Recombinant adeno-associated virus reduces CST changes in neovascular AMD

Abstract

Provided herein is a method of treating an ocular neovascular disease in an individual by reducing variation in central zone retinal thickness (CST) or central retinal thickness (CRT), the method comprising administering to the eye of the individual a unit dose of a recombinant adeno-associated virus (rAAV) particle, the rAAV particle comprising: a) a nucleic acid encoding a polypeptide comprising an amino acid sequence having at least about 95% identity to the amino acid sequence of SEQ ID NO: 35 and flanking AAV2 inverted terminal repeats (ITRs); and b) an AAV2 capsid protein comprising the amino acid sequence LGETTRP (SEQ ID NO: 14) inserted between positions 587 and 588 of the AAV2 capsid protein, the numbering of the amino acid residues corresponding to that of the AAV2 VP1 capsid protein.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority under 35 U.S.C. § 119(e) to U.S. Provisional Application No. 63 / 310,865, filed February 16, 2022, and U.S. Provisional Application No. 63 / 316,705, filed March 4, 2022, each of which is incorporated by reference in its entirety.

[0002] Sequence Listing Description An official copy of the Sequence Listing is submitted herewith as a WIPO standard ST.26 format XML file with the file name "17234-034WO1.xml," created on February 6, 2023, and 49 kilobytes in size. This Sequence Listing, submitted through the USPTO Patent Center, is a part of the present specification and is incorporated herein by reference in its entirety.

[0003] The present disclosure relates to methods of treating ocular neovascular diseases and disorders by reducing variation in central retinal thickness (CST) or central retinal thickness (CRT) in an individual, comprising administering to the eye of the individual a single unit dose of recombinant adeno-associated virus (rAAV) particles encoding an anti-VEGF agent (e.g., aflibercept). [Background technology]

[0004] Age-related macular degeneration (AMD) is a degenerative eye disease that affects the macula, a small, light-sensitive area in the center of the retina responsible for reading and fine vision. Pathologies affecting the macula impair central vision while leaving peripheral vision intact. In severe cases, the disease can lead to central blindness. AMD is a significant cause of vision loss in the US population over age 65, with an estimated prevalence of any form of AMD among those over age 40 of 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. wAMD is characterized by choroidal neovascularization (CNV), which is the infiltration of abnormal blood vessels from the underlying choroid layer into the retina, resulting in retinal cell damage and central blindness. This abnormal angiogenic process is regulated by growth factors, particularly vascular endothelial growth factor (VEGF). The standard treatments for wAMD are 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 a leading cause of vision loss in the working-age population. DR can be nonproliferative (NPDR), which is characterized by no new blood vessel growth, or proliferative (PDR), which is characterized by new abnormal blood vessel growth within the retina or choroid. Diabetic macular edema (DME), a complication of DR, is another example of an eye disease affecting the macula. DME affects up to 10% of diabetic patients and is caused by fluid accumulation in the macula. DME is the most frequent cause of vision loss in DR patients. Available treatments for 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 can prevent the ligands from binding to vascular endothelial growth factor receptors (VEGFR), VEGFR-1, and VEGFR-2, thereby inhibiting angiogenesis and reducing vascular permeability. Aflibercept consists of domain 2 of VEGFR-1 and domain 3 of VEGFR-2 fused to the Fc fragment of IgG1.

[0007] Central retinal thickness (CST) is a measure of macular thickness in the central subfield region surrounding the fovea. In nAMD, CST increases due to fluid accumulation. Previous studies and treatments have shown a correlation between CST variation and visual acuity.

[0008] Previous studies based on clinical trial data have shown that greater retinal thickness fluctuations over the course of intravitreal anti-vascular endothelial growth factor (anti-VEGF) therapy for neovascular age-related macular degeneration (nAMD) are associated with poorer visual outcomes (Lai et al. J.Pers. Med. 2021,11,1024). Lai et al. demonstrated that, when intravitreal anti-VEGF therapy (bevacizumab, ranibizumab, or aflibercept) was administered for the treatment of nAMD, a significant correlation was found between CST fluctuations and visual acuity at 2 years, calculated using spectral-domain optical coherence tomography (OCT) central subfield thickness (CST) fluctuations, indicating that eyes with lower CST fluctuations at 2 years had better visual acuity. Eyes with the least CST fluctuations were associated with the greatest mean visual acuity improvement at 2 years. These findings suggest that eyes receiving anti-VEGF therapy for nAMD with more stable OCT CST fluctuations during the follow-up period are associated with better visual outcomes. Thus, comparative data from the AMD Treatment Trial (CATT) and the Age-Related Choroidal Neovascularization (IVAN) Trial of VEGF inhibition showed that greater CST variability was associated with an increased risk of fibrosis and macular atrophy, which may result in decreased visual acuity (VA).

[0009] Current standard-of-care anti-VEGF agents, such as aflibercept, require re-administration every 4–8 weeks via intravitreal (IVT) injection to achieve optimal treatment outcomes and maintain vision. Adherence to such regimens is burdensome for patients, their caregivers, and healthcare systems, and most patients fall out of compliance with optimal regimens over time, which correlates with vision loss (Khanani AM, et al.). Additionally, complications exist, including endophthalmitis, retinal detachment, traumatic cataracts, and elevated intraocular pressure (IOP), and the risk of these complications may increase with repeated IVT injections (Falavarjani et al., (2013) Eye (Lond), 27(7):787–794). The art needs a treatment for ocular neovascular diseases such as wAMD, DR or DME that is effective, reduces the risk of adverse effects, and is suitable for long-term patient compliance.In this regard, the art also needs a more effective method for reducing the variability of CST. [Prior art documents] [Non-patent literature]

[0010] [Non-Patent Document 1] Klein et al.,(2011)Arch Ophthalmol,129(1):75-80 [Non-patent document 2] Lai et al. J.Pers. Med. 2021,11,1024 [Non-patent document 3] Falavarjani et al.,(2013)Eye(Lond),27(7):787-794 Summary of the Invention

[0011] In one aspect, provided herein is a method of treating an ocular neovascular disease in an individual by reducing variation in central retinal thickness (CST), the method comprising administering about 6×10 11 The method includes administering a unit dose of recombinant adeno-associated virus (rAAV) particles equal to or less than a vector genome (vg), wherein the individual is a human, and the rAAV particles comprise: (a) nucleic acid encoding a polypeptide comprising an amino acid sequence having at least about 95% identity to the amino acid sequence of SEQ ID NO: 35 and flanking AAV2 inverted terminal repeats (ITRs), and (b) an AAV2 capsid protein having a sequence comprising or consisting of the amino acid sequence LGETTRP (SEQ ID NO: 14) inserted between positions 587 and 588 of the AAV2 capsid protein, where the numbering of the amino acid residues corresponds to that of the AAV2 VP1 capsid protein. In some embodiments, the method includes reducing retinal fluid in an eye of the individual.

[0012] In one aspect, provided herein is a method of treating an ocular neovascular disease in an individual by reducing variation in central retinal thickness (CST), the method comprising administering about 6×10 11 The method includes administering a unit dose of recombinant adeno-associated virus (rAAV) particles equal to or less than a vector genome (vg), wherein the individual is a human, and the rAAV particles comprise: (a) nucleic acid encoding a polypeptide comprising an amino acid sequence having at least about 95% identity to the amino acid sequence of SEQ ID NO: 35 and flanking AAV2 inverted terminal repeats (ITRs), and (b) an AAV2 capsid protein having a sequence comprising or consisting of the amino acid sequence LGETTRP (SEQ ID NO: 14) inserted between positions 587 and 588 of the AAV2 capsid protein, where the numbering of the amino acid residues corresponds to that of the AAV2 VP1 capsid protein. In some embodiments, the method includes reducing retinal fluid in an eye of the individual.

[0013] In another aspect, provided herein is a method for reducing CST variation in the eye of an individual with an ocular neovascular disease, the method comprising administering a unit dose of rAAV particles to one eye of the individual, wherein the individual is human, and the rAAV particles comprise: (a) nucleic acid encoding a polypeptide comprising an amino acid sequence having at least about 95% identity to the amino acid sequence of SEQ ID NO: 35 and flanking AAV2 inverted terminal repeats (ITRs); and (b) an AAV2 capsid protein having a sequence comprising or consisting of the amino acid sequence LGETTRP (SEQ ID NO: 14) inserted between positions 587 and 588 of the AAV2 capsid protein, where the numbering of the amino acid residues corresponds to that of the AAV2 VP1 capsid protein. In some embodiments, the individual has received at least one treatment with an anti-VEGF agent within about 2 to 12 weeks prior to administering the unit dose of rAAV particles. In some embodiments, the amount or presence of retinal fluid in one eye of the individual is resistant to pretreatment with an anti-VEGF agent. In some embodiments, the anti-VEGF agent is aflibercept. In some embodiments, the retinal fluid in one eye is reduced by at least about 60%. In some embodiments, the retinal fluid in one eye is reduced by about 80% compared to the level of retinal fluid in one eye of the individual before administering the rAAV to the individual. In some embodiments, the retinal fluid is subretinal fluid (SRF) or intraretinal fluid (IRF). In some embodiments, the unit dose of rAAV particles is about 6 x 10 per eye. 11 vector genome (vg / eye) or less.

[0014] In another aspect, provided herein is a method of treating an ocular neovascular disease in an individual by reducing variation in CST, the method comprising: (a) administering an anti-VEGF agent to one eye of the individual; and (b) administering, after administration of the anti-VEGF agent, about 6×10 VEGF cells to one eye of the individual. 11The method includes administering a unit dose of recombinant adeno-associated virus (rAAV) particles equal to or less than a vector genome (vg), wherein the individual is a human, and the rAAV particles comprise: (i) a nucleic acid encoding a polypeptide comprising an amino acid sequence having at least about 95% identity to the amino acid sequence of SEQ ID NO: 35 and flanking AAV2 inverted terminal repeats (ITRs); and (ii) an AAV2 capsid protein having a sequence comprising the amino acid sequence LGETTRP (SEQ ID NO: 14) inserted between positions 587 and 588 of the AAV2 capsid protein, where the numbering of the amino acid residues corresponds to that of the AAV2 VP1 capsid protein. In some embodiments, the method includes administering the unit dose of the rAAV particles to one eye of the individual about one week or about seven days after administration of an anti-VEGF agent. In some embodiments, the method includes administering the unit dose of the rAAV particles to one eye of the individual about one week to about two weeks after administration of the anti-VEGF agent. In some embodiments, the method comprises administering a unit dose of rAAV particles to one eye of the individual about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, about 14 days, or about 15 days after administration of the anti-VEGF agent. In some embodiments, the method comprises administering an anti-VEGF agent to one eye of the individual on day 1 and administering a unit dose of rAAV particles to one eye of the individual on day 8. In some embodiments, the anti-VEGF agent comprises aflibercept. In some embodiments, the aflibercept is administered at a dose of about 2 mg via intravitreal injection. In some embodiments, the method further comprises administering topical steroid therapy. In some embodiments, the topical steroid therapy is difluprednate therapy. In some embodiments, the topical steroid treatment comprises administering a topical steroid about four times per day for about four weeks, followed by administering a topical steroid about three times per day for about one week, followed by administering a topical steroid about twice per day for about one week, followed by administering a topical steroid about once per day for about one week, starting at the time of administration of the anti-VEGF agent and continuing thereafter.In some embodiments, the topical steroid treatment comprises administering the topical steroid about four times daily for about one month, followed by administering the topical steroid about three times daily for about one month, followed by administering the topical steroid about twice daily for about one month, followed by administering the topical steroid about once daily for about one month, with the timing starting at the time of administration of the anti-VEGF agent and continuing thereafter. In some embodiments, the topical steroid treatment comprises administering the topical steroid about four times daily for about one month, followed by administering the topical steroid about three times daily for about one month, followed by administering the topical steroid about twice daily for about one month, followed by administering the topical steroid about once daily for about one month, with the timing starting at the time of administration of the rAAV particles and continuing thereafter. In some embodiments, the topical steroid comprises 0.05% difluprednate at a dose of about 1 μg to about 3 μg. In some embodiments, the topical steroid comprises 0.05% difluprednate at a dose of about 2.5 μg.

[0015] In some embodiments that may be combined with any of the preceding embodiments, the unit dose of rAAV particles is about 6 x 10 per eye. 10 ~Approx. 2×10 11 In some embodiments, the unit dose of rAAV particles is about 2 x 10 per eye. 11 or about 6 x 10 10 In some embodiments that may be combined with any of the preceding embodiments, the unit dose of rAAV particles is about 6 x 10 per eye. 10 ~Approx. 6×10 11 In some embodiments that may be combined with any of the preceding embodiments, the unit dose of rAAV particles is about 6 x 10 per eye. 10 ~Approx. 2×10 11 In some embodiments that may be combined with any of the preceding embodiments, the unit dose of rAAV particles is about 2 x 10 per eye. 11 Approximately ~6×10 11In some embodiments, the unit dose of rAAV particles is about 2 x 10 per eye. 11 or about 6 x 10 10 In some embodiments, the unit dose of rAAV particles is about 2 x 10 11 In some embodiments, the unit dose of rAAV particles is about 6 x 10 vg / eye. 11 vg / eye.

[0016] In some embodiments that may be combined with any of the preceding embodiments, the individual has one or more symptoms of an ocular neovascular disease in the contralateral eye.

[0017] In some embodiments that may be combined with any of the preceding embodiments, the methods provided herein further include administering a unit dose of rAAV particles to the contralateral eye of the individual. In some embodiments, the unit dose of rAAV particles is administered to the contralateral eye up to about two weeks after administering the unit dose of rAAV particles to one eye. In some embodiments, the unit dose of rAAV particles is administered to the contralateral eye on the same day as administering the unit dose of rAAV particles to one eye, or the unit dose of rAAV particles is administered to the contralateral eye about one day to about 14 days after administering the unit dose of rAAV particles to one eye. In some embodiments, the unit dose of rAAV particles administered to the contralateral eye of the individual contains equal to or less than the unit dose of rAAV particles administered to one eye of the individual. In some embodiments, the unit dose of rAAV particles is administered to the contralateral eye at least about two weeks after administering the unit dose of rAAV particles to one eye. In some embodiments, the unit dose of rAAV particles administered to the contralateral eye of an individual contains more vector genomes per eye (vg / eye) than the unit dose of rAAV particles administered to one eye of the individual.

[0018] In some embodiments that may be combined with any of the preceding embodiments, the nucleic acid comprises the nucleic acid sequence of SEQ ID NO: 40, or a sequence having at least 85% identity thereto.

[0019] In some embodiments that may be combined with any of the preceding embodiments, the polypeptide comprises the amino acid sequence of SEQ ID NO: 35. In some embodiments that may be combined with any of the preceding embodiments, the polypeptide comprises the amino acid sequence of SEQ ID NO: 41. In some embodiments, the polypeptide is aflibercept.

[0020] In some embodiments that may be combined with any of the preceding embodiments, the nucleic acid further comprises a first enhancer region, a promoter region, a 5' UTR region, a second enhancer region, and a polyadenylation site. In some embodiments, the nucleic acid comprises, in 5' to 3' order, (a) a first enhancer region, (b) a promoter region, (c) a 5' UTR region, (d) a nucleic acid encoding a polypeptide comprising an amino acid sequence having at least about 95% identity to the amino acid sequence of SEQ ID NO: 35, (e) a second enhancer region, and (f) a polyadenylation site, and is flanked by AAV2 inverted terminal repeats (ITRs). In some embodiments, the first enhancer region comprises a CMV sequence comprising a sequence of SEQ ID NO: 22 or a sequence having at least 85% identity thereto. In some embodiments, the promoter region comprises a CMV sequence comprising a sequence of SEQ ID NO: 23 or a sequence having at least 85% identity thereto. In some embodiments, the nucleic acid encoding the polypeptide comprises the nucleic acid sequence of SEQ ID NO: 40 or a sequence having at least 85% identity thereto. In some embodiments, the polypeptide comprises the amino acid sequence of SEQ ID NO: 35 or a sequence having at least 85% identity thereto. In some embodiments, the polypeptide comprises the amino acid sequence of SEQ ID NO: 41 or a sequence having at least 85% identity thereto. In some embodiments, the polypeptide is aflibercept. In some embodiments, the 5'UTR region comprises, in 5' to 3' order, a TPL sequence comprising the sequence of SEQ ID NO: 24 or a sequence having at least 85% identity thereto, and an eMLP sequence comprising the sequence of SEQ ID NO: 25 or a sequence having at least 85% identity thereto. In some embodiments, the second enhancer region comprises a complete EES sequence comprising the sequence of SEQ ID NO: 26 or a sequence having at least 85% identity thereto. In some embodiments, the polyadenylation site comprises an HGH polyadenylation site comprising the sequence of SEQ ID NO: 27 or a sequence having at least 85% identity thereto.In some embodiments, the nucleic acid further comprises: (a) a first enhancer region comprising a CMV sequence comprising the sequence of SEQ ID NO: 22 or a sequence having at least 85% identity thereto; (b) a promoter region comprising a CMV sequence comprising the sequence of SEQ ID NO: 23 or a sequence having at least 85% identity thereto; (c) a 5'UTR region comprising, in 5' to 3' order, a TPL sequence comprising the sequence of SEQ ID NO: 24 or a sequence having at least 85% identity thereto; and an eMLP sequence comprising the sequence of SEQ ID NO: 25 or a sequence having at least 85% identity thereto; (d) a second enhancer region comprising a complete EES sequence comprising the sequence of SEQ ID NO: 26 or a sequence having at least 85% identity thereto; and (e) an HGH polyadenylation site comprising the sequence of SEQ ID NO: 27 or a sequence having at least 85% identity thereto. In some embodiments, the nucleic acid comprises AAV ITRs on both sides of the element.

[0021] In some embodiments that may be combined with any of the preceding embodiments, the nucleic acid comprises the sequence of SEQ ID NO: 39, or a sequence having at least 85% identity thereto.

[0022] In some embodiments that may be combined with any of the preceding embodiments, the rAAV particles comprise an AAV2 VP1 capsid protein comprising a GH loop comprising the amino acid sequence of SEQ ID NO: 38, or an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 38. In some embodiments, the rAAV particles comprise an AAV2 VP1 capsid protein comprising a GH loop comprising an amino acid sequence having any of at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 38.

[0023] In some embodiments that may be combined with any of the preceding embodiments, the rAAV particles comprise an AAV2 VP1 capsid protein comprising the amino acid sequence of SEQ ID NO: 37, or an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 37. In some embodiments, the rAAV particles comprise an AAV2 VP1 capsid protein comprising an amino acid sequence having any of at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 37.

[0024] In some embodiments that may be combined with any of the preceding embodiments, the AAV2 capsid protein comprises or consists of the amino acid sequence of AAV2 VP1 comprising the sequence of SEQ ID NO: 13, wherein LGETTRP (SEQ ID NO: 14) is inserted between positions 587 and 588 of the capsid protein. In some embodiments, the AAV2 capsid protein comprises the amino acid sequence of AAV2 VP1 comprising the sequence of SEQ ID NO: 13, wherein LALGETTRPA (SEQ ID NO: 1) is inserted between positions 587 and 588 of the capsid protein, where the numbering of the amino acid residues corresponds to the AAV2 VP1 capsid protein. In some embodiments, the AAV2 capsid protein comprises the amino acid sequence of AAV2 VP1 comprising the sequence of SEQ ID NO: 13, wherein LALGETTRPA (SEQ ID NO: 1) is inserted between positions 587 and 588 of the capsid protein. In some embodiments, the AAV2 capsid protein comprises the amino acid sequence of LALGETTRPA (SEQ ID NO: 1) inserted between positions 587 and 588 of the capsid protein, where the numbering of the amino acid residues corresponds to the AAV2 VP1 capsid protein. In some embodiments, the AAV2 capsid protein comprises the amino acid sequence of AAV2 VP1 comprising the sequence of SEQ ID NO: 13, with LALGETTRPA (SEQ ID NO: 1) inserted between positions 587 and 588 of the capsid protein.

[0025] In some embodiments that may be combined with any of the preceding embodiments, administration of the unit dose of rAAV particles to one eye and / or the contralateral eye is by intravitreal administration.

[0026] In some embodiments that may be combined with any of the preceding embodiments, the unit dose of rAAV particles is present in a pharmaceutical formulation. In some embodiments, the pharmaceutical formulation comprises rAAV particles, sodium chloride, sodium phosphate, and a surfactant. In some embodiments, the pharmaceutical formulation comprises about 150 to about 200 mM sodium chloride, about 1 to about 10 mM sodium dihydrogen phosphate, about 1 to about 10 mM sodium dihydrogen phosphate, about 0.0005% (w / v) to about 0.005% (w / v) poloxamer 188, and about 6 x 10 sucrose per mL. 13 ~Approx. 6×10 10 In some embodiments, the pharmaceutical formulation comprises rAAV particles containing about 180 mM sodium chloride, about 5 mM sodium dihydrogen phosphate, about 5 mM sodium dihydrogen phosphate, about 6 x 10 12 vg / mL rAAV particles, and about 0.001% (w / v) poloxamer 188, wherein the pharmaceutical formulation has a pH of about 7.3. In some embodiments, the pharmaceutical formulation contains about 180 mM sodium chloride, about 5 mM sodium dihydrogen phosphate, about 5 mM sodium dihydrogen phosphate, about 2 x 10 12 vg / mL rAAV particles, and about 0.001% (w / v) poloxamer 188, and the pharmaceutical formulation has a pH of about 7.3. In some embodiments, the pharmaceutical formulation contains about 180 mM sodium chloride, about 5 mM sodium dihydrogen phosphate, about 5 mM sodium dihydrogen phosphate, about 6 x 10 11 vg / mL rAAV particles, and about 0.001% (w / v) poloxamer 188, and the pharmaceutical formulation has a pH of about 7.3.

[0027] In some embodiments that may be combined with any of the preceding embodiments, a unit dose of rAAV particles comprises a volume of about 25 μL to about 250 μL. In some embodiments, a unit dose of rAAV particles comprises a volume of about 100 μL. In some embodiments, a unit dose of rAAV particles comprises a volume of about 30 μL. In some embodiments that may be combined with any of the preceding embodiments, a unit dose of rAAV particles administered to one eye and / or the contralateral eye comprises a volume of about 25 μL to about 250 μL. In some embodiments, a unit dose of rAAV particles administered to one eye and / or the contralateral eye comprises a volume of about 100 μL. In some embodiments, a unit dose of rAAV particles administered to one eye and / or the contralateral eye comprises a volume of about 30 μL.

[0028] In some embodiments that may be combined with any of the preceding embodiments, the individual has previously been treated for an ocular neovascular disease with an anti-VEGF agent. In some embodiments, the individual has received one or two injections of an anti-VEGF agent into one eye and / or the contralateral eye prior to administering rAAV particles to the one eye and / or the contralateral eye. In some embodiments, the individual has not previously been treated for an ocular neovascular disease with an anti-VEGF agent. In some embodiments, the anti-VEGF agent is aflibercept.

[0029] In some embodiments that may be combined with any of the preceding embodiments, 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, branch retinal vein occlusion, diabetic macular edema, diabetic retinal ischemia, ischemic retinopathy, diabetic retinal edema, or any combination thereof.

[0030] In some embodiments that may be combined with any of the preceding embodiments, the unit dose of rAAV particles is administered in combination with steroid therapy. In some embodiments, the steroid therapy is corticosteroid therapy. In some embodiments, the steroid therapy is systemic steroid therapy. In some embodiments, the steroid therapy is oral steroid therapy. In some embodiments, the steroid therapy is prednisone therapy. In some embodiments, the oral prednisone therapy comprises administering prednisone at a dose of about 60 mg per day for a total of 6 days starting 3 days prior to administration of the unit dose of rAAV particles to one eye and / or the contralateral eye, followed by administering prednisone at a dose of about 40 mg per day for a total of 3 days, followed by administering prednisone at a dose of about 20 mg per day for a total of 2 days, followed by administering prednisone at a dose of about 10 mg per day for a total of 2 days. In some embodiments, the steroid therapy is topical steroid therapy. In some embodiments, the steroid therapy is difluprednate therapy. In some embodiments, a steroid is administered before, during, and / or after administration of a unit dose of rAAV particles, hi some embodiments, a steroid is administered before, during, and / or after administration of a unit dose of rAAV particles to one eye and / or the contralateral eye.

[0031] In some embodiments that may be combined with any of the preceding embodiments, the steroid treatment is topical steroid treatment, and the topical steroid treatment is daily steroid treatment for up to about 4 weeks, up to about 6 weeks, or up to about 8 weeks after administration of the unit dose of rAAV particles. In some embodiments, the topical steroid treatment comprises about four administrations of topical steroid at about week 1, about three administrations of topical steroid at about week 2, about two administrations of topical steroid at about week 3, and about one administration of topical steroid at about week 4, timing initiated at the time of administration of the unit dose of rAAV particles and continued thereafter. In some embodiments, the topical steroid treatment comprises administering topical steroid about four times daily for about three weeks after administration of the unit dose of rAAV particles, followed by administering topical steroid about three times daily for about one week, followed by administering topical steroid about twice daily for about one week, followed by administering topical steroid about once daily for about one week. In some embodiments, the topical steroid treatment comprises administering topical steroid about four times per day for about four weeks, followed by administering topical steroid about three times per day for about one week, followed by administering topical steroid about twice per day for about one week, followed by administering topical steroid about once per day for about one week, timing initiated at the time of administration of the unit dose of rAAV particles and continuing thereafter. In some embodiments, the topical steroid treatment comprises administering topical steroid about four times per day for about one month, followed by administering topical steroid about three times per day for about one month, followed by administering topical steroid about twice per day for about one month, followed by administering topical steroid about once per day for about one month, timing initiated at the time of administration of the unit dose of rAAV particles and continuing thereafter. In some embodiments, the topical steroid treatment comprises administering topical steroid about four times per day for about one month, followed by administering topical steroid about three times per day for about one month, followed by administering topical steroid about twice per day for about one month, followed by administering topical steroid about once per day for about one month, starting about one week before administering the unit dose of rAAV particles and continuing thereafter.In some embodiments, the topical steroid comprises 0.05% difluprednate at a dose of about 1 μg to about 3 μg, hi some embodiments, the topical steroid comprises 0.05% difluprednate at a dose of about 2.5 μg.

[0032] In some embodiments, the ocular neovascular disease is wet age-related macular degeneration (wAMD).

[0033] In some embodiments that may be combined with any of the preceding embodiments, administering a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual maintains or reduces retinal thickness compared to the retinal thickness before administering the unit dose of rAAV particles. In some embodiments, the maintenance or reduction in retinal thickness compared to the retinal thickness before administering the unit dose of rAAV particles exists for about 30 weeks, about 34 weeks, about 44 weeks, about 6 months, about 1 year, about 1.5 years, about 2 years, about 3 years, about 5 years, about 10 years, or more after administering the unit dose of rAAV particles to one eye and / or the contralateral eye of the individual. In some embodiments, administering a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual reduces retinal thickness compared to the retinal thickness before administering the unit dose of rAAV particles. In some embodiments, the reduction in retinal thickness is at least about 10% compared to the retinal thickness before administering the unit dose of rAAV particles. In some embodiments, the retinal thickness is central zone retinal thickness (CST) or central retinal thickness (CRT).

[0034] In some embodiments that may be combined with any of the preceding embodiments, administering a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual maintains or reduces macular volume compared to the macular volume before administering the unit dose of rAAV particles. In some embodiments, the maintenance or reduction in macular volume compared to the macular volume before administering the unit dose of rAAV particles exists for about 30 weeks, about 34 weeks, about 44 weeks, about 6 months, about 1 year, about 1.5 years, about 2 years, about 3 years, about 5 years, about 10 years, or more after administering the unit dose of rAAV particles to one eye and / or the contralateral eye of the individual. In some embodiments, administering a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual reduces macular volume compared to the macular volume before administering the unit dose of rAAV particles. In some embodiments, the reduction in macular volume is at least about 10% compared to the macular volume before administering the unit dose of rAAV particles.

[0035] In some embodiments that may be combined with any of the preceding embodiments, administering a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual maintains or improves visual acuity compared to the visual acuity before administering the unit dose of rAAV particles. In some embodiments, the maintenance or improvement in visual acuity compared to the visual acuity before administering the unit dose of rAAV particles exists for about 30 weeks, about 34 weeks, about 44 weeks, about 6 months, about 1 year, about 1.5 years, about 2 years, about 3 years, about 5 years, about 10 years, or more after administering the unit dose of rAAV particles to one eye and / or the contralateral eye of the individual. In some embodiments, administering a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual improves visual acuity compared to the visual acuity before administering the unit dose of rAAV particles. In some embodiments, the visual acuity is best-corrected visual acuity (BCVA).

[0036] In some embodiments that may be combined with any of the preceding embodiments, administration of a single unit dose of rAAV particles to one eye and / or the contralateral eye of an individual results in a therapeutic effect (e.g., treatment of an ocular neovascular disease, reduction of retinal fluid, maintenance or reduction of retinal thickness, maintenance or reduction of macular volume, and / or maintenance or improvement of visual acuity). In some embodiments, administration of a single unit dose of rAAV particles to one eye and / or the contralateral eye of an individual results in a therapeutic effect that exists for about 30 weeks or more, about 34 weeks or more, about 44 weeks or more, about 6 months or more, about 1 year or more, about 1.5 years or more, or about 2 years, about 3 years, about 5 years, about 10 years, or more after administration of the unit dose of rAAV particles to one eye and / or the contralateral eye of the individual.

[0037] In some embodiments that may be combined with any of the preceding embodiments, administering a unit dose of rAAV particles to one eye and / or the contralateral eye of a plurality of individuals results in at least about 50% of the plurality of individuals not requiring anti-VEGF rescue therapy. In some embodiments, after administration of the unit dose of rAAV particles, at least about 50% of the plurality of individuals are free from the need for anti-VEGF rescue therapy for at least about 20 weeks, at least about 36 weeks, at least about 52 weeks, at least about 56 weeks, or longer.

[0038] In some embodiments that may be combined with any of the preceding embodiments, administration of a single unit dose of rAAV particles to one eye and / or the contralateral eye of a plurality of individuals results in at least about 67% of the plurality of individuals not requiring anti-VEGF rescue therapy. In some embodiments, after administration of the unit dose of rAAV particles, at least about 67% of the plurality of individuals are free from the need for anti-VEGF rescue therapy for at least about 20 weeks, at least about 36 weeks, at least about 52 weeks, at least about 60 weeks, at least about 64 weeks, or at least about 66 weeks.

[0039] In some embodiments that may be combined with any of the preceding embodiments, administering a unit dose of rAAV particles to one eye and / or the contralateral eye of a plurality of individuals results in at least about 78% of the plurality of individuals not requiring anti-VEGF rescue therapy. In some embodiments, after administration of the unit dose of rAAV particles, at least about 78% of the plurality of individuals are free from the need for anti-VEGF rescue therapy for at least about 20 weeks, at least about 36 weeks, or longer.

[0040] In some embodiments that may be combined with any of the preceding embodiments, administering a unit dose of rAAV particles to one eye and / or the contralateral eye of a plurality of individuals results in 100% of the plurality of individuals not requiring anti-VEGF rescue therapy. In some embodiments, after administration of the unit dose of rAAV particles, 100% of the plurality of individuals are not requiring anti-VEGF rescue therapy for at least about 64 weeks, at least about 68 weeks, at least about 72 weeks, at least about 76 weeks, at least about 80 weeks, at least about 84 weeks, or more.

[0041] In some embodiments that may be combined with any of the preceding embodiments, administering a unit dose of rAAV particles to one eye and / or the contralateral eye of a plurality of individuals reduces the annual anti-VEGF injection rate by at least about 80%, at least about 85%, at least about 87%, at least about 90%, at least about 95%, at least about 99%, or 100% compared to the annual anti-VEGF injection rate before administering the unit dose of rAAV particles.

[0042] In some embodiments, the ocular neovascular disease is diabetic macular edema (DME). In some embodiments, administration of a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual improves the diabetic retinopathy severity scale (DRSS) by two or three stages. In some embodiments, administration of a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual improves the diabetic retinopathy severity scale (DRSS) by two stages. In some embodiments, administration of a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual improves the diabetic retinopathy severity scale (DRSS) by three stages.

[0043] Incorporation by Reference All references cited herein, including patent applications and publications, are incorporated by reference in their entirety.

[0044] 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, in which: [Brief explanation of the drawings]

[0045] [Figure 1A] 1 shows a schematic diagram of the investigational drug and Phase I study described in Examples 1 and 2. The figure shows a schematic diagram of AAV2.7m8-aflibercept. AAV2.7m8-aflibercept is a replication-deficient recombinant adeno-associated virus (rAAV) vector containing an AAV2.7m8 protein capsid and a vector genome containing an expression cassette for a codon-optimized version of aflibercept cDNA under the control of a ubiquitous chimeric promoter (C11). The AAV2.7m8-aflibercept vector genome also contains two AAV2 inverted terminal repeats (ITRs) flanking the aflibercept cDNA expression cassette. [Figure 1B] 1 shows a schematic diagram of the investigational drug and Phase I study described in Examples 1 and 2. The figure summarizes the study design for the Phase I study described in Examples 1 and 2. [Figure 1C] A diagram of the AAV2.7m8-aflibercept vector genome (SEQ ID NO: 39) is shown. The vector genome contains two inverted terminal repeats (ITRs) of AAV serotype 2 (positions 1-145 and 3772-3916 of SEQ ID NO: 39), and an expression cassette composed of a CMV promoter (positions 180-693 of SEQ ID NO: 39), a 5' untranslated region (UTR) composed of an adenovirus tripartite leader sequence and a synthetic intron (positions 694-1314 of SEQ ID NO: 39), a Kozak sequence (positions 1329-1340 of SEQ ID NO: 39), codon-optimized aflibercept cDNA (positions 1338-2714 of SEQ ID NO: 39), a 3' UTR composed of a human scaffold attachment region (positions 2717-3527 of SEQ ID NO: 39), and a human growth hormone polyadenylation / transcription termination signal (positions 3546-3748 of SEQ ID NO: 39). AAV = adeno-associated virus, CMV = cytomegalovirus, GH = growth hormone, ITR = inverted terminal repeat, polyA = polyadenylation, SAR = scaffold attachment region, TPL = tripartite leader sequence. [Figure 2A] Optical coherence tomography (OCT) images and retinal thickness maps obtained from OCT images are shown for subjects in Cohort 1 of the study described in Example 1. OCT images were taken at the indicated times before and after (Day 1) administration of AAV2.7m8-aflibercept. The treatment interval for anti-VEGF IVT is indicated for all subjects. The figure shows OCT images and retinal thickness maps obtained from Subject 1 at five visits at the indicated time points before aflibercept injection at screening. OCT images were taken immediately before treatment with aflibercept standard therapy. Subject 1 required aflibercept IVT every 5 to 7 weeks and exhibited refractory subretinal fluid and pigment epithelial detachment (PED) despite aflibercept standard therapy. [Figure 2B]Optical coherence tomography (OCT) images and retinal thickness maps obtained from the OCT images are shown for subjects in Cohort 1 of the study described in Example 1. OCT images were taken at the indicated times before and after AAV2.7m8-aflibercept administration (Day 1). The treatment intervals for anti-VEGF IVT are shown for all subjects. The figure shows OCT images and retinal thickness maps obtained from Subject 1 at the time of aflibercept injection at screening (Day -7), AAV2.7m8-aflibercept injection (Day 1), and follow-up visits at the indicated time points. Subject 1 did not require rescue injections after AAV2.7m8-aflibercept injection. Subject 1 showed resolution of subretinal fluid from Week 4 onward and remained free of subretinal and intraretinal fluid (remained dry). [Figure 2C] Optical coherence tomography (OCT) images and retinal thickness maps obtained from the OCT images are shown for subjects in Cohort 1 of the study described in Example 1. OCT images were taken at the indicated times before and after administration of AAV2.7m8-aflibercept (Day 1). The treatment intervals for anti-VEGF IVT are shown for all subjects. The figure shows OCT images and retinal thickness maps obtained from Subject 2 at the five visits indicated before the screening aflibercept injection. The OCT images were taken immediately before treatment with aflibercept standard therapy. Subject 2 required six aflibercept IVT treatments during the eight months prior to AAV2.7m8-aflibercept treatment to maintain retinal anatomy. [Figure 2D]Optical coherence tomography (OCT) images and retinal thickness maps obtained from the OCT images are shown for subjects in Cohort 1 of the study described in Example 1. OCT images were taken at the indicated times before and after AAV2.7m8-aflibercept administration (Day 1). The treatment intervals for anti-VEGF IVT are shown for all subjects. The figure shows OCT images and retinal thickness maps obtained from Subject 2 at the time of aflibercept injection at screening (Day -7), AAV2.7m8-aflibercept injection (Day 1), and follow-up visits at the indicated times. Subject 2 did not require rescue injections after AAV2.7m8-aflibercept injection. Subject 2 demonstrated stable retinal anatomy without subretinal or intraretinal fluid through Week 24. [Figure 2E] Optical coherence tomography (OCT) images and retinal thickness maps obtained from the OCT images are shown for subjects in Cohort 1 of the study described in Example 1. OCT images were taken at the indicated times before and after (Day 1) administration of AAV2.7m8-aflibercept. The treatment intervals for anti-VEGF IVT are shown for all subjects. The figure shows OCT images and retinal thickness maps obtained from subject 3 at four visits at the indicated time points before aflibercept injection at screening. The OCT image taken at Week -27 is not shown. The OCT images were taken immediately before treatment with aflibercept standard therapy. Subject 3 exhibited subretinal fluid, which increased when the interval between aflibercept IVT treatments was increased from 5 weeks to 7 weeks. [Figure 2F]Optical coherence tomography (OCT) images and retinal thickness maps obtained from the OCT images are shown for subjects in Cohort 1 of the study described in Example 1. OCT images were taken at the indicated times before and after AAV2.7m8-aflibercept administration (Day 1). The treatment intervals for anti-VEGF IVT are indicated for all subjects. The figure shows OCT images and retinal thickness maps obtained from Subject 3 at the time of aflibercept injection at screening (Day -7), AAV2.7m8-aflibercept injection (Day 1), and follow-up visits at the indicated time points. Subject 3 did not require rescue injections after AAV2.7m8-aflibercept injection. Subject 3 demonstrated resolution of refractory subretinal fluid by Week 8, with stable retinal structure through Week 24. [Figure 2G] Optical coherence tomography (OCT) images and retinal thickness maps obtained from the OCT images are shown for subjects in Cohort 1 of the study described in Example 1. OCT images were taken at the indicated times before and after (Day 1) administration of AAV2.7m8-aflibercept. The treatment intervals for anti-VEGF IVT are shown for all subjects. The figure shows OCT images and retinal thickness maps obtained from Subject 4 at the indicated visits before aflibercept injection at screening. The OCT images were taken immediately before treatment with ranibizumab 0.5 mg IVT standard of care. Subject 4 presented with subretinal fluid refractory to ranibizumab IVT injection. [Figure 2H]Optical coherence tomography (OCT) images and retinal thickness maps obtained from the OCT images are shown for subjects in Cohort 1 of the study described in Example 1. OCT images were taken at the indicated times before and after AAV2.7m8-aflibercept administration (Day 1). The treatment intervals for anti-VEGF IVT are indicated for all subjects. The figure shows OCT images and retinal thickness maps obtained from Subject 4 at the time of aflibercept injection at screening (Day -14), AAV2.7m8-aflibercept injection (Day 1), and follow-up visits at the indicated time points. Subject 4 did not require rescue injections after AAV2.7m8-aflibercept injection. Subject 4 demonstrated resolution of refractory subretinal fluid by Week 8, with stable retinal structure by Week 24. [Figure 2I] Optical coherence tomography (OCT) images and retinal thickness maps obtained from the OCT images are shown for subjects in Cohort 1 of the study described in Example 1. OCT images were taken at the indicated times before and after (Day 1) administration of AAV2.7m8-aflibercept. The treatment intervals for anti-VEGF IVT are shown for all subjects. The figure shows OCT images and retinal thickness maps obtained from subject 5 at the indicated visits before aflibercept injection at screening. The OCT images were taken immediately before treatment with aflibercept standard therapy. [Figure 2J]Optical coherence tomography (OCT) images and retinal thickness maps obtained from the OCT images are shown for subjects in Cohort 1 of the study described in Example 1. OCT images were taken at the indicated times before and after AAV2.7m8-aflibercept administration (Day 1). The treatment intervals for anti-VEGF IVT are shown for all subjects. The figure shows OCT images and retinal thickness maps obtained from Subject 5 at the time of aflibercept injection at screening (Day -14), AAV2.7m8-aflibercept injection (Day 1), and follow-up visits at the indicated times. Subject 5 did not require rescue injections after AAV2.7m8-aflibercept injection. The subretinal fluid and PED present during AAV2.7m8-aflibercept treatment resolved over time, the retinal anatomy stabilized, and no subretinal or intraretinal fluid remained present through Week 24. [Figure 2K] Optical coherence tomography (OCT) images and retinal thickness maps obtained from the OCT images are shown for subjects in Cohort 1 of the study described in Example 1. OCT images were taken at the indicated times before and after (Day 1) administration of AAV2.7m8-aflibercept. The treatment intervals for anti-VEGF IVT are shown for all subjects. The figure shows OCT images and retinal thickness maps obtained from subject 6 at five visits at the indicated time points before aflibercept injection at screening. OCT images were taken immediately before treatment with either bevacizumab 1.5 mg IVT standard treatment or ranibizumab 0.5 mg IVT standard treatment, as indicated. The retinal appearance of subject 6 was consistent with polypoidal choroidal vasculopathy (PCV). [Figure 2L]Optical coherence tomography (OCT) images and retinal thickness maps obtained from OCT images are shown for subjects in Cohort 1 of the study described in Example 1. OCT images were taken at the indicated times before and after AAV2.7m8-aflibercept administration (Day 1). The treatment intervals for anti-VEGF IVT are indicated for all subjects. The figure shows OCT images and retinal thickness maps obtained from Subject 6 at the time of aflibercept injection at screening (Day -10), AAV2.7m8-aflibercept injection (Day 1), and follow-up visits at the indicated time points. Subject 6 did not require rescue injections after AAV2.7m8-aflibercept injection. Subject 6 did not exhibit an increase in subretinal fluid and achieved some anatomical improvement by Week 24. Subject 6's contralateral eye received standard-of-care aflibercept injections every 4 weeks during the course of the study and showed retinal morphology similar to that of the AAV2.7m8-aflibercept-treated eye. [Figure 3] Figure 1 shows the change in mean central subretinal thickness (CST) at the indicated time points for subjects in Cohort 1 of the study described in Example 1. Error bars represent 90% confidence intervals calculated using the T-distribution. Baseline (BL) represents measurements taken prior to the screening aflibercept injection, 7-15 days (e.g., 7-14 days) prior to AAV2.7m8-aflibercept treatment on Day 1. At 24 weeks post-AAV2.7m8-aflibercept treatment, subjects showed a mean change in CRT of -52.7 μm (90% CI -86.5, -18.8). BL = baseline, D = day, W = week. The Day 1 visit occurred 7-14 days after the baseline visit. [Figure 4]Figure 1 shows mean best-corrected visual acuity (BCVA) measurements based on the Early Treatment Diabetic Retinopathy Study (ETDRS) letter count assessment for subjects in Cohort 1 of the study described in Example 1 at the indicated time points. Error bars indicate 90% confidence intervals calculated using a T-distribution. Baseline (BL) indicates measurements taken prior to the screening aflibercept injection, 7-15 days (e.g., 7-14 days) prior to AAV2.7m8-aflibercept treatment on Day 1. At 24 weeks post-AAV2.7m8-aflibercept treatment, subjects had an average change in BCVA of -2 letters (90% CI -9.1, 5.1). BL = baseline, D = day, W = week. The Day 1 visit occurred 7-14 days after the baseline visit. [Figure 5] 1 shows the nucleic acid sequence of aflibercept (SEQ ID NO: 36). [Figure 6] Figure 1 shows plots of anterior chamber and vitreous cell counts for subjects 1-6 in the study described in Example 1 after treatment with AAV2.7m8-aflibercept. The steroid treatment administered to each patient is indicated below each plot. Aqueous humor cell count categories were based on the Standardization of Uveitis Nomenclature (SUN) criteria (Jabs, DA et al., J Ophthalmol. 2005;140:509-516). Vitreous cell count categories were based on the National Institutes of Health (NIH) guidelines. For aqueous humor cells, a cell count of 0.5+ indicated 1-5 cells, a cell count of 1+ indicated 6-15 cells, a cell count of 2+ indicated 16-25 cells, a cell count of 3+ indicated 26-50 cells, and a cell count of 4+ indicated >50 cells. For vitreous cells, a cell count value of 0.5+ indicates 1-10 cells, a cell count value of 1+ indicates 11-20 cells, a cell count value of 2+ indicates 21-30 cells, a cell count value of 3+ indicates 31-100 cells, and a cell count value of 4+ indicates >100 cells. For the analysis shown in this figure, rare cells were captured as 0.5+. [Figure 7A]Optical coherence tomography (OCT) images and retinal thickness maps obtained from OCT images are shown for subjects 1-6 in Cohort 1 of the study described in Example 1 at a median follow-up period of 34 weeks. Additionally, for each of subjects 1-6, the change in BCVA from baseline, the number of anti-VEGF IVT injections in the 8 months prior to AAV2.7m8-aflibercept administration, and the number of rescue anti-VEGF IVT injections administered during the study are shown. The actual week in which the OCT images and retinal thickness maps were obtained for each subject is shown (Subject 1 = Week 44, Subject 2 = Week 40, Subject 3 = Week 36, Subject 4 = Week 32, Subject 5 = Week 28, and Subject 6 = Week 28). No subjects required rescue anti-VEGF IVT injections during the study, and no retreatment criteria were met at any time during the 44-week follow-up period. No subjects showed signs of disease reactivation on OCT imaging. [Figure 7B] Optical coherence tomography (OCT) images and retinal thickness maps obtained from OCT images are shown for subjects 1-6 in Cohort 1 of the study described in Example 1 at a median follow-up period of 34 weeks. Additionally, for each of subjects 1-6, the change in BCVA from baseline, the number of anti-VEGF IVT injections in the 8 months prior to AAV2.7m8-aflibercept administration, and the number of rescue anti-VEGF IVT injections administered during the study are shown. The actual week in which the OCT images and retinal thickness maps were obtained for each subject is shown (Subject 1 = Week 44, Subject 2 = Week 40, Subject 3 = Week 36, Subject 4 = Week 32, Subject 5 = Week 28, and Subject 6 = Week 28). No subjects required rescue anti-VEGF IVT injections during the study, and no retreatment criteria were met at any time during the 44-week follow-up period. No subjects showed signs of disease reactivation on OCT imaging. [Figure 8]Plots of aqueous humor and vitreous cell counts are shown for subjects 1-6 in Cohort 1 of the study described in Example 1 after treatment with AAV2.7m8-aflibercept up to a median follow-up of 44 weeks (range, 40-52 weeks). The steroid treatment administered to each subject is indicated below each plot. Aqueous humor cell grade categories were based on the Standardization of Uveitis Nomenclature (SUN) criteria (Jabs, DA et al., J Ophthalmol. 2005;140:509-516). Vitreous cell grade categories were based on the National Institutes of Health (NIH) guidelines. For aqueous humor cells, a cell grade of 0.5+ indicates 1-5 cells, a cell grade of 1+ indicates 6-15 cells, a cell grade of 2+ indicates 16-25 cells, a cell grade of 3+ indicates 26-50 cells, and a cell grade of 4+ indicates >50 cells. For vitreous cells, a cell grade of 0.5+ indicates 1-10 cells, a cell grade of 1+ indicates 11-20 cells, a cell grade of 2+ indicates 21-30 cells, a cell grade of 3+ indicates 31-100 cells, and a cell grade of 4+ indicates >100 cells. Rare cells were captured as a cell grade of 0.5+. [Figure 9A] Optical coherence tomography (OCT) images and retinal thickness maps obtained from the OCT images are shown for subjects 1-6 in Cohort 1 of the study described in Example 1 at a median follow-up period of 44 weeks. Changes from baseline in BCVA, ETDRS letter count, and CST are also shown. The actual week in which the OCT image and retinal thickness map were obtained for each subject is indicated. The asterisk indicates that subject 4 underwent gas bubble injection retinal detachment repair to repair spontaneous pseudophakic macular rhegmatogenous retinal detachment (RRD) (FIG. 9B). Subject 4 is still undergoing follow-up, and the OCT images and BCVA values ​​for subject 4 correspond to the last findings before the retinal detachment. [Figure 9B]Optical coherence tomography (OCT) images and retinal thickness maps obtained from the OCT images are shown for subjects 1-6 in Cohort 1 of the study described in Example 1 at a median follow-up period of 44 weeks. Changes from baseline in BCVA, ETDRS letter count, and CST are also shown. The actual week in which the OCT image and retinal thickness map were obtained for each subject is indicated. The asterisk indicates that subject 4 underwent gas bubble injection retinal detachment repair to repair spontaneous pseudophakic macular rhegmatogenous retinal detachment (RRD) (FIG. 9B). Subject 4 is still undergoing follow-up, and the OCT images and BCVA values ​​for subject 4 correspond to the last findings before the retinal detachment. [Figure 10] Figure 1 shows mean best-corrected visual acuity (BCVA) measurements based on the Early Treatment Diabetic Retinopathy Study (ETDRS) visual acuity assessment for subjects in Cohort 2 of the study described in Examples 2 and 3 at the indicated time points. Error bars indicate 90% confidence intervals for the mean absolute BCVA calculated using a T-distribution. Baseline (BL) indicates measurements taken prior to the screening aflibercept injection, 7-15 days (e.g., 7-14 days) prior to AAV2.7m8-aflibercept treatment on Day 1. At 24 weeks post-AAV2.7m8-aflibercept treatment, subjects had a mean change in BCVA of -4.8 letters. BL = baseline, D = day, W = week. The Day 1 visit occurred 7-15 days after the baseline visit. [Figure 11] Figure 1 shows the mean central retinal thickness (CST) at the indicated time points for subjects in Cohort 2 of the study described in Examples 2 and 3. Error bars represent the 90% confidence interval of the mean absolute CST calculated using the T-distribution. Baseline (BL) indicates measurements taken prior to the screening aflibercept injection, 7-15 days (e.g., 7-14 days) prior to AAV2.7m8-aflibercept treatment on Day 1. At 24 weeks post-AAV2.7m8-aflibercept treatment, subjects had a mean change in BCVA of -27.8 μm. BL = baseline, D = day, W = week. The Day 1 visit occurred 7-15 days after the baseline visit. [Figure 12]Plots of aqueous humor and vitreous cell counts are shown for subjects in Cohort 2 of the study described in Examples 2 and 3 after treatment with AAV2.7m8-aflibercept up to a 24-week follow-up period. The steroid treatment administered to each subject is indicated below each plot, along with the frequency of topical steroid (difluprednate) eye drop administration (e.g., 1x = once daily, 2x = twice daily, 3x = three times daily, 4x = four times daily). Aqueous humor cell grade categories were based on the Standardization of Uveitis Nomenclature (SUN) criteria (Jabs, DA et al., J. Ophthalmol. 2005;140:509-516). Vitreous cell grade categories were based on the National Institutes of Health (NIH) guidelines. For aqueous humor cells, a cell grade of 0.5+ indicates 1–5 cells, a cell grade of 1+ indicates 6–15 cells, a cell grade of 2+ indicates 16–25 cells, a cell grade of 3+ indicates 26–50 cells, and a cell grade of 4+ indicates >50 cells. For vitreous cells, a cell grade of 0.5+ indicates 1–10 cells, a cell grade of 1+ indicates 11–20 cells, a cell grade of 2+ indicates 21–30 cells, a cell grade of 3+ indicates 31–100 cells, and a cell grade of 4+ indicates >100 cells. For this analysis, rare cells were captured as a cell grade of 0.5+. [Figure 13A] Optical coherence tomography (OCT) images and retinal thickness maps obtained from the OCT images are shown for subjects 1-6 in cohort 2 of the study described in Examples 2 and 3 over a 24-week follow-up period. The change from baseline in BCVA, the change from baseline in CST, and the number of rescue anti-VEGF injections administered during the 24-week follow-up period are also shown. The asterisk indicates that subject 3 received three rescue anti-VEGF injections, including the one at week 24, all of which were due to a loss of ≥10 letters from baseline in BCVA. This loss was attributed to intraretinal or subretinal fluid. [Figure 13B]

[0023] Figure 1 shows optical coherence tomography (OCT) images and retinal thickness maps obtained from the OCT images from subjects 1-6 in cohort 2 of the study described in Examples 2 and 3 over a 24-week follow-up period. Also shown are the change from baseline in BCVA, the change from baseline in CST, and the number of rescue anti-VEGF injections administered over the 24-week follow-up period. The asterisk indicates that subject 5 received three rescue anti-VEGF injections, the last of which was administered at week 20, due to an increase in central retinal thickness of >75 μm from baseline. [Figure 14] 1 is a Swimmer's Lane plot showing the number of anti-VEGF injections for subjects in cohorts 1 and 2 of the study described in Examples 1-3. The x-axis represents time in weeks relative to the time AAV2.7m8-aflibercept was administered. The y-axis represents each individual subject in cohorts 1 and 2. Circles represent anti-VEGF IVT injections administered before and after treatment with AAV2.7m8-aflibercept. The asterisk indicates that subject 6 in cohort 2 was diagnosed with nAMD 6.4 months before AAV2.7m8-aflibercept administration. The vertical line bisecting the plot indicates the first day AAV2.7m8-aflibercept was administered. The right side of the bisecting vertical line represents each of the 61 subsequent study visits. [Figure 15A]

[0033] Figure 1 shows plots of aqueous humor and vitreous cell counts measured by slit-lamp examination after treatment with AAV2.7m8-aflibercept for subjects in Cohorts 1, 2, and 3 of the study described in Examples 1-5. The figure shows plots of aqueous humor and vitreous cell counts for subjects in Cohort 1 up to a 64-week follow-up period (median = 60 weeks, range = 52-64 weeks). The steroid treatment administered to each subject is indicated below each plot. The grade categories for aqueous humor cells were based on the System of Uveitis Nomenclature (SUN) criteria: Jabs DA, et al. J Ophthalmol 2005;140:509-516. The grade categories for vitreous cells were based on the National Institutes of Health (NIH) guidelines: aqueous humor cells: 0.5+ = 1–5 cells; 1+ = 6–15 cells; 2+ = 16–25 cells; 3+ = 26–50 cells; 4+ = >50 cells. Vitreous cells: 0.5+ = 1–10 cells; 1+ = 11–20 cells; 2+ = 21–30 cells; 3+ = 31–100 cells; 4+ = >100 cells. For this analysis, rare cells were captured as 0.5+. QID = four times daily; TID = three times daily; BID = twice daily; QD = once daily; QOD = once every other day; "QD>>" indicates that the subject is receiving topical steroids once daily on an ongoing basis, "BID>>" indicates that the subject is receiving topical steroids twice daily on an ongoing basis, and "QID>>" indicates that the subject is receiving topical steroids four times daily on an ongoing basis. [Figure 15B]

[0033] Figure 1 shows plots of aqueous humor and vitreous cell counts measured by slit-lamp examination after treatment with AAV2.7m8-aflibercept for subjects in Cohorts 1, 2, and 3 of the study described in Examples 1-5. The figure shows plots of aqueous humor and vitreous cell counts for subjects in Cohort 2 up to a 40-week follow-up period (median = 36 weeks, range = 32-40 weeks). The steroid treatment administered to each subject is indicated below each plot. The grade categories for aqueous humor cells were based on the System of Uveitis Nomenclature (SUN) criteria: Jabs DA, et al. J Ophthalmol 2005;140:509-516. The grade categories for vitreous cells were based on the National Institutes of Health (NIH) guidelines: aqueous humor cells: 0.5+ = 1–5 cells; 1+ = 6–15 cells; 2+ = 16–25 cells; 3+ = 26–50 cells; 4+ = >50 cells. Vitreous cells: 0.5+ = 1–10 cells; 1+ = 11–20 cells; 2+ = 21–30 cells; 3+ = 31–100 cells; 4+ = >100 cells. For this analysis, rare cells were captured as 0.5+. QID = four times daily; TID = three times daily; BID = twice daily; QD = once daily; QOD = once every other day; "QD>>" indicates that the subject is receiving topical steroids once daily on an ongoing basis, "BID>>" indicates that the subject is receiving topical steroids twice daily on an ongoing basis, and "QID>>" indicates that the subject is receiving topical steroids four times daily on an ongoing basis. [Figure 15C]

[0023] Figure 1 shows plots of aqueous humor and vitreous cell counts measured by slit-lamp examination after treatment with AAV2.7m8-aflibercept for subjects in Cohorts 1, 2, and 3 of the study described in Examples 1-5. The figure shows plots of aqueous humor and vitreous cell counts up to a 20-week follow-up period for subjects in Cohort 3. The steroid treatment administered to each subject is indicated below each plot. The grade categories for aqueous humor cells were based on the System of Uveitis Nomenclature (SUN) criteria: Jabs DA, et al. J Ophthalmol 2005;140:509-516. The grade categories for vitreous cells were based on the National Institutes of Health (NIH) guidelines: aqueous humor cells: 0.5+ = 1–5 cells; 1+ = 6–15 cells; 2+ = 16–25 cells; 3+ = 26–50 cells; 4+ = >50 cells. Vitreous cells: 0.5+ = 1–10 cells; 1+ = 11–20 cells; 2+ = 21–30 cells; 3+ = 31–100 cells; 4+ = >100 cells. For this analysis, rare cells were captured as 0.5+. QID = four times daily; TID = three times daily; BID = twice daily; QD = once daily; QOD = once every other day; "QD>>" indicates that the subject is receiving topical steroids once daily on an ongoing basis, "BID>>" indicates that the subject is receiving topical steroids twice daily on an ongoing basis, and "QID>>" indicates that the subject is receiving topical steroids four times daily on an ongoing basis. [Figure 16A] Figure 1 shows the mean BCVA and mean CST from baseline to week 52 for subjects in Cohort 1 of the study described in Examples 1-5 (n=6). The figure shows the mean BCVA (ETDRS letter count) from baseline to week 52. The asterisk indicates one subject had a low BCVA score at weeks 44 and 48 due to retinal detachment. In the figure, error bars indicate the 90% confidence intervals of the mean absolute BCVA and CST values ​​using the T-distribution. BL = baseline, D = day, W = week. [Figure 16B]Figure 1 shows the mean BCVA and mean CST from baseline to week 52 for subjects in Cohort 1 of the study described in Examples 1-5 (n=6). The figure shows the mean CST (μm) from baseline to week 52. An asterisk indicates that one subject had no CST data at weeks 44 and 48 due to retinal detachment. In the figure, error bars indicate the 90% confidence intervals of the mean absolute BCVA and CST values ​​using the T-distribution. BL = baseline, D = day, W = week. [Figure 17A] Figure 1 shows the mean BCVA and mean CST from baseline to week 36 for subjects in Cohort 2 of the study described in Examples 1-5 (n=6). The figure shows the mean BCVA (ETDRS letter count) from baseline to week 36. In the figure, error bars indicate the 90% confidence intervals of the mean absolute BCVA and CST values ​​using a T-distribution. BL = baseline, D = day, W = week. One subject missed the week 36 visit. [Figure 17B] Figure 1 shows the mean BCVA and mean CST from baseline to week 36 for subjects in Cohort 2 of the study described in Examples 1-5 (n=6). The figure shows the mean CST (μm) from baseline to week 36. In the figure, error bars indicate the 90% confidence intervals of the mean absolute BCVA and CST values ​​using the T-distribution. BL = baseline, D = day, W = week. One subject did not visit at week 36. [Figure 18A] Figure 1 shows the mean BCVA and mean CST from baseline to week 20 for five subjects in Cohort 3 of the study described in Examples 1-5. The figure shows the mean BCVA (ETDRS letter count) from baseline to week 20. In the figure, error bars indicate the 90% confidence intervals of the mean absolute BCVA and CST values ​​using a T-distribution. BL = baseline, D = day, W = week. One subject missed the visit at week 36. [Figure 18B]Figure 1 shows the mean BCVA and mean CST from baseline to week 20 for five subjects in Cohort 3 of the study described in Examples 1-5. The figure shows the mean CST (μm) from baseline to week 20. In the figure, error bars indicate the 90% confidence intervals of the mean absolute BCVA and CST values ​​using the T-distribution. BL = baseline, D = day, W = week. One subject did not visit at week 36. [Figure 19] 1 is a Swimmer's Lane plot showing the number of anti-VEGF injections administered to subjects in cohorts 1, 2, and 3 of the study described in Examples 1-5. The x-axis represents time in weeks relative to the time AAV2.7m8-aflibercept was administered. The y-axis represents each individual subject in cohorts 1-3. Circles represent anti-VEGF IVT injections administered before and after treatment with AAV2.7m8-aflibercept. The vertical line bisecting the plot indicates day 1 on which AAV2.7m8-aflibercept was administered. To the right of the bisecting vertical line are each of the subsequent study visits. [Figure 20A] Figure 1 shows OCT images and retinal thickness maps obtained from subject 4 in cohort 1 of the study described in Examples 1-5, taken at the indicated times before and after administration of AAV2.7m8-aflibercept. The figures show OCT images and retinal thickness maps for subject 4 in cohort 1 at the indicated times (weeks) before administration of AAV2.7m8-aflibercept, during which the subject received an anti-VEGF IVT injection (ranibizumab). The times of administration of the screening anti-VEGF IVT injection ("screening anti-VEGF IVT") and AAV2.7m8-aflibercept ("AAV2.7m8-aflibercept") are shown. BCVA (ETDRS character count) and CST (μm) are shown at the indicated time points before and after administration of AAV2.7m8-aflibercept. An asterisk indicates that the subject had a retinal detachment event unrelated to AAV2.7m8-aflibercept. [Figure 20B]Figure 1 shows OCT images and retinal thickness maps obtained from the OCT images of subject 4 in cohort 1 of the study described in Examples 1-5, taken at the indicated times before and after administration of AAV2.7m8-aflibercept. The figures show OCT images and retinal thickness maps of subject 4 in cohort 1 before and after administration of AAV2.7m8-aflibercept at the indicated times (weeks). The times of administration of screening anti-VEGF IVT injection ("screening anti-VEGF IVT") and AAV2.7m8-aflibercept ("AAV2.7m8-aflibercept") are shown. BCVA (ETDRS character count) and CST (μm) are shown at the indicated time points before and after administration of AAV2.7m8-aflibercept. An asterisk indicates that the subject had a retinal detachment event unrelated to AAV2.7m8-aflibercept. [Figure 21A] The figures show OCT images and retinal thickness maps obtained from subject 5 in cohort 3 of the study described in Examples 1-5, taken at the indicated times before and after administration of AAV2.7m8-aflibercept. The figures show OCT images and retinal thickness maps for subject 5 in cohort 3 at the indicated times (weeks) before administration of AAV2.7m8-aflibercept, during which the subject received an anti-VEGF IVT injection (aflibercept). The times of administration of the screening anti-VEGF IVT injection ("screening anti-VEGF IVT") and AAV2.7m8-aflibercept ("AAV2.7m8-aflibercept") are shown. BCVA (ETDRS character count) and CST (μm) are shown at the indicated time points before and after administration of AAV2.7m8-aflibercept. [Figure 21B]

[0023] Figures show OCT images and retinal thickness maps obtained from the OCT images of subject 5 in cohort 3 of the study described in Examples 1-5, taken at the indicated times before and after administration of AAV2.7m8-aflibercept. The figures show OCT images and retinal thickness maps of subject 5 in cohort 3 before and after administration of AAV2.7m8-aflibercept at the indicated times (weeks). The times of administration of screening anti-VEGF IVT injection ("screening anti-VEGF IVT") and AAV2.7m8-aflibercept ("AAV2.7m8-aflibercept") are shown. BCVA (ETDRS letter count) and CST (μm) are shown at the indicated time points before and after administration of AAV2.7m8-aflibercept. [Figure 22A]

[0023] Figure 1 shows plots of aqueous humor and vitreous cell counts measured by slit-lamp examination after treatment with AAV2.7m8-aflibercept for subjects in Cohorts 1, 2, 3, and 4 of the study described in Examples 1-5 and 7. The figure shows plots of aqueous humor and vitreous cell counts up to an 80-week follow-up period for subjects in Cohort 1. The steroid treatment administered to each subject is indicated below each plot. The grade categories for aqueous humor cells were based on the System of Uveitis Nomenclature (SUN) criteria: Jabs DA, et al. J Ophthalmol 2005;140:509-516. The grade categories for vitreous cells were based on the National Institutes of Health (NIH) guidelines: aqueous humor cells: 0.5+ = 1–5 cells; 1+ = 6–15 cells; 2+ = 16–25 cells; 3+ = 26–50 cells; 4+ = >50 cells. Vitreous cells: 0.5+ = 1–10 cells; 1+ = 11–20 cells; 2+ = 21–30 cells; 3+ = 31–100 cells; 4+ = >100 cells. For this analysis, rare cells were captured as 0.5+. QID = four times daily; TID = three times daily; BID = twice daily; QD = once daily; QOD = once every other day; "QD>>" indicates that the subject is continuously receiving topical steroids once daily, "BID>>" indicates that the subject is continuously receiving topical steroids twice daily, "TID>>" indicates that the subject is continuously receiving topical steroids three times daily, and "QID>>" indicates that the subject is continuously receiving topical steroids four times daily. [Figure 22B]

[0023] Figure 1 shows plots of aqueous humor and vitreous cell counts measured by slit-lamp examination after treatment with AAV2.7m8-aflibercept for subjects in Cohorts 1, 2, 3, and 4 of the study described in Examples 1-5 and 7. The figure shows plots of aqueous humor and vitreous cell counts up to a 56-week follow-up period for subjects in Cohort 2. The steroid treatment administered to each subject is indicated below each plot. The grade categories for aqueous humor cells were based on the System of Uveitis Nomenclature (SUN) criteria: Jabs DA, et al. J Ophthalmol 2005;140:509-516. The grade categories for vitreous cells were based on the National Institutes of Health (NIH) guidelines: aqueous humor cells: 0.5+ = 1–5 cells; 1+ = 6–15 cells; 2+ = 16–25 cells; 3+ = 26–50 cells; 4+ = >50 cells. Vitreous cells: 0.5+ = 1–10 cells; 1+ = 11–20 cells; 2+ = 21–30 cells; 3+ = 31–100 cells; 4+ = >100 cells. For this analysis, rare cells were captured as 0.5+. QID = four times daily; TID = three times daily; BID = twice daily; QD = once daily; QOD = once every other day; "QD>>" indicates that the subject is continuously receiving topical steroids once daily, "BID>>" indicates that the subject is continuously receiving topical steroids twice daily, "TID>>" indicates that the subject is continuously receiving topical steroids three times daily, and "QID>>" indicates that the subject is continuously receiving topical steroids four times daily. [Figure 22C]

[0023] Figure 1 shows plots of aqueous humor and vitreous cell counts measured by slit-lamp examination after treatment with AAV2.7m8-aflibercept for subjects in Cohorts 1, 2, 3, and 4 of the study described in Examples 1-5 and 7. The figure shows plots of aqueous humor and vitreous cell counts up to a 36-week follow-up period for subjects in Cohort 3. In the figure, the steroid treatment administered to each subject is indicated below each plot. The grade categories for aqueous humor cells were based on the System of Uveitis Nomenclature (SUN) criteria: Jabs DA, et al. J Ophthalmol 2005;140:509-516. The grade categories for vitreous cells were based on the National Institutes of Health (NIH) guidelines: aqueous humor cells: 0.5+ = 1–5 cells; 1+ = 6–15 cells; 2+ = 16–25 cells; 3+ = 26–50 cells; 4+ = >50 cells. Vitreous cells: 0.5+ = 1–10 cells; 1+ = 11–20 cells; 2+ = 21–30 cells; 3+ = 31–100 cells; 4+ = >100 cells. For this analysis, rare cells were captured as 0.5+. QID = four times daily; TID = three times daily; BID = twice daily; QD = once daily; QOD = once every other day; "QD>>" indicates that the subject is continuously receiving topical steroids once daily, "BID>>" indicates that the subject is continuously receiving topical steroids twice daily, "TID>>" indicates that the subject is continuously receiving topical steroids three times daily, and "QID>>" indicates that the subject is continuously receiving topical steroids four times daily. [Figure 22D]

[0023] Figure 1 shows plots of aqueous humor and vitreous cell counts measured by slit-lamp examination after treatment with AAV2.7m8-aflibercept for subjects in Cohorts 1, 2, 3, and 4 of the study described in Examples 1-5 and 7. The figure shows plots of aqueous humor and vitreous cell counts up to an 8-week follow-up period for subjects in Cohort 4. In the figure, the steroid treatment administered to each subject is indicated below each plot. The grade categories for aqueous humor cells were based on the System of Uveitis Nomenclature (SUN) criteria: Jabs DA, et al. J Ophthalmol 2005;140:509-516. The grade categories for vitreous cells were based on the National Institutes of Health (NIH) guidelines: aqueous humor cells: 0.5+ = 1–5 cells; 1+ = 6–15 cells; 2+ = 16–25 cells; 3+ = 26–50 cells; 4+ = >50 cells. Vitreous cells: 0.5+ = 1–10 cells; 1+ = 11–20 cells; 2+ = 21–30 cells; 3+ = 31–100 cells; 4+ = >100 cells. For this analysis, rare cells were captured as 0.5+. QID = four times daily; TID = three times daily; BID = twice daily; QD = once daily; QOD = once every other day; "QD>>" indicates that the subject is continuously receiving topical steroids once daily, "BID>>" indicates that the subject is continuously receiving topical steroids twice daily, "TID>>" indicates that the subject is continuously receiving topical steroids three times daily, and "QID>>" indicates that the subject is continuously receiving topical steroids four times daily. [Figure 23A] Figure 1 shows the mean BCVA and mean CST from baseline to week 72 for subjects in Cohort 1 of the study described in Examples 1-5 and 7 (n=6). The figure shows the mean BCVA (ETDRS letter count) from baseline to week 72. One subject had low BCVA scores at weeks 44 and 48 due to retinal detachment. *n=5 from weeks 56 to 72. In the figure, error bars indicate the 90% confidence intervals of the mean absolute BCVA and CST values ​​using the T-distribution. BL=baseline, D=day, W=week. [Figure 23B]Figure 1 shows the mean BCVA and mean CST from baseline to week 72 for subjects in Cohort 1 of the study described in Examples 1-5 and 7 (n=6). The figure shows the mean CST (μm) from baseline to week 72. One subject had missing CST data at weeks 44 and 48 due to retinal detachment. *n=5 from weeks 56 to 72. In the figure, error bars indicate the 90% confidence interval of the mean absolute BCVA and CST values ​​using the T-distribution. BL=baseline, D=day, W=week. [Figure 24A] Figure 1 shows the mean BCVA and mean CST from baseline to week 52 for subjects in Cohort 2 of the study described in Examples 1-5 and 7 (n=6). The figure shows the mean BCVA (ETDRS letter count) from baseline to week 52. *n=5 at weeks 36 and 40. In the figure, error bars indicate the 90% confidence intervals of the mean absolute BCVA and CST values ​​using the T-distribution. BL=baseline, D=day, W=week. [Figure 24B] Figure 1 shows the mean BCVA and mean CST from baseline to week 52 for subjects in Cohort 2 of the study described in Examples 1-5 and 7 (n=6). The figure shows the mean CST (μm) from baseline to week 52. *n=5 at weeks 36 and 40. In the figure, error bars indicate the 90% confidence intervals of the mean absolute BCVA and CST values ​​using the T-distribution. BL=baseline, D=day, W=week. [Figure 25A] Figure 1 shows the mean BCVA and mean CST from baseline to week 20 for subjects in Cohort 3 of the study described in Examples 1-5 and 7 (n=9). The figure shows the mean BCVA (ETDRS letter count) from baseline to week 20. *n=8 at weeks 4, 16, and 20. In the figure, error bars indicate the 90% confidence intervals of the mean absolute BCVA and CST values ​​using the T-distribution. BL=baseline, D=day, W=week. [Figure 25B]Figure 1 shows the mean BCVA and mean CST from baseline to week 20 for subjects in Cohort 3 of the study described in Examples 1-5 and 7 (n=9). The figure shows the mean CST (μm) from baseline to week 20. *n=8 at weeks 4, 16, and 20. In the figure, error bars indicate the 90% confidence intervals of the mean absolute BCVA and CST values ​​using the T-distribution. BL=baseline, D=day, W=week. [Figure 26] This is a Swimmer's Lane plot showing the number of anti-VEGF injections administered to subjects in cohorts 1, 2, 3, and 4 of the study described in Examples 1-5 and 7. The x-axis represents time in weeks compared to the time AAV2.7m8-aflibercept was administered. The y-axis represents each individual subject in cohorts 1-4. Circles represent anti-VEGF IVT injections administered before and after treatment with AAV2.7m8-aflibercept. The vertical line bisecting the plot indicates the first day AAV2.7m8-aflibercept was administered. To the right of the bisecting vertical line are each subsequent study visit. Five subjects were diagnosed less than one year before the AAV2.7m8-aflibercept injection (one subject each in cohorts 2 and 3, and three subjects in cohort 4). In cohort 4, data prior to day 1 are incomplete for subject 2 due to reassignment and for subject 5 because the subject entered a clinical trial with unknown drug(s) after diagnosis. [Figure 27]

[0023] Figures 1A-1C show the mean annual anti-VEGF injection rates for subjects in Cohorts 1-3 of the studies described in Examples 1-5 and 7. Figure 1A shows a comparison of the mean annual anti-VEGF injection rates for subjects in Cohort 1 (receiving a "high dose" of 6 x 10 vg / eye; n = 6) and subjects in Cohorts 2 and 3 (receiving a "low dose" of 2 x 10 vg / eye; n = 15). Figure 1B shows a comparison of the mean annual anti-VEGF injection rates for subjects in each of Cohorts 1 (n = 6), 2 (n = 6), and 3 (n = 9). In A-B, annualized rate (pre) = (number of anti-VEGF IVT injections in the 12 months prior to AAV2.7m8-aflibercept) / (number of days from first anti-VEGF IVT injection in the past 12 months to AAV2.7m8-aflibercept injection / 365.25), and annualized rate (post) = (number of anti-VEGF IVT injections since AAV2.7m8-aflibercept) / (number of days from AAV2.7m8-aflibercept to last study follow-up / 365.25). [Figure 28A]

[0033] Figures show OCT images and retinal thickness maps obtained from subject 5 in cohort 3 of the study described in Examples 1-5 and 7, taken at the indicated times before and after administration of AAV2.7m8-aflibercept. The figures show OCT images and retinal thickness maps for subject 5 in cohort 3 at the indicated times (weeks) before administration of AAV2.7m8-aflibercept, during which the subject received an anti-VEGF IVT injection (aflibercept). The times of administration of the screening anti-VEGF IVT injection ("aflibercept IVT") and AAV2.7m8-aflibercept are shown. BCVA (ETDRS letter count) and CST (μm) are shown at the indicated time points before and after administration of AAV2.7m8-aflibercept. [Figure 28B]

[0023] Figures show OCT images and retinal thickness maps obtained from the OCT images of subject 5 in cohort 3 of the study described in Examples 1-5 and 7, taken at the indicated times before and after administration of AAV2.7m8-aflibercept. The figures show OCT images and retinal thickness maps for subject 5 in cohort 3, taken before and at the indicated times (weeks) after administration of AAV2.7m8-aflibercept. The times of screening anti-VEGF IVT injection ("aflibercept IVT") and administration of AAV2.7m8-aflibercept are shown. BCVA (ETDRS letter count) and CST (μm) are shown at the indicated time points before and after administration of AAV2.7m8-aflibercept. [Figure 29] FIG. 1 shows a diagram of the design of the study described in Example 6 evaluating the durability of a single intravitreal (IVT) injection of AAV2.7m8-aflibercept in subjects with diabetic macular edema (DME). DRSS = Diabetic Retinopathy Severity Score; OCT = Optical Coherence Tomography; CST = Central Space Retinal Thickness; *All subjects receive a 7-week course of difluprednate eye drops starting QID and tapering to QD. **PE = Primary Endpoint Assessment. ***EOS = End of Study Assessment. [Figure 30] Two figures are shown for CST measured using four different treatment groups: (1) ranibizumab 0.5 mg monthly (Rq4); intravitreal aflibercept injection (IAI) 0.5 mg monthly (0.5q4); IAI 2 mg monthly (2q4); and IAI 2 mg every 2 months (2q8) after three initial monthly doses. [Figure 31] This case study demonstrates the effectiveness of a single intravitreal (IVT) injection of AAV2.7m8 2x10vg / eye in eliminating fluid and CST fluctuations. The patient was a 90-year-old woman who underwent 21 IVTs prior to the study and 9 IVTs in the last 12 months prior to AAV2.7m8 injection. Optical computed tomography (OCT) images and a graph showing CST fluctuations before and after AAV2.7m8 administration are shown. [Figure 32]The CST annual percent change is shown (+ or -50 μm). A change is defined as the change from one scheduled test week (Week A) to the subsequent scheduled test week (Week B) and meets the following conditions: 1. The magnitude of the change is equal to or exceeds the variability criterion. 2. The direction of change from the first scheduled week (Week A) remains the same for the scheduled test weeks during the transition period between the first and second scheduled weeks (Weeks A and B). 3. The direction of change from the first scheduled week (Week A) changes in the scheduled test week immediately following the second scheduled week (Week B). Annual percent change before AAV2.7m8 = (number of changes since first prior observation) / (number of days from first prior observation to last prior observation / 365.25). Annual percent change after AAV2.7m8 = (number of changes since AAV2.7m8 injection) / (number of days from AAV2.7m8 to last test follow-up / 365.25). The figure also shows a reduction in CST variation in subjects with documented baseline anti-AAV2.7m8 neutralizing antibody (NAb) titer levels <1:125. [Figure 33] The annualized rate of positive CST variation (+50 μm) is shown. Variation is defined as the change from one scheduled test week (Week A) to a subsequent scheduled test week (Week B) and meets the following conditions: 1. The magnitude of change is equal to or greater than the variation criterion. 2. The direction of change from the first scheduled week (Week A) is positive and remains the same for the scheduled test weeks during the transition period between the first and second scheduled weeks (Weeks A and B). 3. The direction of change from the first scheduled week (Week A) changes in the scheduled test week immediately following the second scheduled week (Week B). Annualized rate of variation before AAV2.7m8 = (number of variations since first prior observation) / (number of days from first prior observation to last prior observation / 365.25). Annualized rate of variation after AAV2.7m8 = (number of variations since AAV2.7m8 injection) / (number of days from AAV2.7m8 to last test follow-up / 365.25). The figure also shows a reduction in CST variation in subjects with documented baseline anti-AAV2.7m8 neutralizing antibody (NAb) titer levels <1:125. [Figure 34]The figure shows the annual CST variation (+ or -50 μm) over a period approximately 6 months longer than that shown in Figure 32. CST variation remains reduced. The figure also shows a reduction in CST variation in subjects with documented baseline anti-AAV2.7m8 neutralizing antibody (NAb) titer levels <1:125. DETAILED DESCRIPTION OF THE INVENTION

[0046] Some aspects are described below with reference to example applications for illustration. It should be understood that numerous specific details, relationships, and methods are described to fully understand the features described herein. However, one skilled in the relevant art will readily recognize that the features described herein can be implemented without one or more specific details or in other ways. The features described herein are not limited to the illustrated order of acts or events, as some acts may occur in a different order and / or concurrently with other acts or events. Furthermore, not all illustrated acts or events are necessarily required to implement a methodology in accordance with features described herein.

[0047] definition Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0048] The terms used herein are for the purpose of describing particular examples only and are not intended to be limiting. As used herein, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly dictates otherwise. Furthermore, when the terms "including," "includes," "having," "has," "with," or variations thereof are used in either the detailed description and / or the claims, such terms are intended to be inclusive in a similar sense to the term "comprising." The term "comprising," as used herein, is synonymous with "including" or "containing" and is inclusive, i.e., open-ended.

[0049] As used herein, the reference to "or" is intended to encompass "and / or" unless expressly stated otherwise. As used herein, the term "about" of a numerical value refers to that numerical value ±10%. As used herein, the term "about + number" refers to a number ±10% of that number. Reference to "about + numerical value or parameter" herein includes (and describes) embodiments that refer to the value or parameter itself.

[0050] The terms "subject," "patient," or "individual" refer to primates, e.g., humans and non-human primates, e.g., African green monkeys and rhesus monkeys. In some embodiments, the subject is a human.

[0051] The terms "treat," "treating," "treatment," "improve," or "ameliorating," and other grammatical equivalents, as used herein, refer to alleviating, attenuating, or ameliorating an ocular neovascular disease or disorder or a symptom of an ocular neovascular disease or disorder, preventing additional symptoms of an ocular neovascular disease or disorder, ameliorating or preventing the underlying metabolic cause of a symptom, inhibiting an ocular neovascular disease or disorder, e.g., halting the development of an ocular neovascular disease or disorder, alleviating an ocular neovascular disease or disorder, causing regression of an ocular neovascular disease or disorder, or halting a symptom of an ocular neovascular disease or disorder, and are intended to include prevention. The above terms further include achieving a therapeutic benefit and / or a prophylactic benefit. The term "therapeutic benefit" refers to the eradication or amelioration of the ocular neovascular disease or disorder being treated. In some embodiments, a therapeutic benefit is also achieved by eradicating or ameliorating one or more physiological symptoms associated with an ocular neovascular disease or disorder, such that an improvement is observed in a subject even though the subject still suffers from the disease or disorder. For a prophylactic benefit, a pharmaceutical composition is administered to a subject at risk of developing an ocular neovascular disease or disorder, or a subject who experiences one or more physiological symptoms of an ocular neovascular disease or disorder, even if the subject has not been diagnosed with the disease or disorder.

[0052] The terms "administer," "administering," "administration," and the like, as used herein, may refer to methods used to enable delivery of a therapeutic agent or pharmaceutical composition to the site where a biological effect is desired. These methods include intravitreal or subretinal injection into the eye.

[0053] The terms "effective amount," "therapeutically effective amount," or "pharmaceutically effective amount," as used herein, may refer to the amount of at least one pharmaceutical composition or compound being administered that is sufficient to result in some relief from one or more symptoms of the ocular disease or disorder being treated. An "effective amount," "therapeutically effective amount," or "pharmaceutically effective amount" of a pharmaceutical composition may be administered to a subject in need thereof as a unit dose (described in more detail elsewhere herein).

[0054] The term "pharmaceutically acceptable," as used herein, can refer to a substance, such as a carrier or diluent, that does not abrogate the biological activity or properties of the compounds disclosed herein and that is relatively non-toxic (i.e., that when administered to an individual, it not only does not produce undesired biological effects, but also does not interact in a deleterious way with any of the components of the composition that contains it).

[0055] The term "pharmaceutical composition" or simply "composition," as used herein, may refer to a biologically active compound optionally mixed with at least one pharmaceutically acceptable chemical ingredient such as, but not limited to, a carrier, stabilizer, diluent, dispersant, suspending agent, thickener, excipient, and the like.

[0056] As used herein, "AAV vector" or "rAAV vector" refers to an adeno-associated virus (AAV) vector or recombinant AAV (rAAV) vector that contains a polynucleotide sequence not of AAV origin (e.g., a polynucleotide heterologous to AAV, such as a nucleic acid sequence encoding a therapeutic transgene, e.g., aflibercept) for transduction into a target cell or tissue. Typically, the heterologous polynucleotide is flanked by at least one, and typically two, AAV inverted terminal repeats (ITRs). The term rAAV vector encompasses both rAAV vector particles and rAAV vector plasmids. rAAV vectors may be single-stranded (ssAAV) or self-complementary (scAAV).

[0057] "AAV virus" or "AAV virus 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 derived from wild-type AAV or is a variant AAV capsid protein (e.g., an AAV capsid protein with an insertion, such as the insertion of the 7m8 amino acid sequence described below). When the particle comprises a heterologous polynucleotide (e.g., a polynucleotide other than the wild-type AAV genome, such as a transgene to be delivered to a target cell or tissue), it is referred to as an "rAAV particle," "rAAV vector particle," or "rAAV vector." Thus, because such a vector is contained within the rAAV particle, production of the rAAV particle necessarily involves production of the rAAV vector.

[0058] The term "packaging," as used herein, may refer to a series of intracellular events that can result in the assembly and encapsidation of rAAV particles.

[0059] AAV "rep" and "cap" genes refer to polynucleotide sequences that encode the replication and encapsidation proteins of adeno-associated virus. AAV rep and cap are referred to herein as AAV "packaging genes."

[0060] The term "polypeptide" may encompass proteins, peptides, fragments, mutants, derivatives and analogues thereof, both naturally occurring and non-naturally occurring (an example of a non-naturally occurring protein is a fusion protein). A polypeptide may be a monomer, dimer, trimer, or polymer. Furthermore, a polypeptide may comprise several different domains, each having one or more different activities. For the avoidance of doubt, a "polypeptide" may be of any length greater than two amino acids.

[0061] 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 (where a single amino acid is changed to another amino acid at a position), one or more insertions and / or deletions (where one or more amino acids are inserted or deleted, respectively, in the sequence of the reference protein), and / or truncations of the amino acid sequence at either or both the amino or carboxy termini. A variant may have similar or different biological activity compared to the reference protein or the unmodified protein.

[0062] In some embodiments, a variant may have, for example, at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% overall sequence homology with its corresponding reference protein. In some embodiments, a variant may have at least about 90% overall sequence homology with the wild-type protein. In some embodiments, a variant exhibits at least about 95%, at least about 98%, at least about 99%, at least about 99.5%, or at least about 99.9% overall sequence identity.

[0063] As used herein, "recombinant" can refer to a biomolecule, such as a gene or protein, that (1) has been removed from the environment in which it naturally occurs; (2) is not linked to all or a portion of a polynucleotide with which the gene naturally occurs; (3) is operably linked to a polynucleotide with which the biomolecule is not linked in nature; or (4) is not naturally occurring. The term "recombinant" can be used in reference to cloned DNA isolates, chemically synthesized polynucleotide analogs, or polynucleotide analogs biologically synthesized by heterologous systems, as well as proteins and / or mRNA encoded by such nucleic acids. Thus, for example, a protein synthesized by a microorganism is recombinant if, for example, it is synthesized from mRNA synthesized from a recombinant gene present in the cell.

[0064] The term "anti-VEGF agent" includes any therapeutic agent, including a protein, polypeptide, peptide, fusion protein, multimeric protein, gene product, antibody, human monoclonal antibody, antibody fragment, aptamer, small molecule, kinase inhibitor, receptor or receptor fragment, or nucleic acid molecule, that can reduce, interfere with, disrupt, block, and / or inhibit the activity or function of endogenous VEGF and / or endogenous VEGF receptor (VEGFR), or VEGF-VEGFR interaction or pathway in vivo. An anti-VEGF agent may be any one of known therapeutic agents that can reduce the growth or formation of new blood vessels and / or edema or swelling when delivered to a cell, tissue, or subject in vivo, e.g., ranibizumab, brolucizumab, or bevacizumab. In some embodiments, an anti-VEGF agent may be naturally occurring, non-naturally occurring, or synthetic. In some embodiments, an anti-VEGF agent may be derived from a naturally occurring molecule that has subsequently been modified or mutated to confer anti-VEGF activity. In some embodiments, the anti-VEGF agent is a fusion protein or chimeric protein. In such proteins, a functional domain or polypeptide is artificially fused to a moiety or polypeptide to create a fusion protein or chimeric protein that can sequester VEGF in vivo or function as a VEGFR decoy. In some embodiments, the anti-VEGF agent is a fusion protein or chimeric protein that blocks endogenous VEGFR from interacting with its ligand.

[0065] As used herein, unless otherwise required, "VEGF" may refer to any isoform of VEGF, including, but not limited to, VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E, VEGF-F, or any combination, or any functional fragment or variant thereof. "VEGF" may refer to any member of the VEGF family, including VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E, VEGF-F, members i.e., VEGF-A, placental growth factor (PGF), VEGF-B, VEGF-C, and VEGF-D, or any combination, functional fragment, or variant thereof, unless otherwise required. As used herein, "VEGF receptor" or "VEGFR" or "VEGF-R" may be used to refer to any one of the receptors for VEGF, including, but not limited to, VEGFR-1 (or Flt-1), VEGFR-2 (or Flk-1 / KDR), and VEGFR-3 (or Flt-4). VEGFRs may be membrane-bound or soluble, or functional fragments or truncated forms of the receptor. Examples of anti-VEGF agents include, but are not limited to, ranibizumab, bevacizumab, brolucizumab, or any combination, variant, or functional fragment thereof.

[0066] "Operably linked" or "operably linked" or "coupled" can refer to the juxtaposition of genetic components wherein the components are in a relationship permitting them to operate in their expected manner. For example, a promoter may be operably linked to a coding region if the promoter helps initiate transcription of the coding sequence. Intervening residues may be present between the promoter and coding region so long as this functional relationship is maintained.

[0067] The term "expression vector" or "expression construct" or "cassette" or "plasmid" or simply "vector" may include any type of genetic construct, including AAV or rAAV vectors, that contain a nucleic acid or polynucleotide encoding a gene product, wherein some or all of the nucleic acid encoding the sequence in the gene product is transcribable and that is compatible with gene therapy. The transcript can be translated into a protein. In some embodiments, the transcript is partially or not translated. In certain aspects, expression includes both transcription of the gene and translation of mRNA into a gene product. In other aspects, expression includes only transcription of the nucleic acid encoding the gene of interest. An expression vector may also contain regulatory elements operably linked to the coding region to facilitate expression of the protein in the target cell. The combination of regulatory elements and the gene or genes to which the regulatory elements are operably linked for expression is sometimes referred to as an "expression cassette," many of which are known and available in the art or can be readily constructed from components available in the art.

[0068] The term "heterologous" can refer to an entity that differs in genotype from the rest of the entity to which it is compared. For example, a polynucleotide from a different species that is introduced into a plasmid or vector by genetic engineering techniques can be a heterologous polynucleotide. A promoter that has been removed from its native coding sequence and is operably linked to a coding sequence to which it is not naturally associated can be a heterologous promoter.

[0069] As used herein, "7m8" refers to the amino acid sequence LALGETTRPA (SEQ ID NO: 1).

[0070] "7m8 variant" refers to an rAAV, which may be of any serotype, that has the amino acid sequence LALGETTRPA (SEQ ID NO: 1) inserted into the solvent-exposed GH loop of the capsid protein.

[0071] When 7m8 is inserted into rAAV2 (also referred to as AAV2.7m8), the amino acid sequence LALGETTRPA (SEQ ID NO: 1) is inserted into the GH loop within amino acids 570-611 of the AAV2 capsid protein, e.g., between positions 587 and 588 of the AAV2 capsid protein VP1. In some cases, when 7m8 is inserted into rAAV2 (also referred to as AAV2.7m8), the amino acid sequence LALGETTRPA (SEQ ID NO: 1) is inserted into the GH loop of the AAV2 capsid protein, e.g., between positions 587 and 588 of the AAV2 VP1 comprising the sequence of SEQ ID NO: 13. When 7m8 is inserted into rAAV1 (also referred to as AAV1.7m8), the amino acid sequence LALGETTRPA (SEQ ID NO: 1) is inserted into the GH loop within amino acids 571-612 of the AAV1 capsid protein, e.g., between amino acids 590 and 591 of the AAV1 capsid protein. When 7m8 is inserted into rAAV5 (also referred to as AAV5.7m8), the amino acid sequence LALGETTRPA (SEQ ID NO: 1) is inserted into the GH loop within amino acids 560-601 of the AAV5 capsid protein, e.g., between amino acids 575 and 576 of the AAV5 capsid protein. When 7m8 is inserted into rAAV6 (also referred to as AAV6.7m8), the amino acid sequence LALGETTRPA (SEQ ID NO: 1) is inserted into the GH loop within amino acids 571-612 of the AAV6 capsid protein, e.g., between amino acids 590 and 591 of the AAV6 capsid protein. When 7m8 is inserted into rAAV7 (also referred to as AAV7.7m8), the amino acid sequence LALGETTRPA (SEQ ID NO: 1) is inserted into the GH loop within amino acids 572-613 of the AAV7 capsid protein, e.g., between amino acids 589 and 590 of the AAV7 capsid protein. When 7m8 is inserted into rAAV8 (also referred to as AAV8.7m8), the amino acid sequence LALGETTRPA (SEQ ID NO: 1) is inserted into the GH loop within amino acids 573 to 614 of the AAV8 capsid protein, for example, between amino acids 590 and 591 of the AAV8 capsid protein.When 7m8 is inserted into rAAV9 (also referred to as AAV9.7m8), the amino acid sequence LALGETTRPA (SEQ ID NO: 1) is inserted into the GH loop of the AAV9 capsid protein, for example, between amino acids 588 and 589 of the AAV9 capsid protein. When 7m8 is inserted into rAAV10 (also referred to as AAV10.7m8), the amino acid sequence LALGETTRPA (SEQ ID NO: 1) is inserted into the GH loop within amino acids 573-614 of the AAV10 capsid protein, for example, between amino acids 589 and 590 of the AAV10 capsid protein.

[0072] As used herein, the term "fluctuation" refers to the change in central retinal thickness or central retinal thickness from a scheduled test week (Week A) to a subsequent scheduled test week (Week B) that meets the following conditions: 1. The magnitude of the change is equal to or exceeds the fluctuation criterion. 2. The direction of change from the first scheduled week (Week A) remains the same for the scheduled test weeks in the transition period that occur between the first and second scheduled weeks (Weeks A and B). 3. The direction of change from the first scheduled week (Week A) changes in the scheduled test week immediately following the second scheduled week (Week B).

[0073] As used herein, "annualized rate of change" is categorized and defined for the "pre-AAV2.7m8" injection period (which is calculated as (number of changes since first past observation) / (number of days from first past observation to last past observation / 365.25)) and the "post-AAV2.7m8" injection period (which is calculated as (number of changes since AAV2.7m8 injection) / (number of days from AAV2.7m8 to last study follow-up / 365.25)).

[0074] Overview Current treatments for ocular neovascular diseases such as wAMD (e.g., aflibercept recombinant protein, ranibizumab recombinant protein) require lifelong IVT administration approximately every 4–8 weeks. This treatment may increase the risk of inflammation, infection, and other adverse effects in some patients. Furthermore, current treatments create compliance issues due to repeated and / or frequent visits to the clinic to administer the treatment, especially in elderly patients who are most affected by wAMD. Reducing the frequency of administration is associated with vision loss and exacerbation of the ocular disease or condition. Taking advantage of the ability of AAV vectors to efficiently transduce target retinal cells after IVT injection, therapeutic genes have been successfully transferred to photoreceptors, retinal pigment epithelium, and inner retina to treat a variety of retinal diseases. Thus, administration of rAAV particles encoding anti-VEGF agents (e.g., aflibercept) can provide long-term and / or sustained release of the anti-VEGF agent in vivo.

[0075] Remarkably, rAAV particles encoding aflibercept were delivered to the eyes of individuals with ocular neovascular disease at a dose of 6 × 10 per eye. 11 Administration of a single low unit dose of vector genome (vg) resulted in disease stabilization and robust anatomical responses in all treated individuals (see Example 1). Furthermore, visual acuity stabilized in all treated individuals, and a single low unit dose (6 x 10) of rAAV particles encoding aflibercept resulted in stabilization of visual acuity in all treated individuals. 11 vg / eye), no individual required rescue anti-VEGF therapy (e.g., aflibercept IVT injections). Furthermore, administration of a single unit dose of rAAV particles encoding aflibercept to the eyes of individuals with ocular neovascular disease unexpectedly caused a reduction (e.g., elimination) of symptoms, including intraretinal and subretinal fluid, that were refractory to prior anti-VEGF therapy (e.g., chronic IVT injections of aflibercept, ranibizumab, or bevacizumab).

[0076] Therefore, the present disclosure provides a 6×10 11

[0003] Provided herein are methods for treating ocular neovascular disease in individuals by administering a single unit dose of rAAV particles encoding an anti-VEGF agent (e.g., aflibercept) at or below 1000 mg / eye. Additionally, the present disclosure provides methods for reducing retinal fluid in individuals with ocular neovascular disease by administering a single unit dose of rAAV particles encoding an anti-VEGF agent (e.g., aflibercept). The methods disclosed herein reduce or eliminate the need for repeated IVT injections while providing long-term efficacy, thereby addressing the issues of non-adherence and lack of active participation in treatment. Additionally, the methods provided herein reduce adverse effects associated with multiple IVT injections.

[0077] Treatment method Provided herein are methods for treating an ocular neovascular disease in an individual, the method comprising administering a unit dose of recombinant adeno-associated virus (rAAV) particles to the eye of the individual.

[0078] Also provided herein is a method for reducing retinal fluid in the eye of an individual with an ocular neovascular disease, the method comprising administering a unit dose of rAAV particles to the eye of the individual.

[0079] Also provided herein is a method for treating an ocular neovascular disease in an individual, the method comprising administering an anti-VEGF agent (e.g., aflibercept) to the eye of the individual, and, after administration of the anti-VEGF agent, administering a unit dose of recombinant adeno-associated virus (rAAV) particles to the eye of the individual.

[0080] 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, branch retinal vein occlusion, diabetic macular edema, diabetic retinal ischemia, ischemic retinopathy, diabetic retinal edema, or any combination thereof.

[0081] In some embodiments, the term ocular neovascular disease also encompasses VEGF-driven pre-vascular diseases that progress to neovascular forms if left untreated. In some embodiments, the ocular neovascular disease is non-proliferative diabetic retinopathy, which is a pre-vascular disease.

[0082] In some embodiments, the individual is human. In some embodiments, the individual has received at least one prior treatment (e.g., at least one, at least two, at least three, at least four, at least five, or more treatments) with an anti-VEGF agent (e.g., bevacizumab, brolucizumab, ranibizumab, faricimab, abicipar pegol, conbercept, OPT-302, KSI-301, injectable sunitinib malate (GB-102), PAN-90806 (PanOptica), and / or aflibercept) for an ocular neovascular disease within the past 8 weeks, the past 9 weeks, about the past 10 weeks, about the past 11 weeks, about the past 12 weeks, about the past 13 weeks, about the past 14 weeks, about the past 15 weeks, or about the past 16 weeks prior to administration of the unit dose of rAAV particles. In some embodiments, the individual has had a meaningful response to prior treatment with an anti-VEGF agent (e.g., bevacizumab, brolucizumab, ranibizumab, faricimab, abicipar pegol, conbercept, OPT-302, KSI-301, injectable sunitinib malate (GB-102), PAN-90806 (PanOptica), and / or aflibercept). In some embodiments, the anti-VEGF agent is aflibercept, a functional variant thereof, or a functional fragment thereof. In some embodiments, the anti-VEGF agent comprises a polypeptide comprising an amino acid sequence having at least about 95% identity to the amino acid sequence of SEQ ID NO: 35. In some embodiments, the retinal fluid in the individual's eye is intraretinal fluid (IRF) and / or subretinal fluid (SRF). In some embodiments, the amount or presence of retinal fluid in the eye of the individual is refractory to previous treatment with an anti-VEGF agent (e.g., bevacizumab, brolucizumab, ranibizumab, faricimab, abicipar pegol, conbercept, OPT-302, KSI-301, injectable sunitinib malate (GB-102), PAN-90806 (PanOptica), and / or aflibercept). In some embodiments, the anti-VEGF agent is aflibercept, a functional variant thereof, or a functional fragment thereof.In some embodiments, the anti-VEGF agent comprises a polypeptide comprising an amino acid sequence having at least about 95% identity to the amino acid sequence of SEQ ID NO:35.

[0083] In some embodiments, the ocular neovascular disease is diabetic macular edema (DME). In some embodiments, the individual is human. In some embodiments, the individual has type 1 or type 2 diabetes. In some embodiments, the individual has visual impairment due to diabetic macular edema extending to the fovea. In some embodiments, the individual's eye to be administered with the rAAV particles, prior to administration of the unit dose of rAAV particles, has a visual acuity (BCVA) of about 78 to 50 ETDRS letters (e.g., any of 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, or 78 ETDRS letters). In some embodiments, the individual's eye to be administered with the rAAV particles has a BCVA of about 20 / 32 to about 20 / 100 (Snellen converted visual acuity) prior to administration of the unit dose of rAAV particles. In some embodiments, the individual's eye to be administered with the rAAV particles has a central retinal thickness (CST) of ≧325 μm using Heidelberg Spectralis®, when an IRF including the fovea (fovea 1 mm) is present. In some embodiments, the individual's eye to be administered with the rAAV particles has a decrease in visual acuity primarily due to diabetic macular edema prior to administration of the unit dose of rAAV particles. In some embodiments, the individual has been diagnosed with diabetic macular edema in the eye receiving the rAAV particles within about 6 months or less prior to administration of the unit dose of rAAV particles, e.g., within any of about 6 months or less, about 5 months or less, about 4 months or less, about 3 months or less, about 2 months or less, or about 1 month or less prior to administration of the unit dose of rAAV particles. In some embodiments, prior to administration of the unit dose of rAAV particles, the individual has received zero, one, or two previous treatments for DME in the eye receiving the rAAV particles, e.g., zero, one, or two intravitreal injections of an anti-VEGF agent, such as aflibercept.In some embodiments, the individual has received previous treatment with an anti-VEGF agent in the eye receiving 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 eye receiving the rAAV particles has shown a meaningful response in terms of central retinal thickness to the previous treatment with the anti-VEGF agent, e.g., at least a 10% reduction in central retinal thickness, prior to administration of the unit dose of rAAV particles. In some embodiments, the individual has not experienced an adverse reaction to the previous treatment with an anti-VEGF agent prior to administration of the unit dose of rAAV particles. In some embodiments, the individual does not have neutralizing antibodies against AAV2.7m8 prior to administration of the unit dose of rAAV particles. In some embodiments, the individual's anti-AAV2.7m8 neutralizing antibody titer does not exceed 1:125 prior to administration of the unit dose of rAAV particles, e.g., within about 6 months prior to administration of the unit dose of rAAV particles. In some embodiments, the individual has no history of allergy to aflibercept, corticosteroids, or fluorescein dyes or sodium fluorescein (e.g., used in angiography) prior to administration of the unit dose of rAAV particles. In some embodiments, the individual has a history of mild allergy to aflibercept, corticosteroids, or fluorescein dyes or sodium fluorescein (e.g., used in angiography) prior to administration of the unit dose of rAAV particles, and the allergy is treatable. In some embodiments, the individual does not have uncontrolled diabetes, e.g., an HbA1c greater than 10%, prior to administration of the unit dose of rAAV particles. In some embodiments, the individual does not have a history of diabetic ketoacidosis within about 3 months prior to administration of the unit dose of rAAV particles. In some embodiments, the individual has not started intensive insulin treatment, e.g., via an insulin pump or multiple daily insulin injections, prior to administration of the unit dose of rAAV particles.In some embodiments, the individual is not scheduled to begin intensive insulin treatment, e.g., via an insulin pump or multiple daily insulin injections, within about three months of administration of the unit dose of rAAV particles. In some embodiments, the individual does not have a history of systemic autoimmune disease requiring treatment with systemic steroids or immunosuppressive therapies, e.g., methotrexate or adalimumab, prior to administration of the unit dose of rAAV particles. In some embodiments, the individual has not received systemic drugs known to cause macular edema, such as fingolimod, tamoxifen, chloroquine, or hydroxychloroquine, prior to administration of the unit dose of rAAV particles. In some embodiments, the individual has not received systemic anti-VEGF therapy prior to administration of the unit dose of rAAV particles. In some embodiments, the individual does not have a high risk of proliferative diabetic retinopathy (PDR) in the eye receiving the rAAV particles prior to administration of the unit dose of rAAV particles. In some embodiments, PDR is defined as any vitreous or preretinal hemorrhage, neovascularization elsewhere in >1 / 2 the papillary area within an area equivalent to a standard ETDRS 7 visual field on clinical examination, or neovascularization of the optic nerve of >1 / 3 the papillary area on clinical examination. In some embodiments, the individual has not undergone focal or grid laser photocoagulation in the eye receiving the rAAV particles prior to administration of the unit dose of rAAV particles. In some embodiments, the individual has not undergone any previous panretinal photocoagulation (PRP) in the eye receiving the rAAV particles prior to administration of the unit dose of rAAV particles. In some embodiments, the individual has not undergone anti-VEGF therapy (e.g., an IVT injection of aflibercept) in the eye receiving the rAAV particles prior to administration of the unit dose of rAAV particles. In some embodiments, the individual has not received anti-VEGF therapy (e.g., an IVT injection of aflibercept) in the eye receiving the rAAV particles 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., IVT injections of aflibercept) in the eye receiving the rAAV particles prior to administration of the unit dose of rAAV particles. In some embodiments, the individual has no history of anterior segment neovascularization (e.g., iris neovascularization [NVI] or neovascular glaucoma [NVG]), significant vitreous hemorrhage, fibrovascular tissue proliferation, or tractional retinal detachment in the eye receiving the rAAV particles prior to administration of the unit dose of rAAV particles. In some embodiments, the individual has no structural abnormalities of the fovea that contribute to macular edema or visual impairment (e.g., thick, hard exudates, pigmentary abnormalities, foveal atrophy, vitreomacular traction, or epiretinal membrane) in the eye receiving the unit dose of rAAV particles prior to administration of the unit dose of rAAV particles. In some embodiments, structural abnormalities of the fovea are assessed by clinical examination or OCT. In some embodiments, the individual has no history of retinal disease other than diabetic retinopathy (e.g., age-related macular degeneration (in either eye), retinal vein occlusion, retinal artery occlusion, or pathological myopia) in the eye receiving the rAAV particles prior to administration of the unit dose of rAAV particles. In some embodiments, the individual has no history of ocular disease other than diabetic macular edema, e.g., a history of significant cataract or macular traction, or evidence of posterior subcapsular cataract, in the eye receiving the rAAV particles prior to administration of the unit dose of rAAV particles. In some embodiments, the individual has no history of cataract extraction or yttrium aluminum garnet (YAG) capsulotomy in the eye receiving the rAAV particles within at least about 3 months prior to administration of the unit dose of rAAV particles. In some embodiments, the individual has no history of retinal detachment (with or without repair) in the eye receiving the rAAV particles prior to administration of the unit dose of rAAV particles.In some embodiments, the individual has no history of trabeculectomy, glaucoma shunting, or minimally invasive glaucoma surgery (MIGS) in the eye receiving the rAAV particles prior to administration of the unit dose of rAAV particles. In some embodiments, the individual has no history of vitrectomy or other filtration surgery in the eye receiving the rAAV particles prior to administration of the unit dose of rAAV particles. In some embodiments, the individual has no aphakia or the presence of an anterior chamber intraocular lens in the eye receiving the rAAV particles prior to administration of the unit dose of rAAV particles. In some embodiments, the individual has no uncontrolled ocular hypertension or glaucoma, e.g., an IOP greater than 22 mmHg despite treatment with anti-glaucoma drugs or current use of more than two IOP-lowering drugs, in the eye receiving the unit dose of rAAV particles prior to administration of the unit dose of rAAV particles. In some embodiments, the individual has no history of intraocular or periocular steroid treatment for any ocular disease (e.g., IVT Triesence, Iluvien, or Ozurdex) in the eye receiving the rAAV particles prior to administration of the unit dose of rAAV particles. In some embodiments, the individual has not undergone refractive surgery in the eye receiving the rAAV particles within at least about 90 days prior to administration of the unit dose of rAAV particles. In some embodiments, the individual has not undergone previous penetrating keratoplasty, endothelial keratoplasty, or ocular radiation in the eye receiving the rAAV particles prior to administration of the unit dose of rAAV particles. In some embodiments, the individual has not undergone any previous vitreoretinal surgery in the eye receiving the rAAV particles prior to administration of the unit dose of rAAV particles. In some embodiments, the individual has no history of uveitis or intraocular inflammation, e.g., evidence of or greater than mild, expected post-operative inflammation that has resolved, prior to administration of the unit dose of rAAV particles.In some embodiments, the individual has no history of elevated IOP associated with topical steroid administration prior to administration of the unit dose of rAAV particles. In some embodiments, the individual has no history of ocular herpes simplex virus (HSV), varicella-zoster virus (VZV), or cytomegalovirus (CMV), including viral uveitis, retinitis, or keratitis, prior to administration of the unit dose of rAAV particles. In some embodiments, the individual has no evidence of any external ocular infection, including conjunctivitis, chalazion, or significant blepharitis, prior to administration of the unit dose of rAAV particles. In some embodiments, the individual has no history of ocular toxoplasmosis prior to administration of the unit dose of rAAV particles.

[0084] In some embodiments, the unit dose is expressed as the number of vector genomes (vg). In some embodiments, the unit dose is about 6 x 10 11 The rAAV particles are no more than 10 vector genomes (vg). In some embodiments, the unit dose is expressed as vector genomes (vg) / eye (vg / eye). In some embodiments, the unit dose is about 6 x 10 11 In some embodiments, the unit dose of the rAAV particles is about 6×10 vg / eye or less. 10 ~Approx. 2×10 11 In some embodiments, the unit dose of the rAAV particles is about 2 x 10 vg / eye. 11 or about 6 x 10 10 vg / eye.

[0085] 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.

[0086] In some embodiments, the administration of the unit dose of rAAV particles to the contralateral eye of the individual is up to about 2 weeks (e.g., about day 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14) after the administration of 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 approximately the same as (e.g., less than 1% high or low, less than 5% high or low, less than 10% high or low, or less than 20% high or low) or lower (e.g., about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, or about 90% lower) than the unit dose of rAAV particles administered to one eye of the individual.

[0087] In some embodiments, administration of 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 least about 4 years, at least about 5 years, or longer) after administration of 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 than the unit dose of rAAV particles administered to the unilateral eye of the individual (e.g., about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 125%, about 150%, about 175%, about 200%, about 225%, about 250%, about 275%, about 300%, or greater).

[0088] In some embodiments, the rAAV particles comprise: a) a nucleic acid encoding a polypeptide comprising an amino acid sequence having at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.99%, or 100% identity to the amino acid sequence of SEQ ID NO: 35, flanked by AAV2 inverted terminal repeats (ITRs); and b) an AAV2 capsid protein comprising the amino acid sequence LGETTRP (SEQ ID NO: 14) inserted between positions 587 and 588 of the capsid protein, where the numbering of the amino acid residues corresponds to that of the AAV2 VP1 capsid protein. The sequence of SEQ ID NO: 35 is shown below: SDTGRPFVEMYSEIPEIIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVLS PSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHEKDKTHTCPPCP APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 35)

[0089] In some embodiments, the rAAV particle comprises: a) a nucleic acid encoding a polypeptide comprising an amino acid sequence having at least about 95% identity to the amino acid sequence of SEQ ID NO: 35, flanked by AAV2 inverted terminal repeats (ITRs); and b) an AAV2 capsid protein comprising the amino acid sequence LGETTRP (SEQ ID NO: 14) inserted between positions 587 and 588 of the capsid protein, wherein the numbering of the amino acid residues corresponds to that of the AAV2 VP1 capsid protein.

[0090] In some embodiments, the rAAV particles comprise a nucleic acid encoding a polypeptide comprising an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.99%, or 100% identity to the amino acid sequence of SEQ ID NO: 35, and flanked by AAV2 inverted terminal repeats (ITRs). In some embodiments, the rAAV particles comprise a nucleic acid encoding a polypeptide comprising an amino acid sequence having at least about 95% identity to the amino acid sequence of SEQ ID NO: 35, 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 or functional fragment thereof.

[0091] In some embodiments, the rAAV particles comprise a codon-optimized sequence encoding an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.99%, or 100% identity to the amino acid sequence of SEQ ID NO: 35, and comprise a nucleic acid flanked by AAV2 inverted terminal repeats (ITRs). In some embodiments, the rAAV particles comprise a codon-optimized sequence encoding an amino acid sequence having at least about 95% identity to the amino acid sequence of SEQ ID NO: 35, and comprise a nucleic acid flanked by AAV2 inverted terminal repeats (ITRs). In some embodiments, the rAAV particles comprise a codon-optimized sequence encoding an amino acid sequence having 100% identity to the amino acid sequence of SEQ ID NO: 35, and comprise a nucleic acid flanked by AAV2 inverted terminal repeats (ITRs).

[0092] In some embodiments, the rAAV particles comprise a nucleic acid comprising the cDNA sequence of aflibercept, or a functional variant 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 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.

[0093] In some embodiments, the nucleic acid further comprises (a) a first enhancer region comprising a CMV sequence, (b) a promoter region comprising a CMV sequence, (c) a 5'UTR region comprising, in 5' to 3' order, a TPL sequence and an eMLP sequence, (d) a second enhancer region comprising a full-length EES sequence, and (e) an HGH polyadenylation site. In some embodiments, the enhancer region comprising a CMV sequence comprises the sequence of SEQ ID NO: 22. In some embodiments, the promoter region comprising a CMV sequence comprises the sequence of SEQ ID NO: 23. In some embodiments, the TPL sequence comprises the sequence of SEQ ID NO: 24. In some embodiments, the eMLP sequence comprises the sequence of SEQ ID NO: 25. In some embodiments, the second enhancer region comprising a full-length EES sequence comprises the sequence of SEQ ID NO: 26. In some embodiments, the HGH polyadenylation site comprises the sequence of SEQ ID NO: 27.

[0094] In some embodiments, the rAAV particles comprise an AAV2 capsid protein comprising the amino acid sequence LGETTRP (SEQ ID NO: 14) inserted between positions 587 and 588 of AAV2 VP1 comprising the sequence of SEQ ID NO: 13. The sequence of SEQ ID NO: 13 is shown below. MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPGYKYLGPFNGLD KGEPVNEADAAALEHDKAYDRQLDSGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQ AKKRVLEPLGLVEEPVKTAPGKKRPVEHSPVEPDSSSGTGKAGQQPARKRLNFGQTGDAD SVPDPQPLGQPPAAPSGLGTNTMATGSGAPMADNNEGADGVGNSSGNWHCDSTWMGDRVI TTSTRTWALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLI NNNWGFRPKRLNFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQG CLPPFPADVFMVPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEDVPF HSSYAHSQSLDRLMNPLIDQYLYYLSRTNTPSGTTTQSRLQFSQAGASDIRDQSRNWLPG PCYRQQRVSKTSADNNNSEYSWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVL IFGKQGSEKTNVDIEKVMITDEEIRTTNPVATEQYGSVSTNLQRGNRQAATADVNTQGV LPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPSTT FSAAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSVNVDFTVDTNGVY SEPRPIGTRYLTRNL (SEQ ID NO: 13)

[0095] In some embodiments, the rAAV particles comprise an AAV2 capsid protein comprising the amino acid sequence LALGETTRPA (SEQ ID NO: 1) inserted between positions 587 and 588 of the capsid protein, where the numbering of the amino acid residues corresponds to that of the AAV2 VP1 capsid protein. In some embodiments, the rAAV particles comprise an AAV2 capsid protein comprising the amino acid sequence LALGETTRPA (SEQ ID NO: 1) inserted between positions 587 and 588 of AAV2 VP1, which comprises the sequence of SEQ ID NO: 13.

[0096] In some embodiments, the rAAV particles comprise an AAV2 capsid protein having one of the following amino acid sequences inserted between positions 587 and 588 of the capsid protein: LALGETTRPA (SEQ ID NO: 1), LANETITRPA (SEQ ID NO: 2), LAKAGQANNA (SEQ ID NO: 3), LAKDPKTTNA (SEQ ID NO: 4), KDTDTTR (SEQ ID NO: 5), RAGGSVG (SEQ ID NO: 6), AVDTTKF (SEQ ID NO: 7), STGKVPN (SEQ ID NO: 8), LAKDTDTTRA (SEQ ID NO: 9). In some embodiments, the rAAV particles comprise an AAV2 capsid protein comprising any of the following: LARAGGSVGA (SEQ ID NO: 10), LAAVDTTKFA (SEQ ID NO: 11), LASTGKVPNA (SEQ ID NO: 12), LGETTRP (SEQ ID NO: 14), NETITRP (SEQ ID NO: 15), KAGQANN (SEQ ID NO: 16), KDPKTTN (SEQ ID NO: 17), KDTDTTR (SEQ ID NO: 18), RAGGSVG (SEQ ID NO: 19), AVDTTKF (SEQ ID NO: 20), and STGKVPN (SEQ ID NO: 21), where the numbering of the amino acid residues corresponds to the AAV2 VP1 capsid protein. In some embodiments, the rAAV particles comprise an AAV2 capsid protein comprising the sequence of SEQ ID NO: 13. It contains an AAV2 capsid protein containing any of the following amino acid sequences inserted between positions 587 and 588 of VP1: LALGETTRPA (SEQ ID NO: 1); LANETITRPA (SEQ ID NO: 2), LAKAGQANNA (SEQ ID NO: 3), LAKDPKTTNA (SEQ ID NO: 4), KDTDTTR (SEQ ID NO: 5), RAGGSVG (SEQ ID NO: 6), AVDTTKF (SEQ ID NO: 7), STGKVPN (SEQ ID NO: 8), LAKDTDTTRA (SEQ ID NO: 9), LARAGGSVGA (SEQ ID NO: 10), LAAVDTTKFA (SEQ ID NO: 11), LASTGKVPNA (SEQ ID NO: 12), LGETTRP (SEQ ID NO: 14), NETITRP (SEQ ID NO: 15), KAGQANN (SEQ ID NO: 16), KDPKTTN (SEQ ID NO: 17), KDTDTTR (SEQ ID NO: 18), RAGGSVG (SEQ ID NO: 19), AVDTTKF (SEQ ID NO: 20), and STGKVPN (SEQ ID NO: 21).

[0097] In some embodiments, the unit dose of rAAV particles is administered to one eye and / or the contralateral eye of the individual by intravitreal (IVT) injection, intraocular administration, or intraretinal injection. In some embodiments, the unit dose of rAAV particles is administered to one eye and / or the contralateral eye of the individual by intravitreal (IVT) injection.

[0098] In some embodiments, the unit dose of rAAV particles is present in a pharmaceutical formulation. In some embodiments, the pharmaceutical formulation comprises the rAAV particles, one or more osmolality or ionic strength adjusters, one or more buffering agents, one or more surfactants, and one or more solvents. In some embodiments, the osmolality or ionic strength adjuster is sodium chloride. In some embodiments, the one or more buffering agents are sodium dihydrogen phosphate and / or sodium hydrogen phosphate. In some embodiments, the surfactant is poloxamer 188. In some embodiments, the solvent is water. In some embodiments, the pharmaceutical formulation comprises the rAAV particles, sodium chloride, sodium phosphate, and a surfactant. In some embodiments, the pharmaceutical formulation comprises about 1 x 10 10 vg / mL ~ approx. 1×10 13 In some embodiments, the pharmaceutical formulation comprises about 6 x 10 vg / mL of rAAV particles. 11 vg / mL ~ approx. 6×10 12vg / mL rAAV particles. In some embodiments, the pharmaceutical formulation contains about 150 mM to about 200 mM sodium chloride (e.g., about 150 mM, about 160 mM, about 170 mM, about 180 mM, about 190 mM, or about 200 mM). In some embodiments, the pharmaceutical formulation contains about 1 mM to about 10 mM sodium dihydrogen phosphate (e.g., about 1 mM, about 2 mM, about 3 mM, about 4 mM, about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, or about 10 mM). In some embodiments, the pharmaceutical formulation contains about 1 mM to about 10 mM sodium dihydrogen phosphate (e.g., about 1 mM, about 2 mM, about 3 mM, about 4 mM, about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, or about 10 mM). In some embodiments, the pharmaceutical formulation contains about 0.0005% (w / v) to about 0.005% (w / v) poloxamer 188 (e.g., about 0.0005% (w / v), 0.0006% (w / v), 0.0007% (w / v), 0.0008% (w / v), 0.0009% (w / v), 0.001% (w / v), 0.002% (w / v), 0.003% (w / v), 0.004% (w / v), or about 0.005% (w / v)). In some embodiments, the pH of the pharmaceutical formulation is about 7.0 to about 7.5 (e.g., about 7.0, about 7.1, about 7.2, about 7.3, about 7.4, or about 7.5). In some embodiments, the pharmaceutical formulation contains about 6×10 12 In some embodiments, the pharmaceutical formulation comprises about 6 x 10 vg / mL rAAV particles, about 180 mM sodium chloride, about 5 mM sodium dihydrogen phosphate, about 5 mM sodium dihydrogen phosphate, and about 0.001% (w / v) poloxamer 188, and the pH of the pharmaceutical formulation is about 7.3. 11 vg / mL rAAV particles, about 180 mM sodium chloride, about 5 mM sodium dihydrogen phosphate, about 5 mM sodium dihydrogen phosphate, and about 0.001% (w / v) poloxamer 188, and the pH of the pharmaceutical formulation is about 7.3.

[0099] In some embodiments, the unit dose of rAAV particles comprises a volume of about 25 μL to about 250 μL (e.g., about 25 μL, about 30 μL, about 40 μL, about 50 μL, about 60 μL, about 70 μL, about 80 μL, about 90 μL, about 100 μL, about 110 μL, about 120 μL, about 130 μL, about 140 μL, about 150 μL, about 160 μL, about 170 μL, about 180 μL, about 190 μL, about 200 μL, about 210 μL, about 220 μL, about 230 μL, about 240 μL, or about 250 μL). In some embodiments, the concentration of rAAV particles in the pharmaceutical formulation is adjusted so that the volume of the unit dose of rAAV particles administered to the eye of the individual is between about 25 μL and about 250 μL. In some embodiments, the unit dose of rAAV particles comprises a volume of about 100 μL, hi some embodiments, the unit dose of rAAV particles comprises a volume of about 30 μL.

[0100] In some embodiments, the unit dose of rAAV particles is administered in combination with steroid therapy. In some embodiments, the steroid therapy is corticosteroid therapy. In some embodiments, the steroid therapy is systemic steroid therapy. In some embodiments, the steroid therapy is oral steroid therapy. In some embodiments, the steroid therapy is prednisone therapy. In some embodiments, the steroid therapy is ophthalmic steroid therapy. In some embodiments, the ophthalmic steroid therapy is topical steroid therapy (e.g., eye drops), periocular steroid therapy (e.g., subtenon, subconjunctival), intravitreal steroid therapy, or suprachoroidal steroid therapy. In some embodiments, the topical steroid therapy is difluprednate therapy, medrysone therapy, loteprednol therapy, prednisolone therapy, fluocinolone therapy, triamcinolone therapy, rimexolone therapy, dexamethasone therapy, fluorometholone therapy, fluocinolone therapy, rimexolone therapy, or prednisone therapy. In some embodiments, the topical steroid treatment is difluprednate treatment. In some embodiments, the topical 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.

[0101] In some embodiments, the steroid treatment is an ophthalmic steroid therapeutic agent (e.g., difluprednate). In some embodiments, the ophthalmic steroid treatment (e.g., difluprednate) is daily steroid treatment for up to about 4 weeks, about 6 weeks, or about 8 weeks from administration of the unit dose of rAAV particles. In some embodiments, the ophthalmic steroid treatment comprises about four administrations of ophthalmic steroid at about week 1, about three administrations of ophthalmic steroid at about week 2, about two administrations of ophthalmic steroid at about week 3, and about one administration of ophthalmic steroid at about week 4, starting at the time of administration of the unit dose of rAAV particles and continuing thereafter. In some embodiments, the ophthalmic steroid is about 0.005% to about 0.5% difluprednate. In some embodiments, the ophthalmic steroid is about 0.005%, about 0.006%, about 0.007%, about 0.008%, about 0.009%, about 0.01%, about 0.02%, about 0.03%, about 0.4%, about 0.05%, about 0.06%, about 0.07%, about 0.08%, about 0.09%, or about 0.1% difluprednate. In some embodiments, the ophthalmic steroid is 0.05% difluprednate. In some embodiments, a dose of 0.05% difluprednate is one drop of ophthalmic solution. In some embodiments, one drop is about 50 μl (e.g., about 25 μl to about 50 μl, about 50 μl to about 100 μl). In some embodiments, the dose of difluprednate comprises about 1 μg to about 5 μg, or about 2 μg to about 3 μg, or about 2.5 μg of difluprednate. In some embodiments, the dose of difluprednate comprises about 2.5 μg of difluprednate.

[0102] In some embodiments, the unit dose of rAAV particles is administered in combination with steroid therapy. In some embodiments, the steroid therapy is corticosteroid therapy. In some embodiments, the steroid therapy is systemic steroid therapy. In some embodiments, the steroid therapy is oral steroid therapy. In some embodiments, the steroid therapy is prednisone therapy. In some embodiments, the steroid therapy is ophthalmic steroid therapy. In some embodiments, the ophthalmic steroid therapy is topical steroid therapy (e.g., eye drops), periocular steroid therapy (e.g., sub-Tenon, subconjunctival), intravitreal steroid therapy, or suprachoroidal steroid therapy. In some embodiments, the ophthalmic steroid therapy is a glucocorticoid, including but not limited to, an anti-inflammatory glucocorticoid. In some embodiments, the topical steroid therapy is a glucocorticoid, including but not limited to, an anti-inflammatory glucocorticoid. In some embodiments, the topical steroid treatment is difluprednate, medrysone, loteprednol, prednisolone, fluocinolone, triamcinolone, rimexolone, dexamethasone, fluorometholone, fluocinolone, rimexolone, or prednisone. Anti-inflammatory glucocorticoids may include, but are not limited to, difluprednate, dexamethasone, prednisolone, triamcinolone, fluorometholone, rimexolone, fluocinolone, loteprednol, and generic versions thereof. In some embodiments, the topical steroid treatment is difluprednate. "Dexamethasone" refers to dexamethasone and generic versions of dexamethasone, as well as pharmaceutical compositions containing dexamethasone and generic versions of dexamethasone.Pharmaceutical compositions containing dexamethasone include, but are not limited to, Ozurdex™, Maxidex™, Decadron™, Dexamethasone Intensol™, Ocu-Dex™, Dexycu™, Dextza™, and Zodex™. Ozurdex™ is a pharmaceutical composition containing dexamethasone. "Difluprednate" refers to pharmaceutical compositions containing difluprednate, generic versions of difluprednate, and generic versions of difluprednate or difluprednate. Pharmaceutical compositions containing difluprednate include, but are not limited to, Durezol™ and difluprednate emulsion. "Triamcinolone" refers to pharmaceutical compositions containing triamcinolone, generic versions of triamcinolone, and generic versions of triamcinolone or triamcinolone. Pharmaceutical compositions comprising triamcinolone include, but are not limited to, Triesence™, Xpere™, and Trivalis™. In some embodiments, the topical 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.

[0103] In some embodiments, the steroid treatment is an ophthalmic steroid therapeutic agent (e.g., difluprednate). In some embodiments, the ophthalmic steroid treatment (e.g., difluprednate) is daily topical steroid treatment for up to about 4 weeks, about 6 weeks, or about 8 weeks from administration of the unit dose of rAAV particles. In some embodiments, the topical steroid treatment comprises about four administrations of topical steroid at about week 1, about three administrations of topical steroid at about week 2, about two administrations of topical steroid at about week 3, and about one administration of topical steroid at about week 4, starting at the time of administration of the unit dose of rAAV particles and continuing thereafter. In some embodiments, the topical steroid treatment comprises administering the topical steroid about four times per day (i.e., QID) for about three weeks after administration of the unit dose of rAAV particles, followed by administering the topical steroid about three times per day (i.e., TID) for about one week thereafter, administering the topical steroid about twice per day (i.e., BID) for about one week thereafter, and administering the topical steroid about once per day (i.e., QD) for about one week thereafter. In some embodiments, the topical steroid comprises about 1 μg to about 3 μg doses of 0.05% difluprednate. In some embodiments, the topical steroid comprises about 2.5 μg doses of 0.05% difluprednate. In some embodiments, the topical steroid comprises about 0.005% to about 0.5% difluprednate. In some embodiments, the topical steroid is about 0.005%, about 0.006%, about 0.007%, about 0.008%, about 0.009%, about 0.01%, about 0.02%, about 0.03%, about 0.04%, about 0.05%, about 0.06%, about 0.07%, about 0.08%, about 0.09%, or about 0.1% difluprednate. In some embodiments, the topical steroid is 0.05% difluprednate. In some embodiments, the dose of 0.05% difluprednate is one drop of ophthalmic solution. In some embodiments, one drop is about 50 μl (e.g., about 25 μl to about 50 μl, about 50 μl to about 100 μl).In some embodiments, the dose of difluprednate comprises about 1 μg to about 5 μg, or about 2 μg to about 3 μg, or about 2.5 μg of difluprednate. In some embodiments, the dose of difluprednate comprises about 2.5 μg of difluprednate.

[0104] In some embodiments, after administration of a unit dose of rAAV particles to one eye and / or the other eye of an individual, the retinal fluid (e.g., SRF and / or IRF) in the eye of the individual is reduced by more than about 5%, more than about 10%, more than about 15%, more than about 20%, more than about 30%, more than about 40%, more than about 50%, more than about 60%, more than about 70%, more than about 80%, more than about 90%, or about 100%. In some embodiments, after administration of a unit dose of rAAV particles to one and / or the other eye of an individual, retinal fluid (e.g., SRF and / or IRF) in the eye of the individual is reduced by more than about 5%, more than about 10%, more than about 15%, more than about 20%, more than about 30%, more than about 40%, more than about 50%, more than about 60%, more than about 70%, more than about 80%, more than about 90%, or about 100% compared to the level of retinal fluid in the eye of the individual before administration of the unit dose of rAAV particles. In some embodiments, after administration of a unit dose of rAAV particles to one and / or the other eye of an individual, retinal fluid (e.g., SRF and / or IRF) in the eye of the individual is reduced by about 100% compared to the level of retinal fluid in the eye of the individual before administration of the unit dose of rAAV particles.

[0105] In some embodiments, the methods provided herein further comprise monitoring the level of retinal fluid (e.g., SRF and / or IRF) in the individual's eye and / or the contralateral eye after administration of the unit dose of rAAV particles. In some embodiments, a decrease in retinal fluid (e.g., SRF and / or IRF) in the eye is first observed at 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 in 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 6 weeks, at least 8 weeks, at least 12 weeks, at least 16 weeks, at least 20 weeks, at least 24 weeks, at least 28 weeks, at least 32 weeks, at least 36 weeks, at least 40 weeks, at least 44 weeks, at least 48 weeks, at least 52 weeks, at least 56 weeks, at least 60 weeks, at least 64 weeks, at least 68 weeks, at least 72 weeks, at least 76 weeks, at least 80 weeks, at least 84 weeks, at least 88 weeks, at least 92 weeks, at least 96 weeks, at least 100 weeks, at least 104 weeks, or longer, following administration of the unit dose of rAAV particles.

[0106] In some embodiments, the reduction in intraocular retinal fluid (e.g., SRF and / or IRF) is measured by any method known in the art. In some embodiments, the reduction in intraocular retinal fluid (e.g., SRF and / or IRF) is measured by optical coherence tomography (OCT), spectral domain OCT (SD-OCT), OCT angiography, fluorescein angiography, or by direct retinal observation. In some embodiments, the reduction in intraocular retinal fluid (e.g., SRF and / or IRF) is measured by optical coherence tomography (OCT). In some embodiments, the reduction in intraocular retinal fluid (e.g., SRF and / or IRF) is measured by spectral domain OCT (SD-OCT). In some embodiments, the reduction in intraocular retinal fluid (e.g., SRF and / or IRF) is measured by OCT angiography. In some embodiments, the reduction in intraocular retinal fluid (e.g., SRF and / or IRF) is measured by fluorescein angiography. In some embodiments, the reduction in retinal fluid (eg, SRF and / or IRF) in the eye is measured by direct retinal observation.

[0107] In some embodiments, treatment of an ocular neovascular disease in an individual following administration of a unit dose of rAAV particles to one eye and / or the contralateral eye was assessed based on the level of retinal fluid (e.g., intraretinal fluid (IRF) and / or subretinal fluid (SRF)) compared to the level of retinal fluid (e.g., SRF and / or IRF) before administration of the unit dose of rAAV particles to one eye and / or the contralateral eye (e.g., as described above). In some embodiments, the retinal fluid is subretinal fluid (SRF) or intraretinal fluid (IRF). In some embodiments, the retinal fluid is subretinal fluid (SRF). In some embodiments, the retinal fluid is intraretinal fluid (IRF). In some embodiments, if a decrease in retinal fluid (e.g., IRF and / or SRF) is observed after administration of a unit dose of rAAV particles to one eye and / or the contralateral eye compared to the level of retinal fluid (e.g., IRF and / or SRF) before administration of the unit dose of rAAV particles to one eye and / or the contralateral eye, a decision is made to treat the ocular neovascular disease in the individual (e.g., as described above) after administration of the unit dose of rAAV particles to one eye and / or the contralateral eye. In some embodiments, the ocular neovascular disease is wAMD.

[0108] In some embodiments, administration of a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual maintains or reduces retinal thickness compared to the retinal thickness before administration of the unit dose of rAAV particles. In some embodiments, administration of a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual reduces retinal thickness compared to the retinal thickness before administration of the unit dose of rAAV particles. In some embodiments, the retinal thickness is central retinal thickness (CST) or central retinal thickness (CRT). In some embodiments, administration of a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual reduces retinal thickness by more than about 5%, more than about 10%, more than about 15%, more than about 20%, more than about 25%, more than about 30%, more than about 35%, more than about 40%, more than about 45%, more than about 50%, more than about 55%, more than about 60%, more than about 65%, more than about 70%, more than about 75%, more than about 80%, more than about 85%, more than about 90%, more than about 95%, more than about 99%, or about 100% compared to the retinal thickness before administration of the unit dose of rAAV particles. In some embodiments, retinal thickness (e.g., CST or CRT) is measured by OCT or SD-OCT.

[0109] In some embodiments, administering a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual reduces the central retinal zone thickness (CRT) or central retinal thickness (CST) by about 10 μm to about 100 μm (e.g., by any of greater than about 10 μm, greater than about 15 μm, greater than about 20 μm, greater than about 25 μm, greater than about 30 μm, greater than about 35 μm, greater than about 40 μm, greater than about 45 μm, greater than about 50 μm, greater than about 55 μm, greater than about 60 μm, greater than about 65 μm, greater than about 70 μm, greater than about 75 μm, greater than about 80 μm, greater than about 85 μm, greater than about 90 μm, greater than about 95 μm, greater than about 100 μm, or more). In some embodiments, administering a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual reduces central retinal thickness (CRT) or central retinal thickness (CST) by about 5 μm to about 50 μm (e.g., about 5 μm, about 10 μm, about 15 μm, about 20 μm, about 25 μm, about 30 μm, about 35 μm, about 40 μm, about 45 μm, or about 50 μm). In some embodiments, administering a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual reduces central retinal thickness (CRT) or central retinal thickness (CST) by about 5 μm to about 40 μm (e.g., about 5 μm, about 10 μm, about 15 μm, about 20 μm, about 25 μm, about 30 μm, about 35 μm, or about 40 μm). In some embodiments, administration of a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual results in a reduction in central retinal thickness (CRT) or central retinal thickness (CST) of about 5 μm to about 30 μm (e.g., about 5 μm, about 6 μm, about 7 μm, about 8 μm, about 9 μm, about 10 μm, about 11 μm, about 12 μm, about 13 μm, about 14 μm, about 15 μm, about 16 μm, about 17 μm, about 18 μm, about 19 μm, about 20 μm, about 21 μm, about 22 μm, about 23 μm, about 24 μm, about 25 μm, about 26 μm, about 27 μm, about 28 μm, about 29 μm, or about 30 μm).In some embodiments, administration of a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual results in a reduction in central retinal thickness (CRT) or central retinal thickness (CST) of about 5 μm to about 25 μm (e.g., about 5 μm, about 6 μm, about 7 μm, about 8 μm, about 9 μm, about 10 μm, about 11 μm, about 12 μm, about 13 μm, about 14 μm, about 15 μm, about 16 μm, about 17 μm, about 18 μm, about 19 μm, about 20 μm, about 21 μm, about 22 μm, about 23 μm, about 24 μm, or about 25 μm). In some embodiments, administration of a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual results in a reduction of central retinal thickness (CRT) or central retinal thickness (CST) by about 5 μm to about 20 μm (e.g., about 5 μm, about 6 μm, about 7 μm, about 8 μm, about 9 μm, about 10 μm, about 11 μm, about 12 μm, about 13 μm, about 14 μm, about 15 μm, about 16 μm, about 17 μm, about 18 μm, about 19 μm, or about 20 μm). In some embodiments, administration of a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual results in a reduction of central retinal thickness (CRT) or central retinal thickness (CST) by about 18 μm to about 75 μm. In some embodiments, administration of a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual results in a decrease of about 18.5 μm in central retinal thickness (CRT) or central retinal thickness (CST). In some embodiments, administration of a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual results in a decrease of about 21.0 μm in central retinal thickness (CRT) or central retinal thickness (CST). In some embodiments, administration of a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual results in a decrease of about 8.3 μm in central retinal thickness (CRT) or central retinal thickness (CST). In some embodiments, administration of a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual results in a decrease of about 25.5 μm in central retinal thickness (CRT) or central retinal thickness (CST). In some embodiments, administration of a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual results in a reduction of central retinal thickness (CRT) or central retinal thickness (CST) of about 24.8 μm.In some embodiments, administration of a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual reduces central retinal thickness (CRT) or central retinal thickness (CST) by about 75 μm. In some embodiments, administration of a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual reduces central retinal thickness (CRT) or central retinal thickness (CST) by about 100 μm or more (e.g., by any of about 100 μm or more, about 110 μm or more, about 120 μm or more, about 130 μm or more, about 140 μm or more, about 150 μm or more, about 160 μm or more, about 170 μm or more, about 180 μm or more, about 190 μm or more, or about 200 μm or more). In some embodiments, administration of a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual reduces the central retinal zone thickness (CRT) or central retinal thickness (CST) by about 10 μm to about 200 μm (e.g., 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 200 μm). In some embodiments, administration of a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual reduces the central retinal zone thickness (CRT) or central retinal thickness (CST) by about 50 μm to about 200 μm (e.g., 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 200 μm). In some embodiments, administration of a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual reduces the central retinal zone thickness (CRT) or central retinal thickness (CST) by about 60 μm to about 200 μm (e.g., 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 200 μm).In some embodiments, administration of a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual reduces the central retinal zone thickness (CRT) or central retinal thickness (CST) by about 70 μm to about 200 μm (e.g., 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 200 μm). In some embodiments, administration of a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual reduces the central retinal zone thickness (CRT) or central retinal thickness (CST) by about 80 μm to about 200 μm (e.g., 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 200 μm). In some embodiments, administration of a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual reduces the central retinal zone thickness (CRT) or central retinal thickness (CST) by about 90 μm to about 200 μm (e.g., 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 200 μm). In some embodiments, administration of a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual reduces the central retinal zone thickness (CRT) or central retinal thickness (CST) by about 100 μm to about 200 μm (e.g., 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 200 μm). In some embodiments, administration of a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual results in a reduction in central retinal zone thickness (CRT) or central retinal thickness (CST) of about 110 μm to about 200 μm (e.g., 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 200 μm).In some embodiments, administration of a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual results in a reduction in central retinal zone thickness (CRT) or central retinal thickness (CST) of about 115 μm to about 200 μm (e.g., 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 200 μm). In some embodiments, administration of a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual reduces the central retinal zone thickness (CRT) or central retinal thickness (CST) by about 120 μm to about 200 μm (e.g., 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 200 μm). In some embodiments, administration of a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual reduces the central retinal zone thickness (CRT) or central retinal thickness (CST) by about 125 μm to about 200 μm (e.g., 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 200 μm). In some embodiments, administration of a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual reduces the central retinal zone thickness (CRT) or central retinal thickness (CST) by about 130 μm to about 200 μm (e.g., 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 200 μm).In some embodiments, administration of a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual reduces the central retinal zone thickness (CRT) or central retinal thickness (CST) by about 135 μm to about 200 μm (e.g., 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 200 μm). In some embodiments, administration of a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual reduces the central retinal zone thickness (CRT) or central retinal thickness (CST) by about 140 μm to about 200 μm (e.g., about 140 μm, about 145 μm, about 150 μm, about 155 μm, about 160 μm, about 165 μm, about 170 μm, about 175 μm, about 180 μm, about 185 μm, about 190 μm, about 195 μm, or 200 μm). In some embodiments, administration of a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual reduces the central retinal zone thickness (CRT) or central retinal thickness (CST) by about 145 μm to about 200 μm (e.g., In some embodiments, administration of a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual reduces the central retinal zone thickness (CRT) or central retinal thickness (CST) by about 150 μm to about 200 μm (e.g., 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 200 μm). In some embodiments, administration of a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual reduces the central retinal zone thickness (CRT) or central retinal thickness (CST) by about 155 μm to about 200 μm (e.g., 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 200 μm). In some embodiments, administration of a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual reduces the central retinal zone thickness (CRT) or central retinal thickness (CST) by about 160 μm to about 200 μm (e.g., 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 200 μm). In some embodiments, administration of a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual reduces the central retinal zone thickness (CRT) or central retinal thickness (CST) by about 165 μm to about 200 μm (e.g., about 165 μm, about 170 μm, about 175 μm, about 180 μm, about 185 μm, about 190 μm, about 195 μm, or 200 μm). In some embodiments, administration of a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual reduces the central retinal zone thickness (CRT) or central retinal thickness (CST) by about 170 μm to about 200 μm (e.g., about 170 μm, about 175 μm, about 180 μm, about 185 μm, about 190 μm, about 195 μm, or 200 μm).In some embodiments, administration of a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual reduces the central retinal zone thickness (CRT) or central retinal thickness (CST) by about 175 μm to about 200 μm (e.g., about 175 μm, about 180 μm, about 185 μm, about 190 μm, about 195 μm, or 200 μm). In some embodiments, administration of a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual reduces the central retinal zone thickness (CRT) or central retinal thickness (CST) by about 180 μm to about 200 μm (e.g., about 180 μm, about 185 μm, about 190 μm, about 195 μm, or 200 μm). In some embodiments, administration of a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual results in a decrease of central regional retinal thickness (CRT) or central retinal thickness (CST) of about 21.0 μm compared to the central regional retinal thickness (CRT) or central retinal thickness (CST) before administration of the unit dose of rAAV particles. In some embodiments, administration of a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual results in a decrease of central regional retinal thickness (CRT) or central retinal thickness (CST) of about 8.3 μm compared to the central regional retinal thickness (CRT) or central retinal thickness (CST) before administration of the unit dose of rAAV particles. In some embodiments, administration of a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual results in a decrease of central regional retinal thickness (CRT) or central retinal thickness (CST) of about 26.2 μm compared to the central regional retinal thickness (CRT) or central retinal thickness (CST) before administration of the unit dose of rAAV particles. In some embodiments, administration of a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual results in a decrease in central regional retinal thickness (CRT) or central retinal thickness (CST) of about 24.8 μm compared to the CRT or CST before administration of the unit dose of rAAV particles. In some embodiments, administration of a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual results in a decrease in central regional retinal thickness (CRT) or central retinal thickness (CST) of about 40.8 μm compared to the CRT or CST before administration of the unit dose of rAAV particles.In some embodiments, administration of a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual results in a decrease of central regional retinal thickness (CRT) or central retinal thickness (CST) of about 30.0 μm compared to the central regional retinal thickness (CRT) or central retinal thickness (CST) before administration of the unit dose of rAAV particles. In some embodiments, administration of a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual results in a decrease of central regional retinal thickness (CRT) or central retinal thickness (CST) of about 118.6 μm compared to the central regional retinal thickness (CRT) or central retinal thickness (CST) before administration of the unit dose of rAAV particles. In some embodiments, administration of a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual results in a decrease of central regional retinal thickness (CRT) or central retinal thickness (CST) of about 119.0 μm compared to the central regional retinal thickness (CRT) or central retinal thickness (CST) before administration of the unit dose of rAAV particles. In some embodiments, administration of a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual results in a decrease of central regional retinal thickness (CRT) or central retinal thickness (CST) by about 137.8 μm compared to the central regional retinal thickness (CRT) or central retinal thickness (CST) before administration of the unit dose of rAAV particles. In some embodiments, administration of a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual results in a decrease of central regional retinal thickness (CRT) or central retinal thickness (CST) by about 152.7 μm compared to the central regional retinal thickness (CRT) or central retinal thickness (CST) before administration of the unit dose of rAAV particles. In some embodiments, administration of a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual results in a decrease of central regional retinal thickness (CRT) or central retinal thickness (CST) by about 153.3 μm compared to the central regional retinal thickness (CRT) or central retinal thickness (CST) before administration of the unit dose of rAAV particles. In some embodiments, administration of a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual results in a decrease in central retinal thickness (CRT) or central retinal thickness (CST) of about 149.8 μm compared to the central retinal thickness (CRT) or central retinal thickness (CST) before administration of the unit dose of rAAV particles.

[0110] In some embodiments, administration of a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual results in a change in central retinal region thickness (CRT) or central retinal thickness (CST) of about -200 μm to about +40 μm (e.g., about -200 μm, about -180 μm, about -160 μm, about -140 μm, about -120 μm, about -100 μm, about -80 μm, about -60 μm, about -40 μm, about -20 μm, about 0 μm, about +5 μm, about +10 μm, about +15 μm, about +20 μm, about +25 μm, about +30 μm, about +35 μm, or about +40 μm) compared to the retinal thickness before administration of the unit dose of rAAV particles. In some embodiments, administration of a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual reduces central retinal thickness (CRT) or central retinal thickness (CST) by about 8 μm, about 11 μm, about 16 μm, about 29 μm, about 33 μm, about 38 μm, about 55 μm, about 61 μm, or about 117 μm. In some embodiments, administration of a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual reduces central retinal thickness (CRT) or central retinal thickness (CST) by about 27.8 μm or about 30.8 μm. In some embodiments, administration of a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual increases central retinal thickness (CRT) or central retinal thickness (CST) by about 4 μm, about 12 μm, or about 32 μm. In some embodiments, administration of a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual results in a change in central regional retinal thickness (CRT) or central retinal thickness (CST) of about -21.0 μm compared to the CRT or CST before administration of the unit dose of rAAV particles. In some embodiments, administration of a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual results in a change in central regional retinal thickness (CRT) or central retinal thickness (CST) of about -8.3 μm compared to the CRT or CST before administration of the unit dose of rAAV particles.In some embodiments, administration of a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual results in a change in central regional retinal thickness (CRT) or central retinal thickness (CST) of about -26.2 μm compared to the central regional retinal thickness (CRT) or central retinal thickness (CST) before administration of the unit dose of rAAV particles. In some embodiments, administration of a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual results in a change in central regional retinal thickness (CRT) or central retinal thickness (CST) of about -24.8 μm compared to the central regional retinal thickness (CRT) or central retinal thickness (CST) before administration of the unit dose of rAAV particles. In some embodiments, administration of a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual results in a change in central regional retinal thickness (CRT) or central retinal thickness (CST) of about -40.8 μm compared to the central regional retinal thickness (CRT) or central retinal thickness (CST) before administration of the unit dose of rAAV particles. In some embodiments, administration of a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual results in a change in central regional retinal thickness (CRT) or central retinal thickness (CST) of about -30.0 μm compared to the CRT or CST before administration of the unit dose of rAAV particles. In some embodiments, administration of a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual results in a change in central regional retinal thickness (CRT) or central retinal thickness (CST) of about -118.6 μm compared to the CRT or CST before administration of the unit dose of rAAV particles. In some embodiments, administration of a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual results in a change in central regional retinal thickness (CRT) or central retinal thickness (CST) of about -119.0 μm compared to the CRT or CST before administration of the unit dose of rAAV particles. In some embodiments, administration of a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual results in a change in central regional retinal thickness (CRT) or central retinal thickness (CST) of about -137.8 μm compared to the CRT or CST before administration of the unit dose of rAAV particles.In some embodiments, administration of a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual results in a change in central regional retinal thickness (CRT) or central retinal thickness (CST) of about -152.7 μm compared to the CRT or CST before administration of the unit dose of rAAV particles. In some embodiments, administration of a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual results in a change in central regional retinal thickness (CRT) or central retinal thickness (CST) of about -153.3 μm compared to the CRT or CST before administration of the unit dose of rAAV particles. In some embodiments, administration of a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual results in a change in central retinal thickness (CRT) or central retinal thickness (CST) of approximately -149.8 μm compared to the central retinal thickness (CRT) or central retinal thickness (CST) before administration of the unit dose of rAAV particles.

[0111] In some embodiments, after administration of a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual, at any time within 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, there is a change (e.g., a decrease) in central zone retinal thickness (CRT) or central retinal thickness (CST) compared to the central zone retinal thickness (CRT) or central retinal thickness (CST) before administration of the unit dose of rAAV particles. In some embodiments, after administration of a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual, at any time within 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, 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, there is a change (e.g., a decrease) in central zone retinal thickness (CRT) or central retinal thickness (CST) compared to the CRT or CST before administration of the unit dose of rAAV particles.

[0112] In some embodiments, administration of a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual reduces central regional retinal thickness (CRT) or central retinal thickness (CST) by more than about 5%, more than about 10%, more than about 15%, more than about 20%, more than about 25%, more than about 30%, more than about 35%, more than about 40%, more than about 45%, more than about 50%, more than about 55%, more than about 60%, more than about 65%, more than about 70%, more than about 75%, more than about 80%, more than about 85%, more than about 90%, more than about 95%, more than about 99%, or about 100% compared to the retinal thickness before administration of the unit dose of rAAV particles. In some embodiments, administration of a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual results in at least about a 10% decrease in central retinal thickness (CRT) or central retinal thickness (CST) compared to the retinal thickness before administration of the unit dose of rAAV particles. In some embodiments, administration of a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual results in about a 15% or greater decrease in central retinal thickness (CRT) or central retinal thickness (CST) compared to the retinal thickness before administration of the unit dose of rAAV particles.

[0113] In some embodiments, administration of a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual results in an increase in central region retinal thickness (CRT) or central retinal thickness (CST) of less than about 40 μm (e.g., less than about 40 μm, less than about 35 μm, less than about 30 μm, less than about 25 μm, less than about 20 μm, less than about 15 μm, less than about 10 μm, less than about 5 μm, less than about 1 μm, or less than any of these) compared to the retinal thickness before administration of the unit dose of rAAV particles. In some embodiments, administration of a unit dose of rAAV particles to one eye and / or the contralateral eye of an individual results in an increase in central region retinal thickness (CRT) or central retinal thickness (CST) of less than about 32 μm compared to the retinal thickness before administration of the unit dose of rAAV particles.

[0114] In some embodiments, the maintenance, reduction, or increase in retinal thickness compared to the retinal thickness before administration of the unit dose of rAAV particles is present for about 30 weeks or more after administration of the unit dose of rAAV particles to one eye and / or the contralateral eye of the individual. In some embodiments, the maintenance, reduction, or increase in retinal thickness compared to the retinal thickness before administration of the unit dose of rAAV particles is present for about 30 weeks, about 34 weeks, about 44 weeks, about 6 months, about 1 year, about 1.5 years, about 2 years, about 3 years, about 5 years, about 10 years, or more after administration of the unit dose of rAAV particles to one eye and / or the contralateral eye of the individual.

[0115] In some embodiments, administering the unit dose of rAAV particles to one eye and / or the contralateral eye of the individual maintains or reduces macular volume compared to the macular volume before administration of the unit dose of rAAV particles. In some embodiments, administering the unit dose of rAAV particles to one eye and / or the contralateral eye of the individual reduces macular volume compared to the macular volume before administration of the unit dose of rAAV particles. In some embodiments, administering the unit dose of rAAV particles to one eye and / or the contralateral eye of the individual reduces macular volume by more than about 5%, more than about 10%, more than about 15%, more than about 20%, more than about 25%, more than about 30%, more than about 35%, more than about 40%, more than about 45%, or more than about 50% compared to the macular volume before administration of the unit dose of rAAV particles. In some embodiments, administering the unit dose of rAAV particles to one eye and / or the contralateral eye of the individual reduces macular volume by at least about 10% compared to the macular volume before administration of the unit dose of rAAV particles. In some embodiments, the macular volume is measured by optical coherence tomography (OCT) or standard deviation-octometric (SD-OCT). In some embodiments, administering the unit dose of rAAV particles to one eye and / or the contralateral eye of the individual reduces macular volume by at least about 10% compared to the macular volume before administration of the unit dose of rAAV particles. In some embodiments, administering the unit dose of rAAV particles to one eye and / or the contralateral eye of the individual reduces macular volume by about 15% or more compared to the macular volume before administration of the unit dose of rAAV particles. In some embodiments, the macular volume is measured by OCT or SD-OCT.

[0116] In some embodiments, the macular volume compared to the macular volume before administration of the unit dose of rAAV particles is maintained or reduced about 30 weeks or more after administration of the unit dose of rAAV particles to one eye and / or the contralateral eye of the individual. In some embodiments, the macular volume compared to the macular volume before administration of the unit dose of rAAV particles is maintained or reduced any time point after about 30 weeks, about 34 weeks, about 44 weeks, about 6 months, about 1 year, about 1.5 years, about 2 years, about 3 years, about 5 years, about 10 years, or more after administration of the unit dose of rAAV particles to one eye and / or the contralateral eye of the individual.

[0117] In some embodiments, administering the unit dose of rAAV particles to one eye and / or the contralateral eye of the individual maintains or improves visual acuity compared to the visual acuity before administration of the unit dose of rAAV particles. In some embodiments, administering the unit dose of rAAV particles to one eye and / or the contralateral eye of the individual improves visual acuity compared to the visual acuity before administration of the unit dose of rAAV particles. In some embodiments, administration of the unit dose of rAAV particles to one eye and / or the contralateral eye of the individual improves visual acuity by 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 before administration of the unit dose of rAAV particles. In some embodiments, the visual acuity is best-corrected visual acuity (BCVA). In some embodiments, administration of the unit dose of rAAV particles to one eye and / or the contralateral eye of the individual improves BCVA compared to the BCVA before administration of the unit dose of rAAV particles. In some embodiments, BCVA is expressed as an ETDRS score, which corresponds to the number of letters read correctly (Vitale et al., (2016) JAMA Opthalmol 134(9):1041:1047).

[0118] In some embodiments, administration of the unit dose of rAAV particles to one eye and / or the contralateral eye of the individual improves BCVA by at least 15 ETDRS characters (Vitale et al., (2016) JAMA Opthalmol 134(9):1041:1047) (e.g., at least about 15 letters, at least about 20 letters, at least about 30 letters, at least about 40 letters, at least about 50 letters, at least about 60 letters, or about 70 letters) compared to BCVA before administration of the unit dose of rAAV particles. In some embodiments, administration of the unit dose of rAAV particles to one eye and / or the contralateral eye of the individual improves BCVA by about 1 to about 15 ETDRS characters (e.g., about 1 letter, about 2 letters, about 3 letters, about 4 letters, about 5 letters, about 6 letters, about 7 letters, about 8 letters, about 9 letters, about 10 letters, about 11 letters, about 12 letters, about 13 letters, about 14 letters, or about 15 letters) compared to the BCVA before administration of the unit dose of rAAV particles. In some embodiments, administration of the unit dose of rAAV particles to one eye and / or the contralateral eye of the individual improves BCVA by about 5 ETDRS characters compared to the BCVA before administration of the unit dose of rAAV particles. In some embodiments, administration of the unit dose of rAAV particles to one eye and / or the contralateral eye of the individual improves BCVA by about 1, about 2, about 3, about 4, about 5, about 6, or about 7 ETDRS letters compared to the BCVA before administration of the unit dose of rAAV particles. In some embodiments, administration of the unit dose of rAAV particles to one eye and / or the contralateral eye of the individual improves BCVA by about 3 ETDRS letters or more compared to the BCVA before administration of the unit dose of rAAV particles. In some embodiments, administration of the unit dose of rAAV particles to one eye and / or the contralateral eye of the individual improves BCVA by about 4 ETDRS letters or more compared to the BCVA before administration of the unit dose of rAAV particles.In some embodiments, administering the unit dose of rAAV particles to one eye and / or the contralateral eye of the individual improves BCVA by about 5.1 ETDRS letters or more compared to the BCVA before administration of the unit dose of rAAV particles. In some embodiments, administering the unit dose of rAAV particles to one eye and / or the contralateral eye of the individual improves BCVA by about 6.4 ETDRS letters or more compared to the BCVA before administration of the unit dose of rAAV particles. In some embodiments, administering the unit dose of rAAV particles to one eye and / or the contralateral eye of the individual improves BCVA by about 6.8 ETDRS letters compared to the BCVA before administration of the unit dose of rAAV particles. In some embodiments, administering the unit dose of rAAV particles to one eye and / or the contralateral eye of the individual improves BCVA by about 8.8 ETDRS letters compared to the BCVA before administration of the unit dose of rAAV particles. In some embodiments, administration of the unit dose of rAAV particles to one eye and / or the contralateral eye of the individual improves BCVA by about 2.3 ETDRS letters compared to BCVA before administration of the unit dose of rAAV particles.

[0119] In some embodiments, administration of the unit dose of rAAV particles to one eye and / or the contralateral eye of the individual maintains BCVA compared to the BCVA before administration of the unit dose of rAAV particles, and the individual experiences a decrease in the number of ETDRS letters of less than 15 letters (Vitale et al., (2016) JAMA Opthalmol 134(9):1041:1047) (e.g., 15 letters or less, 14 letters or less, 13 letters or less, 12 letters or less, 11 letters or less, 10 letters or less, 9 letters or less, 8 letters or less, 7 letters or less, 6 letters or less, 5 letters or less, 4 letters or less, 3 letters or less, 2 letters or less, 1 letter, or 0 letters). In some embodiments, administration of the unit dose of rAAV particles to one eye and / or the contralateral eye of the individual maintains BCVA compared to the BCVA before administration of the unit dose of rAAV particles, and the individual experiences a decrease in the number of ETDRS letters of about 2 letters. In some embodiments, administration of the unit dose of rAAV particles to one eye and / or the contralateral eye of the individual maintains BCVA and reduces the number of ETDRS letters in the individual by about 1 letter, about 2 letters, about 3 letters, about 4 letters, about 5 letters, about 6 letters, about 7 letters, about 8 letters, or about 9 letters compared to the BCVA before administration of the unit dose of rAAV particles. In some embodiments, administration of the unit dose of rAAV particles to one eye and / or the contralateral eye of the individual maintains BCVA and reduces the number of ETDRS letters in the individual by 0 letters compared to the BCVA before administration of the unit dose of rAAV particles. In some embodiments, administration of the unit dose of rAAV particles to one eye and / or the contralateral eye of the individual maintains BCVA and reduces the number of ETDRS letters in the individual by about 1 letter compared to the BCVA before 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 the contralateral eye of the individual maintains BCVA and reduces the individual's ETDRS letters by about 2.7 letters compared to the BCVA before administration of the unit dose of rAAV particles.In some embodiments, administering the unit dose of rAAV particles to one eye and / or the contralateral eye of the individual maintains BCVA and reduces the individual's ETDRS by about 2.8 letters compared to the BCVA before administration of the unit dose of rAAV particles. In some embodiments, administering the unit dose of rAAV particles to one eye and / or the contralateral eye of the individual maintains BCVA and reduces the individual's ETDRS by about 2 letters or less compared to the BCVA before administration of the unit dose of rAAV particles. In some embodiments, administering the unit dose of rAAV particles to one eye and / or the contralateral eye of the individual maintains BCVA and reduces the individual's ETDRS by about 3.2 letters or less compared to the BCVA before administration of the unit dose of rAAV particles. In some embodiments, administration of the unit dose of rAAV particles to one eye and / or the contralateral eye of the individual maintains BCVA compared to the BCVA before administration of the unit dose of rAAV particles, and the reduction in the number of ETDRS letters in the individual is about 15 to about 0 letters (e.g., about 15 letters, about 14 letters, about 13 letters, about 12 letters, about 11 letters, about 10 letters, about 9 letters, about 8 letters, about 7 letters, about 6 letters, about 5 letters, about 4 letters, about 3 letters, about 2 letters, about 1 letter, or 0 letters). In some embodiments, administration of the unit dose of rAAV particles to one eye and / or the contralateral eye of the individual maintains BCVA compared to the BCVA before administration of the unit dose of rAAV particles, and the individual's ETDRS letter count is reduced by about 10 to about 0 letters (e.g., about 10 letters, about 9 letters, about 8 letters, about 7 letters, about 6 letters, about 5 letters, about 4 letters, about 3 letters, about 2 letters, about 1 letter, or 0 letters). In some embodiments, administration of the unit dose of rAAV particles to one eye and / or the contralateral eye of the individual maintains BCVA compared to the BCVA before administration of the unit dose of rAAV particles, and the individual's ETDRS letter count is reduced by about 5 to about 0 letters (e.g., about 5 letters, about 4 letters, about 3 letters, about 2 letters, about 1 letter, or 0 letters).In some embodiments, administration of the unit dose of rAAV particles to one eye and / or the contralateral eye of the individual maintains BCVA compared to the BCVA before administration of the unit dose of rAAV particles, and the individual's ETDRS letter count is reduced by about 4 to about 0 letters (e.g., about 4 letters, about 3 letters, about 2 letters, about 1 letter, or 0 letters). In some embodiments, administration of the unit dose of rAAV particles to one eye and / or the contralateral eye of the individual maintains BCVA compared to the BCVA before administration of the unit dose of rAAV particles, and the individual's ETDRS letter count is reduced by about 3 to about 0 letters (e.g., about 3 letters, about 2 letters, about 1 letter, or 0 letters). In some embodiments, administration of the unit dose of rAAV particles to one eye and / or the contralateral eye of the individual maintains BCVA compared to the BCVA before administration of the unit dose of rAAV particles, and the individual's ETDRS letter count is reduced by about 2 to about 0 letters (e.g., about 2 letters, about 1 letter, or 0 letters). In some embodiments, administration of the unit dose of rAAV particles to one eye and / or the contralateral eye of the individual maintains BCVA compared to the BCVA before administration of the unit dose of rAAV particles, and the individual's ETDRS letter count is reduced by about 1 to about 0 letters.

[0120] In some embodiments, administration of the unit dose of rAAV particles to one eye and / or the contralateral eye of the individual results in a change in BCVA of about -20 to +7 or more ETDRS characters (e.g., any of the following ranges: -20, -19, -18, -17, -16, -15, -14, -13, -12, -11, -10, -9, -8, -7, -6, -5, -4, -3, -2, -1, 0, +1, +2, +3, +4, +5, +6, +7, +8, +9, +10, +11, +12, +13, +14, +15, +16, +17, +18, +19, +20, or more) compared to the BCVA before administration of the unit dose of rAAV particles. In some embodiments, administration of the unit dose of rAAV particles to one eye and / or the contralateral eye of the individual results in an increase in BCVA of about 16, 7, or 5 ETDRS letters compared to the BCVA before administration of the unit dose of rAAV particles. In some embodiments, administration of the unit dose of rAAV particles to one eye and / or the contralateral eye of the individual results in a decrease in BCVA of about 19, 14, 7, 6, 5, 4, 3, 2, or 1 ETDRS letter compared to the BCVA before administration of the unit dose of rAAV particles. In some embodiments, administration of the unit dose of rAAV particles to one eye and / or 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 before administration of the unit dose of rAAV particles. In some embodiments, administration of the unit dose of rAAV particles to one eye and / or the contralateral eye of the individual results in a decrease in BCVA of about 2 ETDRS letters or less compared to the BCVA before administration of the unit dose of rAAV particles. In some embodiments, administration of the unit dose of rAAV particles to one eye and / or the contralateral eye of the individual results in a decrease in BCVA of about 3.2 ETDRS letters or less compared to the BCVA before administration of the unit dose of rAAV particles.

[0121] In some embodiments, administration of the unit dose of rAAV particles to one eye and / or the contralateral eye of the individual results in a change in BCVA of about -15 to +7 or more (e.g., any of the following ranges: -15, -14, -13, -12, -11, -10, -9, -8, -7, -6, -5, -4, -3, -2, -1, 0, +1, +2, +3, +4, +5, +6, +7, +8, +9, +10, +11, +12, +13, +14, +15, +16, +17, +18, +19, +20, or more) in ETDRS characters compared to the BCVA before administration of the unit dose of rAAV particles. In some embodiments, administration of the unit dose of rAAV particles to one eye and / or the contralateral eye of the individual results in a change in BCVA of about -10 to +7 or more (e.g., any of the following ETDRS characters: -10, -9, -8, -7, -6, -5, -4, -3, -2, -1, 0, +1, +2, +3, +4, +5, +6, +7, +8, +9, +10, +11, +12, +13, +14, +15, +16, +17, +18, +19, +20, or more) compared to the BCVA before administration of the unit dose of rAAV particles. In some embodiments, administration of the unit dose of rAAV particles to one eye and / or the contralateral eye of the individual results in a change in BCVA of about -5 to +7 or more (e.g., any of the following ranges: -5, -4, -3, -2, -1, 0, +1, +2, +3, +4, +5, +6, +7, +8, +9, +10, +11, +12, +13, +14, +15, +16, +17, +18, +19, +20, or more) in ETDRS letters compared to the BCVA before administration of the unit dose of rAAV particles. In some embodiments, administration of the unit dose of rAAV particles to one eye and / or the contralateral eye of the individual results in a change in BCVA of about -4 to +7 or more ETDRS letters (e.g., any of the ranges 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) compared to the BCVA before administration of the unit dose of rAAV particles.In some embodiments, administration of the unit dose of rAAV particles to one eye and / or the contralateral eye of the individual results in a change in BCVA of about -3 to +7 or more (e.g., any of the following ranges: -3, -2, -1, 0, +1, +2, +3, +4, +5, +6, +7, +8, +9, +10, +11, +12, +13, +14, +15, +16, +17, +18, +19, +20, or more) in ETDRS letters compared to the BCVA before administration of the unit dose of rAAV particles. In some embodiments, administration of the unit dose of rAAV particles to one eye and / or the contralateral eye of the individual results in a change in BCVA of about -2 to +7 or more (e.g., any of the following ranges: -2, -1, 0, +1, +2, +3, +4, +5, +6, +7, +8, +9, +10, +11, +12, +13, +14, +15, +16, +17, +18, +19, +20, or more) in ETDRS letters compared to the BCVA before administration of the unit dose of rAAV particles. In some embodiments, administration of the unit dose of rAAV particles to one eye and / or the contralateral eye of the individual results in a change in BCVA of about -1 to +7 or more (e.g., any of the following ETDRS characters: -1, 0, +1, +2, +3, +4, +5, +6, +7, +8, +9, +10, +11, +12, +13, +14, +15, +16, +17, +18, +19, +20, or more) compared to the BCVA before administration of the unit dose of rAAV particles. In some embodiments, administration of the unit dose of rAAV particles to one eye and / or the contralateral eye of the individual results in a change in BCVA of about 0 to +7 or more (e.g., any of the following in a range: 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 before administration of the unit dose of rAAV particles.In some embodiments, administration of the unit dose of rAAV particles to one eye and / or the contralateral eye of the individual results in a change in BCVA of about +1 to +7 or more (e.g., +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 before administration of the unit dose of rAAV particles. In some embodiments, administration of the unit dose of rAAV particles to one eye and / or the contralateral eye of the individual results in a change in BCVA of about +2 to +7 or more (e.g., +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 before 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 the contralateral eye of the individual results in a change in BCVA of about +3 to +7 or more (e.g., +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 before administration of the unit dose of rAAV particles. In some embodiments, administration of the unit dose of rAAV particles to one eye and / or the contralateral eye of the individual results in a change in BCVA of about +4 to +7 or more (e.g., +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 before administration of the unit dose of rAAV particles.In some embodiments, administration of the unit dose of rAAV particles to one eye and / or the contralateral eye of the individual results in a change in BCVA of about +5 to +7 or more ETDRS letters (e.g., +5, +6, +7, +8, +9, +10, +11, +12, +13, +14, +15, +16, +17, +18, +19, +20, or more) compared to the BCVA before administration of the unit dose of rAAV particles. In some embodiments, administration of the unit dose of rAAV particles to one eye and / or the contralateral eye of the individual results in a change in BCVA of about +6 or about +7 ETDRS letters compared to the BCVA before administration of the unit dose of rAAV particles.

[0122] In some embodiments, administration of the unit dose of rAAV particles to one eye and / or the contralateral eye of the individual results in transient inflammation (e.g., inflammation driven by aqueous humor cells and / or vitreous cells, aqueous flare, posterior synechiae, hypodilation of the pupil). In some embodiments, administration of the unit dose of rAAV particles to one eye and / or the contralateral eye of the individual results in inflammation (e.g., inflammation driven by aqueous humor cells and / or vitreous cells, aqueous flare, posterior synechiae, hypodilation of the pupil) that improves after administration of oral and / or topical steroid therapy and / or mydriatic medication. In some embodiments, administration of the unit dose of rAAV particles to one eye and / or the contralateral eye of the individual results in inflammation (e.g., inflammation driven by aqueous humor cells and / or vitreous cells) that resolves after administration of oral and / or topical steroid therapy. Inflammation (e.g., aqueous humor and / or vitreous cell-driven inflammation, aqueous humor flare, posterior synechiae, hypodilation) can be measured using any method known in the art, such as slit lamp examination.

[0123] In some embodiments, the visual acuity (e.g., BCVA) compared to the visual acuity before administration of the unit dose of rAAV particles is maintained or improved at any time point after about 1 day, about 1 week, about 2 weeks, about 4 weeks, about 8 weeks, about 12 weeks, about 16 weeks, about 20 weeks, about 24 weeks, about 28 weeks, about 32 weeks, about 36 weeks, about 40 weeks, about 44 weeks, about 48 weeks, about 52 weeks, about 56 weeks, about 60 weeks, about 64 weeks, about 68 weeks, about 72 weeks, about 76 weeks, about 80 weeks, about 84 weeks, about 88 weeks, about 92 weeks, about 96 weeks, about 100 weeks, about 104 weeks, about 108 weeks, or more, after administration of the unit dose of rAAV particles to one eye and / or the contralateral eye of the individual. In some embodiments, the visual acuity (e.g., BCVA) compared to the visual acuity before administration of the unit dose of rAAV particles is maintained or improved at about 30 weeks or more after administration of the unit dose of rAAV particles to one eye and / or the contralateral eye of the individual. In some embodiments, the visual acuity (e.g., BCVA) compared to the visual acuity before administration of the unit dose of rAAV particles is maintained or improved at any time point about 30 weeks, about 34 weeks, about 44 weeks, about 6 months, about 1 year, about 1.5 years, about 2 years, about 3 years, about 5 years, about 10 years, or more after administration of the unit dose of rAAV particles to one eye and / or the contralateral eye of the individual.

[0124] In some embodiments, treatment of an ocular neovascular disease in an individual after administration of the unit dose of rAAV particles to one eye and / or the contralateral eye is evaluated based on best-corrected visual acuity (BCVA) in one eye and / or the contralateral eye. In some embodiments, BCVA is expressed as an ETDRS score, which corresponds to the number of letters correctly read (Vitale et al., (2016) JAMA Opthalmol 134(9):1041:1047). In some embodiments, if the individual's ETDRS score decreases by less than 15 letters (e.g., 15 letters or less, 14 letters or less, 13 letters or less, 12 letters or less, 11 letters or less, 10 letters or less, 9 letters or less, 8 letters or less, 7 letters or less, 6 letters or less, 5 letters or less, 4 letters or less, 3 letters or less, 2 letters or less, 1 letter, or 0 letters) compared to before administration of the unit dose of rAAV particles, the individual is determined to have maintained their vision and / or visual acuity. In some embodiments, an individual is determined to have improved vision and / or visual acuity if the individual's ETDRS score increases by at least 15 letters (e.g., at least about 15, at least about 20, at least about 30, at least about 40, at least about 50, at least about 60, or about 70 letters) compared to before administration of the unit dose of rAAV particles to one eye and / or the contralateral eye.

[0125] In some embodiments, treatment of an ocular neovascular disease in an individual after administration of the unit dose of rAAV particles to one eye and / or the contralateral eye is assessed based on the central retinal thickness (CST) or central retinal thickness (CRT) in one eye and / or the contralateral eye. In some embodiments, the CST or CRT is measured by SD-OCT. In some embodiments, treatment of an ocular neovascular disease in an individual after administration of the unit dose of rAAV particles to one eye and / or the contralateral eye is determined to be achieved if the CST or CRT assessed by SD-OCT decreases after administration of the unit dose of rAAV particles to one eye and / or the contralateral eye compared to before administration of the unit dose of rAAV particles to one eye and / or the contralateral eye. In some embodiments, an individual is determined to have been treated for an ocular neovascular disease after administration of the unit dose of rAAV particles to one eye and / or the contralateral eye if the CST or CRT assessed by SD-OCT is maintained after administration of the unit dose of rAAV particles to one eye and / or the contralateral eye compared to before administration of the unit dose of rAAV particles to one eye and / or the contralateral eye.

[0126] In some embodiments, treatment of an ocular neovascular disease in an individual after administration of the unit dose of rAAV particles to one eye and / or the contralateral eye is assessed based on the macular volume in the one eye and / or the contralateral eye. In some embodiments, the macular volume is measured by SD-OCT. In some embodiments, treatment of an ocular neovascular disease in an individual after administration of the unit dose of rAAV particles to one eye and / or the contralateral eye is determined to be achieved if the macular volume assessed by SD-OCT decreases after administration of the unit dose of rAAV particles to one eye and / or the contralateral eye compared to before administration of the unit dose of rAAV particles to the one eye and / or the contralateral eye. In some embodiments, if the macular volume assessed by SD-OCT is maintained after administration of the unit dose of rAAV particles to one eye and / or the contralateral eye compared to before administration of the unit dose of rAAV particles to one eye and / or the contralateral eye, it is determined that the individual has been treated for an ocular neovascular disease after administration of the unit dose of rAAV particles to one eye and / or the contralateral eye.

[0127] In some embodiments, treatment of an ocular neovascular disease in an individual after administration of the unit dose of rAAV particles to one eye and / or the contralateral eye is assessed based on retinal thickness (e.g., central retinal thickness (CRT) or central zone retinal thickness (CST)) and macular volume in one eye and / or the contralateral eye. In some embodiments, the CST and macular volume are measured by SD-OCT. In some embodiments, treatment of an ocular neovascular disease in an individual after administration of the unit dose of rAAV particles to one eye and / or the contralateral eye is determined to have been achieved if the CST and macular volume assessed by SD-OCT are reduced after administration of the unit dose of rAAV particles to one eye and / or the contralateral eye compared to before administration of the unit dose of rAAV particles to one eye and / or the contralateral eye. In some embodiments, if the CST and macular volume assessed by SD-OCT are maintained after administration of the unit dose of rAAV particles to one eye and / or the contralateral eye compared to before administration of the unit dose of rAAV particles to one eye and / or the contralateral eye, it is determined that the individual has been treated for an ocular neovascular disease after administration of the unit dose of rAAV particles to one eye and / or the contralateral eye.

[0128] In some embodiments, treatment of an ocular neovascular disease in an individual after administration of the unit dose of rAAV particles to 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 to 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 to one eye and / or the contralateral eye is determined if the individual requires less than one rescue therapy treatment (e.g., aflibercept injections) every 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, or more after administration of the unit dose of rAAV particles to one eye and / or the contralateral eye.

[0129] In some embodiments, an individual is determined to be treated for an ocular neovascular disease after administration of the unit dose of rAAV particles to one eye and / or the contralateral eye if, after administration of the unit dose of rAAV particles to one eye and / or the contralateral eye, the individual does not require treatment with any rescue therapeutic agent (e.g., an aflibercept injection) for any period 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.

[0130] In some embodiments, the individual does not require treatment with any rescue therapeutic agent (e.g., an 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 to one eye and / or the contralateral eye. In some embodiments, the individual does not require any rescue therapeutic treatment (e.g., an aflibercept injection) for at least about 12 months after administration of the unit dose of rAAV particles to one eye and / or the contralateral eye. In some embodiments, the individual does not require any rescue therapeutic treatment (e.g., an aflibercept injection) for at least about 10 months after administration of the unit dose of rAAV particles to one eye and / or the contralateral eye. In some embodiments, the individual does not require any rescue therapeutic treatment (e.g., an aflibercept injection) for at least about 7 months after administration of the unit dose of rAAV particles to one eye and / or the contralateral eye. In some embodiments, the individual does not require any rescue therapeutic treatment (e.g., an aflibercept injection) for at least about 6 months after administration of the unit dose of rAAV particles to one eye and / or the contralateral eye. In some embodiments, the individual does not require treatment with any rescue therapeutic agent (e.g., an aflibercept injection) for at least about two months after administration of the unit dose of rAAV particles to one eye and / or the contralateral eye.In some embodiments, the individual does not require treatment with any rescue therapeutic agent (e.g., an aflibercept injection) for at least about one month after administration of the unit dose of rAAV particles to one eye and / or the contralateral eye.

[0131] In some embodiments, administration of a single unit dose of rAAV particles to one eye and / or the contralateral eye of a plurality of individuals results in at least about 50% (e.g., at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, or 100%) of the plurality of individuals not requiring anti-VEGF rescue treatment (e.g., aflibercept injections). In some embodiments, administering a single unit dose of rAAV particles to one eye and / or the contralateral eye of a plurality of individuals results in at least about 67% of the individuals (e.g., at least about 67%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, or 100%) not requiring anti-VEGF rescue therapy (e.g., aflibercept injections). In some embodiments, administering a single unit dose of rAAV particles to one eye and / or the contralateral eye of a plurality of individuals results in at least about 50% of the individuals not requiring anti-VEGF rescue therapy (e.g., aflibercept injections). In some embodiments, administering a single unit dose of rAAV particles to one eye and / or the contralateral eye of a plurality of individuals results in at least about 78% of the individuals not requiring anti-VEGF rescue therapy (e.g., aflibercept injections). In some embodiments, administering a single unit dose of rAAV particles to one eye and / or the contralateral eye of a plurality of individuals results in at least about 80% of the plurality of individuals not requiring anti-VEGF rescue therapy (e.g., aflibercept injections). In some embodiments, administering a single unit dose of rAAV particles to one eye and / or the contralateral eye of a plurality of individuals results in at least about 82% of the plurality of individuals not requiring anti-VEGF rescue therapy (e.g., aflibercept injections).In some embodiments, by administering a single unit dose of rAAV particles to one eye and / or the contralateral eye of multiple individuals, 100% of the multiple individuals do not require anti-VEGF rescue therapy (e.g., aflibercept injections).

[0132] In some embodiments, administration of a single unit dose of rAAV particles to one eye and / or the contralateral eye of a plurality of individuals results in at least about 50% of the individuals (e.g., at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, or 100%) experiencing a cytotoxic effect at least about 4 weeks after administration of the rAAV particles, e.g., at least about 4 weeks, at least about 8 weeks, at least about 12 weeks, at least about 16 weeks, at least about 2 weeks, at least about 30 weeks, at least about 40 weeks, at least about 50 weeks, at least about 60 weeks, at least about 70%, at least about 80%, at least about 95%, at least about 99%, or at least about 100%). Anti-VEGF rescue therapy (e.g., aflibercept injections) is not required for any of the following periods: 0 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. In some embodiments, administration of a single unit dose of rAAV particles to one eye and / or the contralateral eye of multiple individuals results in at least about 50% of the multiple individuals not requiring anti-VEGF rescue therapy (e.g., aflibercept injections) for about 52 weeks or more, or about 56 weeks or more following administration of the rAAV particles.In some embodiments, administration of a single unit dose of rAAV particles to one eye and / or the contralateral eye of a plurality of individuals results in at least about 67% (e.g., at least about 67%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, or 100%) of the plurality of individuals not requiring anti-VEGF rescue therapy (e.g., aflibercept injections) for at least about 20 weeks after administration of the rAAV particles, e.g., any of at least about 20 weeks, at least about 24 weeks, at least about 28 weeks, at least about 32 weeks, at least about 36 weeks, at least about 40 weeks, at least about 44 weeks, at least about 48 weeks, at least about 52 weeks, at least about 56 weeks, at least about 60 weeks, at least about 64 weeks, at least about 66 weeks, or more after administration of the rAAV particles. In some embodiments, administration of a single unit dose of rAAV particles to one eye and / or the contralateral eye of a plurality of individuals results in at least about 78% of the plurality of individuals experiencing a response to rAAV for at least about 4 weeks, e.g., at least about 4 weeks, at least about 8 weeks, at least about 12 weeks, at least about 16 weeks, at least about 20 weeks, at least about 24 weeks, at least about 28 weeks, at least about 32 weeks, at least about 36 weeks, at least about 40 weeks, at least about 50 weeks, at least about 51 weeks, at least about 52 weeks, at least about 53 weeks, at least about 54 weeks, at least about 55 weeks, at least about 56 weeks, at least about 57 weeks, at least about 58 weeks, at least about 59 weeks, at least about 60 weeks, at least about 61 weeks, at least about 62 weeks, at least about 63 weeks, at least about 64 weeks, at least about 65 weeks, at least about 66 weeks, at least about 67 weeks, at least about 68 weeks, at least about 69 weeks, at least about 70 weeks, at least about 71 weeks, at least about 72 weeks, at least about 73 weeks, at least about 74 weeks, at least about 75 weeks, at least about 76 weeks, at least about 77 weeks, at least about 78 weeks, at least about 79 weeks, at least about 80 weeks, at least about 81 weeks, at least about 82 weeks, at least about 83 weeks, at least about 84 weeks, at least about 85 weeks, at least about 86 weeks, at least about 87 weeks, at least about 88 weeks, at least about 89 weeks, at least about 90 weeks, at least about 91 weeks, at least about 92 weeks, at least about 93 weeks, at least about 94 weeks, at least about 95 Anti-VEGF rescue therapy (e.g., aflibercept injections) is not required for any of the following periods: 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.In some embodiments, administration of a single unit dose of rAAV particles to one eye and / or the contralateral eye of multiple individuals results in at least about 78% of the multiple individuals not requiring anti-VEGF rescue therapy (e.g., aflibercept injections) for about 20 weeks or more, or about 36 weeks or more following administration of the rAAV particles. In some embodiments, administration of a single unit dose of rAAV particles to the one eye and / or the contralateral eye of a plurality of individuals results in at least about 80% of the plurality of individuals not requiring anti-VEGF rescue therapy (e.g., aflibercept injections) for at least about 20 weeks after administration of the rAAV particles, e.g., any of at least about 20 weeks, at least about 24 weeks, at least about 28 weeks, at least about 32 weeks, at least about 36 weeks, at least about 40 weeks, at least about 44 weeks, at least about 48 weeks, at least about 52 weeks, at least about 56 weeks, at least about 60 weeks, at least about 64 weeks, at least about 66 weeks, or more after administration of the rAAV particles. In some embodiments, administration of a single unit dose of rAAV particles to one eye and / or the contralateral eye of a plurality of individuals results in at least about 82% of the plurality of individuals not requiring anti-VEGF rescue therapy (e.g., aflibercept injections) for at least about 20 weeks after administration of the rAAV particles, e.g., any of at least about 20 weeks, at least about 24 weeks, at least about 28 weeks, at least about 32 weeks, at least about 36 weeks, at least about 40 weeks, at least about 44 weeks, at least about 48 weeks, at least about 52 weeks, at least about 56 weeks, at least about 60 weeks, at least about 64 weeks, at least about 66 weeks, or more after administration of the rAAV particles.In some embodiments, administration of a single unit dose of rAAV particles to one eye and / or the contralateral eye of a plurality of individuals results in 100% of the plurality of individuals not requiring anti-VEGF rescue therapy (e.g., aflibercept injections) for at least about 20 weeks after administration of the rAAV particles, e.g., any of at least about 20 weeks, at least about 24 weeks, at least about 28 weeks, at least about 32 weeks, at least about 36 weeks, at least about 40 weeks, at least about 44 weeks, at least about 48 weeks, at least about 52 weeks, at least about 56 weeks, at least about 60 weeks, at least about 64 weeks, at least about 66 weeks, or more after administration of the rAAV particles. In some embodiments, administration of a single unit dose of rAAV particles to one eye and / or the contralateral eye of a plurality of individuals results in 100% of the plurality of individuals experiencing a response to the rAAV particles for at least about 4 weeks, e.g., at least about 4 weeks, at least about 8 weeks, at least about 12 weeks, at least about 16 weeks, at least about 20 weeks, at least about 24 weeks, at least about 28 weeks, at least about 32 weeks, at least about 36 weeks, at least about 40 weeks, at least about 44 weeks, at least about 48 weeks, at least about 50 weeks, at least about 52 weeks, at least about 54 weeks, at least about 56 weeks, at least about 58 weeks, at least about 59 weeks, at least about 60 weeks, at least about 61 weeks, at least about 62 weeks, at least about 63 weeks, at least about 64 weeks, at least about 65 weeks, at least about 66 weeks, at least about 67 weeks, at least about 68 weeks, at least about 69 weeks, at least about 70 weeks, at least about 71 weeks, at least about 72 weeks, at least about 73 weeks, at least about 74 weeks, at least about 75 weeks, at least about 76 weeks, at least about 77 weeks, at least about 78 weeks, at least about 79 weeks, at least about 80 weeks, at least about 81 weeks, at least about 82 weeks, at least about 83 weeks, at least about 84 weeks, at least about 85 weeks, at least about 86 weeks, at least about 87 weeks, at least about 88 weeks, at least about 89 weeks, at least about 90 weeks, at least about 91 weeks, at least about 92 weeks, at least about 93 weeks, at least about 94 weeks, at least about 95 weeks, at least about 96 weeks, at least about 97 weeks, at least about 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. In some embodiments, by administering a single unit dose of rAAV particles to one eye and / or the contralateral eye of a plurality of individuals, 100% of the plurality of individuals do not require anti-VEGF rescue therapy (e.g., aflibercept injections) for at least about 64 weeks or more, 72 weeks or more, or 84 weeks or more following administration of the rAAV particles.

[0133] In some embodiments, administration of a unit dose of rAAV particles to one eye and / or the contralateral eye of a plurality of individuals results in the proportion of the plurality of individuals requiring rescue therapy (e.g., an aflibercept injection) in one eye and / or the contralateral eye of 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). In some embodiments, administration of a unit dose of rAAV particles to one eye and / or the contralateral eye of a plurality of individuals results in the proportion of the plurality of individuals requiring rescue therapy (e.g., an aflibercept injection) in one eye and / or the contralateral eye of about 50% or less (e.g., 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). In some embodiments, administration of a unit dose of rAAV particles to one eye and / or the contralateral eye of a plurality of individuals results in less than about 30% of the individuals requiring rescue treatment (e.g., aflibercept injections) in the one eye and / or the contralateral eye (e.g., less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 10%, less than about 5%, less than about 2.5%, less than about 1%, or less than about 0.5%). In some embodiments, administration of a unit dose of rAAV particles to one eye and / or the contralateral eye of a plurality of individuals results in less than about 30% of the individuals requiring rescue treatment (e.g., aflibercept injections) in the one eye and / or the contralateral eye (e.g., aflibercept injections). In some embodiments, administering a unit dose of rAAV particles to one eye and / or the contralateral eye of a plurality of individuals results in less than about 20% of the individuals requiring rescue therapy (e.g., an aflibercept injection) in the one eye and / or the contralateral eye.In some embodiments, administering a unit dose of rAAV particles to one eye and / or the contralateral eye of a plurality of individuals results in 0% of the individuals requiring any rescue treatment (e.g., aflibercept injection) in the one eye and / or the contralateral eye.

[0134] In some embodiments, administration of a unit dose of rAAV particles to one eye and / or the contralateral eye of a plurality of individuals provides a therapeutic effect for at least about 4 weeks after administration of the rAAV particles, e.g., at least about 4 weeks, at least about 8 weeks, at least about 12 weeks, at least about 16 weeks, at least about 20 weeks, at least about 24 weeks, at least about 28 weeks, at least about 32 weeks, at least about 36 weeks, at least about 40 weeks, at least about 44 weeks, at least about 48 weeks, at least about 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, or at least about 80 weeks after administration of the rAAV particles. The percentage of the plurality of individuals requiring any rescue treatment (e.g., aflibercept injections) for any period of 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 is 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).In some embodiments, administration of a unit dose of rAAV particles to one eye and / or the contralateral eye of a plurality of individuals results in less than about 30% (e.g., less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 10%, less than about 5%, less than about 2.5%, less than about 1%, or less than about 0.5%) of the plurality of individuals requiring rescue therapy (e.g., aflibercept injections) for at least about 20 weeks after administration of the rAAV particles, e.g., at least about 20 weeks, at least about 24 weeks, at least about 28 weeks, at least about 32 weeks, at least about 36 weeks, at least about 40 weeks, at least about 44 weeks, at least about 48 weeks, at least about 52 weeks, at least about 56 weeks, at least about 60 weeks, at least about 64 weeks, at least about 66 weeks, or more) after administration of the rAAV particles. In some embodiments, administration of a unit dose of rAAV particles to the upper eye and / or the contralateral eye of a plurality of individuals results in less than about 30% of the individuals requiring any rescue therapy (e.g., an aflibercept injection) in the upper eye and / or the contralateral eye for at least about 20 weeks after administration of the rAAV particles, e.g., at least about 20 weeks, at least about 24 weeks, at least about 28 weeks, at least about 32 weeks, at least about 36 weeks, at least about 40 weeks, at least about 44 weeks, at least about 48 weeks, at least about 52 weeks, at least about 56 weeks, at least about 60 weeks, at least about 64 weeks, at least about 66 weeks, or more, after administration of the rAAV particles.In some embodiments, administration of a unit dose of rAAV particles to the upper eye and / or the contralateral eye of a plurality of individuals results in less than about 20% of the plurality of individuals requiring any rescue therapy (e.g., an aflibercept injection) in the upper eye and / or the contralateral eye for at least about 20 weeks after administration of the rAAV particles, e.g., at least about 20 weeks, at least about 24 weeks, at least about 28 weeks, at least about 32 weeks, at least about 36 weeks, at least about 40 weeks, at least about 44 weeks, at least about 48 weeks, at least about 52 weeks, at least about 56 weeks, at least about 60 weeks, at least about 64 weeks, at least about 66 weeks, or more, after administration of the rAAV particles. In some embodiments, administration of a unit dose of rAAV particles to the upper eye and / or the contralateral eye of a plurality of individuals results in 0% of the plurality of individuals requiring any rescue therapy (e.g., an aflibercept injection) in the upper eye and / or the contralateral eye for at least about 20 weeks after administration of the rAAV particles, e.g., at least about 20 weeks, at least about 24 weeks, at least about 28 weeks, at least about 32 weeks, at least about 36 weeks, at least about 40 weeks, at least about 44 weeks, at least about 48 weeks, at least about 52 weeks, at least about 56 weeks, at least about 60 weeks, at least about 64 weeks, at least about 66 weeks, or more, after administration of the rAAV particles.In some embodiments, administration of a unit dose of rAAV particles to one eye and / or the contralateral eye of a plurality of individuals provides a therapeutic effect for at least about 4 weeks after administration of the rAAV particles, e.g., at least about 4 weeks, at least about 8 weeks, at least about 12 weeks, at least about 16 weeks, at least about 20 weeks, at least about 24 weeks, at least about 28 weeks, at least about 32 weeks, at least about 36 weeks, at least about 40 weeks, at least about 44 weeks, at least about 48 weeks, at least about 52 weeks, at least about 60 weeks, at least about 70 weeks, at least about 80 weeks, at least about 90 weeks, at least about 100 weeks, at least about 110 weeks, at least about 120 weeks, at least about 130 weeks, at least about 140 weeks, at least about 150 weeks, at least about 160 weeks, at least about 200 weeks, at least about 240 weeks, at least about 280 weeks, at least about 320 weeks, at least about 360 weeks, at least about 40 ...60 weeks, at least about 180 weeks, at least about 250 weeks, at least about 260 weeks, at least about 270 weeks, at least about 300 weeks, at least about 310 weeks, at least about 320 weeks, at least about 330 weeks, at least about 340 weeks, at least about 350 weeks, at least about 360 weeks, at least about 370 weeks, at least about 380 weeks, at least about 4 The percentage of the plurality of individuals requiring any rescue treatment (e.g., aflibercept injection) in one eye and / or the contralateral eye for any of 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 is 0%.

[0135] In some embodiments, administering the unit dose of rAAV particles to one eye and / or the contralateral eye of a plurality of individuals results in a reduction in the average annual anti-VEGF injection rate by at least about 80%, at least about 85%, at least about 87%, at least about 90%, at least about 95%, at least about 99%, or 100%, compared to the average annual anti-VEGF injection rate before administration of the unit dose of rAAV particles. In some embodiments, administering the unit dose of rAAV particles to one eye and / or the contralateral eye of a plurality of individuals results in a reduction in the average annual anti-VEGF injection rate by about 87% or more, compared to the average annual anti-VEGF injection rate before administration of the unit dose of rAAV particles. In some embodiments, administering the unit dose of rAAV particles to one eye and / or the contralateral eye of a plurality of individuals results in a reduction in the average annual anti-VEGF injection rate by 100%, compared to the average annual anti-VEGF injection rate before administration of the unit dose of rAAV particles.

[0136] In some embodiments, the average annual anti-VEGF injection rate prior to administration of the unit dose of rAAV particles is calculated according to the following formula: The annual rate before administration of the unit dose of rAAV particles is calculated as follows: (number of anti-VEGF injections in the 12 months before administration of the unit dose of rAAV particles) / (number of days from the first anti-VEGF injection to administration of the unit dose of rAAV particles in the 12 months before administration of the unit dose of rAAV particles / 365.25).

[0137] In some embodiments, the average annual anti-VEGF injection rate after administration of the unit dose of rAAV particles is calculated according to the following formula: The annual rate after administration of the unit dose of rAAV particles is calculated as follows: (number of anti-VEGF injections after administration of the unit dose of rAAV particles) / (number of days after administration of the unit dose of rAAV particles / 365.25).

[0138] In some embodiments, an individual is determined to require rescue treatment (e.g., an intravitreal injection of an anti-VEGF such as an aflibercept injection) after administration of rAAV particles if the individual exhibits a 10-letter or greater decrease in BCVA (e.g., using an ETDRS protocol) for the eye and / or contralateral eye receiving rAAV particles compared to the BCVA for the eye and / or contralateral eye receiving rAAV particles attributable to intraretinal or subretinal fluid (e.g., as measured by SD-OCT) prior to administration of the rAAV particles. In some embodiments, if the individual shows an increase of more than 75 μm in central retinal thickness (e.g., CST or CRT) in the eye and / or contralateral eye receiving rAAV particles compared to the central retinal thickness of the eye and / or contralateral eye receiving rAAV particles before administration of the rAAV particles, as measured, for example, by SD-OCT, the individual is determined to need rescue therapy (e.g., intravitreal anti-VEGF injection such as an aflibercept injection) after administration of the rAAV particles. In some embodiments, if the individual shows sight-threatening hemorrhage due to AMD in the eye and / or contralateral eye receiving rAAV particles, the individual is determined to need rescue therapy (e.g., intravitreal anti-VEGF injection such as an aflibercept injection) after administration of the rAAV particles.

[0139] In some embodiments, the rescue treatment comprises administering a standard anti-VEGF treatment. Such standard anti-VEGF treatment comprises one or more anti-VEGF treatments (e.g., intravitreal anti-VEGF injections). In some embodiments, the rescue treatment comprises one or more IVT injections of aflibercept. In some embodiments, the rescue treatment comprises one or more IVT injections of aflibercept comprising about 2 mg of aflibercept.

[0140] In some embodiments, treatment of an ocular neovascular disease in an individual after administration of a unit dose of rAAV particles to one eye and / or the contralateral eye is evaluated based on the level of retinal fluid compared to the level before administration of the unit dose of rAAV particles to one eye and / or the contralateral eye. In some embodiments, if a decrease in retinal fluid is observed after administration of a unit dose of rAAV particles to one eye and / or the contralateral eye compared to the level before administration of the unit dose of rAAV particles to one eye and / or the contralateral eye, it is determined that the individual is treated for ocular neovascular disease after administration of a unit dose of rAAV particles to one eye and / or the contralateral eye. In some embodiments, the ocular neovascular disease is wAMD.

[0141] In some embodiments, treatment of an ocular neovascular disease in an individual after administration of the unit dose of rAAV particles to one eye and / or the contralateral eye is assessed based on resolution of pigment epithelial detachment (PED) compared to the PED before administration of the unit dose of rAAV particles to 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 to one eye and / or the contralateral eye is determined to be achieved if resolution of PED is observed after administration of the unit dose of rAAV particles to one eye and / or the contralateral eye compared to the PED before administration of the unit dose of rAAV particles to one eye and / or the contralateral eye. In some embodiments, the ocular neovascular disease is wAMD.

[0142] In some embodiments, treatment of an ocular neovascular disease in an individual after administration of the unit dose of rAAV particles to one eye and / or the contralateral eye is assessed based on the growth of choroidal neovascularization (CNV) lesions as determined by fluorescein angiography. In some embodiments, treatment of an ocular neovascular disease in an individual after administration of the unit dose of rAAV to one eye and / or the contralateral eye is determined to be achieved if the CNV lesions shrink (e.g., by more than about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, or 100%) after administration of the unit dose of rAAV to one eye and / or the contralateral eye compared to the CNV lesions present before administration of the unit dose of rAAV to the one eye and / or the contralateral eye. In some embodiments, an individual is determined to have been treated for an ocular neovascular disease after administration of a unit dose of rAAV to one eye and / or the other eye if the CNV lesions do not grow (e.g., grow by less than about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 15%, or about 20%) after administration of a unit dose of rAAV to one eye and / or the other eye compared to the CNV lesions present before administration of the unit dose of rAAV to one eye and / or the other eye. In some embodiments, the ocular neovascular disease is AMD.

[0143] In some embodiments, treatment of an ocular neovascular disease in an individual after administration of the unit dose of rAAV particles to one eye and / or the contralateral eye is assessed based on the anatomical features of the one eye and / or the contralateral eye using any method known in the art (e.g., SD-OCT, OCT, fluorescein angiography, digital color fundus photography, etc.). In some embodiments, if an improvement in the anatomical features of the one eye and / or the contralateral eye is observed after administration of the unit dose of rAAV particles to one eye and / or the contralateral eye, it is determined that the individual has been treated for an ocular neovascular disease after administration of the unit dose of rAAV to one eye and / or the contralateral eye. In some embodiments, the ocular neovascular disease is wAMD.

[0144] In some embodiments, treatment of an ocular neovascular disease in an individual after administration of the unit dose of rAAV particles to one eye and / or the contralateral eye is evaluated by ophthalmologic examination, intraocular pressure (e.g., using a Goldman tonometer or a Tono-pen), indirect ophthalmoscopy, examination of one eye and / or the contralateral eye and ocular adnexa, eyelid and / or pupillary responsiveness, ptosis, abnormal pupil shape, anisocoria, abnormal response to light, afferent Evaluation is based on pupillary defects, slit lamp examination (including examination of the eyelid, conjunctiva, cornea, lens, iris, and anterior chamber), abnormalities of the vitreous body, optic nerve, peripheral retina, and posterior segment of the retinal vasculature, SD-OCT, fluorescein angiography, digital color fundus photography (including images of the retina, optic nerve head, 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 zone retinal 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.

[0145] The unit dose of rAAV can be administered to one eye and / or the contralateral eye of an individual by any method known in the art. For example, the unit dose of rAAV may be administered to one eye and / or the contralateral eye of an individual by intraocular infusion or intravitreal injection. In some embodiments, the unit dose of rAAV is administered to one eye and / or the contralateral eye of an individual by intraocular administration. In some embodiments, the unit dose of rAAV particles is administered to one eye and / or the contralateral eye of an individual by intravitreal injection (IVT) or subretinal injection. In some embodiments, the unit dose of rAAV particles is administered to one eye and / or the contralateral eye of an individual by IVT injection. In some embodiments, the unit dose of rAAV is administered by intravitreal injection using aseptic techniques. In some embodiments, the unit dose of rAAV is administered by intravitreal injection using aseptic techniques using povidone-iodine.

[0146] In some embodiments, the individual has not received prior treatment for an ocular neovascular disease. In some embodiments, the individual has not received prior treatment for an ocular neovascular disease in one eye and / or the contralateral eye. In some embodiments, the individual has not received prior treatment with an anti-VEGF agent (e.g., bevacizumab, brolucizumab, ranibizumab, faricimab, abicipar pegol, conbercept, OPT-302, KSI-301, injectable sunitinib maleate (GB-102), PAN-90806 (PanOptica), and / or aflibercept). In some embodiments, the individual has not received prior treatment with an anti-VEGF agent (e.g., bevacizumab, brolucizumab, ranibizumab, faricimab, abicipar pegol, conbercept, OPT-302, KSI-301, injectable sunitinib maleate (GB-102), PAN-90806 (PanOptica), and / or aflibercept) in one eye and / or the other eye. In some embodiments, the individual has not received prior aflibercept treatment. In some embodiments, the individual has not received prior aflibercept treatment in one eye and / or the other eye.

[0147] steroid treatment In some embodiments, the unit dose of rAAV particles is administered in combination with steroid therapy. In some embodiments, the steroid therapy is corticosteroid therapy. Exemplary corticosteroids include aclometasone, amcinonide, beclomethasone, betamethasone, budesonide, ciclesonide, clobetasol, clobetasone, clocortolone, cloprednol, cortivazol, deflazacort, deoxycorticosterone, desonide, desoximetasone, dexamethasone, diflorasone, diflucortolone, difluprednate, fluchloron, fludrocortisone, fludroxycortide, flumethasone, flunisolide, fluocinolone acetonide, fluocinonide, fluocortin, fluocortolone, and 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 urobetasol. In some embodiments, the steroid treatment is systemic steroid treatment. In some embodiments, the steroid treatment is oral steroid treatment. In some embodiments, the steroid treatment is ophthalmic steroid treatment. In some embodiments, the ophthalmic steroid treatment is topical steroid treatment (e.g., eye drops), periocular steroid treatment (e.g., subtenon, subconjunctival), intravitreal steroid treatment, or suprachoroidal steroid treatment. In some embodiments, the topical steroid treatment is difluprednate treatment, medrysone treatment, loteprednol treatment, prednisolone treatment, fluocinolone treatment, triamcinolone treatment, rimexolone treatment, dexamethasone treatment, fluorometholone treatment, fluocinolone treatment, rimexolone treatment, or prednisone treatment.In some embodiments, the ophthalmic steroid treatment is difluprednate treatment. In some embodiments, the steroid treatment is prednisone treatment. In some embodiments, the steroid treatment is difluprednate treatment.

[0148] In some embodiments, the steroid treatment includes systemic steroid treatment and topical steroid treatment. In some embodiments, the systemic steroid treatment is oral steroid treatment. In some embodiments, the systemic steroid treatment is prednisone treatment. In some embodiments, the topical steroid treatment is difluprednate treatment. In some embodiments, the systemic steroid treatment and topical steroid treatment are administered simultaneously (e.g., on the same day). In some embodiments, the systemic steroid treatment and topical steroid treatment are administered separately (e.g., on different days).

[0149] In some embodiments, the steroid is administered before, during, and / or after administration of a unit dose of rAAV particles. In some embodiments, the steroid is administered before, during, and after administration of a unit dose of rAAV particles. In some embodiments, the steroid is administered during and after administration of a unit dose of rAAV particles. In some embodiments, the steroid is administered before administration of the unit dose of rAAV particles. In some embodiments, the steroid is administered during administration of a unit dose of rAAV particles. In some embodiments, the steroid is administered before and during administration of a unit dose of rAAV particles. In some embodiments, the steroid is administered after administration of a unit dose of rAAV particles. In some embodiments, the steroid is administered during and after administration of a unit dose of rAAV particles. In some embodiments, the steroid is administered before and / or after administration of a unit dose of rAAV particles. In some embodiments, the steroid is administered before and after administration of a unit dose of rAAV particles.

[0150] In some embodiments, the steroid treatment is systemic steroid treatment. In some embodiments, the systemic steroid treatment is oral steroid treatment.

[0151] In some embodiments, the steroid treatment is oral prednisone treatment. In some embodiments, the oral prednisone treatment is initiated before administration of the unit dose of rAAV particles. In some embodiments, the initial oral prednisone treatment is administered at a dose of about 40 mg, about 45 mg, about 50 mg, about 55 mg, about 60 mg, about 65 mg, or about 70 mg of prednisone per day about 7 days, about 6 days, about 5 days, about 4 days, about 3 days, about 2 days, about 1 day, or 0 days before administration of the unit dose of rAAV particles, and is continued for about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, or about 10 days or more. In some embodiments, the initial oral prednisone treatment is administered at a dose of about 60 mg of prednisone per day about 3 days before administration of the unit dose of rAAV and continues for about 3 days.

[0152] In some embodiments, the initial oral prednisone therapy is followed by a tapered dose of oral prednisone therapy. In some embodiments, the tapered dose of oral prednisone therapy is administered at a dose of about 20 mg, about 25 mg, about 30 mg, about 35 mg, about 40 mg, about 45 mg, or about 50 mg of prednisone per day for a total of about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, or about 7 days, followed by a dose of about 10 mg, about 15 mg, about 20 mg, or about 25 mg of prednisone per day for about 1 day, about 2 days, about 3 days, or about 4 days, followed by a dose of about 5 mg, about 10 mg, or about 15 mg of prednisone per day for about 1 day, about 2 days, about 3 days, or about 4 days. In some embodiments, the prednisone dose taper is administered at a dose of about 40 mg prednisone per day for 3 days, followed by a dose of about 20 mg prednisone per day for 2 days, followed by a dose of about 10 mg prednisone per day for 2 days.

[0153] In some embodiments, the initial oral prednisone treatment is initiated 3 days prior to administration of the unit dose of rAAV particles at a dose of 60 mg prednisone per day for a total of 6 days, followed by 40 mg prednisone per day for a total of 3 days, then 20 mg prednisone per day for 2 days, then 10 mg prednisone per day for 2 days.

[0154] In some embodiments, the steroid treatment is ophthalmic steroid treatment. In some embodiments, the ophthalmic steroid treatment is difluprednate treatment. In some embodiments, the steroid treatment is administered before, during, and / or after administration of 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.

[0155] In some embodiments, the steroid treatment is ophthalmic steroid treatment, e.g., topical steroid treatment. In some embodiments, the ophthalmic steroid treatment, e.g., topical steroid treatment, is daily steroid treatment for up to 4 weeks, up to 6 weeks, up to 8 weeks, up to 3 months, up to 4 months, up to 5 months, or up to 6 months after administration of the unit dose of rAAV particles. In some embodiments, the topical steroid treatment comprises about four administrations of topical steroid at about week 1, about three administrations of topical steroid at about week 2, about two administrations of topical steroid at about week 3, and about one administration of topical steroid at about week 4, starting at the time of administration of the unit dose of rAAV particles and continuing thereafter. In some embodiments, the topical steroid treatment comprises about four administrations of topical steroid per day (i.e., QID) for about three weeks following administration of the unit dose of rAAV particles, about three administrations of topical steroid per day (i.e., TID) for about one week thereafter, about two administrations of topical steroid per day (i.e., BID) for about one week thereafter, and about one administration of topical steroid per day (i.e., QD) for about one week thereafter. In some embodiments, the ophthalmic steroid treatment is extended at the discretion of the treating physician.

[0156] In some embodiments, the ophthalmic steroid is about 0.005% to about 0.5% difluprednate. In some embodiments, the ophthalmic steroid is about 0.005%, about 0.006%, about 0.007%, about 0.008%, about 0.009%, about 0.01%, about 0.02%, about 0.03%, about 0.4%, about 0.05%, about 0.06%, about 0.07%, about 0.08%, about 0.09%, or about 0.1% difluprednate. In some embodiments, the ophthalmic steroid is 0.05% difluprednate. In some embodiments, a dose of 0.05% difluprednate is one drop of ophthalmic solution. In some embodiments, one drop is about 50 μl (e.g., about 25 μl to about 50 μl, or about 50 μl to about 100 μl). In some embodiments, the dose of difluprednate comprises about 1 μg to about 5 μg, or about 2 μg to about 3 μg, or about 2.5 μg of difluprednate. In some embodiments, the dose of difluprednate comprises about 2.5 μg of difluprednate.

[0157] In some embodiments, the topical steroid treatment comprises 7 weeks of topical steroid treatment, e.g., 0.05% difluprednate. In some embodiments, the topical steroid treatment comprises about 4 times per day (i.e., QID) for about 4 weeks, followed by about 3 times per day (i.e., TID) for about 1 week, followed by about 2 times per day (i.e., BID) for about 1 week, and then about 1 time per day (i.e., QD) for about 1 week, starting about 1 week before administration of the unit dose of rAAV particles. In some embodiments, the topical steroid treatment comprises administering topical steroid about four times per day for about 28 days (i.e., QID), followed by administering topical steroid about three times per day for about 7 days (i.e., TID), followed by administering topical steroid about twice per day for about 7 days (i.e., BID), and then administering topical steroid about once per day for about 7 days (i.e., QD), starting about 7 days before administration of the unit dose of rAAV particles. In some embodiments, the topical steroid treatment comprises administration of topical steroid about four times per day (i.e., QID) from day 1 to about day 28, then about three times per day (i.e., TID) from about day 29 to about day 35, then about two times per day (i.e., BID) from about day 36 to about day 42, and then about one time per day (i.e., QD) from about day 43 to about day 49, starting on day 1. In some embodiments, the topical steroid treatment is continued if inflammation is present.

[0158] In some embodiments, the methods of treatment provided herein include administering an anti-VEGF agent (e.g., an IVT injection of aflibercept) to one eye of the individual prior to administering the unit dose of rAAV particles to the other eye of the individual. In some embodiments, the anti-VEGF agent is administered about 7 days or about 1 week before administering the unit dose of rAAV particles. In some embodiments, the anti-VEGF agent is administered on about day 1 and the unit dose of rAAV particles is administered on about day 8. In some embodiments, the topical steroid treatment includes 7 weeks of topical steroid treatment, for example, 0.05% difluprednate. In some embodiments, the topical steroid treatment comprises administering the topical steroid about four times per day (i.e., QID) for about four weeks, followed by about three times per day (i.e., TID) for about one week, followed by about two times per day (i.e., BID) for about one week, and about once per day (i.e., QD) for about one week, starting at the time of administration of the anti-VEGF agent. In some embodiments, the topical steroid treatment comprises administering the topical steroid about four times per day (i.e., QID) for about 28 days, followed by about three times per day (i.e., TID) for about seven days, followed by about two times per day (i.e., BID) for about seven days, and about once per day (i.e., QD) for about seven days, starting at the time of administration of the anti-VEGF agent. In some embodiments, the topical steroid treatment comprises administering topical steroid about four times per day (i.e., QID) from day 1 to about day 28, then administering topical steroid about three times per day (i.e., TID) from about day 29 to about day 35, then administering topical steroid about two times per day (i.e., BID) from about day 36 to about day 42, and then administering topical steroid about once per day (i.e., QD) from about day 43 to about day 49, starting on day 1. In some embodiments, topical steroid treatment is continued if inflammation is present.

[0159] In some embodiments, the topical steroid treatment comprises 4 months of topical steroid treatment, e.g., 0.05% difluprednate. In some embodiments, the topical steroid treatment comprises about 4 times per day (i.e., QID) for about 1 month, followed by about 3 times per day (i.e., TID) for about 1 month, followed by about 2 times per day (i.e., BID) for about 1 month, and then about 1 time per day (i.e., QD) for about 1 month, starting about 1 week before administration of the unit dose of rAAV particles. In some embodiments, the topical steroid treatment comprises administering topical steroid about four times per day for about 30 days (i.e., QID), followed by administering topical steroid about three times per day for about 30 days (i.e., TID), followed by administering topical steroid about twice per day for about 30 days (i.e., BID), and then administering topical steroid about once per day for about 30 days (i.e., QD), starting about 7 days before administration of the unit dose of rAAV particles. In some embodiments, the topical steroid treatment comprises administration of topical steroid about four times per day (i.e., QID) from day 1 to about day 30, followed by administration of topical steroid about three times per day (i.e., TID) from about day 31 to about day 60, followed by administration of topical steroid about two times per day (i.e., BID) from about day 61 to about day 90, and then administration of topical steroid about once per day (i.e., QD) from about day 91 to about day 120, starting on day 1. In some embodiments, topical steroid treatment is continued if inflammation is present.

[0160] In some embodiments, the methods of treatment provided herein include administering an anti-VEGF agent (e.g., an IVT injection of aflibercept) to one eye of the individual prior to administering the unit dose of rAAV particles to the other eye of the individual. In some embodiments, the anti-VEGF agent is administered about 7 days or about 1 week before administering the unit dose of rAAV particles. In some embodiments, the anti-VEGF agent is administered on about day 1, and the unit dose of rAAV particles is administered on about day 8. In some embodiments, the topical steroid treatment comprises 4 months of topical steroid treatment, for example, 0.05% difluprednate. In some embodiments, the topical steroid treatment comprises about four administrations of topical steroid per day for about one month (i.e., QID), followed by about three administrations of topical steroid per day for about one month (i.e., TID), followed by about two administrations of topical steroid per day for about one month (i.e., BID), and about one administration of topical steroid per day for about one month (i.e., QD), timing initiated at the time of administration of the anti-VEGF agent and continuing thereafter. In some embodiments, the topical steroid treatment comprises about four administrations of topical steroid per day for about 30 days (i.e., QID), followed by about three administrations of topical steroid per day for about 30 days (i.e., TID), followed by about two administrations of topical steroid per day for about 30 days (i.e., BID), and about one administration of topical steroid per day for about 30 days (i.e., QD), timing initiated at the time of administration of the anti-VEGF agent and continuing thereafter. In some embodiments, the topical steroid treatment comprises administering topical steroid about four times per day (i.e., QID) from day 1 to about day 30, then administering topical steroid about three times per day (i.e., TID) from about day 31 to about day 60, then administering topical steroid about two times per day (i.e., BID) from about day 61 to about day 90, and then administering topical steroid about once per day (i.e., QD) from about day 91 to about day 120, starting on day 1.In some embodiments, if inflammation is present, treatment with topical steroids is continued.

[0161] Vectors for delivering transgenes to target cells In some embodiments, the recombinant adeno-associated virus (rAAV) particle comprises a recombinant viral vector derived from an adeno-associated virus (AAV) that has been modified to be replication-deficient in a subject (e.g., a human or non-human primate). In some embodiments, the adeno-associated virus (AAV) is a recombinant AAV (rAAV).

[0162] AAV or rAAV is a small, non-enveloped, single-stranded DNA virus. rAAV is a non-pathogenic human parvovirus that can be engineered to depend on helper viruses, including adenovirus, herpes simplex virus, vaccinia virus, and CMV, for replication.

[0163] Because exposure to wild-type (wt) AAV is not associated with or known to cause any human pathology and is common in the general population, AAV or rAAV is a suitable delivery system for gene therapy. AAV and rAAV used for gene therapy to deliver anti-VEGF agents, such as aflibercept, can be of any serotype. In some embodiments, the methods of the present disclosure can use any suitable AAV serotype, including AAV1, AAV2, AAV2.5, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, rh10, AAV-DJ, and any hybrid or chimeric AAV thereof. In some embodiments, the serotype used is based on the viral tropism or infectivity of the intended target cells. In some embodiments, several AAV vectors can be generated to allow for the selection of the optimal serotype for use with an anti-VEGF agent transgene (e.g., an aflibercept transgene).

[0164] In some embodiments, the methods of the present disclosure provide for the use of pseudotyped AAV. Pseudotyped AAV particles contain the AAV genomic inverted terminal repeats (ITRs) of one AAV serotype encapsidated by an AAV capsid of another AAV serotype. Generally, pseudotyped AAVs are designated "AAV# / #," where the first "#" indicates the AAV ITR serotype and the second "#" indicates the capsid serotype. For example, an AAV particle containing AAV2 ITRs and an AAV1 capsid is designated "AAV2 / 1."

[0165] In some embodiments, the rAAV particle comprises a nucleic acid, e.g., a heterologous nucleic acid. In some embodiments, the nucleic acid encodes a transgene, e.g., an anti-VEGF agent (e.g., aflibercept). In some embodiments, the encoded transgene, e.g., an anti-VEGF agent, is under the transcriptional control of a promoter that initiates transcription of the nucleic acid. In some embodiments, the promoter is a "ubiquitous" promoter. In some embodiments, the promoter is a "strong" or constitutively active promoter, e.g., a cytomegalovirus (CMV) promoter, an elongation factor 1 alpha (EFla) promoter, a glyceraldehyde 3-phosphate dehydrogenase (GAPDH) promoter, or a connexin 36 (or "Cx36") promoter. In some embodiments, the promoter is a tissue-specific promoter that is activated in specific tissues or cells, e.g., retinal cells, to reduce potential toxicity or undesirable effects on non-targeted cells. In some aspects, several AAV vectors may be generated to allow for selection of the optimal serotype and promoter for use with the anti-VEGF agent transgene (e.g., an aflibercept transgene). In some embodiments, the nucleic acid is flanked by AAV inverted terminal repeats (ITRs). In some embodiments, the nucleic acid is flanked by AAV2 ITRs.

[0166] In some embodiments, the AAV vector comprises a polynucleotide cassette for enhancing expression of a transgene (e.g., an anti-VEGF agent such as aflibercept) in target cells (e.g., retinal cells). In some embodiments, the polynucleotide cassette comprises, in 5' to 3' order, (a) a first enhancer region (SEQ ID NO: 22) comprising a CMV sequence; (b) a promoter region (SEQ ID NO: 23) comprising a CMV sequence; (c) a 5' UTR region comprising, in 5' to 3' order, TPL and eMLP sequences (SEQ ID NO: 24 and SEQ ID NO: 25, respectively); (d) a coding sequence encoding a peptide or polypeptide (e.g., an anti-VEGF agent such as aflibercept); (e) a second enhancer region comprising the full-length EES sequence (SEQ ID NO: 26); and (f) an HGH polyadenylation site (SEQ ID NO: 27). In certain of these embodiments, the polynucleotide cassette comprises one or more sequences selected from SEQ ID NOs: 28-32, or sequences having at least 85% identity thereto. In certain of these embodiments, the 5' arm of the polynucleotide cassette comprises or consists of SEQ ID NO: 33, or a sequence having at least 85% identity thereto. In certain of these embodiments, the 3' arm of the polynucleotide cassette comprises or consists of SEQ ID NO: 34, or a sequence having at least 85% identity thereto. The nucleic acid sequences of SEQ ID NOs: 22-34 are shown below. ACTTACGGTA AATGGCCCGC CTGGCTGACC GCCCAACGAC CCCCGCCCAT TGACGTCAAT AATGACGTAT GTTCCCATAG TAACGCCAAT AGGGACTTTC CATTGACGTC AATGGGTGGA GTATTTACGG TAAACTGCCC ACTTGGCAGT ACATCAAGTG TATCATATGC CAAGTCCGCC CCCTATTGAC GTCAATGACG GTAAATGGCC CGCCTGGCAT TATGCCCAGT ACATGACCTT ACGGGACTTT CCTACTTGGC AGTACATCTA CGTATTAGTC ATCGCTATTA CCA (SEQ ID NO: 22) TGCTGATGCG GTTTTGGCAG TACACCAATG GGCGTGGATA GCGGTTTGAC TCACGGGGAT TTCCAAGTCT CCACCCCATT GACGTCAATG GGAGTTTGTT TTGGCACCAA AATCAACGGG ACTTTCCAAA ATGTCGTAAT AACCCCGCCC CGTTGACGCA AATGGGCGGT AGGCGTGTAC GGTGGGAGGT CTATATAAGC AGAGCTCGTT TAGTGAACCG (SEQ ID NO: 23) CTCACTCTCT TCCGCATCGC TGTCTGCGAG GGCCAGCTGT TGGGCTCGCG GTTGAGGACA AACTCTTCGC GGTCTTTCCA GTACTCTTGG ATCGGAAACC CGTCGGCCTC CGAACGGTAC TCCGCCACCG AGGGACCTGA GCGAGTCCGC ATCGACCGGA TCGGAAAACC TCTCGAGAAA GGCGTCTAAC CAGTCACAGT CGCAAGGTAG GCTGAGCACC GTGGCGGGCG GCAGCGGGTG GCGGTCGGGG TTGTTTCTGG CGGAGGTGCT GCTGATGATG TAATTAAAGT AGGCGGTCTT GAGACGGCGG ATGGTCGA (SEQ ID NO: 24) CCAGCTGTTG GGGTGAGTAC TCCCTCTCAA AAGCGGGCAT TACTTCTGCG CTAAGATTGT CAGTTTCCAA AAACGAGGAG GATTTGATAT TCACCTGGCC CG (SEQ ID NO: 25) CTGTTCTCAT CACATCATAT CAAGGTTATA TACCATCAAT ATTGCCACAG ATGTTACTTA GCCTTTTAAT ATTTCTCTAA TTTAGTGTAT ATGCAATGAT AGTTCTCTGA TTTCTGAGAT TGAGTTTCTC ATGTGTAATG ATTATTTAGA GTTTCTCTTT CATCTGTTCA AATTTTTGTC TAGTTTTATT TTTTACTGAT TTGTAAGACT TCTTTTTATA ATCTGCATAT TACAATTCTC TTTACTGGGG TGTTGCAAAT ATTTTCTGTC ATTCTATGGC CTGACTTTTC TTAATGGTTT TTTAATTTTA AAAATAAGTC TTAATATTCA TGCAATCTAA TTAACAATCT TTTCTTTGTG GTTAGGACTT TGAGTCATAA GAAATTTTTC TCTACACTGA AGTCATGATG GCATGCTTCT ATATTATTTT CTAAAAGATT TAAAGTTTTG CCTTCTCCAT TTAGACTTAT AATTCACTGG AATTTTTTTG TGTGTATGGT ATGACATATG GGTTCCCTTT TATTTTTTAC ATATAAATAT ATTTCCCTGT TTTTCTAAAA AAGAAAAAGA TCATCATTTT CCCATTGTAA AATGCCATAT TTTTTTCATA GGTCACTTAC ATATATCAAT GGGTCTGTTT CTGAGCTCTA CTCTATTTTA TCAGCCTCAC TGTCTATCCC CACACATCTC ATGCTTTGCT CTAAATCTTG ATATTTAGTG GAACATTCTT TCCCATTTTG TTCTACAAGA ATATTTTTGT TATTGTCTTT GGGCTTTCTA TATACATTTT GAAATGAGGT TGACAAGTTA (SEQ ID NO: 26) CTGCCCGGGT GGCATCCCTG TGACCCCTCC CCAGTGCCTC TCCTGGCCCT GGAAGTTGCC ACTCCAGTGC CCACCAGCCT TGTCCTAATA AAATTAAGTT GCATCATTTT GTCTGACTAG GTGTCCTTCT ATAATATTAT GGGGTGGAGG GGGGTGGTAT GGAGCAAGGG GCCCAAGTTG GGAAGAAACC TGTAGGGCCT GC (SEQ ID NO: 27) AGGCGGTCTT GAGACGGCGG ATGGTCGAGG TGAGGTGTGG CAGGCTTGAG ATCCAGCTGT TGGGGTGA (SEQ ID NO: 28) CGCTGTTTTG ACCTCCATAG TGGACACCGG GACCGATCCA GCCTCCGCGT CTCAGGGGAG ATCTCGTTTA GTGAACCGTC AGATCCTCAC TCTCTTCCGC ATCGCTGTCT GCGAGGGCCA GCTGTTGGG (SEQ ID NO: 29) TTGATATTCA CCTGGCCCGA TCTGGCCATA CACTTG (SEQ ID NO: 30) CCCAGGTCCA AGTTTAAACG CC (SEQ ID NO: 31) TCTTTGGGCT TTCTATATAC ATTTTGAAAT GAGGTTGACA AGTTACCTAG GAAAACTGTC TTCCTGCCCG GGTGGCA (SEQ ID NO: 32) CTCTGGAGAC GACTTACGGT AAATGGCCCG CCTGGCTGAC CGCCCAACGA CCCCCGCCCA TTGACGTCAA TAATGACGTA TGTTCCCATA GTAACGCCAA TAGGGACTTT CCATTGACGT CAATGGGTGG AGTATTTACG GTAAACTGCC CACTTGGCAG TACATCAAGT GTATCATATG CCAAGTCCGC CCCCTATTGA CGTCAATGAC GGTAAATGGC CCGCCTGGCA TTATGCCCAG TACATGACCT TACGGGACTT TCCTACTTGG CAGTACATCT ACGTATTAGT CATCGCTATT ACCATGCTGA TGCGGTTTTG GCAGTACACC AATGGGCGTG GATAGCGGTT TGACTCACGG GGATTTCCAA GTCTCCACCC CATTGACGTC AATGGGAGTT TGTTTTGGCA CCAAAATCAA CGGGACTTTC CAAAATGTCG TAATAACCCC GCCCCGTTGA CGCAAATGGG CGGTAGGCGT GTACGGTGGG AGGTCTATAT AAGCAGAGCT CGTTTAGTGA ACCGTCAGAT CGCCTGGAGA GGCCATCCAC GCTGTTTTGA CCTCCATAGT GGACACCGGG ACCGATCCAG CCTCCGCGTC TCAGGGGAGA TCTCGTTTAG TGAACCGTCA GATCCTCACT CTCTTCCGCA TCGCTGTCTG CGAGGGCCAG CTGTTGGGCT CGCGGTTGAG GACAAACTCT TCGCGGTCTT TCCAGTACTC TTGGATCGGA AACCCGTCGG CCTCCGAACG GTACTCCGCC ACCGAGGGAC CTGAGCGAGT CCGCATCGAC CGGATCGGAA AACCTCTCGA GAAAGGCGTC TAACCAGTCA CAGTCGCAAG GTAGGCTGAG CACCGTGGCG GGCGGCAGCG GGTGGCGGTC GGGGTTGTTT CTGGCGGAGG TGCTGCTGATGATGTAATTA AAGTAGGCGG TCTTGAGACG GCGGATGGTC GAGGTGAGGT GTGGCAGGCT TGAGATCCAG CTGTTGGGGT GAGTACTCCC TCTCAAAAGC GGGCATTACT TCTGCGCTAA GATTGTCAGT TTCCAAAAAC GAGGAGGATT TGATATTCAC CTGGCCCGAT CTGGCCATAC ACTTGAGTGA CAATGACATC CACTTTGCCT TTCTCTCCAC AGGTGTCCAC TCCCAGGTCC AAGTTTAAAC GCCGCCACCA TG (SEQ ID NO: 33) ACTGTTCTCA TCACATCATA TCAAGGTTAT ATACCATCAA TATTGCCACA GATGTTACTT AGCCTTTTAA TATTTCTCTA ATTTAGTGTA TATGCAATGA TAGTTCTCTG ATTTCTGAGA TTGAGTTTCT CATGTGTAAT GATTATTTAG AGTTTCTCTT TCATCTGTTC AAATTTTTTGT CTAGTTTTAT TTTTTACTGA TTTGTAAGAC TTCTTTTTAT AATCTGCATA TTACAATTCT CTTTACTGGG GTGTTGCAAA TATTTTCTGT CATTCTATGG CCTGACTTTT CTTAATGGTT TTTTAATTTT AAAAATAAGT CTTAATATTC ATGCAATCTA ATTAACAATC TTTTCTTTGT GGTTAGGACT TTGAGTCATA AGAAATTTTT CTCTACACTG AAGTCATGAT GGCATGCTTC TATATTATTT TCTAAAAGAT TTAAAGTTTT GCCTTCTCCA TTTAGACTTA TAATTCACTG GAATTTTTTT GTGTGTATGG TATGACATAT GGGTTCCCTT TTATTTTTTA CATATAAATA TATTTCCCTG TTTTTCTAAA AAAGAAAAAG ATCATCATTT TCCCATTGTA AAATGCCATA TTTTTTTCAT AGGTCACTTA CATATATCAA TGGGTCTGTT TCTGAGCTCT ACTCTATTTT ATCAGCCTCA CTGTCTATCC CCACACATCT CATGCTTTGC TCTAAATCTT GATATTTAGT GGAACATTCT TTCCCATTTT GTTCTACAAG AATATTTTTG TTATTGTCTT TGGGCTTTCT ATATACATTT TGAAATGAGG TTGACAAGTT ACCTAGGAAA ACTGTCTTCC TGCCCGGGTG GCATCCCTGT GACCCCTCCC CAGTGCCTCT CCTGGCCCTG GAAGTTGCCA CTCCAGTGCC CACCAGCCTTGTCCTAATAA AATTAAGTTG CATCATTTTG TCTGACTAGG TGTCCTTCTA TAATATTATG GGGTGGAGGG GGGTGGTATG GAGCAAGGGG CCCAAGTTGG GAAGAAACCT GTAGGGCCTG CGAAGACAGT CAG (SEQ ID NO: 34)

[0167] In some embodiments, the polynucleotide cassette comprises or consists of SEQ ID NO: 39 or a sequence having at least 85% identity thereto.

[0168] SEQ ID NO:39 comprises, from 5' to 3', an AAV serotype 2 inverted terminal repeat (ITR) comprising nucleotides 1-145 of SEQ ID NO:39; a CMV promoter comprising nucleotides 180-693 of SEQ ID NO:39; a 5' untranslated region (UTR) comprising an adenoviral tripartite leader sequence and a synthetic intron, comprising nucleotides 694-1314 of SEQ ID NO:39; a Kozak sequence comprising nucleotides 1329-1340 of SEQ ID NO:39; a codon-optimized aflibercept cDNA sequence comprising nucleotides 1338-2714 of SEQ ID NO:39; a 3' UTR comprising a human scaffold attachment region, comprising nucleotides 2717-3527 of SEQ ID NO:39; a human growth hormone polyadenylation / transcription termination signal comprising nucleotides 3546-3748 of SEQ ID NO:39; and an AAV serotype 2 inverted terminal repeat (ITR) comprising nucleotides 3772-3916 of SEQ ID NO:39. See, for example, Figure 1C.

[0169] Additional polynucleotide cassettes for enhancing expression of a transgene (e.g., a transgene encoding an anti-VEGF agent such as aflibercept) in target cells (e.g., retinal cells) are disclosed in WO2018 / 170473, the contents of which relating to polynucleotide cassettes for enhancing expression of a transgene in target cells are incorporated herein by reference.

[0170] In some embodiments, the rAAV particles comprise a variant capsid protein with increased infectivity of target cells, such as retinal cells, and are used to increase transduction of retinal cells or to increase targeting of gene delivery to retinal cells in an individual. In some embodiments, the rAAV particles comprise an amino acid modification in the capsid protein GH loop / loop IV of the AAV capsid protein. In some embodiments, the modification site is a solvent-accessible portion of the GH loop / loop IV of the AAV capsid protein. For a description of the GH loop / loop IV of the AAV capsid, see, for example, van Vliet et al. (2006) Mol. Ther. 14:809; Padron et al. (2005) J. Virol. 79:5047; and Shen et al. (2007) Mol. Ther. 15:1955. Several AAV capsid variants are known, including the 7m8 variant. In some embodiments, the rAAV particles comprise a variant AAV capsid protein that comprises an insertion of a sequence of 5 to 11 amino acids, e.g., 7 amino acids, in the GH loop of the capsid protein compared to the corresponding parent AAV capsid protein, and the variant capsid protein confers increased infectivity of retinal cells compared to the infectivity of retinal cells by AAV particles comprising the corresponding parent or unmodified AAV capsid protein.In some embodiments, any one of the following amino acid sequences may be inserted into the GH loop of the capsid protein: LALGETTRPA (SEQ ID NO: 1); LANETITRPA (SEQ ID NO: 2), LAKAGQANNA (SEQ ID NO: 3), LAKDPKTTNA (SEQ ID NO: 4), KDTDTTR (SEQ ID NO: 5), RAGGSVG (SEQ ID NO: 6), AVDTTKF (SEQ ID NO: 7), STGKVPN (SEQ ID NO: 8), LAKDTDTTRA (SEQ ID NO: 9), LARAGGSVGA (SEQ ID NO: 10), LAAVDTTKFA (SEQ ID NO: 11), and LASTGKVPNA (SEQ ID NO: 12), LGETTRP (SEQ ID NO: 14), NETITRP (SEQ ID NO: 15), KAGQANN (SEQ ID NO: 16), KDPKTTN (SEQ ID NO: 17), KDTDTTR (SEQ ID NO: 18), RAGGSVG (SEQ ID NO: 19), AVDTTKF (SEQ ID NO: 20), and STGKVPN (SEQ ID NO: 21). In some embodiments, any one of the amino acid sequences set forth in SEQ ID NOS: 1-12 and 14-21 is inserted into the solvent-exposed GH loop of the VP1 capsid protein in rAAV. Further details regarding amino acid sequences that can be inserted into the GH loop of a capsid protein to facilitate transduction of a nucleic acid of interest into retinal cells, for example after IVT injection, are described in WO2012145601, US9587282, US10202657, and US10214785, the contents of which relating to amino acid sequences that can be inserted into the GH loop of a capsid protein are incorporated herein by reference.

[0171] In some embodiments, the rAAV particles have one of the following amino acid sequences inserted between positions 587 and 588 of the AAV2 capsid protein; between amino acids 590 and 591 of the AAV1 capsid protein; between amino acids 575 and 576 of the AAV5 capsid protein; between amino acids 590 and 591 of the AAV6 capsid protein; between amino acids 589 and 590 of the AAV7 capsid protein; between amino acids 590 and 591 of the AAV8 capsid protein; between amino acids 588 and 589 of the AAV9 capsid protein; or between amino acids 589 and 590 of the AAV10 capsid protein: LALGETTRPA (SEQ ID NO: 1); LANETITRPA (SEQ ID NO: 2), LAKAGQANNA (SEQ ID NO: 3), or LALGETTRPA (SEQ ID NO: 4) inserted between positions 587 and 588 of the AAV2 capsid protein; between amino acids 590 and 591 of the AAV1 capsid protein; between amino acids 575 and 576 of the AAV5 capsid protein; between amino acids 590 and 591 of the AAV6 capsid protein; between amino acids 589 and 590 of the AAV7 capsid protein; 3), LAKDPKTTNA (SEQ ID NO: 4), KDTDTTR (SEQ ID NO: 5), RAGGSVG (SEQ ID NO: 6), AVDTTKF (SEQ ID NO: 7), STGKVPN (SEQ ID NO: 8), LAKDTDTTRA (SEQ ID NO: 9), LARAGGSVGA (SEQ ID NO: 10), LAAVDTTKFA (SEQ ID NO: 11), and LASTGKVPNA (SEQ ID NO: 12), LGETTRP (SEQ ID NO: 14), NETITRP (SEQ ID NO: 15), KAGQANN (SEQ ID NO: 16), KDPKTTN (SEQ ID NO: 17), KDTDTTR (SEQ ID NO: 18), RAGGSVG (SEQ ID NO: 19), AVDTTKF (SEQ ID NO: 20), and STGKVPN (SEQ ID NO: 21). In some embodiments, the rAAV particles comprise an AAV2 capsid protein comprising the amino acid sequence LALGETTRPA (SEQ ID NO: 1) inserted between positions 587 and 588 of the capsid protein, where the numbering of the amino acid residues corresponds to that of the AAV2 VP1 capsid protein. In some embodiments, the rAAV particles comprise an AAV2 capsid protein comprising the amino acid sequence LALGETTRPA (SEQ ID NO: 1) inserted between positions 587 and 588 of AAV2 VP1, which comprises the sequence of SEQ ID NO: 13.

[0172] In some embodiments, the rAAV particles comprise a 7m8 variant capsid protein derived from AAV2, which comprises the amino acid sequence LALGETTRA (SEQ ID NO: 1) inserted in the GH loop of the AAV2 VP1 protein between positions 587 and 588 of AAV2 VP1. In some embodiments, the rAAV particles comprise an AAV2 VP1 capsid protein comprising a GH loop comprising the amino acid sequence of SEQ ID NO: 38, or an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 38. In some embodiments, the rAAV particles comprise an AAV2 VP1 capsid protein comprising a GH loop comprising an amino acid sequence having any of at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 38. FSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTNTPSGTTTQSRLQFSQAGASDI RDQSRNWLPGPCYRQQRVSKTSADNNNSEYSWTGATKYHLNGRDSLVNPGPAMASHKDDE EKFFPQSGVLIFGKQGSEKTNVDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQRGNLAL GETTRPARQAATADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLK HPPPQILIKN (SEQ ID NO: 38)

[0173] In some embodiments, the rAAV particles comprise a 7m8 variant capsid protein from AAV2 that includes the amino acid sequence LALGETTRPA (SEQ ID NO: 1) inserted between positions 587 and 588 of AAV2 VP1. The sequence of the 7m8 variant capsid protein from AAV2 that includes the amino acid sequence LALGETTRPA (SEQ ID NO: 1) inserted between positions 587 and 588 of AAV2 VP1 is shown below: MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPGYKYLGPFNGLDKGEPVNEADAAALEHDKAYDRQLDSGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRVLEPLGLVEEPVKTAPGKKRPVEHSPVEPDSSSGTGKAGQQPARKRLNFGQTGDADSVPDPQ PLGQPPAAPSGLGTNTMATGSGAPMADNNEGADGVGNSSGNWHCDSTWMGDRVITTSTRTWALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMV PQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTNTPSGTTTQSRLQFSQAGASDIRDQSRNWLPGPCYRQQRVSKTSADNNNSEYSWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVLIFGKQGSEKTNVDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQRGNLALGETTRPARQAATADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPSTTFSAAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTRYLTRNL (SEQ ID NO: 37)

[0174] In some embodiments, the rAAV particles comprise capsid protein VP1, wherein the capsid protein comprises the amino acid sequence LGETTRP (SEQ ID NO: 14) inserted between positions 587 and 588 of the capsid protein, where the numbering of the amino acid residues corresponds to that of the AAV2 VP1 capsid protein. In some embodiments, the rAAV particles comprise capsid protein VP2, wherein the capsid protein comprises the amino acid sequence LGETTRP (SEQ ID NO: 14) inserted between positions 587 and 588 of the capsid protein, where the numbering of the amino acid residues corresponds to that of the AAV2 VP1 capsid protein. In some embodiments, the rAAV particles comprise capsid protein VP3, the capsid protein comprising the amino acid sequence LGETTRP (SEQ ID NO: 14) inserted between positions 587 and 588 of the capsid protein, where the numbering of the amino acid residues corresponds to that of the AAV2 VP1 capsid protein. In some embodiments, the rAAV particles comprise capsid proteins VP1, VP2, and VP3, each of VP1, VP2, and VP3 comprising the amino acid sequence LGETTRP (SEQ ID NO: 14) inserted between positions 587 and 588 of the capsid protein, where the numbering of the amino acid residues corresponds to that of the AAV2 VP1 capsid protein.

[0175] In some embodiments, the rAAV particles comprise capsid protein VP1, the capsid protein VP1 comprising the amino acid sequence LALGETTRPA (SEQ ID NO: 1) inserted between positions 587 and 588 of the capsid protein, where the numbering of the amino acid residues corresponds to that of the AAV2 VP1 capsid protein. In some embodiments, the rAAV particles comprise capsid protein VP2, the capsid protein VP2 comprising the amino acid sequence LALGETTRPA (SEQ ID NO: 1) inserted between positions 587 and 588 of the capsid protein, where the numbering of the amino acid residues corresponds to that of the AAV2 VP1 capsid protein. In some embodiments, the rAAV particles comprise capsid protein VP3, the capsid protein comprising the amino acid sequence LALGETTRPA (SEQ ID NO: 1) inserted between positions 587 and 588 of the capsid protein, where the numbering of the amino acid residues corresponds to that of the AAV2 VP1 capsid protein. In some embodiments, the rAAV particles comprise capsid proteins VP1, VP2, and VP3, each of VP1, VP2, and VP3 comprising the amino acid sequence LALGETTRPA (SEQ ID NO: 1) inserted between positions 587 and 588 of the capsid protein, where the numbering of the amino acid residues corresponds to that of the AAV2 VP1 capsid protein.

[0176] In some embodiments, the recombinant viruses and / or plasmids used to generate the rAAV viruses contain other transcriptional or regulatory elements, such as a polyA (polyadenylation) sequence, an untranslated region (UTR), a 3'UTR, or a termination sequence. In some embodiments, two or more genes are expressed from the vector or plasmid using an internal ribosome entry site (IRES) or similar element that allows for the simultaneous expression of two or more proteins or creates a multigene or polycistronic mRNA.

[0177] In some embodiments, the rAAV and / or the plasmid used to generate the rAAV comprises one or more of the following nucleic acid elements: a first ITR sequence; a promoter sequence; an intron sequence; a first UTR sequence; a heterologous nucleic acid encoding an anti-VEGF agent (e.g., aflibercept); a second UTR sequence; a polyA sequence; and a second ITR sequence. In some embodiments, a linker sequence(s) is inserted between two or more of the nucleic acid elements. In some embodiments, the heterologous nucleic acid encoding a therapeutic polypeptide encodes, for example, aflibercept (or a functional fragment or functional variant thereof).

[0178] In some embodiments, the vector is a targeted vector, particularly a targeted rAAV (e.g., AAV2.7m8) that exhibits higher infectivity of specific cells, such as retinal cells (e.g., photoreceptors, retinal ganglion cells, Müller cells, bipolar cells, amacrine cells, horizontal cells, or retinal pigment epithelial cells). Viral vectors for use in the present disclosure may also include vectors that exhibit low toxicity and / or low immunogenicity in individuals and express a therapeutically effective amount of the anti-VEGF agent (e.g., aflibercept) in individuals, e.g., humans. Any suitable method known in the art can be used for biochemical purification of recombinant viruses (e.g., rAAV) for preparing, for example, pharmaceutical compositions described elsewhere herein. Recombinant AAV viruses can be harvested directly from cells or from culture media containing cells. Viruses can be purified using various biochemical means, such as gel filtration, filtration, chromatography, affinity purification, density gradient ultracentrifugation, or size exclusion. In some embodiments, the virus is lyophilized.

[0179] In some embodiments, the rAAV particles comprise a 7m8 variant capsid protein, e.g., rAAV2.7m8, and a nucleic acid sequence encoding an anti-VEGF agent (e.g., aflibercept, or a functional fragment or variant thereof). In some embodiments, the rAAV particles (e.g., 7m8 variants) exhibit 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% increased infectivity of retinal cells compared to AAV particles comprising the corresponding parent or unmodified AAV capsid protein. In some embodiments, the increase in infectivity of the retinal cells is any of 5% to 100%, 5% to 95%, 5% to 90%, 5% to 85%, 5% to 80%, 5% to 75%, 5% to 70%, 5% to 65%, 5% to 60%, 5% to 55%, 5% to 50%, 5% to 45%, 5% to 40%, 5% to 35%, 5% to 30%, 5% to 25%, 5% to 20%, 5% to 15%, or 5% to 10% compared to AAV particles containing the corresponding parent or unmodified AAV capsid proteins.

[0180] In some embodiments, the increase in infectivity of the rAAV variant, e.g., rAAV2.7m8, for retinal cells is at least 1-fold, at least 1.1-fold, at least 1.2-fold, at least 1.3-fold, at least 1.4-fold, at least 1.5-fold, at least 1.6-fold, at least 1.7-fold, at least 1.8-fold, at least 1.9-fold, or at least 2-fold compared to AAV particles comprising the corresponding parental or unmodified AAV capsid proteins. In some embodiments, the increase in infectivity is at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, or at least 10-fold compared to AAV particles comprising the corresponding parental AAV capsid proteins. In some embodiments, the increase in infectivity is at least 15-fold, at least 20-fold, at least 25-fold, at least 30-fold, at least 35-fold, at least 40-fold, at least 45-fold, at least 50-fold, at least 55-fold, at least 60-fold, at least 65-fold, at least 70-fold, at least 75-fold, at least 80-fold, at least 85-fold, at least 90-fold, or at least 100-fold compared to AAV particles comprising the corresponding parent or unmodified AAV capsid proteins.

[0181] In some embodiments, the increase in infectivity of the rAAV variant, e.g., rAAV2.7m8, for retinal cells is 10-fold to 100-fold, 10-fold to 95-fold, 10-fold to 90-fold, 10-fold to 85-fold, 10-fold to 80-fold, 10-fold to 75-fold, 10-fold to 70-fold, 10-fold to 65-fold, 10-fold to 60-fold, 10-fold to 55-fold, 10-fold to 50-fold, 10-fold to 45-fold, 10-fold to 40-fold, 10-fold to 35-fold, 10-fold to 30-fold, 10-fold to 25-fold, 10-fold to 20-fold, or 10-fold to 15-fold compared to AAV particles comprising the corresponding parent or unmodified AAV capsid proteins.

[0182] In some embodiments, the increase in infectivity of retinal cells is 2x to 20x, 2x to 19x, 2x to 18x, 2x to 17x, 2x to 16x, 2x to 15x, 2x to 14x, 2x to 13x, 2x to 12x, 2x to 11x, 2x to 10x, 2x to 9x, 2x to 8x, 2x to 7x, 2x to 6x, 2x to 5x, 2x to 4x, or 2x to 3x compared to AAV particles containing the corresponding parent or unmodified AAV capsid proteins.

[0183] In some embodiments, the amino acid modifications of the capsid protein described herein can increase the ability of rAAV particles to penetrate the inner limiting membrane (ILM) of an individual's, e.g., a human's, eye, compared to the ability of AAV particles containing the corresponding parent or unmodified AAV capsid protein to penetrate the ILM of the subject's eye. In some embodiments, the increase in the ability of an rAAV variant, e.g., rAAV2.7m8, to penetrate the ILM is at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 100%, compared to AAV particles containing the corresponding parent or unmodified AAV capsid protein. In some embodiments, the increase in ability to cross the ILM is between 5% and 100%, between 5% and 95%, between 5% and 90%, between 5% and 85%, between 5% and 80%, between 5% and 75%, between 5% and 70%, between 5% and 65%, between 5% and 60%, between 5% and 55%, between 5% and 50%, between 5% and 45%, between 5% and 40%, between 5% and 35%, between 5% and 30%, between 5% and 25%, between 5% and 20%, between 5% and 15%, or between 5% and 10% compared to the parent or unmodified AAV capsid protein.

[0184] In some embodiments, the increase in the ability of an rAAV variant, such as rAAV2.7m8, to cross the ILM is at least 1-fold, at least 1.1-fold, at least 1.2-fold, at least 1.3-fold, at least 1.4-fold, at least 1.5-fold, at least 1.6-fold, at least 1.7-fold, at least 1.8-fold, at least 1.9-fold, or at least 2-fold compared to AAV particles comprising the corresponding parental AAV capsid proteins. In some embodiments, the increase in the ability to cross the ILM is at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, or at least 10-fold compared to AAV particles comprising the corresponding parental AAV capsid proteins. In some embodiments, the increase in ability to cross the ILM is at least 15-fold, at least 20-fol...

Claims

1. A drug for reducing variations in central retinal thickness (CST) or central retinal thickness (CRT) in a human individual with an ocular neovascular disease, the drug comprising a unit dose of recombinant adeno-associated virus (rAAV) particles, the rAAV particles being administered to one eye of the individual, the rAAV particles comprising: (a) a nucleic acid encoding a polypeptide comprising an amino acid sequence having at least about 95% identity to the amino acid sequence of SEQ ID NO: 35, and flanked by AAV2 inverted terminal repeats (ITRs); and (b) an AAV2 capsid protein comprising the amino acid sequence LGETTRP (SEQ ID NO: 14) inserted between positions 587 and 588 of said capsid protein, wherein the numbering of the amino acid residues corresponds to that of the AAV2 VP1 capsid protein. and optionally, an anti-VEGF agent is further administered to the one eye of the individual prior to administering the rAAV particles.

2. the individual has received at least one treatment with an anti-VEGF agent in the approximately 1-12 weeks prior to administering the unit dose of rAAV particles; and / or the level of CST or CRT variation in the eye of the individual is refractory to previous treatment with an anti-VEGF agent; Optionally, the anti-VEGF agent comprises aflibercept, e.g., administered by intravitreal injection at a dose of about 2 mg.

3. The method of claim 1, wherein the level of variation in the CST or CRT after administration of rAAV particles to the one eye is reduced by about 40% to about 90% compared to the level of variation in the CST or CRT in the one eye of the individual before administering the rAAV to the individual, and optionally the reduction is about 45% to about 85% or about 50 to about 90%.

4. The method of any one of claims 1 to 3, wherein the unit dose of rAAV particles is administered to the one eye of the individual after administration of the anti-VEGF agent to the one eye, the unit dose of rAAV particles is administered to the one eye of the individual about 1 to 12 weeks after administration of the anti-VEGF agent, or the anti-VEGF agent is administered to the one eye of the individual on day 1 and the unit dose of rAAV particles is administered to the one eye of the individual on day 8, and optionally the anti-VEGF agent comprises aflibercept, and is administered, for example, by intravitreal injection, at a dose of about 2 mg.

5. The unit dose of the rAAV particles is (a) Approximately 1 x 10 per eye １０ ~Approx. 6×10 １１ vector genome (vg / eye); (b) Approximately 6 × 10 １０ ~ Approximately 6 x 10 １１ vg / eye; (c) Approximately 6 × 10 １０ ~ Approximately 2 × 10 １１ vg / eye; (d) Approximately 2 × 10 １１ ~ Approximately 6 x 10 １１ vg / eye; (e) Approximately 2 x 10 １１ Or about 6 x 10 １１ vg / eye; (f) Approximately 2 × 10 １１ vg / eye; (g) Approximately 6 x 10 １１ vg / eye; or (h) Approximately 6 × 10 １０ vg / eye The drug according to any one of claims 1 to 3,

6. (a) the individual has one or more symptoms of an ocular neovascular disease in the contralateral eye; and / or (b) the unit dose of rAAV particles is administered to the contralateral eye of the individual, and optionally (i) the unit dose of rAAV particles is administered to the contralateral eye up to about two weeks after administration of the unit dose of rAAV particles to the one eye, e.g., the unit dose of rAAV particles is administered to the contralateral eye on the same day as administration of the unit dose of rAAV particles to the one eye, or the unit dose of rAAV particles is administered to the contralateral eye about 1 day to about 14 days after administration of the unit dose of rAAV particles to the one eye; and / or (ii) the unit dose of the rAAV particles administered to the contralateral eye of the individual contains the same or fewer vector genomes per eye (vg / eye) as the unit dose of the rAAV particles administered to the one eye of the individual; or (iii) the unit dose of rAAV particles is administered to the contralateral eye at least about two weeks after administration of the unit dose of rAAV particles to the one eye, and optionally, the unit dose of rAAV particles administered to the contralateral eye of the individual comprises more vector genomes per eye (vg / eye) than the unit dose of rAAV particles administered to the one eye of the individual. The drug according to any one of claims 1 to 3.

7. (a) the nucleic acid comprises the nucleic acid sequence of SEQ ID NO: 40 or a sequence having at least 85% identity thereto; and / or (b) the polypeptide comprises the amino acid sequence of SEQ ID NO: 35 or SEQ ID NO: 41, and / or (c) the polypeptide is aflibercept; The drug according to any one of claims 1 to 3.

8. (a) the nucleic acid further comprises a first enhancer region, a promoter region, a 5'UTR region, a second enhancer region, and a polyadenylation site, optionally 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, the promoter region comprises a CMV sequence comprising the sequence of SEQ ID NO: 23 or a sequence having at least 85% identity thereto, and the 5'UTR region comprises, in 5' to 3' order, the sequence of SEQ ID NO: 24 or a TPL sequence comprising 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, wherein the second enhancer region comprises a full-length EES sequence comprising the sequence of SEQ ID NO: 26 or a sequence having at least 85% identity thereto, and the polyadenylation site comprises an HGH polyadenylation site comprising the sequence of SEQ ID NO: 27 or a sequence having at least 85% identity thereto; and / or (b) the nucleic acid is, in 5' to 3' order: (i) a first enhancer region, optionally comprising a CMV sequence comprising the sequence of SEQ ID NO: 22 or a sequence having at least 85% identity thereto; (ii) a promoter region, optionally comprising a CMV sequence comprising the sequence of SEQ ID NO: 23 or a sequence having at least 85% identity thereto; (iii) a 5'UTR region optionally 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; (iv) a nucleic acid encoding a polypeptide comprising an amino acid sequence having at least about 95% identity to the amino acid sequence of SEQ ID NO: 35, optionally wherein the nucleic acid encoding the polypeptide comprises the nucleic acid sequence of SEQ ID NO: 40, or a sequence having at least 85% identity thereto, e.g., the polypeptide comprises the amino acid sequence of SEQ ID NO: 35, the amino acid sequence of SEQ ID NO: 41, or a sequence having at least 95% identity thereto, or is aflibercept; (v) a second enhancer region, optionally comprising a full-length EES sequence comprising the sequence of SEQ ID NO: 26 or a sequence having at least 85% identity thereto; and (vi) a polyadenylation site, optionally comprising an HGH polyadenylation site comprising the sequence of SEQ ID NO: 27 or a sequence having at least 85% identity thereto. and flanked by AAV2 inverted terminal repeats (ITRs); and / or (c) the nucleic acid comprises the sequence of SEQ ID NO: 39 or a sequence having at least 85% identity thereto; The drug according to any one of claims 1 to 3.

9. (a) the AAV2 capsid protein comprises the amino acid sequence LALGETTRPA (SEQ ID NO: 1) inserted between positions 587 and 588 of the capsid protein, the numbering of the amino acid residues corresponding to that of the AAV2 VP1 capsid protein; and / or (b) 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; and / or (c) 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; The drug according to any one of claims 1 to 3.

10. The agent of any one of claims 1 to 3, wherein the rAAV particle comprises an AAV2 VP1 capsid protein comprising a GH loop comprising the amino acid sequence of SEQ ID NO:38 or an amino acid sequence having at least 90% sequence identity to SEQ ID NO:

38.

11. the unit dose of rAAV particles (a) administered intravitreally to said eye and / or said contralateral eye; and / or (b) in a pharmaceutical formulation, optionally comprising the rAAV particles, sodium chloride, sodium phosphate, and a surfactant, e.g., the pharmaceutical formulation (i) about 150 to about 200 mM sodium chloride, about 1 to about 10 mM sodium dihydrogen phosphate, about 1 to about 10 mM sodium dihydrogen phosphate, about 0.0005% (w / v) to about 0.005% (w / v) poloxamer 188, and about 6 x 10 per mL １３ ~Approx. 6×10 １０ vector genome (vg) (vg / mL) of rAAV particles, wherein the pharmaceutical formulation has a pH of about 7.0 to about 7.5; (ii) about 180 mM sodium chloride, about 5 mM sodium dihydrogen phosphate, about 5 mM sodium dihydrogen phosphate, about 6 x 10 １２ vg / mL rAAV particles, and about 0.001% (w / v) poloxamer 188, wherein the pharmaceutical formulation has a pH of about 7.3; (iii) about 180 mM sodium chloride, about 5 mM sodium dihydrogen phosphate, about 5 mM sodium dihydrogen phosphate, about 2 x 10 １２ vg / mL rAAV particles and about 0.001% (w / v) poloxamer 188, wherein the pharmaceutical formulation has a pH of about 7.3; or (iv) about 180 mM sodium chloride, about 5 mM sodium dihydrogen phosphate, about 5 mM sodium dihydrogen phosphate, about 6 x 10 １１ vg / mL rAAV particles, and about 0.001% (w / v) poloxamer 188, wherein the pharmaceutical formulation has a pH of about 7.

3. The drug according to any one of claims 1 to 3.

12. 4. The method of claim 1, wherein the unit dose of rAAV particles administered to the one eye and / or the contralateral eye has a volume of about 25 μL to about 250 μL, and optionally, the unit dose of rAAV particles administered to the one eye and / or the contralateral eye comprises a volume of about 100 μL or about 30 μL.

13. (a) the individual has previously been treated for the ocular neovascular disease with an anti-VEGF agent, and optionally, the individual has received one or two injections of an anti-VEGF agent into the eye and / or the contralateral eye prior to administering the rAAV particles to the eye and / or the contralateral eye; or (b) the individual has not previously been treated for the ocular neovascular disease with an anti-VEGF agent; Optionally, the anti-VEGF agent is aflibercept. The drug according to any one of claims 1 and 3.

14. The unit dose of rAAV particles is administered in combination with steroid treatment, and optionally (a) the steroid treatment is a topical steroid treatment, for example, the steroid treatment is a difluprednate treatment, for example, difluprednate 0.05% at a dose of about 1 μg to about 3 μg or at a dose of about 2.5 μg; and / or (b) the steroid is administered before, during, and / or after administration of the unit dose of rAAV particles to the one eye and / or the contralateral eye; and / or (c) the steroid treatment is a topical steroid treatment; (i) the topical steroid treatment is daily steroid treatment for up to about 4 weeks, up to about 6 weeks, or up to about 8 weeks after administration of the unit dose of rAAV particles, e.g., the topical steroid treatment comprises administering topical steroid about four times daily for about week 1, administering topical steroid about three times daily for about week 2, administering topical steroid about twice daily for about week 3, and administering topical steroid about once daily for about week 4, with timing starting at the time of administration of the unit dose of rAAV particles and continuing thereafter; or (ii) the topical steroid treatment comprises, after administration of the unit dose of rAAV particles, administering a topical steroid about four times per day for about three weeks, followed by administering a topical steroid about three times per day for about one week, followed by administering a topical steroid about twice per day for about one week, followed by administering a topical steroid about once per day for about one week; or (iii) the topical steroid treatment comprises administering a topical steroid about four times per day for about four weeks, followed by administering a topical steroid about three times per day for about one week, followed by administering a topical steroid about twice per day for about one week, followed by administering a topical steroid about once per day for about one week, wherein the timing begins about one week prior to administration of the unit dose of rAAV particles, or if administered, administration of an anti-VEGF agent; or (iv) the topical steroid treatment comprises administering a topical steroid about four times per day for about four weeks, followed by administering a topical steroid about three times per day for about one week, followed by administering a topical steroid about twice per day for about one week, followed by administering a topical steroid about once per day for about one week, with timing commencing at the time of administration of the anti-VEGF agent and continuing thereafter; The drug according to any one of claims 1 to 3.

15. 4. The medicament of any one of claims 1 to 3, wherein the medicament is for treating an ocular neovascular disease or for improving vision in an individual suffering from an ocular neovascular disease, and optionally the ocular neovascular disease is selected from wet age-related macular degeneration (AMD), retinal neovascularization, choroidal neovascularization, diabetic retinopathy, proliferative diabetic retinopathy, retinal vein occlusion, central retinal vein occlusion, branch retinal vein occlusion, diabetic macular edema (DME), diabetic retinal ischemia, ischemic retinopathy, diabetic retinal edema, or any combination thereof, and further optionally the ocular neovascular disease is wet age-related macular degeneration (AMD) or diabetic macular edema (DME).

Images