KR20220062353A - Methods of Treating Ocular Neovascular Disease Using AAV2 Variants Encoding Aflibercept - Google Patents

Abstract

A method comprising administering to the eye of a subject a unit dose of a recombinant adeno-associated virus (rAAV) particle, wherein the rAAV particle comprises a) an amino acid sequence having at least about 95% identity to the amino acid sequence of SEQ ID NO:35 LGETTRP ( Provided is a method of treating an ocular neovascular disease in a subject comprising an AAV2 capsid protein comprising SEQ ID NO: 14).

Description

Methods of Treating Ocular Neovascular Disease Using AAV2 Variants Encoding Aflibercept

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is filed under 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 (November 2019) 18), U.S. Provisional Patent Application No. 62/959,784 (filed on January 10, 2020), U.S. Provisional Patent Application No. 62/971,835 (filed on February 7, 2020), U.S. Provisional Patent Application No. 63/019,190 (filed on January 10, 2020) 1), 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 disclosures of each of which The entirety of which is incorporated herein by reference.

Field

The present disclosure relates to an ocular neovascular disease in a subject comprising administering to the eye of the subject a single unit dose of a recombinant adeno-associated virus (rAAV) particle encoding an anti-VEGF agent (eg, aflibercept); It relates to a method of treating a disorder.

Submission of Sequence Listing in ASCII Text File

The following submission of an ASCII text file is incorporated herein by reference in its entirety: Computer-readable form (CRF) of the Sequence Listing (filename: 627002001241SEQLIST.TXT, dated recorded: September 9, 2020, size: 41 KB ).

Age-related macular degeneration (AMD) is a degenerative eye disease affecting the macula, a small area of light sensitivity within the center of the retina responsible for reading and precision vision. Conditions affecting the macula reduce central vision while leaving peripheral vision intact. In severe cases, this disease can lead to central blindness. AMD is a notable cause of blindness among people over the age of 65 in the US population, with an estimated prevalence of any AMD among people over age 40 of approximately 6.5% (Klein et al., (2011) Arch Ophthalmol, 129(1) ):75-80). Neovascular or exudative or wet AMD (nAMD, wAMD or nwAMD) is an advanced form of AMD. A hallmark of wAMD is choroidal neovascularization (CNV), the infiltration of abnormal blood vessels in the retina from the basal choroidal layer, which results in retinal cell damage and central blindness. This aberrant angiogenic process is regulated by growth factors, particularly vascular endothelial growth factor (VEGF). Standard management of wAMD is a class of molecules that bind and sequester VEGF, such as ranibizumab (Lucentis) and aflibercept (Eylea).

Diabetic retinopathy (DR) is a major complication of diabetes and a leading cause of vision loss in the working-age population. DR may be non-proliferative DR without new blood vessel growth (NPDR) or proliferative DR with new abnormal blood vessel growth in the retina or choroid (PDR). Diabetic macular edema (DME) is a complication of DR and is another example of an eye disease affecting the macula. DME affects up to 10% of people with diabetes and is caused by fluid build-up in the macula. DME is the most frequent cause of vision loss in people with DR. Available therapies for treating DME include lasers and anti-vascular endothelial growth factor (anti-VEGF) drugs such as aflibercept.

Aflibercept is a recombinant fusion protein that acts as a decoy receptor for vascular endothelial growth factor subtypes A and B (VEGF-A and VEGF-B) and placental growth factor (PGF). By binding to these ligands, aflibercept may prevent them from binding to vascular endothelial growth factor receptor (VEGFR), VEGFR-1 and VEGFR-2, thereby inhibiting angiogenesis and reducing vascular permeability. Aflibercept consists of domain 2 of VEGFR-1 and domain 3 of VEGFR-2 fused with an Fc fragment of IgG1.

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 results and to maintain vision. Adherence to these regimens places a burden on the patient, his/her caregivers, and health care system, and most patients deviate from adherence to optimal regimens over time, which correlates with loss of vision (Khanani AM, et al.) . Additionally, complications exist including endophthalmitis, retinal detachment, traumatic cataracts and elevated intraocular pressure (IOP); The risk of these complications is likely to increase with repeated IVT injections (Falavarjani et al., (2013) Eye (Lond), 27(7):787-794).

Accordingly, there is a need in the art for a therapy of an ocular neovascular disease such as wAMD, DR or DME that is effective, reduces the risk of side effects, and can be adhered to by patients for a very long time.

In one aspect, the method comprises administering to one eye of the individual a unit dose of no more than about 6 x 10 ¹¹ vector genomes (vg) recombinant adeno-associated virus (rAAV) particles, wherein the individual is a human, wherein the rAAV particles are (a) a nucleic acid encoding a polypeptide comprising an amino acid sequence having at least about 95% identity to the amino acid sequence of SEQ ID NO: 35 and flanked by an AAV2 inverted terminal repeat (ITR), and (b) comprising an AAV2 capsid protein comprising or consisting of the amino acid sequence LGETTRP (SEQ ID NO: 14) inserted between positions 587 and 588 of the capsid protein, wherein the amino acid residue numbering corresponds to the AAV2 VP1 capsid protein; Provided herein are methods of treating an ocular neovascular disease in a subject. In some embodiments, the method comprises reducing retinal fluid in the eye of the individual.

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

In another aspect, (a) administering an anti-VEGF agent to one eye of the subject; (b) administering to one eye of the subject a unit dose of no more than about 6 x 10 ¹¹ vector genomes (vg) recombinant adeno-associated virus (rAAV) particles after administration of the anti-VEGF agent, wherein the subject is a human wherein the rAAV particle comprises (i) a nucleic acid encoding a polypeptide comprising an amino acid sequence having at least about 95% identity to the amino acid sequence of SEQ ID NO: 35 and flanked by an AAV2 inverted terminal repeat (ITR); and (ii) an AAV2 capsid protein comprising the amino acid sequence LGETTRP (SEQ ID NO: 14) inserted between positions 587 and 588 of the capsid protein, wherein the amino acid residue numbering corresponds to the AAV2 VP1 capsid protein. Provided herein is a method of treating an ocular neovascular disease in In some embodiments, the method comprises administering a unit dose of the rAAV particles to one eye of the individual about 1 week or about 7 days after administration of the anti-VEGF agent. In some embodiments, the method comprises administering a unit dose of the rAAV particle to one eye of the individual about 1 to about 2 weeks after administration of the anti-VEGF agent. In some embodiments, the method comprises administering the anti-VEGF agent for 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 and administering the unit dose of the rAAV particles to one eye of the subject after about 10 days, about 11 days, about 12 days, about 13 days, about 14 days, or about 15 days. In some embodiments, the method comprises administering the anti-VEGF agent to one eye of the individual on day 1 and administering a unit dose of the rAAV particles to one eye of the individual on day 8. In some embodiments, the anti-VEGF agent comprises aflibercept. In some embodiments, aflibercept is administered by intravitreal injection at a dose of about 2 mg. In some embodiments, the method further comprises administering topical steroid treatment. In some embodiments, the topical steroid treatment is difluprednate treatment. In some embodiments, the topical steroid treatment is about 4 administrations of the topical steroid per day for about 4 weeks, followed by about 3 administrations of the topical steroid per day for about 1 week, followed by the topical steroids per day for about 1 week. about twice administration of a drug, followed by about once administration of a topical steroid per day for about one week; The timing begins with administration of the anti-VEGF agent and thereafter. In some embodiments, the topical steroid treatment is about 4 administrations of the topical steroid per day for about 1 month, followed by about 3 administrations of the topical steroid per day for about 1 month, followed by the topical steroids per day for about 1 month. about twice administration of a drug, followed by about once administration of a topical steroid per day for about 1 month; The timing begins with administration of the anti-VEGF agent and thereafter. In some embodiments, the topical steroid treatment is about 4 administrations of the topical steroid per day for about 1 month, followed by about 3 administrations of the topical steroid per day for about 1 month, followed by the topical steroids per day for about 1 month. about twice administration of a drug, followed by about once administration of a topical steroid per day for about 1 month; The time period begins with administration of the rAAV particles and thereafter. In some embodiments, the topical steroid comprises 0.05% difluprednate in a dose of about 1 μg to about 3 μg. In some embodiments, the topical steroid comprises 0.05% difluprednate at a dose of about 2.5 μg.

In some embodiments that may be combined with any of the above embodiments, the unit dose of the rAAV particles is from about 6 x 10 ¹⁰ to about 2 x 10 ¹¹ vector genomes per eye (vg/eye). In some embodiments, the unit dose of rAAV particles is about 2 x 10 ¹¹ or about 6 x 10 ¹⁰ vector genomes per eye (vg/eye). In some embodiments that may be combined with any of the embodiments above, the unit dose of rAAV particles is from about 6 x 10 ¹⁰ to about 6 x 10 ¹¹ vector genomes per eye (vg/eye). In some embodiments that may be combined with any of the above embodiments, the unit dose of the rAAV particles is from about 6 x 10 ¹⁰ to about 2 x 10 ¹¹ vector genomes per eye (vg/eye). In some embodiments that may be combined with any of the above embodiments, the unit dose of rAAV particles is from about 2 x 10 ¹¹ to about 6 x 10 ¹¹ vector genomes per eye (vg/eye). In some embodiments, the unit dose of rAAV particles is about 2 x 10 ¹¹ or about 6 x 10 ¹⁰ vector genomes per eye (vg/eye). In some embodiments, the unit dose of rAAV particles is about 2×10 ¹¹ vg/eye. In some embodiments, the unit dose of rAAV particles is about 6×10 ¹¹ vg/eye.

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

In some embodiments that may be combined with any of the embodiments above, the methods provided herein further comprise administering a unit dose of the rAAV particles to the contralateral eye of the individual. In some embodiments, administering the unit dose of rAAV particles to the contralateral eye is up to about 2 weeks after administration of the unit dose of rAAV particles to one eye. In some embodiments, administering the unit dose of rAAV particles to the contralateral eye is on the same day as administering the unit dose of rAAV particles to one eye; or administering the unit dose of rAAV particles to the contralateral eye is from about 1 day to about 14 days after administration of the unit dose of rAAV particles to one eye. In some embodiments, the unit dose of rAAV particles administered to the contralateral eye of the individual comprises vector genomes per eye (vg/eye) equal to or less than the unit dose of rAAV particles administered to one eye of the individual. In some embodiments, 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 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 one eye of the individual.

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

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

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

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

In some embodiments that may be combined with any of the above embodiments, the rAAV particle comprises a GH loop comprising the amino acid sequence of SEQ ID NO: 38 or an amino acid sequence with at least 90% sequence identity to SEQ ID NO: 38 AAV2 VP1 capsid protein. In some embodiments, the rAAV particles are 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% to SEQ ID NO: 38 , an AAV2 VP1 capsid protein comprising a GH loop comprising an amino acid sequence having either at least 99% or 100% sequence identity.

In some embodiments that may be combined with any of the above embodiments, the rAAV particle is an AAV2 VP1 capsid protein comprising the amino acid sequence of SEQ ID NO: 37 or an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 37 includes In some embodiments, the rAAV particle is 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% relative to SEQ ID NO:37. , an AAV2 VP1 capsid protein comprising an amino acid sequence having either at least 99% or 100% sequence identity.

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

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

In some embodiments that may be combined with any of the embodiments above, the unit dose of the rAAV particles is in a pharmaceutical formulation. In some embodiments, the pharmaceutical formulation comprises rAAV particles, sodium chloride, sodium phosphate and a surfactant. In some embodiments, the pharmaceutical formulation comprises about 150 to about 200 mM sodium chloride, about 1 to about 10 mM sodium phosphate monobasic, about 1 to about 10 mM sodium phosphate dibasic, about 0.0005% (w/v) to about 0.005% (w/v) poloxamer 188 and rAAV particles per mL of about 6 x 10 ¹³ to about 6 x 10 ¹⁰ vector genomes (vg) (vg/mL), wherein the pharmaceutical formulation has a pH of about 7.0 to about 7.5 has In some embodiments, the pharmaceutical formulation comprises about 180 mM sodium chloride, about 5 mM sodium phosphate monobasic, about 5 mM sodium phosphate dibasic, about 6×10 ¹² vg/mL of rAAV particles and about 0.001% (w/v) poloc Samer 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 sodium phosphate monobasic, about 5 mM sodium phosphate dibasic, about 2×10 ¹² vg/mL of rAAV particles and about 0.001% (w/v) poloc Samer 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 sodium phosphate monobasic, about 5 mM sodium phosphate dibasic, about 6×10 ¹¹ vg/mL of rAAV particles and about 0.001% (w/v) poloc. Samer 188, wherein the pharmaceutical formulation has a pH of about 7.3.

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

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

In some embodiments that may be combined with any of the above embodiments, the ocular neovascular disease is wet age-related macular degeneration (AMD), retinal neovascularization, choroidal neovascularization diabetic retinopathy, proliferative diabetic retinopathy. , retinal vein occlusion, central retinal vein occlusion, branch retinal vein occlusion, diabetic macular edema, diabetic retinal ischemia, ischemic retinopathy, diabetic retinal edema, or any combination thereof.

In some embodiments that may be combined with any of the embodiments above, 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 systemic steroid treatment. In some embodiments, the steroid treatment is oral steroid treatment. In some embodiments, the steroid treatment is prednisone treatment. In some embodiments, oral prednisone treatment begins 3 days prior to administering the unit dose of rAAV particles to one eye and/or the contralateral eye, administering prednisone at a dose of about 60 mg per day for a total of 6 days, followed by total Prednisone is administered at a dose of about 40 mg per day for 3 days followed by prednisone at a dose of about 20 mg per day for a total of 2 days, followed by a dose of about 10 mg per day for a total of 2 days. and administering prednisone. In some embodiments, the steroid treatment is topical steroid treatment. In some embodiments, the steroid treatment is difluprednate treatment. In some embodiments, the steroid is administered before, during, and/or after administration of the unit dose of the rAAV particles. In some embodiments, the steroid is administered before, during and/or after administration of the unit dose of the rAAV particles to one eye and/or the contralateral eye.

In some embodiments that may be combined with any of the above embodiments, the steroid treatment is topical steroid treatment, wherein the topical steroid treatment is for up to about 4 weeks, up to about 6 weeks, or up to about 8 weeks from administration of the unit dose of the rAAV particles. It's a daily steroid treatment. In some embodiments, topical steroid treatment comprises about 4 administrations of the topical steroid in about 1 week, about 3 administrations of the topical steroid in about 2 weeks, about 2 administrations of the topical steroid in about 3 weeks, and a drug including about 1 administration of topical steroid in 4 weeks; The period begins with administration of a unit dose of rAAV particles and thereafter. In some embodiments, topical steroid treatment is about 4 administrations of the topical steroid per day for about 3 weeks following administration of the unit dose of rAAV particles, followed by about 3 administrations of the topical steroid per day for about 1 week, followed by about about twice administration of the topical steroid per day for one week, followed by about once administration of the topical steroid per day for about one week. In some embodiments, the topical steroid treatment is about 4 administrations of the topical steroid per day for about 4 weeks, followed by about 3 administrations of the topical steroid per day for about 1 week, followed by the topical steroids per day for about 1 week. about twice administration of a drug, followed by about once administration of a topical steroid per day for about 1 week; The phase begins about 1 week prior to administration of a unit dose of rAAV particles. In some embodiments, topical steroid treatment is about 4 administrations of the topical steroid per day for about 1 month, followed by about 3 administrations of the topical steroid per day for about 1 month, followed by topical steroids per day for about 1 month. about twice administration of a drug, followed by about once administration of a topical steroid per day for about 1 month; The phase begins about administration of a unit dose of rAAV particles. In some embodiments, topical steroid treatment is about 4 administrations of the topical steroid per day for about 1 month, followed by about 3 administrations of the topical steroid per day for about 1 month, followed by topical steroids per day for about 1 month. about twice administration of a drug, followed by about once administration of a topical steroid per day for about 1 month; The phase begins about 1 week prior to administration of a unit dose of rAAV particles. In some embodiments, the topical steroid comprises 0.05% difluprednate in a dose of about 1 μg to about 3 μg. In some embodiments, the topical steroid comprises 0.05% difluprednate at a dose of about 2.5 μg.

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

In some embodiments that may be combined with any of the above embodiments, administering a unit dose of rAAV particles to one and/or contralateral eye of the individual results in a reduction in retinal thickness compared to retinal thickness prior to administration of the unit dose of rAAV particles. maintain or decrease. In some embodiments, the maintenance or reduction of retinal thickness as compared to retinal thickness prior to administration of the unit dose of the rAAV particles is about 30 weeks, about 34 weeks after administration of the unit dose of the rAAV particles to one eye and/or the contralateral eye of the individual. , 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. In some embodiments, administering the unit dose of rAAV particles to one and/or the contralateral eye of the individual results in a decrease in retinal thickness compared to retinal thickness prior to administration of the unit dose of rAAV particles. In some embodiments, the reduction in retinal thickness is at least about 10% compared to the retinal thickness prior to administration of the unit dose of the rAAV particles. In some embodiments, the retinal thickness is central subfield thickness (CST) or central retinal thickness (CRT).

In some embodiments that may be combined with any of the embodiments above, administering a unit dose of rAAV particles to one and/or the contralateral eye of the individual results in an increase in macular volume compared to macular volume prior to administration of the unit dose of rAAV particles. maintain or decrease. In some embodiments, maintenance or reduction of macular volume as compared to macular volume prior to administration of the unit dose of rAAV particles is at about 30 weeks, about 34 weeks after administration of the unit dose of rAAV particles to one eye and/or the contralateral eye of the individual. , 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. In some embodiments, administering the unit dose of rAAV particles to one and/or 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 the rAAV particles.

In some embodiments that may be combined with any of the embodiments above, administering a unit dose of rAAV particles to one and/or the contralateral eye of the individual results in maintenance of or cause improvement In some embodiments, the maintenance or improvement of visual acuity as compared to visual acuity prior to administration of the unit dose of the rAAV particles is about 30 weeks, about 34 weeks, about 30 weeks after administration of the unit dose of the rAAV particles to one and/or the contralateral eye of the individual. 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. In some embodiments, administering the unit dose of rAAV particles to one and/or the contralateral eye of the individual results in an improvement in visual acuity as compared to visual acuity prior to administration of the unit dose of rAAV particles. In some embodiments, the visual acuity is maximum corrected visual acuity (BCVA).

In some embodiments that may be combined with any of the embodiments above, administering a single unit dose of rAAV particles to one and/or the contralateral eye of the individual provides a therapeutic benefit (e.g., treatment of ocular neovascular disease; reduction of retinal fluid, maintenance or reduction of retinal thickness, maintenance or reduction of macular volume and/or maintenance or improvement of vision). In some embodiments, administering a single unit dose of rAAV particles to one and/or contralateral eye of the individual is at least about 30 weeks, about 34 weeks after administration of the unit dose of rAAV particles to one and/or contralateral eye of the individual. It provides a therapeutic benefit that exists after at least about a week, at least about 44 weeks, at least about 6 months, at least about 1 year, at least about 1.5 years, or at least about 2 years, about 3 years, about 5 years, about 10 years or more.

In some embodiments that may be combined with any of the embodiments above, administering a unit dose of rAAV particles to one eye and/or the contralateral eye of the plurality of individuals results in at least about 50% of the individuals of the plurality of individuals receiving anti-VEGF Do not require salvage treatment. In some embodiments, at least about 50% of the individuals of the plurality of individuals have anti-VEGF 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 the rAAV particles. No salvage treatment required.

In some embodiments that may be combined with any of the embodiments above, administering a single unit dose of rAAV particles to one eye and/or the contralateral eye of the plurality of individuals results in at least about 67% of the individuals of the plurality of individuals anti- Do not require VEGF rescue therapy. In some embodiments, at least about 67% of the individuals of the plurality of individuals are 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 after administration of the unit dose of the rAAV particles. No anti-VEGF rescue treatment is required for about 66 weeks.

In some embodiments that may be combined with any of the embodiments above, administering a unit dose of rAAV particles to one eye and/or the contralateral eye of the plurality of individuals results in at least about 78% of the individuals of the plurality of individuals anti-VEGF Do not require salvage treatment. In some embodiments, at least about 78% of the individuals of the plurality are not in need of anti-VEGF rescue treatment for at least about 20 weeks, at least about 36 weeks or more after administration of the unit dose of the rAAV particles.

In some embodiments that may be combined with any of the above embodiments, administering a unit dose of rAAV particles to one eye and/or the contralateral eye of the plurality of individuals results in 100% of the individuals of the plurality of individuals receiving anti-VEGF rescue treatment do not require In some embodiments, 100% of the individuals of the plurality of individuals are 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 after administration of the unit dose of the rAAV particles. No anti-VEGF rescue treatment is required for a week or longer.

In some embodiments that may be combined with any of the embodiments above, administering the unit dose of rAAV particles to one and/or the contralateral eye of the plurality of individuals comprises an annual anti-VEGF injection rate prior to administration of the unit dose of rAAV particles. 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% of the annual anti-VEGF injection rate as compared to

In some embodiments, the ocular neovascular disease is diabetic macular edema (DME). In some embodiments, administering the unit dose of the rAAV particles to one and/or the contralateral eye of the individual results in a two- or three-step improvement in the diabetic retinopathy severity scale (DRSS). In some embodiments, administering the unit dose of the rAAV particles to one and/or the contralateral eye of the individual results in a two-step improvement in the diabetic retinopathy severity scale (DRSS). In some embodiments, administering the unit dose of the rAAV particles to one and/or the contralateral eye of the individual results in a three-step improvement in the diabetic retinopathy severity scale (DRSS).

incorporated by reference

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

The novel features of the invention are specifically set forth in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description and accompanying drawings, which set forth exemplary embodiments in which the principles of the invention may be employed:
1A-1B provide schematics of the investigational drugs and Phase I studies described in Examples 1 and 2. 1A is a schematic diagram of AAV2.7m8-aflibercept. AAV2.7m8-aflibercept is a recombinant, replication-deficient adeno-associated virus (rAAV) vector containing the AAV2.7m8 protein capsid and a codon-optimized version of the aflibercept cDNA under the control of a ubiquitous chimeric promoter (C11). is a vector genome containing an expression cassette of The AAV2.7m8-aflibercept vector genome also contains two AAV2 inverted terminal repeat sequences (ITRs) flanked by the aflibercept cDNA expression cassette. 1B is a diagram summarizing the study design for the Phase I study described in Examples 1 and 2.
1C provides a diagram of the AAV2.7m8-aflibercept vector genome (SEQ ID NO:39). The vector genome consists of two inverted terminal repeats (ITRs) of AAV serotype 2 (positions 1-145 and 3772-3916 of SEQ ID NO: 39), an expression cassette containing a CMV promoter (positions of SEQ ID NO: 39) 180-693), a 5' untranslated region (UTR) (positions 694-1314 of SEQ ID NO: 39), a Kozak sequence (positions 1329- of SEQ ID NO: 39) containing an adenovirus three-part leader sequence and a synthetic intron 1340), codon-optimized aflibercept cDNA (positions 1338-2714 of SEQ ID NO: 39), a 3' UTR containing a human scaffold attachment region (positions 2717-3527 of SEQ ID NO: 39) and human growth hormonal polyadenylation/transcriptional stop signal (positions 3546-3748 of SEQ ID NO:39). AAV = adeno-associated virus; CMV = cytomegalovirus; GH = growth hormone; ITR = inverted terminal repeat; polyA = polyadenylation; SAR = scaffold-attached region; TPL = 3 part leader sequence.
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. FIG. OCT images were taken at the indicated times before and after administration of AAV2.7m8-aflibercept (day 1). Anti-VEGF IVT treatment intervals are shown for all subjects. 2A provides OCT images taken from subject 1 and retinal thickness maps derived from OCT images at 5 clinic visits at indicated times prior to screening aflibercept injection. OCT images were taken immediately prior to treatment with aflibercept standard care. Subject 1 required aflibercept IVT every 5-7 weeks and exhibited refractory subretinal fluid and pigmented epithelial detachment (PED) despite aflibercept standard of care treatment. 2B shows OCT images taken from subject 1 and retinas derived from OCT images at screening aflibercept injection (day -7), AAV2.7m8-aflibercept injection (day 1) and follow-up visits at the indicated times. Provides a thickness map. Subject 1 did not require any rescue injections after AAV2.7m8-aflibercept injection. Subject 1 showed resolution of subretinal fluid starting at week 4 and remained free of subretinal and intraretinal fluid (kept dry). 2C provides OCT images taken from subject 2 and retinal thickness maps derived from OCT images at 5 clinic visits at the indicated times prior to screening aflibercept injection. OCT images were taken immediately prior to treatment with aflibercept standard care. Subject 2 required 6 aflibercept IVT treatments within 8 months prior to AAV2.7m8-aflibercept treatment to maintain retinal anatomy. 2D shows retinal derived OCT images and OCT images taken from subject 2 at screening aflibercept injection (day -7), AAV2.7m8-aflibercept injection (day 1) and follow-up visits at the indicated times. Provides a thickness map. Subject 2 did not require any rescue injections after AAV2.7m8-aflibercept injection. Subject 2 showed stable retinal anatomy without subretinal or intraretinal fluid by week 24. 2E provides OCT images taken from subject 3 and retinal thickness maps derived from OCT images at 4 clinic visits at indicated times prior to screening aflibercept injection. OCT images taken at week-27 are not shown. OCT images were taken immediately prior to treatment with aflibercept standard care. Subject 3 showed increased subretinal fluid when the interval between aflibercept IVTs was increased from 5 weeks to 7 weeks. 2F shows OCT images taken from subject 3 and retinas derived from OCT images at screening aflibercept injection (day -7), AAV2.7m8-aflibercept injection (day 1) and follow-up visits at the indicated times. Provides a thickness map. Subject 3 did not require any rescue injections after AAV2.7m8-aflibercept injection. Subject 3 showed resolution of refractory subretinal fluid by week 8 and stable retinal anatomy by week 24. 2G provides OCT images taken from subject 4 and retinal thickness maps derived from OCT images at 5 clinic visits at indicated times prior to screening aflibercept injection. OCT images were taken immediately prior to treatment with ranibizumab 0.5 mg IVT standard care. Subject 4 presented with subretinal fluid refractory to ranibizumab IVT injection. 2H shows OCT images taken from subject 4 and retinas derived from OCT images at screening aflibercept injection (day-14), AAV2.7m8-aflibercept injection (day 1) and follow-up visits at the indicated times. Provides a thickness map. Subject 4 did not require any rescue injections after AAV2.7m8-aflibercept injection. Subject 4 showed resolution of refractory subretinal fluid by week 8 and stable retinal anatomy by week 24. 2I provides OCT images taken from subject 5 and retinal thickness maps derived from OCT images at 5 clinic visits at the indicated times prior to screening aflibercept injection. OCT images were taken immediately prior to treatment with aflibercept standard care. 2J shows OCT images taken from subject 5 and retinas derived from OCT images at screening aflibercept injection (day-14), AAV2.7m8-aflibercept injection (day 1) and follow-up visits at the indicated times. Provides a thickness map. Subject 5 did not require any rescue injections after AAV2.7m8-aflibercept injection. Subretinal fluid and PED present at the time of AAV2.7m8-aflibercept treatment resolved over time, retinal anatomy remained stable until week 24, and there was no subretinal or intraretinal fluid. 2K provides OCT images taken from subject 6 and retinal thickness maps derived from OCT images at 5 clinic visits at indicated times prior to screening aflibercept injection. As indicated, OCT images were taken immediately prior to treatment with bevacizumab 1.5 mg IVT standard care or ranibizumab 0.5 mg IVT standard care. The appearance of the retina of subject 6 was consistent with polypoid choroidal angiopathy (PCV). 2L shows OCT images taken from subject 6 and retinas derived from OCT images at screening aflibercept injection (day-10), AAV2.7m8-aflibercept injection (day 1) and follow-up visits at the indicated times. Provides a thickness map. Subject 6 did not require any rescue injections after AAV2.7m8-aflibercept injection. Subject 6 did not show any increase in subretinal fluid and achieved some anatomical improvement by week 24. The contralateral eye of subject 6 received standard care aflibercept injections every 4 weeks over the course of the trial and exhibited retinal morphology similar to the AAV2.7m8-aflibercept-treated eye.
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 represent 90% confidence intervals calculated using the T-distribution. Baseline (BL) represents measurements taken prior to screening aflibercept injection on Day 1, 7-15 days (eg, 7-14 days) of AAV2.7m8-aflibercept treatment. After 24 weeks of AAV2.7m8-aflibercept treatment, subjects had a mean CRT change of -52.7 μm (90% CI -86.5, -18.8). BL = baseline; D = days; W = week. The Day 1 visit occurred 7-14 days after the baseline visit.
4 shows mean maximum corrected visual acuity (BCVA) measurements based on Early Treatment Diabetic Retinopathy Study (ETDRS) character assessment for subjects in Cohort 1 of the study described in Example 1 at the time points indicated. Error bars represent 90% confidence intervals calculated using the T-distribution. Baseline (BL) represents measurements taken prior to screening aflibercept injection on Day 1, 7-15 days (eg, 7-14 days) of AAV2.7m8-aflibercept treatment. After 24 weeks of AAV2.7m8-aflibercept treatment, subjects exhibited a mean change in BCVA of -2 characters (90% CI -9.1, 5.1). BL = baseline; D = days; W = week. The Day 1 visit occurred 7-14 days after the baseline visit.
5 provides the nucleic acid sequence of aflibercept (SEQ ID NO: 36).
6 shows a plot of anterior cell and vitreous cell counts after treatment with AAV2.7m8-aflibercept for subjects 1-6 of the study described in Example 1. FIG. The steroid treatment administered to each patient is shown below each plot. The aqueous humor cell count category was based on the Uveitis Nomenclature Normalization (SUN) criteria (Jabs, DA et al., J Ophthalmol. 2005;140:509-516). The vitreous cell count categories were based on the National Institutes of Health (NIH) guidelines. For aqueous humor cells, a cell count value of 0.5+ represents 1-5 cells; A cell number value of 1+ represents 6-15 cells; A cell number value of 2+ represents 16-25 cells; A cell number value of 3+ represents 26-50 cells; A cell number value of 4+ indicates >50 cells. For vitreous cells, a cell count value of 0.5+ represents 1-10 cells; A cell number value of 1+ represents 11-20 cells; A cell number value of 2+ represents 21-30 cells; A cell number value of 3+ represents 31-100 cells; A cell number value of 4+ indicates >100 cells. Rare cells were captured as 0.5+ for the analysis presented in this figure.
7A-7B show retinal thickness maps derived from optical coherence tomography (OCT) images and OCT images taken from subjects 1-6 in cohort 1 of the study described in Example 1 at a median follow-up time of 34 weeks. Additionally, BCVA change from baseline, number of anti-VEGF IVT injections within 8 months prior to administration of AAV2.7m8-aflibercept, and number of rescue anti-VEGF IVT injections administered during the study were also provided for each of subjects 1-6 do. Actual weeks at which OCT images and retinal thickness maps were obtained are shown for each subject (subject 1 = week 44; subject 2 = week 40; subject 3 = week 36; subject 4 = week 32; subject 5 = week 28 weeks; 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 up to 44 weeks. None of the subjects showed signs of disease reactivation on OCT imaging.
8 is a plot of aqueous humor and vitreous cell counts after treatment with AAV2.7m8-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 (weeks 40-52). show The steroid treatment administered to each subject is shown below each plot. The aqueous humor cell grade category was based on the Standardization of Uveitis Nomenclature (SUN) criteria (Jabs, DA et al. J Ophthalmol. 2005; 140:509-516). The vitreous cell grade categories were based on the National Institutes of Health (NIH) guidelines. For aqueous humor cells, a cell grade of 0.5+ represents 1-5 cells; A cell grade of 1+ represents 6-15 cells; A cell grade of 2+ represents 16-25 cells; A cell grade of 3+ represents 26-50 cells; A cell grade of 4+ indicates >50 cells. For vitreous cells, a cell grade of 0.5+ represents 1-10 cells; A cell grade of 1+ represents 11-20 cells; A cell grade of 2+ represents 21-30 cells; A cell grade of 3+ represents 31-100 cells; A cell grade of 4+ indicates >100 cells. Rare cells were captured as a cell grade of 0.5+.
9A-9B show retinal thickness maps derived from optical coherence tomography (OCT) images and OCT images taken from subjects 1-6 in cohort 1 of the study described in Example 1 at a median follow-up time of 44 weeks. Changes in BCVA ETDRS letter and CST from baseline are also provided. Actual weeks obtained from OCT images and retinal thickness maps for each subject are shown. Asterisks indicate that subject 4 underwent retinal detachment repair by bubble injection to repair spontaneous intraocular intraocular lens detachment lacunar retinal detachment (RRD) ( FIG. 9B ). Subject 4 remains under follow-up; OCT images and BCVA values for subject 4 correspond to the last observation before retinal detachment.
10 shows mean best corrected visual acuity (BCVA) measurements based on Early Treatment Diabetic Retinopathy Study (ETDRS) character assessment for subjects in Cohort 2 of the studies described in Examples 2 and 3 at the indicated time points. Error bars represent 90% confidence intervals for mean absolute BCVA calculated using the T-distribution. Baseline (BL) represents measurements taken prior to screening aflibercept injection on Day 1, 7-15 days (eg, 7-14 days) of AAV2.7m8-aflibercept treatment. After 24 weeks of AAV2.7m8-aflibercept treatment, subjects exhibited a mean BCVA change of -4.8 characters. BL = baseline; D = days; W = week. The Day 1 visit occurred 7-15 days after the baseline visit.
11 shows the mean central retinal thickness (CST) for subjects in Cohort 2 of the studies described in Examples 2 and 3 at the indicated time points. Error bars represent 90% confidence intervals for mean absolute CST calculated using the T-distribution. Baseline (BL) represents measurements taken prior to screening aflibercept injection on Day 1, 7-15 days (eg, 7-14 days) of AAV2.7m8-aflibercept treatment. After 24 weeks of AAV2.7m8-aflibercept treatment, subjects exhibited a mean CST change of -27.8 μm. BL = baseline; D = days; W = week. The Day 1 visit occurred 7-15 days after the baseline visit.
12 shows a plot of aqueous humor and vitreous cell counts after treatment with AAV2.7m8-aflibercept for subjects in Cohort 2 of the studies described in Examples 2 and 3 up to a follow-up time of 24 weeks. The steroid treatment administered to each subject is shown below each plot; The frequency of topical steroid (difluprednate) eye drops administration is shown (eg, 1x = once per day; 2x = twice per day; 3x = 3 times per day; 4x = 4 per day. episode). The aqueous humor cell grade category was based on the Standardization of Uveitis Nomenclature (SUN) criteria (Jabs, DA et al. J Ophthalmol. 2005; 140:509-516). The vitreous cell grade categories were based on the National Institutes of Health (NIH) guidelines. For aqueous humor cells, a cell grade of 0.5+ represents 1-5 cells; A cell grade of 1+ represents 6-15 cells; A cell grade of 2+ represents 16-25 cells; A cell grade of 3+ represents 26-50 cells; A cell grade of 4+ indicates >50 cells. For vitreous cells, a cell grade of 0.5+ represents 1-10 cells; A cell grade of 1+ represents 11-20 cells; A cell grade of 2+ represents 21-30 cells; A cell grade of 3+ represents 31-100 cells; A cell grade of 4+ indicates >100 cells. Rare cells were captured as a cell grade of 0.5+ for this analysis.
13A-13B show retinal thickness maps derived from optical coherence tomography (OCT) images and OCT images taken from subjects 1-6 in cohort 2 of the studies described in Examples 2 and 3 up to a follow-up time of 24 weeks. Changes in BCVA from baseline, change in CST from baseline and number of rescue anti-VEGF injections administered during the 24-week follow-up period are also provided. In FIG. 13A , the asterisk indicates that subject 3 received three rescue anti-VEGF injections, including at week 24, all due to loss of >10 characters of BCVA from baseline. This was due to intraretinal or subretinal fluid. In FIG. 13B , the asterisk indicates that subject 5 received 3 rescue anti-VEGF injections, the last of which occurred at week 20 due to an increase of >75 μm in central subfield thickness from baseline.
14 is a swimmer 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 versus time when AAV2.7m8-aflibercept was administered. The y-axis shows each individual subject in Cohorts 1 and 2. Circular shapes represent anti-VEGF IVT injections administered before and after treatment with AAV2.7m8-aflibercept. Asterisks indicate 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 represents day 1, when AAV2.7m8-aflibercept was administered. To the right of the bisecting vertical is each subsequent 61 study visits.
15A-15C are plots of aqueous humor and vitreous cell counts measured by slit lamp test after treatment with AAV2.7m8-aflibercept for subjects in cohorts 1, 2 and 3 of the study described in Examples 1-5. shows 15A shows a plot of aqueous humor and vitreous cell counts for subjects in Cohort 1 up to a follow-up time of 64 weeks (median = 60 weeks; range = 52-64 weeks). 15B shows a plot of aqueous humor 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). 15C shows a plot of aqueous humor and vitreous cell counts for subjects in Cohort 3 up to a follow-up time of 20 weeks. 15A-15C , the steroid treatment administered to each subject is shown below each plot; The aqueous humor cell grade categories were based on the Uveitis Nomenclature Normalization (SUN) criteria: Jabs DA, et al. J Ophthalmol 2005;140:509-516]; The vitreous cell grade categories were based on the National Institutes of Health (NIH) guidelines; aqueous humor cells: 0.5+ = 1-5 cells; 1+ = 6-15 cells; 2+ = 16-25 cells; 3+ = 26-50 cells; 4+ = >50 cells; vitreous cells: 0.5+ = 1-10 cells; 1+ = 11-20 cells; 2+ = 21-30 cells; 3+ = 31-100 cells; 4+ = >100 cells; Rare cells were captured as 0.5+ for this analysis. QID = 4 times per day; TID = 3 times 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 a day; "QID >>" indicates that the subject continues to receive topical steroids 4 times per day.
16A-16B provide the mean BCVA and mean CST for subjects in Cohort 1 (n=6) of the studies described in Examples 1-5 from baseline to Week 52. 16A shows the mean BCVA from baseline to Week 52 (ETDRS letters). Asterisk indicates that one subject had a low BCVA score at weeks 44 and 48 due to retinal detachment. 16B shows the mean CST (μm) from baseline to Week 52. Asterisks indicate that one subject had no CST data at weeks 44 and 48 due to retinal detachment. 16A-16B , error bars represent 90% confidence intervals for mean absolute BCVA and CST values using T-distribution; BL = baseline; D = days; W = week.
17A-17B provide the mean BCVA and mean CST for subjects in Cohort 2 (n=6) of the studies described in Examples 1-5 from baseline to Week 36. 17A shows the mean BCVA (ETDRS letters) from baseline to Week 36. 17B shows the mean CST (μm) from baseline to Week 36. 17A-17B , error bars represent 90% confidence intervals for mean absolute BCVA and CST values using T-distribution; BL = baseline; D = days; W = week; One subject missed the Week 36 visit.
18A-18B provide the mean BCVA and mean CST for 5 subjects in Cohort 3 of the studies described in Examples 1-5 from baseline to Week 20. 18A shows the mean BCVA (ETDRS letters) from baseline to Week 20. 18B shows the mean CST (μm) from baseline to week 20. 18A-18B , error bars represent 90% confidence intervals for mean absolute BCVA and CST values using T-distribution; BL = baseline; D = days; W = week; One subject missed the Week 36 visit.
19 is a swimmer lane plot showing the number of anti-VEGF injections administered to subjects in cohorts 1, 2 and 3 of the studies described in Examples 1-5. The x-axis represents time in weeks versus time when AAV2.7m8-aflibercept was administered. The y-axis shows each individual subject in cohorts 1-3. Circular shapes represent anti-VEGF IVT injections administered before and after treatment with AAV2.7m8-aflibercept. The vertical line bisecting the plot represents day 1, when AAV2.7m8-aflibercept was administered. To the right of the dichotomy is each follow-up study visit.
20A-20B are derived from optical coherence tomography (OCT) images and OCT images taken from subject 4 of cohort 1 of the study described in Examples 1-5 at the indicated times before and after administration of AAV2.7m8-aflibercept. shows the retinal thickness map. 20A is an OCT image and retina for subject 4 of cohort 1 at the indicated time (weeks) prior to administration of AAV2.7m8-aflibercept during which subjects were administered anti-VEGF IVT injection (ranibizumab). Show the thickness map. 20B shows OCT images and retinal thickness maps for subject 4 of cohort 1 at the indicated times (weeks) before and after administration of AAV2.7m8-aflibercept. The times of administration of screening anti-VEGF IVT injections (“screening anti-VEGF IVT”) and AAV2.7m8-aflibercept (“AAV2.7m8-aflibercept”) are shown. BCVA (ETDRS letters) and CST (μm) at each indicated time before and after administration of AAV2.7m8-aflibercept are presented. Asterisks indicate that the subject had retinal detachment events not related to AAV2.7m8-aflibercept.
21A-21B are derived from optical coherence tomography (OCT) images and OCT images taken from subject 5 of cohort 3 of the study described in Examples 1-5 at the indicated times before and after administration of AAV2.7m8-aflibercept. shows the retinal thickness map. 21A is an OCT image and retina for subject 5 of cohort 3 at the indicated time weeks prior to administration of AAV2.7m8-aflibercept, during which subjects were administered anti-VEGF IVT injection (aflibercept). Show the thickness map. 21B shows OCT images and retinal thickness maps for subject 5 of cohort 3 at the indicated times (weeks) before and after administration of AAV2.7m8-aflibercept. The times of administration of screening anti-VEGF IVT injections (“screening anti-VEGF IVT”) and AAV2.7m8-aflibercept (“AAV2.7m8-aflibercept”) are shown. BCVA (ETDRS letters) and CST (μm) at each indicated time before and after administration of AAV2.7m8-aflibercept are presented.
22A-22D show aqueous humor cells and vitreous as measured by slit lamp examination after treatment with AAV2.7m8-aflibercept for subjects in cohorts 1, 2, 3 and 4 of the studies described in Examples 1-5 and 7; A plot of cell numbers is shown. 22A shows a plot of aqueous humor and vitreous cell counts for subjects in Cohort 1 up to a follow-up time of 80 weeks. 22B shows a plot of aqueous humor and vitreous cell counts for subjects in Cohort 2 up to a follow-up time of 56 weeks. 22C shows a plot of aqueous humor and vitreous cell counts for subjects in Cohort 3 up to a follow-up time of 36 weeks. 22D shows a plot of aqueous humor cell and vitreous cell counts for subjects in Cohort 4 up to 8 weeks of follow-up time. 22A-22D , the steroid treatment administered to each subject is shown below each plot; The aqueous humor cell grade categories were based on the Uveitis Nomenclature Normalization (SUN) criteria: Jabs DA, et al. J Ophthalmol 2005;140:509-516]; The vitreous cell grade categories were based on the National Institutes of Health (NIH) guidelines; aqueous humor cells: 0.5+ = 1-5 cells; 1+ = 6-15 cells; 2+ = 16-25 cells; 3+ = 26-50 cells; 4+ = >50 cells; vitreous cells: 0.5+ = 1-10 cells; 1+ = 11-20 cells; 2+ = 21-30 cells; 3+ = 31-100 cells; 4+ = >100 cells; Rare cells were captured as 0.5+ for this analysis. QID = 4 times per day; TID = 3 times 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 a day; "TID >>" indicates that the subject continues to receive topical steroids 3 times per day; "QID >>" indicates that the subject continues to receive topical steroids 4 times per day.
23A-23B provide the mean BCVA and mean CST for subjects in Cohort 1 (n=6) of the studies described in Examples 1-5 and 7 from baseline to Week 72. 23A shows the mean BCVA from baseline to week 72 (ETDRS letters). One subject had a low BCVA score at weeks 44 and 48 due to retinal detachment. *n = 5 (weeks 56 through 72). 23B shows the mean CST (μm) from baseline to week 72. One subject had no CST data at weeks 44 and 48 due to retinal detachment. *n = 5 (weeks 56 through 72). 23A-23B , error bars represent 90% confidence intervals for mean absolute BCVA and CST values using T-distribution; BL = baseline; D = days; W = week.
24A-24B provide the mean BCVA and mean CST for subjects in Cohort 2 (n=6) of the studies described in Examples 1-5 and 7 from baseline to Week 52. 24A shows the mean BCVA from baseline to Week 52 (ETDRS letters). *n = 5 (for Weeks 36 and 40). 24B shows the mean CST (μm) from baseline to Week 52. *n = 5 (for Weeks 36 and 40). 24A-24B , error bars represent 90% confidence intervals for mean absolute BCVA and CST values using T-distribution; BL = baseline; D = days; W = week.
25A-25B provide the mean BCVA and mean CST for subjects in Cohort 3 (n=9) of the studies described in Examples 1-5 and 7 from baseline to Week 20. 25A shows the mean BCVA (ETDRS letters) from baseline to Week 20. *n = 8 (for Weeks 4, 16 and 20). 25B shows the mean CST (μm) from baseline to week 20. *n = 8 (for Weeks 4, 16 and 20). 25A-25B , error bars represent 90% confidence intervals for mean absolute BCVA and CST values using T-distribution; BL = baseline; D = days; W = week.
26 is a swimmer lane plot showing the number of anti-VEGF injections administered to subjects in cohorts 1, 2, 3 and 4 of the studies described in Examples 1-5 and 7. The x-axis represents time in weeks versus time when AAV2.7m8-aflibercept was administered. The y-axis shows each individual subject in cohorts 1-4. Circular shapes represent anti-VEGF IVT injections administered before and after treatment with AAV2.7m8-aflibercept. The vertical line bisecting the plot represents day 1, when AAV2.7m8-aflibercept was administered. To the right of the dichotomy is each follow-up study visit. 5 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 prior to Day 1 are incomplete due to relocation for Subject 2 and incomplete for Subject 5 because the subject participated in a clinical trial with unknown drug(s) after diagnosis.
27A-27B show the mean annual anti-VEGF injection rates for subjects in Cohorts 1-3 of the studies described in Examples 1-5 and 7; 27A shows subjects in Cohort 1 (6×10 ¹¹ vg/eye administered “high dose”; n=6) and subjects in Cohorts 2 and 3 (2×10 ¹¹ vg/eye “low dose” administered; A comparison of the mean annual anti-VEGF injection rates for n = 15) is provided. 27B provides a comparison of mean annual anti-VEGF injection rates for subjects in each of cohorts 1 (n = 6), 2 (n = 6) and 3 (n = 9). 27A-27B , annual ratio (prior) = (number of anti-VEGF IVT injections within 12 months prior to AAV2.7m8-aflibercept) / (AAV2.7m8-sick from first anti-VEGF IVT injection within last 12 months) days until libercept injection / 365.25); and annual rate (since) = (number of anti-VEGF IVT injections since AAV2.7m8-aflibercept) / (days from AAV2.7m8-aflibercept to last study follow-up / 365.25).
28A-28B are optical coherence tomography (OCT) images and OCT images taken from subject 5 of cohort 3 of the studies described in Examples 1-5 and 7 at the indicated times before and after administration of AAV2.7m8-aflibercept. shows the retinal thickness map derived from 28A is an OCT image and retina for subject 5 of cohort 3 at the indicated time (weeks) prior to administration of AAV2.7m8-aflibercept, during which subjects were administered anti-VEGF IVT injection (aflibercept). Show the thickness map. 28B shows OCT images and retinal thickness maps for subject 5 of cohort 3 at the indicated times (weeks) before and after administration of AAV2.7m8-aflibercept. The time of administration of screening anti-VEGF IVT injection (“aflibercept IVT”) and AAV2.7m8-aflibercept is shown. BCVA (ETDRS letters) and CST (μm) at each indicated time before and after administration of AAV2.7m8-aflibercept are presented.
29 shows a diagram of the study design described in Example 6 evaluating the persistence 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 = center subfield thickness. *All subjects will receive a 7-week course of difluprednate eye drops starting at QID and tapering to QD. **PE = primary endpoint assessment. ***EOS = End of study evaluation.

Several aspects are described below with reference to exemplary applications for purposes of illustration. It should be understood that numerous specific details, relationships and methods are set forth in order to provide a thorough understanding of the features described herein. However, one of ordinary skill in the art will readily recognize that features described herein may be practiced without one or more of the specific details or otherwise. Because some acts may occur in a different order and/or concurrently with other acts or events, the features described herein are not limited by the illustrated order of acts or events. Additionally, not all illustrated acts or events are required to practice a methodology in accordance with features described herein.

Justice

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

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 form is intended to also include the plural form unless the context clearly indicates otherwise. Additionally, where the terms “comprising,” “comprises,” “having,” “having,” “having,” or variations thereof, are used in the specification and/or claims, such terms refer to the terms “comprising” and It is intended to be inclusive in a similar manner. As used herein, the term “comprising” is synonymous with “comprising” or “containing” and is inclusive or open-ended.

Any reference to “or” herein is intended to encompass “and/or” unless otherwise stated. As used herein, the term “about” a number refers to a number plus or minus 10% of that number. The term “about” a range refers to the range minus 10% of its lowest value and plus 10% of its maximum value. Reference herein to “about” a value or parameter includes (and describes) embodiments that relate to that value or parameter per se.

The terms “subject,” “patient,” or “individual” refer to primates, such as humans and non-human primates, such as African green monkeys and rhesus monkeys. In some embodiments, the subject is a human.

As used herein, the terms “treat”, “treating”, “treatment”, “ameliorate” or “ameliorating” and other grammatical equivalents include alleviating the symptoms of an ocular neovascular disease or disorder or ocular neovascular disease or disorder. , weakening or ameliorating, preventing further symptoms of an ocular neovascular disease or disorder, ameliorating or preventing an underlying metabolic cause of a symptom, inhibiting an ocular neovascular disease or disorder, e.g., ocular neoplasia refers to stopping the development of a vascular disease or disorder, alleviating the ocular neovascular disease or disorder, causing regression of the ocular neovascular disease or disorder, or arresting the symptoms of the ocular neovascular disease or disorder, , is intended to include prevention. The term further includes achieving a therapeutic benefit and/or a prophylactic benefit. The term “therapeutic benefit” refers to the eradication or amelioration of the ocular neovascular disease or disorder being treated. In addition, the therapeutic benefit is, in some embodiments, 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 even though the subject still suffers from the ocular neovascular disease or disorder. is achieved by For prophylactic benefit, the pharmaceutical composition is administered to a subject at risk of developing an ocular neovascular disease or disorder, or in a subject reporting one or more of the physiological symptoms of an ocular neovascular disease or disorder, wherein a diagnosis of such disease or disorder is made. Even if not given, it is administered.

As used herein, the terms “administer,” “administering,” “administration,” and the like, may refer to a method used to enable delivery of a therapeutic agent or pharmaceutical composition to a desired site of biological action. These methods include intravitreal or subretinal injection into the eye.

As used herein, the term “effective amount,” “therapeutically effective amount,” or “pharmaceutically effective amount” means sufficient amount of at least one pharmaceutical composition or compound to be administered that will alleviate to some extent one or more of the symptoms of the ocular disease or disorder being treated. quantity can be referred to. An “effective amount,” “therapeutically effective amount,” or “pharmaceutically effective amount” of a pharmaceutical composition may be administered as a unit dose (as described in further detail elsewhere herein) to a subject in need thereof.

As used herein, the term “pharmaceutically acceptable” may refer to a material, such as a carrier or diluent, that does not abrogate the biological activity or properties of a compound disclosed herein and which is relatively nontoxic (ie, when the material is administered to an individual). , it does not cause undesirable biological effects and does not interact in a deleterious manner with any of the components of the composition in which it is contained).

As used herein, the term “pharmaceutical composition” or simply “composition” refers to a biological composition optionally admixed with at least one pharmaceutically acceptable chemical ingredient, such as, but not limited to, carriers, stabilizers, diluents, dispersants, suspending agents, thickeners, excipients, and the like. may refer to an active compound.

As used herein, "AAV vector" or "rAAV vector" refers to a polynucleotide sequence that is not of AAV origin (e.g., a polynucleotide heterologous to AAV, such as a therapeutic transgene, for transduction into a target cell or target tissue; adeno-associated virus (AAV) vector or recombinant AAV (rAAV) vector comprising, for example, a nucleic acid sequence encoding aflibercept. In general, a heterologous polynucleotide is flanked by at least one, usually two, AAV inverted terminal repeat sequences (ITRs). The term rAAV vector encompasses both rAAV vector particles and rAAV vector plasmids. rAAV vectors can be single-stranded (ssAAV) or self-complementary (scAAV).

“AAV virus” or “AAV viral particle” or “rAAV vector particle” or “rAAV particle” refers to a viral particle comprising at least one AAV capsid protein and a polynucleotide rAAV vector. In some cases, the at least one AAV capsid protein is from wild-type AAV or is a variant AAV capsid protein (eg, an AAV capsid protein having an insertion, eg, an insertion of a 7m8 amino acid sequence as set forth below). When the particle comprises a heterologous polynucleotide (eg, a polynucleotide other than the wild-type AAV genome, such as a transgene to be delivered to a target cell or target tissue), it is a "rAAV particle", "rAAV vector particle" or " rAAV vectors". Thus, production of rAAV particles necessarily includes production of rAAV vectors, since such vectors are contained within rAAV particles.

As used herein, the term “packaging” may refer to a series of intracellular events that can result in assembly and encapsidation of rAAV particles.

The AAV “rep” and “cap” genes refer to polynucleotide sequences encoding the replication and encapsidation proteins of adeno-associated viruses. AAV rep and cap are referred to herein as AAV “packaging genes”.

The term “polypeptide” can encompass both naturally occurring and non-naturally occurring proteins (eg, fusion proteins), peptides, fragments, mutants, derivatives and analogs thereof. Polypeptides may be monomers, dimers, trimers or polymers. Additionally, a polypeptide may comprise a number of different domains, each having one or more distinct activities. For the avoidance of doubt, a “polypeptide” may be any length of more than two amino acids.

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

In some embodiments, a variant is, e.g., at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98 relative to its corresponding reference protein. % or 99% total sequence homology. In some embodiments, a variant may have at least about 90% overall sequence homology to a 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.

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

The term "anti-VEGF agent" refers to a protein capable of reducing, interfering, disrupting, blocking and/or inhibiting the activity or function of endogenous VEGF and/or endogenous VEGF receptor (VEGFR) or VEGF-VEGFR interaction or pathway in vivo. , polypeptide, peptide, fusion protein, multimeric protein, gene product, antibody, human monoclonal antibody, antibody fragment, aptamer, small molecule, kinase inhibitor, receptor or receptor fragment, or any therapeutic agent, including a nucleic acid molecule . An anti-VEGF agent is a known therapeutic agent capable of reducing new blood vessel growth or formation and/or edema or swelling when delivered into a cell, tissue or subject in vivo, for example ranibizumab, brolucizumab or beva It may be any one of shizumab. In some embodiments, the anti-VEGF agent may be naturally occurring, non-naturally occurring, or synthetic. In some embodiments, the anti-VEGF agent may be derived from a naturally occurring molecule that has been subsequently modified or mutated to confer anti-VEGF activity. In some embodiments, the anti-VEGF agent is a fusion or chimeric protein. In such proteins, a functional domain or polypeptide is artificially fused to a moiety or polypeptide to create a fusion or chimeric protein that can sequester VEGF in vivo or function as a VEGFR decoy. In some embodiments, the anti-VEGF agent is a fusion or chimeric protein that blocks endogenous VEGFR from interacting with its ligand.

As used herein, "VEGF", unless otherwise required, VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E, VEGF-F or any combination or any functional fragment or variant thereof may refer to any isoform of VEGF, including but not limited to. Unless otherwise required, "VEGF" refers to any member of the VEGF family, including members of VEGF-A, placental growth factor (PGF), VEGF-B, VEGF-C and VEGF-D or any combination, functional fragment or variant thereof. can refer to members of As used herein, “VEGF receptor” or “VEGFR” or “VEGF-R” refers to VEGFR-1 (or Flt-1), VEGFR-2 (or Flk-1/KDR) and VEGFR-3 (or Flt-4). It can be used to refer to any one of the receptors of VEGF, including but not limited thereto. The VEGFR may be a membrane-bound or soluble form or a functional fragment or truncated product of the receptor. Examples of anti-VEGF agents include, but are not limited to, ranibizumab, bevacizumab, brolucizumab or any combination, variant or functional fragment thereof.

“Operably linked” or “operably linked” or “coupled” may refer to the juxtaposition of genetic elements, wherein the elements are in a relationship that permits them to function in the expected manner. For example, a promoter may be operably linked to a coding region if the promoter helps initiating transcription of the coding sequence. As long as this functional relationship is maintained, there may be intervening residues between the promoter and the coding region.

The term “expression vector” or “expression construct” or “cassette” or “plasmid” or simply “vector” refers to a nucleic acid or Gene constructs of any type, including AAV or rAAV vectors, containing polynucleotides. Transcripts can be translated into proteins. In some embodiments, the transcript is partially translated or untranslated. In certain aspects, expression includes both transcription of a gene and translation of mRNA into a gene product. In another aspect, expression comprises only transcription of a nucleic acid encoding a gene of interest. The expression vector may also include control elements operably linked to a coding region to facilitate expression of the protein in a target cell. A control element and a gene or combination of genes operably linked for expression may sometimes be referred to as an "expression cassette," many of which are known and available in the art and are available from or from components available in the art. can be easily built.

The term “heterologous” may refer to an individual that is genotypically distinct from that of the rest of the individual to be compared. For example, a polynucleotide introduced by genetic engineering techniques into a plasmid or vector derived from a different species may be a heterologous polynucleotide. A promoter removed from the native coding sequence and operably linked to a coding sequence not found to be naturally linked may be a heterologous promoter.

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

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

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

survey

Current therapies for ocular neovascular diseases such as wAMD (eg, aflibercept recombinant protein, ranibizumab recombinant protein) require lifetime IVT administration approximately every 4-8 weeks. This may increase the risk of inflammation, infection and other side effects in some patients. Additionally, current therapies pose a challenge of compliance, particularly in elderly patients most affected by wAMD, due to repeated and/or frequent visits to clinics for administration of therapy. Reduced dosing frequency is associated with loss of vision and worsening of eye diseases or conditions. The ability of AAV vectors to efficiently transduce target retinal cells after IVT injection has been exploited to treat various retinal diseases by successfully delivering therapeutic genes into photoreceptors, retinal pigment epithelium and inner retina. Accordingly, administration of rAAV particles encoding an anti-VEGF agent (eg, aflibercept) may provide prolonged and/or sustained release of the anti-VEGF agent in vivo.

Surprisingly, administration of a single low unit dose of rAAV particles encoding aflibercept of 6 x 10 ¹¹ vector genomes (vg) per eye to the eyes of individuals with ocular neovascular disease resulted in stabilization and robustness of the disease in all treated individuals. followed by an anatomical response (see Example 1). Additionally, following administration of a single low unit dose of 6×10 ¹¹ vg/eye of rAAV particles encoding aflibercept, visual acuity was stabilized in all treated subjects, and salvage anti-VEGF treatment (eg, aflibercept None of the subjects required sept IVT injections. Moreover, administration of a single unit dose of rAAV particles encoding aflibercept to the eye of an individual with ocular neovascular disease is unexpectedly associated with prior anti-VEGF treatment (eg, aflibercept, ranibizumab, or bevacizumab). Chronic IVT injection of Mab) resulted in reduction (eg, resolution) of symptoms, including intraretinal and subretinal fluid refractory.

Accordingly, the present disclosure provides a method of treating an ocular neovascular disease in a subject by administering a single unit dose of rAAV particles encoding an anti-VEGF agent (eg, aflibercept) of 6×10 ¹¹ vg/eye or less. provides Additionally, the present disclosure provides a method of reducing retinal fluid in the eye of an individual having an ocular neovascular disease by administering a single unit dose of rAAV particles encoding an anti-VEGF agent (eg, aflibercept). do. The methods disclosed herein address non-compliance and non-compliance issues by reducing or eliminating the need for repeated IVT injections while providing long-term efficacy. Additionally, the methods provided herein reduce side effects associated with multiple IVT injections.

treatment method

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

Also provided herein is a method of reducing retinal fluid in an eye of a subject having an ocular neovascular disease comprising administering to the eye a unit dose of rAAV particles.

Also, administering an anti-VEGF agent (eg, aflibercept) to the eye of the subject, and administering a unit dose of recombinant adeno-associated virus (rAAV) particles to the eye of the subject after administration of the anti-VEGF agent Provided herein is a method of treating an ocular neovascular disease in a subject comprising:

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

In some embodiments, the term ocular neovascular disease also encompasses VEGF-induced pre-neovascular disease, which, if left untreated, progresses to a neovascular form. In some embodiments, the ocular neovascular disease is pre-neovascular disease, non-proliferative diabetic retinopathy.

In some embodiments, the individual is a human. In some embodiments, the individual has about the last 8 weeks, about the last 9 weeks, about the last 10 weeks, about the last 11 weeks, about the last 12 weeks, about the last 13 weeks, about the last 14 weeks, anti-VEGF agent (eg, bevacizumab, brolucizumab, ranibizumab, parisimab, abicipar pegol, convercept, OPT-302, KSI- 301, injectable sunitinib maleate (GB-102), PAN-90806 (PanOptica) and/or aflibercept) at least one prior treatment for ocular neovascular disease (e.g. , at least 1, at least 2, at least 3, at least 4, at least 5 or more treatments). In some embodiments, the individual is an anti-VEGF agent (eg, bevacizumab, brolucizumab, ranibizumab, parisimab, abicipar pegol, Convercept, OPT-302, KSI-301, injection Significant responses to prior treatment with possible sunitinib maleate (GB-102), PAN-90806 (panoptica) and/or aflibercept) were shown. In some embodiments, the anti-VEGF agent is aflibercept, a functional variant thereof, or a functional fragment thereof. In some embodiments, the anti-VEGF agent comprises a polypeptide comprising an amino acid sequence having at least about 95% identity to the amino acid sequence of SEQ ID NO:35. In some embodiments, the retinal fluid in the individual's eye is intraretinal fluid (IRF) and/or subretinal fluid (SRF). In some embodiments, the amount or presence of retinal fluid in the eye of the individual is an anti-VEGF agent (eg, bevacizumab, brolucizumab, ranibizumab, parisimab, abicipar pegol, convercept , OPT-302, KSI-301, injectable sunitinib maleate (GB-102), PAN-90806 (panoptica) and/or aflibercept). In some embodiments, the anti-VEGF agent is aflibercept, a functional variant thereof, or a functional fragment thereof. In some embodiments, the anti-VEGF agent comprises a polypeptide comprising an amino acid sequence having at least about 95% identity to the amino acid sequence of SEQ ID NO:35.

In some embodiments, the ocular neovascular disease is diabetic macular edema (DME). In some embodiments, the individual is a human. In some embodiments, the individual has type 1 or type 2 diabetes. In some embodiments, the individual has visual impairment due to centrally involved diabetic macular edema. In some embodiments, the individual has about 78 to 50 ETDRS letters (e.g., 50, 51, 52, 53, 54, 55, 56, 57, ETDRS letters of any of 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, or 78) (BCVA). In some embodiments, the individual has a visual acuity (Snellen counterpart) of from about 20/32 to about 20/100 in the eye to which the rAAV particles are administered prior to administration of the unit dose of the rAAV particles. In some embodiments, the individual has a central sub of >325 μm when using Heidelberg Spectralis® in the presence of central-involving IRF (central 1 mm) in the eye to which the rAAV particles are administered prior to administration of the unit dose of the rAAV particles. field thickness (CST). In some embodiments, the individual has a decrease in vision in the eye to which the rAAV particles are administered prior to administration of the unit dose of the rAAV particles, primarily due to diabetic macular edema. In some embodiments, the individual administers the unit dose of rAAV particles for no more than about 6 months, e.g., about 6 months, about 5 months, about 4 months, about 3 months, about 2 months, Diabetic macular edema was diagnosed in an eye to which rAAV particles were administered about 1 month or less prior. In some embodiments, the individual has used 0, 1, or 2 prior treatments for DME, e.g., an anti-VEGF agent, e.g., aflibercept, in the eye to which the rAAV particles are administered prior to administration of the unit dose of the rAAV particles. 0, 1 or 2 intravitreal injections were received. In some embodiments, the individual received prior treatment with an anti-VEGF agent in the eye to which the rAAV particles are administered at least about 60 days (ie, about 2 months) prior to administration of the unit dose of the rAAV particles. In some embodiments, the individual has a significant response in central subfield thickness, e.g., at least a central subfield thickness, to prior treatment with an anti-VEGF agent in the eye to which the rAAV particles are administered prior to administration of the unit dose of the rAAV particles. showed a 10% reduction. In some embodiments, the individual did not experience an adverse reaction to prior treatment with an anti-VEGF agent prior to administration of the unit dose of rAAV particles. In some embodiments, the individual does not have neutralizing antibodies 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, eg, 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, a corticosteroid or fluorescein dye or sodium fluorescein (eg, used in angiography) prior to administration of the unit dose of the rAAV particles. In some embodiments, the individual has a history of mild allergy to aflibercept, a corticosteroid or fluorescein dye or sodium fluorescein (e.g., used in angiography) prior to administration of the unit dose of rAAV particles, Allergies are treatable here. In some embodiments, the individual does not have uncontrolled diabetes, eg, greater than 10% HbA1C, prior to administration of the unit dose of the 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 the rAAV particles. In some embodiments, the individual has not initiated intensive insulin treatment prior to administration of the unit dose of the rAAV particles, eg, with an insulin pump or multiple daily insulin injections. In some embodiments, the individual does not plan to initiate intensive insulin treatment within about 3 months after administration of the unit dose of the rAAV particles, eg, with an insulin pump or multiple daily insulin injections. In some embodiments, the individual does not have a history of systemic autoimmune disease requiring treatment with systemic steroids or immunosuppressive treatment, eg, methotrexate or adalimumab, prior to administration of the unit dose of rAAV particles. In some embodiments, the individual is not receiving a systemic drug known to cause macular edema, such as fingolimod, tamoxifen, chloroquine, or hydroxychloroquine, prior to administration of the unit dose of the rAAV particles. In some embodiments, the individual is not receiving systemic anti-VEGF treatment prior to administration of the unit dose of the rAAV particles. In some embodiments, the individual does not have high risk proliferative diabetic retinopathy (PDR) in the eye to which the rAAV particles are administered prior to administration of the unit dose of the rAAV particles. In some embodiments, the PDR is any vitreous or preretinal hemorrhage, neovascularization elsewhere in the >1/2-disc area within an area equivalent to standard ETDRS 7-field on clinical examination, or >1/ on clinical examination Defined as neovascularization of the disc in the 3-disc region. In some embodiments, the individual has not undergone topical or grating laser photocoagulation in the eye to which the rAAV particles are administered prior to administration of the unit dose of the rAAV particles. In some embodiments, the individual has not received any prior panretinal photocoagulation (PRP) in the eye to which the rAAV particles are administered prior to administration of the unit dose of the rAAV particles. In some embodiments, the individual has not received anti-VEGF therapy (eg, aflibercept IVT injection) in the eye to which the rAAV particles are administered prior to administration of the unit dose of the rAAV particles. In some embodiments, the individual has not received anti-VEGF therapy (eg, aflibercept IVT injection) in the eye to which the rAAV particles are administered for at least 60 days prior to administration of the unit dose of the rAAV particles. In some embodiments, the individual has not received more than two anti-VEGF treatments (eg, aflibercept IVT injections) in the eye to which the rAAV particles are administered prior to administration of the unit dose of the rAAV particles. In some embodiments, the individual has anterior segment neovascularization (eg, neovascularization of the iris [NVI] or neovascular glaucoma [NVG]) in the eye to which the rAAV particles are administered prior to administration of the unit dose of the rAAV particles. There is no history of either vitreous hemorrhage, fibrovascular hyperplasia or traction retinal detachment. In some embodiments, the individual prior to administration of the unit dose of the rAAV particles has a structural abnormality in the fovea that contributes to macular edema or visual impairment in the eye to which the rAAV particles are administered (e.g., dense hard exudate, pigment abnormalities, foveal atrophy) , either vitreous macular traction or epiretinal membrane). In some embodiments, structural abnormalities in the fovea are assessed on clinical examination or OCT. In some embodiments, the individual has a retinal disease other than diabetic retinopathy (e.g., age-related macular degeneration (in either eye), retinal veins in the eye to which the rAAV particles are administered prior to administration of the unit dose of the rAAV particles. occlusion, retinal artery occlusion, or pathological myopia). In some embodiments, the individual has prior to administration of the unit dose of the rAAV particles a history of an ocular disease other than diabetic macular edema in the eye to which the rAAV particles are administered, e.g., evidence of significant cataracts or macular traction or subcapsular cataracts. don't have In some embodiments, the individual does not have a history of cataract extraction or yttrium aluminum garnet (YAG) capsulotomy in the eye to which the rAAV particles are administered within at least about 3 months prior to administration of the unit dose of the rAAV particles. In some embodiments, the individual does not have a history of retinal detachment (with or without repair) in the eye to which the rAAV particles are administered prior to administration of the unit dose of the 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 to which the rAAV particles are administered prior to administration of the unit dose of the rAAV particles. In some embodiments, the individual does not have a history of vitrectomy or other filtration surgery in the eye to which the rAAV particles are administered prior to administration of the unit dose of the rAAV particles. In some embodiments, the individual does not have the presence of an aphakic or anterior intraocular lens in the eye to which the rAAV particles are administered prior to administration of the unit dose of the rAAV particles. In some embodiments, the individual has, prior to administration of the unit dose of the rAAV particles, uncontrolled ocular hypertension in the eye to which the rAAV particles are administered, despite current use of treatment with an anti-glaucoma medication or >2 IOP lowering medications. or glaucoma (eg, IOP >22 mmHg). In some embodiments, the individual is administered intraocular or periocular steroid treatment (e.g., IVT Triessence, Ilubien, or Ozurdex) for any ocular condition in the eye to which the rAAV particles are administered prior to administration of the unit dose of the rAAV particles. ) does not have a history of In some embodiments, the individual has not undergone refractive surgery in the eye to which the rAAV particles are administered within at least about 90 days prior to administration of the unit dose of the rAAV particles. In some embodiments, the individual has not received prior penetrating keratoplasty, endothelial keratoplasty, or ocular radiation in the eye to which the rAAV particles are administered prior to administration of the unit dose of the rAAV particles. In some embodiments, the individual has not had any prior vitreous retinal surgery in the eye to which the rAAV particles are administered prior to administration of the unit dose of the rAAV particles. In some embodiments, the individual does not have a history of uveitis or intraocular inflammation, eg, greater than or equal to grade trace, except for mild, expected postoperative inflammation that resolved prior to administration of the unit dose of rAAV particles. In some embodiments, the individual does not have a history of elevated IOP associated with topical steroid administration prior to administration of the unit dose of rAAV particles. In some embodiments, the individual has a history of ocular herpes simplex virus (HSV), including viral uveitis, retinitis, or keratitis, varicella-zoster virus (VZV), or cytomegalovirus (CMV) prior to administration of the unit dose of the rAAV particles. do not have In some embodiments, the individual does not have evidence of any of the external ocular infections, including conjunctivitis, polyps, or significant blepharitis prior to administration of the unit dose of the rAAV particles. In some embodiments, the individual does not have a history of ocular toxoplasmosis prior to administration of the unit dose of rAAV particles.

In some embodiments, a unit dose is expressed as the number of vector genomes (vg). In some embodiments, the unit dose is about 6×10 ¹¹ vector genomes (vg) or less of rAAV particles. In some embodiments, unit dose is expressed as number of vector genomes per eye (vg) (vg/eye). In some embodiments, the unit dose is about 6 x 10 ¹¹ vg/eye or less of rAAV particles. In some embodiments, the unit dose of the rAAV particles is from about 6 x 10 ¹⁰ to about 2 x 10 ¹¹ vg/eye. In some embodiments, the unit dose of rAAV particles is about 2 x 10 ¹¹ or about 6 x 10 ¹⁰ vg/eye.

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

In some embodiments, administering the unit dose of rAAV particles to the contralateral eye of the individual comprises administering the unit dose of rAAV particles to one eye for up to about 2 weeks (e.g., about 0 days, 1 day, 2 days, 3 days) days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days or 14 days). In some embodiments, the unit dose of rAAV particles administered to the individual's contralateral eye is approximately equal to (e.g., less than 1% higher or lower, 5 less than % 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).

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

In some embodiments, the rAAV particle comprises a) 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 a nucleic acid encoding a polypeptide comprising an amino acid sequence having 100% identity and flanked by an AAV2 inverted terminal repeat (ITR), and b) with position 587 of the capsid protein, wherein the amino acid residue numbering corresponds to the AAV2 VP1 capsid protein; and an AAV2 capsid protein comprising the amino acid sequence LGETTRP (SEQ ID NO: 14) inserted between 588. The sequence of SEQ ID NO: 35 is provided below:

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

In some embodiments, the rAAV particle is 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 and a nucleic acid encoding a polypeptide comprising an amino acid sequence having 98%, at least about 99%, at least about 99.99% or 100% identity and flanked by an AAV2 inverted terminal repeat (ITR). In some embodiments, the rAAV particle comprises a nucleic acid encoding a polypeptide comprising an amino acid sequence having at least about 95% identity to the amino acid sequence of SEQ ID NO: 35 and flanked by AAV2 inverted terminal repeats (ITRs). do. 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 a functional fragment thereof.

In some embodiments, the rAAV particle is 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 a nucleic acid comprising a codon-optimized sequence encoding an amino acid sequence having 98%, at least about 99%, at least about 99.99% or 100% identity and flanked by an AAV2 inverted terminal repeat (ITR). In some embodiments, the rAAV particle comprises a codon-optimized sequence encoding an amino acid sequence having at least about 95% identity to the amino acid sequence of SEQ ID NO: 35 and flanked by an AAV2 inverted terminal repeat (ITR). contains nucleic acids. In some embodiments, the rAAV particle comprises a nucleic acid comprising a codon-optimized sequence encoding an amino acid sequence having 100% identity to the amino acid sequence of SEQ ID NO: 35 and flanked by AAV2 inverted terminal repeats (ITRs). includes

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

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

In some embodiments, the rAAV particle comprises an AAV2 capsid protein comprising the amino acid sequence LGETTRP (SEQ ID NO: 14) inserted between positions 587 and 588 of AAV2 VP1 comprising the sequence of SEQ ID NO: 13. The sequence of SEQ ID NO: 13 is provided below:

In some embodiments, the rAAV particle comprises an AAV2 capsid protein comprising the amino acid sequence LALGETTRPA (SEQ ID NO: 1) inserted between positions 587 and 588 of the capsid protein, wherein the amino acid residue numbering corresponds to an AAV2 VP1 capsid protein. . In some embodiments, the rAAV particle comprises an AAV2 capsid protein comprising the amino acid sequence LALGETTRPA (SEQ ID NO: 1) inserted between positions 587 and 588 of AAV2 VP1 comprising the sequence of SEQ ID NO: 13.

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

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

In some embodiments, the unit dose of rAAV particles is in a pharmaceutical formulation. In some embodiments, the pharmaceutical formulation comprises rAAV particles, one or more osmotic or ionic strength agents, one or more buffers, 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 at least one buffer is 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 rAAV particles, sodium chloride, sodium phosphate and a surfactant. In some embodiments, the pharmaceutical formulation comprises about 1× ^{10 10} vg/mL to about 1×10 ¹³ vg/mL of rAAV particles. In some embodiments, the pharmaceutical formulation comprises about 6x10 ¹¹ vg/mL to about 6x10 ¹² vg/mL of rAAV particles. In some embodiments, the pharmaceutical formulation comprises about 150 mM to about 200 mM sodium chloride (e.g., any of about 150 mM, about 160 mM, about 170 mM, about 180 mM, about 190 mM, or about 200 mM). do. In some embodiments, the pharmaceutical formulation comprises about 1 mM to about 10 mM sodium phosphate monobasic (e.g., about 1 mM, about 2 mM, about 3 mM, about 4 mM, about 5 mM, about 6 mM, about 7 mM , about 8 mM, about 9 mM or about 10 mM). In some embodiments, the pharmaceutical formulation is about 1 mM to about 10 mM sodium phosphate dibasic (e.g., about 1 mM, about 2 mM, about 3 mM, about 4 mM, about 5 mM, about 6 mM, about 7 mM , about 8 mM, about 9 mM or about 10 mM). In some embodiments, the pharmaceutical formulation comprises from about 0.0005% (w/v) to about 0.005% (w/v) poloxamer 188 (e.g., about 0.0005% (w/v), 0.0006% (w/v), 0.0007% (w/v), 0.0008% (w/v), 0.0009% (w/v), 0.001% (w/v), 0.002% (w/v), 0.003% (w/v), 0.004% (w/v) or about 0.005% (w/v)). In some embodiments, the pharmaceutical formulation has a pH of about 7.0 to about 7.5 (eg, 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 6x10 ¹² vg/mL of rAAV particles, about 180 mM sodium chloride, about 5 mM sodium phosphate monobasic, about 5 mM sodium phosphate dibasic, 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 6x10 ¹¹ vg/mL of rAAV particles, about 180 mM sodium chloride, about 5 mM sodium phosphate monobasic, about 5 mM sodium phosphate dibasic, and about 0.001% (w/v) poloxamer 188 wherein the pharmaceutical formulation has a pH of about 7.3.

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

In some embodiments, 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 systemic steroid treatment. In some embodiments, the steroid treatment is oral steroid treatment. In some embodiments, the steroid treatment is prednisone treatment. In some embodiments, the steroid treatment is an ophthalmic steroid treatment. In some embodiments, the ophthalmic steroid treatment is topical steroid treatment (eg, eye drops), periocular steroid treatment (eg, subtenon, subconjunctival), intravitreal steroid treatment, or suprachoroidal steroid treatment. In some embodiments, the topical steroid treatment is difluprednate treatment, medrisone treatment, loteprednol treatment, prednisolone treatment, fluocinolone treatment, triamcinolone treatment, rimexolone treatment, dexamethasone treatment, fluorometholone treatment, fluoci Nolon treatment, rimexolone treatment, or prednisone treatment. In some embodiments, the topical steroid treatment is difluprednate treatment. In some embodiments, the steroid treatment is administered before, during, and/or after administration of the unit dose of the rAAV particles. In some embodiments, the steroid treatment is administered prior to administration of the unit dose of the rAAV particles. In some embodiments, the steroid treatment is administered during administration of a unit dose of the rAAV particles. In some embodiments, the steroid treatment is administered following administration of a unit dose of the rAAV particles. In some embodiments, the steroid treatment is administered prior to and during administration of the unit dose of the rAAV particles. In some embodiments, the steroid treatment is administered before and after administration of the unit dose of the rAAV particles. In some embodiments, the steroid treatment is administered during and after administration of the unit dose of the rAAV particles. In some embodiments, the steroid treatment is administered before, during, and after administration of the unit dose of the rAAV particles.

In some embodiments, the steroid treatment is an ophthalmic steroid treatment (eg, difluprednate). In some embodiments, the ophthalmic steroid treatment (eg, difluprednate) is daily steroid treatment for up to about 4 weeks, about 6 weeks, or about 8 weeks following administration of the unit dose of rAAV particles. In some embodiments, the ophthalmic steroid treatment is about 4 administrations of the ophthalmic steroid in about 1 week, about 3 administrations of the ophthalmic steroid in about 2 weeks, and about 2 administrations of the ophthalmic steroid in about 3 weeks. administration and about once administration of the ophthalmic steroid at about the fourth week; The period begins with administration of a unit dose of rAAV particles and thereafter. In some embodiments, the ophthalmic steroid is from about 0.005% to about 0.5% difluprednate. In some embodiments, the ophthalmic steroid is about 0.005%, about 0.006%, about 0.007%, about 0.008%, about 0.009%, about 0.01%, about 0.02%, about 0.03%, about 0.4%, about 0.05%, about any of 0.06%, about 0.07%, about 0.08%, about 0.09%, or about 0.1% difluprednate. In some embodiments, the ophthalmic steroid is difluprednate 0.05%. In some embodiments, the dose of difluprednate 0.05% is one drop of the ophthalmic solution. In some embodiments, a drop is about 50 μl (eg, about 25 μl to about 50 μl, about 50 μl to about 100 μl). In some embodiments, the dose of difluprednate comprises from about 1 μg to about 5 μg or from about 2 μg to about 3 μg or about 2.5 μg difluprednate. In some embodiments, the dose of difluprednate comprises about 2.5 μg difluprednate.

In some embodiments, the steroid treatment is an ophthalmic steroid treatment (eg, difluprednate). In some embodiments, the ophthalmic steroid treatment (eg, difluprednate) is daily topical steroid treatment for up to about 4 weeks, about 6 weeks, or about 8 weeks following administration of a unit dose of rAAV particles. In some embodiments, topical steroid treatment comprises about 4 administrations of the topical steroid in about 1 week, about 3 administrations of the topical steroid in about 2 weeks, about 2 administrations of the topical steroid in about 3 weeks, and a drug including about 1 administration of topical steroid in 4 weeks; The period begins with administration of a unit dose of rAAV particles and thereafter. In some embodiments, topical steroid treatment comprises administration of a unit dose of rAAV particles, followed by administration of about 4 doses of topical steroid per day (i.e., QID) for about 3 weeks, followed by administration of about 3 doses of topical steroid per day for about 1 week. single administration (i.e., TID), followed by about 2 administrations of the topical steroid per day for about 1 week (i.e. BID), followed by about 1 administration of the topical steroid per day for about 1 week (i.e., QD). include In some embodiments, the topical steroid comprises 0.05% difluprednate in a dose of about 1 μg to about 3 μg. In some embodiments, the topical steroid comprises 0.05% difluprednate at a dose of about 2.5 μg. In some embodiments, the topical steroid is from about 0.005% to about 0.5% difluprednate. In some embodiments, the topical steroid is about 0.005%, about 0.006%, about 0.007%, about 0.008%, about 0.009%, about 0.01%, about 0.02%, about 0.03%, about 0.04%, about 0.05%, about 0.06 %, about 0.07%, about 0.08%, about 0.09%, or about 0.1% difluprednate. In some embodiments, the topical steroid is difluprednate 0.05%. In some embodiments, the dose of difluprednate 0.05% is one drop of the ophthalmic solution. In some embodiments, a drop is about 50 μl (eg, about 25 μl to about 50 μl, about 50 μl to about 100 μl). In some embodiments, the dose of difluprednate comprises from about 1 μg to about 5 μg or from about 2 μg to about 3 μg or about 2.5 μg difluprednate. In some embodiments, the dose of difluprednate comprises about 2.5 μg difluprednate.

In some embodiments, the retinal fluid (e.g., SRF and/or IRF) in the eye of the individual is reduced by about 5%, about 10%, reduced by more than any of about 15%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100%. In some embodiments, the retinal fluid (eg, SRF and/or IRF) in the eye of the individual is administered to one eye and/or the contralateral side of the individual, followed by administration of the unit dose of the rAAV particle, followed by administration of the unit dose of the rAAV particle. About 5%, about 10%, about 15%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80 compared to the retinal fluid level in the eye of the whole subject. %, greater than about 90%, or about any of 100%. In some embodiments, the retinal fluid (eg, SRF and/or IRF) in the eye of the individual is administered to one eye and/or the contralateral side of the individual, followed by administration of the unit dose of the rAAV particle, followed by administration of the unit dose of the rAAV particle. It is reduced by about 100% compared to the retinal fluid level in the entire subject's eye.

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

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

In some embodiments, treatment of an ocular neovascular disease in an individual after administration of a unit dose of rAAV particles to one eye and/or the contralateral eye is administered to the retina prior to administration of a unit dose of rAAV particles to one eye and/or the contralateral eye. assessed based on the level of retinal fluid (eg, SRF and/or IRF) compared to the level of fluid (eg, subretinal fluid (SRF) and/or intraretinal fluid (IRF)) (eg, 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 a unit dose of rAAV particles to one eye and/or the contralateral eye is administered to the retina prior to administration of a unit dose of rAAV particles to one eye and/or the contralateral eye. a decrease in retinal fluid (eg, IRF and/or SRF) after administration of a unit dose of rAAV particles to one eye and/or the contralateral eye compared to the level of fluid (eg, IRF and/or SRF) It is determined when observed (eg, as described above). In some embodiments, the ocular neovascular disease is wAMD.

In some embodiments, administering a unit dose of rAAV particles to one and/or the contralateral eye of the individual results in maintenance or reduction of retinal thickness as compared to retinal thickness prior to administration of the unit dose of rAAV particles. In some embodiments, administering the unit dose of rAAV particles to one and/or the contralateral eye of the individual results in a decrease in retinal thickness compared to retinal thickness prior to administration of the unit dose of rAAV particles. In some embodiments, the retinal thickness is central subfield thickness (CST) or central retinal thickness (CRT). In some embodiments, administering the unit dose of rAAV particles to one and/or the contralateral eye of the individual is about 5%, about 10%, about 15%, about 5%, about 10%, about 15%, about retinal thickness prior to administration of the unit dose of rAAV particles. 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% , causing a reduction in retinal thickness of greater than any of about 85%, about 90%, about 95%, about 99%, or about 100%. In some embodiments, retinal thickness (eg, CST or CRT) is determined by OCT or SD-OCT.

In some embodiments, administering a unit dose of rAAV particles to one and/or contralateral eye of the individual is from about 10 μm to about 100 μm (e.g., about 10 μm, about 15 μm, about 20 μm, about 25 μm, about 30 μm, about 35 μm, about 40 μm, about 45 μm, about 50 μm, about 55 μm, about 60 μm, about 65 μm, about 70 μm, about 75 μm, about 80 μm, about 85 μm, causing a decrease in central retinal thickness (CRT) or central subfield thickness (CST) of any of about 90 μm, about 95 μm, about 100 μm or more). In some embodiments, administering a unit dose of rAAV particles to one and/or contralateral eye of the individual is from about 5 μm to about 50 μm (e.g., about 5 μm, about 10 μm, about 15 μm, about 20 causing a decrease in central retinal thickness (CRT) or central subfield thickness (CST) of any of μm, about 25 μm, about 30 μm, about 35 μm, about 40 μm, about 45 μm or about 50 μm). In some embodiments, administering a unit dose of rAAV particles to one and/or the contralateral eye of the individual is between about 5 μm and about 40 μm (e.g., about 5 μm, about 10 μm, about 15 μm, about 20 causing a decrease in central retinal thickness (CRT) or central subfield thickness (CST) of any of μm, about 25 μm, about 30 μm, about 35 μm or about 40 μm). In some embodiments, administering a unit dose of rAAV particles to one and/or contralateral eye of the individual is between about 5 μm and about 30 μm (e.g., about 5 μm, about 6 μm, about 7 μm, about 8 μm, about 9 μm, about 10 μm, about 11 μm, about 12 μm, about 13 μm, about 14 μm, about 15 μm, about 16 μm, about 17 μm, about 18 μm, about 19 μm, about 20 μm, central retinal thickness (CRT) of any of 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) or a decrease in the central subfield thickness (CST). In some embodiments, administering a unit dose of rAAV particles to one and/or the contralateral eye of the individual is between about 5 μm and about 25 μm (e.g., about 5 μm, about 6 μm, about 7 μm, about 8 μm, about 9 μm, about 10 μm, about 11 μm, about 12 μm, about 13 μm, about 14 μm, about 15 μm, about 16 μm, about 17 μm, about 18 μm, about 19 μm, about 20 μm, causing a decrease in central retinal thickness (CRT) or central subfield thickness (CST) of about 21 μm, about 22 μm, about 23 μm, about 24 μm, or about 25 μm). In some embodiments, administering a unit dose of the rAAV particles to one and/or the contralateral eye of the individual is between about 5 μm and about 20 μm (e.g., about 5 μm, about 6 μm, about 7 μm, about 8 of about 9 μm, about 10 μm, about 11 μm, about 12 μm, about 13 μm, about 14 μm, about 15 μm, about 16 μm, about 17 μm, about 18 μm, about 19 μm, or about 20 μm. either) of the central retinal thickness (CRT) or the central subfield thickness (CST). In some embodiments, administering a unit dose of rAAV particles to one and/or the contralateral eye of the individual results in a decrease in central retinal thickness (CRT) or central subfield thickness (CST) from about 18 μm to about 75 μm. do. In some embodiments, administering a unit dose of rAAV particles to one and/or the contralateral eye of the individual results in a decrease in central retinal thickness (CRT) or central subfield thickness (CST) of about 18.5 μm. In some embodiments, administering a unit dose of rAAV particles to one and/or the contralateral eye of the individual results in a decrease in central retinal thickness (CRT) or central subfield thickness (CST) of about 21.0 μm. In some embodiments, administering a unit dose of rAAV particles to one and/or the contralateral eye of the individual results in a decrease in central retinal thickness (CRT) or central subfield thickness (CST) of about 8.3 μm. In some embodiments, administering a unit dose of rAAV particles to one and/or the contralateral eye of the individual results in a decrease in central retinal thickness (CRT) or central subfield thickness (CST) of about 25.5 μm. In some embodiments, administering a unit dose of rAAV particles to one and/or the contralateral eye of the individual results in a decrease in central retinal thickness (CRT) or central subfield thickness (CST) of about 24.8 μm. In some embodiments, administering a unit dose of rAAV particles to one and/or the contralateral eye of the individual results in a decrease in central retinal thickness (CRT) or central subfield thickness (CST) of about 75 μm. In some embodiments, administering a unit dose of rAAV particles to one and/or contralateral eye of the individual is at least about 100 μm (e.g., at least about 100 μm, at least about 110 μm, at least about 120 μm, at least about 130 central retinal thickness (CRT) or center of any of greater than or equal to about μm, greater than about 140 μm, greater than about 150 μm, greater than about 160 μm, greater than about 170 μm, greater than about 180 μm, greater than about 190 μm, or greater than about 200 μm It causes a decrease in the subfield thickness (CST). In some embodiments, administering a unit dose of rAAV particles to one and/or contralateral eye of the individual is between about 10 μm and about 200 μm (e.g., about 10 μm, about 20 μm, about 30 μm, about 40 μm, about 50 μm, about 60 μm, about 70 μm, about 80 μm, about 90 μm, about 100 μm, about 110 μm, about 120 μm, about 130 μm, about 140 μm, about 150 μm, about 160 μm, causing a decrease in central retinal thickness (CRT) or central subfield thickness (CST) of any of about 170 μm, about 180 μm, about 190 μm, or about 200 μm). In some embodiments, administering a unit dose of rAAV particles to one and/or contralateral eye of the individual is from about 50 μm to about 200 μm (e.g., about 50 μm, about 60 μm, about 70 μm, about 80 of about 90 μm, about 100 μm, about 110 μm, about 120 μm, about 130 μm, about 140 μm, about 150 μm, about 160 μm, about 170 μm, about 180 μm, about 190 μm, or about 200 μm either) of the central retinal thickness (CRT) or the central subfield thickness (CST). In some embodiments, administering a unit dose of rAAV particles to one and/or the contralateral eye of the individual is between about 60 μm and about 200 μm (e.g., about 60 μm, about 70 μm, about 80 μm, about 90 μm, about 100 μm, about 110 μm, about 120 μm, about 130 μm, about 140 μm, about 150 μm, about 160 μm, about 170 μm, about 180 μm, about 190 μm, or about 200 μm) It causes a decrease in central retinal thickness (CRT) or central subfield thickness (CST). In some embodiments, administering a unit dose of rAAV particles to one and/or contralateral eye of the individual is from about 70 μm to about 200 μm (e.g., about 70 μm, about 80 μm, about 90 μm, about 100 central retinal thickness (any of 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) CRT) or central subfield thickness (CST). In some embodiments, administering a unit dose of the rAAV particles to one and/or the contralateral eye of the individual is between about 80 μm and about 200 μm (e.g., about 80 μm, about 90 μm, about 100 μm, about 110). central retinal thickness (CRT) or center of any of 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) It causes a decrease in the subfield thickness (CST). In some embodiments, administering a unit dose of rAAV particles to one and/or the contralateral eye of the individual is from about 90 μm to about 200 μm (e.g., about 90 μm, about 100 μm, about 110 μm, about 120 central retinal thickness (CRT) or central subfield thickness (any of 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) CST) decreases. In some embodiments, administering a unit dose of rAAV particles to one and/or contralateral eye of the individual is between about 100 μm and about 200 μm (e.g., about 100 μm, about 110 μm, about 120 μm, about 130 reduction in central retinal thickness (CRT) or central subfield thickness (CST) in any of μm, about 140 μm, about 150 μm, about 160 μm, about 170 μm, about 180 μm, about 190 μm or about 200 μm) causes In some embodiments, administering a unit dose of rAAV particles to one and/or contralateral eye of the individual is between about 110 μm and about 200 μm (e.g., about 110 μm, about 120 μm, about 130 μm, about 140 μm, about 150 μm, about 160 μm, about 170 μm, about 180 μm, about 190 μm, or about 200 μm) of the central retinal thickness (CRT) or central subfield thickness (CST). In some embodiments, administering a unit dose of rAAV particles to one and/or the contralateral eye of the individual is between about 115 μm and about 200 μm (e.g., about 115 μm, about 120 μm, about 125 μm, about 130). μm, about 135 μm, about 140 μm, about 145 μm, about 150 μm, about 155 μm, about 160 μm, about 165 μm, about 170 μm, about 175 μm, about 180 μm, about 185 μm, about 190 μm, causing a decrease in central retinal thickness (CRT) or central subfield thickness (CST) of either about 195 μm or about 200 μm). In some embodiments, administering a unit dose of rAAV particles to one and/or the contralateral eye of the individual is between about 120 μm and about 200 μm (e.g., about 120 μm, about 125 μm, about 130 μm, about 135). μm, about 140 μm, about 145 μm, about 150 μm, about 155 μm, about 160 μm, about 165 μm, about 170 μm, about 175 μm, about 180 μm, about 185 μm, about 190 μm, about 195 μm, or about 200 μm) of central retinal thickness (CRT) or central subfield thickness (CST). In some embodiments, administering a unit dose of rAAV particles to one and/or contralateral eye of the individual is from about 125 μm to about 200 μm (e.g., about 125 μm, about 130 μm, about 135 μm, about 140 of about 145 μm, about 150 μm, about 155 μm, about 160 μm, about 165 μm, about 170 μm, about 175 μm, about 180 μm, about 185 μm, about 190 μm, about 195 μm, or about 200 μm. either) of the central retinal thickness (CRT) or the central subfield thickness (CST). In some embodiments, administering a unit dose of rAAV particles to one and/or contralateral eye of the individual is between about 130 μm and about 200 μm (e.g., about 130 μm, about 135 μm, about 140 μm, about 145 μm, about 150 μm, about 155 μm, about 160 μm, about 165 μm, about 170 μm, about 175 μm, about 180 μm, about 185 μm, about 190 μm, about 195 μm, or about 200 μm) It causes a decrease in central retinal thickness (CRT) or central subfield thickness (CST). In some embodiments, administering a unit dose of rAAV particles to one and/or the contralateral eye of the individual is from about 135 μm to about 200 μm (e.g., about 135 μm, about 140 μm, about 145 μm, about 150 central retinal thickness (any of 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) CRT) or central subfield thickness (CST). In some embodiments, administering a unit dose of rAAV particles to one and/or the contralateral eye of the individual is between about 140 μm and about 200 μm (e.g., about 140 μm, about 145 μm, about 150 μm, about 155). central retinal thickness (CRT) or center of any of 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) It causes a decrease in the subfield thickness (CST). In some embodiments, administering a unit dose of rAAV particles to one and/or the contralateral eye of the individual comprises from about 145 μm to about 200 μm of central retinal thickness (CRT) or central subfield thickness (CST) (e.g., , about 145 μm, about 150 μm, about 155 μm, about 160 μm, about 165 μm, about 170 μm, about 175 μm, about 180 μm, about 185 μm, about 190 μm, about 195 μm, or about 200 μm ) causes a decrease in In some embodiments, administering a unit dose of rAAV particles to one and/or contralateral eye of the individual is between about 150 μm and about 200 μm (e.g., about 150 μm, about 155 μm, about 160 μm, about 165). reduction in central retinal thickness (CRT) or central subfield thickness (CST) of any of μm, about 170 μm, about 175 μm, about 180 μm, about 185 μm, about 190 μm, about 195 μm or about 200 μm) causes In some embodiments, administering a unit dose of rAAV particles to one and/or the contralateral eye of the individual is between about 155 μm and about 200 μm (e.g., about 155 μm, about 160 μm, about 165 μm, about 170 μm, about 175 μm, about 180 μm, about 185 μm, about 190 μm, about 195 μm, or about 200 μm) of central retinal thickness (CRT) or central subfield thickness (CST). In some embodiments, administering a unit dose of rAAV particles to one and/or the contralateral eye of the individual is between about 160 μm and about 200 μm (e.g., about 160 μm, about 165 μm, about 170 μm, about 175 μm). μm, about 180 μm, about 185 μm, about 190 μm, about 195 μm or about 200 μm) of the central retinal thickness (CRT) or central subfield thickness (CST). In some embodiments, administering a unit dose of rAAV particles to one and/or the contralateral eye of the individual is between about 165 μm and about 200 μm (e.g., about 165 μm, about 170 μm, about 175 μm, about 180 causing a decrease in central retinal thickness (CRT) or central subfield thickness (CST) of any of μm, about 185 μm, about 190 μm, about 195 μm, or about 200 μm. In some embodiments, administering a unit dose of rAAV particles to one and/or contralateral eye of the individual is from about 170 μm to about 200 μm (e.g., about 170 μm, about 175 μm, about 180 μm, about 185 causing a decrease in central retinal thickness (CRT) or central subfield thickness (CST) of any of μm, about 190 μm, about 195 μm, or about 200 μm). In some embodiments, administering a unit dose of rAAV particles to one and/or the contralateral eye of the individual is between about 175 μm and about 200 μm (e.g., about 175 μm, about 180 μm, about 185 μm, about 190). μm, about 195 μm, or about 200 μm) of central retinal thickness (CRT) or central subfield thickness (CST). In some embodiments, administering a unit dose of rAAV particles to one and/or contralateral eye of the individual is from about 180 μm to about 200 μm (e.g., about 180 μm, about 185 μm, about 190 μm, about 195 μm). μm or about 200 μm) of central retinal thickness (CRT) or central subfield thickness (CST). In some embodiments, administering the unit dose of rAAV particles to one and/or the contralateral eye of the individual comprises about central retinal thickness (CRT) or central subfield thickness (CST) prior to administration of the unit dose of rAAV particles. It causes a decrease in central retinal thickness (CRT) or central subfield thickness (CST) of 21.0 μm. In some embodiments, administering the unit dose of rAAV particles to one and/or the contralateral eye of the individual comprises about central retinal thickness (CRT) or central subfield thickness (CST) prior to administration of the unit dose of rAAV particles. It causes a decrease in central retinal thickness (CRT) or central subfield thickness (CST) of 8.3 μm. In some embodiments, administering the unit dose of rAAV particles to one and/or the contralateral eye of the individual comprises about central retinal thickness (CRT) or central subfield thickness (CST) prior to administration of the unit dose of rAAV particles. It causes a decrease in central retinal thickness (CRT) or central subfield thickness (CST) of 26.2 μm. In some embodiments, administering the unit dose of rAAV particles to one and/or the contralateral eye of the individual comprises about central retinal thickness (CRT) or central subfield thickness (CST) prior to administration of the unit dose of rAAV particles. It causes a decrease in central retinal thickness (CRT) or central subfield thickness (CST) of 24.8 μm. In some embodiments, administering the unit dose of rAAV particles to one and/or the contralateral eye of the individual comprises about central retinal thickness (CRT) or central subfield thickness (CST) prior to administration of the unit dose of rAAV particles. It causes a decrease in central retinal thickness (CRT) or central subfield thickness (CST) of 40.8 μm. In some embodiments, administering the unit dose of rAAV particles to one and/or the contralateral eye of the individual comprises about central retinal thickness (CRT) or central subfield thickness (CST) prior to administration of the unit dose of rAAV particles. It causes a decrease in central retinal thickness (CRT) or central subfield thickness (CST) of 30.0 μm. In some embodiments, administering the unit dose of rAAV particles to one and/or the contralateral eye of the individual comprises about central retinal thickness (CRT) or central subfield thickness (CST) prior to administration of the unit dose of rAAV particles. It causes a decrease in central retinal thickness (CRT) or central subfield thickness (CST) of 118.6 μm. In some embodiments, administering the unit dose of rAAV particles to one and/or the contralateral eye of the individual comprises about central retinal thickness (CRT) or central subfield thickness (CST) prior to administration of the unit dose of rAAV particles. It causes a decrease in central retinal thickness (CRT) or central subfield thickness (CST) of 119.0 μm. In some embodiments, administering the unit dose of rAAV particles to one and/or the contralateral eye of the individual comprises about central retinal thickness (CRT) or central subfield thickness (CST) prior to administration of the unit dose of rAAV particles. It causes a decrease in central retinal thickness (CRT) or central subfield thickness (CST) of 137.8 μm. In some embodiments, administering the unit dose of rAAV particles to one and/or the contralateral eye of the individual comprises about central retinal thickness (CRT) or central subfield thickness (CST) prior to administration of the unit dose of rAAV particles. Cause a decrease in central retinal thickness (CRT) or central subfield thickness (CST) of 152.7 μm. In some embodiments, administering the unit dose of rAAV particles to one and/or the contralateral eye of the individual comprises about central retinal thickness (CRT) or central subfield thickness (CST) prior to administration of the unit dose of rAAV particles. results in a decrease in central retinal thickness (CRT) or central subfield thickness (CST) of 153.3 μm. In some embodiments, administering a unit dose of rAAV particles to one and/or the contralateral eye of the individual results in a decrease in central retinal thickness (CRT) or central subfield thickness (CST) of the rAAV particles. Administration of the unit dose results in a decrease in central retinal thickness (CRT) or central subfield thickness (CST) of about 149.8 μm compared to central retinal thickness (CRT) or central subfield thickness (CST) prior to administration.

In some embodiments, administering a unit dose of rAAV particles to one and/or contralateral eye of the individual comprises from about -200 μm to about +40 μm (e.g., compared to the retinal thickness prior to administration of the unit dose of rAAV particles). , about -200 μm, about -180 μm, about -160 μm, about -140 μm, about -120 μm, about -100 μm, about -80 μm, about -60 μm, about -40 μm, about -20 μm , about any of 0 μm, about +5 μm, about +10 μm, about +15 μm, about +20 μm, about +25 μm, about +30 μm, about +35 μm, or about +40 μm) It causes changes in retinal thickness (CRT) or central subfield thickness (CST). In some embodiments, administering a unit dose of rAAV particles to one and/or the contralateral eye of the individual comprises about 8 μm, about 11 μm, about 16 μm, about 29 μm, about 33 μm, about 38 μm, about 55 causing a decrease in central retinal thickness (CRT) or central subfield thickness (CST) of any of μm, about 61 μm, or about 117 μm. In some embodiments, administering a unit dose of rAAV particles to one and/or the contralateral eye of the individual results in a decrease in central retinal thickness (CRT) or central subfield thickness (CST) of either about 27.8 μm or about 30.8 μm. causes In some embodiments, administering a unit dose of rAAV particles to one and/or the contralateral eye of the individual results in a central retinal thickness (CRT) or central subfield thickness (CRT) of any of about 4 μm, about 12 μm, or about 32 μm ( CST) increases. In some embodiments, administering the unit dose of rAAV particles to one and/or the contralateral eye of the individual comprises about central retinal thickness (CRT) or central subfield thickness (CST) prior to administration of the unit dose of rAAV particles. Cause a change in central retinal thickness (CRT) or central subfield thickness (CST) of -21.0 μm. In some embodiments, administering the unit dose of rAAV particles to one and/or the contralateral eye of the individual comprises about central retinal thickness (CRT) or central subfield thickness (CST) prior to administration of the unit dose of rAAV particles. Cause a change in central retinal thickness (CRT) or central subfield thickness (CST) of -8.3 μm. In some embodiments, administering the unit dose of rAAV particles to one and/or the contralateral eye of the individual comprises about central retinal thickness (CRT) or central subfield thickness (CST) prior to administration of the unit dose of rAAV particles. Cause a change in central retinal thickness (CRT) or central subfield thickness (CST) of -26.2 μm. In some embodiments, administering the unit dose of rAAV particles to one and/or the contralateral eye of the individual comprises about central retinal thickness (CRT) or central subfield thickness (CST) prior to administration of the unit dose of rAAV particles. Causes a change in central retinal thickness (CRT) or central subfield thickness (CST) of -24.8 μm. In some embodiments, administering the unit dose of rAAV particles to one and/or the contralateral eye of the individual comprises about central retinal thickness (CRT) or central subfield thickness (CST) prior to administration of the unit dose of rAAV particles. Causes a change in central retinal thickness (CRT) or central subfield thickness (CST) of -40.8 μm. In some embodiments, administering the unit dose of rAAV particles to one and/or the contralateral eye of the individual comprises about central retinal thickness (CRT) or central subfield thickness (CST) prior to administration of the unit dose of rAAV particles. Cause a change in central retinal thickness (CRT) or central subfield thickness (CST) of -30.0 μm. In some embodiments, administering the unit dose of rAAV particles to one and/or the contralateral eye of the individual comprises about central retinal thickness (CRT) or central subfield thickness (CST) prior to administration of the unit dose of rAAV particles. Cause a change in central retinal thickness (CRT) or central subfield thickness (CST) of -118.6 μm. In some embodiments, administering the unit dose of rAAV particles to one and/or the contralateral eye of the individual comprises about central retinal thickness (CRT) or central subfield thickness (CST) prior to administration of the unit dose of rAAV particles. −119.0 μm induces a change in central retinal thickness (CRT) or central subfield thickness (CST). In some embodiments, administering the unit dose of rAAV particles to one and/or the contralateral eye of the individual comprises about central retinal thickness (CRT) or central subfield thickness (CST) prior to administration of the unit dose of rAAV particles. Causes a change in central retinal thickness (CRT) or central subfield thickness (CST) of -137.8 μm. In some embodiments, administering the unit dose of rAAV particles to one and/or the contralateral eye of the individual comprises about central retinal thickness (CRT) or central subfield thickness (CST) prior to administration of the unit dose of rAAV particles. -152.7 μm in central retinal thickness (CRT) or central subfield thickness (CST). In some embodiments, administering the unit dose of rAAV particles to one and/or the contralateral eye of the individual comprises about central retinal thickness (CRT) or central subfield thickness (CST) prior to administration of the unit dose of rAAV particles. -153.3 μm in central retinal thickness (CRT) or central subfield thickness (CST). In some embodiments, administering the unit dose of rAAV particles to one and/or the contralateral eye of the individual comprises about central retinal thickness (CRT) or central subfield thickness (CST) prior to administration of the unit dose of rAAV particles. Causes a change in central retinal thickness (CRT) or central subfield thickness (CST) of -149.8 μm.

In some embodiments, a change in central retinal thickness (CRT) or central subfield thickness (CST) as compared to central retinal thickness (CRT) or central subfield thickness (CST) prior to administration of the unit dose of the rAAV particle (e.g., , decrease) is about 1 day, about 1 week, about 2 weeks, about 4 weeks, about 8 weeks, about 16 weeks, about 24 weeks, after any of about 30 weeks, about 32 weeks, about 34 weeks, about 40 weeks, about 44 weeks, about 48 weeks, about 52 weeks or more. In some embodiments, a change in central retinal thickness (CRT) or central subfield thickness (CST) as compared to central retinal thickness (CRT) or central subfield thickness (CST) prior to administration of the unit dose of the rAAV particle (e.g., , decrease) is about 1 day, about 1 week, about 2 weeks, about 4 weeks, about 8 weeks, about 12 weeks, about 16 weeks, after administration of the unit dose of the rAAV particles to one eye and/or the contralateral eye of the subject; 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 after any of 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 .

In some embodiments, administering the unit dose of rAAV particles to one and/or the contralateral eye of the individual is about 5%, about 10%, about 15%, about 5%, about 10%, about 15%, about retinal thickness prior to administration of the unit dose of rAAV particles. 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% , cause a decrease in central retinal thickness (CRT) or central subfield thickness (CST) of greater than any of about 85%, about 90%, about 95%, about 99%, or about 100%. In some embodiments, administering the unit dose of rAAV particles to one and/or the contralateral eye of the individual results in a central retinal thickness (CRT) or central retinal thickness (CRT) of at least about 10% compared to the retinal thickness prior to administration of the unit dose of rAAV particles. It causes a decrease in the subfield thickness (CST). In some embodiments, administering a unit dose of rAAV particles to one and/or contralateral eye of the individual results in a central retinal thickness (CRT) or central sub-retinal thickness (CRT) of at least about 15% compared to the retinal thickness prior to administration of the unit dose of rAAV particles. It causes a decrease in the field thickness (CST).

In some embodiments, administering the unit dose of rAAV particles to one and/or the contralateral eye of the individual is less than about 40 μm (e.g., less than about 40 μm) compared to the retinal thickness prior to administration of the unit dose of rAAV particles. , any of less than about 35 μm, less than about 30 μm, less than about 25 μm, less than about 20 μm, less than about 15 μm, less than about 10 μm, less than about 5 μm, less than about 1 μm or less) of the central retina of the central retina. It causes an increase in thickness (CRT) or center subfield thickness (CST). In some embodiments, administering the unit dose of rAAV particles to one and/or the contralateral eye of the individual results in a central retinal thickness (CRT) or central retinal thickness (CRT) of about 32 μm or less compared to the retinal thickness prior to administration of the unit dose of rAAV particles. It causes an increase in the subfield thickness (CST).

In some embodiments, the maintenance, decrease, or increase in retinal thickness as compared to retinal thickness prior to administration of the unit dose of the rAAV particles occurs at least about 30 weeks after administration of the unit dose of the rAAV particles to one eye and/or the contralateral eye of the individual. exist. In some embodiments, the maintenance, decrease or increase in retinal thickness as compared to retinal thickness prior to administration of the unit dose of the rAAV particles is about 30 weeks after administration of the unit dose of the rAAV particles to one eye and/or the contralateral eye of the individual, about after any of 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.

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

In some embodiments, the maintenance or reduction of the macular volume compared to the macular volume prior to administration of the unit dose of the rAAV particle is at least about 30 weeks after administration of the unit dose of the rAAV particle to one and/or the contralateral eye of the individual. . In some embodiments, the maintenance or reduction of the macular volume compared to the macular volume prior to administration of the unit dose of the rAAV particles is about 30 weeks, about 34 weeks after administration of the unit dose of the rAAV particles to one eye and/or the contralateral eye of the individual. , 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.

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

In some embodiments, administering a unit dose of rAAV particles to one and/or contralateral eye of the individual results in at least 15 ETDRS letters (Vitale et al., (2016) compared to BCVA prior to administration of the unit dose of rAAV particles. 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 characters ) to induce an improvement in BCVA. In some embodiments, administering a unit dose of rAAV particles to one and/or the contralateral eye of the individual results from about 1 to about 15 (e.g., about 1, of any of 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 characters. Causes improvement in BCVA. In some embodiments, administering a unit dose of rAAV particles to one and/or contralateral eye of the individual results in an improvement in BCVA of about 5 ETDRS letters compared to BCVA prior to administration of the unit dose of rAAV particles. In some embodiments, administering the unit dose of rAAV particles to one and/or the contralateral eye of the individual comprises about 1, about 2, about 3, about 4, about 5 compared to BCVA prior to administration of the unit dose of rAAV particles. , resulting in an improvement of the BCVA of the ETDRS letter of either about 6 or about 7. In some embodiments, administering a unit dose of rAAV particles to one and/or contralateral eye of the individual results in an improvement in BCVA of about 3 or more letters of ETDRS compared to BCVA prior to administration of the unit dose of rAAV particles. In some embodiments, administering a unit dose of rAAV particles to one and/or contralateral eye of the individual results in an improvement in BCVA of about 4 or more letters of ETDRS compared to BCVA prior to administration of the unit dose of rAAV particles. In some embodiments, administering a unit dose of rAAV particles to one and/or contralateral eye of the individual results in an improvement in BCVA of at least about 5.1 ETDRS letters as compared to BCVA prior to administration of the unit dose of rAAV particles. In some embodiments, administering a unit dose of rAAV particles to one and/or contralateral eye of the individual results in an improvement in BCVA of at least about 6.4 ETDRS letters as compared to BCVA prior to administration of the unit dose of rAAV particles. In some embodiments, administering the unit dose of rAAV particles to one and/or the contralateral eye of the individual results in an improvement in BCVA of about 6.8 ETDRS characters compared to BCVA prior to administration of the unit dose of rAAV particles. In some embodiments, administering the unit dose of rAAV particles to one and/or the contralateral eye of the individual results in an improvement in BCVA of about 8.8 ETDRS characters compared to BCVA prior to administration of the unit dose of rAAV particles. In some embodiments, administering the unit dose of rAAV particles to one and/or the contralateral eye of the individual results in an improvement in BCVA of about 2.3 ETDRS letters compared to BCVA prior to administration of the unit dose of rAAV particles.

In some embodiments, administering a unit dose of rAAV particles to one and/or the contralateral eye of the individual results in maintenance of BCVA, wherein the individual has less than 15 doses compared to BCVA prior to administration of the unit dose of rAAV particles. ETDRS characters (Vitale et al., (2016) JAMA Opthalmol 134(9):1041:1047) (e.g. 15 or less, 14 or less, 13 or less, 12 or less, 11 or less, 10 or less) , 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, 2 or less, 1 or 0 characters). In some embodiments, administering a unit dose of rAAV particles to one and/or the contralateral eye of the individual results in maintenance of BCVA, wherein the individual is about 2 letters compared to BCVA prior to administration of the unit dose of rAAV particles. to lose In some embodiments, administering a unit dose of rAAV particles to one and/or contralateral eye of the individual results in maintenance of BCVA, wherein the individual is about 1, about 1, compared to BCVA prior to administration of the unit dose of rAAV particles. ETDRS characters of any of 2, about 3, about 4, about 5, about 6, about 7, about 8, or about 9 are lost. In some embodiments, administering a unit dose of rAAV particles to one and/or the contralateral eye of the individual results in maintenance of BCVA, wherein the individual has zero letters compared to BCVA prior to administration of the unit dose of rAAV particles. lose In some embodiments, administering a unit dose of rAAV particles to one and/or the contralateral eye of the individual results in maintenance of BCVA, wherein the individual is about 1 letter compared to BCVA prior to administration of the unit dose of rAAV particles. to lose In some embodiments, administering a unit dose of rAAV particles to one and/or contralateral eye of the individual results in maintenance of BCVA, wherein the individual is about 2.7 letters compared to BCVA prior to administration of the unit dose of rAAV particles. to lose In some embodiments, administering a unit dose of rAAV particles to one and/or the contralateral eye of the individual results in maintenance of BCVA, wherein the individual is about 2.8 characters compared to BCVA prior to administration of the unit dose of rAAV particles. to lose In some embodiments, administering a unit dose of rAAV particles to one and/or the contralateral eye of the individual results in maintenance of BCVA, wherein the individual is about 2 or less compared to BCVA prior to administration of the unit dose of rAAV particles. lose the character of In some embodiments, administering the unit dose of rAAV particles to one eye and/or the contralateral eye of the individual results in maintenance of BCVA, wherein the individual has about 3.2 or less compared to BCVA prior to administration of the unit dose of rAAV particles. lose the character of In some embodiments, administering a unit dose of rAAV particles to one and/or the contralateral eye of the individual results in maintenance of BCVA, wherein the individual is from about 15 to about BCVA compared to BCVA prior to administration of the unit dose of rAAV particles. 0 characters (e.g., 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 any of 1 or 0 characters). In some embodiments, administering a unit dose of rAAV particles to one and/or the contralateral eye of the individual results in maintenance of BCVA, wherein the individual is from about 10 to about BCVA compared to BCVA prior to administration of the unit dose of rAAV particles. 0 characters (eg, any of about 10, about 9, about 8, about 7, about 6, about 5, about 4, about 3, about 2, about 1, or 0 characters) are lost. In some embodiments, administering a unit dose of rAAV particles to one and/or contralateral eye of the individual results in maintenance of BCVA, wherein the individual is from about 5 to about BCVA compared to BCVA prior to administration of the unit dose of rAAV particles. 0 characters (eg, any of about 5, about 4, about 3, about 2, about 1, or 0 characters) are lost. In some embodiments, administering a unit dose of rAAV particles to one and/or contralateral eye of the individual results in maintenance of BCVA, wherein the individual is from about 4 to about BCVA compared to BCVA prior to administration of the unit dose of rAAV particles. 0 characters (eg, any of about 4, about 3, about 2, about 1, or 0 characters) are lost. In some embodiments, administering the unit dose of rAAV particles to one and/or the contralateral eye of the individual results in maintenance of BCVA, wherein the individual is about 3 to about BCVA compared to BCVA prior to administration of the unit dose of rAAV particles. 0 characters (eg, any of about 3, about 2, about 1, or 0 characters) are lost. In some embodiments, administering a unit dose of rAAV particles to one and/or the contralateral eye of the individual results in maintenance of BCVA, wherein the individual is from about 2 to about BCVA compared to BCVA prior to administration of the unit dose of rAAV particles. 0 characters (eg, any of about 2, about 1, or 0 characters) are lost. In some embodiments, administering a unit dose of rAAV particles to one and/or the contralateral eye of the individual results in maintenance of BCVA, wherein the individual is from about 1 to about BCVA compared to BCVA prior to administration of the unit dose of rAAV particles. 0 characters are lost.

In some embodiments, administering a unit dose of rAAV particles to one and/or contralateral eye of the individual results from about -20 to +7 or more (e.g., BCVA) prior to administration of the unit dose of rAAV particles. , -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) causes a change in BCVA of the ETDRS character. In some embodiments, administering a unit dose of rAAV particles to one and/or contralateral eye of the individual results in a BCVA of about any of 16, 7, or 5 ETDRS letters compared to the BCVA prior to administration of the unit dose of rAAV particles. causes an increase in In some embodiments, administering the unit dose of rAAV particles to one and/or the contralateral eye of the individual results in about 19, 14, 7, 6, 5, 4, 3 compared to BCVA prior to administration of the unit dose of rAAV particles. , causing a decrease in BCVA of either 2 or 1 of the ETDRS letters. In some embodiments, administering the unit dose of rAAV particles to one and/or the contralateral eye of the individual results in a decrease in BCVA of about 4.8 or about 0.8 ETDRS letters as compared to BCVA prior to administration of the unit dose of rAAV particles. do. In some embodiments, administering a unit dose of rAAV particles to one and/or contralateral eye of the individual results in a decrease in BCVA of no more than about 2 ETDRS letters as compared to BCVA prior to administration of the unit dose of rAAV particles. . In some embodiments, administering the unit dose of rAAV particles to one and/or the contralateral eye of the individual results in a decrease in BCVA of no more than about 3.2 ETDRS letters as compared to BCVA prior to administration of the unit dose of rAAV particles. .

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

In some embodiments, administering a unit dose of rAAV particles to one and/or the contralateral eye of the individual may result in transient inflammation (e.g., inflammation induced by aqueous humor cells and/or vitreous cells, aqueous humor clouding, post-adhesion, poor pupil dilation). In some embodiments, administering a unit dose of rAAV particles to one and/or contralateral eye of the individual results in inflammation (e.g., inflammation induced by aqueous humor cells and/or vitreous cells, aqueous humor clouding, posterior adhesions, poor pupil dilation), which improves after oral and/or topical steroid treatment and/or administration of a pupil dilator. In some embodiments, administering a unit dose of rAAV particles to one and/or the contralateral eye of the individual results in inflammation (eg, inflammation induced by aqueous humor cells and/or vitreous cells), which can be administered orally and and/or resolves after administration of topical steroid therapy. Inflammation (e.g., inflammation induced by aqueous humor cells and/or vitreous cells, aqueous humor blurring, posterior adhesions, poor pupillary dilatation) can be measured using any method known in the art, such as the slit lamp test. there is.

In some embodiments, the maintenance or improvement of visual acuity compared to visual acuity (eg, BCVA) prior to administration of the unit dose of the rAAV particles is about 1 after administration of the unit dose of the rAAV particles to one and/or the contralateral eye of the individual. days, 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 after any of weeks, about 96 weeks, about 100 weeks, about 104 weeks, about 108 weeks or more. In some embodiments, the maintenance or improvement of visual acuity compared to visual acuity (eg, BCVA) prior to administration of the unit dose of the rAAV particles is about 30 after administration of the unit dose of the rAAV particles to one and/or the contralateral eye of the individual. Exists after more than a week. In some embodiments, the maintenance or improvement of visual acuity compared to visual acuity (eg, BCVA) prior to administration of the unit dose of the rAAV particles is about 30 after administration of the unit dose of the rAAV particles to one and/or the contralateral eye of the individual. after any of 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.

In some embodiments, the treatment of an ocular neovascular disease in an individual after administration of a unit dose of rAAV particles to one eye and/or the contralateral eye is based on best corrected visual acuity (BCVA) in one eye and/or the contralateral eye. is evaluated In some embodiments, BCVA is expressed as an ETDRS score corresponding to the number of characters read correctly (Vitale et al., (2016) JAMA Opthalmol 134(9):1041:1047). In some embodiments, fewer than 15 characters (e.g., 15 or less, 14 or less, 13 No more, 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 loss of any of the dog's characters), the subject is determined to have sight and/or maintenance of sight. In some embodiments, the individual has at least 15 characters (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 any of about 70 characters), the subject is determined to have an improvement in vision and/or visual acuity.

In some embodiments, treatment of an ocular neovascular disease in an individual after administration of a unit dose of rAAV particles to one eye and/or the contralateral eye results in a central subfield thickness (CST) or central subfield thickness (CST) in one eye and/or the contralateral eye. It is evaluated based on retinal thickness (CRT). 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 a unit dose of rAAV particles to one eye and/or the contralateral eye comprises a CST or CRT assessed by SD-OCT in one eye and/or the contralateral eye. a decrease after administration of the unit dose of rAAV particles to one eye and/or the contralateral eye compared to before administration of the unit dose of rAAV particles to the eye. In some embodiments, treatment of an ocular neovascular disease in an individual after administration of a unit dose of rAAV particles to one eye and/or the contralateral eye comprises a CST or CRT assessed by SD-OCT in one eye and/or the contralateral eye. maintained after administration of a unit dose of rAAV particles to one eye and/or the contralateral eye compared to before administration of the unit dose of rAAV particles to the eye.

In some embodiments, treatment of an ocular neovascular disease in an individual following administration of a unit dose of rAAV particles to one eye and/or the contralateral eye is assessed based on macular volume in one 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 a unit dose of rAAV particles to one eye and/or the contralateral eye results in a macular volume assessed by SD-OCT in one eye and/or the contralateral eye. a decrease after administration of the unit dose of rAAV particles to one eye and/or the contralateral eye compared to before administration of the unit dose of rAAV particles. In some embodiments, treatment of an ocular neovascular disease in an individual after administration of a unit dose of rAAV particles to one eye and/or the contralateral eye results in a macular volume assessed by SD-OCT in one eye and/or the contralateral eye. maintained after administration of a unit dose of rAAV particles to one eye and/or the contralateral eye compared to prior administration of the unit dose of rAAV particles.

In some embodiments, treatment of an ocular neovascular disease in an individual after administration of a unit dose of rAAV particles to one eye and/or the contralateral eye is achieved by administering a unit dose of rAAV particles to one eye and/or the contralateral eye, including retinal thickness (e.g., central retina) in one eye and/or the contralateral eye. thickness (CRT) or central subfield thickness (CST)) and macular volume. In some embodiments, CST and macular volume are determined by SD-OCT. In some embodiments, treatment of an ocular neovascular disease in an individual after administration of a unit dose of rAAV particles to one eye and/or the contralateral eye is such that the CST and macular volume assessed by SD-OCT are in one eye and/or the contralateral eye. a decrease after administration of the unit dose of rAAV particles to one eye and/or the contralateral eye compared to prior to administration of the unit dose of rAAV particles to the eye. In some embodiments, treatment of an ocular neovascular disease in an individual after administration of a unit dose of rAAV particles to one eye and/or the contralateral eye is such that the CST and macular volume assessed by SD-OCT are in one eye and/or the contralateral eye. maintained after administration of a unit dose of rAAV particles to one eye and/or the contralateral eye compared to prior administration of the unit dose of rAAV particles to the eye.

In some embodiments, the treatment of an ocular neovascular disease in an individual following administration of a unit dose of rAAV particles to one eye and/or contralateral eye is administered to the individual following administration of a unit dose of rAAV particles to one eye and/or contralateral eye. is assessed based on the number of rescue therapy treatments (eg, aflibercept injections) needed. In some embodiments, treatment of an ocular neovascular disease in an individual following administration of a unit dose of rAAV particles to one eye and/or the contralateral eye comprises administering a unit dose of rAAV particles to one eye and/or the contralateral eye in the individual. After less than 1 rescue therapy treatment (e.g., aflibercept injection) in any of 4 weeks, every 5 weeks, every 6 weeks, every 7 weeks, every 8 weeks, every 9 weeks, every 10 weeks or more ) is determined when necessary.

In some embodiments, the treatment of an ocular neovascular disease in an individual following administration of a unit dose of rAAV particles to one eye and/or the contralateral eye comprises administering a unit dose of rAAV particles to one eye and/or the contralateral eye in the individual. 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 Any rescue therapy treatment (e.g., for any of 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) aflibercept injection) is also not required.

In some embodiments, the individual is at least about 24 months, at least about 23 months, at least about 22 months, at least about 21 months, at least about 20 months, at least about 19 months, at least about 18 months, at least about 17 months, at least about 16 months, at least about 15 months, at least about 14 months, at least about 13 months, at least about 12 months, at least about 11 months, at least about 10 months, at least about 9 months, at least about 8 months, at least about 7 months, at least about 6 months, at least about 5 months, at least about 4 months, at least about 3 months, at least about 2 months, at least about 1 month, at least about 3 weeks, at least No salvage therapy treatment (eg, aflibercept injection) is required for either about 2 weeks or at least about 1 week. In some embodiments, the individual does not require any salvage therapy treatment (eg, aflibercept injection) for at least about 12 months after administration of the unit dose of rAAV particles to one eye and/or the contralateral eye. In some embodiments, the individual does not require any salvage therapy treatment (eg, aflibercept injection) for at least about 10 months after administration of the unit dose of rAAV particles to one eye and/or the contralateral eye. In some embodiments, the individual does not require any salvage therapy treatment (eg, aflibercept injection) for at least about 7 months after administration of the unit dose of rAAV particles to one eye and/or the contralateral eye. In some embodiments, the individual does not require any salvage therapy treatment (eg, aflibercept injection) for at least about 6 months after administration of the unit dose of rAAV particles to one eye and/or the contralateral eye. In some embodiments, the individual does not require any salvage therapy treatment (eg, aflibercept injection) for at least about 2 months after administration of the unit dose of rAAV particles to one eye and/or the contralateral eye. In some embodiments, the individual does not require any salvage therapy treatment (eg, aflibercept injection) for at least about 1 month after administration of the unit dose of rAAV particles to one eye and/or the contralateral eye.

In some embodiments, administering a single unit dose of rAAV particles to one eye and/or the contralateral eye of a plurality of individuals results in at least about 50% (e.g., at least about 50%, at least about 55%, any of 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% ) do not require anti-VEGF rescue treatment (eg, aflibercept injection). In some embodiments, administering a single unit dose of rAAV particles to one and/or contralateral eye of the plurality of individuals results in at least about 67% (e.g., at least about 67%, at least about 70%, Any of 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 subjects are subject to anti-VEGF rescue treatment (e.g., ill Libercept injection) is not required. In some embodiments, administering a single unit dose of rAAV particles to one eye and/or contralateral eye of a plurality of individuals results in at least about 50% of individuals of the plurality of individuals receiving anti-VEGF rescue treatment (eg, afliber Sept injection) is not required. In some embodiments, administering a single unit dose of rAAV particles to one eye and/or contralateral eye of a plurality of individuals results in at least about 78% of individuals of the plurality of individuals receiving anti-VEGF rescue treatment (eg, afliber Sept injection) is not required. In some embodiments, administering a single unit dose of rAAV particles to one eye and/or contralateral eye of a plurality of individuals results in at least about 80% of individuals of the plurality of individuals receiving anti-VEGF rescue treatment (e.g., afliber Sept injection) is not required. In some embodiments, administering a single unit dose of rAAV particles to one and/or contralateral eye of a plurality of individuals results in at least about 82% of individuals of the plurality of individuals receiving anti-VEGF rescue treatment (eg, afliber Sept injection) is not required. In some embodiments, administering a single unit dose of rAAV particles to one and/or contralateral eye of the plurality of individuals results in at least 100% of the individuals of the plurality of individuals receiving anti-VEGF rescue treatment (eg, aflibercept). injection) is not required.

In some embodiments, administering a single unit dose of rAAV particles to one and/or contralateral eye of a plurality of individuals results in at least about 50% (e.g., at least about 50%, at least about 55%, any of 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% ) at least about 4 weeks after administration of the rAAV particles, for example at least about 4 weeks, at least about 8 weeks, at least about 12 weeks, at least about 16 weeks, at least about 20 weeks, at least about 24 weeks after administration of the rAAV particles. weeks, at least about 28 weeks, at least about 32 weeks, at least about 36 weeks, at least about 40 weeks, at least about 44 weeks, at least about 48 weeks, at least about 52 weeks, at least about 56 weeks, at least about 60 weeks, at least about 64 weeks weeks, at least about 68 weeks, at least about 72 weeks, at least about 76 weeks, at least about 80 weeks, at least about 84 weeks, at least about 88 weeks, at least about 92 weeks, at least about 96 weeks, at least about 100 weeks, at least about 104 weeks do not require anti-VEGF rescue treatment (eg, aflibercept injection) for any of a week, at least about 108 weeks or longer. In some embodiments, administering a single unit dose of the rAAV particles to one and/or contralateral eye of the plurality of individuals results in at least about 50% of the individuals of the plurality of individuals having at least about 52 weeks or about 56 weeks following administration of the rAAV particles. Do not require anti-VEGF rescue treatment (eg, aflibercept injection) for more than a week. In some embodiments, administering a single unit dose of rAAV particles to one eye and/or the contralateral eye of the plurality of individuals results in at least about 67% (e.g., at least about 67%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, or 100%) of the subjects at least about 20 weeks after administration of the rAAV particles, e.g. For example, at least about 20 weeks, at least about 24 weeks, at least about 28 weeks, at least about 32 weeks, at least about 36 weeks, at least about 40 weeks, at least about 44 weeks, at least about 48 weeks, at least about 52 weeks after administration of the rAAV particles. do not require anti-VEGF rescue treatment (eg, aflibercept injection) for any of a week, at least about 56 weeks, at least about 60 weeks, at least about 64 weeks, at least about 66 weeks or more. In some embodiments, administering a single unit dose of rAAV particles to one eye and/or contralateral eye of a plurality of individuals results in at least about 78% of individuals of the plurality of individuals administering the rAAV particles at least about 4 weeks after administration of the rAAV particles, e.g. For example, at least about 4 weeks, at least about 8 weeks, at least about 12 weeks, at least about 16 weeks, at least about 20 weeks, at least about 24 weeks, at least about 28 weeks, at least about 32 weeks, at least about 36 weeks after administration of the rAAV particles. , at least about 40 weeks, at least about 44 weeks, at least about 48 weeks, at least about 52 weeks, at least about 56 weeks, at least about 60 weeks, at least about 64 weeks, at least about 68 weeks, at least about 72 weeks, at least about 76 weeks anti-VEGF for any of at least about 80 weeks, at least about 84 weeks, at least about 88 weeks, at least about 92 weeks, at least about 96 weeks, at least about 100 weeks, at least about 104 weeks, at least about 108 weeks or more do not require salvage treatment (eg, aflibercept injection). In some embodiments, administering a single unit dose of rAAV particles to one eye and/or contralateral eye of a plurality of individuals results in at least about 78% of individuals of the plurality of individuals having at least about 20 weeks or about 36 weeks following administration of the rAAV particles. Do not require anti-VEGF rescue treatment (eg, aflibercept injection) for more than a week. In some embodiments, administering a single unit dose of rAAV particles to one and/or contralateral eyes of a plurality of individuals results in at least about 80% of individuals of the plurality of individuals administering the rAAV particles for at least about 20 weeks, e.g., For example, at least about 20 weeks, at least about 24 weeks, at least about 28 weeks, at least about 32 weeks, at least about 36 weeks, at least about 40 weeks, at least about 44 weeks, at least about 48 weeks, at least about 52 weeks after administration of the rAAV particles. , do not require anti-VEGF rescue treatment (eg, aflibercept injection) for any of at least about 56 weeks, at least about 60 weeks, at least about 64 weeks, at least about 66 weeks or more. In some embodiments, administering a single unit dose of rAAV particles to one and/or contralateral eyes of a plurality of individuals results in at least about 82% of individuals of the plurality of individuals administering the rAAV particles for at least about 20 weeks, e.g., For example, at least about 20 weeks, at least about 24 weeks, at least about 28 weeks, at least about 32 weeks, at least about 36 weeks, at least about 40 weeks, at least about 44 weeks, at least about 48 weeks, at least about 52 weeks after administration of the rAAV particles. , do not require anti-VEGF rescue treatment (eg, aflibercept injection) for any of at least about 56 weeks, at least about 60 weeks, at least about 64 weeks, at least about 66 weeks or more. In some embodiments, administering a single unit dose of rAAV particles to one eye and/or contralateral eye of a plurality of individuals results in 100% of individuals of the plurality of individuals administering the rAAV particles at least about 20 weeks after administration of the rAAV particles, e.g., rAAV at least about 20 weeks, at least about 24 weeks, at least about 28 weeks, at least about 32 weeks, at least about 36 weeks, at least about 40 weeks, at least about 44 weeks, at least about 48 weeks, at least about 52 weeks, at least do not require anti-VEGF rescue treatment (eg, aflibercept injection) for any of about 56 weeks, at least about 60 weeks, at least about 64 weeks, at least about 66 weeks or more. In some embodiments, administering a single unit dose of rAAV particles to one eye and/or contralateral eye of a plurality of individuals ensures that 100% of individuals of the plurality of individuals are at least about 4 weeks after administration of the rAAV particles, e.g., rAAV at least about 4 weeks, at least about 8 weeks, at least about 12 weeks, at least about 16 weeks, at least about 20 weeks, at least about 24 weeks, at least about 28 weeks, at least about 32 weeks, at least about 36 weeks, at least about 40 weeks, at least about 44 weeks, at least about 48 weeks, at least about 52 weeks, at least about 56 weeks, at least about 60 weeks, at least about 64 weeks, at least about 68 weeks, at least about 72 weeks, at least about 76 weeks, at least about 76 weeks, at least anti-VEGF rescue treatment for any of about 80 weeks, at least about 84 weeks, at least about 88 weeks, at least about 92 weeks, at least about 96 weeks, at least about 100 weeks, at least about 104 weeks, at least about 108 weeks or more (e.g., aflibercept injection) is not required. In some embodiments, administering a single unit dose of rAAV particles to one and/or contralateral eye of a plurality of individuals results in 100% of individuals in the plurality of individuals at least about 64 weeks, at least 72 weeks, or Do not require anti-VEGF rescue treatment (eg, aflibercept injection) for any of 84 weeks or longer.

In some embodiments, administering a unit dose of rAAV particles to one eye and/or the contralateral eye of a plurality of individuals requires any salvage treatment (eg, aflibercept injection) in one eye and/or the contralateral eye. About 78% or less (eg, about 78% or less, about 75% or less, about 70% or less, about 65% or less, about 60% or less, about 55% or less, about 50% or less 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 or less, about 1% or less, or about 0.5% or less). In some embodiments, administering a unit dose of rAAV particles to one eye and/or the contralateral eye of a plurality of individuals requires any salvage treatment (eg, aflibercept injection) in one eye and/or the contralateral eye. about 50% or less (e.g., about 50% or less, about 45% or less, about 40% or less, about 35% or less, about 30% or less, about 25% or less, about 20% or less) of the plurality of individuals or less, about 15% or less, about 10% or less, about 5% or less, about 2.5% or less, about 1% or less, or about 0.5% or less). In some embodiments, administering a unit dose of rAAV particles to one eye and/or the contralateral eye of a plurality of individuals requires any salvage treatment (eg, aflibercept injection) in one eye and/or the contralateral eye. less than about 30% (e.g., about 30%, about 25%, about 20%, about 15%, about 10%, about 5%, about 2.5%, about 1%, or about less than any of 0.5%). In some embodiments, administering a unit dose of rAAV particles to one eye and/or the contralateral eye of a plurality of individuals requires any salvage treatment (eg, aflibercept injection) in one eye and/or the contralateral eye. to be less than about 30% of the plurality of individuals. In some embodiments, administering a unit dose of rAAV particles to one eye and/or the contralateral eye of a plurality of individuals requires any salvage treatment (eg, aflibercept injection) in one eye and/or the contralateral eye. to be less than about 20% of the plurality of individuals. In some embodiments, administering a unit dose of rAAV particles to one eye and/or the contralateral eye of a plurality of individuals requires any salvage treatment (eg, aflibercept injection) in one eye and/or the contralateral eye. Make sure that the number of objects is 0% of the plurality of objects.

In some embodiments, administering the unit dose of the rAAV particles to one and/or contralateral eyes of the plurality of individuals comprises at least about 4 weeks after administration of the rAAV particles, e.g., at least about 4 weeks after administration of the rAAV particles, at least about 8 weeks, at least about 12 weeks, at least about 16 weeks, at least about 20 weeks, at least about 24 weeks, at least about 28 weeks, at least about 32 weeks, at least about 36 weeks, at least about 40 weeks, at least about 44 weeks, at least about 48 weeks, at least about 52 weeks, at least about 56 weeks, at least about 60 weeks, at least about 64 weeks, at least about 68 weeks, at least about 72 weeks, at least about 76 weeks, at least about 80 weeks, at least about 84 weeks, at least about 84 weeks, at least any salvage treatment (e.g., aflibercept injection) for any of 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 about 78% or less (e.g., about 78% or less, about 75% or less, about 70% or less, about 65% or less, about 60% or less, about 55% or less, about 50% or less) of the plurality of individuals in need thereof % or less, about 45% or less, about 40% or less, about 35% or less, about 30% or less, about 25% or less, about 20% or less, about 15% or less, about 10% or less, about 5% or less, about 2.5 % or less, about 1% or less, or about 0.5% or less). In some embodiments, administering a unit dose of rAAV particles to one and/or contralateral eyes of a plurality of individuals comprises at least about 20 weeks after administration of the rAAV particles, e.g., at least about 20 weeks after administration of the rAAV particles, at least about 24 weeks, at least about 28 weeks, at least about 32 weeks, at least about 36 weeks, at least about 40 weeks, at least about 44 weeks, at least about 48 weeks, at least about 52 weeks, at least about 56 weeks, at least about 60 weeks, at least about 60 weeks, at least Less than about 30% of the plurality of individuals (eg, about 30%) in need of any rescue treatment (eg, aflibercept injection) for any of about 64 weeks, at least about 66 weeks or longer %, about 25%, about 20%, about 15%, about 10%, about 5%, about 2.5%, about 1% or about 0.5%). In some embodiments, administering a unit dose of rAAV particles to one and/or contralateral eyes of a plurality of individuals comprises at least about 20 weeks after administration of the rAAV particles, e.g., at least about 20 weeks after administration of the rAAV particles, at least about 24 weeks, at least about 28 weeks, at least about 32 weeks, at least about 36 weeks, at least about 40 weeks, at least about 44 weeks, at least about 48 weeks, at least about 52 weeks, at least about 56 weeks, at least about 60 weeks, at least about 60 weeks, at least About 30% of the plurality of individuals in need of any salvage treatment (eg, aflibercept injection) in one eye and/or the contralateral eye for any of about 64 weeks, at least about 66 weeks or longer to be less than In some embodiments, administering a unit dose of rAAV particles to one and/or contralateral eyes of a plurality of individuals comprises at least about 20 weeks after administration of the rAAV particles, e.g., at least about 20 weeks after administration of the rAAV particles, at least about 24 weeks, at least about 28 weeks, at least about 32 weeks, at least about 36 weeks, at least about 40 weeks, at least about 44 weeks, at least about 48 weeks, at least about 52 weeks, at least about 56 weeks, at least about 60 weeks, at least about 60 weeks, at least About 20% of the plurality of individuals in need of any salvage treatment (eg, aflibercept injection) in one eye and/or the contralateral eye for any of about 64 weeks, at least about 66 weeks or longer to be less than In some embodiments, administering a unit dose of rAAV particles to one and/or contralateral eyes of a plurality of individuals comprises at least about 20 weeks after administration of the rAAV particles, e.g., at least about 20 weeks after administration of the rAAV particles, at least about 24 weeks, at least about 28 weeks, at least about 32 weeks, at least about 36 weeks, at least about 40 weeks, at least about 44 weeks, at least about 48 weeks, at least about 52 weeks, at least about 56 weeks, at least about 60 weeks, at least about 60 weeks, at least 0% of the plurality of subjects in need of any salvage treatment (eg, aflibercept injection) in one eye and/or the contralateral eye for any of about 64 weeks, at least about 66 weeks or longer do. In some embodiments, administering the unit dose of the rAAV particles to one and/or contralateral eyes of the plurality of individuals comprises at least about 4 weeks after administration of the rAAV particles, e.g., at least about 4 weeks after administration of the rAAV particles, at least about 8 weeks, at least about 12 weeks, at least about 16 weeks, at least about 20 weeks, at least about 24 weeks, at least about 28 weeks, at least about 32 weeks, at least about 36 weeks, at least about 40 weeks, at least about 44 weeks, at least about 48 weeks, at least about 52 weeks, at least about 56 weeks, at least about 60 weeks, at least about 64 weeks, at least about 68 weeks, at least about 72 weeks, at least about 76 weeks, at least about 80 weeks, at least about 84 weeks, at least about 84 weeks, at least any salvage treatment in one eye and/or the contralateral eye for any of 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 For example, 0% of subjects needing aflibercept injection).

In some embodiments, administering the unit dose of rAAV particles to one and/or contralateral eye of the plurality of individuals results in at least about 80%, at least about an average annual anti-VEGF injection rate prior to administration of the unit dose of rAAV particles, a reduction in the mean annual anti-VEGF injection rate of any of about 85%, at least about 87%, at least about 90%, at least about 95%, at least about 99%, or 100%. In some embodiments, administering the unit dose of rAAV particles to one and/or contralateral eye of the plurality of individuals results in an average annualized rate of at least about 87% compared to the average annual anti-VEGF injection rate prior to administration of the unit dose of rAAV particles. causes a decrease in the anti-VEGF injection rate. In some embodiments, administering a unit dose of rAAV particles to one and/or contralateral eyes of a plurality of individuals results in an average annual anti-VEGF injection rate of 100% compared to the average annual anti-VEGF injection rate prior to administration of the unit dose of rAAV particles. - Causes a decrease in the VEGF injection rate.

In some embodiments, the average annual anti-VEGF injection rate prior to administration of a unit dose of rAAV particles is calculated according to the formula:

Annual rate prior to administration of a unit dose of rAAV particles = (number of anti-VEGF injections within 12 months prior to administration of a unit dose of rAAV particles) / (From 1 anti-VEGF injection within the last 12 months prior to administration of a unit dose of rAAV particles days until administration of a unit dose of rAAV particles / 365.25)

In some embodiments, the average annual anti-VEGF injection rate following administration of a unit dose of rAAV particles is calculated according to the formula:

Annual rate after administration of a unit dose of rAAV particles = (number of anti-VEGF injections since administration of a unit dose of rAAV particles) / (days from administration of a unit dose of rAAV particles / 365.25).

In some embodiments, the individual has administered the rAAV particles in one eye and/or compared to BCVA (eg, using the ETDRS protocol) in one eye and/or the contralateral eye to which the rAAV particles are administered prior to administration of the rAAV particles. or if the subject exhibits a loss of 10 or more letters of BCVA due to intraretinal or subretinal fluid in the contralateral eye (e.g., as determined by SD-OCT), the subject may undergo rescue treatment (e.g., eg, anti-VEGF intravitreal injection, such as aflibercept injection). In some embodiments, the individual administers the rAAV particles as compared to a central subfield thickness in one eye and/or the contralateral eye to which the rAAV particles are administered prior to the administration of the rAAV particles, e.g., as determined by SD-OCT. If the subject exhibits an increase in central subfield thickness (e.g., CST or CRT) of greater than 75 μm in one and/or the contralateral eye, the subject is subject to salvage treatment (e.g., anti-VEGF) following administration of the rAAV particles. is determined to require an intravitreal injection, such as aflibercept injection. In some embodiments, if the individual exhibits vision-threatening bleeding due to AMD in one eye and/or the contralateral eye to which the rAAV particles have been administered, the individual is administered salvage treatment (e.g., anti-VEGF free) after administration of the rAAV particles. is determined to require in vivo injection, such as aflibercept injection).

In some embodiments, salvage treatment comprises administration of standard of care anti-VEGF therapy. Such standard of care anti-VEGF therapy includes one or more anti-VEGF treatments (eg, anti-VEGF intravitreal injection). In some embodiments, the salvage treatment comprises one or more aflibercept IVT injections. In some embodiments, the salvage treatment comprises one or more aflibercept IVT injections comprising about 2 mg of aflibercept.

In some embodiments, treatment of an ocular neovascular disease in an individual after administration of a unit dose of rAAV particles to one eye and/or the contralateral eye is administered to the retina prior to administration of a unit dose of rAAV particles to one eye and/or the contralateral eye. Assessed based on retinal fluid level compared to fluid level. In some embodiments, treatment of an ocular neovascular disease in an individual after administration of a unit dose of rAAV particles to one eye and/or the contralateral eye is administered to the retina prior to administration of a unit dose of rAAV particles to one eye and/or the contralateral eye. It is determined when a decrease in retinal fluid is observed following administration of a unit dose of rAAV particles to one eye and/or the contralateral eye compared to the fluid level. In some embodiments, the ocular neovascular disease is wAMD.

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

In some embodiments, the treatment of an ocular neovascular disease in an individual following administration of a unit dose of rAAV particles to one eye and/or the contralateral eye is associated with choroidal neovascularization (CNV) lesion growth as determined by fluorescein angiography. evaluated on the basis of In some embodiments, the treatment of an ocular neovascular disease in an individual following administration of a unit dose of rAAV particles to one eye and/or the contralateral eye comprises administering a unit dose of rAAV particles to one eye and/or the contralateral eye, wherein the CNV lesion is in one eye and/or the contralateral eye. (e.g., about 5%, about 10%, about 20%, about 30%, about 40%) , more than any of about 50%, about 60%, about 70%, about 80%, or 100%). In some embodiments, the treatment of an ocular neovascular disease in an individual following administration of a unit dose of rAAV particles to one eye and/or the contralateral eye comprises administering a unit dose of rAAV particles to one eye and/or the contralateral eye, wherein the CNV lesion is in one eye and/or the contralateral eye. No growth (e.g., about 1%, about 2%, about 3%, about 4%, less than any of about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 15%, or about 20%). In some embodiments, the ocular neovascular disease is wAMD.

In some embodiments, treatment of an ocular neovascular disease in an individual following administration of a unit dose of rAAV particles to one eye and/or the contralateral eye is administered by any method known in the art (e.g., SD-OCT, OCT, fluorescein angiography, digital color fundus imaging, etc.) based on the anatomical features of one eye and/or the contralateral eye. In some embodiments, the treatment of an ocular neovascular disease in an individual following administration of a unit dose of rAAV particles to one eye and/or contralateral eye is administered to one eye and/or contralateral eye followed by administration of a unit dose of rAAV particles to one eye and/or the contralateral eye. It is determined when an improvement in the anatomical features of the eye and/or the contralateral eye is observed. In some embodiments, the ocular neovascular disease is wAMD.

In some embodiments, treatment of an ocular neovascular disease in an individual after administration of a unit dose of rAAV particles to one eye and/or to the contralateral eye comprises an ophthalmic examination, intraocular pressure (eg, a Goldmann applanation tonometer or tono-pen), indirect ophthalmoscopy, examination of one eye and/or contralateral eye and appendages, ptosis and/or pupillary reactivity, ptosis, abnormal pupil shape, unequal pupil, abnormal response to light , afferent pupil defect, slit-lamp examination (including those for eyelid, conjunctiva, cornea, lens, iris and anterior chamber), vitreous, optic nerve, peripheral retina and posterior segment abnormalities of retinal vasculature, SD-OCT, fluorescein vessels Assessed based on contrast, digital color fundus imaging (including imaging 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 assess retinal thickness (eg, central retinal thickness or central subfield thickness), macular volume, and/or the presence of fluid (eg, subretinal or intraretinal fluid). do. In some embodiments, the ocular neovascular disease is wAMD.

A unit dose of rAAV particles may be administered to one and/or the contralateral eye of a subject by any method known in the art. For example, a unit dose of rAAV particles may be administered by intraocular or intravitreal injection into one and/or contralateral eye of a subject. In some embodiments, administering the unit dose of the rAAV particles to one and/or the contralateral eye of the individual is intraocular administration. In some embodiments, administering the unit dose of rAAV particles to one and/or the contralateral eye of the individual is by intravitreal injection (IVT) or subretinal injection. In some embodiments, administering the unit dose of rAAV particles to one and/or the contralateral eye of the individual is by IVT injection. In some embodiments, aseptic technique is used to administer a unit dose of rAAV particles by intravitreal injection. In some embodiments, aseptic technique with providone-iodine is used to administer unit doses of rAAV particles by intravitreal injection.

In some embodiments, the individual has not received prior treatment for ocular neovascular disease. In some embodiments, the individual has not received prior treatment for ocular neovascular disease in one eye and/or the contralateral eye. In some embodiments, the individual is an anti-VEGF agent (eg, bevacizumab, brolucizumab, ranibizumab, parisimab, abicipar pegol, Convercept, OPT-302, KSI-301, injection No prior treatment with possible sunitinib maleate (GB-102), PAN-90806 (panoptica) and/or aflibercept). In some embodiments, the individual has an anti-VEGF agent (eg, bevacizumab, brolucizumab, ranibizumab, parisimab, abicipar pegol, Convercept, OPT) in one eye and/or the contralateral eye. -302, KSI-301, injectable sunitinib maleate (GB-102), PAN-90806 (panoptica) and/or aflibercept). In some embodiments, the individual has not received prior aflibercept treatment. In some embodiments, the individual has not received prior aflibercept treatment in one eye and/or the contralateral eye.

steroid treatment

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, but are not limited to, alclomethasone, amcinonide, beclomethasone, betamethasone, budesonide, ciclesonide, clobetasol, clobetasone, clocortolone, cloprednol, cortibazole, Deplazacort, deoxycorticosterone, desonide, desoxymethasone, dexamethasone, diflorasone, diflucortolone, difluprednate, fluchlorolone, fludrocortisone, fludroxycortide, flumethasone Son, flunisolide, fluocinolone acetonide, fluocinonide, fluocortin, flucortolone, fluorometholone, fluferolone, fluticasone, fupredniden, formocortal, halcinonide, halo Methasone, hydrocortisone acetonate, hydrocortisone butephrate, hydrocortisone butyrate, loteprednol, medrisone, meprednisone, methylprednisolone, methylprednisolone acetonate, mometasone furoate, paramethasone, prednicarbate , prednisone, prednisolone, prednylidene, rimexolone, thixocortol, triamcinolone and ulobetasol. In some embodiments, the steroid treatment is systemic steroid treatment. In some embodiments, the steroid treatment is oral steroid treatment. In some embodiments, the steroid treatment is an ophthalmic steroid treatment. In some embodiments, the ophthalmic steroid treatment is topical steroid treatment (eg, eye drops), periocular steroid treatment (eg, subtenon, subconjunctival), intravitreal steroid treatment, or suprachoroidal steroid treatment. In some embodiments, the topical steroid treatment is difluprednate treatment, medrisone treatment, loteprednol treatment, prednisolone treatment, fluocinolone treatment, triamcinolone treatment, rimexolone treatment, dexamethasone treatment, fluorometholone treatment, fluoci Nolon treatment, rimexolone treatment, or prednisone treatment. In some embodiments, the ophthalmic steroid treatment is difluprednate treatment. In some embodiments, the steroid treatment is prednisone treatment. In some embodiments, the steroid treatment is difluprednate treatment.

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

In some embodiments, the steroid is administered before, during, and/or after administration of the unit dose of the rAAV particles. In some embodiments, the steroid is administered before, during, and after administration of the unit dose of the rAAV particles. In some embodiments, the steroid is administered during and after administration of the unit dose of the rAAV particles. In some embodiments, the steroid is administered prior to administration of the unit dose of the rAAV particles. In some embodiments, the steroid is administered during administration of a unit dose of the rAAV particles. In some embodiments, the steroid is administered prior to and during administration of the unit dose of the rAAV particles. In some embodiments, the steroid is administered following administration of a unit dose of the rAAV particles. In some embodiments, the steroid is administered during and after administration of the unit dose of the rAAV particles. In some embodiments, the steroid is administered before and/or after administration of the unit dose of the rAAV particles. In some embodiments, the steroid is administered before and after administration of the unit dose of the rAAV particles.

In some embodiments, the steroid treatment is systemic steroid treatment. In some embodiments, the systemic steroid treatment is oral steroid treatment.

In some embodiments, the steroid treatment is oral prednisone treatment. In some embodiments, oral prednisone treatment is initiated prior to administration of the unit dose of the rAAV particles. In some embodiments, the initial oral prednisone treatment is 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 of administering the unit dose of the rAAV particles. 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 1 day before, about 3 days, about 4 days, continued for any of about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, or about 10 days or more. In some embodiments, the initial oral prednisone treatment is administered at a dose of about 60 mg of prednisone on the 1st day about 3 days prior to administering the unit dose of rAAV, and continues for about 3 days.

In some embodiments, the initial oral prednisone treatment is followed by a tapering of the oral prednisone treatment dose. In some embodiments, the oral prednisone therapeutic dose tapering is about 20 mg per day for a total of about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, or about 7 days, administered at a dose of any of about 25 mg, about 30 mg, about 35 mg, about 40 mg, about 45 mg, or about 50 mg of prednisone, followed by about 1 day, about 2 days, about 3 days, or about 4 days. administered at a dose of about 10 mg, about 15 mg, about 20 mg or about 25 mg of prednisone per day for any of which is followed by about 1 day, about 2 days, about 3 days, or about 4 days. It is administered at a dose of 5 mg, about 10 mg or about 15 mg of prednisone. In some embodiments, the prednisone dose tapering is a dose of about 40 mg prednisone per day for 3 days, followed by a dose of about 20 mg prednisone per day for 2 days, followed by a dose of about 10 mg prednisone per day for 2 days. administered in a dose of

In some embodiments, the initial oral prednisone treatment is a 60 mg prednisone dose per day for a total of 6 days, followed by a prednisone dose of 40 mg per day for a total of 3 days, followed by 2 doses of prednisone 3 days prior to administering the unit dose of the rAAV particles. Prednisone dose of 20 mg per day for 1 day followed by 10 mg prednisone dose per day for 2 days.

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

In some embodiments, the steroid treatment is an ophthalmic steroid treatment, eg, topical steroid treatment. In some embodiments, the ophthalmic steroid treatment, e.g., topical steroid treatment, is 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 5 months after administration of the unit dose of the rAAV particles. Daily steroid treatment for 6 months. In some embodiments, topical steroid treatment comprises about 4 administrations of the topical steroid in about 1 week, about 3 administrations of the topical steroid in about 2 weeks, about 2 administrations of the topical steroid in about 3 weeks, and a drug including about 1 administration of topical steroid in 4 weeks; The period begins with administration of a unit dose of rAAV particles and thereafter. In some embodiments, topical steroid treatment comprises administration of a unit dose of rAAV particles, followed by administration of about 4 doses of topical steroid per day (i.e., QID) for about 3 weeks, followed by administration of about 3 doses of topical steroid per day for about 1 week. single administration (i.e., TID), followed by about 2 administrations of the topical steroid per day for about 1 week (i.e. BID), followed by about 1 administration of the topical steroid per day for about 1 week (i.e., QD). include In some embodiments, the ophthalmic steroid treatment is extended at the discretion of the treating physician.

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

In some embodiments, topical steroid treatment comprises 7 weeks topical steroid treatment, eg, 0.05% difluprednate. In some embodiments, topical steroid treatment comprises about 4 administrations of the topical steroid per day (ie, QID) for about 4 weeks, followed by about 3 administrations of the topical steroid per day for about 1 week (ie, TID), followed by about twice administration of the topical steroid per day for about 1 week (ie, BID), followed by about once administration of the topical steroid per day for about 1 week (ie, QD); The phase begins about 1 week prior to administration of a unit dose of rAAV particles. In some embodiments, topical steroid treatment comprises about 4 administrations of the topical steroid per day (ie, QID) for about 28 days, followed by about 3 administrations of the topical steroid per day for about 7 days (ie, TID); followed by about twice administration of the topical steroid per day for about 7 days (ie, BID), followed by about once administration of the topical steroid per day for about 7 days (ie, QD); The phase begins about 7 days prior to administration of a unit dose of rAAV particles. In some embodiments, topical steroid treatment is about 4 administrations of the topical steroid per day (i.e., QID) on days 1 to about 28, followed by topical steroids on day 1 from about days 29 to about 35. about three administrations (i.e., TID) of the drug, followed by about two administrations of the topical steroid per day (i.e., BID) on days about 36 to about 42, followed by 1 on days 43 to about 49. including about once a day (ie, QD) of a topical steroid; The period begins on day 1. In some embodiments, topical steroid treatment is continued if inflammation is present.

In some embodiments, the methods of treatment provided herein comprise administering an anti-VEGF agent (eg, aflibercept IVT injection) to one eye of the individual prior to administering a unit dose of rAAV particles to one eye of the individual. do. In some embodiments, the anti-VEGF agent is administered about 7 days or about 1 week prior to administration of the unit dose of the rAAV particles. In some embodiments, the anti-VEGF agent is administered on about the first day and the unit dose of the rAAV particles is administered on about the eighth day. In some embodiments, topical steroid treatment comprises 7 weeks topical steroid treatment, eg, 0.05% difluprednate. In some embodiments, topical steroid treatment comprises about 4 administrations of the topical steroid per day (ie, QID) for about 4 weeks, followed by about 3 administrations of the topical steroid per day for about 1 week (ie, TID), followed by about twice administration of the topical steroid per day for about 1 week (ie, BID), followed by about once administration of the topical steroid per day for about 1 week (ie, QD); The timing begins with administration of the anti-VEGF agent and thereafter. In some embodiments, topical steroid treatment comprises about 4 administrations of the topical steroid per day (ie, QID) for about 28 days, followed by about 3 administrations of the topical steroid per day for about 7 days (ie, TID); followed by about twice administration of the topical steroid per day for about 7 days (ie, BID), followed by about once administration of the topical steroid per day for about 7 days (ie, QD); The timing begins with administration of the anti-VEGF agent and thereafter. In some embodiments, topical steroid treatment is about 4 administrations of the topical steroid per day (i.e., QID) on days 1 to about 28, followed by topical steroids on day 1 from about days 29 to about 35. about three administrations (i.e., TID) of the drug, followed by about two administrations of the topical steroid per day (i.e., BID) on days about 36 to about 42, followed by 1 on days 43 to about 49. including about once a day (ie, QD) of a topical steroid; The period begins on day 1. In some embodiments, topical steroid treatment is continued if inflammation is present.

In some embodiments, the topical steroid treatment comprises 4 months topical steroid treatment, eg, 0.05% difluprednate. In some embodiments, topical steroid treatment comprises about 4 administrations of the topical steroid per day (ie, QID) for about 1 month, followed by about 3 administrations of the topical steroid per day for about 1 month (ie, TID); followed by about twice administration of the topical steroid per day for about 1 month (ie, BID), followed by about 1 administration of the topical steroid per day for about 1 month (ie, QD); The phase begins about 1 week prior to administration of a unit dose of rAAV particles. In some embodiments, topical steroid treatment comprises about 4 administrations of the topical steroid per day (ie, QID) for about 30 days, followed by about 3 administrations of the topical steroid per day for about 30 days (ie, TID); followed by about twice daily administration of the topical steroid (ie, BID) for about 30 days, followed by about 1 administration of the topical steroid per day for about 30 days (ie, QD); The phase begins about 7 days prior to administration of a unit dose of rAAV particles. In some embodiments, topical steroid treatment is about 4 administrations (i.e., QID) of the topical steroid per day on days 1 to about 30, followed by topical steroids on day 1 from about days 31 to about 60. about three administrations (i.e., TID) of the drug, followed by about two administrations of the topical steroid per day (i.e., BID) from about days 61 to about 90 days, followed by 1 administration from about days 91 to about 120 days. including about once a day (ie, QD) of a topical steroid; The period begins on day 1. In some embodiments, topical steroid treatment is continued if inflammation is present.

In some embodiments, the methods of treatment provided herein comprise administering an anti-VEGF agent (eg, aflibercept IVT injection) to one eye of the individual prior to administering a unit dose of rAAV particles to one eye of the individual. do. In some embodiments, the anti-VEGF agent is administered about 7 days or about 1 week prior to administration of the unit dose of the rAAV particles. In some embodiments, the anti-VEGF agent is administered on about the first day and the unit dose of the rAAV particles is administered on about the eighth day. In some embodiments, the topical steroid treatment comprises 4 months topical steroid treatment, eg, 0.05% difluprednate. In some embodiments, topical steroid treatment comprises about 4 administrations of the topical steroid per day (ie, QID) for about 1 month, followed by about 3 administrations of the topical steroid per day for about 1 month (ie, TID); followed by about twice administration of the topical steroid per day for about 1 month (ie, BID), followed by about 1 administration of the topical steroid per day for about 1 month (ie, QD); The timing begins with administration of the anti-VEGF agent and thereafter. In some embodiments, topical steroid treatment comprises about 4 administrations of the topical steroid per day (ie, QID) for about 30 days, followed by about 3 administrations of the topical steroid per day for about 30 days (ie, TID); followed by about twice daily administration of the topical steroid (ie, BID) for about 30 days, followed by about 1 administration of the topical steroid per day for about 30 days (ie, QD); The timing begins with administration of the anti-VEGF agent and thereafter. In some embodiments, topical steroid treatment is about 4 administrations (i.e., QID) of the topical steroid per day on days 1 to about 30, followed by topical steroids on day 1 from about days 31 to about 60. about three administrations (i.e., TID) of the drug, followed by about two administrations of the topical steroid per day (i.e., BID) from about days 61 to about 90 days, followed by 1 administration from about days 91 to about 120 days. including about once a day (ie, QD) of a topical steroid; The period begins on day 1. In some embodiments, topical steroid treatment is continued if inflammation is present.

A vector for delivering a transgene to a target cell

In some embodiments, the recombinant adeno-associated virus (rAAV) particle comprises a recombinant viral vector derived from an adeno-associated virus (AAV) that has been altered to be replication-defective in a subject (eg, a human or non-human primate). . In some embodiments, the adeno-associated virus (AAV) is a recombinant AAV (rAAV).

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

As exposure to wild-type (wt) AAV is not associated with or known to cause any human pathology, and is common in the general population, AAV or rAAV is a suitable delivery system for gene therapy. The AAV and rAAV used in gene therapy for the delivery of anti-VEGF agents, such as aflibercept, may be of any serotype. In some embodiments, the methods of the present disclosure comprise AAV1, AAV2, AAV2.5, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, rh10, AAV-DJ and any hybrids thereof or The use of any suitable AAV serotype is provided, including chimeric AAV. In some embodiments, the serotype used is based on the tropism of the virus or the infectivity of the target cell of interest. In some embodiments, several AAV vectors can be generated that allow selection of the most optimal serotype for use with an anti-VEGF agent transgene (eg, aflibercept transgene).

In some embodiments, the methods of the present disclosure provide for the use of pseudotyped AAV. Pseudotyped AAV particles include those in which the AAV genome inverted terminal repeat (ITR) of one AAV serotype is encapsidated by the AAV capsid of another AAV serotype. Typically, pseudotyped AAVs are designated "AAV#/#", where the first "#" represents the AAV ITR serotype and the second "#" represents the capsid serotype. For example, an AAV particle comprising an AAV2 ITR and an AAV1 capsid would be designated "AAV2/1".

In some embodiments, the rAAV particle comprises a nucleic acid, eg, a heterologous nucleic acid. In some embodiments, the nucleic acid encodes a transgene, eg, an anti-VEGF agent (eg, aflibercept). In some embodiments, the encoded transgene, eg, an anti-VEGF agent, is under the transcriptional control of a promoter that initiates transcription of the nucleic acid. In some embodiments, the promoter is a “ubiquitous” promoter. In some embodiments, the promoter is a "strong" or constitutively active promoter, such as the cytomegalovirus (CMV) promoter, elongation factor 1 alpha (EFla) promoter, glyceraldehyde 3-phosphate dehydrogenase (GAPDH). ) promoter or connexin36 (or "Cx36") promoter. In some embodiments, the promoter is a tissue-specific promoter that is activated in a specific tissue or cell, such as a retinal cell, to reduce potential toxic or undesirable effects on non-targeting cells. In some aspects, several AAV vectors can be generated that allow selection of the most optimal serotype and promoter for use with an anti-VEGF agent transgene (eg, aflibercept transgene). In some embodiments, the nucleic acid is flanked by AAV inverted terminal repeats (ITRs). In some embodiments, the nucleic acid is flanked by AAV2 ITRs.

In some embodiments, the AAV vector comprises a polynucleotide cassette for enhanced expression of a transgene (eg, an anti-VEGF agent, such as aflibercept) in a target cell (eg, retinal cells). In some embodiments, the polynucleotide cassette comprises in 5' to 3' order: (a) a first enhancer region comprising a CMV sequence (SEQ ID NO: 22); (b) a promoter region comprising a CMV sequence (SEQ ID NO: 23); (c) a 5'UTR region comprising TPL and eMLP sequences (SEQ ID NO: 24 and SEQ ID NO: 25, respectively) in 5' to 3' order; (d) a coding sequence encoding a peptide or polypeptide (eg, an anti-VEGF agent, such as aflibercept); (e) a second enhancer region comprising the entire EES sequence (SEQ ID NO: 26); and (f) an HGH polyadenylation site (SEQ ID NO: 27). In certain of these embodiments, the polynucleotide cassette comprises one or more sequences selected from SEQ ID NOs: 28-32 or a sequence having at least 85% identity thereto. In certain of these embodiments, the 5' arm of the polynucleotide cassette comprises or consists of SEQ ID NO: 33 or a sequence having at least 85% identity thereto. In certain of these embodiments, the 3' arm of the polynucleotide cassette comprises or consists of SEQ ID NO: 34 or a sequence having at least 85% identity thereto. The nucleic acid sequences of SEQ ID NOs: 22-34 are provided below:

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

SEQ ID NO:39 shown above is an inverted terminal repeat (ITR) of AAV serotype 2 comprising nucleotides 1-145 of SEQ ID NO:39 in 5' to 3' direction; a CMV promoter comprising nucleotides 180-693 of SEQ ID NO:39; a 5' untranslated region (UTR) comprising an adenoviral tripartite leader sequence and a synthetic intron 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 comprising the human scaffold attachment region and comprising nucleotides 2717-3527 of SEQ ID NO:39; a human growth hormone polyadenylation/transcriptional 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, for example, FIG. 1C.

Additional polynucleotide cassettes for enhanced expression of a transgene (eg, a transgene encoding an anti-VEGF agent, such as aflibercept) in target cells (eg retinal cells) are disclosed in WO2018/170473, The contents of polynucleotide cassettes for enhanced expression of a transgene in a target cell are incorporated herein by reference.

In some embodiments, the rAAV particle comprises a variant capsid protein having increased infectivity of a target cell, e.g., a retinal cell, and increases transduction of a retinal cell in an individual or provides for targeting of gene transfer to a retinal cell. used to increase In some embodiments, the rAAV particle comprises an amino acid modification in the capsid protein GH loop/loop IV of the AAV capsid protein. In some embodiments, the modification site is a solvent-accessible portion of the GH loop/loop IV of an AAV capsid protein. For a description of the GH loop/loop IV of the AAV capsid, see, eg, van Vliet et al. (2006) Mol. Ther. 14:809; Padron et al. (2005) J. Virol. 79:5047; and Shen et al. (2007) Mol. Ther. 15:1955]. Several AAV capsid variants are known, including the 7m8 variant. In some embodiments, the rAAV particle comprises a variant AAV capsid protein comprising an insertion of a sequence of 5 to 11 amino acids, e.g., 7 amino acids, into the GH loop of the capsid protein relative to the corresponding parent AAV capsid protein, wherein the variant capsid protein confers increased infectivity of the retinal cells compared to the infectivity of the retinal cells by AAV particles comprising the corresponding parental or unmodified AAV capsid protein. In some embodiments, any one of the following amino acid sequences may be inserted into the GH loop of a capsid protein: LALGETTRPA (SEQ ID NO: 1); LANETITRPA (SEQ ID NO: 2), LAKAGQANNA (SEQ ID NO: 3), LAKDPKTTNA (SEQ ID NO: 4), KDTDTTR (SEQ ID NO: 5), RAGGSVG (SEQ ID NO: 6), AVDTTKF (SEQ ID NO: 6) : 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: 18) 19), AVDTTKF (SEQ ID NO: 20) and STGKVPN (SEQ ID NO: 21). In some embodiments, any one of the amino acid sequences set forth in SEQ ID NOs: 1-12 and 14-21 is inserted into the solvent-exposed GH loop of the VP1 capsid protein in rAAV. Further details regarding amino acid sequences that can be inserted into the GH loop of a capsid protein to facilitate transduction of, for example, a nucleic acid of interest into retinal cells after IVT injection are provided in WO2012145601, US9587282, US10202657 and US10214785 and the content of amino acid sequences capable of being inserted into the GH loop of the capsid protein is incorporated herein by reference.

In some embodiments, the rAAV particle is positioned 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 the following amino acid sequence inserted between amino acids 589 and 590 of the AAV10 capsid protein: LALGETTRPA (SEQ ID NO: 1); LANETITRPA (SEQ ID NO: 2), LAKAGQANNA (SEQ ID NO: 3), LAKDPKTTNA (SEQ ID NO: 4), KDTDTTR (SEQ ID NO: 5), RAGGSVG (SEQ ID NO: 6), AVDTTKF (SEQ ID NO: 6) : 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: 18) 19), AVDTTKF (SEQ ID NO: 20) and STGKVPN (SEQ ID NO: 21), for example AAV capsid proteins, eg, AAV2 capsid proteins. In some embodiments, the rAAV particle comprises an AAV2 capsid protein comprising the amino acid sequence LALGETTRPA (SEQ ID NO: 1) inserted between positions 587 and 588 of the capsid protein, wherein the amino acid residue numbering corresponds to an AAV2 VP1 capsid protein. . In some embodiments, the rAAV particle comprises an AAV2 capsid protein comprising the amino acid sequence LALGETTRPA (SEQ ID NO: 1) inserted between positions 587 and 588 of AAV2 VP1 comprising the sequence of SEQ ID NO: 13.

In some embodiments, the rAAV particle comprises a 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 AAV2 VP1. In some embodiments, the rAAV particle comprises an AAV2 VP1 capsid protein comprising a GH loop comprising the amino acid sequence of SEQ ID NO:38 or an amino acid sequence having at least 90% sequence identity to SEQ ID NO:38. In some embodiments, the rAAV particles are 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% to SEQ ID NO: 38 , an AAV2 VP1 capsid protein comprising a GH loop comprising an amino acid sequence having either at least 99% or 100% sequence identity.

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

In some embodiments, the rAAV particle comprises 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 particle comprises 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 particle comprises 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 particle comprises capsid proteins VP1, VP2 and VP3, wherein each of VP1, VP2 and VP3 has 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 the AAV2 VP1 capsid protein.

In some embodiments, the rAAV particle comprises 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 particle comprises 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 particle comprises a capsid protein VP3 comprising the amino acid sequence LALGETTRPA (SEQ ID NO: 1) inserted between positions 587 and 588 of the capsid protein, wherein the amino acid residue numbering corresponds to an AAV2 VP1 capsid protein. . In some embodiments, the rAAV particle comprises capsid proteins VP1, VP2 and VP3, wherein each of VP1, VP2 and VP3 has 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 the AAV2 VP1 capsid protein.

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

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

In some embodiments, the vector is more than a targeting vector, particularly of a specific cell, such as a retinal cell (e.g., a photoreceptor, a retinal ganglion cell, a Muller cell, a bipolar cell, an amacrine cell, a horizontal cell, or a retinal pigment epithelial cell). It is a targeting rAAV (eg, AAV2.7m8) that exhibits high infectivity. Viral vectors for use in the present disclosure include those that exhibit low toxicity and/or low immunogenicity in an individual and express a therapeutically effective amount of an anti-VEGF agent (eg, aflibercept) in an individual, eg, a human. can do. Any suitable method known in the art can be used, for example, for the biochemical purification of a recombinant virus (eg, rAAV) for the preparation of a pharmaceutical composition described elsewhere herein. Recombinant AAV virus can be harvested directly from cells or from the culture medium containing the cells. Viruses can be purified using a variety of biochemical means, such as gel filtration, filtration, chromatography, affinity purification, gradient ultracentrifugation, or size exclusion methods. In some embodiments, the virus is lyophilized.

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

In some embodiments, the increase in retinal cell infectivity of the rAAV variant, e.g., rAAV2.7m8, is at least 1-fold, at least 1.1-fold, at least 1.2-fold, any of at least 1.3 times, at least 1.4 times, at least 1.5 times, at least 1.6 times, at least 1.7 times, at least 1.8 times, at least 1.9 times, or at least 2 times. In some embodiments, the increase in infectivity is at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, as compared to an AAV particle comprising a corresponding parental AAV capsid protein. , at least 9 times or at least 10 times. In some embodiments, the increase in infectivity is at least 15-fold, at least 20-fold, at least 25-fold, at least 30-fold, at least 35-fold, at least 40-fold, any of 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.

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

In some embodiments, the increase in retinal cell infectivity is between 2 fold and 20 fold, between 2 fold and 19 fold, between 2 fold and 18 fold, between 2 fold and 17 fold compared to an AAV particle comprising a corresponding parental or unmodified AAV capsid protein. 2x to 16x, 2x to 15x, 2x to 14x, 2x to 13x, 2x to 12x, 2x to 11x, 2x to 10x, 2x to 9x, 2 to 8 times, 2 to 7 times, 2 to 6 times, 2 to 5 times, 2 to 4 times, or 2 to 3 times.

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

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

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

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

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

In some embodiments, the rAAV particle comprises 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% of any of the amino acid sequence of SEQ ID NO:35. and a nucleic acid encoding a polypeptide comprising an amino acid sequence with identity and flanked by an AAV2 inverted terminal repeat (ITR). In some embodiments, the rAAV particle comprises a nucleic acid encoding a polypeptide comprising an amino acid sequence having at least about 95% identity to the amino acid sequence of SEQ ID NO: 35 and flanked by AAV2 inverted terminal repeats (ITRs). do. In some embodiments, the rAAV particle comprises 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 particle comprises a nucleic acid encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:35. In some embodiments, the rAAV particle comprises a nucleic acid encoding aflibercept and flanked by an AAV2 inverted terminal repeat (ITR). The sequence of SEQ ID NO: 35 is provided below:

In some embodiments, the rAAV particle is 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 a nucleic acid having sequence homology of any of 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%, wherein the nucleic acid has an AAV2 inversion The terminal repeats (ITRs) are flanked. The sequence of SEQ ID NO: 36 is provided in FIG. 5 . In some embodiments, the rAAV particle is at least about 75%, at least about 80%, at least about 81%, at least about 82%, at least about 83% to a nucleic acid sequence of aflibercept (eg, SEQ ID NO:36). %, 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% of a nucleic acid having sequence homology wherein the nucleic acid is flanked by an AAV2 inverted terminal repeat (ITR). 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 particle is 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 a nucleic acid having sequence homology of any of 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.9% or 100%, wherein the nucleic acid has an AAV2 inverted end The repeat (ITR) is flanked. In some embodiments, the rAAV particle comprises a nucleic acid comprising the nucleic acid sequence of SEQ ID NO:40. In some embodiments, the rAAV particle comprises a nucleic acid comprising the nucleic acid sequence of SEQ ID NO: 40, wherein the nucleic acid is flanked by an AAV2 inverted terminal repeat (ITR).

In some embodiments, the rAAV particle has at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identity to the amino acid sequence of SEQ ID NO:41. and a nucleic acid encoding a polypeptide comprising an amino acid sequence having an AAV2 inverted terminal repeat (ITR) flanked by it. In some embodiments, the rAAV particle comprises a nucleic acid encoding a polypeptide comprising an amino acid sequence having at least about 95% identity to the amino acid sequence of SEQ ID NO: 41 and flanked by an AAV2 inverted terminal repeat (ITR). do. In some embodiments, the rAAV particle comprises 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 particle comprises a nucleic acid encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:41.

In some embodiments, the nucleic acid sequence of aflibercept is codon-optimized for expression in a primate or human subject. Construction of synthetic genes corresponding to aflibercept amino acid sequences is described in the literature, eg, 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 that "VEGF-Trap _R1R2 (corresponding to aflibercept) cDNA was generated as a synthetic gene by IDT (Coralville, Iowa)"). Any method known in the art can be used to generate cDNA of aflibercept for use in gene therapy or rAAV described herein, taking into account the available amino acid sequences of aflibercept.

Codon optimization can be accomplished by any method known in the art. Codon optimization can be achieved by selecting at least one codon (e.g., about 1, 2, 3, 4, 5, 10, 15, 20, 25, 50, 100 or more codons or more) of the native sequence, Altering a nucleic acid sequence for enhanced expression of a gene in a target of interest or host cell, e.g., a human retinal cell, by replacing the codon with a codon used more frequently or most frequently in the host cell while maintaining the amino acid sequence. refers to the process. Codon usage tables are readily available including, for example: the GenScript Codon Usage Frequency Table tool under www(dot)genscript(dot)com/tools/codon-frequency-table; Codon usage database under 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)].

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

In some embodiments, the rAAV particle comprises a nucleic acid encoding aflibercept. In some embodiments, the polypeptide is aflibercept.

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

In some embodiments, the amino acid sequence of aflibercept is at least 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, any of 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9% or 100% homology. In some embodiments, the nucleic acid sequence encoding aflibercept disclosed herein is compared to the corresponding cDNA sequence of the aflibercept amino acid sequence identified above and the nucleic acid sequence of aflibercept (eg, SEQ ID NO: 36) ) between at least 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94 %, 95%, 96%, 97%, 98%, 99%, 99.9% or 100% sequence homology. In some embodiments, aflibercept is at least 80%, 85%, 90%, 91%, 92 to aflibercept (eg, in terms of its secondary, tertiary, and quaternary structure or conformation). %, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9% or 100% are spatially homologous. In some embodiments, aflibercept is up to 80%, 85%, 90%, 91% (eg, in secondary, tertiary, and quaternary structures or conformations) relative to aflibercept used in standard of care. , 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9% or 100% are spatially homologous.

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

Aflibercept is a 115 kDa fusion protein that can be glycosylated. Aflibercept contains an IgG backbone fused to the extracellular VEGF receptor sequences of human VEGFR-1 and VEGFR-2 and functions like a soluble decoy receptor by binding to VEGF-A with greater affinity than its native or endogenous receptor. . See, eg, Stewart MW. Aflibercept (VEGF Trap-eye): the newest anti-VEGF drug. Br. J. Ophthalmol. 2012 Sep;96(9):1157-8]. The high affinity of aflibercept for VEGF interferes with 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 factors PIGF and VEGF-B by aflibercept may also contribute to the treatment of ocular diseases or disorders characterized by abnormal (eg, excessive) angiogenesis and/or neovascularization. PIGF has been associated with angiogenesis, and certain ocular diseases or disorders, such as wet AMD, may be associated with elevated levels of PIGF. VEGF-B overexpression may be associated with disruption of the blood-retinal barrier and retinal neovascularization. Thus, inhibition of VEGF-A, VEGF-B and PIGF may all contribute to the efficacy of aflibercept.

Method for preparing a vector for delivering a transgene to a target cell

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

Volume

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

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

In some embodiments, administering the unit dose of rAAV particles to the contralateral eye of 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 one eye. am. In some embodiments, administering the unit dose of rAAV particles to the contralateral eye of the individual comprises administering the unit dose of rAAV particles to one eye for up to about 2 weeks (e.g., about 0 days, 1 day, 2 days, 3 days) days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days or 14 days). In some embodiments, administering the unit dose of rAAV particles to the contralateral eye of the individual comprises administering the unit dose of rAAV particles to one eye for up to about 2 weeks (e.g., about 0 days, 1 day, 2 days, 3 days) days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days or 14 days), and administered to the subject's contralateral eye. is approximately equal to the unit dose of rAAV particles administered to one eye of the subject (e.g., less than 1% higher or lower, less than 5% higher or lower, or 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). In some embodiments, administering the unit dose of rAAV particles to the contralateral eye of the individual is up to about 2 weeks after administration of the unit dose of rAAV particles to one eye, wherein the unit dose of rAAV particles administered to the contralateral eye of the individual is approximately equal to a unit dose of rAAV particles administered to one eye of an individual (e.g., less than 1% higher or lower, less than 5% higher or lower, less than 10% higher or lower, or or less than 20% higher or lower). In some embodiments, administering the unit dose of rAAV particles to the contralateral eye of the individual is up to about 2 weeks after administration of the unit dose of rAAV particles to one eye, wherein the unit dose of rAAV particles administered to the contralateral eye of the individual is lower than the unit dose of rAAV particles administered to one eye of the subject (e.g., about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70 %, about 80% or about 90% lower).

In some embodiments, the unit dose of rAAV particles is administered to one eye and/or the contralateral eye of the individual. In some embodiments, a unit dose of a rAAV particle is expressed as the number of vector genomes (vg). In some embodiments, the unit dose is about 6×10 ¹¹ vector genomes (vg) or less of rAAV particles. In some embodiments, a unit dose is about 1x10 ¹⁰ to about 2x10 ¹⁰ , about 2x10 ¹⁰ to about 3x10 ¹⁰ , about 3x10 ¹⁰ to about 4x10 ¹⁰ , about 4x10 ¹⁰ to about 5x10 ¹⁰ , about 5x10 ¹⁰ to about 6x10 ¹⁰ , about 6x10 ¹⁰ to about 7x10 ¹⁰ , about 7x10 ¹⁰ to about 8x10 ¹⁰ , about 8x10 ¹⁰ to about 9x10 ¹⁰ , about 9x10 ¹⁰ to about 10x10 ¹⁰ , about 1x10 ¹¹ to about 2x10 ¹¹ , about 2x10 ¹¹ to about 3x10 ¹¹ , about 3x10 ¹¹ to about 4x10 ¹¹ , about 4x10 ¹¹ to about 5x10 ¹¹ or about 5x10 ¹¹ to about 6x10 ¹¹ vg of rAAV particles, including rAAV particles of any value within these ranges. In some embodiments, the unit dose is from about 6x10 ¹⁰ vector genomes (vg) to about 2x10 ¹¹ vg of rAAV particles. In some embodiments, the unit dose is about 6x10 ¹⁰ vg to about 2x10 ¹¹ vg, about 7x10 ¹⁰ vg to about 2x10 ¹¹ vg, about 8x10 ¹⁰ vg to about 2x10 ¹¹ vg, about 9x10 ¹⁰ vg to about 2x10 ¹¹ vg, about 10x10 ¹⁰ vg to about 2x10 ¹¹ vg or about 1x10 ¹¹ vg to about 2x10 ¹¹ vg of rAAV particles. In some embodiments, the unit dose is from about 6x10 ¹⁰ vg to about 2x10 ¹¹ vg of rAAV particles. In some embodiments, the unit dose is about 6x10 ¹⁰ vg to about 7x10 ¹⁰ vg, about 7x10 ¹⁰ vg to about 8x10 ¹⁰ vg, about 8x10 ¹⁰ vg to about 9x10 ¹⁰ vg, about 9x10 ¹⁰ vg to about 10x10 ¹⁰ vg, about 10x10 ¹⁰ vg to about 1x10 ¹¹ vg or about 1x10 ¹¹ vg to about 2x10 ¹¹ vg of rAAV particles. In some embodiments, the unit dose is about 6x10 ¹⁰ vg, about 7x10 ¹⁰ vg, about 8x10 ¹⁰ vg, about 9x10 ¹⁰ vg, about 10x10 ¹⁰ vg, about 1x10 ¹¹ vg, or about 2x10 ¹¹ vg of rAAV particles. In some embodiments, the unit dose is about 6x10 ¹⁰ vg or about 2x10 ¹¹ vg of rAAV particles. In some embodiments, the unit dose is about 6x10 ¹⁰ vg of rAAV particles. In some embodiments, the unit dose is about 6 x 10 ¹⁰ vg, about 2 x 10 ¹¹ vg, or about 6 x 10 ¹¹ vg. In some embodiments, the unit dose is about 6 x 10 ¹⁰ vg. In some embodiments, the unit dose is about 2 x 10 ¹¹ vg. In some embodiments, the unit dose is about 6 x 10 ¹¹ vg.

In some embodiments, the unit dose of rAAV particles is administered to one eye and/or the contralateral eye of the individual. In some embodiments, unit dose is expressed as number of vector genomes per eye (vg) (vg/eye). In some embodiments, the unit dose is about 6 x 10 ¹¹ vg/eye or less of rAAV particles. In some embodiments, a unit dose is about 1x10 ¹⁰ to about 2x10 ¹⁰ , about 2x10 ¹⁰ to about 3x10 ¹⁰ , about 3x10 ¹⁰ to about 4x10 ¹⁰ , about 4x10 ¹⁰ to about 5x10 ¹⁰ , about 5x10 ¹⁰ to about 6x10 ¹⁰ , about 6x10 ¹⁰ to about 7x10 ¹⁰ , about 7x10 ¹⁰ to about 8x10 ¹⁰ , about 8x10 ¹⁰ to about 9x10 ¹⁰ , about 9x10 ¹⁰ to about 10x10 ¹⁰ , about 1x10 ¹¹ to about 2x10 ¹¹ , about 2x10 ¹¹ to about 3x10 ¹¹ , about 3x10 ¹¹ to about 4x10 ¹¹ , about 4x10 ¹¹ to about 5x10 ¹¹ or about 5x10 ¹¹ to about 6x10 ¹¹ vg/eye of rAAV particles, including rAAV particles of any value within these ranges. In some embodiments, the unit dose is from about 6x10 ¹⁰ vg/eye to about 2x10 ¹¹ vg/eye of rAAV particles. In some embodiments, the unit dose is about 6x10 ¹⁰ vg/eye to about 2x10 ¹¹ vg/eye, about 7x10 ¹⁰ vg/eye to about 2x10 ¹¹ vg/eye, about 8x10 ¹⁰ vg/eye to about 2x10 ¹¹ vg/eye, about 9x10 ¹⁰ vg/eye to about 2x10 ¹¹ vg/eye, about 10x10 ¹⁰ vg/eye to about 2x10 ¹¹ vg/eye or about 1x10 ¹¹ vg/eye to about 2x10 ¹¹ vg/eye rAAV particles. In some embodiments, the unit dose is from about 6x10 ¹⁰ vg/eye to about 2x10 ¹¹ vg/eye of rAAV particles. In some embodiments, the unit dose is about 6x10 ¹⁰ vg/eye to about 7x10 ¹⁰ vg/eye, about 7x10 ¹⁰ vg/eye to about 8x10 ¹⁰ vg/eye, about 8x10 ¹⁰ vg/eye to about 9x10 ¹⁰ vg/eye, about 9x10 ¹⁰ vg/eye to about 10x10 ¹⁰ vg/eye, about 10x10 ¹⁰ vg/eye to about 1x10 ¹¹ vg/eye or about 1x10 ¹¹ vg/eye to about 2x10 ¹¹ vg/eye rAAV particles. In some embodiments, the unit dose is about 6x10 ¹⁰ vg/eye, about 7x10 ¹⁰ vg/eye, about 8x10 ¹⁰ vg/eye, about 9x10 ¹⁰ vg/eye, about 10x10 ¹⁰ vg/eye, about 1x10 ¹¹ vg/eye or about 2x10 ¹¹ vg/eye of rAAV particles. In some embodiments, the unit dose is about 6x10 ¹⁰ vg/eye or about 2x10 ¹¹ vg/eye of rAAV particles. In some embodiments, the unit dose is about 6x10 ¹⁰ vg/eye of rAAV particles. In some embodiments, the unit dose is about 6×10 ¹⁰ vg/eye, about 2×10 ¹¹ vg/eye, or about 6×10 ¹¹ vg/eye. In some embodiments, the unit dose is about 6 x 10 ¹⁰ vg/eye. In some embodiments, the unit dose is about 2 x 10 ¹¹ vg/eye. In some embodiments, the unit dose is about 6 x 10 ¹¹ vg/eye.

In some embodiments, the unit dose of rAAV particles is administered to one eye and/or the contralateral eye of the individual. In some embodiments, E is the abbreviation for base 10 for exponentiation, and xEy refers to x times the y power of base 10/exponent. In some embodiments, a unit dose is expressed as the number of vector genomes (vg). In some embodiments, the unit dose is about 6E ¹¹ vector genomes (vg) or less of rAAV particles. In some embodiments, the unit dose is from about 1E ¹⁰ to about 2E ¹⁰ , from about 2E ¹⁰ to about 3E ¹⁰ , from about 3E ¹⁰ to about 4E ¹⁰ , from about 4E ¹⁰ to about 5E ¹⁰ , from about 5E ¹⁰ to about 6E ¹⁰ , about 6E ¹⁰ to about 7E ¹⁰ , about 7E ¹⁰ to about 8E ¹⁰ , about 8E ¹⁰ to about 9E ¹⁰ , about 9E ¹⁰ to about 10E ¹⁰ , about 1E ¹¹ to about 2E ¹¹ , about 2E ¹¹ to about 3E ¹¹ , about 3E ¹¹ to about 4E ¹¹ , about 4E ¹¹ to about 5E ¹¹ or about 5E ¹¹ to about 6E ¹¹ vg of rAAV particles, including rAAV particles of any value within these ranges. In some embodiments, the unit dose is from about 6E ¹⁰ vector genomes (vg) to about 2E ¹¹ vg of rAAV particles. In some embodiments, the unit dose is about 6E ¹⁰ vg to about 2E ¹¹ vg, about 7E ¹⁰ vg to about 2E ¹¹ vg, about 8E ¹⁰ vg to about 2E ¹¹ vg, about 9E ¹⁰ vg to about 2E ¹¹ vg, about 10E ¹⁰ vg to about 2E ¹¹ vg or about 1E ¹¹ vg to about 2E ¹¹ vg of rAAV particles. In some embodiments, the unit dose is about 6E ¹⁰ vg to about 2E ¹¹ vg of rAAV particles. In some embodiments, the unit dose is about 6E ¹⁰ vg to about 7E ¹⁰ vg, about 7E ¹⁰ vg to about 8E ¹⁰ vg, about 8E ¹⁰ vg to about 9E ¹⁰ vg, about 9E ¹⁰ vg to about 10E ¹⁰ vg, about 10E ¹⁰ vg to about 1E ¹¹ vg or about 1E ¹¹ vg to about 2E ¹¹ vg of rAAV particles. In some embodiments, the unit dose is about 6E ¹⁰ vg, about 7E ¹⁰ vg, about 8E ¹⁰ vg, about 9E ¹⁰ vg, about 10E ¹⁰ vg, about 1E ¹¹ vg, or about 2E ¹¹ vg of rAAV particles. In some embodiments, the unit dose is about 6E ¹⁰ vg or about 2E ¹¹ vg of rAAV particles. In some embodiments, the unit dose is about 6E ¹⁰ vg of rAAV particles. In some embodiments, the unit dose is about 6E ¹⁰ vg, about 2E ¹¹ vg, or about 6E ¹¹ vg. In some embodiments, the unit dose is about 6E ¹⁰ vg. In some embodiments, the unit dose is about 2E ¹¹ vg. In some embodiments, the unit dose is about 6E ¹¹ vg.

In some embodiments, the unit dose of rAAV particles is administered to one eye and/or the contralateral eye of the individual. In some embodiments, unit dose is expressed as number of vector genomes per eye (vg) (vg/eye). In some embodiments, the unit dose is about 6E ¹¹ vg/eye or less of rAAV particles. In some embodiments, the unit dose is from about 1E ¹⁰ to about 2E ¹⁰ , from about 2E ¹⁰ to about 3E ¹⁰ , from about 3E ¹⁰ to about 4E ¹⁰ , from about 4E ¹⁰ to about 5E ¹⁰ , from about 5E ¹⁰ to about 6E ¹⁰ , about 6E ¹⁰ to about 7E ¹⁰ , about 7E ¹⁰ to about 8E ¹⁰ , about 8E ¹⁰ to about 9E ¹⁰ , about 9E ¹⁰ to about 10E ¹⁰ , about 1E ¹¹ to about 2E ¹¹ , about 2E ¹¹ to about 3E ¹¹ , about 3E ¹¹ to about 4E ¹¹ , about 4E ¹¹ to about 5E ¹¹ or about 5E ¹¹ to about 6E ¹¹ vg/eye of rAAV particles, including rAAV particles of any value within these ranges. In some embodiments, the unit dose is from about 6E ¹⁰ vg/eye to about 2E ¹¹ vg/eye of rAAV particles. In some embodiments, the unit dose is about 6E ¹⁰ vg/eye to about 2E ¹¹ vg/eye, about 7E ¹⁰ vg/eye to about 2E ¹¹ vg/eye, about 8E ¹⁰ vg/eye to about 2E ¹¹ vg/eye, about 9E ¹⁰ vg/eye to about 2E ¹¹ vg/eye, about 10E ¹⁰ vg/eye to about 2E ¹¹ vg/eye or about 1E ¹¹ vg/eye to about 2E ¹¹ vg/eye rAAV particles. In some embodiments, the unit dose is from about 6E ¹⁰ vg/eye to about 2E ¹¹ vg/eye of rAAV particles. In some embodiments, the unit dose is about 6E ¹⁰ vg/eye to about 7E ¹⁰ vg/eye, about 7E ¹⁰ vg/eye to about 8E ¹⁰ vg/eye, about 8E ¹⁰ vg/eye to about 9E ¹⁰ vg/eye, about 9E ¹⁰ vg/eye to about 10E ¹⁰ vg/eye, about 10E ¹⁰ vg/eye to about 1E ¹¹ vg/eye or about 1E ¹¹ vg/eye to about 2E ¹¹ vg/eye rAAV particles. In some embodiments, the unit dose is about 6E ¹⁰ vg/eye, about 7E ¹⁰ vg/eye, about 8E ¹⁰ vg/eye, about 9E ¹⁰ vg/eye, about 10E ¹⁰ vg/eye, about 1E ¹¹ vg/eye, or about 2E ¹¹ vg/eye of rAAV particles. In some embodiments, the unit dose is about 6E ¹⁰ vg/eye or about 2E ¹¹ vg/eye of rAAV particles. In some embodiments, the unit dose is about 6E ¹⁰ vg/eye of rAAV particles. In some embodiments, the unit dose is about 6E ¹⁰ vg/eye, about 2E ¹¹ vg/eye, or about 6E ¹¹ vg/eye. In some embodiments, the unit dose is about 6E ¹⁰ vg/eye. In some embodiments, the unit dose is about 2E ¹¹ vg/eye. In some embodiments, the unit dose is about 6E ¹¹ vg/eye.

In some embodiments, the unit dose of rAAV particles is administered to one and/or the contralateral eye of the individual. In some embodiments, the unit dose of rAAV particles is a unit dose sufficient to cause expression of a therapeutic protein (eg, an anti-VEGF agent, such as aflibercept) in the vitreous humor. In some embodiments, the unit dose of the rAAV particles is about 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 in vitreous humor. A unit dose sufficient to achieve a concentration of a Therapeutic protein (eg, an anti-VEGF agent, such as aflibercept) of any one (including any range between these values). In some embodiments, the unit dose of rAAV particles is a unit dose sufficient to cause expression of aflibercept in the vitreous humor. In some embodiments, the unit dose of the rAAV particles is about 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 in vitreous humor. A unit dose sufficient to achieve a concentration of any one of aflibercept (including any range between these values).

In some embodiments, the unit dose of rAAV particles administered to one and/or contralateral eye of the individual is sufficient to cause expression of a therapeutic protein (eg, an anti-VEGF agent, such as aflibercept) in aqueous humor. is the capacity. In some embodiments, the unit dose of the rAAV particles is 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 in aqueous humor (any range therebetween). a unit dose sufficient to achieve a concentration of a therapeutic protein (eg, an anti-VEGF agent, such as aflibercept) of In some embodiments, the unit dose of rAAV particles is a unit dose sufficient to cause expression of aflibercept in aqueous humor. In some embodiments, the unit dose of the rAAV particles is 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 in aqueous humor (any range therebetween). inclusive) is a unit dose sufficient to achieve a concentration of aflibercept.

In some embodiments, the unit dose of rAAV particles is administered to one and/or the contralateral eye of the individual. In some embodiments, the unit dose of rAAV particles is a unit dose sufficient to cause expression of a therapeutic protein (eg, an anti-VEGF agent, such as aflibercept) in the retina. In some embodiments, the unit dose of the rAAV particles is 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 in the retina. A unit dose sufficient to achieve a concentration of a therapeutic protein (eg, an anti-VEGF agent, such as aflibercept) (including any range between these values). 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 the rAAV particles is 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 in the retina. A unit dose sufficient to achieve a concentration of aflibercept (including any range between these values).

In some embodiments, the unit dose of rAAV particles is administered to one and/or the contralateral eye of the individual. In some embodiments, the unit dose of rAAV particles is a unit dose sufficient to cause expression of a therapeutic protein (eg, an anti-VEGF agent, such as aflibercept) in the choroid. In some embodiments, the unit dose of the rAAV particles is 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 in the choroid ( a unit dose sufficient to achieve a concentration of the therapeutic protein (eg, an anti-VEGF agent, such as aflibercept) of any one of (including any range between these values). In some embodiments, the unit dose of rAAV particles is a unit dose sufficient to cause expression of aflibercept in the choroid. In some embodiments, the unit dose of the rAAV particles is 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 in the choroid ( a unit dose sufficient to achieve a concentration of any one of aflibercept (including any range between these values).

In some embodiments, the unit dose of rAAV particles is administered to one and/or the contralateral eye of the individual. In some embodiments, the unit dose of rAAV particles is a therapeutically effective dose.

In some embodiments, a unit dose of rAAV particles is a therapeutically effective dose if the unit dose is sufficient to cause maintenance or reduction of retinal thickness as compared to retinal thickness prior to administration of the unit dose of rAAV particles. In some embodiments, a unit dose of rAAV particles is a therapeutically effective dose if the unit dose is sufficient to cause a decrease in retinal thickness as compared to retinal thickness prior to administration of the unit dose of rAAV particles. In some embodiments, the retinal thickness is central subfield thickness (CST) or central retinal thickness (CRT). In some embodiments, the unit dose of rAAV particles is such that the unit dose is about 5%, about 10%, about 15%, about 20%, about 25%, about 30% compared to the thickness of the retina prior to administration of the unit dose of rAAV particles. , 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 A therapeutically effective dose is sufficient to cause a reduction in retinal thickness of greater than any of 95%, about 99%, or about 100%. In some embodiments, retinal thickness (eg, CST or CRT) is determined by OCT or SD-OCT.

In some embodiments, a unit dose of rAAV particles is a therapeutically effective dose if the unit dose is sufficient to cause maintenance or reduction of macular volume as compared to macular volume prior to administration of the unit dose of rAAV particles. In some embodiments, a unit dose of rAAV particles is a therapeutically effective dose if the unit dose is sufficient to cause a decrease in macular volume compared to the macular volume prior to administration of the unit dose of rAAV particles. In some embodiments, the unit dose of rAAV particles is such that the unit dose is about 5%, about 10%, about 15%, about 20%, about 25%, about 30% compared to the macular volume prior to administration of the unit dose of rAAV particles. , is a therapeutically effective dose if it is sufficient to cause a reduction in macular volume of greater than about any of 35%, about 40%, about 45%, about 50%, about 55%, about 60%, or about 65%. In some embodiments, the macular volume is determined by OCT or SD-OCT.

In some embodiments, a unit dose of rAAV particles is a therapeutically effective dose if the unit dose is sufficient to cause maintenance or improvement of visual acuity as compared to visual acuity prior to administration of the unit dose of rAAV particles. In some embodiments, a unit dose of rAAV particles is a therapeutically effective dose if the unit dose is sufficient to cause an improvement in visual acuity as compared to visual acuity prior to administration of the unit dose of rAAV particles. In some embodiments, the unit dose of rAAV particles is such that the unit dose comprises 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 A dose is therapeutically effective if it is sufficient to cause an improvement in visual acuity of more than either % or more. In some embodiments, the visual acuity is maximum corrected visual acuity (BCVA). In some embodiments, a unit dose of rAAV particles is a therapeutically effective dose if the unit dose is sufficient to cause an improvement in BCVA as compared to BCVA prior to administration of the unit dose of rAAV particles. In some embodiments, BCVA is expressed as an ETDRS score corresponding to the number of characters read correctly (Vitale et al., (2016) JAMA Opthalmol 134(9):1041:1047). In some embodiments, the unit dose of rAAV particles is such that the unit dose is at least 15 ETDRS characters (Vitale et al., (2016) JAMA Opthalmol 134(9):1041:1047 compared to BCVA prior to administration of the unit dose of rAAV particles) ) (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 characters) sufficient to cause an improvement in BCVA. It is a therapeutically effective dose in some cases. In some embodiments, the unit dose of rAAV particles is such that the individual has fewer than 15 ETDRS letters (Vitale et al., (2016) JAMA Opthalmol 134(9):1041: compared to BCVA prior to administration of the unit dose of rAAV particles): 1047) (eg 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 characters) is a therapeutically effective dose if the unit dose is sufficient to cause maintenance of BCVA.

In some embodiments, the unit dose of rAAV particles is a therapeutically effective dose if the individual is determined to have maintenance of vision after administration of the unit dose of rAAV particles. In some embodiments, the unit dose of rAAV particles is a therapeutically effective dose if the individual is determined to have an improvement in vision after administration of the unit dose of rAAV particles. In some embodiments, the unit dose of rAAV particles is a therapeutically effective dose if, after administration of the unit dose of rAAV particles, the CST or CRT assessed by SD-OCT decreases compared to prior to administration of the unit dose of rAAV particles. In some embodiments, a unit dose of rAAV particles is a therapeutically effective dose if, after administration of the unit dose of rAAV particles, the CST or CRT assessed by SD-OCT is maintained compared to prior to administration of the unit dose of rAAV particles.

In some embodiments, the unit dose of rAAV particles is a therapeutically effective dose if, after administration of the unit dose of rAAV particles, the macular volume is reduced as compared to prior to administration of the unit dose of rAAV particles. In some embodiments, a unit dose of rAAV particles is a therapeutically effective dose if, after administration of the unit dose of rAAV particles, the macular volume is maintained compared to prior to administration of the unit dose of rAAV particles.

In some embodiments, the unit dose of the rAAV particles is determined such that the retinal thickness (eg, central retinal thickness (CRT) or central subfield thickness (CST)) and macular volume after administration of the unit dose of rAAV particles is equal to the unit dose of rAAV particles. It is a therapeutically effective dose when it is reduced compared to before administration of In some embodiments, the unit dose of the rAAV particles is determined such that the retinal thickness (eg, central retinal thickness (CRT) or central subfield thickness (CST)) and macular volume after administration of the unit dose of rAAV particles is equal to the unit dose of rAAV particles. It is a therapeutically effective dose if it is maintained compared to before administration.

In some embodiments, the unit dose of rAAV particles is administered after administration of the unit dose of rAAV particles to about every 4 weeks, every 5 weeks, or every 6 weeks after the individual administers the unit dose of rAAV particles to one eye and/or the contralateral eye. , every 7 weeks, every 8 weeks, every 9 weeks, every 10 weeks or more, if less than one rescue therapy treatment (eg, aflibercept injection) is required. In some embodiments, the unit dose of rAAV particles is administered to the individual at least about 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 A therapeutically effective dose if no salvage therapy treatment (eg, aflibercept injection) is required for any of a week, at least 110 weeks or more.

In some embodiments, the unit dose of rAAV particles is after administration of the unit dose of rAAV particles, wherein the individual has a decrease in retinal fluid level as compared to retinal fluid levels prior to administration of the unit dose of rAAV particles to one eye and/or the contralateral eye. If determined to be a therapeutically effective dose. In some embodiments, the unit dose of rAAV particles is administered to the individual after administration of the unit dose of rAAV particles as compared to retinal fluid levels prior to administration of the unit dose of rAAV particles to one eye and/or the contralateral eye. If determined to be a therapeutically effective dose. In some embodiments, the unit dose of rAAV particles is administered in one eye after administration of the unit dose of rAAV particles compared to the level of IRF and/or SRF before the individual administers the unit dose of rAAV particles to one eye and/or the contralateral eye. and/or a decrease in IRF and/or SRF in the contralateral eye.

In some embodiments, the unit dose of rAAV particles is a resolution of PED as compared to pigmented epithelial detachment (PED) after administration of the unit dose of rAAV particles and before the individual administers the unit dose of rAAV particles to one eye and/or the contralateral eye. is a therapeutically effective dose if determined to have

In some embodiments, the unit dose of rAAV particles is such that after administration of the unit dose of rAAV particles, the CNV lesion contracts compared to a CNV lesion present before administration of the unit dose of rAAV particles to one eye and/or the contralateral eye. It is a therapeutically effective dose. In some embodiments, the unit dose of rAAV particles is about 5%, compared to a CNV lesion present after administration of the unit dose of rAAV particles, wherein the CNV lesion is present before administration of the unit dose of rAAV particles to one eye and/or the contralateral eye, A dose is therapeutically effective if it contracts by more than any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%. In some embodiments, the unit dose of rAAV particles is administered when the CNV lesion does not grow after administration of the unit dose of rAAV particles compared to CNV lesions present prior to administration of the unit dose of rAAV particles to one eye and/or the contralateral eye. is a therapeutically effective dose. In some embodiments, the unit dose of rAAV particles is about 1%, compared to a CNV lesion present after administration of the unit dose of rAAV particles, wherein the CNV lesion is present prior to administration of the unit dose of rAAV particles to one eye and/or the contralateral eye, A dose is a therapeutically effective dose if it does not grow by more than about any of 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, or 20%.

In some embodiments, the unit dose of rAAV particles is such that after administration of the unit dose of rAAV particles, the individual has an improvement in anatomical features compared to anatomical features of one eye and/or the contralateral eye before administration of the unit dose of rAAV particles. If determined to be a therapeutically effective dose. In some embodiments, the unit dose of rAAV particles is administered to the individual after administration of the unit dose of rAAV particles as compared to the anatomical features of one eye and/or the contralateral eye before administration of the unit dose of rAAV particles and/or stabilization of anatomical features and/or or a therapeutically effective dose if determined to have maintenance.

In some embodiments, the unit dose of rAAV particles is therapeutically effective if administration of the dose to one and/or the contralateral eye of the individual reduces, arrests or prevents at least one symptom of an ocular neovascular disease or disorder. Do. In the case of ocular neovascular diseases or disorders characterized by abnormal (eg, excessive) angiogenesis, these symptoms include, for example, visual distortions (eg, color vision impairment, blurred vision, deterioration of central vision) and loss of vision. including but not limited to. In some embodiments, the unit dose of rAAV particles administered to one eye and/or the contralateral eye of the individual is such that administration of the unit dose to one and/or the contralateral eye of the individual is a symptom of one or more clinical features of an ocular neovascular disease. A dose that is therapeutically effective if it results in maintenance, partial remission, or complete remission. For example, a unit dose of rAAV particles administered to one and/or contralateral eye of a subject may be determined by any method known in the art for which administration of the dose to one and/or contralateral eye of a subject is determined by any method known in the art. It is therapeutically effective when it causes complete resolution, partial resolution, or maintenance of ocular neovascular disease. In some embodiments, the unit dose of rAAV particles administered to one and/or contralateral eye of the individual is such that administration of the dose to one and/or contralateral eye of the individual results in a maximum corrected visual acuity (BCVA) (eg, ETDRS). Based on score; Vitale et al., (2016) JAMA Opthalmol 134(9):1041:1047), central retinal thickness as determined by SD-OCT, units of rAAV particles in one eye and/or contralateral eye the number of salvage therapy treatments (eg, aflibercept injection) required by the individual after administration of the dose, the presence of intraretinal fluid (IRF) and/or subretinal fluid (SRF), resolution of pigmented epithelial detachment (PED) , choroidal neovascularization (CNV) lesion growth, by anatomical features based on any method known in the art (eg, SD-OCT, OCT, fluorescein angiography, digital color fundus imaging, etc.) It is therapeutically effective if it causes complete resolution, partial resolution, or maintenance of ocular neovascular disease at the time of evaluation. In some embodiments, the unit dose of rAAV particles administered to one and/or contralateral eye of the individual is determined by administration of the dose to one and/or contralateral eye of the individual according to ophthalmologic examination, intraocular pressure (e.g., Goldman apparition). using a tonometer or tono-pen), indirect ophthalmoscopy, examination of one and/or contralateral eye and appendages, ptosis and/or pupillary reactivity, ptosis, abnormal pupil shape, unequal pupil, abnormal response to light, afferent Nerve Pupil Defects, Slit-Lamp Exams (including those for eyelid, conjunctiva, cornea, lens, iris and anterior chamber), vitreous, optic nerve, peripheral retina and posterior segment abnormalities of retinal vasculature, SD-OCT, fluorescein angiography, of ocular neovascular disease as assessed by digital color fundus imaging (including imaging of the retina, optic disc and/or macula), aqueous humor sampling, vitreous humor sampling, OCT-angiography (OCT-A), refraction and visual acuity (BCVA) It is therapeutically effective when it causes complete remission, partial remission, or maintenance.

In some embodiments, the unit dose of rAAV particles administered to one eye of the individual is equal to the unit dose of rAAV particles administered to the contralateral eye of the individual. In some embodiments, the unit dose of rAAV particles administered to one eye of the individual is different from the unit dose of rAAV particles administered to the contralateral eye of the individual. In some embodiments, the unit dose of rAAV particles administered to one eye of the individual is higher than the unit dose of rAAV particles administered to the contralateral eye of the individual, e.g., 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% , more than any of about 275%, about 300% or more. In some embodiments, the unit dose of rAAV particles administered to the contralateral eye of the individual is higher than the unit dose of rAAV particles administered to one eye of the individual, e.g., 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% , more than any of about 275%, about 300% or more. In some embodiments, the unit dose of rAAV particles is expressed as the number of vector genomes per eye (vg) (vg/eye). In some embodiments, the unit dose of rAAV particles is about 6 x 10 ¹¹ vg/eye or less of rAAV particles. In some embodiments, the unit dose of rAAV particles is from about 1x10 ¹⁰ to about 2x10 ¹⁰ , from about 2x10 ¹⁰ to about 3x10 ¹⁰ , from about 3x10 ¹⁰ to about 4x10 ¹⁰ , from about 4x10 ¹⁰ to about 5x10 ¹⁰ , from about 5x10 ¹⁰ to about 6x10 ¹⁰ , about 6x10 ¹⁰ to about 7x10 10 , about 7x10 ¹⁰ to about 8x10 ¹⁰ , about 8x10 ¹⁰ to about 9x10 ¹⁰ , about 9x10 ¹⁰ to about ^{10x10 10 ,} about 1x10 ¹¹ to about 2x10 ¹¹ , about 2x10 ¹¹ to about 3x10 ¹¹ , about 3x10 ¹¹ to about 4x10 ¹¹ , about 4x10 ¹¹ to about 5x10 ¹¹ or about 5x10 ¹¹ to about 6x10 ¹¹ vg/eye rAAV particles, including rAAV particles of any value within these ranges. In some embodiments, the unit dose of rAAV particles is from about 6x10 ¹⁰ vg/eye to about 2x10 ¹¹ vg/eye of rAAV particles. In some embodiments, the unit dose of the rAAV particles is about 6x10 ¹⁰ vg/eye to about 2x10 ¹¹ vg/eye, about 7x10 ¹⁰ vg/eye to about 2x10 ¹¹ vg/eye, about 8x10 ¹⁰ vg/eye to about 2x10 ¹¹ vg rAAV particles per eye, about 9x10 ¹⁰ vg/eye to about 2x10 ¹¹ vg/eye, about 10x10 ¹⁰ vg/eye to about 2x10 ¹¹ vg/eye or about 1x10 ¹¹ vg/eye to about 2x10 ¹¹ vg/eye. In some embodiments, the unit dose of rAAV particles is from about 6x10 ¹⁰ vg/eye to about 2x10 ¹¹ vg/eye of rAAV particles. In some embodiments, the unit dose of the rAAV particles is about 6x10 ¹⁰ vg/eye to about 7x10 ¹⁰ vg/eye, about 7x10 ¹⁰ vg/eye to about 8x10 ¹⁰ vg/eye, about 8x10 ¹⁰ vg/eye to about 9x10 ¹⁰ vg rAAV particles per eye, about 9x10 ¹⁰ vg/eye to about 10x10 ¹⁰ vg/eye, about 10x10 ¹⁰ vg/eye to about 1x10 ¹¹ vg/eye or about 1x10 ¹¹ vg/eye to about 2x10 ¹¹ vg/eye. In some embodiments, the unit dose of the rAAV particles is about 6x10 ¹⁰ vg/eye, about 7x10 ¹⁰ vg/eye, about 8x10 ¹⁰ vg/eye, about 9x10 ¹⁰ vg/eye, about 10x10 ¹⁰ vg/eye, about 1x10 ¹¹ vg /eye or about 2x10 ¹¹ vg/eye of rAAV particles. In some embodiments, the unit dose of rAAV particles is about 6x10 ¹⁰ vg/eye or about 2x10 ¹¹ vg/eye of rAAV particles. In some embodiments, the unit dose of rAAV particles is about 6x10 ¹⁰ vg/eye of rAAV particles. In some embodiments, the unit dose of the rAAV particles is about 6×10 ¹⁰ vg/eye, about 2×10 ¹¹ vg/eye, or about 6×10 ¹¹ vg/eye. In some embodiments, the unit dose of rAAV particles is about 6×10 ¹⁰ vg/eye. In some embodiments, the unit dose of rAAV particles is about 2×10 ¹¹ vg/eye. In some embodiments, the unit dose of rAAV particles is about 6×10 ¹¹ vg/eye.

In some embodiments, the unit dose of rAAV particles administered to one eye of the individual and the unit dose of rAAV particles administered to the contralateral eye of the individual are administered simultaneously. In some embodiments, the unit dose of rAAV particles administered to one eye of the individual and the unit dose of rAAV particles administered to the contralateral eye of the individual are administered at different times. In some embodiments, the unit dose administered to the contralateral eye is at least about 1 hour, at least about 2 hours, at least about 4 hours, at least about 8 hours, at least about 12 hours, at least about 24 hours after administration of the unit dose to one eye. , at least about 1 day, at least about 2 days, at least about 3 days, at least about 4 days, at least about 5 days, at least about 6 days, at least about 7 days, at least about 1 week, at least about 2 weeks, at least about 3 weeks , after at least about 4 weeks or more. In some embodiments, the unit dose administered to the contralateral eye is administered at least about 2 weeks after administration of the unit dose to one eye.

In some embodiments, a single unit dose of rAAV particles is administered to one eye and/or the contralateral eye of the individual. In some embodiments, a single unit dose of rAAV particles administered to one eye and/or the contralateral eye is a therapeutically effective dose. In some embodiments, more than one dose (e.g., more than about any of 2, 3, 4, 5 or more unit doses) of the rAAV particles is administered to one eye and/or the contralateral eye of the individual. is administered In some embodiments, more than one dose of rAAV particles administered to one eye and/or the contralateral eye is a therapeutically effective dose.

In some embodiments, anti-VEGF treatment, e.g., IVT injection with an anti-VEGF agent, such as aflibercept, is administered at a unit dose of rAAV particles (e.g., from about 2 x 10 ¹¹ vg/eye to about 6 x 10 ¹¹ vg/unit dose of rAAV particles of the eye) is administered to one eye and/or the contralateral eye to which the rAAV particles are administered at least about 1 week, eg, at least about 7 days prior to administration. In some embodiments, the anti-VEGF treatment, e.g., an IVT injection with an anti-VEGF agent, such as aflibercept, is administered to the eye on about day 1, and a unit dose of rAAV particles, e.g., about 2 x 10 A unit dose of ¹¹ vg/eye to about 6×10 ¹¹ vg/eye of rAAV particles is administered to the eye on about day 8. In some embodiments, the unit dose of rAAV particles is about 2×10 ¹¹ vg/eye or about 6×10 ¹¹ vg/eye of rAAV particles. In some embodiments, the ocular neovascular disease is diabetic macular edema.

pharmaceutical formulation

In some embodiments, the unit dose of rAAV particles is in a pharmaceutical formulation. In some embodiments, the pharmaceutical formulation comprises rAAV particles, one or more osmotic or ionic strength agents, one or more buffers, 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 at least one buffer is 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 rAAV particles, sodium chloride, sodium phosphate monobasic, sodium phosphate dibasic, and a surfactant.

In some embodiments, the pharmaceutical formulation comprises about 1x10 ¹⁰ vg/mL to about 1x10 ¹³ vg/mL of rAAV particles, about 150 mM to about 200 mM sodium chloride, about 1 mM to about 10 mM sodium phosphate monobasic, about 1 mM to about 10 mM sodium phosphate dibasic and about 0.0005% (w/v) to about 0.005% (w/v) poloxamer 188, wherein the pharmaceutical formulation has a pH of about 7.0 to about 7.5. In some embodiments, the pharmaceutical formulation comprises about 6x10 ¹¹ vg/mL to about 6x10 ¹² vg/mL of rAAV particles, about 150 mM to about 200 mM sodium chloride, about 1 mM to about 10 mM sodium phosphate monobasic, about 1 mM to about 10 mM sodium phosphate dibasic and about 0.0005% (w/v) to about 0.005% (w/v) poloxamer 188, wherein the pharmaceutical formulation has a pH of about 7.0 to about 7.5. In some embodiments, the pharmaceutical formulation comprises about 6x10 ¹¹ vg/mL of rAAV particles, about 150 mM to about 200 mM sodium chloride, about 1 mM to about 10 mM sodium phosphate monobasic, about 1 mM to about 10 mM sodium phosphate dibasic and from about 0.0005% (w/v) to about 0.005% (w/v) poloxamer 188, wherein the pharmaceutical formulation has a pH from about 7.0 to about 7.5. In some embodiments, the pharmaceutical formulation comprises about 6x10 ¹² vg/mL of rAAV particles, about 150 mM to about 200 mM sodium chloride, about 1 mM to about 10 mM sodium phosphate monobasic, about 1 mM to about 10 mM sodium phosphate dibasic and from about 0.0005% (w/v) to about 0.005% (w/v) poloxamer 188, wherein the pharmaceutical formulation has a pH from about 7.0 to about 7.5.

In some embodiments, the rAAV particles in the pharmaceutical formulation are present at a concentration of about 1x10 ¹⁰ vg/ml to about 1x10 ¹³ vg/ml. In some embodiments, the rAAV particles in the pharmaceutical formulation are present at a concentration of about 1x10 ⁰⁹ vg/ml to about 6x10 ¹⁴ vg/ml. In certain embodiments, the rAAV particles in the pharmaceutical formulation are from about 1x10 ⁰⁹ vg/ml to about 2x10 ⁰⁹ vg/ml, from about 2x10 ⁰⁹ vg/ml to about 3x10 ⁰⁹ , from about 3x10 ⁰⁹ vg/ml to about 4x10 ⁰⁹ , about 4x10 ⁰⁹ vg/ml to about 5x10 ⁰⁹ , about 5x10 ⁰⁹ vg/ml to about 6x10 ⁰⁹ , about 6x10 ⁰⁹ vg/ml to about 7x10 ⁰⁹ , about 7x10 ⁰⁹ vg/ml to about 8x10 ⁰⁹ , about 8x10 ⁰⁹ vg/ml to about 9x10 ⁰⁹ , about 9x10 ⁰⁹ vg/ml to about 10x10 ⁰⁹ , about 10x10 ⁰⁹ vg/ml to about 1x10 ¹⁰ , about 1x10 ¹⁰ vg/ml to about 2x10 ¹⁰ , about 2x10 ¹⁰ vg/ml to about 3x10 ¹⁰ , about 3x10 ¹⁰ vg /ml to about 4x10 ¹⁰ , about 4x10 ¹⁰ vg/ml to about 5x10 ¹⁰ , about 5x10 ¹⁰ vg/ml to about 6x10 ¹⁰ , about 6x10 ¹⁰ vg/ml to about 7x10 ¹⁰ , about 7x10 ¹⁰ vg/ml to about 8x10 ¹⁰ , about 8x10 ¹⁰ vg/ml to about 9x10 ¹⁰ , about 9x10 ¹⁰ vg/ml to about 10x10 ¹⁰ , about 10x10 ¹⁰ vg/ml to about 1x10 ¹¹ , about 1x10 ¹¹ vg/ml to about 2x10 ¹¹ , about 2x10 ¹¹ vg/ ml to about 3x10 ¹¹ , about 3x10 ¹¹ vg/ml to about 4x10 ¹¹ , about 4x10 ¹¹ vg/ml to about 5x10 ¹¹ , about 5x10 ¹¹ vg/ml to about 6x10 ¹¹ , about 6x10 ¹¹ vg/ml to about 7x10 ¹¹ , about 7x10 ¹¹ vg/ml to about 8x10 ¹¹ , about 8x10 ¹¹ vg/ml to about 9x10 ¹¹ , about 9x10 ¹¹ vg/ml to about 10x10 ¹¹ , about 1x10 ¹² vg/ml to about 2x10 ¹² , about 2x10 ¹² vg/ml to about 3x10 ¹² , about 3x10 ¹² vg/ml to about 4x10 ¹² , about 4x10 ¹² vg/ml to about 5x10 ¹² , about 5x10 ¹² vg/ml to about 6x10 ¹² , about 6x10 ¹² vg/ml to about 7x10 ¹² , about 7x10 ¹² vg/ml to about 8x10 ¹² , about 8x10 ¹² vg/ml to about 9x10 ¹² , about 9x10 ¹² vg/ml to about 10x10 ¹² , about 1x10 ¹³ vg/ml to about 2x10 ¹³ , about 2x10 ¹³ vg/ml to about 3x10 ¹³ , about 3x10 ¹³ vg/ml to about 4x10 ¹³ , about 4x10 ¹³ vg/ml to about 5x10 ¹³ , about 5x10 ¹³ vg/ml to about 6x10 ¹³ , about 6x10 ¹³ vg/ml to about 7x10 ¹³ , about 7x10 ¹³ vg/ml to about 8x10 ¹³ , about 8x10 ¹³ vg/ml to about 9x10 ¹³ , about 9x10 ¹³ vg/ml to about 10x10 ¹³ , about 1x10 ¹⁴ vg/ml to about 2x10 ¹⁴ , about 2x10 ¹⁴ vg/ml to about 3x10 ¹⁴ , about 3x10 ¹⁴ vg/ml to about 4x10 ¹⁴ , about 4x10 ¹⁴ vg/ml to about 5x10 ¹⁴ or about 5x10 ¹⁴ vg/ml to about 6x10 ¹⁴ vg/mL. In some embodiments, the pharmaceutical formulation comprises about 6x10 ¹¹ vg/mL to about 6x10 ¹² vg/mL of rAAV particles. In some embodiments, the pharmaceutical formulation comprises about 6x10 ¹² vg/mL of rAAV particles. In some embodiments, the pharmaceutical formulation comprises about 6x10 ¹¹ vg/mL of rAAV particles.

In some embodiments, sodium chloride in the pharmaceutical formulation is present at a concentration of about 150 mM to about 200 mM. In certain embodiments, sodium chloride in the pharmaceutical formulation is present at a concentration of about 150 mM, about 160 mM, about 170 mM, about 180 mM, about 190 mM, or about 200 mM. In certain embodiments, sodium chloride in the pharmaceutical formulation is present at a concentration of about 180 mM.

In some embodiments, the monobasic sodium phosphate is present in the pharmaceutical formulation at a concentration of about 1 mM to about 10 mM. In some embodiments, sodium phosphate monobasic is about 1 mM, about 2 mM, about 3 mM, about 4 mM, about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM or about in the pharmaceutical formulation. It is present at a concentration of any of 10 mM. In certain embodiments, sodium phosphate monobasic is present in the pharmaceutical formulation at a concentration of about 5 mM.

In some embodiments, the sodium phosphate dibasic is present in the pharmaceutical formulation at a concentration of about 1 mM to about 10 mM. In some embodiments, sodium phosphate dibasic is about 1 mM, about 2 mM, about 3 mM, about 4 mM, about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM or about in the pharmaceutical formulation. It is present at a concentration of any of 10 mM. In certain embodiments, sodium phosphate dibasic is present in the pharmaceutical formulation at a concentration of about 5 mM.

In some embodiments, poloxamer 188 is present in the pharmaceutical formulation at a concentration of about 0.0005% (w/v) to about 0.005% (w/v). In some embodiments, poloxamer 188 is about 0.0005% (w/v), about 0.0006% (w/v), about 0.0007% (w/v), about 0.0008% (w/v), about 0.0009 in the pharmaceutical formulation. % (w/v), about 0.001% (w/v), about 0.002% (w/v), about 0.003% (w/v), about 0.004% (w/v), or about 0.005% (w/v) ) in any of the concentrations. In certain embodiments, poloxamer 188 is present in the pharmaceutical formulation at a concentration of about 0.001% (w/v).

In some embodiments, the pharmaceutical formulation has a pH of about 7.0 to about 7.5. In some embodiments, the pharmaceutical formulation has a pH of about 7.0, about 7.1, about 7.2, about 7.3, about 7.4, or about 7.5. In certain embodiments, the pharmaceutical formulation has a pH of about 7.3. In some embodiments, hydrochloric acid and sodium hydroxide are used to adjust the pH of the pharmaceutical formulation.

In some embodiments, the pharmaceutical formulation comprises about 6x10 ¹² vg/mL of rAAV particles, about 180 mM sodium chloride, about 5 mM sodium phosphate monobasic, about 5 mM sodium phosphate dibasic, 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 6x10 ¹¹ vg/mL of rAAV particles, about 180 mM sodium chloride, about 5 mM sodium phosphate monobasic, about 5 mM sodium phosphate dibasic, 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 is suitable for administration to one eye and/or the contralateral eye of an individual, eg, a human patient, via intravitreal (IVT) injection to achieve the desired therapeutic or prophylactic effect. In some embodiments, the pharmaceutical formulation is supplied as a reconstituted homogeneous solution. In some embodiments, the solution is a suspension. In some embodiments, the pharmaceutical formulation is supplied as a frozen suspension and thawed prior to administration to one and/or contralateral eye of an individual. In some embodiments, the solution is isotonic.

In other embodiments, for example, the pharmaceutical composition comprising an AAV2.7m8 vector comprising a nucleic acid sequence encoding an anti-VEGF agent (eg, aflibercept or a functional fragment or variant thereof) is supplied in lyophilized form. and reconstituted prior to administration to one and/or the contralateral eye of the subject. In some embodiments, a method provided herein encodes an anti-VEGF agent (eg, aflibercept or a functional fragment or variant thereof) and comprises a rAAV (eg, AAV2.7m8) prior to administration to a subject. and reconstituting, dissolving or solubilizing the lyophilized pharmaceutical composition in a buffer. In some embodiments, such lyophilized pharmaceutical compositions comprise one or more of the following: a cryoprotectant, a surfactant, a salt, a stabilizer, or any combination thereof.

In some embodiments, the pharmaceutical formulation is a homogeneous solution. In some embodiments, the homogenous solution is supplied in a pre-filled syringe. In some embodiments, the pharmaceutical formulation is supplied as a suspension. In some embodiments, the suspension is a solution. In some embodiments, the suspension is refrigerated. In some embodiments, the suspension is frozen. In some embodiments, the methods provided herein ensure that the active ingredient(s) are dissolved and/or evenly distributed in solution prior to administration to one and/or the contralateral eye of an individual (eg, via IVT injection). warming the refrigerated suspension to room temperature and/or stirring the suspension to In some embodiments, the methods provided herein ensure that the active ingredient(s) are dissolved and/or evenly distributed in solution prior to administration to one and/or the contralateral eye of an individual (eg, via IVT injection). It further comprises thawing the frozen suspension and warming and/or stirring the suspension to room temperature to ensure. In some embodiments, the suspension is diluted prior to administration to a subject (eg, via IVT injection). In some embodiments, the suspension is supplied as a pre-filled syringe.

In some embodiments, the pharmaceutical formulation is provided as a frozen suspension. In some embodiments, suspensions include pharmaceutically acceptable excipients such as surfactants, glycerol, non-ionic surfactants, buffers, glycols, salts, and any combinations thereof.

In some embodiments, the suspension is a solution. In some embodiments, the suspension comprises micelles.

In some embodiments, for storage stability and handling convenience, it comprises a nucleic acid sequence encoding an anti-VEGF agent (eg, aflibercept or a functional fragment or variant thereof) and a rAAV (eg, AAV2.7m8) The pharmaceutical formulations used are formulated as lyophilized, lyophilized or vacuum dried powders for reconstitution with saline, buffer or water prior to administration to one and/or the contralateral eye of a subject. Alternatively, pharmaceutical formulations are formulated as aqueous solutions, such as suspensions or homogeneous solutions. The pharmaceutical formulation may contain a rAAV particle comprising a nucleic acid sequence encoding aflibercept. Various excipients such as phosphate, PBS or Tris buffer, glycol, glycerol, saline, surfactant (eg, pluronic or polysorbate) or any combination thereof can be used to stabilize the pharmaceutical formulation. Additionally, cryoprotectants such as alcohols may be used as stabilizers under freezing or drying conditions. In some embodiments, the gene therapy is provided as a suspension, refrigerated suspension, or frozen suspension.

In some embodiments, a suspension of a pharmaceutical formulation as disclosed herein is about 20 μL, 30 μL, 40 μL, 50 μL, 60 μL, 70 μL, 80 μL, 90 μL, 100 μL, 200 μL, 300 μL, 400 μL. have a volume of any of μL, 500 μL, 600 μL, 700 μL, 800 μL, 900 μL, or 1000 μL. In some embodiments, a suspension of a pharmaceutical formulation as disclosed herein has a volume of about 250 μL. In some embodiments, a suspension of a pharmaceutical formulation as disclosed herein comprises 0.1-0.5 mL, 0.1-0.2 mL, 0.3-0.5 mL, 0.5-1.0 mL, 0.5-0.7 mL, 0.6-0.8 mL, 0.8-1 mL, 0.9 to 1.1 mL, 1.0 to 1.2 mL or 1.0 to 1.5 mL. In other embodiments, the volume is 0.1 mL, 0.2 mL, 0.3 mL, 0.4 mL, 0.5 mL, 0.6 mL, 0.7 mL, 0.8 mL, 0.9 mL, 1.0 mL, 1.1 mL, 1.2 mL, 1.3 mL, 1.4 mL or 1.5 mL or less. In some embodiments, a suspension of a pharmaceutical formulation as disclosed herein has a volume of about 0.25 mL.

In some embodiments, suspensions of pharmaceutical formulations as disclosed herein are provided with ready-to-use closures (eg, closures made of chlorobutyl) and sealed (eg, with a sterile aluminum tear-off sealant) sealed sterile. It is provided as a sterile-filtered frozen suspension in ready-to-use vials (eg, 0.5 mL vials; eg, Crystal Zenith® vials). In some embodiments, suspensions of pharmaceutical formulations as disclosed herein are provided with ready-to-use closures (eg, closures made of chlorobutyl) and sealed (eg, with a sterile aluminum tear-off sealant) sealed sterile. Provided as a sterile-filtered frozen suspension in ready-to-use vials (e.g., 0.5 mL vials; e.g., Crystal Zenith® vials), wherein the vials are 0.1-0.5 mL, 0.1-0.2 mL, 0.2-0.3 mL , containing a suspension of the pharmaceutical formulation in a volume of 0.3 to 0.4 mL or 0.4 mL to 0.5 mL. In some embodiments, suspensions of pharmaceutical formulations as disclosed herein are provided with ready-to-use closures (eg, closures made of chlorobutyl) and sealed (eg, with a sterile aluminum tear-off sealant) sealed sterile. It is provided as a sterile-filtered frozen suspension in a ready-to-use vial (eg, a 0.5 mL vial; eg, a Crystal Zenith® vial), wherein the vial contains a volume of about 0.25 mL of the suspension of the pharmaceutical formulation.

In some embodiments, the pharmaceutical formulations disclosed herein are designed, engineered, or adapted for administration to primates (eg, non-human primates and human subjects) via intravitreal or subretinal injection. In some embodiments, a pharmaceutical formulation comprising a rAAV particle comprising a nucleic acid sequence encoding an anti-VEGF agent (eg, aflibercept) is formulated for intravitreal injection into an eye of an individual. In some embodiments, the pharmaceutical composition is about 25 μL, 30 μL, 35 μL, 40 μL, 45 μL, 50 μL, 55 μL, 60 μL, 65 μL, 70 μL, 75 μL, 80 μL, 85 μL, 90 μL , 95 µL, 100 µL, 110 µL, 120 µL, 130 µL, 140 µL, 150 µL, 160 µL, 170 µL, 180 µL, 190 µL, 200 µL, 210 µL, 220 µL, 230 µL, 240 µL, or 250 µL formulated or reconstituted at a concentration that permits intravitreal injection of a volume of no more than any of the μL or exactly any of the above values. In some embodiments, the unit dose of the pharmaceutical formulation is about 25 μL, 30 μL, 35 μL, 40 μL, 45 μL, 50 μL, 55 μL, 60 μL, 65 μL, 70 μL, 75 μL, 80 μL, 85 μL , 90 µL, 95 µL, 100 µL, 110 µL, 120 µL, 130 µL, 140 µL, 150 µL, 160 µL, 170 µL, 180 µL, 190 µL, 200 µL, 210 µL, 220 µL, 230 µL, 240 volume of no more than any of μL or 250 μL, or exactly no more than any of the above values. In some embodiments, the methods disclosed herein comprise about 25 solutions or suspensions of a pharmaceutical formulation comprising a nucleic acid sequence encoding an anti-VEGF agent (eg, aflibercept) and a rAAV (eg, AAV2.7m8). µL, 30 µL, 35 µL, 40 µL, 45 µL, 50 µL, 55 µL, 60 µL, 65 µL, 70 µL, 75 µL, 80 µL, 85 µL, 90 µL, 95 µL, 100 µL, 110 µL, Intravitreal injection in a volume of any of 120 µL, 130 µL, 140 µL, 150 µL, 160 µL, 170 µL, 180 µL, 190 µL, 200 µL, 210 µL, 220 µL, 230 µL, 240 µL, or 250 µL include In some embodiments, the methods disclosed herein comprise about 30 solutions or suspensions of a pharmaceutical formulation comprising a nucleic acid sequence encoding an anti-VEGF agent (eg, aflibercept) and a rAAV (eg, AAV2.7m8). Intravitreal injection in a volume of μL or about 100 μL. In some embodiments, the methods disclosed herein comprise about 30 solutions or suspensions of a pharmaceutical formulation comprising a nucleic acid sequence encoding an anti-VEGF agent (eg, aflibercept) and a rAAV (eg, AAV2.7m8). Include an intravitreal injection in a volume of μL. In some embodiments, the methods disclosed herein comprise about 100 solutions or suspensions of a pharmaceutical formulation comprising a nucleic acid sequence encoding an anti-VEGF agent (eg, aflibercept) and a rAAV (eg, AAV2.7m8). Include an intravitreal injection in a volume of μL.

In some embodiments, the AAV2.7m8 particle comprising the nucleic acid sequence of an anti-VEGF agent (eg, aflibercept) transgene described herein is a component of a gene therapy pharmaceutical formulation. In some embodiments, rAAV particles of any serotype comprising a 7m8 variant capsid protein as described herein are used to prepare a freeze suspension or a freeze-dried or lyophilized formulation composition. In some embodiments, the gene therapy is formulated as a refrigerated or frozen suspension. In some embodiments, the rAAV particle is rAAV2. In some embodiments, the lyophilisate or suspension of the pharmaceutical formulation comprises rAAV2 comprising a 7m8 variant capsid protein and a DNA sequence encoding an anti-VEGF agent (eg, aflibercept). In some embodiments, the suspension is refrigerated or frozen.

In some embodiments, administering the unit dose of rAAV particles to one and/or the contralateral eye of the individual is by intravitreal (IVT) injection. For IVT injection, the rAAV particles can be delivered in the form of a suspension in a pharmaceutical formulation (eg, as described herein). Initially, a local anesthetic is applied to the surface of the eye, followed by an ophthalmic antiseptic solution. The eye is kept open with or without the instrument, and the rAAV particles are injected under direct observation through the sclera by a short, narrow needle, e.g., a 30-gauge needle, into the vitreous cavity of one and/or the contralateral eye of the subject. do. Typically, from about 25 μL to about 250 μL (e.g., about 25 μL, about 30 μL, about 40 μL, about 50 μL, about 60 μL, about 70 μL, about 80 μL, about 90 μL, about 100 μL , about 110 μL, about 120 μL, about 130 μL, about 140 μL, about 150 μL, about 160 μL, about 170 μL, about 180 μL, about 190 μL, about 200 μL, about 210 μL, about 220 μL, about A volume of the rAAV particle suspension (any of 230 μL, about 240 μL, or about 250 μL) may be delivered to the eye by IVT injection. In some embodiments, a unit dose of rAAV particles comprises a volume of about 100 μL. In some embodiments, a unit dose of rAAV particles comprises a volume of about 30 μL. In some embodiments, the IVT injection is performed in combination with removal of vitreous humor. In some embodiments, a vitrectomy may be performed, wherein the entire volume of the vitreous gel is replaced by injection of a rAAV particle suspension (eg, about 4 mL of rAAV particle suspension). A vitrectomy is performed using a cannula of an appropriate orifice size (eg, 20 gauge to 27 gauge), wherein the volume of vitrectomy gel removed is the fluid from the infusion cannula, e.g., saline, isotonic solution, rAAV particles It is replaced by injection of the suspension. IVT administration is generally well tolerated. At the end of the procedure, there is sometimes mild redness at the injection site. There is occasional tenderness, but most patients do not report any pain. No eye patch or eye shield is required after this procedure, and activity is not restricted. Sometimes, antibiotic eye drops are prescribed for several days to help prevent infection.

In some embodiments, the pharmaceutical formulation is administered to an individual (eg, human or non-human primate) via IVT injection for the treatment of an ocular disease or disorder characterized by abnormal (eg, excessive) angiogenesis or neovascularization. ) to be administered to one eye and/or the contralateral eye (eg, a therapeutically effective dose). In some embodiments, the pharmaceutical formulation comprises a unit dose (eg, a therapeutically effective dose) as described in further detail elsewhere herein. In some embodiments, the volume of a unit dose (eg, a therapeutically effective dose) of a viral vector (eg, a rAAV vector disclosed herein) administered to a subject is about 25 μL, 30 μL, 35 μL, 40 μL, 45 µL, 50 µL, 55 µL, 60 µL, 65 µL, 70 µL, 75 µL, 80 µL, 85 µL, 90 µL, 95 µL, 100 µL, 110 µL, 120 µL, 130 µL, 140 µL, 150 µL , 160 μL, 170 μL, 180 μL, 190 μL, 200 μL, 210 μL, 220 μL, 230 μL, 240 μL or 250 μL, inclusive of any range between these values. Minimizing the volume of a unit dose to be administered to a subject can eliminate or alleviate changes in intraocular pressure and other side effects associated with IVT injections (eg, elevated intraocular pressure, inflammation, irritation, or pain).

Pharmaceutical formulations suitable for ocular use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions, suspensions or dispersions. For intravitreal administration, suitable carriers include physiological saline, bacteriostatic water, phosphate buffered saline (PBS) and/or isotonic agents such as glycerol. In certain embodiments, the pharmaceutical formulation is sterile and is fluid to the extent that easy syringeability or injectability exists. In certain embodiments, the pharmaceutical formulation is stable under the conditions of manufacture and storage and is preserved against the contaminating action of microorganisms such as bacteria and fungi. In some embodiments, the pharmaceutical composition may include isotonic agents, such as salts or glycerol. In some embodiments, a surfactant or stabilizer is added to the pharmaceutical composition to prevent agglomeration.

In some embodiments, the pharmaceutical formulation contains an excipient or carrier. A carrier is a solvent or dispersion medium containing, for example, water, saline, ethanol, a polyol (eg, glycerol, propylene glycol and liquid polyethylene glycol, etc.) and any combination thereof. Proper fluidity can be achieved, for example, by the use of coatings such as lecithin, by the maintenance of the required particle size in the case of dispersions and by surfactants such as polysorbates (eg Tween™, polysorbate 20). , polysorbate 80), sodium dodecyl sulfate (sodium lauryl sulfate), lauryl dimethyl amine oxide, cetyltrimethylammonium bromide (CTAB), polyethoxylated alcohol, polyoxyethylene sorbitan, octoxynol (Triton X100™), N,N-dimethyldodecylamine-N-oxide, hexadecyltrimethylammonium bromide (HTAB), polyoxyl 10 lauryl ether, Brij 721™, bile salt (de Sodium oxycholate, sodium cholate), pluronic acid (F-68, F-127), polyoxyl castor oil (Cremophor™), nonylphenol ethoxylate (Tergitol™), cyclodextrin and ethylbenzethonium chloride (Hyamine™). Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, cresol, thimerosal, and the like. In many embodiments, isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol and/or sodium chloride are included in the pharmaceutical formulation. Prolonged absorption of the internal composition may be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin. In some embodiments, the pharmaceutical carrier comprises sodium phosphate, sodium chloride, polysorbate and sucrose. In some embodiments, the pharmaceutical formulation comprises a surfactant, eg, a non-ionic surfactant, such as a polysorbate, poloxamer, or pluronic. In some embodiments, the addition of a non-ionic surfactant reduces agglomeration in the pharmaceutical composition.

Also provided herein are kits comprising at least one pharmaceutical agent described herein. In some embodiments, the kit comprises a frozen suspension of the pharmaceutical formulation (eg, 1 unit dose in a vial). In some embodiments, a kit comprises a lyophilized or freeze-dried pharmaceutical formulation disclosed herein (eg, one unit dose in a vial) and a solution for dissolving, diluting and/or reconstituting a lyophilized pharmaceutical composition. In some embodiments, the solution for reconstitution or dilution is supplied as a pre-filled syringe. In some embodiments, the kit comprises a freeze-dried or lyophilized pharmaceutical composition comprising rAAV (eg, AAV2.7m8) and a solution for reconstitution of the pharmaceutical composition to a desired concentration or volume. In some embodiments, the kit comprises a buffer to help prevent aggregation upon reconstitution of the pharmaceutical compositions disclosed herein. In some embodiments, the pharmaceutical composition is provided in a pre-filled syringe. In some embodiments, the kit comprises a dual-chamber syringe or container, wherein one of the chambers contains a buffer for dissolving or diluting the pharmaceutical composition. In some embodiments, the kit comprises a syringe for injection. In some embodiments, the reconstituted solution is filtered prior to administration. In some embodiments, the kit comprises a filter or filter syringe for filtering the reconstituted pharmaceutical composition prior to administration to a patient. In some embodiments, the kit is equipped with a ready-to-use stopper (e.g., made of chlorobutyl) and sealed (e.g., with a sterile aluminum tear-off sealant) and sealed sterile ready-to-use vials (e.g., , a suspension of a pharmaceutical formulation comprising rAAV particles as disclosed herein provided as a sterile-filtered frozen suspension in a 0.5 mL vial; eg, a Crystal Zenith® vial). In some embodiments, the kit is equipped with a ready-to-use stopper (e.g., made of chlorobutyl) and sealed (e.g., with a sterile aluminum tear-off sealant) and sealed sterile ready-to-use vials (e.g., , a suspension of a pharmaceutical formulation comprising rAAV particles as disclosed herein provided as a sterile-filtered frozen suspension in a 0.5 mL vial; e.g., a Crystal Zenith® vial, wherein the vial contains 0.1 to 0.5 mL; contain a volume of 0.1 to 0.2 mL, 0.2 to 0.3 mL, 0.3 to 0.4 mL or 0.4 mL to 0.5 mL of the suspension of the pharmaceutical formulation. In some embodiments, the kit is equipped with a ready-to-use stopper (e.g., made of chlorobutyl) and sealed (e.g., with a sterile aluminum tear-off sealant) and sealed sterile ready-to-use vials (e.g., , a suspension of a pharmaceutical formulation comprising rAAV particles as disclosed herein provided as a sterile-filtered frozen suspension in a 0.5 mL vial; e.g., a Crystal Zenith® vial), wherein the vial contains a volume of about 0.25 mL Contains suspensions of pharmaceutical formulations. In some embodiments, the kit includes instructions for use; Further comprising instructions for treating ocular neovascular disease, eg, using the rAAV particles disclosed herein.

ocular neovascular disease

In one aspect, the present disclosure provides a method of treating an ocular neovascular disease in a subject. In another aspect, the present disclosure provides a method of reducing retinal fluid in the eye of a subject having an ocular neovascular disease.

In some embodiments, the ocular neovascular disease is age-related macular degeneration (AMD), wet-AMD, retinal neovascularization, choroidal neovascularization diabetic retinopathy, proliferative diabetic retinopathy, retinal vein occlusion, central retina venous occlusion, branch retinal vein occlusion, diabetic macular edema, diabetic retinal ischemia, ischemic retinopathy, diabetic retinal edema, or any combination thereof. In some embodiments, the ocular neovascular disease is active choroidal neovascularization (CNV) secondary to age-related macular degeneration (AMD).

In some embodiments, the ocular neovascular disease is relapsed and/or persistent wAMD. In some embodiments, the ocular neovascular disease is active subfoveal CNV secondary to AMD. In some embodiments, active subfoveal CNV secondary to AMD occupies >50% of total lesion size. In some embodiments, active subfoveal CNV secondary to AMD is leakage in fluorescein angiography (FA), fluid in spectral domain optical coherence tomography (SD-OCT), and/or subretinal in color fundus imaging. Occupies >50% of the total lesion size with evidence of bleeding. In some embodiments, active subfoveal CNV secondary to AMD is leakage in fluorescein angiography (FA), fluid in spectral domain optical coherence tomography (SD-OCT), and/or subretinal in color fundus imaging. Occupies >50% of the total lesion size with evidence of bleeding, and the overall dimension of the lesion does not exceed the area of the 12 macular photocoagulation study discs. In some embodiments, one eye and/or the contralateral eye of the individual has 78-25 (e.g., about 78, about 75, about 70, about 65, about 60) unit doses of the rAAV particles of the present disclosure prior to administration. , less than about any of 55, about 50, about 45, about 40, about 35, about 30, or about 25). In some embodiments, one eye and/or the contralateral eye of the individual is about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40 prior to administration of the unit dose of the rAAV particles of the present disclosure. , about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, or about 100 or more (BCVA) was shown.

In some embodiments, the individual had polypoid choroidal vasculopathy (PCV) in one eye and/or the contralateral eye prior to administration of the unit dose of rAAV particles.

In some embodiments, the ETDRS character evaluation is performed at about 0.5 meters, about 1 meter, about 2 meters, about 3 meters, or about 4 meters. In some embodiments, the ETDRS text evaluation is performed at about 4 meters.

In some embodiments, the individual is administered an anti-VEGF agent (eg, bevacizumab, brolucizumab, rani) within about the last 12 weeks (eg, about 3 or about 4 months) prior to administration of the unit dose of the rAAV particles. bizumab, parisimab, abicipar pegol, convercept, OPT-302, KSI-301, injectable sunitinib maleate (GB-102), PAN-90806 (panoptica) and/or afliber sept) (eg, at least 1, at least 2, at least 3, at least 4, at least 5 or more treatments). In some embodiments, the individual administers the antibiotic to one eye and/or the contralateral eye for about the last 12 weeks (e.g., about 3 or about 4 months) prior to administering the unit dose of the rAAV particles to one and/or the contralateral eye. -VEGF agents (eg, bevacizumab, brolucizumab, ranibizumab, parisimab, abicipar pegol, convercept, OPT-302, KSI-301, injectable sunitinib maleate (GB) -102), PAN-90806 (Panoptica) and/or aflibercept) received 2 or 3 prior treatments. In some embodiments, the individual has an anti-VEGF agent (eg, bevacizumab, brolucizumab, ranibizumab, parisimab, abicipar pegol, convercept, OPT) in one eye and/or the contralateral eye. -302, KSI-301, injectable sunitinib maleate (GB-102), PAN-90806 (panoptica) and/or aflibercept) at least about 1 time, at least about 5 times, at least about 10 times times, at least about 20 times, at least about 30 times, at least about 40 times, at least about 50 times, at least about 60 times, at least about 70 times, at least about 80 times, at least about 90 times, at least about 100 times, at least about 110 times times, at least about 120 or more prior treatments. In some embodiments, the individual has a calculated anti-VEGF agent (eg, bevacizumab, brolucizumab, ranibizumab and/or aflibercept) injection interval in one eye and/or the contralateral eye of about 2 weeks, about 3 weeks, 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 9 weeks, about 10 weeks, about 11 weeks, about 12 weeks or more. In some embodiments, the individual has a calculated anti-VEGF (eg, bevacizumab, brolucizumab, ranibizumab and/or aflibercept) injection interval in one eye and/or the contralateral eye of about 5- 7 weeks, about 4-10 weeks, about 4-7 weeks, or about 4-6 weeks. In some embodiments, the individual administers the unit dose of the rAAV particles to one and/or the contralateral eye for at least about 5 days, at least about 6 days, at least about 7 days, at least about 8 days, at least about 9 days, at least about 10 days, at least about 11 days, at least about 12 days, at least about 13 days, at least about 14 days, at least about 15 days, at least about 16 days, at least about 17 days, at least about 18 days, at least about 19 days, or at least about an anti-VEGF agent (e.g., bevacizumab, brolucizumab, ranibizumab, parisimab, abicipar pegol, convercept, OPT- 302, KSI-301, injectable sunitinib maleate (GB-102), PAN-90806 (panoptica) and/or aflibercept). In some embodiments, the individual administers an anti-VEGF agent (e.g., an anti-VEGF agent to one eye and/or the contralateral eye about 7 days, about 10 days, or about 14 days before administering the unit dose of the rAAV particles to one eye and/or the contralateral eye. For example, bevacizumab, brolucizumab, ranibizumab, parisimab, abicipar pegol, convercept, OPT-302, KSI-301, injectable sunitinib maleate (GB-102), PAN- 90806 (Panoptica) and/or aflibercept). In some embodiments, the prior treatment comprises an intraocular, subretinal, or intravitreal injection with an anti-VEGF agent. In some embodiments, the anti-VEGF agent is bevacizumab, brolucizumab, ranibizumab, parisimab, abicipar pegol, convercept, OPT-302, KSI-301, injectable sunitinib maleate (GB-102), PAN-90806 (Panoptica) and/or aflibercept. In some embodiments, the anti-VEGF agent is aflibercept. In some embodiments, the individual administers an anti-VEGF agent (e.g., bevacizumab, Brolucizumab, ranibizumab, parisimab, abicipar pegol, convercept, OPT-302, KSI-301, injectable sunitinib maleate (GB-102), PAN-90806 (panoptica) and/or aflibercept) 1 to 20 times (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20) prior treatment. In some embodiments, the individual administers an anti-VEGF agent (e.g., bevacizumab, Brolucizumab, ranibizumab, parisimab, abicipar pegol, convercept, OPT-302, KSI-301, injectable sunitinib maleate (GB-102), PAN-90806 (panoptica) and/or about 9 or about 10 prior treatments with aflibercept).

In some embodiments, the individual has a meaningful response to prior treatment with an anti-VEGF agent. In some embodiments, the anti-VEGF agent is aflibercept, a functional variant thereof, or a functional fragment thereof. In some embodiments, the anti-VEGF agent comprises a polypeptide comprising an amino acid sequence having at least about 95% identity to the amino acid sequence of SEQ ID NO:35. In some embodiments, the individual has prior anti-VEGF treatment for ocular neovascular disease in one eye and/or the contralateral eye prior to administering a unit dose of the rAAV particles to one eye and/or the contralateral eye (eg, afliber sep, a functional variant thereof or a functional fragment thereof). In some embodiments, the anti-VEGF treatment is at least about 5 days, at least about 6 days, at least as compared to central retinal thickness (CRT) or central subfield thickness (CST) at initial diagnosis in one and/or the contralateral eye. about 7 days, at least about 8 days, at least about 9 days, at least about 10 days, at least about 11 days, at least about 12 days, at least about 13 days, at least about 14 days, at least about 15 days, at least about 16 days, at least >30% (eg, at least 30%, at least 40%, at least 50%, at least 60%, at least of central retinal thickness (CRT) or central subfield thickness (CST) after about 17 days or more If a reduction of any of 70%, at least 80%, at least 90% or 100% is observed, the subject is subject to prior anti-VEGF treatment for ocular neovascular disease (eg, aflibercept, a functional variant thereof). or a functional fragment thereof). In some embodiments, about 7 days, about 10 days of anti-VEGF treatment compared to central retinal thickness (CRT) or central subfield thickness (CST) prior to administration of prior anti-VEGF treatment in one eye and/or the contralateral eye. ≥30% (eg, at least 30%, at least 40%, at least 50%, at least 60%, at least) of central retinal thickness (CRT) or central subfield thickness (CST) after more than one day or about 14 days If a reduction of any of 70%, at least 80%, at least 90% or 100% is observed, the subject is subject to prior anti-VEGF treatment for ocular neovascular disease (eg, aflibercept, a functional variant thereof). or a functional fragment thereof).

In some embodiments, the central subfield thickness and/or central retinal thickness is determined by SD-OCT in one eye and/or the contralateral eye. The central subfield thickness is the average thickness of the retina across the central subfield of the ETDRS grating, which is 1 mm diameter centered on the fovea.

In some embodiments, the individual has a central subfield thickness and/or central retinal thickness of ≧20% (e.g., 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 any of 100%) is observed. is determined to have a meaningful response to prior treatment with an anti-VEGF agent (eg, aflibercept, a functional variant thereof, or a functional fragment thereof). In some embodiments, the individual has at least about 5 days, at least about 6 days of administration of the prior treatment with the anti-VEGF agent as compared to central subfield thickness and/or central retinal thickness prior to administration of the prior treatment with the anti-VEGF agent. days, at least about 7 days, at least about 8 days, at least about 9 days, at least about 10 days, at least about 11 days, at least about 12 days, at least about 13 days, at least about 14 days, at least about 15 days, at least about 16 days ≥20% of central subfield thickness and/or central retinal thickness (e.g., 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 100%) of an anti-VEGF agent for ocular neovascular disease (eg, aflibercept, a functional variant thereof) or a functional fragment thereof). In some embodiments, the individual has about 7 days, about 10 days of administration of the prior treatment with the anti-VEGF agent, as compared to central subfield thickness and/or central retinal thickness prior to administration of the prior treatment with the anti-VEGF agent. or ≥20% of central subfield thickness and/or central retinal thickness after about 14 days (e.g., at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80 %, at least any of 90% or 100%), prior use of an anti-VEGF agent for ocular neovascular disease (eg, aflibercept, a functional variant thereof or a functional fragment thereof) determined to have a meaningful response to treatment. In some embodiments, the central subfield thickness and/or central retinal thickness is determined by SD-OCT in one eye and/or the contralateral eye. In some embodiments, if normalization of CST is observed without observable vascular effusion after anti-VEGF treatment in one eye and/or the contralateral eye, the individual is treated with an anti-VEGF agent for ocular neovascular disease (eg, ill libercept, a functional variant thereof, or a functional fragment thereof)). Normalization refers to CST values that are normal for that class of patients (eg, based on age, sex, etc.).

In some embodiments, central retinal thickness (CRT) or central subfield thickness (CST) prior to administration of anti-VEGF treatment (eg, aflibercept) as determined by SD-OCT in one eye and/or the contralateral eye ), anti-VEGF treatment at least about 5 days, at least about 6 days, at least about 7 days, at least about 8 days, at least about 9 days, at least about 10 days, at least about 11 days, at least about 12 days, at least >20% of central retinal thickness (CRT) or central subfield thickness (CST) (e.g., after about 13 days, at least about 14 days, at least about 15 days, at least about 16 days, at least about 17 days or more) For example, if a decrease in any of 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 100%) is observed, the subject is determined to have a meaningful response to prior anti-VEGF treatment for ocular neovascular disease (eg, aflibercept, a functional variant thereof, or a functional fragment thereof). In some embodiments, the anti-VEGF treatment compared to the central retinal thickness (CRT) or central subfield thickness (CST) prior to administration of the anti-VEGF treatment, as determined by SD-OCT in one eye and/or the contralateral eye. >20% (eg, at least 20%, at least 30%, at least 40%, at least 50% of central retinal thickness (CRT) or central subfield thickness (CST) after about 7 days, about 10 days, or about 14 days , at least 60%, at least 70%, at least 80%, at least 90% or any of 100%) is observed, the subject is subject to prior anti-VEGF treatment for ocular neovascular disease (e.g., ill libercept, a functional variant thereof or a functional fragment thereof).

In some embodiments, the individual has not received prior treatment for ocular neovascular disease. In some embodiments, the individual has not received prior treatment for ocular neovascular disease in one eye and/or the contralateral eye. In some embodiments, the individual has not received prior anti-VEGF treatment. In some embodiments, the individual has not received prior anti-VEGF treatment in one eye and/or the contralateral eye. In some embodiments, the individual has not received prior aflibercept treatment. In some embodiments, the individual has not received prior aflibercept treatment in one eye and/or the contralateral eye.

In some embodiments, the ocular disease or disorder treated according to the methods described herein is diabetic macular edema. Diabetic macular edema (DME) is a swelling of the retina in diabetes due to leakage of fluid from blood vessels within the macula. The macula is the central part of the retina, a small area rich in cones, special nerve endings that detect color, and rely on daytime vision. As macular edema develops, blurring occurs in the middle or just next to the central visual field. Vision loss due to diabetic macular edema may progress over a period of several months, making it impossible to focus clearly. Common symptoms of DME are blurred vision, floaters, and double vision, which, if left untreated, can eventually lead to blindness. In some embodiments, the methods and pharmaceutical compositions as disclosed herein are used to treat DME. In some embodiments, treatment of DME is assessed by measuring refraction or visual acuity (eg, BCVA using ETDRS letters). In some embodiments, visual acuity is measured starting at a distance of about 4 meters prior to dilation of one eye and/or the contralateral eye. In some embodiments, vision is considered maintained if an individual loses less than 15 characters on the ETDRS score after administration of a unit dose of rAAV particles compared to before administration of the unit dose of rAAV particles. In some embodiments, the treatment of DME is assessed by SD-OCT and/or OCT-A. In some embodiments, treatment of DME is assessed by measuring central subfield thickness and/or macular volume using SD-OCT. In some embodiments, the treatment of DME is administered after administration of a unit dose of rAAV particles by the number of treatments (eg, aflibercept rescue treatment) with an anti-VEGF agent (eg, aflibercept) administered. is evaluated In some embodiments, two centroids recorded prior to administration of the unit dose of rAAV particles (eg, on Day 1) and about 3 weeks after administration of the unit dose of rAAV particles (eg, at Week 4). If an increase in CST of >50 μm as assessed by SD-OCT occurs compared to the lower of the subfield thickness (CST) measurements, then at least one aflibercept rescue treatment is administered after administration of a unit dose of rAAV particles. is administered In some embodiments, two BCVAs recorded prior to administration of the unit dose of rAAV particles (eg, on Day 1) and about 3 weeks after administration of the unit dose of rAAV particles (eg, at Week 4). If a loss of >5 characters in BCVA occurs due to worsening of DME disease activity compared to the higher of the measurements, one or more aflibercept rescue treatments are administered following administration of a unit dose of rAAV particles. In some embodiments, the treatment of DME is assessed using the Diabetic Retinopathy Severity Scale (DRSS) compared to DRSS prior to administration of a unit dose of rAAV particles, eg, using ultra-wide color fundus imaging. In some embodiments, treatment of DME is assessed by development of a visual threatening complication (eg, anterior segment neovascularization, diabetic macular edema, high risk PDR development, vitreous hemorrhage, or traction retinal detachment). In some embodiments, the visual threat complication is assessed by ultra-wide imaging and clinical examination. In some embodiments, administering the unit dose of the rAAV particles to one and/or the contralateral eye of the individual results in a two- or three-step improvement in the diabetic retinopathy severity scale (DRSS). In some embodiments, administering the unit dose of the rAAV particles to one and/or the contralateral eye of the individual results in a two-step improvement in the diabetic retinopathy severity scale (DRSS). In some embodiments, administering the unit dose of the rAAV particles to one and/or the contralateral eye of the individual results in a three-step improvement in the diabetic retinopathy severity scale (DRSS).

In some embodiments, the ocular disease or disorder treated according to the methods described herein is retinal vein occlusion. Retinal vein occlusion is a blockage of the small veins that carry blood from the retina. The retina is a layer of tissue at the back of the inner eye that converts optical images into nerve signals and sends them to the brain. Retinal vein occlusion is most often caused by hardening of the arteries (atherosclerosis) and the formation of blood clots. Blockage of smaller veins within the retina (branch veins or BRVOs) often occurs where retinal arteries that are thickened or hardened by atherosclerosis cross and put pressure on the retinal veins. Symptoms of retinal vein occlusion may include sudden clouding or loss of vision in all or part of one eye.

In some embodiments, the ocular disease or disorder treated according to the methods described herein is choroidal neovascularization (CNV), also known as wet age-related macular degeneration (wAMD). Choroidal neovascularization may involve the growth of new blood vessels originating from the choroid into the subretinal pigment epithelium (sub-RPE) or subretinal space through disruption of Bruck's membrane, which may be a major cause of vision loss. CNV can cause a sudden deterioration of notable central vision within a few weeks. Other symptoms may include color disturbances and metamorphosis (distortion in which straight lines appear wavy). Bleeding of new blood vessels can accelerate the onset of symptoms of CNV. CNV may also include a feeling of tightness behind the eye.

The advanced "wet" form of AMD (neovascular or exudative) can frequently cause rapid and often substantial loss of central vision in patients. In the wet form of AMD, choroidal neovascularization forms and develops into a network of blood vessels that can grow under and through the retinal pigment epithelium. As this is accompanied by plasma leakage and/or hemorrhage into the subretinal space, central vision can be suddenly and severely lost when it occurs in the macula. The present disclosure contemplates the treatment or prevention of AMD, wet AMD. In some embodiments, the methods and pharmaceutical compositions as disclosed herein are used to treat AMD.

In some embodiments, the methods described herein include bevacizumab, brolucizumab, ranibizumab, parisimab, abicipar pegol, convercept, OPT-302, KSI-301, injectable sunitinib maleate (GB-102), PAN-90806 (Panoptica) and/or aflibercept for the prophylaxis or treatment of an ocular disease or disorder in a subject who has received prior treatment. In some embodiments, the methods described herein are used to prevent or treat an ocular disease or disorder that is responsive to treatment with bevacizumab, brolucizumab, ranibizumab, and/or aflibercept.

In some embodiments, the individual administers the unit dose of the rAAV particles to one eye and/or the contralateral eye for at least 1 day, at least 1 week, at least 1 month, at least 2 months, at least 4 months, at least 6 months, at least 12 months. , at least 18 months, at least 24 months, at least 30 months, at least 36 months, at least 42 months, at least 48 months, at least 54 months, at least 60 months, at least 66 months, at least 72 months, at least 78 months, at least 84 months, at least was diagnosed with ocular neovascular disease at least 90 months, 96 months, at least 102 months, at least 108 months, at least 114 months, at least 120 months, at least 126 months, at least 132 months or more.

The following description is presented to enable any person skilled in the art to make and use the various embodiments. Descriptions of specific devices, techniques, and applications are provided by way of example only. Various modifications to the embodiments described herein will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments and applications without departing from the spirit and scope of the various embodiments. Accordingly, the various embodiments are not intended to be limited to the examples described and presented herein, but are to be accorded the scope consistent with the claims.

Example

Example 1: An open-label phase 1 study of AAV2.7m8-aflibercept at a dose of 6×10 ¹¹ vg/eye in neovascular (wet) age-related macular degeneration.

This example describes AAV2.7m8, a rAAV vector containing the VEGF inhibitor aflibercept and AAV2.7m8 protein capsid, for the treatment of age-related macular degeneration (AMD) with choroidal neovascularization (wet AMD; wAMD). -Describe an open-label phase 1 study of aflibercept.

I. Research Purpose

A. Primary purpose

The primary objective of this study was to evaluate the safety and tolerability of a single intravitreal (IVT) injection of AAV2.7m8-aflibercept in subjects with wAMD.

primary endpoint

The primary endpoints of this study were the type, severity, and incidence of ocular and systemic adverse events (AEs).

B. Secondary purpose

The secondary objectives of this study were:

● To evaluate the effect of AAV2.7m8-aflibercept on maximum corrected visual acuity (BCVA).

● To evaluate the effect of AAV2.7m8-aflibercept on central subfield thickness (CST), also known as central retinal thickness (CRT).

● To assess the need for rescue therapy from Weeks 4 to 104.

● To evaluate the effect of AAV2.7m8-aflibercept on the presence of intraretinal fluid (IRF) and subretinal fluid (SRF).

● To evaluate the effect of AAV2.7m8-aflibercept on the resolution of PED in patients with pigmented epithelial detachment (PED) at baseline.

secondary endpoint

The secondary endpoints of this study were:

● Mean change in BCVA from baseline over time, assessed by the ETDRS character over time, from Day 8 to Week 104 compared to baseline.

● Percentage of subjects with a BCVA gain of ≧15 ETDRS characters from baseline over time, assessed from Day 8 to Week 104 compared to baseline.

● Percentage of subjects with a decrease in BCVA of ≤15 ETDRS characters from baseline over time, assessed from Day 8 to Week 104 compared to baseline.

• Mean change in CST and macular volume from baseline over time, assessed from Day 8 to Week 104 compared to baseline.

● Mean number of aflibercept injections over time, assessed from week 4 to week 104.

• Percentage of subjects requiring injections of aflibercept over time, assessed from week 4 to week 104.

● Percentage of subjects without IRF over time, assessed from Day 8 to Week 104.

● Percentage of subjects without SRF over time, assessed from Day 8 to Week 104.

● Percentage of subjects with PED at baseline, assessed from Day 8 to Week 104, without PED over time.

II. study subject

The subjects in this study were diagnosed with active choroidal neovascularization (CNV) secondary to age-related macular degeneration (AMD), had a history of recent responsiveness to anti-VEGF therapy, and received frequent injections of anti-VEGF therapy. was needed

Only one eye was selected as the study eye for the duration of the study. If both eyes met all inclusion and exclusion criteria, the eye with the worst BCVA assessed at screening was selected as the study eye. If both eyes meet all inclusion and exclusion criteria and the BCVA values are the same for both eyes, the subject chooses to select his/her non-predominant eye for treatment, or chooses the right eye as the study eye by default did

A. Inclusion Criteria

Subjects meeting the following inclusion criteria were enrolled in this study:

● Male or female subjects, age ≥ 50 years.

The study eye had the following evidence, along with previous or current evidence, that active subfoveal CNV secondary to AMD occupies >50% of total lesion size:

○ Leakage in fluorescein angiography (FA), subretinal hemorrhage in fluid or color fundus photographs on spectral domain optical coherence tomography (SD-OCT); and

○ The overall dimension of the lesion did not exceed the area of the 12 macular photocoagulation study discs.

● Subjects were on active anti-VEGF treatment for wAMD with at least 2 injections within 4 months prior to screening.

● Vision of study eye at screening visit (prior to aflibercept injection):

○ 78-25 BCVA ETDRS.

● Vision of the non-study eye:

○ ≥35 BCVA ETDRS.

● showed a significant anti-VEGF response identified by the clinical investigator and defined as follows:

o reduction of ≥ 30% in central subfield thickness from initial diagnosis as assessed using SD-OCT; or

o reduction of ≥ 20% of central subfield thickness from screening as assessed using SD-OCT; or

○ Normalization of CST without observable vascular effusion.

The VEGF reactivity was confirmed by the clinical investigator for the purpose of confirming the anti-VEGF response at the first visit before administration of AAV2.7m8-aflibercept. Subjects determined not to have a significant anti-VEGF response were not screened and were not enrolled in this study.

B. Exclusion Criteria

Subjects meeting the following exclusion criteria were not enrolled in this study:

neutralizing antibody

• Although there is no established correlation between the levels of neutralizing antibodies (NAb) against AAV in serum and vitreous humor (Lukason et al., (2011) Mol Ther 19 (2):260-265), AAV2 as a precaution. Subjects were screened for NAb against the 7m8 vector. Subjects with anti-AAV2.7m8 neutralizing antibody titer levels > 1:125 recorded within 6 months prior to administration of AAV2.7m8-aflibercept were excluded from this study.

CNV lesions

● Known history or evidence of the following CNV lesion features:

○ Fibrosis or atrophy, rupture of the retinal epithelium in the fovea in the study eye, or any condition that prevents vision improvement.

○ Scarring or fibrosis constituting >50% of the total lesion area.

○ Lesion size including blood, scarring and neovascularization as assessed by fluorescein angiography (FA) > 12 macular photocoagulation study disc areas (30.5 mm ² ).

○ Subretinal hemorrhage ≥ 50% of the total lesion area, or the presence of subfoveal blood, an area ≥ 1 disc in size in the study eye (if subfoveal blood is present, the fovea is surrounded by 270 degrees by visible CNV) .

Eye condition (retina/posterior eye)

● History of significant preretinal or vitreous macular traction (VMT) syndrome in the study eye at the time of administration of AAV2.7m8-aflibercept, or full-thickness macular foramen (Gass stage 2 or greater) in the study eye.

● With diabetic retinopathy (in either eye), retinal vein occlusion, uveitis, suspected retinal hemangioma hyperplasia, polypoid choroidopathy or other causes (eg ocular histoplasmosis, trauma or pathological myopia). History of retinal disease other than wAMD in the study eye, including CNV due to or any other vascular disease in the eye (a benign condition of the vitreous or peripheral retina is non-exclusive).

● History of retinal detachment (with or without repair) in the study eye.

Other conditions (non-retinal)

● Known history or evidence of significant non-retinal disease or media opacification in the study eye that may impair vision during the course of the study, requiring surgery and/or interfering with proper visualization or imaging of the retina (eg, central corneal scarring, significant cataracts, corneal dystrophy, scleramalacia).

● Uncontrolled ocular hypertension or glaucoma (defined as intraocular pressure [IOP] > 22 mmHg despite treatment with anti-glaucoma medications) in the study eye at the time of AAV2.7m8-aflibercept administration or > at the time of screening Use of two IOP lowering medications.

• Activity or history of ocular or periocular infection in either eye within 4 weeks prior to administration of AAV2.7m8-aflibercept.

● Eye surgery/procedure

• Any previous intraocular or periocular surgery on the study eye within 6 months of administration of AAV2.7m8-aflibercept or any planned major surgical procedure within 6 months of administration of AAV2.7m8-aflibercept. Blepharoplasty > 1 month after AAV2.7m8-aflibercept was not an exclusion criterion.

● History of vitrectomy, trabeculectomy, or other filtration surgery in the study eye.

● Yttrium aluminum garnet (YAG) retrocapsulectomy within 3 months prior to AAV2.7m8-aflibercept.

• Any prior treatment with photodynamic therapy or retinal laser and any prior treatment radiation for treatment of wAMD in the study eye area.

General/systemic conditions

● Unless otherwise specified, history or evidence of any of the following cardiovascular diseases within 6 months of administration:

○ History of severe heart disease (eg, New York Heart Association [NYHA] functional class III or IV) or clinical evidence of unstable angina.

○ Acute coronary syndrome, myocardial infarction or coronary revascularization.

○ Ventricular arrhythmias or uncontrolled arrhythmias requiring ongoing treatment.

○ Uncontrolled hypertension, defined as mean systolic blood pressure (SBP) ≥160 mmHg or mean diastolic blood pressure (DBP) ≥100 mmHg, despite the use of BP-lowering medications within the pre-dose screening period.

○ History of cerebrovascular accident or transient ischemic attack.

● Any history of ongoing bleeding disorder or International Normalized Ratio (INR) >3.0. Use of aspirin or other anticoagulants (eg, factor Xa inhibitors) was not an exclusion criterion. The INR was repeated during the screening period to confirm that the eligibility criteria were met.

● Evidence of uncontrolled diabetes with HbA1c >7.0% within the screening period prior to administration of AAV2.7m8-aflibercept.

● History of malignancy within the past 5 years, except for:

○ Local basal cell or squamous cell carcinoma of the skin.

○ Intraepithelial carcinoma of the cervix or breast.

○ Papillary, non-invasive bladder cancer.

○ Stage I and II prostate cancer clinically indicated by stable prostate-specific antigen (PSA) when observed for 6 months.

○ Any other cancer that has been in complete remission for at least 2 years or has been considered cured by surgery.

● Positive HIV, hepatitis B or hepatitis C (unless treated with documented remedies).

Evidence of any type of systemically active infection considered clinically significant by the investigator based on clinical examination and/or temperature >38.5°C within 36 hours prior to administration of AAV2.7m8-aflibercept; or doubt.

● Known severe allergies to:

○ Fluorescein dye or sodium fluorescein used for angiography (treatable mild allergies are acceptable); or

○ Aflibercept.

● Women who are pregnant, breastfeeding or planning to become pregnant during the study.

● Other significant laboratory abnormalities or medical conditions that compromised the safety of the subject from the investigator's point of view.

medicine

• Use of systemic anti-inflammatory steroids or immunosuppressive medications (other than protocol-specified prednisone) within 5 half-lives prior to administration of AAV2.7m8-aflibercept. Inhaled or topical steroids and nonsteroidal anti-inflammatory drugs (NSAIDs) were permitted.

● Received any of the following:

○ Investigational drug within 30 days or 5 half-lives (whichever is longer) prior to administration of AAV2.7m8-aflibercept.

○ Prior gene therapy.

III. Investigational drugs

AAV2.7m8-aflibercept, an investigational medicinal product (IMP), was a recombinant replication-deficient adeno-associated virus (rAAV) vector containing an AAV2.7m8 protein capsid derived by directed evolution in vivo to the AAV2 capsid ( Dalkara et al., (2013) Sci Transl Med 5(189):189ra76; US2014/0364338). AAV2.7m8-aflibercept carried the expression cassette of a codon-optimized version of the aflibercept cDNA under the control of a ubiquitous chimeric promoter ( FIG. 1A ) (see WO2018170473A1). AAV2.7m8-aflibercept was developed in Sf9 cells using two different baculoviruses, one encoding genes for the AAV2 Rep and AAV2.7m8 Cap proteins and the other encoding the human aflibercept cDNA expression cassette. It was prepared by using a baculovirus expression vector system.

AAV2.7m8-aflibercept was supplied as a sterile-filtered frozen suspension in sterile ready-to-use 0.5 mL crystal Zenith vials containing 0.25 mL of IMP formulated as shown in Table 1.

Table 1: AAV2.7m8-aflibercept investigational drug formulation for Cohort 1.

IV. study design

A. Dosage and method of administration

cohort 1

Subjects in Cohort 1 received a single IVT injection of AAV2.7m8-aflibercept 6×10 ¹¹ Administered in an injection volume of 100 μL at a dose of 1 vg/eye.

AAV2.7m8-aflibercept vials were removed from the freezer storage at ≤ -60°C and thawed at room temperature. AAV2.7m8-aflibercept was administered via IVT injection. Aseptic technique with povidone-iodine under local or subconjunctival anesthesia was used. Post-injection care and medication regimens were provided based on trial standard care.

B. Study Visit

Screening (Day-15 to Day -7)

As shown in FIG. 1B , subjects received a single IVT injection of 2 mg of aflibercept on days -15 to -7 (eg, days -14 to -7) during screening, which is the standard consistent with management. Subjects received routine clinical trial standard post-injection care.

Study Day 1

On study day 1 (7-15 days after IVT injection of aflibercept, eg 7-14 days), study subjects indicated that they were responsive to anti-VEGF therapy prior to administration of AAV2.7m8-aflibercept. An SD-OCT study was performed to confirm that Anti-VEGF reactivity was confirmed by clinical investigators. As described above (see inclusion criteria), only subjects identified as having a meaningful anti-VEGF response were eligible for enrollment in this study.

Subjects responsive to anti-VEGF therapy were sequentially enrolled in the study cohort, who received a single AAV2.7m8-aflibercept IVT injection in the study eye. As described above, only one eye was selected as the study eye for the duration of the study.

After administration of AAV2.7m8-aflibercept

Subjects will undergo clinical evaluation and treatment (if necessary) on Days 3 and 8, during Weeks 2, 4, 6 and 8 and every 4 weeks thereafter (i.e., Week 12, Weeks 16, 20 and 24) returned. A safety and efficacy analysis of Cohort 1 was performed at Week 24.

Beginning at Week 4, subjects were eligible to receive a rescue injection of aflibercept 2 mg IVT if there was evidence of increased disease activity per retreatment criteria (see below). The presence of any of the following required resumption of standard anti-VEGF treatment with 2 mg IVT aflibercept:

● Loss of ≥ 10 characters of BCVA from baseline (using the ETDRS protocol) and intraretinal or subretinal fluid observed by SD-OCT and determined to be the cause of BCVA loss by the clinical investigator.

● Increase in central subfield thickness > 75 μm from baseline as assessed by SD-OCT.

● Presence of vision-threatening bleeding due to macular degeneration.

Subjects were re-evaluated for safety and efficacy at week 52 following administration of AAV2.7m8-aflibercept. The follow-up period continues until week 104 after administration of AAV2.7m8-aflibercept.

C. Corticosteroid Therapy

To reduce the possible risk of post-injection ocular inflammation, subjects are administered prophylactic corticosteroid therapy (eg, prednisone) and closely monitored for ocular and systemic tolerability of the vector.

Subjects in Cohort 1 received prophylactic 13-day oral corticosteroid therapy starting with prednisone at 60 mg/day for a total of 6 days from 3 days before AAV2.7m8-aflibercept treatment (days -3) to 3 days after treatment with AAV2.7m8-aflibercept. administered. After that, the prednisone tapering was continued for 7 days. A summary of oral prednisone therapy is provided in Table 2.

Table 2: Oral prednisone therapy.

The immune response was limited upon exposure to capsid antigen by designing to initiate immunosuppression (ie, prednisone) prior to AAV2.7m8-aflibercept IVT injection. Subjects self-administered prednisone for a 13-day regimen.

Subjects received topical or oral corticosteroids (prednisone) as needed during Weeks 2-24 of the study.

D. Prohibited Medications and Treatments

The following medications were contraindicated during the study:

● Any systemic anti-VEGF agent, including bevacizumab.

• Any anti-VEGF agent in the study eye other than study drug or aflibercept injection 2 mg, depending on rescue anti-VEGF injection criteria in this study.

● IVT steroids in the study eye.

● Immunosuppressive drugs. Systemic, inhaled or topical steroids and NSAIDs were permitted.

● Use and participation in any other investigational studies.

● Cataract surgery in the study eye can be performed if clinically present and scheduled >90 days post IVT administration and/or >7 days post last injection of aflibercept.

● Subjects who develop AMD in the non-study eye may receive standard of care therapy in the non-study eye.

E. Summary of Study Design for Cohort 1

Six subjects were enrolled in Cohort 1.

Subjects in Cohort 1 received a single IVT injection of AAV2.7m8-aflibercept 6×10 ¹¹ Administered at a dose of 1 vg/eye. The first (surveillance) subject enrolled in Cohort 1 received an IVT injection of AAV2.7m8-aflibercept, which was assessed for 29 days before being dosed to subsequent 5 subjects in the cohort (Subjects 2-6).

A summary of the study design for Cohort 1 is provided in Table 3.

Table 3: Summary of study design for Cohort 1.

F. Study Duration

The duration of subject participation in this study is approximately 108 weeks for each subject. This includes a 4 week screening period and an additional 104 week study period.

Upon completion or discontinuation of the study, where appropriate, subjects are provided with the opportunity to enroll in a long-term follow-up study to further evaluate the safety of such gene therapy.

V. Study Evaluation

A. General Physical Examination and Vital Signs

Relevant medical and ophthalmic history of each subject was collected and recorded. A general physical examination consisted of height (screening only), weight and vital signs.

Vital signs consisted of blood pressure, pulse rate measurements, body temperature and respiration rate. A 12-lead electrocardiogram (ECG) was taken for each subject. The following clinical laboratory and antibody tests were performed: chemistry, complete blood count (CBC), coagulation studies, urinalysis, serological evidence of HIV or hepatitis and pregnancy tests.

At the Week 104, End of Study (EOS) and/or Early Termination visit, a physical will evaluate whether any changes have occurred in the subject's physical condition since the screening examination.

B. Immune Response and Aflibercept Expression

Total anti-AAV2.7m8 antibody was measured. A reporter gene-based transduction interference assay assessed by cutpoints was used to determine neutralizing anti-AAV2.7m8 antibodies in the subject's serum.

An ELISA-based cutpoint antibody assay was used to measure the humoral immune response to anti-aflibercept antibodies in serum.

Serum was collected to determine the presence of aflibercept protein.

Cellular immunity to the AAV2.7m8 capsid protein and aflibercept protein was determined using the ELISPOT assay.

C. Full Ophthalmic Examination and Other Assessment Methods

Study evaluations included ophthalmologic examination, intraocular pressure (IOP) and indirect ophthalmoscopy.

Ophthalmic examinations include external examination of the eye and appendages, commercial screening and slitting for ptosis/pupillary reactivity (including, but not limited to, ptosis, abnormal pupil shape, unequal pupil, abnormal response to light and afferent pupil defect). -Lamp examination (eyelid, conjunctiva, cornea, lens, iris, anterior chamber) was done. The slit-lamp examination examined anterior ocular structures and was used to grade any findings. If any observations were noted during the slit-lamp examination, at any visit, severity was graded by the Investigator and the observation was described as clinically significant or not clinically significant.

IOP measurements were performed using a Goldman applanation tonometer or Tono-Pen™. IOP measurements were performed prior to any IVT injections and prior to eye dilatation. The Day 1 visit required IOP measurements pre- and post-injection (30 min post-injection).

Extended indirect ophthalmoscopy included evaluation of posterior segment abnormalities of the vitreous, optic nerve, peripheral retina, and retinal vasculature. If any findings were noted during ophthalmoscopy, at any visit, severity was rated by the investigator and the findings were described as clinically significant or not clinically significant. The Day 1 visit required pre- and post-injection indirect ophthalmoscopic evaluations.

Spectral domain optical coherence tomography (SD-OCT)

Depth-resolved tissue structure information encoded by the magnitude and delay of back-scattered light was obtained by spectral analysis of the interference fringe pattern using SD-OCT.

fluorescein angiography

Fluorescein angiographic imaging was used to confirm patient eligibility for study enrollment, to evaluate the efficacy of CNV lesion growth, and to assess leakage compared to baseline.

digital color fundus photography

Color fundus images of the retina, optic disc, and macula were taken.

Optical coherence tomography angiography (OCT-A)

OCT-A imaging (swept-source or spectral-domain) was used to obtain information on blood flow as well as volumetric three-dimensional maps of the retina and choroid.

refraction and vision

Refraction and BCVA were measured by a trained and certified optometrist at the study site. Visual acuity was measured at a starting distance of 4 meters prior to eye dilation.

D. Safety Assessment

To mitigate the risks associated with IVT administration of AAV2.7m8-aflibercept, subjects were closely monitored on the day of IVT AAV2.7m8-aflibercept administration and after treatment thereafter.

The safety of AAV2.7m8-aflibercept was assessed through collection of AEs, vital signs, physical and eye exams, ECG, pregnancy tests, and laboratory evaluations.

Intensive monitoring of subjects was performed in the first 8 weeks of the study, followed by regular safety assessments for safety and efficacy thereafter. All subjects had their visual acuity tested by the Early Treatment Diabetic Retinopathy Study (ETDRS) character assessment at each study visit, and standard care aflibercept IVT injection was used as rescue treatment.

The severity or intensity of AEs was graded on the following scale:

● Mild: AEs were noteworthy, but did not significantly impair the subject's daily activities.

● Moderate: AE reduced or impaired normal daily activities, but did not neutralize it.

● Severe: AE disables and prevents performance of normal daily activities.

Safety is assessed over 104 weeks following administration of study treatment. Upon completion of the end-of-study (EOS) visit, it is proposed that subjects enroll in a long-term extension study to further evaluate the safety and persistence of transgene expression.

E. Efficacy Assessment

The efficacy of AAV2.7m8-aflibercept in the treatment of wAMD was assessed by the following scale. The main evaluation time point was 24 weeks. Baseline values of BCVA and SD-OCT refer to values taken during the screening visit on days-15 to -7 (eg, days-14 to -7) prior to aflibercept injection. For analysis, baseline values were used for comparison.

Vision was assessed primarily via BCVA, expressed as an ETDRS score (number of characters read correctly) (Vitale et al., (2016) JAMA Ophtalmol 134(9):1041-1047). Subjects were classified as visual retention if they lost less than 15 characters on the ETDRS score compared to baseline. Calculated endpoints included mean change from baseline, percent gain of at least 15 characters compared to baseline, and percent loss of at least 15 characters compared to baseline.

Leakage was assessed compared to baseline by performing FA to assess CNV lesions using standard techniques.

Retinal thickness (e.g., central retinal thickness or central subfield thickness), macular volume and fluid (e.g., subretinal fluid and retinal) compared to baseline values by performing SD-OCT using approved equipment and standard techniques. the presence of internal fluid).

The number of aflibercept injections given following AAV2.7m8-aflibercept treatment per subject over time from week 4 to week 104 was determined. Additionally, the time to first aflibercept injection following treatment with AAV2.7m8-aflibercept and the proportion of subjects who did not require aflibercept rescue treatment were determined.

The proportion of subjects without IRF over time from week 4 to week 104 was determined.

The proportion of subjects without SRF over time from week 4 to week 104 was determined.

Among subjects with PED at baseline, the proportion of subjects without PED over time from week 4 to week 104 was determined.

F. Statistics

The safety population included all subjects who received AAV2.7m8-aflibercept, and they were analyzed according to the dose received.

All other safety parameters were summarized by cohort. The Medical Dictionary for Regulatory Activities (MedDRA, version 21) classification was used to encode the AEs to provide the preferred term (PT) and organ system classification (SOC) for each event. SAEs and AEs leading to study withdrawal were listed separately.

Efficacy analyzes included all subjects. Efficacy analysis endpoints will be assessed and descriptive statistics will be calculated per cohort. The main evaluation time point was 24 weeks. Efficacy was assessed according to dose received and aggregated.

VI. result

A. Subject Characteristics

All 6 subjects enrolled in Cohort 1 were diagnosed with wAMD. At the time of enrollment, subjects had high requirements for anti-VEGF treatment (eg, requiring frequent anti-VEGF treatment), a functional visual field of approximately 20/50, some excessive central subfield thickness on OCT, - was receiving regular IVT injections of VEGF treatment and responded to therapy. Disease characteristics and treatment histories for all subjects are provided in Tables 4-5.

As shown in Table 4, subjects in Cohort 1 were diagnosed with wAMD between approximately 1 year (Subject 5) and at least approximately 10 years (Subject 4) prior to administration of AAV2.7m8-aflibercept. Subjects received a wide range of prior anti-VEGF IVT injections in the study eye from 7 (Subject 5) to 109 (Subject 4) prior injections. Mean anti-VEGF IVT injection intervals calculated prior to this study ranged from every 4 weeks to every 10 weeks. All subjects received 2 or 3 anti-VEGF injections within 4 months prior to screening for enrollment in this study. Subjects were administered AAV2.7m8-aflibercept either 7 days (subjects 1-3) or 14 days (subjects 4-6) post screening anti-VEGF injection prior to study start.

Table 4: Baseline characteristics of subjects in Cohort 1.

Table 5: Disease characteristics and treatment history of study subjects in Cohort 1.

B. Safety

During 24 weeks after administration of AAV2.7m8-aflibercept, no SAEs occurred and no AEs met the criteria for dose limiting toxicity (DLT). No drug-related non-ocular AEs were also observed. Ocular inflammation was observed in all subjects and was manageable with topical steroids. In addition, no vasculitis, retinitis or choroiditis were observed. 19 ocular AEs considered potentially related to AAV2.7m8-aflibercept were observed; 14 were mild and 5 were moderate (2 AEs were intermediate uveitis, 1 AE was vitreous cells, and 2 AEs were anterior cells). One patient had 2 anterior cell events (1 mild event and 1 moderate event). Mild to moderate intraocular inflammation responsive to topical or oral corticosteroids was frequently observed during initial follow-up. OCT images showed persistent fluid release in most subjects, and there were no signs of exacerbation. Visual acuity was generally stable. A summary of all safety events related to AAV2.7m8-aflibercept up to week 24 is provided in Table 6.

Table 6: All safety events related to AAV2.7m8-aflibercept.

All subjects received oral prednisone at 60 mg for 6 days starting on day -3, followed by a 7 day tapering course of prednisone. Clinical evaluation of ocular cell inflammation revealed that no clinically significant inflammation occurred early after AAV2.7m8-aflibercept. Additionally, as shown in FIG. 6 , no exacerbation or new inflammation was observed when subjects received steroid eye drops. By week 24, anterior cell inflammation resolved or improved. The observed cellular inflammation was generally mild. The aqueous humor cell count category presented in Figure 6 was based on standardized (SUN) criteria for uveitis nomenclature (Jabs, DA et al., J Ophthalmol. 2005;140:509-516), whereas the vitreous cell count category was determined by the National Institutes of Health (NIH). It was based on guidelines.

Taken together, the safety assessment showed that AAV2.7m8-aflibercept was well tolerated and no DLTs or SAEs were reported. All safety events were mild to moderate, and most were inflammation-related. Continued AE follow-up showed that mean visual acuity remained stable and no anti-VEGF rescue injection was required. Thus, the results indicate that AAV2.7m8-aflibercept administered as a single IVT injection at a dose of 6x10 ¹¹ vg/eye to wAMD patients who previously required frequent anti-VEGF injections has an acceptable safety profile.

C. Efficacy

After AAV2.7m8-aflibercept administration, all 6 subjects showed stabilization of disease activity based on OCT assessment. As shown in FIGS. 2A-2L , OCT images taken before and after treatment with AAV2.7m8-aflibercept showed that a robust anatomical response was evident in all 6 subjects in Cohort 1. For example, subject 1 (eg, compare FIGS. 2A and 2B ), subject 2 (eg, compare FIGS. 2C and 2D ), subject 3 (eg, compare FIGS. 2E and 2F ) ), in Subject 4 (eg, compare FIGS. 2G and 2H ) and Subject 5 (eg, compare FIGS. 2I and 2J ), subretinal fluid persisted on standard-of-care anti-VEGF treatment, but AAV2. It resolved after administration of 7m8-aflibercept and remained resolved. Subject 6 showed retinal morphology consistent with polypoid choroidal angiopathy (PCV) ( FIG. 2K ), some fluid persisted after treatment with AAV2.7m8-aflibercept, but no evidence of disease progression ( FIG. 2L ). ). Additionally, as shown in FIG. 3 , none of the subjects showed an increase in central subfield thickness (CST), and a mean decrease of −52.7 μm was observed (90% CI −86.5, −18.8). Increased CST is an indicator of disease progression in wAMD. Importantly, none of the subjects received or required standard care aflibercept rescue injection (“rescue injection”).

Maximum corrected visual acuity (BCVA) was measured based on the ETDRS letter assessment in all subjects throughout the study. As shown in Figure 4, BCVA was stable across subjects in Cohort 1 with a mean -2 letter reduction (90% CI -9.1, 5.1).

At a median follow-up time of 34 weeks after administration of AAV2.7m8-aflibercept, no subjects showed signs of disease reactivation on OCT imaging ( FIGS. 7A-7B ). Additionally, no subjects required any rescue anti-VEGF IVT injections, and no subjects met retreatment criteria at any time point during the follow-up period up to 44 weeks. Finally, for an additional follow-up period after week 24, BCVA was maintained (ie, no patient lost or acquired more than 10 ETDRS letters) and the anatomical improvement observed at week 24 (ie, retinal resolution of subretinal and intraretinal fluids and reduction of CST) were also maintained, and no safety concerns occurred.

A summary of safety and efficacy outcomes evaluated at a median follow-up time of 34 weeks is provided in Table 7. A summary of efficacy outcomes assessed at a median follow-up time of 44 weeks is provided in Table 8. One patient experienced a macular-separated retinal detachment not related to study treatment. For that patient, the CST and BCVA mean values and ranges presented in Table 8 were calculated using the last observation before dissection.

Table 7: Summary of safety and efficacy results at a median follow-up time of 34 weeks.

Table 8: Summary of efficacy results at a median follow-up time of 44 weeks.

A summary of further analyzes of efficacy outcomes at a median follow-up time of 44 weeks (range 40-52 weeks) following administration of AAV2.7m8-aflibercept is provided in Table 9.

Table 9: Summary of efficacy results at follow-up times of 40-52 weeks (median 44 weeks).

Cellular inflammatory events and steroid treatment given to each subject were analyzed up to a follow-up time of 52 weeks. As shown in FIG. 8 , subjects 1 and 5 had no cellular responses after day 3. Both subjects began topical steroids at the second 6 months of the study for aqueous humor cell (AC) clouding and posterior adhesions. Both subjects received topical steroids and dilated eye drops, and subject 1 stopped receiving topical steroid eye drops. Subjects 2 and 6 showed early onset inflammation by the first month after administration of AAV2.7m8-aflibercept, with AC response and some leakage into vitreous cells. Inflammation in both subjects resolved after treatment with topical steroids. Both subjects continued to use eye drops twice daily (BID). Subjects 3 and 4 experienced a more delayed onset of cellular inflammation after topical steroids were discontinued (up to 2 to 3 months after administration of AAV2.7m8-aflibercept). Inflammation in these subjects was responsive to topical steroids. Subject 3 stopped receiving topical steroid eye drops. Subject 4 experienced an unrelated retinal detachment and is receiving BID eye drops.

Overall, inflammation was low-grade, and no early clinically significant inflammation was reported. All inflammations were responsive to topical steroids and tended to resolve over time. Inflammation was mainly induced by aqueous humor cellular processes.

9A-9B show OCT imaging of subjects 1-6 from week 40 to week 52 of follow-up. No subjects showed signs of disease reactivation on OCT imaging, and BCVA and anatomical improvements were maintained. Additionally, no subjects required any rescue anti-VEGF IVT injections or met retreatment criteria.

D. Conclusion

The current standard of care regimen for wAMD is anti-VEGF IVT injections, typically long-term required, about every 4-8 weeks. Compliance with these regimens can be difficult for patients, caregivers, and healthcare systems, leading to loss of vision due to suboptimal dosing and undertreatment.

The results presented in this example show that AAV2.7m8-aflibercept provided a clear benefit in improving retinal anatomy and stabilizing vision while exhibiting an acceptable safety profile. Specifically, patients maintained vision during the study, AAV2.7m8-aflibercept was shown to be safe and well tolerated, and the observed inflammation was generally mild and responsive to steroid eye drops.

Although subjects in Cohort 1 of this study previously required frequent anti-VEGF injections to slow or prevent progression of their disease (Table 4), anti-VEGF after administration of AAV2.7m8-aflibercept during this study. It was noted that no rescue injection was required.

It was noted that subretinal fluid was present on OCT scans of subjects receiving standard of care anti-VEGF treatment for >20 weeks and continued 1-2 weeks after screening aflibercept IVT injection. This fluid was present even though the subject was treated repeatedly and was therefore refractory to an IVT bolus of standard of care anti-VEGF protein. Unexpectedly, this refractory SRF resolved after treatment with AAV2.7m8-aflibercept, an outcome not expected from preclinical studies.

The observed safety and efficacy of AAV2.7m8-aflibercept administered at a dose of 6x10 ¹¹ vg/eye was similar to that of AAV2.7m8-aflibercept administered at lower doses with topical corticosteroids as described in Example 2. Continued this phase 1 study to evaluate safety and efficacy.

Example 2: An open-label phase 1 study of AAV2.7m8-aflibercept at doses lower than 6×10 ¹¹ vg/eye with topical corticosteroids in neovascular (wet) age-related macular degeneration.

The following examples are of the phase 1 study described in Example 1 to evaluate the safety and efficacy of AAV2.7m8-aflibercept administered at doses lower than 6x10 ¹¹ vg/eye in combination with topical corticosteroids in subjects with wAMD. Write a continuation.

I. Study Objectives and Endpoints

Primary objectives, secondary objectives, and primary and secondary endpoints were as described in Section I of Example 1.

II. study subject

The study subjects in this study were as described in Section II of Example 1.

III. Investigational drugs

The investigational drug is AAV2.7m8-aflibercept as detailed in Section III of Example 1 and as shown in Figure 1a, with the exception of AAV2.7m8 to maintain a suitable injection volume as shown in Table 10 below. - The concentration of aflibercept was varied.

Table 10: AAV2.7m8-aflibercept investigational drug formulations for cohorts 2-4.

IV. study design

A. Dosage and method of administration

The doses described below of AAV2.7m8-aflibercept were administered as described in Section IV of Example 1.

Study Cohorts 2-4

Cohort 2: Six subjects diagnosed with wAMD are enrolled in Cohort 2. Subjects in Cohort 2 will receive a single IVT injection of AAV2.7m8-aflibercept with prophylactic oral prednisone therapy at a dose of 2 of 2×10 ¹¹ vg/eye.

Cohort 3: Nine subjects diagnosed with wAMD are enrolled in Cohort 3. Subjects in Cohort 3 will receive a single IVT injection of AAV2.7m8-aflibercept with prophylactic topical corticosteroid therapy at a dose of 2 of 2×10 ¹¹ vg/eye.

Cohort 4: Nine subjects diagnosed with wAMD are enrolled in Cohort 4. A single IVT injection of AAV2.7m8-aflibercept with topical corticosteroid therapy to subjects in Cohort 4 when signs of choroidal neovascularization effusions requiring rescue therapy are observed in the majority of subjects in Cohorts 2 and 3 is administered at a dose 3 of 6x10 ¹⁰ vg/eye (Cohort 4b). Single IVT of AAV2.7m8-aflibercept with topical corticosteroid therapy to subjects in Cohort 4, if no signs of choroidal neovascularization effusions requiring rescue therapy were observed in the majority of subjects in Cohorts 2 and 3 Injections are administered at dose 1 of 6x10 ¹¹ vg/eye (Cohort 4a).

B. Study Visit

Study visits are as described in Section IV of Example 1 and in FIG. 1B .

C. Corticosteroid Therapy

Subjects in Cohort 2 are administered a prophylactic 13-day oral corticosteroid regimen as detailed for the cohort in Example 1:

Cohort 2: Subjects in Cohort 2 were treated with AAV2.7m8-aflibercept 3 days before and 3 days after AAV2.7m8-aflibercept with prophylactic 13-day oral corticosteroid therapy starting with 60 mg prednisone for a total of 6 days. A single IVT injection of Sept is administered at a dose of 2 of 2 x 10 ¹¹ vg/eye. This is followed by a 7-day prednisone tapering. A summary of oral prednisone therapy for Cohort 2 is provided in Table 2 of Example 1.

Subjects in Cohorts 3 and 4 are administered a prophylactic tapering regimen of topical corticosteroids (difluprednate 0.05% eye drops) as described below:

Cohort 3: Subjects in Cohort 3 will receive a single IVT injection of AAV2.7m8-aflibercept with prophylactic topical 4-week tapering corticosteroid therapy at a dose of 2 of 2×10 ¹¹ vg/eye.

Cohort 4: Subjects in Cohort 4b will receive a single IVT injection of AAV2.7m8-aflibercept at dose 3 of 6x10 ¹⁰ vg/eye with prophylactic topical 4-week tapering corticosteroid therapy, which may be prolonged at the treating physician's discretion. do. Subjects in cohort 4a will receive a single IVT injection of AAV2.7m8-aflibercept at dose 3 of 6x10 ¹¹ vg/eye with prophylactic topical 4-week tapering difluprednate therapy which may be prolonged at the discretion of the treating physician .

A summary of topical difluprednate therapy is provided in Table 11A below.

Table 11A: Summary of topical difluprednate therapy.

Subjects in Cohorts 3 and 4 may also be administered a 6-week prophylactic corticosteroid regimen, which may be extended at the treating physician's discretion. A summary of the 6-week prophylactic corticosteroid regimen is provided in Table 11B.

Table 11B. Summary of 6-week topical difluprednate therapy.

Design to initiate immunosuppression immediately following exposure to AAV2.7m8-aflibercept IVT injection limits the immune response upon exposure to capsid antigen. Subjects received a drop of difluprednate 0.05% solution (2.5%) in the conjunctival sac of the treated eye 4 times a day for 1 week starting on the day of injection of AAV2.7m8-aflibercept (Day 1, post-injection) μg difluprednate) is self-administered. This is followed by three times a day for the second week, twice a day for the third week and once a day for the fourth week. The tapering corticosteroid therapy may be extended at the treating physician's discretion.

D. Prohibited Medications and Treatments

Prohibited medications and treatments for this study were as described in Section IV of Example 1.

E. Summary of Study Design for Cohorts 2-4

A summary of the study design for cohorts 2-4 is provided in Table 12.

Table 12: Summary of study design for cohorts 2-4.

F. Study Duration

The duration of subject participation in this study was as described in Section IV of Example 1.

V. Study Evaluation

The evaluations performed in this study were as described in Section V of Example 1. Additionally, samples are taken from aqueous humor and vitreous humor.

waterproof sampling

At Screening (before aflibercept injection) and Day 1 (before AAV2.7m8-aflibercept injection), Weeks 12, 24, 36, 52, 76, 88, Weeks Aqueous samples are obtained at week 104 and at any visit requiring first aflibercept (Eylea) rescue treatment or early termination. The aqueous humor samples are analyzed for aflibercept, VEGF-A and NAb concentrations.

vitreous fluid sampling

A sample of vitreous humor is obtained when a vitrectomy is performed at any time during the study. The vitreous humor samples are analyzed primarily for aflibercept concentration. The remaining samples are analyzed for VEGF-A concentration.

VI. result

A. Subject Characteristics

Disease characteristics and treatment history are recorded for all subjects. In particular, number of days or years of neovascular AMD diagnosis, number of prior anti-VEGF injections in study eye, mean calculated anti-VEGF injection interval before study, number of anti-VEGF injections received within 4 months prior to screening, screening aflibercept IVT dose Record the date and AAV2.7m8-aflibercept injection date.

B. Safety

Subjects are closely monitored on the day of IVT AAV2.7m8-aflibercept administration and after treatment thereafter.

Assess the safety of AAV2.7m8-aflibercept through collection of vital signs, physical and eye exams, ECG, pregnancy tests, and laboratory evaluations.

The incidence, severity, and relationship to treatment of AEs, SAEs and DLTs are evaluated in all subjects.

The incidence, severity, and relationship to treatment of intraocular inflammation are evaluated in all subjects.

Visual acuity is assessed in all subjects via BCVA, expressed as an ETDRS score (number of characters read correctly) (Vitale et al., (2016) JAMA Ophtalmol 134(9):1041-1047).

Requirements for anti-VEGF rescue injections are monitored in all subjects.

The presence of correlations between pre-existing anti-AAV NAb-levels and AEs, SAEs and DLTs in the subject's serum is determined.

To evaluate the effect of steroid prophylaxis (topical or oral) on the development and resolution of AEs, SAEs, DLTs and intraocular inflammation.

Standard of care aflibercept IVT injection is used as rescue treatment.

C. Efficacy Assessment

Disease stabilization is assessed in all subjects. Retinal thickness, central subfield thickness, fluid and other anatomical features are measured (eg, by OCT imaging) before and after administration of study treatment.

The efficacy of AAV2.7m8-aflibercept in the treatment of wAMD is assessed by the following scale. The main evaluation time point is 24 weeks. Baseline values of BCVA and SD-OCT refer to values taken during the screening visit on days-15 to -7 (eg, days-14 to -7) prior to aflibercept injection. Baseline values are used for comparison for analysis.

Vision is primarily assessed via BCVA, expressed as an ETDRS score (number of characters read correctly) (Vitale et al., (2016) JAMA Ophtalmol 134(9):1041-1047). A subject is classified as visual retention if it loses less than 15 characters on the ETDRS score compared to baseline. Calculated endpoints include mean change from baseline, percent gain of at least 15 characters compared to baseline, and percent loss of at least 15 characters compared to baseline.

Evaluate leakage compared to baseline by performing FA to assess CNV lesions using standard techniques.

Retinal thickness (e.g., central retinal thickness or central subfield thickness), macular volume and fluid (e.g., subretinal fluid and retinal) compared to baseline values by performing SD-OCT using approved equipment and standard techniques. ) and fluid is assessed.

Determine the number of aflibercept injections given following AAV2.7m8-aflibercept treatment per subject, over time from week 4 to week 104. Additionally, the time to the first aflibercept injection following treatment with AAV2.7m8-aflibercept and the proportion of subjects who did not require aflibercept retreatment are determined.

Determine the proportion of subjects without IRF over time from week 4 to week 104.

Determine the proportion of subjects without SRF over time from week 4 to week 104.

The proportion of subjects with PED at baseline, over time from Week 4 to Week 104, without PED is determined.

Example 3: Results of an open-label phase 1 study of AAV2.7m8-aflibercept at doses lower than 6×10 ¹¹ vg/eye with topical corticosteroids in neovascular (wet) age-related macular degeneration.

This example presents the results of a phase 1 study described in Example 2 that evaluated the safety and efficacy of AAV2.7m8-aflibercept administered at doses lower than 6x10 ¹¹ vg/eye in combination with topical corticosteroids in subjects with wAMD. Write down

result

cohort 2

Baseline characteristics of subjects in Cohort 2 are provided in Table 13.

Table 13. Baseline characteristics of subjects in Cohort 2.

As described in Example 2, subjects in Cohort 2 were given AAV2 with prophylactic 13-day oral corticosteroid therapy starting with 60 mg prednisone for a total of 6 days before and 3 days after AAV2.7m8-aflibercept treatment. A single IVT injection of .7m8-aflibercept was administered at a dose of 2 of 2×10 ¹¹ vg/eye. After that, the prednisone tapering was continued for 7 days. A summary of oral prednisone therapy for Cohort 2 is provided in Table 2 of Example 1.

As shown in Figure 10, 24 weeks after administration of AAV2.7m8-aflibercept, BCVA was stable across subjects in Cohort 2 with a mean reduction of -4.8 ETDRS characters. Additionally, as shown in FIG. 11 , subjects exhibited improved central retinal thickness in OCT with a mean reduction in CST of 27.8 μm. 4 of 6 subjects (66%) did not receive rescue anti-VEGF injections at a follow-up time of 24 weeks.

Cellular inflammatory events and steroid treatment given for each subject were analyzed up to a follow-up time of 24 weeks. As shown in Figure 12, inflammation was responsive to topical steroids. Subjects 4 and 5 had no clinically significant cellular inflammation (subject 5 had <5 AC cells at week 20) and did not require steroid treatment. Subjects 2 and 3 exhibited 1/2+ aqueous humor and vitreous cell grades, or 1-5 cells observed in aqueous humor and 1-10 cells observed in vitreous resolved after a short course of topical steroids. Subject 1 had an initial inflammatory response that responded rapidly to steroid eye drops after cessation of oral steroids; Subjects maintained steroid eye drops 4 times per day (QID). Subject 6 had AC cell responses that worsened on BID eye drops and resolved with QID eye drops. The aqueous humor cell grade category was based on the Standardization of Uveitis Nomenclature (SUN) criteria (Jabs, DA et al. J Ophthalmol. 2005;140:509-516). The vitreous cell grade categories were based on the National Institutes of Health (NIH) guidelines.

Taken together, the inflammation was generally mild. Oral steroids did not provide long enough coverage in certain subjects. However, all inflammatory events were responsive to topical steroids.

OCT imaging 24 weeks after administration of AAV2.7m8-aflibercept showed anatomical improvement and maintenance of visual acuity (BCVA). 13A-13B , Subjects 1, 2, 4, and 6 showed resolution of fluid. Subject 3 received 3 rescue aflibercept injections for the loss of 10 BCVA letters, all due to retinal fluid. Subject 5 all received 3 rescue aflibercept injections for an increase in CST of 75 μm.

By week 24 after administration of AAV2.7m8-aflibercept, 4 of 6 subjects had not received any anti-VEGF rescue injections. As shown in the swimmer lane plot of FIG. 14 , subjects 3 and 5 each received three anti-VEGF IVT injections during a 24-week follow-up period.

Taken together, the results for Cohort 2 after 24 weeks of administration of AAV2.7m8-aflibercept show that AAV2.7m8-aflibercept has robust efficacy. In particular, mean BCVA was maintained and retinal anatomy and CST improved in subjects not requiring rescue anti-VEGF injection. Additionally, AAV2.7m8-aflibercept was safe, well tolerated, and low-grade inflammation was responsive to steroid eye drops.

Example 4: Overview of the results of an open label phase 1 study of AAV2.7m8-aflibercept in neovascular (wet) age-related macular degeneration.

This example provides an overview of the results of an open label phase 1 study of AAV2.7m8-aflibercept for the treatment of age-related macular degeneration with choroidal neovascularization (AMD) described in Examples 1-3. .

safety

Results available for Cohort 1 to a median follow-up time of 44 weeks and Cohort 2 to a median follow-up time of 24 weeks show that AAV2.7m8-aflibercept was well tolerated. Specifically, no AAV2.7m8-aflibercept or procedure-related serious adverse events were reported. Additionally, no AAV2.7m8-aflibercept-related systemic adverse events or adverse events meeting the criteria for dose-limiting toxicity were reported. AAV2.7m8-aflibercept-related adverse events were mild (71%) or moderate (29%). Low-grade inflammation was commonly reported and responsive to steroid eye drops. No vasculitis, retinitis or choroiditis were reported. One serious adverse event of spontaneous intraocular lens retinal detachment not related to AAV2.7m8-aflibercept has been reported. Retinal detachment was surgically repaired and the subject was kept under follow-up.

efficacy

Results available for Cohort 1 to a median follow-up time of 44 weeks and Cohort 2 to a median follow-up time of 24 weeks show that AAV2.7m8-aflibercept had robust efficacy. In Cohort 1, subjects did not receive anti-VEGF rescue injections. In Cohort 2, 4 of 6 subjects did not receive anti-VEGF rescue injections. Taken together, in 10 of 12 subjects (83%) in Cohorts 1 and 2 who did not receive anti-VEGF rescue injections, mean BCVA was maintained and mean CST improved.

A summary of the available safety and efficacy results for Cohorts 1 and 2 is provided in Table 14.

Table 14. Summary of safety and efficacy results for subjects in Cohorts 1 and 2.

Example 5: Additional results of an open label phase 1 study of AAV2.7m8-aflibercept in neovascular (wet) age-related macular degeneration.

This example describes the results for Cohorts 1, 2 and 3 of the Phase 1 studies described in Examples 1-4 that evaluated the safety and efficacy of a single intravitreal injection of AAV2.7m8-aflibercept in subjects with wAMD. do. Safety and efficacy outcomes were assessed for Cohort 1 at a median follow-up time of 60 weeks (range 52-64 weeks), for Cohort 2 at a median follow-up time of 36 weeks (range 32-40 weeks) and for Cohort 3 up to 20 weeks follow-up. provided in time.

result

Subjects in Cohort 1 received a single IVT injection of AAV2.7m8-aflibercept at a dose of 6×10 ¹¹ vg/eye. Cohort 2 subjects received a single IVT injection of AAV2.7m8-aflibercept at a dose of 2×10 ¹¹ vg/eye. Subjects in Cohorts 1 and 2 also received prophylactic oral prednisone therapy (see Table 2).

Cohort 3 subjects received a single IVT injection of AAV2.7m8-aflibercept with 6 weeks of prophylactic topical corticosteroid therapy at a dose of 2×10 ¹¹ vg/eye (see Table 11B). Topical corticosteroid therapy started on the same day as administration of AAV2.7m8-aflibercept (administered after IVT injection of AAV2.7m8-aflibercept) 4 times a day (QID for 3 weeks) for 3 weeks corticosteroid eye drops administered (0.05% difluprednate [2.5 μg]) followed by corticosteroid eye drops administered 3 times a day (TID for 1 week) for 1 week followed by 2 times a day for 1 week (1 week) BID) administered corticosteroid eye drops followed by 3-week tapering of corticosteroid eye drops administered once daily (QD for 1 week) for 1 week.

object characteristics

Baseline subject characteristics for subjects in Cohorts 1, 2 and 3 are provided in Table 15.

Table 15. Baseline characteristics of subjects in Cohorts 1-3.

As shown in Table 15, subjects in cohorts 1-3 previously required frequent anti-VEGF injections to maintain vision. Baseline characteristics for subjects in cohorts 1-3 were similar age, similar mean prior anti-VEGF injections, and frequent injections to maintain a relatively high baseline time of about 65 ETDRS characters across cohorts (eg, AAV2. A similar need for 7m8-aflibercept (>9 injections in the preceding year) was well balanced. Subjects in Cohort 3 were, on average, younger and had fewer prior anti-VEGF injections and a thicker mean baseline CST since initial diagnosis.

Safety Results

As shown in Table 16, no AAV2.7m8-aflibercept- or procedure-related serious adverse events (SAEs) occurred across cohorts 1-3. Additionally, no AAV2.7m8-aflibercept-related systemic adverse events (AEs) were observed. A SAE of retinal detachment not related to study treatment occurred, which was repaired and resolved surgically (previous cataract extraction and artificial lens implantation were major risk factors). Two subjects had an adverse event of elevated intraocular pressure (IOP), which resolved. One subject had two mild IOP elevation events (highest IOP was 24 mmHg), both of which were treated with Combigan® eye drops. A second subject was receiving Combigan® for ocular hypertension and the IOP AE resolved spontaneously within 1 month without change to treatment.

Subjects in Cohort 3 (administered with prophylactic steroid eye drops) had fewer ocular adverse events.

Table 16. Adverse events in Cohorts 1, 2 and 3.

As shown in FIGS. 15A-15C , mild and low-grade inflammation primarily affecting the anterior segment was common across cohorts 1-3 and was responsive to topical steroids. No clinical or fluorescein (fluorescein angiography of the posterior pole) evidence of vasculitis, retinitis or choroiditis was observed.

3 subjects in Cohort 1 continue to be administered with topical steroids, but only 1 subject has any evidence of inflammatory cells. Subject 2 received topical steroids once per day; Subject 4 received topical steroids twice daily; Subject 6 receives topical steroids once per day. Taken together, none of the subjects in Cohort 1 had an inflammatory cell count greater than 2+. Additionally, subjects in Cohort 1 had an inflammatory cell count of 2+ for only one time point.

Two subjects in Cohort 2 continue to be administered topical steroids and have evidence of mild inflammatory cells. Subject 1 receives topical steroid once per day and subject 6 receives topical steroid once per day. Subjects 1 and 6 had some breakthrough inflammation of up to 3+ inflammatory cell counts, but both subjects resolved quickly with topical steroid treatment.

Subjects in Cohort 3 received a 6-week prophylactic steroid eye drop regimen. This regimen minimized initial ocular inflammation ( FIG. 15C ). Certain subjects in Cohort 3 will also continue to be administered topical steroids. Subject 3 of Cohort 3 is receiving topical steroids 4 times per day. Subject 5 was no longer receiving topical steroids, but was administered topical steroids as follows: 4 times a day for 3 weeks following administration of AAV2.7m8-aflibercept followed by 2 times a day for 1 week; Then starting at week 5, 4 times a day for 1 week, then reduced from 3 times a day for 1 week to 2 times a day for 1 week and then once a day for 1 week. Subjects 7-9 in Cohort 3 continue on a 6-week prophylactic steroid eye drop regimen. In addition to the prophylactic steroid therapy described, subject 6 was hospitalized for chronic obstructive pulmonary disease (COPD) after treatment with AAV2.7m8-aflibercept and was prescribed oral steroids. COPD was not found to be associated with AAV2.7m8-aflibercept.

efficacy

16A shows the mean BCVA (ETDRS letters) from baseline to Week 52 for subjects in Cohort 1. At a median follow-up time of 60 weeks (range of 52-64 weeks), subjects in Cohort 1 had a mean BCVA change of -2.7 characters from baseline, and 100% of subjects (6/6) did not receive rescue anti-VEGF injections. didn't A clear decrease in mean BCVA at weeks 44 and 48 was due to one subject experiencing a SAE of retinal detachment. 16B shows the mean CST (μm) from baseline to Week 52 for subjects in Cohort 1. At a median follow-up time of 60 weeks (range of 52-64 weeks), subjects in Cohort 1 had a mean CST change of -26.2 μm from baseline.

17A shows the mean BCVA (ETDRS letters) from baseline to Week 36 for subjects in Cohort 2. At a median follow-up time of 36 weeks (range of 32-40 weeks), subjects in Cohort 2 had a mean BCVA change of -2.8 characters from baseline, and 67% of subjects (4/6) did not receive rescue anti-VEGF injections. didn't Subjects who did not receive rescue anti-VEGF injection had a mean BCVA change from baseline of +2.3 characters. 17B shows the mean CST (μm) from baseline to Week 36 for subjects in Cohort 2. At a median follow-up time of 36 weeks (range of 32-40 weeks), subjects in Cohort 2 had a mean CST change of -40.8 μm from baseline. Additionally, subjects who did not receive rescue anti-VEGF injection had a mean CST change of -30.0 μm from baseline. Taken together, in Cohort 2, CST improvement was maintained through Week 36.

18A shows the mean BCVA (ETDRS letters) from baseline to Week 20 for 5 subjects in Cohort 3. At a follow-up time of 20 weeks, 5 subjects from Cohort 3 had a mean BCVA change of +6.8 characters from baseline, and 80% (4/5) of 5 subjects from Cohort 3 received rescue anti-VEGF injections. did not receive Subjects who did not receive rescue anti-VEGF injection had a mean BCVA change from baseline of +8.8 characters. Taken together, in 5 subjects in Cohort 3, BCVA improved by Week 20 (ie, a mean improvement of 6.8 characters for 5 subjects in Cohort 3). 18B shows the mean CST from baseline to Week 20 for 5 subjects in Cohort 3. At a follow-up time of 20 weeks, 5 subjects from Cohort 3 had a mean CST change of -137.8 μm from baseline. Additionally, subjects who did not receive rescue anti-VEGF injection had a mean CST change of -149.8 μm from baseline. Taken together, in 5 subjects in Cohort 3, mean CST improved by week 20, with a substantial decrease in CST (ie, a mean decrease of 137.8 μm for 5 subjects in Cohort 3).

As shown in the swimmer lane plot of FIG. 19 , subjects in cohort 1 did not receive rescue anti-VEGF injections at a median follow-up time of 60 weeks (range 52-64 weeks). Subjects 3 and 5 of Cohort 2 each received 4 anti-VEGF rescue injections (aflibercept). Subject 3 of cohort 3 received two anti-VEGF rescue injections (aflibercept).

Subject Case Study

At baseline, subject 4 (62 years old male) of cohort 1 had received 109 prior anti-VEGF injections (9 within the last 12 months prior to AAV2.7m8-aflibercept administration) and was persistent even with frequent anti-VEGF injections. had subretinal fluid ( FIG. 20A ). As shown in FIG. 20B , some decrease in subretinal fluid was observed in subject 4 after screening IVT aflibercept injection. After administration of AAV2.7m8-aflibercept, the subretinal fluid continued to resolve and the retina was dry by week 12. This effect continued without signs of fluid recurrence until the subject experienced a diagnosed macular involvement unrelated retinal detachment at week 44. Due to macular involvement, visual acuity was reduced and the subject underwent vitrectomy and successful retinal detachment repair surgery using vesicles. However, due to air bubbles, OCT imaging was not possible at week 48 and the subject could not read any characters on the visual acuity chart. As the bubbles dissolved, vision improved and there were no signs of wet AMD disease activity or fluid recurrence. Fundus topography was slightly altered due to retinal detachment, and the fovea was manually positioned to match the preoperative reference scan in the retinal thickness map presented in Figure 20B at weeks 44, 52 and 60. At week 60, the retina remained dry, vision was within 10 characters of baseline (BCVA), and the subject remained without rescue injection.

At baseline, subject 5 (82 years old male) of cohort 3 received 19 prior anti-VEGF injections (9 within the last 12 months prior to AAV2.7m8-aflibercept dosing) and persisted even with frequent anti-VEGF injections. had subretinal fluid ( FIG. 21A ). As shown in FIG. 21B , Subject 5 was responsive to screening anti-VEGF injections, but subretinal fluid returned within 2 weeks. Thus, subject 5 is an example of a subject that was responsive to anti-VEGF treatment but required frequent injections to reduce fluid. Based on subject 5's medical history, subject would require bolus anti-VEGF injections on a monthly basis (eg, about once every 4 weeks). However, after 1 week of AAV2.7m8-aflibercept administration, the subretinal fluid did not increase, and no fluid was observed at 12 weeks, 16 weeks and 20 weeks after AAV2.7m8-aflibercept administration. These results demonstrate that the disease course in subject 5 was altered by a single administration of AAV2.7m8-aflibercept.

Argument

The results described in this example showed that AAV2.7m8-aflibercept was well tolerated and exhibited robust efficacy across Cohorts 1, 2 and 3. In particular, these results showed that a single dose of AAV2.7m8-aflibercept reduced the anti-VEGF injection burden in subjects in cohorts 1, 2 and 3.

Results for subjects in Cohort 1 receiving AAV2.7m8-aflibercept at a dose of 6x10 ¹¹ vg/eye required anti-VEGF rescue injections 1 year and thereafter after administration of AAV2.7m8-aflibercept. One subject was shown to be zero, thus demonstrating long-term persistence of efficacy of AAV2.7m8-aflibercept. In addition, the majority of subjects in cohorts 2 and 3 who received AAV2.7m8-aflibercept at a dose of 2x10 ¹¹ vg/eye (a dose 3-fold lower than cohort 1) also remained without anti-VEGF rescue injection.

Taken together, of 17 subjects (including 5 subjects from Cohort 3, followed for 20 weeks) who received AAV2.7m8-aflibercept in Cohorts 1-3, 14 (82%) received anti-VEGF rescue injections. was maintained without, the mean BCVA was maintained, and there was an improvement in the mean CST.

Results from cohorts 1-3 also showed that all adverse events related to AAV2.7m8-aflibercept were mild to moderate, ocular inflammation observed after administration of AAV2.7m8-aflibercept was of low grade, and that steroid eye drops showed that it was reactive. Additionally, subjects in Cohort 3 who received the 6-week prophylactic steroid eye drop regimen had minimal inflammation, thus demonstrating that the 6 week prophylactic steroid eye drop regimen was effective in minimizing the initial ocular inflammation. No events of vasculitis, retinitis or choroiditis were observed.

Example 6: A Phase 2, multicenter, randomized, double-shielded, active-controlled study of AAV2.7m8-aflibercept in subjects with diabetic macular edema.

This example describes a phase 2, multicenter, randomized, double-shielded, active-controlled study evaluating the persistence of a single intravitreal (IVT) injection of AAV2.7m8-aflibercept in subjects with diabetic macular edema. .

I. Study Objectives and Endpoints

A. Primary purpose

The primary objective of this study was to evaluate the persistence of a single IVT injection of AAV2.7m8-aflibercept.

B. Secondary purpose

The secondary objectives of this study included:

● Evaluation of safety and tolerability of AAV2.7m8-aflibercept.

● Evaluation of the effect of AAV2.7m8-aflibercept on macular edema.

● Evaluation of the effect of AAV2.7m8-aflibercept on maximum corrected visual acuity (BCVA).

● Evaluation of the effect of AAV2.7m8-aflibercept on the Diabetic Retinopathy Severity Scale (DRSS) score.

● Assessment of need for rescue aflibercept (2 mg IVT).

● Evaluation of the effect of a previous dose of aflibercept (2 mg IVT) prior to administration of AAV2.7m8-aflibercept.

● Evaluation of the effect of AAV2.7m8-aflibercept on the development of visual threatening complications (anterior segment neovascularization, vitreous hemorrhage or traction retinal detachment).

C. Primary endpoint

The primary endpoint of this study is the time to exacerbation of diabetic macular edema (DME) disease activity in the study eye, defined by the occurrence of any of the following:

● Increase in central subfield thickness (CST) of >50 μm as assessed by SD-OCT compared to the lower of the two CST measurements recorded on day 1 or week 4.

● Loss of >5 characters in BCVA due to worsening of DME disease activity compared to the higher of the two BCVA measures recorded at Day 1 or Week 4, whichever is higher.

D. Secondary endpoints

The secondary endpoints for this study (unless otherwise specified) are based on outcome measures for the study eye and include:

● Incidence and severity of ocular and non-ocular adverse events (AEs).

● Changes in CST and macular volume from baseline over time to Week 48.

● Change in BCVA from baseline over time to Week 48.

● Frequency of rescue aflibercept (2 mg IVT) in the study eye over time during the study.

● Incidence of 2-step and 3-step improvement in DRSS score over time to Week 48.

● Incidence of stage 2- and stage 3-exacerbations of DRSS scores over time to Week 48.

● Development of visual threatening complications (anterior segment neovascularization, vitreous hemorrhage, or any other high-risk proliferative diabetic retinopathy (DR) or traction retinal detachment) over time up to week 48.

● Incidence of CST <300 μm over time up to week 48.

● Incidence of clinically significant findings by physical examination, ophthalmologic examination, imaging, and laboratory evaluation over time to Week 48.

II. study group

A. Inclusion Criteria

Subjects with newly diagnosed DME (i.e., diagnosed with DME within 6 months of screening) who received up to two prior injections of anti-VEGF therapy in the study eye are included in this study if they meet the following inclusion criteria: :

● Age ≥ 18 years old.

● Type 1 or Type 2 diabetes.

● Visual impairment due to centrally involved diabetic macular edema.

● Time of screening:

o Study Eye: BCVA 78 to 50 ETDRS characters (inclusive) (approximate Snellen counterparts 20/32 to 20/100).

○ Non-study eye: BCVA 35 or greater ETDRS characters (approximately 20/200 or greater Snellen correspondence).

● CST of ≥ 325 μm of study eye at screening visit when using Heidelberg Spectralis® in the presence of central-involving IRF (central 1 mm).

● Decreased vision in the study eye determined primarily to be due to DME.

● Early diagnosis of DME within 6 months of screening.

● Up to 2 prior injections (0, 1 or 2) of anti-VEGF in the study eye.

○ If prior anti-VEGF was administered to the study eye, there must be a significant CST response (eg, ≥ 10% reduction), and there must be no adverse reaction to anti-VEGF (eg, inflammation).

● Minimum 60-day interval between the last anti-VEGF injection in the study eye and Day 1 randomization.

B. Exclusion Criteria

Subjects meeting any of the following criteria are excluded from this study:

● Anti-AAV2.7m8 neutralizing antibody titers > 1:125 recorded within 6 months prior to randomization.

● Prior ocular gene therapy.

● History of allergy to aflibercept, corticosteroids or fluorescein dyes or sodium fluorescein used for angiography (mild treatable allergies are acceptable).

● History or evidence of any of the following cardiovascular diseases within 6 months of administration:

○ Clinical evidence of severe heart disease (eg, New York Heart Association [NYHA] functional class III or IV) or unstable angina.

○ Acute coronary syndrome, myocardial infarction or coronary revascularization, cerebrovascular accident (CVA), transient ischemic attack (TIA).

○ Ventricular arrhythmias or uncontrolled arrhythmias requiring ongoing treatment.

○ Uncontrolled hypertension, defined as systolic blood pressure (SBP) >160 mmHg or diastolic blood pressure (DBP) >100 mmHg, despite the use of BP-lowering medications within the screening period. If BP-lowering medication is required, subjects must receive a stable dose of the same medication for 30 consecutive days prior to randomization.

● Any history of ongoing bleeding disorders. The use of aspirin or other anticoagulants (eg, factor Xa inhibitors) is not an exclusion criterion.

● Uncontrolled diabetes, defined as HbA1C >10%; or a history of diabetic ketoacidosis within 3 months prior to randomization; or subjects who initiate or plan to do so within the next 3 months on intensive insulin therapy (pump or multiple daily injections) within the last 3 months.

● History of systemic autoimmune disease requiring treatment with systemic steroids or immunosuppressive therapy (eg methotrexate, adalimumab).

• Systemic drugs known to cause macular edema (eg fingolimod, tamoxifen, chloroquine/hydroxychloroquine) or any prior systemic anti-VEGF therapy.

• Known to be positive for HIV or active viral hepatitis (unless healing is documented after treatment for hepatitis C); Known history of syphilis.

• Known severe renal impairment as indicated by estimated CrCl <30 mL/min (by Cockcroft-Gault calculations); The need or expected need for hemodialysis during the study period.

● Any febrile disease within 1 week prior to randomization.

Additionally, subjects who meet any of the following ocular exclusion criteria in the study eye are excluded from this study:

● Any vitreous or preretinal hemorrhage, neovascularization elsewhere in the >1/2-disc area within an area equivalent to the standard ETDRS 7-field on clinical examination, or discs in the >1/3-disc area on clinical examination high-risk proliferative diabetic retinopathy (PDR) at screening, defined as neovascularization of

● Any prior focal or grating laser photocoagulation or any prior panretinal photocoagulation (PRP) in the study eye.

• Any anti-VEGF therapy in the previous 60 days prior to randomization (up to 2 prior anti-VEGF injections were allowed, but could not be made in the previous 60 days).

● History of anterior segment neovascularization (eg, neovascularization of the iris [NVI] or neovascular glaucoma [NVG]), significant vitreous hemorrhage, fibrovascular proliferation, or traction retinal detachment.

● On clinical examination or OCT, laboratory evidence of structural abnormalities in the fovea (eg, dense hard exudate, pigment abnormalities, foveal atrophy, vitreous macular retraction or epiretinal membrane) that are thought to contribute to macular edema or visual impairment. .

● History of retinal disease other than diabetic retinopathy, including age-related macular degeneration (in either eye), retinal vein occlusion, retinal artery occlusion, pathological myopia, etc. in the study eye.

● Any current ocular disease other than DME or a history of such ocular disease (e.g., For significant cataract, macular traction) or any evidence of subcapsular cataract.

● History of cataract extraction or yttrium aluminum garnet (YAG) capsulotomy within 3 months prior to Day 1.

● History of retinal detachment (with or without repair) in the study eye.

● History of trabeculectomy or glaucoma shunt or minimally invasive glaucoma surgery (MIGS).

● History of vitrectomy or other filtration surgery.

● Presence of an aphakic or anterior intraocular lens.

● Uncontrolled ocular hypertension or glaucoma (defined as IOP >22 mmHg despite treatment with antiglaucoma medications) in the study eye at the time of randomization or current use of >2 IOP lowering medications.

• Any history of guidance or periocular steroid treatment for any ocular condition (eg, IVT Triessence, Ilubien or Ozurdex).

● Refractive correction surgery within the 90-day period prior to screening.

● Previous penetrating keratoplasty, endothelial keratoplasty, or ocular radiation.

● Any prior vitreoretinal surgery.

Additionally, subjects who meet any of the following ocular exclusion criteria in the study eye or in the non-study eye (ie, "other eyes") are excluded from this study:

• Any history of uveitis or intraocular inflammation (grade trace or higher), except for mild expected postoperative inflammation that resolved.

● History of elevated IOP associated with topical steroid administration.

● Known history of ocular herpes simplex virus (HSV), varicella-zoster virus (VZV) or cytomegalovirus (CMV), including viral uveitis, retinitis, or keratitis.

● Evidence of external ocular infection, including conjunctivitis, polyps, or significant blepharitis.

● History of ocular toxoplasmosis.

III. study design

A. Study Treatment

This is a multicenter, randomized, double-shielded, controlled, parallel-group study to evaluate the efficacy, safety and tolerability of a single 0.10 mL IVT injection of AAV2.7m8-aflibercept. A clinical study of two doses of AAV2.7m8-aflibercept.

Subjects who have an initial diagnosis of DME within 6 months of screening and have received up to two prior injections of anti-VEGF therapy are eligible for enrollment.

Approximately 33 eligible subjects will receive one of two doses of AAV2.7m8-aflibercept (6 x 10 ¹¹ vg/eye or 2 x 10 ¹¹ vg/eye) or receive a simulated ocular injection with a previous aflibercept injection. Randomized into receiving control arm. Subjects assigned to receive AAV2.7m8-aflibercept are further randomized to receive prior aflibercept or sham ocular injection. The study areas are summarized below:

● Arm 1 (n=6): Subjects receive AAV2.7m8-aflibercept at a dose of 6×10 ¹¹ vg/eye along with previous aflibercept dose.

● Arm 2 (n=6): Subjects receive AAV2.7m8-aflibercept at a dose of 6×10 ¹¹ vg/eye without prior aflibercept dose.

● Arm 3 (n=6): Subjects receive AAV2.7m8-aflibercept at a dose of 2×10 ¹¹ vg/eye along with a previous dose of aflibercept.

● Arm 4 (n=6): Subjects receive AAV2.7m8-aflibercept at a dose of 2×10 ¹¹ vg/eye without prior aflibercept dose.

● Arm 5 (n=9): Subjects receive aflibercept only (active control).

To maintain masking of treatment assignment, subjects assigned to either the prior aflibercept-free arm on Day 1 or the AAV2.7m8-aflibercept-free arm on Day 8 will receive a simulated ocular injection at the corresponding visit. receive Only one eye per subject is selected as the study eye. If both eyes are eligible, the eye with worse BCVA is selected as the study eye.

Both AAV2.7m8-aflibercept and aflibercept are administered via IVT injection. Do not perform IVT injections if active inflammation is present. Aseptic technique with povidone-iodine is used under local or subconjunctival anesthesia. The simulated ocular injection procedure is performed using an empty syringe (using a blunt end) without a needle pressed against the eye under the same conditions to mimic the injection.

A summary of the divisions in this study is provided in Table 17.

Table 17. Research Divisions.

After the IVT injections assigned on Days 1 and 8, all subjects were evacuated from Day 1 to Weeks 2, 4, and then every 4 weeks until Week 48 (e.g., Weeks 8, 12, Week 16, etc.) are followed. To maintain shielding, both subjects and assessment performing personnel are screened for treatment assignments throughout the study.

All subjects are followed for 48 weeks post randomization.

An overview of the study design is provided in FIG. 29 .

B. Prophylactic topical steroid therapy

All subjects receive a prophylactic 7-week topical corticosteroid regimen of difluprednate (0.05%; eg, duresol) starting on Day 1. Subjects received difluprednate 4 times a day (QID) for 4 weeks (ie, days 1 to 28), then 3 times a day (TID) for 1 week (ie, for 7 days) , then self-administering twice a day (BID) for 1 week (ie, for 7 days) and finally once a day (QD) for 1 week (ie, for 7 days). Diminishment does not begin in the presence of active inflammation. Extend this therapy if signs of inflammation develop. A summary of difluprednate therapy is provided in Table 18.

Table 18. Difluprednate therapy.

C. Relief Treatment

Beginning at week 8, subjects receive rescue aflibercept (2 mg IVT) if any of the following are met:

• Increase of >50 μm in CST compared to the lower of the two CST measurements recorded on day 1 or week 4 as assessed by SD-OCT.

● Loss of >5 characters in BCVA due to worsening of DME disease activity compared to the higher of the two BCVA measures recorded at Day 1 or Week 4, whichever is higher.

Do not inject aflibercept into an eye with active inflammation. A minimum of 21 days is required between rescue aflibercept injections.

D. Medication and Treatment

The following medications are contraindicated during the study:

● Any systemic anti-VEGF agent, including bevacizumab.

● Systemic drugs known to cause macular edema (eg fingolimod, tamoxifen, chloroquine/hydroxychloroquine).

• Any anti-VEGF agent other than study drug or aflibercept IVT 2 mg in the study eye.

● IVT steroids in the study eye (eg Ozurdex or Ilubien or Triessence).

● Systemic immunosuppressive drugs (eg, intravenous steroids, methotrexate, azathioprine, cyclosporine, adalimumab, infliximab, etanercept). Inhaled or topical steroids and NSAIDs are permitted.

Subjects who develop high-risk PDR in the study eye receive salvage aflibercept followed by panretinal photocoagulation (PRP).

Subjects with visually significant cataracts are not enrolled in the study, but if cataracts develop during the study, cataract surgery in the study eye will appear clinically and after >90 days of AAV2.7m8-aflibercept administration and/or May be performed if scheduled > 7 days after the last injection of aflibercept.

Subjects who develop DME in the other eye (non-study eye) may receive standard of care therapy.

IV. study evaluation

A. General Physical Examination and Vital Signs

A general physical examination (PE) is performed at screening and at the end of study (EOS) or early termination visit. It consists of a physical system examination of general appearance, nervous system, HEENT (head, eyes, ears, nose and throat), neck, cardiovascular, respiratory, abdomen, extremities, skin, weight and height. At the EOS or Early Termination visit, the physical evaluates whether any changes have occurred in the subject's physical condition since the screening examination. A targeted physical examination is performed as needed for evaluation of AEs.

Vital signs consist of blood pressure, pulse rate, body temperature and respiration rate. A 12-lead electrocardiogram (ECG) is performed for each subject at screening and at the EOS or early termination visit.

B. Laboratory Tests, Vector Expression and Immune Response

The following clinical laboratory tests are performed for the study: chemistry, complete blood count, HbA1C, urinalysis and HLA-B27 genotyping.

A sample of the subject (both blood and/or aqueous humor) is collected to measure:

• Total antibody to AAV2.7m8: Serum to total anti-AAV2.7m8 antibody is measured in an ELISA assay.

• Neutralizing Antibody to AAV2.7m8: Serum to neutralizing anti-AAV2.7m8 antibody is measured in a cell-based assay.

• Anti-aflibercept antibody: Serum for the humoral immune response to aflibercept is measured in an ELISA assay.

• Aflibercept protein expression: Serum and aqueous humor samples are collected and determined in a mesoscale discovery assay for the presence of aflibercept protein.

• Cell-mediated immune response: Cellular immunity to the AAV2.7m8 capsid and aflibercept protein is measured in an ELISPOT assay.

C. Full Ophthalmic Examination

Study evaluations included ophthalmologic examination, intraocular pressure (IOP) and indirect ophthalmoscopy.

Ophthalmic examinations include external examination of the eye and appendages, commercial screening and slitting for ptosis/pupillary reactivity (including, but not limited to, ptosis, abnormal pupil shape, unequal pupil, abnormal response to light and afferent pupil defect). -Lamp examination (eyelid, conjunctiva, cornea, lens, iris, anterior chamber) consists of. The slit-lamp examination is used to examine anterior ocular structures and to grade any findings. If any observations were noted during the slit-lamp examination, at any visit, the severity is graded and the observation is described as clinically significant or not clinically significant.

IOP measurements are performed using a Goldman applanation tonometer or Tono-Pen™. The same IOP measurement method is used throughout the study for each individual subject. IOP measurements are performed prior to any IVT injections and eye dilatation using the same method throughout the study. Day 1 and Day 8 visits require pre- and post-injection (30 min post-injection) IOP measurements.

Extended indirect ophthalmoscopy involves the evaluation of posterior segment abnormalities of the vitreous, optic nerve, peripheral retina, and retinal vasculature. If any findings were noted during ophthalmoscopy, at any visit, the severity is graded and the finding is described as clinically significant or not clinically significant. Day 1 and Day 8 visits require pre- and post-injection indirect ophthalmoscopic evaluations.

D. Refraction and Vision

Measure refraction and BCVA. Optometry is taken at a starting distance of 4 meters before the eyes are dilated.

E. Imaging

Spectral domain optical coherence tomography (SD-OCT) is an interferometric technique that provides depth-resolved tissue structure information encoded as magnitude and delay of back-scattered light by spectral analysis of an interference fringe pattern. If subjects received anti-VEGF injections at visits prior to study randomization, OCTs from these visits are collected and forwarded to a central reading center.

Optical coherence tomography angiography (OCT-A) is an imaging technique that provides information about blood flow as well as volumetric three-dimensional maps of the retina and choroid. There are two types of OCT-A, swept-source and spectral-domain. Use swept-source imaging when available. If a swept-source device is not available and a spectrum-domain device is available, it uses a spectrum domain device.

A standardized procedure for acquisition of ultra-wide fundus digital photographic images of the retina, optic disc, and macula is followed. Additionally, pictures of the iris are taken prior to expansion.

Standardized procedures for examining retinal circulation and vascular permeability are followed using dye-tracing methods. This involves injection of sodium fluorescein into the systemic circulation, after which an angiogram is obtained by digital photography of the emitted fluorescence after illumination of the retina with blue light at a wavelength of 490 nm.

F. Laboratory, biomarkers and other biological specimens

A waterproofing sample is collected and analyzed for levels of aflibercept, VEGF-A, neutralizing antibody (NAb) and additional biomarkers. A sample of vitreous humor is obtained and analyzed for aflibercept concentration and other biomarkers.

G. Safety

Report all clinically significant adverse events (AEs) following study treatment administration. Each subject is followed for any ongoing study treatment-related AEs and/or serious AEs (SAEs) a) until the end of the AE reporting period 30 days after the last study visit, or b) until resolved or stable. Any clinically significant safety assessment associated with DME is not reported as an AE or SAE unless determined to be more severe than expected for the subject's condition. Disease progression under the study is captured as an efficacy outcome.

Adverse events of special interest to this study include:

● Vision-threatening Adverse Event: An adverse event is considered to be vision-threatening if one or more of the following criteria are met:

○ Causes a reduction of ≥ 30 characters in BCVA compared to previous visits.

○ Requires surgical or medical intervention (ie, conventional surgery, vitrectomy) to prevent permanent vision loss.

○ Causes severe intraocular inflammation (ie endophthalmitis, 4+ anterior cells/cloudy or 4+ vitreous cells).

Report all above-listed vision-threatening adverse events as serious adverse events, listing the underlying cause (if known) of the event as a major event term.

H. Efficacy

The efficacy of AAV2.7m8-aflibercept in the treatment of DME is assessed by the following scale. Baseline values for BCVA and SD-OCT endpoints refer to pretreatment measurements taken at the Day 1 visit when administering aflibercept IVT or sham ocular injection.

● BCVA: Vision is assessed primarily through BCVA, expressed as an ETDRS score (number of characters read correctly). A subject is classified as visual retention if it loses less than 15 characters on the ETDRS score compared to baseline. Calculated endpoints include mean change from baseline, percent gain of at least 15 characters compared to baseline, and percent loss of at least 15 characters compared to baseline.

● Central Subfield Thickness: Perform SD-OCT using approved equipment and standard techniques to evaluate thickness and fluid compared to baseline values. Endpoints include CST and macular volume.

● Aflibercept Retreatment: The incidence and timing of given aflibercept injections following AAV2.7m8-aflibercept treatment over time.

● For each time point, the Diabetic Retinopathy Severity Scale (DRSS) is determined using ultra-wide color fundus imaging and compared to Day 1.

● Visual threatening complications (anterior segment neovascularization, diabetic macular edema, high-risk PDR development, vitreous hemorrhage or traction retinal detachment) as determined by ultra-wide-angle imaging and clinical examination.

I. Statistical Analysis

The main analysis population included all randomized subjects receiving study treatment (AAV2.7m8-aflibercept IVT or sham ocular injection) on Day 8. All safety and efficacy parameters are descriptively summarized by treatment arm. Mean, standard deviation (SD), median and range are given for continuous variables; Frequency counts and percentages are provided for categorical variables. Confidence intervals for means and percentages are provided at both the 90% and 95% levels. A Kaplan-Meier survival assay is used to derive the median time to first onset of DME disease exacerbation. All rescue aflibercept (2 mg IVT) received by each subject during the study is summarized using a statistical model for recurrent events. The mean cumulative function (MCF) curve over time is plotted against the mean cumulative number of injections. The effect of treatment on changes in BCVA and CST over time is investigated using a mixed-effect model for repeated measures (MMRM). We investigate the effect of treatment on changes in DRSS over time using a generalized mixed model for categorical outcomes. Interim analyzes (IA) are made after all subjects have been followed for 24 weeks.

Example 7: Additional results of an open label phase 1 study of AAV2.7m8-aflibercept in neovascular (wet) age-related macular degeneration.

This example presents results for cohorts 1, 2, 3 and 4 of the Phase 1 studies described in Examples 1-5 evaluating the safety and efficacy of a single intravitreal injection of AAV2.7m8-aflibercept in subjects with wAMD. Write down Safety and efficacy outcomes were as follows: Cohort 1 at a median follow-up time of 72 weeks (range of 64-84 weeks), Cohort 2 at a median follow-up time of 52 weeks (range of 52-56 weeks), and a median follow-up time of 36 weeks (range of 20-40 weeks) ) and for Cohort 4 at a median follow-up time of 4 weeks (range of 2-8 weeks).

result

Subjects in Cohort 1 received a single IVT injection of AAV2.7m8-aflibercept at a dose of 6×10 ¹¹ vg/eye. Cohort 2 subjects received a single IVT injection of AAV2.7m8-aflibercept at a dose of 2×10 ¹¹ vg/eye. Subjects in Cohorts 1 and 2 also received prophylactic oral prednisone therapy (see Table 2).

Cohort 3 subjects received a single IVT injection of AAV2.7m8-aflibercept at a dose of 2×10 ¹¹ vg/eye. Cohort 4 subjects received a single IVT injection of AAV2.7m8-aflibercept at a dose of 6×10 ¹¹ vg/eye. Subjects in cohorts 3 and 4 were administered 6 weeks of prophylactic topical corticosteroid therapy (see Example 5 and Table 11B).

object characteristics

Baseline characteristics for subjects in cohorts 1-4 were well balanced, with similar age and mean prior anti-VEGF injections, and a similar need for frequent injections to maintain a baseline view of approximately 65 ETDRS characters across cohorts. . Some subjects in Cohort 4 were diagnosed more recently than in other cohorts, eg, less than 12 months prior to administration of AAV2.7m8-aflibercept, and thus the mean number of prior anti-VEGF injections is lower. Taken together, subjects in cohorts 1-4 had good vision, and some fluids were frequently treated with anti-VEGF injections, making them difficult to treat. Baseline subject characteristics for subjects in Cohorts 1-4 are provided in Table 19.

Table 19. Baseline characteristics of subjects in Cohorts 1-4.

safety

There were no deaths, discontinuation of treatment, or AAV2.7m8-aflibercept-related non-ocular adverse events.

When observed, inflammation was responsive to and managed by topical steroids. No clinical or fluorescein (fluorescein angiography of the posterior pole) evidence of posterior inflammation was observed. In addition, no vasculitis, retinitis, choroiditis, vascular occlusion or endophthalmitis occurred.

All AAV2.7m8-aflibercept-related ocular adverse events were mild (78%) or moderate (22%).

One adverse event of special interest (AESI) of recurrent moderate uveitis occurred and was considered AAV2.7m8-aflibercept related (Cohort 1). Recurrent moderate uveitis AESI was responsive to steroid eye drops. Additionally, a treatment-unrelated ocular serious adverse event (SAE) of retinal detachment occurred, which was repaired and resolved surgically (Cohort 1).

Two subjects had a mild adverse event of mild intraocular pressure (IOP) elevation, which resolved. One subject from Cohort 1 had two mild IOP elevations (up to 24 mmHg), both of which were treated with Combigan® eye drops. A second subject was receiving Combigan® for ocular hypertension and the IOP adverse event resolved spontaneously within 1 month without change to treatment.

Overall, AAV2.7m8-aflibercept was well tolerated, and there were no systemic adverse events attributable to AAV2.7m8-aflibercept.

A summary of the safety outcomes in Cohorts 1-4 of this study is provided in Table 20.

Table 20. Adverse events in cohorts 1-4.

AAV2.7m8-aflibercept-related adverse events (reported in MedDRA preferred term) that occurred in Cohorts 1-4 are provided in Table 21.

Table 21. AAV2.7m8-aflibercept-related AEs in preferred terms in cohorts 1-4.

Ocular adverse events (reported in MedDRA preferred term) with a frequency of >2 events are provided in Table 22.

Table 22. Ocular AEs (≧2 events) in preferred terms.

Non-ocular adverse events (reported in MedDRA preferred term) that occurred with a frequency of >2 events are provided in Table 23.

Table 23. Non-ocular AEs (≧2 events) in preferred terms.

A summary of ocular and non-ocular serious, mild and moderate adverse events observed in Cohorts 1-4 of this study is provided in Table 24.

Table 24. Adverse events in cohorts 1-4.

22A-22D , when observed, inflammation was predominantly in the anterior segment and was responsive to, and managed by, topical steroids. No evidence of posterior inflammation was observed, and there were no cases of vasculitis.

As shown in FIG. 22A , two subjects in Cohort 1 (Subjects 2 and 6) who had previously received QD steroid eye drops and had no active inflammation subsequently discontinued eye drops. Subject 2 had recurrent moderate uveitis after discontinuation, and steroid eye drops were restarted. Inflammation improved and subject resumed tapering steroid eye drops. At the time of data cut, subject 4 received BID steroid eye drops and had minor inflammation after retinal detachment surgery. Subject 6 did not receive any steroid eye drops and had no inflammation. Taken together, inflammation in subjects in Cohort 1 resolved, and only 2 of 6 subjects continued steroid eye drops at the time of data cut. Results from Cohort 1 showed that all subjects were responsive to topical steroids and had a steroid tapering trend at the time of data cut.

In Cohort 2, 4 of 6 subjects were free of inflammation, and 2 of 6 subjects continued to receive steroid eye drops at the time of data cut ( FIG. 22B ). Subject 3 received topical steroids approximately 46 weeks after cataract surgery. At the time of data cut, subject 1 had received steroid eye drops QD and subject 6 had low grade inflammation and continued to receive steroid eye drops. Taken together, inflammation in subjects in Cohort 2 was resolving, and all subjects were past the 52-week (1 year) milestone at the time of data cut.

In Cohort 3, 6 of 9 subjects were free of inflammation at the time of data cut ( FIG. 22C ), all beyond the 20-week milestone. At the time of data cut, subjects 2, 3 and 4 were receiving steroid eye drops. Subject 4 started treatment with BID Durezol at week 24 due to poor pupillary dilatation and posterior adhesions possibly associated with subclinical inflammation. 3 of 9 subjects continued to receive steroid eye drops at the time of data cut.

As shown in FIG. 22D , subjects in Cohort 4 showed minimal or zero initial inflammation with steroid eye drops, consistent with the results observed for Cohort 3. Subjects 1, 2 and 3 all had inflammation and continued to receive steroid eye drops at the time of data cut.

The 6-week prophylactic topical steroid therapy administered in cohorts 3 and 4 showed less active evidence of inflammation compared to oral steroid therapy administered in cohorts 1 and 2 ( FIGS. 22A-22B ) ( FIGS. 22C-22D ).

efficacy

23A shows the mean BCVA (ETDRS letters) from baseline to Week 72 for subjects in Cohort 1. At a median follow-up time of 72 weeks (range of 64-84 weeks), subjects in Cohort 1 had a mean BCVA change of -3.2 characters from baseline, and 100% of subjects (6/6) did not receive rescue anti-VEGF injections. didn't A clear decrease in mean BCVA at weeks 44 and 48 was due to one subject experiencing a SAE of retinal detachment. Taken together, BCVA scores in subjects in Cohort 1 remained stable through week 72. 23B shows the mean CST (μm) from baseline to Week 72 for subjects in Cohort 1. At a median follow-up time of 72 weeks (range of 64-84 weeks), subjects in Cohort 1 had a mean CST change of -21.0 μm from baseline. Taken together, CST measurements in Cohort 1 showed that anatomy was maintained through week 72 for all subjects. The drop and change of error bars observed at weeks 68 and 72 in FIG. 23B was always due to the exclusion of one subject with a very thick retina.

24A shows the mean BCVA (ETDRS letters) from baseline to Week 52 for subjects in Cohort 2. At a median follow-up time of 52 weeks (range of 52-56 weeks), subjects in Cohort 2 had a mean BCVA change of -2.0 characters from baseline, and 50% of subjects (3/6) did not receive rescue anti-VEGF injections. didn't Subjects who did not receive rescue anti-VEGF injection had a mean BCVA change of +0 characters from baseline. Taken together, BCVA scores in subjects in Cohort 2 remained stable after 52 weeks. 24B shows the mean CST (μm) from baseline to Week 52 for subjects in Cohort 2. At a median follow-up time of 52 weeks (range of 52-56 weeks), subjects in Cohort 2 had a mean CST change of -24.8 μm from baseline. Additionally, subjects who did not receive rescue anti-VEGF injection had a mean CST change from baseline of -8.3 μm. Taken together, CST measurements in Cohort 2 showed that the anatomy was maintained after week 52.

25A shows the mean BCVA (ETDRS letters) from baseline to Week 20 for subjects in Cohort 3. At a median follow-up time of 36 weeks (range 20-40 weeks), subjects in Cohort 3 had a mean BCVA change of +4.0 characters from baseline, and 78% of subjects from Cohort 3 (7/9) had rescue anti- No VEGF injections were received. Subjects who did not receive rescue anti-VEGF injection had a mean BCVA change from baseline of +6.4 characters. Taken together, BCVA scores in subjects in Cohort 3 improved. 25B shows the mean CST (μm) from baseline to Week 20 for subjects in Cohort 3. At a median follow-up time of 36 weeks (range 20-40 weeks), subjects in Cohort 3 had a mean CST change of -118.6 μm from baseline. Additionally, subjects who did not receive rescue anti-VEGF injection had a mean CST change of -152.7 μm from baseline. Taken together, mean CST continued to improve in all 9 subjects in Cohort 3.

As shown in the swimmer lane plot of FIG. 26 , subjects in Cohort 1 did not receive rescue anti-VEGF injections at a median follow-up time of 72 weeks (range of 64-84 weeks). In Cohort 2 (median follow-up time of 52 weeks; range of 52-56 weeks), subject 2 received 1 anti-VEGF rescue injection (aflibercept) and subjects 3 and 5 each received 6 anti-VEGF rescue injections (ill libercept) was obtained. In Cohort 3 (median follow-up time of 36 weeks; range 20-40 weeks), subject 2 received 1 anti-VEGF rescue injection (aflibercept) and subject 3 received 4 anti-VEGF rescue injections (aflibercept). received Taken together, 10 of 15 subjects in cohort 2-3 remained without rescue injection. Taken together, even with rescue injections in certain cohorts, there was a dramatic reduction in anti-VEGF injection burden.

A substantial decrease in the annual anti-VEGF injection rate was observed in subjects in Cohorts 1-3 ( FIGS. 27A-27B ). Specifically, subjects in Cohort 1 did not receive any rescue anti-VEGF injections (100% reduction), and subjects in Cohorts 2 and 3 combined had an 87% decrease in mean annual anti-VEGF injection rates ( FIG. 27A ).

case study

At baseline, subject 5 of cohort 3 (82 years old male with wAMD in the right eye) received 19 prior anti-VEGF injections (9 within the last 12 months prior to AAV2.7m8-aflibercept dosing). Subject 5 had persistent subretinal fluid even with frequent anti-VEGF injections every 5 weeks ( FIG. 28A ). The central white area of the thickness map of FIG. 28A reflects the very thick macula.

As shown in FIG. 28B , subject 5 received a screening aflibercept injection 2 weeks prior to the planned AAV2.7m8-aflibercept injection. Subject 5 showed an excellent response to screening aflibercept injection, with resolution of IRF and improvement of SRF. Thus, subject 5 was responsive to anti-VEGF treatment but required very frequent injections to reduce fluid. One week after AAV2.7m8-aflibercept injection, fluid improved. If this subject was only receiving bolus aflibercept injections, the fluid would begin to return within 4-5 weeks and the OCT image would look like a baseline OCT image with significant fluid present. However, as shown in FIG. 28B , fluid and vision did not improve after 12, 16, 20, 28 and 36 weeks of AAV2.7m8-aflibercept injection. Thus, in subject 5, AAV2.7m8-aflibercept injection addressed the treatment burden by eliminating the need for injection, improved vision and controlled disease activity. These results demonstrate that the disease course in subject 5 was altered by a single administration of AAV2.7m8-aflibercept.

Argument

The results described in this example showed that AAV2.7m8-aflibercept continued to be well tolerated and had a favorable safety profile in all cohorts (n = 30). All AAV2.7m8-aflibercept-related ocular adverse events were mild (78%) or moderate (22%), and ocular inflammation, when observed, was responsive to steroid eye drops.

Additionally, the results described in this example showed that AAV2.7m8-aflibercept had robust and sustained efficacy.

All subjects administered AAV2.7m8-aflibercept at a dose of 6×10 ¹¹ vg/eye remained without rescue anti-VEGF injection, and AAV2.7m8-aflibercept at a dose of 2×10 ¹¹ vg/eye Of the 15 subjects administered, 10 remained without rescue anti-VEGF injection, with a clear dose response observed. All subjects in Cohort 1 (dose of 6×10 ¹¹ vg/eye) remained without rescue injection 15 months after administration of AAV2.7m8-aflibercept.

The results described in this example also showed that a single IVT injection of AAV2.7m8-aflibercept resulted in a substantial reduction in the annual anti-VEGF injection rate. Mean BCVA generally improved and remained, and mean CST generally improved.

Results from Cohort 4 showed that, consistent with the results observed in Cohort 3, a 6-week prophylactic regimen of steroid eye drops was effective in minimizing initial ocular inflammation.

Although this disclosure has been described in some detail by way of illustration and example for purposes of clarity of understanding, the description and examples should not be construed as limiting the scope of the disclosure. The disclosures of all patents and scientific literature cited herein are expressly incorporated by reference in their entirety.

SEQUENCE LISTING <110> Adverum Biotechnologies, Inc. <120> METHODS OF TREATING OCULAR NEOVASCULAR DISEASES USING AAV2 VARIANTS ENCODING AFLIBERCEPT <130> 62700-20012.41 <140> Not Yet Assigned <141> Concurrently Herewith <150> PCT/US2019/062066 <151> 2019-11-18 <150> US 63/063,203 <151> 2020-08-07 <150> US 63/030,819 <151> 2020-05-27 <150> US 63/019,190 <151> 2020-05-01 <150> US 62/971,835 < 151> 2020-02-07 <150> US 62/959,784 <151> 2020-01-10 <150> US 62/913,648 <151> 2019-10-10 <150> US 62/899,070 <151> 2019-09 -11 <160> 41 <170> FastSEQ for Windows Version 4.0 <210> 1 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 1 Leu Ala Leu Gly Glu Thr Thr Arg Pro Ala 1 5 10 <210> 2 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 2 Leu Ala Asn Glu Thr Ile Thr Arg Pro Ala 1 5 10 <210 > 3 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 3 Leu Ala Lys Ala Gly Gln Ala Asn Asn Ala 1 5 10 <210> 4 <211> 10 <212 > P RT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 4 Leu Ala Lys Asp Pro Lys Thr Thr Asn Ala 1 5 10 <210> 5 <211> 7 <212> PRT <213> Artificial Sequence <220 > <223> Synthetic Construct <400> 5 Lys Asp Thr Asp Thr Thr Arg 1 5 <210> 6 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 6 Arg Ala Gly Gly Ser Val Gly 1 5 <210> 7 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 7 Ala Val Asp Thr Thr Lys Phe 1 5 <210> 8 < 211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 8 Ser Thr Gly Lys Val Pro Asn 1 5 <210> 9 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 9 Leu Ala Lys Asp Thr Asp Thr Thr Arg Ala 1 5 10 <210> 10 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 10 Leu Ala Arg Ala Gly Gly Ser Val Gly Ala 1 5 10 <210> 11 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 11 Leu Ala Ala Val Asp Thr Thr Lys Phe Ala 1 5 10 <210> 12 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 12 Leu Ala Ser Thr Gly Lys Val Pro Asn Ala 1 5 10 <210> 13 <211> 735 <212> PRT <213> Adeno-associated virus - 2 <400> 13 Met Ala Ala Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Thr Leu Ser 1 5 10 15 Glu Gly Ile Arg Gln Trp Trp Lys Leu Lys Pro Gly Pro Pro Pro Pro 20 25 30 Lys Pro Ala Glu Arg His Lys Asp Asp Ser Arg Gly Leu Val Leu Pro 35 40 45 Gly Tyr Lys Tyr Leu Gly Pro Phe Asn Gly Leu Asp Lys Gly Glu Pro 50 55 60 Val Asn Glu Ala Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp 65 70 75 80 Arg Gln Leu Asp Ser Gly Asp Asn Pro Tyr Leu Lys Tyr Asn His Ala 85 90 95 Asp Ala Glu Phe Gln Glu Arg Leu Lys Glu Asp Thr Ser Phe Gly Gly 100 105 110 Asn Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg Val Leu Glu P ro 115 120 125 Leu Gly Leu Val Glu Glu Pro Val Lys Thr Ala Pro Gly Lys Lys Arg 130 135 140 Pro Val Glu His Ser Pro Val Glu Pro Asp Ser Ser Ser Gly Thr Gly 145 150 155 160 Lys Ala Gly Gln Gln Pro Ala Arg Lys Arg Leu Asn Phe Gly Gln Thr 165 170 175 Gly Asp Ala Asp Ser Val Pro Asp Pro Gln Pro Leu Gly Gln Pro Pro 180 185 190 Ala Ala Pro Ser Gly Leu Gly Thr Asn Thr Met Ala Thr Gly Ser Gly 195 200 205 Ala Pro Met Ala Asp Asn Asn Glu Gly Ala Asp Gly Val Gly Asn Ser 210 215 220 Ser Gly Asn Trp His Cys Asp Ser Thr Trp Met Gly Asp Arg Val Ile 225 230 235 240 Thr Thr Ser Thr Arg Thr Trp Ala Leu Pro Thr Tyr Asn Asn His Leu 245 250 255 Tyr Lys Gln Ile Ser Ser Gln Ser Gly Ala Ser Asn A sp Asn His Tyr 260 265 270 Phe Gly Tyr Ser Thr Pro Trp Gly Tyr Phe Asp Phe Asn Arg Phe His 275 280 285 Cys His Phe Ser Pro Arg Asp Trp Gln Arg Leu Ile Asn Asn Asn Trp 290 295 295 300 Gly Phe Arg Pro Lys Arg Leu Asn Phe Lys Leu Phe Asn Ile Gln Val 305 310 315 320 Lys Glu Val Thr Gln Asn Asp Gly Thr Thr Thr Ile Ala Asn Asn Leu 325 330 335 Thr Ser Thr Val Gln Val Phe Thr Asp Ser Glu Tyr Gln Leu Pro Tyr 340 345 350 Val Leu Gly Ser Ala His Gln Gly Cys Leu Pro Pro Phe Pro Ala Asp 355 360 365 Val Phe Met Val Pro Gln Tyr Gly Tyr Leu Thr Leu Asn Asn Gly Ser 370 375 380 Gln Ala Val Gly Arg Ser Ser Phe Tyr Cys Leu Glu Tyr Phe Pro Ser 385 390 395 400 Gln Met Leu Arg Thr Gly Asn Asn Phe T hr Phe Ser Tyr Thr Phe Glu 405 410 415 Asp Val Pro Phe His Ser Ser Tyr Ala His Ser Gln Ser Leu Asp Arg 420 425 430 Leu Met Asn Pro Leu Ile Asp Gln Tyr Leu Tyr Tyr Leu Ser Arg Thr 435 440 445 Asn Thr Pro Ser Gly Thr Thr Thr Gln Ser Arg Leu Gln Phe Ser Gln 450 455 460 Ala Gly Ala Ser Asp Ile Arg Asp Gln Ser Arg Asn Trp Leu Pro Gly 465 470 475 480 Pro Cys Tyr Arg Gln Gln Arg Val Ser Lys Thr Ser Ala Asp Asn Asn 485 490 495 Asn Ser Glu Tyr Ser Trp Thr Gly Ala Thr Lys Tyr His Leu Asn Gly 500 505 510 Arg Asp Ser Leu Val Asn Pro Gly Pro Ala Met Ala Ser His Lys Asp 515 520 525 Asp Glu Glu Lys Phe Phe Pro Gln Ser Gly Val Leu Ile Phe Gly Lys 530 535 540 Gln Gly Ser Glu Lys Thr Asn Val Asp Ile Glu L ys Val Met Ile Thr 545 550 555 560 Asp Glu Glu Glu Ile Arg Thr Thr Asn Pro Val Ala Thr Glu Gln Tyr 565 570 575 Gly Ser Val Ser Thr Asn Leu Gln Arg Gly Asn Arg Gln Ala Ala Thr 580 585 590 Ala Asp Val Asn Thr Gln Gly Val Leu Pro Gly Met Val Trp Gln Asp 595 600 605 Arg Asp Val Tyr Leu Gln Gly Pro Ile Trp Ala Lys Ile Pro His Thr 610 615 620 Asp Gly His Phe His Pro Ser Pro Leu Met Gly Gly Phe Gly Leu Lys 625 630 635 640 His Pro Pro Pro Gln Ile Leu Ile Lys Asn Thr Pro Val Pro Ala Asn 645 650 655 Pro Ser Thr Thr Phe Ser Ala Ala Lys Phe Ala Ser Phe Ile Thr Gln 660 665 670 Tyr Ser Thr Gly Gln Val Ser Val Glu Ile Glu Trp Glu Leu Gln Lys 675 680 685 Glu Asn Ser Lys Arg Trp Asn Pro G lu Ile Gln Tyr Thr Ser Asn Tyr 690 695 700 Asn Lys Ser Val Asn Val Asp Phe Thr Val Asp Thr Asn Gly Val Tyr 705 710 715 720 Ser Glu Pro Arg Pro Ile Gly Thr Arg Tyr Leu Thr Arg Asn Leu 725 730 735 < 210> 14 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 14 Leu Gly Glu Thr Thr Arg Pro 1 5 <210> 15 <211> 7 <212> PRT < 213> Artificial Sequence <220> <223> Synthetic Construct <400> 15 Asn Glu Thr Ile Thr Arg Pro 1 5 <210> 16 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 16 Lys Ala Gly Gln Ala Asn Asn 1 5 <210> 17 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 17 Lys Asp Pro Lys Thr Thr Asn 1 5 <210> 18 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 18 Lys Asp Thr Asp Thr Thr Arg 1 5 <210> 19 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 19 Arg Ala Gly Gly Ser Val Gly 1 5 <210> 20 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 20 Ala Val Asp Thr Thr Lys Phe 1 5 <210> 21 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 21 Ser Thr Gly Lys Val Pro Asn 1 5 <210> 22 <211> 293 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Construct <400> 22 acttacggta aatggcccgc ctggctgacc gcccaacgac ccccgcccat tgacgtcaat 60 aatgacgtat gttcccatag taacgccaat agggactttc cattgacgtc aatgggtgga 120 gtatttacgg taaactgccc acttggcagt acatcaagtg tatcatatgc caagtccgcc 180 ccctattgac gtcaatgacg gtaaatggcc cgcctggcat tatgcccagt acatgacctt 240 acgggacttt cctacttggc agtacatcta cgtattagtc atcgctatta cca 293 <210> 23 <211> 220 <212> DNA <213> Artificial Sequence <220> < 223> Synthetic Construct <400> 23 tgctgatgcg gttttggcag tacaccaatg ggcgtg gata gcggtttgac tcacggggat 60 ttccaagtct ccaccccatt gacgtcaatg ggagtttgtt ttggcaccaa aatcaacggg 120 actttccaaa atgtcgtaat aaccccgccc cgttgacgca aatgggcggt aggcgtgtac 180 ggtgggaggt ctatataagc agagctcgtt tagtgaaccg 220 <210> 24 <211> 318 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Construct <400 > 24 ctcactctct tccgcatcgc tgtctgcgag ggccagctgt tgggctcgcg gttgaggaca 60 aactcttcgc ggtctttcca gtactcttgg atcggaaacc cgtcggcctc cgaacggtac 120 tccgccaccg agggacctga gcgagtccgc atcgaccgga tcggaaaacc tctcgagaaa 180 ggcgtctaac cagtcacagt cgcaaggtag gctgagcacc gtggcgggcg gcagcgggtg 240 gcggtcgggg ttgtttctgg cggaggtgct gctgatgatg taattaaagt aggcggtctt 300 gagacggcgg atggtcga 318 <210> 25 <211> 102 <212 > DNA <213> Artificial Sequence <220> <223> Synthetic Construct <400> 25 ccagctgttg gggtgagtac tccctctcaa aagcgggcat tacttctgcg ctaagattgt 60 cagtttccaa aaacgaggag gatttgatat 213 Sequence tcacctggcc <211> <210> DNA 26 Artificial cg < 810 <212> 220> <223> Synthetic Construct <400> 26 c tgttctcat cacatcatat caaggttata taccatcaat attgccacag atgttactta 60 gccttttaat atttctctaa tttagtgtat atgcaatgat agttctctga tttctgagat 120 tgagtttctc atgtgtaatg attatttaga gtttctcttt catctgttca aatttttgtc 180 tagttttatt ttttactgat ttgtaagact tctttttata atctgcatat tacaattctc 240 tttactgggg tgttgcaaat attttctgtc attctatggc ctgacttttc ttaatggttt 300 tttaatttta aaaataagtc ttaatattca tgcaatctaa ttaacaatct tttctttgtg 360 gttaggactt tgagtcataa gaaatttttc tctacactga agtcatgatg gcatgcttct 420 atattatttt ctaaaagatt taaagttttg ccttctccat ttagacttat aattcactgg 480 aatttttttg tgtgtatggt atgacatatg ggttcccttt tattttttac atataaatat 540 atttccctgt ttttctaaaa aagaaaaaga tcatcatttt cccattgtaa aatgccatat 600 ttttttcata ggtcacttac atatatcaat gggtctgttt ctgagctcta ctctatttta 660 tcagcctcac tgtctatccc cacacatctc atgctttgct ctaaatcttg atatttagtg 720 gaacattctt tcccattttg ttctacaaga atatttttgt tattgtcttt gggctttcta 780 tatacatttt gaaatgaggt tgacaagtta 810 <210> 27 < 211> 202 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Construct <400> 27 ctgcccgggt ggcatccctg tgacccctcc ccagtgcctc tcctggccct ggaagttgcc 60 actccagtgc ccaccagcct tgtcctaata aaattaagtt gcatcatttt gtctgactag 120 gtgtccttct ataatattat ggggtggagg ggggtggtat ggagcaaggg gcccaagttg 180 ggaagaaacc tgtagggcct gc 202 <210> 28 <211> 68 <212> DNA < 213> Artificial Sequence <220> <223> Synthetic Construct <400> 28 aggcggtctt gagacggcgg atggtcgagg tgaggtgtgg caggcttgag atccagctgt 60 tggggtga 68 <210> 29 <211> 129 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Construct <400> 29 cgctgttttg acctccatag tggacaccgg gaccgatcca gcctccgcgt ctcaggggag 60 atctcgttta gtgaaccgtc agatcctcac tctcttccgc atcgctgtct gcgagggcca 120 gctgtttg cctggcccga tctggccata cacttg 36 <210> 31 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Construct <400> 31 cccaggtcca agtttaaacg cc 22 <210> 32 <211> 77 <212> DNA < 213> A rtificial Sequence <220> <223> Synthetic Construct <400> 32 tctttgggct ttctatatac attttgaaat gaggttgaca agttacctag gaaaactgtc 60 ttcctgcccg ggtggca 77 <210> 33 <211> 1172 <212> DNA <213> Artificial Sequence 400> 33 ctctggagac gacttacggt aaatggcccg cctggctgac cgcccaacga cccccgccca 60 ttgacgtcaa taatgacgta tgttcccata gtaacgccaa tagggacttt ccattgacgt 120 caatgggtgg agtatttacg gtaaactgcc cacttggcag tacatcaagt gtatcatatg 180 ccaagtccgc cccctattga cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag 240 tacatgacct tacgggactt tcctacttgg cagtacatct acgtattagt catcgctatt 300 accatgctga tgcggttttg gcagtacacc aatgggcgtg gatagcggtt tgactcacgg 360 ggatttccaa gtctccaccc cattgacgtc aatgggagtt tgttttggca ccaaaatcaa 420 cgggactttc caaaatgtcg taataacccc gccccgttga cgcaaatggg cggtaggcgt 480 gtacggtggg aggtctatat aagcagagct cgtttagtga accgtcagat cgcctggaga 540 ggccatccac gctgttttga cctccatagt ggacaccggg accgatccag cctccgcgtc 600 tcaggggaga tctcgtttag tgaaccgtca gatcctcact ctcttccgc a tcgctgtctg 660 cgagggccag ctgttgggct cgcggttgag gacaaactct tcgcggtctt tccagtactc 720 ttggatcgga aacccgtcgg cctccgaacg gtactccgcc accgagggac ctgagcgagt 780 ccgcatcgac cggatcggaa aacctctcga gaaaggcgtc taaccagtca cagtcgcaag 840 gtaggctgag caccgtggcg ggcggcagcg ggtggcggtc ggggttgttt ctggcggagg 900 tgctgctgat gatgtaatta aagtaggcgg tcttgagacg gcggatggtc gaggtgaggt 960 gtggcaggct tgagatccag ctgttggggt gagtactccc tctcaaaagc gggcattact 1020 tctgcgctaa gattgtcagt ttccaaaaac gaggaggatt tgatattcac ctggcccgat 1080 ctggccatac acttgagtga caatgacatc cactttgcct ttctctccac aggtgtccac 1140 tcccaggtcc aagtttaaac gccgccacca tg 1172 <210> 34 <211> 1043 <212> DNA <213> Synthetic Sequence t cagtgstruct <acacat ca cata t 34 t tagstruct <ttacat t ac t t t agstruct <ttacat tgat tgact tagstruct <ttacct 223> agccttttaa tatttctcta atttagtgta tatgcaatga tagttctctg atttctgaga 120 ttgagtttct catgtgtaat gattatttag agtttctctt tcatctgttc aaatttttgt 180 ctagttttat ttttttactact ttatttt ttat attatactga tctttg tactggg gtgttgcaaa tattttctgt cattctatgg cctgactttt cttaatggtt 300 ttttaatttt aaaaataagt cttaatattc atgcaatcta attaacaatc ttttctttgt 360 ggttaggact ttgagtcata agaaattttt ctctacactg aagtcatgat ggcatgcttc 420 tatattattt tctaaaagat ttaaagtttt gccttctcca tttagactta taattcactg 480 gaattttttt gtgtgtatgg tatgacatat gggttccctt ttatttttta catataaata 540 tatttccctg tttttctaaa aaagaaaaag atcatcattt tcccattgta aaatgccata 600 tttttttcat aggtcactta catatatcaa tgggtctgtt tctgagctct actctatttt 660 atcagcctca ctgtctatcc ccacacatct catgctttgc tctaaatctt gatatttagt 720 ggaacattct ttcccatttt gttctacaag aatatttttg ttattgtctt tgggctttct 780 atatacattt tgaaatgagg ttgacaagtt acctaggaaa actgtcttcc tgcccgggtg 840 gcatccctgt gacccctccc cagtgcctct cctggccctg gaagttgcca ctccagtgcc 900 caccagcctt gtcctaataa aattaagttg catcattttg tctgactagg tgtccttcta 960 taatattatg gggtggaggg gggtggtatg gagcaagggg cccaagttgg gaagaaacct 1020 gtagggcctg cgaagacagt cag 1043 <210> 35 < 211> 431 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 35 Ser Asp Thr Gly Arg Pro Phe Val Glu Met Tyr Ser Glu Ile Pro Glu 1 5 10 15 Ile Ile His Met Thr Glu Gly Arg Glu Leu Val Ile Pro Cys Arg Val 20 25 30 Thr Ser Pro Asn Ile Thr Val Thr Leu Lys Lys Phe Pro Leu Asp Thr 35 40 45 Leu Ile Pro Asp Gly Lys Arg Ile Ile Trp Asp Ser Arg Lys Gly Phe 50 55 60 Ile Ile Ser Asn Ala Thr Tyr Lys Glu Ile Gly Leu Leu Thr Cys Glu 65 70 75 80 Ala Thr Val Asn Gly His Leu Tyr Lys Thr Asn Tyr Leu Thr His Arg 85 90 95 Gln Thr Asn Thr Ile Ile Asp Val Val Leu Ser Pro Ser His Gly Ile 100 105 110 Glu Leu Ser Val Gly Glu Lys Leu Val Leu Asn Cys Thr Ala Arg Thr 115 120 125 Glu Leu Asn Val Gly Ile Asp Phe Asn Trp Glu Tyr Pro Ser Ser Lys 130 135 140 His Gln His Lys Lys Leu Val Asn Arg Asp Leu Lys Thr Gln Ser Gly 145 150 155 160 Ser Glu Met Lys Lys Phe Leu Ser Thr Leu Thr Ile Asp Gly Val Thr 165 170 175 Arg Ser Asp Gln Gly Leu Tyr Thr Cys Ala Ala Ser Ser Gly Leu Met 180 185 190 Thr Lys Lys Asn Ser Thr Phe Val Arg Val His Glu Lys Asp Lys Thr 195 200 205 His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser 210 215 220 Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg 225 230 235 240 Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro 245 250 255 Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 260 265 270 Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val 275 280 285 Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 290 295 300 Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr 305 310 315 320 Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu 325 330 335 Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys 340 345 350 Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 355 360 365 Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp 370 375 380 Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser 385 390 395 400 Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 405 410 415 Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 420 425 430 <210> 36 <211> 1377 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Construct <400> 36 atggtcagct actgggacac cggggtcctg ctgtgcgcgc tgctcagctg tctgcttctc 60 acaggatcta gttccggaag tga taccggt agacctttcg tagagatgta cagtgaaatc 120 cccgaaatta tacacatgac tgaaggaagg gagctcgtca ttccctgccg ggttacgtca 180 cctaacatca ctgttacttt aaaaaagttt ccacttgaca ctttgatccc tgatggaaaa 240 cgcataatct gggacagtag aaagggcttc atcatatcaa atgcaacgta caaagaaata 300 gggcttctga cctgtgaagc aacagtcaat gggcatttgt ataagacaaa ctatctcaca 360 catcgacaaa ccaatacaat catagatgtg gttctgagtc cgtctcatgg aattgaacta 420 tctgttggag aaaagcttgt cttaaattgt acagcaagaa ctgaactaaa tgtggggatt 480 gacttcaact gggaataccc ttcttcgaag catcagcata agaaacttgt aaaccgagac 540 ctaaaaaccc agtctgggag tgagatgaag aaatttttga gcaccttaac tatagatggt 600 gtaacccgga gtgaccaagg attgtacacc tgtgcagcat ccagtgggct gatgaccaag 660 aagaacagca catttgtcag ggtccatgaa aaggacaaaa ctcacacatg cccaccgtgc 720 ccagcacctg aactcctggg gggaccgtca gtcttcctct tccccccaaa acccaaggac 780 accctcatga tctcccggac ccctgaggtc acatgcgtgg tggtggacgt gagccacgaa 840 gaccctgagg tcaagttcaa ctggtacgtg gacggcgtgg aggtgcataa tgccaagaca 900 aagccgcggg aggagcagta caacagcacg taccgtgtgg t cagcgtcct caccgtcctg 960 caccaggact ggctgaatgg caaggagtac aagtgcaagg tctccaacaa agccctccca 1020 gcccccatcg agaaaaccat ctccaaagcc aaagggcagc cccgagaacc acaggtgtac 1080 accctgcccc catcccggga tgagctgacc aagaaccagg tcagcctgac ctgcctggtc 1140 aaaggcttct atcccagcga catcgccgtg gagtgggaga gcaatgggca gccggagaac 1200 aactacaaga ccacgcctcc cgtgctggac tccgacggct ccttcttcct ctacagcaag 1260 ctcaccgtgg acaagagcag gtggcagcag gggaacgtct tctcatgctc cgtgatgcat 1320 gaggctctgc acaaccacta cacgcagaag agcctctccc tgtctccggg taaatga 1377 <210> 37 <211> 745 <212> PRT <213> Adeno-associated virus - 2 <400> 37 Met Ala Ala Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Thr Leu Ser 1 5 10 15 Glu Gly Ile Arg Gln Trp Trp Lys Leu Lys Pro Gly Pro Pro Pro Pro 20 25 30 Lys Pro Ala Glu Arg His Lys Asp Asp Ser Arg Gly Leu Val Leu Pro 35 40 45 Gly Tyr Lys Tyr Leu Gly Pro Phe Asn Gly Leu Asp Lys Gly Glu Pro 50 55 60 Val Asn Glu Ala Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp 65 70 75 80 Arg Gln Leu Asp Ser Gly Asp Asn Pro Tyr Leu Lys Tyr Asn His Ala 85 90 95 Asp Ala Glu Phe Gln Glu Arg Leu Lys Glu Asp Thr Ser Phe Gly Gly 100 105 110 Asn Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg Val Leu Glu Pro 115 120 125 Leu Gly Leu Val Glu Glu Pro Val Lys Thr Ala Pro Gly Lys Lys Arg 130 135 140 Pro Val Glu His Ser Pro Val Glu Pro Asp Ser Ser Ser Gly Thr Gly 145 150 155 160 Lys Ala Gly Gln Gln Pro Ala Arg Lys Arg Leu Asn Phe Gly Gln Thr 165 170 175 Gly Asp Ala Asp Ser Val Pro Asp Pro Gln Pro Leu Gly Gln Pro Pro 180 185 190 Ala Ala Pro Ser Gly Leu Gly Thr Asn Thr Met Ala Thr Gly Ser Gly 195 200 205 Ala Pro Met Ala Asp Asn Asn Glu Gly Ala Asp Gly Val Gly Asn Ser 210 215 220 Ser Gly Asn Trp His Cys Asp Ser Thr Trp Met Gly Asp Arg Val Ile 225 230 235 240 Thr Thr Ser Thr Arg Thr Trp Ala Leu Pro Thr Tyr Asn Asn His Leu 245 250 255 Tyr Lys Gln Ile Ser Ser Gln Ser Gly Ala Ser Asn Asp Asn His Tyr 260 265 270 Phe Gly Tyr Ser Thr Pro Trp Gly Tyr Phe Asp Phe Asn Arg Phe His 275 280 285 Cys His Phe Ser Pro Arg Asp Trp Gln Arg Leu Ile Asn Asn Asn Trp 290 295 300 Gly Phe Arg Pro Lys Arg Leu Asn Phe Lys Leu Phe Asn Ile Gln Val 305 310 315 320 Lys Glu Val Thr Gln Asn Asp Gly Thr Thr Thr Ile Ala Asn Asn Leu 325 330 335 Thr Ser Thr Val Gln Val Phe Thr Asp Ser Glu Tyr Gln Leu Pro Tyr 340 345 350 Val Leu Gly Ser Ala His Gln Gly Cys Leu Pro Pro Phe Pro Ala Asp 355 360 365 Val Phe Met Val Pro Gln Tyr Gly Tyr Leu Thr Leu Asn Asn Gl y Ser 370 375 380 Gln Ala Val Gly Arg Ser Ser Phe Tyr Cys Leu Glu Tyr Phe Pro Ser 385 390 395 400 Gln Met Leu Arg Thr Gly Asn Asn Phe Thr Phe Ser Tyr Thr Phe Glu 405 410 415 Asp Val Pro Phe His Ser Ser Tyr Ala His Ser Gln Ser Leu Asp Arg 420 425 430 Leu Met Asn Pro Leu Ile Asp Gln Tyr Leu Tyr Tyr Leu Ser Arg Thr 435 440 445 Asn Thr Pro Ser Gly Thr Thr Thr Gln Ser Arg Leu Gln Phe Ser Gln 450 455 460 Ala Gly Ala Ser Asp Ile Arg Asp Gln Ser Arg Asn Trp Leu Pro Gly 465 470 475 480 Pro Cys Tyr Arg Gln Gln Arg Val Ser Lys Thr Ser Ala Asp Asn Asn 485 490 495 Asn Ser Glu Tyr Ser Trp Thr Gly Ala Thr Lys Tyr His Leu Asn Gly 500 505 510 Arg Asp Ser Leu Val Asn Pro Gly Pro Ala Met Al a Ser His Lys Asp 515 520 525 Asp Glu Glu Lys Phe Phe Pro Gln Ser Gly Val Leu Ile Phe Gly Lys 530 535 540 Gln Gly Ser Glu Lys Thr Asn Val Asp Ile Glu Lys Val Met Ile Thr 545 550 555 560 Asp Glu Glu Glu Ile Arg Thr Thr Asn Pro Val Ala Thr Glu Gln Tyr 565 570 575 Gly Ser Val Ser Thr Asn Leu Gln Arg Gly Asn Leu Ala Leu Gly Glu 580 585 590 Thr Thr Arg Pro Ala Arg Gln Ala Ala Thr Ala Asp Val Asn Thr Gln 595 600 605 Gly Val Leu Pro Gly Met Val Trp Gln Asp Arg Asp Val Tyr Leu Gln 610 615 620 Gly Pro Ile Trp Ala Lys Ile Pro His Thr Asp Gly His Phe His Pro 625 630 635 640 Ser Pro Leu Met Gly Gly Phe Gly Leu Lys His Pro Pro Pro Gln Ile 645 650 655 Leu Ile Lys Asn Thr Pro Val Pro Al a Asn Pro Ser Thr Thr Phe Ser 660 665 670 Ala Ala Lys Phe Ala Ser Phe Ile Thr Gln Tyr Ser Thr Gly Gln Val 675 680 685 Ser Val Glu Ile Glu Trp Glu Leu Gln Lys Glu Asn Ser Lys Arg Trp 690 695 700 Asn Pro Glu Ile Gln Tyr Thr Ser Asn Tyr Asn Lys Ser Val Asn Val 705 710 715 720 Asp Phe Thr Val Asp Thr Asn Gly Val Tyr Ser Glu Pro Arg Pro Ile 725 730 735 Gly Thr Arg Tyr Leu Thr Arg Asn Leu 740 745 < 210> 38 <211> 250 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 38 Phe Ser Tyr Thr Phe Glu Asp Val Pro Phe His Ser Ser Tyr Ala His 1 5 10 15 Ser Gln Ser Leu Asp Arg Leu Met Asn Pro Leu Ile Asp Gln Tyr Leu 20 25 30 Tyr Tyr Leu Ser Arg Thr Asn Thr Pro Ser Gly Thr Thr Thr Gln Ser 35 40 45 Arg Leu Gln Phe Ser Gln Ala Gly Ala Ser Asp Ile Arg Asp Gln Ser 50 55 60 Arg Asn Trp Leu Pro Gly Pro Cys Tyr Arg Gln Gln Arg Val Ser Lys 65 70 75 80 Thr Ser Ala Asp Asn Asn Asn Ser Glu Tyr Ser Trp Thr Gly Ala Thr 85 90 95 Lys Tyr His Leu Asn Gly Arg Asp Ser Leu Val Asn Pro Gly Pro Ala 100 105 110 Met Ala Ser His Lys Asp Asp Glu Glu Lys Phe Phe Pro Gln Ser Gly 115 120 125 Val Leu Ile Phe Gly Lys Gln Gly Ser Glu Lys Thr Asn Val Asp Ile 130 135 140 Glu Lys Val Met Ile Thr Asp Glu Glu Glu Ile Arg Thr Thr Asn Pro 145 150 155 160 Val Ala Thr Glu Gln Tyr Gly Ser Val Ser Thr Asn Leu Gln Arg Gly 165 170 175 Asn Leu Ala Leu Gly Glu Thr Thr Arg Pro Ala Arg Gln Ala Ala Thr 180 185 190 Ala Asp Val Asn Thr Gln Gly Val Leu Pro Gly Met Val Trp Gln Asp 195 200 205 Arg Asp Val Tyr Leu Gln Gly Pro Ile Trp Ala Lys Ile Pro His Thr 210 21 5 220 Asp Gly His Phe His Pro Ser Pro Leu Met Gly Gly Phe Gly Leu Lys 225 230 235 240 His Pro Pro Pro Gln Ile Leu Ile Lys Asn 245 250 <210> 39 <211> 3916 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Construct <400> 39 gcgcgctcgc tcgctcactg aggccgcccg ggcaaagccc gggcgtcggg cgacctttgg 60 tcgcccggcc tcagtgagcg agcgagcgcg cagagaggga gtggccaact ccatcactag 120 gggttccttg tagttaatga ttaacccgcc atgctactta tctacgtact ctggagacga 180 cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt gacgtcaata 240 atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca atgggtggag 300 tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc aagtccgccc 360 cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta catgacctta 420 cgggactttc ctacttggca gtacatctac gtattagtca tcgctattac catgctgatg 480 cggttttggc agtacaccaa tgggcgtgga tagcggtttg actcacgggg atttccaagt 540 ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg ggactttcca 600 aaatgtcgta ataaccccgc cccgttgacg caaatgggcg gtaggcgtgt acggtgggag 660 gtctatataa gcagagctcg tttagtgaac cgtcagatcg cctggagagg ccatccacgc 720 tgttttgacc tccatagtgg acaccgggac cgatccagcc tccgcgtctc aggggagatc 780 tcgtttagtg aaccgtcaga tcctcactct cttccgcatc gctgtctgcg agggccagct 840 gttgggctcg cggttgagga caaactcttc gcggtctttc cagtactctt ggatcggaaa 900 cccgtcggcc tccgaacggt actccgccac cgagggacct gagcgagtcc gcatcgaccg 960 gatcggaaaa cctctcgaga aaggcgtcta accagtcaca gtcgcaaggt aggctgagca 1020 ccgtggcggg cggcagcggg tggcggtcgg ggttgtttct ggcggaggtg ctgctgatga 1080 tgtaattaaa gtaggcggtc ttgagacggc ggatggtcga ggtgaggtgt ggcaggcttg 1140 agatccagct gttggggtga gtactccctc tcaaaagcgg gcattacttc tgcgctaaga 1200 ttgtcagttt ccaaaaacga ggaggatttg atattcacct ggcccgatct ggccatacac 1260 ttgagtgaca atgacatcca ctttgccttt ctctccacag gtgtccactc ccaggtccaa 1320 gtttaaacgc cgccaccatg gtgtcatact gggatactgg agtcttgctt tgtgccctgc 1380 tgtcctgcct cctcctgact ggctccagct cgggctcaga taccggtcgc cccttcgtgg 1440 agatgtactc cgagatcccg gaaattatcc acatgactga ggggcgcgaa cttgtgatcc 1500 cctgccgggt caccagcccg aacattactg tgactttgaa gaagttcccc ctggacaccc 1560 tgattccgga tgggaagaga attatctggg attcacggaa gggattcatc atcagcaacg 1620 cgacctacaa ggaaattggc ctcctcactt gcgaagccac tgtgaacgga cacttgtaca 1680 agaccaacta cctgacccac cgccaga cca acaccatcat cgacgtcgtc ctgtcccctt 1740 cgcacgggat cgagctctcg gtgggagaga agttggtgct taactgcacc gcccggacgg 1800 aactgaatgt gggaatcgac ttcaactggg aatacccgtc cagcaagcat cagcataaga 1860 agctggtgaa ccgggacctc aagactcagt ccggcagcga aatgaagaag ttcctgtcga 1920 ccctcactat tgacggagtg accagatccg accagggcct ctacacttgc gccgcttcca 1980 gcggactcat gaccaagaag aacagcactt tcgtgagggt gcatgagaag gacaagaccc 2040 acacgtgtcc gccgtgccca gccccagagc tgctgggagg cccttccgtg ttcctgtttc 2100 cgcccaagcc aaaggatacc ctgatgatct caaggacccc tgaggtcaca tgcgtcgtgg 2160 tggatgtgtc gcacgaggac cctgaagtca aattcaattg gtatgtggac ggagtggaag 2220 tccacaacgc gaaaaccaag ccgagagaag aacagtacaa ttccacctac cgggtggtgt 2280 cggtgctgac tgtgctgcac caggactggc tcaacggaaa ggagtacaag tgcaaggtgt 2340 ccaacaaggc tctgcccgca cctattgaaa agaccatctc caaggccaag ggtcaacctc 2400 gcgagcctca ggtgtacact ctgcctccaa gccgggacga actgactaag aaccaagtct 2460 ctctgacctg tttggtgaag ggcttctacc cgtcagacat cgcagtggag tgggagtcaa 2520 acggtcagcc ggagaacaac tacaaaacaa cc ccccccgt gctggactcc gacggctcct 2580 tcttcctgta ctccaagctt accgtggata agagccgctg gcaacagggc aacgtgtttt 2640 cctgctccgt catgcacgaa gccctgcaca accattatac ccagaagtcc ctgtcgctgt 2700 cccccgggaa atagtgactg ttctcatcac atcatatcaa ggttatatac catcaatatt 2760 gccacagatg ttacttagcc ttttaatatt tctctaattt agtgtatatg caatgatagt 2820 tctctgattt ctgagattga gtttctcatg tgtaatgatt atttagagtt tctctttcat 2880 ctgttcaaat ttttgtctag ttttattttt tactgatttg taagacttct ttttataatc 2940 tgcatattac aattctcttt actggggtgt tgcaaatatt ttctgtcatt ctatggcctg 3000 acttttctta atggtttttt aattttaaaa ataagtctta atattcatgc aatctaatta 3060 acaatctttt ctttgtggtt aggactttga gtcataagaa atttttctct acactgaagt 3120 catgatggca tgcttctata ttattttcta aaagatttaa agttttgcct tctccattta 3180 gacttataat tcactggaat ttttttgtgt gtatggtatg acatatgggt tcccttttat 3240 tttttacata taaatatatt tccctgtttt tctaaaaaag aaaaagatca tcattttccc 3300 attgtaaaat gccatatttt tttcataggt cacttacata tatcaatggg tctgtttctg 3360 agctctactc tattttatca gcctcactgt ctatcccc ac acatctcatg ctttgctcta 3420 aatcttgata tttagtggaa cattctttcc cattttgttc tacaagaata tttttgttat 3480 tgtctttggg ctttctatat acattttgaa atgaggttga caagttacct aggaaaactg 3540 tcttcctgcc cgggtggcat ccctgtgacc cctccccagt gcctctcctg gccctggaag 3600 ttgccactcc agtgcccacc agccttgtcc taataaaatt aagttgcatc attttgtctg 3660 actaggtgtc cttctataat attatggggt ggaggggggt ggtatggagc aaggggccca 3720 agttgggaag aaacctgtag ggcctgcgta cgtagataag tagcatggcg ggttaatcat 3780 taactacaag gaacccctag tgatggagtt ggccactccc tctctgcgcg ctcgctcgct 3840 cactgaggcc gggcgaccaa aggtcgcccg acgcccgggc tttgcccggg cggcctcagt 3900 gagcgagcga gcgcgc 3916 <210> 40 <211> 1377 <212> DNA <213> Artificial Sequence <400> 40 atggtgtcat actgggatac tggagtcttg ctttgtgccc tgctgtcctg cctcctcctg 60 actggctcca gctcgggctc agataccggt cgccccttcg tggagatgta ctccgagatc 120 ccggaaatta tccacatgac tgaggggcgc gaacttgtga tcccctgccg ggtcaccagc 180 ccgaacatta ctgtgacttt gaagaagttc cccctggaca ccctgattcc ggatgggaag 240 agaattatct gggattcacg gaagggattc atcatcagca acgcgaccta caaggaaatt 300 ggcctcctca cttgcgaagc cactgtgaac ggacacttgt acaagaccaa ctacctgacc 360 caccgccaga ccaacaccat catcgacgtc gtcctgtccc cttcgcacgg gatcgagctc 420 tcggtgggag agaagttggt gcttaactgc accgcccgga cggaactgaa tgtgggaatc 480 gacttcaact gggaataccc gtccagcaag catcagcata agaagctggt gaaccgggac 540 ctcaagactc agtccggcag cgaaatgaag aagttcctgt cgaccctcac tattgacgga 600 gtgaccagat ccgaccaggg cctctacact tgcgccgctt ccagcggact catgaccaag 660 aagaacagca ctttcgtgag ggtgcatgag aaggacaaga cccacacgtg tccgccgtgc 720 ccagccccag agctgctggg aggcccttcc gtgttcctgt ttccgcccaa gccaaaggat 780 accctgatga tctcaaggac ccctgaggtc acatgcgtcg tggtggatgt gtcgcaggag 840 gaccctgaag t caaattcaa ttggtatgtg gacggagtgg aagtccacaa cgcgaaaacc 900 aagccgagag aagaacagta caattccacc taccgggtgg tgtcggtgct gactgtgctg 960 caccaggact ggctcaacgg aaaggagtac aagtgcaagg tgtccaacaa ggctctgccc 1020 gcacctattg aaaagaccat ctccaaggcc aagggtcaac ctcgcgagcc tcaggtgtac 1080 actctgcctc caagccggga cgaactgact aagaaccaag tctctctgac ctgtttggtg 1140 aagggcttct acccgtcaga catcgcagtg gagtgggagt caaacggtca gccggagaac 1200 aactacaaaa caaccccccc cgtgctggac tccgacggct ccttcttcct gtactccaag 1260 cttaccgtgg ataagagccg ctggcaacag ggcaacgtgt tttcctgctc cgtcatgcac 1320 gaagccctgc acaaccatta tacccagaag tccctgtcgc tgtcccccgg Thrgaatag 1377 <210> 41 <211> 458 <212> PRT <213> Synthetic Val Tyr Val<223> As Met Ser Tyr Tyr <223> Artificial Sequence <220> <223> Leu Leu Cys Ala Leu Leu Ser 1 5 10 15 Cys Leu Leu Leu Thr Gly Ser Ser Ser Gly Ser Asp Thr Gly Arg Pro 20 25 30 Phe Val Glu Met Tyr Ser Glu Ile Pro Glu Ile Ile His Met Thr Glu 35 40 45 Gly Arg Glu Leu Val Ile Pro Cys Arg Val Thr Ser P ro Asn Ile Thr 50 55 60 Val Thr Leu Lys Lys Phe Pro Leu Asp Thr Leu Ile Pro Asp Gly Lys 65 70 75 80 Arg Ile Ile Trp Asp Ser Arg Lys Gly Phe Ile Ile Ser Asn Ala Thr 85 90 95 Tyr Lys Glu Ile Gly Leu Leu Thr Cys Glu Ala Thr Val Asn Gly His 100 105 110 Leu Tyr Lys Thr Asn Tyr Leu Thr His Arg Gln Thr Asn Thr Ile Ile 115 120 125 Asp Val Val Leu Ser Pro Ser His Gly Ile Glu Leu Ser Val Gly Glu 130 135 140 Lys Leu Val Leu Asn Cys Thr Ala Arg Thr Glu Leu Asn Val Gly Ile 145 150 155 160 Asp Phe Asn Trp Glu Tyr Pro Ser Ser Lys His Gln His Lys Lys Leu 165 170 175 Val Asn Arg Asp Leu Lys Thr Gln Ser Gly Ser Glu Met Lys Lys Phe 180 185 190 Leu Ser Thr Leu Thr Ile Asp Gly Val Thr Arg Ser Asp Gln Gly Leu 195 200 205 Tyr Thr Cys Ala Ala Ser Ser Gly Leu Met Thr Lys Lys Asn Ser Thr 210 215 220 Phe Val Arg Val His Glu Lys Asp Lys Thr His Thr Cys Pro Cys 225 230 235 240 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 245 250 255 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 260 265 270 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 275 280 285 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 290 295 300 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 305 310 315 320 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 325 330 335 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 340 345 350 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu 355 360 365 Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 370 375 380 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 385 390 395 400 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 405 410 415 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 420 425 430 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 435 440 445 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 450 455

Claims (106)

comprising administering to one eye of the individual a unit dose of no more than about 6 x 10 ¹¹ vector genome (vg) recombinant adeno-associated virus (rAAV) particles, wherein the individual is a human, wherein the rAAV particles are
a) a nucleic acid encoding a polypeptide comprising an amino acid sequence having at least about 95% identity to the amino acid sequence of SEQ ID NO: 35 and flanked by AAV2 inverted terminal repeats (ITRs), and
b) an AAV2 capsid protein comprising the amino acid sequence LGETTRP (SEQ ID NO: 14) inserted between positions 587 and 588 of the capsid protein, wherein the amino acid residue numbering corresponds to the AAV2 VP1 capsid protein
A method of treating an ocular neovascular disease in a subject, comprising: The method of claim 1 , wherein the retinal fluid in the eye of a subject having an ocular neovascular disease is reduced. A method comprising administering a unit dose of rAAV particles to one eye of a subject having an ocular neovascular disease, wherein the subject is a human, wherein the rAAV particles are
a) a nucleic acid encoding a polypeptide comprising an amino acid sequence having at least about 95% identity to the amino acid sequence of SEQ ID NO: 35 and flanked by AAV2 inverted terminal repeats (ITRs), and
b) an AAV2 capsid protein comprising the amino acid sequence LGETTRP (SEQ ID NO: 14) inserted between positions 587 and 588 of the capsid protein, wherein the amino acid residue numbering corresponds to the AAV2 VP1 capsid protein
A method of reducing retinal fluid in the eye of an individual having an ocular neovascular disease comprising a. 4. The method of claim 3, wherein the individual has received at least one anti-VEGF agent treatment within about 12 weeks prior to administration of the unit dose of the rAAV particles. 5. The method of claim 3 or 4, wherein the amount or presence of retinal fluid in one eye of the subject is refractory to prior treatment with the anti-VEGF agent. 6. The method of claim 4 or 5, wherein the anti-VEGF agent is aflibercept. 7. The method of any one of claims 2-6, wherein retinal fluid in one eye is reduced by at least about 60%. 8. The method of any one of claims 2-7, wherein the retinal fluid in one eye is reduced by about 80% compared to the retinal fluid level in one eye of the subject prior to administering the rAAV to the subject. 9. The method according to any one of claims 2 to 8, wherein the retinal fluid is subretinal fluid (SRF) or intraretinal fluid (IRF). 10. The method of any one of claims 3-9, wherein the unit dose of rAAV particles is no more than about 6×10 ¹¹ vector genomes per eye (vg/eye). (a) administering an anti-VEGF agent to one eye of the subject;
(b) administering to one eye of the subject a unit dose of no more than about 6 x 10 ¹¹ vector genomes (vg) recombinant adeno-associated virus (rAAV) particles after administration of the anti-VEGF agent, wherein the subject is a human , wherein the rAAV particles are
(i) a nucleic acid encoding a polypeptide comprising an amino acid sequence having at least about 95% identity to the amino acid sequence of SEQ ID NO: 35 and flanked by an AAV2 inverted terminal repeat (ITR), and
(ii) an AAV2 capsid protein comprising the amino acid sequence LGETTRP (SEQ ID NO: 14) inserted between positions 587 and 588 of the capsid protein, wherein the amino acid residue numbering corresponds to the AAV2 VP1 capsid protein
A method of treating an ocular neovascular disease in a subject, comprising: The method of claim 11 , comprising administering a unit dose of rAAV particles to one eye of the subject about 1 week or about 7 days after administration of the anti-VEGF agent. 13. The method of claim 11 or 12, comprising administering the anti-VEGF agent to one eye of the subject on day 1 and administering a unit dose of the rAAV particles to one eye of the subject on day 8. 14. The method of any one of claims 11-13, wherein the anti-VEGF agent comprises aflibercept. 15. The method of claim 14, wherein aflibercept is administered by intravitreal injection at a dose of about 2 mg. 16. The method of any one of claims 1-15, wherein the unit dose of the rAAV particles is from about 6 x 10 ¹⁰ to about 6 x 10 ¹¹ vector genomes per eye (vg/eye). 17. The method of any one of claims 1 to 16, wherein the unit dose of the rAAV particles is from about 6 x 10 ¹⁰ to about 2 x 10 ¹¹ vector genomes per eye (vg/eye). 17. The method of any one of claims 1-16, wherein the unit dose of the rAAV particles is from about 2 x 10 ¹¹ to about 6 x 10 ¹¹ vector genomes per eye (vg/eye). The method of claim 18 , wherein the unit dose of the rAAV particles is about 2×10 ¹¹ or about 6×10 ¹¹ vector genomes per eye (vg/eye). The method of claim 19 , wherein the unit dose of rAAV particles is about 2×10 ¹¹ vector genomes per eye (vg/eye). The method of claim 19 , wherein the unit dose of rAAV particles is about 6×10 ¹¹ vector genomes per eye (vg/eye). 22. The method of any one of claims 1-21, wherein the subject has one or more symptoms of an ocular neovascular disease in the contralateral eye. 23. The method of any one of claims 1-22, further comprising administering a unit dose of the rAAV particles to the contralateral eye of the subject. 24. The method of claim 23, wherein administering the unit dose of rAAV particles to the contralateral eye is up to about 2 weeks after administration of the unit dose of rAAV particles to one eye. 25. The method of claim 24,
(a) administering the unit dose of rAAV particles to the contralateral eye is on the same day as administering the unit dose of rAAV particles to one eye; or
(b) administering the unit dose of rAAV particles to the contralateral eye is from about 1 day to about 14 days after administration of the unit dose of rAAV particles to one eye;
Way. 26. The method of claim 24 or 25, wherein the unit dose of rAAV particles administered to the contralateral eye of the individual comprises vector genomes per eye (vg/eye) equal to or less than the unit dose of rAAV particles administered to one eye of the individual. how to do it. 24. The method of claim 23, wherein administering the unit dose of rAAV particles to the contralateral eye is at least about 2 weeks after administration of the unit dose of rAAV particles to one eye. 28. The method of claim 27, wherein 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 one eye of the individual. 29. The method of any one of claims 1-28, wherein the nucleic acid comprises the nucleic acid sequence of SEQ ID NO: 40 or a sequence having at least 85% identity thereto. 30. The method of any one of claims 1-29, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO:35. 31. The method of any one of claims 1-30, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO:41. 32. The method of any one of claims 1-31, wherein the polypeptide is aflibercept. 33. The method of any one of claims 1-32, wherein the nucleic acid further comprises a first enhancer region, a promoter region, a 5'UTR region, a second enhancer region and a polyadenylation site. 34. The method of any one of claims 1-33, wherein the nucleic acid is in 5' to 3' order.
(a) a first enhancer region;
(b) a promoter region;
(c) a 5'UTR region;
(d) a nucleic acid encoding a polypeptide comprising an amino acid sequence having at least about 95% identity to the amino acid sequence of SEQ ID NO:35;
(e) a second enhancer region; and
(f) polyadenylation sites
wherein the AAV2 inverted terminal repeat (ITR) is flanked. 35. The method of claim 33 or 34, wherein the first enhancer region comprises a CMV sequence comprising the sequence of SEQ ID NO: 22 or a sequence having at least 85% identity thereto. 36. The method according to any one of claims 33 to 35, wherein the promoter region comprises a CMV sequence comprising the sequence of SEQ ID NO: 23 or a sequence having at least 85% identity thereto. 37. The method of any one of claims 34-36, wherein the nucleic acid encoding the polypeptide comprises the nucleic acid sequence of SEQ ID NO: 40 or a sequence having at least 85% identity thereto. 38. The method of any one of claims 34-37, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 35 or a sequence having at least 95% identity thereto. 39. The method of any one of claims 34-38, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 41 or a sequence having at least 95% identity thereto. 40. The method of any one of claims 34-39, wherein the polypeptide is aflibercept. 41. The TPL sequence and sequence according to any one of claims 33 to 40, wherein the 5'UTR region comprises in 5' to 3' order the sequence of SEQ ID NO: 24 or a sequence having at least 85% identity thereto. A method comprising an eMLP sequence comprising the sequence of identification number 25 or a sequence having at least 85% identity thereto. 42. The method of any one of claims 33-41, wherein the second enhancer region comprises the entire EES sequence comprising the sequence of SEQ ID NO:26 or a sequence having at least 85% identity thereto. 43. The method of any one of claims 33-42, wherein the polyadenylation site comprises an HGH polyadenylation site comprising the sequence of SEQ ID NO: 27 or a sequence having at least 85% identity thereto. . 33. The method of any one of claims 1-32, wherein the nucleic acid 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 TPL sequence comprising, in 5' to 3' order, the sequence of SEQ ID NO: 24 or a sequence having at least 85% identity thereto and the sequence of SEQ ID NO: 25 or a sequence having at least 85% identity thereto a 5'UTR region comprising an eMLP sequence comprising; (d) a second enhancer region comprising the entire EES sequence comprising the sequence of SEQ ID NO:26 or a sequence having at least 85% identity thereto; and (e) an HGH polyadenylation site comprising the sequence of SEQ ID NO: 27 or a sequence having at least 85% identity thereto. 45. The method of any one of claims 1-44, wherein the nucleic acid comprises the sequence of SEQ ID NO:39 or a sequence having at least 85% identity thereto. 46. The method of any one of claims 1-45, wherein the AAV2 capsid protein comprises the amino acid sequence LALGETTRPA (SEQ ID NO: 1) inserted between positions 587 and 588 of the capsid protein, wherein the amino acid residue numbering is AAV2 VP1 corresponding to a capsid protein. 47. The method of any one of claims 1-46, wherein the AAV2 capsid protein comprises the amino acid sequence LGETTRP (SEQ ID NO: 14) inserted between positions 587 and 588 of AAV2 VP1 comprising the sequence of SEQ ID NO: 13 How to include. 48. The method of any one of claims 1-47, wherein the AAV2 capsid protein comprises the amino acid sequence LALGETTRPA (SEQ ID NO: 1) inserted between positions 587 and 588 of AAV2 VP1 comprising the sequence of SEQ ID NO: 13 How to include. 47. The method of any one of claims 1-46, wherein the rAAV particle comprises a GH loop comprising the amino acid sequence of SEQ ID NO:38 or an amino acid sequence having at least 90% sequence identity to SEQ ID NO:38. A method comprising an AAV2 VP1 capsid protein comprising: 50. The method of any one of claims 1-49, wherein administering the unit dose of the rAAV particles to one and/or the contralateral eye is by intravitreal administration. 51. The method of any one of claims 1-50, wherein the unit dose of the rAAV particles is in the pharmaceutical formulation. 52. The method of claim 51, wherein the pharmaceutical formulation comprises rAAV particles, sodium chloride, sodium phosphate and a surfactant. 53. The method of claim 52, wherein the pharmaceutical formulation is about 150 to about 200 mM sodium chloride, about 1 to about 10 mM sodium phosphate monobasic, about 1 to about 10 mM sodium phosphate dibasic, about 0.0005% (w/v) to about 0.005 % (w/v) poloxamer 188 and from about 6 x 10 ¹³ to about 6 x 10 ¹⁰ vector genomes (vg) (vg/mL) per mL of rAAV particles, wherein the pharmaceutical formulation comprises from about 7.0 to about 7.5 of rAAV particles. having a pH. 54. The method of claim 53, wherein the pharmaceutical formulation comprises about 180 mM sodium chloride, about 5 mM sodium phosphate monobasic, about 5 mM sodium phosphate dibasic, about 6 x 10 ¹² vg/mL of rAAV particles and about 0.001% (w/v) A method comprising poloxamer 188, wherein the pharmaceutical formulation has a pH of about 7.3. 54. The method of claim 53, wherein the pharmaceutical formulation comprises about 180 mM sodium chloride, about 5 mM sodium phosphate monobasic, about 5 mM sodium phosphate dibasic, about 2 x 10 ¹² vg/mL of rAAV particles and about 0.001% (w/v) A method comprising poloxamer 188, wherein the pharmaceutical formulation has a pH of about 7.3. 54. The method of claim 53, wherein the pharmaceutical formulation comprises about 180 mM sodium chloride, about 5 mM sodium phosphate monobasic, about 5 mM sodium phosphate dibasic, about 6 x 10 ¹¹ vg/mL of rAAV particles and about 0.001% (w/v) A method comprising poloxamer 188, wherein the pharmaceutical formulation has a pH of about 7.3. 57. The method of any one of claims 1-56, wherein the unit dose of rAAV particles is administered to one eye and/or the contralateral eye in a volume of about 25 μL to about 250 μL. 58. The method of claim 57, wherein the unit dose of rAAV particles administered to one eye and/or the contralateral eye comprises a volume of about 100 μL. 58. The method of claim 57, wherein the unit dose of rAAV particles administered to one eye and/or the contralateral eye comprises a volume of about 30 μL. 60. The method of any one of claims 1-59, wherein the subject has received prior treatment for ocular neovascular disease with an anti-VEGF agent. 61. The method of claim 60, wherein the subject has received one or two injections of the anti-VEGF agent in one eye and/or the contralateral eye prior to administration of the rAAV particles to the eye and/or the contralateral eye. 60. The method of any one of claims 1-3 and 7-59, wherein the subject has not received prior treatment for ocular neovascular disease with an anti-VEGF agent. 63. The method of any one of claims 60-62, wherein the anti-VEGF agent is aflibercept. 64. The method of any one of claims 1-63, wherein the ocular neovascular disease is wet age-related macular degeneration (AMD), retinal neovascularization, choroidal neovascularization diabetic retinopathy, proliferative diabetic retinopathy, Retinal vein occlusion, central retinal vein occlusion, branch retinal vein occlusion, diabetic macular edema, diabetic retinal ischemia, ischemic retinopathy, diabetic retinal edema, or any combination thereof. 65. The method of any one of claims 1-10 and 16-64, wherein the unit dose of rAAV particles is administered in combination with steroid treatment. 66. The method of claim 65, wherein the steroid treatment is a corticosteroid treatment. 67. The method of claim 65 or 66, wherein the steroid treatment is systemic steroid treatment. 68. The method of any one of claims 65-67, wherein the steroid treatment is oral steroid treatment. 69. The method of any one of claims 65-68, wherein the steroid treatment is prednisone treatment. 67. The method of claim 65 or 66, wherein the steroid treatment is topical steroid treatment. 71. The method of claim 70, wherein the steroid treatment is difluprednate treatment. 72. The method of any one of claims 65-71, wherein the steroid is administered before, during and/or after administration of the unit dose of the rAAV particles to one eye and/or the contralateral eye. 73. The method of any one of claims 70-72, wherein the steroid treatment is topical steroid treatment and the topical steroid treatment is for up to about 4 weeks, up to about 6 weeks, or up to about 8 weeks from administration of the unit dose of the rAAV particles. How to be a daily steroid treatment. 74. The method of claim 73, wherein the topical steroid treatment comprises about 4 administrations of the topical steroid per day in about the first week, about 3 administrations of the topical steroid per day in about the second week, and about 3 administrations of the topical steroid per day in about the third week. about 2 administrations of the topical steroid and about 1 administration of the topical steroid per day at about the fourth week; wherein the timing begins with administration of a unit dose of rAAV particles and thereafter. 74. The method of claim 73, wherein the topical steroid treatment comprises about 4 administrations of the topical steroid per day for about 3 weeks after administration of the unit dose of rAAV particles, followed by about 3 administrations of the topical steroid per day for about 1 week, followed by administration of the unit dose of the rAAV particles. A method comprising administering about twice a day of the topical steroid for about 1 week, followed by about 1 administration of the topical steroid per day for about 1 week. 74. The method of claim 73, wherein the topical steroid treatment comprises about 4 administrations of the topical steroid per day for about 4 weeks, followed by about 3 administrations of the topical steroid per day for about 1 week, followed by topical administration of the topical steroid per day for about 1 week. about twice administration of the steroid, followed by about once administration of the topical steroid per day for about 1 week; wherein the timing begins about 1 week prior to administration of the unit dose of rAAV particles. 65. The method of any one of claims 11-64, further comprising administering a topical steroid treatment. 78. The method of claim 77, wherein the topical steroid treatment is difluprednate treatment. 79. The method of claim 77 or 78, wherein the topical steroid treatment comprises about 4 administrations of the topical steroid per day for about 4 weeks, followed by about 3 administrations of the topical steroid per day for about 1 week, followed by about 1 week. about 2 administrations of the topical steroid per day followed by about 1 administration of the topical steroid per day for about 1 week; wherein the timing begins with administration of the anti-VEGF agent and thereafter. 80. The method of any one of claims 71-79, wherein the topical steroid comprises 0.05% difluprednate in a dose of about 1 μg to about 3 μg. 81. The method of any one of claims 71-80, wherein the topical steroid comprises a dose of about 2.5 μg of difluprednate 0.05%. 82. The method of any one of claims 1-81, wherein administering the unit dose of rAAV particles to one and/or the contralateral eye of the subject maintains retinal thickness compared to retinal thickness prior to administration of the unit dose of rAAV particles. or causing a decrease. 83. The method of claim 82, wherein administering the unit dose of rAAV particles to one and/or the contralateral eye of the subject results in a decrease in retinal thickness as compared to retinal thickness prior to administration of the unit dose of rAAV particles. 84. The method of claim 83, wherein the reduction in retinal thickness is at least about 10% compared to retinal thickness prior to administration of the unit dose of the rAAV particles. 85. The method of any one of claims 82-84, wherein the retinal thickness is a central subfield thickness (CST) or a central retinal thickness (CRT). 86. The method of any one of claims 1-85, wherein administering the unit dose of the rAAV particles to one and/or the contralateral eye of the subject maintains the macular volume compared to the macular volume prior to administration of the unit dose of the rAAV particles. or causing a decrease. 87. The method of claim 86, wherein administering the unit dose of rAAV particles to one and/or the contralateral eye of the subject results in a decrease in macular volume compared to macular volume prior to administration of the unit dose of rAAV particles. 88. The method of claim 87, wherein the reduction in macular volume is at least about 10% compared to macular volume prior to administration of the unit dose of the rAAV particles. 89. The method of any one of claims 1-88, wherein administering the unit dose of rAAV particles to one and/or the contralateral eye of the subject maintains or improves visual acuity as compared to visual acuity prior to administration of the unit dose of rAAV particles. a method that causes 91. The method of claim 89, wherein administering the unit dose of rAAV particles to one and/or the contralateral eye of the subject results in an improvement in visual acuity as compared to visual acuity prior to administration of the unit dose of rAAV particles. 91. The method of claim 89 or 90, wherein the visual acuity is maximum corrected visual acuity (BCVA). 92. The method of any one of claims 1-91, wherein administering the unit dose of the rAAV particles to one and/or contralateral eye of the plurality of individuals results in at least about 50% of the subjects of the plurality of individuals receiving anti-VEGF rescue a method that does not require treatment. 93. The method of claim 92, wherein at least about 50% of the subjects of the plurality of subjects anti- 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 the rAAV particles. and no VEGF rescue treatment is required. 92. The method of any one of claims 1-91, wherein administering the unit dose of the rAAV particles to one eye and/or the contralateral eye of the plurality of individuals results in at least about 67% of the subjects of the plurality of individuals receiving anti-VEGF rescue a method that does not require treatment. 95. The method of claim 94, wherein at least about 67% of the subjects of the plurality of subjects are 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 and no anti-VEGF rescue treatment is required for at least about 66 weeks. 92. The method of any one of claims 1-91, wherein administering the unit dose of the rAAV particles to one eye and/or the contralateral eye of the plurality of individuals results in at least about 78% of the subjects of the plurality of individuals receiving anti-VEGF rescue a method that does not require treatment. 97. The method of claim 96, wherein at least about 78% of the subjects of the plurality are not in need of anti-VEGF rescue treatment for at least about 20 weeks, at least about 36 weeks or more after administration of the unit dose of the rAAV particles. Way. 92. The method of any one of claims 1-91, wherein administering the unit dose of the rAAV particles to one and/or the contralateral eye of the plurality of individuals results in 100% of the subjects of the plurality of subjects receiving anti-VEGF rescue treatment. How to avoid needing it. 99. The method of claim 98, wherein 100% of the subjects of the plurality of subjects are 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 after administration of the unit dose of the rAAV particles. and no anti-VEGF rescue treatment is required for 84 weeks or longer. 101. The method of any one of claims 1-99, wherein administering the unit dose of rAAV particles to one and/or the contralateral eye of the plurality of subjects comprises an annual anti-VEGF injection rate prior to administration of the unit dose of rAAV particles and and 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% reduction in the annual anti-VEGF injection rate as compared. 101. The method of any one of claims 1-10, 16-76 and 80-100, wherein the ocular neovascular disease is wet age-related macular degeneration (AMD). 94. The ocular neovascular disease of any one of claims 1-3, 11-64, 77-83, 85-87 and 89-91. The method is diabetic macular edema (DME). 103. The method of claim 102, wherein administering the unit dose of the rAAV particles to one and/or the contralateral eye of the subject results in a two- or three-step improvement in the diabetic retinopathy severity scale (DRSS). A unit dose of no more than about 6 x 10 ¹¹ vector genomes (vg) recombinant adeno-associated virus (rAAV) particles for use in a method of treating an ocular neovascular disease in a subject, said method comprising said unit in one eye of a subject administering a dose, wherein the subject is a human, wherein the rAAV particles
a) a nucleic acid encoding a polypeptide comprising an amino acid sequence having at least about 95% identity to the amino acid sequence of SEQ ID NO: 35 and flanked by AAV2 inverted terminal repeats (ITRs), and
b) an AAV2 capsid protein comprising the amino acid sequence LGETTRP (SEQ ID NO: 14) inserted between positions 587 and 588 of the capsid protein, wherein the amino acid residue numbering corresponds to the AAV2 VP1 capsid protein
A unit dose of no more than about 6 x 10 ¹¹ vector genomes (vg) recombinant adeno-associated virus (rAAV) particles for use in a method of treating an ocular neovascular disease in an individual, comprising: A unit dose of rAAV particles for use in a method of reducing retinal fluid in an eye of a subject having an ocular neovascular disease, the method comprising administering the unit dose to one eye of the subject, wherein the subject is a human and , where the rAAV particles are
a) a nucleic acid encoding a polypeptide comprising an amino acid sequence having at least about 95% identity to the amino acid sequence of SEQ ID NO: 35 and flanked by AAV2 inverted terminal repeats (ITRs), and
b) an AAV2 capsid protein comprising the amino acid sequence LGETTRP (SEQ ID NO: 14) inserted between positions 587 and 588 of the capsid protein, wherein the amino acid residue numbering corresponds to the AAV2 VP1 capsid protein
A unit dose of rAAV particles for use in a method of reducing retinal fluid in the eye of a subject having an ocular neovascular disease, comprising: 107. The unit dose of claim 104 or 105, wherein said method further comprises administering an anti-VEGF agent to said one eye of the subject prior to administration of said unit dose.

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