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WO2024254052A1 - Formulations de virus adéno-associés - Google Patents

Formulations de virus adéno-associés Download PDF

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Publication number
WO2024254052A1
WO2024254052A1 PCT/US2024/032381 US2024032381W WO2024254052A1 WO 2024254052 A1 WO2024254052 A1 WO 2024254052A1 US 2024032381 W US2024032381 W US 2024032381W WO 2024254052 A1 WO2024254052 A1 WO 2024254052A1
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Prior art keywords
pharmaceutical composition
composition according
aav
nucleotide sequence
buffering
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Inventor
Catherine T. BARGLOW
Johnny C. GONZALES
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4D Molecular Therapeutics Inc
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4D Molecular Therapeutics Inc
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14141—Use of virus, viral particle or viral elements as a vector
    • C12N2750/14143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14151—Methods of production or purification of viral material

Definitions

  • AAV formulations intended for human administration should be not only safe, sterile, and of good manufacturing practice (GMP) grade, these formulations should also exhibit and promote the long-term stability of the AAV, minimizing loss of AAV potency during the manufacture, packaging, and storage processes. Though the efforts to design such AAV formulations have been great, there still remains a need for improved AAV formulation.
  • GMP manufacturing practice
  • the formulations are suitable for long-term storage of AAV, minimizing loss of AAV potency, and advantageously prevent subvisible particle formation and prevent adsorption to the surfaces of the containers in which the AAV are packaged and stored and of the machinery used during manufacture.
  • the pharmaceutical composition of the present disclosure comprises: adeno-associated virus (AAV) and about 5 mM to about 20 mM of a buffering agent, about 100 mM to about 250 mM of a pharmaceutically acceptable salt, about 0.0001% (w/v) to about 0.01% (w/v) of a non-ionic surfactant and preferably has a pH of about 7.0 to about 9.0.
  • AAV adeno-associated virus
  • the pharmaceutical composition of the present disclosure comprises: 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 has a pH of about 7.0 to about 9.0.
  • AAV e.g., AAV2 or a variant thereof
  • the invention provides a liquid formulation.
  • the formulation is lyophilized from a liquid formulation.
  • the lyophilized pharmaceutical composition of the present disclosure is lyophilized from a liquid formulation.
  • methods of preparing a pharmaceutical composition comprising AAV are provided.
  • the method comprises 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 a pH of between about 7.0 to about 9.0, thereby obtaining a pharmaceutical composition comprising AAV.
  • Methods of storing a composition comprising AAV are also provided.
  • Methods for delivering a heterologous nucleic acid comprising a nucleotide sequence encoding a gene product to a mammalian subject are provided.
  • the method comprises administering to the mammal a pharmaceutical composition as herein described.
  • the heterologous nucleic acid is delivered to a retinal cell of the subject, e.g., a photoreceptor cell (e.g., rods; cones), a retinal ganglion cell (RGC), a glial cell (e.g., a Müller glial cell, a microglial cell), a bipolar cell, an amacrine cell, a horizontal cell, and/or a retinal pigmented epithelium (RPE) cell of the subject.
  • a retinal cell of the subject e.g., a photoreceptor cell (e.g., rods; cones), a retinal ganglion cell (RGC), a glial cell (e.g., a Müller glial cell, a microglial cell), a bipolar cell, an amacrine cell, a horizontal cell, and/or a retinal pigmented epithelium (RPE) cell of the subject.
  • RPE retinal pigmented epithelium
  • the disorder is an ocular disorder.
  • the disorder is a VEGF-associated ocular disease such as wet or dry age-related macular degeneration (AMD) or geographic atrophy secondary to AMD.
  • the pharmaceutical composition of the present disclosure comprises: AAV and about 8-12 mM Tris buffer, about 160-200 mM sodium chloride, about 0.0005%-0.01% (w/v) Pluronic F68 and preferably comprises a pH of about 7.4 to 8.1.
  • the invention provides a liquid formulation.
  • the formulation is lyophilized from a liquid formulation.
  • 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 comprises a pH of about 7.6.
  • the invention provides a liquid formulation.
  • the formulation is lyophilized from a liquid formulation.
  • 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 comprises a pH of about 7.7.
  • the invention provides a liquid formulation.
  • the formulation is lyophilized from a liquid formulation.
  • 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 comprises a pH of about 7.8.
  • the invention provides a liquid formulation.
  • the formulation is lyophilized from a liquid formulation.
  • 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 comprises a pH of about 7.9.
  • the invention provides a liquid formulation.
  • the formulation is lyophilized from a liquid formulation.
  • 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 comprises a pH of about 8.0.
  • the invention provides a liquid formulation.
  • the formulation is lyophilized from a liquid formulation.
  • 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 comprises a pH of about 8.1.
  • the invention provides a liquid formulation.
  • the formulation is lyophilized from a liquid formulation.
  • the pharmaceutical composition of the present disclosure comprises: AAV and about 9 mM to about 20 mM buffering agent; about 140 to about 200 mM pharmaceutically acceptable salt; about 0.001% (w/v) to about 0.01% (w/v) non-ionic surfactant; and a pH of about 7.3 to 8.6.
  • the pharmaceutical composition of the present disclosure comprises: AAV and about 9-20 mM Tris buffer, about 140-200 mM sodium chloride, about 0.001%-0.01% (w/v) Pluronic F68 and comprises a pH of about 7.3 to 8.6.
  • the invention provides a liquid formulation.
  • the formulation is lyophilized from a liquid formulation.
  • the pharmaceutical composition does not comprise a divalent cation and/or does not comprise a sugar or sugar alcohol.
  • the AAV formulations provided herein are suitable for pharmaceutical administration.
  • the AAV is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, or AAV10.
  • the AAV is AAV 2, AAV 5, AAV 8, or AAV 9.
  • the AAV is AAV2 or a variant of AAV2.
  • the AAV is a recombinant AAV (rAAV) comprising a heterologous nucleic acid encoding a gene product and a capsid protein comprising a peptide insertion of from about 7 amino acids to about 20 amino acids (a “heterologous peptide” or “peptide insertion”) in the GH-loop of the capsid protein, preferably in a surface-exposed region of the GH-loop, relative to a corresponding parental AAV capsid protein.
  • rAAV recombinant AAV
  • the peptide is inserted following any of the amino acids in positions 584-591 in VP1 of AAV2 or a corresponding position in another AAV serotype (i.e., the insertion site is between amino acids 587 and 588 of VP1 of AAV2 or is between amino acids 588 and 589, between amino acids 584 and 585, between amino acids 585 and 586, between amino acids 586 and 587, between amino acids 590 and 591, or is between amino acids 591 and 592 of AAV2 or the corresponding positions in the capsid protein of another AAV serotype).
  • the insertion site is between amino acids 587 and 588 of VP1 of AAV2 or is between amino acids 588 and 589, between amino acids 584 and 585, between amino acids 585 and 586, between amino acids 586 and 587, between amino acids 590 and 591, or is between amino acids 591 and 592 of AAV2 or the corresponding positions in the capsid protein of another AAV serotype).
  • the insertion site is between amino acids 587 and 588 of VP1 of AAV2 or is between amino acids 588 and 589 of AAV2 or the corresponding positions in the capsid protein of another AAV serotype.
  • the capsid protein further comprises one or more amino acid substitutions relative to VP1 capsid of AAV2 or one or more corresponding substitutions in another AAV serotype, preferably wherein the capsid protein further comprises a P34A amino acid substitution relative to VP1 capsid of AAV2 or the corresponding substitution in another AAV serotype.
  • DESCRIPTION OF THE DRAWINGS [0022] Figure 1 Representative background membrane images for 3-day storage at 37°C.
  • Figure 2 Aflibercept expression, from pH 6.0-7.0, after 2-week storage at 25°C/60% relative humidity (RH).
  • Figure 3 Aflibercept expression, from pH 7.0-7.8, after 2-week storage at 25°C/60% relative humidity (RH).
  • Figure 4 illustrates colloidal stability (> 90% Area (10-100 d.nm) by Intensity) of the tested formulations across a range of sodium chloride concentrations
  • Figure 5 illustrates Final Fluorescence values by Differential Scanning Fluorimetry (DSF) at the specified sodium chloride concentrations following 0.45 Pm filtration.
  • DSF Differential Scanning Fluorimetry
  • FIG. 6 illustrates the percentage recovery after 3 Freeze-Thaw (3XFT) by DSF at the specified sodium chloride concentrations.
  • AAV refers to adeno-associated virus in both naturally occurring and recombinant forms (rAAV), and encompasses mutant forms of AAV.
  • AAV further includes, but is not limited to, AAV type 1, AAV type 2, AAV type 3, AAV type 4, AAV type 5, AAV type 6, AAV type 7, AAV type 8, AAV type 9, AAV type 10, avian AAV, bovine AAV, canine AAV, equine AAV, ovine AAV, primate AAV, and non-primate AAV.
  • the AAV is AAV2 or a variant thereof.
  • a "pharmaceutically acceptable salt” is a salt that can be formulated into a compound or conjugate for pharmaceutical use including, e.g., metal salts (sodium, potassium, magnesium, calcium, etc.) and salts of ammonia or organic amines that is safe for administration to a subject (e.g., a human) in a drug formulation (see, for example, Berge, et al. “Pharmaceutical Salts,” J. Pharm. Sci.1977; 66:1, which is incorporated herein by reference in its entirety and for all purposes.).
  • Suitable “pharmaceutically acceptable salts” include, but are not limited to, metal salts such as sodium, potassium and cesium salts; alkaline earth metal salts such as calcium and magnesium salts; organic amine salts such as triethylamine, guanidine and N-substituted guanidine salts, acetamidine and N-substituted acetamidine, pyridine, picoline, ethanolamine, triethanolamine, dicyclohexylamine, and N,N'-dibenzylethylenediamine salts.
  • metal salts such as sodium, potassium and cesium salts
  • alkaline earth metal salts such as calcium and magnesium salts
  • organic amine salts such as triethylamine, guanidine and N-substituted guanidine salts, acetamidine and N-substituted acetamidine, pyridine, picoline, ethanolamine, triethanolamine, dicyclohexylamine, and
  • “Pharmaceutically acceptable salts” include, but are not limited to inorganic acid salts such as the hydrochloride, hydrobromide, sulfate, phosphate; organic acid salts such as trifluoroacetate and maleate salts; sulfonates such as methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, camphor sulfonate and naphthalenesulfonate; amino acid salts such as arginate, alaninate, asparginate and glutamate; and carbohydrate salts such as gluconate and galacturonate.
  • inorganic acid salts such as the hydrochloride, hydrobromide, sulfate, phosphate
  • organic acid salts such as trifluoroacetate and maleate salts
  • sulfonates such as methanesulfonate, ethanesulf
  • Non-limiting examples of pharmaceutically acceptable salts include, without limitation, sodium salts, ammonium salts, potassium salts (e.g, sodium, ammonium, and potassium chloride; sodium, ammonium, and potassium acetate; sodium, ammonium, and potassium citrate; sodium, ammonium, and potassium phosphate; sodium, ammonium, and potassium fluoride; sodium, ammonium, and potassium bromide; and sodium, ammonium, and potassium iodide).
  • isolated designates a biological material (cell, nucleic acid or protein) that has been removed from its original environment (the environment in which it is naturally present). For example, a polynucleotide present in the natural state in a plant or an animal is not isolated, however the same polynucleotide separated from the adjacent nucleic acids in which it is naturally present, is considered “isolated.”
  • a "coding region” or “coding sequence” is a portion of polynucleotide which consists of codons translatable into amino acids.
  • a "stop codon” (TAG, TGA, or TAA) is typically not translated into an amino acid, it can be considered to be part of a coding region, but any flanking sequences, for example promoters, ribosome binding sites, transcriptional terminators, introns, and the like, are not part of a coding region.
  • the boundaries of a coding region are typically determined by a start codon at the 5' terminus, encoding the amino terminus of the resultant polypeptide, and a translation stop codon at the 3' terminus, encoding the carboxyl terminus of the resulting polypeptide.
  • Two or more coding regions can be present in a single polynucleotide construct, e.g., on a single vector, or in separate polynucleotide constructs, e.g., on separate (different) vectors. It follows, then that a single vector can contain just a single coding region, or comprise two or more coding regions.
  • a single vector can contain just a single coding region, or comprise two or more coding regions.
  • the term "regulatory region” refers to nucleotide sequences located upstream (5' non-coding sequences), within, or downstream (3' non-coding sequences) of a coding region, and which influence the transcription, RNA processing, stability, or translation of the associated coding region.
  • Regulatory regions can include promoters, translation leader sequences, introns, polyadenylation recognition sequences, RNA processing sites, effector binding sites and stem-loop structures. If a coding region is intended for expression in a eukaryotic cell, a polyadenylation signal and transcription termination sequence will usually be located 3' to the coding sequence.
  • nucleic acid is interchangeable with “polynucleotide” or “nucleic acid molecule” and a polymer of nucleotides is intended.
  • a polynucleotide which encodes a gene product e.g., a polypeptide, an interfering RNA such as a primary miRNA, short hairpin RNA (shRNA), or a small interfering RNA (siRNA)
  • a promoter and/or other transcription or translation control elements operably associated with one or more coding regions.
  • a coding region for a gene product e.g., a polypeptide
  • a coding region for a gene product is associated with one or more regulatory regions in such a way as to place expression of the gene product under the influence or control of the regulatory region(s).
  • a coding region and a promoter are "operably associated" if induction of promoter function results in the transcription of mRNA encoding the gene product encoded by the coding region, and if the nature of the linkage between the promoter and the coding region does not interfere with the ability of the promoter to direct the expression of the gene product or interfere with the ability of the DNA template to be transcribed.
  • Other transcription control elements besides a promoter, for example enhancers, operators, repressors, and transcription termination signals, can also be operably associated with a coding region to direct gene product expression.
  • Transcriptional control sequences refer to DNA regulatory sequences, such as promoters, enhancers, terminators, and the like, that provide for the expression of a coding sequence in a host cell.
  • transcription control regions include, without limitation, transcription control regions which function in vertebrate cells, such as, but not limited to, promoter and enhancer segments from cytomegaloviruses (the immediate early promoter, in conjunction with intron-A), simian virus 40 (the early promoter), and retroviruses (such as Rous sarcoma virus).
  • transcription control regions include those derived from vertebrate genes such as actin, heat shock protein, bovine growth hormone and rabbit beta-globin, as well as other sequences capable of controlling gene expression in eukaryotic cells. Additional suitable transcription control regions include tissue-specific promoters and enhancers as well as lymphokine-inducible promoters (e.g., promoters inducible by interferons or interleukins).
  • tissue-specific promoters and enhancers as well as lymphokine-inducible promoters (e.g., promoters inducible by interferons or interleukins).
  • lymphokine-inducible promoters e.g., promoters inducible by interferons or interleukins.
  • translation control elements include, but are not limited to ribosome binding sites, translation initiation and termination codons, and elements derived from picornaviruses (particularly an internal ribosome entry site, or IRES, also referred to as a CITE sequence).
  • RNA messenger RNA
  • tRNA transfer RNA
  • shRNA small hairpin RNA
  • siRNA small interfering RNA
  • expression produces a "gene product.”
  • a gene product can be either a nucleic acid, e.g., a messenger RNA produced by transcription of a gene, or a polypeptide which is translated from a transcript.
  • Gene products described herein further include nucleic acids with post transcriptional modifications, e.g., polyadenylation or splicing, or polypeptides with post translational modifications, e.g., methylation, glycosylation, the addition of lipids, association with other protein subunits, or proteolytic cleavage.
  • “Promoter” and “promoter sequence” are used interchangeably and refer to a DNA sequence capable of controlling the expression of a coding sequence or functional RNA.
  • a coding sequence is located 3' to a promoter sequence. Promoters can be derived in their entirety from a native gene, or be composed of different elements derived from different promoters found in nature, or even comprise synthetic DNA segments.
  • promoters can direct the expression of a gene in different tissues or cell types, or at different stages of development, or in response to different environmental or physiological conditions. Promoters that cause a gene to be expressed in most cell types at most times are commonly referred to as “constitutive promoters.” Promoters that cause a gene to be expressed in a specific cell type are commonly referred to as “cell-specific promoters” or “tissue- specific promoters.” Promoters that cause a gene to be expressed at a specific stage of development or cell differentiation are commonly referred to as “developmentally-specific promoters” or “cell differentiation-specific promoters.” Promoters that are induced and cause a gene to be expressed following exposure or treatment of the cell with an agent, biological molecule, chemical, ligand, light, or the like that induces the promoter are commonly referred to as “inducible promoters” or “regulatable promoters.” It is further recognized that since in most cases the exact boundaries of regulatory sequences have not been completely defined
  • Plasmid refers to an extra-chromosomal element often carrying a gene that is not part of the central metabolism of the cell, and usually in the form of circular double- stranded DNA molecules.
  • Such elements can be autonomously replicating sequences, genome integrating sequences, phage or nucleotide sequences, linear, circular, or supercoiled, of a single- or double-stranded DNA or RNA, derived from any source, in which a number of nucleotide sequences have been joined or recombined into a unique construction which is capable of introducing a promoter fragment and DNA sequence for a selected gene product along with appropriate 3' untranslated sequence into a cell.
  • a polynucleotide or polypeptide has a certain percent "sequence identity" to another polynucleotide or polypeptide, meaning that, when aligned, that percentage of bases or amino acids are the same when comparing the two sequences. Sequence similarity can be determined in a number of different manners. To determine sequence identity, sequences can be aligned using the methods and computer programs, including BLAST, available over the world wide web at ncbi.nlm.nih.gov/BLAST/. Another alignment algorithm is FASTA, available in the Genetics Computing Group (GCG) package, from Madison, Wis., USA.
  • GCG Genetics Computing Group
  • amino acid substitution and its synonyms described above are intended to encompass modification of an amino acid sequence by replacement of an amino acid with another, substituting, amino acid. The substitution may be a conservative substitution.
  • amino acids having hydrophobic nonacidic side chains amino acids having hydrophobic acidic side chains, amino acids having hydrophilic nonacidic side chains, amino acids having hydrophilic acidic side chains, and amino acids having hydrophilic basic side chains.
  • Common properties may also be amino acids having hydrophobic side chains, amino acids having aliphatic hydrophobic side chains, amino acids having aromatic hydrophobic side chains, amino acids with polar neutral side chains, amino acids with electrically charged side chains, amino acids with electrically charged acidic side chains, and amino acids with electrically charged basic side chains.
  • the effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and/or may be therapeutic in terms of a partial or complete cure for a disease and/or adverse effect attributable to the disease.
  • Treatment covers any treatment of a disease in a mammal, particularly in a human, and includes: (a) preventing the disease (and/or symptoms caused by the disease) from occurring in a subject which may be predisposed to the disease or at risk of acquiring the disease but has not yet been diagnosed as having it; (b) inhibiting the disease (and/or symptoms caused by the disease), i.e., arresting its development; and (c) relieving the disease (and/or symptoms caused by the disease), i.e., causing regression of the disease (and/or symptoms caused by the disease), i.e., ameliorating the disease and/or one or more symptoms of the disease.
  • the terms "individual,” “host,” “subject,” and “patient” are used interchangeably herein, and refer to a mammal, including, but not limited to, humans; non- human primates, including simians; mammalian sport animals (e.g., horses); mammalian farm animals (e.g., sheep, goats, etc.); mammalian pets (dogs, cats, etc.); and rodents (e.g., mice, rats, etc.).
  • the term "effective amount” as used herein is an amount sufficient to effect beneficial or desired clinical results. An effective amount can be administered in one or more administrations.
  • an effective amount of a compound is an amount that is sufficient to palliate, ameliorate, stabilize, reverse, prevent, slow or delay the progression of (and/or symptoms associated with) a particular disease state (e.g., a disorder associated with complement dysfunction).
  • an effective amount of an infectious rAAV virion is an amount of the infectious rAAV virion that is able to effectively deliver a heterologous nucleic acid to a target cell (or target cells) of the individual.
  • Effective amounts may be determined preclinically by, e.g., detecting in the cell or tissue the gene product (RNA, protein) that is encoded by the heterologous nucleic acid sequence using techniques that are well understood in the art, e.g. RT-PCR, western blotting, ELISA, fluorescence or other reporter readouts, and the like. Effective amounts may be determined clinically by, e.g. detecting a change in the onset or progression of disease using methods known in the art, e.g.6-minute walk test, left ventricular ejection fraction, hand-held dynamometry, Vignos Scale and the like as described herein and as known in the art.
  • the present disclosure provides formulations, e.g., pharmaceutical compositions, compatible for human administration which also are suitable for long-term storage of AAV and minimizing loss of AAV potency.
  • the formulations provided herein are advantageous, because the formulations prevent subvisible particle formation, and retain significant AAV activity when stored for extended periods of time.
  • the pharmaceutical compositions provided herein reduce or retard degradation and/or aggregation.
  • the present invention provides formulations of AAV comprising a therapeutically effective amount or dose of an AAV, a pharmaceutically acceptable salt, a non-ionic surfactant, and one or more buffering agents providing a pH as herein described to the formulation.
  • the AAV formulations provided herein are suitable for pharmaceutical administration.
  • the AAV is AAV2 or a variant thereof.
  • AAV Formulations and Compositions [0049]
  • the pharmaceutical composition of the present disclosure comprises: adeno-associated virus (AAV) and about 5 mM to about 20 mM of a buffering agent, about 100 mM to about 250 mM of a pharmaceutically acceptable salt, about 0.0001% (w/v) to about 0.01% (w/v) of a non-ionic surfactant, and a pH of at least about 7.0.
  • AAV adeno-associated virus
  • the pharmaceutical composition of the present disclosure comprises: adeno-associated virus (AAV) and about 5 mM to about 20 mM of a buffering agent, about 100 mM to about 200 mM of a pharmaceutically acceptable salt, about 0.001% (w/v) to about 0.01% (w/v) of a non-ionic surfactant, and a pH of between about 7.0 and about 9.0.
  • AAV adeno-associated virus
  • the pharmaceutical composition of the present disclosure comprises: adeno-associated virus (AAV) and about 5 mM to about 20 mM of a buffering agent, about 150 mM to about 200 mM of a pharmaceutically acceptable salt, about 0.001% (w/v) to about 0.01% (w/v) of a non-ionic surfactant, and a pH of at least about 7.4.
  • AAV adeno-associated virus
  • the pharmaceutical composition of the present disclosure comprises: adeno-associated virus (AAV) and about 10 mM to about 20 mM of a buffering agent, about 150 mM to about 200 mM of a pharmaceutically acceptable salt, about 0.001% (w/v) to about 0.01% (w/v) of a non-ionic surfactant, and a pH of at least about 7.4
  • AAV adeno-associated virus
  • the composition is a sterile composition.
  • sterile it is meant that there are substantially no immunogenic components in the composition, such as for example substantially no microbes (e.g., fungi, bacteria, viruses, spore forms, etc.).
  • the invention provides a liquid formulation.
  • the formulation is lyophilized from a liquid formulation.
  • the pharmaceutical composition of the present disclosure comprises about 5 mM to about 20 mM, about 5 mM to about 15 mM, about 10 mM to about 20 mM, or about 15 mM to about 25 mM of a buffering agent.
  • the pharmaceutical composition comprises about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, about 10 mM, about 11 mM, about 12 mM, about 13 mM, about 14 mM, about 15 mM, about 16 mM, about 17 mM, about 18 mM, about 19 mM, about 20 mM, about 21 mM, about 22 mM, about 23 mM, about 24 mM, or about 25 mM of a buffering agent.
  • buffering agents include without limitation, phosphate buffers, histidine, sodium citrate, HEPES, Tris, Bicine, glycine, N-glycylglycine, sodium acetate, sodium carbonate, glycyl glycine, lysine, arginine, sodium phosphate, and mixtures thereof.
  • the buffer is a Tris buffer.
  • the pharmaceutical composition of the present disclosure comprises about 5 mM to about 25 mM, about 5 mM to about 15 mM, about 10 mM to about 20 mM, or about 15 mM to about 25 mM Tris.
  • the pharmaceutical composition comprises about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, about 10 mM, about 11 mM, about 12 mM, about 13 mM, about 14 mM, about 15 mM, about 16 mM, about 17 mM, about 18 mM, about 19 mM, about 20 mM, about 21 mM, about 22 mM, about 23 mM, about 24 mM, or about 25 mM Tris.
  • the pharmaceutical composition comprises about 8 mM Tris to about 22 mM Tris, or about 9 mM Tris to about 21 mM Tris, or about 10 mM Tris to about 20 mM Tris. In certain embodiments, the pharmaceutical composition comprises about 10 mM Tris.
  • the pharmaceutical composition of the present disclosure comprises about 100 mM to about 250 mM, about 110 mM to about 240 mM, about 120 mM to about 230 mM, about 130 mM to about 220 mM, about 140 mM to about 210 mM, about 145 mM to about 205 mM, about 140 mM to about 200 mM, about 145 mM to about 200 mM, about 150 mM to about 200 mM, about 155 mM to about 195 mM, about 160 mM to about 195 mM, about 165 mM to about 195 mM, about 170 mM to about 195 mM, about 175 mM to about 195 mM, about 175 mM to about 194 mM, about 175 mM to about 193 mM, about 175 mM to about 192 mM, about 175 mM to about 191 m
  • the composition comprises about 150 mM, about 155 mM, about 160 mM, about 175 mM, about 180 mM, about 185 mM, about 190 mM, or about 200 mM of a pharmaceutically acceptable salt.
  • the pharmaceutically acceptable salt is a sodium salt (e.g., sodium chloride).
  • pharmaceutical compositions containing a non-ionic detergent are provided.
  • non-ionic surfactants that may be used in the formulations disclosed herein are known in the art of pharmaceutical science, and include, without limitation, Polysorbate 80 (Tween 80; PS80), Polysorbate 20 (Tween 20; PS20), and various poloxamers or pluronics, including Pluronic F-68, and BRIJ 35, or mixtures thereof.
  • the non-ionic surfactant used in the present pharmaceutical compositions is a pluronic, particularly Pluronic F-68.
  • the pharmaceutical composition of the present disclosure comprises about 0.001% (w/v) to about 0.01% (w/v) or about 0.0025% (w/v) to about 0.0075% (w/v) non-ionic surfactant.
  • the pharmaceutical composition comprises about 0.001% (w/v), about 0.0015% (w/v), about 0.002% (w/v), about 0.0025% (w/v), about 0.003% (w/v), about 0.0035% (w/v), about 0.004% (w/v), about 0.0045% (w/v), about 0.005% (w/v), about 0.0055% (w/v), about 0.006% (w/v), about 0.0065% (w/v), about 0.007% (w/v), about 0.0075% (w/v), about 0.008% (w/v), about 0.0085% (w/v), about 0.009% (w/v), about 0.0095% (w/v), about 0.001% (w/v) non-ionic surfactant.
  • the pharmaceutical composition of the present disclosure comprises about 0.005% (w/v) non-ionic surfactant.
  • the pharmaceutical composition of the present disclosure comprises about 0.001% (w/v) to about 0.01% (w/v) or about 0.0025% (w/v) to about 0.0075% (w/v) Pluronic F68.
  • the pharmaceutical composition of the present disclosure comprises about 0.005% (w/v) Pluronic F68.
  • the pharmaceutical composition of the present disclosure comprises: adeno-associated virus (AAV) and about 5 mM to about 25 mM Tris, about 100 mM to about 250 mM sodium salt, about 0.001% (w/v) to about 0.01% (w/v) non-ionic surfactant, and a pH of between 7.0 and 9.0.
  • the pharmaceutically acceptable salt is present at about 150 nM to about 210 mM.
  • the pharmaceutically acceptable salt is present at about 170 nM to about 200 mM.
  • the pharmaceutically acceptable salt is sodium chloride.
  • the present disclosure also provides a pharmaceutical composition that can be liquid or lyophilized (e.g., lyophilized from a liquid formulation) comprising adeno-associated virus (AAV) and about 10 mM Tris buffer, about 180 mM sodium chloride, about 0.005% (w/v) Pluronic F68 and the pH of the pharmaceutical composition is about 7.9 ⁇ 0.3.
  • AAV adeno-associated virus
  • Pluronic F68 Pluronic F68
  • the pharmaceutical composition of the present disclosure has a pH of about 7.0, about 7.1, about 7.2, about 7.2, about 7.3, about 7.4, about 7.5, about 7.6, about 7.7, about 7.8, about 7.9, about 8.0, about 8.1, about 8.2, about 8.3, about 8.4, about 8.5, about 8.6, about 8.7, about 8.8, about 8.9, or about 9.0.
  • the pH of the pharmaceutical composition is above about 7.3 and below about 9.0 or below about 8.5.
  • the pH of the pharmaceutical composition is about 7.8 to about 9.0 or is about 7.9.
  • the pH of the pharmaceutical composition is from about 7.3 to about 8.6.
  • the formulations or pharmaceutical compositions of the present disclosure comprise additional pharmaceutically acceptable ingredients.
  • the formulations or pharmaceutical compositions comprise any one or a combination of the following: acidifying agents, additives, adsorbents, aerosol propellants, air displacement agents, alkalizing agents, anticaking agents, anticoagulants, antimicrobial preservatives, antioxidants, antiseptics, bases, binders, buffering agents, chelating agents, coating agents, coloring agents, desiccants, detergents, diluents, disinfectants, disintegrants, dispersing agents, dissolution enhancing agents, dyes, emollients, emulsifying agents, emulsion stabilizers, fillers, film forming agents, flavor enhancers, flavoring agents, flow enhancers, gelling agents, granulating agents, humectants, lubricants, mucoadhesives, ointment bases, ointments,
  • the formulations or pharmaceutical compositions of the present disclosure comprise any one or a combination of the following components: acacia, acesulfame potassium, acetyltributyl citrate, acetyltriethyl citrate, agar, albumin, alcohol, dehydrated alcohol, denatured alcohol, dilute alcohol, aleuritic acid, alginic acid, aliphatic polyesters, alumina, aluminum hydroxide, aluminum stearate, amylopectin, ⁇ -amylose, ascorbic acid, ascorbyl palmitate, aspartame, bacteriostatic water for injection, bentonite, bentonite magma, benzalkonium chloride, benzethonium chloride, benzoic acid, benzyl alcohol, benzyl benzoate, bronopol, butylated hydroxyanisole, butylated hydroxytoluene, butylparaben, butylparaben sodium, calcium alginate
  • the formulations or pharmaceutical compositions of the present disclosure do not comprise one or a combination of the above ingredients. In exemplary embodiments, the formulations or pharmaceutical compositions of the present disclosure comprises none of these ingredients. In exemplary aspects, the pharmaceutical composition of the present disclosure does not comprise dextran. In exemplary aspects, the pharmaceutical composition of the present disclosure does not comprise calcium chloride. In other exemplary aspects, the pharmaceutical composition of the present disclosure does not comprise a sugar or a sugar alcohol (e.g., does not comprise sucrose, trehalose, or mannitol). In related exemplary aspects, the pharmaceutical composition does not comprise glycine. [0070] AAV [0071] In exemplary embodiments, the pharmaceutical composition of the present disclosure comprises AAV.
  • the AAV may be of any AAV serotype.
  • the AAV is of AAV1 serotype, AAV2 serotype, AAV3 serotype, AAV4 serotype, AAV5 serotype, AAV6 serotype, AAV7 serotype, AAV8 serotype, AAV9 serotype, or AAV10 serotype.
  • the AAV is of AAV2 serotype or a variant thereof.
  • the AAV is an rAAV as described in U.S.
  • Patent Publication No.2020/0282077 which is incorporated herein by reference in its entirety, in particular an rAAV comprising a capsid protein according to paragraphs 171-179 of U.S. Patent Publication No.2020/0282077 and/or comprising a heterologous nucleic acid according to paragraphs 222- 248 of U.S. Patent Publication No.2020/0282077.
  • the variant capsid protein when present in an AAV virion, confers increased infectivity of a retinal cell compared to the infectivity of a retinal cell by an AAV virion comprising the corresponding parental capsid protein.
  • the “GH loop,” or loop IV, of the AAV capsid protein it is meant the solvent- accessible portion referred to in the art as the GH loop, or loop IV, of AAV capsid protein.
  • the GH loop/loop IV of AAV capsid see, e.g., van Vliet et al. (2006) Mol. Ther.14:809; Padron et al. (2005) J. Virol.79:5047; and Shen et al. (2007) Mol.
  • the insertion site can be within about amino acids 570-611 of AAV2 VP1.
  • the peptide insertion has from 1 to 3 spacer amino acids (Y 1 - Y3) at the amino and/or carboxyl terminus of the amino acid sequence ISDQTKH (SEQ ID NO:1).
  • Exemplary spacer amino acids include, without limitation, leucine (L), alanine (A), glycine (G), serine (S), threonine (T), and proline (P).
  • a peptide insertion comprises 2 spacer amino acids at the N-terminus and 2 spacer amino acids at the C- terminus.
  • a peptide insertion comprises 2 spacer amino acids at the N- terminus and 1 spacer amino acids at the C-terminus.
  • the peptide insertion comprises or consists of the amino acid sequence LAISDQTKHA (SEQ ID NO:2).
  • the variant AAV capsid protein comprises a peptide insertion comprising the amino acid sequence ISDQTKH (SEQ ID NO:1) and further comprises one or more amino acid substitutions relative to a corresponding parental AAV capsid protein. Representative examples of amino acid substitutions may be found at e.g., col.26, lines 40-65 of U.S. Patent No.11,576,983, the entire contents of which are incorporated herein by reference.
  • the variant AAV capsid protein comprises a peptide insertion comprising the amino acid sequence ISDQTKH (SEQ ID NO:1) and further comprises a P34A amino acid substitution relative to VP1 capsid of AAV2 or the corresponding substitution in another AAV serotype.
  • the variant capsid protein may comprise one or more features disclosed in U.S. Patent No.11,576,983, in particular, one or more features disclosed at column 26, line 66 to column 29, line 50 of U.S. Patent No.11,576,983.
  • the variant capsid protein comprises the following amino acid sequence or comprises an amino acid sequence at least 80%, at least 90%, least 95%, at least 98%, or at least 99% identical to the following amino acid sequence: MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKAAERHKDDSRGLVLPGYKYLGPFNGLDKGE PVNEADAAALEHDKAYDRQLDSGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRV LEPLGLVEEPVKTAPGKKRPVEHSPVEPDSSSGTGKAGQQPARKRLNFGQTGDADSVPDPQPLG QPPAAPSGLGTNTMATGSGAPMADNNEGADGVGNSSGNWHCDSTWMGDRVITTSTRTWALPT YNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFK LFNIQVKEVTQNDGTTTIANNLTSTVQVF
  • the variant AAV capsid protein of SEQ ID NO:3 contains the following modifications relative to native AAV2 capsid: (i) a proline (P) to alanine (A) mutation at amino acid position 34, which is located inside the assembled capsid (VP1 protein only), and (ii) an insertion of 10 amino acids (leucine-alanine-isoleucine-serine-aspartic acid-glutamine-threonine- lysine-histidine-alanine/LAISDQTKHA (SEQ ID NO:2)) at amino acid position 588, which is present in VP1, VP2, and VP3.
  • the capsid comprises a variant capsid protein comprising a sequence at least 90%, at least 95%, at least 98%, at least 99% identical to SEQ ID NO:3 and comprising a P34A substitution and an LAISDQTKHA (SEQ ID NO:2) peptide insertion at amino acid position 588.
  • a pharmaceutical composition as herein described comprises an rAAV encapsulating a heterologous nucleic acid comprising a nucleotide sequence encoding one or more gene products.
  • the one or more gene products are selected from an interfering RNA (e.g., a microRNA) and a polypeptide.
  • the heterologous nucleic acid encodes aflibercept and optionally further encodes an interfering RNA that decreases the expression of VEGF-C.
  • the heterologous nucleic acid comprises a nucleotide sequence described in U.S. Patent Publication No.2024/0131195A1, the entire contents of which are incorporated herein by reference, in particular at Table 2.
  • the rAAV comprises a heterologous nucleic acid comprising the following nucleotide sequence encoding aflibercept, codon-optimized for expression in humans: ATGGTTTCTTACTGGGACACCGGCGTGCTGCTGTGTGCCCTGCTTTCTTGTCTGCTGCTGACC GGCTCTAGCAGCGGCTCTGATACCGGCAGACCCTTCGTGGAAATGTACAGCGAGATCCCCGA GATCATCCACATGACCGAGGGCAGAGAGCTGGTCATCCCTTGCAGAGTGACAAGCCCCAAC ATCACCGTGACTCTGAAGAAGTTCCCTGGACACACTGATCCCCGACGGCAAGAGAATCAT CTGGGACAGCCGGAAGGGCTTCATCATCAGCAACGCCACCTACAAAGAGATCGGCCTGCTG ACCTGTGAAGCCACCGTGAATGGCCACCTGTACAAGACCAACTACCTGACACACAGACAGA CCAACACCATCATCGACGTGGTGCTGAGCCCTAGCCACGGCATT
  • the aflibercept gene product comprises the following amino acid sequence or a sequence at least 90%, 95%, 97%, 98%, or at least 99% identical thereto: MVSYWDTGVLLCALLSCLLLTGSSSGSDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTL KKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVLSP SHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTID GVTRSDQGLYTCAASSGLMTKKNSTFVRVHEKDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMI SRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLN GKEYKCKVSNKALPAPIE
  • the pharmaceutical composition comprises an rAAV comprising a heterologous nucleic acid comprising the following sequence (encoding aflibercept + human VEGF-C interfering RNA) or a sequence at least 80%, at least 90%, at least 95%, at least 98% or at least 99% identical thereto: TTGGCCACTCCCTCTCTGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCC GGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCGCAGAGAGGGAGTGGCCAACTCCATCACT AGGGGTTCCTATCGATTGAATTCCCCGGGGATCCACTAGTTATTAATAGTAATCAATTACGGGGTCATT AGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGC CCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTC CATTGACGTCAA
  • the pharmaceutical composition comprises an rAAV comprising a heterologous nucleic acid comprising a nucleotide sequence encoding a wild type or codon- optimized human Rab escort protein-1 (REP1) protein, e.g., as described in U.S. Patent No. 11,357,870, the entire contents of which are hereby incorporated by reference.
  • REP1 human Rab escort protein-1
  • the heterologous nucleic acid encoding REP1 comprises the following codon-optimized nucleotide sequence or a sequence 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:
  • the heterologous nucleic acid encodes human RPGR ORF15 and comprises the following codon-optimized nucleotide sequence or a sequence 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: ATGAGAGAACCCGAGGAACTGATGCCCGACTCTGGCGCCGTGTTTACCTTCGGCAAGAGCAAGTT CGCCGAGAACAACCCCGGCAAGTTCTGGTTCAAGAACGACGTGCCAGTGCACCTGAGCTGCGGA GATGAACACTCTGCCGTGGTCACCGGCAACAACAAGCTGTACATGTTCGGCAGCAACAACTGGG GCCAGCTCGGCCTGGGATCTAAGTCTGCCATCAGCAAGCCTACCTGCGTGAAGGCCCTGAAGCCT GAGAAAGTGAAACTGGCCGCCTGCGGCAGAAATC
  • the soluble hfH protein variant comprises an amino acid sequence as set forth in U.S. Patent No.10,988,519, the entire contents of which are incorporated herein by reference.
  • the heterologous nucleic acid encodes an fH variant having the following amino acid sequence: MRLLAKIICLMLWAICVAEDCNELPPRRNTEILTGSWSDQTYPEGTQAIYKCRPGYRSLG NIIMVCRKGEWVALNPLRKCQKRPCGHPGDTPFGTFTLTGGNVFEYGVKAVYTCNEGY QLLGEINYRECDTDGWTNDIPICEVVKCLPVTAPENGKIVSSAMEPDREYHFGQAVRFV CNSGYKIEGDEEMHCSDDGFWSKEKPKCVEISCKSPDVINGSPISQKIIYKENERFQYKC NMGYEYSERGDAVCTESGWRPLPSCEEKSTLKPCDYPDIKHGGLYHENMRRPYFPVAV GKYYSYYY
  • the pharmaceutical composition comprises about 1 x 10 11 to about 1 x 10 12 vector particles or vector genomes.
  • the pharmaceutical composition maintains a relative potency in liquid form of at least 90% over 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.
  • the composition comprising AAV is storage stable 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.
  • greater than 80% of the initial amount of AAV e.g., the amount of AAV in the composition prior to storage
  • is potent after the storage period e.g., a storage period of about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, or about 6 months or longer.
  • greater than 90% of the initial amount of AAV is potent after the storage period (e.g., a storage period of about 1 month, about 2 months, 3 months, about 4 months, about 5 months, or about 6 months or longer). In exemplary aspects, greater than 95% of the initial amount of AAV is potent after the storage period (e.g., a storage period of about 3 months, about 4 months, about 5 months, or about 6 months or longer).
  • the biopotency of the AAV at the end of the storage period is substantially the same as the biopotency of the AAV at the beginning of the storage period.
  • the biopotency of the AAV at the end of the storage period is increased relative to the biopotency of the AAV at the beginning of the storage period.
  • the appearance of the composition at the end of the storage period is substantially the same as the composition at the beginning of the storage period.
  • the appearance of the composition at the end of the storage period is characterized by having no visible particles.
  • the particle concentration of the composition at the end of the storage period is substantially the same as the particle concentration of the composition at the beginning of the storage period.
  • the particle concentration of the composition at the end of the storage period is determined by microflow imaging (MFI).
  • the pharmaceutical composition is maintained at about -60°C or below for a substantial portion (e.g., the duration) of the storage period. In other embodiments, the pharmaceutical composition is maintained at from about 2°C to about 8°C for a substantial portion (e.g., the duration) of the storage period. [0096] In some embodiments, a method for treating a disorder in a subject in need thereof is provided, the method comprising administering to the subject a pharmaceutical composition as described herein in an amount effective to treat the disorder.
  • the pharmaceutical composition is administered intraocularly to a human with a VEGF-related ocular disorder in an amount effective to treat the VEGF-related ocular disorder, preferably wherein the pharmaceutical composition is administered via intravitreal, subretinal and/or suprachoroidal injection, more preferably via a single intravitreal injection.
  • the disorder is selected from those listed at paragraphs 218 and 246 of US Patent Application Publication No.2020/0282077.
  • the formulations disclosed herein may be formulated for administration via known methods, such as intraocular administration (e.g., via intravitreal, subretinal and/or suprachoroidal administration).
  • the pharmaceutical composition is administered intraocularly to a human, preferably wherein the pharmaceutical composition is administered via intravitreal, subretinal and/or suprachoroidal injection, more preferably via a single intravitreal injection.
  • the treatment regimen includes administering one or more doses over an extended period of time.
  • a single dose e.g., a single dosage unit
  • the initial dose may be followed by one or more doses administered to the subject at a subsequent time.
  • more than one dose e.g., more than one dosage unit
  • the initial doses may be followed by one or more doses administered to the subject at a subsequent time.
  • a single dose (e.g., a single dosage unit) may be administered to the subject, and the single dose may be followed by a single dose administered to the subject at a subsequent time. Additional single doses may be administered at subsequent points in time.
  • a single dose (e.g., a single dosage unit) may be administered to the subject, and the single dose may be followed by two doses administered to the subject at a subsequent time. Additional single or multiple doses may be administered at subsequent points in time.
  • dosage units of the present disclosure can be administered prior to, concurrent with, or subsequent to other active agents for treating related or unrelated conditions, e.g., in combination therapy.
  • aspects of the present disclosure further include combination therapies.
  • the subject method includes administering a therapeutically effective amount of one or more additional active agents.
  • combination therapy is meant that a AAV composition (e.g., as described herein) can be used in a combination with another therapeutic agent to treat a single disease or condition.
  • a compound of the present disclosure is administered concurrently with the administration of another therapeutic agent, which can be administered as a component of a composition including the compound of the present disclosure or as a component of a different composition.
  • a composition including a compound of the present disclosure is administered prior or subsequent to administration of another therapeutic agent.
  • Example 1 [00101] The below sections summarize key findings that led to identification of an improved rAAV formulation (10 mM Tris buffer, pH 7.9, 180 mM NaCl, 0.005% Pluronic F68) which exhibits reduced risk of subvisible particle formation as well as improved liquid storage stability, which is consistent with the stabilization provided by increased ionic strength and reduced rAAV surface adsorption by use of surfactant and the importance of optimizing formulation pH.
  • Tris buffer component Tris Hydrochloride/Tromethamine
  • rAAV comprising a capsid protein of SEQ ID NO:3 and a nucleic acid comprising the nucleotide sequence of SEQ ID NO:6, was buffer-exchanged into 5 different buffers (Form 1-5) and subjected to various forced degradation conditions.
  • Form 4 showed markedly decreased colloidal stability (i.e., increased poly-dispersity index (PDI) and decreased % area, 10-100 d.nm, via dynamic light scattering (DLS) and increased SVPs) at most conditions tested.
  • PDI poly-dispersity index
  • DLS dynamic light scattering
  • 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)
  • Form 5 (10 mM Tris buffer, pH 8.0, 180 mM NaCl, 0.005% F68) showed improved colloidal stability, relative to the Form 1 control (DPBS, 0.005% F68), at most conditions.
  • rAAV (comprising a capsid protein of SEQ ID NO:3 and a nucleic acid comprising the nucleotide sequence of SEQ ID NO:6) was buffer-exchanged into Phosphate or Tris buffer at a pH 7.0, 7.4 and 7.8.
  • AFLIB aflibercept
  • All three formulations contained rAAV, comprising a capsid protein of SEQ ID NO:3 and a nucleic acid comprising the nucleotide sequence of SEQ ID NO:6, at a target concentration of ⁇ 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.
  • Tris Buffer Temperature Dependence Example 3 The long-term stability of rAAV, comprising a capsid protein of SEQ ID NO:3 and a nucleic acid comprising the nucleotide sequence of SEQ ID NO:6, formulated in 10 mM Tris, 180 mM NaCl, 0.005% Pluronic F68 has, subsequently, been evaluated at both ⁇ -60°C and 2- 8°C.
  • product development lots 4DER000096 and 4DER000097 (2-mL CZ vial filled with rAAV, comprising a capsid protein of SEQ ID NO:3 and a nucleic acid comprising the nucleotide sequence of SEQ ID NO:6, formulated in 10 mM Tris, 180 mM NaCl, 0.005% Pluronic F68), were evaluated in an abbreviated long-term stability study conducted at ⁇ -60°C and 2-8°C. pH, subvisible particles, genomic titer, transgene expression (protein-based) and monodispersion results can be found in Table 10 and Table 11 below. Results for clinical lot 4D2210031 can be found in Table 12. [00125] Table 10: Stability at ⁇ -60°C and 2-8°C [00126] Table 11: Stability at ⁇ -60°C and 2-8°C

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Abstract

L'invention concerne des compositions pharmaceutiques comprenant le virus adéno-associé (AAV). Selon certains aspects, les compositions pharmaceutiques comprennent environ 5 mM à environ 20 mM de tampon Tris, environ 100 mM à environ 250 mM de chlorure de sodium, environ 0,001 % (p/v) à environ 0,01 % (p/v) de Pluronic F68, et présentent un pH compris entre 7,0 et 9,0. La présente invention concerne également des procédés de préparation d'une composition pharmaceutique contenant des AAV, des procédés de traitement d'un trouble associé au VEGF chez un sujet et des procédés de stockage de compositions d'AAV.
PCT/US2024/032381 2023-06-09 2024-06-04 Formulations de virus adéno-associés Pending WO2024254052A1 (fr)

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Citations (4)

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Publication number Priority date Publication date Assignee Title
US20210380643A1 (en) * 2017-09-20 2021-12-09 4D Molecular Therapeutics Inc. Adeno-associated virus variant capsids and methods of use thereof
US20210395772A1 (en) * 2020-04-27 2021-12-23 4D Molecular Therapeutics Inc. Adeno-associated variants, formulations and methods for pulmonary delivery
US20220090129A1 (en) * 2018-12-05 2022-03-24 Abeona Therapeutics Inc. Recombinant adeno-associated viral vector for gene delivery
US20220143115A1 (en) * 2019-04-19 2022-05-12 Regenxbio Inc. Adeno-Associated Virus Vector Formulations and Methods

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20210380643A1 (en) * 2017-09-20 2021-12-09 4D Molecular Therapeutics Inc. Adeno-associated virus variant capsids and methods of use thereof
US20220090129A1 (en) * 2018-12-05 2022-03-24 Abeona Therapeutics Inc. Recombinant adeno-associated viral vector for gene delivery
US20220143115A1 (en) * 2019-04-19 2022-05-12 Regenxbio Inc. Adeno-Associated Virus Vector Formulations and Methods
US20210395772A1 (en) * 2020-04-27 2021-12-23 4D Molecular Therapeutics Inc. Adeno-associated variants, formulations and methods for pulmonary delivery

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