Improvement of recombinant polypeptide and virus production

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 a method comprising transfecting a host cell in a culture medium containing dextran sulfate.

[0002] Cross - Reference to Related Applications This application claims the benefit of U.S. Patent Application No. 63 / 139,992, filed on January 21, 2021, which is hereby incorporated by reference in its entirety.

Background Art

[0003] Background Recombinant adeno - associated virus (AAV) - based vectors are currently the most widely used developing gene therapy products. The reasons for the preference for using the rAAV vector system are, in part, the absence of diseases associated with wild - type viruses, the ability of AAV transduction in both non - dividing and dividing cells, and the observation of long - term robust transgene expression in clinical trials, which indicates great potential for delivery in gene therapy applications. In addition, various natural and recombinant rAAV vector serotypes specifically target various tissues, organs, and cells and help avoid any existing immunity to the vector, thus expanding the therapeutic applications of AAV - based gene therapy. Before recombinant virus particles can be more widely adopted in late - stage clinical and commercial use, there is a need to develop new methods for the large - scale production of recombinant virus particles.

[0004] Therefore, in the art, there is a need to improve the productivity and yield of methods for generating rAAV particles on a large scale.

Summary of the Invention

[0005] Summary In one embodiment, the present disclosure provides a method for translocating cells, comprising: (a) preparing a cell culture containing cells, wherein the culture contains dextran sulfate in a concentration between about 0.1 mg / L and about 10 mg / L; and (b) translocating cells by adding a composition comprising one or more polynucleotides and a translocating reagent to the culture. In some embodiments, the cell culture is a suspension culture. In some embodiments, the cell culture is a suspension culture containing suspension-adaptive HEK cells. In some embodiments, the translocating reagent comprises polyethyleneimine (PEI).

[0006] In a further embodiment, the Disclosure provides a method for producing recombinant polypeptides, comprising: (a) preparing a cell culture containing cells suitable for the production of recombinant polypeptides, wherein the culture contains dextran sulfate in a concentration between about 0.1 mg / L and about 10 mg / L; (b) translocating cells to the culture from (a) by adding a composition containing one or more polynucleotides encoding a polypeptide and a translocation reagent; and (c) maintaining the cell culture containing the translocated cells under conditions that enable the production of recombinant polypeptides. In some embodiments, the recombinant polypeptide is an antibody or its antigen-binding fragment, a bispecific antibody, an enzyme, a fusion protein, or an Fc fusion protein.

[0007] In a further embodiment, the Disclosure provides a method for generating recombinant viral particles, comprising: (a) preparing a cell culture containing cells suitable for the generation of recombinant viral particles, wherein the culture contains dextran sulfate in a concentration between about 0.1 mg / L and about 10 mg / L; (b) translocating the cells by adding a composition to the culture from (a) that contains one or more polynucleotides containing genes necessary for the generation of recombinant viral particles and a translocation reagent; and (c) maintaining the cell culture containing the translocated cells under conditions that enable the generation of recombinant viral particles. In some embodiments, the recombinant virus is recombinant adeno-associated virus (rAAV). In some embodiments, rAAV is 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. It contains capsid proteins of PHP.B, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, or AAV.HSC16 serotypes. In some embodiments, rAAV contains capsid proteins of AAV8 or AAV9 serotype. In some embodiments, the cell culture is a suspension culture containing suspension-adaptive HEK cells. In some embodiments, the translocation reagent contains polyethyleneimine (PEI).

[0008] In one embodiment, the present disclosure provides a method for transposing cells, comprising: (a) culturing cells in a cell culture, the culture containing an initial dextran sulfate concentration between about 1 mg / L and about 20 mg / L and a final dextran sulfate concentration between about 0.1 mg / L and about 10 mg / L; and (b) transposing cells by adding a composition containing one or more polynucleotides and a transposition reagent to the culture from (a). In some embodiments, the cell culture is a suspension culture. In some embodiments, the cell culture is a suspension culture containing suspension-adaptive HEK cells. In some embodiments, the transposition reagent contains polyethyleneimine (PEI).

[0009] In a further embodiment, the Disclosure provides a method for producing recombinant polypeptides, comprising: (a) culturing cells suitable for the production of recombinant polypeptides in a cell culture, wherein the culture contains an initial dextran sulfate concentration between about 1 mg / L and about 20 mg / L and a final dextran sulfate concentration between about 0.1 mg / L and about 10 mg / L; (b) translocating the cells to the culture from (a) by adding a composition comprising one or more polynucleotides encoding a polypeptide and a translocation reagent; and (c) maintaining the cell culture containing the translocated cells under conditions that enable the production of recombinant polypeptides. In some embodiments, the recombinant polypeptide is an antibody or its antigen-binding fragment, a bispecific antibody, an enzyme, a fusion protein, or an Fc fusion protein.

[0010] In a further embodiment, the Disclosure provides a method for generating recombinant virus particles, comprising: (a) culturing cells suitable for the generation of recombinant virus particles in a cell culture for about 1 to about 5 days, wherein the culture contains an initial dextran sulfate concentration between about 1 mg / L and about 20 mg / L and a final dextran sulfate concentration between about 0.1 mg / L and about 10 mg / L; (b) translocating the cells to the culture from (a) by adding a composition containing one or more polynucleotides containing genes necessary for the generation of recombinant virus particles and a translocation reagent; and (c) maintaining the cell culture containing the translocated cells under conditions that enable the generation of recombinant virus particles. In some embodiments, the recombinant virus is recombinant adeno-associated virus (rAAV). In some embodiments, rAAV is 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. It contains capsid proteins of PHP.B, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, or AAV.HSC16 serotypes. In some embodiments, rAAV contains capsid proteins of AAV8 or AAV9 serotype. In some embodiments, the cell culture is a suspension culture containing suspension-adaptive HEK cells. In some embodiments, the translocation reagent contains polyethyleneimine (PEI).

[0011] In a further embodiment, the Disclosure provides a method for improving the production of recombinant polypeptides, comprising: (a) preparing a cell culture containing cells suitable for the production of recombinant polypeptides, wherein the culture contains dextran sulfate in a concentration between about 0.1 mg / L and about 10 mg / L; (b) translocating cells to the culture from (a) by adding a composition containing one or more polynucleotides encoding a polypeptide and a translocation reagent; and (c) maintaining the cell culture containing the translocated cells under conditions that enable the production of recombinant polypeptides. In some embodiments, the method disclosed herein produces more polypeptides than a method comprising translocating cells in a culture that does not contain dextran sulfate. In some embodiments, the recombinant polypeptide is an antibody or its antigen-binding fragment, a bispecific antibody, an enzyme, a fusion protein, or an Fc fusion protein.

[0012] In a further embodiment, the Disclosure provides a method for improving the production of recombinant virus particles, comprising: (a) preparing a cell culture containing cells suitable for the production of recombinant virus particles, wherein the culture contains dextran sulfate in a concentration between about 0.1 mg / L and about 10 mg / L; (b) translocating the cells to the culture from (a) by adding a composition containing one or more polynucleotides containing genes necessary for the production of recombinant virus particles and a translocation reagent; and (c) maintaining the cell culture containing the translocated cells under conditions that enable the production of recombinant virus particles. In some embodiments, the method disclosed herein produces more recombinant virus particles than a method that involves translocating cells in a culture that does not contain dextran sulfate. In some embodiments, the recombinant virus is recombinant adeno-associated virus (rAAV). In some embodiments, rAAV is 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. It contains capsid proteins of PHP.B, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, or AAV.HSC16 serotypes. In some embodiments, rAAV contains capsid proteins of AAV8 or AAV9 serotype. In some embodiments, the cell culture is a suspension culture containing suspension-adaptive HEK cells. In some embodiments, the translocation reagent contains polyethyleneimine (PEI).

[0013] In some embodiments, this disclosure provides the following:

[0014] [1.] A method for transmuting cells, a) Preparing a cell culture containing the cells, wherein the culture contains dextran sulfate in a concentration of approximately 0.1 mg / L to approximately 10 mg / L, b) Translocation into the cells by adding a composition containing one or more polynucleotides and a translocation reagent to the culture in (a) The method, including the method described above.

[0015] [2.] A method for generating recombinant polypeptides, a) Preparing a cell culture containing cells suitable for the production of the recombinant polypeptide, wherein the culture contains dextran sulfate in a concentration of approximately 0.1 mg / L to approximately 10 mg / L, b) The cell is transmigrated by adding a composition containing one or more polynucleotides encoding the polypeptide and a transtransfer reagent to the culture in a), c) Maintaining the cell culture containing the transfused cells under conditions that enable the production of the recombinant polypeptide. The method, including the method described above.

[0016] [3.] The method according to 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 generating recombinant virus particles, a) Preparing a cell culture containing cells suitable for generating the recombinant virus particles, wherein the culture contains dextran sulfate in a concentration of approximately 0.1 mg / L to approximately 10 mg / L, b) The cell translocation is performed by adding a composition to the culture of a) the above, which contains one or more polynucleotides containing genes necessary for generating the recombinant virus particles and a translocation reagent. c) Maintaining the cell culture containing the transfused cells under conditions that enable the generation of recombinant virus particles. The method, including the method described above.

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

[0019] The method according to any one of claims [1] to [4], wherein the culture in [6.]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] The method according to any one of claims [1] to [4], wherein the culture in [7.]a) contains about 2 mg / L of dextran sulfate.

[0021] [8.] A method for translocating cells, a) Culturing the cells in a cell culture medium, wherein the culture medium contains an initial dextran sulfate concentration between approximately 1 mg / L and approximately 20 mg / L and a final dextran sulfate concentration between approximately 0.1 mg / L and approximately 10 mg / L. b) by adding a composition containing one or more polynucleotides and a translocation reagent to the culture in a), translocation is performed on the cells. The method, including the method described above.

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

[0023] [10.] The method according to claim [9], wherein said 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 generating recombinant virus particles, a) culturing cells suitable for the production of said recombinant virus particles in a cell culture for about 1 day to about 5 days, wherein said culture comprises an initial dextran sulfate concentration between about 1 mg / L and about 20 mg / L and a final dextran sulfate concentration between about 0.1 mg / L and about 10 mg / L, said culturing; b) transfecting said cells by adding to the culture of a) a composition comprising one or more polynucleotides containing the genes necessary for the production of said recombinant virus particles and a transfection reagent; c) maintaining the cell culture comprising the transfected cells under conditions that allow production of the recombinant virus particles The method comprising.

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

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

[0026] [13.] The method according to 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 approximately 4 mg / L.

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

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

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

[14] , wherein the final concentration of dextran sulfate 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 concentration of dextran sulfate is approximately 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 according to any one of claims [4] to [7] and

[11] to

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

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

[11] to

[18] , wherein the recombinant virus particle is an 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 The method according to claim

[20] , comprising a capsid protein of 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.

[0035] [22.] The method according to claim

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

[0036] [23.] The method according to claim

[20] , wherein the rAAV particles comprise a capsid protein of the AAV8 or AAV9 serotype.

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

[20] to

[23] , wherein the rAAV particles include a genome containing a transgene.

[0038] [25.] The method according to claim

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

[0039] [26.] The method according to claim

[25] , wherein the regulating element comprises one or more of an enhancer, a promoter, and a poly-A region.

[0040] [27.] The method according to claim

[24] or claim

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

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

[24] to

[27] , wherein the transgene encodes anti-VEGF Fab, idulonidase (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 aforementioned transgenes include gamma-sarcoglycan, Rab escort protein 1 (REP1 / CHM), retinoid isomerohydrolase (RPE65), cyclic nucleotide gate channel alpha-3 (CNGA3), cyclic nucleotide gate channel beta-3 (CNGB3), aromatic L-amino acid decarboxylase (AADC), lysosome-associated membrane protein 2 isoform B (LAMP2B), factor VIII, factor IX, retinitis pigmentosa GTPase regulator (RPGR), retinosoxin (RS1), and sarcoplasmic reticulum calcium. Mu ATPase (SERCA2a), aflibercept, battenin (CLN3), transmembrane ER protein (CLN6), glutamate decarboxylase (GAD), glial cell line-derived neurotrophic factor (GDNF), aquaporin 1 (AQP1), dystrophin, minidystrophin, microdystrophin, myotubularin 1 (MTM1), follistatin (FST), glucose-6-phosphatase (G6Pase), apolipoprotein A2 (APOA2), uridine diphosphate glucuronosyltransferase 1A1 (UGT 1A1), arylsulfatase B (ARSB), N-acetyl-alpha-glucosaminidase (NAGLU), alpha-glucosidase (GAA), alpha-galactosidase (GLA), beta-galactosidase (GLB1), lipoprotein lipase (LPL), alpha-1-antitrypsin (AAT), phosphodiesterase 6B (PDE6B), ornithine carbamoyltransferase 9OTC), survival motor neuron (SMN1), survival motor neuron (SMN2), neurturin (NRTN), neurotroph The method according to any one of claims

[24] to

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

[0043] [30.] The one or more polynucleotides a) rAAV genome to be packaged, b) Adenovirus helper function required for packaging, c) Sufficient AAV rep protein for packaging, and d) Sufficient AAV cap protein for packaging The method according to any one of claims

[20] to

[29] , which codes for

[0044] [31.] The method according to claim

[30] , wherein the one or more polynucleotides comprise 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 function.

[0045] [32.] The method according to 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 according to any one of claims

[20] to

[28] , further comprising recovering the rAAV particles.

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

[20] to

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

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

[20] to

[33] , wherein the cell culture produces at least about 1.1 times, 1.2 times, 1.3 times, 1.4 times, 1.5 times, 1.6 times, 1.7 times, 1.8 times, 1.9 times, or 2 times more rAAV particles measured as GC / ml than the reference method of a) in which the culture 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 according to claim

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

[0051] [38.] The method according to claim

[36] or claim

[37] , wherein the cells include 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 according to claim

[36] or claim

[37] , wherein the cells comprise HEK293 cells.

[0053] [40.] The method according to claim

[36] or claim

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

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

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

[0055] [42.] The method according to claim

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

[0056] [43.] The method according to claim

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

[0057] [44.] The method according to claim 41, wherein the trait transfer reagent comprises polyethyleneimine (PEI).

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

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

[0059] [46.] The method according to claim

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

[0060] [47.] The method according to claim

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

[0061] [48.] The method according to claim

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

[0062] [49.] The method according to claim

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

[0063] [50.] A composition comprising isolated rAAV particles produced by the method described in any one of claims

[20] to

[49] .

[0064] Further other features and advantages of the compositions and methods described herein will become clearer from the following detailed description when read in conjunction with the accompanying drawings. [Brief explanation of the drawing]

[0065] [Figure 1] Initial shaking flask screening of dextran sulfate for use before and during translocation. 1:2,500, 1:500, 1:10,000, 1:20,000, 1:40,000, and 1:80,000 represent dilution ratios of the 25 g / L dextran sulfate stock solution present at the time of translocation, 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. [Figure 2]Initial shaking flask screening of dextran sulfate for use before and during translocation. 6K, 7K, 8K, 9K, 10K, 12K, and 15K indicate dilution ratios of the 25 g / L dextran sulfate stock solution present at the time of translocation, 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. [Figure 3] Bench-scale 2L dextran sulfate titration for translocation: genome titer. 6K, 7K, 8K, 9K, and 10K indicate dilution ratios of the 25g / L dextran sulfate stock solution present at translocation, corresponding to concentrations of 4.2mg / L, 3.6mg / L, 3.1mg / L, 2.8mg / L, and 2.5mg / L, respectively. [Figure 4] Bench-scale 2L dextran sulfate titration for translocation: cell imaging. 1:6,000, 1:7,000, 1:8,000, 1:9,000, and 1:10,000 represent dilution ratios of the 25 g / L dextran sulfate stock solution present at translocation, 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 5] Bench-scale 2L dextran sulfate titration for translocation: viable cell density. 1:6,000, 1:7,000, 1:8,000, 1:9,000, and 1:10,000 represent dilution ratios of the 25 g / L dextran sulfate stock solution present at translocation, 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 titration for translocation: cell viability. 1:6,000, 1:7,000, 1:8,000, 1:9,000, and 1:10,000 represent dilution ratios of the 25 g / L dextran sulfate stock solution present at the time of translocation, 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 generation in a bench-scale 5L reactor using dextran sulfate at a concentration of 2 mg / L during translocation. [Figure 8] AAV8 generation was observed in a 2L bench-scale reactor using different commercially available media. Dextran sulfate was present at a concentration of 2 mg / L during translocation. [Figure 9] AAV8 generation in a shaking flask using different host cell clones. Dextran sulfate was present at 2 mg / L during translocation. [Figure 10] AAV9 generation in a bench-scale 5L reactor using dextran sulfate during translocation. [Figure 11] Adding dextran sulfate to a high-density seed train before translocation increases AAV titer. [Figure 12] Adding dextran sulfate to the seed train before translocation increases AAV titer. [Modes for carrying out the invention]

[0066] Detailed explanation Surprisingly, it was found that dextran sulfate can increase rAAV titer through a transient transduction-based synthesis method. This finding was unexpected, as dextran sulfate is known to inhibit transient transduction. For example, Geng et al. (2007), on page 55, conclude that dextran sulfate completely inhibits PEI-mediated transduction. Similarly, the recently published PALL® Biotech “Guide for DNA Transfection in iCELLis® 500 and iCELLis 500+ Bioreactors for Large Scale Gene Therapy Vector Manufacturing” (“2020 Guide”), on page 9, teaches that dextran sulfate inhibits PEI-mediated transduction. 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 generation by transient translocation-based methods would be inhibited, or at least less productive, by the presence of dextran sulfate in the cell culture during translocation. In contrast, as discussed in the examples, the presence of dextran sulfate in the translocation medium resulted in a surprising increase in rAAV generation. Increased rAAV generation was observed in processes for generating rAAV particles containing different capsid serotypes or transgenes using different cell culture media, host cell clones, and production volumes.

[0067] These remarkable findings were used to develop methods for transtransferring cells, generating recombinant polypeptides, generating recombinant viral particles (e.g., recombinant adeno-associated virus (rAAV) particles), improving the generation of recombinant polypeptides, and improving the generation of recombinant viral particles (e.g., rAAV particles) as described herein. In some embodiments, the method involves transtransferring cells by adding a composition comprising one or more polynucleotides and a transtransfer 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 that grow attached to microcarriers or macrocarriers in a stirred bioreactor. In some embodiments, the cell culture is a suspension cell culture comprising suspension-adaptive HEK293 cells. In some embodiments, the recombinant viral particles are recombinant adeno-associated virus (rAAV) particles. In some embodiments, rAAV is 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. It contains capsid proteins of PHP.B, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, or AAV.HSC16 serotypes. In some embodiments, rAAV contains capsid proteins of AAV8 or AAV9 serotypes.

[0068] Given that a very large number of rAAV particles are required to prepare a single therapeutic dose, any increase in rAAV yield reduces the product cost per unit dose. Increased virus yield can, in turn, reduce not only the cost of consumables required for rAAV particle production, but also the capital investment costs associated with constructing industrial virus purification facilities.

[0069] definition Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art in which this disclosure relates. To facilitate understanding of the methods of this disclosure, several terms and expressions are defined below.

[0070] For example, modifying the amount of a component in a composition, the concentration of a component in a composition, the flow rate, the rAAV particle yield, the feed volume, the salt concentration, similar values, and ranges thereof, “about” as used in the methods provided herein means variations in numerical quantities that may result from, for example, typical measurement and handling procedures used to prepare a concentrate or working solution; accidental errors in these procedures; differences in the manufacture, source, or purity of the components used in preparing the composition or performing the method; and similar considerations. The term “about” also includes amounts that differ as a composition or mixture having a particular initial concentration ages. The term “about” also includes amounts that differ as a composition or mixture having a particular initial concentration is mixed or processed. Whether modified by the term “about,” a claim includes an equivalent of that quantity. In some embodiments, the term “about” means a range of about 10 to 20 percent more or less than the number or range indicated. In further embodiments, “about” means plus or minus 10 percent of the number or range indicated. For example, “about 10%” indicates a range of 9% to 11%.

[0071] The term "sulfated dextran" refers to a sulfated polysaccharide comprising a polymer backbone of α-1,6 glycosidic bonds between glucose monomers and branching from α-1,3 bonds. Sulfated dextran is commercially available, for example, from MilliporeSigma (Saint Louis, Mo.). "Sulfated dextran" should be understood to encompass both the free acid and its salts. In some embodiments, sulfated dextran is a salt. In some embodiments, sulfated dextran is a free acid. In some embodiments, sulfated dextran is a salt containing a monovalent cation. In some embodiments, sulfated dextran is a Li, Na, K, Rb, or Cs salt. In some embodiments, sulfated dextran is a Na salt. In some embodiments, sulfated dextran contains about 10% to about 25% sulfur. In some embodiments, sulfated dextran contains about 15% to about 20% sulfur. In some embodiments, each glucosyl residue of sulfated dextran contains an average of 1 to 3 sulfate groups. In some embodiments, each glucosyl residue of dextran sulfate contains an average of 2 to 3 sulfate groups. In some embodiments, dextran sulfate contains about 17% sulfur, which corresponds to about 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 approximately 3 kDa to approximately 25 kDa. In some embodiments, the average molecular weight of dextran sulfate is approximately 3 kDa to approximately 10 kDa. In some embodiments, the average molecular weight of dextran sulfate is approximately 4 kDa to approximately 25 kDa. In some embodiments, the average molecular weight of dextran sulfate is approximately 4 kDa to approximately 10 kDa.In some embodiments, the average molecular weight of dextran sulfate is approximately 5 kDa to approximately 25 kDa. In some embodiments, the average molecular weight of dextran sulfate is approximately 5 kDa to approximately 10 kDa. In some embodiments, the average molecular weight of dextran sulfate is approximately 5 kDa. In some embodiments, dextran sulfate is a sodium salt having an average molecular weight between approximately 3 kDa and 10 kDa. In some embodiments, dextran sulfate is a sodium salt having an average molecular weight of approximately 5 kDa. In some embodiments, dextran sulfate is a sodium salt containing approximately 15% to 20% sulfur and having an average molecular weight between approximately 3 kDa and 10 kDa. In some embodiments, dextran sulfate is a sodium salt containing approximately 17% sulfur and having an average molecular weight of approximately 5 kDa.

[0072] "AAV" is an abbreviation for adeno-associated virus and can be used to refer to the virus itself or its modifiers, derivatives, or pseudotypes. Unless otherwise required, the term encompasses all subtypes, as well as both the native and recombinant forms. The abbreviation "rAAV" refers to recombinant adeno-associated virus. The term "AAV" includes AAV type 1 (AAV1), AAV type 2 (AAV2), AAV type 3 (AAV3), AAV type 4 (AAV4), AAV type 5 (AAV5), AAV type 6 (AAV6), AAV type 7 (AAV7), AAV type 8 (AAV8), AAV type 9 (AAV9), avian AAV, bovine AAV, canine AAV, equine AAV, primate AAV, non-primate AAV, and sheep AAV, as well as their modifiers, derivatives, or pseudotypes. "Primate AAV" refers to AAV that infects primates, "non-primate AAV" refers to AAV that infects non-primate mammals, and "bovine AAV" refers to AAV that infects bovine mammals, and so on.

[0073] The term "recombination" as applied to AAV particles means that the AAV particle is the product of one or more procedures that result in an AAV particle construct that is essentially different from the AAV particle itself.

[0074] Recombinant adeno-associated virus particles, or "rAAV particles," refer to viral particles composed of at least one AAV capsid protein and a capsidized polynucleotide rAAV vector genome containing heterologous polynucleotides (i.e., polynucleotides other than the wild-type AAV genome, e.g., a transgene to be delivered to mammalian cells). rAAV particles can be any AAV serotype (e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, or AAV10, or their derivatives / modifiers / pseudotypes), including any modifiers, derivatives, or pseudotypes. Such AAV serotypes and derivatives / modifiers / pseudotypes, as well as methods for generating such serotypes / derivatives / modifiers / pseudotypes, are known in the art (see, for example, Asokan et al., Mol.Ther. 20(4):699-708 (2012)).

[0075] The rAAV particles of this disclosure may be any serotype or any combination of serotypes (for example, a population of rAAV particles containing two or more serotypes (for example, two or more of rAAV2, rAAV8, and rAAV9 particles)). In some embodiments, the rAAV particles are rAAV1, rAAV2, rAAV3, rAAV4, rAAV5, rAAV6, rAAV7, rAAV8, rAAV9, rAAV10, or other rAAV particles, or two or more combinations thereof. In some embodiments, the rAAV particles are rAAV8 or rAAV9 particles.

[0076] In some embodiments, the rAAV particles have an AAV capsid protein 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, modifiers, or pseudotypes thereof. In some embodiments, the rAAV particles have an AAV capsid protein of a serotype consisting of AAV8, AAV9, or derivatives, modifiers, or pseudotypes thereof.

[0077] The term “cell culture” refers to cells grown attached to or in suspension, bioreactors, roller bottles, hyperstacks, microspheres, macrospheres, flasks, etc., as well as the components of the supernatant or suspension itself, including, but not limited to, rAAV particles, cells, cell debris, cell contaminants, colloidal particles, biomolecules, host cell proteins, nucleic acids, lipids, and flocculants. Large-scale approaches such as bioreactors (including suspension cultures and adherent cells grown attached to microcarriers or macrocarriers in agitated bioreactors) are also encompassed by the term “cell culture.” Cell culture procedures for both large-scale and small-scale protein production are also included in this disclosure. 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 agitated 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 an alternating tangential flow (ATF) supported high-density perfusion culture.

[0078] As used herein, the terms “purify,” “separate,” “isolate,” “remove,” “isolate,” or “isolate” mean increasing the degree of purity of a target product (e.g., rAAV particles and rAAV genome) from a sample containing the target product and one or more impurities. Typically, the degree of purity of a target product is increased by removing (completely or incompletely) at least one impurity from the sample. In some embodiments, the degree of purity of rAAV in a sample is increased by removing (completely or incompletely) one or more impurities from the sample using the methods described herein.

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

[0080] Whenever embodiments described herein using the phrase "including" should be understood as also providing other similar embodiments described in terms of "consisting of" and / or "essentially consisting of".

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

[0082] Where embodiments of this disclosure describe the Markush Group or other alternative groups, the methods of this disclosure encompass not only the entire group as a whole, but also each individual member of the group, all possible subgroups of the main group, and even the main group in which one or more group members are absent. The methods of this disclosure also anticipate the express exclusion of one or more group members in the methods of this disclosure.

[0083] Methods for transmuting cells In one embodiment, the present disclosure provides a method for translocating cells, comprising: (a) preparing a cell culture containing cells, wherein the culture contains dextran sulfate in a concentration between about 0.1 mg / L and about 10 mg / L; and (b) translocating cells by adding a composition comprising one or more polynucleotides and a translocation reagent to the culture from (a).

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

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

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

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

[0088] In some embodiments, the initial dextran sulfate concentration is between approximately 1 mg / L and approximately 10 mg / L, between approximately 1 mg / L and approximately 5 mg / L, between approximately 2 mg / L and approximately 10 mg / L, between approximately 3 mg / L and approximately 10 mg / L, or between approximately 3 mg / L and approximately 5 mg / L of dextran sulfate. In some embodiments, the initial dextran sulfate concentration is between approximately 1 mg / L and approximately 10 mg / L of dextran sulfate. In some embodiments, the initial dextran sulfate concentration is between approximately 2 mg / L and approximately 10 mg / L of dextran sulfate. In some embodiments, the initial dextran sulfate concentration is between approximately 3 mg / L and approximately 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 of dextran sulfate. In some embodiments, the starting dextran sulfate concentration is about 2 mg / L of dextran sulfate. In some embodiments, the starting dextran sulfate concentration is about 3 mg / L of dextran sulfate. In some embodiments, the starting dextran sulfate concentration is about 4 mg / L of dextran sulfate. In some embodiments, the starting dextran sulfate concentration is about 5 mg / L of dextran sulfate. In some embodiments, the starting dextran sulfate concentration is about 6 mg / L of dextran sulfate. In some embodiments, the starting dextran sulfate concentration is about 7 mg / L of dextran sulfate. In some embodiments, the starting dextran sulfate concentration is about 8 mg / L of dextran sulfate.

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

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

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

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

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

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

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

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

[0098] In some embodiments, one or more polynucleotides contain genes necessary for the generation of recombinant virus particles. In some embodiments, the recombinant virus particles are recombinant adenovirus particles. In some embodiments, the recombinant virus particles are recombinant adeno-associated virus (rAAV) particles.

[0099] Any suitable translocation reagent known in the art can be used to translocate cells. In some embodiments, the translocation reagent comprises a cationic organic carrier. See, for example, Gigante et al., Medchemcomm 10(10):1692-1718(2019); Damen et al., Medchemcomm 9(9):1404-1425(2018) (each of these is incorporated herein by reference in whole). In some embodiments, the cationic organic carrier comprises lipids, e.g., DOTMA, DOTAP, helper lipids (Dope, cholesterol), and combinations thereof. In some embodiments, the cationic organic carrier comprises polyvalent cationic lipids, e.g., DOSPA, DOGS, and mixtures thereof. In some embodiments, the cationic organic carrier comprises bipolar lipids, or bora amphiphiles (bola). In some embodiments, the cationic organic carrier comprises biologically reductive and / or dimerizable lipids. 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 polymers, 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 includes peptides, such as basic amino acid-rich peptides (CWL18), cell-permeable peptides (CPP) (Arg-rich peptides (octaarginine, TAT)), nuclear localization signals (NLS) (SV40), and targeting (RGD).In some embodiments, the cationic organic carrier comprises a polymer (e.g., PEI) combined with a cationic liposome. Paris et al., Molecules 25(14):3277 (2020) (the whole of which is incorporated herein by reference). In some embodiments, the translocation reagent comprises calcium phosphate, a highly branched organic compound (dendrimer), a cationic polymer (e.g., DEAE dextran or polyethyleneimine (PEI)), and lipofection.

[0100] In some embodiments, the translocation 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 translocation reagent comprises polyethyleneimine (PEI), linear PEI, branched PEI, or mixtures thereof. In some embodiments, the translocation reagent comprises polyethyleneimine (PEI). In some embodiments, the translocation reagent comprises linear PEI. In some embodiments, the translocation reagent comprises branched PEI. In some embodiments, the translocation reagent comprises polyethyleneimine (PEI) having a molecular weight between about 5 and about 25 kDa. In some embodiments, the translocation reagent comprises PEGylated polyethyleneimine (PEI). In some embodiments, the translocation reagent comprises modified polyethyleneimine (PEI) to which hydrophobic moieties (e.g., cholesterol, choline, alkyl groups, and several amino acids) are attached.

[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 contains 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 cells include mammalian cells or insect cells. In some embodiments, the cells include mammalian cells. In some embodiments, the cells include HEK293 cells, HEK-derived cells, CHO cells, CHO-derived cells, HeLa cells, SF-9 cells, BHK cells, Vero cells, and / or PerC6 cells. In some embodiments, the cells include HEK293 cells.

[0103] In some embodiments, the cells include suspension-adaptive cells. In some embodiments, the cells include suspension-adaptive 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 include 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 include suspension-adapted HEK293 cells. In some embodiments, the cells include suspension-adapted CHO cells.

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

[0105] While not bound by any particular theory, the methods disclosed herein increase the efficiency of translocation, and as a result, cells translocated according to the methods disclosed herein are more likely to contain one or more polynucleotides than control cells translocated in cell cultures without dextran sulfate. In some embodiments, the methods disclosed herein increase the efficiency of translocation 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 without dextran sulfate. Methods for measuring translocation efficiency are well known in the art. In some embodiments, translocation efficiency is measured using a reporter transgene construct (e.g., a reporter transgene encoding a fluorescent protein (e.g., GFP)).

[0106] Method for generating recombinant virus particles In one embodiment, the Disclosure provides a method for generating recombinant viral particles, comprising: (a) preparing a cell culture containing cells suitable for the generation of recombinant viral particles, wherein the culture contains dextran sulfate in a concentration between approximately 0.1 mg / L and approximately 10 mg / L; (b) translocating the cells to the culture from (a) by adding a composition containing one or more polynucleotides containing genes necessary for the generation of recombinant viral particles and a translocation reagent; and (c) maintaining the cell culture containing the translocated cells under conditions that enable the generation of recombinant viral particles. In some embodiments, the culture from a) contains dextran sulfate in a concentration between approximately 1 mg / L and approximately 3 mg / L. In some embodiments, the recombinant viral particles are recombinant adeno-associated virus (rAAV) particles. In some embodiments, the one or more polynucleotides contain one or more helper genes, rep genes, cap genes, and transgenes (e.g., a target gene or the rAAV genome to be packaged). In some embodiments, one or more polynucleotides comprise a mixture of three polynucleotides (one encoding cap and rep genes, one encoding adenovirus helper functions necessary for packaging (e.g., adenovirus E1a, E1b, E4, E2a, and VA genes), and one encoding the rAAV genome to be packaged). In some embodiments, the 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, 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 contains HEK293 cells adapted for 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 translocation reagent contains a cationic polymer. In some embodiments, the translocation reagent contains PEI.

[0107] In some embodiments, the Disclosure provides a method for increasing the production of recombinant virus particles, comprising: (a) preparing a cell culture containing cells suitable for the production of recombinant virus particles, wherein the culture contains dextran sulfate in a concentration between about 0.1 mg / L and about 10 mg / L; (b) translocating the cells to the culture from (a) by adding a composition containing one or more polynucleotides containing genes necessary for the production of recombinant virus particles and a translocation reagent; and (c) maintaining the cell culture containing the translocated cells under conditions that enable the production of recombinant virus particles. In some embodiments, the culture from a) contains dextran sulfate in a concentration between about 1 mg / L and about 3 mg / L. In some embodiments, the culture from a) contains dextran sulfate in a concentration of about 2 mg / L. In some embodiments, the recombinant virus particles are recombinant adeno-associated virus (rAAV) particles. In some embodiments, the one or more polynucleotides contain one or more helper genes, rep genes, cap genes, and transgenes (e.g., the target gene or the rAAV genome to be packaged). In some embodiments, one or more polynucleotides comprise a mixture of three polynucleotides (one encoding cap and rep genes, one encoding adenovirus helper functions necessary for packaging (e.g., adenovirus E1a, E1b, E4, E2a, and VA genes), and one encoding the rAAV genome to be packaged). In some embodiments, the rAAV particles are AAV8 or AAV9 particles. In some embodiments, the rAAV particles have an AAV capsid protein of a serotype selected from the group consisting of AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.PHB, and AAV.7m8. In some embodiments, the rAAV particles have AAV capsid proteins with high sequence homology to AAV8 or AAV9, such as AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, and AAV.hu37.In some embodiments, the cell culture is a suspension culture. In some embodiments, the cell culture contains HEK293 cells adapted for 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 translocation reagent contains a cationic polymer. In some embodiments, the translocation reagent contains PEI.

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

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

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

[0111] In some embodiments, the Disclosure provides a method for generating recombinant virus particles, comprising: (a) culturing cells suitable for the generation of recombinant virus particles in a cell culture for about 1 to about 5 days, wherein the culture contains an initial dextran sulfate concentration between about 1 mg / L and about 20 mg / L and a final dextran sulfate concentration between about 0.1 mg / L and about 10 mg / L; (b) translocating the cells to the culture from (a) by adding a composition containing one or more polynucleotides containing genes necessary for the generation of recombinant virus particles and a translocation reagent; and (c) maintaining the cell culture containing the translocated cells under conditions that enable the generation of recombinant virus particles. In some embodiments, the initial dextran sulfate concentration is between about 3 mg / L and about 6 mg / L of dextran sulfate, and the final dextran sulfate concentration is between about 1 mg / L and about 3 mg / L of dextran sulfate. In some embodiments, the starting dextran sulfate concentration is approximately 4 mg / L of dextran sulfate, and the final dextran sulfate concentration is approximately 2 mg / L of dextran sulfate. In some embodiments, the recombinant virus particles are recombinant adeno-associated virus (rAAV) particles. In some embodiments, one or more polynucleotides comprise one or more helper genes, rep genes, cap genes, and transgenes (e.g., the target gene or the rAAV genome to be packaged). In some embodiments, one or more polynucleotides comprise a mixture of three polynucleotides (one encoding the cap and rep genes, one encoding the adenovirus helper function required for packaging (e.g., adenovirus E1a, E1b, E4, E2a, and VA genes), and one encoding the rAAV genome to be packaged). In some embodiments, the 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, 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 contains HEK293 cells adapted for 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 translocation reagent contains a cationic polymer. In some embodiments, the translocation reagent includes PEI.

[0112] In some embodiments, the Disclosure provides a method for increasing the production of recombinant virus particles, comprising: (a) culturing cells suitable for the production of recombinant virus particles in a cell culture for about 1 to about 5 days, wherein the culture contains an initial dextran sulfate concentration between about 1 mg / L and about 20 mg / L and a final dextran sulfate concentration between about 0.1 mg / L and about 10 mg / L; (b) translocating the cells to the culture from (a) by adding a composition containing one or more polynucleotides containing genes necessary for the production of recombinant virus particles and a translocation reagent; and (c) maintaining the cell culture containing the translocated cells under conditions that enable the production of recombinant virus particles. In some embodiments, the initial dextran sulfate concentration is between about 3 mg / L and about 6 mg / L of dextran sulfate, and the final dextran sulfate concentration is between about 1 mg / L and about 3 mg / L of dextran sulfate. In some embodiments, the starting dextran sulfate concentration is approximately 4 mg / L of dextran sulfate, and the final dextran sulfate concentration is approximately 2 mg / L of dextran sulfate. In some embodiments, the recombinant virus particles are recombinant adeno-associated virus (rAAV) particles. In some embodiments, one or more polynucleotides comprise one or more helper genes, rep genes, cap genes, and transgenes (e.g., the target gene or the rAAV genome to be packaged). In some embodiments, one or more polynucleotides comprise a mixture of three polynucleotides (one encoding the cap and rep genes, one encoding the adenovirus helper function required for packaging (e.g., adenovirus E1a, E1b, E4, E2a, and VA genes), and one encoding the rAAV genome to be packaged). In some embodiments, the 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, 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 contains HEK293 cells adapted for 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 translocation reagent contains a cationic polymer. In some embodiments, the translocation reagent includes PEI.

[0113] In some embodiments, the initial dextran sulfate concentration is between approximately 1 mg / L and approximately 10 mg / L, between approximately 1 mg / L and approximately 5 mg / L, between approximately 2 mg / L and approximately 10 mg / L, between approximately 3 mg / L and approximately 10 mg / L, or between approximately 3 mg / L and approximately 5 mg / L of dextran sulfate. In some embodiments, the initial dextran sulfate concentration is between approximately 1 mg / L and approximately 10 mg / L of dextran sulfate. In some embodiments, the initial dextran sulfate concentration is between approximately 2 mg / L and approximately 10 mg / L of dextran sulfate. In some embodiments, the initial dextran sulfate concentration is between approximately 3 mg / L and approximately 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 of dextran sulfate. In some embodiments, the starting dextran sulfate concentration is about 2 mg / L of dextran sulfate. In some embodiments, the starting dextran sulfate concentration is about 3 mg / L of dextran sulfate. In some embodiments, the starting dextran sulfate concentration is about 4 mg / L of dextran sulfate. In some embodiments, the starting dextran sulfate concentration is about 5 mg / L of dextran sulfate. In some embodiments, the starting dextran sulfate concentration is about 6 mg / L of dextran sulfate. In some embodiments, the starting dextran sulfate concentration is about 7 mg / L of dextran sulfate. In some embodiments, the starting dextran sulfate concentration is about 8 mg / L of dextran sulfate.

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

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

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

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

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

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

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

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

[0123] In some embodiments, recombinant virus particles are rAAV particles. In some embodiments, 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 The rAAV particles contain capsid proteins of serotypes 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. In some embodiments, the rAAV particles contain capsid proteins of serotypes AAV8, AAV9, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, or AAV.hu37. In some embodiments, the rAAV particles contain the AAV8 serotype capsid protein. In some embodiments, the rAAV particles contain the AAV9 serotype capsid protein.

[0124] In some embodiments, recombinant viral particles contain a transgene. Various viral transgene expression systems suitable for use in specific host cells are known to those skilled in the art. It should be understood that any viral transgene expression system can be used according to the methods disclosed herein. In some embodiments, the transgene contains a regulatory element responsively bound to a polynucleotide encoding a polypeptide. In some embodiments, the regulatory element includes one or more of an enhancer, a promoter, and a poly(A) region. In some embodiments, the regulatory element and the polynucleotide encoding the polypeptide are heterogeneous.

[0125] In some embodiments, the transgene encodes anti-VEGF Fab, idulonidase (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).In some embodiments, the transgenes include gamma-sarcoglycan, Rab escort protein 1 (REP1 / CHM), retinoid isomerohydrolase (RPE65), cyclic nucleotide gate channel alpha-3 (CNGA3), cyclic nucleotide gate channel beta-3 (CNGB3), aromatic L-amino acid decarboxylase (AADC), lysosome-associated membrane protein 2 isoform B (LAMP2B), factor VIII, factor IX, retinitis pigmentosa GTPase regulator (RPGR), and retinoskin (RS 1) Sarcoplasmic reticulum calcium ATPase (SERCA2a), aflibercept, battenin (CLN3), transmembrane ER protein (CLN6), glutamate decarboxylase (GAD), glial cell line-derived neurotrophic factor (GDNF), aquaporin 1 (AQP1), dystrophin, minidystrophin, microdystrophin, myotubularin 1 (MTM1), follistatin (FST), glucose-6-phosphatase (G6Pase), apolipoprotein A2 (APOA2), uridine diphosphate glucuronosyl Transferase 1A1 (UGT1A1), arylsulfatase B (ARSB), N-acetyl-alpha-glucosaminidase (NAGLU), alpha-glucosidase (GAA), alpha-galactosidase (GLA), beta-galactosidase (GLB1), lipoprotein lipase (LPL), alpha-1-antitrypsin (AAT), phosphodiesterase 6B (PDE6B), ornithine carbamoyltransferase 9OTC), survival motor neuron (SMN1), survival motor neuron (SMN2), The recombinant virus particles encode neuroturin (NRTN), neurotrophin-3 (NT-3 / NTF3), porphobilinogen deaminase (PBGD), nerve growth factor (NGF), mitochondrial-coded NADH:ubiquinone oxidoreductase core subunit 4 (MT-ND4), protective protein cathepsin A (PPCA), dyspherin, MER proto-oncogene tyrosine kinase (MERTK), cystic fibrosis membrane conductance regulator (CFTR), or tumor necrosis factor receptor (TNFR)-immunoglobulin (IgG1)Fc fusion. In some embodiments, the recombinant virus particles are rAAV particles.In some embodiments, the rAAV particles contain the AAV8 serotype capsid protein. In some embodiments, the rAAV particles contain the AAV9 serotype capsid protein.

[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-CoV-1 or SARS-CoV-2. In some embodiments, the transgene encodes the spike protein or an immunogenic fragment of SARS-CoV-1 or SARS-CoV-2. In some embodiments, the transgene encodes the spike protein or an immunogenic fragment of SARS-CoV-2. In some embodiments, the transgene encodes the receptor-binding domain of the SARS-CoV-2 spike protein. In some embodiments, the recombinant virus particle is an rAAV particle. In some embodiments, the recombinant virus particle is a recombinant adenovirus particle. In some embodiments, the recombinant virus particle is a recombinant chimpanzee adenovirus particle.

[0127] Recombinant virus particle production systems based on translocation 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 these is incorporated herein by reference in whole). The methods disclosed herein can be used to produce recombinant virus particles in translocation-based production systems. In some embodiments, the recombinant virus particles are recombinant dengue virus, recombinant Ebola virus, recombinant human papillomavirus (HPV), recombinant human immunodeficiency virus (HIV), recombinant adeno-associated virus (AAV), recombinant lentivirus, recombinant influenza virus, recombinant varicella-stomatitis virus (VSV), recombinant poliovirus, recombinant adenovirus, recombinant retrovirus, recombinant vaccinia, recombinant reovirus, recombinant measles virus, recombinant Newcastle disease virus (NDV), recombinant herpes zoster virus (HZV), recombinant herpes simplex virus (HSV), or recombinant baculovirus. In some embodiments, the recombinant virus particles are recombinant adeno-associated virus (AAV), recombinant lentivirus, or recombinant influenza virus. In some embodiments, the recombinant virus particles are recombinant lentivirus. In some embodiments, the recombinant virus particles are recombinant influenza virus. In some embodiments, the recombinant virus particles are recombinant baculovirus. In some embodiments, the recombinant virus particles are 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 translocation reagent known in the art for translocation into cells 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 for suspension culture). In some embodiments, the methods disclosed herein include translocation into cells using chemically based translocation methods. In some embodiments, the methods disclosed herein include translocation into cells using cationic organic carriers. See, for example, Gigante et al., Medchemcomm 10(10):1692-1718(2019); Damen et al., Medchemcomm 9(9):1404-1425(2018) (each of these is incorporated herein by reference in whole). In some embodiments, the cationic organic carriers include lipids, e.g., DOTMA, DOTAP, helper lipids (Dope, cholesterol), and combinations thereof. In some embodiments, the cationic organic carrier comprises polyvalent cationic lipids, such as DOSPA, DOGS, and mixtures thereof. In some embodiments, the cationic organic carrier comprises bipolar lipids, or bora amphiphiles (bola). In some embodiments, the cationic organic carrier comprises biologically reducing and / or dimerizable lipids. In some embodiments, the cationic organic carrier comprises gemin surfactants. In some embodiments, the cationic organic carrier comprises Lipofectin®, Transfectam®, Lipofectamine®, Lipofectamine 2000®, or Lipofectamin PLUS 2000®.In some embodiments, the cationic organic carrier includes polymers, 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 includes peptides, such as peptides rich in basic amino acids (CWL). 18 The reagents include a cell-permeable peptide (CPP) (Arg-rich peptide (octaarginine, TAT)), a nuclear localization signal (NLS) (SV40), and a targeting agent (RGD). In some embodiments, the cationic organic carrier comprises a polymer (e.g., PEI) combined with a cationic liposome. Paris et al., Molecules 25(14):3277 (2020) (the whole of which is incorporated herein by reference). In some embodiments, the translocation reagent comprises calcium phosphate, a highly branched organic compound (dendrimer), a cationic polymer (e.g., DEAE dextran or polyethyleneimine (PEI)), and lipofection.

[0129] In some embodiments, the translocation 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 translocation reagent comprises polyethyleneimine (PEI), linear PEI, branched PEI, or mixtures thereof. In some embodiments, the translocation reagent comprises polyethyleneimine (PEI). In some embodiments, the translocation reagent comprises linear PEI. In some embodiments, the translocation reagent comprises branched PEI. In some embodiments, the translocation reagent comprises polyethyleneimine (PEI) having a molecular weight between about 5 and about 25 kDa. In some embodiments, the translocation reagent comprises PEGylated polyethyleneimine (PEI). In some embodiments, the translocation reagent comprises modified polyethyleneimine (PEI) to which hydrophobic moieties (e.g., cholesterol, choline, alkyl groups, and several amino acids) are attached.

[0130] A composition comprising one or more polynucleotides and a translocation reagent can be prepared by any method known to those skilled in the art. In some embodiments, the composition is prepared by mixing one or more polynucleotides with at least one translocation reagent, which includes diluting each of the translocation reagents and one or more polynucleotides in a sterile liquid (e.g., a tissue culture medium) and mixing the diluted translocation reagents and the diluted one or more polynucleotides. In some embodiments, the tissue culture medium used for diluting the translocation reagents and / or one or more polynucleotides does not contain dextran sulfate. Those skilled in the art will understand that the dilution and mixing are carried out to produce a composition comprising the translocation reagents and polynucleotides in desired ratios and concentrations. In some embodiments, the dilution and mixing of at least one translocation reagent and one or more polynucleotides produces a composition comprising the translocation reagents and polynucleotides in a weight ratio between about 1:5 and 5:1. In some embodiments, the weight ratio of translocation reagents and polynucleotides is between about 1:3 and 3:1. In some embodiments, the weight ratio of the translocation reagent to the polynucleotide is between approximately 1:3 and 1:1. In some embodiments, the weight ratio of the translocation reagent to the polynucleotide is between approximately 1:2 and 1:1.5. In some embodiments, the weight ratio of the translocation reagent to the polynucleotide is approximately 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 the translocation reagent to the polynucleotide is approximately 1:2. In some embodiments, the weight ratio of the translocation reagent to the polynucleotide is approximately 1:1.75. In some embodiments, the weight ratio of the translocation reagent to the polynucleotide is approximately 1:1.5. In some embodiments, the weight ratio of the translocation reagent to the polynucleotide is approximately 1:1.25. In some embodiments, the weight ratio of the translocation reagent to the polynucleotide is approximately 1:1. In some embodiments, the weight ratio of the translocation reagent to the polynucleotide is approximately 1.25:1.In some embodiments, the weight ratio of the translocation reagent to the polynucleotide is approximately 1.5:1. In some embodiments, the weight ratio of the translocation reagent to the polynucleotide is approximately 1.75:1. In some embodiments, the weight ratio of the translocation reagent to the polynucleotide is approximately 2:1. In some embodiments, one or more polynucleotides contain three plasmids. In some embodiments, one or more polynucleotides contain two plasmids. In some embodiments, one or more polynucleotides contain one plasmid. In some embodiments, the recombinant virus is recombinant AAV, and one or more polynucleotides contain a mixture of three polynucleotides (one encoding the cap and rep genes, one encoding the adenovirus helper function required for packaging (e.g., adenovirus E1a, E1b, E4, E2a, and VA genes), and one encoding the rAAV genome to be packaged). In some embodiments, the 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 translocation reagent is PEI.

[0131] In some embodiments, a composition comprising a translocation reagent and one or more polynucleotides is incubated before being added to a culture to enable the formation of a polynucleotide:translocation reagent complex. In some embodiments, incubation is performed at room temperature. In some embodiments, incubation includes shaking the composition, for example, in a shaker between about 100 rpm and about 200 rpm. In some embodiments, incubation is performed between about 5 minutes and about 20 minutes. In some embodiments, incubation is performed between about 10 minutes and about 15 minutes. In some embodiments, incubation is performed for 15 minutes or less. In some embodiments, incubation is performed for 10 minutes or less. In some embodiments, incubation is performed for about 5 minutes, about 10 minutes, or about 15 minutes. In some embodiments, incubation is performed for about 10 minutes. In some embodiments, the translocation reagent comprises PEI.

[0132] In some embodiments, the volume of the composition added to the culture, which includes one or more polynucleotides containing genes necessary for the production of recombinant virus particles and a translocation reagent, is between about 5% and about 20% of the culture volume. In some embodiments, the volume of the added composition is between about 7% and about 15% of the culture volume. In some embodiments, the volume of the added composition is about 10% of the culture volume. In some embodiments, one or more polynucleotides contain genes necessary for the production of recombinant AAV particles. In some embodiments, the translocation reagent contains PEI. In some embodiments, the culture contains HEK293 cells (e.g., HEK293 cells adapted for suspension culture).

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

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

[0136] In some embodiments, the culture contains between approximately 2 x 10⁶ and approximately 10⁷ viable cells / ml. In some embodiments, the culture contains between approximately 3 x 10⁶ and approximately 8 x 10⁶ viable cells / ml. In some embodiments, the culture contains approximately 3 x 10⁶ viable cells / ml. In some embodiments, the culture contains approximately 4 x 10⁶ viable cells / ml. In some embodiments, the culture contains approximately 5 x 10⁶ viable cells / ml. In some embodiments, the culture contains approximately 6 x 10⁶ viable cells / ml. In some embodiments, the culture contains approximately 7 x 10⁶ viable cells / ml. In some embodiments, the culture contains approximately 8 x 10⁶ viable cells / ml. In some embodiments, the culture contains HEK293 cells (e.g., HEK293 cells adapted for suspension culture).

[0137] In some embodiments, the cells include mammalian cells or insect cells. In some embodiments, the cells include mammalian cells. In some embodiments, the cells include HEK293 cells, HEK-derived cells, CHO cells, CHO-derived cells, HeLa cells, SF-9 cells, BHK cells, Vero cells, and / or PerC6 cells. In some embodiments, the cells include HEK293 cells.

[0138] In some embodiments, the culture is maintained for about 2 to 10 days after adding a composition containing one or more polynucleotides containing genes necessary for the generation of recombinant virus particles and a translocation reagent. In some embodiments, the culture is maintained for about 5 to 14 days or longer after adding the composition. In some embodiments, the culture is maintained for about 2 to 7 days after adding the composition. In some embodiments, the culture is maintained for about 3 to 5 days after adding the composition. In some embodiments, the culture is maintained for about 2, 3, 4, 5, 6, or 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 generation of rAAV particles for continuous collection. In some embodiments, the culture contains HEK293 cells (e.g., HEK293 cells adapted for suspension culture).

[0139] In some embodiments, the methods disclosed herein increase the generation of recombinant viral particles (e.g., rAAV particles) compared to a reference method that involves translocation into cells in a dextran sulfate-free cell culture. In some embodiments, the methods disclosed herein generate at least about 1.1 times, 1.2 times, 1.3 times, 1.4 times, 1.5 times, 1.6 times, 1.7 times, 1.8 times, 1.9 times, or 2 times more viral particles than a reference method that involves translocation into cells in a dextran sulfate-free cell culture. 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 that involves translocation into cells in a dextran sulfate-free cell culture. 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 which involves translocation into cells in a cell culture that does not contain dextran sulfate. In some embodiments, the methods disclosed herein generate at least about 10% more viral particles than the reference method. In some embodiments, the methods disclosed herein generate at least about 20 percent more viral particles than the reference method. In some embodiments, the methods disclosed herein generate at least about 20 percent more viral particles than the reference method. In some embodiments, the methods disclosed herein generate at least about 20 percent more viral particles than the reference method. In some embodiments, the methods disclosed herein generate at least about 70 percent more viral particles than the reference method. In some embodiments, the methods disclosed herein generate at least about 100 percent more viral particles than the reference method. In some embodiments, the methods disclosed herein increase the generation of recombinant viruses by at least about 50%, at least about 75%, or at least about 100%.In some embodiments, the methods disclosed herein increase the production of recombinant virus by at least about twofold, at least about threefold, or at least about fivefold. In some embodiments, the methods disclosed herein increase the production of rAAV by at least about twofold. In some embodiments, the increase in production is quantified by comparing the recombinant virus (e.g., rAAV) titer of the produced culture. In some embodiments, the recombinant virus (e.g., rAAV) titer is measured as genome copies (GC) per milliliter of the produced culture. In some embodiments, the recombinant virus is rAAV. In some embodiments, the rAAV particles contain a capsid protein from an AAV capsid serotype selected from AAV8 and AAV9. In some embodiments, the rAAV particles have the AAV capsid serotype of AAV8. In some embodiments, the rAAV particles have the AAV capsid serotype of AAV9. In some embodiments, the rAAV particles have a capsid serotype selected from the group consisting of AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.PHB, and AAV.7m8. In some embodiments, the rAAV particles have a capsid protein with high sequence homology to AAV8 or AAV9, such as AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, and AAV.hu37.

[0140] In some embodiments, the methods disclosed herein increase the yield of rAAV particles while maintaining or improving the quality attributes of rAAV particles and compositions containing them. In some embodiments, the quality of rAAV particles and compositions containing them is evaluated by quantifying the concentration of rAAV particles (e.g., GC / ml), the percentage of particles containing copies of the rAAV genome, the proportion of particles without the genome, the infectivity of the rAAV particles, the stability of the rAAV particles, and the concentration of residual host cell proteins or residual host cell nucleic acids (e.g., host cell genomic DNA, plasmids encoding rep and cap genes, plasmids encoding helper functions, plasmids encoding the rAAV genome). In some embodiments, the quality of rAAV particles or compositions containing them produced by the methods disclosed herein is the same as the quality of rAAV particles or compositions produced by a reference method comprising a single step of mixing, incubating, and transferring the same volume of polynucleotide:transfer reagent complex. In some embodiments, the quality of rAAV particles or compositions containing the same produced by the methods disclosed herein is better than the quality of rAAV particles or compositions produced by a reference method which involves a single step of mixing, incubating, and transferring the same volume of polynucleotide:transfer reagent complex.

[0141] In some embodiments, the methods disclosed herein produce rAAV particles between approximately 1 × 10⁻¹⁰ GC / ml and approximately 1 × 10⁻¹³ GC / ml. In some embodiments, the methods disclosed herein produce rAAV particles between approximately 1 × 10⁻¹⁰ GC / ml and approximately 1 × 10⁻¹¹ GC / ml. In some embodiments, the methods disclosed herein produce rAAV particles between approximately 5 × 10⁻¹⁰ GC / ml and approximately 1 × 10⁻¹² GC / ml. In some embodiments, the methods disclosed herein produce rAAV particles between approximately 5 × 10⁻¹⁰ GC / ml and approximately 1 × 10⁻¹³ GC / ml. In some embodiments, the methods disclosed herein produce rAAV particles between approximately 1 × 10⁻¹¹ GC / ml and approximately 1 × 10⁻¹³ GC / ml. In some embodiments, the methods disclosed herein produce rAAV particles between approximately 5 × 10⁻¹⁰ GC / ml and approximately 5 × 10⁻¹² GC / ml. In some embodiments, the methods disclosed herein produce rAAV particles between approximately 1 × 10⁻¹¹ GC / ml and approximately 5 × 10⁻¹² GC / ml. In some embodiments, the methods disclosed herein produce rAAV particles greater than approximately 1 × 10⁻¹¹ GC / ml. In some embodiments, the methods disclosed herein produce rAAV particles greater than approximately 5 × 10⁻¹¹ GC / ml. In some embodiments, the methods disclosed herein produce rAAV particles greater than approximately 1 × 10⁻¹² GC / ml. In some embodiments, the rAAV particles contain a capsid protein from an AAV capsid serotype selected from AAV8 and AAV9. In some embodiments, the rAAV particles have the AAV capsid serotype of AAV8. In some embodiments, the rAAV particles have the AAV capsid serotype of AAV9. In some embodiments, the rAAV particles contain a capsid protein from an AAV capsid serotype selected from the group consisting of AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.PHB, and AAV.7m8.In some embodiments, the rAAV particles include capsid proteins with high sequence homology to AAV8 or AAV9, such as AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, and AAV.hu37.

[0142] In some embodiments, the methods disclosed herein produce at least about 5 × 10⁻¹⁰ GC / ml of rAAV particles. In some embodiments, the methods disclosed herein produce at least about 1 × 10⁻¹¹ GC / ml of rAAV particles. In some embodiments, the methods disclosed herein produce at least about 5 × 10⁻¹¹ GC / ml of rAAV particles. In some embodiments, the methods disclosed herein produce at least about 1 × 10⁻¹² GC / ml of rAAV particles. In some embodiments, the methods disclosed herein produce at least about 5 × 10⁻¹² GC / ml of rAAV particles. In some embodiments, the methods disclosed herein produce at least about 1 × 10⁻¹³ GC / ml of rAAV particles. In some embodiments, the methods disclosed herein produce at least about 5 × 10⁻¹³ GC / ml of rAAV particles. In some embodiments, the methods disclosed herein produce at least about 5 × 10⁻¹³ GC / ml of rAAV particles. In some embodiments, the rAAV particles comprise a capsid protein from an AAV capsid serotype selected from AAV8 and AAV9. In some embodiments, the rAAV particles have the AAV capsid serotype of AAV8. In some embodiments, the rAAV particles have the AAV capsid serotype of AAV9. In some embodiments, the rAAV particles contain a capsid protein from an AAV capsid serotype selected from the group consisting of AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.PHB, and AAV.7m8. In some embodiments, the rAAV particles contain a capsid protein with high sequence homology to AAV8 or AAV9, such as AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, and AAV.hu37.

[0143] In the art, numerous cell culture-based systems for the generation of rAAV particles are known, and any of these can be used in carrying out the methods disclosed herein. An rAAV-generating culture for generating rAAV virus particles requires (1) suitable host cells (e.g., human 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 adenoviruses (e.g., temperature-sensitive adenoviruses), herpesviruses, baculoviruses, or plasmid constructs that provide helper functions; (3) AAV rep and cap genes and gene products; (4) transgenes adjacent to the AAV ITR sequence (e.g., therapeutic transgenes); and (5) suitable media and media components to support rAAV generation.

[0144] Those skilled in the art are aware of numerous methods by which rAAV can be generated or packaged by introducing AAV rep and cap genes, AAV helper genes (e.g., adenovirus E1a, E1b, E4, E2a, and VA genes), and rAAV genomes (containing one or more target genes adjacent to reverse terminal repeats (ITRs)) into cells. The expression "adenovirus helper function" means multiple viral helper genes that are expressed intracellularly (as RNA or protein) to enable AAV to grow efficiently within the cell. Those skilled in the art understand that helper viruses, including adenoviruses and herpes simplex viruses (HSV), promote AAV replication, and that certain genes providing essential functions have been identified, and that, for example, helpers can induce changes in the cellular environment that promote such AAV gene expression and replication. In some embodiments of the methods disclosed herein, the AAV rep and cap genes, helper genes, and rAAV genome are introduced into cells by transduction of one or more plasmid vectors encoding the AAV rep and cap genes, helper genes, and rAAV genome.

[0145] Molecular biology techniques for developing plasmids or viral vectors encoding AAV rep and cap genes, helper genes, and / or the rAAV genome are commonly known in the art. In some embodiments, the AAV rep and cap genes are encoded by a single plasmid vector. In some embodiments, AAV helper genes (e.g., adenovirus E1a, E1b, E4, E2a, and VA genes) are encoded by a single plasmid vector. In some embodiments, the E1a or E1b gene is stably expressed by the host cell, and the remaining AAV helper genes are introduced into the cell by transduction with a single viral vector. In some embodiments, the E1a and E1b genes are stably expressed by the host cell, and the E4, E2a, and VA genes are introduced into the cell by transduction with a single plasmid vector. In some embodiments, one or more helper genes are stably expressed by the host cell, and one or more helper genes are introduced into the cell by transduction with a single plasmid vector. In some embodiments, helper genes are stably expressed by host cells. In some embodiments, the AAV rep and cap genes are encoded by a single viral vector. In some embodiments, AAV helper genes (e.g., adenovirus E1a, E1b, E4, E2a, and VA genes) are encoded by a single viral vector. In some embodiments, the E1a or E1b gene is stably expressed by host cells, and the remaining AAV helper genes are introduced into cells by transduction using a single viral vector. In some embodiments, the E1a and E1b genes are stably expressed by host cells, and the E4, E2a, and VA genes are introduced into cells by transduction using a single viral vector. In some embodiments, one or more helper genes are stably expressed by host cells, and one or more helper genes are introduced into cells by transduction using a single viral vector.In some embodiments, the AAV rep and cap genes, the adenovirus helper function required for packaging, and the rAAV genome to be packaged are introduced into cells by transduction using one or more polynucleotides, for example, a vector. In some embodiments, the method disclosed herein involves transduction into cells of a mixture of three polynucleotides (one encoding the cap and rep genes, one encoding the adenovirus helper function required for packaging (e.g., adenovirus E1a, E1b, E4, E2a, and VA genes), and one encoding the rAAV genome to be packaged). In some embodiments, the AAV cap gene is the AAV8 or AAV9 cap gene. In some embodiments, the AAV cap gene is the AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.PHB, or AAV.7m8 cap gene. In some embodiments, the AAV cap gene encodes a capsid protein with high sequence homology to AAV8 or AAV9, such as AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, and AAV.hu37. In some embodiments, the vector encoding the rAAV genome to be packaged contains the target gene adjacent to the AAV ITR.In some embodiments, AAV ITR is AAV1, AAV2, rAAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, 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 It is from .5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, or AAV.HSC16, or another AAV serotype.

[0146] Any combination of vectors can be used to introduce AAV rep and cap genes, AAV helper genes, and the rAAV genome into cells into which rAAV particles are generated or packaged. In some embodiments of the methods disclosed herein, a first plasmid vector encoding the rAAV genome containing a target gene adjacent to an AAV reverse terminal repeat (ITR), a second vector encoding the AAV rep and cap genes, and a third vector encoding the helper gene can be used. In some embodiments, a mixture of the three vectors is simultaneously translocated into the cell.

[0147] In some embodiments, a combination of translocation and infection is used by using a viral vector in conjunction with a plasmid vector.

[0148] In some embodiments, one or more of the rep and cap genes, as well as an AAV helper gene, are constitutively expressed by the cell and do not require transduction or transfection into the cell. In some embodiments, the cell constitutively expresses the rep and / or cap gene. In some embodiments, the cell constitutively expresses one or more AAV helper genes. In some embodiments, the cell constitutively expresses E1a. In some embodiments, the cell contains a stable transgene encoding the rAAV genome.

[0149] In some embodiments, the AAV rep, cap, and helper genes (e.g., Ela gene, E1b gene, E4 gene, E2a gene, or VA gene) can be any AAV serotype. Similarly, the AAV ITR can also be any AAV serotype. For example, in some embodiments, AAV ITR is AAV1, AAV2, rAAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, 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 These are from AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, or AAV.HSC16, or other AAV serotypes (e.g., hybrid serotypes having sequences from two or more serotypes). In some embodiments, the AAV cap gene is from the AAV9 or AAV8 cap gene.In some embodiments, the AAV cap gene is AAV1, AAV2, rAAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, 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 These are from AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, or AAV.HSC16, or other AAV serotypes (e.g., hybrid serotypes having sequences from two or more serotypes). In some embodiments, the AAV rep and cap genes for rAAV particle generation are from different serotypes. For example, the rep gene is from AAV2, while the cap gene is from AAV9.

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

[0151] Recombinant virus (e.g., rAAV) cultures can be routinely grown under a variety of conditions suitable for the specific host cell being used (over a wide temperature range, for varying lengths of time, etc.). As is known in the art, rAAV virus cultures can be adapted to 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, and TCM cells. These include 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 disposable systems such as spinner flasks, agitated tank bioreactors, and wave bag systems. Numerous suspension cultures for generating rAAV particles are known in the art, including, for example, the cultures disclosed in U.S. Patent No. 6,995,006, No. 9,783,826, and U.S. Patent Application Publication No. 20120122155 (each of which is incorporated herein by reference in whole). In some embodiments, the recombinant virus is 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 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 for growth in suspension culture. In some embodiments, the methods disclosed herein use HEK293 cells adapted for growth in suspension culture. In some embodiments, recombinant virus particles are recombinant AAV particles.

[0153] In some embodiments, the cell cultures disclosed herein are suspension cultures. In some embodiments, the large-scale suspension cell cultures disclosed herein contain HEK293 cells adapted for growth in suspension culture. In some embodiments, the cell cultures disclosed herein contain serum-free medium, animal component-free medium, or chemically defined medium. In some embodiments, the cell cultures disclosed herein contain serum-free medium. In some embodiments, suspension-adapted cells are cultured in a shaking flask, spinner flask, cell bag, or bioreactor.

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

[0155] In some embodiments, the large-scale suspension cell cultures disclosed herein include high-density cell cultures. In some embodiments, the culture has a total cell density between about 1 × 10⁻⁶ cells / ml and about 30 × 10⁻⁶ 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 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, It contains capsid proteins from AAV capsid serotypes selected from AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, and AAV.HSC16. In some embodiments, rAAV particles are AAV1, AAV2, rAAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, 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 This includes capsid proteins that are derivatives, modifiers, or pseudotypes of 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 proteins.

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

[0158] In some embodiments, the rAAV particles contain a capsid protein from an AAV capsid serotype selected from the group consisting of AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.PHB, and AAV.7m8. In some embodiments, the rAAV particles contain a capsid protein with high sequence homology to AAV8 or AAV9, such as AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, and AAV.hu37.

[0159] In some embodiments, the rAAV particles contain a capsid protein that is a derivative, modifier, or pseudotype of the AAV8 capsid protein or the AAV9 capsid protein. In some embodiments, the rAAV particles contain a capsid protein that is at least 80% identical to the VP1, VP2, and / or VP3 sequences of the AAV8 capsid protein, for example, 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, etc., i.e., up to 100% identical to the AAV8 capsid protein.

[0160] In some embodiments, the rAAV particles contain a capsid protein that is a derivative, modifier, or pseudotype of the AAV9 capsid protein. In some embodiments, the rAAV particles contain a capsid protein that is at least 80% identical to the VP1, VP2, and / or VP3 sequences of the AAV9 capsid protein, for example, 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, etc., i.e., up to 100% identical to the AAV9 capsid protein.

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

[0162] In additional embodiments, the rAAV particles include a mosaic capsid. In additional embodiments, the rAAV particles include pseudotype rAAV particles. In additional embodiments, the rAAV particles include a capsid containing a capsid protein chimera of two or more AAV capsid serotypes.

[0163] rAAV particles The method provided is suitable for use in the generation of any isolated recombinant AAV particles. Thus, rAAV can be any serotype, modifier, or derivative known in the art, or any combination thereof (e.g., a population of rAAV particles containing two or more serotypes (e.g., two or more of rAAV2, rAAV8, and rAAV9 particles)). In some embodiments, rAAV particles are 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, A 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 of these.

[0164] In some embodiments, rAAV particles are 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 It possesses a capsid protein from an AAV serotype selected from 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 their derivatives, modifications, or pseudotypes.In some embodiments, rAAV particles are, for example, 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, A The capsid protein contains a capsid protein that is at least 80% identical to the VP1, VP2, and / or VP3 sequences of an AAV capsid serotype selected from 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, for example, 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, etc., i.e., up to 100% identical.

[0165] In some embodiments, rAAV particles are 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 This includes capsid proteins from AAV capsid serotypes selected from YF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, or AAV.HSC16, or their derivatives, modifiers, or pseudotypes.In some embodiments, rAAV particles are, for example, 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, A The capsid protein contains a capsid protein that is at least 80% identical to the VP1, VP2, and / or VP3 sequences of an AAV capsid serotype selected from 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, for example, 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, etc., i.e., up to 100% identical.

[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 is incorporated herein by reference in its entirety). In certain embodiments, the rAAV particles comprise a capsid having one of the amino acid inserts: LGETTRP or LALGETTRP, as described in U.S. Patents 9,193,956, 9,458,517, and 9,587,282, and U.S. Patent Application Publication 2016 / 0376323 (each of which is incorporated herein by reference in its entirety). In some embodiments, the rAAV particles include the capsid of AAV.7m8, as described in U.S. Patent Nos. 9,193,956, 9,458,517, and 9,587,282, and U.S. Patent Application Publication No. 2016 / 0376323 (each of which is incorporated herein by reference in whole). In some embodiments, the rAAV particles include any AAV capsid disclosed in U.S. Patent No. 9,585,971, e.g., AAV.PHP.B. In some embodiments, the rAAV particles include any AAV capsid disclosed in U.S. Patent No. 9,840,719 and WO2015 / 013313 (each of which is incorporated herein by reference in whole), e.g., AAV.Rh74 and RHM4-1. In some embodiments, the rAAV particles include any AAV capsid disclosed in WO2014 / 172669 (which is incorporated herein by reference in its entirety), e.g., AAV rh.74. In some embodiments, the rAAV particles include the AAV2 / 5 capsid as described in Georgiadis et al., 2016, Gene Therapy 23:857-862 and Georgiadis et al., 2018, Gene Therapy 25:450 (each of which is incorporated herein by reference in its entirety). In some embodiments, the rAAV particles include any AAV capsid disclosed in WO2017 / 070491 (which is incorporated herein by reference in its entirety), e.g., AAV2tYF.In some embodiments, the rAAV particles include a capsid of AAVLK03 or AAV3B, as described in Puzzo et al., 2017, Sci. Transl. Med. 29(9):418 (each of which is incorporated herein by reference in whole). In some embodiments, the rAAV particles include any AAV capsid disclosed in U.S. Patent Nos. 8,628,966, U.S. 8,927,514, U.S. 9,923,120, and WO2016 / 049230, e.g., HSC1, HSC2, HSC3, HSC4, HSC5, HSC6, HSC7, HSC8, HSC9, HSC10, HSC11, HSC12, HSC13, HSC14, HSC15, or HSC16 (each of which is incorporated herein by reference in whole).

[0167] In some embodiments, rAAV particles are used in the following patents and patent applications (each of which is incorporated herein by reference in whole): U.S. Patent Nos. 7,282,199, 7,906,111, 8,524,446, 8,999,678, 8,628,966, 8,927,514, 8,734,809, U.S. Patent Nos. 9,284,357, 9,409,953, 9,169,299, 9,193, This includes AAV capsids disclosed in any of the following: Patent Nos. 956, 9458517, and 9,587,282; U.S. Patent Application Publications 2015 / 0374803, 2015 / 0126588, 2017 / 0067908, 2013 / 0224836, 2016 / 0215024, 2017 / 0051257; and International Patent Application Nos. PCT / US2015 / 034799 and PCT / EP2015 / 053335. In some embodiments, the rAAV particles have a capsid protein that is at least 80% identical to the VP1, VP2, and / or VP3 sequences of the AAV capsid disclosed in any of the following patents and patent applications (each of which is incorporated herein by reference in whole): for example, 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, etc., i.e., up to 100% identical: U.S. Patents 7,282,199, 7,906,111, 8,524,446, 8,9 U.S. Patent Applications Nos. 99,678, 8,628,966, 8,927,514, 8,734,809, U.S. Patent Nos. 9,284,357, 9,409,953, 9,169,299, 9,193,956, 9,458,517, and 9,587,282, U.S. Patent Application Publication No. U.S.2015 Patent applications No. / 0374803, No. 2015 / 0126588, No. 2017 / 0067908, No. 2013 / 0224836, No. 2016 / 0215024, No. 2017 / 0051257, and international patent applications PCT / US2015 / 034799 and PCT / EP2015 / 053335.

[0168] In some embodiments, rAAV particles are used in International Patent Application Publications WO2003 / 052051 (see, e.g., SEQ ID NO: 2), WO2005 / 033321 (see, e.g., SEQ ID NOs: 123 and 88), WO03 / 042397 (see, e.g., SEQ ID NOs: 2, 81, 85, and 97), WO2006 / 068888 (see, e.g., SEQ ID NOs: 1 and 3-6), WO2006 / 110689 (see, e.g., SEQ ID NOs: 5-38), WO2009 / 1 The capsid protein is disclosed in Patent No. 04964 (see, for example, SEQ ID NOs. 1-5, 7, 9, 20, 22, 24, and 31), WO2010 / 127097 (see, for example, SEQ ID NOs. 5-38), and WO2015 / 191508 (see, for example, SEQ ID NOs. 80-294), and U.S. Patent Application Publication No. 20150023924 (see, for example, SEQ ID NOs. 1, 5-10) (each of which is incorporated herein by reference in its entirety). In some embodiments, the rAAV particles have a capsid protein that is at least 80% identical to the VP1, VP2 and / or VP3 sequences of the AAV capsid disclosed below, e.g., 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, i.e., up to 100% identical: International Patent Application Publication WO2003 / 052051 (see, e.g., SEQ ID NO: 2), WO2005 / 033321 (see, e.g., SEQ ID NOs: 123 and 88), WO03 / 04239 See, for example, Sequence IDs 2, 81, 85, and 97; WO2006 / 068888 (see, for example, Sequence IDs 1 and 3-6); WO2006 / 110689 (see, for example, Sequence IDs 5-38); WO2009 / 104964 (see, for example, Sequence IDs 1-5, 7, 9, 20, 22, 24, and 31); WO2010 / 127097 (see, for example, Sequence IDs 5-38); and WO2015 / 191508 (see, for example, Sequence IDs 80-294); and U.S. Patent Application Publication No. 20150023924 (see, for example, Sequence IDs 1, 5-10).

[0169] Nucleic acid sequences of AAV-based viral vectors, as well as methods for producing recombinant AAV and AAV capsids, are taught, for example, in: U.S. Patents Nos. 7,282,199, 7,906,111, 8,524,446, 8,999,678, 8,628,966, 8,927,514, 8,734,809, U.S. Patents Nos. 9,284,357, 9,409,953, 9,169,299, 9,193,956, 9458517, and 9,587,282, U.S. Patent Application Publications 2015 / 0374803 and 2015 / 0126588. Patent applications No. 2017 / 0067908, No. 2013 / 0224836, No. 2016 / 0215024, No. 2017 / 0051257, International Patent Application No. PCT / US2015 / 034799, No. PCT / EP2015 / 053335, No. WO2003 / 052051, No. WO2005 Patent applications Nos. / 033321, WO03 / 042397, WO2006 / 068888, WO2006 / 110689, WO2009 / 104964, WO2010 / 127097, and WO2015 / 191508, as well as U.S. Patent Application Publication No. 20150023924.

[0170] The provided method is suitable for use in the generation of recombinant AAV encoding a transgene. In certain embodiments, the transgenes are from Tables 1A-1C. In some embodiments, the rAAV genome comprises a vector containing the following components: (1) AAV reverse terminal repeats adjacent to the expression cassette, (2) regulatory elements, e.g., a) promoter / enhancer, b) polyA signal, and c) introns, optionally, and (3) a nucleic acid sequence encoding the transgene. In other embodiments for expressing intact or substantially intact monoclonal antibodies (mAbs), the rAAV genome comprises a vector containing the following components: (1) AAV reverse terminal repeats adjacent to the expression cassette, (2) regulatory elements, e.g., a) promoter / enhancer, b) polyA signal, and c) introns, optionally, and (3) a nucleic acid sequence encoding the light chain Fab and heavy chain Fab of the antibody, or at least the heavy chain or light chain Fab, and optionally the heavy chain Fc region. In yet another embodiment for expressing intact or substantially intact mAbs, the rAAV genome comprises a vector comprising: (1) AAV reverse terminal repeats adjacent to the expression cassette; (2) regulatory elements, e.g., a) promoter / enhancer, b) polyA signaling, and c) introns of choice;(3) Nucleic acid sequences encoding heavy chain Fab of the following: anti-VEGF (e.g., sevacizumab, ranibizumab, bevacizumab, and brolucizumab), anti-EpoR (e.g., LKA-651), anti-ALK1 (e.g., askrinbakumab), anti-C5 (e.g., tesidolumab and eculizumab), anti-CD105 (e.g., carotuximab), anti-CC1Q (e.g., ANX-007), anti-TNFα (e.g., adalimumab, infliximab, and golim) Anti-IL-5 (e.g., mepolizumab), anti-IL-12 / IL-23 (e.g., ustekinumab), anti-IL-19 (e.g., inebilizumab), anti-IL-19 (e.g., inebilizumab), anti-IL-19 (e.g., elezanumab), anti-IL-19 (e.g., lezanumab), anti-IL-19 (e.g., inebilizumab), anti-IL-19 (e.g., inebilizumab), anti-IL-19 (e.g., elezan mAb (e.g., etrolizumab), anti-SOST mAb (e.g., romosozumab), anti-pKal mAb (e.g., lanadelmab), anti-ITGA4 (e.g., natalizumab), anti-ITGA4B7 (e.g., vedolizumab), anti-BLyS (e.g., belimumab), anti-PD-1 (e.g., nivolumab and pembrolizumab), anti-RANKL (e.g., densomab), anti-PCSK9 (e.g., alirocumab and evolocumab), anti-ANGPTL3 (e.g., evinacumab*), anti-OxPL (e.g., E06), anti-fD (e.g., lamparizumab), or anti-MMP9 (e.g., andecaliximab); optionally, an Fc polypeptide of the same isotype as the native form of the therapeutic antibody, e.g., IgG isotype amino acid sequence IgG1, IgG2, or IgG4, or modified Fc thereof;Furthermore, nucleic acid sequences encoding the light chains of the following: anti-VEGF (e.g., sevacizumab, ranibizumab, bevacizumab, and brolucizumab), anti-EpoR (e.g., LKA-651), anti-ALK1 (e.g., askrinbakumab), anti-C5 (e.g., tesidolumab and eculizumab), anti-CD105 or anti-ENG (e.g., carotuximab), anti-CC1Q (e.g., ANX-007), anti-TNFα (e.g., adalimumab, infliximab, and goli Mumab), anti-RGMa (e.g., elezanumab), anti-TTR (e.g., NI-301 and PRX-004), anti-CTGF (e.g., pamlevlumab), anti-IL6R (e.g., satralizumab and sarilumab), anti-IL4R (e.g., dupilumab), anti-IL17A (e.g., ixekizumab and secukinumab), anti-IL-5 (e.g., mepolizumab), anti-IL12 / IL23 (e.g., ustekinumab), anti-CD19 (e.g., inebilizumab), anti-ITGF7 mAbs (e.g., etrolizumab), anti-SOST mAbs (e.g., romosozumab), anti-pKal mAbs (e.g., lanadelmab), anti-ITGA4 (e.g., natalizumab), anti-ITGA4B7 (e.g., vedolizumab), anti-BLyS (e.g., belimumab), anti-PD-1 (e.g., nivolumab and pembrolizumab), anti-RANKL (e.g., densomab), anti-PCSK9 (e.g., alirocumab and evolocumab), anti-ANGPTL3 (e.g., evinacumab), anti-OxPL (e.g., E06), anti-fD (e.g., lampalizumab), or anti-MMP9 (e.g., andecaliximab). At this time, the heavy chain (Fab and optionally the Fc region) and light chain are separated by self-cleaving furin(F) / F2A or a flexible linker to ensure that the heavy chain and light chain polypeptides are expressed in equal amounts.

[0171] [Table 1A] TIFF0007866562000002.tif236165TIFF0007866562000003.tif236165TIFF0007866562000004.tif236165TIFF0007866562000005.tif139165

[0172] Table 1B TIFF0007866562000007.tif214165TIFF0007866562000008.tif143165

[0173]

Table 1C

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

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

[0176] In additional embodiments, the rAAV particles contain a capsid comprising a capsid protein chimera of two or more AAV capsid serotypes. In some embodiments, the capsid proteins are 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, It is a chimera of two or more AAV capsid proteins selected from AAV serotypes: 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 AAVs (ssAAVs) may be used. In certain embodiments, self-complementary vectors, such as scAAVs, may be used (see, for example, Wu, 2007, Human Gene Therapy, 18(2):171-82; McCarty et al, 2001, Gene Therapy, Vol. 8, Number 16:1248-1254; and U.S. Patents 6,596,535, 7,125,717, and 7,456,683 (each of which is incorporated herein by reference in whole)).

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

[0179] In some embodiments, the rAAV particles contain a capsid protein that is a derivative, modifier, or pseudotype of the AAV8 capsid protein or the AAV9 capsid protein. In some embodiments, the rAAV particles contain a capsid protein that is at least 80% identical to the VP1, VP2, and / or VP3 sequences of the AAV8 capsid protein, for example, 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, etc., i.e., up to 100% identical to the AAV8 capsid protein.

[0180] In some embodiments, the rAAV particles contain a capsid protein that is a derivative, modifier, or pseudotype of the AAV9 capsid protein. In some embodiments, the rAAV particles contain a capsid protein that is at least 80% identical to the VP1, VP2, and / or VP3 sequences of the AAV9 capsid protein, for example, 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, etc., i.e., up to 100% identical to the AAV9 capsid protein.

[0181] In additional embodiments, rAAV particles include mosaic capsids. Mosaic AAV particles consist of a mixture of viral capsid proteins from different serotypes of AAV. In some embodiments, rAAV particles include AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B, AA It contains a mosaic capsid containing a capsid protein of a serotype selected from V2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, and AAV.HSC16. In some embodiments, the rAAV particles include a mosaic capsid containing a capsid protein of a serotype selected from AAV1, AAV2, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAVrh.8, AAVrh.10, AAVrh.37, AAVrh.20, and AAVrh.74.

[0182] In additional embodiments, the rAAV particles include pseudotyped rAAV particles. In some embodiments, the pseudotyped rAAV particles include (a) a nucleic acid vector containing AAV ITR, and (b) 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 It contains a capsid composed of capsid proteins derived from HP.B, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, and AAV.HSC16). In additional embodiments, the rAAV particles include pseudotyped rAAV particles composed of capsid proteins of AAV serotypes 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 include pseudotyped rAAV particles containing the AAV8 capsid protein. In additional embodiments, the rAAV particles include pseudotyped rAAV particles composed of the AAV9 capsid protein. 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, for example, Duan et al., J. Virol., 75:7662-7671 (2001); Halbert et al., J. Virol., 74:1524-1532 (2000); Zolotukhin et al., Methods 28:158-167 (2002); and Auricchio et al., Hum. Molec. Genet. 10:3075-3081 (2001)).

[0183] In additional embodiments, the rAAV particles contain a capsid comprising a capsid protein chimera of two or more AAV capsid serotypes. In some embodiments, the capsid proteins include AAV8 capsid protein and AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, and AAV16, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B, A It includes an AAV capsid protein chimera with one or more AAV capsid proteins selected from AAV serotypes selected from AV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, and AAV.HSC16. In some embodiments, the rAAV particles contain an AAV capsid protein chimera, which is an AAV8 capsid protein and one or more AAV capsid proteins from AAV serotypes selected from AAV1, AAV2, AAV5, AAV6, AAV7, AAV9, AAV10, rAAVrh10, AAVrh.8, AAVrh.10, AAVrh.37, AAVrh.20, and AAVrh.74.In some embodiments, rAAV particles contain AAV9 capsid protein and 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 comprises an AAV capsid protein chimera with a capsid protein of one or more AAV capsid serotypes selected from AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, and AAV.HSC16. In some embodiments, the rAAV particles contain an AAV capsid protein chimera, which is an AAV9 capsid protein and a capsid protein of one or more AAV capsid serotypes selected from AAV1, AAV2, AAV3, AAV4, AAV5, AA6, AAV7, AAV8, AAV9, AAVrh.8, AAVrh.10, AAVrh.37, AAVrh.20, and AAVrh.74.

[0184] Method 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 rAAV particles from a feed containing impurities (e.g., an rAAV-producing culture). In some embodiments, the method for producing a formulation comprising isolated recombinant adeno-associated virus (rAAV) particles disclosed herein comprises (a) isolating rAAV particles from a feed containing impurities (e.g., an rAAV-producing culture), and (b) formulation the isolated rAAV particles to produce a formulation.

[0185] In some embodiments, the Disclosure further provides a method for producing a formulation comprising isolated recombinant adeno-associated virus (rAAV) particles in a pharmaceutical unit dose, the method comprising isolating rAAV particles from a feed containing impurities (e.g., an rAAV-producing 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 for isolating rAAV particles includes downstream processing, e.g., collection of cell cultures, clarification of the collected cell cultures (e.g., by centrifugation or deep filtration), tangential flow filtration, affinity chromatography, anion exchange chromatography, cation exchange chromatography, size exclusion chromatography, hydrophobic interaction chromatography, hydroxyl apatite chromatography, sterile filtration, or any combination(s) of these. In some embodiments, the downstream processing includes at least two, at least three, at least four, at least five, or at least six of the following: collection of cell cultures, clarification of the collected cell cultures (e.g., by centrifugation or deep filtration), tangential flow filtration, affinity chromatography, anion exchange chromatography, cation exchange chromatography, size exclusion chromatography, hydrophobic interaction chromatography, hydroxyl apatite chromatography, and sterile filtration. In some embodiments, the downstream process includes collecting the cell culture, clarifying the collected cell culture (e.g., by deep filtration), sterile filtration, tangential flow filtration, affinity chromatography, and anion exchange chromatography. In some embodiments, the downstream process includes clarifying the collected cell culture, sterile filtration, tangential flow filtration, affinity chromatography, and anion exchange chromatography. In some embodiments, the downstream process includes clarifying the collected cell culture by deep filtration, sterile filtration, tangential flow filtration, affinity chromatography, and anion exchange chromatography. In some embodiments, the clarification of the collected cell culture includes sterile filtration. In some embodiments, the downstream process does not include centrifugation. In some embodiments, the rAAV particles contain the AAV8 serotype capsid protein. In some embodiments, the rAAV particles contain the AAV9 serotype capsid protein.

[0187] In some embodiments, a method for isolating rAAV particles produced according to the method disclosed herein includes collecting a cell culture, clarifying the collected cell culture (e.g., by deep filtration), a first sterile filtration, a first tangential flow filtration, affinity chromatography, anion exchange chromatography (e.g., monolithic anion exchange chromatography or AEX chromatography using a quaternary amine ligand), a second tangential flow filtration, and a second sterile filtration. In some embodiments, a method for isolating rAAV particles produced according to the method disclosed herein includes clarification of the collected cell culture, a first sterile filtration, a first tangential flow filtration, affinity chromatography, anion exchange chromatography (e.g., monolithic anion exchange chromatography or AEX chromatography using a quaternary amine ligand), a second tangential flow filtration, and a second sterile filtration.In some embodiments, a method for isolating rAAV particles produced according to the method disclosed herein includes clarification of the collected cell culture by deep filtration, a first sterile filtration, a first tangential flow filtration, affinity chromatography, anion exchange chromatography (e.g., monolithic anion exchange chromatography or AEX chromatography using a quaternary amine ligand), a second tangential flow filtration, and a second sterile filtration. In some embodiments, a method for isolating rAAV particles disclosed herein includes clarification of the collected cell culture by deep filtration, a first sterile filtration, affinity chromatography, anion exchange chromatography (e.g., monolithic anion exchange chromatography or AEX chromatography using a quaternary amine ligand), tangential flow filtration, and a second sterile filtration. In some embodiments, the method does not include centrifugation. In some embodiments, clarification of the collected cell culture includes sterile filtration. In some embodiments, the rAAV particles contain the capsid protein of the AAV8 serotype. In some embodiments, the rAAV particles contain the capsid protein of the AAV9 serotype.

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

[0189] rAAV-producing cultures can be routinely grown under various conditions suitable for the specific host cells being used (over a wide temperature range, over varying lengths of time, etc.). As is known in the art, rAAV-producing cultures include adhesion-dependent cultures that can be cultured in suitable adhesion-dependent containers (e.g., roller bottles, hollow fiber filters, microcarriers, and packed or fluidized bed bioreactors). Furthermore, rAAV vector-producing cultures can be grown with suspension-adaptive host cells, such as HeLa cells, HEK293 cells, HEK293-derived cells (e.g., HEK293T cells, HEK293F cells), Vero cells, CHO cells, CHO-K1 cells, CHO-derived cells, EB66 cells, BSC cells, HepG2 cells, LLC-MK cells, CV-1 cells, COS cells, MDBK cells, MDCK cells, CRFK cells, RAF cells, RK cells, TCMK-1 cells, LLC The cells may also include PK cells, PK15 cells, LLC-RK cells, MDOK cells, BHK cells, BHK-21 cells, NS-1 cells, MRC-5 cells, WI-38 cells, BHK cells, 3T3 cells, 293 cells, RK cells, Per.C6 cells, chicken embryo cells, or SF-9 cells, which can be cultured in a variety of ways, including, for example, spinner flasks, agitated tank bioreactors, and disposable systems such as Wave bag systems. In some embodiments, the cells are HEK293 cells. In some embodiments, the cells are HEK293 cells adapted for growth in suspension culture. Numerous suspension cultures for generating rAAV particles are known in the Art, including, for example, the cultures disclosed in U.S. Patent No. 6,995,006, No. 9,783,826, and U.S. Patent Application Publication No. 20120122155 (each of which is incorporated herein by reference in whole).

[0190] In some embodiments, the rAAV-producing culture comprises a high-density cell culture. In some embodiments, the culture has a total cell density between approximately 1 × 10⁻⁶ cells / ml and approximately 30 × 10⁻⁶ cells / ml. In some embodiments, more than approximately 50% of the cells are viable cells. In some embodiments, the cells are HeLa cells, HEK293 cells, HEK293-derived cells (e.g., HEK293T cells, HEK293F cells), Vero cells, or SF-9 cells. In further embodiments, the cells are HEK293 cells. In further embodiments, the cells are HEK293 cells adapted for growth in suspension culture.

[0191] In additional embodiments of the provided method, the rAAV-producing culture includes a suspension culture containing rAAV particles. Numerous suspension cultures for producing rAAV particles are known in the Art, including, for example, the cultures disclosed in U.S. Patent No. 6,995,006, No. 9,783,826, and U.S. Patent Application Publication No. 20120122155 (each of which is incorporated herein by reference in whole). In some embodiments, the suspension culture includes a culture of mammalian cells or insect cells. In some embodiments, the suspension culture includes cultures of HeLa cells, HEK293 cells, HEK293-derived cells (e.g., HEK293T cells, HEK293F cells), Vero cells, CHO cells, CHO-K1 cells, CHO-derived cells, EB66 cells, BSC cells, HepG2 cells, LLC-MK cells, CV-1 cells, COS cells, MDBK cells, MDCK cells, CRFK cells, RAF cells, RK cells, TCMK-1 cells, LLCPK cells, PK15 cells, LLC-RK cells, MDOK cells, BHK cells, BHK-21 cells, NS-1 cells, MRC-5 cells, WI-38 cells, BHK cells, 3T3 cells, 293 cells, RK cells, Per.C6 cells, chicken embryo cells, or SF-9 cells. In some embodiments, the suspension culture includes a culture of HEK293 cells.

[0192] In some embodiments, a method for generating rAAV particles comprises preparing a cell culture containing cells capable of generating rAAV, adding a histone deacetylase (HDAC) inhibitor to the cell culture to a final concentration between about 0.1 mM and about 20 mM, and maintaining the cell culture under conditions that enable the generation of rAAV particles. In some embodiments, the HDAC inhibitor comprises a short-chain fatty acid or a salt thereof. In some embodiments, the HDAC inhibitor comprises butyric acid (e.g., sodium butyrate), valproic acid (e.g., sodium valproate), propionic acid (e.g., sodium propionate), or a combination thereof.

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

[0194] Recombinant AAV particles can be collected from rAAV-producing cultures by collecting the productive culture containing host cells, or by collecting the consumed medium from the productive culture, provided that the cells are cultured under conditions known in the art to induce the release of rAAV particles from intact host cells into the culture medium. Recombinant AAV particles can also be collected from rAAV-producing cultures by lysing the host cells of the productive culture. Suitable methods for lysing cells are also known in the art, and include, for example, multiple freeze / thaw cycles, sonication, microfluidization, and treatment with chemicals (e.g., surfactants and / or proteases).

[0195] At collection, rAAV-producing cultures may contain one or more of the following: (1) host cell proteins; (2) host cell DNA; (3) plasmid DNA; (4) helper viruses; (5) helper virus proteins; (6) helper virus DNA; and (7) culture medium components (e.g., serum proteins, amino acids, transferrin, and other low molecular weight proteins). rAAV-producing cultures may also contain product-related impurities, such as inactive vector forms, empty viral capsids, aggregated viral particles or capsids, misfolded viral capsids, and degraded viral particles.

[0196] In some embodiments, the rAAV-producing culture collection is clarified to remove host cell debris. In some embodiments, the producing culture collection is clarified by filtration through a series of deep filters. Clarification can also be achieved by various other standard techniques known in the art, for example, by centrifugation or by filtration through any cellulose acetate filter with a pore size of 0.2 mm or larger known in the art. In some embodiments, clarification of the collected cell culture includes sterile filtration. In some embodiments, the producing culture collection is clarified by centrifugation. In some embodiments, clarification of the producing culture collection does not include centrifugation.

[0197] In some embodiments, the collected cell culture is clarified using filtration. In some embodiments, the clarification of the collected cell culture includes deep filtration. In some embodiments, the clarification of the collected cell culture further includes deep filtration and sterile filtration. In some embodiments, the collected cell culture is clarified using a filter train containing one or more different filtration media. In some embodiments, the filter train contains one deep filtration medium. In some embodiments, the filter train contains one or more deep filtration media. In some embodiments, the filter train contains two deep filtration media. In some embodiments, the filter train contains one sterile filtration medium. In some embodiments, the filter train contains two deep filtration media and one sterile filtration medium. In some embodiments, the deep filtration medium is a porous deep filter. In some embodiments, the filter train contains Clarisolve® 20MS, Millistak+® C0HC, and sterile-grade filtration media. In some embodiments, the filter train includes Clarisolve® 20MS, Millistak+® C0HC, and Sartopore® 2 XLG 0.2 μm. In some embodiments, the collected cell culture is pretreated before contact with the deep filter. In some embodiments, the pretreatment includes adding salt to the collected cell culture. In some embodiments, the pretreatment includes adding a chemical flocculant to the collected cell culture. In some embodiments, the collected cell culture is not pretreated before contact with the deep filter.

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

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

[0200] Aseptic filtration encompasses filtration using a sterile-grade filter medium. In some embodiments, the sterile-grade filter medium is a 0.2 or 0.22 μm pore filter. In some embodiments, the sterile-grade filter medium contains polyethersulfone (PES). In some embodiments, the sterile-grade filter medium contains polyvinylidene fluoride (PVDF). In some embodiments, the sterile-grade filter medium has a hydrophilic heterogeneous double-layer design. In some embodiments, the sterile-grade filter medium has a hydrophilic heterogeneous double-layer design of a 0.8 μm pre-filter and a 0.2 μm final filter membrane. In some embodiments, the sterile-grade filter medium has a hydrophilic heterogeneous double-layer design of a 1.2 μm pre-filter and a 0.2 μm final filter membrane. In some embodiments, the sterile-grade filter medium is a 0.2 or 0.22 μm pore filter. In further embodiments, the sterile-grade filter medium is a 0.2 μm pore filter. In some embodiments, the sterile-grade filter medium is a combination of nominal pore sizes of Sartopore® 2 XLG 0.2 μm, Durapore® PVDF membrane 0.45 μm, or Sartoguard® PES 1.2 μm + 0.2 μm. In some embodiments, the sterile-grade filter medium is Sartopore® 2 XLG 0.2 μm.

[0201] In some embodiments, the clarified feed is concentrated via tangential flow filtration ("TFF") before being applied to a chromatographic medium, such as an affinity chromatography medium. Large-scale concentrations of viruses using TFF ultrafiltration are described in Paul et al., Human Gene Therapy 4:609-615 (1993). The TFF concentration of the clarified feed allows for chromatographic application of technically controllable amounts of the clarified feed and enables more rational column sizing without requiring long recirculation times. In some embodiments, the clarified feed is concentrated between at least 2x and at least 10x. In some embodiments, the clarified feed is concentrated between at least 10x and at least 20x. In some embodiments, the clarified feed is concentrated between at least 20x and at least 50x. In some embodiments, the clarified feed is concentrated to about 20x. Those skilled in the art will also recognize that TFF may be used to remove small molecule impurities (e.g., cell culture impurities including culture medium components, serum albumin, or other serum proteins) from a feed clarified via diafiltration. In some embodiments, the clarified feed is subjected to diafiltration to remove small molecule impurities. In some embodiments, the diafiltration includes using a diafiltration volume of buffer between about 3 and about 10. In some embodiments, the diafiltration includes using a diafiltration volume of buffer between about 5. Those skilled in the art will also recognize that TFF may be used at any step of the purification process where it is desirable to exchange the buffer before carrying out the next step in the purification process. In some embodiments, the method for isolating rAAV from a clarified feed disclosed herein includes the use of TFF for buffer exchange.

[0202] Affinity chromatography can be used to isolate rAAV particles from a composition. In some embodiments, affinity chromatography is used to isolate rAAV particles from a clarified feed. In some embodiments, affinity chromatography is used to isolate rAAV particles from a feed that has been clarified by tangential flow filtration. Suitable affinity chromatography media are known in the art and are not limited to AVB Sepharose®, POROS® CaptureSelect® AAVX affinity resin, POROS® CaptureSelect® AAV9 affinity resin, and POROS® CaptureSelect® AAV8 affinity resin. In some embodiments, the affinity chromatography medium is POROS® CaptureSelect® AAV9 affinity resin. In some embodiments, the affinity chromatography medium is POROS® CaptureSelect® AAV8 affinity resin. In some embodiments, the affinity chromatography medium is POROS® CaptureSelect® AAVX affinity resin.

[0203] Anion exchange chromatography can be used to isolate rAAV particles from a composition. In some embodiments, anion exchange chromatography is used after affinity chromatography as a final concentration and polishing step. Suitable anion exchange chromatography media are known in the art and are not limited to, but include UNOsphere® Q (Biorad, Hercules, Calif.) and N-charged amino or imino resins, e.g., POROS® 50 PI, or any DEAE, TMAE, tertiary or quaternary amine, or PEI-based resins known in the art (U.S. Patent No. 6,989,264; Brument et al., Mol. Therapy 6(5):678-686 (2002); Gao et al., Hum. Gene Therapy 11:2079-2091 (2000)). In some embodiments, the anion exchange chromatography media contains a quaternary amine. In some embodiments, the anion exchange medium is a monolithic anion exchange chromatography resin. In some embodiments, the monolithic anion exchange chromatography medium comprises a glycidyl methacrylate-ethylenedimethacrylate polymer or a styrene-divinylbenzene polymer. In some embodiments, the monolithic anion exchange chromatography medium is selected from the group consisting of CIMmultus® QA-1 advanced composite column (quaternary amine), CIMmultus® DEAE-1 advanced composite column (diethylamino), CIM® QA disc (quaternary amine), CIM® DEAE, and CIM® EDA disc (ethylenediamino). In some embodiments, the monolithic anion exchange chromatography medium is a CIMmultus® QA-1 advanced composite column (quaternary amine). In some embodiments, the monolithic anion exchange chromatography medium is a CIM® QA disc (quaternary amine). In some embodiments, the anion exchange chromatography medium is CIM QA (BIA Separations, Slovenia).In some embodiments, the anion exchange chromatography medium is BIA CIM® QA-80 (column volume 80 mL). Those skilled in the art will understand that a wash buffer of suitable ionic strength can be identified so that impurities (including, but not limited to, impurities introduced by upstream purification steps) are removed while the rAAV maintains its binding to the resin.

[0204] In some embodiments, anion exchange chromatography is carried out according to the method disclosed in WO2019 / 241535 (which is incorporated herein by reference in its entirety).

[0205] In some embodiments, a method for isolating rAAV particles includes quantifying the vector genome titer, capsid titer, and / or the complete capsid:empty capsid ratio in a composition containing the isolated rAAV particles. In some embodiments, the vector genome titer is quantified by quantitative PCR (qPCR), digital PCR (dPCR), or droplet digital PCR (ddPCR). In some embodiments, the capsid titer is quantified by serotype-specific ELISA. In some embodiments, the complete 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 complete capsid:empty capsid ratio are quantified by spectrophotometric measurement, for example, by measuring the absorbance of the composition at 260 nm and at 280 nm. In some embodiments, the rAAV particles are not denatured before measuring the absorbance of the composition. In some embodiments, the rAAV particles are denatured before 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 peak absorbance. Several methods for measuring the absorbance of the composition at 260 nm and 280 nm are known in the art. A method for quantifying the vector genome titer and capsid titer of a composition containing isolated recombinant rAAV particles is disclosed in WO2019 / 212922 (which is incorporated herein by reference in its entirety).

[0207] In additional embodiments, the disclosure provides compositions comprising isolated rAAV particles produced according to the methods disclosed herein. In some embodiments, the compositions are pharmaceutical compositions comprising a pharmaceutically acceptable carrier.

[0208] As used herein, the term “pharmaceutically acceptable” means a bioacceptable formulation, gas, liquid, or solid, or mixture thereof, suitable for one or more routes of administration, in vivo delivery, or contact. A “pharmaceutically acceptable” composition is a material that is not biologically or otherwise undesirable, for example, that can be administered to a subject without causing substantially undesirable biological effects. Such a pharmaceutical composition can therefore be used, for example, when administering rAAV isolated according to the methods of this disclosure to a subject. Such compositions include solvents (aqueous or non-aqueous), solutions (aqueous or non-aqueous), emulsions (e.g., oil in water or water in oil), suspensions, syrups, elixirs, dispersions and suspension media, coatings, isotonic and absorption enhancers or retarders, which are suitable for pharmaceutical administration or in vivo contact or delivery. Aqueous and non-aqueous solvents, solutions, and suspensions may include suspending agents and thickeners. Such pharmaceutically acceptable carriers include tablets (coated or uncoated), capsules (hard or soft), microbeads, powders, granules, and crystals. Complementary active compounds (e.g., preservatives, antimicrobial agents, antiviral agents, and antifungal agents) can also be incorporated into the composition. Pharmaceutical compositions can be formulated to be compatible with specific routes of administration or delivery, as described herein or as known to those skilled in the art. Therefore, pharmaceutical compositions may include carriers, diluents, or excipients suitable for administration via various routes.The rAAV particles, method, and suitable pharmaceutical compositions and delivery systems of the present invention are known in the art (for example, Remington: The Science and Practice of Pharmacy (2003) 20th ed., Mack Publishing Co., Easton, Pa.; Remington's Pharmaceutical Sciences (1990) 18th ed., Mack Publishing Co., Easton, Pa.; The Merck Index (1996) 12th ed., Merck Publishing Group, Whitehouse, NJ; Pharmaceutical Principles of Solid Dosage Forms (1993), Technonic Publishing Co., Inc., Lancaster, Pa.; Ansel and Stoklosa, Pharmaceutical Calculations (2001) 11th ed., Lippincott Williams & Wilkins, Baltimore, Md.; and Poznansky et al., Drug Delivery Systems). (See 1980, RLJuliano, ed., Oxford, NY, pp. 253–315).

[0209] In some embodiments, the composition is a pharmaceutical unit dose. “Unit dose” means a physically separate unit suitable as a unit drug dose for the target being treated. Each unit contains a predetermined amount, optionally together with a pharmaceutical carrier (excipient, diluent, vehicle, or filler), and is calculated to produce a desired effect (e.g., prophylactic or therapeutic effect) when administered in one or more doses. Unit dosage forms may be, for example, in ampoules and vials, and these may include liquid compositions or compositions in a freeze-dried or lyophilized state, for example, by adding a sterile liquid carrier before in vivo administration or delivery. Individual unit dosage forms may be included in multi-dose kits or containers. Recombinant vectors (e.g., AAV) sequences, plasmids, vector genomes, and recombinant viral particles, as well as these pharmaceutical compositions, may be packaged in single or multiple unit dose forms to facilitate administration and ensure uniform drug dose. In some embodiments, the compositions 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, AAV2.5, A The rAAV particles contain AAV capsid proteins from AAV capsid serotypes selected from AV2tYF, 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] Method for generating recombinant polypeptides In one embodiment, the Disclosure provides a method for producing recombinant polypeptides, comprising: (a) preparing a cell culture containing cells suitable for the production of recombinant polypeptides, wherein the culture contains dextran sulfate in a concentration between about 0.1 mg / L and about 10 mg / L; (b) translocating cells to the culture from a) by adding a composition containing one or more polynucleotides encoding a polypeptide and a translocation reagent; and (c) maintaining the cell culture containing the translocated cells under conditions that enable the production of recombinant polypeptides.

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

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

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

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

[0215] In some embodiments, the initial dextran sulfate concentration is between approximately 1 mg / L and approximately 10 mg / L, between approximately 1 mg / L and approximately 5 mg / L, between approximately 2 mg / L and approximately 10 mg / L, between approximately 3 mg / L and approximately 10 mg / L, or between approximately 3 mg / L and approximately 5 mg / L of dextran sulfate. In some embodiments, the initial dextran sulfate concentration is between approximately 1 mg / L and approximately 10 mg / L of dextran sulfate. In some embodiments, the initial dextran sulfate concentration is between approximately 2 mg / L and approximately 10 mg / L of dextran sulfate. In some embodiments, the initial dextran sulfate concentration is between approximately 3 mg / L and approximately 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 of dextran sulfate. In some embodiments, the starting dextran sulfate concentration is about 2 mg / L of dextran sulfate. In some embodiments, the starting dextran sulfate concentration is about 3 mg / L of dextran sulfate. In some embodiments, the starting dextran sulfate concentration is about 4 mg / L of dextran sulfate. In some embodiments, the starting dextran sulfate concentration is about 5 mg / L of dextran sulfate. In some embodiments, the starting dextran sulfate concentration is about 6 mg / L of dextran sulfate. In some embodiments, the starting dextran sulfate concentration is about 7 mg / L of dextran sulfate. In some embodiments, the starting dextran sulfate concentration is about 8 mg / L of dextran sulfate.

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

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

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

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

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

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

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

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

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

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

[0227] The term "antibody fragment" refers to a portion of an intact antibody, or any functional domain of the antibody (e.g., an antigen-binding fragment or its single chain, an effector domain or a 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, "antibody fragment" includes an antigen-binding site or an epitope-binding site.

[0228] As used herein, the terms “Fc region” or simply “Fc” should be understood to mean the carboxyl terminus or portion thereof of the constant region of an immunoglobulin chain, preferably the constant region of an immunoglobulin heavy chain. For example, an immunoglobulin Fc region may 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 subclass 1, 2, 3, or 4). Other classes of immunoglobulins, IgA (Igα), IgD (Igδ), IgE (Igε), and IgM (Igμ), may also be used. To achieve specific results, selecting a specific immunoglobulin heavy chain constant region sequence from a particular class and subclass of immunoglobulin is considered to be within the scope of the art. In some embodiments, a portion of the DNA construct encoding the immunoglobulin Fc region preferably includes at least a portion of the hinge domain, and preferably at least a portion of the CH3 domain of Fc gamma or a homologous domain of IgA, IgD, IgE, or IgM. Furthermore, amino acid substitutions or deletions within the immunoglobulin heavy chain constant region are intended to be useful in carrying out the methods and compositions disclosed herein. For example, an amino acid substitution can be introduced into the upper CH2 region to create an Fc variant with reduced affinity for the Fc receptor (Cole, J. Immunol. 159:3613 (1997)).

[0229] Various recombinant expression systems suitable for generating recombinant polypeptides within specific host cells are known to those skilled in the art. It should be understood that any recombinant expression system can be used for the generation of recombinant polypeptides according to the methods disclosed herein.

[0230] Any suitable translocation reagent known in the art for translocation into cells can be used in the production of recombinant polypeptides according to the methods disclosed herein. In some embodiments, the translocation reagent comprises a cationic organic carrier. See, for example, Gigante et al., Medchemcomm 10(10):1692-1718(2019); Damen et al., Medchemcomm 9(9):1404-1425(2018) (each of these is incorporated herein by reference in whole). In some embodiments, the cationic organic carrier comprises lipids, e.g., DOTMA, DOTAP, helper lipids (Dope, cholesterol), and combinations thereof. In some embodiments, the cationic organic carrier comprises polyvalent cationic lipids, e.g., DOSPA, DOGS, and mixtures thereof. In some embodiments, the cationic organic carrier comprises bipolar lipids, or bora amphiphile (bola). In some embodiments, the cationic organic carrier comprises a biologically reducing and / or dimerizable lipid. In some embodiments, the cationic organic carrier comprises a gemin 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 polymers, e.g., 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 includes peptides, such as basic amino acid-rich peptides (CWL18), cell-permeable peptides (CPP) (Arg-rich peptides (octaarginine, TAT)), nuclear localization signals (NLS) (SV40), and targeting (RGD).In some embodiments, the cationic organic carrier comprises a polymer (e.g., PEI) combined with a cationic liposome. Paris et al., Molecules 25(14):3277(2020) (the entire text is incorporated herein by reference). In some embodiments, the translocation reagent comprises calcium phosphate, a highly branched organic compound (dendrimer), a cationic polymer (e.g., DEAE dextran or polyethyleneimine (PEI)), and lipofection.

[0231] In some embodiments, the translocation 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 translocation reagent comprises polyethyleneimine (PEI), linear PEI, branched PEI, or mixtures thereof. In some embodiments, the translocation reagent comprises polyethyleneimine (PEI). In some embodiments, the translocation reagent comprises linear PEI. In some embodiments, the translocation reagent comprises branched PEI. In some embodiments, the translocation reagent comprises polyethyleneimine (PEI) having a molecular weight between about 5 and about 25 kDa. In some embodiments, the translocation reagent comprises PEGylated polyethyleneimine (PEI). In some embodiments, the translocation reagent comprises modified polyethyleneimine (PEI) to which hydrophobic moieties (e.g., cholesterol, choline, alkyl groups, and several amino acids) are attached.

[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 cells include mammalian cells or insect cells. In some embodiments, the cells include mammalian cells. In some embodiments, the cells include HEK293 cells, HEK-derived cells, CHO cells, CHO-derived cells, HeLa cells, SF-9 cells, BHK cells, Vero cells, and / or PerC6 cells. In some embodiments, the cells include HEK293 cells.

[0234] In some embodiments, the cells include suspension-adaptive cells. In some embodiments, the cells include suspension-adaptive 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 include 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 include suspension-adapted HEK293 cells. In some embodiments, the cells include suspension-adapted CHO cells.

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

[0236] While not bound by any particular theory, the methods disclosed herein increase the efficiency of translocation, resulting in cells translocated according to the methods disclosed herein producing more recombinant polypeptides than control cells translocated in cell cultures without 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 polypeptides than a control method using cell cultures without 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 the catalytic activity of recombinantly expressed polypeptides).

[0237] In some embodiments, the methods for producing recombinant polypeptides disclosed herein further include isolating the polypeptides. Various methods for isolating recombinant expression polypeptides are known to those skilled in the art. It should be understood that any known method for isolating recombinant expression polypeptides can be used in accordance with the methods disclosed herein. In some embodiments, the method for isolating recombinant expression polypeptides includes collecting cell cultures, clarifying the collected cell cultures (e.g., by centrifugation or deep filtration), tangential flow filtration, affinity chromatography, anion exchange chromatography, cation exchange chromatography, size exclusion chromatography, hydrophobic interaction chromatography, hydroxyl apatite chromatography, sterile filtration, or any combination(s) of these. In some embodiments, the downstream process includes collecting cell cultures, clarifying the collected cell cultures (e.g., by centrifugation or deep filtration), tangential flow filtration, affinity chromatography, anion exchange chromatography, cation exchange chromatography, size exclusion chromatography, hydrophobic interaction chromatography, hydroxyl apatite chromatography, and sterile filtration, at least two, at least three, at least four, at least five, or at least six of these. [Examples]

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

[0239] Despite the teaching that dextran sulfate inhibits translocation, the inventors investigated the effect of dextran sulfate on AAV titer in a transient translocation-based AAV production system. Recombinant AAV was produced by transient translocation of HEK293 cells. Briefly, HEK293 cells were grown in a 250 ml shaking flask for 48 hours in a medium containing 0.3–10 mg / L of dextran sulfate. Polyethyleneimine (PEI) and a mixture of three plasmids encoding adenovirus helper function, transgenes, and AAV Cap / Rep were translocated into the cells. The translocated culture was maintained for 5 days after translocation to enable AAV production. The AAV titer in the culture supernatant was quantified using a PCR-based method. The titers obtained using 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 an increase in AAV titer. This finding is unexpected given the clear prior art instruction that dextran sulfate inhibits transient translocation, but it is consistent with the finding that dextran sulfate at concentrations of 10 mg / L or higher (Figure 1) inhibited AAV production. Dextran sulfate at a concentration of 0.313 mg / L had no significant effect on AAV titer (Figure 1).

[0240] Example 2: Effect of dextran sulfate on AAV titer in a bench-scale 2L reactor. The effect of dextran sulfate on transfusion-based AAV generation was investigated in a bench-scale reactor. Transient transfusion of HEK293 cells generated recombinant AAV8 containing transgene 2. Briefly, HEK293 cells were grown in a 2L reactor for 3 days in a medium containing various concentrations of dextran sulfate. Polyethyleneimine (PEI) and a mixture of three plasmids encoding adenovirus helper function, transgene, and AAV Cap / Rep were transfused into the cells. The transfused culture was maintained for 4 days after transfusion to enable AAV generation. AAV particles were collected from the culture supernatant or from the culture after cell lysis (Figure 3). Cell density and cell viability were quantified daily (Figures 5 and 6). Cell morphology was evaluated on day 4 (Figure 4). A dextran sulfate concentration range of 2.5–4.2 mg / L did not inhibit translocation in a 2 L reactor and was beneficial to cell morphology, including increased viability and live cell density.

[0241] Example 3: Effect of dextran sulfate on AAV titer in a bench-scale 5L reactor. The effect of dextran sulfate on transfusion-based AAV generation was investigated in a bench-scale reactor. Transient transfusion of HEK293 cells generated recombinant AAV8 containing transgene 2. Briefly, HEK293 cells were grown in a 5L reactor for 3 days in a medium containing 4 mg / L dextran sulfate. Prior to transfusion, the culture was diluted 1:1 with fresh medium to obtain a 2 mg / L dextran sulfate concentration. Transfusion was performed into the cells using a mixture of polyethyleneimine (PEI) and three plasmids encoding adenovirus helper function, transgene, and AAV Cap / Rep. The transfused culture was maintained for 4 days after transfusion to allow AAV generation. AAV particles were recovered from the culture supernatant or from the culture after cell lysis. The AAV supernatant or lysis titer increased by an average of 35–40% with the addition of dextran sulfate. Figure 7.

[0242] Example 4: Effect of dextran sulfate on AAV titer in different culture media. The effect of dextran sulfate on translocation-based AAV generation was investigated in different commercially available culture media (M1, M2, and M3 in Figure 8). Transient translocation of HEK293 cells generated recombinant AAV8 containing transgene 2. Briefly, HEK293 cells were grown in different culture media containing 4 mg / L dextran sulfate in a 2 L reactor for 3 days. Before translocation, the culture was diluted 1:1 with fresh medium to obtain a 2 mg / L dextran sulfate concentration. Polyethyleneimine (PEI) and a mixture of three plasmids encoding adenovirus helper function, transgene, and AAV Cap / Rep were translocated into the cells. The translocated culture was maintained for 4 days after translocation to allow AAV generation. AAV particles were collected from the culture supernatant or from the culture after cell lysis. Figure 8. In M1, M2, and M3 culture media, the inclusion of dextran sulfate in the culture medium increased the titer recovered from cell lysis by 25%, 130%, and 10%, respectively.

[0243] Example 5: Effect of dextran sulfate on AAV titer when using different host cell clones. The effect of dextran sulfate on transfusion-based AAV generation was investigated using different HEK293 host cell clones. Transient transfusion of different HEK293 cell clones generated recombinant AAV8 containing transgene 2. Briefly, HEK293 cell clones were grown in a culture medium containing 4 mg / L dextran sulfate in a shaking flask for 3 days. Before transfusion, the culture was diluted 1:1 with fresh medium to obtain a dextran sulfate concentration of 2 mg / L. Polyethyleneimine (PEI) and a mixture of three plasmids encoding adenovirus helper function, transgene, and AAV Cap / Rep were transfused into the cells. The transfused culture was maintained for 4 days after transfusion to allow AAV generation. AAV particles were collected from the culture supernatant. Figure 9. AAV8 titer increased with the inclusion of dextran sulfate in all five HEK cell clones examined. AAV8 titers increased by an average of 18% in five different HEK cell clones upon inclusion of dextran sulfate.

[0244] Example 6: Effect of dextran sulfate on AAV9 titer in a bench-scale 5L reactor. The effect of dextran sulfate on transfusion-based AAV9 generation was investigated in a bench-scale reactor. Transient transfusion of HEK293 cells generated recombinant AAV9 containing transgene 3. Briefly, HEK293 cells were grown in a medium containing 4 mg / L dextran sulfate in a 5 L reactor for 3 days. Before transfusion, the culture was diluted 1:1 with fresh medium to obtain a dextran sulfate concentration of 2 mg / L. Polyethyleneimine (PEI) and a mixture of three plasmids encoding adenovirus helper function, transgene, and AAV Cap / Rep were transfused into the cells. The transfused culture was maintained for 5 days after transfusion to allow AAV generation. Figure 10. The titer of the AAV9 supernatant increased by an average of 30% with the inclusion of dextran sulfate.

[0245] Example 7: Effect of dextran sulfate on AAV titer when used both in the seed cell train before translocation and during the culture stage (transgene 3). Recombinant AAV9 containing transgene 3 was generated by transient transfusion of HEK293 cells in a 200L culture. HEK cells were propagated using a seed train that included a high-density perfusion culture step in the presence of 4 mg / L dextran sulfate. HEK seed cells were inoculated into a 200L culture, and the dextran sulfate concentration was adjusted to 2 mg / L in the culture before transfusion. Polyethyleneimine (PEI) and a mixture of three plasmids encoding adenovirus helper function, transgene, and AAV Cap / Rep were transfused into the cells. The transfused culture was maintained for 5 days after transfusion to allow AAV generation. AAV particles were collected from either the culture supernatant (black bars in Figure 11) or the culture after cell lysis (gray bars in Figure 11). HEK seed cells propagated in the absence of dextran sulfate were inoculated into the control culture. Figure 11. When dextran sulfate was used both during seed cell proliferation and during translocation of the resulting culture, the AAV9 titer increased by 30%.

[0246] Example 8: Effect of dextran sulfate on AAV titer when used both in the seed train before translocation and during the production culture (transgene 1). The effect of dextran sulfate in a seed train on translocation-based AAV generation was investigated in a bench-scale reactor. Transient translocation of HEK293 cells generated recombinant AAV8 containing transgene 1. Briefly, HEK293 cells were propagated for 5 passages (18 days) in medium with or without dextran sulfate. The cells were then propagated for 3 days in a triple-repeat 2L reactor in medium with or without 4 mg / L dextran sulfate (seed train). Before translocation, the cultures were diluted 1:1 with fresh medium to obtain dextran sulfate concentrations of 2 mg / L or 0, respectively. Polyethyleneimine (PEI) and a mixture of three plasmids encoding adenovirus helper function, transgene, and AAV Cap / Rep were translocated into the cells. The translocated cultures were maintained for 4 days after translocation to allow AAV generation. AAV particles were recovered from the culture after cell lysis. The AAV solubility titer increased by an average of 10–15% when dextran sulfate was included in the seed train and the resulting culture (statistically significant difference, p<0.05). Figure 12.

[0247] While the methods described herein have been explained in conjunction with what is considered to be the most practical and preferred embodiments, it should be understood that the methods encompassed herein are not to be limited to the disclosed embodiments, but rather are intended to cover a variety of modifications and equivalent arrangements that fall within the spirit and scope of the appended claims.

[0248] All publications, patents, patent applications, internet sites, and accession number / database sequences (including both polynucleotide and polypeptide sequences) cited herein are incorporated herein by reference in whole for any purpose to the same extent that each individual publication, patent, patent application, internet site, or accession number / database sequence is incorporated by reference specifically and individually.

Claims

1. A method for transmitter transfer into cells, a) Preparing a suspension cell culture containing the aforementioned cells, wherein the culture contains dextran sulfate in a concentration of 1 mg / L to 3 mg / L, b) Translocation into the cells by adding a composition containing one or more polynucleotides and a translocation reagent to the culture of (a) Includes, The aforementioned translocation reagent contains a polymer. The aforementioned method.

2. A method for generating recombinant polypeptides, a) Preparing a suspension cell culture containing cells suitable for the production of the recombinant polypeptide, wherein the culture contains dextran sulfate in a concentration of 1 mg / L to 3 mg / L, b) Transplanting the polypeptide into the cells by adding a composition containing one or more polynucleotides encoding the polypeptide and a transtransfer reagent to the culture of a), c) Maintaining the cell culture containing the transfused cells under conditions that enable the production of the recombinant polypeptide. Includes, The aforementioned translocation reagent contains a polymer. The aforementioned method.

3. The method according to 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.

4. A method for generating recombinant virus particles, a) Preparing a suspension cell culture containing cells suitable for generating the recombinant virus particles, wherein the culture contains dextran sulfate in a concentration of 1 mg / L to 3 mg / L, b) Transtransferring the virus into the cells by adding a composition containing one or more polynucleotides containing genes necessary for generating the recombinant virus particles and a transtransfer reagent to the culture of a), c) Maintaining the cell culture containing the transfused cells under conditions that enable the generation of the recombinant virus particles. Includes, The aforementioned translocation reagent contains a polymer. The aforementioned method.

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

6. The method according to claim 4 or 5, wherein the recombinant virus particles are rAAV particles.

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. The method according to claim 6, comprising a capsid protein of serotype 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.

8. The one or more polynucleotides a) rAAV genome to be packaged, b) Adenovirus helper function necessary for packaging, c) Sufficient AAV rep protein for packaging, and d) Sufficient AAV cap protein for packaging The method according to claim 6 or 7, which codes for...

9. The method according to claim 8, 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 function.

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

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

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

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

14. The method according to any one of claims 1 to 12, wherein the cells include 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.

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

16. The method according to any one of claims 1 to 12, wherein the cells include CHO cells or CHO-K1 cells.

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

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

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

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

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