Methods for treating diabetic macular edema with adeno-associated virus

Abstract

Pharmaceutical compositions comprising adeno-associated virus (AAV) and their use in treating diabetic macular edema 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 Treating Diabetic Macular Edema with Adeno- Associated VirusCROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Patent Application Serial Nos. 63 / 671,910, filed July 16, 2024, and 63 / 743,293, filed January 9, 2025, the entire contents of each of which are incorporated herein by reference.SEQUENCE LISTING SUBMISSION VIA EFS-WEB

[0092] A computer readable XML file, entitled “090400-5030 WO Sequence Listing” created on July 14, 2025, with a file size of 18,874 bytes contains the sequence listing for this application and is hereby incorporated by reference in its entiretyBACKGROUND OF THE INVENTION

[0003] Diabetic retinopathy (DR) is a major complication of diabetes mellitus and is a leading cause of visual loss in the working age population. DR may be non-proliferative (NPDR), with no new blood vessel growth, or proliferative DR (PDR), with new abnormal blood vessel growth within the retina or choroid. Diabetic macular edema (DME) is a complication of DR and is another example of an ocular disease affecting the macula. DME affects up to 10% of people with diabetes and is caused by fluid accumulation in the macula. DME is the most frequent cause of sight loss in people with DR. Available therapies for treating DME include laser and anti-vascular endothelial growth factor (anti- VEGF) drugs 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 need 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, andmost 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 (IOP); the risks of these complications are likely to increase with repeated IVT injections.[0(106] Therefore, there is a need in the art for therapies for ocular neovascular diseases such as DME, 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 diabetic macular edema (DME) with improved efficacy and / or reduced adverse side effects.

[0008] In some embodiments, the present disclosure provides methods for treating DME comprising intraocularly administering to the eye of a subject a single unit dose of a first pharmaceutical composition comprising a recombinant adeno-associated virus (rAAV) comprising a 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 10!° vector genomes per eye to about 6x 1010vector genomes per eye, preferably wherein the unit dose comprises about l >:10i 0vector genomes per eye or about 3x 10‘° vector genomes per eye.

[0009] In other embodiments, the present disclosure provides methods for treating DME 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 a 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 IxlO10vector genomes per eye to about 6x 1010vector genomes per eye, preferably wherein the umt dose comprises about lxlOinvector genomes per eye or about 3* 1010vector genomes per eye, and (ii) administering one or more corticosteroids to the eye of the subject before, simultaneous with and / or after administration of the first pharmaceutical composition, wherein at least one corticosteroid is administered topically.

[0010] In preferred embodiments, the one or more corticosteroids comprises difluprednate. In some preferred embodiments, the one or more corticosteroids comprises difluprednate ophthalmic emulsion 0.05%.

[0011] In other embodiments, a method for treating a subject with DME 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 1 x 101'3vector genomes per eye to about 6X1010vector genomes per eye, preferably wherein the unit dose comprises about 1 xJO10vector genomes per eye or about 3>: 10!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.

[0012] 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.

[0013] In some preferred embodiments, the rAAV comprises a heterologous nucleic acid encoding one or more anti-VEGF gene products, 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. In certain embodiments, the peptide is inserted following any of the amino acids in positions 584-591 in VP1 of AAV2 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 AA V serotype. In some embodiments, the capsid protein furthercomprises 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 substitation in another AAV serotype. In preferred embodiments, the one or more anti-VEGF gene products comprises aflibercept. In particularly preferred embodiments, the one or more anti-VEGF gene products comprises aflibercept and an artificial miRNA that reduces expression of VEGF-C.DESCRIPTION OF THE DRAWINGS

[0014] FIG. 1 illustrates aspects of a Phase II clinical study that enrolled DME patients with high CST and employed stringent supplemental criteria, with a focus on safety and dose selection. The 32 week interim data is described in Example 2.

[0015] FIG. 2 illustrates the mean change in BCVA (ETDRS leters) from baseline in DME patients administered a single 3E10 vg / eye (top) or 1E10 vg / eye (botom) intravitreal dose of rAAV (comprising a capsid of SEQ ID NO:4 and a nucleic acid of SEQ ID NO:9) and three loading doses of aflibercept at 32 weeks. Supplemental injections of aflibercept are shown below the graph. Patients receiving the 3E10 vg / eye dose demonstrated improved and sustained visual acuity from baseline of +8.4 letters at 32 weeks.

[0016] FIG. 3 illustrates mean change in CST (urn) from baseline in DME patients administered a single 3E10 vg / eye or IE 10 vg / eye intravitreal dose of rAAV (comprising a capsid of SEQ ID NO:4 and a nucleic acid of SEQ ID NO:9) and three loading doses of aflibercept at 32 weeks. Patients receiving the 3E10 vg / eye dose demonstrated improved and sustained anatomic control from baseline of -194 pm at 32 weeks.

[0017] FIG. 4 illustrates the mean cumulative number of aflibercept injections (not including the loading doses) from weeks 8 to 32 in DME patients receiving a 3E10 vg / eye dose or 1E10 vg / eye dose of rAAV (comprising a capsid of SEQ ID NO:4 and a nucleic acid of SEQ ID NO:9) compared to standard aflibercept treatment (2mg Q8W). Patients receiving the 3E10 vg-'eye treatment demonstrated an 86% reduction in treatment burden vs. aflibercept 2mg Q8W.

[0018] FIG. 5 illustrates the proposed dosing regimen for a phase III clinical trial on treatment naive patients with a diagnosis of DME within 6 months of screening, CST>325pmand confirmed on-study response to aflibercept with a single 3E10 vg / eye dosage of an rAAV comprising a capsid of SEQ ID NO:4 and a nucleic acid of SEQ ID NO:9.DETAILED DESCRIPTION OF THE INVENTION

[0019] Definitions

[0020] 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, z\AV type 8, AAV type 9, AAV type 10, avian AAV, bovine AAV, canine AAV, equine AAV, ovine AAV, primate AAV, and non- primate AW. In certain embodiments, the AAV is AAV2 or a variant thereof.

[0021] 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 drag formulation (see, for example, Berge, et al. “Pharmaceutical Salts,” J. Phann. Sci.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).

[0022] 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."

[0023] 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 die 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.

[0024] 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.

[0025] As used herein, the term “nucleic acid” is interchangeable with “polynucleotide” or “nucleic acid molecule” and a polymer of nucleotides is intended.

[0026] 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 regulatory' 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 with 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.

[0027] "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, without 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 well 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).

[0028] 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).

[0029] 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 withoutlimitation transcription of the polynucleotide into messenger RNA (mRNA), transfer RNA (tRNA), small hairpin RNA (shRNA), small interfering RNA (siRNA) or any other RNA product, and the translation of an mRN A 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.

[0030] “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 regulators' sequences have not been completely defined, DNA fragments of different lengths can have identical promoter activity.

[0031] 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 of interleukin-5. Gene, 79(2), 269-277, the contents of which are incorporated herein by reference.

[6032] 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.

[0033] 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).

[1134] 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 sidechains, amino acids having hydrophilic acidic side chains, and amino acids having hydrophilic basic side chains. Common properties may also be amino acids having hydrophobic 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 electrically 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.

[0035] 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.

[0036] 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.).

[0037] 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) aparticular disease stale (e.g., a disorder associated with complement dysfunction). Accordingly, an effective amount of an infectious rAAV virion is an amount of the infectious rAAV 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, band-held dynamometry, Vignos Scale and the like as described herein and as known in the art.

[0038] 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 1 1.5. The tenn "about" also accounts for typical error or imprecision in measurement of values.Detailed Description[0(139] Provided herein are methods for the treatment of DME in a subject in need thereof, the methods comprising intraocularly administering to an eye of the subject a single unit doseI Iof a first pharmaceutical composition comprising an rAAV of serotype 2 or a variant thereof encapsulating a heterologous nucleic acid encoding one or more anti-VEGF gene products operably linked to a promoter, wherein the unit dose comprises about I x 1010vector genomes per eye to about 6><10!0vector genomes per eye, preferably wherein the unit dose comprises about 1 x 1010vector genomes per eye or about 3 * 10!0vector genomes per eye. In some aspects, the unit dose comprises about lxlO!O, about 1.5xl0‘°, about 2xlO10, about 2.5xiO10, about 3x1 Oio, about 3.5x10% about 4x10’°, about 4.5xl0]u, about 5xlOw, about 5.5x10’° or about 6x10’° vector genomes per eye.

[0040] Also provided herein are methods for the treatment of DME in a subject in need thereof, the methods comprising (i) 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-VEGF gene products, wherein the unit dose comprises 1 riO10vector genomes per eye to 6*10wvector genomes per eye, preferably wherein the unit dose comprises 1* IO10vector genomes per eye or 3* IO10vector genomes per eye, and (ii) administering one or more corticosteroids to the eye of the subject.

[0941] Also provided herein are methods for the treatment of DME in a subject in need thereof, the methods comprising (i) 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-VEGF gene products, wherein the unit dose comprises about 1 x] 0!0vector genomes per eye to about 6xl0i0vector genomes per eye. preferably wherein the unit dose comprises about IxlO10vector genomes per eye or about 3* IO10vector genomes per eye, and (ii) administering to the eye of the subject of one or more doses of a second pharmaceutical composition comprising an anti-angiogenic protein. In preferred embodiments, the heterologous nucleic acid comprises a nucleotide sequence encoding aflibercept and the second pharmaceutical composition comprises aflibercept protein. Preferably, one or more corticosteroids are also administered to the subject.

[0042] Corticosteroid Administration Regimens

[0043] In certain embodiments, a method of treating diabetic macular edema (DME) in a subject is provided, the method compri sing: (i) intraocularly administering to an eye of thesubject a single unit dose of a first pharmaceutical composition comprising an rAA.V, 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 about 1 x] O10vector genomes per eye to about 6XIO10vector genomes per eye, preferably wherein the unit dose comprises about lx]O10vector genomes per eye or about 3xl010vector genomes per eye, and (ii) topically administering one or more corticosteroids to the eye of the subject before, simultaneous with and / or after administration of the first pharmaceutical composition, wherein at least one corticosteroid is administered topically. 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. In preferred embodiments, multiple doses of the one or more corticosteroids are administered to the eye of the subject.

[0044] Preferably, the one or more corticosteroids are administered to the subject in multiple doses, in which the one or more corticosteroids are administered to the subject for an administration period. The term “administration period” means the time period during which the one or more corticosteroids are administered to the subject.

[8045] In some embodiments, the one or more corticosteroids are administered to the eye of the subject for an admini stration period of at least 4 weeks, at least 8 weeks, at least 12 weeks, at least 16 weeks, or at least about 20 weeks, or at least 21 weeks, preferably wherein the dose of the one or more corticosteroids is tapered over the administration period. In related aspects, the subject is intraocularly administered consecutive multiple daily doses of the one or more corticosteroids for at least three weeks, for at least six weeks, for at least nine weeks, for at least twelve weeks, for at least fifteen weeks, for at least eighteen weeks or for at least twenty-one weeks during the administration period.

[0046] In some preferred embodiments, the subject is intraocularly administered the one or more corticosteroids for an administration period that is initiated prior to administration of the first pharmaceutical composition. In particularly preferred embodiments, 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.[0047j In related aspects, the subject is administered four doses of the one or more corticosteroids 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 the one or more corticosteroids continues until at least about four weeks after administering the first pharmaceutical composition.

[0048] In some preferred embodiments, the one or more corticosteroids comprises difluprednate, preferably difluprednate ophthalmic emulsion 0.05% or corticosteroids equivalent to difluprednate ophthalmic emulsion 0.05%. Difluprednate ophthalmic emulsion 0.05% is marketed as DUREZOL® in the United States.

[0049] In a preferred embodiment, the method comprises the following administration 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(11) 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(Hi) 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 every other day for about 28 days administered topically, preferably wherein the administration period is about 1 15 days.

[0050] In another preferred embodiment, the method comprises the following administration schedule:(I) corticosteroids equivalent to difluprednate ophthalmic emulsion 0.05% or difluprednate ophthalmic emulsion 0.05% six times per day for about three weeks administered topically; followed by(ii) corticosteroids equivalent to difluprednate ophthalmic emulsion 0.05% or difluprednate ophthalmic emulsion 0.05% five times per day for about three weeks administered topically; followed by(lit) corticosteroids equivalent to difluprednate ophthalmic emulsion 0.05% or difluprednate ophthalmic emulsion 0.05% four times per day for about three weeks administered topically; followed by(iv) corticosteroids equivalent to difluprednate ophthalmic emulsion 0.05% or difluprednate ophthalmic emulsion 0.05% three times per day for about three weeks administered topically; followed by(v) corticosteroids equivalent to difluprednate ophthalmic emulsion 0.05% or difluprednate ophthalmic emulsion 0.05% twice per day for about three weeks administered topically; followed by(vi) corticosteroids equivalent to difluprednate ophthalmic emulsion 0.05% or difluprednate ophthalmic emulsion 0.05% once per day for about three weeks administered topically; followed by(vii) corticosteroids equivalent to difluprednate ophthalmic emulsion 0.05% or difluprednate ophthalmic emulsion 0.05% every other day for about three weeks administered topically, and optionally wherein a first dose of corticosteroid comprising corticosteroids equivalent to difluprednate ophthalmic emulsion 0.05% or difluprednate ophthalmic emulsion 0.05% four times per day for 3 days is administered topically about 3 days prior to administering the first pharmaceutical composition.

[0051] 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 someaspects, intraocular inflammation is measured as Grade >1 inflammation episodes as assessed by Standardization of Uveitis Nomenclature (SUN).

[0052] Any of the above embodiments may be combined with any embodiment of a supplemental aflibercept regimen as described below,

[0053] Supplemental Aflibercept Hegimens

[8054] In certain aspects, an eye of the subject is administered one or more doses of an anti-VEGF agent 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 IxlO10to about 3x10’° vector genomes (vg) of the rAAV, more preferably delivers about 1x10i0or about 3x1010vg of the rAA V to the eye of the subject.

[8055] In some aspects, aflibercept protein comprises the following amino acid sequence:MVSYWDTGVLLCALLSCLLLTGSSSGSDTGRPFVEMYSEIPEIIHMTEGRELVIPCRV TSPNITVTLKKFPLDTLIPDGKRIIWDSRK.GFIISNATYKEIGLLTCEATVNGHLYKTNY LTHRQTNTIIDVVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKL VNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHEKD KTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNY KTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 1).

[0056] 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).

[0057] 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, ALT-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.

[0058] In some embodiments, a method of treating DME 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-associaled virus (rAAV), saidrAAV 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, wherein the unit dose comprises about [zio3uvector genomes per eye to about 6xlO1Qvector genomes per eye, preferably wherein the unit dose comprises about l*10!0vector genomes per eye or about 3x10;0vector genomes per eye, 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.

[0059] In some aspects, 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 bevacizurnab, brolucizuniab, 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).

[0860] In some embodiments, the subject is administered one or more doses of the second pharmaceutical composition (comprising aflibercept protein or an equivalent thereof) about 12 weeks prior to administering the first pharmaceutical composition and / or about 8 weeks prior to administering the first pharmaceutical composition, and-'or about 4 weeks prior to administering the first pharmaceutical composition and / or about 1 week prior to administering the first pharmaceutical composition and / or about 2 weeks after administering the first pharmaceutical composition and / or about 4 weeks after administering the first pharmaceutical composition. In preferred embodiments, an eye of the subject is administereda first dose of the second pharmaceutical composition about 12 weeks prior to administration of the first pharmaceutical composition, a second dose of the second pharmaceutical composition about 8 weeks prior to administration of the first pharmaceutical composition, a third dose about 4 weeks prior to administration of the first pharmaceutical composition, a fourth dose about 1 week prior to administration of the first pharmaceutical composition and fifth dose of the second pharmaceutical composition about 4 weeks after the first pharmaceutical composition is administered to the subject.

[0061] 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 82 to about 86 days prior to administering the first pharmaceutical composition, preferably about 84 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 -82 to Day -86, preferably at about Week -12.

[0062] 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 54 to about 58 days prior to administering the first pharmaceutical composition, preferably about 56 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 -54 to Day -58, preferably at about Week -8.1n related embodiments, the eye of the subject is administered a third dose of the second pharmaceutical composition (comprising aflibercept protein or an equivalent thereof) about 26 to about 30 days prior to administering the first pharmaceutical composition, preferably about 28 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 -26 to Day -30, preferably at about Week -4.

[0063] In related embodiments, the eye of the subject is administered a fourth dose of the second pharmaceutical composition (comprising aflibercept protein or an equivalent thereof) about 5 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 thesecond pharmaceutical composition is administered at Day -5 to Day -7, preferably at about Week -1.

[9064] In related embodiments, the eye of the subject is administered a fifth dose of the second pharmaceutical composition (comprising aflibercept protein or an equivalent thereof) about 26 to about 30 days 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 1 and the second pharmaceutical composition is administered at Day 26 to Day 30, preferably at about Week 4..[W965] In other embodiments, the subject is administered at least one additional (e.g., a fourth, a fifth, a sixth, and so on) dose of the second pharmaceutical composition (comprising aflibercept protein) if the subject is identified as a candidate for receiving at least one additional dose of the second pharmaceutical composition. In some aspects, the subject is identified as a candidate for receiving at least one additional dose of the second pharmaceutical composition if an increase from baseline in retinal central subfield thickness (CST) >50 pm is observed in the subject. Preferably, an increase from baseline in retinal central subfield thickness (CST) >50 pm is measured at least about eight weeks after administering the first pharmaceutical composition. Preferably, the baseline CST in the subject is measured within 24 hours of receiving the first pharmaceutical composition.

[0066] In some embodiments, a subject identified as a candidate for receiving at least one additional dose of the second pharmaceutical composition is administered additional doses of the second pharmaceutical composition at about four week intervals until the CST in the subject decreases by >30pm (preferably the decrease is maintained in the subject for at least 4 weeks) or until the subject’s CST is <325 gm.

[0067] hi preferred embodiments, the first and / or second pharmaceutical compositions are administered intravitreally to the subject. In particularly preferred embodiments, the first and second pharmaceutical composition are administered intravitreally to the subject.

[0068] 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 first dose ofcorticosteroid is administered about three days prior to administering the first pharmaceutical / composition:(1) 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; followed by(ill) 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 every other day for about 28 days administered topically, preferably wherein the administration period is about 115 days.

[0069] 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 rAA V. 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).

[0679] 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.

[0071] 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.

[0072] First Pharmaceutical Composition

[0073] Adeno- Associated Virus

[9074] In exemplary7embodiments, the first pharmaceutical composition of the present disclosure comprises AAV of serotype 2 or a variant thereof.

[0075] 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 AA V (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.

[0076] In some aspects, the pharmaceutical composition comprises an rAA V 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 AA V virion comprising the corresponding parental capsid protein.

[0077] 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) Moi. 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 570-61 1 of AAV2 VP1.17 / 934,801. In certain embodiments, the peptide is inserted following any of the amino acidsin positions 584-591 in VP1 of AAV2 (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). 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.

[0078] 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-terniinus 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 LA1SDQTKHA (SEQ ID NO:3).

[0079] 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 or more 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.

[9080] In some preferred embodiments, the variant AA\?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.

[0081] 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.

[0082] 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 FNGLDKGEPVNEADAAALEHDKAYDRQLDSGDNPYLK.YNHADAEFQERI-KEDTSF GGNLGRAVFQAKKRVLEPLGLVEEPVKTAPGKKRPVEHSPVEPDSSSGTGKAGQQP ARKRLNFGQTGDADSVPDPQPLGQPPAAPSGLGTNTMATGSGAPMADNNEGADGV GNSSGNWHCDSTWMGDRVITTSTRTWALPTYNNHLYKQISSQSGASNDNHYFGYST PWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTQNDGTTTIA NNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMVPQYGYLTLNNGSQAVGR SSFYCLEYFPSQMLRTGNNFTFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSR INTPSGTTTQSRLQFSQAGASDIRDQSRNWLPGPCYRQQRVSKTSADNNNSEYSWT GATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGXTIFGKQGSEKTNVDIEKVMIT DEEEIRTTNP VATEQYG SVSTNLQRGNLAISDQTKHARQAAT ADVN TQGVLPGM VW QDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPSTTFSAA KFASFITQYSTGQVSVEIEWELQKENSKRWMPEIQYTSNYNKSVNVDFTVDTNGVYS EPRPIGTRYLTRNL (SEQ ID N0:4).

[0083] The variant AAV capsid protein of SEQ ID N0:4 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 (leucine-alanine-isoleucine-serine-aspartic acid-glutamine- threonine-lysine-histidine-alanine / LAISDQTKIIA (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 least 99% identical to SEQ ID NO:4 and comprising a P34A substitution and an LA1SDQTKHA (SEQ ID NO:3) peptide insertion at amino acid position 588.

[0884] A first pharmaceutical composition as herein described comprises an rAAV encapsulating a heterologous nucleic acid comprising a nucleotide sequence encoding one or more anii-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.

[0085] 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 toAATAACGTCTTGCTGAGGTAG (SEQ ID NO:6). In related aspects, the nucleic acid comprises a nucleotide sequence encoding an artificial miRNA comprising at least 90% sequence identity withGACTTCTTAACCCAACAGAAGGCTCGAGAAGGTATATTGCTGTTGACAGTGAGCGGCTACCTCAGCAAGACGTTATTTAGTGAAGCCACAGATGTAAATAAC GTCTTGCTGAGGTAGCTGCCTACTGCCTCGGACTTCAAGGGGCTAGAATT (SEQ ID NO:7).

[0086] In other preferred aspects, the rAAV comprises a heterologous nucleic acid comprising the following nucleotide sequence encoding aflibercept, codon-optimized for expression in humans:ATGGTTTCTTACTGGGACACCGGCGTGCTGCTGTGTGCCCTGCTTTCTTGTCTGCTGCTGA CCGGCTCTAGCAGCGGCTCTGATACCGGCAGACCCTTCGTGGAAATGTACAGCGAGATC CCCGAGATCATCCACATGACCGAGGGCAGAGAGCTGGTCATCCCTTGCAGAGTGACAAG CCCCAACATCACCGTGACTCTGAAGAAGTTCCCTCTGGACACACTGATCCCCGACGGCAA GAGAATCATCTGGGACAGCCGGAAGGGCTTCATCATCAGCAACGCCACCTACAAAGAGA TCGGCCTGCTGACCTGTGAAGCCACCGTGAATGGCCACCTGTACAAGACCAACTACCTGA CACACAGACAGACCAACACCATCATCGACGTGGTGCTGAGCCCTAGCCACGGCATTGAA CTGTCTGTGGGCGAGAAGCTGGTGCTGAACTGTACCGCCAGAACCGAGCTGAACGTGGG CATCGACTTCAACTGGGAGTACCCCAGCAGCAAGCACCAGCACAAGAAACTGGTCAACC GGGACCTGAAAACCCAGAGCGGCAGCGAGATGAAGAAATTCCTGAGCACCCTGACCATC GACGGCGTGACCAG A AGTGACCAGGGCCTGTACACATGTGCCGCCAGCTCTGGCCTGAT GACCAAGAAAAACAGCACCTTCGTGCGGGTGCACGAGAAGGACAAGACCCACACCTGTC CTCCATGTCCTGCTCCAGAACTGCTCGGCGGACCTTCCGTGTTCCTGTTTCCTCCAAAGCC TAAGGACACCCTGATGATCAGCAGAACCCCTGAAGTGACCTGCGTGGTGGTGGATGTGT CCCACGAGGATCCCGAAGTGAAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAAC GCCAAGACCAAGCCTAGAGAGGAACAGTACAATAGCACCTACAGAGTGGTGTCCGTGCT GACCGTGCTGCACCAGGATTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTCCAACA AGGCCCTGCCTGCTCCTATCGAGAAAACCATCTCCAAGGCCAAGGGCCAGCCTAGGGAA CCCCAGGTTTACACACTGCCTCCAAGCAGGGACGAGCTGACAAAGAACCAGGTGTCCCT GACCTGCCTGGTCAAGGGCTTCTACCCTTCCGATATCGCCGTGGAATGGGAGAGCAATGG CCAGCCTGAGAACAACTACAAGACAACCCCTCCTGTGCTGGACAGCGACGGCTCATTCTT CCTGTACAGCAAGCTGACAGTGGACAAGAGCAGATGGCAGCAGGGCAACGTGTTCAGCT GCTCCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGAGCCTGTCTC CTGGCAAA (SEQ ID NO:8 )

[0087] 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.

[0088] In particularly preferred aspects, the pharmaceutical composition comprises an rAAV comprising a heterologous nucleic acid comprising the following sequence (encoding aflibercepi + 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:TTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTC ACTGAGGCCGGGCGACC AAAGG TCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCA GAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCTATCGATTGAATTCCCCGG GGATCCACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCAT ATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCC CAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCA ATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACT TGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGA CGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCT ACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTCGAGGTGAGCC CCACGTTCTGCTTCACTCTCCCCATCTCCCCCCCCTCCCCACCCCCAATTTTGTAT TTATTTATTTTTTAATTATTTTGTGCAGCGATGGGGGCGGGGGGGGGGGGGGGGC GQGCGCCAGGCGGGGCGGGGCGGGGCGAGGGGGGGGGCGGGGCGAGGCGGAG AGGTGCGGCGGCAGCCAATCAGAGCGGCGCGCTCCGAAAGTTTCCTTTTATGGCGAGGCGGOGGCGGCGGCGCCCC 1 ATAAAAAGCGAAGCGCGCGGCGGGCGGGGA GTCGCTGCGACGCTGCCTTCGCC CCGTGCCCCGCTCCGCCGCCGCCTCGCGCCGC CCGCCCCGGCTCTGACTGACCGCGTTACTCCCACAGGTGAGCGGGCGGGACGGC CCTTCTCCTCCGGGCTGTAATTAGCGCITGGTTTAATGACGGCTTGTTTCTTTTCT GTGGCTGCGTGAAAGCCTTGAGGGGCTCCGGGAGGGCCCTTTGTGCGGGGGGAG CGGCTCGGGGGGTGCGTGCGTGTGTGTGTGCGTGGGGAGCGCCGCGTGCGGCTC CGGG€TGCCCGG€GGCTG'rGA.GCGCTGCGGG€GCGGCG€GGGGCITIGTGGGGT CCGCAGTGTGCGCGAGGGGAGCGCGGCCGGGGGCGGTGCCCCGCGGTGCGGGG GGGGCTGCGAGGGGAACAAAGGCTGCG I GCGGGGTG I GTGCGTGGGGGGG I GA GCAGGGGGTGTGGGCGCGTCGGTCGGGC TGCAACCV ( ( ( c ! ( ■( ACCCCCCTCCCC GAGTTGCTGAGCACGGCCCGGCTTCGGGTGCGGGG< ' ft Co I A(XXKKJCGTGGGG CGGGGCTCGCCGTGCCGGGCGGGGGGI'CJGCGGCAG< j 1 GG( »G I GCCGGGCGGGGCGGGGCCGCCTCGGGCCGGGGAGGGCTCGGGGGAGGGGCGCGGCGGCCCCCG GAGCGCCGGCGCGACTTCTTAACCCAACAGAAGGCTCGAGAAGGTATATTGCTG TTGACAGTGAGCGGCTACCTCAGCAAGACGTTATTTAGTGAAGCCACAGATGTA AATAACGTCTTGCTGAGGTAGCTGCCTACTGCCTCGGACTTCAAGGGGCTAGAAT TCGCGGCTGTCGAGGCGCGGCGAGCCGCAGCCATTGCCTTTTATGGTAATCGTGC GAGAGGGCGCAGGGACTTCCTTTGTCCCAAATCTGTGCGGAGCCGAAATCTGGG AGGCGCCGCCGCACCCCCTCTAGCGGGCGCGGGGCGAAGCGGTGCGGCGCCGGC AGGAAGGAAATGGGCGGGGAGGGCCTTCGTGCGTCGCCGCGCCGCCGTCCCCTT Cl (.X.'Cl Cl CCAGCCICGGGCfC ft it C GCGUGGGGACGGCI GCC rrCGGGGGGGACGGGGCAGGGCGGGGTTCGGC 1 I C fGGCGTGTGACCGGCGGCTCTAGAGCCTCT GCTAACCATGTTCATGCCTTCTTCTTTTTCCTACAGTCTAGAGTCGACCTGCAGGT GGATATCTTGGCTAGCACGCCACCATGGTTTCTTACTGGGACACCGGCGTGCTGCTGTGTGCCCTGCTTTCTTGTCTGCTGCTGACCGGCTCTAGCAGCGGCTCTGATACC GGCAGACCCTTCGTGGAAATGTACAGCGAGATCCCCGAGATCATCCACATGACC GAGGGCAGAGAGC I GGTCAT CCC I TGCAGAG IGACAAGCCCCAACATCACCGTG ACTCTGAAGAAGTTCCCTCTGGACACACTGATCCCCGACGGCAAGAGAATCATCT GGGACAGCCGGAAGGGCTTCATCATCAGCAACGCCACCTACAAAGAGATCGGCC TGCTGACCTGTGAAGCCACCGTGAATGGCCACCTGTACAAGACCAACTACCTGA CACACAGACAGACCAACACCATCATCGACGTGGTGCTGAGCCCTAGCCACGGCA TTGAACTGTCTGTGGGCGAGAAGCTGGTGCTGAACTGTACCGCCAGAACCGAGC TGAACGTGGGCATCGACTTCAACTGGGAGTACCCCAGCAGCAAGCACCAGCACA AGA4ACTGGTCAACCGGGACCTGAAAACCCAGAGCGGCAGCGAGATGAAGAAA TTCCTGAGCACCCTGACCATCGACGGCGTGACCAGAAGTGACCAGGGCCTGTAC ACATGTGCCGCCAGCTCTGGCCTGATGACCAAGAAAAACAGCACCTTCGTGCGG GTGCACGAGAAGGACAAGACCCACACCTGTCCTCCATGTCCTGCTCCAGAACTG CTCGGCGGACCTTCCGTGTTCCTGTTTCCTCCAAAGCCTAAGGACACCCTGATGA TCAGCAGAACCCCTGAAGTGACCTGCGTGGTGGTGGATGTGTCCCACGAGGATC CCGAAGTGAAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGA CCAAGCCTAGAGAGGAACAGTACAATAGCACCTACAGAGTGGTGTCCGTGCTGA CCGTGCTGCACCAGGATTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTCCA ACAAGGCCCTGCCTGCTCCTATCGAGAAAACCATCTCCAAGGCCAAGGGCCAGC CTAGGGAACCCCAGGTTTACACACTGCCTCCAAGCAGGGACGAGCTGACAAAGA ACCAGGTGTCCCTGACCTGCCTGGTCAAGGGCTTCTACCCTTCCGATATCGCCGT GGAATGGGAGAGCAATGGCCAGCCTGAGAACAACTACAAGACAACCCCTCCTGT GCTGGACAGCGACGGCTCATTCTTCCTGTACAGCAAGCTGACAGTGGACAAGAG CAGATGGCAGCAGGGCAACGTGTTCAGCTGCTCCGTGATGCACGAGGCCCTGCA CAACCACTACACCCAGAAGTCCCTGAGCCTGTCTCCTGGCAAATGAGCCACGCGT AACACGTGGGGGATCCAGACATGATAAGATACATTGATGAGTTTGGACAAACCA CAACTAGAATGCAGTGAAAAAAATGCTTTA'nTGTGAAATTTGTGATGCTATTGC TTTATTTGTAACCATTATAAGCTGCAATAAACAAGTTAACAACAACAATTGCATT CATTTTATGTTTCAGGTTCAGGGGGAGGTGTGGGAGGTTTTTTAAAGCAAGTAAA ACCTCTACAAATGTGGTATGGCTGATTATGATCAATGCATGGCCGGCCGGAGGA ACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAG GCCGCCCGGGCAAAGCCCGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTG AGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAA (SEQ ID NO:9).

[0089] In particularly preferred embodiments, an amount of the pharmaceutical composition effective to deliver a unit dose of about 1x10'° to about 6x10’*°, preferably about 3 x 10i0vg 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.

[0090] Formulations

[0091] 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 apharmaceutically 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.

[0092] 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.(0093] 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.

[0094] 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 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

[0095] 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.).

[0096] In some embodiments, the invention provides a liquid formulation. In certain embodiments, the formulation is lyophilized from a liquid formulation.

[0097] 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 11 mM, about 12 mM, about 13 mM, about 14 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.

[0098] 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.

[0099] In exemplary aspects, the first pharmaceutical composi tion 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, 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 Tris. In some embodiments, the first pharmaceutical composition comprises about 8 mM Tris to about 22 mM Tris, or about 9 mM Tris 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.

[80180] 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).8

[0101] 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.[(J0102 ] 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), about0.0045% (w / v), about 0.005% (w / v), about 0.0055% (w / v), about 0.006% (w / v), about0.0065% (w / v), about 0.007% (w / v), about 0.0075% (w / v), about 0.008% (w / v), about0.0085% (w / v), about 0.009% (w / v), about 0.0095% (w / v), about 0.001% (w / v) non-ionic surfactant. In certain embodiments, the first pharmaceutical composition of the present disclosure comprises about 0.005% (w / v) non-ionic surfactant.

[90103] 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.

[0104] 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 certainembodiments, the pharmaceutically acceptable salt is present at about 170 nM to about 200 mM. In certain embodiments, the pharmaceutically acceptable salt is sodium chloride.

[90105] 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.

[0106] pH

[0107] 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 is about 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.

[0108] Additional components

[90109] 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- misciblecosolvents, 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, aleuritic 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, benzyd benzoate, bronopol, butylated hydroxyanisole, butylated hydroxy toluene, 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, carboxvmethyl cellulose calcium, carboxymethyl cellulose sodium, j3-carotene, carrageenan, castor oil, hydrogenated castor oil, cationic emulsifying wax, cellulose acetate, cellulose acetate phthalate, ethyl cellulose, microcrystalline cellulose, powdered cellulose, silicified microcrystailine cellulose, sodium carboxymethyl cellulose, cetosteaiyl alcohol, cetrimide, cetyl alcohol, chlorhexidine, chlorobutanol, chlorocresol, cholesterol, chlorhexidine acetate, chlorhexidine gluconate, chlorhexidine hydrochloride, chlorodifluoroetharte (HCFC), chlorodifluoromeihane, chlorofluorocarbons (CFC)chlorophenoxy ethanol, chloroxylenol, corn syrup solids, anhydrous citric acid, citric acid monohydrate, cocoa butter, coloring agents, com oil, cottonseed oil, cresol, m-cresol, o-cresol, p-cresol, croscannellose sodium, crospovidone, cyclamic acid, cyclodextrins, dextrates, dextrin, dextrose, dextrose anhydrous, diazolidinyi urea, dibutyl phthalate, dibutyl sebacate, diethanolamine, diethyl phthalate, difiuoroethane (HFC), dirnethyl-p-cyclodextrin, cydodextrin-type 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 disodimn, edtic acid, eglurnine, 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 (MFC), hexadecyltrimethylammonium bromide, high fructose syrup, human serum albumin, hydrocarbons (HC), 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 carboxymethylcellulose sodium, 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, polyethylenepolyoxypropyl ene-block polymers, polymethacrylates, polyoxyethylene alkyl ethers, polyoxyethylene castor oil derivatives, polyoxyethylene sorbitol fatty acid 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, hi basic, 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 (KFC), theobroma oil, thimerosal, titanium dioxide, alpha tocopherol, tocopheryl acetate, alpha tocopheryl acid succinate, beta-tocopherol, delta- tocopherol, gamma-tocopherol, tragacanth, triacetm, 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 gurn, xylitol, zein, zinc propionate, zinc salts, zine 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 pusposes, 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.[001 ID] 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.

[0111] In exemplary embodiments, 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, thepharmaceutical composition of the present disclosure does not comprise calcium chloride. In other exemplary 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.

[0112] 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 wherein the pharmaceutical composition is administered via intravitreal, subretinal and / or suprachoroidal injection, more preferably via a single intravitreal injection.[0(1113] 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 more doses 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.

[0114] 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 therapeuticagent to treat a single disease or condition. In certain embodiments, a compound of the present disclosure is administered concurrently wi th 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.

[0115] Additional embodiments of the invention are provided below at items 1-48:1. A method of treating diabetes macular edema (DME) in a subject 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 of serotype 2 or a variant thereof and (ii) 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 IxlO10to about 6xl0!0, preferably about 3xl0!0, vector genomes.2. The method according to item 1, 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 ( Y j -Y3) at the amino and / or carboxyl terminus of SEQ ID NO:2.3. The method according to item 2, wherein the insertion peptide consists of the sequence LAISDQTKHA (SEQ ID NO:3).4. The method according to item 2 or 3, wherein the peptide is inserted following 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.5. The method according to any one of items 2 to 4, 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, SI09T, R144K, R.144M, Q164K, T176P, LI 881,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 5, 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 any one of items 1 to 6, wherein the heterologous nucleic acid comprises a nucleotide sequence encoding a polypeptide of SEQ ID NO: 1. The method according to item 7, wherein the nucleotide sequence encoding a polypeptide of SEQ ID NO: 1 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 1 to 8, 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 9, 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. The method according to item 10, 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 is 100% identical to SEQ ID NO: 8, wherein the nucleotide sequence of (i) and (ii) are operably linked to a promoter , The method according to item 11, 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. The method according to item 12, wherein the rAAV comprises a capsid protein comprising an amino acid sequence at least 98% 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:9.14. The method according to item 13, wherein the rAAV comprises a capsid protein of SEQ ID NO:4 and a heterologous nucleic acid of SEQ ID NO:9.15. The method according to any one of items 1 to 14, wherein the first pharmaceutical composition is administered intravitreally.16. The method according to any one of items 1-15, 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 NaCI; 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 9.0.17. The method according to item 16, 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 8.6.18. The method according to item 17 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.19. The method according to item 18, wherein the pH of the buffering composition is about 7.6 to about 8.2 at 25 °C.20. The method according to any one of items 1 to 19, wherein the method further comprises administering one or more corticosteroids to the eye of the subject before, simultaneous with and / or after administration of the first pharmaceutical composition, wherein at least one corticosteroid is administered topically.21. The method according to item 20, wherein the subject is administered multiple doses of the one or more corticosteroids.The method according to item 20 or 21, 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, or at least about 21 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 20 to 22, 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 or for at least sixteen weeks during the administration period. The method according to any one of items 20 to 22, wherein the subject is administered consecutive multiple daily doses of the one or more corticosteroids for at least three weeks, for at least six weeks or for at least nine weeks or for at least twelve weeks or for at least fifteen weeks or for at least eighteen weeks or for at least twenty-one weeks during the administration period. The method according to any one of items 20 to 24, 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 20 to 25, wherein the subject is administered four 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 20 to 26, wherein the one or more corticosteroids comprises difluprednate ophthalmic emulsion 0.05% or corticosteroids equivalent to difluprednate ophthalmic emulsion 0.05%. The method according to any one of items 20 to 27, 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 byb. 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 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 115 days. The method according to any one of items 20 to 28, wherein the subject is administered one or more corticosteroids to the subject according to the following schedule: a. corticosteroids equivalent to difluprednate ophthalmic emulsion 0.05% or difluprednate ophthalmic emulsion 0.05% six times per day for about three weeks administered topically; followed by b. corticosteroids equivalent to difluprednate ophthalmic emulsion 0.05% or difluprednate ophthalmic emulsion 0.05% five times per day for about three weeks administered topically; followed by c. corticosteroids equivalent to difluprednate ophthalmic emulsion 0.05% or difluprednate ophthalmic emulsion 0.05% four times per day for about three weeks administered topically; followed by d. corticosteroids equivalent to difluprednate ophthalmic emulsion 0.05% or difluprednate ophthalmic emulsion 0.05% three times per day for about three weeks administered topically; followed by e. corticosteroids equivalent to difluprednate ophthalmic emulsion 0.05% or difluprednate ophthalmic emulsion 0.05% twice per day for about three weeks administered topically; followed by f. corticosteroids equivalent to difluprednate ophthalmic emulsion 0.05% or difluprednate ophthalmic emulsion 0.05% once per day for about three weeks administered topically; followed byg. corticosteroids equivalent to difluprednate ophthalmic emulsion 0.05% or difluprednate ophthalmic emulsion 0.05% every other day for about three weeks administered topically; and optionally wherein a first dose of corticosteroid comprising corticosteroids equivalent to difluprednate ophthalmic emulsion 0.05% or difluprednate ophthalmic emulsion 0.05% four times per day for 3 days is administered topically about 3 days prior to administering the first pharmaceutical composition. The method according to any one of items 1 to 29, wherein the method further comprises administering to the subject at least one dose of a second pharmaceutical composition comprising aflibercept protein or a biosimilar thereof before, simultaneous with and / or after administration of the first pharmaceutical composition. The method according of item 30, wherein the second pharmaceutical composition comprises 2mg of aflibercept protein or 2mg of aflibercept protein biosimilar. The method according to item 30 or 31, wherein the subject is administered one or more dose of the second pharmaceutical composition about 12 weeks prior to administering the first pharmaceutical composition and / or about 8 weeks prior to administering the first pharmaceutical composition and / or about 4 weeks prior to administering the first pharmaceutical composition and / or about 1 week prior to administering the first pharmaceutical composition and / or about 4 weeks after administering the first pharmaceutical composition. The item according to any one of items 30 to 32, wherein the subject is administered a first dose of the second pharmaceutical composition about 1 1 weeks to about 13 weeks, preferably about 12 weeks, prior to administering the first pharmaceutical composition, a second dose of the second pharmaceutical composition about 7 weeks to about 9 weeks, preferably about 8 weeks, prior to administering the first pharmaceutical composition, a third dose of the second pharmaceutical composition about 5 weeks to about 6 weeks, preferably about 4 weeks, prior to administering the first pharmaceutical composition, a fourth dose of the second pharmaceutical composition about 1 week to about 2 weeks, preferably about 1 week prior to administering the first pharmaceutical composition and a fifth dose of the second pharmaceutical composition about 3 weeks to about 5 weeks, preferably about four weeks, after administering the first pharmaceutical composition.The method according to item 33, wherein the subject is administered a first dose of the second pharmaceutical composition about 12 weeks prior to administering the first pharmaceutical composition, a second dose of the second pharmaceutical composition about 8 weeks prior to administering the first pharmaceutical composition, a third dose of the second pharmaceutical composition about 4 weeks prior to administering the first pharmaceutical composition, a fourth dose of the second pharmaceutical composition about 1 week prior to administering the first pharmaceutical composition and a fifth dose of the second pharmaceutical composition about 4 weeks after administering the first pharmaceutical composition. The method according to any one of items 30 to 34, wherein the subject is administered at least one dose of the second pharmaceutical composition if the subject is determined to have an increase from baseline in retinal central subfield thickness (CST) >50pm at least eight weeks after the first pharmaceutical composition is administered. The method according to item 35, wherein the baseline for CST is measured in the subject within 24 hours of receiving the first pharmaceutical composition. The method according to item 35 or 36, wherein the subject is administered additional doses of the second pharmaceutical composition at four week intervals until the CST in the subject decreases by >30pm or the subject’s CST is <325pm. The method according to any one of items 1 to 37, wherein the first pharmaceutical composition is administered intravitreally. The method according to any one of items 30 to 38, wherein the second pharmaceutical composition is administered intravitreally, The method according to any one of items 1 to 39, wherein the first pharmaceutical composition comprises a buffering composition comprising: about 5 mM to about 20 mM of a buffering agent, preferably a Tris buffer; about 100 mM to about 250 mM of a pharmaceutically acceptable salt, preferably NaCl; and 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 9.0.41. The method according to item 40, wherein the buffering composition comprises: about 9 mM to about 20 mM of the buffering agent; about 140 to about 200 mM of the pharmaceutically acceptable salt; and 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 8.6.42. The method according to item 41, 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.43. The method according to item 42, wherein the pH of the buffering composition is about 7.6 to about 8.2 at 25 °C,44. A method of treating diabetic macular edema 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 3xl010vector 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 12 weeks prior to administration of the first pharmaceutical composition,(ii) one dose of the second pharmaceutical composition is administered about 8 weeks prior to administration of the first pharmaceutical composition,(lit) one dose of the second pharmaceutical composition is administered about 4 weeks prior to administration of the first pharmaceutical composition,(iv) one dose of the second pharmaceutical composition is administered about 1 week prior to administration of the first pharmaceutical composition, and(v) one dose of the second pharmaceutical composition is administered about 4 weeks after administration of the first pharmaceutical composition.45. The method according to item 44, wherein one or more additional 2 mg doses of the second pharmaceutical composition are administered to the subject at least eight weeks after administration of the first pharmaceutical composition, if the subject exhibits an increase in CST_>50 pm compared to a reference value.46. The method according to item 45, wherein the CST reference value is the CST measurement in the subj ect wi thin 24 hours of administration of the first pharmaceutical composition.47. The method according to any one of item s 44 to 46, 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:(vi) 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(vii) 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(viii) 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(ix) 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 1 15 days.48. The method according to any one of items 44 to 47, wherein the subject is naive to treatment with a VEGF inhibitor and / or has a CST > 325 pm.EXAMPLES

[0116] 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 mod ifications thereof will be apparent to one skilled in the art from reading this application.Example 1A Phase 2 Randomized, Active-Controlled. Double-masked Trial of Intravitreal Gene Therapy in Adults with Diabetic Macular Edema

[0117] METHODS

[0118] Study Design

[0119] A prospective, multicenter, Phase 2 randomized, active-controlled, double-masked dose-ranging trial is underway to evaluate the safety and efficacy of an rAAV comprising a capsid of SEQ ID NO:4 and a nucleic acid of SEQ ID NO:9 (hereinafter “the rAAV”) in adults with DME who are either naive to or have prior exposure to anti-VEGF therapy. The rAAV will be administered as a single IVT injection to a designated “study eye.”

[0120] The trial will be conducted in two parts (termed Part 1 and Part 2) in adults with aflibercept-responsive DME (CTS > 350 pm). Part 1 (Dose Confirmation) is an open-label sequential cohort enrollment in which 2 doses of the rAAV will be tested (rAAV Dose 1 ; m=6-9) (rAAV Dose 2; ir=6-9). Part 2 (Expansion) is randomized, controlled, and masked to treatment, arm with the following groups: rAAV Dose 1 (n=l 8); rAAV Dose 2 (n=18); and aflibercept (2 mg; QW8; n=18).

[0121] Study Endpoints

[0122] The trial has been designed with an appropriate population, sufficient size, and duration to initially characterize the safety and tolerability' of the rAAV in adults with DME,evaluate the pre-specified endpoints and select an appropriate dose level for further evaluation.

[0123] The primary endpoint for this study is annualized number of aflibercept injections in the study eye. Key secondary endpoints include:* Mean cumulative number of aflibercept injections over time« Change from baseline in BCVA as assessed using the ETDRS Visual Acuity Chart at Weeks 52 and 104» Change from baseline in CST measured by spectral domain optical coherence tomography (SD-OCT) at Weeks 52 and 104» Percentage of subjects with a >2 and >3-Step Diabetic Retinopathy Severity (DRS) improvement from baseline on the Early Treatment Diabetic Retinopathy Study Diabetic Retinopathy Severity Scale (ETDRS-DRSS) at Weeks 52 and 104

[0124] Study Objectives

[0125] 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 DME* Identity1,the appropriate dose level of the r AAV for further evaluation« Characterize long-term safety and clinical activity of gene therapy with the rAAV

[0126] Enrollment Plan

[0127] Part 1 will consi st of up to 18 subjects (n=6-9 per rAAV dose level) in sequentially enrolled, open-label cohorts. Analysis of available safety data will be performed once all subjects in Part 1 complete at least the Week 12 visit (or otherwise discontinue participation), The DSMC will review cumulative data and recommend whether or not to proceed to Part 2 and if so with which rAAV dose level(s).

[0128] In Part 2, up to two dose levels of the rAAV (as deemed safe, well-tolerated, and potentially active in Part 1) will expand to enroll an additional 18 subjects per arm.Approximately 72 subjects may be enrolled in the trial,

[0129] In Past 2, eligible subjects will be randomized on Day 1 in a 1:1:1 allocation to one of the following planned arms:« rAAV Dose Level 1® rAAV Dose Level 2» Aflibercept control (2 mg)

[0130] Randomization will be stratified by anti-VEGF treatment-naive vs. treatment- experienced status.[1)0131 ] In Part 2, subjects, site personnel performing best-corrected visual acuity (BCVA) assessments, photographers, and reading center personnel involved in image assessment will be masked to treatment arm for the duration of the trial. There will be a minimum of two investigators at each site: an unmasked Investigator will administer interventions (but will remain masked to the rAAV dose level); a masked Investigator will perform required masked study procedures. The Sponsor and its representatives and tire site pharmacist or designee will be unmasked to treatment assignment (including rAA V dose level).

[0132] An independent Data and Safety Monitoring Committee (DSMC) will be unmasked and will evaluate safety throughout the trial and advise on study progress.

[0133] Interventions and Follow-up

[0134] Each subject will complete a Screening Period to assess initial eligibility. The study eye will be selected during the Screening Period based on protocol eligibility criteria (including reading center eligibility). If both eyes are eligible, the study eye will be determined by the Investigator and subject prior to Week -8.

[0135] During the Aflibercept Run-in Period, subjects will receive one aflibercept injection in the study eye at Week —8 and a second aflibercept injection at Week -4. SD-OCT will be performed early in the Day 1 visit window (between Days -7 to -5) to determine if the study eye is responsive to aflibercept, i.e. improvement of >40 pm in central subfield thickness (CST) relative to Week -8, as assessed by an independent reading center.

[0136] During the Treatment Period, all subjects with confirmed eligibility will initiate a topical corticosteroid regimen at Day -3 and will complete the protocol-specified regimen and tapering schedule. On Day 1, subjects will receive one injection of the rAAV at the assigned / randomized dose level in the study eye. Subjects randomized to the aflibercept control arm in Part 2 will receive a sham injection on Day 1. At Week 2, all subjects will receive a third aflibercept injection in the study eye.[001371 In Part 2, all subjects randomized to the aflibercept control arm will receive aflibercept once every 8 weeks (Q8W) starting at Week 8. In Part 2 only, subjects randomized to rAAV arms will receive sham injections Q8W.[0(1138] Starting at Week 8, alt subjects will be evaluated to receive supplemental aflibercept at 4week intervals. Supplemental aflibercept may be administered if the following criterion is met: increase in CST >50pm compared with Day 1 (as assessed by the independent reading center). Following a supplemental aflibercept injection, the subject will continue to receive additional supplemental aflibercept injections at approximately 4-week intervals until change in CST (compared to previous OCT) is <30pm in 2 consecutive visits OR CST is <325pm, as confirmed by an independent reading center.

[0139] All subjects will complete a 2-year (104 weeks) Follow-up Period to assess safety and clinical outcomes. Following the Week 104 visit, subjects may be enrolled in a long-term follow-up study under a separate protocol (in accordance with current regulatory guidelines).

[0149] Aflibercept, rAAV, and prophylactic corticosteroids will be administered to subjects participating in the trial as summarized in Table 1 below.Table 1: rAAV-C002 Dosing Schedule [

[0141] Prophylactic Corticosteroids[DO 142] Prophylactic corticosteroids will be administered to the study eye for all subjects.

[0143] The planned prophylactic corticosteroid regimen is specified in tire Table 2 below:Table 2: Prophylactic Corticosteroid Regimen[1)0144] The Sponsor, in consultation with Investigators and the DSMC, may provide recommendations for modifications io the planned prophylactic corticosteroid regimen based on emerging data from trial participants. These modifications may include, but are not limited to, adjustments to duration / tapering schedule or other topical corticosteroids, modified initiation of prophylactic corticosteroids.

[0145] Modifications to Corticosteroid Regimen

[68146] Clinically significant new, breakthrough (defined as an increase in IOI that in the judgment of the Investigator requires an increase in intensity of treatment), or rebound IOI (defined as IOI that recurs following cessation of corticosteroid treatment) requiring therapeutic intervention as determined by the Investigator should be managed per clinical standard of care.[01)147] At the Investigator’s discretion, persistent +0.5 anterior chamber cell (per Standardization of Uveitis Nomenclature [SUN] criteria) that is asymptomatic and not associated with a decrease in visual acuity may be observed without treatment.

[0148] While management of IOI is ultimately up to the Investigator, the Sponsor recommends the following regimen:* Topical difluprednate ophthalmic emulsion 0.05% (marketed as Durezol® in the United States) in the study eye for 21 weeks as follows: o Six times per day * 3 weeks o Fi ve times per day x 3 weeks o QID x 3 weekso TIDx3 weeks o BIDx3 weeks o QD x 3 weeks o QOD x 3 weeks o Discontinue♦ If breakthrough or rebound IOI persists or recurs following a second course of topical difluprednate treatment, IVT steroid therapy may be considered as a next step following consultation with the Medical Monitor.Subjects will be asked to record compliance with the steroid regimen in a SteroidDiary.

[0149] RESULTS

[0150] As of the data cut-off of 24 June 2024, 22 subjects have been dosed in Part 1 with rAAV at the following dose levels: 5x10svg / eye, n=l, IX10'° vg / eye, n=12, and 3 x 1010vg / eye, n:;:9.

[0151] Ophthalmic exams were performed at each visit to evaluate for IOI (white blood cells, flare, and haze). As of 24 June 2024. all subjects have completed the 16-week protocol-specified prophylactic topical corticosteroid regimen. With follow-up ranging from 20 to 36 weeks, there have been no examination findings or signs and symptoms consistent with intraocular inflammation in any subject.

[0152] CONCLUSION

[0153] The safety profile of the rAAV to date has been favorable, with no evidence of IOI observed to date using the steroid immunoprophylaxis, durezol 16-week taper.Example 2A Phase 2 Randomized, Active-Controlled, Double-masked Trial of Intravitreal Gene Therapy in Adults with Diabetic Macular Edema - Interim 32 Week Results

[0154] Brief Sanimairy of Results in patients with diabetic macular edema (DME) receiving a single intravitreal high dose (3x1010vg / eye, n;;:9) of the rAAV:® improved and sustained BCVA: +8.4 letters• improved and sustained anatomic control (CST): - 194 pm* 86% reduction in injection burden vs. aflibercept 2 mg Q8W o 7 of 8 patients who completed the loading doses per protocol (at -8 weeks, -4 weeks and +2 weeks) received 0-1 additional injections

[0155] No intraocular inflammation or hypotony was observed at any timepoint or dose level of the rAAV. All patients completed 16-week topical steroid taper on schedule and did not require farther steroids.

[0156] The key objectives of the study were to evaluate the safety and tolerability of a single TVT injection of the rAAV in adults with DME and to identify the dose level of 4D- 150 for farther evaluation in Part 2 & Phase 3. The key eligibility was diagnosis of DME within 2 years of screening and CST > 350 pM (includes treatment naive patients). The criteria was confirmed anti-VEGF response (CST decline > 40 pM at Week -1 versus Week - 8), where CST is defined as thickness of 1 mm area from ILM to BM.[0(1157] The treatment regimen is depicted at Figure 1.

[0158] Notably the disease activity criteria for supplemental aflibercept (CST increase > 50 pm compared to baseline measured at Day 1 and supplemental aflibercept injections until CST decreases by > 30 pm on 2 consecutive visits or CST' is < 325 pm) were stringent compared to selected criteria from other trials (e.g., Eylea). Additionally, the trial was designed with fewer loading doses and enrolled population relatively more severe to prior selected studies (e.g., Eylea).

[0159] Baseline CST, BCVA and prior treatment status across dose arms is shown in the table below:1 patient in 1E10 vg / eye arm with early termination due to death (unrelated to treatment) prior to completion of a post-baseline assessment1 patient in 1E10 vg / eye group missed Week 24-32 visits

[0160] Aflibercept 2mg Q8W through 40 weeks improves CST by --140-170 pm with -30 pm fluctuations and with 7 injections.

[60161] Administration of the single dose of rAAV at 3E10 vg / eye demonstrated improved and sustained visual acuity from baseline of +8.4 leters at 32 Weeks (see Figure 2) and demonstrated improved and sustained anatomic control from baseline of - 194 pm at 32 Weeks (see Figure 3).

[0162] Following the loading phase, the 3E10 vg / eye dose of rAAV demonstrated 86% reduction in treatment burden vs. aflibercept 2mg Q8 W, with a dose response in favor of 3E10 vg / eye (see Figure 4).

[60163] Following a single intravitreal dose (3E10 vg / eye) of the rAAV, robust and durable expression of aflibercept (aqueous humor) was observed in supplemental injection-free patients during follow up ranging from at least 1 to 2 years, with aflibercept expression remaining positive and stable in 100% of patients during the period.

[60164] While the materials and methods of this invention 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 find 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 diabetic macular edema in a human subject, 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 of SEQ ID NO:9 or a sequence at least 95% identical thereto, wherein the unit dose comprises about IxlO10to about. 6xlO10vector genomes (vg), preferably about 3xlO50vg of the rAAV; and administering to the eye of the subject 5 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 the 5 doses of the second pharmaceutical composition are administered to the subject no earlier than about four months prior to administration of the first pharmaceutical composition and no later than about one month after administration of the first pharmaceutical composition.

2. The method according to claim 1, wherein(i) one dose of the second pharmaceutical composition is administered about 13 weeks prior to administration of the first pharmaceutical composition,(ii) one dose of the second pharmaceutical composi tion is administered about 9 weeks prior to administration of the first pharmaceutical composition,(ill) one dose of the second pharmaceutical composition is administered about 5 weeks prior to administration of the first pharmaceutical composition,(iv) one dose of the second pharmaceutical composition is administered about 1 week prior to administration of the first pharmaceutical composition, and(v) one dose of the second pharmaceutical composition is administered about 4 weeks after administration of the first pharmaceutical composition.

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 eight weeks after administration of the first pharmaceutical composition, if the subject exhibits an increase in CST >50 pm compared to a reference value.

4. The method according to claim 3, wherein the CST reference value is the CST measurement in the subject within 24 hours of administration of the first pharmaceutical composition.

5. The method according to any one of claims 1 to 4. further comprising 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,6. The method according to claim 5, wherein the one or more corticosteroids are administered to the eye of 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:(s) corticosteroids equivalent to difluprednate ophthalmic emulsion 0.05% or difluprednate ophthalmic emulsion 0.05% four times per day for about 28 days or 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; followed 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 every other day for about 28 days administered topically, preferably wherein the administration period is about 140-143 days.

7. The method according to any one of claims 1 to 4, wherein the subject is naive to treatment with a VEGF inhibitor and / or has a CST > 325 pm.

8. The method according to any one of claims 1-7, 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.

9. The method according to claim 8, 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.

10. The method according to claim 9, 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.

11. The method according to claim 10, wherein the pH of the buffering composition is about 7.6 to about 8.2 at 25 °C.

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