Improved production of recombinant polypeptides and viruses

Abstract

Provided herein are improved methods for producing recombinant polypeptides or viral particles. In some embodiments, the methods for producing recombinant polypeptides or viral particles disclosed herein include providing a cell culture comprising cells capable of producing recombinant polypeptides or viral particles and dextran sulfate, and transfecting the cells by adding to the culture a composition comprising one or more polynucleotides and a transfection reagent. In some embodiments, the recombinant viral particles are recombinant AAV (rAAV) particles.

Description

[Technical field]

[0001] The present disclosure relates to methods that include transfecting host cells in a culture medium that includes dextran sulfate.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Patent Application No. 63 / 139,992, filed January 21, 2021, which is incorporated by reference in its entirety. [Background technology]

[0003] background Recombinant adeno-associated virus (AAV)-based vectors are currently the most widely used gene therapy products under development. The use of rAAV vector systems is favored in part due to the lack of disease associated with wild-type viruses, the ability of AAV to transduce non-dividing as well as dividing cells, and the resulting long-term robust transgene expression observed in clinical trials, indicating great potential for delivery in gene therapy indications. In addition, various natural and recombinant rAAV vector serotypes can specifically target different tissues, organs, and cells, helping to circumvent any pre-existing immunity to the vector, thus expanding the therapeutic application of AAV-based gene therapy. Before replication-defective virus (e.g., AAV)-based gene therapy can be more widely adopted for late-stage clinical and commercial use, new methods for large-scale production of recombinant viral particles need to be developed.

[0004] Thus, there is a need in the art for improved productivity and yield of methods for large-scale production of rAAV particles. Summary of the Invention

[0005] overview In one aspect, the disclosure provides a method of transfecting cells, the method comprising: (a) providing a cell culture comprising cells, the culture comprising between about 0.1 mg / L and about 10 mg / L dextran sulfate; and (b) transfecting the cells by adding to the culture a composition comprising one or more polynucleotides and a transfection reagent. In some embodiments, the cell culture is a suspension culture. In some embodiments, the cell culture is a suspension culture comprising suspension-adapted HEK cells. In some embodiments, the transfection reagent comprises polyethyleneimine (PEI).

[0006] In a further aspect, the disclosure provides a method of producing a recombinant polypeptide comprising: (a) providing a cell culture comprising cells suitable for producing a recombinant polypeptide, the culture comprising between about 0.1 mg / L and about 10 mg / L dextran sulfate; (b) transfecting the cells by adding to the culture of (a) a composition comprising one or more polynucleotides encoding a polypeptide and a transfection reagent; and (c) maintaining the cell culture comprising the transfected cells under conditions that allow for the production of the recombinant polypeptide. In some embodiments, the recombinant polypeptide is an antibody or antigen-binding fragment thereof, a bispecific antibody, an enzyme, a fusion protein, or an Fc-fusion protein.

[0007] In a further aspect, the disclosure provides a method of producing recombinant viral particles, the method comprising: (a) providing a cell culture comprising cells suitable for producing recombinant viral particles, the culture comprising between about 0.1 mg / L and about 10 mg / L dextran sulfate; (b) transfecting the cells by adding to the culture of (a) a composition comprising one or more polynucleotides comprising genes required for the production of recombinant viral particles and a transfection reagent; and (c) maintaining the cell culture comprising the transfected cells under conditions that allow for the production of recombinant viral particles. In some embodiments, the recombinant virus is a recombinant adeno-associated virus (rAAV). In some embodiments, the rAAV is selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV. The capsid protein of the 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 serotype. In some embodiments, the rAAV comprises a capsid protein of an AAV8 or AAV9 serotype. In some embodiments, the cell culture is a suspension culture comprising suspension-adapted HEK cells. In some embodiments, the transfection reagent comprises polyethylenimine (PEI).

[0008] In one aspect, the disclosure provides a method of transfecting cells, comprising: (a) culturing cells in a cell culture, the culture comprising an initial dextran sulfate concentration of between about 1 mg / L and about 20 mg / L and a final dextran sulfate concentration of between about 0.1 mg / L and about 10 mg / L; and (b) transfecting the cells by adding to the culture of (a) a composition comprising one or more polynucleotides and a transfection reagent. In some embodiments, the cell culture is a suspension culture. In some embodiments, the cell culture is a suspension culture comprising suspension-adapted HEK cells. In some embodiments, the transfection reagent comprises polyethyleneimine (PEI).

[0009] In a further aspect, the disclosure provides a method of producing a recombinant polypeptide comprising: (a) culturing in a cell culture cells suitable for producing the recombinant polypeptide, the culture comprising an initial dextran sulfate concentration of between about 1 mg / L and about 20 mg / L and a final dextran sulfate concentration of between about 0.1 mg / L and about 10 mg / L; (b) transfecting the cells by adding to the culture of (a) a composition comprising one or more polynucleotides encoding a polypeptide and a transfection reagent; and (c) maintaining the cell culture comprising the transfected cells under conditions that allow for the production of the recombinant polypeptide. In some embodiments, the recombinant polypeptide is an antibody or antigen-binding fragment thereof, a bispecific antibody, an enzyme, a fusion protein, or an Fc-fusion protein.

[0010] In a further aspect, the disclosure provides a method of producing recombinant viral particles, comprising: (a) culturing cells suitable for the production of recombinant viral particles in a cell culture for about 1 day to about 5 days, the culture comprising an initial dextran sulfate concentration of between about 1 mg / L and about 20 mg / L and a final dextran sulfate concentration of between about 0.1 mg / L and about 10 mg / L; (b) transfecting the cells by adding to the culture of (a) a composition comprising one or more polynucleotides comprising genes required for the production of recombinant viral particles and a transfection reagent; and (c) maintaining the cell culture comprising the transfected cells under conditions that allow the production of recombinant viral particles. In some embodiments, the recombinant virus is a recombinant adeno-associated virus (rAAV). In some embodiments, the rAAV is selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV. The capsid protein of the 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 serotype. In some embodiments, the rAAV comprises a capsid protein of an AAV8 or AAV9 serotype. In some embodiments, the cell culture is a suspension culture comprising suspension-adapted HEK cells. In some embodiments, the transfection reagent comprises polyethylenimine (PEI).

[0011] In a further aspect, the disclosure provides a method of improving the production of a recombinant polypeptide, comprising: (a) providing a cell culture comprising cells suitable for the production of a recombinant polypeptide, the culture comprising between about 0.1 mg / L and about 10 mg / L of dextran sulfate; (b) transfecting the cells by adding to the culture of (a) a composition comprising one or more polynucleotides encoding a polypeptide and a transfection reagent; and (c) maintaining the cell culture comprising the transfected cells under conditions that allow the production of the recombinant polypeptide. In some embodiments, the methods disclosed herein produce more polypeptides than methods comprising transfecting cells in a culture that does not contain dextran sulfate. In some embodiments, the recombinant polypeptide is an antibody or antigen-binding fragment thereof, a bispecific antibody, an enzyme, a fusion protein, or an Fc-fusion protein.

[0012] In a further aspect, the disclosure provides a method of improving the production of recombinant viral particles, comprising: (a) providing a cell culture comprising cells suitable for the production of recombinant viral particles, the culture comprising between about 0.1 mg / L and about 10 mg / L dextran sulfate; (b) transfecting the cells by adding to the culture of (a) a composition comprising one or more polynucleotides comprising genes required for the production of recombinant viral particles and a transfection reagent; and (c) maintaining the cell culture comprising the transfected cells under conditions that allow the production of recombinant viral particles. In some embodiments, the methods disclosed herein produce more recombinant viral particles than methods comprising transfecting cells in a culture that does not contain dextran sulfate. In some embodiments, the recombinant virus is a recombinant adeno-associated virus (rAAV). In some embodiments, the rAAV is selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV. The capsid protein of the 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 serotype. In some embodiments, the rAAV comprises a capsid protein of an AAV8 or AAV9 serotype. In some embodiments, the cell culture is a suspension culture comprising suspension-adapted HEK cells. In some embodiments, the transfection reagent comprises polyethylenimine (PEI).

[0013] In some embodiments, the present disclosure provides:

[0014] [1.] A method for transfecting a cell, comprising: a) providing a cell culture comprising the cells, the culture comprising between about 0.1 mg / L and about 10 mg / L of dextran sulfate; b) transfecting said cells by adding to said culture of (a) a composition comprising one or more polynucleotides and a transfection reagent; The method comprising:

[0015] [2.] A method for producing a recombinant polypeptide, comprising: a) providing a cell culture comprising cells suitable for producing the recombinant polypeptide, said culture comprising between about 0.1 mg / L and about 10 mg / L dextran sulfate; b) transfecting the cells by adding to the culture of a) a composition comprising one or more polynucleotides encoding the polypeptide and a transfection reagent; c) maintaining said cell culture containing said transfected cells under conditions that allow production of said recombinant polypeptide; The method comprising:

[0016] [3.] The method of claim [2], wherein the polypeptide is an antibody or an antigen-binding fragment thereof, a bispecific antibody, an enzyme, a fusion protein, or an Fc fusion protein.

[0017] [4.] A method for producing recombinant viral particles, comprising: a) providing a cell culture comprising cells suitable for producing the recombinant viral particles, the culture comprising between about 0.1 mg / L and about 10 mg / L dextran sulfate; b) transfecting the cells by adding to the culture of a) a composition comprising one or more polynucleotides comprising genes necessary for generating the recombinant viral particles and a transfection reagent; c) maintaining said cell culture containing said transfected cells under conditions that allow the production of said recombinant viral particles; The method comprising:

[0018] [5.] The method of any one of claims [1] to [4], wherein the culture of a) contains between about 0.5 mg / L and about 10 mg / L, between about 0.5 mg / L and about 5 mg / L, between about 0.5 mg / L and about 3 mg / L, between about 1 mg / L and about 10 mg / L, between about 1 mg / L and about 5 mg / L, between about 1 mg / L and about 4 mg / L, or between about 1 mg / L and about 3 mg / L of dextran sulfate.

[0019] [6.] The method according to any one of claims [1] to [4], wherein the culture of a) contains about 0.5 mg / L, about 1 mg / L, about 1.5 mg / L, about 2 mg / L, about 2.5 mg / L, about 3 mg / L, about 4 mg / L, or about 5 mg / L of dextran sulfate.

[0020] [7.] The method according to any one of claims [1] to [4], wherein the culture of a) contains approximately 2 mg / L of dextran sulfate.

[0021] [8.] A method for transfecting a cell, comprising: a) culturing the cells in a cell culture, the culture comprising an initial dextran sulfate concentration of between about 1 mg / L and about 20 mg / L and a final dextran sulfate concentration of between about 0.1 mg / L and about 10 mg / L; b) transfecting said cells by adding to said culture of a) a composition comprising one or more polynucleotides and a transfection reagent; The method comprising:

[0022] [9.] A method for producing a recombinant polypeptide, comprising the steps of: a) culturing in a cell culture a cell suitable for producing the recombinant polypeptide, the culture comprising an initial dextran sulfate concentration of between about 1 mg / L and about 20 mg / L and a final dextran sulfate concentration of between about 0.1 mg / L and about 10 mg / L; b) transfecting the cells by adding to the culture of a) a composition comprising one or more polynucleotides encoding the polypeptide and a transfection reagent; c) maintaining said cell culture containing said transfected cells under conditions that allow production of said recombinant polypeptide; The method comprising:

[0023] [10.] The method of claim [9], wherein the polypeptide is an antibody or an antigen-binding fragment thereof, a bispecific antibody, an enzyme, a fusion protein, or an Fc fusion protein.

[0024] [11.] A method for producing recombinant viral particles, comprising: a) culturing cells suitable for producing the recombinant viral particles in cell culture for about 1 to about 5 days, the culture comprising an initial dextran sulfate concentration of between about 1 mg / L and about 20 mg / L and a final dextran sulfate concentration of between about 0.1 mg / L and about 10 mg / L; b) transfecting the cells by adding to the culture of a) a composition comprising one or more polynucleotides comprising genes necessary for generating the recombinant viral particles and a transfection reagent; c) maintaining said cell culture containing said transfected cells under conditions that allow the production of said recombinant viral particles; The method comprising:

[0025] [12.] The method of any one of claims [8] to

[11] , wherein the initial dextran sulfate concentration is between about 1 mg / L and about 10 mg / L, between about 1 mg / L and about 5 mg / L, between about 2 mg / L and about 10 mg / L, between about 3 mg / L and about 10 mg / L, or between about 3 mg / L and about 5 mg / L.

[0026] [13.] The method of any one of claims [8] to

[11] , wherein the initial dextran sulfate concentration is about 2 mg / L, about 3 mg / L, about 4 mg / L, about 5 mg / L, about 6 mg / L, about 7 mg / L, about 8 mg / L, about 9 mg / L, or about 10 mg / L.

[0027] [14.] The method according to any one of claims [8] to

[11] , wherein the initial dextran sulfate concentration is about 4 mg / L.

[0028] [15.] The method of any one of claims [8] to

[14] , wherein the final dextran sulfate concentration is between about 0.5 mg / L and about 10 mg / L, between about 0.5 mg / L and about 5 mg / L, between about 0.5 mg / L and about 5 mg / L, between about 0.5 mg / L and about 3 mg / L, between about 1 mg / L and about 10 mg / L, between about 1 mg / L and about 5 mg / L, between about 1 mg / L and about 4 mg / L, or between about 1 mg / L and about 3 mg / L.

[0029] [16.] The method according to any one of claims [8] to

[14] , wherein the final dextran sulfate concentration is about 0.5 mg / L, about 1 mg / L, about 1.5 mg / L, about 2 mg / L, about 2.5 mg / L, about 3 mg / L, about 4 mg / L, or about 5 mg / L.

[0030] [17.] The method according to any one of claims [8] to

[14] , wherein the final dextran sulfate concentration is about 2 mg / L.

[0031] [18.] The method according to any one of claims [8] to

[11] , wherein the initial dextran sulfate concentration is about 4 mg / L and the final dextran sulfate concentration is about 2 mg / L.

[0032] [19.] The method described in any one of claims [4] to [7] and

[11] to

[18] , wherein the recombinant viral particle is a recombinant adeno-associated viral particle (rAAV) particle or a recombinant lentiviral particle.

[0033] [20.] The method described in any one of claims [4] to [7] and

[11] to

[18] , wherein the recombinant viral particle is a rAAV particle.

[0034] [21.] The rAAV particles are AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, 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 serotype capsid protein.

[0035] [22.] The method of claim

[20] , wherein the rAAV particles comprise capsid proteins of AAV8, AAV9, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, or AAV.hu37 serotypes.

[0036] [23.] The method of claim

[20] , wherein the rAAV particles contain capsid proteins of AAV8 or AAV9 serotypes.

[0037] [24.] The method of any one of claims

[20] to

[23] , wherein the rAAV particle contains a genome including an introduced gene.

[0038] [25.] The method of claim

[24] , wherein the introduced gene comprises a regulatory element operably linked to a polynucleotide encoding a polypeptide.

[0039] [26.] The method of claim

[25] , wherein the regulatory element comprises one or more of an enhancer, a promoter, and a polyA region.

[0040] [27.] The method of claim

[24] or claim

[25] , wherein the regulatory element and the polynucleotide encoding the polypeptide are heterologous.

[0041] [28.] The method of any one of claims

[24] to

[27] , wherein the introduced gene 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).

[0042] [29.] The transgene is selected from the group consisting of gamma-sarcoglycan, Rab escort protein 1 (REP1 / CHM), retinoid isomerohydrolase (RPE65), cyclic nucleotide-gated channel alpha 3 (CNGA3), cyclic nucleotide-gated channel beta 3 (CNGB3), aromatic L-amino acid decarboxylase (AADC), lysosomal-associated membrane protein 2 isoform B (LAMP2B), factor VIII, factor IX, retinitis pigmentosa GTPase regulator (RPGR), retinoschisin (RS1), sarcoplasmic reticulum calcium (SRC), and sarcoplasmic reticulum calcium phosphate (SRP). ATPase (SERCA2a), aflibercept, battenin (CLN3), transmembrane ER protein (CLN6), glutamic acid decarboxylase (GAD), glial cell line-derived neurotrophic factor (GDNF), aquaporin 1 (AQP1), dystrophin, mini-dystrophin, micro-dystrophin, myotubularin 1 (MTM1), follistatin (FST), glucose-6-phosphatase (G6Pase), apolipoprotein A2 (APOA2), uridine diphosphate glucuronosyltransferase 1A1 (UGT 1A1), arylsulfatase B (ARSB), N-acetylglucosaminidase (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 The method of any one of claims

[24] to

[27] , wherein the nucleic acid encoding the polypeptide encodes: cystic fibrosis transmembrane conductance regulator (CFTR), tumor necrosis factor receptor (TNFR)-immunoglobulin (IgG1) Fc fusion protein, or a polypeptide encoding mitochondrial-encoded NADH:ubiquinone oxidoreductase core subunit 4 (MT-ND4), protective protein cathepsin A (PPCA), dysferlin, MER proto-oncogene tyrosine kinase (MERTK), or a polypeptide encoding cystic fibrosis transmembrane conductance regulator (CFTR).

[0043] [30.] The one or more polynucleotides are a) the rAAV genome to be packaged; b) adenovirus helper functions necessary for packaging; c) sufficient AAV rep protein for packaging; and d) AAV cap protein sufficient for packaging The method according to any one of claims

[20] to

[29] , wherein the

[0044] [31.] The method of claim

[30] , wherein the one or more polynucleotides include a polynucleotide encoding the rAAV genome, a polynucleotide encoding the AAV rep protein and the AAV cap protein, and a polynucleotide encoding the adenovirus helper functions.

[0045] [32.] The method of claim

[30] or claim

[31] , wherein the adenovirus helper function comprises at least one of the adenovirus E1a gene, E1b gene, E4 gene, E2a gene, and VA gene.

[0046] [33.] The method of any one of claims

[20] to

[28] , further comprising recovering the rAAV particles.

[0047] [34.] The method of any one of claims

[20] to

[33] , wherein the cell culture produces between about 5 x 10e+10 GC / ml and about 1 x 10e+12 GC / ml of rAAV particles.

[0048] [35.] The method of any one of claims

[20] to

[33] , wherein the cell culture produces at least about 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, or 2-fold more rAAV particles, measured as GC / ml, than a reference method in which the culture of a) does not contain dextran sulfate.

[0049] [36.] The method according to any one of claims [1] to

[35] , wherein the cell culture is a suspension cell culture.

[0050] [37.] The method of claim

[36] , wherein the cell culture comprises suspension-adapted cells.

[0051] [38.] The method of claim

[36] or claim

[37] , wherein the cells comprise HEK293 cells, HEK-derived cells, CHO cells, CHO-derived cells, HeLa cells, SF-9 cells, BHK cells, Vero cells, and / or PerC6 cells, or a combination thereof.

[0052] [39.] The method of claim

[36] or claim

[37] , wherein the cells comprise HEK293 cells.

[0053] [40.] The method of claim

[36] or claim

[37] , wherein the cells comprise CHO cells or CHO-K1 cells.

[0054] [41.] The method of any one of claims [1] to

[40] , wherein the transfection reagent comprises a lipid, a polymer, a peptide, or a combination thereof.

[0055] [42.] The method of claim

[41] , wherein the transfection reagent comprises a lipid, and the lipid comprises DOTMA, DOTAP, DOSPA, DOGS, or a combination thereof.

[0056] [43.] The method of claim

[41] , wherein the transfection reagent comprises a polymer, and the polymer comprises poly(L-lysine) (PLL), polyethyleneimine (PEI), a polysaccharide, poly[2-(dimethylamino)ethyl methacrylate] (PDMAEMA), a dendrimer, or a combination thereof.

[0057] [44.] The method of claim 41, wherein the transfection reagent comprises polyethyleneimine (PEI).

[0058] [45.] The method of any one of claims [1] to

[44] , wherein the cell culture has a volume of between about 50 liters and about 20,000 liters.

[0059] [46.] The method of claim

[45] , wherein the cell culture has a volume of between about 50 liters and about 5,000 liters.

[0060] [47.] The method of claim

[45] , wherein the cell culture has a volume of between about 50 liters and about 2,000 liters.

[0061] [48.] The method of claim

[45] , wherein the cell culture has a volume of between about 50 liters and about 1,000 liters.

[0062] [49.] The method of claim

[41] , wherein the cell culture has a volume of between about 50 liters and about 500 liters.

[0063] [50.] A composition comprising isolated rAAV particles produced by a method according to any one of claims

[20] to

[49] .

[0064] Further features and advantages of the compositions and methods described herein will become more apparent from the following detailed description, taken in conjunction with the accompanying drawings. [Brief description of the drawings]

[0065] [Figure 1] Initial shake flask screen of dextran sulfate for use before and during transfection. 1:2,500, 1:500, 1:10,000, 1:20,000, 1:40,000, and 1:80,000 represent dilutions of the 25 g / L dextran sulfate stock solution present at the time of transfection, corresponding to concentrations of 10 mg / L, 5 mg / L, 2.5 mg / L, 1.25 mg / L, 625 g / L, and 313 g / L, respectively. [Diagram 2]Initial shake flask screen of dextran sulfate for use before and during transfection. 6K, 7K, 8K, 9K, 10K, 12K, and 15K represent dilutions of the 25 g / L dextran sulfate stock solution present at the time of transfection, corresponding to concentrations of 4.2 mg / L, 3.6 mg / L, 3.1 mg / L, 2.8 mg / L, 2.5 mg / L, 2.1 mg / L, and 1.7 mg / L, respectively. [Diagram 3] Bench-scale 2L dextran sulfate titration for transfection: genomic titer. 6K, 7K, 8K, 9K, and 10K represent dilution factors of the 25 g / L dextran sulfate stock solution present at the time of transfection, corresponding to concentrations of 4.2 mg / L, 3.6 mg / L, 3.1 mg / L, 2.8 mg / L, and 2.5 mg / L, respectively. [Figure 4] Bench-scale 2L dextran sulfate titrations for transfection: cell imaging. 1:6,000, 1:7,000, 1:8,000, 1:9,000, and 1:10,000 represent dilution factors of the 25 g / L dextran sulfate stock solution present at the time of transfection, corresponding to concentrations of 4.2 mg / L, 3.6 mg / L, 3.1 mg / L, 2.8 mg / L, and 2.5 mg / L, respectively. [Diagram 5] Bench-scale 2L dextran sulfate titration for transfection: Viable cell density. 1:6,000, 1:7,000, 1:8,000, 1:9,000, and 1:10,000 represent dilution factors of the 25 g / L dextran sulfate stock solution present at the time of transfection, corresponding to concentrations of 4.2 mg / L, 3.6 mg / L, 3.1 mg / L, 2.8 mg / L, and 2.5 mg / L, respectively. [Figure 6] Bench-scale 2L dextran sulfate titrations for transfection: cell viability. 1:6,000, 1:7,000, 1:8,000, 1:9,000, and 1:10,000 represent dilution factors of the 25 g / L dextran sulfate stock solution present at the time of transfection, corresponding to concentrations of 4.2 mg / L, 3.6 mg / L, 3.1 mg / L, 2.8 mg / L, and 2.5 mg / L, respectively. [Figure 7]AAV8 production in a bench-scale 5 L reactor using dextran sulfate at a concentration of 2 mg / L during transfection. [Figure 8] AAV8 production in a 2 L bench-scale reactor using different commercial media. Dextran sulfate was present at 2 mg / L during transfection. [Figure 9] AAV8 production in shake flasks using different host cell clones. Dextran sulfate was present at 2 mg / L during transfection. [Figure 10] AAV9 production in a bench-scale 5 L reactor using dextran sulfate during transfection. [Figure 11] Inclusion of dextran sulfate in high density seed trains prior to transfection increases AAV titers. [Figure 12] Inclusion of dextran sulfate in the seed train prior to transfection increases AAV titers. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0066] Detailed Description Surprisingly, it was found that dextran sulfate can increase rAAV titers in transient transfection-based production methods. This finding was unexpected because dextran sulfate is known to inhibit transient transfection. For example, Geng et al. (2007) conclude on page 55 that dextran sulfate completely inhibits PEI-mediated transfection. Similarly, the recently published “Guide for DNA Transfection in iCELLis® 500 and iCELLis 500+ Bioreactors for Large Scale Gene Therapy Vector Manufacturing” ("2020 Guide") by PALL® Biotech teaches on page 9 that dextran sulfate inhibits PEI-mediated transfection. For example, a person skilled in the art considering the teachings of Geng et al. (2007) and the 2020 Guide would reasonably expect that rAAV production by transient transfection-based methods would be inhibited or at least less productive due to the presence of dextran sulfate in the cell culture during transfection. In contrast, as discussed in the Examples, the presence of dextran sulfate in the transfection medium surprisingly increased rAAV production relative to the presence of dextran sulfate. Increased rAAV production was observed in the process of producing rAAV particles containing different capsid serotypes or transgenes using different cell culture media, host cell clones, and production volumes.

[0067] These surprising discoveries were used to develop the methods of transfecting cells, producing recombinant polypeptides, producing recombinant viral particles (e.g., recombinant adeno-associated virus (rAAV) particles), improving the production of recombinant polypeptides, and improving the production of recombinant viral particles (e.g., rAAV particles) described herein. In some embodiments, the methods include transfecting cells by adding a composition comprising one or more polynucleotides and a transfection reagent to a culture comprising cells and dextran sulfate. In some embodiments, the cell culture is a suspension cell culture. In some embodiments, the cell culture comprises adherent cells grown attached to microcarriers or macrocarriers in a stirred bioreactor. In some embodiments, the cell culture is a suspension cell culture comprising suspension adapted HEK293 cells. In some embodiments, the recombinant viral particles are recombinant adeno-associated virus (rAAV) particles. In some embodiments, the rAAV is selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV. In some embodiments, the rAAV comprises capsid proteins of an AAV8 or AAV9 serotype.

[0068] Given that a large number of rAAV particles are required to prepare one therapeutic unit dose, any increase in rAAV yield reduces the cost of product per unit dose. Increased virus yields can correspondingly reduce the cost of consumables required to generate rAAV particles, as well as the capital investment costs associated with building industrial virus purification facilities.

[0069] definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. To facilitate understanding of the methods of this disclosure, several terms and expressions are defined below.

[0070] For example, the term "about" as used in the methods provided herein to modify the amount of a component in a composition, the concentration of a component in a composition, flow rate, rAAV particle yield, feed volume, salt concentration, similar values, and ranges thereof, refers to variations in numerical quantities that may occur, for example, due to typical measuring and handling procedures used to make concentrates or use solutions; due to inadvertent errors in these procedures; due to differences in the manufacture, source, or purity of components used in making the composition or carrying out the method; and due to similar considerations. The term "about" also encompasses amounts that vary due to aging of a composition or mixture having a particular initial concentration. The term "about" also encompasses amounts that vary due to mixing or processing of a composition or mixture having a particular initial concentration. Whether or not modified by the term "about," the claims include the equivalent of the amount. In some embodiments, the term "about" refers to a range of about 10-20% more or less than the indicated number or range. In further embodiments, "about" refers to plus or minus 10% of the indicated number or range. For example, "about 10%" refers to a range of 9% to 11%.

[0071] The term "dextran sulfate" refers to a sulfated polysaccharide that includes a polymeric backbone of α-1,6 glycosidic linkages between glucose monomers and branches from α-1,3 linkages. Dextran sulfate can be commercially obtained, for example, from MilliporeSigma (Saint Louis, Mo.). "Dextran sulfate" is understood to include both the free acid and its salts. In some embodiments, dextran sulfate is a salt. In some embodiments, dextran sulfate is a free acid. In some embodiments, dextran sulfate is a salt containing a monovalent cation. In some embodiments, dextran sulfate is a Li, Na, K, Rb, or Cs salt. In some embodiments, dextran sulfate is a Na salt. In some embodiments, dextran sulfate contains about 10% to about 25% sulfur. In some embodiments, dextran sulfate contains about 15% to about 20% sulfur. In some embodiments, each glucosyl residue of dextran sulfate contains an average of 1 to 3 sulfate groups. In some embodiments, each glucosyl residue of dextran sulfate contains an average of 2-3 sulfate groups. In some embodiments, dextran sulfate contains about 17% sulfur, which corresponds to approximately 2.3 sulfate groups per glucosyl residue. In some embodiments, the average molecular weight of dextran sulfate is about 3 kDa to about 500 kDa, about 3 kDa to about 250 kDa, about 3 kDa to about 100 kDa, about 3 kDa to about 50 kDa, about 3 kDa to about 25 kDa, or about 3 kDa to about 10 kDa. In some embodiments, the average molecular weight of dextran sulfate is about 5 kDa to about 500 kDa, about 5 kDa to about 250 kDa, about 5 kDa to about 100 kDa, about 5 kDa to about 50 kDa, about 5 kDa to about 25 kDa, or about 5 kDa to about 10 kDa. In some embodiments, the average molecular weight of dextran sulfate is about 3 kDa to about 25 kDa. In some embodiments, the average molecular weight of dextran sulfate is about 3 kDa to about 10 kDa. In some embodiments, the average molecular weight of dextran sulfate is about 4 kDa to about 25 kDa. In some embodiments, the average molecular weight of dextran sulfate is about 4 kDa to about 10 kDa.In some embodiments, the average molecular weight of dextran sulfate is about 5 kDa to about 25 kDa. In some embodiments, the average molecular weight of dextran sulfate is about 5 kDa to about 10 kDa. In some embodiments, the average molecular weight of dextran sulfate is about 5 kDa. In some embodiments, the dextran sulfate is a sodium salt having an average molecular weight between about 3 kDa and 10 kDa. In some embodiments, the dextran sulfate is a sodium salt having an average molecular weight of about 5 kDa. In some embodiments, the dextran sulfate is a sodium salt, contains about 15% to 20% sulfur, and has an average molecular weight between about 3 kDa and 10 kDa. In some embodiments, the dextran sulfate is a sodium salt, contains about 17% sulfur, and has an average molecular weight of about 5 kDa.

[0072] "AAV" is an abbreviation for adeno-associated virus and can be used to refer to the virus itself or any modification, derivative, or pseudotype thereof. The term encompasses all subtypes and both natural and recombinant forms, unless otherwise required. 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 ovine AAV, as well as modifications, derivatives, or pseudotypes thereof. "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.

[0073] "Recombinant" as applied to an AAV particle means that the AAV particle is the product of one or more procedures that result in an AAV particle construct that is distinct from the AAV particle itself.

[0074] Recombinant adeno-associated viral particle "rAAV particle" refers to a viral particle composed of at least one AAV capsid protein and an encapsidated polynucleotide rAAV vector genome that contains a heterologous polynucleotide (i.e., a polynucleotide other than a wild-type AAV genome, e.g., a transgene to be delivered to a mammalian cell). The rAAV particle can be any AAV serotype (e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, or AAV10, or derivatives / modifications / pseudotypes thereof), including any modification, derivative, or pseudotype. Such AAV serotypes and derivatives / modifications / pseudotypes, as well as methods for generating such serotypes / derivatives / modifications / pseudotypes, are known in the art (see, e.g., Asokan et al., Mol. Ther. 20(4):699-708 (2012)).

[0075] The rAAV particles of the disclosure can be of any serotype, or any combination of serotypes (e.g., an rAAV particle population comprising two or more serotypes (e.g., comprising 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 a combination of two or more thereof. In some embodiments, the rAAV particles are rAAV8 or rAAV9 particles.

[0076] 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, AAV16, or derivatives, modifications, or pseudotypes thereof. In some embodiments, the rAAV particles have AAV capsid proteins of a serotype selected from the group consisting of AAV8, AAV9, or derivatives, modifications, or pseudotypes thereof.

[0077] The term "cell culture" refers to cells grown adherent 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 adherent cells grown attached to microcarriers or macrocarriers in stirred bioreactors, are also encompassed by the term "cell culture." Cell culture procedures for both large-scale and small-scale production of proteins are encompassed by the present disclosure. In some embodiments, the term "cell culture" refers to cells grown in suspension. In some embodiments, the term "cell culture" refers to adherent cells grown attached to microcarriers or macrocarriers in stirred bioreactors. In some embodiments, the term "cell culture" refers to cells grown in perfusion culture. In some embodiments, the term "cell culture" refers to cells grown in alternating tangential flow (ATF) supported high density perfusion cultures.

[0078] As used herein, the terms "purifying," "purifying," "separating," "separating," "isolate," "isolating," "isolating," or "isolation" refer to increasing the degree of purity of a target product (e.g., rAAV particles and rAAV genomes) from a sample that contains the 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 by using the methods described herein.

[0079] As used in this disclosure and claims, the singular forms "a," "an," and "the" include the plural forms unless the context clearly dictates otherwise.

[0080] Whenever embodiments are described herein using the term "comprising," it should be understood that other similar embodiments described in terms of "consisting of" and / or "consisting essentially of" are also provided. Also, when embodiments are described herein using the term "consisting essentially of," it should be understood that other similar embodiments described in terms of "consisting of" are also provided.

[0081] The term "and / or," when used herein in phrases such as "A and / or B," is intended to include both A and B, A or B, A (alone), and B (alone). Similarly, the term "and / or," when used in phrases such as "A, B, and / or C," is intended to encompass each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0082] When embodiments of the present disclosure are described in terms of a Markush group or other alternative grouping, the methods of the present disclosure include not only the entire group listed as a whole, but also each member of the group individually, all possible subgroups of the main group, and the main group in the absence of one or more of the group members. The methods of the present disclosure also contemplate the explicit exclusion of any one or more of the group members in the methods of the present disclosure.

[0083] Methods for transfecting cells In one aspect, the disclosure provides a method of transfecting cells, the method comprising: (a) providing a cell culture comprising cells, the culture comprising between about 0.1 mg / L and about 10 mg / L of dextran sulfate; and (b) transfecting the cells by adding to the culture of (a) a composition comprising one or more polynucleotides and a transfection reagent.

[0084] In some embodiments, the culture of a) comprises between about 0.5 mg / L and about 10 mg / L, between about 0.5 mg / L and about 5 mg / L, between about 0.5 mg / L and about 3 mg / L, between about 1 mg / L and about 10 mg / L, between about 1 mg / L and about 5 mg / L, between about 1 mg / L and about 4 mg / L, or between about 1 mg / L and about 3 mg / L dextran sulfate. In some embodiments, the culture of a) comprises between about 0.5 mg / L and about 5 mg / L dextran sulfate. In some embodiments, the culture of a) comprises between about 1 mg / L and about 5 mg / L dextran sulfate. In some embodiments, the culture of a) comprises between about 1 mg / L and about 3 mg / L dextran sulfate.

[0085] In some embodiments, the culture of a) comprises about 0.5 mg / L, about 1 mg / L, about 1.5 mg / L, about 2 mg / L, about 2.5 mg / L, about 3 mg / L, about 4 mg / L, or about 5 mg / L dextran sulfate. In some embodiments, the culture of a) comprises about 1 mg / L dextran sulfate. In some embodiments, the culture of a) comprises about 1.5 mg / L dextran sulfate. In some embodiments, the culture of a) comprises about 2 mg / L dextran sulfate. In some embodiments, the culture of a) comprises about 2.5 mg / L dextran sulfate. In some embodiments, the culture of a) comprises about 3 mg / L dextran sulfate. In some embodiments, the culture of a) comprises about 3.5 mg / L dextran sulfate. In some embodiments, the culture of a) comprises about 4 mg / L dextran sulfate.

[0086] In some embodiments, the culture of a) contains about 2 mg / L dextran sulfate.

[0087] In some embodiments, the disclosure provides a method of transfecting cells, the method comprising: (a) culturing cells in a cell culture, the culture comprising an initial dextran sulfate concentration of between about 1 mg / L and about 20 mg / L and a final dextran sulfate concentration of between about 0.1 mg / L and about 10 mg / L; and (b) transfecting the cells by adding to the culture of (a) a composition comprising one or more polynucleotides and a transfection reagent.

[0088] In some embodiments, the starting dextran sulfate concentration is between about 1 mg / L and about 10 mg / L, between about 1 mg / L and about 5 mg / L, between about 2 mg / L and about 10 mg / L, between about 3 mg / L and about 10 mg / L, or between about 3 mg / L and about 5 mg / L of dextran sulfate. In some embodiments, the starting dextran sulfate concentration is between about 1 mg / L and about 10 mg / L of dextran sulfate. In some embodiments, the starting dextran sulfate concentration is between about 2 mg / L and about 10 mg / L of dextran sulfate. In some embodiments, the starting dextran sulfate concentration is between about 3 mg / L and about 6 mg / L of dextran sulfate.

[0089] In some embodiments, the starting dextran sulfate concentration is about 2 mg / L, about 3 mg / L, about 4 mg / L, about 5 mg / L, about 6 mg / L, about 7 mg / L, about 8 mg / L, about 9 mg / L, or about 10 mg / L dextran sulfate. In some embodiments, the starting dextran sulfate concentration is about 2 mg / L dextran sulfate. In some embodiments, the starting dextran sulfate concentration is about 3 mg / L dextran sulfate. In some embodiments, the starting dextran sulfate concentration is about 4 mg / L dextran sulfate. In some embodiments, the starting dextran sulfate concentration is about 5 mg / L dextran sulfate. In some embodiments, the starting dextran sulfate concentration is about 6 mg / L dextran sulfate. In some embodiments, the starting dextran sulfate concentration is about 7 mg / L dextran sulfate. In some embodiments, the starting dextran sulfate concentration is about 8 mg / L dextran sulfate.

[0090] In some embodiments, the starting dextran sulfate concentration is about 4 mg / L dextran sulfate.

[0091] In some embodiments, the final dextran sulfate concentration is between about 0.5 mg / L and about 10 mg / L, between about 0.5 mg / L and about 5 mg / L, between about 0.5 mg / L and about 3 mg / L, between about 1 mg / L and about 10 mg / L, between about 1 mg / L and about 5 mg / L, between about 1 mg / L and about 4 mg / L, or between about 1 mg / L and about 3 mg / L dextran sulfate. In some embodiments, the final dextran sulfate concentration is between about 0.5 mg / L and about 5 mg / L dextran sulfate. In some embodiments, the final dextran sulfate concentration is between about 1 mg / L and about 5 mg / L dextran sulfate. In some embodiments, the final dextran sulfate concentration is between about 1 mg / L and about 3 mg / L dextran sulfate.

[0092] In some embodiments, the final dextran sulfate concentration is about 0.5 mg / L, about 1 mg / L, about 1.5 mg / L, about 2 mg / L, about 2.5 mg / L, about 3 mg / L, about 4 mg / L, or about 5 mg / L dextran sulfate. In some embodiments, the final dextran sulfate concentration is about 1 mg / L dextran sulfate. In some embodiments, the final dextran sulfate concentration is about 1.5 mg / L dextran sulfate. In some embodiments, the final dextran sulfate concentration is about 2 mg / L dextran sulfate. In some embodiments, the final dextran sulfate concentration is about 2.5 mg / L dextran sulfate. In some embodiments, the final dextran sulfate concentration is about 3 mg / L dextran sulfate. In some embodiments, the final dextran sulfate concentration is about 3.5 mg / L dextran sulfate. In some embodiments, the final dextran sulfate concentration is about 4 mg / L dextran sulfate.

[0093] In some embodiments, the final dextran sulfate concentration is about 2 mg / L dextran sulfate.

[0094] In some embodiments, the starting dextran sulfate concentration is between about 1 mg / L and about 10 mg / L, between about 1 mg / L and about 5 mg / L, between about 2 mg / L and about 10 mg / L, between about 3 mg / L and about 10 mg / L, or between about 3 mg / L and about 5 mg / L of dextran sulfate and the final dextran sulfate concentration is between about 0.5 mg / L and about 10 mg / L, between about 0.5 mg / L and about 5 mg / L, between about 0.5 mg / L and about 3 mg / L, between about 1 mg / L and about 10 mg / L, between about 1 mg / L and about 5 mg / L, between about 1 mg / L and about 4 mg / L, or between about 1 mg / L and about 3 mg / L of dextran sulfate. In some embodiments, the starting dextran sulfate concentration is between about 3 mg / L and about 6 mg / L dextran sulfate and the final dextran sulfate concentration is between about 1 mg / L and about 3 mg / L dextran sulfate.

[0095] In some embodiments, the starting dextran sulfate concentration is about 2 mg / L, about 3 mg / L, about 4 mg / L, about 5 mg / L, about 6 mg / L, about 7 mg / L, about 8 mg / L, about 9 mg / L, or about 10 mg / L of dextran sulfate and the final dextran sulfate concentration is about 0.5 mg / L, about 1 mg / L, about 1.5 mg / L, about 2 mg / L, about 2.5 mg / L, about 3 mg / L, about 4 mg / L, or about 5 mg / L of dextran sulfate.

[0096] In some embodiments, the starting dextran sulfate concentration is about 4 mg / L of dextran sulfate and the final dextran sulfate concentration is about 2 mg / L of dextran sulfate.

[0097] In some embodiments, the one or more polynucleotides comprise a transgene. In some embodiments, the transgene comprises a regulatory element operably linked to the polynucleotide encoding the polypeptide. In some embodiments, the polypeptide comprises an antibody or an antigen-binding fragment thereof, a bispecific antibody, an enzyme, a fusion protein, or an Fc-fusion protein. In some embodiments, the polypeptide comprises an antibody or an antigen-binding fragment thereof.

[0098] In some embodiments, the one or more polynucleotides comprise genes necessary for the generation of a recombinant viral particle. In some embodiments, the recombinant viral particle is a recombinant adenovirus particle. In some embodiments, the recombinant viral particle is a recombinant adeno-associated virus (rAAV) particle.

[0099] Any suitable transfection reagent known in the art can be used to transfect cells. In some embodiments, the transfection reagent comprises a cationic organic carrier. See, e.g., Gigante et al., Medchemcomm 10(10):1692-1718(2019); Damen et al. Medchemcomm 9(9):1404-1425(2018), each of which is incorporated herein by reference in its entirety. In some embodiments, the cationic organic carrier comprises a lipid, e.g., DOTMA, DOTAP, helper lipid (Dope, cholesterol), and combinations thereof. In some embodiments, the cationic organic carrier comprises a multivalent cationic lipid, e.g., DOSPA, DOGS, and mixtures thereof. In some embodiments, the cationic organic carrier comprises a bipolar lipid, or bolaamphiphile (bora). In some embodiments, the cationic organic carrier comprises a biologically reducible and / or dimerizable lipid. In some embodiments, the cationic organic carrier comprises a gemini surfactant. In some embodiments, the cationic organic carrier comprises Lipofectin™, Transfectam™, Lipofectamine™, Lipofectamine 2000™, or Lipofectamin PLUS 2000™. In some embodiments, the cationic organic carrier comprises a polymer, such as poly(L-lysine) (PLL), polyethyleneimine (PEI), polysaccharides (chitosan, dextran, cyclodextrin (CD)), poly[2-(dimethylamino)ethyl methacrylate] (PDMAEMA), and dendrimers (polyamidoamine (PAMAM), poly(propyleneimine) (PPI)). In some embodiments, the cationic organic carrier comprises a peptide, such as a peptide rich in basic amino acids (CWL18), a cell penetrating peptide (CPP) (Arg-rich peptide (octaarginine, TAT)), a nuclear localization signal (NLS) (SV40), and a targeting signal (RGD).In some embodiments, the cationic organic carrier comprises a polymer (e.g., PEI) in combination with a cationic liposome. Paris et al., Molecules 25(14):3277(2020) (hereby incorporated by reference in its entirety). In some embodiments, the transfection reagent comprises calcium phosphate, highly branched organic compounds (dendrimers), cationic polymers (e.g., DEAE dextran or polyethyleneimine (PEI)), lipofection.

[0100] In some embodiments, the transfection reagent comprises poly(L-lysine) (PLL), polyethyleneimine (PEI), linear PEI, branched PEI, dextran, cyclodextrin (CD), poly[2-(dimethylamino)ethyl methacrylate] (PDMAEMA), polyamidoamine (PAMAM), poly(propyleneimine) (PPI), or mixtures thereof. In some embodiments, the transfection reagent comprises polyethyleneimine (PEI), linear PEI, branched PEI, or mixtures thereof. In some embodiments, the transfection reagent comprises polyethyleneimine (PEI). In some embodiments, the transfection reagent comprises linear PEI. In some embodiments, the transfection reagent comprises branched PEI. In some embodiments, the transfection reagent comprises polyethyleneimine (PEI) having a molecular weight between about 5 and about 25 kDa. In some embodiments, the transfection reagent comprises PEGylated polyethyleneimine (PEI). In some embodiments, the transfection reagent comprises a modified polyethyleneimine (PEI) with hydrophobic moieties attached (eg, cholesterol, choline, alkyl groups, and some amino acids).

[0101] Any cell culture system known in the art can be used. In some embodiments, the cell culture is a suspension cell culture. In some embodiments, the cell culture is an adherent cell culture. In some embodiments, the cell culture comprises adherent cells grown attached to microcarriers or macrocarriers in a stirred bioreactor. In some embodiments, the cell culture is a perfusion culture. In some embodiments, the cell culture is an alternating tangential flow (ATF) supported high density perfusion culture.

[0102] In some embodiments, the cell comprises a mammalian cell or an insect cell. In some embodiments, the cell comprises a mammalian cell. In some embodiments, the cell comprises 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, and / or a PerC6 cell. In some embodiments, the cell comprises a HEK293 cell.

[0103] In some embodiments, the cells include suspension-adapted cells. In some embodiments, the cells include suspension-adapted 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 cells comprise suspension-adapted HEK293 cells, HEK293-derived cells (e.g., HEK293T cells, HEK293F cells), CHO cells, CHO-K1 cells, or CHO-derived cells. In some embodiments, the cells comprise suspension-adapted HEK293 cells. In some embodiments, the cells comprise suspension-adapted CHO cells.

[0104] In some embodiments, the cell culture has a volume between about 50 liters and about 20,000 liters. In some embodiments, the cell culture has a volume between about 50 liters and about 5,000 liters. In some embodiments, the cell culture has a volume between about 50 liters and about 2,000 liters. In some embodiments, the cell culture has a volume between about 50 liters and about 1,000 liters. In some embodiments, the cell culture has a volume between about 50 liters and about 500 liters.

[0105] Without being bound to any particular theory, the methods disclosed herein increase the efficiency of transfection, such that cells transfected according to the methods disclosed herein are more likely to contain one or more polynucleotides than control cells transfected in cell cultures that do not contain dextran sulfate. In some embodiments, the methods disclosed herein increase the efficiency of transfection by at least about 10%, at least about 20%, at least about 30%, at least about 40%, or at least about 50% compared to a control method using cell cultures that do not contain dextran sulfate. Methods for measuring transfection efficiency are well known in the art. In some embodiments, transfection efficiency is measured using a reporter transgene construct (e.g., a reporter transgene encoding a fluorescent protein (e.g., GFP)).

[0106] Methods for generating recombinant viral particles In one aspect, the disclosure provides a method for producing recombinant viral particles, comprising: (a) providing a cell culture comprising cells suitable for producing recombinant viral particles, the culture comprising between about 0.1 mg / L and about 10 mg / L dextran sulfate; (b) transfecting the cells by adding to the culture of (a) a composition comprising one or more polynucleotides comprising genes required for the production of recombinant viral particles and a transfection reagent; and (c) maintaining the cell culture comprising the transfected cells under conditions allowing the production of recombinant viral particles. In some embodiments, the culture of (a) comprises between about 1 mg / L and about 3 mg / L dextran sulfate. In some embodiments, the recombinant viral particles are recombinant adeno-associated viral (rAAV) particles. In some embodiments, the one or more polynucleotides comprise one or more helper genes, a rep gene, a cap gene, and a transgene (e.g., a gene of interest or a rAAV genome to be packaged). In some embodiments, the one or more polynucleotides comprise a mixture of three polynucleotides: one encoding the cap and rep genes, one encoding the adenovirus helper functions required for packaging (e.g., the adenovirus E1a, E1b, E4, E2a, and VA genes), and one encoding the rAAV genome to be packaged. In some embodiments, the rAAV particles are AAV8 or AAV9 particles. 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, e.g., AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, and AAV.hu37. In some embodiments, the cell culture is a suspension culture.In some embodiments, the cell culture comprises HEK293 cells adapted to growth in suspension culture. In some embodiments, the cell culture has a volume between about 400 liters and about 5,000 liters. In some embodiments, the transfection reagent comprises a cationic polymer. In some embodiments, the transfection reagent comprises PEI.

[0107] In some embodiments, the disclosure provides a method for increasing production of recombinant viral particles, comprising: (a) providing a cell culture comprising cells suitable for producing recombinant viral particles, the culture comprising between about 0.1 mg / L and about 10 mg / L dextran sulfate; (b) transfecting the cells by adding to the culture of (a) a composition comprising one or more polynucleotides comprising genes required for the production of recombinant viral particles and a transfection reagent; and (c) maintaining the cell culture comprising the transfected cells under conditions allowing the production of recombinant viral particles. In some embodiments, the culture of (a) comprises between about 1 mg / L and about 3 mg / L dextran sulfate. In some embodiments, the culture of (a) comprises about 2 mg / L dextran sulfate. In some embodiments, the recombinant viral particles are recombinant adeno-associated viral (rAAV) particles. In some embodiments, the one or more polynucleotides comprise one or more helper genes, a rep gene, a cap gene, and a transgene (e.g., a gene of interest or a rAAV genome to be packaged). In some embodiments, the one or more polynucleotides comprise a mixture of three polynucleotides: one encoding the cap and rep genes, one encoding the adenovirus helper functions required for packaging (e.g., the adenovirus E1a, E1b, E4, E2a, and VA genes), and one encoding the rAAV genome to be packaged. In some embodiments, the rAAV particles are AAV8 or AAV9 particles. 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.In some embodiments, the cell culture is a suspension culture. In some embodiments, the cell culture comprises HEK293 cells adapted to growth in suspension culture. In some embodiments, the cell culture has a volume between about 400 liters and about 5,000 liters. In some embodiments, the transfection reagent comprises a cationic polymer. In some embodiments, the transfection reagent comprises PEI.

[0108] In some embodiments, the culture of a) comprises between about 0.5 mg / L and about 10 mg / L, between about 0.5 mg / L and about 5 mg / L, between about 0.5 mg / L and about 3 mg / L, between about 1 mg / L and about 10 mg / L, between about 1 mg / L and about 5 mg / L, between about 1 mg / L and about 4 mg / L, or between about 1 mg / L and about 3 mg / L dextran sulfate. In some embodiments, the culture of a) comprises between about 0.5 mg / L and about 5 mg / L dextran sulfate. In some embodiments, the culture of a) comprises between about 1 mg / L and about 5 mg / L dextran sulfate. In some embodiments, the culture of a) comprises between about 1 mg / L and about 3 mg / L dextran sulfate. In some embodiments, the culture of a) comprises about 2 mg / L dextran sulfate.

[0109] In some embodiments, the culture of a) comprises about 0.5 mg / L, about 1 mg / L, about 1.5 mg / L, about 2 mg / L, about 2.5 mg / L, about 3 mg / L, about 4 mg / L, or about 5 mg / L dextran sulfate. In some embodiments, the culture of a) comprises about 1 mg / L dextran sulfate. In some embodiments, the culture of a) comprises about 1.5 mg / L dextran sulfate. In some embodiments, the culture of a) comprises about 2 mg / L dextran sulfate. In some embodiments, the culture of a) comprises about 2.5 mg / L dextran sulfate. In some embodiments, the culture of a) comprises about 3 mg / L dextran sulfate. In some embodiments, the culture of a) comprises about 3.5 mg / L dextran sulfate. In some embodiments, the culture of a) comprises about 4 mg / L dextran sulfate.

[0110] In some embodiments, the culture of a) contains about 2 mg / L dextran sulfate.

[0111] In some embodiments, the disclosure provides a method of producing recombinant viral particles, comprising: (a) culturing cells suitable for producing recombinant viral particles in cell culture for about 1 day to about 5 days, the culture comprising an initial dextran sulfate concentration of between about 1 mg / L and about 20 mg / L and a final dextran sulfate concentration of between about 0.1 mg / L and about 10 mg / L; (b) transfecting the cells by adding to the culture of (a) a composition comprising one or more polynucleotides comprising genes required for the production of recombinant viral particles and a transfection reagent; and (c) maintaining the cell culture comprising the transfected cells under conditions that allow the production of recombinant viral particles. In some embodiments, the initial dextran sulfate concentration is between about 3 mg / L and about 6 mg / L dextran sulfate and the final dextran sulfate concentration is between about 1 mg / L and about 3 mg / L dextran sulfate. In some embodiments, the starting dextran sulfate concentration is about 4 mg / L dextran sulfate and the final dextran sulfate concentration is about 2 mg / L dextran sulfate. In some embodiments, the recombinant viral particles are recombinant adeno-associated virus (rAAV) particles. In some embodiments, the one or more polynucleotides include one or more helper genes, rep genes, cap genes, and transgenes (e.g., genes of interest or rAAV genomes to be packaged). In some embodiments, the one or more polynucleotides include a mixture of three polynucleotides, one encoding the cap and rep genes, one encoding the adenovirus helper functions required for packaging (e.g., adenovirus E1a genes, E1b genes, E4 genes, E2a genes, and VA genes), and one encoding the rAAV genome to be packaged. 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 an AAV 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 cell culture is a suspension culture. In some embodiments, the cell culture comprises HEK293 cells adapted to growth in suspension culture. In some embodiments, the cell culture has a volume between about 400 liters and about 5,000 liters. In some embodiments, the transfection reagent comprises a cationic polymer. In some embodiments, the transfection reagent comprises PEI.

[0112] In some embodiments, the disclosure provides a method for increasing production of recombinant viral particles, comprising: (a) culturing cells suitable for production of recombinant viral particles in cell culture for about 1 day to about 5 days, the culture comprising an initial dextran sulfate concentration of between about 1 mg / L and about 20 mg / L and a final dextran sulfate concentration of between about 0.1 mg / L and about 10 mg / L; (b) transfecting the cells by adding to the culture of (a) a composition comprising one or more polynucleotides comprising genes required for production of recombinant viral particles and a transfection reagent; and (c) maintaining the cell culture comprising the transfected cells under conditions that allow for production of recombinant viral particles. In some embodiments, the initial dextran sulfate concentration is between about 3 mg / L and about 6 mg / L dextran sulfate and the final dextran sulfate concentration is between about 1 mg / L and about 3 mg / L dextran sulfate. In some embodiments, the starting dextran sulfate concentration is about 4 mg / L dextran sulfate and the final dextran sulfate concentration is about 2 mg / L dextran sulfate. In some embodiments, the recombinant viral particles are recombinant adeno-associated virus (rAAV) particles. In some embodiments, the one or more polynucleotides include one or more helper genes, rep genes, cap genes, and transgenes (e.g., genes of interest or rAAV genomes to be packaged). In some embodiments, the one or more polynucleotides include a mixture of three polynucleotides, one encoding the cap and rep genes, one encoding the adenovirus helper functions required for packaging (e.g., adenovirus E1a genes, E1b genes, E4 genes, E2a genes, and VA genes), and one encoding the rAAV genome to be packaged. 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 an AAV 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 cell culture is a suspension culture. In some embodiments, the cell culture comprises HEK293 cells adapted to growth in suspension culture. In some embodiments, the cell culture has a volume between about 400 liters and about 5,000 liters. In some embodiments, the transfection reagent comprises a cationic polymer. In some embodiments, the transfection reagent comprises PEI.

[0113] In some embodiments, the starting dextran sulfate concentration is between about 1 mg / L and about 10 mg / L, between about 1 mg / L and about 5 mg / L, between about 2 mg / L and about 10 mg / L, between about 3 mg / L and about 10 mg / L, or between about 3 mg / L and about 5 mg / L of dextran sulfate. In some embodiments, the starting dextran sulfate concentration is between about 1 mg / L and about 10 mg / L of dextran sulfate. In some embodiments, the starting dextran sulfate concentration is between about 2 mg / L and about 10 mg / L of dextran sulfate. In some embodiments, the starting dextran sulfate concentration is between about 3 mg / L and about 6 mg / L of dextran sulfate.

[0114] In some embodiments, the starting dextran sulfate concentration is about 2 mg / L, about 3 mg / L, about 4 mg / L, about 5 mg / L, about 6 mg / L, about 7 mg / L, about 8 mg / L, about 9 mg / L, or about 10 mg / L dextran sulfate. In some embodiments, the starting dextran sulfate concentration is about 2 mg / L dextran sulfate. In some embodiments, the starting dextran sulfate concentration is about 3 mg / L dextran sulfate. In some embodiments, the starting dextran sulfate concentration is about 4 mg / L dextran sulfate. In some embodiments, the starting dextran sulfate concentration is about 5 mg / L dextran sulfate. In some embodiments, the starting dextran sulfate concentration is about 6 mg / L dextran sulfate. In some embodiments, the starting dextran sulfate concentration is about 7 mg / L dextran sulfate. In some embodiments, the starting dextran sulfate concentration is about 8 mg / L dextran sulfate.

[0115] In some embodiments, the starting dextran sulfate concentration is about 4 mg / L dextran sulfate.

[0116] In some embodiments, the final dextran sulfate concentration is between about 0.5 mg / L and about 10 mg / L, between about 0.5 mg / L and about 5 mg / L, between about 0.5 mg / L and about 3 mg / L, between about 1 mg / L and about 10 mg / L, between about 1 mg / L and about 5 mg / L, between about 1 mg / L and about 4 mg / L, or between about 1 mg / L and about 3 mg / L dextran sulfate. In some embodiments, the final dextran sulfate concentration is between about 0.5 mg / L and about 5 mg / L dextran sulfate. In some embodiments, the final dextran sulfate concentration is between about 1 mg / L and about 5 mg / L dextran sulfate. In some embodiments, the final dextran sulfate concentration is between about 1 mg / L and about 3 mg / L dextran sulfate.

[0117] In some embodiments, the final dextran sulfate concentration is about 0.5 mg / L, about 1 mg / L, about 1.5 mg / L, about 2 mg / L, about 2.5 mg / L, about 3 mg / L, about 4 mg / L, or about 5 mg / L dextran sulfate. In some embodiments, the final dextran sulfate concentration is about 1 mg / L dextran sulfate. In some embodiments, the final dextran sulfate concentration is about 1.5 mg / L dextran sulfate. In some embodiments, the final dextran sulfate concentration is about 2 mg / L dextran sulfate. In some embodiments, the final dextran sulfate concentration is about 2.5 mg / L dextran sulfate. In some embodiments, the final dextran sulfate concentration is about 3 mg / L dextran sulfate. In some embodiments, the final dextran sulfate concentration is about 3.5 mg / L dextran sulfate. In some embodiments, the final dextran sulfate concentration is about 4 mg / L dextran sulfate.

[0118] In some embodiments, the final dextran sulfate concentration is about 2 mg / L dextran sulfate.

[0119] In some embodiments, the starting dextran sulfate concentration is between about 1 mg / L and about 10 mg / L, between about 1 mg / L and about 5 mg / L, between about 2 mg / L and about 10 mg / L, between about 3 mg / L and about 10 mg / L, or between about 3 mg / L and about 5 mg / L of dextran sulfate and the final dextran sulfate concentration is between about 0.5 mg / L and about 10 mg / L, between about 0.5 mg / L and about 5 mg / L, between about 0.5 mg / L and about 3 mg / L, between about 1 mg / L and about 10 mg / L, between about 1 mg / L and about 5 mg / L, between about 1 mg / L and about 4 mg / L, or between about 1 mg / L and about 3 mg / L of dextran sulfate. In some embodiments, the starting dextran sulfate concentration is between about 3 mg / L and about 6 mg / L dextran sulfate and the final dextran sulfate concentration is between about 1 mg / L and about 3 mg / L dextran sulfate.

[0120] In some embodiments, the starting dextran sulfate concentration is about 2 mg / L, about 3 mg / L, about 4 mg / L, about 5 mg / L, about 6 mg / L, about 7 mg / L, about 8 mg / L, about 9 mg / L, or about 10 mg / L of dextran sulfate and the final dextran sulfate concentration is about 0.5 mg / L, about 1 mg / L, about 1.5 mg / L, about 2 mg / L, about 2.5 mg / L, about 3 mg / L, about 4 mg / L, or about 5 mg / L of dextran sulfate.

[0121] In some embodiments, the starting dextran sulfate concentration is about 4 mg / L of dextran sulfate and the final dextran sulfate concentration is about 2 mg / L of dextran sulfate.

[0122] In some embodiments, the recombinant viral particle is a recombinant adeno-associated virus (rAAV) particle. In some embodiments, the recombinant viral particle is a recombinant adenovirus (e.g., human adenovirus or chimpanzee adenovirus) particle. In some embodiments, the recombinant viral particle is a recombinant lentivirus particle.

[0123] In some embodiments, the recombinant viral particle is a rAAV particle. In some embodiments, the rAAV particle is selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV. In some embodiments, the rAAV particles comprise capsid proteins of an 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 serotype. In some embodiments, the rAAV particles comprise capsid proteins of an AAV8, AAV9, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, or AAV.hu37 serotype. In some embodiments, the rAAV particles comprise capsid proteins of the AAV 8 serotype. In some embodiments, the rAAV particles comprise capsid proteins of the AAV 9 serotype.

[0124] In some embodiments, the recombinant viral particle comprises a transgene. A variety of viral transgene expression systems suitable for use in a particular host cell are known to those skilled in the art. It is understood that any viral transgene expression system can be used in accordance with the methods disclosed herein. In some embodiments, the transgene comprises a regulatory element operably linked to the polynucleotide encoding the polypeptide. In some embodiments, the regulatory element comprises one or more of an enhancer, a promoter, and a polyA region. In some embodiments, the regulatory element and the polynucleotide encoding the polypeptide are heterologous.

[0125] In some embodiments, the transgene encodes an anti-VEGF Fab, iduronidase (IDUA), iduronate 2-sulfatase (IDS), low density lipoprotein receptor (LDLR), tripeptidyl peptidase 1 (TPP1), or the non-membrane-bound splice variant of VEGF receptor 1 (sFlt-1).In some embodiments, the transgene is selected from the group consisting of gamma-sarcoglycan, Rab escort protein 1 (REP1 / CHM), retinoid isomerohydrolase (RPE65), cyclic nucleotide-gated channel alpha 3 (CNGA3), cyclic nucleotide-gated channel beta 3 (CNGB3), aromatic L-amino acid decarboxylase (AADC), lysosomal associated membrane protein 2 isoform B (LAMP2B), factor VIII, factor IX, retinitis pigmentosa GTPase regulator (RPGR), retinoschisin (RS 1), sarcoplasmic reticulum calcium ATPase (SERCA2a), aflibercept, battenin (CLN3), transmembrane ER protein (CLN6), glutamic acid decarboxylase (GAD), glial cell line-derived neurotrophic factor (GDNF), aquaporin 1 (AQP1), dystrophin, mini-dystrophin, micro-dystrophin, myotubularin 1 (MTM1), follistatin (FST), glucose-6-phosphatase (G6Pase), apolipoprotein A2 (APOA2), uridine diphosphate glucuronosyl Transferase 1A1 (UGT1A1), arylsulfatase B (ARSB), N-acetylglucosaminidase (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), Encodes neurturin (NRTN), neurotrophin-3 (NT-3 / NTF3), porphobilinogen deaminase (PBGD), nerve growth factor (NGF), mitochondrial encoded NADH:ubiquinone oxidoreductase core subunit 4 (MT-ND4), protective protein cathepsin A (PPCA), dysferlin, MER proto-oncogene tyrosine kinase (MERTK), cystic fibrosis transmembrane conductance regulator (CFTR), or tumor necrosis factor receptor (TNFR)-immunoglobulin (IgG1) Fc fusion. In some embodiments, the recombinant viral particle is a rAAV particle.In some embodiments, the rAAV particles comprise capsid proteins of the AAV 8 serotype. In some embodiments, the rAAV particles comprise capsid proteins of the AAV 9 serotype.

[0126] In some embodiments, the transgene encodes a heterologous viral polypeptide. In some embodiments, the viral polypeptide is a coronavirus polypeptide. In some embodiments, the coronavirus is SARS-CoV1 or SARS-CoV2. In some embodiments, the transgene encodes a spike protein of SARS-CoV1 or SARS-CoV2, or an immunogenic fragment thereof. In some embodiments, the transgene encodes a spike protein of SARS-CoV2, or an immunogenic fragment thereof. In some embodiments, the transgene encodes a receptor binding domain of the SARS-CoV2 spike protein. In some embodiments, the recombinant viral particle is a rAAV particle. In some embodiments, the recombinant viral particle is a recombinant adenovirus particle. In some embodiments, the recombinant viral particle is a recombinant chimpanzee adenovirus particle.

[0127] Transfection-based recombinant virus particle production systems are known to those skilled in the art. See, for example, Reiser et al., Gene Ther 7(11):910-3(2000); Dull et al., J Virol.72(11):8463-8471(1998); Hoffmann et al., PNAS 97(11)6108-6113(2000); Milian et al., Vaccine 35(26):3423-3430 (2017), each of which is incorporated herein by reference in its entirety. The methods disclosed herein can be used to produce recombinant virus particles in transfection-based production systems. In some embodiments, the recombinant viral particle is a recombinant dengue virus, a recombinant Ebola virus, a recombinant human papilloma virus (HPV), a recombinant human immunodeficiency virus (HIV), a recombinant adeno-associated virus (AAV), a recombinant lentivirus, a recombinant influenza virus, a recombinant vesicular stomatitis virus (VSV), a recombinant poliovirus, a recombinant adenovirus, a recombinant retrovirus, a recombinant vaccinia, a recombinant reovirus, a recombinant measles, a recombinant Newcastle disease virus (NDV), a recombinant varicella zoster virus (HZV), a recombinant herpes simplex virus (HSV), or a recombinant baculovirus. In some embodiments, the recombinant viral particle is a recombinant adeno-associated virus (AAV), a recombinant lentivirus, or a recombinant influenza virus. In some embodiments, the recombinant viral particle is a recombinant lentivirus. In some embodiments, the recombinant viral particle is a recombinant influenza virus. In some embodiments, the recombinant viral particle is a recombinant baculovirus. In some embodiments, the recombinant viral particle is a recombinant adeno-associated virus (AAV). 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.

[0128] Any suitable transfection reagent for transfecting cells known in the art can be used to generate recombinant viral particles (e.g., rAAV particles) according to the methods disclosed herein. In some embodiments, the cells are HEK293 cells (e.g., HEK293 cells adapted to suspension culture). In some embodiments, the methods disclosed herein include transfecting the cells using a chemical-based transfection method. In some embodiments, the methods disclosed herein include transfecting the cells using a cationic organic carrier. See, e.g., Gigante et al., Medchemcomm 10(10):1692-1718(2019); Damen et al. Medchemcomm 9(9):1404-1425(2018), each of which is incorporated herein by reference in its entirety. In some embodiments, the cationic organic carrier includes a lipid, e.g., DOTMA, DOTAP, helper lipid (Dope, cholesterol), and combinations thereof. In some embodiments, the cationic organic carrier comprises a polyvalent cationic lipid, such as DOSPA, DOGS, and mixtures thereof. In some embodiments, the cationic organic carrier comprises a bipolar lipid, or a bolaamphiphile (Bola). In some embodiments, the cationic organic carrier comprises a biologically reducible and / or dimerizable lipid. In some embodiments, the cationic organic carrier comprises a gemini surfactant. In some embodiments, the cationic organic carrier comprises Lipofectin™, Transfectam™, Lipofectamine™, Lipofectamine 2000™, or Lipofectamin PLUS 2000™.In some embodiments, the cationic organic carrier comprises a polymer, such as poly(L-lysine) (PLL), polyethyleneimine (PEI), polysaccharides (chitosan, dextran, cyclodextrin (CD)), poly[2-(dimethylamino)ethyl methacrylate] (PDMAEMA), and dendrimers (polyamidoamine (PAMAM), poly(propyleneimine) (PPI)). In some embodiments, the cationic organic carrier comprises a peptide, such as a peptide rich in basic amino acids (CWL. 18 ), cell penetrating peptide (CPP) (Arg-rich peptide (octaarginine, TAT)), nuclear localization signal (NLS) (SV40), and targeting (RGD). In some embodiments, the cationic organic carrier comprises a polymer (e.g., PEI) in combination with a cationic liposome. Paris et al., Molecules 25(14):3277(2020) (incorporated herein by reference in its entirety). In some embodiments, the transfection reagent comprises calcium phosphate, highly branched organic compounds (dendrimer), cationic polymers (e.g., DEAE dextran or polyethyleneimine (PEI)), lipofection.

[0129] In some embodiments, the transfection reagent comprises poly(L-lysine) (PLL), polyethyleneimine (PEI), linear PEI, branched PEI, dextran, cyclodextrin (CD), poly[2-(dimethylamino)ethyl methacrylate] (PDMAEMA), polyamidoamine (PAMAM), poly(propyleneimine) (PPI), or mixtures thereof. In some embodiments, the transfection reagent comprises polyethyleneimine (PEI), linear PEI, branched PEI, or mixtures thereof. In some embodiments, the transfection reagent comprises polyethyleneimine (PEI). In some embodiments, the transfection reagent comprises linear PEI. In some embodiments, the transfection reagent comprises branched PEI. In some embodiments, the transfection reagent comprises polyethyleneimine (PEI) having a molecular weight between about 5 and about 25 kDa. In some embodiments, the transfection reagent comprises PEGylated polyethyleneimine (PEI). In some embodiments, the transfection reagent comprises a modified polyethyleneimine (PEI) with hydrophobic moieties attached (eg, cholesterol, choline, alkyl groups, and some amino acids).

[0130] The composition comprising one or more polynucleotides and a transfection reagent can be prepared by any method known to one of skill in the art. In some embodiments, the composition is prepared by mixing one or more polynucleotides with at least one transfection reagent, which includes diluting each of the transfection reagent and one or more polynucleotides in a sterile liquid (e.g., tissue culture medium) and mixing the diluted transfection reagent and the diluted one or more polynucleotides. In some embodiments, the tissue culture medium used to dilute the transfection reagent and / or one or more polynucleotides does not contain dextran sulfate. One of skill in the art will appreciate that the dilution and mixing is performed to produce a composition comprising the transfection reagent and polynucleotides in a desired ratio and concentration. In some embodiments, the dilution and mixing of the at least one transfection reagent and one or more polynucleotides produces a composition comprising the transfection reagent and polynucleotides in a weight ratio of between about 1:5 and 5:1. In some embodiments, the weight ratio of the transfection reagent and polynucleotides is between about 1:3 and 3:1. In some embodiments, the weight ratio of transfection reagent and polynucleotide is between about 1:3 and 1:1. In some embodiments, the weight ratio of transfection reagent and polynucleotide is between about 1:2 and 1:1.5. In some embodiments, the weight ratio of transfection reagent and polynucleotide is about 1:5, 1:4, 1:3, 1:2.5, 1:2, 1:1.75, 1:1.5, 1:1.25, 1:1, 1.25:1, 1:5:1, 1:75:1, 2:1, 2:5:1, 3:1, 4:1, or 5:1. In some embodiments, the weight ratio of transfection reagent and polynucleotide is about 1:2. In some embodiments, the weight ratio of transfection reagent and polynucleotide is about 1:1.75. In some embodiments, the weight ratio of transfection reagent and polynucleotide is about 1:1.5. In some embodiments, the weight ratio of transfection reagent to polynucleotide is about 1:1.25. In some embodiments, the weight ratio of transfection reagent to polynucleotide is about 1:1. In some embodiments, the weight ratio of transfection reagent to polynucleotide is about 1.25:1.In some embodiments, the weight ratio of transfection reagent to polynucleotide is about 1.5:1. In some embodiments, the weight ratio of transfection reagent to polynucleotide is about 1.75:1. In some embodiments, the weight ratio of transfection reagent to polynucleotide is about 2:1. In some embodiments, the one or more polynucleotides comprise three plasmids. In some embodiments, the one or more polynucleotides comprise two plasmids. In some embodiments, the one or more polynucleotides comprise one plasmid. In some embodiments, the recombinant virus is a recombinant AAV and the one or more polynucleotides comprise a mixture of three polynucleotides, one encoding the cap and rep genes, one encoding the adenovirus helper functions required for packaging (e.g., the adenovirus E1a, E1b, E4, E2a, and VA genes), and one encoding the rAAV genome to be packaged. 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 an AAV 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 transfection reagent is PEI.

[0131] In some embodiments, a composition comprising a transfection reagent and one or more polynucleotides is incubated prior to addition to the culture to allow for the formation of polynucleotide:transfection reagent complexes. In some embodiments, the incubation is at room temperature. In some embodiments, the incubation comprises shaking the composition, for example, in a shaker, at between about 100 rpm and about 200 rpm. In some embodiments, the incubation is for between about 5 minutes and about 20 minutes. In some embodiments, the incubation is for about 10 minutes to about 15 minutes. In some embodiments, the incubation is for 15 minutes or less. In some embodiments, the incubation is for 10 minutes or less. In some embodiments, the incubation is for about 5 minutes, about 10 minutes, or about 15 minutes. In some embodiments, the incubation is for about 10 minutes. In some embodiments, the incubation is for about 10 minutes. In some embodiments, the transfection reagent comprises PEI.

[0132] In some embodiments, the volume of the composition comprising one or more polynucleotides comprising genes required for the generation of recombinant viral particles and a transfection reagent added to the culture is between about 5% and about 20% of the volume of the culture. In some embodiments, the volume of the composition added is between about 7% and about 15% of the volume of the culture. In some embodiments, the volume of the composition added is about 10% of the volume of the culture. In some embodiments, the one or more polynucleotides comprise genes required for the generation of recombinant AAV particles. In some embodiments, the transfection reagent comprises PEI. In some embodiments, the culture comprises HEK293 cells (e.g., HEK293 cells adapted to suspension culture).

[0133] In some embodiments, the culture has a volume between about 400 liters and about 20,000 liters. In some embodiments, the culture has a volume between about 500 liters and about 20,000 liters. In some embodiments, the culture has a volume between about 700 liters and about 20,000 liters. In some embodiments, the culture has a volume between about 1,000 liters and about 20,000 liters. In some embodiments, the culture has a volume between about 400 liters and about 10,000 liters. In some embodiments, the culture has a volume between about 500 liters and about 10,000 liters. In some embodiments, the culture has a volume between about 700 liters and about 10,000 liters. In some embodiments, the culture has a volume between about 1,000 liters and about 10,000 liters. In some embodiments, the culture has a volume between about 400 liters and about 5,000 liters. In some embodiments, the culture has a volume between about 500 liters and about 5,000 liters. In some embodiments, the culture has a volume between about 700 liters and about 5,000 liters. In some embodiments, the culture has a volume between about 1,000 liters and about 5,000 liters. In some embodiments, the culture comprises HEK293 cells (e.g., HEK293 cells adapted to suspension culture).

[0134] In some embodiments, the culture has a volume between about 200 liters and about 5,000 liters. In some embodiments, the culture has a volume between about 200 liters and about 2,000 liters. In some embodiments, the culture has a volume between about 200 liters and about 1,000 liters. In some embodiments, the culture has a volume between about 200 liters and about 500 liters. In some embodiments, the culture comprises HEK293 cells (e.g., HEK293 cells adapted to suspension culture).

[0135] In some embodiments, the culture has a volume of about 200 liters. In some embodiments, the culture has a volume of about 300 liters. In some embodiments, the culture has a volume of about 400 liters. In some embodiments, the culture has a volume of about 500 liters. In some embodiments, the culture has a volume of about 750 liters. In some embodiments, the culture has a volume of about 1,000 liters. In some embodiments, the culture has a volume of about 2,000 liters. In some embodiments, the culture has a volume of about 3,000 liters. In some embodiments, the culture has a volume of about 5,000 liters. In some embodiments, the culture comprises HEK293 cells (e.g., HEK293 cells adapted to suspension culture).

[0136] In some embodiments, the culture comprises between about 2x10E+6 and about 10E+7 viable cells / ml. In some embodiments, the culture comprises between about 3x10E+6 and about 8x10E+6 viable cells / ml. In some embodiments, the culture comprises about 3x10E+6 viable cells / ml. In some embodiments, the culture comprises about 4x10E+6 viable cells / ml. In some embodiments, the culture comprises about 5x10E+6 viable cells / ml. In some embodiments, the culture comprises about 6x10E+6 viable cells / ml. In some embodiments, the culture comprises about 7x10E+6 viable cells / ml. In some embodiments, the culture comprises about 8x10E+6 viable cells / ml. In some embodiments, the culture comprises HEK293 cells (e.g., HEK293 cells adapted to suspension culture).

[0137] In some embodiments, the cell comprises a mammalian cell or an insect cell. In some embodiments, the cell comprises a mammalian cell. In some embodiments, the cell comprises 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, and / or a PerC6 cell. In some embodiments, the cell comprises a HEK293 cell.

[0138] In some embodiments, the culture is maintained for about 2 to about 10 days after adding a composition comprising one or more polynucleotides comprising genes required for the production of recombinant viral particles and a transfection reagent. In some embodiments, the culture is maintained for about 5 to about 14 days or more after adding the composition. In some embodiments, the culture is maintained for about 2 to about 7 days after adding the composition. In some embodiments, the culture is maintained for about 3 to about 5 days after adding the composition. In some embodiments, the culture is maintained for about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, or about 7 days after adding the composition. In some embodiments, the culture is maintained for about 5 days after adding the composition. In some embodiments, the cell culture is maintained for about 6 days after adding the composition. In some embodiments, the cell culture is maintained under conditions that allow for the production of rAAV particles for continued harvest. In some embodiments, the culture comprises HEK293 cells (e.g., HEK293 cells adapted to suspension culture).

[0139] In some embodiments, the methods disclosed herein increase the production of recombinant viral particles (e.g., rAAV particles) compared to a reference method involving transfecting cells in a cell culture that does not contain dextran sulfate. In some embodiments, the methods disclosed herein generate at least about 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, or 2-fold more viral particles than a reference method involving transfecting cells in a cell culture that does not contain dextran sulfate. In some embodiments, the methods disclosed herein generate at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% more viral particles than a reference method involving transfecting cells in a cell culture that does not contain dextran sulfate. In some embodiments, the methods disclosed herein generate at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% more virus particles than a reference method comprising transfecting cells in a cell culture that does not contain dextran sulfate. In some embodiments, the methods disclosed herein generate at least about 10% more virus particles than the reference method. In some embodiments, the methods disclosed herein generate at least about 20 percent more virus particles than the reference method. In some embodiments, the methods disclosed herein generate at least about 20 percent more virus particles than the reference method. In some embodiments, the methods disclosed herein generate at least about 20 percent more virus particles than the reference method. In some embodiments, the methods disclosed herein generate at least about 70 percent more virus particles than the reference method. In some embodiments, the methods disclosed herein generate at least about 100 percent more virus particles than the reference method. In some embodiments, the methods disclosed herein increase production of recombinant virus by at least about 50%, at least about 75%, or at least about 100%.In some embodiments, the methods disclosed herein increase recombinant virus production 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 production by at least about 2-fold. In some embodiments, the increase in production is quantified by comparing the recombinant virus (e.g., rAAV) titers of the production cultures. In some embodiments, the recombinant virus (e.g., rAAV) titers are measured as genome copies (GC) per milliliter of production culture. In some embodiments, the recombinant virus is rAAV. In some embodiments, the rAAV particles comprise capsid proteins from an AAV capsid serotype selected from AAV8 and AAV9. In some embodiments, the rAAV particles have an AAV capsid serotype of AAV8. In some embodiments, the rAAV particles have an 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 capsid proteins with high sequence homology to AAV8 or AAV9, e.g., AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, and AAV.hu37.

[0140] In some embodiments, the methods disclosed herein increase the production of rAAV particles while maintaining or improving the quality attributes of the rAAV particles and compositions comprising the same. In some embodiments, the quality of the rAAV particles and compositions comprising the same is assessed by quantifying the concentration of the rAAV particles (e.g., GC / ml), the percentage of particles containing copies of the rAAV genome, the proportion of particles that do not contain a genome, the infectivity of the rAAV particles, the stability of the rAAV particles, the concentration of residual host cell proteins or the concentration of 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 the rAAV particles or compositions comprising the same produced by the methods disclosed herein is the same as the quality of the rAAV particles or compositions produced by a reference method that includes a single step of mixing, incubating, and transferring the same volume of polynucleotide:transfection reagent complex. In some embodiments, the quality of the rAAV particles, or compositions comprising same, produced by the methods disclosed herein is better than the quality of the rAAV particles or compositions produced by a reference method that involves a single step of mixing, incubating, and transferring the same volume of polynucleotide:transfection reagent complex.

[0141] In some embodiments, the methods disclosed herein generate rAAV particles between about 1×10e+10 GC / ml and about 1×10e+13 GC / ml. In some embodiments, the methods disclosed herein generate rAAV particles between about 1×10e+10 GC / ml and about 1×10e+11 GC / ml. In some embodiments, the methods disclosed herein generate rAAV particles between about 5×10e+10 GC / ml and about 1×10e+12 GC / ml. In some embodiments, the methods disclosed herein generate rAAV particles between about 5×10e+10 GC / ml and about 1×10e+13 GC / ml. In some embodiments, the methods disclosed herein generate rAAV particles between about 1×10e+11 GC / ml and about 1×10e+13 GC / ml. In some embodiments, the methods disclosed herein generate rAAV particles between about 5×10e+10 GC / ml and about 5×10e+12 GC / ml. In some embodiments, the methods disclosed herein produce rAAV particles between about 1×10e+11 GC / ml and about 5×10e+12 GC / ml. In some embodiments, the methods disclosed herein produce rAAV particles greater than about 1×10e+11 GC / ml. In some embodiments, the methods disclosed herein produce rAAV particles greater than about 5×10e+11 GC / ml. In some embodiments, the methods disclosed herein produce rAAV particles greater than about 1×10e+12 GC / ml. In some embodiments, the rAAV particles comprise capsid proteins from an AAV capsid serotype selected from AAV8 and AAV9. In some embodiments, the rAAV particles have an AAV capsid serotype of AAV8. In some embodiments, the rAAV particles have an AAV capsid serotype of AAV9. In some embodiments, the rAAV particles comprise capsid proteins 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 comprise 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.

[0142] In some embodiments, the methods disclosed herein generate rAAV particles of at least about 5×10e+10 GC / ml. In some embodiments, the methods disclosed herein generate rAAV particles of at least about 1×10e+11 GC / ml. In some embodiments, the methods disclosed herein generate rAAV particles of at least about 5×10e+11 GC / ml. In some embodiments, the methods disclosed herein generate rAAV particles of at least about 1×10e+12 GC / ml. In some embodiments, the methods disclosed herein generate rAAV particles of at least about 5×10e+12 GC / ml. In some embodiments, the methods disclosed herein generate rAAV particles of at least about 1×10e+13 GC / ml. In some embodiments, the methods disclosed herein generate rAAV particles of at least about 5×10e+13 GC / ml. In some embodiments, the rAAV particles comprise capsid proteins from an AAV capsid serotype selected from AAV8 and AAV9. In some embodiments, the rAAV particles have an AAV capsid serotype of AAV8. In some embodiments, the rAAV particles have an AAV capsid serotype of AAV9. In some embodiments, the rAAV particles comprise capsid proteins 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 comprise capsid proteins with high sequence homology to AAV8 or AAV9, e.g., AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, and AAV.hu37.

[0143] Numerous cell culture-based systems for the production of rAAV particles are known in the art, any of which can be used to practice the methods disclosed herein. rAAV production cultures for producing rAAV viral particles require (1) suitable host cells (e.g., including human-derived cell lines (e.g., HeLa, A549, or HEK293 cells and their derivatives (HEK293T cells, HEK293F cells)), mammalian cell lines (e.g., Vero), CHO cells or CHO-derived cells); (2) suitable helper virus functions provided by wild-type or mutant adenovirus (e.g., temperature-sensitive adenovirus), herpes virus, baculovirus, or plasmid constructs providing helper functions; (3) AAV rep and cap genes and gene products; (4) a transgene (e.g., a therapeutic transgene) flanked by AAV ITR sequences; and (5) suitable media and media components to support rAAV production.

[0144] Those skilled in the art are aware of numerous ways in which AAV rep and cap genes, AAV helper genes (e.g., adenovirus E1a, E1b, E4, E2a, and VA genes), and rAAV genomes (including one or more genes of interest flanked by inverted terminal repeats (ITRs)) can be introduced into cells to produce or package rAAV. The phrase "adenovirus helper functions" refers to multiple viral helper genes that are expressed in cells (as RNA or protein) so that AAV grows efficiently in the cells. Those skilled in the art understand that helper viruses, including adenovirus and herpes simplex virus (HSV), facilitate AAV replication, and that certain genes that provide essential functions have been identified, for example, helpers can induce changes to the cellular environment that facilitate such AAV gene expression and replication. In some embodiments of the methods disclosed herein, the AAV rep and cap genes, helper genes, and the rAAV genome are introduced into cells by transfection with one or more plasmid vectors encoding the AAV rep and cap genes, helper genes, and the rAAV genome.

[0145] Molecular biology techniques for developing plasmids or viral vectors encoding AAV rep and cap genes, helper genes, and / or rAAV genomes are generally known in the art. In some embodiments, the AAV rep and cap genes are encoded by a single plasmid vector. In some embodiments, the AAV helper genes (e.g., the adenovirus E1a, E1b, E4, E2a, and VA genes) are encoded by a single plasmid vector. In some embodiments, the E1a or E1b genes are stably expressed by the host cell, and the remaining AAV helper genes are introduced into the cell by transfection 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 transfection 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 transfection with a single plasmid vector. In some embodiments, the helper genes are stably expressed by the host cell. In some embodiments, the AAV rep and cap genes are encoded by a single viral vector. In some embodiments, the AAV helper genes (e.g., the adenovirus E1a, E1b, E4, E2a, and VA genes) are encoded by a single viral vector. In some embodiments, the E1a or E1b genes are stably expressed by the host cell, and the remaining AAV helper genes are introduced into the cell by transfection 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 transfection with a single viral 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 transfection with a single viral vector.In some embodiments, the AAV rep and cap genes, the adenoviral helper functions necessary for packaging, and the rAAV genome to be packaged are introduced into a cell by transfection with one or more polynucleotides, e.g., vectors. In some embodiments, the methods disclosed herein involve transfecting a cell with a mixture of three polynucleotides, one encoding the cap and rep genes, one encoding the adenoviral helper functions necessary for packaging (e.g., the adenoviral E1a, E1b, E4, E2a, and VA genes), and one encoding the rAAV genome to be packaged. In some embodiments, the AAV cap gene is an AAV8 or AAV9 cap gene. In some embodiments, the AAV cap gene is an 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, e.g., 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 a gene of interest flanked by AAV ITRs.In some embodiments, the AAV ITRs are selected from the group consisting of AAV1, AAV2, rAAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, 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, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, or AAV.HSC16, or from other AAV serotypes.

[0146] Any combination of vectors can be used to introduce the AAV rep and cap genes, AAV helper genes, and the rAAV genome into the cells in which the rAAV particles are produced or packaged. In some embodiments of the methods disclosed herein, a first plasmid vector can be used that encodes a rAAV genome that includes a gene of interest flanked by AAV inverted terminal repeats (ITRs), a second vector that encodes the AAV rep and cap genes, and a third vector that encodes the helper genes. In some embodiments, a mixture of the three vectors is co-transfected into the cells.

[0147] In some embodiments, a combination of transfection and infection is used by using both a viral vector in addition to a plasmid vector.

[0148] In some embodiments, one or more of the rep and cap genes and the AAV helper genes are constitutively expressed by the cell and do not need to be transfected or transduced into the cell. In some embodiments, the cell constitutively expresses the rep and / or cap genes. 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.

[0149] In some embodiments, the AAV rep, cap, and helper genes (e.g., Ela, E1b, E4, E2a, or VA genes) can be of any AAV serotype. Similarly, the AAV ITRs can be of any AAV serotype. For example, in some embodiments, the AAV ITR is AAV1, AAV2, rAAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, 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, 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 more than one serotype). In some embodiments, the AAV cap gene is from an AAV9 or AAV8 cap gene.In some embodiments, the AAV cap gene is selected from the group consisting of AAV1, AAV2, rAAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, 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, AAV3 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.

[0150] Any suitable medium known in the art can be used for the production of recombinant viral particles (e.g., rAAV particles) according to the methods disclosed herein. Such media include, but are not limited to, Modified Eagle's Medium (MEM), Dulbecco's Modified Eagle's Medium (DMEM), and media produced by Hyclone Laboratories and JRH, including Sf-900 II SFM medium as described in U.S. Pat. No. 6,723,551, which is incorporated herein by reference in its entirety. In some embodiments, the medium comprises Dynamis™ medium, FreeStyle™ 293 Expression Medium, or Expi293™ Expression Medium from Invitrogen / ThermoFisher. In some embodiments, the medium comprises Dynamis™ medium. In some embodiments, the methods disclosed herein use cell cultures comprising serum-free medium, animal component-free medium, or chemically defined medium. In some embodiments, the medium is an animal component-free medium. In some embodiments, the medium comprises serum. In some embodiments, the medium comprises fetal bovine serum. In some embodiments, the medium is a glutamine-free medium. In some embodiments, the medium includes glutamine. In some embodiments, the medium is supplemented with one or more of nutrients, salts, buffers, and additives (e.g., antifoam agents). In some embodiments, the medium is supplemented with glutamine. In some embodiments, the medium is supplemented with serum. In some embodiments, the medium is supplemented with fetal bovine serum. In some embodiments, the medium is supplemented with a poloxamer, e.g., Kolliphor® P 188 Bio. In some embodiments, the medium is a basal medium. In some embodiments, the medium is a feed medium.

[0151] Recombinant virus (e.g., rAAV) production cultures can be routinely grown under a variety of conditions (over a wide range of temperatures, for various lengths of time, etc.) appropriate for the particular host cell being utilized. As is known in the art, rAAV virus production cultures can include suspension-adapted 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, TCM cells, and the like. K-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, and SF-9 cells, which can be cultured in a variety of ways, including, for example, spinner flasks, stirred tank bioreactors, and disposable systems such as the Wave bag system. Numerous suspension cultures for producing rAAV particles are known in the art, including, for example, those disclosed in U.S. Patent Nos. 6,995,006, 9,783,826, and U.S. Patent Publication No. 20120122155, each of which is incorporated herein by reference in its entirety. In some embodiments, the recombinant virus is a recombinant AAV.

[0152] Any cell or cell line known in the art to produce recombinant viral particles (e.g., rAAV particles) can be used in any one of the methods disclosed herein. In some embodiments, the methods disclosed herein for producing recombinant viral particles (e.g., rAAV particles) or increasing the production of recombinant viral particles (e.g., rAAV particles) use 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, LLC-MK cells, MDCK cells, RAF cells, RK cells, TCMK-1 cells, PK15 cells, BHK cells, BHK-21 cells, NS-1 cells, BHK cells, 293 cells, RK cells, Per.C6 cells, chicken embryo cells, or SF-9 cells. In some embodiments, the methods disclosed herein use mammalian cells. In some embodiments, the methods disclosed herein use insect cells, such as SF-9 cells. In some embodiments, the methods disclosed herein use cells adapted to grow in suspension culture. In some embodiments, the methods disclosed herein use HEK293 cells adapted to grow in suspension culture. In some embodiments, the recombinant viral particles are recombinant AAV particles.

[0153] In some embodiments, the cell culture disclosed herein is a suspension culture. In some embodiments, the large-scale suspension cell culture disclosed herein comprises HEK293 cells adapted to growth in suspension culture. In some embodiments, the cell culture disclosed herein comprises serum-free medium, animal component-free medium, or chemically defined medium. In some embodiments, the cell culture disclosed herein comprises serum-free medium. In some embodiments, the suspension-adapted cells are cultured in shake flasks, spinner flasks, cell bags, or bioreactors.

[0154] In some embodiments, the cell cultures disclosed herein comprise serum-free media, animal component-free media, or chemically defined media. In some embodiments, the cell cultures disclosed herein comprise serum-free media.

[0155] In some embodiments, the large-scale suspension cell culture disclosed herein comprises a high-density cell culture. In some embodiments, the culture has a total cell density between about 1×10E+06 cells / ml and about 30×10E+06 cells / ml. In some embodiments, more than about 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.

[0156] 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 are selected from the group consisting of AAV1, AAV2, rAAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, 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.Hu37 ... The capsid protein comprises an AAV capsid serotype 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, the rAAV particles comprise AAV1, AAV2, rAAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, 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, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, or AAV.HSC16 capsid protein.

[0157] In some embodiments, the rAAV particles comprise capsid proteins from an AAV capsid serotype selected from AAV8 and AAV9. In some embodiments, the rAAV particles have an AAV capsid serotype of AAV8. In some embodiments, the rAAV particles have an AAV capsid serotype of AAV9.

[0158] In some embodiments, the rAAV particles comprise capsid proteins 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 comprise 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.

[0159] In some embodiments, the rAAV particles comprise capsid proteins that are derivatives, modifications, or pseudotypes of the AAV8 capsid protein or the AAV9 capsid protein. In some embodiments, the rAAV particles comprise capsid proteins that are at least 80% identical to the VP1, VP2, and / or VP3 sequences of the AAV8 capsid protein, e.g., 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, etc., i.e., up to 100% identical to the AAV8 capsid protein.

[0160] In some embodiments, the rAAV particles comprise capsid proteins that are derivatives, modifications, or pseudotypes of the AAV9 capsid protein. In some embodiments, the rAAV particles comprise capsid proteins that are at least 80% identical to the VP1, VP2, and / or VP3 sequences of the AAV9 capsid protein, e.g., 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, etc., i.e., up to 100% identical to the AAV9 capsid protein.

[0161] In some embodiments, the rAAV particles comprise capsid proteins that have at least 80% identity or more, e.g., 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, etc., i.e., up to 100% identity, to the VP1, VP2, and / or VP3 sequences of an AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.PHB, or AAV.7m8 capsid protein. In some embodiments, the rAAV particles comprise AAV capsid proteins with high sequence homology to AAV8 or AAV9, e.g., capsid proteins having at least 80% identity or more, e.g., 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, etc., i.e., up to 100% identity, to the VP1, VP2, and / or VP3 sequences of AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, and AAV.hu37.

[0162] In additional embodiments, the rAAV particles comprise mosaic capsids. In additional embodiments, the rAAV particles comprise pseudotyped rAAV particles. In additional embodiments, the rAAV particles comprise capsids that include capsid protein chimeras of two or more AAV capsid serotypes.

[0163] rAAV particles The provided methods are suitable for use in the generation of any isolated recombinant AAV particle, such that the rAAV can be of any serotype, modification, or derivative known in the art, or any combination thereof (e.g., a population of rAAV particles comprising two or more serotypes, e.g., comprising two or more of rAAV2, rAAV8, and rAAV9 particles). In some embodiments, the rAAV particles comprise AAV1, AAV2, rAAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, 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. 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, or AAV.HSC16, or other rAAV particles, or a combination of two or more thereof.

[0164] In some embodiments, the rAAV particles include AAV1, AAV1, AAV2, rAAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, 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, AAV 2tYF, 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 a derivative, modification, or pseudotype thereof.In some embodiments, the rAAV particle may be any of the following: AAV1, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, 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, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, AAV.HSC16, 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, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC16, AAV.HSC The capsid protein is at least 80% identical to the VP1, VP2, and / or VP3 sequences of an AAV capsid serotype selected from AV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, or AAV.HSC16, e.g., 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, etc., i.e., up to 100% identical.

[0165] In some embodiments, the rAAV particles comprise AAV1, AAV1, AAV2, rAAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, 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 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 a derivative, modification, or pseudotype thereof.In some embodiments, the rAAV particles may be any of the following: AAV1, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, 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, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, AAV.HSC16, 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, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC16, AAV.HSC18, A The capsid protein is at least 80% identical to the VP1, VP2, and / or VP3 sequences of an AAV capsid serotype selected from AV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, or AAV.HSC16, e.g., 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, etc., i.e., up to 100% identical.

[0166] 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 are incorporated by reference in their entireties. In certain embodiments, the rAAV particles comprise a capsid with one of the amino acid insertions: LGETTRP or LALGETTRP, as described in U.S. Patent Nos. 9,193,956, 9458517, and 9,587,282, and U.S. Patent Application Publication No. 2016 / 0376323, each of which is incorporated by reference in its entirety. In some embodiments, the rAAV particles comprise an AAV.7m8 capsid, 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 by reference in its entirety. In some embodiments, the rAAV particles comprise any AAV capsid disclosed in U.S. Patent No. 9,585,971, such as AAV.PHP.B. In some embodiments, the rAAV particles comprise any AAV capsid disclosed in U.S. Patent No. 9,840,719 and WO2015 / 013313, each of which is incorporated by reference in its entirety, such as AAV.Rh74 and RHM4-1. In some embodiments, the rAAV particles comprise any of the AAV capsids disclosed in WO2014 / 172669 (incorporated herein by reference in its entirety), such as AAV rh.74. In some embodiments, the rAAV particles comprise an 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 comprise any of the AAV capsids disclosed in WO2017 / 070491 (incorporated herein by reference in its entirety), such as AAV2tYF.In some embodiments, the rAAV particles comprise an AAVLK03 or AAV3B capsid as described in Puzzo et al., 2017, Sci. Transl. Med. 29(9):418, which are incorporated by reference in their entireties. In some embodiments, the rAAV particles comprise any AAV capsid disclosed in U.S. Patent Nos. 8,628,966, 8,927,514, 9,923,120, and WO2016 / 049230, such as HSC1, HSC2, HSC3, HSC4, HSC5, HSC6, HSC7, HSC8, HSC9, HSC10, HSC11, HSC12, HSC13, HSC14, HSC15, or HSC16, each of which is incorporated by reference in its entirety.

[0167] In some embodiments, the rAAV particles are disclosed in accordance with the following patents and patent applications, each of which is incorporated by reference in its entirety: U.S. Pat. Nos. 7,282,199, 7,906,111, 8,524,446, 8,999,678, 8,628,966, 8,927,514, 8,734,809, US Pat. Nos. 9,284,357, 9,409,953, 9,169,299, 9,193, Nos. 956, 9458517, and 9,587,282, U.S. Patent Application Publication Nos. 2015 / 0374803, 2015 / 0126588, 2017 / 0067908, 2013 / 0224836, 2016 / 0215024, 2017 / 0051257, and International Patent Application Nos. PCT / US2015 / 034799 and PCT / EP2015 / 053335. In some embodiments, the rAAV particles have capsid proteins that are at least 80% identical or greater, e.g., 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, etc., i.e., up to 100% identical, to the VP1, VP2, and / or VP3 sequences of the AAV capsid disclosed in any of the following patents and patent applications, each of which is incorporated by reference in its entirety herein: U.S. Patent Nos. 7,282,199, 7,906,111, 8,524,446, 8,924,446, and 8,932,446. Nos. 99,678, 8,628,966, 8,927,514, 8,734,809, US9,284,357, 9,409,953, 9,169,299, 9,193,956, 9458517, and 9,587,282, U.S. Patent Application Publication No. US2015 / 0374803, 2015 / 0126588, 2017 / 0067908, 2013 / 0224836, 2016 / 0215024, 2017 / 0051257, as well as International Patent Application Nos. PCT / US2015 / 034799 and PCT / EP2015 / 053335.

[0168] In some embodiments, the rAAV particles are prepared using the methods described in International Patent Application Publication Nos. 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 No. WO 2010 / 127097 (see, e.g., SEQ ID NOs: 5-38), and WO 2015 / 191508 (see, e.g., SEQ ID NOs: 80-294), and U.S. Patent Application Publication No. 20150023924 (see, e.g., SEQ ID NOs: 1, 5-10), the contents of each of which are incorporated herein by reference in their entireties. In some embodiments, the rAAV particles have capsid proteins that are at least 80% identical or greater, e.g., 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, etc., i.e., up to 100% identical, to the VP1, VP2 and / or VP3 sequences of the AAV capsids disclosed in International Patent Application Publication Nos. WO 2003 / 052051 (see, e.g., SEQ ID NO: 2), WO 2005 / 033321 (see, e.g., SEQ ID NOs: 123 and 88), WO 03 / 04239, and the like. No. 7 (see, for example, SEQ ID NOs: 2, 81, 85, and 97), WO2006 / 068888 (see, for example, SEQ ID NOs: 1 and 3-6), WO2006 / 110689 (see, for example, SEQ ID NOs: 5-38), WO2009 / 104964 (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).

[0169] Nucleic acid sequences for AAV-based viral vectors, as well as methods for producing recombinant AAV and AAV capsids, are taught, for example, in U.S. Pat. Nos. 7,282,199, 7,906,111, 8,524,446, 8,999,678, 8,628,966, 8,927,514, 8,734,809, US Pat. Nos. 9,284,357, 9,409,953, 9,169,299, 9,193,956, 9458517, and 9,587,282; U.S. Patent Application Publication Nos. 2015 / 0374803, 2015 / 0126588, and the like. 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 / 033321, WO03 / 042397, WO2006 / 068888, WO2006 / 110689, WO2009 / 104964, WO2010 / 127097, and WO2015 / 191508, and U.S. Patent Application Publication No. 20150023924.

[0170] The provided methods are suitable for use in the generation of recombinant AAV encoding a transgene. In certain embodiments, the transgene is from Tables 1A-1C. In some embodiments, the rAAV genome comprises a vector comprising the following components: (1) AAV inverted terminal repeats flanking an expression cassette; (2) regulatory control elements, such as a) promoter / enhancer, b) polyA signal, and c) optionally an intron; 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 comprising the following components: (1) AAV inverted terminal repeats flanking an expression cassette; (2) regulatory control elements, such as a) promoter / enhancer, b) polyA signal, and c) optionally an intron; and (3) a nucleic acid sequence encoding the light chain Fab and heavy chain Fab of an antibody, or at least the heavy chain or light chain Fab, and optionally the heavy chain Fc region. In yet other embodiments for expressing an intact or substantially intact mAb, the rAAV genome comprises a vector containing the following components: (1) AAV inverted terminal repeats flanking the expression cassette; (2) regulatory control elements, e.g., a) promoter / enhancer, b) polyA signal, and c) optionally introns;and (3) nucleic acid sequences encoding the heavy chain Fabs of the following: anti-VEGF (e.g., sevacizumab, ranibizumab, bevacizumab, and brolucizumab), anti-EpoR (e.g., LKA-651), anti-ALK1 (e.g., asclinbacumab), 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 golimumab), and anti-VEGF (e.g., serotinib, serotinib, and serotinib). mab), anti-RGMa (e.g., elezanumab), anti-TTR (e.g., NI-301 and PRX-004), anti-CTGF (e.g., pamrevlumab), 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 mAb (e.g., etrolizumab), anti-SOST mAb (e.g., romosozumab), anti-pKal mAb (e.g., lanadelumab), 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); optionally an Fc polypeptide of the same isotype as the native form of the therapeutic antibody, e.g., the IgG isotype amino acid sequence IgG1, IgG2, or IgG4, or a modified Fc thereof;and nucleic acid sequences encoding the light chains of: anti-VEGF (e.g., sevacizumab, ranibizumab, bevacizumab, and brolucizumab), anti-EpoR (e.g., LKA-651), anti-ALK1 (e.g., asclinbacumab), 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 golitinib), anti-IL-5 (e.g., mepolizumab), anti-IL12 / IL23 (e.g., ustekinumab), anti-CD19 (e.g., inebilizumab), anti-ITGF7 (e.g., pamrevlumab), anti-IL6R (e.g., satralizumab and sarilumab), anti-IL4R (e.g., dupilumab), anti-IL17A (e.g., ixekizumab and secukinumab), ... mAb (e.g., etrolizumab), anti-SOST mAb (e.g., romosozumab), anti-pKal mAb (e.g., lanadelumab), 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). Here, the heavy chain (Fab and optionally the Fc region) and the light chain are separated by a self-cleaving furin (F) / F2A or a flexible linker, ensuring that the heavy and light chain polypeptides are expressed in equal amounts;

[0171] [Table 1A] TIFF2024503895000002.tif236165TIFF2024503895000003.tif236165TIFF2024503895000004.tif236165TIFF2024503895000005.tif139165

[0172]

Table 1B

[0173]

Table 1C

[0174] In some embodiments, the rAAV particle is a rAAV viral vector encoding an anti-VEGF Fab. In a particular embodiment, the rAAV particle is a rAAV8-based viral vector encoding an anti-VEGF Fab. In a more particular embodiment, the rAAV particle is a rAAV8-based viral vector encoding ranibizumab. In some embodiments, the rAAV particle is a rAAV viral vector encoding iduronidase (IDUA). In a particular embodiment, the rAAV particle is a rAAV9-based viral vector encoding IDUA. In some embodiments, the rAAV particle is a rAAV viral vector encoding iduronate 2-sulfatase (IDS). In a particular embodiment, the rAAV particle is a rAAV9-based viral vector encoding IDS. In some embodiments, the rAAV particle is a rAAV viral vector encoding low density lipoprotein receptor (LDLR). In a particular embodiment, the rAAV particle is a rAAV8-based viral vector encoding LDLR. In some embodiments, the rAAV particle is a rAAV viral vector encoding tripeptidyl peptidase 1 (TPP1) protein. In certain embodiments, the rAAV particle is a rAAV9-based viral vector encoding TPP1. In some embodiments, the rAAV particle is a rAAV viral vector encoding the non-membrane-bound splice variant of VEGF receptor 1 (sFlt-1).In some embodiments, the rAAV particles contain gamma-sarcoglycan, Rab escort protein 1 (REP1 / CHM), retinoid isomerohydrolase (RPE65), cyclic nucleotide-gated channel alpha 3 (CNGA3), cyclic nucleotide-gated channel beta 3 (CNGB3), aromatic L-amino acid decarboxylase (AADC), lysosomal-associated membrane protein 2 isoform B (LAMP2B), factor VIII, factor IX, retinitis pigmentosa GTPase regulator (RPGR), retinoschisin (RS 1), sarcoplasmic reticulum calcium ATPase (SERCA2a), aflibercept, battenin (CLN3), transmembrane ER protein (CLN6), glutamic acid decarboxylase (GAD), glial cell line-derived neurotrophic factor (GDNF), aquaporin 1 (AQP1), dystrophin, microdystrophin, myotubularin 1 (MTM1), follistatin (FST), glucose-6-phosphatase (G6Pase), apolipoprotein A2 (APOA2), uridine diphosphate glucuronosyltransferase 1A1 (UGT1A1), arylsulfatase B (ARSB), N-acetylglucosaminidase (NAGLU), alpha-glucosidase (GAA), alpha-galactosidase (GLA), beta-galactosidase (GLB1), lipoprotein lipase (LPL), alpha 1-antitrypsin (AAT), phosphodiesterase 6B (PDE6B), ornithine carbamoyltransferase 9 (OTC), survival motor neuron (SMN1), survival motor neuron (SMN2), neurturin (NRTN) , neurotrophin-3 (NT-3 / NTF3), porphobilinogen deaminase (PBGD), nerve growth factor (NGF), mitochondrially encoded NADH:ubiquinone oxidoreductase core subunit 4 (MT-ND4), protective protein cathepsin A (PPCA), dysferlin, MER proto-oncogene tyrosine kinase (MERTK), cystic fibrosis transmembrane conductance regulator (CFTR), or tumor necrosis factor receptor (TNFR)-immunoglobulin (IgG1) Fc fusion.

[0175] In additional embodiments, the rAAV particles comprise pseudotyped AAV capsids. In some embodiments, the pseudotyped AAV capsids are rAAV2 / 8 or rAAV2 / 9 pseudotyped AAV capsids. Methods for producing and using pseudotyped rAAV particles are known in the art (see, e.g., Duan et al., J. Virol., 75:7662-7671 (2001); Halbert et al., J. Virol., 74:1524-1532 (2000); Zolotukhin et al., Methods 28:158-167 (2002); and Auricchio et al., Hum. Molec. Genet. 10:3075-3081, (2001)).

[0176] In additional embodiments, the rAAV particles comprise capsids that include capsid protein chimeras of two or more AAV capsid serotypes. In some embodiments, the capsid proteins are selected from the group consisting of AAV1, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, 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 two or more AAV capsid protein chimeras from an AAV serotype 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, or AAV.HSC16.

[0177] In certain embodiments, single-stranded AAV (ssAAV) can be used. In certain embodiments, self-complementary vectors, such as scAAV, can be used (see, e.g., Wu, 2007, Human Gene Therapy, 18(2):171-82; McCarty et al, 2001, Gene Therapy, Vol. 8, Number 16:1248-1254; and U.S. Patent Nos. 6,596,535, 7,125,717, and 7,456,683, each of which is incorporated herein by reference in its entirety).

[0178] In some embodiments, the rAAV particles comprise capsid proteins from an AAV capsid serotype selected from AAV8 or AAV9. In some embodiments, the rAAV particles have an AAV capsid serotype of AAV8. In some embodiments, the rAAV particles have an AAV capsid serotype of AAV9.

[0179] In some embodiments, the rAAV particles comprise capsid proteins that are derivatives, modifications, or pseudotypes of the AAV8 capsid protein or the AAV9 capsid protein. In some embodiments, the rAAV particles comprise capsid proteins that are at least 80% identical to the VP1, VP2, and / or VP3 sequences of the AAV8 capsid protein, e.g., 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, etc., i.e., up to 100% identical to the AAV8 capsid protein.

[0180] In some embodiments, the rAAV particles comprise capsid proteins that are derivatives, modifications, or pseudotypes of the AAV9 capsid protein. In some embodiments, the rAAV particles comprise capsid proteins that are at least 80% identical to the VP1, VP2, and / or VP3 sequences of the AAV9 capsid protein, e.g., 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, etc., i.e., up to 100% identical to the AAV9 capsid protein.

[0181] In additional embodiments, the rAAV particle comprises a mosaic capsid. Mosaic AAV particles are composed of a mixture of viral capsid proteins from different serotypes of AAV. In some embodiments, the rAAV particle comprises a mosaic capsid of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, 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 and a mosaic capsid comprising capsid proteins of a serotype selected from AAV.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, and AAV.HSC16. In some embodiments, the rAAV particles comprise a mosaic capsid comprising capsid proteins of a serotype selected from AAV1, AAV2, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAVrh.8, AAVrh.10, AAVhu.37, AAVrh.20, and AAVrh.74.

[0182] In additional embodiments, the rAAV particles comprise pseudotyped rAAV particles. In some embodiments, the pseudotyped rAAV particles comprise (a) a nucleic acid vector comprising AAV ITRs, and (b) an AAVx (e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.P The capsid comprises a capsid composed of capsid proteins derived from AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, and AAV.HSC16). In additional embodiments, the rAAV particles comprise pseudotyped rAAV particles comprised of capsid proteins of an AAV serotype selected from AAV1, AAV2, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAVrh.8, and AAVrh.10, AAVhu.37, AAVrh.20, and AAVrh.74. In additional embodiments, the rAAV particles comprise pseudotyped rAAV particles comprised of AAV8 capsid proteins. In additional embodiments, the rAAV particles comprise pseudotyped rAAV particles comprised of AAV9 capsid proteins. In some embodiments, the pseudotyped rAAV8 or rAAV9 particles are rAAV2 / 8 or rAAV2 / 9 pseudotyped particles.Methods for generating and using pseudotyped rAAV particles are known in the art (see, e.g., Duan et al., J. Virol., 75:7662-7671 (2001); Halbert et al., J. Virol., 74:1524-1532 (2000); Zolotukhin et al., Methods 28:158-167 (2002); and Auricchio et al., Hum. Molec. Genet. 10:3075-3081, (2001)).

[0183] In additional embodiments, the rAAV particles comprise capsids that include capsid protein chimeras of two or more AAV capsid serotypes. In some embodiments, the capsid protein comprises an AAV8 capsid protein and a chimera of any of the following 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, AAV.Anc80L65 ... and AAV capsid protein chimeras with one or more AAV capsid proteins from an AAV serotype 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, the rAAV particles comprise an AAV capsid protein chimera of an AAV8 capsid protein and one or more AAV capsid proteins from an AAV serotype selected from AAV1, AAV2, AAV5, AAV6, AAV7, AAV9, AAV10, rAAVrh10, AAVrh.8, AAVrh.10, AAVhu.37, AAVrh.20, and AAVrh.74.In some embodiments, the rAAV particles comprise an AAV9 capsid protein and one or more of the following: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, 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, and AAV.HSC16. In some embodiments, the rAAV particles comprise an AAV capsid protein chimera of AAV9 capsid protein and capsid protein of one or more AAV capsid serotypes selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh.8, AAVrh.10, AAVhu.37, AAVrh.20, and AAVrh.74.

[0184] Methods for isolating rAAV particles In some embodiments, the disclosure provides a method for producing a composition comprising isolated recombinant adeno-associated virus (rAAV) particles, the method comprising isolating the rAAV particles from an impure feed (e.g., an rAAV production culture). In some embodiments, a method for producing a formulation comprising the isolated recombinant adeno-associated virus (rAAV) particles disclosed herein comprises (a) isolating the rAAV particles from an impure feed (e.g., an rAAV production culture), and (b) formulating the isolated rAAV particles to produce the formulation.

[0185] In some embodiments, the disclosure further provides a method for producing a formulation comprising a pharmaceutical unit dose of isolated recombinant adeno-associated virus (rAAV) particles, the method comprising isolating the rAAV particles from an impure feed (e.g., an rAAV production culture) and formulating the isolated rAAV particles.

[0186] The isolated rAAV particles can be isolated using methods known in the art. In some embodiments, the method of isolating rAAV particles includes downstream processing, such as harvesting the cell culture, clarifying the harvested cell culture (e.g., by centrifugation or depth filtration), tangential flow filtration, affinity chromatography, anion exchange chromatography, cation exchange chromatography, size exclusion chromatography, hydrophobic interaction chromatography, hydroxylapatite chromatography, sterile filtration, or any combination (or combinations) thereof. In some embodiments, the downstream processing includes at least two, at least three, at least four, at least five, or at least six of harvesting the cell culture, clarifying the harvested cell culture (e.g., by centrifugation or depth filtration), tangential flow filtration, affinity chromatography, anion exchange chromatography, cation exchange chromatography, size exclusion chromatography, hydrophobic interaction chromatography, hydroxylapatite chromatography, and sterile filtration. In some embodiments, downstream processing includes harvesting the cell culture, clarification of the harvested cell culture (e.g., by depth filtration), sterile filtration, tangential flow filtration, affinity chromatography, and anion exchange chromatography. In some embodiments, downstream processing includes clarification of the harvested cell culture, sterile filtration, tangential flow filtration, affinity chromatography, and anion exchange chromatography. In some embodiments, downstream processing includes clarification of the harvested cell culture by depth filtration, sterile filtration, tangential flow filtration, affinity chromatography, and anion exchange chromatography. In some embodiments, clarification of the harvested cell culture includes sterile filtration. In some embodiments, downstream processing does not include centrifugation. In some embodiments, the rAAV particles include capsid proteins of the AAV8 serotype. In some embodiments, the rAAV particles include capsid proteins of the AAV9 serotype.

[0187] In some embodiments, a method of isolating rAAV particles produced according to the methods disclosed herein comprises harvesting the cell culture, clarification of the harvested cell culture (e.g., by depth filtration), a first sterile filtration, a first tangential flow filtration, affinity chromatography, anion exchange chromatography (e.g., monolith anion exchange chromatography with a quaternary amine ligand or AEX chromatography), a second tangential flow filtration, and a second sterile filtration. In some embodiments, a method of isolating rAAV particles disclosed herein comprises harvesting the cell culture, clarification of the harvested cell culture (e.g., by depth filtration), a first sterile filtration, affinity chromatography, anion exchange chromatography (e.g., monolith anion exchange chromatography with a quaternary amine ligand or AEX chromatography), tangential flow filtration, and a second sterile filtration. In some embodiments, methods of isolating rAAV particles produced according to the methods disclosed herein include clarification of the harvested cell culture, a first sterile filtration, a first tangential flow filtration, affinity chromatography, anion exchange chromatography (e.g., monolith anion exchange chromatography with a quaternary amine ligand or AEX chromatography), a second tangential flow filtration, and a second sterile filtration. In some embodiments, methods of isolating rAAV particles disclosed herein include clarification of the harvested cell culture, a first sterile filtration, affinity chromatography, anion exchange chromatography (e.g., monolith anion exchange chromatography with a quaternary amine ligand or AEX chromatography), tangential flow filtration, and a second sterile filtration.In some embodiments, the method of isolating rAAV particles produced according to the methods disclosed herein comprises clarification of the harvested cell culture by depth filtration, a first sterile filtration, a first tangential flow filtration, affinity chromatography, anion exchange chromatography (e.g., monolith anion exchange chromatography with a quaternary amine ligand or AEX chromatography), a second tangential flow filtration, and a second sterile filtration. In some embodiments, the method of isolating rAAV particles disclosed herein comprises clarification of the harvested cell culture by depth filtration, a first sterile filtration, affinity chromatography, anion exchange chromatography (e.g., monolith anion exchange chromatography with a quaternary amine ligand or AEX chromatography), a second tangential flow filtration, and a second sterile filtration. In some embodiments, the method does not include centrifugation. In some embodiments, the clarification of the harvested cell culture comprises sterile filtration. In some embodiments, the rAAV particles comprise capsid proteins of the AAV8 serotype. In some embodiments, the rAAV particles comprise capsid proteins of the AAV9 serotype.

[0188] A number of cell culture-based systems are known in the art for transfection, stable cell line generation, and production of rAAV particles, including infectious hybrid virus production systems (including adenovirus-AAV hybrids, herpesvirus-AAV hybrids, and baculovirus-AAV hybrids). Any rAAV production culture for generating rAAV viral particles requires (1) a suitable host cell, including, for example, a human-derived cell line (e.g., HeLa, A549, or HEK293 cells and their derivatives (HEK293T cells, HEK293F cells)), a mammalian cell line (e.g., Vero), or, in the case of a baculovirus production system, an insect-derived cell line (e.g., SF-9); (2) suitable helper virus functions provided by wild-type or mutant adenovirus (e.g., temperature sensitive adenovirus), herpesvirus, baculovirus, or a plasmid construct providing helper functions; (3) AAV rep and cap genes and gene products; (4) a transgene (e.g., a therapeutic transgene) flanked by AAV ITR sequences; and (5) suitable media and media components to support rAAV production. Suitable media known in the art can be used for the production of rAAV vectors. Such media include, but are not limited to, Modified Eagle's Medium (MEM), Dulbecco's Modified Eagle's Medium (DMEM), and media produced by Hyclone Laboratories and JRH, including Sf-900 II SFM medium as described in U.S. Pat. No. 6,723,551, which is incorporated herein by reference in its entirety.

[0189] rAAV production cultures can be routinely grown under a variety of conditions (over a wide range of temperatures, for various lengths of time, etc.) appropriate for the particular host cell being utilized. As known in the art, rAAV production cultures include attachment-dependent cultures that can be cultured in suitable attachment-dependent vessels (e.g., roller bottles, hollow fiber filters, microcarriers, and packed-bed or fluidized-bed bioreactors). rAAV vector production cultures can also be grown in suspension-adapted 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 cells, RK ... 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 may be cultured in a variety of ways, including, for example, spinner flasks, stirred tank bioreactors, and disposable systems such as the Wave bag system. In some embodiments, the cells are HEK293 cells. In some embodiments, the cells are HEK293 cells adapted to growth in suspension culture. Numerous suspension cultures for producing rAAV particles are known in the art, including, for example, those disclosed in U.S. Pat. Nos. 6,995,006, 9,783,826, and U.S. Patent Publication No. 20120122155, each of which is incorporated herein by reference in its entirety.

[0190] In some embodiments, the rAAV production culture comprises a high density cell culture. In some embodiments, the culture has a total cell density between about 1×10E+06 cells / ml and about 30×10E+06 cells / ml. In some embodiments, more than about 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 to growth in suspension culture.

[0191] In additional embodiments of the provided methods, the rAAV production culture comprises a suspension culture comprising rAAV particles. Numerous suspension cultures for producing rAAV particles are known in the art, including, for example, those disclosed in U.S. Patent Nos. 6,995,006, 9,783,826, and U.S. Patent Publication No. 20120122155, each of which is incorporated herein by reference in its entirety. In some embodiments, the suspension culture comprises a culture of mammalian cells or insect cells. In some embodiments, the suspension culture comprises a culture 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 comprises a culture of HEK293 cells.

[0192] In some embodiments, a method for the production of rAAV particles includes providing a cell culture containing cells capable of producing rAAV, adding a histone deacetylase (HDAC) inhibitor to the cell culture to a final concentration of between about 0.1 mM and about 20 mM, and maintaining the cell culture under conditions that allow the production 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.

[0193] In some embodiments, the rAAV particles are produced as disclosed in WO2020 / 033842, which is incorporated by reference in its entirety.

[0194] Recombinant AAV particles can be collected from rAAV production culture by harvesting the production culture containing the host cells, or by harvesting the spent medium from the production culture, provided that the cells are cultured under conditions known in the art to cause rAAV particle release from intact host cells into the medium. Recombinant AAV particles can also be collected from rAAV production culture by lysing the host cells of the production 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., detergents and / or proteases).

[0195] At harvest, the rAAV production culture can contain one or more of the following: (1) host cell proteins; (2) host cell DNA; (3) plasmid DNA; (4) helper virus; (5) helper virus proteins; (6) helper virus DNA; and (7) media components (including, for example, serum proteins, amino acids, transferrin, and other low molecular weight proteins). The rAAV production culture can further contain product-associated impurities, such as inactive vector forms, empty viral capsids, aggregated viral particles or capsids, misfolded viral capsids, and disassembled viral particles.

[0196] In some embodiments, the rAAV production culture harvest is clarified to remove host cell debris. In some embodiments, the production culture harvest is clarified by filtration through a series of depth filters. Clarification can also be achieved by a variety of other standard techniques known in the art, such as centrifugation or filtration through any cellulose acetate filter with a pore size of 0.2 mm or greater known in the art. In some embodiments, clarification of the harvested cell culture comprises sterile filtration. In some embodiments, the production culture harvest is clarified by centrifugation. In some embodiments, clarification of the production culture harvest does not comprise centrifugation.

[0197] In some embodiments, the harvested cell culture is clarified using filtration. In some embodiments, the clarification of the harvested cell culture comprises depth filtration. In some embodiments, the clarification of the harvested cell culture further comprises depth filtration and sterile filtration. In some embodiments, the harvested cell culture is clarified using a filter train comprising one or more different filtration media. In some embodiments, the filter train comprises one depth filtration medium. In some embodiments, the filter train comprises one or more depth filtration media. In some embodiments, the filter train comprises two depth filtration media. In some embodiments, the filter train comprises one sterile filtration medium. In some embodiments, the filter train comprises two depth filtration media and one sterile filtration medium. In some embodiments, the depth filter medium is a porous depth filter. In some embodiments, the filter train comprises Clarisolve® 20MS, Millistak+® COHC, and sterile grade filtration media. In some embodiments, the filter train comprises Clarisolve® 20MS, Millistak+® COHC, and Sartopore® 2 XLG 0.2 μm. In some embodiments, the harvested cell culture is pretreated prior to contacting with the depth filter. In some embodiments, the pretreatment comprises adding salt to the harvested cell culture. In some embodiments, the pretreatment comprises adding a chemical flocculant to the harvested cell culture. In some embodiments, the harvested cell culture is not pretreated prior to contacting with the depth filter.

[0198] In some embodiments, the production culture harvest is clarified by filtration, as disclosed in WO2019 / 212921, which is incorporated by reference in its entirety.

[0199] In some embodiments, rAAV production culture harvests are treated with a nuclease (e.g., Benzonase®) or endonuclease (e.g., endonuclease from Serratia marcescens) to digest high molecular weight DNA present in the production culture. Nuclease or endonuclease digestion can be routinely performed under standard conditions known in the art. For example, nuclease digestion is performed at a final concentration of 1-2.5 units / mL of Benzonase® at temperatures ranging from ambient to 37° C. for periods ranging from 30 minutes to several hours.

[0200] Sterile filtration encompasses filtration using a sterile grade filter medium. In some embodiments, the sterile grade filtration medium is a 0.2 or 0.22 μm pore filter. In some embodiments, the sterile grade filtration medium comprises polyethersulfone (PES). In some embodiments, the sterile grade filtration medium comprises polyvinylidene fluoride (PVDF). In some embodiments, the sterile grade filtration medium has a hydrophilic heterogeneous bilayer design. In some embodiments, the sterile grade filtration medium has a hydrophilic heterogeneous bilayer design of a 0.8 μm prefilter and a 0.2 μm final filter membrane. In some embodiments, the sterile grade filtration medium has a hydrophilic heterogeneous bilayer design of a 1.2 μm prefilter and a 0.2 μm final filter membrane. In some embodiments, the sterile grade filtration medium is a 0.2 or 0.22 μm pore filter. In further embodiments, the sterile grade filtration medium is a 0.2 μm pore filter. In some embodiments, the sterile grade filtration medium is a combination of Sartopore® 2 XLG 0.2 μm, Durapore™ PVDF membrane 0.45 μm, or Sartoguard® PES 1.2 μm+0.2 μm nominal pore size. In some embodiments, the sterile grade filtration medium is Sartopore® 2 XLG 0.2 μm.

[0201] In some embodiments, the clarified feed is concentrated via tangential flow filtration ("TFF") before being applied to a chromatography medium, such as an affinity chromatography medium. Large-scale concentration of viruses using TFF ultrafiltration is described in Paul et al., Human Gene Therapy 4:609-615 (1993). The TFF concentration of the clarified feed allows for a technically manageable amount of clarified feed to be chromatographed and also allows for more rational sizing of columns without the need for long recirculation times. In some embodiments, the clarified feed is concentrated between at least 2-fold and at least 10-fold. In some embodiments, the clarified feed is concentrated between at least 10-fold and at least 20-fold. In some embodiments, the clarified feed is concentrated between at least 20-fold and at least 50-fold. In some embodiments, the clarified feed is concentrated about 20-fold. Those skilled in the art will also recognize that TFF may be used to remove small molecule impurities (e.g., cell culture impurities including media components, serum albumin, or other serum proteins) from the clarified feed via diafiltration. In some embodiments, the clarified feed is subjected to diafiltration to remove small molecule impurities. In some embodiments, the diafiltration involves using between about 3 and about 10 diafiltration volumes of buffer. In some embodiments, the diafiltration involves using about 5 diafiltration volumes of buffer. Those skilled in the art will also recognize that TFF may be used at any step of the purification process where buffer exchange is desired prior to performing the next step in the purification process. In some embodiments, the methods for isolating rAAV from a clarified feed disclosed herein involve the use of TFF to exchange buffer.

[0202] Affinity chromatography can be used to isolate rAAV particles from the composition. In some embodiments, affinity chromatography is used to isolate rAAV particles from a clarified feed. In some embodiments, affinity chromatography is used to isolate rAAV particles from a clarified feed that has been subjected to tangential flow filtration. Suitable affinity chromatography media are known in the art and include, but 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 media is POROS™ CaptureSelect™ AAV9 affinity resin. In some embodiments, the affinity chromatography media is POROS™ CaptureSelect™ AAV8 affinity resin. In some embodiments, the affinity chromatography medium is POROS™ CaptureSelect™ AAVX affinity resin.

[0203] Anion exchange chromatography can be used to isolate rAAV particles from the 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 include, but are not limited to, UNOsphere™ Q (Biorad, Hercules, Calif.), and N-charged amino or imino resins, such as POROS™ 50 PI, or any DEAE, TMAE, tertiary or quaternary amine, or PEI-based resins known in the art (U.S. Pat. No. 6,989,264; Brument et al., Mol. Therapy 6(5):678-686 (2002); Gao et al., Hum. Gene Therapy 11:2079-2091 (2000)). In some embodiments, the anion exchange chromatography media comprises a quaternary amine. In some embodiments, the anion exchange media is a monolithic anion exchange chromatography resin. In some embodiments, the monolithic anion exchange chromatography medium comprises a glycidyl methacrylate-ethylene dimethacrylate 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 Disk (Quaternary Amine), CIM® DEAE, and CIM® EDA Disk (Ethylene Diamino). In some embodiments, the monolithic anion exchange chromatography medium is CIMmultus™ QA-1 Advanced Composite Column (Quaternary Amine). In some embodiments, the monolithic anion exchange chromatography medium is CIM® QA Disk (Quaternary Amine). In some embodiments, the anion exchange chromatography medium is CIM QA (BIA Separations, Slovenia).In some embodiments, the anion exchange chromatography medium is BIA CIM® QA-80 (column volume 80 mL). One of skill in the art can appreciate that a wash buffer of suitable ionic strength can be identified such that impurities (including but not limited to impurities that may be introduced by upstream purification steps) are removed while the rAAV remains bound to the resin.

[0204] In some embodiments, anion exchange chromatography is performed according to the methods disclosed in WO2019 / 241535, which is incorporated by reference in its entirety.

[0205] In some embodiments, the method of isolating rAAV particles comprises quantifying vector genome titer, capsid titer, and / or full capsid:empty capsid ratio in a composition comprising the isolated rAAV particles. In some embodiments, the vector genome titer is quantified by quantitative PCR (qPCR) or digital PCR (dPCR) or droplet digital PCR (ddPCR). In some embodiments, the capsid titer is quantified by serotype-specific ELISA. In some embodiments, the full capsid:empty capsid ratio is quantified by analytical ultracentrifugation (AUC) or transmission electron microscopy (TEM).

[0206] In some embodiments, the vector genome titer, capsid titer, and / or the ratio of full capsids:empty capsids are quantified spectrophotometrically, for example, by measuring the absorbance of the composition at 260 nm and by measuring the absorbance of the composition at 280 nm. In some embodiments, the rAAV particles are not denatured prior to measuring the absorbance of the composition. In some embodiments, the rAAV particles are denatured prior to measuring the absorbance of the composition. In some embodiments, the absorbance of the composition at 260 nm and 280 nm is quantified using a spectrophotometer. In some embodiments, the absorbance of the composition at 260 nm and 280 nm is quantified using HPLC. In some embodiments, the absorbance is the peak absorbance. Several methods for measuring the absorbance of a composition at 260 nm and 280 nm are known in the art. Methods for quantifying vector genome titer and capsid titer of compositions containing isolated recombinant rAAV particles are disclosed in WO2019 / 212922, which is incorporated by reference in its entirety.

[0207] In additional embodiments, the present disclosure provides a composition comprising an isolated rAAV particle produced according to the methods disclosed herein. In some embodiments, the composition is a pharmaceutical composition comprising a pharma- ceutically acceptable carrier.

[0208] As used herein, the term "pharmaceutical acceptable" refers to a biologically acceptable formulation, gas, liquid, or solid, or mixture thereof, suitable for one or more routes of administration, in vivo delivery or contact. A "pharmaceutical acceptable" composition is a material that is not biologically or otherwise undesirable, e.g., the material can be administered to a subject without causing substantial undesirable biological effects. Thus, such pharmaceutical compositions can be used, for example, in administering rAAV isolated according to the methods of the present 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, dispersion and suspension media, coatings, isotonicity and absorption enhancers or retarders that are compatible with pharmaceutical administration or in vivo contact or delivery. Aqueous and non-aqueous solvents, solutions, and suspensions can include suspending agents and thickening agents. Such pharmaceutical acceptable carriers include tablets (coated or uncoated), capsules (hard or soft), microbeads, powders, granules, and crystals. Supplementary active compounds (e.g., preservatives, antimicrobial agents, antiviral agents, and antifungal agents) can also be incorporated into the compositions. Pharmaceutical compositions can be formulated to be compatible with a particular route of administration or delivery, as described herein or known to those skilled in the art. Thus, pharmaceutical compositions include carriers, diluents, or excipients suitable for administration by various routes.Pharmaceutical compositions and delivery systems suitable for the rAAV particles and methods and uses of the invention are known in the art (e.g., Remington: The Science and Practice of Pharmacy (2003) 20th ed., Mack Publishing Co., Easton, Pa.; Remington's Pharmaceutical Sciences (1990) 18th ed., Mack Publishing Co., Easton, Pa.; The Merck Index (1996) 12th ed., Merck Publishing Group, Whitehouse, NJ; Pharmaceutical Principles of Solid Dosage Forms (1993), Technonic Publishing Co., Inc., Lancaster, Pa.; Ansel and Stoklosa, Pharmaceutical Calculations (2001) 11th ed., Lippincott Williams & Wilkins, Baltimore, Md.; and Poznansky et al., Drug Delivery Systems (1980), R.L. Juliano, ed., Oxford, NY, pp. 253-315).

[0209] In some embodiments, the composition is a pharmaceutical unit dose. "Unit dose" refers to a physically discrete unit suitable as a unitary dosage for a subject to be treated. Each unit contains a predetermined amount, optionally with a pharmaceutical carrier (excipient, diluent, vehicle, or filler), 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, which may contain liquid compositions, or compositions in a freeze-dried or lyophilized state, for example, a sterile liquid carrier may be added prior to administration or delivery in vivo. Individual unit dosage forms may be included in a multi-dose kit or container. Recombinant vector (e.g., AAV) sequences, plasmids, vector genomes, and recombinant viral particles, as well as pharmaceutical compositions thereof, may be packaged in single or multiple unit dosage forms for ease of administration and uniformity of dosage. In some embodiments, the composition comprises AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, 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, AAV. The rAAV particles include AAV capsid proteins from an AAV capsid serotype 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.

[0210] Methods for Producing Recombinant Polypeptides In one aspect, the disclosure provides a method of producing a recombinant polypeptide, the method comprising: (a) providing a cell culture comprising cells suitable for production of the recombinant polypeptide, the culture comprising between about 0.1 mg / L and about 10 mg / L of dextran sulfate; (b) transfecting the cells by adding to the culture of a) a composition comprising one or more polynucleotides encoding the polypeptide and a transfection reagent; and (c) maintaining the cell culture comprising the transfected cells under conditions that allow for production of the recombinant polypeptide.

[0211] In some embodiments, the culture of a) comprises between about 0.5 mg / L and about 10 mg / L, between about 0.5 mg / L and about 5 mg / L, between about 0.5 mg / L and about 3 mg / L, between about 1 mg / L and about 10 mg / L, between about 1 mg / L and about 5 mg / L, between about 1 mg / L and about 4 mg / L, or between about 1 mg / L and about 3 mg / L dextran sulfate. In some embodiments, the culture of a) comprises between about 0.5 mg / L and about 5 mg / L dextran sulfate. In some embodiments, the culture of a) comprises between about 1 mg / L and about 5 mg / L dextran sulfate. In some embodiments, the culture of a) comprises between about 1 mg / L and about 3 mg / L dextran sulfate.

[0212] In some embodiments, the culture of a) comprises about 0.5 mg / L, about 1 mg / L, about 1.5 mg / L, about 2 mg / L, about 2.5 mg / L, about 3 mg / L, about 4 mg / L, or about 5 mg / L dextran sulfate. In some embodiments, the culture of a) comprises about 1 mg / L dextran sulfate. In some embodiments, the culture of a) comprises about 1.5 mg / L dextran sulfate. In some embodiments, the culture of a) comprises about 2 mg / L dextran sulfate. In some embodiments, the culture of a) comprises about 2.5 mg / L dextran sulfate. In some embodiments, the culture of a) comprises about 3 mg / L dextran sulfate. In some embodiments, the culture of a) comprises about 3.5 mg / L dextran sulfate. In some embodiments, the culture of a) comprises about 4 mg / L dextran sulfate.

[0213] In some embodiments, the culture of a) contains about 2 mg / L dextran sulfate.

[0214] In some embodiments, the disclosure provides a method of producing a recombinant polypeptide comprising: (a) culturing in a cell culture cells suitable for production of the recombinant polypeptide, the culture comprising an initial dextran sulfate concentration of between about 1 mg / L and about 20 mg / L and a final dextran sulfate concentration of between about 0.1 mg / L and about 10 mg / L; (b) transfecting the cells by adding to the culture of a) a composition comprising one or more polynucleotides encoding the polypeptide and a transfection reagent; and (c) maintaining the cell culture comprising the transfected cells under conditions that allow for production of the recombinant polypeptide.

[0215] In some embodiments, the starting dextran sulfate concentration is between about 1 mg / L and about 10 mg / L, between about 1 mg / L and about 5 mg / L, between about 2 mg / L and about 10 mg / L, between about 3 mg / L and about 10 mg / L, or between about 3 mg / L and about 5 mg / L of dextran sulfate. In some embodiments, the starting dextran sulfate concentration is between about 1 mg / L and about 10 mg / L of dextran sulfate. In some embodiments, the starting dextran sulfate concentration is between about 2 mg / L and about 10 mg / L of dextran sulfate. In some embodiments, the starting dextran sulfate concentration is between about 3 mg / L and about 6 mg / L of dextran sulfate.

[0216] In some embodiments, the starting dextran sulfate concentration is about 2 mg / L, about 3 mg / L, about 4 mg / L, about 5 mg / L, about 6 mg / L, about 7 mg / L, about 8 mg / L, about 9 mg / L, or about 10 mg / L dextran sulfate. In some embodiments, the starting dextran sulfate concentration is about 2 mg / L dextran sulfate. In some embodiments, the starting dextran sulfate concentration is about 3 mg / L dextran sulfate. In some embodiments, the starting dextran sulfate concentration is about 4 mg / L dextran sulfate. In some embodiments, the starting dextran sulfate concentration is about 5 mg / L dextran sulfate. In some embodiments, the starting dextran sulfate concentration is about 6 mg / L dextran sulfate. In some embodiments, the starting dextran sulfate concentration is about 7 mg / L dextran sulfate. In some embodiments, the starting dextran sulfate concentration is about 8 mg / L dextran sulfate.

[0217] In some embodiments, the starting dextran sulfate concentration is about 4 mg / L dextran sulfate.

[0218] In some embodiments, the final dextran sulfate concentration is between about 0.5 mg / L and about 10 mg / L, between about 0.5 mg / L and about 5 mg / L, between about 0.5 mg / L and about 3 mg / L, between about 1 mg / L and about 10 mg / L, between about 1 mg / L and about 5 mg / L, between about 1 mg / L and about 4 mg / L, or between about 1 mg / L and about 3 mg / L dextran sulfate. In some embodiments, the final dextran sulfate concentration is between about 0.5 mg / L and about 5 mg / L dextran sulfate. In some embodiments, the final dextran sulfate concentration is between about 1 mg / L and about 5 mg / L dextran sulfate. In some embodiments, the final dextran sulfate concentration is between about 1 mg / L and about 3 mg / L dextran sulfate.

[0219] In some embodiments, the final dextran sulfate concentration is about 0.5 mg / L, about 1 mg / L, about 1.5 mg / L, about 2 mg / L, about 2.5 mg / L, about 3 mg / L, about 4 mg / L, or about 5 mg / L dextran sulfate. In some embodiments, the final dextran sulfate concentration is about 1 mg / L dextran sulfate. In some embodiments, the final dextran sulfate concentration is about 1.5 mg / L dextran sulfate. In some embodiments, the final dextran sulfate concentration is about 2 mg / L dextran sulfate. In some embodiments, the final dextran sulfate concentration is about 2.5 mg / L dextran sulfate. In some embodiments, the final dextran sulfate concentration is about 3 mg / L dextran sulfate. In some embodiments, the final dextran sulfate concentration is about 3.5 mg / L dextran sulfate. In some embodiments, the final dextran sulfate concentration is about 4 mg / L dextran sulfate.

[0220] In some embodiments, the final dextran sulfate concentration is about 2 mg / L dextran sulfate.

[0221] In some embodiments, the starting dextran sulfate concentration is between about 1 mg / L and about 10 mg / L, between about 1 mg / L and about 5 mg / L, between about 2 mg / L and about 10 mg / L, between about 3 mg / L and about 10 mg / L, or between about 3 mg / L and about 5 mg / L of dextran sulfate and the final dextran sulfate concentration is between about 0.5 mg / L and about 10 mg / L, between about 0.5 mg / L and about 5 mg / L, between about 0.5 mg / L and about 3 mg / L, between about 1 mg / L and about 10 mg / L, between about 1 mg / L and about 5 mg / L, between about 1 mg / L and about 4 mg / L, or between about 1 mg / L and about 3 mg / L of dextran sulfate. In some embodiments, the starting dextran sulfate concentration is between about 3 mg / L and about 6 mg / L dextran sulfate and the final dextran sulfate concentration is between about 1 mg / L and about 3 mg / L dextran sulfate.

[0222] In some embodiments, the starting dextran sulfate concentration is about 2 mg / L, about 3 mg / L, about 4 mg / L, about 5 mg / L, about 6 mg / L, about 7 mg / L, about 8 mg / L, about 9 mg / L, or about 10 mg / L of dextran sulfate and the final dextran sulfate concentration is about 0.5 mg / L, about 1 mg / L, about 1.5 mg / L, about 2 mg / L, about 2.5 mg / L, about 3 mg / L, about 4 mg / L, or about 5 mg / L of dextran sulfate.

[0223] In some embodiments, the starting dextran sulfate concentration is about 4 mg / L of dextran sulfate and the final dextran sulfate concentration is about 2 mg / L of dextran sulfate.

[0224] In some embodiments, the one or more polynucleotides comprise a transgene. In some embodiments, the transgene comprises a regulatory element operably linked to a polynucleotide encoding a polypeptide.

[0225] In some embodiments, the polypeptide comprises an antibody or antigen-binding fragment thereof, a bispecific antibody, an enzyme, a fusion protein, or an Fc-fusion protein. In some embodiments, the polypeptide comprises an antibody or an antigen-binding fragment thereof. In some embodiments, the polypeptide comprises a fusion protein (e.g., an Fc-fusion protein). In some embodiments, the polypeptide comprises an enzyme.

[0226] The term "antibody" as used herein encompasses whole antibodies and antibody fragments (including any functional domain of an antibody, e.g., an antigen-binding fragment or single chain thereof, an effector domain, a salvage receptor binding epitope, or portions thereof). A typical antibody has at least two heavy chains (H chains) and two light chains (L chains) interconnected by disulfide bonds. Each heavy chain is composed of a heavy chain variable region (VH) and a heavy chain constant region. In some embodiments, the heavy chain constant region comprises three domains, CH1, CH2, and CH3. Each light chain is composed of a light chain variable region (VL) and a light chain constant region. In some embodiments, the light chain constant region comprises one domain, C1. The VH and VL regions can be further subdivided into regions of hypervariability called complementarity determining regions (CDRs), which are interspersed with more conserved regions called framework regions (FWs). Each VH and VL is composed of three CDRs and four FWs, arranged from amino-terminus to carboxy-terminus in the following order: FW1, CDR1, FW2, CDR2, FW3, CDR3, FW4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant regions of the antibody may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. Non-limiting types of antibodies of the present disclosure include classic antibodies, scFvs, and combinations thereof.

[0227] The term "antibody fragment" refers to a portion of an intact antibody and any functional domain of an antibody (e.g., an antigen-binding fragment or single chain thereof, an effector domain or portion thereof). Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, Fv fragments, linear antibodies, single chain antibodies, and multispecific antibodies formed from antibody fragments. As used herein, an "antibody fragment" comprises an antigen-binding site or an epitope-binding site.

[0228] As used herein, the term "Fc region" or simply "Fc" is understood to mean the carboxyl-terminal portion of an immunoglobulin chain constant region, preferably an immunoglobulin heavy chain constant region, or a portion thereof. For example, an immunoglobulin Fc region can include (1) a CH1 domain, a CH2 domain, and a CH3 domain, (2) a CH1 domain and a CH2 domain, (3) a CH1 domain and a CH3 domain, (4) a CH2 domain and a CH3 domain, or (5) a combination of two or more domains and an immunoglobulin hinge region. In some embodiments, the Fc region includes at least an immunoglobulin hinge region, a CH2 domain, and a CH3 domain, and preferably lacks a CH1 domain. In some embodiments, the class of immunoglobulin from which the heavy chain constant region is derived is IgG (Igγ) (γ subclasses 1, 2, 3, or 4). Other classes of immunoglobulins, IgA (Igα), IgD (Igδ), IgE (Igε), and IgM (Igμ), can also be used. It is considered within the level of ordinary skill in the art to select a particular immunoglobulin heavy chain constant region sequence from a particular immunoglobulin class and subclass to achieve a particular result. In some embodiments, the portion of the DNA construct encoding the immunoglobulin Fc region preferably includes at least a portion of the hinge domain, preferably at least a portion of the CH3 domain of Fc gamma or a homologous domain of either IgA, IgD, IgE, or IgM. It is further contemplated that substitutions or deletions of amino acids within the immunoglobulin heavy chain constant region are useful in the practice of the methods and compositions disclosed herein. As an example, amino acid substitutions may be introduced in the upper CH2 region to create an Fc variant with reduced affinity for the Fc receptor (Cole, J. Immunol. 159:3613 (1997)).

[0229] A variety of recombinant expression systems suitable for producing recombinant polypeptides in a particular host cell are known to those skilled in the art. It is understood that any recombinant expression system can be used to produce a recombinant polypeptide according to the methods disclosed herein.

[0230] Any suitable transfection reagent for transfecting cells known in the art can be used for producing recombinant polypeptides according to the methods disclosed herein. In some embodiments, the transfection reagent comprises a cationic organic carrier. See, e.g., Gigante et al., Medchemcomm 10(10):1692-1718(2019); Damen et al. Medchemcomm 9(9):1404-1425(2018), each of which is incorporated herein by reference in its entirety. In some embodiments, the cationic organic carrier comprises a lipid, e.g., DOTMA, DOTAP, helper lipid (Dope, cholesterol), and combinations thereof. In some embodiments, the cationic organic carrier comprises a multivalent cationic lipid, e.g., DOSPA, DOGS, and mixtures thereof. In some embodiments, the cationic organic carrier comprises a bipolar lipid, or a bolaamphiphile (Bola). In some embodiments, the cationic organic carrier comprises a biologically reducible and / or dimerizable lipid. In some embodiments, the cationic organic carrier comprises a gemini surfactant. In some embodiments, the cationic organic carrier comprises Lipofectin™, Transfectam™, Lipofectamine™, Lipofectamine 2000™, or Lipofectamin PLUS 2000™. In some embodiments, the cationic organic carrier comprises a polymer, such as poly(L-lysine) (PLL), polyethyleneimine (PEI), polysaccharides (chitosan, dextran, cyclodextrin (CD)), poly[2-(dimethylamino)ethyl methacrylate] (PDMAEMA), and dendrimers (polyamidoamine (PAMAM), poly(propyleneimine) (PPI)). In some embodiments, the cationic organic carrier comprises a peptide, such as a peptide rich in basic amino acids (CWL18), a cell penetrating peptide (CPP) (Arg-rich peptide (octaarginine, TAT)), a nuclear localization signal (NLS) (SV40), and a targeting signal (RGD).In some embodiments, the cationic organic carrier comprises a polymer (e.g., PEI) in combination with a cationic liposome. Paris et al., Molecules 25(14):3277(2020) (hereby incorporated by reference in its entirety). In some embodiments, the transfection reagent comprises calcium phosphate, highly branched organic compounds (dendrimers), cationic polymers (e.g., DEAE dextran or polyethyleneimine (PEI)), lipofection.

[0231] In some embodiments, the transfection reagent comprises poly(L-lysine) (PLL), polyethyleneimine (PEI), linear PEI, branched PEI, dextran, cyclodextrin (CD), poly[2-(dimethylamino)ethyl methacrylate] (PDMAEMA), polyamidoamine (PAMAM), poly(propyleneimine) (PPI), or mixtures thereof. In some embodiments, the transfection reagent comprises polyethyleneimine (PEI), linear PEI, branched PEI, or mixtures thereof. In some embodiments, the transfection reagent comprises polyethyleneimine (PEI). In some embodiments, the transfection reagent comprises linear PEI. In some embodiments, the transfection reagent comprises branched PEI. In some embodiments, the transfection reagent comprises polyethyleneimine (PEI) having a molecular weight between about 5 and about 25 kDa. In some embodiments, the transfection reagent comprises PEGylated polyethyleneimine (PEI). In some embodiments, the transfection reagent comprises a modified polyethyleneimine (PEI) with hydrophobic moieties attached (eg, cholesterol, choline, alkyl groups, and some amino acids).

[0232] Any cell culture system known in the art can be used for the production of recombinant polypeptides according to the methods disclosed herein. In some embodiments, the cell culture is a suspension cell culture. In some embodiments, the cell culture is an adherent cell culture. In some embodiments, the cell culture comprises adherent cells grown attached to microcarriers or macrocarriers in a stirred bioreactor. In some embodiments, the cell culture is a perfusion culture. In some embodiments, the cell culture is an alternating tangential flow (ATF) supported high density perfusion culture.

[0233] In some embodiments, the cell comprises a mammalian cell or an insect cell. In some embodiments, the cell comprises a mammalian cell. In some embodiments, the cell comprises 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, and / or a PerC6 cell. In some embodiments, the cell comprises a HEK293 cell.

[0234] In some embodiments, the cells include suspension-adapted cells. In some embodiments, the cells include suspension-adapted 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 cells comprise suspension-adapted HEK293 cells, HEK293-derived cells (e.g., HEK293T cells, HEK293F cells), CHO cells, CHO-K1 cells, or CHO-derived cells. In some embodiments, the cells comprise suspension-adapted HEK293 cells. In some embodiments, the cells comprise suspension-adapted CHO cells.

[0235] In some embodiments, the cell culture has a volume between about 50 liters and about 20,000 liters. In some embodiments, the cell culture has a volume between about 50 liters and about 5,000 liters. In some embodiments, the cell culture has a volume between about 50 liters and about 2,000 liters. In some embodiments, the cell culture has a volume between about 50 liters and about 1,000 liters. In some embodiments, the cell culture has a volume between about 50 liters and about 500 liters.

[0236] Without being bound to any particular theory, the methods disclosed herein increase the efficiency of transfection, such that cells transfected according to the methods disclosed herein produce more recombinant polypeptide than control cells transfected in cell cultures that do not contain dextran sulfate. In some embodiments, the methods disclosed herein produce at least about 10%, at least about 20%, at least about 30%, at least about 40%, or at least about 50% more recombinant polypeptide than control methods using cell cultures that do not contain dextran sulfate. Methods for measuring recombinant polypeptide production are well known in the art. In some embodiments, recombinant polypeptide production is measured using Western blotting, ELIS assays, or functional assays (e.g., assays for measuring catalytic activity of recombinantly expressed polypeptides).

[0237] In some embodiments, the method of producing a recombinant polypeptide disclosed herein further comprises isolating the polypeptide. Various methods for isolating a recombinantly expressed polypeptide are known to those skilled in the art. It is understood that any known method for isolating a recombinantly expressed polypeptide can be used according to the methods disclosed herein. In some embodiments, the method of isolating a recombinantly expressed polypeptide comprises harvesting the cell culture, clarifying the harvested cell culture (e.g., by centrifugation or depth filtration), tangential flow filtration, affinity chromatography, anion exchange chromatography, cation exchange chromatography, size exclusion chromatography, hydrophobic interaction chromatography, hydroxylapatite chromatography, sterile filtration, or any combination(s) thereof. In some embodiments, the downstream processing comprises at least two, at least three, at least four, at least five, or at least six of harvesting the cell culture, clarifying the harvested cell culture (e.g., by centrifugation or depth filtration), tangential flow filtration, affinity chromatography, anion exchange chromatography, cation exchange chromatography, size exclusion chromatography, hydrophobic interaction chromatography, hydroxylapatite chromatography, and sterile filtration. EXAMPLES

[0238] Example 1: Dextran sulfate surprisingly increases AAV production in a transient transfection-based system. The inventors have surprisingly found that dextran sulfate can increase AAV titer in transient transfection-based production methods. Alternating tangential flow (ATF)-supported high-density perfusion culture technology was tested to generate seed cells for large-scale transient transfection-based AAV production cultures. When suspension-adapted HEK cells from a high-density perfusion reactor were used to seed the production culture, recombinant AAV production was reduced 5-fold. A possible cause of the reduced titer is increased aggregation of seed cells resulting from high-density perfusion culture, which may have led to variability in seeding density and growth rate, as well as inaccurate transfection reagent concentrations. Although cell culture additives such as dextran sulfate were known to reduce aggregation, these agents are known to interfere with transient transfection, and therefore the use of these agents has not been considered a viable option in the production of cells for transient transfection. For example, Geng et al. (2007) conclude on page 55 that dextran sulfate completely inhibits PEI-mediated transfection. Similarly, a recently published "Guide for DNA Transfection in iCELLis® 500 and iCELLis 500+ Bioreactors for Large Scale Gene Therapy Vector Manufacturing" by PALL® Biotech teaches on page 9 that dextran sulfate inhibits PEI-mediated transfection.

[0239] Despite teachings that dextran sulfate inhibits transfection, we tested the effect of dextran sulfate on AAV titers in a transient transfection-based AAV production system. Recombinant AAV was produced by transient transfection of HEK293 cells. Briefly, HEK293 cells were grown in medium containing 0.3-10 mg / L dextran sulfate for 48 hours in 250 ml shake flasks. Cells were transfected with a mixture of polyethylenimine (PEI) and three plasmids encoding adenoviral helper functions, a transgene, and AAV Cap / Rep. Transfected cultures were maintained for 5 days post-transfection to allow AAV production. A PCR-based method was used to quantitate AAV titers in culture supernatants. Titers obtained with recombinant AAV8 containing transgene 1 and transgene 2 are shown in Figures 1 and 2, respectively. Surprisingly, the presence of dextran sulfate at concentrations between 0.652 mg / L and 2.5 mg / L (Figure 1) and between 1.7 mg / L and 3.6 mg / L resulted in increased AAV titers. This finding is unexpected in light of the clear teaching of the prior art that dextran sulfate inhibits transient transfection, but is consistent with the finding that dextran sulfate at 10 mg / L or higher (Figure 1) inhibited AAV production. Dextran sulfate had no significant effect on AAV titers when used at 0.313 mg / L (Figure 1).

[0240] Example 2: Effect of dextran sulfate on AAV titer in a bench-scale 2 L reactor. The effect of dextran sulfate on transfection-based AAV production was investigated in bench-scale reactors. Recombinant AAV8 containing transgene 2 was produced by transient transfection of HEK293 cells. Briefly, HEK293 cells were grown in medium containing various concentrations of dextran sulfate for 3 days in a 2L reactor. Cells were transfected with a mixture of polyethylenimine (PEI) and three plasmids encoding adenovirus helper functions, the transgene, and AAV Cap / Rep. Transfected cultures were maintained for 4 days after transfection to allow AAV production. AAV particles were harvested from culture supernatants or from cultures after cell lysis. Figure 3. Cell density and cell viability were quantified daily. Figures 5 and 6. Cell morphology was assessed on day 4 (Figure 4). A dextran sulfate concentration range of 2.5-4.2 mg / L did not inhibit transfection in a 2 L reactor and was beneficial to cell morphology, including increased viability and viable cell density.

[0241] Example 3: Effect of dextran sulfate on AAV titer in a bench-scale 5 L reactor. The effect of dextran sulfate on transfection-based AAV production was investigated in bench-scale reactors. Recombinant AAV8 containing transgene 2 was produced by transient transfection of HEK293 cells. Briefly, HEK293 cells were grown in medium containing 4 mg / L dextran sulfate for 3 days in a 5 L reactor. Prior to transfection, cultures were diluted 1:1 with fresh medium to obtain a dextran sulfate concentration of 2 mg / L. Cells were transfected with a mixture of polyethylenimine (PEI) and three plasmids encoding adenoviral helper functions, the transgene, and AAV Cap / Rep. Transfected cultures were maintained for 4 days post-transfection to allow AAV production. AAV particles were harvested from culture supernatants or from cultures following cell lysis. AAV supernatant or lysis titers were increased by an average of 35-40%, respectively, by the addition of dextran sulfate. Figure 7.

[0242] Example 4: Effect of dextran sulfate on AAV titers in different culture media. The effect of dextran sulfate on transfection-based AAV production was investigated in different commercially available culture media (M1, M2, and M3 in FIG. 8). Recombinant AAV8 containing transgene 2 was produced by transient transfection of HEK293 cells. Briefly, HEK293 cells were grown in different culture media containing 4 mg / L dextran sulfate for 3 days in a 2L reactor. Prior to transfection, the culture was diluted 1:1 with fresh medium to obtain a dextran sulfate concentration of 2 mg / L. The cells were transfected with a mixture of polyethylenimine (PEI) and three plasmids encoding adenovirus helper functions, the transgene, and AAV Cap / Rep. The transfected cultures were maintained for 4 days after transfection to allow AAV production. AAV particles were harvested from the culture supernatant or from the culture after cell lysis. FIG. 8. Inclusion of dextran sulfate in the culture increased the titers recovered from cell lysis by 25%, 130%, and 10% in M1, M2, and M3 media, respectively.

[0243] Example 5: Effect of dextran sulfate on AAV titers using different host cell clones. The effect of dextran sulfate on transfection-based AAV production was investigated using different HEK293 host cell clones. Recombinant AAV8 containing transgene 2 was generated by transient transfection of different HEK293 cell clones. Briefly, HEK293 cell clones were expanded in culture medium containing 4 mg / L dextran sulfate for 3 days in shake flasks. Prior to transfection, cultures were diluted 1:1 with fresh medium to obtain a dextran sulfate concentration of 2 mg / L. Cells were transfected with a mixture of polyethylenimine (PEI) and three plasmids encoding adenoviral helper functions, the transgene, and AAV Cap / Rep. Transfected cultures were maintained for 4 days after transfection to allow AAV production. AAV particles were harvested from the culture supernatant. Figure 9. AAV8 titers were increased by inclusion of dextran sulfate in all five HEK cell clones examined. AAV8 titers were increased by an average of 18% by inclusion of dextran sulfate in five different HEK cell clones.

[0244] Example 6: Effect of dextran sulfate on AAV9 titers in a bench-scale 5 L reactor. The effect of dextran sulfate on transfection-based AAV9 production was investigated in bench-scale reactors. Recombinant AAV9 containing transgene 3 was produced by transient transfection of HEK293 cells. Briefly, HEK293 cells were grown in medium containing dextran sulfate at a concentration of 4 mg / L for 3 days in a 5 L reactor. Prior to transfection, the culture was diluted 1:1 with fresh medium to obtain a dextran sulfate concentration of 2 mg / L. Cells were transfected with a mixture of polyethylenimine (PEI) and three plasmids encoding adenovirus helper functions, the transgene, and AAV Cap / Rep. Transfected cultures were maintained for 5 days post-transfection to allow AAV production. Figure 10. AAV9 supernatant titers increased by an average of 30% with the inclusion of dextran sulfate.

[0245] Example 7: Effect of dextran sulfate on AAV titers when used both in the seed cell train prior to transfection and in the production culture (transgene 3). Recombinant AAV9 containing transgene 3 was produced by transient transfection of HEK293 cells in 200L production cultures. HEK cells were expanded using a seed train including a high density perfusion culture step in the presence of 4mg / L dextran sulfate. 200L production cultures were inoculated with HEK seed cells and the dextran sulfate concentration was adjusted to 2mg / L in the production culture prior to transfection. Cells were transfected with a mixture of polyethylenimine (PEI) and three plasmids encoding adenovirus helper functions, the transgene, and AAV Cap / Rep. Transfected cultures were maintained for 5 days after transfection to allow AAV production. AAV particles were harvested either from the culture supernatant (black bars in FIG. 11) or from the culture after cell lysis (gray bars in FIG. 11). Control production cultures were inoculated with HEK seed cells expanded in the absence of dextran sulfate. FIG. 11. AAV9 titers increased by 30% when dextran sulfate was used both during seed cell expansion and during transfection of production cultures.

[0246] Example 8: Effect of dextran sulfate on AAV titer when used both in the seed train prior to transfection and in the production culture (transgene 1). The effect of dextran sulfate in the seed train on transfection-based AAV production in bench-scale reactors was investigated. Recombinant AAV8 containing transgene 1 was generated by transient transfection of HEK293 cells. Briefly, HEK293 cells were expanded for 5 passages (18 days) in medium with or without dextran sulfate. Cells were then expanded in medium with or without 4 mg / L dextran sulfate (0) (seed train) for 3 days in triplicate 2L reactors. Prior to transfection, cultures were diluted 1:1 with fresh medium to obtain dextran sulfate concentrations of 2 mg / L or 0, respectively. Cells were transfected with a mixture of polyethylenimine (PEI) and three plasmids encoding adenovirus helper functions, the transgene, and AAV Cap / Rep. Transfected cultures were maintained for 4 days post-transfection to allow AAV production. AAV particles were recovered from the cultures after cell lysis. AAV lytic titers were increased by an average of 10-15% by including dextran sulfate in the seed train and production cultures (statistically significant p<0.05). Figure 12.

[0247] While the methods of this disclosure have been described in conjunction with what are presently considered to be the most practical and preferred embodiments, it is to be understood that the methods encompassed by this disclosure are not to be limited to the disclosed embodiments, but on the contrary, are intended to cover various modifications and equivalent arrangements included within the spirit and scope of the appended claims.

[0248] All publications, patents, patent applications, internet sites, and accession number / database sequences (including both polynucleotide and polypeptide sequences) cited in this specification are hereby incorporated by reference in their entirety for all purposes to the same extent as if each individual publication, patent, patent application, internet site, or accession number / database sequence was specifically and individually incorporated by reference.

Claims

1. 1. A method of transfecting a cell, comprising: a) providing a suspension cell culture comprising the cells, the culture comprising between about 1 mg / L and about 3 mg / L of dextran sulfate; b) transfecting said cells by adding to said culture of (a) a composition comprising one or more polynucleotides and a transfection reagent; Including, the transfection reagent comprises a polymer; The method.

2. 1. A method for producing a recombinant polypeptide comprising the steps of: a) providing a suspension cell culture comprising cells suitable for producing the recombinant polypeptide, said culture comprising between about 1 mg / L and about 3 mg / L of dextran sulfate; b) transfecting the cells by adding to the culture of a) a composition comprising one or more polynucleotides encoding the polypeptide and a transfection reagent; c) maintaining said cell culture containing said transfected cells under conditions that allow the production of said recombinant polypeptide; Including, the transfection reagent comprises a polymer; The method.

3. The method of claim 2, wherein the polypeptide is an antibody or antigen-binding fragment thereof, a bispecific antibody, an enzyme, a fusion protein, or an Fc-fusion protein.

4. 1. A method for producing recombinant viral particles, comprising: a) providing a suspension cell culture comprising cells suitable for producing the recombinant viral particles, the culture comprising between about 1 mg / L and about 3 mg / L dextran sulfate; b) transfecting said cells by adding to said culture of a) a composition comprising one or more polynucleotides comprising genes necessary for generating said recombinant viral particles and a transfection reagent; c) maintaining the cell culture containing the transfected cells under conditions that allow the production of the recombinant viral particles; Including, the transfection reagent comprises a polymer; The method.

5. The method according to any one of claims 1 to 4, wherein the culture of a) contains about 2 mg / L of dextran sulfate.

6. The method of claim 4 or 5, wherein the recombinant viral particle is a rAAV particle.

7. The rAAV particles may be AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, 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.

7. The method of claim 6, wherein the capsid protein of the 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.

8. the one or more polynucleotides a) the rAAV genome to be packaged; b) adenovirus helper functions necessary for packaging; c) AAV rep proteins sufficient for packaging, and d) AAV cap protein sufficient for packaging The method of claim 6 or 7, wherein the compound is encoded by

9. 9. The method of claim 8, wherein the one or more polynucleotides comprise a polynucleotide encoding the rAAV genome, a polynucleotide encoding the AAV rep proteins and the AAV cap proteins, and a polynucleotide encoding the adenovirus helper functions.

10. 10. The method of claim 8 or claim 9, wherein the adenovirus helper functions include at least one of the adenovirus E1a gene, E1b gene, E4 gene, E2a gene, and VA gene.

11. The method of any one of claims 6 to 10, further comprising recovering the rAAV particles.

12. 12. The method of any one of claims 6-11, wherein the cell culture produces at least about 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, or 2-fold more rAAV particles, measured as GC / ml, than a reference method in which the culture of a) does not contain dextran sulfate.

13. The method of any one of claims 1 to 12, wherein the cell culture comprises suspension-adapted cells.

14. 13. The method of any one of claims 1 to 12, wherein the cells comprise HEK293 cells, HEK-derived cells, CHO cells, CHO-derived cells, HeLa cells, SF-9 cells, BHK cells, Vero cells, and / or PerC6 cells, or combinations thereof.

15. The method of any one of claims 1 to 12, wherein the cells comprise HEK293 cells.

16. The method of any one of claims 1 to 12, wherein the cell comprises a CHO cell or a CHO-K1 cell.

17. 17. The method of any one of claims 1 to 16, wherein the transfection reagent comprises a polymer, the polymer comprising poly(L-lysine) (PLL), polyethyleneimine (PEI), a polysaccharide, poly[2-(dimethylamino)ethyl methacrylate] (PDMAEMA), a dendrimer, or a combination thereof.

18. The method of any one of claims 1 to 16, wherein the transfection reagent comprises polyethyleneimine (PEI).

19. The method of any one of claims 1 to 18, wherein the cell culture has a volume of between about 50 liters and about 20,000 liters.

20. 20. The method of claim 19, wherein the cell culture has a volume of between about 50 liters and about 5,000 liters.

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