Adeno-associated virus preparations

Abstract

Compositions and methods for the formulation and use of recombinant adeno-associated virus (rAAV) are described. In certain embodiments, the present application discloses recombinant adeno-associated virus (rAAV or AAV) formulations that provide one or more of the following: maintaining stable freeze-thaw and lyophilization (lyophilization) performance, and enabling long-term storage at temperatures above -80°C while maintaining important quality attributes such as genome recovery, retained titer, minimized aggregation and degradation, improved vector quality, retained viral protein (VP) ratios, maximum chemical stability (minimal deamidation and oxidation), and / or increased thermodynamic stability.

Description

[Technical Field]

[0001] FIELD OF THE INVENTION FIELD OF THE INVENTION This application relates to compositions and methods for the formulation and use of gene therapy pharmaceuticals. In certain embodiments, this application discloses recombinant adeno-associated virus (rAAV or AAV) formulations that provide one or more of the following: maintaining stable freeze-thaw and lyophilization (freeze-drying) performance, and allowing for long-term storage at temperatures above -80°C while maintaining important quality attributes such as genome recovery, maintained potency, minimized aggregation and degradation, improved vector quality, maintained viral protein (VP) ratios, maximum chemical stability (minimal deamidation and oxidation), and / or improved thermodynamic stability.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 63 / 477,017, filed December 23, 2022, and U.S. Provisional Application No. 63 / 612,443, filed December 20, 2023.

[0003] Reference to an electronically submitted sequence listing The contents of the electronic sequence listing (065830_18WO.xml; size: 5,457 bytes; creation date: December 20, 2023) are incorporated herein by reference in their entirety. [Background technology]

[0004] background Challenges to the stability of rAAV formulations include protein degradation, aggregation, and loss of efficacy. Significant formulation degradation pathways include (1) freeze / thaw-induced unfolding and loss of activity; and (2) aggregation at low ionic strength. Potential degradation mechanisms include physical instability, aggregation, surface adsorption, chemical instability, disulfide formation / exchange, deamidation, oxidation, and isomerization (Srivastavaz et al., Journal of Pharmaceutical Sciences, volume 110, issue 7, 2021, pages 2609-2624).

[0005] Current rAAV-based therapeutics are generally formulated for cryopreservation, e.g., at -80°C ± 10°C. Such formulations contain components, e.g., specific salts, phosphate buffer species, and surfactants, at concentrations that provide an osmolality compatible with the expected route of administration to prevent aggregation, improve stability, and minimize product surface losses. However, these formulations can exhibit unpredictable performance under freeze-thaw conditions, e.g., due to the inclusion of a phosphate buffer-based matrix, which can also result in pH shifts. In addition, the lack of a sugar component in conventional formulations hinders their adaptability for lyophilization, which is critical for targeting higher shelf storage temperatures, e.g., 2-8°C.

[0006] One particular challenge in the formulation of rAAV therapeutics is the high ionic strength formulations (often with NaCl concentrations of 150 mM or greater) common within the rAAV field, which result in high baseline osmolality, low glass transition temperatures (T g ') and limit the inclusion of additional excipients such as sugars.

[0007] WO2020214929 and WO2018128689 include examples of rAAV formulations.

[0008] In view of the above, there is a need in the art for new stable rAAV formulations that possess one or more of the following properties: are suitable for lyophilization, can maintain predictable freeze-thaw performance, exhibit enhanced long-term shelf stability in various forms, e.g., frozen, liquid, or lyophilized at elevated temperatures, and are compatible with various routes of administration, including systemic, ocular, and CNS: Summary of the Invention

[0009] Summary of the Invention In certain embodiments, the present application relates to compositions and methods for the formulation and use of gene therapy products. Specifically, the present application relates to rAAV formulations that are compatible with at least one or all of lyophilization, maintain predictable freeze-thaw performance, and exhibit enhanced long-term stability at -80°C, -40°C, -20°C, 2-8°C, and 25°C.

[0010] In one general aspect, the present application provides a therapeutic pharmaceutical agent; (a) 1 to 100 mM of one or more salts; (b) a buffer; (c) 0.001 to 0.05 wt. % of one or more nonionic surfactants; (d) 3 to 10 wt. % of one or more sugars; (e) water; (f) optionally, one or more amino acids; and The present invention relates to a stable formulation in which the pH of the composition is 4.5 to 7.5 and 60% or more of the pharmaceutical agent is recovered after freezing and thawing the formulation.

[0011] In another general aspect, the application provides a therapeutic pharmaceutical agent; (a) 1 to 100 mM of one or more salts; (b) a buffer; (c) 0.001 to 0.05 wt. % of one or more nonionic surfactants; (d) 3 to 10 wt. % of one or more sugars; (e) water; (f) optionally, one or more amino acids; and The composition has a pH of 4.5 to 7.5, and relates to a safe formulation in which 60% or more of the pharmaceutical agent is recovered after the formulation is lyophilized.

[0012] Another general aspect of the present application is (a) a recombinant adeno-associated virus (AAV); (b) 25 mM sodium chloride; (c) 5 mM magnesium chloride; (d) 25 mM sodium acetate; (e) 0.01% by weight of poloxamer 188; (f) 5.4 wt. % sucrose; (g) water; and (h) A stable formulation having a pH of 5 to 5.5.

[0013] Another general aspect of the present application is (a) a recombinant adeno-associated virus (AAV); (b) 10 mM Tris (Tris(hydroxymethyl)aminomethane); (c) 75 mM sodium chloride; (e) 0.005% by weight of poloxamer 188; (d) 5% by weight of sucrose; (e) water; and (f) For stable formulations having a pH of 7.3.

[0014] In another general aspect, the present application provides a method for manufacturing a method of a medical device comprising: (a) recombinant adeno-associated virus (rAAV); (b) 10 mM sodium citrate; (c) 75 mM sodium chloride; (d) 0.001 wt% poloxamer 188; (e) 7 wt% sucrose; (f) water; and (g) For stable formulations having a pH of 6.0.

[0015] Another general aspect of the present application relates to a method for reducing degradation of a therapeutic pharmaceutical agent after freezing and thawing the therapeutic pharmaceutical agent, the method comprising: (a) a therapeutic drug; (b) 1 to 100 mM of one or more salts; (c) a buffer; (d) 0.001 to 0.05 wt. % of one or more nonionic surfactants; (e) 3 to 10 wt. % of one or more sugars; (f) water, (g) pH 4.5 to 7.5, and (h) optionally preparing a composition comprising one or more amino acids; freezing the composition for a period of time at a temperature of greater than -80°C to -10°C, e.g., -70°C to -15°C, -60°C to -20°C, -50°C to -30°C, or -45°C to -35°C, wherein 60% or more of the therapeutic agent is recovered after thawing compared to the same product stored at -80°C for the same period of time; Includes.

[0016] Another general aspect of the present application relates to a method for reducing degradation of a therapeutic pharmaceutical agent after freezing and thawing the therapeutic pharmaceutical agent, the method comprising: (a) a therapeutic product; and (b) 25 mM sodium chloride; (c) 5 mM magnesium chloride; (d) 25 mM sodium acetate; (e) 0.01% by weight of poloxamer 188; (f) 5.4 wt. % sucrose; (g) water, (h) preparing a composition having a pH of 5 to 5.5; freezing the composition for a period of time at a temperature of greater than -80°C to -10°C, e.g., -70°C to -15°C, -60°C to -20°C, -50°C to -30°C, or -45°C to -35°C, wherein 60% or more of the therapeutic agent is recovered after thawing compared to the same product stored at -80°C for the same period of time; Includes.

[0017] Another general aspect of the present application relates to a method for maintaining at least 50% relative potency of a therapeutic pharmaceutical after freezing and thawing the therapeutic pharmaceutical, the method comprising: (a) the therapeutic product; and (b) 25 mM sodium chloride; (c) 5 mM magnesium chloride; (d) 25 mM sodium acetate; (e) 0.01% by weight of poloxamer 188; (f) 5.4 wt. % sucrose; (g) water, (h) preparing a composition having a pH of 5 to 5.5; freezing the composition for a period of time at a temperature greater than -60°C to -10°C, for example -60°C to -10°C, -50°C to -20°C, -45°C to -25°C, or -30°C to -40°C, wherein after thawing, the pharmaceutical product exhibits at least 50% relative potency compared to the same product stored at -80°C for the same period of time; Includes.

[0018] Another general aspect of the present application relates to a method for reducing deamination of amino acids on the capsid of an rAAV, the method comprising: (a) rAAV, and (b) 25 mM sodium chloride; (c) 5 mM magnesium chloride; (d) 25 mM sodium acetate; (e) 0.01% by weight of poloxamer 188; (f) 5.4 wt. % sucrose; (g) water; and (h) preparing a composition having a pH of 5 to 5.5.

[0019] Another general aspect of the present application relates to a method for reducing degradation of a therapeutic pharmaceutical agent after lyophilizing the therapeutic pharmaceutical agent, the method comprising: (a) a therapeutic product; and (b) 10 mM Tris; (c) 75 mM sodium chloride; (d) 0.005% by weight of poloxamer 188; (e) 5% by weight of sucrose; (f) water, (g) preparing a composition having a pH of 7.3; storing the lyophilized composition for a period of time at a temperature between -20°C and 8°C, such as at a temperature between -20°C and 0°C or 0°C and 8°C, wherein 60% or more of the therapeutic agent is recovered after reconstitution of the lyophilized product compared to the same product stored at -80°C without lyophilization; Includes.

[0020] Another general aspect of the present application relates to a method for maintaining at least 50% relative potency of a therapeutic pharmaceutical after lyophilizing the therapeutic pharmaceutical, the method comprising: (a) a therapeutic product; and (b) 10 mM Tris; (c) 75 mM sodium chloride; (d) 0.005% by weight of poloxamer 188; (e) 5% by weight of sucrose; (f) water; and (g) preparing a composition having a pH of 7.3; storing the lyophilized composition for a period of time at a temperature between -20°C and 8°C, for example, at a temperature between -20°C and 0°C or 0°C and 8°C, wherein the pharmaceutical product exhibits at least 50% relative potency after reconstitution of the lyophilized product compared to the same product stored at -80°C without being lyophilized; Includes.

[0021] Another general aspect of the present application relates to a method for reducing deamination of amino acids on the capsid of an rAAV, the method comprising: (a) rAAV, and (b) 10 mM Tris; (c) 75 mM sodium chloride; (d) 0.005% by weight of poloxamer 188; (e) 5% by weight of sucrose; (f) water; and (g) preparing a composition having a pH of 7.3; Includes.

[0022] Another general aspect of the present application relates to a method for reducing degradation of a therapeutic pharmaceutical agent after lyophilizing the therapeutic pharmaceutical agent, the method comprising: (a) a therapeutic product; and (b) 10 mM sodium citrate; (c) 75 mM sodium chloride; (d) 0.001 wt% poloxamer 188; (e) 7 wt% sucrose; (f) water, (g) preparing a composition having a pH of 6.0; storing the lyophilized composition for a period of time at a temperature between -20°C and 8°C, such as at a temperature between -20°C and 0°C or 0°C and 8°C, wherein 60% or more of the therapeutic agent is recovered after reconstitution of the lyophilized product compared to the same product stored at -80°C without lyophilization; Includes.

[0023] Another general aspect of the present application relates to a method for maintaining at least 50% relative potency of a therapeutic pharmaceutical after lyophilizing the therapeutic pharmaceutical, the method comprising: (a) a therapeutic product; and (b) 10 mM sodium citrate; (c) 75 mM sodium chloride; (d) 0.001 wt% poloxamer 188; (e) 7 wt% sucrose; (f) water, (g) preparing a composition having a pH of 6.0; storing the lyophilized composition at a temperature between -20°C and 8°C, for example, at a temperature between -20°C and 0°C or 0°C and 8°C, for a period of time, wherein the pharmaceutical product exhibits at least 50% relative potency after reconstitution of the lyophilized product compared to the same product stored at -80°C without being lyophilized; Includes.

[0024] Another general aspect of the present application relates to a method for reducing deamination of amino acids on the capsid of an rAAV, the method comprising: (a) rAAV, and (b) 10 mM sodium citrate; (c) 75 mM sodium chloride; (d) 0.001 wt% poloxamer 188; (e) 7 wt% sucrose; (f) water; and (g) preparing a composition having a pH of 6.0; Includes.

[0025] Another general aspect of the present application relates to a method for reducing low molecular weight species (LMW) after freeze-drying a product, the method comprising: (a) a therapeutic product; and (b) 10 mM Tris; (c) 75 mM sodium chloride; (d) 0.005% by weight of poloxamer 188; (e) 5% by weight of sucrose; (f) water; and (g) preparing a composition having a pH of 7.3; storing the lyophilized composition at a temperature between -20°C and 8°C, for example at a temperature between -20°C and 0°C or 0°C and 8°C, for a period of time, wherein the lyophilized product is reconstituted and the LMW species is less than 5% by weight compared to the same product without lyophilization, as measured by analytical ultracentrifugation; Includes.

[0026] Another general aspect of the present application relates to a method for reducing low molecular weight species (LMW) after freeze-drying a product, the method comprising: (a) a therapeutic product; and (b) 10 mM sodium citrate; (c) 75 mM sodium chloride; (d) 0.001 wt% poloxamer 188; (e) 7 wt% sucrose; (f) water, (g) preparing a composition having a pH of 6.0; storing the lyophilized composition at a temperature between -20°C and 8°C, for example at a temperature between -20°C and 0°C or 0°C and 8°C, for a period of time, wherein the lyophilized product is reconstituted and the LMW species is less than 5% by weight compared to the same product without lyophilization, as measured by analytical ultracentrifugation; Includes.

[0027] Another general aspect of the present application relates to a method for reducing viral protein degradation after freezing a product, the method comprising: (a) rAAV, and (b) 25 mM sodium chloride; (c) 5 mM magnesium chloride; (d) 25 mM sodium acetate; (e) 0.01% by weight of poloxamer 188; (f) 5.4 wt. % sucrose; (g) water; and (h) preparing a composition having a pH of 5 to 5.5; storing the frozen composition at a temperature of -80°C up to -10°C, for example -70°C to -15°C, -60°C to -20°C, -50°C to -30°C, or -45°C to -35°C for a period of time, wherein the VP1:VP3 ratio changes by less than 20% after thawing the frozen product; Includes.

[0028] Another general aspect of the present application relates to a method for reducing viral protein degradation after lyophilizing a product, the method comprising: (a) a therapeutic product; and (b) 10 mM Tris; (c) 75 mM sodium chloride; (d) 0.005% by weight of poloxamer 188; (e) 5% by weight of sucrose; (f) water; and (g) preparing a composition having a pH of 7.3; storing the lyophilized composition for a period of time at a temperature between -20°C and 8°C, for example, at a temperature between -20°C and 0°C or 0°C and 8°C, wherein the reconstituted lyophilized product has a VP1:VP3 ratio that changes by less than 20% compared to the sample before lyophilization; Includes.

[0029] Another general aspect of the present application relates to a method for reducing viral protein degradation after lyophilizing a product, the method comprising: (a) a therapeutic product; and (b) 10 mM sodium citrate; (c) 75 mM sodium chloride; (d) 0.001 wt% poloxamer 188; (e) 7 wt% sucrose; (f) water, (g) preparing a composition having a pH of 6.0; storing the lyophilized composition for a period of time at a temperature between -20°C and 8°C, for example, at a temperature between -20°C and 0°C or 0°C and 8°C, wherein the reconstituted lyophilized product has a VP1:VP3 ratio that changes by less than 20% compared to the sample before lyophilization; Includes.

[0030] Another general aspect of the present application relates to a method for reducing a subvisible particle (SVP) concentration of a therapeutic pharmaceutical after freeze-thawing the therapeutic pharmaceutical, the method comprising: (a) a therapeutic product; and (b) 25 mM sodium chloride; (c) 5 mM magnesium chloride; (d) 25 mM sodium acetate; (e) 0.01% by weight of poloxamer 188; (f) 5.4 wt. % sucrose; (g) water, (h) preparing a composition having a pH of 5 to 5.5; freezing the composition at −20° C. for at least one year, wherein after thawing, the composition has an SVP concentration of less than 100 particles / mL for particles 10 μm or larger and less than 10 particles / mL for particles 25 μm or larger; Includes.

[0031] Another general aspect of the present application relates to a method for reducing subvisible particle (SVP) concentrations of a therapeutic pharmaceutical product after five freeze-thaw cycles, the method comprising: (a) a therapeutic product; and (b) 25 mM sodium chloride; (c) 5 mM magnesium chloride; (d) 25 mM sodium acetate; (e) 0.01% by weight of poloxamer 188; (f) 5.4 wt. % sucrose; (g) water, (h) preparing a composition having a pH of 5 to 5.5; freezing the composition for a period of time at a temperature greater than -80°C to -10°C, e.g., at a temperature of -70°C to -15°C, -60°C to -20°C, -50°C to -30°C, or -45°C to -35°C, wherein the composition is thawed and refrozen five times, and upon the fifth thaw, the SVP concentration is less than 100 particles / mL for particles 10 μm or larger and less than 10 particles / mL for particles 25 μm or larger; Includes.

[0032] Another general aspect of the present application relates to a method for reducing subvisible particle (SVP) concentrations in a therapeutic pharmaceutical, the method comprising: (a) a therapeutic product; and (b) 10 mM Tris; (c) 75 mM sodium chloride; (d) 0.005% by weight of poloxamer 188; (e) 5% by weight of sucrose; (f) water; and (g) preparing a composition having a pH of 7.3; storing the lyophilized composition for a period of time at a temperature between -20°C and 8°C, for example at a temperature between -20°C and 0°C or 0°C and 8°C, wherein the formulation has been subjected to one lyophilization cycle and the lyophilized composition has an SVP of less than 100 particles / mL for particles of 10 μm or larger and less than 10 particles / mL for particles of 25 μm or larger; Includes.

[0033] Another general aspect of the present application relates to a method for reducing subvisible particle (SVP) concentrations in a therapeutic pharmaceutical, the method comprising: (a) a therapeutic product; and (b) 10 mM sodium citrate; (c) 75 mM sodium chloride; (d) 0.001 wt% poloxamer 188; (e) 7 wt% sucrose; (f) water, (g) preparing a composition having a pH of 6.0; storing the lyophilized composition for a period of time at a temperature between -20°C and 8°C, for example at a temperature between -20°C and 0°C or 0°C and 8°C, wherein the formulation has been subjected to one lyophilization cycle and the lyophilized composition has an SVP of less than 100 particles / mL for particles of 10 μm or larger and less than 10 particles / mL for particles of 25 μm or larger; Includes.

[0034] According to an embodiment of the present application, the therapeutic pharmaceutical is rAAV. The rAAV may comprise a capsid derived from one or more AAVs selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV9.47, AAV9(hu14), AAV10, AAV11, AAV12, Rh8, Rh10, Rh74, AAV3B, AAV-2i8, RHM4-1, DJ, DJ8, NP59, Anc-80, and variants thereof. The rAAV may comprise a transgene encoding a polypeptide, or a nucleic acid selected from the group consisting of siRNA, antisense molecules, miRNA, ribozymes, and shRNA.

[0035] According to embodiments of the present application, the rAAV used in embodiments of the present application may be any of the following: GAA (acid alpha-glucosidase), ATP7B (copper-transporting ATPase 2), alpha-galactosidase A (GLA), ASS1 (argininosuccinate synthase), beta-glucocerebrosidase, beta-hexosaminidase A, SERPING1 (C1 protease inhibitor or C1 esterase inhibitor), glucose-6-phosphatase, CFTR (cystic fibrosis transmembrane conductance regulator), blood clotting (clotting) factors (e.g., factor XIII, factor IX, factor VIII, factor X, Factor VII, factor VIIa, protein C), gain-of-function blood coagulation factors, antibodies, retinal pigment epithelium-specific 65kDa protein (RPE65), erythropoietin, LDL (low-density lipoprotein) receptor, lipoprotein lipase (LPL), ornithine transcarbamylase (OTC), beta-globin, alpha-globin, spectrin, alpha-antitrypsin, adenosine deaminase (ADA), metal transporters (ATP7A or ATP7), sulfamidase, enzymes involved in lysosomal storage diseases (ARSA), hypoxanthine guanine phosphatase Phoribosyltransferase, β-25 glucocerebrosidase, sphingomyelinase, lysosomal hexosaminidase, branched-chain ketoacid dehydrogenase, hormones, growth factors, insulin-like growth factor 1 or 2, platelet-derived growth factor (PDGF), epidermal growth factor (EGF), nerve growth factor (NGF), neurotrophic factor-3 and -4, brain-derived neurotrophic factor (BDNF), glial cell line-derived growth factor (GDNF), transforming growth factor α and β, cytokines, α-interferon, β-interferon, interferon-γ, Interleukin-2 (IL-2), interleukin-4 (IL-4), interleukin-12 (IL-12), granulocyte-macrophage colony-stimulating factor (GM-CSF), lymphotoxin (LT), suicide gene product, herpes simplex virus thymidine kinase, cytosine deaminase (CD), diphtheria toxin (DT), cytochrome P450 (CYP), deoxycytidine kinase (DCK), tumor necrosis factor (TNF), drug resistance proteins, tumor suppressor proteins (e.g., p53, Rb, Wt-1, NF1, von Hippel-Lindau (VHL),adenomatous polyposis coli (APC), peptides with immunomodulatory properties, tolerogenic or immunogenic peptides or proteins tregitopes or hCDR1 (edorazides), insulin, glucokinase (GCK), guanylate cyclase 2D (GUCY2D) (or Leber congenital amaurosis (LCA) associated with GUCY2D variants (GUCY2D-LCA)), Rab escort protein 1 (REP1) (choroideremia (CHM) encodes REP1), LCA5 (Leber congenital amaurosis 5) (LCA-lebercillin), ornithine ketoacid aminotransferase (gyrate atrophy), retinoschisin 1 (X-linked retinoschisis), USH1C (Usher syndrome 1C), X-linked retinitis pigmentosa GTPase (XLRP), M Transgenes encoding ERTK (AR form of RP: retinitis pigmentosa), DFNB1 (connexin 26 deafness), ACHM2, 3 and 4 (amongria), PKD-1+ or PKD-2 (polycystic kidney disease), TPP1 (tripeptidyl peptidase 1), CLN2 (neuronal ceroid lipofuscinosis 2), sulfatase, N-acetylglucosamine-1-phosphate transferase, cathepsin A, GM2-AP (ganglioside GM2 activator), NPC1 (NPC intracellular cholesterol transporter 1), VPC2, sphingolipid activating protein, one or more zinc finger nucleases for genome editing, one or more donor sequences used as repair templates for genome editing, or variants thereof,

[0036] The foregoing and other objects, aspects, features, and advantages of the present disclosure will become more apparent and may be better understood by referring to the following description taken in conjunction with the accompanying drawings. [Brief explanation of the drawings]

[0037] [Figure 1]Vector recovery, calculated as a percentage of TO titer, for each formulation at all temperatures (5°C, -20°C, -40°C, -80°C) and time points tested (2 weeks to 6 months) is shown. F1 (Formulation 1) = 25 mM Na acetate, 25 mM NaCl, 5 mM MgCl, pH 5.2, 5.4% sucrose, 0.01% Pluronic™ F-68 (poloxamer 188); F2 (Formulation 2) = 10 mM Na phosphate, 180 mM NaCl, pH 7.3, 0.001% poloxamer 188; and F3 (Formulation 3) = 10 mM Na phosphate, 60 mM NaCl, 3% sucrose, pH 7.3, 0.001% poloxamer 188. [Figure 2] The IEX peak residence times for each formulation are shown at all temperatures (5°C, -20°C, -40°C, -80°C) and time points tested (2 weeks to 6 months). [Figure 3] The total high molecular weight (HMW) species of all samples reported by SEC-MALS (size exclusion chromatography-multi-angle light scattering) analysis are shown for each of the formulations at all temperatures (5°C, -20°C, -40°C, -80°C) and time points tested (2 weeks to 6 months). [Figure 4] VP capsid protein ratios for all samples reported by CE-SDS (capillary electrophoresis with sodium dodecyl sulfate) are shown for each of the formulations at all temperatures (5 °C, -20 °C, -40 °C, -80 °C) and time points tested (2 weeks to 6 months). [Figure 5] Fluorescence intensity data and Tm (melting point) for all TO samples from intrinsic fluorescence differential scanning fluorimetry (DSF) are shown. 1 and 2 indicate replicate measurements within each formulation sample. The dotted arrow indicates the peak shift seen in F1. [Figure 6] Normalized exogenous DSF fluorescence intensity for different TO formulations is shown. [Figure 7]High accuracy (HIAC) light obscuration measurements of samples showing invisible particle counts are shown for each of the formulations at all temperatures (5°C, -20°C, -40°C, -80°C) and time points tested (2 weeks to 6 months). [Figure 8] 1 shows peptide mapping data indicating the level of deamidation and oxidation at each hotspot residue in the formulation at the specific temperatures and time points tested. [Figure 9] Relative potency values ​​are shown for all samples relative to the TO sample for each formulation at all temperatures (5°C, -20°C, -40°C, -80°C) and time points tested (2 months and 6 months). [Figure 10] Figure 1 shows an analysis of matrix conditions that affect rAAV titer during fluid stress at 30° C. Favorable conditions, with increased % remaining after stress, are a combination of pH<7.3, NaCl<100 mM, and MgCl<10 mM. [Figure 11] The effects of specific buffers and excipients on subvisible particle (SVP) formation during rAAV freeze-thaw are shown. Figures 11A-11B show that the number and size of SVPs are highly dependent on the buffer species, freeze-thaw rate, and the proportion of rAAV particles that do not contain a therapeutic genome (i.e., the "empty" rAAV fraction). Figures 11C-11D show optimized storage conditions (i.e., the presence of sugars and surfactants as cryoprotectants) with a significant reduction in SVP formation, as well as the buffer species, freeze-thaw rate, and the empty rAAV fraction. [Figure 12] 1 shows the pH of rAAV in formulation F1 measured in thawed solution after long-term storage. [Figure 13A] Figure 13A shows the titer of rAAV in formulation F1 during long-term storage. Titer of intact viral genome measured by droplet digital PCR after each time and temperature combination. Figure 13B shows the total vector genome recovered. [Figure 13B] FIG. 13B shows the titers normalized by the initial measured titer (T0). [Figure 14]Figure 1 shows the cell-based potency of rAAV in formulation F1 during long-term storage. In vitro potency measured by cell-based functional assay. All measurements are relative to the potency measured prior to long-term stability (T0 conditions). [Figure 15A] Figure 15A shows the cell-based infectivity of rAAV in formulation F1 during long-term storage. Infectivity is measured by a cell-based assay for each time and temperature combination. Figure 15B shows the vector-particle concentration to infectious vector titer ratio, or PI ratio. [Figure 15B] Figure 15B shows the infectious titers. [Figure 16A] Figure 16A shows the total capsid and empty:full ratio of rAAV in formulation F1 measured by absorbance and at 280 nm and 260 nm during long-term storage. Figure 16B shows the total capsid concentration. [Figure 16B] FIG. 16B shows the ratio of empty:full viral capsids. [Figure 17A] Figure 17A shows the ratio of capsid viral proteins of rAAV in formulation F1 during long-term storage. Figure 17A shows the ratio of viral proteins normalized to viral protein 1 (VP1), including viral protein 2 (VP2), viral protein 3 (VP3), and a VP3 variant called pre-VP3. [Figure 17B] Figure 17B shows the percentage of mass attributed to the combination of VP1, VP2, and VP3. [Figure 17C] Figure 17C shows the percentage of protein attributable to degradation species that are not VP1, VP2, or VP3. [Figure 18]

[0023] Figure 1 shows oligomeric high molecular weight rAAV impurities in Formulation F1 during long-term storage. Impurities were measured by analytical size exclusion chromatography and are reported as percent area by absorbance at 280 nm. [Figure 19] Deamidation of rAAV capsids in formulation F1 during long-term storage is shown, reporting key amino acids N57, N254, N255, N263, and N514. Deamidation was measured by peptide mapping mass spectrometry. [Figure 20] The percent recovery of reconstituted rAAV after each lyophilization cycle is shown. Recovery is the viral genome recovery by qPCR, normalized by titer before lyophilization. rAAV was lyophilized in two formulations, F4 and F5, defined below, using secondary drying (SD) at either 25°C or 30°C: F4 (Formulation 4) = 10 mM Tris, 75 mM NaCl, 5% sucrose, 0.005% poloxamer 188, pH 7.3; F5 (Formulation 5) = 10 mM sodium citrate, 75 mM NaCl, 7% sucrose, 0.001% poloxamer, pH 6.0. [Figure 21] Figure 1 shows the cell-based potency of reconstituted rAAV after a lyophilization cycle. Potency was normalized to the pre-lyophilization sample. rAAV was lyophilized in formulation F4 and secondary dried at 25°C or 30°C. [Figure 22] Figure 1 shows oligomeric high molecular weight species in reconstituted rAAV solutions after lyophilization as measured by SEC-MALS. rAAV was lyophilized in formulations F4 and F5 using secondary drying (SD) at either 25°C or 30°C. Filtered TO indicates the 0.2 μm filtered sample before lyophilization. [Figure 23] The ratios of capsid proteins (VP1, VP2, VP3, and pre-VP3 variants) in the reconstituted rAAV solution after lyophilization are shown. Viral protein abundance was measured by CE-SDS and normalized to the amount of VP1 in each sample. TO indicates the value before lyophilization. [Figure 24] The titer recovery of reconstituted rAAV during long-term storage and relative humidity (RH) is shown. rAAV is lyophilized in F4 and stored between -20°C and 35°C for up to 6 months. The titer is measured by QPCR, and the recovery rate is calculated based on the titer before lyophilization (TO liquid). [Figure 25] Cell-based potency of reconstituted rAAV is shown. rAAV is lyophilized in F4 and stored between -20°C and 35°C for up to 6 months. Potency is normalized to the potency before lyophilization (TO liquid). [Figure 26A]Figure 26A shows the percentage of oligomeric high molecular weight (HMW) in reconstituted rAAV as assessed by SEC-MALS. rAAV was lyophilized in F4 and stored between -20°C and 35°C for up to 6 months. Figure 26B shows the percent monomer. [Figure 26B] Figure 26B shows the percent HMW. [Figure 27] The proportion of high molecular weight (HMW) in reconstituted rAAV was assessed by analytical ultracentrifugation, and the variation of HMW, partial capsid (partial), and low molecular weight (LMH) species was reported. rAAV was lyophilized in F4 and stored between -20°C and 35°C for up to 6 months. [Figure 28] The concentration of subvisible particles in reconstituted rAAV is shown, as assessed by light obscuration (HIAC). rAAV is lyophilized in F4 and stored between -20°C and 35°C for up to 6 months. Particle concentrations are shown as >5 μm, >10 μm, and >25 μm. TO liquid refers to the sample before lyophilization, and day 0 after lyophilization refers to the sample after the lyophilization cycle. [Figure 29] The ratios of rAAV capsid proteins (VP1, VP2, VP3, and pre-VP variants) in the reconstituted solution are shown. rAAV was lyophilized in F4 and stored between -20°C and 35°C for up to 6 months. Viral protein abundance was measured by CE-SDS and normalized to the amount of VP1 in each sample. T0 liquid represents the sample before lyophilization. DETAILED DESCRIPTION OF THE INVENTION

[0038] Detailed Description Various publications, articles, and patents are cited or described in the Background and throughout this specification, and each of these references is incorporated herein by reference in its entirety. The discussion of documents, acts, materials, devices, articles and the like which has been included in the present specification is for the purpose of providing a context for the present invention. Such discussion is not an admission that any or all of these matters form part of the prior art with respect to any invention disclosed or claimed.

[0039] In certain embodiments, the present disclosure relates to compositions and methods for the formulation and use of gene therapy products. In particular, the present application discloses stable rAAV formulations that preferably have one or more of the following properties, which maintain predictable (stable) freeze-thaw and lyophilization (lyophilization) performance and allow for longer shelf life at temperatures above -60°C, while maintaining important quality attributes such as genome recovery, maintained potency, minimal aggregation and degradation, improved vector quality, maximal chemical stability (e.g., minimal deamidation and oxidation), and / or maximal thermodynamic stability:

[0040] In certain embodiments, the expected stability period is at least 6 months, preferably 1 year, and most preferably 2 years. If accelerated conditions (usually temperatures significantly higher or lower than normal storage conditions) show acceptable changes within the observation period, it is reasonable to extrapolate stability results to the next observation period (typically intervals of 6 to 12, 18 to 24, 36 to 48, etc.). For frozen storage conditions without accelerated stability conditions, if there are no changes within the current time frame, the next time frame is also expected to be acceptable. (Compendial guidance; European Medicines Agency, Note For Guidance On Evaluation Of Stability Data, CPMP / ICH / 420 / 02, August 2003; and USP <787> ).

[0041] Unless otherwise defined, all technical and scientific terms used herein generally have their ordinary meaning in the art to which this invention belongs, within the context of this disclosure and within the specific context in which each term is used. Certain terms are explained below or elsewhere herein to provide additional guidance to the practitioner in describing the disclosed compositions and methods and how to make and use them.

[0042] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. As used herein, the use of the words "a" or "an" can mean "one," but is also consistent with the meanings of "one or more," "at least one," and "one or more than one."

[0043] Throughout this specification, including the claims that follow, unless the context otherwise requires, the word "comprise," and variations such as "comprises" and "comprising," will be understood to imply the inclusion of the stated elements or steps, or group of elements or steps, but not the exclusion of any other elements or steps, or group of elements or steps. As used herein, the term "comprising" can be replaced with the terms "containing" or "including," and, as used herein, may also be replaced by the term "having."

[0044] As used herein, "consisting of" excludes any element, step, or ingredient not recited in a claim, where such element, step, or ingredient pertains to the claimed invention. As used herein, "consisting essentially of" does not exclude materials or steps that do not materially affect the basic and novel characteristics of the claim. Whenever used herein in the context of aspects or embodiments of the invention, any of the foregoing terms "comprising," "containing," "including," and "having" can be replaced with the terms "consisting of" or "consisting essentially of" to vary the scope of the disclosure.

[0045] As used herein, the conjunction term "and / or" between multiple listed elements is understood to encompass both individual and combined alternatives. For example, when two elements are joined by "and / or," the first alternative refers to the applicability of the first element without the second element. The second alternative refers to the applicability of the second element without the first element. The third alternative refers to the applicability of the first and second elements. Any one of these alternatives is understood to be within the meaning and therefore meets the requirements of the term "and / or" as used herein. The simultaneous applicability of multiple alternatives is also understood to be within the meaning and therefore meets the requirements of the term "and / or."

[0046] All of the features disclosed in this specification may be combined in any combination. Each feature disclosed in this specification may be replaced by an alternative feature serving the same, equivalent, or similar purpose.

[0047] The terms "about" or "approximately" can mean within an acceptable error range for a particular value as determined by one of ordinary skill in the art, which will depend to some extent on the limitations of the method by which the value is measured or determined, i.e., the measurement system. For example, "about" can mean within 3 or more than 3 standard deviations, according to practice in the art. Alternatively, "about" can mean a range of up to 20%, preferably up to 10%, more preferably up to 5%, and more preferably even up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, preferably within 5-fold, and more preferably within 2-fold of a value. With respect to formulation ingredients and pH, "about" refers to plus or minus 10% of the indicated value.

[0048] The term "vector" refers to a carrier nucleic acid molecule that can be manipulated by inserting or incorporating a nucleic acid. Examples of "vectors" include, but are not limited to, plasmids, viruses including rAAV vectors, or other vehicles that can deliver nucleic acid molecules. Vectors can be used for genetic engineering to introduce or transfer polynucleotides into cells and transcribe or translate the inserted polynucleotides within the cells. An "expression vector" is a specialized vector that contains a gene or nucleic acid sequence with the regulatory regions necessary for expression in a host cell.

[0049] Viral vectors are derived from or based on one or more nucleic acid elements comprising a viral genome, and may also include a viral capsid that encapsulates the nucleic acid. A particular viral vector is an adeno-associated virus (AAV) vector, referred to herein as "rAAV."

[0050] The term "recombinant" as a modifier of a composition means that the composition has been manipulated or engineered in a manner that is not generally found in nature. Recombinant compositions include recombinant vectors, such as recombinant AAV vectors, recombinant polynucleotides or polypeptides, or recombinant cells or animals. The term "recombinant" as a modifier of a nucleic acid or vector indicates a combination of elements that do not occur in nature. Examples of recombinant nucleic acids include recombinant viral vector nucleic acids that provide 5' and / or 3' viral elements, and expression cassettes that contain one or more elements not naturally associated with the 5' and / or 3' elements, as well as expression cassettes that may contain different recombination components, such as heterologous promoters, polyA, introns, and spacers. Similarly, viral vectors, such as rAAV vectors, may contain naturally occurring or modified capsids that encapsulate recombinant viral vector nucleic acids. A specific example of a recombinant AAV vector would be an rAAV vector in which a nucleic acid sequence not normally present in the wild-type AAV genome (heterologous nucleic acid sequence) has been inserted into the AAV genome. Although the term "recombinant" is not always used herein with respect to sequences such as rAAV vectors, polynucleotides, etc., recombinant forms that include rAAV vectors, polynucleotides, etc. are expressly included despite such omission.

[0051] Viral vector nucleic acids contain 5' and / or 3' viral elements that provide viral packaging and may provide additional activities such as self-priming, DNA replication, promoter activity, genome integration, or episomal binding. The 5' and 3' elements are generally located at or near the 5' and 3' ends of the recombinant viral vector nucleic acid and may be naturally occurring sequences or modified versions of naturally occurring sequences. (Naso et al., (2017) BioDrugs, 31(4), 317-334; and Bulcha et al., (2021) Sig. Transduct. Target Ther. 6:53(2021)).

[0052] Additional elements of rAAV vectors include, but are not limited to, transcription termination signals or stop codons, one or more copies of AAV ITR sequences, or 5' or 3' untranslated regions (e.g., polyadenylation (polyA) sequences) flanking sequences such as introns. Nucleic acid elements include expression vector components containing transgenes along with regulatory elements that provide and / or promote translation, nuclear export of nucleic acids, and translation. Additional elements include, for example, filler or stuffer polynucleotide sequences, for example, to improve packaging and reduce the presence of contaminating nucleic acids. For shorter sequences, the inclusion of a stuffer or filler sequence can be used to adjust the length of the total nucleic acid sequence to the size of the viral genomic sequence that is acceptable for AAV vector packaging into viral particles. In various embodiments, the filler or stuffer nucleic acid sequence is a non-translated (non-protein-coding) segment of nucleic acid.

[0053] When the wild-type heterologous nucleic acid or transgene is too large to be packaged within an AAV vector particle and AAV delivery is desired, the heterologous nucleic acid can be provided in a modified, fragmented, or truncated form for packaging within and delivery by an AAV vector, ultimately providing a functional protein or nucleic acid product, such as a therapeutic protein or nucleic acid product.

[0054] Recombinant AAV vector nucleic acid can be derived from the wild-type (wt or wild-type) genome of AAV by using molecular methods to modify the wild-type genome. For example, rAAV can be obtained by removing part of the natural nucleic acid sequence from the AAV genome and replacing it with a non-natural nucleic acid sequence called heterologous nucleic acid. Alternatively, wild-type ITR or its variant can be added to the 3' and / or 5' end of the heterologous sequence. Typically, for AAV, one or both inverted terminal repeat (ITR) sequences of the AAV genome are maintained in the rAAV vector. rAAV is distinguished from the AAV genome because in rAAV, all or part of the AAV genome is replaced with a non-natural sequence or otherwise modified with respect to the AAV genome nucleic acid. Therefore, the incorporation of a non-natural sequence defines the AAV vector as a "recombinant" vector, which can be called a "rAAV vector."

[0055] Reference to a viral vector can provide viral nucleic acid and can also provide capsids (also called particles). The term "rAAV" can be used to refer to a recombinant viral vector nucleic acid enclosed in a capsid and / or a recombinant viral vector nucleic acid. Viral vectors can be used, for example, to infect (transduce) cells ex vivo, in vitro, or in vivo.

[0056] "Vector genome" refers to the portion of the recombinant plasmid sequence that is ultimately packaged or encapsidated to form a viral (e.g., rAAV) particle. When a recombinant plasmid is used to construct or manufacture a recombinant vector, the vector genome does not include portions of the "plasmid" that do not correspond to the vector genome sequence of the recombinant plasmid. This non-vector portion of the recombinant plasmid is called the "plasmid backbone," which is important for plasmid cloning and amplification, a process necessary for propagation and recombinant virus production, but is not itself packaged or enclosed in a viral (e.g., rAAV) particle. Thus, vector "genome" refers to the nucleic acid packaged or encapsidated by the virus (e.g., the nucleic acid packaged in an rAAV).

[0057] The terms "nucleic acid" and "polynucleotide" are used interchangeably herein to refer to all forms of nucleic acids, oligonucleotides, including deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). Nucleic acids include genomic DNA, cDNA, and antisense DNA, as well as spliced ​​or unspliced ​​mRNA, rRNA, tRNA, and inhibitory DNA or RNA. Inhibitory RNAs include, but are not limited to, small or short hairpin (sh) RNA, microRNA (miRNA), small or short interfering (si) RNA, trans-splicing RNA, or antisense RNA. Nucleic acids include naturally occurring polynucleotides, synthetic polynucleotides, and intentionally modified or altered polynucleotides (e.g., variant nucleic acids). Nucleic acids include, but are not limited to, cDNA, genomic DNA, RNA, and fragments thereof, which may be single-stranded or double-stranded.

[0058] Polynucleotides can be single-, double-, or triple-stranded, linear or circular, and can be of any length. When discussing nucleic acids, the sequence or structure of a particular polynucleotide may be described herein according to the convention of providing the sequence in the 5' to 3' direction.

[0059] As used herein, viral protein (VP) refers to at least one of the three subunit proteins (or any variant thereof) comprising the AAV capsid, designated viral protein 1, viral protein 2, and viral protein 3 (VP1, VP2, VP3), and pre-VP3. Decreased viral peptide protein degradation is typically observed as a decrease in the relative abundance of VP1 and / or VP2 compared to VP3. Viral protein degradation can be expressed as an increase in the relative abundance of VP3 (and similar species, including pre-VP3) compared to VP1 or VP2. One non-limiting example is determining and then comparing the VP1:VP3 ratio, where VP3 includes pre-VP3 (e.g., VP3 + pre-VP3), before and after storage conditions. If the VP1:VP3 ratio is higher after storage, degradation is present. To avoid confusion, in this application, when VP3 is calculated when determining degradation, the value is VP3 + pre-VP3.

[0060] "Transgene" is used herein to refer to a nucleic acid intended or introduced into a cell or organism. A transgene includes any heterologous nucleic acid contained in a vector (particularly an rAAV) that encodes a polypeptide or protein or encodes an inhibitory RNA. A transgene can directly increase the level of a target protein by encoding an RNA that the host cell machinery processes and translates into the target protein. A transgene can indirectly increase the level of a target protein by encoding a regulatory protein that promotes target protein expression, or by encoding an RNA that inhibits proteins and / or nucleic acids that serve to repress the target protein. A transgene can be a gene of interest (GOI) as described below.

[0061] Heterologous nucleic acids can be introduced or transferred into cells via vectors such as rAAV, "transduction" or "transfection." The term "transducer" and its grammatical variants refer to the introduction of a vehicle carrying a nucleic acid into a cell or host organism. Finally, the introduced nucleic acid can be extrachromosomal or only transiently present in the recipient cell or host organism.

[0062] A "transduced cell" is a cell into which a transgene or heterologous nucleic acid has been introduced. Thus, a "transduced" cell refers to a genetic change in a cell after incorporation of an exogenous molecule, e.g., a nucleic acid (e.g., a transgene), into the cell. Thus, a "transduced cell" is a cell into which a "nucleic acid" or "polynucleotide" has been introduced, or its progeny into which an exogenous nucleic acid has been introduced. The cell can proliferate and transcribe the introduced nucleic acid, and, if the nucleic acid encodes a protein, can subsequently translate the nucleic acid transcript. With respect to gene therapy uses and methods, the transduced cell can be within a subject.

[0063] "Expression control element" is a type of regulatory element and refers to a nucleic acid sequence(s) that influences the expression of an operably linked nucleic acid. Typically, such elements are included to facilitate proper heterologous polynucleotide transcription and, if necessary, translation (e.g., promoters, enhancers, splicing signals for introns, maintaining the correct reading frame of the gene to allow in-frame translation of mRNA, and stop codons, etc.). Such elements typically act in cis, referred to as "cis-acting" elements, but can also act in trans.

[0064] The term "operably linked" means that the regulatory sequence is positioned in an appropriate position relative to the nucleic acid sequence so that it affects the expression of the nucleic acid sequence. This same definition can be applied to the arrangement of nucleic acid sequences and transcriptional control elements (e.g., promoters, enhancers, and termination elements) in an expression vector, such as an rAAV vector.

[0065] As used herein, "E:F ratio" or "empty AAV fraction" refers to the ratio of empty rAAV particles that are not packaged with an rAAV genome to complete rAAV particles that are packaged with an rAAV genome.

[0066] "Degradation" refers to a general set of chemical and physical changes that accumulate over time and reduce the safety and / or efficacy of a vector. These may include, but are not limited to, genome excretion, vector aggregation, capsid protein fragmentation, and capsid protein deamidation. Furthermore, in some embodiments of the present invention, degradation products are referred to as high molecular weight species (HMW) or low molecular weight species (LMW).

[0067] In some embodiments, the heterologous nucleic acid encoding the protein (e.g., a therapeutic protein) is provided in a modified or truncated form, or the heterologous nucleic acid is provided in multiple constructs, each delivered by a separate multiple AAV vector.

[0068] In certain embodiments, the heterologous nucleic acid is provided as a truncated variant that includes removal of portions unnecessary for function, such that the encoding heterologous polynucleotide is of reduced size for packaging into an AAV vector, while maintaining the function of the encoded protein (e.g., a therapeutic protein).

[0069] In certain embodiments, the heterologous nucleic acid is provided in a split AAV vector, each providing nucleic acid encoding a different portion of a protein (e.g., a therapeutic protein), thus delivering multiple portions of the protein (e.g., a therapeutic protein) that assemble and function within the cell.

[0070] In other aspects, the heterologous nucleic acid is provided by a dual AAV vector using overlapping, trans-splicing, or hybrid trans-splicing dual vector techniques. In certain embodiments, two overlapping AAV vectors are used that combine intracellularly to generate a complete expression cassette from which a full-length protein (e.g., a therapeutic protein) is expressed.

[0071] The term "therapeutic pharmaceutical agent" refers to any drug administered to a patient that produces a clinically meaningful response for a given indication. Such pharmaceutical agents are biologics and include, but are not limited to, antibodies, modified cells, cell or gene therapies, viruses, bacteria, nanoparticles, nucleic acids, and biological molecules.

[0072] The term "isolated" when used as a composition modification agent means that the composition is provided in a different environment from that in which it occurs in nature.For example, the composition can be completely or at least partially prepared or separated from the in vivo environment in which it naturally exists.In general, isolated compositions are substantially free from one or more materials that they normally associate with in nature, such as one or more proteins, nucleic acids, lipids, carbohydrates, cell membranes.

[0073] The term "isolated" does not exclude further combinations; for example, isolated rAAV sequences, or isolated rAAV particles encapsulating rAAV sequences, may be provided as pharmaceutical formulations. The term "isolated" also does not exclude alternative physical forms of the composition, such as hybrid / chimeric, multimeric / oligomeric, modified (e.g., phosphorylated, glycosylated, lipidated) or derivatized forms, or forms expressed in host cells that provide an environment different from that in which they naturally occur.

[0074] The term "substantially pure" refers to a preparation that contains at least 50-60% by weight of a compound or molecule of interest (e.g., nucleic acid, oligonucleotide, protein, capsid, rAAV particle, etc.). Purity is measured by methods appropriate for the particular compound or molecule of interest (e.g., chromatographic methods, agarose or polyacrylamide gel electrophoresis, HPLC (high performance liquid chromatography) analysis, etc.). In some embodiments, an rAAV preparation is substantially pure whole rAAV particles when whole particles comprise at least 70% by weight of the preparation.

[0075] When referring to a particular nucleotide or amino acid sequence, the phrase "consisting essentially of" means a sequence having the characteristics of a given SEQ ID NO. For example, when used in reference to an amino acid sequence, the phrase includes the sequence itself and molecular modifications that do not affect the basic and novel characteristics of the sequence.

[0076] Nucleic acid molecules, expression vectors (e.g., AAV vector genomes), plasmids containing nucleic acids encoding the modified or variant AAV capsids of the invention, and heterologous nucleic acids can be prepared using recombinant DNA technology methods. The availability of nucleotide sequence information allows for the preparation of the nucleic acid molecules of the invention by a variety of means. For example, nucleic acid sequences can be generated using a variety of standard cloning and recombinant DNA techniques, through cellular expression and chemical synthesis techniques. The purity of polynucleotides can be determined by sequencing, gel electrophoresis, and the like. For example, nucleic acids can be isolated using hybridization or computer-based database screening techniques. These techniques include, but are not limited to, (1) hybridization of genomic DNA or cDNA libraries with probes to detect homologous nucleotide sequences; (2) antibody screening to detect polypeptides with common structural features, e.g., using expression libraries; (3) polymerase chain reaction (PCR) on genomic DNA or cDNA using primers capable of annealing to the nucleic acid sequence of interest; (4) computer searches of sequence databases for related sequences; and (5) differential screening of subtraction nucleic acid libraries.

[0077] Ionic strength is a measure of the effective salt composition and electrostatic screening of a solution. The effect of ionic strength on a formulation can include affecting electrostatic interactions and colloidal stability in solution. Ionic strength is typically estimated as shown in Equation 1, where C i is the concentration of each ionic species, and Z i is the charge of the species. In certain embodiments, "low" ionic strength is between about 25-50 mM and "moderate" ionic strength between 50-95 mM. TIFF2026502876000002.tif20164

[0078] I. Viral Vectors The viral vector that comprises viral nucleic acid enclosed in protein capsid can deliver viral vector nucleic acid to cells or tissue.Depending on specific vector, viral vector can further comprise viral envelope.The examples of viral vector that can be used for gene delivery include adenovirus vector, rAAV, retrovirus vector and herpes simplex vector.

[0079] Different serotypes exist for different types of viruses. Different serotypes can confer different activities, such as cell and tissue tropism and the potential to elicit a host immune response. The term "serotype" broadly refers to both serologically distinct viruses and serologically indistinguishable viruses that may exist within subgroups or variants of a given serotype. Serological specificity can be determined based on the lack of cross-reactivity between antibodies against one capsid and another. Such differences in cross-reactivity are usually due to differences in capsid protein sequences / antigenic determinants (e.g., resulting from differences in the VP1, VP2, and / or VP3 sequences of AAV serotypes).

[0080] As more naturally occurring virus isolates are discovered and / or capsid variants are generated, they may or may not be serologically distinct from any of the currently existing serotypes. Thus, if a new virus does not have serological differences, it is a subgroup or variant of the corresponding serotype.

[0081] IA adenovirus vector Adenoviruses are non-enveloped, double-stranded DNA viruses. Recombinant adenoviral vectors contain recombinant adenoviral nucleic acid that lacks one or more proteins involved in viral replication and further contain an adenoviral capsid. Recombinant adenoviral vectors can contain different amounts of adenoviral DNA. The adenovirus (Ad) genome is flanked at its ends by hairpin-like inverted terminal repeats (ITRs), which vary in length from 30 to 371 bp. The ITRs function as self-priming structures that promote primase-independent DNA replication. Packaging of the viral genome requires a packaging signal located on the left arm of the genome. (Liu and Seol (2020) BMB Reports; 53(11):565-575; and Bulcha et al., (2021) Sig. Transduct. Target Ther. 6:53.)

[0082] In certain embodiments, the recombinant adenoviral vector is a third-generation vector, also referred to as "gutless" or "helper-dependent." Gutless vectors can be produced from recombinant adenoviral nucleic acid that is free of all or substantially all viral sequences except for the ITRs and packaging signal. Gutless adenoviral vectors are high-capacity vectors that can accommodate DNA inserts up to approximately 36 kb. Preferred recombinant adenoviral nucleic acids are approximately 27 kb to approximately 37 kb. Stuffer sequences can be added to the recombinant adenoviral nucleic acid to increase nucleic acid size and capsid integration. Preferred stuffer sequences avoid coding sequences, repetitive sequences, recombination sequences, and immunogenic sequences. (Liu and Seol (2020) BMB Reports; 53(11):565-575; and Bulcha et al., (2021) Sig. Transduct. Target Ther. 6:53, and Sandig et al., PNAS (2000) 97(3):1002-1007, each of which is incorporated herein by reference in its entirety).

[0083] In certain embodiments, recombinant adenoviral vectors can be produced based on rare human or chimpanzee serotypes. The use of chimpanzee and rare human serotypes can help reduce the host immune response to the recombinant adenoviral vector due to pre-existing immunity. (Guo et al., (2018) Human vaccines & immunotherapeutics, 14(7):1679-1685, and Bulcha et al., (2021) Sig. Transduct. Target Ther. 6:53.)

[0084] Adenoviral vectors can be produced, for example, by using appropriate helper viruses or plasmids and cell lines to provide the viral proteins necessary for vector production in trans (Liu and Seol (2020) BMB Reports; 53(11):565-575; and Bulcha et al., (2021) Sig. Transduct. Target Ther. 6:53.).

[0085] IB recombinant AAV vector Recombinant adeno-associated virus vectors are based on adeno-associated viruses. Adeno-associated viruses are single-stranded DNA viruses containing a 4.7 kb genome flanked by 145 nt ITRs at both ends of the genome. ITR activity is important for self-priming and packaging, and may also provide additional activities such as promoter activity. AAV 5' and 3' ITRs can vary in size, and the 5' and 3' inverted repeats do not need to be exact inverted repeats.

[0086] The rAAV vector comprises an AAV recombinant nucleic acid and a viral capsid. The rAAV recombinant nucleic acid lacks one or more AAV proteins involved in viral replication. In certain embodiments, the rAAV vector comprises an AAV 5' and / or 3' ITR together with a DNA insert. In certain embodiments, the rAAV nucleic acid comprises a 5' ITR and / or a 3' ITR independently selected from the 5' and 3' ITRs provided in AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh.10, AAVrh.74, and AAV3B ITRs. In further embodiments, the 5' and 3' ITRs are present, and both ITRs are derived from the same serotype genome.

[0087] Recombinant adeno-associated virus vectors typically accept DNA inserts, generally ranging in size from about 4 kb to about 5.2 kb. If necessary, a stuffer sequence can be used to increase the size and packaging efficiency of the rAAV nucleic acid. In certain embodiments, the rAAV nucleic acid, including the stuffer, is less than 5.5 kb. In further embodiments, the rAAV nucleic acid comprising the stuffer is less than 5.2 kb, less than 5.1 kb, less than 5.0 kb, less than 4.9 kb, less than 4.8 kb, less than 4.7 kb, less than 4.6 kb, between 4 kb and 5.2 kb, between 3.0 kb and 5.5 kb, between 4.0 kb and 5.0 kb, or between 4.3 kb and 4.8 kb, or about 4.2 kb, about 4.3 kb, about 4.4 kb, about 4.5 kb, about 4.6 kb, about 4.7 kb, about 4.8 kb, about 4.9 kb, or about 5.0 kb. Preferred stuffer sequences avoid coding sequences, repetitive sequences, recombination sequences, and immunogenic sequences.

[0088] In certain embodiments, the rAAV is a self-complementary adeno-associated viral vector (scAAV) or a short hairpin adeno-associated viral vector (shAAV). scAAV and shAAV provide double-stranded rAAV nucleic acid that can be incorporated into the AAV capsid. scAAV and shAAV contain inverted dimer repeats that provide intramolecular double-stranded DNA. scAAV can be generated by mutating the ITR terminal resolution site so that rep cannot nick the terminal resolution site. shAAV can utilize a short hairpin to generate double-stranded AAV nucleic acid. scAAV and shAAV, which are double-stranded DNA, offer the advantage of avoiding the DNA synthesis step required for single-stranded rAAV nucleic acid during cell entry. A potential disadvantage of scAAV and shAAV is that the size of the DNA insert that can be incorporated is reduced by about half compared to single-stranded rAAV nucleic acid. (U.S. Pat. No. 10,457,940; Xie et al., Mol Ther. (2017) 25(6):1363-1374; and McCarty Mol. Ther. (2008) 16(10):1648-1656; each of which is incorporated by reference herein in its entirety).

[0089] Naturally occurring AAV capsids contain the viral proteins VP1, VP2, and VP3 in a ratio of approximately 1:1:10. AAV vectors can be produced where all three viral proteins are based on a particular serotype, or where one, two, or all three viral proteins are based on different serotypes.

[0090] The recombinant AAV capsid and nucleic acid can be based on the same serotype (or subgroup or variant), or can be different from each other. In certain embodiments, the rAAV nucleic acid has the same serotype genome (e.g., ITRs) as the capsid protein.

[0091] In different embodiments, the rAAV capsid is at least 80%, at least 85%, at least 90%, at least 100%, at least 120%, at least 140%, at least 160%, at least 18 ... The VP1 of SEQ ID NO: 1 comprises a protein having 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.9%, or 100% identical sequence identity to any of the capsids. A recombinant AAV capsid comprising VP1 of SEQ ID NO: 1 is described, for example, in U.S. Patent No. 9,840,719, which is incorporated herein by reference.

[0092] In certain embodiments, the AAV capsid comprises VP1, VP2, and VP3, each independently having at least 80%, at least 90%, at least 95%, or 100% sequence identity to VP1, VP2, or VP3 of any of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh.74, AAV3B, AAV-2i8, AAVrh.10, AAVrh.8, AAVHSC, AAV-B1, AAV-AS, AAV1 / rh.10, and variants thereof (e.g., capsid variants such as amino acid insertions, additions, substitutions, and deletions). See, for example, U.S. Pat. Nos. 9,909,142 and 9,840,719, and U.S. Patent Publication No. 2013 / 0059732, which disclose RHM4-1, RHM15-1, RHM15-2, RHM15-3 / RHM15-5, RHM15-4, and RHM15-6.

[0093] In certain embodiments, the rAAV capsid comprises a VP1 consisting of the nucleotide sequence of SEQ ID NO: 1. In further embodiments, the AAV capsid further comprises a VP2 comprising the nucleotide sequence of SEQ ID NO: 2. In preferred embodiments, the AAV capsid comprises a VP1 comprising the sequence of SEQ ID NO: 1, a VP2 comprising the sequence of SEQ ID NO: 2, and a VP3 comprising the sequence of SEQ ID NO: 3.

[0094] The AAV genome contains two major genes: rep and cap. Transcription from the rep gene is initiated from two distinct promoters and produces nonstructural proteins called Rep78, Rep68, Rep52, and Rep40. The rep proteins function in genome replication and / or capsid formation. The cap gene encodes the structural proteins (VP1, VP2, and Vp3) that make up the capsid, the nonstructural assembly activating protein (APP), which functions in capsid assembly, and membrane-associated accessory proteins thought to be involved in the productive phase of the replication cycle. (Maurer and Weitzman (2020) Hum. Gene Ther. 31(9-10):499-511, incorporated herein by reference in its entirety.)

[0095] AAV requires helper virus functions to complete its replication cycle. Helper virus functions can be provided by different viruses in permissive cell lines. Permissive cell lines are cell lines that can support viral replication. Examples of helper viruses for AAV include adenovirus, HSV-1, HPV-16, and HBoV1, which can be used in conjunction with permissive primate cells, for example. For example, baculovirus can be used in conjunction with permissive insect cells such as sf9. (Maurer and Weitzman (2020) Hum. Gene Ther. (2020) 31 (9-10): 499-511, and Meier et al., (2020) Viruses 19; 12 (6): 662, both of which are incorporated herein by reference in their entirety.)

[0096] Recombinant AAV can be produced, for example, by using an appropriate helper virus or plasmid and cell line to provide the viral proteins required for vector production in trans. In certain embodiments, rAAV is produced using an rAAV vector genome plasmid. The plasmid contains that portion of the rAAV nucleic acid that is ultimately packaged or encapsidated to form a viral (e.g., rAAV) vector. The "plasmid backbone" contains elements important for propagation and recombinant virus production. Except for possible 3'ITR and / or 5'ITR cloning remnants, the plasmid backbone itself is not packaged or encapsidated into viral particles.

[0097] The vector genome plasmid may contain regions such as an origin of replication and a selection marker. Additional sites that may be present include cloning sites.

[0098] II. Preparations In certain embodiments, the present disclosure relates to compositions and methods for formulating and using gene therapy products. In certain embodiments, the present application relates to rAAV formulations that maintain predictable (stable) freeze-thaw and lyophilization (lyophilization) performance, allowing for long-term shelf storage at temperatures above -60°C while maintaining genome recovery and critical quality attributes. In certain embodiments, the selection of specific components of an rAAV formulation can affect certain critical quality attributes. For example, but not by way of limitation, the inventors have shown that a pH of about 4.5 to about 8 in the formulation can be advantageous for enhancing stability and minimizing degradation at various storage temperatures (e.g., 2-8°C, -20°C) and under stress conditions. Additionally, the formulations described herein employ lower ionic strength than conventional rAAV formulations, which, contrary to generally accepted principles, allows for well-maintained storage at higher temperatures without compromising quality, stability, or functionality (see Wright, J. Frasier, et al., "Identification of factors that contribute to recombinant AAV2 particle aggregation and methods to prevent its occurrence during vector purification and formulation," Molecular Therapy, (2005) 12:1; Rodrigues, Gerard A., et al., "Pharmaceutical development of AAV-based gene therapy products for the eye," Pharmaceutical Research, (2019) 36:29; and Srivastava, Arvind, et al. "Manufacturing challenges and rational formulation development for AAV viral vectors," Journal of Pharmaceutical Sciences 110.7 (2021):2609-2624).

[0099] In certain embodiments, the inventors disclose low ionic strength (20-50 mM) and medium ionic strength (50-95 mM) formulations that maintain key critical quality attributes. These attributes are known to correlate with safety and efficacy, as well as extended shelf life (>1.5 years). The extended shelf life of rAAV formulations with CQA profiles such as those demonstrated herein is needed in the field given the challenges of global supply and distribution in liquid and frozen states.

[0100] In some embodiments, the disclosed formulations are suitable for lyophilization, further extending shelf life and ease of supply and distribution. In contrast to conventional rAAV formulations stored at -80°C, the low ionic strength, sugar-containing, and low pH (pH 5.2) formulation (F1) exhibits excellent stability and quality retention at temperatures such as -20°C, -40°C, and -80°C over long-term storage periods (i.e., 6 months to 3 years). The medium ionic strength formulation (F3) exhibits sufficient stability and quality retention at temperatures such as -20°C, -40°C, and -80°C over long-term storage periods (i.e., 6 months to 3 years).

[0101] In certain embodiments, the ratio of empty particles to full rAAV particles (empty:full) in the preparation is preferably 1:9 or less, including 1:10, 1:15, 1:20, 1:25, 1:30, 1:35, 1:40, 1:45, 1:50 or less, or any ratio therebetween, more preferably 1:49 or more. In other embodiments, the ratio of empty particles to full rAAV particles in the preparation is preferably 3:7 or less, including 3:8, 3:9, 3:10, 3:11, 3:12 or less, or any ratio therebetween. In other embodiments, the ratio of empty particles to full rAAV particles in the preparation is preferably 0:1 or less.

[0102] In certain embodiments, the rAAV formulations of the present disclosure comprise the following: a plurality of rAAV genomes, a salt or mixture of salts to adjust the ionic strength of the formulation, a buffer, a sugar, and a surfactant. For example, in certain embodiments, the plurality of rAAV genomes comprises 1 x 10 9 , 1x10 10, 1x10 11 , 1x10 12 , 1x10 13 , 1x10 14 , 1x10 15 Approximately 1x10 vg / mL or any concentration therebetween 9 ~approx. 1x10 15 It is present at a concentration of 10 ...

[0103] In certain embodiments, the buffer in the rAAV formulation of the present disclosure is a non-phosphate buffer. In certain embodiments, the non-phosphate buffer is sodium acetate. In certain embodiments, the non-phosphate buffer is Tris (tris(hydroxymethyl)aminomethane), HEPES, sodium citrate, succinate, and mixtures thereof.

[0104] In certain embodiments, the non-phosphate buffering agent is present at a concentration of about 1 mM to about 50 mM (for low ionic strength), e.g., 1 mM, 5 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, 50 mM, or any concentration therebetween. In certain embodiments, the non-phosphate buffering agent is present at a concentration of about 50 mM to about 100 mM (for moderate ionic strength), including 50 mM, 55 mM, 60 mM, 65 mM, 70 mM, 75 mM, 80 mM, 85 mM, 90 mM, 95 mM, 100 mM, or any concentration therebetween. In certain embodiments, the non-phosphate buffering agent is sodium acetate, present at a concentration of about 25 mM.

[0105] In certain embodiments, the salt that determines the ionic strength of the formulations of the present disclosure is sodium chloride. In certain embodiments, sodium chloride is present at a concentration of about 1 mM to about 180 mM, including 1 mM, 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, 100 mM, 110 mM, 120 mM, 130 mM, 140 mM, 150 mM, 160 mM, 170 mM, 180 mM, or any concentration therebetween. In certain embodiments, sodium chloride is preferably present at a concentration of about 60 mM. In certain embodiments, sodium chloride is most preferably present at a concentration of about 25 mM.

[0106] In certain embodiments, the salt used as an excipient in the formulations of the present disclosure is magnesium chloride. In certain embodiments, magnesium chloride is present at a concentration of about 1 mM to about 10 mM, including 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, or any concentration therebetween. In certain embodiments, magnesium chloride is present at a concentration of about 5 mM.

[0107] In certain embodiments, the rAAV formulations of the present application contain a sugar. In certain embodiments, the sugar content of the formulation is about 0% to about 20%, including 0%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, or any concentration therebetween. In certain embodiments, the sugar content of the formulation is preferably about 5.4%. In certain embodiments, the sugar is sucrose. In certain embodiments, the sugar content of the formulation, including the sugar content, is about 3%.

[0108] In certain embodiments, the rAAV formulations of the present disclosure include a surfactant. In certain embodiments, the surfactant is present at a concentration of about 0-0.05%, including 0%, 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, or any concentration therebetween. In certain embodiments, the surfactant is poloxamer 188 (Pluronic® f-68 or P188). In certain embodiments, the surfactant is selected from polysorbate 20 (PS20), polysorbate 80 (PS80), or a combination thereof. In certain embodiments, poloxamer 188 is most preferably present at a concentration of about 0.01%. In certain embodiments, poloxamer 188 is present at a concentration of about 0.001%.

[0109] In certain embodiments, the rAAV formulations of the present disclosure contain a stabilizer. In certain embodiments, the stabilizer is an amino acid. In certain embodiments, the amino acid is arginine or histidine.

[0110] In certain embodiments, the rAAV formulations of the present disclosure are prepared to have a pH of about 4.5 to about 8 (including 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, or any pH in between). In certain embodiments, the pH is most preferably about 5.2. In certain embodiments, the pH is about 7.4.

[0111] In certain embodiments, an rAAV formulation of the disclosure comprises about 25 mM Na acetate, about 25 mM NaCl, about 5 mM Mg2Cl, a pH of about 5.2, about 5.4% sucrose, and about 0.01% poloxamer 188, and is referred to herein as "Formulation 1" or "F1."

[0112] In certain embodiments, the buffer in the rAAV formulation of the present disclosure is a phosphate buffer. In certain embodiments, the phosphate buffer is sodium phosphate. In certain embodiments, the phosphate buffer is present at a concentration of about 1 mM to about 50 mM, including 1 mM, 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, or any concentration therebetween. In certain embodiments, the phosphate buffer is sodium phosphate present at about 10 mM.

[0113] In certain embodiments, an rAAV formulation of the present disclosure comprises about 10 mM sodium phosphate, 60 mM sodium chloride, a pH of about 7.4, 3% sucrose, 0.001% poloxamer 188, and is referred to herein as "Formulation 3" or "F3."

[0114] In certain embodiments, the formulation maintains its pH within + / - 0.1 when stored at -40°C for 6 months. In certain embodiments, the formulation maintains its pH within + / - 0.1 when stored at -40°C for 2 months. In certain embodiments, the formulation maintains its pH within + / - 0.1 when stored at -20°C for 6 months. In certain embodiments, the formulation maintains its pH within + / - 0.1 when stored at -20°C for 2 months. In certain embodiments, the formulation maintains its pH within + / - 0.1 when stored at 5°C for 6 months. In certain embodiments, the formulation maintains its pH within + / - 0.1 when stored at 5°C for 2 months.

[0115] In certain embodiments of the present disclosure, the rAAV formulation exhibits a glass transition temperature of approximately −39°C for the frozen concentrate. In certain embodiments, it is beneficial to store the biologic below the glass transition temperature to minimize mobility and enhance stability. Increasing the Tg' (glass transition temperature) also allows for storage at temperatures above −80°C, which is advantageous and highly desirable given the ease of supply chain practices, including handling / transportation and storage at higher temperatures. It is also a viable approach because it maximizes sterility assurance (i.e., −80°C is generally below the glass transition of plastics and rubbers, so low-temperature storage can compromise container closure systems, which generally makes plastics brittle at these low temperatures).

[0116] In certain embodiments, rAAV formulations of the present disclosure are prepared such that the formulations exhibit similar or better recovery of the vector genome (genome recovery) after storage under different conditions compared to the genome recovery of the same formulation before storage. In certain embodiments, rAAV formulations of the present disclosure are prepared such that the formulations exhibit about 50% to about 150% (including 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, or any percentage therebetween) genome recovery after storage at 5°C for about 1 day, 3 days, 5 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 2 years, or 3 years compared to the genome recovery of the same formulation before storage. Genome recovery can be measured as described in the Examples below.

[0117] In certain embodiments, the formulation exhibits genomic recovery of about 50% to about 150% (including 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, or 150%, or any percentage therebetween) after storage at -40°C for about 1 day, 3 days, 5 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 2 years, or 3 years compared to the genomic recovery of the same formulation before storage.

[0118] In certain embodiments, the formulation exhibits genomic recovery of about 50% to about 150% (including 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, or 150%, or any percentage therebetween) after storage at -20°C for about 1 day, 3 days, 5 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 2 years, or 3 years compared to the genomic recovery of the same formulation before storage.

[0119] In certain embodiments, the formulation exhibits genomic recovery of about 50% to about 150% (including 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, or 150%, or any percentage therebetween) after storage at 5° C. for about 1 day, 3 days, 5 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 2 years, or 3 years compared to the genomic recovery of the same formulation before storage.

[0120] In certain embodiments, rAAV formulations of the present disclosure are prepared such that the formulation maintains similar or better potency (relative potency) after storage under different conditions compared to that of the same formulation before storage. In certain embodiments, rAAV formulations of the present disclosure are prepared such that the formulation exhibits a relative potency of about 50% to about 150% (including 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, or any percentage therebetween) after storage at 5°C for about 1 day, 3 days, 5 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 2 years, or 3 years compared to the relative potency of the same formulation before storage.

[0121] In certain embodiments, the formulation exhibits a relative potency of about 50% to about 150% (including 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, or any percentage therebetween) after storage at −20° C. for about 1 day, 3 days, 5 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 2 years, or 3 years, compared to the relative potency of the same formulation before storage.

[0122] In certain embodiments, the formulations exhibit a relative potency of about 50% to about 150% (including 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, or any percentage therebetween) after storage at −40° C. for about 1 day, 3 days, 5 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 2 years, or 3 years compared to the relative potency of the same formulation before storage.

[0123] In certain embodiments, the formulation exhibits a relative potency of about 50% to about 150% (including 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, or 150%, or any percentage therebetween) after storage at −80° C. for about 1 day, 3 days, 5 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 2 years, or 3 years, compared to the relative potency of the same formulation before storage.

[0124] In certain embodiments, the rAAV formulations of the present disclosure are prepared such that the formulation is suitable for intravenous, intraarterial, intraocular, intrathecal, or intracerebral injection. In certain embodiments, the formulation is liquid. In certain embodiments, the formulation is suitable for lyophilization. In certain embodiments, the formulation is contained in a unit-dose container. In certain embodiments, the unit-dose container is a vial. In certain embodiments, the vial is a sealed glass vial. In certain embodiments, the vial is a cyclic olefin polymer vial, also known as a CZ vial.

[0125] In certain embodiments, the rAAV formulations of the present disclosure comprise various viral strains or serotypes. In certain non-limiting embodiments, the rAAV is prepared using the methods described in Pulicherla et al. al., Mol. Ther., 19(6) 1070-1078 (2011) (describing, among other things, AAV9 variants including AAV9.47), U.S. Pat. No. 7,906,111 (describing, among other things, AAV9(hu14)), U.S. Pat. No. 10,532,111 (describing, among other things, NP59), U.S. Pat. No. 10,738,087 (describing, among other things, Anc80), WO 2012 / 145601, WO 2013 / 158879, WO 2015 / 013313, WO 2018 / 156654, U.S. Pat. No. 9,840,719 (describing RHM4-1), U.S. Pat. No. 7,749,492, U.S. Pat. rAAV vectors can be based on any AAV genome, including, but not limited to, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV9.47, AAV9(hu14), AAV10, AAV11, AAV12, Rh8, Rh10, Rh74, AAV3B, AAV-2i8, RHM4-1, DJ, DJ8, NP59, Anc-80, and variants thereof, including variants of the AAV capsids described in U.S. Patent No. 7,588,772 (depicting DJ and DJ8) and U.S. Patent No. 9,587,282 (all of which are incorporated herein by reference in their entireties). Thus, rAAV vectors contain gene / protein sequences identical to those characteristic of specific serotypes, as well as mixed serotypes.

[0126] In certain embodiments, the pH of the formulation is adjusted prior to administration.

[0127] III. rAAV containing the gene of interest (GOI) In certain embodiments, the rAAV contains a gene of interest (GOI). In certain non-limiting embodiments, the GOI contains a nucleic acid sequence encoding a therapeutic protein or an inhibitory nucleic acid sequence. In certain embodiments, the GOI can be introduced / transferred by a vector, including rAAV transduction or transfection into cells. In certain embodiments, the introduced GOI can also exist extrachromosomally or only transiently in the recipient cell or host organism.

[0128] In certain embodiments, the GOI encodes a protein (e.g., a therapeutic protein) that is provided in a modified or truncated form, or the GOI is provided in multiple constructs delivered by separate multiple AAV vectors.

[0129] In certain embodiments, the GOI is provided as a truncated variant that maintains the functionality of the encoded protein (e.g., a therapeutic protein), including the removal of portions unnecessary for function, such that the GOI is of a reduced size for packaging into an AAV vector.

[0130] In certain embodiments, the GOI is provided in a split AAV vector, each providing nucleic acid encoding a different portion of a protein (e.g., a therapeutic protein), thus delivering multiple portions of a protein (e.g., a therapeutic protein) that assemble and function in a mammalian rAAV production cell of the present disclosure.

[0131] In certain embodiments, the GOI is provided by a dual AAV vector using overlapping, trans-splicing, or hybrid trans-splicing dual vector techniques. In certain embodiments, two overlapping AAV vectors are used that combine in a mammalian rAAV production cell of the present disclosure to generate a complete expression cassette from which a full-length protein (e.g., a therapeutic protein) is expressed.

[0132] Non-limiting examples of heterologous nucleic acids encoding gene products (e.g., therapeutic proteins) useful according to the present invention include those that may be used to treat diseases or disorders "hemostasis" or blood clotting disorders, including hemophilia A, hemophilia A patients with inhibitory antibodies, hemophilia B, clotting factor deficiencies VII, VIII, IX and X, XI, V, XII, II, von Willebrand factor, combined FV / FVIII deficiency, thalassemia, vitamin K epoxide reductase CI deficiency, and gamma-carboxylase deficiency. These may be used to treat diseases or disorders including, but not limited to, anemia, bleeding associated with trauma, injury, thrombosis, thrombocytopenia, stroke, coagulopathy, disseminated intravascular coagulation (DIC); hyperanticoagulation associated with heparin, low molecular weight heparin, pentasaccharides, warfarin, and small molecule antithrombotic agents (i.e., FXa inhibitors); and platelet disorders, such as Bernard-Soulier syndrome (BSS), Glanzmann thromboblastemia (GT), and storage pool deficiency.

[0133] In certain embodiments, the individual has a disease or disorder that affects or originates in the central nervous system (CNS). In certain embodiments, the disease is a neurological disease. In certain embodiments, the CNS or neurodegenerative disease is Alzheimer's disease (AD), Huntington's disease (HD), amyotrophic lateral sclerosis (ALS), hereditary spastic hemiplegia, primary lateral sclerosis (PLS), spinal muscular atrophy (SMA), Parkinson's disease (spinal and bulbar muscular atrophy or SBMA), a polyglutamine repeat disease, or Parkinson's disease. In certain embodiments, the CNS or neurodegenerative disease is a polyglutamine repeat disease. In certain embodiments, the polyglutamine repeat disease is spinocerebellar ataxia (SCA1, SCA2, SCA3, SCA6, SCA7, or SCA17).

[0134] In certain embodiments, the rAAV particles present in the immediately disclosed formulations are selected from the group consisting of insulin, glucagon, growth hormone (GH), parathyroid hormone (PTH), growth hormone-releasing factor (GRF), follicle-stimulating hormone (FSH), luteinizing hormone (LH), human chorionic gonadotropin (hCG), vascular endothelial growth factor (VEGF), angiopoietin, angiostatin, granulocyte colony-stimulating factor (GCSF), erythropoietin (EPO), connective tissue growth factor (CTGF), basic fibroblast growth factor (bFGF), acidic fibroblast growth factor (aFGF), epidermal growth factor (EGF), transforming growth factor α (TGF), and erythropoietin (EPO). a), platelet-derived growth factor (PDGF), insulin growth factor I and II (IGF-I and IGF-II), TGFp, activin, inhibin, bone morphogenetic protein (BMP), nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophins NT-3 and NT4 / 5, ciliary neurotrophic factor (CNTF), glial cell line-derived neurotrophic factor (GDNF), neuroturin, agrin, netrin-1 and netrin-2, hepatocyte growth factor (HGF), ephrin, noggin, sonic hedgehog, and tyrosine hydroxylase.

[0135] In certain embodiments, the rAAV particles present in the formulations of the instant disclosure comprise a heterologous nucleic acid encoding a gene product selected from the group consisting of thrombopoietin (TPO), interleukins (IL1 through IL-17), monocyte chemotactic protein, leukemia inhibitory factor, granulocyte-macrophage colony-stimulating factor, Fas ligand, tumor necrosis factors alpha and beta, interferons alpha, beta and gamma, stem cell factor, flk-2 / flt3 ligand, IgG, IgM, IgA, IgD and IgE, chimeric immunoglobulins, humanized antibodies, single-chain antibodies, T cell receptors, chimeric T cell receptors, single-chain T cell receptors, class I and class II MHC molecules.

[0136] In certain embodiments, the rAAV particles present in the immediately disclosed formulations are selected from the group consisting of carbamoyl synthetase I, ornithine transcarbamylase, argininosuccinate synthetase, argininosuccinate lyase, arginase, fumaryl acetate hydrolase, phenylalanine hydroxylase, alpha-1 antitrypsin, glucose-6-phosphatase, porphobilinogen deaminase, factor V, factor VIII, factor IX, cystathionate β-synthase, branched-chain keto acid decarboxylase, arginase, arginase, arginine ... The heterologous nucleic acid encoding a gene product selected from the group consisting of: bumin, isovaleryl-CoA dehydrogenase, propionyl-CoA carboxylase, methylmalonyl-CoA mutase, glutaryl-CoA dehydrogenase, insulin, β-glucosidase, pyruvate carboxylate, hepatic phosphorylase, phosphorylase kinase, glycine decarboxylase (GLDC), RPE65, H-protein, cystic fibrosis transmembrane conductance regulator (CFTR) sequence, and dystrophin cDNA sequence.

[0137] In certain embodiments, the rAAV particles present in the formulations of the present disclosure comprise a heterologous nucleic acid encoding a polypeptide, a nucleic acid encoding a protein or transcribed into a transcript of interest, or a nucleic acid selected from the group consisting of an siRNA, an antisense molecule, an miRNA, a ribozyme, and an shRNA.

[0138] In certain embodiments, the rAAV particles present in the formulations of the instant disclosure are used to treat Pompe disease for GAA (acid alpha-glucosidase), Wilson disease for ATP7B (copper-transporting ATPase 2), Fabry disease for α-galactosidase, citrullinemia type 1 for ASS1 (argininosuccinate synthase), Gaucher disease type 1 for β-glucocerebrosidase, Tay-Sachs disease for β-hexosaminidase, hereditary angioedema (HAE) (also known as C1 inhibitor deficiency type I and type II), and glycogen-6-phosphatase for glycogen storage disease type I (GSDI).

[0139] In certain embodiments, the heterologous nucleic acid present in the rAAV present in the immediately disclosed formulation is selected from the group consisting of CFTR (cystic fibrosis transmembrane conductance regulator), blood clotting (clotting) factors (such as factor XIII, factor IX, factor VIII, factor X, factor VII, factor VIIa, protein C, etc.), gain-of-function blood clotting factors, antibodies, retinal pigment epithelium-specific 65 kDa protein (RPE65), erythropoietin, LDL (low density lipoprotein) receptor, lipoprotein lipase (LPL), ornithine transcarbamylase (OTC), β Globin, α-globin, spectrin, α-antitrypsin, adenosine deaminase (ADA), metal transporters (ATP7A or ATP7), sulfamidase, enzymes involved in lysosomal storage diseases (ARSA), hypoxanthine guanine phosphoribosyltransferase, β-25 glucocerebrosidase, sphingomyelinase, lysosomal hexosaminidase, branched-chain ketoacid dehydrogenase, hormones, growth factors, insulin-like growth factor 1 or 2, platelet-derived growth factor (PDGF), epithelial growth factor (EGF), Growth factor (EGF), nerve growth factor (NGF), neurotrophic factors-3 and -4, brain-derived neurotrophic factor (BDNF), glial cell line-derived growth factor (GDNF), transforming growth factors α and β, cytokines, α-interferon, β-interferon, interferon-γ, interleukin-2 (IL-2), interleukin-4 (IL-4), interleukin-12 (IL-12), granulocyte-macrophage colony-stimulating factor (GM-CSF), lymphotoxin (LT), suicide gene product, herpes simplex Antiviral thymidine kinase, cytosine deaminase (CD), diphtheria toxin (DT), cytochrome P450 (CYP), deoxycytidine kinase (DCK), tumor necrosis factor (TNF), drug resistance proteins, tumor suppressor proteins (e.g., p53, Rb, Wt-1, NF1, von Hippel-Lindau (VHL), adenomatous polyposis coli (APC)), peptides with immunomodulatory properties, tolerogenic or immunogenic peptides or proteins Tregitope or hCDR1 (edorazide), insulin, glucokinase (GCK),Guanylate cyclase 2D (GUCY2D) (or Leber congenital amaurosis (LCA) associated with GUCY2D variants (GUCY2D-LCA)), Rab escort protein 1 (REP1) (choroideremia (CHM) encodes REP1), LCA5 (Leber congenital amaurosis 5) (LCA-lebercillin), ornithine ketoacid aminotransferase (gyrate atrophy), retinoschisin 1 (X-linked retinoschisis), USH1C (Usher syndrome 1C), X-linked retinitis pigmentosa GTPase (XLRP), MERTK (AR form of RP: retinitis pigmentosa), DFNB1 (connexin 26 deafness), ACHM 2, 3, and 4 (achromatopsia), PKD-1+ or PKD-2 (polycystic kidney disease), TPP1 (tripeptidyl peptidase 1), CLN2 (neuronal ceroid lipofuscinosis 2), sulfatase, N-acetylglucosamine-1-phosphate transferase, cathepsin A, GM2-AP (ganglioside GM2 activator), NPC1 (NPC intracellular cholesterol transporter 1), VPC2, sphingolipid activating protein, one or more zinc finger nucleases for genome editing, one or more donor sequences used as repair templates for genome editing, and variants thereof.

[0140] Nucleic acid molecules, cloning vectors, expression vectors (e.g., vector genomes), and plasmids can be prepared using recombinant DNA technology methods. The availability of nucleotide sequence information allows for the preparation of nucleic acid molecules by a variety of means. For example, heterologous nucleic acid encoding factor IX (FIX) containing vectors or plasmids can be produced using a variety of standard cloning and recombinant DNA techniques, via cellular expression or in vitro translation and chemical synthesis techniques. The purity of polynucleotides can be determined by sequencing, gel electrophoresis, and the like. For example, nucleic acids can be isolated using hybridization or computer-based database screening techniques. Such techniques include, but are not limited to, (1) hybridization of genomic DNA or cDNA libraries with probes to detect homologous nucleotide sequences; (2) antibody screening to detect polypeptides sharing structural features, e.g., using expression libraries; (3) polymerase chain reaction (PCR) on genomic DNA or cDNA using primers capable of annealing to the nucleic acid sequence of interest; (4) computer searches of sequence databases for related sequences; and (5) differential screening of subtraction nucleic acid libraries. IV. Sequence TIFF2026502876000003.tif123153 TIFF2026502876000004.tif101153 TIFF2026502876000005.tif96153

[0141] Embodiment The present application includes, but is not limited to, the following numbered embodiments.

[0142] Embodiment 1. A stable formulation comprising, together with a therapeutic pharmaceutical agent: (a) 1 to 100 mM of one or more salts; (b) a buffer; (c) 0.001 to 0.05 wt. % of one or more nonionic surfactants; (d) 1 to 10 wt. % of one or more sugars; (e) water; (f) optionally, one or more amino acids; and The pH of the composition is 4.5 to 7.5 (e.g., any pH of 4.5, 5, 5.5, 6, 6.5, 7, 7.5, or any percentage therebetween), and after freezing and thawing the formulation, 60% or more (e.g., 60%, 70%, 80%, 90%, 100%, or any percentage therebetween) of the drug product is recovered; Stable formulation.

[0143] Embodiment 2. The stable formulation of embodiment 1, wherein the therapeutic pharmaceutical agent is a recombinant adeno-associated virus (AAV or rAAV).

[0144] Embodiment 3. The stable formulation of any one of embodiments 1-2, wherein the buffer comprises a non-phosphate buffer.

[0145] Embodiment 4. The stable formulation of embodiment 3, wherein the non-phosphate buffering agent is sodium acetate.

[0146] Embodiment 5. The stable formulation of any one of embodiments 3-4, wherein the non-phosphate buffering agent is present in the formulation at a concentration of 1 mM to about 50 mM.

[0147] Embodiment 6. The stable formulation of any one of embodiments 1-5, wherein the one or more salts comprise 10-100 mM sodium chloride.

[0148] Embodiment 7. The stable formulation of embodiment 6, wherein the one or more salts comprise a first salt and a second salt.

[0149] Embodiment 8 The stable formulation of embodiment 7, wherein the first salt is sodium chloride and the second salt is magnesium chloride.

[0150] Embodiment 9. The stable formulation of embodiment 6, wherein sodium chloride is present in the formulation at a concentration of about 25 mM.

[0151] Embodiment 10. The stable formulation of embodiment 8, wherein magnesium chloride is present in the formulation at a concentration of about 1 mM to about 10 mM.

[0152] Embodiment 11 The stable formulation of embodiment 10, wherein the magnesium chloride is present at a concentration of about 5 mM.

[0153] Embodiment 12. The stable formulation of any one of embodiments 1 to 11, wherein the sugar is sucrose.

[0154] Embodiment 13. The stable formulation of embodiment 12, wherein the sucrose content of the formulation is 5.4%.

[0155] Embodiment 14. The stable formulation of any one of embodiments 1 to 13, wherein the non-ionic surfactant is present in the formulation at a concentration of about 0.01%.

[0156] Embodiment 15. The stable formulation of embodiment 14, wherein the surfactant is poloxamer 188, polysorbate 20, polysorbate 80, or poloxamer 407.

[0157] Embodiment 16. The stable formulation of embodiment 15, wherein the surfactant is a polysorbate selected from the group consisting of polysorbate 20 or polysorbate 80.

[0158] Embodiment 17. The stable formulation of embodiment 1, wherein the pH is about 5.2.

[0159] Embodiment 18. The stable formulation of any one of embodiments 1-2, wherein the buffer is a phosphate buffer.

[0160] Embodiment 19. The stable formulation of embodiment 18, wherein the phosphate buffer is sodium phosphate.

[0161] Embodiment 20. The stable formulation of embodiment 19, wherein the sodium phosphate is present at about 10 mM.

[0162] Embodiment 21. The stable formulation of any one of embodiments 1 to 20, wherein the formulation is lyophilized.

[0163] Embodiment 22. The stable formulation of any one of embodiments 1 to 20, wherein the formulation is a liquid.

[0164] Embodiment 23. The therapeutic pharmaceutical agent is selected from the group consisting of GAA (acid alpha-glucosidase), ATP7B (copper-transporting ATPase 2), alpha-galactosidase A (GLA), ASS1 (argininosuccinate synthase), beta-glucocerebrosidase, beta-hexosaminidase A, SERPING1 (C1 protease inhibitor or C1 esterase inhibitor), glucose-6-phosphatase, CFTR (cystic fibrosis transmembrane conductance regulator), blood clotting (clotting) factors (e.g., factor XIII, factor IX, factor VIII, factor X, factor VII, factor VIIa, protein C), gain-of-function blood coagulation factors, antibodies, retinal pigment epithelium-specific 65kDa protein (RPE65), erythropoietin, LDL (low-density lipoprotein) receptor, lipoprotein lipase (LPL), ornithine transcarbamylase (OTC), β-globin, α-globin, spectrin, α-antitrypsin, adenosine deaminase (ADA), metal transporters (ATP7A or ATP7), sulfamidase, enzymes involved in lysosomal storage diseases (ARSA), hypoxanthine guanine phosphoribosyltransferase, β-25 glycosaminoglycans, Brosidase, sphingomyelinase, lysosomal hexosaminidase, branched-chain ketoacid dehydrogenase, hormones, growth factors, insulin-like growth factor 1 or 2, platelet-derived growth factor (PDGF), epidermal growth factor (EGF), nerve growth factor (NGF), neurotrophic factors-3 and -4, brain-derived neurotrophic factor (BDNF), glial cell line-derived growth factor (GDNF), transforming growth factors α and β, cytokines, α-interferon, β-interferon, interferon-γ, interleukin-2 (IL-2), interleukins -4 (IL-4), interleukin 12 (IL-12), granulocyte-macrophage colony-stimulating factor (GM-CSF), lymphotoxin (LT), suicide gene products, herpes simplex virus thymidine kinase, cytosine deaminase (CD), diphtheria toxin (DT), cytochrome P450 (CYP), deoxycytidine kinase (DCK), tumor necrosis factor (TNF), drug resistance proteins, tumor suppressor proteins (e.g., p53, Rb, Wt-1, NF1, von Hippel-Lindau (VHL), adenomatous polyposis coli (APC)), peptides with immunomodulatory properties,Tolerogenic or immunogenic peptide or protein tregitopes or hCDR1 (edorazides), insulin, glucokinase (GCK), guanylate cyclase 2D (GUCY2D) (or Leber congenital amaurosis (LCA) associated with GUCY2D variants (GUCY2D-LCA)), Rab escort protein 1 (REP1) (choroideremia (CHM) encodes REP1), LCA5 (Leber congenital amaurosis 5) (LCA-lebercillin), ornithine ketoacid aminotransferase (gyrate atrophy), retinoschisin 1 (X-linked retinoschisis), USH1C (Usher syndrome 1C), X-linked retinitis pigmentosa GTPase (XLRP), MERTK (AR form of RP: retinitis pigmentosa), DFNB1 (connexin The stable formulation of any one of embodiments 1 to 22, wherein the stable formulation is capable of increasing intracellular protein levels of one or more of the following: 1) IL-26 deafness), ACHM2, 3 and 4 (achromatopsia), PKD-1+ or PKD-2 (polycystic kidney disease), TPP1 (tripeptidyl peptidase 1), CLN2 (neuronal ceroid lipofuscinosis 2), sulfatase, N-acetylglucosamine-1-phosphate transferase, cathepsin A, GM2-AP (ganglioside GM2 activator), NPC1 (NPC intracellular cholesterol transporter 1), VPC2, sphingolipid activating protein, one or more zinc finger nucleases for genome editing, one or more donor sequences used as repair templates for genome editing, or variants thereof.

[0165] Embodiment 24. A stable formulation comprising: (a) recombinant adeno-associated virus (rAAV); (b) 25 mM sodium chloride; (c) 5 mM magnesium chloride; (d) 25 mM sodium acetate; (e) 0.01% by weight of poloxamer 188; (f) 5.4 wt. % sucrose; (g) water; and (h) pH is 5 to 5.5, for example, pH 5, 5.1, 5.2, 5.3, 5.4, 5.5, or any pH therebetween; Stable formulation.

[0166] Embodiment 25. A stable formulation comprising: (a) recombinant adeno-associated virus (rAAV); (b) 10 mM Tris; (c) 75 mM sodium chloride; (d) 0.005% by weight of poloxamer 188; (e) 5% by weight of sucrose; (f) water; and (g) A stable formulation having a pH of 7.3.

[0167] Embodiment 26. A stable formulation comprising: (a) recombinant adeno-associated virus (rAAV); (b) 10 mM sodium citrate; (c) 75 mM sodium chloride; (d) 0.001 wt% poloxamer 188; (e) 7 wt% sucrose; (f) water; and (g) pH 6.0; Stable formulation.

[0168] Embodiment 27. The stable formulation of embodiments 24-26, wherein the rAAV comprises a capsid derived from one or more AAVs selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV9.47, AAV9(hu14), AAV10, AAV11, AAV12, Rh8, Rh10, Rh74, AAV3B, AAV-2i8, RHM4-1 (VP1, SEQ ID NO:1 or VP2, SEQ ID NO:2 or VP3, SEQ ID NO:3), DJ, DJ8, NP59, Anc-80, and variants thereof.

[0169] Embodiment 28. The stable formulation of embodiments 24-27, wherein the rAAV comprises a transgene encoding a polypeptide or a nucleic acid selected from the group consisting of siRNA, antisense molecules, miRNA, ribozymes and shRNA.

[0170] Embodiment 29. The rAAV is selected from the group consisting of GAA (acid alpha-glucosidase), ATP7B (copper-transporting ATPase 2), alpha-galactosidase A (GLA), ASS1 (argininosuccinate synthase), beta-glucocerebrosidase, beta-hexosaminidase A, SERPING1 (C1 protease inhibitor or C1 esterase inhibitor), glucose-6-phosphatase, CFTR (cystic fibrosis transmembrane conductance regulator), blood clotting (clotting) factors (e.g., factor XIII, factor IX, factor VIII, factor X, factor VII, factor VIIa, protein C), and the like. ), gain-of-function blood coagulation factors, antibodies, retinal pigment epithelium-specific 65kDa protein (RPE65), erythropoietin, LDL (low-density lipoprotein) receptor, lipoprotein lipase (LPL), ornithine transcarbamylase (OTC), β-globin, α-globin, spectrin, α-antitrypsin, adenosine deaminase (ADA), metal transporters (ATP7A or ATP7), sulfamidase, enzymes involved in lysosomal storage diseases (ARSA), hypoxanthine guanine phosphoribosyltransferase, β-25 glucose Levosidase, sphingomyelinase, lysosomal hexosaminidase, branched-chain ketoacid dehydrogenase, hormones, growth factors, insulin-like growth factor 1 or 2, platelet-derived growth factor (PDGF), epidermal growth factor (EGF), nerve growth factor (NGF), neurotrophic factors-3 and -4, brain-derived neurotrophic factor (BDNF), glial cell line-derived growth factor (GDNF), transforming growth factors α and β, cytokines, α-interferon, β-interferon, interferon-γ, interleukin-2 (IL-2), interleukins -4 (IL-4), interleukin 12 (IL-12), granulocyte-macrophage colony-stimulating factor (GM-CSF), lymphotoxin (LT), suicide gene products, herpes simplex virus thymidine kinase, cytosine deaminase (CD), diphtheria toxin (DT), cytochrome P450 (CYP), deoxycytidine kinase (DCK), tumor necrosis factor (TNF), drug resistance proteins, tumor suppressor proteins (e.g., p53, Rb, Wt-1, NF1, von Hippel-Lindau (VHL), adenomatous polyposis coli (APC)), peptides with immunomodulatory properties,Tolerogenic or immunogenic peptide or protein tregitope or hCDR1 (edorazide), insulin, glucokinase (GCK), guanylate cyclase 2D (GUCY2D) (or Leber congenital amaurosis (LCA) associated with GUCY2D variants (GUCY2D-LCA)), Rab escort protein 1 (REP1) (choroideremia (CHM) encodes REP1), LCA5 (Leber congenital amaurosis 5) (LCA-lebercillin), ornithine ketoacid aminotransferase (gyrate atrophy), retinoschisin 1 (X-linked retinoschisis), USH1C (Usher syndrome 1C), X-linked retinitis pigmentosa GTPase (XLRP), MERTK (AR form of RP: retinitis pigmentosa), 29. The stable formulation of embodiment 28, comprising a transgene encoding DFNB1 (connexin 26 deafness), ACHM2, 3 and 4 (achromatosis), PKD-1+ or PKD-2 (polycystic kidney disease), TPP1 (tripeptidyl peptidase 1), CLN2 (neuronal ceroid lipofuscinosis 2), sulfatase, N-acetylglucosamine-1-phosphate transferase, cathepsin A, GM2-AP (ganglioside GM2 activator), NPC1 (NPC intracellular cholesterol transporter 1), VPC2, sphingolipid activating protein, one or more zinc finger nucleases for genome editing, one or more donor sequences used as repair templates for genome editing, or variants thereof.

[0171] Embodiment 30. The stable formulation of embodiment 29, wherein the rAAV comprises a transgene encoding GAA (acid alpha-glucosidase).

[0172] Embodiment 31 The stable formulation of embodiment 29, wherein the rAAV comprises a transgene encoding ATP7B (copper-transporting ATPase 2).

[0173] Embodiment 32. The stable formulation of embodiment 29, wherein the rAAV comprises a transgene encoding alpha-galactosidase A (GLA).

[0174] Embodiment 33. The stable formulation of embodiment 29, wherein the rAAV comprises a transgene encoding ASS1 (argininosuccinate synthase).

[0175] Embodiment 34 The stable formulation of embodiment 29, wherein the rAAV comprises a transgene encoding β-glucocerebrosidase.

[0176] Embodiment 35 The stable formulation of embodiment 29, wherein the rAAV comprises a transgene encoding β-hexosaminidase A.

[0177] Embodiment 36. The stable formulation of embodiment 29, wherein the AAV comprises a transgene encoding SERPING1 (C1 protease inhibitor or C1 esterase inhibitor).

[0178] Embodiment 37. The stable formulation of embodiment 29, wherein the AAV comprises a transgene encoding glucose-6-phosphatase.

[0179] Embodiment 38. The stable formulation of embodiment 29, wherein the AAV comprises a transgene encoding CFTR (cystic fibrosis transmembrane conductance regulator).

[0180] Embodiment 39. The stable formulation of embodiment 29, wherein the AAV comprises a transgene encoding a blood clotting (clotting) factor (e.g., Factor XIII, Factor IX, Factor VIII, Factor X, Factor VII, Factor VIIa, Protein C).

[0181] Embodiment 40. The stable formulation of any of embodiments 1-39, wherein after the composition is frozen at temperatures above -80°C and down to -10°C for a period of time, 60% or more of the therapeutic agent or rAAV is recovered after thawing compared to the same product stored at -80°C for the same period of time.

[0182] Embodiment 41. The stable formulation of any of embodiments 1 to 39, wherein after the composition has been frozen for a period of time at a temperature greater than -80°C and up to -10°C, e.g., at a temperature of -70°C to -15°C, -60°C to -20°C, -50°C to -30°C, or -45°C to -35°C, the pharmaceutical agent or rAAV exhibits at least 50% relative potency compared to the same product stored at -80°C for the same period of time.

[0183] Embodiment 42. The stable formulation of any of embodiments 1 to 39, wherein the pharmaceutical agent or rAAV exhibits a glass transition temperature of the frozen concentrate of -45°C to -35°C, such as -45°C, -44°C, -43°C, -42°C, -41°C, -40°C, -39°C, -38°C, -37°C, -36°C, or -35°C, after the composition has been frozen at a temperature greater than -80°C and up to -10°C for a period of time.

[0184] Embodiment 43. The stable formulation of any one of embodiments 1 to 39, wherein the formulation is lyophilized.

[0185] Embodiment 44. The stable formulation of any one of embodiments 1 to 42, wherein the formulation is a liquid.

[0186] Embodiment 45. A lyophilized composition, the composition being substantially dehydrated, which can be reconstituted with a diluent to form the formulation of any one of Embodiments 1 to 44.

[0187] Embodiment 46. The lyophilized composition of embodiment 45, wherein reconstituting comprises mixing the lyophilized composition with water for injection (WFI).

[0188] Embodiment 47. The stable formulation of any one of embodiments 1 to 43 or the lyophilized composition of any one of embodiments 44 to 46, wherein the formulation or composition is present in a unit-dose container.

[0189] Embodiment 48. The stable formulation or lyophilized composition of embodiment 47, wherein the unit-dose container is a vial.

[0190] Embodiment 49. The stable formulation or lyophilized composition of embodiment 48, wherein the vial is a sealed glass vial.

[0191] Embodiment 50. A method for reducing degradation of a therapeutic pharmaceutical agent after freeze-thawing, the method comprising: (a) a therapeutic drug; (b) 1 to 100 mM of one or more salts; (c) a buffer; (d) 0.001 to 0.05 wt. % of one or more nonionic surfactants; (e) 3 to 10 wt. % of one or more sugars; (f) water, (g) pH 4.5 to 7, and (h) optionally preparing a composition comprising one or more amino acids; freezing the composition for a period of time at a temperature above -80°C to -10°C, e.g., -70°C to -15°C, -60°C to -20°C, -50°C to -30°C, or -45°C to -35°C, wherein upon thawing, 60% or more of the therapeutic agent is recovered compared to the same product stored at -80°C for the same period of time; A method comprising:

[0192] Embodiment 51. A method for reducing degradation of a therapeutic pharmaceutical product after freeze-thawing the product, comprising: (a) a therapeutic product; and (b) 25 mM sodium chloride; (c) 5 mM magnesium chloride; (d) 25 mM sodium acetate; (e) 0.01% by weight of poloxamer 188; (f) 5.4 wt. % sucrose; (g) water, (h) preparing a composition having a pH of 5 to 5.5; freezing the composition for a period of time at a temperature greater than -80°C and up to -10°C, e.g., -70°C to -15°C, -60°C to -20°C, -50°C to -30°C, or -45°C to -35°C, wherein upon thawing, 60% or more of the therapeutic agent is recovered compared to the same product stored at -80°C for the same period of time; A method comprising:

[0193] Embodiment 52. A method for preserving at least 50% of the relative potency of a therapeutic pharmaceutical product after freeze-thawing, the method comprising: (a) a therapeutic product; and (b) 25 mM sodium chloride; (c) 5 mM magnesium chloride; (d) 25 mM sodium acetate; (e) 0.01% by weight of poloxamer 188; (f) 5.4 wt. % sucrose; (g) water, (h) preparing a composition having a pH of 5 to 5.5; freezing the composition for a period of time at a temperature greater than -60°C to -10°C, e.g., -60°C to -10°C, -50°C to -20°C, -45°C to -25°C, or -30°C to -40°C, wherein upon thawing, the pharmaceutical product exhibits at least 50% relative potency compared to the same product stored at -80°C for the same period of time; A method comprising:

[0194] Embodiment 53. A method for reducing degradation of a therapeutic pharmaceutical agent, the method comprising: (a) a therapeutic product; and (b) 10 mM Tris; (c) 75 mM sodium chloride; (d) 0.005% by weight of poloxamer 188; (e) 5% by weight of sucrose; (f) water, (g) preparing a composition having a pH of 7.3; lyophilizing the composition and storing it for a period of time at a temperature between -20°C and 8°C, e.g., -20°C to 0°C or 0°C to 8°C, wherein 60% or more of the therapeutic agent is recovered after reconstitution of the lyophilized composition compared to the same product stored at -80°C without lyophilization; A method comprising:

[0195] Embodiment 54. A method for reducing degradation of a therapeutic pharmaceutical agent, the method comprising: (a) a therapeutic product; and (b) 10 mM sodium citrate; (c) 75 mM sodium chloride; (d) 0.001 wt% poloxamer 188; (e) 7 wt% sucrose; (f) water, (g) preparing a composition having a pH of 6.0; lyophilizing and storing the composition at a temperature between -20°C and 8°C, e.g., between -20°C and 0°C or between 0°C and 8°C, for a period of time, wherein 60% or more of the therapeutic agent is recovered after reconstitution of the lyophilized composition compared to the same product stored at -80°C without lyophilization; A method comprising:

[0196] Embodiment 55. A method of maintaining at least 50% relative efficacy of a therapeutic pharmaceutical agent, the method comprising: (a) a therapeutic product; and (b) 10 mM Tris; (c) 75 mM sodium chloride; (d) 0.005% by weight of poloxamer 188; (e) 5% by weight of sucrose; (f) water, (g) preparing a composition having a pH of 7.3; freeze-drying the composition and storing it for a period of time at a temperature between -20°C and 8°C, e.g., at a temperature between -20°C and 0°C or 0°C and 8°C, wherein the pharmaceutical product exhibits at least 50% relative potency after reconstitution of the freeze-dried composition compared to the same pharmaceutical product stored at -80°C without being freeze-dried; A method comprising:

[0197] Embodiment 56. A method of maintaining at least 50% relative efficacy of a therapeutic pharmaceutical agent, the method comprising: (a) a therapeutic product; and (b) 10 mM sodium citrate; (c) 75 mM sodium chloride; (d) 0.001 wt% poloxamer 188; (e) 7 wt% sucrose; (f) water, (g) preparing a composition having a pH of 6.0; freeze-drying and storing the composition at a temperature between -20°C and 8°C, e.g., at a temperature between -20°C and 0°C or 0°C and 8°C, for a period of time, wherein the pharmaceutical product exhibits at least 50% relative potency after reconstitution of the lyophilized composition compared to the same formulation stored at -80°C without being freeze-dried; A method comprising:

[0198] Embodiment 57. The method of any one of embodiments 50 to 56, wherein the therapeutic agent is a recombinant adeno-associated virus (rAAV).

[0199] Embodiment 58. A method for reducing deamination of amino acids on the capsid of an rAAV, the method comprising: (a) rAAV, and (b) 25 mM sodium chloride; (c) 5 mM magnesium chloride; (d) 25 mM sodium acetate; (e) 0.01% by weight of poloxamer 188; (f) 5.4 wt. % sucrose; (g) water; and (h) preparing a composition having a pH of 5 to 5.5; A method comprising:

[0200] Embodiment 59. A method for reducing viral protein degradation of an rAAV product after freezing and thawing the composition, comprising: (a) rAAV, and (b) 25 mM sodium chloride; (c) 5 mM magnesium chloride; (d) 25 mM sodium acetate; (e) 0.01% by weight of poloxamer 188; (f) 5.4 wt. % sucrose; (g) water; and (h) preparing a composition having a pH of 5 to 5.5; storing the frozen composition for a period of time at a temperature between -80°C and -10°C, e.g., -70°C to -15°C, -60°C to -20°C, -50°C to -30°C, or -45°C to -35°C, wherein the change in the ratio of VP1:VP3 in the thawed composition is less than 20% compared to before freezing the composition; A method comprising:

[0201] Embodiment 60. A method for reducing deamination of amino acids on the capsid of an rAAV, the method comprising: (a) rAAV, and (b) 10 mM Tris; (c) 75 mM sodium chloride; (d) 0.005% by weight of poloxamer 188; (e) 5% by weight of sucrose; (f) water; and (g) preparing a composition having a pH of 7.3; A method comprising:

[0202] Embodiment 61. A method for reducing deamination of amino acids on the capsid of rAAV, comprising: (a) rAAV, and (b) 10 mM sodium citrate; (c) 75 mM sodium chloride; (d) 0.001 wt% poloxamer 188; (e) 7 wt% sucrose; (f) water; and (g) preparing a composition having a pH of 6.0; A method comprising:

[0203] Embodiment 62. A method for reducing the concentration of subvisible particles (SVPs) in an rAAV product after the product has been frozen and thawed, the method comprising: (a) rAAV, and (b) 25 mM sodium chloride; (c) 5 mM magnesium chloride; (d) 25 mM sodium acetate; (e) 0.01% by weight of poloxamer 188; (f) 5.4 wt. % sucrose; (g) water, (h) preparing a composition having a pH of 5 to 5.5; freezing the composition at −20° C. for at least one year, wherein after thawing, the composition has an SVP concentration of less than 100 particles / mL for particles 10 μm or larger and less than 10 particles / mL for particles 25 μm or larger; A method comprising:

[0204] Embodiment 63. A method for reducing the concentration of subvisible particles (SVPs) in an rAAV product after the product has been frozen and thawed, the method comprising: (a) rAAV, and (b) 25 mM sodium chloride; (c) 5 mM magnesium chloride; (d) 25 mM sodium acetate; (e) 0.01% by weight of poloxamer 188; (f) 5.4 wt. % sucrose; (g) water, (h) preparing a composition having a pH of 5 to 5.5; freezing the composition at −20° C. for at least one year, wherein after thawing, the composition has an SVP concentration of less than 50 particles / mL for particles 10 μm or larger and less than 5 particles / mL for particles 25 μm or larger; A method comprising:

[0205] Embodiment 64. A method for reducing the subvisible particle (SVP) concentration of an rAAV product after said product has been freeze-thawed for five cycles, the method comprising: (a) rAAV, and (b) 25 mM sodium chloride; (c) 5 mM magnesium chloride; (d) 25 mM sodium acetate; (e) 0.01% by weight of poloxamer 188; (f) 5.4 wt. % sucrose; (g) water, (h) preparing a composition having a pH of 5 to 5.5; freezing the composition for a period of time at a temperature greater than -80°C to -10°C, e.g., -70°C to -15°C, -60°C to -20°C, -50°C to -30°C, or -45°C to -35°C, wherein the composition is thawed and refrozen five times, and upon the fifth thaw, the SVP concentration is less than 100 particles / mL for particles 10 μm or larger and less than 10 particles / mL for particles 25 μm or larger; A method comprising:

[0206] Embodiment 65. A method for reducing the subvisible particle (SVP) concentration of an rAAV product after said product has been freeze-thawed for five cycles, the method comprising: (a) rAAV, and (b) 25 mM sodium chloride; (c) 5 mM magnesium chloride; (d) 25 mM sodium acetate; (e) 0.01% by weight of poloxamer 188; (f) 5.4 wt. % sucrose; (g) water, (h) preparing a composition having a pH of 5 to 5.5; freezing the composition for a period of time at a temperature greater than -80°C to -10°C, e.g., -70°C to -15°C, -60°C to -20°C, -50°C to -30°C, or -45°C to -35°C, wherein the composition is thawed and refrozen five times, and upon the fifth thaw, the SVP concentration is less than 50 particles / mL for particles 10 μm or larger and less than 5 particles / mL for particles 25 μm or larger; A method comprising:

[0207] Embodiment 66. A method for reducing high molecular weight species (HMW) in an rAAV product after five freeze-thaw cycles of said product, comprising: (a) rAAV, and (b) 25 mM sodium chloride; (c) 5 mM magnesium chloride; (d) 25 mM sodium acetate; (e) 0.01% by weight of poloxamer 188; (f) 5.4 wt. % sucrose; (g) water, (h) preparing a composition having a pH of 5 to 5.5; freezing the composition for a period of time at a temperature greater than -80°C to -10°C, e.g., -70°C to -15°C, -60°C to -20°C, -50°C to -30°C, or -45°C to -35°C, wherein the composition is thawed and refrozen five times, and upon the fifth thaw, the HMW species gain less than 3% by weight compared to the same product at TO as measured by analytical ultracentrifugation; A method comprising:

[0208] Embodiment 67. A method for reducing high molecular weight species (HMW) in an rAAV product after said product has been frozen and thawed, comprising: (a) rAAV, and (b) 25 mM sodium chloride; (c) 5 mM magnesium chloride; (d) 25 mM sodium acetate; (e) 0.01% by weight of poloxamer 188; (f) 5.4 wt. % sucrose; (g) water, (h) preparing a composition having a pH of 5 to 5.5; freezing the composition at -20°C for at least one year, wherein after thawing, HMW species have increased by less than 3% by weight compared to the same product at TO as measured by analytical ultracentrifugation; A method comprising:

[0209] Embodiment 68. A method for reducing viral protein degradation of an rAAV product after lyophilization of the product, comprising: (a) rAAV, and (b) 10 mM Tris; (c) 75 mM sodium chloride; (d) 0.005% by weight of poloxamer 188; (e) 5% by weight of sucrose; (f) water; and (g) preparing a composition having a pH of 7.3; storing the lyophilized composition for a period of time at a temperature between -20°C and 8°C, for example, at a temperature between -20°C and 0°C or 0°C and 8°C, wherein the reconstituted lyophilized product has a VP1:VP3 ratio that is less than 20% different from that before lyophilization; A method comprising:

[0210] Embodiment 69. A method for reducing viral protein degradation of an rAAV product after lyophilization of said product, comprising: (a) rAAV, and (b) 10 mM sodium citrate; (c) 75 mM sodium chloride; (d) 0.001 wt% poloxamer 188; (e) 7 wt% sucrose; (f) water, (g) preparing a composition having a pH of 6.0; storing the lyophilized composition for a period of time at a temperature between -20°C and 8°C, for example, at a temperature between -20°C and 0°C or 0°C and 8°C, wherein the reconstituted lyophilized product has a VP1:VP3 ratio that is less than 20% different from that before lyophilization; A method comprising:

[0211] Embodiment 70. A method for reducing the concentration of subvisible particles (SVPs) of an rAAV product, comprising: (a) rAAV, and (b) 10 mM Tris; (c) 75 mM sodium chloride; (d) 0.005% by weight of poloxamer 188; (e) 5% by weight of sucrose; (f) water; and (g) preparing a composition having a pH of 7.3; storing the lyophilized composition for a period of time at a temperature between -20°C and 8°C, for example at a temperature between -20°C and 0°C or 0°C and 8°C, wherein the formulation has been subjected to one lyophilization cycle and the lyophilized composition has an SVP of less than 100 particles / mL for particles of 10 μm or larger and less than 10 particles / mL for particles of 25 μm or larger; A method comprising:

[0212] Embodiment 71. A method for reducing the concentration of subvisible particles (SVPs) of an rAAV product, comprising: (a) rAAV, and (b) 10 mM Tris; (c) 75 mM sodium chloride; (d) 0.005% by weight of poloxamer 188; (e) 5% by weight of sucrose; (f) water; and (g) preparing a composition having a pH of 7.3; storing the lyophilized composition for a period of time at a temperature between -20°C and 8°C, for example at a temperature between -20°C and 0°C or 0°C and 8°C, wherein the formulation has been subjected to one lyophilization cycle and the lyophilized composition has an SVP of less than 50 particles / mL for particles of 10 μm or larger and less than 5 particles / mL for particles of 25 μm or larger; A method comprising:

[0213] Embodiment 72. A method for reducing the concentration of subvisible particles (SVPs) of an rAAV product, comprising: (a) rAAV, and (b) 10 mM sodium citrate; (c) 75 mM sodium chloride; (d) 0.001 wt% poloxamer 188; (e) 7 wt% sucrose; (f) water, (g) preparing a composition having a pH of 6.0; storing the lyophilized composition for a period of time at a temperature between -20°C and 8°C, for example at a temperature between -20°C and 0°C or 0°C and 8°C, wherein the formulation has been subjected to one lyophilization cycle and the lyophilized composition has an SVP of less than 100 particles / mL for particles of 10 μm or larger and less than 10 particles / mL for particles of 25 μm or larger; A method comprising:

[0214] Embodiment 73. A method for reducing the concentration of subvisible particles (SVPs) of an rAAV product, comprising: (a) rAAV, and (b) 10 mM sodium citrate; (c) 75 mM sodium chloride; (d) 0.001 wt% poloxamer 188; (e) 7 wt% sucrose; (f) water, (g) preparing a composition having a pH of 6.0; storing the lyophilized composition for a period of time at a temperature between -20°C and 8°C, for example at a temperature between -20°C and 0°C or 0°C and 8°C, wherein the formulation has been subjected to one lyophilization cycle and the lyophilized composition has an SVP of less than 50 particles / mL for particles of 10 μm or larger and less than 5 particles / mL for particles of 25 μm or larger; A method comprising:

[0215] Embodiment 74. A method for reducing low molecular weight species (LMW) in an rAAV product after lyophilization of the product, the method comprising: (a) rAAV, and (b) 10 mM sodium citrate; (c) 75 mM sodium chloride; (d) 0.001 wt% poloxamer 188; (e) 7 wt% sucrose; (f) water, (g) preparing a composition having a pH of 6.0; storing the lyophilized composition at a temperature between -20°C and 8°C, for example at a temperature between -20°C and 0°C or 0°C and 8°C, for a period of time, wherein the lyophilized product is reconstituted and the LMW species is less than 5% by weight compared to the same product without lyophilization, as measured by analytical ultracentrifugation; A method comprising:

[0216] Embodiment 75. A method for reducing low molecular weight species (LMW) in an rAAV product after lyophilization of said product, comprising: (a) rAAV, and (b) 10 mM Tris; (c) 75 mM sodium chloride; (d) 0.005% by weight of poloxamer 188; (e) 5% by weight of sucrose; (f) water; and (g) preparing a composition having a pH of 7.3; storing the lyophilized composition at a temperature between -20°C and 8°C, for example at a temperature between -20°C and 0°C or 0°C and 8°C, for a period of time, wherein the lyophilized product is reconstituted and the LMW species is less than 5% by weight compared to the same product without lyophilization, as measured by analytical ultracentrifugation; A method comprising:

[0217] Embodiment 76. A method for reducing high molecular weight species (HMW) in an rAAV product after said product has been lyophilized, comprising the steps of: (a) rAAV, and (b) 10 mM sodium citrate; (c) 75 mM sodium chloride; (d) 0.001 wt% poloxamer 188; (e) 7 wt% sucrose; (f) water, (g) preparing a composition having a pH of 6.0; storing the lyophilized composition for a period of time at a temperature between -20°C and 8°C, for example at a temperature between -20°C and 0°C or 0°C and 8°C, wherein the lyophilized product is reconstituted and the HMW species increase by less than 3% by weight compared to the same product without lyophilization, as measured by analytical ultracentrifugation; A method comprising:

[0218] Embodiment 77. A method for reducing high molecular weight (HMW) in an rAAV product after lyophilization of said product, comprising: (a) rAAV, and (b) 10 mM Tris; (c) 75 mM sodium chloride; (d) 0.005% by weight of poloxamer 188; (e) 5% by weight of sucrose; (f) water; and (g) preparing a composition having a pH of 7.3; storing the lyophilized composition at a temperature between -20°C and 8°C, for example at a temperature between -20°C and 0°C or 0°C and 8°C, for a period of time such that the lyophilized product is reconstituted and the HMW species increase by less than 3% by weight compared to the same product without lyophilization, as measured by analytical ultracentrifugation; A method comprising:

[0219] Embodiment 78. The method of embodiments 57 to 77, wherein the rAAV comprises a capsid derived from one or more AAVs selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV9.47, AAV9(hu14), AAV10, AAV11, AAV12, Rh8, Rh10, Rh74, AAV3B, AAV-2i8, RHM4-1 (VP1, SEQ ID NO: 1 or VP2, SEQ ID NO: 2 or VP3, SEQ ID NO: 3), DJ, DJ8, NP59, Anc-80, and variants thereof.

[0220] Embodiment 79. The method of any one of embodiments 57 to 78, wherein the rAAV comprises a transgene encoding a polypeptide or a nucleic acid selected from the group consisting of siRNA, antisense molecules, miRNA, ribozymes and shRNA.

[0221] Embodiment 80. The rAAV is selected from the group consisting of GAA (acid alpha-glucosidase), ATP7B (copper-transporting ATPase 2), alpha-galactosidase A (GLA), ASS1 (argininosuccinate synthase), beta-glucocerebrosidase, beta-hexosaminidase A, SERPING1 (C1 protease inhibitor or C1 esterase inhibitor), glucose-6-phosphatase, CFTR (cystic fibrosis transmembrane conductance regulator), blood clotting (clotting) factors (e.g., factor XIII, factor IX, factor VIII, factor X, factor VII, factor VIIa, protein C), and the like. ), gain-of-function blood coagulation factors, antibodies, retinal pigment epithelium-specific 65kDa protein (RPE65), erythropoietin, LDL (low-density lipoprotein) receptor, lipoprotein lipase (LPL), ornithine transcarbamylase (OTC), β-globin, α-globin, spectrin, α-antitrypsin, adenosine deaminase (ADA), metal transporters (ATP7A or ATP7), sulfamidase, enzymes involved in lysosomal storage diseases (ARSA), hypoxanthine guanine phosphoribosyltransferase, β-25 glucose Levosidase, sphingomyelinase, lysosomal hexosaminidase, branched-chain ketoacid dehydrogenase, hormones, growth factors, insulin-like growth factor 1 or 2, platelet-derived growth factor (PDGF), epidermal growth factor (EGF), nerve growth factor (NGF), neurotrophic factors-3 and -4, brain-derived neurotrophic factor (BDNF), glial cell line-derived growth factor (GDNF), transforming growth factors α and β, cytokines, α-interferon, β-interferon, interferon-γ, interleukin-2 (IL-2), interleukins -4 (IL-4), interleukin 12 (IL-12), granulocyte-macrophage colony-stimulating factor (GM-CSF), lymphotoxin (LT), suicide gene products, herpes simplex virus thymidine kinase, cytosine deaminase (CD), diphtheria toxin (DT), cytochrome P450 (CYP), deoxycytidine kinase (DCK), tumor necrosis factor (TNF), drug resistance proteins, tumor suppressor proteins (e.g., p53, Rb, Wt-1, NF1, von Hippel-Lindau (VHL), adenomatous polyposis coli (APC)), peptides with immunomodulatory properties,Tolerogenic or immunogenic peptide or protein tregitope or hCDR1 (edorazide), insulin, glucokinase (GCK), guanylate cyclase 2D (GUCY2D) (or Leber congenital amaurosis (LCA) associated with GUCY2D variant (GUCY2D-LCA)), Rab escort protein 1 (REP1) (choroideremia (CHM) encodes REP1), LCA5 (Leber congenital amaurosis 5) (LCA-lebercillin), ornithine ketoacid aminotransferase (gyrate atrophy), retinoschisin 1 (X-linked retinoschisis), USH1C (Usher syndrome 1C), X-linked retinitis pigmentosa GTPase (XLRP), MERTK (AR form of RP: retinitis pigmentosa) 80. The method of embodiment 79, comprising a transgene encoding one or more of the following: DFNB1 (connexin 26 deafness), ACHM2, 3 and 4 (achromatopsia), PKD-1+ or PKD-2 (polycystic kidney disease), TPP1 (tripeptidyl peptidase 1), CLN2 (neuronal ceroid lipofuscinosis 2), sulfatase, N-acetylglucosamine-1-phosphate transferase, cathepsin A, GM2-AP (ganglioside GM2 activator), NPC1 (NPC intracellular cholesterol transporter 1), VPC2, sphingolipid activating protein, one or more zinc finger nucleases for genome editing, one or more donor sequences used as repair templates for genome editing, or variants thereof.

[0222] Embodiment 81. The method of embodiment 80, wherein the rAAV comprises a transgene encoding GAA (acid alpha-glucosidase).

[0223] Embodiment 82. The method of embodiment 80, wherein the rAAV comprises a transgene encoding ATP7B (copper-transporting ATPase 2).

[0224] Embodiment 83 The method of embodiment 80, wherein the rAAV comprises a transgene encoding alpha-galactosidase A (GLA).

[0225] Embodiment 84 The method of embodiment 80, wherein the rAAV comprises a transgene encoding ASS1 (argininosuccinate synthase).

[0226] Embodiment 85 The method of embodiment 80, wherein the rAAV comprises a transgene encoding β-glucocerebrosidase.

[0227] Embodiment 86 The method of embodiment 80, wherein the rAAV comprises a transgene encoding β-hexosaminidase A.

[0228] Embodiment 87 The method of embodiment 80, wherein the rAAV comprises a transgene encoding SERPING1 (C1 protease inhibitor or C1 esterase inhibitor).

[0229] Embodiment 88 The method of embodiment 80, wherein the rAAV comprises a transgene encoding glucose-6-phosphatase.

[0230] Embodiment 89. The method of embodiment 80, wherein the rAAV comprises a transgene encoding CFTR (cystic fibrosis transmembrane conductance regulator).

[0231] Embodiment 90. The method of embodiment 80, wherein the rAAV comprises a transgene encoding a blood clotting (clotting) factor (e.g., Factor XIII, Factor IX, Factor VIII, Factor X, Factor VII, Factor VIIa, Protein C).

[0232] Embodiment 91. The formulation and method of any of embodiments 1 to 90, wherein the pharmaceutical agent is rAAV, and the rAAV comprises a capsid comprising a VP1 comprising the nucleotide sequence of SEQ ID NO: 1. In a further embodiment, the rAAV capsid further comprises a VP2 comprising the nucleotide sequence of SEQ ID NO: 2. In a preferred embodiment, the rAAV capsid comprises a VP1 comprising the nucleotide sequence of SEQ ID NO: 1, a VP2 comprising the nucleotide sequence of SEQ ID NO: 2, and a VP3 comprising the nucleotide sequence of SEQ ID NO: 3.

[0233] Additional Aspects and Embodiments Examples of additional aspects, embodiments, and combinations thereof include the following.

[0234] 1. A recombinant adeno-associated virus (rAAV) formulation comprising: a) Approximately 1 × 10 9 ~Approx. 1×10 15 vg / mL of rAAV and b) an ionic strength of about 40 mM to about 90 mM; c) a buffer; and d) 0.0005 to 0.05 wt. % of one or more nonionic surfactants; e) 1 to 10% by weight of one or more sugars; f) water, g) A component having a pH of about 5.0 to about 7.5.

[0235] 2. The rAAV formulation according to claim 1, wherein the formulation comprises the following components: a) 20 mM to 30 mM Na acetate, b) 10 mM to 50 mM NaCl; c) 1 mM MgCl2 to 9.5 mM MgCl2; d) 3% to 8% sucrose; e) 0.0005% to 0.05% Poloxamer 188 (P188), f) water, The formulation has a pH of 5.0 to 6.0.

[0236] 3. The rAAV formulation according to claim 2, wherein the formulation comprises the following components: a) about 25 mM Na acetate; b) about 25 mM NaCl; c) about 5 mM MgCl2; and d) about 5.4% sucrose; e) 0.01% P188; f) water, The formulation has a pH of about 5.2.

[0237] 4. The rAAV formulation of 1, wherein the formulation comprises the following components: a) 5mM to 20mM Tris; b) 50 mM to 90 mM NaCl; c) 3% to 8% by weight of sucrose; d) 0.001% to 0.01% (by weight) of poloxamer 188 (P188); e) water, The formulation has a pH of 7.1 to 7.5.

[0238] 5. The rAAV formulation of claim 4, wherein the formulation comprises the following components: a) about 10 mM Tris; b) about 75 mM NaCl; c) about 5% sucrose; d) about 0.005% P188; e) water, The formulation has a pH of about 7.3.

[0239] 6. The rAAV formulation of 1, wherein the formulation comprises the following components: a) 5mM to 20mM sodium citrate; b) 50 mM to 90 mM NaCl; c) 5% to 9% by weight of sucrose; d) 0.0005% to 0.01% (by weight) of poloxamer 188 (P188); e) water, The formulation has a pH of 5.5 to 6.5.

[0240] 7. The rAAV formulation of 6, wherein the formulation comprises the following components: a) about 10 mM Na citrate; b) about 75 mM NaCl; c) about 7% by weight sucrose; d) approximately 0.001% P188; e) water, The formulation has a pH of about 6.0.

[0241] 8. The rAAV formulation of any one of 1 to 7, wherein the formulation consists essentially of the components.

[0242] 9. The rAAV formulation of any one of 1 to 7, wherein the formulation consists of the components.

[0243] 10. The rAAV formulation of any one of 1 to 9, comprising a capsid comprising VP1 comprising the sequence of SEQ ID NO: 1, VP2 comprising the sequence of SEQ ID NO: 2, and VP3 comprising the sequence of SEQ ID NO: 3.

[0244] 11. rAAV is approximately 1 × 10 10 ~Approx. 1×10 14 11. The rAAV formulation of any one of 1 to 10, provided in vg / mL.

[0245] 12. rAAV is approximately 1 × 10 13 ~Approx. 4×10 13 12. The rAAV formulation described in 11, provided in vg / mL.

[0246] 13. The rAAV formulation of any one of 1 to 12, wherein the rAAV comprises a recombinant viral nucleic acid encoding GAA or GLA.

[0247] 14. A lyophilized recombinant adeno-associated virus (rAAV) formulation produced by lyophilizing the rAAV formulation of any one of 1 to 13. [Example]

[0248] The following examples are merely illustrative of the subject matter of the present disclosure and should not be construed as limiting in any way.

[0249] Example 1: Long-term stability of rAAV in low ionic strength formulations A. Material Sodium acetate trihydrate, sodium chloride, and magnesium chloride hexahydrate were purchased from Fisher Scientific (Waltham, MA). Sodium phosphate dibasic and monobasic salts were obtained from JT Baker (Radnor, PA), and sucrose was obtained from Pfanstiehl (Waukegan, IL). Kolliphor® P188 BIO (Pluronic® F68) was purchased from BASF (Ludwigshafen, Germany). The AAV capsid of RHM4-1 was prepared as described in U.S. Pat. No. 9,840,719 (VP1 of SEQ ID NO: 1, VP2 of SEQ ID NO: 2, and VP3 of SEQ ID NO: 3), which is incorporated herein by reference. Multiple AAV vectors of RHM4-1 capsid were prepared, each carrying a different transgene. One rAAV was produced containing a transgene encoding acid alpha-glucosidase (GAA), as described in U.S. Patent No. 20210222141, the contents of which are incorporated herein by reference. This GAA-containing vector is referred to herein as AAV-GAA. Another type of vector with the same capsid contains a transgene encoding alpha-galactosidase A (GLA), as described in International Publication No. WO 2022 / 155665, the contents of which are incorporated herein by reference. This GLA-containing vector is referred to herein as AAV-GLA.

[0250] B. Preparation of Formulations Formulations were made by buffer exchange with the corresponding buffer (100 kDa molecular weight cut-off (MWCO)) in 24-well plates. All formulations were diluted to 2×10 with the corresponding buffer and surfactant. 13 vg / mL ~ 3 × 10 13The formulations were diluted to a target titer of 2E13 vg / mL (2E13 vg / mL to 3E13 vg / mL) and subsequently finally filtered through a 0.22 μm syringe filter (EMD Millipore, Burlington, MA). The final formulations, designated F1 (Formulation 1), F2 (Formulation 2), and F3 (Formulation 3) (Table 1), were filled into sterile 5 mL CZ vials (West Pharmaceuticals, Upper Darby, PA) at volumes ranging from 1.3 to 3 mL, sealed, and placed in stability chambers corresponding to conditions including -80°C, -20°C, -40°C, 5°C, and 25°C, respectively. At each time point shown in Table 2, from time point 0 (referred to as TO) through 2 weeks (2w), 2 months (2m), and 6 months (6m), samples were withdrawn from the stability testing conditions and aliquoted into 2 mL sterile CZ vials (West Pharmaceuticals, Upper Darby, PA) for testing of critical quality attributes (CQAs) by various assays (listed below).

[0251] [Table 1]

[0252] [Table 2]

[0253] C. Visual Inspection All samples were visually inspected under a light meter to assess appearance and identify the presence of visible particles.

[0254] All samples of F1 (low pH and low ionic strength), F2 (conventional pH and conventional ionic strength), and F3 (moderate ionic strength and conventional pH) at all test conditions, including T0, 25°C-2 weeks and 2 months, -80°C-2 months, -20°C-2 months and 6 months, -40°C-2 months and 6 months, and 5°C-2 months and 6 months, were revealed to be clear, colorless solutions without visible particles.

[0255] D. Glass transition (Tg') of frozen concentrate The glass transition temperatures of the freeze-concentrates (Tg') of all T0 formulation samples were measured by low-temperature differential scanning calorimetry (LT-DSC) USP (United States Pharmacopeial Convention). <891> The thermal analysis was determined by the method of: LT-DSC was tracked at a rate of 10 °C / min with 9.8–18.6 mg of sample solution using a TA Instruments Q2000. The heat evolution or uptake of the sample during the cooling and warming steps of the thermal event reflected the energy difference recorded as a measurement.

[0256] The glass transitions of the frozen concentrates (Tg') analyzed by LT-DSC are shown in Table 3. The Tg' values ​​for F1 and F3 were found to be -38.85 °C and -41.96 °C, respectively. As can be seen, the Tg' values ​​could not be obtained for the platform formulation (F2), which has historically been reported to be approximately -65 °C. The glass transition temperature of F1 was higher than that of F2. It is beneficial to store biologics below their glass transition to minimize mobility and enhance stability. Increasing the Tg' also allows for storage at temperatures other than -80 °C, which is advantageous and highly desirable given the ease of supply chain operations, including handling / transportation at higher temperatures. It is also a viable approach to maximize sterility assurance (low-temperature storage can compromise container closure systems because -80 °C is generally below the glass transition of plastics and rubber, and plastics generally become brittle at these low temperatures).

[0257] [Table 3]

[0258] E. Vector genome titer by qPCR A qPCR assay was performed to measure vector genome copy numbers for all samples. Samples were first incubated with DNAse at room temperature for 15 minutes to digest unencapsulated vector genome DNA. The DNAse reaction was stopped, and samples were then incubated at 95°C to open capsids and then diluted to fall within a standard range based on estimated titers. Titer recovery (%) was calculated based on the titer measurements and comparison with the TO value.

[0259] Figure 1 and Table 4 show the titer recoveries for all samples relative to the TO titer. Vector titer recoveries were observed to be maintained for F1 at all storage conditions, including 5°C, -20°C, -40°C, and -80°C, for longer timepoints, including 2 to 6 months (87 to 107% genome recovery). The data indicate an estimated shelf life of greater than 1.5 years at 2 to 8°C when a common acceptance standard of 60 to 140% is used. Similarly, a shelf life of greater than 2 years is supported at all freezing temperatures, including -20°C. Vector titer recoveries for F3 were also maintained at all storage conditions, including 5°C, -20°C, -40°C, and -80°C, for longer timepoints, including 2 to 6 months (86 to 107% genome recovery). However, for the F2 formulation (high ionic strength), the titer recovery decreased to approximately 73.7% and 62.52% at 25°C for 2 weeks and 2 months, respectively, while samples from all other conditions showed higher titer recoveries.High ionic strength formulations (i.e., those with NaCl >150 mM) are commonly used for rAAV preparations, and previous reports have demonstrated the recovery and stability of such formulations (see Wright, J. Frasier, et al., “Identification of factors that contribute to recombinant AAV2 particle aggregation and methods to prevent its occurrence during vector purification and formulation”, Molecular Therapy, (2005) 12:1; Rodrigues, Gerard A., et al., “Pharmaceutical development of AAV-based gene therapy products for the eye”, Pharmaceutical Research, (2019) 36:29; and Srivastava, Arvind, et al. “Manufacturing challenges and rational formulation development for AAV viral vectors”, Journal of Pharmaceutical Sciences 110.7 (2021): 2609-2624). High ionic strength formulations are used in commercially available AAV products (e.g., see ZOLGENSMA® package insert; LUXTURNA® package insert). Here, we demonstrate that titer recovery is improved and maintained within the storage period of studies using moderate ionic strength formulations (50-95 mM; 60 mM F3) compared to conventional high ionic strength formulations (e.g., F2). Titer recovery was most improved and maintained using low ionic strength formulations (<50 mM; 25 mM F1) at low pH (<7). Titer recovery is also likely to be maintained when estimated beyond 1.5 years.

[0260] [Table 4]

[0261] F. % Full Capsid Measurement by Ion Exchange Chromatography (IEX) Ion exchange chromatography (IEX) assays were performed using standard methods and UV at 280 nm. The rAAV samples contained primarily intact (full) rAAV, with a blank-to-full ratio of 3:7 or less. Samples recovered for all formulations at all stability conditions were reported to be enriched exclusively in full particles. Changes in residence time were observed for F2 and F3 formulations over a 2-month period at 25°C (Figure 2), indicating possible deamidation, which was further confirmed by peptide mapping data. However, neither changes in residence time nor deamidation were observed in F1 samples across all test conditions. Deamidation is highly undesirable, as it inversely correlates with rAAV efficacy and transduction (Giles, April R., et al. “Deamidation of amino acids on the surface of adeno-associated viral capsids leads to charge heterogeneity and altered vector function.” Molecular Therapy. 2018. 26(12):2848-2862).

[0262] G. SEC-MALS for flocculation Size exclusion chromatography was used to examine the monomer molecular weight, hydrodynamic radius, and concentrations of empty capsids, full capsids, and total capsids. The abundance of monomers and high molecular weight (HMW) species was equal to their percentage of the total integrated UV signal at 280 nm. SEC-MALS data, as shown in Figure 3, revealed that the abundance of HMW species, representing impurities and degradants, was lower in the low pH and low ionic strength formulation F1 and the moderately low ionic strength formulation F3 compared to the conventional pH and ionic strength formulation F2. This result indicates better quality and stability for F1 and F3 than for F2. For F2, the HMW% was relatively high for all stability conditions.

[0263] H. Capsid Protein Purity CE-SDS Samples containing rAAV were denatured with sodium dodecyl sulfate (SDS) and β-mercaptoethanol (BME) at 70° C. The denatured samples were subjected to capillary electrophoresis, and the separated proteins were detected by absorbance at 220 nm.

[0264] The VP protein ratio was not significantly affected for all formulations under all stability conditions, as can be seen in Figure 4. Thus, capsid protein purity was maintained across all formulations and was not adversely affected by lower pH and / or ionic strength.

[0265] I. Size Measurement by Dynamic Light Scattering (DLS) DLS was performed to study the intensity and volume particle size distribution (PSD) and to measure the mean hydrodynamic size (Z-average). The polydispersity index (PDI) was also measured, which serves as an indicator of aggregation.

[0266] The reported DLS data are shown in Table 5, which demonstrate that vector quality and stability were maintained for the low pH and / or low ionic strength F1 and F3 formulations under different conditions. Particle size was maintained at approximately 24-30 nm for both F1 and F3 formulations. The PDI value for F1 was slightly higher than that for F2 and F3, but this is not due to vector aggregation but rather to complex species within the buffer matrix (as confirmed by buffer scans).

[0267] [Table 5]

[0268] J. Intrinsic Fluorescence Differential Scanning Fluorescence (DSF) for Thermal Stability To study the thermal unfolding of viral proteins, we assessed the changes in the intrinsic fluorescence signal of tryptophan residues present in rAAV capsids during a thermal gradient. Data corresponding to the change in the fluorescence intensity ratio 350 / 330 nm and the viral protein melting temperature (Tm) of the TO samples of all formulations were measured to investigate their thermodynamic stability using 10 μL samples in replicates in sealed capillaries.

[0269] The intrinsic fluorescence DSF data shown in Figure 5 showed that the melting temperature (Tm) of F1 changed by approximately 5°C relative to F2 and F3, indicating a significant change in the unfolding of the viral proteins. F1 exhibited the highest Tm, indicating superior thermodynamic or physical stability.

[0270] K. External Fluorescence Differential Scanning Fluorescence (DSF) for Thermal Stability Genome release from rAAV samples stained with the DNA-binding dye SYBR® Gold upon heat stress induction was detected by extrinsic fluorescence (DSF). To examine the thermodynamic stability of the rAAV formulations, changes in the onset of DNA uncoating temperature were assessed for TO samples.

[0271] Exogenous DSF assays using a DNA-binding dye (SYBR® Gold) showed that the onset of DNA uncoating temperature for F1 was observed to shift by approximately 5°C compared to F2 and F3 (Figure 6). This shift highlights the greater thermodynamic stability (low ionic strength and low pH) of the F1 formulation and supports our conclusion that capsids are structurally most compacted near pH 5.5, which coincides with the isoelectric point.

[0272] L. Light blocking for evaluation of invisible particles Subvisible particulates were measured with a light obscuration (HIAC) dependent particle counter. Particle measurements in buckets of different sizes, including >5 μm, >10 μm, and >25 μm, were recorded as cumulative counts / mL. HIAC data for subvisible particle counts across all size ranges did not indicate any significant concerns (Figure 7). All values ​​were based on the USP (United States Pharmacopeial Convention) <787> It meets the standards for the acceptable number of invisible particles in therapeutic protein injections.

[0273] M. Peptide Mapping for Post-Translational Modifications (PTMs) Proteins in the RHM4-1 capsid were denatured, disulfides reduced to free thiols, and irreversibly alkylated. Aliquots of the samples were enzymatically digested separately with trypsin and Asp-N, respectively. Peptides from each sample generated from the enzymatic digests were separated by reverse-phase liquid chromatography and detected by mass spectrometry. The raw data were processed using a bioinformatics pipeline, and sequence coverage was mapped by comparing identified peptides of the RHM4-1 capsid protein with theoretical peptides. Each peptide was identified using intact molecular weight and sequence-specific fragment ion data. The levels of PTMs, including deamidation and oxidation, were determined.

[0274] As can be seen in Figure 8, F1 did not show significant levels of deamidation compared to F2 and F3, with higher levels observed in the 2-week and 2-month 25°C samples (30-35%), in contrast to F1 (2.5%). Significant oxidation was observed in the 2-month 25°C stressed samples of F2 and F3 (20-30%), in contrast to F1 (9%).

[0275] N. Potency by cell-based assay Samples containing engineered AAV vectors encoding GAA (AAV-GAA), as described in US Patent Application Publication No. 20210222141, were tested in a potency assay measuring GAA activity in cells transduced with the GAA vector. Cells were seeded into 96-well plates and transduced with a GAA vector reference standard (RS), a positive control sample (PC), and a test article (TA). Three days after transduction, the enzymatic activity (cleavage to release a fluorophore) of secreted GAA by the cells was measured spectrophotometrically using a fluorescence microplate reader. The fluorescence of each reference standard and test sample dilution was plotted against the vector genome concentration, and dose-response curves were fitted using linear regression software. The test sample line was compared to the reference standard line using slope ratio analysis, and the relative potency of the sample is reported as a percentage of the reference standard (%RP). A time 0 (T0) sample stored at -80°C for 6 months served as the control for all formulations.

[0276] No effect of the formulation buffer on cell viability was observed. The potency data in Figure 9 show that F1 maintained high relative potency for 2 and 6 months for conditions including -20°C, -40°C, and -80°C, indicating the feasibility of storing rAAV in the F1 formulation at these temperatures for a shelf life of at least 2 years. Formulations F2 and F3 maintained high relative potency at 5°C, -20°C, -40°C, and -80°C. The medium ionic strength formulation, F3, is expected to maintain potency when stored at 2-8°C for at least 2 years (estimated from current data trends).

[0277] O.Overview The overall findings of the long-term stability study confirm that the low-ionic-strength, sucrose-based surfactant-containing, low-pH formulation (F1) performed best for most CQAs (i.e., had the best CQA profile). The favorable data for F1, including maintenance of potency and efficacy, reduced degradation, reduced aggregation, reduced impurities, and no potential for deamidation compared to the F2 and F3 formulations, also support an extended shelf life at higher temperatures, including -20°C and -40°C (vs. -80°C), using the F1 formulation. Furthermore, the presence of sucrose and Tg data highlight the stabilizing effect of sugar and support improved storage at frozen temperatures and amenability for lyophilization.

[0278] Those skilled in the art understand that no single CQA is definitive; rather, the CQA profile of each formulation indicates which formulation is best for a particular purpose or product. Here, low ionic strength and low pH formulation F1 has the best CQA profile compared to conventional, generally recognized higher physiological pH (7.3) and ionic strength (>150 mM NaCl) formulations for long-term frozen storage and thawing of rAAV products. The addition of surfactants is also expected to minimize potential adsorption losses to manufacturing and administration components, thereby ensuring high and improved dose precision.

[0279] Example 2: Optimized excipients suppress degradation during short-term stability A.rAAV solution preparation Various matrix solutions were prepared using various buffers, amino acids, salts, detergents, and sugar additives. The engineered rAAV product was prepared in the matrix by either dilution or buffer exchange, as described above in Example 1. The rAAV solutions were then exposed to short-term stress for 1 to 30 days via controlled exposure to temperature (25-35°C).

[0280] B. Forced deterioration of the liquid phase Loss of genome titer is a prominent degradation mechanism of rAAV, reducing therapeutic efficacy and increasing immunogenicity. Short-term storage at elevated temperatures accelerates this degradation process compared to cold storage, allowing for the evaluation of key conditions for enhancing liquid storage stability within an experimentally feasible timeframe.

[0281] rAAV was diluted into matrices of varying pH, ionic strength, and additional additives in 96-well microtiter plates. The solutions were sealed and incubated at 30–35°C for short-term stress. After the rAAV solutions were exposed to stress, the solutions were cooled to 4°C to minimize further changes and analyzed using ion exchange-high performance liquid chromatography (IEX-HPLC) to assess changes in titer.

[0282] An example of the change in rAAV genome titer during forced degradation is shown for rAAV in Figure 10 and analyzed using a partition coefficient model. The change in titer is measured by the relative change in area of ​​the intact, complete rAAV vector by HPLC. This analysis identifies three preferred conditions for preserving vector titer during incubation: pH < 7.3, NaCl concentration < 100 mM, and MgCl2 concentration < 10 mM. All of these characteristics reduce titer loss during rAAV exposure. In certain embodiments, a combination of all three attributes of F1 may be preferred to best reduce rAAV titer loss.

[0283] A summary of statistical analyses to identify significant parameters, shown in Table 6, supports these findings. The effects of conditions (pH, ionic strength (expressed as sodium chloride concentration), magnesium, arginine, and histidine) on preventing titer loss were determined using analysis of variance (ANOVA). Effects were generally judged to be "strong" or "moderate" when the effect probability (p) was less than 0.01 or between 0.01 and 0.1, respectively. Conditions that improved titer loss included a pH below 7.0, low salt concentrations (NaCl concentration <100 mM, MgCl concentration <10 mM), and the addition of arginine and histidine. Significance was determined by analysis using ANOVA and partition models. Preferred ranges indicate the preferred range for a given attribute.

[0284] [Table 6]

[0285] C. Formation of invisible particles during freezing and thawing The freezing and thawing process (FT) during virus production is a common source of viral aggregation and subvisible particle formation (SVP), increasing drug immunogenicity and patient risk. Predictable particle formation is required to manage batch-to-batch variability in product composition across major manufacturing scales, as well as downstream manufacturing processes such as pooling, frozen product storage, and freezing for lyophilization. The effect of FT on SVP formation in rAAV products (as described above) was studied by diluting the rAAV product into matrices containing various buffers, surfactants, salts, and other excipients. Over 100 experimental conditions were tested, with strategic parameter variations enabling the creation of statistical models describing SVP formation. Solutions were sealed and exposed to FT at controlled temperature ramp rates, and SVP formation was monitored. SVP size and abundance were then fitted to empirical statistical models to understand the general behavior observed across the tested conditions.

[0286] A statistical model of SVP formation in a non-optimized matrix is ​​shown in Figures 11A and 11B. The number and size of SVPs are highly dependent on FT velocity, empty rAAV content, and buffer components. In the field, controlling each of these parameters during the production process is difficult. Empty rAAV content refers to the percentage of empty particles (AAVs without a complete genome) in a sample. Empty rAAV content can vary significantly between batches, ranging from 90% full to over 80% empty between batches and projects within a given serotype. Freeze-thaw rates can vary more than 10-fold with changes in container size and freezing or thawing temperature. Buffer components are often highly limited by compatibility with critical processing steps and / or patient administration routes. Such dependence of SVP formation on processing and manufacturing conditions can result in undesirable and unpredictable SVP levels in pharmaceutical products, which can vary depending on processing steps, processing scale, storage temperature, batch-to-batch product composition, and route of patient administration.

[0287] SVP formation with improved and predictable properties is demonstrated in Figures 11C and 11D. The addition of sugars and detergents is important for reducing overall SVP formation and for eliminating SVP formation independent of FT rate, empty rAAV content, and buffer components at both neutral and acidic pH. Improvements in SVP formation are obtained with 0.001-0.05% detergent, or more preferably, in the range of 0.01-0.05%. Improvements in SVP formation are also observed with the addition of sugars to 1-6%, more preferably, 2-4%. A statistical analysis of the effects supporting these findings is shown in Table 7.

[0288] [Table 7-1]

[0289] Together, these forced degradation studies explore the important effects of ionic strength, buffer species, pH, detergents, sugars, vector purity (e.g., percentage of empty capsids), and freeze-thaw rate on overall rAAV stability. Together, they reveal the following: (a) moderate ionic strength can mitigate both vector genome loss and vector aggregation; (b) both weakly acidic pH (5-6.5) and neutral pH (6.5-7.5) can stabilize rAAV; (c) a combination of non-ionic detergents (preferably >0.005% v / v) and sugars (preferably >1.5%) produces freezing characteristics where particle aggregation is unaffected by freezing rate or empty capsid concentration, all common buffer components being equivalent. Based on the results of Example 2, we developed formulations F4 and F5 (F4 (formulation 4) = 10 mM Tris, 75 mM NaCl, 5% sucrose, 0.005% poloxamer 188, pH 7.3; F5 (formulation 5) = 10 mM citrate, 75 mM NaCl, 7% sucrose, 0.001% poloxamer, pH 6.0). Formulations suitable for lyophilization conditions include (a) a moderate ionic strength of 50-95 mM, (b) a pH of 5-7.5, (c) a non-volatile buffer capable of buffering in the pH range of 5-7.5, and a minimal sugar concentration sufficient to provide a stable lyophilized cake.

[0290] Example 3: Long-term stability of AAV-GLA in low ionic strength formulations A. Formulation composition and stability test of AAV-GLA A RHM4-1 capsid vector containing a transgene encoding α-galactosidase A (GLA), (AAV-GLA) (using VP1 of SEQ ID NO: 1, VP2 of SEQ ID NO: 2, and VP3 of SEQ ID NO: 3, as set forth in U.S. Pat. No. 9,840,719) was prepared (as described in WO 2022 / 155665) and formulated in Formulation F1 (25 mM sodium acetate, 25 mM NaCl, 5 mM MgCl, 5.4% sucrose, 0.01% coliform P188, pH 5.2) as a sterile, single-dose, preservative-free aqueous solution in 5 mL CZ vials (catalog number: 19550209, West Pharmaceutical Services).

[0291] AAV-GLA in formulation F1 was monitored for long-term stability at storage conditions including 3, 6, 9, and 12 months at -80°C ± 10°C, 6 and 12 months at -20°C ± 10°C, and 1, 3, and 6 months at 5°C ± 3°C. The study also evaluated the stability of the material at accelerated stability conditions such as 25°C / 60% RH for 2 weeks and 1 month.

[0292] B. pH of AAV-GLA during storage The pH of AAV-GLA during storage was measured potentiometrically. The analytical procedure was as per USP <791> and Ph.Eur.2.2.3. The post-storage measurements (Figure 12) were consistent across all conditions and time points compared to TO and were within the intended specifications for pH.

[0293] C. Vector genome titer by ddPCR The viral genome titer of AAV-GLA in formulation F1 during storage was measured using droplet digital polymerase chain reaction (ddPCR). Samples were added to a ddPCR solution containing transgene-specific primers and a fluorescent probe. Each sample was divided into uniform nanoliter-sized droplets, and target and background DNA were randomly distributed among the droplets during the fractionation process. The droplets were transferred to a 96-well plate for PCR amplification, amplifying the transgene segment. A droplet reader read each droplet and determined the percentage of positive droplets. The percentage of positive droplets was used to calculate the copy number concentration of the target DNA. The vector genome concentration results for each test article and control were reported as vector genome copies per mL of product (vg / mL).

[0294] Vector titer and recovery rate (Figure 13) shows the titer recovery rate for all samples relative to the TO titer. Genomic titer recovery was confirmed to be maintained at each time point: 3 months (97%), 6 months (78%), 9 months (74%), and 12 months (80%) under the target storage conditions of -80°C. Long-term storage at 2-8°C for up to 6 months and at -20°C for up to 12 months also showed high recovery rates (75-101%). Therefore, titer recovery rates were shown to be maintained at 70% or higher for samples up to 12 months old.

[0295] D. In Vitro Efficacy Cell-based potency assays were designed to reflect the mechanism of action of α-Gal A protein relative to a reference standard. Potency was measured by assessing the enzymatic activity resulting from expression of α-Gal A from AAV-GLA-transduced reporter cells, and enzymatic activity was assessed using a fluorogenic reporter.

[0296] Potency (Figure 14) was maintained within at least 65% across all samples at -20°C, -80°C, and 1 month and 2-8°C. The maximum 35% loss is within the typical variability of cell-based potency assays, with no consistent decline at storage temperatures of -20°C and -80°C. Together, these results demonstrate suitability for storage at -20°C to -80°C for at least 1 year.

[0297] E. In vitro infectivity A cell-based infectivity assay was used to evaluate the in vitro infection activity of AAV-GLA. This cell-based method involved co-infection of AAV packaging cells carrying the AAV rep and cap genes with AAV-GLA and Ad5. The readout incorporated 50% endpoint determination of vector DNA by ddPCR using the same primer and probe reagents as in the vector genome concentration ddPCR assay. Data analysis and calculation of infectious titers were based on the Karber method. Data were reported as the ratio of vector particle concentration to infectious vector titer.

[0298] The PI ratio (vector particle concentration to infectious vector titer ratio, Figure 15A) and infectious titer (Figure 15B) show the variability typical of in vitro cell-based assays. However, the behavior is consistent with the in vitro cell-based potency (Figure 14), with no consistent loss of infectivity. Importantly, infectious titers were found to be maintained at all timepoints, up to 6 months at 2-8°C, and up to 12 months at -20°C and -80°C.

[0299] F. Capsid and empty capsid concentrations: Full ratio by UV absorbance Absorbance at 280 nm and 260 nm was used to assess total capsids and the ratio of empty capsid particles to total capsid particles (E:F) in AAV-GLA preparations. In all measurements, total capsids (Figure 16A) ranged from 5.5 to 5.9 x 10 13 The E:F ratio (Figure 16A) was ≦0.25 for all samples and did not change significantly over the 12-month storage period.

[0300] G. Capsid purity and capsid ratio by CE-SDS Capillary electrophoresis with sodium dodecyl sulfate (CE-SDS) was used to assess the relative abundance and purity of viral capsid proteins. This measures the abundance of VP1, VP2, and VP3 viral proteins, capsid protein variants (pre-VP3), as well as important additional peptide impurities and peptide fragments. AAV-GLA particles were reduced and denatured. Reduced capsid proteins were separated by size using capillary electrophoresis (CE) based on the electrophoretic mobility of protein molecules. Capsid protein stoichiometry and capsid protein purity were determined based on the corrected peak areas of VP1, VP2, VP3, and the VP3 variant pre-VP3.

[0301] The relative ratios of VP proteins (Figure 17A) fluctuate within 20% of the measured TO value, with no consistent increase or decrease during storage. The total purity of VP proteins (percentages associated with VP1, VP2, and VP3, Figure 17B) is greater than 98%, with a slight decrease from TO to 96% associated only with storage at 25°C. Similarly, the ratios of peptide impurities are consistently less than 2% for all samples, except for a slight increase to approximately 4% in the 25°C sample. Figure 17C shows the average impurity percentages. Based on the data, we found that protein purity is maintained for at least one year under all storage conditions, including 2-8°C, -20°C, and -80°C.

[0302] Percentage of full capsids by H.IEX Capsid charge and charge variants were characterized by analytical anion exchange chromatography (AEX). Empty and full capsid particles were eluted from an analytical AEX column, and absorbance was monitored at 280 nm. The E:F ratio was determined by the ratio of the empty capsid peak area to the full capsid peak area after correcting for the difference in extinction coefficient due to the encapsidated DNA in full capsids versus empty capsids.

[0303] [Table 7-2]

[0304] The IEX results are summarized in Table 7. Samples searched at all stability conditions reported E:F ratios ≤0.2 (LOD) and A260 / 280 ratios of 1.3-1.4 consistently for all samples throughout the 12-month period. There was no significant change in retention time for all samples, further suggesting no signs of deamidation (further supported by peptide mapping data).

[0305] I. Aggregation by SEC-MALS Oligomeric aggregates were assessed by analytical size exclusion by multi-angle light scattering (SEC-MALS), which separates species based on hydrodynamic volume. An in-line MALS detector was used to determine molecular weight and hydrodynamic radius in a single run. The relative abundance of high molecular weight species (Figure 18) was very similar in all samples over the course of one year of storage, with monomer abundance levels (<1%) being very similar.

[0306] J. Evaluation of invisible particles by light obscuration A low-volume 1 mL method was used to assess subvisible particulates using light obscuration (HIAC). Particle measurements in different size buckets, such as >5 μm, >10 μm, and >25 μm, are shown in Table 8 as cumulative counts / mL. In all conditions, particulate levels were very low, typically at least 10-fold lower than the official limits for all low-volume products. Furthermore, no appreciable increase was observed for all samples during storage for up to one year.

[0307] [Table 8]

[0308] K. Particle size and particle size distribution by DLS DLS data (Table 9), in which particle size distribution and polydispersity were assessed by dynamic light scattering, showed no discernible aggregation for all samples over the 12-month period, a main peak size (radius in nm) ranging from 25 to 32 nm, a main peak polydispersity index (PDI) of ≤0.3, and a percent mass composition of monomer of ≥98%.

[0309] [Table 9]

[0310] L. Peptide Mapping for Post-Translational Modifications (PTMs) Chemical degradation of key peptides was analyzed by mass spectrometry using limited proteolysis. RHM4-1 capsid protein samples were denatured, disulfides reduced to free thiols, and then irreversibly alkylated. Samples were enzymatically digested, separated by reversed-phase liquid chromatography, and detected by mass spectrometry. Raw data were processed using a bioinformatics pipeline, and sequence coverage was mapped by comparing identified and theoretical peptides of RHM4-1 capsid protein. Each peptide was identified using intact molecular weight and sequence-specific fragment ion data and analyzed for post-translational modifications, including deamidation and oxidation.

[0311] Deamidation levels (Figure 19) were observed to be 8% or less across all asparagine residues for all samples from all conditions, with no clear increase in deamidation for any residue.

[0312] M. Overview The overall findings of the long-term stability study confirm that the low ionic strength, sucrose-based, high Pluronic concentration, low pH formulation exhibits no change in analytical attributes over a one-year period at -20°C. Many properties, including aggregation, particle formation, and vector genome titer, also showed no change at accelerated stability conditions (4°C and 25°C) for up to six months. Together, this indicates great stability that can be reasonably tolerated for at least one year between -20°C and -80°C, and at least two years.

[0313] Example 4. Stability of lyophilized AAV-GAA in medium ionic strength formulations A. Stability after freeze-drying cycle As described in Example 2, formulation development supported modifications to F1 to create medium ionic strength formulations F4 and F5 (listed in Table 10). The stability of AAV-GAA under lyophilization cycles was tested in medium ionic strength formulations F4 and F5. rAAV solutions were prepared by buffer exchange and addition of key excipients to an aqueous solution of purified AAV-GAA, followed by 0.2 μm filtration and filling into glass vials. The solutions were frozen at -50°C and warmed to -35°C for primary drying and 30°C to 25°C for secondary drying. The lyophilized cakes were then reconstituted in deionized water and tested for changes in key quality attributes.

[0314] [Table 10]

[0315] B. Vector Recovery After Lyophilization After the lyophilization cycle, the lyophilized cakes were reconstituted and assayed for encapsulated genome titer to confirm any changes in titer due to the lyophilization process. Vector genome titers were measured using quantitative polymerase chain reaction (qPCR). Figure 20 shows titers before and after reconstitution for secondary drying at 25°C versus 35°C. Titers were normalized to the pre-lyophilization titer (TO). AAV-GAA showed a 15% increase in titer for F4 and a 5% increase for F5 (within the experimental error of a typical qPCR assay, indicating no degradation due to the lyophilization cycle in either F4 or F5).

[0316] C. Potency after lyophilization After the lyophilization cycle, the cakes were reconstituted and assayed for in vitro cell potency to verify changes in potency due to the lyophilization process. Figure 21 shows the relative cell potency after lyophilization normalized to the potency before lyophilization. The vectors showed less than 10% relative potency loss, indicating acceptable degradation due to the lyophilization process in these solution matrices.

[0317] D. High molecular weight after freeze-drying After the lyophilization cycle, the cakes were reconstituted and assayed for high molecular weight species using SEC-MALS. Figure 22 shows the percentage of high molecular weight species as determined by area percent absorbance at 280 nm. All samples showed less than a 0.5% increase in high molecular weight species, indicating that the lyophilization process has minimal effect on aggregated species.

[0318] E. VP protein integrity after lyophilization After the lyophilization cycle, the cakes were reconstituted and assayed for changes in capsid proteins. Figure 23 shows the relative ratios of VP1, VP2, VP3, and pre-VP3 proteins. All ratios are normalized to VP1 occupancy, with pre-VP3 representing a common degradation species formed from the degradation of VP2 and VP3. The F5 formulation with secondary drying at 30°C shows some fragmentation, with an approximately 15% increase in VP3 and pre-VP3. All other conditions show no significant change from TO.

[0319] F. Long-term stability of lyophilized AAV-GAA formulation F4 The AAV-GAA in formulation F4 was lyophilized and monitored for long-term stability. The rAAV solution was prepared by buffer exchange of the purified AAV-GAA vector, diluted to a target titer of 1.5E13 vg / mL, added sugar and surfactant from a high-concentration stock, and the final solution was filtered through a 0.22 μm filter. The prepared rAAV solution was then frozen at -50°C and lyophilized by primary drying at -35°C and secondary drying at 25°C or 30°C. The vials were then stoppered and sealed under filtered nitrogen (NF). The sealed vials were stored as described in Table 11. After the time periods indicated in Table 11, the samples were reconstituted and aliquoted into 2 mL sterile vials for testing of critical quality attributes by various assays.

[0320] [Table 11]

[0321] G. Vector genome titer by qPCR. The titer of lyophilized AAV-GAA (Figure 24) during storage was assessed by qPCR. The titers of all samples were comparable to or higher than that of the TO control (no lyophilization). Thus, vector titer recovery was maintained for up to 6 months (100-114% genome recovery) under all storage conditions, including 35°C / 65% RH, 25°C / 60% RH, -20°C, and 2-8°C, with no apparent decrease after up to 6 months of storage at room temperature.

[0322] H. Potency by Cell-Based Assay In vitro cell-based potency (Figure 25) assesses the functional activity of lyophilized AAV-GAA during storage. The lyophilized formulation maintains at least 60% potency at 2-8°C and -20°C. This is comparable to the control TO, as the in vitro potency assay has a large relative standard error of at least 20%. Some loss of potency is observed at 35°C / 65% RH (approximately 50% loss at 0.5 months) and 25°C / 60% RH (52% after 3 months, 63% after 6 months). This loss is within acceptable limits for these accelerated stability conditions and at temperatures significantly higher than storage temperatures (-20°C to 4°C).

[0323] I. Oligomer Aggregation (SEC-MALS) The formation of oligomeric high molecular weight species (HMW) in lyophilized AAV-GAA was assessed by size-exclusion chromatography using multi-angle light scattering and reported as a percentage of the total integrated UV signal at 280. As shown in Figures 26A-C, the relative abundances were all between 2 and 3% for all samples before lyophilization and during storage, indicating no change in the monomeric state and oligomeric aggregation throughout the study. From this, it is reasonable to estimate that HMW formation is negligible for at least one year when stored at 4°C.

[0324] J. Fragmentation and Aggregation (Analytical Ultracentrifugation) Analytical ultracentrifugation is a sensitive method for detecting high molecular weight species (HMW) and capsid fragments (low molecular weight species, LMW), which are important degradation products that can form during storage. Lyophilized AAV-GAA was evaluated by monitoring the AUC using a CsCl gradient, the sedimentation profile using absorbance at 230 nm, and modeling using the Lamm equation.

[0325] Figure 27 shows the HMW, partial, and LMW populations over the storage period. For both HMW and partial, all samples changed within 2% of the pre-lyophilized sample, indicating no significant change. LMW species were below the detection limit (approximately 5%) for most samples, indicating no fragmentation upon lyophilization or storage for at least 6 months. Storage at 4°C is expected to result in negligible changes in LMW, HMW, and E:F for at least 1 year.

[0326] K. Invisible particle formation Subvisible particulates are a highly regulated characteristic for parenteral therapeutics. Subvisible particulates were measured by light obscuration using a 1 mL low volume method. Particle measurements are shown in Figure 28 in different size brackets of 10 um and 25 um and are presented as cumulative counts / mL. Measured levels were below 50 particles / mL for all samples, including the 35°C and 25°C accelerated stress conditions, all of which met USP <787> This is more than 100 times lower than the official guidelines.

[0327] L.CE-SDS The capsid-viral protein ratio was assessed for reconstituted lyophilized AAV-GAA. The amount of VP protein was monitored by capillary electrophoresis under reducing conditions using SDS detergent. The VP ratio normalized to VP1 occupancy (Figure 29) showed no change across all storage conditions between -20°C and 25°C. Compared to the pre-lyophilization condition, the 35°C / 65% RH condition showed an approximately 10% increase in pre-vp3 + vp3 abundance, indicating some fragmentation of VP proteins at this condition. However, this change is within the acceptable range for promoting stability. No significant changes were observed across all sample time points compared to the TO liquid (before lyophilization).

[0328] M. Overview of Freeze-Drying Stability Lyophilization cycles and long-term storage were tested for AAV-GAA. Across two medium ionic strength formulations, F4 and F5, lyophilization cycles had minimal impact on rAAV titer, potency, and key attributes, including capsid fragmentation, aggregation, and subvisible particles. Long-term stability of formulation F4 demonstrated no change in titer, aggregation, capsid fragmentation, and VP protein fragmentation over 6 months at 4°C. In vitro potency varied within 2 standard deviations (40%) of assay error for up to 6 months of storage at 4°C, with only a significant decrease at 3 months for accelerated stability conditions. Thus, these medium ionic strength formulations adequately stabilize rAAV during lyophilization and storage at -20°C to 4°C for at least 1 year.

Claims

1. A stable formulation comprising, together with a therapeutic drug, a) 1-100 mM of one or more salts; b) a buffer; and c) 0.001 to 0.05 wt. % of one or more nonionic surfactants; d) 1 to 10% by weight of one or more sugars; e) water; and f) optionally, one or more amino acids; and The pH of the composition is 4.5 to 7.5, and 60% or more of the pharmaceutical product is recovered after freezing and thawing the formulation. Stable formulation.

2. 10. The stable formulation of claim 1, wherein the therapeutic agent is a recombinant adeno-associated virus (rAAV).

3. 3. The stable formulation of any one of claims 1-2, wherein the buffer comprises a non-phosphate buffer.

4. 4. The stable formulation of claim 3, wherein the non-phosphate buffer is sodium acetate.

5. 5. The stable formulation of any one of claims 3 to 4, wherein the non-phosphate buffering agent is present in the formulation at a concentration of 1 mM to about 50 mM.

6. 6. The stable formulation of any one of claims 1 to 5, wherein the one or more salts comprise 10 to 100 mM sodium chloride.

7. 7. The stable formulation of claim 6, wherein the one or more salts comprise a first salt and a second salt.

8. 8. The stable formulation of claim 7, wherein the first salt is sodium chloride and the second salt is magnesium chloride.

9. 7. The stable formulation of claim 6, wherein the sodium chloride is present in the formulation at a concentration of about 25 mM.

10. 9. The stable formulation of claim 8, wherein the magnesium chloride is present in the formulation at a concentration of about 1 mM to about 10 mM.

11. 11. The stable formulation of claim 10, wherein the magnesium chloride is present at a concentration of about 5 mM.

12. The stable formulation of any one of claims 1 to 11, wherein the sugar is sucrose.

13. 13. The stable formulation of claim 12, wherein the sucrose content in the formulation is 5.4%.

14. 14. The stable formulation of any one of claims 1 to 13, wherein the non-ionic surfactant is present in the formulation at a concentration of about 0.01%.

15. 15. The stable formulation of claim 14, wherein the surfactant is poloxamer 188, polysorbate 20, polysorbate 80, or poloxamer 407.

16. 16. The stable formulation of claim 15, wherein the surfactant is a polysorbate selected from the group consisting of polysorbate 20 and polysorbate 80.

17. 2. The stable formulation of claim 1, wherein the pH is about 5.

2.

18. The stable formulation of any one of claims 1 to 2, wherein the buffer is a phosphate buffer.

19. 19. The stable formulation of claim 18, wherein the phosphate buffer is sodium phosphate.

20. 20. The stable formulation of claim 19, wherein the sodium phosphate is present at about 10 mM.

21. 21. The stable formulation of any one of claims 1 to 20, wherein the formulation is lyophilized.

22. 21. The stable formulation of any one of claims 1 to 20, wherein the formulation is a liquid.

23. The therapeutic agent is selected from the group consisting of GAA (acid α-glucosidase), ATP7B (copper transporting ATPase 2), α-galactosidase A (GLA), ASS1 (argininosuccinate synthase), β-glucocerebrosidase, β-hexosaminidase A, SERPING1 (C1 protease inhibitor or C1 esterase inhibitor), glucose-6-phosphatase, CFTR (cystic fibrosis transmembrane conductance regulator), blood clotting (clotting) factors (e.g., Factor XIII, Factor IX, Factor VIII, Factor X, Factor VII, Factor VIIa, Protein C), Gain-of-function blood coagulation factors, antibodies, retinal pigment epithelium-specific 65 kDa protein (RPE65), erythropoietin, LDL (low density lipoprotein) receptor, lipoprotein lipase (LPL), ornithine transcarbamylase (OTC), β-globin, α-globin, spectrin, α-antitrypsin, adenosine deaminase (ADA), metal transporters (ATP7A or ATP7), sulfamidase, enzymes involved in lysosomal storage diseases (ARSA), hypoxanthine guanine phosphoribosyltransferase, β-25 glucocerebrosidase enzymes, sphingomyelinase, lysosomal hexosaminidase, branched-chain ketoacid dehydrogenase, hormones, growth factors, insulin-like growth factor 1 or 2, platelet-derived growth factor (PDGF), epidermal growth factor (EGF), nerve growth factor (NGF), neurotrophic factors-3 and -4, brain-derived neurotrophic factor (BDNF), glial cell line-derived growth factor (GDNF), transforming growth factors α and β, cytokines, α-interferon, β-interferon, interferon-γ, interleukin-2 (IL-2), interleukin-4 (IL-4), interleukin 12 (IL-12), granulocyte macrophage colony-stimulating factor (GM-CSF), lymphotoxin (LT), suicide gene products, herpes simplex virus thymidine kinase, cytosine deaminase (CD), diphtheria toxin (DT), cytochrome P450 (CYP), deoxycytidine kinase (DCK), tumor necrosis factor (TNF), drug resistance proteins, tumor suppressor proteins (e.g., p53, Rb, Wt-1, NF1, von Hippel-Lindau (VHL), adenomatous polyposis coli (APC)), peptides with immunomodulatory properties,Tolerogenic or immunogenic peptide or protein tregitope or hCDR1 (edorazide), insulin, glucokinase (GCK), guanylate cyclase 2D (GUCY2D) (or Leber congenital amaurosis (LCA) associated with GUCY2D variant (GUCY2D-LCA)), Rab escort protein 1 (REP1) (choroideremia (CHM) encodes REP1), LCA5 (Leber congenital amaurosis 5) (LCA-lebercillin), ornithine ketoacid aminotransferase (gyrate atrophy), retinoschisin 1 (X-linked retinoschisis), USH1C (Usher syndrome 1C), X-linked retinitis pigmentosa GTPase (XLRP), MERTK (AR form of RP: retinitis pigmentosa), DFNB1 (conjunctivitis The stable formulation of any one of claims 1 to 21, which is capable of increasing intracellular protein levels of one or more of the following: Syn 26 hearing loss, ACHM2, 3 and 4 (achromatopsia), PKD-1+ or PKD-2 (polycystic kidney disease), TPP1 (tripeptidyl peptidase 1), CLN2 (neuronal ceroid lipofuscinosis 2), sulfatase, N-acetylglucosamine-1-phosphate transferase, cathepsin A, GM2-AP (ganglioside GM2 activator), NPC1 (NPC intracellular cholesterol transporter 1), VPC2, sphingolipid activating protein, one or more zinc finger nucleases for genome editing, one or more donor sequences used as repair templates for genome editing, or variants thereof.

24. A stable formulation comprising: a) a recombinant adeno-associated virus (rAAV); and b) 25 mM sodium chloride; c) 5 mM magnesium chloride; d) 25 mM sodium acetate; e) 0.01% by weight of poloxamer 188; f) 5.4% by weight of sucrose; g) water, and h) pH is 5 to 5.5; Stable formulation.

25. A stable formulation comprising: (a) a recombinant adeno-associated virus (rAAV); and (b) 10 mM Tris; (c) 75 mM sodium chloride; (d) 0.005 wt% Poloxamer 188; and (e) 5% by weight of sucrose; (f) water; (g) pH 7.3; Stable formulation.

26. 1. A stable formulation, comprising: (a) a recombinant adeno-associated virus (rAAV); and (b) 10 mM sodium citrate; (c) 75 mM sodium chloride; (d) 0.001 wt% poloxamer 188; and (e) 7% by weight sucrose; (f) water; (g) pH 6.0; Stable formulation.

27. 27. The stable formulation of any one of claims 24 to 26, wherein the rAAV comprises a capsid derived from one or more AAV selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV9.47, AAV9(hu14), AAV10, AAV11, AAV12, Rh8, Rh10, Rh74, AAV3B, AAV-2i8, RHM4-1 (VP1, SEQ ID NO: 1 or VP2, SEQ ID NO: 2 or VP3, SEQ ID NO: 3), DJ, DJ8, NP59, Anc-80, and variants thereof.

28. 28. The stable formulation of any one of claims 24 to 27, wherein the rAAV comprises a transgene encoding a polypeptide or a nucleic acid selected from the group consisting of siRNA, an antisense molecule, miRNA, a ribozyme, and shRNA.

29. The rAAV may encode GAA (acid α-glucosidase), ATP7B (copper-transporting ATPase 2), α-galactosidase A (GLA), ASS1 (argininosuccinate synthase), β-glucocerebrosidase, β-hexosaminidase A, SERPING1 (C1 protease inhibitor or C1 esterase inhibitor), glucose-6-phosphatase, CFTR (cystic fibrosis transmembrane conductance regulator), blood clotting (clotting) factors (e.g., factor XIII, factor IX, factor VIII, factor X, factor VII, factor VIIa, protein C), or a functional protein. Acquired blood coagulation factors, antibodies, retinal pigment epithelium-specific 65 kDa protein (RPE65), erythropoietin, LDL (low density lipoprotein) receptor, lipoprotein lipase (LPL), ornithine transcarbamylase (OTC), β-globin, α-globin, spectrin, α-antitrypsin, adenosine deaminase (ADA), metal transporters (ATP7A or ATP7), sulfamidase, enzymes involved in lysosomal storage diseases (ARSA), hypoxanthine guanine phosphoribosyltransferase, β-25 glucocerebromoglycerin sphingomyelinase, lysosomal hexosaminidase, branched-chain ketoacid dehydrogenase, hormones, growth factors, insulin-like growth factor 1 or 2, platelet-derived growth factor (PDGF), epidermal growth factor (EGF), nerve growth factor (NGF), neurotrophic factor-3 and -4, brain-derived neurotrophic factor (BDNF), glial cell line-derived growth factor (GDNF), transforming growth factor α and β, cytokines, α-interferon, β-interferon, interferon-γ, interleukin-2 (IL-2), interleukin- 4 (IL-4), interleukin 12 (IL-12), granulocyte-macrophage colony-stimulating factor (GM-CSF), lymphotoxin (LT), suicide gene products, herpes simplex virus thymidine kinase, cytosine deaminase (CD), diphtheria toxin (DT), cytochrome P450 (CYP), deoxycytidine kinase (DCK), tumor necrosis factor (TNF), drug resistance proteins, tumor suppressor proteins (e.g., p53, Rb, Wt-1, NF1, von Hippel-Lindau (VHL), adenomatous polyposis coli (APC)), peptides with immunomodulatory properties,Tolerogenic or immunogenic peptide or protein tregitope or hCDR1 (edorazide), insulin, glucokinase (GCK), guanylate cyclase 2D (GUCY2D) (or Leber congenital amaurosis (LCA) associated with GUCY2D variant (GUCY2D-LCA)), Rab escort protein 1 (REP1) (choroideremia (CHM) encodes REP1), LCA5 (Leber congenital amaurosis 5) (LCA-lebercillin), ornithine ketoacid aminotransferase (gyrate atrophy), retinoschisin 1 (X-linked retinoschisis), USH1C (Usher syndrome 1C), X-linked retinitis pigmentosa GTPase (XLRP), MERTK (AR form of RP: retinitis pigmentosa), D 29. The stable formulation of claim 28, comprising the transgene encoding FNB1 (connexin 26 deafness), ACHM2, 3, and 4 (achromatosis), PKD-1+ or PKD-2 (polycystic kidney disease), TPP1 (tripeptidyl peptidase 1), CLN2 (neuronal ceroid lipofuscinosis 2), sulfatase, N-acetylglucosamine-1-phosphate transferase, cathepsin A, GM2-AP (ganglioside GM2 activator), NPC1 (NPC intracellular cholesterol transporter 1), VPC2, sphingolipid activating protein, one or more zinc finger nucleases for genome editing, one or more donor sequences used as repair templates for genome editing, or variants thereof.

30. 29. The stable formulation of claim 28, wherein the rAAV comprises the transgene encoding GAA (acid alpha-glucosidase).

31. 29. The stable formulation of claim 28, wherein the rAAV comprises the transgene encoding ATP7B (copper-transporting ATPase 2).

32. 29. The stable formulation of claim 28, wherein the rAAV comprises the transgene encoding alpha-galactosidase A (GLA).

33. 29. The stable formulation of claim 28, wherein the rAAV comprises the transgene encoding ASS1 (argininosuccinate synthase).

34. 29. The stable formulation of claim 28, wherein the rAAV comprises the transgene encoding β-glucocerebrosidase.

35. 29. The stable formulation of claim 28, wherein the rAAV comprises the transgene encoding β-hexosaminidase A.

36. 29. The stable formulation of claim 28, wherein the rAAV comprises the transgene encoding SERPING1 (C1 protease inhibitor or C1 esterase inhibitor).

37. 29. The stable formulation of claim 28, wherein the rAAV comprises the transgene encoding glucose-6-phosphatase.

38. 29. The stable formulation of claim 28, wherein the rAAV comprises the transgene encoding CFTR (cystic fibrosis transmembrane conductance regulator).

39. 29. The stable formulation of claim 28, wherein the rAAV comprises the transgene encoding a blood clotting (clotting) factor (e.g., Factor XIII, Factor IX, Factor VIII, Factor X, Factor VII, Factor Vila, Protein C).

40. 40. The stable formulation of any one of claims 1-39, wherein after the composition is frozen at a temperature above -80°C to -10°C for a period of time, 60% or more of the therapeutic agent or rAAV is recovered after thawing compared to the same product stored at -80°C for the same period of time.

41. 40. The stable formulation of any one of claims 1 to 39, wherein after the composition has been frozen for a period of time at a temperature of greater than -80°C to -10°C, e.g., at a temperature of -70°C to -15°C, -60°C to -20°C, -50°C to -30°C, or -45°C to -35°C, the pharmaceutical agent or rAAV exhibits at least 50% relative potency compared to the same product stored at -80°C for the same period of time.

42. 42. The stable formulation of any one of claims 1 to 41, wherein the pharmaceutical agent or rAAV exhibits a glass transition temperature of the frozen concentrate of -45°C to -35°C, e.g., -45°C, -44°C, -43°C, -42°C, -41°C, -40°C, -39°C, -38°C, -37°C, -36°C or -35°C, after the composition has been frozen at a temperature above -80°C to -10°C.

43. 43. The stable formulation of any one of claims 1 to 42, wherein the composition is a liquid.

44. 40. The stable formulation of any one of claims 1 to 39, wherein the composition is lyophilized.

45. 40. A lyophilized composition, said composition being substantially dehydrated, said lyophilized composition being capable of being reconstituted with a diluent to form the formulation of any one of claims 1 to 39.

46. 46. ​​The lyophilized composition of claim 45, wherein the reconstituting comprises mixing the lyophilized composition with water for injection (WFI).

47. 47. The stable formulation of any one of claims 1 to 43 or the lyophilized composition of any one of claims 44 to 46, wherein the formulation or composition is present in a unit dose container.

48. 48. The stable formulation or lyophilized composition of claim 47, wherein the unit dose container is a vial.

49. 49. The stable formulation or lyophilized composition of claim 48, wherein the vial is a sealed glass vial.

50. 1. A method for reducing post-freeze-thaw degradation of a therapeutic pharmaceutical, said method comprising: (a) the therapeutic pharmaceutical; and (b) 1-100 mM of one or more salts; (c) a buffer; and (d) 0.001 to 0.05 wt. % of one or more nonionic surfactants; (e) 3 to 10 wt. % of one or more sugars; (f) water; (g) a pH of 4.5 to 7.5; and (h) optionally preparing a composition comprising one or more amino acids; freezing the composition for a period of time at a temperature above -80°C to -10°C, e.g., -70°C to -15°C, -60°C to -20°C, -50°C to -30°C, or -45°C to -35°C, wherein upon thawing, 60% or more of the therapeutic agent is recovered compared to the same product stored at -80°C for the same period of time; A method comprising:

51. 1. A method for reducing post-freeze-thaw degradation of a therapeutic pharmaceutical, said method comprising: (a) the therapeutic product; and (b) 25 mM sodium chloride; (c) 5 mM magnesium chloride; (d) 25 mM sodium acetate; (e) 0.01% by weight of poloxamer 188; (f) 5.4% by weight of sucrose; (g) water; (h) preparing a composition having a pH of 5 to 5.5; freezing the composition for a period of time at a temperature above -80°C to -10°C, e.g., -70°C to -15°C, -60°C to -20°C, -50°C to -30°C, or -45°C to -35°C, wherein upon thawing, 60% or more of the therapeutic agent is recovered compared to the same product stored at -80°C for the same period of time; A method comprising:

52. 1. A method for preserving at least 50% of the relative potency of a therapeutic pharmaceutical product after freezing and thawing said pharmaceutical product, said method comprising: (a) the therapeutic product; and (b) 25 mM sodium chloride; (c) 5 mM magnesium chloride; (d) 25 mM sodium acetate; (e) 0.01% by weight of poloxamer 188; (f) 5.4% by weight of sucrose; (g) water; (h) preparing a composition having a pH of 5 to 5.5; freezing the composition for a period of time at a temperature above -60°C to -10°C, e.g., -60°C to -10°C, -50°C to -20°C, -45°C to -25°C, or -30°C to -40°C, wherein upon thawing, the pharmaceutical product exhibits at least 50% relative potency compared to the same product stored at -80°C for the same period of time; A method comprising:

53. 1. A method for reducing degradation of a therapeutic pharmaceutical, said method comprising: (a) the therapeutic product; and (b) 10 mM Tris; (c) 75 mM sodium chloride; (d) 0.005% by weight of poloxamer 188; (e) 5% by weight of sucrose; (f) water; (g) preparing a composition having a pH of 7.3; lyophilizing the composition and storing it at a temperature between −20° C. and 8° C., e.g., −20° C. to 0° C. or 0° C. to 8° C., for a period of time, wherein 60% or more of the therapeutic agent is recovered after reconstitution of the lyophilized composition compared to the same product stored at −80° C. without lyophilization; A method comprising:

54. 1. A method for reducing degradation of a therapeutic pharmaceutical, the method comprising: (a) the therapeutic product; and (b) 10 mM sodium citrate; (c) 75 mM sodium chloride; (d) 0.001 wt% poloxamer 188; and (e) 7% by weight of sucrose; (f) water; (g) preparing a composition having a pH of 6.0; lyophilizing the composition and storing it at a temperature between −20° C. and 8° C., e.g., −20° C. to 0° C. or 0° C. to 8° C., for a period of time, wherein 60% or more of the therapeutic agent is recovered after reconstitution of the lyophilized composition compared to the same product stored at −80° C. without lyophilization; A method comprising:

55. 1. A method for retaining at least 50% relative efficacy of a therapeutic pharmaceutical agent, said method comprising: (a) the therapeutic product; and (b) 10 mM Tris; (c) 75 mM sodium chloride; (d) 0.005% by weight of poloxamer 188; (e) 5% by weight of sucrose; (f) water; and (g) preparing a composition having a pH of 7.3; lyophilizing the composition and storing it for a period of time at a temperature between −20° C. and 8° C., e.g., −20° C. to 0° C. or 0° C. to 8° C., wherein the pharmaceutical product exhibits at least 50% relative potency after reconstitution of the lyophilized composition compared to the same formulation stored at −80° C. without lyophilization; A method comprising:

56. 1. A method for retaining at least 50% relative efficacy of a therapeutic pharmaceutical agent, said method comprising: (a) the therapeutic product; and (b) 10 mM sodium citrate; (c) 75 mM sodium chloride; (d) 0.001 wt% poloxamer 188; and (e) 7% by weight of sucrose; (f) water; (g) preparing a composition having a pH of 6.0; freeze-drying and storing the composition at a temperature between −20° C. and 8° C., e.g., −20° C. to 0° C. or 0° C. to 8° C., for a period of time, wherein the pharmaceutical product exhibits at least 50% relative potency after reconstitution of the lyophilized composition compared to the same formulation stored at −80° C. without lyophilization; A method comprising:

57. 57. The method of any one of claims 50 to 56, wherein the therapeutic agent is a recombinant adeno-associated virus (rAAV).

58. 1. A method for reducing deamination of amino acids on the capsid of an rAAV, the method comprising: (a) the rAAV; and (b) 25 mM sodium chloride; (c) 5 mM magnesium chloride; (d) 25 mM sodium acetate; (e) 0.01% by weight of poloxamer 188; (f) 5.4% by weight of sucrose; (g) water; and (h) preparing a composition having a pH of 5 to 5.5; A method comprising:

59. 1. A method for reducing viral protein degradation of an rAAV product after freezing and thawing said composition, said method comprising: (a) the rAAV; and (b) 25 mM sodium chloride; (c) 5 mM magnesium chloride; (d) 25 mM sodium acetate; (e) 0.01% by weight of poloxamer 188; (f) 5.4% by weight of sucrose; (g) water; and (h) preparing a composition having a pH of 5 to 5.5; storing the frozen composition for a period of time at a temperature between -80°C and -10°C, for example, at a temperature of -70°C to -15°C, -60°C to -20°C, -50°C to -30°C, or -45°C to -35°C, wherein the change in the ratio of VP1:VP3 in the thawed composition is less than 20% compared to before freezing the composition; A method comprising:

60. 1. A method for reducing deamination of amino acids on the capsid of an rAAV, the method comprising: (a) the rAAV; and (b) 10 mM Tris; (c) 75 mM sodium chloride; (d) 0.005% by weight of poloxamer 188; (e) 5% by weight of sucrose; (f) water; and (g) preparing a composition having a pH of 7.3; A method comprising:

61. 1. A method for reducing deamination of amino acids on the capsid of an rAAV, the method comprising: (a) the rAAV; and (b) 10 mM sodium citrate; (c) 75 mM sodium chloride; (d) 0.001 wt% poloxamer 188; and (e) 7% by weight of sucrose; (f) water; and (g) preparing a composition having a pH of 6.0; A method comprising:

62. 1. A method for reducing the concentration of subvisible particles (SVPs) in an rAAV product after the product has been frozen and thawed, the method comprising: (a) the rAAV; and (b) 25 mM sodium chloride; (c) 5 mM magnesium chloride; (d) 25 mM sodium acetate; (e) 0.01% by weight of poloxamer 188; (f) 5.4% by weight of sucrose; (g) water; (h) preparing a composition having a pH of 5 to 5.5; freezing the composition at −20° C. for at least one year, wherein after thawing, the composition has an SVP concentration of less than 100 particles / mL for particles 10 mm or larger and less than 10 particles / mL for particles 25 mm or larger; A method comprising:

63. 1. A method for reducing the concentration of subvisible particles (SVPs) in an rAAV product after the product has been frozen and thawed, the method comprising: (a) the rAAV; and (b) 25 mM sodium chloride; (c) 5 mM magnesium chloride; (d) 25 mM sodium acetate; (e) 0.01% by weight of poloxamer 188; (f) 5.4% by weight of sucrose; (g) water; (h) preparing a composition having a pH of 5 to 5.5; freezing the composition at −20° C. for at least one year, wherein after thawing, the composition has an SVP concentration of less than 50 particles / mL for particles 10 μm or larger and less than 5 particles / mL for particles 25 μm or larger; A method comprising:

64. 1. A method for reducing the subvisible particle (SVP) concentration of an rAAV product after the product has been freeze-thawed for five cycles, the method comprising: (a) the rAAV; and (b) 25 mM sodium chloride; (c) 5 mM magnesium chloride; (d) 25 mM sodium acetate; (e) 0.01% by weight of poloxamer 188; (f) 5.4% by weight of sucrose; (g) water; (h) preparing a composition having a pH of 5 to 5.5; freezing the composition for a period of time at a temperature of greater than -80°C to -10°C, e.g., -70°C to -15°C, -60°C to -20°C, -50°C to -30°C, or -45°C to -35°C, wherein the composition is thawed and refrozen five times, and upon the fifth thaw, the SVP concentration is less than 100 particles / mL for particles 10 μm or larger and less than 10 particles / mL for particles 25 μm or larger; A method comprising:

65. 1. A method for reducing the subvisible particle (SVP) concentration of an rAAV product after the product has been freeze-thawed for five cycles, the method comprising: (a) the rAAV; and (b) 25 mM sodium chloride; (c) 5 mM magnesium chloride; (d) 25 mM sodium acetate; (e) 0.01% by weight of poloxamer 188; (f) 5.4% by weight of sucrose; (g) water; (h) preparing a composition having a pH of 5 to 5.5; freezing the composition for a period of time at a temperature of greater than -80°C to -10°C, e.g., -70°C to -15°C, -60°C to -20°C, -50°C to -30°C, or -45°C to -35°C, wherein the composition is thawed and refrozen five times, and upon the fifth thaw, the SVP concentration is less than 50 particles / mL for particles 10 μm or larger and less than 5 particles / mL for particles 25 μm or larger; A method comprising:

66. 1. A method for reducing high molecular weight species (HMW) in an rAAV product after said product has been freeze-thawed for five cycles, said method comprising: (a) the rAAV; and (b) 25 mM sodium chloride; (c) 5 mM magnesium chloride; (d) 25 mM sodium acetate; (e) 0.01% by weight of poloxamer 188; (f) 5.4% by weight of sucrose; (g) water; (h) preparing a composition having a pH of 5 to 5.5; freezing the composition for a period of time at a temperature of greater than -80°C to -10°C, e.g., -70°C to -15°C, -60°C to -20°C, -50°C to -30°C, or -45°C to -35°C, wherein the composition is thawed and refrozen five times, and upon the fifth thaw, HMW species increase by less than 3% by weight compared to the same product at TO as measured by analytical ultracentrifugation; A method comprising:

67. 1. A method for reducing high molecular weight species (HMW) in an rAAV product after the product has been frozen and thawed, the method comprising: (a) the rAAV; and (b) 25 mM sodium chloride; (c) 5 mM magnesium chloride; (d) 25 mM sodium acetate; (e) 0.01% by weight of poloxamer 188; (f) 5.4% by weight of sucrose; (g) water; (h) preparing a composition having a pH of 5 to 5.5; freezing the composition at −20° C. for at least one year, wherein after thawing, HMW species have increased by less than 3% by weight compared to the same product at TO as measured by analytical ultracentrifugation; A method comprising:

68. 1. A method for reducing viral protein degradation of an rAAV product after the product has been lyophilized, the method comprising: (a) the rAAV; and (b) 10 mM Tris; (c) 75 mM sodium chloride; (d) 0.005% by weight of poloxamer 188; (e) 5% by weight of sucrose; (f) water; and (g) preparing a composition having a pH of 7.3; storing the freeze-dried composition for a period of time at a temperature between -20°C and 8°C, for example at a temperature between -20°C and 0°C or 0°C and 8°C, wherein the VP1:VP3 ratio in the reconstituted freeze-dried product changes by less than 20% compared to before freeze-drying; A method comprising:

69. 1. A method for reducing viral protein degradation of an rAAV product after the product has been lyophilized, the method comprising: (a) the rAAV; and (b) 10 mM sodium citrate; (c) 75 mM sodium chloride; (d) 0.001 wt% poloxamer 188; and (e) 7% by weight of sucrose; (f) water; (g) preparing a composition having a pH of 6.0; storing the freeze-dried composition for a period of time at a temperature between -20°C and 8°C, for example at a temperature between -20°C and 0°C or 0°C and 8°C, wherein the VP1:VP3 ratio in the reconstituted freeze-dried product changes by less than 20% compared to before freeze-drying; A method comprising:

70. 1. A method for reducing subvisible particle (SVP) concentrations of an rAAV product, the method comprising: (a) the rAAV; and (b) 10 mM Tris; (c) 75 mM sodium chloride; (d) 0.005% by weight of poloxamer 188; (e) 5% by weight of sucrose; (f) water; and (g) preparing a composition having a pH of 7.3; storing the lyophilized composition for a period of time at a temperature between -20°C and 8°C, for example at a temperature between -20°C and 0°C or 0°C and 8°C, wherein the formulation has been subjected to one lyophilization cycle and the lyophilized composition has an SVP of less than 100 particles / mL for particles 10 μm or larger and less than 10 particles / mL for particles 25 μm or larger; A method comprising:

71. 1. A method for reducing subvisible particle (SVP) concentrations of an rAAV product, the method comprising: (a) the rAAV; and (b) 10 mM Tris; (c) 75 mM sodium chloride; (d) 0.005% by weight of poloxamer 188; (e) 5% by weight of sucrose; (f) water; and (g) preparing a composition having a pH of 7.3; storing the lyophilized composition for a period of time at a temperature between -20°C and 8°C, for example at a temperature between -20°C and 0°C or 0°C and 8°C, wherein the formulation has been subjected to one lyophilization cycle and the lyophilized composition has an SVP of less than 50 particles / mL for particles 10 μm or larger and less than 5 particles / mL for particles 25 μm or larger; A method comprising:

72. 1. A method for reducing subvisible particle (SVP) concentrations of an rAAV product, the method comprising: (a) the rAAV; and (b) 10 mM sodium citrate; (c) 75 mM sodium chloride; (d) 0.001 wt% poloxamer 188; and (e) 7% by weight of sucrose; (f) water; (g) preparing a composition having a pH of 6.0; storing the lyophilized composition for a period of time at a temperature between -20°C and 8°C, for example at a temperature between -20°C and 0°C or 0°C and 8°C, wherein the formulation has been subjected to one lyophilization cycle and the lyophilized composition has an SVP of less than 100 particles / mL for particles 10 μm or larger and less than 10 particles / mL for particles 25 μm or larger; A method comprising:

73. 1. A method for reducing subvisible particle (SVP) concentrations of an rAAV product, the method comprising: (a) the rAAV; and (b) 10 mM sodium citrate; (c) 75 mM sodium chloride; (d) 0.001 wt% poloxamer 188; and (e) 7% by weight of sucrose; (f) water; (g) preparing a composition having a pH of 6.0; storing the lyophilized composition for a period of time at a temperature between -20°C and 8°C, for example at a temperature between -20°C and 0°C or 0°C and 8°C, wherein the formulation has been subjected to one lyophilization cycle and the lyophilized composition has an SVP of less than 50 particles / mL for particles 10 μm or larger and less than 5 particles / mL for particles 25 μm or larger; A method comprising:

74. 1. A method for reducing low molecular weight species (LMW) in an rAAV product after the product has been lyophilized, the method comprising: (a) the rAAV; and (b) 10 mM sodium citrate; (c) 75 mM sodium chloride; (d) 0.001 wt% poloxamer 188; and (e) 7% by weight of sucrose; (f) water; (g) preparing a composition having a pH of 6.0; storing the lyophilized composition for a period of time at a temperature between -20°C and 8°C, for example at a temperature between -20°C and 0°C or 0°C and 8°C, wherein the lyophilized product is reconstituted and the LMW species is less than 5% by weight compared to the same product without lyophilization, as measured by analytical ultracentrifugation; A method comprising:

75. 1. A method for reducing low molecular weight species (LMW) in an rAAV product after the product has been lyophilized, the method comprising: (a) the rAAV; and (b) 10 mM Tris; (c) 75 mM sodium chloride; (d) 0.005% by weight of poloxamer 188; (e) 5% by weight of sucrose; (f) water; and (g) preparing a composition having a pH of 7.3; storing the lyophilized composition for a period of time at a temperature between -20°C and 8°C, for example at a temperature between -20°C and 0°C or 0°C and 8°C, wherein the lyophilized product is reconstituted and the LMW species is less than 5% by weight compared to the same product without lyophilization, as measured by analytical ultracentrifugation; A method comprising:

76. 1. A method for reducing high molecular weight species (HMW) in an rAAV product after the product has been lyophilized, the method comprising: (a) the rAAV; and (b) 10 mM sodium citrate; (c) 75 mM sodium chloride; (d) 0.001 wt% poloxamer 188; and (e) 7% by weight sucrose; (f) water; (g) preparing a composition having a pH of 6.0; storing the lyophilized composition for a period of time at a temperature between -20°C and 8°C, for example at a temperature between -20°C and 0°C or 0°C and 8°C, wherein the lyophilized product is reconstituted and HMW species increase by less than 3% by weight compared to the same product without lyophilization, as measured by analytical ultracentrifugation; A method comprising:

77. 1. A method for reducing high molecular weight (HMW) in an rAAV product after lyophilization of the product, the method comprising: (a) the rAAV; and (b) 10 mM Tris; (c) 75 mM sodium chloride; (d) 0.005% by weight of poloxamer 188; (e) 5% by weight of sucrose; (f) water; and (g) preparing a composition having a pH of 7.3; storing the lyophilized composition for a period of time at a temperature between -20°C and 8°C, for example at a temperature between -20°C and 0°C or 0°C and 8°C, wherein the lyophilized product is reconstituted and HMW species increase by less than 3% by weight compared to the same product without lyophilization, as measured by analytical ultracentrifugation; A method comprising:

78. 78. The method of claims 57-77, wherein the rAAV comprises a capsid derived from one or more AAV selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV9.47, AAV9(hu14), AAV10, AAV11, AAV12, Rh8, Rh10, Rh74, AAV3B, AAV-2i8, RHM4-1 (VP1, SEQ ID NO: 1 or VP2, SEQ ID NO: 2 or VP3, SEQ ID NO: 3), DJ, DJ8, NP59, Anc-80, and variants thereof.

79. 79. The method of any one of claims 57 to 78, wherein the rAAV comprises a transgene encoding a polypeptide or a nucleic acid selected from the group consisting of an siRNA, an antisense molecule, an miRNA, a ribozyme, and an shRNA.

80. The rAAV may encode GAA (acid α-glucosidase), ATP7B (copper-transporting ATPase 2), α-galactosidase A (GLA), ASS1 (argininosuccinate synthase), β-glucocerebrosidase, β-hexosaminidase A, SERPING1 (C1 protease inhibitor or C1 esterase inhibitor), glucose-6-phosphatase, CFTR (cystic fibrosis transmembrane conductance regulator), blood clotting (clotting) factors (e.g., factor XIII, factor IX, factor VIII, factor X, factor VII, factor VIIa, protein C), or a functional protein. Acquired blood coagulation factors, antibodies, retinal pigment epithelium-specific 65 kDa protein (RPE65), erythropoietin, LDL (low density lipoprotein) receptor, lipoprotein lipase (LPL), ornithine transcarbamylase (OTC), β-globin, α-globin, spectrin, α-antitrypsin, adenosine deaminase (ADA), metal transporters (ATP7A or ATP7), sulfamidase, enzymes involved in lysosomal storage diseases (ARSA), hypoxanthine guanine phosphoribosyltransferase, β-25 glucocerebromoglycerin sphingomyelinase, lysosomal hexosaminidase, branched-chain ketoacid dehydrogenase, hormones, growth factors, insulin-like growth factor 1 or 2, platelet-derived growth factor (PDGF), epidermal growth factor (EGF), nerve growth factor (NGF), neurotrophic factor-3 and -4, brain-derived neurotrophic factor (BDNF), glial cell line-derived growth factor (GDNF), transforming growth factor α and β, cytokines, α-interferon, β-interferon, interferon-γ, interleukin-2 (IL-2), interleukin- 4 (IL-4), interleukin 12 (IL-12), granulocyte-macrophage colony-stimulating factor (GM-CSF), lymphotoxin (LT), suicide gene products, herpes simplex virus thymidine kinase, cytosine deaminase (CD), diphtheria toxin (DT), cytochrome P450 (CYP), deoxycytidine kinase (DCK), tumor necrosis factor (TNF), drug resistance proteins, tumor suppressor proteins (e.g., p53, Rb, Wt-1, NF1, von Hippel-Lindau (VHL), adenomatous polyposis coli (APC)), peptides with immunomodulatory properties,Tolerogenic or immunogenic peptide or protein tregitope or hCDR1 (edorazide), insulin, glucokinase (GCK), guanylate cyclase 2D (GUCY2D) (or Leber congenital amaurosis (LCA) associated with GUCY2D variant (GUCY2D-LCA)), Rab escort protein 1 (REP1) (choroideremia (CHM) encodes REP1), LCA5 (Leber congenital amaurosis 5) (LCA-lebercillin), ornithine ketoacid aminotransferase (gyrate atrophy), retinoschisin 1 (X-linked retinoschisis), USH1C (Usher syndrome 1C), X-linked retinitis pigmentosa GTPase (XLRP), MERTK (AR form of RP: retinitis pigmentosa) 80. The method of claim 79, wherein the transgene encodes one or more of the following: DFNB1 (connexin 26 deafness), ACHM2, 3 and 4 (achromatosis), PKD-1+ or PKD-2 (polycystic kidney disease), TPP1 (tripeptidyl peptidase 1), CLN2 (neuronal ceroid lipofuscinosis 2), sulfatase, N-acetylglucosamine-1-phosphate transferase, cathepsin A, GM2-AP (ganglioside GM2 activator), NPC1 (NPC intracellular cholesterol transporter 1), VPC2, sphingolipid activating protein, one or more zinc finger nucleases for genome editing, one or more donor sequences used as repair templates for genome editing, or variants thereof.

81. 81. The method of claim 80, wherein the rAAV comprises the transgene encoding GAA (acid alpha-glucosidase).

82. 81. The method of claim 80, wherein the rAAV comprises the transgene encoding ATP7B (copper transporting ATPase 2).

83. 81. The method of claim 80, wherein the rAAV comprises the transgene encoding alpha galactosidase A (GLA).

84. 81. The method of claim 80, wherein the rAAV comprises the transgene encoding ASS1 (argininosuccinate synthase).

85. 81. The method of claim 80, wherein the rAAV comprises the transgene encoding β-glucocerebrosidase.

86. 81. The method of claim 80, wherein the rAAV comprises the transgene encoding beta-hexosaminidase A.

87. 81. The method of claim 80, wherein the rAAV comprises the transgene encoding SERPING1 (C1 protease inhibitor or C1 esterase inhibitor).

88. 81. The method of claim 80, wherein the rAAV comprises the transgene encoding glucose-6-phosphatase.

89. 81. The method of claim 80, wherein the rAAV comprises the transgene encoding CFTR (cystic fibrosis transmembrane conductance regulator).

90. 81. The method of claim 80, wherein the rAAV comprises the transgene encoding a blood clotting (clotting) factor (e.g., Factor XIII, Factor IX, Factor VIII, Factor X, Factor VII, Factor Vila, Protein C).

91. 79. The method of claim 27 or 78, wherein the selected rAAV comprises VP1, SEQ ID NO: 1 or VP2, SEQ ID NO: 2 or VP3, SEQ ID NO:

3.

92. 81. The method of claim 80, wherein the selected rAAV comprises VP1, SEQ ID NO: 1 or VP2, SEQ ID NO: 2 or VP3, SEQ ID NO:

3.

93. 27. The formulation of any one of claims 2 and 24-26, wherein the rAAV comprises a capsid comprising VP1 of SEQ ID NO:

1.

94. 94. The formulation of claim 93, wherein the capsid further comprises VP2 comprising SEQ ID NO:

2.

95. 95. The formulation of claim 94, wherein the capsid further comprises VP3 of SEQ ID NO:

3.

96. 93. The formulation or method of any one of claims 1 to 92, wherein the pharmaceutical agent is a rAAV, and the rAAV comprises a capsid comprising VP1 of SEQ ID NO:

1.

97. 97. The formulation of claim 96, wherein the capsid further comprises VP2 comprising SEQ ID NO:

2.

98. 99. The formulation of claim 98, wherein the capsid further comprises VP3 of SEQ ID NO: 3.

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