Adeno-associated virus preparations

Abstract

Pharmaceutical compositions containing adeno-associated virus (AAV) are provided herein. In some embodiments, the pharmaceutical composition comprises about 5 mM to about 20 mM Tris buffer, about 100 mM to about 250 mM sodium chloride, about 0.001% (w / v) to about 0.01% (w / v) Pluronic® F68, and has a pH between 7.0 and 9.0. Methods for preparing AAV-containing pharmaceutical compositions, methods for treating VEGF-related disorders in subjects, and methods for storing AAV compositions are also provided.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims the interests of U.S. Provisional Patent Application No. 63 / 507,266, filed on 9 June 2023, the full disclosure of which is incorporated herein by reference.

[0002] Sequence listing submission via EFS-WEB A computer-readable XML file titled "090400-5024-WO-Sequence-Listing," with a file size of approximately 49,743 bytes and created on or around June 3, 2024, contains the sequence listing of this application, and the entire file is incorporated herein by reference. [Background technology]

[0003] Background of the Invention AAV formulations intended for human administration are understood to be not only safe, sterile, and meet Good Manufacturing Practice (GMP) grade for pharmaceuticals and quasi-drugs, but also to exhibit and promote the long-term stability of AAV and minimize the loss of AAV efficacy during manufacturing, packaging, and storage processes. While considerable effort has been made to design such AAV formulations, improved AAV formulations are still needed. [Overview of the Initiative]

[0004] Summary of the Invention Formulations suitable for human administration that address the unmet needs described herein are described herein. Advantageously, in some embodiments, the formulations are suitable for long-term storage of AAV, minimize loss of AAV efficacy, advantageously prevent the formation of subvisible particles, and prevent adsorption of AAV to the surfaces of containers in which it is packaged and stored, as well as to machinery used during manufacturing.

[0005] In some embodiments, the pharmaceutical compositions of the present disclosure comprise adeno-associated virus (AAV), about 5 mM to about 20 mM of a buffer, about 100 mM to about 250 mM of a pharmaceutically acceptable salt, and about 0.0001% (w / v) to about 0.01% (w / v) of a nonionic surfactant, and preferably have a pH of about 7.0 to about 9.0.

[0006] In some embodiments, the pharmaceutical compositions of the present disclosure comprise AAV (e.g., AAV2 or a variant thereof), about 5 mM to about 20 mM Tris buffer, about 100 mM to about 250 mM sodium chloride, and about 0.0001% (w / v) to about 0.01% (w / v) Pluronic® F68, and preferably have a pH of about 7.0 to about 9.0.

[0007] In some embodiments, the present invention provides liquid formulations. In certain embodiments, the formulation is lyophilized from the liquid formulation. In certain embodiments, the lyophilized pharmaceutical composition of the present disclosure is lyophilized from the liquid formulation.

[0008] In other embodiments, a method for preparing a pharmaceutical composition containing AAV is provided. In a particular embodiment, the method includes the steps of combining about 5 mM to about 20 mM Tris buffer, about 100 mM to about 250 mM sodium chloride, about 0.0001% (w / v) to about 0.01% (w / v) Pluronic® F68, and AAV, and adjusting the pH to between about 7.0 and about 9.0 to obtain a pharmaceutical composition containing AAV. A method for storing the composition containing AAV is also provided.

[0009] The present invention provides a method for delivering heterologous nucleic acids, including nucleotide sequences encoding gene products, to mammalian subjects. In exemplary embodiments, the method comprises the step of administering a pharmaceutical composition described herein to a mammal. In some embodiments, the heterologous nucleic acids are delivered to target retinal cells, such as target photoreceptor cells (e.g., rods; cones), retinal ganglion cells (RGCs), glial cells (e.g., Müllerian glial cells, microglia), bipolar cells, amacrine cells, horizontal cells, and / or retinal pigment epithelium (RPE) cells.

[0010] This disclosure provides a method for treating a subject for a disorder treatable by gene therapy. In exemplary embodiments, the method includes administering to a subject an amount of a pharmaceutical composition described herein that is effective in treating the disorder. In some embodiments, the disorder is an ocular disorder. In particularly preferred embodiments, the disorder is a VEGF-related ocular disease, such as wet or dry age-related macular degeneration (AMD) or geographic atrophy secondary to AMD.

[0011] In certain embodiments, the pharmaceutical composition of the present disclosure comprises AAV and about 8–12 mM Tris buffer, about 160–200 mM sodium chloride, and about 0.0005%–0.01% (w / v) Pluronic® F68, preferably with a pH of about 7.4–8.1. In some embodiments, the present invention provides a liquid formulation. In certain embodiments, the formulation is lyophilized from the liquid formulation.

[0012] In certain embodiments, the pharmaceutical composition of the present disclosure comprises AAV and about 8–12 mM, preferably about 10 mM, Tris buffer, about 160–200 mM, preferably about 180 mM, about 0.0005%–0.01% (w / v) Pluronic® F68, and has a pH of about 7.6. In some embodiments, the present invention provides a liquid formulation. In certain embodiments, the formulation is lyophilized from the liquid formulation.

[0013] In certain embodiments, the pharmaceutical composition of the present disclosure comprises AAV and about 8–12 mM, preferably about 10 mM, Tris buffer, about 160–200 mM, preferably about 180 mM, about 0.0005%–0.01% (w / v) Pluronic® F68, and has a pH of about 7.7. In some embodiments, the present invention provides a liquid formulation. In certain embodiments, the formulation is lyophilized from the liquid formulation.

[0014] In certain embodiments, the pharmaceutical composition of the present disclosure comprises AAV and about 8 - 12 mM, preferably about 10 mM, Tris buffer, about 160 - 200 mM, preferably about 180 mM, sodium chloride, about 0.0005% - 0.01% (w / v) Pluronic® F68, and has a pH of about 7.8. In some embodiments, the present invention provides a liquid formulation. In certain embodiments, the formulation is lyophilized from the liquid formulation.

[0015] In certain embodiments, the pharmaceutical composition of the present disclosure comprises AAV and about 8 - 12 mM, preferably about 10 mM, Tris buffer, about 160 - 200 mM, preferably about 180 mM, sodium chloride, about 0.0005% - 0.01% (w / v) Pluronic® F68, and has a pH of about 7.9. In some embodiments, the present invention provides a liquid formulation. In certain embodiments, the formulation is lyophilized from the liquid formulation.

[0016] In certain embodiments, the pharmaceutical composition of the present disclosure comprises AAV and about 8 - 12 mM, preferably about 10 mM, Tris buffer, about 160 - 200 mM, preferably about 180 mM, sodium chloride, about 0.0005% - 0.01% (w / v) Pluronic® F68, and has a pH of about 8.0. In some embodiments, the present invention provides a liquid formulation. In certain embodiments, the formulation is lyophilized from the liquid formulation.

[0017] In certain embodiments, the pharmaceutical composition of the present disclosure comprises AAV and about 8 - 12 mM, preferably about 10 mM, Tris buffer, about 160 - 200 mM, preferably about 180 mM, sodium chloride, about 0.0005% - 0.01% (w / v) Pluronic® F68, and has a pH of about 8.1. In some embodiments, the present invention provides a liquid formulation. In certain embodiments, the formulation is lyophilized from the liquid formulation.

[0018] In some embodiments, the pharmaceutical compositions of the present disclosure comprise AAV and about 9 mM to about 20 mM buffer; about 140 to about 200 mM pharmaceutically acceptable salt; about 0.001% (w / v) to about 0.01% (w / v) nonionic surfactant; and a pH of about 7.3 to 8.6. In certain embodiments, the pharmaceutical compositions of the present disclosure comprise AAV and about 9 to 20 mM Tris buffer, about 140 to 200 mM sodium chloride, about 0.001% to 0.01% (w / v) Pluronic® F68; and a pH of about 7.3 to 8.6. In some embodiments, the present invention provides liquid formulations. In certain embodiments, the formulations are lyophilized from the liquid formulations.

[0019] In certain embodiments, the pharmaceutical composition does not contain divalent cations and / or sugars or sugar alcohols.

[0020] In general, the AAV formulations provided herein are suitable for pharmaceutically administered substances. In certain embodiments, the AAV is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, or AAV10. In certain embodiments, the AAV is AAV2, AAV5, AAV8, or AAV9. In certain embodiments, the AAV is AAV2 or a variant of AAV2.

[0021] In some preferred embodiments, the AAV is a recombinant AAV (rAAV) comprising a gene product and a heterogeneous nucleic acid encoding a capsid protein, which includes a peptide insertion of about 7 to about 20 amino acids ("heterogeneous peptide" or "peptide insertion") within the GH loop of the capsid protein relative to the corresponding parental AAV capsid protein, preferably within the surface-exposed region of the GH loop. In certain embodiments, the peptide is inserted after any of the amino acids at positions 584–591 of VP1 in AAV2 or at the corresponding position in another AAV serotype (i.e., the insertion site is between amino acids 587 and 588 of VP1 in AAV2, or 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 between amino acids 591 and 592 in AAV2 or at the corresponding position 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 between amino acids 588 and 589 of AAV2, or at a corresponding position in the capsid protein of another AAV serotype. In some embodiments, the capsid protein further comprises one or more amino acid substitutions for the VP1 capsid of AAV2 or one or more corresponding substitutions in another AAV serotype, and preferably, the capsid protein further comprises a P34A amino acid substitution for the VP1 capsid of AAV2 or a corresponding substitution in another AAV serotype. [Brief explanation of the drawing]

[0022] [Figure 1] Figure 1. Typical background film image after 3 days of storage at 37°C.

[0023] [Figure 2] Figure 2. Aflibercept expression at pH 6.0-7.0 after 2 weeks of storage at 25°C / 60% relative humidity (RH).

[0024] [Figure 3]Figure 3. Aflibercept expression at pH 7.0-7.8 after 2 weeks of storage at 25°C / 60% relative humidity (RH).

[0025] [Figure 4] Figure 4 shows the colloidal stability (area exceeding 90% by intensity (diameter 10-100 nm)) of the tested formulations over a range of sodium chloride concentrations.

[0026] [Figure 5] Figure 5 shows the final fluorescence values ​​obtained by differential scanning fluorescence (DSF) at the specified sodium chloride concentration after 0.45 μm filtration.

[0027] [Figure 6] Figure 6 shows the recovery rate after three freeze-thaw cycles (3×FT) using DSF at the specified sodium chloride concentration. [Modes for carrying out the invention]

[0028] Detailed description of the invention definition

[0029] As used herein, the term “AAV” refers to adeno-associated viruses in both naturally occurring and recombinant (rAAV) forms, encompassing variant forms of AAV. The term AAV further includes, but is not limited to, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, avian AAV, bovine AAV, canine AAV, equine AAV, sheep AAV, primate AAV, and non-primate AAV. In certain embodiments, AAV is AAV2 or a variant thereof.

[0030] A “pharmaceutically acceptable salt” is a salt that can be formulated into a compound or conjugate for a pharmaceutical use that is safe for administration to a subject (e.g., human) in a pharmaceutical formulation (e.g., including metal salts (sodium, potassium, magnesium, calcium, etc.) and salts of ammonia or organic amines) (see, e.g., Berge, et al. “Pharmaceutical Salts,” J.Pharm.Sci.1977;66:1 (the whole of which is incorporated herein by reference for all purposes)). Preferred “pharmaceutically acceptable salts” include, but are not limited to, metal salts, e.g., sodium salts, potassium salts, and cesium salts; alkaline earth metal salts, e.g., calcium salts and magnesium salts; and organic amine salts, e.g., triethylamine salts, guanidine salts, and N-substituted guanidine salts, acetamidine and N-substituted acetamidine salts, pyridine salts, picoline salts, ethanolamine salts, triethanolamine salts, dicyclohexylamine salts, and N,N'-dibenzylethylenediamine salts. "Pharmacologically acceptable salts" (of a basic nitrogen center) include, but are not limited to, inorganic salts, e.g., hydrochlorides, hydrobroms, sulfates, phosphates; organic salts, e.g., trifluoroacetates and maleates; sulfonates, e.g., methanesulfonates, ethanesulfonates, benzenesulfonates, p-toluenesulfonates, camphorsulfonates, and naphthalenesulfonates; amino acid salts, e.g., arginates, alaninates, asparaginates, and glutamates; and carbohydrate salts, e.g., gluconates and galacturonates. The selection and use of pharmaceutically acceptable salts are well known in the art; see, for example, Stahl and Wermuth, Pharmaceutical Salts: Properties, Selection, and Use, 2nd Revised edition, Wiley, Hoboken, NJ (the whole is incorporated herein by reference for all purposes).Non-limiting examples of pharmaceutically acceptable salts include, but are not limited to, sodium salts, ammonium salts, and potassium salts (e.g., sodium chloride, ammonium chloride, and potassium chloride; sodium acetate, ammonium acetate, and potassium acetate; sodium citrate, ammonium citrate, and potassium citrate; sodium phosphate, ammonium phosphate, and potassium phosphate; sodium fluoride, ammonium fluoride, and potassium fluoride; sodium bromide, ammonium bromide, and potassium bromide; and sodium iodide, ammonium iodide, and potassium iodide).

[0031] The term "isolated" refers to biological material (cells, nucleic acids, or proteins) that has been removed from its original environment (the environment in which it naturally exists). For example, a polynucleotide present in the natural state of a plant or animal is not isolated, but the same polynucleotide separated from a naturally occurring adjacent nucleic acid is considered "isolated."

[0032] As used herein, “coding region” or “coding sequence” is a portion of a polynucleotide consisting of codons that can be translated into amino acids. “Stop codons” (TAG, TGA, or TAA) are typically not translated into amino acids but can be considered part of a coding region, while any adjacent sequences, such as promoters, ribosome-binding sites, transcription terminators, or introns, are not part of a coding region. The boundaries of a coding region are typically determined by a 5' start codon encoding the amino terminus of the resulting polypeptide and a 3' translation stop codon encoding the carboxyl terminus of the resulting polypeptide. Two or more coding regions may exist in a single polynucleotide construct, for example, on a single vector, or in separate polynucleotide constructs, for example, on separate (different) vectors. Thus, a single vector may contain only one coding region, or it may contain two or more coding regions.

[0033] As used herein, the term “regulatory region” refers to a nucleotide sequence located upstream (5' non-coding sequence), within, or downstream (3' non-coding sequence) of a coding region that affects the transcription, RNA processing, stability, or translation of the associated coding region. Regulatory regions may include promoters, translational leader sequences, introns, polyadenylation recognition sequences, RNA processing sites, effector binding sites, and stem-loop structures. When a coding region is intended for expression in eukaryotic cells, polyadenylation signals and transcription termination sequences are typically located 3' of the coding sequence.

[0034] As used herein, the term “nucleic acid” is interchangeable with “polynucleotide” or “nucleic acid molecule,” and refers to polymers of nucleotides.

[0035] A gene product, such as a polynucleotide encoding a polypeptide, a primary miRNA, a short hairpin RNA (shRNA), or an interfering RNA such as a small interfering RNA (siRNA), may contain promoters and / or other transcriptional or translational regulatory elements that are operably associated with one or more coding regions. In an operable association, the coding region of a gene product, such as a polypeptide, associates with one or more regulatory regions to place the expression of the gene product under the influence or control of the regulatory regions. For example, if the induction of promoter function results in the transcription of mRNA encoding the gene product encoded by the coding region, and the nature of the linkage between the promoter and the coding region does not interfere with the promoter's ability to direct the expression of the gene product or the ability of the DNA template to be transcribed, then the coding region and promoter are "operably associated". Other transcriptional regulatory elements other than promoters, such as enhancers, operators, repressors, and transcription termination signals, can also operably associate with coding regions to direct gene product expression.

[0036] "Transcriptional regulatory sequences" refer to DNA regulatory sequences such as promoters, enhancers, and terminators that provide expression for coding sequences in host cells. Various transcriptional regulatory regions are known to those skilled in the art. These include, but are not limited to, transcriptional regulatory regions that function in vertebrate cells, e.g., cytomegalovirus (pre-initial promoter in combination with intron-A), Simianvirus 40 (initial promoter), and promoter and enhancer segments derived from retroviruses (such as Roussarcoma virus). Other transcriptional regulatory regions include those derived from vertebrate genes, e.g., actin, heat shock proteins, bovine growth hormone, and rabbit betaglobin, as well as other sequences that can regulate gene expression in eukaryotic cells. Further suitable transcriptional regulatory regions include tissue-specific promoters and enhancers, as well as lymphokine-inducible promoters (e.g., promoters that can be induced by interferon or interleukin).

[0037] Similarly, various translational regulatory elements are known to those skilled in the art. These include, but are not limited to, elements derived from ribosome binding sites, translation start and termination codons, and picornaviruses (in particular, internal ribosome entry sites or IRESs, also known as CITE sequences).

[0038] As used herein, the term “expression” refers to the process by which a polynucleotide produces a gene product, such as RNA or polypeptide. This includes, but is not limited to, the transcription of a 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 mRNA into polypeptides. Expression produces a “gene product.” As used herein, a gene product may be either a nucleic acid, such as messenger RNA produced by the transcription of a gene, or a polypeptide translated from a transcript. Gene products as described herein further include nucleic acids having post-transcriptional modifications, such as polyadenylation or splicing, or polypeptides having post-translational modifications, such as methylation, glycosylation, lipid addition, association with other protein subunits, or proteolytic cleavage.

[0039] "Promoter" and "promoter sequence" refer to interchangeable DNA sequences that can control the expression of a coding sequence or functional RNA. Generally, the coding sequence is located 3' of the promoter sequence. A promoter may be entirely derived from a native gene, or it may consist of different elements derived from different naturally occurring promoters, or it may contain synthetic DNA segments. It is understood by those skilled in the art that different promoters can direct gene expression in different tissues or cell types, at different developmental stages, or in response to different environmental or physiological conditions. In most cases, promoters that express a gene in most cell types are generally called "constitutive promoters." Promoter that expresses a gene in a specific cell type are generally called "cell-specific promoters" or "tissue-specific promoters." Promoter that expresses a gene at a specific stage of development or cell differentiation are generally called "development-specific promoters" or "cell differentiation-specific promoters." Promoter that is induced and expresses a gene after exposure or treatment of cells with promoter-inducing drugs, biological molecules, chemicals, ligands, light, etc. are generally called "inducible promoters" or "modulatory promoters." In most cases, the precise boundaries of regulatory sequences are not fully defined, and it is further recognized that DNA fragments of different lengths can have the same promoter activity.

[0040] The term "plasmid" refers to extrachromosomal elements that often carry genes that are not part of the cell's central metabolism, and typically take the form of a circular double-stranded DNA molecule. Such elements can be single-stranded or double-stranded DNA or RNA autonomous replication sequences, genomic integration sequences, phages or nucleotide sequences originating from any source, and can be linear, circular or supercoiled, where some nucleotide sequences are ligated or recombined into a unique construct that can introduce promoter fragments and DNA sequences for selected gene products into the cell, along with appropriate 3' untranslated sequences.

[0041] A polynucleotide or polypeptide has a certain percentage of "sequence identity" with another polynucleotide or polypeptide, meaning that when aligned, the percentage of bases or amino acids in the two sequences is the same when compared. Sequence similarity can be determined in several different ways. To determine sequence identity, sequences can be aligned using methods and computer programs, including BLAST, which is available on the World Wide Web at ncbi.nlm.nih.gov / BLAST / . Another alignment algorithm is FASTA, available in the Genetics Computing Group (GCC) package from Madison, Wisconsin, 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 allow gaps in sequences. Smith-Waterman is one type of algorithm that allows gaps in sequence alignment. See Meth.Mol.Biol.70:173-187(1997). Additionally, the GAP program, which uses the Needleman and Wunsch alignment methods, can be used to align sequences. See J.Mol.Biol.48:443-453(1970).

[0042] The term "amino acid substitution" and its synonyms used above are intended to encompass the modification of an amino acid sequence by substituting one amino acid with another. Substitutions may be conservative or non-conservative. When referring to two amino acids, the term "conservative" is intended to mean that the amino acids share common properties recognized by those skilled in the art. For example, amino acids with hydrophobic non-acidic side chains, amino acids with hydrophobic acidic side chains, amino acids with hydrophilic non-acidic side chains, amino acids with hydrophilic acidic side chains, and amino acids with hydrophilic basic side chains. Common properties may also include amino acids with hydrophobic side chains, amino acids with aliphatic hydrophobic side chains, amino acids with aromatic hydrophobic side chains, amino acids with polar neutral side chains, amino acids with charged side chains, amino acids with charged acidic side chains, and amino acids with charged basic side chains. Both naturally occurring and non-naturally occurring amino acids are known in the art and can be used as amino acid substitutions in embodiments. Methods for substituting amino acids are well known to those skilled in the art and include, but are not limited to, mutations in the nucleotide sequence encoding the amino acid sequence. In this specification, references to “one or more” are intended to encompass, for example, individual embodiments 1, 2, 3, 4, 5, 6, or more.

[0043] As used herein, terms such as “treatment” and “to treat” refer to obtaining a desired pharmacological and / or physiological effect. The effect may be prophylactic in that it completely or partially prevents the disease or its symptoms, and / or therapeutic in that it partially or completely cures the disease and / or adverse effects resulting from the disease. As used herein, “treatment” encompasses any treatment of a disease in mammals, in particular humans, and includes (a) preventing the development of the disease in subjects who are susceptible to the disease (and / or symptoms caused by the disease) or at risk of acquiring the disease but have not yet been diagnosed as having it; (b) inhibiting the disease (and / or symptoms caused by the disease), i.e., stopping its development; and (c) reducing 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., remission of one or more symptoms of the disease.

[0044] The terms “individual,” “host,” “subject,” and “patient” are used interchangeably herein and are not limited to, but refer to: humans; mammals, including non-human primates such as monkeys; mammalian sports animals (e.g., horses); mammalian domestic animals (e.g., sheep, goats, etc.); mammalian pets (e.g., dogs, cats, etc.); and rodents (e.g., mice, rats, etc.).

[0045] As used herein, the term “effective dose” is a quantity sufficient to produce a beneficial or desired clinical outcome. An effective dose may be administered in one or more doses. For the purposes of this disclosure, an effective dose of a compound (e.g., infectious rAAV virion) is a quantity sufficient to alleviate, improve, stabilize, reverse, prevent, delay or slow the progression (and / or associated symptoms) of a particular medical condition (e.g., a disorder associated with complement dysfunction). Thus, an effective dose of infectious rAAV virion is the amount of infectious rAAV virion that can effectively deliver heterologous nucleic acid to target cells (or target cells) of an individual. An effective dose may be determined preclinically by detecting the gene product (RNA, protein) encoded by the heterologous nucleic acid sequence within a cell or tissue using, for example, techniques well understood in the art, such as RT-PCR, Western blotting, ELISA, fluorescence or other reporter readout. The effective dose can be clinically determined, for example, by detecting changes in disease onset or progression using methods known in the art, such as the 6-minute walk test described herein and known in the art, left ventricular ejection fraction, handheld dynamometry, and the Vignos scale.

[0046] Detailed explanation This disclosure provides formulations suitable for human administration, such as pharmaceutical compositions, which are also suitable for long-term storage of AAV and minimizing the loss of AAV efficacy. The formulations provided herein are advantageous because they prevent the formation of particles invisible to the naked eye and retain significant AAV activity when stored for extended periods. In certain embodiments, the pharmaceutical compositions provided herein reduce or delay degradation and / or aggregation.

[0047] In certain embodiments, the present invention provides a formulation of AAV comprising a therapeutically effective amount or dose of AAV, a pharmaceutically acceptable salt, a nonionic surfactant, and one or more buffers that provide the formulation with the pH described herein. Generally, the AAV formulations provided herein are suitable for pharmaceutically administered. In certain embodiments, the AAV is AAV2 or a variant thereof.

[0048] AAV formulations and compositions

[0049] In exemplary embodiments, the pharmaceutical compositions of the present disclosure comprise adeno-associated virus (AAV), about 5 mM to about 20 mM of a buffer, about 100 mM to about 250 mM of a pharmaceutically acceptable salt, about 0.001% (w / v) to about 0.01% (w / v) of a nonionic surfactant, and a pH of at least about 7.0.

[0050] In exemplary embodiments, the pharmaceutical compositions of the present disclosure include adeno-associated virus (AAV), about 5 mM to about 20 mM of a buffer, 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 nonionic surfactant, and a pH between about 7.0 and about 9.0.

[0051] In exemplary embodiments, the pharmaceutical compositions of the present disclosure comprise adeno-associated virus (AAV), about 5 mM to about 20 mM of a buffer, 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 nonionic surfactant, and a pH of at least about 7.4.

[0052] In exemplary embodiments, the pharmaceutical compositions of the present disclosure comprise adeno-associated virus (AAV), about 10 mM to about 20 mM of a buffer, 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 nonionic surfactant, and a pH of at least about 7.4.

[0053] In certain embodiments, a composition is a sterile composition. "Sterile" means that the composition is substantially free of immunogenic components, for example, substantially free of microorganisms (e.g., fungi, bacteria, viruses, spore forms, etc.).

[0054] In some embodiments, the present invention provides a liquid formulation. In certain embodiments, the formulation is freeze-dried from the liquid formulation.

[0055] In exemplary embodiments, the pharmaceutical compositions of the present disclosure include 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 embodiments, the pharmaceutical compositions include 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.

[0056] Pharmaceutically acceptable buffers include, but are not limited to, phosphate buffer, histidine, sodium citrate, HEPES, Tris, Bicine, glycine, N-glycylglycine, sodium acetate, sodium carbonate, glycylglycine, lysine, arginine, sodium phosphate, and mixtures thereof. In some preferred embodiments, the buffer is Tris buffer.

[0057] In exemplary embodiments, the pharmaceutical compositions of this disclosure include Tris in amounts of 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. In exemplary embodiments, the pharmaceutical compositions include Tris in amounts of 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. In some embodiments, the pharmaceutical composition contains 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 a particular embodiment, the pharmaceutical composition contains about 10 mM Tris.

[0058] In exemplary embodiments, the pharmaceutical compositions of this disclosure are approximately 100 mM to approximately 250 mM, approximately 110 mM to approximately 240 mM, approximately 120 mM to approximately 230 mM, approximately 130 mM to approximately 220 mM, approximately 140 mM to approximately 210 mM, approximately 145 mM to approximately 205 mM, approximately 140 mM to approximately 200 mM, approximately 145 mM to approximately 200 mM, approximately 150 mM to approximately 200 mM, approximately 155 mM to approximately 195 mM, approximately 160 mM to approximately 195 mM, approximately 165 mM to approximately 195 mM, approximately 170 mM to approximately 195 mM, and approximately 175 mM. Contains pharmaceutically acceptable salts (as defined above) in the following concentrations: mM to approximately 195 mM, approximately 175 mM to approximately 194 mM, approximately 175 mM to approximately 193 mM, approximately 175 mM to approximately 192 mM, approximately 175 mM to approximately 191 mM, approximately 176 mM to approximately 190 mM, approximately 177 mM to approximately 189 mM, approximately 177 mM to approximately 188 mM, approximately 177 mM to approximately 187 mM, approximately 177 mM to approximately 186 mM, approximately 178 to approximately 184, approximately 178 to approximately 183, approximately 178 to approximately 182, approximately 179 to approximately 181, or approximately 180 mM. In exemplary embodiments, the composition contains pharmaceutically acceptable salts in concentrations of 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. In certain embodiments, the pharmaceutically acceptable salt is a sodium salt (e.g., sodium chloride).

[0059] In certain embodiments, a pharmaceutical composition comprising a nonionic detergent is provided. Pharmaceutically acceptable nonionic surfactants that may be used in the formulations disclosed herein are known in the field of pharmaceutical science and include, but are not limited to, polysorbate 80 (Tween® 80; PS80), polysorbate 20 (Tween® 20; PS20), and various poloxamers or Pluronic® (including Pluronic® F-68), and BRIJ® 35, or mixtures thereof. In preferred embodiments, the nonionic surfactant used in the pharmaceutical composition is Pluronic®, particularly Pluronic® F-68.

[0060] In certain embodiments, the pharmaceutical compositions of this disclosure contain about 0.001% (w / v) to about 0.01% (w / v) or about 0.0025% (w / v) to about 0.0075% (w / v) of nonionic surfactants. In exemplary embodiments, the pharmaceutical compositions contain 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), and about 0.00 The composition contains 55% (w / v), approximately 0.006% (w / v), approximately 0.0065% (w / v), approximately 0.007% (w / v), approximately 0.0075% (w / v), approximately 0.008% (w / v), approximately 0.0085% (w / v), approximately 0.009% (w / v), approximately 0.0095% (w / v), and approximately 0.001% (w / v) nonionic surfactants. In certain embodiments, the pharmaceutical composition of the Disclosure contains approximately 0.005% (w / v) nonionic surfactants.

[0061] In exemplary embodiments, the pharmaceutical compositions of this disclosure contain 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 embodiments, the pharmaceutical compositions contain 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). ), approximately 0.0055% (w / v), approximately 0.006% (w / v), approximately 0.0065% (w / v), approximately 0.007% (w / v), approximately 0.0075% (w / v), approximately 0.008% (w / v), approximately 0.0085% (w / v), approximately 0.009% (w / v), approximately 0.0095% (w / v), and approximately 0.001% (w / v) Pluronic® F68. In certain embodiments, the pharmaceutical composition of this disclosure contains approximately 0.005% (w / v) Pluronic® F68.

[0062] In exemplary embodiments, the pharmaceutical compositions of this disclosure include adeno-associated virus (AAV), 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) nonionic surfactant, and a pH between 7.0 and 9.0. In certain embodiments, the pharmaceutically acceptable salt is present at about 150 nM to about 210 mM. In certain embodiments, the pharmaceutically acceptable salt is present at about 170 nM to about 200 mM. In certain embodiments, the pharmaceutically acceptable salt is sodium chloride.

[0063] This disclosure also provides a pharmaceutical composition, which may be a liquid or lyophilized (e.g., lyophilized from a liquid formulation) containing adeno-associated virus (AAV) and about 10 mM Tris buffer, about 180 mM sodium chloride, and about 0.005% (w / v) Pluronic® F68, wherein the pH of the pharmaceutical composition is about 7.9 ± 0.3.

[0064] pH

[0065] In exemplary embodiments, the pharmaceutical compositions of this disclosure have 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 embodiments, the pH of the pharmaceutical composition is greater than about 7.3 and less than about 9.0 or less than about 8.5. In certain embodiments, the pH of the pharmaceutical composition is between about 7.8 and about 9.0 or about 7.9. In other embodiments, the pH of the pharmaceutical composition is between about 7.3 and about 8.6.

[0066] Additional components

[0067] In some embodiments, the formulations or pharmaceutical compositions of the present disclosure include additional pharmaceutically acceptable components. In exemplary embodiments, the formulations or pharmaceutical compositions include any one or combination of the following: acidifiers, additives, adsorbents, aerosol propellants, air replacement agents, alkalizing agents, anticaking agents, anticoagulants, antimicrobial preservatives, antioxidants, disinfectants, bases, binders, buffers, chelating agents, coating agents, colorants, drying agents, cleaning agents, diluents, disinfectants, disintegrants, dispersants, dissolution enhancers, dyes, emollients, emulsifiers, emulsifier stabilizers, and fillers. Film-forming agents, flavor enhancers, flavoring substances, flow enhancers, gelling agents, granulating agents, wetting agents, lubricants, mucilage agents, ointment bases, ointments, oily vehicles, organic bases, fragrance bases, pigments, plasticizers, abrasives, preservatives, metal ion sequestering agents, skin penetration agents, solubilizers, solvents, stabilizers, suppository bases, surfactants, suspending agents, sweeteners, therapeutic agents, thickeners, isotonic agents, toxic agents, viscosity enhancers, water absorbents, water-miscible cosolvents, water softeners, or wetting agents. In some embodiments, the formulations or pharmaceutical compositions of the present disclosure include any one or combination of the following components: acacia, acesulfame potassium, acetyl tributyl citrate, triethyl acetyl citrate, agar, albumin, alcohol, anhydrous alcohol, denatured alcohol, dilute alcohol, aloylitic acid, alginic acid, aliphatic polyester, alumina, aluminum hydroxide, aluminum stearate, amylopectin, α-amylose, ascorbic acid, ascorbyl palmitate, aspartame, distilled water for bacteriostatic injection, bentonite, bentonite magma, benzalkonium chloride, benzethonium chloride, benzoic acid, benzalkonium chloride Dioxide alcohol, benzyl benzoate, bronopol, butylated hydroxyanisole, butylated hydroxytoluene, butylparaben, sodium butylparaben, calcium alginate, calcium ascorbate, calcium carbonate, calcium cyclamate, anhydrous calcium hydrogen phosphate, dicalcium phosphate, tricalcium phosphate, calcium propionate, calcium silicate, calcium sorbate, calcium stearate, calcium sulfate, calcium sulfate hemihydrate, rapeseed oil, carbomer, carbon dioxide, calcium carboxymethylcellulose, sodium carboxymethylcellulose, beta-carotene, carrageenan, castor oil,Hydrogenated castor oil, cationic emulsion wax, cellulose acetate, cellulose acetate phthalate, ethylcellulose, microcrystalline cellulose, powdered cellulose, silicified microcrystalline cellulose, sodium carboxymethylcellulose, cetostearyl alcohol, cetrimide, cetyl alcohol, chlorhexidine, chlorobutanol, chlorocresol, cholesterol, chlorhexidine acetate, chlorhexidine gluconate, chlorhexidine hydrochloride, chlorodifluoroethane (HCFC), chlorodifluoromethane, chlorofluorocarbon (CFC) chloro Phenoxyethanol, chlorooxylenol, corn syrup solids, anhydrous citric acid, citric acid monohydrate, cocoa butter, coloring agents, corn oil, cottonseed oil, cresol, m-cresol, o-cresol, p-cresol, croscarmellose sodium, crospovidone, cyclamic acid, cyclodextrin, dextrate, dextrin, dextrose, anhydrous dextrose, diazolidinyl urea, dibutyl phthalate, dibutyl sebacate, diethanolamine, diethyl phthalate, difluoroethane (HFC), dimethyl-β-cyclodextrin Trin, cyclodextrin-type compounds, e.g., Captisol®, dimethyl ether, dimethyl phthalate, dipotassium edetate, disodium edetate, disodium hydrogen phosphate, doxert calcium, doxert potassium, doxert sodium, dodecyl gallate, dodecyltrimethylammonium bromide, disodium calcium edetate, edetate, eglumine, ethyl alcohol, ethylcellulose, ethyl gallate, ethyl laurate, ethyl maltol, ethyl oleate, ethylparaben, potassium ethylparaben, ethyl Sodium ethylparaben, ethyl vanillin, fructose, fructose solution, pulverized fructose, pyrogen-free fructose, powdered fructose, fumaric acid, gelatin, glucose, glucose solution, saturated vegetable fatty acid glyceride mixture, glycerin, glyceryl behenate, glyceryl monooleate, glyceryl monostearate, self-emulsifying glyceryl monostearate, glyceryl palmitostearate, glycine, glycol, glycoflor, guar gum, heptafluoropropane (HFC), hexadecyltrimethylammonium bromide,High-fructose syrup, human serum albumin, hydrocarbons (HCl), dilute hydrochloric acid, hydrogenated vegetable oil type II, hydroxyethylcellulose, 2-hydroxyethyl-β-cyclodextrin, hydroxypropylcellulose, low-substituted hydroxypropylcellulose, 2-hydroxypropyl-β-cyclodextrin, hydroxypropyl methylcellulose, hydroxypropyl methylcellulose phthalate, imidourea, indigo carmine, ion exchanger, iron oxide, isopropyl alcohol, isopropyl myristate, isopropyl palmitate, isotonic saline, kaolin Phosphorus, lactic acid, lactitol, lactose, lanolin, lanolin alcohol, anhydrous lanolin, lecithin, aluminum magnesium silicate, magnesium carbonate, regular magnesium carbonate, anhydrous magnesium carbonate, magnesium carbonate hydroxide, magnesium hydroxide, magnesium lauryl sulfate, magnesium oxide, magnesium silicate, magnesium stearate, magnesium trisilicate, anhydrous magnesium trisilicate, malic acid, malt, maltitol, maltitol solution, maltodextrin, maltol, maltose, mannitol, medium-chain triglycerides, meg Lumin, menthol, methylcellulose, methyl methacrylate, methyl oleate, methylparaben, potassium methylparaben, sodium methylparaben, microcrystalline cellulose and sodium carboxymethylcellulose, mineral oil, light oil, mineral oil and lanolin alcohol, oil, olive oil, monoethanolamine, montmorillonite, octyl gallate, oleic acid, palmitic acid, paraffin, peanut oil, petrolatum, petrolatum and lanolin alcohol, pharmaceutical glazing agents, phenol, liquefied phenol, phenoxyethanol, phenoxy Ropanol, phenylethyl alcohol, phenylmercury acetate, phenylmercury borate, phenylmercury(II) nitrate, polariline, polariline potassium, poloxamer, polydextrose, polyethylene glycol, polyethylene oxide, polyacrylate, polyethylene-polyoxypropylene-block polymer, polymethacrylate, polyoxyethylene alkyl ether, polyoxyethylene castor oil derivative, polyoxyethylene sorbitol fatty acid ester, polyoxyethylene stearate, polyvinyl alcohol, polyvinylpyrrolidone,Potassium alginate, potassium benzoate, potassium bicarbonate, potassium bisulfite, potassium chloride, potassium citrate, anhydrous potassium citrate, potassium hydrogen phosphate, potassium metabisulfite, potassium dihydrogen phosphate, potassium propionate, potassium sorbate, povidone, propanol, propionic acid, propylene carbonate, propylene glycol, propylene glycol alginate, propyl gallate, propylparaben, potassium propylparaben, sodium propylparaben, protamine sulfate, Rapeseed oil, Ringer's solution, saccharin, ammonium saccharin, calcium saccharin, sodium saccharin, safflower oil, saponite, serum protein, sesame oil, colloidal silica, colloidal silicon dioxide, sodium alginate, sodium ascorbate, sodium benzoate, sodium bicarbonate, sodium bisulfite, sodium chloride, anhydrous sodium citrate, sodium citrate dihydrate, sodium chloride, sodium cyclamate, sodium edetate (sodium edentate), sodium dodecyl sulfate, sodium lauryl sulfate, sodium metabisulfite, sodium phosphate, dibasic sodium phosphate, monobasic sodium phosphate, tribasic sodium phosphate, anhydrous sodium propionate, sodium propionate, sodium sorbate, sodium starch glycolate, sodium stearyl fumarate, sodium sulfite, sorbic acid, sorbitan ester (sorbitan fatty acid ester), sorbitol, sorbitol solution 70%, soybean oil, whale wax, starch, corn starch, potato starch, pregelatinized starch, sterilizable maize starch, stearic acid, purified stearic acid, stearyl alcohol, sucrose, sugar, tableting sugar, powdered sugar, sugar spheres, invert sugar, Sugartab, Sunset Yellow FCF, synthetic paraffin, talc, tartaric acid, tartradazine, tetrafluoroethane (HFC), cocoa butter, tyromesal, titanium dioxide, alpha-tocopherol, tocopheryl acetate, alpha-tocopheryl succinate, beta-tocopherol, delta-tocopherol, gamma-tocopherol, tragacanth, triacetin, tributyl citrate, triethanolamine, triethyl citrate, trimethyl-β-cyclodextrin, trimethyltetradecylammonium bromide, Tris buffer, trisodium edetateedentate), vanillin, type I hydrogenated vegetable oil, water, soft water, hard water, carbon dioxide-free water, pyrogen-free water, distilled water for injection, sterile water for inhalation, sterile distilled water for injection, sterile water for irrigation, wax, anionic emulsifying wax, carnauba wax, cationic emulsifying wax, cetyl ester wax, microcrystalline wax, nonionic emulsifying wax, suppository wax, white wax, yellow wax, white petrolatum, lanolin fat, xanthan gum, xylitol, zein, zinc propionate, zinc salts, zinc stearate, or any excipients listed in Handbook of Pharmaceutical Excipients, Third Edition, AHKibbe (Pharmaceutical Press, London, UK, 2000) (the whole of which is incorporated for reference). Remington's Pharmaceutical Sciences, Sixteenth Edition, EW Martin (Mack Publishing Co., Easton, Pa., 1980) (which is incorporated in its entirety for all intended purposes) discloses various components used in the formulation of pharmaceutically acceptable compositions and known techniques for their preparation. Any conventional agent is intended for use in a pharmaceutical composition unless it is incompatible with the pharmaceutical composition.

[0068] In some embodiments, the formulation or pharmaceutical composition comprises a sugar or a sugar alcohol. In some embodiments, the sugar or sugar alcohol is sucrose, trehalose, mannitol, or a combination thereof.

[0069] In exemplary embodiments, the formulations or pharmaceutical compositions of the Disclosure do not contain one or a combination of the above-mentioned components. In exemplary embodiments, the formulations or pharmaceutical compositions of the Disclosure do not contain any of these components. In exemplary embodiments, the pharmaceutical compositions of the Disclosure do not contain dextran. In exemplary embodiments, the pharmaceutical compositions of the Disclosure do not contain calcium chloride. In other exemplary embodiments, the pharmaceutical compositions of the Disclosure do not contain sugars or sugar alcohols (e.g., sucrose, trehalose, or mannitol). In related exemplary embodiments, the pharmaceutical compositions do not contain glycine.

[0070] AAV

[0071] In exemplary embodiments, the pharmaceutical composition of the present disclosure comprises AAV. AAV may be any AAV serotype. In exemplary embodiments, AAV may be AAV1 serotype, AAV2 serotype, AAV3 serotype, AAV4 serotype, AAV5 serotype, AAV6 serotype, AAV7 serotype, AAV8 serotype, AAV9 serotype, or AAV10 serotype. In exemplary embodiments, AAV may be AAV2 serotype or a variant thereof.

[0072] In certain embodiments, the AAV is an rAAV described in U.S. Patent Application Publication 2020 / 0282077 (the entirety of which is incorporated herein by reference), in particular an rAAV comprising a capsid protein as defined in paragraphs 171–179 of U.S. Patent Application Publication 2020 / 0282077 and / or an rAAV comprising heterologous nucleic acids as defined in paragraphs 222–248 of U.S. Patent Application Publication 2020 / 0282077.

[0073] In some embodiments, the pharmaceutical composition comprises an rAAV containing a mutant AAV capsid encapsulating a heterologous nucleic acid encoding a gene product, wherein the mutant AAV capsid comprises a mutant AAV capsid protein containing an insertion of about 7 to about 20 amino acids ("heterologous peptide" or "peptide insertion") within the GH loop of the parent AAV capsid protein, the peptide comprising the amino acid sequence ISDQTKH (SEQ ID NO: 1). Preferably, when the mutant capsid protein is present in an AAV virion, it increases the infectivity of retinal cells compared to the infectivity of retinal cells by an AAV virion containing the corresponding parent capsid protein.

[0074] The "GH loop" or loop IV of the AAV capsid protein refers in this art to the solvent-accessible portion of the AAV capsid protein known as the GH loop or loop IV. For more information on the GH loop / loop IV of the AAV capsid, see, for example, van Vliet et al. (2006) Mol. Ther. 14:809; Padron et al. (2005) J. Virol. 79:5047; and Shen et al. (2007) Mol. Ther. 15:1955. Therefore, for example, the insertion site may be within the range of approximately amino acids 570-611 of AAV2 VP1.

[0075] In some embodiments, the peptide insertion has 1 to 3 spacer amino acids (Y1 to Y3) at the amino and / or carboxyl terminals of the amino acid sequence ISDQTKH (SEQ ID NO: 1). Exemplary spacer amino acids include, but are not limited to, leucine (L), alanine (A), glycine (G), serine (S), threonine (T), and proline (P). In certain embodiments, the peptide insertion has 2 spacer amino acids at the N-terminus and 2 spacer amino acids at the C-terminus. In other embodiments, the peptide insertion has 2 spacer amino acids at the N-terminus and 1 spacer amino acid at the C-terminus. In preferred embodiments, the peptide insertion includes or is derived from the amino acid sequence LAISDQTKHA (SEQ ID NO: 2).

[0076] In some embodiments, the variant AAV capsid protein comprises a peptide insertion containing the amino acid sequence ISDQTKH (SEQ ID NO: 1) and further comprises one or more amino acid substitutions compared to the corresponding parent AAV capsid protein. Typical examples of amino acid substitutions are described, for example, in column 26, lines 40-65 of U.S. Patent No. 11,576,983, the entire contents of which are incorporated herein by reference.

[0077] In some preferred embodiments, the variant AAV capsid protein comprises a peptide insertion containing the amino acid sequence ISDQTKH (SEQ ID NO: 1), and further comprises a P34A amino acid substitution for the VP1 capsid of AAV2 or a corresponding substitution in another AAV serotype.

[0078] In other embodiments, the variant capsid protein may include one or more features disclosed in U.S. Patent No. 11,576,983, particularly one or more features disclosed in columns 26, lines 66-29, and line 50 of U.S. Patent No. 11,576,983.

[0079] In a particularly preferred embodiment, the mutant capsid protein contains the following amino acid sequence, or contains an amino acid sequence that is at least 80%, at least 90%, at least (least)95%, at least 98%, or at least 99% identical to the following amino acid sequence: [ka] [ka]

[0080] The mutant AAV capsid protein of SEQ ID NO: 3 includes the following modifications compared to the native AAV2 capsid: (i) a mutation from proline (P) to alanine (A) at amino acid position 34 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 / LAISDQTKHA (SEQ ID NO: 2)) at amino acid position 588 present in VP1, VP2, and VP3. In some embodiments, the capsid includes a mutant capsid protein that contains at least 90%, at least 95%, at least 98%, or at least 99% identical sequence to SEQ ID NO: 3, and includes the P34A substitution at amino acid position 588 and the LAISDQTKHA (SEQ ID NO: 2) peptide insertion.

[0081] In some embodiments, the pharmaceutical compositions described herein include rAAVs encapsulating heterogeneous nucleic acids comprising nucleotide sequences encoding one or more gene products. In some embodiments, the one or more gene products are selected from interfering RNAs (e.g., microRNAs) and polypeptides. In particularly preferred embodiments, the heterogeneous nucleic acids encode aflibercept and, optionally, further encode interfering RNA that reduces VEGF-C expression. In relevant embodiments, the heterogeneous nucleic acids include nucleotide sequences particularly those listed in Table 2 of U.S. Patent Application Publication No. 2024 / 0131195A1 (the entirety of which is incorporated herein by reference).

[0082] In some embodiments, rAAV comprises heterogeneous nucleic acids including the following nucleotide sequence encoding aflibercept that has been codon-optimized for expression in humans: [ka] [ka]

[0083] 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: 4, and / or includes a stop codon (e.g., TGA) at the end of the sequence. In some embodiments, the aflibercept gene product includes the following amino acid sequence or a sequence that is at least 90%, 95%, 97%, 98%, or at least 99% identical thereto: [ka]

[0084] In a related embodiment, the pharmaceutical composition includes an rAAV comprising a heterogeneous nucleic acid containing the following sequence (encoding aflibercept + human VEGF-C interfering RNA) or a sequence identical to it by at least 80%, at least 90%, at least 95%, at least 98%, or at least 99%: [ka] [ka]

[0085] In relevant embodiments, the pharmaceutical composition comprises a heterogeneous nucleic acid comprising a nucleotide sequence encoding a wild-type or codon-optimized human Rab escort protein-1 (REP1) protein as described in, for example, U.S. Patent No. 11,357,870 (the entirety of which is incorporated herein by reference). In some preferred embodiments, the heterogeneous nucleic acid encoding REP1 comprises the following codon-optimized nucleotide sequences or sequences that are at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 97%, at least 98%, or at least 99% identical thereto: [ka] [ka]

[0086] In other related embodiments, the pharmaceutical composition comprises a heterologous nucleic acid (rAAV) comprising a nucleotide sequence encoding a wild-type or codon-optimized retinitis pigmentosa GTPase regulatory factor (RPGR) protein as described, for example, U.S. Patent No. 11,345,930 (the entirety of which is incorporated herein by reference). In some preferred embodiments, the heterologous nucleic acid comprises a codon-optimized nucleotide sequence encoding human RPGR ORF15 or a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 97%, at least 98%, or at least 99% identical thereto: [ka] [ka] [ka]

[0087] In other related embodiments, the pharmaceutical composition comprises a heterologous nucleic acid comprising a nucleotide sequence encoding an engineered human complement regulator H(fH) gene operably linked to an expression regulatory sequence, wherein the human fH(hfH) gene encodes a soluble hfH protein variant that retains complement regulatory function, and the fH variant comprises short consensus repeats (SCRs) 1, 2, 3, 4, 19, and 20. In some embodiments, the soluble hfH protein variant comprises an amino acid sequence described in U.S. Patent No. 10,988,519 (the entirety of which is incorporated herein by reference). In particularly preferred embodiments, the heterologous nucleic acid encodes an fH variant having the following amino acid sequence: [ka]

[0088] In some embodiments, heterologous nucleic acids include the following nucleotide sequences or nucleotide sequences that are at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical thereto: [ka] [ka] [ka]

[0089] In relevant embodiments, heterologous nucleic acids include the following nucleotide sequences or nucleotide sequences that are at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical thereto: [ka] [ka] [ka]

[0090] In relevant embodiments, heterologous nucleic acids include the following nucleotide sequences or nucleotide sequences that are at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical thereto: [ka] [ka] [ka]

[0091] In related embodiments, the heterologous nucleic acid comprises the following nucleotide sequence or a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical thereto: [Chemical formula] [Chemical formula] [Chemical formula]

[0092] In some embodiments, the pharmaceutical composition comprises 1×10 12 , 11 , 12 , 12 , 12 , 12 , 12 , 12 , 13 , 12 , 12 ~1×10 15 vector particles (vp) or vector genomes (vg), 1×10 10 ~1×10 13 vector particles or vector genomes, or about 1×10 10 , about 2×10 10 , 3×10 10 , about 4×10 10 , about 5×10 10 , about 6×10 10 , about 7×10 10 , about 8×10 10 , about 9×10 10 , about 1×10 11 , about 2×10 11 , about 3×10 11 , about 4×10 11 , about 5×10 11 , about 6×10 11 , about 7×10 11 , about 8×10 11 , about 9×10 11 , about 1×10 12 , about 2×10 12 , about 3×10 12 , about 4×10 12 , about 5×10 12 , about 6×10 12 , about 7×10 12 , about 8×10 12 , about 9×10 12 [[ID=8​It contains vector particles or vector genomes. In some embodiments, the pharmaceutical composition contains about 1 × 10⁶ 11 ~Approx. 1×10 12 Contains individual vector particles or vector genomes.

[0093] In some embodiments, the pharmaceutical composition maintains at least 90% relative potency in liquid form for a period of at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, at least 7 weeks, at least 8 weeks, at least 9 weeks, at least 10 weeks, at least 3 months, at least 4 months, at least 5 months, or at least 6 months at 25°C and 60% relative humidity.

[0094] In exemplary embodiments, a composition containing AAV exhibits storage stability as a liquid for at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, or at least 12 months. In exemplary embodiments, more than 80% of the initial amount of AAV (e.g., the amount of AAV in the composition before storage) becomes effective after the storage period (e.g., about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, or about 6 months, or longer). In exemplary embodiments, more than 90% of the initial amount of AAV becomes effective after the storage period (e.g., about 1 month, about 2 months, 3 months, about 4 months, about 5 months, or about 6 months, or longer). In exemplary embodiments, more than 95% of the initial amount of AAV becomes effective after a storage period (e.g., approximately 3 months, 4 months, 5 months, or 6 months, or longer). In exemplary embodiments, the biological potency of the AAV at the end of the storage period is substantially the same as that of the AAV at the beginning of the storage period. In exemplary embodiments, the biological potency of the AAV at the end of the storage period increases compared to that of the AAV at the beginning of the storage period. In exemplary embodiments, the appearance of the composition at the end of the storage period is substantially the same as that of the composition at the beginning of the storage period. In exemplary embodiments, the appearance of the composition at the end of the storage period is characterized by the absence of visible particles. In exemplary embodiments, the particle concentration of the composition at the end of the storage period is substantially the same as that of the composition at the beginning of the storage period. In exemplary embodiments, the particle concentration of the composition at the end of the storage period is determined by microflow imaging (MFI).

[0095] In some embodiments, the pharmaceutical composition is maintained at approximately -60°C or below for a significant portion of the storage period (e.g., duration). In other embodiments, the pharmaceutical composition is maintained at approximately 2°C to approximately 8°C for a significant portion of the storage period (e.g., duration).

[0096] In some embodiments, a method is provided for treating a disorder in a subject requiring treatment of the disorder, the method comprising the step of administering to the subject an amount of a pharmaceutical composition described herein that is effective in treating the disorder. In some preferred embodiments, the pharmaceutical composition is administered intraocularly to a human having a VEGF-related ocular disorder in an amount effective in treating the VEGF-related ocular disorder, preferably via intravitreous, subretinal, and / or parachoroidal injection, more preferably via a single intravitreous injection. In other embodiments, the disorder is selected from those enumerated in paragraphs 218 and 246 of U.S. Patent Application Publication No. 2020 / 0282077.

[0097] The formulations disclosed herein may be formulated for administration via known methods such as intraocular administration (e.g., via intravitreal, subretinal, and / or choroidal administration). In some preferred embodiments, the pharmaceutical composition is administered intraocularly to a human, preferably via intravitreal, subretinal, and / or choroidal injection, more preferably via a single intravitreal injection.

[0098] In some embodiments, the treatment regimen includes the administration of one or more doses over a long period. In certain cases, a single dose (e.g., a single dosing unit) is administered to the subject, and one or more doses may be administered to the subject at subsequent time points after the initial dose. In some examples, more than one dose (e.g., more than one dosing unit) is administered to the subject, and one or more doses may be administered to the subject at subsequent time points after the initial dose. For example, a single dose (e.g., a single dosing unit) may be administered to the subject, and after the single dose, a single dose may be administered to the subject at a subsequent time point. An additional single dose may be administered at a subsequent time point. In other cases, a single dose (e.g., a single dosing unit) may be administered to the subject, and after the single dose, two doses may be administered to the subject at a subsequent time point. An additional single or multiple dose may be administered at a subsequent time point.

[0099] In certain embodiments, the drug units of the Disclosure may be administered, for example, in combination therapy, before, simultaneously with, or after other active agents for treating related or unrelated symptoms. The administration of separate pharmaceutical compositions may be simultaneous or at different times (e.g., consecutively, in any order, on the same day or on different days), insofar as the therapeutically effective effect of the combination of these substances is induced in the subject being treated. Thus, embodiments of the Disclosure further include combination therapy. In certain embodiments, the method of the Subject Matter includes the step of administering a therapeutically effective amount of one or more additional active agents. Combination therapy means that an AAV composition (e.g., as described herein) can be used in combination with another therapeutic agent to treat a single disease or symptom. In certain embodiments, the compounds of the Disclosure are administered simultaneously with the administration of another therapeutic agent, and the other therapeutic agent may be administered as a component of a composition containing the compounds of the Disclosure or as a component of a different composition. In certain embodiments, a composition containing the compounds of the Disclosure is administered before or after the administration of another therapeutic agent. [Examples]

[0100] Examples The following embodiments illustrate preferred embodiments of the present invention and are not intended to limit the scope of the invention. While the present invention has been described in relation to its preferred embodiments, various modifications will be apparent to those skilled in the art from reading this application.

[0101] Example 1 The following section summarizes the key findings that led to the identification of an improved rAAV formulation (10 mM Tris buffer, pH 7.9, 180 mM NaCl, 0.005% Pluronic® F68), which shows a reduced risk of microscopic particle formation and improved liquid storage stability, consistent with the importance of stabilization provided by increased ionic strength, reduction of rAAV surface adsorption through the use of surfactants, and optimization of formulation pH. Wright, JF et al. Mol Ther. 2005 Jul;12(1):171-8; Patricio MI, Mol Ther Methods Clin Dev. 2019 Nov 20;17:99-106; Srivastava, A. et al. Journal of Pharmaceutical Sciences, Volume 110, Issue 7, 2021. The tendency for rAAV (containing the capsid protein of SEQ ID NO: 3 and nucleic acid including the nucleotide sequence of SEQ ID NO: 6) to increase in stability at higher pH levels was the initial motivation for selecting the Tris buffer component (Tris hydrochloride / tromethamine) in improved rAAV formulations. However, at least one study also showed that Tris buffer reduced susceptibility to chemical degradation (e.g., deamidation and oxidation) compared to Hepes and phosphate formulations prepared with sodium chloride and Pluronic® F68 at the same target pH and equivalent concentrations.

[0102] [Table 1]

[0103] rAAV containing the capsid protein of SEQ ID NO: 3 and nucleic acids including the nucleotide sequence of SEQ ID NO: 6 was buffer-exchanged in five different buffers (Forms 1-5) and subjected to various forced degradation conditions. A summary of the titer, DLS, and subvisible particle test results can be found in Table 2. The titer was similar under most conditions, except after storage at 37°C for 3 days. Interestingly, the titer at 37°C suggested increased thermal stability in Form 4 (10 mM histidine, pH 6, 180 mM NaCl, 0.005% F68). However, Form 4 showed a significant decrease in colloidal stability under most conditions tested (i.e., increased polydispersity index (PDI) and decreased % area (diameter 10-100 nm) due to dynamic light scattering (DLS), as well as increased SVP). Conversely, Form 2 (10 mM sodium phosphate, pH 7.0, 170 mM NaCl, 0.005% F68), Form 3 (10 mM sodium phosphate, pH 7.0, 180 mM NaCl, 0.005% F68), and Form 5 (10 mM Tris buffer, pH 8.0, 180 mM NaCl, 0.005% F68) showed improved colloidal stability compared to the Form 1 control (DPBS, 0.005% F68) under most conditions. To further illustrate the higher risk of subvisible particle formation shown by Forms 1 and 4, representative background membrane images (Figure 1) were included under 3-day storage conditions. These results suggest that pH 7 or pH 8 formulations containing increased sodium chloride are more likely to yield improvements over the buffer (DPBS, 0.005% F68).

[0104] [Table 2]

[0105] pH optimization

[0106] The following studies aimed to further evaluate pH dependence. In the first study, rAAV (containing the capsid protein of SEQ ID NO: 3 and the nucleic acid containing the nucleotide sequence of SEQ ID NO: 6) was buffer-exchanged in six different formulations containing sodium phosphate buffer in the pH range of 6.0–7.0. In the second study, rAAV (containing the capsid protein of SEQ ID NO: 3 and the nucleic acid containing the nucleotide sequence of SEQ ID NO: 6) was buffer-exchanged in phosphate buffer or Tris buffer at pH 7.0, 7.4, and 7.8. These tests (along with subsequent formulation development experiments) included testing of aflibercept (AFLIB) expression using a modified form of the release assay adapted for high-throughput sample analysis. Aflibercept expression was reported as relative potency (%RP) or AFLIB expression compared to a standard. Similar to previous results, the first of these studies demonstrated a decrease in colloidal stability when the pH decreased from 7.0 to 6.0 (Table 3). A decrease in relative potency (Figure 2) was also observed for low-pH formulations in response to heat stress. Conversely, a reduced risk of subvisible particle formation at 25°C / 60%RH and comparable or better 2-week storage stability were observed for rAAV formulated in 10 mM sodium phosphate, pH 7.0, 175 mM NaCl, 0.005% F68 compared to rAAV formulated in Form 1 (DPBS, 0.005% F68). Results from a second study (Table 4) demonstrated a further reduction in the risk of subvisible particles as the pH increased from 7.0 to 7.8. Most interestingly, rAAV formulated in 10 mM Tris buffer, pH 7.8, 180 mM NaCl, 0.005% F68 not only showed a low subvisible particle count under all conditions but also demonstrated the best relative potency (98.2%) after 2 weeks of storage at 25°C / 60%RH (Figure 3).

[0107] [Table 3]

[0108] [Table 4]

[0109] The results of the previous study suggested that formulation of rAAV (e.g., containing the capsid protein of SEQ ID NO: 3) in 10 mM Tris buffer, pH 7.8, 180 mM NaCl, and 0.005% F68 not only satisfies the primary objective (i.e., reduction of subvisible particles) but also improves liquid storage stability. Therefore, pH 7.8 was re-examined (in phosphate, Tris, and Hepes) and compared with Form 1 (DPBS, 0.005% F68) during storage at 25°C / 60%RH. The relative potency results can be found in Table 5. All four formulations exhibited very similar behavior, and there was a possibility of slight improvement associated with the pH 7.8 formulations (Forms 1-3) after 2 weeks of storage at 25°C / 60%RH. Furthermore, mass spectrometry results (Table 6) did not reveal any new degradation products for the pH 7.8 formulations at T=0. However, the phosphate and Hepes pH 7.8 formulations appeared to show an increased deamidation rate compared to Form 1 during 2 weeks of storage at 25°C / 60%RH. Conversely, the Tris (pH 7.8) formulation showed a deamidation profile very similar to Form 1, while also exhibiting a reduced risk of oxidation.

[0110] [Table 5]

[0111] [Table 6]

[0112] Confirmatory forced disassembly study

[0113] Overall, the subvisible particle data, relative potency, and chemical stability all suggested that Tris-based buffers were a suitable choice for improved rAAV formulations. Therefore, the intention of the next formulation development study was to conduct forced degradation experiments using the bracket method (Tris, pH 7.8 ± 0.3) in a representative container capping system (i.e., 0.3 mL filled into a 2 mL CZ vial). Samples were tested after T=0, three freeze-thaw cycles (3 × FT), and two weeks of storage at 2–8°C and 25°C / 60% RH. Results can be found in Table 8. The FDA states that "GT vector-based final products should be tested for particulate matter, and the test methods and shipping standards should be those specified in the USP <789> It demands that they "should comply." See https: / / www.fda.gov / media / 124641 / download. In the testing of particles invisible to the naked eye, 11 out of 12 samples tested were subject to USP <789> The results met the limits. Furthermore, 100% area (volume diameter 10-100 nm) was observed in 11 of the 12 tested samples. These two results seemed to confirm an increase in physical stability within the tested range. Relative potency also proved to be very similar for all three formulations. Each showed a decrease in aflibercept expression after 2 weeks of storage at 25°C / 60%RH and relative potency of over 100% after 2 weeks of storage at 2-8°C, respectively.

[0114] [Table 7]

[0115] [Table 8-1]

[0116] The results of this confirmatory development study ultimately led to the selection of the formulation initially described as 10 mM Tris buffer, pH 8.0, 180 mM NaCl, 0.005% F68. However, it was confirmed that most pH tests performed during formulation development were measured at room temperature (typically in the range of approximately 19–21°C). Therefore, in efforts to improve the robustness of the diluent manufacturing (particularly the known temperature dependence exhibited by Tris buffer), all future pH tests (in-process and final) should be conducted according to the USP <791> It was determined that the test should be performed at 25°C. Therefore, three preparations of lead formulation buffer were prepared and pH tests were performed at 20°C and 25°C (Table 8). As expected, the pH decreased by approximately 0.1 pH units when measured at 25°C. This prompted corresponding changes to the target pH (7.9 ± 0.3) and the buffer description (10 mM Tris buffer, pH 7.9, 180 mM NaCl, 0.005% F68).

[0117] [Table 8-2]

[0118] Example 2 The effect of sodium chloride concentration (140–200 mM) was evaluated in the following formulations after T=0 and three freeze-thaw cycles (3×FT). All three formulations contained rAAV, which includes the capsid protein of SEQ ID NO: 3 and the nucleotide sequence of SEQ ID NO: 6, at a target concentration of approximately 1.6E12 vg / mL. 20 mM Tris, pH 8 1 140 mM NaCl, 0.005% F68 20 mM Tris, pH 8 1 180 mM NaCl, 0.005% F68 20 mM Tris, pH 8 1 200 mM NaCl, 0.005% F68 1 pH 8 is the target pH at room temperature. This is expected to be approximately 0.1 pH units lower (i.e., 7.9) when measured at 25°C.

[0119] Samples tested with the Wyatt DynaProIII DLS plate reader (Figure 4) demonstrated similar colloidal stability (i.e., over 90% of the area (diameter 10–100 nm) based on intensity) across the evaluated sodium chloride range.

[0120] The samples were then filtered with 0.45 μm, spiked with 20×Sybr Gold, and tested by differential scanning fluorescence (Uncle instrument, Unchained Labs). The final fluorescence (final FL) values ​​reported in Figure 5 represent the fluorescence emission signal in the 500–650 nm range collected at 15°C after the samples were subjected to thermal melting (15–95°C). The results suggest similar amounts of accessible ssDNA (i.e., relative titer) present in all three formulations at T=0. The 3×FT samples showed slightly higher signal recovery at sodium chloride concentrations of 180 mM or higher. However, none of the filtered 3×FT stressed samples showed a recovery rate of less than 78% (Figure 6).

[0121] Many of the above study results include pH values ​​measured at room temperature. However, as shown in Table 8, Tris buffer exhibits temperature dependence. This temperature dependence was also captured in individual tests in which Tris (pH 7.8) buffer was measured at room temperature (the same temperature at which the buffer was initially prepared and tested) and after equilibration at either 2–8°C or 25°C / 60%RH stability chambers. The results indicate that a pH shift of approximately -0.1 units should be expected for 10 mM Tris buffer prepared at room temperature and then stored at 25°C (consistent with Table 8). Furthermore, a pH shift of 0.3 units is expected for 10 mM Tris buffer prepared at room temperature and then stored at 2–8°C. This, in turn, means that rAAV containing the capsid protein of Sequence ID No. 3 demonstrated stability in Tris-buffered saline solutions ranging from approximately pH 7.3 (pH 7.4 preparation stored at 25°C / 60%RH) to 8.6 (pH 8.3 preparation stored at 2-8°C), as shown in Table 9 below.

[0122] [Table 9]

[0123] Example 3 The long-term stability of rAAV containing the capsid protein of SEQ ID NO: 3 and the nucleic acid containing the nucleotide sequence of SEQ ID NO: 6, formulated in 10 mM Tris, 180 mM NaCl, and 0.005% Pluronic® F68, was subsequently evaluated at -60°C and 2–8°C.

[0124] In short, product development lots 4DER000096 and 4DER000097 (2-mL CZ vials filled with rAAV containing nucleic acids including the capsid protein of SEQ ID NO: 3 and the nucleotide sequence of SEQ ID NO: 6 formulated in 10 mM Tris, 180 mM NaCl, and 0.005% Pluronic® F68) were evaluated in simplified long-term stability studies conducted at -60°C and 2–8°C. The results for pH, subvisible particles, genomic titer, transgene expression (protein-based), and monodispersity can be found in Tables 10 and 11 below. The results for clinical lot 4D2210031 can be found in Table 12.

[0125] [Table 10]

[0126] [Table 11]

[0127] [Table 12]

[0128] The results show that rAAV containing the capsid protein of SEQ ID NO: 3 and the nucleic acid containing the nucleotide sequence of SEQ ID NO: 6, formulated in 10 mM Tris, 180 mM NaCl, and 0.005% Pluronic® F68, is stable for more than 12 months at -60°C and for 3 months or longer at 2–8°C. [Table 13]

[0129] While the materials and methods of the present invention have been described in relation to preferred embodiments, it will be apparent to those skilled in the art that modifications can be applied to the methods described herein without departing from the concept, spirit, and scope of the present invention. All such similar substitutions and modifications, which will be apparent to those skilled in the art, are considered to fall within the spirit, scope, and concept of the present invention.

Claims

1. A pharmaceutical composition comprising adeno-associated virus (AAV) and a buffering composition, (i) A buffer solution of approximately 5 mM to approximately 20 mM, preferably Tris buffer solution; (ii) A pharmaceutically acceptable salt, preferably NaCl, in a concentration of about 100 mM to about 250 mM; and (iii) Nonionic surfactant in a concentration of approximately 0.0001% (w / v) to approximately 0.01% (w / v) Includes, A pharmaceutical composition wherein the buffer composition has a pH of approximately 7.0 to approximately 9.

0.

2. The buffer composition is (i) The buffering agent in a volume of approximately 7 mM to approximately 15 mM; (ii) the pharmaceutically acceptable salt in a concentration of about 150 mM to about 200 mM; and (iii) The nonionic surfactant in an amount of approximately 0.0001% (w / v) to approximately 0.01% (w / v). Includes, The pharmaceutical composition according to claim 1, wherein the buffer composition has a pH greater than 7.

0.

3. The buffer composition is (i) The buffering agent in a volume of approximately 7 mM to approximately 12 mM; (ii) the pharmaceutically acceptable salt in a concentration of about 150 to about 200 mM; and (iii) The nonionic surfactant in an amount of approximately 0.0005% (w / v) to approximately 0.01% (w / v). Includes, The pharmaceutical composition according to claim 1 or 2, wherein the buffer composition has a pH greater than 7.0 and less than about 8.

5.

4. The buffer composition is (i) The buffering agent in a volume of approximately 7 mM to approximately 12 mM; (ii) the pharmaceutically acceptable salt in a concentration of about 150 to about 200 mM; and (iii) The nonionic surfactant in an amount of approximately 0.0005% (w / v) to approximately 0.01% (w / v). Includes, The pharmaceutical composition according to any one of claims 1 to 3, wherein the buffer composition has a pH of about 7.5 to about 8.

3.

5. The buffer composition is (i) The buffering agent in a volume of approximately 9 mM to approximately 20 mM; (ii) the pharmaceutically acceptable salt in a concentration of about 140 to about 200 mM; and (iii) Approximately 0.001% (w / v) to approximately 0.01% (w / v) of the aforementioned nonionic surfactant Includes, The pharmaceutical composition according to claim 1, wherein the buffer composition has a pH of about 7.3 to 8.

6.

6. The buffer composition is (i) The buffering agent in an amount of approximately 10 mM; (ii) the pharmaceutically acceptable salt at approximately 180 mM; (iii) Approximately 0.005% of the nonionic surfactant Includes, The pharmaceutical composition according to any one of claims 1 to 5, wherein the buffer composition has a pH of about 7.

9.

7. The pharmaceutical composition according to any one of claims 1 to 6, wherein the buffering agent is a Tris buffer.

8. The pharmaceutical composition according to claim 7, wherein the pharmaceutical composition contains 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, or about 20 mM Tris buffer.

9. The pharmaceutical composition according to any one of claims 1 to 8, wherein the pharmaceutically acceptable salt is selected from the group consisting of sodium salts, ammonium salts, potassium salts, and combinations thereof.

10. The pharmaceutical composition according to claim 9, wherein the pharmaceutically acceptable salt is NaCl.

11. The pharmaceutical composition according to claim 1, comprising approximately 100–250 mM, approximately 110–240 mM, approximately 120–230 mM, approximately 130–220 mM, approximately 140–220 mM, approximately 150–210 mM, approximately 150–200 mM, approximately 160–200 mM, approximately 170–190 mM, approximately 175–190 mM, approximately 175–185 mM, or approximately 180 mM of NaCl.

12. The pharmaceutical composition according to claim 1, wherein the buffer composition has a pH of about 7.1, 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, or about 8.

7.

13. The pharmaceutical composition according to any one of claims 1 to 12, wherein the nonionic surfactant is a nonionic block copolymer, preferably a polyoxyethylene-polyoxypropylene block copolymer, more preferably Pluronic® F68, and preferably the pharmaceutical composition contains about 0.001%, about 0.002%, about 0.003%, about 0.004%, about 0.005%, about 0.006%, about 0.007%, about 0.008%, about 0.009%, or about 0.01% of Pluronic® F68.

14. The pharmaceutical composition according to any one of claims 1 to 13, wherein the pharmaceutical composition exhibits a reduction in invisible particle formation and / or improved liquid storage stability compared to a pharmaceutical composition comprising phosphate-buffered saline (PBS) buffer (8 mM Na₂HPO₄, 1.5 mM KH₂PO₄, 2.7 mM KCl, 138 mM NaCl, 0.9 mM CaCl₂, and 0.5 mM MgCl₂, pH 7.0; DPBS) and Pluronic® F68.

15. The pharmaceutical composition according to any one of claims 1 to 14, wherein the AAV comprises a serotype 2 capsid protein (AAV2 capsid) or a variant thereof.

16. The pharmaceutical composition according to claim 15, wherein the AAV comprises a mutant capsid protein, the mutant capsid protein comprises a heterologous peptide of 7, 8, 9, 10, or 11 amino acids in length that is covalently inserted into the GH loop of the capsid protein compared to the AAV2 capsid, and the peptide insertion comprises the amino acid sequence ISDQTKH (SEQ ID NO: 1).

17. The inserted peptide has 1 to 3 spacer amino acids (Y) at the amino and / or carboxyl terminals of the amino acid sequence ISDQTKH (SEQ ID NO: 1). １ ~Y ３ The pharmaceutical composition according to claim 16, wherein the inserted peptide is preferably LAISDQTKHA (SEQ ID NO: 2).

18. The pharmaceutical composition according to claim 17, wherein the peptide is inserted after any of the amino acids at positions 584-591 in VP1 of AAV2 or at a corresponding position in another AAV serotype, preferably the insertion position is between amino acids 587 and 588 of VP1 of AAV2, or between amino acids 588 and 589 of AAV2, or at a corresponding position in the capsid protein of another AAV serotype.

19. The mutant capsid protein has one or more amino acid substitutions compared to VP1 of AAV2, preferably the following amino acid substitutions: MIL, L15P, P34A, N57D, N66K, R81Q, Q101R, S109T, R144K, R144M, Q164K, T176P, L188I, S196Y, G226E, G236V, I240T, P250S, N A pharmaceutical composition according to any one of claims 16 to 18, comprising one or more of the amino acid substitutions 312K, P363L, D368H, N449D, T456K, S463Y, D472N, R484C, A524T, P535S, N551S, A593E, 1698V, V708I, V719M, S721L, and L735Q, more preferably P34A.

20. The pharmaceutical composition according to claim 19, wherein the mutant capsid protein is at least 90% identical, at least 95% identical, at least 98% identical, or 100% identical to the full length of the amino acid sequence described in SEQ ID NO:

3.

21. The pharmaceutical composition according to any one of claims 1 to 20, wherein the AAV is a recombinant AAV (rAAV) comprising a heterogeneous nucleic acid containing a nucleotide sequence encoding a gene product.

22. The pharmaceutical composition according to claim 21, wherein the gene product is an interfering RNA and / or polypeptide, and the nucleotide sequence encoding the gene product is operably linked to a promoter.

23. The pharmaceutical composition according to claim 22, wherein the heterogeneous nucleic acid comprises a nucleotide sequence encoding the polypeptide of Sequence ID No.

5.

24. The pharmaceutical composition according to claim 23, wherein the nucleotide sequence encoding the polypeptide of SEQ ID NO: 5 is at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to that of SEQ ID NO:

4.

25. The pharmaceutical composition according to any one of claims 22 to 24, wherein the nucleotide sequence encodes an interfering RNA that reduces the expression of VEGF-C.

26. The pharmaceutical composition according to any one of claims 23 to 25, wherein the heterogeneous nucleic acid comprises a nucleotide sequence that is at least 70% identical to the nucleotide sequence described in Sequence ID No.

6.

27. A pharmaceutical composition according to any one of claims 23 to 26 for use in treating neovascular (wet) age-related macular degeneration, macular edema after retinal vein occlusion, diabetic macular edema, and / or diabetic retinopathy.

28. The pharmaceutical composition according to claim 22, wherein the heterogeneous nucleic acid comprises a nucleotide sequence encoding the Rab escort protein-1 (REP1) protein.

29. The pharmaceutical composition according to claim 28, wherein the nucleotide sequence encoding the REP1 protein is at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to SEQ ID NO:

7.

30. The pharmaceutical composition according to claim 28 or 29 for use in treating coloideremia.

31. The pharmaceutical composition according to claim 22, wherein the heterogeneous nucleic acid comprises a nucleotide sequence encoding a retinitis pigmentosa GTPase regulatory factor (RPGR) protein.

32. The pharmaceutical composition according to claim 31, wherein the nucleotide sequence encoding the RPGR protein is at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to SEQ ID NO:

8.

33. The pharmaceutical composition according to claim 31 or 32 for use in treating X-linked retinitis pigmentosa.

34. The pharmaceutical composition according to claim 22, wherein the heterogeneous nucleic acid comprises a nucleotide sequence encoding a complement regulator H (CFH) protein or a fragment thereof.

35. The pharmaceutical composition according to claim 34, wherein the nucleotide sequence encoding the CFH protein or a fragment thereof has at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% sequence identity with respect to the amino acid sequence described in Sequence ID No.

9.

36. The pharmaceutical composition according to claim 35, wherein the nucleotide sequence encoding the CFH protein or a fragment thereof comprises the amino acid sequence described in Sequence ID No.

9.

37. The rAAV according to any one of claims 34 to 36, wherein the rAAV has at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% sequence identity with respect to the nucleotide sequence described in any one of sequence numbers 10 to 13, or comprises a heterogeneous nucleic acid containing a nucleotide sequence that is identical to the nucleotide sequence described in any one of sequence numbers 10 to 13.

38. A pharmaceutical composition according to any one of claims 34 to 37 for use in treating dry age-related macular degeneration and / or geographic atrophy.

39. The pharmaceutical composition according to any one of claims 21 to 38, wherein the nucleic acid further comprises a 5'ITR, a promoter operably linked to the nucleotide sequence, a polyadenylated sequence, and a 3'ITR.

40. The pharmaceutical composition according to claim 39, wherein the promoter is a ubiquitous promoter, and preferably the promoter is a CAG or CBA promoter.

41. The pharmaceutical composition according to claim 39, wherein the promoter is a tissue-specific promoter, and preferably the promoter is a rhodopsin kinase promoter.

42. The aforementioned pharmaceutical composition is 1 × 10 ８ ~1 x 10 １５ Vector particles (vp), preferably 1 × 10⁻¹⁶ １０ ~1 x 10 １３ vp or 1 × 10 １１ ~1 x 10 １２ A pharmaceutical composition according to any one of claims 1 to 41, comprising a unit dose of vp.

43. The pharmaceutical composition according to any one of claims 1 to 42, wherein the pharmaceutical composition is a storage-stable composition.

44. The pharmaceutical composition according to claim 43, wherein the pharmaceutical composition is stable as a liquid at a temperature of about -60°C or below for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or at least 24 months.

45. The pharmaceutical composition according to claim 43 or 44, wherein the pharmaceutical composition is stable as a liquid at a temperature of about 2 to 8°C for at least 2 weeks, at least 3 weeks, at least 1 month, at least 5 weeks, at least 6 weeks, at least 7 weeks, at least 2 months, at least 9 weeks, at least 10 weeks, at least 11 weeks, at least 3 months, at least 13 weeks, at least 14 weeks, at least 15 weeks, at least 4 months, at least 17 weeks, at least 18 weeks, at least 19 weeks, at least 5 months, or at least 6 months.

46. The pharmaceutical composition according to any one of claims 43 to 45, wherein the pharmaceutical composition is in liquid form and maintains a relative potency of at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or at least 24 months.

47. The pharmaceutical composition according to any one of claims 43 to 46, wherein the buffer composition comprises about 10 mM Tris, about 180 mM NaCl, and about 0.005% Pluronic® F68, and the pH of the buffer composition is about 7.4 to about 8.4 at 25°C.

48. The pharmaceutical composition according to claim 47, wherein the pH of the buffer composition is about 7.6 to about 8.2 at 25°C.

49. The pharmaceutical composition according to any one of claims 1 to 48, wherein the pharmaceutical composition does not contain a divalent cation and does not contain sugar or sugar alcohol.

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