Adeno-associated viral vector formulations and methods

Abstract

Provided herein are formulations comprising recombinant AAV particles. In some embodiments, the formulations are frozen or lyophilized. Also provided herein are methods for reducing rAAV genome release from rAAV particles.

Description

[Technical field]

[0001] 1. CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 62 / 836,115, filed April 19, 2019, which is incorporated by reference in its entirety herein.

[0002] References to Electronically Submitted Sequence Listings This application incorporates by reference the sequence listing submitted herewith as a text file, which has the file name "12656-131-228_Sequence_Listing.txt", a creation date of April 16, 2020, and a file size of 70,092 bytes.

[0003] 2. Introduction Stable formulations of recombinant adeno-associated virus (rAAV) products and methods for their formulation are described. The formulations and methods are suitable for delivering therapeutic rAAV products to human subjects to treat a variety of diseases and disorders. [Background technology]

[0004] 3. Background of the invention Recombinant adeno-associated virus (AAV)-based vectors are currently the most widely used gene therapy products in development. rAAV vector systems are suitable for use in part due to the lack of disease associated with wild-type viruses, AAV's ability to transduce dividing as well as non-dividing cells, and the long-term robust transgene expression observed in clinical trials, which indicates great potential for delivery in gene therapy indications. In addition, the different serotypes of natural and recombinant rAAV vectors allow them to specifically target different tissues, organs, and cells, and help evade any pre-existing immunity to the vector, expanding the therapeutic applications of AAV-based gene therapy.

[0005] AAV products must not only demonstrate safety and efficacy, but also remain stable and potent during manufacture, transportation, storage, and administration. When commercializing any pharmaceutical product, it would be advantageous to identify a formulation that provides long-term stability, regardless of whether the transportation or storage requirements are at ambient temperature, under refrigerated conditions, or under frozen conditions. In the case of rAAV products, the formulation must be able to withstand the stresses introduced by various conditions and still maintain product quality. Thus, improved formulations containing recombinant AAV particles are needed. Summary of the Invention

[0006] The present disclosure provides formulations comprising recombinant adeno-associated virus (rAAV) particles and a buffer, a cryoprotectant and lyoprotectant excipient, and an amorphous salt. In some embodiments, provided herein are stable formulations comprising recombinant adeno-associated virus (rAAV) particles and a buffer, a sugar, and an amorphous salt. In some embodiments, the formulation is suitable for lyophilization.

[0007] In some embodiments, the buffer comprises Tris, sucrose, and less than about 100 mM sodium citrate. In some embodiments, the formulation further comprises a non-ionic surfactant, such as poloxamer 188. In some embodiments, the formulation further comprises a plasticizer or stabilizer, such as glycerol. In some embodiments, the rAAV is selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV-11, AAV-12, AAV-13, AAV-14, AAV-15, and AAV-16, rAAV.rh8, rAAV.rh10, rAAV.rh20, rAAV.rh39, rAAV.Rh74, rAAV.RHM4-1, AAV.hu37, rAAV.Anc80, rAAV.Anc80L65, rAAV.7m8 , rAAV.PHP.B, rAAV2.5, rAAV2tYF, rAAV3B, rAAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, or AAV.HSC16 capsid proteins. In some embodiments, the rAAV comprises capsid proteins of an AAV-8 or AAV-9 serotype.

[0008] The present disclosure provides a formulation comprising recombinant adeno-associated virus (rAAV) particles and a buffer, a sugar, and a salt comprising sodium citrate. In some embodiments, the buffer comprises Tris, sucrose, and less than about 100 mM sodium citrate. In some embodiments, the formulation further comprises a non-ionic surfactant, e.g., poloxamer 188. In some embodiments, the formulation further comprises a plasticizer or stabilizer, e.g., glycerol. In some embodiments, the rAAV is selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV-11, AAV-12, AAV-13, AAV-14, AAV-15, and AAV-16, rAAV.rh8, rAAV.rh10, rAAV.rh20, rAAV.rh39, rAAV.Rh74, rAAV.RHM4-1, AAV.hu37, rAAV.Anc80, rAAV.Anc80L65, rAAV.7m8 , rAAV.PHP.B, rAAV2.5, rAAV2tYF, rAAV3B, rAAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, or AAV.HSC16 capsid proteins. In some embodiments, the rAAV comprises capsid proteins of an AAV-8 or AAV-9 serotype.

[0009] In some embodiments, the formulation is suitable for lyophilization. In some embodiments, the pharmaceutical composition is a lyophilized composition of the liquid compositions disclosed herein. In some embodiments, the pharmaceutical composition is a reconstituted lyophilized formulation.

[0010] In some embodiments, the formulation is a liquid formulation. In some embodiments, the formulation is a frozen formulation. In some embodiments, the formulation is a lyophilized formulation of a liquid formulation disclosed herein. In some embodiments, the formulation is a reconstituted lyophilized formulation.

[0011] In some embodiments, the formulation is a lyophilized formulation having a residual moisture content of about 1% to about 7%.

[0012] The present disclosure also provides methods for producing stable formulations comprising recombinant adeno-associated virus (rAAV) particles.

[0013] The present disclosure also provides methods for reducing rAAV genome release from rAAV particles.

[0014] The present disclosure also provides for the use of sugars to reduce rAAV genome release from rAAV particles.

[0015] The present disclosure also provides for the use of plasticizers to reduce rAAV genome release from rAAV particles.

[0016] The present disclosure also provides a method for treating a disease or disorder in a subject in need of such treatment, the method comprising administering a formulation comprising the recombinant AAV particles disclosed herein.

[0017] In some embodiments, the present disclosure provides: [1] Recombinant adeno-associated virus (rAAV) particles, and a) a buffering agent, b) sugar, and c) Salts containing sodium citrate A stable formulation comprising:

[0018] [2] The formulation according to [1], wherein the buffer comprises about 1 mM to about 50 mM Tris.

[0019] [3] The formulation according to [2], comprising about 1 mM to about 30 mM, about 1 mM to about 20 mM, about 5 mM to about 30 mM, about 5 mM to about 20 mM, about 10 mM to about 30 mM, about 10 mM to about 20 mM, or about 20 mM to about 50 mM Tris.

[0020] [4] The formulation of [2], comprising about 1 mM, about 2 mM, about 3 mM, about 5 mM, about 10 mM, about 15 mM, about 20 mM, about 25 mM, about 30 mM, or about 40 mM Tris.

[0021] [5] The formulation of [2], comprising about 5 mM Tris.

[0022] [6] The formulation according to any one of [1] to [4], having a pH of about 6.5 to 8.0.

[0023] [7] The formulation described in [6], having a pH of about 7.2 to 7.8.

[0024] [8] The formulation of [6], wherein the pH is about 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, or 7.8.

[0025] [9] The formulation described in [6], having a pH of about 7.5.

[0026]

[10] The formulation according to any one of [1] to [9], comprising about 50 mM to about 400 mM sugar.

[0027]

[11] The formulation of

[10] , comprising about 50 mM to about 350 mM, about 50 mM to about 300 mM, about 50 mM to about 250 mM, about 50 mM to about 200 mM, or about 50 mM to about 150 mM sugar.

[0028]

[12] The formulation of

[10] , comprising about 100 mM to about 400 mM, about 150 mM to about 400 mM, about 200 mM to about 400 mM, about 250 mM to about 400 mM, or about 300 mM to about 400 mM sugar.

[0029]

[13] The formulation of

[10] , comprising about 100 mM to about 300 mM, about 150 mM to about 250 mM, about 200 mM to about 300 mM, or about 250 mM to about 350 mM sugar.

[0030]

[14] The formulation of

[10] , comprising about 50 mM, about 100 mM, about 150 mM, about 160 mM, about 170 mM, about 180 mM, about 190 mM, about 200 mM, about 210 mM, about 220 mM, about 230 mM, about 240 mM, about 250 mM, about 260 mM, about 270 mM, about 280 mM, about 290 mM, about 300 mM, or about 350 mM sugar.

[0031]

[15] The formulation of

[10] , comprising about 190 mM to about 230 mM, about 170 mM to about 250 mM, or about 150 mM to about 270 mM sugar.

[0032]

[16] The formulation of

[10] , comprising about 210 mM sugar.

[0033]

[17] The formulation according to any one of [1] to

[16] , wherein the sugar is a non-reducing sugar.

[0034]

[18] The formulation described in

[17] , wherein the non-reducing sugar is sucrose, trehalose, or raffinose.

[0035]

[19] The formulation described in

[17] , wherein the non-reducing sugar is sucrose.

[0036]

[20] The formulation according to any one of [1] to

[16] , wherein the sugar is a reducing sugar.

[0037]

[21] The formulation described in

[20] , wherein the reducing sugar is glucose, fructose, mannose, galactose, or lactose.

[0038]

[22] The formulation described in

[20] , wherein the reducing sugar is glucose.

[0039]

[23] The formulation of any one of [1] to

[22] , comprising less than about 100 mM sodium citrate.

[0040]

[24] The formulation of

[23] , comprising about 10 mM to about 100 mM sodium citrate.

[0041]

[25] The formulation of

[23] , comprising about 10 mM to about 100 mM, about 20 mM to about 100 mM, about 30 mM to about 100 mM, about 40 mM to about 100 mM, about 50 mM to about 100 mM, about 10 mM to about 80 mM, about 10 mM to about 60 mM, about 10 mM to about 50 mM, about 10 mM to about 40 mM, or about 10 mM to about 30 mM sodium citrate.

[0042]

[26] The formulation of

[23] , comprising about 10 mM to about 50 mM, about 20 mM to about 60 mM, about 30 mM to about 70 mM, or about 10 mM to about 30 mM sodium citrate.

[0043]

[27] The formulation of

[23] , comprising about 10 mM, about 20 mM, about 30 mM, about 40 mM, about 50 mM, or about 60 mM sodium citrate.

[0044]

[28] The formulation of

[23] , comprising about 20 mM sodium citrate.

[0045]

[29] The formulation of any one of [1] to

[28] , further comprising about 0.0005% to about 0.01% of a nonionic surfactant.

[0046]

[30] The formulation of

[29] , comprising about 0.002% nonionic surfactant.

[0047]

[31] The formulation of

[28] or

[29] , wherein the nonionic surfactant comprises poloxamer 188, poloxamer 407, polysorbate 80, polysorbate 20, Pluronic F-68, or BRIJ 35.

[0048]

[32] The formulation described in

[31] , wherein the nonionic surfactant comprises poloxamer 188.

[0049]

[33] Recombinant adeno-associated virus (rAAV) particles, and a) about 1 mM to about 25 mM Tris, b) about 50 mM to about 400 mM sugar; c) about 10 mM to about 100 mM sodium citrate, and d) about 0.0005% to about 0.01% of a nonionic surfactant; A stable formulation comprising: The formulation has a pH of about 7.2 to about 7.8.

[0050]

[34] Recombinant adeno-associated virus (rAAV) particles, and a) about 2 mM to about 10 mM Tris, b) about 150 mM to about 250 mM sugar; c) about 10 mM to about 20 mM sodium citrate, and d) about 0.001% to about 0.005% of a nonionic surfactant; A stable formulation comprising: The formulation has a pH of about 7.2 to about 7.8.

[0051]

[35] Recombinant adeno-associated virus (rAAV) particles, and a) about 5 mM Tris, b) about 210 mM sugar; c) about 20 mM sodium citrate, and d) about 0.002% of a nonionic surfactant; A stable formulation comprising: The formulation has a pH of about 7.5.

[0052]

[36] The formulation according to any one of

[33] to

[35] , wherein the sugar is a non-reducing sugar.

[0053]

[37] The formulation described in

[36] , wherein the non-reducing sugar is sucrose, trehalose, or raffinose.

[0054]

[38] The formulation described in

[17] , wherein the non-reducing sugar is sucrose.

[0055]

[39] A formulation described in any one of [1] to

[38] , comprising about 1.0E+11 genome copies / mL (GC / mL) and about 1.0E+15 GC / mL of rAAV particles.

[0056]

[40] The formulation of

[39] , comprising about 1.0E+11GC / mL, about 1.0E+12GC / mL, about 1.0E+13GC / mL, about 1.0E+14GC / mL, or about 1.0E+15GC / mL of rAAV particles.

[0057]

[41] The rAAV particles include AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV-11, AAV-12, AAV-13, AAV-14, AAV-15 and AAV-16, rAA V.rh8, rAAV.rh10, rAAV.rh20, rAAV.rh39, rAAV.Rh74, rAAV.RHM4-1, AAV.hu37, rAAV.Anc80, rAAV.Anc80L65, rAAV.7m8, rAAV.PHP.B, The formulation of any one of [1] to

[40] , comprising a capsid protein of rAAV2.5, rAAV2tYF, rAAV3B, rAAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, or AAV.HSC16.

[0058]

[42] The formulation described in

[41] , wherein the rAAV particles contain capsid proteins of the AAV-8 or AAV-9 serotype.

[0059]

[43] The formulation of any one of [1] to

[42] , further comprising a plasticizer selected from the group consisting of glycerol, xylitol, sorbitol, or mannitol.

[0060]

[44] The formulation according to any one of [1] to

[42] , further comprising glycerol.

[0061]

[45] The formulation of

[44] , comprising about 0.1% to about 5% glycerol.

[0062]

[46] The formulation of

[44] , comprising about 0.1% to about 2% glycerol.

[0063]

[47] The formulation of

[44] , comprising about 0.25% to about 2% glycerol.

[0064]

[48] ​​The formulation described in any one of [1] to

[47] , which is a liquid formulation.

[0065]

[49] The formulation according to any one of [1] to

[47] , which is a frozen formulation.

[0066]

[50] The formulation according to any one of [1] to

[47] , which is a lyophilized formulation or a reconstituted lyophilized formulation.

[0067]

[51] The formulation according to

[50] , wherein the residual moisture content is about 1% to about 7%.

[0068]

[52] The formulation according to

[51] , wherein the residual water content is about 1% to about 7%, about 2% to about 7%, about 3% to about 7%, about 4% to about 7%, about 5% to about 7%, about 1% to about 6%, about 1% to about 5%, about 1% to about 4%, or about 1% to about 3%.

[0069]

[53] The formulation according to

[51] , wherein the residual moisture content is about 3% to about 7%, about 3% to about 6%, or about 3% to about 5%.

[0070]

[54] The formulation of

[51] , wherein the residual moisture content is about 3%, about 3.5%, about 4%, about 4.5%, about 5%, about 5.5%, or about 6%.

[0071]

[55] The formulation of any one of [1] to

[54] , wherein the % relative potency of the rAAV particles is at least about 60%, at least about 70%, or at least about 80% after storage of the formulation at room temperature for 3 months, wherein the reference rAAV particles are stored at -70°C in Dulbecco's phosphate buffered saline (DPBS) containing 0.001% poloxamer 188 buffer.

[0072]

[56] The formulation of any one of [1] to

[54] , wherein the relative potency of the rAAV particles is at least about 60%, at least about 70%, or at least about 80% after storage of the formulation at room temperature for 6 months, wherein the reference rAAV particles are stored at -70°C in DPBS containing 0.001% poloxamer 188 buffer.

[0073]

[57] The formulation of any one of [1] to

[54] , wherein the relative potency of the rAAV particles is at least about 30%, at least about 40%, at least about 50%, or at least 60% after storage of the formulation at 35°C for 1 week, wherein the reference rAAV particles are stored at -70°C in DPBS containing 0.001% poloxamer 188 buffer.

[0074]

[58] The formulation of any one of [1] to

[54] , wherein the relative potency of the rAAV particles is at least about 30%, at least about 40%, at least about 50%, or at least 60% after storage of the formulation at 35°C for 2 weeks, wherein the reference rAAV particles are stored at -70°C in DPBS containing 0.001% poloxamer 188 buffer.

[0075]

[59] The formulation of any one of [1] to

[54] , wherein the relative potency of the rAAV particles is at least about 30%, at least about 40%, at least about 50%, or at least 60% after storage of the formulation at 35°C for 4 weeks, wherein the reference rAAV particles are stored at -70°C in DPBS containing 0.001% poloxamer 188 buffer.

[0076]

[60] A method for producing a stable formulation comprising recombinant adeno-associated virus (rAAV) particles, the method comprising mixing rAAV particles with a buffer, sugar, salt, optionally a plasticizer, and optionally a non-ionic surfactant of the formulation described in any one of [1] to

[47] , thereby producing the formulation comprising rAAV.

[0077]

[61] A method for reducing rAAV genome release from rAAV particles, comprising producing a formulation comprising rAAV particles, a buffer, a sugar, a salt, and optionally a non-ionic surfactant, wherein rAAV genome release from the rAAV particles after three freeze-thaw cycles is reduced compared to rAAV genome release in a formulation that does not contain the sugar.

[0078]

[62] A method for reducing rAAV genome release from rAAV particles, comprising producing a formulation comprising rAAV particles, a buffer, a sugar, a salt, and optionally a non-ionic surfactant, wherein rAAV genome release from the rAAV particles after lyophilization and reconstitution is reduced compared to rAAV genome release in a formulation that does not contain the sugar.

[0079]

[63] The formulation of

[61] or

[62] , further comprising freeze-drying the formulation to a residual water content of about 1% to about 5%.

[0080]

[64] A method for reducing rAAV genome release from rAAV particles, comprising producing a formulation comprising rAAV particles, a buffer, a sugar, a salt, a plasticizer, and optionally a non-ionic surfactant, wherein rAAV genome release from the rAAV particles after three freeze-thaw cycles is reduced compared to rAAV genome release in a formulation that does not contain the sugar.

[0081]

[65] A method for reducing rAAV genome release from rAAV particles, comprising producing a formulation comprising rAAV particles, a buffer, a sugar, a salt, a plasticizer, and optionally a non-ionic surfactant, wherein rAAV genome release from the rAAV particles after lyophilization and reconstitution is reduced compared to rAAV genome release in a formulation that does not contain the sugar.

[0082]

[66] Use of a sugar to reduce rAAV genome release from rAAV particles, comprising producing a formulation comprising rAAV particles, a buffer, a sugar, a salt, and optionally a non-ionic surfactant, wherein rAAV genome release from the rAAV particles after three freeze-thaw cycles is reduced compared to rAAV genome release in a formulation that does not contain the sugar.

[0083]

[67] Use of a sugar to reduce rAAV genome release from rAAV particles, comprising producing a formulation comprising rAAV particles, a buffer, a sugar, a salt, and optionally a non-ionic surfactant, wherein rAAV genome release from the rAAV particles after lyophilization and reconstitution is reduced compared to rAAV genome release in a formulation that does not contain the sugar.

[0084]

[68] The use of

[66] or

[67] , further comprising lyophilizing the formulation to a residual water content of about 1% to about 7%.

[0085]

[69] Use of a plasticizer to reduce rAAV genome release from rAAV particles, comprising producing a formulation comprising rAAV particles, a buffer, a sugar, a salt, a plasticizer, and optionally a non-ionic surfactant, wherein rAAV genome release from the rAAV particles after three freeze-thaw cycles is reduced compared to rAAV genome release in a formulation that does not contain the sugar.

[0086]

[70] Use of a plasticizer to reduce rAAV genome release from rAAV particles, comprising producing a formulation comprising rAAV particles, a buffer, a sugar, a salt, a plasticizer, and optionally a non-ionic surfactant, wherein rAAV genome release from the rAAV particles after lyophilization and reconstitution is reduced compared to rAAV genome release in a formulation that does not contain the sugar.

[0087]

[71] The method according to any one of

[61] to

[65] or the use according to any one of

[66] to

[70] , wherein rAAV genome release is determined by measuring relative fluorescence in the presence of a DNA-specific fluorescent stain.

[0088]

[72] The method of any one of

[61] to

[65] and

[71] or the use of any one of

[66] to

[71] , wherein freezing-induced rAAV genome release is reduced by at least about 10%, 20%, 50%, 80%, or 90%.

[0089]

[73] The method according to any one of

[61] to

[65] and

[71] or the use according to any one of

[66] to

[71] , wherein freezing-induced rAAV genome release is substantially eliminated.

[0090]

[74] The method according to any one of

[61] to

[65] and

[71] to

[73] or the use according to any one of

[66] to

[73] , wherein the sugar is a non-reducing sugar.

[0091]

[75] The method according to

[74] or the use according to

[74] , wherein the non-reducing sugar is sucrose, trehalose, or raffinose.

[0092]

[76] The method according to

[74] or the use according to

[74] , wherein the non-reducing sugar is sucrose.

[0093]

[77] The method according to any one of

[61] to

[65] and

[71] to

[73] or the use according to any one of

[66] to

[73] , wherein the sugar is a reducing sugar.

[0094]

[78] The method according to

[77] or the use according to

[77] , wherein the reducing sugar is glucose, fructose, mannose, galactose or lactose.

[0095]

[79] The method according to

[77] or the use according to

[77] , wherein the reducing sugar is glucose.

[0096]

[80] The method according to any one of

[61] to

[65] and

[71] to

[79] or the use according to any one of

[66] to

[79] , wherein the plasticizer comprises glycerol.

[0097]

[81] The formulation is one according to any one of [1] to

[47] , the method according to any one of

[61] to

[65] and

[71] to

[80] or the use according to any one of

[66] to

[80] .

[0098] In some embodiments, the methods disclosed herein include producing a stable formulation comprising recombinant adeno-associated virus (rAAV) particles, the rAAV particles being produced by isolating the rAAV particles from an impure feed (e.g., an rAAV production culture), and the method of isolating the rAAV particles includes one or more processing steps. In some embodiments, the processing is at least one of harvesting the cell culture, clarification of the harvested cell culture (e.g., by centrifugation or depth filtration), tangential flow filtration, affinity chromatography, anion exchange chromatography, cation exchange chromatography, size exclusion chromatography, hydrophobic interaction chromatography, and sterile filtration. In further embodiments, the processing includes at least two, at least three, at least four, at least five, or at least six of harvesting the cell culture, clarification of the harvested cell culture (e.g., by centrifugation or depth filtration), tangential flow filtration, affinity chromatography, anion exchange chromatography, cation exchange chromatography, size exclusion chromatography, hydrophobic interaction chromatography, and sterile filtration. In some embodiments, the processing does not include centrifugation of the harvested cell culture.

[0099] The disclosure provides a method of producing a stable formulation comprising isolated recombinant adeno-associated virus (rAAV) particles, the method comprising: (a) isolating the rAAV particles from an impure supply by one or more of centrifugation, depth filtration, tangential flow filtration, ultrafiltration, affinity chromatography, size exclusion chromatography, ion exchange chromatography, and hydrophobic interaction chromatography, and formulating the isolated rAAV particles to produce a stable formulation.

[0100] The disclosure provides methods for producing a stable pharmaceutical unit dosage form comprising isolated recombinant adeno-associated virus (rAAV) particles, the method comprising: (a) isolating the rAAV particles from an impure supply by one or more of centrifugation, depth filtration, tangential flow filtration, ultrafiltration, affinity chromatography, size exclusion chromatography, ion exchange chromatography, and hydrophobic interaction chromatography, and formulating the isolated rAAV particles.

[0101] In certain embodiments, provided herein are recombinant adeno-associated virus (rAAV) particles, and a) about 5 mM Tris, b) about 210 mM sucrose; c) about 20 mM sodium citrate, and d) about 0.002% (w / v) poloxamer 188; It is a stable formulation comprising:

[0102] In certain embodiments, provided herein are recombinant adeno-associated virus (rAAV) particles, and a) about 5 mM Tris, b) about 210 mM sucrose; c) about 20 mM sodium citrate; d) about 0.002% (w / v) poloxamer 188, and e) about 0.25% (w / v) glycerol; It is a stable formulation comprising:

[0103] In certain embodiments, provided herein are recombinant adeno-associated virus (rAAV) particles, and a) about 5 mM Tris, b) about 210 mM sucrose; c) about 20 mM sodium citrate; d) about 0.002% (w / v) poloxamer 188, and e) about 0.5% (w / v) sorbitol; It is a stable formulation comprising:

[0104] In certain embodiments, provided herein are recombinant adeno-associated virus (rAAV) particles, and a) about 5 mM Tris, b) about 30 mM sodium sulfate; c) about 263 mM sucrose, and d) about 0.005% (w / v) poloxamer 188; It is a stable formulation comprising:

[0105] In certain embodiments, provided herein are recombinant adeno-associated virus (rAAV) particles, and a) Tris, b) sucrose, c) sodium citrate, and d) Poloxamer 188 and A stable formulation comprising: However, the formulation has an ionic strength of 60 mM to 150 mM.

[0106] In certain embodiments, provided herein are recombinant adeno-associated virus (rAAV) particles, and a) Tris, b) sucrose, c) sodium citrate, d) Poloxamer 188, and e) glycerol and A stable formulation comprising: However, the formulation has an ionic strength of 60 mM to 150 mM.

[0107] In certain embodiments, provided herein are recombinant adeno-associated virus (rAAV) particles, and a) Tris, b) sucrose, c) sodium citrate, d) Poloxamer 188, and e) Sorbitol A stable formulation comprising: However, the formulation has an ionic strength of 60 mM to 150 mM.

[0108] In certain embodiments, provided herein are recombinant adeno-associated virus (rAAV) particles, and a) Tris, b) sodium sulfate, c) sucrose, and d) Poloxamer 188 and A stable formulation comprising: However, the formulation has an ionic strength of 60 mM to 150 mM.

[0109] In certain embodiments, provided herein are recombinant adeno-associated virus (rAAV) particles, and a) about 5 mM Tris, b) about 210 mM sucrose; c) about 20 mM sodium citrate, and d) about 0.002% (w / v) poloxamer 188; It is a stable formulation comprising:

[0110] In certain embodiments, provided herein are recombinant adeno-associated virus (rAAV) particles, and a) about 5 mM Tris, b) about 210 mM sucrose; c) about 20 mM sodium citrate; d) about 0.002% (w / v) poloxamer 188, and e) about 0.25% (w / v) glycerol; It is a stable formulation comprising:

[0111] In certain embodiments, provided herein are recombinant adeno-associated virus (rAAV) particles, and a) about 5 mM Tris, b) about 210 mM sucrose; c) about 20 mM sodium citrate; d) about 0.002% (w / v) poloxamer 188, and e) about 0.5% (w / v) sorbitol; It is a stable formulation comprising:

[0112] In certain embodiments, provided herein are recombinant adeno-associated virus (rAAV) particles, and a) about 5 mM Tris, b) about 30 mM sodium sulfate; c) about 263 mM sucrose, and d) about 0.005% (w / v) poloxamer 188; It is a stable formulation comprising:

[0113] In certain embodiments, provided herein are recombinant adeno-associated virus (rAAV) particles, and a) Tris, b) sucrose, c) sodium citrate, and d) Poloxamer 188 and A stable formulation comprising: However, the formulation has an ionic strength of 60 mM to 150 mM.

[0114] In certain embodiments, provided herein are recombinant adeno-associated virus (rAAV) particles, and a) Tris, b) sucrose, c) sodium citrate, d) Poloxamer 188, and e) glycerol and A stable formulation comprising: However, the formulation has an ionic strength of 60 mM to 150 mM.

[0115] In certain embodiments, provided herein are recombinant adeno-associated virus (rAAV) particles, and a) Tris, b) sucrose, c) sodium citrate, d) Poloxamer 188, and e) Sorbitol A stable formulation comprising: However, the formulation has an ionic strength of 60 mM to 150 mM.

[0116] In certain embodiments, provided herein are recombinant adeno-associated virus (rAAV) particles, and a) Tris, b) sodium sulfate, c) sucrose, and d) Poloxamer 188 and A stable formulation comprising: However, the formulation has an ionic strength of 60 mM to 150 mM.

[0117] In certain embodiments, provided herein are recombinant adeno-associated virus (rAAV) particles, and a) about 5 mM Tris, b) about 210 mM sucrose; c) about 20 mM sodium citrate, and d) about 0.002% (w / v) poloxamer 188; It is a stable formulation comprising:

[0118] In certain embodiments, provided herein are recombinant adeno-associated virus (rAAV) particles, and a) about 5 mM Tris, b) about 210 mM sucrose; c) about 20 mM sodium citrate; d) about 0.002% (w / v) poloxamer 188, and e) about 0.25% (w / v) glycerol; It is a stable formulation comprising:

[0119] In certain embodiments, provided herein are recombinant adeno-associated virus (rAAV) particles, and a) about 5 mM Tris, b) about 210 mM sucrose; c) about 20 mM sodium citrate; d) about 0.002% (w / v) poloxamer 188, and e) about 0.5% (w / v) sorbitol; It is a stable formulation comprising:

[0120] In certain embodiments, provided herein are recombinant adeno-associated virus (rAAV) particles, and a) about 5 mM Tris, e) about 30 mM sodium sulfate; f) about 263 mM sucrose, and g) about 0.005% (w / v) poloxamer 188; It is a stable formulation comprising:

[0121] In certain embodiments, provided herein are recombinant adeno-associated virus (rAAV) particles, and a) Tris, b) sucrose, c) sodium citrate, and d) Poloxamer 188 and A stable formulation comprising: However, the formulation has an ionic strength of 60 mM to 150 mM.

[0122] In certain embodiments, provided herein are recombinant adeno-associated virus (rAAV) particles, and a) Tris, b) sucrose, c) sodium citrate, d) Poloxamer 188, and e) glycerol and A stable formulation comprising: However, the formulation has an ionic strength of 60 mM to 150 mM.

[0123] In certain embodiments, provided herein are recombinant adeno-associated virus (rAAV) particles, and a) Tris, b) sucrose, c) sodium citrate, d) Poloxamer 188, and e) Sorbitol A stable formulation comprising: However, the formulation has an ionic strength of 60 mM to 150 mM.

[0124] In certain embodiments, provided herein are recombinant adeno-associated virus (rAAV) particles, and a) Tris, b) sodium sulfate, c) sucrose, and d) Poloxamer 188 and A stable formulation comprising: However, the formulation has an ionic strength of 60 mM to 150 mM.

[0125] In certain embodiments, provided herein are recombinant adeno-associated virus (rAAV) particles, and a) Tris, b) sucrose, c) sodium citrate, and d) Poloxamer 188 and A stable formulation comprising: However, the formulation has an ionic strength of less than 200 mM.

[0126] In certain embodiments, provided herein are recombinant adeno-associated virus (rAAV) particles, and a) Tris, b) sucrose, c) sodium citrate, d) Poloxamer 188, and e) glycerol and A stable formulation comprising: However, the formulation has an ionic strength of less than 200 mM.

[0127] In certain embodiments, provided herein are recombinant adeno-associated virus (rAAV) particles, and a) Tris, b) sucrose, c) sodium citrate, d) Poloxamer 188, and e) Sorbitol A stable formulation comprising: However, the formulation has an ionic strength of less than 200 mM.

[0128] In certain embodiments, provided herein are recombinant adeno-associated virus (rAAV) particles, and a) Tris, b) sodium sulfate, c) sucrose, and d) Poloxamer 188 and A stable formulation comprising: However, the formulation has an ionic strength of less than 200 mM.

[0129] In certain embodiments, the stable formulation does not include mannitol.

[0130] In certain embodiments, the stable formulation comprises less than about 150 mM, about 140 mM, about 130 mM, about 120 mM, about 110 mM, about 100 mM, about 90 mM, about 80 mM, about 70 mM, about 60 mM, about 50 mM, about 40 mM, about 30 mM, about 20 mM, or about 10 mM mannitol.

[0131] In certain embodiments, the stable formulation comprises sucrose at a concentration of about 210 mM. In certain embodiments, the stable formulation comprises sucrose at a concentration of about 263 mM. In certain embodiments, the stable formulation comprises sucrose at a concentration of about 409 mM. In certain embodiments, the stable formulation comprises sucrose at a concentration of about 14.6 mM. In certain embodiments, the stable formulation comprises sucrose at a concentration of about 45 mM. In certain embodiments, the stable formulation comprises sucrose at a concentration of about 0 mM to about 20 mM. In certain embodiments, the stable formulation comprises sucrose at a concentration of about 20 mM to about 50 mM. In certain embodiments, the stable formulation comprises sucrose at a concentration of about 50 mM to about 100 mM. In certain embodiments, the stable formulation comprises sucrose at a concentration of about 100 mM to about 200 mM. In certain embodiments, the stable formulation comprises sucrose at a concentration of about 200 mM to about 300 mM. In certain embodiments, the stable formulation comprises sucrose at a concentration of about 300 mM to about 400 mM. In certain embodiments, the stable formulation comprises sucrose at a concentration of about 400 mM to about 500 mM. In certain embodiments, the stable formulation comprises sucrose at a concentration of about 500 mM to about 600 mM. In certain embodiments, the stable formulation comprises sucrose at a concentration of about 600 mM to about 700 mM.

[0132] In certain embodiments, the stable formulation comprises sorbitol at a concentration of about 0.5%. In certain embodiments, the stable formulation comprises sorbitol at a concentration of about 0.25%. In certain embodiments, the stable formulation comprises sorbitol at a concentration of about 0.10%.

[0133] In certain embodiments, the stable formulation contains sorbitol at a concentration of about 0% to about 0.10%. In certain embodiments, the stable formulation contains sorbitol at a concentration of about 0.10% to about 0.20%. In certain embodiments, the stable formulation contains sorbitol at a concentration of about 0.20% to about 0.30%. In certain embodiments, the stable formulation contains sorbitol at a concentration of about 0.30% to about 0.40%. In certain embodiments, the stable formulation contains sorbitol at a concentration of about 0.40% to about 0.50%. In certain embodiments, the stable formulation contains sorbitol at a concentration of about 0.50% to about 0.60%. In certain embodiments, the stable formulation contains sorbitol at a concentration of about 0.60% to about 0.70%. In certain embodiments, the stable formulation contains sorbitol at a concentration of about 0.70% to about 0.80%. In certain embodiments, the stable formulation comprises sorbitol at a concentration of about 0.80% to about 0.90%. In certain embodiments, the stable formulation comprises sorbitol at a concentration of about 0.90% to about 1.00%.

[0134] In certain embodiments, the stable formulation comprises glycerol at a concentration of about 0.5%. In certain embodiments, the stable formulation comprises glycerol at a concentration of about 0.25%. In certain embodiments, the stable formulation comprises glycerol at a concentration of about 0.10%.

[0135] In certain embodiments, the stable formulation comprises glycerol at a concentration of about 0% to about 0.10%. In certain embodiments, the stable formulation comprises glycerol at a concentration of about 0.10% to about 0.20%. In certain embodiments, the stable formulation comprises glycerol at a concentration of about 0.20% to about 0.30%. In certain embodiments, the stable formulation comprises glycerol at a concentration of about 0.30% to about 0.40%. In certain embodiments, the stable formulation comprises glycerol at a concentration of about 0.40% to about 0.50%. In certain embodiments, the stable formulation comprises glycerol at a concentration of about 0.50% to about 0.60%. In certain embodiments, the stable formulation comprises glycerol at a concentration of about 0.60% to about 0.70%. In certain embodiments, the stable formulation comprises glycerol at a concentration of about 0.70% to about 0.80%. In certain embodiments, the stable formulation comprises glycerol at a concentration of about 0.80% to about 0.90%. In certain embodiments, the stable formulation comprises glycerol at a concentration of about 0.90% to about 1.00%.

[0136] In certain embodiments, the stable formulation comprises poloxamer 188 at a concentration of about 0.001% (weight / volume, 0.01 g / L). In certain embodiments, the stable formulation comprises poloxamer 188 at a concentration of about 0.002% (weight / volume, 0.02 g / L). In certain embodiments, the stable formulation comprises poloxamer 188 at a concentration of about 0.005% (weight / volume, 0.05 g / L).

[0137] In certain embodiments, the stable formulation comprises poloxamer 188 at a concentration of about 0.0005% (weight / volume, 0.005 g / L) to about 0.05% (weight / volume, 0.5 g / L). In certain embodiments, the stable formulation comprises poloxamer 188 at a concentration of about 0.0001% (weight / volume, 0.001 g / L) to about 0.01% (weight / volume, 0.1 g / L). In certain embodiments, the stable formulation comprises poloxamer 188 at a concentration of about 0.0005% (weight / volume, 0.005 g / L) to about 0.001% (weight / volume, 0.01 g / L). In certain embodiments, the stable formulation comprises poloxamer 188 at a concentration of about 0.001% (weight / volume, 0.01 g / L) to about 0.05% (weight / volume, 0.5 g / L). In certain embodiments, the stable formulation comprises poloxamer 188 at a concentration of about 0.0005% (weight / volume, 0.005 g / L). In certain embodiments, the stable formulation comprises poloxamer 188 at a concentration of about 0.0006% (weight / volume, 0.006 g / L). In certain embodiments, the stable formulation comprises poloxamer 188 at a concentration of about 0.0007% (weight / volume, 0.007 g / L). In certain embodiments, the stable formulation comprises poloxamer 188 at a concentration of about 0.0008% (weight / volume, 0.008 g / L). In certain embodiments, the stable formulation comprises poloxamer 188 at a concentration of about 0.0009% (weight / volume, 0.009 g / L). In certain embodiments, the stable formulation comprises poloxamer 188 at a concentration of about 0.001% (weight / volume, 0.01 g / L). In certain embodiments, the stable formulation comprises poloxamer 188 at a concentration of about 0.002% (weight / volume, 0.02 g / L). In certain embodiments, the stable formulation comprises poloxamer 188 at a concentration of about 0.003% (weight / volume, 0.03 g / L). In certain embodiments, the stable formulation comprises poloxamer 188 at a concentration of about 0.004% (weight / volume, 0.04 g / L). In certain embodiments, the stable formulation comprises poloxamer 188 at a concentration of about 0.005% (weight / volume, 0.05 g / L). In certain embodiments, the stable formulation comprises poloxamer 188 at a concentration of about 0.01% (weight / volume, 0.1 g / L).In one particular embodiment, the stable formulation comprises poloxamer 188 at a concentration of about 0.05% (weight / volume, 0.5 g / L).

[0138] In some embodiments, the present disclosure provides a stable formulation comprising a recombinant adeno-associated virus (AAV), a salt excipient, and a sugar. In some embodiments, the stable formulation also comprises a surfactant. In some embodiments, the stable formulation also comprises poloxamer 188. In some embodiments, the stable formulation is suitable for lyophilization.

[0139] In some embodiments, provided herein is a stable formulation comprising recombinant adeno-associated virus (rAAV) particles and a buffer, a sugar, and an amorphous salt having an ionic strength of 60 mM to 150 mM, the formulation being suitable for lyophilization. In some embodiments, the stable formulation also comprises a surfactant. In some embodiments, the stable formulation also comprises poloxamer 188. In some embodiments, the formulation is a pre-lyophilized formulation.

[0140] In some embodiments, provided herein is a stable formulation comprising recombinant adeno-associated virus (rAAV) particles and a buffer, a sugar, and an amorphous salt having an ionic strength of 30 mM to 100 mM, the formulation being suitable for lyophilization. In some embodiments, the stable formulation also comprises a surfactant. In some embodiments, the stable formulation also comprises poloxamer 188. In some embodiments, the formulation is a pre-lyophilized formulation.

[0141] In some embodiments, provided herein is a stable formulation comprising recombinant adeno-associated virus (rAAV) particles and a buffer, a sugar, and an amorphous salt with an ionic strength greater than 200 mM, the formulation being suitable for lyophilization. In some embodiments, the stable formulation also comprises a surfactant. In some embodiments, the stable formulation also comprises poloxamer 188. In some embodiments, the formulation is a pre-lyophilized formulation.

[0142] In some embodiments, provided herein is a stable formulation comprising recombinant adeno-associated virus (rAAV) particles, a buffer, a sugar, an amorphous salt having an ionic strength of 60 mM to 150 mM, and a surfactant, the formulation being suitable for lyophilization. In some embodiments, the stable formulation also comprises a surfactant. In some embodiments, the stable formulation also comprises poloxamer 188. In some embodiments, the formulation is a pre-lyophilized formulation.

[0143] In some embodiments, provided herein is a stable formulation comprising recombinant adeno-associated virus (rAAV) particles, a buffer, a sugar, an amorphous salt having an ionic strength of 30 mM to 100 mM, and a surfactant, the formulation being suitable for lyophilization. In some embodiments, the stable formulation also comprises a surfactant. In some embodiments, the stable formulation also comprises poloxamer 188. In some embodiments, the formulation is a pre-lyophilized formulation.

[0144] In some embodiments, provided herein is a stable formulation comprising recombinant adeno-associated virus (rAAV) particles, a buffer, a sugar, and an amorphous salt with an ionic strength greater than 200 mM, and a surfactant, the formulation being suitable for lyophilization. In some embodiments, the stable formulation also comprises a surfactant. In some embodiments, the stable formulation also comprises poloxamer 188. In some embodiments, the formulation is a pre-lyophilized formulation.

[0145] In some embodiments, the formulations disclosed herein contain about 0.0001% to about 0.5% of a non-ionic surfactant. In some embodiments, the formulations disclosed herein contain about 0.0005% to about 0.1% of a non-ionic surfactant. In some embodiments, the formulations disclosed herein contain about 0.001%, about 0.002%, about 0.003%, about 0.004%, about 0.005%, about 0.007%, or about 0.01% of a non-ionic surfactant.

[0146] In some embodiments, the formulations disclosed herein contain about 0.0001% to about 0.5% poloxamer 188. In some embodiments, the formulations disclosed herein contain about 0.0005% to about 0.1% poloxamer 188. In some embodiments, the formulations disclosed herein contain about 0.001%, about 0.002%, about 0.003%, about 0.004%, about 0.005%, about 0.007%, or about 0.01% poloxamer 188.

[0147] In some embodiments, the formulations disclosed herein comprise about 0.0005% poloxamer 188. In some embodiments, the formulations disclosed herein comprise about 0.001% poloxamer 188. In some embodiments, the formulations disclosed herein comprise about 0.002% poloxamer 188. In some embodiments, the formulations disclosed herein comprise about 0.003% poloxamer 188. In some embodiments, the formulations disclosed herein comprise about 0.004% poloxamer 188. In some embodiments, the formulations disclosed herein comprise about 0.005% poloxamer 188. In some embodiments, the formulations disclosed herein comprise about 0.008% poloxamer 188.

[0148] In some embodiments, the salt is a pharma- ceutically acceptable salt. In some embodiments, the salt is an amorphous salt that does not crystallize. In some embodiments, the salt is a sodium salt. In some embodiments, the salt is sodium citrate, sodium acetate, or sodium chloride. In some embodiments, the salt is sodium citrate, sodium sulfate, ammonium sulfate, or magnesium sulfate. In some embodiments, the salt is a multivalent salt, which is composed of multiple charged ions with higher ionic strength (per excipient molecule added compared to monovalent sodium chloride) and can inhibit AAV aggregation while minimizing crystallization.

[0149] In some embodiments, the salt excipient is added or adjusted to achieve the desired ionic strength. In some embodiments, the amorphous salt has an ionic strength of about 150 mM, about 145 mM, about 140 mM, about 135 mM, about 130 mM, about 125 mM, about 120 mM, about 115 mM, about 110 mM, about 110 mM, about 105 mM, or about 100 mM or less. In certain embodiments, the stable formulation has a buffer ionic strength of about 150 mM, about 145 mM, about 140 mM, about 135 mM, about 130 mM, about 125 mM, about 120 mM, about 115 mM, about 110 mM, about 105 mM, or about 100 mM or less. In some embodiments, the rAAV particles in the formulation are rAAV8 particles or rAAV9 particles.

[0150] In some embodiments, the stable formulation has an ionic strength of 150 mM, 145 mM, 140 mM, 135 mM, 130 mM, 125 mM, 120 mM, 115 mM, or 110 mM or less. In certain embodiments, the stable formulation has a buffer ionic strength of 150 mM, 145 mM, 140 mM, 135 mM, 130 mM, 125 mM, 120 mM, 115 mM, or 110 mM or less. In some embodiments, the rAAV particles in the formulation have AAV8 capsids.

[0151] In certain embodiments, the stable formulation has an ionic strength of greater than 60 mM. In certain embodiments, the stable formulation has an ionic strength of about 60 mM to 150 mM. In certain embodiments, the stable formulation has an ionic strength of about 60 mM to 115 mM. In certain embodiments, the stable formulation has an ionic strength of about 60 mM to 100 mM. In certain embodiments, the stable formulation has an ionic strength of about 60 mM. In certain embodiments, the stable formulation has an ionic strength of about 65 mM. In certain embodiments, the stable formulation has an ionic strength of about 70 mM. In certain embodiments, the stable formulation has an ionic strength of about 75 mM. In certain embodiments, the stable formulation has an ionic strength of about 80 mM. In certain embodiments, the stable formulation has an ionic strength of about 85 mM. In certain embodiments, the stable formulation has an ionic strength of about 90 mM. In some embodiments, the rAAV particles in the formulation have AAV8 capsids.

[0152] In certain embodiments, the stable formulation has an ionic strength of more than 30 mM. In certain embodiments, the stable formulation has an ionic strength of about 30 mM to 100 mM. In certain embodiments, the stable formulation has an ionic strength of about 30 mM. In certain embodiments, the stable formulation has an ionic strength of about 35 mM. In certain embodiments, the stable formulation has an ionic strength of about 40 mM. In certain embodiments, the stable formulation has an ionic strength of about 45 mM. In certain embodiments, the stable formulation has an ionic strength of about 50 mM. In certain embodiments, the stable formulation has an ionic strength of about 55 mM. In certain embodiments, the stable formulation has an ionic strength of about 60 mM. In certain embodiments, the stable formulation has an ionic strength of about 65 mM. In certain embodiments, the stable formulation has an ionic strength of about 70 mM. In certain embodiments, the stable formulation has an ionic strength of about 75 mM. In certain embodiments, the stable formulation has an ionic strength of about 80 mM. In certain embodiments, the stable formulation has an ionic strength of about 85 mM. In certain embodiments, the stable formulation has an ionic strength of about 90 mM. In certain embodiments, the stable formulation has an ionic strength of about 95 mM. In certain embodiments, the stable formulation has an ionic strength of about 100 mM. In some embodiments, the rAAV particles in the formulation have AAV9 capsids.

[0153] In certain embodiments, the stable formulation has an ionic strength of about 60 mM to 150 mM. In certain embodiments, the stable formulation has an ionic strength of about 60 mM to 115 mM. In certain embodiments, the stable formulation has an ionic strength of about 65 mM to 95 mM. In certain embodiments, the stable formulation has an ionic strength of about 70 mM to 90 mM. In certain embodiments, the stable formulation has an ionic strength of about 75 mM to 85 mM. In some embodiments, the rAAV particles in the formulation have AAV8 capsids.

[0154] In certain embodiments, the stable formulation has an ionic strength of about 30 mM to 100 mM. In certain embodiments, the stable formulation has an ionic strength of about 35 mM to 95 mM. In certain embodiments, the stable formulation has an ionic strength of about 40 mM to 90 mM. In certain embodiments, the stable formulation has an ionic strength of about 45 mM to 85 mM. In certain embodiments, the stable formulation has an ionic strength of about 50 mM to 80 mM. In certain embodiments, the stable formulation has an ionic strength of about 55 mM to 75 mM. In certain embodiments, the stable formulation has an ionic strength of about 60 mM to 70 mM. In some embodiments, the rAAV particles in the formulation do not have AAV8 capsids. In some embodiments, the rAAV particles in the formulation have AAV9 capsids.

[0155] In some embodiments, the stable formulation has an ionic strength of greater than about 200 mM. In some embodiments, the rAAV particles in the formulation have AAV2 capsids. In some embodiments, the rAAV particles in the formulation do not have rAAV8 or rAAV9 capsids.

[0156] In some embodiments, the stable formulation has an ionic strength of greater than 200 mM, 210 mM, 220 mM, 230 mM, 240 mM, 250 mM, 260 mM, 270 mM, 280 mM, 290 mM, or 300 mM. In some embodiments, the rAAV particles in the formulation have AAV2 capsids. In some embodiments, the rAAV particles in the formulation do not have rAAV8 or rAAV9 capsids.

[0157] In certain embodiments, the stable formulation has an ionic strength of about 200 mM to about 210 mM. In certain embodiments, the stable formulation has an ionic strength of about 210 mM to about 220 mM. In certain embodiments, the stable formulation has an ionic strength of about 220 mM to about 230 mM. In certain embodiments, the stable formulation has an ionic strength of about 230 mM to about 240 mM. In certain embodiments, the stable formulation has an ionic strength of about 240 mM to about 250 mM. In certain embodiments, the stable formulation has an ionic strength of about 250 mM to about 260 mM. In certain embodiments, the stable formulation has an ionic strength of about 260 mM to about 270 mM. In certain embodiments, the stable formulation has an ionic strength of about 270 mM to about 280 mM. In certain embodiments, the stable formulation has an ionic strength of about 280 mM to about 290 mM. In certain embodiments, the stable formulation has an ionic strength of about 290 mM to about 300 mM. In some embodiments, the rAAV particles in the formulation have no AAV8 or AAV9 capsids.

[0158] In some embodiments, the formulations disclosed herein contain about 0.0001% to about 0.5% of a non-ionic surfactant. In some embodiments, the formulations disclosed herein contain about 0.0005% to about 0.1% of a non-ionic surfactant. In some embodiments, the formulations disclosed herein contain about 0.001%, about 0.002%, about 0.003%, about 0.004%, about 0.005%, about 0.007%, or about 0.01% of a non-ionic surfactant.

[0159] In some embodiments, the formulations disclosed herein contain about 0.0001% to about 0.5% poloxamer 188. In some embodiments, the formulations disclosed herein contain about 0.0005% to about 0.1% poloxamer 188. In some embodiments, the formulations disclosed herein contain about 0.001%, about 0.002%, about 0.003%, about 0.004%, about 0.005%, about 0.007%, or about 0.01% poloxamer 188.

[0160] In some embodiments, the formulations disclosed herein comprise about 0.0005% poloxamer 188. In some embodiments, the formulations disclosed herein comprise about 0.001% poloxamer 188. In some embodiments, the formulations disclosed herein comprise about 0.002% poloxamer 188. In some embodiments, the formulations disclosed herein comprise about 0.003% poloxamer 188. In some embodiments, the formulations disclosed herein comprise about 0.004% poloxamer 188. In some embodiments, the formulations disclosed herein comprise about 0.005% poloxamer 188. In some embodiments, the formulations disclosed herein comprise about 0.008% poloxamer 188.

[0161] In some embodiments, the salt is a pharma- ceutically acceptable salt. In some embodiments, the salt is an amorphous salt that does not crystallize. In some embodiments, the salt is a sodium salt. In some embodiments, the salt is sodium citrate, sodium acetate, or sodium chloride. In some embodiments, the salt is sodium citrate, sodium sulfate, ammonium sulfate, or magnesium sulfate. In some embodiments, the salt is a multivalent salt, which is composed of multiple charged ions with higher ionic strength (per molecule of excipient added compared to monovalent sodium chloride) and can inhibit AAV aggregation while minimizing crystallization.

[0162] In some embodiments, the stable formulation has an ionic strength of about 200 mM, about 195 mM, 180 mM, 175 mM, 170 mM, 165 mM, 160 mM, 155 mM, 150 mM, 145 mM, 140 mM, 135 mM, 130 mM, 125 mM, 120 mM, 115 mM, or 110 mM or less.

[0163] In some embodiments, salt excipients are added or adjusted to achieve the desired ionic strength. In some embodiments, the stable formulation has an ionic strength of about 200 mM, about 190 mM, about 180 mM, about 170 mM, about 160 mM, about 155 mM, about 150 mM, about 145 mM, about 140 mM, about 135 mM, about 130 mM, about 125 mM, about 120 mM, about 115 mM, or about 110 mM or less. In certain embodiments, the stable formulation has a buffer ionic strength of about 150 mM, about 145 mM, about 140 mM, about 135 mM, about 130 mM, about 125 mM, about 120 mM, about 115 mM, or about 110 mM or less. In some embodiments, the rAAV particles in the formulation are rAAV8 particles. In some embodiments, the rAAV particles in the formulation have AAV9 capsids.

[0164] In certain embodiments, the stable formulation has an ionic strength of about 60 mM to 150 mM. In certain embodiments, the stable formulation has an ionic strength of about 60 mM to 115 mM. In certain embodiments, the stable formulation has an ionic strength of about 60 mM to 100 mM. In certain embodiments, the stable formulation has an ionic strength of about 60 mM. In certain embodiments, the stable formulation has an ionic strength of about 65 mM. In certain embodiments, the stable formulation has an ionic strength of about 70 mM. In certain embodiments, the stable formulation has an ionic strength of about 75 mM. In certain embodiments, the stable formulation has an ionic strength of about 80 mM. In certain embodiments, the stable formulation has an ionic strength of about 85 mM. In certain embodiments, the stable formulation has an ionic strength of about 90 mM. In some embodiments, the rAAV particles in the formulation have AAV8 capsids.

[0165] In certain embodiments, the stable formulation has an ionic strength of about 30 mM to 100 mM. In certain embodiments, the stable formulation has an ionic strength of about 30 mM. In certain embodiments, the stable formulation has an ionic strength of about 35 mM. In certain embodiments, the stable formulation has an ionic strength of about 40 mM. In certain embodiments, the stable formulation has an ionic strength of about 45 mM. In certain embodiments, the stable formulation has an ionic strength of about 50 mM. In certain embodiments, the stable formulation has an ionic strength of about 55 mM. In certain embodiments, the stable formulation has an ionic strength of about 60 mM. In certain embodiments, the stable formulation has an ionic strength of about 65 mM. In certain embodiments, the stable formulation has an ionic strength of about 70 mM. In certain embodiments, the stable formulation has an ionic strength of about 75 mM. In certain embodiments, the stable formulation has an ionic strength of about 80 mM. In certain embodiments, the stable formulation has an ionic strength of about 85 mM. In certain embodiments, the stable formulation has an ionic strength of about 90 mM. In certain embodiments, the stable formulation has an ionic strength of about 95 mM. In certain embodiments, the stable formulation has an ionic strength of about 100 mM. In some embodiments, the rAAV particles in the formulation have AAV9 capsids.

[0166] In certain embodiments, the stable formulation has an ionic strength of about 60 mM to 150 mM. In certain embodiments, the stable formulation has an ionic strength of about 60 mM to 140 mM. In certain embodiments, the stable formulation has an ionic strength of about 60 mM to 130 mM. In certain embodiments, the stable formulation has an ionic strength of about 60 mM to 120 mM. In certain embodiments, the stable formulation has an ionic strength of about 60 mM to 115 mM. In certain embodiments, the stable formulation has an ionic strength of about 65 mM to 95 mM. In certain embodiments, the stable formulation has an ionic strength of about 70 mM to 90 mM. In certain embodiments, the stable formulation has an ionic strength of about 75 mM to 85 mM. In some embodiments, the rAAV particles in the formulation have AAV8 capsids.

[0167] In certain embodiments, the stable formulation has an ionic strength of about 30 mM to 100 mM. In certain embodiments, the stable formulation has an ionic strength of about 35 mM to 95 mM. In certain embodiments, the stable formulation has an ionic strength of about 40 mM to 90 mM. In certain embodiments, the stable formulation has an ionic strength of about 45 mM to 85 mM. In certain embodiments, the stable formulation has an ionic strength of about 50 mM to 80 mM. In certain embodiments, the stable formulation has an ionic strength of about 55 mM to 75 mM. In certain embodiments, the stable formulation has an ionic strength of about 60 mM to 70 mM. In some embodiments, the rAAV particles in the formulation have AAV9 capsids.

[0168] In certain embodiments, the pH of the stable formulation is about 7.4.

[0169] In certain embodiments, the pH of the stable formulation is about 6.0 to 8.8. In certain embodiments, the pH of the stable formulation is about 6.0 to 9.0. In certain embodiments, the pH of the stable formulation is about 6.0. In certain embodiments, the pH of the stable formulation is about 6.1. In certain embodiments, the pH of the stable formulation is about 6.2. In certain embodiments, the pH of the stable formulation is about 6.3. In certain embodiments, the pH of the stable formulation is about 6.4. In certain embodiments, the pH of the stable formulation is about 6.5. In certain embodiments, the pH of the stable formulation is about 6.6. In certain embodiments, the pH of the stable formulation is about 6.7. In certain embodiments, the pH of the stable formulation is about 6.8. In certain embodiments, the pH of the stable formulation is about 6.9. In certain embodiments, the pH of the stable formulation is about 7.0. In certain embodiments, the pH of the stable formulation is about 7.1. In certain embodiments, the pH of the stable formulation is about 7.2. In certain embodiments, the pH of the stable formulation is about 7.3. In certain embodiments, the pH of the stable formulation is about 7.4. In certain embodiments, the pH of the stable formulation is about 7.5. In certain embodiments, the pH of the stable formulation is about 7.6. In certain embodiments, the pH of the stable formulation is about 7.7. In certain embodiments, the pH of the stable formulation is about 7.8. In certain embodiments, the pH of the stable formulation is about 7.9. In certain embodiments, the pH of the stable formulation is about 8.0. In certain embodiments, the pH of the stable formulation is about 8.1. In certain embodiments, the pH of the stable formulation is about 8.2. In certain embodiments, the pH of the stable formulation is about 8.3. In certain embodiments, the pH of the stable formulation is about 8.4. In certain embodiments, the pH of the stable formulation is about 8.5. In certain embodiments, the pH of the stable formulation is about 8.6. In certain embodiments, the pH of the stable formulation is about 8.7. In certain embodiments, the pH of the stable formulation is about 8.8. In certain embodiments, the pH of the stable formulation is about 8.9.In certain embodiments, the pH of the stable formulation is about 9.0.

[0170] In certain embodiments, the vector genome concentration (VGC) of the stable formulation is about 3×10 9 GC / mL, 4×10 9 GC / mL, 5×10 9 GC / mL, 6×10 9 GC / mL, 7×10 9 GC / mL, 8×10 9 GC / mL, 9×10 9 GC / mL, approximately 1×10 10 GC / mL, approximately 2×10 10 GC / mL, approximately 3×10 10 GC / mL, approximately 4×10 10 GC / mL, approximately 5×10 10 GC / mL, approximately 6×10 10 GC / mL, approximately 7×10 10 GC / mL, approximately 8×10 10 GC / mL, approximately 9×10 10 GC / mL, approximately 1×10 11 GC / mL, approximately 2×10 11 GC / mL, approximately 3×10 11 GC / mL, approximately 4×10 11 GC / mL, approximately 5×10 11 GC / mL, approximately 6×10 11 GC / mL, approximately 7×10 11 GC / mL, approximately 8×10 11 GC / mL, approximately 9×10 11 GC / mL, approximately 1×10 12 GC / mL, approximately 2×10 12 GC / mL, approximately 3×10 12 GC / mL, approximately 4×10 12 GC / mL, approximately 5×10 12 GC / mL, approximately 6×10 12 GC / mL, approximately 7×10 12 GC / mL, approximately 8×10 12 GC / mL, approximately 9×10 12 GC / mL, approximately 1×10 13 GC / mL, approximately 1×10 13 GC / mL, approximately 2×10 13 GC / mL, or approximately 3 x 1013 GC / mL.

[0171] In certain embodiments, recombinant adeno-associated virus (rAAV) particles in a stable formulation are at least about 2%, about 5%, about 7%, about 10%, about 12%, about 15%, about 17%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 100%, about 2-fold, about 3-fold, about 5-fold, about 10-fold, about 100-fold, or greater than about 1000-fold more stable to a lyophilization or reconstitution process than the same rAAV particles in a reference formulation. In certain embodiments, the stability of the rAAV particles is determined by the assay(s) disclosed in Section 6.8.

[0172] In certain embodiments, the rAAV particles in the stable formulation have at least about 2%, about 5%, about 7%, about 10%, about 12%, about 15%, about 17%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 100%, about 2-fold, about 3-fold, about 5-fold, about 10-fold, about 100-fold, or more than about 1000-fold higher in infectivity than the same rAAV particles in a reference formulation. In certain embodiments, the viral infectivity of the rAAV particles is determined by the assay(s) disclosed in Section 6.8. In certain embodiments, the infectivity is measured before or after lyophilization. In certain embodiments, the infectivity is measured before or after reconstitution of the lyophilized formulation.

[0173] In certain embodiments, the rAAV particles in the stable formulation exhibit at least about 2%, about 5%, about 7%, about 10%, about 12%, about 15%, about 17%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 100%, about 2-fold, about 3-fold, about 5-fold, about 10-fold, about 100-fold, or about 1000-fold less aggregation than the same rAAV particles in a reference formulation. In certain embodiments, aggregation of the rAAV particles is determined by the assay(s) disclosed in Section 6.8. In certain embodiments, aggregation is measured before or after lyophilization. In certain embodiments, aggregation is measured before or after reconstitution of the lyophilized formulation.

[0174] In certain embodiments, the stable formulation is lyophilized prior to storage.

[0175] In certain embodiments, the stable formulation is reconstituted after storage.

[0176] In certain embodiments, the formulation is stored at -80°C, -70°C, -20°C, 4°C, 20°C, 25°C, 30°C, 35°C, 37°C, or 40°C.

[0177] In certain embodiments, the formulation is stored for about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, 12 months, about 15 months, about 18 months, about 24 months, about 2 years, about 3 years, or about 4 years.

[0178] In certain embodiments, recombinant adeno-associated virus (rAAV) particles in a stable formulation are at least about 2%, about 5%, about 7%, about 10%, about 12%, about 15%, about 17%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 100%, about 2-fold, about 3-fold, about 5-fold, about 10-fold, about 100-fold, or about 1000-fold more stable to a lyophilization or reconstitution process than the same rAAV particles in a reference formulation. In certain embodiments, the stability of the rAAV particles is determined by the assay(s) disclosed in Section 6.8.

[0179] In certain embodiments, the rAAV particles in the stable formulation have at least about 2%, about 5%, about 7%, about 10%, about 12%, about 15%, about 17%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 100%, about 2-fold, about 3-fold, about 5-fold, about 10-fold, about 100-fold, or about 1000-fold higher infectivity than the same rAAV particles in a reference formulation. In certain embodiments, the viral infectivity of the rAAV particles is determined by the assay(s) disclosed in Section 6.8. In certain embodiments, the infectivity is measured before or after lyophilization. In certain embodiments, the infectivity is measured before or after reconstitution of the lyophilized formulation.

[0180] In certain embodiments, the rAAV particles in the stable formulation exhibit at least about 2%, about 5%, about 7%, about 10%, about 12%, about 15%, about 17%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 100%, about 2-fold, about 3-fold, about 5-fold, about 10-fold, about 100-fold, or about 1000-fold less aggregation than the same rAAV particles in a reference formulation. In certain embodiments, aggregation of the rAAV particles is determined by the assay(s) disclosed in Section 6.8. In certain embodiments, aggregation is measured before or after lyophilization. In certain embodiments, aggregation is measured before or after reconstitution of the lyophilized formulation.

[0181] In certain embodiments, the stable formulation is lyophilized prior to storage.

[0182] In certain embodiments, the stable formulation is reconstituted after storage.

[0183] In certain embodiments, the formulation is stored at -80°C, -70°C, -20°C, 4°C, 20°C, 25°C, 30°C, 35°C, 37°C, or 40°C.

[0184] In certain embodiments, the formulation is stored for about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, 12 months, about 15 months, about 18 months, about 24 months, about 2 years, about 3 years, about 4 years, or about 5 years. In certain embodiments, the rAAV particles in a stable formulation are at least about 2%, about 5%, about 7%, about 10%, about 12%, about 15%, about 17%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 100%, about 2-fold, about 3-fold, about 5-fold, about 10-fold, about 100-fold, or more than about 1000-fold more stable than the same rAAV particles in a reference formulation stored under the same conditions after the formulation has been stored for a period of time, e.g., about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, 12 months, about 15 months, about 18 months, about 24 months, about 2 years, about 3 years, about 4 years, or about 5 years. In certain embodiments, the stability of the rAAV particles over a period of time is determined by the assay(s) disclosed in Section 6.8.

[0185] In certain embodiments, the rAAV particles in a stable formulation are at least about 2%, about 5%, about 7%, about 10%, about 12%, about 15%, about 17%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 100%, about 2-fold, about 3-fold, about 5-fold, about 10-fold, about 100-fold, or more than about 1000-fold more stable than the same rAAV particles in a reference formulation after the formulation is stored for a period of time, e.g., at least about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, 12 months, about 15 months, about 18 months, about 24 months, about 2 years, about 3 years, about 4 years, or about 5 years. In certain embodiments, the stability of the rAAV particles over a period of time is determined by the assay(s) disclosed in Section 6.8.

[0186] In certain embodiments, the rAAV particles in the stable formulation have an in vitro relative potency (IVRP) that is at least about 2%, about 5%, about 7%, about 10%, about 12%, about 15%, about 17%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 100%, about 2-fold, about 3-fold, about 5-fold, about 10-fold, about 100-fold, or about 1000-fold higher than the same rAAV particles in a reference formulation. In certain embodiments, the in vitro relative potency (IVRP) of the rAAV particles is determined by the assay(s) disclosed in Section 6.8. In certain embodiments, the IVRP is measured before or after lyophilization. In certain embodiments, the IVRP is measured before or after reconstitution of the lyophilized formulation.

[0187] In certain embodiments, the rAAV particles in the stable formulation have at least about 2%, about 5%, about 7%, about 10%, about 12%, about 15%, about 17%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 100%, about 2-fold, about 3-fold, about 5-fold, about 10-fold, about 100-fold, or about 1000-fold less free DNA than the same rAAV particles in a reference formulation. In certain embodiments, the free DNA of the rAAV particles is determined by the assay(s) disclosed in Section 6.8. In certain embodiments, the free DNA is measured before or after lyophilization. In certain embodiments, the free DNA is measured before or after reconstitution of the lyophilized formulation.

[0188] In certain embodiments, the rAAV particles in the stable formulation have at least about 2%, about 5%, about 7%, about 10%, about 12%, about 15%, about 17%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 100%, about 2-fold, about 3-fold, about 5-fold, about 10-fold, about 100-fold, or about 1000-fold less rAAV genome release than the same rAAV particles in the reference formulation. In certain embodiments, the rAAV genome release is determined by measuring the relative fluorescence in the presence of a DNA-specific fluorescent stain. In certain embodiments, the rAAV genome release is determined by the assay(s) disclosed in Section 6.8. In certain embodiments, the rAAV genome release is measured before or after lyophilization. In certain embodiments, the rAAV genome release is measured before or after reconstitution of the lyophilized formulation.

[0189] In certain embodiments, the rAAV particles in a stable formulation have a change in size of at most about 20%, about 15%, about 10%, about 8%, about 5%, about 4%, about 3%, about 2%, or about 1% after the formulation is stored for a period of time, such as about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, about 15 months, about 18 months, about 24 months, about 2 years, about 3 years, about 4 years, or about 5 years. In certain embodiments, the rAAV particle size is determined by the assay(s) disclosed in Section 6.8. In certain embodiments, the size is measured before or after a freeze / thaw cycle.

[0190] In certain embodiments, the rAAV particles in a stable formulation have a change in size of at most 20%, 15%, 10%, 8%, 5%, 4%, 3%, 2%, or about 1% after storage of the formulation for a period of time, e.g., at least about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, about 15 months, about 18 months, about 24 months, about 2 years, about 3 years, about 4 years, or about 5 years. In certain embodiments, the rAAV particle size is determined by the assay(s) disclosed in Section 6.8. In certain embodiments, the size is measured before or after lyophilization. In certain embodiments, the size is measured before or after reconstitution of the lyophilized formulation.

[0191] In certain embodiments, the rAAV particles in a stable formulation are at least about 2%, about 5%, about 7%, about 10%, about 12%, about 15%, about 17%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 100%, about 2-fold, about 3-fold, about 5-fold, about 10-fold, about 100-fold, or about 1000-fold more stable than the same rAAV particles in a reference formulation when stored at -80° C., -70° C., -20° C., 4° C., 20° C., 25° C., 30° C., 35° C., 37° C., or 40° C. In certain embodiments, the stability of the rAAV particles is determined by the assay(s) disclosed in Section 6.8.

[0192] In certain embodiments, the rAAV particles in the stable formulation are incubated at -80°C, -70°C, -20°C, 4°C, 20°C, 25°C, 30°C, 35°C, 37°C, or 40°C for a period of time, e.g., about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, about 13 months, about 14 months, about 15 months, about 16 months, about 17 months, about 18 months, about 19 months, about 20 months, about 21 months, about 22 months, about 23 months, about 24 months, about 25 months, about 26 months, about 27 months, about 28 months, about 29 months, about 30 months, about 31 months, about 32 months, about 33 months, about 34 months, about 35 months, about 36 months, about 37 months, about 38 months, about 39 months, about 40 ... When stored for about 15 months, about 18 months, about 24 months, about 2 years, about 3 years, about 4 years, or about 5 years, the infectivity is at least 2%, 5%, 7%, 10%, 12%, 15%, 17%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 100%, 2-fold, 3-fold, 5-fold, 10-fold, 100-fold, or 1000-fold greater than the same rAAV particles in a reference formulation. In certain embodiments, the viral infectivity of the rAAV particles is determined by the assay(s) disclosed in Section 6.8.

[0193] In certain embodiments, the rAAV particles in the stable formulation are incubated at -80°C, -70°C, -20°C, 4°C, 20°C, 25°C, 30°C, 35°C, 37°C, or 40°C for a period of time, such as at least about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, about 14 months, about 16 months, about 18 months, about 19 months, about 20 months, about 21 months, about 22 months, about 23 months, about 24 months, about 25 months, about 26 months, about 27 months, about 28 months, about 29 months, about 30 months, about 31 months, about 32 months, about 33 months, about 34 months, about 35 months, about 36 months, about 37 months, about 38 months, about 39 months, about 40 months, about 40 months, about 41 months, about 42 months, about 43 months, about 44 months, about 45 months, about 46 months, about 47 months, about 48 months, about 49 months, about 50 months, about 50 months, about 51 months, about 52 months, about 53 months, about 54 months, about 55 months, about 56 months, about 57 months, about 58 months, about 59 months, about 60 months, about 61 months, about 62 months, about 63 months, about 64 months, about 65 months, about 66 months, about 67 months, about 68 months, about 69 months, about 70 months, The infectivity is at least 2%, 5%, 7%, 10%, 12%, 15%, 17%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 100%, 2-fold, 3-fold, 5-fold, 10-fold, 100-fold, or 1000-fold greater than the same rAAV particles in a reference formulation when stored for 2 months, about 15 months, about 18 months, about 24 months, about 2 years, about 3 years, about 4 years, or about 5 years. In certain embodiments, the viral infectivity of the rAAV particles is determined by the assay(s) disclosed in Section 6.8.

[0194] In certain embodiments, the rAAV particles in the stable formulation are incubated at -80°C, -70°C, -20°C, 4°C, 20°C, 25°C, 30°C, 35°C, 37°C, or 40°C for a period of time, e.g., about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, about 13 months, about 14 months, about 15 months, about 16 months, about 17 months, about 18 months, about 19 months, about 20 months, about 21 months, about 22 months, about 23 months, about 24 months, about 25 months, about 26 months, about 27 months, about 28 months, about 29 months, about 30 months, about 31 months, about 32 months, about 33 months, about 34 months, about 35 months, about 36 months, about 37 months, about 38 months, about 39 months, about 40 ... When stored for about 15 months, about 15 months, about 18 months, about 24 months, about 2 years, about 3 years, about 4 years, or about 5 years, there is at least 2%, 5%, 7%, 10%, 12%, 15%, 17%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 100%, 2-fold, 3-fold, 5-fold, 10-fold, 100-fold, or 1000-fold less aggregation than the same rAAV particles in a reference formulation. In certain embodiments, aggregation of rAAV particles is identified by the assay(s) disclosed in Section 6.8.

[0195] In certain embodiments, the rAAV particles in the stable formulation are incubated at -80°C, -70°C, -20°C, 4°C, 20°C, 25°C, 30°C, 35°C, 37°C, or 40°C for a period of time, such as at least about 11 weeks, about 2 weeks, about 3 weeks, about 4 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about When stored for 12 months, about 15 months, about 18 months, about 24 months, about 2 years, about 3 years, about 4 years, or about 5 years, there is at least 2%, 5%, 7%, 10%, 12%, 15%, 17%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 100%, 2-fold, 3-fold, 5-fold, 10-fold, 100-fold, or 1000-fold less aggregation than the same rAAV particles in a reference formulation. In certain embodiments, aggregation of rAAV particles is identified by the assay(s) disclosed in Section 6.8.

[0196] In certain embodiments, the rAAV particles in the stable formulation are incubated at -80°C, -70°C, -20°C, 4°C, 20°C, 25°C, 30°C, 35°C, 37°C, or 40°C for a period of time, e.g., about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, about 13 months, about 14 months, about 15 months, about 16 months, about 17 months, about 18 months, about 19 months, about 20 months, about 21 months, about 22 months, about 23 months, about 24 months, about 25 months, about 26 months, about 27 months, about 28 months, about 29 months, about 30 months, about 31 months, about 32 months, about 33 months, about 34 months, about 35 months, about 36 months, about 37 months, about 38 months, about 39 months, about 40 ... The stability is at least 2%, 5%, 7%, 10%, 12%, 15%, 17%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 100%, 2-fold, 3-fold, 5-fold, 10-fold, 100-fold, or 1000-fold greater than the same rAAV particles in a reference formulation when stored for about 1 month, about 15 months, about 18 months, about 24 months, about 2 years, about 3 years, about 4 years, or about 5 years. In certain embodiments, the stability of the rAAV particles over a period of time is determined by the assay(s) disclosed in Section 6.8.

[0197] In certain embodiments, the rAAV particles in the stable formulation are incubated at -80°C, -70°C, -20°C, 4°C, 20°C, 25°C, 30°C, 35°C, 37°C, or 40°C for a period of time, such as at least about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, about 14 months, about 16 months, about 18 months, about 19 months, about 20 months, about 21 months, about 22 months, about 23 months, about 24 months, about 25 months, about 26 months, about 27 months, about 28 months, about 29 months, about 30 months, about 31 months, about 32 months, about 33 months, about 34 months, about 35 months, about 36 months, about 37 months, about 38 months, about 39 months, about 40 months, about 40 months, about 41 months, about 42 months, about 43 months, about 44 months, about 45 months, about 46 months, about 47 months, about 48 months, about 49 months, about 50 months, about 50 months, about 51 months, about 52 months, about 53 months, about 54 months, about 55 months, about 56 months, about 57 months, about 58 months, about 59 months, about 60 months, about 61 months, about 62 months, about 63 months, about 64 months, about 65 months, about 66 months, about 67 months, about 68 months, about 69 months, about 70 months, The stability is at least 2%, 5%, 7%, 10%, 12%, 15%, 17%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 100%, 2-fold, 3-fold, 5-fold, 10-fold, 100-fold, or 1000-fold greater than the same rAAV particles in a reference formulation when stored for 2 months, about 15 months, about 18 months, about 24 months, about 2 years, about 3 years, about 4 years, or about 5 years. In certain embodiments, the stability of the rAAV particles over a period of time is determined by the assay(s) disclosed in Section 6.8.

[0198] In certain embodiments, the rAAV particles in a stable formulation exhibit improved in vivo stability compared to the same rAAV particles in a reference formulation when stored at -80°C, -70°C, -20°C, 4°C, 20°C, 25°C, 30°C, 35°C, 37°C, or 40°C for a period of time, e.g., 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, about 15 months, about 18 months, about 24 months, about 2 years, about 3 years, about 4 years, or about 5 years. The in vitro relative potency (IVRP) is at least 2%, 5%, 7%, 10%, 12%, 15%, 17%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 100%, 2-fold, 3-fold, 5-fold, 10-fold, 100-fold, or greater than 1000. In certain embodiments, the in vitro relative potency (IVRP) of the rAAV particles is determined by the assay(s) disclosed in Section 6.8.

[0199] In certain embodiments, the rAAV particles in a stable formulation exhibit a reduced molecular weight (MME) of about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, about 15 months, about 18 months, about 24 months, about 2 years, about 3 years, about 4 years, or about 5 years when stored at -80°C, -70°C, -20°C, 4°C, 20°C, 25°C, 30°C, 35°C, 37°C, or 40°C for a period of time, such as at least about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, about 15 months, about 18 months, about 24 months, about 2 years, about 3 years, about 4 years, or about 5 years, compared to the same rAAV particles in a reference formulation. The in vitro relative potency (IVRP) is at least 2%, 5%, 7%, 10%, 12%, 15%, 17%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 100%, 2-fold, 3-fold, 5-fold, 10-fold, 100-fold, or 1000-fold higher. In certain embodiments, the in vitro relative potency (IVRP) of the rAAV particles is determined by the assay(s) disclosed in Section 6.8.

[0200] In certain embodiments, the rAAV particles in the stable formulation exhibit a reduced in vivo stability compared to the same rAAV particles in a reference formulation when stored at -80°C, -70°C, -20°C, 4°C, 20°C, 25°C, 30°C, 35°C, 37°C, or 40°C for a period of time, e.g., about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, about 15 months, about 18 months, about 24 months, about 2 years, about 3 years, about 4 years, or about 5 years. The in vitro relative potency (IVRP) is at least 2%, 5%, 7%, 10%, 12%, 15%, 17%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 100%, 2-fold, 3-fold, 5-fold, 10-fold, 100-fold, or greater than 1000. In certain embodiments, the in vitro relative potency (IVRP) of the rAAV particles is determined by the assay(s) disclosed in Section 6.8.

[0201] In certain embodiments, the rAAV particles in a stable formulation exhibit a reduced molecular weight (MME) of about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, about 15 months, about 18 months, about 24 months, about 2 years, about 3 years, about 4 years, or about 5 years when stored at -80°C, -70°C, -20°C, 4°C, 20°C, 25°C, 30°C, 35°C, 37°C, or 40°C for a period of time, such as at least about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, about 15 months, about 18 months, about 24 months, about 2 years, about 3 years, about 4 years, or about 5 years, compared to the same rAAV particles in a reference formulation. The in vitro relative potency (IVRP) is at least 2%, 5%, 7%, 10%, 12%, 15%, 17%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 100%, 2-fold, 3-fold, 5-fold, 10-fold, 100-fold, or 1000-fold higher. In certain embodiments, the in vitro relative potency (IVRP) of the rAAV particles is determined by the assay(s) disclosed in Section 6.8.

[0202] In certain embodiments, the rAAV particles in the stable formulation are incubated at -80°C, -70°C, -20°C, 4°C, 20°C, 25°C, 30°C, 35°C, 37°C, or 40°C for a period of time, e.g., about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, about 13 months, about 14 months, about 15 months, about 16 months, about 17 months, about 18 months, about 19 months, about 20 months, about 21 months, about 22 months, about 23 months, about 24 months, about 25 months, about 26 months, about 27 months, about 28 months, about 29 months, about 30 months, about 31 months, about 32 months, about 33 months, about 34 months, about 35 months, about 36 months, about 37 months, about 38 months, about 39 months, about 40 ... When stored for about 15 months, about 18 months, about 24 months, about 2 years, about 3 years, about 4 years, or about 5 years, the rAAV particles have at least 2%, 5%, 7%, 10%, 12%, 15%, 17%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 100%, 2-fold, 3-fold, 5-fold, 10-fold, or 1000-fold less free DNA than the same rAAV particles in a reference formulation. In certain embodiments, the free DNA of the rAAV particles is determined by the assay(s) disclosed in Section 6.8.

[0203] In certain embodiments, the rAAV particles in the stable formulation are incubated at -80°C, -70°C, -20°C, 4°C, 20°C, 25°C, 30°C, 35°C, 37°C, or 40°C for a period of time, such as at least about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, about 13 months, about 14 months, about 15 months, about 16 months, about 17 months, about 18 months, about 19 months, about 20 months, about 21 months, about 22 months, about 23 months, about 24 months, about 25 months, about 26 months, about 27 months, about 28 months, about 29 months, about 30 months, about 31 months, about 32 months, about 33 months, about 34 months, about 35 months, about 36 months, about 37 months, about 38 months, about 39 months, about 40 months, about 40 months, about 41 months, about 42 months, about 43 months, about 44 months, about 45 months, about 46 months, about 47 months, about 48 months, about 49 months, about 50 months, about 50 months, about 51 months, about 52 months, about 53 months, about 54 months, about 55 months, about 56 months, about 57 months, about 58 months, about 59 months, about 60 months, about 61 months, about 62 months, about 63 months, about 64 months, about 65 months, about 66 months, about 67 months, When stored for about 15 months, about 18 months, about 24 months, about 2 years, about 3 years, about 4 years, or about 5 years, the rAAV particles have at least 2%, 5%, 7%, 10%, 12%, 15%, 17%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 100%, 2-fold, 3-fold, 5-fold, 10-fold, 100-fold, or 1000-fold less free DNA than the same rAAV particles in a reference formulation. In certain embodiments, the free DNA of the rAAV particles is determined by the assay(s) disclosed in Section 6.8.

[0204] In certain embodiments, the rAAV particles in a stable formulation change in particle size by at most 20%, 15%, 10%, 8%, 5%, 4%, 3%, 2%, or 1% when stored for a period of time, e.g., about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, about 15 months, about 18 months, about 24 months, about 2 years, about 3 years, about 4 years, or about 5 years, at -80° C., -70° C., -20° C., 4° C., 20° C., 25° C., 30° C., 35° C., 37° C., or 40° C. In certain embodiments, the rAAV particle size is determined by the assay(s) disclosed in Section 6.8.

[0205] In certain embodiments, the rAAV particles in a stable formulation change in particle size by at most 20%, 15%, 10%, 8%, 5%, 4%, 3%, 2%, or 1% when compared to the same rAAV particles in a reference formulation when stored for a period of time, e.g., at least about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, about 15 months, about 18 months, about 24 months, about 2 years, about 3 years, about 4 years, or about 5 years, at -80° C., -70° C., -20° C., 4° C., 20° C., 25° C., 30° C., 35° C., 37° C., or 40° C. In certain embodiments, the rAAV particle size is determined by the assay(s) disclosed in Section 6.8.

[0206] In certain embodiments, the rAAV particle size is determined by the assay(s) disclosed in Section 6.8.

[0207] In certain embodiments, the reference formulation is DPBS with 0.001% poloxamer 188 buffer.

[0208] In certain embodiments, the reference formulation is a sugar-free formulation. In certain embodiments, the reference formulation is a plasticizer-free formulation.

[0209] In certain embodiments, the formulation is frozen in the lyophilization process to a temperature of about -20° C. In certain embodiments, the frozen formulation, when frozen at -20° C., maintains a pH of about pH 6 to about pH 9. In certain embodiments, the frozen formulation, when frozen at -20° C., maintains a pH value within plus or minus 1 unit of the pH value before freezing.

[0210] In certain embodiments, the glass transition temperature (Tg) of the lyophilized cake of the formulation is greater than 35°C.

[0211] In certain embodiments, the glass transition temperature (Tg') of a maximally freeze-concentrated solution of the formulation is greater than -40°C.

[0212] In certain embodiments, the moisture content is about 0.5% to about 1%. In certain embodiments, the moisture content is about 1% to about 2%. In certain embodiments, the moisture content is about 2% to about 3%. In certain embodiments, the moisture content is about 3% to about 4%. In certain embodiments, the moisture content is about 4% to about 5%.

[0213] In certain embodiments, the moisture content is about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.8%, about 1.0%, about 1.2%, about 1.2%, about 1.3%, about 1.4%, about 1.5%, about 1.6%, about 1.7%, about 1.8%, about 1.9%, about 2.0%, about 3.0%, about 4.0%, or about 5.0%.

[0214] In some embodiments, the formulations disclosed herein include a plasticizer or stabilizer. Pharmaceutically acceptable stabilizers or plasticizers include, but are not limited to, glycerol, xylitol, and sorbitol. In some embodiments, the formulations disclosed herein include glycerol, xylitol, sorbitol, or a combination thereof. In some embodiments, the formulations disclosed herein include glycerol.

[0215] In some embodiments, the formulations disclosed herein contain from about 0.1% to about 5% of a plasticizer or stabilizer. In some embodiments, the formulations disclosed herein contain from about 0.1% to about 2% of a plasticizer or stabilizer. In some embodiments, the formulations disclosed herein contain from about 0.25% to about 2% of a plasticizer.

[0216] In some embodiments, the formulations disclosed herein contain from about 0.1% to about 5% glycerol. In some embodiments, the formulations disclosed herein contain from about 0.1% to about 2% glycerol. In some embodiments, the formulations disclosed herein contain from about 0.25% to about 2% glycerol.

[0217] In some embodiments, the term "about" refers to plus or minus 10% of a given value or range. In some embodiments, the term "about" refers to a range of about 10-20% greater or less than the stated number or range. In certain embodiments, the term "about" includes the exact number recited.

[0218] Still other features and advantages of the compositions and methods described herein will become more apparent from the following detailed description taken in conjunction with the accompanying drawings.

[0219] 4.1 Illustrative Embodiments 4.1.1 Set 1 1. Recombinant adeno-associated virus (rAAV) particles, and a) a buffering agent, b) sugar, and c) Sodium citrate and A stable formulation comprising:

[0220] 2. The formulation of clause 1, wherein said buffer comprises about 1 mM to about 50 mM Tris.

[0221] 3. The formulation of clause 2 comprising about 1 mM to about 30 mM, about 1 mM to about 20 mM, about 5 mM to about 30 mM, about 5 mM to about 20 mM, about 10 mM to about 30 mM, about 10 mM to about 20 mM, or about 20 mM to about 50 mM Tris.

[0222] 4. The formulation of clause 2 comprising about 1 mM, about 2 mM, about 3 mM, about 5 mM, about 10 mM, about 15 mM, about 20 mM, about 25 mM, about 30 mM, or about 40 mM Tris.

[0223] 5. The formulation of clause 2 comprising about 5 mM Tris.

[0224] 6. The formulation according to any one of clauses 1 to 4, wherein the pH is about 6.5 to 8.0.

[0225] 7. The formulation according to clause 6, wherein the pH is about 7.2 to 7.8.

[0226] 8. The formulation of clause 6, wherein the pH is about 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, or 7.8.

[0227] 9. The formulation of clause 6, wherein the pH is about 7.5.

[0228] 10. The formulation of any one of clauses 1 to 9, comprising about 50 mM to about 400 mM sugar.

[0229] 11. The formulation of clause 10, comprising about 50 mM to about 350 mM, about 50 mM to about 300 mM, about 50 mM to about 250 mM, about 50 mM to about 200 mM, or about 50 mM to about 150 mM sugar.

[0230] 12. The formulation of clause 10, comprising about 100 mM to about 400 mM, about 150 mM to about 400 mM, about 200 mM to about 400 mM, about 250 mM to about 400 mM, or about 300 mM to about 400 mM sugar.

[0231] 13. The formulation of clause 10, comprising about 100 mM to about 300 mM, about 150 mM to about 250 mM, about 200 mM to about 300 mM, or about 250 mM to about 350 mM sugar.

[0232] 14. The formulation of clause 10, comprising about 50 mM, about 100 mM, about 150 mM, about 160 mM, about 170 mM, about 180 mM, about 190 mM, about 200 mM, about 210 mM, about 220 mM, about 230 mM, about 240 mM, about 250 mM, about 260 mM, about 270 mM, about 280 mM, about 290 mM, about 300 mM, or about 350 mM of sugar.

[0233] 15. The formulation of clause 10, comprising about 190 mM to about 230 mM, about 170 mM to about 250 mM, or about 150 mM to about 270 mM sugar.

[0234] 16. The formulation of clause 10, comprising about 210 mM sugar.

[0235] 17. The formulation according to any one of clauses 1 to 16, wherein the sugar is a non-reducing sugar.

[0236] 18. The formulation of clause 17, wherein the non-reducing sugar is sucrose, trehalose, or raffinose.

[0237] 19. The formulation according to clause 17, wherein the non-reducing sugar is sucrose.

[0238] 20. The formulation according to any one of clauses 1 to 16, wherein the sugar is a reducing sugar.

[0239] 21. The formulation according to clause 20, wherein the reducing sugar is glucose, fructose, mannose, galactose, or lactose.

[0240] 22. The formulation according to clause 20, wherein the reducing sugar is glucose.

[0241] 23. The formulation of any one of clauses 1 to 22, comprising less than about 100 mM sodium citrate.

[0242] 24. The formulation of clause 23, comprising about 10 mM to about 100 mM sodium citrate.

[0243] 25. The formulation of clause 23, comprising about 10 mM to about 100 mM, about 20 mM to about 100 mM, about 30 mM to about 100 mM, about 40 mM to about 100 mM, about 50 mM to about 100 mM, about 10 mM to about 80 mM, about 10 mM to about 60 mM, about 10 mM to about 50 mM, about 10 mM to about 40 mM, or about 10 mM to about 30 mM sodium citrate.

[0244] 26. The formulation of clause 23, comprising about 10 mM to about 50 mM, about 20 mM to about 60 mM, about 30 mM to about 70 mM, or about 10 mM to about 30 mM sodium citrate.

[0245] 27. The formulation of clause 23, comprising about 10 mM, about 20 mM, about 30 mM, about 40 mM, about 50 mM, or about 60 mM sodium citrate.

[0246] 28. The formulation of clause 23, comprising about 20 mM sodium citrate.

[0247] 29. The formulation of any one of clauses 1 to 28, further comprising about 0.0005% to about 0.01% of a non-ionic surfactant.

[0248] 30. The formulation of clause 29, comprising about 0.002% of a non-ionic surfactant.

[0249] 31. The formulation according to clause 28 or clause 29, wherein the non-ionic surfactant comprises poloxamer 188, poloxamer 407, polysorbate 80, polysorbate 20, Pluronic F-68, or BRIJ 35.

[0250] 32. The formulation according to clause 31, wherein the non-ionic surfactant comprises poloxamer 188.

[0251] 33. Recombinant adeno-associated virus (rAAV) particles, and a) about 1 mM to about 25 mM Tris, b) about 50 mM to about 400 mM sugar; c) about 10 mM to about 100 mM sodium citrate, and d) about 0.0005% to about 0.01% of a nonionic surfactant; A stable formulation comprising: The formulation has a pH of about 7.2 to about 7.8.

[0252] 34. Recombinant adeno-associated virus (rAAV) particles, and a) about 2 mM to about 10 mM Tris, b) about 150 mM to about 250 mM sugar; c) about 10 mM to about 20 mM sodium citrate, and d) about 0.001% to about 0.005% of a nonionic surfactant; A stable formulation comprising: The formulation has a pH of about 7.2 to about 7.8.

[0253] 35. Recombinant adeno-associated virus (rAAV) particles, and a) about 5 mM Tris, b) about 210 mM sugar; c) about 20 mM sodium citrate, and d) about 0.002% of a nonionic surfactant; A stable formulation comprising: The formulation has a pH of about 7.5.

[0254] 36. The formulation according to any one of clauses 33 to 35, wherein the sugar is a non-reducing sugar.

[0255] 37. The formulation according to clause 36, wherein the non-reducing sugar is sucrose, trehalose, or raffinose.

[0256] 38. The formulation according to clause 17, wherein the non-reducing sugar is sucrose.

[0257] 39. The formulation of any one of clauses 1 to 38, comprising about 1.0E+11 genome copies / mL (GC / mL) to about 1.0E+15 GC / mL of rAAV particles.

[0258] 40. The formulation of clause 39, comprising about 1.0E+11 GC / mL, about 1.0E+12 GC / mL, about 1.0E+13 GC / mL, about 1.0E+14 GC / mL, or about 1.0E+15 GC / mL of rAAV particles.

[0259] 41. The rAAV particles include AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV-11, AAV-12, AAV-13, AAV-14, AAV-15 and AAV-16, rAAV .rh8, rAAV.rh10, rAAV.rh20, rAAV.rh39, rAAV.Rh74, rAAV.RHM4-1, AAV.hu37, rAAV.Anc80, rAAV.Anc80L65, rAAV.7m8, rAAV.PHP.B, The formulation of any one of clauses 1 to 40, comprising a capsid protein of rAAV2.5, rAAV2tYF, rAAV3B, rAAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, or AAV.HSC16.

[0260] 42. The formulation of clause 41, wherein the rAAV particles comprise capsid proteins of the AAV-8 or AAV-9 serotype.

[0261] 43. The formulation according to any one of clauses 1 to 42, further comprising a plasticizer selected from the group consisting of glycerol, xylitol, sorbitol, or mannitol.

[0262] 44. The formulation according to any one of clauses 1 to 42, further comprising glycerol.

[0263] 45. The formulation of clause 44, comprising about 0.1% to about 5% glycerol.

[0264] 46. ​​The formulation of clause 44, comprising about 0.1% to about 2% glycerol.

[0265] 47. The formulation of clause 44, comprising about 0.25% to about 2% glycerol.

[0266] 48. A formulation according to any one of clauses 1 to 47, which is a liquid formulation.

[0267] 49. The formulation according to any one of clauses 1 to 47, which is a frozen formulation.

[0268] 50. The formulation according to any one of clauses 1 to 47, which is a lyophilized formulation or a reconstituted lyophilized formulation.

[0269] 51. The formulation according to clause 50, having a residual moisture content of about 1% to about 7%.

[0270] 52. The formulation according to clause 51, wherein the residual moisture content is about 1% to about 7%, about 2% to about 7%, about 3% to about 7%, about 4% to about 7%, about 5% to about 7%, about 1% to about 6%, about 1% to about 5%, about 1% to about 4%, or about 1% to about 3%.

[0271] 53. The formulation according to clause 51, wherein the residual moisture content is from about 3% to about 7%, from about 3% to about 6%, or from about 3% to about 5%.

[0272] 54. The formulation according to clause 51, wherein the residual moisture content is about 3%, about 3.5%, about 4%, about 4.5%, about 5%, about 5.5%, or about 6%.

[0273] 55. The formulation of any one of clauses 1 to 54, wherein the % relative potency of the rAAV particles is at least about 60%, at least about 70%, or at least about 80% after storage of the formulation at room temperature for 3 months, wherein the reference rAAV particles are stored at -70°C in DPBS containing 0.001% poloxamer 188 buffer.

[0274] 56. The formulation of any one of clauses 1 to 54, wherein the % relative potency of the rAAV particles is at least about 60%, at least about 70%, or at least about 80% after storage of the formulation at room temperature for 6 months, wherein the reference rAAV particles are stored at -70°C in Dulbecco's phosphate buffered saline (DPBS) containing 0.001% poloxamer 188 buffer.

[0275] 57. The formulation of any one of clauses 1 to 54, wherein the % relative potency of the rAAV particles is at least about 30%, at least about 40%, at least about 50%, or at least 60% after storage of the formulation at 35°C for 1 week, wherein the reference rAAV particles are stored at -70°C in DPBS containing 0.001% poloxamer 188 buffer.

[0276] 58. The formulation of any one of clauses 1 to 54, wherein the % relative potency of the rAAV particles is at least about 30%, at least about 40%, at least about 50%, or at least 60% after storage of the formulation at 35°C for 2 weeks, wherein the reference rAAV particles are stored at -70°C in DPBS containing 0.001% poloxamer 188 buffer.

[0277] 59. The formulation of any one of clauses 1 to 54, wherein the % relative potency of the rAAV particles is at least about 30%, at least about 40%, at least about 50%, or at least 60% after storage of the formulation at 35° C. for 4 weeks, wherein the reference rAAV particles are stored at -70° C. in DPBS containing 0.001% poloxamer 188 buffer.

[0278] 60. A method for producing a stable formulation comprising recombinant adeno-associated virus (rAAV) particles, the method comprising mixing rAAV particles with a buffer, a sugar, a salt, optionally a plasticizer, and optionally a non-ionic surfactant of a formulation described in any one of clauses 1 to 47, thereby producing the formulation comprising rAAV.

[0279] 61. A method for reducing rAAV genome release from rAAV particles, comprising producing a formulation comprising rAAV particles, a buffer, a sugar, a salt, and optionally a non-ionic surfactant, wherein rAAV genome release from the rAAV particles after three freeze-thaw cycles is reduced compared to rAAV genome release in a formulation that does not contain the sugar.

[0280] 62. A method for reducing rAAV genome release from rAAV particles, comprising producing a formulation comprising rAAV particles, a buffer, a sugar, a salt, and optionally a non-ionic surfactant, wherein rAAV genome release from the rAAV particles after lyophilization and reconstitution is reduced compared to rAAV genome release in a formulation that does not contain the sugar.

[0281] 63. The method of claim 61 or 62, further comprising lyophilizing the formulation to a residual moisture content of about 1% to about 5%.

[0282] 64. A method for reducing rAAV genome release from rAAV particles, comprising producing a formulation comprising rAAV particles, a buffer, a sugar, a salt, a plasticizer, and optionally a non-ionic surfactant, wherein rAAV genome release from the rAAV particles after three freeze-thaw cycles is reduced compared to rAAV genome release in a formulation that does not contain the sugar.

[0283] 65. A method for reducing rAAV genome release from rAAV particles, comprising producing a formulation comprising rAAV particles, a buffer, a sugar, a salt, a plasticizer, and optionally a non-ionic surfactant, wherein rAAV genome release from the rAAV particles after lyophilization and reconstitution is reduced compared to rAAV genome release in a formulation that does not contain the sugar.

[0284] 66. Use of a sugar to reduce rAAV genome release from rAAV particles, comprising producing a formulation comprising rAAV particles, a buffer, a sugar, a salt, and optionally a non-ionic surfactant, wherein rAAV genome release from the rAAV particles after three freeze-thaw cycles is reduced compared to rAAV genome release in a formulation that does not contain the sugar.

[0285] 67. Use of a sugar to reduce rAAV genome release from rAAV particles, comprising producing a formulation comprising rAAV particles, a buffer, a sugar, a salt, and optionally a non-ionic surfactant, wherein rAAV genome release from the rAAV particles after lyophilization and reconstitution is reduced compared to rAAV genome release in a formulation that does not contain the sugar.

[0286] 68. The use according to clause 66 or 67, further comprising lyophilizing the formulation to a residual moisture content of about 1% to about 7%.

[0287] 69. Use of a plasticizer to reduce rAAV genome release from rAAV particles, comprising producing a formulation comprising rAAV particles, a buffer, a sugar, a salt, a plasticizer, and optionally a non-ionic surfactant, wherein rAAV genome release from the rAAV particles after three freeze-thaw cycles is reduced compared to rAAV genome release in a formulation that does not contain the sugar.

[0288] 70. Use of a plasticizer to reduce rAAV genome release from rAAV particles, comprising producing a formulation comprising rAAV particles, a buffer, a sugar, a salt, a plasticizer, and optionally a non-ionic surfactant, wherein rAAV genome release from the rAAV particles after lyophilization and reconstitution is reduced compared to rAAV genome release in a formulation that does not contain the sugar.

[0289] 71. The method according to any one of clauses 61 to 65 or the use according to any one of clauses 66 to 70, wherein rAAV genome release is determined by measuring relative fluorescence in the presence of a DNA-specific fluorescent stain.

[0290] 72. The method according to any one of clauses 61 to 65 and 71 or the use according to any one of clauses 66 to 71, wherein freezing-induced rAAV genome release is reduced by at least about 10%, 20%, 50%, 80%, or 90%.

[0291] 73. The method according to any one of clauses 61 to 65 and 71 or the use according to any one of clauses 66 to 71, wherein freezing-induced rAAV genome release is substantially eliminated.

[0292] 74. The method according to any one of clauses 61 to 65 and 71 to 73 or the use according to any one of clauses 66 to 73, wherein the sugar is a non-reducing sugar.

[0293] 75. The method according to clause 74 or the use according to clause 74, wherein the non-reducing sugar is sucrose, trehalose, or raffinose.

[0294] 76. The method according to clause 74 or the use according to clause 74, wherein the non-reducing sugar is sucrose.

[0295] 77. The method according to any one of clauses 61 to 65 and 71 to 73 or the use according to any one of clauses 66 to 73, wherein the sugar is a reducing sugar.

[0296] 78. The method according to clause 77 or the use according to clause 77, wherein the reducing sugar is glucose, fructose, mannose, galactose or lactose.

[0297] 79. The method according to clause 77 or the use according to clause 77, wherein the reducing sugar is glucose.

[0298] 80. The method of any one of clauses 61 to 65 and 71 to 79, wherein the plasticizer comprises glycerol. Or the use of any one of clauses 66 to 79.

[0299] 81. The method according to any one of clauses 61 to 65 and 71 to 80 or the use according to any one of clauses 66 to 80, wherein the formulation is according to any one of clauses 1 to 47.

[0300] 4.1.2 Set 2 1. Recombinant adeno-associated virus (rAAV) particles, and a) a buffering agent, b) sugar, and c) Amorphous salts and A stable formulation comprising: The formulation is suitable for lyophilization.

[0301] 2. The formulation of clause 1, wherein said buffer comprises about 1 mM to about 50 mM Tris.

[0302] 3. The formulation of clause 2 comprising about 1 mM to about 30 mM, about 1 mM to about 20 mM, about 5 mM to about 30 mM, about 5 mM to about 20 mM, about 10 mM to about 30 mM, about 10 mM to about 20 mM, or about 20 mM to about 50 mM Tris.

[0303] 4. The formulation of clause 2 comprising about 1 mM, about 2 mM, about 3 mM, about 5 mM, about 10 mM, about 15 mM, about 20 mM, about 25 mM, about 30 mM, or about 40 mM Tris.

[0304] 5. The formulation of clause 2 comprising about 5 mM Tris.

[0305] 6. Recombinant adeno-associated virus (rAAV) particles, and a) a buffering agent, b) sugar, and c) Amorphous salts with an ionic strength greater than 60 mM A stable formulation comprising: The formulation is suitable for lyophilization.

[0306] 7. The formulation of clause 6, wherein the rAAV particles have AAV8 capsids.

[0307] 8. Recombinant adeno-associated virus (rAAV) particles, and a) a buffering agent, b) sugar, and c) Amorphous salts having an ionic strength of 60 mM to 150 mM; A stable formulation comprising: The formulation is suitable for lyophilization.

[0308] 9. The formulation of clause 8, wherein the rAAV particles have AAV8 capsids.

[0309] 10. Recombinant adeno-associated virus (rAAV) particles, and a) a buffering agent, b) sugar, and c) Amorphous salts having an ionic strength of 30 to 100 mM; A stable formulation comprising: The formulation is suitable for lyophilization.

[0310] 11. The formulation of clause 10, wherein the rAAV particles have rAAV9 capsids.

[0311] 12. Recombinant adeno-associated virus (rAAV) particles, and a) a buffering agent, b) sugar, and c) Amorphous salts with an ionic strength greater than 200 mM A stable formulation comprising: The formulation is suitable for lyophilization.

[0312] 13. The formulation of clause 12, wherein the rAAV particles do not have rAAV8 or rAAV9 capsids.

[0313] 14. The formulation according to any one of clauses 1 to 13, wherein the amorphous salt is sodium citrate.

[0314] 15. The formulation according to any one of clauses 1 to 13, wherein the amorphous salt is sodium sulfate.

[0315] 16. The formulation according to any one of clauses 1 to 13, wherein the amorphous salt is ammonium sulfate.

[0316] 17. The formulation according to any one of clauses 1 to 13, wherein the amorphous salt is magnesium sulfate.

[0317] 18. The formulation of any one of clauses 1 to 13, wherein the amorphous salt is sodium citrate, sodium sulfate, ammonium sulfate, magnesium sulfate, or a combination thereof.

[0318] 19. A formulation according to any one of clauses 1 to 13, comprising sodium citrate.

[0319] 20. A formulation according to any one of clauses 1 to 13, comprising sodium sulfate.

[0320] 21. A formulation according to any one of clauses 1 to 13, comprising ammonium sulfate.

[0321] 22. A formulation according to any one of clauses 1 to 13, comprising magnesium sulfate.

[0322] 23. A formulation according to any one of clauses 1 to 13, comprising sodium citrate, sodium sulfate, ammonium sulfate, magnesium sulfate, or a combination thereof.

[0323] 24. The formulation of any one of clauses 1 to 13, wherein the formulation comprises about 10 mM, about 20 mM, about 30 mM, about 40 mM, about 50 mM, about 60 mM, about 70 mM, about 80 mM, about 90 mM, about 100 mM, about 120 mM, about 140 mM, about 150 mM, or about 200 mM sodium sulfate.

[0324] 25. A formulation according to any one of clauses 1 to 24, having a pH of about 6.5 to 8.0.

[0325] 26. The formulation according to clause 25, having a pH of about 7.2 to 7.8.

[0326] 27. The formulation of clause 25, wherein the pH is about 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, or 7.8.

[0327] 28. The formulation according to clause 25, wherein the pH is about 7.5.

[0328] 29. The formulation of any one of clauses 1 to 28, comprising about 50 mM to about 400 mM sugar.

[0329] 30. The formulation of clause 29, comprising about 50 mM to about 350 mM, about 50 mM to about 300 mM, about 50 mM to about 250 mM, about 50 mM to about 200 mM, or about 50 mM to about 150 mM sugar.

[0330] 31. The formulation of clause 29, comprising about 100 mM to about 400 mM, about 150 mM to about 400 mM, about 200 mM to about 400 mM, about 250 mM to about 400 mM, or about 300 mM to about 400 mM sugar.

[0331] 32. The formulation of clause 29, comprising about 100 mM to about 300 mM, about 150 mM to about 250 mM, about 200 mM to about 300 mM, or about 250 mM to about 350 mM sugar.

[0332] 33. The formulation of clause 29, comprising about 50 mM, about 100 mM, about 150 mM, about 160 mM, about 170 mM, about 180 mM, about 190 mM, about 200 mM, about 210 mM, about 220 mM, about 230 mM, about 240 mM, about 250 mM, about 260 mM, about 270 mM, about 280 mM, about 290 mM, about 300 mM, or about 350 mM of sugar.

[0333] 34. The formulation of clause 29, comprising about 190 mM to about 230 mM, about 170 mM to about 250 mM, or about 150 mM to about 270 mM sugar.

[0334] 35. The formulation of clause 29, comprising about 210 mM sugar.

[0335] 36. The formulation according to any one of clauses 1 to 35, wherein the sugar is a non-reducing sugar.

[0336] 37. The formulation according to clause 36, wherein the non-reducing sugar is sucrose, trehalose, or raffinose.

[0337] 38. The formulation according to clause 36, wherein the non-reducing sugar is sucrose.

[0338] 39. The formulation according to any one of clauses 1 to 35, wherein the sugar is a reducing sugar.

[0339] 40. The formulation of clause 39, wherein the reducing sugar is glucose, fructose, mannose, galactose, or lactose.

[0340] 41. The formulation of any one of clauses 1 to 40, comprising less than about 100 mM sodium citrate.

[0341] 42. The formulation of clause 41, comprising about 10 mM to about 100 mM sodium citrate.

[0342] 43. The formulation of clause 41 comprising about 10 mM to about 100 mM, about 20 mM to about 100 mM, about 30 mM to about 100 mM, about 40 mM to about 100 mM, about 50 mM to about 100 mM, about 10 mM to about 80 mM, about 10 mM to about 60 mM, about 10 mM to about 50 mM, about 10 mM to about 40 mM, or about 10 mM to about 30 mM sodium citrate.

[0343] 44. The formulation of clause 41, comprising about 10 mM to about 50 mM, about 20 mM to about 60 mM, about 30 mM to about 70 mM, or about 10 mM to about 30 mM sodium citrate.

[0344] 45. The formulation of clause 41, comprising about 10 mM, about 20 mM, about 30 mM, about 40 mM, about 50 mM, or about 60 mM sodium citrate.

[0345] 46. ​​The formulation of clause 41, comprising about 20 mM sodium citrate.

[0346] 47. The formulation of any one of clauses 1 to 46, further comprising about 0.0005% to about 0.01% of a non-ionic surfactant.

[0347] 48. The formulation according to clause 47, comprising about 0.002% of a non-ionic surfactant.

[0348] 49. The formulation according to clause 46 or clause 47, wherein the non-ionic surfactant comprises poloxamer 188, poloxamer 407, polysorbate 80, polysorbate 20, Pluronic F-68, or BRIJ 35.

[0349] 50. The formulation according to clause 49, wherein the non-ionic surfactant comprises poloxamer 188.

[0350] 51. Recombinant adeno-associated virus (rAAV) particles, and a) about 1 mM to about 25 mM Tris, b) about 50 mM to about 400 mM sugar; c) about 10 mM to about 100 mM sodium citrate, and d) about 0.0005% to about 0.01% of a nonionic surfactant; A stable formulation comprising: The formulation has a pH of about 7.2 to about 7.8.

[0351] 52. Recombinant adeno-associated virus (rAAV) particles, and a) about 2 mM to about 10 mM Tris, b) about 150 mM to about 250 mM sugar; c) about 10 mM to about 20 mM sodium citrate, and d) about 0.001% to about 0.005% of a nonionic surfactant; A stable formulation comprising: The formulation has a pH of about 7.2 to about 7.8.

[0352] 53. Recombinant adeno-associated virus (rAAV) particles, and a) about 5 mM Tris, b) about 210 mM sugar; c) about 20 mM sodium citrate, and d) about 0.002% of a nonionic surfactant; A stable formulation comprising: The formulation has a pH of about 7.5.

[0353] 54. The formulation according to any one of clauses 51 to 53, wherein the sugar is a non-reducing sugar.

[0354] 55. The formulation according to clause 54, wherein the non-reducing sugar is sucrose, trehalose, or raffinose.

[0355] 56. The formulation according to clause 55, wherein the non-reducing sugar is sucrose.

[0356] 57. Recombinant adeno-associated virus (rAAV) particles, and a) about 5 mM Tris, b) about 210 mM sucrose; c) about 20 mM sodium citrate, and d) about 0.002% (w / v) poloxamer 188; A stable formulation comprising:

[0357] 58. Recombinant adeno-associated virus (rAAV) particles, and a) about 5 mM Tris, b) about 210 mM sucrose; c) about 20 mM sodium citrate; d) about 0.002% (w / v) poloxamer 188, and e) about 0.25% (w / v) glycerol; A stable formulation comprising:

[0358] 59. Recombinant adeno-associated virus (rAAV) particles, and a) about 5 mM Tris, b) about 210 mM sucrose; c) about 20 mM sodium citrate; d) about 0.002% (w / v) poloxamer 188, and e) about 0.5% (w / v) sorbitol; A stable formulation comprising:

[0359] 60. Recombinant adeno-associated virus (rAAV) particles, and a) about 5 mM Tris, b) about 30 mM sodium sulfate; c) about 263 mM sucrose, and d) about 0.005% (w / v) poloxamer 188; A stable formulation comprising:

[0360] 61. A formulation according to any one of clauses 1 to 60, suitable for lyophilization.

[0361] 62. The formulation according to any one of clauses 1 to 61, wherein the pH is about 7.5.

[0362] 63. The formulation according to any one of clauses 1 to 61, wherein the pH is about 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, or 7.8.

[0363] 64. The formulation according to any one of clauses 1 to 61, wherein the pH is about 7.1.

[0364] 65. A formulation according to any one of clauses 1 to 61, having a pH of about 6.5 to 8.0.

[0365] 66. A formulation according to any one of clauses 1 to 61, having a pH of about 7.2 to about 7.8.

[0366] 67. The formulation of any one of clauses 1 to 66, comprising about 1.0E+11 genome copies / mL (GC / mL) to about 1.0E+15 GC / mL of rAAV particles.

[0367] 68. The formulation of clause 67, comprising about 1.0E+11 GC / mL, about 1.0E+12 GC / mL, about 1.0E+13 GC / mL, about 1.0E+14 GC / mL, or about 1.0E+15 GC / mL of rAAV particles.

[0368] 69. The rAAV particles include AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV-11, AAV-12, AAV-13, AAV-14, AAV-15 and AAV-16, rAAV .rh8, rAAV.rh10, rAAV.rh20, rAAV.rh39, rAAV.Rh74, rAAV.RHM4-1, AAV.hu37, rAAV.Anc80, rAAV.Anc80L65, rAAV.7m8, rAAV.PHP.B, The formulation of any one of clauses 1 to 68, comprising a capsid protein of rAAV2.5, rAAV2tYF, rAAV3B, rAAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, or AAV.HSC16.

[0369] 70. The formulation of clause 69, wherein the rAAV particles comprise capsid proteins of the AAV-8 or AAV-9 serotype.

[0370] 71. The formulation according to any one of clauses 1 to 70, further comprising a stabilizer selected from the group consisting of glycerol or sorbitol.

[0371] 72. The formulation according to any one of clauses 1 to 70, further comprising glycerol.

[0372] 73. The formulation of any one of clauses 1 to 70, comprising about 0.1% to about 5% glycerol.

[0373] 74. The formulation of any one of clauses 1 to 70, comprising about 0.1% to about 2% glycerol.

[0374] 75. The formulation of any one of clauses 1 to 70, comprising about 0.25% to about 2% glycerol.

[0375] 76. A formulation according to any one of clauses 1 to 70, which does not contain mannitol.

[0376] 77. The formulation of any one of clauses 1 to 70, comprising less than 150 mM, 100 mM, 50 mM, 20 mM, or 10 mM mannitol.

[0377] 78. The formulation according to any one of clauses 1 to 77, which is a pre-lyophilized formulation.

[0378] 79. A formulation as described in clause 1, having a residual moisture content of about 1% to about 7%.

[0379] 80. The formulation according to clause 79, wherein the residual moisture content is about 1% to about 7%, about 2% to about 7%, about 3% to about 7%, about 4% to about 7%, about 5% to about 7%, about 1% to about 6%, about 1% to about 5%, about 1% to about 4%, or about 1% to about 3%.

[0380] 81. The formulation according to clause 79, wherein the residual moisture content is from about 3% to about 7%, from about 3% to about 6%, or from about 3% to about 5%.

[0381] 82. The formulation according to clause 79, wherein the residual moisture content is about 3%, about 3.5%, about 4%, about 4.5%, about 5%, about 5.5%, or about 6%.

[0382] 83. The formulation according to clause 79, wherein the water content is from about 1% to about 2%.

[0383] 84. The formulation according to clause 79, wherein the water content is about 1.5%, about 1.4%, about 1.3%, about 1.2%, or about 1.1%.

[0384] 85. The formulation according to clause 79, wherein the water content is about 1%.

[0385] 86. The formulation according to any one of clauses 1 to 78, wherein the glass transition temperature (Tg) of the lyophilized cake of the formulation is greater than 35°C.

[0386] 87. The formulation according to any one of clauses 1 to 78, wherein the glass transition temperature (Tg') of a maximum freeze-concentrated solution of the formulation is greater than -40°C.

[0387] 88. The formulation of any one of clauses 1 to 78, wherein the relative potency percent of the rAAV particles is at least about 60%, at least about 70%, or at least about 80% after storage of the formulation at room temperature for 3 months, wherein the reference rAAV particles are stored at -70°C in DPBS containing 0.001% poloxamer 188 buffer.

[0388] 89. The formulation of any one of clauses 1 to 78, wherein the relative potency percent of the rAAV particles is at least about 60%, at least about 70%, or at least about 80% after storage of the formulation at room temperature for 6 months, wherein the reference rAAV particles are stored at -70°C in Dulbecco's phosphate buffered saline (DPBS) containing 0.001% poloxamer 188 buffer.

[0389] 90. The formulation of any one of clauses 1 to 78, wherein the relative potency percent of the rAAV particles is at least about 30%, at least about 40%, at least about 50%, or at least 60% after storage of the formulation at 35°C for 1 week, wherein the reference rAAV particles are stored at -70°C in DPBS containing 0.001% poloxamer 188 buffer.

[0390] 91. The formulation of any one of clauses 1 to 78, wherein the % relative potency of the rAAV particles is at least about 30%, at least about 40%, at least about 50%, or at least 60% after storage of the formulation at 35°C for 2 weeks, wherein the reference rAAV particles are stored at -70°C in DPBS containing 0.001% poloxamer 188 buffer.

[0391] 92. The formulation of any one of clauses 1 to 78, wherein the relative potency percent of the rAAV particles is at least about 30%, at least about 40%, at least about 50%, or at least 60% after storage of the formulation at 35°C for 4 weeks, wherein the reference rAAV particles are stored at -70°C in DPBS containing 0.001% poloxamer 188 buffer.

[0392] 93. The formulation according to any one of clauses 87 to 92, wherein the formulation is lyophilized prior to storage.

[0393] 94. The formulation according to clause 94, wherein the lyophilized formulation is reconstituted after storage.

[0394] 95. The formulation of any one of clauses 1 to 78, wherein the relative potency % of the rAAV particles is at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or at least about 99% immediately after lyophilization.

[0395] 96. The level of rAAV particle aggregation in the formulation is compared with that in the reference formulation. r The formulation of any one of clauses 1 to 78, wherein the level of AAV particle aggregation is reduced by about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100% compared to the level of AAV particle aggregation.

[0396] 97. The formulation of any one of clauses 1 to 78, wherein the stability of the formulation is assessed by vector genome content or viral titer assay, and the formulation has at least about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, or about 200% more genome content after storage for 1 day, 2 days, 3 days, 4 days, 5 days, 6 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, when compared to the genome content of a reference formulation stored under identical conditions.

[0397] 98. The formulation of any one of clauses 1 to 78, wherein the stability of the formulation is assessed by measuring the relative potency of the formulation, and the formulation has a relative potency that is at least about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, or about 200% higher after storage for 1 day, 2 days, 3 days, 4 days, 5 days, 6 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, when compared to the genome content of a reference formulation stored under identical conditions.

[0398] 99. The formulation according to any one of clauses 1 to 78, wherein the stability of the formulation is assessed by reduction in infectivity, and the formulation has at least about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, or about 200% less after storage for 1 day, 2 days, 3 days, 4 days, 5 days, 6 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, when compared to the reduction in infectivity of a reference formulation stored under identical conditions.

[0399] 100. The formulation of any one of clauses 1 to 78, wherein the stability of the formulation is assessed by rAAV genome release, which is determined by measuring relative fluorescence in the presence of a DNA-specific fluorescent stain, and wherein the formulation has a relative fluorescence level that is at least about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, or about 200% lower after storage for 1 day, 2 days, 3 days, 4 days, 5 days, 6 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, when compared to the relative fluorescence level of a reference formulation stored under identical conditions.

[0400] 101. The formulation according to any one of clauses 97 to 100, wherein the formulation is lyophilized prior to storage.

[0401] 102. The formulation according to clause 101, wherein the lyophilized formulation is reconstituted after storage.

[0402] 103. The formulation of any one of clauses 97 to 102, wherein the formulation is stored at -80°C, -70°C, -20°C, 4°C, 20°C, 25°C, 30°C, 35°C, or 40°C.

[0403] 104. The formulation according to any one of clauses 96 to 103, wherein the reference formulation is DPBS containing 0.001% poloxamer 188 buffer.

[0404] 105. The formulation according to any one of clauses 96 to 104, wherein the reference formulation is a sugar-free formulation.

[0405] 106. The formulation according to any one of clauses 96 to 105, wherein the reference formulation is a formulation that does not contain a plasticizer.

[0406] 107. The formulation of any one of clauses 1 to 106, wherein the formulation is frozen to a temperature of about -20°C during the freeze-drying process.

[0407] 108. The formulation of any one of clauses 1 to 107, wherein the frozen formulation maintains a pH of about pH 6 to about pH 9 when frozen at -20°C.

[0408] 109. The formulation of any one of clauses 1 to 107, wherein the frozen formulation, when frozen at -20°C, maintains a pH value within plus or minus 1 unit of the pH value before freezing.

[0409] 110. The formulation according to any one of clauses 1 to 109, which is a stabilized aqueous rAAV formulation for lyophilization.

[0410] 111. A method for producing a stable formulation comprising recombinant adeno-associated virus (rAAV) particles, the method comprising mixing rAAV particles with a buffer, sugar, salt, optionally a plasticizer, and optionally a non-ionic surfactant of a formulation described in any one of clauses 1 to 108, thereby producing the formulation comprising rAAV.

[0411] 112. A method for reducing rAAV genome release from rAAV particles, comprising producing a formulation comprising rAAV particles, a buffer, a sugar, a salt, and optionally a non-ionic surfactant, wherein rAAV genome release from the rAAV particles after three freeze-thaw cycles is reduced compared to rAAV genome release in a formulation that does not contain the sugar.

[0412] 113. A method for reducing rAAV genome release from rAAV particles, comprising producing a formulation comprising rAAV particles, a buffer, a sugar, a salt, and optionally a non-ionic surfactant, wherein rAAV genome release from the rAAV particles after lyophilization and reconstitution is reduced compared to rAAV genome release in a formulation that does not contain the sugar.

[0413] 114. The method of clause 112 or 113, further comprising lyophilizing the formulation to a residual moisture content of about 1% to about 5%.

[0414] 115. A method for reducing rAAV genome release from rAAV particles, comprising producing a formulation comprising rAAV particles, a buffer, a sugar, a salt, a plasticizer, and optionally a non-ionic surfactant, wherein rAAV genome release from the rAAV particles after three freeze-thaw cycles is reduced compared to rAAV genome release in a formulation that does not contain the sugar.

[0415] 116. A method for reducing rAAV genome release from rAAV particles, comprising producing a formulation comprising rAAV particles, a buffer, a sugar, a salt, a plasticizer, and optionally a non-ionic surfactant, wherein rAAV genome release from the rAAV particles after lyophilization and reconstitution is reduced compared to rAAV genome release in a formulation that does not contain the sugar.

[0416] 117. A method for using sugar to reduce rAAV genome release from rAAV particles, comprising producing a formulation comprising rAAV particles, a buffer, sugar, a salt, and optionally a non-ionic surfactant, wherein rAAV genome release from the rAAV particles after three freeze-thaw cycles is reduced compared to rAAV genome release in a formulation that does not contain the sugar.

[0417] 118. A method for reducing rAAV genome release from rAAV particles, comprising producing a formulation comprising rAAV particles, a buffer, a sugar, a salt, and optionally a non-ionic surfactant, wherein rAAV genome release from the rAAV particles after lyophilization and reconstitution is reduced compared to rAAV genome release in a formulation that does not contain the sugar.

[0418] 119. The method of clause 117 or 118, further comprising lyophilizing the formulation to a residual moisture content of about 1% to about 7%.

[0419] 120. Use of a plasticizer to reduce rAAV genome release from rAAV particles, comprising producing a formulation comprising rAAV particles, a buffer, a sugar, a salt, a plasticizer, and optionally a non-ionic surfactant, wherein rAAV genome release from the rAAV particles after three freeze-thaw cycles is reduced compared to rAAV genome release in a formulation that does not contain the sugar.

[0420] 121. Use of a plasticizer to reduce rAAV genome release from rAAV particles, comprising producing a formulation comprising rAAV particles, a buffer, a sugar, a salt, a plasticizer, and optionally a non-ionic surfactant, wherein rAAV genome release from the rAAV particles after lyophilization and reconstitution is reduced compared to rAAV genome release in a formulation that does not contain the sugar.

[0421] 122. The method according to any one of clauses 112 to 116 or the use according to any one of clauses 117 to 121, wherein rAAV genome release is determined by measuring relative fluorescence in the presence of a DNA-specific fluorescent stain.

[0422] 123. The method according to any one of clauses 112 to 116 and 122 or the use according to any one of clauses 117 to 121, wherein freezing-induced rAAV genome release is reduced by at least about 10%, 20%, 50%, 80%, or 90%.

[0423] 124. The method according to any one of clauses 112 to 116 and 122 or the use according to any one of clauses 117 to 121, wherein freezing-induced rAAV genome release is substantially eliminated.

[0424] 125. The method according to any one of clauses 112 to 116 and 122 to 124 or the use according to any one of clauses 117 to 124, wherein the sugar is a non-reducing sugar.

[0425] 126. The method according to clause 125 or the use according to clause 125, wherein the non-reducing sugar is sucrose, trehalose, or raffinose.

[0426] 127. The method according to clause 125 or the use according to clause 125, wherein the non-reducing sugar is sucrose.

[0427] 128. The method according to any one of clauses 112 to 116 and 122 to 124 or the use according to any one of clauses 117 to 124, wherein the sugar is a reducing sugar.

[0428] 129. The method according to clause 122 or the use according to clause 122, wherein the reducing sugar is glucose, fructose, mannose, galactose or lactose.

[0429] 130. The method according to clause 128 or the use according to clause 128, wherein the reducing sugar is glucose.

[0430] 131. The method according to any one of clauses 111 to 117 and 122 to 130 or the use according to any one of clauses 82 to 105, wherein the plasticizer comprises glycerol.

[0431] 132. The method according to any one of clauses 111 to 117 and 122 to 131 or the use according to any one of clauses 82 to 106, wherein the formulation is according to any one of clauses 1 to 110.

[0432] 133. A method for producing a stable lyophilized formulation of an rAAV product, comprising the step of subjecting a pre-lyophilized formulation to lyophilization, wherein the pre-lyophilized formulation is one described in any one of claims 1 to 110.

[0433] 134. A method for treating or preventing a disease, comprising administering to a subject in need of treatment or prevention a therapeutically effective amount of an rAAV formulation that has been reconstituted from a stable lyophilized formulation, wherein the pre-lyophilized formulation of the stable lyophilized formulation is as defined in any one of claims 1 to 110. [Brief description of the drawings]

[0434] 5. Brief description of the drawings [Figure 1] Viral particle aggregation is affected by ionic strength. (A) Viral particle aggregation observed upon dilution into buffers of different ionic strength. Salt 1 is sodium chloride and salt 2 is sodium citrate. (B) Viral particle aggregation increases over time. [Diagram 2] Minimum ionic strength to prevent aggregation. Effective diameter of AAV8 particles at 1.8E+13GC / mL prepared with different NaCl concentrations. Based on the particle diameter, it appears that an ionic strength of 90 mM or higher is required to prevent particle aggregation. [Diagram 3] The minimum ionic strength is serotype dependent. Effective diameter of AAV8 (open squares) and AAV9 (open triangles) adjusted with different NaCl concentrations. The vector concentration was 6.0E+11 GC / mL. [Figure 4] Prevention of aggregation is necessary but not sufficient to ensure a stable formulation. Size distribution of virus particles after storage at -80°C for 6 months (6 months, -80°C) or at 25°C for 6 months (6 months, 25°C) is shown. [Diagram 5] Detection of AAV genome release in the presence of SYBR gold, a dye that exhibits a significant increase in fluorescence intensity when bound to DNA. Solutions contain 9.7E+11 GC / mL AAV8 particles in Tris buffer containing poloxamer 188 in the presence of either (A) NaCl or (B) sodium citrate. The solid line is a non-frozen control, and the dashed line is after three freeze-thaw cycles. [Figure 6]DNA release is increased in the presence of salts that crystallize during freezing. Salt 1 is sodium citrate and salt 2 is sodium chloride. DNA release in control samples and after three freeze-thaw cycles (3xF / T) is shown. Buffer 1 is Tris, buffer 2 is sodium phosphate, and buffer 3 is histidine. [Figure 7] Detection of AAV genome release in the presence of SYBR gold. Solutions contain 1E+12GC / mL AAV8 in either phosphate buffered saline (PBS) (A) or PBS with 4% sucrose (B). The solid line is the non-frozen control, and the dashed line is after 10 freeze-thaw cycles. [Figure 8] The addition of amorphous sugar inhibits cosolute crystallization and results in less DNA release. Shown are DNA release after control samples and three freeze-thaw cycles (3×F / T) in buffers containing different ratios of crystalline mannitol and amorphous sucrose components. [Figure 9] The reduction in GC leads to a concomitant decrease in relative potency after lyophilization. The relative potency and viral DNA titer of the formulations after lyophilization are shown. The sample labeled "C" contained crystalline bulking agent. [Figure 10] The pH of the freezing solution affects the stability of the lyophilized solid. The pH of the liquid and freezing solution is indicated by color. The relative potency of the lyophilized formulations after 6 months storage at 25° C. is shown. [Figure 11] Percent relative potency of AAV8 formulations after non-lyophilized control ("Liquid"), after lyophilization ("Post Lyo"), and after 3 and 6 months of storage at room temperature. Formulations are either Tris buffer (solid black) or phosphate buffer (solid grey). [Figure 12] Percent relative potency of lyophilized AAV8 formulations during storage at 35° C. Residual moisture content is 5.8% (circles), 4.7% (triangles), 3.6% (diamonds), and 2.1% (squares). [Figure 13] Relative percent potency of lyophilized AAV8 formulations during storage at 5°C (triangles) or room temperature (squares). [Figure 14]Detection of AAV genome release in the presence of SYBR gold. Solutions contained formulation buffer only (thick solid line) or 2.4E+11 GC / mL AAV8 either before lyophilization (solid line) or after lyophilization to different residual moisture levels: 4.7% (dotted line), 3.6% (dashed line), and 2.1% (dash-dotted line), followed by reconstitution with water to the original volume. As the residual moisture decreases, more AAV genomes are released. [Figure 15] Comparison of AAV stability of formulations 1A, 6A, and 7A after lyophilization. A shows free DNA release due to AAV capsid damage. Higher fluorescence intensity indicates more free DNA. B shows logarithm of viral titer reduction. C shows in vitro relative potency. [Figure 16] Thermal transitions of formulation 4A characterized by low-temperature DSC. [Figure 17] Comparison of formulations 1A, 2A, 3A, 4A, and 5A after 2 weeks at 35° C. A shows free DNA release due to AAV capsid damage. B shows virus titer. C shows in vitro relative potency. [Figure 18] Particle size of AAV after dilution in NaCl or Na2SO4 solutions of different ionic strengths: A, diameter at TO, B, diameter after 8 hours, C, diameter after 24 hours at room temperature in the presence of increasing sucrose concentrations in the presence of 30 mM Na2SO4. [Figure 19] 1 shows the pH shift of dPBS, Formulation 4A, and Formulation 5A when frozen to -20°C. [Figure 19] 1 shows the pH shift of dPBS, Formulation 4A, and Formulation 5A when frozen to -20°C. [Figure 20] Clustal multiple sequence alignment of AAV capsids 1-9 (SEQ ID NOs: 41-51). Amino acid substitutions (indicated in bold in the last line) can be introduced into AAV9 and AAV8 capsids by "recruiting" amino acid residues from corresponding positions in other aligned AAV capsids. Sequence regions designated "HVR" = hypervariable regions. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0435] 6. Detailed Description Provided herein are formulations comprising rAAV particles. In some embodiments, the formulations disclosed herein comprise a buffer, one or more stabilizers (e.g., sugars, polyols, amino acids), salts, and optionally a non-ionic surfactant. In some embodiments, the formulations disclosed herein are frozen or lyophilized formulations. In some embodiments, the formulations disclosed herein remain amorphous during freezing. In some embodiments, the formulations comprising rAAV particles disclosed herein are stable and do not significantly lose infectivity when stored at room temperature for extended periods of time.

[0436] In some embodiments, provided herein are stable formulations, methods of treatment involving the stable formulations, and kits involving the stable formulations.

[0437] In some embodiments, the formulations described in Chapter 6.2 are formulated such that they have one or more of the functional properties described in Chapter 6.2. In some embodiments, the stable formulations described in Chapter 6.2 are formulated such that they have one or more of the properties described in Chapter 6.2. In certain embodiments, the formulations provided herein have various advantages, such as improved stability after lyophilization and improved long-term stability under various conditions. Also provided herein are assays that may be used in Chapter 6.8. The inventors have surprisingly found that the minimum ionic strength required to prevent aggregation is AAV serotype dependent. AAV8 aggregation can be prevented at an ionic strength of less than 200 mM, and the ionic strength required for AAV9 is lower than that of AAV8. The inventors have further surprisingly found that crystallization of the formulation components during freezing promotes the reduction of rAAV genomes from rAAV particles. To the best of the inventors' knowledge, there have been no published reports of this type of genome reduction or of methods to prevent genome reduction through formulation development. The inventors have found that genome loss can be mitigated by either of two formulation approaches: (a) formulating with a salt that does not crystallize instead of a salt that does crystallize during freezing, and (b) including a component (e.g., sugar) that inhibits crystallization in the formulation. In some embodiments, a further uniqueness of the compositions disclosed herein is the use of sodium citrate in combination with sucrose, which provides unique advantages in the lyophilization process compared to other salts. In some embodiments, the formulations disclosed herein use a buffer that prevents pH shift during freezing.

[0438] The inventors further surprisingly found that, in some embodiments, making a lyophilized rAAV formulation as dry as possible does not provide the greatest stability in the dry state. As a result, in some embodiments, the stable rAAV formulations disclosed herein contain a residual water content of about 1%-7%. Also, in some embodiments, without wishing to be held to a particular theory, the stable rAAV formulations disclosed herein contain a plasticizer (e.g., glycerol) that may act as a substitute for water in the dry state.

[0439] 6.1 Definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. To facilitate understanding of the disclosed methods, several terms and phrases are defined below.

[0440] "About" modifies, for example, amounts of components in a composition, concentrations of components in a composition, flow rates, rAAV particle yields, feed rates, salt concentrations, and similar values ​​and ranges thereof used in the methods provided herein, where the term refers to variations in quantity that may occur, for example, through typical measuring and handling procedures used to make concentrates or use solutions, through unintentional errors in such procedures, through differences in the manufacture, source, or purity of components used to make the compositions or carry out the methods, and from similar considerations. The term "about" also encompasses amounts that differ due to aging of a composition having a particular initial concentration or mixture. The term "about" also encompasses amounts that differ due to mixing or processing of a composition having a particular initial concentration or mixture. Whether or not modified by the term "about," the claims include equivalents to the amount in question. In some embodiments, the term "about" refers to a range of about 10-20% greater or less than the indicated number or range. In further embodiments, "about" refers to plus or minus 10% of the indicated number or range. For example, "about 10%" refers to a range of 9% to 11%. In certain embodiments, the term "about" is inclusive of the exact number recited.

[0441] "AAV" is an abbreviation for adeno-associated virus and can be used to refer to the virus itself or to modifications, derivatives, or pseudotypes thereof. The term encompasses all subtypes and both native and recombinant forms, unless otherwise required. The abbreviation "rAAV" refers to recombinant adeno-associated virus. The term "AAV" includes AAV type 1 (AAV-1), AAV type 2 (AAV-2), AAV type 3 (AAV-3), AAV type 4 (AAV-4), AAV type 5 (AAV-5), AAV type 6 (AAV-6), AAV type 7 (AAV-7), AAV type 8 (AAV-8), AAV type 9 (AAV-9), avian AAV, bovine AAV, canine AAV, equine AAV, primate AAV, non-primate AAV, and ovine AAV, as well as modifications, derivatives, or pseudotypes thereof. "Primate AAV" refers to AAV that infects primates, "non-primate AAV" refers to AAV that infects non-primate mammals, and "bovine AAV" refers to AAV that infects non-primate mammals. V」 indicates an AAV that infects bovine mammals, etc.

[0442] "Recombinant," as applied to an AAV particle, means that the AAV particle is the product of one or more techniques that result in an AAV particle construct that is distinct from naturally occurring AAV particles.

[0443] A recombinant adeno-associated virus particle "rAAV particle" refers to a virus particle composed of at least one AAV capsid protein and a polynucleotide rAAV vector containing a heterologous polynucleotide (i.e., a polynucleotide other than a wild-type AAV genome, e.g., a transgene to be delivered to a mammalian cell) enclosed in the capsid. The rAAV particle can be of any AAV serotype, including any modification, derivative, or pseudotype (e.g., AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, or AAV-10, or derivatives / modifications / pseudotypes thereof). Such AAV serotypes and derivatives / modifications / pseudotypes, as well as methods for generating such serotypes / derivatives / modifications / pseudotypes, are known in the art (see, e.g., Asokan et al., Mol. Ther. 20(4):699-708 (2012)).

[0444] The rAAV particles of the present disclosure can be of any serotype, or any combination of serotypes (e.g., a population of rAAV particles that includes two or more serotypes, such as two or more of rAAV2 particles, rAAV8 particles, and rAAV9 particles). In some embodiments, the rAAV particles are rAAV1, rAAV2, rAAV3, rAAV4, rAAV5, rAAV6, rAAV7, rAAV8, rAAV9, rAAV10, or other rAAV particles, or combinations of two or more thereof. In some embodiments, the rAAV particles are rAAV8 particles or rAAV9 particles.

[0445] In some embodiments, the rAAV particles have an AAV capsid protein of a serotype selected from the group consisting of AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10, AAV-11, AAV-12, AAV-13, AAV-14, AAV-15, and AAV-16, or a derivative, modification, or pseudotype thereof. In some embodiments, the rAAV particles have an AAV capsid protein of an AAV-8, AAV-9 serotype, or a derivative, modification, or pseudotype thereof.

[0446] The terms "purifying," "purification," "separate," "separating," "separation," "isolate," "isolating," or "isolation," as used herein, refer to increasing the purity of rAAV particles from a sample containing a product of interest and one or more impurities. Typically, the purity of the product of interest is increased by removing (fully or partially) at least one impurity from the sample. In some embodiments, the purity of rAAV in a sample is increased by removing (fully or partially) one or more impurities from the sample using the methods described herein.

[0447] As used in this disclosure and claims, the singular forms "a," "an," and "the" include the plural forms unless the context clearly dictates otherwise.

[0448] It is to be understood that whenever an embodiment is described herein using the word "comprising," other similar embodiments described with "consisting of" and / or "consisting essentially of" are also provided. It is also to be understood that whenever an embodiment is described herein using the word "consisting essentially of," other similar embodiments described with "consisting of" are also provided.

[0449] The term "and / or," when used herein, for example in a phrase such as "A and / or B," is intended to include both A and B; A or B; A (alone); and B (alone). Similarly, the term "and / or," when used in a phrase such as "A, B, and / or C," is intended to encompass each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0450] When embodiments of the present disclosure are described in terms of Markush groups or other groupings of alternatives, the disclosed methods include not only the entire recited group in its entirety, but also each individual member of the group and all possible subgroups of the main group, as well as the main group absent one or more of the group members. The disclosed methods also envision the explicit exclusion of any one or more of the group members of the disclosed methods.

[0451] 6.2 Formulations containing rAAV particles In some embodiments, the disclosure provides a formulation comprising recombinant adeno-associated virus (rAAV) particles, as well as a buffer, a sugar, and a salt. In some embodiments, the salt is a pharma- ceutically acceptable salt. In some embodiments, the salt is an amorphous salt that does not crystallize. In some embodiments, the salt is a sodium salt. In some embodiments, the salt is sodium citrate, sodium acetate, or sodium chloride. In some embodiments, the salt is sodium citrate, sodium sulfate, ammonium sulfate, or magnesium sulfate. Multivalent salts are composed of multiply charged ions with higher ionic strength (per molecule of excipient added compared to monovalent sodium chloride), which can inhibit AAV aggregation while minimizing crystallization. Thus, rAAV formulation compositions containing salts other than monovalent sodium chloride that maintain the amorphous matrix of the composition may be useful.

[0452] 6.2.1 Salt In some embodiments, salt excipients are added or adjusted to achieve the desired ionic strength.

[0453] In some embodiments, provided herein is a stable formulation comprising recombinant adeno-associated virus (rAAV) particles, as well as a buffer, a sugar, and an amorphous salt, and having an ionic strength of 60 mM to 150 mM, the formulation being suitable for lyophilization. In some embodiments, the stable formulation also comprises a surfactant. In some embodiments, the stable formulation also comprises poloxamer 188.

[0454] In some embodiments, provided herein are stable formulations comprising recombinant adeno-associated virus (rAAV) particles and a buffer, a sugar, and an amorphous salt, the formulation having an ionic strength of greater than 200 mM, the formulation being suitable for lyophilization. In some embodiments, the stable formulation also comprises a surfactant. In some embodiments, the stable formulation also comprises poloxamer 188.

[0455] In some embodiments, provided herein are stable formulations comprising recombinant adeno-associated virus (rAAV) particles and a buffer, a sugar, and an amorphous salt, with an ionic strength of greater than 200 mM, 210 mM, 220 mM, 230 mM, 240 mM, 250 mM, 260 mM, 270 mM, 280 mM, 290 mM, or 300 mM, which formulations are suitable for lyophilization. In some embodiments, the stable formulations also comprise a surfactant. In some embodiments, the stable formulations also comprise poloxamer 188.

[0456] In some embodiments, the salt is a pharma- ceutically acceptable salt. In some embodiments, the salt is an amorphous salt that does not crystallize. In some embodiments, the salt is a sodium salt. In some embodiments, the salt is sodium citrate, sodium acetate, or sodium chloride. In some embodiments, the salt is sodium citrate, sodium sulfate, ammonium sulfate, or magnesium sulfate. In some embodiments, the salt is a multivalent salt, which is composed of multiple charged ions with higher ionic strength (per excipient molecule added compared to monovalent sodium chloride) and can inhibit AAV aggregation while minimizing crystallization.

[0457] In some embodiments, the present disclosure provides a stable formulation comprising a recombinant adeno-associated virus (AAV), a salt excipient, and a sugar. In some embodiments, the stable formulation also comprises a surfactant. In some embodiments, the stable formulation also comprises poloxamer 188. In some embodiments, the stable formulation is suitable for lyophilization.

[0458] In some embodiments, provided herein is a stable formulation comprising recombinant adeno-associated virus (rAAV) particles and a buffer, a sugar, and an amorphous salt having an ionic strength of 60 mM to 150 mM, the formulation being suitable for lyophilization. In some embodiments, the stable formulation also comprises a surfactant. In some embodiments, the stable formulation also comprises poloxamer 188. In some embodiments, the formulation is a pre-lyophilized formulation.

[0459] In some embodiments, provided herein is a stable formulation comprising recombinant adeno-associated virus (rAAV) particles and a buffer, a sugar, and an amorphous salt having an ionic strength of 30 mM to 100 mM, the formulation being suitable for lyophilization. In some embodiments, the stable formulation also comprises a surfactant. In some embodiments, the stable formulation also comprises poloxamer 188. In some embodiments, the formulation is a pre-lyophilized formulation.

[0460] In some embodiments, provided herein is a stable formulation comprising recombinant adeno-associated virus (rAAV) particles and a buffer, a sugar, and an amorphous salt having an ionic strength greater than 200 mM, the formulation being suitable for lyophilization. In some embodiments, the stable formulation also comprises a surfactant. In some embodiments, the stable formulation also comprises poloxamer 188. In some embodiments, the formulation is a pre-lyophilized formulation.

[0461] In some embodiments, provided herein is a stable formulation comprising recombinant adeno-associated virus (rAAV) particles, a buffer, a sugar, an amorphous salt having an ionic strength of 60 mM to 150 mM, and a surfactant, the formulation being suitable for lyophilization. In some embodiments, the stable formulation also comprises a surfactant. In some embodiments, the stable formulation also comprises poloxamer 188. In some embodiments, the formulation is a pre-lyophilized formulation.

[0462] In some embodiments, provided herein is a stable formulation comprising recombinant adeno-associated virus (rAAV) particles, a buffer, a sugar, an amorphous salt having an ionic strength of 30 mM to 100 mM, and a surfactant, the formulation being suitable for lyophilization. In some embodiments, the stable formulation also comprises a surfactant. In some embodiments, the stable formulation also comprises poloxamer 188. In some embodiments, the formulation is a pre-lyophilized formulation.

[0463] In some embodiments, provided herein is a stable formulation comprising recombinant adeno-associated virus (rAAV) particles, a buffer, a sugar, and an amorphous salt having an ionic strength greater than 200 mM, and a surfactant, the formulation being suitable for lyophilization. In some embodiments, the stable formulation also comprises a surfactant. In some embodiments, the stable formulation also comprises poloxamer 188. In some embodiments, the formulation is a pre-lyophilized formulation.

[0464] In some embodiments, the formulations disclosed herein contain about 0.0001% to about 0.5% of a non-ionic surfactant. In some embodiments, the formulations disclosed herein contain about 0.0005% to about 0.1% of a non-ionic surfactant. In some embodiments, the formulations disclosed herein contain about 0.001%, about 0.002%, about 0.003%, about 0.004%, about 0.005%, about 0.007%, or about 0.01% of a non-ionic surfactant.

[0465] In some embodiments, the formulations disclosed herein contain about 0.0001% to about 0.5% poloxamer 188. In some embodiments, the formulations disclosed herein contain about 0.0005% to about 0.1% poloxamer 188. In some embodiments, the formulations disclosed herein contain about 0.001%, about 0.002%, about 0.003%, about 0.004%, about 0.005%, about 0.007%, or about 0.01% poloxamer 188.

[0466] In some embodiments, the formulations disclosed herein comprise about 0.0005% poloxamer 188. In some embodiments, the formulations disclosed herein comprise about 0.001% poloxamer 188. In some embodiments, the formulations disclosed herein comprise about 0.002% poloxamer 188. In some embodiments, the formulations disclosed herein comprise about 0.003% poloxamer 188. In some embodiments, the formulations disclosed herein comprise about 0.004% poloxamer 188. In some embodiments, the formulations disclosed herein comprise about 0.005% poloxamer 188. In some embodiments, the formulations disclosed herein comprise about 0.008% poloxamer 188.

[0467] In some embodiments, the salt is a pharma- ceutically acceptable salt. In some embodiments, the salt is an amorphous salt that does not crystallize. In some embodiments, the salt is a sodium salt. In some embodiments, the salt is sodium citrate, sodium acetate, or sodium chloride. In some embodiments, the salt is sodium citrate, sodium sulfate, ammonium sulfate, or magnesium sulfate. In some embodiments, the salt is a multivalent salt, which is composed of multiple charged ions with higher ionic strength (per excipient molecule added compared to monovalent sodium chloride) and can inhibit AAV aggregation while minimizing crystallization.

[0468] In some embodiments, the salt excipient is added or adjusted to achieve the desired ionic strength. In some embodiments, the amorphous salt has an ionic strength of about 150 mM, about 145 mM, about 140 mM, about 135 mM, about 130 mM, about 125 mM, about 120 mM, about 115 mM, about 110 mM, about 110 mM, about 105 mM, or about 100 mM or less. In certain embodiments, the stable formulation has a buffer ionic strength of about 150 mM, about 145 mM, about 140 mM, about 135 mM, about 130 mM, about 125 mM, about 120 mM, about 115 mM, about 110 mM, about 105 mM, or about 100 mM or less. In some embodiments, the rAAV particles in the formulation are rAAV8 particles or rAAV9 particles.

[0469] In some embodiments, the stable formulation has an ionic strength of 150 mM, 145 mM, 140 mM, 135 mM, 130 mM, 125 mM, 120 mM, 115 mM, or 110 mM or less. In certain embodiments, the stable formulation has a buffer ionic strength of 150 mM, 145 mM, 140 mM, 135 mM, 130 mM, 125 mM, 120 mM, 115 mM, or 110 mM or less. In some embodiments, the rAAV particles in the formulation have AAV8 capsids.

[0470] In certain embodiments, the stable formulation has an ionic strength of greater than 60 mM. In certain embodiments, the stable formulation has an ionic strength of about 60 mM to 150 mM. In certain embodiments, the stable formulation has an ionic strength of about 60 mM to 115 mM. In certain embodiments, the stable formulation has an ionic strength of about 60 mM to 100 mM. In certain embodiments, the stable formulation has an ionic strength of about 60 mM. In certain embodiments, the stable formulation has an ionic strength of about 65 mM. In certain embodiments, the stable formulation has an ionic strength of about 70 mM. In certain embodiments, the stable formulation has an ionic strength of about 75 mM. In certain embodiments, the stable formulation has an ionic strength of about 80 mM. In certain embodiments, the stable formulation has an ionic strength of about 85 mM. In certain embodiments, the stable formulation has an ionic strength of about 90 mM. In some embodiments, the rAAV particles in the formulation have AAV8 capsids.

[0471] In certain embodiments, the stable formulation has an ionic strength of more than about 30 mM. In certain embodiments, the stable formulation has an ionic strength of about 30 mM to 100 mM. In certain embodiments, the stable formulation has an ionic strength of about 30 mM. In certain embodiments, the stable formulation has an ionic strength of about 35 mM. In certain embodiments, the stable formulation has an ionic strength of about 40 mM. In certain embodiments, the stable formulation has an ionic strength of about 45 mM. In certain embodiments, the stable formulation has an ionic strength of about 50 mM. In certain embodiments, the stable formulation has an ionic strength of about 55 mM. In certain embodiments, the stable formulation has an ionic strength of about 60 mM. In certain embodiments, the stable formulation has an ionic strength of about 65 mM. In certain embodiments, the stable formulation has an ionic strength of about 70 mM. In certain embodiments, the stable formulation has an ionic strength of about 75 mM. In certain embodiments, the stable formulation has an ionic strength of about 80 mM. In certain embodiments, the stable formulation has an ionic strength of about 85 mM. In certain embodiments, the stable formulation has an ionic strength of about 90 mM. In certain embodiments, the stable formulation has an ionic strength of about 95 mM. In certain embodiments, the stable formulation has an ionic strength of about 100 mM. In some embodiments, the rAAV particles in the formulation have AAV9 capsids.

[0472] In certain embodiments, the stable formulation has an ionic strength of about 60 mM to 115 mM. In certain embodiments, the stable formulation has an ionic strength of about 65 mM to 95 mM. In certain embodiments, the stable formulation has an ionic strength of about 70 mM to 90 mM. In certain embodiments, the stable formulation has an ionic strength of about 75 mM to 85 mM. In some embodiments, the rAAV particles in the formulation have AAV8 capsids.

[0473] In certain embodiments, the stable formulation has an ionic strength of about 30 mM to 100 mM. In certain embodiments, the stable formulation has an ionic strength of about 35 mM to 95 mM. In certain embodiments, the stable formulation has an ionic strength of about 40 mM to 90 mM. In certain embodiments, the stable formulation has an ionic strength of about 45 mM to 85 mM. In certain embodiments, the stable formulation has an ionic strength of about 50 mM to 80 mM. In certain embodiments, the stable formulation has an ionic strength of about 55 mM to 75 mM. In certain embodiments, the stable formulation has an ionic strength of about 60 mM to 70 mM. In some embodiments, the rAAV particles in the formulation do not have AAV8 capsids. In some embodiments, the rAAV particles in the formulation have AAV9 capsids.

[0474] In some embodiments, the stable formulation has an ionic strength of greater than about 200 mM. In some embodiments, the rAAV particles in the formulation have AAV2 capsids. In some embodiments, the rAAV particles in the formulation have no AAV8 or AAV9 capsids.

[0475] In some embodiments, the stable formulation has an ionic strength of greater than 200 mM, 210 mM, 220 mM, 230 mM, 240 mM, 250 mM, 260 mM, 270 mM, 280 mM, 290 mM, or 300 mM. In some embodiments, the rAAV particles in the formulation have AAV2 capsids.

[0476] In certain embodiments, the stable formulation has an ionic strength of about 200 mM to about 210 mM. In certain embodiments, the stable formulation has an ionic strength of about 210 mM to about 220 mM. In certain embodiments, the stable formulation has an ionic strength of about 220 mM to about 230 mM. In certain embodiments, the stable formulation has an ionic strength of about 230 mM to about 240 mM. In certain embodiments, the stable formulation has an ionic strength of about 240 mM to about 250 mM. In certain embodiments, the stable formulation has an ionic strength of about 250 mM to about 260 mM. In certain embodiments, the stable formulation has an ionic strength of about 260 mM to about 270 mM. In certain embodiments, the stable formulation has an ionic strength of about 270 mM to about 280 mM. In certain embodiments, the stable formulation has an ionic strength of about 280 mM to about 290 mM. In certain embodiments, the stable formulation has an ionic strength of about 290 mM to about 300 mM. In some embodiments, the rAAV particles in the formulation have no AAV8 or AAV9 capsids.

[0477] In some embodiments, the formulations disclosed herein contain less than about 100 mM salt. In some embodiments, the formulations disclosed herein contain from about 10 mM to about 100 mM salt.

[0478] In some embodiments, the formulations disclosed herein include from about 10 mM to about 100 mM, from about 20 mM to about 100 mM, from about 30 mM to about 100 mM, from about 40 mM to about 100 mM, from about 50 mM to about 100 mM, from about 10 mM to about 80 mM, from about 10 mM to about 60 mM, from about 10 mM to about 50 mM, from about 10 mM to about 40 mM, or from about 10 mM to about 30 mM of salt.

[0479] In some embodiments, the formulations disclosed herein contain from about 10 mM to about 50 mM, from about 20 mM to about 60 mM, from about 30 mM to about 70 mM, or from about 10 mM to about 30 mM of salt.

[0480] In some embodiments, the formulations disclosed herein comprise about 10 mM, about 20 mM, about 30 mM, about 40 mM, about 50 mM, or about 60 mM salt.

[0481] In some embodiments, the formulations disclosed herein comprise about 20 mM salt.

[0482] In some embodiments, the formulations disclosed herein contain less than about 100 mM sodium citrate. In some embodiments, the formulations disclosed herein contain from about 10 mM to about 100 mM sodium citrate.

[0483] In some embodiments, the formulations disclosed herein include from about 10 mM to about 100 mM sodium citrate. In some embodiments, the formulations disclosed herein include from about 20 mM to about 100 mM sodium citrate. In some embodiments, the formulations disclosed herein include from about 30 mM to about 100 mM sodium citrate. In some embodiments, the formulations disclosed herein include from about 40 mM to about 100 mM sodium citrate. In some embodiments, the formulations disclosed herein include from about 50 mM to about 100 mM sodium citrate. In some embodiments, the formulations disclosed herein include from about 10 mM to about 80 mM sodium citrate. In some embodiments, the formulations disclosed herein include from about 10 mM to about 60 mM sodium citrate. In some embodiments, the formulations disclosed herein include from about 10 mM to about 50 mM sodium citrate. In some embodiments, the formulations disclosed herein include from about 10 mM to about 40 mM sodium citrate.In some embodiments, the formulations disclosed herein include from about 10 mM to about 30 mM sodium citrate.

[0484] In some embodiments, the formulations disclosed herein include from about 10 mM to about 50 mM sodium citrate. In some embodiments, the formulations disclosed herein include from about 20 mM to about 60 mM sodium citrate. In some embodiments, the formulations disclosed herein include from about 30 mM to about 70 mM sodium citrate. In some embodiments, the formulations disclosed herein include from about 10 mM to about 30 mM sodium citrate.

[0485] In some embodiments, the formulations disclosed herein comprise about 10 mM sodium citrate. In some embodiments, the formulations disclosed herein comprise about 20 mM sodium citrate. In some embodiments, the formulations disclosed herein comprise about 30 mM sodium citrate. In some embodiments, the formulations disclosed herein comprise about 40 mM sodium citrate. In some embodiments, the formulations disclosed herein comprise about 50 mM sodium citrate. In some embodiments, the formulations disclosed herein comprise about 60 mM sodium citrate.

[0486] In some embodiments, the formulations disclosed herein contain about 20 mM sodium citrate.

[0487] 6.2.2 Buffers Buffers are well known in the art and include, without limitation, phosphate buffer, histidine, sodium citrate, HEPES, Tris, Bicine, glycine, N-glycylglycine, sodium acetate, sodium carbonate, glycylglycine, lysine, arginine, sodium phosphate, and mixtures thereof. In certain embodiments, the buffer is histidine (e.g., L-histidine). In some embodiments, the buffer is a pharma- ceutically acceptable buffer. In some embodiments, the buffer comprises Tris.

[0488] In some embodiments, the formulations disclosed herein comprise about 1 mM to about 50 mM of buffering agent. In some embodiments, the formulations disclosed herein comprise about 1 mM to about 30 mM, about 1 mM to about 20 mM, about 5 mM to about 30 mM, about 5 mM to about 20 mM, about 10 mM to about 30 mM, about 10 mM to about 20 mM, or about 20 mM to about 50 mM of buffering agent. In some embodiments, the formulations disclosed herein comprise about 1 mM, about 2 mM, about 3 mM, about 5 mM, about 10 mM, about 15 mM, about 20 mM, about 25 mM, about 30 mM, or about 40 mM of buffering agent. In some embodiments, the formulations disclosed herein comprise about 5 mM of buffering agent.

[0489] In some embodiments, the formulations disclosed herein contain about 1 mM to about 50 mM Tris. In some embodiments, the formulations disclosed herein contain about 1 mM to about 30 mM, about 1 mM to about 20 mM, about 5 mM to about 30 mM, about 5 mM to about 20 mM, about 10 mM to about 30 mM, about 10 mM to about 20 mM, or about 20 mM to about 50 mM Tris. In some embodiments, the formulations disclosed herein contain about 1 mM, about 2 mM, about 3 mM, about 5 mM, about 10 mM, about 15 mM, about 20 mM, about 25 mM, about 30 mM, or about 40 mM Tris. In some embodiments, the formulations disclosed herein contain about 5 mM Tris.

[0490] In some embodiments, the formulations disclosed herein include a buffering agent capable of providing the formulations disclosed herein with substantially the same pH in the liquid and frozen states.

[0491] In some embodiments, the formulations disclosed herein have a pH of about 6.5 to 8.0. In some embodiments, the formulations disclosed herein have a pH of about 7.0 to 8.0. In some embodiments, the formulations disclosed herein have a pH of about 7.2 to 7.8.

[0492] In some embodiments, the formulations disclosed herein have a pH of about 6.6. In some embodiments, the formulations disclosed herein have a pH of about 6.8. In some embodiments, the formulations disclosed herein have a pH of about 7.0. In some embodiments, the formulations disclosed herein have a pH of about 7.2. In some embodiments, the formulations disclosed herein have a pH of about 7.3. In some embodiments, the formulations disclosed herein have a pH of about 7.4. In some embodiments, the formulations disclosed herein have a pH of about 7.5. In some embodiments, the formulations disclosed herein have a pH of about 7.6. In some embodiments, the formulations disclosed herein have a pH of about 7.7. In some embodiments, the formulations disclosed herein have a pH of about 7.8.

[0493] In some embodiments, the formulations disclosed herein have a pH of about 7.5.

[0494] In some embodiments, the formulations disclosed herein have substantially the same pH in the liquid and frozen states.

[0495] In some embodiments, the formulations disclosed herein have a pH of about 6.5-8.0 in the liquid and frozen state. In some embodiments, the formulations disclosed herein have a pH of about 7.0-8.0 in the liquid and frozen state. In some embodiments, the formulations disclosed herein have a pH of about 7.2-7.8 in the liquid and frozen state. In some embodiments, the formulations disclosed herein have a pH of about 6.6, 6.8, 7.0, or 7.2 in the liquid and frozen state. In some embodiments, the formulations disclosed herein have a pH of about 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, or 7.8 in the liquid and frozen state. In some embodiments, the formulations disclosed herein have a pH of about 7.5 in the liquid and frozen state.

[0496] 6.2.3 sugar In some embodiments, the formulations disclosed herein include a non-reducing sugar. In some embodiments, the non-reducing sugar is sucrose, trehalose, raffinose, or a combination thereof. In some embodiments, the formulations disclosed herein include sucrose.

[0497] In some embodiments, the formulations disclosed herein include a reducing sugar. In some embodiments, the reducing sugar is glucose, fructose, mannose, galactose, lactose, or a combination thereof. In some embodiments, the formulations disclosed herein include dextrose.

[0498] In some embodiments, the formulations disclosed herein contain from about 50 mM to about 400 mM sugar. In some embodiments, the formulations disclosed herein contain from about 50 mM to about 350 mM, from about 50 mM to about 300 mM, from about 50 mM to about 250 mM, from about 50 mM to about 200 mM, or from about 50 mM to about 150 mM sugar.

[0499] In some embodiments, the formulations disclosed herein contain from about 100 mM to about 400 mM, from about 150 mM to about 400 mM, from about 200 mM to about 400 mM, from about 250 mM to about 400 mM, or from about 300 mM to about 400 mM sugar.

[0500] In some embodiments, the formulations disclosed herein contain from about 100 mM to about 300 mM, from about 150 mM to about 250 mM, from about 200 mM to about 300 mM, or from about 250 mM to about 350 mM sugar.

[0501] In some embodiments, the formulations disclosed herein comprise about 50 mM, about 100 mM, about 150 mM, about 160 mM, about 170 mM, about 180 mM, about 190 mM, about 200 mM, about 210 mM, about 220 mM, about 230 mM, about 240 mM, about 250 mM, about 260 mM, about 270 mM, about 280 mM, about 290 mM, about 300 mM, or about 350 mM sugar.

[0502] In some embodiments, the formulations disclosed herein contain from about 190 mM to about 230 mM, from about 170 mM to about 250 mM, or from about 150 mM to about 270 mM sugar.

[0503] In some embodiments, the formulations disclosed herein comprise about 210 mM sugar.

[0504] In some embodiments, the formulations disclosed herein comprise about 50 mM to about 400 mM sucrose. In some embodiments, the formulations disclosed herein comprise about 50 mM to about 350 mM sucrose. In some embodiments, the formulations disclosed herein comprise about 50 mM to about 300 mM sucrose. In some embodiments, the formulations disclosed herein comprise about 50 mM to about 250 mM sucrose. In some embodiments, the formulations disclosed herein comprise about 50 mM to about 200 mM sucrose. In some embodiments, the formulations disclosed herein comprise about 50 mM to about 150 mM sucrose.

[0505] In some embodiments, the formulations disclosed herein comprise from about 100 mM to about 400 mM sucrose. In some embodiments, the formulations disclosed herein comprise from about 150 mM to about 400 mM sucrose. In some embodiments, the formulations disclosed herein comprise from about 200 mM to about 400 mM sucrose. In some embodiments, the formulations disclosed herein comprise from about 250 mM to about 400 mM sucrose. In some embodiments, the formulations disclosed herein comprise from about 300 mM to about 400 mM sucrose.

[0506] In some embodiments, the formulations disclosed herein contain about 100 mM to about 300 mM sucrose. In some embodiments, the formulations disclosed herein contain about 150 mM to about 250 mM sucrose. In some embodiments, the formulations disclosed herein contain about 200 mM to about 300 mM sucrose. In some embodiments, the formulations disclosed herein contain about 250 mM to about 350 mM sucrose.

[0507] In some embodiments, the formulations disclosed herein comprise about 50 mM sucrose. In some embodiments, the formulations disclosed herein comprise about 100 mM sucrose. In some embodiments, the formulations disclosed herein comprise about 150 mM sucrose. In some embodiments, the formulations disclosed herein comprise about 160 mM sucrose. In some embodiments, the formulations disclosed herein comprise about 170 mM sucrose. In some embodiments, the formulations disclosed herein comprise about 180 mM sucrose. In some embodiments, the formulations disclosed herein comprise about 190 mM sucrose. In some embodiments, the formulations disclosed herein comprise about 200 mM sucrose. In some embodiments, the formulations disclosed herein comprise about 210 mM sucrose. In some embodiments, the formulations disclosed herein comprise about 220 mM sucrose. In some embodiments, the formulations disclosed herein comprise about 230 mM sucrose. In some embodiments, the formulations disclosed herein comprise about 240 mM sucrose. In some embodiments, the formulations disclosed herein comprise about 250 mM sucrose. In some embodiments, the formulations disclosed herein comprise about 260 mM sucrose. In some embodiments, the formulations disclosed herein comprise about 270 mM sucrose. In some embodiments, the formulations disclosed herein comprise about 280 mM sucrose. In some embodiments, the formulations disclosed herein comprise about 290 mM sucrose. In some embodiments, the formulations disclosed herein comprise about 300 mM sucrose. In some embodiments, the formulations disclosed herein comprise about 350 mM sucrose.

[0508] In some embodiments, the formulations disclosed herein contain about 190 mM to about 230 mM sucrose. In some embodiments, the formulations disclosed herein contain about 170 mM to about 250 mM sucrose. In some embodiments, the formulations disclosed herein contain about 150 mM to about 270 mM sucrose.

[0509] In some embodiments, the formulations disclosed herein contain about 210 mM sucrose.

[0510] In some embodiments, the formulations disclosed herein include a specific ratio of sugar and salt. In some embodiments, the ratio of sugar to salt is a molar ratio. In some embodiments, the molar ratio of sugar to salt is about 1, about 2, about 3, about 5, about 10, about 20, about 40, or about 60. In some embodiments, the molar ratio of sugar to salt is about 5 to about 20. In some embodiments, the molar ratio of sugar to salt is about 10. In some embodiments, the sugar includes sucrose and the salt includes sodium citrate.

[0511] In some embodiments, the formulations disclosed herein include salt and sugar in a particular ratio. In some embodiments, the ratio of salt to sugar is a weight to weight (w:w) ratio. In some embodiments, the w:w ratio of salt to sugar is about 3:1, about 1:1, or about 1:3. In some embodiments, the w:w ratio of salt to sugar is about 3:1 to about 1:3. In some embodiments, the w:w ratio of salt to sugar is about 1:1. In some embodiments, the sugar includes sucrose and the salt includes sodium citrate.

[0512] 6.2.4 Surfactants In some embodiments, the formulations disclosed herein further comprise a non-ionic surfactant. Pharmaceutically acceptable non-ionic surfactants include, without limitation, poloxamer 188, poloxamer 407, polysorbate 80, polysorbate 20, Pluronic F-68, or BRIJ 35. In some embodiments, the formulations disclosed herein comprise poloxamer 188, poloxamer 407, polysorbate 80, polysorbate 20, Pluronic F-68, BRIJ 35, or combinations thereof. In some embodiments, the formulations disclosed herein comprise poloxamer 188.

[0513] In some embodiments, the formulations disclosed herein further comprise about 0.0001% to about 0.5% of a non-ionic surfactant. In some embodiments, the formulations disclosed herein further comprise about 0.0005% to about 0.1% of a non-ionic surfactant. In some embodiments, the formulations disclosed herein further comprise about 0.001%, about 0.002%, about 0.003%, about 0.004%, about 0.005%, about 0.007%, or about 0.01% of a non-ionic surfactant.

[0514] In some embodiments, the formulations disclosed herein further comprise about 0.0001% to about 0.5% poloxamer 188. In some embodiments, the formulations disclosed herein further comprise about 0.0005% to about 0.1% poloxamer 188. In some embodiments, the formulations disclosed herein further comprise about 0.001%, about 0.002%, about 0.003%, about 0.004%, about 0.005%, about 0.007%, or about 0.01% poloxamer 188.

[0515] In some embodiments, the formulations disclosed herein further comprise about 0.0005% poloxamer 188. In some embodiments, the formulations disclosed herein further comprise about 0.001% poloxamer 188. In some embodiments, the formulations disclosed herein further comprise about 0.002% poloxamer 188. In some embodiments, the formulations disclosed herein further comprise about 0.003% poloxamer 188. In some embodiments, the formulations disclosed herein further comprise about 0.004% poloxamer 188. In some embodiments, the formulations disclosed herein further comprise about 0.005% poloxamer 188. In some embodiments, the formulations disclosed herein further comprise about 0.008% poloxamer 188.

[0516] 6.2.5 Stabilizers or plasticizers In some embodiments, the formulations disclosed herein further comprise a plasticizer or stabilizer. Pharmaceutically acceptable stabilizers or plasticizers include, but are not limited to, glycerol, xylitol, and sorbitol. In some embodiments, the formulations disclosed herein further comprise glycerol, xylitol, sorbitol, or a combination thereof. In some embodiments, the formulations disclosed herein further comprise glycerol.

[0517] In some embodiments, the formulations disclosed herein further comprise from about 0.1% to about 5% of a plasticizer or stabilizer. In some embodiments, the formulations disclosed herein further comprise from about 0.1% to about 2% of a plasticizer or stabilizer. In some embodiments, the formulations disclosed herein further comprise from about 0.25% to about 2% of a plasticizer.

[0518] In some embodiments, the formulations disclosed herein further comprise about 0.1% to about 5% glycerol. In some embodiments, the formulations disclosed herein further comprise about 0.1% to about 2% glycerol. In some embodiments, the formulations disclosed herein further comprise about 0.25% to about 2% glycerol.

[0519] 6.2.6 Formulation Characteristics In some embodiments, the formulations disclosed herein have a higher glass transition temperature than the corresponding pure sugar solution and pure salt solution. In some embodiments, the formulations disclosed herein comprise sucrose and sodium citrate and have a higher glass transition temperature than the corresponding pure sucrose and pure sodium citrate salt. In some embodiments, the formulations disclosed herein comprise about 50 mM to about 400 mM sucrose and about 10 mM to 100 mM sodium citrate.

[0520] In some embodiments, the formulations disclosed herein comprise between about 1.0E+11 GC / mL and about 1.0E+14 GC / mL of rAAV particles. In some embodiments, the formulations disclosed herein comprise between about 1.0E+11 GC / mL and about 1.0E+15 GC / mL of rAAV particles. In some embodiments, the formulations disclosed herein comprise between about 1.0E+11 GC / mL, about 1.0E+12 GC / mL, about 1.0E+13 GC / mL, about 1.0E+14 GC / mL, or about 1.0E+15 GC / mL of rAAV particles. In some embodiments, the rAAV particles include any of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV-11, AAV-12, AAV-13, AAV-14, AAV-15 and AAV-16, rAAV.rh8, rAAV.rh10, rAAV.rh20, rAAV.rh39, rAAV.Rh74, rAAV.RHM4-1, AAV.hu37, rAAV.Anc80, rAAV.Anc80L65, rAAV.7m8, rAAV. In some embodiments, the rAAV particles comprise capsid proteins of an AAV-8 serotype, an AAV-9 serotype, or a combination thereof.

[0521] In some embodiments, the formulations disclosed herein are liquid formulations.

[0522] In some embodiments, the formulations disclosed herein are frozen formulations.

[0523] In some embodiments, the formulations disclosed herein are lyophilized formulations obtained by lyophilizing the liquid formulations disclosed herein.

[0524] In some embodiments, the formulations disclosed herein are reconstituted lyophilized formulations.

[0525] In some embodiments, the formulations disclosed herein are pre-lyophilized formulations.

[0526] In some embodiments, the formulations disclosed herein are lyophilized formulations having a residual moisture content of about 1% to about 7%. In some embodiments, the residual moisture content is determined using Karl Fischer titration. In some embodiments, the residual moisture content is about 1% to about 7%. In some embodiments, the residual moisture content is about 2% to about 7%. In some embodiments, the residual moisture content is about 3% to about 7%. In some embodiments, the residual moisture content is about 4% to about 7%. In some embodiments, the residual moisture content is about 5% to about 7%. In some embodiments, the residual moisture content is about 1% to about 6%. In some embodiments, the residual moisture content is about 1% to about 5%. In some embodiments, the residual moisture content is about 1% to about 4%. In some embodiments, the residual moisture content is about 1% to about 3%.

[0527] In some embodiments, the formulations disclosed herein are lyophilized formulations having a residual moisture content of about 1% to about 7%. In some embodiments, the residual moisture content is about 3% to about 7%. In some embodiments, the residual moisture content is about 3% to about 6%. In some embodiments, the residual moisture content is about 3% to about 5%.

[0528] In some embodiments, the formulations disclosed herein are lyophilized formulations having a residual moisture content of about 1% to about 7%. In some embodiments, the residual moisture content is about 3%. In some embodiments, the residual moisture content is about 3.5%. In some embodiments, the residual moisture content is about 4%. In some embodiments, the residual moisture content is about 4.5%. In some embodiments, the residual moisture content is about 5%. In some embodiments, the residual moisture content is about 5.5%. In some embodiments, the residual moisture content is about 6%.

[0529] In some embodiments of the formulations disclosed herein, the relative potency of the rAAV particles is at least about 60%, at least about 70%, or at least about 80% after the formulation is stored at room temperature for 3 months. In some embodiments of the formulations disclosed herein, the relative potency of the rAAV particles is at least about 60% after the formulation is stored at room temperature for 3 months. In some embodiments of the formulations disclosed herein, the relative potency of the rAAV particles is at least about 70% after the formulation is stored at room temperature for 3 months. In some embodiments of the formulations disclosed herein, the relative potency of the rAAV particles is at least about 60%, at least about 70%, or at least about 80% after the formulation is stored at room temperature for 6 months. In some embodiments of the formulations disclosed herein, the relative potency of the rAAV particles is at least about 60% after the formulation is stored at room temperature for 6 months. In some embodiments of the formulations disclosed herein, the relative potency of the rAAV particles is at least about 70% after the formulation is stored at room temperature for 6 months. In some embodiments of the formulations disclosed herein, the relative potency of the rAAV particles is at least about 30%, at least about 40%, at least about 50%, or at least 60% after the formulation is stored at 35° C. for 1 week. In some embodiments of the formulations disclosed herein, the relative potency of the rAAV particles is at least about 30% after the formulation is stored at 35° C. for 1 week. In some embodiments of the formulations disclosed herein, the relative potency of the rAAV particles is at least about 50% after the formulation is stored at 35° C. for 1 week. In some embodiments of the formulations disclosed herein, the relative potency of the rAAV particles is at least about 30%, at least about 40%, at least about 50%, or at least 60% after the formulation is stored at 35° C. for 2 weeks. In some embodiments of the formulations disclosed herein, the relative potency of the rAAV particles is at least about 30% after the formulation is stored at 35° C. for 2 weeks. In some embodiments of the formulations disclosed herein, the relative % potency of the rAAV particles is at least about 50% after the formulation is stored for 2 weeks at 35° C. In some embodiments of the formulations disclosed herein, the relative % potency of the rAAV particles is at least about 30%, at least about 40%, at least about 50%, or at least 60% after the formulation is stored for 4 weeks at 35° C.In some embodiments of the formulations disclosed herein, the relative potency of the rAAV particles is at least about 30% after the formulation is stored at 35° C. for 4 weeks. In some embodiments of the formulations disclosed herein, the relative potency of the rAAV particles is at least about 50% after the formulation is stored at 35° C. for 4 weeks. In some embodiments, the reference rAAV particles are stored at −70° C. in DPBS containing 0.001% poloxamer 188 buffer. In some embodiments, the relative potency is determined as disclosed in PCT International Application No. PCT / US19 / 56042, filed October 14, 2019, entitled “METHODS FOR MEASURING THE INFECTIVITY OF REPLICATION DEFECTIVE VIRAL VECTORS AND VIRUSES,” which is incorporated herein by reference in its entirety. In some embodiments, the formulation is a frozen formulation. In some embodiments, the formulation is a lyophilized formulation. In some embodiments, the formulation is a pre-lyophilized formulation. In some embodiments, the formulation is a liquid formulation. In some embodiments, the formulation comprises rAAV particles and about 1 mM to about 25 mM Tris, about 50 mM to about 400 mM sucrose, about 10 mM to about 100 mM sodium citrate, and about 0.0005% to about 0.01% non-ionic surfactant, and the formulation has a pH of about 7.2 to about 7.8.

[0530] In some embodiments, the formulations disclosed herein comprise rAAV particles, as well as about 1 mM to about 25 mM buffer, about 50 mM to about 400 mM sugar, about 10 mM to about 100 mM sodium citrate, and about 0.0005% to about 0.01% non-ionic surfactant, and the formulation has a pH of about 7.2 to about 7.8. In some embodiments, the buffer comprises Tris, and the sugar comprises sucrose. In some embodiments, the buffer comprises Tris, the sugar comprises sucrose, and the non-ionic surfactant comprises poloxamer 188. In some embodiments, the buffer comprises Tris, the sugar comprises sucrose, the non-ionic surfactant comprises poloxamer 188, and the formulation further comprises a plasticizer (e.g., about 0.1% to about 2% glycerol). In some embodiments, the formulation is a frozen formulation. In some embodiments, the formulation is a lyophilized formulation. In some embodiments, the formulation is a liquid formulation. In some embodiments, the rAAV particles comprise rAAV-8, rAAV-9, or a combination thereof. In some embodiments, the formulation comprises about 1.0E+11 GC / mL to about 1.0E+14 GC / mL of rAAV particles. In some embodiments, the formulation comprises about 1.0E+11 GC / mL to about 1.0E+15 GC / mL of rAAV particles.

[0531] In some embodiments, the formulations disclosed herein comprise rAAV particles, as well as about 2 mM to about 10 mM buffer, about 150 mM to about 250 mM sugar, about 10 mM to about 20 mM sodium citrate, and about 0.001% to about 0.005% non-ionic surfactant, and the formulation has a pH of about 7.2 to about 7.8. In some embodiments, the buffer comprises Tris, and the sugar comprises sucrose. In some embodiments, the buffer comprises Tris, the sugar comprises sucrose, and the non-ionic surfactant comprises poloxamer 188. In some embodiments, the buffer comprises Tris, the sugar comprises sucrose, the non-ionic surfactant comprises poloxamer 188, and the formulation further comprises a plasticizer (e.g., about 0.1% to about 2% glycerol). In some embodiments, the formulation is a frozen formulation. In some embodiments, the formulation is a lyophilized formulation. In some embodiments, the formulation is a liquid formulation. In some embodiments, the rAAV particles comprise rAAV-8, rAAV-9, or a combination thereof. In some embodiments, the formulation comprises about 1.0E+11 GC / mL to about 1.0E+14 GC / mL of rAAV particles. In some embodiments, the formulation comprises about 1.0E+11 GC / mL to about 1.0E+15 GC / mL of rAAV particles.

[0532] In some embodiments, the formulations disclosed herein comprise rAAV particles and about 5 mM buffer, about 210 mM sugar, about 20 mM sodium citrate, and about 0.002% non-ionic surfactant, and the formulation has a pH of about 7.5. In some embodiments, the buffer comprises Tris, and the sugar comprises sucrose. In some embodiments, the buffer comprises Tris, the sugar comprises sucrose, and the non-ionic surfactant comprises poloxamer 188. In some embodiments, the buffer comprises Tris, the sugar comprises sucrose, the non-ionic surfactant comprises poloxamer 188, and the formulation further comprises a plasticizer (e.g., about 0.1% to about 2% glycerol). In some embodiments, the formulation is a frozen formulation. In some embodiments, the formulation is a lyophilized formulation. In some embodiments, the formulation is a liquid formulation. In some embodiments, the rAAV particles comprise rAAV-8, rAAV-9, or a combination thereof. In some embodiments, the formulation contains about 1.0E+11 GC / mL to about 1.0E+14 GC / mL of rAAV particles. In some embodiments, the formulation contains about 1.0E+11 GC / mL to about 1.0E+15 GC / mL of rAAV particles.

[0533] Formulations disclosed herein can include rAAV particles that include capsid proteins from any AAV capsid serotype. In some embodiments, the rAAV particles include AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV-11, AAV-12, AAV-13, AAV-14, AAV-15, and AAV-16, rAAV.rh8, rAAV.rh10, rAAV.rh20, rAAV.rh39, rAAV.Rh74, rAAV.RHM4-1, AAV.hu37, rAAV.Anc80, rAAV.Anc80L65, rAAV.7m8, rAAV.Anc80L65 ... , rAAV.PHP.B, rAAV2.5, rAAV2tYF, rAAV3B, rAAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, or AAV.HSC16. In some embodiments, the rAAV particles include any of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV-11, AAV-12, AAV-13, AAV-14, AAV-15, and AAV-16, rAAV.rh8, rAAV.rh10, rAAV.rh20, rAAV.rh39, rAAV.Rh74, rAAV.RHM4-1, AAV.hu37, rAAV.Anc80, rAAV.Anc80L65, rAAV.7m8, rAAV.PHP.B, These include capsid proteins that are derivatives, modifications, or pseudotypes of rAAV2.5, rAAV2tYF, rAAV3B, rAAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, or AAV.HSC16 capsid proteins.

[0534] In some embodiments, the rAAV particles comprise capsid proteins from an AAV capsid serotype selected from AAV-8 and AAV-9. In some embodiments, the rAAV particles have an AAV capsid serotype of AAV-8. In some embodiments, the rAAV particles have an AAV capsid serotype of AAV-9.

[0535] In some embodiments, the rAAV particles comprise capsid proteins that are derivatives, modifications, or pseudotypes of AAV-8 or AAV-9 capsid proteins. In some embodiments, the rAAV particles comprise capsid proteins having at least 80% or more identical to the VP1, VP2, and / or VP3 sequences of the AAV-8 capsid protein, e.g., 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, etc., i.e., up to 100% identical AAV-8 capsid protein.

[0536] In some embodiments, the rAAV particle composition comprises a capsid protein that is a derivative, modification, or pseudotype of the AAV-9 capsid protein. In some embodiments, the rAAV particles in the feeding composition comprise a capsid protein having at least 80% or more identical to the VP1, VP2, and / or VP3 sequences of the AAV-9 capsid protein, e.g., 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, etc., i.e., up to 100% identical AAV-9 capsid protein.

[0537] In further embodiments, the rAAV particles comprise mosaic capsids. In further embodiments, the rAAV particles comprise pseudotyped rAAV particles. In further embodiments, the rAAV particles comprise capsids that include chimeric capsid proteins of two or more AAV capsid serotypes.

[0538] In some embodiments, the formulations disclosed herein are stable formulations that include a pharma- ceutically acceptable carrier.

[0539] As used herein, the term "pharmaceutical acceptable" refers to a biologically acceptable formulation, gas, liquid, or solid, or mixture thereof, suitable for one or more routes of administration, in vivo delivery, or contact. A "pharmaceutical acceptable" composition is a material that is free from biological or other undesirable properties, e.g., such a material can be administered to a subject without causing substantial undesirable biological effects. That is, such a stable formulation can be used, for example, to administer rAAV isolated according to the methods of the present disclosure to a subject. Supplementary active compounds (e.g., preservatives, antibacterial agents, antiviral agents, and antifungal agents) can be incorporated into the composition. Pharmaceutical compositions can be formulated to be compatible with a particular route of administration or delivery, as described herein or known to those of skill in the art. Thus, pharmaceutical compositions include carriers, diluents, or excipients suitable for administration by various routes.The rAAV particles and methods of the invention, as well as pharmaceutical compositions and delivery suitable for use therein, are known in the art (see, e.g., Remington: The Science and Practice of Pharmacy (2003) 20th ed., Mack Publishing Co., Easton, Pa.; Remington's Pharmaceutical Sciences (1990) 18th ed., Mack Publishing Co., Easton, Pa.; The Merck Index (1996) 12th ed., Merck Publishing Group, Whitehouse, NJ; Pharmaceutical Principles of Solid Dosage Forms (1993), Technonic Publishing Co., Inc., Lancaster, Pa.; Ansel and Stoklosa, Pharmaceutical Calculations (2001) 11th ed., Lippincott Williams & Wilkins, Baltimore, Md.; and Poznansky et al., Drug Delivery Systems (1980), R.L. Juliano, ed., Oxford, NY, (see pp. 253-315).

[0540] In some embodiments, the formulations disclosed herein are pharma- ceutically acceptable compositions suitable for administration to a patient in need of such a formulation by known methods, such as intravenous administration (e.g., as a bolus or by continuous infusion over a period of time), or by intramuscular, intraperitoneal, intracerebrospinal, subcutaneous, intraarticular, intrasynovial, intrathecal, oral, topical, or inhalation routes. In some embodiments, the formulations disclosed herein are suitable for systemic or local administration. Systemic administration includes, but is not limited to, oral, subdermal, intraperitoneal, subcutaneous, nasal, sublingual, or rectal routes of administration. Local administration includes, but is not limited to, topical, subcutaneous, intramuscular, subretinal, intrathecal, and intraperitoneal routes of administration.

[0541] In some embodiments, the formulations disclosed herein are pharmaceutical unit doses. A "unit dose" refers to a physically discrete unit suitable for unitary administration to a subject to be treated, each unit containing a predetermined amount, optionally with a pharmaceutical carrier (excipient, diluent, vehicle, or filler), calculated to produce a desired effect (e.g., a prophylactic or therapeutic effect) when administered in one or more doses. Unit dosage forms may be, for example, in ampoules and vials, which may contain liquid compositions, or compositions in a freeze-dried or lyophilized state, for example, to which a sterile liquid carrier may be added prior to administration or in vivo delivery. Individual unit dosage forms may be included in multi-dose kits or containers. Recombinant vector (e.g., AAV) sequences, plasmids, vector genomes, and recombinant viral particles, as well as pharmaceutical compositions thereof, may be packaged in single or multiple unit dosage forms for ease of administration and uniformity of dosage. In some embodiments, the composition comprises any of the following: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV-11, AAV-12, AAV-13, AAV-14, AAV-15, and AAV-16, rAAV.rh8, rAAV.rh10, rAAV.rh20, rAAV.rh39, rAAV.Rh74, rAAV.RHM4-1, AAV.hu37, rAAV.Anc80, rAAV.Anc80L65, rAAV.7m8, rAAV.P In some embodiments, the AAV capsid serotype is AAV-8. In some embodiments, the AAV capsid serotype is AAV-9.

[0542] In some embodiments, the disclosure provides a method of producing a formulation comprising recombinant adeno-associated virus (rAAV) particles as disclosed herein, the method comprising mixing rAAV particles with a buffer, sugar, salt, optional plasticizer, and optional non-ionic surfactant of a formulation as disclosed herein, thereby producing said formulation comprising rAAV. Methods for mixing the components of a formulation to produce a formulation are known to those of skill in the art.

[0543] In certain embodiments, provided herein are recombinant adeno-associated virus (rAAV) particles, and a) about 5 mM Tris, b) about 210 mM sucrose; c) about 20 mM sodium citrate, and d) about 0.002% (w / v) poloxamer 188; It is a stable formulation comprising:

[0544] In certain embodiments, provided herein are recombinant adeno-associated virus (rAAV) particles, and a) about 5 mM Tris, b) about 210 mM sucrose; c) about 20 mM sodium citrate; d) about 0.002% (w / v) poloxamer 188, and e) about 0.25% (w / v) glycerol; It is a stable formulation comprising:

[0545] In certain embodiments, provided herein are recombinant adeno-associated virus (rAAV) particles, and a) about 5 mM Tris, b) about 210 mM sucrose; c) about 20 mM sodium citrate; d) about 0.002% (w / v) poloxamer 188, and e) about 0.5% (w / v) sorbitol; It is a stable formulation comprising:

[0546] In certain embodiments, provided herein are recombinant adeno-associated virus (rAAV) particles, and a) about 5 mM Tris, b) about 30 mM sodium sulfate; c) about 263 mM sucrose, and d) about 0.005% (w / v) poloxamer 188; It is a stable formulation comprising:

[0547] In certain embodiments, the stable formulation does not include mannitol.

[0548] In certain embodiments, the stable formulation comprises less than 150 mM, 140 mM, 130 mM, 120 mM, 110 mM, 100 mM, 90 mM, 80 mM, 70 mM, 60 mM, 50 mM, 40 mM, 30 mM, 20 mM, or 10 mM mannitol.

[0549] In certain embodiments, the stable formulation comprises sucrose at a concentration of about 210 mM. In certain embodiments, the stable formulation comprises sucrose at a concentration of about 263 mM. In certain embodiments, the stable formulation comprises sucrose at a concentration of about 409 mM. In certain embodiments, the stable formulation comprises sucrose at a concentration of about 14.6 mM. In certain embodiments, the stable formulation comprises sucrose at a concentration of about 45 mM. In certain embodiments, the stable formulation comprises sucrose at a concentration of about 0 mM to about 20 mM. In certain embodiments, the stable formulation comprises sucrose at a concentration of about 20 mM to about 50 mM. In certain embodiments, the stable formulation comprises sucrose at a concentration of about 50 mM to about 100 mM. In certain embodiments, the stable formulation comprises sucrose at a concentration of about 100 mM to about 200 mM. In certain embodiments, the stable formulation comprises sucrose at a concentration of about 200 mM to about 300 mM. In certain embodiments, the stable formulation comprises sucrose at a concentration of about 300 mM to about 400 mM. In certain embodiments, the stable formulation comprises sucrose at a concentration of about 400 mM to about 500 mM. In certain embodiments, the stable formulation comprises sucrose at a concentration of about 500 mM to about 600 mM. In certain embodiments, the stable formulation comprises sucrose at a concentration of about 600 mM to about 700 mM.

[0550] In certain embodiments, the stable formulation comprises sorbitol at a concentration of about 0.5%. In certain embodiments, the stable formulation comprises sorbitol at a concentration of about 0.25%. In certain embodiments, the stable formulation comprises sorbitol at a concentration of about 0.10%.

[0551] In certain embodiments, the stable formulation contains sorbitol at a concentration of about 0% to about 0.10%. In certain embodiments, the stable formulation contains sorbitol at a concentration of about 0.10% to about 0.20%. In certain embodiments, the stable formulation contains sorbitol at a concentration of about 0.20% to about 0.30%. In certain embodiments, the stable formulation contains sorbitol at a concentration of about 0.30% to about 0.40%. In certain embodiments, the stable formulation contains sorbitol at a concentration of about 0.40% to about 0.50%. In certain embodiments, the stable formulation contains sorbitol at a concentration of about 0.50% to about 0.60%. In certain embodiments, the stable formulation contains sorbitol at a concentration of about 0.60% to about 0.70%. In certain embodiments, the stable formulation contains sorbitol at a concentration of about 0.70% to about 0.80%. In certain embodiments, the stable formulation comprises sorbitol at a concentration of about 0.80% to about 0.90%. In certain embodiments, the stable formulation comprises sorbitol at a concentration of about 0.90% to about 1.00%.

[0552] In certain embodiments, the stable formulation comprises glycerol at a concentration of about 0.5%. In certain embodiments, the stable formulation comprises glycerol at a concentration of about 0.25%. In certain embodiments, the stable formulation comprises glycerol at a concentration of about 0.10%.

[0553] In certain embodiments, the stable formulation comprises glycerol at a concentration of about 0% to about 0.10%. In certain embodiments, the stable formulation comprises glycerol at a concentration of about 0.10% to about 0.20%. In certain embodiments, the stable formulation comprises glycerol at a concentration of about 0.20% to about 0.30%. In certain embodiments, the stable formulation comprises glycerol at a concentration of about 0.30% to about 0.40%. In certain embodiments, the stable formulation comprises glycerol at a concentration of about 0.40% to about 0.50%. In certain embodiments, the stable formulation comprises glycerol at a concentration of about 0.50% to about 0.60%. In certain embodiments, the stable formulation comprises glycerol at a concentration of about 0.60% to about 0.70%. In certain embodiments, the stable formulation comprises glycerol at a concentration of about 0.70% to about 0.80%. In certain embodiments, the stable formulation comprises glycerol at a concentration of about 0.80% to about 0.90%. In certain embodiments, the stable formulation comprises glycerol at a concentration of about 0.90% to about 1.00%.

[0554] In certain embodiments, the stable formulation comprises poloxamer 188 at a concentration of about 0.001% (weight / volume, 0.01 g / L). In certain embodiments, the stable formulation comprises poloxamer 188 at a concentration of about 0.002% (weight / volume, 0.02 g / L). In certain embodiments, the stable formulation comprises poloxamer 188 at a concentration of about 0.005% (weight / volume, 0.05 g / L).

[0555] In certain embodiments, the stable formulation comprises poloxamer 188 at a concentration of about 0.0005% (weight / volume, 0.005 g / L) to about 0.05% (weight / volume, 0.5 g / L). In certain embodiments, the stable formulation comprises poloxamer 188 at a concentration of about 0.0001% (weight / volume, 0.001 g / L) to about 0.01% (weight / volume, 0.1 g / L). In certain embodiments, the stable formulation comprises poloxamer 188 at a concentration of about 0.0005% (weight / volume, 0.005 g / L) to about 0.001% (weight / volume, 0.01 g / L). In certain embodiments, the stable formulation comprises poloxamer 188 at a concentration of about 0.001% (weight / volume, 0.01 g / L) to about 0.05% (weight / volume, 0.5 g / L). In certain embodiments, the stable formulation comprises poloxamer 188 at a concentration of about 0.0005% (weight / volume, 0.005 g / L). In certain embodiments, the stable formulation comprises poloxamer 188 at a concentration of about 0.0006% (weight / volume, 0.006 g / L). In certain embodiments, the stable formulation comprises poloxamer 188 at a concentration of about 0.0007% (weight / volume, 0.007 g / L). In certain embodiments, the stable formulation comprises poloxamer 188 at a concentration of about 0.0008% (weight / volume, 0.008 g / L). In certain embodiments, the stable formulation comprises poloxamer 188 at a concentration of about 0.0009% (weight / volume, 0.009 g / L). In certain embodiments, the stable formulation comprises poloxamer 188 at a concentration of about 0.001% (weight / volume, 0.01 g / L). In certain embodiments, the stable formulation comprises poloxamer 188 at a concentration of about 0.002% (weight / volume, 0.02 g / L). In certain embodiments, the stable formulation comprises poloxamer 188 at a concentration of about 0.003% (weight / volume, 0.03 g / L). In certain embodiments, the stable formulation comprises poloxamer 188 at a concentration of about 0.004% (weight / volume, 0.04 g / L). In certain embodiments, the stable formulation comprises poloxamer 188 at a concentration of about 0.005% (weight / volume, 0.05 g / L). In certain embodiments, the stable formulation comprises poloxamer 188 at a concentration of about 0.01% (weight / volume, 0.1 g / L).In one particular embodiment, the stable formulation comprises poloxamer 188 at a concentration of about 0.05% (weight / volume, 0.5 g / L).

[0556] In certain embodiments, the pH of the pharmaceutical composition is about 7.4.

[0557] In certain embodiments, the pH of the pharmaceutical composition is about 6.0 to 8.8. In certain embodiments, the pH of the pharmaceutical composition is about 6.0 to 9.0. In certain embodiments, the pH of the pharmaceutical composition is about 6.0. In certain embodiments, the pH of the pharmaceutical composition is about 6.1. In certain embodiments, the pH of the pharmaceutical composition is about 6.2. In certain embodiments, the pH of the pharmaceutical composition is about 6.3. In certain embodiments, the pH of the pharmaceutical composition is about 6.4. In certain embodiments, the pH of the pharmaceutical composition is about 6.5. In certain embodiments, the pH of the pharmaceutical composition is about 6.6. In certain embodiments, the pH of the pharmaceutical composition is about 6.7. In certain embodiments, the pH of the pharmaceutical composition is about 6.8. In certain embodiments, the pH of the pharmaceutical composition is about 6.9. In certain embodiments, the pH of the pharmaceutical composition is about 7.0. In certain embodiments, the pH of the pharmaceutical composition is about 7.1. In certain embodiments, the pH of the pharmaceutical composition is about 7.2. In certain embodiments, the pH of the pharmaceutical composition is about 7.3. In certain embodiments, the pH of the pharmaceutical composition is about 7.4. In certain embodiments, the pH of the pharmaceutical composition is about 7.5. In certain embodiments, the pH of the pharmaceutical composition is about 7.6. In certain embodiments, the pH of the pharmaceutical composition is about 7.7. In certain embodiments, the pH of the pharmaceutical composition is about 7.8. In certain embodiments, the pH of the pharmaceutical composition is about 7.9. In certain embodiments, the pH of the pharmaceutical composition is about 8.0. In certain embodiments, the pH of the pharmaceutical composition is about 8.1. In certain embodiments, the pH of the pharmaceutical composition is about 8.2. In certain embodiments, the pH of the pharmaceutical composition is about 8.3. In certain embodiments, the pH of the pharmaceutical composition is about 8.4. In certain embodiments, the pH of the pharmaceutical composition is about 8.5. In certain embodiments, the pH of the pharmaceutical composition is about 8.6. In certain embodiments, the pH of the pharmaceutical composition is about 8.7. In certain embodiments, the pH of the pharmaceutical composition is about 8.8.In certain embodiments, the pH of the pharmaceutical composition is about 8.9. In certain embodiments, the pH of the pharmaceutical composition is about 9.0.

[0558] In certain embodiments, the vector genome concentration (VGC) of the stable formulation is about 3×10 9 GC / mL, 4×10 9 GC / mL, 5×10 9 GC / mL, 6×10 9 GC / mL, 7×10 9 GC / mL, 8×10 9 GC / mL, 9×10 9 GC / mL, approximately 1×10 10 GC / mL, approximately 2×10 10 GC / mL, approximately 3×10 10 GC / mL, approximately 4×10 10 GC / mL, approximately 5×10 10 GC / mL, approximately 6×10 10 GC / mL, approximately 7×10 10 GC / mL, approximately 8×10 10 GC / mL, approximately 9×10 10 GC / mL, approximately 1×10 11 GC / mL, approximately 2×10 11 GC / mL, approximately 3×10 11 GC / mL, approximately 4×10 11 GC / mL, approximately 5×10 11 GC / mL, approximately 6×10 11 GC / mL, approximately 7×10 11 GC / mL, approximately 8×10 11 GC / mL, approximately 9×10 11 GC / mL, approximately 1×10 12 GC / mL, approximately 2×10 12 GC / mL, approximately 3×10 12 GC / mL, approximately 4×10 12 GC / mL, approximately 5×10 12 GC / mL, approximately 6×10 12 GC / mL, approximately 7×10 12 GC / mL, approximately 8×10 12 GC / mL, approximately 9×10 12 GC / mL, approximately 1×10 13 GC / mL, approximately 1×10 13 GC / mL, approximately 2×1013 GC / mL, or approximately 3 x 10 13 GC / mL.

[0559] 6.3 Methods for reducing rAAV genome release from rAAV particles In some embodiments, the disclosure provides a method of reducing rAAV genome release from rAAV particles, the method comprising producing a formulation as disclosed herein comprising rAAV particles, a buffer, a sugar, a salt, and optionally a non-ionic surfactant, wherein the rAAV genome release from the rAAV particles after three freeze-thaw cycles is reduced compared to the rAAV genome release in a formulation that does not comprise sugar. In some embodiments, the formulation is a frozen formulation. In some embodiments, the formulation is a lyophilized formulation. In some embodiments, the salt comprises sodium citrate. In some embodiments, the salt comprises sodium citrate and the sugar comprises sucrose. In some embodiments, the method of reducing rAAV genome release disclosed herein further comprises lyophilizing the formulation to a residual moisture content of about 1% to about 5%.

[0560] In some embodiments, the disclosure provides a method of reducing rAAV genome release from rAAV particles, the method comprising producing a formulation as disclosed herein comprising rAAV particles, a buffer, a sugar, a salt, and optionally a non-ionic surfactant, wherein the rAAV genome release from the rAAV particles after lyophilization and reconstitution is reduced compared to the rAAV genome release in a formulation that does not comprise sugar. In some embodiments, the formulation is a frozen formulation. In some embodiments, the formulation is a lyophilized formulation. In some embodiments, the salt comprises sodium citrate. In some embodiments, the salt comprises sodium citrate and the sugar comprises sucrose. In some embodiments, the method of reducing rAAV genome release disclosed herein further comprises lyophilizing the formulation to a residual moisture content of about 1% to about 5%.

[0561] In some embodiments, the disclosure provides a method of reducing rAAV genome release from rAAV particles, the method comprising producing a formulation as disclosed herein comprising rAAV particles, a buffer, a sugar, a salt, a plasticizer, and optionally a non-ionic surfactant, wherein the rAAV genome release from the rAAV particles after three freeze-thaw cycles is reduced compared to the rAAV genome release in a formulation that does not comprise sugar. In some embodiments, the formulation is a frozen formulation. In some embodiments, the formulation is a lyophilized formulation. In some embodiments, the salt comprises sodium citrate. In some embodiments, the salt comprises sodium citrate, the sugar comprises sucrose. In some embodiments, the salt comprises sodium citrate, the sugar comprises sucrose, and the plasticizer comprises glycerol. In some embodiments, the method of reducing rAAV genome release disclosed herein further comprises lyophilizing the formulation.

[0562] In some embodiments, the disclosure provides a method of reducing rAAV genome release from rAAV particles, the method comprising producing a formulation disclosed herein comprising rAAV particles, a buffer, a sugar, a salt, a plasticizer, and optionally a non-ionic surfactant, wherein the rAAV genome release from the rAAV particles after lyophilization and reconstitution is reduced compared to the rAAV genome release in a formulation without sugar. In some embodiments, the formulation is a frozen formulation. In some embodiments, the formulation is a lyophilized formulation. In some embodiments, the salt comprises sodium citrate. In some embodiments, the salt comprises sodium citrate, the sugar comprises sucrose. In some embodiments, the salt comprises sodium citrate, the sugar comprises sucrose, and the plasticizer comprises glycerol. In some embodiments, the method of reducing rAAV genome release disclosed herein further comprises lyophilizing the formulation.

[0563] In some embodiments, the disclosure provides a use of a sugar to reduce rAAV genome release from rAAV particles, the use comprising producing a formulation as disclosed herein comprising rAAV particles, a buffer, a sugar, a salt, and optionally a non-ionic surfactant, wherein the rAAV genome release from the rAAV particles after three freeze-thaw cycles is reduced compared to the rAAV genome release in a formulation without the sugar. In some embodiments, the formulation is a frozen formulation. In some embodiments, the formulation is a lyophilized formulation. In some embodiments, the salt comprises sodium citrate. In some embodiments, the salt comprises sodium citrate and the sugar comprises sucrose. In some embodiments, the use of a sugar to reduce rAAV genome release as disclosed herein further comprises lyophilizing the formulation to a residual moisture content of about 1% to about 7%.

[0564] In some embodiments, the disclosure provides a use of a sugar to reduce rAAV genome release from rAAV particles, the use comprising producing a formulation as disclosed herein comprising rAAV particles, a buffer, a sugar, a salt, and optionally a non-ionic surfactant, wherein the rAAV genome release from the rAAV particles after lyophilization and reconstitution is reduced compared to the rAAV genome release in a formulation without sugar. In some embodiments, the formulation is a frozen formulation. In some embodiments, the formulation is a lyophilized formulation. In some embodiments, the salt comprises sodium citrate. In some embodiments, the salt comprises sodium citrate and the sugar comprises sucrose. In some embodiments, the use of a sugar to reduce rAAV genome release as disclosed herein further comprises lyophilizing the formulation to a residual moisture content of about 1% to about 7%.

[0565] In some embodiments, the disclosure provides a use of a plasticizer to reduce rAAV genome release from rAAV particles, the use comprising producing a formulation as disclosed herein comprising rAAV particles, a buffer, a sugar, a salt, a plasticizer, and optionally a non-ionic surfactant, wherein rAAV genome release from the rAAV particles after three freeze-thaw cycles is reduced compared to the rAAV genome release in a formulation without sugar. In some embodiments, the formulation is a frozen formulation. In some embodiments, the formulation is a lyophilized formulation. In some embodiments, the salt comprises sodium citrate. In some embodiments, the salt comprises sodium citrate, the sugar comprises sucrose. In some embodiments, the salt comprises sodium citrate, the sugar comprises sucrose, and the plasticizer comprises glycerol. In some embodiments, the use of a sugar to reduce rAAV genome release as disclosed herein further comprises lyophilizing the formulation.

[0566] In some embodiments, the disclosure provides a use of a plasticizer to reduce rAAV genome release from rAAV particles, the use comprising producing a formulation as disclosed herein comprising rAAV particles, a buffer, a sugar, a salt, a plasticizer, and optionally a non-ionic surfactant, wherein rAAV genome release from the rAAV particles after lyophilization and reconstitution is reduced compared to rAAV genome release in a formulation without sugar. In some embodiments, the formulation is a frozen formulation. In some embodiments, the formulation is a lyophilized formulation. In some embodiments, the salt comprises sodium citrate. In some embodiments, the salt comprises sodium citrate, the sugar comprises sucrose. In some embodiments, the salt comprises sodium citrate, the sugar comprises sucrose, and the plasticizer comprises glycerol. In some embodiments, the use of a sugar to reduce rAAV genome release as disclosed herein further comprises lyophilizing the formulation.

[0567] In some embodiments of the methods of reducing rAAV genome release or the use of compounds to reduce rAAV genome release disclosed herein, rAAV genome release is determined by measuring relative fluorescence in the presence of a DNA-specific fluorescent stain. In some embodiments, the DNA-specific fluorescent stain is SYBR gold.

[0568] In some embodiments of the methods of reducing rAAV genome release or the use of compounds for reducing rAAV genome release disclosed herein, freezing-induced rAAV genome release is reduced by at least about 10%, 20%, 50%, 80%, or 90%. In some embodiments, freezing-induced rAAV genome release is reduced by at least about 20%. In some embodiments, freezing-induced rAAV genome release is reduced by at least about 50%. In some embodiments, freezing-induced rAAV genome release is reduced by at least about 80%. In some embodiments, freezing-induced rAAV genome release is substantially eliminated.

[0569] In some embodiments of the methods of reducing rAAV genome release or the use of compounds for reducing rAAV genome release disclosed herein, the sugar is a non-reducing sugar. In some embodiments, the non-reducing sugar is sucrose, trehalose, raffinose, or a combination thereof. In some embodiments, the non-reducing sugar is sucrose.

[0570] In some embodiments of the methods of reducing rAAV genome release or the use of a compound for reducing rAAV genome release disclosed herein, the sugar is a reducing sugar. In some embodiments, the reducing sugar is glucose, fructose, mannose, galactose, lactose, or a combination thereof. In some embodiments, the reducing sugar is glucose.

[0571] In some embodiments of the methods of reducing rAAV genome release or the use of compounds to reduce rAAV genome release disclosed herein, the plasticizer comprises glycerol, xylitol, sorbitol, mannitol, or a combination thereof. In some embodiments, the plasticizer is glycerol.

[0572] 6.4 rAAV particles The provided formulation is suitable for containing any isolated recombinant AAV particle and is suitable for use in a method of treating a disease or disorder in a subject in need of the treatment, comprising administering any isolated recombinant AAV particle.The provided method is also suitable for formulating any isolated recombinant AAV particle and reducing rAAV genome release from any isolated recombinant AAV particle.Therefore, the rAAV can be of any serotype, any modification or derivative thereof known in the art, or any combination thereof known in the art (e.g., a rAAV particle population that includes two or more serotypes, such as two or more of rAAV2 particles, rAAV8 particles, and rAAV9 particles). In some embodiments, the rAAV particles include AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV-11, AAV-12, AAV-13, AAV-14, AAV-15 and AAV-16, rAAV.rh8, rAAV.rh10, rAAV.rh20, rAAV.rh39, rAAV.Rh74, rAAV.RHM4-1, AAV.hu37, rAAV.Anc80, rAAV.Anc80L65, rAAV.7m8, rAAV.PHP. B, rAAV2.5, rAAV2tYF, rAAV3B, rAAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, or AAV.HSC16, or other rAAV particles, or a combination of two or more thereof.

[0573] In some embodiments, the rAAV particles include any of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV-11, AAV-12, AAV-13, AAV-14, AAV-15 and AAV-16, rAAV.rh8, rAAV.rh10, rAAV.rh20, rAAV.rh39, rAAV.Rh74, rAAV.RHM4-1, AAV.hu37, rAAV.Anc80, rAAV.Anc80L65, rAAV.7m8, rAAV.PHP.B, rAAV2. and / or AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, or AAV.HSC16, or a derivative, modification, or pseudotype thereof.In some embodiments, rAAV particles include, for example, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV-11, AAV-12, AAV-13, AAV-14, AAV-15 and AAV-16, rAAV.rh8, rAAV.rh10, rAAV.rh20. , rAAV.rh39, rAAV.Rh74, rAAV.RHM4-1, AAV.hu37, rAAV.Anc80, rAAV.Anc80L65, rAAV. 7m8, rAAV.PHP.B, rAAV2.5, rAAV2tYF, rAAV3B, rAAV.LK03, AAV.HSC1, AAV.HSC2, AAV.H AAV.HSC16, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, or AAV.HSC16, or a capsid protein that is at least 80% identical, e.g., 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, etc., i.e., up to 100% identical, to the VP1, VP2, and / or VP3 sequences of an AAV capsid serotype selected from AAV.HSC16, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, or AAV.HSC16.

[0574] In some embodiments, the rAAV particles include any of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV-11, AAV-12, AAV-13, AAV-14, AAV-15, and AAV-16, rAAV.rh8, rAAV.rh10, rAAV.rh20, rAAV.rh39, rAAV.Rh74, rAAV.RHM4-1, AAV.hu37, rAAV.Anc80, rAAV.Anc80L65, rAAV.7m8, rAAV.PHP.B, rAAV2.5, rAAV.Anc80L65, rAAV.Anc80L65, rAAV.7m8, rAAV.PHP.B, rAAV.AAV.Anc80L65, r ... , rAAV2tYF, rAAV3B, rAAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, or AAV.HSC16, or a derivative, modification, or pseudotype thereof.In some embodiments, the rAAV particles may be any of the following: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV-11, AAV-12, AAV-13, AAV-14, AAV-15, and AAV-16, rAAV.rh8, rAAV.rh10, rAAV.rh20, rAAV.rh39, rAAV.Rh74, rAAV.RHM4-1, AAV.hu37, rAAV.Anc80, rAAV.Anc80L65, rAAV.7m8, rAAV.PHP.B, rAAV2.5, rAAV2tYF, rAAV3B, rAAV.LK03, AAV.HSC1, AAV.HSC2, AAV. The capsid proteins include those that are at least 80% identical to the VP1, VP2, and / or VP3 sequences of an AAV capsid serotype selected from AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, or AAV.HSC16, e.g., 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, etc., i.e., up to 100% identical.

[0575] In certain embodiments, the rAAV particles comprise Anc80 or Anc80L65 as described in Zinn et al., 2015, Cell Rep. 12(6): 1056-1068, which are incorporated by reference in their entirety. In certain embodiments, the rAAV particles comprise one of the amino acid insertions LGETTRP or LALGETTRP as described in U.S. Patent Nos. 9,193,956, 9458517, and 9,587,282, and U.S. Patent Application Publication No. 2016 / 0376323, each of which is incorporated by reference in its entirety. In certain embodiments, the rAAV particles comprise AAV.7m8 as described in U.S. Patent Nos. 9,193,956, 9,458,517, and 9,587,282, and U.S. Patent Application Publication No. 2016 / 0376323, each of which is incorporated herein by reference in its entirety. In certain embodiments, the rAAV particles comprise any AAV disclosed in U.S. Patent No. 9,585,971, such as AAV-PHP.B. In certain embodiments, the rAAV particles comprise any AAV disclosed in U.S. Patent No. 9,840,719 and WO 2015 / 013313, such as AAV.Rh74 and RHM4-1, each of which is incorporated herein by reference in its entirety. In certain embodiments, the rAAV particles comprise any AAV disclosed in WO 2014 / 172669, such as AAV rh.74, which is incorporated herein by reference in its entirety. In certain embodiments, the rAAV particles comprise AAV2 / 5 as described in Georgiadis et al., 2016, Gene Therapy 23: 857-862 and Georgiadis et al., 2018, Gene Therapy 25: 450, each of which is incorporated herein by reference in its entirety. In certain embodiments, the rAAV particles comprise any AAV disclosed in WO 2017 / 070491, such as AAV2tYF, which is incorporated herein by reference in its entirety.In certain embodiments, the rAAV particles comprise AAVLK03 or AAV3B as described in Puzzo et al., 2017, Sci. Transl. Med. 29(9): 418, which is incorporated by reference in its entirety. In certain embodiments, the rAAV particles comprise any AAV disclosed in U.S. Patent Nos. 8,628,966, 8,927,514, 9,923,120, and WO 2016 / 049230, such as HSC1, HSC2, HSC3, HSC4, HSC5, HSC6, HSC7, HSC8, HSC9, HSC10, HSC11, HSC12, HSC13, HSC14, HSC15, or HSC16, each of which is incorporated by reference in its entirety.

[0576] In certain embodiments, the rAAV particles comprise AAV as disclosed in any of the following patents and patent applications, each of which is incorporated by reference in its entirety: U.S. Pat. Nos. 7,282,199, 7,906,111, 8,524,446, 8,999,678, 8,628,966, 8,927,514, 8,734,809, 9,284,357, and 9,409,953. Nos. 9,169,299, 9,193,956, 9458517, and 9,587,282, U.S. Patent Application Publication Nos. 2015 / 0374803, 2015 / 0126588, 2017 / 0067908, 2013 / 0224836, 2016 / 0215024, 2017 / 0051257, and International Patent Application Nos. PCT / US2015 / 034799 and PCT / EP2015 / 053335. In some embodiments, the rAAV particles have capsid proteins that are at least 80% identical or greater, e.g., 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, etc., or up to 100% identical, to the VP1, VP2, and / or VP3 sequences of the AAV capsid disclosed in any of the following patents and patent applications, each of which is incorporated by reference in its entirety herein: U.S. Pat. Nos. 7,282,199; 7,906,111; 8,524,446; and 8,999,677. Nos. 8, 8,628,966, 8,927,514, 8,734,809, 9,284,357, 9,409,953, 9,169,299, 9,193,956, 9458517, and 9,587,282; U.S. Patent Application Publication No. 2015 / 037 Nos. 4803, 2015 / 0126588, 2017 / 0067908, 2013 / 0224836, 2016 / 0215024, and 2017 / 0051257, as well as International Patent Application Nos. PCT / US2015 / 034799 and PCT / EP2015 / 053335.

[0577] In some embodiments, the rAAV particles can be prepared using any of the methods described in International Publication Nos. WO 2003 / 052051 (see, e.g., SEQ ID NO: 2), WO 2005 / 033321 (see, e.g., SEQ ID NOs: 123 and 88), WO 03 / 042397 (see, e.g., SEQ ID NOs: 2, 81, 85, and 97), WO 2006 / 068888 (see, e.g., SEQ ID NOs: 1 and 3-6), WO 2006 / 110689, (see, e.g., SEQ ID NOs: 5-38), WO 2009 / 104964 (see, e.g., SEQ ID NOs: 1-5, 7, 9, 20, 22, 24, and 31), WO 2010 / 127097 (see, e.g., SEQ ID NOs: 5-38), and WO No. 2015 / 191508 (see, e.g., SEQ ID NOs: 80-294), as well as U.S. Patent Publication No. 20150023924 (see, e.g., SEQ ID NOs: 1, 5-10), the contents of each of which are incorporated herein by reference in their entirety. In some embodiments, the rAAV particles can be prepared using any of the methods described in International Publication Nos. WO 2003 / 052051 (see, e.g., SEQ ID NO: 2), WO 2005 / 033321 (see, e.g., SEQ ID NOs: 123 and 88), WO 03 / 042397 (see, e.g., SEQ ID NOs: 2, 81, 85, and 97), WO 2006 / 068888 (see, e.g., SEQ ID NOs: 1 and 3-6), WO 2006 / 110689 (see, e.g., SEQ ID NOs: 5-38), WO 2009 / 104964 (see, e.g., SEQ ID NOs: 1-5, 7, 9, 20, 22, 24, and 31), WO 2010 / 127097 (see, e.g., SEQ ID NOs: 5-38), and WO AAV capsid proteins that are at least 80% identical, e.g., 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, etc., i.e., up to 100% identical, to the VP1, VP2, and / or VP3 sequences of the AAV capsid disclosed in U.S. Patent Publication No. 2015 / 191508 (see, e.g., SEQ ID NOs:80-294), and U.S. Patent Publication No. 20150023924 (see, e.g., SEQ ID NOs:1, 5-10).

[0578] Nucleic acid sequences for AAV-based viral vectors, as well as methods for producing recombinant AAV and AAV capsids, are described in, e.g., U.S. Patent Nos. 7,282,199, 7,906,111, 8,524,446, 8,999,678, 8,628,966, 8,927,514, 8,734,809, 9,284,357, 9,409,953, 9,169,299, 9, Nos. 193,956, 9458517, and 9,587,282, U.S. Patent Application Publication Nos. 2015 / 0374803, 2015 / 0126588, 2017 / 0067908, 2013 / 0224836, 2016 / 0215024, and 2017 / 0051257, International Patent Application Nos. PCT / US2015 / 034799, PCT / EP2015 / 053335, WO No. 2003 / 052051, WO 2005 / 033321, WO 03 / 042397, WO 2006 / 068888, WO 2006 / 110689, WO 2009 / 104964, WO 2010 / 127097, and WO 2015 / 191508, as well as U.S. Application Publication No. 20150023924.

[0579] The provided methods are suitable for use in the production of recombinant AAV encoding a transgene. In some embodiments, provided herein is an AAV viral vector encoding an anti-VEGF Fab. In certain embodiments, provided herein is an AAV8-based viral vector encoding an anti-VEGF Fab. In more specific embodiments, provided herein is an AAV8-based viral vector encoding ranibizumab. In some embodiments, provided herein is an AAV viral vector encoding iduronidase (IDUA). In certain embodiments, provided herein is an AAV9-based viral vector encoding IDUA. In some embodiments, provided herein is an AAV viral vector encoding iduronate 2-sulfatase (IDS). In certain embodiments, provided herein is an AAV9-based viral vector encoding IDS. In some embodiments, provided herein is an AAV viral vector encoding low density lipoprotein receptor (LDLR). In certain embodiments, provided herein is an AAV8-based viral vector encoding LDLR. In some embodiments, provided herein are AAV viral vectors encoding tripeptidyl peptidase 1 (TPP1) protein. In certain embodiments, provided herein are AAV9-based viral vectors encoding TPP.

[0580] In further embodiments, the rAAV particles comprise pseudotyped rAAV particles. In some embodiments, the pseudotyped AAV is rAAV2 / 8 or rAAV2 / 9 pseudotyped AAV. Methods for making and using pseudotyped rAAV particles are known in the art (see, e.g., Duan et al., J. Virol., 75:7662-7671 (2001); Halbert et al., J. Virol., 74:1524-1532 (2000); Zolotukhin et al., Methods 28:158-167 (2002); and Auricchio et al., Hum. Molec. Genet. 10:3075-3081, (2001)).

[0581] In further embodiments, the rAAV particles comprise capsids that include chimeric capsid proteins of two or more AAV capsid serotypes, including AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV-11, AAV-12, AAV-13, AAV-14, AAV-15, and AAV-16, rAAV.rh8, rAAV.rh10, rAAV.rh20, rAAV.rh39, rAAV.Rh74, rAAV.RHM4-1, AAV.hu37, rAAV.Anc80, rAAV.Anc80L65, rAAV.7m8, rAAV.PHP.B. , rAAV2.5, rAAV2tYF, rAAV3B, rAAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, or AAV.HSC16.

[0582] In certain embodiments, single-stranded AAV (ssAAV) can be used. In certain embodiments, self-complementary vectors, such as scAAV, can be used (see, e.g., Wu, 2007, Human Gene Therapy, 18(2):171-82; McCarty et al, 2001, Gene Therapy, Vol. 8, Number 16, Pages 1248-1254; and U.S. Patent Nos. 6,596,535, 7,125,717, and 7,456,683, each of which is incorporated herein by reference in its entirety).

[0583] In some embodiments, the rAAV particles may be selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV-11, AAV-12, AAV-13, AAV-14, AAV-15, and AAV-16, rAAV.rh8, rAAV.rh10, rAAV.rh20, rAAV.rh39, rAAV.Rh74, rAAV.RHM4-1, AAV.hu37, rAAV.Anc80, rAAV.Anc80L65, rAAV.7m8, rAAV.PHP.B, rAAV2 .5, rAAV2tYF, rAAV3B, rAAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, or AAV.HSC16, or a derivative, modification, or pseudotype thereof.In some embodiments, rAAV particles include, for example, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV-11, AAV-12, AAV-13, AAV-14, AAV-15 and AAV-16, rAAV.rh8, rAAV.rh10, rAAV.rh20. , rAAV.rh39, rAAV.Rh74, rAAV.RHM4-1, AAV.hu37, rAAV.Anc80, rAAV.Anc80L65, rAAV. 7m8, rAAV.PHP.B, rAAV2.5, rAAV2tYF, rAAV3B, rAAV.LK03, AAV.HSC1, AAV.HSC2, AAV.H AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, or AAV.HSC16, including capsid proteins that are at least 80% identical, e.g., 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, etc., i.e., up to 100% identical, to the VP1, VP2, and / or VP3 sequences of an AAV capsid serotype selected from AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, or AAV.HSC16.

[0584] In some embodiments, the rAAV particles comprise capsid proteins from an AAV capsid serotype selected from AAV-8 or AAV-9. In some embodiments, the rAAV particles have an AAV capsid serotype of AAV-8. In some embodiments, the rAAV particles have an AAV capsid serotype of AAV-9.

[0585] In some embodiments, the rAAV particles comprise capsid proteins that are derivatives, modifications, or pseudotypes of AAV-8 or AAV-9 capsid proteins. In some embodiments, the rAAV particles comprise capsid proteins having at least 80% or more identity, e.g., 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, etc., i.e., up to 100% identity, to the VP1, VP2, and / or VP3 sequences of the AAV-8 capsid protein.

[0586] In some embodiments, the rAAV particles comprise capsid proteins that are derivatives, modifications, or pseudotypes of the AAV-9 capsid proteins. In some embodiments, the rAAV particles comprise capsid proteins having at least 80% or more identity, e.g., 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, etc., i.e., up to 100% identity, to the VP1, VP2, and / or VP3 sequences of the AAV-9 capsid proteins.

[0587] In further embodiments, the rAAV particles comprise a mosaic capsid. The mosaic AAV particles are composed of a mixture of viral capsid proteins from different serotypes of AAV. In some embodiments, the rAAV particles comprise a mosaic capsid comprising capsid proteins from a serotype selected from AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10, rAAVrhlO, AAV-11, AAV-12, AAV-13, AAV-14, AAV-15, and AAV-16. In some embodiments, the rAAV particles comprise a mosaic capsid comprising capsid proteins from a serotype selected from AAV-1, AAV-2, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10, AAVr.8, and AAVr.10.

[0588] In further embodiments, the rAAV particles comprise pseudotyped rAAV particles. In some embodiments, the pseudotyped rAAV particles comprise (a) a nucleic acid vector comprising AAV ITRs, and (b) a capsid comprised of capsid proteins derived from an AAVx (e.g., AAV-1, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10, AAV-11, AAV-12, AAV-13, AAV-14, AAV-15, and AAV-16). In further embodiments, the rAAV particles are selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV-11, AAV-12, AAV-13, AAV-14, AAV-15 and AAV-16, rAAV.rh8, rAAV.rh10, rAAV.rh20, rAAV.rh39, rAAV.Rh74, rAAV.RHM4-1, AAV.hu37, rAAV.Anc80, rAAV.Anc80L65, rAAV.7m8, rAAV.PHP. In a further embodiment, the rAAV particle comprises a pseudotyped rAAV particle comprised of a capsid protein of an AAV serotype selected from AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, and AAV.HSC16. In a further embodiment, the rAAV particle comprises a pseudotyped rAAV particle comprised of an AAV-8 capsid protein. In a further embodiment, the rAAV particle comprises a pseudotyped rAAV particle comprised of an AAV-9 capsid protein. In some embodiments, the pseudotyped rAAV8 or rAAV9 particles are rAAV2 / 8 or rAAV2 / 9 pseudotyped particles.Methods for making and using pseudotyped rAAV particles are known in the art (see, e.g., Duan et al., J. Virol., 75:7662-7671 (2001); Halbert et al., J. Virol., 74:1524-1532 (2000); Zolotukhin et al., Methods 28:158-167 (2002); and Auricchio et al., Hum. Molec. Genet. 10:3075-3081, (2001)).

[0589] In further embodiments, the rAAV particles comprise capsids that include capsid proteins that are chimeric of two or more AAV capsid serotypes. In some embodiments, the rAAV particles include capsids that include AAV-8 capsid proteins and capsid proteins that are chimeric of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV-11, AAV-12, AAV-13, AAV-14, AAV-15, and AAV-16, rAAV.rh8, rAAV.rh10, rAAV.rh20, rAAV.rh39, rAAV.Rh74, rAAV.RHM4-1, AAV.hu37, rAAV.Anc80, rAAV.Anc80L65, rAAV.7m8, rAAV.PHP.B, rAAV. and AAV capsid proteins that are chimeric with one or more AAV capsid proteins from an AAV serotype selected from AAV2.5, rAAV2tYF, rAAV3B, rAAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, and AAV.HSC16. In some embodiments, the rAAV particles comprise an AAV capsid protein that is a chimera of AAV-8 capsid protein and one or more AAV capsid proteins from an AAV serotype selected from AAV-1, AAV-2, AAV-5, AAV-6, AAV-7, AAV-9, AAV-10, rAAVrh10, AAVrh.8, and AAVrh.10.In some embodiments, the rAAV particles comprise an AAV-9 capsid protein and one or more of the following: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV-11, AAV-12, AAV-13, AAV-14, AAV-15, and AAV-16, rAAV.rh8, rAAV.rh10, rAAV.rh20, rAAV.rh39, rAAV.Rh74, rAAV.RHM4-1, AAV.hu37, rAAV.Anc80, rAAV.Anc80L65, rAAV.7m8, rAAV.PHP.B, rAAV. and AAV capsid proteins that are chimeric with capsid proteins of one or more AAV capsid serotypes selected from AAV2.5, rAAV2tYF, rAAV3B, rAAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, and AAV.HSC16. In some embodiments, the rAAV particles comprise an AAV capsid protein that is a chimera of an AAV-9 capsid protein and a capsid protein of one or more AAV capsid serotypes selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh.8, and AAVrh.10.

[0590] 6.5 rAAV particle isolation method In some embodiments, the disclosure provides a method of producing a formulation comprising the isolated recombinant adeno-associated virus (rAAV) particles disclosed herein, the method comprising: (a) isolating rAAV particles from a feed comprising impurities (e.g., a rAAV production culture), and (b) formulating the isolated rAAV particles using a method disclosed herein to produce the formulation. In some embodiments, the method of producing a formulation comprising the isolated recombinant adeno-associated virus (rAAV) particles disclosed herein comprises: (a) isolating rAAV particles from a feed comprising impurities (e.g., a rAAV production culture), (b) determining the genomic titer of the isolated rAAV particles, and (c) formulating the isolated rAAV particles using a method disclosed herein to produce the formulation.

[0591] In some embodiments, the disclosure further provides a method of producing a pharmaceutical unit dosage form of a formulation comprising the isolated recombinant adeno-associated virus (rAAV) particles disclosed herein, the method comprising isolating the rAAV particles from a supply comprising impurities (e.g., a rAAV production culture), determining the genomic titer of the isolated rAAV particles, and formulating the isolated rAAV particles using the methods disclosed herein. In some embodiments, the method of producing a pharmaceutical unit dosage form of a formulation comprising the isolated recombinant adeno-associated virus (rAAV) particles disclosed herein comprises isolating the rAAV particles from a supply comprising impurities (e.g., a rAAV production culture), and formulating the isolated rAAV particles using the methods disclosed herein.

[0592] The isolated rAAV particles can be isolated using methods known in the art. In some embodiments, the method of isolating the rAAV particles includes downstream processing, such as harvesting the cell culture, clarification of the harvested cell culture (e.g., by centrifugation or depth filtration), tangential flow filtration, affinity chromatography, anion exchange chromatography, cation exchange chromatography, size exclusion chromatography, hydrophobic interaction chromatography, hydroxylapatite chromatography, sterile filtration, or any combination (or combinations) thereof. In some embodiments, the downstream processing includes at least two, at least three, at least four, at least five, or at least six of the following: harvesting the cell culture, clarification of the harvested cell culture (e.g., by centrifugation or depth filtration), tangential flow filtration, affinity chromatography, anion exchange chromatography, cation exchange chromatography, size exclusion chromatography, hydrophobic interaction chromatography, hydroxylapatite chromatography, and sterile filtration. In some embodiments, downstream processing includes harvesting the cell culture, clarification of the harvested cell culture (e.g., by depth filtration), sterile filtration, tangential flow filtration, affinity chromatography, and anion exchange chromatography. In some embodiments, downstream processing includes clarification of the harvested cell culture, sterile filtration, tangential flow filtration, affinity chromatography, and anion exchange chromatography. In some embodiments, downstream processing includes clarification of the harvested cell culture by depth filtration, sterile filtration, tangential flow filtration, affinity chromatography, and anion exchange chromatography. In some embodiments, clarification of the harvested cell culture includes sterile filtration. In some embodiments, downstream processing does not include centrifugation. In some embodiments, the rAAV particles include capsid proteins of the AAV-8 serotype. In some embodiments, the rAAV particles include capsid proteins of the AAV-9 serotype.

[0593] In some embodiments, the method for isolating rAAV particles includes harvesting the cell culture, clarifying the harvested cell culture (e.g., by depth filtration), a first sterile filtration, a first tangential flow filtration, affinity chromatography, monolith anion exchange chromatography, a second tangential flow filtration, and a second sterile filtration. In some embodiments, the method for isolating rAAV particles includes clarification of the harvested cell culture, a first sterile filtration, a first tangential flow filtration, affinity chromatography, monolith anion exchange chromatography, a second tangential flow filtration, and a second sterile filtration. In some embodiments, the method for isolating rAAV particles includes clarification of the harvested cell culture by depth filtration, a first sterile filtration, a first tangential flow filtration, affinity chromatography, monolith anion exchange chromatography, a second tangential flow filtration, and a second sterile filtration. In some embodiments, the method does not include centrifugation. In some embodiments, the clarification of the harvested cell culture includes sterile filtration. In some embodiments, the rAAV particles include capsid proteins of the AAV-8 serotype. In some embodiments, the rAAV particles comprise capsid proteins of the AAV-9 serotype.

[0594] Numerous methods are known in the art for producing rAAV particles, including transfection, production of stable cell lines, and infectious hybrid virus production systems, including adenovirus-AAV hybrids, herpesvirus-AAV hybrids, and baculovirus-AAV hybrids. All rAAV production cultures for rAAV viral particle production require: (1) stable host cells, such as human-derived cell lines such as HeLa, A549, or HEK293 cells and their derivatives (HEK293T cells, HEK293F cells), mammalian cell lines such as Vero, or insect-derived cell lines such as SF-9 in the case of baculovirus production systems; (2) suitable helper virus functions, provided by wild-type or mutant adenoviruses (e.g., temperature-sensitive adenoviruses), herpes viruses, baculoviruses, or plasmid constructs providing helper functions; (3) AAV rep and cap genes and gene products; (4) a transgene (e.g., a therapeutic transgene) flanked by AAV ITR sequences; and (5) suitable media and media components to support rAAV production. Suitable media known in the art can be used for the production of rAAV vectors. Such media include, without limitation, media manufactured by Hyclone Laboratories and JRH, including Modified Eagle Medium (MEM), Dulbecco's Modified Eagle Medium (DMEM), and Sf-900 II SFM medium as described in U.S. Pat. No. 6,723,551, which is incorporated herein by reference in its entirety.

[0595] rAAV production cultures can be routinely grown under a variety of conditions (over a wide range of temperatures, for varying lengths of time, etc.) appropriate for the particular host cells utilized. As is known in the art, rAAV production cultures include attachment-dependent cultures, which can be cultured in suitable attachment-dependent vessels, such as roller bottles, hollow fiber filters, microcarriers, and packed-bed or fluidized-bed bioreactors. rAAV vector production cultures can also include host cells suitable for suspension culture, such as HeLa, HEK293, Vero, and derivatives thereof, as well as SF-9 cells, which can be cultured in a variety of ways, such as spinner flasks, stirred-tank bioreactors, and disposable systems such as the Wave bag system. Numerous suspension cultures for the production of rAAV particles are known in the art, including, for example, those disclosed in U.S. Pat. Nos. 6,995,006, 9,783,826, and U.S. Patent Application Publication No. 20120122155, each of which is incorporated by reference in its entirety herein.

[0596] In some embodiments, a method of producing rAAV particles includes providing a cell culture comprising cells capable of producing rAAV; adding a histone deacetylase (HDAC) inhibitor to the cell culture at a final concentration of about 0.1 mM to about 20 mM; and maintaining the cell culture under conditions permissive for production of rAAV particles. In some embodiments, the HDAC inhibitor comprises a short chain fatty acid or a salt thereof. In some embodiments, the HDAC inhibitor comprises a butyrate (e.g., sodium butyrate), a valproate (e.g., sodium valproate), a propionate (e.g., sodium propionate), or a combination thereof.

[0597] In some embodiments, the rAAV particles are produced as disclosed in U.S. PCT International Publication No. WO2020 / 033842, published February 13, 2020, and entitled "SCALABLE METHOD FOR RECOMBINANT AAV PRODUCTION," which is incorporated by reference in its entirety herein.

[0598] Recombinant AAV particles can be prepared by harvesting a production culture containing the host cells or by harvesting spent medium from a production culture. A The rAAV production culture can be harvested, provided that when the medium is harvested, the cells have been cultured under conditions known in the art to cause the release of rAAV particles from intact host cells into the medium. The recombinant AAV particles are released from the rAAV medium by lysis of the host cells of the production culture. A Cells may also be harvested from V production cultures. Suitable methods for lysing cells are known in the art and include, for example, multiple freeze-thaw cycles, sonication, microfluidization, and treatment with chemicals such as detergents and / or proteases.

[0599] At harvest, rAAV production cultures may contain one or more of the following: (1) host cell proteins, (2) host cell DNA, (3) plasmid DNA, (4) helper virus, (5) helper virus proteins, (6) helper virus DNA, and (7) media components, including, for example, serum proteins, amino acids, transferrin, and other low molecular weight proteins. rAAV production cultures may further contain product-associated impurities, such as inactive vector, empty viral capsids, aggregated viral particles or capsids, misfolded viral capsids, and degraded viral particles.

[0600] In some embodiments, the rAAV production culture harvest is clarified to remove host cell debris. In some embodiments, the production culture harvest is clarified by filtration through a series of depth filtration filters. Clarification can also be achieved by a variety of other standard techniques known in the art, such as centrifugation or filtration through any cellulose acetate filter with a pore size of 0.2 mm or greater known in the art. In some embodiments, clarification of the harvested cell culture comprises sterile filtration. In some embodiments, the production culture harvest is clarified by centrifugation. In some embodiments, clarification of the cell culture does not comprise centrifugation.

[0601] In some embodiments, the harvested cell culture is clarified using filtration. In some embodiments, the clarification of the harvested cell culture comprises depth filtration. In some embodiments, the clarification of the harvested cell culture further comprises depth filtration and sterile filtration. In some embodiments, the harvested cell culture is clarified using a filter train comprising one or more different filtration media. In some embodiments, the filter train comprises a depth filtration medium. In some embodiments, the filter train comprises one or more depth filtration media. In some embodiments, the filter train comprises two depth filtration media. In some embodiments, the filter train comprises a sterile filtration medium. In some embodiments, the filter train comprises two depth filtration media and a sterile filtration medium. In some embodiments, the depth filtration filter media is a porous depth filtration filter. In some embodiments, the filter train comprises Clarisolve® 20MS, Millistak+® COHC, and a sterilizing grade filter media. In some embodiments, the filter train comprises Clarisolve® 20MS, Millistak+® COHC, and Sartopore® 2 XLG 0.2 μm. In some embodiments, the harvested cell culture is pretreated before contacting with the depth filter. In some embodiments, the pretreatment comprises adding salt to the harvested cell culture. In some embodiments, the pretreatment comprises adding a chemical flocculant to the harvested cell culture. In some embodiments, the harvested cell culture is not pretreated before contacting with the depth filter.

[0602] In some embodiments, the production culture harvest is clarified by filtration as disclosed in PCT International Publication No. WO2019212921A1, published November 8, 2019, and entitled "SCALABLE CLARIFICATION PROCESS FOR RECOMBINANT AAV PRODUCTION," which is incorporated by reference in its entirety.

[0603] In some embodiments, the rAAV production culture harvest is treated with a nuclease (e.g., Benzonase®) or an endonuclease (e.g., Serratia marcescens endonuclease) to digest high molecular weight DNA present in the production culture. Nuclease or endonuclease digestion can be routinely performed under standard conditions known in the art. For example, nuclease digestion is performed at a final concentration of 1-2.5 units / ml of Benzonase® at temperatures ranging from room temperature to 37° C. for a period of 30 minutes to several hours.

[0604] Sterile filtration encompasses filtration using a sterilizing grade filter medium. In some embodiments, the sterilizing grade filter medium is a 0.2 or 0.22 μm pore size filter. In some embodiments, the sterilizing grade filter medium comprises polyethersulfone (PES). In some embodiments, the sterilizing grade filter medium comprises polyvinylidene fluoride (PVDF). In some embodiments, the sterilizing grade filter medium has a hydrophilic heterogeneous bilayer design. In some embodiments, the sterilizing grade filter medium has a hydrophilic heterogeneous bilayer design of a 0.8 μm pre-filter and a 0.2 μm final filter membrane. In some embodiments, the sterilizing grade filter medium has a hydrophilic heterogeneous bilayer design of a 1.2 μm pre-filter and a 0.2 μm final filter membrane. In some embodiments, the sterilizing grade filter medium is a 0.2 or 0.22 μm pore size filter. In a further embodiment, the sterilizing grade filter medium is a 0.2 μm pore size filter. In some embodiments, the sterilizing grade filter media is Sartopore® 2 XLG 0.2 μm, Durapore™ PVDF membrane 0.45 μm, or Sartoguard® PES 1.2 μm + 0.2 μm nominal pore size combination. In some embodiments, the sterilizing grade filter media is Sartopore® 2 XLG 0.2 μm.

[0605] In some embodiments, the clarified feed is concentrated by tangential flow filtration ("TFF") before being applied to a chromatography medium, such as an affinity chromatography medium. Large-scale concentration of viruses using TFF ultrafiltration is described in Paul et al., Human Gene Therapy 4:609-615 (1993). TFF concentration of the clarified feed allows for a technically manageable volume of the clarified feed to be chromatographed, allowing for more reasonable column dimensions without the need for lengthy recirculation times. In some embodiments, the clarified feed is concentrated at least 2-fold to at least 10-fold. In some embodiments, the clarified feed is concentrated at least 10-fold to at least 20-fold. In some embodiments, the clarified feed is concentrated at least 20-fold to at least 50-fold. In some embodiments, the clarified feed is concentrated about 20-fold. As will be appreciated by one of skill in the art, TFF can also be used to remove small molecule impurities (e.g., cell culture contaminants, including media components, serum albumin, or other serum proteins) to form a clarified feed via diafiltration. In some embodiments, the clarified feed is subjected to diafiltration to remove small molecule impurities. In some embodiments, the diafiltration involves the use of about 3 to about 10 diafiltration volumes of buffer. In some embodiments, the diafiltration involves the use of about 5 diafiltration volumes of buffer. As will also be appreciated by one of skill in the art, TFF can also be used at any step in the purification process where it is desirable to exchange buffer prior to performing the next step in the purification process. In some embodiments, the methods of isolating rAAV from a clarified feed disclosed herein involve the use of TFF to exchange buffer.

[0606] Affinity chromatography can be used to isolate rAAV particles from the composition. In some embodiments, affinity chromatography is used to isolate rAAV particles from a clarified feed. In some embodiments, affinity chromatography is used to isolate rAAV particles from a clarified feed that has been subjected to tangential flow filtration. Suitable affinity chromatography media are known in the art, and include, without limitation, AVB Sepharose™, POROS™ CaptureSelect™ AAVX affinity resin, POROS™ CaptureSelect™ AAV9 affinity resin, and POROS™ CaptureSelect™ AAV8 affinity resin. In some embodiments, the affinity chromatography media is POROS™ CaptureSelect™ AAV9 affinity resin. In some embodiments, the affinity chromatography media is POROS™ CaptureSelect™ AAV8 affinity resin. In some embodiments, the affinity chromatography medium is POROS™ CaptureSelect™ AAVX affinity resin.

[0607] Anion exchange chromatography can be used to isolate rAAV particles from the composition. In some embodiments, after affinity chromatography, anion exchange chromatography is used as a final concentration and purification step. Suitable anion exchange chromatography media are known in the art, including, without limitation, Unosphere Q (Biorad, Hercules, Calif.), and N-charged amino or imino resins, such as POROS 50 PI, or any DEAE, TMAE, tertiary or quaternary amine, or PEI-based resins known in the art (U.S. Pat. No. 6,989,264; Brument et al., Mol. Therapy 6(5):678-686 (2002); Gao et al., Hum. Gene Therapy 11:2079-2091 (2000)). In some embodiments, the anion exchange chromatography media comprises a quaternary amine. In some embodiments, the anion exchange media is a monolithic anion exchange chromatography resin. In some embodiments, the monolithic anion exchange chromatography medium comprises glycidyl methacrylate-ethylene dimethacrylate or styrene-divinylbenzene polymer. In some embodiments, the monolithic anion exchange chromatography medium is selected from the group consisting of CIMmultus™ QA-1 Advanced Composite Column (quaternary amine), CIMmultus™ DEAE-1 Advanced Composite Column (diethylamino), CIM® QA Disk (quaternary amine), CIM® DEAE, and CIM® EDA Disk (ethylenediamino). In some embodiments, the monolithic anion exchange chromatography medium is CIMmultus™ QA-1 Advanced Composite Column (quaternary amine). In some embodiments, the monolithic anion exchange chromatography medium is CIM® QA Disk (quaternary amine). In some embodiments, the anion exchange chromatography medium is CIM QA (BIA Separations, Slovenia).In some embodiments, the anion exchange chromatography medium is BIA CIM® QA-80 (column volume 80 mL). As one of skill in the art can appreciate, a wash buffer of appropriate ionic strength can be identified such that the rAAV remains bound to the resin while impurities are washed away, including, but not limited to, those that may be introduced by upstream purification steps.

[0608] In some embodiments, anion exchange chromatography is performed according to the methods disclosed in PCT International Published Application No. WO2019 / 241535, published December 19, 2019, and entitled "Anion Exchange Chromatography for Recombinant AAV production," which is incorporated by reference in its entirety.

[0609] In some embodiments, the method of isolating rAAV particles comprises determining the vector genome titer, capsid titer, and / or the ratio of full to empty capsids in a composition comprising the isolated rAAV particles. In some embodiments, the vector genome titer is determined by quantitative PCR (qPCR) or digital PCR (dPCR) or droplet digital PCR (ddPCR). In some embodiments, the capsid titer is determined by serotype-specific ELISA. In some embodiments, the ratio of full to empty capsids is determined by analytical ultracentrifugation (AUC) or transmission electron microscopy (TEM).

[0610] In some embodiments, the vector genome titer, capsid titer, and / or the ratio of full to empty capsids are determined spectrophotometrically, for example, by measuring the absorbance of the composition at 260 nm and measuring the absorbance of the composition at 280 nm. In some embodiments, the rAAV particles are not denatured prior to measuring the absorbance of the composition. In some embodiments, the rAAV particles are denatured prior to measuring the absorbance of the composition. In some embodiments, the absorbance of the composition at 260 nm and 280 nm is determined using a spectrophotometer. In some embodiments, the absorbance of the composition at 260 nm and 280 nm is determined using HPLC. In some embodiments, the absorbance is peak absorbance. Several methods for measuring the absorbance of a composition at 260 nm and 280 nm are known in the art. Methods for determining the vector genome titer and capsid titer of compositions comprising isolated recombinant rAAV particles are disclosed in PCT International Publication No. WO2019212922A1, published November 7, 2019, entitled "Systems and methods of spectrophotometry for the determination of genome content, capsid content and full / empty ratios of adeno-associated virus particles," which is incorporated by reference in its entirety herein.

[0611] 6.6 Kit In some embodiments, the present disclosure provides a kit comprising a formulation disclosed herein that can be used to perform a therapy for a disease or disorder in a subject in need of such treatment, the therapy comprising administration of isolated recombinant AAV particles. In some embodiments, the kit comprises at least one formulation comprising a formulation comprising rAAV particles disclosed herein in one or more containers. In some embodiments, the kit comprises all of the components necessary and / or sufficient to perform a therapy for a disease or disorder in a subject in need of such treatment, the therapy comprising administration of isolated recombinant AAV particles. As will be readily apparent to one of skill in the art, the formulation comprising rAAV particles disclosed herein can be readily incorporated into one of the established kit formats, which are well known in the art.

[0612] In order that the disclosure provided herein may be readily understood and put to practical use, certain embodiments will now be illustrated by the following non-limiting examples.

[0613] 6.7 Functional characteristics In certain embodiments, the formulations or stable formulations described herein are suitable for intravenous administration, subcutaneous administration, intramuscular injection, suprachoroidal injection (e.g., via a suprachoroidal drug delivery device such as a microinjector with a microneedle), subretinal injection via a transvitreal approach (surgical procedure), juxtascleral administration, intravitreal administration, subconjunctival administration, intraretinal administration, subretinal administration via the suprachoroidal space (e.g., a surgical procedure via a subretinal drug delivery device with a catheter that can be inserted into the suprachoroidal space and advanced toward the posterior pole, where the microneedle injects into the subretinal space), and / or posterior juxtascleral depot surgery (e.g., via a juxtascleral drug delivery device with a cannula that can be inserted at its tip and maintained in direct attachment to the scleral surface).

[0614] In certain embodiments, the pharmaceutical composition has a desired density suitable for intravenous administration, subcutaneous administration, intramuscular injection, suprachoroidal injection (e.g., via a suprachoroidal drug delivery device such as a microinjector with a microneedle), subretinal injection via a transvitreal approach (surgical procedure), juxtascleral administration, intravitreal administration, subconjunctival administration, intraretinal administration, subretinal administration via the suprachoroidal space (e.g., via a subretinal drug delivery device with a catheter that can be inserted into the suprachoroidal space and advanced toward the posterior pole, where the microneedle injects into the subretinal space), and / or posterior juxtascleral depot surgery (e.g., via a juxtascleral drug delivery device with a cannula that can be inserted at its tip and maintained in direct attachment to the scleral surface).

[0615] In certain embodiments, the pharmaceutical composition has a desired viscosity suitable for intravenous administration, subcutaneous administration, intramuscular injection, suprachoroidal injection (e.g., via a suprachoroidal drug delivery device such as a microinjector with a microneedle), juxtascleral administration, intravitreal administration, subconjunctival administration, intraretinal administration, subretinal injection via a transvitreal approach (surgical procedure), subretinal administration via the suprachoroidal space (e.g., via a subretinal drug delivery device with a catheter that can be inserted into the suprachoroidal space and advanced toward the posterior pole, where the microneedle injects into the subretinal space), and / or posterior juxtascleral depot surgery (e.g., via a juxtascleral drug delivery device with a cannula that can be inserted at its tip and maintained in direct attachment to the scleral surface).

[0616] In certain embodiments, recombinant adeno-associated virus (rAAV) particles in a stable formulation are at least about 2%, about 5%, about 7%, about 10%, about 12%, about 15%, about 17%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 100%, about 2-fold, about 3-fold, about 5-fold, about 10-fold, about 100-fold, or about 1000-fold more stable to the lyophilization or reconstitution process than the same rAAV particles in a reference formulation. In certain embodiments, the stability of the rAAV particles is determined by the assay(s) disclosed in Section 6.8.

[0617] In certain embodiments, the rAAV particles in the stable formulation have at least about 2%, about 5%, about 7%, about 10%, about 12%, about 15%, about 17%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 100%, about 2-fold, about 3-fold, about 5-fold, about 10-fold, about 100-fold, or about 1000-fold higher infectivity than the same rAAV particles in a reference formulation. In certain embodiments, the viral infectivity of the rAAV particles is determined by the assay(s) disclosed in Section 6.8. In certain embodiments, the infectivity is measured before or after lyophilization. In certain embodiments, the infectivity is measured before or after reconstitution of the lyophilized formulation.

[0618] In certain embodiments, the rAAV particles in the stable formulation exhibit at least about 2%, about 5%, about 7%, about 10%, about 12%, about 15%, about 17%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 100%, about 2-fold, about 3-fold, about 5-fold, about 10-fold, about 100-fold, or about 1000-fold less aggregation than the same rAAV particles in a reference formulation. In certain embodiments, aggregation of the rAAV particles is determined by the assay(s) disclosed in Section 6.8. In certain embodiments, aggregation is measured before or after lyophilization. In certain embodiments, aggregation is measured before or after reconstitution of the lyophilized formulation.

[0619] In certain embodiments, the stable formulation is lyophilized prior to storage.

[0620] In certain embodiments, the stable formulation is reconstituted after storage.

[0621] In certain embodiments, the formulation is stored at -80°C, -70°C, -20°C, 4°C, 20°C, 25°C, 30°C, 35°C, 37°C, or 40°C.

[0622] In certain embodiments, the formulation is stored for about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, 12 months, about 15 months, about 18 months, about 24 months, about 2 years, about 3 years, about 4 years, or about 5 years. In certain embodiments, the rAAV particles in a stable formulation are at least about 2%, about 5%, about 7%, about 10%, about 12%, about 15%, about 17%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 100%, about 2-fold, about 3-fold, about 5-fold, about 10-fold, about 100-fold, or about 1000-fold more stable than the same rAAV particles in a reference formulation stored under the same conditions after the formulation has been stored for a period of time, e.g., about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, 12 months, about 15 months, about 18 months, about 24 months, about 2 years, about 3 years, about 4 years, or about 5 years. In certain embodiments, the stability of the rAAV particles over a period of time is determined by the assay(s) disclosed in Section 6.8.

[0623] In certain embodiments, the rAAV particles in a stable formulation are at least about 2%, about 5%, about 7%, about 10%, about 12%, about 15%, about 17%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 100%, about 2-fold, about 3-fold, about 5-fold, about 10-fold, about 100-fold, or about 1000-fold more stable than the same rAAV particles in a reference formulation after the formulation is stored for a period of time, e.g., at least about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, 12 months, about 15 months, about 18 months, about 24 months, about 2 years, about 3 years, about 4 years, or about 5 years. In certain embodiments, the stability of the rAAV particles over a period of time is determined by the assay(s) disclosed in Section 6.8.

[0624] In certain embodiments, the rAAV particles in the stable formulation have an in vitro relative potency (IVRP) that is at least about 2%, about 5%, about 7%, about 10%, about 12%, about 15%, about 17%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 100%, about 2-fold, about 3-fold, about 5-fold, about 10-fold, about 100-fold, or about 1000-fold higher than the same rAAV particles in a reference formulation. In certain embodiments, the in vitro relative potency (IVRP) of the rAAV particles is determined by the assay(s) disclosed in Section 6.8. In certain embodiments, the IVRP is measured before or after lyophilization. In certain embodiments, the IVRP is measured before or after reconstitution of the lyophilized formulation.

[0625] In certain embodiments, the rAAV particles in the stable formulation have at least about 2%, about 5%, about 7%, about 10%, about 12%, about 15%, about 17%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 100%, about 2-fold, about 3-fold, about 5-fold, about 10-fold, about 100-fold, or about 1000-fold less free DNA than the same rAAV particles in a reference formulation. In certain embodiments, the free DNA of the rAAV particles is determined by the assay(s) disclosed in Section 6.8. In certain embodiments, the free DNA is measured before or after lyophilization. In certain embodiments, the free DNA is measured before or after reconstitution of the lyophilized formulation.

[0626] In certain embodiments, the rAAV particles in the stable formulation have at least about 2%, about 5%, about 7%, about 10%, about 12%, about 15%, about 17%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 100%, about 2-fold, about 3-fold, about 5-fold, about 10-fold, about 100-fold, or about 1000-fold less rAAV genome release than the same rAAV particles in the reference formulation. In certain embodiments, the rAAV genome release is determined by measuring the relative fluorescence in the presence of a DNA-specific fluorescent stain. In certain embodiments, the rAAV genome release is determined by the assay(s) disclosed in Section 6.8. In certain embodiments, the rAAV genome release is measured before or after lyophilization. In certain embodiments, the rAAV genome release is measured before or after reconstitution of the lyophilized formulation.

[0627] In certain embodiments, the rAAV particles in a stable formulation have a change in size of at most about 20%, about 15%, about 10%, about 8%, about 5%, about 4%, about 3%, about 2%, or about 1% after the formulation is stored for a period of time, such as about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, about 15 months, about 18 months, about 24 months, about 2 years, about 3 years, about 4 years, or about 5 years. In certain embodiments, the size of the rAAV particles is determined by the assay(s) disclosed in Section 6.8. In certain embodiments, the size is measured before or after a freeze-thaw cycle.

[0628] In certain embodiments, the rAAV particles in a stable formulation have a change in dimension of at most 20%, 15%, 10%, 8%, 5%, 4%, 3%, 2%, or 1% after storage of the formulation for a period of time, e.g., at least about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, about 15 months, about 18 months, about 24 months, about 2 years, about 3 years, about 4 years, or about 5 years. In certain embodiments, the dimensions of the rAAV particles are determined by the assay(s) disclosed in Section 6.8. In certain embodiments, the dimensions are measured before or after lyophilization. In certain embodiments, the dimensions are measured before or after reconstitution of the lyophilized formulation.

[0629] In certain embodiments, the rAAV particles in a stable formulation are at least about 2%, about 5%, about 7%, about 10%, about 12%, about 15%, about 17%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 100%, about 2-fold, about 3-fold, about 5-fold, about 10-fold, about 100-fold, or about 1000-fold more stable than the same rAAV particles in a reference formulation when stored at -80° C., -70° C., -20° C., 4° C., 20° C., 25° C., 30° C., 35° C., 37° C., or 40° C. In certain embodiments, the stability of the rAAV particles is determined by the assay(s) disclosed in Section 6.8.

[0630] In certain embodiments, the rAAV particles in the stable formulation can be incubated at -80°C, -70°C, -20°C, 4°C, 20°C, 25°C, 30°C, 35°C, 37°C, or 40°C for a period of time, such as about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, about 14 months, about 16 months, about 18 months, about 19 months, about 20 months, about 21 months, about 22 months, about 23 months, about 24 months, about 25 months, about 26 months, about 27 months, about 28 months, about 29 months, about 30 months, about 31 months, about 32 months, about 33 months, about 34 months, about 35 months, about 36 months, about 37 months, about 38 months, about 39 months, about 40 months, about 40 months, about 40 months, about 41 months, about 42 months, about 43 months, about 44 months, about 45 months, about 46 months, about 47 months, about 48 months, about 49 months, about 50 months, about 50 months, about 51 months, about 52 months, about 53 months, about 54 months, about 55 months, about 56 months, about 57 months, about 58 months, about 59 months, about 60 months, about 61 months, about 62 months, about 63 months, about 64 months, about 65 months, about 66 months, about 67 months, about 68 months, about 69 months, about The infectivity is at least 2%, 5%, 7%, 10%, 12%, 15%, 17%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 100%, 2-fold, 3-fold, 5-fold, 10-fold, 100-fold, or 1000-fold greater than the same rAAV particles in a reference formulation when stored for 2 months, about 15 months, about 18 months, about 24 months, about 2 years, about 3 years, about 4 years, or about 5 years. In certain embodiments, the viral infectivity of the rAAV particles is determined by the assay(s) disclosed in Section 6.8.

[0631] In certain embodiments, the rAAV particles in the stable formulation can be incubated at -80°C, -70°C, -20°C, 4°C, 20°C, 25°C, 30°C, 35°C, 37°C, or 40°C for a period of time, such as at least about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, about 13 months, about 14 months, about 15 months, about 16 months, about 17 months, about 18 months, about 19 months, about 20 months, about 21 months, about 22 months, about 23 months, about 24 months, about 25 months, about 26 months, about 27 months, about 28 months, about 29 months, about 30 months, about 31 months, about 32 months, about 33 months, about 34 months, about 35 months, about 36 months, about 37 months, about 38 months, about 39 months, about 40 ... When stored for about 12 months, about 15 months, about 18 months, about 24 months, about 2 years, about 3 years, about 4 years, or about 5 years, the infectivity is at least 2%, 5%, 7%, 10%, 12%, 15%, 17%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 100%, 2-fold, 3-fold, 5-fold, 10-fold, 100-fold, or 1000-fold greater than the same rAAV particles in a reference formulation. In certain embodiments, the viral infectivity of the rAAV particles is determined by the assay(s) disclosed in Section 6.8.

[0632] In certain embodiments, the rAAV particles in the stable formulation can be incubated at -80°C, -70°C, -20°C, 4°C, 20°C, 25°C, 30°C, 35°C, 37°C, or 40°C for a period of time, such as about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, about 14 months, about 16 months, about 18 months, about 19 months, about 20 months, about 21 months, about 22 months, about 23 months, about 24 months, about 25 months, about 26 months, about 27 months, about 28 months, about 29 months, about 30 months, about 31 months, about 32 months, about 33 months, about 34 months, about 35 months, about 36 months, about 37 months, about 38 months, about 39 months, about 40 months, about 40 months, about 40 months, about 41 months, about 42 months, about 43 months, about 44 months, about 45 months, about 46 months, about 47 months, about 48 months, about 49 months, about 50 months, about 50 months, about 51 months, about 52 months, about 53 months, about 54 months, about 55 months, about 56 months, about 57 months, about 58 months, about 59 months, about 60 months, about 61 months, about 62 months, about 63 months, about 64 months, about 65 months, about 66 months, about 67 months, about 68 months, about 69 months, about When stored for 2 months, about 15 months, about 18 months, about 24 months, about 2 years, about 3 years, about 4 years, or about 5 years, there is at least 2%, 5%, 7%, 10%, 12%, 15%, 17%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 100%, 2-fold, 3-fold, 5-fold, 10-fold, 100-fold, or 1000-fold less aggregation than the same rAAV particles in a reference formulation. In certain embodiments, aggregation of rAAV particles is identified by the assay(s) disclosed in Section 6.8.

[0633] In certain embodiments, the rAAV particles in the stable formulation can be incubated at -80°C, -70°C, -20°C, 4°C, 20°C, 25°C, 30°C, 35°C, 37°C, or 40°C for a period of time, such as at least about 11 weeks, about 2 weeks, about 3 weeks, about 4 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, or about 12 months. , about 12 months, about 15 months, about 18 months, about 24 months, about 2 years, about 3 years, about 4 years, or about 5 years, there is at least 2%, 5%, 7%, 10%, 12%, 15%, 17%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 100%, 2-fold, 3-fold, 5-fold, 10-fold, 100-fold, or 1000-fold less aggregation than the same rAAV particles in a reference formulation when stored for about 12 months, about 15 months, about 18 months, about 24 months, about 2 years, about 3 years, about 4 years, or about 5 years. In certain embodiments, aggregation of rAAV particles is identified by the assay(s) disclosed in Section 6.8.

[0634] In certain embodiments, the rAAV particles in the stable formulation can be incubated at -80°C, -70°C, -20°C, 4°C, 20°C, 25°C, 30°C, 35°C, 37°C, or 40°C for a period of time, such as about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, about 14 months, about 16 months, about 18 months, about 19 months, about 20 months, about 21 months, about 22 months, about 23 months, about 24 months, about 25 months, about 26 months, about 27 months, about 28 months, about 29 months, about 30 months, about 31 months, about 32 months, about 33 months, about 34 months, about 35 months, about 36 months, about 37 months, about 38 months, about 39 months, about 40 months, about 40 months, about 40 months, about 41 months, about 42 months, about 43 months, about 44 months, about 45 months, about 46 months, about 47 months, about 48 months, about 49 months, about 50 months, about 50 months, about 51 months, about 52 months, about 53 months, about 54 months, about 55 months, about 56 months, about 57 months, about 58 months, about 59 months, about 60 months, about 61 months, about 62 months, about 63 months, about 64 months, about 65 months, about 66 months, about 67 months, about 68 months, about 69 months, about The stability is at least 2%, 5%, 7%, 10%, 12%, 15%, 17%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 100%, 2-fold, 3-fold, 5-fold, 10-fold, 100-fold, or 1000-fold greater than the same rAAV particles in a reference formulation when stored for 2 months, about 15 months, about 18 months, about 24 months, about 2 years, about 3 years, about 4 years, or about 5 years. In certain embodiments, the stability of the rAAV particles over a period of time is determined by the assay(s) disclosed in Section 6.8.

[0635] In certain embodiments, the rAAV particles in the stable formulation can be incubated at -80°C, -70°C, -20°C, 4°C, 20°C, 25°C, 30°C, 35°C, 37°C, or 40°C for a period of time, such as at least about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, about 13 months, about 14 months, about 15 months, about 16 months, about 17 months, about 18 months, about 19 months, about 20 months, about 21 months, about 22 months, about 23 months, about 24 months, about 25 months, about 26 months, about 27 months, about 28 months, about 29 months, about 30 months, about 31 months, about 32 months, about 33 months, about 34 months, about 35 months, about 36 months, about 37 months, about 38 months, about 39 months, about 40 ... The stability is at least 2%, 5%, 7%, 10%, 12%, 15%, 17%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 100%, 2-fold, 3-fold, 5-fold, 10-fold, 100-fold, or 1000-fold greater than the same rAAV particles in a reference formulation when stored for about 12 months, about 15 months, about 18 months, about 24 months, about 2 years, about 3 years, about 4 years, or about 5 years. In certain embodiments, the stability of the rAAV particles over a period of time is determined by the assay(s) disclosed in Section 6.8.

[0636] In certain embodiments, the rAAV particles in a stable formulation exhibit improved in vivo stability compared to the same rAAV particles in a reference formulation when stored at -80°C, -70°C, -20°C, 4°C, 20°C, 25°C, 30°C, 35°C, 37°C, or 40°C for a period of time, such as 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, about 15 months, about 18 months, about 24 months, about 2 years, about 3 years, about 4 years, or about 5 years. The in vitro relative potency (IVRP) is at least 2%, 5%, 7%, 10%, 12%, 15%, 17%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 100%, 2-fold, 3-fold, 5-fold, 10-fold, 100-fold, or greater than 1000. In certain embodiments, the in vitro relative potency (IVRP) of the rAAV particles is determined by the assay(s) disclosed in Section 6.8.

[0637] In certain embodiments, the rAAV particles in a stable formulation exhibit a reduced molecular weight (Mn) of in comparison to the same rAAV particles in a reference formulation when stored at -80°C, -70°C, -20°C, 4°C, 20°C, 25°C, 30°C, 35°C, 37°C, or 40°C for a period of time, such as at least about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, about 15 months, about 18 months, about 24 months, about 2 years, about 3 years, about 4 years, or about 5 years. The in vitro relative potency (IVRP) is at least 2%, 5%, 7%, 10%, 12%, 15%, 17%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 100%, 2-fold, 3-fold, 5-fold, 10-fold, 100-fold, or 1000-fold higher. In certain embodiments, the in vitro relative potency (IVRP) of the rAAV particles is determined by the assay(s) disclosed in Section 6.8.

[0638] In certain embodiments, the rAAV particles in a stable formulation exhibit improved in vivo stability compared to the same rAAV particles in a reference formulation when stored at -80°C, -70°C, -20°C, 4°C, 20°C, 25°C, 30°C, 35°C, 37°C, or 40°C for a period of time, e.g., about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, about 15 months, about 18 months, about 24 months, about 2 years, about 3 years, about 4 years, or about 5 years. The in vitro relative potency (IVRP) is at least 2%, 5%, 7%, 10%, 12%, 15%, 17%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 100%, 2-...

Claims

1. Recombinant adeno-associated virus (rAAV) particles; a) Tris, b) sugar, and c) a salt that does not crystallize during freezing of the formulation; A stable formulation comprising: The formulation is suitable for lyophilization; and The formulation, wherein the rAAV particles comprise AAV8 capsid proteins.

2. 2. The formulation of claim 1, wherein the salt has an ionic strength of 60 mM or greater.

3. The formulation of claim 1 or 2 having an ionic strength of less than 200 mM.

4. The formulation of any one of claims 1 to 3, wherein the salt has an ionic strength of from 60 mM to 150 mM.

5. The formulation comprises: a) 1 mM to 50 mM Tris, or b) Tris at 1 mM to 30 mM, 1 mM to 20 mM, 5 mM to 30 mM, 5 mM to 20 mM, 10 mM to 30 mM, 10 mM to 20 mM, or 20 mM to 50 mM, or c) 1 mM, 2 mM, 3 mM, 5 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, or 40 mM Tris The formulation according to any one of claims 1 to 4, comprising:

6. The formulation of claim 5, comprising 5 mM Tris.

7. The formulation of claim 1, wherein the salt is a multivalent salt containing one or more multiply charged ions.

8. The formulation of claim 1, wherein the salt is a citrate or sulfate.

9. 9. The formulation of any one of claims 1 to 8, wherein the salt is sodium citrate, sodium sulfate, ammonium sulfate, magnesium sulfate, or a combination thereof.

10. 10. The formulation of claim 1, wherein the formulation comprises 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, 100 mM, 120 mM, 140 mM, 150 mM, or 200 mM sodium sulfate.

11. The pH is, a) 6.5-8.0, b) 7.2 to 7.8, c) 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, or 7.8; d) 7.5, or e) 7.1 The formulation according to any one of claims 1 to 10, wherein

12. a) 50 mM to 400 mM sugar; b) 50 mM to 350 mM, 50 mM to 300 mM, 50 mM to 250 mM, 50 mM to 200 mM, or 50 mM to 150 mM sugar; c) 100 mM to 400 mM, 150 mM to 400 mM, 200 mM to 400 mM, 250 mM to 400 mM, or 300 mM to 400 mM sugar; d) 100 mM to 300 mM, 150 mM to 250 mM, 200 mM to 300 mM, or 250 mM to 350 mM sugar, or e) 50 mM, 100 mM, 150 mM, 160 mM, 170 mM, 180 mM, 190 mM, 200 mM, 210 mM, 220 mM, 230 mM, 240 mM, 250 mM, 260 mM, 270 mM, 280 mM, 290 mM, 300 mM, or 350 mM sugar.

12. The formulation of claim 1 , comprising:

13. The formulation of claim 12, comprising 190 mM to 230 mM, 170 mM to 250 mM, or 250 mM to 270 mM sugar.

14. The formulation of claim 13, comprising 210 mM sugar.

15. a) the sugar is a non-reducing sugar, or b) the sugar is a reducing sugar; 15. A formulation according to any one of claims 1 to 14.

16. The formulation of claim 15, wherein the non-reducing sugar is selected from the group consisting of sucrose, trehalose, and raffinose; or the reducing sugar is selected from the group consisting of glucose, fructose, mannose, galactose, and lactose.

17. 17. The formulation of any one of claims 1 to 16, comprising sucrose.

18. a) less than 100 mM sodium citrate; b) 10 mM to 100 mM sodium citrate; c) 10 mM to 100 mM, 20 mM to 100 mM, 30 mM to 100 mM, 40 mM to 100 mM, 50 mM to 100 mM, 10 mM to 80 mM, 10 mM to 60 mM, 10 mM to 50 mM, 10 mM to 40 mM, or 10 mM to 30 mM sodium citrate; d) 10 mM to 50 mM, 20 mM to 60 mM, 30 mM to 70 mM, or 10 mM to 30 mM sodium citrate, or e) 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, or 60 mM sodium citrate 18. The formulation of any one of claims 1 to 17, comprising:

19. The formulation of claim 18, comprising 20 mM sodium citrate.

20. moreover, a) 0.0005% to 0.01% of a non-ionic surfactant, or b) 0.002% non-ionic surfactant 20. The formulation of any one of claims 1 to 19, comprising:

21. 21. The formulation of claim 20, wherein the non-ionic surfactant comprises poloxamer 188, poloxamer 407, polysorbate 80, polysorbate 20, Pluronic F-68, or BRIJ 35.

22. 22. The formulation of any one of claims 1 to 21, further comprising a stabilizer selected from the group consisting of glycerol or sorbitol.

23. The formulation comprises: a) 0.1% to 5% glycerol; b) 0.1% to 2% glycerol, or c) 0.25% to 2% glycerol 23. The formulation of claim 22, comprising:

24. a) the formulation does not contain mannitol, or b) the formulation comprises less than 10 mM, 20 mM, 50 mM, 100 mM or 150 mM mannitol; 24. A formulation according to any one of claims 1 to 23.

25. A. a) 1 mM to 25 mM Tris; b) 50 mM to 400 mM sugar; c) 10 mM to 100 mM sodium citrate, and d) 0.0005% to 0.01% of a non-ionic surfactant and having a pH of 7.2 to 7.8; or B. a) 2 mM to 10 mM Tris; b) 150 mM to 250 mM sugar; c) 10 mM to 20 mM sodium citrate, and d) 0.001% to 0.005% of a non-ionic surfactant and having a pH of 7.2 to 7.8; 25. A formulation according to any one of claims 1 to 24.

26. A. a) 5 mM Tris; b) 210 mM sugar; c) 20 mM sodium citrate, and d) 0.002% non-ionic surfactant and having a pH of 7.5; B. a) 5 mM Tris; b) 210 mM sucrose; c) 20 mM sodium citrate, and d) 0.002% (w / v) Poloxamer 188 Contains or C. a) 5 mM Tris; b) 210 mM sucrose; c) 20 mM sodium citrate; d) 0.002% (w / v) poloxamer 188, and e) 0.25% (w / v) glycerol Contains or D. a) 5 mM Tris; b) 210 mM sucrose; c) 20 mM sodium citrate; d) 0.002% (w / v) poloxamer 188, and e) 0.5% (w / v) sorbitol Contains or E. a) 5 mM Tris; b) 30 mM sodium sulfate; c) 263 mM sucrose, and d) 0.005% (w / v) Poloxamer 188 26. The formulation of claim 25, comprising:

27. A. rAAV particles between 1.0E+11 genome copies / mL (GC / mL) and 1.0E+15 GC / mL, or B. rAAV particles of 1.0E+11GC / mL, 1.0E+12GC / mL, 1.0E+13GC / mL, 1.0E+14GC / mL, or 1.0E+15GC / mL 27. The formulation of any one of claims 1 to 26, comprising:

28. The remaining moisture content is a) 1% to 7%, b) 1% to 7%, 2% to 7%, 3% to 7%, 4% to 7%, 5% to 7%, 1% to 6%, 1% to 5%, 1% to 4%, or 1% to 3%, c) 3% to 7%, 3% to 6%, or 3% to 5%, d) 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, or 6%; e) 1% to 2%, f) 1.5%, 1.4%, 1.3%, 1.2%, or 1.1%, or g) 1% 28. The formulation of any one of claims 1 to 27, wherein

29. a) the glass transition temperature (Tg) of a lyophilized cake of said formulation is greater than 35° C.; or b) the glass transition temperature (Tg') of the maximum freeze-concentrated solution of said formulation is greater than -40°C; 29. A formulation according to any one of claims 1 to 28.

30. a) the % relative potency of the rAAV particles is at least 60%, at least 70%, or at least 80% after storage of the formulation at room temperature for 3 months, where the reference rAAV particles are stored in DPBS with 0.001% poloxamer 188 buffer at -70°C; b) the % relative potency of the rAAV particles is at least 60%, at least 70%, or at least 80% after storage of the formulation at room temperature for 6 months, where the reference rAAV particles are stored at -70°C in Dulbecco's Phosphate Buffered Saline (DPBS) with 0.001% Poloxamer 188 buffer; c) the % relative potency of the rAAV particles is at least 30%, at least 40%, at least 50%, or at least 60% after one week of storage of the formulation at 35° C., where the reference rAAV particles are stored in DPBS with 0.001% poloxamer 188 buffer at −70° C.; d) the % relative potency of the rAAV particles is at least 30%, at least 40%, at least 50%, or at least 60% after storage of the formulation for 2 weeks at 35° C., where the reference rAAV particles are stored in DPBS with 0.001% poloxamer 188 buffer at −70° C.; or e) the % relative potency of the rAAV particles is at least 30%, at least 40%, at least 50%, or at least 60% after storage of the formulation at 35° C. for 4 weeks, where the reference rAAV particles are stored at −70° C. in DPBS with 0.001% poloxamer 188 buffer; 30. A formulation according to any one of claims 1 to 29.

31. 31. The formulation of any one of claims 1 to 30, wherein the formulation is for lyophilization prior to storage.

32. The formulation of claim 31, wherein the formulation is for reconstitution after storage.

33. A. the % relative potency of the rAAV particles immediately after lyophilization is at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99%; B. The level of rAAV particle aggregation of the formulation is reduced by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% when compared to the level of rAAV particle aggregation in a reference formulation; or C. a) the stability of the formulation is assessed by vector genome content or viral titer assay, and the formulation has at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or 200% more genome content after storing the formulation for 1 day, 2 days, 3 days, 4 days, 5 days, 6 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, when compared to the genome content of a reference formulation stored under the same conditions; b) the stability of the formulation is assessed by measuring the relative potency of the formulation, wherein the formulation has a relative potency that is at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or 200% greater after storing the formulation for 1 day, 2 days, 3 days, 4 days, 5 days, 6 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, when compared to the genome content of a reference formulation stored under the same conditions; c) the stability of the formulation is assessed by loss of infectivity, and the formulation exhibits at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or 200% less loss in infectivity after storage of the formulation for 1 day, 2 days, 3 days, 4 days, 5 days, 6 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, when compared to the loss in infectivity of a reference formulation stored under the same conditions; or d) the stability of the formulation is assessed by rAAV genome release, which is determined by measuring relative fluorescence in the presence of a DNA-specific fluorescent stain, and the formulation has a relative fluorescence level that is at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or 200% lower after storing the formulation for 1 day, 2 days, 3 days, 4 days, 5 days, 6 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, when compared to the relative fluorescence level of a reference formulation stored under the same conditions; The reference formulation is (i) DPBS with 0.001% Poloxamer 188 buffer; (ii) a sugar-free formulation, or (iii) Plasticizer-free formulations 33. The formulation of any one of claims 1 to 32, wherein

34. a) the formulation is stored at −80° C., −70° C., −20° C., 4° C., 20° C., 25° C., 30° C., 35° C., or 40° C.; b) the formulation is frozen at a temperature of −20° C. in the process of lyophilization; c) the frozen formulation maintains a pH between pH 6 and pH 9 when frozen at −20° C.; d) the frozen formulation, when frozen to -20°C, maintains a pH value within plus or minus one unit of the pre-freezing pH value; or e) the formulation is a stabilized aqueous formulation of rAAV for lyophilization; 34. A formulation according to any one of claims 1 to 33.

35. 35. A method for producing a stable formulation comprising recombinant adeno-associated virus (rAAV) particles, the method comprising mixing rAAV particles with Tris, sugar, and salt of a formulation described in any one of claims 1 to 34, thereby producing the formulation comprising rAAV.

36. 1. A method for reducing rAAV genome release from rAAV particles, comprising: a) producing a formulation according to any one of claims 1 to 34, wherein rAAV genome release from the rAAV particles after three freeze-thaw cycles is reduced compared to rAAV genome release in a formulation that does not contain sugar; or b) producing a formulation according to any one of claims 1 to 34, wherein rAAV genome release from the rAAV particles after lyophilization and reconstitution is reduced compared to rAAV genome release in a formulation that does not contain sugar.

37. 35. A method for producing a stable lyophilized formulation of an rAAV product, comprising subjecting a pre-lyophilized formulation to lyophilization, wherein the pre-lyophilized formulation is one described in any one of claims 1 to 34.

38. 35. The formulation of any one of claims 1 to 34 for use in a method for treating or preventing a disease, the formulation being for lyophilization and reconstitution prior to administration to a subject in need thereof.

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