodiments, the rAAV particle comprises a nucleic acid comprising the cDNA sequence of aflibercept or its functional variant or its functional fragment, and flanked by AAV2 inverted repeat repeats (ITRs).In some embodiments, the rAAV particle comprises a nucleic acid comprising the codon-optimized cDNA sequence of aflibercept or its functional variant or its functional fragment, and flanked by AAV2 inverted repeat repeats (ITRs).In some embodiments, the rAAV particle comprises a nucleic acid comprising the nucleic acid sequence of SEQ ID NO:36.

[0129] 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 order from 5' to 3', a TPL sequence and an eMLP sequence; (d) a second enhancer region comprising a full-length EES sequence; and (e) an HGH polyadenylation site. In some embodiments, the enhancer region comprising a CMV sequence comprises the sequence of SEQ ID NO:22. In some embodiments, the promoter region comprising a 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 a 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.

[0130] 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: (SEQ ID NO:13)

[0131] 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 the AAV2 VP1 capsid protein, which comprises the sequence of SEQ ID NO: 13.

[0132] 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), LAGGSVGA (SEQ ID NO:11), LAGGSVGA (SEQ ID NO:12), LAGGSVGA (SEQ ID NO:13), LAGGSVGA (SEQ ID NO:14), LAGGSVGA (SEQ ID NO:15), LAGGSVGA (SEQ ID NO:16), LAGGSVGA (SEQ ID NO:17), LAGGSVGA (SEQ ID NO:18), LAGGSVGA (SEQ ID NO:19), LAGGSVGA (SEQ ID NO:20), LAGGSVGA (SEQ ID NO:21), LAGGSVGA (SEQ ID NO:22), LAGGSVGA (SEQ ID NO:23), LAGGSVGA (SEQ ID NO:24), LAGGSVGA (SEQ ID NO:25), LAGGSVGA (SEQ ID NO:26), LAGGSVGA (SEQ ID NO:27), LAGGSVGA (SEQ ID NO:28), LAGGSVGA (SEQ ID NO:29), LAGGSVGA (SEQ ID NO:30), LAGGSVGA (SEQ ID NO:31), LAGGSVGA (SEQ ID NO:32), LAGGSVGA (SEQ ID NO:33), LAGGSVGA (SEQ ID NO:34), LAGGSVGA (SEQ In some embodiments, the rAAV particles comprise an AAV2 capsid protein comprising any of the following: sequence number 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), where the amino acid residue numbering corresponds to the AAV2 VP1 capsid protein. The AAV2 capsid protein includes 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).

[0133] In some embodiments, the administration of a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual is by intravitreal (IVT) injection, intraocular administration, or intraretinal injection. In some embodiments, the administration of a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual is by intravitreal (IVT) injection.

[0134] In some embodiments, the unit dose of the 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 1×10 10 vg / mL ~ approx. 1×10 13 In some embodiments, the pharmaceutical formulation comprises about 6×10 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% (w / v) to about 0.005% (w / v) poloxamer 188 (e.g., about 0.0005% (w / v), 0.0006% (w / v), 0.0007% (w / v), 0.0008% (w / v), 0.0009% (w / v), 0.001% (w / v), 0.002% (w / v), 0.003% (w / v), 0.004% (w / v), or about 0.005% (w / v)). 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 comprises about 6×10 11 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.

[0135] 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 such 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 the rAAV particles comprises a volume of about 100 μL. In some embodiments, a unit dose of the rAAV particles comprises a volume of about 30 μL.

[0136] 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 ophthalmic steroid treatment is a glucocorticoid, including but not limited to an anti-inflammatory glucocorticoid. In some embodiments, the topical steroid treatment is a glucocorticoid, including but not limited to an anti-inflammatory glucocorticoid. 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. Anti-inflammatory glucocorticoids can include, but are not limited to, difluprednate, dexamethasone, prednisolone, triamcinolone, fluorometholone, rimexolone, fluocinolone, loteprednol, and bioequivalents thereof. In some embodiments, the topical steroid treatment is difluprednate treatment. By "dexamethasone" is intended dexamethasone, biosimilars of dexamethasone, bioisosteres of dexamethasone, and pharmaceutical compositions containing dexamethasone, biosimilars of dexamethasone, or bioisosteres of dexamethasone. Pharmaceutical compositions containing dexamethasone include, but are not limited to, Ozurdex™, Maxidex™, Decadron™, Dexamethasone Intensol™, Ocu-Dex™, Dexycu™, Dextenza™, and Zodex™.Ozurdex™ is a pharmaceutical composition that includes dexamethasone. By "difluprednate" is intended difluprednate, biosimilars of difluprednate, bioequivalents of difluprednate, and pharmaceutical compositions that include difluprednate, biosimilars of difluprednate, or bioequivalents of difluprednate. Pharmaceutical compositions that include difluprednate include, but are not limited to, Durezol™, and difluprednate emulsion. By "triamcinolone" is intended triamcinolone, biosimilars of triamcinolone, bioequivalents of triamcinolone, and pharmaceutical compositions that include triamcinolone, biosimilars of triamcinolone, or bioequivalents of triamcinolone. Pharmaceutical compositions that include triamcinolone include, but are not limited to, Triesence™, Xipere™, and Trivaris™. 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.

[0137] 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 a 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 includes about 4 administrations of ophthalmic steroid at about week 1, about 3 administrations of ophthalmic steroid at about week 2, about 2 administrations of ophthalmic steroid at about week 3, and about 1 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.

[0138] 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 a 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, about 4 administrations per day (i.e., QID) of topical steroid for about 3 weeks, followed by about 3 administrations per day (i.e., TID) of topical steroid for about 1 week, followed by about 2 administrations per day (i.e., BID) of topical steroid for about 1 week, followed by about 1 administration per day (i.e., QD) of topical steroid for about 1 week. In some embodiments, the topical steroid comprises about 1 μg to about 3 μg dose of 0.05% difluprednate. In some embodiments, the topical steroid comprises about 2.5 μg dose 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 from about 1 μg to about 5 μg, or from 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.

[0139] In some aspects, the invention provides a method of treating an intraocular neovascular disease in an individual, comprising administering recombinant adeno-associated virus (rAAV) particles to one eye of the 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, 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, the amino acid residue numbering corresponding to that of the AAV2 VP1 capsid protein, and wherein a unit dose of the rAAV particles is administered in combination with oral steroid treatment and topical steroid treatment, or oral steroid treatment and intravitreal (IVT) steroid treatment. In some embodiments, the oral steroid treatment and topical steroid treatment, or oral steroid treatment and IVT is corticosteroid treatment. In some embodiments, the topical steroid treatment is a difluprednate treatment. In some embodiments, the topical steroid treatment comprises difluprednate 0.05% at a dose of about 1 μg to about 3 μg. In some embodiments, the topical steroid treatment comprises difluprednate 0.05% at a dose of about 2.5 μg. In some embodiments, the topical steroid treatment comprises administration of topical steroid about four times per day from about day 7 prior to administration of a unit dose of rAAV particles to about day 35 after administration of a unit dose of rAAV particles, administration of topical steroid about three times per day from about day 36 to about day 63 after administration of a unit dose of rAAV particles, administration of topical steroid about two times per day from about day 64 to about day 91 after administration of a unit dose of rAAV particles, administration of topical steroid about once per day from about day 92 to about day 119 after administration of a unit dose of rAAV particles, and administration of topical steroid about once every other day starting from about day 120 to about day 147 after administration of a unit dose of rAAV particles.In some embodiments, the topical steroid treatment comprises administration of topical steroid about 4 times per day from about 1 week prior to administration of a unit dose of the rAAV particles to about 5 weeks after administration of a unit dose of the rAAV particles, administration of topical steroid about 3 times per day from about 6 weeks to about 9 weeks after administration of a unit dose of the rAAV particles, administration of topical steroid about 2 times per day from about 10 weeks to about 13 weeks after administration of a unit dose of the rAAV particles, administration of topical steroid about once per day from about 14 weeks to about 17 weeks after administration of a unit dose of the rAAV particles, and administration of topical steroid about once every other day from about 18 to about 21 weeks after administration of a unit dose of the rAAV particles. In some embodiments, the oral steroid treatment is prednisone treatment. In some embodiments, oral steroid treatment is administered at a dose of about 0.8 mg / kg / day to about 1.00 mg / kg / day for 1 week prior to and 2 weeks after administration of a unit dose of rAAV particles, about 0.59 mg / kg / day to about 0.75 mg / kg / day in weeks 3 to 5 after administration of a unit dose of rAAV particles, about 0.4 mg / kg / day to about 0.6 mg / kg / day in week 6 after administration of a unit dose of rAAV particles, about 0.25 mg / kg / day to about 0.375 mg / kg / day in week 7 after administration of a unit dose of rAAV particles, about 0.14 mg / kg / day to about 0.28 mg / kg / day in week 8 after administration of a unit dose of rAAV particles, and about 0.063 mg / kg / day to about 0.125 mg / kg / day in week 9 after administration of a unit dose of rAAV particles. In some embodiments, oral steroid treatment is administered at a dose of about 1.00 mg / kg / day for 1 week prior to and 2 weeks after administration of a unit dose of rAAV particles, about 0.75 mg / kg / day or less in weeks 3 to 5 after administration of a unit dose of rAAV particles, about 0.6 mg / kg / day or less in week 6 after administration of a unit dose of rAAV particles, about 0.375 mg / kg / day or less in week 7 after administration of a unit dose of rAAV particles, about 0.28 mg / kg / day or less in week 8 after administration of a unit dose of rAAV particles, and about 0.125 mg / kg / day or less in week 9 after administration of a unit dose of rAAV particles.In some embodiments, the oral steroid treatment is administered at a dose of about 0.125 mg / kg / day to about 0.14 mg / kg / day for the first 4 days of the 9th week after administration of a unit dose of rAAV particles, and if the individual weighs 70 kg or more, at a dose of about 0.0625 mg / kg / day to about 0.070 mg / kg / day for the first 4 days of the 9th week after administration of a unit dose of rAAV particles. In some embodiments, the IVT steroid treatment is a triamcinolone acetonide treatment, a dexamethasone treatment. In some embodiments, the IVT steroid treatment is a treatment such as, but not limited to, Ozurdex®, Iluvien®, or Triesence®. In some embodiments, the IVT steroid treatment is about 2 mg of triamcinolone acetonide treatment. In some embodiments, the IVT steroid treatment comprises administration of IVT steroid treatment one week prior to administration of a unit dose of rAAV particles and 12 weeks after administration of a unit dose of rAAV particles. In some embodiments, the IVT steroid treatment is about 0.7 mg of dexamethasone. In some embodiments, the IVT steroid treatment is an intravitreal implant one week prior to administration of a unit dose of rAAV particles. In some embodiments, oral steroid treatment and / or topical steroid treatment is administered before, during and / or after administration of a unit dose of rAAV particles.

[0140] In some aspects, the invention provides a unit dose of recombinant adeno-associated virus (rAAV) particles for use in a method of treating an intraocular neovascular disease in an individual, the method comprising administering the unit dose to one eye of the 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, the nucleic acid flanking the AAV2 inverted terminal repeat (ITR); 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, and wherein the unit dose of rAAV particles is administered in combination with oral steroid treatment and topical steroid treatment, or oral steroid treatment and intravitreal (IVT) steroid treatment.

[0141] In some embodiments, the retinal fluid (e.g., SRF and / or IRF) of the individual's eye is reduced by greater than any of about 5%, about 10%, about 15%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 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) of the individual's eye is reduced by greater than any of about 5%, about 10%, about 15%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or 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 prior to administration of the unit dose of rAAV particles. In some embodiments, 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.

[0142] In some embodiments, the methods provided herein further include monitoring the levels of retinal fluid (e.g., SRF and / or IRF) in one eye and / or the contralateral eye of the individual following administration of a unit dose of the rAAV particles. In some embodiments, a reduction in retinal fluid (e.g., SRF and / or IRF) in the eye is first observed after any of the following periods: 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, following administration of the unit dose of the 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.

[0143] 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) of the eye is determined by direct retinal observation.

[0144] In some embodiments, whether an intraocular neovascular disease is treated in an individual following 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., SRF and / or IRF) compared to the level of retinal fluid (e.g., intraretinal fluid (IRF) and / or subretinal fluid (SRF)) prior to 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, 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 (e.g., as described above) 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 contralateral eye compared to the level of retinal fluid (e.g., IRF and / or SRF) before administration of the unit dose of rAAV particles to the one eye and / or the contralateral eye. In some embodiments, the intraocular neovascular disease is wAMD.

[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 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 zone 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.

[0146] 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 central foveal 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, 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 reduction in central foveal thickness (CRT) or central zone 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 reduction in central foveal thickness (CRT) or central zone 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 reduction in central foveal 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 reduction in central foveal 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 reduction in central foveal thickness (CRT) or central zone retinal thickness (CST) of about 5 μm to about 20 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, 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 reduction in central foveal thickness (CRT) or central zone 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 opposite eye of an individual results in a decrease of about 18.5 μm in central retinal thickness (CRT) or central zone retinal thickness (CST). In some embodiments, administering a unit dose of rAAV particles to one eye and / or the opposite eye of an individual results in a decrease of about 21.0 μm in central retinal thickness (CRT) or central zone retinal thickness (CST). In some embodiments, administering a unit dose of rAAV particles to one eye and / or the opposite eye of an individual results in a decrease of about 8.3 μm in central retinal thickness (CRT) or central zone retinal thickness (CST). In some embodiments, administering a unit dose of rAAV particles to one eye and / or the opposite eye of an individual results in a decrease of about 25.5 μm in central retinal thickness (CRT) or central zone 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 in central foveal 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 about 75 μm in central retinal thickness (CRT) or central zone 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 100 μm or more in central retinal thickness (CRT) or central zone retinal thickness (CST) (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 reduction in central foveal 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 reduction in central foveal 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 reduction in central foveal 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 reduction in central foveal 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 reduction in central foveal 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 reduction in central foveal 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 reduction in central foveal 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 reduction in central foveal 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 reduction in central foveal 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 reduction in central foveal 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 reduction in central foveal 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 reduction in central foveal 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 reduction in central foveal 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 170 μm, about 180 μm, about 190 μm, about 200 μm, or about 210 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 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 reduction in central foveal 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 reduction in central foveal 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 reduction in central foveal thickness (CRT) or central zone retinal 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 reduction in central foveal thickness (CRT) or central zone retinal 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 reduction in central foveal thickness (CRT) or central zone 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 reduction in central foveal thickness (CRT) or central zone 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 foveal thickness (CRT) or central zone retinal thickness (CST) of about 180 μm to about 200 μm (e.g., any of 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 zone retinal thickness (CST) of about 21.0 μm compared to the central foveal thickness (CRT) or central zone retinal thickness (CST) prior to 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 foveal thickness (CRT) or central regional retinal thickness (CST) of about 8.3 μm compared to the central foveal 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 foveal thickness (CRT) or central regional retinal thickness (CST) of about 26.2 μm compared to the central foveal 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 foveal thickness (CRT) or central regional retinal thickness (CST) of about 24.8 μm compared to the central foveal 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 foveal thickness (CRT) or central regional retinal thickness (CST) of about 40.8 μm compared to the central foveal 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 foveal thickness (CRT) or central regional retinal thickness (CST) of about 30.0 μm compared to the central foveal 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 foveal thickness (CRT) or central regional retinal thickness (CST) of about 118.6 μm compared to the central foveal 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 foveal thickness (CRT) or central regional retinal thickness (CST) of about 119.0 μm compared to the central foveal 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 foveal thickness (CRT) or central regional retinal thickness (CST) of about 137.8 μm compared to the central foveal 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 foveal thickness (CRT) or central zone retinal thickness (CST) of about 152.7 μm compared to the central foveal thickness (CRT) or central zone 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 foveal thickness (CRT) or central regional retinal thickness (CST) of about 153.3 μm compared to the central foveal 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 foveal thickness (CRT) or central regional retinal thickness (CST) of about 149.8 μm compared to the central foveal thickness (CRT) or central regional retinal thickness (CST) before administration of the unit dose of rAAV particles.

[0147] 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 foveal 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 opposite eye of an individual results in a decrease in central foveal thickness (CRT) or central zone 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 opposite eye of an individual results in a decrease in central foveal thickness (CRT) or central zone 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 opposite eye of an individual results in an increase in central foveal thickness (CRT) or central zone 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 foveal thickness (CRT) or central regional retinal thickness (CST) of about -21.0 μm compared to the central foveal 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 foveal thickness (CRT) or central regional retinal thickness (CST) of about -8.3 μm compared to the central foveal 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 foveal thickness (CRT) or central regional retinal thickness (CST) of about -26.2 μm compared to the central foveal 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 foveal thickness (CRT) or central regional retinal thickness (CST) of about -24.8 μm compared to the central foveal 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 foveal thickness (CRT) or central regional retinal thickness (CST) of about -40.8 μm compared to the central foveal 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 foveal thickness (CRT) or central regional retinal thickness (CST) of about -30.0 μm compared to the central foveal 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 foveal thickness (CRT) or central regional retinal thickness (CST) of about -118.6 μm compared to the central foveal 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 foveal thickness (CRT) or central regional retinal thickness (CST) of about -119.0 μm compared to the central foveal 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 foveal thickness (CRT) or central zone retinal thickness (CST) of about -137.8 μm compared to the central foveal thickness (CRT) or central zone 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 foveal thickness (CRT) or central regional retinal thickness (CST) of about -152.7 μm compared to the central foveal 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 foveal thickness (CRT) or central regional retinal thickness (CST) of about -153.3 μm compared to the central foveal 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 foveal thickness (CRT) or central zone retinal thickness (CST) of about -149.8 μm compared to the central foveal thickness (CRT) or central zone retinal thickness (CST) before administration of the unit dose of rAAV particles.

[0148] In some embodiments, the 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 a unit dose of the 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 a unit dose of the 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 a unit dose of the rAAV particles is observed 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 a unit dose of the 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.

[0149] 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 central retinal thickness (CRT) or central zone retinal thickness (CST) of greater than 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, administering a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual results in a reduction in central retinal thickness (CRT) or central zone retinal thickness (CST) of 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 greater compared to the retinal thickness before administration of the unit dose of rAAV particles.

[0150] 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 zone 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, less than about 1 μm, or less than that) 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 zone retinal thickness (CST) of about 32 μm or less compared to the retinal thickness before administration of the unit dose of rAAV particles.

[0151] In some embodiments, the maintenance, reduction, or increase in retinal thickness compared to the retinal thickness before administration of a unit dose of rAAV particles is present at about 30 weeks or more after administration of a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual. In some embodiments, the maintenance, reduction, or increase in retinal thickness compared to the retinal thickness before administration of a 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 a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual.

[0152] 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 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.

[0153] In some embodiments, the maintenance or reduction in macular volume compared to the macular volume before administration of a unit dose of rAAV particles is present at about 30 weeks or more after administration of a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual. In some embodiments, the maintenance or reduction in macular volume compared to the macular volume before administration of a 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 a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual.

[0154] In some 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, administering a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual results in an improvement of visual acuity compared to the visual acuity 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 of visual acuity 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 greater than any of the above, 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). 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).

[0155] 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 Opthal mol 134(9):1041:1047) (e.g., at least about 15 letters, at least about 20 letters, at least about 30 letters, at least about 40 letters, at least about 50 letters, at least about 60 letters, or about 70 letters) on the ETDRS compared to the BCVA before administration of the unit dose of the 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 opposite eye of an individual results in an improvement in BCVA of about 5.1 letters or more in 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 opposite eye of an individual results in an improvement in BCVA of about 6.4 letters or more in 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 opposite eye of an individual results in an improvement in BCVA of about 6.8 letters in 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 opposite eye of an individual results in an improvement in BCVA of about 8.8 letters in 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 the rAAV particles.

[0156] 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 prior to administration of the unit dose of rAAV particles, and wherein the individual experiences a reduction in ETDRS letters of less than 15 letters (Vitale et al., (2016) JAMA Opthalmol 134(9):1041:1047) (e.g., 15 letters or less, 14 letters or less, 13 letters or less, 12 letters or less, 11 letters or less, 10 letters or less, 9 letters or less, 8 letters or less, 7 letters or less, 6 letters or less, 5 letters or less, 4 letters or less, 3 letters or less, 2 letters 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 a maintained BCVA compared to the BCVA before administration of the unit dose of rAAV particles, where 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 a maintained BCVA compared to the BCVA before administration of the unit dose of rAAV particles, where 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 a maintained BCVA compared to the BCVA before administration of the unit dose of rAAV particles, where 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, where the individual has a letter reduction of about 1. 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, where 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 a maintained BCVA compared to the BCVA before administration of the unit dose of rAAV particles, where 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 a maintained BCVA compared to the BCVA before administration of the unit dose of rAAV particles, where the individual has a letter reduction of about 2 letters 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 a maintained BCVA compared to the BCVA before administration of the unit dose of rAAV particles, where the individual has a letter reduction of about 3.2 letters 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, and wherein the individual experiences a letter reduction of about 15 to 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, where the individual's letter reduction is 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, where the individual's letter reduction is 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 a maintained BCVA compared to the BCVA before administration of the unit dose of rAAV particles, where the individual's letter reduction is about 4 to 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 a maintained BCVA compared to the BCVA before administration of the unit dose of rAAV particles, where the individual's letter reduction is 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 a maintained BCVA compared to the BCVA before administration of the unit dose of rAAV particles, where the individual's letter reduction is 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 maintenance of BCVA compared to the BCVA before administration of the unit dose of rAAV particles, where the individual experiences a letter reduction of about 1 to about 0 letters.

[0157] 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 on the ETDRS of about -20 to +7 letters or more (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) 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 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 a decrease 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 a decrease 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.

[0158] 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 letters, 0 letters, +1 letters, +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 (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) 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 +2 to +7 letters or more (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) 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 +3 to +7 letters or more (e.g., any of +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., any of +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.

[0159] 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., aqueous humor and / or vitreous cell-driven inflammation, aqueous humor flare, posterior iris synechiae, hypodilation). 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., aqueous humor and / or vitreous cell-driven inflammation, aqueous humor flare, posterior iris synechiae, hypodilation) that improves following 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., aqueous humor and / or vitreous cell-driven inflammation) that resolves following administration of oral and / or topical steroid treatment. Inflammation (e.g., inflammation driven by aqueous humor cells and / or vitreous cells, aqueous humor flare, posterior synechiae, hypodilation) can be measured using any method known in the art, such as slit lamp examination.

[0160] In some embodiments, the maintenance or improvement in visual acuity (e.g., BCVA) compared to visual acuity prior to administration of a unit dose of the rAAV particles is present at any time point after 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 following administration of a unit dose of the 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 a unit dose of rAAV particles is present at about 30 weeks or more after administration of a 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 a 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 a unit dose of rAAV particles to one eye and / or the contralateral eye of the individual.

[0161] In some embodiments, treatment of an ocular neovascular disease in an individual following administration of a unit dose of rAAV particles to one eye and / or the contralateral eye is assessed based on best corrected visual acuity (BCVA) in one eye and / or the contralateral 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 the individual has a decrease in ETDRS score of less than 15 letters (e.g., any of 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, 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 the individual has an increase of at least 15 letters (e.g., any of 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 about 70 letters) compared to before administration of a unit dose of rAAV particles to one eye and / or the contralateral eye.

[0162] 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 opposite eye is assessed based on the central zone retinal thickness (CST) or central foveal retinal thickness (CRT) in one eye and / or the opposite 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 opposite eye is determined if the CST or CRT assessed by SD-OCT is decreased after administration of a unit dose of rAAV particles to one eye and / or the opposite eye compared to before administration of a unit dose of rAAV particles to one eye and / or the opposite 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 CST or CRT 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.

[0163] 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 opposite eye is assessed based on the macular volume in one eye and / or the opposite eye. In some embodiments, the macular volume is determined by SD-OCT. 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 opposite eye is determined if the macular volume assessed by SD-OCT is decreased after administration of a unit dose of rAAV particles to one eye and / or the opposite eye compared to before administration of a unit dose of rAAV particles to one eye and / or the opposite 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 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.

[0164] In some embodiments, whether an intraocular neovascular disease has been treated in an individual following administration of a unit dose of rAAV particles to one eye and / or the contralateral eye is assessed based on 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 following 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 decreased after administration of a unit dose of rAAV particles to one eye and / or the contralateral eye compared to before administration of a unit dose of rAAV particles to 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.

[0165] 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 opposite eye is evaluated based on the number of rescue therapeutic treatments (e.g., aflibercept injections) required by the individual after administration of a unit dose of rAAV particles to one eye and / or the opposite eye. In some embodiments, if an individual requires less than one rescue therapeutic treatment (e.g., aflibercept injection) every 4 weeks, every 5 weeks, every 6 weeks, every 7 weeks, every 8 weeks, every 9 weeks, every 10 weeks, or more after administration of a unit dose of rAAV particles to one eye and / or the opposite eye, it is determined that an intraocular neovascular disease is treated in an individual after administration of a unit dose of rAAV particles to one eye and / or the opposite eye.

[0166] In some embodiments, an intraocular neovascular disease is determined to be treated in an individual following 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 the following periods: 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 following administration of a unit dose of rAAV particles to one eye and / or the contralateral eye.

[0167] 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 treatment (e.g., aflibercept injection) 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 treatment (e.g., aflibercept injection) for at least about one month after administration of a unit dose of rAAV particles to one eye and / or the contralateral eye.

[0168] 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% of the individuals in the plurality (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%) not needing anti-VEGF rescue treatment (e.g., 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 in the plurality not needing anti-VEGF rescue treatment (e.g., 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 in the plurality not needing anti-VEGF rescue treatment (e.g., aflibercept injection). In some embodiments, administration of a single unit dose of rAAV particles to one eye and / or the opposite eye of a plurality of individuals results in at least about 80% of the individuals in the plurality not needing anti-VEGF rescue treatment (e.g., aflibercept injection). In some embodiments, administration of a single unit dose of rAAV particles to one eye and / or the opposite eye of a plurality of individuals results in at least about 82% of the individuals in the plurality not needing anti-VEGF rescue treatment (e.g., aflibercept injection). In some embodiments, administration of a single unit dose of rAAV particles to one eye and / or the opposite eye of a plurality of individuals results in 100% of the individuals in the plurality not needing anti-VEGF rescue treatment (e.g., aflibercept injection).

[0169] 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% of the individuals within the plurality (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%) remaining healthy for 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 38 weeks, at least about 39 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 weeks, at least about 72 weeks, at least about 75 weeks, at least about 75 weeks, at 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 in 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 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 78% of the individuals in the plurality experiencing amelioration for 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 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 70 weeks, at least about 72 weeks, at least about 74 weeks, at least about 76 weeks, at least about 78 weeks, at least about 79 weeks, at least about 80 weeks, at least about 82 weeks, at least about 84 weeks, at least about 86 weeks, at least about 88 weeks, at least about 90 weeks, at least about 92 weeks, at least about 94 weeks, at least about 96 weeks, at least about 98 weeks, at least about 99 weeks, at least about 100 weeks, at least about 102 weeks, at least about 104 weeks, at least about 106 weeks, at least about 108 weeks, at least about 108 weeks, at least about 112 weeks, at least about 116 weeks, at least about 120 weeks, at least about 122 weeks, at least about 124 No anti-VEGF rescue treatment (e.g., aflibercept injections) is 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 for 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 viable for 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 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 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 82 weeks, at least about 84 weeks, at least about 85 weeks, at least about 86 weeks, at least about 87 weeks, at least about 89 weeks, at least about 90 weeks, at least about 92 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 99 weeks, at least about 100 weeks, at least about 102 weeks, at least about 106 weeks, at least about 108 weeks, at 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 in 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.

[0170] 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 in 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 some 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 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 in the plurality requiring some 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 in the plurality (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 for the one eye and / or the contralateral eye (e.g., an aflibercept injection). 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 for the one eye and / or the contralateral eye (e.g., an aflibercept injection).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 any rescue treatment (e.g., 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 0% of the individuals in the plurality requiring any rescue treatment (e.g., aflibercept injection) to the one eye and / or the contralateral eye.

[0171] 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 in 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) having a cytological or phenotypic response at least about 4 weeks after administration of the rAAV particles, e.g., rAAV. Any rescue treatment (e.g., aflibercept injections) is required for any of 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, following 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 in the plurality requiring any 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 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 any rescue treatment (e.g., an aflibercept injection) to 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 any rescue treatment (e.g., an aflibercept injection) to 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 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., 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 the rAAV particles to one eye and / or contralateral eye of a plurality of individuals results in 0% of the individuals in the plurality remaining responsive to 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 48 weeks, at least about 50 weeks, at least about 55 weeks, at least about 60 weeks, at least about 65 weeks, at least about 70 weeks, at least about 75 weeks, at least about 80 weeks, at least about 85 weeks, at least about 90 weeks, at least about 95 weeks, at least about 100 weeks, at least about 10 ... Any rescue treatment (e.g., aflibercept injection) is required 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.

[0172] 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 prior to 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 prior to 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 prior to administration of the unit dose of rAAV particles.

[0173] 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 prior to administration of a unit dose of rAAV particles) / (number of days from first anti-VEGF injection during the 12 months prior to administration of a unit dose of rAAV particles to administration of a unit dose of rAAV particles / 365.25)

[0174] In some embodiments, the average annual anti-VEGF injection rate following 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).

[0175] In some embodiments, an individual is determined to require rescue treatment following administration of the rAAV particles (e.g., an intravitreal anti-VEGF injection, such as an aflibercept injection) 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 receiving the rAAV particles and / or the contralateral eye compared to the BCVA for one eye receiving the rAAV particles and / or the contralateral eye prior to administration of the rAAV particles. In some embodiments, the individual is determined to require rescue treatment (e.g., intravitreal injection of anti-VEGF, such as aflibercept injection) after administration of rAAV particles if the eye and / or the contralateral eye receiving the rAAV particles shows an increase in central zone retinal thickness (e.g., CST or CRT) of greater than 75 μm, as determined, for example, by SD-OCT, compared to the central zone retinal thickness of the eye and / or the contralateral eye receiving the rAAV particles before administration of the rAAV particles. In some embodiments, the individual is determined to require rescue treatment (e.g., intravitreal injection of anti-VEGF, such as aflibercept injection) after administration of rAAV particles if the eye and / or the contralateral eye receiving the rAAV particles shows vision-threatening hemorrhage due to AMD.

[0176] In some embodiments, rescue treatment comprises administering standard anti-VEGF treatment.Such standard anti-VEGF treatment comprises one or more anti-VEGF treatments (e.g., anti-VEGF intravitreal injections).In some embodiments, rescue treatment comprises one or more aflibercept IVT injections.In some embodiments, rescue treatment comprises one or more aflibercept IVT injections, including about 2mg aflibercept.

[0177] 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 opposite 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 opposite 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 opposite eye compared to the level of retinal fluid before administration of a unit dose of rAAV particles to one eye and / or the opposite eye, it is determined that an intraocular neovascular disease is treated in an individual after administration of a unit dose of rAAV particles to one eye and / or the opposite eye. In some embodiments, the intraocular neovascular disease is wAMD.

[0178] 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 opposite 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 opposite eye. In some embodiments, it is determined that an intraocular neovascular disease is treated in an individual after administration of a unit dose of rAAV particles to one eye and / or the opposite eye if a resolution of PED is observed after administration of a unit dose of rAAV particles to one eye and / or the opposite eye compared to the PED before administration of a unit dose of rAAV particles to one eye and / or the opposite eye. In some embodiments, the intraocular neovascular disease is wAMD.

[0179] 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 lesion shrinks (e.g., by greater than any of 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 lesion present before administration of a 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 following administration of a unit dose of rAAV particles to one eye and / or the contralateral eye if the CNV lesion does 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%) following administration of a unit dose of rAAV particles to one eye and / or the contralateral eye compared to the CNV lesion present prior to administration of the unit dose of rAAV particles to the one eye and / or the contralateral eye. In some embodiments, the intraocular neovascular disease is wAMD.

[0180] 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 other eye is assessed based on anatomical features of the one eye and / or the other eye based on any method known in the art (e.g., SD-OCT, OCT, fluorescein angiography, digital color fundus photography, etc.). In some embodiments, if an improvement is observed in the anatomical features of the one eye and / or the other eye 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 is 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.

[0181] In some embodiments, whether an intraocular neovascular disease is treated in an individual following administration of a unit dose of rAAV particles to one eye and / or the contralateral eye is determined by ophthalmic 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 pupillary responsiveness, ptosis, abnormal pupil shape, anisocoria, abnormal response to light, The evaluation is based on afferent pupillary defect, slit lamp examination (including examination of eyelid, conjunctiva, cornea, lens, iris, and anterior chamber), abnormality of vitreous body, optic nerve, peripheral retina, and posterior segment of retinal vasculature, SD-OCT, fluorescein angiography, digital color fundus photography (including images of 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., foveal retinal thickness or central zone 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.

[0182] The unit dose of rAAV particles may be administered to one eye and / or the opposite eye of an individual by any method known in the art. For example, the unit dose of rAAV particles may be administered to one eye and / or the opposite eye of an individual by intraocular or intravitreal injection. In some embodiments, the administration of the unit dose of rAAV particles to one eye and / or the opposite eye of an individual is intraocular. In some embodiments, the administration of the unit dose of rAAV particles to one eye and / or the opposite eye of an individual is by intravitreal injection (IVT) or subretinal injection. In some embodiments, the administration of the unit dose of rAAV particles to one eye and / or the opposite 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 providone iodine.

[0183] In some embodiments, the individual has not been previously treated for intraocular neovascular disease. In some embodiments, the individual has not been previously treated for intraocular neovascular disease in one eye and / or the opposite eye. In some embodiments, the individual has not been previously treated with anti-VEGF agents (e.g., bevacizumab, brolucizumab, ranibizumab, faricimab, abicipar pegol, conbercept, OPT-302, KSI-301, sunitinib maleate for injection (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, sunitinib maleate for injection (GB-102), PAN-90806 (PanOptica), and / or aflibercept) in one eye and / or the opposite 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 opposite eye.

[0184] Steroid treatment 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. Exemplary corticosteroids include aclomethasone, amcinomide, beclomethasone, betamethasone, budesonide, ciclesonide, clobetasol, clobetasone, clocortolone, cloprednol, cortivazol, deflazacort, deoxycorticosterone, desonide, desoximetasone, dexamethasone, diflorasone, diflucortolone, difluprednate, fluclorolone, fludrocortisone, fludroxycortide, flumethasone, flunisolide, fluocinolone acetonide, fluocinonide, fluocortin, fluocortolone, fluorometholone, flupeparin ... The steroid treatment includes, but is not limited to, lorone, fluticasone, fuprednidene, formocortal, halcinonide, halometasone, 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 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 ophthalmic steroid treatment is a difluprednate treatment. In some embodiments, the steroid treatment is a prednisone treatment. In some embodiments, the steroid treatment is a difluprednate treatment.

[0185] In some embodiments, the steroid treatment comprises a systemic steroid treatment and a topical steroid treatment. In some embodiments, the systemic steroid treatment is an oral steroid treatment. In some embodiments, the systemic steroid treatment is a prednisone treatment. In some embodiments, the topical steroid treatment is a difluprednate treatment. In some embodiments, the systemic steroid treatment and the topical steroid treatment are administered simultaneously (e.g., on the same day). In some embodiments, the systemic steroid treatment and the topical steroid treatment are administered separately (e.g., on different days).

[0186] In some embodiments, the steroid treatment is an oral treatment, such as, but not limited to, prednisone treatment, methylprednisolone treatment, budesonide treatment, triamcinolone treatment, hydrocortisone treatment, and betamethasone treatment. In some embodiments, the oral steroid treatment (e.g., prednisone) is a daily steroid treatment for up to about 4 weeks, about 6 weeks, about 8 weeks, or about 9 weeks after administration of a unit dose of rAAV particles. In some embodiments, the oral steroid treatment (e.g., prednisone) is about 0.8 mg / kg / day to about 1.00 mg / kg / day for 1 week prior to administration of a unit dose of rAAV particles to 2 weeks after administration, about 0.59 mg / kg / day to about 0.75 mg / kg / day for the 3rd to 5th weeks after administration of a unit dose of rAAV particles, and about 0.4 mg / kg / day for the 6th week after administration of a unit dose of rAAV particles. g / day to about 0.6 mg / kg / day, less than about 0.25 mg / kg / day to about 0.375 mg / kg / day in the 7th week after administration of a unit dose of rAAV particles, less than about 0.14 mg / kg / day to about 0.28 mg / kg / day in the 8th week after administration of a unit dose of rAAV particles, and less than about 0.063 mg / kg / day to about 0.125 mg / kg / day in the 9th week after administration of a unit dose of rAAV particles. In some embodiments, oral steroid treatment is administered at a dose of about 1.00 mg / kg / day starting 1 week prior to and continuing for 2 weeks after administration of a unit dose of rAAV particles, about 0.75 mg / kg / day or less in weeks 3 to 5 after administration of a unit dose of rAAV particles, about 0.6 mg / kg / day or less in week 6 after administration of a unit dose of rAAV particles, about 0.375 mg / kg / day or less in week 7 after administration of a unit dose of rAAV particles, about 0.28 mg / kg / day or less in week 8 after administration of a unit dose of rAAV particles, and about 0.125 mg / kg / day or less in week 9 after administration of a unit dose of rAAV particles. In some embodiments, oral steroid treatment is administered at a dose of about 0.125 mg / kg / day to about 0.14 mg / kg / day for the first 4 days of the 9th week after administration of a unit dose of rAAV particles, or, if the individual weighs 70 kg or more, at a dose of about 0.0625 mg / kg / day to about 0.070 mg / kg / day for the first 4 days of the 9th week after administration of a unit dose of rAAV particles.

[0187] 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.

[0188] In some embodiments, the steroid treatment is systemic steroid treatment. In some embodiments, the systemic steroid treatment is oral steroid treatment.

[0189] In some embodiments, the steroid treatment is oral prednisone treatment. In some embodiments, the 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.

[0190] In some embodiments, after the initial oral prednisone treatment, the dose of oral prednisone treatment is tapered.In some embodiments, the tapered dose of oral prednisone treatment is administered at a dose of about 20mg, about 25mg, about 30mg, about 35mg, about 40mg, about 45mg, or about 50mg / 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, then at a dose of about 10mg, about 15mg, about 20mg, or about 25mg / day of prednisone for a total of about 1 day, about 2 days, about 3 days, or about 4 days, then at a dose of about 5mg, about 10mg, or about 15mg / day of prednisone for a total of about 1 day, about 2 days, about 3 days, or about 4 days. In some embodiments, the prednisone dose taper 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.

[0191] 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, then a dose of 20 mg / day of prednisone for 2 days, then a dose of 10 mg / day of prednisone for 2 days.

[0192] In some embodiments, the steroid treatment is an oral treatment (e.g., prednisone). In some embodiments, the oral steroid treatment (e.g., prednisone) is a daily steroid treatment for up to about 4 weeks, about 6 weeks, about 8 weeks, or about 9 weeks after administration of a unit dose of rAAV particles. In some embodiments, the oral steroid treatment (e.g., prednisone) is about 0.8 mg / kg / day to about 1.00 mg / kg / day for 1 week prior to administration of a unit dose of rAAV particles and for 2 weeks after administration, about 0.59 mg / kg / day to about 0.75 mg / kg / day for weeks 3 to 5 after administration of a unit dose of rAAV particles, and about 0.4 mg / kg / day for weeks 6 after administration of a unit dose of rAAV particles. g / day to about 0.6 mg / kg / day, less than about 0.25 mg / kg / day to about 0.375 mg / kg / day in the 7th week after administration of a unit dose of rAAV particles, less than about 0.14 mg / kg / day to about 0.28 mg / kg / day in the 8th week after administration of a unit dose of rAAV particles, and less than about 0.063 mg / kg / day to about 0.125 mg / kg / day in the 9th week after administration of a unit dose of rAAV particles. In some embodiments, oral steroid treatment is administered at a dose of about 1.00 mg / kg / day for 1 week prior to and 2 weeks after administration of a unit dose of rAAV particles, about 0.75 mg / kg / day or less in weeks 3 to 5 after administration of a unit dose of rAAV particles, about 0.6 mg / kg / day or less in week 6 after administration of a unit dose of rAAV particles, about 0.375 mg / kg / day or less in week 7 after administration of a unit dose of rAAV particles, about 0.28 mg / kg / day or less in week 8 after administration of a unit dose of rAAV particles, and about 0.125 mg / kg / day or less in week 9 after administration of a unit dose of rAAV particles. In some embodiments, oral steroid treatment is administered at a dose of about 0.125 mg / kg / day to about 0.14 mg / kg / day for the first 4 days of the 9th week after administration of a unit dose of rAAV particles, or, if the individual weighs 70 kg or more, at a dose of about 0.0625 mg / kg / day to about 0.070 mg / kg / day for the first 4 days of the 9th week after administration of a unit dose of rAAV particles.

[0193] In some embodiments, the steroid treatment is an ophthalmic steroid treatment. In some embodiments, the ophthalmic steroid treatment is a 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.

[0194] In some embodiments, the steroid treatment is an eye drop steroid treatment, e.g., a topical steroid treatment. In some embodiments, the eye drop 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 includes about 4 doses of topical steroid at about week 1, about 3 doses of topical steroid at about week 2, about 2 doses of topical steroid at about week 3, and about 1 dose 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 about 4 administrations of topical steroid per day (i.e., QID) for about 3 weeks following administration of a unit dose of rAAV particles, followed by about 3 administrations of topical steroid per day (i.e., TID) for about 1 week, followed by about 2 administrations of topical steroid per day (i.e., BID) for about 1 week, and then about 1 administration of topical steroid per day (i.e., QD) for about 1 week. In some embodiments, the ophthalmic steroid treatment is extended at the discretion of the treating physician.

[0195] 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.

[0196] 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 prior to administration of the unit dose of rAAV particles. In some embodiments, the topical steroid treatment comprises about 4 administrations of topical steroid per day for about 28 days (i.e., QID), followed by about 3 administrations of topical steroid per day for about 7 days (i.e., TID), followed by about 2 administrations of topical steroid per day for about 7 days (i.e., BID), and about 1 administration of topical steroid per day for about 7 days (i.e., QD), timing starting about 7 days prior to administration of a unit dose of rAAV particles. In some embodiments, topical steroid treatment comprises about 4 administrations of topical steroid per day (i.e., QID) from day 1 to about day 28, followed by about 3 administrations of topical steroid per day (i.e., TID) from about day 29 to about day 35, followed by about 2 administrations of topical steroid per day (i.e., BID) from about day 36 to about day 42, and then about 1 administration of topical steroid per day (i.e., QD) from about day 43 to about day 49, timing starting on day 1. In some embodiments, topical steroid treatment is continued if inflammation is present.

[0197] 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 prior to 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 prior to 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 4 doses of topical steroid per day (i.e., QID) for about 4 weeks, followed by about 3 doses of topical steroid per day (i.e., TID) for about 1 week, followed by about 2 doses of topical steroid per day (i.e., BID) for about 1 week, and followed by about 1 dose of topical steroid per day (i.e., QD) for about 1 week, timing starting from and following administration of the anti-VEGF agent. In some embodiments, the topical steroid treatment comprises about 4 doses of topical steroid per day (i.e., QID) for about 28 days, followed by about 3 doses of topical steroid per day (i.e., TID) for about 7 days, followed by about 2 doses of topical steroid per day (i.e., BID) for about 7 days, and followed by about 1 dose of topical steroid per day (i.e., QD) for about 7 days, timing starting from and following administration of the anti-VEGF agent. In some embodiments, topical steroid treatment comprises about 4 administrations of topical steroid per day (i.e., QID) from day 1 to about day 28, followed by about 3 administrations of topical steroid per day (i.e., TID) from about day 29 to about day 35, followed by about 2 administrations of topical steroid per day (i.e., BID) from about day 36 to about day 42, and then about 1 administration of topical steroid per day (i.e., QD) from about day 43 to about day 49, timing starting on day 1. In some embodiments, topical steroid treatment is continued if inflammation is present.

[0198] In some embodiments, the topical steroid treatment comprises 4 months of topical steroid treatment, e.g., 0.05% difluprednate. In some embodiments, the topical steroid treatment comprises about 4 doses of topical steroid per day for about 1 month (i.e., QID), followed by about 3 doses of topical steroid per day for about 1 month (i.e., TID), followed by about 2 doses of topical steroid per day for about 1 month (i.e., BID), followed by about 1 dose of topical steroid per day for about 1 month (i.e., QD), starting about 1 week prior to administration of the unit dose of rAAV particles. In some embodiments, the topical steroid treatment comprises about 4 administrations of topical steroid per day for about 30 days (i.e., QID), followed by about 3 administrations of topical steroid per day for about 30 days (i.e., TID), followed by about 2 administrations of topical steroid per day for about 30 days (i.e., BID), and about 1 administration of topical steroid per day for about 30 days (i.e., QD), timing starting about 7 days prior to administration of a unit dose of rAAV particles. In some embodiments, topical steroid treatment comprises about 4 administrations of topical steroid per day (i.e., QID) from day 1 to about day 30, followed by about 3 administrations of topical steroid per day (i.e., TID) from about day 31 to about day 60, followed by about 2 administrations of topical steroid per day (i.e., BID) from about day 61 to about day 90, and then about 1 administration of topical steroid per day (i.e., QD) from about day 91 to about day 120, timing starting on day 1. In some embodiments, topical steroid treatment is continued if inflammation is present.

[0199] 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 prior to 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 prior to 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 4 doses of topical steroid per day (i.e., QID) for about 1 month, followed by about 3 doses of topical steroid per day (i.e., TID) for about 1 month, followed by about 2 doses of topical steroid per day (i.e., BID) for about 1 month, and followed by about 1 dose of topical steroid per day (i.e., QD) for about 1 month, timing starting from and following administration of the anti-VEGF agent. In some embodiments, the topical steroid treatment comprises about 4 doses of topical steroid per day (i.e., QID) for about 30 days, followed by about 3 doses of topical steroid per day (i.e., TID) for about 30 days, followed by about 2 doses of topical steroid per day (i.e., BID) for about 30 days, and followed by about 1 dose of topical steroid per day (i.e., QD) for about 30 days, timing starting from and following administration of the anti-VEGF agent. In some embodiments, topical steroid treatment comprises about 4 administrations of topical steroid per day (i.e., QID) from day 1 to about day 30, followed by about 3 administrations of topical steroid per day (i.e., TID) from about day 31 to about day 60, followed by about 2 administrations of topical steroid per day (i.e., BID) from about day 61 to about day 90, and then about 1 administration of topical steroid per day (i.e., QD) from about day 91 to about day 120, timing starting on day 1. In some embodiments, topical steroid treatment is continued if inflammation is present.

[0200] Vectors for delivering transgenes to target cells 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).

[0201] 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.

[0202] 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 tropism of the virus or the 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).

[0203] In some embodiments, the method of the present disclosure provides for the use of pseudotyped AAV. Pseudotyped AAV particles comprise the AAV genome inverted terminal repeats (ITRs) of one AAV serotype encapsidated by the AAV capsid of another AAV serotype. Typically, pseudotyped AAV is designated as "AAV# / #", where the first "#" indicates the AAV ITR serotype and the second "#" indicates the serotype of the capsid. For example, an AAV particle comprising AAV2 ITR and AAV1 capsid is designated as "AAV2 / 1".

[0204] 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, e.g., 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 a particular tissue or cell, e.g., a retinal cell, 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.

[0205] In some embodiments, the AAV vector comprises a polynucleotide cassette to enhance expression of a transgene (e.g., an anti-VEGF agent such as aflibercept) in a target cell (e.g., a retinal cell). 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, a TPL and eMLP sequence (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 a 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 TTAATATTCA TGCAATCTAA TTAACAATCT TTTCTTTGTG GTTAGGACTT TGAGTCATAA GAAATTTTTC TCTACACTGA AGTCATGATG GCATGCTTCT ATATTATTTT CTAAAAGATT TAAAGTTTTG CCTTCTCCAT TTAGACTTAT 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 TGCTGCTGATGATGTAATTA 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 AAATTTTTTGT 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 GAATTTTTTT 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 CACCAGCCTTGTCCTAATAA AATTAAGTTG CATCATTTTG TCTGACTAGG TGTCCTTCTA TAATATTATG GGGTGGAGGG GGGTGGTATG GAGCAAGGGG CCCAAGTTGG GAAGAAACCT GTAGGGCCTG CGAAGACAGT CAG (SEQ ID NO: 34)

[0206] In some embodiments, the polynucleotide cassette comprises or consists of SEQ ID NO: 39, or a sequence having at least 85% identity thereto.

[0207] SEQ ID NO:39, as above, comprises, in the 5' to 3' direction, an AAV serotype 2 inverted terminal repeat (ITR) comprising nucleotides 1 to 145 of SEQ ID NO:39; a CMV promoter comprising nucleotides 180 to 693 of SEQ ID NO:39; a 5' untranslated region (UTR) comprising an adenoviral tripartite leader sequence and a synthetic intron, comprising nucleotides 694 to 1314 of SEQ ID NO:39; a Kozak sequence comprising nucleotides 1329 to 1340 of SEQ ID NO:39; a codon-optimized aflibercept cDNA sequence comprising nucleotides 1338 to 2714 of SEQ ID NO:39; a 3' UTR comprising a human scaffold attachment region, comprising nucleotides 2717 to 3527 of SEQ ID NO:39; a human growth hormone polyadenylation / transcription termination signal comprising nucleotides 3546 to 3748 of SEQ ID NO:39; and an AAV serotype 2 inverted terminal repeat (ITR) comprising nucleotides 3772 to 3916 of SEQ ID NO:39.

[0208] 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.

[0209] In some embodiments, the rAAV particles comprise variant capsid proteins with increased infectivity of target cells, such as retinal cells, and are used to increase transduction into retinal cells or to increase targeting of gene delivery to retinal cells in an individual. In some embodiments, the rAAV particles comprise amino acid modifications 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 a description 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 et al. (2007) Mol. Ther. 15:1955. Several AAV capsid variants are known, including the 7m8 variant. In some embodiments, the rAAV particles comprise a variant AAV capsid protein that comprises an insertion of between 5 amino acids and 11 amino acids, e.g., a 7 amino acid sequence, in the GH loop of the capsid protein compared to a 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.

[0210] In some embodiments, the rAAV particles contain the following amino acid sequences inserted at the following positions: 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. The AAV capsid proteins include any one of 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), such as AAV2 capsid proteins. 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 the AAV2 VP1 capsid protein, which comprises the sequence of SEQ ID NO: 13.

[0211] In some embodiments, the rAAV particles comprise a 7m8 variant capsid protein from AAV2 that comprises 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 VP1 capsid protein that comprises a GH loop that comprises 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 comprises a GH loop that comprises 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. FSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTNTPSGTTTQSRLQFSQAGASDIRDQSRNWLPGPCYRQQRVSKTSADNNNSEYSWTGATKYHLNGRDSLVNPGPAMASHKDDE EKFFPQSGVLIFGKQGSEKTNVDIEKVMITDEEIRTTNPVATEQYGSVSTNLQRGNLALGETTRPARQAATADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLK HPPPQILIKN (SEQ ID NO: 38)

[0212] In some embodiments, the rAAV particles comprise a 7m8 variant capsid protein from AAV2 that comprises the amino acid sequence LALGETTRPA (SEQ ID NO: 1) inserted between AAV2 VP1 positions 587 and 588. The sequence of the 7m8 variant capsid protein from AAV2 that comprises the amino acid sequence LALGETTRPA (SEQ ID NO: 1) inserted between AAV2 VP1 positions 587 and 588 is provided below: (SEQ ID NO:37)

[0213] 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.

[0214] 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.

[0215] 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, a 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.

[0216] In some embodiments, the rAAV and / or the plasmid used to generate the rAAV comprises one or more of the following nucleic acid elements: a first ITR sequence; a promoter sequence; an intron sequence; a first UTR sequence; a heterologous nucleic acid encoding an anti-VEGF agent (e.g., aflibercept); a second UTR sequence; a polyA sequence; and a second ITR sequence. In some embodiments, a linker sequence(s) is inserted between two or more of the nucleic acid elements. In some embodiments, the heterologous nucleic acid encoding a therapeutic polypeptide encodes, for example, aflibercept (or a functional fragment or variant thereof).

[0217] 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, Muller cells, bipolar cells, amacrine cells, horizontal cells, or retinal pigment epithelial cells). A viral vector for use in the present disclosure may include a vector that exhibits low toxicity and / or low immunogenicity in an individual and expresses a therapeutically effective amount of an anti-VEGF agent (e.g., aflibercept) in an individual, such as a human. Any suitable method known in the art can be used for biochemical purification of recombinant virus (e.g., rAAV), for example, for preparing pharmaceutical compositions described elsewhere herein. Recombinant AAV virus can be harvested directly from cells or from culture medium containing cells. Virus 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.

[0218] 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 any increase between 5%-100%, between 5%-95%, between 5%-90%, between 5%-85%, between 5%-80%, between 5%-75%, between 5%-70%, between 5%-65%, between 5%-60%, between 5%-55%, between 5%-50%, between 5%-45%, between 5%-40%, between 5%-35%, between 5%-30%, between 5%-25%, between 5%-20%, between 5%-15%, or between 5%-10%, compared to AAV particles containing the corresponding parent or unmodified AAV capsid protein.

[0219] In some embodiments, the increase in infectivity of the rAAV variant, such as rAAV2.7m8, to retinal cells 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.

[0220] 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 parent or unmodified AAV capsid proteins.

[0221] 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 parent or unmodified AAV capsid protein.

[0222] In some embodiments, the amino acid modifications of the capsid protein described herein may confer an increased ability to cross the inner limiting membrane (ILM) of an individual's, e.g., human, eye, as compared to the ability of AAV particles containing the corresponding parent or unmodified AAV capsid protein to cross the ILM of the subject's eye. In some embodiments, the increased ability of the rAAV variant, e.g., rAAV2.7m8, to cross 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% as 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 an increase of 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 the parent or unmodified AAV capsid protein.

[0223] In some embodiments, the increased ability of the rAAV variant, such as 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 containing 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 containing the corresponding parental AAV capsid proteins. In some embodiments, the increase in ability to cross the ILM is either 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.

[0224] In some embodiments, the increase in 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 parent or unmodified AAV capsid proteins.

[0225] In some embodiments, the increase in 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 parent or unmodified AAV capsid proteins.

[0226] In some embodiments, rAAV.7m8 comprising aflibercept-encoding nucleic acid is used for gene therapy. In some embodiments, AAV2 or rAAV2 is used to deliver a nucleic acid sequence encoding an anti-VEGF agent (e.g., aflibercept) to a subject's eye or retinal cells via intravitreal or subretinal injection. In some embodiments, AAV2 or rAAV2 is used to deliver a nucleic acid sequence encoding an anti-VEGF agent (e.g., aflibercept) to a subject's eye or retinal cells via intravitreal injection. In some embodiments, rAAV2.7m8 is used to deliver a nucleic acid sequence of an anti-VEGF agent (e.g., aflibercept) to a subject's retinal cells. In some embodiments, a heterologous nucleic acid (e.g., a nucleic acid encoding an anti-VEGF agent such as aflibercept) is integrated into a target cell genome (e.g., a retinal cell genome) to result in long-term expression of, for example, an anti-VEGF agent (e.g., 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).

[0227] 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 sequences (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 sequences (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 sequences (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 sequences (ITRs). The sequence of SEQ ID NO:35 is provided below: SDTGRPFVEMYSEIPEIIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVLSPS HGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHEKDKTHTCPPCPAPEL LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 35)

[0228] In some embodiments, the 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 at least about 100% sequence identity to the nucleic acid sequence of SEQ ID NO:36, and having AAV2 inverted terminal repeats (ITRs) on both ends. In some embodiments, the rAAV particle comprises 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 homology with the nucleic acid sequence of aflibercept (e.g., SEQ ID NO: 36), and 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, 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 100% sequence identity to the nucleic acid sequence of SEQ ID NO: 40, the nucleic acid flanked by AAV2 inverted terminal repeats (ITRs). In some embodiments, the rAAV particles comprise a nucleic acid comprising a nucleic acid sequence of SEQ ID NO: 40. In some embodiments, the rAAV particles comprise a nucleic acid comprising a nucleic acid sequence of SEQ ID NO: 40, the nucleic acid flanked by AAV2 inverted terminal repeats (ITRs).

[0229] 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, flanked by AAV2 inverted repeat sequences (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, flanked by AAV2 inverted repeat sequences (ITRs). In some embodiments, the rAAV particles comprise a nucleic acid encoding a polypeptide comprising an amino acid sequence of SEQ ID NO:41, flanked by AAV2 inverted repeat sequences (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)

[0230] In some embodiments, the nucleic acid sequence of aflibercept is The synthetic sequence corresponding to the aflibercept amino acid sequence has been codon-optimized for expression. The construction of the gene was carried out in accordance with the literature, for example, 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 (corresponding to aflibercept) cDNA was produced as a synthetic gene by IDT (Coralville, IA) as described in "Aflibercept cDNA was produced 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 produce aflibercept cDNA for use in gene therapy or rAAV as described herein.

[0231] 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 the codon that is more frequently or most frequently used in host cell, while maintaining native amino acid sequence, in order to enhance the expression of gene in target or host cell of interest, such as human retina cell. Codon usage tables are readily available, including, 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).

[0232] 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.

[0233] In some embodiments, the rAAV particles comprise a nucleic acid encoding aflibercept. In some embodiments, the polypeptide is aflibercept.

[0234] As used herein, "aflibercept" refers to a polypeptide or protein sequence or a functional fragment or 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 with 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.

[0235] 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 a...

Claims

1. 1. A method for treating an intraocular neovascular disease in an individual, comprising administering to an individual about 2×10 １１ 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: 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, the nucleic acid being 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. A method comprising:

2. The method of claim 1 , wherein retinal fluid in the eye of the individual having an intraocular neovascular disease is reduced.

3. 1. A method for reducing retinal fluid in an eye of an individual having an ocular neovascular disease, comprising administering a unit dose of rAAV particles to one eye of the individual, the individual being a human, the rAAV particles comprising: 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, the nucleic acid being 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. A method comprising:

4. The method of claim 3, wherein the individual received at least one treatment with an anti-VEGF agent about 12 weeks prior to administering the unit dose of the rAAV particles.

5. 5. The method of claim 3 or claim 4, wherein the amount or presence of retinal fluid in said one eye of said individual is refractory to previous treatment with an anti-VEGF agent.

6. 6. The method of claim 4 or claim 5, wherein the anti-VEGF agent is aflibercept.

7. 7. The method of any one of claims 2-6, wherein the retinal fluid in said one eye is reduced by at least about 60%.

8. 8. The method of any one of claims 2-7, wherein the retinal fluid in said one eye is reduced by about 80% compared to the level of retinal fluid in said one eye of said individual prior to administration of the rAAV to said individual.

9. The method according to any one of claims 2 to 8, wherein the retinal fluid is subretinal fluid (SRF) or intraretinal fluid (IRF).

10. The unit dose of the rAAV particles is about 6×10 １１ 10. The method of any one of claims 3 to 9, wherein the vector genome / eye (vg / eye) or less.

11. 1. A method for treating an intraocular neovascular disease in an individual, comprising: (a) administering an anti-VEGF agent to one eye of the individual; (b) about 6×10 recombinant adeno-associated virus (rAAV) particles after administration of the anti-VEGF agent; １１ administering a unit dose of a vector genome (vg) or less to said one eye of said 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, the nucleic acid being 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. A method comprising:

12. 12. The method of claim 11, comprising administering a 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.

13. 13. The method of claim 11 or claim 12, comprising administering the anti-VEGF agent to said one eye of the individual on day 1 and administering a unit dose of the rAAV particles to said one eye of the individual on day 8.

14. The method of any one of claims 11 to 13, wherein the anti-VEGF agent comprises aflibercept.

15. 15. The method of claim 14, wherein the aflibercept is administered by intravitreal injection at a dose of about 2 mg.

16. The unit dose of the rAAV particles is about 6×10 １０ ~Approx. 6×10 １１ The method according to any one of claims 1 to 15, wherein the vector is a genome / eye (vg / eye).

17. The unit dose of the rAAV particles is about 6×10 １０ ~Approx. 2×10 １１ The method according to any one of claims 1 to 16, wherein the vector is a vector genome / eye (vg / eye).

18. The unit dose of the rAAV particles is about 2×10 １１ ~Approx. 6×10 １１ The method according to any one of claims 1 to 16, wherein the vector is a vector genome / eye (vg / eye).

19. The unit dose of the rAAV particles is about 2×10 １１ Or about 6 x 10 １１ 20. The method of claim 18, wherein the vector is a genome / eye (vg / eye).

20. The unit dose of the rAAV particles is about 2×10 １１ 20. The method of claim 19, wherein the vector genome / eye (vg / eye).

21. The unit dose of the rAAV particles is about 6×10 １１ 20. The method of claim 19, wherein the vector genome / eye (vg / eye).

22. The method of any one of claims 1 to 21, wherein the individual has one or more symptoms of an intraocular neovascular disease in the contralateral eye.

23. 23. The method of any one of claims 1-22, further comprising administering a unit dose of the rAAV particles to the contralateral eye of the individual.

24. 24. The method of claim 23, wherein the step of administering a unit dose of the rAAV particles to the contralateral eye is up to about 2 weeks after the step of administering a unit dose of the rAAV particles to the one eye.

25. (a) the step of administering a unit dose of the rAAV particles to the contralateral eye is on the same day as the step of administering a unit dose of the rAAV particles to the one eye; or 25. The method of claim 24, wherein (b) the step of administering a unit dose of the rAAV particles to the contralateral eye is between about 1 day and about 14 days after the step of administering a unit dose of the rAAV particles to one eye.

26. 26. The method of claim 24 or claim 25, wherein the unit dose of the 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 the rAAV particles administered to the one eye of the individual.

27. 24. The method of claim 23, wherein the step of administering a unit dose of the rAAV particles to the contralateral eye is at least about two weeks after the step of administering a unit dose of the rAAV particles to one eye.

28. 28. The method of claim 27, wherein 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 the one eye of the individual.

29. The method according to any one of claims 1 to 28, wherein the nucleic acid comprises the nucleic acid sequence of SEQ ID NO: 40 or a sequence having at least 85% identity thereto.

30. The method of any one of claims 1 to 29, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO:

35.

31. The method of any one of claims 1 to 30, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO:

41.

32. The method of any one of claims 1 to 31, wherein the polypeptide is aflibercept.

33. The method of any one of claims 1 to 32, wherein the nucleic acid further comprises a first enhancer region, a promoter region, a 5'UTR region, a second enhancer region, and a polyadenylation site.

34. The nucleic acid comprises, in 5' to 3' order: (a) a first enhancer region; (b) a promoter region; (c) 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 flanked by AAV2 inverted terminal repeats (ITRs).

35. 35. The method of claim 33 or claim 34, wherein 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.

36. 36. The method of any one of claims 33 to 35, wherein the promoter region comprises a CMV sequence comprising the sequence of SEQ ID NO: 23 or a sequence having at least 85% identity thereto.

37. The method of any one of claims 34 to 36, wherein the nucleic acid encoding a polypeptide comprises the nucleic acid sequence of SEQ ID NO: 40 or a sequence having at least 85% identity thereto.

38. The method according to any one of claims 34 to 37, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 35 or a sequence having at least 95% identity thereto.

39. The method of any one of claims 34 to 38, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 41 or a sequence having at least 95% identity thereto.

40. The method of any one of claims 34 to 39, wherein the polypeptide is aflibercept.

41. The method of any one of claims 33 to 40, wherein the 5'UTR region comprises, in order from 5' to 3', a TPL sequence comprising a sequence of SEQ ID NO: 24 or a sequence having at least 85% identity thereto, and an eMLP sequence comprising a sequence of SEQ ID NO: 25 or a sequence having at least 85% identity thereto.

42. The method of any one of claims 33 to 41, wherein 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.

43. 43. The method of any one of claims 33 to 42, wherein 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.

44. 33. The method of any one of claims 1 to 32, wherein the nucleic acid further comprises: (a) a first enhancer region comprising a CMV sequence comprising a sequence of SEQ ID NO:22 or a sequence having at least 85% identity thereto; (b) a promoter region comprising a CMV sequence comprising a 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 a sequence of SEQ ID NO:24 or a sequence having at least 85% identity thereto, and an eMLP sequence comprising a 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 a sequence of SEQ ID NO:26 or a sequence having at least 85% identity thereto; and (e) an HGH polyadenylation site comprising a sequence of SEQ ID NO:27 or a sequence having at least 85% identity thereto.

45. The method according to any one of claims 1 to 44, wherein the nucleic acid comprises the sequence SEQ ID NO: 39 or a sequence having at least 85% identity thereto.

46. 46. ​​The method of any one of claims 1 to 45, wherein the AAV2 capsid protein comprises the amino acid sequence LALGETTRPA (SEQ ID NO:1) inserted between positions 587 and 588 of the capsid protein, wherein the amino acid residue numbering corresponds to AAV2 VP1 capsid protein.

47. 47. The method of any one of claims 1 to 46, wherein the AAV2 capsid protein comprises 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.

48. 48. The method of any one of claims 1 to 47, wherein the AAV2 capsid protein comprises 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.

49. 47. The method of any one of claims 1-46, wherein 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.

50. 50. The method of any one of claims 1 to 49, wherein administration of a unit dose of rAAV particles to the one eye and / or the contralateral eye is by intravitreal administration.

51. The method of any one of claims 1 to 50, wherein the unit dose of the rAAV particles is in a pharmaceutical formulation.

52. 52. The method of claim 51, wherein the pharmaceutical formulation comprises the rAAV particles, sodium chloride, sodium phosphate and a surfactant.

53. 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% (w / v) to about 0.005% (w / v) poloxamer 188, and about 6×10 １３ ~Approx. 6×10 １０ 53. The method of claim 52, wherein the pharmaceutical formulation comprises vector genome (vg) / mL (vg / mL) of the rAAV particles and has a pH of about 7.0 to about 7.

5.

54. The pharmaceutical formulation comprises about 180 mM sodium chloride, about 5 mM sodium dihydrogen phosphate, about 5 mM sodium hydrogen phosphate, approximately 6 x 10 １２ 54. The method of claim 53, comprising about 0.001% (weight / volume) poloxamer 188, and about 0.001% (weight / volume) poloxamer 188, and wherein the pharmaceutical formulation has a pH of about 7.

3.

55. The pharmaceutical formulation comprises about 180 mM sodium chloride, about 5 mM sodium dihydrogen phosphate, about 5 mM sodium dihydrogen phosphate, about 2×10 １２ 54. The method of claim 53, comprising about 0.001% (weight / volume) poloxamer 188, and about 0.001% (weight / volume) poloxamer 188, and wherein the pharmaceutical formulation has a pH of about 7.

3.

56. The pharmaceutical formulation comprises about 180 mM sodium chloride, about 5 mM sodium dihydrogen phosphate, about 5 mM sodium dihydrogen phosphate, about 6×10 １１ 54. The method of claim 53, comprising about 0.001% (weight / volume) poloxamer 188, and about 0.001% (weight / volume) poloxamer 188, and wherein the pharmaceutical formulation has a pH of about 7.

3.

57. 57. The method of any one of claims 1-56, wherein the unit dose of rAAV particles is administered to the one eye and / or the contralateral eye in a volume of about 25 μL to about 250 μL.

58. 58. The method of claim 57, wherein the unit dose of the rAAV particles administered to the one eye and / or the contralateral eye comprises a volume of about 100 μL.

59. 58. The method of claim 57, wherein the unit dose of rAAV particles administered to the one eye and / or the contralateral eye comprises a volume of about 30 μL.

60. The method of any one of claims 1 to 59, wherein the individual has undergone previous treatment for the intraocular neovascular disease with an anti-VEGF agent.

61. The method of claim 60, wherein the individual has received one or two injections of an anti-VEGF agent into the one eye and / or the contralateral eye prior to administration of the rAAV particles to the one eye and / or the contralateral eye.

62. The method of any one of claims 1-3 or 7-59, wherein the individual has not previously been treated for the intraocular neovascular disease with an anti-VEGF agent.

63. 63. The method of any one of claims 60 to 62, wherein the anti-VEGF agent is aflibercept.

64. 64. The method of any one of claims 1 to 63, wherein 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.

65. The method of any one of claims 1-10 or 16-64, wherein the unit dose of rAAV particles is administered in combination with steroid treatment.

66. 66. The method of claim 65, wherein the steroid treatment is a corticosteroid treatment.

67. 67. The method of claim 65 or claim 66, wherein the steroid treatment is systemic steroid treatment.

68. 68. The method of any one of claims 65 to 67, wherein the steroid treatment is oral steroid treatment.

69. 69. The method of any one of claims 65 to 68, wherein the steroid treatment is prednisone treatment.

70. 67. The method of claim 65 or claim 66, wherein the steroid treatment is a topical steroid treatment.

71. 71. The method of claim 70, wherein the steroid treatment is difluprednate treatment.

72. 72. The method of any one of claims 65-71, wherein the steroid is administered before, during, and / or after administration of a unit dose of the rAAV particles to the one eye and / or the contralateral eye.

73. 73. The method of any one of claims 70-72, wherein the steroid treatment is a topical steroid treatment, and the topical steroid treatment is a daily steroid treatment for up to about 4 weeks, up to about 6 weeks, or up to about 8 weeks from administration of a unit dose of the rAAV particles.

74. The method of claim 73, wherein the topical steroid treatment comprises about 4 administrations of topical steroid per day for about week 1, about 3 administrations of topical steroid per day for about week 2, about 2 administrations of topical steroid per day for about week 3, and about 1 administration of topical steroid per day for about week 4, timing starting from and following administration of a unit dose of the rAAV particles.

75. 74. The method of claim 73, wherein the topical steroid treatment comprises administering a topical steroid about four times per day for about three weeks after administration of a unit dose of the rAAV particles, followed by administering a topical steroid about three times per day for about one week, followed by administering a topical steroid about twice per day for about one week, and followed by administering a topical steroid about once per day for about one week.

76. 74. The method of claim 73, wherein 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 beginning about one week prior to administration of the unit dose of rAAV particles.

77. 65. The method of any one of claims 11 to 64, further comprising administering topical steroid treatment.

78. 78. The method of claim 77, wherein the topical steroid treatment is a difluprednate treatment.

79. 79. The method of claim 77 or claim 78, wherein the topical steroid treatment comprises about four administrations of topical steroid per day for about four weeks, followed by about three administrations of topical steroid per day for about one week, followed by about two administrations of topical steroid per day for about one week, and then about one administration of topical steroid per day for about one week, timing starting with and following administration of the anti-VEGF agent.

80. 80. The method of any one of claims 71-79, wherein the topical steroid comprises about 1 μg to about 3 μg dose of 0.05% difluprednate.

81. 81. The method of any one of claims 71-80, wherein the topical steroid comprises about a 2.5 μg dose of 0.05% difluprednate.

82. 82. The method of any one of claims 1-81, wherein the step of administering a unit dose of the rAAV particles to the one eye and / or the contralateral eye of the individual results in a maintenance or reduction in retinal thickness compared to the retinal thickness prior to administration of the unit dose of the rAAV particles.

83. 83. The method of claim 82, wherein the step of administering a unit dose of the rAAV particles to the one eye and / or the contralateral eye of the individual results in a decrease in retinal thickness compared to retinal thickness prior to administration of the unit dose of the rAAV particles.

84. The method of claim 83, wherein the reduction in retinal thickness is at least about 10% compared to the retinal thickness before administration of the unit dose of the rAAV particles.

85. The method according to any one of claims 82 to 84, wherein the retinal thickness is central zone retinal thickness (CST) or central foveal retinal thickness (CRT).

86. 86. The method of any one of claims 1-85, wherein the step of administering a unit dose of the rAAV particles to the one eye and / or the contralateral eye of the individual results in a maintenance or reduction in macular volume compared to the macular volume prior to administration of the unit dose of the rAAV particles.

87. 87. The method of claim 86, wherein the step of administering a unit dose of the rAAV particles to the one eye and / or the contralateral eye of the individual results in a reduction in macular volume compared to the macular volume prior to administration of the unit dose of the rAAV particles.

88. The method of claim 87, wherein the reduction in macular volume is at least about 10% compared to the macular volume prior to administration of the unit dose of the rAAV particles.

89. 89. The method of any one of claims 1-88, wherein the step of administering a unit dose of the rAAV particles to the one eye and / or the contralateral eye of the individual results in the maintenance or improvement of visual acuity compared to visual acuity prior to administration of the unit dose of the rAAV particles.

90. 90. The method of claim 89, wherein the step of administering a unit dose of the rAAV particles to the one eye and / or the contralateral eye of the individual results in improved vision compared to vision prior to administration of the unit dose of the rAAV particles.

91. 91. The method of claim 89 or claim 90, wherein the visual acuity is best corrected visual acuity (BCVA).

92. 92. The method of any one of claims 1-91, wherein administration of a unit dose of the rAAV particles to one eye and / or contralateral eye of a plurality of individuals results in at least about 50% of individuals within the plurality not requiring anti-VEGF rescue treatment.

93. The method of claim 92, wherein at least about 50% of the individuals in the plurality do not require 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 a unit dose of the rAAV particles.

94. 92. The method of any one of claims 1-91, wherein administration of a unit dose of the rAAV particles to one eye and / or contralateral eye of a plurality of individuals results in at least about 67% of individuals within the plurality not requiring anti-VEGF rescue treatment.

95. The method of claim 94, wherein at least about 67% of the individuals in the plurality do not require 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 a unit dose of the rAAV particles.

96. 92. The method of any one of claims 1-91, wherein administration of a unit dose of the rAAV particles to one eye and / or contralateral eye of a plurality of individuals results in at least about 78% of individuals within the plurality not requiring anti-VEGF rescue treatment.

97. 97. The method of claim 96, wherein at least about 78% of the individuals in the plurality do not require anti-VEGF rescue treatment for at least about 20 weeks, at least about 36 weeks, or longer after administration of a unit dose of the rAAV particles.

98. 92. The method of any one of claims 1-91, wherein administration of a unit dose of the 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.

99. The method of claim 98, wherein 100% of the individuals in the plurality do not require 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.

100. 100. The method of any one of claims 1-99, wherein administration of a unit dose of the rAAV particles to one eye and / or a contralateral eye of a plurality of 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 prior to administration of the unit dose of the rAAV particles.

101. 100. The method of any one of claims 1-10, 16-76, or 80-100, wherein the intraocular neovascular disease is exudative age-related macular degeneration (AMD).

102. 100. The method of any one of claims 1-3, 11-64, 77-83, 85-87, or 89-91, wherein the intraocular neovascular disease is diabetic macular edema (DME).

103. The method of claim 102, wherein the step of administering a unit dose of the rAAV particles to the one eye and / or the contralateral eye of the individual results in a two or three stage improvement in the Diabetic Retinopathy Severity Scale (DRSS).

104. About 6×10 recombinant adeno-associated virus (rAAV) particles for use in a method for treating intraocular neovascular disease in an individual. １１ a unit dose of a vector genome (vg) or less, the method comprising administering the unit dose to one eye of the individual, the individual being a human, and the rAAV particles comprising: 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, the nucleic acid being 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. A unit dose comprising:

105. 1. A unit dose of rAAV particles for use in a method for reducing retinal fluid in an eye of an individual having an ocular neovascular disease, the method comprising administering the unit dose to one eye of the individual, the individual being a human, and the rAAV particles comprising: 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, the nucleic acid being 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. A unit dose comprising:

106. 106. A unit dose for use according to claim 104 or claim 105, wherein the method further comprises the step of administering an anti-VEGF agent to said one eye of said individual prior to administration of said unit dose.

107. 1. A method of treating an intraocular neovascular disease in an individual, comprising administering recombinant adeno-associated virus (rAAV) particles to one eye of the 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, the nucleic acid being 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. wherein the unit dose of rAAV particles is administered in combination with oral steroid treatment and topical steroid treatment, or oral steroid treatment and intravitreal (IVT) steroid treatment.

108. 108. The method of claim 107, wherein the oral steroid treatment and the topical steroid treatment, or the oral steroid treatment and the IVT, is a corticosteroid treatment.

109. The method of claim 107 or claim 108, wherein the topical steroid treatment is a difluprednate treatment.

110. 110. The method of any one of claims 107 to 109, wherein the topical steroid treatment comprises difluprednate 0.05% at a dose of about 1 μg to about 3 μg.

111. 111. The method of any one of claims 107 to 110, wherein the topical steroid treatment comprises difluprednate 0.05% at a dose of about 2.5 μg.

112. The topical steroid treatment comprises: a. administration of a topical steroid about 4 times per day from about 7 days prior to administration of the unit dose of the rAAV particles to about 35 days after administration of the unit dose of the rAAV particles; b. administration of a topical steroid about three times per day from about day 36 to about day 63 following administration of the unit dose of the rAAV particles; c. administration of a topical steroid about twice per day from about day 64 to about day 91 following administration of the unit dose of the rAAV particles; d. administration of a topical steroid about once per day from about day 92 to about day 119 after administration of the unit dose of the rAAV particles; and e. administration of a topical steroid about once every other day starting about 120 days until about 147 days after administration of the unit dose of the rAAV particles; The method of any one of claims 107 to 111, comprising:

113. The topical steroid treatment comprises: a. administration of a topical steroid about 4 times per day from about 1 week prior to administration of a unit dose of the rAAV particles to about 5 weeks after administration of a unit dose of the rAAV particles; b. administration of a topical steroid about three times per day from about 6 weeks to about 9 weeks after administration of the unit dose of the rAAV particles; c. administration of a topical steroid about twice per day from about 10 weeks to about 13 weeks after administration of the unit dose of the rAAV particles; d. administration of a topical steroid about once per day from about 14 weeks to about 17 weeks after administration of the unit dose of the rAAV particles; and e. Administration of a topical steroid about once every other day about 18 to about 21 weeks after administration of the unit dose of the rAAV particles. The method of any one of claims 107 to 112, comprising:

114. 114. The method of any one of claims 107 to 113, wherein the oral steroid treatment is a prednisone treatment.

115. said oral steroid treatment comprising: a. from about 0.8 mg / kg / day to about 1.00 mg / kg / day for one week prior to and two weeks after administration of a unit dose of the rAAV particles; b. from about 0.59 mg / kg / day to about 0.75 mg / kg / day 3-5 weeks after administration of the unit dose of the rAAV particles; c. from about 0.4 mg / kg / day to about 0.6 mg / kg / day at week 6 after administration of the unit dose of the rAAV particles; d. from about 0.25 mg / kg / day to about 0.375 mg / kg / day at week 7 after administration of the unit dose of the rAAV particles; e. less than about 0.14 mg / kg / day to about 0.28 mg / kg / day at week 8 following administration of a unit dose of the rAAV particles; and f. less than about 0.063 mg / kg / day to about 0.125 mg / kg / day 9 weeks after administration of the unit dose of the rAAV particles The method of claim 114, wherein the dose is

116. said oral steroid treatment comprising: a. about 1.00 mg / kg / day or less for one week prior to and two weeks after administration of a unit dose of the rAAV particles; b. about 0.75 mg / kg / day or less for 3-5 weeks following administration of a unit dose of the rAAV particles; c. about 0.6 mg / kg / day or less at week 6 after administration of the unit dose of the rAAV particles; d. about 0.375 mg / kg / day or less at week 7 after administration of the unit dose of the rAAV particles; e. about 0.28 mg / kg / day or less at week 8 following administration of the unit dose of the rAAV particles; and f. about 0.125 mg / kg / day or less for the 9th week following administration of the unit dose of the rAAV particles The method of claim 114 or 115, wherein the dose is

117. 117. The method of any one of claims 114-116, wherein the oral steroid treatment is administered at a dose of about 0.125 mg / kg / day to about 0.14 mg / kg / day for the first 4 days of the 9th week after administration of a unit dose of the rAAV particles, and if the individual weighs 70 kg or more, at a dose of about 0.0625 mg / kg / day to about 0.070 mg / kg / day for the first 4 days of the 9th week after administration of a unit dose of the rAAV particles.

118. 118. The method of any one of claims 107-117, wherein the IVT steroid treatment is triamcinolone acetonide treatment, or dexamethasone treatment.

119. 119. The method of claim 118, wherein the IVT steroid treatment is about 2 mg of triamcinolone acetonide treatment.

120. The method of claim 118 or 119, wherein the IVT steroid treatment comprises administration of the IVT steroid treatment one week prior to administration of the unit dose of the rAAV particles and 12 weeks after administration of the unit dose of the rAAV particles.

121. 119. The method of claim 118, wherein the IVT steroid treatment is about 0.7 mg of dexamethasone.

122. The method of claim 121, wherein the IVT steroid treatment is an intravitreal implant one week prior to administration of the unit dose of the rAAV particles.

123. The method of any one of claims 107 to 122, wherein the oral steroid treatment and / or the topical steroid treatment is administered before, during and / or after administration of a unit dose of the rAAV particles.

124. 1. A unit dose of recombinant adeno-associated virus (rAAV) particles for use in a method of treating an intraocular neovascular disease in an individual, the method comprising administering the unit dose to one eye of the individual, the individual being a human, and the rAAV particles comprising: 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, the nucleic acid being 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. wherein the unit dose of rAAV particles is administered in combination with oral steroid treatment and topical steroid treatment, or oral steroid treatment and intravitreal (IVT) steroid treatment.

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