Methods of treating ocular neovascular diseases using AAV2 variants encoding aflibercept

Abstract

Provided are methods for treating an ocular neovascular disease in an individual, comprising administering a unit dose of recombinant adeno-associated virus (rAAV) particles to an eye of the individual, wherein the rAAV particles comprise: a) a nucleic acid encoding a polypeptide comprising an amino acid sequence with at least about 95% identity to the amino acid sequence of SEQ ID NO: (35) and flanked by AAV2 inverted terminal repeats (ITRs), and b) an AAV2 capsid protein comprising an amino acid sequence LGETTRP (SEQ ID NO: (14)) inserted between positions (587) and (588) of the capsid protein, wherein the amino acid residue numbering corresponds to an AAV2 VP1 capsid protein.

Description

(19) *EP004696318A1* (11) EP 4 696 318 A1 (12) EUROPEAN PATENT APPLICATION (43) Date of publication: 18.02.2026 Bulletin 2026 / 08 (21) Application number: 25180005.8 (22) Date of filing: 10.09.2020 (51) International Patent Classification (IPC): A61K 38 / 17 (2006.01) A61K 31 / 00 (2006.01) A61K 35 / 761 (2015.01) C12N 7 / 00 (2006.01) C12N 15 / 86 (2006.01) A61P 27 / 02 (2006.01) (52) Cooperative Patent Classification (CPC): (C-Sets available) C12N 7 / 00; A61K 9 / 0048; A61K 31 / 573; A61K 31 / 713; A61K 35 / 761; A61K 38 / 179; A61K 45 / 06; A61P 27 / 02; C12N 15 / 86 (Cont.) (84) Designated Contracting States: AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR (30) Priority: 11.09.2019 US 201962899070 P 10.10.2019 US 201962913648 P 18.11.2019 PCT / US2019 / 062066 10.01.2020 US 202062959784 P 07.02.2020 US 202062971835 P 01.05.2020 US 202063019190 P 27.05.2020 US 202063030819 P 07.08.2020 US 202063063203 P (62) Document number(s) of the earlier application(s) in accordance with Art. 76 EPC: 20780467.5 / 4 027 983 (71) Applicant: Adverum Biotechnologies, Inc. Redwood City, CA 94063 (US) (72) Inventors: • GASMI, Mehdi Redwood City, 94063 (US) • KISS, Szilard Redwood City, 94063 (US) • OSBORNE, Aaron Redwood City, 94063 (US) • TURPCU, Adam Redwood City, 94063 (US) (74) Representative: EIP Fairfax House 15 Fulwood Place London WC1V 6HU (GB) Remarks: •This application was filed on 30.05.2025 as a divisional application to the application mentioned under INID code 62. •Claims filed after the date of filing of the application (Rule 68(4) EPC). (54) METHODS OF TREATING OCULAR NEOVASCULAR DISEASES USING AAV2 VARIANTS ENCODING AFLIBERCEPT (57) Provided are methods for treating an ocular neovascular disease in an individual, comprising admin- istering a unit dose of recombinant adeno-associated virus (rAAV) particles to an eye of the individual, wherein the rAAV particles comprise: a) a nucleic acid encoding a polypeptide comprising an amino acid sequence with at least about 95% identity to the amino acid sequence of SEQ ID NO: (35) and flanked by AAV2 inverted terminal repeats (ITRs), and b) an AAV2 capsid protein compris- ing an amino acid sequence LGETTRP (SEQ ID NO: (14)) inserted between positions (587) and (588) of the capsid protein, wherein the amino acid residue number- ing corresponds to an AAV2 VP1 capsid protein. EP 4 69 6 31 8 A 1 Processed by Luminess, 75001 PARIS (FR) (Cont. next page) (52) Cooperative Patent Classification (CPC): (Cont.) C-Sets A61K 31 / 573, A61K 2300 / 00; A61K 31 / 713, A61K 2300 / 00; A61K 38 / 179, A61K 2300 / 00 2 EP 4 696 318 A1 Description CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 62 / 899,070 filed September 11, 2019, U.S. Provisional Patent Application No. 62 / 913,648 filed October 10, 2019, International Application No. PCT / US2019 / 062066 filed November 18, 2019, U.S. Provisional Patent Application No. 62 / 959,784 filed January 10, 2020, U.S. Provisional Patent Application No. 62 / 971,835 filed February 7, 2020, U.S. Provisional Patent Application No. 63 / 019,190 filed May1, 2020, U.S. Provisional Patent Application No. 63 / 030,819 filed May 27, 2020 and U.S. Provisional Patent Application No. 63 / 063,203 filed August 7, 2020, the disclosure of each of which are hereby incorporated by reference in its entirety. FIELD

[0002] The present disclosure relates to methods of treating ocular neovascular disease and disorders in an individual that comprise administering a single unit dose of a recombinant adeno associated virus (rAAV) particles encoding an anti- VEGF agent (e.g., aflibercept) to an eye of an individual. SUBMISSION OF SEQUENCE LISTING ON ASCII TEXT FILE

[0003] The content of the following submission on ASCII text file is incorporated herein by reference in its entirety: a computer readable form (CRF) of the Sequence Listing (file name: 627002001241SEQLIST.TXT, date recorded: September 9, 2020, size: 41 KB). BACKGROUND

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

[0005] Diabetic retinopathy (DR) is a major complication of diabetes mellitus, and is a leading cause of visual loss in the workingagepopulation.DRmaybenon-proliferative (NPDR),with nonewblood vessel growth, or proliferativeDR (PDR), with new abnormal blood vessel growth within the retina or choroid. Diabetic macular edema (DME) is a complication of DR, and is another example of an ocular disease affecting the macula. DME affects up to 10% of people with diabetes and is caused by fluid accumulation in the macula. DME is the most frequent cause of sight loss in people with DR. Available therapies for treatingDME include laser andanti-vascular endothelial growth factor (anti-VEGF)drugssuchasaflibercept.

[0006] Aflibercept is a recombinant fusion protein that acts as a decoy receptor for vascular endothelial growth factor subtypesAandB(VEGF-AandVEGF-B)andplacental growth factor (PGF).Bybinding to these ligands,aflibercept isable to prevent them from binding to vascular endothelial growth factor receptors (VEGFR), VEGFR‑1 and VEGFR‑2, to suppress neovascularization and decrease vascular permeability. Aflibercept consists of domain 2 of VEGFR‑1 and domain 3 of VEGFR‑2 fused with the Fc fragment of IgG1.

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

[0008] Therefore, there isaneed in theart for therapies forocular neovascular diseasessuchaswAMD,DR,orDMEthat are effective, reduce the risk of adverse effects, and are amenable to high long-term patient compliance. 3 EP 4 696 318 A1 5 10 15 20 25 30 35 40 45 50 55 SUMMARY OF THE DISCLOSURE

[0009] In oneaspect, providedherein is amethod for treatinganocular neovascular disease in an individual, themethod comprisingadministeringaunit doseof about6×1011 vectorgenomes (vg) or lessof recombinantadeno-associatedvirus (rAAV) particles to one eye of the individual, wherein the individual is a human, and wherein the rAAV particles comprise: (a) a nucleic acid encoding a polypeptide comprising an amino acid sequencewith at least about 95% identity to the amino acid sequence of SEQ ID NO: 35 and flanked by AAV2 inverted terminal repeats (ITRs), and (b) an AAV2 capsid protein comprising, or consisting of, an aminoacid sequenceLGETTRP (SEQ IDNO: 14) inserted betweenpositions587and588 of the capsid protein, wherein the amino acid residue numbering corresponds to an AAV2 VP1 capsid protein. In some embodiments, the method comprises reducing retinal fluid in an eye of the individual.

[0010] In another aspect, provided herein is a method for reducing retinal fluid in an 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 a human, and wherein the rAAV particles comprise: (a) a nucleic acid encoding a polypeptide comprising an amino acid sequence with at least about 95% identity to the amino acid sequence of SEQ ID NO: 35 and flanked by AAV2 inverted terminal repeats (ITRs), and (b) an AAV2 capsid protein comprising, or consisting of, an amino acid sequence LGETTRP (SEQ ID NO: 14) inserted between positions 587 and 588 of the capsid protein, wherein the amino acid residue numbering corresponds to an AAV2 VP1 capsid protein. In some embodiments, the individual has receivedat least one treatment of ananti-VEGFagent inabout last 12weeksprior toadministrationof theunit doseof rAAV particles. In some embodiments, the amount or presence of retinal fluid in the one eye of the individual is refractory to prior treatment with an anti-VEGF agent. In some embodiments, the anti-VEGF agent is aflibercept. In some embodiments, the retinal fluid in theoneeye is reducedbyat least about60%. Insomeembodiments, the retinal fluid in theoneeye is reduced by about 80% compared to the level of retinal fluid in the one eye of the individual prior to administration of 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 × 1011 vector genomes per eye (vg / eye) or less.

[0011] In another aspect, provided herein is a method for treating an ocular neovascular disease in an individual, the method comprising: (a) administering an anti-VEGF agent to one eye of the individual; and (b) administering a unit dose of about 6 × 1011 vector genomes (vg) or less of recombinant adeno-associated virus (rAAV) particles to the one eye of the individual after administration of the anti-VEGF agent, wherein the individual is a human, and wherein the rAAV particles comprise: (i) a nucleic acid encoding a polypeptide comprising an amino acid sequence with at least about 95% identity to the amino acid sequence of SEQ ID NO: 35 and flanked by AAV2 inverted terminal repeats (ITRs), and (ii) an AAV2 capsid protein comprising an amino acid sequence LGETTRP (SEQ ID NO: 14) inserted between positions 587 and 588 of the capsid protein, wherein the amino acid residue numbering corresponds to an AAV2 VP1 capsid protein. In some embodiments, the method comprises administering the unit dose of rAAV particles to the one eye of the individual about 1 week or about 7 days after administration of the anti-VEGF agent. In some embodiments, the method comprises administering the unit dose of rAAV particles to the one eye of the individual about 1 week to about 2 weeks after administration of the anti-VEGF agent. In some embodiments, the method comprises administering the unit dose of rAAV particles to the oneeye 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 the anti-VEGF agent to the one eye of the individual on Day 1, and administering the unit dose of rAAV particles to the one eye of the individual on 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 by intravitreal injection. In some embodiments, the method further comprises administering a topical steroid treatment. In someembodiments, the topical steroid treatment is adifluprednate treatment. In some embodiments, the topical steroid treatment comprises about four administrations of topical steroid per day for about fourweeks, followedbyabout threeadministrations of topical steroid per day for about oneweek, followedby about twoadministrations of topical steroid per day for about oneweek, and followedbyabout oneadministration of topical steroid per day for about one week; timing starting with and following administration of the anti-VEGF agent. In some embodiments, the topical steroid treatment comprises about four administrations of topical steroid per day for about one month, followed by about three administrations of topical steroid per day for about one month, followed by about two administrations of topical steroid per day for about one month, and followed by about one administration of topical steroid per day for about one month; timing starting with and following administration of the anti-VEGF agent. In some embodiments, the topical steroid treatment comprises about four administrations of topical steroid per day for about one month, followed by about three administrations of topical steroid per day for about one month, followed by about two administrations of topical steroid per day for about one month, and followed by about one administration of topical steroid per day for about onemonth; timing starting with and following administration of the rAAVparticles. In someembodiments, the topical steroid comprises difluprednate 0.05% at a dose of about 1µg to about 3 µg. In some embodiments, the topical steroid comprises difluprednate 0.05% at a dose of about 2.5µg.

[0012] In some embodiments that may be combined with any of the preceding embodiments, the unit dose of rAAV 4 EP 4 696 318 A1 5 10 15 20 25 30 35 40 45 50 55 particles is about6×1010 toabout 2×1011 vector genomespereye (vg / eye). In someembodiments, theunit doseof rAAV particles is about 2 × 1011 or about 6 × 1010 vector genomes per eye (vg / eye). In some embodiments that may be combined with any of the preceding embodiments, the unit dose of rAAV particles is between about 6 × 1010 to about 6 × 1011 vector genomes per eye (vg / eye). In some embodiments that may be combined with any of the preceding embodiments, the unit dose of rAAV particles is between about 6 × 1010 to about 2 × 1011 vector genomes per eye (vg / eye). In some embodiments that may be combined with any of the preceding embodiments, the unit dose of rAAV particles is between about 2 × 1011 to about 6 × 1011 vector genomes per eye (vg / eye). In some embodiments, the unit doseof rAAVparticles is about2×1011orabout6×1010 vectorgenomespereye (vg / eye). In someembodiments, theunit dose of rAAV particles is about 2 × 1011 vg / eye. In some embodiments, the unit dose of rAAV particles is about 6 × 1011 vg / eye.

[0013] 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.

[0014] In some embodiments that may be combined with any of the preceding 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 administering the unit dose of rAAV particles to the contralateral eye is up to about 2 weeks after administering theunit doseof rAAVparticles to theoneeye. In someembodiments, theadministering theunit doseof rAAV particles to the contralateral eye is on the same day as the administering the unit dose of rAAV particles to the one eye; or the administering the unit dose of rAAV particles to the contralateral eye is between about 1 day to about 14 days after administering the unit dose of rAAV particles to the one eye. In some embodiments, the unit dose of rAAV particles administered to the contralateral eye of the individual comprises the same or less vector genomes per eye (vg / eye) than the unit dose of rAAV particles administered to the one eye of the individual. In some embodiments, the administering the unit dose of rAAV particles to the contralateral eye is at least about 2 weeks after administering the unit dose of rAAV particles to the one eye. In some embodiments, the 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.

[0015] In someembodiments thatmaybe combinedwith anyof the precedingembodiments, the nucleic acid comprises the nucleic acid sequence of SEQ ID NO: 40 or a sequence having at least 85% identity thereto.

[0016] In someembodiments thatmaybecombinedwith anyof the precedingembodiments, the polypeptide comprises the amino acid sequence of SEQ ID NO: 35. In some embodiments that may be combined with any of the preceding embodiments, the polypeptide comprises the amino acid sequence of SEQ ID NO: 41. In some embodiments, the polypeptide is aflibercept.

[0017] In some embodiments 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 the 5’ to 3’ order: (a) a first enhancer region; (b) a promoter region; (c) a 5’UTR region; (d) a nucleic acid encoding a polypeptide comprising an amino acid sequence with at least about 95% identity to the amino acid sequence of SEQ ID NO: 35; (e) a second enhancer region; and (f) a polyadenylation site; and flanked by AAV2 inverted terminal repeats (ITRs). In some embodiments, the first enhancer region comprises a CMV sequence comprising the sequence of SEQ ID NO: 22 or a sequence having at least 85% identity thereto. In some embodiments, thepromoter regioncomprisesaCMVsequencecomprising thesequenceofSEQ IDNO:23or asequence having at least 85% identity thereto. In someembodiments, the nucleic acid encoding a polypeptide comprises the nucleic acid sequence of SEQ ID NO: 40 or a sequence having at least 85% identity thereto. In some embodiments, the polypeptide comprises the amino acid sequence of SEQ ID NO: 35 or a sequence having at least 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, aTPLsequence comprising the sequenceofSEQ IDNO:24or a sequencehavingat least 85% identity thereto, and an eMLP sequence comprising the sequence of SEQ ID NO: 25 or a sequence having at least 85% identity thereto. In some embodiments, the second enhancer region comprises a full EES sequence comprising the sequence of SEQ ID NO: 26 or a sequence having at least 85% identity thereto. In some embodiments, the polyadenyla- tion site comprises a HGH polyadenylation site comprising the sequence of SEQ ID NO: 27 or a sequence having at least 85% identity thereto. In some embodiments, the nucleic acid further comprises (a) a first enhancer region comprising a CMV sequence comprising the sequence of SEQ ID NO: 22 or a sequence having at least 85% identity thereto; (b) a promoter region, comprising a CMV sequence comprising the sequence of SEQ ID NO: 23 or a sequence having at least 85% identity thereto; (c) a 5’UTR region comprising, in 5’ to 3’ order, a TPL sequence comprising the sequence of SEQ ID NO: 24 or a sequence having at least 85% identity thereto, and an eMLP sequence comprising the sequence of SEQ ID NO: 25 or a sequence having at least 85% identity thereto; (d) a second enhancer region comprising a full EES sequence comprising the sequence of SEQ ID NO: 26 or a sequence having at least 85% identity thereto; and (e) a HGH polyadenylation site comprising the sequence of SEQ ID NO: 27 or a sequence having at least 85% identity thereto. In some embodiments, the nucleic acid comprises AAV ITRs flanking the elements. 5 EP 4 696 318 A1 5 10 15 20 25 30 35 40 45 50 55

[0018] In someembodiments thatmaybe combinedwith anyof the precedingembodiments, the nucleic acid comprises the sequence of SEQ ID NO: 39 or a sequence having at least 85% identity thereto.

[0019] In some embodiments that may be combined with any of the preceding embodiments, the rAAV particles comprise an AAV2VP1 capsid protein comprising aGH loop that comprises the amino acid sequence ofSEQ IDNO: 38 or an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 38. In some embodiments, the rAAV particles comprise an AAV2 VP1 capsid protein comprising a GH loop that comprises an amino acid sequence having any of at least 90%,at least 91%,at least 92%,at least 93%,at least 94%,at least 95%,at least 96%,at least 97%,at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 38.

[0020] 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 havingat least 90%sequence identity toSEQIDNO:37. In someembodiments, the rAAVparticles compriseanAAV2VP1 capsid protein comprising an amino acid sequence having any of at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 37.

[0021] In some embodiments that may be combined with any of the preceding embodiments, the AAV2 capsid protein comprises, or consists of, theaminoacidsequenceLGETTRP(SEQ IDNO:14) insertedbetweenpositions587and588of theAAV2 VP1comprising the sequence of SEQ ID NO: 13. In some embodiments, theAAV2 capsid protein comprises the amino acid sequence LALGETTRPA (SEQ ID NO: 1) inserted between positions 587 and 588 of the capsid protein, wherein the amino acid residue numbering corresponds to an AAV2 VP1 capsid protein. In some embodiments, the AAV2 capsid protein comprises the amino acid sequence LALGETTRPA (SEQ ID NO: 1) inserted between positions 587 and 588 of the AAV2 VP1 comprising the sequence of SEQ ID NO: 13. In some embodiments, the AAV2 capsid protein comprises the amino acid sequence LALGETTRPA (SEQ ID NO: 1) inserted between positions 587 and 588 of the capsid protein, wherein the amino acid residue numbering corresponds to an AAV2 VP1 capsid protein. In some embodiments, the AAV2 capsid protein comprises the amino acid sequence LALGETTRPA (SEQ ID NO: 1) inserted between positions 587 and 588 of the AAV2 VP1 comprising the sequence of SEQ ID NO: 13.

[0022] In some embodiments that may be combined with any of the preceding embodiments, the administration of the unit dose of rAAV particles to the one eye and / or the contralateral eye is by intravitreal administration.

[0023] In some embodiments that may be combined with any of the preceding embodiments, the unit dose of rAAV particles is in a pharmaceutical formulation. In some embodiments, the pharmaceutical formulation comprises the rAAV particles, sodium chloride, sodium phosphate and a surfactant. In some embodiments, the pharmaceutical formulation comprises about 150 to about 200 mM sodium chloride, about 1 to about 10 mM monobasic sodium phosphate, about 1 to about10mMdibasic sodiumphosphate, about 0.0005%(w / v) toabout0.005%(w / v)poloxamer188, andabout6×1013 to about 6 × 1010 vector genomes (vg) per mL (vg / mL) of the rAAV particles, wherein the pharmaceutical formulation has a pH of about 7.0 to about 7.5. In some embodiments, the pharmaceutical formulation comprises about 180 mM sodium chloride, about 5 mM monobasic sodium phosphate, about 5 mM dibasic sodium phosphate, about 6 × 1012 vg / mL of the rAAV particles, and about 0.001% (w / v) poloxamer 188, wherein the pharmaceutical formulation has a pH of about 7.3. In some embodiments, the pharmaceutical formulation comprises about 180 mM sodium chloride, about 5 mM monobasic sodiumphosphate, about 5 mM dibasic sodiumphosphate, about 2× 1012 vg / mL of the rAAVparticles, and about 0.001% (w / v) poloxamer 188, wherein the pharmaceutical formulation has a pH of about 7.3. In some embodiments, the pharmaceutical formulation comprises about 180 mM sodium chloride, about 5 mM monobasic sodium phosphate, about 5 mM dibasic sodium phosphate, about 6 × 1011 vg / mL of the rAAV particles, and about 0.001% (w / v) poloxamer 188, wherein the pharmaceutical formulation has a pH of about 7.3.

[0024] In some embodiments that may be combined with any of the preceding embodiments, the unit dose of rAAV particles comprises a volume of about 25 µL to about 250 µL. In some embodiments, the unit dose of rAAV particles comprises a volume of about 100µL. In some embodiments, the unit dose of rAAV particles comprises a volume of about 30µL. In someembodiments thatmaybecombinedwithanyof theprecedingembodiments, theunit doseof rAAVparticles administered to the one eye and / or to the contralateral eye comprises a volume of about 25 µL to about 250 µL. In some embodiments, the unit dose of rAAV particles administered to the one eye and / or to the contralateral eye comprises a volume of about 100µL. In some embodiments, the unit dose of rAAV particles administered to the one eye and / or to the contralateral eye comprises a volume of about 30µL.

[0025] In some embodiments that may be combined with any of the preceding embodiments, the individual received prior treatment for the ocular neovascular disease with an anti-VEGF agent. In some embodiments, the individual has received 1 or 2 injections of an anti-VEGF agent in the one eye and / or in the contralateral eye prior to administration of the rAAV particles in the one eye and / or in the contralateral eye. In some embodiments, the individual has not received prior treatment for the ocular neovascular disease with an anti-VEGF agent. In some embodiments, the anti-VEGF agent is aflibercept.

[0026] In some embodiments that may be combined with any of the preceding embodiments, the ocular neovascular disease iswetage-relatedmaculardegeneration (AMD), retinal neovascularization, choroidal neovascularizationdiabetic 6 EP 4 696 318 A1 5 10 15 20 25 30 35 40 45 50 55 retinopathy, proliferative diabetic retinopathy, retinal vein occlusion, central retinal vein occlusion, branched retinal vein occlusion, diabetic macular edema, diabetic retinal ischemia, ischemic retinopathy, diabetic retinal edema, or any combination thereof.

[0027] 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 treatment. In some embodiments, the steroid treatment is a corticosteroid treatment. In some embodiments, the steroid treatment is a systemic steroid treatment. In some embodi- ments, the steroid treatment is an oral steroid treatment. In some embodiments, the steroid treatment is a prednisone treatment. In some embodiments, the oral prednisone treatment comprises administering prednisone at a dose of about 60 mg per day for a total of 6 days starting at 3 days before administration of the unit dose of rAAV particles to the one eye and / or to 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, and followed by administering prednisone at a dose of about 10 mg per day for a total of 2 days. In some embodiments, the steroid treatment is a topical steroid treatment. In some embodiments, the steroid treatment is a difluprednate treatment. In some embodiments, the steroid is administered before, during and / or after administration of the unit dose of rAAV particles. In some embodiments, the steroid is administeredbefore, duringand / or after administrationof theunit doseof rAAVparticles to theoneeyeand / or to the contralateral eye.

[0028] In some embodiments that may be combined with any of the preceding embodiments, the steroid treatment is a topical steroid treatment and the topical steroid treatment is a daily steroid treatment for up to about 4 weeks, up to about 6 weeks, or up toabout 8weeks fromadministering theunit doseof rAAVparticles. In someembodiments, the topical steroid treatment comprises about four administrations of topical steroid on about week 1, about three administrations of topical steroid on about week 2, about two administrations of topical steroid on about week 3, and about one administration of topical steroid on about week 4; timing starting with and following administration of the unit dose of rAAV particles. In some embodiments, the topical steroid treatment comprises about four administrations of topical steroid per day for about 3 weeks after administration of the unit dose of rAAV particles, followed by about 3 administrations of topical steroid per day for about 1 week, followed by about 2 administrations of topical steroid per day for about 1 week, and followed by about 1 administration of topical steroid per day for about 1 week. In some embodiments, the topical steroid treatment comprises about four administrationsof topical steroid per day for about fourweeks, followedbyabout threeadministrationsof topical steroidperday forabout oneweek, followedbyabout twoadministrationsof topical steroidper day foraboutoneweek, and followedbyaboutoneadministrationof topical steroidperday foraboutoneweek; timingstartingataboutoneweekprior to administration of the unit dose of rAAVparticles. In some embodiments, the topical steroid treatment comprises about four administrationsof topical steroidper day forabout onemonth, followedbyabout threeadministrationsof topical steroidper day for aboutonemonth, followedbyabout twoadministrationsof topical steroidper day for about onemonth, and followed byabout oneadministration of topical steroid per day for about onemonth; timing starting at about the administration of the unit dose of rAAV particles. In some embodiments, the topical steroid treatment comprises about four administrations of topical steroid per day for about onemonth, followed byabout three administrations of topical steroid per day for about one month, followed by about two administrations of topical steroid per day for about one month, and followed by about one administrationof topical steroidper day for about onemonth; timingstartingat about oneweekprior toadministrationof the unit dose of rAAV particles. In some embodiments, the topical steroid comprises difluprednate 0.05% at a dose of about 1µg to about 3 µg. In some embodiments, the topical steroid comprises difluprednate 0.05% at a dose of about 2.5µg.

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

[0030] In some embodiments that may be combined with any of the preceding embodiments, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in maintenance or a decrease of retinal thickness compared to the retinal thickness prior to administration of the unit dose of rAAV particles. In some embodiments, the maintenance or the decrease of retinal thickness compared to the retinal thickness prior to admin- istration of the unit dose of rAAV particles is present at about 30 weeks, about 34 weeks, about 44 weeks, about 6 months, about 1 year, about 1.5 years, about 2 years, about 3 years, about 5 years, about 10 years, or more after administration of the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual. In some embodiments, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in a decrease in retinal thickness compared to the retinal thickness prior to administration of the unit dose of rAAV particles. In some embodiments, the decrease in retinal thickness is at least about 10% compared to the retinal thickness prior to administration of the unit dose of rAAV particles. In some embodiments, retinal thickness is central subfield thickness (CST) or central retinal thickness (CRT).

[0031] In some embodiments that may be combined with any of the preceding embodiments, the administering the unit doseof rAAVparticles to theoneeyeand / or to thecontralateral eyeof the individual results inmaintenanceoradecrease in macular volume compared to the macular volume prior to administration of the unit dose of rAAV particles. In some embodiments, the maintenance or the decrease in macular volume compared to the macular volume prior to adminis- tration of the unit dose of rAAV particles is present at 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 7 EP 4 696 318 A1 5 10 15 20 25 30 35 40 45 50 55 the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual. In some embodiments, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in a decrease in macular volume compared to the macular volume prior to administration of the unit dose of rAAV particles. In some embodiments, the decrease in macular volume is at least about 10% compared to the macular volume prior to administration of the unit dose of rAAV particles.

[0032] In some embodiments that may be combined with any of the preceding embodiments, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in maintenance or an improvement of visual acuity compared to thevisual acuity prior toadministrationof theunit doseof rAAVparticles. In some embodiments, the maintenance or the improvement of visual acuity compared to the visual acuity prior to administration of the unit dose of rAAV particles is present at about 30 weeks, about 34 weeks, about 44 weeks, about 6 months, about 1 year, about 1.5 years, about 2 years, about 3 years, about 5 years, about 10 years, or more after administration of the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual. In some embodiments, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in an improvement of visual acuity compared to thevisual acuity prior toadministrationof theunit doseof rAAVparticles. In some embodiments, visual acuity is best corrected visual acuity (BCVA).

[0033] In some embodiments that may be combined with any of the preceding embodiments, administration of a single unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual provides a therapeutic benefit (e.g., treatment of anocular neovascular disease, reduction of retinal fluid,maintenanceor adecreaseof retinal thickness, maintenance or a decrease in macular volume, and / or maintenance or an improvement of visual acuity). In some embodiments, administration of a single unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual provides a therapeutic benefit that is present at about 30 weeks or more, about 34 weeks or more, about 44 weeks or more, about 6 months or more, about 1 year or more, about 1.5 years or more, or about 2 years, about 3 years, about 5 years, about 10 years, or more after administration of the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual.

[0034] In some embodiments that may be combined with any of the preceding embodiments, administration of the unit dose of rAAVparticles to the oneeyeand / or to the contralateral eye of a plurality of individuals results in at least about 50% of the individuals in the plurality not requiring ananti-VEGF rescue treatment. In someembodiments, at least about 50%of the individuals in the plurality do not require an anti-VEGF rescue treatment for at least about 20 weeks, at least about 36 weeks, at least about 52 weeks, at least about 56 weeks, or more after administration of the unit dose of rAAV particles.

[0035] In some embodiments that may be combined with any of the preceding embodiments, administration of a single unit dose of rAAV particles to the one eye and / or to the contralateral eye of a plurality of individuals results in at least about 67% of the individuals in the plurality not requiring an anti-VEGF rescue treatment. In some embodiments, at least about 67% of the individuals in the plurality do not require an anti-VEGF rescue treatment for at least about 20 weeks, at least about 36 weeks, at least about 52 weeks, at least about 60 weeks, at least about 64weeks, or at least about 66weeks after administration of the unit dose of rAAV particles.

[0036] In some embodiments that may be combined with any of the preceding embodiments, administration of the unit dose of rAAVparticles to the oneeyeand / or to the contralateral eye of a plurality of individuals results in at least about 78% of the individuals in the plurality not requiring ananti-VEGF rescue treatment. In someembodiments, at least about 78%of the individuals in the plurality do not require an anti-VEGF rescue treatment for at least about 20 weeks, at least about 36 weeks, or more after administration of the unit dose of rAAV particles.

[0037] In some embodiments that may be combined with any of the preceding embodiments, administration of the unit dose of rAAV particles to the one eye and / or to the contralateral eye of a plurality of individuals results in 100% of the individuals in the plurality not requiring an anti-VEGF rescue treatment. In some embodiments, 100% of the individuals in the plurality do not require an anti-VEGF rescue treatment for at least about 64 weeks, at least about 68 weeks, at least about 72 weeks, at least about 76 weeks, at least about 80 weeks, at least about 84 weeks, or more after administration of the unit dose of rAAV particles.

[0038] In some embodiments that may be combined with any of the preceding embodiments, administration of the unit dose of rAAV particles to the one eye and / or to the contralateral eye of a plurality of individuals results in a reduction in the annualized anti-VEGF injection rate of at least about 80%, at least about 85%, at least about 87%, at least about 90%, at least about 95%, at least about 99%, or 100% compared to the annualized anti-VEGF injection rate prior to administration of the unit dose of rAAV particles.

[0039] In some embodiments, the ocular neovascular disease is diabetic macular edema (DME). In some embodi- ments, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in a 2-step or in a 3-step improvement in Diabetic Retinopathy Severity Scale (DRSS). In some embodiments, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in a 2- step improvement in Diabetic Retinopathy Severity Scale (DRSS). In some embodiments, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in a 3-step improvement in Diabetic Retinopathy Severity Scale (DRSS). 8 EP 4 696 318 A1 5 10 15 20 25 30 35 40 45 50 55 INCORPORATION BY REFERENCE

[0040] All references cited herein, including patent applications and publications, are incorporated by reference in their entirety. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which: FIGS. 1A‑1B provide schematics of the investigational medicinal product and the phase I study described in Examples 1 and 2. FIG. 1A is a schematic of AAV2.7m8-aflibercept. AAV2.7m8-aflibercept is a recombinant, replication-deficient adeno-associated viral (rAAV) vector containing the AAV2.7m8 protein capsid and a vector genomecontaininganexpressioncassetteof a codon-optimizedversionof theaflibercept cDNAunder thecontrol of a ubiquitous chimeric promoter (C11). The AAV2.7m8-aflibercept vector genome also contains two AAV2 inverted terminal repeat sequences (ITR) flanking the aflibercept cDNA expression cassette. FIG. 1B is a diagram summar- izing the study design for the phase I study described in Examples 1 and 2. FIG. 1C provides a diagram of the AAV2.7m8-aflibercept vector genome (SEQ ID NO: 39). The vector genome comprises two inverted terminal repeats (ITRs) of AAV serotype 2 (at positions 1‑145 and 3772‑3916 of SEQ ID NO: 39), an expression cassette comprised of the CMV promoter (at positions 180‑693 of SEQ ID NO: 39), a 5’ Untranslated Region (UTR) comprised of Adenovirus Tripartite Leader Sequence and Synthetic Intron (at positions 694‑1314 of SEQ ID NO: 39), a Kozak sequence (at positions 1329‑1340 of SEQ ID NO: 39), a codon-optimized aflibercept cDNA (at positions 1338‑2714 of SEQ ID NO: 39), a 3’ UTR comprised of human scaffold attachment region (at positions 2717‑3527 of SEQ ID NO: 39), and human growth hormone polyadenylation / transcription stop signal (at positions 3546‑3748 of SEQ ID NO: 39). AAV = adeno-associated virus; CMV = cytomegalovirus; GH = growth hormone; ITR = inverted terminal repeat; polyA = polyadenylation; SAR = scaffold-attached region; TPL = tripartite leader sequence. FIGS. 2A‑2L show optical coherence tomography (OCT) images and retinal thickness maps derived from OCT images taken from subjects in Cohort 1 of the study described in Example 1. The OCT images were taken at the indicated times before and after administration of AAV2.7m8-aflibercept (Day 1). The anti-VEGF IVT treatment interval is indicated for all subjects. FIG. 2A provides OCT images and retinal thickness maps derived from OCT images taken from Subject 1 at five office visits at the times indicated prior to the Screening aflibercept injection. OCT images were taken immediately prior to treatment with aflibercept standard of care. Subject 1 required aflibercept IVT every 5‑7 weeks and exhibited refractory subretinal fluid and a pigment epithelial detachment (PED) despite aflibercept standard of care treatment. FIG. 2B provides OCT images and retinal thickness maps derived from OCTimages taken fromSubject 1at theScreeningaflibercept injection (Day ‑7), at theAAV2.7m8-aflibercept injection (Day 1), and at follow-up visits at the times indicated. Subject 1 did not require any rescue injections after the AAV2.7m8-aflibercept injection. Subject 1 exhibited resolution of subretinal fluid beginning at week 4, and remained free of subretinal and intraretinal fluid (remained dry). FIG. 2C provides OCT images and retinal thickness maps derived from OCT images taken from Subject 2 at five office visits at the times indicated prior to the Screening aflibercept injection. OCT images were taken immediately prior to treatment with aflibercept standard of care. Subject 2 required six aflibercept IVT treatments in the 8 months prior to AAV2.7m8-aflibercept treatment to maintain retinal anatomy.FIG. 2D providesOCT images and retinal thicknessmapsderived from OCTimages taken fromSubject 2 at theScreeningaflibercept injection (Day ‑7), at theAAV2.7m8-aflibercept injection (Day1), andat follow-upvisits at the times indicated. Subject 2 did not require any rescue injections after the AAV2.7m8-aflibercept injection. Subject 2 exhibited stable retinal anatomywith no subretinal or intraretinal fluid through week 24.FIG. 2E providesOCT images and retinal thickness maps derived from OCT images taken from Subject 3 at four office visits at the times indicated prior to the Screening aflibercept injection. OCT images taken on Week ‑27 are not shown. OCT images were taken immediately prior to treatment with aflibercept standard of care. Subject 3 exhibited subretinal fluid, which increased when the interval between aflibercept IVTwas increased from 5 to 7 weeks.FIG. 2F provides OCT images and retinal thickness maps derived from OCT images taken from Subject 3 at the Screening aflibercept injection (Day ‑7), the AAV2.7m8-aflibercept injection (Day 1), and at follow-up visits at the times indicated. Subject 3 did not require any rescue injections after the AAV2.7m8-aflibercept injection. Subject 3 exhibited resolution of refractory subretinal fluid by week 8, and stable retinal anatomy through week 24. FIG. 2G provides OCT images and retinal thickness maps derived from OCT images taken from Subject 4 at five office visits at the times indicated prior to the Screening aflibercept injection. OCT images were taken immediately prior to treatment with ranibizumab 0.5 mg IVTstandard of 9 EP 4 696 318 A1 5 10 15 20 25 30 35 40 45 50 55 care. Subject 4 exhibited subretinal fluid that was refractory to ranibizumab IVT injections. FIG. 2H provides OCT images and retinal thickness maps derived from OCT images taken from Subject 4 at the Screening aflibercept injection (Day ‑14), theAAV2.7m8-aflibercept injection (Day1), andat follow-up visits at the times indicated. Subject 4 did not require any rescue injections after the AAV2.7m8-aflibercept injection. Subject 4 exhibited resolution of refractory subretinal fluid by week 8, and stable retinal anatomy through week 24. FIG. 2I provides OCT images and retinal thickness maps derived from OCT images taken from Subject 5 at five office visits at the times indicated prior to theScreeningaflibercept injection.OCTimageswere taken immediatelyprior to treatmentwithaflibercept standardof care. FIG. 2J provides OCT images and retinal thickness maps derived from OCT images taken from Subject 5 at the Screening aflibercept injection (Day ‑14), the AAV2.7m8-aflibercept injection (Day 1), and at follow-up visits at the times indicated. Subject 5 did not require any rescue injections after the AAV2.7m8-aflibercept injection. Subretinal fluid and PEDs present at the time of AAV2.7m8-aflibercept treatment resolved overtime, and retinal anatomy remained stable and free of subretinal or intraretinal fluid through week 24. FIG. 2K provides OCT images and retinal thickness maps derived from OCT images taken from Subject 6 at five office visits at the times indicated prior to the Screening aflibercept injection. OCT images were taken immediately prior to treatment with either bevacizumab 1.5 mg IVT standard of care or ranibizumab 0.5 mg IVT standard of care, as indicated. The appearance of Subject 6’s retina was consistent with polypoidal choroidal vasculopathy (PCV). FIG. 2L provides OCT images and retinal thickness maps derived from OCT images taken from Subject 6 at the Screening aflibercept injection (Day ‑10), the AAV2.7m8-aflibercept injection (Day 1), and at follow-up visits at the times indicated. Subject 6 did not require any rescue injectionsafter theAAV2.7m8-aflibercept injection.Subject 6didnot exhibit any increase in subretinal fluidand achieved some anatomical improvement through week 24. The contralateral eye of Subject 6 received standard of care aflibercept injections every 4 weeks over the course of the trial, and exhibited similar retinal morphology to the AAV2.7m8-aflibercept-treated eye. FIG. 3 shows the change in mean central retinal thickness (CST) for subjects in Cohort 1 of the study described in Example 1 at the indicated time points. Error bars indicate the 90% confidence interval, calculated using the T- distribution. Baseline (BL) indicates the measurement taken prior to the Screening aflibercept injection 7 to 15 days (e.g., 7‑14days)prior toAAV2.7m8-aflibercept treatment onday1.At24weekspostAAV2.7m8-aflibercept treatment, subjects on average exhibited a change in CRTof ‑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. FIG. 4 shows the mean best corrected visual acuity (BCVA) measurements based on Early Treatment Diabetic Retinopathy Study (ETDRS) letters assessments for subjects in Cohort 1 of the study described in Example 1 at the indicated time points. Error bars indicate the 90% confidence interval, calculated using the T-distribution. Baseline (BL) indicates themeasurement takenprior to theScreening aflibercept injection 7 to 15days (e.g., 7‑14days) prior to AAV2.7m8-aflibercept treatment on day 1. At 24 weeks post AAV2.7m8-aflibercept treatment, subjects on average exhibited a 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. FIG. 5 provides the nucleic acid sequence of aflibercept (SEQ ID NO: 36). FIG. 6 shows plots of anterior chamber cell and vitreous cell counts following treatment with AAV2.7m8-aflibercept for subjects 1‑6 of the study described in Example 1. The steroid treatment administered to each patient is indicated below each plot. Aqueous 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 National Institutes ofHealth (NIH) guidelines. For aqueous cells, a cell count value of 0.5+ indicates 1‑5 cells; a cell count value of 1+ indicates 6‑15 cells; a cell count value of 2+ indicates 16‑25 cells; a cell count value of 3+ indicates 26‑50 cells; and a cell count value of 4+ indicates >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; anda cell count value of 4+ indicates >100 cells. Rare cellswere capturedas0.5+ for the analysis shown in this figure. FIGS. 7A‑7B show optical coherence tomography (OCT) images and retinal thickness maps derived from OCT images taken fromsubjects 1‑6 inCohort 1 of the studydescribed inExample1at amedian followup timeof 34weeks. In addition, the change in BCVA from Baseline, the number of anti-VEGF IVT injections in the 8 months prior to administration of AAV2.7m8-aflibercept, and the number of administered rescue anti-VEGF IVT injections during the study are also provided for each of subjects 1‑6. The actual week during which the OCT images and retinal thickness maps were obtained for each subject are indicated (Subject 1 = Week 44; Subject 2 = Week 40; Subject 3 = Week 36; Subject 4 = Week 32; Subject 5 = Week 28; and Subject 6 = Week 28). No subjects required rescue anti-VEGF IVT injections during the study and no retreatment criteria were met at any point during the follow-up period of up to 44 weeks. No subjects exhibited signs of disease re-activation on OCT imaging. FIG. 8 showsplots of aqueous andvitreous cell counts following treatmentwithAAV2.7m8-aflibercept for subjects 1‑6 in Cohort 1 of the study described in Example 1 up to a median follow-up time of 44 weeks (40‑52 weeks). The steroid treatment administered toeachsubject is indicatedbeloweachplot. Aqueouscell gradecategorieswerebasedon the 10 EP 4 696 318 A1 5 10 15 20 25 30 35 40 45 50 55 Standardization of Uveitis Nomenclature (SUN) criteria (Jabs, DA et al. J Ophthalmol. 2005; 140:509‑516). Vitreous cell grade categories were based on National Institutes of Health (NIH) guidelines. For aqueous cells, a cell grade of 0.5+ indicates1‑5cells; a cell gradeof 1+ indicates6‑15cells; a cell gradeof 2+ indicates16‑25cells; a cell gradeof 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 gradeof 1+ indicates11‑20cells; a cell gradeof 2+ indicates21‑30cells; a cell gradeof 3+ indicates31‑100 cells; and a cell grade of 4+ indicates >100 cells. Rare cells were captured as a cell grade of 0.5+. FIGS. 9A‑9B show optical coherence tomography (OCT) images and retinal thickness maps derived from OCT images taken fromsubjects1‑6 inCohort 1of thestudydescribed inExample1atamedian follow-up timeof 44weeks. The change in BCVA ETDRS letters and CST from Baseline are also provided. The actual weeks during which the OCT images and retinal thickness maps were obtained for each subject are indicated. The asterisk indicates that Subject 4 underwent retinal detachment repair with gas bubble injection in order to repair a spontaneous pseudo- phakic inferior macula off rhegmatogenous retinal detachment (RRD) (FIG. 9B). Subject 4 remains under follow-up; OCT images and BCVA values for Subject 4 correspond to the last observations prior to retinal detachment. FIG. 10 shows the mean best corrected visual acuity (BCVA) measurements based on Early Treatment Diabetic RetinopathyStudy (ETDRS) lettersassessments for subjects inCohort 2of thestudydescribed inExamples2and3at the indicated timepoints. Error bars indicate the90%confidence interval of themeanabsoluteBCVAcalculated using the T-distribution. Baseline (BL) indicates the measurement taken prior to the Screening aflibercept injection 7 to 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 exhibited amean change in BCVAof ‑4.8 letters. BL =baseline; D =day;W =week. The day 1 visit occurred 7‑15 days after the baseline visit. FIG. 11 shows the mean central retinal thickness (CST) for subjects in Cohort 2 of the study described in Examples 2 and3at the indicated timepoints.Error bars indicate the90%confidence interval of themeanabsoluteCSTcalculated using theT-distribution.Baseline (BL) indicates themeasurement takenprior to theScreeningaflibercept injection7 to 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 exhibited a mean change in CSTof ‑27.8 µm. BL = baseline; D = day; W = week. The day 1 visit occurred 7‑15 days after the baseline visit. FIG. 12 shows plots of aqueous cell and vitreous cell counts following treatment with AAV2.7m8-aflibercept for subjects in Cohort 2 of the study described in Examples 2 and 3 up to a follow-up time of 24 weeks. The steroid treatment administered to each subject is indicated below each plot; the frequency of topical steroid (difluprednate) eye drop administration is indicated (e.g., 1x = once per day; 2x = twice per day; 3x = 3 times per day; 4x = 4 times per day). Aqueouscell grade categorieswerebasedon theStandardization ofUveitisNomenclature (SUN) criteria (Jabs, DAet al. JOphthalmol. 2005;140:509‑516).Vitreouscell gradecategorieswerebasedonNational InstitutesofHealth (NIH)guidelines. For aqueouscells, a cell gradeof 0.5+ indicates1‑5cells; a cell gradeof 1+ indicates6‑15cells; a cell grade of 2+ indicates 16‑25cells; a cell grade of 3+ indicates 26‑50 cells; anda 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+ for this analysis. FIGS. 13A‑13B show optical coherence tomography (OCT) images and retinal thickness maps derived from OCT images taken from subjects 1‑6 in Cohort 2 of the study described in Examples 2 and 3 up to a follow-up time of 24 weeks. The change in BCVA from Baseline, the change in CST from baseline, and the number of rescue anti-VEGF injections administered during the 24-week follow up period are also provided. InFIG. 13A, the asterisks indicate that Subject 3 received 3 rescue anti-VEGF injections, including at week 24, all due to loss of ≥10 letters in BCVA from baseline. This was attributed to intraretinal or subretinal fluid. In FIG. 13B, the asterisks indicate that Subject 5 received 3 rescue anti-VEGF injections, the last of which occurred on week 20, due to an increase in central subfield thickness of >75µm from baseline. FIG. 14 is a Swimmer’s Lane Plot showing the number of anti-VEGF injections for subjects in Cohorts 1 and 2 of the study described in Examples 1‑3. The x-axis represents time in weeks relative to the time AAV2.7m8-aflibercept was administered. The y-axis shows each individual subject in Cohorts 1 and 2. The circular shapes represent anti-VEGF IVT injections administered before andafter treatment withAAV2.7m8-aflibercept. Theasterisk indicates that Subject 6 of Cohort 2 was diagnosed with nAMD 6.4 months prior to administration of AAV2.7m8-aflibercept. The vertical line bisecting the plot indicates day 1, when AAV2.7m8-aflibercept was administered. To the right of the bisecting vertical line are each of the subsequent 61 study visits. FIGS. 15A‑15C show plots of aqueous cell and vitreous cell counts as measured by slit lamp examination following treatment with AAV2.7m8-aflibercept for subjects in Cohorts 1, 2 and 3 of the study described in Examples 1‑5. FIG. 15A shows plots of aqueous cell and vitreous cell counts for subjects in Cohort 1 up to a follow up time of 64 weeks (median=60weeks; range=52‑64weeks).FIG. 15B showsplots of aqueous cell and vitreous cell counts for subjects in Cohort 2 up to a follow up time of 40 weeks (median = 36 weeks; range = 32‑40 weeks). FIG. 15C shows plots of aqueous cell and vitreous cell counts for subjects inCohort 3 up to a followup timeof 20weeks. InFIGS. 15A‑15C, the 11 EP 4 696 318 A1 5 10 15 20 25 30 35 40 45 50 55 steroid treatment administered to each subject is indicated below each plot; aqueous cell grade categories were basedon theStandardizationofUveitisNomenclature (SUN)criteria: JabsDA,etal. JOphthalmol2005;140:509‑516; vitreous cell grade categories were based on National Institutes of Health (NIH) guidelines; aqueous cells: 0.5+ = 1‑5 cells; 1+=6‑15cells; 2+=16‑25cells; 3+=26‑50cells; 4+=>50cells; vitreous cells: 0.5+=1‑10cells; 1+=11‑20cells; 2+ = 21‑30 cells; 3+ = 31‑100 cells; 4+ = >100 cells; rare cells were captured as 0.5+ for this analysis. QID = four times per day; TID = three time per day; BID = twice per day; QD = once per day; QOD = once every other day; "QD »" indicates that the subject continues to receive topical steroids once per day; "BID >>" indicates that the subject continues to receive topical steroids twice per day; and "QID >>" indicates that the subject continues to receive topical steroids four times per day. FIGS. 16A‑16B provide the mean BCVA and mean CST for subjects in Cohort 1 of the study described in Examples 1‑5 (n=6) frombaselineup toWeek52.FIG.16A shows themeanBCVA(ETDRS letters) frombaselineup toWeek52. The asterisk indicates that one subject had low BCVA scores at Weeks 44 and 48 due to retinal detachment.FIG. 16B shows the mean CST (µm) from baseline up to Week 52. The asterisk indicates that one subject had no CST data at Weeks 44and48due to retinal detachment. InFIGS. 16A‑16B, the error bars indicate the90%confidence intervals of the mean absolute BCVA and CST values using the T-distribution; BL = baseline; D = day; W = week. FIGS. 17A‑17B provide the mean BCVA and mean CST for subjects in Cohort 2 of the study described in Examples 1‑5 (n=6) frombaselineup toWeek36.FIG.17A shows themeanBCVA(ETDRS letters) frombaselineup toWeek36. FIG. 17B shows the mean CST (µm) from baseline up to Week 36. InFIGS. 17A‑17B, the 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 missed the Week 36 visit. FIGS. 18A‑18B provide the mean BCVA and mean CST for five subjects in Cohort 3 of the study described in Examples 1‑5 from baseline up to Week 20. FIG. 18A shows the mean BCVA (ETDRS letters) from baseline up to Week20.FIG. 18B shows themeanCST (µm) frombaselineup toWeek20. InFIGS. 18A‑18B, theerror 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 missed the Week 36 visit. FIG. 19 is aSwimmer’sLanePlot showing thenumberof anti-VEGF injectionsadministered to subjects inCohorts1, 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 shows each individual subject in Cohorts 1‑3. The circular shapes represent anti-VEGF IVT injections administered before and after treatment with AAV2.7m8-aflibercept. The vertical line bisecting the plot indicates Day 1, when AAV2.7m8-aflibercept was administered. To the right of the bisecting vertical line are each of the subsequent study visits. FIGS. 20A‑20B show optical coherence tomography (OCT) images and retinal thickness maps derived from OCT images taken from Subject 4 in Cohort 1 of the study described in Examples 1‑5 at the indicated times before andafter administration of AAV2.7m8-aflibercept. FIG. 20A shows OCT images and retinal thickness maps for Subject 4 in Cohort 1 at the indicated times (weeks) prior to administration of AAV2.7m8-aflibercept, during which the subject was administered anti-VEGF IVT injections (ranibizumab). FIG. 20B shows OCT images and retinal thickness maps for Subject 4 in Cohort 1 at the indicated times (weeks) before and after administration of AAV2.7m8-aflibercept. The times of administration of the screening anti-VEGF IVT injection ("Screening anti-VEGF IVT") and of AAV2.7m8- aflibercept ("AAV2.7m8-aflibercept") are indicated. The BCVA (ETDRS letters) and CST (µm) at each indicated time before and after administration of AAV2.7m8-aflibercept are provided. The asterisk indicates that the subject had a retinal detachment event unrelated to AAV2.7m8-aflibercept. FIGS. 21A‑21B show optical coherence tomography (OCT) images and retinal thickness maps derived from OCT images taken from Subject 5 in Cohort 3 of the study described in Examples 1‑5 at the indicated times before andafter administration of AAV2.7m8-aflibercept. FIG. 21A shows OCT images and retinal thickness maps for Subject 5 in Cohort 3 at the indicated times (weeks) prior to administration of AAV2.7m8-aflibercept, during which the subject was administered anti-VEGF IVT injections (aflibercept). FIG. 21B shows OCT images and retinal thickness maps for Subject 5 in Cohort 3 at the indicated times (weeks) before and after administration of AAV2.7m8-aflibercept. The times of administration of the screening anti-VEGF IVT injection ("Screening anti-VEGF IVT") and of AAV2.7m8- aflibercept ("AAV2.7m8-aflibercept") are indicated. The BCVA (ETDRS letters) and CST (µm) at each indicated time before and after administration of AAV2.7m8-aflibercept are provided. FIGS. 22A‑22D show plots of aqueous cell and vitreous cell counts as measured by slit lamp examination following treatment with AAV2.7m8-aflibercept for subjects in Cohorts 1, 2, 3, and 4 of the study described in Examples 1‑5 and 7. FIG. 22A shows plots of aqueous cell and vitreous cell counts for subjects in Cohort 1 up to a follow up time of 80 weeks. FIG. 22B shows plots of aqueous cell and vitreous cell counts for subjects in Cohort 2 up to a follow up time of 56 weeks.FIG. 22C shows plots of aqueous cell and vitreous cell counts for subjects in Cohort 3 up to a follow up time of 36 weeks. FIG. 22D shows plots of aqueous cell and vitreous cell counts for subjects in Cohort 4 up to a follow up time of 8 weeks. In FIGS. 22A‑22D, the steroid treatment administered to each subject is indicated below each plot; aqueous cell grade categorieswerebasedon theStandardization ofUveitisNomenclature (SUN) criteria: JabsDA, et 12 EP 4 696 318 A1 5 10 15 20 25 30 35 40 45 50 55 al. J Ophthalmol 2005;140:509‑516; vitreous cell grade categories were based on National Institutes of Health (NIH) guidelines; aqueous 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+=1‑10cells; 1+=11‑20cells; 2+=21‑30cells; 3+=31‑100cells; 4+=>100cells; rarecellswerecapturedas 0.5+ for this analysis.QID= four timesper day; TID= three timeper day; BID= twice per day;QD=onceper day; QOD = once every other day; "QD »" indicates that the subject continues to receive topical steroids once per day; "BID >>" indicates that the subject continues to receive topical steroids twice per day; "TID >>" indicates that the subject continues to receive topical steroids three times per day; and "QID >>" indicates that the subject continues to receive topical steroids four times per day. FIGS. 23A‑23B provide the mean BCVA and mean CST for subjects in Cohort 1 of the study described in Examples 1‑5 and 7 (n=6) from baseline up to Week 72. FIG. 23A shows the mean BCVA (ETDRS letters) from baseline up to Week 72. One subject had low BCVA scores at Weeks 44 and 48 due to retinal detachment. *n = 5 from Week 56 to Week 72. FIG. 23B shows the mean CST (µm) from baseline up to Week 72. One subject had no CST data at Weeks 44 and 48 due to retinal detachment. *n = 5 from Week 56 to Week 72. In FIGS. 23A‑23B, the error bars indicate the 90%confidence intervals of themeanabsoluteBCVAandCSTvaluesusing theT-distribution; BL=baseline;D=day; W = week. FIGS. 24A‑24B provide the mean BCVA and mean CST for subjects in Cohort 2 of the study described in Examples 1‑5 and 7 (n=6) from baseline up to Week 52. FIG. 24A shows the mean BCVA (ETDRS letters) from baseline up to Week52. *n=5 forWeek36andWeek40.FIG.24B shows themeanCST (µm) frombaselineup toWeek52. *n=5 for Week 36 and Week 40. In FIGS. 24A‑24B, the 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. FIGS. 25A‑25B provide the mean BCVA and mean CST for subjects in Cohort 3 of the study described in Examples 1‑5 and 7 (n=9) from baseline up to Week 20. FIG. 25A shows the mean BCVA (ETDRS letters) from baseline up to Week 20. *n = 8 for Weeks 4, 16, and 20. FIG. 25B shows the mean CST (µm) from baseline up to Week 20. *n = 8 for Weeks 4, 16, and 20. In FIGS. 25A‑25B, the 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. FIG. 26 is a Swimmer’s Lane Plot showing the number of anti-VEGF injections administered to subjects in Cohorts 1, 2, 3, and 4 of the study described in Examples 1‑5 and 7. The x-axis represents time in weeks relative to the time AAV2.7m8-aflibercept was administered. The y-axis shows each individual subject in Cohorts 1‑4. The circular shapes represent anti-VEGF IVT injections administered before and after treatment with AAV2.7m8-aflibercept. The vertical line bisecting the plot indicates Day 1, when AAV2.7m8-aflibercept was administered. To the right of the bisecting vertical line are each of the subsequent study visits. Five subjects were diagnosed <1 year prior to AAV2.7m8-aflibercept injection (1 subject in each of Cohorts 2 and 3, and 3 subjects in Cohort 4). In Cohort 4, data beforeDay1are incomplete forSubject 2 due to relocation, andSubject 5because the subject participated in a clinical trial with unknown medication(s) after diagnosis. FIGS. 27A‑27B show themeanannualized anti-VEGF injection rate for subjects in Cohorts 1‑3 of the study described inExamples1‑5and7.FIG. 27Aprovidesacomparisonof themeanannualizedanti-VEGF injection rates for subjects in Cohort 1 (administered the "High Dose" of 6 × 1011 vg / eye; n = 6) and for subjects in Cohorts 2 and 3 (administered the "Low Dose" of 2 × 1011 vg / eye; n = 15). FIG. 27B provides a comparison of the mean annualized anti-VEGF injection rates for subjects in each of Cohorts 1 (n = 6), 2 (n = 6), and 3 (n = 9). In FIGS. 27A‑27B, Annualized rate (Prior) = (numberof anti-VEGF IVTinjections in the12monthsprior toAAV2.7m8-aflibercept) / (days from thefirst anti- VEGF IVTinjection in thepast 12months to theAAV2.7m8-aflibercept injection / 365.25); andAnnualized rate (Post)= (number of anti-VEGF IVT injections since AAV2.7m8-aflibercept) / (days fromAAV2.7m8-aflibercept to the last study follow-up / 365.25). FIGS. 28A‑28B show optical coherence tomography (OCT) images and retinal thickness maps derived from OCT images taken fromSubject 5 inCohort 3of thestudydescribed inExamples1‑5and7at the indicated timesbeforeand after administrationofAAV2.7m8-aflibercept.FIG.28AshowsOCTimagesand retinal thicknessmaps forSubject 5 in Cohort 3 at the indicated times (weeks) prior to administration of AAV2.7m8-aflibercept, during which the subject was administered anti-VEGF IVT injections (aflibercept). FIG. 28B shows OCT images and retinal thickness maps for Subject 5 in Cohort 3 at the indicated times (weeks) before and after administration of AAV2.7m8-aflibercept. The times of administration of the screening anti-VEGF IVT injection ("Aflibercept IVT") and of AAV2.7m8-aflibercept are indicated. The BCVA (ETDRS letters) and CST (µm) at each indicated time before and after administration of AAV2.7m8-aflibercept are provided. FIG. 29 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 Subfield Thickness. *All subjects receive a 7-week course of difluprednate eye drops, starting at QID and tapering to QD. **PE= Primary Endpoint assessment. ***EOS= End of Study assessment. 13 EP 4 696 318 A1 5 10 15 20 25 30 35 40 45 50 55 DETAILED DESCRIPTION

[0042] Several aspects are described below with reference to example applications for illustration. It should be understood that numerous specific details, relationships, and methods are set forth to provide a full understanding of the features described herein. Onehavingordinary skill in the relevant art, however, will readily recognize that the features describedherein canbepracticedwithout oneormoreof the specific details orwithothermethods. The featuresdescribed herein are not limited by the illustrated ordering of acts or events, as some acts can occur in different orders and / or concurrently with other acts or events. Furthermore, not all illustrated acts or events are required to implement a methodology in accordance with the features described herein. Definitions

[0043] Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0044] The terminology used herein is for the purpose of describing particular examples only and is not intended to be limiting. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, unless the context clearly indicatesotherwise.Furthermore, to theextent that the terms "including", "includes", "having", "has", "with", or variants thereof are used in either the detailed description and / or the claims, such termsare intended to be inclusive in a manner similar to the term "comprising". The term "comprising" as used herein is synonymous with "including" or "containing", and is inclusive or open-ended.

[0045] Any reference to "or" herein is intended to encompass "and / or" unless otherwise stated. As usedherein, the term "about" a number refers to that number plus or minus 10% of that number. The term "about" a range refers to that range minus10%of its lowest valueandplus10%of its greatest value.Reference to "about" a valueor parameter herein includes (and describes) embodiments that are directed to that value or parameter per se.

[0046] The term "subject", "patient", or "individual" refers to primates, such as humans and non-human primates, e.g., African green monkeys and rhesus monkeys. In some embodiments, the subject is a human.

[0047] The terms "treat," "treating", "treatment," "ameliorate" or "ameliorating" and other grammatical equivalents as used herein, refer to alleviating, abating or ameliorating an ocular neovascular disease or disorder, or symptoms of the ocular neovascular disease or disorder, preventing additional symptoms of the ocular neovascular disease or disorder, ameliorating or preventing the underlying metabolic causes of symptoms, inhibiting the ocular neovascular disease or disorder, e.g., arresting the development of the ocular neovascular disease or disorder, relieving the ocular neovascular disease or disorder, causing regression of the ocular neovascular disease or disorder, or stopping the symptoms of the ocular neovascular disease or disorder, and are intended to include prophylaxis. The terms further include achieving a therapeutic benefit and / or a prophylactic benefit. The term "therapeutic benefit" refers to eradication or amelioration of the ocular neovascular disease or disorder being treated. Also, a therapeutic benefit is achieved with the eradication or amelioration of one or more of the physiological symptoms associated with the ocular neovascular disease or disorder such that an improvement is observed in the subject, notwithstanding that, in some embodiments, the subject is still afflicted with the ocular neovascular disease or disorder. For prophylactic benefit, the pharmaceutical compositions are administered to a subject at risk of developing the ocular neovascular disease or disorder, or to a subject reporting one or more of the physiological symptoms of the ocular neovascular disease or disorder, even if a diagnosis of the disease or disorder has not been made.

[0048] The terms "administer," "administering", "administration," and the like, as used herein, can refer to the methods that are used to enable delivery of therapeutics or pharmaceutical compositions to the desired site of biological action. These methods include intravitreal or subretinal injection to an eye.

[0049] The terms "effective amount", "therapeutically effective amount" or "pharmaceutically effective amount" as used herein, can refer to a sufficient amount of at least onepharmaceutical composition or compoundbeing administeredwhich will relieve to some extent one or more of the symptoms of the ocular disease or disorder being treated. An "effective amount", "therapeutically effective amount" or "pharmaceutically effective amount" of a pharmaceutical composition may be administered to a subject in need thereof as a unit dose (as described in further detail elsewhere herein).

[0050] The term "pharmaceutically acceptable" as usedherein, can refer to amaterial, suchasa carrier or diluent,which doesnot abrogate thebiological activity or properties of a compounddisclosedherein, and is relatively nontoxic (i.e., when the material is administered to an individual it does not cause undesirable biological effects nor does it interact in a deleterious manner with any of the components of the composition in which it is contained).

[0051] The term "pharmaceutical composition," or simply "composition" as used herein, can refer to a biologically active compound, optionally mixed with at least one pharmaceutically acceptable chemical component, such as, though not limited to carriers, stabilizers, diluents, dispersing agents, suspending agents, thickening agents, excipients and the like.

[0052] An "AAV vector" or "rAAV vector" as used herein refers to an adeno-associated virus (AAV) vector or a recombinant AAV (rAAV) vector comprising a polynucleotide sequence not of AAV origin (e.g., a polynucleotide hetero- 14 EP 4 696 318 A1 5 10 15 20 25 30 35 40 45 50 55 logous to AAV such as a nucleic acid sequence that encodes a therapeutic transgene, e.g., aflibercept) for transduction intoa target cell or to a target tissue. Ingeneral, theheterologouspolynucleotide is flankedbyat least one, andgenerally by two, AAV inverted terminal repeat sequences (ITRs). The term rAAV vector encompasses both rAAV vector particles and rAAV vector plasmids. A rAAV vector may be either single-stranded (ssAAV) or self-complementary (scAAV).

[0053] An"AAVvirus"or "AAVviral particle"or "rAAVvectorparticle" or "rAAVparticle" refers toaviral particle comprising at least one AAV capsid protein and a polynucleotide rAAV vector. In some cases, the at least one AAV capsid protein is fromawild typeAAVor is a variantAAVcapsidprotein (e.g., anAAVcapsidproteinwithan insertion, e.g., an insertionof the 7m8 amino sequence as set forth below). If the particle comprises a heterologous polynucleotide (e.g., a polynucleotide other than a wild-type AAV genome such as a transgene to be delivered to a target cell or target tissue), it is referred to as a "rAAV particle", "rAAV vector particle" or a "rAAV vector". Thus, production of rAAV particles necessarily includes production of a rAAV vector, as such a vector contained within a rAAV particle.

[0054] The term "packaging" as used herein can refer to a series of intracellular events that can result in the assembly and encapsidation of a rAAV particle.

[0055] AAV "rep" and "cap" genes refer to polynucleotide sequencesencoding replicationandencapsidation proteins of adeno-associated virus. AAV rep and cap are referred to herein as AAV "packaging genes."

[0056] The term "polypeptide" can encompass both naturally occurring and non-naturally occurring proteins (e.g., a fusionprotein), peptides, fragments,mutants, derivativesandanalogs thereof. Apolypeptidemaybemonomeric, dimeric, trimeric, or polymeric. Further, a polypeptide may comprise a number of different domains, each of which has one or more distinct activities. For the avoidance of doubt, a "polypeptide" may be any length greater two amino acids.

[0057] 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 asanative orwild typeprotein. Avariantmayhaveoneormoreaminoacid point substitutions, inwhich a single aminoacid at a position hasbeenchanged to another aminoacid, oneormore insertionsand / or deletions, inwhich oneormoreamino acids are inserted or deleted, respectively, in the sequence of the reference protein, and / or truncations of the amino acid sequence at either or both the amino or carboxy termini. A variant can have the same or a different biological activity compared to the reference protein, or the unmodified protein.

[0058] In some embodiments, a variant can have, for example, at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% overall sequence homology to its counterpart reference protein. In some embodiments, a variant can have at least about 90% overall sequence homology to the wild-type protein. In some embodiments, a variant exhibits at least about 95%, at least about 98%, at least about 99%, at least about 99.5%, or at least about 99.9% overall sequence identity.

[0059] Asused herein, "recombinant" can refer to a biomolecule, e.g., a geneor protein, that (1) has been removed from itsnaturallyoccurringenvironment, (2) isnotassociatedwithall or aportionofapolynucleotide inwhich thegene is found in nature, (3) isoperatively linked toapolynucleotidewhich it isnot linked to innature, or (4)doesnot occur innature.The term "recombinant" can be used in reference to cloned DNA isolates, chemically synthesized polynucleotide analogs, or polynucleotide analogs that are biologically synthesized by heterologous systems, as well as proteins and / or mRNAs encodedby suchnucleic acids. Thus, for example, a protein synthesized byamicroorganism is recombinant, for example, if it is synthesized from an mRNA synthesized from a recombinant gene present in the cell.

[0060] The term "anti-VEGF agent" includes any therapeutic agent, including proteins, polypeptides, peptides, fusion protein, multimeric proteins, gene products, antibody, human monoclonal antibody, antibody fragment, aptamer, small molecule, kinase inhibitor, receptor or receptor fragment, or nucleic acid molecule, that can reduce, interfere with, disrupt, blockand / or inhibit theactivity or functionof anendogenousVEGFand / oranendogenousVEGF receptor (VEGFR), or the VEGF-VEGFR interactionorpathway in vivo.Ananti-VEGFagentcanbeanyoneof theknown therapeuticagents that can reduce new blood vessel growth or formation and / or oedem, or swelling, when delivered into a cell, tissue, or a subject in vivo, e.g., ranibizumab, brolucizumab, or bevacizumab. In some embodiments, an anti-VEGF agent can be naturally occurring, non-naturally occurring, or synthetic. In some embodiments, an anti-VEGF agent can be derived from a naturally occurring molecule that was subsequently modified or mutated to confer an anti‑ VEGF activity. In some embodiments, an anti-VEGFagent is a fusion or chimeric protein. In suchproteins, functional domains or polypeptides are artificially fused to a moiety or a polypeptide to make a fusion or chimeric protein that can sequester VEGF in vivo or function as a VEGFR decoy. In some embodiments, an anti-VEGF agent is a fusion or chimeric protein that blocks endogenous VEGFR from interacting with its ligands.

[0061] Asusedherein, "VEGF" can refer to any isoformofVEGF, unless required otherwise, including, but not limited to, VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E, VEGF-F, or any combination, or any functional fragment or variant thereof. Unless required otherwise, "VEGF" can refer to any member of the VEGF family, including members: VEGF-A, placenta growth factor (PGF), VEGF-B, VEGF-C, and VEGF-D, or any combination, functional fragment, or variant thereof. As used herein, "VEGF receptor" or "VEGFR" or "VEGF-R" can be used to refer to any one of the receptors of VEGF, including, but not limited to, VEGFR‑1 (or Flt‑1), VEGFR‑2 (or Flk‑1 / KDR), and VEGFR‑3 (or Flt‑4). VEGFR can be 15 EP 4 696 318 A1 5 10 15 20 25 30 35 40 45 50 55 a membrane bound or soluble form, or a functional fragment or truncation of a receptor. Examples of anti-VEGF agents include, but are not limited to, ranibizumab, bevacizumab, brolucizumab, or any combination, variant, or functional fragment thereof.

[0062] "Operatively linked" or "operably linked" or "coupled" can refer to a juxtaposition of genetic elements,wherein the elements are in a relationship permitting them to operate in an expected manner. For instance, a promoter can be operatively linked to a coding region if the promoter helps initiate transcription of the coding sequence. There may be intervening residues between the promoter and coding region so long as this functional relationship is maintained.

[0063] The term "expression vector" or "expression construct" or "cassette" or "plasmid" or simply "vector" can include any type of genetic construct, including AAVor rAAV vectors, containing a nucleic acid or polynucleotide coding for a gene product in which part or all of the nucleic acid encoding sequence is capable of being transcribed and is adapted for gene therapy. The transcript can be translated into a protein. In some embodiments, the transcript is partially translated or not translated. In certain aspects, expression includes both transcription of a gene and translation of mRNA into a gene product. In other aspects, expression only includes transcription of the nucleic acid encoding genes of interest. An expression vector can also comprise control elements operatively linked to the encoding region to facilitate expression of the protein in target cells. The combination of control elements and a gene or genes to which they are operably linked for expression can sometimes be referred to as an "expression cassette," a large number of which are known and available in the art or can be readily constructed from components that are available in the art.

[0064] The term "heterologous" can refer to an entity that is genotypically distinct from that of the rest of the entity to which it is being compared. For example, a polynucleotide introduced by genetic engineering techniques into a plasmid or vector derived from a different species can be a heterologous polynucleotide. A promoter removed from its native coding sequence and operatively linked to a coding sequence with which it is not naturally found linked can be a heterologous promoter.

[0065] As used herein, "7m8" refers to the amino acid sequence LALGETTRPA (SEQ ID NO: 1).

[0066] "7m8 variant" refers to a rAAV, which can be of any serotype, with the amino acid sequence LALGETTRPA (SEQ ID NO: 1) inserted in the solvent exposed GH loop of the capsid protein.

[0067] When 7m8 is inserted in a rAAV2 (also referred to as AAV2.7m8), the amino acid sequence LALGETTRPA (SEQ ID NO: 1) is inserted into the GH loop within amino acids 570‑611 of the AAV2 capsid protein, e.g., between positions 587 and 588of theAAV2 capsid protein, VP1. In some cases, when 7m8 is inserted in a rAAV2 (also referred to as AAV2.7m8), the amino acid sequence LALGETTRPA (SEQ ID NO: 1) is inserted into the GH loop of the AAV2 capsid protein, e.g., betweenpositions587and588ofAAV2VP1comprising the sequenceofSEQ IDNO:13.When7m8 is inserted in a rAAV1 (also referred to asAAV1.7m8), theaminoacid sequenceLALGETTRPA (SEQ IDNO:1) is inserted into theGH loopwithin aminoacids571‑612of theAAV1capsidprotein, e.g., betweenaminoacids590and591of theAAV1capsidprotein.When 7m8 is inserted in a rAAV5 (also referred to as AAV5.7m8), the amino acid sequence LALGETTRPA (SEQ ID NO: 1) is inserted into theGH loopwithin aminoacids560‑601of theAAV5capsidprotein, e.g., betweenaminoacids575and576of the AAV5 capsid protein. When 7m8 is inserted in a rAAV6 (also referred to as AAV6.7m8), the amino acid sequence LALGETTRPA (SEQ ID NO: 1) is inserted into the GH loop within amino acids 571 to 612 of the AAV6 capsid protein, e.g., between amino acids 590 and 591 of the AAV6 capsid protein. When 7m8 is inserted in a rAAV7 (also referred to as AAV7.7m8), theaminoacid sequenceLALGETTRPA (SEQ IDNO:1) is inserted into theGH loopwithin aminoacids572 to 613 of the AAV7 capsid protein, e.g., between amino acids 589 and 590 of the AAV7 capsid protein. When 7m8 is inserted in a rAAV8 (also referred to asAAV8.7m8), the aminoacid sequenceLALGETTRPA (SEQ IDNO: 1) is inserted into theGH loop within amino acids 573 to 614 of the AAV8 capsid protein, e.g., between amino acids 590 and 591 of the AAV8 capsid protein. When 7m8 is inserted in a rAAV9 (also referred to as AAV9.7m8), the amino acid sequence LALGETTRPA (SEQ ID NO: 1) is inserted into the GH loop of the AAV9 capsid protein, e.g., between amino acids 588 and 589 of the AAV9 capsid protein. When 7m8 is inserted in a rAAV10 (also referred to as AAV10.7m8), the amino acid sequence LALGETTRPA (SEQ ID NO: 1) is inserted into the GH loop within amino acids 573 to 614 of the AAV10 capsid protein, e.g., between amino acids 589 and 590 of the AAV10 capsid protein. Overview

[0068] Current therapies (e.g., aflibercept recombinant protein, ranibizumab recombinant protein) for ocular neovas- cular diseases such as wAMD require lifelong IVTadministration approximately every 4‑8 weeks. This can increase the risk of inflammation, infection, and other adverse effects in some patients. Further, current therapies create compliance challenges due to repeated and / or frequent trips to medical offices for administration of the therapy, especially in elderly patients, who are most affected with wAMD. Reduction in frequency of administration is associated with vision loss and deterioration of the eye disease or condition. The ability of AAV vectors to efficiently transduce target retinal cells following IVTinjectionhasbeenexploited tosuccessfully transfer therapeuticgenes intophotoreceptors, retinal pigmentepithelium, and the inner retina to treat a variety of retinal diseases. Thus, administration of rAAV particles encoding an anti-VEGF agent (e.g., aflibercept) can provide prolonged and / or sustained release of the anti-VEGF agent in vivo. 16 EP 4 696 318 A1 5 10 15 20 25 30 35 40 45 50 55

[0069] Surprisingly, administration of a single low unit dose of 6 × 1011 vector genomes (vg) per eye of rAAV particles encoding aflibercept to the eyes of individuals with an ocular neovascular disease led to stabilization of the disease and a robust anatomical response in all treated individuals (SeeExample 1). In addition, visual acuity was stabilized in all treated individuals and none of the individuals required rescue anti-VEGF treatment (e.g., aflibercept IVT injections) after administration of the single low unit dose of 6 × 1011 vg / eye of rAAV particles encoding aflibercept. Moreover, administration of the single unit dose of rAAV particles encoding aflibercept to the eyes of individuals with an ocular neovascular diseaseunexpectedly causeda reduction (e.g., resolution) of symptoms, including intraretinal and subretinal fluid that were refractory to prior anti-VEGF treatments (e.g., chronic IVT injections of aflibercept, ranibizumab, or bevacizumab).

[0070] Accordingly, the present disclosure provides methods of treating an ocular neovascular disease in an individual by administering a single unit dose of 6 × 1011 vg / eye or less of rAAV particles encoding an anti-VEGF agent (e.g., aflibercept). In addition, thepresent disclosureprovidesmethods for reducing retinal fluid in the eyeof an individualwithan 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 non-compliance and non-adherence problem. In addition, the methods provided herein reduce the adverse effects associated with multiple IVT injections. Methods of Treatment

[0071] Provided herein is a method for treating an ocular neovascular disease in an individual, the method comprising administering a unit dose of recombinant adeno-associated virus (rAAV) particles to an eye of the individual.

[0072] 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 an eye of the individual.

[0073] Also provided herein is a method for treating an ocular neovascular disease in an individual, the method comprisingadministeringananti-VEGFagent (e.g., aflibercept) toaneyeof the individual, andadministeringaunit doseof recombinant adeno-associated virus (rAAV) particles to the eye of the individual after administration of the anti-VEGF agent.

[0074] In someembodiments, the ocular neovascular disease iswet age-relatedmacular degeneration (wAMD), retinal neovascularization, choroidal neovascularization diabetic retinopathy, proliferative diabetic retinopathy, retinal vein occlusion, central retinal vein occlusion, branched retinal vein occlusion, diabetic macular edema, diabetic retinal ischemia, ischemic retinopathy, diabetic retinal edema, or any combination thereof.

[0075] In some embodiments, the term ocular neovascular disease also encompasses VEGF-driven pre-neovascular diseaseswhich, if left untreated, progress to aneovascular form. In someembodiments, theocular neovascular disease is the pre-neovascular disease, non-proliferative diabetic retinopathy.

[0076] In someembodiments, the individual is a human. In someembodiments, the individual receivedat least oneprior treatment (e.g., at least one, at least two, at least three, at least four, at least 5 or more treatments) for the ocular neovascular disease with an anti-VEGF agent (e.g., bevacizumab, brolucizumab, ranibizumab, faricimab, abicipar pegol, conbercept, OPT‑302, KSI‑301, injectable sunitinib maleate (GB‑102), PAN‑90806 (PanOptica), and / or aflibercept) in about the last 8 weeks, about the last 9 weeks, about the last 10 weeks, about the last 11 weeks, about the last 12 weeks, about the last 13weeks, about the last 14weeks, about the last 15weeks, or about the last 16weeksprior to administration of the unit dose of rAAV particles. In some embodiments, the individual demonstrated a meaningful response to a prior treatment with anti-VEGF agent (e.g., bevacizumab, brolucizumab, ranibizumab, faricimab, abicipar pegol, conbercept, OPT‑302, KSI‑301, injectable sunitinib maleate (GB‑102), PAN‑90806 (PanOptica), and / or aflibercept). In some embodi- ments, the anti-VEGF agent is aflibercept, a functional variant thereof, or a functional fragment thereof. In some embodiments, the anti-VEGF agent comprises a polypeptide comprising an amino acid sequence with at least about 95% identity to the amino acid sequence of SEQ ID NO: 35. In some embodiments, the retinal fluid in the eye of an individual is intraretinal fluid (IRF) and / or subretinal fluid (SRF). In some embodiments, the amount or presence of retinal fluid in the eye of the individual is refractory to prior treatment with an anti-VEGF agent (e.g., bevacizumab, brolucizumab, ranibizumab, faricimab, abicipar pegol, conbercept, OPT‑302, KSI‑301, injectable sunitinib maleate (GB‑102), PAN‑90806 (PanOptica), and / or aflibercept). In some embodiments, the anti-VEGF agent is aflibercept, a functional variant thereof, or a functional fragment thereof. In some embodiments, the anti-VEGF agent comprises a polypeptide comprising an amino acid sequence with at least about 95% identity to the amino acid sequence of SEQ ID NO: 35.

[0077] In some embodiments, the ocular neovascular disease is diabetic macular edema (DME). In some embodi- ments, the individual is a human. In some embodiments, the individual has type 1 or type 2 diabetes mellitus. In some embodiments, the individual has vision impairment that is due to center involving diabetic macular edema. In some embodiments, the individual has visual acuity (BCVA) of between about 78 to 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 the eye administered the rAAV particles prior to administration of the unit dose of rAAV particles. In some embodiments, the 17 EP 4 696 318 A1 5 10 15 20 25 30 35 40 45 50 55 individual has visual acuity (Snellen equivalent) of between about 20 / 32 to about 20 / 100 in the eye administered the rAAV particles prior to administration of the unit dose of rAAV particles. In some embodiments, the individual has a central subfield thickness (CST) of ≥ 325µm using Heidelberg Spectralis® with center-involving IRF (center 1 mm) in the eye administered the rAAV particles prior to administration of the unit dose of rAAV particles. In some embodiments, the individual hasadecrease in vision in theeyeadministered the rAAVparticlesprior toadministrationof theunit doseof rAAV particles that is primarily due todiabeticmacular edema. Insomeembodiments, the individualwasdiagnosedwithdiabetic macular edema in the eye administered the rAAV particles about 6 months or less prior to administration of the unit dose of rAAV particles, e.g., any of about 6 months, about 5 months, about 4 months, about 3 months, about 2 months, about 1 month, or less, prior to administration of the unit dose of rAAVparticles. In someembodiments, the individual received 0, 1, or 2 prior treatments for DME in the eye administered the rAAV particles, e.g., 0, 1, or 2 intravitreal injections with an anti- VEGF agent, e.g., aflibercept, prior to administration of the unit dose of rAAV particles. In some embodiments, the individual received the prior treatments with an anti-VEGF agent in the eye administered the rAAV particles at least about 60days (i.e.,about2months)prior toadministrationof theunit doseof rAAVparticles. In someembodiments, the individual exhibited a meaningful response in central subfield thickness to the prior treatments with an anti-VEGF agent in the eye administered the rAAV particles prior to administration of the unit dose of rAAV particles, for example, at least a 10% reduction in central subfield thickness. In some embodiments, the individual did not experience an adverse reaction to the prior treatments with an anti-VEGF agent prior to administration of the unit dose of rAAV particles. In some embodiments, the individual does not have neutralizing antibodies to AAV2.7m8 prior to administration of the unit dose of rAAV particles. In some embodiments, the individual does not have an anti-AAV2.7m8 neutralizing antibody titer of greater than 1:125 prior to administration of the unit dose of rAAV particles, e.g., within about 6 months prior to administration of the unit dose of rAAV particles. In some embodiments, the individual does not have a history of allergy to aflibercept, corticosteroids, or fluorescein dye or sodium fluorescein (e.g., used in angiography) prior to administration of the unit dose of rAAV particles. In someembodiments, the individual hasahistoryofmild allergy to aflibercept, corticosteroid, or fluorescein dyeor sodium fluorescein (e.g., used in angiography) prior to administration of the unit dose of rAAV particles, wherein the allergy is amenable to treatment. In some embodiments, the individual does not have uncontrolled diabetes, e.g., HbA1C of greater than 10%, prior to administration of the unit dose of rAAV particles. In some embodiments, the individual does not have a history of diabetic ketoacidosis within about 3 months prior to administration of the unit dose of rAAV particles. In some embodiments, the individual has not initiated intensive insulin treatment, e.g., with an insulin pump or multiple daily insulin injections, prior to administration of the unit dose of rAAV particles. In some embodiments, the individual does not plan to initiate intensive insulin treatment, e.g., with an insulin pump or multiple daily insulin injections, within about 3 months after administration of the unit dose of rAAV particles. In some embodiments, the individual does not have a history of systemic autoimmunedisease that requires treatmentwith systemic steroidsor immunosuppressive treatments, e.g.,methotrexate or adalimumab, prior to administration of the unit dose of rAAV particles. In some embodiments, the individual is not being administered a systemic drug known to cause macular edema, such as fingolimod, tamoxifen, chloroquine, or hydro- xychloroquine, prior to administration of the unit dose of rAAV particles. In some embodiments, the individual is not being administered a systemic anti-VEGF treatment prior to administration of the unit dose of rAAV particles. In some embodiments, the individual does not have high-risk proliferative diabetic retinopathy (PDR) in the eye administered the rAAV particles prior to administration of the unit dose of rAAV particles. In some embodiments, PDR is defined as any vitreous or preretinal hemorrhage, neovascularization elsewhere >1 / 2-disc area within an area equivalent to standard ETDRS 7-field on clinical examination, or neovascularization of disc > 1 / 3-disc area on clinical examination. In some embodiments, the individual does not have focal or grid laser photocoagulation in the eye administered the rAAV particles prior to administration of the unit dose of rAAV particles. In some embodiments, the individual does not have any prior pan retinal photocoagulation (PRP) in the eye administered the rAAV particles prior to administration of the unit dose of rAAV particles. In some embodiments, the individual has not received an anti-VEGF therapy (e.g., aflibercept IVT injections) in theeyeadministered the rAAVparticlesprior toadministrationof theunit doseof rAAVparticles. In someembodiments, the individual has not received an anti-VEGF therapy (e.g., aflibercept IVT injections) in the eye administered the rAAV particles for at least 60 days prior to administration of the unit dose of rAAV particles. In some embodiments, the individual has not received more than two anti-VEGF treatments (e.g., aflibercept IVT injections) in the eye administered the rAAV particles prior to administration of the unit dose of rAAV particles. In some embodiments, the individual does not have a history of any of anterior segment neovascularization (e.g., neovascularization of the iris [NVI] or neovascular glaucoma [NVG]), significant vitreous hemorrhage, fibrovascular proliferation, or tractional retinal detachment in the eye adminis- tered the rAAVparticlesprior toadministrationof theunit doseof rAAVparticles. In someembodiments, the individual does not have structural abnormalities at the fovea (e.g., any of dense hard exudates, pigment abnormalities, foveal atrophy, vitreomacular traction or epiretinal membrane) in the eye administered the rAAV particles that contribute to macular edema or visual impairment prior to administration of the unit dose of rAAV particles. In some embodiments, structural abnormalities at the fovea are assessed on clinical examination or OCT. In some embodiments, the individual does not have a history of retinal disease other than diabetic retinopathy (e.g., age-related macular degeneration (in either eye), retinal vein occlusion, retinal arterial occlusion, or pathologic myopia) in the eye administered the rAAV particles prior to 18 EP 4 696 318 A1 5 10 15 20 25 30 35 40 45 50 55 administration of the unit dose of rAAV particles. In some embodiments, the individual does not have history of ocular disease other than diabetic macular edema in the eye administered the rAAV particles, e.g., a significant cataract or macular traction, or evidence of posterior subcapsular cataract, prior to administration of the unit dose of rAAV particles. In some embodiments, the individual does not have history of cataract extraction or Yttrium Aluminum Garnet (YAG) capsulotomy in the eye administered the rAAV particles within at least about 3 months prior to administration of the unit dose of rAAV particles. In some embodiments, the individual does not have a history of retinal detachment (with or without repair) in the eye administered the rAAV particles prior to administration of the unit dose of rAAV particles. In some embodiments, the individual does not have a history of any of trabeculectomy, glaucoma shunt, or minimally invasive glaucoma surgery (MIGS) in the eye administered the rAAV particles prior to administration of the unit dose of rAAV particles. In some embodiments, the individual does not have a history of vitrectomy or other filtration surgery in the eye administered the rAAV particles prior to administration of the unit dose of rAAV particles. In some embodiments, the individual does not have aphakia or presence of an anterior chamber intraocular lens in the eye administered the rAAV particles prior to administration of the unit dose of rAAV particles. In some embodiments, the individual does not have uncontrolled ocular hypertension or glaucoma in the eye administered the rAAV particles, e.g., IOP >22 mmHg despite treatment with anti-glaucoma medication or current use of >2 IOP lowering medications, prior to administration of the unit dose of rAAV particles. In some embodiments, the individual does not have a history of intraocular or periocular steroid treatment for any ocular condition (e.g., IVTTriesence, Iluvien or Ozurdex) in the eye administered the rAAVparticles prior to administration of the unit dose of rAAV particles. In some embodiments, the individual has not had refractive surgery in the eye administered the rAAV particles within at least about 90 days prior to administration of the unit dose of rAAV particles. In some embodiments, the individual does not have previous penetrating keratoplasty, endothelial keratoplasty, or ocular radiation in the eye administered the rAAV particles prior to administration of the unit dose of rAAV particles. In some embodiments, the individual has not had any prior vitreoretinal surgery in the eye administered the rAAV particles prior to administration of the unit dose of rAAV particles. In some embodiments, the individual does not have a history of uveitis or intraocular inflammation, e.g., grade trace or above except mild anticipated post-operative inflammation that resolved, prior to administration of the unit dose of rAAV particles. In some embodiments, the individual does not have a history of IOP elevation that is related to topical steroid administration prior to administration of the unit dose of rAAV particles. In some embodiments, the individual does not have a history of ocular Herpes Simplex Virus (HSV), Varicella- zoster virus (VZV), or Cytomegalovirus (CMV), including viral uveitis, retinitis or keratitis prior to administration of the unit dose of rAAV particles. In some embodiments, the individual does not have evidence of any of external ocular infection, including conjunctivitis, chalazion, or significant blepharitis prior to administration of the unit dose of rAAV particles. In some embodiments, the individual does not have history of ocular toxoplasmosis prior to administration of the unit dose of rAAV particles.

[0078] In some embodiments, the unit dose is expressed as the number of vector genomes (vg). In some embodiments, the unit dose is about 6 × 1011 vector genomes (vg) or less of the rAAV particles. In some embodiments, the unit dose is expressed as the number of vector genomes (vg) per eye (vg / eye). In some embodiments, the unit dose is about 6 × 1011 vg / eye or less of the rAAV particles. In some embodiments, the unit dose of rAAV particles is about 6 × 1010 to about 2 × 1011 vg / eye. In some embodiments, the unit dose of rAAV particles is about 2 × 1011 or about 6 × 1010 vg / eye.

[0079] 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.

[0080] In someembodiments, theadministering theunit doseof rAAVparticles to thecontralateral eyeof the individual is up to about 2 weeks (e.g., about 0 days, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, or 14 days) after administering the unit dose of rAAV particles to the one eye. In some embodiments, the unit dose of rAAV particles administered to the contralateral eye of the individual is about the same as (e.g., less than 1% higher or lower, less than 5% higher or lower, less than 10% higher or lower, or less than 20% higher or lower) or lower (e.g., about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, or about 90% lower) than the unit dose of rAAV particles administered to the one eye of the individual.

[0081] In someembodiments, the administering theunit doseof rAAVparticles 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 8months, at least about 9months, at least about 10months, at least about 11months, at least about 12months, 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 more) after administering the unit dose of rAAV particles to the one eye. In some embodiments, the unit dose of rAAV particles administered to the contralateral eye of the individual is higher (e.g., any of about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 125%, about 19 EP 4 696 318 A1 5 10 15 20 25 30 35 40 45 50 55 150%, about 175%, about 200%, about 225%, about 250%, about 275%, about 300%, or more, higher) than the unit dose of rAAV particles administered to the one eye of the individual.

[0082] In some embodiments, the rAAV particles comprise a) a nucleic acid encoding a polypeptide comprising an amino acid sequence with at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.99%, or 100% identity to the amino acid sequence of SEQ ID NO: 35 and flanked by AAV2 inverted terminal repeats (ITRs), and b) an AAV2 capsid protein comprising an amino acid sequence LGETTRP (SEQ ID NO: 14) inserted between positions 587 and 588 of the capsid protein, wherein the amino acid residue numbering corresponds to an AAV2 VP1 capsid protein. The sequence of SEQ ID NO: 35 is provided below:

[0083] In some embodiments, the rAAV particles comprise a) a nucleic acid encoding a polypeptide comprising an amino acid sequence with at least about 95% identity to the amino acid sequence of SEQ ID NO: 35 and flanked by AAV2 inverted terminal repeats (ITRs), and b) an AAV2 capsid protein comprising an amino acid sequence LGETTRP (SEQ ID NO: 14) inserted between positions 587 and 588 of the capsid protein, wherein the amino acid residue numbering corresponds to an AAV2 VP1 capsid protein.

[0084] In some embodiments, the rAAV particles comprise a nucleic acid encoding a polypeptide comprising an amino acid sequence with at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.99%, or 100% identity to the amino acid sequence of SEQ ID NO: 35 and flanked by AAV2 inverted terminal repeats (ITRs). In some embodiments, the rAAV particles comprise a nucleic acid encoding a polypeptide comprising an amino acid sequence with at least about 95% identity to the amino acid sequence of SEQ ID NO: 35 and flanked by AAV2 inverted terminal repeats (ITRs). In some embodiments, the polypeptide comprises the amino acid sequence of SEQ ID NO: 35. In some embodiments, the polypeptide is aflibercept or a functional variant thereof or functional fragment thereof.

[0085] In some embodiments, the rAAV particles comprise a nucleic acid comprising a codon-optimized sequence encoding an amino acid sequence with at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.99%, or 100% identity to the amino acid sequence of SEQ ID NO: 35 and flanked by AAV2 inverted terminal repeats (ITRs). In some embodiments, the rAAV particles comprise a nucleic acid comprising a codon-optimized sequence encoding an amino acid sequence with at least about 95% identity to the amino acid sequence of SEQ ID NO: 35 and flanked by AAV2 inverted terminal repeats (ITRs). In some embodiments, the rAAV particles comprise a nucleic acid comprising a codon-optimized sequence encoding an amino acid sequence with 100% identity to the amino acid sequence of SEQ ID NO: 35 and flanked by AAV2 inverted terminal repeats (ITRs).

[0086] In some embodiments, the rAAV particles comprise a nucleic acid comprising the cDNA sequence of aflibercept or a functional variant thereof or functional fragment thereof andflankedbyAAV2 inverted terminal repeats (ITRs). In some embodiments, the rAAV particles comprise a nucleic acid comprising a codon-optimized cDNA sequence of aflibercept or a functional variant thereof or functional fragment thereof and flanked by AAV2 inverted terminal repeats (ITRs). In some embodiments, the rAAV particles comprise a nucleic acid comprising the nucleic acid sequence of SEQ ID NO: 36.

[0087] In some embodiments, the nucleic acid further comprises (a) a first enhancer region comprising a CMV sequence; (b) a promoter region comprising a CMV sequence; (c) a 5’UTR region comprising, in the 5’ to 3’ order, a TPL sequence and an eMLP sequence; (d) a second enhancer region comprising a full EES sequence; and (e) a HGH polyadenylation site. In someembodiments, theenhancer regioncomprisingaCMVsequencecomprises thesequenceof SEQ IDNO:22. In someembodiments, thepromoter region comprisingaCMVsequencecomprises the sequenceofSEQ 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 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.

[0088] In someembodiments, the rAAVparticles compriseanAAV2capsid protein comprising theaminoacid sequence LGETTRP (SEQ IDNO: 14) inserted between positions 587and588of theAAV2VP1comprising the sequenceof SEQ ID 20 EP 4 696 318 A1 5 10 15 20 25 30 35 40 45 50 55 NO: 13. The sequence of SEQ ID NO: 13 is provided below:

[0089] In someembodiments, the rAAVparticles compriseanAAV2capsid protein comprising theaminoacid sequence LALGETTRPA (SEQ ID NO: 1) inserted between positions 587 and 588 of the capsid protein, wherein the amino acid residue numbering corresponds to an AAV2 VP1 capsid protein. In some embodiments, the rAAV particles comprise an AAV2 capsid protein comprising the amino acid sequence LALGETTRPA (SEQ ID NO: 1) inserted between positions 587 and 588 of the AAV2 VP1 comprising the sequence of SEQ ID NO: 13.

[0090] In some embodiments, the rAAV particles comprise an AAV2 capsid protein comprising any of the following amino acid sequences inserted between positions 587 and 588 of the capsid protein, wherein the amino acid residue numbering corresponds to an AAV2 VP1 capsid protein: LALGETTRPA (SEQ ID NO: 1); LANETITRPA (SEQ ID NO: 2), LAKAGQANNA (SEQ ID NO: 3), LAKDPKTTNA (SEQ ID NO: 4), KDTDTTR (SEQ ID NO: 5), RAGGSVG (SEQ IDNO: 6), AVDTTKF (SEQ IDNO: 7), STGKVPN (SEQ IDNO: 8), LAKDTDTTRA (SEQ IDNO: 9), LARAGGSVGA (SEQ IDNO: 10), LAAVDTTKFA (SEQ ID NO: 11), LASTGKVPNA (SEQ ID NO: 12), LGETTRP (SEQ ID NO: 14), NETITRP (SEQ ID NO: 15), KAGQANN(SEQ IDNO:16), KDPKTTN (SEQ IDNO:17), KDTDTTR (SEQ IDNO:18),RAGGSVG(SEQ IDNO:19), AVDTTKF (SEQ ID NO: 20), and STGKVPN (SEQ ID NO: 21). In some embodiments, the rAAV particles comprise an AAV2 capsid protein comprising any of the following amino acid sequences inserted between positions 587 and 588 of the AAV2 VP1 comprising the sequence of SEQ ID NO: 13: LALGETTRPA (SEQ ID NO: 1); LANETITRPA (SEQ ID NO: 2), LAKAGQANNA (SEQ ID NO: 3), LAKDPKTTNA (SEQ ID NO: 4), KDTDTTR (SEQ ID NO: 5), RAGGSVG (SEQ IDNO: 6), AVDTTKF (SEQ IDNO: 7), STGKVPN (SEQ IDNO: 8), LAKDTDTTRA (SEQ IDNO: 9), LARAGGSVGA (SEQ IDNO: 10), LAAVDTTKFA (SEQ ID NO: 11), LASTGKVPNA (SEQ ID NO: 12), LGETTRP (SEQ ID NO: 14), NETITRP (SEQ ID NO: 15), KAGQANN(SEQ IDNO:16), KDPKTTN (SEQ IDNO:17), KDTDTTR (SEQ IDNO:18),RAGGSVG(SEQ IDNO:19), AVDTTKF (SEQ ID NO: 20), and STGKVPN (SEQ ID NO: 21).

[0091] In some embodiments, the administration of the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual is by intravitreal (IVT) injection, intraocular administration, or intraretinal injection. In some embodiments, the administration of the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual is by intravitreal (IVT) injection.

[0092] In some embodiments, the unit dose of rAAV particles is in a pharmaceutical formulation. In some embodiments, the pharmaceutical formulation comprises the rAAV particles, one or more osmotic or ionic strength agents, one or more buffering agents, one or more surfactants, and one or more solvents. In some embodiments, the osmotic or ionic strength agent is sodium chloride. In some embodiments, the one or more buffering agents are sodium phosphate monobasic and / or sodium phosphate dibasic. In some embodiments, the surfactant is Poloxamer 188. In some embodiments, the solvent is water. In some embodiments, the pharmaceutical formulation comprises the rAAV particles, sodium chloride, sodium phosphate and a surfactant. In some embodiments, the pharmaceutical formulation comprises about 1×1010 vg / mL to about 1×1013 vg / mL of rAAV particles. In some embodiments, the pharmaceutical formulation comprises about 6×1011vg / mL toabout 6×1012vg / mLof rAAVparticles. In someembodiments, thepharmaceutical formulation comprises about 150mMtoabout 200mMsodiumchloride (e.g., anyof about 150mM,about 160mM,about 170mM,about 180mM, about 190 mM, or about 200 mM). In some embodiments, the pharmaceutical formulation comprises about 1 mM to about 21 EP 4 696 318 A1 5 10 15 20 25 30 35 40 45 50 55 10 mM monobasic sodium phosphate (e.g., about 1mM, about 2 mM, about 3 mM, about 4 mM, about 5 mM, about 6 mM, about 7mM,about 8mM,about 9mM,or about 10mM). In someembodiments, the pharmaceutical formulation comprises about 1 mM to about 10 mM dibasic sodium phosphate (e.g., about 1mM, about 2 mM, about 3 mM, about 4 mM, about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, or about 10 mM). In some embodiments, the pharmaceutical formulation comprises about 0.0005% (w / v) to about 0.005% (w / v) poloxamer 188 (e.g., any of about 0.0005% (w / v), 0.0006%(w / v) , 0.0007%(w / v) , 0.0008%(w / v), 0.0009%(w / v), 0.001%(w / v) , 0.002%(w / v) , 0.003%(w / v) , 0.004%(w / v), or about 0.005%(w / v)). In someembodiments, thepharmaceutical formulationhasapHofabout 7.0 toabout 7.5 (e.g., any of about 7.0, about 7.1, about 7.2, about 7.3, about 7.4, or about 7.5). In some embodiments, the pharmaceutical formulation comprises about 6×1012 vg / mL of rAAV particles, about 180 mM sodium chloride, about 5 mM monobasic sodium phosphate, about 5 mM dibasic sodium phosphate, and about 0.001% (w / v) poloxamer 188, wherein the pharmaceutical formulation has a pH of about 7.3. In some embodiments, the pharmaceutical formulation comprises about 6×1011 vg / mLof rAAVparticles, about 180mMsodiumchloride, about 5mMmonobasic sodiumphosphate, about 5 mMdibasic sodiumphosphate, and about 0.001% (w / v) poloxamer 188,wherein the pharmaceutical formulation has apH of about 7.3.

[0093] In some embodiments, the unit dose of rAAV particles comprises a volume of between about 25 µL to about 250 µL (e.g., any of about 25 µL, about 30 µL, about 40 µL, about 50 µL, about 60 µL, about 70 µL, about 80 µL, about 90 µL, about 100µL, about 110µL, about 120 µL, about 130 µL, about 140 µL, about 150 µL, about 160µL, about 170µL, 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, theconcentrationof rAAVparticles in thepharmaceutical formulation isadjustedsuch that thevolumeof the unit dose of rAAV particles administered to an eye of the individual is between about 25 µL to about 250 µL. In some embodiments, the unit dose of rAAVparticles comprises a volumeof about 100µL. In someembodiments, the unit dose of rAAV particles comprises a volume of about 30 µL.

[0094] In some embodiments, the unit dose of rAAV particles is administered in combination with steroid treatment. In some embodiments, the steroid treatment is a corticosteroid treatment. In some embodiments, the steroid treatment is a systemic steroid treatment. In some embodiments, the steroid treatment is an oral steroid treatment. In some embodi- ments, the steroid treatment is a prednisone treatment. In some embodiments, the steroid treatment is an ophthalmic steroid treatment. In some embodiments, the ophthalmic steroid treatment is a topical steroid treatment (e.g., a drop), a periocular steroid treatment (e.g., subtenons, subconjunctival), an intravitreal steroid treatment, or a superchoroidal steroid treatment. In some embodiments, the topical steroid treatment is a difluprednate treatment, a medrysone treatment, a loteprednol treatment, a prednisolone treatment, a fluocinolone treatment, a triamcinolone treatment, a rimexolone treatment, a dexamethasone treatment, a fluorometholone treatment, a fluocinolone treatment, a rimexolone treatment, or a prednisone treatment. In some embodiments, the topical steroid treatment is a difluprednate treatment. In some embodiments, 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.

[0095] In some embodiments, the steroid treatment is an ophthalmic steroid treatment (e.g., difluprednate). In some embodiments, the ophthalmic steroid treatment (e.g., difluprednate) is a daily steroid treatment for up to about 4 weeks, about 6weeks, or about 8weeks fromadministering theunit doseof rAAVparticles. In someembodiments, theophthalmic steroid treatment comprisesabout four administrationsof ophthalmic steroidonaboutweek1, about threeadministrations of ophthalmic steroid on about week 2, about two administrations of ophthalmic steroid on about week 3, and about one administration of ophthalmic steroid on about week 4; timing starting with and following administration of the unit dose of rAAV particles. In some embodiments, the ophthalmic steroid is about 0.005% to about 0.5% difluprednate. In some embodiments, theophthalmic steroid is anyof about 0.005%,about 0.006%,about 0.007%,about 0.008%,about 0.009%, about 0.01%, about 0.02%, about 0.03%, about 0.4%, about 0.05%, about 0.06%, about 0.07%, about 0.08%, about 0.09%, or about 0.1% difluprednate. In some embodiments, the ophthalmic steroid is difluprednate 0.05%. In some embodiments, a dose of difluprednate 0.05% is one drop of ophthalmic solution. In some embodiments, one drop is about 50 µl (e.g., about 25 µl to about 50 µl, about 50 µl to about 100 µl). In some embodiments, a dose of difluprednate comprises about 1 µg to about 5 µg, or about 2 µg to about 3 µg, or about 2.5 µg difluprednate. In some embodiments, a dose of difluprednate comprises about 2.5 µg difluprednate.

[0096] In some embodiments, the steroid treatment is an ophthalmic steroid treatment (e.g., difluprednate). In some embodiments, the ophthalmic steroid treatment (e.g., difluprednate) is a daily topical steroid treatment for up to about 4 weeks, about 6 weeks, or about 8 weeks from administering the unit dose of rAAV particles. In some embodiments, the 22 EP 4 696 318 A1 5 10 15 20 25 30 35 40 45 50 55 topical steroid treatment comprises about four administrations of topical steroid on about week 1, about three admin- istrations of topical steroid on about week 2, about two administrations of topical steroid on about week 3, and about one administration of topical steroid on about week 4; timing starting with and following administration of the unit dose of rAAV particles. In some embodiments, the topical steroid treatment comprises about four administrations of topical steroid (i.e., QID)per day for about3weeksafter administrationof theunit doseof rAAVparticles, followedbyabout3administrationsof topical steroid per day (i.e., TID) for about 1 week, followed by about 2 administrations of topical steroid per day (i.e.,BID) for about 1 week, and followed by about 1 administration of topical steroid per day (i.e., QD) for about 1 week. In some embodiments, the topical steroid comprises difluprednate 0.05% at a dose of about 1 µg to about 3 µg. In some embodiments, the topical steroid comprises difluprednate 0.05% at a dose of about 2.5µg. In some embodiments, the topical steroid is about 0.005% to about 0.5% difluprednate. In some embodiments, the topical steroid is any of about 0.005%, about 0.006%, about 0.007%, about 0.008%, about 0.009%, about 0.01%, about 0.02%, about 0.03%, about 0.04%, about 0.05%, about 0.06%, about 0.07%, about 0.08%, about 0.09%, or about 0.1% difluprednate. In some embodiments, the topical steroid is difluprednate 0.05%. In someembodiments, a doseof difluprednate 0.05% is onedrop of ophthalmic solution. In someembodiments, one drop is about 50µl (e.g., about 25µl to about 50µl, about 50µl to about 100µl). In some embodiments, a dose of difluprednate comprises about 1 µg to about 5µg, or about 2µg to about 3µg, or about 2.5 µg difluprednate. In some embodiments, a dose of difluprednate comprises about 2.5 µg difluprednate.

[0097] In some embodiments, the retinal fluid in the eye of the individual (e.g., SRF and / or IRF) is reduced by more than any of about 5%, about 10%, about 15%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100% after administration of the unit dose of rAAV particles to the one eye and / or to the contralateral of the individual. In some embodiments, the retinal fluid in the eye of the individual (e.g., SRF and / or IRF) is reduced by more than any of about 5%, about 10%, about 15%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100% after administration of the unit dose of rAAV particles to the one eye and / or to the contralateral of the individual compared to the level of retinal fluid in the eye of the individual prior to administration of the unit dose or rAAV particles. In some embodiments, the retinal fluid in the eye of the individual (e.g., SRF and / or IRF) is reduced by about 100% after administration of the unit dose of rAAV particles to the one eye and / or to thecontralateral of the individual compared to the level of retinal fluid in theeyeof the individual prior toadministrationof the unit dose or rAAV particles.

[0098] In some embodiments, the methods provided herein further comprise monitoring the level of retinal fluid (e.g., SRF and / or IRF) in the one eye and / or the contralateral eye of the individual after administration of the unit dose of rAAV particles. In someembodiments, the reduction of retinal fluid (e.g., SRFand / or IRF) in theeye is first observedanyof about 1day, about 3days, about 8days, about 2weeks, about 4weeks, about 6weeks, about 8weeks, about 12weeks, about 16 weeks, about 20 weeks, about 24 weeks, about 28 weeks, about 32 weeks, about 36 weeks, about 40 weeks, about 44 weeks, about 48 weeks, about 52 weeks, about 56 weeks, about 60 weeks, about 64 weeks, about 68 weeks, about 72 weeks, about 76 weeks, about 80 weeks, about 84 weeks, about 88 weeks, about 92 weeks, about 96 weeks, about 100 weeks, about 104 weeks, or more after administration of the unit dose of rAAV particles. In some embodiments, the reduction of retinal fluid (e.g., SRF and / or IRF) in the eye continues or is maintained for at least 1 week, at least 2 weeks, at least 4 weeks, at least 6weeks, at least 8 weeks, at least 12 weeks, at least 16weeks, at least 20 weeks, at least 24 weeks, at least 28 weeks, at least 32 weeks, at least 36 weeks, at least 40 weeks, at least 44 weeks, at least 48 weeks, at least 52 weeks, at least 56 weeks, at least 60 weeks, at least 64 weeks, at least 68 weeks, at least 72 weeks, at least 76 weeks, at least 80 weeks, at least 84 weeks, at least 88 weeks, at least 92 weeks, at least 96 weeks, at least 100 weeks, at least 104 weeks, or more after administration of the unit dose of rAAV particles.

[0099] In someembodiments, the reduction of retinal fluid (e.g., SRFand / or IRF) in the eye is determinedbyanymethod known in the art. In some embodiments, the reduction of retinal fluid (e.g., SRF and / or IRF) in the eye is determined by optical coherence tomography (OCT), spectral domain OCT (SD-OCT), OCTangiography, fluorescein angiography, or by direct retinal observation. In some embodiments, the reduction of retinal fluid (e.g., SRF and / or IRF) in the eye is determinedbyoptical coherence tomography (OCT). In someembodiments, the reductionof retinal fluid (e.g., SRFand / or IRF) in the eye is determinedby spectral domainOCT (SD-OCT). In someembodiments, the reduction of retinal fluid (e.g., SRF and / or IRF) in the eye is determined by OCTangiography. In some embodiments, the reduction of retinal fluid (e.g., SRF and / or IRF) in the eye is determined by fluorescein angiography. In some embodiments, the reduction of retinal fluid (e.g., SRF and / or IRF) in the eye is determined by direct retinal observation.

[0100] In someembodiments, treatment of an ocular neovascular disease in an individual after administration of the unit dose of rAAV particles to the one eye and / or the contralateral eye is assessed based on the level of retinal fluid (e.g., intraretinal fluid (IRF) and / or subretinal fluid (SRF)) compared the level of retinal fluid (e.g., SRF and / or IRF) prior to administration of the unit dose of rAAV particles to the one eye and / or the contralateral eye (e.g., as described above). In some embodiments, the retinal fluid is subretinal fluid (SRF) or intraretinal fluid (IRF). In some embodiments, the retinal fluid is subretinal fluid (SRF). In some embodiments, the retinal fluid is intraretinal fluid (IRF). In some embodiments, treatment of an ocular neovascular disease in an individual after administration of the unit dose of rAAVparticles to theone eye and / or the contralateral eye is determined if a reduction in retinal fluid (e.g., IRF and / or SRF) is observed after 23 EP 4 696 318 A1 5 10 15 20 25 30 35 40 45 50 55 administration of the unit doseof rAAVparticles to theoneeyeand / or the contralateral eye compared to the levels of retinal fluid (e.g., IRF and / or SRF) prior to administration of the unit dose of rAAV particles in the one eye and / or the contralateral eye (e.g., as described above). In some embodiments, the ocular neovascular disease is wAMD.

[0101] In some embodiments, the administering the unit dose of rAAVparticles to the one eyeand / or to the contralateral eye of the individual results in maintenance or a decrease of retinal thickness compared to the retinal thickness prior to administration of the unit doseof rAAVparticles. In someembodiments, theadministering theunit doseof rAAVparticles to theoneeyeand / or to the contralateral eyeof the individual results in adecreaseof retinal thickness compared to the retinal thickness prior to administration of the unit dose of rAAV particles. In some embodiments, retinal thickness is central subfield thickness (CST) or central retinal thickness (CRT). In someembodiments, the administering theunit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in a decrease of retinal thickness of more than any of about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or about 100% compared to the retinal thickness prior to administration of the unit dose of rAAV particles. In some embodiments, the retinal thickness (e.g., CST or CRT) is determined by OCT or SD-OCT.

[0102] In some embodiments, the administering the unit dose of rAAVparticles to the one eyeand / or to the contralateral eye of the individual results in a decrease of central retinal thickness (CRT) or central subfield thickness (CST) of about 10 µm to about 100µm (e.g., more than any of about 10µm, about 15µm, about 20µm, about 25µm, about 30µm, about 35 µm, about 40 µm, about 45 µm, about 50 µm, about 55 µm, about 60 µm, about 65 µm, about 70 µm, about 75 µm, about 80µm,about 85µm,about 90µm,about 95µm,about 100µm,ormore). In someembodiments, theadministering theunit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in a decrease of central retinal thickness (CRT) or central subfield thickness (CST) of about 5 µm to about 50 µm (e.g., any of about 5 µm, about 10 µm, about 15µm,about 20µm, about 25µm, about 30µm,about 35µm, about 40µm, about 45µm,or about 50µm). In some embodiments, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in adecreaseof central retinal thickness (CRT)or central subfield thickness (CST) of about 5µmtoabout 40µm(e.g., anyof about 5µm,about 10µm,about 15µm,about 20µm,about 25µm,about 30µm,about 35µm,or about 40 µm). In some embodiments, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in a decrease of central retinal thickness (CRT) or central subfield thickness (CST) of about 5 µm to about 30 µm (e.g., any of about 5 µm, about 6 µm, about 7 µm, about 8 µm, about 9 µm, about 10µm, 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, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in a decrease of central retinal thickness (CRT) or central subfield thickness (CST) of about 5 µm to about 25 µm (e.g., any of about 5 µm, about 6 µm, about 7 µm, about 8 µm, about 9 µm, about 10 µm, about 11 µm, about 12 µm, about 13 µm, about 14 µm, about 15 µm, about 16 µm, about 17 µm, about 18 µm, about 19 µm, about 20 µm, about 21 µm, about 22 µm, about 23 µm, about 24 µm, or about 25 µm). In some embodiments, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in a decrease of central retinal thickness (CRT) or central subfield thickness (CST) of about 5 µm to about 20 µm (e.g., any of about5µm,about6µm,about7µm,about8µm,about9µm,about10µm,about11µm,about12µm,about13µm,about 14 µm, about 15 µm, about 16 µm, about 17 µm, about 18 µm, about 19 µm, or about 20 µm). In some embodiments, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in a decrease of central retinal thickness (CRT) or central subfield thickness (CST) of between about 18µm to about 75µm. In some embodiments, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in a decrease of central retinal thickness (CRT) or central subfield thickness (CST) of about 18.5 µm. In some embodiments, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in a decrease of central retinal thickness (CRT) or central subfield thickness (CST) of about 21.0 µm. In some embodiments, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in a decrease of central retinal thickness (CRT) or central subfield thickness (CST) of about 8.3 µm. In some embodiments, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in a decrease of central retinal thickness (CRT) or central subfield thickness (CST) of about 25.5 µm. In some embodiments, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in a decrease of central retinal thickness (CRT) or central subfield thickness (CST) of about 24.8 µm. In some embodiments, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in a decrease of central retinal thickness (CRT) or central subfield thickness (CST) of about 75 µm. In some embodiments, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in a decrease of central retinal thickness (CRT) or central subfield thickness (CST) of about 100 µm or more (e.g., any of about 100 µm or more, about 110µm or more, about 120 µm or more, about 130 µm or more, about 140 µmormore, about 150µmormore, about 160µmormore, about 170µmormore, about 180µmormore, about 190µmor more, or about 200 µm or more). In some embodiments, the administering the unit dose of rAAV particles to the one eye 24 EP 4 696 318 A1 5 10 15 20 25 30 35 40 45 50 55 and / or to the contralateral eye of the individual results in a decrease of central retinal thickness (CRT) or central subfield thickness (CST) of between about 10 µm to about 200 µm (e.g., any of about 10 µm, about 20 µm, about 30 µm, about 40 µm, about 50 µm, about 60 µm, about 70 µm, about 80 µm, about 90 µm, about 100 µm, about 110 µm, about 120 µm, about 130 µm, about 140 µm, about 150 µm, about 160 µm, about 170 µm, about 180 µm, about 190 µm, or about 200 µm). In someembodiments, the administering the unit dose of rAAVparticles to the oneeyeand / or to the contralateral eye of the individual results in a decrease of central retinal thickness (CRT) or central subfield thickness (CST) of between about 50µm to about 200µm (e.g., any of about 50µm, about 60µm, about 70µm, about 80µm, about 90µm, about 100 µm, about 110 µm, about 120 µm, about 130 µm, about 140 µm, about 150 µm, about 160 µm, about 170 µm, about 180 µm, about 190 µm, or about 200 µm). In some embodiments, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in a decrease of central retinal thickness (CRT) or central subfield thickness (CST) of between about 60 µm to about 200 µm (e.g., any of about 60 µm, about 70 µm, about 80 µm, about 90 µm, about 100 µm, about 110 µm, about 120 µm, about 130 µm, about 140 µm, about 150 µm, about 160 µm, about 170 µm, about 180 µm, about 190 µm, or about 200 µm). In some embodiments, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in a decrease of central retinal thickness (CRT) or central subfield thickness (CST) of between about 70 µm to about 200 µm (e.g., any of about 70 µm, about 80 µm, about 90 µm, about 100 µm, about 110 µm, about 120 µm, about 130 µm, about 140 µm, about 150 µm, about 160 µm, about 170 µm, about 180 µm, about 190 µm, or about 200 µm). In some embodiments, the administering theunit dose of rAAVparticles to theoneeyeand / or to the contralateral eye of the individual results in adecrease of central retinal thickness (CRT) or central subfield thickness (CST) of between about 80µm to about 200µm (e.g., any of about 80 µm, about 90 µm, about 100 µm, about 110 µm, about 120 µm, about 130 µm, about 140 µm, about 150 µm, about 160 µm,about170µm,about 180µm,about 190µm,or about200µm). In someembodiments, theadministering theunit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in a decrease of central retinal thickness (CRT) or central subfield thickness (CST) of between about 90 µm to about 200 µm (e.g., any of about 90 µm, about 100 µm, about 110 µm, about 120 µm, about 130 µm, about 140 µm, about 150 µm, about 160 µm, about 170 µm, about 180µm,about 190µm,or about 200µm). In someembodiments, theadministering theunit doseof rAAVparticles to theoneeyeand / or to thecontralateral eyeof the individual results inadecreaseof central retinal thickness (CRT)or central subfield thickness (CST) of between about 100 µm to about 200 µm (e.g., any of about 100 µm, about 110 µm, about 120 µm,about 130µm,about140µm,about150µm,about 160µm,about170µm,about 180µm,about190µm,orabout200 µm). In someembodiments, the administering the unit dose of rAAVparticles to the oneeyeand / or to the contralateral eye of the individual results in a decrease of central retinal thickness (CRT) or central subfield thickness (CST) of between about 110 µm to about 200 µm (e.g., any of about 110 µm, about 120 µm, about 130 µm, about 140 µm, about 150 µm, about 160 µm, about 170 µm, about 180 µm, about 190 µm, or about 200 µm). In some embodiments, the administering theunit dose of rAAVparticles to theoneeyeand / or to the contralateral eye of the individual results in adecrease of central retinal thickness (CRT) or central subfield thickness (CST) of between about 115 µm to about 200 µm (e.g., any of about 115 µm, about 120 µm, about 125 µm, about 130 µm, about 135 µm, about 140 µm, about 145 µm, about 150 µm, about 155 µm, about 160 µm, about 165 µm, about 170 µm, about 175 µm, about 180 µm, about 185 µm, about 190 µm, about 195µm,orabout 200µm). In someembodiments, theadministering theunit doseof rAAVparticles to theoneeyeand / or to the contralateral eye of the individual results in a decrease of central retinal thickness (CRT) or central subfield thickness (CST) of between about 120µm toabout 200µm (e.g., any of about 120µm, about 125µm,about 130µm, about 135µm, about 140 µm, about 145 µm, about 150 µm, about 155 µm, about 160 µm, about 165 µm, about 170 µm, about 175 µm, about 180 µm, about 185 µm, about 190 µm, about 195 µm, or about 200 µm). In some embodiments, the administering theunit dose of rAAVparticles to theoneeyeand / or to the contralateral eye of the individual results in adecrease of central retinal thickness (CRT) or central subfield thickness (CST) of between about 125 µm to about 200 µm (e.g., any of about 125 µm, about 130 µm, about 135 µm, about 140 µm, about 145 µm, about 150 µm, about 155 µm, about 160 µm, about 165 µm, about 170 µm, about 175 µm, about 180 µm, about 185 µm, about 190 µm, about 195 µm, or about 200 µm). In some embodiments, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in a decrease of central retinal thickness (CRT) or central subfield thickness (CST) of betweenabout 130 µmtoabout200µm(e.g., anyofabout130µm,about135µm,about140µm,about145µm,about150µm,about155µm, about160µm,about165µm,about 170µm,about175µm,about 180µm,about185µm,about190µm,about 195µm,or about 200 µm). In some embodiments, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in a decrease of central retinal thickness (CRT) or central subfield thickness (CST) of between about 135µm toabout 200µm (e.g., any of about 135µm, about 140µm,about 145µm, about 150µm, about 155 µm, about 160 µm, about 165 µm, about 170 µm, about 175 µm, about 180 µm, about 185 µm, about 190 µm, about 195 µm, or about 200 µm). In some embodiments, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in a decrease of central retinal thickness (CRT) or central subfield thickness (CST)ofbetweenabout140µmtoabout200µm(e.g., anyofabout140µm,about145µm,about150µm,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 195µm,orabout 200µm). In someembodiments, theadministering theunit doseof rAAVparticles to theoneeyeand / or to 25 EP 4 696 318 A1 5 10 15 20 25 30 35 40 45 50 55 the contralateral eye of the individual results in a decrease of central retinal thickness (CRT) or central subfield thickness (CST) of between about 145µm toabout 200µm (e.g., any of about 145µm, about 150µm,about 155µm, about 160µm, about 165 µm, about 170 µm, about 175 µm, about 180 µm, about 185 µm, about 190 µm, about 195 µm, or about 200 µm). In someembodiments, the administering the unit dose of rAAVparticles to the oneeyeand / or to the contralateral eye of the individual results in a decrease of central retinal thickness (CRT) or central subfield thickness (CST) of between about 150 µm to about 200 µm (e.g., any of about 150 µm, about 155 µm, about 160 µm, about 165 µm, about 170 µm, about 175 µm, about 180 µm, about 185 µm, about 190 µm, about 195 µm, or about 200 µm). In some embodiments, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in a decrease of central retinal thickness (CRT) or central subfield thickness (CST) of between about 155 µm to about 200 µm (e.g., any of about 155 µm, about 160 µm, about 165 µm, about 170 µm, about 175 µm, about 180 µm, about 185 µm, about 190µm,about 195µm,or about 200µm). In someembodiments, theadministering theunit doseof rAAVparticles to theoneeyeand / or to thecontralateral eyeof the individual results inadecreaseof central retinal thickness (CRT)or central subfield thickness (CST) of between about 160 µm to about 200 µm (e.g., any of about 160 µm, about 165 µm, about 170 µm, about 175µm, about 180µm, about 185µm, about 190µm, about 195µm, or about 200µm). In some embodiments, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in a decrease of central retinal thickness (CRT) or central subfield thickness (CST) of between about 165 µm to about 200 µm (e.g., any of about 165 µm, about 170 µm, about 175 µm, about 180 µm, about 185 µm, about 190 µm, about 195 µm, or about 200 µm). In some embodiments, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in a decrease of central retinal thickness (CRT) or central subfield thickness (CST) of between about 170µm toabout 200µm (e.g., any of about 170µm, about 175µm,about 180µm, about 185µm, about 190µm,about 195µm,or about 200µm). In someembodiments, theadministering theunit doseof rAAVparticles to theoneeyeand / or to thecontralateral eyeof the individual results inadecreaseof central retinal thickness (CRT)or central subfield thickness (CST) of between about 175 µm to about 200 µm (e.g., any of about 175 µm, about 180 µm, about 185 µm, about 190 µm, about 195 µm, or about 200 µm). In some embodiments, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in a decrease of central retinal thickness (CRT) or central subfield thickness (CST) of betweenabout 180µmtoabout 200µm(e.g., anyof about 180µm,about 185 µm, about 190 µm, about 195 µm, or about 200 µm). In some embodiments, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in a decrease of central retinal thickness (CRT) or central subfield thickness (CST) of about 21.0 µm compared to the central retinal thickness (CRT) or central subfield thickness (CST) prior to administration of theunit doseof rAAVparticles. In someembodiments, theadministering theunit dose of rAAVparticles to theoneeyeand / or to the contralateral eye of the individual results in adecrease of central retinal thickness (CRT)or central subfield thickness (CST)of about 8.3µmcompared to the central retinal thickness (CRT) or central subfield thickness (CST) prior to administration of the unit dose of rAAV particles. In some embodiments, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in a decrease of central retinal thickness (CRT) or central subfield thickness (CST) of about 26.2 µm compared to the central retinal thickness (CRT)or central subfield thickness (CST)prior toadministrationof theunit doseof rAAVparticles. In some embodiments, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in a decrease of central retinal thickness (CRT) or central subfield thickness (CST) of about 24.8 µm compared to the central retinal thickness (CRT) or central subfield thickness (CST) prior to administration of the unit dose of rAAV particles. In some embodiments, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in a decrease of central retinal thickness (CRT) or central subfield thickness (CST) of about 40.8 µm compared to the central retinal thickness (CRT) or central subfield thickness (CST) prior to administration of the unit doseof rAAVparticles. In someembodiments, theadministering theunit doseof rAAVparticles to theoneeyeand / or to thecontralateral eyeof the individual results inadecreaseof central retinal thickness (CRT)or central subfield thickness (CST) of about 30.0 µm compared to the central retinal thickness (CRT) or central subfield thickness (CST) prior to administration of the unit dose of rAAV particles. In some embodiments, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in a decrease of central retinal thickness (CRT) or central subfield thickness (CST) of about 118.6 µm compared to the central retinal thickness (CRT) or central subfield thickness (CST) prior to administration of the unit dose of rAAV particles. In some embodiments, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in a decrease of central retinal thickness (CRT) or central subfield thickness (CST) of about 119.0 µm compared to the central retinal thickness (CRT)or central subfield thickness (CST)prior toadministrationof theunit doseof rAAVparticles. In some embodiments, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in a decrease of central retinal thickness (CRT) or central subfield thickness (CST) of about 137.8 µm compared to the central retinal thickness (CRT) or central subfield thickness (CST) prior to administration of the unit dose of rAAV particles. In some embodiments, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in a decrease of central retinal thickness (CRT) or central subfield thickness (CST) of about 152.7 µm compared to the central retinal thickness (CRT) or central subfield thickness (CST) prior to 26 EP 4 696 318 A1 5 10 15 20 25 30 35 40 45 50 55 administration of the unit doseof rAAVparticles. In someembodiments, theadministering theunit doseof rAAVparticles to theoneeyeand / or to thecontralateral eyeof the individual results inadecreaseof central retinal thickness (CRT)or central subfield thickness (CST) of about 153.3 µm compared to the central retinal thickness (CRT) or central subfield thickness (CST) prior to administration of the unit dose of rAAV particles. In some embodiments, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in a decrease of central retinal thickness (CRT) or central subfield thickness (CST) of about 149.8 µm compared to the central retinal thickness (CRT) or central subfield thickness (CST) prior to administration of the unit dose of rAAV particles.

[0103] In some embodiments, the administering the unit dose of rAAVparticles to the one eyeand / or to the contralateral eyeof the individual results inachange in thecentral retinal thickness (CRT)or central subfield thickness (CST)ofbetween about ‑200 µm to about +40 µm (e.g., any about ‑200 µm, about ‑180 µm, about ‑160 µm, about ‑140 µm, about ‑120 µm, about ‑100µm,about ‑80µm,about ‑60µm,about - 40µm,about ‑20µm,about 0µm,about +5µm,about +10µm,about +15 µm, about +20 µm, about +25 µm, about +30 µm, about +35 µm, or about +40 µm) compared to the retinal thickness prior to administration of the unit dose of rAAV particles. In some embodiments, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in a decrease in the central retinal thickness (CRT) or central subfield thickness (CST) of any of about 8 µm, about 11 µm, about 16 µm, about 29 µm, about 33 µm, about 38 µm, about 55µm, about 61µm, or about 117µm. In some embodiments, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in a decrease in the central retinal thickness (CRT) or central subfield thickness (CST) of any of about 27.8 µm or about 30.8 µm. In some embodiments, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in an increase in the central retinal thickness (CRT) or central subfield thickness (CST) of any of about 4 µm, about 12 µm, or about 32 µm. In some embodiments, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in a change in the central retinal thickness (CRT) or central subfield thickness (CST) of about ‑21.0 µm compared to the central retinal thickness (CRT) or central subfield thickness (CST) prior to administration of the unit doseof rAAVparticles. In someembodiments, theadministering theunit doseof rAAVparticles to the one eye and / or to the contralateral eye of the individual results in a change in the central retinal thickness (CRT) or central subfield thickness (CST) of about ‑8.3 µm compared to the central retinal thickness (CRT) or central subfield thickness (CST) prior to administration of the unit dose of rAAV particles. In some embodiments, the administering the unit doseof rAAVparticles to theoneeyeand / or to thecontralateral eyeof the individual results in a change in thecentral retinal thickness (CRT) or central subfield thickness (CST) of about ‑26.2 µm compared to the central retinal thickness (CRT) or central subfield thickness (CST) prior to administration of the unit dose of rAAV particles. In some embodiments, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in a change in the central retinal thickness (CRT) or central subfield thickness (CST) of about ‑24.8µmcompared to the central retinal thickness (CRT)or central subfield thickness (CST)prior toadministrationof theunit doseof rAAVparticles. In some embodiments, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in a change in the central retinal thickness (CRT) or central subfield thickness (CST) of about ‑40.8 µm compared to the central retinal thickness (CRT) or central subfield thickness (CST) prior to administration of the unit dose of rAAV particles. In some embodiments, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in a change in the central retinal thickness (CRT) or central subfield thickness (CST) of about ‑30.0 µm compared to the central retinal thickness (CRT) or central subfield thickness (CST) prior to administration of the unit doseof rAAVparticles. In someembodiments, theadministering theunit doseof rAAVparticles to the one eye and / or to the contralateral eye of the individual results in a change in the central retinal thickness (CRT) or central subfield thickness (CST) of about ‑118.6 µm compared to the central retinal thickness (CRT) or central subfield thickness (CST) prior to administration of the unit dose of rAAV particles. In some embodiments, the administering the unit doseof rAAVparticles to theoneeyeand / or to thecontralateral eyeof the individual results in a change in thecentral retinal thickness (CRT) or central subfield thickness (CST) of about ‑119.0 µm compared to the central retinal thickness (CRT) or central subfield thickness (CST) prior to administration of the unit dose of rAAV particles. In some embodiments, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in a change in the central retinal thickness (CRT) or central subfield thickness (CST) of about ‑137.8 µm compared to the central retinal thickness (CRT) or central subfield thickness (CST) prior to administration of the unit dose of rAAV particles. In someembodiments, theadministering theunit doseof rAAVparticles to theoneeyeand / or to thecontralateral eyeof the individual results in a change in the central retinal thickness (CRT) or central subfield thickness (CST) of about ‑152.7 µm compared to the central retinal thickness (CRT) or central subfield thickness (CST) prior to administration of the unit dose of rAAV particles. In some embodiments, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in a change in the central retinal thickness (CRT) or central subfield thickness (CST) of about ‑153.3 µm compared to the central retinal thickness (CRT) or central subfield thickness (CST) prior to administration of the unit doseof rAAVparticles. In someembodiments, theadministering theunit doseof rAAVparticles to the one eye and / or to the contralateral eye of the individual results in a change in the central retinal thickness (CRT) or central subfield thickness (CST) of about ‑149.8 µm compared to the central retinal thickness (CRT) or central subfield 27 EP 4 696 318 A1 5 10 15 20 25 30 35 40 45 50 55 thickness (CST) prior to administration of the unit dose of rAAV particles.

[0104] In some embodiments, the change (e.g., the decrease) in the central retinal thickness (CRT) or central subfield thickness (CST) compared to the central retinal thickness (CRT) or central subfield thickness (CST) prior to administration of the unit dose of rAAV particles is present at any of about 1 day, about 1 week, about 2 weeks, about 4 weeks, about 8 weeks, about 16 weeks, about 24 weeks, about 30 weeks, about 32 weeks, about 34 weeks, about 40 weeks, about 44 weeks, about 48 weeks, about 52 weeks, or more after administration of the unit dose of rAAV particles to the one eye and / or to thecontralateral eyeof the individual. In someembodiments, the change (e.g., thedecrease) in the central retinal thickness (CRT) or central subfield thickness (CST) compared to the central retinal thickness (CRT) or central subfield thickness (CST) prior to administration of the unit dose of rAAV particles is present at any of about 1 day, about 1 week, about 2 weeks, about 4 weeks, about 8 weeks, about 12 weeks, about 16 weeks, about 20 weeks, about 24 weeks, about 28 weeks, about 32 weeks, about 36 weeks, about 40 weeks, about 44 weeks, about 48 weeks, about 52 weeks, about 56 weeks, about 60 weeks, about 64 weeks, about 68 weeks, about 72 weeks, about 76 weeks, about 80 weeks, about 84 weeks, about 88 weeks, about 92 weeks, about 96 weeks, about 100 weeks, about 104 weeks, about 108 weeks, or more after administration of the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual.

[0105] In some embodiments, the administering the unit dose of rAAVparticles to the one eyeand / or to the contralateral eyeof the individual results inadecreaseof central retinal thickness (CRT)or central subfield thickness (CST)ofmore than any of about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or about 100% compared to the retinal thickness prior to administration of the unit dose of rAAV particles. In some embodiments, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in a decrease of central retinal thickness (CRT) or central subfield thickness (CST) of at least about 10% compared to the retinal thickness prior to administration of the unit dose of rAAV particles. In some embodiments, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in a decrease of central retinal thickness (CRT) or central subfield thickness (CST) of about 15% or more compared to the retinal thickness prior to administration of the unit dose of rAAV particles.

[0106] In some embodiments, the administering the unit dose of rAAVparticles to the one eyeand / or to the contralateral eyeof the individual results in an increaseof central retinal thickness (CRT)or central subfield thickness (CST) of less than about 40µm(e.g., anyof less thanabout 40µm, less thanabout 35µm, less thanabout 30µm, less thanabout 25µm, less than about 20 µm, less than about 15 µm, less than about 10 µm, less than about 5 µm, less than about 1 µm, or less) compared to the retinal thickness prior to administration of the unit dose of rAAV particles. In some embodiments, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in an increase of central retinal thickness (CRT) or central subfield thickness (CST) of about 32 µm or less compared to the retinal thickness prior to administration of the unit dose of rAAV particles.

[0107] In some embodiments, the maintenance, the decrease, or the increase of retinal thickness compared to the retinal thickness prior to administration of the unit dose of rAAV particles is present at about 30 weeks or more after administration of the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual. In some embodiments, the maintenance, the decrease, or the increase of retinal thickness compared to the retinal thickness prior to administration of the unit dose of rAAV particles is present at any of about 30 weeks, about 34 weeks, about 44 weeks, about 6 months, about 1 year, about 1.5 years, about 2 years, about 3 years, about 5 years, about 10 years, or more, after administration of the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual.

[0108] In some embodiments, the administering the unit dose of rAAVparticles to the one eyeand / or to the contralateral eye of the individual results in maintenance or a decrease in macular volume compared to the macular volume prior to administration of the unit doseof rAAVparticles. In someembodiments, theadministering theunit doseof rAAVparticles to the one eye and / or to the contralateral eye of the individual results in a decrease in macular volume compared to the macular volume prior to administration of the unit dose of rAAV particles. In some embodiments, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in a decrease in macular volume of more than any of about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, or about 50% compared to the macular volume prior to administration of the unit dose of rAAV particles. In some embodiments, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in a decrease in macular volume of at least about 10% compared to the macular volume prior to administration of the unit dose of rAAV particles. In some embodiments, the macular volume is determined by OCTor SD- OCT. In someembodiments, the administering theunit doseof rAAVparticles to theoneeyeand / or to the contralateral eye of the individual results in a decrease in macular volume of at least about 10% compared to the macular volume prior to administration of the unit doseof rAAVparticles. In someembodiments, theadministering theunit doseof rAAVparticles to the one eye and / or to the contralateral eye of the individual results in a decrease in macular volume of about 15% or more compared to the macular volume prior to administration of the unit dose of rAAV particles. In some embodiments, the macular volume is determined by OCT or SD-OCT.

[0109] In some embodiments, the maintenance or the decrease in macular volume compared to the macular volume 28 EP 4 696 318 A1 5 10 15 20 25 30 35 40 45 50 55 prior to administration of the unit dose of rAAVparticles is present at about 30weeksormoreafter administration of the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual. In some embodiments, the maintenanceor the decrease inmacular volumecompared to themacular volumeprior to administration of theunit doseof rAAV particles is present at any of about 30 weeks, about 34 weeks, about 44 weeks, about 6 months, about 1 year, about 1.5 years, about 2 years, about 3 years, about 5 years, about 10 years, ormore after administration of the unit doseof rAAV particles to the one eye and / or to the contralateral eye of the individual.

[0110] In some embodiments, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in maintenance or an improvement of visual acuity compared to the visual acuity prior to administration of the unit doseof rAAVparticles. In someembodiments, theadministering theunit doseof rAAVparticles to the one eye and / or to the contralateral eye of the individual results in an improvement of visual acuity compared to the visual acuity prior to administration of the unit dose of rAAV particles. In some embodiments, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in an improvement of visual acuity of more than any of about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about80%,about90%,about100%,about125%,about150%,about175%,about200%,about225%,about250%,about 275%,about 300%,ormore, compared to thevisual acuity prior toadministrationof theunit doseof rAAVparticles. In some embodiments, visual acuity is best corrected visual acuity (BCVA). In some embodiments, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in an improvement of BCVA compared to theBCVAprior to administration of theunit doseof rAAVparticles. In someembodiments, 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).

[0111] In some embodiments, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in an improvement of BCVA of at least 15 ETDRS letters (Vitale et al., (2016) JAMA Opthalmol 134(9):1041:1047) (e.g., at least about 15, at least about 20, at least about 30, at least about 40, at least about 50, at least about 60, or about 70 letters) compared to the BCVA prior to administration of the unit dose of rAAV particles. In some embodiments, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results inan improvement ofBCVAofbetweenabout1 toabout 15 (e.g., anyofabout 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, or about 15) ETDRS letters compared to the BCVA prior to administration of the unit dose of rAAV particles. In some embodiments, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in an improvement of BCVA of about 5 ETDRS letters compared to the BCVA prior to administration of the unit dose of rAAV particles. In some embodiments, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in an improvement of BCVA of any of about 1, about 2, about 3, about 4, about 5, about 6, or about 7 ETDRS letters compared to the BCVA prior to administration of the unit dose of rAAV particles. In some embodiments, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in an improvement of BCVA of about 3 ETDRS letters or more compared to the BCVA prior to administration of the unit dose of rAAV particles. In some embodiments, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in an improvement of BCVA of about 4 ETDRS letters or more compared to the BCVA prior to administration of the unit dose of rAAV particles. In some embodiments, the administering the unit dose of rAAVparticles to theoneeyeand / or to thecontralateral eyeof the individual results inan improvementofBCVAofabout5.1 ETDRS letters or more compared to the BCVA prior to administration of the unit dose of rAAV particles. In some embodiments, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in an improvement of BCVA of about 6.4 ETDRS letters or more compared to the BCVA prior to administration of the unit doseof rAAVparticles. In someembodiments, theadministering theunit doseof rAAVparticles to the one eye and / or to the contralateral eye of the individual results in an improvement of BCVA of about 6.8 ETDRS letters compared to the BCVA prior to administration of the unit dose of rAAV particles. In some embodiments, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in an improvement of BCVA of about 8.8 ETDRS letters compared to the BCVA prior to administration of the unit dose of rAAV particles. In some embodiments, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in an improvement of BCVA of about 2.3 ETDRS letters compared to the BCVA prior to administration of the unit dose of rAAV particles.

[0112] In some embodiments, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in maintenance of BCVA, wherein the individual loses fewer than 15 ETDRS letters (Vitale et al., (2016) JAMA Opthalmol 134(9):1041:1047) (e.g., any of 15 or less, 14 or less, 13 or less, 12 or less, 11 or less, 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, 2 or less, 1, or 0 letters) compared to theBCVAprior to administrationof theunit doseof rAAVparticles. In someembodiments, theadministering theunit doseof rAAVparticles to the oneeyeand / or to the contralateral eye of the individual results in maintenance of BCVA,wherein the individual loses about 2 letters compared to the BCVA prior to administration of the unit dose of rAAV particles. In some embodiments, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in 29 EP 4 696 318 A1 5 10 15 20 25 30 35 40 45 50 55 maintenance of BCVA, wherein the individual loses any of about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, or about 9 ETDRS letters compared to the BCVA prior to administration of the unit dose of rAAV particles. In some embodiments, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in maintenance of BCVA, wherein the individual loses 0 letters compared to the BCVA prior to administration of the unit dose of rAAV particles. In some embodiments, the administering the unit dose of rAAV particles to the oneeyeand / or to the contralateral eye of the individual results in maintenance of BCVA,wherein the individual loses about 1 letter compared to the BCVA prior to administration of the unit dose of rAAV particles. In some embodiments, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in maintenance of BCVA, wherein the individual loses about 2.7 letters compared to the BCVA prior to administration of the unit doseof rAAVparticles. In someembodiments, theadministering theunit doseof rAAVparticles to theoneeyeand / or to the contralateral eye of the individual results in maintenance of BCVA, wherein the individual loses about 2.8 letters compared to the BCVA prior to administration of the unit dose of rAAV particles. In some embodiments, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in maintenance of BCVA, wherein the individual loses about 2 letters or less compared to the BCVA prior to administration of the unit dose of rAAV particles. In some embodiments, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in maintenance of BCVA, wherein the individual loses about 3.2 letters or less compared to the BCVA prior to administration of the unit dose of rAAV particles. In some embodiments, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in maintenance of BCVA,wherein the individual losesbetweenabout 15 toabout 0 letters (e.g., anyof about 15, about 14, about 13, about 12, about 11, about 10, about 9, about 8, about 7, about 6, about 5, about 4, about 3, about 2, about 1, or 0 letters) compared to theBCVAprior toadministrationof theunit doseof rAAVparticles. In someembodiments, theadministering theunit doseof rAAV particles to the one eye and / or to the contralateral eye of the individual results in maintenance of BCVA, wherein the individual losesbetweenabout 10 toabout 0 letters (e.g., anyof about 10, about 9, about 8, about 7, about 6, about 5, about 4, about 3, about 2, about 1, or 0 letters) compared to the BCVA prior to administration of the unit dose of rAAV particles. In some embodiments, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in maintenance of BCVA, wherein the individual loses between about 5 to about 0 letters (e.g., any of about 5, about 4, about 3, about 2, about 1, or 0 letters) compared to the BCVA prior to administration of the unit dose of rAAV particles. In some embodiments, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results inmaintenance of BCVA,wherein the individual loses betweenabout 4 to about 0 letters (e.g., any of about 4, about 3, about 2, about 1, or 0 letters) compared to the BCVA prior to administration of the unit doseof rAAVparticles. In someembodiments, theadministering theunit doseof rAAVparticles to theoneeyeand / or to the contralateral eye of the individual results inmaintenance of BCVA,wherein the individual loses betweenabout 3 to about 0 letters (e.g., any of about 3, about 2, about 1, or 0 letters) compared to the BCVA prior to administration of the unit dose of rAAV particles. In some embodiments, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results inmaintenance of BCVA,wherein the individual loses betweenabout 2 to about 0 letters (e.g., any of about 2, about 1, or 0 letters) compared to the BCVA prior to administration of the unit dose of rAAV particles. In someembodiments, theadministering theunit doseof rAAVparticles to theoneeyeand / or to the contralateral eye of the individual results in maintenance of BCVA, wherein the individual loses between about 1 to about 0 letters compared to the BCVA prior to administration of the unit dose of rAAV particles.

[0113] In some embodiments, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in a change inBCVAof betweenabout ‑20 to +7ormore (e.g., anyof ‑20, ‑19, ‑18, ‑17, ‑16, ‑15, ‑14, ‑13, ‑12, ‑11, ‑10, ‑9, ‑8, ‑7, ‑6, ‑5, ‑4, ‑3, ‑2, ‑1, 0, +1, +2, +3, +4, +5, +6, +7, +8, +9, +10, +11, +12, +13, +14, +15, +16, +17, +18, +19, +20, or more) ETDRS letters, compared to the BCVA prior to administration of the unit dose of rAAV particles. In someembodiments, theadministering theunit doseof rAAVparticles to theoneeyeand / or to the contralateral eye of the individual results in an increase in BCVA of about any of 16, 7 or 5 ETDRS letters compared to the BCVA prior to administration of the unit doseof rAAVparticles. In someembodiments, theadministering theunit doseof rAAVparticles to theoneeyeand / or to the contralateral eyeof the individual results in adecrease inBCVAof about anyof 19, 14, 7, 6, 5, 4, 3, 2, or 1 ETDRS letters compared to the BCVA prior to administration of the unit dose of rAAV particles. In some embodiments, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in a decrease in BCVA of about 4.8 or about 0.8 ETDRS letters compared to the BCVA prior to administration of the unit doseof rAAVparticles. In someembodiments, theadministering theunit doseof rAAVparticles to the one eye and / or to the contralateral eye of the individual results in a decrease in BCVA of about 2 ETDRS letters or less compared to the BCVA prior to administration of the unit dose of rAAV particles. In some embodiments, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in a decrease in BCVA of about 3.2 ETDRS letters or less compared to the BCVA prior to administration of the unit dose of rAAV particles.

[0114] In some embodiments, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in a change in BCVAof between about ‑15 to +7ormore (e.g., any of ‑15, ‑14, ‑13, ‑12, ‑11, ‑10, ‑9, ‑8, ‑7, ‑6, ‑5, ‑4, ‑3, ‑2, ‑1, 0, +1, +2, +3, +4, +5, +6, +7, +8, +9, +10, +11, +12, +13, +14, +15, +16, +17, +18, +19, +20, or 30 EP 4 696 318 A1 5 10 15 20 25 30 35 40 45 50 55 more) ETDRS letters, compared to the BCVA prior to administration of the unit dose of rAAV particles. In some embodiments, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in a change in BCVA of between about ‑10 to +7 or more (e.g., any of ‑10, ‑9, ‑8, ‑7, - 6, ‑5, ‑4, ‑3, ‑2, ‑1, 0, +1, +2, +3, +4, +5, +6, +7, +8, +9, +10, +11, +12, +13, +14, +15, +16, +17, +18, +19, +20, or more) ETDRS letters, compared to the BCVA prior to administration of the unit dose of rAAV particles. In some embodiments, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in a change in BCVA of between about ‑5 to +7 or more (e.g., any of ‑5, ‑4, ‑3, ‑2, ‑1, 0, +1, +2, +3, +4, +5, +6, +7, +8, +9, +10, +11, +12, +13, +14, +15, +16, +17, +18, +19, +20, or more) ETDRS letters, compared to the BCVA prior to administration of the unit dose of rAAV particles. In some embodiments, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eyeof the individual results in a change inBCVAof betweenabout ‑4 to +7ormore (e.g., anyof ‑4, ‑3, ‑2, ‑1, 0, +1, +2, +3, +4, +5, +6, +7, +8, +9, +10, +11, +12, +13, +14, +15, +16, +17, +18, +19, +20, or more) ETDRS letters, compared to the BCVA prior to administration of the unit dose of rAAV particles. In some embodiments, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in a change in BCVA of betweenabout ‑3 to+7ormore (e.g., anyof ‑3, ‑2, ‑1,0,+1,+2,+3,+4,+5,+6,+7,+8,+9,+10,+11,+12,+13,+14,+15,+16, +17, +18, +19, +20, or more) ETDRS letters, compared to the BCVA prior to administration of the unit dose of rAAV particles. In someembodiments, theadministering theunit doseof rAAVparticles to theoneeyeand / or to the contralateral eye of the individual results in a change in BCVA of between about ‑2 to +7 or more (e.g., any of ‑2, ‑1, 0, +1, +2, +3, +4, +5, +6, +7, +8, +9,+10, +11,+12,+13,+14, +15,+16,+17,+18,+19,+20, ormore)ETDRS letters, compared to theBCVAprior to administrationof theunit doseof rAAVparticles. In someembodiments, theadministering theunit doseof rAAVparticles to theoneeyeand / or to thecontralateral eyeof the individual results in a change inBCVAofbetweenabout ‑1 to+7ormore (e.g., anyof ‑1,0, +1,+2,+3, +4,+5,+6, +7,+8,+9, +10,+11,+12,+13,+14,+15,+16,+17,+18,+19,+20, ormore)ETDRS letters, compared to the BCVA prior to administration of the unit dose of rAAV particles. In some embodiments, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in a change inBCVAof betweenabout 0 to+7ormore (e.g., anyof 0, +1, +2, +3, +4, +5, +6, +7, +8, +9, +10,+11, +12, +13, +14, +15, +16, +17, +18, +19, +20, or more) ETDRS letters, compared to the BCVA prior to administration of the unit dose of rAAV particles. In some embodiments, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eyeof the individual results ina change inBCVAofbetweenabout+1 to+7ormore (e.g., anyof +1,+2, +3,+4, +5, +6, +7, +8, +9, +10, +11, +12, +13, +14, +15, +16, +17, +18, +19, +20, or more) ETDRS letters, compared to the BCVA prior to administration of the unit dose of rAAV particles. In some embodiments, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in a change in BCVA of between about +2 to +7 or more (e.g., any of +2, +3, +4, +5, +6, +7, +8, +9, +10, +11, +12, +13, +14, +15, +16, +17, +18, +19, +20, or more) ETDRS letters, compared to theBCVAprior to administration of the unit dose of rAAVparticles. In some embodiments, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in a change in BCVA of between about +3 to +7 or more (e.g., any of +3, +4, +5, +6, +7, +8, +9, +10, +11, +12, +13, +14, +15, +16, +17, +18, +19, +20, or more) ETDRS letters, compared to the BCVA prior to administration of the unit dose of rAAV particles. In someembodiments, theadministering theunit doseof rAAVparticles to theoneeyeand / or to the contralateral eye of the individual results in a change inBCVAof betweenabout +4 to +7ormore (e.g., any of +4, +5, +6, +7, +8, +9, +10, +11, +12, +13,+14, +15,+16, +17,+18, +19,+20, ormore)ETDRS letters, compared to theBCVAprior toadministration of the unit dose of rAAV particles. In some embodiments, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in a change in BCVA of between about +5 to +7 or more (e.g., any of +5, +6, +7, +8, +9, +10, +11, +12, +13, +14, +15, +16, +17, +18, +19, +20, or more) ETDRS letters, compared to the BCVA prior to administration of the unit dose of rAAV particles. In some embodiments, the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in a change in BCVA of about +6 or about +7 ETDRS letters, compared to the BCVA prior to administration of the unit dose of rAAV particles.

[0115] In some embodiments, administration of the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in transient inflammation (e.g., inflammation driven by aqueous cells and / or vitreous cells, aqueous flare, posterior synchiae, poor pupil dilation). In some embodiments, administration of the unit dose of rAAV particles to theoneeyeand / or to the contralateral eye of the individual results in inflammation (e.g., inflammationdriven by aqueous cells and / or vitreous cells, aqueous flare, posterior synchiae, poor pupil dilation) that is improved after administration of oral and / or topical steroid treatment and / or mydryatics. In some embodiments, administration of the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in inflammation (e.g., inflammation driven by aqueous cells and / or vitreous cells) that resolves after administration of oral and / or topical steroid treatment. Inflammation (e.g., inflammation driven by aqueous cells and / or vitreous cells, aqueous flare, posterior synchiae, poor pupil dilation) may be measured using any method known in the art, such as the slit lamp exam.

[0116] In some embodiments, the maintenance or the improvement of visual acuity (e.g., BCVA) compared to the visual acuityprior toadministrationof theunit doseof rAAVparticles is presentat anyofabout1day, about1week,about2weeks, about 4weeks, 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 31 EP 4 696 318 A1 5 10 15 20 25 30 35 40 45 50 55 weeks, about 64 weeks, about 68 weeks, about 72 weeks, about 76 weeks, about 80 weeks, about 84 weeks, about 88 weeks, about 92 weeks, about 96 weeks, about 100 weeks, about 104 weeks, about 108 weeks, or more, after administration of the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual. In some embodiments, the maintenance or the improvement of visual acuity (e.g., BCVA) compared to the visual acuity prior to administrationof theunit doseof rAAVparticles is present at about 30weeksormoreafter administration of theunit doseof rAAV particles to the one eye and / or to the contralateral eye of the individual. In some embodiments, the maintenance or the improvement of visual acuity (e.g., BCVA) compared to the visual acuity prior to administration of the unit dose of rAAV particles is present at any of about 30 weeks, about 34 weeks, about 44 weeks, about 6 months, about 1 year, about 1.5 years, about 2 years, about 3 years, about 5 years, about 10 years, or more, after administration of the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual.

[0117] In someembodiments, treatment of an ocular neovascular disease in an individual after administration of the unit doseof rAAVparticles in theoneeyeand / or thecontralateral eye isassessedbasedonbest correctedvisual acuity (BCVA) in the one eye and / or the contralateral eye. In some embodiments, BCVA is expressed as an ETDRS score, which corresponds to the number of letters correctly read (Vitale et al., (2016) JAMA Opthalmol 134(9):1041:1047). In some embodiments, an individual is determined to have maintenance of vision and / or visual acuity if the individual loses fewer than15 letters inanETDRSscore (e.g., anyof15or less, 14or less, 13or less, 12or less, 11or less, 10or less, 9or less, 8or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, 2 or less, 1, or 0 letters) compared to prior to administration of the unit dose of rAAV particles in the one eye and / or the contralateral eye. In some embodiments, an individual is determined to have an improvement of vision and / or visual acuity if the individual gains at least 15 letters (e.g., any of at least about 15, at least about 20, at least about 30, at least about 40, at least about 50, at least about 60, or about 70 letters) comparted to prior to administration of the unit dose of rAAV particles in the one eye and / or the contralateral eye.

[0118] In someembodiments, treatment of an ocular neovascular disease in an individual after administration of the unit dose of rAAVparticles in theoneeyeand / or the contralateral eye is assessedbasedon central subfield thickness (CST) or central retinal thickness (CRT) in the one eye and / or the contralateral eye. In some embodiments, CST or CRT is determined by SD-OCT. In some embodiments, treatment of an ocular neovascular disease in an individual after administration of the unit dose of rAAV particles in the one eye and / or the contralateral eye is determined if the CST or CRTassessed by SD-OCT is decreased after administration of the unit dose of rAAV particles in the one eye and / or the contralateral eye compared to prior to administration of the unit dose of rAAV particles in the one eye and / or the contralateral eye. In some embodiments, treatment of an ocular neovascular disease in an individual after administration of the unit dose of rAAV particles in the one eye and / or the contralateral eye is determined if the CSTor CRTassessed by SD-OCT is maintained after administration of the unit dose of rAAV particles in the one eye and / or the contralateral eye compared to prior to administration of the unit dose of rAAV particles in the one eye and / or the contralateral eye.

[0119] In someembodiments, treatment of an ocular neovascular disease in an individual after administration of the unit dose of rAAV particles in the one eye and / or the contralateral eye is assessed based on macular volume in the one eye and / or the contralateral eye. In some embodiments, macular volume is determined by SD-OCT. In some embodiments, treatment of an ocular neovascular disease in an individual after administration of the unit dose of rAAVparticles in the one eye and / or the contralateral eye is determined if the macular volume assessed by SD-OCT is decreased after admin- istration of the unit dose of rAAV particles in the one eye and / or the contralateral eye compared to prior to administration of the unit dose of rAAV particles in the one eye and / or the contralateral eye. In some embodiments, treatment of an ocular neovascular disease in an individual after administration of the unit dose of rAAV particles in the one eye and / or the contralateral eye is determined if the macular volume assessed by SD-OCT is maintained after administration of the unit dose of rAAV particles in the one eye and / or the contralateral eye compared to prior to administration of the unit dose of rAAV particles in the one eye and / or the contralateral eye.

[0120] In someembodiments, treatment of an ocular neovascular disease in an individual after administration of the unit dose of rAAV particles in the one eye and / or the contralateral eye is assessed based on retinal thickness (e.g., central retinal thickness (CRT) or central subfield thickness (CST)) and macular volume in the one eye and / or the contralateral eye. In some embodiments, CSTand macular volume are determined by SD-OCT. In some embodiments, treatment of an ocular neovascular disease in an individual after administration of the unit dose of rAAV particles in the one eye and / or the contralateral eye is determined if theCSTandmacular volumeassessedbySD-OCTare decreasedafter administration of the unit dose of rAAV particles in the one eye and / or the contralateral eye compared to prior to administration of the unit dose of rAAV particles in the one eye and / or the contralateral eye. In some embodiments, treatment of an ocular neovascular disease in an individual after administration of the unit dose of rAAV particles in the one eye and / or the contralateral eye isdetermined if theCSTandmacular volumeassessedbySD-OCTaremaintainedafter administrationof the unit dose of rAAV particles in the one eye and / or the contralateral eye compared to prior to administration of the unit dose of rAAV particles in the one eye and / or the contralateral eye.

[0121] In someembodiments, treatment of an ocular neovascular disease in an individual after administration of the unit dose of rAAV particles in the one eye and / or the contralateral eye is assessed based on the number of rescue therapy treatments (e.g., aflibercept injections) required by the individual after administration of the unit dose of rAAV particles in 32 EP 4 696 318 A1 5 10 15 20 25 30 35 40 45 50 55 the one eye and / or the contralateral eye. In some embodiments, treatment of an ocular neovascular disease in an individual after administration of the unit dose of rAAV particles in the one eye and / or the contralateral eye is determined if an individual requires less than one rescue therapy treatment (e.g., aflibercept injection) any of every 4 weeks, every 5 weeks, every 6 weeks, every 7 weeks, every 8 weeks, every 9 weeks, every 10 weeks, or more, after administration of the unit dose of rAAV particles in the one eye and / or the contralateral eye.

[0122] In someembodiments, treatment of an ocular neovascular disease in an individual after administration of the unit dose of rAAVparticles in theoneeyeand / or the contralateral eye is determined if an individual does not require any rescue therapy treatment (e.g., aflibercept injection) for any of at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, at least 7 weeks, at least 8 weeks, at least 9 weeks, at least 10 weeks, at least 15 weeks, at least 20 weeks, at least 30 weeks, at least 40 weeks, at least 50 weeks, at least 60 weeks, at least 70 weeks, at least 80 weeks, at least 90 weeks, at least 100 weeks, at least 110 weeks, or more, after administration of the unit dose of rAAV particles in the one eye and / or the contralateral eye.

[0123] In some embodiments, the individual does not require any rescue therapy treatment (e.g., aflibercept injection) for any of at least about 24 months, at least about 23 months, at least about 22 months, at least about 21 months, at least about 20months, at least about 19months, at least about 18months, at least about 17months, at least about 16months, 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 10months, at least about 9months, at least about 8months, at least about 7months, at least about 6 months, at least about 5 months, at least about 4 months, at least about 3 months, at least about 2 months, at least about 1 month, at least about 3 weeks, at least about 2 weeks, or at least about 1 week after administration of the unit dose of rAAV particles in the one eye and / or the contralateral eye. In some embodiments, the individual does not require any rescue therapy treatment (e.g., aflibercept injection) for at least about 12 months after administration of the unit dose of rAAV particles in the one eye and / or the contralateral eye. In some embodiments, the individual does not require any rescue therapy treatment (e.g., aflibercept injection) for at least about 10 months after administration of the unit dose of rAAV particles in the one eye and / or the contralateral eye. In some embodiments, the individual does not require any rescue therapy treatment (e.g., aflibercept injection) for at least about 7 months after administration of the unit dose of rAAV particles in the one eye and / or the contralateral eye. In some embodiments, the individual does not require any rescue therapy treatment (e.g., aflibercept injection) for at least about 6 months after administration of the unit dose of rAAV particles in the one eye and / or the contralateral eye. In some embodiments, the individual does not require any rescue therapy treatment (e.g., aflibercept injection) for at least about 2 months after administration of the unit dose of rAAV particles in the one eye and / or the contralateral eye. In some embodiments, the individual does not require any rescue therapy treatment (e.g., aflibercept injection) for at least about 1 month after administration of the unit dose of rAAV particles in the one eye and / or the contralateral eye.

[0124] In some embodiments, administration of a single unit dose of rAAV particles to the one eye and / or to the contralateral eye of a plurality of individuals results in at least about 50% (e.g., any of at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, or 100%) of the individuals in the plurality not requiring an anti-VEGF rescue treatment (e.g., aflibercept injection). In some embodiments, administration of a single unit dose of rAAV particles to the one eye and / or to the contralateral eye of a plurality of individuals results in at least about 67% (e.g., any of at least about 67%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, or 100%) of the individuals in the plurality not requiring an anti-VEGF rescue treatment (e.g., aflibercept injection). In some embodiments, administration of a single unit dose of rAAV particles to the one eye and / or to the contralateral eye of a plurality of individuals results in at least about 50% of the individuals in the plurality not requiring an anti-VEGF rescue treatment (e.g., aflibercept injection). In some embodiments, administration of a singleunit doseof rAAVparticles to theoneeyeand / or to thecontralateral eyeof aplurality of individuals results inat least about 78% of the individuals in the plurality not requiring an anti-VEGF rescue treatment (e.g., aflibercept injection). In someembodiments, administration of a single unit dose of rAAVparticles to theoneeyeand / or to the contralateral eyeof a plurality of individuals results in at least about 80% of the individuals in the plurality not requiring an anti-VEGF rescue treatment (e.g., aflibercept injection). In some embodiments, administration of a single unit dose of rAAV particles to the one eye and / or to the contralateral eye of a plurality of individuals results in at least about 82% of the individuals in the plurality not requiring an anti-VEGF rescue treatment (e.g., aflibercept injection). In some embodiments, administration of a single unit dose of rAAV particles to the one eye and / or to the contralateral eye of a plurality of individuals results in 100% of the individuals in the plurality not requiring an anti-VEGF rescue treatment (e.g., aflibercept injection).

[0125] In some embodiments, administration of a single unit dose of rAAV particles to the one eye and / or to the contralateral eye of a plurality of individuals results in at least about 50% (e.g., any of at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, or 100%) of the individuals in the plurality not requiring an anti-VEGFrescue treatment (e.g., aflibercept injection) for at least about 4weeksafter administrationof the rAAVparticles, e.g., any of at least about 4 weeks, at least about 8 weeks, at least about 12 weeks, at least about 16 weeks, at least about 33 EP 4 696 318 A1 5 10 15 20 25 30 35 40 45 50 55 20weeks, at least about 24weeks, at least about 28weeks, at least about 32weeks, at least about 36weeks, at least about 40weeks, at least about 44weeks, at least about 48weeks, at least about 52weeks, at least about 56weeks, at least about 60weeks, at least about 64weeks, at least about 68weeks, at least about 72weeks, at least about 76weeks, at least about 80weeks, at least about 84weeks, at least about 88weeks, at least about 92weeks, at least about 96weeks, at least about 100 weeks, at least about 104 weeks, at least about 108 weeks, or more after administration of the rAAV particles. In some embodiments, administration of a single unit dose of rAAV particles to the one eye and / or to the contralateral eye of a plurality of individuals results in at least about 50% of the individuals in the plurality not requiring an anti-VEGF rescue treatment (e.g., aflibercept injection) for about 52 weeks or more, or about 56 weeks or more, after administration of the rAAV particles. In some embodiments, administration of a single unit dose of rAAV particles to the one eye and / or to the contralateral eye of a plurality of individuals results in at least about 67% (e.g., any of at least about 67%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, or 100%) of the individuals in the plurality not requiring an anti-VEGF rescue treatment (e.g., aflibercept injection) for at least about 20 weeks after administration of the rAAV particles, e.g., any of at least about 20 weeks, at least about 24 weeks, at least about 28weeks, at least about 32weeks, at least about 36weeks, at least about 40weeks, at least about 44 weeks, at least about 48weeks, at least about 52weeks, at least about 56weeks, at least about 60weeks, at least about 64 weeks, at least about 66weeks, ormoreafter administrationof the rAAVparticles. In someembodiments, administrationof a singleunit doseof rAAVparticles to theoneeyeand / or to thecontralateral eyeof aplurality of individuals results inat least about 78% of the individuals in the plurality not requiring an anti-VEGF rescue treatment (e.g., aflibercept injection) for at least about 4weeks after administration of the rAAVparticles, e.g., any of at least about 4weeks, at least about 8weeks, at least about 12weeks, at least about 16weeks, at least about 20weeks, at least about 24weeks, at least about 28weeks, at least about 32weeks, at least about 36weeks, at least about 40weeks, at least about 44weeks, at least about 48weeks, at least about 52weeks, at least about 56weeks, at least about 60weeks, at least about 64weeks, at least about 68weeks, at least about 72weeks, at least about 76weeks, at least about 80weeks, at least about 84weeks, at least about 88weeks, at least about 92 weeks, at least about 96 weeks, at least about 100 weeks, at least about 104 weeks, at least about 108 weeks, or more, after administration of the rAAV particles. In some embodiments, administration of a single unit dose of rAAV particles to the one eye and / or to the contralateral eye of a plurality of individuals results in at least about 78% of the individuals in the plurality not requiring an anti-VEGF rescue treatment (e.g., aflibercept injection) for about 20 weeks or more, or about 36 weeks or more, after administration of the rAAV particles. In some embodiments, administration of a single unit dose of rAAV particles to the one eye and / or to the contralateral eye of a plurality of individuals results in at least about 80% of the individuals in the plurality not requiring an anti-VEGF rescue treatment (e.g., aflibercept injection) for at least about 20 weeks after administration of the rAAV particles, e.g., any of at least about 20 weeks, at least about 24 weeks, at least about 28weeks, at least about 32weeks, at least about 36weeks, at least about 40weeks, at least about 44 weeks, at least about 48weeks, at least about 52weeks, at least about 56weeks, at least about 60weeks, at least about 64 weeks, at least about 66weeks, ormoreafter administrationof the rAAVparticles. In someembodiments, administrationof a singleunit doseof rAAVparticles to theoneeyeand / or to thecontralateral eyeof aplurality of individuals results inat least about 82% of the individuals in the plurality not requiring an anti-VEGF rescue treatment (e.g., aflibercept injection) for at least about 20 weeks after administration of the rAAV particles, e.g., any of at least about 20 weeks, at least about 24 weeks, at least about 28weeks, at least about 32weeks, at least about 36weeks, at least about 40weeks, at least about 44 weeks, at least about 48weeks, at least about 52weeks, at least about 56weeks, at least about 60weeks, at least about 64 weeks, at least about 66 weeks, or more, after administration of the rAAV particles. In some embodiments, administration of a single unit dose of rAAV particles to the one eye and / or to the contralateral eye of a plurality of individuals results in 100% of the individuals in the plurality not requiring an anti-VEGF rescue treatment (e.g., aflibercept injection) for at least about 20 weeks after administration of the rAAV particles, e.g., any of at least about 20 weeks, at least about 24 weeks, at least about 28weeks, at least about 32weeks, at least about 36weeks, at least about 40weeks, at least about 44weeks, at least about 48weeks, at least about 52weeks, at least about 56weeks, at least about 60weeks, at least about 64weeks, at least about 66 weeks, or more, after administration of the rAAV particles. In some embodiments, administration of a single unit dose of rAAV particles to the one eye and / or to the contralateral eye of a plurality of individuals results in 100% of the individuals in the plurality not requiring an anti-VEGF rescue treatment (e.g., aflibercept injection) for at least about 4 weeks after administration of the rAAV particles, e.g., any of any of at least about 4 weeks, at least about 8 weeks, at least about 12weeks, at least about 16weeks, at least about 20weeks, at least about 24weeks, at least about 28weeks, at least about 32weeks, at least about 36weeks, at least about 40weeks, at least about 44weeks, at least about 48weeks, at least about 52weeks, at least about 56weeks, at least about 60weeks, at least about 64weeks, at least about 68weeks, at least about 72weeks, at least about 76weeks, at least about 80weeks, at least about 84weeks, at least about 88weeks, 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 moreafteradministrationof the rAAVparticles. In someembodiments, administrationofasingleunit doseof rAAVparticles to the oneeyeand / or to the contralateral eye of a plurality of individuals results in 100%of the individuals in the plurality not requiring an anti-VEGF rescue treatment (e.g., aflibercept injection) for any of about 64 weeks or more, 72 weeks or more, or 84 weeks or more, after administration of the rAAV particles. 34 EP 4 696 318 A1 5 10 15 20 25 30 35 40 45 50 55

[0126] In some embodiments, administration of a unit dose of rAAV particles in the one eye and / or the contralateral eye of aplurality of individuals results inabout 78%or less (e.g., anyof about78%or less, about 75%or less, about70%or less, about 65% or less, about 60% or less, about 55% or less, about 50% or less, about 45% or less, about 40% or less, about 35% or less, about 30% or less, about 25% or less, about 20% or less, about 15% or less, about 10% or less, about 5% or less, about 2.5% or less, about 1% or less, or about 0.5% or less) of the individuals in the plurality requiring any rescue treatment (e.g., aflibercept injection) in theoneeyeand / or the contralateral eye. In someembodiments, administration of a unit doseof rAAVparticles in theoneeyeand / or thecontralateral eyeof aplurality of individuals results inabout 50%or less (e.g., any of about 50% or less, about 45% or less, about 40% or less, about 35% or less, about 30% or less, about 25% or less, about 20% or less, about 15% or less, about 10% or less, about 5% or less, about 2.5% or less, about 1% or less, or about 0.5% or less) of the individuals in the plurality requiring any rescue treatment (e.g., aflibercept injection) in the one eye and / or the contralateral eye. In some embodiments, administration of a unit dose of rAAV particles in the one eye and / or the contralateral eye of a plurality of individuals results in less than about 30% (e.g., less than any of about 30%, about 25%, about 20%, about 15%, about 10%, about 5%, about 2.5%, about 1%, or about 0.5%) of the individuals in the plurality requiring any rescue treatment (e.g., aflibercept injection) in the one eye and / or the contralateral eye. In some embodiments, administration of a unit dose of rAAV particles in the one eye and / or the contralateral eye of a plurality of individuals results in less than about 30% of the individuals in the plurality requiring any rescue treatment (e.g., aflibercept injection) in the oneeyeand / or the contralateral eye. In some embodiments, administration of a unit dose of rAAVparticles in the oneeye and / or the contralateral eye of a plurality of individuals results in less than about 20%of the individuals in the plurality requiring any rescue treatment (e.g., aflibercept injection) in the one eye and / or the contralateral eye. In some embodiments, administration of a unit dose of rAAV particles in the one eye and / or the contralateral eye of a plurality of individuals results in 0% of the individuals in the plurality requiring any rescue treatment (e.g., aflibercept injection) in the one eye and / or the contralateral eye.

[0127] In some embodiments, administration of a unit dose of rAAV particles in the one eye and / or the contralateral eye of aplurality of individuals results inabout 78%or less (e.g., anyof about78%or less, about 75%or less, about70%or less, about 65% or less, about 60% or less, about 55% or less, about 50% or less, about 45% or less, about 40% or less, about 35% or less, about 30% or less, about 25% or less, about 20% or less, about 15% or less, about 10% or less, about 5% or less, about 2.5% or less, about 1% or less, or about 0.5% or less) of the individuals in the plurality requiring any rescue treatment (e.g., aflibercept injection) for at least about 4 weeks after administration of the rAAV particles, e.g., any of at least about 4 weeks, at least about 8 weeks, at least about 12 weeks, at least about 16 weeks, at least about 20 weeks, at least about 24weeks, at least about 28weeks, at least about 32weeks, at least about 36weeks, at least about 40weeks, at least about 44weeks, at least about 48weeks, at least about 52weeks, at least about 56weeks, at least about 60weeks, at least about 64weeks, at least about 68weeks, at least about 72weeks, at least about 76weeks, at least about 80weeks, at least about 84 weeks, at least about 88 weeks, at least about 92 weeks, at least about 96 weeks, at least about 100 weeks, at least about 104 weeks, at least about 108 weeks, or more, after administration of the rAAV particles. In some embodiments, administration of a unit dose of rAAV particles in the one eye and / or the contralateral eye of a plurality of individuals results in less than about 30% (e.g., less than any of about 30%, about 25%, about 20%, about 15%, about 10%, about 5%, about 2.5%, about 1%, or about 0.5%) of the individuals in the plurality requiring any rescue treatment (e.g., aflibercept injection) for at least about 20 weeks after administration of the rAAV particles, e.g., any of at least about 20weeks, at least about 24weeks, at least about 28weeks, at least about 32weeks, at least about 36weeks, at least about 40weeks, at least about 44weeks, at least about 48weeks, at least about 52weeks, at least about 56weeks, at least about 60 weeks, at least about 64 weeks, at least about 66 weeks, or more, after administration of the rAAV particles. In some embodiments, administration of a unit dose of rAAV particles in the one eye and / or the contralateral eye of a plurality of individuals results in less than about 30% of the individuals in the plurality requiring any rescue treatment (e.g., aflibercept injection) in the one eye and / or the contralateral eye for at least about 20 weeks after administration of the rAAV particles, e.g., any of at least about 20 weeks, at least about 24 weeks, at least about 28 weeks, at least about 32 weeks, at least about 36weeks, at least about 40weeks, at least about 44weeks, at least about 48weeks, at least about 52weeks, at least about 56 weeks, at least about 60 weeks, at least about 64 weeks, at least about 66 weeks, or more, after administration of the rAAV particles. In some embodiments, administration of a unit dose of rAAV particles in the one eye and / or the contralateral eye of a plurality of individuals results in less than about 20% of the individuals in the plurality requiring any rescue treatment (e.g., aflibercept injection) in the one eye and / or the contralateral eye for at least about 20 weeks after administration of the rAAV particles, e.g., any of at least about 20 weeks, at least about 24 weeks, at least about 28 weeks, at least about 32weeks, at least about 36weeks, at least about 40weeks, at least about 44weeks, at least about 48weeks, at least about 52weeks, at least about 56weeks, at least about 60weeks, at least about 64weeks, at least about 66weeks, or more after administration of the rAAV particles. In some embodiments, administration of a unit dose of rAAV particles in the one eye and / or the contralateral eye of a plurality of individuals results in 0% of the individuals in the plurality requiring any rescue treatment (e.g., aflibercept injection) in theoneeyeand / or thecontralateral eye forat least about20weeksafter 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 32weeks, at least about 36weeks, at least about 40weeks, at least about 44weeks, at least about 48weeks, 35 EP 4 696 318 A1 5 10 15 20 25 30 35 40 45 50 55 at least about 52weeks, at least about 56weeks, at least about 60weeks, at least about 64weeks, at least about 66weeks, or more after administration of the rAAV particles. In some embodiments, administration of a unit dose of rAAV particles in the one eye and / or the contralateral eye of a plurality of individuals results in 0% of the individuals in the plurality requiring any rescue treatment (e.g., aflibercept injection) in the one eye and / or the contralateral eye for at least about 4 weeks after administration of the rAAV particles, e.g., any of at least about 4 weeks, at least about 8 weeks, at least about 12 weeks, at least about 16weeks, at least about 20weeks, at least about 24weeks, at least about 28weeks, at least about 32weeks, at least about 36weeks, at least about 40weeks, at least about 44weeks, at least about 48weeks, at least about 52weeks, at least about 56weeks, at least about 60weeks, at least about 64weeks, at least about 68weeks, at least about 72weeks, at least about 76weeks, at least about 80weeks, at least about 84weeks, at least about 88weeks, at least about 92weeks, at least about 96 weeks, at least about 100 weeks, at least about 104 weeks, at least about 108 weeks, or more, after administration of the rAAV particles.

[0128] In some embodiments, administration of the unit dose of rAAV particles to the one eye and / or to the contralateral eyeof a plurality of individuals results in a reduction in themeanannualizedanti-VEGF injection rateof anyof 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 mean annualized anti-VEGF injection rate prior to administration of the unit dose of rAAV particles. In some embodiments, administration of the unit dose of rAAV particles to the one eye and / or to the contralateral eye of a plurality of individuals results in a reduction in the mean annualized anti-VEGF injection rate of about 87% or more compared to the mean annualized anti-VEGF injection rate prior to administration of the unit dose of rAAV particles. In some embodiments, administration of the unit dose of rAAV particles to the one eye and / or to the contralateral eye of a plurality of individuals results in a reduction in the mean annualized anti-VEGF injection rate of 100% compared to the mean annualized anti-VEGF injection rate prior to administration of the unit dose of rAAV particles.

[0129] In some embodiments, the mean annualized anti-VEGF injection rate prior to administration of the unit dose of rAAV particles is calculated according to the formula: Annualized rate prior to administration of the unit dose of rAAV particles = (number of anti-VEGF injections in 12 months prior toadministrationof theunit doseof rAAVparticles) / (days from thefirst anti-VEGF injection in thepast 12monthsprior to administration of the unit dose of rAAV particles to the administration of the unit dose of rAAV particles / 365.25)

[0130] In someembodiments, themeanannualizedanti-VEGF injection rateafteradministrationof theunit doseof rAAV particles is calculated according to the formula: Annualized rate after administration of the unit dose of rAAV particles = (number of anti-VEGF injections since admin- istration of the unit dose of rAAV particles) / (days from administration of the unit dose of rAAV particles / 365.25).

[0131] In some embodiments, an individual is determined to require a rescue treatment (e.g., anti-VEGF intravitreal injection, such as aflibercept injection) after administration of the rAAV particles if the individual exhibits loss of 10 or more letters in BCVA (e.g., using the ETDRS protocol) in the one eye and / or the contralateral eye administered the rAAV particles that is attributed to intraretinal or subretinal fluid (e.g., as determined by SD-OCT) compared to the BCVA in the one eye and / or the contralateral eye administered the rAAV particles prior to administration of the rAAV particles. In some embodiments, an individual is determined to require a rescue treatment (e.g., anti-VEGF intravitreal injection, such as aflibercept injection) after administration of the rAAV particles if the individual exhibits an increase in central subfield thickness (e.g., CST or CRT) greater than 75 µm in the one eye and / or the contralateral eye administered the rAAV particles compared to the central subfield thickness in the one eye and / or the contralateral eye administered the rAAV particles prior to administration of the rAAVparticles, e.g., as determinedbySD-OCT. In someembodiments, an individual is determined to require a rescue treatment (e.g., anti-VEGF intravitreal injection, such as aflibercept injection) after administration of the rAAV particles if the individual exhibits vision-threatening hemorrhage due to AMD in the one eye and / or the contralateral eye administered the rAAV particles.

[0132] In some embodiments, a rescue treatment comprises administration of a standard of care anti-VEGF therapy. Such standard of care anti-VEGF therapy comprises one or more anti-VEGF treatments (e.g., anti-VEGF intravitreal injections). In some embodiments, a rescue treatment comprises one or more aflibercept IVT injections. In some embodiments, a rescue treatment comprises one or more aflibercept IVT injections comprising about 2 mg of aflibercept.

[0133] In someembodiments, treatment of an ocular neovascular disease in an individual after administration of the unit dose of rAAV particles in the one eye and / or the contralateral eye is assessed based on the level of retinal fluid compared the level of retinal fluidprior toadministrationof theunit doseof rAAVparticles in theoneeyeand / or thecontralateral eye. In some embodiments, treatment of an ocular neovascular disease in an individual after administration of the unit dose of rAAV particles in the one eye and / or the contralateral eye is determined if a reduction in retinal fluid is observed after administration of the unit dose of rAAV particles in the one eye and / or the contralateral eye compared to the level of retinal fluid prior to administration of the unit dose of rAAV particles in the one eye and / or the contralateral eye. In some embodiments, the ocular neovascular disease is wAMD.

[0134] In someembodiments, treatment of an ocular neovascular disease in an individual after administration of the unit dose of rAAVparticles in the one eyeand / or the contralateral eye is assessed based on the resolution of pigment epithelial detachment (PED) compared to PED prior to administration of the unit dose of rAAV particles in the one eye and / or the 36 EP 4 696 318 A1 5 10 15 20 25 30 35 40 45 50 55 contralateral eye. In some embodiments, treatment of an ocular neovascular disease in an individual after administration of the unit dose of rAAV particles in the one eye and / or the contralateral eye is determined if resolution of PED after administration of the unit dose of rAAV particles in the one eye and / or the contralateral eye is observed, compared to PED prior to administration of the unit dose of rAAV particles in the one eye and / or the contralateral eye. In some embodiments, the ocular neovascular disease is wAMD.

[0135] In someembodiments, treatment of an ocular neovascular disease in an individual after administration of the unit dose of rAAV particles in the one eye and / or the contralateral eye is assessed based on choroidal neovascularization (CNV) lesion growth as determined by fluorescein angiography. In some embodiments, treatment of an ocular neovas- cular disease in an individual after administration of the unit dose of rAAV particles in the one eye and / or the contralateral eye is determined if CNV lesions shrink (e.g., by more than any of about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, or 100%) after administration of the unit dose of rAAV particles in the one eye and / or the contralateral eye compared to CNV lesions present prior to administration of the unit dose of rAAV particles in the one eye and / or the contralateral eye. In some embodiments, treatment of an ocular neovascular disease in an individual after administration of theunit doseof rAAVparticles in the oneeyeand / or the contralateral eye is determined if CNV lesions do not grow (e.g., grow less than any of about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about7%,about8%,about9%,about10%,about15%,orabout20%)afteradministrationof theunit doseof rAAVparticles in theoneeyeand / or the contralateral eyecompared toCNV lesionspresent prior toadministrationof theunit doseof rAAV particles in the one eye and / or the contralateral eye. In some embodiments, the ocular neovascular disease is wAMD.

[0136] In someembodiments, treatment of an ocular neovascular disease in an individual after administration of the unit dose of rAAV particles in the one eye and / or the contralateral eye is assessed based on the anatomical features of the one eye and / or the contralateral eye based on any methods known in the art (e.g., SD-OCT, OCT, fluorescein angiography, digital color fundus photography, etc.). In some embodiments, treatment of an ocular neovascular disease in an individual after administration of the unit dose of rAAV particles in the one eye and / or the contralateral eye is determined if an improvement in anatomical features of the one eye and / or the contralateral eye is observed after administration of the unit doseof rAAVparticles in theoneeyeand / or thecontralateral eye. In someembodiments, theocularneovascular disease is wAMD.

[0137] In someembodiments, treatment of an ocular neovascular disease in an individual after administration of the unit dose of rAAV particles in the one eye and / or the contralateral eye is assessed based on ophthalmologic examination, intraocular pressure (e.g., using a Goldmann applanation tonometer or Tono-pen), indirect ophthalmoscopy, examination of the one eye and / or the contralateral eye and adnexa, eyelid and / or pupil responsiveness, belpharoptosis, abnormal pupil shape, unequal pupils, abnormal reaction to light, afferent pupillary defects, slit-lamp examination (including of the eyelids, conjunctiva, cornea, lens, iris, andanterior chamber), posterior segmentabnormalitiesof thevitreous, opticnerve, peripheral retina, and retinal vasculature, SD-OCT, fluorescein angiography, digital color fundus photography (including images of the retina, optic disc, and / or macula), aqueous humor sampling, vitreous humor sampling, OCT-angiography (OCT-A), refraction and / or visual acuity (BCVA). In some embodiments, SD-OCT is performed to evaluate retinal thickness (e.g., central retinal thickness or central subfield thickness), macular volume, and / or the presence of fluid (e.g., subretinal fluid or intraretinal fluid). In some embodiments, the ocular neovascular disease is wAMD.

[0138] Theunit doseof rAAVparticlesmaybeadministered to theoneeyeand / or to thecontralateral eyeof the individual by any method known in the art. For example, the unit dose of rAAV particles may be administered to the one eye and / or to the contralateral eye of the individual intraocularly, or by intravitreal injection. In some embodiments, the administration of the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual is intraocular. In some embodiments, the administration of the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual is by intravitreal injection (IVT) or subretinal injection. In some embodiments, the administration of the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual is by IVT injection. In some embodiments, aseptic technique is employed to administer a unit dose of rAAV particles by intravitreal injection. In some embodiments, aseptic technique with providone-iodine is employed to administer a unit dose of rAAV particles by intravitreal injection.

[0139] In some embodiments, the individual has not received a prior treatment for an ocular neovascular disease. In some embodiments, the individual has not received a prior treatment in the one eye and / or the contralateral eye for an ocular neovascular disease. In some embodiments, the individual has not received a prior treatment with an anti-VEGF agent (e.g., bevacizumab, brolucizumab, ranibizumab, faricimab, abicipar pegol, conbercept, OPT‑302, KSI‑301, injectable sunitinib maleate (GB‑102), PAN‑90806 (PanOptica), and / or aflibercept). In some embodiments, the individual has not received a prior treatment with an anti-VEGF agent (e.g., bevacizumab, brolucizumab, ranibizumab, faricimab, abicipar pegol, conbercept, OPT‑302, KSI‑301, injectable sunitinib maleate (GB‑102), PAN‑90806 (PanOptica), and / or aflibercept) in the one eye and / or the contralateral eye. In some embodiments, the individual has not received a prior aflibercept treatment. In some embodiments, the individual has not received a prior aflibercept treatment in the one eye and / or the contralateral eye. 37 EP 4 696 318 A1 5 10 15 20 25 30 35 40 45 50 55 Steroid Treatments

[0140] In some embodiments, the unit dose of rAAV particles is administered in combination with steroid treatment. In some embodiments, the steroid treatment is a corticosteroid treatment. Exemplary corticosteroids include, without limitation, aclometasone, amcinomide, beclometasone, betamethasone, budesonide, ciclesonide, clobetasol, clobeta- sone, clocortolone, cloprednol, cortivazol, deflazacort, deoxycorticosterone, desonide desoximetasone, dexametha- sone, diflorasone, diflucortolone, difluprednate, fluclorolone, fludrocortisone, fludroxycortide, flumetasone, flunisolide, fluocinolone acetonide, fluocinonide, fluocortin, fluocortolone, fluorometholone, fluperolone, fluticasone, fuprednidene, formocortal, halcinonide, halometasone, hydrocortisone aceponate, hydrocortisone buteprate, hydrocortisone butyrate, loteprednol, medrysone, meprednisone, methylprednisolone, methylprednisolone aceponate, mometasone furoate, paramethasone, prednicarbate, prednisone, prednisolone, prednylidene, remexolone, tixocortol, triamcinolone, and ulobetasol. In some embodiments, the steroid treatment is a systemic steroid treatment. In some embodiments, the steroid treatment is an oral steroid treatment. In some embodiments, the steroid treatment is an ophthalmic steroid treatment. In some embodiments, the ophthalmic steroid treatment is a topical steroid treatment (e.g. a drop), a periocular steroid treatment (e.g., subtenons, subconjunctival), an intravitreal steroid treatment, or a superchoroidal steroid treatment. In some embodiments, the topical steroid treatment is a difluprednate treatment, a medrysone treatment, a loteprednol treatment, a prednisolone treatment, a fluocinolone treatment, a triamcinolone treatment, a rimexolone treatment, adexamethasone treatment, afluorometholone treatment, a fluocinolone treatment, a rimexolone treatment, or a prednisone treatment. In some embodiments, the ophthalmic steroid treatment is a difluprednate treatment. In some embodiments, the steroid treatment is a prednisone treatment. In some embodiments, the steroid treatment is a difluprednate treatment.

[0141] In some embodiments, the steroid treatment comprises a systemic steroid treatment and a topical steroid treatment. In some embodiments, the systemic steroid treatment is an oral steroid treatment. In some embodiments, the systemic steroid treatment is a prednisone treatment. In some embodiments, the topical steroid treatment is a diflu- prednate treatment. In some embodiments, the systemic steroid treatment and the topical steroid treatment are administered simultaneously (e.g., on the same day). In some embodiments, the systemic steroid treatment and the topical steroid treatment are administered separately (e.g., on different days).

[0142] In some embodiments, the steroid is administered before, during, and / or after administration of the unit dose of rAAV particles. In some embodiments, the steroid is administered before, during, and after administration of the unit dose of rAAV particles. In some embodiments, the steroid is administered during, and after administration of the unit dose of rAAV particles. In some embodiments, the steroid is administered before administration of the unit dose of rAAV particles. In some embodiments, the steroid is administered during administration of the unit dose of rAAV particles. In some embodiments, the steroid is administered before and during administration of the unit dose of rAAV particles. In some embodiments, the steroid is administeredafter administration of theunit doseof rAAVparticles. In someembodiments, the steroid is administeredduringandafter administrationof the unit doseof rAAVparticles. In someembodiments, the steroid is administered before and / or after administration of the unit dose of rAAV particles. In some embodiments, the steroid is administered before and after administration of the unit dose of rAAV particles.

[0143] In someembodiments, the steroid treatment is a systemic steroid treatment. In someembodiments, the systemic steroid treatment is an oral steroid treatment.

[0144] In some embodiments, the steroid treatment is an oral prednisone treatment. In some embodiments, the oral prednisone treatment is initiated prior to administration of the unit dose of rAAV particles. In some embodiments, an initial oral prednisone treatment is administered at a dose of any 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 any of 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 rAAVparticles, and is continued for any of 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, an 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 is continued for about 3 days.

[0145] In some embodiments, the initial oral prednisone treatment is followed by an oral prednisone treatment dose taper. In some embodiments, the oral prednisone treatment dose taper is administered at a dose of any of about 20 mg, about25mg,about30mg,about35mg,about40mg,about45mg,orabout50mgofprednisoneperday fora total ofanyof 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 any of about 1 day, about 2 days, about 3 days, or about 4days, followedbyadoseof about 5mg, about 10mg, or about 15mgof prednisoneper day for about 1day, about 2 days, about 3 days, or about 4 days. In someembodiments, the prednisonedose taper is administered at a doseof any of about 40 mg of prednisone per day for 3 days, followed by a dose of about 20 mg of prednisone per day for 2 days, followed by a dose of about 10 mg of prednisone per day for 2 days.

[0146] In some embodiments, an initial oral prednisone treatment is initiated 3 days before administration of the unit dose of rAAV particles at a dose of 60 mg of prednisone per day for a total of 6 days, followed by a dose of 40 mg of 38 EP 4 696 318 A1 5 10 15 20 25 30 35 40 45 50 55 prednisone per day for a total of 3 days, followed by a dose of 20 mg of prednisone per day for 2 days, followed by a dose of 10 mg of prednisone per day for 2 days.

[0147] In some embodiments, the steroid treatment is an ophthalmic steroid treatment. In some embodiments, the ophthalmic steroid treatment is a difluprednate treatment. In some embodiments, the steroid treatment is administered before, during, and / or after administrationof theunit doseof rAAVparticles. In someembodiments, 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 administeredduring, andafter administration of theunit dose of rAAVparticles. In someembodiments, the steroid treatment is administered before, during, and after administration of the unit dose of rAAV particles.

[0148] In some embodiments, the steroid treatment is an ophthalmic steroid treatment, e.g., a topical steroid treatment. In some embodiments, the ophthalmic steroid treatment, e.g., a topical steroid treatment, is a daily steroid treatment for up to 4 weeks, up to 6 weeks, up to 8 weeks, up to 3 months, up to 4 months, up to 5 months, or up to 6 months after administration of the unit dose of rAAVparticles. In some embodiments, the topical steroid treatment comprises about four administrations of topical steroid on about week 1, about three administrations of topical steroid on about week 2, about two administrations of topical steroid on about week 3, and about one administration of topical steroid on about week 4; timing starting with and following administration of the unit dose of rAAV particles. In some embodiments, the topical steroid treatment comprises about four administrations of topical steroid per day (i.e., QID) for about 3 weeks after administration of the unit dose of rAAVparticles, followed by about 3 administrations of topical steroid per day (i.e.,TID) for about 1 week, followed by about 2 administrations of topical steroid per day (i.e., BID) for about 1 week, and followed by about 1 administration of topical steroid per day (i.e.,QD) for about 1 week. In some embodiments, the ophthalmic steroid treatment is extended at the discretion of the treating physician.

[0149] In some embodiments, the ophthalmic steroid is about 0.005% to about 0.5% difluprednate. In some embodi- ments, the ophthalmic steroid is any of about 0.005%, about 0.006%, about 0.007%, about 0.008%, about 0.009%, about 0.01%, about 0.02%, about 0.03%, about 0.4%, about 0.05%, about 0.06%, about 0.07%, about 0.08%, about 0.09%, or about 0.1% difluprednate. In some embodiments, the ophthalmic steroid is difluprednate 0.05%. In some embodiments, a dose of difluprednate 0.05% is one drop of ophthalmic solution. In some embodiments, one drop is about 50µl (e.g., about 25µl to about 50µl, or about 50µl to about 100µl). In someembodiments, a doseof difluprednate comprisesabout 1µg to about 5 µg, or about 2 µg to about 3 µg, or about 2.5 µg difluprednate. In some embodiments, a dose of difluprednate comprises about 2.5 µg difluprednate.

[0150] In some embodiments, the topical steroid treatment comprises a 7-week topical steroid treatment, e.g., 0.05% difluprednate. In some embodiments, the topical steroid treatment comprises about four administrations of topical steroid per day (i.e., QID) for about four weeks, followed by about three administrations of topical steroid per day (i.e., TID) for about one week, followed by about two administrations of topical steroid per day (i.e., BID) for about one week, and followed by about one administration of topical steroid per day (i.e.,QD) for about one week; timing starting at about one week prior to administration of the unit dose of rAAV particles. In some embodiments, the topical steroid treatment comprises about four administrations of topical steroid per day (i.e., QID) for about 28 days, followed by about three administrationsof topical steroidperday (i.e.,TID) for about7days, followedbyabout twoadministrationsof topical steroid per day (i.e.,BID) for about 7 days, and followed byabout one administration of topical steroid per day (i.e.,QD) for about 7 days; timing starting at about 7 days prior to administration of the unit dose of rAAV particles. In some embodiments, the topical steroid treatment comprises about four administrations of topical steroid per day (i.e.,QID) on Day 1 to about Day 28, followed by about three administrations of topical steroid per day (i.e., TID) on about Day 29 to about Day 35, followed by about two administrations of topical steroid per day (i.e., BID) on about Day 36 to about Day 42, and followed by about one administration of topical steroid per day (i.e.,QD) on about Day 43 to about Day 49; timing starting at Day 1. In some embodiments, the topical steroid treatment is continued if inflammation is present.

[0151] In some embodiments, the methods of treatment provided herein comprise administering an anti-VEGF agent (e.g., an aflibercept IVT injection) to one eye of the individual prior to administration of the unit dose of rAAV particles to the oneeyeof the individual. In someembodiments, theanti-VEGFagent is administeredabout 7daysor about 1weekprior to administrationof theunit doseof rAAVparticles. In someembodiments, theanti-VEGFagent is administeredonaboutDay 1 and the unit dose of rAAV particles is administered on about Day 8. In some embodiments, the topical steroid treatment comprises a 7-week topical steroid treatment, e.g., 0.05% difluprednate. In some embodiments, the topical steroid treatment comprisesabout four administrationsof topical steroidper day (i.e.,QID) for about fourweeks, followedbyabout three administrations of topical steroid per day (i.e., TID) for about one week, followed by about two administrations of topical steroid per day (i.e., BID) for about one week, and followed by about one administration of topical steroid per day (i.e., QD) for about one week; timing starting with and following administration of the anti-VEGF agent. In some embodiments, the topical steroid treatment comprises about four administrations of topical steroid per day (i.e., QID) 39 EP 4 696 318 A1 5 10 15 20 25 30 35 40 45 50 55 for about 28 days, followed by about three administrations of topical steroid per day (i.e.,TID) for about 7 days, followed by about twoadministrations of topical steroid per day (i.e.,BID) for about 7 days, and followedby about one administration of topical steroid per day (i.e.,QD) for about 7 days; timing starting with and following administration of the anti-VEGF agent. In some embodiments, the topical steroid treatment comprises about four administrations of topical steroid per day (i.e., QID)onDay1 toaboutDay28, followedbyabout threeadministrationsof topical steroid per day (i.e.,TID) onaboutDay29 to about Day 35, followed by about two administrations of topical steroid per day (i.e., BID) on about Day 36 to about Day 42, and followed by about one administration of topical steroid per day (i.e.,QD) on about Day 43 to about Day 49; timing starting at Day 1. In some embodiments, the topical steroid treatment is continued if inflammation is present.

[0152] In some embodiments, the topical steroid treatment comprises a 4-month topical steroid treatment, e.g., 0.05% difluprednate. In some embodiments, the topical steroid treatment comprises about four administrations of topical steroid per day (i.e., QID) for about one month, followed by about three administrations of topical steroid per day (i.e., TID) for about one month, followed by about two administrations of topical steroid per day (i.e., BID) for about one month, and followed by about one administration of topical steroid per day (i.e.,QD) for about one month; timing starting at about one week prior to administration of the unit dose of rAAV particles. In some embodiments, the topical steroid treatment comprises about four administrations of topical steroid per day (i.e., QID) for about 30 days, followed by about three administrations of topical steroid per day (i.e., TID) for about 30 days, followed by about two administrations of topical steroidper day (i.e.,BID) for about 30days, and followedbyabout oneadministrationof topical steroidper day (i.e.,QD) for about 30 days; timing starting at about 7 days prior to administration of the unit dose of rAAV particles. In some embodiments, the topical steroid treatment comprises about four administrations of topical steroid per day (i.e., QID) on Day 1 to about Day 30, followed by about three administrations of topical steroid per day (i.e., TID) on about Day 31 to about Day 60, followed by about two administrations of topical steroid per day (i.e.,BID) on about Day 61 to about Day 90, and followed by about one administration of topical steroid per day (i.e., QD) on about Day 91 to about Day 120; timing starting at Day 1. In some embodiments, the topical steroid treatment is continued if inflammation is present.

[0153] In some embodiments, the methods of treatment provided herein comprise administering an anti-VEGF agent (e.g., an aflibercept IVT injection) to one eye of the individual prior to administration of the unit dose of rAAV particles to the oneeyeof the individual. In someembodiments, theanti-VEGFagent is administeredabout 7daysor about 1weekprior to administrationof theunit doseof rAAVparticles. In someembodiments, theanti-VEGFagent is administeredonaboutDay 1 and the unit dose of rAAV particles is administered on about Day 8. In some embodiments, the topical steroid treatment comprises a 4-month topical steroid treatment, e.g., 0.05% difluprednate. In some embodiments, the topical steroid treatment comprisesabout four administrationsof topical steroid per day (i.e.,QID) for about onemonth, followedbyabout three administrations of topical steroid per day (i.e., TID) for about one month, followed by about two administrations of topical steroid per day (i.e., BID) for about one month, and followed by about one administration of topical steroid per day (i.e., QD) for about one month; timing starting with and following administration of the anti-VEGF agent. In some embodiments, the topical steroid treatment comprises about four administrations of topical steroid per day (i.e., QID) forabout30days, followedbyabout threeadministrationsof topical steroidperday (i.e.,TID) for about30days, followedby about two administrations of topical steroid per day (i.e.,BID) for about 30 days, and followed by about one administration of topical steroid per day (i.e., QD) for about 30 days; timing starting with and following administration of the anti-VEGF agent. In some embodiments, the topical steroid treatment comprises about four administrations of topical steroid per day (i.e.,QID) on Day 1 to about Day 30, followed by about three administrations of topical steroid per day (i.e., TID) on about Day31 toaboutDay60, followedbyabout twoadministrationsof topical steroid per day (i.e.,BID) onaboutDay61 toabout Day 90, and followed by about one administration of topical steroid per day (i.e.,QD) on about Day 91 to about Day 120; timing starting at Day 1. In some embodiments, the topical steroid treatment is continued if inflammation is present. Vectors for Delivering Transgenes to Target Cells

[0154] In some embodiments, the recombinant adeno-associated virus (rAAV) particles comprise a recombinant viral vector derived from adeno-associated virus (AAV) that has been altered so that it is replication-defective in the subject (e.g., a human or a non-human primate). In some embodiments, the adeno-associated virus (AAV) is a recombinant AAV (rAAV).

[0155] AAV or rAAV are small non-enveloped single-stranded DNA viruses. rAAVs are non-pathogenic human parvoviruses and can be made to be dependent on helper viruses, including adenovirus, herpes simplex virus, vaccinia virus and CMV, for replication.

[0156] Exposure to wild type (wt) AAV is not associated or known to cause any human pathologies and is common in the general population, making AAVor rAAVa suitable delivery system for gene therapy. AAVand rAAVused for gene therapy for delivery of an anti-VEGF agent, e.g., aflibercept, can be of any serotype. In some embodiments, the methods of the disclosureprovide for useof any suitableAAVserotype, includingAAV1,AAV2,AAV2.5,AAV3,AAV4,AAV5,AAV6,AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, rh10, AAV-DJ, and any hybrid or chimeric AAV thereof. In some embodiments, the serotype used is based on tropism of the virus, or infectivity of a target cell of interest. In some embodiments, several AAV 40 EP 4 696 318 A1 5 10 15 20 25 30 35 40 45 50 55 vectorsmaybe generated to allow selection of themost optimal serotype for use with an anti-VEGFagent transgene (e.g., aflibercept transgene).

[0157] In some embodiments, the methods of the present disclosure provide for the use of pseudotyped AAV. Pseudotyped AAV particles comprise AAV genome inverted terminal repeats (ITRs) of one AAV serotype encapsidated by an AAV capsid of another AAV serotype. Typically, pseudotyped AAV is designated as "AAV# / #", where the first "#" indicates the AAV ITR serotype and the second "#" indicates capsid serotype. For example, an AAV particle comprising AAV2 ITRs and an AAV1 capsid would be designated "AAV2 / 1".

[0158] In some embodiments, the rAAV particles comprise a nucleic acid, e.g., a heterologous nucleic acid. In some embodiments, the nucleic acid encodes a transgene, e.g., an anti-VEGF agent (e.g., aflibercept). In some embodiments, the encoded transgene, e.g., anti-VEGF agent, is under the transcriptional control of a promoter that initiates transcription of the nucleic acid. In some embodiments, the promoter is a "ubiquitous" promoter. In some embodiments, the promoter is a "strong" or constitutively active promoter, e.g., a cytomegalovirus (CMV) promoter, an elongation factor 1 alpha (EFla) promoter, a glyceraldehyde 3-phosphate dehydrogenase (GAPDH) promoter, or a connexin36 (or "Cx36") promoter. In some embodiments, the promoter is a tissue-specific promoter that is activated in specific tissues or cells, such as retinal cells, to reducepotential toxicity or undesirable effects to non-targeted cells. In someaspects, severalAAVvectorsmaybe generated to allow selection of the most optimal serotype and promoter for use with the anti-VEGF agent transgene (e.g., aflibercept transgene). In someembodiments, the nucleic acid is flankedbyAAV inverted terminal repeats (ITRs). In some embodiments, the nucleic acid is flanked by AAV2 ITRs.

[0159] In some embodiments, the AAV vector comprises a polynucleotide cassette for enhanced expression of a transgene (e.g., an anti-VEGF agent such as aflibercept) in a target cell (e.g., a retinal cell). In some embodiments, the polynucleotide cassette comprises in 5’ to 3’ order: (a) a first enhancer region comprising a CMV sequence (SEQ ID NO: 22); (b) a promoter region, comprising a CMV sequence (SEQ ID NO: 23); (c) a 5’UTR region comprising, in 5’ to 3’ order, TPLandeMLPsequences (SEQIDNO:24andSEQIDNO:25, respectively); (d) acodingsequenceencodingapeptideor polypeptide (e.g., an anti-VEGF agent such as aflibercept); (e) a second enhancer region comprising a full EES sequence (SEQ IDNO:26); and (f) aHGHpolyadenylation site (SEQ IDNO:27). In certainof theseembodiments, thepolynucleotide cassette comprises one or more sequences selected from SEQ ID NOs: 28‑32, or a sequence with at least 85% identity thereto. In certain of theseembodiments, the5’ armof thepolynucleotide cassette comprisesor consists ofSEQ IDNO:33 or a sequence with at least 85% identity thereto. In certain of theseembodiments, the 3’ armof the polynucleotide cassette comprises or consists of SEQ ID NO: 34 or a sequence with at least 85% identity thereto. The nucleic acid sequences of SEQ ID NOs: 22‑34 are provided below: 41 EP 4 696 318 A1 5 10 15 20 25 30 35 40 45 50 55 TTGATATTCA CCTGGCCCGA TCTGGCCATA CACTTG (SEQ OD NO: 30) CCCAGGTCCA AGTTTAAACG CC (SEQ ID NO: 31) 42 EP 4 696 318 A1 5 10 15 20 25 30 35 40 45 50 55

[0160] In someembodiments, thepolynucleotidecassette comprisesor consistsofSEQIDNO:39orasequencewithat least 85% identity thereto. 43 EP 4 696 318 A1 5 10 15 20 25 30 35 40 45 50 55 44 EP 4 696 318 A1 5 10 15 20 25 30 35 40 45 50 55

[0161] SEQIDNO:39shownabovecomprises, in the5’ to3’ direction, an inverted terminal repeat (ITR) ofAAVserotype 2comprisingnucleotides1‑145ofSEQIDNO:39; aCMVpromoter comprisingnucleotides180‑693ofSEQIDNO:39; a5’ Untranslated Region (UTR), including an Adenovirus Tripartite Leader Sequence and Synthetic Intron, and comprising nucleotides 694‑1314 of SEQ ID NO: 39; a Kozak sequence comprising nucleotides 1329‑1340 of SEQ ID NO: 39; a codon-optimized aflibercept cDNA sequence comprising nucleotides 1338‑2714 of SEQ ID NO: 39; a 3’ UTR including a human scaffold attachment region and comprising nucleotides 2717‑3527 of SEQ ID NO: 39; a human growth hormone polyadenylation / transcription stop signal comprising nucleotides 3546‑3748 of SEQ ID NO: 39; and an inverted terminal repeat (ITR) of AAV serotype 2 comprising nucleotides 3772‑3916 of SEQ ID NO: 39. See, e.g., FIG. 1C.

[0162] Additional polynucleotide cassettes for enhanced expression of a transgene (e.g., a transgeneencoding an anti- VEGF agent such as aflibercept) in a target cell (such as a retinal cell) are disclosed in WO2018 / 170473, the contents of which related to polynucleotide cassettes for enhanced expression of a transgene in a target cell are incorporated herein by reference.

[0163] In some embodiments, the rAAV particles comprise a variant capsid protein having increased infectivity of target cells, e.g. retinal cells, are used to increase transduction of retinal cells or to increase targeting of gene delivery to retinal cells in an individual. In someembodiments, the rAAVparticle comprisesanaminoacidmodification inacapsidproteinGH loop / loop IVof theAAV capsid protein. In some embodiments, the site of modification is a solvent-accessible portion of the GH loop / loop IVof the AAV capsid protein. For a description of the GH loop / loop IVof AAV capsid, see, e.g., van Vliet et al. (2006) Mol. Ther. 14:809; Padron et al. (2005) J. Virol. 79:5047; and Shen et al. (2007) Mol. Ther. 15:1955. Several AAV capsid variants are known, including the 7m8 variant. In some embodiments, a rAAV particle comprises a variant AAV capsid protein that comprises an insertion of from 5 amino acids to 11 amino acids, e.g., 7 amino acid sequence, in the GH loop of a capsid protein relative to a corresponding parental AAV capsid protein, and wherein the variant capsid protein confers increased infectivity of a retinal cell compared to the infectivity of the retinal cell by an AAV particle comprising the corresponding parental or unmodified AAV capsid protein. In some embodiments, any one of the following amino acid sequences can be inserted in the GH loop of a capsid protein: LALGETTRPA (SEQ ID NO: 1); LANETITRPA (SEQ ID NO: 2), LAKAGQANNA (SEQ IDNO: 3), LAKDPKTTNA (SEQ ID NO: 4), KDTDTTR (SEQ IDNO: 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 45 EP 4 696 318 A1 5 10 15 20 25 30 35 40 45 50 55 sequences set forth in SEQ ID NOs: 1‑12 and 14‑21 is inserted in the solvent-exposed GH loop of VP1 capsid protein in a rAAV. Additional details regarding amino acid sequences that can be inserted into the GH loop of a capsid protein, e.g., to facilitate transduction of a nucleic acid of interest to a retinal cell following IVT injection, are provided in WO2012145601, US9587282, US10202657, and US10214785, the contents of which related to amino acid sequences that can be inserted into the GH loop of a capsid protein are incorporated herein by reference.

[0164] In some embodiments, the rAAV particles comprise an AAV capsid protein, e.g., an AAV2 capsid protein, that includes any one of the following amino acid sequences: LALGETTRPA (SEQ ID NO: 1); LANETITRPA (SEQ ID NO: 2), LAKAGQANNA (SEQ ID NO: 3), LAKDPKTTNA (SEQ ID NO: 4), KDTDTTR (SEQ ID NO: 5), RAGGSVG (SEQ IDNO: 6), AVDTTKF (SEQ IDNO: 7), STGKVPN (SEQ IDNO: 8), LAKDTDTTRA (SEQ IDNO: 9), LARAGGSVGA (SEQ IDNO: 10), LAAVDTTKFA (SEQ ID NO: 11), and LASTGKVPNA (SEQ ID NO: 12), LGETTRP (SEQ ID NO: 14), NETITRP (SEQ ID NO:15), KAGQANN(SEQ IDNO:16), KDPKTTN (SEQ IDNO:17),KDTDTTR (SEQ IDNO:18),RAGGSVG(SEQ IDNO: 19), AVDTTKF (SEQ ID NO: 20), and STGKVPN (SEQ ID NO: 21) inserted at the following positions: between positions 587 and 588 of the AAV2 capsid protein; between amino acids 590 and 591 of the AAV1 capsid protein; between amino acids 575 and 576 of the AAV5 capsid protein; between amino acids 590 and 591 of the AAV6 capsid protein; between amino acids 589 and 590 of the AAV7 capsid protein; between amino acids 590 and 591 of the AAV8 capsid protein; between amino acids 588 and 589 of the AAV9 capsid protein; or between amino acids 589 and 590 of the AAV10 capsid protein. In some embodiments, the rAAV particles comprise AAV2 capsid proteins comprising an amino acid sequence LALGETTRPA (SEQ ID NO: 1) inserted between positions 587 and 588 of the capsid protein, wherein the amino acid residuenumbering corresponds toanAAV2VP1capsidprotein. In someembodiments, the rAAVparticles compriseAAV2 capsid proteins comprising the amino acid sequence LALGETTRPA (SEQ ID NO: 1) inserted between positions 587 and 588 of the AAV2 VP1 comprising the sequence of SEQ ID NO: 13.

[0165] In some embodiments, rAAV particles comprise the 7m8 variant capsid protein from AAV2 comprising the amino acid sequence LALGETTRPA (SEQ ID NO: 1) inserted in the GH loop of the AAV2 VP1 protein between positions 587 and 588 of the AAV2 VP1. In some embodiments, the rAAV particles comprise an AAV2 VP1 capsid protein comprising a GH loop that comprises the amino acid sequence of SEQ ID NO: 38 or an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 38. In some embodiments, the rAAV particles comprise an AAV2 VP1 capsid protein comprising a GH loop that comprises an amino acid sequence having any of at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 38.

[0166] In some embodiments, rAAV particles comprise the 7m8 variant capsid protein from AAV2 comprising the amino acid sequence LALGETTRPA (SEQ ID NO: 1) inserted between positions 587 and588 of theAAV2 VP1. The sequence of the 7m8 variant capsid protein from AAV2 comprising the amino acid sequence LALGETTRPA (SEQ ID NO: 1) is inserted between positions 587 and 588 of the AAV2 VP1 is provided below: 46 EP 4 696 318 A1 5 10 15 20 25 30 35 40 45 50 55

[0167] In some embodiments, the rAAV particles comprise a capsid protein VP1 comprising the amino acid sequence LGETTRP (SEQ ID NO: 14) inserted between positions 587 and 588 of the capsid protein, wherein the amino acid residue numbering corresponds to an AAV2 VP1 capsid protein. In some embodiments, the rAAV particles comprise a capsid protein VP2 comprising the amino acid sequence LGETTRP (SEQ ID NO: 14) inserted between positions 587 and 588 of the capsid protein, wherein the amino acid residue numbering corresponds to an AAV2 VP1 capsid protein. In some embodiments, the rAAV particles comprise a capsid protein VP3 comprising the amino acid sequence LGETTRP (SEQ ID NO: 14) inserted between positions 587 and 588 of the capsid protein, wherein the amino acid residue numbering corresponds to an AAV2 VP1 capsid protein. In some embodiments, the rAAV particles comprise capsid proteins VP1, VP2, and VP3, wherein each of VP1, VP2, and VP3 comprise the amino acid sequence LGETTRP (SEQ ID NO: 14) inserted between positions 587 and 588 of the capsid protein, wherein the amino acid residue numbering corresponds to an AAV2 VP1 capsid protein.

[0168] In some embodiments, the rAAV particles comprise a capsid protein VP1 comprising the amino acid sequence LALGETTRPA (SEQ ID NO: 1) inserted between positions 587 and 588 of the capsid protein, wherein the amino acid residue numbering corresponds to an AAV2 VP1 capsid protein. In some embodiments, the rAAV particles comprise a capsid protein VP2 comprising the amino acid sequence LALGETTRPA (SEQ ID NO: 1) inserted between positions 587 and 588 of the capsid protein, wherein the amino acid residue numbering corresponds to an AAV2 VP1 capsid protein. In some embodiments, the rAAV particles comprise a capsid protein VP3 comprising the amino acid sequence LALGETTR- PA (SEQ ID NO: 1) inserted between positions 587 and 588 of the capsid protein, wherein the amino acid residue numbering corresponds to an AAV2 VP1 capsid protein. In some embodiments, the rAAV particles comprise capsid proteinsVP1,VP2, andVP3,whereineachofVP1,VP2,andVP3comprise theaminoacid sequenceLALGETTRPA(SEQ ID NO: 1) inserted between positions 587 and 588 of the capsid protein, wherein the amino acid residue numbering corresponds to an AAV2 VP1 capsid protein.

[0169] In some embodiments, a recombinant virus and / or plasmid used to generate a rAAV virus comprises other transcriptional or regulatory elements, such as a poly A (polyadenylation) sequence, untranslated regions (UTRs), 3’ UTRs, or termination sequences. In some embodiments, more than one gene is expressed from the vector or plasmid using internal ribosome entry site (IRES) or similar element that allows co-expression of two or more proteins or create multigene, or polycistronic mRNA.

[0170] In some embodiments, the rAAV and / or plasmid used to generate the rAAV comprises one or more of the following nucleic acid elements: a first ITR sequence; a promoter sequence; an intron sequence; a first UTR sequence; a heterologous nucleic acid encoding an anti-VEGF agent (e.g., aflibercept); a second UTR sequence; a polyA sequence; and a second ITR sequence. In some embodiments, linker sequence(s) are inserted between two or more of the nucleic acid elements. In some embodiments, the heterologous nucleic acid encodes a therapeutic polypeptide, e.g., encodes aflibercept (or a functional fragment or functional variant thereof).

[0171] In some embodiments, the vector is a targeted vector, especially a targeted rAAV (e.g., AAV2.7m8) that shows higher infectivity of a specific cell, such as a retinal cell (e.g., a photoreceptor, a retinal ganglion cell, a Müller cell, a bipolar cell, an amacrine cell, a horizontal cell, or a retinal pigmented epithelium cell). Viral vectors for use in the disclosure can include those that exhibit low toxicity and / or low immunogenicity in an individual and expresses therapeutically effective quantities of the anti-VEGF agent (e.g., aflibercept) in an individual, e.g., a human. Any suitable method known in the art 47 EP 4 696 318 A1 5 10 15 20 25 30 35 40 45 50 55 can be used in the biochemical purification of recombinant viruses (e.g., rAAV), e.g., for the preparation of pharmaceutical compositions described elsewhere herein. Recombinant AAV viruses can be harvested directly from cells, or from the culture media comprising cells. Virus can be purified using various biochemical means, such as gel filtration, filtration, chromatography, affinity purification, gradient ultracentrifugation, or size exclusion methods. In some embodiments, the virus is lyophilized.

[0172] In someembodiments, the rAAVparticles comprise a 7m8variant capsid protein, e.g., rAAV2.7m8, and a nucleic acid sequence that encodesananti-VEGFagent (e.g., aflibercept, or a functional fragment or functional variant thereof). In some embodiments, the rAAV particles (e.g., the 7m8 variant) have an increase in retinal cell infectivity of any of at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 100% as compared to an AAV particle comprising the corresponding parental or unmodified AAV capsid protein. In some embodiments, the increase in infectivity of retinal cells is an increase of any of between 5% to 100%, between 5% to 95%, between 5% to 90%, between 5% to 85%, between 5% to 80%, between 5% to 75%, between 5% to 70%, between 5% to 65%, between 5% to 60%, between 5% to 55%, between 5% to 50%, between 5% to 45%, between 5% to 40%, between 5% to 35%, between 5% to 30%, between 5% to 25%, between 5% to 20%, between 5% to 15%, or between 5% to 10%, as compared to an AAV particle comprising the corresponding parental or unmodified AAV capsid protein.

[0173] In someembodiments, the increase in retinal cell infectivity of a rAAVvariant, e.g., rAAV2.7m8, is anyof at least 1- fold, at least 1.1-fold, at least 1.2-fold, at least 1.3-fold, at least 1.4-fold, at least 1.5-fold, at least 1.6-fold, at least 1.7-fold, at least 1.8-fold, at least 1.9-fold, or at least 2-fold compared to an AAV particle comprising the corresponding parental or unmodified AAV capsid protein. In some embodiments, the increase in infectivity is any of at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, or at least 10-fold, as compared toan AAV particle comprising the corresponding parental AAV capsid protein. In some embodiments, the increase in infectivity is any of at least 15-fold, at least 20-fold, at least 25-fold, at least 30-fold, at least 35-fold, at least 40-fold, at least 45-fold, at least 50-fold, at least 55-fold, at least 60-fold, at least 65-fold, at least 70-fold, at least 75-fold, at least 80-fold, at least 85- fold, at least 90-fold, or at least 100-fold compared toanAAVparticle comprising the correspondingparental or unmodified AAV capsid protein.

[0174] In some embodiments, the increase in retinal cell infectivity of a rAAV variant, e.g., rAAV2.7m8, is between 10- fold to 100-fold, between 10-fold to 95-fold, between 10-fold to 90-fold, between 10-fold to 85-fold, between 10-fold to 80- fold, between 10-fold to 75-fold, between 10-fold to 70-fold, between 10-fold to 65-fold, between 10-fold to 60-fold, between10-fold to 55-fold, between10-fold to 50-fold, between10-fold to 45-fold, between10-fold to 40-fold, between10- fold to 35-fold, between 10-fold to 30-fold, between 10-fold to 25-fold, between 10-fold to 20-fold, or between10-fold to 15- fold, as compared to an AAV particle comprising the corresponding parental or unmodified AAV capsid protein.

[0175] In someembodiments, the increase in retinal cell infectivity is between2-fold to20-fold, between2-fold to19-fold, between 2-fold to 18-fold, between 2-fold to 17-fold, between 2-fold to 16-fold, between 2-fold to 15-fold, between 2-fold to 14-fold, between 2-fold to 13-fold, between 2-fold to 12-fold, between 2-fold to 11-fold, between 2-fold to 10-fold, between 2-fold to 9-fold, between 2-fold to 8-fold, between 2-fold to 7-fold, between 2-fold to 6-fold, between 2-fold to 5-fold, between 2-fold to 4-fold, or between 2-fold to 3-fold, as compared to an AAV particle comprising the corresponding parental or unmodified AAV capsid protein.

[0176] In some embodiments, an amino acid modification of a capsid protein described herein can confer an increase in anability to crossan internal limitingmembrane (ILM) in aneyeof an individual, e.g., a human, as compared to theability of an AAV particle comprising the corresponding parental or unmodified AAV capsid protein to cross the ILM in the eye of the subject. In someembodiments, the increase in theability to cross the ILMofa rAAVvariant, e.g., rAAV2.7m8, isan increase of anyof at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 100% as compared to an AAV particle comprising the corresponding parental or unmodified AAV capsid protein. In some embodiments, the increase in the ability to cross the ILM is an increase of between 5% to 100%, between 5% to 95%, between 5% to 90%, between 5% to 85%, between 5% to 80%, between 5% to 75%, between 5% to 70%, between 5% to 65%, between 5% to 60%, between 5% to 55%, between 5% to 50%, between 5% to 45%, between 5% to 40%, between 5% to 35%, between 5% to 30%, between 5% to 25%, between 5% to 20%, between 5% to 15%, or between 5% to 10%, as compared to the parental or unmodified AAV capsid protein.

[0177] In some embodiments, the increase in the ability to cross the ILM of a rAAV variant, e.g., rAAV2.7m8, is any of at least 1-fold, at least 1.1-fold, at least 1.2-fold, at least 1.3-fold, at least 1.4-fold, at least 1.5-fold, at least 1.6-fold, at least 1.7-fold, at least 1.8-fold, at least 1.9-fold, or at least 2-fold compared to an AAV particle comprising the corresponding parental AAV capsid protein. In some embodiments, the increase in the ability to cross the ILM is any of at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, or at least 10-fold, as compared to an AAV particle comprising the corresponding parental AAV capsid protein. In some embodiments, the increase in the ability to cross the ILM is any of at least 15-fold, at least 20-fold, at least 25-fold, at least 30-fold, at least 35- fold, at least 40-fold, at least 45-fold, at least 50-fold, at least 55-fold, at least 60-fold, at least 65-fold, at least 70-fold, at least 75-fold, at least 80-fold, at least 85-fold, at least 90-fold, or at least 100-fold compared to an AAV particle comprising 48 EP 4 696 318 A1 5 10 15 20 25 30 35 40 45 50 55 the corresponding parental or unmodified AAV capsid protein.

[0178] In some embodiments, the increase in the ability to cross the ILM of a rAAV variant, e.g., rAAV2.7m8, is between 10-fold to 100-fold, between 10-fold to 95-fold, between 10-fold to 90-fold, between 10-fold to 85-fold, between 10-fold to 80-fold, between 10-fold to 75-fold, between 10-fold to 70-fold, between 10-fold to 65-fold, between 10-fold to 60-fold, between10-fold to 55-fold, between10-fold to 50-fold, between10-fold to 45-fold, between10-fold to 40-fold, between10- fold to 35-fold, between 10-fold to 30-fold, between 10-fold to 25-fold, between 10-fold to 20-fold, or between10-fold to 15- fold as compared to an AAV particle comprising the corresponding parental or unmodified AAV capsid protein.

[0179] In some embodiments, the increase in the ability to cross the ILM of a rAAV variant, e.g., rAAV2.7m8, is between 2-fold to 20-fold, between 2-fold to 19-fold, between 2-fold to 18-fold, between 2-fold to 17-fold, between 2-fold to 16-fold, between 2-fold to 15-fold, between 2-fold to 14-fold, between 2-fold to 13-fold, between 2-fold to 12-fold, between 2-fold to 11-fold, between 2-fold to 10-fold, between 2-fold to 9-fold, between 2-fold to 8-fold, between 2-fold to 7-fold, between 2- fold to 6-fold, between 2-fold to 5-fold, between 2-fold to 4-fold, or between 2-fold to 3-fold, as compared to an AAV particle comprising the corresponding parental or unmodified AAV capsid protein.

[0180] In some embodiments, rAAV.7m8comprising nucleic acid encoding aflibercept is used for gene therapy. In some embodiments, AAV2 or rAAV2 is used to deliver a nucleic acid sequence encoding an anti-VEGF agent (e.g., aflibercept) into an eye or retinal cells of a subject via intravitreal or subretinal injection. In some embodiments, AAV2 or rAAV2 is used todeliveranucleicacid sequenceencodingananti-VEGFagent (e.g., aflibercept) intoaneyeor retinal cells of asubject via intravitreal injection. In some embodiments, rAAV2.7m8 is used to deliver the nucleic acid sequence of the anti-VEGF agent (e.g., aflibercept) into the retinal cells of a subject. In some embodiments, the heterologous nucleic acid (e.g., a nucleic acid that encodes an anti-VEGF agent such as aflibercept) integrates into the target cell genome (e.g., retinal cell genome), resulting in long-term expression of, e.g., the anti-VEGF agent (such as aflibercept), in the target cell. In some embodiments, the viral vector delivers a plasmid or other extrachromosomal genetic element that comprises the heterologous nucleic acid (e.g., a nucleic acid that encodes an anti-VEGF agent such as aflibercept) to the target cell (e.g., retinal cell).

[0181] In some embodiments, the rAAV particles comprise a nucleic acid encoding a polypeptide comprising an amino acid sequence with any of at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% identity to the amino acid sequence of SEQ ID NO: 35 and flanked by AAV2 inverted terminal repeats (ITRs). In some embodiments, the rAAV particles comprise a nucleic acid encoding a polypeptide comprising an amino acid sequence with at least about 95% identity to the amino acid sequence of SEQ ID NO: 35 and flanked by AAV2 inverted terminal repeats (ITRs). In some embodiments, the rAAV particles comprise a nucleic acid encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 35 and flanked by AAV2 inverted terminal repeats (ITRs). In some embodiments, the rAAV particles comprise a nucleic acid encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 35. In some embodiments, the rAAV particles comprise a nucleic acid encoding aflibercept and flanked by AAV2 inverted terminal repeats (ITRs). The sequence of SEQ ID NO: 35 is provided below:

[0182] In some embodiments, the rAAV particles comprise a nucleic acid with any of at least about 75%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.9%, or at least about 100%sequence homology to the nucleic acid sequence of SEQ IDNO:36, andwherein thenucleic acid is flankedbyAAV2 inverted terminal repeats (ITRs). ThesequenceofSEQ ID NO: 36 is provided in FIG. 5. In some embodiments, the rAAV particles comprise a nucleic acid with any of at least about 75%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.9%, or at least about 100% sequence homology to the nucleic acid sequence of aflibercept (e.g., SEQ ID NO: 36), and wherein the nucleic acid is flanked by AAV2 inverted 49 EP 4 696 318 A1 5 10 15 20 25 30 35 40 45 50 55 terminal repeats (ITRs). In some embodiments, the nucleic acid sequence of aflibercept is derived from its amino acid sequence. In some embodiments, the nucleic acid sequence of aflibercept is codon optimized to improve its expression in a subject. In some embodiments, the rAAV particles comprise a nucleic acid with any of at least about 75%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.9%, or 100% sequence homology to the nucleic acid sequence of SEQ ID NO: 40, and wherein the nucleic acid is flanked by AAV2 inverted terminal repeats (ITRs). In some embodiments, the rAAV particles compriseanucleic acid comprising thenucleic acid sequenceofSEQ IDNO:40. In someembodiments, the rAAV particles comprise a nucleic acid comprising the nucleic acid sequence of SEQ ID NO: 40, and wherein the nucleic acid is flanked by AAV2 inverted terminal repeats (ITRs).

[0183] In some embodiments, the rAAV particles comprise a nucleic acid encoding a polypeptide comprising an amino acid sequence with any of at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%,or 100% identity to theaminoacid sequenceofSEQ IDNO:41andflankedbyAAV2 inverted terminal repeats (ITRs). In some embodiments, the rAAV particles comprise a nucleic acid encoding a polypeptide comprising an amino acid sequence with at least about 95% identity to the amino acid sequence of SEQ ID NO: 41 and flanked by AAV2 inverted terminal repeats (ITRs). In some embodiments, the rAAV particles comprise a nucleic acid encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 41 and flanked by AAV2 inverted terminal repeats (ITRs). In some embodiments, the rAAV particles comprise a nucleic acid encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 41. 50 EP 4 696 318 A1 5 10 15 20 25 30 35 40 45 50 55

[0184] In some embodiments, the nucleic acid sequence of aflibercept is codon-optimized for expression in a primate or a human subject. Construction of a synthetic gene corresponding to the aflibercept amino acid sequence has been described in literature, e.g., Kanda A, Noda K, Saito W, Ishida S. Aflibercept Traps Galectin‑1, an Angiogenic Factor Associated with Diabetic Retinopathy. Scientific Reports 5:17946 (2015) (describing "VEGF-TrapR1R2 (corresponding to aflibercept) cDNAwas generated as a synthetic gene by IDT (Coralville, IA)"). Given the available amino acid sequence of aflibercept, anymethodknown in theart canbeused togenerate thecDNAofaflibercept for use inagene therapyora rAAV described herein.

[0185] Codon optimization can be achieved with any method known in the art. Codon optimization refers to a process of modifying a nucleic acid sequence for enhanced expression of a gene in target or host cells of interest, e.g., human retinal cells, by replacing at least one codon (e.g., about or more than 1, 2, 3, 4, 5, 10, 15, 20, 25, 50, 100 or more codons) of a native sequence with codons that are used more frequently or are most frequently used in the host cell while maintaining the native amino acid sequence. Codon usage tables are readily available, including for examples, GenScript Codon Usage Frequency Table Tool at www(dot)genscript(dot)com / tools / codon-frequency-table; Codon Usage Database at www(dot)kazusa(dot)or(dot)jp / codon / ; and Nakamura, Y., et al. "Codon usage tabulated from the international DNA sequence databases: status for the year 2000" Nucl. Acids Res. 28:292 (2000).

[0186] Homology refers to the percent conservation of residues of an alignment between two sequences, including, but not limited to functional fragments, sequences comprising insertions, deletions, substitutions, pseudofragments, pseu- dogenes, splice variants or artificially optimized sequences.

[0187] In some embodiments, the rAAV particles comprise a nucleic acid encoding aflibercept. In some embodiments, the polypeptide is aflibercept.

[0188] As used herein, "aflibercept" refers to a polypeptide or protein sequence, or a functional fragment or variant or mutant thereof, with any of at least 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more, or 100% homology to the aflibercept amino acid sequence identified above (SEQ ID NO: 35). Homology refers to the percent conservation of residues of an alignment between two sequences, including, but not limited to functional fragments, sequences comprising insertions, deletions, substitutions, pseudofrag- ments, pseudogenes, splice variants or artificially optimized sequences.

[0189] In someembodiments, theaminoacid sequenceof aflibercept is anyof at least 75%,80%,81%,82%,83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% homologous to the afliberceptaminoacidsequenceofSEQIDNO:35. Insomeembodiments, thenucleicacid sequenceencodingaflibercept disclosed herein is compared to the corresponding cDNA sequence of the aflibercept amino acid sequence identified above, and shows any of at least 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%,99%, 99.9%, or 100%sequencehomologybetween thenucleic acid sequencesof aflibercept (e.g., SEQ ID NO: 36). In some embodiments, aflibercept is any of at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% spatially homologous to aflibercept (e.g., in terms of its secondary, tertiary, and quaternary structure or conformation). In some embodiments, aflibercept is any of at most 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% spatially homologous to the aflibercept used in the standard of care (e.g., secondary, tertiary, and quaternary structure or conformation).

[0190] In some embodiments, the aflibercept gene product, or aflibercept transgene, as included in a gene therapy based on a rAAV, comprises a capsid variant as disclosed herein (e.g., the 7m8 variant), encodes a protein, fusion protein, orpolypeptide thathasanyofat least 75%,at least80%,at least 81%,at least 82%,at least 83%,at least 84%,at least85%, at least 86%,at least 87%,at least 88%,at least 89%,at least 90%,at least 91%,at least 92%,at least 93%,at least 94%,at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% homology to the above amino acid sequence of SEQ ID NO: 35, or between the corresponding cDNA sequences of aflibercept (e.g., cDNA of aflibercept sequence used in a gene therapy compared to SEQ ID NO: 36). In some embodiments, the methods compositions disclosed herein comprise a functional fragment of aflibercept, or a variant or mutant thereof. In some embodiments, the nucleic acid sequence of aflibercept is modified or codon-optimized to enhance its activity, expression, stability, and / or 51 EP 4 696 318 A1 5 10 15 20 25 30 35 40 45 50 55 solubility in vivo.

[0191] Aflibercept is a 115 kDa fusion protein, which can be glycosylated. Aflibercept comprises an IgG backbone fused to extracellular VEGF receptor sequences of the human VEGFR‑1 and VEGFR‑2, and functions like a soluble decoy receptor by binding VEGF-A with a greater affinity than its natural or endogenous receptors. See, for example, Stewart MW. Aflibercept (VEGF Trap-eye): the newest anti-VEGF drug. Br. J. Ophthalmol. 2012 Sep;96(9):1157‑8. Aflibercept’s high affinity for VEGF interferes or disrupts subsequent binding and activation of native or endogenous VEGF receptors. Reduced VEGF activity can lead to decreased angiogenesis and vascular permeability. Inhibition of placental growth factor PIGF and VEGF-B by aflibercept may also contribute to the treatment of ocular diseases or disorders characterized by abnormal (e.g., excessive) angiogenesis and / or neovascularization. PIGF has beenassociatedwith angiogenesis and certain ocular diseases or disorders, such as wet AMD, may be associated with elevated levels of PIGF. VEGF-B overexpression canbe associated with breakdown of the blood-retinal barrier and retinal angiogenesis. Thus, inhibition of VEGF-A, VEGF-B, and PIGF may all contribute to the efficacy of aflibercept. Methods for Preparation of Vectors for Delivering Transgenes to Target Cells

[0192] In some embodiments, the rAAV particles are manufactured using any method known in the art. In some embodiments, the rAAVparticlesaremanufacturedusingabaculovirusexpression vector system inSf9 cells.Sf9 cells are an insect cell culturecell linecommonlyused for recombinant proteinproductionusingbaculovirus. In someembodiments, the rAAV particles are manufactured using two baculoviruses in Sf9 cells. In some embodiments, the rAAV particles are manufactured using two baculoviruses in Sf9 cells, wherein a first baculovirus encodes the genes for AAV2 Rep and AAV2.7m8 Cap proteins and a second baculovirus encodes an anti-VEGF agent. In some embodiments, the rAAV particles are manufactured using two baculoviruses in Sf9 cells, wherein a first baculovirus encodes the genes for AAV2 Rep and AAV2.7m8 Cap proteins and a second baculovirus encodes an aflibercept (e.g., human aflibercept) cDNA expression cassette. In some embodiments, the rAAV particles are manufactured using two baculoviruses in Sf9 cells, wherein a first baculovirus encodes the genes for AAV2 Rep and AAV2.7m8 Cap proteins and a second baculovirus comprisesanucleicacidencodingapolypeptidecomprisinganaminoacidsequencewithat least about95% 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. Doses

[0193] 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.

[0194] In someembodiments, the administering theunit doseof rAAVparticles 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 8months, at least about 9months, at least about 10months, at least about 11months, at least about 12months, 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, ormore)afteradministering theunit doseof rAAVparticles to theoneeye. Insomeembodiments, theadministering theunit doseof rAAVparticles to the contralateral eye is at least about 2weeksafter administering theunit doseof rAAVparticles to the one eye and the unit dose of rAAV particles administered to the contralateral eye of the individual is higher (e.g., any of about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 125%, about 150%, about 175%, about 200%, about 225%, about 250%, about 275%, about 300%, or more, higher) than the unit dose of rAAV particles administered to the one eye of the individual.

[0195] In someembodiments, theadministering theunit doseof rAAVparticles to thecontralateral eyeof the individual is up to about 1 week, up to about 2 weeks, up to about 3 weeks, or up to about 4 weeks after administering the unit dose of rAAV particles to the one eye. In some embodiments, the administering the unit dose of rAAV particles to the contralateral eye of the individual is up to about 2weeks (e.g., about 0 days, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10days, 11 days, 12days, 13days, or 14days) after administering the unit dose of rAAVparticles to the oneeye. In someembodiments, theadministering theunit doseof rAAVparticles to thecontralateral eyeof the individual is up to about 2weeks (e.g., about 0days, 1 day, 2days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9days, 10days, 11days, 12days, 13 days, or 14 days) after administering the unit dose of rAAV particles to the one eye and the unit dose of rAAV particles administered to the contralateral eyeof the individual is about the sameas (e.g., less than1%higher or lower, less than 5% higheror lower, less than10%higheror lower, or less than20%higheror lower), or lower (e.g., about5%,about10%,about 52 EP 4 696 318 A1 5 10 15 20 25 30 35 40 45 50 55 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, or about 90% lower) than the unit dose of rAAVparticles administered to theoneeyeof the individual. In someembodiments, theadministering theunit doseof rAAV particles to thecontralateral eyeof the individual is up toabout2weeksafter administering theunit doseof rAAVparticles to the one eye and the unit dose of rAAV particles administered to the contralateral eye of the individual is about the same (e.g., less than 1% higher or lower, less than 5% higher or lower, less than 10% higher or lower, or less than 20% higher or lower) as the unit dose of rAAV particles administered to the one eye of the individual. In some embodiments, the administering the unit dose of rAAV particles to the contralateral eye of the individual is up to about 2 weeks after administering the unit dose of rAAV particles to the one eye and the unit dose of rAAV particles administered to the contralateral eye of the individual is 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 theunit dose of rAAVparticles administered to theoneeyeof the individual.

[0196] In someembodiments, theunit doseof rAAVparticles is administered tooneeyeand / or to thecontralateral eyeof the individual. In someembodiments, theunit doseof rAAVparticles is expressedas thenumberof vectorgenomes (vg). In some embodiments, the unit dose is about 6 × 1011 vector genomes (vg) or less of the rAAV particles. In some embodiments, the unit dose is about 1×1010 to about 2×1010, between about 2×1010 to about 3×1010, between about 3×1010 to about 4×1010, between about 4×1010 to about 5×1010, between about 5×1010 to about 6×1010, between about 6×1010 to about 7×1010, between about 7×1010 to about 8×1010, between about 8×1010 to about 9×1010, between about 9×1010 to about 10×1010, between about 1×1011 to about 2×1011, between about 2×1011 to about 3×1011, between about 3×1011 to about 4×1011, between about 4×1011 to about 5× 1011, or between about 5×1011 to about 6×1011 vg of the rAAV particles, including any value within these ranges, of the rAAV particles. In some embodiments, the unit dose is about 6×1010 vector genomes (vg) to about 2×1011 vg of the rAAV particles. In some embodiments, theunit dose isabout 6×1010vg toabout 2×1011vg, about 7×1010vg toabout2×1011 vg,about 8×1010vg to about 2×1011 vg, about 9×1010 vg to about 2×1011 vg, about 10×1010 vg to about 2×1011 vg, or about 1×1011 vg to about 2×1011 vg of the rAAV particles. In some embodiments, the unit dose is about 6×1010 vg to about 2×1011 vg of the rAAV particles. In some embodiments, the unit dose is about 6×1010 vg to about 7×1010 vg, about 7×1010 vg to about 8×1010 vg, about 8×1010 vg to about 9×1010 vg, about 9×1010 vg to about 10×1010 vg, about 10×1010 vg to about 1×1011 vg, or about 1×1011 vg to about 2×1011 vg of the rAAV particles. In some embodiments, the unit dose is about 6×1010 vg, about 7×1010 vg, about 8×1010 vg, about 9×1010 vg, about 10×1010 vg, about 1×1011 vg, or about 2×1011 vg of the rAAV particles. In some embodiments, the unit dose is about 6×1010 vg or about 2×1011 vg of the rAAV particles. In someembodiments, the unit dose is about 6×1010 vgof the rAAVparticles. In some embodiments, the unit dose is about 6 × 1010 vg, about 2 × 1011 vg, or about 6× 1011 vg. In some embodiments, the unit dose is about 6 × 1010 vg. In some embodiments, the unit dose is about 2 × 1011 vg. In some embodiments, the unit dose is about 6× 1011 vg.

[0197] In someembodiments, theunit doseof rAAVparticles is administered tooneeyeand / or to thecontralateral eyeof the individual. In someembodiments, theunit dose is expressedas thenumber of vector genomes (vg) per eye (vg / eye). In some embodiments, the unit dose is about 6 × 1011 vg / eye or less of the rAAV particles. In some embodiments, the unit dose is about 1×1010 to about 2×1010, betweenabout 2×1010 to about 3×1010, betweenabout 3×1010 to about 4×1010, between about 4×1010 to about 5×1010, between about 5×1010 to about 6×1010, between about 6×1010 to about 7×1010, between about 7×1010 to about 8×1010, between about 8×1010 to about 9×1010, between about 9×1010 to about 10×1010, between about 1×1011 to about 2×1011, betweenabout 2×1011 to about 3×1011, betweenabout 3×1011 to about 4×1011, between about 4×1011 to about 5×1011, or between about 5×1011 to about 6×1011 vg / eye of the rAAV particles, including any value within these ranges, of the rAAV particles. In some embodiments, the unit dose is about 6×1010 vg / eye to about 2×1011 vg / eye of the rAAV particles. In some embodiments, the unit dose is about 6×1010 vg / eye to about 2×1011 vg / eye, about 7×1010 vg / eye to about 2×1011 vg / eye, about 8×1010 vg / eye to about 2×1011 vg / eye, about 9×1010 vg / eye to about 2×1011 vg / eye, about 10×1010 vg / eye to about 2×1011 vg / eye, or about 1×1011 vg / eye to about 2×1011 vg / eye of the rAAV particles. In some embodiments, the unit dose is about 6×1010 vg / eye to about 2×1011 vg / eye of the rAAV particles. In some embodiments, the unit dose is about 6×1010 vg / eye to about 7×1010 vg / eye, about 7×1010 vg / eye to about 8×1010 vg / eye, about 8×1010 vg / eye to about 9×1010 vg / eye, about 9×1010 vg / eye to about 10×1010 vg / eye, about 10×1010 vg / eye to about 1×1011 vg / eye, or about 1×1011 vg / eye to about 2×1011 vg / eye of the rAAV particles. In some embodiments, the unit dose is about 6×1010 vg / eye, about 7×1010 vg / eye, about 8×1010 vg / eye, about 9×1010 vg / eye, about 10×1010 vg / eye, about 1×1011 vg / eye, or about 2×1011 vg / eyeof the rAAVparticles. In some embodiments, the unit dose is about 6×1010 vg / eye or about 2×1011 vg / eye of the rAAV particles. In some embodiments, the unit dose is about 6×1010 vg / eye of the rAAV particles. In some embodiments, the unit dose is about 6 × 1010 vg / eye, about 2 × 1011 vg / eye, or about 6× 1011 vg / eye. In some embodiments, the unit dose is about 6 × 1010 vg / eye. In some embodiments, the unit dose is about 2 × 1011 vg / eye. In some embodiments, the unit dose is about 6× 1011 vg / eye.

[0198] In someembodiments, theunit doseof rAAVparticles is administered tooneeyeand / or to thecontralateral eyeof the individual. In someembodiments,E isa shorthand forbase10 for exponentiation, andxEy refers to xmultipliedbybase 10 to the y power / exponent. In some embodiments, the unit dose is expressed as the number of vector genomes (vg). In some embodiments, the unit dose is about 6E11 vector genomes (vg) or less of the rAAV particles. In some embodiments, 53 EP 4 696 318 A1 5 10 15 20 25 30 35 40 45 50 55 the unit dose is about 1E10 to about 2E10, between about 2E10 to about 3E10, between about 3E10 to about 4E10, between about 4E10 to about 5E10, between about 5E10 to about 6E10, between about 6E10 to about 7E10, between about 7E10 to about 8E10, between about 8E10 to about 9E10, between about 9E10 to about 10E10, between about 1E11 to about 2E11, betweenabout 2E11 toabout3E11, betweenabout 3E11 toabout4E11, betweenabout 4E11 toabout5E11, or betweenabout 5E11 to about 6E11 vg of the rAAV particles, including any value within these ranges, of the rAAV particles. In some embodiments, the unit dose is about 6E10 vector genomes (vg) to about 2E11 vg of the rAAV particles. In some embodiments, the unit dose is about 6E10 vg to about 2E11 vg, about 7E10 vg to about 2E11 vg, about 8E10 vg to about 2E11 vg, about 9E10 vg to about 2E11 vg, about 10E10 vg to about 2E11 vg, or about 1E11 vg to about 2E11 vg of the rAAV particles. In some embodiments, the unit dose is about 6E10 vg to about 2E11 vg of the rAAV particles. In some embodiments, the unit dose is about 6E10 vg to about 7E10 vg, about 7E10 vg to about 8E10 vg, about 8E10 vg to about 9E10 vg, about 9E10 vg to about 10E10 vg, about 10E10 vg to about 1E11 vg, or about 1E11 vg to about 2E11 vg of the rAAV particles. In some embodiments, the unit dose is about 6E10 vg, about 7E10 vg, about 8E10 vg, about 9E10 vg, about 10E10 vg, about 1E11 vg, or about 2E11 vg of the rAAV particles. In some embodiments, the unit dose is about 6E10 vg or about 2E11 vg of the rAAV particles. In some embodiments, the unit dose is about 6E10 vg of the rAAV particles. In some embodiments, theunit dose isabout 6E10 vg, about 2E11 vg, or about 6E11 vg. In someembodiments, theunit dose is about 6E10 vg. In some embodiments, the unit dose is about 2E11 vg. In some embodiments, the unit dose is about 6E11 vg.

[0199] In someembodiments, theunit doseof rAAVparticles is administered tooneeyeand / or to thecontralateral eyeof the individual. In someembodiments, theunit dose is expressedas thenumber of vector genomes (vg) per eye (vg / eye). In someembodiments, theunit dose is about 6E11 vg / eyeor lessof the rAAVparticles. In someembodiments, theunit dose is about 1E10 to about 2E10, between about 2E10 to about 3E10, between about 3E10 to about 4E10, between about 4E10 to about 5E10, between about 5E10 to about 6E10, between about 6E10 to about 7E10, between about 7E10 to about 8E10, betweenabout 8E10 to about 9E10, betweenabout 9E10 to about 10E10, betweenabout 1E11 to about 2E11, betweenabout 2E11 to about 3E11, between about 3E11 to about 4E11, between about 4E11 to about 5E11, or between about 5E11 to about 6E11 vg / eyeof the rAAVparticles, includinganyvaluewithin these ranges, of the rAAVparticles. In someembodiments, the unit dose isabout6E10vg / eye toabout2E11vg / eyeof the rAAVparticles. In someembodiments, theunit dose isabout6E10 vg / eye to about 2E11 vg / eye, about 7E10 vg / eye to about 2E11 vg / eye, about 8E10 vg / eye to about 2E11 vg / eye, about 9E10 vg / eye to about 2E11 vg / eye, about 10E10 vg / eye to about 2E11 vg / eye, or about 1E11 vg / eye to about 2E11 vg / eye of the rAAV particles. In some embodiments, the unit dose is about 6E10 vg / eye to about 2E11 vg / eye of the rAAV particles. In some embodiments, the unit dose is about 6E10 vg / eye to about 7E10 vg / eye, about 7E10 vg / eye to about 8E10 vg / eye, about 8E10 vg / eye to about 9E10 vg / eye, about 9E10 vg / eye to about 10E10 vg / eye, about 10E10 vg / eye to about 1E11 vg / eye, or about 1E11 vg / eye to about 2E11 vg / eye of the rAAV particles. In some embodiments, the unit dose is about 6E10 vg / eye, about 7E10 vg / eye, about 8E10 vg / eye, about 9E10 vg / eye, about 10E10 vg / eye, about 1E11 vg / eye, or about 2E11 vg / eye of the rAAV particles. In some embodiments, the unit dose is about 6E10 vg / eye or about 2E11 vg / eye of the rAAV particles. In some embodiments, the unit dose is about 6E10 vg / eye of the rAAV particles. In some embodiments, the unit dose is about 6E10 vg / eye, about 2E11 vg / eye, or about 6E11 vg / eye. In some embodiments, the unit dose is about 6E10 vg / eye. In some embodiments, the unit dose is about 2E11 vg / eye. In some embodiments, the unit dose is about 6E11 vg / eye.

[0200] In someembodiments, theunit doseof rAAVparticles is administered tooneeyeand / or to thecontralateral eyeof the individual. In some embodiments, the unit dose of rAAV particles is a unit dose sufficient to cause expression of the therapeutic protein (e.g., ananti-VEGFagent suchasaflibercept) in the vitreous fluid. In someembodiments, theunit dose of rAAV particles is a unit dose sufficient to achieve a concentration of the therapeutic protein (e.g., an anti-VEGF agent such as aflibercept) at about any one of 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5,7, 7.5, 8, 8.5, 9, 9.5, 10 µg / ml, or more, including any range in between these values, in the vitreous fluid. In some embodiments, the unit dose of rAAV particles is a unit dose sufficient to cause expression of aflibercept in the vitreous fluid. In some embodiments, the unit dose of rAAV particles is a unit dose sufficient to achieve a concentration of aflibercept at about any one of 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5,7, 7.5, 8, 8.5, 9, 9.5, 10 µg / ml, or more, including any range in between these values, in the vitreous fluid.

[0201] In some embodiments, the unit dose of rAAV particles administered to the oneeye and / or to the contralateral eye of the individual is a unit dose sufficient to cause expression of the therapeutic protein (e.g., an anti-VEGF agent such as aflibercept) in the aqueous fluid. In some embodiments, the unit dose of rAAV particles is a unit dose sufficient to achieve a concentration of the therapeutic protein (e.g., ananti-VEGFagent suchasaflibercept) of at least about 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95, 1.0 µg / ml, or more, including any range in between these values, in the aqueous fluid. In some embodiments, the unit dose of rAAV particles is a unit dose sufficient to cause expression of aflibercept in the aqueous fluid. In some embodiments, the unit dose of rAAV particles is a unit dose sufficient to achieve a concentration of aflibercept of at least about 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95, 1.0 µg / ml, or more, including any range in between these values, in the aqueous fluid.

[0202] In someembodiments, theunit doseof rAAVparticles is administered tooneeyeand / or to thecontralateral eyeof the individual. In some embodiments, the unit dose of rAAV particles is a unit dose sufficient to cause expression of the therapeutic protein (e.g., an anti-VEGF agent such as aflibercept) in the retina. In some embodiments, the unit dose of 54 EP 4 696 318 A1 5 10 15 20 25 30 35 40 45 50 55 rAAV particles is a unit dose sufficient to achieve a concentration of the therapeutic protein (e.g., an anti-VEGF agent such as aflibercept) of at least about 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5,7, 7.5, 8, 8.5, 9, 9.5, 10 µg / g, or more, including any range in between these values, in the retina. In some embodiments, the unit dose of rAAV particles is a unit dose sufficient to cause expression of aflibercept in the retina. In some embodiments, the unit dose of rAAV particles is a unit dose sufficient to achieve a ...

Claims

1. A unit dose of recombinant adeno-associated virus (rAAV) particles for use in a method for reducing retinal fluid in an 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 by intravitreal administration, wherein the individual is a human, and wherein the rAAV particles comprise: a) a nucleic acid encoding a polypeptide comprising an amino acid sequence with at least about 95% identity to the amino acid sequence of SEQ ID NO: 35 and flanked by AAV2 inverted terminal repeats (ITRs), and b) an AAV2 capsid protein comprising an amino acid sequence LGETTRP (SEQ ID NO: 14) inserted between positions 587 and 588 of the capsid protein, wherein the amino acid residue numbering corresponds to an AAV2 VP1 capsid protein.

2. The unit dose of rAAV particles of claim 1, wherein: (a) the individual has received at least one treatment of an anti-VEGF agent in about 12 weeks prior to administration of the unit dose of rAAV particles, and / or the amount or presence of retinal fluid in the one eye of the individual is refractory to prior treatment with an anti-VEGF agent, optionally wherein the anti-VEGF agent is aflibercept; (b) the retinal fluid in the one eye is reduced by at least about 60% compared to the level of retinal fluid in the one eye of the individual prior to administration of the rAAV particles to the individual; (c) the retinal fluid in the one eye is reduced by about 80% compared to the level of retinal fluid in the one eye of the individual prior to administration of the rAAV particles to the individual; and / or (d) the retinal fluid is subretinal fluid (SRF) or intraretinal fluid (IRF).

3. The unit dose of rAAV particles of claim 1, wherein the method further comprises administering an anti-VEGF agent to the one eye of the individual prior to administration of the unit dose of rAAV particles; optionally wherein: (a) the method comprises administering the unit dose of rAAV particles to the one eye of the individual about 1 week or about 7 days after administration of the anti-VEGF agent; (b) the method comprises administering the anti-VEGF agent to the one eye of the individual on Day 1, and administering the unit dose of rAAV particles to the one eye of the individual on Day 8; and / or (c) the anti-VEGF agent comprises aflibercept, optionally wherein the aflibercept is administered at a dose of about 2 mg by intravitreal injection.

4. The unit dose of rAAV particles of any one of claims 1-3, wherein: (a) the unit dose of rAAV particles is between about 6 × 1010 to about 6 × 1011 vector genomes per eye (vg / eye); or (b) the unit dose of rAAV particles is between about 6 × 1010 to about 2 × 1011 vg / eye, or between about 2 × 1011 to about 6 × 1011 vg / eye, optionally wherein the unit dose of rAAV particles is about 6 × 1010, about 2 × 1011, about 6 × 1011 vg / eye.

5. The unit dose of rAAV particles of any one of claims 1-4, wherein: (a) the individual has one or more symptoms of an ocular neovascular disease in the contralateral eye; and / or (b) the method further comprises administering a unit dose of rAAV particles to the contralateral eye of the individual by intravitreal administration, optionally wherein: i) the administering the unit dose of rAAV particles to the contralateral eye is up to about 2 weeks after administering the unit dose of rAAV particles to the one eye, optionally wherein: (1) the administering the unit dose of rAAV particles to the contralateral eye is on the same day as the administering the unit dose of rAAV particles to the one eye, or between about 1 day to about 14 days after administering the unit dose of rAAV particles to the one eye, and / or (2) the unit dose of rAAV particles administered to the contralateral eye comprises the same or less vector genomes per eye (vg / eye) than the unit dose of rAAV particles administered to the one eye; or ii) the administering the unit dose of rAAV particles to the contralateral eye is at least about 2 weeks after administering the unit dose of rAAV particles to the one eye, optionally wherein the unit dose of rAAV particles administered to the contralateral eye comprises more vector genomes per eye (vg / eye) than the unit dose of rAAV particles administered to the one eye.

6. The unit dose of rAAV particles of any one of claims 1-5, wherein: (a) the nucleic acid comprises the nucleic acid sequence of SEQ ID NO: 40 or a sequence having at least 85% identity thereto; (b) the polypeptide comprises the amino acid sequence of SEQ ID NO: 35 or SEQ ID NO: 41; or (c) the polypeptide is aflibercept.

7. The unit dose of rAAV particles of any one of claims 1-6, wherein the nucleic acid further comprises a first enhancer region, a promoter region, a 5'UTR region, a second enhancer region, and a polyadenylation site, 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, 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, the second enhancer region comprises a full EES sequence comprising the sequence of SEQ ID NO: 26 or a sequence having at least 85% identity thereto, 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.

8. The unit dose of rAAV particles of any one of claims 1-5, wherein the nucleic acid comprises, in the 5' to 3' order: (a) a first enhancer region; (b) a promoter region; (c) a 5'UTR region; (d) a nucleic acid encoding a polypeptide comprising an amino acid sequence with at least about 95% identity to the amino acid sequence of SEQ ID NO: 35; (e) a second enhancer region; and (f) a polyadenylation site; and flanked by AAV2 inverted terminal repeats (ITRs); 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, the nucleic acid encoding a polypeptide comprises the nucleic acid sequence of SEQ ID NO: 40 or a sequence having at least 85% identity thereto, the polypeptide comprises the amino acid sequence of SEQ ID NO: 35 or SEQ ID NO: 41, or a sequence having at least 95% identity thereto, or the polypeptide is aflibercept, 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, the second enhancer region comprises a full EES sequence comprising the sequence of SEQ ID NO: 26 or a sequence having at least 85% identity thereto, and / or the polyadenylation site comprises a HGH polyadenylation site comprising the sequence of SEQ ID NO: 27 or a sequence having at least 85% identity thereto.

9. The unit dose of rAAV particles of any one of claims 1-8, wherein: (a) the nucleic acid comprises the sequence of SEQ ID NO: 39 or a sequence having at least 85% identity thereto; (b) the AAV2 capsid protein comprises the amino acid sequence LALGETTRPA (SEQ ID NO: 1) inserted between positions 587 and 588 of the capsid protein, wherein the amino acid residue numbering corresponds to an AAV2 VP1 capsid protein; (c) 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; (d) 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; and / or (e) the rAAV particles comprise an AAV2 VP1 capsid protein comprising a GH loop that comprises the amino acid sequence of SEQ ID NO: 38 or an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 38.

10. The unit dose of rAAV particles of any one of claims 1-9, wherein: (a) the unit dose of rAAV particles is in a pharmaceutical formulation, optionally wherein the pharmaceutical formulation comprises the rAAV particles, sodium chloride, sodium phosphate and a surfactant, further optionally wherein the pharmaceutical formulation comprises about 150 to about 200 mM sodium chloride, about 1 to about 10 mM monobasic sodium phosphate, about 1 to about 10 mM dibasic sodium phosphate, about 0.0005% (w / v) to about 0.005% (w / v) poloxamer 188, and about 6 × 1013 to about 6 × 1010 vector genomes (vg) per mL (vg / mL) of the rAAV particles, wherein the pharmaceutical formulation has a pH of about 7.0 to about 7.5, and optionally wherein: i) the pharmaceutical formulation comprises about 180 mM sodium chloride, about 5 mM monobasic sodium phosphate, about 5 mM dibasic sodium phosphate, about 6 ×1012 vg / mL of the rAAV particles, and about 0.001% (w / v) poloxamer 188, wherein the pharmaceutical formulation has a pH of about 7.3, or ii) the pharmaceutical formulation comprises about 180 mM sodium chloride, about 5 mM monobasic sodium phosphate, about 5 mM dibasic sodium phosphate, about 2 ×1012 vg / mL of the rAAV particles, and about 0.001% (w / v) poloxamer 188, wherein the pharmaceutical formulation has a pH of about 7.3, or iii) the pharmaceutical formulation comprises about 180 mM sodium chloride, about 5 mM monobasic sodium phosphate, about 5 mM dibasic sodium phosphate, about 6 ×1011 vg / mL of the rAAV particles, and about 0.001% (w / v) poloxamer 188, wherein the pharmaceutical formulation has a pH of about 7.3; (b) the unit dose of rAAV particles administered to the one eye and / or to the contralateral eye is in a volume of about 25 µL to about 250 µL, optionally wherein the unit dose of rAAV particles administered to the one eye and / or to the contralateral eye comprises a volume of about 100 µL or about 30 µL; (c) the individual: i) received prior treatment for the ocular neovascular disease with an anti-VEGF agent, optionally wherein the individual has received 1 or 2 injections of an anti-VEGF agent in the one eye and / or in the contralateral eye prior to administration of the rAAV particles in the one eye and / or in the contralateral eye, or ii) has not received prior treatment for the ocular neovascular disease with an anti-VEGF agent, optionally wherein the anti-VEGF agent is aflibercept; and / or (d) the ocular neovascular disease is wet age-related macular degeneration (AMD), retinal neovascularization, choroidal neovascularization diabetic retinopathy, proliferative diabetic retinopathy, retinal vein occlusion, central retinal vein occlusion, branched retinal vein occlusion, diabetic macular edema, diabetic retinal ischemia, ischemic retinopathy, diabetic retinal edema, or any combination thereof.

11. The unit dose of rAAV particles of any one of claims 1-10, wherein: (a) the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in: (i) maintenance or a decrease of retinal thickness compared to the retinal thickness prior to administration of the unit dose of rAAV particles, optionally wherein: 1) the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in a decrease in retinal thickness compared to the retinal thickness prior to administration of the unit dose of rAAV particles, 2) the decrease in retinal thickness is at least about 10% compared to the retinal thickness prior to administration of the unit dose of rAAV particles, and / or 3) retinal thickness is central subfield thickness (CST) or central retinal thickness (CRT); (ii) maintenance or a decrease in macular volume compared to the macular volume prior to administration of the unit dose of rAAV particles, optionally wherein: 1) the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in a decrease in macular volume compared to the macular volume prior to administration of the unit dose of rAAV particles, and / or 2) the decrease in macular volume is at least about 10% compared to the macular volume prior to administration of the unit dose of rAAV particles; and / or (iii) maintenance or an improvement of visual acuity compared to the visual acuity prior to administration of the unit dose of rAAV particles, optionally wherein: 1) the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in an improvement of visual acuity compared to the visual acuity prior to administration of the unit dose of rAAV particles, and / or 2) visual acuity is best corrected visual acuity (BCVA); and / or (b) administration of the unit dose of rAAV particles to the one eye and / or to the contralateral eye of a plurality of individuals results in: (i) at least about 50% of the individuals in the plurality not requiring an anti-VEGF rescue treatment, optionally wherein at least about 50% of the individuals in the plurality do not require an anti-VEGF rescue treatment for at least about 20 weeks, at least about 36 weeks, at least about 52 weeks, at least about 56 weeks, or more after administration of the unit dose of rAAV particles, at least about 67% of the individuals in the plurality not requiring an anti-VEGF rescue treatment, optionally wherein at least about 67% of the individuals in the plurality do not require an anti-VEGF rescue treatment for at least about 20 weeks, at least about 36 weeks, at least about 52 weeks, at least about 60 weeks, at least about 64 weeks, or at least about 66 weeks after administration of the unit dose of rAAV particles, at least about 78% of the individuals in the plurality not requiring an anti-VEGF rescue treatment, optionally wherein at least about 78% of the individuals in the plurality do not require an anti-VEGF rescue treatment for at least about 20 weeks, at least about 36 weeks, or more after administration of the unit dose of rAAV particles, or 100% of the individuals in the plurality not requiring an anti-VEGF rescue treatment, optionally wherein 100% of the individuals in the plurality do not require an anti-VEGF rescue treatment for at least about 64 weeks, at least about 68 weeks, at least about 72 weeks, at least about 76 weeks, at least about 80 weeks, at least about 84 weeks, or more after administration of the unit dose of rAAV particles, and / or (ii) a reduction in the annualized anti-VEGF injection rate of at least about 80%, at least about 85%, at least about 87%, at least about 90%, at least about 95%, at least about 99%, or 100% compared to the annualized anti-VEGF injection rate prior to administration of the unit dose of rAAV particles.

12. The unit dose of rAAV particles of any one of claims 1-11, wherein the ocular neovascular disease is: (a) wet age-related macular degeneration (AMD); or (b) diabetic macular edema (DME), optionally wherein the administering the unit dose of rAAV particles to the one eye and / or to the contralateral eye of the individual results in a 2-step or in a 3-step improvement in Diabetic Retinopathy Severity Scale (DRSS).

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