Methods of treating ocular neovascular diseases using AAV2 variants encoding aflibercept

Abstract

Provided are methods for treating an ocular neovascular disease in an individual, comprising administering a unit dose of recombinant adeno-associated virus (rAAV) particles to an eye of the individual, wherein the rAAV particles comprise: a) a nucleic acid encoding a polypeptide comprising an amino acid sequence with 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 an 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 an AAV2 VP1 capsid protein.

Description

Attorney Docket No.62700-20017.40 METHODS OF TREATING OCULAR NEOVASCULAR DISEASES USING AAV2 VARIANTS ENCODING AFLIBERCEPT CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This patent application claims priority benefits of U.S. Provisional Application No. 63 / 672,195 filed on July 16, 2024, U.S. Provisional Application No.63 / 721,473 filed on November 16, 2024, and U.S. Provisional Application No.63 / 741,552 filed on January 3, 2025, the content of each of which is incorporated herein by reference in their entirety. REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0002] The content of the electronic sequence listing (627002001740seqlist.xml; Size: 52,410 bytes; and Date of Creation: July 14, 2025) is herein incorporated by reference in its entirety. FIELD OF THE APPLICATION

[0003] The present disclosure relates to methods of treating ocular neovascular disease and disorders in an individual that comprise administering a single unit dose of a recombinant adeno associated virus (rAAV) particles encoding an anti-VEGF agent (e.g., aflibercept) to an eye of an individual. BACKGROUND OF THE APPLICATION

[0004] Age-related macular degeneration (AMD) is a degenerative ocular disease affecting the macula, a light sensitive, small area in the center of the retina that is responsible for reading and fine vision. Conditions affecting the macula reduce central vision while leaving peripheral vision intact. In severe cases, the disease can lead to central blindness. AMD is a notable cause of vision loss in the US population among persons 65 years and older, and the estimated prevalence of any AMD among persons over 40 years of age is approximately 6.5% (Klein et al., (2011) Arch Ophthalmol, 129(1):75-80). Neovascular or exudative or wet AMD (nAMD, wAMD, or nwAMD) is an advanced form of AMD. The hallmark of wAMD is choroidal neovascularization (CNV), which is the infiltration of abnormal blood vessels in the retina from the underlying choroid layer, resulting in retinal cell damage and central blindness. This abnormal angiogenic process is modulated by growth factors, in particular, vascular endothelial growth factor (VEGF). The standard of care of wAMD is a class of molecules that bind to and sequester VEGF, such as ranibizumab (Lucentis) and aflibercept (Eylea).

[0005] Diabetic retinopathy (DR) is a major complication of diabetes mellitus, and is a leading cause of visual loss in the working age population. DR may be non-proliferative (NPDR), with no new blood vessel growth, or proliferative DR (PDR), with new abnormal blood vessel growth within the retina or choroid. Diabetic macular edema (DME) is a complication of DR, and is 1sf-6242343Attorney Docket No.62700-20017.40 another example of an ocular disease affecting the macula. DME affects up to 10% of people with diabetes and is caused by fluid accumulation in the macula. DME is the most frequent cause of sight loss in people with DR. Available therapies for treating DME include laser 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 is able to prevent them from binding to vascular endothelial growth factor receptors (VEGFR), VEGFR-1 and VEGFR-2, to suppress neovascularization and decrease vascular permeability. Aflibercept consists of domain 2 of VEGFR-1 and domain 3 of VEGFR-2 fused with the Fc fragment of IgG1.

[0007] Current standard of care anti-VEGF agents such as aflibercept need to be re- administered via intravitreal (IVT) injection every 4 to 8 weeks to achieve optimal therapeutic outcomes and maintain visual acuity. Compliance with such a regimen is burdensome to patients, their caregivers, and the healthcare system, and most patients fall out of compliance with the optimal regimen over time, which is correlated with vision loss (Khanani AM, et al.). In addition, there are complications including endophthalmitis, retinal detachments, traumatic cataract, and elevated intraocular pressure (IOP); the risks of these complications are likely to increase with repeated IVT injections (Falavarjani et al., (2013) Eye (Lond), 27(7):787-794).

[0008] Therefore, there is a need in the art for therapies for ocular neovascular diseases such as wAMD, DR, or DME that are effective, reduce the risk of adverse effects, and are amenable to high long-term patient compliance. BRIEF SUMMARY OF THE APPLICATION

[0009] The present invention in one aspect provides a method for treating an ocular neovascular disease in an individual, the method comprising administering (i) a single unit dose of recombinant adeno-associated virus (rAAV) particles to one eye of the individual, wherein the individual is a human, wherein the rAAV particles comprise: a) a nucleic acid encoding a polypeptide comprising an amino acid sequence with 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 an 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 an AAV2 VP1 capsid protein, and (ii) a topical steroid treatment beginning prior to the administration of the single unit dose of rAAV to the one eye, wherein the topical steroid treatment comprises (a) about four administrations of the topical steroid per day daily for about 6 weeks, (b) followed by about three administrations of the topical steroid per day 2sf-6242343Attorney Docket No.62700-20017.40 daily for about 5 weeks, (c) followed by about two administrations of the topical steroid per day daily for about 12 weeks, and (d) followed by about one administration of the topical steroid per day daily for about 4 weeks. In some embodiments, the method further comprises one administration of the topical steroid every other day for about 2 weeks following step (ii)(d).

[0010] In some embodiments according to the method described above, an initial topical steroid is administered on Day 1 and the unit dose of rAAV particles is administered on any one of Day 4, Day 5, Day 6, Day 7, Day 8, Day 9, Day 10, or Day 11.

[0011] In some embodiments according to any of the methods described above, the topical steroid treatment is a difluprednate treatment. In some embodiments, the difluprednate treatment is for ophthalmic use. In some embodiments, each administration of the topical steroid comprises a dose of about 1 µg to about 3 µg of the difluprednate. In some embodiments, each administration of the topical steroid comprises a dose of about 2.5 µg of the difluprednate.

[0012] In some embodiments according to any of the methods described above, the method of treating results in reducing a retinal fluid in the eye of the individual with an ocular neovascular disease. In some embodiments, the retinal fluid is subretinal fluid (SRF) or intraretinal fluid (IRF).

[0013] In some embodiments according to any of the methods described above, the individual has received one or more treatment of an anti-VEGF agent from about 1 week to about 8 weeks prior to administration of the unit dose of rAAV particles. In some embodiments, the method further comprises administering a first loading dose of the anti-VEGF agent to the one eye of the individual from about Day -56 to about Day -49 and a second loading dose of the anti-VEGF agent to the one eye of the individual from about Day -28 to about Day -21. In some embodiments, the amount or presence of retinal fluid in the one eye of the individual is refractory to prior treatment with an anti-VEGF agent. In some embodiments, the anti-VEGF agent is aflibercept.

[0014] In some embodiments according to any of the methods described above, the unit dose of rAAV particles is between about 6 × 1010to about 6 × 1011vector genomes per eye (vg / eye). In some embodiments, the unit dose of rAAV particles is about 6 × 1010vector genomes per eye (vg / eye).

[0015] In some embodiments according to any of the methods described above, the method comprises administering the unit dose of rAAV particles to the one eye of the individual about 4 days to about 11 days after administration of the anti-VEGF agent. In some embodiments, the method comprises administering the anti-VEGF agent to the one eye of the individual on Day 1, and administering the unit dose of rAAV particles to the one eye of the individual on any one of 3sf-6242343Attorney Docket No.62700-20017.40 Day 4, Day 5, Day 6, Day 7, Day 8, Day 9, Day 10, or Day 11. In some embodiments, the method further comprises administering one or more rescue anti-VEGF agents after administering the rAAV particles. In some embodiments, the anti-VEGF agent comprises aflibercept. In some embodiments, the aflibercept is administered at a dose of about 2 mg by intravitreal injection.

[0016] In some embodiments according to any of the methods described above, the individual has one or more symptoms of an ocular neovascular disease in the contralateral eye. In some embodiments, the method further comprises administering a unit dose of rAAV particles to the contralateral eye of the individual. In some embodiments, the administering the unit dose of rAAV particles to the contralateral eye is up to about 2 weeks after administering the unit dose of rAAV particles to the one eye. In some embodiments, (a) the administering the unit dose of rAAV particles to the contralateral eye is on the same day as the administering the unit dose of rAAV particles to the one eye; or (b) the administering the unit dose of rAAV particles to the contralateral eye is between about 1 day to about 3 years after administering the unit dose of rAAV particles to the one eye. In some embodiments, the unit dose of rAAV particles administered to the contralateral eye of the individual comprises the same vector genomes per eye (vg / eye) than the unit dose of rAAV particles administered to the one eye of the individual. In some embodiments, the administering the unit dose of rAAV particles to the contralateral eye is at least about 2 weeks after administering the unit dose of rAAV particles to the one eye. In some embodiments, the individual has a baseline Best Corrected Visual Acuity (BCVA) score of between about 35 to about 78 Early Treatment Diabetic Retinopathy Study (ETDRS) letters.

[0017] In some embodiments according to any of the methods described above, the nucleic acid comprises the nucleic acid sequence of SEQ ID NO: 40 or a sequence having at least 85% identity thereto. In some embodiments, the polypeptide comprises the amino acid sequence of SEQ ID NO: 35. In some embodiments, the polypeptide comprises the amino acid sequence of SEQ ID NO: 41. In some embodiments, the polypeptide is aflibercept.

[0018] In some embodiments according to any of the methods described above, 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 the 5’ to 3’ order: (a) a first enhancer region; (b) a promoter region; (c) a 5'UTR region; (d) a nucleic acid encoding a polypeptide comprising an amino acid sequence with 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). In some embodiments, the first enhancer region comprises a CMV sequence comprising the sequence of 4sf-6242343Attorney Docket No.62700-20017.40 SEQ ID NO: 22 or a sequence having at least 85% identity thereto. In some embodiments, the promoter region comprises a CMV sequence comprising the sequence of SEQ ID NO: 23 or a sequence having at least 85% identity thereto. In some embodiments, the nucleic acid sequence comprises the sequence of SEQ ID NO: 43 or a sequence having at least 85% identity thereto. In some embodiments, 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. In some embodiments, the polypeptide comprises the amino acid sequence of SEQ ID NO: 35 or a sequence having at least 95% identity thereto. In some embodiments, the polypeptide comprises the amino acid sequence of SEQ ID NO: 41 or a sequence having at least 95% identity thereto. In some embodiments, the polypeptide is aflibercept. In some embodiments, the 5'UTR region comprises, in 5' to 3' order, a TPL sequence comprising the sequence of SEQ ID NO: 24 or a sequence having at least 85% identity thereto, and an eMLP sequence comprising the sequence of SEQ ID NO: 25 or a sequence having at least 85% identity thereto. In some embodiments, the second enhancer region comprises a full EES sequence comprising the sequence of SEQ ID NO: 26 or a sequence having at least 85% identity thereto. In some embodiments, the polyadenylation site comprises a HGH polyadenylation site comprising the sequence of SEQ ID NO: 27 or a sequence having at least 85% identity thereto. In some embodiments, the nucleic acid comprises the sequence of SEQ ID NO: 39 or a sequence having at least 85% identity thereto. 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, wherein the amino acid residue numbering corresponds to an AAV2 VP1 capsid protein. In some embodiments, the AAV2 capsid protein comprises the amino acid sequence LGETTRP (SEQ ID NO: 14) inserted between positions 587 and 588 of the 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 AAV2 VP1 comprising the sequence of SEQ ID NO: 13. In some embodiments, the rAAV particles comprise an AAV2 VP1 capsid protein comprising 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.

[0019] In some embodiments according to any of the methods described above, the administration of the unit dose of rAAV particles to the one eye and / or the contralateral eye is by intravitreal administration.

[0020] In some embodiments according to any of the methods described above, the unit dose of rAAV particles is in a pharmaceutical formulation. In some embodiments, the pharmaceutical formulation comprises the rAAV particles, sodium chloride, sodium phosphate and a surfactant. 5sf-6242343Attorney Docket No.62700-20017.40 In some embodiments, the pharmaceutical formulation comprises about 150 to about 200 mM sodium chloride, about 1 to about 10 mM monobasic sodium phosphate, about 1 to about 10 mM dibasic sodium phosphate, about 0.0005% (w / v) to about 0.005% (w / v) poloxamer 188, and about 6 × 1011to about 6 × 1012vector genomes (vg) per mL (vg / mL) of the rAAV particles, wherein the pharmaceutical formulation has a pH of about 7.0 to about 7.5. In some embodiments, the pharmaceutical formulation comprises about 180 mM sodium chloride, about 5 mM monobasic sodium phosphate, about 5 mM dibasic sodium phosphate, about 1.2 × 1012vg / mL of the rAAV particles, and about 0.001% (w / v) poloxamer 188, wherein the pharmaceutical formulation has a pH of about 7.3. In some embodiments, the pharmaceutical formulation comprises about 180 mM sodium chloride, about 1.8 mM monobasic sodium phosphate, about 8.2 mM dibasic sodium phosphate, about 1.2 × 1012vg / mL of the rAAV particles, and about 0.001% (w / v) poloxamer 188, wherein the pharmaceutical formulation has a pH of about 7.3.

[0021] In some embodiments according to any of the methods described above, the unit dose of rAAV particles administered to the one eye and / or to the contralateral eye in 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 to 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 to the contralateral eye comprises a volume of about 50 μL. In some embodiments, the unit dose of rAAV particles administered to the one eye and / or to the contralateral eye comprises a volume of about 30 μL.

[0022] In some embodiments according to any of the methods described above, the individual received prior treatment for the ocular neovascular disease with an anti-VEGF agent. In some embodiments, the individual has received 1 or 2 injections of an anti-VEGF agent in the one eye and / or in the contralateral eye prior to administration of the rAAV particles in the one eye and / or in the contralateral eye. In some embodiments, the individual has not received prior treatment for the ocular neovascular disease with an anti-VEGF agent. In some embodiments, the anti-VEGF agent is aflibercept.

[0023] In some embodiments according to any of the methods described above, the ocular neovascular disease is wet age-related macular degeneration (AMD), retinal neovascularization, choroidal neovascularization diabetic retinopathy, proliferative diabetic retinopathy, retinal vein occlusion, central retinal vein occlusion, branched retinal vein occlusion, diabetic macular edema, diabetic retinal ischemia, ischemic retinopathy, diabetic retinal edema, or any combination thereof. In some embodiments, the ocular neovascular disease is wet age-related 6sf-6242343Attorney Docket No.62700-20017.40 macular degeneration (AMD). In some embodiments, the ocular neovascular disease is diabetic macular edema (DME).

[0024] In some embodiments according to any of the methods described above, the topical steroid comprises a difluprednate ophthalmic emulsion 0.05%. In some embodiments, the difluprednate ophthalmic emulsion 0.05% is in a pharmaceutical formulation. In some embodiments, the pharmaceutical formulation comprises about 0.5 mg difluprednate per mL of the ophthalmic emulsion, wherein the pharmaceutical formulation has a pH of about 5.2 to about 5.8, In some embodiments, the pharmaceutical formulation further comprises boric acid, castor oil, edetate disodium, glycerin, polysorbate 80, sodium acetate, sodium hydroxide, and sorbic acid 0.1%. In some embodiments, the administration is about one drop of the difluprednate ophthalmic emulsion 0.05% eyedrop per administration. INCORPORATION BY REFERENCE

[0025] All references cited herein, including patent applications and publications, are incorporated by reference in their entirety. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:

[0027] FIG.1 depicts a study schema for a Phase 3 study comprising an 8-week screening period, 56-week disease assessment period, and a long-term follow-up period for up to an additional 4 years. The study schema includes Arm 1 (study arm; single IVT injection of AAV2.7m8-aflibercept 6 × 1010vg / eye) and Arm 2 (control arm; aflibercept [Eylea®] 2 mg IVT Q8W). Participants will enter the study at SV1 (Day -56) and receive a first loading dose of aflibercept 2 mg. Participants will receive a second loading dose of aflibercept 2 mg on Day -28 and a third loading dose of aflibercept 2 mg on D1. Starting at Day 1, participants in Arm 1 will receive difluprednate ophthalmic emulsion 0.05% eyedrops with tapering regimen (solid circle under W1) while participants in Arm 2 will receive emulsified lubricant eye drops for masking purposes (encircled “S” under W1). Eligible participants in Arm 1 will receive supplemental aflibercept 2 mg at Weeks 8, 16, 24, 32, 40, 48, and 56. Arm 1 participants who do not receive supplemental aflibercept will receive a sham. The visits during which participants may receive sham or supplemental aflibercept (if criteria are met) are labeled with an encircled “S” for Arm 1. In addition to scheduled aflibercept administration at Weeks 8, 16, 24, 32, 40, 48, and 56 (solid 7sf-6242343Attorney Docket No.62700-20017.40 circles), participants in Arm 2 who meet any of the supplemental aflibercept criteria at Weeks 4, 12, 20, 28, 36, 44, or 52 will be eligible to receive aflibercept 2 mg. All empty circles indicate administration of supplemental aflibercept 2 mg (if criteria are met). During the second year on study, participants will have scheduled visits every 4 weeks and may receive aflibercept 2 mg if any of the supplemental treatment criteria are met at any scheduled visit. During the third, fourth, and fifth years on study, participants will have scheduled visits every 12 weeks and may receive aflibercept 2 mg if any of the supplemental treatment criteria are met at any scheduled or unscheduled visit. BCVA: best-corrected visual acuity; D: day; IVT: intravitreal(ly); Q4W: every 4 weeks; Rand: randomization; SV: screening visit; W: Week.

[0028] FIG.2 depicts a diagram of the AAV2.7m8-aflibercept vector genome (SEQ ID NO: 39). The vector genome comprises two inverted terminal repeats (ITRs) of AAV serotype 2 (at positions 1-145 and 3772-3916 of SEQ ID NO: 39), an expression cassette comprised of the CMV promoter (at positions 180-693 of SEQ ID NO: 39), a 5’ Untranslated Region (UTR) comprised of Adenovirus Tripartite Leader Sequence and Synthetic Intron (at positions 694-1314 of SEQ ID NO: 39), a Kozak sequence (at positions 1329-1340 of SEQ ID NO: 39), a codon- optimized aflibercept cDNA (at positions 1338-2714 of SEQ ID NO: 39), a 3’ UTR comprised of human scaffold attachment region (at positions 2717-3527 of SEQ ID NO: 39), and human growth hormone polyadenylation / transcription stop signal (at positions 3546-3748 of SEQ ID NO: 39). AAV = adeno-associated virus; CMV = cytomegalovirus; GH = growth hormone; ITR = inverted terminal repeat; polyA = polyadenylation; SAR = scaffold-attached region; TPL = tripartite leader sequence.

[0029] FIG.3 depicts the nucleic acid sequence of aflibercept (SEQ ID NO: 36).

[0030] FIG.4 depicts a study schema for a Phase 3 study comprising an 8-week screening period, 56-week disease assessment period, and a long-term follow-up period for up to an additional 4 years. The study schema includes Arm 1 (study arm; single IVT injection of AAV2.7m8-aflibercept 6 × 1010vg / eye) and Arm 2 (control arm; aflibercept 2 mg IVT Q8W). Participants will enter the study at SV1 (Day -56 to -49) and receive a first loading dose of aflibercept 2 mg. Participants will receive a second loading dose of aflibercept 2 mg on Day -28 and a third loading dose of aflibercept 2 mg on D1. Starting on Day 1, all participants will receive difluprednate ophthalmic emulsion 0.05% eyedrops with tapering regimen (solid circle under W1). The downward-sloping “difluprednate prophylaxis” line depicts that the difluprednate dosing is a tapering regimen across 27 weeks. At Week 1, eligible participants will be randomized in a 1:1 ratio to Arm 1 or Arm 2. Starting at Week 4, particpants in both study arms will be eligible to receive aflibercept 2 mg at any scheduled visit, at which aflibercept is not 8sf-6242343Attorney Docket No.62700-20017.40 required to be administered per protocol (i.e., any scheduled visit for Arm 1 and Weeks 4, 12, 20, 28, 36, 44, or 52 for those randomized to Arm 2). Participants will be eligible if they meet any of the supplemental treatment criteria. During the second year on study, participants will have scheduled visits every 4 weeks and may receive aflibercept 2 mg if any of the supplemental treatment criteria are met at any visit. During the third, fourth, and fifth years on study, participants will have scheduled visits every 12 weeks and may receive aflibercept 2 mg if any of the supplemental treatment criteria are met at any scheduled or unscheduled visit. BCVA: best- corrected visual acuity; D: day; IVT: intravitreal(ly); Q4W: every 4 weeks; SV: screening visit; W: week; vg: vector genome. DETAILED DESCRIPTION OF THE APPLICATION

[0031] Several aspects are described below with reference to example applications for illustration. It should be understood that numerous specific details, relationships, and methods are set forth to provide a full understanding of the features described herein. One having ordinary skill in the relevant art, however, will readily recognize that the features described herein can be practiced without one or more of the specific details or with other methods. The features described herein are not limited by the illustrated ordering of acts or events, as some acts can occur in different orders and / or concurrently with other acts or events. Furthermore, not all illustrated acts or events are required to implement a methodology in accordance with the features described herein. Definitions

[0032] Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0033] The terminology used herein is for the purpose of describing particular examples only and is 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, to the extent that the terms “including”, “includes”, “having”, “has”, “with”, or variants thereof are used in either the detailed description and / or the 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.

[0034] Any reference to “or” herein is intended to encompass “and / or” unless otherwise stated. As used herein, the term “about” a number refers to that number plus or minus 10% of that number. The term “about” a range refers to that range minus 10% of its lowest value and plus 10% of its greatest value. Reference to “about” a value or parameter herein includes (and describes) embodiments that are directed to that value or parameter per se. 9sf-6242343Attorney Docket No.62700-20017.40

[0035] The term “subject”, “patient”, or “individual” refers to primates, such as humans and non-human primates, e.g., African green monkeys and rhesus monkeys. In some embodiments, the subject is a human.

[0036] The terms “treat,” “treating”, “treatment,” “ameliorate” or “ameliorating” and other grammatical equivalents as used herein, refer to alleviating, abating or ameliorating an ocular neovascular disease or disorder, or symptoms of the ocular neovascular disease or disorder, preventing additional symptoms of the ocular neovascular disease or disorder, ameliorating or preventing the underlying metabolic causes of symptoms, inhibiting the ocular neovascular disease or disorder, e.g., arresting the development of the ocular neovascular disease or disorder, relieving the ocular neovascular disease or disorder, causing regression of the ocular neovascular disease or disorder, or stopping the symptoms of the ocular neovascular disease or disorder, and are intended to include prophylaxis. The terms further include achieving a therapeutic benefit and / or a prophylactic benefit. The term “therapeutic benefit” refers to eradication or amelioration of the ocular neovascular disease or disorder being treated. Also, a therapeutic benefit is achieved with the eradication or amelioration of one or more of the physiological symptoms associated with the ocular neovascular disease or disorder such that an improvement is observed in the subject, notwithstanding that, in some embodiments, the subject is still afflicted with the ocular neovascular disease or disorder. For prophylactic benefit, the pharmaceutical compositions are administered to a subject at risk of developing the ocular neovascular disease or disorder, or to a subject reporting one or more of the physiological symptoms of the ocular neovascular disease or disorder, even if a diagnosis of the disease or disorder has not been made.

[0037] The terms “administer,” “administering”, “administration,” and the like, as used herein, can refer to the methods that are used to enable delivery of therapeutics or pharmaceutical compositions to the desired site of biological action. These methods include intravitreal or subretinal injection to an eye.

[0038] The terms “effective amount”, “therapeutically effective amount” or “pharmaceutically effective amount” as used herein, can refer to a sufficient amount of at least one pharmaceutical composition or compound being administered which will relieve to some extent one or more of the symptoms of the ocular disease or disorder being treated. An “effective amount”, “therapeutically effective amount” or “pharmaceutically effective amount” of a pharmaceutical composition may be administered to a subject in need thereof as a unit dose (as described in further detail elsewhere herein).

[0039] The term “pharmaceutically acceptable” as used herein, can refer to a material, such as a carrier or diluent, which does not abrogate the biological activity or properties of a compound 10sf-6242343Attorney Docket No.62700-20017.40 disclosed herein, and is relatively nontoxic (i.e., when the material is administered to an individual it does not cause undesirable biological effects nor does it interact in a deleterious manner with any of the components of the composition in which it is contained).

[0040] The term “pharmaceutical composition,” or simply “composition” as used herein, can refer to a biologically active compound, optionally mixed with at least one pharmaceutically acceptable chemical component, such as, though not limited to carriers, stabilizers, diluents, dispersing agents, suspending agents, thickening agents, excipients and the like.

[0041] An “AAV vector” or “rAAV vector” as used herein refers to an adeno-associated virus (AAV) vector or a recombinant AAV (rAAV) vector comprising a polynucleotide sequence not of AAV origin (e.g., a polynucleotide heterologous to AAV such as a nucleic acid sequence that encodes a therapeutic transgene, e.g., aflibercept) for transduction into a target cell or to a target tissue. In general, the heterologous polynucleotide is flanked by at least one, and generally by two, AAV inverted terminal repeat sequences (ITRs). The term rAAV vector encompasses both rAAV vector particles and rAAV vector plasmids. A rAAV vector may be either single-stranded (ssAAV) or self-complementary (scAAV).

[0042] An “AAV virus” or “AAV viral particle” or “rAAV vector particle” or “rAAV particle” refers to a viral particle comprising at least one AAV capsid protein and a polynucleotide rAAV vector. In some cases, the at least one AAV capsid protein is from a wild type AAV or is a variant AAV capsid protein (e.g., an AAV capsid protein with an insertion, e.g., an insertion of the 7m8 amino sequence as set forth below). If the particle comprises a heterologous polynucleotide (e.g., a polynucleotide other than a wild-type AAV genome such as a transgene to be delivered to a target cell or target tissue), it is referred to as a “rAAV particle”, “rAAV vector particle” or a “rAAV vector”. Thus, production of rAAV particles necessarily includes production of a rAAV vector, as such a vector contained within a rAAV particle.

[0043] The term “packaging” as used herein can refer to a series of intracellular events that can result in the assembly and encapsidation of a rAAV particle.

[0044] AAV “rep” and “cap” genes refer to polynucleotide sequences encoding replication and encapsidation proteins of adeno-associated virus. AAV rep and cap are referred to herein as AAV “packaging genes.”

[0045] The term “polypeptide” can encompass both naturally occurring and non-naturally occurring proteins (e.g., a fusion protein), peptides, fragments, mutants, derivatives and analogs thereof. A polypeptide may be monomeric, dimeric, trimeric, or polymeric. Further, a polypeptide may comprise a number of different domains, each of which has one or more distinct 11sf-6242343Attorney Docket No.62700-20017.40 activities. For the avoidance of doubt, a “polypeptide” may be any length greater two amino acids.

[0046] As used herein, “polypeptide variant” or simply “variant” refers to a polypeptide whose sequence contains an 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, in which a single amino acid at a position has been changed to another amino acid, one or more insertions and / or deletions, in which one or more amino acids are inserted or deleted, respectively, in the sequence of the reference protein, and / or truncations of the amino acid sequence at either or both the amino or carboxy termini. A variant can have the same or a different biological activity compared to the reference protein, or the unmodified protein.

[0047] In some embodiments, a variant can have, for example, at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% overall sequence homology to its counterpart reference protein. In some embodiments, a variant can have at least about 90% overall sequence homology to 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.

[0048] As used herein, “recombinant” can refer to a biomolecule, e.g., a gene or protein, that (1) has been removed from its naturally occurring environment, (2) is not associated with all or a portion of a polynucleotide in which the gene is found in nature, (3) is operatively linked to a polynucleotide which it is not linked to in nature, or (4) does not occur in nature. The term “recombinant” can be used in reference to cloned DNA isolates, chemically synthesized polynucleotide analogs, or polynucleotide analogs that are 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, for example, if it is synthesized from an mRNA synthesized from a recombinant gene present in the cell.

[0049] The term “anti-VEGF agent” includes any therapeutic agent, including proteins, polypeptides, peptides, fusion protein, multimeric proteins, gene products, 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 an endogenous VEGF and / or an endogenous VEGF receptor (VEGFR), or the VEGF-VEGFR interaction or pathway in vivo. An anti-VEGF agent can be any one of the known therapeutic agents that can reduce new blood vessel growth or formation and / or 12sf-6242343Attorney Docket No.62700-20017.40 oedem, or swelling, when delivered into a cell, tissue, or a subject in vivo, e.g., ranibizumab, brolucizumab, or bevacizumab. In some embodiments, an anti-VEGF agent can be naturally occurring, non-naturally occurring, or synthetic. In some embodiments, an anti-VEGF agent can be derived from a naturally occurring molecule that was subsequently modified or mutated to confer an anti-VEGF activity. In some embodiments, an anti-VEGF agent is a fusion or chimeric protein. In such proteins, functional domains or polypeptides are artificially fused to a moiety or a polypeptide to make a fusion or chimeric protein that can sequester VEGF in vivo or function as a VEGFR decoy. In some embodiments, an anti-VEGF agent is a fusion or chimeric protein that blocks endogenous VEGFR from interacting with its ligands.

[0050] As used herein, “VEGF” can refer to any isoform of VEGF, unless required otherwise, including, but not limited to, VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E, VEGF-F, or any combination, or any functional fragment or variant thereof. Unless required otherwise, “VEGF” can refer to any member of the VEGF family, including members: VEGF-A, placenta 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” can 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 can be a membrane bound or soluble form, or a functional fragment or truncation of a receptor. Examples of anti-VEGF agents include, but are not limited to, ranibizumab, bevacizumab, brolucizumab, or any combination, variant, or functional fragment thereof.

[0051] “Operatively linked” or “operably linked” or “coupled” can refer to a juxtaposition of genetic elements, wherein the elements are in a relationship permitting them to operate in an expected manner. For instance, a promoter can be operatively linked to a coding region if the promoter helps initiate transcription of the coding sequence. There may be intervening residues between the promoter and coding region so long as this functional relationship is maintained.

[0052] The term “expression vector” or “expression construct” or “cassette” or “plasmid” or simply “vector” can include any type of genetic construct, including AAV or rAAV vectors, containing a nucleic acid or polynucleotide coding for a gene product in which part or all of the nucleic acid encoding sequence is capable of being transcribed and is adapted for gene therapy. The transcript can be translated into a protein. In some embodiments, the transcript is partially translated or not translated. In certain aspects, expression includes both transcription of a gene and translation of mRNA into a gene product. In other aspects, expression only includes transcription of the nucleic acid encoding genes of interest. An expression vector can also comprise control elements operatively linked to the encoding region to facilitate expression of 13sf-6242343Attorney Docket No.62700-20017.40 the protein in target cells. The combination of control elements and a gene or genes to which they are operably linked for expression can sometimes be referred to as an “expression cassette,” a large number of which are known and available in the art or can be readily constructed from components that are available in the art.

[0053] The term “heterologous” can refer to an entity that is genotypically distinct from that of the rest of the entity to which it is being compared. For example, a polynucleotide introduced by genetic engineering techniques into a plasmid or vector derived from a different species can be a heterologous polynucleotide. A promoter removed from its native coding sequence and operatively linked to a coding sequence with which it is not naturally found linked can be a heterologous promoter.

[0054] As used herein, “7m8” refers to the amino acid sequence LALGETTRPA (SEQ ID NO: 1).

[0055] “7m8 variant” refers to a rAAV, which can be of any serotype, with the amino acid sequence LALGETTRPA (SEQ ID NO: 1) inserted in the solvent exposed GH loop of the capsid protein.

[0056] When 7m8 is inserted in a 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, e.g., between positions 587 and 588 of the AAV2 capsid protein, VP1. In some cases, when 7m8 is inserted in a 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, e.g., between positions 587 and 588 of AAV2 VP1 comprising the sequence of SEQ ID NO: 13. When 7m8 is inserted in a 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, e.g., between amino acids 590 and 591 of the AAV1 capsid protein. When 7m8 is inserted in a 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 in a 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 to 612 of the AAV6 capsid protein, e.g., between amino acids 590 and 591 of the AAV6 capsid protein. When 7m8 is inserted in a 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 to 613 of the AAV7 capsid protein, e.g., between amino acids 589 and 590 of the AAV7 capsid protein. When 7m8 is inserted in a rAAV8 (also referred to as AAV8.7m8), the amino 14sf-6242343Attorney Docket No.62700-20017.40 acid sequence LALGETTRPA (SEQ ID NO: 1) is inserted into the GH loop within amino acids 573 to 614 of the AAV8 capsid protein, e.g., between amino acids 590 and 591 of the AAV8 capsid protein. When 7m8 is inserted in a 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 in a 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 to 614 of the AAV10 capsid protein, e.g., between amino acids 589 and 590 of the AAV10 capsid protein. Methods of Treatment

[0057] Provided herein is a method for treating an ocular neovascular disease in an individual, the method comprising administering (i) a single unit dose of recombinant adeno-associated virus (rAAV) particles to one eye of the individual, wherein the individual is a human, wherein the rAAV particles comprise: a) a nucleic acid encoding a polypeptide comprising an amino acid sequence with 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 an 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 an AAV2 VP1 capsid protein, and (ii) a topical steroid treatment beginning prior to the administration of the single unit dose of rAAV to the one eye, wherein the topical steroid treatment comprises (a) about four administrations of the topical steroid per day daily for about 6 weeks, (b) followed by about three administrations of the topical steroid per day daily for about 5 weeks, (c) followed by about two administrations of the topical steroid per day daily for about 12 weeks, and (d) followed by about one administration of the topical steroid per day daily for about 4 weeks. In some embodiments, the method further comprises one administration of the topical steroid every other day for about 2 weeks following step (ii)(d).

[0058] In some embodiments, the ocular neovascular disease is wet age-related macular degeneration (wAMD), retinal neovascularization, choroidal neovascularization diabetic retinopathy, proliferative diabetic retinopathy, retinal vein occlusion, central retinal vein occlusion, branched retinal vein occlusion, diabetic macular edema, diabetic retinal ischemia, ischemic retinopathy, diabetic retinal edema, or any combination thereof. In some embodiments, the ocular neovascular disease is wet age-related macular degeneration (AMD). In some embodiments, the ocular neovascular disease is diabetic macular edema (DME). In some embodiments, the individual has wet age-related macular degeneration (AMD), retinal neovascularization, choroidal neovascularization diabetic retinopathy, proliferative diabetic 15sf-6242343Attorney Docket No.62700-20017.40 retinopathy, retinal vein occlusion, central retinal vein occlusion, branched retinal vein occlusion, diabetic macular edema, diabetic retinal ischemia, ischemic retinopathy, diabetic retinal edema, or any combination thereof. In some embodiments, the individual has wet age-related macular degeneration (AMD). In some embodiments, the individual has diabetic macular edema (DME).

[0059] In some embodiments, the term ocular neovascular disease also encompasses VEGF- driven pre-neovascular diseases which, if left untreated, progress to a neovascular form. In some embodiments, the ocular neovascular disease is the pre-neovascular disease, non-proliferative diabetic retinopathy.

[0060] In some embodiments, the individual is a human. In some embodiments, the individual received one or more prior treatment (e.g., at least one, at least two, at least three, at least four, at least 5 or more treatments) for the ocular 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) from about 1 week to about 8 weeks prior to administration of the unit dose of rAAV particles. In some embodiments, the individual demonstrated a meaningful response to a prior treatment with 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 with at least about 95% identity to the amino acid sequence of SEQ ID NO: 35. In some embodiments, the retinal fluid in the eye of an individual is intraretinal fluid (IRF) and / or subretinal fluid (SRF). In some embodiments, the amount or presence of retinal fluid in the eye of the individual is refractory to prior treatment with an anti-VEGF agent (e.g., bevacizumab, brolucizumab, ranibizumab, faricimab, abicipar pegol, conbercept, OPT-302, KSI-301, injectable sunitinib maleate (GB-102), PAN-90806 (PanOptica), and / or aflibercept). In 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 with at least about 95% identity to the amino acid sequence of SEQ ID NO: 35.

[0061] In some embodiments, the ocular 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 mellitus. In some embodiments, the individual has vision impairment that is due to center involving diabetic macular edema. In some embodiments, the individual has visual acuity (BCVA) of between about 78 to 35 ETDRS letters (e.g., any of 35, 36, 37, 38, 39, 40, 41, 16sf-6242343Attorney Docket No.62700-20017.40 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 5354, 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 letters) in the eye administered the rAAV particles prior to administration of the unit dose of rAAV particles. In some embodiments, the individual has visual acuity (Snellen equivalent) of between about 20 / 32 to about 20 / 200 in the eye administered the rAAV particles prior to administration of the unit dose of rAAV particles. In some embodiments, the individual has a central subfield thickness (CST) of ≥ 325μm using Heidelberg Spectralis® with center-involving IRF (center 1 mm) in the eye administered the rAAV particles prior to administration of the unit dose of rAAV particles. In some embodiments, the individual has a decrease in vision in the eye administered the rAAV particles prior to administration of the unit dose of rAAV particles that is primarily due to diabetic macular edema. In some embodiments, the individual was diagnosed with diabetic macular edema in the eye administered the rAAV particles about 6 months or less prior to administration of the unit dose of 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 rAAV particles. In some embodiments, the individual received 0, 1, or 2 prior treatments for DME in the eye administered the rAAV particles, e.g., 0, 1, or 2 intravitreal injections with an anti-VEGF agent, e.g., aflibercept, prior to administration of the unit dose of rAAV particles. In some embodiments, the individual was diagnosed with age-related macular degeneration (e.g., nAMD, wAMD, or nwAMD). In some embodiments, in the individual wad diagnosed with choroidal neovascularization secondary to nAMD. In some embodiments, the individual was diagnosed with age-related macular degeneration in the eye administered the rAAV particles about 6 months or less prior to administration of the unit dose of 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 rAAV particles. In some embodiments, the individual received 0, 1, or 2 prior treatments for AMD in the eye administered the rAAV particles, e.g., 0, 1, or 2 intravitreal injections with an anti-VEGF agent, e.g., aflibercept, prior to administration of the unit dose of rAAV particles. In some embodiments, the individual received the prior treatments with an anti-VEGF agent in the eye administered the rAAV particles at least about 60 days (i.e., about 2 months) prior to administration of the unit dose of rAAV particles. In some embodiments, the individual exhibited a meaningful response in central subfield thickness to the prior treatments with an anti-VEGF agent in the eye administered the rAAV particles prior to administration of the unit dose of rAAV particles, for example, at least a 10% reduction in central subfield thickness. In some embodiments, the individual did not experience an adverse reaction to the prior treatments with an anti-VEGF agent prior to administration of the unit dose 17sf-6242343Attorney Docket No.62700-20017.40 of rAAV particles. In some embodiments, the individual does not have neutralizing antibodies to AAV2.7m8 prior to administration of the unit dose of rAAV particles. In some embodiments, the individual does not have an anti-AAV2.7m8 neutralizing antibody titer of greater than 1:125 prior to administration of the unit dose of rAAV particles, e.g., within about 6 months prior to administration of the unit dose of rAAV particles. In some embodiments, the individual does not have a history of allergy to aflibercept, corticosteroids, or fluorescein dye or sodium fluorescein (e.g., used in angiography) prior to administration of the unit dose of rAAV particles. In some embodiments, the individual has a history of mild allergy to aflibercept, corticosteroid, or fluorescein dye or sodium fluorescein (e.g., used in angiography) prior to administration of the unit dose of rAAV particles, wherein the allergy is amenable to treatment. In some embodiments, the individual does not have uncontrolled diabetes, e.g., HbA1C of greater than 10%, prior to administration of the unit dose of rAAV particles. In some embodiments, the individual does not have a history of diabetic ketoacidosis within about 3 months prior to administration of the unit dose of rAAV particles. In some embodiments, the individual has not initiated intensive insulin treatment, e.g., with an insulin pump or multiple daily insulin injections, prior to administration of the unit dose of rAAV particles. In some embodiments, the individual does not plan to initiate intensive insulin treatment, e.g., with an insulin pump or multiple daily insulin injections, within about 3 months after administration of the unit dose of rAAV particles. In some embodiments, the individual does not have a history of systemic autoimmune disease that requires treatment with systemic steroids or immunosuppressive treatments, e.g., methotrexate or adalimumab, prior to administration of the unit dose of rAAV particles. In some embodiments, the individual is not being administered a systemic drug known to cause macular edema, such as fingolimod, tamoxifen, chloroquine, or hydroxychloroquine, prior to administration of the unit dose of rAAV particles. In some embodiments, the individual is not being administered a systemic anti-VEGF treatment prior to administration of the unit dose of rAAV particles. In some embodiments, the individual does not have high-risk proliferative diabetic retinopathy (PDR) in the eye administered the rAAV particles prior to administration of the unit dose of rAAV particles. In some embodiments, PDR is defined as any vitreous or preretinal hemorrhage, neovascularization elsewhere >1 / 2-disc area within an area equivalent to standard ETDRS 7-field on clinical examination, or neovascularization of disc > 1 / 3-disc area on clinical examination. In some embodiments, the individual does not have focal or grid laser photocoagulation in the eye administered the rAAV particles prior to administration of the unit dose of rAAV particles. In some embodiments, the individual does not have any prior pan retinal photocoagulation (PRP) in the eye administered the rAAV particles prior to administration of the unit dose of rAAV 18sf-6242343Attorney Docket No.62700-20017.40 particles. In some embodiments, the individual has not received an anti-VEGF therapy (e.g., aflibercept IVT injections) in the eye administered the rAAV particles prior to administration of the unit dose of rAAV particles. In some embodiments, the individual has not received an anti- VEGF therapy (e.g., aflibercept IVT injections) in the eye administered the rAAV particles for at least 60 days prior to administration of the unit dose of rAAV particles. In some embodiments, the individual has not received more than two anti-VEGF treatments (e.g., aflibercept IVT injections) in the eye administered the rAAV particles prior to administration of the unit dose of rAAV particles. In some embodiments, the individual does not have a history of any of anterior segment neovascularization (e.g., neovascularization of the iris [NVI] or neovascular glaucoma [NVG]), significant vitreous hemorrhage, fibrovascular proliferation, or tractional retinal detachment in the eye administered the rAAV particles prior to administration of the unit dose of rAAV particles. In some embodiments, the individual does not have structural abnormalities at the fovea (e.g., any of dense hard exudates, pigment abnormalities, foveal atrophy, vitreomacular traction or epiretinal membrane) in the eye administered the rAAV particles that contribute to macular edema or visual impairment prior to administration of the unit dose of rAAV particles. In some embodiments, structural abnormalities at the fovea are assessed on clinical examination or OCT. In some embodiments, the individual does not have a history of retinal disease other than diabetic retinopathy (e.g., age-related macular degeneration (in either eye), retinal vein occlusion, retinal arterial occlusion, or pathologic myopia) in the eye administered the rAAV particles prior to administration of the unit dose of rAAV particles. In some embodiments, the individual does not have history of ocular disease other than diabetic macular edema in the eye administered the rAAV particles, e.g., a significant cataract or macular traction, or evidence of posterior subcapsular cataract, prior to administration of the unit dose of rAAV particles. In some embodiments, the individual does not have history of cataract extraction or Yttrium Aluminum Garnet (YAG) capsulotomy in the eye administered the rAAV particles within at least about 3 months prior to administration of the unit dose of rAAV particles. In some embodiments, the individual does not have a history of retinal detachment (with or without repair) in the eye administered the rAAV particles prior to administration of the unit dose of rAAV particles. In some embodiments, the individual does not have a history of any of trabeculectomy, glaucoma shunt, or minimally invasive glaucoma surgery (MIGS) in the eye administered the rAAV particles prior to administration of the unit dose of rAAV particles. In some embodiments, the individual does not have a history of vitrectomy or other filtration surgery in the eye administered the rAAV particles prior to administration of the unit dose of rAAV particles. In some embodiments, the individual does not have aphakia or presence of an anterior chamber 19sf-6242343Attorney Docket No.62700-20017.40 intraocular lens in the eye administered the rAAV particles prior to administration of the unit dose of rAAV particles. In some embodiments, the individual does not have uncontrolled ocular hypertension or glaucoma in the eye administered the rAAV particles, e.g., IOP >22 mmHg despite treatment with anti-glaucoma medication or current use of >2 IOP lowering medications, 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) in the eye administered the rAAV particles prior to administration of the unit dose of rAAV particles. In some embodiments, the individual has not had refractive surgery in the eye administered 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 in the eye administered the rAAV particles prior to administration of the unit dose of rAAV particles. In some embodiments, the individual has not had any prior vitreoretinal surgery in the eye administered the rAAV particles prior to administration of the unit dose of rAAV particles. In some embodiments, the individual does not have a history of uveitis or intraocular inflammation, e.g., grade trace or above except mild anticipated post-operative inflammation that resolved, prior to administration of the unit dose of rAAV particles. In some embodiments, the individual does not have a history of IOP elevation that is related to topical steroid administration prior to administration of the unit dose of rAAV particles. In some embodiments, the individual does not have a history of ocular Herpes Simplex Virus (HSV), Varicella-zoster virus (VZV), or Cytomegalovirus (CMV), including viral uveitis, retinitis or keratitis prior to administration of the unit dose of rAAV particles. In some embodiments, the individual does not have evidence of any of external ocular infection, including conjunctivitis, chalazion, or significant blepharitis prior to administration of the unit dose of rAAV particles. In some embodiments, the individual does not have history of ocular toxoplasmosis prior to administration of the unit dose of rAAV particles. In some embodiments, the individual is treatment naïve (i.e., no history of anti-VEGF treatment). In some embodiments, the individual is treatment-experienced. In some embodiments, the individual has a history of at least about 1 (e.g., at least about any of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15) anti-VEGF treatment in the six months prior to administration of the unit dose of rAAV particles and did not receive an anti-VEGF IVT injection in the study eye within 28 days prior to Screening Visit 1 (Day -56 to -49). In some embodiments, the anti- VEGF treatment is selected from the group consisting of Beovu®(brolucizumab-dbll), Eylea®(aflibercept), Eylea®HD (aflibercept), Lucentis®(ranibizumab), Susvimo™ (ranibizumab injection), and Vabysmo®(faricimab-svoa injection). In some embodiments, Day -56 is 56 days 20sf-6242343Attorney Docket No.62700-20017.40 prior to the initial administration of a topical steroid treatment (e.g., difluprednate) on Day 1 and / or an anti-VEGF agent (e.g., aflibercept protein) on Day 1. In some embodiments, Day -49 is 49 days prior to the initial administration of a topical steroid treatment (e.g., difluprednate) on Day 1 and / or an anti-VEGF agent (e.g., aflibercept protein) on Day 1.

[0062] In some embodiments, the unit dose is expressed as the number of vector genomes (vg). In some embodiments, the unit dose is about 6 × 1011vector genomes (vg) or less of the rAAV particles. In some embodiments, the unit dose is expressed as the number of vector genomes (vg) per eye (vg / eye). In some embodiments, the unit dose is about 6 × 1011vg / eye or less of the rAAV particles. In some embodiments, the unit dose of rAAV particles is about 6 × 1010to about 2 × 1011vg / eye. In some embodiments, the unit dose of rAAV particles is about 2 × 1011or about 6 × 1010vg / eye.

[0063] In some embodiments, the unit dose of 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 methods provided herein further comprise administering a unit dose of rAAV particles to the contralateral eye of the individual. In some embodiments, the one eye of the individual is the right eye and the contralateral eye is the left eye. In some embodiments, the one eye of the individual is the left eye and the contralateral eye is the right eye.

[0064] In some embodiments, the administering the unit dose of rAAV particles to the contralateral eye of the individual is up to about 2 weeks (e.g., about 0 days, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, or 14 days) after administering the unit dose of rAAV particles to the one eye. In some embodiments, the unit dose of rAAV particles administered to the contralateral eye of the individual is about the same as (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 the one eye of the individual.

[0065] In some embodiments, the administering the 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 21sf-6242343Attorney Docket No.62700-20017.40 least about 4 years, at least about 5 years, or more) after administering the unit dose of rAAV particles to the one eye. In some embodiments, the unit dose of rAAV particles administered to the contralateral eye of the individual is higher (e.g., any of about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 125%, about 150%, about 175%, about 200%, about 225%, about 250%, about 275%, about 300%, or more, higher) than the unit dose of rAAV particles administered to the one eye of the individual.

[0066] In some embodiments, the rAAV particles comprise a) a nucleic acid encoding a polypeptide comprising an amino acid sequence with at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.99%, or 100% identity to the amino acid sequence of SEQ ID NO: 35 and flanked by AAV2 inverted terminal repeats (ITRs), and b) an AAV2 capsid protein comprising an amino acid sequence LGETTRP (SEQ ID NO: 14) or LALGETTRPA (SEQ ID NO: 1) inserted between positions 587 and 588 of the capsid protein, wherein the amino acid residue numbering corresponds to an AAV2 VP1 capsid protein. The sequence of SEQ ID NO: 35 is provided below: SDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFI ISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVLSPSHGIELSVGEKLVLNCTARTEL NVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKK NSTFVRVHEKDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFF LYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 35)

[0067] In some embodiments, the rAAV particles comprise a) a nucleic acid encoding a polypeptide comprising an amino acid sequence with 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 an amino acid sequence LGETTRP (SEQ ID NO: 14) or LALGETTRPA (SEQ ID NO: 1) inserted between positions 587 and 588 of the capsid protein, wherein the amino acid residue numbering corresponds to an AAV2 VP1 capsid protein.

[0068] In some embodiments, the rAAV particles comprise a nucleic acid encoding a polypeptide comprising an amino acid sequence with 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 22sf-6242343Attorney Docket No.62700-20017.40 NO: 35 and 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 with at least about 95% identity to the amino acid sequence of SEQ ID NO: 35 and flanked by AAV2 inverted terminal repeats (ITRs). In some embodiments, the polypeptide comprises the amino acid sequence of SEQ ID NO: 35. In some embodiments, the polypeptide is aflibercept or a functional variant thereof or functional fragment thereof.

[0069] In some embodiments, the rAAV particles comprise a nucleic acid comprising a codon- optimized sequence encoding an amino acid sequence with at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.99%, or 100% identity to the amino acid sequence of SEQ ID NO: 35 and flanked by AAV2 inverted terminal repeats (ITRs). In some embodiments, the rAAV particles comprise a nucleic acid comprising a codon-optimized sequence encoding an amino acid sequence with at least about 95% identity to the amino acid sequence of SEQ ID NO: 35 and flanked by AAV2 inverted terminal repeats (ITRs). In some embodiments, the rAAV particles comprise a nucleic acid comprising a codon-optimized sequence encoding an amino acid sequence with 100% identity to the amino acid sequence of SEQ ID NO: 35 and flanked by AAV2 inverted terminal repeats (ITRs).

[0070] In some embodiments, the rAAV particles comprise a nucleic acid comprising the cDNA sequence of aflibercept or a functional variant thereof or functional fragment thereof and flanked by AAV2 inverted terminal repeats (ITRs). In some embodiments, the rAAV particles comprise a nucleic acid comprising a codon-optimized cDNA sequence of aflibercept or a functional variant thereof or functional fragment thereof and flanked by AAV2 inverted terminal repeats (ITRs). In some embodiments, the rAAV particles comprise a nucleic acid comprising the nucleic acid sequence of SEQ ID NO: 36.

[0071] 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 the 5' to 3' order, a TPL sequence and an eMLP sequence; (d) a second enhancer region comprising a full EES sequence; and (e) a 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 first enhancer region comprising a CMV sequence and the promoter region comprising a CMV sequence comprise the sequence of SEQ ID NO: 43. 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. 23sf-6242343Attorney Docket No.62700-20017.40 In some embodiments, the second enhancer region comprising a full 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.

[0072] In some embodiments, the rAAV particles comprise an AAV2 capsid protein comprising the amino acid sequence LGETTRP (SEQ ID NO: 14) or LALGETTRPA (SEQ ID NO: 1) inserted between positions 587 and 588 of the AAV2 VP1 comprising the sequence of SEQ ID NO: 13. The sequence of SEQ ID NO: 13 is provided below: MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPGYKYLGPFNGLD KGEPVNEADAAALEHDKAYDRQLDSGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQ AKKRVLEPLGLVEEPVKTAPGKKRPVEHSPVEPDSSSGTGKAGQQPARKRLNFGQTGDAD SVPDPQPLGQPPAAPSGLGTNTMATGSGAPMADNNEGADGVGNSSGNWHCDSTWMGDRVI TTSTRTWALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLI NNNWGFRPKRLNFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQG CLPPFPADVFMVPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEDVPF HSSYAHSQSLDRLMNPLIDQYLYYLSRTNTPSGTTTQSRLQFSQAGASDIRDQSRNWLPG PCYRQQRVSKTSADNNNSEYSWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVL IFGKQGSEKTNVDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQRGNRQAATADVNTQGV LPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPSTT FSAAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSVNVDFTVDTNGVY SEPRPIGTRYLTRNL (SEQ ID NO: 13)

[0073] 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, wherein the amino acid residue numbering corresponds to an 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 comprising the sequence of SEQ ID NO: 13.

[0074] In some embodiments, the rAAV particles comprise an AAV2 capsid protein comprising any of the following amino acid sequences inserted between positions 587 and 588 of the capsid protein, wherein the amino acid residue numbering corresponds to an AAV2 VP1 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), LASTGKVPNA (SEQ ID NO: 12), LGETTRP (SEQ ID NO: 14), NETITRP (SEQ ID NO: 15), KAGQANN 24sf-6242343Attorney Docket No.62700-20017.40 (SEQ ID NO: 16), KDPKTTN (SEQ ID NO: 17), KDTDTTR (SEQ ID NO: 18), RAGGSVG (SEQ ID NO: 19), AVDTTKF (SEQ ID NO: 20), STGKVPN (SEQ ID NO: 21), and ISDQTKH (SEQ ID NO: 42). In some embodiments, the rAAV particles comprise an AAV2 capsid protein comprising any of the following amino acid sequences inserted between positions 587 and 588 of the AAV2 VP1 comprising the sequence of SEQ ID NO: 13: 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), STGKVPN (SEQ ID NO: 21), and ISDQTKH (SEQ ID NO: 42).

[0075] In some embodiments, the administration of the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual is by intravitreal (IVT) injection, intraocular administration, or intraretinal injection. In some embodiments, the administration of the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual is by intravitreal (IVT) injection.

[0076] In some embodiments, the unit dose of rAAV particles is in a pharmaceutical formulation. In some embodiments, the pharmaceutical formulation comprises the rAAV particles, one or more osmotic or ionic strength agents, one or more buffering agents, one or more surfactants, and one or more solvents. In some embodiments, the osmotic or ionic strength agent is sodium chloride. In some embodiments, the one or more buffering agents are sodium phosphate monobasic and / or sodium phosphate dibasic. In some embodiments, the surfactant is Poloxamer 188. In some embodiments, the solvent is water. 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 1×1010vg / mL to about 1×1013vg / mL of rAAV particles. In some embodiments, the pharmaceutical formulation comprises about 6×1011vg / mL to about 6×1012vg / mL of rAAV particles. In some embodiments, the pharmaceutical formulation comprises about 150 mM to about 200 mM sodium chloride (e.g., any of 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 monobasic sodium phosphate (e.g., about 1mM, 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 25sf-6242343Attorney Docket No.62700-20017.40 mM to about 10 mM dibasic sodium phosphate (e.g., about 1mM, 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., any of 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., any of 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 ×1012vg / mL of rAAV particles, about 180 mM sodium chloride, about 5 mM monobasic sodium phosphate, about 5 mM dibasic sodium phosphate, and about 0.001% (w / v) poloxamer 188, wherein the pharmaceutical formulation has a pH of about 7.3, and wherein the pH is adjusted with NaOH to be about a pH of about 7.3. In some embodiments, the pharmaceutical formulation comprises about 6 ×1011vg / mL of rAAV particles, about 180 mM sodium chloride, about 5 mM monobasic sodium phosphate, about 5 mM dibasic sodium phosphate, and about 0.001% (w / v) poloxamer 188, wherein the pharmaceutical formulation has a pH of about 7.3, and wherein the pH is adjusted with NaOH to be about a pH of about 7.3. In some embodiments, the pharmaceutical formulation comprises about 1.2 ×1012vg / mL of rAAV particles, about 180 mM sodium chloride, about 5 mM monobasic sodium phosphate, about 5 mM dibasic sodium phosphate, and about 0.001% (w / v) poloxamer 188, wherein the pharmaceutical formulation has a pH of about 7.3, and wherein the pH is adjusted with NaOH to be about a pH of about 7.3. In some embodiments, the pharmaceutical formulation comprises about 180 mM sodium chloride, about 1.8 mM monobasic sodium phosphate, about 8.2 mM dibasic sodium phosphate, about 1.2 × 1012vg / mL of the rAAV particles, and about 0.001% (w / v) poloxamer 188, wherein the pharmaceutical formulation has a pH of about 7.

[0077] In some embodiments, the unit dose of rAAV particles comprises a volume of between about 25 μL to 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 the unit dose of rAAV particles administered to an eye of the individual is between 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 administered to the one eye and / or to the contralateral eye comprises a 26sf-6242343Attorney Docket No.62700-20017.40 volume of about 50 μL. In some embodiments, the unit dose of rAAV particles comprises a volume of about 30 μL.

[0078] In some embodiments, the unit dose of rAAV particles is administered in combination with steroid treatment. In some embodiments, the steroid treatment is a corticosteroid 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., a drop). In some embodiments, the topical steroid treatment is a difluprednate treatment, a medrysone treatment, a loteprednol treatment, a prednisolone treatment, a fluocinolone treatment, a triamcinolone treatment, a rimexolone treatment, a dexamethasone treatment, a fluorometholone treatment, a fluocinolone treatment, a rimexolone treatment, or a prednisone treatment. In some embodiments, the topical steroid treatment is a difluprednate treatment. In some embodiments, the steroid treatment is administered before, during, and / or after administration of the unit dose of rAAV particles. In some embodiments, the steroid treatment is administered before administration of the unit dose of rAAV particles. In some embodiments, the steroid treatment is administered during administration of the unit dose of rAAV particles. In some embodiments, the steroid treatment is administered after administration of the unit dose of rAAV particles. In some embodiments, the steroid treatment is administered before and during administration of the unit dose of rAAV particles. In some embodiments, the steroid treatment is administered before and after administration of the unit dose of rAAV particles. In some embodiments, the steroid treatment is administered during, and after administration of the unit dose of rAAV particles. In some embodiments, the steroid treatment is administered before, during, and after administration of the unit dose of rAAV particles.

[0079] In some embodiments, the steroid treatment is an ophthalmic steroid treatment (e.g., difluprednate). In some embodiments, the ophthalmic steroid treatment (e.g., difluprednate) comprises (a) about four administrations of the topical steroid per day daily for about 6 weeks, (b) followed by about three administrations of the topical steroid per day daily for about 5 weeks, (c) followed by about two administrations of the topical steroid per day daily for about 12 weeks, and (d) followed by about one administration of the topical steroid per day daily for about 4 weeks, wherein an initial topical steroid is administered on Day 1 and the unit dose of rAAV particles is administered on any one of Day 4, Day 5, Day 6, Day 7, Day 8, Day 9, Day 10, or Day 11. In some embodiments, the ophthalmic steroid treatment (e.g., difluprednate) further comprises one administration of the topical steroid every other day for about 2 weeks following step (ii)(d). In some embodiments, the ophthalmic steroid is about 0.005% to about 0.5% difluprednate. In some embodiments, the ophthalmic steroid is any of about 0.005%, about 27sf-6242343Attorney Docket No.62700-20017.40 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 difluprednate 0.05%. In some embodiments, a dose of difluprednate 0.05% 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, a 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, a dose of difluprednate comprises about 2.5 µg difluprednate.

[0080] 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 25 weeks, up to 26 weeks, up to 27 weeks, up to 28 weeks, up to 29 weeks, up to 30 weeks, up to 31 weeks, up to 32 weeks, up to 33 weeks, up to 34 weeks, or up to 35 weeks, wherein the topical steroid treatment is administered on Day 1 and the unit dose of rAAV particles is administered on any one of Day 4, Day 5, Day 6, Day 7, Day 8, Day 9, Day 10, or Day 11. In some embodiments, the ophthalmic steroid treatment (e.g., difluprednate) is a daily topical steroid treatment for at least 25 weeks, at least 26 weeks, at least 27 weeks, at least 28 weeks, at least 29 weeks, at least 30 weeks, at least 31 weeks, at least 32 weeks, at least 33 weeks, at least 34 weeks, or at least 35 weeks, wherein the topical steroid treatment is administered on Day 1 and the unit dose of rAAV particles is administered on any one of Day 4, Day 5, Day 6, Day 7, Day 8, Day 9, Day 10, or Day 11. In some embodiments, the topical steroid treatment begins prior to the administration of the single unit dose of rAAV to the one eye, wherein the topical steroid treatment comprises (a) about four administrations of the topical steroid per day daily (i.e., QID) for about 6 weeks, (b) followed by about three administrations of the topical steroid per day daily (i.e., TID) for about 5 weeks, (c) followed by about two administrations of the topical steroid per day daily (i.e., BID) for about 12 weeks, and (d) followed by about one administration of the topical steroid per day daily (i.e., QD) for about 4 weeks. In some embodiments, the topical steroid treatment further comprises about one administration of the topical steroid every other day (i.e., QOD) for about 2 weeks following step (d). In some embodiments, the dosing timeline of the rAAV particles, topical steroid, the anti- VEGF agent loading dose(s), supplemental aflibercept, and / or sham injection is based on the administration timeline shown in FIG.4. In some embodiments, the topical steroid comprises difluprednate 0.05% at a dose of about 1µg to about 3 µg. In some embodiments, the topical steroid comprises difluprednate 0.05% at a dose of about 2.5µg. In some embodiments, the topical steroid is about 0.005% to about 0.5% difluprednate. In some embodiments, the topical 28sf-6242343Attorney Docket No.62700-20017.40 steroid is any of 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 difluprednate 0.05%. In some embodiments, a dose of difluprednate 0.05% 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, a 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, a dose of difluprednate comprises about 2.5 µg difluprednate.

[0081] In some embodiments, the retinal fluid in the eye of the individual (e.g., SRF and / or IRF) is reduced by more 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 the unit dose of rAAV particles to the one eye and / or to the contralateral of the individual. In some embodiments, the retinal fluid in the eye of the individual (e.g., SRF and / or IRF) is reduced by more 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 the unit dose of rAAV particles to the one eye and / or to the contralateral of the individual compared to the level of retinal fluid in the eye of the individual prior to administration of the unit dose or rAAV particles. In some embodiments, the retinal fluid in the eye of the individual (e.g., SRF and / or IRF) is reduced by about 100% after administration of the unit dose of rAAV particles to the one eye and / or to the contralateral of the individual compared to the level of retinal fluid in the eye of the individual prior to administration of the unit dose or rAAV particles.

[0082] In some embodiments, the methods provided herein further comprise monitoring the level of retinal fluid (e.g., SRF and / or IRF) in the one eye and / or the contralateral eye of the individual after administration of the unit dose of rAAV particles. In some embodiments, the reduction of retinal fluid (e.g., SRF and / or IRF) in the eye is first observed any of 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 more after administration of the unit dose of rAAV particles. In some embodiments, the reduction of retinal fluid (e.g., SRF and / or IRF) in the eye continues or is maintained for at least 1 week, at least 2 weeks, at least 4 weeks, at least 6 weeks, at least 8 29sf-6242343Attorney Docket No.62700-20017.40 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 more after administration of the unit dose of rAAV particles.

[0083] In some embodiments, the reduction of retinal fluid (e.g., SRF and / or IRF) in the eye is determined by any method known in the art. In some embodiments, the reduction of retinal fluid (e.g., SRF and / or IRF) in the eye is determined by optical coherence tomography (OCT), spectral domain OCT (SD-OCT), OCT angiography, fluorescein angiography, or by direct retinal observation. In some embodiments, the reduction of retinal fluid (e.g., SRF and / or IRF) in the eye is determined by optical coherence tomography (OCT). In some embodiments, the reduction of retinal fluid (e.g., SRF and / or IRF) in the eye is determined by spectral domain OCT (SD- OCT). In some embodiments, the reduction of retinal fluid (e.g., SRF and / or IRF) in the eye is determined by OCT angiography. In some embodiments, the reduction of retinal fluid (e.g., SRF and / or IRF) in the eye is determined by fluorescein angiography. In some embodiments, the reduction of retinal fluid (e.g., SRF and / or IRF) in the eye is determined by direct retinal observation.

[0084] In some embodiments, treatment of an ocular neovascular disease in an individual after administration of the unit dose of rAAV particles to the one eye and / or the contralateral eye is assessed based on the level of retinal fluid (e.g., intraretinal fluid (IRF) and / or subretinal fluid (SRF)) compared the level of retinal fluid (e.g., SRF and / or IRF) prior to administration of the unit dose of rAAV particles to the 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, treatment of an ocular neovascular disease in an individual after administration of the unit dose of rAAV particles to the one eye and / or the contralateral eye is determined if a reduction in retinal fluid (e.g., IRF and / or SRF) is observed after administration of the unit dose of rAAV particles to the one eye and / or the contralateral eye compared to the levels of retinal fluid (e.g., IRF and / or SRF) prior to administration of the unit dose of rAAV particles in the one eye and / or the contralateral eye (e.g., as described above). In some embodiments, the ocular neovascular disease is wAMD.

[0085] In some embodiments, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in an improved mean change from 30sf-6242343Attorney Docket No.62700-20017.40 baseline. In some embodiments, the baseline is an initial measurement or condition serving as a reference point for comparison of the measurement or condition over time. In some embodiments, the baseline for key endpoint measurements is taken prior to the administration of aflibercept at Screening Visit 1 (Day -56 to Day -49). In some embodiments, the baseline for best-corrected visual acuity (BCVA) is measured prior to the administration of aflibercept at Screening Visit 1 (Day -56 to Day -49). In some embodiments, the baseline for mean BCVA is measured prior to the administration of aflibercept at Screening Visit 1 (Day -56 to Day -49). In some embodiments, the baseline for central subfield thickness (CST) is measured prior to the administration of aflibercept at Screening Visit 1 (Day -56 to Day -49). In some embodiments, the baseline for mean CST is calculated from data collected prior to the administration of aflibercept at Screening Visit 1 (Day -56 to Day -49). In some embodiments, the percentage of participants with CST ≤ 300 is calculated from data collected prior to the administration of aflibercept at Screening Visit 1 (Day -56 to Day -49). In some embodiments, the mean number of CST fluctuations > 50 μm is calculated from data collected prior to the administration of aflibercept at Screening Visit 1 (Day -56 to Day -49). In some embodiments, the percentage of participants with CST fluctuations > 50 μm is calculated from data collected prior to the administration of aflibercept at Screening Visit 1 (Day -56 to Day -49). In some embodiments, the baseline for the area of choroidal neovascularization (CNV) lesion is measured prior to the administration of aflibercept at Screening Visit 1 (Day -56 to Day -49). In some embodiments, the baseline for macular volume is measured prior to the administration of aflibercept at Screening Visit 1 (Day -56 to Day -49). In some embodiments, the baseline percentage of participants without intraretinal fluid (IRF) is calculated from data collected prior to the administration of aflibercept at Screening Visit 1 (Day -56 to Day -49). In some embodiments, the baseline percentage of participants without subretinal fluid (SRF) is calculated from data collected prior to the administration of aflibercept at Screening Visit 1 (Day -56 to Day -49). In some embodiments, the baseline percentage of participants without IRF and / or SRF is calculated from data collected prior to the administration of aflibercept at Screening Visit 1 (Day -56 to Day -49). In some embodiments, the baseline mean in patient-reported outcome (NEI VFQ-25) total and subscale scores is calculated from data collected prior to the administration of aflibercept at Screening Visit 1 (Day -56 to Day -49). In some embodiments, the baseline least squares is measured prior to the administration of aflibercept at Screening Visit 1 (Day -56 to Day -49). In some embodiments, the baseline least squares mean is calculated from data collected prior to the administration of aflibercept at Screening Visit 1 (Day -56 to Day -49). In some embodiments, Day -56 is 56 days prior to the initial administration of a topical steroid treatment (e.g., 31sf-6242343Attorney Docket No.62700-20017.40 difluprednate) on Day 1 and / or an anti-VEGF agent (e.g., aflibercept protein) on Day 1. In some embodiments, Day -49 is 49 days prior to the initial administration of a topical steroid treatment (e.g., difluprednate) on Day 1 and / or an anti-VEGF agent (e.g., aflibercept protein) on Day 1.

[0086] In some embodiments, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in an improved mean change from baseline in best-corrected visual acuity (BCVA) based on an average BCVA at Weeks 52 and 56 compared to the mean change from baseline in BCVA of an individual administered aflibercept to the one eye and / or to the contralateral eye of the individual. In some embodiments, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in a decrease in mean number of aflibercept IVT injections received from Week 4 to Week 56 compared to the mean number of aflibercept IVT injections received from Week 4 to Week 56 of an individual administered aflibercept to the one eye and / or to the contralateral eye of the individual. In some embodiments, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in a greater percentage of individuals who gain at least 5, 10, or 15 letters in BCVA from baseline compared to each post-baseline visit through Week 56, compared to the corresponding percentage for individuals administered aflibercept to the one eye and / or to the contralateral eye of the individuals. In some embodiments, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in an improved mean change from baseline to each post-baseline visit through Week 56 compared to the mean change from baseline to each post-baseline visit through Week 56 of an individual administered aflibercept to the one eye and / or to the contralateral eye of the individual. In some embodiments, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in a greater number and percentage of individuals with BCVA of 73 letters or more at each visit starting at Week 4, compared to the corresponding number and corresponding percentage for individuals administered aflibercept to the one eye and / or to the contralateral eye of the individuals. In some embodiments, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in a decrease in mean change in CST from baseline to Week 56 compared to the mean change in CST from baseline to Week 56 of an individual administered aflibercept to the one eye and / or to the contralateral eye of the individual. In some embodiments, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in a decrease in mean change in CST from baseline to each post-baseline visit compared to the mean change in CST from baseline to each post-baseline visit of an individual administered aflibercept to the one eye and / or to the 32sf-6242343Attorney Docket No.62700-20017.40 contralateral eye of the individual. In some embodiments, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in a greater percentage of individuals with CST ≤ 300 μm from baseline to each post-baseline visit through Week 56 compared to the corresponding percentage for individuals administered aflibercept to the one eye and / or to the contralateral eye of the individuals. In some embodiments, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in a decrease in the mean number of CST flucutations > 50 μm from baseline over time through Week 56 compared to the mean number of CST flucutations > 50 μm from baseline over time through Week 56 of an individual administered aflibercept to the one eye and / or to the contralateral eye of the individual. In some embodiments, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results a lower percentage of individuals with CST fluctuations > 50 μm from baseline over time through Week 56 compared to the corresponding percentage for individuals administered aflibercept to the one eye and / or to the contralateral eye of the individuals. In some embodiments, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results a higher percent reduction in mean rate of annualized anti-VEGF injections after 1 year of study treatment relative to the annualized number of anti-VEGF injections received in the year prior to screen compared to the corresponding percentage for individuals administered aflibercept to the one eye and / or to the contralateral eye of the individuals. In some embodiments, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in a higher percentage of individuals who are supplemental aflibercept injection-free from Week 4 through Week 56 compared to the corresponding percentage for individuals administered aflibercept to the one eye and / or to the contralateral eye of the individuals. In some embodiments, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in a higher percentage of individuals who received 0 or 1 supplemental aflibercept injection from Week 4 through Week 56 compared to the corresponding percentage for individuals administered aflibercept to the one eye and / or to the contralateral eye of the individuals. In some embodiments, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in a decreased mean change in area of choroidal neovascularization (CNV) lesion from baseline to week 56 compared to the corresponding mean change for individuals administered aflibercept to the one eye and / or to the contralateral eye of the individuals. In some embodiments, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in a decreased mean change in 33sf-6242343Attorney Docket No.62700-20017.40 macular volume from baseline to Week 56 compared to the corresponding mean change for individuals administered aflibercept to the one eye and / or to the contralateral eye of the individuals. In some embodiments, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in a greater percentage of individuals without intraretinal fluid (IRF) from baseline to each post-baseline visit compared to the corresponding percentage for individuals administered aflibercept to the one eye and / or to the contralateral eye of the individuals. In some embodiments, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in a greater percentage of individuals without subretinal fluid (SRF) from baseline to each post-baseline visit compared to the corresponding percentage for individuals administered aflibercept to the one eye and / or to the contralateral eye of the individuals. In some embodiments, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in a greater percentage of individuals without IRF and / or SRF from baseline to each post- baseline visit compared to the corresponding percentage for individuals administered aflibercept to the one eye and / or to the contralateral eye of the individuals. In some embodiments, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in decreased time to no IRF and SRF compared to the corresponding time for individuals administered aflibercept to the one eye and / or to the contralateral eye of the individuals. In some embodiments, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in increased time to sutain dry retina (defined as no IRF or SRF, maintained for 2 consecutive visits) compared to the corresponding time for individuals administered aflibercept to the one eye and / or to the contralateral eye of the individuals. In some embodiments, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in decreased incidence, severity, duration, and type of ocular and non-ocular adverse events compared to the corresponding incidence, severity, duration, and type of ocular and non-ocular adverse events for individuals administered aflibercept to the one eye and / or to the contralateral eye of the individuals. In some embodiments, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in improved mean change in patient-reported outcome (25-Item National Eye Institute Visual Function Questionnaire [NEI VFQ-25]) total and subscale scores from baseline to Weeks 28 and 56 compared to the corresponding mean change for individuals administered aflibercept to the one eye and / or to the contralateral eye of the individuals. 34sf-6242343Attorney Docket No.62700-20017.40

[0087] In some embodiments, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in maintenance or a decrease of retinal thickness compared to the retinal thickness prior to administration of the unit dose of rAAV particles. In some embodiments, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in a decrease of retinal thickness compared to the retinal thickness prior to administration of the unit dose of rAAV particles. In some embodiments, retinal thickness is central subfield thickness (CST) or central retinal thickness (CRT). In some embodiments, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in a decrease of retinal thickness of more 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 prior to 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.

[0088] In some embodiments, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in a decrease of central retinal thickness (CRT) or central subfield thickness (CST) of about 10 μm to about 100 μm (e.g., more than 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, or more). In some embodiments, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in a decrease of central retinal thickness (CRT) or central subfield thickness (CST) of about 5 μm to about 50 μm (e.g., any of 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, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in a decrease of central retinal thickness (CRT) or central subfield thickness (CST) of about 5 μm to about 40 μm (e.g., any of 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, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in a decrease of central retinal thickness (CRT) or central subfield 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 35sf-6242343Attorney Docket No.62700-20017.40 26 μm, about 27 μm, about 28 μm, about 29 μm, or about 30 μm). In some embodiments, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in a decrease of central retinal thickness (CRT) or central subfield 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, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in a decrease of central retinal thickness (CRT) or central subfield 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, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in a decrease of central retinal thickness (CRT) or central subfield thickness (CST) of between about 18 μm to about 75 μm. In some embodiments, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in a decrease of central retinal thickness (CRT) or central subfield thickness (CST) of about 18.5 μm. In some embodiments, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in a decrease of central retinal thickness (CRT) or central subfield thickness (CST) of about 21.0 μm. In some embodiments, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in a decrease of central retinal thickness (CRT) or central subfield thickness (CST) of about 8.3 μm. In some embodiments, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in a decrease of central retinal thickness (CRT) or central subfield thickness (CST) of about 25.5 μm. In some embodiments, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in a decrease of central retinal thickness (CRT) or central subfield thickness (CST) of about 24.8 μm. In some embodiments, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in a decrease of central retinal thickness (CRT) or central subfield thickness (CST) of about 75 μm. In some embodiments, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in a decrease of central retinal thickness (CRT) or central subfield thickness (CST) of about 100 μm or more (e.g., any of about 100 μm or more, about 110 μm or more, about 120 μm or more, about 130 μm or more, about 140 μm or more, 36sf-6242343Attorney Docket No.62700-20017.40 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, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in a decrease of central retinal thickness (CRT) or central subfield thickness (CST) of between 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, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in a decrease of central retinal thickness (CRT) or central subfield thickness (CST) of between 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, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in a decrease of central retinal thickness (CRT) or central subfield thickness (CST) of between 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, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in a decrease of central retinal thickness (CRT) or central subfield thickness (CST) of between 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, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in a decrease of central retinal thickness (CRT) or central subfield thickness (CST) of between 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, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in a decrease of central retinal thickness (CRT) or central subfield thickness (CST) of between 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, the administering the unit dose of rAAV 37sf-6242343Attorney Docket No.62700-20017.40 particles to the one eye and / or to the contralateral eye of the individual results in a decrease of central retinal thickness (CRT) or central subfield thickness (CST) of between 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, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in a decrease of central retinal thickness (CRT) or central subfield thickness (CST) of between 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, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in a decrease of central retinal thickness (CRT) or central subfield thickness (CST) of between 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, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in a decrease of central retinal thickness (CRT) or central subfield thickness (CST) of between 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, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in a decrease of central retinal thickness (CRT) or central subfield thickness (CST) of between 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, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in a decrease of central retinal thickness (CRT) or central subfield thickness (CST) of between 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, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in a decrease of central retinal thickness (CRT) or central subfield thickness (CST) of between about 135 μm to about 200 μm (e.g., any 38sf-6242343Attorney Docket No.62700-20017.40 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, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in a decrease of central retinal thickness (CRT) or central subfield thickness (CST) of between about 140 μm to about 200 μm (e.g., any of 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, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in a decrease of central retinal thickness (CRT) or central subfield thickness (CST) of between about 145 μm to about 200 μm (e.g., any of 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, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in a decrease of central retinal thickness (CRT) or central subfield thickness (CST) of between 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, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in a decrease of central retinal thickness (CRT) or central subfield thickness (CST) of between 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, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in a decrease of central retinal thickness (CRT) or central subfield thickness (CST) of between 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, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in a decrease of central retinal thickness (CRT) or central subfield thickness (CST) of between 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, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in a decrease of central retinal thickness (CRT) or central subfield thickness (CST) of between about 170 μm to about 200 μm (e.g., any of about 170 μm, about 175 μm, about 180 μm, about 185 μm, about 190 μm, 39sf-6242343Attorney Docket No.62700-20017.40 about 195 μm, or about 200 μm). In some embodiments, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in a decrease of central retinal thickness (CRT) or central subfield thickness (CST) of between about 175 μm to about 200 μm (e.g., any of about 175 μm, about 180 μm, about 185 μm, about 190 μm, about 195 μm, or about 200 μm). In some embodiments, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in a decrease of central retinal thickness (CRT) or central subfield thickness (CST) of between 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, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in a decrease of central retinal thickness (CRT) or central subfield thickness (CST) of about 21.0 μm compared to the central retinal thickness (CRT) or central subfield thickness (CST) prior to administration of the unit dose of rAAV particles. In some embodiments, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in a decrease of central retinal thickness (CRT) or central subfield thickness (CST) of about 8.3 μm compared to the central retinal thickness (CRT) or central subfield thickness (CST) prior to administration of the unit dose of rAAV particles. In some embodiments, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in a decrease of central retinal thickness (CRT) or central subfield thickness (CST) of about 26.2 μm compared to the central retinal thickness (CRT) or central subfield thickness (CST) prior to administration of the unit dose of rAAV particles. In some embodiments, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in a decrease of central retinal thickness (CRT) or central subfield thickness (CST) of about 24.8 μm compared to the central retinal thickness (CRT) or central subfield thickness (CST) prior to administration of the unit dose of rAAV particles. In some embodiments, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in a decrease of central retinal thickness (CRT) or central subfield thickness (CST) of about 40.8 μm compared to the central retinal thickness (CRT) or central subfield thickness (CST) prior to administration of the unit dose of rAAV particles. In some embodiments, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in a decrease of central retinal thickness (CRT) or central subfield thickness (CST) of about 30.0 μm compared to the central retinal thickness (CRT) or central subfield thickness (CST) prior to administration of the unit dose of rAAV particles. In some embodiments, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in a decrease of 40sf-6242343Attorney Docket No.62700-20017.40 central retinal thickness (CRT) or central subfield thickness (CST) of about 118.6 μm compared to the central retinal thickness (CRT) or central subfield thickness (CST) prior to administration of the unit dose of rAAV particles. In some embodiments, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in a decrease of central retinal thickness (CRT) or central subfield thickness (CST) of about 119.0 μm compared to the central retinal thickness (CRT) or central subfield thickness (CST) prior to administration of the unit dose of rAAV particles. In some embodiments, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in a decrease of central retinal thickness (CRT) or central subfield thickness (CST) of about 137.8 μm compared to the central retinal thickness (CRT) or central subfield thickness (CST) prior to administration of the unit dose of rAAV particles. In some embodiments, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in a decrease of central retinal thickness (CRT) or central subfield thickness (CST) of about 152.7 μm compared to the central retinal thickness (CRT) or central subfield thickness (CST) prior to administration of the unit dose of rAAV particles. In some embodiments, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in a decrease of central retinal thickness (CRT) or central subfield thickness (CST) of about 153.3 μm compared to the central retinal thickness (CRT) or central subfield thickness (CST) prior to administration of the unit dose of rAAV particles. In some embodiments, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in a decrease of central retinal thickness (CRT) or central subfield thickness (CST) of about 149.8 μm compared to the central retinal thickness (CRT) or central subfield thickness (CST) prior to administration of the unit dose of rAAV particles.

[0089] In some embodiments, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in a change in the central retinal thickness (CRT) or central subfield thickness (CST) of between about -200 μm to about +40 μm (e.g., any 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 prior to administration of the unit dose of rAAV particles. In some embodiments, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in a decrease in the central retinal thickness (CRT) or central subfield thickness (CST) of any of about 8 μm, about 11 μm, about 16 μm, about 29 41sf-6242343Attorney Docket No.62700-20017.40 μm, about 33 μm, about 38 μm, about 55 μm, about 61 μm, or about 117 μm. In some embodiments, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in a decrease in the central retinal thickness (CRT) or central subfield thickness (CST) of any of about 27.8 μm or about 30.8 μm. In some embodiments, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in an increase in the central retinal thickness (CRT) or central subfield thickness (CST) of any of about 4 μm, about 12 μm, or about 32 μm. In some embodiments, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in a change in the central retinal thickness (CRT) or central subfield thickness (CST) of about -21.0 μm compared to the central retinal thickness (CRT) or central subfield thickness (CST) prior to administration of the unit dose of rAAV particles. In some embodiments, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in a change in the central retinal thickness (CRT) or central subfield thickness (CST) of about -8.3 μm compared to the central retinal thickness (CRT) or central subfield thickness (CST) prior to administration of the unit dose of rAAV particles. In some embodiments, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in a change in the central retinal thickness (CRT) or central subfield thickness (CST) of about -26.2 μm compared to the central retinal thickness (CRT) or central subfield thickness (CST) prior to administration of the unit dose of rAAV particles. In some embodiments, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in a change in the central retinal thickness (CRT) or central subfield thickness (CST) of about -24.8 μm compared to the central retinal thickness (CRT) or central subfield thickness (CST) prior to administration of the unit dose of rAAV particles. In some embodiments, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in a change in the central retinal thickness (CRT) or central subfield thickness (CST) of about -40.8 μm compared to the central retinal thickness (CRT) or central subfield thickness (CST) prior to administration of the unit dose of rAAV particles. In some embodiments, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in a change in the central retinal thickness (CRT) or central subfield thickness (CST) of about -30.0 μm compared to the central retinal thickness (CRT) or central subfield thickness (CST) prior to administration of the unit dose of rAAV particles. In some embodiments, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in a change in the central retinal thickness (CRT) or central subfield thickness (CST) of about - 42sf-6242343Attorney Docket No.62700-20017.40 118.6 μm compared to the central retinal thickness (CRT) or central subfield thickness (CST) prior to administration of the unit dose of rAAV particles. In some embodiments, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in a change in the central retinal thickness (CRT) or central subfield thickness (CST) of about -119.0 μm compared to the central retinal thickness (CRT) or central subfield thickness (CST) prior to administration of the unit dose of rAAV particles. In some embodiments, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in a change in the central retinal thickness (CRT) or central subfield thickness (CST) of about -137.8 μm compared to the central retinal thickness (CRT) or central subfield thickness (CST) prior to administration of the unit dose of rAAV particles. In some embodiments, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in a change in the central retinal thickness (CRT) or central subfield thickness (CST) of about -152.7 μm compared to the central retinal thickness (CRT) or central subfield thickness (CST) prior to administration of the unit dose of rAAV particles. In some embodiments, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in a change in the central retinal thickness (CRT) or central subfield thickness (CST) of about -153.3 μm compared to the central retinal thickness (CRT) or central subfield thickness (CST) prior to administration of the unit dose of rAAV particles. In some embodiments, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in a change in the central retinal thickness (CRT) or central subfield thickness (CST) of about -149.8 μm compared to the central retinal thickness (CRT) or central subfield thickness (CST) prior to administration of the unit dose of rAAV particles.

[0090] In some embodiments, the change (e.g., the decrease) in the central retinal thickness (CRT) or central subfield thickness (CST) compared to the central retinal thickness (CRT) or central subfield thickness (CST) prior to administration of the unit dose of rAAV particles is present at any of about 1 day, about 1 week, about 2 weeks, about 4 weeks, about 8 weeks, about 16 weeks, about 24 weeks, about 30 weeks, about 32 weeks, about 34 weeks, about 40 weeks, about 44 weeks, about 48 weeks, about 52 weeks, or more after administration of the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual. In some embodiments, the change (e.g., the decrease) in the central retinal thickness (CRT) or central subfield thickness (CST) compared to the central retinal thickness (CRT) or central subfield thickness (CST) prior to administration of the unit dose of rAAV particles is present at any of about 1 day, about 1 week, about 2 weeks, about 4 weeks, about 8 weeks, about 12 weeks, about 43sf-6242343Attorney Docket No.62700-20017.40 16 weeks, about 20 weeks, about 24 weeks, about 28 weeks, about 32 weeks, about 36 weeks, about 40 weeks, about 44 weeks, about 48 weeks, about 52 weeks, about 56 weeks, about 60 weeks, about 64 weeks, about 68 weeks, about 72 weeks, about 76 weeks, about 80 weeks, about 84 weeks, about 88 weeks, about 92 weeks, about 96 weeks, about 100 weeks, about 104 weeks, about 108 weeks, or more after administration of the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual.

[0091] In some embodiments, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in a decrease of central retinal thickness (CRT) or central subfield thickness (CST) of more 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 prior to administration of the unit dose of rAAV particles. In some embodiments, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in a decrease of central retinal thickness (CRT) or central subfield thickness (CST) of at least about 10% compared to the retinal thickness prior to administration of the unit dose of rAAV particles. In some embodiments, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in a decrease of central retinal thickness (CRT) or central subfield thickness (CST) of about 15% or more compared to the retinal thickness prior to administration of the unit dose of rAAV particles.

[0092] In some embodiments, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in an increase of central retinal thickness (CRT) or central subfield thickness (CST) of less than about 40 μm (e.g., any of 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) compared to the retinal thickness prior to administration of the unit dose of rAAV particles. In some embodiments, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in an increase of central retinal thickness (CRT) or central subfield thickness (CST) of about 32 μm or less compared to the retinal thickness prior to administration of the unit dose of rAAV particles.

[0093] In some embodiments, the maintenance, the decrease, or the increase of retinal thickness compared to the retinal thickness prior to administration of the unit dose of rAAV particles is present at about 30 weeks or more after administration of the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual. In some embodiments, the maintenance, 44sf-6242343Attorney Docket No.62700-20017.40 the decrease, or the increase of retinal thickness compared to the retinal thickness prior to administration of the unit dose of rAAV particles is present at any of about 30 weeks, about 34 weeks, about 44 weeks, about 6 months, about 1 year, about 1.5 years, about 2 years, about 3 years, about 5 years, about 10 years, or more, after administration of the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual.

[0094] In some embodiments, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in maintenance or a decrease in macular volume compared to the macular volume prior to administration of the unit dose of rAAV particles. In some embodiments, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in a decrease in macular volume compared to the macular volume prior to administration of the unit dose of rAAV particles. In some embodiments, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in a decrease in macular volume of more than any of about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, or about 50% compared to the macular volume prior to administration of the unit dose of rAAV particles. In some embodiments, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in a decrease in macular volume of at least about 10% compared to the macular volume prior to administration of the unit dose of rAAV particles. In some embodiments, the macular volume is determined by OCT or SD-OCT. In some embodiments, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in a decrease in macular volume of at least about 10% compared to the macular volume prior to administration of the unit dose of rAAV particles. In some embodiments, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in a decrease in macular volume of about 15% or more compared to the macular volume prior to administration of the unit dose of rAAV particles. In some embodiments, the macular volume is determined by OCT or SD-OCT.

[0095] In some embodiments, the maintenance or the decrease in macular volume compared to the macular volume prior to administration of the unit dose of rAAV particles is present at about 30 weeks or more after administration of the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual. In some embodiments, the maintenance or the decrease in macular volume compared to the macular volume prior to administration of the unit dose of rAAV particles is present at any of 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, 45sf-6242343Attorney Docket No.62700-20017.40 or more after administration of the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual.

[0096] Best Corrected Visual Acuity (BCVA) is a measure of how well an individual who has any refractive error, such as nearsightedness or farsightedness, can see with corrective lenses. BCVA is measured using a standardized visual acuity chart that consists of different-sized letter or symbols arranged in rows representing a different level of visual acuity. Examples of standardized visual acuity charts include, but are not limited to, Snellen, Random E, LogMAR, Times New Roman, Jaeger, and Early Treatment Diabetic Retinopathy Study charts. When measuring BCVA, an individual is asked to read letters or symbols from the visual acuity chart at a specific distance. BCVA is expressed as a fraction where the numerator represents the distance at which an individual can read the visual acuity chart and the denominator represents the distance at which an individual with normal vision can read the same line on the visual acuity chart.

[0097] In some embodiments, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in maintenance or an improvement of visual acuity compared to the visual acuity prior to administration of the unit dose of rAAV particles. In some embodiments, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in an improvement of visual acuity compared to the visual acuity prior to administration of the unit dose of rAAV particles. In some embodiments, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in an improvement of visual acuity of more than any of about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 125%, about 150%, about 175%, about 200%, about 225%, about 250%, about 275%, about 300%, or more, compared to the visual acuity prior to administration of the unit dose of rAAV particles. In some embodiments, visual acuity is best corrected visual acuity (BCVA). In some embodiments, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in an improvement of BCVA compared to the BCVA prior to 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 correctly read (Vitale et al., (2016) JAMA Opthalmol 134(9):1041:1047).

[0098] In some embodiments, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in an improvement of BCVA of at least 15 ETDRS letters (Vitale et al., (2016) JAMA Opthalmol 134(9):1041:1047) (e.g., at least about 15, at least about 20, at least about 30, at least about 40, at least about 50, at least about 60, or about 46sf-6242343Attorney Docket No.62700-20017.40 70 letters) compared to the BCVA prior to administration of the unit dose of rAAV particles. In some embodiments, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in an improvement of BCVA of between about 1 to about 15 (e.g., any of about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, or about 15) ETDRS letters compared to the BCVA prior to administration of the unit dose of rAAV particles. In some embodiments, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in an improvement of BCVA of about 5 ETDRS letters compared to the BCVA prior to administration of the unit dose of rAAV particles. In some embodiments, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in an improvement of BCVA of any of about 1, about 2, about 3, about 4, about 5, about 6, or about 7 ETDRS letters compared to the BCVA prior to administration of the unit dose of rAAV particles. In some embodiments, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in an improvement of BCVA of about 3 ETDRS letters or more compared to the BCVA prior to administration of the unit dose of rAAV particles. In some embodiments, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in an improvement of BCVA of about 4 ETDRS letters or more compared to the BCVA prior to administration of the unit dose of rAAV particles. In some embodiments, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in an improvement of BCVA of about 5.1 ETDRS letters or more compared to the BCVA prior to administration of the unit dose of rAAV particles. In some embodiments, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in an improvement of BCVA of about 6.4 ETDRS letters or more compared to the BCVA prior to administration of the unit dose of rAAV particles. In some embodiments, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in an improvement of BCVA of about 6.8 ETDRS letters compared to the BCVA prior to administration of the unit dose of rAAV particles. In some embodiments, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in an improvement of BCVA of about 8.8 ETDRS letters compared to the BCVA prior to administration of the unit dose of rAAV particles. In some embodiments, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in an improvement of BCVA of about 2.3 ETDRS letters compared to the BCVA prior to administration of the unit dose of rAAV particles. 47sf-6242343Attorney Docket No.62700-20017.40

[0099] In some embodiments, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in maintenance of BCVA, wherein the individual loses fewer than 15 ETDRS letters (Vitale et al., (2016) JAMA Opthalmol 134(9):1041:1047) (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 the BCVA prior to administration of the unit dose of rAAV particles. In some embodiments, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in maintenance of BCVA, wherein the individual loses about 2 letters compared to the BCVA prior to administration of the unit dose of rAAV particles. In some embodiments, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in maintenance of BCVA, wherein the individual loses any of about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, or about 9 ETDRS letters compared to the BCVA prior to administration of the unit dose of rAAV particles. In some embodiments, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in maintenance of BCVA, wherein the individual loses 0 letters compared to the BCVA prior to administration of the unit dose of rAAV particles. In some embodiments, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in maintenance of BCVA, wherein the individual loses about 1 letter compared to the BCVA prior to administration of the unit dose of rAAV particles. In some embodiments, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in maintenance of BCVA, wherein the individual loses about 2.7 letters compared to the BCVA prior to administration of the unit dose of rAAV particles. In some embodiments, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in maintenance of BCVA, wherein the individual loses about 2.8 letters compared to the BCVA prior to administration of the unit dose of rAAV particles. In some embodiments, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in maintenance of BCVA, wherein the individual loses about 2 letters or less compared to the BCVA prior to administration of the unit dose of rAAV particles. In some embodiments, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in maintenance of BCVA, wherein the individual loses about 3.2 letters or less compared to the BCVA prior to administration of the unit dose of rAAV particles. In some embodiments, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in maintenance of BCVA, wherein the individual loses between about 15 to 48sf-6242343Attorney Docket No.62700-20017.40 about 0 letters (e.g., any of about 15, about 14, about 13, about 12, about 11, about 10, about 9, about 8, about 7, about 6, about 5, about 4, about 3, about 2, about 1, or 0 letters) compared to the BCVA prior to administration of the unit dose of rAAV particles. In some embodiments, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in maintenance of BCVA, wherein the individual loses between about 10 to about 0 letters (e.g., any of about 10, about 9, about 8, about 7, about 6, about 5, about 4, about 3, about 2, about 1, or 0 letters) compared to the BCVA prior to administration of the unit dose of rAAV particles. In some embodiments, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in maintenance of BCVA, wherein the individual loses between about 5 to about 0 letters (e.g., any of about 5, about 4, about 3, about 2, about 1, or 0 letters) compared to the BCVA prior to administration of the unit dose of rAAV particles. In some embodiments, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in maintenance of BCVA, wherein the individual loses between about 4 to about 0 letters (e.g., any of about 4, about 3, about 2, about 1, or 0 letters) compared to the BCVA prior to administration of the unit dose of rAAV particles. In some embodiments, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in maintenance of BCVA, wherein the individual loses between about 3 to about 0 letters (e.g., any of about 3, about 2, about 1, or 0 letters) compared to the BCVA prior to administration of the unit dose of rAAV particles. In some embodiments, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in maintenance of BCVA, wherein the individual loses between about 2 to about 0 letters (e.g., any of about 2, about 1, or 0 letters) compared to the BCVA prior to administration of the unit dose of rAAV particles. In some embodiments, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in maintenance of BCVA, wherein the individual loses between about 1 to about 0 letters compared to the BCVA prior to administration of the unit dose of rAAV particles.

[0100] In some embodiments, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in a change in BCVA of between about - 20 to +7 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) ETDRS letters, compared to the BCVA prior to administration of the unit dose of rAAV particles. In some embodiments, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in an increase in BCVA of 49sf-6242343Attorney Docket No.62700-20017.40 about any of 16, 7 or 5 ETDRS letters compared to the BCVA prior to administration of the unit dose of rAAV particles. In some embodiments, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in a decrease in BCVA of about any of 19, 14, 7, 6, 5, 4, 3, 2, or 1 ETDRS letters compared to the BCVA prior to administration of the unit dose of rAAV particles. In some embodiments, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in a decrease in BCVA of about 4.8 or about 0.8 ETDRS letters compared to the BCVA prior to administration of the unit dose of rAAV particles. In some embodiments, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in a decrease in BCVA of about 2 ETDRS letters or less compared to the BCVA prior to administration of the unit dose of rAAV particles. In some embodiments, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in a decrease in BCVA of about 3.2 ETDRS letters or less compared to the BCVA prior to administration of the unit dose of rAAV particles.

[0101] In some embodiments, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in a change in BCVA of between about - 15 to +7 or more (e.g., any of -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) ETDRS letters, compared to the BCVA prior to administration of the unit dose of rAAV particles. In some embodiments, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in a change in BCVA of between about - 10 to +7 or more (e.g., any of -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) ETDRS letters, compared to the BCVA prior to administration of the unit dose of rAAV particles. In some embodiments, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in a change in BCVA of between about -5 to +7 or more (e.g., any of -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) ETDRS letters, compared to the BCVA prior to administration of the unit dose of rAAV particles. In some embodiments, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in a change in BCVA of between about -4 to +7 or more (e.g., any of -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) ETDRS letters, compared to the BCVA prior to administration of the unit dose of rAAV particles. In some embodiments, the administering the unit dose of rAAV particles to the one eye and / or to 50sf-6242343Attorney Docket No.62700-20017.40 the contralateral eye of the individual results in a change in BCVA of between about -3 to +7 or more (e.g., any of -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) ETDRS letters, compared to the BCVA prior to administration of the unit dose of rAAV particles. In some embodiments, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in a change in BCVA of between about -2 to +7 or more (e.g., any of -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) ETDRS letters, compared to the BCVA prior to administration of the unit dose of rAAV particles. In some embodiments, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in a change in BCVA of between about -1 to +7 or more (e.g., any of -1, 0, +1, +2, +3, +4, +5, +6, +7, +8, +9, +10, +11, +12, +13, +14, +15, +16, +17, +18, +19, +20, or more) ETDRS letters, compared to the BCVA prior to administration of the unit dose of rAAV particles. In some embodiments, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in a change in BCVA of between about 0 to +7 or more (e.g., any of 0, +1, +2, +3, +4, +5, +6, +7, +8, +9, +10, +11, +12, +13, +14, +15, +16, +17, +18, +19, +20, or more) ETDRS letters, compared to the BCVA prior to administration of the unit dose of rAAV particles. In some embodiments, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in a change in BCVA of between about +1 to +7 or more (e.g., any of +1, +2, +3, +4, +5, +6, +7, +8, +9, +10, +11, +12, +13, +14, +15, +16, +17, +18, +19, +20, or more) ETDRS letters, compared to the BCVA prior to administration of the unit dose of rAAV particles. In some embodiments, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in a change in BCVA of between about +2 to +7 or more (e.g., any of +2, +3, +4, +5, +6, +7, +8, +9, +10, +11, +12, +13, +14, +15, +16, +17, +18, +19, +20, or more) ETDRS letters, compared to the BCVA prior to administration of the unit dose of rAAV particles. In some embodiments, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in a change in BCVA of between about +3 to +7 or more (e.g., any of +3, +4, +5, +6, +7, +8, +9, +10, +11, +12, +13, +14, +15, +16, +17, +18, +19, +20, or more) ETDRS letters, compared to the BCVA prior to administration of the unit dose of rAAV particles. In some embodiments, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in a change in BCVA of between about +4 to +7 or more (e.g., any of +4, +5, +6, +7, +8, +9, +10, +11, +12, +13, +14, +15, +16, +17, +18, +19, +20, or more) ETDRS letters, compared to the BCVA prior to administration of the unit dose of rAAV particles. In some 51sf-6242343Attorney Docket No.62700-20017.40 embodiments, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in a change in BCVA of between about +5 to +7 or more (e.g., any of +5, +6, +7, +8, +9, +10, +11, +12, +13, +14, +15, +16, +17, +18, +19, +20, or more) ETDRS letters, compared to the BCVA prior to administration of the unit dose of rAAV particles. In some embodiments, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in a change in BCVA of about +6 or about +7 ETDRS letters, compared to the BCVA prior to administration of the unit dose of rAAV particles.

[0102] In some embodiments, administration of the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in transient inflammation (e.g., inflammation driven by aqueous cells and / or vitreous cells, aqueous flare, posterior synchiae, poor pupil dilation). In some embodiments, administration of the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in inflammation (e.g., inflammation driven by aqueous cells and / or vitreous cells, aqueous flare, posterior synchiae, poor pupil dilation) that is improved after administration of a topical steroid treatment. In some embodiments, administration of the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in inflammation (e.g., inflammation driven by aqueous cells and / or vitreous cells) that resolves after administration of a topical steroid treatment. Inflammation (e.g., inflammation driven by aqueous cells and / or vitreous cells, aqueous flare, posterior synchiae, poor pupil dilation) may be measured using any method known in the art, such as the slit lamp exam.

[0103] In some embodiments, the maintenance or the improvement of visual acuity (e.g., BCVA) compared to the visual acuity prior to administration of the unit dose of rAAV particles is present at any of about 1 day, about 1 week, about 2 weeks, about 4 weeks, about 8 weeks, about 12 weeks, about 16 weeks, about 20 weeks, about 24 weeks, about 28 weeks, about 32 weeks, about 36 weeks, about 40 weeks, about 44 weeks, about 48 weeks, about 52 weeks, about 56 weeks, about 60 weeks, about 64 weeks, about 68 weeks, about 72 weeks, about 76 weeks, about 80 weeks, about 84 weeks, about 88 weeks, about 92 weeks, about 96 weeks, about 100 weeks, about 104 weeks, about 108 weeks, or more, after administration of the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual. In some embodiments, the maintenance or the improvement of visual acuity (e.g., BCVA) compared to the visual acuity prior to administration of the unit dose of rAAV particles is present at about 30 weeks or more after administration of the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual. In some embodiments, the maintenance or the improvement of 52sf-6242343Attorney Docket No.62700-20017.40 visual acuity (e.g., BCVA) compared to the visual acuity prior to administration of the unit dose of rAAV particles is present at any of about 30 weeks, about 34 weeks, about 44 weeks, about 6 months, about 1 year, about 1.5 years, about 2 years, about 3 years, about 5 years, about 10 years, or more, after administration of the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual.

[0104] In some embodiments, treatment of an ocular neovascular disease in an individual after administration of the unit dose of rAAV particles in the one eye and / or the contralateral eye is assessed based on best corrected visual acuity (BCVA) in the one eye and / or the contralateral eye. In some embodiments, BCVA is expressed as an ETDRS score, which corresponds to the number of letters correctly read (Vitale et al., (2016) JAMA Opthalmol 134(9):1041:1047). In some embodiments, an individual is determined to have maintenance of vision and / or visual acuity if the individual loses fewer than 15 letters in an ETDRS score (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 prior to administration of the unit dose of rAAV particles in the one eye and / or the contralateral eye. In some embodiments, an individual is determined to have an improvement of vision and / or visual acuity if the individual gains 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) comparted to prior to administration of the unit dose of rAAV particles in the one eye and / or the contralateral eye.

[0105] In some embodiments, treatment of an ocular neovascular disease in an individual after administration of the unit dose of rAAV particles in the one eye and / or the contralateral eye is assessed based on central subfield thickness (CST) or central retinal thickness (CRT) in the one eye and / or the contralateral eye. In some embodiments, CST or CRT is determined by SD-OCT. In some embodiments, treatment of an ocular neovascular disease in an individual after administration of the unit dose of rAAV particles in the one eye and / or the contralateral eye is determined if the CST or CRT assessed by SD-OCT is decreased after administration of the unit dose of rAAV particles in the one eye and / or the contralateral eye compared to prior to administration of the unit dose of rAAV particles in the one eye and / or the contralateral eye. In some embodiments, treatment of an ocular neovascular disease in an individual after administration of the unit dose of rAAV particles in the one eye and / or the contralateral eye is determined if the CST or CRT assessed by SD-OCT is maintained after administration of the unit dose of rAAV particles in the one eye and / or the contralateral eye compared to prior to administration of the unit dose of rAAV particles in the one eye and / or the contralateral eye. 53sf-6242343Attorney Docket No.62700-20017.40

[0106] In some embodiments, treatment of an ocular neovascular disease in an individual after administration of the unit dose of rAAV particles in the one eye and / or the contralateral eye is assessed based on macular volume in the one eye and / or the contralateral eye. In some embodiments, macular volume is determined by SD-OCT. In some embodiments, treatment of an ocular neovascular disease in an individual after administration of the unit dose of rAAV particles in the one eye and / or the contralateral eye is determined if the macular volume assessed by SD-OCT is decreased after administration of the unit dose of rAAV particles in the one eye and / or the contralateral eye compared to prior to administration of the unit dose of rAAV particles in the one eye and / or the contralateral eye. In some embodiments, treatment of an ocular neovascular disease in an individual after administration of the unit dose of rAAV particles in the one eye and / or the contralateral eye is determined if the macular volume assessed by SD-OCT is maintained after administration of the unit dose of rAAV particles in the one eye and / or the contralateral eye compared to prior to administration of the unit dose of rAAV particles in the one eye and / or the contralateral eye.

[0107] In some embodiments, treatment of an ocular neovascular disease in an individual after administration of the unit dose of rAAV particles in the one eye and / or the contralateral eye is assessed based on retinal thickness (e.g., central retinal thickness (CRT) or central subfield thickness (CST)) and macular volume in the one eye and / or the contralateral eye. In some embodiments, CST and macular volume are determined by SD-OCT. In some embodiments, treatment of an ocular neovascular disease in an individual after administration of the unit dose of rAAV particles in the one eye and / or the contralateral eye is determined if the CST and macular volume assessed by SD-OCT are decreased after administration of the unit dose of rAAV particles in the one eye and / or the contralateral eye compared to prior to administration of the unit dose of rAAV particles in the one eye and / or the contralateral eye. In some embodiments, treatment of an ocular neovascular disease in an individual after administration of the unit dose of rAAV particles in the one eye and / or the contralateral eye is determined if the CST and macular volume assessed by SD-OCT are maintained after administration of the unit dose of rAAV particles in the one eye and / or the contralateral eye compared to prior to administration of the unit dose of rAAV particles in the one eye and / or the contralateral eye.

[0108] In some embodiments, treatment of an ocular neovascular disease in an individual after administration of the unit dose of rAAV particles in the one eye and / or the contralateral eye is assessed based on the number of rescue therapy treatments (e.g., aflibercept injections) required by the individual after administration of the unit dose of rAAV particles in the one eye and / or the contralateral eye. In some embodiments, treatment of an ocular neovascular disease in an 54sf-6242343Attorney Docket No.62700-20017.40 individual after administration of the unit dose of rAAV particles in the one eye and / or the contralateral eye is determined if an individual requires less than one rescue therapy treatment (e.g., aflibercept injection) any of 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 the unit dose of rAAV particles in the one eye and / or the contralateral eye.

[0109] In some embodiments, treatment of an ocular neovascular disease in an individual after administration of the unit dose of rAAV particles in the one eye and / or the contralateral eye is determined if an individual does not require any rescue therapy treatment (e.g., aflibercept injection) for any of at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, at least 7 weeks, at least 8 weeks, at least 9 weeks, at least 10 weeks, at least 15 weeks, at least 20 weeks, at least 30 weeks, at least 40 weeks, at least 50 weeks, at least 60 weeks, at least 70 weeks, at least 80 weeks, at least 90 weeks, at least 100 weeks, at least 110 weeks, or more, after administration of the unit dose of rAAV particles in the one eye and / or the contralateral eye.

[0110] In some embodiments, the individual does not require any rescue therapy treatment (e.g., aflibercept injection) 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 the unit dose of rAAV particles in the 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 12 months after administration of the unit dose of rAAV particles in the 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 10 months after administration of the unit dose of rAAV particles in the 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 7 months after administration of the unit dose of rAAV particles in the 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 6 months after administration of the unit dose of rAAV particles in the one eye and / or the contralateral eye. In some embodiments, the individual does not require 55sf-6242343Attorney Docket No.62700-20017.40 any rescue therapy treatment (e.g., aflibercept injection) for at least about 2 months after administration of the unit dose of rAAV particles in the 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 1 month after administration of the unit dose of rAAV particles in the one eye and / or the contralateral eye.

[0111] In some embodiments, administration of a single unit dose of rAAV particles to the one eye and / or to the 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 in the plurality not requiring an anti- VEGF rescue treatment (e.g., aflibercept injection). In some embodiments, administration of a single unit dose of rAAV particles to the one eye and / or to the 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 an anti-VEGF rescue treatment (e.g., aflibercept injection). In some embodiments, administration of a single unit dose of rAAV particles to the one eye and / or to the contralateral eye of a plurality of individuals results in at least about 50% of the individuals in the plurality not requiring an anti-VEGF rescue treatment (e.g., aflibercept injection). In some embodiments, administration of a single unit dose of rAAV particles to the one eye and / or to the contralateral eye of a plurality of individuals results in at least about 78% of the individuals in the plurality not requiring an anti-VEGF rescue treatment (e.g., aflibercept injection). In some embodiments, administration of a single unit dose of rAAV particles to the one eye and / or to the contralateral eye of a plurality of individuals results in at least about 80% of the individuals in the plurality not requiring an anti-VEGF rescue treatment (e.g., aflibercept injection). In some embodiments, administration of a single unit dose of rAAV particles to the one eye and / or to the contralateral eye of a plurality of individuals results in at least about 82% of the individuals in the plurality not requiring an anti-VEGF rescue treatment (e.g., aflibercept injection). In some embodiments, administration of a single unit dose of rAAV particles to the one eye and / or to the contralateral eye of a plurality of individuals results in 100% of the individuals in the plurality not requiring an anti-VEGF rescue treatment (e.g., aflibercept injection).

[0112] In some embodiments, administration of a single unit dose of rAAV particles to the one eye and / or to the 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 56sf-6242343Attorney Docket No.62700-20017.40 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 an anti- VEGF rescue treatment (e.g., aflibercept injection) for at least about 4 weeks after administration of the rAAV particles, e.g., 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 more after administration of the rAAV particles. In some embodiments, administration of a single unit dose of rAAV particles to the one eye and / or to the contralateral eye of a plurality of individuals results in at least about 50% of the individuals in the plurality not requiring an anti-VEGF rescue treatment (e.g., aflibercept injection) for about 52 weeks or more, or about 56 weeks or more, after administration of the rAAV particles. In some embodiments, administration of a single unit dose of rAAV particles to the one eye and / or to the 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 an anti-VEGF rescue treatment (e.g., aflibercept injection) 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 more after administration of the rAAV particles. In some embodiments, administration of a single unit dose of rAAV particles to the one eye and / or to the contralateral eye of a plurality of individuals results in at least about 78% of the individuals in the plurality not requiring an anti-VEGF rescue treatment (e.g., aflibercept injection) for at least about 4 weeks after administration of the rAAV particles, e.g., 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 57sf-6242343Attorney Docket No.62700-20017.40 least about 96 weeks, at least about 100 weeks, at least about 104 weeks, at least about 108 weeks, or more, after administration of the rAAV particles. In some embodiments, administration of a single unit dose of rAAV particles to the one eye and / or to the contralateral eye of a plurality of individuals results in at least about 78% of the individuals in the plurality not requiring an anti-VEGF rescue treatment (e.g., aflibercept injection) for about 20 weeks or more, or about 36 weeks or more, after administration of the rAAV particles. In some embodiments, administration of a single unit dose of rAAV particles to the one eye and / or to the contralateral eye of a plurality of individuals results in at least about 80% of the individuals in the plurality not requiring an anti-VEGF rescue treatment (e.g., aflibercept injection) 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 more after administration of the rAAV particles. In some embodiments, administration of a single unit dose of rAAV particles to the one eye and / or to the contralateral eye of a plurality of individuals results in at least about 82% of the individuals in the plurality not requiring an anti-VEGF rescue treatment (e.g., aflibercept injection) 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 more, after administration of the rAAV particles. In some embodiments, administration of a single unit dose of rAAV particles to the one eye and / or to the contralateral eye of a plurality of individuals results in 100% of the individuals in the plurality not requiring an anti-VEGF rescue treatment (e.g., aflibercept injection) 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 more, after administration of the rAAV particles. In some embodiments, administration of a single unit dose of rAAV particles to the one eye and / or to the contralateral eye of a plurality of individuals results in 100% of the individuals in the plurality not requiring an anti-VEGF rescue treatment (e.g., aflibercept injection) for at least about 4 weeks after administration of the rAAV particles, e.g., any of 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 58sf-6242343Attorney Docket No.62700-20017.40 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 more after administration of the rAAV particles. In some embodiments, administration of a single unit dose of rAAV particles to the one eye and / or to the contralateral eye of a plurality of individuals results in 100% of the individuals in the plurality not requiring an anti-VEGF rescue treatment (e.g., aflibercept injection) for any of about 64 weeks or more, 72 weeks or more, or 84 weeks or more, after administration of the rAAV particles.

[0113] In some embodiments, administration of a unit dose of rAAV particles in the one eye and / or the contralateral eye of a plurality of individuals results in about 78% or less (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) of the individuals in the plurality requiring any rescue treatment (e.g., aflibercept injection) in the one eye and / or the contralateral eye. In some embodiments, administration of a unit dose of rAAV particles in the 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 any rescue treatment (e.g., aflibercept injection) in the one eye and / or the contralateral eye. In some embodiments, administration of a unit dose of rAAV particles in the one eye and / or the 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 injection) in the one eye and / or the contralateral eye. In some embodiments, administration of a unit dose of rAAV particles in the 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., aflibercept injection) in the one eye and / or the contralateral eye. In some embodiments, administration of a unit dose of rAAV particles in the 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) in the 59sf-6242343Attorney Docket No.62700-20017.40 one eye and / or the contralateral eye. In some embodiments, administration of a unit dose of rAAV particles in the 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) in the one eye and / or the contralateral eye.

[0114] In some embodiments, administration of a unit dose of rAAV particles in the one eye and / or the contralateral eye of a plurality of individuals results in about 78% or less (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) of the individuals in the plurality requiring any rescue treatment (e.g., aflibercept injection) for at least about 4 weeks after administration of the rAAV particles, e.g., 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 more, after administration of the rAAV particles. In some embodiments, administration of a unit dose of rAAV particles in the one eye and / or the 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 injection) 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 more, after administration of the rAAV particles. In some embodiments, administration of a unit dose of rAAV particles in the 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., aflibercept injection) in the 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 60sf-6242343Attorney Docket No.62700-20017.40 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 more, after administration of the rAAV particles. In some embodiments, administration of a unit dose of rAAV particles in the 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) in the 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 more after administration of the rAAV particles. In some embodiments, administration of a unit dose of rAAV particles in the 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) in the 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 more after administration of the rAAV particles. In some embodiments, administration of a unit dose of rAAV particles in the 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) in the one eye and / or the contralateral eye for at least about 4 weeks after administration of the rAAV particles, e.g., 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 more, after administration of the rAAV particles.

[0115] In some embodiments, administration of the unit dose of rAAV particles to the one eye and / or to the contralateral eye of a plurality of individuals results in a reduction in the mean annualized anti-VEGF injection rate of any 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 61sf-6242343Attorney Docket No.62700-20017.40 mean annualized anti-VEGF injection rate prior to administration of the unit dose of rAAV particles. In some embodiments, administration of the unit dose of rAAV particles to the one eye and / or to the contralateral eye of a plurality of individuals results in a reduction in the mean annualized anti-VEGF injection rate of about 87% or more compared to the mean annualized anti-VEGF injection rate prior to administration of the unit dose of rAAV particles. In some embodiments, administration of the unit dose of rAAV particles to the one eye and / or to the contralateral eye of a plurality of individuals results in a reduction in the mean annualized anti- VEGF injection rate of 100% compared to the mean annualized anti-VEGF injection rate prior to administration of the unit dose of rAAV particles.

[0116] In some embodiments, the mean annualized anti-VEGF injection rate prior to administration of the unit dose of rAAV particles is calculated according to the formula: Annualized rate prior to administration of the unit dose of rAAV particles = (number of anti- VEGF injections in 12 months prior to administration of the unit dose of rAAV particles) / (days from the first anti-VEGF injection in the past 12 months prior to administration of the unit dose of rAAV particles to the administration of the unit dose of rAAV particles / 365.25)

[0117] In some embodiments, the mean annualized anti-VEGF injection rate after administration of the unit dose of rAAV particles is calculated according to the formula: Annualized rate after administration of the unit dose of rAAV particles = (number of anti-VEGF injections since administration of the unit dose of rAAV particles) / (days from administration of the unit dose of rAAV particles / 365.25).

[0118] In some embodiments, an individual is determined to require a rescue treatment (e.g., anti-VEGF intravitreal injection, such as aflibercept injection) after administration of the rAAV particles if the individual exhibits loss of 10 or more letters in BCVA (e.g., using the ETDRS protocol) in the one eye and / or the contralateral eye administered the rAAV particles that is attributed to intraretinal or subretinal fluid (e.g., as determined by SD-OCT) compared to the BCVA in the one eye and / or the contralateral eye administered the rAAV particles prior to administration of the rAAV particles. In some embodiments, an individual is determined to require a rescue treatment (e.g., anti-VEGF intravitreal injection, such as aflibercept injection) after administration of the rAAV particles if the individual exhibits an increase in central subfield thickness (e.g., CST or CRT) greater than 75 μm in the one eye and / or the contralateral eye administered the rAAV particles compared to the central subfield thickness in the one eye and / or the contralateral eye administered the rAAV particles prior to administration of the rAAV particles, e.g., as determined by SD-OCT. In some embodiments, an individual is determined to require a rescue treatment (e.g., anti-VEGF intravitreal injection, such as aflibercept injection) 62sf-6242343Attorney Docket No.62700-20017.40 after administration of the rAAV particles if the individual exhibits vision-threatening hemorrhage due to AMD in the one eye and / or the contralateral eye administered the rAAV particles.

[0119] In some embodiments, a rescue treatment comprises administration of a standard of care anti-VEGF therapy. Such standard of care anti-VEGF therapy comprises one or more anti-VEGF treatments (e.g., anti-VEGF intravitreal injections). In some embodiments, a rescue treatment comprises one or more aflibercept IVT injections. In some embodiments, a rescue treatment comprises one or more aflibercept IVT injections comprising about 2 mg of aflibercept.

[0120] In some embodiments, treatment of an ocular neovascular disease in an individual after administration of the unit dose of rAAV particles in the one eye and / or the contralateral eye is assessed based on the level of retinal fluid compared the level of retinal fluid prior to administration of the unit dose of rAAV particles in the one eye and / or the contralateral eye. In some embodiments, treatment of an ocular neovascular disease in an individual after administration of the unit dose of rAAV particles in the one eye and / or the contralateral eye is determined if a reduction in retinal fluid is observed after administration of the unit dose of rAAV particles in the one eye and / or the contralateral eye compared to the level of retinal fluid prior to administration of the unit dose of rAAV particles in the one eye and / or the contralateral eye. In some embodiments, the ocular neovascular disease is wAMD.

[0121] In some embodiments, treatment of an ocular neovascular disease in an individual after administration of the unit dose of rAAV particles in the one eye and / or the contralateral eye is assessed based on the resolution of pigment epithelial detachment (PED) compared to PED prior to administration of the unit dose of rAAV particles in the one eye and / or the contralateral eye. In some embodiments, treatment of an ocular neovascular disease in an individual after administration of the unit dose of rAAV particles in the one eye and / or the contralateral eye is determined if resolution of PED after administration of the unit dose of rAAV particles in the one eye and / or the contralateral eye is observed, compared to PED prior to administration of the unit dose of rAAV particles in the one eye and / or the contralateral eye. In some embodiments, the ocular neovascular disease is wAMD.

[0122] In some embodiments, treatment of an ocular neovascular disease in an individual after administration of the unit dose of rAAV particles in the one eye and / or the contralateral eye is assessed based on choroidal neovascularization (CNV) lesion growth as determined by fluorescein angiography. In some embodiments, treatment of an ocular neovascular disease in an individual after administration of the unit dose of rAAV particles in the one eye and / or the contralateral eye is determined if CNV lesions shrink (e.g., by more than any of about 5%, about 63sf-6242343Attorney Docket No.62700-20017.40 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, or 100%) after administration of the unit dose of rAAV particles in the one eye and / or the contralateral eye compared to CNV lesions present prior to administration of the unit dose of rAAV particles in the one eye and / or the contralateral eye. In some embodiments, treatment of an ocular neovascular disease in an individual after administration of the unit dose of rAAV particles in the one eye and / or the contralateral eye is determined if CNV lesions do not grow (e.g., grow less than any of about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 15%, or about 20%) after administration of the unit dose of rAAV particles in the one eye and / or the contralateral eye compared to CNV lesions present prior to administration of the unit dose of rAAV particles in the one eye and / or the contralateral eye. In some embodiments, the ocular neovascular disease is wAMD.

[0123] In some embodiments, treatment of an ocular neovascular disease in an individual after administration of the unit dose of rAAV particles in the one eye and / or the contralateral eye is assessed based on the anatomical features of the one eye and / or the contralateral eye based on any methods known in the art (e.g., SD-OCT, OCT, fluorescein angiography, digital color fundus photography, etc.). In some embodiments, treatment of an ocular neovascular disease in an individual after administration of the unit dose of rAAV particles in the one eye and / or the contralateral eye is determined if an improvement in anatomical features of the one eye and / or the contralateral eye is observed after administration of the unit dose of rAAV particles in the one eye and / or the contralateral eye. In some embodiments, the ocular neovascular disease is wAMD.

[0124] In some embodiments, treatment of an ocular neovascular disease in an individual after administration of the unit dose of rAAV particles in the one eye and / or the contralateral eye is assessed based on ophthalmologic examination, intraocular pressure (e.g., using a Goldmann applanation tonometer or Tono-pen), indirect ophthalmoscopy, examination of the one eye and / or the contralateral eye and adnexa, eyelid and / or pupil responsiveness, belpharoptosis, abnormal pupil shape, unequal pupils, abnormal reaction to light, afferent pupillary defects, slit-lamp examination (including of the eyelids, conjunctiva, cornea, lens, iris, and anterior chamber), posterior segment abnormalities of the vitreous, optic nerve, peripheral retina, and retinal vasculature, SD-OCT, fluorescein angiography, digital color fundus photography (including images of the retina, optic disc, and / or macula), aqueous humor sampling, vitreous humor sampling, OCT-angiography (OCT-A), refraction and / or visual acuity (BCVA). In some embodiments, SD-OCT is performed to evaluate retinal thickness (e.g., central retinal thickness or central subfield thickness), macular volume, and / or the presence of fluid (e.g., subretinal fluid or intraretinal fluid). In some embodiments, the ocular neovascular disease is wAMD. 64sf-6242343Attorney Docket No.62700-20017.40

[0125] The unit dose of rAAV particles may be administered to the one eye and / or to the contralateral eye of the individual by any method known in the art. For example, the unit dose of rAAV particles may be administered to the one eye and / or to the contralateral eye of the individual intraocularly, or by intravitreal injection. In some embodiments, the administration of the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual is intraocular. In some embodiments, the administration of the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual is by intravitreal injection (IVT) or subretinal injection. In some embodiments, the administration of the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual is by IVT injection. In some embodiments, aseptic technique is employed to administer a unit dose of rAAV particles by intravitreal injection. In some embodiments, aseptic technique with providone-iodine is employed to administer a unit dose of rAAV particles by intravitreal injection.

[0126] In some embodiments, the individual has not received a prior treatment for an ocular neovascular disease. In some embodiments, the individual has not received a prior treatment in the one eye and / or the contralateral eye for an ocular neovascular disease. In some embodiments, the individual has not received a prior treatment with an anti-VEGF agent (e.g., bevacizumab, brolucizumab, ranibizumab, faricimab, abicipar pegol, conbercept, OPT-302, KSI-301, injectable sunitinib maleate (GB-102), PAN-90806 (PanOptica), and / or aflibercept). In some embodiments, the individual has not received a prior treatment with an anti-VEGF agent (e.g., bevacizumab, brolucizumab, ranibizumab, faricimab, abicipar pegol, conbercept, OPT-302, KSI-301, injectable sunitinib maleate (GB-102), PAN-90806 (PanOptica), and / or aflibercept) in the one eye and / or the contralateral eye. In some embodiments, the individual has not received a prior aflibercept treatment. In some embodiments, the individual has not received a prior aflibercept treatment in the one eye and / or the contralateral eye.

[0127] In some embodiments, the method of treating an ocular neovascular disease described herein further comprises selecting a human individual suitable for such treatment. In some embodiments, In some embodiments, the human individual meets one or more of the criteria: 1) male or female with a diagnosis of CNV secondary to nAMD in the study eye, with nAMD disease activity (i.e., any fluid [IRF or SRF] present on SD-OCT or CST > 300 μm); 2) at least 50 years old at Screening Visit 1 (Day -56); 3) ETDRS BCVA letter score ≥ 40 and ≤ 78 (approximate Snellen equivalent of 20 / 160 to 20 / 32) in the study eye at Screening Visit 1 (Day - 56); 4) ETDRS BCVA letter score ≥ 40 (approximate Snellen equivalent of 20 / 160 or better) in the non-study eye at Screening Visit 1 (Day -56); 5) previously treated with anti-VEGF therapy but has not received an anti-VEGF IVT injection in the study eye within 21 days prior to 65sf-6242343Attorney Docket No.62700-20017.40 Screening Visit 1 (Day -56); 6) demonstrated a meaningful anatomic response to anti-VEGF therapy, as determined by the CRC based on 2 SD-OCT images, wherein the meaningful anatomic response is defined as either: reduction of ≥ 10% in CST (values will be rounded to the nearest whole number), OR any evidence of reduction in overall fluid (SRF / IRF); 7) women of childbearing potential (i.e., postmenarcheal, has not reached a postmenopausal state [> 12 continuous months of amenorrhea with no identified cause other than menopause], and has not undergone surgical sterilization [removal of both ovaries and / or uterus]) have a negative pregnancy test during screening and agree to use an acceptable form of contraception that results in a failure rate < 1% per year (e.g., bilateral tubal ligation; surgical sterilization of the participant’s sole male partner; oral, implantable, or injectable hormonal contraceptives that inhibit ovulation; hormone-releasing intrauterine devices; copper intrauterine devices) or agree to remain abstinent (refrain from heterosexual intercourse). Contraception methods that do not result in a failure rate of < 1% per year (e.g., male or female condom with or without spermicide; cap, diaphragm, or sponge with spermicide) are not acceptable. The reliability of sexual abstinence should be evaluated in relation to the preferred and usual lifestyle of the participant. In some embodiments, periodic abstinence (e.g., calendar, ovulation, symptothermal, or postovulation methods) and withdrawal are not acceptable methods of contraception; and 8) sexually active male participants agree to use an acceptable form of contraception that results in a failure rate < 1% per year during the study, if their female partners are of childbearing potential. In some embodiments, Day -56 is 56 days prior to the initial administration of a topical steroid treatment (e.g., difluprednate) on Day 1 and / or an anti-VEGF agent (e.g., aflibercept protein) on Day 1. In some embodiments, Day -49 is 49 days prior to the initial administration of a topical steroid treatment (e.g., difluprednate) on Day 1 and / or an anti-VEGF agent (e.g., aflibercept protein) on Day 1.

[0128] In some embodiments, the method of treating an ocular neovascular disease described herein further comprises selecting a human individual suitable for such treatment. In some embodiments, In some embodiments, the human individual meets one or more of the criteria: 1) male or female with a diagnosis of CNV secondary to nAMD in the study eye, with nAMD disease activity (i.e., any fluid [IRF or SRF] present on SD-OCT) at Screening Visit 1 (Day -56 to -49); 2) at least 50 years old at Screening Visit 1 (Day -56 to -49); 3) ETDRS BCVA letter score ≥ 35 and ≤ 78 (approximate Snellen equivalent of 20 / 200 to 20 / 32) in the study eye at Screening Visit 1 (Day -56 to -49) with no loss of vision ≥5 ETDRS letters from Screening Visit 1 (Day -56 to -49) to Day 1; 4) ETDRS BCVA letter score ≥ 35 (approximate Snellen equivalent of 20 / 200 or better) in the non-study eye at Screening Visit 1 (Day -56 to -49); 5) demonstrated a 66sf-6242343Attorney Docket No.62700-20017.40 meaningful anatomic response to anti-VEGF therapy, as determined by the CRC based on SD- OCT images of the study eye collected during screening and at Day 1. A meaningful anatomic response is defined as either complete resolution of fluid (SRF / IRF) at Day 1, OR reduction in CST at Day 1 from Screening Visit 1 of 10% for participants with CST > 300 μm at Screening Visit 1 or 5% for participants with CST ≤ 300 μm at Screening Visit 1; and 6) able to reliably use eye drops per protocol, according to the Investigator’s judgment. In some embodiments, Day -56 is 56 days prior to the initial administration of a topical steroid treatment (e.g., difluprednate) on Day 1 and / or an anti-VEGF agent (e.g., aflibercept protein) on Day 1. In some embodiments, Day -49 is 49 days prior to the initial administration of a topical steroid treatment (e.g., difluprednate) on Day 1 and / or an anti-VEGF agent (e.g., aflibercept protein) on Day 1.

[0129] In some embodiments, the method of treating an ocular neovascular disease described herein further comprises excluding a human individual not suitable for such treatment. In some embodiments, a human individual not suitable for the method of treatment described herein meets one or more of the general criteria: 1) history of a medical condition (systemic disease, metabolic dysfunction, physical examination finding, or clinical laboratory finding) giving reasonable suspicion of a condition that contraindicates the use of AAV2.7m8-aflibercept, compromises the participant’s ability to comply with the planned study activities, or that might affect the interpretation of the results of the study or render the participant at high risk for treatment complications in the opinion of the Investigator. History of severe COVID-19 infection may meet this exclusion criteria if, in the opinion of the Investigator, it is likely to lead to any of the complications listed above; 2) received any prior gene therapy; 3) received any non-gene therapy IMP or medical device in the study eye within 3 months of Screening Visit 1 (Day -56) or 5 half-lives of the IMP prior to dosing with AAV2.7m8-aflibercept, whichever is longer; 4) history of allergy, hypersensitivity, or contraindications to the use of any product or its excipients administrated within this protocol including aflibercept, corticosteroids (i.e., Ozurdex®, difluprednate), or fluorescein dye or sodium fluorescein used in angiography (mild allergy amenable to treatment is allowable); 5) evidence of poorly controlled diabetes or glycated hemoglobin (HbA1c) ≥ 8.0% during screening; 6) female participants who are pregnant or breastfeeding or who intend to become pregnant or breastfeed in the future; 7) history or evidence of any of the following cardiovascular diseases: (a) myocardial infarction in the 6- month period prior to Day 1, (b) uncontrolled hypertension defined as systolic blood pressure > 160 mmHg or diastolic blood pressure > 100 mmHg, despite using blood pressure-lowering medication within the screening period. If blood pressure-lowering medications are required, participant should be on a stable dose of the same medication continuously for 30 days prior to 67sf-6242343Attorney Docket No.62700-20017.40 Day 1, and / or (c) stroke in the 6-month period prior to Day 1; 8) any history of ongoing bleeding disorders. The use of aspirin or other anticoagulants (e.g., Factor Xa inhibitors) is permitted; 9) use of systemic corticosteroids for 30 or more consecutive days within the 90 days prior to Day 1, with the exception of low stable doses of corticosteroids (defined as ≤ 10 mg prednisolone / prednisolone or equivalent dose used for 90 days or more prior to Day 1). Inhaled, nasal or dermal steroids are permitted; 10) systemic anti-VEGF therapy during the 3-month period prior to Screening Visit 1 (Day -56); 11) history of malignancy within the last 5 years, except for the following adequately treated: (a) local basal cell or squamous cell carcinoma of the skin, (b) carcinoma in situ of the cervix or breast, (c) papillary, non-invasive bladder cancer, (d) prostate cancer Stage 1 and 2 for which observation is clinically indicated with stable prostate- specific antigen for 6 months, and / or (d) any other cancer that has been in complete remission for at least 2 years or considered surgically cured; 12) positive for human immunodeficiency virus (HIV) infection or hepatitis B or C virus at screening (unless having received a documented cure for hepatitis C virus); 13) history of COVID-19 or syphilis within 6 weeks prior to Week 1; and 14) any febrile illness within 1 week prior to Day 1. In some embodiments, Day -56 is 56 days prior to the initial administration of a topical steroid treatment (e.g., difluprednate) on Day 1 and / or an anti-VEGF agent (e.g., aflibercept protein) on Day 1.

[0130] In some embodiments, the method of treating an ocular neovascular disease described herein further comprises excluding a human individual not suitable for such treatment. In some embodiments, a human individual not suitable for the method of treatment described herein meets one or more of the ocular criteria: 1) any active ocular or periocular infection (e.g., infectious conjunctivitis, keratitis, scleritis, endophthalmitis, infectious blepharitis, uveitis) in the study eye from Screening Visit 1 (Day -56) to Day 1; 2) evidence of the total area of scar or macular fibrosis making up ≥ 50% of the total lesion area, atrophy, or other structural damage in the center of the fovea in the study eye as confirmed by the CRC at Screening Visit 1 (Day -56); 3) central subfield of the study eye affected by fibrosis or geographic atrophy, assessed by color fundus photography (CFP) at Screening Visit 1 (Day -56) and confirmed by the CRC; 4) evidence of subretinal hemorrhage that is either 50% or more of the total lesion area, or if the blood is under the fovea and is ≥ 1 disc area in size in the study eye, as confirmed by the CRC at Screening Visit 1 (Day -56) (if blood is under the fovea, fovea is surrounded 270 degrees by visible CNV); 5) any history or evidence of a concurrent intraocular condition in the study eye, including retinal diseases other than nAMD, that, in the judgment of the Investigator, could reduce the potential for visual improvement, confound assessment of the macula or require medical or surgical intervention during the study; 6) history of or evidence of the following in the 68sf-6242343Attorney Docket No.62700-20017.40 study eye: (a) intraocular or refractive surgery within 90 days prior to Day 1, (b) any previous penetrating keratoplasty or vitrectomy, (c) any previous panretinal photocoagulation, and / or (d) any previous submacular surgery, other surgical intervention or laser treatment for age-related macular degeneration; 7) any history or evidence of retinal detachment (with or without repair) or retinal pigment epithelium rip / tear in the study eye, as determined by the Investigator during screening or at Day 1; 8) aphakia and / or complete or partial absence of posterior capsule at Screening Visit 1 (Day -56) or cataract extraction or yttrium aluminum garnet (YAG) laser capsulotomy in the study eye within 3 months prior to Day -56 (partial absence of the posterior capsule due to YAG laser capsulotomy performed > 3 months prior to Day -56 is acceptable); 9) uncontrolled ocular hypertension or glaucoma in the study eye at the Screening Visit 1 (Day -56) and Day 1 (defined as IOP > 22 mmHg despite treatment with antiglaucoma medication, or according to Investigator’s judgment) or current use of ≥ 2 IOP lowering medications; 10) any history of uveitis or IOI (grade trace or above) except mild anticipated post-operative inflammation that resolved in either eye; 11) any history of intraocular or periocular steroid treatment for any ocular condition (e.g., IVT Triesence®, Iluvien®, or Ozurdex®) within 6 months prior to Screening Visit 1 (Day -56); 12) any history of IOP elevation related to topical steroid administration in either eye; 13) known history of ocular herpes simplex virus, varicella-zoster virus, or cytomegalovirus, including viral uveitis, retinitis, or keratitis in either eye; and 14) previous therapeutic radiation near the region of the study eye. In some embodiments, Day -56 is 56 days prior to the initial administration of a topical steroid treatment (e.g., difluprednate) on Day 1 and / or an anti-VEGF agent (e.g., aflibercept protein) on Day 1.

[0131] In some embodiments, the method of treating an ocular neovascular disease described herein further comprises excluding a human individual not suitable for such treatment. In some embodiments, a human individual not suitable for the method of treatment described herein meets one or more of the general criteria: 1) history of a medical condition (systemic disease, metabolic dysfunction, physical examination finding, or clinical laboratory finding) giving reasonable suspicion of a condition that contraindicates the use of AAV2.7m8-aflibercept, compromises the participant’s ability to comply with the planned study activities, or that might affect the interpretation of the results of the study or render the participant at high risk for treatment complications in the opinion of the Investigator. History of severe COVID-19 infection may meet this exclusion criteria if, in the opinion of the Investigator, it is likely to lead to any of the complications listed above; 2) received any prior gene therapy; 3) received any non-gene therapy IMP or medical device in the study eye within 3 months of Screening Visit 1 (Day -56 to Day -49) or 5 half-lives of the IMP prior to dosing with AAV2.7m8-aflibercept, whichever is 69sf-6242343Attorney Docket No.62700-20017.40 longer; 4) history of allergy, hypersensitivity, or contraindications to the use of any product or its excipients administrated within this protocol including aflibercept, corticosteroids (i.e., difluprednate), or fluorescein dye or sodium fluorescein used in angiography (mild allergy amenable to treatment is allowable); 5) evidence of poorly controlled diabetes or glycated hemoglobin (HbA1c) ≥ 8.0% during screening from Screening Visit 1 (Day -56 to Day -49) to Week 1; 6) female participants who are pregnant or breastfeeding or who intend to become pregnant or breastfeed in the future; 7) history or evidence of any of the following cardiovascular diseases: 1) myocardial infarction in the 6-month period prior to Day 1, b) uncontrolled hypertension defined as systolic blood pressure > 160 mmHg or diastolic blood pressure > 100 mmHg, despite using blood pressure-lowering medication within the screening period. If blood pressure-lowering medications are required, participant should be on a stable dose of the same medication continuously for 30 days prior to Day 1; and / or c) stroke in the 6-month period prior to Day 1; 8) any history of ongoing bleeding disorders. The use of aspirin or other anticoagulants (e.g., Factor Xa inhibitors) is permitted; 9) use of systemic immunosuppressive drugs (e.g., intravenous steroids, methotrexate, azathioprine, cyclosporin, secukinumab, denosumab, and anti-tumor necrosis factors [TNFs], such as adalimumab, infliximab, etanercept) within the 90 days prior to Screening Visit 1 (Day -56 to Day -49). Low stable doses of corticosteroids (defined as ≤ 10 mg prednisolone / prednisolone or equivalent dose) are permitted, as well as any inhaled, nasal or dermal steroid use; 10) received anti-VEGF IVT injection in the study eye within 28 days prior to Screening Visit 1 (Day -56 to -49) or systemic anti-VEGF therapy during the 3-month period prior to Screening Visit 1 (Day -56 to Day -49); 11) history of malignancy within the last 5 years prior to Screening Visit 1 (Day -56 to Day -49), except for the following adequately treated: a) local basal cell or squamous cell carcinoma of the skin, b) carcinoma in situ of the cervix or breast, c) papillary, non-invasive bladder cancer, d) prostate cancer Stage 1 and 2 for which observation is clinically indicated with stable prostate-specific antigen for 6 months, and / or e) any other cancer that has been in complete remission for at least 2 years or considered surgically cured; 12) positive for human immunodeficiency virus (HIV) infection or hepatitis B or C virus at Screening Visit 1 (Day -56 to Day -49) (unless having received a documented cure for hepatitis C virus); 13) history of COVID-19 or syphilis within 6 weeks prior to Week 1; and 14) any febrile illness within 1 week prior to Day 1. In some embodiments, Day - 56 is 56 days prior to the initial administration of a topical steroid treatment (e.g., difluprednate) on Day 1 and / or an anti-VEGF agent (e.g., aflibercept protein) on Day 1. In some embodiments, Day -49 is 49 days prior to the initial administration of a topical steroid treatment (e.g., difluprednate) on Day 1 and / or an anti-VEGF agent (e.g., aflibercept protein) Day 1. 70sf-6242343Attorney Docket No.62700-20017.40

[0132] In some embodiments, the method of treating an ocular neovascular disease described herein further comprises excluding a human individual not suitable for such treatment. In some embodiments, a human individual not suitable for the method of treatment described herein meets one or more of the ocular criteria: 1) any active ocular or periocular infection (e.g., infectious conjunctivitis, keratitis, scleritis, endophthalmitis, infectious blepharitis, uveitis) in the study eye from Screening Visit 1 (Day -56 to Day -49) to Week 1; 2) evidence of the total area of scar or macular fibrosis making up ≥ 50% of the total lesion area, atrophy, or other structural damage in the center of the fovea in the study eye as confirmed by the CRC at Screening Visit 1 (Day -56 to Day -49); 3) central subfield of the study eye affected by fibrosis or geographic atrophy, assessed by color fundus photography (CFP) at Screening Visit 1 (Day -56 to Day -49) and confirmed by the CRC; 4) evidence of subretinal hemorrhage that is either 50% or more of the total lesion area, or if the blood is under the fovea and is ≥ 1 disc area in size in the study eye, as confirmed by the CRC at Screening Visit 1 (Day -56 to Day -49) (if blood is under the fovea, fovea is surrounded 270 degrees by visible CNV); 5) any history or evidence of a concurrent intraocular condition in the study eye, including retinal diseases other than nAMD, that, in the judgment of the Investigator, could reduce the potential for visual improvement, confound assessment of the macula or require medical or surgical intervention during the study; 6) history of or evidence of the following in the study eye: a) intraocular or refractive surgery within 90 days prior to Screening Visit 1 (Day -56 to Day -49), b) any previous penetrating keratoplasty or vitrectomy, c) any previous panretinal photocoagulation, and / or d) any previous submacular surgery, other surgical intervention (including port delivery system) or laser treatment for age- related macular degeneration; 7) any history or evidence of retinal detachment (with or without repair) or retinal pigment epithelium rip / tear in the study eye, as determined by the Investigator during screening or at Day 1; 8) aphakia and / or complete or partial absence of posterior capsule at Screening Visit 1 (Day -56 to Day -49) or cataract extraction or yttrium aluminum garnet (YAG) laser capsulotomy in the study eye within 3 months prior to Screening Visit 1 (partial absence of the posterior capsule due to YAG laser capsulotomy performed > 3 months prior to Screening Visit 1 is acceptable); 9) uncontrolled ocular hypertension or glaucoma in the study eye from Screening Visit 1 (Day -56 to Day -49) to Week 1 (defined as IOP > 22 mmHg despite treatment with antiglaucoma medication, or according to Investigator’s judgment) or current use of ≥ 2 IOP lowering medications or normal tension glaucoma / suspect in the study eye; 10) any history of IOP elevation related to topical steroid administration in either eye; 11) any history of uveitis or inflammation (grade trace or above) except mild anticipated post-operative inflammation that resolved in either eye; 12) Any history of intraocular or periocular steroid 71sf-6242343Attorney Docket No.62700-20017.40 treatment for any ocular condition (e.g., IVT Triesence®, Iluvien®, or Ozurdex®) within 6 months prior to Screening Visit 1 (Day -56 to Day -49); 13) any history of treatment with complement inhibitors (e.g., pegcetacoplan and avacincaptad pegol) for geographic atrophy in the study eye; 14) known history of ocular herpes simplex virus, varicella-zoster virus, or cytomegalovirus, including viral uveitis, retinitis, or keratitis in either eye; and 15) previous therapeutic radiation near the region of the study eye. In some embodiments, Day -56 is 56 days prior to the initial administration of a topical steroid treatment (e.g., difluprednate) on Day 1 and / or an anti-VEGF agent (e.g., aflibercept protein) on Day 1. In some embodiments, Day -49 is 49 days prior to the initial administration of a topical steroid treatment (e.g., difluprednate) on Day 1 and / or an anti-VEGF agent (e.g., aflibercept protein) on Day 1.

[0133] In some embodiments, the method of treating an ocular neovascular disease described herein further comprises excluding a human individual not suitable for such treatment. In some embodiments, a human individual not suitable for the method of treatment described herein meets one or more of the general criteria: 1) history of a medical condition (systemic disease, metabolic dysfunction, physical examination finding, or clinical laboratory finding) giving reasonable suspicion of a condition that contraindicates the use of AAV2.7m8-aflibercept, compromises the participant’s ability to comply with the planned study activities, or that might affect the interpretation of the results of the study or render the participant at high risk for treatment complications in the opinion of the Investigator. History of severe COVID-19 infection may meet this exclusion criteria if, in the opinion of the Investigator, it is likely to lead to any of the complications listed above; 2) received any prior gene therapy; 3) received any non-gene therapy IMP or medical device in the study eye within 3 months of Screening Visit 1 (Day -56 to Day -49) or 5 half-lives of the IMP prior to dosing with AAV2.7m8-aflibercept, whichever is longer; 4) history of allergy, hypersensitivity, or contraindications to the use of any product or its excipients administrated within this protocol including aflibercept, corticosteroids (i.e., difluprednate), or fluorescein dye or sodium fluorescein used in angiography (mild allergy amenable to treatment is allowable); 5) evidence of poorly controlled diabetes or glycated hemoglobin (HbA1c) ≥ 8.0% during screening from Screening Visit 1 (Day -56 to Day -49) to Week 1; 6) female participants who are pregnant or breastfeeding or who intend to become pregnant or breastfeed in the future; 7) history or evidence of any of the following cardiovascular diseases: 1) myocardial infarction in the 6-month period prior to Day 1, b) uncontrolled hypertension defined as systolic blood pressure > 160 mmHg or diastolic blood pressure > 100 mmHg, despite using blood pressure-lowering medication within the screening period. If blood pressure-lowering medications are required, participant should be on a stable dose of the same 72sf-6242343Attorney Docket No.62700-20017.40 medication continuously for 30 days prior to Day 1; and / or c) stroke in the 6-month period prior to Day 1; 8) any history of ongoing bleeding disorders. The use of aspirin or other anticoagulants (e.g., Factor Xa inhibitors) is permitted; 9) use of systemic immunosuppressive drugs (e.g., intravenous steroids, methotrexate, azathioprine, cyclosporin, secukinumab, and anti-tumor necrosis factors [TNFs], such as adalimumab, infliximab, etanercept) within the 90 days prior to Screening Visit 1 (Day -56 to Day -49). Low stable doses of corticosteroids (defined as ≤ 10 mg prednisolone / prednisolone or equivalent dose) are permitted, as well as any inhaled, nasal or dermal steroid use; 10) received anti-VEGF IVT injection in the study eye within 28 days prior to Screening Visit 1 (Day -56 to -49) or systemic anti-VEGF therapy during the 3-month period prior to Screening Visit 1 (Day -56 to Day -49); 11) history of malignancy within the last 5 years prior to Screening Visit 1 (Day -56 to Day -49), except for the following adequately treated: a) local basal cell or squamous cell carcinoma of the skin, b) carcinoma in situ of the cervix or breast, c) papillary, non-invasive bladder cancer, d) prostate cancer Stage 1 and 2 for which observation is clinically indicated with stable prostate-specific antigen for 6 months, and / or e) any other cancer that has been in complete remission for at least 2 years or considered surgically cured; 12) positive for human immunodeficiency virus (HIV) infection or hepatitis B virus (HBV) or hepatitis C virus (HCV) at Screening Visit 1 (Day -56 to Day -49) as follows: a. HIV-1 and / or HIV-2 antibody positive, b. Hepatitis B surface antigen positive, and / or c. Hepatitis C antibody positive unless participant was previously documented to having achieved cure or has negative HCV RNA test without treatment (i.e., spontaneous clearance); 13) history of COVID- 19 or syphilis within 6 weeks prior to Week 1; and 14) any febrile illness within 1 week prior to Day 1. In some embodiments, Day -56 is 56 days prior to the initial administration of a topical steroid treatment (e.g., difluprednate) on Day 1 and / or an anti-VEGF agent (e.g., aflibercept protein) on Day 1. In some embodiments, Day -49 is 49 days prior to the initial administration of a topical steroid treatment (e.g., difluprednate) on Day 1 and / or an anti-VEGF agent (e.g., aflibercept protein) Day 1

[0134] In some embodiments, the method of treating an ocular neovascular disease described herein further comprises excluding a human individual not suitable for such treatment. In some embodiments, a human individual not suitable for the method of treatment described herein meets one or more of the ocular criteria: 1) any active ocular or periocular infection (e.g., infectious conjunctivitis, keratitis, scleritis, endophthalmitis, infectious blepharitis, uveitis) in the study eye from Screening Visit 1 (Day -56 to Day -49) to Week 1; 2) evidence of the total area of scar or macular fibrosis making up ≥ 50% of the total lesion area, atrophy, or other structural damage in the center of the fovea in the study eye as confirmed by the CRC at Screening Visit 1 73sf-6242343Attorney Docket No.62700-20017.40 (Day -56 to Day -49); 3) central subfield of the study eye affected by fibrosis or geographic atrophy, assessed by color fundus photography (CFP) at Screening Visit 1 (Day -56 to Day -49) and confirmed by the CRC; 4) evidence of subretinal hemorrhage that is either 50% or more of the total lesion area, or if the blood is under the fovea and is ≥ 1 disc area in size in the study eye, as confirmed by the CRC at Screening Visit 1 (Day -56 to Day -49) (if blood is under the fovea, fovea is surrounded 270 degrees by visible CNV); 5) any history or evidence of a concurrent intraocular condition in the study eye, including retinal diseases other than nAMD, that, in the judgment of the Investigator, could reduce the potential for visual improvement, confound assessment of the macula or require medical or surgical intervention during the study; 6) history of or evidence of the following in the study eye: a) intraocular or refractive surgery within 90 days prior to Screening Visit 1 (Day -56 to Day -49), b) any previous penetrating keratoplasty or vitrectomy, c) any previous panretinal photocoagulation, and / or d) any previous submacular surgery, other surgical intervention (including port delivery system) or laser treatment for age- related macular degeneration; 7) any history or evidence of retinal detachment (with or without repair) or retinal pigment epithelium rip / tear in the study eye, as determined by the Investigator during screening or at Day 1; 8) aphakia and / or complete or partial absence of posterior capsule at Screening Visit 1 (Day -56 to Day -49) or cataract extraction or yttrium aluminum garnet (YAG) laser capsulotomy in the study eye within 3 months prior to Screening Visit 1 (partial absence of the posterior capsule due to YAG laser capsulotomy performed > 3 months prior to Screening Visit 1 is acceptable); 9) uncontrolled ocular hypertension or glaucoma in the study eye from Screening Visit 1 (Day -56 to Day -49) to Week 1 (defined as IOP > 22 mmHg despite treatment with antiglaucoma medication, or according to Investigator’s judgment) or current use of ≥ 2 IOP lowering medications or normal tension glaucoma / suspect in the study eye or history of any of the following procedures in the study eye: a. Incisional glaucoma surgery (i.e., glaucoma drainage implant / shunt or trabeculectomy), b. Ocular angle-based surgery (i.e., goniotomy or canaloplasty), c. Minimally Invasive Glaucoma Surgery (MIGS) in the study eye, and / or d. Angle-based glaucoma surgery (i.e., ALT or SLT); 10) any history of IOP elevation related to topical steroid administration in either eye; 11) any history of uveitis or inflammation (grade trace or above) except mild anticipated post-operative inflammation that resolved in either eye; 12) Any history of intraocular or periocular steroid treatment for any ocular condition (e.g., IVT Triesence®, Iluvien®, or Ozurdex®) within 6 months prior to Screening Visit 1 (Day -56 to Day -49); 13) any history of treatment with complement inhibitors (e.g., pegcetacoplan and avacincaptad pegol) for geographic atrophy in the study eye; 14) known history of ocular herpes simplex virus, varicella-zoster virus, or cytomegalovirus, including viral uveitis, retinitis, or 74sf-6242343Attorney Docket No.62700-20017.40 keratitis in either eye; and 15) previous therapeutic radiation near the region of the study eye. In some embodiments, Day -56 is 56 days prior to the initial administration of a topical steroid treatment (e.g., difluprednate) on Day 1 and / or an anti-VEGF agent (e.g., aflibercept protein) on Day 1. In some embodiments, Day -49 is 49 days prior to the initial administration of a topical steroid treatment (e.g., difluprednate) on Day 1 and / or an anti-VEGF agent (e.g., aflibercept protein) on Day 1. Steroid Treatments

[0135] In some embodiments, the unit dose of rAAV particles is administered in combination with steroid treatment. In some embodiments, the steroid treatment is a corticosteroid treatment. Exemplary corticosteroids include, without limitation, aclometasone, amcinomide, beclometasone, betamethasone, budesonide, ciclesonide, clobetasol, clobetasone, clocortolone, cloprednol, cortivazol, deflazacort, deoxycorticosterone, desonide desoximetasone, dexamethasone, diflorasone, diflucortolone, difluprednate, fluclorolone, fludrocortisone, fludroxycortide, flumetasone, flunisolide, fluocinolone acetonide, fluocinonide, fluocortin, fluocortolone, fluorometholone, fluperolone, 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 ulobetasol. 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. a drop). In some embodiments, the topical steroid treatment is a difluprednate treatment. In some embodiments, the ophthalmic steroid treatment is a difluprednate treatment. In some embodiments, the steroid treatment is a difluprednate treatment. In some embodiments, the difluprednate is Durezol®or Amneal Pharmaceutical’s difluprednate ophthalmic emulsion. In some embodiments, the difluprednate treatment begins prior to the administration of the single unit dose of rAAV to the one eye, wherein the topical steroid treatment comprises (a) about four administrations of the topical steroid per day daily (i.e., QID) for about 6 weeks, (b) followed by about three administrations of the topical steroid per day daily (i.e., TID) for about 5 weeks, (c) followed by about two administrations of the topical steroid per day daily (i.e., BID) for about 12 weeks, and (d) followed by about one administration of the topical steroid per day daily (i.e., QD) for about 4 weeks, wherein the difluprednate is administered on Day 1 and the unit dose of rAAV particles is administered on any one of Day 4, Day 5, Day 6, Day 7, Day 8, Day 9, Day 10, or Day 11. In some embodiments, the difluprednate treatment further comprises about one administration of the topical steroid 75sf-6242343Attorney Docket No.62700-20017.40 every other day (i.e., QOD) for about 2 weeks following step (d). In some embodiments, the dosing timeline of the rAAV particles, topical steroid, the anti-VEGF agent loading dose(s), supplemental aflibercept, and / or sham injection is based on the administration timeline shown in FIG.4.

[0136] In some embodiments, the steroid is administered before, during, and / or after administration of the unit dose of rAAV particles. In some embodiments, the steroid is administered before, during, and after administration of the unit dose of rAAV particles. In some embodiments, the steroid is administered during, and after administration of the unit dose of rAAV particles. In some embodiments, the steroid is administered before administration of the unit dose of rAAV particles. In some embodiments, the steroid is administered during administration of the unit dose of rAAV particles. In some embodiments, the steroid is administered before and during administration of the unit dose of rAAV particles. In some embodiments, the steroid is administered after administration of the unit dose of rAAV particles. In some embodiments, the steroid is administered during and after administration of the unit dose of rAAV particles. In some embodiments, the steroid is administered before and / or after administration of the unit dose of rAAV particles. In some embodiments, the steroid is administered before and after administration of the unit dose of rAAV particles.

[0137] 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 the unit dose of rAAV particles. In some embodiments, the steroid treatment is administered before administration of the unit dose of rAAV particles. In some embodiments, the steroid treatment is administered during administration of the unit dose of rAAV particles. In some embodiments, the steroid treatment is administered after administration of the unit dose of rAAV particles. In some embodiments, the steroid treatment is administered before and during administration of the unit dose of rAAV particles. In some embodiments, the steroid treatment is administered before and after administration of the unit dose of rAAV particles. In some embodiments, the steroid treatment is administered during, and after administration of the unit dose of rAAV particles. In some embodiments, the steroid treatment is administered before, during, and after administration of the unit dose of rAAV particles.

[0138] In some embodiments, the steroid treatment is an ophthalmic steroid treatment, e.g., a topical steroid treatment. In some embodiments, the topical steroid treatment begins prior to the administration of the single unit dose of rAAV to the one eye. In some embodiments, the ophthalmic steroid treatment, e.g., a topical steroid treatment, is a daily steroid treatment for up 76sf-6242343Attorney Docket No.62700-20017.40 to 25 weeks, up to 26 weeks, up to 27 weeks, up to 28 weeks, up to 29 weeks, up to 30 weeks, up to 31 weeks, up to 32 weeks, up to 33 weeks, up to 34 weeks, or up to 35 weeks, wherein the topical steroid treatment is administered on Day 1 and the unit dose of rAAV particles is administered on any one of Day 4, Day 5, Day 6, Day 7, Day 8, Day 9, Day 10, or Day 11. In some embodiments, the ophthalmic steroid treatment, e.g., a topical steroid treatment, is a daily steroid treatment for at least 25 weeks, at least 26 weeks, at least 27 weeks, at least 28 weeks, at least 29 weeks, at least 30 weeks, at least 31 weeks, at least 32 weeks, at least 33 weeks, at least 34 weeks, or at least 35 weeks, wherein the topical steroid treatment is administered on Day 1 and the unit dose of rAAV particles is administered on any one of Day 4, Day 5, Day 6, Day 7, Day 8, Day 9, Day 10, or Day 11. In some embodiments, the topical steroid treatment begins prior to the administration of the single unit dose of rAAV to the one eye, wherein the topical steroid treatment comprises (a) about four administrations of the topical steroid per day daily (i.e., QID) for about 6 weeks, (b) followed by about three administrations of the topical steroid per day daily (i.e., TID) for about 5 weeks, (c) followed by about two administrations of the topical steroid per day daily (i.e., BID) for about 12 weeks, and (d) followed by about one administration of the topical steroid per day daily (i.e., QD) for about 4 weeks. In some embodiments, the ophthalmic steroid treatment is extended at the discretion of the treating physician. In some embodiments, the topical steroid treatment further comprises about one administration of the topical steroid every other day (i.e., QOD) for about 2 weeks following step (d). In some embodiments, the dosing timeline of the rAAV particles, topical steroid, the anti- VEGF agent loading dose(s), supplemental aflibercept, and / or sham injection is based on the administration timeline shown in FIG.4.

[0139] In some embodiments, the ophthalmic steroid is about 0.005% to about 0.5% difluprednate. In some embodiments, the ophthalmic steroid is any of 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 difluprednate 0.05%. In some embodiments, the topical and / or ophthalmic steroid comprises a difluprednate ophthalmic emulsion 0.05%. In some embodiments, a dose of difluprednate 0.05% is one drop of ophthalmic solution. In some embodiments, the administration is about one drop of the difluprednate ophthalmic emulsion 0.05% eyedrop per administration. 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, a 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, a dose of difluprednate comprises about 2.5 µg 77sf-6242343Attorney Docket No.62700-20017.40 difluprednate. In some embodiments, the difluprednate ophthalmic emulsion 0.05% is in a pharmaceutical formulation. In some embodiments, wherein the pharmaceutical formulation comprises about 0.5 mg difluprednate per mL of the ophthalmic emulsion, wherein the pharmaceutical formulation has a pH of about 5.2 to about 5.8. In some embodiments, the pharmaceutical formulation further comprises boric acid, castor oil, edetate disodium, glycerin, polysorbate 80, sodium acetate, sodium hydroxide, and sorbic acid 0.1%.

[0140] In some embodiments, the topical steroid treatment comprises a 27-week topical steroid treatment, e.g., 0.05% difluprednate. In some embodiments, the topical steroid treatment begins prior to the administration of the single unit dose of rAAV to the one eye, wherein the topical steroid treatment comprises (a) about four administrations of the topical steroid per day daily (i.e., QID) for about 6 weeks, (b) followed by about three administrations of the topical steroid per day daily (i.e., TID) for about 5 weeks, (c) followed by about two administrations of the topical steroid per day daily (i.e., BID) for about 12 weeks, and (d) followed by about one administration of the topical steroid per day daily (i.e., QD) for about 4 weeks, wherein an initial topical steroid is administered on Day 1 and the unit dose of rAAV particles is administered on any one of Day 4, Day 5, Day 6, Day 7, Day 8, Day 9, Day 10, or Day 11. In some embodiments, the topical steroid treatment is continued if inflammation is present. In some embodiments, the topical steroid treatment further comprises about one administration of the topical steroid every other day (i.e., QOD) for about 2 weeks following step (d). In some embodiments, the dosing timeline of the rAAV particles, topical steroid, the anti-VEGF agent loading dose(s), supplemental aflibercept, and / or sham injection is based on the administration timeline shown in FIG.4. Anti-VEGF Agent Treatment

[0141] In some embodiments, the methods of treatment provided herein comprise administering an anti-VEGF agent (e.g., an aflibercept IVT injection) to one eye of the individual prior to administration of the unit dose of rAAV particles to the one eye of the individual. In some embodiments, the anti-VEGF agent is administered about 1 week to about 8 weeks prior to administration of the unit dose of rAAV particles. In some embodiments, the method further comprises administering a first loading dose of the anti-VEGF agent to the one eye of the individual from about Day -56 to about Day -49 and a second loading dose of the anti-VEGF agent to the one eye of the individual from about Day -28 to about Day -21. In some embodiments, the anti-VEGF agent is administered on about Day 1 and the unit dose of rAAV particles is administered on any one of about Day 4, Day 5, Day 6, Day 7, Day 8, Day 9, Day 10, or Day 11. In some embodiments, the topical steroid treatment comprises a 27-week topical 78sf-6242343Attorney Docket No.62700-20017.40 steroid treatment, e.g., 0.05% difluprednate. In some embodiments, the topical steroid treatment comprises (a) about four administrations of the topical steroid per day daily (i.e., QID) for about 6 weeks, (b) followed by about three administrations of the topical steroid per day daily (i.e., TID) for about 5 weeks, (c) followed by about two administrations of the topical steroid per day daily (i.e., BID) for about 12 weeks, and (d) followed by about one administration of the topical steroid per day daily (i.e., QD) for about 4 weeks, wherein the anti-VEGF agent is administered on about Day 1 and the unit dose of rAAV particles is administered on any one of about Day 4, Day 5, Day 6, Day 7, Day 8, Day 9, Day 10, or Day 11. In some embodiments, the topical steroid treatment is continued if inflammation is present. In some embodiments, the topical steroid treatment further comprises about one administration of the topical steroid every other day (i.e., QOD) for about 2 weeks following step (d). In some embodiments, the dosing timeline of the rAAV particles, topical steroid, the anti-VEGF agent loading dose(s), supplemental aflibercept, and / or sham injection is based on the administration timeline shown in FIG.4. In some embodiments, about Day -56 is about 56 days prior to the initial administration of a topical steroid treatment (e.g., difluprednate) on Day 1 and / or an anti-VEGF agent (e.g., aflibercept protein) on Day 1. In some embodiments, about Day -49 is about 49 days prior to the initial administration of a topical steroid treatment (e.g., difluprednate) on Day 1 and / or an anti- VEGFagent (e.g., aflibercept protein) on Day 1. In some embodiments, about Day -28 is about 28 days prior to the initial administration of a topical steroid treatment (e.g., difluprednate) on Day 1 and / or an anti-VEGF agent (e.g., aflibercept protein) on Day 1. In some embodiments, about Day -21 is about 21 days prior to the initial administration of a topical steroid treatment (e.g., difluprednate) on Day 1 and / or an anti-VEGF agent (e.g., aflibercept protein) on Day 1.

[0142] In some embodiments, the anti-VEGF agent (e.g., an aflibercept IVT injection) is supplemented to the one eye of the individual at any one of about Week 4, Week 8, Week 12, Week 16, Week 20, Week 24, Week 28, Week 32, Week 36, Week 40, Week 44, Week 48, Week 52, or Week 56. Vectors for Delivering Transgenes to Target Cells

[0143] In some embodiments, the recombinant adeno-associated virus (rAAV) particles comprise a recombinant viral vector derived from adeno-associated virus (AAV) that has been altered so that it is replication-defective in the subject (e.g., a human or a non-human primate). In some embodiments, the adeno-associated virus (AAV) is a recombinant AAV (rAAV).

[0144] AAV or rAAV are small non-enveloped single-stranded DNA viruses. rAAVs are non- pathogenic human parvoviruses and can be made to be dependent on helper viruses, including adenovirus, herpes simplex virus, vaccinia virus and CMV, for replication. 79sf-6242343Attorney Docket No.62700-20017.40

[0145] Exposure to wild type (wt) AAV is not associated or known to cause any human pathologies and is common in the general population, making AAV or rAAV a suitable delivery system for gene therapy. AAV and rAAV used for gene therapy for delivery of an anti-VEGF agent, e.g., aflibercept, can be of any serotype. In some embodiments, the methods of the disclosure provide for 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 tropism of the virus, or infectivity of a target cell of interest. In some embodiments, several AAV vectors may be generated to allow selection of the most optimal serotype for use with an anti-VEGF agent transgene (e.g., aflibercept transgene).

[0146] In some embodiments, the methods of the present disclosure provide for the use of pseudotyped AAV. Pseudotyped AAV particles comprise AAV genome inverted terminal repeats (ITRs) of one AAV serotype encapsidated by an 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 capsid serotype. For example, an AAV particle comprising AAV2 ITRs and an AAV1 capsid would be designated “AAV2 / 1”.

[0147] In some embodiments, the rAAV particles comprise 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., 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 connexin36 (or “Cx36”) promoter. In some embodiments, the promoter is a tissue-specific promoter that is activated in specific tissues or cells, such as retinal cells, to reduce potential toxicity or undesirable effects to non-targeted cells. In some aspects, several AAV vectors may be generated to allow selection of the most optimal serotype and promoter for use with the anti-VEGF agent transgene (e.g., 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.

[0148] In some embodiments, the AAV vector comprises a polynucleotide cassette for enhanced 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, 80sf-6242343Attorney Docket No.62700-20017.40 comprising a CMV sequence (SEQ ID NO: 23); (c) a 5'UTR region comprising, in 5' to 3' order, TPL and eMLP sequences (SEQ ID NO: 24 and SEQ ID NO: 25, respectively); (d) a coding sequence encoding a peptide or polypeptide (e.g., an anti-VEGF agent such as aflibercept); (e) a second enhancer region comprising a full EES sequence (SEQ ID NO: 26); and (f) a 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 a sequence with 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 with 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 with 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 81sf-6242343Attorney Docket No.62700-20017.40 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 OD NO: 30) CCCAGGTCCA AGTTTAAACG CC (SEQ ID NO: 31) TCTTTGGGCT TTCTATATAC ATTTTGAAAT GAGGTTGACA AGTTACCTAG GAAAACTGTC TTCCTGCCCG GGTGGCA (SEQ ID NO: 32) CTCTGGAGAC GACTTACGGT AAATGGCCCG CCTGGCTGAC CGCCCAACGA CCCCCGCCCA TTGACGTCAA TAATGACGTA TGTTCCCATA GTAACGCCAA TAGGGACTTT CCATTGACGT CAATGGGTGG AGTATTTACG GTAAACTGCC CACTTGGCAG TACATCAAGT GTATCATATG CCAAGTCCGC CCCCTATTGA CGTCAATGAC GGTAAATGGC CCGCCTGGCA TTATGCCCAG TACATGACCT TACGGGACTT TCCTACTTGG CAGTACATCT ACGTATTAGT CATCGCTATT ACCATGCTGA TGCGGTTTTG GCAGTACACC AATGGGCGTG GATAGCGGTT TGACTCACGG GGATTTCCAA GTCTCCACCC CATTGACGTC AATGGGAGTT TGTTTTGGCA CCAAAATCAA CGGGACTTTC CAAAATGTCG TAATAACCCC GCCCCGTTGA CGCAAATGGG CGGTAGGCGT GTACGGTGGG AGGTCTATAT AAGCAGAGCT CGTTTAGTGA ACCGTCAGAT CGCCTGGAGA GGCCATCCAC GCTGTTTTGA CCTCCATAGT GGACACCGGG ACCGATCCAG CCTCCGCGTC TCAGGGGAGA TCTCGTTTAG TGAACCGTCA GATCCTCACT CTCTTCCGCA TCGCTGTCTG CGAGGGCCAG CTGTTGGGCT CGCGGTTGAG GACAAACTCT TCGCGGTCTT TCCAGTACTC TTGGATCGGA AACCCGTCGG CCTCCGAACG GTACTCCGCC ACCGAGGGAC CTGAGCGAGT CCGCATCGAC CGGATCGGAA AACCTCTCGA GAAAGGCGTC TAACCAGTCA CAGTCGCAAG GTAGGCTGAG CACCGTGGCG GGCGGCAGCG GGTGGCGGTC GGGGTTGTTT CTGGCGGAGG TGCTGCTGAT GATGTAATTA AAGTAGGCGG TCTTGAGACG GCGGATGGTC GAGGTGAGGT GTGGCAGGCT TGAGATCCAG CTGTTGGGGT GAGTACTCCC TCTCAAAAGC GGGCATTACT TCTGCGCTAA GATTGTCAGT TTCCAAAAAC GAGGAGGATT TGATATTCAC CTGGCCCGAT 82sf-6242343Attorney Docket No.62700-20017.40 CTGGCCATAC ACTTGAGTGA CAATGACATC CACTTTGCCT TTCTCTCCAC AGGTGTCCAC TCCCAGGTCC AAGTTTAAAC GCCGCCACCA TG (SEQ ID NO: 33) ACTGTTCTCA TCACATCATA TCAAGGTTAT ATACCATCAA TATTGCCACA GATGTTACTT AGCCTTTTAA TATTTCTCTA ATTTAGTGTA TATGCAATGA TAGTTCTCTG ATTTCTGAGA TTGAGTTTCT CATGTGTAAT GATTATTTAG AGTTTCTCTT TCATCTGTTC AAATTTTTGT CTAGTTTTAT TTTTTACTGA TTTGTAAGAC TTCTTTTTAT AATCTGCATA TTACAATTCT CTTTACTGGG GTGTTGCAAA TATTTTCTGT CATTCTATGG CCTGACTTTT CTTAATGGTT TTTTAATTTTCTTAATATTC ATGCAATCTA ATTAACAATC TTTTCTTTGT GGTTAGGACT TTGAGTCATA AGAAATTTTT CTCTACACTG AAGTCATGAT GGCATGCTTC TATATTATTT TCTAAAAGAT TTAAAGTTTT GCCTTCTCCA TTTAGACTTA TAATTCACTG GAATTTTTTT GTGTGTATGG TATGACATAT GGGTTCCCTT TTATTTTTTA CATATAAATA TATTTCCCTG TTTTTCTAAAATCATCATTT TCCCATTGTA AAATGCCATA TTTTTTTCAT AGGTCACTTA CATATATCAA TGGGTCTGTT TCTGAGCTCT ACTCTATTTT ATCAGCCTCA CTGTCTATCC CCACACATCT CATGCTTTGC TCTAAATCTT GATATTTAGT GGAACATTCT TTCCCATTTT GTTCTACAAG AATATTTTTG TTATTGTCTT TGGGCTTTCT ATATACATTT TGAAATGAGG TTGACAAGTT ACCTAGGAAA ACTGTCTTCC TGCCCGGGTG GCATCCCTGT GACCCCTCCC CAGTGCCTCT CCTGGCCCTG GAAGTTGCCA CTCCAGTGCC CACCAGCCTT GTCCTAATAA AATTAAGTTG CATCATTTTG TCTGACTAGG TGTCCTTCTA TAATATTATG GGGTGGAGGG GGGTGGTATG GAGCAAGGGG CCCAAGTTGG GAAGAAACCT GTAGGGCCTG CGAAGACAGT CAG (SEQ ID NO: 34)

[0149] In some embodiments, the nucleic acid comprises the sequence of SEQ ID NO: 43 or a sequence having at least 85% identity thereto. acttacggtaaatggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaataatga cgtatgttcccatagtaacgccaatagggactttccattgacgtcaatgggtggagtatttacgg taaactgcccacttggcagtacatcaagtgtatcatatgccaagtccgccccctattgacgtcaa tgacggtaaatggcccgcctggcattatgcccagtacatgaccttacgggactttcctacttggc agtacatctacgtattagtcatcgctattaccatgctgatgcggttttggcagtacaccaatggg cgtggatagcggtttgactcacggggatttccaagtctccaccccattgacgtcaatgggagttt gttttggcaccaaaatcaacgggactttccaaaatgtcgtaataaccccgccccgttgacgcaaa tgggcggtaggcgtgtacggtgggaggtctatataagcagagctcgtttagtgaaccgt (SEQ ID NO: 43)

[0150] In some embodiments, the polynucleotide cassette comprises or consists of SEQ ID NO: 39 or a sequence with at least 85% identity thereto. 83sf-6242343Attorney Docket No.62700-20017.40 gcgcgctcgctcgctcactgaggccgcccgggcaaagcccgggcgtcgggcgacctttggtcgcc cggcctcagtgagcgagcgagcgcgcagagagggagtggccaactccatcactaggggttccttg tagttaatgattaacccgccatgctacttatctacgtactctggagacgacttacggtaaatggc ccgcctggctgaccgcccaacgacccccgcccattgacgtcaataatgacgtatgttcccatagt aacgccaatagggactttccattgacgtcaatgggtggagtatttacggtaaactgcccacttgg cagtacatcaagtgtatcatatgccaagtccgccccctattgacgtcaatgacggtaaatggccc gcctggcattatgcccagtacatgaccttacgggactttcctacttggcagtacatctacgtatt agtcatcgctattaccatgctgatgcggttttggcagtacaccaatgggcgtggatagcggtttg actcacggggatttccaagtctccaccccattgacgtcaatgggagtttgttttggcaccaaaat caacgggactttccaaaatgtcgtaataaccccgccccgttgacgcaaatgggcggtaggcgtgt acggtgggaggtctatataagcagagctcgtttagtgaaccgtcagatcgcctggagaggccatc cacgctgttttgacctccatagtggacaccgggaccgatccagcctccgcgtctcaggggagatc tcgtttagtgaaccgtcagatcctcactctcttccgcatcgctgtctgcgagggccagctgttgg gctcgcggttgaggacaaactcttcgcggtctttccagtactcttggatcggaaacccgtcggcc tccgaacggtactccgccaccgagggacctgagcgagtccgcatcgaccggatcggaaaacctct cgagaaaggcgtctaaccagtcacagtcgcaaggtaggctgagcaccgtggcgggcggcagcggg tggcggtcggggttgtttctggcggaggtgctgctgatgatgtaattaaagtaggcggtcttgag acggcggatggtcgaggtgaggtgtggcaggcttgagatccagctgttggggtgagtactccctc tcaaaagcgggcattacttctgcgctaagattgtcagtttccaaaaacgaggaggatttgatatt cacctggcccgatctggccatacacttgagtgacaatgacatccactttgcctttctctccacag gtgtccactcccaggtccaagtttaaacgccgccaccatggtgtcatactgggatactggagtct tgctttgtgccctgctgtcctgcctcctcctgactggctccagctcgggctcagataccggtcgc cccttcgtggagatgtactccgagatcccggaaattatccacatgactgaggggcgcgaacttgt gatcccctgccgggtcaccagcccgaacattactgtgactttgaagaagttccccctggacaccc tgattccggatgggaagagaattatctgggattcacggaagggattcatcatcagcaacgcgacc tacaaggaaattggcctcctcacttgcgaagccactgtgaacggacacttgtacaagaccaacta cctgacccaccgccagaccaacaccatcatcgacgtcgtcctgtccccttcgcacgggatcgagc tctcggtgggagagaagttggtgcttaactgcaccgcccggacggaactgaatgtgggaatcgac ttcaactgggaatacccgtccagcaagcatcagcataagaagctggtgaaccgggacctcaagac tcagtccggcagcgaaatgaagaagttcctgtcgaccctcactattgacggagtgaccagatccg accagggcctctacacttgcgccgcttccagcggactcatgaccaagaagaacagcactttcgtg agggtgcatgagaaggacaagacccacacgtgtccgccgtgcccagccccagagctgctgggagg cccttccgtgttcctgtttccgcccaagccaaaggataccctgatgatctcaaggacccctgagg tcacatgcgtcgtggtggatgtgtcgcacgaggaccctgaagtcaaattcaattggtatgtggac ggagtggaagtccacaacgcgaaaaccaagccgagagaagaacagtacaattccacctaccgggt ggtgtcggtgctgactgtgctgcaccaggactggctcaacggaaaggagtacaagtgcaaggtgt ccaacaaggctctgcccgcacctattgaaaagaccatctccaaggccaagggtcaacctcgcgag cctcaggtgtacactctgcctccaagccgggacgaactgactaagaaccaagtctctctgacctg tttggtgaagggcttctacccgtcagacatcgcagtggagtgggagtcaaacggtcagccggaga acaactacaaaacaaccccccccgtgctggactccgacggctccttcttcctgtactccaagctt accgtggataagagccgctggcaacagggcaacgtgttttcctgctccgtcatgcacgaagccct gcacaaccattatacccagaagtccctgtcgctgtcccccgggaaatagtgactgttctcatcac atcatatcaaggttatataccatcaatattgccacagatgttacttagccttttaatatttctct aatttagtgtatatgcaatgatagttctctgatttctgagattgagtttctcatgtgtaatgatt atttagagtttctctttcatctgttcaaatttttgtctagttttattttttactgatttgtaaga cttctttttataatctgcatattacaattctctttactggggtgttgcaaatattttctgtcatt 84sf-6242343Attorney Docket No.62700-20017.40 ctatggcctgacttttcttaatggttttttaattttaaaaataagtcttaatattcatgcaatct aattaacaatcttttctttgtggttaggactttgagtcataagaaatttttctctacactgaagt catgatggcatgcttctatattattttctaaaagatttaaagttttgccttctccatttagactt ataattcactggaatttttttgtgtgtatggtatgacatatgggttcccttttattttttacata taaatatatttccctgtttttctaaaaaagaaaaagatcatcattttcccattgtaaaatgccat atttttttcataggtcacttacatatatcaatgggtctgtttctgagctctactctattttatca gcctcactgtctatccccacacatctcatgctttgctctaaatcttgatatttagtggaacattc tttcccattttgttctacaagaatatttttgtta ttgtctttgggctttctatatacattttgaaatgaggttgacaagttacctaggaaaactgtctt cctgcccgggtggcatccctgtgacccctccccagtgcctctcctggccctggaagttgccactc cagtgcccaccagccttgtcctaataaaattaagttgcatcattttgtctgactaggtgtccttc tataatattatggggtggaggggggtggtatggagcaaggggcccaagttgggaagaaacctgta gggcctgcgtacgtagataagtagcatggcgggttaatcattaactacaaggaacccctagtgat ggagttggccactccctctctgcgcgctcgctcgctcactgaggccgggcgaccaaaggtcgccc gacgcccgggctttgcccgggcggcctcagtgagcgagcgagcgcgc (SEQ ID NO: 39)

[0151] SEQ ID NO: 39 shown above comprises, in the 5’ to 3’ direction, an inverted terminal repeat (ITR) of AAV serotype 2 comprising nucleotides 1-145 of SEQ ID NO: 39; a CMV promoter comprising nucleotides 180-693 of SEQ ID NO: 39; a 5’ Untranslated Region (UTR), including an Adenovirus Tripartite Leader Sequence and Synthetic Intron, and comprising nucleotides 694-1314 of SEQ ID NO: 39; a Kozak sequence comprising nucleotides 1329-1340 of SEQ ID NO: 39; a codon-optimized aflibercept cDNA sequence comprising nucleotides 1338- 2714 of SEQ ID NO: 39; a 3’ UTR including a human scaffold attachment region and comprising nucleotides 2717-3527 of SEQ ID NO: 39; a human growth hormone polyadenylation / transcription stop signal comprising nucleotides 3546-3748 of SEQ ID NO: 39; and an inverted terminal repeat (ITR) of AAV serotype 2 comprising nucleotides 3772-3916 of SEQ ID NO: 39. See, e.g., FIG.2.

[0152] Additional polynucleotide cassettes for enhanced expression of a transgene (e.g., a transgene encoding an anti-VEGF agent such as aflibercept) in a target cell (such as a retinal cell) are disclosed in WO2018 / 170473, the contents of which related to polynucleotide cassettes for enhanced expression of a transgene in a target cell are incorporated herein by reference.

[0153] In some embodiments, the rAAV particles comprise a variant capsid protein having increased infectivity of target cells, e.g. retinal cells, are used to increase transduction of retinal cells or to increase targeting of gene delivery to retinal cells in an individual. In some embodiments, the rAAV particle comprises an amino acid modification in a capsid protein GH loop / loop IV of the AAV capsid protein. In some embodiments, the site of modification 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 AAV capsid, see, e.g., 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 85sf-6242343Attorney Docket No.62700-20017.40 capsid variants are known, including the 7m8 variant. In some embodiments, a rAAV particle comprises a variant AAV capsid protein that comprises an insertion of from 5 amino acids to 11 amino acids, e.g., 7 amino acid sequence, in the GH loop of a capsid protein relative to a corresponding parental AAV capsid protein, and wherein the variant capsid protein confers increased infectivity of a retinal cell compared to the infectivity of the retinal cell by an AAV particle comprising the corresponding parental or unmodified AAV capsid protein. In some embodiments, any one of the following amino acid sequences can be inserted in the GH loop of a 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), STGKVPN (SEQ ID NO: 21), and ISDQTKH (SEQ ID NO: 42). In some embodiments, any one of the amino acid sequences set forth in SEQ ID NOs: 1-12 and 14-21 is inserted in the solvent-exposed GH loop of VP1 capsid protein in a rAAV. Additional details regarding amino acid sequences that can be inserted into the GH loop of a capsid protein, e.g., to facilitate transduction of a nucleic acid of interest to a retinal cell following IVT injection, are provided in WO2012145601, US9587282, US10202657, and US10214785, the contents of which related to amino acid sequences that can be inserted into the GH loop of a capsid protein are incorporated herein by reference.

[0154] In some embodiments, the rAAV particles comprise an AAV capsid protein, e.g., an AAV2 capsid protein, that includes any one of the following amino acid sequences: 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), STGKVPN (SEQ ID NO: 21), and ISDQTKH (SEQ ID NO: 42) 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; 86sf-6242343Attorney Docket No.62700-20017.40 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. In some embodiments, the rAAV particles comprise AAV2 capsid proteins comprising an 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 an AAV2 VP1 capsid protein. In some embodiments, the rAAV particles comprise AAV2 capsid proteins comprising the amino acid sequence LALGETTRPA (SEQ ID NO: 1) inserted between positions 587 and 588 of the AAV2 VP1 comprising the sequence of SEQ ID NO: 13.

[0155] In some embodiments, rAAV particles comprise the 7m8 variant capsid protein from AAV2 comprising the amino acid sequence LALGETTRPA (SEQ ID NO: 1) inserted in the GH loop of the AAV2 VP1 protein between positions 587 and 588 of the AAV2 VP1. In some embodiments, the rAAV particles comprise an AAV2 VP1 capsid protein comprising 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 comprising 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. FSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTNTPSGTTTQSRLQFSQAGASDI RDQSRNWLPGPCYRQQRVSKTSADNNNSEYSWTGATKYHLNGRDSLVNPGPAMASHK DDE EKFFPQSGVLIFGKQGSEKTNVDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQRGNLAL GETTRPARQAATADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGF GLK HPPPQILIKN (SEQ ID NO: 38)

[0156] In some embodiments, rAAV particles comprise the 7m8 variant capsid protein from AAV2 comprising the amino acid sequence LALGETTRPA (SEQ ID NO: 1) inserted between positions 587 and 588 of the AAV2 VP1. The sequence of the 7m8 variant capsid protein from AAV2 comprising the amino acid sequence LALGETTRPA (SEQ ID NO: 1) is inserted between positions 587 and 588 of the AAV2 VP1 is provided below: MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPGYKYLGPFNG LDKGEPVNEADAAALEHDKAYDRQLDSGDNPYLKYNHADAEFQERLKEDTSFGGNLG 87sf-6242343Attorney Docket No.62700-20017.40 RAVFQAKKRVLEPLGLVEEPVKTAPGKKRPVEHSPVEPDSSSGTGKAGQQPARKRLNFG QTGDADSVPDPQPLGQPPAAPSGLGTNTMATGSGAPMADNNEGADGVGNSSGNWHCD STWMGDRVITTSTRTWALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHC HFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEY QLPYVLGSAHQGCLPPFPADVFMVPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTG NNFTFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTNTPSGTTTQSRLQFSQAG ASDIRDQSRNWLPGPCYRQQRVSKTSADNNNSEYSWTGATKYHLNGRDSLVNPGPAM ASHKDDEEKFFPQSGVLIFGKQGSEKTNVDIEKVMITDEEEIRTTNPVATEQYGSVSTNL QRGNLALGETTRPARQAATADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHP SPLMGGFGLKHPPPQILIKNTPVPANPSTTFSAAKFASFITQYSTGQVSVEIEWELQKENS KRWNPEIQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTRYLTRNL (SEQ ID NO: 37)

[0157] In some embodiments, the rAAV particles comprise a capsid protein VP1 comprising the amino acid sequence LGETTRP (SEQ ID NO: 14) or LALGETTRPA (SEQ ID NO: 1) inserted between positions 587 and 588 of the capsid protein, wherein the amino acid residue numbering corresponds to an 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) or LALGETTRPA (SEQ ID NO: 1) inserted between positions 587 and 588 of the capsid protein, wherein the amino acid residue numbering corresponds to an 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) or LALGETTRPA (SEQ ID NO: 1) inserted between positions 587 and 588 of the capsid protein, wherein the amino acid residue numbering corresponds to an AAV2 VP1 capsid protein. In some embodiments, the rAAV particles comprise capsid proteins VP1, VP2, and VP3, wherein each of VP1, VP2, and VP3 comprise the amino acid sequence LGETTRP (SEQ ID NO: 14) or LALGETTRPA (SEQ ID NO: 1) inserted between positions 587 and 588 of the capsid protein, wherein the amino acid residue numbering corresponds to an AAV2 VP1 capsid protein.

[0158] 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, wherein the amino acid residue numbering corresponds to an 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, wherein the amino acid residue numbering corresponds to an AAV2 VP1 capsid protein. In some embodiments, the rAAV particles comprise a capsid protein 88sf-6242343Attorney Docket No.62700-20017.40 VP3 comprising 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 an AAV2 VP1 capsid protein. In some embodiments, the rAAV particles comprise capsid proteins VP1, VP2, and VP3, wherein each of VP1, VP2, and VP3 comprise 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 an AAV2 VP1 capsid protein.

[0159] In some embodiments, a recombinant virus and / or plasmid used to generate a rAAV virus comprises other transcriptional or regulatory elements, such as a poly A (polyadenylation) sequence, untranslated regions (UTRs), 3’ UTRs, or termination sequences. In some embodiments, more than one gene is expressed from the vector or plasmid using internal ribosome entry site (IRES) or similar element that allows co-expression of two or more proteins or create multigene, or polycistronic mRNA.

[0160] In some embodiments, the rAAV and / or 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, linker sequence(s) are inserted between two or more of the nucleic acid elements. In some embodiments, the heterologous nucleic acid encodes a therapeutic polypeptide, e.g., encodes aflibercept (or a functional fragment or functional variant thereof).

[0161] In some embodiments, the vector is a targeted vector, especially a targeted rAAV (e.g., AAV2.7m8) that shows higher infectivity of a specific cell, such as a retinal cell (e.g., a photoreceptor, a retinal ganglion cell, a Müller cell, a bipolar cell, an amacrine cell, a horizontal cell, or a retinal pigmented epithelium cell). Viral vectors for use in the disclosure can include those that exhibit low toxicity and / or low immunogenicity in an individual and expresses therapeutically effective quantities of the anti-VEGF agent (e.g., aflibercept) in an individual, e.g., a human. Any suitable method known in the art can be used in the biochemical purification of recombinant viruses (e.g., rAAV), e.g., for the preparation of pharmaceutical compositions described elsewhere herein. Recombinant AAV viruses can be harvested directly from cells, or from the culture media comprising cells. Virus can be purified using various biochemical means, such as gel filtration, filtration, chromatography, affinity purification, gradient ultracentrifugation, or size exclusion methods. In some embodiments, the virus is lyophilized.

[0162] In some embodiments, the rAAV particles comprise a 7m8 variant capsid protein, e.g., rAAV2.7m8, and a nucleic acid sequence that encodes an anti-VEGF agent (e.g., aflibercept, or a 89sf-6242343Attorney Docket No.62700-20017.40 functional fragment or functional variant thereof). In some embodiments, the rAAV particles (e.g., the 7m8 variant) have an increase in retinal cell infectivity of any 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% as compared to an AAV particle comprising the corresponding parental or unmodified AAV capsid protein. In some embodiments, the increase in infectivity of retinal cells is an increase of any of between 5% to 100%, between 5% to 95%, between 5% to 90%, between 5% to 85%, between 5% to 80%, between 5% to 75%, between 5% to 70%, between 5% to 65%, between 5% to 60%, between 5% to 55%, between 5% to 50%, between 5% to 45%, between 5% to 40%, between 5% to 35%, between 5% to 30%, between 5% to 25%, between 5% to 20%, between 5% to 15%, or between 5% to 10%, as compared to an AAV particle comprising the corresponding parental or unmodified AAV capsid protein.

[0163] In some embodiments, the increase in retinal cell infectivity of a rAAV variant, e.g., rAAV2.7m8, is any of 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 an AAV particle comprising the corresponding parental or unmodified AAV capsid protein. In some embodiments, the increase in infectivity is any of 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, as compared to an AAV particle comprising the corresponding parental AAV capsid protein. In some embodiments, the increase in infectivity is any of 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 an AAV particle comprising the corresponding parental or unmodified AAV capsid protein.

[0164] In some embodiments, the increase in retinal cell infectivity of a rAAV variant, e.g., rAAV2.7m8, is between 10-fold to 100-fold, between 10-fold to 95-fold, between 10-fold to 90- fold, between 10-fold to 85-fold, between 10-fold to 80-fold, between 10-fold to 75-fold, between 10-fold to 70-fold, between 10-fold to 65-fold, between 10-fold to 60-fold, between 10- fold to 55-fold, between 10-fold to 50-fold, between 10-fold to 45-fold, between 10-fold to 40- fold, between 10-fold to 35-fold, between 10-fold to 30-fold, between 10-fold to 25-fold, between 10-fold to 20-fold, or between 10-fold to 15-fold, as compared to an AAV particle comprising the corresponding parental or unmodified AAV capsid protein.

[0165] In some embodiments, the increase in retinal cell infectivity is between 2-fold to 20-fold, between 2-fold to 19-fold, between 2-fold to 18-fold, between 2-fold to 17-fold, between 2-fold 90sf-6242343Attorney Docket No.62700-20017.40 to 16-fold, between 2-fold to 15-fold, between 2-fold to 14-fold, between 2-fold to 13-fold, between 2-fold to 12-fold, between 2-fold to 11-fold, between 2-fold to 10-fold, between 2-fold to 9-fold, between 2-fold to 8-fold, between 2-fold to 7-fold, between 2-fold to 6-fold, between 2-fold to 5-fold, between 2-fold to 4-fold, or between 2-fold to 3-fold, as compared to an AAV particle comprising the corresponding parental or unmodified AAV capsid protein.

[0166] In some embodiments, an amino acid modification of a capsid protein described herein can confer an increase in an ability to cross an internal limiting membrane (ILM) in an eye of an individual, e.g., a human, as compared to the ability of an AAV particle comprising the corresponding parental or unmodified AAV capsid protein to cross the ILM in the eye of the subject. In some embodiments, the increase in the ability to cross the ILM of a rAAV variant, e.g., rAAV2.7m8, is an increase of any 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% as compared to an AAV particle comprising the corresponding parental or unmodified AAV capsid protein. In some embodiments, the increase in the ability to cross the ILM is an increase of between 5% to 100%, between 5% to 95%, between 5% to 90%, between 5% to 85%, between 5% to 80%, between 5% to 75%, between 5% to 70%, between 5% to 65%, between 5% to 60%, between 5% to 55%, between 5% to 50%, between 5% to 45%, between 5% to 40%, between 5% to 35%, between 5% to 30%, between 5% to 25%, between 5% to 20%, between 5% to 15%, or between 5% to 10%, as compared to the parental or unmodified AAV capsid protein.

[0167] In some embodiments, the increase in the ability to cross the ILM of a rAAV variant, e.g., rAAV2.7m8, is any of 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 an AAV particle comprising the corresponding parental AAV capsid protein. In some embodiments, the increase in the ability to cross the ILM is any of 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, as compared to an AAV particle comprising the corresponding parental AAV capsid protein. In some embodiments, the increase in the ability to cross the ILM is any of 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 an AAV particle comprising the corresponding parental or unmodified AAV capsid protein.

[0168] In some embodiments, the increase in the ability to cross the ILM of a rAAV variant, e.g., rAAV2.7m8, is between 10-fold to 100-fold, between 10-fold to 95-fold, between 10-fold to 91sf-6242343Attorney Docket No.62700-20017.40 90-fold, between 10-fold to 85-fold, between 10-fold to 80-fold, between 10-fold to 75-fold, between 10-fold to 70-fold, between 10-fold to 65-fold, between 10-fold to 60-fold, between 10- fold to 55-fold, between 10-fold to 50-fold, between 10-fold to 45-fold, between 10-fold to 40- fold, between 10-fold to 35-fold, between 10-fold to 30-fold, between 10-fold to 25-fold, between 10-fold to 20-fold, or between 10-fold to 15-fold as compared to an AAV particle comprising the corresponding parental or unmodified AAV capsid protein.

[0169] In some embodiments, the increase in the ability to cross the ILM of a rAAV variant, e.g., rAAV2.7m8, is between 2-fold to 20-fold, between 2-fold to 19-fold, between 2-fold to 18- fold, between 2-fold to 17-fold, between 2-fold to 16-fold, between 2-fold to 15-fold, between 2- fold to 14-fold, between 2-fold to 13-fold, between 2-fold to 12-fold, between 2-fold to 11-fold, between 2-fold to 10-fold, between 2-fold to 9-fold, between 2-fold to 8-fold, between 2-fold to 7-fold, between 2-fold to 6-fold, between 2-fold to 5-fold, between 2-fold to 4-fold, or between 2-fold to 3-fold, as compared to an AAV particle comprising the corresponding parental or unmodified AAV capsid protein.

[0170] In some embodiments, rAAV.7m8 comprising nucleic acid encoding aflibercept 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) into an eye or retinal cells of a subject 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) into an eye or retinal cells of a subject via intravitreal injection. In some embodiments, rAAV2.7m8 is used to deliver the nucleic acid sequence of the anti-VEGF agent (e.g., aflibercept) into the retinal cells of a subject. In some embodiments, the heterologous nucleic acid (e.g., a nucleic acid that encodes an anti-VEGF agent such as aflibercept) integrates into the target cell genome (e.g., retinal cell genome), resulting in long-term expression of, e.g., the anti-VEGF agent (such as aflibercept), in the target cell. In some embodiments, the viral vector delivers a plasmid or other extrachromosomal genetic element that comprises the heterologous nucleic acid (e.g., a nucleic acid that encodes an anti-VEGF agent such as aflibercept) to the target cell (e.g., retinal cell).

[0171] In some embodiments, the rAAV particles comprise a nucleic acid encoding a polypeptide comprising an amino acid sequence with any of 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 terminal repeats (ITRs). In some embodiments, the rAAV particles comprise a nucleic acid encoding a polypeptide comprising an amino acid sequence with at least about 95% identity to the amino acid sequence of SEQ ID NO: 35 and flanked by AAV2 inverted terminal repeats (ITRs). In 92sf-6242343Attorney Docket No.62700-20017.40 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 terminal repeats (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 terminal repeats (ITRs). The sequence of SEQ ID NO: 35 is provided below: SDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFI ISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVLSPSHGIELSVGEKLVLNCTARTEL NVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKK NSTFVRVHEKDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFF LYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 35)

[0172] In some embodiments, the rAAV particles comprise a nucleic acid with 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 homology to the nucleic acid sequence of SEQ ID NO: 36, and wherein the nucleic acid is flanked by AAV2 inverted terminal repeats (ITRs). The sequence of SEQ ID NO: 36 is provided in FIG.3. In some embodiments, the rAAV particles comprise a nucleic acid with 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 homology to the nucleic acid sequence of aflibercept (e.g., SEQ ID NO: 36), and wherein the nucleic acid is flanked by AAV2 inverted terminal repeats (ITRs). In some embodiments, the nucleic acid sequence of aflibercept is derived from its amino acid sequence. In some embodiments, the nucleic acid sequence of aflibercept is codon optimized to improve its expression in a subject. In some embodiments, the rAAV particles comprise a nucleic acid with any of at least about 75%, at least about 80%, at least about 81%, at least about 82%, at 93sf-6242343Attorney Docket No.62700-20017.40 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 homology to the nucleic acid sequence of SEQ ID NO: 40, and wherein the nucleic acid is flanked by AAV2 inverted terminal repeats (ITRs). In some embodiments, the rAAV particles comprise a nucleic acid comprising the nucleic acid sequence of SEQ ID NO: 40. In some embodiments, the rAAV particles comprise a nucleic acid comprising the nucleic acid sequence of SEQ ID NO: 40, and wherein the nucleic acid is flanked by AAV2 inverted terminal repeats (ITRs). atggtgtcatactgggatactggagtcttgctttgtgccctgctgtcctgcctcctcctgactgg ctccagctcgggctcagataccggtcgccccttcgtggagatgtactccgagatcccggaaatta tccacatgactgaggggcgcgaacttgtgatcccctgccgggtcaccagcccgaacattactgtg actttgaagaagttccccctggacaccctgattccggatgggaagagaattatctgggattcacg gaagggattcatcatcagcaacgcgacctacaaggaaattggcctcctcacttgcgaagccactg tgaacggacacttgtacaagaccaactacctgacccaccgccagaccaacaccatcatcgacgtc gtcctgtccccttcgcacgggatcgagctctcggtgggagagaagttggtgcttaactgcaccgc ccggacggaactgaatgtgggaatcgacttcaactgggaatacccgtccagcaagcatcagcata agaagctggtgaaccgggacctcaagactcagtccggcagcgaaatgaagaagttcctgtcgacc ctcactattgacggagtgaccagatccgaccagggcctctacacttgcgccgcttccagcggact catgaccaagaagaacagcactttcgtgagggtgcatgagaaggacaagacccacacgtgtccgc cgtgcccagccccagagctgctgggaggcccttccgtgttcctgtttccgcccaagccaaaggat accctgatgatctcaaggacccctgaggtcacatgcgtcgtggtggatgtgtcgcacgaggaccc tgaagtcaaattcaattggtatgtggacggagtggaagtccacaacgcgaaaaccaagccgagag aagaacagtacaattccacctaccgggtggtgtcggtgctgactgtgctgcaccaggactggctc aacggaaaggagtacaagtgcaaggtgtccaacaaggctctgcccgcacctattgaaaagaccat ctccaaggccaagggtcaacctcgcgagcctcaggtgtacactctgcctccaagccgggacgaac tgactaagaaccaagtctctctgacctgtttggtgaagggcttctacccgtcagacatcgcagtg gagtgggagtcaaacggtcagccggagaacaactacaaaacaaccccccccgtgctggactccga cggctccttcttcctgtactccaagcttaccgtggataagagccgctggcaacagggcaacgtgt tttcctgctccgtcatgcacgaagccctgcacaaccattatacccagaagtccctgtcgctgtcc cccgggaaatag (SEQ ID NO: 40)

[0173] In some embodiments, the rAAV particles comprise a nucleic acid encoding a polypeptide comprising an amino acid sequence with any of 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 94sf-6242343Attorney Docket No.62700-20017.40 acid sequence of SEQ ID NO: 41 and 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 with at least about 95% identity to the amino acid sequence of SEQ ID NO: 41 and flanked by AAV2 inverted terminal repeats (ITRs). In some embodiments, the rAAV particles comprise a nucleic acid encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 41 and flanked by AAV2 inverted terminal repeats (ITRs). In some embodiments, the rAAV particles comprise a nucleic acid encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 41. MVSYWDTGVLLCALLSCLLLTGSSSGSDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITV TLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDV VLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLST LTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHEKDKTHTCPPCPAPELLGGPSVFLFPPKPKD TLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWL NGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAV EWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS PGK (SEQ ID NO: 41)

[0174] In some embodiments, the nucleic acid sequence of aflibercept is codon-optimized for expression in a primate or a human subject. Construction of a synthetic gene corresponding to the aflibercept amino acid sequence has been described in literature, e.g., Kanda A, Noda K, Saito W, Ishida S. Aflibercept Traps Galectin-1, an Angiogenic Factor Associated with Diabetic Retinopathy. Scientific Reports 5:17946 (2015) (describing “VEGF-TrapR1R2(corresponding to aflibercept) cDNA was generated as a synthetic gene by IDT (Coralville, IA)”). Given the available amino acid sequence of aflibercept, any method known in the art can be used to generate the cDNA of aflibercept for use in a gene therapy or a rAAV described herein.

[0175] Codon optimization can be achieved with any method known in the art. Codon optimization refers to a process of modifying a nucleic acid sequence for enhanced expression of a gene in target or host cells of interest, e.g., human retinal cells, by replacing at least one codon (e.g., about or more than 1, 2, 3, 4, 5, 10, 15, 20, 25, 50, 100 or more codons) of a native sequence with codons that are used more frequently or are most frequently used in the host cell while maintaining the native amino acid sequence. Codon usage tables are readily available, including for examples, 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). 95sf-6242343Attorney Docket No.62700-20017.40

[0176] Homology refers to the percent conservation of residues of an alignment between two sequences, including, but not limited to functional fragments, sequences comprising insertions, deletions, substitutions, pseudofragments, pseudogenes, splice variants or artificially optimized sequences.

[0177] In some embodiments, the rAAV particles comprise a nucleic acid encoding aflibercept. In some embodiments, the polypeptide is aflibercept.

[0178] As used herein, “aflibercept” refers to a polypeptide or protein sequence, or a functional fragment or variant or mutant thereof, with any of at least 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more, or 100% homology to the aflibercept amino acid sequence identified above (SEQ ID NO: 35). Homology refers to the percent conservation of residues of an alignment between two sequences, including, but not limited to functional fragments, sequences comprising insertions, deletions, substitutions, pseudofragments, pseudogenes, splice variants or artificially optimized sequences.

[0179] In some embodiments, the amino acid sequence of aflibercept is any of 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 sequence encoding aflibercept disclosed herein is compared to the corresponding cDNA sequence of the aflibercept amino acid sequence identified above, and shows any of 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% sequence homology between the nucleic acid sequences of aflibercept (e.g., SEQ ID NO: 36). In some embodiments, aflibercept is any of at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% spatially homologous to aflibercept (e.g., in terms of its secondary, tertiary, and quaternary structure or conformation). In some embodiments, aflibercept is any of at most 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% spatially homologous to the aflibercept used in the standard of care (e.g., secondary, tertiary, and quaternary structure or conformation).

[0180] In some embodiments, the aflibercept gene product, or aflibercept transgene, as included in a gene therapy based on a rAAV, comprises a capsid variant as disclosed herein (e.g., the 7m8 variant), encodes a protein, fusion protein, or polypeptide that has any of at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% homology to the above amino acid sequence of SEQ ID NO: 35, or between the corresponding 96sf-6242343Attorney Docket No.62700-20017.40 cDNA sequences of aflibercept (e.g., cDNA of aflibercept sequence used in a gene therapy compared to SEQ ID NO: 36). In some embodiments, the methods compositions disclosed herein comprise a functional fragment of aflibercept, or a variant or mutant thereof. In some embodiments, the nucleic acid sequence of aflibercept is modified or codon-optimized to enhance its activity, expression, stability, and / or solubility in vivo.

[0181] Aflibercept is a 115 kDa fusion protein, which can be glycosylated. Aflibercept comprises an IgG backbone fused to extracellular VEGF receptor sequences of the human VEGFR-1 and VEGFR-2, and functions like a soluble decoy receptor by binding VEGF-A with a greater affinity than its natural or endogenous receptors. See, for example, Stewart MW. Aflibercept (VEGF Trap-eye): the newest anti-VEGF drug. Br. J. Ophthalmol.2012 Sep;96(9):1157-8. Aflibercept’s high affinity for VEGF interferes or disrupts subsequent binding and activation of native or endogenous VEGF receptors. Reduced VEGF activity can lead to decreased angiogenesis and vascular permeability. Inhibition of placental growth factor PIGF and VEGF-B by aflibercept may also contribute to the treatment of ocular diseases or disorders characterized by abnormal (e.g., excessive) angiogenesis and / or neovascularization. PIGF has been associated with angiogenesis and certain ocular diseases or disorders, such as wet AMD, may be associated with elevated levels of PIGF. VEGF-B overexpression can be associated with breakdown of the blood-retinal barrier and retinal angiogenesis. Thus, inhibition of VEGF-A, VEGF-B, and PIGF may all contribute to the efficacy of aflibercept. Methods for Preparation of Vectors for Delivering Transgenes to Target Cells

[0182] In some embodiments, the rAAV particles are manufactured using any method known in the art. In some embodiments, the rAAV particles are manufactured using a baculovirus expression vector system in Sf9 cells. Sf9 cells are an insect cell culture cell line commonly used for recombinant protein production using baculovirus. In some embodiments, the rAAV particles are manufactured using two baculoviruses in Sf9 cells. In some embodiments, the rAAV particles are manufactured using two baculoviruses in Sf9 cells, wherein a first baculovirus encodes the genes for AAV2 Rep and AAV2.7m8 Cap proteins and a second baculovirus encodes an anti- VEGF agent. In some embodiments, the rAAV particles are manufactured using two baculoviruses in Sf9 cells, wherein a first baculovirus encodes the genes for AAV2 Rep and AAV2.7m8 Cap proteins and a second baculovirus encodes an aflibercept (e.g., human aflibercept) cDNA expression cassette. In some embodiments, the rAAV particles are manufactured using two baculoviruses in Sf9 cells, wherein a first baculovirus encodes the genes for AAV2 Rep and AAV2.7m8 Cap proteins and a second baculovirus comprises a nucleic acid encoding a polypeptide comprising an amino acid sequence with at least about 95% identity to 97sf-6242343Attorney Docket No.62700-20017.40 the amino acid sequence of SEQ ID NO: 35 and flanked by AAV2 inverted terminal repeats (ITRs). In some embodiments, the polypeptide comprises the amino acid sequence of SEQ ID NO: 35. In some embodiments, the polypeptide is aflibercept. Unit Doses for rAAV particles

[0183] In some embodiments, the unit dose of 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.

[0184] In some embodiments, the unit dose of rAAV particles is administered to one eye of the individual. In some embodiments, the unit dose of rAAV particles is expressed as the number of vector genomes (vg). In some embodiments, the unit dose is about 6 × 1011vector genomes (vg) or less of the rAAV particles. In some embodiments, the unit dose is about 1×1010to about 2×1010, between about 2×1010to about 3×1010, between about 3×1010to about 4×1010, between about 4×1010to about 5×1010, between about 5×1010to about 6×1010, between about 6×1010to about 7×1010, between about 7×1010to about 8×1010, between about 8×1010to about 9×1010, between about 9×1010to about 10×1010, between about 1×1011to about 2×1011, between about 2×1011to about 3×1011, between about 3×1011to about 4×1011, between about 4×1011to about 5×1011, or between about 5×1011to about 6×1011vg of the rAAV particles, including any value within these ranges, of the rAAV particles. In some embodiments, the unit dose is about 6×1010vector genomes (vg) to about 2...

Claims

Attorney Docket No.62700-20017.40 CLAIMS 1. A method for treating an ocular neovascular disease in an individual, the method comprising administering (i) a single unit dose of recombinant adeno-associated virus (rAAV) particles to one eye of the individual, wherein the individual is a human, wherein the rAAV particles comprise: a) a nucleic acid encoding a polypeptide comprising an amino acid sequence with 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 an 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 an AAV2 VP1 capsid protein, and (ii) a topical steroid treatment beginning prior to the administration of the single unit dose of rAAV to the one eye, wherein the topical steroid treatment comprises (a) about four administrations of the topical steroid per day daily for about 6 weeks, (b) followed by about three administrations of the topical steroid per day daily for about 5 weeks, (c) followed by about two administrations of the topical steroid per day daily for about 12 weeks, and (d) followed by about one administration of the topical steroid per day daily for about 4 weeks.

2. The method of claim 1, wherein the method further comprises one administration of the topical steroid every other day for about 2 weeks following step (ii)(d).

3. The method of claim 1 or 2, wherein an initial topical steroid is administered on Day 1 and the unit dose of rAAV particles is administered on any one of Day 4, Day 5, Day 6, Day 7, Day 8, Day 9, Day 10, or Day 11.

4. The method of any one of claims 1-3, wherein the topical steroid treatment is a difluprednate treatment.

5. The method of claim 4, wherein the difluprednate treatment is for ophthalmic use.

6. The method of any one of claims 1-5, wherein each administration of the topical steroid comprises a dose of about 1 µg to about 3 µg of the difluprednate. 170sf-6242343Attorney Docket No.62700-20017.40 7. The method of any one of claims 1-6, wherein each administration of the topical steroid comprises a dose of about 2.5 µg of the difluprednate.

8. The method of any one of claims 1-7, wherein the method of treating results in reducing a retinal fluid in the eye of the individual with an ocular neovascular disease.

9. The method of claim 8, wherein the retinal fluid is subretinal fluid (SRF) or intraretinal fluid (IRF).

10. The method of any one of claims 1-9, wherein the individual has received one or more treatment of an anti-VEGF agent from about 1 week to about 8 weeks prior to administration of the unit dose of rAAV particles.

11. The method of any one of claims 3-10, further comprising administering a first loading dose of the anti-VEGF agent to the one eye of the individual from about Day -56 to about Day -49 and a second loading dose of the anti-VEGF agent to the one eye of the individual from about Day -28 to about Day -21.

12. The method of claim 8 or 9, wherein the amount or presence of retinal fluid in the one eye of the individual is refractory to prior treatment with an anti-VEGF agent.

13. The method of any one of claims 10-12, wherein the anti-VEGF agent is aflibercept.

14. The method of any one of claims 1-13, wherein the unit dose of rAAV particles is between about 6 × 1010to about 6 × 1011vector genomes per eye (vg / eye).

15. The method of any one of claims 1-14, wherein the unit dose of rAAV particles is about 6 × 1010vector genomes per eye (vg / eye).

16. The method of any one of claims 1-15, comprising administering the unit dose of rAAV particles to the one eye of the individual about 4 days to about 11 days after administration of the anti-VEGF agent. 171sf-6242343Attorney Docket No.62700-20017.40 17. The method of claim 16, comprising administering the anti-VEGF agent to the one eye of the individual on Day 1, and administering the unit dose of rAAV particles to the one eye of the individual on any one of Day 4, Day 5, Day 6, Day 7, Day 8, Day 9, Day 10, or Day 11.

18. The method of any one of claims 1-17, further comprising administering one or more rescue anti-VEGF agents after administering the rAAV particles.

19. The method of any one of claims 10-18, wherein the anti-VEGF agent comprises aflibercept.

20. The method of claim 19, wherein the aflibercept is administered at a dose of about 2 mg by intravitreal injection.

21. The method of any one of claims 1-20, wherein the individual has one or more symptoms of an ocular neovascular disease in the contralateral eye.

22. The method of any one of claims 1-21, further comprising administering a unit dose of rAAV particles to the contralateral eye of the individual.

23. The method of claim 22, wherein the administering the unit dose of rAAV particles to the contralateral eye is up to about 2 weeks after administering the unit dose of rAAV particles to the one eye.

24. The method of claim 22, wherein: (a) the administering the unit dose of rAAV particles to the contralateral eye is on the same day as the administering the unit dose of rAAV particles to the one eye; or (b) the administering the unit dose of rAAV particles to the contralateral eye is between about 1 day to about 3 years after administering the unit dose of rAAV particles to the one eye.

25. The method of claim 23 or 24, wherein the unit dose of rAAV particles administered to the contralateral eye of the individual comprises the same vector genomes per eye (vg / eye) than the unit dose of rAAV particles administered to the one eye of the individual. 172sf-6242343Attorney Docket No.62700-20017.40 26. The method of claim 22, wherein the administering the unit dose of rAAV particles to the contralateral eye is at least about 2 weeks after administering the unit dose of rAAV particles to the one eye.

27. The method of any one of claims 1-26, wherein the individual has a baseline Best Corrected Visual Acuity (BCVA) score of between about 35 to about 78 Early Treatment Diabetic Retinopathy Study (ETDRS) letters.

28. The method of any one of claims 1-27, wherein the nucleic acid comprises the nucleic acid sequence of SEQ ID NO: 40 or a sequence having at least 85% identity thereto.

29. The method of any one of claims 1-28, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO:

35.

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

41.

31. The method of any one of claims 1-30, wherein the polypeptide is aflibercept.

32. The method of any one of claims 1-31, 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.

33. The method of any one of claims 1-32, wherein the nucleic acid comprises, in the 5’ to 3’ order: (a) a first enhancer region; (b) a promoter region; (c) a 5'UTR region; (d) a nucleic acid encoding a polypeptide comprising an amino acid sequence with 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). 173sf-6242343Attorney Docket No.62700-20017.40 34. The method of claim 32 or 33, 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.

35. The method of any one of claims 32-34, 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.

36. The method of any one of claims 33-35, 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.

37. The method of any one of claims 33-36, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 35 or a sequence having at least 95% identity thereto.

38. The method of any one of claims 33-37, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 41 or a sequence having at least 95% identity thereto.

39. The method of any one of claims 33-38, wherein the polypeptide is aflibercept.

40. The method of any one of claims 32-39, wherein the 5'UTR region comprises, in 5' to 3' order, a TPL sequence comprising the sequence of SEQ ID NO: 24 or a sequence having at least 85% identity thereto, and an eMLP sequence comprising the sequence of SEQ ID NO: 25 or a sequence having at least 85% identity thereto.

41. The method of any one of claims 32-40, wherein the second enhancer region comprises a full EES sequence comprising the sequence of SEQ ID NO: 26 or a sequence having at least 85% identity thereto.

42. The method of any one of claims 32-41, wherein the polyadenylation site comprises a HGH polyadenylation site comprising the sequence of SEQ ID NO: 27 or a sequence having at least 85% identity thereto. 174sf-6242343Attorney Docket No.62700-20017.40 43. The method of any one of claims 1-31, wherein the nucleic acid further comprises (a) a first enhancer region comprising a CMV sequence comprising the sequence of SEQ ID NO: 22 or a sequence having at least 85% identity thereto; (b) a promoter region, comprising a CMV sequence comprising the sequence of SEQ ID NO: 23 or a sequence having at least 85% identity thereto; (c) a 5'UTR region comprising, in 5' to 3' order, a TPL sequence comprising the sequence of SEQ ID NO: 24 or a sequence having at least 85% identity thereto, and an eMLP sequence comprising the sequence of SEQ ID NO: 25 or a sequence having at least 85% identity thereto; (d) a second enhancer region comprising a full EES sequence comprising the sequence of SEQ ID NO: 26 or a sequence having at least 85% identity thereto; and (e) a HGH polyadenylation site comprising the sequence of SEQ ID NO: 27 or a sequence having at least 85% identity thereto.

44. The method of any one of claims 1-43, wherein the nucleic acid comprises the sequence of SEQ ID NO: 39 or a sequence having at least 85% identity thereto.

45. The method of any one of claims 1-44, 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 an AAV2 VP1 capsid protein.

46. The method of any one of claims 1-45, wherein the AAV2 capsid protein comprises the amino acid sequence LGETTRP (SEQ ID NO: 14) inserted between positions 587 and 588 of the AAV2 VP1 comprising the sequence of SEQ ID NO:

13.

47. The method of any one of claims 1-46, wherein the AAV2 capsid protein comprises the amino acid sequence LALGETTRPA (SEQ ID NO: 1) inserted between positions 587 and 588 of the AAV2 VP1 comprising the sequence of SEQ ID NO:

13.

48. The method of any one of claims 1-45, wherein the rAAV particles comprise an AAV2 VP1 capsid protein comprising 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.

49. The method of any one of claims 1-48, wherein the administration of the unit dose of rAAV particles to the one eye and / or the contralateral eye is by intravitreal administration. 175sf-6242343Attorney Docket No.62700-20017.40 50. The method of any one of claims 1-49, wherein the unit dose of rAAV particles is in a pharmaceutical formulation.

51. The method of claim 50, wherein the pharmaceutical formulation comprises the rAAV particles, sodium chloride, sodium phosphate and a surfactant.

52. The method of claim 51, wherein the pharmaceutical formulation comprises about 150 to about 200 mM sodium chloride, about 1 to about 10 mM monobasic sodium phosphate, about 1 to about 10 mM dibasic sodium phosphate, about 0.0005% (w / v) to about 0.005% (w / v) poloxamer 188, and about 6 × 1011to about 6 × 1012vector genomes (vg) per mL (vg / mL) of the rAAV particles, wherein the pharmaceutical formulation has a pH of about 7.0 to about 7.

5.

53. The method of claim 52, wherein the pharmaceutical formulation comprises about 180 mM sodium chloride, about 5 mM monobasic sodium phosphate, about 5 mM dibasic sodium phosphate, about 1.2 × 1012vg / mL of the rAAV particles, and about 0.001% (w / v) poloxamer 188, wherein the pharmaceutical formulation has a pH of about 7.

3.

54. The method of claim 52, wherein the pharmaceutical formulation comprises about 180 mM sodium chloride, about 1.8 mM monobasic sodium phosphate, about 8.2 mM dibasic sodium phosphate, about 1.2 × 1012vg / mL of the rAAV particles, and about 0.001% (w / v) poloxamer 188, wherein the pharmaceutical formulation has a pH of about 7.

3.

55. The method of any one of claims 1-52, wherein the unit dose of rAAV particles administered to the one eye and / or to the contralateral eye in a volume of about 25 μL to about 250 μL.

56. The method of claim 55, wherein the unit dose of rAAV particles administered to the one eye and / or to the contralateral eye comprises a volume of about 100 μL.

57. The method of claims 53, 54, and 55, wherein the unit dose of rAAV particles administered to the one eye and / or to the contralateral eye comprises a volume of about 50 μL. 176sf-6242343Attorney Docket No.62700-20017.40 58. The method of claim 55, wherein the unit dose of rAAV particles administered to the one eye and / or to the contralateral eye comprises a volume of about 30 μL.

59. The method of any one of claims 1-58, wherein the individual received prior treatment for the ocular neovascular disease with an anti-VEGF agent.

60. The method of claim 59, wherein the individual has received 1 or 2 injections of an anti-VEGF agent in the one eye and / or in the contralateral eye prior to administration of the rAAV particles in the one eye and / or in the contralateral eye.

61. The method of any one of claims 1-9, 12-15, and 21-58, wherein the individual has not received prior treatment for the ocular neovascular disease with an anti-VEGF agent.

62. The method of any one of claims 59-61, wherein the anti-VEGF agent is aflibercept.

63. The method of any one of claims 1-62, wherein the ocular neovascular disease is wet age-related macular degeneration (AMD), retinal neovascularization, choroidal neovascularization diabetic retinopathy, proliferative diabetic retinopathy, retinal vein occlusion, central retinal vein occlusion, branched retinal vein occlusion, diabetic macular edema, diabetic retinal ischemia, ischemic retinopathy, diabetic retinal edema, or any combination thereof.

64. The method of claim 63, wherein the ocular neovascular disease is wet age-related macular degeneration (AMD).

65. The method of claim 63, wherein the ocular neovascular disease is diabetic macular edema (DME).

66. The method of claims 1-65, wherein the topical steroid comprises a difluprednate ophthalmic emulsion 0.05%.

67. The method of claim 66, wherein the difluprednate ophthalmic emulsion 0.05% is in a pharmaceutical formulation. 177sf-6242343Attorney Docket No.62700-20017.40 68. The method of claim 67, wherein the pharmaceutical formulation comprises about 0.5 mg difluprednate per mL of the ophthalmic emulsion, wherein the pharmaceutical formulation has a pH of about 5.2 to about 5.

8.

69. The method of claim 68, wherein the pharmaceutical formulation further comprises boric acid, castor oil, edetate disodium, glycerin, polysorbate 80, sodium acetate, sodium hydroxide, and sorbic acid 0.1%.

70. The method of claims 66-69, wherein the administration is about one drop of the difluprednate ophthalmic emulsion 0.05% eyedrop per administration.

71. The method of claim 32 or 33, wherein the nucleic acid comprises the sequence of SEQ ID NO: 43 or a sequence having at least 85% identity thereto. 178sf-6242343

Images