AAV vector formulation

Abstract

Disclosed herein are aqueous compositions comprising rAAV vectors and an excipient that stabilizes the rAAV vector, wherein the excipient comprises a gum isolated from an Acacia tree, including Senegalia senegal and Vachellia seyal.

Description

AAV VECTOR FORMU LATIONCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority under 35 U.S.C. § 119(e) to U.S. Provisional Application Serial No. 63 / 514967 filed on July 21, 2023, the disclosure of which is expressly incorporated herein.FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

[0002] This invention was made with government support under HL160472 awarded by National Institutes of Health. The Government has certain rights in the invention.BACKGROUND

[0003] Gene therapy is the delivery of exogenous nucleic acids to correct or replace defective genes. Among viral vectors available for gene therapy, adeno-associated virus (AAV) has become the most popular modality in biotechnology today. AAV is a small icosahedral non-pathogenic parvovirus with a non-enveloped protein capsid. Recombinant AAV vectors (rAAVs) are composed of the same capsid sequence and structure as found in wild-type AAVs (wtAAVs). However, rAAVs comprise genomes that are devoid of all AAV protein-coding sequences and have therapeutic gene expression cassettes in their place. The only sequences of viral origin are the inverted-terminal-repeats (ITRs), which are needed to guide genome replication and packaging during vector production. The complete removal of viral coding sequences maximizes the packaging capacity of rAAVs and contributes to their low immunogenicity and cytotoxicity when delivered in vivo. The ITR-flanked rAAV genome can be cloned into plasmids and can be conveniently manipulated using standard molecular cloning techniques.

[0004] rAAVs have gained much attention in the past 20 years due to their non- pathogenic nature, low host integration, and broad tropism that can be tailored to deliver therapeutic genes to treat a variety of illnesses / disorders. Hundreds of AAV- related Phasel / I I clinical trials are currently being conducted.

[0005] Despite the approved AAVs and success of numerous clinical trials, challenges and limitations of rAAV vectors remain. One limitation relates to the stability of the rAAV vectors. The manufacturing production and shelf-life of AAV vectors still rely on cold storage (e.g. keeping the viral vectors at -20°C or -80°C) like many vaccines. Yet, even under such storage conditions, AAV degradation still occurs after being frozen, and / or after each freeze-thaw cycle. Accordingly, compositions that can stabilized AAV vectors are desirable. As disclosed herein the addition of an inexpensive excipient (at very low concentrations) has been discovered to significantly stabilize AAV vectors and slow down the degradation of AAV vectors after many freeze-thaw cycles. Even after being exposed to room temperature for half a month, AAVs retained their ability to effect gene transfer in vivo more efficiently than the AAVs controls suspended in commonly used phosphate buffer saline (PBS).SUMMARY

[0006] In accordance with one embodiment of the present disclosure, a method is provided for stabilizing an adenovirus or adeno-associated virus (AAV), including rAAVs, and improving their transfection and transduction efficacy. The method comprises the step of combining the AAV / rAAV with an excipient comprising a gum (or fractional components thereof) isolated from an Acacia tree, including for example Acacia tree species of Senegalia Senegal and / or Vachellia seya\. In one embodiment the method comprises combining a solution comprising an rAAV with gum arabic wherein the gum arabic is present in an amount selected from a range of about 0.1% to about 5% w / v, optionally wherein the gum arabic is present at about 1% w / v.

[0007] In accordance with one embodiment, a stabilized adenovirus or AAV vector formulation is provided wherein the composition comprises an adenovirus or AAV vector and an excipient that stabilizes the adenovirus or AAV vector during storage and / or exposure to elevated temperatures, including temperatures greater than - 20°C, above 0°C, and up to room temperature (about 20°C to about 22°C) or higher. In accordance with one embodiment an inexpensive excipient, comprising natural gum isolated from acacia trees, is mixed with an adenovirus or AAV formulation to significantly stabilize and slow down the degradation of adenovirus or AAV vectorsduring storage, exposed to freeze-thaw cycles, and / or exposed to room temperature, relative to adenovirus or AAV comprising compositions lacking the natural gum excipient. In one embodiment the natural gum excipient is gum arabic, wherein the gum arabic is present in an amount selected from a range of about 0.1% to about 5% w / v, optionally wherein the gum arabic is present at about 1% w / v. In accordance with one embodiment a composition is provided comprising an rAAV vector, a natural gum excipient from an acacia tree, optionally gum arabic, and a pharmaceutically acceptable carrier.

[0008] Compositions comprising AAV vectors suspended in an aqueous carrier comprising a natural gum component, including those isolated from acacia trees, retained their functional ability to conduct gene transfer in vivo, relative to the controls comprising AAVs suspended in commonly used phosphate buffer saline (PBS). A solution comprising 1% of gum arabic has been discovered to provide enhanced stability to AAV vectors (relative to other standard excipients) and conferred the highest efficacy of gene transfer both in vitro and in vivo compared to counterpart PBS control, sucrose additives, and another excipient T1 (see Table 1), even in the absence of freeze thaw cycles or long term storage.

[0009] In accordance with one embodiment a composition is provided comprising an AAV vector suspended in an aqueous carrier, wherein the aqueous carrier comprises gum arabic. Optionally, the aqueous carrier can be a pharmaceutically acceptable carrier comprising the gum arabic, and in some embodiments the carrier further comprises a buffer. In accordance with one embodiment the composition comprisesan AAV vector suspended in an aqueous carrier that comprises a final concentration (%w / v) of about 0.01% to about 5% gum arabic.

[0010] In accordance with one embodiment a pharmaceutical composition is provided comprising a recombinant adeno-associated virus in aqueous solution that comprises 0.5, 1.0, or 2% w / v gum arabic and a pharmaceutically acceptable carrier, wherein the composition is sterilized and optionally stored within various package containers. In other embodiments the pharmaceutical compositions can be further packaged as part of a kit that includes a disposable device for administering the composition to a patient. The containers or kits may be labeled for storage at ambient room temperature or at refrigerated temperature.BRIEF DESCRIPTION OF DRAWINGS

[0011] Figs. 1A-1D provide data for the preservation of AAV9 infectivity when combined with different additives versus time left at room temperature. AAV9 packaged scAAV-CB-Gluc was formulated in different additives and used to infect GM16095 cells. Fig. 1A shows the results for AAV9 infectivity when stored in PBS and NaCI; Fig. IB shows the results for AAV9 infectivity when stored in PBS and sucrose; Fig. 1C shows the results for AAV9 infectivity when stored in PBS and pullulan (Tl); and Fig. ID shows the results for AAV9 infectivity when stored in PBS and gum arabic (T2); Shown are transduction data of Gaussia luciferase measured 48hr post infection. N = 3 per group.

[0012] Figs. 2A and 2B show the thermal stability of AAV8 (Fig. 2A) and AAV9 (Fig. 2B) in different additives measured by infectivity after being heated at 56°C for lOmin. GM16095 cells were transduced and gene expression (either Flow cytometry or Glue assay) was performed at 72hr post injection.

[0013] Fig. 3. presents data demonstrating AAV9 infectivity preserved by different additives versus number of freeze-thaw cycles. N =3 per group.

[0014] Figs. 4A and 4B present data showing AAV8 infectivity preserved by different additives vs time over 2 weeks left at room temperature (Fig. 4A) or versus 3 freezethaw cycles (Fig 4B). Fresh = freshly thawed right before infection of GM16095 cells as a control group.

[0015] Fig. 5. presents data from an in vivo study with AAV9-TTR-cohFIX after 2 weeks left at room temperature in different excipients (except group Fresh was freshly thawed AAV right before injection as a control group). Human Factor IX expressed in C3H / HeJ-HB mice at week 3 post injection (dose = lEllvg / mouse).

[0016] Fig. 6 shows the structure of the main gum arable polysaccharide, wherein n is an integer greater than 2.

[0017] Figs. 7A-7D: Transduction efficiency of AAV9-GLUC in different excipients / additives being stored at room temperature for 3 weeks. Data shown as GLUC assay measurements at 48hr post infection with an MOI = 25k on GM16095 cells; n = 3 wells / group; dotted line is background signal. Base buffer in all samples was PBS plus different excipients as indicated: NaCI (Fig. 7A), sucrose (Fig. 7B), T1 (Fig. 7C), and T2 (Fig. 7D).

[0018] Figs. 8A-8E: AAV9's transduction efficiency versus time and elevated temperatures in PBS plus different excipients. Transduction efficiency of AAV9 formulated in different buffers was measured as relative fluorescent units (RFU) from transduction of GM16095 cells: versus storage time at 37°C (Fig. 8A), versus Freeze / Thaw (F / T) cycles (Fig. 8B), at five F / T cycles (Fig. 8C), and versus heated at 65°C for 3min (Figs. 8D and 8E). Data in (Fig. 8E) are normalized from data in (Fig. 8D). N = 3 wells / group. Two-way ANOVA.

[0019] Figs. 9A-9F: Transduction efficiency of AAV5 and AAV8 in different excipients over multiple F / T cycles. Transduction efficiency on GM16095 cells measured at 72hr post infection, based on the transgene GFP (flow cytometry) delivered by AAV5 after F / T cycles (Figs. 9A, 9C) or by AAV8 (Figs. 9B, 9D), and after storage at 37°C for 15 days by AAV5 (Fig. 9E) or AAV8 (Fig. 9F). N = 3 wells / group. Two-way ANOVA.

[0020] Figs. 10A-10B: Transduction efficiency of AAV8 and AAV9 in different excipients after being thawed out of -20°C versus heated at 56°C for 10 min. Transduction efficiency on GM16095 cells measured at 72hr post infection, based on the transgene delivered by AAV8 (Fig. 10A, %GFP positive cells from flow cytometry) and AAV9 (Fig. 10B, RFU of Gaussia luciferase secreted). N = 3 wells / group. One-way ANOVA.

[0021] Fig. 11A-11G In vivo transduction efficiency of AAV8-CB-eGFP stored at room temperature for 2 weeks in different excipients. The study design is shown in Fig.11A. Direct GFP expression from WT BALB / c whole liver cells, measured by flow cytometry at week-2 post IV-injection as % positive cells (Fig. 11B). Representative dot plots of the gated population from different formulations are shown (Figs. 11C- 11G). N = 3-5 mice / group. Dose = lEllvg / mouse. One-way ANOVA.

[0022] Fig. 12A-12D: In vivo comparison of systemic human-FIX expression in hemophilia B mice delivered by AAV9 stored in different conditions. The study design is shown in Fig. 12A. Comparison of plasma ALT activity levels is shown in Fig. 12B. Transduction efficiency of AAV9-TTR-cohFIX stored at room temperature for 2 weeks in different additives before IV injection + a fresh PBS AAV control (without room temperature incubation) is shown in Fig. 12C. Fold change comparison is shown for l%T2-2wkRT versus PBS-2wkRT is shown in Fig. 12D. N = 3-5 mice / group; dose = lEllvg / mouse. One-way ANOVA. *See Methods section for a Note regarding Panel C data.DETAILED DESCRIPTIONDEFINITIONS

[0023] In describing and claiming the invention, the following terminology will be used in accordance with the definitions set forth below.

[0024] The term "about" as used herein means greater or lesser than the value or range of values stated by 10 percent but is not intended to limit any value or range of values to only this broader definition. Each value or range of values preceded by the term "about" is also intended to encompass the embodiment of the stated absolute value or range of values.

[0025] As used herein, the term "purified" and like terms relate to the isolation of a molecule or compound in a form that is substantially free of contaminants normally associated with the molecule or compound in a native or natural environment. As used herein, the term "purified" does not require absolute purity; rather, it is intended as a relative definition. The term "purified polypeptide" is used herein to describe a polypeptide which has been separated from other compounds including, but not limited to nucleic acid molecules, lipids and carbohydrates.

[0026] The term "isolated" requires that the referenced material be removed from its original environment (e.g., the natural environment if it is naturally occurring). For example, a naturally-occurring polynucleotide present in a living animal is not isolated, but the same polynucleotide, separated from some or all of the coexisting materials in the natural system, is isolated.

[0027] As used herein, the term "pharmaceutically acceptable carrier" includes any of the standard pharmaceutical carriers, such as a phosphate buffered saline solution, water, emulsions such as an oil / water or water / oil emulsion, and various types of wetting agents. The term also encompasses any of the agents approved by a regulatory agency of the US Federal government or listed in the US Pharmacopeia for use in animals, including humans.

[0028] The term "AAV vector" as used herein defines a delivery vehicle comprising an AAV capsid protein and an associated nucleic acid sequence, wherein the vector delivers the associated nucleic acid sequence into the interior of a cell upon contact with the cell.

[0029] The term "operably linked to" refers to the functional relationship of a nucleic acid with another nucleic acid sequence. Promoters, enhancers, transcriptional and translational stop sites, and other signal sequences are examples of nucleic acid sequences that can operably linked to other sequences. For example, operable linkage of DNA to a transcriptional control element refers to the physical and functional relationship between the DNA and promoter such that the transcription of such DNA is initiated from the promoter by an RNA polymerase that specifically recognizes, binds to and transcribes the DNA.

[0030] The term "tropism" as used herein in reference to viruses, defines the target cellular types that a virus preferentially, or has the capacity to, infect and establish a successful infection.

[0031] The term "chemical modification" as used herein defines a reaction where a protein is contacted with a reagent to produce a modified protein that comprises a chemical moiety covalent linked to the native protein.

[0032] As used herein the term gum arabic is intended to cover any natural exudate isolated from an Acacia tree. A fractional component of gum arabic is defined as any derivative product of gum arabic that stabilizes an AAV vector present in an aqueous formulation relative to a control aqueous formulation comprising the AAV vector but lacking the gum arabic component. In one embodiment gum arabic comprises about 20-50% Galactose, about 10-40% Arabinose about 5-30% Rhamnose and about 5 to 40% Glucuronic acid. In one embodiment gum arabic comprises a branched polysaccharide composed of a backbone of (1, 3)-lin ked |3-d-galactopyranosyl units and 2 to 5 side chains consisting of (1, 3 [-linked -d-galactopyranosyl units (see Fig. 6).

[0033] As used herein the term "pullulan" comprises a polysaccharide polymer composed of maltotriose units (oc-1,4- glucan oc-1,6 glucan), whereas consecutive maltotriose units are connected to each other by cc-1,6 glycosidic bond.EMBODIMENTS

[0034] As disclosed herein compositions and methods are provided for the stabilization and storage of viral vectors, including for example AAV delivery vehicles. In accordance with the present disclosure, a viral vector stabilizing composition is provided wherein the composition comprises an aqueous solution containing a natural exudate isolated from an Acacia tree present in an amount selected from the range of about 0.1% to about 5.0% w / v, about 0.1% to about 3.0% w / v, or about 0.1% to about 2.0% w / v of the final aqueous solution. The excipient can be added to any solution (or formulation) containing a viral vector (including for example AAV vectors) to stabilize the viral vector. In one embodiment the excipient is an aqueous solution comprising about 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7% , 0.8%, 1.0%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 2.0%, 2.5%, 3.0%, 3.5%, 4.0%, 4.5% or about 5.0% w / v of the final aqueous solution

[0035] The viral vector stabilizing composition can be combined with a viral vector, such as an adeno-associated virus (AAV) or an rAAV vector to enhance the stability and transfection efficacy of the viral vector. In one embodiment a composition is provided comprising a viral vector and an excipient in an effective amount to stabilize the viral vector, wherein the excipient is an exudate isolated from an Acacia tree. In one embodiment the AAV stabilizing excipient comprises a gum isolated from an Acacia tree, including gums isolated from the Acacia tree species of Senegalia Senegal and / or Vachellia seyal. In one embodiment the AAV stabilizing excipient of the present invention is gum arabic present in an amount selected from the range of about 0.1% to about 2% w / v, optionally present in an amount of about 1% w / v. In one embodiment an adeno-associated virus (AAV) stabilizing composition is provided wherein the composition comprises an aqueous solution containing an rAAV vector and gum arabic.

[0036] In one embodiment a method of increasing the efficacy of transfection of an AAV vector is provided, wherein the method comprises adding a natural gum isolated from the Acacia tree species of Senegalia Senegal and / or Vachellia seyal to a pharmaceutical composition comprising an AAV vector. In one embodiment the natural gum is gum arabic.

[0037] In one embodiment the excipient of the present compositions is present in an amount sufficient to stabilize the AAV vector to exposure to elevated temperatures, including extended exposure times to room temperature, e.g., 20°C, as well as exposure to repeated freeze thaw cycles. In one embodiment the excipient is present in the aqueous AAV formulations at a w / v percentage selected from 0.1%, 0.5%, 1.0%, 1.5% 2.0%, 3.0%, 4.0% or 5.0%.

[0038] Gum Arabic or acacia gum (AG) is an exudate produced by Acacia trees, which shields them against insects, molds, and drought. Highly water soluble, gum arabic has been under extensive use since the dawn of civilization with an extensive array of applications, notably as a binder and emulsifier. This natural hydrocolloid is composed of a complex mixture of biopolymers. In order to understand gum arabic interfacial properties, its composition and associated structures have been extensively studied. The gum was first identified as a heterogeneous mixture of protein and polysaccharides differing in their molecular weight and hydrophobicity.

[0039] AG is a complex polysaccharide, either neutral or slightly acidic, found as a mixed calcium salt of a polysaccharide acid (Arabic acid). The backbone is composed of 1,3-1 i n ked |3-D-galactopyranosyl units. The side chains are composed of two or five 1,3-linked |3-D- galactopyranosyl units, joined to the main chain by 1,6-linkages. Both the main and the side chains contain units of oc-L-arabinofuranosyl, a-L-rhamnopyranosyl, [3-D-glucuronopyranosyl and 4-O-methyl-P-D-glucuropyranosyl, the last two mostly as end units. Idris et al. (1998) reported AG to be comprised of 39-42% galactose, 24-27% arabinose, 12- 16% rhamnose, 15-16% glucuronic acid, 1.5-2.6% protein, 0.22-0.39% nitrogen, and 16.0% moisture. The chemical composition and physical chemical properties of AG can vary with its origin (Acacia Senegal or Acacia seyal), the age of the trees from which it was obtained, climatic conditions and soil environment, and the process submitted after its harvest. AG is a highly heterogeneous material that can be separated into three main fractions by hydrophobic interaction chromatography (HIC). These different values may change depending on gum origin, age, storage conditions, etc. Ray et al. (1995) fractionated AG by both HIC and gel permeation chromatography (GPC); their results were in broad agreement with those of Randall et al. (1989) and Renard et al. (2006). The main amino acids present in the proteinaceous component of AG and arabinogalactan protein complex (AGP) were hydroxyproline, serine and proline, whereas in GP, aspartic acid was the most abundant (Islam, et al., 1997). Osman et al. (1993) fractionated AG by HIC to yield four fractions, all of which had a similar carbohydrate composition, but differed in their content of protein, amino acid composition and molecular weight distribution. All four fractions reacted with an array of anti-arabinogalactan-protein monoclonal antibodies via anti-carbohydrate epitopesand were precipitated by Yariv's reagent, which indicated that all four fractions belonged to the arabinogalactan protein complex family (AGP's family). AGP-type macromolecules represent about 94-96% of total compounds found in Acacia Senegal or seyal gums. Minor components are mainly minerals (~3-5%) including Na, K, Ca, Mg, and trace metals such as Zn, Fe, Pb and Cu ions. Small concentrations of tannins, around 0.4% can be found giving more or less colored gums, which is especially remarkable with Acacia seyal. Variability in tannins content was reported both for A. Senegal (0.3-0.6%) or A. seyal (0.6-1.2%) gums but only on five samples. Others emphasized that tannins can be found in AGs but not on Acacia Senegal var. Senegal gums which is clearly in contradiction with data from Minzhi (2002, 2003). Renard et al. (2006) identified traces of trans ferulic acid, ferulic acid and 8-5' non cyclic diferulic acid in AG and AGP and GP fractions. AGs also contain traces of lipids and enzymes such as oxidases and peroxidases, diastases and pectinases.

[0040] Gum arabic is inexpensive, and so being used an excipient for AAV vector formulation would be greatly economical with tremendous stabilizing effects.

[0041] In brief, gum arabic is a mixture of polysaccharides and amino acids with some of the main ingredients as followed in Table 3.

[0042] In accordance with embodiment 1, an adeno-associated virus (AAV) stabilizing composition is provided wherein the composition comprises

[0043] an AAV vector; and

[0044] an aqueous solution comprising a gum isolated from an Acacia tree.

[0045] In accordance with embodiment 2, the gum of embodiment 1 is isolated from an Acacia tree species of Senegalia Senegal and / or Vachellia seyal.

[0046] In accordance with embodiment 3, the gum of embodiment 1 is isolated from an Acacia tree species of Senegalia Senegal.

[0047] In accordance with embodiment 4, the gum of embodiment 1 is isolated from ad Acacia tree species of Vachellia seyal.

[0048] In accordance with embodiment 5, the gum of embodiment 1 is a mixture of gums isolated from Acacia tree species of Senegalia Senegal and Vachellia seyal mixed at a ratio selected from the group consisting of 75:25, 50:50 and 25:75.

[0049] In accordance with embodiment 6, the composition of any one of embodiments 1 to5 is provided wherein the aqueous solution comprises

[0050] 20-50% Galactose;

[0051] 10-40% Arabinose;

[0052] 5-30% Rhamnose; and

[0053] 5-40% Glucuronic acid.

[0054] In accordance with embodiment 7, the composition of any one of embodiments 1 to6 is provided wherein the aqueous solution comprises gum arabic, or fractional component thereof.

[0055] In accordance with embodiment 8, an adeno-associated virus (AAV) stabilizing composition is provided wherein the composition comprises

[0056] an AAV vector; and

[0057] a composition comprising a branched polysaccharide composed of a backbone of (1, 3)-lin ked -d-galactopyranosyl units and 2 to 5 side chains consisting of (1, 3)-linked -d-galactopyranosyl units (see Fig. 6).

[0058] In accordance with embodiment 9, the adeno-associated virus (AAV) stabilizing composition of embodiment 8 is provided wherein the composition comprises gum arabic.

[0059] In accordance with embodiment 10, a method of improving the transfection efficiency of an AAV vector is provided, wherein the method comprises admixing the AAV vector with an aqueous solution comprising a gum isolated from an Acacia tree, optionally wherein the gum is isolated from an Acacia tree species of Senegalia Senegal and / or Vachellia seyal.

[0060] In accordance with embodiment 11, a method of embodiment 10 is provided wherein the aqueous solution comprises a gum isolated from an Acacia tree species of Senegalia Senegal.

[0061] In accordance with embodiment 12, a method of embodiment 10 is provided wherein the aqueous solution comprises a gum isolated from an Acacia tree species of Vachellia seyal.

[0062] In accordance with embodiment 13, a method of embodiment 10 is provided wherein the aqueous solution comprises a mixture of gums isolated from Acacia tree species of Senegalia Senegal and Vachellia seyal mixed at a ratio selected from the group consisting of 75:25, 50:50 and 25:75.

[0063] In accordance with embodiment 14, a method of any one of embodiments 10 to 13 is provided wherein the aqueous solution comprises20-50% Galactose;10-40% Arabinose;5-30% Rhamnose; and5-40% Glucuronic acid.

[0064] In accordance with embodiment 15, a method of embodiment 10 is provided wherein the aqueous solution comprises gum arabic, or fractional component thereof.

[0065] In accordance with embodiment 16, a method of embodiment 10 is provided wherein the aqueous solution comprises a branched polysaccharide composed of a backbone of (1, 3)-lin ked P-d-galactopyranosyl units and 2 to 5 side chains consisting of (1, 3)-linked P-d-galactopyranosyl units (see Fig. 6).

[0066] In accordance with embodiment 17, the composition of any one of embodiments 1- 9 or the method of any one of embodiments 10 to 16 is provided wherein the AAV is a recombinant adeno-associated virus (rAAV).

[0067] In accordance with embodiment 18, the composition of any one of embodiments 1- 9 or the method of any one of embodiments 10 to 16 is provided wherein the aqueous solution comprises a pharmaceutically acceptable carrier.

[0068] In accordance with embodiment 19, the composition of any one of embodiments 1- 9 or the method of any one of embodiments 10 to 16 is provided wherein the aqueous solution comprises gum arabic present in an amount selected from a range of about 0.1% to about 2%, optionally from a range of about 0.5% to about 1%.

[0069] In accordance with embodiment 20, the composition of any one of embodiments 1- 9 or the method of any one of embodiments 10 to 16 is provided wherein the AAV vector comprises capsid protein and said capsid protein comprises a non-native chemical moiety covalently linked to said capsid protein, wherein said modification alters the tropism of the modified rAAV relative to the original unmodified rAAV.

[0070] In accordance with embodiment 21, the AAV vector of embodiment 20 is provided wherein the capsid protein of the AAV vector is attached to a chemical moiety selected from the group consisting of 5-(Dimethylamino)naphthalene-l-sulfonyl chloride (De), and N- ethylmaleimide (NEM).

[0071] In accordance with embodiment 22, the AAV vector of embodiment 20 is provided wherein the chemical moiety is 5-(Dimethylamino)naphthalene-l-sulfonyl chloride (De) andthe altered tropism is an enhanced preferential uptake of the chemical modified AAV, relative to the original unmodified AAV, by muscle tissue.

[0072]

[0062] In accordance with embodiment 23, the AAV vector of embodiment 20 is provided wherein the chemical moiety is N-ethylmaleimide (NEM) and the altered tropism is an enhanced preferential uptake of the chemical modified AAV, relative to the original unmodified AAV, by bone tissue.

[0073] In accordance with embodiment 24, the composition of any one of embodiments 1- 9 or the method of any one of embodiments 10 to 16 is provided wherein the AAV vector is a recombinant adeno-associated virus (rAAV) selected from serotypes: AAV2, AAV5, AAV5x, AAV8, and AAV9.

[0074] In accordance with embodiment 25 a method of stabilizing an adeno-associated virus is provided wherein said method comprises combining the adeno-associated virus with an aqueous excipient comprising a gum isolated from an Acacia tree.

[0075] In accordance with embodiment 26, the method of embodiment 25 is provided wherein the gum is isolated from Acacia Senegal or Acacia seyal.

[0076] In accordance with embodiment 27 , the method of embodiment 25 is provided wherein the gum is gum arabic.

[0077] In accordance with embodiment 28, the method of any one of embodiments 25-27 is provided wherein the adeno-associated virus is a recombinant adeno-associated virus (rAAV).

[0078] In accordance with embodiment 29, the method of any one of embodiments 25-28 is provided wherein the excipient comprises a pharmaceutically acceptable carrier, optionally comprising a buffer that stabilizes the composition at physiological pH.

[0079] In accordance with embodiment 30, the method of any one of embodiments 25-29 is provided wherein the gum is added to a final concentration selected from a range of about 0.1% to about 5% w / v, optionally wherein the gum is added to a final concentration of about 1% w / v.

[0080] In accordance with embodiment 32 a kit for preparing a composition comprising a stabilized rAVV is provided, wherein said kit comprises

[0081] a container comprising an rAVV; and

[0082] a container comprising a solution of gum arabic, optionally with instructional materials for use.

[0083] In accordance with embodiment 33, the kit of embodiment 32 is provided further comprising a disposable device for administering the composition comprising a stabilized rAVV to a patient, optionally wherein the device comprises a needle and a syringe.EXAMPLESEXAMPLE 1

[0084] Data below are results from in vitro and in vivo experiments utilizing different excipients in PBS for AAV formulations to study their stabilizing effects on AAV vectors (infectivity).

[0085] In brief, in vitro data show that excipient T2 (gum arabic) was best at stabilizing AAV9 and AAV8 from freeze-thaw cycles and exposure to room temperature for up to 3 weeks (Figs. 1 - 4). Excipient T2 also outperformed another test excipient T1 (pullulan) and 20-30% sucrose at protecting AAV vectors from degradation from freeze-thaw stress and thermal stress (being heating at 56°C for 10 min as shown in Fig. 2). To confirm this, in vivo diseased C3H / HeJ-HB mice (hemophilic B mice) were IV-injected with AAV9 being left at room temperature for 2 weeks in different excipients. Fig. 5 shows the results; 1% T2 formulation did stabilized and preserved the AAV9's infectivity to be the best gene transfer as a measure of human-Factor9 at week 3 post-injection, compared to other excipients tested, or even the freshly thawed vector.EXAMPLE 2

[0086] One of the most revolutionary advances in modern medicine is human gene therapy - the delivery of a gene of interest into the human body to treat a specific disease. The cell & gene therapy market is estimated at over $9 billion in 2023 and is predicted to hit $45 billion by 2028. Having many desirable attributes and low pathogenicity, recombinant adeno- associated virus (AAV) has become a leading platform in gene therapy. Breakthrough AAV products have made their way to the clinic include the treatments of hemophilia B (HEMGENIX), spinal muscular atrophy (ZOLGENSMA), and Leber congenital amaurosis (LUXTURNA). Despite significant clinical successes, development of AAV drugs still faces many technical challenges including the costly manufacturing production, storage, shipping, and handling. Like other viral vectors and vaccines, current manufacturing production and shelf-life of AAVs rely on the complex cold-chain storages to keep them cold, frozen, and away from light. Yet, AAV aggregation and degradation still occur after a limited time or even after each freeze-thaw cycle. This cold-chain problem accounts for a substantial cost of all biopharma logistic spending. In developing countries, it's even a bigger problem for shipping and storage due to the lack of infrastructure for the required temperature- controlled systems.

[0087] To achieve an optimal therapeutic gene transfer, AAV vectors must be formulated in a way to maximize their stability and transduction efficiency. In a previous study, thermal stability and capsid melting temperature differ from serotype to serotype. Any concentrated vector stocks are prone to aggregation and sorption on to surfaces of container (glass or plastic) and administration devices (syringes), which can reduce infectious particles and lead to significant losses of therapeutic efficacy, especially enduring the freeze-thaw stress. Mechanisms for these processes mostly involve intra-molecular forces, hydrophobicity, ionic strength, electrostatic interactions of the capsid proteins in different solution conditions. A rational design for AAV formulation is often studying solution conditions where additives or excipients are added to improve AAV stability and minimize physical and chemical instability. However, there is a lack of knowledge for such rational and systemic methods of excipient selection for each AAV serotype. To bridge this knowledge gap, the present study evaluated multiple excipients (NaCI, sucrose, pullulan, and gum arabic) in a based buffer of phosphate- buffered saline (PBS) for AAV5, AAV8, and AAV9 formulation development.

[0088] Sucrose was chosen because it is the most common effective anti-freezing additive for viral vaccine stabilization (examples: influenza, anthrax, MMR, and COVID-19). Two other test excipients were included as T1 and 72 which were pullulan and gum arabic, respectively (See Table 1 for detailed formulations). Pullulan is a polysaccharide polymer produced by the fungus Aureobasidium pullulans. Gum arabic is the extract from the exudate produced by acacia trees which was identified as a heterogenous mixture of polysaccharides and proteins. Pullulan (71) and gum arabic (72) were chosen due to their nature derived, water solubility, similarity in water tension, low viscosity, high stability, safe status in pharmaceutical & food industries, and being inexpensive. Using in vitro and in vivo measurements of AAV transduction or transduction efficiency, significant differences were observed in vector stability over weeks of being stored at room temperature, 37°C, and through many freeze-thaw cycles among the different excipients tested, with 1% 72 performing best.

[0089] Materials and Methods

[0090] Materials

[0091] Sucrose, pullulan, and gum arabic powder were purchased from Thermo scientific. IX DMEM and PBS were purchased from Gibco. Plasmids were purified from GenElute HP Maxiprep Kits purchased from Sigma.

[0092] rAAV Production and Purifications

[0093] Suspension cell line HEK293F was used for rAAV vector production. The cells were cultured in Viral production medium (Thermo) in shaker flasks at 120rpm at 37°C, 8% CO2.Cell viability was maintained at >95%, and transfection was performed when the cell density was at about 2 - 2.5 E6 cells / mL using the standard triple-plasmid transfection method of pRepCap (serotype 5,8 and 9), pHelper, and pITR-AAV (pds-CB-EGFP, CB-GLUC, or TTR- cohFIX) at an equal molar ratio. These cells were collected at 72hr post transfection. Centrifugation at 3000g for 15 min was performed to separate the cells and media. The cells were resuspended in lysis buffer of 50mM Tris-HCI (+0.15M NaCI, pH 8.2), went through 3 cycles of freeze / thaw to release rAAV viruses, and centrifuged at 3000g for 20 min to collect the supernatant. This supernatant was combined with the cell media and treated with DNase I (lOU / mL) for lhr at 37°C to digest contaminant DNAs to improve the quality of the crude lysate. rAAV viruses in the crude were purified using liquid chromatography with an affinity column (POROS AAVX CaptureSelect, Thermo #A36651).

[0094] In vitro cell culture & transduction assays

[0095] GM16095 human fibroblasts cells were purchased from Coriell Institute, and grown in 10%FBS DMEM with 1% antibiotics, at 37°C with 5% CO2. The cells were sub-cultured once they reach 90% confluency. For transduction assays, the cells were counted and seeded in a 96-well plate 24hr before transduction with rAAVs. Based on the AAV transgene of each experiment, the cells were trypsinized for flow cytometry to determine direct GFP+ positive or measured for Gaussia Luciferase activity (using the Pierce Gaussia Luciferase Glow Assay Kit, Thermo #16161, followed the manufacture's protocol).

[0096] Animal in vivo experiments

[0097] All mice were maintained in the laboratory animal resource center at Indiana University-Purdue University, Indianapolis (IUPUI). All animal experimental protocols were approved and performed as per the guidelines of Indiana University's Institutional Biosafety Committees (IBC) and Institutional Animal Care and Use Committee (IACUC). Each specific experiment (specified in each Results Figure) includes either WT Balb / c mice (purchased from the Jackson Lab) or hemophilia B C3H / HeJ-HB mice (in house breed). All mice were treated with rAAVs (purified by either iodixanol centrifugation or affinity chromatography) at 8-10-week-old; negative control group was mock injected with PBS. AAV dosage was lEllvg vg / mouse. For the Balb / c mice, at sacrifice (week 2 post injection), their livers were harvested for further flow cytometry analysis. For the C3H / HeJ-HB mice, plasma samples were collected by retro-orbital eye bleed into 0.38% sodium citrate; *At week 8 after the bleed, the group of PBS-2wkRT was accidently sacrificed, therefore, the lack of its data at the final timepoint of week-10.

[0098] Flow cytometry analysis

[0099] Mouse livers were processed at day of harvest and measured for eGFP signal based on a previously described protocol. Simple gating for unfixed, single cells from liver was set for direct fluorescence of GFP+ cells from negative control GFP- cells using the Attune NxTFlow Cytometer.

[0100] Statistics

[0101] GraphPad Prism 9.5.0 software was used to calculate all statistics. Unless otherwise stated, data are presented as means ± the standard deviations with annotations of * p < 0.05; ** p < 0.01; *** p < 0.001; *** p < 0.0001.

[0102] Results

[0103] AAV transduction efficiency maintained in vitro

[0104] Throughout the entirety of this study, self-complementary AAV vectors (AAV9, AAV8, and AAV5) were tested in the same based buffer (lx phosphate buffered saline, PBS, pH 7.2) plus different excipients at various concentrations (NaCI, sucrose, additive Tl, or T2). To evaluate how these different excipients affected rAAV transgene expression, the inventors designed vectors to express a variety of reporter genes including engineered green fluorescent protein (eGFP), Gaussia luciferase (GLUC), and human Factor IX (h FIX).

[0105] First, AAV9-CB-GLUC was formulated in different excipients and stored at room temperature (25°C) for up to 3 weeks or 21 days. The vector's transduction efficiency was measured by the expression of the reporter gene Gaussia luciferase (GLUC) in transduced human fibroblast cells, GM16095. As shown in Fig. 7A, the resulting transduction efficiency of AAV9 in just PBS (with no additives added) dropped to ~7000 RFU compared to the freshly thawed control at day 0 of ~29000 RFU over the span of 3 days left at room temperature. That was over 75% reduction in transduction efficiency or transduction efficiency mostly because of thermal degradation of the viral capsid proteins. Sucrose, at a higher concentration of 20-40%, improved the AAV9's thermal stability over 21 days (19000 over 29000 RFU, Fig. 7B). Additive Tl, at around 1% in PBS, was shown to best stabilize AAV9 over 21 days, similarly to 20% of sucrose (65% transduction efficiency remained, Fig. 7C). Additive T2, at around 1% in PBS, was shown to best stabilize AAV9 during the first 7 days, as 95% transduction efficiency slowly decreased to about 65% at day 21 (Fig. 7D). Interestingly, higher concentrations >2% of T2, provided less protection of AAV9 transduction efficiency. To summarize our first experiment, 20% sucrose, 1% Tl, and 1% T2 were found to be the best thermal stabilizers for AAV9.

[0106] To better address the thermal stabilizing effect of these additives, AAV9 was stored at an elevated temperature of 37°C, over the span of half a month. As a result, 1% Tl excipient was shown to be the worst agent to maintain the vector transduction efficiency atday 15, while the best stabilizing agent was 1% T2 (Fig. 8A). The other two groups (20% sucrose and PBS alone) moderately maintained AAV transduction efficiency. Motivated to test their protection against freeze-thawing stress, these same four groups of AAV9 formulations were evaluated for the transduction efficiency maintained in vitro. As shown in Fig. 8B, significant loss of AAV transduction efficiency was found after 5 freeze / thaw cycles in PBS without any additives (~90% loss), while 20% sucrose and 1% T1 stabilized transduction efficiency to ~57% (100K over 175K RFU, at 5 F / T cycles). At the same time, more than 85% of the transduction efficiency was surprisingly preserved by 1% T2 excipient (150K over 175K RFU). Fig. 8C shows the normalized transduction efficiency of these formulations at 5 F / T cycles as a function of cell fraction infected with significant differences found among all four groups. To further test how stability of AAV9 at extreme temperatures is altered by 1% T2, these formulations were heated at 65°C for 3 minutes right before a transduction assay. The results are shown in Figs. 8D and 8E, as 60% of the transduction efficiency was maintained by 2% T2 while the others could only preserve <25%. Thermal melting temperature of AAV9 both full and empty capsids were reported to be 77°C, and the data here demonstrate that 1% T2 potentially raised its melting temperature to be much higher, therefore protecting the viral particles at these elevated temperatures and extending AAV9 lifetime.

[0107] To test if this stabilizing effect of 1% T2 is serotype dependent, two other AAV serotypes were analyzed: AAV5 and AAV8 (scITR-CB-eGFP). Similar studies were performed to evaluate the transduction efficiency of different AAV formulations as a function of gene expression versus F / T cycles. As shown in Figs. 9A-9F, 1% T2 also best stabilized AAV5 (Fig. 9A and Fig. 9C) and AAV8 (Fig. 9B and Fig. 9D) against 5 to 10 freeze-thaw cycles significantly among all other groups evaluated, preserving almost 99% of transduction efficiency. To evaluate the thermal stabilizing effect of these additives, storage of both AAV5 and AAV8 at 37°C was performed over 15 days before infecting GM 16095 cells. As a result, 1% T2 excipient was shown to be the second-best agent to maintain AAV5's transduction efficiency at day 15, while the best stabilizing agent was 20% sucrose (Fig. 9E). For AAV8, 1% T2 excipient was shown to be the best stabilizer, while the second-best stabilizing agent was 20% sucrose at day 15 (Fig. 9F). The other two formulation groups (1% T1 and PBS alone) completely lost the transduction efficiency of both AAV5 and AAV8 after being stored for 15 days at 37°C.

[0108] To mimic a realistic situation where AAV-based drugs are often aliquoted and stored at freezing temperature ( -20°C), we tested the maintained transduction efficiency of AAV8 and AAV9 pre-titered aliquots (in different formulations) by thawing them out once. Inparallel, the same groups were heated at 56°C for lOmin right before transduction assays to quickly determine their thermal stability. Upon analysis, the best excipient protecting both AAV8 and AAV9 from one F / T, and F / T once plus heating at 56°C was 1%T2 (Fig. 10A and 10B). The second-best excipient for AAV8 was 20% sucrose, while that for AAV9 was 1% Tl. The buffer PBS alone had the lowest stabilizing effect for both AAV8 and AAV9 against even one F / T cycle as their corresponded transduction efficiency was the lowest. These data highlighted the significant power of excipients in AAV formulations.

[0109] AAV transduction efficiency maintained in vivo

[0110] To establish the feasibility of these excipients for AAV-based drugs in the clinic, we performed in vivo studies with AAV8 expressing eGFP formulated with the different excipients. In order to determine the long-term stabilizing impact of these excipients, aliquots of AAV8 were incubated in either PBS alone or PBS plus excipients (20% sucrose, 1% Tl, or 1% T2) for 2 weeks at room temperature before systemic tail-vein injection into WT BALB / c mice (study design shown in Fig. 11A). At week-2 post injection, the mice were sacrificed and whole livers were collected for digestion and quantitative analysis of GFP+ cells by flow cytometry. As shown in Fig. 11B, mice injected with AAV8 stored in 1% T2 gave the most significant increase in the gene expression as shown by %GFP positive liver cells. This is more than 20-fold higher GFP expression than that of the PBS and 20%-sucrose groups. The two groups of PBS and 20% sucrose gave almost no expression of GFP, while 1% Tl gave some GFP expression (about one fifth of the 1% T2 group). Representative dot plots of the gated population are shown in Figs. 11C-11G. This data demonstrates the superior thermally stabilizing effect of 1% T2 on AAV8 translated from in vitro to in vivo.

[0111] In the second in vivo study, a hemophilia B mouse model, (C3H / HeJ-HB) was used to monitor a longer-term expression of a codon-optimized human Factor IX (h FIX) carried by AAV9 formulated in different excipients. In a similar design, the AAV9 were incubated (in either PBS alone, 1% Tl, or 1% T2) at room temperature for 2 weeks before injection into the mice. This time, another group of fresh AAV9 (without room temperature incubation) was added as a second control group for a more comprehensive comparison. The study design is shown in Fig. 12A, as at various time points post-AAV-injection (up to week 8-10), retro-orbital bleeding was performed to determine the circulating h F IX expressed by the different groups tested. In addition, alanine transaminase (ALT) activity was also monitored as indicators of toxicity stress caused by each group. As a result, shown in Fig. 12B, at weeks 1, 3, and 5 PI, no significant levels of serum ALT were found by all groups, which indicated that the excipients used (1% Tl and 1% T2) were well tolerated for the AAV injections. The expression of h FIX delivered by each group is shown in Figure 12C. Being stored at roomtemperature for 2 weeks, the AAV9 in PBS gave the lowest hF IX expression (group PBS- 2wkRT), while the other groups (1% Tl-2wkRT and 1% T2-2wkRT) gave significantly higher h FIX expression. Remarkably, the transduction efficiency of the AAV9 in 1% T2 for 2 weeks storage at room temperature, was greatly preserved as the highest h F IX expression was detected up to week 10 PI. It was just as infectious as the fresh AAV group (PBS-fresh) since no significant difference was found between the two groups at all time points evaluated. The vast difference in gene expression by the 1% T2 group versus PBS-group was shown in Figure 12D with a normalized fold change comparison. The long-term stabilizing effect of 1% T2 outperformed the based buffer PBS alone by more than 20 folds after 2 months of systemic injection, highlighting the immense potential of excipient T2 in AAV formulations.

[0112] Crucial to the success of clinical gene transfer is the research and development of vector capsid, its DNA cargo, and the final product formulations that ensure safety, consistency, and potency of the desired therapeutic results. Extensive research has been focusing on the AAV capsids and their nucleic acid cargo, while formulation design of the final product has not been significantly explored. Our finding here provides better understanding of AAV formulations using different excipients and presents simple solutions to the cold-chain problem of AAV biologies. As the results show, the common excipient sucrose (at high concentration ~20%) might be effective as a stabilizer for many vaccines, but not the best option for AAV products, especially for AAV8 and AAV9. Being inexpensive and natural, compounds like pullulan and gum arabic have great promise as excipients for AAV formulations. At incredibly low concentrations (<1%), gum arabic was able to preserve the in vivo transduction efficiency of AAV8 and AAV9 remarkably, even after being stored at room temperature for half a month. Current FDA-approved AAV formulations using Trisbased or PBS-based solutions only allow limited storage at room temperatures: Luxturna (4hr), Zolgensma (8hr), and Hemgenix (24hr, 5% sucrose). The results presented here clearly show that T1 and T2 formulations for AAVs are superior.

Claims

Claims1. An adeno-associated virus (AAV) stabilizing composition, said composition comprising an AAV vector; and an aqueous solution containing a gum isolated from an Acacia tree.

2. The composition of claim 1 wherein the gum is isolated from Acacia Senegal or Acacia seyal.

3. The composition of claim 1 wherein the gum is gum arabic.

4. The composition of claim 1 wherein the AAV is a recombinant adeno-associated virus (rAAV).

5. The composition of claim 3 wherein the aqueous solution comprises a pharmaceutically acceptable carrier.

6. The composition of claim 3 wherein the aqueous solution comprises a buffer that stabilizes the composition at physiological pH.

7. The composition of claim 3 wherein the gum arabic is present in an amount selected from a range of about 0.1% to about 5% w / v.

8. The composition of claim 7 wherein the gum arabic is present at about 1% w / v.

9. The composition of any one of claims 1-8 wherein said recombinant adeno- associated virus (rAAV) is modified to comprise a non-native chemical moiety covalently linked to a capsid protein of said rAAV, wherein said modification alters the tropism of the rAAV comprising the non-native chemical moiety relative to the rAAV lacking the non-native chemical moiety.

10. The composition of claim 9 wherein the non-native chemical moiety is selected from the group consisting of 5-(Dimethylamino)naphthalene-l-sulfonyl chloride (De), and N- ethylmaleimide (NEM).

11. The composition of claim 9 wherein the chemical moiety is 5- (Dimethylamino)naphthalene-l-sulfonyl chloride (De) and the altered tropism is an enhanced preferential uptake of the chemical modified AAV, relative to the original unmodified AAV, by muscle tissue.

12. The composition of claim 9 wherein the chemical moiety is N-ethylmaleimide (NEM) and the altered tropism is an enhanced preferential uptake of the chemical modified AAV, relative to the original unmodified AAV, by bone tissue.

13. The composition of any one of claims 1-8 wherein the rAAV is a recombinant adeno- associated virus (rAAV) selected from serotypes: AAV2, AAV5, AAV5x, AAV8, and AAV9.

14. A method of stabilizing an adeno-associated virus, said method comprising combining the adeno-associated virus with an excipient comprising a gum isolated from an Acacia tree.

15. The method of claim 14 wherein the gum is isolated from Acacia Senegal or Acacia seyal.

16. The method of claim 14 wherein the gum is gum arabic.

17. The method of claim 14 wherein the adeno-associated virus is a recombinant adeno- associated virus (rAAV).

18. The method of any one of claims 14-17 wherein the excipient comprises a pharmaceutically acceptable carrier, optionally comprising a buffer that stabilizes the composition at physiological pH.

19. The method of claim 16 wherein the gum arabic is added to a final concentration selected from a range of about 0.1% to about 5% w / v.

20. The method of claim 19 wherein the gum arabic is added to a final concentration of about 1% w / v.

21. A kit for preparing a composition comprising a stabilized rAVV, said kit comprising a container comprising an rAVV; and a container comprising a solution of gum arabic.

22. The kit of claim 21 further comprising a disposable device for administering the composition comprising a stabilized rAVV to a patient, optionally wherein the device comprises a needle and a syringe.

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