A method for treating intraocular neovascularization using the AAV2 variant encoding aflibercept.

Abstract

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

Description

[Technical Field]

[0001] Cross-references to related applications This application claims the interests of U.S. Provisional Patent Application No. 62 / 899,070, filed on 11 September 2019, and U.S. Provisional Patent Application No. 62 / 913,648, filed on 10 October 2019, the entirety of each disclosure thereof is thus incorporated herein by reference.

[0002] Submission of sequence listings in ASCII text files. The contents of the following submission in ASCII text file are incorporated herein by reference in their entirety: a computer-readable (CRF) sequence listing (filename: 627002001240SEQLIST.TXT, date: November 18, 2019, size: 29KB).

[0003] field This disclosure relates to a method for treating intraocular neovascular disease and disorders in an individual, comprising the step of administering a single dose of recombinant adeno-associated virus (rAAV) particles encoding an anti-VEGF agent (e.g., aflibercept) to the individual's eye. [Background technology]

[0004] background Age-related macular degeneration (AMD) is a degenerative eye disease that affects the macula, a light-sensitive area in the center of the retina responsible for reading and fine vision. The condition affecting the macula reduces central vision, while peripheral vision remains unaffected. In severe cases, the disease can lead to central blindness. AMD is a prominent cause of vision loss in the US population aged 65 and older, with an estimated prevalence of approximately 6.5% of any AMD in people over 40 years of age (Klein et al., (2011) Arch Ophthalmol, 129(1):75-80). Neovascular, exudative, or exudative AMD (nAMD, wAMD, or nwAMD) is a progressive form of AMD. A typical feature of wAMD is choroidal neovascularization (CNV), which is an abnormal infiltration of blood vessels from the underlying choroidal layer into the retina, leading to retinal cell damage and central blindness. This abnormal angiogenesis process is modulated by growth factors, particularly vascular endothelial growth factor (VEGF).

[0005] The standard treatment for wAMD is a class of molecules that bind to and sequester VEGF, such as ranibizumab (Lucentis) and aflibercept (Eylea). For example, aflibercept is a recombinant fusion protein that acts as a decoy receptor for vascular endothelial growth factor subtypes A and B (VEGF-A and VEGF-B) and placental growth factor (PGF). By binding to these ligands, aflibercept can prevent these ligands from binding to vascular endothelial growth factor receptors (VEGFR), VEGFR-1 and VEGFR-2, thereby suppressing angiogenesis and reducing vascular permeability. Aflibercept consists of domain 2 of VEGFR-1 and domain 3 of VEGFR-2 fused to the Fc fragment of IgG1.

[0006] Current standard treatments for anti-VEGF agents require readmission via intravitreal injection (IVT) every 4–8 weeks to achieve optimal therapeutic outcomes and maintain vision. Compliance with such regimens is burdensome for patients, their caregivers, and the healthcare system, and most patients deviate from optimal regimen compliance over time, which correlates with vision loss (Khanani AM, et al.). In addition, complications exist, including endophthalmitis, retinal detachment, traumatic cataracts, and elevated intraocular pressure (IOP), and the risk of these complications may be increased by repeated IVT injections (Falavarjani et al., (2013) Eye (Lond), 27(7):787-794). [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Klein et al., (2011) Arch Ophthalmol, 129(1):75-80 [Non-Patent Document 2] Falavarjani et al., (2013) Eye (Lond), 27(7):787-794 [Overview of the project] [Problems that the invention aims to solve]

[0008] Therefore, there is a need in this field for effective treatments of intraocular neovascular diseases such as wAMD that reduce the risk of adverse effects and are readily accepted by patients for long-term, high compliance. [Means for solving the problem]

[0009] Summary of this disclosure In one embodiment, a method for treating intraocular neovascular disease in an individual, comprising approximately 6 × 10⁶ recombinant adeno-associated virus (rAAV) particles, as described herein. 11A method is provided comprising the step of administering a vector genome (vg) or less than a unit dose to one eye of an individual, wherein the individual is human, and the rAAV particles comprise an AAV2 capsid protein comprising (a) a nucleic acid having at least about 95% identity with the amino acid sequence of SEQ ID NO: 35 and AAV2 terminal inverse sequences (ITRs) at both ends, 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 method comprises the step of reducing retinal fluid in the individual's eye.

[0010] In another embodiment, this specification provides a method for reducing retinal fluid in the eye of an individual having an intraocular neovascular disease, comprising the step of administering a unit dose of rAAV particles to one eye of the individual, wherein the individual is human, and the rAAV particles comprise an AAV2 capsid protein comprising (a) a nucleic acid having an amino acid sequence having at least about 95% identity with the amino acid sequence of SEQ ID NO: 35 and having AAV2 terminal inverse sequences (ITRs) at both ends, 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 individual has received at least one treatment with an anti-VEGF agent in the last 12 weeks or so prior to the administration of a unit dose of rAAV particles. In some embodiments, the amount or presence of retinal fluid in one eye of the individual is refractory to the previous treatment with an anti-VEGF agent. In some embodiments, the anti-VEGF agent is aflibercept. In some embodiments, the retinal fluid of one eye is reduced by at least about 60%. In some embodiments, the retinal fluid of one eye is reduced by about 80% compared to the level of retinal fluid of one eye in the individual before administration of rAAV to the individual. In some embodiments, the retinal fluid is subretinal fluid (SRF) or intraretinal fluid (IRF). In some embodiments, the unit dose of rAAV particles is about 6 × 10⁻⁶ 11The vector genome / eye (vg / eye) is less than or equal to that.

[0011] In some embodiments that may be combined with any of the prior embodiments, the unit dose of rAAV particles is approximately 6 × 10⁻⁶ 10 ~Approx. 2×10 11 The vector is genome / eye (vg / eye). In some embodiments, the unit dose of rAAV particles is approximately 2 × 10⁻⁶. 11 Or approximately 6 x 10 10 This is a vector genome / eye (vg / eye).

[0012] In some embodiments, which may be combined with any of the prior embodiments, the method provided herein further includes the step of administering a unit dose of rAAV particles to the contralateral eye of an individual. In some embodiments, the step of administering a unit dose of rAAV particles to the contralateral eye occurs at most about two weeks after the step of administering a unit dose of rAAV particles to one eye. In some embodiments, the unit dose of rAAV particles administered to the contralateral eye of an individual contains the same or fewer vector genomes / eye (vg / eye) as the unit dose of rAAV particles administered to one eye of the individual. In some embodiments, the step of administering a unit dose of rAAV particles to the contralateral eye occurs at least about two weeks after the step of administering a unit dose of rAAV particles to one eye. In some embodiments, the unit dose of rAAV particles administered to the contralateral eye of an individual contains more vector genomes / eye (vg / eye) than the unit dose of rAAV particles administered to one eye of the individual.

[0013] In some embodiments, which may be combined with any of the prior embodiments, the polypeptide comprises the amino acid sequence of SEQ ID NO: 35. In some embodiments, the polypeptide is aflibercept.

[0014] In some embodiments, which may be combined with any of the prior 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 nucleic acid encoding a polypeptide having an amino acid sequence having at least about 95% identity with the amino acid sequence of SEQ ID NO: 35, with AAV2 terminal inversion sequences (ITRs) at both ends; (d) a 5'UTR region; (e) a second enhancer region; and (f) a polyadenylation site. In some embodiments, the first enhancer region comprises a CMV sequence having 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 having the sequence of SEQ ID NO: 23 or a sequence having at least 85% identity thereto. In some embodiments, the polypeptide includes the amino acid sequence of SEQ ID NO: 35 or a sequence having at least 85% identity thereto. In some embodiments, the polypeptide is aflibercept. In some embodiments, the 5'UTR region includes a TPL sequence containing the sequence of SEQ ID NO: 24 or a sequence having at least 85% identity thereto, in order from 5' to 3', and an eMLP sequence containing the sequence of SEQ ID NO: 25 or a sequence having at least 85% identity thereto. In some embodiments, the second enhancer region includes a full-length EES sequence containing the sequence of SEQ ID NO: 26 or a sequence having at least 85% identity thereto. In some embodiments, the polyadenylation site includes an HGH polyadenylation site containing the sequence of SEQ ID NO: 27 or a sequence having at least 85% identity thereto.In some embodiments, the nucleic acid further comprises: (a) a first enhancer region comprising a CMV sequence containing the sequence of SEQ ID NO: 22 or a sequence having at least 85% identity thereto; (b) a promoter region comprising a CMV sequence containing the sequence of SEQ ID NO: 23 or a sequence having at least 85% identity thereto; (c) a 5'UTR region comprising a TPL sequence containing the sequence of SEQ ID NO: 24 or a sequence having at least 85% identity thereto, and an eMLP sequence containing the sequence of SEQ ID NO: 25 or a sequence having at least 85% identity thereto, in order from 5' to 3'; (d) a second enhancer region comprising a full-length EES sequence containing the sequence of SEQ ID NO: 26 or a sequence having at least 85% identity thereto; and (e) an HGH polyadenylation site containing the sequence of SEQ ID NO: 27 or a sequence having at least 85% identity thereto. In some embodiments, the nucleic acid comprises AAV ITRs at both ends of the element.

[0015] In some embodiments, which may be combined with any of the prior embodiments, the AAV2 capsid protein includes the amino acid sequence LGETTRP (SEQ ID NO: 14) inserted between positions 587 and 588 of AAV2 VP1 containing the sequence of SEQ ID NO: 13. In some embodiments, the AAV2 capsid protein includes the amino acid sequence LALGETTRPA (SEQ ID NO: 1) inserted between positions 587 and 588 of the capsid protein, with amino acid residue numbering corresponding to the AAV2 VP1 capsid protein. In some embodiments, the AAV2 capsid protein includes the amino acid sequence LALGETTRPA (SEQ ID NO: 1) inserted between positions 587 and 588 of AAV2 VP1 containing the sequence of SEQ ID NO: 13.

[0016] In some embodiments that may be combined with any of the prior embodiments, the administration of a unit dose of rAAV particles to one eye and / or the opposite eye is by intravitreous administration.

[0017] In some embodiments that can be combined with any of the previous embodiments, the unit dose of 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 dihydrogen phosphate, about 1 to about 10 mM disodium hydrogen phosphate, about 0.0005% (weight / volume) to about 0.005% (weight / volume) poloxamer 188, and about 6×10 13 ~about 6×10 10 vector genomes (vg) / mL (vg / mL) of rAAV particles, and the pharmaceutical formulation has a pH of about 7.0 to about 7.5. In some embodiments, the pharmaceutical formulation comprises about 180 mM sodium chloride, about 5 mM sodium dihydrogen phosphate, about 5 mM disodium hydrogen phosphate, about 6×10 12 vg / mL of rAAV particles, and about 0.001% (weight / volume) poloxamer 188, and the pharmaceutical formulation has a pH of about 7.3. In some embodiments, the pharmaceutical formulation comprises about 180 mM sodium chloride, about 5 mM sodium dihydrogen phosphate, about 5 mM disodium hydrogen phosphate, about 6×10 11 vg / mL of rAAV particles, and about 0.001% (weight / volume) poloxamer 188, and the pharmaceutical formulation has a pH of about 7.3.

[0018] In some embodiments that can be combined with any of the previous embodiments, the unit dose of rAAV particles comprises a volume of about 25 μL to about 250 μL. In some embodiments, the unit dose of rAAV particles comprises a volume of about 100 μL. In some embodiments, the unit dose of rAAV particles comprises a volume of about 30 μL.

[0019] In some embodiments that can be combined with any of the previous embodiments, the individual has received a previous treatment for an intraocular neovascular disease with an anti-VEGF agent. In some embodiments, the anti-VEGF agent is aflibercept.

[0020] In some embodiments that may be combined with any of the prior embodiments, the intraocular neovascular disease is exudative age-related macular degeneration (AMD), retinal neovascularization, choroidal neovascularization, diabetic retinopathy, proliferative diabetic retinopathy, retinal vein occlusion, central retinal vein occlusion, branch retinal vein occlusion, diabetic macular edema, diabetic retinal ischemia, ischemic retinopathy, diabetic retinal edema, or any combination thereof.

[0021] In some embodiments, which may be combined with any of the prior embodiments, a unit dose of rAAV particles is administered in combination with a steroid treatment. In some embodiments, the steroid treatment is a corticosteroid treatment. In some embodiments, the steroid treatment is a systemic steroid treatment. In some embodiments, the steroid treatment is an oral steroid treatment. In some embodiments, the steroid treatment is a prednisone treatment. In some embodiments, the steroid treatment is a topical steroid treatment. In some embodiments, the steroid treatment is a difluprednate treatment. In some embodiments, the steroid is administered before, during, and / or after administration of a unit dose of rAAV particles.

[0022] In some embodiments, which may be combined with any of the prior embodiments, the steroid treatment is a topical steroid treatment, which is a daily steroid treatment for up to about 4 weeks, up to about 6 weeks, or up to about 8 weeks, starting from the administration of a unit dose of rAAV particles. In some embodiments, the topical steroid treatment includes about 4 administrations of topical steroids in about week 1, about 3 administrations of topical steroids in about week 2, about 2 administrations of topical steroids in about week 3, and about 1 administration of topical steroids in about week 4, with the timing starting from and after the administration of a unit dose of rAAV particles. In some embodiments, the topical steroid contains about 1 μg to about 3 μg of 0.05% difluprednate. In some embodiments, the topical steroid contains about 2.5 μg of 0.05% difluprednate.

[0023] In some embodiments, which may be combined with any of the prior embodiments, the step of administering a unit dose of rAAV particles to one eye and / or the opposite eye of an individual results in maintenance or reduction of retinal thickness compared to the retinal thickness before administration of the unit dose of rAAV particles. In some embodiments, the step of administering a unit dose of rAAV particles to one eye and / or the opposite eye of an individual results in a decrease in retinal thickness compared to the retinal thickness before administration of the unit dose of rAAV particles. In some embodiments, the decrease in retinal thickness is at least about 10% compared to the retinal thickness before administration of the unit dose of rAAV particles. In some embodiments, the retinal thickness is central region retinal thickness (CST) or foveal retinal thickness (CRT).

[0024] In some embodiments, which may be combined with any of the prior embodiments, the step of administering a unit dose of rAAV particles to one eye and / or the opposite eye of an individual results in maintenance or reduction of macular volume compared to the macular volume before administration of the unit dose of rAAV particles. In some embodiments, the step of administering a unit dose of rAAV particles to one eye and / or the opposite eye of an individual results in a decrease in macular volume compared to the macular volume before 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 before administration of the unit dose of rAAV particles.

[0025] In some embodiments, which may be combined with any of the prior embodiments, the step of administering a unit dose of rAAV particles to one eye and / or the opposite eye of an individual results in maintenance or improvement of visual acuity compared to visual acuity before administration of the unit dose of rAAV particles. In some embodiments, the step of administering a unit dose of rAAV particles to one eye and / or the opposite eye of an individual results in maintenance or improvement of visual acuity compared to visual acuity before administration of the unit dose of rAAV particles. In some embodiments, the step of administering a unit dose of rAAV particles to one eye and / or the opposite eye of an individual results in improvement of visual acuity compared to visual acuity before administration of the unit dose of rAAV particles. In some embodiments, visual acuity is best corrected visual acuity (BCVA). Embedding by reference

[0026] All references, including patent applications and publications, cited herein are incorporated herein by reference in their entirety.

[0027] Novel features of the present invention are described in detail in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by referring to the following detailed description and accompanying drawings, which describe exemplary embodiments in which the principles of the present invention are utilized. The present invention provides, for example, the following items: (Item 1) A method for treating intraocular neovascular disease in an individual, comprising approximately 6 × 10⁶ recombinant adeno-associated virus (rAAV) particles. 11 The procedure includes the step of administering a vector genome (vg) or a unit dose less than that to one eye of the individual, wherein the individual is human and the rAAV particles are a) A nucleic acid encoding a polypeptide containing an amino acid sequence having at least approximately 95% identity with the amino acid sequence of SEQ ID NO: 35, and having AAV2 terminal inverse sequences (ITRs) at both ends, and b) A method comprising an AAV2 capsid protein having the amino acid sequence LGETTRP (SEQ ID NO: 14) inserted between positions 587 and 588 of the capsid protein, wherein the amino acid residue numbering corresponds to that of an AAV2 VP1 capsid protein. (Item 2) The method according to item 1, wherein the retinal fluid of the eye of the individual having intraocular neovascular disease is reduced. (Item 3) A method for reducing retinal fluid in the eye of an individual with intraocular neovascular disease, comprising the step of administering a unit dose of rAAV particles to one eye of the individual, wherein the individual is a human and the rAAV particles are a) A nucleic acid encoding a polypeptide containing an amino acid sequence having at least approximately 95% identity with the amino acid sequence of SEQ ID NO: 35, and having AAV2 terminal inverse sequences (ITRs) at both ends, and b) A method comprising an AAV2 capsid protein having the amino acid sequence LGETTRP (SEQ ID NO: 14) inserted between positions 587 and 588 of the capsid protein, wherein the amino acid residue numbering corresponds to that of an AAV2 VP1 capsid protein. (Item 4) The method according to item 3, wherein the individual has received at least one treatment with an anti-VEGF agent approximately 12 weeks prior to administering the unit dose of rAAV particles. (Item 5) The method according to item 3 or item 4, wherein the amount or presence of retinal fluid in the one eye of the individual is refractory to prior treatment with an anti-VEGF agent. (Item 6) The method according to item 4 or item 5, wherein the anti-VEGF agent is aflibercept. (Item 7) The method according to any one of items 2 to 6, wherein the retinal fluid in the one eye is reduced by at least about 60%. (Item 8) The method according to any one of items 2 to 7, wherein the retinal fluid of the one eye is reduced by about 80% compared to the level of retinal fluid of the one eye of the individual before administration of the rAAV to the individual. (Item 9) The method according to any one of items 2 to 8, wherein the retinal fluid is subretinal fluid (SRF) or intraretinal fluid (IRF). (Item 10) The unit dose of rAAV particles is approximately 6 × 10 11 A vector genome / eye (vg / eye) or less, as described in any one of items 2-9. (Item 11) The unit dose of rAAV particles is approximately 6 × 10 10 ~Approx. 2×10 11 A vector genome / eye (vg / eye) as described in any one of items 1 to 10. (Item 12) The unit dose of rAAV particles is approximately 2 × 10 11 Or approximately 6 x 10 10 A vector genome / eye (vg / eye) as described in any one of items 1 to 11. (Item 13) The method according to any one of items 1 to 12, further comprising the step of administering a unit dose of rAAV particles to the eye opposite the individual. (Item 14) The method according to item 13, wherein the step of administering the unit dose of rAAV particles to the opposite eye occurs at most about two weeks after the step of administering the unit dose of rAAV particles to the one eye. (Item 15) The method according to item 14, wherein the unit dose of rAAV particles administered to the opposite eye of the individual is the same as or less than the unit dose of rAAV particles administered to the one eye of the individual, comprising a vector genome / eye (vg / eye). (Item 16) The method according to item 13, wherein the step of administering the unit dose of rAAV particles to the opposite eye is at least about two weeks after the step of administering the unit dose of rAAV particles to the one eye. (Item 17) The method according to item 16, wherein the unit dose of rAAV particles administered to the opposite eye of the individual contains more vector genomes / eye (vg / eye) than the unit dose of rAAV particles administered to one eye of the individual. (Item 18) The method according to any one of items 1 to 17, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 35. (Item 19) The method according to any one of items 1 to 18, wherein the polypeptide is aflibercept. (Item 20) The method according to any one of items 1 to 19, 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. (Item 21) The nucleic acids are arranged in the order from 5' to 3': (a) First enhancer region; (b) Promoter area; (c) A nucleic acid encoding a polypeptide having an amino acid sequence that is at least approximately 95% identical to the amino acid sequence of SEQ ID NO: 35; (d) 5'UTR region; (e) Second enhancer region; and (f) Polyadenylation site; The method according to any one of items 1 to 20, comprising and having an AAV2 terminal inversion sequence (ITR) at both ends. (Item 22) The method according to item 20 or item 21, wherein the first enhancer region comprises a CMV sequence having at least 85% identity with the sequence of sequence number 22. (Item 23) The method according to any one of items 20 to 22, wherein the promoter region includes a CMV sequence that includes the sequence of sequence number 23, or a sequence having at least 85% identity thereto. (Item 24) The method according to any one of items 20 to 23, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 35 or a sequence having at least 85% identity thereto. (Item 25) The method according to any one of items 20 to 24, wherein the polypeptide is aflibercept. (Item 26) The method according to any one of items 20 to 25, wherein the 5'UTR region includes, in order from 5' to 3', a TPL sequence containing the sequence of sequence number 24 or a sequence having at least 85% identity thereto, and an eMLP sequence containing the sequence of sequence number 25 or a sequence having at least 85% identity thereto. (Item 27) The method according to any one of items 20 to 26, wherein the second enhancer region comprises a full-length EES sequence containing the sequence of sequence number 26 or a sequence having at least 85% identity thereto. (Item 28) The method according to any one of items 20 to 27, wherein the polyadenylation site comprises an HGH polyadenylation site having the sequence of SEQ ID NO: 27 or a sequence having at least 85% identity thereto. (Item 29) The method according to any one of items 1 to 19, wherein the nucleic acid further comprises: (a) a first enhancer region comprising a CMV sequence comprising the sequence of SEQ ID NO: 22 or a sequence having at least 85% identity thereto; (b) a promoter region comprising a CMV sequence comprising the sequence of SEQ ID NO: 23 or a sequence having at least 85% identity thereto; (c) a 5'UTR region comprising, in order from 5' to 3', a TPL sequence comprising the sequence of SEQ ID NO: 24 or a sequence having at least 85% identity thereto, and an eMLP sequence comprising the sequence of SEQ ID NO: 25 or a sequence having at least 85% identity thereto; (d) a second enhancer region comprising a full-length EES sequence comprising the sequence of SEQ ID NO: 26 or a sequence having at least 85% identity thereto; and (e) an HGH polyadenylation site comprising the sequence of SEQ ID NO: 27 or a sequence having at least 85% identity thereto. (Item 30) The method according to any one of items 1 to 29, wherein the AAV2 capsid protein includes the amino acid sequence LALGETTRPA (SEQ ID NO: 1) inserted between positions 587 and 588 of the capsid protein, and the amino acid residue numbering corresponds to the AAV2 VP1 capsid protein. (Item 31) The method according to any one of items 1 to 30, wherein the AAV2 capsid protein includes the amino acid sequence LGETTRP (sequence number 14) inserted between positions 587 and 588 of AAV2 VP1 containing the sequence of sequence number 13. (Item 32) The method according to any one of items 1 to 31, wherein the AAV2 capsid protein includes the amino acid sequence LALGETTRPA (sequence number 1) inserted between positions 587 and 588 of AAV2 VP1 containing the sequence of sequence number 13. (Item 33) The method according to any one of items 1 to 32, wherein the administration of the unit dose of rAAV particles to the one eye and / or the opposite eye is by intravitreous administration. (Item 34) The method according to any one of items 1 to 33, wherein the unit dose of rAAV particles is present in the pharmaceutical preparation. (Item 35) The method according to item 34, wherein the pharmaceutical formulation comprises the rAAV particles, sodium chloride, sodium phosphate, and a surfactant. (Item 36) The aforementioned pharmaceutical preparation contains approximately 150-200 mM sodium chloride, approximately 1-10 mM sodium dihydrogen phosphate, approximately 1-10 mM sodium hydrogen phosphate, approximately 0.0005% (weight / volume)-0.005% (weight / volume) poloxamer 188, and approximately 6 × 10 13 ~Approx. 6×10 10 The method according to item 35, comprising the rAAV particles in vector genome (vg) / mL (vg / mL), wherein the pharmaceutical formulation has a pH of approximately 7.0 to approximately 7.5. (Item 37) The aforementioned pharmaceutical preparation contains approximately 180 mM sodium chloride, approximately 5 mM sodium dihydrogen phosphate, approximately 5 mM sodium hydrogen phosphate, and approximately 6 × 10 12 The method according to item 36, comprising vg / mL of rAAV particles and about 0.001% (weight / volume) poloxamer 188, wherein the pharmaceutical formulation has a pH of about 7.3. (Item 38) The aforementioned pharmaceutical preparation contains approximately 180 mM sodium chloride, approximately 5 mM sodium dihydrogen phosphate, approximately 5 mM sodium hydrogen phosphate, and approximately 6 × 10 11 The method according to item 36, comprising vg / mL of rAAV particles and about 0.001% (weight / volume) poloxamer 188, wherein the pharmaceutical formulation has a pH of about 7.3. (Item 39) The method according to any one of items 1 to 38, wherein the unit dose of rAAV particles comprises a volume of about 25 μL to about 250 μL. (Item 40) The method according to item 39, wherein the unit dose of rAAV particles comprises a volume of approximately 100 μL. (Item 41) The method according to item 39, wherein the unit dose of rAAV particles comprises a volume of approximately 30 μL. (Item 42) The method according to any one of items 1 to 41, wherein the individual has received prior treatment for the intraocular neovascular disease with an anti-VEGF agent. (Item 43) The method according to item 42, wherein the anti-VEGF agent is aflibercept. (Item 44) The method according to any one of items 1 to 43, wherein the intraocular neovascular disease is exudative age-related macular degeneration (AMD), retinal neovascularization, choroidal neovascularization, diabetic retinopathy, proliferative diabetic retinopathy, retinal vein occlusion, central retinal vein occlusion, branch retinal vein occlusion, diabetic macular edema, diabetic retinal ischemia, ischemic retinopathy, diabetic retinal edema, or any combination thereof. (Item 45) The method according to any one of items 1 to 44, wherein the unit dose of rAAV particles is administered in combination with a steroid treatment. (Item 46) The method according to item 45, wherein the steroid treatment is a corticosteroid treatment. (Item 47) The method according to item 45 or item 46, wherein the steroid treatment is a systemic steroid treatment. (Item 48) The method according to any one of items 45 to 47, wherein the steroid treatment is an oral steroid treatment. (Item 49) The method according to any one of items 45 to 48, wherein the steroid treatment is prednisone treatment. (Item 50) The method according to item 45 or item 46, wherein the steroid treatment is a topical steroid treatment. (Item 51) The method according to item 50, wherein the steroid treatment is difluprednate treatment. (Item 52) The method according to any one of items 45 to 51, wherein the steroid is administered before, during, and / or after administration of the unit dose of rAAV particles. (Item 53) The method according to any one of items 50 to 52, wherein the steroid treatment is a topical steroid treatment, and the topical steroid treatment is a daily steroid treatment for up to about 4 weeks, up to about 6 weeks, or up to about 8 weeks from the administration of the unit dose of rAAV particles. (Item 54) The method according to item 53, wherein the topical steroid treatment comprises approximately four administrations of topical steroids in approximately week 1, approximately three administrations of topical steroids in approximately week 2, approximately two administrations of topical steroids in approximately week 3, and approximately one administration of topical steroids in approximately week 4, with the timing starting with and after the administration of the unit dose of rAAV particles. (Item 55) The method according to item 54, wherein the topical steroid contains approximately 1 μg to approximately 3 μg of 0.05% difluprednate. (Item 56) The method according to item 54, wherein the topical steroid comprises approximately 2.5 μg of 0.05% difluprednate. (Item 57) The method according to any one of items 1 to 56, wherein the step of administering the unit dose of rAAV particles to the one eye and / or the opposite eye of the individual results in maintenance or reduction of retinal thickness compared to the retinal thickness before administration of the unit dose of rAAV particles. (Item 58) The method according to item 57, wherein the step of administering the unit dose of rAAV particles to the one eye and / or the opposite eye of the individual results in a reduction in retinal thickness compared to the retinal thickness before administration of the unit dose of rAAV particles. (Item 59) The method according to item 57 or item 58, wherein the reduction in retinal thickness is at least about 10% compared to the retinal thickness before administration of the unit dose of rAAV particles. (Item 60) The method described in any one of items 57-59, wherein the retinal thickness is the central regional retinal thickness (CST) or the foveal retinal thickness (CRT). (Item 61) The method according to any one of items 1 to 60, wherein the step of administering the unit dose of rAAV particles to the one eye and / or the opposite eye of the individual results in maintenance or reduction of macular volume compared to the macular volume before administration of the unit dose of rAAV particles. (Item 62) The method according to item 61, wherein the step of administering the unit dose of rAAV particles to the one eye and / or the opposite eye of the individual results in a reduction in macular volume compared to the macular volume before administration of the unit dose of rAAV particles. (Item 63) The method according to item 62, wherein the reduction in macular volume is at least about 10% compared to the macular volume before administration of the unit dose of rAAV particles. (Item 64) The method according to any one of items 1 to 63, wherein the step of administering the unit dose of rAAV particles to the one eye and / or the opposite eye of the individual results in maintenance or improvement of visual acuity compared to the visual acuity before administration of the unit dose of rAAV particles. (Item 65) The method according to any one of items 1 to 64, wherein the step of administering the unit dose of rAAV particles to the one eye and / or the opposite eye of the individual results in an improvement in visual acuity compared to the visual acuity before administration of the unit dose of rAAV particles. (Item 66) The method described in any one of items 64-65, wherein visual acuity is best corrected visual acuity (BCVA). [Brief explanation of the drawing]

[0028] [Figure 1A] Figures 1A and 1B present schematic diagrams of the investigational drug and the Phase I trials described in Examples 1 and 2. Figure 1A is a schematic diagram of AAV2.7m8-aflibercept. AAV2.7m8-aflibercept is a recombinant replication-deficient adeno-associated virus (rAAV) vector containing an AAV2.7m8 protein capsid and a vector genome containing an expression cassette (C11) of a codon-optimized version of aflibercept cDNA under the control of a ubiquitous chimeric promoter. The AAV2.7m8-aflibercept vector genome also contains two AAV2 terminal inversion sequences (ITRs) at both ends of the aflibercept cDNA expression cassette. Figure 1B is a diagram summarizing the study design of the Phase I trials described in Examples 1 and 2. [Figure 1B] Same as above.

[0029] [Figure 2A]Figures 2A–2L show optical coherence tomography (OCT) images and retinal thickness maps extracted from OCT images obtained from subjects in Cohort 1 of the study described in Example 1. OCT images were taken at the indicated time points before and after administration of AAV2.7m8-aflibercept (day 1). The anti-VEGF IVT treatment interval is shown for all subjects. Figure 2A shows OCT images and retinal thickness maps extracted from OCT images obtained from subject 1 at the indicated time points during five visits prior to the aflibercept injection at screening. OCT images were taken immediately before treatment with standard aflibercept therapy. Subject 1 required aflibercept IVT every 5–7 weeks and showed refractory subretinal fluid and pigment epithelial detachment (PED) despite treatment with standard aflibercept therapy. Figure 2B presents OCT images and retinal thickness maps extracted from OCT images taken from Subject 1 at the time of screening aflibercept injection (-7 days), at the time of AAV2.7m8-aflibercept injection (1 day), and at follow-up visits at the indicated time points. Subject 1 did not require any rescue injections after AAV2.7m8-aflibercept injection. Subject 1 showed subretinal fluid dissipation starting at week 4, and remained subretinal and intraretinal fluid-free (dry). Figure 2C presents OCT images and retinal thickness maps extracted from OCT images taken from Subject 2 at five visits at the indicated time points prior to screening aflibercept injection. OCT images were taken immediately before treatment with standard aflibercept therapy. Subject 2 required six aflibercept IVT treatments to maintain the anatomical structure of the retina during the 8 months prior to treatment with AAV2.7m8-aflibercept. Figure 2D presents OCT images and retinal thickness maps extracted from OCT images obtained from Subject 2 at the time of screening aflibercept injection (-7 days), at the time of AAV2.7m8-aflibercept injection (1 day), and at follow-up visits at the indicated time points. Subject 2 did not require any rescue injections after AAV2.7m8-aflibercept injection. Subject 2 showed stable retinal anatomical structure up to week 24, with no subretinal or intraretinal fluid present.Figure 2E presents OCT images and retinal thickness maps extracted from OCT images taken at four visits at indicated time points prior to the aflibercept injection at screening from Subject 3. -The OCT image taken at 27 weeks is not shown. OCT images were taken immediately before treatment with standard aflibercept therapy. Subject 3 showed subretinal fluid, which increased as the interval between aflibercept IVTs increased from 5 weeks to 7 weeks. Figure 2F presents OCT images and retinal thickness maps extracted from OCT images taken at the time of aflibercept injection at screening (-7 days), at the time of AAV2.7m8-aflibercept injection (1 day), and at follow-up visits at indicated time points from Subject 3. Subject 3 did not require any rescue injections after the AAV2.7m8-aflibercept injection. Subject 3 showed dissipation of refractory subretinal fluid by 8 weeks and stable retinal anatomical structure until 24 weeks. Figure 2G presents OCT images and retinal thickness maps extracted from OCT images taken at five visits at indicated time points prior to the screening aflibercept injection in Subject 4. OCT images were taken immediately before treatment with standard ranibizumab 0.5 mg IVT. Subject 4 showed refractory subretinal fluid to ranibizumab IVT injection. Figure 2H presents OCT images and retinal thickness maps extracted from OCT images taken at the time of the screening aflibercept injection (-14 days), the time of the AAV2.7m8-afibercept injection (1 day), and at follow-up visits at indicated time points in Subject 4. Subject 4 did not require any rescue injections after the AAV2.7m8-afibercept injection. Subject 4 showed resolution of refractory subretinal fluid by week 8 and stable retinal anatomical structure until week 24. Figure 2I shows OCT images and retinal thickness maps extracted from OCT images taken during five visits to Subject 5 at the indicated time points prior to aflibercept injection during screening. OCT images were taken immediately before treatment with standard aflibercept therapy.Figure 2J presents OCT images and retinal thickness maps derived from the OCT images obtained from Subject 5 at the time of aflibercept injection during screening (-14 days), at the time of AAV2.7m8-aflibercept injection (day 1), and at follow-up visits at the indicated time points. Subject 5 did not require any rescue injections after AAV2.7m8-aflibercept injection. Subretinal fluid and PED were present during treatment with AAV2.7m8-aflibercept and dissipated over time, and the retinal anatomical structure remained stable until week 24, with no subretinal fluid or intraretinal fluid present. Figure 2K presents OCT images and retinal thickness maps derived from the OCT images obtained from Subject 6 at five visits at the indicated time points before aflibercept injection during screening. The OCT images were obtained immediately before treatment with either bevacizumab 1.5 mg IVT standard treatment or ranibizumab 0.5 mg IVT standard treatment as shown. The appearance of Subject 6's retina was consistent with polypoidal choroidal vasculopathy (PCV). Figure 2L presents OCT images and retinal thickness maps derived from the OCT images obtained from Subject 6 at the time of aflibercept injection during screening (-10 days), at the time of AAV2.7m8-aflibercept injection (day 1), and at follow-up visits at the indicated time points. Subject 6 did not require any rescue injections after AAV2.7m8-aflibercept injection. Subject 6 showed no increase in subretinal fluid until week 24, and some anatomical improvement was achieved. The contralateral eye of Subject 6 received aflibercept injection, which is the standard treatment, every 4 weeks over the course of the study and showed a similar retinal morphology to the eye treated with AAV2.7m8-aflibercept. [Figure 2B] The same as above. [Figure 2C] The same as above. [Figure 2D] The same as above. [Figure 2E] The same as above. [Figure 2F] The same as above. [Figure 2G] The same as above. [Figure 2H] The same as above. [Figure 2I] The same as above. [Figure 2J] Same as above. [Figure 2K] Same as above. [Figure 2L] Same as above.

[0030] [Figure 3] Figure 3 shows the mean foveal retinal thickness (CST) change at the indicated time point for subjects in Cohort 1 of the study described in Example 1. Error bars indicate 90% confidence intervals calculated using the T distribution. Baseline (BL) represents the measurement taken before aflibercept injection at screening 7–15 days (e.g., 7–14 days) prior to treatment with AAV2.7m8-aflibercept on day 1. At 24 weeks after treatment with AAV2.7m8-aflibercept, the mean change in CRT shown by subjects was -52.7 μm (90% CI -86.5, -18.8). BL = Baseline; D = Day; W = Week. The day 1 visit was performed 7–14 days after the baseline visit.

[0031] [Figure 4] Figure 4 shows the mean best-corrected visual acuity (BCVA) measurements based on Early Treatment Diabetic Retinopathy Study (ETDRS) letter assessment at indicated time points for subjects in Cohort 1 of the study described in Example 1. Error bars indicate 90% confidence intervals calculated using the T-distribution. Baseline (BL) represents the measurement taken before aflibercept injection at screening 7–15 days (e.g., 7–14 days) prior to treatment with AAV2.7m8-aflibercept on day 1. At 24 weeks after treatment with AAV2.7m8-aflibercept, the change in BCVA shown by subjects was a mean -2 letters (90% CI -9.1, 5.1). BL = Baseline; D = Day; W = Week. The day 1 visit took place 7–14 days after the baseline visit.

[0032] [Figure 5] Figure 5 shows the nucleic acid sequence of aflibercept (SEQ ID NO: 36).

[0033] [Figure 6] Figure 6 shows plots of anterior chamber cell and vitreous cell counts after treatment with AAV2.7m8-aflibercept for subjects 1-6 of the study described in Example 1. The steroid treatment administered to each patient is shown below each plot. Aqueous humor cell count categories were based on the Standardization of Uveitis Nomenclature (SUN) criteria (Jabs, DA et al., J Ophthalmol. 2005; 140: 509-516). Vitreous cell count categories were based on National Institutes of Health (NIH) guidelines. For aqueous humor cells, a cell count of 0.5+ indicates 1-5 cells, a cell count of 1+ indicates 6-15 cells, a cell count of 2+ indicates 16-25 cells, a cell count of 3+ indicates 26-50 cells, and a cell count of 4+ indicates >50 cells. For vitreous cells, a cell count of 0.5+ indicates 1-10 cells, 1+ indicates 11-20 cells, 2+ indicates 21-30 cells, 3+ indicates 31-100 cells, and 4+ indicates >100 cells. In the analysis shown in this figure, rare cells were identified as 0.5+.

[0034] [Figure 7A]Figures 7A–7B show optical coherence tomography (OCT) images and retinal thickness maps extracted from OCT images obtained at the median follow-up time of week 34 from subjects 1–6 of Cohort 1 of the study described in Example 1. Furthermore, the change from baseline in BCVA, the number of anti-VEGF IVT injections in the 8 months prior to AAV2.7m8-aflibercept administration, and the number of rescue anti-VEGF IVT injections administered during the study are also presented for each of subjects 1–6. The actual week in which the OCT image and retinal thickness map were obtained is shown for each subject (Subject 1 = week 44; Subject 2 = week 40; Subject 3 = week 36; Subject 4 = week 32; Subject 5 = week 28; and Subject 6 = week 28). No subjects required rescue anti-VEGF IVT injections during the study, and no retreatment criteria were met at any point during the maximum 44-week follow-up period. No subjects showed signs of disease reactivation on OCT imaging. [Figure 7B] Same as above. [Modes for carrying out the invention]

[0035] Detailed explanation Several embodiments are described below with reference to applications as illustrative examples. It should be understood that numerous specific details, relationships, and methods are provided to offer a full understanding of the features described herein. However, those skilled in the art will readily recognize that the features described herein can be practiced without one or more of the specific details, or by other means. The features described herein are not limited to the illustrated order of actions or events, as some actions may occur in a different order and / or simultaneously with other actions or events. Furthermore, not all illustrated actions or events are necessarily required to implement the methodology in accordance with the features described herein. definition

[0036] Unless otherwise defined, all technical terms used herein have the same meaning as those commonly understood by those skilled in the art.

[0037] The terms used herein are for illustrative purposes only and are not intended to be limiting. Where used herein, the singular forms “a,” “an,” and “the” are intended to include the plural forms as well, unless the text clearly indicates otherwise. Furthermore, where the terms “including,” “includes,” “having,” “has,” and “with,” or their variations thereof, are used in any part of the detailed description and / or claims, such terms are intended to be comprehensive in a similar manner to the term “comprising.” Where used herein, the term “comprising” is synonymous with “including” or “containing,” and is either comprehensive or non-limiting.

[0038] In this specification, any reference to “or” is intended to include “and / or” unless otherwise indicated. Where used herein, the number in the term “about” refers to a number plus or minus 10% of that number. The range of the term “about” refers to a range from minus 10% of its lowest value to plus 10% of its highest value.

[0039] 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 human.

[0040] The terms “to treat,” “treating,” “treatment,” “improving,” or “improving,” and other grammatical equivalents, as used herein, are intended to mean reducing, weakening, or improving an intraocular neovascular disease or disorder, or the symptoms of an intraocular neovascular disease or disorder, preventing additional symptoms of an intraocular neovascular disease or disorder, improving or preventing the underlying metabolic causes of symptoms, inhibiting an intraocular neovascular disease or disorder, for example, stopping the development of an intraocular neovascular disease or disorder, alleviating an intraocular neovascular disease or disorder, causing regression of an intraocular neovascular disease or disorder, or stopping the symptoms of an intraocular neovascular disease or disorder, and are intended to include prevention. The terms further include achieving therapeutic benefits and / or preventive benefits. The term “therapeutic benefit” means the eradication or improvement of the intraocular neovascular disease or disorder being treated. Similarly, therapeutic benefits are also achieved by the elimination or improvement of one or more physiological symptoms associated with intraocular neovascular disease or disorder, thereby, in some embodiments, an improvement is observed in the subject even though the subject still suffers from the disease or disorder. In the case of preventive benefits, the pharmaceutical composition is administered to subjects at risk of developing intraocular neovascular disease or disorder, or to subjects reporting one or more physiological symptoms of intraocular neovascular disease or disorder, even if a diagnosis of the disease or disorder has not been made.

[0041] The terms “administer,” “administering,” and “dosing,” as used herein, may refer to methods used to enable the delivery of a therapeutic agent or pharmaceutical composition to a desired site of biological action. These methods include intravitreous or subretinal injection into the eye.

[0042] The terms “effective dose,” “therapeutic dose,” or “pharmaceutical dose,” as used herein, may refer to a sufficient amount of at least one pharmaceutical composition or compound administered to alleviate, to some extent, one or more symptoms of the eye disease or disorder being treated. The “effective dose,” “therapeutic dose,” or “pharmaceutical dose” of a pharmaceutical composition may be administered as a unit dose to a subject requiring it (as further described elsewhere herein).

[0043] When used herein, the term "pharmaceutically acceptable" may refer to a material, such as a carrier or diluent, that does not preclude the biological activity or properties of the compounds disclosed herein and is relatively non-toxic (i.e., does not cause undesirable biological effects when administered to an organism and does not adversely interact with any of the components of the composition in which it is contained).

[0044] The terms “pharmaceutical composition” or simply “composition” as used herein may refer to a bioactive compound optionally mixed with at least one pharmaceutically acceptable chemical component, such as, but not limited to, a carrier, stabilizer, diluent, dispersant, suspending agent, concentrator, excipient, etc.

[0045] When used herein, “AAV vector” or “rAAV vector” refers to an adeno-associated virus (AAV) vector or recombinant AAV (rAAV) vector containing a polynucleotide sequence that is not of AAV origin (e.g., a polynucleotide heterologous to AAV, such as a nucleic acid sequence encoding a therapeutic transgene, e.g., aflibercept) for transduction into target cells or target tissues. Generally, heterologous polynucleotides have at least one AAV terminal inversion sequence (ITR) adjacent to them, and generally have two AAV terminal inversion sequences (ITRs) at both ends. The term rAAV vector encompasses both rAAV vector particles and rAAV vector plasmids. rAAV vectors can be either single-stranded (ssAAV) or self-complementary (scAAV).

[0046] The terms "AAV virus," "AAV virus particle," "rAAV vector particle," or "rAAV particle" refer to a viral particle containing at least one AAV capsid protein and a polynucleotide rAAV vector. In some examples, at least one AAV capsid protein is derived from wild-type AAV or is a variant AAV capsid protein (e.g., an AAV capsid protein having an insertion, e.g., an insertion of the 7m8 amino sequence as described below). If the particle contains heterologous polynucleotides (e.g., polynucleotides other than those in the wild-type AAV genome, such as transgenes delivered to target cells or tissues), it is called an "rAAV particle," "rAAV vector particle," or "rAAV vector." Thus, the production of rAAV particles necessarily includes the production of rAAV vectors, since such vectors are contained within the rAAV particles.

[0047] As used herein, the term "packaging" may refer to a series of intracellular events that can result in the assembly and capsid formation of rAAV particles.

[0048] The AAV "rep" and "cap" genes refer to polynucleotide sequences that encode the replication and capsid-forming proteins of adeno-associated virus. In this specification, AAV rep and cap are referred to as AAV "packaging genes."

[0049] The term "polypeptide" can encompass both naturally occurring and non-naturally occurring proteins (e.g., fusion proteins), peptides, fragments, mutants, derivatives, and analogs thereof. Polypeptides can be monomers, dimers, trimers, or polymers. Furthermore, a polypeptide may contain several distinct domains, each having one or more distinct activities. To avoid misunderstanding, a "polypeptide" can be of any length longer than two amino acids.

[0050] As used herein, “polypeptide variant” or simply “variant” refers to a polypeptide whose sequence contains an amino acid modification. In some embodiments, the modification is an insertion, duplication, deletion, rearrangement, or substitution of one or more amino acids compared to the amino acid sequence of a reference protein or polypeptide, e.g., a native or wild-type protein. A variant may have point substitutions of one or more amino acids, where a single amino acid at one position is changed to another; one or more insertions and / or deletions, where one or more amino acids are inserted or deleted, respectively, in the sequence of the reference protein; and / or cleavage of an amino acid sequence at either or both of the amino-terminus and / or carboxyl-terminus. A variant may have the same or different biological activity compared to the reference protein or an unmodified protein.

[0051] In some embodiments, the variant may have, for example, at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% overall sequence homology with its relative reference protein. In some embodiments, the variant may have at least about 90% overall sequence homology with the wild-type protein. In some embodiments, the 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.

[0052] As used herein, “recombinant” can mean a biomolecule, such as a gene or protein, that (1) has been removed from its naturally occurring environment, (2) whose gene is not associated with all or some of the naturally occurring polynucleotides, (3) is operably ligated to a polynucleotide that is not ligated in nature, or (4) is not naturally occurring. The term “recombinant” can be used in relation to cloned DNA isolates, chemically synthesized polynucleotide analogs, or polynucleotide analogs biologically synthesized by heterologous systems, as well as proteins and / or mRNA encoded by such nucleic acids. Thus, for example, a protein synthesized by a microorganism is recombinant if, for example, it is synthesized from mRNA synthesized from a recombinant gene present in a cell.

[0053] The term “anti-VEGF agent” includes any therapeutic agent, including proteins, polypeptides, peptides, fusion proteins, multimeric proteins, gene products, antibodies, human monoclonal antibodies, antibody fragments, aptamers, small molecules, kinase inhibitors, receptors or receptor fragments, or nucleic acid molecules, that can reduce, interfere with, disrupt, block, and / or inhibit the activity or function of endogenous VEGF and / or endogenous VEGF receptors (VEGFR), or the VEGF-VEGFR interaction or pathway in vivo. An anti-VEGF agent may be any one of known therapeutic agents that, when delivered to cells, tissues, or subjects in vivo, can reduce the growth or formation of new blood vessels and / or edema or swelling, such as ranibizumab, brolucizumab, or bevacizumab. In some embodiments, the anti-VEGF agent may be naturally occurring, not naturally occurring, or synthetic. In some embodiments, the anti-VEGF agent may be derived from a naturally occurring molecule that has subsequently been modified or mutated to confer anti-VEGF activity. In some embodiments, the anti-VEGF agent may be a fusion protein or chimeric protein. In such proteins, a functional domain or polypeptide is artificially fused to a portion or polypeptide to create a fusion protein or chimeric protein that can sequester VEGF in vivo or function as a VEGFR decoy. In some embodiments, the anti-VEGF agent is a fusion protein or chimeric protein that blocks endogenous VEGFR from interacting with its ligand.

[0054] As used herein, “VEGF” may refer to any isoform of VEGF, including but not limited to VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E, VEGF-F, or any combination thereof, or any functional fragment or variant thereof, unless otherwise required. Unless otherwise required, “VEGF” may refer to any member of the VEGF family, including members: VEGF-A, placental growth factor (PGF), VEGF-B, VEGF-C, and VEGF-D, or any combination thereof, functional fragment, or variant thereof. As used herein, “VEGF receptor” or “VEGFR” or “VEGF-R” may be used to refer to any one of the receptors of VEGF, including but not limited to VEGFR-1 (or Flt-1), VEGFR-2 (or Flk-1 / KDR), and VEGFR-3 (or Flt-4). VEGFR may be membrane-bound or soluble, or a functional fragment or cleavage 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.

[0055] "Operatally linked," "operably linked," or "coupled" can refer to the juxtaposition of gene elements in a relationship that allows them to operate in the expected manner. For example, a promoter can be operably linked to a coding region if the promoter helps initiate transcription of the coding sequence. Intervening residues may exist between the promoter and the coding region as long as this functional relationship is maintained.

[0056] The terms “expression vector,” “expression construct,” “cassette,” “plasmid,” or simply “vector” may include any type of gene product, including AAV or rAAV vectors containing nucleic acids or polynucleotides encoding a gene product suitable for gene therapy, in which some or all of the nucleic acids encoding the sequence are transcribable. The transcript may be translated into a protein. In some embodiments, the transcript may be partially translated or not translated at all. In certain embodiments, expression includes both the transcription of the gene and the translation of the mRNA into a gene product. In other embodiments, expression includes only the transcription of the nucleic acid encoding the gene of interest. Expression vectors may also include regulatory elements operably ligated to the coding region to facilitate protein expression in target cells. The regulatory elements and combinations of genes or multiple genes to which they are operably ligated for expression may sometimes be called “expression cassettes,” many of which are known and available in the art or can be readily constructed from components available in the art.

[0057] The term "heterogeneous" can refer to an entity whose genotype differs from the rest of the entity being compared. For example, a polynucleotide introduced into a plasmid or vector from a different species by genetic engineering techniques may be a heterogeneous polynucleotide. A promoter that has been removed from its native coding sequence and operably ligated to a coding sequence with which it is not found naturally may be a heterogeneous promoter.

[0058] As used herein, "7m8" refers to the amino acid sequence LALGETTRPA (SEQ ID NO: 1).

[0059] The "7m8 variant" refers to any possible serotype of rAAV in which the amino acid sequence LALGETTRPA (SEQ ID NO: 1) is inserted into the GH loop exposed to the solvent of the capsid protein.

[0060] When 7m8 is inserted into rAAV2 (also known as AAV2.7m8), the amino acid sequence LALGETTRPA (sequence number 1) is inserted into the GH loop of the AAV2 capsid protein between amino acids 570-611, for example, between positions 587 and 588 of the AAV2 capsid protein VP1. In some cases, when 7m8 is inserted into rAAV2 (also known as AAV2.7m8), the amino acid sequence LALGETTRPA (sequence number 1) is inserted into the GH loop of the AAV2 capsid protein, for example, between positions 587 and 588 of the AAV2 VP1 containing the sequence of sequence number 13. When 7m8 is inserted into rAAV1 (also known as AAV1.7m8), the amino acid sequence LALGETTRPA (SEQ ID NO: 1) is inserted into the GH loop of the AAV1 capsid protein between amino acids 571-612, for example, between amino acids 590 and 591. When 7m8 is inserted into rAAV5 (also known as AAV5.7m8), the amino acid sequence LALGETTRPA (SEQ ID NO: 1) is inserted into the GH loop of the AAV5 capsid protein between amino acids 560-601, for example, between amino acids 575 and 576. When 7m8 is inserted into rAAV6 (also called AAV6.7m8), the amino acid sequence LALGETTRPA (SEQ ID NO: 1) is inserted into the GH loop between amino acids 571-612 of the AAV6 capsid protein, for example, between amino acids 590 and 591. When 7m8 is inserted into rAAV7 (also called AAV7.7m8), the amino acid sequence LALGETTRPA (SEQ ID NO: 1) is inserted into the GH loop between amino acids 572-613 of the AAV7 capsid protein, for example, between amino acids 589 and 590. When 7m8 is inserted into rAAV8 (also called AAV8.7m8), the amino acid sequence LALGETTRPA (SEQ ID NO: 1) is inserted into the GH loop between amino acids 573-614 of the AAV8 capsid protein, for example, between amino acids 590 and 591.When 7m8 is inserted into rAAV9 (also known as AAV9.7m8), the amino acid sequence LALGETTRPA (SEQ ID NO: 1) is inserted into the GH loop of the AAV9 capsid protein, for example, between amino acids 588 and 589. When 7m8 is inserted into rAAV10 (also known as AAV10.7m8), the amino acid sequence LALGETTRPA (SEQ ID NO: 1) is inserted into the GH loop within amino acids 573-614 of the AAV10 capsid protein, for example, between amino acids 589 and 590. overview

[0061] Current treatments for intraocular neovascularization, such as wAMD (e.g., aflibercept), require lifetime in vitro vein therapy (IVT) administration approximately every 4–8 weeks. This can increase the risk of inflammation, infection, and other adverse effects in some patients. Furthermore, current treatments present compliance issues, particularly for elderly patients who are most affected by wAMD, due to repeated and / or frequent visits to the clinic for treatment. Reducing the number of administrations is associated with vision loss and worsening of the eye disease or condition. The ability of AAV vectors to efficiently transduce target retinal cells after IVT injection has made them available for successfully transferring therapeutic genes into photoreceptors, retinal pigment epithelium, and the inner retina to treat a variety of retinal diseases. Thus, administration of rAAV particles encoding anti-VEGF agents (e.g., aflibercept) can provide long-term and / or sustained in vivo release of anti-VEGF agents.

[0062] Surprisingly, the rAAV particle encoding aflibercept has a 6 × 10⁻¹⁰ 11 A single low dose of vector genome (vg) / eye administered to the eyes of individuals with intraocular neovascular disease resulted in disease stabilization and a robust anatomical response in all treated individuals (see Example 1). In addition, 6 × 10⁶ rAAV particles encoding aflibercept were administered. 11Following a single low-dose administration of vg / eye, visual acuity stabilized in all treated individuals, and none required rescue anti-VEGF treatment (e.g., IVT injection of aflibercept). Furthermore, single-dose administration of rAAV particles encoding aflibercept to the eyes of individuals with intraocular neovascular disease unexpectedly resulted in a reduction (e.g., resolution) of symptoms, including intraretinal and subretinal fluid, which had been refractory to previous anti-VEGF treatments (e.g., long-term IVT injections of aflibercept, ranibizumab, or bevacizumab).

[0063] Therefore, this disclosure relates to 6 × 10⁶ rAAV particles encoding an anti-VEGF agent (e.g., aflibercept). 11 This disclosure provides a method for treating intraocular neovascularization in an individual by administering a single dose of vg / eye or less. In addition, this disclosure provides a method for reducing retinal fluid in the eye of an individual with intraocular neovascularization by administering a single dose of rAAV particles encoding an anti-VEGF agent (e.g., aflibercept). The methods disclosed herein address issues of non-compliance and non-adherence while reducing or eliminating the need for repeated IVT injections and providing long-term efficacy. In addition, the methods provided herein reduce adverse effects associated with multiple IVT injections. Treatment method

[0064] This specification provides a method for treating intraocular neovascular disease in an individual, comprising the step of administering a unit dose of recombinant adeno-associated virus (rAAV) particles to the eye of the individual.

[0065] Similarly, this specification provides a method for reducing retinal fluid in the eye of an individual having an intraocular neovascular disease, comprising the step of administering a unit dose of rAAV particles to the eye of the individual.

[0066] In some embodiments, intraocular neovascular disease is exudative age-related macular degeneration (wAMD), retinal neovascularization, choroidal neovascularization, diabetic retinopathy, proliferative diabetic retinopathy, retinal vein occlusion, central retinal vein occlusion, branch retinal vein occlusion, diabetic macular edema, diabetic retinal ischemia, ischemic retinopathy, diabetic retinal edema, or any combination thereof.

[0067] In some embodiments, the individual is human. In some embodiments, the individual received at least one prior treatment for intraocular neovascular disease with an anti-VEGF agent (e.g., bevacizumab, brolucizumab, ranibizumab, falisimab, abisipalpegol, and / or aflibercept) during approximately the last 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, 15 weeks, or 16 weeks prior to the administration of a unit dose of rAAV particles. In some embodiments, the individual showed a meaningful response to the prior treatment with an anti-VEGF agent (e.g., bevacizumab, brolucizumab, ranibizumab, falisimab, abisipalpegol, and / or aflibercept). In some embodiments, the anti-VEGF agent is aflibercept, its functional variant, or a functional fragment thereof. In some embodiments, the anti-VEGF agent comprises a polypeptide having an amino acid sequence having at least about 95% identity with the amino acid sequence of SEQ ID NO: 35. In some embodiments, the retinal fluid of the individual's eye is intraretinal fluid (IRF) and / or subretinal fluid (SRF). In some embodiments, the amount or presence of retinal fluid of the individual's eye is refractory to prior treatment with an anti-VEGF agent (e.g., bevacizumab, brolucizumab, ranibizumab, falisimab, abisipalpegol, and / or aflibercept). In some embodiments, the anti-VEGF agent is aflibercept, its functional variant, or a functional fragment thereof. In some embodiments, the anti-VEGF agent comprises a polypeptide having an amino acid sequence having at least about 95% identity with the amino acid sequence of SEQ ID NO: 35.

[0068] In some embodiments, the unit dose is expressed as the number of vector genomes (vg). In some embodiments, the unit dose is approximately 6 × 10⁶ of rAAV particles. 11 The vector genome (vg) or less. In some embodiments, the unit dose is expressed as vector genome count (vg) / eye (vg / eye). In some embodiments, the unit dose is approximately 6 × 10⁻⁶ of rAAV particles. 11 The dose is vg / eye or less. In some embodiments, the unit dose of rAAV particles is about 6 × 10⁻⁶. 10 ~Approx. 2×10 11 The unit dose is vg / eye. In some embodiments, the unit dose of rAAV particles is approximately 2 × 10⁻⁶. 11 Or approximately 6 x 10 10 vg / eye

[0069] In some embodiments, a unit dose of rAAV particles is administered to one eye of an individual. In some embodiments, one eye of an individual is either the right or left eye. In some embodiments, one eye of an individual is the right eye. In some embodiments, one eye of an individual is the left eye. In some embodiments, the method provided herein further includes the step of administering a unit dose of rAAV particles to the opposite eye of an individual. In some embodiments, one eye of an individual is the right eye and the opposite eye is the left eye. In some embodiments, one eye of an individual is the left eye and the opposite eye is the right eye.

[0070] In some embodiments, the step of administering a unit dose of rAAV particles to the contralateral eye of the individual occurs up to approximately two weeks (e.g., about day 0, day 1, day 2, day 3, day 4, day 5, day 6, day 7, day 8, day 9, day 10, day 11, day 12, day 13, or day 14) after the step of administering a unit dose of rAAV particles to one eye of the individual. In some embodiments, the unit dose of rAAV particles administered to the contralateral eye of the individual is approximately the same as the unit dose of rAAV particles administered to one eye of the individual (e.g., less than 1% higher or lower, less than 5% higher or lower, less than 10% higher or lower, or less than 20% higher or lower) or lower (e.g., about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, or about 90% lower).

[0071] In some embodiments, the step of administering a unit dose of rAAV particles to the opposite eye occurs at least about two weeks (for example, at least about two weeks, at least about three weeks, at least about four weeks, at least about one month, at least about two months, at least about three months, at least about four months, at least about five months, at least about six months, at least about seven months, at least about eight months, at least about nine months, at least about ten months, at least about eleven months, at least about twelve months, at least about one year, at least about two years, at least about three years, at least about four years, at least about five years, or longer) after the step of administering a unit dose of rAAV particles to one eye. In some embodiments, the unit dose of rAAV particles administered to the opposite eye of an individual is higher than the unit dose of rAAV particles administered to one eye of the individual (e.g., higher than about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 125%, about 150%, about 175%, about 200%, about 225%, about 250%, about 275%, about 300%, or any higher percentage).

[0072] In some embodiments, the rAAV particles include a) a nucleic acid having AAV2 terminal inversion sequences (ITRs) at both ends, encoding a polypeptide containing an amino acid sequence having at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.99%, or 100% identity with the amino acid sequence of SEQ ID NO: 35, and b) an AAV2 capsid protein containing 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. The sequence of SEQ ID NO: 35 is provided below: SDTGRPFVEMYSEIPEIIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVLSPS HGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHEKDKTHTCPPCPAPEL LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG(Sequence ID 35)

[0073] In some embodiments, the rAAV particles include a) a nucleic acid encoding a polypeptide having an amino acid sequence having at least about 95% identity with the amino acid sequence of SEQ ID NO: 35 and having AAV2 terminal inverse sequences (ITRs) at both ends, and b) an AAV2 capsid protein having the amino acid sequence LGETTRP (SEQ ID NO: 14) inserted between positions 587 and 588 of the capsid protein, wherein the amino acid residue numbering corresponds to that of the AAV2 VP1 capsid protein.

[0074] In some embodiments, the rAAV particles encode a polypeptide comprising an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.99%, or 100% identity with the amino acid sequence of SEQ ID NO: 35, and include nucleic acids having AAV2 terminal inverse sequences (ITRs) at both ends. In some embodiments, the rAAV particles encode a polypeptide comprising an amino acid sequence having at least about 95% identity with the amino acid sequence of SEQ ID NO: 35, and include nucleic acids having AAV2 terminal inverse sequences (ITRs) at both ends. 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.

[0075] In some embodiments, the rAAV particles include a codon-optimized sequence encoding an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.99%, or 100% identity with the amino acid sequence of SEQ ID NO: 35, and include nucleic acids having AAV2 terminal inversion sequences (ITRs) at both ends. In some embodiments, the rAAV particles include a codon-optimized sequence encoding an amino acid sequence having at least about 95% identity with the amino acid sequence of SEQ ID NO: 35, and include nucleic acids having AAV2 terminal inversion sequences (ITRs) at both ends. In some embodiments, the rAAV particles include a codon-optimized sequence encoding an amino acid sequence having 100% identity with the amino acid sequence of SEQ ID NO: 35, and include nucleic acids having AAV2 terminal inversion sequences (ITRs) at both ends.

[0076] In some embodiments, the rAAV particles include a nucleic acid comprising a cDNA sequence of aflibercept or a functional variant or a functional fragment thereof, with AAV2 terminal inversion sequences (ITRs) at both ends. In some embodiments, the rAAV particles include a nucleic acid comprising a codon-optimized cDNA sequence of aflibercept or a functional variant or a functional fragment thereof, with AAV2 terminal inversion sequences (ITRs) at both ends. In some embodiments, the rAAV particles include a nucleic acid comprising the nucleic acid sequence of SEQ ID NO: 36.

[0077] In some embodiments, the nucleic acid further comprises (a) a first enhancer region containing a CMV sequence; (b) a promoter region containing a CMV sequence; (c) a 5'UTR region containing a TPL sequence and an eMLP sequence in the order of 5' to 3'; (d) a second enhancer region containing a full-length EES sequence; and (e) an HGH polyadenylation site. In some embodiments, the enhancer region containing the CMV sequence includes the sequence of SEQ ID NO: 22. In some embodiments, the promoter region containing the CMV sequence includes the sequence of SEQ ID NO: 23. In some embodiments, the TPL sequence includes the sequence of SEQ ID NO: 24. In some embodiments, the eMLP sequence includes the sequence of SEQ ID NO: 25. In some embodiments, the second enhancer region containing a full-length EES sequence includes the sequence of SEQ ID NO: 26. In some embodiments, the HGH polyadenylation site includes the sequence of SEQ ID NO: 27.

[0078] In some embodiments, the rAAV particles contain an AAV2 capsid protein containing the amino acid sequence LGETTRP (SEQ ID NO: 14) inserted between positions 587 and 588 of AAV2 VP1 containing the sequence of SEQ ID NO: 13. The sequence of SEQ ID NO: 13 is provided below: MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPGYKYLGPFNGLD KGEPVNEADAAALEHDKAYDRQLDSGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQ AKKRVLEPLGLVEEPVKTAPGKKRPVEHSPVEPDSSSGTGKAGQQPARKRLNFGQTGDAD SVPDPQPLGQPPAAPSGLGTNTMATGSGAPMADNNEGADGVGNSSGNWHCDSTWMGDRVI TTSTRTWALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLI NNNWGFRPKRLNFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQG CLPPFPADVFMVPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEDVPF HSSYAHSQSLDRLMNPLIDQYLYYLSRTNTPSGTTTQSRLQFSQAGASDIRDQSRNWLPG PCYRQQRVSKTSADNNNSEYSWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVL IFGKQGSEKTNVDIEKVMITDEEIRTTNPVATEQYGSVSTNLQRGNRQAATADVNTQGV LPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPSTT FSAAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSVNVDFTVDTNGVY SEPRPIGTRYLTRNL(Sequence ID 13)

[0079] In some embodiments, the rAAV particles include an AAV2 capsid protein containing 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 that of the AAV2 VP1 capsid protein. In some embodiments, the rAAV particles include an AAV2 capsid protein containing the amino acid sequence LALGETTRPA (SEQ ID NO: 1) inserted between positions 587 and 588 of the AAV2 VP1 containing the sequence of SEQ ID NO: 13.

[0080] In some embodiments, the rAAV particles have the following amino acid sequence inserted between positions 587 and 588 of the capsid protein: LALGETTRPA (SEQ ID NO: 1); LANETITRPA (SEQ ID NO: 2), LAKAGQANNA (SEQ ID NO: 3), LAKDPKTTNA (SEQ ID NO: 4), KDTDTTR (SEQ ID NO: 5), RAGGSVG (SEQ ID NO: 6), AVDTTKF (SEQ ID NO: 7), STGKVPN (SEQ ID NO: 8), LAKDTDTTRA (SEQ ID NO: 9), LARAGGSVGA (SEQ ID NO: 8). The AAV2 capsid protein comprises any of the following: sequence number 10), LAAVDTTKFA (sequence number 11), LASTGKVPNA (sequence number 12), LGETTRP (sequence number 14), NETITRP (sequence number 15), KAGQANN (sequence number 16), KDPKTTN (sequence number 17), KDTDTTR (sequence number 18), RAGGSVG (sequence number 19), AVDTTKF (sequence number 20), and STGKVPN (sequence number 21), wherein the amino acid residue numbering corresponds to the AAV2 VP1 capsid protein. In some embodiments, the rAAV particle comprises an AAV2 containing the sequence of sequence number 13. The AAV2 capsid protein contains one of the following amino acid sequences inserted between positions 587 and 588 of VP1: LALGETTRPA (SEQ ID NO: 1); LANETITRPA (SEQ ID NO: 2), LAKAGQANNA (SEQ ID NO: 3), LAKDPKTTNA (SEQ ID NO: 4), KDTDTTR (SEQ ID NO: 5), RAGGSVG (SEQ ID NO: 6), AVDTTKF (SEQ ID NO: 7), STGKVPN (SEQ ID NO: 8), LAKDTDTTRA (SEQ ID NO: 9), LARAGGSVGA (SEQ ID NO: 10), LAAVDTTKFA (SEQ ID NO: 11), LASTGKVPNA (SEQ ID NO: 12), LGETTRP (SEQ ID NO: 14), NETITRP (SEQ ID NO: 15), KAGQANN (SEQ ID NO: 16), KDPKTTN (SEQ ID NO: 17), KDTDTTR (SEQ ID NO: 18), RAGGSVG (SEQ ID NO: 19), AVDTTKF (SEQ ID NO: 20), and STGKVPN (SEQ ID NO: 21).

[0081] In some embodiments, a unit dose of rAAV particles is administered to one eye and / or the opposite eye of an individual by intravitreal (IVT) injection, intraocular injection, or intraretinal injection.

[0082] In some embodiments, the unit dose of rAAV particles is present in the pharmaceutical formulation. In some embodiments, the pharmaceutical formulation comprises rAAV particles, one or more osmotic pressure or ionic strength modifiers, one or more buffers, one or more surfactants, and one or more solvents. In some embodiments, the osmotic pressure or ionic strength modifier is sodium chloride. In some embodiments, one or more buffers are sodium dihydrogen phosphate and / or sodium hydrogen phosphate. In some embodiments, the surfactant is poloxamer 188. In some embodiments, the solvent is water. In some embodiments, the pharmaceutical formulation comprises rAAV particles, sodium chloride, sodium phosphate, and a surfactant. In some embodiments, the pharmaceutical formulation is approximately 1 × 10⁻⁶ 10 vg / mL ~ approx. 1×10 13 It contains vg / mL of rAAV particles. In some embodiments, the pharmaceutical formulation contains approximately 6 × 10 11 vg / mL ~ approx. 6×10 12The formulation contains vg / mL of rAAV particles. In some embodiments, the pharmaceutical formulation contains about 150 mM to about 200 mM sodium chloride (e.g., any of about 150 mM, about 160 mM, about 170 mM, about 180 mM, about 190 mM, or about 200 mM). In some embodiments, the pharmaceutical formulation contains about 1 mM to about 10 mM sodium dihydrogen phosphate (e.g., about 1 mM, about 2 mM, about 3 mM, about 4 mM, about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, or about 10 mM). In some embodiments, the pharmaceutical formulation contains about 1 mM to about 10 mM sodium hydrogen phosphate (e.g., about 1 mM, about 2 mM, about 3 mM, about 4 mM, about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, or about 10 mM). In some embodiments, the pharmaceutical formulation contains about 0.0005% (weight / volume) to about 0.005% (weight / volume) of poloxamer 188 (e.g., any of about 0.0005% (weight / volume), 0.0006% (weight / volume), 0.0007% (weight / volume), 0.0008% (weight / volume), 0.0009% (weight / volume), 0.001% (weight / volume), 0.002% (weight / volume), 0.003% (weight / volume), 0.004% (weight / volume), or about 0.005% (weight / volume)). In some embodiments, the pharmaceutical formulation has a pH of about 7.0 to about 7.5 (e.g., any of about 7.0, about 7.1, about 7.2, about 7.3, about 7.4, or about 7.5). In some embodiments, the pharmaceutical formulation contains about 6 × 10 12 The pharmaceutical formulation contains vg / mL of rAAV particles, approximately 180 mM sodium chloride, approximately 5 mM sodium dihydrogen phosphate, approximately 5 mM sodium hydrogen phosphate, and approximately 0.001% (weight / volume) poloxamer 188, and has a pH of approximately 7.3. In some embodiments, the pharmaceutical formulation has a pH of approximately 6 × 10 11 The pharmaceutical formulation contains vg / mL rAAV particles, approximately 180 mM sodium chloride, approximately 5 mM sodium dihydrogen phosphate, approximately 5 mM sodium hydrogen phosphate, and approximately 0.001% (weight / volume) poloxamer 188, and has a pH of approximately 7.3.

[0083] In some embodiments, the unit dose of rAAV particles contains a volume between approximately 25 μL and approximately 250 μL (for example, approximately 25 μL, approximately 30 μL, approximately 40 μL, approximately 50 μL, approximately 60 μL, approximately 70 μL, approximately 80 μL, approximately 90 μL, approximately 100 μL, approximately 110 μL, approximately 120 μL, approximately 130 μL, approximately 140 μL, approximately 150 μL, approximately 160 μL, approximately 170 μL, approximately 180 μL, approximately 190 μL, approximately 200 μL, approximately 210 μL, approximately 220 μL, approximately 230 μL, approximately 240 μL, or approximately 250 μL). In some embodiments, the concentration of rAAV particles in the pharmaceutical formulation is adjusted so that the volume of the unit dose of rAAV particles administered to the eye of an individual is between approximately 25 μL and approximately 250 μL. In some embodiments, a unit dose of rAAV particles contains a volume of approximately 100 μL. In some embodiments, a unit dose of rAAV particles contains a volume of approximately 30 μL.

[0084] In some embodiments, a unit dose of rAAV particles is administered in combination with a steroid treatment. In some embodiments, the steroid treatment is a corticosteroid treatment. In some embodiments, the steroid treatment is a systemic steroid treatment. In some embodiments, the steroid treatment is an oral steroid treatment. In some embodiments, the steroid treatment is a prednisone treatment. In some embodiments, the steroid treatment is an ophthalmic steroid treatment. In some embodiments, the ophthalmic steroid treatment is a topical steroid treatment (e.g., eye drops), a periorbital steroid treatment (e.g., sub-Tenon's capsule, subconjunctival), an intravitreal steroid treatment, or a 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, fluocinolone treatment, rimexolone treatment, or prednisone treatment. In some embodiments, the topical steroid treatment is difluprednate treatment. In some embodiments, steroid treatment is administered before, during, and / or after administration of a unit dose of rAAV particles. In some embodiments, steroid treatment is administered before administration of a unit dose of rAAV particles. In some embodiments, steroid treatment is administered during administration of a unit dose of rAAV particles. In some embodiments, steroid treatment is administered after administration of a unit dose of rAAV particles. In some embodiments, steroid treatment is administered before and during administration of a unit dose of rAAV particles. In some embodiments, steroid treatment is administered before and after administration of a unit dose of rAAV particles. In some embodiments, steroid treatment is administered during and after administration of a unit dose of rAAV particles. In some embodiments, steroid treatment is administered before, during, and after administration of a unit dose of rAAV particles.

[0085] In some embodiments, the steroid treatment is an ophthalmic steroid treatment (e.g., difluprednate). In some embodiments, the ophthalmic steroid treatment (e.g., difluprednate) is a daily steroid treatment for up to approximately 4 weeks, approximately 6 weeks, or approximately 8 weeks following the administration of a unit dose of rAAV particles. In some embodiments, the ophthalmic steroid treatment includes approximately 4 administrations of ophthalmic steroids in approximately week 1, approximately 3 administrations of ophthalmic steroids in approximately week 2, approximately 2 administrations of ophthalmic steroids in approximately week 3, and approximately 1 administration of ophthalmic steroids in approximately week 4, with the timing starting from and after the administration of the unit dose of rAAV particles. In some embodiments, the ophthalmic steroid is approximately 0.005% to approximately 0.5% difluprednate. In some embodiments, the ophthalmic steroid is one of the following: about 0.005%, about 0.006%, about 0.007%, about 0.008%, about 0.009%, about 0.01%, about 0.02%, about 0.03%, about 0.4%, about 0.05%, about 0.06%, about 0.07%, about 0.08%, about 0.09%, or about 0.1% difluprednate. In some embodiments, the ophthalmic steroid is 0.05% difluprednate. In some embodiments, the dose of 0.05% difluprednate is one drop of ophthalmic solution. In some embodiments, one drop is about 50 μl (e.g., about 25 μl to about 50 μl, about 50 μl to about 100 μl). In some embodiments, the dose of difluprednate includes about 1 μg to about 5 μg, or about 2 μg to about 3 μg, or about 2.5 μg of difluprednate. In some embodiments, the dose of difluprednate includes about 2.5 μg of difluprednate.

[0086] In some embodiments, the steroid treatment is an ophthalmic steroid treatment (e.g., difluprednate). In some embodiments, the ophthalmic steroid treatment (e.g., difluprednate) is a daily topical steroid treatment for up to about 4 weeks, about 6 weeks, or about 8 weeks, following the administration of a unit dose of rAAV particles. In some embodiments, the topical steroid treatment includes about 4 doses of topical steroid in about week 1, about 3 doses of topical steroid in about week 2, about 2 doses of topical steroid in about week 3, and about 1 dose of topical steroid in about week 4, with the timing starting from and after the administration of the unit dose of rAAV particles. In some embodiments, the topical steroid contains about 1 μg to about 3 μg dose of 0.05% difluprednate. In some embodiments, the topical steroid contains about 2.5 μg dose of 0.05% difluprednate. In some embodiments, the topical steroid contains about 0.005% to about 0.5% difluprednate. In some embodiments, the topical steroid is one of the following: about 0.005%, about 0.006%, about 0.007%, about 0.008%, about 0.009%, about 0.01%, about 0.02%, about 0.03%, about 0.4%, about 0.05%, about 0.06%, about 0.07%, about 0.08%, about 0.09%, or about 0.1% difluprednate. In some embodiments, the topical steroid is 0.05% difluprednate. In some embodiments, the dose of 0.05% difluprednate is one drop of ophthalmic solution. In some embodiments, one drop is about 50 μl (e.g., about 25 μl to about 50 μl, about 50 μl to about 100 μl). In some embodiments, the dose of difluprednate includes about 1 μg to about 5 μg, or about 2 μg to about 3 μg, or about 2.5 μg of difluprednate. In some embodiments, the dose of difluprednate includes about 2.5 μg of difluprednate.

[0087] In some embodiments, the retinal fluid (e.g., SRF and / or IRF) of an individual's eye is reduced by more than approximately 5%, approximately 10%, approximately 15%, approximately 20%, approximately 30%, approximately 40%, approximately 50%, approximately 60%, approximately 70%, approximately 80%, approximately 90%, or approximately 100% after administration of a unit dose of rAAV particles to one eye and / or the opposite eye of the individual. In some embodiments, the retinal fluid of an individual's eye (e.g., SRF and / or IRF) is reduced by approximately 100% after administration of a unit dose of rAAV particles to one eye and / or the opposite eye of the individual, compared to the level of retinal fluid in the individual's eye before administration of a unit dose of rAAV particles.

[0088] In some embodiments, the method provided herein further includes the step of monitoring the levels of retinal fluid (e.g., SRF and / or IRF) in one eye and / or the other eye of an individual after administration of a unit dose of rAAV particles. In some embodiments, the reduction in retinal fluid (e.g., SRF and / or IRF) of the eye is first observed after any of the following periods of time: about 1 day, about 3 days, about 8 days, about 2 weeks, about 4 weeks, about 6 weeks, about 8 weeks, about 12 weeks, about 16 weeks, about 20 weeks, about 24 weeks, about 28 weeks, about 32 weeks, about 36 weeks, about 40 weeks, about 44 weeks, about 48 weeks, about 52 weeks, about 56 weeks, about 60 weeks, about 64 weeks, about 68 weeks, about 72 weeks, about 76 weeks, about 80 weeks, about 84 weeks, about 88 weeks, about 92 weeks, about 96 weeks, about 100 weeks, about 104 weeks, or longer. In some embodiments, the reduction of retinal fluid (e.g., SRF and / or IRF) of the eye persists or is maintained for at least 1 week, at least 2 weeks, at least 4 weeks, at least 6 weeks, at least 8 weeks, at least 12 weeks, at least 16 weeks, at least 20 weeks, at least 24 weeks, at least 28 weeks, at least 32 weeks, at least 36 weeks, at least 40 weeks, at least 44 weeks, at least 48 weeks, at least 52 weeks, at least 56 weeks, at least 60 weeks, at least 64 weeks, at least 68 weeks, at least 72 weeks, at least 76 weeks, at least 80 weeks, at least 84 weeks, at least 88 weeks, at least 92 weeks, at least 96 weeks, at least 100 weeks, at least 104 weeks, or longer, after administration of a unit dose of rAAV particles.

[0089] In some embodiments, the reduction of retinal fluid (e.g., SRF and / or IRF) is determined by any method known in the Art. In some embodiments, the reduction of retinal fluid (e.g., SRF and / or IRF) is determined by optical coherence tomography (OCT), spectral domain OCT (SD-OCT), OCT angiography, fluorescein angiography, or direct retinal observation. In some embodiments, the reduction of retinal fluid (e.g., SRF and / or IRF) is determined by optical coherence tomography (OCT). In some embodiments, the reduction of retinal fluid (e.g., SRF and / or IRF) is determined by spectral domain OCT (SD-OCT). In some embodiments, the reduction of retinal fluid (e.g., SRF and / or IRF) is determined by OCT angiography. In some embodiments, the reduction of retinal fluid (e.g., SRF and / or IRF) is determined by fluorescein angiography. In some embodiments, the reduction of retinal fluid (e.g., SRF and / or IRF) in the eye is determined by direct retinal observation.

[0090] In some embodiments, whether intraocular neovascular disease has been treated in an individual after administration of a unit dose of rAAV particles to one eye and / or the opposite eye is assessed based on the level of retinal fluid (e.g., SRF and / or IRF) compared to the level of retinal fluid (e.g., intraretinal fluid (IRF) and / or subretinal fluid (SRF)) before administration of a unit dose of AAV particles to one eye and / or the opposite eye (e.g., as described above). In some embodiments, retinal fluid is subretinal fluid (SRF) or intraretinal fluid (IRF). In some embodiments, retinal fluid is subretinal fluid (SRF). In some embodiments, retinal fluid is intraretinal fluid (IRF). In some embodiments, if a reduction in retinal fluid (e.g., IRF and / or SRF) is observed after administration of a unit dose of rAAV particles to one eye and / or the opposite eye, compared to the level of retinal fluid (e.g., IRF and / or SRF) before administration of a unit dose of rAAV particles to one eye and / or the opposite eye, it is determined that intraocular neovascularization has been treated in the individual after administration of a unit dose of rAAV particles to one eye and / or the opposite eye (e.g., as described above). In some embodiments, intraocular neovascularization is wAMD.

[0091] In some embodiments, the step of administering a unit dose of rAAV particles to one eye and / or the opposite eye of an individual results in maintenance or reduction of retinal thickness compared to the retinal thickness before administration of the unit dose of rAAV particles. In some embodiments, the step of administering a unit dose of rAAV particles to one eye and / or the opposite eye of an individual results in a decrease in retinal thickness compared to the retinal thickness before administration of the unit dose of rAAV particles. In some embodiments, retinal thickness is central region retinal thickness (CST) or foveal retinal thickness (CRT). In some embodiments, the step of administering a unit dose of rAAV particles to one eye and / or the opposite eye of an individual results in a reduction of retinal thickness of approximately 5%, approximately 10%, approximately 15%, approximately 20%, approximately 25%, approximately 30%, approximately 35%, approximately 40%, approximately 45%, approximately 50%, approximately 55%, approximately 60%, approximately 65%, approximately 70%, approximately 75%, approximately 80%, approximately 85%, approximately 90%, approximately 95%, approximately 99%, or approximately 100% compared to the retinal thickness before administration of the unit dose of rAAV particles. In some embodiments, retinal thickness (e.g., CST or CRT) is determined by OCT or SD-OCT. In some embodiments, the step of administering a unit dose of rAAV particles to one eye and / or the opposite eye of an individual results in a reduction of approximately 10 μm to approximately 100 μm (e.g., approximately 10 μm, approximately 15 μm, approximately 20 μm, approximately 25 μm, approximately 30 μm, approximately 35 μm, approximately 40 μm, approximately 45 μm, approximately 50 μm, approximately 55 μm, approximately 60 μm, approximately 65 μm, approximately 70 μm, approximately 75 μm, approximately 80 μm, approximately 85 μm, approximately 90 μm, approximately 95 μm, approximately 100 μm, or any larger μm). In some embodiments, the step of administering a unit dose of rAAV particles to one eye and / or the opposite eye of an individual results in a reduction of foveal retinal thickness (CRT) or central region retinal thickness (CST) of approximately 5%, approximately 10%, approximately 15%, approximately 20%, approximately 25%, approximately 30%, approximately 35%, approximately 40%, approximately 45%, approximately 50%, approximately 55%, approximately 60%, approximately 65%, approximately 70%, approximately 75%, approximately 80%, approximately 85%, approximately 90%, approximately 95%, approximately 99%, or approximately 100% compared to the retinal thickness before administration of the unit dose of rAAV particles.In some embodiments, the step of administering a unit dose of rAAV particles to one eye and / or the opposite eye of an individual results in a reduction of at least about 10% in foveal retinal thickness (CRT) or central regional retinal thickness (CST) compared to the retinal thickness before administration of the unit dose of rAAV particles. In some embodiments, the step of administering a unit dose of rAAV particles to one eye and / or the opposite eye of an individual results in a reduction of about 15% or more in foveal retinal thickness (CRT) or central regional retinal thickness (CST) compared to the retinal thickness before administration of the unit dose of rAAV particles.

[0092] In some embodiments, the step of administering a unit dose of rAAV particles to one eye and / or the opposite eye of an individual results in maintenance or reduction of macular volume compared to the macular volume before administration of the unit dose of rAAV particles. In some embodiments, the step of administering a unit dose of rAAV particles to one eye and / or the opposite eye of an individual results in a decrease in macular volume compared to the macular volume before administration of the unit dose of rAAV particles. In some embodiments, the step of administering a unit dose of rAAV particles to one eye and / or the opposite eye of an individual results in a decrease in macular volume of about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, or about 50% greater than the macular volume before administration of the unit dose of rAAV particles. In some embodiments, the step of administering a unit dose of rAAV particles to one eye and / or the opposite eye of an individual results in a reduction of at least about 10% of macular volume compared to the macular volume before administration of the unit dose of rAAV particles. In some embodiments, macular volume is determined by OCT or SD-OCT. In some embodiments, the step of administering a unit dose of rAAV particles to one eye and / or the opposite eye of an individual results in a reduction of at least about 10% of macular volume compared to the macular volume before administration of the unit dose of rAAV particles. In some embodiments, the step of administering a unit dose of rAAV particles to one eye and / or the opposite eye of an individual results in a reduction of about 15% or more of macular volume compared to the macular volume before administration of the unit dose of rAAV particles. In some embodiments, macular volume is determined by OCT or SD-OCT.

[0093] In some embodiments, the step of administering a unit dose of rAAV particles to one eye and / or the opposite eye of an individual results in maintenance or improvement of visual acuity compared to visual acuity before administration of the unit dose of rAAV particles. In some embodiments, the step of administering a unit dose of rAAV particles to one eye and / or the opposite eye of an individual results in improvement of visual acuity compared to visual acuity before administration of the unit dose of rAAV particles. In some embodiments, the step of administering a unit dose of rAAV particles to one eye and / or the opposite eye of an individual results in an improvement of visual acuity of about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 125%, about 150%, about 175%, about 200%, about 225%, about 250%, about 275%, about 300%, or greater than that percentage compared to visual acuity before administration of the unit dose of rAAV particles. In some embodiments, visual acuity is defined as best corrected visual acuity (BCVA). In some embodiments, the step of administering a unit dose of rAAV particles to one eye and / or the opposite eye of an individual results in an improvement in BCVA compared to the BCVA before administration of the unit dose of rAAV particles. In some embodiments, BCVA is expressed as an ETDRS score, which corresponds to the number of letters read exactly (Vitale et al., (2016) JAMA Opthalmol 134(9):1041:1047). In some embodiments, the step of administering a unit dose of rAAV particles to one eye and / or the opposite eye of an individual results in an improvement of at least 15 ETDRS characters of BCVA (Vitale et al., (2016) JAMA Opthalmol 134(9):1041:1047) (e.g., at least about 15, at least about 20, at least about 30, at least about 40, at least about 50, at least about 60, or about 70 characters) compared to the BCVA before administration of a unit dose of rAAV particles. In some embodiments, the step of administering a unit dose of rAAV particles to one eye and / or the opposite eye of an individual results in an improvement of about 5 ETDRS characters of BCVA compared to the BCVA before administration of a unit dose of rAAV particles.In some embodiments, the step of administering a unit dose of rAAV particles to one eye and / or the opposite eye of an individual results in a maintenance of BCVA, where the individual has a reduction of less than 15 ETDRS characters (Vitale et al., (2016) JAMA Opthalmol 134(9):1041:1047) (e.g., less than 15 characters, less than 14 characters, less than 13 characters, less than 12 characters, less than 11 characters, less than 10 characters, less than 9 characters, less than 8 characters, less than 7 characters, less than 6 characters, less than 5 characters, less than 4 characters, less than 3 characters, less than 2 characters, 1 character, or 0 characters) compared to the BCVA before administration of the unit dose of rAAV particles. In some embodiments, the step of administering a unit dose of rAAV particles to one eye and / or the opposite eye of an individual results in a maintenance of BCVA, in which the individual has a decrease of approximately 2 letters compared to the BCVA before administration of the unit dose of rAAV particles.

[0094] In some embodiments, whether intraocular neovascular disease was treated in an individual after administration of a unit dose of rAAV particles to one eye and / or the opposite eye is assessed based on the best corrected visual acuity (BCVA) in one eye and / or the opposite eye. In some embodiments, BCVA is expressed as an ETDRS score, which corresponds to the number of letters read exactly (Vitale et al., (2016) JAMA Opthalmol 134(9):1041:1047). In some embodiments, an individual is determined to have maintained vision and / or visual acuity if they have a decrease of less than 15 characters (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 character, or 0 characters) in their ETDRS score compared to before administration of a unit dose of rAAV particles to one eye and / or the other eye. In some embodiments, an individual is determined to have improved vision and / or visual acuity if they have an increase of 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 about 70 characters) compared to before administration of a unit dose of rAAV particles to one eye and / or the other eye.

[0095] In some embodiments, whether intraocular neovascular disease has been treated in an individual after administration of a unit dose of rAAV particles to one eye and / or the opposite eye is assessed based on central region retinal thickness (CST) or foveal retinal thickness (CRT) in one eye and / or the opposite eye. In some embodiments, CST or CRT is determined by SD-OCT. In some embodiments, if the CST or CRT assessed by SD-OCT decreases after administration of a unit dose of rAAV particles to one eye and / or the opposite eye compared to before administration of the unit dose of rAAV particles to one eye and / or the opposite eye, it is determined that intraocular neovascular disease has been treated in the individual after administration of a unit dose of rAAV particles to one eye and / or the opposite eye. In some embodiments, if CST or CRT, as assessed by SD-OCT, is maintained after administration of a unit dose of rAAV particles to one eye and / or the opposite eye compared to before administration of a unit dose of rAAV particles to one eye and / or the opposite eye, it is determined that intraocular neovascular disease was treated in the individual after administration of a unit dose of rAAV particles to one eye and / or the opposite eye.

[0096] In some embodiments, whether intraocular neovascularization has been treated in an individual after administration of a unit dose of rAAV particles to one eye and / or the opposite eye is assessed based on the macular volume in one eye and / or the opposite eye. In some embodiments, the macular volume is determined by SD-OCT. In some embodiments, if the macular volume assessed by SD-OCT decreases after administration of a unit dose of rAAV particles to one eye and / or the opposite eye compared to before administration of the unit dose of rAAV particles to one eye and / or the opposite eye, it is determined that intraocular neovascularization has been treated in the individual after administration of a unit dose of rAAV particles to one eye and / or the opposite eye. In some embodiments, if macular volume, as assessed by SD-OCT, is maintained after administration of a unit dose of rAAV particles to one eye and / or the opposite eye compared to before administration of a unit dose of rAAV particles to one eye and / or the opposite eye, it is determined that intraocular neovascular disease was treated in the individual after administration of a unit dose of rAAV particles to one eye and / or the opposite eye.

[0097] In some embodiments, whether intraocular neovascularization has been treated in an individual after administration of a unit dose of rAAV particles to one eye and / or the opposite eye is assessed based on retinal thickness (e.g., foveal retinal thickness (CRT) or central region retinal thickness (CST)) and macular volume in one eye and / or the opposite eye. In some embodiments, CST and macular volume are determined by SD-OCT. In some embodiments, if the CST and macular volume assessed by SD-OCT decrease after administration of a unit dose of rAAV particles to one eye and / or the opposite eye compared to before administration of the unit dose of rAAV particles to one eye and / or the opposite eye, it is determined that intraocular neovascularization has been treated in the individual after administration of a unit dose of rAAV particles to one eye and / or the opposite eye. In some embodiments, if CST and macular volume, as assessed by SD-OCT, are maintained after administration of a unit dose of rAAV particles to one eye and / or the opposite eye compared to before administration of a unit dose of rAAV particles to one eye and / or the opposite eye, it is determined that intraocular neovascular disease was treated in the individual after administration of a unit dose of rAAV particles to one eye and / or the opposite eye.

[0098] In some embodiments, whether intraocular neovascularization has been treated in an individual after administration of a unit dose of rAAV particles to one eye and / or the opposite eye is assessed based on the number of rescue treatments (e.g., aflibercept injections) required by the individual after administration of a unit dose of rAAV particles to one eye and / or the opposite eye. In some embodiments, if the individual requires fewer than one rescue treatment (e.g., aflibercept injection) every 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks or more after administration of a unit dose of rAAV particles to one eye and / or the opposite eye, it is determined that intraocular neovascularization has been treated in the individual after administration of a unit dose of rAAV particles to one eye and / or the opposite eye.

[0099] In some embodiments, if an individual does not require any rescue treatment (e.g., aflibercept injection) for any period of at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, at least 7 weeks, at least 8 weeks, at least 9 weeks, at least 10 weeks, at least 15 weeks, at least 20 weeks, at least 30 weeks, at least 40 weeks, at least 50 weeks, at least 60 weeks, at least 70 weeks, at least 80 weeks, at least 90 weeks, at least 100 weeks, at least 110 weeks, or longer, it is determined that intraocular neovascular disease has been treated in the individual after administration of a unit dose of rAAV particles to one eye and / or the opposite eye.

[0100] In some embodiments, whether intraocular neovascularization (IMD) has been treated in an individual after administration of a unit dose of rAAV particles to one eye and / or the opposite eye is assessed based on the level of retinal fluid compared to the level of retinal fluid before administration of a unit dose of rAAV particles to one eye and / or the opposite eye. In some embodiments, if a reduction in retinal fluid is observed after administration of a unit dose of rAAV particles to one eye and / or the opposite eye compared to the level of retinal fluid before administration of a unit dose of rAAV particles to one eye and / or the opposite eye, it is determined that IMD has been treated in the individual after administration of a unit dose of rAAV particles to one eye and / or the opposite eye. In some embodiments, IMD is wAMD.

[0101] In some embodiments, whether intraocular neovascularization (IMD) has been treated in an individual after administration of a unit dose of rAAV particles to one eye and / or the opposite eye is assessed based on the dissipation of pigment epithelial exfoliation (PED) compared to the PED before administration of a unit dose of rAAV particles to one eye and / or the opposite eye. In some embodiments, if dissipation of PED is observed after administration of a unit dose of rAAV particles to one eye and / or the opposite eye compared to the PED before administration of a unit dose of rAAV particles to one eye and / or the opposite eye, it is determined that IMD has been treated in the individual after administration of a unit dose of rAAV particles to one eye and / or the opposite eye. In some embodiments, IMD is wAMD.

[0102] In some embodiments, whether intraocular neovascular disease has been treated in an individual after administration of a unit dose of rAAV particles to one eye and / or the opposite eye is assessed based on the growth of choroidal neovascularization (CNV) lesions determined by fluorescein angiography. In some embodiments, if the CNV lesion shrinks (e.g., by more than approximately 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 100%) after administration of a unit dose of rAAV particles to one eye and / or the opposite eye compared to the CNV lesion present before administration of a unit dose of rAAV particles to one eye and / or the opposite eye, it is determined that intraocular neovascular disease has been treated in the individual after administration of a unit dose of rAAV particles to one eye and / or the opposite eye. In some embodiments, if the CNV lesion does not grow after administration of a unit dose of rAAV particles to one eye and / or the opposite eye compared to the CNV lesion present before administration of a unit dose of rAAV particles to one eye and / or the opposite eye (e.g., less than about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, or 20%), it is determined that the intraocular neovascularization has been treated in the individual after administration of a unit dose of rAAV particles to one eye and / or the opposite eye. In some embodiments, the intraocular neovascularization is wAMD.

[0103] In some embodiments, whether intraocular neovascularization has been treated in an individual after administration of a unit dose of rAAV particles to one eye and / or the opposite eye is assessed based on the anatomical features of one eye and / or the opposite eye based on any method known in the art (e.g., SD-OCT, OCT, fluorescein angiography, digital color fundus photography, etc.). In some embodiments, if improvement is observed in the anatomical features of one eye and / or the opposite eye after administration of a unit dose of rAAV particles to one eye and / or the opposite eye, it is determined that intraocular neovascularization has been treated in the individual after administration of a unit dose of rAAV particles to one eye and / or the opposite eye. In some embodiments, intraocular neovascularization is wAMD.

[0104] In some embodiments, whether intraocular neovascular disease was treated in an individual after administration of a unit dose of rAAV particles to one eye and / or the opposite eye is determined by ophthalmological examination, intraocular pressure (e.g., using a Goldmann applanation tonometer or Tono-pen), indirect ophthalmoscopic fundus examination, examination of one eye and / or the opposite eye and ocular adnexa, responsiveness of the eyelids and / or pupils, ptosis, abnormal pupillary shape, pupillary inequality, abnormal response to light, Assessment is based on concentric pupillary disorders, slit-lamp examination (including examination of the eyelids, conjunctiva, cornea, lens, iris, and anterior chamber), abnormalities of the vitreous humor, optic nerve, peripheral retina, and posterior segment of the retinal vascular structure, SD-OCT, fluorescein angiography, digital color fundus photography (including images of the retina, optic disc, and / or macula), aqueous humor sampling, vitreous fluid sampling, OCT-angiography (OCT-A), and refractive and / or visual acuity (BCVA). In some embodiments, SD-OCT is performed to assess the presence of retinal thickness (e.g., foveal retinal thickness or central region retinal thickness), macular volume, and / or fluid (e.g., subretinal fluid or intraretinal fluid). In some embodiments, intraocular neovascular disease is wAMD.

[0105] A unit dose of rAAV particles may be administered to one eye and / or the opposite eye of an individual by any method known in the art. For example, a unit dose of rAAV particles may be administered intraocularly or intravitreally to one eye and / or the opposite eye of an individual. In some embodiments, the administration of a unit dose of rAAV particles to one eye and / or the opposite eye of an individual is intraocular. In some embodiments, the administration of a unit dose of rAAV particles to one eye and / or the opposite eye of an individual is by intravitreal injection (IVT) or subretinal injection. In some embodiments, the administration of a unit dose of rAAV particles to one eye and / or the opposite eye of an individual is by IVT injection. In some embodiments, a unit dose of rAAV particles is administered by intravitreal injection using an aseptic technique. In some embodiments, a unit dose of rAAV particles is administered by intravitreal injection using an aseptic technique with povidone iodine.

[0106] In some embodiments, the individual has not received prior treatment for intraocular neovascularization. In some embodiments, the individual has not received prior treatment for intraocular neovascularization in one eye and / or the opposite eye. In some embodiments, the individual has not received prior treatment with an anti-VEGF agent (e.g., bevacizumab, brolucizumab, ranibizumab, falisimab, abisipalpegol, and / or aflibercept). In some embodiments, the individual has not received prior treatment with an anti-VEGF agent (e.g., bevacizumab, brolucizumab, ranibizumab, falisimab, abisipalpegol, and / or aflibercept) in one eye and / or the opposite eye. In some embodiments, the individual has not received prior aflibercept treatment. In some embodiments, the individual has not received prior aflibercept treatment in one eye and / or the opposite eye. Steroid treatment

[0107] In some embodiments, a unit dose of rAAV particles is administered in combination with a steroid treatment. In some embodiments, the steroid treatment is a corticosteroid treatment. Exemplary corticosteroids include acromethasone, amcinomide, beclomethasone, betamethasone, budesonide, ciclesonide, clobetasol, clobetasone, crocortol, cloprednol, cortivazole, deflazacort, deoxycorticosterone, desonide, dexoximethasone, dexamethasone, diflorasone, diflucortol, difluprednate, fluchlorolone, fludrocortisone, fludroxortide, flumetasone, flunisolide, fluocinolone acetonide, fluocinonide, fluocortin, fluocortolone, fluorometholone, and fluperolone. This includes, but is not limited to, lorone, fluticasone, fuprednidene, formocortal, halcinonide, halomethasone, hydrocortisone aceponate, hydrocortisone butyrate, hydrocortisone butyrate, loteprednol, medrisone, meprednisone, methylprednisolone, methylprednisolone aceponate, mometasone furoate, paramethasone, prednicarbate, prednisone, prednisolone, prednylidene, remexolone, thixocortol, triamcinolone, and urobetasol. In some embodiments, the steroid treatment is a systemic steroid treatment. In some embodiments, the steroid treatment is an oral steroid treatment. In some embodiments, the steroid treatment is an ophthalmic steroid treatment. In some embodiments, ophthalmic steroid treatment is topical steroid treatment (e.g., eye drops), periorbital steroid treatment (e.g., sub-Tenon's capsule, subconjunctival), intravitreous steroid treatment, or suprachoroidal steroid treatment. In some embodiments, topical steroid treatment is difluprednate treatment, medrisone treatment, loteprednol treatment, prednisolone treatment, fluocinolone treatment, triamcinolone treatment, rimexolone treatment, dexamethasone treatment, fluorometholone treatment, fluocinolone treatment, rimexolone treatment, or prednisone treatment.In some embodiments, the ophthalmic steroid treatment is difluprednate treatment. In some embodiments, the steroid treatment is prednisone treatment. In some embodiments, the steroid treatment is difluprednate treatment.

[0108] In some embodiments, steroid treatment includes systemic steroid treatment and topical steroid treatment. In some embodiments, systemic steroid treatment is oral steroid treatment. In some embodiments, systemic steroid treatment is prednisone treatment. In some embodiments, topical steroid treatment is difluprednate treatment. In some embodiments, systemic steroid treatment and topical steroid treatment are administered simultaneously (e.g., on the same day). In some embodiments, systemic steroid treatment and topical steroid treatment are administered separately (e.g., on different days).

[0109] In some embodiments, steroids are administered before, during, and / or after the administration of a unit dose of rAAV particles. In some embodiments, steroids are administered before, during, and after the administration of a unit dose of rAAV particles. In some embodiments, steroids are administered during and after the administration of a unit dose of rAAV particles. In some embodiments, steroids are administered before the administration of a unit dose of rAAV particles. In some embodiments, steroids are administered during the administration of a unit dose of rAAV particles. In some embodiments, steroids are administered before and during the administration of a unit dose of rAAV particles. In some embodiments, steroids are administered after the administration of a unit dose of rAAV particles. In some embodiments, steroids are administered during and after the administration of a unit dose of rAAV particles. In some embodiments, steroids are administered before and / or after the administration of a unit dose of rAAV particles. In some embodiments, steroids are administered before and after the administration of a unit dose of rAAV particles.

[0110] In some embodiments, the steroid treatment is a systemic steroid treatment. In some embodiments, the systemic steroid treatment is an oral steroid treatment. In some embodiments, the steroid treatment is an oral prednisone treatment. In some embodiments, the oral prednisone treatment is initiated before the administration of a unit dose of rAAV particles. In some embodiments, the initial oral prednisone treatment is administered at a dose of approximately 40 mg, approximately 45 mg, approximately 50 mg, approximately 55 mg, approximately 60 mg, approximately 65 mg, or approximately 70 mg / day of prednisone, either approximately 7 days, approximately 6 days, approximately 5 days, approximately 4 days, approximately 3 days, approximately 2 days, approximately 1 day, or 0 days prior to the administration of a unit dose of rAAV particles, and lasts for approximately 3 days, approximately 4 days, approximately 5 days, approximately 6 days, approximately 7 days, approximately 8 days, approximately 9 days, or approximately 10 days, or longer. In some embodiments, the initial oral prednisone treatment is administered at a dose of approximately 60 mg / day of prednisone, approximately 3 days before the administration of the unit dose of rAAV, and lasts for approximately 3 days.

[0111] In some embodiments, the dose of oral prednisone is gradually reduced after the initial oral prednisone treatment. In some embodiments, the dose reduction of oral prednisone is administered at a dose of approximately 20 mg, 25 mg, 30 mg, 35 mg, 40 mg, 45 mg, or 50 mg / day of prednisone for a total of approximately 1, 2, 3, 4, 5, 6, or 7 days, followed by a dose of approximately 10 mg, 15 mg, 20 mg, or 25 mg / day of prednisone for approximately 1, 2, 3, or 4 days, followed by a dose of approximately 5 mg, 10 mg, or 15 mg / day of prednisone for approximately 1, 2, 3, or 4 days. In some embodiments, the dose reduction of prednisone is performed by administering any dose of prednisone of approximately 40 mg / day for 3 days, followed by approximately 20 mg / day for 2 days, and then approximately 10 mg / day for 2 days.

[0112] In some embodiments, the initial oral prednisone treatment is initiated three days before administration of a unit dose of rAAV particles, starting with a dose of 60 mg / day of prednisone for a total of six days, followed by a dose of 40 mg / day of prednisone for a total of three days, then a dose of 20 mg / day of prednisone for two days, and then a dose of 10 mg / day of prednisone for two days.

[0113] In some embodiments, the steroid treatment is an ophthalmic steroid treatment. In some embodiments, the ophthalmic steroid treatment is a difluprednate treatment. In some embodiments, the steroid treatment is administered before, during, and / or after administration of a unit dose of rAAV particles. In some embodiments, the steroid treatment is administered before administration of a unit dose of rAAV particles. In some embodiments, the steroid treatment is administered during administration of a unit dose of rAAV particles. In some embodiments, the steroid treatment is administered after administration of a unit dose of rAAV particles. In some embodiments, the steroid treatment is administered before and during administration of a unit dose of rAAV particles. In some embodiments, the steroid treatment is administered before and after administration of a unit dose of rAAV particles. In some embodiments, the steroid treatment is administered during and after administration of a unit dose of rAAV particles. In some embodiments, the steroid treatment is administered before, during, and after administration of a unit dose of rAAV particles.

[0114] In some embodiments, the steroid treatment is ophthalmic steroid treatment. In some embodiments, the ophthalmic steroid treatment is daily steroid treatment for up to 4 weeks, up to 6 weeks, or up to 8 weeks after administration of a unit dose of rAAV particles. In some embodiments, the topical steroid treatment includes approximately 4 administrations of topical steroids in approximately week 1, approximately 3 administrations of topical steroids in approximately week 2, approximately 2 administrations of topical steroids in approximately week 3, and approximately 1 administration of topical steroids in approximately week 4, with the timing starting from and after administration of the unit dose of rAAV particles. In some embodiments, the ophthalmic steroid treatment is extended at the discretion of the treating physician.

[0115] In some embodiments, the ophthalmic steroid is approximately 0.005% to approximately 0.5% difluprednate. In some embodiments, the ophthalmic steroid is any of approximately 0.005%, approximately 0.006%, approximately 0.007%, approximately 0.008%, approximately 0.009%, approximately 0.01%, approximately 0.02%, approximately 0.03%, approximately 0.4%, approximately 0.05%, approximately 0.06%, approximately 0.07%, approximately 0.08%, approximately 0.09%, or approximately 0.1% difluprednate. In some embodiments, the ophthalmic steroid is 0.05% difluprednate. In some embodiments, the dose of 0.05% difluprednate is one drop of ophthalmic solution. In some embodiments, one drop is approximately 50 μl (e.g., approximately 25 μl to approximately 50 μl, approximately 50 μl to approximately 100 μl). In some embodiments, the dose of difluprednate includes about 1 μg to about 5 μg, or about 2 μg to about 3 μg, or about 2.5 μg of difluprednate. In some embodiments, the dose of difluprednate includes about 2.5 μg of difluprednate. Vectors for delivering transgenes to target cells

[0116] In some embodiments, recombinant adeno-associated virus (rAAV) particles include a recombinant viral vector derived from adeno-associated virus (AAV) that has been modified to be replication-deficient in the subject (e.g., human or non-human primate). In some embodiments, adeno-associated virus (AAV) is recombinant AAV (rAAV).

[0117] AAV, or rAAV, is a small, non-enveloped, single-stranded DNA virus. rAAV is a non-pathogenic human parvovirus and can be constructed to rely on helper viruses, including adenoviruses, herpes simplex viruses, vaccinia viruses, and CMV, for replication.

[0118] Since exposure to wild-type (wt) AAV is known not to be associated with or cause any human pathological conditions and is common in the general population, AAV or rAAV are suitable delivery systems for gene therapy. Any serotype may be used for gene therapy to deliver anti-VEGF agents, such as aflibercept. In some embodiments, the methods of this disclosure provide the use of any suitable AAV serotype, including AAV1, AAV2, AAV2.5, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, rh10, AAV-DJ, and any hybrid or chimeric AAV thereof. In some embodiments, the serotype used is based on the viral directionality or the infectivity of the target cells of interest. In some embodiments, several AAV vectors may be generated to allow for the selection of the most optimal serotype for use with anti-VEGF agent transgenes (e.g., aflibercept transgenes).

[0119] In some embodiments, the methods of this disclosure provide the use of pseudotyped AAVs. Pseudotyped AAV particles contain an AAV genome terminal inversion (ITR) of one AAV serotype capsided by an AAV capsid of another AAV serotype. Typically, pseudotyped AAVs are designated as "AAV# / #", where the first "#" indicates the AAV ITR serotype and the second "#" indicates the serotype of the capsid. For example, an AAV particle containing an AAV2 ITR and an AAV1 capsid would be designated "AAV2 / 1".

[0120] In some embodiments, rAAV particles contain nucleic acids, e.g., heterologous nucleic acids. In some embodiments, the nucleic acid encodes a transgene, e.g., an anti-VEGF agent (e.g., aflibercept). In some embodiments, the encoded transgene, e.g., an anti-VEGF agent, is under the transcriptional control of a promoter that initiates transcription of the nucleic acid. In some embodiments, the promoter is a “ubiquitous” promoter. In some embodiments, the promoter is a “potent” or constitutively active promoter, e.g., a cytomegalovirus (CMV) promoter, an elongation factor 1 alpha (EFla) promoter, a glyceraldehyde 3-phosphate dehydrogenase (GAPDH) promoter, or a connexin 36 (or “Cx36”) promoter. In some embodiments, the promoter is a tissue-specific promoter that is activated in specific tissues or cells, e.g., retinal cells, to reduce potential toxicity or undesirable effects on non-targeted cells. In some embodiments, several AAV vectors may be generated to allow for the selection of the most optimal serotype and promoter for use with an anti-VEGF agent transgene (e.g., aflibercept transgene). In some embodiments, the nucleic acid has AAV terminal inversion sequences (ITRs) at both ends. In some embodiments, the nucleic acid has AAV2 ITRs at both ends.

[0121] In some embodiments, the AAV vector includes a polynucleotide cassette to enhance the expression of a transgene (e.g., an anti-VEGF agent such as aflibercept) in target cells (e.g., retinal cells). In some embodiments, the polynucleotide cassette includes, in 5' to 3' order: (a) a first enhancer region containing a CMV sequence (SEQ ID NO: 22); (b) a promoter region containing a CMV sequence (SEQ ID NO: 23); (c) a 5' UTR region containing 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 (e.g., an anti-VEGF agent such as aflibercept); (e) a second enhancer region containing a full-length EES sequence (SEQ ID NO: 26); and (f) an HGH polyadenylation site (SEQ ID NO: 27). In certain embodiments of these, the polynucleotide cassette includes one or more sequences selected from SEQ ID NOs: 28-32, or sequences having at least 85% identity with them. In certain embodiments of these, the 5' arm of the polynucleotide cassette contains or comprises a sequence having at least 85% identity with SEQ ID NO: 33. In certain embodiments of these, the 3' arm of the polynucleotide cassette contains or comprises a sequence having at least 85% identity with SEQ ID NO: 34. The nucleic acid sequences of SEQ ID NOs: 22-34 are provided below: ACTTACGGTA AATGGCCCGC CTGGCTGACC GCCCAACGAC CCCCGCCCAT TGACGTCAAT AATGACGTAT GTTCCCATAG TAACGCCAAT AGGGACTTTC CATTGACGTC AATGGGTGGA GTATTTACGG TAAACTGCCC ACTTGGCAGT ACATCAAGTG TATCATATGC CAAGTCCGCC CCCTATTGAC GTCAATGACG GTAAATGGCC CGCCTGGCAT TATGCCCAGT ACATGACCTT ACGGGACTTT CCTACTTGGC AGTACATCTA CGTATTAGTC ATCGCTATTA CCA (SEQ ID NO: 22) TGCTGATGCG GTTTTGGCAG TACACCAATG GGCGTGGATA GCGGTTTGAC TCACGGGGAT TTCCAAGTCT CCACCCCATT GACGTCAATG GGAGTTTGTT TTGGCACCAA AATCAACGGG ACTTTCCAAA ATGTCGTAAT AACCCCGCCC CGTTGACGCA AATGGGCGGT AGGCGTGTAC GGTGGGAGGT CTATATAAGC AGAGCTCGTT TAGTGAACCG (SEQ ID NO: 23) CTCACTCTCT TCCGCATCGC TGTCTGCGAG GGCCAGCTGT TGGGCTCGCG GTTGAGGACA AACTCTTCGC GGTCTTTCCA GTACTCTTGG ATCGGAAACC CGTCGGCCTC CGAACGGTAC TCCGCCACCG AGGGACCTGA GCGAGTCCGC ATCGACCGGA TCGGAAAACC TCTCGAGAAA GGCGTCTAAC CAGTCACAGT CGCAAGGTAG GCTGAGCACC GTGGCGGGCG GCAGCGGGTG GCGGTCGGGG TTGTTTCTGG CGGAGGTGCT GCTGATGATG TAATTAAAGT AGGCGGTCTT GAGACGGCGG ATGGTCGA (SEQ ID NO: 24) CCAGCTGTTG GGGTGAGTAC TCCCTCTCAA AAGCGGGCAT TACTTCTGCG CTAAGATTGT CAGTTTCCAA AAACGAGGAG GATTTGATAT TCACCTGGCC CG (SEQ ID NO: 25) CTGTTCTCAT CACATCATAT CAAGGTTATA TACCATCAAT ATTGCCACAG ATGTTACTTA GCCTTTTAAT ATTTCTCTAA TTTAGTGTAT ATGCAATGAT AGTTCTCTGA TTTCTGAGAT TGAGTTTCTC ATGTGTAATG ATTATTTAGA GTTTCTCTTT CATCTGTTCA AATTTTTGTC TAGTTTTATT TTTTACTGAT TTGTAAGACT TCTTTTTATA ATCTGCATAT TACAATTCTC TTTACTGGGG TGTTGCAAAT ATTTTCTGTC ATTCTATGGC CTGACTTTTC TTAATGGTTT TTTAATTTTA AAAATAAGTC TTAATTTCA TGCAATCTAA TTAACAATCT TTTCTTTGTG GTTAGGACTT TGAGTCATAA GAAATTTTTC TCTACACTGA AGTCATGATG GCATGCTTCT ATATTATTTT CTAAAAGATT TAAAGTTTTG CCTTCTCCAT TTAGACTAT AATTCACTGG AATTTTTTTG TGTGTATGGT ATGACATATG GGTTCCCTTT TATTTTTTAC ATATAAATAT ATTTCCCTGT TTTTCTAAAA AAGAAAAAGA TCATCATTTT CCCATTGTAA AATGCCATAT TTTTTTCATA GGTCACTTAC ATATATCAAT GGGTCTGTTT CTGAGCTCTA CTCTATTTTA TCAGCCTCAC TGTCTATCCC CACACATCTC ATGCTTTGCT CTAAATCTTG ATATTTAGTG GAACATTCTT TCCCATTTTG TTCTACAAGA ATATTTTTGT TATTGTCTTT GGGCTTTCTA TATACATTTT GAAATGAGGT TGACAAGTTA (Sequence No. 26) CTGCCCGGGT GGCATCCCTG TGACCCCTCC CCAGTGCCTC TCCTGGCCCT GGAAGTTGCC ACTCCAGTGC CCACCAGCCT TGTCCTAATA AAATTAAGTT GCATCATTTT GTCTGACTAG GTGTCCTTCT ATAATATTAT GGGGTGGAGG GGGGTGGTAT GGAGCAAGGG GCCCAAGTTG GGAAGAAACC TGTAGGGCCT GC (Sequence ID 27) AGGCGGTCTT GAGACGGCGG ATGGTCGAGG TGAGGTGTGG CAGGCTTGAG ATCCAGCTGT TGGGGTGA (Sequence No. 28) CGCTGTTTTG ACCTCCATAG TGGACACCGG GACCGATCCA GCCTCCGCGT CTCAGGGGAG ATCTCGTTTA GTGAACCGTC AGATCCTCAC TCTCTTCCGC ATCGCTGTCT GCGAGGGCCA GCTGTTGGG (Sequence ID 29) TTGATATTCA CCTGGCCCGA TCTGGCCATA CACTTG (Sequence ID 30) CCCAGGTCCA AGTTTAAACG CC (Sequence ID 31) TCTTTGGGCT TTCTATATAC ATTTTGAAAT GAGGTTGACA AGTTACCTAG GAAAACTGTC TTCCTGCCCG GGTGGCA (Sequence No. 32) CTCTGGAGAC GACTTACGGT AAATGGCCCG CCTGGCTGAC CGCCCAACGA CCCCCGCCCA TTGACGTCAA TAATGACGTA TGTTCCCATA GTAACGCCAA TAGGGACTTT CCATTGACGT CAATGGGTGG AGTATTTACG GTAAACTGCC CACTTGGCAG TACATCAAGT GTATCATATG CCAAGTCCGC CCCCTATTGA CGTCAATGAC GGTAAATGGC CCGCCTGGCA TTATGCCCAG TACATGACCT TACGGGACTT TCCTACTTGG CAGTACATCT ACGTATTAGT CATCGCTATT ACCATGCTGA TGCGGTTTTG GCAGTACACC AATGGGCGTG GATAGCGGTT TGACTCACGG GGATTTCCAA GTCTCCACCC CATTGACGTC AATGGGAGTT TGTTTTGGCA CCAAAATCAA CGGGACTTTC CAAAATGTCG TAATAACCCC GCCCCGTTGA CGCAAATGGG CGGTAGGCGT GTACGGTGGG AGGTCTATAT AAGCAGAGCT CGTTTAGTGA ACCGTCAGAT CGCCTGGAGA GGCCATCCAC GCTGTTTTGA CCTCCATAGT GGACACCGGG ACCGATCCAG CCTCCGCGTC TCAGGGGAGA TCTCGTTTAG TGAACCGTCA GATCCTCACT CTCTTCCGCA TCGCTGTCTG CGAGGGCCAG CTGTTGGGCT CGCGGTTGAG GACAAACTCT TCGCGGTCTT TCCAGTACTC TTGGATCGGA AACCCGTCGG CCTCCGAACG GTACTCCGCC ACCGAGGGAC CTGAGCGAGT CCGCATCGAC CGGATCGGAA AACCTCTCGA GAAAGGCGTC TAACCAGTCA CAGTCGCAAG GTAGGCTGAG CACCGTGGCG GGCGGCAGCG GGTGGCGGTC GGGGTTGTTT CTGGCGGAGG TGCTGCTGAT GATGTAATTA AAGTAGGCGG TCTTGAGACG GCGGATGGTC GAGGTGAGGT GTGGCAGGCT TGAGATCCAG CTGTTGGGGT GAGTACTCCC TCTCAAAAGC GGGCATTACT TCTGCGCTAA GATTGTCAGT TTCCAAAAAC GAGGAGGATT TGATATTCAC CTGGCCCGAT CTGGCCATAC ACTTGAGTGA CAATGACATC CACTTTGCCT TTCTCTCCAC AGGTGTCCAC TCCCAGGTCC AAGTTTAAAC GCCGCCACCA TG(SEQ ID NO: 33) ACTGTTCTCA TCACATCATA TCAAGGTTAT ATACCATCAA TATTGCCACA GATGTTACTT AGCCTTTTAA TATTTCTCTA ATTTAGTGTA TATGCAATGA TAGTTCTCTG ATTTCTGAGA TTGAGTTTCT CATGTGTAAT GATTATTTAG AGTTTCTCTT TCATCTGTTC AAATTTTTGT CTAGTTTTAT TTTTTACTGA TTTGTAAGAC TTCTTTTTAT AATCTGCATA TTACAATTCT CTTTACTGGG GTGTTGCAAA TATTTTCTGT CATTCTATGG CCTGACTTTT CTTAATGGTT TTTTAATTTT AAAAATAAGT CTTAATATTC ATGCAATCTA ATTAACAATC TTTTCTTTGT GGTTAGGACT TTGAGTCATA AGAAATTTTT CTCTACACTG AAGTCATGAT GGCATGCTTC TATATTATTT TCTAAAAGAT TTAAAGTTTT GCCTTCTCCA TTTAGACTTA TAATTCACTG GAATTTTTTT GTGTGTATGG TATGACATAT GGGTTCCCTT TTATTTTTTA CATATAAATA TATTTCCCTG TTTTTCTAAA AAAGAAAAAG ATCATCATTT TCCCATTGTA AAATGCCATA TTTTTTTCAT AGGTCACTTA CATATATCAA TGGGTCTGTT TCTGAGCTCT ACTCTATTTT ATCAGCCTCA CTGTCTATCC CCACACATCT CATGCTTTGC TCTAAATCTT GATATTTAGT GGAACATTCT TTCCCATTTT GTTCTACAAG AATATTTTTG TTATTGTCTT TGGGCTTTCT ATATACATTT TGAAATGAGG TTGACAAGTT ACCTAGGAAA ACTGTCTTCC TGCCCGGGTG GCATCCCTGT GACCCCTCCC CAGTGCCTCT CCTGGCCCTG GAAGTTGCCA CTCCAGTGCC CACCAGCCTT GTCCTAATAA AATTAAGTTG CATCATTTTG TCTGACTAGG TGTCCTTCTA TAATATTATG GGGTGGAGGG GGGTGGTATG GAGCAAGGGG CCCAAGTTGG GAAGAAACCT GTAGGGCCTG CGAAGACAGT CAG (SEQ ID NO: 34)

[0122] An additional polynucleotide cassette for enhancing the expression of transgenes (e.g., transgenes encoding anti-VEGF agents such as aflibercept) in target cells (e.g., retinal cells) is disclosed in WO2018 / 170473, and the contents relating thereto to the polynucleotide cassette for enhancing the expression of transgenes in target cells are incorporated herein by reference.

[0123] In some embodiments, rAAV particles contain variant capsid proteins that have increased infectivity to target cells, such as retinal cells, and are used to increase transduction into retinal cells or to increase the targeting of gene delivery to retinal cells in an organism. In some embodiments, rAAV particles contain amino acid modifications to the GH loop / loop IV of the AAV capsid protein. In some embodiments, the modification site is the solvent-accessible portion of the GH loop / loop IV of the AAV capsid protein. For a description of the GH loop / loop IV of the AAV capsid, see, for example, van Vliet et al. (2006) Mol. Ther. 14:809; Padron et al. (2005) J. Virol. 79:5047; and Shen et al. (2007) Mol. Ther. 15:1955. Several AAV capsid variants, including the 7m8 variant, are known. In some embodiments, the rAAV particles contain a variant AAV capsid protein that includes an insertion of 5 to 11 amino acids, e.g., 7 amino acids, into the GH loop of the capsid protein compared to the corresponding parent AAV capsid protein, and the variant capsid protein confers increased infectivity of retinal cells compared to the infectivity of retinal cells by AAV particles containing the corresponding parent or unmodified AAV capsid protein.In some embodiments, one of the following amino acid sequences may be inserted into the GH loop of the capsid protein: LALGETTRPA (SEQ ID NO: 1); LANETITRPA (SEQ ID NO: 2), LAKAGQANNA (SEQ ID NO: 3), LAKDPKTTNA (SEQ ID NO: 4), KDTDTTR (SEQ ID NO: 5), RAGGSVG (SEQ ID NO: 6), AVDTTKF (SEQ ID NO: 7), STGKVPN (SEQ ID NO: 8), LAKDTDTTRA (SEQ ID NO: 9), LARAGGSVGA (SEQ ID NO: 10), LAAVDTTKFA (SEQ ID NO: 11), and LASTGKVPNA (SEQ ID NO: 12), LGETTRP (SEQ ID NO: 14), NETITRP (SEQ ID NO: 15), KAGQANN (SEQ ID NO: 16), KDPKTTN (SEQ ID NO: 17), KDTDTTR (SEQ ID NO: 18), RAGGSVG (SEQ ID NO: 19), AVDTTKF (SEQ ID NO: 20), and STGKVPN (SEQ ID NO: 21). In some embodiments, one of the amino acid sequences described in SEQ ID NOs: 1-12 and 14-21 is inserted into the solvent-exposed GH loop of the VP1 capsid protein in rAAV. Additional details regarding amino acid sequences that can be inserted into the GH loop of the capsid protein to facilitate transduction of the nucleic acid of interest into retinal cells, for example, after IVT injection, are provided in WO2012145601, US9587282, US10202657, and US10214785, the contents relating thereto to amino acid sequences that can be inserted into the GH loop of the capsid protein are incorporated herein by reference.

[0124] In some embodiments, rAAV particles are inserted between the following amino acid sequences at the following positions: between positions 587 and 588 of the AAV2 capsid protein; between amino acids 590 and 591 of the AAV1 capsid protein; between amino acids 575 and 576 of the AAV5 capsid protein; between amino acids 590 and 591 of the AAV6 capsid protein; between amino acids 589 and 590 of the AAV7 capsid protein; between amino acids 590 and 591 of the AAV8 capsid protein; between amino acids 588 and 589 of the AAV9 capsid protein; or between amino acids 589 and 590 of the AAV10 capsid protein: LALGETTRPA (SEQ ID NO: 1); LANETITRPA (SEQ ID NO: 2), LAKAGQANNA (SEQ ID NO: 3), LAKDPK The AAV capsid protein, such as the AAV2 capsid protein, contains one of the following: TTNA (SEQ ID NO: 4), KDTDTTR (SEQ ID NO: 5), RAGGSVG (SEQ ID NO: 6), AVDTTKF (SEQ ID NO: 7), STGKVPN (SEQ ID NO: 8), LAKDTDTTRA (SEQ ID NO: 9), LARAGGSVGA (SEQ ID NO: 10), LAAVDTTKFA (SEQ ID NO: 11), and LASTGKVPNA (SEQ ID NO: 12), LGETTRP (SEQ ID NO: 14), NETITRP (SEQ ID NO: 15), KAGQANN (SEQ ID NO: 16), KDPKTTN (SEQ ID NO: 17), KDTDTTR (SEQ ID NO: 18), RAGGSVG (SEQ ID NO: 19), AVDTTKF (SEQ ID NO: 20), and STGKVPN (SEQ ID NO: 21). In some embodiments, the rAAV particles include an AAV2 capsid protein containing 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 that of the AAV2 VP1 capsid protein. In some embodiments, the rAAV particles include an AAV2 capsid protein containing the amino acid sequence LALGETTRPA (SEQ ID NO: 1) inserted between positions 587 and 588 of the AAV2 VP1 containing the sequence of SEQ ID NO: 13.

[0125] In some embodiments, the rAAV particles contain a 7m8 variant capsid protein from AAV2, which includes the amino acid sequence LALGETTRPA (SEQ ID NO: 1) inserted between positions 587 and 588 of AAV2 VP1. The sequence of the 7m8 variant capsid protein from AAV2, which includes the amino acid sequence LALGETTRPA (SEQ ID NO: 1) inserted between positions 587 and 588 of AAV2 VP1, is provided below: (Sequence ID 37)

[0126] In some embodiments, the rAAV particles include capsid protein VP1, which contains the amino acid sequence LGETTRP (SEQ ID NO: 14) inserted between positions 587 and 588 of the capsid protein, and whose amino acid residue numbering corresponds to that of the AAV2 VP1 capsid protein. In some embodiments, the rAAV particles include capsid protein VP2, which contains the amino acid sequence LGETTRP (SEQ ID NO: 14) inserted between positions 587 and 588 of the capsid protein, and whose amino acid residue numbering corresponds to that of the AAV2 VP1 capsid protein. In some embodiments, the rAAV particles include capsid protein VP3, which contains the amino acid sequence LGETTRP (SEQ ID NO: 14) inserted between positions 587 and 588 of the capsid protein, and whose amino acid residue numbering corresponds to that of the AAV2 VP1 capsid protein. In some embodiments, the rAAV particles include capsid proteins VP1, VP2, and VP3, each containing the amino acid sequence LGETTRP (SEQ ID NO: 14) inserted between positions 587 and 588 of the capsid protein, and the amino acid residue numbering corresponds to the AAV2 VP1 capsid protein.

[0127] In some embodiments, the rAAV particles include capsid protein VP1, which contains the amino acid sequence LALGETTRPA (SEQ ID NO: 1) inserted between positions 587 and 588 of the capsid protein, and whose amino acid residue numbering corresponds to that of the AAV2 VP1 capsid protein. In some embodiments, the rAAV particles include capsid protein VP2, which contains the amino acid sequence LALGETTRPA (SEQ ID NO: 1) inserted between positions 587 and 588 of the capsid protein, and whose amino acid residue numbering corresponds to that of the AAV2 VP1 capsid protein. In some embodiments, the rAAV particles include capsid protein VP3, which contains the amino acid sequence LALGETTRPA (SEQ ID NO: 1) inserted between positions 587 and 588 of the capsid protein, and whose amino acid residue numbering corresponds to that of the AAV2 VP1 capsid protein. In some embodiments, the rAAV particles include capsid proteins VP1, VP2, and VP3, each containing the amino acid sequence LALGETTRPA (SEQ ID NO: 1) inserted between positions 587 and 588 of the capsid protein, and the amino acid residue numbering corresponds to the AAV2 VP1 capsid protein.

[0128] In some embodiments, the recombinant virus and / or plasmid used to generate the rAAV virus includes other transcriptional or regulatory elements, such as poly(A) (polyadenylated) sequences, untranslated regions (UTRs), 3'UTRs, or stop sequences. In some embodiments, one or more genes are expressed from the vector or plasmid using intrasequence ribosome entry sites (IRESs), or similar elements that allow for the simultaneous expression of two or more proteins or create multigene or multicistronic mRNAs.

[0129] In some embodiments, the plasmid used to generate rAAV and / or rAAV includes one or more of the following nucleic acid elements: a first ITR sequence; a promoter sequence; an intron sequence; a first UTR sequence; a heterogeneous nucleic acid encoding an anti-VEGF agent (e.g., aflibercept); a second UTR sequence; a poly(A) sequence; and a second ITR sequence. In some embodiments, linker sequences are inserted between two or more nucleic acid elements. In some embodiments, the heterogeneous nucleic acid encoding the therapeutic polypeptide encodes aflibercept (or a functional fragment or functional variant thereof).

[0130] In some embodiments, the vector is a targeted vector, particularly a targeted rAAV (e.g., AAV2.7m8) that exhibits high infectivity to specific cells, such as retinal cells (e.g., photoreceptors, retinal ganglion cells, Müller cells, bipolar cells, amacrine cells, horizontal cells, or retinal pigment epithelial cells). Viral vectors for use in this disclosure may include vectors that exhibit low toxicity and / or low immunogenicity in organisms and express therapeutically effective amounts of anti-VEGF agents (e.g., aflibercept) in organisms, such as humans. Any suitable method known in the art can be used for the biochemical purification of recombinant viruses (e.g., rAAV) for preparing pharmaceutical compositions, for example, as described elsewhere herein. Recombinant AAV viruses can be isolated directly from cells or from culture media containing cells. Viruses can be purified using a variety of biochemical means, such as gel filtration, filtration, chromatography, affinity purification, density gradient ultracentrifugation, or size exclusion. In some embodiments, the viruses are lyophilized.

[0131] In some embodiments, the rAAV particles include a 7m8 variant capsid protein, e.g., rAAV2.7m8, and a nucleic acid sequence encoding an anti-VEGF agent (e.g., aflibercept, or a functional fragment or functional variant thereof). In some embodiments, the rAAV particles (e.g., the 7m8 variant) have any of the following increased retinal cell infectivity: at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 100% compared to AAV particles containing the corresponding parent or unmodified AAV capsid protein. In some embodiments, the increased infectivity of retinal cells is one of the following increases compared to AAV particles containing the corresponding parent or unmodified AAV capsid protein: between 5% and 100%, between 5% and 95%, between 5% and 90%, between 5% and 85%, between 5% and 80%, between 5% and 75%, between 5% and 70%, between 5% and 65%, between 5% and 60%, between 5% and 55%, between 5% and 50%, between 5% and 45%, between 5% and 40%, between 5% and 35%, between 5% and 30%, between 5% and 25%, between 5% and 20%, between 5% and 15%, or between 5% and 10%.

[0132] In some embodiments, the increased infectivity of an rAAV variant, e.g., rAAV2.7m8, to retinal cells is at least 1-fold, at least 1.1-fold, at least 1.2-fold, at least 1.3-fold, at least 1.4-fold, at least 1.5-fold, at least 1.6-fold, at least 1.7-fold, at least 1.8-fold, at least 1.9-fold, or at least 2-fold compared to AAV particles containing the corresponding parent or unmodified AAV capsid protein. In some embodiments, the increased infectivity is at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, or at least 10-fold compared to AAV particles containing the corresponding parent AAV capsid protein. In some embodiments, the increase in infectivity is at least 15 times, at least 20 times, at least 25 times, at least 30 times, at least 35 times, at least 40 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 compared to AAV particles containing the corresponding parent or unmodified AAV capsid protein.

[0133] In some embodiments, the increased retinal cell infectivity of an rAAV variant, e.g., rAAV2.7m8, is between 10 and 100 times, between 10 and 95 times, between 10 and 90 times, between 10 and 85 times, between 10 and 80 times, between 10 and 75 times, between 10 and 70 times, between 10 and 65 times, between 10 and 60 times, between 10 and 55 times, between 10 and 50 times, between 10 and 45 times, between 10 and 40 times, between 10 and 35 times, between 10 and 30 times, between 10 and 25 times, between 10 and 20 times, or between 10 and 15 times, compared to AAV particles containing the corresponding parent or unmodified AAV capsid protein.

[0134] In some embodiments, the increase in retinal cell infectivity is between 2 to 20 times, between 2 to 19 times, between 2 to 18 times, between 2 to 17 times, between 2 to 16 times, between 2 to 15 times, between 2 to 14 times, between 2 to 13 times, between 2 to 12 times, between 2 to 11 times, between 2 to 10 times, between 2 to 9 times, between 2 to 8 times, between 2 to 7 times, between 2 to 6 times, between 2 to 5 times, between 2 to 4 times, or between 2 to 3 times compared to AAV particles containing the corresponding parent or unmodified AAV capsid protein.

[0135] In some embodiments, the amino acid modifications of the capsid proteins described herein may confer an increased ability to pass through the internal limiting membrane (ILM) of an individual, such as a human eye, compared to the ability of AAV particles containing the corresponding parental or unmodified AAV capsid protein to pass through the ILM of the eye in question. In some embodiments, the increased ability of an rAAV variant, such as rAAV2.7m8, to pass through the ILM is any increase of at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 100% compared to AAV particles containing the corresponding parental or unmodified AAV capsid protein. In some embodiments, the increased ability to pass through ILM is an increase of 5% to 100%, 5% to 95%, 5% to 90%, 5% to 85%, 5% to 80%, 5% to 75%, 5% to 70%, 5% to 65%, 5% to 60%, 5% to 55%, 5% to 50%, 5% to 45%, 5% to 40%, 5% to 35%, 5% to 30%, 5% to 25%, 5% to 20%, 5% to 15%, or 5% to 10% compared to the parent or unmodified AAV capsid protein.

[0136] In some embodiments, the increased ability of an rAAV variant, e.g., rAAV2.7m8, to pass through the ILM is at least 1x, at least 1.1x, at least 1.2x, at least 1.3x, at least 1.4x, at least 1.5x, at least 1.6x, at least 1.7x, at least 1.8x, at least 1.9x, or at least 2x compared to an AAV particle containing the corresponding parent AAV capsid protein. In some embodiments, the increased ability to pass through the ILM is at least 2x, at least 3x, at least 4x, at least 5x, at least 6x, at least 7x, at least 8x, at least 9x, or at least 10x compared to an AAV particle containing the corresponding parent AAV capsid protein. In some embodiments, the increased ability to pass through the ILM is at least 15 times, at least 20 times, at least 25 times, at least 30 times, at least 35 times, at least 40 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 compared to AAV particles containing the corresponding parent or unmodified AAV capsid protein.

[0137] In some embodiments, the increased ability of an rAAV variant, e.g., rAAV2.7m8, to pass through the ILM is between 10 and 100 times, between 10 and 95 times, between 10 and 90 times, between 10 and 85 times, between 10 and 80 times, between 10 and 75 times, between 10 and 70 times, between 10 and 65 times, between 10 and 60 times, between 10 and 55 times, between 10 and 50 times, between 10 and 45 times, between 10 and 40 times, between 10 and 35 times, between 10 and 30 times, between 10 and 25 times, between 10 and 20 times, or between 10 and 15 times compared to AAV particles containing the corresponding parent or unmodified AAV capsid protein.

[0138] In some embodiments, the increased ability of an rAAV variant, e.g., rAAV2.7m8, to pass through the ILM is between 2 and 20 times, between 2 and 19 times, between 2 and 18 times, between 2 and 17 times, between 2 and 16 times, between 2 and 15 times, between 2 and 14 times, between 2 and 13 times, between 2 and 12 times, between 2 and 11 times, between 2 and 10 times, between 2 and 9 times, between 2 and 8 times, between 2 and 7 times, between 2 and 6 times, between 2 and 5 times, between 2 and 4 times, or between 2 and 3 times, compared to AAV particles containing the corresponding parent or unmodified AAV capsid protein.

[0139] In some embodiments, rAAV.7m8 containing the nucleic acid encoding aflibercept is used for gene therapy. In some embodiments, AAV2 or rAAV2 is used to deliver a nucleic acid sequence encoding an anti-VEGF agent (e.g., aflibercept) to target eye or retinal cells via intravitreous or subretinal injection. In some embodiments, AAV2 or rAAV2 is used to deliver a nucleic acid sequence encoding an anti-VEGF agent (e.g., aflibercept) to target eye or retinal cells via intravitreous injection. In some embodiments, rAAV2.7m8 is used to deliver the nucleic acid sequence of an anti-VEGF agent (e.g., aflibercept) to target retinal cells. In some embodiments, a heterologous nucleic acid (e.g., a nucleic acid encoding an anti-VEGF agent such as aflibercept) is integrated into the target cell genome (e.g., retinal cell genome) to result in long-term expression of, for example, an anti-VEGF agent (e.g., aflibercept) in the target cells. In some embodiments, the viral vector delivers a plasmid or other extrachromosomal gene element containing heterologous nucleic acid (e.g., nucleic acid encoding an anti-VEGF agent such as aflibercept) to target cells (e.g., retinal cells).

[0140] In some embodiments, the rAAV particles include a nucleic acid encoding a polypeptide having at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% identity with the amino acid sequence of SEQ ID NO: 35, and having AAV2 terminal inverse sequences (ITRs) at both ends. In some embodiments, the rAAV particles include a nucleic acid encoding a polypeptide having at least about 95% identity with the amino acid sequence of SEQ ID NO: 35, and having AAV2 terminal inverse sequences (ITRs) at both ends. In some embodiments, the rAAV particles include a nucleic acid encoding a polypeptide having the amino acid sequence of SEQ ID NO: 35, and having AAV2 terminal inverse sequences (ITRs) at both ends. In some embodiments, the rAAV particles include a nucleic acid encoding a polypeptide having the amino acid sequence of SEQ ID NO: 35. In some embodiments, the rAAV particles include a nucleic acid encoding aflibercept, and having AAV2 terminal inverse sequences (ITRs) at both ends. The sequence for sequence number 35 is provided below: SDTGRPFVEMYSEIPEIIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVLSPS HGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHEKDKTHTCPPCPAPEL LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG(Sequence ID 35)

[0141] In some embodiments, the rAAV particles are nucleic acids having sequence homology to the nucleic acid sequence of SEQ ID NO: 36 of at least about 75%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.9%, or at least about 100%, and include nucleic acids having AAV2 terminal inversion sequences (ITRs) at both ends. The sequence of SEQ ID NO: 36 is provided in Figure 5. In some embodiments, the rAAV particles are nucleic acids having at least about 75%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.9%, or at least about 100% sequence homology to the nucleic acid sequence of aflibercept (e.g., SEQ ID NO: 36), and include nucleic acids having AAV2 terminal inversion sequences (ITRs) at both ends. 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 the subject.

[0142] In some embodiments, the nucleic acid sequence of aflibercept is codon-optimized for expression in primate or human subjects. Construction of synthetic genes corresponding to the aflibercept amino acid sequence is described in the literature, e.g., Kanda A, Noda K, Saito W, Ishida S. Aflibercept Traps Galectin-1, an Angiogenic Factor Associated with Diabetic Retinopathy. Scientific Reports 5:17946 (2015) ("VEGF-Trap"). R1R2 It is stated that "(the cDNA corresponding to aflibercept) was generated as a synthetic gene by IDT (Coralville, IA)." Considering the available amino acid sequence of aflibercept, cDNA of aflibercept for use in the gene therapy or rAAV described herein can be generated using any method known in the art.

[0143] Codon optimization can be achieved by any method known in the art. Codon optimization refers to the process of modifying a nucleic acid sequence by replacing at least one codon of the native sequence (e.g., about one or more, two, three, four, five, ten, fifteen, twenty, twenty-five, fifty, one hundred, or more codons) with a codon that is more frequently used or most frequently used in the host cell, while maintaining the native amino acid sequence, in order to enhance the expression of a gene of interest or in a host cell, such as human retinal cells. For example, codon usage tables are readily available, including the GenScript Codon Usage Frequency Table Tool at www(dot)genscript(dot)com / tools / codon-frequency-table; the Codon Usage Database at www(dot)kazusa(dot)or(dot)jp / codon / ; and Nakamura, Y., et al. "Codon usage tabulated from the international DNA sequence databases: status for the year 2000" Nucl. Acids Res. 28:292 (2000).

[0144] Homology refers to the percentage conservation of residues in the alignment between two sequences, including but not limited to functional fragments, insertions, deletions, substitutions, pseudofragments, pseudogenes, splice variants, or artificially optimized sequences.

[0145] In some embodiments, the rAAV particles contain a nucleic acid encoding aflibercept. In some embodiments, the polypeptide is aflibercept.

[0146] As used herein, “Aflibercept” means a polypeptide or protein sequence or a functional fragment, variant, or mutant thereof having at least 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher, or 100% homology to the aflibercept amino acid sequence identified above (SEQ ID NO: 35). Homology refers to the percentage conservation of residues in alignment between two sequences, including but not limited to functional fragments, sequences containing insertions, deletions, or substitutions, pseudofragments, pseudogenes, splice variants, or artificially optimized sequences.

[0147] In some embodiments, the amino acid sequence of aflibercept is homologous to the aflibercept amino acid sequence of SEQ ID NO: 35 by 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%. In some embodiments, the nucleic acid sequences encoding aflibercept disclosed herein exhibit 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 with the nucleic acid sequence of aflibercept (e.g., SEQ ID NO: 36) when compared with the corresponding cDNA sequence of the aflibercept amino acid sequence identified above. In some embodiments, aflibercept is spatially homologous (e.g., with respect to its secondary, tertiary, and quaternary structures or conformation) to aflibercept used in standard treatment by at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100%.

[0148] In some embodiments, the aflibercept gene product or aflibercept transgene included in rAAV-based gene therapy comprises a capsid variant (e.g., the 7m8 variant) disclosed herein and encodes a protein, fusion protein, or polypeptide having at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% homology between the above amino acid sequence of SEQ ID NO: 35 or the corresponding cDNA sequence of aflibercept (e.g., cDNA of the aflibercept sequence used in gene therapy compared to SEQ ID NO: 36). In some embodiments, the methods and compositions disclosed herein include a functional fragment of aflibercept, or a variant or mutant thereof. In some embodiments, the nucleic acid sequence of aflibercept is modified or codon-optimized to enhance its activity, expression, stability, and / or solubility in vivo.

[0149] Aflibercept is a glycosylated 115 kDa fusion protein. Aflibercept contains an IgG scaffold fused to the extracellular VEGF receptor sequences of human VEGFR-1 and VEGFR-2, and functions as a soluble decoy receptor by binding to VEGF-A with higher affinity than its native or endogenous receptor. See, for example, Stewart MW. Aflibercept (VEGF Trap-eye): the newest anti-VEGF drug. Br. J. Ophthalmol. 2012 Sep;96(9):1157-8. Aflibercept's high affinity for VEGF interferes with or disrupts the subsequent binding and activation of native or endogenous VEGF receptors. Reduced VEGF activity can lead to decreased angiogenesis and vascular permeability. Inhibition of placental growth factor PIGF and VEGF-B by aflibercept may also contribute to the treatment of eye diseases or disorders characterized by abnormal (e.g., excessive) angiogenesis and / or neovascularization. PIGF is associated with certain eye diseases or disorders, such as angiogenesis and exudative AMD, which may be related to elevated levels of PIGF. Overexpression of VEGF-B may be associated with disruption of the blood-retinal barrier and retinal angiogenesis. Thus, inhibition of VEGF-A, VEGF-B, and PIGF may all contribute to the efficacy of aflibercept. Method for preparing vectors to deliver transgenes to target cells

[0150] In some embodiments, rAAV particles are produced using any method known in the art. In some embodiments, rAAV particles are produced using a baculovirus expression vector system in Sf9 cells. Sf9 cells are an insect cell culture system commonly used for recombinant protein production using baculoviruses. In some embodiments, rAAV particles are produced using two baculoviruses in Sf9 cells. In some embodiments, rAAV particles are produced using two baculoviruses in Sf9 cells, where the first baculovirus encodes the genes for the AAV2 Rep and AAV2.7m8 Cap proteins, and the second baculovirus encodes an anti-VEGF agent. In some embodiments, rAAV particles are produced using two baculoviruses in Sf9 cells, where the first baculovirus encodes the genes for the AAV2 Rep and AAV2.7m8 Cap proteins, and the second baculovirus encodes an aflibercept (e.g., human aflibercept) cDNA expression cassette. In some embodiments, rAAV particles are produced in Sf9 cells using two baculoviruses, the first baculovirus encoding the genes for the AAV2 Rep and AAV2.7m8 Cap proteins, and the second baculovirus encoding a polypeptide containing an amino acid sequence having at least about 95% identity with the amino acid sequence of SEQ ID NO: 35, and comprising nucleic acids having AAV2 terminal inversion sequences (ITRs) at both ends. In some embodiments, the polypeptide contains the amino acid sequence of SEQ ID NO: 35. In some embodiments, the polypeptide is aflibercept. dose

[0151] In some embodiments, a unit dose of rAAV particles is administered to one eye of an individual. In some embodiments, one eye of an individual is either the right or left eye. In some embodiments, one eye of an individual is the right eye. In some embodiments, one eye of an individual is the left eye. In some embodiments, the method provided herein further includes the step of administering a unit dose of rAAV particles to the opposite eye of an individual. In some embodiments, one eye of an individual is the right eye and the opposite eye is the left eye. In some embodiments, one eye of an individual is the left eye and the opposite eye is the right eye.

[0152] In some embodiments, the step of administering a unit dose of rAAV particles to the opposite eye occurs at least about two weeks (for example, at least about two weeks, at least about three weeks, at least about four weeks, at least about one month, at least about two months, at least about three months, at least about four months, at least about five months, at least about six months, at least about seven months, at least about eight months, at least about nine months, at least about ten months, at least about eleven months, at least about twelve months, at least about one year, at least about two years, at least about three years, at least about four years, at least about five years, or longer) after the step of administering a unit dose of rAAV particles to one eye. In some embodiments, the step of administering a unit dose of rAAV particles to the opposite eye occurs at least about two weeks after the step of administering a unit dose of rAAV particles to one eye, and the unit dose of rAAV particles administered to the opposite eye of the individual is higher than the unit dose of rAAV particles administered to one eye of the individual (e.g., higher than about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 125%, about 150%, about 175%, about 200%, about 225%, about 250%, about 275%, about 300%, or any of these higher percentages).

[0153] In some embodiments, the step of administering a unit dose of rAAV particles to the eye opposite the individual occurs up to approximately 1 week, up to approximately 2 weeks, up to approximately 3 weeks, or up to approximately 4 weeks after the step of administering a unit dose of rAAV particles to one eye. In some embodiments, the step of administering a unit dose of rAAV particles to the eye opposite the individual occurs up to approximately 2 weeks (e.g., approximately 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days) after the step of administering a unit dose of rAAV particles to one eye. In some embodiments, the step of administering a unit dose of rAAV particles to the contralateral eye of the individual occurs up to approximately two weeks (e.g., about day 0, day 1, day 2, day 3, day 4, day 5, day 6, day 7, day 8, day 9, day 10, day 11, day 12, day 13, or day 14) after the step of administering a unit dose of rAAV particles to one eye of the individual, and the unit dose of rAAV particles administered to the contralateral eye of the individual is approximately the same as (e.g., less than 1% higher or lower, less than 5% higher or lower, less than 10% higher or lower, or less than 20% higher or lower) or lower (e.g., about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, or about 90% lower) than the unit dose of rAAV particles administered to one eye of the individual. In some embodiments, the step of administering a unit dose of rAAV particles to the contralateral eye of the individual occurs up to approximately two weeks after the step of administering a unit dose of rAAV particles to one eye, and the unit dose of rAAV particles administered to the contralateral eye of the individual is approximately the same as the unit dose of rAAV particles administered to one eye of the individual (e.g., less than 1% higher or lower, less than 5% higher or lower, less than 10% higher or lower, or less than 20% higher or lower). In some embodiments, the step of administering a unit dose of rAAV particles to the contralateral eye of the individual occurs up to approximately two weeks after the step of administering a unit dose of rAAV particles to one eye, and 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 individual (e.g., about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% lower).

[0154] In some embodiments, a unit dose of rAAV particles is administered to one eye and / or the opposite eye of an individual. In some embodiments, a unit dose of rAAV particles is expressed as vector genome number (vg). In some embodiments, a unit dose is approximately 6 × 10⁶ rAAV particles. 11 The vector genome (vg) or less. In some embodiments, the unit dose is approximately 1 × 10⁶ of rAAV particles. 10 ~Approx. 2×10 10 vg, approx. 2×10 10 ~Approx. 3×10 10 During vg, approximately 3 × 10 10 ~Approx. 4×10 10 During vg, approximately 4 × 10 10 ~Approx. 5×10 10 During vg, approximately 5 × 10 10 ~Approx. 6×10 10 During vg, approximately 6 × 10 10 ~Approx. 7×10 10 During vg, approximately 7 × 10 10 ~Approx. 8×10 10 During vg, approximately 8 × 10 10 ~Approx. 9×10 10 During vg, approximately 9 x 10 10 ~About 10×10 10 During vg, approximately 1 × 10 11 ~Approx. 2×10 11 During vg, approximately 2 × 10 11 ~Approx. 3×10 11 During vg, approximately 3 × 10 11 ~Approx. 4×10 11 During vg, approximately 4 × 10 11 ~Approx. 5×10 11 Between vg, or approximately 5 × 10 11 ~Approx. 6×10 11 The values ​​are between vg and include any value within these ranges for rAAV particles. In some embodiments, the unit dose is approximately 6 × 10⁻⁶ of rAAV particles. 10 Vector genome (vg) ~ approximately 2 × 10⁻⁶ 11 The value is vg. In some embodiments, the unit dose is approximately 6 × 10⁶ of rAAV particles. 10 vg~approx. 2×10 11 vg, approx. 7×10 10 vg~approx. 2×10 11 vg, approx. 8×10 10vg ~ about 2×10 11 vg, about 9×10 10 vg ~ about 2×10 11 vg, about 10×10 10 vg ~ about 2×10 11 vg, or about 1×10 11 vg ~ about 2×10 11 vg. In some embodiments, the unit dose is about 6×10 10 vg ~ about 2×10 11 vg. In some embodiments, the unit dose is about 6×10 10 vg ~ about 7×10 10 vg, about 7×10 10 vg ~ about 8×10 10 vg, about 8×10 10 vg ~ about 9×10 10 vg, about 9×10 10 vg ~ about 10×10 10 vg, about 10×10 10 vg ~ about 1×10 11 vg, or about 1×10 11 vg ~ about 2×10 11 vg. In some embodiments, the unit dose is about 6×10 10 vg, about 7×10 10 vg, about 8×10 10 vg, about 9×10 10 vg, about 10×10 10 vg, about 1×10 11 vg, or about 2×10 11 vg. In some embodiments, the unit dose is about 6×10 10 vg or about 2×10 11 vg. In some embodiments, the unit dose is about 6×10 10 vg. In some embodiments, the unit dose is about 6×10 10 vg, about 2×10 11 vg, or about 6×10 11 vg. In some embodiments, the unit dose is about 6×10 10 vg. In some embodiments, the unit dose is about 2×10 11 vg. In some embodiments, the unit dose is about 6×​It is vg.

[0155] In some embodiments, a unit dose of rAAV particles is administered to one eye and / or the opposite eye of an individual. In some embodiments, the unit dose is expressed as vector genome count (vg) / eye (vg / eye). In some embodiments, the unit dose is approximately 6 × 10⁶ rAAV particles. 11 The dose is vg / eye or less. In some embodiments, the unit dose is about 1 × 10⁶ of rAAV particles. 10 ~Approx. 2×10 10 vg / eye, approx. 2×10 10 ~Approx. 3×10 10 vg / interpupillary space, approximately 3 x 10 10 ~Approx. 4×10 10 vg / interpupillary space, approximately 4 x 10 10 ~Approx. 5×10 10 vg / interpupillary space, approximately 5 x 10 10 ~Approx. 6×10 10 vg / interpupillary space, approximately 6 x 10 10 ~Approx. 7×10 10 vg / interpupillary space, approximately 7 x 10 10 ~Approx. 8×10 10 vg / interocular distance, approximately 8 x 10 10 ~Approx. 9×10 10 vg / interpupillary space, approximately 9 x 10 10 ~About 10×10 10 vg / inter-eye space, approximately 1 × 10⁻⁶ 11 ~Approx. 2×10 11 vg / interpupillary space, approximately 2 × 10⁻⁶ 11 ~Approx. 3×10 11 vg / interpupillary space, approximately 3 x 10 11 ~Approx. 4×10 11 vg / interpupillary space, approximately 4 x 10 11 ~Approx. 5×10 11 vg / interocular distance, or approximately 5 x 10 11 ~Approx. 6×10 11 The values ​​are between vg / eye and include any value within these ranges for rAAV particles. In some embodiments, the unit dose is approximately 6 × 10⁶ rAAV particles. 10 vg / eye ~ approx. 2×10 11 The value is vg / eye. In some embodiments, the unit dose is approximately 6 × 10⁶ rAAV particles. 10 vg / eye ~ approx. 2×10 11vg / eye, approx. 7 x 10 10 vg / eye ~ approx. 2×10 11 vg / eye, approx. 8 x 10 10 vg / eye ~ approx. 2×10 11 vg / eye, approx. 9 x 10 10 vg / eye ~ approx. 2×10 11 vg / eye, approx. 10 x 10 10 vg / eye ~ approx. 2×10 11 vg / eye, or approximately 1 × 10⁻⁶ 11 vg / eye ~ approx. 2×10 11 The value is vg / eye. In some embodiments, the unit dose is approximately 6 × 10⁶ rAAV particles. 10 vg / eye ~ approx. 2×10 11 The value is vg / eye. In some embodiments, the unit dose is approximately 6 × 10⁶ rAAV particles. 10 vg / eye ~ approx. 7×10 10 vg / eye, approx. 7 x 10 10 vg / eye ~ approx. 8×10 10 vg / eye, approx. 8 x 10 10 vg / eye ~ approx. 9×10 10 vg / eye, approx. 9 x 10 10 vg / eye ~ approx. 10×10 10 vg / eye, approx. 10 x 10 10 vg / eye ~ approx. 1×10 11 vg / eye, or approximately 1 × 10⁻⁶ 11 vg / eye ~ approx. 2×10 11 The value is vg / eye. In some embodiments, the unit dose is approximately 6 × 10⁶ rAAV particles. 10 vg / eye, approx. 7 x 10 10 vg / eye, approx. 8 x 10 10 vg / eye, approx. 9×10 10 vg / eye, approx. 10 x 10 10 vg / eye, approx. 1×10 11 vg / eye, or approximately 2 × 10 11 The value is vg / eye. In some embodiments, the unit dose is approximately 6 × 10⁶ rAAV particles. 10 vg / eye or approximately 2 x 10 11 The value is vg / eye. In some embodiments, the unit dose is approximately 6 × 10⁶ rAAV particles. 10 The dosage is vg / eye. In some embodiments, the unit dose is approximately 6 × 10⁻⁶ 10 vg / eye, approx. 2×10 11vg / eye, or approximately 6 x 10 11 The unit dose is vg / eye. In some embodiments, the unit dose is approximately 6 × 10⁻⁶. 10 The unit dose is vg / eye. In some embodiments, the unit dose is approximately 2 × 10⁻⁶. 11 The unit dose is vg / eye. In some embodiments, the unit dose is approximately 6 × 10⁻⁶. 11 vg / eye

[0156] In some embodiments, a unit dose of rAAV particles is administered to one eye and / or the opposite eye of an individual. In some embodiments, E is an abbreviation for base 10 exponent, and xEy refers to x multiplied by base 10 power / power exponent y. In some embodiments, the unit dose is expressed as vector genome number (vg). In some embodiments, the unit dose is approximately 6E of rAAV particles. 11 The vector genome (vg) or less. In some embodiments, the unit dose is about 1E of rAAV particles. 10 ~approximately 2E 10 vg, about 2E 10 ~about 3E 10 During vg, approximately 3E 10 ~about 4E 10 During vg, approximately 4E 10 ~about 5E 10 During vg, approximately 5E 10 ~approximately 6E 10 During vg, approximately 6E 10 ~about 7E 10 During vg, approximately 7E 10 ~approximately 8E 10 During vg, approximately 8E 10 ~about 9E 10 During vg, approximately 9E 10 ~approximately 10E 10 During vg, approximately 1E 11 ~approximately 2E 11 During vg, approximately 2E 11 ~about 3E 11 During vg, approximately 3E 11 ~about 4E 11 During vg, approximately 4E 11 ~about 5E 11 Between vg, or about 5E 11 ~approximately 6E 11The range is between vg and includes any value within these ranges for rAAV particles. In some embodiments, the unit dose is about 6E of rAAV particles. 10 Vector genome (vg) ~ approximately 2E 11 It is vg. In some embodiments, the unit dose is about 6E of rAAV particles. 10 vg~about 2E 11 vg, about 7E 10 vg~about 2E 11 vg, about 8E 10 vg~about 2E 11 vg, about 9E 10 vg~about 2E 11 vg, about 10E 10 vg~about 2E 11 vg, or approximately 1E 11 vg~about 2E 11 It is vg. In some embodiments, the unit dose is about 6E of rAAV particles. 10 vg~about 2E 11 It is vg. In some embodiments, the unit dose is about 6E of rAAV particles. 10 vg~about 7E 10 vg, about 7E 10 vg~about 8E 10 vg, about 8E 10 vg~about 9E 10 vg, about 9E 10 vg~about 10E 10 vg, about 10E 10 vg~about 1E 11 vg, or approximately 1E 11 vg~about 2E 11 It is vg. In some embodiments, the unit dose is about 6E of rAAV particles. 10 vg, about 7E 10 vg, about 8E 10 vg, about 9E 10 vg, about 10E 10 vg, about 1E 11 vg, or approximately 2E 11 It is vg. In some embodiments, the unit dose is about 6E of rAAV particles. 10 VG or approximately 2E 11 It is vg. In some embodiments, the unit dose is about 6E of rAAV particles. 10 It is vg. In some embodiments, the unit dose is approximately 6E 10 vg, about 2E11 vg, or approximately 6 × 10 11 It is vg. In some embodiments, the unit dose is approximately 6E. 10 It is vg. In some embodiments, the unit dose is approximately 2E. 11 In some embodiments, the unit dose is approximately 6 × 10⁻⁶. 11 It is vg.

[0157] In some embodiments, a unit dose of rAAV particles is administered to one eye and / or the opposite eye of an individual. In some embodiments, the unit dose is expressed as vector genome count (vg) / eye (vg / eye). In some embodiments, the unit dose is approximately 6E of rAAV particles. 11 The dose is vg / eye or less. In some embodiments, the unit dose is about 1E of rAAV particles. 10 ~approximately 2E 10 vg / eye, approx. 2E 10 ~about 3E 10 vg / interpupillary distance, approximately 3E 10 ~about 4E 10 vg / interpupillary distance, approximately 4E 10 ~about 5E 10 vg / interpupillary distance, approximately 5E 10 ~approximately 6E 10 vg / interpupillary distance, approximately 6E 10 ~about 7E 10 vg / interpupillary distance, approximately 7E 10 ~approximately 8E 10 vg / interpupillary distance, approximately 8E 10 ~about 9E 10 vg / interpupillary distance, approximately 9E 10 ~approximately 10E 10 vg / interpupillary distance, approximately 1E 11 ~approximately 2E 11 vg / interpupillary distance, approximately 2E 11 ~about 3E 11 vg / interpupillary distance, approximately 3E 11 ~about 4E 11 vg / interpupillary distance, approximately 4E 11 ~about 5E 11 vg / between the eyes, or approximately 5E 11 ~approximately 6E 11 The values ​​are between vg / eye and include any value within these ranges for rAAV particles. In some embodiments, the unit dose is approximately 6E of rAAV particles. 10 vg / eye ~ approx. 2E11 The value is vg / eye. In some embodiments, the unit dose is approximately 6E of rAAV particles. 10 vg / eye ~ approx. 2E 11 vg / eye, about 7E 10 vg / eye ~ approx. 2E 11 vg / eye, about 8E 10 vg / eye ~ approx. 2E 11 vg / eye, approx. 9E 10 vg / eye ~ approx. 2E 11 vg / eye, approx. 10E 10 vg / eye ~ approx. 2E 11 vg / eye, or approximately 1E 11 vg / eye ~ approx. 2E 11 The value is vg / eye. In some embodiments, the unit dose is approximately 6E of rAAV particles. 10 vg / eye ~ approx. 2E 11 The value is vg / eye. In some embodiments, the unit dose is approximately 6E of rAAV particles. 10 vg / eye ~ approx. 7E 10 vg / eye, about 7E 10 vg / eye ~ approx. 8E 10 vg / eye, about 8E 10 vg / eye ~ approx. 9E 10 vg / eye, approx. 9E 10 vg / eye~approx. 10E 10 vg / eye, approx. 10E 10 vg / eye ~ approx. 1E 11 vg / eye, or approximately 1E 11 vg / eye ~ approx. 2E 11 The value is vg / eye. In some embodiments, the unit dose is approximately 6E of rAAV particles. 10 vg / eye, about 7E 10 vg / eye, about 8E 10 vg / eye, approx. 9E 10 vg / eye, approx. 10E 10 vg / eye, about 1E 11 vg / eye, or approximately 2E 11 The value is vg / eye. In some embodiments, the unit dose is approximately 6E of rAAV particles. 10 vg / eye or about 2E 11 The value is vg / eye. In some embodiments, the unit dose is approximately 6E of rAAV particles. 10 The value is vg / eye. In some embodiments, the unit dose is approximately 6E 10 vg / eye, approx. 2E 11vg / eye, or approximately 6 x 10 11 The unit dose is vg / eye. In some embodiments, the unit dose is approximately 6E 10 The unit dose is vg / eye. In some embodiments, the unit dose is approximately 2E 11 The unit dose is vg / eye. In some embodiments, the unit dose is approximately 6 × 10⁻⁶. 11 vg / eye

[0158] In some embodiments, a unit dose of rAAV particles is administered to one eye and / or the opposite eye of an individual. In some embodiments, a unit dose of rAAV particles is sufficient to induce the expression of a therapeutic protein (e.g., an anti-VEGF agent such as aflibercept) in the vitreous fluid. In some embodiments, a unit dose of rAAV particles is sufficient to achieve a concentration of a therapeutic protein (e.g., an anti-VEGF agent such as aflibercept) in the vitreous fluid that includes any range between these values, of approximately 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 higher. In some embodiments, a unit dose of rAAV particles is sufficient to induce the expression of aflibercept in the vitreous fluid. In some embodiments, the unit dose of rAAV particles is sufficient to achieve a concentration of aflibercept in vitreous fluid of any one of approximately 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 higher, encompassing any range between these values.

[0159] In some embodiments, a unit dose of rAAV particles administered to one eye and / or the opposite eye of an individual is sufficient to induce the expression of a therapeutic protein (e.g., an anti-VEGF agent such as aflibercept) in the aqueous humor. In some embodiments, a unit dose of rAAV particles is sufficient to achieve a concentration of the therapeutic protein (e.g., an anti-VEGF agent such as aflibercept) in the aqueous humor that includes any range between these values, 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 higher. In some embodiments, a unit dose of rAAV particles is sufficient to induce the expression of aflibercept in the aqueous humor. In some embodiments, the unit dose of rAAV particles is sufficient to achieve a concentration of aflibercept in aqueous humor that includes any range between these values, 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 higher.

[0160] In some embodiments, a unit dose of rAAV particles is administered to one eye and / or the opposite eye of an individual. In some embodiments, a unit dose of rAAV particles is sufficient to induce the expression of a therapeutic protein (e.g., an anti-VEGF agent such as aflibercept) in the retina. In some embodiments, a unit dose of rAAV particles is sufficient to achieve a concentration of a therapeutic protein (e.g., an anti-VEGF agent such as aflibercept) in the retina containing 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 any range between these values. In some embodiments, a unit dose of rAAV particles is sufficient to induce the expression of aflibercept in the retina. In some embodiments, the unit dose of rAAV particles is sufficient to achieve a concentration of aflibercept in the retina of at least about 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10 μg / g, or any range between these values.

[0161] In some embodiments, a unit dose of rAAV particles is administered to one eye and / or the opposite eye of an individual. In some embodiments, a unit dose of rAAV particles is sufficient to induce the expression of a therapeutic protein (e.g., an anti-VEGF agent such as aflibercept) in the choroid. In some embodiments, a unit dose of rAAV particles is sufficient to achieve a concentration of a therapeutic protein (e.g., an anti-VEGF agent such as aflibercept) in the choroid, including any range between these values, of approximately 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 higher. In some embodiments, a unit dose of rAAV particles is sufficient to induce the expression of aflibercept in the choroid. In some embodiments, the unit dose of rAAV particles is sufficient to achieve a concentration of aflibercept in the choroid that includes any range between these values, of approximately 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 higher.

[0162] In some embodiments, a unit dose of rAAV particles is administered to one eye and / or the opposite eye of an individual. In some embodiments, a unit dose of rAAV particles is a therapeutically effective dose.

[0163] 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 compared to the retinal thickness before 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 reduction in retinal thickness compared to the retinal thickness before administration of the unit dose of rAAV particles. In some embodiments, retinal thickness is central region retinal thickness (CST) or foveal retinal thickness (CRT). In some embodiments, a unit dose of rAAV particles is a therapeutically effective dose if the unit dose is sufficient to cause a reduction in retinal thickness of approximately 5%, approximately 10%, approximately 15%, approximately 20%, approximately 25%, approximately 30%, approximately 35%, approximately 40%, approximately 45%, approximately 50%, approximately 55%, approximately 60%, approximately 65%, approximately 70%, approximately 75%, approximately 80%, approximately 85%, approximately 90%, approximately 95%, approximately 99%, or approximately 100% compared to the retinal thickness before administration of a unit dose of rAAV particles. In some embodiments, retinal thickness (e.g., CST or CRT) is determined by OCT or SD-OCT.

[0164] 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 compared to the macular volume before 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 before 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 of about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, or about 65% compared to the macular volume before administration of the unit dose of rAAV particles. In some embodiments, macular volume is determined by OCT or SD-OCT.

[0165] 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 compared to visual acuity before 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 improvement of visual acuity compared to visual acuity before 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 improvement of visual acuity of about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 125%, about 150%, about 175%, about 200%, about 225%, about 250%, about 275%, about 300%, or greater than any of these percentages compared to visual acuity before administration of the unit dose of rAAV particles. In some embodiments, visual acuity is best 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 compared to the BCVA before administration of the unit dose of rAAV particles. In some embodiments, BCVA is expressed as an ETDRS score, which corresponds to the exact number of characters read (Vitale et al., (2016) JAMA Opthalmol 134(9):1041:1047). 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 of at least 15 ETDRS characters (Vitale et al., (2016) JAMA Opthalmol 134(9):1041:1047) (e.g., at least about 15, at least about 20, at least about 30, at least about 40, at least about 50, at least about 60, or about 70 characters) compared to the BCVA before 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 maintenance of BCVA, resulting in a decrease of less than 15 ETDRS characters in an individual compared to the BCVA before administration of the unit dose of rAAV particles (Vitale et al., (2016) JAMA Opthalmol 134(9):1041:1047) (e.g., less than 15 characters, less than 14 characters, less than 13 characters, less than 12 characters, less than 11 characters, less than 10 characters, less than 9 characters, less than 8 characters, less than 7 characters, less than 6 characters, less than 5 characters, less than 4 characters, less than 3 characters, less than 2 characters, less than 1 character, or 0 characters).

[0166] 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 individual is determined to have maintained vision. 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 individual is determined to have improved vision. In some embodiments, a unit dose of rAAV particles is a therapeutically effective dose if, after administration of the unit dose of rAAV particles, CST or CRT as assessed by SD-OCT decreases compared to before 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, CST or CRT as assessed by SD-OCT is maintained compared to before administration of the unit dose of rAAV particles.

[0167] In some embodiments, a unit dose of rAAV particles is a therapeutically effective dose if the macular volume decreases after administration of the unit dose of rAAV particles compared to before administration of the unit dose of rAAV particles. In some embodiments, a unit dose of rAAV particles is a therapeutically effective dose if the macular volume is maintained after administration of the unit dose of rAAV particles compared to before administration of the unit dose of rAAV particles.

[0168] In some embodiments, a unit dose of rAAV particles is a therapeutically effective dose if retinal thickness (e.g., foveal retinal thickness (CRT) or central region retinal thickness (CST)) and macular volume decrease after administration of the unit dose of rAAV particles compared to before administration of the unit dose of rAAV particles. In some embodiments, a unit dose of rAAV particles is a therapeutically effective dose if retinal thickness (e.g., foveal retinal thickness (CRT) or central region retinal thickness (CST)) and macular volume are maintained after administration of the unit dose of rAAV particles compared to before administration of the unit dose of rAAV particles.

[0169] In some embodiments, a unit dose of rAAV particles is a therapeutically effective dose if, after administration of a unit dose of rAAV particles, the individual requires less than one rescue treatment (e.g., aflibercept injection) at intervals of approximately every 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, or longer, after administration of a unit dose of rAAV particles to one eye and / or the other eye. In some embodiments, a unit dose of rAAV particles is a therapeutically effective dose if, after administration of a unit dose of rAAV particles, the individual does not require any rescue treatment (e.g., aflibercept injection) for at least approximately one week, at least two weeks, at least three weeks, at least four weeks, at least five weeks, at least six weeks, at least seven weeks, at least eight weeks, at least nine weeks, at least ten weeks, at least fifteen weeks, at least twenty weeks, at least thirty weeks, at least forty weeks, at least fifty weeks, at least sixty weeks, at least seventy weeks, at least eighty weeks, at least ninety weeks, at least 100 weeks, at least 110 weeks, or longer.

[0170] 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 individual is determined to have a reduction in retinal fluid compared to the level of retinal fluid before administration of the unit dose of rAAV particles to one eye and / or the opposite eye. 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 individual is determined to have maintenance of retinal fluid compared to the level of retinal fluid before administration of the unit dose of rAAV particles to one eye and / or the opposite eye. 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 individual is determined to have a reduction in IRF and / or SRF in one eye and / or the opposite eye compared to the level of IRF and / or SRF before administration of the unit dose of rAAV particles to one eye and / or the opposite eye.

[0171] In some embodiments, a unit dose of rAAV particles is a therapeutically effective dose if it is determined that the individual has resolution of pigment epithelial exfoliation (PED) after administration of a unit dose of rAAV particles compared to PED before administration of a unit dose of rAAV particles to one eye and / or the opposite eye.

[0172] In some embodiments, a unit dose of rAAV particles is a therapeutically effective dose if the CNV lesion shrinks after administration of the unit dose of rAAV particles compared to the CNV lesion present before administration of the unit dose of rAAV particles to one eye and / or the opposite eye. In some embodiments, a unit dose of rAAV particles is a therapeutically effective dose if the CNV lesion shrinks by approximately 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% after administration of the unit dose of rAAV particles compared to the CNV lesion present before administration of the unit dose of rAAV particles to one eye and / or the opposite eye. In some embodiments, a unit dose of rAAV particles is a therapeutically effective dose if the CNV lesion does not grow after administration of the unit dose of rAAV particles compared to the CNV lesion present before administration of the unit dose of rAAV particles to one eye and / or the opposite eye. In some embodiments, a unit dose of rAAV particles is a therapeutically effective dose if, after administration of a unit dose of rAAV particles, the CNV lesion does not grow by more than approximately 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, or 20% compared to the CNV lesion present before administration of a unit dose of rAAV particles to one eye and / or the opposite eye.

[0173] In some embodiments, a unit dose of rAAV particles is a therapeutically effective dose if it is determined that an individual has an improvement in the anatomical features of one eye and / or the opposite eye after administration of a unit dose of rAAV particles compared to the anatomical features before administration of a unit dose of rAAV particles. In some embodiments, a unit dose of rAAV particles is a therapeutically effective dose if it is determined that an individual has stabilization and / or maintenance of the anatomical features of one eye and / or the opposite eye after administration of a unit dose of rAAV particles compared to the anatomical features before administration of a unit dose of rAAV particles.

[0174] In some embodiments, a unit dose of rAAV particles is therapeutically effective if, when administered to one eye and / or the opposite eye of an individual, it reduces, stops, or prevents at least one symptom of intraocular neovascular disease or disorder. In cases of intraocular neovascular disease or disorder characterized by abnormal (e.g., excessive) angiogenesis, such symptoms include, but are not limited to, visual impairment (e.g., color blindness, blurred vision, deterioration of central vision) and visual loss. In some embodiments, a unit dose of rAAV particles administered to one eye and / or the opposite eye of an individual is a therapeutically effective dose if, when administered to one eye and / or the opposite eye of an individual, it results in maintenance, partial resolution, or complete resolution of one or more clinical features of intraocular neovascular disease. For example, a unit dose of rAAV particles administered to one eye and / or the opposite eye of an individual is therapeutically effective if, when measured by any method known in the art, the administration of the dose to one eye and / or the opposite eye of an individual results in complete resolution, partial resolution, or maintenance of intraocular neovascular disease. In some embodiments, a unit dose of rAAV particles administered to one eye and / or the opposite eye of an individual is therapeutically effective if, when measured by any method known in the art, the administration of the dose to one eye and / or the opposite eye results in best corrected visual acuity (BCVA) (e.g., based on the ETDRS score; Vitale et al., (2016) JAMA Opthalmol 134(9):1041:1047), therapeutically effective if it results in complete resolution, partial resolution, or maintenance of intraocular neovascular disease when evaluated by anatomical features based on any method known in the art (e.g., SD-OCT, OCT, fluorescein angiography, digital color fundus photography, etc.), central retinal thickness determined by SD-OCT, number of rescue treatments (e.g., aflibercept injections) required by the individual after administration of a unit dose of rAAV particles to one eye and / or the opposite eye, presence of intraretinal fluid (IRF) and / or subretinal fluid (SRF), resolution of pigment epithelial detachment (PED), growth of choroidal neovascularization (CNV) lesions, and evaluation by any method known in the art (e.g., SD-OCT, OCT, fluorescein angiography, digital color fundus photography, etc.).In some embodiments, the unit dose of rAAV particles administered to one eye and / or the opposite eye of an individual is the dose administered to one eye and / or the opposite eye of an individual, and is used for ophthalmic examination, intraocular pressure (e.g., using a Goldmann applanation tonometer or Tono-pen), indirect ophthalmoscopic examination, examination of one eye and / or the opposite eye and ocular adnexa, responsiveness of the eyelid and / or pupil, ptosis, abnormal pupillary shape, pupillary inequality, abnormal response to light, afferent pupillary disorder, slit-lamp examination (eye It is therapeutically effective if it results in complete resolution, partial resolution, or maintenance of intraocular neovascular disease when evaluated by examination of the eyelids, conjunctiva, cornea, lens, iris, and anterior chamber, abnormalities of the vitreous humor, optic nerve, peripheral retina, and posterior segment of the retinal vascular structure, SD-OCT, fluorescein angiography, digital color fundus photography (including images of the retina, optic nerve head, and / or macula), aqueous humor sampling, vitreous fluid sampling, OCT-angiography (OCT-A), and refractive and visual acuity (BCVA).

[0175] In some embodiments, the unit dose of rAAV particles administered to one eye of an individual is the same as the unit dose of rAAV particles administered to the other eye of the individual. In some embodiments, the unit dose of rAAV particles administered to one eye of an individual is different from the unit dose of rAAV particles administered to the other eye of the individual. In some embodiments, the unit dose of rAAV particles administered to one eye of an individual is higher than the unit dose of rAAV particles administered to the other eye of the individual, for example, higher than 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 any higher percentage. In some embodiments, the unit dose of rAAV particles administered to the opposite eye of an individual is higher than the unit dose of rAAV particles administered to one eye of the individual, for example, about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 125%, 150%, 175%, 200%, 225%, 250%, 275%, 300%, or higher. In some embodiments, the unit dose of rAAV particles is expressed as vector genome count (vg) / eye (vg / eye). In some embodiments, the unit dose of rAAV particles is about 6 × 10⁻⁶ of rAAV particles. 11 The dose is vg / eye or less. In some embodiments, the unit dose of rAAV particles is about 1 × 10⁻⁶ of rAAV particles. 10 ~Approx. 2×10 10 vg / eye, approx. 2×10 10 ~Approx. 3×10 10 vg / interpupillary space, approximately 3 x 10 10 ~Approx. 4×10 10 vg / interpupillary space, approximately 4 x 10 10 ~Approx. 5×10 10 vg / interpupillary space, approximately 5 x 10 10 ~Approx. 6×10 10 vg / interpupillary space, approximately 6 x 10 10 ~Approx. 7×10 10 vg / interpupillary space, approximately 7 x 10 10 ~Approx. 8×10 10 vg / interocular distance, approximately 8 x 10 10 ~Approx. 9×10 10vg / interpupillary space, approximately 9 x 10 10 ~About 10×10 10 vg / inter-eye space, approximately 1 × 10⁻⁶ 11 ~Approx. 2×10 11 vg / interpupillary space, approximately 2 × 10⁻⁶ 11 ~Approx. 3×10 11 vg / interpupillary space, approximately 3 x 10 11 ~Approx. 4×10 11 vg / interpupillary space, approximately 4 x 10 11 ~Approx. 5×10 11 vg / interocular distance, or approximately 5 x 10 11 ~Approx. 6×10 11 The values ​​are between vg / eye and include any value within these ranges for rAAV particles. In some embodiments, the unit dose of rAAV particles is approximately 6 × 10⁻⁶ of rAAV particles. 10 vg / eye ~ approx. 2×10 11 The value is vg / eye. In some embodiments, the unit dose of rAAV particles is approximately 6 × 10⁶ of rAAV particles. 10 vg / eye ~ approx. 2×10 11 vg / eye, approx. 7 x 10 10 vg / eye ~ approx. 2×10 11 vg / eye, approx. 8 x 10 10 vg / eye ~ approx. 2×10 11 vg / eye, approx. 9×10 10 vg / eye ~ approx. 2×10 11 vg / eye, approx. 10 x 10 10 vg / eye ~ approx. 2×10 11 vg / eye, or approximately 1 × 10⁻⁶ 11 vg / eye ~ approx. 2×10 11 The value is vg / eye. In some embodiments, the unit dose of rAAV particles is approximately 6 × 10⁶ of rAAV particles. 10 vg / eye ~ approx. 2×10 11 The value is vg / eye. In some embodiments, the unit dose of rAAV particles is approximately 6 × 10⁶ of rAAV particles. 10 vg / eye ~ approx. 7×10 10 vg / eye, approx. 7 x 10 10 vg / eye ~ approx. 8×10 10 vg / eye, approx. 8 x 10 10 vg / eye ~ approx. 9×10 10 vg / eye, approx. 9×10 10 vg / eye ~ approx. 10×10 10 vg / eye, approx. 10 x 1010 vg / eye ~ approx. 1×10 11 vg / eye, or approximately 1 × 10⁻⁶ 11 vg / eye ~ approx. 2×10 11 The value is vg / eye. In some embodiments, the unit dose of rAAV particles is approximately 6 × 10⁶ of rAAV particles. 10 vg / eye, approx. 7 x 10 10 vg / eye, approx. 8 x 10 10 vg / eye, approx. 9×10 10 vg / eye, approx. 10 x 10 10 vg / eye, approx. 1×10 11 vg / eye, or approximately 2 × 10 11 The value is vg / eye. In some embodiments, the unit dose of rAAV particles is approximately 6 × 10⁶ of rAAV particles. 10 vg / eye or approximately 2 x 10 11 The value is vg / eye. In some embodiments, the unit dose of rAAV particles is approximately 6 × 10⁶ of rAAV particles. 10 The unit dose is vg / eye. In some embodiments, the unit dose of rAAV particles is approximately 6 × 10⁻⁶. 10 vg / eye, approx. 2×10 11 vg / eye, or approximately 6 x 10 11 The value is vg / eye. In some embodiments, the unit dose of rAAV particles is approximately 6 × 10⁻⁶. 10 The value is vg / eye. In some embodiments, the unit dose of rAAV particles is approximately 2 × 10⁻⁶. 11 The value is vg / eye. In some embodiments, the unit dose of rAAV particles is approximately 6 × 10⁻⁶. 11 vg / eye

[0176] In some embodiments, the unit dose of rAAV particles administered to one eye of an individual and the unit dose of rAAV particles administered to the opposite eye of the individual are administered at the same time. In some embodiments, the unit dose of rAAV particles administered to one eye of an individual and the unit dose of rAAV particles administered to the opposite eye of the individual are administered at different times. In some embodiments, the unit dose administered to the opposite eye is administered 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, 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, at least about 4 weeks, or longer than the unit dose administered to one eye. In some embodiments, the unit dose administered to the opposite eye is administered at least about 2 weeks after the unit dose administered to one eye.

[0177] In some embodiments, a single dose of rAAV particles is administered to one eye and / or the opposite eye of an individual. In some embodiments, a single dose of rAAV particles administered to one eye and / or the opposite eye is a therapeutically effective dose. In some embodiments, one or more doses of rAAV particles (e.g., more than about 2, 3, 4, 5, or more than that unit dose) are administered to one eye and / or the opposite eye of an individual. In some embodiments, one or more doses of rAAV particles administered to one eye and / or the opposite eye is a therapeutically effective dose. Pharmaceutical preparations

[0178] In some embodiments, the unit dose of rAAV particles is present in the pharmaceutical formulation. In some embodiments, the pharmaceutical formulation comprises rAAV particles, one or more osmotic pressure or ionic strength modifiers, one or more buffers, one or more surfactants, and one or more solvents. In some embodiments, the osmotic pressure or ionic strength modifier is sodium chloride. In some embodiments, one or more buffers are sodium dihydrogen phosphate and / or sodium hydrogen phosphate. In some embodiments, the surfactant is poloxamer 188. In some embodiments, the solvent is water. In some embodiments, the pharmaceutical formulation comprises rAAV particles, sodium chloride, sodium dihydrogen phosphate, sodium hydrogen phosphate, and a surfactant.

[0179] In some embodiments, the pharmaceutical formulation is approximately 1 × 10 10 vg / mL ~ approx. 1×10 13 The pharmaceutical formulation contains vg / mL of rAAV particles, approximately 150 mM to approximately 200 mM sodium chloride, approximately 1 mM to approximately 10 mM sodium dihydrogen phosphate, approximately 1 mM to approximately 10 mM sodium hydrogen phosphate, and approximately 0.0005% (weight / volume) to approximately 0.005% (weight / volume) poloxamer 188, and has a pH of approximately 7.0 to approximately 7.5. In some embodiments, the pharmaceutical formulation is approximately 6 × 10 11 vg / mL ~ approx. 6×10 12 The pharmaceutical formulation contains vg / mL of rAAV particles, approximately 150 mM to approximately 200 mM sodium chloride, approximately 1 mM to approximately 10 mM sodium dihydrogen phosphate, approximately 1 mM to approximately 10 mM sodium hydrogen phosphate, and approximately 0.0005% (weight / volume) to approximately 0.005% (weight / volume) poloxamer 188, and has a pH of approximately 7.0 to approximately 7.5. In some embodiments, the pharmaceutical formulation is approximately 6 × 10 11 The pharmaceutical formulation contains vg / mL of rAAV particles, approximately 150 mM to approximately 200 mM sodium chloride, approximately 1 mM to approximately 10 mM sodium dihydrogen phosphate, approximately 1 mM to approximately 10 mM sodium hydrogen phosphate, and approximately 0.0005% (weight / volume) to approximately 0.005% (weight / volume) poloxamer 188, and has a pH of approximately 7.0 to approximately 7.5. In some embodiments, the pharmaceutical formulation is approximately 6 × 10 12The pharmaceutical formulation contains vg / mL rAAV particles, approximately 150 mM to 200 mM sodium chloride, approximately 1 mM to 10 mM sodium dihydrogen phosphate, approximately 1 mM to 10 mM sodium hydrogen phosphate, and approximately 0.0005% (weight / volume) to 0.005% (weight / volume) poloxamer 188, and has a pH of approximately 7.0 to 7.5.

[0180] In some embodiments, the rAAV particles in the pharmaceutical formulation are approximately 1 × 10⁶ 10 vg / ml ~ approx. 1×10 13 They are present at a concentration of vg / ml. In some embodiments, the rAAV particles in the pharmaceutical formulation are approximately 1 × 10⁶ 09 vg / ml ~ approx. 6×10 14 They are present at a concentration of vg / ml. In certain embodiments, the rAAV particles in the pharmaceutical formulation are approximately 1 × 10⁶ 09 vg / ml ~ approx. 2×10 09 vg / ml, approx. 2×10 09 vg / ml ~ approx. 3×10 09 , about 3×10 09 vg / ml ~ approx. 4×10 09 , about 4×10 09 vg / ml ~ approx. 5×10 09 , about 5×10 09 vg / ml ~ approx. 6×10 09 , about 6×10 09 vg / ml ~ approx. 7×10 09 , about 7×10 09 vg / ml ~ approx. 8×10 09 , about 8×10 09 vg / ml ~ approx. 9×10 09 , about 9×10 09 vg / ml ~ approx. 10×10 09 , about 10×10 09 vg / ml ~ approx. 1×10 10 , about 1×10 10 vg / ml ~ approx. 2×10 10 , about 2×10 10 vg / ml ~ approx. 3×10 10 , about 3×10 10 vg / ml ~ approx. 4×10 10 , about 4×10 10 vg / ml ~ approx. 5×10 10 , about 5×10 10vg / ml ~ approximately 6×10 10 Approximately 6×10 10 vg / ml ~ approximately 7×10 10 Approximately 7×10 10 vg / ml ~ approximately 8×10 10 Approximately 8×10 10 vg / ml ~ approximately 9×10 10 Approximately 9×10 10 vg / ml ~ approximately 10×10 10 Approximately 10×10 10 vg / ml ~ approximately 1×10 11 Approximately 1×10 11 vg / ml ~ approximately 2×10 11 Approximately 2×10 11 vg / ml ~ approximately 3×10 11 Approximately 3×10 11 vg / ml ~ approximately 4×10 11 Approximately 4×10 11 vg / ml ~ approximately 5×10 11 Approximately 5×10 11 vg / ml ~ approximately 6×10 11 Approximately 6×10 11 vg / ml ~ approximately 7×10 11 Approximately 7×10 11 vg / ml ~ approximately 8×10 11 Approximately 8×10 11 vg / ml ~ approximately 9×10 11 Approximately 9×10 11 vg / ml ~ approximately 10×10 11 Approximately 1×10 12 vg / ml ~ approximately 2×10 12 Approximately 2×10 12 vg / ml ~ approximately 3×10 12 Approximately 3×10 12 vg / ml ~ approximately 4×10 12 Approximately 4×10 12 vg / ml ~ approximately 5×10 12 Approximately 5×10 12 vg / ml ~ approximately 6×10 12 Approximately 6×10 12 vg / ml ~ approximately 7×10 12 Approximately 7×10 12 vg / ml ~ approximately 8×10 12 Approximately 8×10 12 vg / ml ~ approximately 9×10 12 Approximately 9×10 12vg / ml ~ approx. 10×10 12 , about 1×10 13 vg / ml ~ approx. 2×10 13 , about 2×10 13 vg / ml ~ approx. 3×10 13 , about 3×10 13 vg / ml ~ approx. 4×10 13 , about 4×10 13 vg / ml ~ approx. 5×10 13 , about 5×10 13 vg / ml ~ approx. 6×10 13 , about 6×10 13 vg / ml ~ approx. 7×10 13 , about 7×10 13 vg / ml ~ approx. 8×10 13 , about 8×10 13 vg / ml ~ approx. 9×10 13 , about 9×10 13 vg / ml ~ approx. 10×10 13 , about 1×10 14 vg / ml ~ approx. 2×10 14 , about 2×10 14 vg / ml ~ approx. 3×10 14 , about 3×10 14 vg / ml ~ approx. 4×10 14 , about 4×10 14 vg / ml ~ approx. 5×10 14 , or approximately 5 x 10 14 vg / ml ~ approx. 6×10 14 It exists at a concentration of vg / mL. In some embodiments, the pharmaceutical formulation is approximately 6 × 10 11 vg / mL ~ approx. 6×10 12 It contains vg / mL of rAAV particles. In some embodiments, the pharmaceutical formulation contains approximately 6 × 10 12 It contains vg / mL of rAAV particles. In some embodiments, the pharmaceutical formulation contains approximately 6 × 10 11 It contains vg / mL of rAAV particles. In some embodiments, the pharmaceutical formulation contains approximately 6 × 10 11 vg / mL ~ approx. 6×10 12 It contains vg / mL of rAAV particles. In some embodiments, the pharmaceutical formulation contains approximately 6 × 10 12 It contains vg / mL of rAAV particles. In some embodiments, the pharmaceutical formulation contains approximately 6 × 10 11 Contains vg / mL rAAV particles.

[0181] In some embodiments, sodium chloride is present in the pharmaceutical formulation at a concentration of approximately 150 mM to approximately 200 mM. In certain embodiments, sodium chloride is present in the pharmaceutical formulation at a concentration of approximately 150 mM, approximately 160 mM, approximately 170 mM, approximately 180 mM, approximately 190 mM, or approximately 200 mM. In certain embodiments, sodium chloride is present in the pharmaceutical formulation at a concentration of approximately 180 mM.

[0182] In some embodiments, sodium dihydrogen phosphate is present in the pharmaceutical formulation at a concentration of about 1 mM to about 10 mM. In some embodiments, sodium dihydrogen phosphate is present in the pharmaceutical formulation at a concentration of 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 a particular embodiment, sodium dihydrogen phosphate is present in the pharmaceutical formulation at a concentration of about 5 mM.

[0183] In some embodiments, sodium hydrogen phosphate is present in the pharmaceutical formulation at a concentration of about 1 mM to about 10 mM. In some embodiments, sodium hydrogen phosphate is present in the pharmaceutical formulation at any of the following concentrations: 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 a particular embodiment, sodium hydrogen phosphate is present in the pharmaceutical formulation at a concentration of about 5 mM.

[0184] In some embodiments, poloxamer 188 is present in the pharmaceutical formulation at a concentration of approximately 0.0005% (weight / volume) to approximately 0.005% (weight / volume). In some embodiments, poloxamer 188 is present in the pharmaceutical formulation at a concentration of approximately 0.0005% (weight / volume), approximately 0.0006% (weight / volume), approximately 0.0007% (weight / volume), approximately 0.0008% (weight / volume), approximately 0.0009% (weight / volume), approximately 0.001% (weight / volume), approximately 0.002% (weight / volume), approximately 0.003% (weight / volume), approximately 0.004% (weight / volume), or approximately 0.005% (weight / volume). In a particular embodiment, poloxamer 188 is present in the pharmaceutical formulation at a concentration of approximately 0.001% (weight / volume).

[0185] In some embodiments, the pharmaceutical formulation has a pH of approximately 7.0 to approximately 7.5. In some embodiments, the pharmaceutical formulation has a pH of approximately 7.0, approximately 7.1, approximately 7.2, approximately 7.3, approximately 7.4, or approximately 7.5. In certain embodiments, the pharmaceutical formulation has a pH of approximately 7.3. In some embodiments, hydrochloric acid and sodium hydroxide are used to adjust the pH of the pharmaceutical formulation.

[0186] In some embodiments, the pharmaceutical formulation is approximately 6 × 10 12 The pharmaceutical formulation contains vg / mL of rAAV particles, approximately 180 mM sodium chloride, approximately 5 mM sodium dihydrogen phosphate, approximately 5 mM sodium hydrogen phosphate, and approximately 0.001% (weight / volume) poloxamer 188, and has a pH of approximately 7.3. In some embodiments, the pharmaceutical formulation has a pH of approximately 6 × 10 11 The pharmaceutical formulation contains vg / mL rAAV particles, approximately 180 mM sodium chloride, approximately 5 mM sodium dihydrogen phosphate, approximately 5 mM sodium hydrogen phosphate, and approximately 0.001% (weight / volume) poloxamer 188, and has a pH of approximately 7.3.

[0187] In some embodiments, the pharmaceutical formulation is suitable for administration by intravitreal (IVT) injection into one eye and / or the opposite eye of an individual, such as a human patient, to achieve a desired therapeutic or preventive effect. In some embodiments, the pharmaceutical formulation is supplied as a restored homogeneous solution. In some embodiments, the solution is a suspension. In some embodiments, the pharmaceutical formulation is supplied as a frozen suspension and thawed before administration to one eye and / or the opposite eye of an individual. In some embodiments, the solution is isotonic.

[0188] In other embodiments, a pharmaceutical composition comprising an AAV2.7m8 vector containing a nucleic acid sequence encoding an anti-VEGF agent (e.g., a functional fragment or variant thereof) is supplied in a lyophilized form and restored before administration to one eye and / or the opposite eye of an individual. In some embodiments, the method provided herein further includes the step of restoring, dissolving, or solubilizing a lyophilized pharmaceutical composition comprising rAAV (e.g., AAV2.7m8) and encoding an anti-VEGF agent (e.g., aflibercept or a functional fragment or variant thereof) in a buffer before administration to a subject. In some embodiments, such a lyophilized pharmaceutical composition comprises one or more of the following: cryoprotective substances, surfactants, salts, stabilizers, or any combination thereof.

[0189] In some embodiments, the pharmaceutical formulation is a homogeneous solution. In some embodiments, the homogeneous 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 method provided herein further includes the step of warming a refrigerated suspension to room temperature and / or stirring the suspension before administration to one eye and / or the opposite eye of an individual (e.g., by IVT injection) to ensure that the active ingredient(s) dissolve in the solution and / or are uniformly distributed. In some embodiments, the method provided herein further includes the step of thawing a frozen suspension, warming it to room temperature and / or stirring the suspension before administration to one eye and / or the opposite eye of an individual (e.g., by IVT injection) to ensure that the active ingredient(s) dissolve in the solution and / or are uniformly distributed. In some embodiments, the suspension is diluted before administration to a subject (e.g., by IVT injection). In some embodiments, the suspension is supplied as a pre-filled syringe.

[0190] In some embodiments, the pharmaceutical formulation is provided as a frozen suspension. In some embodiments, the suspension comprises pharmaceutically acceptable excipients, such as surfactants, glycerol, nonionic surfactants, buffers, glycols, salts, and any combination thereof.

[0191] In some embodiments, the suspension is a solution. In some embodiments, the suspension contains micelles.

[0192] In some embodiments, for storage stability and ease of handling, a pharmaceutical formulation containing nucleic acid sequences encoding rAAV (e.g., AAV2.7m8) and an anti-VEGF agent (e.g., aflibercept or a functional fragment or variant thereof) is formulated as a lyophilized powder, freeze-dried powder, or vacuum-dried powder, which is then restored with saline, a buffer, or water before administration to one eye and / or the other eye of an individual. Alternatively, the pharmaceutical formulation is formulated as an aqueous solution, such as a suspension or a homogeneous solution. The pharmaceutical formulation may contain rAAV particles containing the nucleic acid sequence encoding aflibercept. The pharmaceutical formulation can be stabilized using various excipients, such as phosphoric acid, PBS, or Tris buffer, glycol, glycerol, saline, surfactants (e.g., Pluronic® or polysorbate), or any combination thereof. Furthermore, cryoprotective substances such as alcohol can be used as stabilizers under freeze or dry conditions. In some embodiments, gene therapeutics are provided as a suspension, refrigerated suspension, or frozen suspension.

[0193] In some embodiments, the suspension of the pharmaceutical formulation disclosed herein has a volume of approximately 20 μL, 30 μL, 40 μL, 50 μL, 60 μL, 70 μL, 80 μL, 90 μL, 100 μL, 200 μL, 300 μL, 400 μL, 500 μL, 600 μL, 700 μL, 800 μL, 900 μL, or 1000 μL. In some embodiments, the suspension of the pharmaceutical formulation disclosed herein has a volume of approximately 250 μL. In some embodiments, the suspension of the pharmaceutical formulation disclosed herein has a volume of 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, 0.9-1.1 mL, 1.0-1.2 mL, or 1.0-1.5 mL. In other embodiments, the volume is 0.1 mL or less, 0.2 mL or less, 0.3 mL or less, 0.4 mL or less, 0.5 mL or less, 0.6 mL or less, 0.7 mL or less, 0.8 mL or less, 0.9 mL or less, 1.0 mL or less, 1.1 mL or less, 1.2 mL or less, 1.3 mL or less, 1.4 mL or less, or 1.5 mL or less. In some embodiments, the suspension of the pharmaceutical formulation disclosed herein has a volume of approximately 0.25 mL.

[0194] In some embodiments, the suspension of the pharmaceutical formulations disclosed herein is provided as a sealed (e.g., using a sterile aluminum peel-off seal) sterile filtered frozen suspension in a sterile ready-to-use vial (e.g., a 0.5 mL vial; e.g., a Crystal Zenith® vial) with a ready-to-use stopper (e.g., a chlorobutyl stopper). In some embodiments, the suspension of the pharmaceutical formulations disclosed herein is provided as a sealed (e.g., using a sterile aluminum peel-off seal) sterile filtered frozen suspension in a sterile ready-to-use vial (e.g., a 0.5 mL vial; e.g., a Crystal Zenith® vial) with a ready-to-use stopper (e.g., a chlorobutyl stopper), wherein the vial contains a volume of the pharmaceutical formulation suspension of 0.1–0.5 mL, 0.1–0.2 mL, 0.2–0.3 mL, 0.3–0.4 mL, or 0.4 mL–0.5 mL. In some embodiments, the suspension of the pharmaceutical formulation disclosed herein is provided as a sealed (e.g., using a sterile aluminum peel-off seal) sterile filtered frozen suspension in a sterile, ready-to-use vial (e.g., a 0.5 mL vial; e.g., a Crystal Zenith® vial) with a ready-to-use stopper (e.g., a chlorobutyl stopper), where the vial contains a volume of approximately 0.25 mL of the pharmaceutical formulation suspension.

[0195] In some embodiments, the pharmaceutical formulations disclosed herein are designed, engineered, or adapted for administration to primates (e.g., non-human primates and human subjects) by intravitreous or subretinal injection. In some embodiments, a pharmaceutical formulation comprising rAAV particles containing nucleic acid sequences encoding an anti-VEGF agent (e.g., aflibercept) is formulated for intravitreous injection into the eye of an individual. In some embodiments, the pharmaceutical composition is expressed in amounts of approximately 25 μL or less, approximately 30 μL or less, approximately 35 μL or less, approximately 40 μL or less, approximately 45 μL or less, approximately 50 μL or less, approximately 55 μL or less, approximately 60 μL or less, approximately 65 μL or less, approximately 70 μL or less, approximately 75 μL or less, approximately 80 μL or less, approximately 85 μL or less, approximately 90 μL or less, approximately 95 μL or less, approximately 100 μL or less, approximately 110 μL or less, approximately 120 μL or less, approximately 130 μL or less, approximately 140 μL or less, approximately 150 μL or less, approximately 160 μL or less, approximately 170 μL or less, approximately 180 μL or less, approximately 190 μL or less, approximately 200 μL or less, approximately 210 μL or less, and approximately 220 μL. The following formulations or reconstitutions are made to a concentration that allows for intravitreal injection in volumes of approximately 230 μL or less, approximately 240 μL or less, or approximately 250 μL or less, or any of the following: 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 or less.In some embodiments, the unit dose of the pharmaceutical formulation is approximately 25 μL or less, approximately 30 μL or less, approximately 35 μL or less, approximately 40 μL or less, approximately 45 μL or less, approximately 50 μL or less, approximately 55 μL or less, approximately 60 μL or less, approximately 65 μL or less, approximately 70 μL or less, approximately 75 μL or less, approximately 80 μL or less, approximately 85 μL or less, approximately 90 μL or less, approximately 95 μL or less, approximately 100 μL or less, approximately 110 μL or less, approximately 120 μL or less, approximately 130 μL or less, approximately 140 μL or less, approximately 150 μL or less, approximately 160 μL or less, approximately 170 μL or less, approximately 180 μL or less, approximately 190 μL or less, approximately 200 μL or less. It contains a volume of approximately 210 μL or less, approximately 220 μL or less, approximately 230 μL or less, approximately 240 μL or less, or approximately 250 μL or less, or any of the following volumes: 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 or less. In some embodiments, the methods disclosed herein include intravitreal injection of a solution or suspension of a pharmaceutical formulation containing nucleic acid sequences encoding rAAV (e.g., AAV2.7m8) and an anti-VEGF agent (e.g., aflibercept) in any volume of approximately 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. In some embodiments, the methods disclosed herein involve intravitreal injection of a solution or suspension of a pharmaceutical formulation containing nucleic acid sequences encoding rAAV (e.g., AAV2.7m8) and an anti-VEGF agent (e.g., aflibercept) in a volume of about 30 μL or about 100 μL.In some embodiments, the method disclosed herein involves intravitreal injection of a solution or suspension of a pharmaceutical formulation containing nucleic acid sequences encoding rAAV (e.g., AAV2.7m8) and an anti-VEGF agent (e.g., aflibercept) in a volume of about 30 μL. In some embodiments, the method disclosed herein involves intravitreal injection of a solution or suspension of a pharmaceutical formulation containing nucleic acid sequences encoding rAAV (e.g., AAV2.7m8) and an anti-VEGF agent (e.g., aflibercept) in a volume of about 100 μL.

[0196] In some embodiments, AAV2.7m8 particles containing the nucleic acid sequence of the anti-VEGF agent (e.g., aflibercept) transgene described herein are components of a gene therapy pharmaceutical formulation. In some embodiments, rAAV particles of any serotype containing the 7m8 variant capsid protein described herein are used to create a frozen 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 particles are rAAV2. In some embodiments, the lyophilized or frozen pharmaceutical formulation contains rAAV2 containing the 7m8 variant capsid protein and the DNA sequence encoding the anti-VEGF agent (e.g., aflibercept). In some embodiments, the suspension is refrigerated or frozen.

[0197] In some embodiments, the administration of a unit dose of rAAV particles to one eye and / or the opposite eye of an individual is by intravitreal (IVT) injection. With regard to IVT injection, rAAV particles can be delivered in the form of a suspension of the pharmaceutical formulation (e.g., as described herein). First, a local anesthetic is applied to the surface of the eye, followed by the application of an eye disinfectant solution. With the eye kept open with or without the use of instruments, the rAAV particles are injected through the sclera into the vitreous cavity of one eye and / or the opposite eye of the individual using a short, fine needle, e.g., a 30-gauge needle, under direct observation. Generally, rAAV particle suspensions in volumes of approximately 25 μL to approximately 250 μL (for example, approximately 25 μL, 30 μL, 40 μL, 50 μL, 60 μL, 70 μL, 80 μL, 90 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) can be delivered to the eye by IVT injection. In some embodiments, the unit dose of rAAV particles contains a volume of approximately 100 μL. In some embodiments, the unit dose of rAAV particles contains a volume of approximately 30 μL. In some embodiments, IVT injection is performed in combination with the removal of vitreous fluid. In some embodiments, vitrectomy can be performed, replacing the entire volume of vitreous gel by injecting an rAAV particle suspension (e.g., approximately 4 mL of rAAV particle suspension). Vitrectomy is performed using a cannula of an appropriate diameter (e.g., 20 gauge to 27 gauge), and the volume of removed vitreous gel is replaced by injecting a fluid from the injection cannula, such as saline, isotonic solution, or rAAV particle suspension. IVT administration is generally well tolerated. Sometimes the injection site becomes slightly red at the end of the procedure. Tenderness may occur occasionally, but the vast majority of patients report no pain. After this procedure, neither an eye patch nor an eye protection bandage is needed, and activity is not restricted. Sometimes, antibiotic eye drops for several days are prescribed to help prevent infection.

[0198] In some embodiments, the pharmaceutical formulation is a unit dose (e.g., a therapeutically effective dose) administered by IVT injection into one eye and / or the opposite eye of an individual (e.g., a human or non-human primate) to treat an eye disease or disorder characterized by abnormal (e.g., excessive) angiogenesis or neovascularization. In some embodiments, the pharmaceutical formulation includes a unit dose (e.g., a therapeutically effective dose) described in further detail elsewhere in this specification. In some embodiments, the volume of a unit dose (e.g., a therapeutically effective dose) of the viral vector (e.g., the rAAV vector disclosed herein) administered to a subject is no more than or equal to one of the following values: approximately 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, including any range between these values. By minimizing the volume of the unit dose administered to the subject, changes in intraocular pressure and other adverse effects associated with IVT injection (e.g., increased intraocular pressure, inflammation, irritation, or pain) can be avoided or mitigated.

[0199] Pharmaceutical formulations suitable for ocular use include sterile aqueous solutions or dispersions and sterile injection solutions, suspensions, or sterile powders for immediate preparation of dispersions. For intravitreous 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 a fluid sterilized to the extent that syringability or injectability exists. In certain embodiments, the pharmaceutical formulation is stable under manufacturing and storage conditions and protected from contamination by microorganisms such as bacteria and fungi. In some embodiments, the pharmaceutical composition may contain salts or isotonic agents such as glycerol. In some embodiments, surfactants or stabilizers are added to the pharmaceutical composition to prevent aggregation.

[0200] In some embodiments, the pharmaceutical formulation contains excipients or carriers. The carrier is a solvent or dispersion medium containing, for example, water, saline solution, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), or any combination thereof. Appropriate fluidity is achieved, for example, by using a coating such as lecithin, and in the case of dispersion, by maintaining the required particle size. Furthermore, polysorbates (e.g., Tween®, Polysorbate 20, Polysorbate 80), sodium dodecyl sulfate (sodium lauryl sulfate), lauryldimethylamine oxide, cetyltrimethylammonium bromide (CTAB), polyethoxylated alcohols, polyoxyethylene sorbitan, octoxynol (Triton X100®), N,N-dimethyldodecylamine-N-oxide, hexadecyltrimethylammonium bromide (HTAB), polyoxyl 10 lauryl ether, Brij The absorption can be maintained by using surfactants such as 721 (trademark), bile salts (sodium deoxycholate, sodium cholate), Pluronic acid (F-68, F-127), polyoxyl castor oil (Cremophor (trademark)), nonylphenol ethoxylate (Tergitol (trademark)), cyclodextrin, and ethylbenzethonium chloride (Hyamine (trademark)). Prevention of microbial action can be achieved by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, cresol, thimerosal, etc. In many embodiments, isotonic agents are included in the pharmaceutical formulation, such as sugars, polyhydric alcohols such as mannitol and sorbitol, and / or sodium chloride. Sustained absorption of the internal composition can be achieved by including absorption-delaying agents, such as aluminum monostearate and gelatin, in the composition. In some embodiments, the pharmaceutical carrier includes sodium phosphate, sodium chloride, polysorbate, and sucrose. In some embodiments, the pharmaceutical formulation includes a surfactant, such as a nonionic surfactant such as polysorbate, poloxamer, or Pluronic®.In some embodiments, the addition of a nonionic surfactant reduces aggregation in the pharmaceutical composition.

[0201] Kits comprising at least one pharmaceutical formulation described herein are also provided herein. In some embodiments, the kit comprises a frozen suspension of the pharmaceutical formulation (e.g., one unit dose in a vial). In some embodiments, the kit comprises a lyophilized or freeze-dried pharmaceutical formulation disclosed herein (e.g., one unit dose in a vial) and a solution for dissolving, diluting, and / or restoring the lyophilized pharmaceutical composition. In some embodiments, the solution for restoration or dilution is supplied as a pre-filled syringe. In some embodiments, the kit comprises a freeze-dried or lyophilized pharmaceutical composition containing rAAV (e.g., AAV2.7m8) and a solution for restoring the pharmaceutical composition to a desired concentration or volume. In some embodiments, the kit comprises a buffer that helps prevent aggregation when restoring the pharmaceutical composition disclosed herein. In some embodiments, the pharmaceutical composition is provided in a pre-filled syringe. In some embodiments, the kit comprises a double-chamber syringe or container, in which case 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 restored solution is filtered before administration. In some embodiments, the kit includes a filter or filter syringe for filtering the restored pharmaceutical composition before administration to a patient. In some embodiments, the kit includes a suspension of a pharmaceutical formulation containing rAAV particles disclosed herein, provided as a sealed (e.g., using a sterile aluminum peel-off seal) sterile filtered frozen suspension in a sterile, ready-to-use vial (e.g., a 0.5 mL vial; e.g., a Crystal Zenith® vial) with a ready-to-use stopper (e.g., a stopper made of chlorobutyl).In some embodiments, the kit comprises a suspension of a pharmaceutical formulation containing rAAV particles disclosed herein, provided as a sealed (e.g., using a sterile aluminum peel-off seal) sterile filtered frozen suspension in a sterile, ready-to-use vial (e.g., a 0.5 mL vial; e.g., a Crystal Zenith® vial) with a ready-to-use stopper (e.g., a chlorobutyl stopper), wherein the vial contains a suspension of the pharmaceutical formulation in a volume of 0.1–0.5 mL, 0.1–0.2 mL, 0.2–0.3 mL, 0.3–0.4 mL, or 0.4 mL–0.5 mL. In some embodiments, the kit comprises a suspension of a pharmaceutical formulation containing the disclosed rAAV particles, provided as a sealed (e.g., using a sterile aluminum peel-off seal) sterile filtered frozen suspension in a sterile, ready-to-use vial (e.g., a 0.5 mL vial; e.g., a Crystal Zenith® vial) with a ready-to-use stopper (e.g., a chlorobutyl stopper), wherein the vial contains a volume of the pharmaceutical formulation suspension of about 0.25 mL. In some embodiments, the kit further comprises instructions for use; e.g., instructions for treating intraocular neovascular disease using the rAAV particles disclosed herein. Intraocular neovascular disease

[0202] In one embodiment, the disclosure provides a method for treating intraocular neovascular disease in an individual. In another embodiment, the disclosure provides a method for reducing retinal fluid in the eye of an individual having intraocular neovascular disease.

[0203] In some embodiments, intraocular neovascularization is age-related macular degeneration (AMD), exudative 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, intraocular neovascularization is active choroidal neovascularization (CNV) secondary to age-related macular degeneration (AMD). In some embodiments, intraocular neovascularization is recurrent and / or persistent wAMD. In some embodiments, intraocular neovascularization is active subfoveal CNV secondary to AMD. In some embodiments, active subfoveal CNV secondary to AMD accounts for ≥50% of the total lesion size. In some embodiments, active subfoveal CNVs secondary to AMD account for ≥50% of the total lesion size and are accompanied by evidence of leakage on fluorescein angiography (FA), exudate on spectral-domain optical coherence tomography (SD-OCT), and / or subretinal hemorrhage on color fundus photography. In some embodiments, active subfoveal CNVs secondary to AMD account for ≥50% of the total lesion size and are accompanied by evidence of leakage on fluorescein angiography (FA), exudate on spectral-domain optical coherence tomography (SD-OCT), and / or subretinal hemorrhage on color fundus photography, and the total size of the lesion does not exceed the 12-macular photocoagulation test disk area. In some embodiments, the best corrected visual acuity (BCVA) based on the ETDRS letter rating demonstrated by one eye and / or the other eye of an individual before administration of a unit dose of the rAAV particles of this disclosure was between 78 and 25 (e.g., less than about 78, about 75, about 70, about 65, about 60, about 55, about 50, about 45, about 40, about 35, about 30, or about 25). In some embodiments, the best corrected visual acuity (BCVA) based on the ETDRS letter rating demonstrated by one eye and / or the other eye of an individual before administration of a unit dose of the rAAV particles of this disclosure was greater than about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, or about 100.

[0204] In some embodiments, individuals had polypoid choroidal vasculopathy (PCV) in one eye and / or the opposite eye prior to administration of a unit dose of rAAV particles.

[0205] In some embodiments, ETDRS character evaluation is performed at approximately 0.5 meters, 1 meter, 2 meters, 3 meters, or 4 meters. In some embodiments, ETDRS character evaluation is performed at approximately 4 meters.

[0206] In some embodiments, individuals have received at least one prior treatment with an anti-VEGF agent (e.g., bevacizumab, brolucizumab, ranibizumab, falisimab, abisipalpegol, and / or aflibercept) in the period of approximately 12 weeks (e.g., approximately 4 months) immediately preceding the administration of a unit dose of rAAV particles. In some embodiments, individuals have received two or three prior treatments with an anti-VEGF agent (e.g., bevacizumab, brolucizumab, ranibizumab, falisimab, abisipalpegol, and / or aflibercept) in one eye and / or the other eye in the period of approximately 12 weeks (e.g., approximately 4 months) immediately preceding the administration of a unit dose of rAAV particles in one eye and / or the other eye. In some embodiments, individuals had received at least about 1, at least about 5, at least about 10, at least about 20, at least about 100, at least about 40, at least about 50, at least about 60, at least about 70, at least about 80, at least about 90, at least about 100, at least about 110, at least about 120, or more prior treatments with an anti-VEGF agent (e.g., bevacizumab, brolucizumab, ranibizumab, falisimab, abisipalpegol, and / or aflibercept) in one eye and / or the opposite eye. In some embodiments, individuals had calculated anti-VEGF agent (e.g., bevacizumab, brolucizumab, ranibizumab, and / or aflibercept) injection intervals for one eye and / or the opposite eye of approximately 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, or longer. In some embodiments, individuals had calculated anti-VEGF agent (e.g., bevacizumab, brolucizumab, ranibizumab, and / or aflibercept) injection intervals for one eye and / or the opposite eye of approximately 5–7 weeks, 4–10 weeks, 4–7 weeks, or 4–6 weeks.In some embodiments, individuals had received prior treatment with an anti-VEGF agent (e.g., bevacizumab, brolucizumab, ranibizumab, falisimab, abisipalpegol, and / or aflibercept) in one eye and / or the other 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 20 days prior to administration of a unit dose of rAAV particles to one eye and / or the other eye. In some embodiments, individuals had received prior treatment with an anti-VEGF agent (e.g., bevacizumab, brolucizumab, ranibizumab, falisimab, abisipalpegol, and / or aflibercept) in one eye and / or the other eye for about 7, 10, or 14 days prior to administration of a unit dose of rAAV particles to one eye and / or the other eye. In some embodiments, the prior treatment includes intravitreal, subretinal, or intravitreal injection of the anti-VEGF agent. In some embodiments, the anti-VEGF agent is bevacizumab, brolucizumab, ranibizumab, falisimab, abisipalpegol, and / or aflibercept. In some embodiments, the anti-VEGF agent is aflibercept.

[0207] In some embodiments, individuals showed a meaningful response to prior treatment with an anti-VEGF agent. In some embodiments, the anti-VEGF agent is aflibercept, its functional variant, or its functional fragment. In some embodiments, the anti-VEGF agent comprises a polypeptide having an amino acid sequence having at least about 95% identity with the amino acid sequence of SEQ ID NO: 35. In some embodiments, individuals showed a meaningful response to prior anti-VEGF treatment for intraocular neovascularization in one eye and / or the opposite eye (e.g., aflibercept, its functional variant, or its functional fragment) prior to administration of a unit dose of rAAV particles to one eye and / or the opposite eye. In some embodiments, an individual is determined to have had a meaningful response to prior anti-VEGF treatment for intraocular neovascular disease (e.g., aflibercept, its functional variant, or its functional fragment) if a reduction of ≥30% (e.g., at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 9%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17, or longer) in foveal retinal thickness (CRT) or central regional retinal thickness (CST) compared to the initial diagnosis for one eye and / or the opposite eye.In some embodiments, an individual is determined to have a meaningful response to prior anti-VEGF treatment for intraocular neovascular disease (e.g., aflibercept, its functional variant, or its functional fragment) if a reduction of ≥30% (e.g., at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or 100%) of foveal retinal thickness (CRT) or central regional retinal thickness (CST) in one eye and / or the opposite eye compared to the CRT or CST before administration of prior anti-VEGF treatment, is observed for a period longer than approximately 7 days, at least 10 days, or at least 14 days after anti-VEGF treatment.

[0208] In some embodiments, the central region retinal thickness and / or foveal retinal thickness are determined by SD-OCT of one eye and / or the opposite eye. The central region retinal thickness is the average thickness of the retina across the central region of an ETDRS grid, which is a 1 mm diameter circle centered on the fovea.

[0209] In some embodiments, an individual is determined to have a meaningful response to prior treatment with an anti-VEGF agent for intraocular neovascular disease (e.g., aflibercept, its functional variant, or its functional fragment) if a decrease 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 100%) of central region retinal thickness and / or foveal retinal thickness is observed compared to the central region retinal thickness and / or foveal retinal thickness before administration of the prior treatment with the anti-VEGF agent. In some embodiments, an individual is determined to have a meaningful response to prior treatment with an anti-VEGF agent (e.g., aflibercept, its functional variant, or its functional fragment) if a decrease 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 17 days, or longer) in central retinal thickness and / or foveal retinal thickness compared to the central retinal thickness and / or foveal retinal thickness before administration of the prior treatment with an anti-VEGF agent is observed for at least about 5 days, at least about 6 days, at least about 7 days, at least 80%, at least 80%, at least 13 days, at least 14 days, at least 15 days, at least 16 days, at least 17 days, or longer after administration of the prior treatment with an anti-VEGF agent.In some embodiments, an individual is determined to have a meaningful response to prior treatment with an anti-VEGF agent (e.g., aflibercept, its functional variant, or its functional fragment) if a decrease 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 100%) of central region retinal thickness and / or foveal retinal thickness compared to the central region retinal thickness and / or foveal retinal thickness before prior treatment with an anti-VEGF agent is observed over a period of approximately 7, 10, or 14 days after administration of the prior treatment with an anti-VEGF agent. In some embodiments, central region retinal thickness and / or foveal retinal thickness is determined by SD-OCT of one eye and / or the opposite eye. In some embodiments, an individual is determined to have a meaningful response to prior treatment with an anti-VEGF agent for intraocular neovascularization (e.g., aflibercept, its functional variant, or its functional fragment) if normalization of the CST is observed after treatment with an anti-VEGF agent in one eye and / or the opposite eye, and there is no observable vascular exudation. Normalization refers to the CST value being normal with respect to the patient's class (e.g., based on age, sex, etc.).

[0210] In some embodiments, individuals are determined by SD-OCT of one eye and / or the opposite eye to have had an intermediate period before administration of anti-VEGF treatment (e.g., aflibercept) 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, or longer after anti-VEGF treatment. A meaningful response to prior anti-VEGF treatment for intraocular neovascular disease (e.g., aflibercept, its functional variant, or its functional fragment) is determined if a decrease 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 100%) of the foveal retinal thickness (CRT) or central retinal thickness (CST) is observed compared to the CRT or CST. In some embodiments, an individual is determined to have a meaningful response to prior anti-VEGF treatment for intraocular neovascular disease (e.g., aflibercept, its functional variant, or its functional fragment) if, as determined by SD-OCT of one eye and / or the opposite eye, a reduction 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 100%) of foveal retinal thickness (CRT) or central regional retinal thickness (CST) compared to pre-treatment with anti-VEGF, over a period of approximately 7, 10, or 14 days after anti-VEGF treatment.

[0211] In some embodiments, the individual has not received prior treatment for intraocular neovascular disease. In some embodiments, the individual has not received prior treatment for intraocular neovascular disease in one eye and / or the opposite 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 opposite eye. In some embodiments, the individual has not received prior aflibercept treatment. In some embodiments, the individual has not received prior aflibercept treatment in one eye and / or the opposite eye.

[0212] In some embodiments, the eye disease or disorder treated according to the methods disclosed herein is diabetic macular edema. Diabetic macular edema (DME) is swelling of the retina caused by leakage of fluid from blood vessels in the macula in diabetes. The macula is the central part of the retina, a narrow area rich in cones, which are specialized nerve endings that detect color and on which daytime vision depends. As macular edema develops, blurring occurs in the center or just to the sides of the central field of vision. Blindness from diabetic macular edema can progress over a period of several months, making it impossible to focus clearly. Common symptoms of DME include blurred vision, floaters, double vision, and, if left untreated, eventual loss of vision. In some embodiments, DME is treated using the methods and pharmaceutical compositions disclosed herein.

[0213] In some embodiments, the eye disease or disorder treated according to the method described herein is retinal vein occlusion. Retinal vein occlusion is the blockage of the small veins that carry blood from the retina. The retina is a layer of tissue at the back of the inside of the eye that converts images of light into nerve signals and sends them to the brain. Retinal vein occlusion is most often caused by arteriosclerosis (atherosclerosis) and the formation of blood clots. Blockage of smaller veins in the retina (branch veins or BRVOs) often occurs where retinal arteries, thickened or hardened by atherosclerosis, cross and put pressure on the retinal veins. Symptoms of retinal vein occlusion may include sudden blurring of vision or loss of vision in all or part of one eye.

[0214] In some embodiments, the eye disease or disorder treated according to the methods described herein is choroidal neovascularization (CNV), also known as exudative age-related macular degeneration (wAMD). Choroidal neovascularization may involve the growth of new blood vessels originating in the choroid and extending from a crease in Bruch's membrane into the subretinal pigment epithelium (sub-RPE) or subretinal space, which can be a major cause of blindness. CNV can result in a sudden and noticeable deterioration of central vision within weeks. Other symptoms may include color vision impairment and degenerative vision (a distortion where straight lines appear wavy). Bleeding from the new blood vessels can accelerate the onset of CNV symptoms. CNV may also include a feeling of pressure at the back of the eye.

[0215] Advanced “exudative” forms of AMD (neovascular or exudative) can frequently cause rapid and often substantial loss of central vision in patients. In exudative forms of AMD, choroidal neovascularization forms and develops into a network of blood vessels that can grow beneath and through the retinal pigment epithelium. This is accompanied by leakage and / or hemorrhage of plasma into the subretinal space, which, if occurring in the macula, can result in sudden and severe loss of central vision. This disclosure is intended for the treatment or prevention of AMD, exudative AMD. In some embodiments, AMD is treated using the methods and pharmaceutical compositions disclosed herein.

[0216] In some embodiments, the methods described herein are used to prevent or treat eye diseases or disorders in subjects that have received prior treatment with bevacizumab, brolucizumab, ranibizumab, falisimab, abisipalpegol, and / or aflibercept. In some embodiments, the methods described herein are used to prevent or treat eye diseases or disorders that are responsive to treatment with bevacizumab, brolucizumab, ranibizumab, and / or aflibercept.

[0217] In some embodiments, the individual was diagnosed with intraocular neovascularization 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 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 earlier, before administration of a unit dose of rAAV particles to one eye and / or the other eye.

[0218] The following descriptions are provided to enable those skilled in the art to create and use various embodiments. Descriptions of specific devices, techniques, and applications are provided merely as examples. Various modifications to the embodiments described herein will be readily apparent to those skilled in the art, and the general principles defined herein can be applied to other embodiments and applications without departing from the spirit and scope of the various embodiments. Accordingly, the various embodiments are not limited to those described and shown herein, but are consistent with the scope not inconsistent with the claims. [Examples]

[0219] (Example 1) 6 x 10 for neovascular (exudative) age-related macular degeneration 11 An open-label Phase 1 trial of AAV2.7m8-aflibercept at a dose of vg / ocular. This example describes an open-label Phase 1 trial of aflibercept, a VEGF inhibitor, and AAV2.7m8-aflibercept, an rAAV vector containing the AAV2.7m8 protein capsid, for the treatment of age-related macular degeneration (AMD) with choroidal neovascularization. I. Purpose of the Examination A. Main purpose

[0220] 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 endpoints

[0221] The primary endpoints of this study were the type, severity, and incidence of ocular and systemic adverse events (AEs). B. Secondary Objectives

[0222] The secondary objectives of this study were as follows: • To evaluate the effect of AAV2.7m8-Aflibercept on best corrected visual acuity (BCVA). • To evaluate the effect of AAV2.7m8-aflibercept on central retinal thickness (CST), which is also known as foveal retinal thickness (CRT). • Assess the need for rescue treatment from week 4 to week 104. • To evaluate the effects 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 epithelial pigment detachment (PED) in patients with PED at baseline. Secondary endpoints

[0223] The secondary endpoints of this study were as follows: • The mean change in BCVA over time from baseline to baseline, as assessed by ETDRS letters from day 8 to week 104. • Percentage of subjects who, evaluated from day 8 to week 104, had a BCVA increase of ≥15 ETDRS characters over time compared to baseline. • Percentage of subjects who, evaluated from day 8 to week 104, had a BCVA reduction of ≤15 ETDRS characters over time compared to baseline. • Mean changes in CST and macular volume over time from baseline, assessed from day 8 to week 104 and compared to baseline. • The average number of aflibercept injections over time, evaluated from week 4 to week 104. • Evaluate the percentage of patients requiring aflibercept injections over time, from week 4 to week 104. • The percentage of subjects without IRF (Intra-Risk Failure) over time, evaluated from day 8 to week 104. • The percentage of subjects without SRF (Surface-Resistant Factor Function) over time, evaluated from day 8 to week 104. • The percentage of subjects who did not have PED at baseline, evaluated from day 8 to week 104, over time. II. Subjects of the Examination

[0224] The subjects of this study were those diagnosed with active choroidal neovascularization (CNV) secondary to age-related macular degeneration (AMD), who had a history of responding to recent anti-VEGF treatment, and who required frequent injections of anti-VEGF drugs.

[0225] Throughout the duration of the study, only one eye was selected to be tested. If both eyes met all inclusion and exclusion criteria, the eye with the worst BCVA as assessed at screening was selected to be tested. If both eyes met all inclusion and exclusion criteria and both eyes had the same BCVA value, the participant could choose to treat the non-dominant eye or, by default, the right eye was selected as the eye to be tested. A. Inclusion Criteria

[0226] Subjects that met the following inclusion criteria were enrolled in this study: • Applicants must be male or female, and aged 50 or older. • There is prior or current evidence of active subfoveal CNV secondary to AMD occupying ≥50% of the total lesion size in the eye being tested, and accompanied by: ○ Leakage on fluorescein angiography (FA), fluid on spectral domain optical coherence tomography (SD-OCT), or subretinal hemorrhage on color fundus photography; and ○ The total dimensions of the lesion must not exceed the area of ​​the 12-macular photocoagulation test disc. • The subject must have received at least two injections of aggressive anti-VEGF treatment for wAMD within the four months prior to screening. Regarding the vision of the eye being tested during the screening visit (before the aflibercept injection): ○BCVA ETDRS score should be between 78 and 25. Regarding the vision of eyes that are not being tested: ○BCVA ETDRS must be ≥35. • A meaningful anti-VEGF response has been confirmed by the principal investigator and demonstrated as follows: ○ Evaluation using SD-OCT shows that the central retinal thickness has decreased by ≥30% from the initial diagnosis; or ○Evaluation using SD-OCT shows that central retinal thickness has decreased by ≥20% since screening; or ○CST is normalized and there is no observable vascular exudation.

[0227] VEGF responsiveness was assessed by the principal investigator on day 1, prior to administration of AAV2.7m8-aflibercept, to confirm the anti-VEGF response. Subjects determined to not have a meaningful anti-VEGF response failed screening and were not enrolled in this study. B. Exclusion criteria

[0228] The following individuals who met the exclusion criteria were not enrolled in this study: neutralizing antibody Although a correlation between serum and vitreous fluid neutralizing antibody (NAb) levels and AAV has not been established (Lukason et al., (2011) Mol Ther 19 (2): 260-265), subjects were screened for NAb levels against the AAV2.7m8 vector as a precaution. Subjects who demonstrated anti-AAV2.7m8 neutralizing antibody titers >1:125 during the 6 months prior to AAV2.7m8-aflibercept administration were excluded from this study. CNV lesions • Known history or evidence for the following CNV lesion characteristics: ○ Fibrosis or atrophy, retinal epithelial tear in the center of the fovea of ​​the eye being tested, or any condition that does not improve vision. ○ Scarring or fibrosis constituting >50% of the total lesion area. ○ Macular photocoagulation test disk area (30.5 mm²) including blood, scarring, and neovascularization, as evaluated by fluorescein angiography (FA). 2 ) lesion size. ○ Subretinal hemorrhage of ≥50% of the total lesion area in the eye being tested, or presence of subfoveal blood of ≥1 disk area size (if blood is subfoveal, the fovea was surrounded by 270 degrees of visible CNV). Eye condition (retina / posterior eye) • History of significant epiretinal membrane or vitreomacular traction (VMT) syndrome in the eye being tested at the time of aflibercept administration, or a history of full-thickness macular hole (Gass Stage 2 and higher) in the eye being tested. • A history of retinal diseases other than wAMD in the eye being tested, including diabetic retinopathy (in either eye), retinal vein occlusion, uveitis, suspected retinal hemangiomatous proliferation, polypoid choroidal disease, or CNV due to other causes (e.g., ocular histoplasmopathy, trauma, or pathological myopia), or any other vascular disease in the eye (benign conditions of the vitreous or peripheral retina were not excluded). • History of retinal detachment (with or without repair) in the eye being tested. Other conditions (non-retinal) • Known history or evidence of significant non-retinal disease or intermediate transparent media opacity in the eye being tested that could impair vision during the course of the test, require surgery, and / or interfere with adequate visualization or imaging of the retina (e.g., central corneal scarring, significant cataract, corneal dystrophy, scleromalacia). • AAV2.7m8 - Uncontrolled ocular hypertension or glaucoma (defined as intraocular pressure [IOP] >22 mmHg despite treatment with antiglaucoma drugs) or use of IOP-lowering agents >2 at the time of screening in the eye being tested for aflibercept. • Active infection of the eye or periocular area, or a history thereof, in either eye within 4 weeks prior to administration of AAV2.7m8-aflibercept. Eye surgery / procedures • Any prior intraocular or periorbital surgery on the eye undergoing a trial within 6 months of AAV2.7m8-aflibercept administration, or any planned major surgical procedure within 6 months of AAV2.7m8-aflibercept administration. Eyelid surgery within 1 month of AAV2.7m8-aflibercept administration was not excluded. • History of vitrectomy, trabeculectomy, or other filtration surgery in the eye being tested. • AAV2.7m8 - Yttrium aluminum garnet (YAG) posterior capsulotomy within 3 months prior to administration of aflibercept. • Any prior treatment and testing of the area of ​​the eye with photodynamic therapy or retinal laser to treat wAMD, or any prior therapeutic radiation exposure within the eye. General / Full Body Condition Unless otherwise specified, a history or evidence of any of the following cardiovascular diseases within 6 months of medication: ○ A history of severe heart disease (e.g., New York Heart Association [NYHA] Functional Class III or IV) or clinical evidence of unstable angina. ○ Acute coronary syndrome, myocardial infarction, or coronary artery regeneration. ○ Ventricular tachyarrhythmias or uncontrolled arrhythmias requiring continued treatment. ○Uncontrolled hypertension, defined as mean systolic blood pressure (SBP) ≥ 160 mmHg or mean diastolic blood pressure (DBP) ≥ 100 mmHg, despite the use of blood pressure-lowering drugs during the pre-medication screening period. ○ A history of cerebrovascular accident or transient ischemic attack. • Any history of ongoing bleeding disorder or an International Normalized Ratio (INR) > 3.0. Use of aspirin or other anticoagulants (e.g., factor Xa inhibitors) was not an exclusion criterion. INR was reported during the screening period to confirm that eligibility criteria were met. • AAV2.7m8 - Evidence of uncontrolled diabetes with HbA1c > 7.0% during the pre-treatment screening period for aflibercept. • History of malignant tumors within the past 5 years, except for the following, provided that the treatment has been adequate: ○Localized basal cell carcinoma or squamous cell carcinoma of the skin. ○ Carcinoma in situ of the cervix or breast. Papillary non-invasive bladder cancer. ○Prostate cancer stages 1 and 2 in which stable prostate-specific antigen (PSA) levels are clinically demonstrated over a 6-month observation period. ○ Any other cancer that has been in complete remission or considered surgically cured for at least two years. • Positive for HIV, hepatitis B, or hepatitis C (unless treated and cured). • Evidence or suspicion of any type of systemic active infection deemed clinically significant by the principal investigator based on clinical laboratory tests and / or body temperature >38.5°C within 36 hours prior to administration of AAV2.7m8-aflibercept. • Known severe allergies to the following: ○Fluorescein dye or sodium fluorescein used in angiography (mild allergies to which the procedure can be applied are tolerated); or ○ Aflibercept. • Women who are pregnant, breastfeeding, or planning to become pregnant during the trial. • Other significant abnormal laboratory values ​​or medical conditions that, in the opinion of the principal investigator, impair the safety of the subjects. Drug application • Prior to administration of AAV2.7m8-aflibercept, use of systemic anti-inflammatory steroids or immunosuppressants with a half-life of 5 (other than protocol-specified prednisone) was permitted. Inhaled or topical steroids and nonsteroidal anti-inflammatory drugs (NSAIDs) were acceptable. • You have received any of the following: ○The investigation drug, AAV2.7m8-aflibercept, was administered within the longer of either 30 days prior to administration or its 5-half-life. ○Previous gene therapy drugs. Search for pharmaceuticals

[0229] The investigational drug (IMP), AAV2.7m8-aflibercept, was a recombinant replication-deficient adeno-associated virus (rAAV) vector containing an AAV2.7m8 protein capsid derived from in vivo directional evolution against the AAV2 capsid (Dalkara et al., (2013) Sci Transl Med 5 (189): 189ra76; US2014 / 0364338). AAV2.7m8-aflibercept possessed an expression cassette of a codon-optimized version of aflibercept cDNA under the control of a ubiquitous chimeric promoter (Figure 1A) (see WO2018170473A1). AAV2.7m8-aflibercept was produced in Sf9 cells using a baculovirus expression vector system that employs two different baculoviruses, one encoding the genes for the AAV2 Rep and AAV2.7m8 Cap proteins, and the other encoding the human aflibercept cDNA expression cassette.

[0230] AAV2.7m8-aflibercept was supplied as a sterile, filtered, frozen suspension in a sterile, ready-to-use 0.5 mL Crystal Zenith vial containing 0.25 mL of IMP, formulated as shown in Table 1. [Table 1] IV. Test Design A. Dosage and method of administration Cohort 1

[0231] In Cohort 1, participants received AAV2.7m8-aflibercept at dose 1, 6 × 10⁶ times. 11 The drug was administered via a single IVT injection at a dose of 100 μL per eye.

[0232] Vials of AAV2.7m8-aflibercept were removed from cryopreservation at ≤-60°C and thawed at room temperature. AAV2.7m8-aflibercept was administered by IVT injection. Aseptic techniques using povidone-iodine were employed in conjunction with local or subconjunctival anesthesia. Post-injection care and drug administration regimens were performed according to institutional standard practices. B. Visiting the examination site Screening (Day 15 to Day 7)

[0233] As shown in Figure 1B, subjects received a single in vitro injection of aflibercept 2 mg in conjunction with standard treatment during screening between day -15 and day -7 (e.g., day -14 to day -7). Subjects received standard post-injection care at a typical facility. Day 1 of the exam

[0234] On day 1 of the trial (between 7 and 15 days after the IVT injection of aflibercept, for example, between 7 and 14 days), subjects underwent an SD-OCT study to confirm their responsiveness to anti-VEGF therapy prior to administration of AAV2.7m8-aflibercept. Anti-VEGF responsiveness was confirmed by the principal investigator. Only subjects who demonstrated a meaningful anti-VEGF response as described above (see inclusion criteria) were eligible to enroll in this trial.

[0235] Subjects responsive to anti-VEGF therapy were sequentially enrolled in the study cohort and received a single AAV2.7m8-aflibercept IVT injection in the eye being tested. As described above, only one eye was selected as the test eye for the duration of the study. AAV2.7m8 - After aflibercept administration

[0236] Participants returned for clinical evaluation and treatment (if necessary) on days 3 and 8, during weeks 2, 4, 6, and 8, and every four weeks thereafter (i.e., weeks 12, 16, 20, and 24). Safety and efficacy analyses for Cohort 1 were performed at week 24.

[0237] Initiated in week 4, if there was evidence of increased disease activity according to the retreatment criteria (see below), the patient was deemed eligible to receive a rescue injection of aflibercept 2 mg IVT. Resumption of standard anti-VEGF treatment with 2 mg IVT aflibercept was justified by the presence of any one of the following: • A decrease of ≥10 letters from baseline in BCVA (using the ETDRS protocol) and intraretinal or subretinal fluid observed by SD-OCT and judged by the principal investigator to be the cause of the BCVA decrease. • As assessed by SD-OCT, an increase of >75 μm from baseline in central retinal thickness. • The presence of vision-threatening bleeding caused by macular degeneration.

[0238] The subjects will be re-evaluated for safety and efficacy at week 52 after administration of AAV2.7m8-aflibercept. The follow-up period will continue until week 104 after administration of AAV2.7m8-aflibercept. C. Corticosteroid regimen

[0239] To reduce the risk of potential post-injection eye inflammation, subjects were administered a prophylactic corticosteroid regimen (e.g., prednisone), and the ocular and systemic tolerability of the vector was closely monitored.

[0240] The subjects of Cohort 1 received a prophylactic 13-day oral corticosteroid regimen. Treatment was initiated with 60 mg / day of prednisone for a total of 6 days, from 3 days before (-3 days) to 3 days after treatment with AAV2.7m8-aflibercept. This was followed by a 7-day tapering of prednisone. A summary of the oral prednisone regimen is shown in Table 2. [Table 2]

[0241] The initiation of immunosuppression (i.e., prednisone) prior to IVT injection of AAV2.7m8-aflibercept was designed to limit the immune response upon exposure to the capsid antigen. Participants self-administered prednisone over a 13-day regimen.

[0242] Participants received topical or oral corticosteroids (prednisone) from week 2 to week 24 of the trial, as needed. D. Prohibited Substances and Treatments

[0243] The following drugs were prohibited during the trial: • All systemic anti-VEGF agents, including bevacizumab. • Any anti-VEGF agent administered to the eye being tested, other than the study drug or aflibercept 2 mg injection in accordance with the rescue anti-VEGF injection criteria for this study. • IVT steroids applied to the eye being tested. • Immunosuppressants. Systemic, inhaled, or topical steroids and NSAIDs were acceptable. • Use of and participation in any other research and investigational reagents. Cataract surgery in the eye being tested may be performed if clinically required and scheduled more than 90 days after IVT administration and / or more than 7 days after the last injection of aflibercept. • Patients with AMD in the eye not being tested can receive standard treatment for the eye not being tested. E. Summary of the study design for Cohort 1

[0244] Six participants were enrolled in Cohort 1.

[0245] In Cohort 1, participants received 6 × 10⁶ doses of AAV2.7m8-aflibercept. 11 The drug was administered via a single in vitro injection at a dose of 1 / vg / ocular. The first (sentinel) subject enrolled in Cohort 1 received an in vitro injection of AAV2.7m8-aflibercept and was evaluated for 29 days before the drug was administered to the next five subjects (subjects 2-6) within the cohort.

[0246] A summary of the study design for Cohort 1 is presented in Table 3. [Table 3] F. Test duration

[0247] The duration of participation in the study for each participant is approximately 108 weeks. This includes a 4-week screening period and an additional 104-week study period.

[0248] Once the trial is completed or discontinued, participants will be offered the opportunity to enroll in a long-term follow-up trial to further evaluate the safety of this gene therapy, if necessary. V. Test Evaluation A. General physical examination and vital signs

[0249] Relevant medical and ophthalmic history was collected and recorded for each subject. A general physical examination consisted of height (only at screening), weight, and vital signs.

[0250] Vital signs included blood pressure, pulse rate, body temperature, and respiratory rate. A 12-lead electrocardiogram (ECG) was obtained for each subject. The following clinical and antibody tests were performed: chemical tests, complete blood count (CBC), coagulation tests, urinalysis, serological evidence for HIV or hepatitis, and pregnancy tests.

[0251] At week 104, at the end of the study (EOS), and / or at the early termination visit, a physical examination will be performed to assess whether there have been any changes in the subject's health status since the screening examination. B. Immune response and aflibercept expression

[0252] Total anti-AAV2.7m8 antibodies were measured. Neutralizing anti-AAV2.7m8 antibodies in the serum of the subjects were determined using a transduction interference assay based on a reporter gene evaluated by a cutpoint.

[0253] The humoral immune response to anti-aflibercept antibodies was measured in serum using an ELISA-based cutpoint antibody assay.

[0254] Serum samples were collected to determine the presence of aflibercept protein.

[0255] Cellular immunity against AAV2.7m8 capsid protein and aflibercept protein was measured using the ELISPOT assay. C. Comprehensive ophthalmological examination and other evaluation methods

[0256] The evaluation included ophthalmological examinations, intraocular pressure (IOP), and indirect optometry.

[0257] The ophthalmic examination consisted of external examination of the eye and adnexa, routine screening for eyelid / pupil responsiveness (including, but not limited to, ptosis, abnormal pupillary shape, pupillary inequality, abnormal response to light, and concentric pupillary disorder), and slit-lamp examination (eyelids, conjunctiva, cornea, lens, iris, and anterior chamber). Slit-lamp examination was used to examine the anterior structures of the eye and to grade any findings. If any findings were observed during slit-lamp examination at any given visit, their severity was graded by the principal investigator and documented as clinically significant or clinically insignificant.

[0258] IOP measurements were performed using a Goldmann applanation tonometer or Tono-pen™. IOP measurements were performed before every IVT injection and before dilating the eye. On the first day visit, IOP measurements were required before and after injection (30 minutes after injection).

[0259] Dilated indirect ophthalmoscopic examinations included evaluation of abnormalities in the vitreous humor, optic nerve, peripheral retina, and the posterior segment of the retinal vascular structure. If any findings were observed during ophthalmoscopic examination at any visit, their severity was graded by the principal investigator and documented as clinically significant or insignificant. Pre- and post-injection indirect ophthalmoscopic evaluations were required at the first visit. Spectral Domain Optical Coherence Tomography (SD-OCT)

[0260] Using SD-OCT, depth-resolved tissue structure information, encoded in the magnitude and delay of backscattered light, was obtained by spectral analysis of interference fringe patterns. Fluorescein angiography

[0261] To evaluate the effectiveness of CNV lesion growth and to assess leakage compared to baseline, fluorescein angiography images were used to confirm patient eligibility for trial enrollment. Digital color fundus photography

[0262] Color fundus images of the retina, optic nerve head, and macula were acquired. Optical coherence tomography (OCT-A)

[0263] OCT-A imaging (wavelength sweep or spectral domain) was used to obtain three-dimensional volume maps of the retina and choroid, as well as information on blood flow. Refraction and visual acuity

[0264] Refraction and BCVA were measured by trained and certified visual acuity testers at the testing site. Visual acuity was measured at a starting distance of 4 meters before dilation of the eyes. D. Safety Evaluation

[0265] 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 the procedure.

[0266] The safety of AAV2.7m8-aflibercept was evaluated by collecting adverse events (AEs), vital signs, physical and ophthalmic examinations, ECG, pregnancy tests, and laboratory assessments.

[0267] Intensive monitoring of participants was conducted during the first eight weeks of the trial, followed by a standard safety assessment regarding safety and efficacy. All participants underwent visual acuity testing using the Early Treatment Diabetic Retinopathy Study (ETDRS) letter assessment at each trial visit, and aflibercept IVT injection, the standard treatment, was used as a rescue measure.

[0268] The severity or intensity of the AE was assessed using the following scale: • Mild cases: While the adverse events (AEs) were noteworthy, they did not significantly impair the subjects' daily activities. • Moderate: AE reduced or impaired normal daily activities, but did not result in incapacitation. • Severe: The patient becomes incapacitated due to AE (explosive adverse event) and is unable to perform normal daily activities.

[0269] Safety will be evaluated over 104 weeks following administration of the test treatment. Upon completion of the End of Study (EOS) visit, participants will be invited to enroll in a long-term extension study to further evaluate the safety and durability of transgene expression. E. Evaluation of effectiveness

[0270] The efficacy of AAV2.7m8-aflibercept for the treatment of wAMD was evaluated using the following criteria. The critical evaluation time point was 24 weeks. Baseline values ​​for BCVA and SD-OCT refer to values ​​obtained before aflibercept injection during screening visits between days -15 and -7 (e.g., days -14 and -7). Baseline values ​​were used for comparison in the analysis.

[0271] Vision was primarily assessed by BCVA, expressed as the ETDRS score (number of characters accurately read) (Vitale et al., (2016) JAMA Ophtalmol 134 (9): 1041-1047). Participants were classified as having maintained vision if they had a decrease of less than 15 characters in their ETDRS score compared to baseline. Calculated endpoints included the mean change from baseline, the percentage of participants with an increase of at least 15 characters compared to baseline, and the percentage of participants with a decrease of 15 characters or more compared to baseline.

[0272] FA was performed, and CNV lesions were evaluated using standard techniques to assess leakage compared to baseline.

[0273] SD-OCT was performed using approved equipment and standard techniques to assess retinal thickness (e.g., foveal retinal thickness or central region retinal thickness), macular volume, and the presence of fluid (e.g., subretinal and intraretinal fluid) compared to baseline values.

[0274] We determined the number of aflibercept injections administered per subject after AAV2.7m8-aflibercept treatment over time from week 4 to week 104. Furthermore, we determined the time from AAV2.7m8-aflibercept treatment to the first aflibercept injection and the percentage of subjects who did not require aflibercept rescue treatment.

[0275] We determined the proportion of subjects without an IRF over time from week 4 to week 104.

[0276] The proportion of subjects without SRF over time from week 4 to week 104 was determined.

[0277] We determined the proportion of subjects who did not have PED during the time course from week 4 to week 104 among those who had PED at baseline. F. Statistical values

[0278] The safety analysis population included all subjects who received AAV2.7m8-aflibercept and were analyzed according to the dose they received.

[0279] All other safety parameters were summarized for each cohort. AEs were coded using the MedDRA (Medical Drug Regulatory Terminology, version 21) classification, with each event assigned a basic term (PT) and a systemic category (SOC). SAEs and AEs leading to study withdrawal were listed separately.

[0280] The efficacy analysis included all participants. Efficacy endpoints were evaluated, and descriptive statistics were calculated for each cohort. The critical evaluation time point was 24 weeks. Efficacy was assessed according to the dose received and overall. VI.Results A. Characteristics of the subject

[0281] All six subjects enrolled in Cohort 1 were diagnosed with wAMD. At enrollment, the subjects had a significant need for anti-VEGF treatment (e.g., required frequent anti-VEGF treatment), functional vision of approximately 20 / 50, some excessive central retinal thickness on OCT, and were receiving regular IVT injections of anti-VEGF treatment and were responding to treatment. The disease characteristics and treatment history of all subjects are presented in Tables 4-5.

[0282] As shown in Table 4, subjects in Cohort 1 were diagnosed with wAMD between approximately one year prior to administration of AAV2.7m8-aflibercept (Subject 5) and approximately 10 years prior or earlier (Subject 4). Subjects had received extensive prior anti-VEGF IVT injections in the eyes being studied from 7 (Subject 5) to 109 (Subject 4) prior to injection. The calculated mean anti-VEGF IVT injection intervals prior to this study ranged from every 4 weeks to every 10 weeks. All subjects had received two or three anti-VEGF injections in the 4 months prior to screening for enrollment in this study. Subjects were administered AAV2.7m8-aflibercept 7 days (Subjects 1-3) or 14 days (Subjects 4-6) after the pre-study screening anti-VEGF injection. [Table 4] [Table 5] B. Safety

[0283] During the 24-week period following administration of AAV2.7m8-aflibercept, no SAEs occurred, and no AEs meeting the criteria for dose-limiting toxicity (DLT) occurred. No drug-related non-ocular AEs were observed. Ocular inflammation was observed in all subjects and was manageable with topical steroids. Furthermore, no vasculitis, retinitis, or choroiditis was observed. Nineteen ocular AEs that appeared to be potentially related to AAV2.7m8-aflibercept were observed, 14 of which were mild and 5 were moderate (2 AEs were intermediate uveitis, 1 was vitreous cell AE, and 2 were anterior chamber cell AE). One patient had two anterior chamber cell events (1 mild and 1 moderate). Mild to moderate intraocular inflammation responsive to topical or oral corticosteroids was frequently observed during initial follow-up. OCT images showed the dissipation of residual fluid in the majority of subjects, and no signs of worsening were observed. Visual acuity was generally stable. A summary of all safety events associated with AAV2.7m8-aflibercept over the 24-week period is presented in Table 6. [Table 6]

[0284] All subjects received oral prednisone 60 mg starting on day 3 for 6 days, followed by a 7-day tapering course. Clinical evaluation of ocular cellular inflammation revealed that no clinically significant inflammation occurred in the early post-AAV2.7m8-aflibercept period. Furthermore, as shown in Figure 6, no exacerbation or new inflammation was observed when subjects received steroid eye drops. Anterior chamber cellular inflammation resolved or improved by week 24. Observed cellular inflammation was generally mild. The aqueous humor cell count categories shown in Figure 6 were based on the Standardization of Uveitis Nomenclature (SUN) criteria (Jabs, DA et al., J Ophthalmol. 2005; 140: 509-516), while the vitreous cell count categories were based on the National Institutes of Health (NIH) guidelines.

[0285] Overall, the safety assessment showed that AAV2.7m8-aflibercept was well-tolerated, with no DLTs or SAEs reported. All safety events were mild to moderate, and the majority were inflammation-related. Continued follow-up of AEs showed that mean visual acuity remained stable, and rescue injections with anti-VEGF were not required. Therefore, the results suggest that 6 × 10⁶ doses should be used in wAMD patients who previously required frequent anti-VEGF injections. 11 The safety profile of AAV2.7m8-aflibercept administered as a single IVT injection at a dose of vg / eye is shown to be acceptable. C. Effectiveness

[0286] Following administration of AAV2.7m8-aflibercept, disease activity stabilization was demonstrated in all six subjects based on OCT evaluation. As shown in Figures 2A-2L, OCT images obtained before and after treatment with AAV2.7m8-aflibercept revealed robust anatomical responses in all six subjects in Cohort 1. For example, in Subject 1 (e.g., compare Figures 2A and 2B), Subject 2 (e.g., compare Figures 2B and 2C), Subject 3 (e.g., compare Figures 2D and 2E), Subject 4 (e.g., compare Figures 2F and 2G), and Subject 5 (e.g., compare Figures 2H and 2I), subretinal fluid persisted after standard anti-VEGF treatment, but it dissipated and remained dissipated after administration of AAV2.7m8-aflibercept. Subject 6 exhibited retinal morphology consistent with polypoidal choroidal vasculopathy (PCV) (Figure 2K), and although some fluid remained after treatment with AAV2.7m8-aflibercept, there was no evidence of disease progression (Figure 2L). Furthermore, as shown in Figure 3, no subjects showed an increase in central retinal thickness (CST), and a mean decrease of -52.7 μm was observed (90% CI -86.5, -18.8). An increase in CST indicates disease progression in wAMD. Importantly, none of the subjects received, nor required, a rescue injection of aflibercept, which is standard treatment.

[0287] Best corrected visual acuity (BCVA) was measured for all participants throughout the study based on ETDRS letter evaluation. As shown in Figure 4, BCVA remained stable across the cohort 1 participants, with a mean decrease of -2 letters (90% CI -9.1, 5.1).

[0288] At 34 weeks after administration of AAV2.7m8-aflibercept, the median follow-up time, no subjects showed signs of disease reactivation on OCT imaging (Figures 7A-7B). Furthermore, no subjects required any rescue anti-VEGF IVT injections, and no subjects met the criteria for retreatment at any point during the maximum 44-week follow-up period. Finally, BCVA was maintained during the additional follow-up period beyond 24 weeks (i.e., no patients had more than 10 ETDRS letters of decrease or increase), the anatomical improvements observed at week 24 (i.e., dissipation of subretinal and intraretinal fluid, as well as reduction in CST) were maintained, and no safety concerns arose.

[0289] Table 7 presents a summary of the safety and efficacy results evaluated at week 34, the median follow-up time. [Table 7] D. Conclusion

[0290] The current standard of care for wAMD is typically anti-VEGF IVT injections, required approximately every 4–8 weeks over a long period. Compliance with this regimen can be challenging for patients, caregivers, and healthcare systems, leading to vision loss due to suboptimal medication and inadequate treatment.

[0291] The results presented in this example demonstrate that AAV2.7m8-aflibercept provides a clear benefit in terms of improving retinal anatomical structure and stabilizing vision, while exhibiting an acceptable safety profile. In particular, patient vision was maintained throughout the study, demonstrating that AAV2.7m8-aflibercept is safe and well-tolerated, and the observed inflammation was generally mild and responsive to steroid eye drops.

[0292] The subjects in Cohort 1 of this study had previously required frequent anti-VEGF injections to slow or prevent disease progression (Table 4), but during this study, they did not require rescue injections with anti-VEGF after administration of AAV2.7m8-aflibercept.

[0293] In subjects receiving standard anti-VEGF treatment for longer than 20 weeks, OCT scans revealed the presence of subretinal fluid, which persisted 1–2 weeks after screening IVT injections of aflibercept. This fluid remained despite repeated treatments and was therefore refractory to the standard anti-VEGF protein IVT bolus. Unexpectedly, this refractory subretinal fluid (SRF) resolved after treatment with AAV2.7m8-aflibercept, a result not predicted from preclinical studies.

[0294] 6×10 11 The observed safety and efficacy of AAV2.7M8-aflibercept administered at a dose of vg / eye led to the continuation of this Phase 1 trial to evaluate the safety and efficacy of AAV2.7M8-aflibercept administered at a low dose with a topical corticosteroid, as described in Example 2.

[0295] (Example 2) For neovascular (exudative) age-related macular degeneration, 6 × 10 11 An open-label phase 1 trial of AAV2.7m8-aflibercept at a lower dose than vg / ocular and topical corticosteroids. In the following examples, subjects with wAMD were treated with topical corticosteroids, 6 × 10 11 This document describes the continuation of the Phase 1 trial described in Example 1 to evaluate the safety and efficacy of AAV2.7m8-aflibercept administered at a lower dose than vg / ocular. I. Study Objectives and Endpoints

[0296] The primary objectives, secondary objectives, and primary and secondary endpoints are as described in Section I of Example 1. II. Subjects of the Examination

[0297] The subjects of this study are as described in Section II of Example 1. Search for pharmaceuticals

[0298] The drug used in the investigation was AAV2.7m8-aflibercept, and the details are described in Section III of Example 1, as shown in Figure 1A, with the exception that the concentration of AAV2.7m8-aflibercept was varied to maintain an appropriate injection dose, as shown in Table 8 below. [Table 8] IV. Test Design A. Dosage and method of administration

[0299] AAV2.7m8-aflibercept was administered in the following doses as described in Section IV of Example 1. Study cohorts 2-4

[0300] Cohort 2: Six individuals diagnosed with wAMD will be enrolled in Cohort 2. These individuals will receive a single IVT injection of AAV2.7m8-aflibercept at dose 2, 2 × 10⁶ times. 11 It is administered via VG / ocular injection, accompanied by a prophylactic oral prednisone regimen.

[0301] Cohort 3: Nine individuals diagnosed with wAMD will be enrolled in Cohort 3. Participants in Cohort 3 will receive a single IVT injection of AAV2.7m8-aflibercept at dose 2, 2 x 10⁶ times. 11 It is administered via VG / ocular injection and accompanied by a prophylactic topical corticosteroid regimen.

[0302] Cohort 4: Nine subjects diagnosed with wAMD will be enrolled in Cohort 4. If signs of choroidal neovascular exudation requiring rescue treatment are observed in the majority of subjects in Cohorts 2 and 3, subjects in Cohort 4 will receive a single IVT injection of AAV2.7m8-aflibercept at dose 3, 6 × 10 times. 10 Administer vg / eye with a topical corticosteroid regimen (Cohort 4b). If the majority of subjects in Cohorts 2 and 3 do not show signs of choroidal neovascular exudation requiring rescue treatment, subjects in Cohort 4 will receive a single IVT injection of AAV2.7m8-aflibercept at dose 1, 6 × 10⁶ times. 11 Administered via VG / ocular injection, accompanied by a topical corticosteroid regimen (Cohort 4a). B. Visiting the examination site

[0303] The test visit was conducted as described in Section IV and Figure 1B of Example 1. C. Corticosteroid regimen

[0304] As described in detail for the cohort in Example 1, subjects in Cohort 2 will be administered a prophylactic 13-day oral corticosteroid regimen:

[0305] Cohort 2: The subjects of Cohort 2 received a single IVT injection of AAV2.7m8-aflibercept at dose 2, 2 × 10⁶ times. 11 The regimen is administered VG / eye, and a prophylactic 13-day oral corticosteroid regimen is started with 60 mg of prednisone and administered for a total of 6 days: 3 days before and 3 days after treatment with AAV2.7m8-aflibercept. This is followed by a 7-day tapering of prednisone. A summary of the oral prednisone regimen for Cohort 2 is presented in Table 2 of Example 1.

[0306] Participants in cohorts 3 and 4 will receive the following prophylactic tapering regimen of topical corticosteroids (0.05% difluprednate eye drops):

[0307] Cohort 3: The subjects of C...

Claims

1. A composition comprising rAAV particles for use in a method for reducing retinal fluid in the eye of an individual with intraocular neovascular disease, wherein the method is about 6 × 10 １１ The procedure comprises the step of administering the composition to one eye of the individual in a unit dose of a vector genome (vg) or less than a vector genome of rAAV particles, wherein the individual is human and the rAAV particles are a) A nucleic acid encoding a polypeptide having an amino acid sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO: 35, and having AAV2 terminal inverse sequences (ITRs) at both ends, and b) An AAV2 capsid protein containing the amino acid sequence LGETTRP (SEQ ID NO: 14) inserted between positions 587 and 588 of the capsid protein, wherein the amino acid residue numbering corresponds to that of the AAV2 VP1 capsid protein, A composition wherein the retinal fluid of one eye is reduced by about 60% compared to the level of retinal fluid of one eye of the individual before administration of the rAAV to the individual.

2. The composition according to claim 1, wherein the individual has received at least one treatment with an anti-VEGF agent about 12 weeks prior to the administration of the composition.

3. The composition according to claim 1 or 2, wherein the amount or presence of retinal fluid in one eye of the individual is refractory to prior treatment with an anti-VEGF agent.

4. The composition according to claim 2 or 3, wherein the anti-VEGF agent is aflibercept.

5. The composition according to any one of claims 1 to 4, wherein the retinal fluid of one eye is reduced by about 80% compared to the level of retinal fluid of one eye of the individual before administration of the rAAV to the individual.

6. The composition according to any one of claims 1 to 5, wherein the retinal fluid is subretinal fluid (SRF) or intraretinal fluid (IRF).

7. The unit dose of rAAV particles is approximately 6 × 10 １１ The composition according to any one of claims 1 to 6, wherein the vector genome / eye (vg / eye) is less than or equal to a vector genome / eye.

8. The unit dose of rAAV particles is approximately 6 × 10 １０ ~Approx. 2×10 １１ The composition according to any one of claims 1 to 7, wherein the vector genome / eye (vg / eye).

9. The unit dose of rAAV particles is approximately 2 × 10 １１ Or approximately 6 x 10 １０ The composition according to any one of claims 1 to 8, wherein the vector genome / eye (vg / eye).

10. The composition according to any one of claims 1 to 9, further comprising the step of administering the composition to the eye opposite the individual in a unit dose of rAAV particles.

11. The composition according to claim 10, wherein the step of administering the composition to the opposite eye occurs at most about two weeks after the step of administering the composition to the one eye.

12. The composition according to claim 11, wherein the unit dose of rAAV particles administered to the opposite eye of the individual comprises the same or a smaller vector genome / eye (vg / eye) as the unit dose of rAAV particles administered to the one eye of the individual.

13. The composition according to claim 10, wherein the step of administering the composition to the opposite eye is performed at least about two weeks after the step of administering the composition to the one eye.

14. The composition according to claim 13, wherein the unit dose of rAAV particles administered to the opposite eye of the individual contains more vector genomes / eye (vg / eye) than the unit dose of rAAV particles administered to one eye of the individual.

15. The composition according to any one of claims 1 to 14, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO:

35.

16. The composition according to any one of claims 1 to 15, wherein the polypeptide is aflibercept.

17. The composition according to any one of claims 1 to 16, 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.

18. The nucleic acids are arranged in the order from 5' to 3': (a) First enhancer region; (b) Promoter region; (c) 5'UTR region; (d) A nucleic acid encoding a polypeptide having an amino acid sequence that is at least about 95% identical to the amino acid sequence of Sequence ID No. 35; (e) Second enhancer region; and (f) Polyadenylated sites; The composition according to any one of claims 1 to 17, comprising and having an AAV2 terminal inversion sequence (ITR) at both ends.

19. The composition according to claim 17 or 18, wherein the first enhancer region comprises a CMV sequence including the sequence of sequence number 22.

20. The composition according to any one of claims 17 to 19, wherein the promoter region comprises a CMV sequence containing the sequence of sequence number 23.

21. The composition according to any one of claims 17 to 20, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO:

35.

22. The composition according to any one of claims 17 to 21, wherein the polypeptide is aflibercept.

23. The composition according to any one of claims 17 to 22, wherein the 5'UTR region includes, in order from 5' to 3', a TPL sequence containing the sequence of sequence number 24 and an eMLP sequence containing the sequence of sequence number 25.

24. The composition according to any one of claims 17 to 23, wherein the second enhancer region comprises a full-length EES sequence including the sequence of sequence number 26.

25. The composition according to any one of claims 17 to 24, wherein the polyadenylated site comprises an HGH polyadenylated site containing the sequence of Sequence ID No.

27.

26. The composition according to any one of claims 1 to 16, wherein the nucleic acid further comprises: (a) a first enhancer region comprising a CMV sequence comprising the sequence of SEQ ID NO: 22; (b) a promoter region comprising a CMV sequence comprising the sequence of SEQ ID NO: 23; (c) a 5'UTR region comprising a TPL sequence comprising the sequence of SEQ ID NO: 24 and an eMLP sequence comprising the sequence of SEQ ID NO: 25, in the order of 5' to 3'; (d) a second enhancer region comprising a full-length EES sequence comprising the sequence of SEQ ID NO: 26; and (e) an HGH polyadenylation site comprising the sequence of SEQ ID NO:

27.

27. The composition according to any one of claims 1 to 26, wherein the AAV2 capsid protein includes the amino acid sequence LALGETTRPA (SEQ ID NO: 1) inserted between positions 587 and 588 of the capsid protein, and the amino acid residue numbering corresponds to the AAV2 VP1 capsid protein.

28. The composition according to any one of claims 1 to 27, wherein the AAV2 capsid protein comprises the amino acid sequence LGETTRP (sequence number 14) inserted between positions 587 and 588 of AAV2 VP1 containing the sequence of sequence number 13.

29. The composition according to any one of claims 1 to 28, wherein the AAV2 capsid protein comprises the amino acid sequence LALGETTRPA (SEQ ID NO: 1) inserted between positions 587 and 588 of AAV2 VP1 containing the sequence of SEQ ID NO:

13.

30. The composition according to any one of claims 1 to 29, wherein the administration of the composition to the one eye and / or the opposite eye is by intravitreous administration.

31. The composition according to any one of claims 1 to 30, wherein the composition is present in a pharmaceutical preparation.

32. The composition according to claim 31, wherein the pharmaceutical preparation comprises the rAAV particles, sodium chloride, sodium phosphate, and a surfactant.

33. The aforementioned pharmaceutical preparation contains approximately 150 to approximately 200 mM sodium chloride, approximately 1 to approximately 10 mM sodium dihydrogen phosphate, approximately 1 to approximately 10 mM sodium hydrogen phosphate, approximately 0.0005% (weight / volume) to approximately 0.005% (weight / volume) poloxamer 188, and approximately 6 × 10 １３ ~Approx. 6×10 １０ The composition according to claim 32, comprising the vector genome (vg) / mL (vg / mL) rAAV particles, wherein the pharmaceutical formulation has a pH of about 7.0 to about 7.

5.

34. The pharmaceutical preparation contains about 180 mM sodium chloride, about 5 mM sodium dihydrogen phosphate, about 5 mM disodium hydrogen phosphate, about 6 × 10 １２ vg / mL of the rAAV particles, and about 0.001% (weight / volume) poloxamer 188, and the pharmaceutical preparation has a pH of about 7.3, the composition according to claim 33.

35. The aforementioned pharmaceutical preparation contains approximately 180 mM sodium chloride, approximately 5 mM sodium dihydrogen phosphate, approximately 5 mM sodium hydrogen phosphate, and approximately 6 × 10 １１ The composition according to claim 33, comprising vg / mL of rAAV particles and about 0.001% (weight / volume) of poloxamer 188, wherein the pharmaceutical formulation has a pH of about 7.

3.

36. The composition according to any one of claims 1 to 35, wherein the unit dose of rAAV particles comprises a volume of about 25 μL to about 250 μL.

37. The composition according to claim 36, wherein the unit dose of rAAV particles contains a volume of about 100 μL.

38. The composition according to claim 36, wherein the unit dose of rAAV particles contains a volume of about 30 μL.

39. The composition according to any one of claims 1 to 38, wherein the individual has received prior treatment for the intraocular neovascular disease with an anti-VEGF agent.

40. The composition according to claim 39, wherein the anti-VEGF agent is aflibercept.

41. The composition according to any one of claims 1 to 40, wherein the intraocular neovascular disease is exudative age-related macular degeneration (AMD), retinal neovascularization, choroidal neovascularization, diabetic retinopathy, proliferative diabetic retinopathy, retinal vein occlusion, central retinal vein occlusion, branch retinal vein occlusion, diabetic macular edema, diabetic retinal ischemia, ischemic retinopathy, diabetic retinal edema, or any combination thereof.

42. The composition according to any one of claims 1 to 41, wherein the composition is administered in combination with a steroid treatment.

43. The composition according to claim 42, wherein the steroid treatment is a corticosteroid treatment.

44. The composition according to claim 42 or 43, wherein the steroid treatment is a systemic steroid treatment.

45. The composition according to any one of claims 42 to 44, wherein the steroid treatment is an oral steroid treatment.

46. The composition according to any one of claims 42 to 45, wherein the steroid treatment is a prednisone treatment.

47. The composition according to claim 42 or 43, wherein the steroid treatment is a topical steroid treatment.

48. The composition according to claim 47, wherein the steroid treatment is difluprednate treatment.

49. The composition according to any one of claims 42 to 48, wherein the steroid is administered before, during, and / or after administration of the composition.

50. The composition according to any one of claims 47 to 49, wherein the steroid treatment is a topical steroid treatment, and the topical steroid treatment is a daily steroid treatment for up to about 4 weeks, up to about 6 weeks, or up to about 8 weeks from the administration of the composition.

51. The composition according to claim 50, wherein the topical steroid treatment comprises approximately four administrations of topical steroids in approximately one week, approximately three administrations of topical steroids in approximately two weeks, approximately two administrations of topical steroids in approximately three weeks, and approximately one administration of topical steroids in approximately four weeks, the timing of which begins with and ends with the administration of the composition.

52. The composition according to claim 51, wherein the topical steroid comprises about 1 μg to about 3 μg of 0.05% difluprednate.

53. The composition according to claim 51, wherein the topical steroid comprises approximately 2.5 μg of 0.05% difluprednate.

54. The composition according to any one of claims 1 to 53, wherein the step of administering the composition to one eye and / or the opposite eye of the individual results in maintenance or reduction of retinal thickness compared to the retinal thickness before administration of the composition.

55. The composition according to claim 54, wherein the step of administering the composition to one eye and / or the opposite eye of the individual results in a reduction in retinal thickness compared to the retinal thickness before administration of the composition.

56. The composition according to claim 54 or 55, wherein the reduction in retinal thickness is at least about 10% compared to the retinal thickness before administration of the composition.

57. The composition according to any one of claims 54 to 56, wherein the retinal thickness is the central region retinal thickness (CST) or the foveal retinal thickness (CRT).

58. The composition according to any one of claims 1 to 57, wherein the step of administering the composition to one eye and / or the opposite eye of the individual results in maintenance or reduction of macular volume compared to the macular volume before administration of the composition.

59. The composition according to claim 58, wherein the step of administering the composition to one eye and / or the opposite eye of the individual results in a reduction in macular volume compared to the macular volume before administration of the composition.

60. The composition according to claim 59, wherein the reduction in macular volume is at least about 10% compared to the macular volume before administration of the composition.

61. The composition according to any one of claims 1 to 60, wherein the step of administering the composition to one eye and / or the opposite eye of the individual results in maintenance or improvement of visual acuity compared to visual acuity before administration of the composition.

62. The composition according to any one of claims 1 to 61, wherein the step of administering the composition to one eye and / or the opposite eye of the individual results in an improvement in visual acuity compared to the visual acuity before administration of the composition.

63. The composition according to any one of claims 61 or 62, wherein the visual acuity is the best corrected visual acuity (BCVA).

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