A scalable method for recombinant AAV generation

Abstract

Provided herein are improved methods for producing recombinant adeno-associated virus (rAAV) particles. In some embodiments, the methods for producing recombinant AAV (rAAV) particles provided herein comprise culturing cells capable of producing rAAV particles in the presence of a histone deacetylase (HDAC) inhibitor. In some embodiments, the methods for producing recombinant AAV (rAAV) particles provided herein comprise culturing cells capable of producing rAAV particles in the presence of a histone deacetylase (HDAC) inhibitor and an increased amount of a sodium salt. [Selection diagram] Figure 4

Description

Technical Field

[0001] Cross - reference to related applications This application claims priority to U.S. Provisional Application No. 62 / 717,212, filed on August 10, 2018, the entire disclosure of which is incorporated herein by reference.

Background Art

[0002] Recombinant adeno - associated virus (rAAV) - based vectors are currently the most widely used developing gene therapy products. The reasons for the preference for the use of rAAV vector systems are, in part, the absence of diseases associated with wild - type viruses, the ability of AAV transduction in both non - dividing and dividing cells, and the observation of long - term robust transgene expression in clinical trials, which indicates great potential for delivery in gene therapy applications. In addition, various natural AAV and recombinant AAV vector serotypes specifically target various tissues, organs, and cells and help avoid any existing immunity to the vector, thus expanding the therapeutic applications of AAV - based gene therapy.

[0003] Histone deacetylase (HDAC) inhibitors are used in translocation protocols for protein expression in recombinant animal cell cultures. For example, Vazquez-Lombardi exemplify the use of HDAC inhibitors ("Enhancer 1 and 2") as co-translocation reagents with an IgG-expressing plasmid, and additionally, the addition of enhancers to the culture on day 2 (Vazquez-Lombardi et al., Nature Protocols, 13(1):99-117 (2018) (Non-Patent Literature 1), published online on December 14, 2017). WO2013166339A1 describes the use of enhancer 1 (valproic acid) and enhancer 2 (sodium propionate) under small-scale culture conditions of less than 50 L, and it has been observed that enhancer 2 alone does not have a strong effect, but when combined with enhancer 1, it can be beneficial for recombinant IgG production. Chun reported that the use of propionic acid and butyrate promoted the production of recombinant B-domain-deficient factor VIII by CHO cells, although both alkanates inhibited cell growth, and rFVIII production reached its peak at approximately 3.5 days (Chun et al., Biotechnology Letters, 25:315-319 (2003) (Non-Patent Literature 2)). Cervera reported using a mixture of transfection enhancers to increase the production of virus-like particles containing a single recombinant polypeptide (e.g., Gag-like virus-like particles) in HEK293 suspension cell culture (Cervera et al, Appl. Microbiol. Biotechnol., 99:9935-9949 (2015) (Non-Patent Literature 3)). However, none of these reports disclose the use of histone deacetylase (HDAC) inhibitors to increase the recombinant generation of viral particles that capsid the genome (for example, cultured cells expressing rAAV particles containing multiple polypeptides and nucleotide genomes).

[0004] Tiernan and Tipper have disclosed the use of the HDAC inhibitor trichostatin A in a method for producing a stable cell line containing the rAAV transgene (WO2018 / 175775 (Patent Document 1)). Tiernan and Tipper have disclosed that a method comprising simultaneous transfection of a recombinant viral vector and an HDAC inhibitor may enhance the integration of the viral vector into the host cell genome and increase the yield of viral vectors collected from host cells. While Tiernan and Tipper have disclosed the use of a stable cell line for the production of rAAV particles, they have neither taught nor suggested the use of an HDAC inhibitor in the rAAV particle production culture.

[0005] Before AAV-based gene therapy can be widely adopted in late-stage clinical and commercial use, it is necessary to develop new methods for large-scale, GMP-compliant rAAV particle production. The main challenge in developing upstream processes is establishing scalable, cost-effective, and GMP-compliant methods for generating rAAV. Using currently approved methods, producing a single unit dose of rAAV particles can cost hundreds of thousands of dollars. Therefore, a GMP-compliant, scalable process for generating rAAV particles is needed. [Prior art documents] [Patent Documents]

[0006] [Patent Document 1] WO2018 / 175775 [Non-patent literature]

[0007] [Non-Patent Document 1] Vazquez-Lombardi et al.,Nature Protocols,13(1):99-117(2018) [Non-Patent Document 2] Chun et al.,Biotechnology Letters,25:315-319(2003) [Non-Patent Document 3] Cervera et al,Appl.Microbiol.Biotechnol.,99:9935-9949(2015) [Overview of the project]

[0008] This disclosure provides a method for producing recombinant AAV (rAAV) particles, comprising culturing cells capable of producing rAAV particles in the presence of an effective amount of a histone deacetylase (HDAC) inhibitor under conditions that enable the production of rAAV particles. In some embodiments, the cells are cultured in the presence of the HDAC inhibitor and a sodium salt at a concentration between about 110 mM and 250 mM. In some embodiments, the method disclosed herein comprises isolating the rAAV particles produced according to the method disclosed herein. In some embodiments, the method disclosed herein comprises collecting the cell culture, clarifying the collected cell culture (e.g., by centrifugation or deep filtration), tangential flow filtration, affinity chromatography, anion exchange chromatography, cation exchange chromatography, size exclusion chromatography, hydrophobic interaction chromatography, sterile filtration, or any combination thereof. In some embodiments, the method disclosed herein does not involve centrifugation. In some embodiments, the methods disclosed herein include collecting a cell culture, clarifying the collected cell culture by deep filtration, a first sterile filtration, a first tangential flow filtration, affinity chromatography, anion exchange chromatography (e.g., monolithic anion exchange chromatography), a second tangential flow filtration, and a second sterile filtration. In some embodiments, a method for isolating rAAV particles produced according to the methods disclosed herein includes clarifying the collected cell culture by deep filtration, a first sterile filtration, a first tangential flow filtration, affinity chromatography, anion exchange chromatography (e.g., monolithic anion exchange chromatography), a second tangential flow filtration, and a second sterile filtration.

[0009] In some embodiments, this disclosure provides the following: [1.] A method for producing rAAV particles, (a) Prepare a cell culture containing cells, (b) The cells are given the following: i. rAAV genome to be packaged, ii. Adenovirus helper function necessary for packaging, iii. Sufficient AAV rep protein for packaging, and iv. Sufficient AAV cap protein for packaging Introducing one or more polynucleotides that encode at least one of the following, (c) Adding an HDAC inhibitor to the cell culture to a final concentration between approximately 0.1 mM and approximately 20 mM, (d) After (b), maintain the cell culture under conditions that enable the generation of the rAAV particles for a period of approximately 2 to 15 days. The method, including the method described above. [2.] The method according to [2], wherein the HDAC inhibitor is a short-chain fatty acid or a salt thereof. [3.] The method according to [1] or [2], wherein the HDAC inhibitor is valproic acid, propionic acid, butyric acid, or a salt thereof. [4.] The method according to [3], wherein the HDAC inhibitor is sodium valproate. [5.] The method according to [3], wherein the HDAC inhibitor is sodium propionate. [6.] The method according to any one of [1] to [5], wherein the cell culture has a final HDAC inhibitor concentration between approximately 0.5 mM and approximately 5 mM. [7.] The method according to any one of [1] to [5], wherein the cell culture has a final HDAC inhibitor concentration between approximately 0.5 mM and approximately 3 mM. [8.] The method according to any one of [1] to [7], wherein the HDAC inhibitor is added after step b). [9.] The method according to [8], wherein the HDAC inhibitor is added approximately 1 hour to approximately 48 hours after step b). [10.] The method according to [8], wherein the HDAC inhibitor is added approximately 12 hours to approximately 36 hours after step b). [11.] The method according to [8], wherein the HDAC inhibitor is added approximately 18 to 30 hours after step b). [12.] The method according to [8], wherein the HDAC inhibitor is added before approximately 48 hours after step b). [13.] The method according to [8], wherein the HDAC inhibitor is added before approximately 36 hours after step b). [14.] The method according to [8], wherein the HDAC inhibitor is added at least about 6 hours after step b). [15.] The method according to [8], wherein the HDAC inhibitor is added at least about 12 hours after step b). [16.] The method according to [8], wherein the HDAC inhibitor is added about 6 hours, about 9 hours, about 12 hours, about 18 hours, about 20 hours, about 24 hours, about 30 hours, about 36 hours, or about 48 hours after step b). [17.] The method according to [8], wherein the HDAC inhibitor is added approximately 20 hours after step b). [18.] The method according to [8], wherein the HDAC inhibitor is added approximately 24 hours after step b). [19.] The method according to any one of [1] to

[18] , further comprising adding to the culture an amount of the sodium salt sufficient to increase the final concentration of the sodium salt by a range of about 20 mM to about 150 mM. [20.] The method according to

[19] , wherein the final concentration of the sodium salt is increased by a range of approximately 20 mM to approximately 50 mM, approximately 40 mM to approximately 80 mM, or approximately 70 mM to approximately 120 mM. [21.] The method according to

[19] wherein the final concentration of the sodium salt is increased by only between approximately 40 mM and approximately 140 mM. [22.] The method according to any one of [1] to

[21] , further comprising adding to the culture an amount of the sodium salt sufficient to increase the final concentration of the sodium salt to between about 120 mM and about 250 mM. [23.] The method according to

[22] , wherein the final concentration of the sodium salt is between about 130 mM and about 160 mM, between about 150 mM and about 190 mM, or between about 180 mM and about 240 mM. [24.] The method according to

[22] , wherein the final concentration of the sodium salt is between about 150 mM and about 240 mM. [25.] The method according to

[22] , wherein the cell culture contains between about 90 mM and about 120 mM of NaCl before adding the sodium salt. [26.] The method according to any one of

[19] to

[25] , wherein the sodium salt is sodium chloride. [27.] The method according to any one of

[19] to

[26] , wherein the HDAC inhibitor and the sodium salt are added separately in any order. [28.] The method according to any one of

[19] to

[27] , wherein the sodium salt is added before b). [29.] The method according to any one of

[19] to

[27] , wherein the sodium salt is added after b). [30.] The method according to any one of

[19] to

[29] , wherein the sodium salt is added after adding the HDAC inhibitor. [31.] The method according to

[30] , wherein the sodium salt is added from about 5 minutes to about 6 hours after adding the HDAC inhibitor. [32.] The method according to

[30] , wherein the sodium salt is added from about 20 minutes to about 2 hours after adding the HDAC inhibitor. [33.] The method according to

[30] , wherein the sodium salt is added before about 2 hours after adding the HDAC inhibitor. [34.] The method according to

[30] , wherein the sodium salt is added before about 1 hour after adding the HDAC inhibitor. [35.] The method according to

[30] , wherein the sodium salt is added at least about 5 minutes after adding the HDAC inhibitor. [36.] The method according to

[30] , wherein the sodium salt is added at least about 20 minutes after adding the HDAC inhibitor. [37.] The method according to any one of [1] to

[36] , wherein the cell culture is maintained for about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, or about 7 days after b). [38.] The method according to

[37] , wherein the cell culture is maintained for about 5 days after b). [39.] The method according to any one of [1] to

[38] , comprising introducing into the cell one or more polynucleotides encoding the following: i. The rAAV genome to be packaged, ii. Adenovirus helper functions necessary for packaging, iii. Sufficient AAV rep proteins for packaging, and iv. Sufficient AAV cap proteins for packaging [40.] The method according to any one of [1] to

[39] , wherein the adenovirus helper functions include at least one of the adenovirus E1a gene, E1b gene, E4 gene, E2a gene, and VA gene. [41.] The method according to any one of [1] to

[40] , wherein introducing the one or more polynucleotides into the cell is performed by transfection. [42.] The method according to any one of [1] to

[41] , wherein the cell is a mammalian cell. [43.] The method according to any one of [1] to

[41] , wherein the cell is an insect cell. [44.] The method according to any one of [1] to

[41] , wherein the cell is a HEK293 cell, a HEK-derived cell, a CHO cell, a CHO-derived cell, a HeLa cell, a SF-9 cell, a BHK cell, a Vero cell, or a PerC6 cell. [45.] The method according to any one of [1] to

[41] , wherein the cell is a HEK293 cell. [46.] The method according to any one of [1] to

[45] , wherein the cell culture is a suspension culture. [47.] The method according to any one of [1] to

[46] , further comprising recovering the rAAV particles. [48.] The method according to any one of [1] to

[47] wherein the cell culture generates rAAV particles exceeding 5 × 10 e + 10 GC / ml. [49.] The method according to any one of [1] to

[48] , wherein the cell culture generates at least about twice as many rAAV particles as measured as GC / ml compared to a culture without the addition of the HDAC inhibitor and sodium salt. [50.] The method according to any one of [1] to

[49] , wherein the cell culture has a volume between approximately 50 liters and approximately 20,000 liters. [51.] A method for producing rAAV particles, (a) Prepare a cell culture containing cells capable of producing rAAV, (b) Adding an HDAC inhibitor to the cell culture to a final concentration between approximately 0.1 mM and approximately 20 mM, (c) Maintaining the cell culture under conditions that enable the generation of the rAAV particles. The method, including the method described above. [52.] The method according to

[51] , wherein the HDAC inhibitor is a short-chain fatty acid or a salt thereof. [53.] The method according to

[52] , wherein the HDAC inhibitor is valproic acid, propionic acid, butyric acid, or a salt thereof. [54.] The method according to

[53] , wherein the HDAC inhibitor is sodium propionate. [55.] The method according to

[53] , wherein the HDAC inhibitor is sodium valproate. [56.] The method according to any one of

[51] to

[55] , wherein the cell culture has a final HDAC inhibitor concentration between approximately 0.5 mM and approximately 5 mM. [57.] The method according to any one of

[51] to

[55] , wherein the cell culture has a final HDAC inhibitor concentration between approximately 0.5 mM and approximately 3 mM. [58.] The method according to any one of

[51] to

[57] , further comprising adding to the culture an amount sufficient to increase the final concentration of the sodium salt by about 20 mM to 150 mM. [59.] The method according to

[58] , wherein the final concentration of the sodium salt is increased by a range of approximately 20 mM to approximately 50 mM, approximately 40 mM to approximately 80 mM, or approximately 70 mM to approximately 120 mM. [60.] The method according to

[58] , wherein the final concentration of the sodium salt is increased by only between approximately 40 mM and approximately 140 mM. [61.] The method according to any one of

[51] to

[60] , further comprising adding to the culture an amount of the sodium salt sufficient to increase the final concentration of the sodium salt to between about 120 mM and about 250 mM. [62.] The method according to

[61] , wherein the final concentration of the sodium salt is between approximately 130 mM and approximately 160 mM, between approximately 150 mM and approximately 190 mM, or between approximately 180 mM and approximately 240 mM. [63.] The method according to

[61] , wherein the final concentration of the sodium salt is between approximately 150 mM and approximately 240 mM. [64.] The method according to any one of

[51] to

[63] , wherein the cell culture contains NaCl between approximately 90 mM and approximately 120 mM before the addition of the sodium salt. [65.] The method according to any one of

[51] to

[64] , wherein the sodium salt is sodium chloride. [66.] The method according to any one of

[51] to

[65] , wherein the HDAC inhibitor and the sodium salt are added separately in any order. [67.] The method according to

[66] , wherein the sodium salt is added after the addition of the HDAC inhibitor. [68.] The method according to

[67] , wherein the sodium salt is added approximately 5 minutes to 6 hours after the addition of the HDAC inhibitor. [69.] The method according to

[67] , wherein the sodium salt is added approximately 20 minutes to approximately 2 hours after the addition of the HDAC inhibitor. [70.] The method according to

[67] , wherein the sodium salt is added no more than approximately 2 hours after the addition of the HDAC inhibitor. [71.] The method according to

[67] , wherein the sodium salt is added no more than approximately one hour after the addition of the HDAC inhibitor. [72.] The method according to

[67] , wherein the sodium salt is added at least about 5 minutes after the addition of the HDAC inhibitor. [73.] The method according to

[67] , wherein the sodium salt is added at least about 20 minutes after the addition of the HDAC inhibitor. [74.] The method according to any one of

[51] to

[73] , wherein the cell culture is maintained under conditions that allow for the generation of the rAAV particles for a period of about 2 to about 10 days or for a period of about 5 to 14 days after b). [75.] The method according to any one of

[51] to

[73] , wherein the cell culture is maintained for about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, or about 7 days after b). [76.] The method according to

[75] , wherein the cell culture is maintained for about 5 days after b). [77.] A method for generating rAAV particles, comprising culturing cells capable of generating rAAV particles in a medium containing an HDAC inhibitor in a concentration of about 0.1 mM to about 20 mM under conditions that enable the generation of the rAAV particles. [78.] The method according to

[77] , wherein the HDAC inhibitor is a short-chain fatty acid or a salt thereof. [79.] The method according to

[78] , wherein the HDAC inhibitor is valproic acid, propionic acid, butyric acid, or a salt thereof. [80.] The method according to

[79] , wherein the HDAC inhibitor is sodium valproate. [81.] The method according to

[79] , wherein the HDAC inhibitor is sodium propionate. [82.] The method according to any one of

[77] to

[81] , wherein the culture medium contains the HDAC inhibitor in a concentration between approximately 0.5 mM and approximately 5 mM. [83.] The method according to any one of

[77] to

[81] , wherein the culture medium contains the HDAC inhibitor in a concentration between approximately 0.5 mM and approximately 3 mM. [84.] The method according to any one of

[77] to

[83] , wherein the culture medium further comprises sodium chloride in a concentration between approximately 120 mM and approximately 250 mM. [85.] The method according to any one of

[77] to

[83] , wherein the culture medium further comprises sodium chloride (NaCl) in a concentration between approximately 130 mM and approximately 160 mM, between approximately 150 mM and approximately 190 mM, or between approximately 180 mM and approximately 240 mM. [86.] The method according to any one of

[77] to

[83] , wherein the culture medium further comprises sodium chloride in a concentration between approximately 150 mM and approximately 240 mM. [87.] To the cells capable of generating rAAV, the following: (a) rAAV genome to be packaged, (b) Adenovirus helper function required for packaging, (c) Sufficient AAV rep protein for packaging, and (d) Sufficient AAV cap protein to package The method according to any one of

[51] to

[86] , wherein one or more polynucleotides encoding at least one of the following are transfected. [88.] To the cells capable of generating rAAV, the following: (a) rAAV genome to be packaged, (b) Adenovirus helper function required for packaging, (c) Sufficient AAV rep protein for packaging, and (d) Sufficient AAV cap protein to package A method according to any one of

[51] to

[86] , wherein one or more polynucleotides encoding are transfected. [89.] The method according to any one of

[51] to

[88] , wherein the cells are mammalian cells or insect cells. [90.] The method according to any one of

[51] to

[88] , wherein the cells are HEK293 cells, HeLa cells, SF-9 cells, BHK cells, Vero cells, or PerC6 cells, and optionally the cells are HEK293 cells. [91.] The method according to any one of

[51] to

[90] , wherein the cell culture is a suspension culture. [92.] The method according to any one of

[77] to

[91] , wherein the culturing under conditions that enable the generation of the rAAV particles is carried out over a period of about 2 to about 10 days or over a period of about 5 to 14 days. [93.] The method according to any one of

[77] to

[91] , wherein the culturing under conditions that enable the generation of the rAAV particles is carried out for about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, or about 7 days. [94.] The method according to

[93] , wherein the culturing under conditions that enable the generation of the rAAV particles is carried out for about 5 days. [95.] The method according to any one of

[51] to

[94] , further comprising recovering the rAAV particles. [96.] The method according to any one of

[51] to

[95] , wherein the cell culture generates rAAV particles between approximately 5 × 10 e + 10 GC / ml and approximately 1 × 10 e + 12 GC / ml. [97.] The method according to any one of

[51] to

[96] , wherein the cell culture generates at least about twice as many rAAV particles as measured as GC / ml compared to a culture without the addition of the HDAC inhibitor and sodium salt. [98.] A method for increasing the production of rAAV particles (a) Prepare a cell culture containing cells, (b) The cells are given the following: i. rAAV genome to be packaged, ii. Adenovirus helper function necessary for packaging, iii. Sufficient AAV rep protein for packaging, and iv. Sufficient AAV cap protein for packaging Introducing one or more polynucleotides that encode at least one of the following, (c) Adding an HDAC inhibitor to the cell culture to a final concentration between approximately 0.1 mM and approximately 20 mM, (d) After (b), maintain the cell culture under conditions that enable the generation of the rAAV particles for a period of approximately 2 to 15 days. The method, including the method described above. [99.] The method according to

[98] , wherein the HDAC inhibitor is sodium valproate. [100.] The method according to

[98] , wherein the HDAC inhibitor is sodium propionate. [101.] The method according to any one of

[98] to

[0100] , further comprising adding to the culture an amount of the sodium salt sufficient to increase the final concentration of the sodium salt by a range of about 40 mM to about 150 mM. [102.] The method according to any one of

[98] to

[0101] , wherein the sodium salt is sodium chloride. [103.] A method for increasing the production of rAAV particles (a) Prepare a cell culture containing cells capable of producing rAAV, (b) Adding an HDAC inhibitor to the cell culture to a final concentration between approximately 0.1 mM and approximately 20 mM, (c) Maintaining the cell culture under conditions that enable the generation of the rAAV particles. The method, including the method described above. [104.] The method according to

[0103] , wherein the HDAC inhibitor is sodium valproate. [105.] The method according to

[0103] , wherein the HDAC inhibitor is sodium propionate. [106.] The method according to any one of

[0103] to

[0105] , further comprising adding to the culture an amount of the sodium salt sufficient to increase the final concentration of the sodium salt to between approximately 120 mM and approximately 250 mM. [107.] The method according to any one of

[0103] to

[0106] , wherein the sodium salt is sodium chloride. [108.] The method according to any one of

[0103] to

[0107] , wherein the cell culture is maintained under conditions that allow for the generation of the rAAV particles for a period of about 2 to about 10 days or for a period of about 5 to 14 days after b). [109.] The method according to any one of

[0103] to

[0107] , wherein the cell culture is maintained for approximately 2 days, approximately 3 days, approximately 4 days, approximately 5 days, approximately 6 days, or approximately 7 days after b). [110.] The method according to

[0109] , wherein the cell culture is maintained for approximately 5 days after b). [111.] A method for increasing the generation of rAAV particles, comprising culturing cells capable of generating rAAV particles in a medium containing an HDAC inhibitor in a concentration of about 0.1 mM to about 20 mM under conditions that enable the generation of the rAAV particles. [112.] The method according to

[0111] , wherein the HDAC inhibitor is sodium valproate. [113.] The method according to

[0111] , wherein the HDAC inhibitor is sodium propionate. [114.] The method according to any one of

[0111] to

[0113] , wherein the culture medium further comprises sodium chloride in a concentration between approximately 120 mM and approximately 250 mM. [115.] The rAAV particles are AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, and AAV16, AAV .rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B, AAV2.5, The method according to any one of [1] to

[0114] , comprising a capsid protein of the AAV2tYF, AAV3B, AAV.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 serotype. [116.] The method according to any one of [1] to

[0114] , wherein the rAAV particles contain a capsid protein of the AAV8, AAV9, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, or AAV.hu37 serotype. [117.] The method according to any one of [1] to

[0114] , wherein the rAAV particles comprise a capsid protein of the AAV8 or AAV9 serotype. [118.] The method according to any one of [1] to

[0117] , wherein the cell culture has a volume between approximately 50 liters and approximately 20,000 liters. [119.] The method according to

[0118] , wherein the cell culture has a volume between approximately 50 liters and approximately 5,000 liters. [120.] The method according to

[0118] , wherein the cell culture has a volume between approximately 50 liters and approximately 2,000 liters. [121.] The method according to

[0118] , wherein the cell culture has a volume between approximately 50 liters and approximately 1,000 liters. [122.] The method according to

[0118] , wherein the cell culture has a volume between approximately 50 liters and approximately 500 liters. A composition comprising isolated rAAV particles produced by the method described in any one of [1] to

[0122] . [124.] The method according to any one of [1]~

[50] or

[98] ~

[0102] , wherein the packaged rAAV genome contains the transgene. [125.] The method according to

[0124] , wherein the introduced gene comprises a regulatory element responsively linked to a polynucleotide encoding a polypeptide. [126.] The method according to

[0125] , wherein the regulating element comprises one or more of an enhancer, a promoter, and a poly-A region. [127.] The method according to

[0125] or

[0126] , wherein the regulatory element and the polynucleotide encoding the polypeptide are heterogeneous. [128.] The method according to any one of

[0124] to

[0127] , wherein the transgene encodes an anti-VEGF Fab, iduronidase (IDUA), iduronate 2-sulfatase (IDS), low-density lipoprotein receptor (LDLR), tripeptidyl peptidase 1 (TPP1), or a non-membrane-bound splice variant of VEGF receptor 1 (sFlt-1). [129.] The aforementioned transgenes include gamma-sarcoglycan, Rab escort protein 1 (REP1 / CHM), retinoid isomerohydrolase (RPE65), cyclic nucleotide gate channel alpha-3 (CNGA3), cyclic nucleotide gate channel beta-3 (CNGB3), aromatic L-amino acid decarboxylase (AADC), lysosome-associated membrane protein 2 isoform B (LAMP2B), factor VIII, factor IX, retinitis pigmentosa GTPase regulator (RPGR), retinosuxin (RS1), and sarcovesicles. Somatic calcium ATPase (SERCA2a), aflibercept, battenin (CLN3), transmembrane ER protein (CLN6), glutamate decarboxylase (GAD), glial cell line-derived neurotrophic factor (GDNF), aquaporin 1 (AQP1), dystrophin, myotubularin 1 (MTM1), follistatin (FST), glucose-6-phosphatase (G6Pase), apolipoprotein A2 (APOA2), uridine diphosphate glucuronosyltransferase 1A1 (UGT1A1), aryl sulf Atase B (ARSB), N-acetyl-alpha-glucosaminidase (NAGLU), alpha-glucosidase (GAA), alpha-galactosidase (GLA), beta-galactosidase (GLB1), lipoprotein lipase (LPL), alpha-1-antitrypsin (AAT), phosphodiesterase 6B (PDE6B), ornithine carbamoyltransferase 9OTC), survival motor neuron (SMN1), survival motor neuron (SMN2), neurturin (NRTN), neurotrophin-3 (N The method according to any one of items

[0124] to

[0127] , encoding T-3 / NTF3), porphobilinogen deaminase (PBGD), nerve growth factor (NGF), mitochondrial code NADH:ubiquinone oxidoreductase core subunit 4 (MT-ND4), protective protein cathepsin A (PPCA), dysferrin, MER proto-oncogene tyrosine kinase (MERTK), cystic fibrosis membrane conductance regulator (CFTR), or tumor necrosis factor receptor (TNFR)-immunoglobulin (IgG1) Fc fusion.

[0010] In some embodiments, the methods disclosed herein further include downstream processing of rAAV particles produced according to any one of the methods of [1] to

[0129] . In some embodiments, the downstream processing is at least one of the following: collection of cell cultures, clarification of the collected cell cultures (e.g., by centrifugation or deep 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 upstream processing includes at least two, at least three, at least four, at least five, or at least six of the following: collection of cell cultures, clarification of the collected cell cultures (e.g., by centrifugation or deep 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 downstream processing does not include centrifugation of the collected cell cultures. [Brief explanation of the drawing]

[0011] [Figure 1] Yield of recombinant virus obtained after the addition of sodium chloride, sodium butyrate, and / or sodium valproate. The final concentrations of the added reagents are shown. The basic medium contained approximately 100 mM sodium chloride, but did not contain sodium butyrate or sodium valproate. [Figure 2] Yield of recombinant virus obtained after the addition of sodium chloride and / or sodium valproate. The final concentration of the added reagent is shown. The basic medium contained approximately 100 mM sodium chloride, but did not contain sodium valproate. [Figure 3] Virus yield from large-scale production of rAAV8 particles using NaCl and sodium propionate. [Figure 4] Yield of rAAV9 obtained after the addition of sodium chloride and / or sodium propionate. [Figure 5] Yield of rAAV9 obtained after the addition of sodium chloride and / or sodium propionate. [Modes for carrying out the invention]

[0012] Detailed explanation In some embodiments, the Disclosure provides a method for producing rAAV particles by culturing cells capable of producing rAAV particles in the presence of an effective amount of a histone deacetylase (HDAC) inhibitor under conditions that produce rAAV particles. In some embodiments, the cells are cultured in the presence of the HDAC inhibitor and a sodium salt at a concentration between approximately 110 mM and 250 mM. The rAAV particles produced by the method disclosed herein are suitable for further downstream processing by collecting and purifying the rAAV particles to produce, for example, isolated rAAV particles and compositions containing them, such as pharmaceutical compositions. The method described provides a flexible, cost-effective, and commercially scalable process that conforms to GMP regulatory requirements for the production of rAAV particles for use in gene therapy applications. The methods described herein are not limited to AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, and AAV16, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B, and AAV2 The method is suitable for any rAAV serotype, including AAV2tYF, AAV3B, AAV.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, as well as their derivatives, modifiers, or pseudotypes. In some embodiments, the method is used to produce rAAV8 particles. In some embodiments, the method is used to produce rAAV8 derivative particles, rAAV8 modifier particles, or rAAV8 pseudotype particles. In some embodiments, the method is used to produce rAAV9 particles.In some embodiments, the method is used to generate rAAV9 derivative particles, rAAV9 modified particles, or rAAV9 pseudotype particles.

[0013] The inventors were surprised to discover that the method disclosed herein more than doubles the rAAV yield. Such results could not have been predicted based on previous knowledge that HDAC inhibitors can increase recombinant polypeptide expression in transfected cells. For cells to produce AAV particles, three capsid proteins and a single-stranded nucleotide genome must be assembled to form a functional viral unit. There was no reason to think that HDAC inhibitors could simultaneously increase the production of all AAV components, including the production of the AAV genome. Furthermore, even if HDAC inhibitors increased the production of viral polypeptides, there was no reason to think that the host cell mechanisms could assemble the increased number of AAV particles. Furthermore, Cervera's report that transfection enhancers can increase the generation of virus-like particles (VLPs) did not provide a reason to believe that HDAC inhibitors could increase rAAV particle generation in host cells, since VLPs contain a single gag protein and no genome (Cervera et al, Appl. Microbiol. Biotechnol., 99: 9935-9949 (2015)). Thus, the generation of VLPs did not require the assembly of multiple polypeptides and nucleotide genomes.

[0014] Given that a very large number of rAAV particles are required to prepare a single unit dose, a more than doubling of rAAV yield would significantly reduce the product cost per unit dose. Increased virus yield would, in turn, reduce not only the cost of consumables required for AAV particle production, but also the capital investment costs associated with constructing industrial virus purification facilities.

[0015] definition Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art in which this disclosure relates. To facilitate understanding of the methods of this disclosure, several terms and expressions are defined below.

[0016] For example, modifying the amount of a component in a composition, the concentration of a component in a composition, the flow rate, the rAAV particle yield, the feed volume, the salt concentration, similar values, and ranges thereof, “about” as used in the methods provided herein means variations in numerical quantities that may result from, for example, typical measurement and handling procedures used to prepare a concentrate or working solution; accidental errors in these procedures; differences in the manufacture, source, or purity of the components used in preparing the composition or performing the method; and similar considerations. The term “about” also includes amounts that differ as a composition or mixture having a particular initial concentration ages. The term “about” also includes amounts that differ as a composition or mixture having a particular initial concentration is mixed or processed. Whether modified by the term “about,” a claim includes an equivalent of that quantity. In some embodiments, the term “about” means a range of about 10 to 20 percent more or less than the number or range indicated. In further embodiments, “about” means plus or minus 10 percent of the number or range indicated. For example, “about 10%” indicates a range of 9% to 11%.

[0017] "AAV" is an abbreviation for adeno-associated virus and can be used to refer to the virus itself or its modifiers, derivatives, or pseudotypes. Unless otherwise required, the term encompasses all subtypes, as well as both the native and recombinant forms. The abbreviation "rAAV" refers to recombinant adeno-associated virus. The term "AAV" includes AAV type 1 (AAV1), AAV type 2 (AAV2), AAV type 3 (AAV3), AAV type 4 (AAV4), AAV type 5 (AAV5), AAV type 6 (AAV6), AAV type 7 (AAV7), AAV type 8 (AAV8), AAV type 9 (AAV9), avian AAV, bovine AAV, canine AAV, equine AAV, primate AAV, non-primate AAV, and sheep AAV, as well as their modifiers, derivatives, or pseudotypes. "Primate AAV" means AAV that infects primates, "non-primate AAV" means AAV that infects non-primate mammals, "bovine AAV" means AAV that infects bovine mammals, and so on. In some embodiments, the AAV particles are AAV1, AAV2, rAAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, and AAV16, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV .7m8, AAV.PHP.B, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AA V.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, or AAV.HSC16.In some embodiments, rAAV particles are AAV1, AAV2, rAAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, and AAV16, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV. It is a derivative, modifier, or pseudotype of PHP.B, AAV2.5, AAV2tYF, AAV3B, AAV.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.

[0018] The term "recombination" as applied to AAV particles means that the AAV particle is the product of one or more procedures that result in an AAV particle construct that is essentially different from the AAV particle itself.

[0019] Recombinant adeno-associated virus particles, or "rAAV particles," refer to viral particles composed of at least one AAV capsid protein and a capsidized polynucleotide rAAV vector containing heterologous polynucleotides (i.e., polynucleotides other than the wild-type AAV genome, e.g., a transgene to be delivered to mammalian cells). rAAV particles can be any AAV serotype (e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, or AAV10, or their derivatives / modifiers / pseudotypes), including any modifiers, derivatives, or pseudotypes. Such AAV serotypes and derivatives / modifiers / pseudotypes, as well as methods for generating such serotypes / derivatives / modifiers / pseudotypes, are known in the art (see, for example, Asokan et al., Mol.Ther. 20(4):699-708 (2012)). In some embodiments, the rAAV particles are AAV1, AAV2, rAAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, and AAV16, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B It contains capsid proteins from AAV capsid serotypes selected from AAV2.5, AAV2tYF, AAV3B, AAV.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, rAAV particles are AAV1, AAV2, rAAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, and AAV16, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B, AAV2. 5. Capsid proteins that are derivatives, modifiers, or pseudotypes of 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.

[0020] The rAAV particles of this disclosure may be any serotype or any combination of serotypes (for example, a population of rAAV particles containing two or more serotypes (for example, two or more of rAAV2, rAAV8, 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 two or more combinations thereof. In some embodiments, the rAAV particles are rAAV8 or rAAV9 particles. In some embodiments, rAAV particles are AAV1, AAV2, rAAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, and AAV16, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP. It contains capsid proteins from two or more serotypes selected from B, AAV2.5, AAV2tYF, AAV3B, AAV.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, rAAV particles are AAV1, AAV2, rAAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, and AAV16, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B, AAV2.5, AAV2t It comprises two or more serotype derivatives, modifiers, or pseudotypes of capsid proteins selected from YF, AAV3B, AAV.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 capsid proteins.

[0021] In some embodiments, the rAAV particles have AAV capsid proteins of a serotype selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, and AAV16, or their derivatives, modifiers, or pseudotypes. In some embodiments, the rAAV particles have AAV capsid proteins of a serotype selected from the group consisting of AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.PHB, and AAV.7m8. In some embodiments, the rAAV particles have AAV capsid proteins with high sequence homology to AAV8 or AAV9, such as AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, and AAV.hu37.

[0022] The term “cell culture” refers to cells grown attached to or in suspension, bioreactors, roller bottles, hyperstacks, microspheres, macrospheres, flasks, etc., as well as the components of the supernatant or suspension itself, including, but not limited to, rAAV particles, cells, cell debris, cell contaminants, colloidal particles, biomolecules, host cell proteins, nucleic acids, lipids, and flocculants. Large-scale approaches such as bioreactors (including suspension cultures and attached cells grown attached to microcarriers or macrocarriers in agitated bioreactors) are also encompassed by the term “cell culture.” Cell culture procedures for both large-scale and small-scale protein production are also included in this disclosure.

[0023] As used herein, the terms “purify,” “remove,” “separate,” “separate,” “separate,” “isolate,” or “isolate” mean increasing the degree of purity of rAAV particles from a sample containing a target product and one or more impurities. Typically, the degree of purity of the target product is increased by removing (completely or incompletely) at least one impurity from the sample. In some embodiments, the degree of purity of rAAV in a sample is increased by removing (completely or incompletely) one or more impurities from the sample using the methods described herein.

[0024] As used in this disclosure and claims, the singular forms "a," "an," and "the" include the plural form unless otherwise explicitly stated in the context.

[0025] Whenever embodiments described herein using the phrase "including" should be understood as also providing other similar embodiments described in terms of "consisting of" and / or "essentially consisting of".

[0026] When the term "and / or" is used herein in expressions such as "A and / or B," it is intended to include both A and B, A or B, A (alone), and B (alone). Similarly, when the term "and / or" is used in expressions such as "A, B, and / or C," it is intended to include 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).

[0027] Where embodiments of this disclosure describe the Markush Group or other alternative groups, the methods of this disclosure encompass not only the entire group as a whole, but also each individual member of the group, all possible subgroups of the main group, and even the main group in which one or more group members are absent. The methods of this disclosure also anticipate the express exclusion of one or more group members in the methods of this disclosure.

[0028] Method for generating rAAV In some embodiments, the Disclosure provides a method for generating rAAV particles, comprising: (a) preparing a cell culture containing cells capable of generating rAAV; (b) adding a histone deacetylase (HDAC) inhibitor to the cell culture to a final concentration between about 0.1 mM and about 20 mM; and (c) maintaining the cell culture under conditions that enable the generation 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 butyric acid (e.g., sodium butyrate), valproic acid (e.g., sodium valproate), propionic acid (e.g., sodium propionate), or a combination thereof. In some embodiments, after the addition of the HDAC inhibitor, the cell culture contains butyric acid (e.g., sodium butyrate), valproic acid (e.g., sodium valproate), or propionic acid (e.g., sodium propionate) between about 0.5 mM and about 10 mM. In some embodiments, the cell culture contains butyric acid (e.g., sodium butyrate) in a concentration of about 0.5 mM to about 5 mM. In some embodiments, the cell culture contains valproic acid (e.g., sodium valproate) in a concentration of about 0.5 mM to about 5 mM. In some embodiments, the cell culture contains propionic acid (e.g., sodium propionate) in a concentration of about 0.5 mM to about 5 mM. In some embodiments, the cell culture contains valproic acid (e.g., sodium valproate) in a concentration of about 0.5 mM to about 3 mM. In some embodiments, the cell culture contains propionic acid (e.g., sodium propionate) in a concentration of about 0.5 mM to about 3 mM. In some embodiments, the HDAC inhibitor is added about 12 hours to about 36 hours after step b). In some embodiments, the method further includes adding a sodium salt to the culture in an amount sufficient to increase the final concentration of the sodium salt (e.g., sodium chloride) by only about 20 mM to 150 mM. In some embodiments, the final concentration of the sodium salt increases only between about 40 mM and 140 mM. In some embodiments, the final concentration of the sodium salt is between about 150 mM and 240 mM.In some embodiments, the sodium salt is sodium chloride. In some embodiments, the sodium salt is added after the addition of the HDAC inhibitor. In some embodiments, the sodium salt is added at least about 20 minutes after the addition of the HDAC inhibitor. In some embodiments, the method further includes recovering the rAAV particles. In some embodiments, the cells capable of producing rAAV particles are HEK293 cells transfected with one or more polynucleotides encoding (i) an rAAV genome to be packaged, (ii) adenovirus helper function necessary for packaging, (iii) enough AAV rep protein for packaging, and (iv) enough AAV cap protein for packaging. In some embodiments, the cell culture is a suspension culture. In some embodiments, cells capable of producing rAAV particles are HEK293 cells transfected with one or more polynucleotides encoding at least one of the following: (i) an rAAV genome to be packaged, (ii) adenovirus helper function required for packaging, (iii) an AAV rep protein sufficient for packaging, and (iv) an AAV cap protein sufficient for packaging. In some embodiments, the cell culture is a suspension culture. In some embodiments, the rAAV particles are AAV8 or AAV9 particles. In some embodiments, the rAAV particles have an AAV capsid protein of a serotype selected from the group consisting of AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.PHB, and AAV.7m8. In some embodiments, the rAAV particles have AAV capsid proteins with high sequence homology to AAV8 or AAV9, such as AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, and AAV.hu37. In some embodiments, the cell culture is maintained for about 2 to 10 days after step b) under conditions that allow for the generation of rAAV particles.In some embodiments, the cell culture is maintained under conditions that allow for the generation of rAAV particles for about 5 to about 14 days or longer after step b). In some embodiments, the cell culture is maintained under conditions that allow for the generation of rAAV particles for continuous collection.

[0029] In some embodiments, the Disclosure provides a method for generating recombinant adeno-associated virus (rAAV) particles, comprising: (a) preparing a cell culture containing cells; (b) introducing into the cells one or more polynucleotides encoding at least one of the following: (i) an rAAV genome to be packaged; (ii) an adenovirus helper function necessary for packaging; (iii) an AAV rep protein sufficient for packaging; and (iv) an AAV cap protein sufficient for packaging; (c) adding an HDAC inhibitor to the cell culture to a final concentration between about 0.1 mM and about 20 mM; and (d) maintaining the cell culture under conditions that enable the generation of the 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 butyric acid (e.g., sodium butyrate), valproic acid (e.g., sodium valproate), propionic acid (e.g., sodium propionate), or a combination thereof. In some embodiments, after adding an HDAC inhibitor, the cell culture contains butyric acid (e.g., sodium butyrate), valproic acid (e.g., sodium valproate), or propionic acid (e.g., sodium propionate) in concentrations between about 0.5 mM and about 10 mM. In some embodiments, the cell culture contains butyric acid (e.g., sodium butyrate) in concentrations between about 0.5 mM and about 5 mM. In some embodiments, the cell culture contains valproic acid (e.g., sodium valproate) in concentrations between about 0.5 mM and about 5 mM. In some embodiments, the cell culture contains propionic acid (e.g., sodium propionate) in concentrations between about 0.5 mM and about 5 mM. In some embodiments, the cell culture contains butyric acid (e.g., sodium butyrate) in concentrations between about 0.5 mM and about 5 mM. In some embodiments, the cell culture contains valproic acid (e.g., sodium valproate) in concentrations between about 0.5 mM and about 3 mM. In some embodiments, the cell culture contains propionic acid (e.g., sodium propionate) in a concentration between approximately 0.5 mM and approximately 3 mM.In some embodiments, the HDAC inhibitor is added approximately 12 to 36 hours after step b). In some embodiments, the method further includes adding a sufficient amount of sodium salt (e.g., sodium chloride) to the culture to increase the final concentration of the sodium salt by about 20 mM to 150 mM. In some embodiments, the final concentration of the sodium salt increases by about 40 mM to 140 mM. In some embodiments, the final concentration of the sodium salt is between about 150 mM and 240 mM. In some embodiments, the sodium salt is sodium chloride. In some embodiments, the sodium salt is added to the cell culture after the HDAC inhibitor has been added. In some embodiments, the sodium salt is added at least about 20 minutes after the HDAC inhibitor has been added. In some embodiments, the method further includes recovering the rAAV particles. In some embodiments, the cells are HEK293 cells. In some embodiments, the cell culture is a suspension culture. In some embodiments, cells are transfected with one or more polynucleotides encoding (i) an rAAV genome to be packaged, (ii) an adenovirus helper function required for packaging, (iii) an AAV rep protein sufficient for packaging, and (iv) an AAV cap protein sufficient for packaging. In some embodiments, the rAAV particles are AAV8 or AAV9 particles. In some embodiments, the rAAV particles have an AAV capsid protein of a serotype selected from the group consisting of AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.PHB, and AAV.7m8.In some embodiments, the rAAV particles have AAV capsid proteins with high sequence homology to AAV8 or AAV9, such as AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, and AAV.hu37. In some embodiments, the cell culture is maintained for about 2 to about 10 days after step b) under conditions that allow for the generation of rAAV particles. In some embodiments, the cell culture is maintained for about 5 to about 14 days or longer after step b) under conditions that allow for the generation of rAAV particles. In some embodiments, the cell culture is maintained under conditions that allow for the generation of rAAV particles for continuous collection.

[0030] In some embodiments, the disclosure provides a method for producing rAAV particles, comprising culturing cells capable of producing rAAV particles in a medium containing an HDAC inhibitor between about 0.1 mM and about 20 mM under conditions that enable the 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 butyric acid (e.g., sodium butyrate), valproic acid (e.g., sodium valproate), propionic acid (e.g., sodium propionate), or a combination thereof. In some embodiments, the medium contains butyric acid (e.g., sodium butyrate), valproic acid (e.g., sodium valproate), or propionic acid (e.g., sodium propionate) between about 0.5 mM and about 10 mM. In some embodiments, the medium contains butyric acid (e.g., sodium butyrate) between about 0.5 mM and about 5 mM. In some embodiments, the medium contains valproic acid (e.g., sodium valproate) between about 0.5 mM and about 5 mM. In some embodiments, the culture medium contains propionic acid (e.g., sodium propionate) between about 0.5 mM and about 5 mM. In some embodiments, the culture medium further contains NaCl between about 120 mM and about 250 mM. In some embodiments, the culture medium contains NaCl between about 150 mM and about 190 mM or between about 180 mM and about 240 mM. In some embodiments, the method further includes recovering the rAAV particles. In some embodiments, the cells capable of producing rAAV particles are HEK293 cells transfected with one or more polynucleotides encoding (i) an rAAV genome to be packaged, (ii) adenovirus helper function necessary for packaging, (iii) enough AAV rep protein for packaging, and (iv) enough AAV cap protein for packaging.In some embodiments, cells capable of producing rAAV particles are HEK293 cells transfected with one or more polynucleotides encoding at least one of the following: (i) an rAAV genome to be packaged, (ii) adenovirus helper function required for packaging, (iii) an AAV rep protein sufficient for packaging, and (iv) an AAV cap protein sufficient for packaging. In some embodiments, the cell culture is a suspension culture. In some embodiments, the rAAV particles are AAV8 or AAV9 particles. In some embodiments, the rAAV particles have an AAV capsid protein of a serotype selected from the group consisting of AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.PHB, and AAV.7m8. In some embodiments, the rAAV particles have AAV capsid proteins with high sequence homology to AAV8 or AAV9, such as AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, and AAV.hu37. In some embodiments, the cell culture is kept under conditions that allow for the generation of rAAV particles for a period of about 2 to about 10 days. In some embodiments, the cell culture is kept under conditions that allow for the generation of rAAV particles for a period of about 5 to about 14 days or longer. In some embodiments, the cell culture is maintained under conditions that allow for the generation of rAAV particles for continuous collection.

[0031] In some embodiments, the Disclosure provides methods for increasing the generation of rAAV particles. In some embodiments, the method for increasing rAAV generation comprises (a) preparing a cell culture containing cells; (b) introducing into the cells one or more polynucleotides encoding at least one of the following: (i) an rAAV genome to be packaged, (ii) an adenovirus helper function necessary for packaging, (iii) an AAV rep protein sufficient for packaging, and (iv) an AAV cap protein sufficient for packaging; (c) adding an HDAC inhibitor to the cell culture to a final concentration between about 0.1 mM and about 20 mM; and (d) maintaining the cell culture under conditions that enable the generation of rAAV particles. In some embodiments, the cell culture is maintained under conditions that enable the generation of rAAV particles for about 2 to about 10 days after step b). In some embodiments, the cell culture is maintained under conditions that enable the generation of rAAV particles for about 5 to about 14 days or longer after step b). In some embodiments, the cell culture is maintained under conditions that allow for the generation of rAAV particles for continuous collection.

[0032] In some embodiments, a method for increasing rAAV production involves culturing cells capable of producing rAAV particles in a medium containing an HDAC inhibitor between about 0.1 mM and about 20 mM under conditions that enable the production of rAAV particles. In some embodiments, the cell culture is kept under conditions that enable the production of rAAV particles for a period of about 2 to about 10 days. In some embodiments, the cell culture is kept under conditions that enable the production of rAAV particles for a period of about 5 to about 14 days or longer after step b). In some embodiments, the cell culture is maintained under conditions that enable the production of rAAV particles for continuous collection.

[0033] In some embodiments, a method for increasing rAAV production includes (a) preparing a cell culture containing cells capable of producing rAAV, (b) adding an HDAC inhibitor to the cell culture to a final concentration between about 0.1 mM and about 20 mM, and (c) maintaining the cell culture under conditions that enable the production of rAAV particles. In some embodiments, the cell culture is kept under conditions that enable the production of rAAV particles for a period of about 2 to about 10 days. In some embodiments, the cell culture is kept under conditions that enable the production of rAAV particles for a period of about 5 to about 14 days or longer. In some embodiments, the cell culture is maintained under conditions that enable the production of rAAV particles for continuous collection.

[0034] Those skilled in the art will understand that any histone deacetylase (HDAC) inhibitor compound can be used in the manner disclosed herein. HDAC inhibitors are a recognized class of compounds in the art. See, for example, Eckschlager et al., Int.J.Mol.Sci.18,1414;doi:10.3390 / ijms18071414(2017); Huber et al., The Journal of Biological Chemistry,286(25):22211-22218(2011). In some embodiments, HDAC inhibitors include HDAC isoform selective inhibitors. In some embodiments, HDAC inhibitors selectively inhibit the activity of one or more HDACs of class I, II, III, and IV. In some embodiments, HDAC inhibitors inhibit the activity of one or more HDACs from class I and class II. In some embodiments, HDAC inhibitors include pan-inhibitors. In some embodiments, the HDAC inhibitor comprises hydroxamic acid, short-chain fatty acid, benzamide, cyclic tetrapeptide, or sirtuin inhibitor. In some embodiments, the HDAC inhibitor comprises short-chain fatty acid or a salt thereof. In some embodiments, the HDAC inhibitor comprises hydroxamic acid. In some embodiments, the HDAC inhibitor comprises hydroxamic acid, such as trichostatin A, suberanilohydroxamic acid (SAHA), bellinostat (PXD101), panaviostat, zivinostat, resminostat, avexinostat, xinostat, rosirinostat, practinostat, and CHR-3996. In some embodiments, HDAC inhibitors include short-chain fatty acids or salts thereof, such as valproic acid, propionic acid, butyric acid, 2,2-dimethylbutyric acid, 2-ethylbutyric acid, pentanoic acid, hexanoic acid, heptanoic acid, octanoic acid, phenylbutyric acid, and salts thereof (Steliou et al., BioResearch Open Access, Vol.1, Issue 4, doi.org / 10.1089 / biores.2012.0223(2012)).In some embodiments, the HDAC inhibitor comprises valproic acid or a salt thereof (e.g., sodium valproate). In some embodiments, the HDAC inhibitor comprises butyric acid or a salt thereof (e.g., sodium butyrate). In some embodiments, the HDAC inhibitor comprises propionic acid or a salt thereof (e.g., sodium propionate). In some embodiments, the HDAC inhibitor comprises benzamides, such as entinostat, tasedinarin, 4SC202, and mosetinostat. In some embodiments, the HDAC inhibitor comprises a cyclic tetrapeptide, such as romidepsin. In some embodiments, the HDAC inhibitor comprises sirtuin inhibitors, such as nicotinamide, syltinol, canbinol, and EX-527. In some embodiments, the HDAC inhibitor comprises [4-(2-amino-phenylcarbamoyl)-benzyl]carbamate pyridine-3-ylmethyl ester and its derivatives, pyroxamide, oxamfratin, apicidine, depsipeptide, depdesin, trapoxin, M344, scriptide, MC 1293, sodium 1-naphthoate, CAY10398, sodium phenylbutyrate, suberoylbis-hydroxamic acid (SBHA), CAY10433, oxamfratin, or HC toxin. In some embodiments, the HDAC inhibitor does not contain pyruvate or a salt thereof. In some embodiments, the HDAC inhibitor does not contain nicotinamide. In some embodiments, the HDAC inhibitor comprises a combination of two or more HDAC inhibitors.

[0035] In some embodiments, the methods disclosed herein include adding an HDAC inhibitor to a cell culture to a final concentration between about 0.1 mM and about 20 mM. In some embodiments, the methods disclosed herein include culturing cells capable of producing rAAV in a medium containing an HDAC inhibitor between about 0.1 mM and about 20 mM. In some embodiments, the cell cultures disclosed herein contain an HDAC inhibitor between about 0.1 mM and about 20 mM. For example, a cell culture containing 2 mM of HDAC inhibitor should be understood to include cells and a medium containing 2 mM of HDAC inhibitor. In some embodiments, the concentration of the HDAC inhibitor is between about 0.5 mM and about 10 mM. In some embodiments, the concentration of the HDAC inhibitor is between about 0.5 mM and about 5 mM. In some embodiments, the concentration of the HDAC inhibitor is between about 0.5 mM and about 3 mM. In some embodiments, the concentration of the HDAC inhibitor is about 0.5 mM, about 1 mM, about 1.5 mM, about 2 mM, about 2.5 mM, about 3 mM, about 3.5 mM, about 4 mM, about 4.5 mM, or about 5 mM. In some embodiments, the concentration of the HDAC inhibitor is about 1.5 mM. In some embodiments, the concentration of the HDAC inhibitor is about 2 mM. In some embodiments, the concentration of the HDAC inhibitor is about 3 mM. In some embodiments, the concentration of the HDAC inhibitor is about 4 mM.

[0036] In some embodiments, the cell culture contains valproic acid or a salt thereof (e.g., sodium valproate) in concentrations ranging from about 0.5 mM to about 10 mM. In some embodiments, the cell culture contains valproic acid or a salt thereof (e.g., sodium valproate) in concentrations ranging from about 0.5 mM to about 5 mM. In some embodiments, the cell culture contains valproic acid or a salt thereof (e.g., sodium valproate) in concentrations ranging from about 0.5 mM to about 3 mM. In some embodiments, the cell culture contains valproic acid or a salt thereof (e.g., sodium valproate) in concentrations ranging from about 0.5 mM, about 1 mM, about 1.5 mM, about 2 mM, about 2.5 mM, about 3 mM, about 3.5 mM, about 4 mM, about 4.5 mM, or about 5 mM. In some embodiments, the cell culture contains about 1.5 mM valproic acid or a salt thereof (e.g., sodium valproate). In some embodiments, the cell culture contains about 2 mM valproic acid or a salt thereof (e.g., sodium valproate). In some embodiments, the cell culture contains about 3 mM valproic acid or a salt thereof (e.g., sodium valproate). In some embodiments, the cell culture contains about 4 mM valproic acid or a salt thereof (e.g., sodium valproate).

[0037] In some embodiments, the cell culture contains butyric acid or a salt thereof (e.g., sodium butyrate) in concentrations ranging from about 0.5 mM to about 10 mM. In some embodiments, the cell culture contains butyric acid or a salt thereof (e.g., sodium butyrate) in concentrations ranging from about 0.5 mM to about 5 mM. In some embodiments, the cell culture contains butyric acid or a salt thereof (e.g., sodium butyrate) in concentrations ranging from about 0.5 mM to about 3 mM. In some embodiments, the cell culture contains about 0.5 mM, about 1 mM, about 1.5 mM, about 2 mM, about 2.5 mM, about 3 mM, about 3.5 mM, about 4 mM, about 4.5 mM, or about 5 mM of butyric acid or a salt thereof (e.g., sodium butyrate). In some embodiments, the cell culture contains about 1.5 mM of butyric acid or a salt thereof (e.g., sodium butyrate). In some embodiments, the cell culture contains about 2 mM of butyric acid or a salt thereof (e.g., sodium butyrate). In some embodiments, the cell culture contains about 3 mM butyric acid or a salt thereof (e.g., sodium butyrate). In some embodiments, the cell culture contains about 4 mM butyric acid or a salt thereof (e.g., sodium butyrate).

[0038] In some embodiments, the cell culture contains propionic acid or a salt thereof (e.g., sodium propionate) in concentrations ranging from about 0.5 mM to about 10 mM. In some embodiments, the cell culture contains propionic acid or a salt thereof (e.g., sodium propionate) in concentrations ranging from about 0.5 mM to about 5 mM. In some embodiments, the cell culture contains propionic acid or a salt thereof (e.g., sodium propionate) in concentrations ranging from about 0.5 mM to about 3 mM. In some embodiments, the cell culture contains propionic acid or a salt thereof (e.g., sodium propionate) in concentrations ranging from about 0.5 mM, about 1 mM, about 1.5 mM, about 2 mM, about 2.5 mM, about 3 mM, about 3.5 mM, about 4 mM, about 4.5 mM, or about 5 mM. In some embodiments, the cell culture contains about 1.5 mM of propionic acid or a salt thereof (e.g., sodium propionate). In some embodiments, the cell culture contains about 2 mM propionic acid or a salt thereof (e.g., sodium propionate). In some embodiments, the cell culture contains about 3 mM propionic acid or a salt thereof (e.g., sodium propionate). In some embodiments, the cell culture contains about 4 mM propionic acid or a salt thereof (e.g., sodium propionate).

[0039] In some embodiments, the methods disclosed herein include preparing a cell culture containing cells, introducing one or more polynucleotides into the cells, and adding an HDAC inhibitor to the cell culture. In some embodiments, the HDAC inhibitor is added before introducing one or more polynucleotides into the cells. In some embodiments, the HDAC inhibitor is added after introducing one or more polynucleotides into the cells. In some embodiments, the HDAC inhibitor is added about 1 hour to about 48 hours or about 12 hours to about 36 hours after introducing one or more polynucleotides into the cells. In some embodiments, the HDAC inhibitor is added about 16 hours to about 30 hours after introducing one or more polynucleotides into the cells. In some embodiments, the HDAC inhibitor is added about 18 hours to about 26 hours after introducing one or more polynucleotides into the cells. In some embodiments, the HDAC inhibitor is added about 18 hours to about 22 hours after introducing one or more polynucleotides into the cells. In some embodiments, the HDAC inhibitor is added about 22 hours to about 26 hours after introducing one or more polynucleotides into the cells. In some embodiments, the HDAC inhibitor is added before approximately 48 hours or before approximately 36 hours after introducing one or more polynucleotides into the cells. In some embodiments, the HDAC inhibitor is added at least approximately 6 hours, at least approximately 9 hours, at least approximately 12 hours, at least approximately 18 hours, or at least approximately 20 hours after introducing one or more polynucleotides into the cells. In some embodiments, the HDAC inhibitor is added at approximately 6 hours, at approximately 9 hours, at approximately 12 hours, at approximately 18 hours, at approximately 20 hours, at approximately 22 hours, at approximately 24 hours, at approximately 30 hours, at approximately 36 hours, or at approximately 48 hours after introducing one or more polynucleotides into the cells. In some embodiments, the HDAC inhibitor is added approximately 18 hours after introducing one or more polynucleotides into the cells. In some embodiments, the HDAC inhibitor is added approximately 20 hours after introducing one or more polynucleotides into the cells.In some embodiments, the HDAC inhibitor is added approximately 22 hours after the cells have been introduced with one or more polynucleotides. In some embodiments, the HDAC inhibitor is added approximately 24 hours after the cells have been introduced with one or more polynucleotides. In some embodiments, introducing one or more polynucleotides into cells includes transtransferring one or more polynucleotides into cells.

[0040] In some embodiments, the cell medium is replaced between introducing one or more polynucleotides into cells and adding an HDAC inhibitor to the cell culture. In some embodiments, the cell medium is supplemented between introducing one or more polynucleotides into cells and adding an HDAC inhibitor to the cell culture. In some embodiments, the cell medium is supplemented between introducing one or more polynucleotides into cells and adding an HDAC inhibitor to the cell culture with one or more nutrients, salts, buffers, and additives (e.g., antifoaming agents). In some embodiments, introducing one or more polynucleotides into cells includes transtransferring one or more polynucleotides into cells.

[0041] In some embodiments, the methods disclosed herein include adding a sodium salt to a cell culture. In some embodiments, the methods disclosed herein include culturing cells in a medium containing a sodium salt. In some embodiments, the cell cultures disclosed herein contain a sodium salt. A cell culture containing a sodium salt should be understood to include cells and a medium containing a sodium salt agent.

[0042] In some embodiments, the sodium salt is an inorganic salt. In some embodiments, the sodium salt is an organic salt. In some embodiments, the sodium salt is a sodium halide containing a halogen (e.g., fluorine, chlorine, bromine, and iodine). In some embodiments, the sodium salt is sodium chloride or sodium bromide. In some embodiments, the sodium salt is sodium chloride, sodium carbonate, sodium phosphate, or sodium sulfate. In some embodiments, the sodium salt is sodium chloride.

[0043] In some embodiments, the cell culture or cell medium contains sodium salts between about 120 mM and about 250 mM. In some embodiments, the cell culture or cell medium contains sodium salts between about 130 mM and about 160 mM, between about 150 mM and about 190 mM, or between about 180 mM and about 240 mM. In some embodiments, the cell culture or cell medium contains sodium salts between about 150 mM and about 240 mM. In some embodiments, the cell culture or cell medium contains sodium salts between about 150 mM and about 190 mM. In some embodiments, the cell culture or cell medium contains sodium salts between about 180 mM and about 240 mM. In some embodiments, the sodium salt is sodium chloride.

[0044] In some embodiments, the cell culture or cell medium contains sodium chloride between about 120 mM and about 250 mM. In some embodiments, the cell culture or cell medium contains sodium chloride between about 130 mM and about 160 mM, between about 150 mM and about 190 mM, or between about 180 mM and about 240 mM. In some embodiments, the cell culture or cell medium contains sodium chloride between about 150 mM and about 240 mM. In some embodiments, the cell culture or cell medium contains sodium chloride between about 150 mM and about 190 mM. In some embodiments, the cell culture or cell medium contains sodium chloride between about 180 mM and about 240 mM. In some embodiments, the sodium salt is sodium chloride. In some embodiments, the final concentration of the sodium salt is increased to about 140 mM sodium chloride. In some embodiments, the final concentration of the sodium salt is increased to about 170 mM sodium chloride. In some embodiments, the final concentration of the sodium salt increases to approximately 200 mM sodium chloride.

[0045] In some embodiments, the methods disclosed herein involve adding a sufficient amount of sodium salt to a cell culture to increase the final concentration of the sodium salt by about 20 mM to about 150 mM. In some embodiments, the final concentration of the sodium salt increases by about 20 mM to about 50 mM. In some embodiments, the final concentration of the sodium salt increases by about 40 mM to about 80 mM. In some embodiments, the final concentration of the sodium salt increases by about 70 mM to about 120 mM. In some embodiments, the final concentration of the sodium salt increases by about 40 mM to about 140 mM. In some embodiments, the final concentration of the sodium salt increases by about 30 mM. In some embodiments, the final concentration of the sodium salt increases by about 60 mM. In some embodiments, the final concentration of the sodium salt increases by about 90 mM. In some embodiments, the sodium salt is sodium chloride.

[0046] In some embodiments, the methods disclosed herein involve adding a sufficient amount of sodium chloride to a cell culture to increase the final concentration of sodium chloride by about 20 mM to about 150 mM. In some embodiments, the final concentration of sodium chloride increases by about 20 mM to about 50 mM. In some embodiments, the final concentration of sodium chloride increases by about 40 mM to about 80 mM. In some embodiments, the final concentration of sodium chloride increases by about 70 mM to about 120 mM. In some embodiments, the final concentration of sodium chloride increases by about 40 mM to about 140 mM. In some embodiments, the final concentration of sodium chloride increases by about 30 mM. In some embodiments, the final concentration of sodium chloride increases by about 60 mM. In some embodiments, the final concentration of sodium chloride increases by about 90 mM.

[0047] In some embodiments, the cell culture contains sodium chloride before adding sodium chloride to further increase the sodium chloride concentration, for example, by 90 mM. In some embodiments, the cell culture contains about 90 mM, about 100 mM, or about 110 mM of sodium chloride before sodium chloride is added to the cell culture to further increase the sodium chloride concentration. If the cell culture contains about 110 mM of sodium chloride and enough sodium chloride is added to the cell culture to increase the sodium chloride concentration by about 90 mM, it should be understood that the final concentration of sodium chloride is about 200 mM. In some embodiments, the sodium chloride is sodium chloride.

[0048] In some embodiments, the cell culture contains about 90 mM, about 100 mM, or about 110 mM sodium chloride before sodium chloride is added to the cell culture to further increase the concentration of sodium chloride.

[0049] In some embodiments, the methods disclosed herein involve adding a sufficient amount of sodium salt to a cell culture to increase the final concentration of the sodium salt from about 120 mM to about 250 mM. In some embodiments, the final concentration of the sodium salt increases from about 130 mM to about 160 mM. In some embodiments, the final concentration of the sodium salt increases from about 150 mM to about 190 mM. In some embodiments, the final concentration of the sodium salt increases from about 180 mM to about 240 mM. In some embodiments, the final concentration of the sodium salt increases from about 150 mM to about 240 mM. In some embodiments, the final concentration of the sodium salt increases to about 140 mM. In some embodiments, the final concentration of the sodium salt increases to about 170 mM. In some embodiments, the final concentration of the sodium salt increases to about 200 mM. In some embodiments, the sodium salt is sodium chloride.

[0050] In some embodiments, the methods disclosed herein include adding a sufficient amount of sodium chloride to a cell culture to increase the final concentration of sodium chloride from about 120 mM to about 250 mM. In some embodiments, the final concentration of sodium chloride increases from about 130 mM to about 160 mM. In some embodiments, the final concentration of sodium chloride increases from about 150 mM to about 190 mM. In some embodiments, the final concentration of sodium chloride increases from about 180 mM to about 240 mM. In some embodiments, the final concentration of sodium chloride increases from about 150 mM to about 240 mM. In some embodiments, the final concentration of sodium chloride increases to about 140 mM. In some embodiments, the final concentration of sodium chloride increases to about 170 mM. In some embodiments, the final concentration of sodium chloride increases to about 200 mM.

[0051] In some embodiments, the method disclosed herein comprises (a) preparing a cell culture containing cells, (b) introducing one or more polynucleotides into the cells, (c) adding an HDAC inhibitor to the cell culture, and (d) adding a sodium salt (e.g., sodium chloride) to the cell culture. It should be understood that (b), (c), and (d) may be carried out in any order. In some embodiments, (b), (c), and (d) are carried out in the order (b)-(c)-(d). In some embodiments, (b), (c), and (d) are carried out in the order (b)-(d)-(c). In some embodiments, (b), (c), and (d) are carried out in the order (d)-(c)-(b). In some embodiments, (b), (c), and (d) are carried out in the order (d)-(b)-(c). In some embodiments, (c) and (d) are carried out simultaneously after (b). In some embodiments, (c) and (d) are carried out simultaneously by adding a composition comprising an HDAC inhibitor and a sodium salt (e.g., sodium chloride) to a cell culture. In some embodiments, (c) and (d) are carried out simultaneously by adding a first composition comprising an HDAC inhibitor and a second composition comprising a sodium salt (e.g., sodium chloride) to a cell culture. In some embodiments, introducing one or more polynucleotides into cells includes transtransferring one or more polynucleotides into cells.

[0052] In some embodiments, the method disclosed herein comprises (a) preparing a cell culture containing cells, (b) introducing one or more polynucleotides into the cells, (c) adding an HDAC inhibitor to the cell culture, and (d) adding a sodium salt (e.g., sodium chloride) to the cell culture, wherein (c) is performed after (b). In some embodiments, (c) is performed about 1 hour to about 48 hours or about 12 hours to about 36 hours after (b). In some embodiments, (c) is performed before about 48 hours or before about 36 hours after (b). In some embodiments, (c) is performed at least about 6 hours, at least about 9 hours, at least about 12 hours, or at least about 18 hours after (b). In some embodiments, (c) is added about 6 hours, 9 hours, 12 hours, 18 hours, 20 hours, 22 hours, 24 hours, 30 hours, 36 hours, or 48 hours after (b). In some embodiments, (c) is carried out about 24 hours after (b). In some embodiments, (c) is carried out about 20 hours after (b). In some embodiments, introducing one or more polynucleotides into cells includes transtransferring one or more polynucleotides into cells.

[0053] In some embodiments, the method disclosed herein comprises (a) preparing a cell culture containing cells, (b) introducing one or more polynucleotides into the cells, (c) adding an HDAC inhibitor to the cell culture, and (d) adding a sodium salt (e.g., sodium chloride) to the cell culture, wherein (b), (c), and (d) are carried out in the order (b)-(c)-(d). In some embodiments, (d) is carried out about 5 minutes to about 6 hours after (c). In some embodiments, (d) is carried out about 20 minutes to about 2 hours after (c). In some embodiments, (d) is carried out before about 2 hours or before about 1 hour after (c). In some embodiments, (d) is carried out at least about 5 minutes, at least about 10 minutes, at least about 20 minutes, at least about 30 minutes, at least about 40 minutes, and at least about 50 minutes after (c). In some embodiments, (d) is performed at least about 30 minutes after (c). In some embodiments, (d) is performed about 10 minutes, about 20 minutes, about 30 minutes, about 40 minutes, about 50 minutes, or about 60 minutes after (c). In some embodiments, (d) is performed about 40 minutes after (c). In some embodiments, introducing one or more polynucleotides into cells includes transtransferring one or more polynucleotides into cells.

[0054] In some embodiments, the method disclosed herein comprises (a) preparing a cell culture containing cells, (b) introducing one or more polynucleotides into the cells, (c) adding an HDAC inhibitor to the cell culture, and (d) adding a sodium salt (e.g., sodium chloride) to the cell culture, wherein (b), (c), and (d) are carried out in the order (b)-(c)-(d). In some embodiments, (c) is carried out about 1 hour to about 48 hours or about 12 hours to about 36 hours after (b). In some embodiments, (c) is carried out before about 48 hours or before about 36 hours after (b). In some embodiments, (c) is carried out at least about 6 hours, at least about 9 hours, at least about 12 hours, or at least about 18 hours after (b). In some embodiments, (c) is added about 6 hours, 9 hours, 12 hours, 18 hours, 20 hours, 22 hours, 24 hours, 30 hours, 36 hours, or 48 hours after (b). In some embodiments, (c) is carried out about 24 hours after (b). In some embodiments, (c) is carried out about 20 hours after (b). In some embodiments, (d) is carried out about 5 minutes to about 6 hours after (c). In some embodiments, (d) is carried out about 20 minutes to about 2 hours after (c). In some embodiments, (d) is carried out before about 2 hours or before about 1 hour after (c). In some embodiments, (d) is carried out at least about 5 minutes, at least about 10 minutes, at least about 20 minutes, at least about 30 minutes, at least about 40 minutes, or at least about 50 minutes after (c). In some embodiments, (d) is performed at least about 30 minutes after (c). In some embodiments, (d) is performed about 10 minutes, about 20 minutes, about 30 minutes, about 40 minutes, about 50 minutes, or about 60 minutes after (c). In some embodiments, (d) is performed about 40 minutes after (c).

[0055] In some embodiments, the methods disclosed herein include preparing a cell culture containing cells, introducing one or more polynucleotides into the cells, adding an HDAC inhibitor to the cell culture, adding a sodium salt (e.g., sodium chloride) to the cell culture, and maintaining the cell culture under conditions that allow for the generation of rAAV particles. In some embodiments, the cell culture is maintained for about 2 to about 10 days after introducing one or more polynucleotides into the cells. In some embodiments, the cell culture is maintained for about 5 to about 14 days or longer after introducing one or more polynucleotides into the cells. In some embodiments, the cell culture is maintained for about 2 to about 7 days after introducing one or more polynucleotides into the cells. In some embodiments, the cell culture is maintained for about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, or about 7 days after introducing one or more polynucleotides into the cells. In some embodiments, the cell culture is maintained for about 5 days after introducing one or more polynucleotides into the cells. In some embodiments, the cell culture is maintained for about 6 days after introducing one or more polynucleotides into the cells. In some embodiments, the cell culture is maintained under conditions that allow for the generation of rAAV particles for continuous collection. In some embodiments, introducing one or more polynucleotides into the cells includes transtransferring one or more polynucleotides into the cells.

[0056] In some embodiments, the methods disclosed herein increase the generation of rAAV particles compared to methods that do not involve the addition of HDAC inhibitors or sodium salts to cell cultures. In some embodiments, the methods disclosed herein increase rAAV generation by at least about 50%, at least about 75%, or at least about 100%. In some embodiments, the methods disclosed herein increase rAAV generation by at least about 2-fold, at least about 3-fold, or at least about 5-fold. In some embodiments, the methods disclosed herein increase rAAV generation by at least about 2-fold. In some embodiments, the increase in generation is quantified by comparing the rAAV titer of the resulting culture. In some embodiments, the rAAV titer is measured as genome copies (GC) per milliliter of the resulting culture. In some embodiments, the rAAV particles contain capsid proteins from AAV capsid serotypes selected from AAV8 and AAV9. In some embodiments, the rAAV particles have the AAV capsid serotype of AAV8. In some embodiments, the rAAV particles have the AAV capsid serotype of AAV9. In some embodiments, the rAAV particles have a capsid serotype selected from the group consisting of AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.PHB, and AAV.7m8. In some embodiments, the rAAV particles have a capsid protein with high sequence homology to AAV8 or AAV9, for example, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, and AAV.hu37.

[0057] In some embodiments, the methods disclosed herein increase the yield of rAAV particles while maintaining or improving the quality attributes of rAAV particles and compositions containing them. In some embodiments, the quality of rAAV particles and compositions containing them is evaluated by quantifying the concentration of rAAV particles (e.g., GC / ml), the percentage of particles containing copies of the rAAV genome, the proportion of particles without the genome, the infectivity of rAAV particles, the stability of rAAV particles, and the concentration of residual host cell proteins or residual host cell nucleic acids (e.g., host cell genomic DNA, plasmids encoding rep and cap genes, plasmids encoding helper functions, plasmids encoding the rAAV genome). In some embodiments, the quality of rAAV particles or compositions containing them produced by the methods disclosed herein is the same as the quality of rAAV particles or compositions produced by methods that do not involve the addition of HDAC inhibitors or sodium salts to the cell culture. In some embodiments, the quality of rAAV particles or compositions containing them produced by the methods disclosed herein is better than the quality of rAAV particles or compositions produced by methods that do not involve the addition of HDAC inhibitors or sodium salts to the cell culture.

[0058] In some embodiments, the methods disclosed herein produce rAAV particles between approximately 1 × 10⁻¹⁰ GC / ml and approximately 1 × 10⁻¹³ GC / ml. In some embodiments, the methods disclosed herein produce rAAV particles between approximately 1 × 10⁻¹⁰ GC / ml and approximately 1 × 10⁻¹¹ GC / ml. In some embodiments, the methods disclosed herein produce rAAV particles between approximately 5 × 10⁻¹⁰ GC / ml and approximately 1 × 10⁻¹² GC / ml. In some embodiments, the methods disclosed herein produce rAAV particles between approximately 5 × 10⁻¹⁰ GC / ml and approximately 1 × 10⁻¹³ GC / ml. In some embodiments, the methods disclosed herein produce rAAV particles between approximately 1 × 10⁻¹¹ GC / ml and approximately 1 × 10⁻¹³ GC / ml. In some embodiments, the methods disclosed herein produce rAAV particles between approximately 5 × 10⁻¹⁰ GC / ml and approximately 5 × 10⁻¹² GC / ml. In some embodiments, the methods disclosed herein produce rAAV particles between approximately 1 × 10⁻¹¹ GC / ml and approximately 5 × 10⁻¹² GC / ml. In some embodiments, the methods disclosed herein produce rAAV particles greater than approximately 1 × 10⁻¹¹ GC / ml. In some embodiments, the methods disclosed herein produce rAAV particles greater than approximately 5 × 10⁻¹¹ GC / ml. In some embodiments, the methods disclosed herein produce rAAV particles greater than approximately 1 × 10⁻¹² GC / ml. In some embodiments, the rAAV particles contain a capsid protein from an AAV capsid serotype selected from AAV8 and AAV9. In some embodiments, the rAAV particles have the AAV capsid serotype of AAV8. In some embodiments, the rAAV particles have the AAV capsid serotype of AAV9. In some embodiments, the rAAV particles contain a capsid protein from an AAV capsid serotype selected from the group consisting of AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.PHB, and AAV.7m8.In some embodiments, the rAAV particles include capsid proteins with high sequence homology to AAV8 or AAV9, such as AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, and AAV.hu37.

[0059] In some embodiments, the methods disclosed herein produce at least about 5 × 10⁻¹⁰ GC / ml of rAAV particles. In some embodiments, the methods disclosed herein produce at least about 1 × 10⁻¹¹ GC / ml of rAAV particles. In some embodiments, the methods disclosed herein produce at least about 5 × 10⁻¹¹ GC / ml of rAAV particles. In some embodiments, the methods disclosed herein produce at least about 1 × 10⁻¹² GC / ml of rAAV particles. In some embodiments, the methods disclosed herein produce at least about 5 × 10⁻¹² GC / ml of rAAV particles. In some embodiments, the methods disclosed herein produce at least about 1 × 10⁻¹³ GC / ml of rAAV particles. In some embodiments, the methods disclosed herein produce at least about 5 × 10⁻¹³ GC / ml of rAAV particles. In some embodiments, the methods disclosed herein produce at least about 5 × 10⁻¹³ GC / ml of rAAV particles. In some embodiments, the rAAV particles comprise a capsid protein from an AAV capsid serotype selected from AAV8 and AAV9. In some embodiments, the rAAV particles have the AAV capsid serotype of AAV8. In some embodiments, the rAAV particles have the AAV capsid serotype of AAV9. In some embodiments, the rAAV particles contain a capsid protein from an AAV capsid serotype selected from the group consisting of AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.PHB, and AAV.7m8. In some embodiments, the rAAV particles contain a capsid protein with high sequence homology to AAV8 or AAV9, such as AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, and AAV.hu37.

[0060] Numerous cell culture-based systems for generating rAAV particles are known in the art and can be used to carry out the methods disclosed herein. Examples of cell culture-based systems include transfusion, stable cell line generation, and infectious hybrid virus generation systems (including adenovirus-AAV hybrids, herpesvirus-AAV hybrids, and baculovirus-AAV hybrids). Any rAAV-producing culture for generating rAAV virus particles requires (1) a suitable host cell (in the case of a baculovirus-producing system, for example, a human cell line (e.g., HeLa, A549, or HEK293 cells and their derivatives (HEK293T cells, HEK293F cells)), a mammalian cell line (e.g., Vero), CHO cells or CHO-derived cells, or an insect cell line (e.g., SF-9)); (2) a suitable helper virus function provided by a wild-type or mutant adenovirus (e.g., a temperature-sensitive adenovirus), a herpesvirus, a baculovirus, or a plasmid construct that provides helper function; (3) AAV rep and cap genes and gene products; (4) a transgene adjacent to the AAV ITR sequence (e.g., a therapeutic transgene); and (5) a suitable medium and medium components to support rAAV production.

[0061] In one embodiment, the Specified provides a method for generating rAAV, comprising: (a) preparing a cell culture comprising insect cells; (b) introducing one or more baculovirus vectors into the cells encoding at least one of the following: i. an rAAV genome to be packaged, ii. an AAV rep protein sufficient for packaging, and iii. an AAV cap protein sufficient for packaging; (c) adding an HDAC inhibitor to the cell culture to a final concentration between about 0.1 mM and about 20 mM; and maintaining the cell culture under conditions that allow for the generation of rAAV particles for a period of about 2 to about 15 days or longer after (b). In some embodiments, the method comprises using a first baculovirus vector encoding the rep and cap genes and a second baculovirus vector encoding the rAAV genome. In some embodiments, the method comprises using a baculovirus encoding the rAAV genome and insect cells expressing the rep and cap genes. In some embodiments, the method involves using a baculovirus vector encoding rep and cap genes and the rAAV genome. In some embodiments, the insect cells are Sf-9 cells. In some embodiments, the insect cells are Sf-9 cells containing one or more stably incorporated heterologous polynucleotides encoding rep and cap genes. In some embodiments, the method further involves adding a sodium salt to the culture in an amount sufficient to increase the final concentration of the sodium salt between about 20 mM and about 150 mM. In some embodiments, the HDAC inhibitor is valproic acid, propionic acid, butyric acid, or a salt thereof.

[0062] In some embodiments, the methods disclosed herein utilize a baculovirus production system. In some embodiments, the baculovirus production system utilizes a first baculovirus encoding rep and cap genes and a second baculovirus encoding the rAAV genome. In some embodiments, the baculovirus production system utilizes a baculovirus encoding the rAAV genome and host cells expressing the rep and cap genes. In some embodiments, the baculovirus production system utilizes a baculovirus encoding the rep and cap genes and the rAAV genome. In some embodiments, the baculovirus production system utilizes Sf-9 cells.

[0063] Those skilled in the art are aware of numerous methods by which rAAV can be generated or packaged by introducing AAV rep and cap genes, AAV helper genes (e.g., adenovirus E1a, E1b, E4, E2a, and VA genes), and rAAV genomes (containing one or more target genes adjacent to reverse terminal repeats (ITRs)) into cells. The expression "adenovirus helper function" means multiple viral helper genes that are expressed intracellularly (as RNA or protein) to enable AAV to grow efficiently within the cell. Those skilled in the art understand that helper viruses, including adenoviruses and herpes simplex viruses (HSV), promote AAV replication, and that certain genes providing essential functions have been identified, and that, for example, helpers can induce changes in the cellular environment that promote such AAV gene expression and replication. In some embodiments of the methods disclosed herein, the AAV rep and cap genes, helper genes, and rAAV genome are introduced into cells by transduction using one or more plasmid vectors encoding the AAV rep and cap genes, helper genes, and rAAV genome. In some embodiments of the methods disclosed herein, the AAV rep and cap genes, helper genes, and rAAV genome can be introduced into cells by transduction using a viral vector encoding the AAV rep and cap genes, helper genes, and rAAV genome, such as an rHSV vector. In some embodiments of the methods disclosed herein, one or more of the AAV rep and cap genes, helper genes, and rAAV genome are introduced into cells by transduction using an rHSV vector. In some embodiments, the rHSV vector encodes the AAV rep and cap genes. In some embodiments, the rHSV vector encodes the helper genes. In some embodiments, the rHSV vector encodes the rAAV genome. In some embodiments, the rHSV vector encodes the AAV rep and cap genes. In some embodiments, the rHSV vector encodes a helper gene and the rAAV genome.In some embodiments, the rHSV vector encodes a helper gene as well as the AAV rep and cap genes.

[0064] In one embodiment, the Specified provides a method for generating rAAV, comprising: (a) preparing a cell culture containing cells; (b) introducing one or more rHSV vectors into the cells encoding at least one of the following: i. an rAAV genome to be packaged, ii. a helper function necessary for packaging the rAAV, iii. an AAV rep protein sufficient for packaging, and iv. an AAV cap protein sufficient for packaging; and (c) adding an HDAC inhibitor to the cell culture to a final concentration between about 0.1 mM and about 20 mM; and maintaining the cell culture under conditions that allow for the generation of rAAV particles for a period of about 2 to about 15 days or longer after (b). In some embodiments, the rHSV vector encodes AAV rep and cap genes. In some embodiments, the rHSV vector encodes a helper function. In some embodiments, the rHSV vector contains one or more endogenous genes encoding a helper function. In some embodiments, the rHSV vector contains one or more heterologous genes encoding a helper function. In some embodiments, the rHSV vector encodes the rAAV genome. In some embodiments, the rHSV vector encodes the AAV rep and cap genes. In some embodiments, the rHSV vector encodes helper function and the rAAV genome. In some embodiments, the rHSV vector encodes helper function and the AAV rep and cap genes. In some embodiments, the cells contain one or more stably incorporated heterologous polynucleotides encoding the rep and cap genes. In some embodiments, the method further includes adding a sodium salt to the culture in an amount sufficient to increase the final concentration of the sodium salt between about 20 mM and about 150 mM. In some embodiments, the HDAC inhibitor is valproic acid, propionic acid, butyric acid, or a salt thereof.

[0065] In one embodiment, the Specified provides a method for generating rAAV, comprising: (a) preparing a cell culture comprising mammalian cells; (b) introducing into the cells one or more polynucleotides encoding at least one of the following: i. an rAAV genome to be packaged, ii. a helper function necessary for packaging the rAAV, iii. an AAV rep protein sufficient for packaging, and iv. an AAV cap protein sufficient for packaging; and (c) adding an HDAC inhibitor to the cell culture to a final concentration between about 0.1 mM and about 20 mM; and maintaining the cell culture under conditions that allow for the generation of the rAAV particles for a period of about 2 to about 15 days or longer after (b). In some embodiments, the helper function is encoded by an adenovirus gene. In some embodiments, the mammalian cells comprise one or more stably incorporated heterologous polynucleotides encoding the rep and cap genes. In some embodiments, the method further includes adding a sodium salt to the culture in an amount sufficient to increase the final concentration of the sodium salt between about 20 mM and about 150 mM. In some embodiments, the HDAC inhibitor is valproic acid, propionic acid, butyric acid, or salts thereof.

[0066] Molecular biology techniques for developing plasmids or viral vectors encoding AAV rep and cap genes, helper genes, and / or the rAAV genome are commonly known in the art. In some embodiments, the AAV rep and cap genes are encoded by a single plasmid vector. In some embodiments, AAV helper genes (e.g., adenovirus E1a, E1b, E4, E2a, and VA genes) are encoded by a single plasmid vector. In some embodiments, the E1a or E1b gene is stably expressed by the host cell, and the remaining AAV helper genes are introduced into the cell by transduction with a single viral vector. In some embodiments, the E1a and E1b genes are stably expressed by the host cell, and the E4, E2a, and VA genes are introduced into the cell by transduction with a single plasmid vector. In some embodiments, one or more helper genes are stably expressed by the host cell, and one or more helper genes are introduced into the cell by transduction with a single plasmid vector. In some embodiments, helper genes are stably expressed by host cells. In some embodiments, the AAV rep and cap genes are encoded by a single viral vector. In some embodiments, AAV helper genes (e.g., adenovirus E1a, E1b, E4, E2a, and VA genes) are encoded by a single viral vector. In some embodiments, the E1a or E1b gene is stably expressed by host cells, and the remaining AAV helper genes are introduced into cells by transduction using a single viral vector. In some embodiments, the E1a and E1b genes are stably expressed by host cells, and the E4, E2a, and VA genes are introduced into cells by transduction using a single viral vector. In some embodiments, one or more helper genes are stably expressed by host cells, and one or more helper genes are introduced into cells by transduction using a single viral vector.In some embodiments, the AAV rep and cap genes, the adenovirus helper function required for packaging, and the rAAV genome to be packaged are introduced into cells by transduction using one or more polynucleotides, for example, a vector. In some embodiments, the method disclosed herein involves transduction into cells of a mixture of three polynucleotides (one encoding the cap and rep genes, one encoding the adenovirus helper function required for packaging (e.g., adenovirus E1a, E1b, E4, E2a, and VA genes), and one encoding the rAAV genome to be packaged). In some embodiments, the AAV cap gene is the AAV8 or AAV9 cap gene. In some embodiments, the AAV cap gene is the AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.PHB, or AAV.7m8 cap gene. In some embodiments, the AAV cap gene encodes a capsid protein with high sequence homology to AAV8 or AAV9, such as AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, and AAV.hu37. In some embodiments, the vector encoding the rAAV genome to be packaged contains the target gene adjacent to the AAV ITR.In some embodiments, AAV ITR is AAV1, AAV2, rAAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, and AAV16, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B, AAV 2.5, AAV2tYF, AAV3B, AAV.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 from another AAV serotype.

[0067] Any combination of vectors can be used to introduce AAV rep and cap genes, AAV helper genes, and the rAAV genome into cells into which rAAV particles are generated or packaged. In some embodiments of the methods disclosed herein, a first plasmid vector encoding the rAAV genome containing a target gene adjacent to an AAV reverse terminal repeat (ITR), a second vector encoding the AAV rep and cap genes, and a third vector encoding the helper gene can be used. In some embodiments, a mixture of the three vectors can be simultaneously translocated into cells.

[0068] In some embodiments, a combination of translocation and infection is used by using a viral vector in conjunction with a plasmid vector.

[0069] In some embodiments, one or more of the rep and cap genes, as well as an AAV helper gene, are constitutively expressed by the cell and do not require transduction or transfection into the cell. In some embodiments, the cell constitutively expresses the rep and / or cap gene. In some embodiments, the cell constitutively expresses one or more AAV helper genes. In some embodiments, the cell constitutively expresses E1a. In some embodiments, the cell contains a stable transgene encoding the rAAV genome.

[0070] In some embodiments, the AAV rep, cap, and helper genes (e.g., Ela gene, E1b gene, E4 gene, E2a gene, or VA gene) can be any AAV serotype. Similarly, the AAV ITR can also be any AAV serotype. For example, in some embodiments, AAV ITR is AAV1, AAV2, rAAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, and AAV16, AAV.rh8, AAV.rh 10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B, AAV2.5, AAV2tYF, AAV3 B, AAV.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 AAV serotypes (e.g., hybrid serotypes having sequences from two or more serotypes). In some embodiments, the AAV cap gene is from the AAV9 or AAV8 cap gene.In some embodiments, the AAV cap gene is AAV1, AAV2, rAAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, and AAV16, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B, AAV2.5, AAV2tYF, AAV These are from 3B, AAV.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 AAV serotypes (e.g., hybrid serotypes having sequences from two or more serotypes). In some embodiments, the AAV rep and cap genes for rAAV particle generation are from different serotypes. For example, the rep gene is from AAV2, while the cap gene is from AAV9.

[0071] Any suitable method known in the art that can be used for translocation of cells can be used for rAAV particle production according to the method disclosed herein. In some embodiments, the method disclosed herein includes translocation of cells using a chemical-based translocation method. In some embodiments, the chemical-based translocation method uses calcium phosphate, a highly branched organic compound (dendrimer), a cationic polymer (e.g., DEAE dextran or polyethyleneimine (PEI)), or lipofection. In some embodiments, the chemical-based translocation method uses a cationic polymer (e.g., DEAE dextran or polyethyleneimine (PEI)). In some embodiments, the chemical-based translocation method uses polyethyleneimine (PEI). In some embodiments, the chemical-based translocation method uses DEAE dextran. In some embodiments, the chemical-based translocation method uses calcium phosphate.

[0072] Any suitable culture medium known in the art can be used for rAAV particle generation according to the methods disclosed herein. Such media include, but are not limited to, modified Eagle medium (MEM), Dulbecco's modified Eagle medium (DMEM), and media produced by Hydrone Laboratories and JRH, including Sf-900 II SFM medium as described in U.S. Patent No. 6,723,551 (which is incorporated herein by reference in its entirety). In some embodiments, the medium includes Invitrogen / ThermoFisher Dynamis® medium, FreeStyle® 293 expression medium, or Expi293® expression medium. In some embodiments, the medium includes Dynamis® medium. In some embodiments, the methods disclosed herein use cell cultures including serum-free medium, animal component-free medium, or chemically defined medium. In some embodiments, the medium is animal component-free medium. In some embodiments, the medium includes serum. In some embodiments, the medium includes fetal bovine serum. In some embodiments, the culture medium is glutamine-free. In some embodiments, the culture medium contains glutamine. In some embodiments, the culture medium is supplemented with one or more of the following: nutrients, salts, buffers, and additives (e.g., antifoaming agents). In some embodiments, the culture medium is supplemented with glutamine. In some embodiments, the culture medium is supplemented with serum. In some embodiments, the culture medium is supplemented with fetal bovine serum. In some embodiments, the culture medium is supplemented with poloxamer, for example, Kolliphor® P 188 Bio. In some embodiments, the culture medium is a basic medium. In some embodiments, the culture medium is a feed medium.

[0073] rAAV-producing cultures can be routinely grown under a variety of conditions suitable for the specific host cells being used (over a wide temperature range, over varying lengths of time, etc.). As is known in the art, rAAV-producing cultures include adhesion-dependent cultures that can be cultured in suitable adhesion-dependent containers (e.g., roller bottles, hollow fiber filters, multi-layer or multi-tray tissue culture flasks (i.e., stacks, e.g., hyperstacks), microcarriers, and packed-bed or fluid-bed bioreactors). Furthermore, rAAV vector-producing cultures can be cultured in suspension-adaptive host cells, such as HeLa cells, HEK293 cells, HEK293-derived cells (e.g., HEK293T cells, HEK293F cells), Vero cells, CHO cells, CHO-K1 cells, CHO-derived cells, EB66 cells, BSC cells, HepG2 cells, LLC-MK cells, CV-1 cells, COS cells, MDBK cells, MDCK cells, CRFK cells, RAF cells, RK cells, TCMK-1 cells, LLC PK cells, PK15 cells, LLC-RK cells, MDOK cells, BHK cells, BHK-21 cells, NS-1 cells, MRC-5 cells, WI-38 cells, BHK cells, 3T3 cells, 293 cells, RK cells, Per.C6 cells, chicken embryo cells, and SF-9 cells may also be included, and these can be cultured in a variety of ways, including disposable systems such as spinner flasks, agitated tank bioreactors, and wave bag systems. Numerous suspension cultures for generating rAAV particles are known in the Art, including, for example, the cultures disclosed in U.S. Patent No. 6,995,006, No. 9,783,826, and U.S. Patent Application Publication No. 20120122155 (each of which is incorporated herein by reference in whole).

[0074] Any cell or cell line known in the art to produce rAAV particles can be used in any of the methods disclosed herein. In some embodiments, the methods disclosed herein for generating or increasing rAAV particles utilize HeLa cells, HEK293 cells, HEK293-derived cells (e.g., HEK293T cells, HEK293F cells), Vero cells, CHO cells, CHO-K1 cells, CHO-derived cells, EB66 cells, BSC cells, HepG2 cells, LLC-MK cells, CV-1 cells, COS cells, MDBK cells, MDCK cells, CRFK cells, RAF cells, RK cells, TCMK-1 cells, LLCPK cells, PK15 cells, LLC-RK cells, MDOK cells, BHK cells, BHK-21 cells, NS-1 cells, MRC-5 cells, WI-38 cells, BHK cells, 3T3 cells, 293 cells, RK cells, Per.C6 cells, chicken embryo cells, or SF-9 cells. In some embodiments, the methods disclosed herein utilize mammalian cells. In some embodiments, the methods disclosed herein use insect cells, for example, SF-9 cells. In some embodiments, the methods disclosed herein use HEK293 cells. In some embodiments, the methods disclosed herein use HEK293 cells adapted for growth in suspension culture.

[0075] In some embodiments, the cell cultures disclosed herein are suspension cultures. In some embodiments, the cell cultures disclosed herein are suspension cultures containing HEK293. In some embodiments, the cell cultures disclosed herein are suspension cultures containing HEK293 cells adapted for growth in suspension culture. In some embodiments, the cell cultures disclosed herein include serum-free medium, animal component-free medium, or chemically defined medium. In some embodiments, the cell cultures disclosed herein include serum-free medium. In some embodiments, suspension-adapted cells are cultured in a shaking flask, spinner flask, cell bag, or bioreactor.

[0076] In some embodiments, the cell cultures disclosed herein include cells attached to a substrate (e.g., a microcarrier) that is suspended in the culture medium. In some embodiments, the cells are HEK293 cells.

[0077] In some embodiments, the cell cultures disclosed herein are adherent cultures. In some embodiments, the cell cultures disclosed herein are adherent cultures containing HEK293. In some embodiments, the cell cultures disclosed herein include serum-free medium, animal component-free medium, or chemically defined medium. In some embodiments, the cell cultures disclosed herein include serum-free medium.

[0078] In some embodiments, the cell cultures disclosed herein include high-density cell cultures. In some embodiments, the culture has a total cell density between approximately 1 × 10⁻⁶ cells / ml and approximately 30 × 10⁻⁶ cells / ml. In some embodiments, more than approximately 50% of the cells are viable cells. In some embodiments, the cells are HeLa cells, HEK293 cells, HEK293-derived cells (e.g., HEK293T cells, HEK293F cells), Vero cells, or SF-9 cells. In further embodiments, the cells are HEK293 cells. In further embodiments, the cells are HEK293 cells adapted for growth in suspension culture.

[0079] The methods disclosed herein can be used to generate rAAV particles containing capsid proteins from any AAV capsid serotype. In some embodiments, the rAAV particles may be AAV1, AAV2, rAAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, and AAV16, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B It contains capsid proteins from AAV capsid serotypes selected from AAV2.5, AAV2tYF, AAV3B, AAV.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, rAAV particles are AAV1, AAV2, rAAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, and AAV16, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B, AAV2. 5. Capsid proteins that are derivatives, modifiers, or pseudotypes of 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.

[0080] In some embodiments, the rAAV particles contain a capsid protein from an AAV capsid serotype selected from AAV8 and AAV9. In some embodiments, the rAAV particles have the AAV capsid serotype of AAV8. In some embodiments, the rAAV particles have the AAV capsid serotype of AAV9.

[0081] In some embodiments, the rAAV particles contain a capsid protein from an AAV capsid serotype selected from the group consisting of AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.PHB, and AAV.7m8. In some embodiments, the rAAV particles contain a capsid protein with high sequence homology to AAV8 or AAV9, such as AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, and AAV.hu37.

[0082] In some embodiments, the rAAV particles contain a capsid protein that is a derivative, modifier, or pseudotype of the AAV8 or AAV9 capsid protein. In some embodiments, the rAAV particles contain a capsid protein that is at least 80% identical to the VP1, VP2, and / or VP3 sequences of the AAV8 capsid protein, for example, 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 AAV8 capsid protein.

[0083] In some embodiments, the rAAV particles contain a capsid protein that is a derivative, modifier, or pseudotype of the AAV9 capsid protein. In some embodiments, the rAAV particles contain a capsid protein that is at least 80% identical to the VP1, VP2, and / or VP3 sequences of the AAV9 capsid protein, for example, 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 AAV9 capsid protein.

[0084] In some embodiments, the rAAV particles contain capsid proteins having at least 80% identity to the VP1, VP2, and / or VP3 sequences of AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.PHB, or AAV.7m8 capsid proteins, for example, 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, etc., i.e., up to 100% identity. In some embodiments, rAAV particles contain AAV capsid proteins with high sequence homology to AAV8 or AAV9, such as AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, and AAV.hu37, with at least 80% identity to the VP1, VP2, and / or VP3 sequences, e.g., 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, i.e., capsid proteins with up to 100% identity.

[0085] In additional embodiments, the rAAV particles include a mosaic capsid. In additional embodiments, the rAAV particles include pseudotype rAAV particles. In additional embodiments, the rAAV particles include a capsid containing a capsid protein chimera of two or more AAV capsid serotypes.

[0086] In some embodiments of the methods disclosed herein, large quantities of cell cultures may be present (e.g., during a commercial manufacturing process). In some embodiments, the methods disclosed herein are suitable for processing large quantities of cell cultures containing rAAV particles. The term “large quantities” means quantities relevant to the commercial and / or industrial production of rAAV particles. In some embodiments, the term “large quantities” means between about 20 liters and about 20,000 liters, between about 50 liters and about 20,000 liters, between about 100 liters and about 20,000 liters, between about 500 liters and about 20,000 liters, between about 1,000 liters and about 20,000 liters, between about 20 liters and about 5,000 liters, between about 50 liters and about 5,000 liters, between about 100 liters and about 3,000 liters, between about 500 liters and about 3,000 liters, and between about 1,500 liters and about 2,500 liters. In some embodiments, the term “large quantities” means between about 50 liters and about 2,000 liters. In some embodiments, the term “large quantity” means between approximately 50 liters and approximately 3,000 liters. In some embodiments, the term “large quantity” means between approximately 50 liters and approximately 5,000 liters. In some embodiments, the term “large quantity” means approximately 200 liters. In some embodiments, the term “large quantity” means approximately 500 liters. In some embodiments, the term “large quantity” means approximately 1,000 liters. In some embodiments, the term “large quantity” means approximately 1,500 liters. In some embodiments, the term “large quantity” means approximately 2,000 liters. In some embodiments, the term “large quantity” means approximately 2,500 liters. In some embodiments, the term “large quantity” means approximately 3,000 liters. In some embodiments, the term “large quantity” means approximately 5,000 liters. In some embodiments, the term “large quantity” means approximately 10,000 liters. In some embodiments, the term “large quantity” means approximately 15,000 liters.In some embodiments, the term “large quantity” means approximately 20,000 liters. In some embodiments, the term “large quantity” means between approximately 10 liters and 1,000 liters, between approximately 10 liters and 100 liters, between approximately 20 liters and 500 liters, between approximately 50 liters and 500 liters, between approximately 100 liters and 1,000 liters, or between approximately 100 liters and 500 liters.

[0087] rAAV particles The method provided is suitable for use in the generation of any isolated recombinant AAV particles. Therefore, rAAVs produced according to the method of this disclosure may be any serotype, modifier, or derivative known in the art, or any combination thereof (e.g., a population of rAAV particles containing two or more serotypes, e.g., AAV1, AAV2, rAAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, and AAV16, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.Anc80, AA This can be a group containing two or more of the following: V.Anc80L65, AAV.7m8, AAV.PHP.B, AAV2.5, AAV2tYF, AAV3B, AAV.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 of these.

[0088] In some embodiments, rAAV particles are AAV1, AAV2, rAAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, and AAV16, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B, AAV2.5, AAV2t It possesses a capsid protein from an AAV serotype selected from YF, AAV3B, AAV.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 their derivatives, modifications, or pseudotypes.In some embodiments, rAAV particles are, for example, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, and AAV16, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.Anc80, rAAV.Anc80L65, AAV.7m8, AAV.PHP.B, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AA It contains a capsid protein that is at least 80% identical to the VP1, VP2, and / or VP3 sequences of an AAV capsid serotype selected from V.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, for example, 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, etc., i.e., up to 100% identical.

[0089] In some embodiments, the rAAV particles are AAV1, AAV2, rAAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, and AAV16, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B, AAV2.5, AAV2tY This includes capsid proteins from AAV capsid serotypes selected from F, AAV3B, AAV.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 their derivatives, modifiers, or pseudotypes.In some embodiments, rAAV particles are, for example, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, and AAV16, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV It contains a capsid protein that is at least 80% identical to the VP1, VP2, and / or VP3 sequences of an AAV capsid serotype selected from .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, for example, 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, etc., i.e., up to 100% identical.

[0090] In some embodiments, the rAAV particles comprise a capsid of Anc80 or Anc80L65, as described in Zinn et al., 2015, Cell Rep. 12(6):1056-1068 (which is incorporated herein by reference in its entirety). In certain embodiments, the rAAV particles comprise a capsid having one of the amino acid inserts: LGETTRP or LALGETTRP, as described in U.S. Patents 9,193,956, 9,458,517, and 9,587,282, and U.S. Patent Application Publication 2016 / 0376323 (each of which is incorporated herein by reference in its entirety). In some embodiments, the rAAV particles include the capsid of 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 some embodiments, the rAAV particles include any AAV capsid disclosed in U.S. Patent No. 9,585,971, e.g., AAV-PHP.B. In some embodiments, the rAAV particles include any AAV capsid disclosed in U.S. Patent No. 9,840,719 and WO2015 / 013313 (each of which is incorporated herein by reference in its entirety), e.g., AAV.Rh74 and RHM4-1. In some embodiments, the rAAV particles include any AAV capsid disclosed in WO2014 / 172669 (which is incorporated herein by reference in its entirety), e.g., AAV rh.74. In some embodiments, the rAAV particles include the AAV2 / 5 capsid 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 some embodiments, the rAAV particles include any AAV capsid disclosed in WO2017 / 070491 (which is incorporated herein by reference in its entirety), e.g., AAV2tYF.In some embodiments, the rAAV particles include a capsid of AAVLK03 or AAV3B, as described in Puzzo et al., 2017, Sci. Transl. Med. 29(9):418. In some embodiments, the rAAV particles include any AAV capsid disclosed in U.S. Patent No. 8,628,966, U.S. Patent No. 8,927,514, U.S. Patent No. 9,923,120, and WO2016 / 049230, e.g., HSC1, HSC2, HSC3, HSC4, HSC5, HSC6, HSC7, HSC8, HSC9, HSC10, HSC11, HSC12, HSC13, HSC14, HSC15, or HSC16 (each of these, in whole, is incorporated herein by reference).

[0091] In some embodiments, rAAV particles are used in the following patents and patent applications (each of which is incorporated herein by reference in whole): 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, U.S. Patent Nos. 9,284,357, 9,409,953, 9,169,299, 9,193, This includes AAV capsids disclosed in any of the following: Patent Nos. 956, 9458517, and 9,587,282; U.S. Patent Application Publications 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 a capsid protein that is at least 80% 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 herein by reference in whole): for example, 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, etc., i.e., up to 100% identical: U.S. Patent No. 7,282,199, No. 7,906,111, No. 8,524,446, No. 8,9 U.S. Patent Applications Nos. 99,678, 8,628,966, 8,927,514, 8,734,809, U.S. Patent Nos. 9,284,357, 9,409,953, 9,169,299, 9,193,956, 9,458,517, and 9,587,282, U.S. Patent Application Publication No. U.S.2015 Patent applications No. / 0374803, No. 2015 / 0126588, No. 2017 / 0067908, No. 2013 / 0224836, No. 2016 / 0215024, No. 2017 / 0051257, and international patent applications PCT / US2015 / 034799 and PCT / EP2015 / 053335.

[0092] In some embodiments, rAAV particles are used in International Patent Application Publications WO2003 / 052051 (see, e.g., SEQ ID NO: 2), WO2005 / 033321 (see, e.g., SEQ ID NOs: 123 and 88), WO03 / 042397 (see, e.g., SEQ ID NOs: 2, 81, 85, and 97), WO2006 / 068888 (see, e.g., SEQ ID NOs: 1 and 3-6), WO2006 / 110689 (see, e.g., SEQ ID NOs: 5-38), WO2009 / 1 The capsid protein is disclosed in Patent No. 04964 (see, for example, SEQ ID NOs. 1-5, 7, 9, 20, 22, 24, and 31), WO2010 / 127097 (see, for example, SEQ ID NOs. 5-38), and WO2015 / 191508 (see, for example, SEQ ID NOs. 80-294), and U.S. Patent Application Publication No. 20150023924 (see, for example, SEQ ID NOs. 1, 5-10) (each of which is incorporated herein by reference in its entirety). In some embodiments, the rAAV particles have a capsid protein that is at least 80% identical to the VP1, VP2 and / or VP3 sequences of the AAV capsid disclosed below, e.g., 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, i.e., up to 100% identical: International Patent Application Publication WO2003 / 052051 (see, e.g., SEQ ID NO: 2), WO2005 / 033321 (see, e.g., SEQ ID NOs: 123 and 88), WO03 / 04239 See, for example, Sequence IDs 2, 81, 85, and 97; WO2006 / 068888 (see, for example, Sequence IDs 1 and 3-6); WO2006 / 110689 (see, for example, Sequence IDs 5-38); WO2009 / 104964 (see, for example, Sequence IDs 1-5, 7, 9, 20, 22, 24, and 31); WO2010 / 127097 (see, for example, Sequence IDs 5-38); and WO2015 / 191508 (see, for example, Sequence IDs 80-294); and U.S. Patent Application Publication No. 20150023924 (see, for example, Sequence IDs 1, 5-10).

[0093] Nucleic acid sequences of AAV-based viral vectors, as well as methods for producing recombinant AAV and AAV capsids, are taught, for example, in: U.S. Patents Nos. 7,282,199, 7,906,111, 8,524,446, 8,999,678, 8,628,966, 8,927,514, 8,734,809, U.S. Patents Nos. 9,284,357, 9,409,953, 9,169,299, 9,193,956, 9458517, and 9,587,282, U.S. Patent Application Publications 2015 / 0374803 and 2015 / 0126588. Patent applications No. 2017 / 0067908, No. 2013 / 0224836, No. 2016 / 0215024, No. 2017 / 0051257, International Patent Application No. PCT / US2015 / 034799, No. PCT / EP2015 / 053335, No. WO2003 / 052051, No. WO2005 Patent applications Nos. / 033321, WO03 / 042397, WO2006 / 068888, WO2006 / 110689, WO2009 / 104964, WO2010 / 127097, and WO2015 / 191508, as well as U.S. Patent Application Publication No. 20150023924.

[0094] The provided method is suitable for use in the generation of recombinant AAV encoding a transgene. In certain embodiments, the transgenes are from Tables 1A-1C. In some embodiments, the rAAV genome comprises a vector containing the following components: (1) AAV reverse terminal repeats adjacent to the expression cassette, (2) regulatory elements, e.g., a) promoter / enhancer, b) polyA signal, and c) introns, optionally, and (3) a nucleic acid sequence encoding the transgene. In other embodiments for expressing intact or substantially intact monoclonal antibodies (mAbs), the rAAV genome comprises a vector containing the following components: (1) AAV reverse terminal repeats adjacent to the expression cassette, (2) regulatory elements, e.g., a) promoter / enhancer, b) polyA signal, and c) introns, optionally, and (3) a nucleic acid sequence encoding the light chain Fab and heavy chain Fab of the antibody, or at least the heavy chain or light chain Fab, and optionally the heavy chain Fc region. In yet another embodiment for expressing intact or substantially intact mAbs, the rAAV genome comprises a vector comprising: (1) AAV reverse terminal repeats adjacent to the expression cassette; (2) regulatory elements, e.g., a) promoter / enhancer, b) polyA signaling, and c) introns of choice;(3) Nucleic acid sequences encoding heavy chain Fab of the following: anti-VEGF (e.g., sevacizumab, ranibizumab, bevacizumab, and brolucizumab), anti-EpoR (e.g., LKA-651), anti-ALK1 (e.g., askrinbakumab), anti-C5 (e.g., tesidolumab and eculizumab), anti-CD105 (e.g., carotuximab), anti-CC1Q (e.g., ANX-007), anti-TNFα (e.g., adalimumab, infliximab, and golim) Anti-IL-5 (e.g., mepolizumab), anti-IL-12 / IL-23 (e.g., ustekinumab), anti-IL-19 (e.g., inebilizumab), anti-IL-19 (e.g., inebilizumab), anti-IL-19 (e.g., elezanumab), anti-IL-19 (e.g., lezanumab), anti-IL-19 (e.g., inebilizumab), anti-IL-19 (e.g., inebilizumab), anti-IL-19 (e.g., elezan mAb (e.g., etrolizumab), anti-SOST mAb (e.g., romosozumab), anti-pKal mAb (e.g., lanadelmab), anti-ITGA4 (e.g., natalizumab), anti-ITGA4B7 (e.g., vedolizumab), anti-BLyS (e.g., belimumab), anti-PD-1 (e.g., nivolumab and pembrolizumab), anti-RANKL (e.g., densomab), anti-PCSK9 (e.g., alirocumab and evolocumab), anti-ANGPTL3 (e.g., evinacumab*), anti-OxPL (e.g., E06), anti-fD (e.g., lamparizumab), or anti-MMP9 (e.g., andecaliximab); optionally, an Fc polypeptide of the same isotype as the native form of the therapeutic antibody, e.g., IgG isotype amino acid sequence IgG1, IgG2, or IgG4, or modified Fc thereof;Furthermore, nucleic acid sequences encoding the light chains of the following: anti-VEGF (e.g., sevacizumab, ranibizumab, bevacizumab, and brolucizumab), anti-EpoR (e.g., LKA-651), anti-ALK1 (e.g., askrinbakumab), anti-C5 (e.g., tesidolumab and eculizumab), anti-CD105 or anti-ENG (e.g., carotuximab), anti-CC1Q (e.g., ANX-007), anti-TNFα (e.g., adalimumab, infliximab, and goli Mumab), anti-RGMa (e.g., elezanumab), anti-TTR (e.g., NI-301 and PRX-004), anti-CTGF (e.g., pamlevlumab), anti-IL6R (e.g., satralizumab and sarilumab), anti-IL4R (e.g., dupilumab), anti-IL17A (e.g., ixekizumab and secukinumab), anti-IL-5 (e.g., mepolizumab), anti-IL12 / IL23 (e.g., ustekinumab), anti-CD19 (e.g., inebilizumab), anti-ITGF7 mAbs (e.g., etrolizumab), anti-SOST mAbs (e.g., romosozumab), anti-pKal mAbs (e.g., lanadelmab), anti-ITGA4 (e.g., natalizumab), anti-ITGA4B7 (e.g., vedolizumab), anti-BLyS (e.g., belimumab), anti-PD-1 (e.g., nivolumab and pembrolizumab), anti-RANKL (e.g., densomab), anti-PCSK9 (e.g., alirocumab and evolocumab), anti-ANGPTL3 (e.g., evinacumab), anti-OxPL (e.g., E06), anti-fD (e.g., lampalizumab), or anti-MMP9 (e.g., andecaliximab). At this time, the heavy chain (Fab and optionally the Fc region) and light chain are separated by self-cleaving furin(F) / F2A or a flexible linker to ensure that the heavy chain and light chain polypeptides are expressed in equal amounts.

[0095] (Table 1A) TIFF0007900915000001.tif211165TIFF0007900915000002.tif252166TIFF0007900915 000003.tif252166TIFF0007900915000004.tif252167TIFF0007900915000005.tif49165

[0096] (Table 1B) TIFF0007900915000006.tif198146TIFF0007900915000007.tif246147TIFF0007900915000008.tif61146

[0097] (Table 1C) TIFF0007900915000009.tif192146TIFF0007900915000010.tif250148TIFF0007900915000011.tif40146

[0098] In some embodiments, rAAV viral vectors encoding anti-VEGF Fab are provided herein. In specific embodiments, rAAV8-based viral vectors encoding anti-VEGF Fab are provided herein. In more specific embodiments, rAAV8-based viral vectors encoding ranibizumab are provided herein. In some embodiments, rAAV viral vectors encoding idulonidase (IDUA) are provided herein. In specific embodiments, rAAV9-based viral vectors encoding IDUA are provided herein. In some embodiments, rAAV viral vectors encoding iduronate 2-sulfatase (IDS) are provided herein. In specific embodiments, rAAV9-based viral vectors encoding IDS are provided herein. In some embodiments, rAAV viral vectors encoding low-density lipoprotein receptor (LDLR) are provided herein. In specific embodiments, rAAV8-based viral vectors encoding LDLR are provided herein. In some embodiments, rAAV viral vectors encoding tripeptidyl peptidase 1 (TPP1) protein are provided herein. In specific embodiments, rAAV9-based viral vectors encoding TPP1 are provided herein. In some embodiments, rAAV viral vectors encoding a non-membrane-bound splice variant of VEGF receptor 1 (sFlt-1) are provided herein.In some embodiments, the following are used herein: gamma-sarcoglycan, Rab escort protein 1 (REP1 / CHM), retinoid isomerohydrolase (RPE65), cyclic nucleotide gate channel alpha-3 (CNGA3), cyclic nucleotide gate channel beta-3 (CNGB3), aromatic L-amino acid decarboxylase (AADC), lysosome-associated membrane protein 2 isoform B (LAMP2B), factor VIII, factor IX, retinitis pigmentosa GTPase regulator (RPGR), retinoskin (R S1) Sarcoplasmic reticulum calcium ATPase (SERCA2a), aflibercept, battenin (CLN3), transmembrane ER protein (CLN6), glutamate decarboxylase (GAD), glial cell line-derived neurotrophic factor (GDNF), aquaporin 1 (AQP1), dystrophin, myotubularin 1 (MTM1), follistatin (FST), glucose-6-phosphatase (G6Pase), apolipoprotein A2 (APOA2), uridine diphosphate glucuronosyltransferase 1A1 (UGT1A1) , arylsulfatase B (ARSB), N-acetyl-alpha-glucosaminidase (NAGLU), alpha-glucosidase (GAA), alpha-galactosidase (GLA), beta-galactosidase (GLB1), lipoprotein lipase (LPL), alpha-1-antitrypsin (AAT), phosphodiesterase 6B (PDE6B), ornithine carbamoyltransferase 9OTC), survival motor neuron (SMN1), survival motor neuron (SMN2), neuronurulin (NRTN), neuron rAAV viral vectors are provided that encode rofin-3 (NT-3 / NTF3), porphobilinogen deaminase (PBGD), nerve growth factor (NGF), mitochondrial-coded NADH:ubiquinone oxidoreductase core subunit 4 (MT-ND4), protective protein cathepsin A (PPCA), dyspherin, MER proto-oncogene tyrosine kinase (MERTK), cystic fibrosis membrane conductance regulator (CFTR), or tumor necrosis factor receptor (TNFR)-immunoglobulin (IgG1)Fc fusion.

[0099] In additional embodiments, the rAAV particles comprise a pseudotype AAV capsid. In some embodiments, the pseudotype AAV capsid is an rAAV2 / 8 or rAAV2 / 9 pseudotype AAV capsid. Methods for generating and using pseudotype rAAV particles are known in the art (see, for example, 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)).

[0100] In additional embodiments, the rAAV particles contain a capsid comprising a capsid protein that is a chimeric of two or more AAV capsid serotypes. In some embodiments, the capsid protein is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, and AAV16, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B, AA It is a chimera of two or more AAV capsid proteins from an AAV serotype selected from V2.5, AAV2tYF, AAV3B, AAV.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.

[0101] In certain embodiments, single-stranded AAVs (ssAAVs) may be used. In certain embodiments, self-complementary vectors, such as scAAVs, may be used (see, for example, Wu, 2007, Human Gene Therapy, 18(2):171-82; McCarty et al, 2001, Gene Therapy, Vol. 8, Number 16:1248-1254; and U.S. Patents 6,596,535, 7,125,717, and 7,456,683 (each of which is incorporated herein by reference in whole)).

[0102] In some embodiments, rAAV particles contain a capsid protein from an AAV capsid serotype selected from AAV8 or AAV9. In some embodiments, rAAV particles contain a capsid protein from an AAV capsid serotype selected from the group consisting of AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.PHB, and AAV.7m8. In some embodiments, rAAV particles contain a capsid protein with high sequence homology to AAV8 or AAV9, e.g., AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, and AAV.hu37. In some embodiments, rAAV particles have an AAV1, or a derivative, modified, or pseudotype AAV capsid serotype thereof. In some embodiments, the rAAV particles have AAV4, or its derivatives, modifiers, or pseudotype AAV capsid serotypes. In some embodiments, the rAAV particles have AAV5, or its derivatives, modifiers, or pseudotype AAV capsid serotypes. In some embodiments, the rAAV particles have AAV8, or its derivatives, modifiers, or pseudotype AAV capsid serotypes. In some embodiments, the rAAV particles have AAV9, or its derivatives, modifiers, or pseudotype AAV capsid serotypes.

[0103] In some embodiments, the rAAV particles contain a capsid protein that is a derivative, modifier, or pseudotype of the AAV8 or AAV9 capsid protein. In some embodiments, the rAAV particles contain a capsid protein that is at least 80% identical to the VP1, VP2, and / or VP3 sequences of the AAV8 capsid protein, for example, 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 AAV8 capsid protein.

[0104] In some embodiments, the rAAV particles contain a capsid protein that is a derivative, modifier, or pseudotype of the AAV9 capsid protein. In some embodiments, the rAAV particles contain a capsid protein that is at least 80% identical to the VP1, VP2, and / or VP3 sequences of the AAV9 capsid protein, for example, 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 AAV8 capsid protein.

[0105] In some embodiments, the rAAV particles contain capsid proteins having at least 80% identity to the VP1, VP2, and / or VP3 sequences of AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.PHB, or AAV.7m8 capsid proteins, for example, 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, etc., i.e., up to 100% identity. In some embodiments, rAAV particles contain AAV capsid proteins with high sequence homology to AAV8 or AAV9, such as AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, and AAV.hu37, with at least 80% identity to the VP1, VP2, and / or VP3 sequences, e.g., 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, i.e., capsid proteins with up to 100% identity.

[0106] In additional embodiments, rAAV particles include mosaic capsids. Mosaic AAV particles consist of a mixture of viral capsid proteins from different serotypes of AAV. In some embodiments, rAAV particles include AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, and AAV16, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B, A It contains a mosaic capsid containing a capsid protein of a serotype selected from AV2.5, AAV2tYF, AAV3B, AAV.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.

[0107] In some embodiments, the rAAV particles contain a mosaic capsid comprising a capsid protein of a serotype selected from AAV1, AAV2, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAVrh.8, and AAVrh.10.

[0108] In additional embodiments, the rAAV particles include pseudotyped rAAV particles. In some embodiments, the pseudotyped rAAV particles include (a) a nucleic acid vector containing AAV ITR and (b) a capsid consisting of a capsid protein derived from AAVx (e.g., AAV1, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, and AAV16). In an additional embodiment, the rAAV particles are AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, and AAV16, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B, AAV2.5, The rAAV particles include pseudotyped rAAV particles composed of capsid proteins of AAV serotypes selected from AAV2tYF, AAV3B, AAV.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 an additional embodiment, the rAAV particles include pseudotyped rAAV particles containing the AAV8 capsid protein. In an additional embodiment, the rAAV particles include pseudotyped rAAV particles composed of the AAV9 capsid protein. In some embodiments, pseudotype rAAV8 or rAAV9 particles are rAAV2 / 8 or rAAV2 / 9 pseudotype particles.Methods for generating and using pseudotype rAAV particles are known in the art (see, for example, 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)).

[0109] In an additional embodiment, the rAAV particle comprises a capsid containing a capsid protein that is a chimeric of two or more AAV capsid serotypes. In a further embodiment, the capsid protein comprises AAV1, AAV2, rAAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, and AAV16, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B, AA In a further embodiment, the capsid protein is a chimera of two or more AAV capsid proteins from AAV serotypes selected from V2.5, AAV2tYF, AAV3B, 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 a further embodiment, the capsid protein is a chimera of two or more AAV capsid proteins from AAV serotypes selected from AAV1, AAV2, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAVrh.8, and AAVrh.10.

[0110] In some embodiments, rAAV particles contain the AAV8 capsid protein and AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, and AAV16, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B, and AAV2 It contains an AAV capsid protein that is a chimera with one or more AAV capsid proteins from AAV serotypes selected from .5, AAV2tYF, AAV3B, AAV.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 contain an AAV capsid protein that is a chimera of the AAV8 capsid protein and one or more AAV capsid proteins from AAV serotypes selected from AAV1, AAV2, AAV5, AAV6, AAV7, AAV9, AAV10, AAVrh.8, and AAVrh.10.

[0111] In some embodiments, rAAV particles contain the AAV9 capsid protein and AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, and AAV16, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B, and AAV2 It contains an AAV capsid protein that is a chimera with a capsid protein of one or more AAV capsid serotypes selected from .5, AAV2tYF, AAV3B, AAV.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.

[0112] In some embodiments, the rAAV particles contain an AAV capsid protein that is a chimera of the AAV9 capsid protein and the capsid protein of one or more AAV capsid serotypes selected from AAV1, AAV2, AAV3, AAV4, AAV5, AA6, AAV7, AAV8, AAV9, AAVrh.8, and AAVrh.10.

[0113] Method for isolating rAAV particles

[0114] The methods for generating rAAV particles disclosed herein (e.g., any one of the methods described in [1] to

[0129] ) can be used in combination with upstream processing for isolating the rAAV particles.

[0115] rAAV particles produced according to the methods disclosed herein (e.g., any one of the methods in [1] to

[0129] ) can be isolated using methods known in the art. In some embodiments, a method for isolating rAAV particles produced according to the methods disclosed herein includes downstream processing, e.g., collection of cell cultures, clarification of the collected cell cultures (e.g., by centrifugation or deep filtration), tangential flow filtration, affinity chromatography, anion exchange chromatography, cation exchange chromatography, size exclusion chromatography, hydrophobic interaction chromatography, hydroxyapatite chromatography, sterile filtration, or any combination(s) of these. 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: collection of cell cultures, clarification of the collected cell cultures (e.g., by centrifugation or deep filtration), tangential flow filtration, affinity chromatography, anion exchange chromatography, cation exchange chromatography, size exclusion chromatography, hydrophobic interaction chromatography, hydroxyapatite chromatography, and sterile filtration. In some embodiments, the downstream process includes collecting the cell culture, clarifying the collected cell culture (e.g., by deep filtration), sterile filtration, tangential flow filtration, affinity chromatography, and anion exchange chromatography. In some embodiments, the downstream process includes clarifying the collected cell culture, sterile filtration, tangential flow filtration, affinity chromatography, and anion exchange chromatography. In some embodiments, the downstream process includes clarifying the collected cell culture by deep filtration, sterile filtration, tangential flow filtration, affinity chromatography, and anion exchange chromatography. In some embodiments, the clarification of the collected cell culture includes sterile filtration. In some embodiments, the downstream process does not include centrifugation. In some embodiments, the rAAV particles contain the AAV8 serotype capsid protein.In some embodiments, the rAAV particles contain a capsid protein of the AAV9 serotype.

[0116] In some embodiments, a method for isolating rAAV particles according to the method disclosed herein includes collecting a cell culture, clarifying the collected cell culture (e.g., by deep filtration), a first sterile filtration, a first tangential flow filtration, affinity chromatography, anion exchange chromatography (e.g., monolithic anion exchange chromatography or AEX chromatography using a quaternary amine ligand), a second tangential flow filtration, and a second sterile filtration. In some embodiments, a method for isolating rAAV particles produced according to the method disclosed herein includes clarification of the collected cell culture, a first sterile filtration, a first tangential flow filtration, affinity chromatography, anion exchange chromatography (e.g., monolithic anion exchange chromatography or AEX chromatography using a quaternary amine ligand), a second tangential flow filtration, and a second sterile filtration.In some embodiments, a method for isolating rAAV particles produced according to the method disclosed herein includes clarification of the collected cell culture by deep filtration, a first sterile filtration, a first tangential flow filtration, affinity chromatography, anion exchange chromatography (e.g., monolithic anion exchange chromatography or AEX chromatography using a quaternary amine ligand), a second tangential flow filtration, and a second sterile filtration. In some embodiments, a method for isolating rAAV particles disclosed herein includes clarification of the collected cell culture by deep filtration, a first sterile filtration, affinity chromatography, anion exchange chromatography (e.g., monolithic anion exchange chromatography or AEX chromatography using a quaternary amine ligand), tangential flow filtration, and a second sterile filtration. In some embodiments, the method does not include centrifugation. In some embodiments, clarification of the collected cell culture includes sterile filtration. In some embodiments, the rAAV particles contain the capsid protein of the AAV8 serotype. In some embodiments, the rAAV particles contain the capsid protein of the AAV9 serotype.

[0117] Numerous methods for generating rAAV particles are known in the art, including transfusion, stable cell line generation, and infectious hybrid virus generation systems (including adenovirus-AAV hybrids, herpesvirus-AAV hybrids, and baculovirus-AAV hybrids). Any rAAV-producing culture for generating rAAV virus particles requires (1) a suitable host cell (in the case of baculoviruses, for example, a human cell line (e.g., HeLa, A549, or HEK293 cells and their derivatives (HEK293T cells, HEK293F cells)), a mammalian cell line (e.g., Vero), or an insect cell line (e.g., SF-9)); (2) a suitable helper virus function provided by a wild-type or mutant adenovirus (e.g., a temperature-sensitive adenovirus), a herpesvirus, a baculovirus, or a plasmid construct providing helper function; (3) AAV rep and cap genes and gene products; (4) a transgene adjacent to the AAV ITR sequence (e.g., a therapeutic transgene); and (5) a suitable medium and medium components to support rAAV production. Suitable media known in the art can be used for the production of rAAV vectors. Such media include, but are not limited to, modified Eagle medium (MEM), Dulbecco's modified Eagle medium (DMEM), and media produced by Hyclone Laboratories and JRH, including Sf-900 II SFM medium as described in U.S. Patent No. 6,723,551 (which is incorporated herein by reference in its entirety).

[0118] rAAV-producing cultures can be routinely grown under various conditions suitable for the specific host cells being used (over a wide temperature range, over varying lengths of time, etc.). As is known in the art, rAAV-producing cultures include adhesion-dependent cultures that can be cultured in suitable adhesion-dependent containers (e.g., roller bottles, hollow fiber filters, microcarriers, and packed or fluidized bed bioreactors). Furthermore, rAAV vector-producing cultures can be grown with suspension-adaptive host cells, such as HeLa cells, HEK293 cells, HEK293-derived cells (e.g., HEK293T cells, HEK293F cells), Vero cells, CHO cells, CHO-K1 cells, CHO-derived cells, EB66 cells, BSC cells, HepG2 cells, LLC-MK cells, CV-1 cells, COS cells, MDBK cells, MDCK cells, CRFK cells, RAF cells, RK cells, TCMK-1 cells, LLC The cells may also include PK cells, PK15 cells, LLC-RK cells, MDOK cells, BHK cells, BHK-21 cells, NS-1 cells, MRC-5 cells, WI-38 cells, BHK cells, 3T3 cells, 293 cells, RK cells, Per.C6 cells, chicken embryo cells, or SF-9 cells, which can be cultured in a variety of ways, including, for example, spinner flasks, agitated tank bioreactors, and disposable systems such as Wave bag systems. In some embodiments, the cells are HEK293 cells. In some embodiments, the cells are HEK293 cells adapted for growth in suspension culture. Numerous suspension cultures for generating rAAV particles are known in the Art, including, for example, the cultures disclosed in U.S. Patent No. 6,995,006, No. 9,783,826, and U.S. Patent Application Publication No. 20120122155 (each of which is incorporated herein by reference in whole).

[0119] In some embodiments, the rAAV-producing culture comprises a high-density cell culture. In some embodiments, the culture has a total cell density between approximately 1 × 10⁻⁶ cells / ml and approximately 30 × 10⁻⁶ cells / ml. In some embodiments, more than approximately 50% of the cells are viable cells. In some embodiments, the cells are HeLa cells, HEK293 cells, HEK293-derived cells (e.g., HEK293T cells, HEK293F cells), Vero cells, or SF-9 cells. In further embodiments, the cells are HEK293 cells. In further embodiments, the cells are HEK293 cells adapted for growth in suspension culture.

[0120] In additional embodiments of the provided method, the rAAV-producing culture includes a suspension culture containing rAAV particles. Numerous suspension cultures for producing rAAV particles are known in the Art, including, for example, the cultures disclosed in U.S. Patent No. 6,995,006, No. 9,783,826, and U.S. Patent Application Publication No. 20120122155 (each of which is incorporated herein by reference in whole). In some embodiments, the suspension culture includes a culture of mammalian cells or insect cells. In some embodiments, the suspension culture includes cultures of HeLa cells, HEK293 cells, HEK293-derived cells (e.g., HEK293T cells, HEK293F cells), Vero cells, CHO cells, CHO-K1 cells, CHO-derived cells, EB66 cells, BSC cells, HepG2 cells, LLC-MK cells, CV-1 cells, COS cells, MDBK cells, MDCK cells, CRFK cells, RAF cells, RK cells, TCMK-1 cells, LLCPK cells, PK15 cells, LLC-RK cells, MDOK cells, BHK cells, BHK-21 cells, NS-1 cells, MRC-5 cells, WI-38 cells, BHK cells, 3T3 cells, 293 cells, RK cells, Per.C6 cells, chicken embryo cells, or SF-9 cells. In some embodiments, the suspension culture includes a culture of HEK293 cells.

[0121] Recombinant AAV particles can be collected from rAAV-producing cultures by collecting the product culture containing host cells, or by collecting the consumed medium from the product culture, provided that the cells are cultured under conditions known in the art to induce the release of rAAV particles from intact host cells into the culture medium. Recombinant AAV particles can also be collected from rAAV-producing cultures by lysing the host cells of the product culture. Suitable methods for lysing cells are also known in the art, and include, for example, multiple freeze / thaw cycles, sonication, microfluidization, and treatment with chemicals (e.g., surfactants and / or proteases).

[0122] At collection, rAAV-producing cultures may contain one or more of the following: (1) host cell proteins; (2) host cell DNA; (3) plasmid DNA; (4) helper viruses; (5) helper virus proteins; (6) helper virus DNA; and (7) culture medium components (e.g., serum proteins, amino acids, transferrin, and other low molecular weight proteins). rAAV-producing cultures may also contain product-related impurities, such as inactive vector forms, empty viral capsids, aggregated viral particles or capsids, misfolded viral capsids, and degraded viral particles.

[0123] In some embodiments, the rAAV-producing culture collection is clarified to remove host cell debris. In some embodiments, the producing culture collection is clarified by filtration through a series of deep filters. Clarification can also be achieved by various other standard techniques known in the art, for example, by centrifugation or by filtration through any cellulose acetate filter with a pore size of 0.2 mm or larger known in the art. In some embodiments, clarification of the collected cell culture includes sterile filtration. In some embodiments, the producing culture collection is clarified by centrifugation. In some embodiments, clarification of the producing culture collection does not include centrifugation.

[0124] In some embodiments, the collected cell culture is clarified using filtration. In some embodiments, the clarification of the collected cell culture includes deep filtration. In some embodiments, the clarification of the collected cell culture further includes deep filtration and sterile filtration. In some embodiments, the collected cell culture is clarified using a filter train containing one or more different filtration media. In some embodiments, the filter train contains one deep filtration medium. In some embodiments, the filter train contains one or more deep filtration media. In some embodiments, the filter train contains two deep filtration media. In some embodiments, the filter train contains one sterile filtration medium. In some embodiments, the filter train contains two deep filtration media and one sterile filtration medium. In some embodiments, the deep filtration medium is a porous deep filter. In some embodiments, the filter train contains Clarisolve® 20MS, Millistak+® C0HC, and sterile-grade filtration media. In some embodiments, the filter train includes Clarisolve® 20MS, Millistak+® C0HC, and Sartopore® 2 XLG 0.2 μm. In some embodiments, the collected cell culture is pretreated before contact with the deep filter. In some embodiments, the pretreatment includes adding salt to the collected cell culture. In some embodiments, the pretreatment includes adding a chemical flocculant to the collected cell culture. In some embodiments, the collected cell culture is not pretreated before contact with the deep filter.

[0125] In some embodiments, the resulting culture collection is clarified by filtration as disclosed in PCT International Patent Application No. PCT / US2019 / 029539, filed on 27 April 2019, entitled “SCALABLE CLARIFICATION PROCESS FOR RECOMBINANT AAV PRODUCTION” (which is incorporated herein by reference in its entirety).

[0126] In some embodiments, rAAV-producing culture collections are treated with a nuclease (e.g., Bensonase®) or endonuclease (e.g., endonuclease from Serratia marcescens) to digest the high molecular weight DNA present in the producing culture. Nuclease or endonuclease digestion can be routinely carried out under standard conditions known in the art. For example, nuclease digestion is carried out for 30 minutes to several hours with a final concentration of 1 to 2.5 units / ml of Bensonase® at temperatures ranging from ambient temperature to 37°C.

[0127] Aseptic filtration encompasses filtration using a sterile-grade filter medium. In some embodiments, the sterile-grade filter medium is a 0.2 or 0.22 μm pore filter. In some embodiments, the sterile-grade filter medium contains polyethersulfone (PES). In some embodiments, the sterile-grade filter medium contains polyvinylidene fluoride (PVDF). In some embodiments, the sterile-grade filter medium has a hydrophilic heterogeneous double-layer design. In some embodiments, the sterile-grade filter medium has a hydrophilic heterogeneous double-layer design of a 0.8 μm pre-filter and a 0.2 μm final filter membrane. In some embodiments, the sterile-grade filter medium has a hydrophilic heterogeneous double-layer design of a 1.2 μm pre-filter and a 0.2 μm final filter membrane. In some embodiments, the sterile-grade filter medium is a 0.2 or 0.22 μm pore filter. In further embodiments, the sterile-grade filter medium is a 0.2 μm pore filter. In some embodiments, the sterile-grade filter medium is a combination of nominal pore sizes of Sartopore® 2 XLG 0.2 μm, Durapore® PVDF membrane 0.45 μm, or Sartoguard® PES 1.2 μm + 0.2 μm. In some embodiments, the sterile-grade filter medium is Sartopore® 2 XLG 0.2 μm.

[0128] In some embodiments, the clarified feed is concentrated via tangential flow filtration ("TFF") before being applied to a chromatographic medium, such as an affinity chromatography medium. Large-scale concentrations of viruses using TFF ultrafiltration are described in Paul et al., Human Gene Therapy 4:609-615 (1993). The TFF concentration of the clarified feed allows for chromatographic application of technically controllable amounts of the clarified feed and enables more rational column sizing without requiring long recirculation times. In some embodiments, the clarified feed is concentrated between at least 2x and at least 10x. In some embodiments, the clarified feed is concentrated between at least 10x and at least 20x. In some embodiments, the clarified feed is concentrated between at least 20x and at least 50x. In some embodiments, the clarified feed is concentrated to about 20x. Those skilled in the art will also recognize that TFF may be used to remove small molecule impurities (e.g., cell culture impurities including culture medium components, serum albumin, or other serum proteins) from a feed clarified via diafiltration. In some embodiments, the clarified feed is subjected to diafiltration to remove small molecule impurities. In some embodiments, the diafiltration includes using a diafiltration volume of buffer between about 3 and about 10. In some embodiments, the diafiltration includes using a diafiltration volume of buffer between about 5. Those skilled in the art will also recognize that TFF may be used at any step of the purification process where it is desirable to exchange the buffer before carrying out the next step in the purification process. In some embodiments, the method for isolating rAAV from a clarified feed disclosed herein includes the use of TFF for buffer exchange.

[0129] Affinity chromatography can be used to isolate rAAV particles from a 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 feed that has been clarified by tangential flow filtration. Suitable affinity chromatography media are known in the art and are not limited to AVB Sepharose®, POROS® CaptureSelect® AAVX affinity resin, POROS® CaptureSelect® AAV9 affinity resin, and POROS® CaptureSelect® AAV8 affinity resin. In some embodiments, the affinity chromatography medium is POROS® CaptureSelect® AAV9 affinity resin. In some embodiments, the affinity chromatography medium is POROS® CaptureSelect® AAV8 affinity resin. In some embodiments, the affinity chromatography medium is POROS® CaptureSelect® AAVX affinity resin.

[0130] Anion exchange chromatography can be used to isolate rAAV particles from a composition. In some embodiments, anion exchange chromatography is used after affinity chromatography as a final concentration and polishing step. Suitable anion exchange chromatography media are known in the art and are not limited to, but include, Unosphere Q (Biorad, Hercules, Calif.) and N-charged amino or imino resins, e.g., POROS 50 PI, or any DEAE, TMAE, tertiary or quaternary amine, or PEI-based resins known in the art (U.S. Patent 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 contains a quaternary amine. In some embodiments, the anion exchange medium is a monolithic anion exchange chromatography resin. In some embodiments, the monolithic anion exchange chromatography medium comprises a glycidyl methacrylate-ethylenedimethacrylate polymer or a 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 disc (quaternary amine), CIM® DEAE, and CIM® EDA disc (ethylenediamino). In some embodiments, the monolithic anion exchange chromatography medium is a CIMmultus® QA-1 advanced composite column (quaternary amine). In some embodiments, the monolithic anion exchange chromatography medium is a CIM® QA disc (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). Those skilled in the art will understand that a wash buffer of suitable ionic strength can be identified so that impurities (including, but not limited to, impurities introduced by upstream purification steps) are removed while the rAAV maintains its binding to the resin.

[0131] In some embodiments, anion exchange chromatography is carried out according to the method disclosed in U.S. Provisional Patent Application No. 62 / 684,835, “Anion Exchange Chromatography for Recombinant AAV production,” filed June 14, 2018 (which is incorporated herein by reference in its entirety).

[0132] In additional embodiments, the disclosure provides compositions comprising isolated rAAV particles produced according to the methods disclosed herein. In some embodiments, the compositions are pharmaceutical compositions comprising a pharmaceutically acceptable carrier.

[0133] As used herein, the term “pharmaceutically acceptable” means a bioacceptable formulation, gas, liquid, or solid, or mixture thereof, suitable for one or more routes of administration, in vivo delivery, or contact. A “pharmaceutically acceptable” composition is a material that is not biologically or otherwise undesirable, for example, that can be administered to a subject without causing substantially undesirable biological effects. Such a pharmaceutical composition can therefore be used, for example, when administering rAAV isolated according to the methods of this disclosure to a subject. Such compositions include solvents (aqueous or non-aqueous), solutions (aqueous or non-aqueous), emulsions (e.g., oil in water or water in oil), suspensions, syrups, elixirs, dispersions and suspension media, coatings, isotonic and absorption enhancers or retarders, which are suitable for pharmaceutical administration or in vivo contact or delivery. Aqueous and non-aqueous solvents, solutions, and suspensions may include suspending agents and thickeners. Such pharmaceutically acceptable carriers include tablets (coated or uncoated), capsules (hard or soft), microbeads, powders, granules, and crystals. Complementary active compounds (e.g., preservatives, antimicrobial agents, antiviral agents, and antifungal agents) can also be incorporated into the composition. Pharmaceutical compositions can be formulated to be compatible with specific routes of administration or delivery, as described herein or as known to those skilled in the art. Therefore, pharmaceutical compositions may include carriers, diluents, or excipients suitable for administration via various routes.The rAAV particles, method, and suitable pharmaceutical compositions and delivery systems of the present invention are known in the art (for example, 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). (See 1980, RLJuliano, ed., Oxford, NY, pp. 253–315).

[0134] In some embodiments, the composition is a pharmaceutical unit dose. “Unit dose” means a physically separate unit suitable as a unit drug dose for the target being treated. Each unit contains a predetermined amount, optionally together with a pharmaceutical carrier (excipient, diluent, vehicle, or filler), and is calculated to produce a desired effect (e.g., prophylactic or therapeutic effect) when administered in one or more doses. Unit dosage forms may be, for example, in ampoules and vials, and these may include liquid compositions or compositions in a freeze-dried or lyophilized state, and a sterile liquid carrier may be added, for example, before in vivo administration or delivery. Individual unit dosage forms may be included in multi-dose kits or containers. Recombinant vectors (e.g., AAV) sequences, plasmids, vector genomes, and recombinant viral particles, as well as these pharmaceutical compositions, may be packaged in single or multiple unit dose forms to facilitate administration and ensure uniformity of dosage. In some embodiments, the compositions are AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, and AAV16, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B, AAV2.5, The rAAV particles contain AAV capsid proteins from AAV capsid serotypes selected from AAV2tYF, AAV3B, AAV.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 AAV capsid serotype is AAV8. In some embodiments, the AAV capsid serotype is AAV9. [Examples]

[0135] Example 1. Effects of sodium chloride, sodium butyrate, and / or sodium valproate on rAAV yield. The effects of sodium chloride, sodium butyrate, and / or sodium valproate on rAAV yield in a HEK293 suspension cell-based process were investigated. HEK293 cells were seeded at a density of 1 × 10⁶ live cells / ml in an advanced microscale bioreactor. The culture medium contained 100 mM NaCl. At 48 hours of ECD (culture duration), polyethyleneimine and a mixture of three plasmids encoding adenovirus helper function, transgenes, and AAV 2 / 8 Rep / Cap were transfused into the cells. 24 hours after transfusion, NaCl, sodium butyrate, and / or sodium valproate were added to the culture. Test conditions included 0, 25 mM, and 90 mM NaCl, 0, 2 mM, and 4 mM sodium butyrate, and 0, 1.5 mM, and 3 mM sodium valproate. A complete factorial combination of conditions was tested using 36 reaction conditions. The supernatant of the culture was collected after 168 hours of ECD, i.e., 5 days after transfusion. The obtained rAAV yield is shown in Figure 1. The virus yield was significantly improved by adding 60 mM sodium chloride and either 4 mM sodium butyrate or 3 mM sodium valproate 1 day after transfusion.

[0136] Example 2. Effects of sodium chloride and / or sodium valproate on rAAV yield. The effects of adding sodium chloride, sodium valproate, and combinations thereof at different time points on rAAV yield were investigated in a HEK293 suspension cell-based process. HEK293 suspension cells at a density of 1 × 10⁶ live cells / ml were seeded in an advanced microscale bioreactor. The culture medium contained 100 mM NaCl. At 48 hours of ECD (culture duration), polyethyleneimine and a mixture of three plasmids encoding adenovirus helper function, transgenes, and AAV 2 / 8 Rep / Cap were transfused into the cells. NaCl and / or sodium valproate were added to the culture at 4, 24, or 48 hours after transfusion. The concentrations of NaCl and sodium valproate tested were 0, 30 mM, and 60 mM NaCl, and 0, 1 mM, and 2 mM sodium valproate. The supernatant of the culture was collected at 168 hours of ECD, i.e., 5 days after translocation. The obtained rAAV yield is shown in Figure 2. Using a design of experiments strategy, 24 different conditions were tested and analyzed. A model created from the DOE (Design of Experiments) tests under 24 conditions predicted that adding 2 mM sodium valproate 4 hours after translocation, followed by 30 mM NaCl 24 hours after translocation, would significantly enhance the virus yield.

[0137] Example 3. Large-scale production of rAAV using a NaCl enhancer. The effect of NaCl on rAAV yield was investigated in a 50-liter culture of HEK293 cells expressing AAV8 particles that capsidize the transgene. HEK suspension cultures were grown using a standard process. Cells were diluted to approximately 4 × 10⁶ live cells. The culture medium used contained 100 mM NaCl. 24 hours after dilution, polyethyleneimine and a mixture of three plasmids encoding adenovirus helper function, the transgene, and AAV Cap / Rev were transfected into the cells. 24 hours after transfection, sufficient NaCl was added to increase the final NaCl concentration by 60 mM (i.e., to approximately 160 mM). The supernatant was collected 4 days after transfection. These treatments yielded 1 × 10⁶ e⁶ genome copies (GC) / ml and 9 × 10⁶ e⁶. This yield is approximately twice as high as that obtained using the same process without increasing the NaCl concentration after translocation.

[0138] Example 4. Large-scale production of AAV using NaCl and sodium propionate. The effect of NaCl on rAAV yield was investigated in a 50-liter culture of HEK293 cells expressing AAV8 particles that capsidize the transgene. HEK suspension cultures were grown using a standard process. Cells were diluted to approximately 4 × 10⁶ live cells. The culture medium used contained 100 mM NaCl. 24 hours after dilution, polyethyleneimine and a mixture of three plasmids encoding adenovirus helper function, the transgene, and AAV 2 / 8 Rep / Cap were transfused into the cells. 24 hours after transfusion, sufficient NaCl was added to increase the final NaCl concentration by 30 mM (i.e., to approximately 130 mM). Then, sufficient sodium propionate (NaPr) was added to increase the final NaPr concentration to 2 mM (NaPr was not present in the medium at the start). The supernatant was collected 4 days after transfusion. These treatments yielded 1 × 10⁻¹¹ genome copies (GC) / ml, which was approximately 1.7 times higher than the yield obtained with the same process without increasing NaCl and NaPr concentrations after translocation (Figure 3). Furthermore, titer increased significantly without compromising quality. Product quality, e.g., complete capsid%, fragmented rAAV%, and residual DNA, e.g., residual 18S (the 18S RNA gene used to estimate residual mammalian genomic DNA), residual E1a, and residual plasmid, was not negatively affected by the scale-up generation process with enhancers compared to a similar process without enhancers.

[0139] Example 5. Effects of sodium chloride and / or sodium propionate on rAAV yield. The effect of sodium chloride and sodium propionate (NaPr) (ThermoFisher Scientific) on rAAV yield in suspension culture of HEK293 cells expressing rAAV9 particles that capsidize transgenes. HEK293 cells were seeded at a density of 1 × 10⁶ live cells / ml in an advanced microscale bioreactor. The culture medium contained 100 mM NaCl. At 48 hours of ECD (culture duration), polyethyleneimine and a mixture of three plasmids encoding adenovirus helper function, transgenes, and AAV 2 / 9 Rep / Cap were transfected into the cells. NaCl, NaPr, an anti-aggregating agent, EFC+, and / or sodium valproate were added to the culture 4, 20, or 36 hours after transfecting. Test conditions included 0, 30 mM, and 60 mM NaCl, and 0, 2 mM, and 4 mM NaPr. Multiple conditions were tested using a design of experiments approach. The supernatant of the culture was collected at 168 hours ECD, i.e., 5 days after transfusion, to obtain rAAV yield. Adding both 30 mM sodium chloride and 2 mM sodium propionate 20 hours after transfusion significantly and synergistically improved the virus yield. The improvement in titer obtained by adding NaCl and / or NaPr is shown in Figure 4 (averaging the effects of other test conditions). Administration of 2 mM sodium propionate without an increase in NaCl resulted in approximately a 1.5-fold increase in titer. Increasing NaCl without sodium propionate resulted in approximately a 1.2-fold increase in titer. On the other hand, combining 2 mM sodium propionate with an increase in NaCl resulted in a yield approximately twice as high as the baseline yield of the same process without increases in NaCl and NaPr concentrations after transfusion. The observed approximately twofold increase is higher than the increase expected if the effects of NaPr and NaCl increases were additive.

[0140] Example 6. Effects of sodium chloride and / or sodium propionate on rAAV yield. The effect of sodium chloride and sodium propionate (ThermoFisher Scientific) on rAAV yield in suspension culture of HEK293 cells expressing rAAV9 particles that capsidize the transgene. HEK293 cells were seeded at a density of 1 × 10⁶ live cells / ml in an advanced microscale bioreactor. The culture medium contained 100 mM NaCl. At 48 hours of ECD (culture duration), polyethyleneimine and a mixture of three plasmids encoding adenovirus helper function, the transgene, and AAV 2 / 9 Rep / Cap were transfected into the cells. Twenty hours after transfecting, NaCl and sodium propionate (NaPr) were added to the culture. The test conditions were 0 and 30 mM NaCl and 0 and 2 mM NaPr. Using an experimental design strategy, combinations of conditions were tested under multiple reaction conditions. The supernatant of the culture was collected at 168 hours of ECD, i.e., 5 days after transfusion, to obtain the rAAV yield. The addition of both 30 mM sodium chloride and 2 mM sodium propionate 20 hours after transfusion significantly improved the virus yield. Figure 5 (averaging the effects of other test conditions) shows the improvement in titer with the addition of NaCl and / or NaPr. This is approximately 1.6 times higher than the yield obtained with the same process without increasing the concentrations of NaCl and NaPr after transfusion.

[0141] While the methods described herein have been explained in conjunction with what is considered to be the most practical and preferred embodiments, it should be understood that the methods encompassed herein are not to be limited to the disclosed embodiments, but rather are intended to cover a variety of modifications and equivalent arrangements that fall within the spirit and scope of the appended claims.

[0142] All publications, patents, patent applications, internet sites, and accession number / database sequences (including both polynucleotide and polypeptide sequences) cited herein are incorporated herein by reference in whole for any purpose to the same extent that each individual publication, patent, patent application, internet site, or accession number / database sequence is incorporated by reference specifically and individually.

Claims

1. A method for generating rAAV particles, (a) Cells, as follows: ・The rAAV genome to be packaged, - Adenovirus helper function necessary for packaging, - Sufficient AAV rep protein for packaging, and - Sufficient AAV cap protein for packaging Translocation involving one or more polynucleotides encoding To prepare a cell culture containing cells capable of producing rAAV, (b) Adding propionic acid or a salt thereof to the cell culture to a final concentration between 0.5 mM and 3 mM, (c) Adding to the culture a sufficient amount of the sodium salt to increase the final concentration of the sodium salt by between 20 mM and 50 mM, (d) Maintaining the cell culture under conditions that enable the generation of the rAAV particles. Includes, Transplanting cells involves the use of chemical-based transplantation methods. Before adding the sodium salt, the cell culture contains NaCl between approximately 90 mM and approximately 120 mM. The propionic acid or its salt and the sodium salt are added separately in any order after the cells have been transfused. The aforementioned method.

2. The method according to claim 1, wherein the sodium salt is sodium chloride.

3. The method according to claim 1 or claim 2, wherein the sodium salt is added after the addition of the propionic acid or a salt thereof.

4. The method according to claim 3, wherein the sodium salt is added 5 minutes to 6 hours after the addition of the propionic acid or a salt thereof.

5. The method according to any one of claims 1 to 4, wherein the propionic acid or a salt thereof is added 1 to 48 hours after translocation of cells.

6. The method according to any one of claims 1 to 5, wherein the cells are HEK293 cells.

7. The method according to any one of claims 1 to 6, wherein the cell culture is a suspension culture.

8. The method according to any one of claims 1 to 7, further comprising recovering the rAAV particles.

9. The method according to any one of claims 1 to 8, wherein the cell culture generates rAAV particles between 5 × 10 e + 10 GC / ml and 1 × 10 e + 12 GC / ml.

10. The method according to any one of claims 1 to 9, wherein the cell culture produces at least twice as many rAAV particles as measured by GC / ml compared to a culture without the addition of propionic acid or its salt and sodium salt.

11. The method according to any one of claims 1 to 10, wherein the rAAV particles contain a capsid protein of serotype AAV8, AAV9, AAV. rh10, AAV. rh20, AAV. rh39, AAV. Rh74, AAV. RHM4-1, or AAV. hu37.

12. The method according to any one of claims 1 to 11, wherein the rAAV comprises a genome containing the transgene.

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