Methods for treating wet age-related macular degeneration with adeno-associated virus

Abstract

Pharmaceutical compositions comprising adeno-associated virus (AAV) and their use in treating wet age-related macular degeneration are provided herein. In some aspects, treatment methods encompass co-administration of one or more corticosteroids and / or supplemental administrations of aflibercept protein.

Description

Methods for Treatise Wet Age*Reiated Macalar Deg^eration with Adeno- Associated VirusCROSS REFERENCE TO RELATED APPLICATIONS pool] This application claims the benefit of U.S. Provisional Patent Application Serial Nos., 63 / 671,894, filed July 16, 2024, 63 / 736,368, filed December 19, 2024, 63 / 744,636, filed January 13, 2025 and 63 / 751,673, filed January 30, 2025, the entire contents of each of which are incorporated herein by reference.SEQUENCE LISTING SUBMISSION VIA EFS-WEB

[0002] A computer readable XML file, entitled “090400-5029 WO Sequence Listing” created on July 14, 2025, with a file size of 18,709 bytes contains the sequence listing for this application and is hereby incorporated by reference in its entirety.BACKGROUND OF THE INVENTION

[0003] Age-related macular degeneration (AMD) is a degenerative ocular disease affecting the macula, a light sensitive, small area in the center of the retina that is responsible for reading and fine vision. Conditions affecting the macula reduce central vision while leaving peripheral vision intact. In severe cases, the disease can lead to central blindness.Neovascular or exudative or wet AMD is an advanced form of AMD. The hallmark of wet AMD is choroidal neovascularization (CNV), which is the infiltration of abnormal blood vessels in the retina from the underlying choroid layer, resulting in retinal cell damage and central blindness. This abnormal angiogenic process is modulated by growth factors, in particular, vascular endothelial growth factor (VEGF). The standard of care of wet AMD is a class of molecules that bind to and sequester VEGF, such as aflibercept (Eylea).

[0004] Aflibercept is a recombinant fusion protein that acts as a decoy receptor for vascular endothelial growth factor subtypes A and B (VEGF-A and VEGF-B) and placental growth factor (PGF). By binding to these ligands, aflibercept is able to prevent them from binding to vascular endothelial growth factor receptors (VEGFR), VEGFR-1 and VEGFR-2, to suppress neovascularization and decrease vascular permeability. Aflibercept consists of domain 2 of VEGFR-1 and domain 3 of VEGFR-2 fused with the Fc fragment of IgGl.

[0005] Aflibercept needs to be readministered via intravitreal (IVT) injection every 4 to 8 weeks to achieve optimal therapeutic outcomes and maintain visual acuity. Compliance with such a regimen is burdensome to patients, their caregivers, and the healthcare system, and most patients fall out of compliance with the optimal regimen over time, which is correlated with vision loss. In addition, there are complications including endophthalmitis, retinal detachments, traumatic cataract, and elevated intraocular pressure (TOP); the risks of these complications are likely to increase with repeated IVT injections.

[0006] Therefore, there is a need in the art for therapies for ocular neovascular diseases such as wet AMD, that are effective, reduce the risk of adverse effects, and are amenable to high long-term patient compliance.SUMMARY OF THE INVENTION

[0007] Described herein are methods for treating wet (neovascular) Age-Related Macular Degeneration (wet AMD) with improved efficacy and / or reduced adverse side effects.

[0008] In some embodiments, the present disclosure provides methods for treating wet AMD comprising (i) intraocularly administering to an eye of the subject a single unit dose of a first pharmaceutical composition comprising a recombinant adeno-associated virus (rAAV) comprising capsid of serotype 2 or a variant thereof and a heterologous nucleic acid encoding one or more anti-VEGF agents operably linked to a promoter, wherein the unit dose comprises about IMO10vector genomes per eye to about 6xlOluvector genomes per eye, preferably wherein the unit dose comprises about 1 xJ 0!Ovector genomes per eye or about 3 x 1010vector genomes per eye, and (ii) topically administering prednisolone acetate or a corticosteroid equivalent to prednisolone acetate to the eye of the subject before, simultaneous with and / or after administration of the first pharmaceutical composition, and administering triamcinolone acetonide or a corticosteroid equivalent to triamcinolone acetonide to the subject by subtenon injection.

[0009] In other embodiments, a method of treating wet AMD in a subject is provided, the method comprising (i) intraocularly administering to a subject a single unit dose of a first pharmaceutical composition comprising a recombinant adeno-associated virus (rAAV) comprising capsid of serotype 2 or a variant thereof and a heterologous nucleic acid encoding one or more anti-VEGF agents operably linked to a promoter, wherein the unit dose comprises about 1 x 1 O10vector genomes per eye to about 6* 10wvector genomes per eye,preferably wherein the unit dose comprises about 1 IO10vector genomes per eye or about 3 * 1 O’0vector genomes per eye, and (ii) administering an intravitreal implant comprising a corticosteroid to the eye of the subject before, simultaneous with or after administration of the first pharmaceutical composition, wherein the corticosteroid is administered by intravitreal implant at least once prior to administering the first pharmaceutical composition and at least once after administering the first pharmaceutical composition. In preferred embodiments, dexamethasone or an equivalent thereof is administered to the subject by intravitreal implant.

[0010] In other embodiments, a method for treating a subject with wet AMD is provided comprising (i) intraocularly administering to an eye of the subject a single unit dose of a first pharmaceutical composition comprising an rAAV comprising a capsid protein of serotype 2 or a variant thereof and a heterologous nucleic acid comprising a nucleotide sequence encoding an anti-VEGF gene product operably linked to a promoter, wherein the unit dose comprises about l ><10’10vector genomes per eye to about 6*10!0vector genomes per eye, preferably wherein the unit dose comprises about I > 10: Ovector genomes per eye or about 3* 1 O’0vector genomes per eye, and (ii) administering to the eye of the subject at least one dose of a second pharmaceutical composition comprising aflibercept protein before, simultaneous with and / or after administration of the first pharmaceutical composition. In some aspects, the unit dose comprises about IxlO10, about 1.5xl0i0, about 2xlO10, about 2.5x10!O, about 3x10!0, about 3.5x1010, about 4xlO’1U, about 4.5x10!°, about 5x10i0, about 5.5x10!0or about 6x1010vector genomes per eye.

[0011] In related aspects, a first pharmaceutical composition as herein described comprises an rAAV of serotype 2 or variant thereof and about 5 mM to about 20 mM of a buffering agent, about 100 mM to about 250 mM of a pharmaceutically acceptable salt, about 0.0001% (w / v) to about 0.01% (w / v) of a non-ionic surfactant and preferably has a pH of about 7.0 to about 9.0.

[0012] In some preferred embodiments, the rAAV comprises a heterologous nucleic acid encoding an anti-VEGF gene product and a capsid protein comprising a peptide insertion of from about 7 amino acids to about 20 amino acids (a “heterologous peptide” or “peptide insertion”) in the GH-loop of the capsid protein, preferably in a surface-exposed region of the GH-loop, relative to a corresponding parental AAV capsid protein, hr certain embodiments, the peptide is inserted following any of the amino acids in positions 584-591 in VP1 ofAAV2 or a corresponding position in another AAV serotype (i.e., the insertion site is between amino acids 587 and 588 of VP1 of AAV2 or is between amino acids 588 and 589, between amino acids 584 and 585, between amino acids 585 and 586, between amino acids 586 and 587, between amino acids 590 and 591, or is between amino acids 591 and 592 of AAV2 or the corresponding positions in the capsid protein of another AAV serotype). In certain preferred embodiments, the insertion site is between amino acids 587 and 588 of VP1 of AAV2 or is between amino acids 588 and 589 of AAV2 or the corresponding positions in the capsid protein of another AAV serotype. In some embodiments, the capsid protein further comprises one or more amino acid substitutions relative to VP1 capsid of AAV2 or one or more corresponding substitutions in another AAV serotype, preferably wherein the capsid protein further comprises a P34A amino acid substitution relative to VP1 capsid of AAV2 or the corresponding substitution in another AAV serotype.DESCRIPTION OF THE DRAWINGS

[0013] FIG. 1 illustrates characteristics (mean values) of the Phase 2b population. BCVA: 72 letters; CST; 329 pm; Recently Diagnosed = 50%; Time Since diagnosis: 1.4 years

[0014] FIGS. 2A-B illustrate durable and stable aflibercept concentrations (ng / mL) in the aqueous humor (AH) of AMD patients following a single intravitreal injection of an rAAV comprising a capsid of SEQ ID NO:4 and a nucleic acid of SEQ ID NO:9. Supplemental injection-free subjects with detectable AH aflibercept in Phase l / 2a 3E10 vg / eye dose group. Robust and durable aflibercept expression was observed in these patients during follow' up ranging from 1 to 2 years. 100% of patients positive for expression remain positive and stable for the indicated period.

[0015] FIG. 2C illustrates ocular biodistribution of aflibercept expressed from the rAAV in patients.

[0016] FIG. 3A illustrates CVA and CST in Phase 2b population through week 52. Improved visual acuity and sustained anatomic control were demonstrated. Over 52-weeks of follow-up patients in the Phase 2b trial demonstrated a mean improvement in visual acuity. This was accompanied by sustained anatomic control over the same time period. All patients in the rAAV arm are represented by the solid line, with the subset of supplemental injection- free patients being represented by the dashed line. FIG. 3B illustrates that 57% of thesepatients were able to achieve these resuits without receiving a supplemental injection, with an additional 13% requiring only one supplemental injection translating to 70% of patients needing 0 or 1 injection over the course of a year. After the last loading dose, patients in the 3E10 subgroup received an average of less than one injection per patient over the course of 52-weck follow-up.

[0017] FIG. 4 illustrates that, while there was no control arm in this cohort, compared to a projected standard 2q8 aflibercept arm that would be used in the Phase 3 setting, the number of injections in the in the 3E10 subgroup represents an 83% reduction in treatment burden after the loading period.

[0018] FIG. 5A illustrates CVA and CST in recently diagnosed Phase 2b population (defined as < 6 months prior to date of screening) through week 52. These patients demonstrated a mean improvement in visual acuity over 52 weeks of follow up. This was accompanied by sustained anatomic over the same time period. All patients in the subgroup arm are represented by the solid blue line, with the subset of recently-diagnosed patient who remained supplemental injection-free being represented by the dashed line. FIG 5B illustrates that 80% of these recently diagnosed patients were able to achieve these results without receiving a supplemental injection, with no patient needing more than 2 injections over the course of a year. / After the last loading dose, patients in the recently diagnosed subgroup received an average of point three injections per patient over the course of 52-week followup.

[0019] FIG. 6 illustrates the mean cumulative number of aflibercept injections in recently diagnosed study subjects, A reduction in treatment burden of 94% compared to standard 2q8 aflibercept arm was observed in this group.

[0020] FIG. 7 illustrates aspects of the Phase 3 trial consisting of two Phase 3 studies, one of w'hich will enroll treatment naive patients and the other which will enroll both treatment naive and treatment experienced (diagnosed within 6-months) patients. Briefly, subjects with BCVA of 25-78 letters and responsive to the three loading doses of aflibercept (at Weeks -5, -1 and +4) are administered 3E10 vg / eye of the rAAV. The comparator is aflibercept 2mg Q8 weeks.DETAILED DESCRIPTION OF THE INVENTION

[0021] Definitions

[0022] As used herein, the term “AAV” refers to adeno-associated virus in both naturally occurring and recombinant forms (rAAV), and encompasses mutant forms of AAV. The term AAV further includes, but is not limited to, AAV type 1, AAV type 2, AAV type 3, AAV type 4, AAV type 5, AAV type 6, AAV type 7, AAV type 8, AAV type 9, AAV type 10, avian AAV, bovine AAV, canine AAV, equine AAV, ovine AAV, primate AAV, and nonprimate AAV. In certain embodiments, the AAV is AAV2 or a variant thereof.

[0023] A "pharmaceutically acceptable salt" is a salt that can be formulated into a compound or conjugate for pharmaceutical use including, e.g., metal salts (sodium, potassium, magnesium, calcium, etc.) and salts of ammonia or organic amines that is safe for administration to a subject (e.g., a human) in a drug formulation (see, for example, Berge, et al. “Pharmaceutical Salts,” J. Pharm. Sei.1977; 66:1, which is incorporated herein by reference in its entirety and for all purposes.). Suitable “pharmaceutically acceptable salts” include, but are not limited to, metal salts such as sodium, potassium and cesium salts; alkaline earth metal salts such as calcium and magnesium salts; organic amine salts such as triethylamine, guanidine and N-substituted guanidine salts, acetamidine and N-substituted acetamidine, pyridine, picoline, ethanolamine, triethanolamine, dicyclohexylamine, and N,N'- dibenzylethylenediamine salts. “Pharmaceutically acceptable salts” (of basic nitrogen centers) include, but are not limited to inorganic acid salts such as the hydrochloride, hydrobromide, sulfate, phosphate; organic acid salts such as trifluoroacetate and maleate salts; sulfonates such as methanesulfonate, ethanesulfonate, benzenesulfonate, p- toluenesulfonate, camphor sulfonate and naphthalenesulfonate; amino acid salts such as arginate, alaninate, asparginate and glutamate; and carbohydrate salts such as gluconate and galacturonate. The selection and use of pharmaceutically acceptable salts is well known in the art, for example, see Stahl and Wermuth, Pharmaceutical Salts: Properties, Selection, and Use, 2nd Revised edition, Wiley, Hoboken, N.J., which is incorporated herein by reference in its entirety and for all purposes. Non-limiting examples of pharmaceutically acceptable salts include, without limitation, sodium salts, ammonium salts, potassium salts (e.g, sodium, ammonium, and potassium chloride; sodium, ammonium, and potassium acetate; sodium, ammonium, and potassium citrate; sodium, ammonium, and potassium phosphate; sodium.ammonium, and potassium fluoride; sodium, ammonium, and potassium bromide; and sodium, ammonium, and potassium iodide).

[0024] The term "isolated" designates a biological material (cell, nucleic acid or protein) that has been removed from its original environment (the environment in which it is naturally present). For example, a polynucleotide present in the natural state in a plant or an animal is not isolated, however the same polynucleotide separated from the adjacent nucleic acids in which it is naturally present, is considered "isolated."

[0025] As used herein, a "coding region" or "coding sequence" is a portion of polynucleotide which consists of codons translatable into amino acids. Although a "stop codon" (TAG, TGA, or TAA) is typically not translated into an amino acid, it can be considered to be part of a coding region, but any flanking sequences, for example promoters, ribosome binding sites, transcriptional terminators, introns, and the like, are not part of a coding region. The boundaries of a coding region are typically determined by a start codon at the 5' terminus, encoding the amino terminus of the resultant polypeptide, and a translation stop codon at the 3* terminus, encoding the carboxyl terminus of the resulting polypeptide. Two or more coding regions can be present in a single polynucleotide construct, e.g., on a single vector, or in separate polynucleotide constructs, e.g., on separate (different) vectors. It follows, then that a single vector can contain just a single coding region, or comprise two or more coding regions.

[0026] As used herein, the term "regulatory region" refers to nucleotide sequences located upstream (5' non-coding sequences), within, or downstream (3' non-coding sequences) of a coding region, and which influence the transcription, RNA processing, stability, or translation of the associated coding region. Regulatory’ regions can include promoters, translation leader sequences, introns, polyadenylation recognition sequences, RNA processing sites, effector binding sites and stem-loop structures. If a coding region is intended for expression in a eukaryotic cell, a polyadenylation signal and transcription termination sequence will usually be located 3' to the coding sequence.

[0027] As used herein, the term “nucleic acid” is interchangeable with “polynucleotide” or “nucleic acid molecule” and a polymer of nucleotides is intended.

[0028] A polynucleotide which encodes a gene product, e.g., a polypeptide, an interfering RNA such as a primary’ miRNA, short hairpin RNA (shRNA), or a small interfering RNA(siRNA), can include a promoter and / or other transcription or translation control elements operably associated with one or more coding regions. In an operable association a coding region for a gene product, e.g., a polypeptide, is associated with one or more regulatory regions in such a way as to place expression of the gene product under the influence or control of the regulator}' region(s). For example, a coding region and a promoter are "operably associated" if induction of promoter function results in the transcription of mRNA encoding the gene product encoded by the coding region, and if the nature of the linkage between the promoter and the coding region does not interfere w'ith the ability of the promoter to direct the expression of the gene product or interfere with the ability of the DNA template to be transcribed. Other transcription control elements, besides a promoter, for example enhancers, operators, repressors, and transcription termination signals, can also be operably associated with a coding region to direct gene product expression.

[0029] "Transcriptional control sequences" refer to DNA regulatory sequences, such as promoters, enhancers, terminators, and the like, that provide for the expression of a coding sequence in a host cell. A variety of transcription control regions are known to those skilled in the art. These include, w'ithout limitation, transcription control regions which function in vertebrate cells, such as, but not limited to, promoter and enhancer segments from cytomegaloviruses (the immediate early promoter, in conjunction with intron-A), simian virus 40 (the early promoter), and retroviruses (such as Rous sarcoma virus). Other transcription control regions include those derived from vertebrate genes such as actin, heat shock protein, bovine growth hormone and rabbit beta-globin, as w'ell as other sequences capable of controlling gene expression in eukaryotic cells. Additional suitable transcription control regions include tissue-specific promoters and enhancers as "well as lymphokine- inducible promoters (e.g., promoters inducible by interferons or interleukins).

[0030] Similarly, a variety of translation control elements are known to those of ordinary skill in the art. These include, but are not limited to ribosome binding sites, translation initiation and termination codons, and elements derived from picornaviruses (particularly an internal ribosome entry' site, or IRES, also referred to as a CITE sequence).

[0031] The term "expression" as used herein refers to a process by which a polynucleotide produces a gene product, for example, an RNA or a polypeptide. It includes without limitation transcription of the polynucleotide into messenger RNA (mRNA), transfer RNA (tRNA), small hairpin RNA (shRNA), small interfering RNA (siRNA) or any other RNAproduct, and the translation of an mRNA into a polypeptide. Expression produces a "gene product.” As used herein, a gene product can be either a nucleic acid, e.g., a messenger RNA produced by transcription of a gene, or a polypeptide which is translated from a transcript. Gene products described herein further include nucleic acids with post transcriptional modifications, e.g., polyadenylation or splicing, or polypeptides with post translational modifications, e.g., methylation, glycosylation, the addition of lipids, association with other protein subunits, or proteolytic cleavage.

[0032] “Promoter" and "promoter sequence" are used interchangeably and refer to a DNA sequence capable of controlling the expression of a coding sequence or functional RNA. In general, a coding sequence is located 3’ to a promoter sequence. Promoters can be derived in their entirety from a native gene, or be composed of different elements derived from different promoters found in nature, or even comprise synthetic DNA segments. It is understood by those skilled in the art that different promoters can direct the expression of a gene in different tissues or cell types, or at different stages of development, or in response to different environmental or physiological conditions. Promoters that cause a gene to be expressed in most cell types at most times are commonly referred to as "constitutive promoters." Preferred constitutive promoters for use in the present methods include, without limitation, CAG promoters and CBA promoters. Promoters that cause a gene to be expressed in a specific cell type are commonly referred to as "cell-specific promoters" or "tissue-specific promoters." Promoters that cause a gene to be expressed at a specific stage of development or cell differentiation are commonly referred to as "developmentally-specific promoters" or "cell differentiation-specific promoters." Promoters that are induced and cause a gene to be expressed following exposure or treatment of the cell with an agent, biological molecule, chemical, ligand, light, or the like that induces the promoter are commonly referred to as "inducible promoters" or "regulatable promoters." It is further recognized that since in most cases the exact boundaries of regulatory sequences have not been completely defined, DNA fragments of different lengths can have identical promoter activity.

[0033] A “CAG promoter” is composed of (C) the cytomegalovirus (CMV) early enhancer element, (A) the promoter, the first exon and the first intron of chicken beta-actin gene, (G) the splice acceptor of the rabbit beta-globin gene. See Miyazaki, J., Takaki, S., Araki, K., Tashiro, F., Tominaga, A., Takatsu, K., & Yamamura, K. (1989). Expression vector system based on the chicken p-actin promoter directs efficient production ofinterleukin-5. Gene, 79(2), 269-277, the contents of which are incorporated herein by reference.

[0034] The term "plasmid" refers to an extra-chromosomal element often carrying a gene that is not part of the central metabolism of the cell, and usually in the form of circular double-stranded DNA molecules. Such elements can be autonomously replicating sequences, genome integrating sequences, phage or nucleotide sequences, linear, circular, or supercoiled, of a single- or double-stranded DNA or RNA, derived from any source, in which a number of nucleotide sequences have been joined or recombined into a unique construction which is capable of introducing a promoter fragment and DNA sequence for a selected gene product along with appropriate 3’ untranslated sequence into a cell.

[0035] A polynucleotide or polypeptide has a certain percent "sequence identity" to another polynucleotide or polypeptide, meaning that, when aligned, that percentage of bases or amino acids are the same when comparing the two sequences. Sequence similarity can be determined in a number of different manners. To determine sequence identity, sequences can be aligned using the methods and computer programs, including BLAST, available over the world wide web at ncbi.nlm.nih.gov / BLAST / . Another alignment algorithm is FASTA, available in the Genetics Computing Group (GCG) package, from Madison, Wis., USA.Other techniques for alignment are described in Methods in Enzymology, vol. 266: Computer Methods for Macromolecular Sequence Analysis (1996), ed. Doolittle, Academic Press, Inc. Of particular interest are alignment programs that permit gaps in the sequence. The Smith- Waterman is one type of algorithm that permits gaps in sequence alignments. See Meth. Mol. Biol. 70: 173-187 (1997). Also, the GAP program using the Needleman and Wunsch alignment method can be utilized to align sequences. See J. Mol. Biol. 48: 443-453 (1970).

[0036] The term "amino acid substitution" and its synonyms described above are intended to encompass modification of an amino acid sequence by replacement of an amino acid with another, substituting, amino acid. The substitution may be a conservative substitution. It may also be a non-conservative substitution. The term conservative, in referring to two amino acids, is intended to mean that the amino acids share a common property recognized by one of skill in the art. For example, amino acids having hydrophobic nonacidic side chains, amino acids having hydrophobic acidic side chains, amino acids having hydrophilic nonacidic side chains, amino acids having hydrophilic acidic side chains, and amino acids having hydrophilic basic side chains. Common properties may also be amino acids havinghydrophobic side chains, amino acids having aliphatic hydrophobic side chains, amino acids having aromatic hydrophobic side chains, amino acids with polar neutral side chains, amino acids with electrically charged side chains, amino acids with electrically charged acidic side chains, and amino acids with eiectricaliy charged basic side chains. Both naturally occurring and non-naturally occurring amino acids are known in the art and may be used as substituting amino acids in embodiments. Methods for replacing an amino acid are well known to the skilled in the art and include, but are not limited to, mutations of the nucleotide sequence encoding the amino acid sequence. Reference to "one or more" herein is intended to encompass the individual embodiments of, for example, 1, 2, 3, 4, 5, 6, or more.

[0037] As used herein, the terms "treatment," "treating," and the like, refer to obtaining a desired pharmacologic and / or physiologic effect. The effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and-'or may be therapeutic in terms of a partial or complete cure for a disease and / or adverse effect attributable to the disease. "Treatment, " as used herein, covers any treatment of a disease in a mammal, particularly in a human, and includes: (a) preventing the disease (and / or symptoms caused by the disease) from occurring in a subject which may be predisposed to the disease or at risk of acquiring the disease but has not yet been diagnosed as having it; (b) inhibiting the disease (and / or symptoms caused by the disease), i.e., arresting its development: and (c) relieving the disease (and / or symptoms caused by the disease), i.e., causing regression of the disease (and / or symptoms caused by the disease), i.e., ameliorating the disease and / or one or more symptoms of the disease.

[0038] The terms "individual," "host," "subject," and "patient" are used interchangeably herein, and refer to a mammal, including, but not limited to, humans; non- human primates, including simians; mammalian sport animals (e.g., horses); mammalian farm animals (e.g., sheep, goats, etc.); mammalian pets (dogs, cats, etc.); and rodents (e.g., mice, rats, etc.).

[0039] The term "effective amount" as used herein is an amount sufficient to effect beneficial or desired clinical results. An effective amount can be administered in one or more administrations. For purposes of this disclosure, an effective amount of a compound (e.g., an infectious rAAV virion) is an amount that is sufficient to palliate, ameliorate, stabilize, reverse, prevent, slow or delay the progression of (and / or symptoms associated with) a particular disease state (e.g., a disorder associated with complement dysfunction). Accordingly, an effective amount of an infectious rAAV virion is an amount of the infectiousrAAV virion that is able to effectively deliver a heterologous nucleic acid to a target cell (or target cells) of the individual. Effective amounts may be determined preclinically by, e.g., detecting in the cell or tissue the gene product (RNA, protein) that is encoded by the heterologous nucleic acid sequence using techniques that are well understood in the art, e.g. RT-PCR, western blotting, ELISA, fluorescence or other reporter readouts, and the like. Effective amounts may be determined clinically by, e.g. detecting a change in the onset or progression of disease using methods known in the art, e.g. 6-minute walk test, left ventricular ejection fraction, hand-held dynamometry, Vignos Scale and the like as described herein and as known in the art.

[0040] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention. Ranges can be expressed herein as from "about" one particular value, and / or to "about" another particular value. When such a range is expressed, another case includes from the one particular value and / or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent "about," it will be understood that the particular value forms another case. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. The term "about" as used herein refers to a range that is 15% plus or minus from a stated numerical value within the context of the particular usage. For example, about 10 would include a range from 8.5 to 11.5. The term "about" also accounts for typical error or imprecision in measurement of values.Detailed Description

[0041] Provided herein are methods for the treatment of wet AMD in a subject in need thereof, the methods comprising intraocularly administering to an eye of the subject a single unit dose of a first pharmaceutical composition comprising an rAAV of serotype 2 or a variant thereof encapsulating a heterologous nucleic acid encoding one or more anti-VEGFgene products, wherein the unit dose comprises about IxlO10vector genomes per eye to about 6* 10'° vector genomes per eye, preferably wherein the unit dose comprises about l x]Osovector genomes per eye or about 3x] 0’;IJvector genomes per eye, and further comprising administering to the eye of the subject one or more corticosteroids and / or administering to the eye of the subject one or more doses of a second pharmaceutical composition, the second pharmaceutical composition comprising an anti-VEGF protein. In preferred embodiments, the heterologous nucleic acid comprises a nucleotide sequence encoding aflibercept and the second pharmaceutical composition comprises aflibercept protein.

[0042] Corticosteroid Administration Regimens

[0043] In certain embodiments, a method of treating wet age-related macular degeneration (wet AMD) in a subject is provided, the method comprising: (i) intraocularly administering to an eye of the subject a single unit dose of a first pharmaceutical composition comprising an rAAV, said rAAV comprising a capsid protein of serotype 2 or a variant thereof and a heterologous nucleic acid comprising a nucleotide sequence encoding at least one anti-VEGF gene product operably linked to a promoter, wherein the unit dose comprises DIO10vector genomes per eye to 6* 10!0vector genomes per eye, preferably wherein the unit dose comprises I x JO10vector genomes per eye or 3x 10!Ovector genomes per eye, and (ii) topically administering prednisolone acetate or a corticosteroid equivalent to prednisolone acetate to the eye of the subject before, simultaneous with and / or after administration of the first pharmaceutical composition, and administering triamcinolone acetonide or a corticosteroid equivalent to triamcinolone acetonide to the subject by periocular injection, preferably by subtenon injection. A “unit dose” refers to the amount of rAAV administered to an eye of the subject in a single dose and is typically independent of the weight of the subject. A single unit dose refers to a single administration of the unit dose to the subject.

[0044] Periocular administration of triamcinolone acetonide preferably consists of a single unit dose of corticosteroids equivalent to about 20 mg to about 60 mg per eye of the subject, preferably equivalent to about 40 mg, triamcinolone acetonide by periocular injection, within about 3 days of administering the first pharmaceutical composition, preferably within about 48 hours of administering the first pharmaceutical composition, more preferably within about 24 hours of administering the first pharmaceutical composition. Periocular injection of triamcinolone acetonide can be performed as a localized deposition of the corticosteroid intothe orbital floor or as subtenon injection of triamcinolone acetonide. Preferably, triamcinolone acetonide is administered by subtenon injection.

[0045] Prednisolone acetate or an equivalent thereof is preferably administered to the subject in multiple doses, in which the prednisolone acetate is administered to the subject for an administration period. The term “administration period” means the time period during which prednisolone acetate is administered to the subject.

[0046] In some embodiments, prednisolone acetate is administered to the subject for an administration period of at least 4 weeks, at least 8 weeks, at least 12 weeks, at least 16 weeks, or at least about 20 weeks, preferably wherein the dose of prednisolone acetate is tapered over the administration period. In related aspects, the subject is administered consecutive daily doses of prednisolone acetate for at least four weeks, for at least eight weeks or for at least twelve weeks during the administration period.

[0047] In some preferred embodiments, the subject is administered prednisolone acetate for an administration period that is initiated prior to administration of the first pharmaceutical composition. In particularly preferred embodiments, a first dose of prednisolone acetate is administered to the subject at least about three days prior to administering the first pharmaceutical composition.

[0048] In related aspects, the subject is administered four doses of prednisolone acetate per day consecutively for at least about 4 weeks beginning at least about three days prior to administering the first pharmaceutical composition. Preferably, administration of prednisolone acetate continues until at least about four weeks after administering the first pharmaceutical composition.

[0049] In a preferred embodiment, the method comprises the following administration schedule, wherein the first dose of prednisolone acetate is administered about three days prior to administering the first pharmaceutical composition:(i) corticosteroids equivalent to prednisolone acetate ophthalmic suspension, USP 1 .0% or prednisolone acetate ophthalmic suspension, USP 1.0% four times per day for about 28 days administered topically; followed by(ii) corticosteroids equivalent to prednisolone acetate ophthalmic suspension, USP 1.0% or prednisolone acetate ophthalmic suspension, USP 1.0% three times per day for about 28 days administered topically; followed by(Hi) corticosteroids equivalent to prednisolone acetate ophthalmic suspension, USP 1.0% or prednisolone acetate ophthalmic suspension, USP 1.0% two times per day for about 28 days administered topically; followed by(iv) corticosteroids equivalent to prednisolone acetate ophthalmic suspension, USP 1 .0% or prednisolone acetate ophthalmic suspension, USP 1 .0% once per day for about 28 days administered topically, wherein the administration period is about 112 days and wherein the subject is administered a single unit dose of corticosteroids equivalent to 40 mg triamcinolone acetonide or is administered a single unit dose of 40 mg triamcinolone by subtenon injection within about 24 hours of administering the first pharmaceutical composition.[0S50] In another preferred embodiment, the method comprises the following administration schedule:(i) corticosteroids equivalent to prednisolone acetate ophthalmic suspension, USP 1.0% or prednisolone acetate ophthalmic suspension, USP 1.0% six times per day for about 3 days administered topically, initiated about three days prior to administering the first pharmaceutical composition; followed by(ii) corticosteroids equivalent to prednisolone acetate ophthalmic suspension, USP 1.0% or prednisolone acetate ophthalmic suspension, USP 1.0% six times per day for about 28 days administered topically initiated within 24 hours of administering the first pharmaceutical composition; followed by(Hi) corticosteroids equivalent to prednisolone acetate ophthalmic suspension, USP 1.0% or prednisolone acetate ophthalmic suspension, USP 1.0% four times per day for about 28 days administered topically; followed by(iv) corticosteroids equivalent to prednisolone acetate ophthalmic suspension, USP 1.0% or prednisolone acetate ophthalmic suspension, USP 1.0% three times per day for about 28 days administered topically; followed by(v) corticosteroids equivalent to prednisolone acetate ophthalmic suspension, USP 1.0% or prednisolone acetate ophthalmic suspension, USP 1.0% two times per day for about 14 days administered topically; followed by(vi) corticosteroids equivalent to prednisolone acetate ophthalmic suspension, USP 1.0% or prednisolone acetate ophthalmic suspension, USP 1.0% once per day for about 14 days administered topically, wherein the administration period is about 115 days and wherein the subject is administered a single unit dose of corticosteroids equivalent to 40 mg triamcinolone acetonide or is administered a single unit dose of 40 mg triamcinolone by subtenon injection within about 24 hours of administering the first pharmaceutical composition.

[0051] In another preferred embodiment, the method comprises the following administration schedule, wherein the first dose of prednisolone acetate is administered about three days prior to administering the first pharmaceutical composition:(i) corticosteroids equivalent to prednisolone acetate ophthalmic suspension, USP1.0% or prednisolone acetate ophthalmic suspension, USP 1 .0% four times per day for about 56 days administered topically; followed by(ii) corticosteroids equivalent to prednisolone acetate ophthalmic suspension, USP1 .0% or prednisolone acetate ophthalmic suspension, USP 1 .0% three times per day for about 28 days administered topically; followed by(in) corticosteroids equivalent to prednisolone acetate ophthalmic suspension, USP 1 .0% or prednisolone acetate ophthalmic suspension, USP 1.0% two times per day for about 14 days administered topically; followed by(iv) corticosteroids equivalent to prednisolone acetate ophthalmic suspension, USP 1.0% or prednisolone acetate ophthalmic suspension, USP 1.0% once per day for about 14 days administered topically, wherein the administration period is about 112 days and wherein the subject is administered a single unit dose of corticosteroids equivalent to 40 mg triamcinolone acetonide or is administered a single unit dose of 40 mg triamcinolone subtenon injection within about 24 hours of administering the first pharmaceutical composition.

[0052] In another embodiment, a method of treating wet AMD in a subject is provided, the method comprising: (i) intraocularly administering to an eye of the subject a single unit dose of a first pharmaceutical composition comprising an rAAV, said rAAV comprising a capsid protein of serotype 2 or a variant thereof and a heterologous nucleic acid comprising a nucleotide sequence encoding aflibercept operably linked to a promoter, and (ii) topically administering prednisolone acetate and optionally administering 40 mg triamcinolone acetonide or a corticosteroid equivalent thereto by subtenon injection, according to the following schedule:(i) prednisolone acetate ophthalmic suspension USP 1.0% administered topically about six times per day for about 4 weeks, followed by(ii) prednisolone acetate ophthalmic suspension USP 1.0% administered topically about five times per day for about 4 weeks, followed by(iii) prednisolone acetate ophthalmic suspension USP 1 .0% administered topically about four times per day for about 4 weeks, followed by(iv) prednisolone acetate ophthalmic suspension USP 1 .0% administered topically about three times per day for about 4 weeks, followed by(v) prednisolone acetate ophthalmic suspension USP 1.0% administered topically about two times per day for about 4 weeks, followed by(vi) prednisolone acetate ophthalmic suspension USP 1.0% administered topically about once per day for about 4 weeks, followed by(vii) prednisolone acetate ophthalmic suspension USP 1.0% administered topically about once every other day for about 4 w'eeks, wherein the administration period is about 28 weeks. In some embodiments, the administration regimen is administered subsequent to a prior topical 0.5% prednisolone acetate administration regimen, for example, if the subject exhibited clinically significant breakthrough during or following the prior treatment regimen. In some aspects, clinically significant breakthrough is defined as an increase in inflammation or rebound inflammation.

[0053] In another embodiment, a method of treating wet AMD in a subject is provided, the method comprising: (i) intraocularly administering to an eye of the subject a single unit dose of a first pharmaceutical composition comprising an rAAV, said rAAV comprising a capsid protein of serotype 2 or a variant thereof and a heterologous nucleic acid comprising a nucleotide sequence encoding aflibercept operably linked to a promoter, and (ii) topically administering difluprednate or a corticosteroid equivalent thereto according to the following schedule difluprednate ophthalmic emulsion 0.05% administered topically about six times per day for about 3 weeks; followed by® difluprednate ophthalmic emulsion 0.05% administered topically about five times per day for about 3 weeks; followed by« difluprednate ophthalmic emulsion 0.05% administered topically about four times per day for about 3 weeks; followed by® difluprednate ophthalmic emulsion 0.05% administered topically about three times per day for about 3 weeks; followed by« difluprednate ophthalmic emulsion 0.05% administered topically about two times per day for about 3 weeks; followed by® difluprednate ophthalmic emulsion 0.05% administered topically about once per day for about 3 weeks; followed by® difluprednate ophthalmic emulsion 0.05% administered topically about every other day for about 3 weeks; wherein the administration period is about 21 weeks. In some embodiments, the administration regimen is administered subsequent to a prior topical difluprednate ophthalmic emulsion 0.05% administration regimen, for example, if the subject exhibited clinically significant breakthrough during or following the prior treatment regimen. In some aspects, clinically significant breakthrough is defined as an increase in inflammation or rebound inflammation in a treated eye of the subject.

[8054] In certain embodiments, a method of treating wet age-related macular degeneration (wet AMD) in a subject is provided, the method comprising: (i) intraocularly administering to the eye of the subject a single unit dose of a first pharmaceutical composition comprising an rAAV, said rAAV comprising a capsid protein of serotype 2 or a variant thereof and a heterologous nucleic acid comprising a nucleotide sequence encoding at least one anti-VEGF gene product operably linked to a promoter, and (ii) administering an intravitreal implant comprising a corticosteroid to the eye of the subject before, simultaneous with or after administration of the first pharmaceutical composition, wherein the corticosteroid is administered by intravitreal implant at least once prior to administering the first pharmaceutical composition and at least once after administering the first pharmaceutical composition. Preferably, the first pharmaceutical composition comprises from about 1x1010to about 6xl0!° vector genomes (vg) of the rAAV, e.g., about 1x10!Oor about 3x1010vg / eye of the subject.

[0055] In preferred embodiments, the intravitreal implant comprises dexamethasone or an equivalent thereof. Intravitreal implants comprising dexamethasone include, without limitation, OZURDEX®. Alternatively, the intravitreal implant may comprise a different corticosteroid such as ILUVIEN® which comprises the corticosteroid fluocinolone acetonide.

[0056] In related aspects, a first intravitreal implant is administered to the subject within about one or about two weeks prior to administering the first pharmaceutical composition. In some preferred aspects, the first intravitreal implant is administered to the subject within about one week prior to administering the first pharmaceutical, such as about seven days, six days, five days, four days, three days, two days or 1 day prior to administering the first pharmaceutical composition. In some aspects, the first intravitreal implant is administered to the subject within about 24 hours of administering the first pharmaceutical composition.

[0057] In other related aspects, at least a second intravitreal implant is administered to the subject following administration of the first intravitreal implant. In some aspects, the second intravitreal implant is administered to the subject about 30 to about 90 days after administering the first intravitreal implant. For example, the second intravitreal implant may be administered to the subject about four weeks, about five weeks, about six weeks, about seven weeks, about eight weeks, about nine weeks, about ten weeks, about eleven weeks, about twelve weeks or more after administering the first intravitreal implant. In preferred aspects, the second intravitreal implant is administered to the subject about 10 weeks to about14 weeks or about 1 1 weeks to about 13 weeks after administering the first intravitreal implant, more preferably about 12 weeks after administering the first intravitreal implant.

[0058] In certain aspects, administration of the first pharmaceutical composition in combination with any embodiment of corticosteroid co-administration substantially reduces or eliminates intraocular inflammation in the subject. In some aspects, intraocular inflammation includes measurements of white blood cells, haze and / or flare. In some aspects, intraocular inflammation is measured as Grade >1 inflammation episodes as assessed by Standardization of Uveitis Nomenclature (SUN).

[0059] Any of the above embodiments may be combined with any embodiment of a supplemental aflibercept regimen as described below.

[0060] Supplemental Aflibercept Regimens

[0061] In certain aspects, one or more doses of an anti-VEGF agent is administered to the eye of the subject prior to, simultaneous with and / or subsequent to receiving a first pharmaceutical composition, preferably wherein the anti-VEGF agent is an aflibercept protein. Preferably, the first pharmaceutical composition delivers a single unit dose of about 1x10’° to about 6xlO10vector genomes (vg) of the rAAV to the eye of the subject, more preferably delivers about 1x10!0or about 3x10i0vg of the rAAV to the eye of the subject.

[0062] In some aspects, aflibercept protein comprises the following amino acid sequence:MVSYWDTGVLLCALLSCLLLTGSSSGSDTGRPFVEMYSEIPEIIIIMTEGRELVIPCRV TSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATWGHLYKTNYLTHRQTNTIIDVVLSPSHGIELSVGEKI.VI.NCTARTELNVGIDFNWEYPSSKHQHKKL VNRDLKTQSGSEMKKFLSTLT1DGVTRSDQGLYTCAASSGLMTKKNSTFVRVHEKD KTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEATCVVVDVSHEDPEVKFNWYV DGVEVHN,\KTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDLAVEWESNGQPENNY KTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:1).

[0063] In other aspects, aflibercept protein comprises amino acids 1 -457 of SEQ ID NO: 1 (i.e., lacks the terminal lysine of SEQ ID NO:1).

[0064] In some aspects, aflibercept protein is administered to the subject as Eylea® or a biosimilar or biobetter thereof. Examples of biosimilars include Yesafili (aflibercept-jbvf) and Opuviz (aflibercept-yszy). Other nonlimiting examples include MYL-1701P, ABP 938,FYB203, SB-15, SOK583A19, CT-P42, z\LT-L9, and OT-702. In some preferred aspects, the aflibercept protein is administered in a dose of about 1 mg to about 10 mg, preferably about 2 mg, by intravitreal injection.

[0065] In some embodim ents, a method of treating wet AMD in a subject is provided comprising (i) intraocularly administering to an eye of the subject a single unit dose of a first pharmaceutical composition comprising a recombinant adeno-associated virus (rAAV), said rAAV comprising a capsid protein of serotype 2 or a variant thereof and a heterologous nucleic acid comprising a nucleotide sequence encoding an anti-VEGF gene product, preferably aflibercept, operably linked to a promoter, and (ii) administering to the eye of the subject at least one dose of a second pharmaceutical composition comprising an anti-VEGF protein, preferably aflibercept or an equivalent thereof, before, simultaneous with and / or after administration of the first pharmaceutical composition.

[0066] In some aspects, the eye of the subject has received one or more doses of an anti- VEGF agent prior to receiving the first pharmaceutical composition. Non-limiting examples of anti-VEGF agents include bevacizumab, brolucizumab, ranibizumab, faricimab, abicipar pegol, conbercept, OPT-302, KSI-301, injectable sunitinib maleate (GB-102), PAN-90806 (PanOptica), and / or aflibercept. In some embodiments, the subject received at least one dose of aflibercept protein within about 18 weeks, 17 weeks, 16 weeks, 15 weeks, 14 weeks, 13 weeks, 12 weeks, 11 -weeks or 10 weeks of receiving the first pharmaceutical composition. In other embodiments, the subject has received from about 6 to about 12 doses of aflibercept protein within about 12 months of receiving the first pharmaceutical composition. In some aspects, identification of the subject as refractory' to a prior dose of aflibercept protein is determined based on a measurement of subretinal and / or intraretinal fluid in the treated eye relative to a baseline (e.g., prior to receiving one or more doses of aflibercept protein). In other embodiments, the subject has not received any prior anti-VEGF treatment prior to receiving the first pharmaceutical composition (e.g., the subject is naive to aflibercept therapy).

[0067] In some embodiments, the eye of the subject is administered a first dose of the second pharmaceutical composition (comprising aflibercept protein or an equivalent thereof) about 30 to about 40 days prior to administering the first pharmaceutical composition, preferably about 35 days prior to administering the first pharmaceutical composition. In other words, the first pharmaceutical composition is administered to the subject at Day 1 andthe second pharmaceutical composition is administered at Day -30 to Day -40. preferably at Week -5.

[0068] In some embodiments, the eye of the subject is administered a second dose of the second pharmaceutical composition (comprising aflibercept protein or an equivalent thereof) about 5 days to about 9 days prior to administering the first pharmaceutical composition, preferably about 7 days prior to administering the first pharmaceutical composition. In other words, the first pharmaceutical composition is administered to the subject at Day 1 and the second pharmaceutical composition is administered at Day -5 to Day -9, preferably at Week - 1.

[0069] In related embodiments, the eye of the subject is administered a third dose of the second pharmaceutical composition about 26 days to about 30 after administering the first pharmaceutical composition, preferably about 28 days after administering the first pharmaceutical composition. In other words, the first pharmaceutical composition is administered to the subject at Day I and the second pharmaceutical composition is administered at Day 26 to Day 30, preferably at W’eek 4.

[0070] In other embodiments, the eye of the subject is administered at least one addi tional dose (e.g., a fourth dose, a fifth dose, a sixth dose and so on) of the second pharmaceutical composition (comprising aflibercept protein) if the subject is identified as a candidate for at least one additional dose. In some aspects, the subject is identified as a candidate for receiving at least one additional dose of the second pharmaceutical composition if one or more of tire following criteria are met: a. loss of >10 letters from baseline best corrected visual acuity (BCVA) b. increase from baseline in retinal central subfield thickness (CST) >75 pm and / or c. presence of or new vision-threatening hemorrhage due to wet AMD.

[0071] Preferably a loss of loss of >10 letters from BCVA is determined to be attributable to intraretinal or subretinal fluid.

[0072] In other embodiments, a subject is identified as a candidate for receiving at least one additional dose of the second pharmaceutical composition if the subject is determined to have significant wet AMD activity' at a timepoint subsequent to administration of the firstpharmaceutical composition. For example, the subject may be determined to have a level of retinal fluid above a predetermined threshold level in an eye treated with the first pharmaceutical composition.

[0073] In preferred embodiments, the first pharmaceutical composition and / or the second pharmaceutical composition(s) are / is administered intravitreally to the subject. In particularly preferred embodiments, the first and second pharmaceutical compositions are administered intravitreally to the subject.

[0074] In some embodiments, the method further comprises (in addition to administering one or more doses of the second pharmaceutical composition) administering to the subject corticosteroids according to the following administration regimen, wherein the subject is administered one or more corticosteroids to the subject according to the following schedule, wherein the first dose of corticosteroid is administered about three days prior to administering the first pharmaceutical composition:(i) corticosteroids equivalent to difluprednate ophthalmic emulsion 0.05% or difluprednate ophthalmic emulsion 0.05% four times per day for about 31 days administered topically; followed by(ii) corticosteroids equivalent to difluprednate ophthalmic emulsion 0.05% or difluprednate ophthalmic emulsion 0.05% three times per day for about 28 days administered topically: follow'ed by(iii) corticosteroids equivalent to difluprednate ophthalmic emulsion 0.05% or difluprednate ophthalmic emulsion 0.05% two times per day for about 28 days administered topically; followed by(iv) corticosteroids equivalent to difluprednate ophthalmic emulsion 0.05% or difluprednate ophthalmic emulsion 0.05% once per day for about 28 days administered topically; followed by(v) corticosteroids equivalent to difluprednate ophthalmic emulsion 0.05% or difluprednate ophthalmic emulsion 0.05% once ever}7other day for about 28 days administered topically, preferably wherein the administration period is about 143 days.

[0075] In aspects of any of the above methods, administering the unit dose of rAAV in the first pharmaceutical composition to an eye of the subject results in maintenance of or a decrease of retinal thickness compared to the retinal thickness prior to administration of the unit dose of rAAV. In certain aspects of the methods, the decrease in retinal thickness is at least about 10% compared to the retinal thickness prior to administration of the unit dose of rAAV. In aspects of certain methods, the retinal thickness is central subfield thickness (CST) or central retinal thickness (CRT).

[0076] In aspects of any of the above methods, administering the unit dose of rAAV in the first pharmaceutical composition to the eye of the subject results in maintenance of or a decrease in macular volume compared to the macular volume prior to administration of the unit dose of rAAV. In an aspect of the methods, administering the unit dose of rAAV to the eye of the subject results in a decrease in macular volume compared to the macular volume prior to administration of the unit dose of rAAV. In aspect of the methods, the decrease in macular volume is at least about 10% compared to the macular volume prior to administration of the unit dose of rAAV particles.

[0077] In aspects of any of the above methods, administering the unit dose of rAAV to the eye of the subject results in maintenance or an improvement of visual acuity' compared to the visual acuity' prior to administration of the unit dose of rAAV. In some aspects of the methods, administering the unit dose of rAAV to the one eye of the subject results in an improvement of visual acuity compared to the visual acuity prior to administration of the unit dose of rAAV particles. In aspects of the methods, visual acuity is best corrected visual acuity (BCVA).

[0078] In aspects of any of the above methods, one or more subjects results do not require an anti-VEGF (e.g., aflibercept) rescue treatment for at least about 24 weeks, at least about 36 weeks, at least about 52 weeks, at least about 56 weeks or more after administration of the unit dose of the rAAV to an eye of the one or more subjects. In some aspects, at least about 50%, at least about 55%, at least about 55%, at least about 60%, or at least about 65% of multiple subjects receiving a single unit dose of the first pharmaceutical composition do not require an anti-VEGF rescue treatment for at least about 24 weeks.

[0079] In aspects of any of the above methods, administration of the unit dose of rAAV to the eye of one or more subjects results in a reduction in the annualized anti-VEFG (e.g.,aflibercept) injection rate of at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 87%, at least about 90%, at least about 95% at least about 99% or 100% compared to the annualized anti-VEGF injection rate prior to administration of the unit dose of rAAV.

[0080] In certain aspects, aflibercept expression is maintained in an eye of a subject with AMD administered a unit dose of a first pharmaceutical composition comprising an rAAV comprising a capsid of SEQ ID NO:4 and a nucleic acid of SEQ ID NO:9 (i.e., a treated eye), for at least about 24 weeks, at least about 28 weeks, at least about 32 weeks, at least about 36 weeks, at least about 40 weeks, at least about 44 weeks, at least about 48 weeks, at least about 52 weeks, at least about 56 weeks, at least about 60 weeks, at least about 64 weeks, at least about 68 weeks, at least about 72 weeks, at least about 76 w'eeks, at least about 80 weeks, at least about 84 weeks, at least about 88 weeks, at least about 92 w'eeks, at least about 96 weeks, at least about 100 weeks, or at least about 104 weeks. In some aspects, aflibercept expression is measured as the concentration of aflibercept in the aqueous humor of the subject, e.g., using ELISA. In some aspects, the subject does not receive any VEGF-A (e.g. aflibercept) treatment beginning about 8 weeks after administration of the first pharmaceutical composition. In some aspects, aflibercept in the treated eye of the subject decrease less than about 40%, less than about 30%, less than about 20%, or less than about 10% relative to an aflibercept level measured in the treated eye of the subject within about 24 weeks after administration of the first pharmaceutical composition.

[0081] First Pharmaceutical Composition

[0082] Adeno-Associated Virus

[0083] In exemplary embodiments, the first pharmaceutical composition of the present disclosure comprises AAV of serotype 2 or a variant thereof.

[0084] In certain embodiments, the AAV is an AAV2 variant as described in U.S. Patent Publication No. 2020 / 0282077, which is incorporated herein by reference in its entirety, in particular a recombinant AAV (rAAV) comprising a capsid protein according to paragraphs 171-179 of U.S. Patent Publication No. 2020 / 0282077 and / or comprising a heterologous nucleic acid according to paragraphs 222-248 of U.S. Patent Publication No. 2020 / 0282077.

[0085] In some aspects, the pharmaceutical composition comprises an rAAV comprising a variant AAV2 capsid encapsulating a heterologous nucleic acid encoding an anti-VEGF gene product, wherein the variant AAV capsid comprises a variant AAV capsid protein comprising an insertion of from about 7 to about 20 amino acids (a “heterologous peptide” or “peptide insertion”) in the GH-loop of a parental AAV capsid protein, wherein the peptide comprises the amino acid sequence ISDQTKH (SEQ ID NO:2). Preferably, the variant capsid protein, when present in an AAV virion, confers increased infectivity of a retinal cell compared to the infectivity of a retinal cell by an AAV virion comprising the corresponding parental capsid protein.

[0086] By the “GH loop,” or loop IV, of the AAV capsid protein it is meant the solvent- accessible portion referred to in the art as the GH loop, or loop IV, of AAV capsid protein. For the GH loop / loop IV of AAV capsid, see, e.g., van Vliet et al. (2006) Mol. Ther. 14:809; Padron et al. (2005) J. Virol. 79:5047; and Shen et al. (2007) Mol. Ther. 15:1955. Thus, for example, the insertion site can be within about amino acids 570-611 of AAV2 VPI.17 / 934,801. In certain embodiments, the peptide is inserted following any of the amino acids in positions 584-591 in VPI of AAV2 (i.e., the insertion site is between amino acids 587 and 588 of VPI of AAV2 or is between amino acids 588 and 589, between amino acids 584 and 585, between amino acids 585 and 586, between amino acids 586 and 587, between amino acids 590 and 591, or is between amino acids 591 and 592 of AAV2). In certain preferred embodiments, the insertion site is between amino acids 587 and 588 of VPI of AAV2 or is between amino acids 588 and 589 of AAV2.

[0087] In some embodiments, the peptide insertion has from 1 to 3 spacer amino acids (Y1-Y3) at the amino and / or carboxyl terminus of the amino acid sequence ISDQTKH (SEQ ID NO:2). Exemplary spacer amino acids include, without limitation, leucine (L), alanine (A), glycine (G), serine (S), threonine (T), and proline (P). In certain embodiments, a peptide insertion comprises 2 spacer amino acids at the N-terminus and 2 spacer amino acids at the C-terminus. In other embodiments, a peptide insertion comprises 2 spacer amino acids at the N-terminus and 1 spacer amino acids at the C-terminus. In preferred embodiments, the peptide insertion comprises or consists of the amino acid sequence LAISDQTKHA (SEQ ID NO:3).

[0088] In some aspects, the variant AAV capsid protein comprises a peptide insertion comprising the amino acid sequence ISDQTKH (SEQ ID NO:2) and further comprises one ormore amino acid substitutions relative to a corresponding parental AAV capsid protein.Representative examples of amino acid substitutions may be found at e.g., col. 26, lines 40- 65 of U.S. Patent No. 11, 576, 983, the entire contents of which are incorporated herein by reference.

[0089] In some preferred embodiments, the variant AAV capsid protein comprises a peptide insertion comprising the amino acid sequence ISDQTKH (SEQ ID NO:2) and further comprises a P34A amino acid substitution relative to VP1 capsid of AAV2 or the corresponding substitution in another AAV serotype.

[0090] In other aspects, the variant capsid protein may comprise one or more features disclosed in U.S. Patent No. 11,576,983, in particular, one or more features disclosed at column 26, line 66 to column 29, line 50 of U.S. Patent No. 11,576,983.

[0091] In a particularly preferred embodiment, the variant capsid protein comprises the following amino acid sequence or comprises an amino acid sequence at least 80%, at least 90%, least 95%, at least 98%, or at least 99% identical to the following amino acid sequence:MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKAAERHKDDSRGLVLPGYKYLGP FNGLDKGEPVNEADAAALEIIDKAYDRQLDSGDNPYLKYNIIADAEFQERLKEDTSF GGNLGRAVFQAKKRVLEPLGLVEEPVKTAPGKKRPVEHSPVEPDSSSGTGKAGQQP ARKRLNFGQTGDADSVPDPQPLGQPPAAPSGLGTNTMATGSGAPMADNNEGADGV GNSSGNVTdCDSTWMGDRVITTSTRTWALPTYNNHLYKQISSQSGASNDNHYFGYST PWGYFDFNRFHCIIFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTQNDGTTTIA NNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMVPQYGYLTLNNGSQAVGR SSFYCLEYFPSQMLRTGNNFTFSYTFEDVPFIISSYAFISQSLDRLMNPLIDQYLYYLSRTNTPSGTTTQSRLQFSQAGASDIRDQSRNWLPGPCYRQQRVSKTSADNNNSEYSWT GATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVLIFGKQGSEKTNVDIEKVMIT DEEEIRTTNPVATEQYGSVSTNLQRGNLAISDQTKHARQAATADVNTQGVLPGMVW QDRDVYLQGPTWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPSTTFSAA KFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSVNVDFTVDTNGVYS EPRPIGTRYLTRNL (SEQ ID NO:4).[0092[ The variant AAV capsid protein of SEQ ID NO:4 contains the following modifications relative to native AAV2 capsid: (i) a proline (P) to alanine (A) mutation at amino acid position 34, which is located inside the assembled capsid (VP1 protein only), and (ii) an insertion of 10 amino acids (leucme-alanine-isoleucme-serine-aspartic acid-glutamine- threonine-lysine-histidine-alanine / LAISDQTKHA (SEQ ID NO:3)) at amino acid position 588, which is present in VP1, VP2, and VP3. In some embodiments, the capsid comprises a variant capsid protein comprising a sequence at least 90%, at least 95%, at least 98%, at least99% identical to SEQ ID NO:4 and comprising a P34A substitution and an LAISDQTKHA (SEQ ID NO:3) peptide insertion at amino acid position 588.

[0093] A first pharmaceutical composition as herein described comprises an rAAV encapsulating a heterologous nucleic acid comprising a nucleotide sequence encoding one or more anti-VEGF gene products. In some aspects, the one or more gene products are selected from an interfering RNA (e.g., a microRNA) and a polypeptide. In particularly preferred embodiments, the heterologous nucleic acid encodes aflibercept and further encodes an interfering RNA that decreases the expression of VEGF-C. In related aspects, the heterologous nucleic acid comprises a nucleotide sequence described in U.S. Patent Publication No. 2024 / 0131195A1, the entire contents of which are incorporated herein by reference, in particular at Table 2.

[0094] In particularly preferred aspects, the heterologous nucleic acid comprises a nucleotide sequence encoding an interfering RNA, preferably an artificial miRNA, comprising a sense strand at least 90% identical to CTACCTCAGCAAGACGTTATT (SEQ ID NO:5) and an antisense strand at least 90% identical to AATAACGTCTTGCTGAGGTAG (SEQ ID NO:6). In related aspects, the nucleic acid comprises a nucleotide sequence encoding an artificial miRNA comprising at least 90% sequence identity withGACTTCTTAACCCAACAGAAGGCTCGAGAAGGTATATTGCTGTTGACAGTGA GCGGCTACCTCAGCAAGACGTTAT1TAGTGAAGCCACAGATGTAAATAAC GTCTTGCTGAGGTAGCTGCCTACTGCCTCGGACTTCAAGGGGCTAGAATTC (SEQ ID NO:7).

[0095] In other preferred aspects, the rAAV comprises a heterologous nucleic acid comprising the following nucleotide sequence encoding aflibercept, codon-optimized for expression in humans:ATGGTTTCTTACTGGGACACCGGCGTGCTGCTGTGTGCCCTGCTTTCTTGTCTGCTGCTOA CCGGCTCTAGCAGCGGCTCTGATACCGGCAGACCCTTCGTGGAAATGTACAGCGAGATC CCCGAGATCATCCACATGACCGAGGGCAGAGAGCTGGTCATCCCTTGCAGAGTGACAAG CCCCAACATCACCGTGACTCTGAAGAAGn'CCCTCTGGACACACTGATCCCCGACGGCAA GAGAATCATCTGGGACAGCCGGAAGGGCTTCATCATCAGCAACGCCACCTACAAAGAGA TCGGCCTGCTGACCTGTGAAGCCACCGTGAATGGCCACCTGTACAAGACCAACTACCTGACACACAGACAGACCAACACCATCATCGACGTGGTGCTGAGCCCTAGCCACGGCATTGAA CTGTCTGTGGGCGAGAAGCTGGTGCTGAACTGTACCGCCAGAACCGAGCTGAACGTGGG CATCGACTTCAACTGGGAGTACCCCAGCAGCAAGCACCAGCACAAGAAACTGGTCAACC GGGACCTGAAAACCCAGAGCGGCAGCGAGATGAAGAAATTCCTGAGCACCCTGACCATC GACGGCGTGACCAGAAGTGACCAGGGCCTGTACACATGTGCCGCCAGCTCTGGCCTGATGACCz\AGAAAzAACAGCACCTTCGTGCGGGTGCACGAGAAGGzA.CAAGACCC;ACz\CCTGTCCTCCATGTCCTGCTCCAGAACTGCTCGGCGGACCTTCCGTGTTCCTGTTTCCTCCAAAGCC TAAGGACACCCTGATGATCAGCAGAACCCCTGAAGTGACCTGCGTGGTGGTGGATGTGT CCCACGAGGATCCCGAAGTGAAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAAC GCCAAGACCAAGCCTAGAGAGGAACAGTACAATAGCACCTACAGAGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGCCCTGCCTGCTCCTATCGAGAAAACCATCTCCAAGGCCAAGGGCCAGCCTAGGGAA CCCCAGGTTTACACACTGCCTCCAAGCAGGGACGAGCTGACAAAGAACCAGGTGTCCCT GACCTGCCTGGTCAAGGGCTTCTACCCTTCCGATATCGCCGTGGAATGGGAGAGCAATGG CCAGCCTGAGAACAACTACAAGACAACCCCTCCTGTGCTGGACAGCGACGGCTCATTCTTCCTGTACAGCAAGCTGACAGTGGACAAGAGCAGATGGCAGCAGGGCAACGTGTTCAGCT GCTCCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGAGCCTGTCTC CTGGCAAA (SEQ ID NO: 8)

[0096] In some embodiments, the sequence is at least 80%, at least 90%, at least 95% or at least 99% identical to the nucleotide sequence of SEQ ID NO:8 and / or comprises a stop codon (e.g. TGA) at the end of the sequence.

[0097] In particularly preferred aspects, the pharmaceutical composition comprises an rAAV comprising a heterologous nucleic acid comprising the following sequence (encoding aflibercept + human VEGF-C interfering RNA) or a sequence at least 80%, at least 90%, at least 95%, at least 98% or at least 99% identical thereto:TTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGG TCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCA GAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCTATCGATTGAATTCCCCGG GGATCCACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCAT ATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCC CAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCA ATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACT TGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGA CGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCT ACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTCGAGGTGAGCC CCACGTTCTGCTTCACTCTCCCCATCTCCCCCCCCTCCCCACCCCCAATTTTGTAT TTATTTATTTTTTAATTATTTTGTGCAGCGATGGGGGCGGGGGGGGGGGGGGGGCGCGCGCCAGGCGGGGCGGGGCGGGGCGAGGGGCGGGGCGGGGCGAGGCGGAGAGGTGCGGCGGCAGCCAATCAGAGCGGCGCGCTCCGAAAGTTTCCTTTTATGGCGAGGCGGCGGCGGCGGCGGCCCTATAAAAAGCGAAGCGCGCGGCGGGCGGGGAGTCGCTGCGACGCTGCCTTCGCCCCGTGCCCCGCTCCGCCGCCGCCTCGCGCCGCCCGCCCCGGCTCTGACTGACCGCGTTACTCCCACAGGTGAGCGGGCGGGACGGCCCTTCTCCTCCGGGCTGTAATTAGCGCTTGGTTTAATGACGGCTTGTTTCTTTTCTGTGGCTGCGTGAAAGCCTrGAGGGGCTCCGGGAGGGCCCTTTGTGCGGGGGGAGCGGCTCGGGGGGTGCG'IGCGTGTGTGTGTGCGTGGGGAGCGCCGCGTGCGGCTCCGCGCTGCCCGGCGGCTGTGAGCGCTGCGGGCGCGGCGCGGGGCTTTGTGCGCTCCGCAGTGTGCGCGAGGGGAGCGCGGCCGGGGGCGGTGCCCCGCGGTGCGGGGGGGGCTGCGAGGGGAACAAAGGCTGCGTGCGGGGTGTGTGCGTGGGGGGGTGAGCAGGGGGTGTGGGCGCGTCGGTCGGGCTGCAACCCCCCCTGCACCCCCCTCCCCGAGTTGCTGAGCACGGCCCGGCTTCGGGTGCGGGGCTCCGTACGGGGCGTGGCGCGGGGCTCGCCGTGCCGGGCGGGGGGTGGCGGCAGGTGGGGGTGCCGGGCGGGGCGGGGCCGCCTCGGGCCGGGGAGGGCrCGGGGGAGGGGCGCGGCGGCCX’CCGGAGCGCCGGCGCGAClTCrFAACCCAACAGAAGGCTCGAGAAGGTATATTGCTGTTGACAGTGAGCGGCTACCTCAGCAAGACGTTATTTAGTGAAGCCACAGATGTAAATAACGTCTTGCTGAGGTAGCTGCCTACTGCCTCGGACTTCAAGGGGCTAGAATTCGCGGCTGTCGAGGCGCGGCGAGCCGCAGCCATTGCCTTTTATGGTAATCGTGCGAGAGGGCGCAGGGACTTCCTITGTCCCAAATCTGTGCGGAGCCGAAATCTGGGAGGCGCCGCCGCACCCCCTCTAGCGGGCGCGGGGCGAAGCGGTGCGGCGCCGGCAGGAAGGAAATGGGCGGGGAGGGCCTTCGTGCGTCGCCGCGCCGCCGTCCCCTTCTCCCTCTCCAGCCTCGGGGCTGTCCGCGGGGGGACGGCTGCCTTCGGGGGGGACGGGGCAGGGCGGGGTTCGGCTTCTGGCGTGTGACCGGCGGCTCTAGAGCCTCTGCTAACCATGTTCATGCCTTCTTCTTTTTCCTACAGTCTAGAGTCGACCTGCAGGTGGATATCTTGGCTAGCACGCCACCATGGTTTCTTACTGGGACACCGGCGTGCTGC TGTGTGCCCTGCTTTCTTGTCTGCTGCTGACCGGCTCTAGC AGCGGCTCTGATA CC GGCAGACCCTTCGTGGAAATGTACAGCGAGATCCCCGAGATCATCCACATGACCGAGGGCAGAGAGCTGGTCATCCCTTGCAGAGTGACAAGCCCCAACATCACCGTGACTCTGAAGAAGTTCCCTCTGGACACACTGATCCCCGACGGCAAGAGAATCATCTGGGACAGCCGGAAGGGCTTCATCATCAGCAACGCCACCTACAAAGAGATCGGCCTGCTGACCTGTGAAGCCACCGTGAATGGCCACCTGTACAAGACCAACTACCTGACACACAGACAGACCAACACCATCATCGACGTGGTGCTGAGCCCTAGCCACGGCATTGAACTGTCTGTGGGCGAGAAGCTGGTGCTGAACTGTACCGCCAGAACCGAGCTGAACGTGGGCATCGACITCAACTGGGAGTACCCCAGCAGCAAGCACCAGCACAAGAAACTGGTCAACCGGGACCTGAAAACCCAGAGCGGCAGCGAGATGAAGAAATTCCTGAGCACCCTGACCATCGACGGCGTGACCAGAAGTGACCAGGGCCTGTACACATGTGCCGCCAGCTCTGGCCTGATGACCAAGAAAAACAGCACCTTCGTGCGGGTGCACGAGAAGGACAAGACCCACACCTGTCCTCCATGTCCTGCTCCAGAACTGCTCGGCGGACCTTCCGTGTTCCTGTTTCCTCCAAAGCCTAAGGACACCCTGATGATCAGCAGAACCCCTGAAGIGACCTGCGTGGTGGTGGA I’GTG I CCCACGAGGA FCCCGAAGTGAAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCTAGAGAGGAACAGTACAATAGCACCTACAGAGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGC.AAAGAGTACAAGTGCAAGGTGTCCAACAAGGCCCTGCCTGCTCCTATCGAGAAAACCATCTCCAAGGCCAAGGGCCAGCCTAGGGAACCCCAGGTTTACACACTGCCTCCAAGCAGGGACGAGCTGACAAAGAACCAGGTGTCCCTGACCTGCCTGGTCAAGGGCITCTACCCTTCCGATATCGCCGTGGAATGGGAGAGCAATGGCCAGCCTGAGAACAACTACAAGACAACCCCTCCTGTGCTGGACAGCGACGGCTCATTC1TCCTGTACAGCAAGCTGACAGTGGACAAGAGCAGATGGCAGCAGGGCAACGTGTTCAGCTGCTCCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGAGCCTGTCTCCTGGCAAATGAGCCACGCGTAACACGTGGGGGATCCAGACATGATAAGATACATTGATGAGTTTGGACAAACCA CAACTAGAATGCAGTGAAAAAAATGCTTTATTTGTGAAATTTGTGATGCTATTGC TTTAITTGTAAC’.CAITATAAGCTGCAATAAACAAGTTAACAACAACAATTGCATT CATTTTATGTTTCAGGTTCAGGGGGAGGTGTGGGAGGTTTTTTAAAGCAAGTAAA ACCTCTACAAATGTGGTATGGCTGATTA TGA'I CAATGCA TGGCCGGCCGGAGGA ACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAG GCCGCCCGGGCAAAGCCCGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTG AGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAA (SEQ ID NO:9).

[0098] In particularly preferred embodiments, an amount of the pharmaceutical composition effective to deliver a unit dose of about 1x1010to about 6x1 O30, preferably about 3 x IO10vg of an rAAV comprising (i) a capsid protein comprising the amino acid sequence set forth as SEQ ID NO:4 and (ii) a heterologous nucleic acid comprising the nucleotide sequence of SEQ ID NO:9, is administered intravitreally to the subject.

[0099] Formulations

[0100] In exemplary aspects, the first pharmaceutical composition of the present disclosure comprises: adeno-associated virus (AAV) of serotype 2 or a variant thereof and about 5 mM to about 20 mM of a buffering agent, about 100 mM to about 250 mM of a pharmaceutically acceptable salt, about 0.0001% (w / v) to about 0.01% (w / v) of a non-ionic surfactant, and a pH of at least about 7.0.

[0101] In exemplary aspects, the first pharmaceutical composition of the present disclosure comprises: AAV of serotype 2 or a variant thereof and about 5 mM to about 20 mM of a buffering agent, about 100 mM to about 200 mM of a pharmaceutically acceptable salt, about 0.001% (w / v) to about 0.01 % (w / v) of a non-ionic surfactant, and a pH of between about 7.0 and about 9.0.

[0102] In exemplary' aspects, the first pharmaceutical composition of the present disclosure comprises: AAV of serotype 2 or a variant thereof and about 5 mM to about 20 mM of a buffering agent, about 150 mM to about 200 mM of a pharmaceutically acceptable salt, about 0.001% (w / v) to about 0.01% (w / v) of a non-ionic surfactant, and a pH of at least about 7.4.

[0103] In exemplary aspects, the first pharmaceutical composition of the present disclosure comprises: AAV of serotype 2 or a variant thereof and about 10 mM to about 20 mM of a buffering agent, about 150 mM to about 200 mM of a pharmaceutically acceptablesalt, about 0.001% (w / v) to about 0.01% (w / v) of a non-ionic surfactant, and a pH of at least about 7.4

[0104] In certain aspects, the composition is a sterile composition. By “sterile” it is meant that there are substantially no immunogenic components in the composition, such as for example substantially no microbes (e.g., fungi, bacteria, viruses, spore forms, etc.).

[0105] In some embodiments, the invention provides a liquid formulation. In certain embodiments, the formulation is lyophilized from a liquid formulation.

[0106] In exemplary aspects, the first pharmaceutical composition of the present disclosure comprises about 5 mM to about 20 mM, about 5 mM to about 15 mM, about 10 mM to about 20 mM, or about 15 mM to about 25 mM of a buffering agent. In exemplary aspects, the pharmaceutical composition comprises about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, about 10 mM, about 1 1 mM, about 12 mM, about 13 mM, about14 mM, about 15 mM, about 16 mM, about 17 mM, about 18 mM, about 19 mM, about 20 mM, about 21 mM, about 22 mM, about 23 mM, about 24 mM, or about 25 mM of a buffering agent.

[0107] Pharmaceutically acceptable buffering agents include without limitation, phosphate buffers, histidine, sodium citrate, HEPES, Tris, Bicine, glycine, N-glycylglycine, sodium acetate, sodium carbonate, glycyl glycine, lysine, arginine, sodium phosphate, and mixtures thereof. In some preferred embodiments, the buffer is a Tris buffer.

[0108] In exemplary aspects, the first pharmaceutical composition of the present disclosure comprises about 5 mM to about 25 mM, about 5 mM to about 15 mM, about 10 mM to about 20 mM, or about 15 mM to about 25 mM Tris. In exemplary aspects, the pharmaceutical composition comprises about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, about 10 mM, about 11 mM, about 12 mM, about 13 mM, about 14 mM, about15 mM, about 16 mM, about 17 mM, about 18 mM, about 19 mM, about 20 mM, about 21 mM, about 22 mM, about 23 mM, about 24 mM, or about 25 mM Tris. In some embodiments, the first pharmaceutical composition comprises about 8 mM Tris to about 22 mM Tris, or about 9 mM Iris to about 21 mM Tris, or about 10 mM Tris to about 20 mM Tris. In certain embodiments, the first pharmaceutical composition comprises about 10 mM Tris.

[0109] In exemplary aspects, the first pharmaceutical composition of the present disclosure comprises about 100 mM to about 250 mM, about 110 mM to about 240 mM, about 120 mM to about 230 mM, about 130 mM to about 220 mM, about 140 mM to about 210 mM, about 145 mM to about 205 mM, about 140 mM to about 200 mM, about 145 mM to about 200 mM, about 150 mM to about 200 mM, about 155 mM to about 195 mM, about 160 mM to about 195 mM, about 165 mM to about 195 mM, about 170 mM to about 195 mM, about 175 mM to about 195 mM, about 175 mM to about 194 mM, about 175 mM to about 193 mM, about 175 mM to about 192 mM, about 175 mM to about 191 mM, about 176 mM to about 190 mM, about 177 mM to about 189 mM, about 177 mM to about 188 mM, about 177 mM to about 187 mM, about 177 mM to about 186 mM, about 178 to about 184, about 178 to about 183, about 178 to about 182, about 179 to about 181 or about 180 mM of a pharmaceutically acceptable salt (as defined above). In exemplary embodiments, the composition comprises about 150 mM, about 155 mM, about 160 mM, about 175 mM, about 180 mM, about 185 mM, about 190 mM, or about 200 mM of a pharmaceutically acceptable salt. In certain embodiments, the pharmaceutically acceptable salt is a sodium salt (e.g., sodium chloride).

[0110] In certain embodiments, pharmaceutical compositions containing a non-ionic detergent are provided. Pharmaceutically acceptable non-ionic surfactants that may be used in the formulations disclosed herein are known in the art of pharmaceutical science, and include, without limitation, Polysorbate 80 (Tween 80; PS80), Polysorbate 20 (Tween 20; PS20), and various poloxamers or pluronics, including Pluronic F-68, and BRU 35, or mixtures thereof. In a preferred embodiment, the non-ionic surfactant used in the present pharmaceutical compositions is a pluronic, particularly Pluronic F-68.

[0111] In certain embodiments, the first pharmaceutical composition of the present disclosure comprises about 0.001% (w / v) to about 0.01% (w / v) or about 0.0025% (w / v) to about 0.0075% (w / v) non-ionic surfactant. In exemplary aspects, the pharmaceutical composition comprises about 0.001% (w / v), about 0.0015% (w / v), about 0.002% (w / v), about 0.0025% (w / v), about 0.003% (w / v), about 0.0035% (w / v), about 0.004% (w / v), about 0.0045% (w / v), about 0.005% (w / v), about 0.0055% (w / v), about 0.006% (w / v), about 0.0065% (w / v), about 0.007% (w / v), about 0.0075% (w / v), about 0.008% (w / v), about 0.0085% (w / v), about 0.009% (w / v), about 0.0095% (w / v), about 0.001% (w / v) non-ionicsurfactant. In certain embodiments, the first pharmaceutical composition of the present disclosure comprises about 0.005% (w / v) non-ionic surfactant.

[0112] In exemplary aspects, the first pharmaceutical composition of the present disclosure comprises about 0.001% (w / v) to about 0.01% (w / v) or about 0.0025% (w / v) to about 0.0075% (w / v) Pluronic F68. In exemplary aspects, the pharmaceutical composition comprises about 0.001% (w / v), about 0.0015% (w / v), about 0,002% (w / v), about 0.0025% (w / v), about 0.003% (w / v), about 0.0035% (w / v), about 0.004% (w / v), about 0.0045% (w / v), about 0.005% (w / v), about 0.0055% (w / v), about 0.006% (w / v), about 0.0065% (w / v), about 0.007% (w / v), about 0.0075% (w / v), about 0.008% (w / v), about 0.0085% (w / v), about 0.009% (w / v), about 0.0095% (w / v), about 0.001% (w / v) Pluronic F68. In certain embodiments, the first pharmaceutical composition of the present disclosure comprises about 0.005% (w / v) Pluronic F68.

[0113] In exemplary aspects, the first pharmaceutical composition of the present disclosure comprises: AAV of serotype 2 or a variant thereof and about 5 mM to about 25 mM Tris, about 100 mM to about 250 mM sodium salt, about 0.001% (w / v) to about 0.01% (w / v) non-ionic surfactant, and a pH of between 7.0 and 9.0. In certain embodiments, the pharmaceutically acceptable salt is present at about 150 nM to about 210 mM. In certain embodiments, the pharmaceutically acceptable salt is present at about 170 nM to about 200 mM. In certain embodiments, the pharmaceutically acceptable salt is sodium chloride.

[0114] The present disclosure also provides a pharmaceutical composition that can be liquid or lyophilized (e.g., lyophilized from a liquid formulation) comprising adeno- associated virus (AAV) and about 10 mM Tris buffer, about 180 mM sodium chloride, about 0.005% (w / v) Pluronic F68 and the pH of the pharmaceutical composition is about 7.9±0.3.

[0115] pH

[0116] In exemplary' embodiments, the first pharmaceutical composition of the present disclosure has a pH of about 7.0, about 7.1, about 7.2, about 7.2, about 7.3, about 7.4, about 7.5, about 7.6, about 7.7, about 7.8, about 7.9, about 8.0, about 8.1, about 8.2, about 8.3, about 8.4, about 8.5, about 8.6, about 8.7, about 8.8, about 8.9, or about 9.0. In exemplary aspects, the first pH of the pharmaceutical composition is above about 7.3 and below' about 9.0 or below about 8.5. In certain embodiments, the pH of the pharmaceutical composition isabout 7.8 to about 9.0 or is about 7.9. In other embodiments, the pH of the pharmaceutical composition is from about 7.3 to about 8.6.

[0117] Additional components

[0118] In some embodiments, the formulations or pharmaceutical compositions of the present disclosure comprise additional pharmaceutically acceptable ingredients. In exemplary aspects, the formulations or pharmaceutical compositions comprise any one or a combination of the following: acidifying agents, additives, adsorbents, aerosol propellants, air displacement agents, alkalizing agents, anticaking agents, anticoagulants, antimicrobial preservatives, antioxidants, antiseptics, bases, binders, buffering agents, chelating agents, coating agents, coloring agents, desiccants, detergents, diluents, disinfectants, disintegrants, dispersing agents, dissolution enhancing agents, dyes, emollients, emulsifying agents, emulsion stabilizers, fillers, film forming agents, flavor enhancers, flavoring agents, flow enhancers, gelling agents, granulating agents, humectants, lubricants, mucoadhesives, ointment bases, ointments, oleaginous vehicles, organic bases, pastille bases, pigments, plasticizers, polishing agents, preservatives, sequestering agents, skin penetrants, solubilizing agents, solvents, stabilizing agents, suppository bases, surface active agents, surfactants, suspending agents, sweetening agents, therapeutic agents, thickening agents, tonicity agents, toxicity agents, viscosity-increasing agents, water-absorbing agents, water- miscible cosolvents, water softeners, or wetting agents. In some embodiments, the formulations or pharmaceutical compositions of the present disclosure comprise any one or a combination of the following components: acacia, acesulfame potassium, acetyltributyl citrate, acetyltriethyl citrate, agar, albumin, alcohol, dehydrated alcohol, denatured alcohol, dilute alcohol, aieuritic acid, alginic acid, aliphatic polyesters, alumina, aluminum hydroxide, aluminum stearate, amylopectin, a-amylose, ascorbic acid, ascorbyl palmitate, aspartame, bacteriostatic water for injection, bentonite, bentonite magma, benzalkonium chloride, benzethonium chloride, benzoic acid, benzyl alcohol, benzyl benzoate, bronopol, butylated hydroxyanisole, butylated hydroxytoluene, butylparaben, butylparaben sodium, calcium alginate, calcium ascorbate, calcium carbonate, calcium cyclamate, dibasic anhydrous calcium phosphate, dibasic dehydrate calcium phosphate, tribasic calcium phosphate, calcium propionate, calcium silicate, calcium sorbate, calcium stearate, calcium sulfate, calcium sulfate hemihydrate, canola oil, carbomer, carbon dioxide, carboxymethyl cellulose calcium, carboxymethyl cellulose sodium, p-carotene, carrageenan, castor oil, hydrogenated castor oil, cationicemulsifying wax. cellulose acetate, cellulose acetate phthalate, ethyl cellulose, microcrystalline cellulose, powdered cellulose, silicified microcrystalline cellulose, sodium carboxymethyl cellulose, cetostearyl alcohol, cetrimide, cetyl alcohol, chlorhexidine, chlorobutanol, chlorocresol, cholesterol, chlorhexidine acetate, chlorhexidine gluconate, chlorhexidine hydrochloride, chlorodifluoroethane (IICFC), chlorodifluoromethane, chlorofluorocarbons (CFC)chlorophenoxyethanoL chloroxylenol, com syrup solids, anhydrous citric acid, citric acid monohydrate, cocoa butter, coloring agents, corn oil, cottonseed oil, cresol, m-cresol, o-cresol, p-cresol, croscannellose sodium, crospovidone, cyclamic acid, cyclodextrins, dextrates, dextrin, dextrose, dextrose anhydrous, diazolidinyl urea, dibutyl phthalate, dibutyl sebacate, diethanolamine, diethyl phthalate, difluoroethane (IIFC), dimethyl-P-cyclodextrin, cyclodextrin-fype compounds such as Captisol®, dimethyl ether, dimethyl phthalate, dipotassium edentate, disodium edentate, disodium hydrogen phosphate, docusate calcium, docusate potassium, docusate sodium, dodecyl gallate, dodecyltrimethylammonium bromide, edentate calcium disodium, edtic acid, eglumine, ethyl alcohol, ethylcellulose, ethyl gallate, ethyl laurate, ethyl maltol, ethyl oleate, ethylparaben, ethylparaben potassium, ethylparaben sodium, ethyl vanillin, fructose, fructose liquid, fructose milled, fructose pyrogen-free, powdered fructose, fumaric acid, gelatin, glucose, liquid glucose, glyceride mixtures of saturated vegetable fatty acids, glycerin, glyceryl behenate, glyceryl monooleate, glyceryl monostearate, self- emulsifying glyceryl monostearate, glyceryl palmitostearate, glycine, glycols, glycofurol, guar gum, heptafluoropropane (HFC), hexadecyltrimethylammonium bromide, high fructose syrup, human serum albumin, hydrocarbons (I IC), dilute hydrochloric acid, hydrogenated vegetable oil type II, hydroxyethyl cellulose, 2-hydroxyethyl-p- cyclodextrin, hydroxypropyl cellulose, low-substituted hydroxypropyl cellulose, 2- hydroxypropyl-p-cyclodextrin, hydroxypropyl methylcellulose, hydroxypropyl methylcellulose phthalate, imidurea, indigo carmine, ion exchangers, iron oxides, isopropyl alcohol, isopropyl myristate, isopropyl palmitate, isotonic saline, kaolin, lactic acid, lactitol. lactose, lanolin, lanolin alcohols, anhydrous lanolin, lecithin, magnesium aluminum silicate, magnesium carbonate, normal magnesium carbonate, magnesium carbonate anhydrous, magnesium carbonate hydroxide, magnesium hydroxide, magnesium lauryl sulfate, magnesium oxide, magnesium silicate, magnesium stearate, magnesium trisilicate, magnesium trisilicate anhydrous, malic acid, malt, maltitol, maltitol solution, maltodextrin, maltol, maltose, mannitol, medium chain triglycerides, meglumine, menthol, methylcellulose, methyl methacrylate, methyl oleate, methylparaben, methylparaben potassium, methylparaben sodium, microcrystalline cellulose and carboxymethylcellulosesodium, mineral oil, light mineral oil, mineral oil and lanolin alcohols, oil, olive oil, monoethanolamine, montmorillonite, octyl gallate, oleic acid, palmitic acid, paraffin, peanut oil, petrolatum, petrolatum and lanolin alcohols, pharmaceutical glaze, phenol, liquified phenol, phenoxyethanol, phenoxypropanol, phenylethyl alcohol, phenylmercuric acetate, phenylmercuric borate, phenylmercuric nitrate, polacrilin, polacrilin potassium, poloxamer, polydextrose, polyethylene glycol, polyethylene oxide, polyacrylates, polyethylene- polyoxypropylene-block polymers, polymethacrylates, polyoxyethylene alkyl ethers, polyoxyethylene castor oil derivatives, polyoxyethylene sorbitol fatty7acid esters, polyoxyethylene stearates, polyvinyl alcohol, polyvinyl pyrrolidone, potassium alginate, potassium benzoate, potassium bicarbonate, potassium bisulfite, potassium chloride, postassium citrate, potassium citrate anhydrous, potassium hydrogen phosphate, potassium metabisulfite, monobasic potassium phosphate, potassium propionate, potassium sorbate, povidone, propanol, propionic acid, propylene carbonate, propylene glycol, propylene glycol alginate, propyl gallate, propylparaben, propylparaben potassium, propylparaben sodium, protamine sulfate, rapeseed oil, Ringer's solution, saccharin, saccharin ammonium, saccharin calcium, saccharin sodium, safflower oil, saponite, serum proteins, sesame oil, colloidal silica, colloidal silicon dioxide, sodium alginate, sodium ascorbate, sodium benzoate, sodium bicarbonate, sodium bisulfite, sodium chloride, anhydrous sodium citrate, sodium citrate dehydrate, sodium chloride, sodium cyclamate, sodium edentate, sodium dodecyl sulfate, sodium lauryl sulfate, sodium metabisulfite, sodium phosphate, dibasic, sodium phosphate, monobasic, sodium phosphate, tribasic, anhydrous sodium propionate, sodium propionate, sodium sorbate, sodium starch glycolate, sodium stearyl fumarate, sodium sulfite, sorbic acid, sorbitan esters (sorbitan fatty esters), sorbitol, sorbitol solution 70%, soybean oil, spermaceti wax, starch, com starch, potato starch, pregelatinized starch, sterilizable maize starch, stearic acid, purified stearic acid, stearyl alcohol, sucrose, sugars, compressible sugar, confectioner’s sugar, sugar spheres, invert sugar, Sugartab, Sunset Yellow FCF, synthetic paraffin, talc, tartaric acid, tartrazine, tetrafluoroethane (HFC), theobroma oil, thimerosal, titanium dioxide, alpha tocopherol, tocopheryl acetate, alpha tocopheryl acid succinate, beta-tocopherol, delta- tocopherol, gamma-tocopherol, tragacanth, triacetin, tributyl citrate, triethanolamine, triethyl citrate, trimethyl-P-cyclodextrin, trimethyltetradecylammonium bromide, tris buffer, trisodium edentate, vanillin, type I hydrogenated vegetable oil, water, soft water, hard water, carbon dioxide-free water, pyrogen-free water, water for injection, sterile water for inhalation, sterile water for injection, sterile water for irrigation, waxes, anionic emulsifying wax, carnauba wax, cationic emulsifying wax, cetyl ester wax, microcrystalline wax.nonionic emulsifying wax, suppository wax, white wax, yellow wax, white petrolatum, wool fat, xanthan gum, xylitol, zein, zinc propionate, zinc salts, zinc stearate, or any excipient in the Handbook of Pharmaceutical Excipients, Third Edition, A. H. Kibbe (Pharmaceutical Press, London, UK, 2000), which is incorporated by reference in its entirety. Remington’s Pharmaceutical Sciences, Sixteenth Edition, E. W. Martin (Mack Publishing Co., Easton, Pa., 1980), which is incorporated by reference in its entirety for all intended purposes, discloses various components used in formulating pharmaceutically acceptable compositions and known techniques for the preparation thereof. Except insofar as any conventional agent is incompatible with the pharmaceutical compositions, its use in pharmaceutical compositions is contemplated.

[0119] In some aspects, the formulations or pharmaceutical compositions comprise a sugar or sugar alcohol. In some aspects, the sugar or sugar alcohol is sucrose, trehalose, mannitol, or a combination thereof.

[0120] In exemplar}7embodiments, the formulations or pharmaceutical compositions of the present disclosure do not comprise one or a combination of the above ingredients. In exemplary' embodiments, the formulations or pharmaceutical compositions of the present disclosure comprises none of these ingredients. In exemplary aspects, the pharmaceutical composition of the present disclosure does not comprise dextran. In exemplary aspects, the pharmaceutical composition of the present disclosure does not comprise calcium chloride. In other exem plary aspects, the pharmaceutical composition of the present disclosure does not comprise a sugar or a sugar alcohol (e.g., does not comprise sucrose, trehalose, or mannitol). In related exemplary aspects, the pharmaceutical composition does not comprise glycine.

[0121] The formulations (e.g., first pharmaceutical composition) disclosed herein may be formulated for administration via known methods, such as intraocular administration (e.g., via intravitreal, subretinal and / or suprachoroidal administration). In some preferred embodiments, the pharmaceutical composition is administered intraocularly to a human, preferably w'herein the pharmaceutical composition is administered via intravitreal, subretinal and / or suprachoroidal injection, more preferably via a single intravitreal injection.

[0122] In some embodiments, the treatment regimen includes administering one or more doses over an extended period of time. In certain cases, a single dose (e.g., a single dosage unit) is administered to the subject, and the initial dose may be followed by one or moredoses administered to the subject at a subsequent time. In some instances, more than one dose (e.g., more than one dosage unit) is administered to the subject, and the initial doses may be followed by one or more doses administered to the subject at a subsequent time. For example, a single dose (e.g., a single dosage unit) may be administered to the subject, and the single dose may be followed by a single dose administered to the subject at a subsequent time. Additional single doses may be administered at subsequent points in time. In other cases, a single dose (e.g., a single dosage unit) may be administered to the subject, and the single dose may be followed by two doses administered to the subject at a subsequent time. Additional single or multiple doses may be administered at subsequent points in time.

[0123] In certain embodiments, dosage units of the present disclosure can be administered prior to, concurrent with, or subsequent to other active agents for treating related or unrelated conditions, e.g., in combination therapy. Administration of separate pharmaceutical compositions can be performed simultaneously or at different times (e.g., sequentially, in either order, on the same day, or on different days), as long as a therapeutically effective effect of the combination of these substances is caused in the subject undergoing therapy. Accordingly, aspects of the present disclosure further include combination therapies. In certain embodiments, the subject method includes administering a therapeutically effective amount of one or more additional active agents. By combination therapy is meant that a AAV composition (e.g., as described herein) can be used in a combination with another therapeutic agent to treat a single disease or condition. In certain embodiments, a compound of the present disclosure is administered concurrently with the administration of another therapeutic agent, which can be administered as a component of a composition including the compound of the present disclosure or as a component of a different composition. In certain embodiments, a composition including a compound of the present disclosure is administered prior or subsequent to administration of another therapeutic agent.

[0124] Some embodiments of the invention are exemplified in the following items 1 to 68:1 . A method of treating wet age-related macular degeneration (wet AMD) in a subject comprising: intraocularly administering to the subject a single unit dose of a first pharmaceutical composition comprising a recombinant adeno-associated virus (rAAV), said rAAV comprising (i) a capsid protein of serotype 2 or a variant thereof and (ii) a heterologousnucleic acid comprising a nucleotide sequence encoding at least one anti-VEGF gene product operably linked to a promoter, wherein the unit dose comprises about IxlO10to about 3xlO10, preferably about IxlO10or about 3xlO10, vector genomes (vg) per eye of the rAAV, and administering one or more corticosteroids comprising prednisolone acetate or a corticosteroid equivalent to prednisolone acetate administered topically to the eye of the subject before, simultaneous with and / or after administration of the first pharmaceutical composition, and administering triamcinolone acetonide or a corticosteroid equivalent to triamcinolone acetonide to the subject by subtenon injection. The method according to item 1, wherein the subject is administered multiple doses of the one or more corticosteroids. The method according to item 1 or 2, wherein the one or more corticosteroids are administered to the subject for an administration period of at least 4 weeks, at least 8 weeks, at least 12 weeks, at least 16 weeks, or at least about 20 weeks, preferably wherein the dose of the one or more corticosteroids is tapered over the administration period. The method according to any one of items 1-3, wherein the subject is administered consecutive multiple daily doses of the one or more corticosteroids for at least four weeks, for at least eight weeks or for at least twelve weeks during the administration period. The method according to any one of items 1-4, wherein a first dose of the one or more corticosteroids is administered to the subject at least about three days prior to administering the first pharmaceutical composition. The method according to any one of items 1 -5, wherein the subject is administered tour doses per day of the corticosteroid consecutively for at least 4 weeks beginning at least about three days prior to administering the first pharmaceutical composition and continuing until at least about four weeks after administering the first pharmaceutical composition.The method according to any one of items 1-6, wherein the subject is topically administered prednisolone acetate and is administered triamcinolone acetonide by subtenon injection. The method according to item 7, wherein administration of the one or more corticosteroids to the subject comprises the following schedule, wherein the first dose of corticosteroid is administered about three days prior to administering the first pharmaceutical composition: a. corticosteroids equivalent to prednisolone acetate ophthalmic suspension, USP 1.0% or prednisolone acetate ophthalmic suspension. USP 1.0% four times per day for about 28 days administered topically; followed by b. corticosteroids equivalent to prednisolone acetate ophthalmic suspension, USP 1 .0% or prednisolone acetate ophthalmic suspension, USP 1.0% three times per day for about 28 days administered topically; followed by c. corticosteroids equivalent to prednisolone acetate ophthalmic suspension, USP 1.0% or prednisolone acetate ophthalmic suspension, USP 1.0% two times per day for about 28 days administered topically; followed by d. corticosteroids equivalent to prednisolone acetate ophthalmic suspension, USP 1.0% or prednisolone acetate ophthalmic suspension, USP 1.0% once per day for about 28 days administered topically, wherein the administration period is about 112 days and wherein the subject is administered a single unit dose of corticosteroids equivalent to 40 mg triamcinolone acetonide or is administered a single unit dose of 40 mg triamcinolone by subtenon injection within about 24 hours of administering the first pharmaceutical composition. The method according to item 7. wherein administration of the one or more corticosteroids to the subject comprises the follow'ing schedule: a. corticosteroids equivalent to prednisolone acetate ophthalmic suspension, USP 1.0% or prednisolone acetate ophthalmic suspension, USP 1.0% six times per day for about 3 days administered topically, initiated about three days prior to administering the first pharmaceutical composition; followed byb. corticosteroids equivalent to prednisolone acetate ophthalmic suspension, USP 1.0% or prednisolone acetate ophthalmic suspension, USP 1.0% six times per day for about 28 days administered topically initiated within 24 hours of administering the first pharmaceutical composition; followed by c. corticosteroids equivalent to prednisolone acetate ophthalmic suspension, USP 1.0% or prednisolone acetate ophthalmic suspension, USP 1.0% four times per day for about 28 days administered topically; followed by d. corticosteroids equivalent to prednisolone acetate ophthalmic suspension, USP 1.0% or prednisolone acetate ophthalmic suspension, USP 1 .0% three times per day for about 28 days administered topically; followed by e. corticosteroids equivalent to prednisolone acetate ophthalmic suspension, USP 1 .0% or prednisolone acetate ophthalmic suspension, USP 1.0% two times per day for about 14 days administered topically; followed by f. corticosteroids equivalent to prednisolone acetate ophthalmic suspension, USP 1.0% or prednisolone acetate ophthalmic suspension, USP 1.0% once per day for about 14 days administered topically, wherein the administration period is about 115 days and wherein the subject is administered a single unit dose of corticosteroids equivalent to 40 mg triamcinolone acetonide or is administered a single unit dose of 40 mg triamcinolone by subtenon injection within about 24 hours of administering the first pharmaceutical composition. The method according to item 7, wherein administration of the one or more corticosteroids to the subject comprises the following schedule, wfierein the first dose of corticosteroid is administered about three days prior to administering the first pharmaceutical composition: a. corticosteroids equivalent to prednisolone acetate ophthalmic suspension, USP 1.0% or prednisolone acetate ophthalmic suspension, USP 1.0% four times per day for about 56 days administered topically; followed byb. corticosteroids equivalent to prednisolone acetate ophthalmic suspension, USP 1.0% or prednisolone acetate ophthalmic suspension, USP 1.0% three times per day for about 28 days administered topically; followed by c. corticosteroids equivalent to prednisolone acetate ophthalmic suspension, USP 1 .0% or prednisolone acetate ophthalmic suspension, USP 1.0% two times per day for about 14 days administered topically; followed by d. corticosteroids equivalent to prednisolone acetate ophthalmic suspension, USP 1.0% or prednisolone acetate ophthalmic suspension, USP 1.0% once per day for about 14 days administered topically, wherein the administration period is about 112 days and wherein the subject is administered a single unit dose of corticosteroids equivalent to 40 mg triamcinolone acetonide or is administered a single unit dose of 40 mg triamcinolone subtenon injection within about 24 hours of administering the first pharmaceutical composition. A m ethod of treating wet AMD in a subject in need thereof, the method comprising: (i) intraocularly administering to an eye of the subject a single unit dose of a first pharmaceutical composition comprising an rAAV, said rAAV comprising a capsid protein of serotype 2 or a variant thereof and a heterologous nucleic acid comprising a nucleotide sequence encoding aflibercept operably linked to a promoter, preferably wherein the unit dose comprises about IxlO10to about 3xl0iQ, preferably about lxl0luor about 3xl0i0, vg of the rAAV per eye and (ii) topically administering prednisolone acetate and optionally administering 40 mg triamcinolone acetonide or a corticosteroid equivalent thereto by subtenon injection, according to the following schedule: a. prednisolone acetate ophthalmic suspension USP 1 .0% administered topically about six times per day for about 4 weeks, followed by b. prednisolone acetate ophthalmic suspension USP 1.0% administered topically about five times per day for about 4 weeks, followed by c. prednisolone acetate ophthalmic suspension USP 1.0% administered topically about four times per day for about 4 weeks, followed byd. prednisolone acetate ophthalmic suspension USP 1.0% administered topically about three times per day for about 4 weeks, followed by e. prednisolone acetate ophthalmic suspension USP 1.0% administered topically about two times per day for about 4 weeks, followed by f. prednisolone acetate ophthalmic suspension USP 1 .0% administered topically about once per day for about 4 weeks, followed by g. prednisolone acetate ophthalmic suspension USP 1.0% administered topically about once every other day for about 4 weeks, wherein the administration period is about 28 weeks. The method according to item 1 1, wherein the subject exhibited clinically significant breakthrough during or following a prior corticosteroid treatment comprising prednisolone acetate. A method of treating wet age-related macular degeneration (wet AMD) comprising in a subject comprising: intraocularly administering to the subject a single unit dose of a first pharmaceutical composition comprising a recombinant adeno-associated virus (rAAV), said rAAV comprising (i) a capsid protein of serotype 2 or a variant thereof and (ii) a heterologous nucleic acid comprising a nucleotide sequence encoding a gene product operably linked to a promoter, preferably wherein the single unit dose comprises about lxI0i0to about 3xlO’10vg or comprises about IxlO10or about 3xlO10vg of the rAAV per eye, and administering an intravitreal implant comprising a corticosteroid to the eye of the subject before, simultaneous with or after administration of the first pharmaceutical composition, wherein the corticosteroid is administered by intravitreal implant at least once prior to administering the first pharmaceutical composition and at least once after administering the first pharmaceutical composition. The method according to item 13. w'herein the corticosteroid is dexamethasone or corticosteroids equivalent to dexamethasone.The method according to item 13 or 14, wherein a first intravitreal implant is administered to the subject about one week prior to administering the first pharmaceutical composition or within about 24 hours of administering the first pharmaceutical composition. The method according to any one of items 13-15, wherein a second intravitreal implant is administered to the subject about 10 weeks to about 14 weeks, preferably about 12 w'eeks, after administering the first pharmaceutical composition. The method according to any one of items 1-16, wherein the subject is administered at least one dose of a second pharmaceutical composition comprising aflibercept protein or a biosimilar thereof, preferably wherein the second pharmaceutical composition comprises about 2 mg of aflibercept protein or 2mg of aflibercept protein biosimilar. The method according to item 17, wherein the subject is administered a dose of the second pharmaceutical composition about 30 days to about 40 days prior to administering the first pharmaceutical composition, preferably about 35 days prior to administering the first pharmaceutical composition. The method according to item 17 or 18, wherein the subject is administered a dose of the second pharmaceutical composition about 5 days to about 9 days prior to administering the first pharmaceutical composition, preferably about 7 days prior to administering the first pharmaceutical composition. The method according to any one of items 17 to 19, wherein the subject is administered a dose of the second pharmaceutical composition about 26 days to about 30 days after administering the first pharmaceutical composition, preferably about 28 days after administering the first pharmaceutical composition. The method according to any one of items 17 to 20, wherein the subject is administered at least one dose of the second pharmaceutical composition subsequent to administration of the first pharmaceutical composition if the subject is determined to have one or more of the following characteristic s: a. loss of >10 leters from baseline best corrected visual acuity (BC VA)b. increase from baseline in retinal central subfield thickness (CST) >75 pm and / or c . presence of or new vision-threatening hemorrhage due to wet AMD, The method according to item 21, wherein the baseline for BCVA and / or CST is measured in the subject within 24 hours of receiving the first pharmaceutical composition. The method according to item 21, wherein the subject is administered at least one dose of the second pharmaceutical composition if the subject is determined to have significant wet AMD activity subsequent to administration of the first pharmaceutical composition. The method according to any one of items 1 to 23, wherein the first pharmaceutical composition is administered intravitreally. The method according to any one of items 18 to 24, wherein the second pharmaceutical composition is administered intravitreally. A method of treating w'et age-related macular degeneration (wet AMD) in a subject comprising: intraocularly administering to the subject a single unit dose of a first pharmaceutical composition comprising a recombinant adeno-associated virus (rAAV), said rAAV comprising (i) a capsid protein of serotype 2 or a variant thereof and (ii) a heterologous nucleic acid comprising a nucleotide sequence encoding a gene product operably linked to a promoter, wherein the unit dose comprises about 1x10!uto about 3xlO10, preferably about 1x1010or about 3x1010, vector genomes (vg) of the rAAV per eye and administering to the subject at least one dose of a second pharmaceutical composition comprising aflibercept protein or a biosimilar thereof, preferably wherein the second pharmaceutical composition comprises about 2 mg of aflibercept protein or 2mg of aflibercept protein biosimilar, before, simultaneous with and / or after administration of the first pharmaceutical composition. The method according to item 26, wherein the subject is administered a dose of the second pharmaceutical composition about 35 days to about 40 days prior toadministering the first pharmaceutical composition, preferably about 35 days prior to administering the first pharmaceutical composition. The method according to item 26 or 27, wherein the subject is administered a dose of the second pharmaceutical composition about 5 days to about 9 days prior to administering the first pharmaceutical composition, preferably about 7 days prior to administering the first pharmaceutical composition. The method according to any one of items 26 to 28, wherein the subject is administered a dose of the second pharmaceutical composition about 26 days to about 30 days after administering the first pharmaceutical composition, preferably about 28 days after administering the first pharmaceutical composition. The method according to any one of items 26 to 29, wherein the subject is administered at least one dose of the second pharmaceutical composition subsequent to administration of the first pharmaceutical composition if the subject is determined to have one or more of the following characteristics: a. loss of >10 letters from baseline best corrected visual acuity (BCVA) b. increase from baseline in retinal central subfield thickness (CST) >75 pm and / or c. presence of or new vision-threatening hemorrhage due to wet AMD. The method according to item 30, wherein the baseline for BCVA and / or CST is measured in the subject within 24 hours of receiving the first pharmaceutical composition. The method according to any one of clams 26 to 31 , wherein the subject is administered at least one dose of the second pharmaceutical composition if the subject is determined to have significant wet AMD activity subsequent to administration of the first pharmaceutical composition. The method according to any one of items 26 to 32, wherein the first pharmaceutical composition is administered intravitreally.The method according to any one of items 26 to 33, wherein the second pharmaceutical composition is administered intravitreally. The method according to any one of items 26 to 34, wherein the subject is administered one or more corticosteroids to the subject according to the following schedule, wherein the first dose of corticosteroid is administered about three days prior to administering the first pharmaceutical composition: a. corticosteroids equivalent to difluprednate ophthalmic emulsion 0.05% or difluprednate ophthalmic emulsion 0.05% four times per day for about 31 days administered topically; followed by b. corticosteroids equivalent to difluprednate ophthalmic emulsion 0.05% or difluprednate ophthalmic emulsion 0.05% three times per day for about 28 days administered topically; followed by c. corticosteroids equivalent to difluprednate ophthalmic emulsion 0.05% or difluprednate ophthalmic emulsion 0.05% two times per day for about 28 days administered topically; followed by d. corticosteroids equivalent to difluprednate ophthalmic emulsion 0.05% or difluprednate ophthalmic emulsion 0.05% once per day for about 28 days administered topically; followed by e. corticosteroids equivalent to difluprednate ophthalmic emulsion 0.05% or difluprednate ophthalmic emulsion 0.05% once every' other day for about 28 days administered topically, preferably wherein the administration period is about 143 days. A method of treating wet AMD in a subject in need thereof, the method comprising: (i) intraocularly administering to an eye of the subject a single unit dose of a first pharmaceutical composition comprising an rAAV, said rAAV comprising a capsid protein of serotype 2 or a variant thereof and a heterologous nucleic acid comprising a nucleotide sequence encoding aflibercept operably linked to a promoter, wherein the unit dose comprises about lxl0!0to about 3xl010vg, preferably about IxlO10vg or about 3x10i0vg of the rAAV per eye, and (ii) topically administering to the eye of thesubject difluprednate or a corticosteroid equivalent thereto according to the following schedule: a. difluprednate ophthalmic emulsion 0.05% administered topically about six times per day for about 3 weeks; followed by b. difluprednate ophthalmic emulsion 0.05% administered topically about five times per day for about 3 weeks; followed by c. difluprednate ophthalmic emulsion 0.05% administered topically about four times per day for about 3 weeks; followed by d. difluprednate ophthalmic emulsion 0.05% administered topically about three times per day for about 3 weeks; followed by e. difluprednate ophthalmic emulsion 0.05% administered topically about two times per day for about 3 weeks; followed by f. difluprednate ophthalmic emulsion 0.05% administered topically about once per day for about 3 weeks; followed by g. difluprednate ophthalmic emulsion 0.05% administered topically about every other day for about 3 weeks; w'herein the administration period is about 21 w'eeks.37. The method according to item 34, wherein the subject exhibited clinically significant breakthrough during or following a prior corticosteroid treatment comprising difluprednate.38. The method according to any one of items 1 to 35, wherein the rAAV comprises (i) a variant capsid protein comprising a heterologous peptide covalently inserted into the GH-loop of the capsid protein relative to AAV2 capsid, the insertion peptide having a length of 10 amino acids and comprising the amino acid sequence ISDQTKH (SEQ ID NO:2) with from 1 to 3 spacer amino acids (Y1-Y3) at the amino and / or carboxyl terminus of SEQ ID NO:2.The method according to item 36, wherein the insertion peptide consists of the sequence LAISDQTKHA (SEQ ID NO:3). The method according to item 36 or 37, wherein the peptide is inserted foilowing any of the amino acids in positions 584-591 in VP1 of AAV2 or a corresponding position in another AAV serotype, preferably wherein the insertion site is between amino acids 587 and 588 of VP1 of AAV2 or is between amino acids 588 and 589 of AAV2 or the corresponding positions in the capsid protein of another AAV serotype. The method according to any one of items 36 to 38, wherein the variant capsid protein comprises one or more amino acid substitution(s) relative to VP1 of AAV2, preferably one or more of the following amino acid substitutions: MIL, L15P, P34A, N57D, N66K, R81Q, Q101R, S109T, R144K, R144M, Q164K, T176P, L188I, S196Y, G226E, G236V, I240T, P250S, N312K, P363L, D368H, N449D, T456K, S463Y, D472N, R484C, A524T, P535S, N551S, A593E, 1698V, V708I, V719M, S721L, and L735Q, more preferably a P34A amino acid substitution. The method according to item 39, wherein the variant capsid protein is at least 90% identical, at least 95% identical, at least 98% identical or 100% identical to the entire length of the amino acid sequence set forth as SEQ ID NO:4. The method according to item 40, wherein the heterologous nucleic acid comprises a nucleotide sequence encoding a polypeptide of SEQ ID NO: 1 . The method according to item 41 , ■wherein the nucleotide sequence encoding a polypeptide of SEQ ID NO:Y is at least 80%, at least 85%, at last 90%, at least 95%, or is 100% identical to SEQ ID NO:8. The method according to any one of items 40 to 42, wherein the heterologous nucleic acid further comprises a nucleotide sequence encoding an interfering RNA that reduces the expression of VEGF-C. The method according to item 43, wherein the nucleotide sequence encoding the interfering RNA comprises a sense strand comprising at least 90% sequence identity' with SEQ ID NO:5 and an antisense strand comprising at least 90% sequence identity with SEQ ID NO:6.41. The method according to item 44, wherein the nucleic acid comprises (i) a nucleotide sequence encoding an artificial miRNA comprising at least 90% sequence identity with the sequence set forth as SEQ ID NO:7 and (ii) a nucleotide sequence at least 95%, or 100% identical to SEQ ID NO:8, wherein the nucleotide sequence of (i) and (ii) are operably linked to a promoter.48. The method according to item 45, wherein the promoter is a CAG promoter, preferably wherein the nucleotide sequence encoding the artificial miRNA is located within the hybrid chicken p-actin and rabbit P-globin intron of the CAG promoter.49. The method according to item 46, wherein the rAAV comprises a capsid protein comprising an amino acid sequence at least 95% identical to SEQ ID NO:4 and a heterologous nucleic acid comprising a nucleotide sequence at least 80% identical to the nucleotide sequence set forth as SEQ ID NO: 950. The method according to item 47, wherein the rAAV comprises a capsid protein of SEQ ID NON and a heterologous nucleic acid of SEQ ID NO:9.51. The method according to any one of items 1 -48, wherein the first pharmaceutical composition comprises a buffering composition comprising:(i) about 5 mM to about 20 mM of a buffering agent, preferably a Tris buffer;(ii) about 100 mM to about 250 mM of a pharmaceutically acceptable salt, preferably NaCl; and(iii) about 0.0001 % (w / v) to about 0.01 % (w / v) of a non-ionic surfactant; wherein the buffering composition has a pH of about 7.0 to about 9.0.52. The method according to item 49, wherein the buffering composition comprises:(i) about 9 mM to about 20 mM of the buffering agent;(ii) about 140 to about 200 mM of the pharmaceutically acceptable salt; and(iii) about 0.001% (w / v) to about 0.01% (w / v) of the non-ionic surfactant; wherein the buffering composition has a pH of about 7.3 to 8.6.53. The method according to item 50 wherein the buffering composition comprises about 10 mM Tris, about 180 mM NaCL, about 0.005% Pluronic F68 and wherein the pH of the buffering composition is about 7.4 to about 8.4 at 25 °C.54. The method according to item 51, wherein the pH of the buffering composition is about 7.6 to about 8.2 at 25 °C.55. A method of treating macular neovascularization secondary to age-related macular degeneration in a human subject in need thereof, the method comprising: intravitreally administering to an eye of the subject a single unit dose of a first pharmaceutical composition comprising a recombinant adeno-associated virus (rAAV), said rAAV comprising (i) a capsid protein comprising the amino acid sequence of SEQ ID NO:4 and (ii) a heterologous nucleic acid comprising the nucleotide of SEQ ID NO:9, wherein the unit dose comprises about 3xl0!0vector genomes (vg) of the rAAV; and administering to the eye of the subject multiple doses of a second pharmaceutical composition comprising aflibercept protein or a biosimilar thereof, the second pharmaceutical composition administered at a dose equivalent to about 2 mg aflibercept, wherein(i) one dose of the second pharmaceutical composition is administered about 5 weeks prior to administration of the first pharmaceutical composition,(ii) one dose of the second pharmaceutical composition is administered about one week prior to administration of the first pharmaceutical composition, and(iii) one dose of the second pharmaceutical composition is administered about four weeks after administration of the first pharmaceutical composition.56. The method according to item 55, wherein one or more additional 2 mg doses of the second pharmaceutical composition are administered to the subject at least twelve weeks after administration of the first pharmaceutical composition, if one or more of the following criteria are met:(i) >5 letter decrease in BCVA compared to a referenceBCVA and a >50-micron increase in CST compared to areference CST resulting from nAMD activity as determined by a qualified professional; or(ii) >10 leter decrease in BCVA compared to a reference BCVA resulting from nAMD activity as determined by a qualified professional, or(ii i) >100-micron increase in CST compared to a reference CST due to nAMD activity' as determined by a qualified professional, or(iv) presence of a new macular hemorrhage (NMH) due to nAMD that, in the opinion of a medical practitioner, is sight-threatening The method according to item 56, wherein the BCVA reference value is the average of BCVA measurements in the subject at four weeks and 8 weeks following administration of the first pharmaceutical composition and / or the CST reference value is the CST measurement in the subject at four weeks following administration of the first pharmaceutical composition. The method according to any one of items 55 to 57, wherein the subject is administered one or more corticosteroids according to the following schedule, wherein the first dose of corticosteroid is administered about three days prior to administering the first pharmaceutical composition: a. corticosteroids equivalent to difluprednaie ophthalmic emulsion 0.05% or difluprednate ophthalmic emulsion 0.05% four times per day for about 31 days administered topically; followed by b. corticosteroids equivalent to difluprednate ophthalmic emulsion 0.05% or difluprednate ophthalmic emulsion 0.05% three times per day for about 28 days administered topically; followed by c. corticosteroids equivalent to difluprednate ophthalmic emulsion 0.05% or difluprednate ophthalmic emulsion 0.05% two times per day for about 28 days administered topically; followed byd. corticosteroids equivalent to difluprednate ophthalmic emulsion 0.05% or difluprednate ophthalmic emulsion 0.05% once per day for about 28 days administered topically; followed by f. corticosteroids equivalent to difluprednate ophthalmic emulsion 0.05% or difluprednate ophthalmic emulsion 0.05% once every other day for about 28 days administered topically, preferably wherein the administration period is about 143 days. The method according to any one of items 1-58, wherein afhbercept levels in a treated eye of the subject remain stable in the subject for at least about 52 weeks, preferably for at least about 104 weeks following administration of the first pharmaceutical composition, preferably wherein an aflibercept level in the treated eye comprises an aflibercept concentration in the aqueous humor of the treated eye. The method according to item 59, wherein the subject does not receive any further administrations of the second composition beginning about 8 weeks after administration of the first composition and continuing at least 24 weeks, at l east 36 weeks, at least 48 weeks, preferably at least 52 weeks after administration of the first composition. The method according to item 59 or 60, wherein aflibercept levels remain stable in the treated eye of the subject from at least about 24 weeks following administration of the first pharmaceutical composition to at least 52 w'eeks following administration of the first pharmaceutical composition, preferably remain stable in the treated eye of the subject to at least 104 weeks following administration of the first pharmaceutical composition. The method according to any one of items 59 to 61, wherein aflibercept levels in the treated eye of the subject decrease less than about 40%, less than about 30%, less than about 20%, or less than about 10% relative to an aflibercept level measured in the treated eye of the subject within about 24 weeks after administration of the first pharmaceutical composition. The method according to any one of items 1-62, wherein the subject received a diagnosis of wet AMD < 6 months prior to administration of the first pharmaceutical composition.64. The method according to any one of items 1-63, wherein the subject exhibits at least an 80%, preferably at least a 90% reduction in anti-VEGF rejections at least 52 weeks after administration of the first pharmaceutical composition compared to a standard anti-VEGF therapy.65. The method according to item 64, wherein the standard anti-VEGF therapy comprises injection of aflibercept protein ever}' eight weeks.66. The method according to any one of items 1-65, wherein the subject received greater than six anti-VEGF treatments prior to administration of the first pharmaceutical composition.67. The method according to any one of items 1-65. wherein the subject received six or fewer anti-VEGF treatments prior to administration of the first pharmaceutical composition.68. The method according to item 67, wherein the subject is naive to treatment with an anti-VEGF therapy.EXAMPLES

[0125] The following examples illustrate preferred embodiments of the present invention and are not intended to limit the scope of the invention in any way. While this invention has been described in relation to its preferred embodiments, various modifications thereof will be apparent to one skilled in the art from reading this application.Example 1A Phase 1 / 2 Dose-Escalation and Randomized, Controlled, Masked Expansion Trial of Intravitreal Gene Therapy in Adults with Neovascular (Wet) Age-Related Macular Degeneration

[0126] METHODS

[0127] Study Design

[0128] A prospective, multicenter. Phase 1 / 2 dose-escalation and randomized, controlled, masked expansion trial in adults with wet AMD undergoing active anti-VEGF treatment who have demonstrated a clinical response consistent with anti-VEGF activity. The trial consistsof Dose Escalation and Dose Expansion phases, concurrent Steroid Optimization cohorts and a Population Extension phase as depicted below. An independent Data and Safety Monitoring Committee (DSMC) will evaluate safety throughout the trial and advise on study progress.

[0129] Study Endpoints

[0130] The trial has been designed with an appropriate population, sufficient size, and duration to initially characterize the safety and tolerability of a recombinant AAV (rAAV) comprising a capsid with the amino acid sequence of SEQ ID NO:4 and a heterologous nucleic acid comprising the nucleotide sequence of SEQ ID NO:9 (hereinafter “the rAAV”) in adults with wet AVID, evaluate the pre-specified endpoints and select an appropriate dose level for further evaluation.

[0131] The primary endpoint for this study is incidence and severity of TEAEs and SAEs, including clinically significant changes in safety parameters. Key secondary endpoints include:• Time to receiving the first supplemental aflibercept injection• Percentage of subjects requiring supplemental aflibercept injections over 52 weeks• Number of supplemental aflibercept injections over 52 weeks• Percent change in annualized mean number of anti-VEGF IVT injections before and after the rAAV injection (or Day 1 for the aflibercept only arm)• Change from baseline in BCVA over time (up to 52 weeks) as assessed using the ETDRS Visual Acuity Chart• Change from baseline in CST over time (up to 52 weeks) measured by SD OCT

[0132] Study Objectives

[0133] The study objectives include:® Evaluate the safety and tolerability of the rAAV following a single dose« Explore the clinical and pharmacodynamic activity and pharmacokinetics of the rAAV in subjects with wet AMD• Identity' the appropriate dose level of the rAAV for further evaluation• Characterize long-term safety and clinical activity of the rAAV gene therapy

[0134] Enrollment Plan

[0135] In Dose Escalation, the safety and tolerability of multiple dose levels of the rAAV will be explored following an open-label, 3+3 dose escalation design. The first subject ineach dose cohort will be a sentinel subject with reduced visual acuity. The starting dose level is 3x1()10vg / eye; dose level(s) of 1 x 10!0vg / eye or lower may also be evaluated based on emerging data. The maximum dose level will not exceed IxlO12vg / eye.

[0136] Once the rAAV dose level has been cleared by the DSMC, Steroid Optimization cohorts) may be opened to evaluate various prophylactic steroid regimens. Up to 40 subjects total may be enrolled to evaluate up to 4 different cleared rAAV dose ievel / steroid regimen permutations (n<10 per cohort). The initial Steroid Optimization cohort will have modified eligibility criteria; additional Steroid Optimization cohorts will receive an additional aflibercept injection on Day 28. Steroid Optimization may continue concurrently with additional Dose Escalation cohorts, Dose Expansion, or Population Extension phases of the trial.

[0137] In Dose Expansion, subjects (n=50) will be randomized 2:2:1 on Day -7 to receive one of 2 dose levels of the r (n=20 for each dose level) based on results from Dose Escalation, or aflibercept at a fixed regimen (n=10). Site personnel performing BCVA assessments, photographers, and the independent reading center will be masked to treatment assignment throughout the 24-month Follow-up Period. Other site personnel (including investigator) and subjects will be unmasked to treatment assignment but will be masked to the rAAV dose level. The Sponsor and its representatives and the site dose preparation / pharmacist will be unmasked to treatment assignment (including the rAAV dose level).

[0138] Following enrollment of the randomized, masked Dose Expansion phase, the openlabel Population Extension will evaluate the rAAV dose level(s) cleared by the DSMC in subjects with wet AMD with a reduced anti-VEGF injection burden relative to the Dose EscalatioiV'Expansion population (as defined by eligibility criteria). Up to 45 subjects total may be sequentially enrolled to evaluate up to 3 cleared rAAV dose levels (n<15 per cohort).

[0139] Interventions

[0140] If both eyes are eligible, the “study eye” to be injected will be determined by the Investigator and subject prior to Day -7. Ail subjects will receive aflibercept (2 mg IVT) in the study eye on Day -7 (>4 weeks from their last IVT anti-VEGF treatment). Subjects receiving the rAAV will begin an assigned prophylactic corticosteroid regimen in the study eye prior to rAAV IVT injection. On Day 1, subjects assigned to receive the rAAV willreceive a single IVT injection of the rAAV at the assigned dose level in the study eye. Subjects in Population Extension and additional Steroid Optimization cohort(s) will receive a second aflibercept injection in the study eye on Day 28. Subjects randomized to the aflibercept comparator arm in Dose Expansion will receive aflibercept approximately every 8 weeks (starting from Day -7). All subjects will be eligible for subsequent supplemental aflibercept therapy if protocol-specified criteria are met. Central subfield thickness (CST) criteria should be confirmed by the reading center prior to administration of supplemental aflibercept.>

[0141] A subject will receive aflibercept (2 mg IVT) if any of the following criteria are met:• Loss of >10 leters from average of the Day -7 and Day 1 BCVA measurements that is attributed to intraretinal or subretinal fluid observed by the Investigator• Increase of >75 pm from average of Day -7 and Day CST measurementsfshould be confirmed by the reading center in expedited fashion prior to administration of supplemental aflibercept)• Presence of or new vision-threatening hemorrhage due to wet AMD as determ ined by Investigator: collect color fundus photography and submit to reading center

[0142] Supplemental aflibercept may also be administered after discussion with the Medical Monitor, if, in the opinion of the Investigator, significant disease activity is present that does not meet the above criteria. Supplemental aflibercept may be administered at unscheduled visits if necessary.

[0143] Prophylactic Corticosteroids

[0144] Prophylactic and concomitant corticosteroids will be administered to all subjects receiving the rAAV (but not subjects randomized to aflibercept treatment). Subjects in the initial Dose Escalation cohorts and subjects initially enrolled in Dose Expansion will receive the corticosteroid regimen as specified in the table below.

[0145] Corticosteroid Regimen (only for subjects receiving the rAAV)BID = twice per day; IVT = intravitreal; QD = once per day; QID = 4 times per day QOD once every other day; TID - 3 times per day; wks = weeks

[0146] The following are alternative steroid regimens evaluated (or to be evaluated) in this study:

[0147] Modifications to Corticosteroid Regimen

[0148] Should the protocol-specified corticosteroid regimen described above prove to be insufficient or poorly tolerated for an individual subject, the Investigator may modify the corticosteroid regimen in consultation with the Medical Monitor. Additional medications or modifications to corticosteroid regimen may be used to mitigate side effects per Investigator discretion.

[0149] Clinically significant breakthrough (defined as an increase in inflammation that in the judgment of the investigator requires an increase in intensity of treatment) or rebound inflammation (defined as inflammation that recurs following cessation of corticosteroid treatment) requiring therapeutic intervention in subjects who receive the rAAV as determinedby the Investigator should be managed per clinical standard of care. While management is ultimately up to the Investigator, the Sponsor recommends the following regimens:» If original prophylactic steroid regimen is topical difluprednate ophthalmic emulsion 0.05% (marketed as Durezol® in the United States), then: o Topical difluprednate ophthalmic emulsion 0.05% in the study eye for 21 weeks as follows:■ Six times per dayx3 weeks■ Five times per day x 3 weeks■ QID x 3 weeks8TID x 3 weeks* BID x 3 weeks» QD x 3 weeks■ QOD x 3 weeks8Discontinue» If original prophylactic steroid regimen is prednisolone acetate ophthalmic suspension, USP 1.0% (with or without subtenon triamcinolone acetonide), then: o Topical prednisolone acetate ophthalmic suspension USP 1.0% in the study for 20 weeks as follows:8Six times per day x 4 weeks a Five times per day x 4 weeks a QID x 4 weeks8TID x 4 weeks8BID x 2 weeks8QD x 2 weeks a Discontinue o Administration of subtenon triamcinolone acetonide 40 mg in addition to or as an alternative to topical prednisolone acetate ophthalmic suspension is optional and up to the discretion of the investigator.® If breakthrough or rebound intraocular inflammation persists or recurs following a second course of local ocular corticosteroid treatment, IVT steroid therapy may be considered as a next step following consultation with the Medical Monitor.

[0150] Oral prednisone may also be considered if necessary' following consultation with the Medical Monitor.

[0151] Results

[0152] As of the data cut-off of 04 June 2024, 132 subjects have been enrolled: 15 in initial Dose Escalation / Expl oration, 51 in Dose Expansion, 32 in Population Extension, 29 inSteroid Optimization, and 5 in the higher dose cohort of 6* 10i0vg / eye. Summary of subjects enrolled to-date is provided in the table below:

[0154] Ophthalmic exams were performed at each visit to evaluate for IOI (white blood cells, flare, and haze). Below is a summary' of the IOI data observed through the cut-off date of 04 June 2024.

[0155] In the initial Dose Exploration (n=l 5) no Grade >1 inflammation episodes as assessed by Standardization of Uveitis Nomenclature (SUN) and National Eye Institute (NEI) scores were reported post rAAV administration. Two subjects dosed at 3>< 1010vg / eye, were reported to have intermittent pigmented cells starting at Week 12. In the first subject who was a sentinel subject, pigmented cells were reported in the anterior chamber and the investigator has been treating with topical steroids. The second subject was reported to have trace pigmented and white blood cell (WBC) in the vitreous but the investigator did not modify prophylactic steroid regimen and has not treated with additional steroids. In Dose Escalation (n=5), in one subject at the 6* 1010vg / eye, 1+ vitreous cells were observed at Week 10. The subject was treated with additional durezol; no cells were observed at Week 12. No inflammation has been reported in the other subjects to-date.

[0156] In Steroid Optimization (n=29), 1+ vitreous cells were reported in a single subject (dosed with 3xl010vg / eye) at the Day 28 visit, The subject was assigned to subtenon triamcinolone on Day 1 and 16-week prednisolone starting at Day -3 for the prophylactic steroid regimen. Following the observation of the vitreous cells, the subject was switched from prednisolone to durezol; trace cells were reported at Week 8 and no cells reported at Week 12. No inflammation has been reported in the other subjects to-date.

[0157] In Population Extension (n=32), in one subject dosed with rAAV 1 x 10i0vg / eye, 1+ vitreous cells (mixed pigmented and unpigmented cells at Week 16: subject completed the prophylactic durezol and did not receive additional treatment. At Week 24, in the same subject, 3+ vitreous cells, 0.5+ anterior chamber cells, moderate (2+) vitreous haze, and faint (1+) anterior chamber flare w'as observed. The next day. 2+ vitreous cells, 0.5+ anterior chamber cells, and moderate (2+) vitreous haze was observed. The subject is being treated with additional durezol. In a second subject dosed with rAAV I * 1010vg / eye, trace pigmented cells were observed in the anterior chamber starting at Day 28; the investigator has been treating with additional durezol. In one subject dosed with rAAV 3* IO50vg / eye; trace vitreous cells were observed starting at Week 8; the subject completed the prophylactic steroid regimen and no additional steroids were given. No inflammation has been reported in the other subjects to-date.

[0158] In Dose Expansion (n=41 receiving rAAV), 1 + mixed pigmented and WBC was reported in the anterior chamber for one subject at an unscheduled visit at Week 17. The subject was treated with additional durezol and no cells were reported at the Week 20 visit. In two subjects, 1+ pigmented cells were reported in the anterior chamber at Week 16 and Week 40. No treatment was initiated (including modification of the prophylactic steroid regimen) and by the next visit, no cells were reported. Additionally, trace cells in the anterior chamber and / or vitreous were reported in 3 subjects; the investigator did not modify the prophylactic steroid regimen.

[0159] 52 Week Results

[0160] Interim results from the Phase 2a randomized dose expansion study showed that the rAAV w'as safe and well tolerated and exhibited durable clinical activity' in adults with neovascular (wet) age-related macular degeneration (nAJvlD) and severe disease activity who required >6 anti-VEGF injections in the prior 12 months.

[0161] Reported here are week 52 results from the rAAV 3 x 1010vg / eye dose group in the Phase 2b population extension cohort, which comprises adults with nAMD who received 1-6 anti-VEGF injections in the prior 12 months.

[0162] The Phase 2b population extension cohort includes adults with nAMD and best corrected visual acuity (BCVA) of 34-83 letters. Participants received IVT AFLB 2 mg on Days -7 and 28. A single IVT injection of rAAV 3xl010vg / eye or 1 *10i0vg / eye wasadministered on Day 1 . Study endpoints include adverse events, supplemental anti-VEGF injections, and change from baseline in BCVA and central subfield thickness (CST),

[80163] A total of 30 participants received IVT 4D-150 at the planned Phase 3 dose of 3x 10i0vg / eye. Demographics and baseline characteristics are summarized at Figure I and in the table below.

[0164] Population characteristics in Phase 2b (Population Extension) are shown in Figure 1 and below

[0165] Table: Baseline Characteristics* Incl udes Day -7 aflibercept injection. BCVA, best corrected visual acuity; ETDRS, Early Treatment Diabetic Retinopathy Study; SD, standard deviation; VEGF, vascular endothelial growth factor.

[0166] As of 15 January 2025, 29 participants had completed 52 weeks of follow up. Treatment with the rAAV was well tolerated; there were no rAAV related serious adverse events and no hypotony, endophthalmitis, occlusive or non-occlusive retinal vasculitis, or choroidal effusions. Mild vitritis was reported in one participant and was transient. The mean cumulative number of supplemental aflibercept injections at w'eek 52 was 0.97; 57% of participants remained injection free and 70% received <1 supplemental injection. Visual acuity and CST remained stable through week 52. The mean ±SD change from baseline in BCVA was +1.0 ±8.5 ETDRS letters and the mean change in CST was -11.0 ±66.2 jun. In recently diagnosed participants (<6 months, n=l 5), mean changes of +2.4 +8.7 ETDRS letters and -10 ±56.6 pm in BCVA and CST, respectively.

[0167] A robust reduction in anti-VEGF injection burden was observed in the Phase 2b trial over 52 weeks. A -83% reduction in anti-VEGF injection burden was observed in the rAAV arm in the overall Phase 2b population vs Q8W aflibercept control (comparator) arm. For subjects in the Phase 2b population with a recent diagnosis, a -94% reduction in anti- VEGF injection burden w'as observed in the rAAV arm vs Q8W aflibercept control arm. The rAAV was well tolerated, with no significant intraocular inflammation (IOI), no serious adverse events and no treatment-related hypotony, endophthalmitis, occlusive / non-occlusive retinal vasculitis, or choroidal effusions observed in the rAAV treated population over the 52 week period.

[0168] These results are summarized in Figures 2-6.

[0169] Aflibercept expression was maintained in patients in the study that remained free of supplemental anti-VEGF injections for the duration of the study. Aflibercept expression (measured by ELISA assay using VEG-A capture / Ab) in the aqueous humor of the treated eye of these patients, measured at 24 weeks, 36 weeks, 52 weeks, 76 weeks, and 104 weeks following administration of the rAAV (3E10 vg / eye dose).

[0170] Conclusion

[0171] The safety profile of the rAAV to date has been favorable, with minimal evidence of IOI observed to date using any of the steroid immunoprophylaxis [durezol taper (20-week taper), subtenon (Day 1) plus prednisolone taper (16-20 weeks), or ozurdex (Day 1 and Week 12)]. The 3E10 dose was well-tolerated across all populations with no rAAV-related serious adverse events. No patients had rAAV-related hypotony, endophthalmitis, vasculitis, choroidal effusions or retinal artery' occlusions during the 52 week period. 2.8% of patients receiving the 3E10 dose experienced transient > +1 VC that resolved by the following visit. Importantly, 99% of patients were able to complete their steroid taper on time.[08.172] Preliminary clinical data from Dose Escalation / Exploration (N=T 5) and Dose Expansion (N=51) suggests a single IVT injection of the rAAV in subjects with nAMD who required frequent anti-VEGF injections in the prior year (6-12 injections) is safe and well- tolerated and demonstrates encouraging signs of activity at all dose levels through Week 36 and Week 24.

[0173] In Dose Escalation / 'Exploration, a dose response was also observed for percentage of subjects injection-free and for reduction in annualized anti-VEGF injection rates; at Week 36, 20% to 80% were supplemental anti-VEGF injection-free and the mean annualized number of injections was reduced by 64% to 81 %. At the 3 >< 1010vg / eye dose level, 80% remained anti-VEGF injection free and the mean annualized injection rate was reduced by 81%.

[0174] In Dose Expansion, at Week 24, 50% to 63% were supplemental anti-VEGF injection-free and the mean annualized number of injections was reduced by 85% to 89%. At the 3xI0iUvg / eye dose level, 63% were supplemental anti-VEGF injection-free and the mean annualized number of injections was reduced by 89%. At Week 52, a -83% reduction in anti-VEGF injection burden compared to 2Q8W aflibercept comparator population was observed in the overall Phase 2b population and a -94% reduction was observed in recently diagnosed (< 6 months from screening) subjects.EXAMPLE 2A Phase 3, Randomized, Double-Masked, Active-Controlled Trial of a Single Intravitreal Injection of rAAV in Adults with Macular Neovascularization Secondary' to Age-Related Macular Degeneration (nAMD)

[0175] The trial is designed to evaluate the efficacy, safety and durability7of a single intravitreal (IVT) injection (3 x 101Gvg) of a recombinant AAV (rAAV) comprising a capsid with the amino acid sequence of SEQ ID NO:4 and a heterologous nucleic acid comprising the nucleotide sequence of SEQ ID NO:9 (hereinafter “the rAAV”) compared with aflibercept (2mg IVT) every 8 weeks (Q8W) in adults nAMD. The primary efficacy objective is to demonstrate non-inferiority of a single injection of the rAAV to aflibercept in the mean change in BCVA from baseline to week 52. The Phase 3 trial will consist of two Phase 3 studies: one will enroll treatment naive patients only and the other will enroll both treatment naive and treatment experienced (diagnosed within 6-months) patients. An overview of the study is provided at Figure 7.

[0176] Design

[0177] The trial (estimated duration = 2 years and 2 months) is a multicenter, randomized, double-masked, active-controlled trial in adults with macular neovascularization (MNV)secondary to age related macular degeneration (nAMD) with no prior exposure to IVT anti- vascular endothelial growth factor (anti-VEGF) therapy (i.e., treatment naive). The objective is to demonstrate non-inferiority of a single injection of the rAAV to the active control in the mean change in BCVA from baseline to week 52. Prospective subjects (at least 50 years of age) will undergo screen evaluation (Screening visit) between Week -7 to -5 to determine eligibility, followed by a run-in period between Week -5 and Day 1 to confirm eligibility. Eligible subjects will be randomized on Day 1. All subjects who are not randomized at Day 1 due to eligibility reasons are considered screen failures. An overview of the Trial Design is illustrated in the Table below:Topical corticosteroids with 20-week taper (starting on Day -3, all subjects)* Supplemental aflibercept (AFLB; 2 mg IVT) administered to any subject where Disease Activity Criteria met at sham or interim visits

[0178] For each subject, one eye will be selected as the “study eye.” If both eyes are eligible, the eye with the worse BCVA at the Screening Visit will be selected as the study eye. During the trial, treatment of the contralateral (i.e., non-study) eye is permitted and should continue per standard of care, including anti-VEGF therapy for nAMD, as applicable.

[0179] To qualify for randomization, subjects will be required to demonstrate anatomic response to aflibercept. Specifically, all subjects will receive one aflibercept injection (2 mg IVT) in the study eye on Week -5 (baseline). At the Week -1 visit; response will be assessed by the Reading Center and defined as: reduction in central subfield thickness (CST) of >15% OR complete resolution of intraretinal fluid (IRF) and / or subretinal fluid (SRF). At Week -1, if the Reading Center has confirmed eligibility (i.e. both aflibercept-responsiveness and per all applicable Reading Center eligibility criteria), the subject will receive a second aflibercept injection in the study eye at Week -1.

[0180] All subjects will begin a 20-week tapered course of topical corticosteroids in the study eye starting on Day -3.

[0181] On Day 1, subjects will be randomized in a 1:1 allocation to one of the following treatment arms: (1) Investigational: rAAV (3 xlO10vg) (n=200) (2) Active Comparator: AFLB (aflibercept 2 mg) (n:::200)

[0182] On Day 1, subjects randomized to the rAAV arm will receive a single IVT injection of 3* IO'10vg of the rAAV in the study eye; subjects randomized to the AFLB arm will receive a sham injection in the study eye. At Week 4, all subjects will receive a third aflibercept injection in the study eye.

[0183] Beginning at Week 12, subjects in the AFLB arm will receive additional AFLB injections following a fixed dose regimen Q8W through Week 100. To maintain masking, subjects randomized to the rAAV arm will receive sham injections at these visits.

[0184] Disease Activity' Criteria have been defined to provide supplemental aflibercept for subjects in the rAAV arm if needed. To maintain masking, all subjects will be evaluated at 4- week intervals beginning at Week 12 for the following Disease Activity Criteria:® >5 letters decrease in BCVA AND a >50-micron increase in CST due to nAMD activity' Or® >10 letters decrease in BCVA due to nAMD activity' Or« >100-micron increase in CST due to nAMD activity Or® Presence of new7macular hemorrhage due to nAMD that, in the opinion of the investigator, is sight-threatening

[0185] If one or more Disease Activity7Criteria are met for a given subject at a Fixed Regimen Visit® Subjects in the rAAV arm will forgo the sham injection and receive supplemental aflibercept (2mg IVT) at the visit® Subjects in the AFLB arm will receive Fixed Regimen AFLB (2 mg IVT) as scheduled for the visit.

[90186] If one or more Disease Activity Criteria are met for a given subject at an Interim Visit (i.e., no Fixed Regimen Q8W AFLB / sham is scheduled):® Subjects in the rAAV arm will receive supplemental aflibercept (2mg IVT) at the visit® Subjects in the AFLB arm will receive supplemental aflibercept (2mg IVT) at the visit.

[0187] During the 104-week Follow-up Period, all subjects will undergo assessments at 4- week intervals to assess safety and clinical outcomes.Ocular Inclusion Criteria1. Treatment naive macular neovascularization (MN V) secondary to nAMD in the study eye2. Active subfoveal MNV or juxtafoveal / extrafoveal MNV with a subfoveal component (where activity is defined as evidence of SRF, IRF, subretinal hyperreflective material, or leakage) identified by fluorescein angiography (FA) or spectral domain optical coherence tomography (SD-OCT) , in the study eye, at the Screening Visit confirmed by the Reading Center3. MNV lesion in the study eye of any type (i.e. predominantly classic, minimally classic, or occult [including polypoidal choroidal vasculopathy and retinal angiomatous proliferation]) at the Screening Visit, confirmed by the Reading Center, which exhibits all of the following characteristics: a. Total lesion size of 9 disc areas or less (inclusive of blood, fibrosis, atrophy or neovascularization) on FA b. MNV component area at least 50% of total lesion size on FA c. MNV exudation (i.e. presence of fluid) on SD-OCT4. CST <500um in the study eye at Screening visit, confirmed by the Reading Center5. Demonstrated clinical response to aflibercept and functional stability in the study eye: a. From Week -5 to Week -7 : a > 15% reduction in CST or complete resolution of IRF and / or SRF, determined by SD-OCT and confirmed by the Reading Centerb. At Day 1 ; BCVA measurement must not have decreased by 15 ETDRS letters or more compared to BCVA at the Screening visit6. BCVA between 25 and 78 ETDRS letters, inclusive (20 / 320-20 / 32 Snellen equivalent) in the study eye at the Screening Visit7. BCVA >34 ETDRS letters (-20 / 200 Snellen equivalent) in the contralateral eye at the Screening Visit8. Study eye amenable to IVT injection identified by the Investigator prior to Week -5

[0188] Interventions

[0189] Aflibercept (2mg IVT) will be administered to all subjects at Week -5, Week -1 and Week 4 (i.e., Aflibercept Run-in Period).

[0190] Corticosteroids: All subjects will initiate topical corticosteroids in the study eye on Day -3 and will complete the protocol-specified regimen and 20-week tapering schedule.

[0191] Randomized interventions

[0192] Investigational: rAAV (3x10wvg) will be administered as a single dose, 50 pL IVT injection on Day 1 . Subjects assigned to AFLB arm will receive a sham injection on Day 1.

[0193] Active Control: Aflibercept (2mg IVT) will be administered Q8W starting at Week 12 in the AFLB arm. Subjects assigned to rAAV arm will receive sham injections at these visits (unless Disease Activity Criteria met).

[0194] Supplemental Aflibercept (2mg IVT) will be administered if a subject meets protocol-specified Disease Activity' Criteria.

[0195] Disease Activity Criteria: Beginning at Week 12, all subjects will be evaluated at 4-week intervals to assess whether the subject meets one or more of the following Disease Activity Criteria. The BCVA reference value is the average of BCVA measurements at Weeks 4 and 8. The CST reference value is the CST measurement at Week 8:® >5 leter decrease in BCVA AND a >50-micron increase in CST due to nAMD activity; or« >10 letter decrease in BCVA due to nAMD activity, or® >100-micron increase in CST due to nAMD activity, or« Presence of new macular hemorrhage (NMH) due to nAMD that, in the opinion of a medical practitioner, is sight-threatening

[9196] Subjects in the rAAV arm who meet the Disease Activity Criteria in any of the scheduled sham-injection visits will forgo the sham injection and receive supplemental aflibercept (2 mg IVT) at the visit.

[0197] ENDPOINTS[0G198j Primary’ Endpoint: Mean change from baseline in BCVA ETDRS leter score at Week 52.

[0199] Secondary Endpoints:® Mean annualized number of aflibercept injections after Week 4 through Weeks 52 and 104♦ Incidence and timing of supplemental aflibercept injections after Week 4 through Weeks 52 and 104® Proportion of subjects not requiring supplemental aflibercept injections after Week 4 through Weeks 42 and 104 in the 4AAV arm® Time to first supplemental aflibercept injection after Week 4 in the rAAV arm® Mean change from baseline in CST over time through Weeks 52 and 104« Mean change from baseline in BCVA ETDRS leter score over time through Weeks 52 and 104

[0200] Exploratory Efficacy Endpoints« Distribution of change from baseline in BCVA ETDRS leter score over time through weeks 52 and 104 in categories of > 15 leter loss, 10-14 leters loss, 5-9 letter loss, <5 letter change, 5-9 leter gain, 10-14 leter gain and >15 letter gain.« Percentage of subjects with absence of intraretinal fluid (IRF), subretinal fluid (SRF) and / or both, over time through Week 52 and 104* Mean change from baseline in Visual Function Questionnaire (VFQ-25) and EQ-5D- 5L score over time

[0201] SrfegSteBids

[0202] All subjects in the study will initiate topical corticosteroids in the study eye on Day -3 and will complete the protocol-specified regimen and 20-week tapering schedule.

[0203] The corticosteroid regimen is shown in the Table belowBID = twice per day; QD = once per day; QID = 4 times per day; QOD = every other day;TID = 3 times per day

[0204] A summary of the interventions and concomitant therapy discussed above is provided in the below table:1 Aflibercept (2 mg IVT) administered Q8W starting at Week 12 in the AFLB arm (Weeks 12, 20, 28, 36, 44, 52, 60, 68, 76, 84, 92 and 100). Subjects assigned to rAAV arm will receive sham injections at the same visits2 Disease Activity Criteria will be evaluated in all subjects beginning at Week 12.[00)51 The investigational product (rAAV) is supplied as a solution intended for IVT administration and is supplied in a 2 mL vial, containing 0.3 mL of product. The investigational product was manufactured, packaged, and labeled according to Good Manufacturing Practice (GMP) regulations. The rAAV (3 x!0lcvg) will be administered as a single dose (50 pl) IVT injection in the study eye on Day 1. The IVT injection procedure will be performed by the injecting (unmasked) Investigator experienced in the procedure per standard of care.

[0206] Aflibercept (marketed as EYLEA®) is a VEGF inhibitor indicated for the treatment of patients with nAMD. The recommended dose of aflibercept is 2 mg (0.05 mL of 40 mg / mL solution) administered by IVT injection Q4W for the first 3 months, followed by 2 mg (0.05 mL of 40 mg / mL solution) via IVT injection Q8W.

[0207] During the Aflibercept Run-in Period, all subjects will receive aflibercept (2 mg IVT) in the study eye on Week ~5, Week -1, and Week 4 per the approved prescribing information. Aflibercept injections should not occur earlier than 21 days following the prior IVT administration.

[0208] Subjects randomized to the AFLB comparator arm will receive aflibercept (2 mg IVT) Q8W beginning at Week 12 through Week 100 as per the approved prescribing information and sham injections on the alternating 4-week interval visits.

[0209] Subjects assigned to rAAV arm will receive sham injections at the same visits (unless Disease Activity Criteria are met).

[0210] WTiile the materials and methods of this in vention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the method described herein without departing from the concept, spirit and scope of the invention. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention.

Claims

CLAIMS1. A method of treating macular neovascularization secondary to age-related macular degeneration in a human subject in need thereof, the method comprising: intravitreally administering to an eye of the subject in need of such treatment, a single unit dose of a first pharmaceutical composition comprising a recombinant adeno- associated virus (rAAV), said rAAV comprising (i) a capsid protein comprising the amino acid sequence of SEQ ID NO:4 or a sequence at least 98% identical thereto and (ii) a heterologous nucleic acid comprising the nucleotide sequence of SEQ ID NO:9 or a sequence at least 95% identical thereto, wherein the unit dose comprises about 3x10i0vector genomes (vg) of the rAAV; and administering to the eye of the subject multiple doses of a second pharmaceutical composition comprising aflibercept protein or a biosimilar thereof, the second pharmaceutical composition administered at a dose equivalent to about 2 mg aflibercept, wherein(i) one dose of the second pharmaceutical composition is administered about 5 weeks prior to administration of the first pharmaceutical composition,(ii) one dose of the second pharmaceutical composition is administered about one week prior to administration of the first pharmaceutical composition, and(iii) one dose of the second pharmaceutical composition is administered about four weeks after administration of the first pharmaceutical composition, and administering to the eye of the subject one or more corticosteroids for a period of at least about 20 weeks beginning 1 -7 days prior to administering the first pharmaceutical composition and during which period the one or more corticosteroids are administered at least every other day.

2. The method according to claim 1, wherein the one or more corticosteroids are administered according to following schedule, wherein the first dose of corticosteroid is administered about three days prior to administering the first pharmaceutical composition:a. corticosteroids equivalent to difluprednate ophthalmic emulsion 0.05% or difluprednate ophthalmic emulsion 0.05% four times per day for about 31 days administered topically; followed by b. corticosteroids equivalent to difluprednate ophthalmic emulsion 0.05% or difluprednate ophthalmic emulsion 0.05% three times per day for about 28 days administered topically; followed by c. corticosteroids equivalent to difluprednate ophthalmic emulsion 0.05% or difluprednate ophthalmic emulsion 0.05% two times per day for about 28 days administered topically; followed by d. corticosteroids equivalent to difluprednate ophthalmic emulsion 0.05% or difluprednate ophthalmic emulsion 0.05% once per day for about 28 days administered topically; followed by g. corticosteroids equivalent to difluprednate ophthalmic emulsion 0.05% or difluprednate ophthalmic emulsion 0.05% once every other day for about 28 days administered topically, preferably wherein the administration period is about 143 days.

3. The method according to claim 1 or 2, wherein one or more additional 2 mg doses of the second pharmaceutical composition are administered to the subject at least twelve ■weeks after administration of the first pharmaceutical composition, if one or more of the following criteria are met:(v) >5 letter decrease in BCVA compared to a reference BCVA and a >50-micron increase in CST compared to a reference CST resulting from nAMD activity as determined by a qualified professional; or(vi) >10 letter decrease in BCVA compared to a reference BCVA resulting from nAMD activity as determined by a qualified professional, or(vii) >100-micron increase in CST compared to a reference CST due to nAMD activity as determined by a qualified professional, or(viii) presence of a new macular hemorrhage (NMH) due to nAMD that, in the opinion of a medical practitioner, is sight-threatening4. The method according to claim 3, wherein the BCVA reference value is the average of BCVA measurements in the subject at four weeks and § weeks following administration of the first pharmaceutical composition and / or the CST reference value is the CST measurement in the subject at four weeks following administration of the first pharmaceutical composition.

5. The method according to any one of claims 1-4, wherein aflibercept levels in a treated eye of the subject remain stable in the subject for at least about 52 weeks, preferably for at least about 104 weeks following administration of the first pharmaceutical composition, preferably wherein an aflibercept level in the treated eye comprises an aflibercept concentration in the aqueous humor of the treated eye.

6. The method according to claim 5, wherein the subject does not receive any further administrations of the second composition beginning about 8 w'eeks after administration of the first composition and continuing at least 24 weeks, at least 36 weeks, at least 48 weeks, preferably at least 52 weeks after administration of the first composition.

7. The method according to claim 5 or 6, wherein aflibercept levels remain stable in the treated eye of the subject from at least about 24 weeks following administration of the first pharmaceutical composition to at least 52 weeks following administration of the first pharmaceutical composition, preferably remain stable in the treated eye of the subject to at least 104 weeks following administration of the first pharmaceutical composition.

8. The method according to any one of claims 5 to 7, wherein aflibercept levels in the treated eye of the subject decrease less than about 40%, less than about 30%, less than about 20%, or less than about 10% relative to an aflibercept level measured in the treated eye of the subject within about 24 weeks after administration of the first pharmaceutical composition.

9. The method according to any one of claims 1-8, wherein the subject received a diagnosis of wet AMD < 6 months prior to administration of the first pharmaceutical composition.

10. The method according to any one of claims 1-9, wherein the subject exhibits at least an 80%, preferably at least a 90% reduction in anti-VEGF rejections at least 52 weeks after administration of the first pharmaceutical composition compared to a standard anti-VEGF therapy.11 . The method according to claim 10, wherein the standard anti-VEGF therapy comprises injection of aflibercept protein every eight weeks.

12. The method according to any one of claims 1-11, wherein the subject received greater than six anti-VEGF treatments prior to administration of the first pharmaceutical composition.

13. The method according to any one of claims 1-11, wherein the subject received six or fewer anti-VEGF treatments prior to administration of the first pharmaceutical composition.

14. The method according to claim 13, wherein the subject is naive to treatment with an anti-VEGF therapy.

15. The method according to any one of claims 1-14, wherein the first pharmaceutical composition comprises a buffering composition comprising:(iv) about 5 mM to about 20 mM of a buffering agent, preferably a Tris buffer;(v) about 100 mM to about 250 mM of a pharmaceutically acceptable salt, preferably NaCI; and(vi) about 0.0001 % (w / v) to about 0.01 % (w / v) of a non-ionic surfactant; wherein the buffering composition has a pH of about 7.0 to about 9.0.

16. The method according to claim 15, wherein the buffering composition comprises:(iv) about 9 mM to about 20 mM of the buffering agent;(v) about 140 to about 200 mM of the pharmaceutically acceptable salt; and(vi) about 0.001% (w / v) to about 0.01% (w / v) of the non-ionic surfactant; wherein the buffering composition has a pH of about 7.3 to 8.6.

17. The method according to claim 16, wherein the buffering composition comprises about 10 mM Tris, about 180 mM NaCL, about 0.005% Plutonic F68 and wherein the pH of the buffering composition is about 7.4 to about 8.4 at 25 "C.

18. The method according to claim 17, wherein the pH of the buffering composition is about 7.6 to about 8.2 at 25 °C.

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