Compositions and methods for recombinant AAV production

Abstract

This specification provides host cells suitable for use in the production of recombinant AAV particles and polypeptides. This specification also provides methods for producing rAAV particles and polypeptides.

Description

Technical Field

[0001] The present disclosure relates to host cells and their use in methods for generating recombinant adeno-associated virus (rAAV) particles and polypeptides.

[0002] Cross-reference to Related Applications This application claims the benefit of U.S. Patent Application No. 63 / 464,619, filed May 7, 2023, 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 and safest gene therapy products in development. Naso et al., BioDrugs 31:317-334 (2017) (Non-Patent Document 1). The use of rAAV vector systems is preferred, in part, because there are no diseases associated with wild-type viruses, AAV transduction is possible in both non-dividing and dividing cells, and long-term robust transgene expression has been observed in clinical trials, indicating 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. However, in addition to the limitation of AAV yield due to relatively low process efficiency, higher doses are required in clinical trials, restricting the wider application of AAV in common diseases such as cancer. There is a need to develop new methods for large-scale production of recombinant virus particles before recombinant virus-based gene therapy can be more widely adopted in late clinical stages and commercial use.

[0004] HEK293 cells are the most widely used cells for recombinant AAV generation. The most common platform involves transtransferring HEK293 cells with three plasmids. One plasmid, often referred to as the trans plasmid, contains the Rep and Cap genes and encodes proteins for viral replication and capsid formation. A second plasmid, often referred to as the helper plasmid, encodes essential adenovirus helper genes (E4, E2A, and virus-associated (VA)RNA). A third plasmid, often referred to as the cis plasmid, contains an expression cassette encoding the target gene flanked by two reverse terminal repeats (ITRs), which is incorporated into rAAV as its genome. In other platforms, HEK293 cells are transtransferred using two plasmids (one encoding the Rep and Cap genes and helper function, and the other encoding an expression cassette flanked by two ITRs).

[0005] HEK293 cells are widely used for recombinant AAV production, partly due to the following advantages: HEK293 cells can be grown in suspension culture in serum-free medium, they are of human origin, and the helper genes E1A and E1B, which are required for AAV production, are endogenously expressed by HEK293 cells. Laura Abaandou et al., Cells, 10(7):1667 (2021) (Non-patent document 2).

[0006] Numerous factors (including host cell density, culture medium, suspension or adherent cell use, shaking flask or bioreactor use, harvesting time, total amount of DNA used for translocation, and optimal ratio of three plasmids) affect recombinant AAV production. See, for example, Grieger et al., Mol Ther. 24(2):287-297 (2016) (Non-patent Literature 3); Zhao et al., Mol Ther Methods Clin Dev. 18:312-320 (2020) (Non-patent Literature 4); Joiner et al., Current Opinion in Chemical Engineering 36(11):100823 (2022) (Non-patent Literature 5). Reduced viability of host cells (e.g., HEK293 cells) after translocation has been identified as one of the key factors limiting recombinant AAV yield. Several factors contribute to the decreased viability of HEK293 cells after translocation, including the occurrence of apoptosis after plasmid DNA uptake (Li et al, Exp Cell Res. 253(2):541-50 (1999) (Non-Patent Literature 6)), virus-induced ER stress and immunodefence (Li et al., Crit Rev Microbiol. 41(2):150-64 (2015) (Non-Patent Literature 7)), and mitochondrial dysfunction and oxidative damage induced by reactive oxygen species (ROS) under high cell density conditions (Koo et al, Cell Metab. 28(2):196-206 (2018) (Non-Patent Literature 8)).

[0007] The effects of genetic modification of host cells on the production of recombinant AAV and polypeptides remain unclear. Therefore, there is a need in the art to improve the productivity and yield of methods for large-scale production of rAAV particles and recombinant polypeptides by providing improved host cells. [Prior art documents] [Non-patent literature]

[0008] [Non-Patent Document 1] Naso et al., BioDrugs 31:317 - 334(2017) [Non - Patent Document 2] Laura Abaandou et al., Cells, 10(7):1667(2021) [Non - Patent Document 3] Grieger et al., Mol Ther. 24(2):287 - 297(2016) [Non - Patent Document 4] Zhao et al., Mol Ther Methods Clin Dev. 18:312 - 320(2020) [Non - Patent Document 5] Joiner et al., Current Opinion in Chemical Engineering 36(11):100823(2022) [Non - Patent Document 6] Li et al, Exp Cell Res. 253(2):541 - 50(1999) [Non - Patent Document 7] Li et al., Crit Rev Microbiol. 41(2):150 - 64(2015) [Non - Patent Document 8] Koo et al, Cell Metab. 28(2):196 - 206(2018) [Summary of the Invention]

[0009] Summary In one embodiment, the disclosure provides recombinant cells suitable for generating recombinant viruses or polypeptides, comprising at least one modification that reduces or eliminates the activity of at least one endogenous gene or gene product in an apoptotic signaling pathway. In some embodiments, the at least one endogenous gene or gene product in an apoptotic signaling pathway is selected from the group consisting of ANGPT1, ARHGEF7, ARHGEF17, BID, BNIP1, C19orf2, CHST11, CUL1, DAP3, DYNLL1, FIS1, HMGB2, IGFBP3, NRG1, TNFRSF10C, TNFSF12, MAL, MAX, and VEGFB genes or gene products. In some embodiments, the at least one endogenous gene or gene product in an apoptotic signaling pathway is selected from the group consisting of ARHGEF7, ARHGEF17, BNIP1, C19orf2, CHST11, DYNLL1, IGFBP3, MAX, and VEGFB genes or gene products. In some embodiments, at least one endogenous gene or gene product in the apoptotic signaling pathway includes the DYNLL1 and / or MAX gene or gene product. In some embodiments, the recombinant virus is recombinant adeno-associated virus (AAV). In some embodiments, the recombinant polypeptide is an antibody. In some embodiments, the cells are HEK293 cells, HEK293-derived cells, CHO cells, CHO-derived cells, HeLa cells, SF-9 cells, BHK cells, Vero cells, CAP cells, or PerC6 cells. In some embodiments, the cells are HEK293 cells. In some embodiments, the cells are HEK293-derived cells.In some embodiments, the cells are recombinant HEK293 cells capable of producing rAAV, and the cells include at least one modification that reduces or eliminates the activity of at least one endogenous gene or gene selected from the group consisting of ANGPT1, ARHGEF7, ARHGEF17, BID, BNIP1, C19orf2, CHST11, CUL1, DAP3, DYNLL1, FIS1, HMGB2, IGFBP3, NRG1, TNFRSF10C, TNFSF12, MAL, MAX, and VEGFB genes or gene products. In some embodiments, the cells are recombinant HEK293 cells capable of producing rAAV, and the cells include at least one modification that reduces or eliminates the activity of at least one endogenous gene or gene selected from the group consisting of the DYNLL1 and MAX genes or gene products. In some embodiments, the cells are recombinant HEK293 cells capable of producing recombinant polypeptides (e.g., antibodies), and the cells include at least one modification that reduces or eliminates the activity of at least one endogenous gene or gene selected from the group consisting of the ANGPT1, ARHGEF7, ARHGEF17, BID, BNIP1, C19orf2, CHST11, CUL1, DAP3, DYNLL1, FIS1, HMGB2, IGFBP3, NRG1, TNFRSF10C, TNFSF12, MAL, MAX, and VEGFB genes or gene products. In some embodiments, the cells are recombinant HEK293 cells capable of producing recombinant polypeptides (e.g., antibodies), and the cells include at least one endogenous gene or at least one modification that reduces or eliminates the activity of a gene selected from the group consisting of ARHGEF7, ARHGEF17, BNIP1, C19orf2, CHST11, DYNLL1, IGFBP3, MAX, and VEGFB genes or gene products.In some embodiments, the cells are HEK293 cells adapted to suspension culture.

[0010] In one embodiment, the disclosure provides recombinant cells capable of producing recombinant viruses (e.g., AAV) or polypeptides (e.g., antibodies), wherein the cells include at least one modification that reduces or eliminates the activity of at least one gene or gene product in an apoptotic signaling pathway. In some embodiments, the at least one endogenous gene or gene product in the apoptotic signaling pathway is selected from the group consisting of ANGPT1, ARHGEF7, ARHGEF17, BID, BNIP1, C19orf2, CHST11, CUL1, DAP3, DYNLL1, FIS1, HMGB2, IGFBP3, NRG1, TNFRSF10C, TNFSF12, MAL, MAX, and VEGFB genes or gene products. In some embodiments, the at least one endogenous gene or gene product in the apoptotic signaling pathway includes the DYNLL1 and / or MAX gene or gene product. In some embodiments, at least one endogenous gene or gene product in the apoptotic signaling pathway comprises the DYNLL1 gene or gene product and a second gene or gene product selected from the group consisting of ANGPT1, ARHGEF7, ARHGEF17, BID, BNIP1, C19orf2, CHST11, CUL1, DAP3, FIS1, HMGB2, IGFBP3, NRG1, TNFRSF10C, TNFSF12, MAL, MAX, and VEGFB. In some embodiments, at least one endogenous gene or gene product in the apoptotic signaling pathway comprises the DYNLL1 gene or gene product and one or more genes or gene products selected from the group consisting of ANGPT1, ARHGEF7, ARHGEF17, BID, BNIP1, C19orf2, CHST11, CUL1, DAP3, FIS1, HMGB2, IGFBP3, NRG1, TNFRSF10C, TNFSF12, MAL, MAX, and VEGFB. In some embodiments, the cells are HEK293 cells. In some embodiments, the cells are HEK293 cell-derived cells.

[0011] In one embodiment, the disclosure provides a cell bank comprising a plurality of cells, wherein each cell has at least one modification that reduces or eliminates the activity of at least one gene or gene product in an apoptotic signaling pathway. In some embodiments, the at least one endogenous gene or gene product in an apoptotic signaling pathway is selected from the group consisting of ANGPT1, ARHGEF7, ARHGEF17, BID, BNIP1, C19orf2, CHST11, CUL1, DAP3, DYNLL1, FIS1, HMGB2, IGFBP3, NRG1, TNFRSF10C, TNFSF12, MAL, MAX, and VEGFB genes or gene products. In some embodiments, the at least one endogenous gene or gene product in an apoptotic signaling pathway includes the DYNLL1 and / or MAX gene or gene product. In some embodiments, at least one endogenous gene or gene product in the apoptotic signaling pathway comprises the DYNLL1 gene or gene product and a second gene or gene product selected from the group consisting of ANGPT1, ARHGEF7, ARHGEF17, BID, BNIP1, C19orf2, CHST11, CUL1, DAP3, FIS1, HMGB2, IGFBP3, NRG1, TNFRSF10C, TNFSF12, MAL, MAX, and VEGFB. In some embodiments, at least one endogenous gene or gene product in the apoptotic signaling pathway comprises the DYNLL1 gene or gene product and one or more genes or gene products selected from the group consisting of ANGPT1, ARHGEF7, ARHGEF17, BID, BNIP1, C19orf2, CHST11, CUL1, DAP3, FIS1, HMGB2, IGFBP3, NRG1, TNFRSF10C, TNFSF12, MAL, MAX, and VEGFB. In some embodiments, the cells are HEK293 cells. In some embodiments, the cells are HEK293-derived cells.

[0012] In one embodiment, the disclosure provides a cell culture comprising a plurality of cells capable of producing recombinant viruses (e.g., AAV) or polypeptides (e.g., antibodies), wherein the cells include at least one modification that reduces or eliminates the activity of at least one gene or gene product in an apoptotic signaling pathway. In some embodiments, the at least one endogenous gene or gene product in the apoptotic signaling pathway is selected from the group consisting of ANGPT1, ARHGEF7, ARHGEF17, BID, BNIP1, C19orf2, CHST11, CUL1, DAP3, DYNLL1, FIS1, HMGB2, IGFBP3, NRG1, TNFRSF10C, TNFSF12, MAL, MAX, and VEGFB genes or gene products. In some embodiments, the at least one endogenous gene or gene product in the apoptotic signaling pathway includes the DYNLL1 and / or MAX gene or gene product. In some embodiments, at least one endogenous gene or gene product in the apoptotic signaling pathway comprises the DYNLL1 gene or gene product and a second gene or gene product selected from the group consisting of ANGPT1, ARHGEF7, ARHGEF17, BID, BNIP1, C19orf2, CHST11, CUL1, DAP3, FIS1, HMGB2, IGFBP3, NRG1, TNFRSF10C, TNFSF12, MAL, MAX, and VEGFB. In some embodiments, at least one endogenous gene or gene product in the apoptotic signaling pathway comprises the DYNLL1 gene or gene product and one or more genes or gene products selected from the group consisting of ANGPT1, ARHGEF7, ARHGEF17, BID, BNIP1, C19orf2, CHST11, CUL1, DAP3, FIS1, HMGB2, IGFBP3, NRG1, TNFRSF10C, TNFSF12, MAL, MAX, and VEGFB. In some embodiments, the cells are HEK293 cells. In some embodiments, the cells are HEK293-derived cells. In some embodiments, the cells are HEK293 cells.In some embodiments, the cells are HEK293-derived cells.

[0013] In one embodiment, the Disclosure provides a method for generating a recombinant virus (e.g., AAV) or polypeptide (e.g., antibody), comprising (a) preparing a cell culture comprising a plurality of cells capable of generating a recombinant virus (e.g., AAV) or polypeptide (e.g., antibody), and (b) maintaining the cell culture under conditions that enable the generation of a recombinant virus (e.g., AAV) or polypeptide (e.g., antibody), wherein the cell comprises at least one modification that reduces or eliminates the activity of at least one gene or gene product in an apoptotic signaling pathway. In some embodiments, the at least one endogenous gene or gene product in an apoptotic signaling pathway is selected from the group consisting of ANGPT1, ARHGEF7, ARHGEF17, BID, BNIP1, C19orf2, CHST11, CUL1, DAP3, DYNLL1, FIS1, HMGB2, IGFBP3, NRG1, TNFRSF10C, TNFSF12, MAL, MAX, and VEGFB genes or gene products. In some embodiments, at least one endogenous gene or gene product in the apoptotic signaling pathway includes the DYNLL1 gene and / or the MAX gene or gene product. In some embodiments, at least one endogenous gene or gene product in the apoptotic signaling pathway includes the DYNLL1 gene or gene product and a second gene or gene product selected from the group consisting of ANGPT1, ARHGEF7, ARHGEF17, BID, BNIP1, C19orf2, CHST11, CUL1, DAP3, FIS1, HMGB2, IGFBP3, NRG1, TNFRSF10C, TNFSF12, MAL, MAX, and VEGFB. In some embodiments, at least one endogenous gene or gene product in the apoptotic signaling pathway comprises the DYNLL1 gene or gene product and one or more genes or gene products selected from the group consisting of ANGPT1, ARHGEF7, ARHGEF17, BID, BNIP1, C19orf2, CHST11, CUL1, DAP3, FIS1, HMGB2, IGFBP3, NRG1, TNFRSF10C, TNFSF12, MAL, MAX, and VEGFB.In some embodiments, the cells are HEK293 cells. In some embodiments, the cells are HEK293-derived cells.

[0014] In one embodiment, the Disclosure provides a method for generating recombinant viruses (e.g., AAV) or polypeptides (e.g., antibodies), comprising culturing cells capable of generating recombinant viruses (e.g., AAV) or polypeptides (e.g., antibodies) under conditions that enable the generation of recombinant viruses (e.g., AAV) or polypeptides (e.g., antibodies), wherein the cells are subjected to at least one modification that reduces or eliminates the activity of at least one gene or gene product in an apoptotic signaling pathway. In some embodiments, the at least one endogenous gene or gene product in an apoptotic signaling pathway is selected from the group consisting of ANGPT1, ARHGEF7, ARHGEF17, BID, BNIP1, C19orf2, CHST11, CUL1, DAP3, DYNLL1, FIS1, HMGB2, IGFBP3, NRG1, TNFRSF10C, TNFSF12, MAL, MAX, and VEGFB genes or gene products. In some embodiments, at least one endogenous gene or gene product in the apoptotic signaling pathway includes the DYNLL1 gene and / or the MAX gene or gene product. In some embodiments, at least one endogenous gene or gene product in the apoptotic signaling pathway includes the DYNLL1 gene or gene product and a second gene or gene product selected from the group consisting of ANGPT1, ARHGEF7, ARHGEF17, BID, BNIP1, C19orf2, CHST11, CUL1, DAP3, FIS1, HMGB2, IGFBP3, NRG1, TNFRSF10C, TNFSF12, MAL, MAX, and VEGFB. In some embodiments, at least one endogenous gene or gene product in the apoptotic signaling pathway comprises the DYNLL1 gene or gene product and one or more genes or gene products selected from the group consisting of ANGPT1, ARHGEF7, ARHGEF17, BID, BNIP1, C19orf2, CHST11, CUL1, DAP3, FIS1, HMGB2, IGFBP3, NRG1, TNFRSF10C, TNFSF12, MAL, MAX, and VEGFB. In some embodiments, the cells are HEK293 cells.In some embodiments, the cells are HEK293 cell-derived cells.

[0015] In one embodiment, the Disclosure provides a method for increasing the production of recombinant virus (e.g., AAV) or polypeptide (e.g., antibody), comprising (a) preparing a cell culture comprising a plurality of cells capable of producing recombinant virus (e.g., AAV) or polypeptide (e.g., antibody), and (b) maintaining the cell culture under conditions that enable the production of recombinant virus (e.g., AAV) or polypeptide (e.g., antibody), wherein the cells are subjected to at least one modification that reduces or eliminates the activity of at least one gene or gene product in an apoptotic signaling pathway. In some embodiments, the at least one endogenous gene or gene product in an apoptotic signaling pathway is selected from the group consisting of ANGPT1, ARHGEF7, ARHGEF17, BID, BNIP1, C19orf2, CHST11, CUL1, DAP3, DYNLL1, FIS1, HMGB2, IGFBP3, NRG1, TNFRSF10C, TNFSF12, MAL, MAX, and VEGFB genes or gene products. In some embodiments, at least one endogenous gene or gene product in the apoptotic signaling pathway includes the DYNLL1 gene and / or the MAX gene or gene product. In some embodiments, at least one endogenous gene or gene product in the apoptotic signaling pathway includes the DYNLL1 gene or gene product and a second gene or gene product selected from the group consisting of ANGPT1, ARHGEF7, ARHGEF17, BID, BNIP1, C19orf2, CHST11, CUL1, DAP3, FIS1, HMGB2, IGFBP3, NRG1, TNFRSF10C, TNFSF12, MAL, MAX, and VEGFB.In some embodiments, at least one endogenous gene or gene product in the apoptotic signaling pathway comprises the DYNLL1 gene or gene product and one or more genes or gene products selected from the group consisting of ANGPT1, ARHGEF7, ARHGEF17, BID, BNIP1, C19orf2, CHST11, CUL1, DAP3, FIS1, HMGB2, IGFBP3, NRG1, TNFRSF10C, TNFSF12, MAL, MAX, and VEGFB. In some embodiments, the cells are HEK293 cells. In some embodiments, the cells are HEK293-derived cells.

[0016] In one embodiment, the Disclosure provides a method for increasing the production of recombinant virus (e.g., AAV) or polypeptide (e.g., antibody), comprising culturing cells capable of producing recombinant virus (e.g., AAV) or polypeptide (e.g., antibody) under conditions that enable the production of recombinant virus (e.g., AAV) or polypeptide (e.g., antibody), wherein the cells are subjected to at least one modification that reduces or eliminates the activity of at least one gene or gene product in an apoptotic signaling pathway. In some embodiments, the at least one endogenous gene or gene product in an apoptotic signaling pathway is selected from the group consisting of ANGPT1, ARHGEF7, ARHGEF17, BID, BNIP1, C19orf2, CHST11, CUL1, DAP3, DYNLL1, FIS1, HMGB2, IGFBP3, NRG1, TNFRSF10C, TNFSF12, MAL, MAX, and VEGFB genes or gene products. In some embodiments, at least one endogenous gene or gene product in the apoptotic signaling pathway includes the DYNLL1 gene and / or the MAX gene or gene product. In some embodiments, at least one endogenous gene or gene product in the apoptotic signaling pathway includes the DYNLL1 gene or gene product and a second gene or gene product selected from the group consisting of ANGPT1, ARHGEF7, ARHGEF17, BID, BNIP1, C19orf2, CHST11, CUL1, DAP3, FIS1, HMGB2, IGFBP3, NRG1, TNFRSF10C, TNFSF12, MAL, MAX, and VEGFB. In some embodiments, at least one endogenous gene or gene product in the apoptotic signaling pathway comprises the DYNLL1 gene or gene product and one or more genes or gene products selected from the group consisting of ANGPT1, ARHGEF7, ARHGEF17, BID, BNIP1, C19orf2, CHST11, CUL1, DAP3, FIS1, HMGB2, IGFBP3, NRG1, TNFRSF10C, TNFSF12, MAL, MAX, and VEGFB. In some embodiments, the cells are HEK293 cells.In some embodiments, the cells are HEK293 cell-derived cells.

[0017] In one embodiment, the present disclosure provides a method for generating rAAV particles, comprising (a) preparing a cell culture comprising a plurality of cells; (b) introducing one or more polynucleotides into the cells that encode at least one of the following: (i) an rAAV genome to be packaged; (ii) an adenovirus helper function required for packaging; (iii) an AAV rep protein sufficient for packaging; and (iv) an AAV cap protein sufficient for packaging; and (c) maintaining the cell culture under conditions that enable the generation of rAAV particles, wherein the cells include at least one modification that reduces or eliminates the activity of at least one gene or gene product in an apoptotic signaling pathway. In some embodiments, at least one endogenous gene or gene product in the apoptosis signaling pathway is selected from the group consisting of ANGPT1, ARHGEF7, ARHGEF17, BID, BNIP1, C19orf2, CHST11, CUL1, DAP3, DYNLL1, FIS1, HMGB2, IGFBP3, NRG1, TNFRSF10C, TNFSF12, MAL, MAX, and VEGFB genes or gene products. In some embodiments, at least one endogenous gene or gene product in the apoptosis signaling pathway includes DYNLL1 and / or the MAX gene or gene product. In some embodiments, at least one endogenous gene or gene product in the apoptotic signaling pathway comprises the DYNLL1 gene or gene product and a second gene or gene product selected from the group consisting of ANGPT1, ARHGEF7, ARHGEF17, BID, BNIP1, C19orf2, CHST11, CUL1, DAP3, FIS1, HMGB2, IGFBP3, NRG1, TNFRSF10C, TNFSF12, MAL, MAX, and VEGFB.In some embodiments, at least one endogenous gene or gene product in the apoptotic signaling pathway comprises the DYNLL1 gene or gene product and one or more genes or gene products selected from the group consisting of ANGPT1, ARHGEF7, ARHGEF17, BID, BNIP1, C19orf2, CHST11, CUL1, DAP3, FIS1, HMGB2, IGFBP3, NRG1, TNFRSF10C, TNFSF12, MAL, MAX, and VEGFB. In some embodiments, the cells are HEK293 cells. In some embodiments, the cells are HEK293-derived cells.

[0018] In one embodiment, the present disclosure provides a method for increasing the generation of rAAV particles, comprising (a) preparing a cell culture comprising a plurality of cells; (b) introducing one or more polynucleotides into the cells that encode at least one of the following: (i) an rAAV genome to be packaged; (ii) an adenovirus helper function required for packaging; (iii) an AAV rep protein sufficient for packaging; and (iv) an AAV cap protein sufficient for packaging; and (c) maintaining the cell culture under conditions that enable the generation of rAAV particles, wherein the cells include at least one modification that reduces or eliminates the activity of at least one gene or gene product in an apoptotic signaling pathway. In some embodiments, at least one endogenous gene or gene product in the apoptosis signaling pathway is selected from the group consisting of ANGPT1, ARHGEF7, ARHGEF17, BID, BNIP1, C19orf2, CHST11, CUL1, DAP3, DYNLL1, FIS1, HMGB2, IGFBP3, NRG1, TNFRSF10C, TNFSF12, MAL, MAX, and VEGFB genes or gene products. In some embodiments, at least one endogenous gene or gene product in the apoptosis signaling pathway includes DYNLL1 and / or the MAX gene or gene product. In some embodiments, at least one endogenous gene or gene product in the apoptotic signaling pathway comprises the DYNLL1 gene or gene product and a second gene or gene product selected from the group consisting of ANGPT1, ARHGEF7, ARHGEF17, BID, BNIP1, C19orf2, CHST11, CUL1, DAP3, FIS1, HMGB2, IGFBP3, NRG1, TNFRSF10C, TNFSF12, MAL, MAX, and VEGFB.In some embodiments, at least one endogenous gene or gene product in the apoptotic signaling pathway comprises the DYNLL1 gene or gene product and one or more genes or gene products selected from the group consisting of ANGPT1, ARHGEF7, ARHGEF17, BID, BNIP1, C19orf2, CHST11, CUL1, DAP3, FIS1, HMGB2, IGFBP3, NRG1, TNFRSF10C, TNFSF12, MAL, MAX, and VEGFB. In some embodiments, the cells are HEK293 cells. In some embodiments, the cells are HEK293-derived cells.

[0019] In one embodiment, the Disclosure provides a method for generating recombinant polypeptides (e.g., antibodies), comprising (a) preparing a cell culture comprising a plurality of cells, (b) introducing one or more polynucleotides encoding recombinant polypeptides (e.g., antibodies) into the cells, and (c) maintaining the cell culture under conditions that enable the generation of recombinant polypeptides (e.g., antibodies), wherein the cells undergo at least one modification that reduces or eliminates the activity of at least one gene or gene product in an apoptotic signaling pathway. In some embodiments, the at least one endogenous gene or gene product in an apoptotic signaling pathway is selected from the group consisting of ANGPT1, ARHGEF7, ARHGEF17, BID, BNIP1, C19orf2, CHST11, CUL1, DAP3, DYNLL1, FIS1, HMGB2, IGFBP3, NRG1, TNFRSF10C, TNFSF12, MAL, MAX, and VEGFB genes or gene products. In some embodiments, at least one endogenous gene or gene product in the apoptotic signaling pathway includes the DYNLL1 gene and / or the MAX gene or gene product. In some embodiments, at least one endogenous gene or gene product in the apoptotic signaling pathway includes the DYNLL1 gene or gene product and a second gene or gene product selected from the group consisting of ANGPT1, ARHGEF7, ARHGEF17, BID, BNIP1, C19orf2, CHST11, CUL1, DAP3, FIS1, HMGB2, IGFBP3, NRG1, TNFRSF10C, TNFSF12, MAL, MAX, and VEGFB. In some embodiments, at least one endogenous gene or gene product in the apoptotic signaling pathway comprises the DYNLL1 gene or gene product and one or more genes or gene products selected from the group consisting of ANGPT1, ARHGEF7, ARHGEF17, BID, BNIP1, C19orf2, CHST11, CUL1, DAP3, FIS1, HMGB2, IGFBP3, NRG1, TNFRSF10C, TNFSF12, MAL, MAX, and VEGFB.In some embodiments, the cells are HEK293 cells. In some embodiments, the cells are HEK293-derived cells.

[0020] In one embodiment, the Disclosure provides a method for increasing the production of recombinant polypeptides (e.g., antibodies), comprising (a) preparing a cell culture comprising a plurality of cells, (b) introducing one or more polynucleotides encoding recombinant polypeptides (e.g., antibodies) into the cells, and (c) maintaining the cell culture under conditions that enable the production of recombinant polypeptides (e.g., antibodies), wherein the cells undergo at least one modification that reduces or eliminates the activity of at least one gene or gene product in an apoptotic signaling pathway. In some embodiments, the at least one endogenous gene or gene product in an apoptotic signaling pathway is selected from the group consisting of ANGPT1, ARHGEF7, ARHGEF17, BID, BNIP1, C19orf2, CHST11, CUL1, DAP3, DYNLL1, FIS1, HMGB2, IGFBP3, NRG1, TNFRSF10C, TNFSF12, MAL, MAX, and VEGFB genes or gene products. In some embodiments, at least one endogenous gene or gene product in the apoptotic signaling pathway includes the DYNLL1 gene and / or the MAX gene or gene product. In some embodiments, at least one endogenous gene or gene product in the apoptotic signaling pathway includes the DYNLL1 gene or gene product and a second gene or gene product selected from the group consisting of ANGPT1, ARHGEF7, ARHGEF17, BID, BNIP1, C19orf2, CHST11, CUL1, DAP3, FIS1, HMGB2, IGFBP3, NRG1, TNFRSF10C, TNFSF12, MAL, MAX, and VEGFB. In some embodiments, at least one endogenous gene or gene product in the apoptotic signaling pathway comprises the DYNLL1 gene or gene product and one or more genes or gene products selected from the group consisting of ANGPT1, ARHGEF7, ARHGEF17, BID, BNIP1, C19orf2, CHST11, CUL1, DAP3, FIS1, HMGB2, IGFBP3, NRG1, TNFRSF10C, TNFSF12, MAL, MAX, and VEGFB.In some embodiments, the cells are HEK293 cells. In some embodiments, the cells are HEK293-derived cells.

[0021] In some embodiments, the present disclosure provides the following. [1.] A recombinant cell capable of generating rAAV, wherein the cell comprises at least one modification that reduces or eliminates the activity of at least one endogenous gene or gene product in the apoptosis signaling pathway. [2.] The cell according to [1], wherein the cell comprises at least two modifications that reduce or eliminate the activity of at least two genes or gene products in the apoptosis signaling pathway. [3.] A cell bank comprising a plurality of cells, wherein the cells comprise at least one modification that reduces or eliminates the activity of at least one endogenous gene or gene product in the apoptosis signaling pathway. [4.] The cell bank according to [3], wherein the cells comprise at least two modifications that reduce or eliminate the activity of at least two genes or gene products in the apoptosis signaling pathway. [5.] A cell culture comprising a plurality of cells capable of generating rAAV, wherein the cells comprise at least one modification that reduces or eliminates the activity of at least one endogenous gene or gene product in the apoptosis signaling pathway. [6.] The cell culture according to [5], wherein the cells comprise at least two modifications that reduce or eliminate the activity of at least two genes or gene products in the apoptosis signaling pathway. [7.] A method for generating rAAV particles, comprising: a) preparing a cell culture comprising a plurality of cells capable of generating rAAV; b) maintaining the cell culture under conditions that allow for the generation of the rAAV particles. and The cells include at least one modification that reduces or removes the activity of at least one endogenous gene or gene product in the apoptotic signaling pathway. The aforementioned method. [8.] A method for generating rAAV particles, comprising culturing cells capable of generating rAAV particles under conditions that enable the generation of the rAAV particles, wherein the cells include at least one modification that reduces or removes the activity of at least one endogenous gene or gene product in an apoptotic signaling pathway. [9.] A method for increasing the production of rAAV particles, a) Prepare a cell culture containing multiple cells capable of producing rAAV, b) Maintain the cell culture under conditions that enable the generation of the rAAV particles. Includes, The cells include at least one modification that reduces or removes the activity of at least one endogenous gene or gene product in the apoptotic signaling pathway. The aforementioned method. [10.] A method for increasing the production of rAAV particles, comprising culturing cells capable of producing rAAV particles under conditions that enable the production of rAAV particles, wherein the cells include at least one modification that reduces or removes the activity of at least one endogenous gene or gene product in an apoptotic signaling pathway. [11.] The method according to any one of [7] to

[10] , wherein the cells are modified to reduce or remove the activity of at least two genes or gene products in an apoptotic signaling pathway. [12.] To the cells capable of generating rAAV, the following: a) The 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 [1] to

[11] , wherein one or more polynucleotides encoding at least one of the following are transfected. [13.] To the cells capable of producing rAAV, the following: a) The 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 A method according to any one of [1] to

[12] , wherein one or more polynucleotides encoding are transfected. [14.] A method for producing rAAV particles, a) Prepare a cell culture containing multiple cells, b) To the cells, the following: i. The packaged rAAV genome, ii. Adenovirus helper function necessary for packaging, iii. Sufficient AAV rep protein for packaging, and iv. Sufficient AAV cap protein for packaging Introducing one or more polynucleotides that encode at least one of the following, c) Maintain the cell culture under conditions that enable the generation of the rAAV particles. Includes, The cells include at least one modification that reduces or removes the activity of at least one endogenous gene or gene product in the apoptotic signaling pathway. The aforementioned method. [15.] A method for increasing the production of rAAV particles, a) Prepare a cell culture containing multiple cells, b) To the cells, the following: i. The packaged rAAV genome, ii. Adenovirus helper function necessary for packaging, iii. Sufficient AAV rep protein for packaging, and iv. Sufficient AAV cap protein for packaging Introducing one or more polynucleotides that encode at least one of the following, c) Maintain the cell culture under conditions that enable the generation of the rAAV particles. Includes, The cells include at least one modification that reduces or removes the activity of at least one endogenous gene or gene product in the apoptotic signaling pathway. The aforementioned method. [16.] The method according to

[14] or

[15] , wherein the cells are modified to reduce or eliminate the activity of at least two genes or gene products in an apoptotic signaling pathway. [17.] The following: i. The packaged rAAV genome, ii. Adenovirus helper function necessary for packaging, iii. Sufficient AAV rep protein for packaging, and iv. Sufficient AAV cap protein for packaging A method according to any one of

[14] to

[16] , comprising introducing one or more polynucleotides encoding a [18.] The method according to any one of

[14] to

[17] , wherein the introduction of one or more polynucleotides into the cells is carried out by transfusion. [19.] The cell, cell bank, cell culture, or method according to any one of [1] to

[18] , wherein the modification includes a mutation in a gene. [20.] The cell, cell bank, cell culture, or method according to

[19] , wherein the gene modification includes a missense mutation, a nonsense mutation, or a frameshift mutation. [21.] The cell, cell bank, cell culture, or method according to

[19] , wherein the gene modification includes a deletion. [22.] The cell, cell bank, cell culture, or method according to any one of

[19] to

[21] , wherein the mutation is a heterozygous mutation. [23.] The cell, cell bank, cell culture, or method according to

[22] , wherein the heterozygous mutation affects one copy of the gene. [24.] The cell, cell bank, cell culture, or method according to

[22] , wherein the heterozygous mutation affects more than one copy of the gene. [25.] The cells, cell bank, cell culture, or method according to

[22] , wherein the heterozygous mutation affects one, two, or three copies of the gene. [26.] The cell, cell bank, cell culture, or method according to any one of

[19] to

[21] , wherein the mutation is a homozygous mutation. [27.] The cell, cell bank, cell culture, or method according to any one of [1] to

[22] , wherein the modification comprises an inhibitory nucleic acid molecule capable of reducing or removing the activity of the gene or gene product. [28.] The cell, cell bank, cell culture, or method according to

[27] , wherein the modification comprises antisense RNA. [29.] The cell, cell bank, cell culture, or method according to

[27] , wherein the modification comprises a small interfering RNA (siRNA), a microRNA (miRNA), or a short hairpin RNA (shRNA). [30.] The cell, cell bank, cell culture, or method according to any one of [1] to

[29] , wherein the at least one endogenous gene or gene product in the apoptotic signaling pathway is selected from the group consisting of ANGPT1, ARHGEF7, ARHGEF17, BID, BNIP1, C19orf2, CHST11, CUL1, DAP3, DYNLL1, FIS1, HMGB2, IGFBP3, NRG1, TNFRSF10C, TNFSF12, MAL, MAX, and VEGFB genes or gene products. [31.] The cell, cell bank, cell culture, or method according to

[30] , wherein the at least one endogenous gene or gene product comprises any two genes or gene products selected from the group consisting of ANGPT1, ARHGEF7, ARHGEF17, BID, BNIP1, C19orf2, CHST11, CUL1, DAP3, DYNLL1, FIS1, HMGB2, IGFBP3, NRG1, TNFRSF10C, TNFSF12, MAL, MAX, and VEGFB genes or gene products. [32.] The cell, cell bank, cell culture, or method according to any one of [1] to

[29] , wherein the at least one endogenous gene or gene product is selected from the group consisting of ARHGEF7, ARHGEF17, BNIP1, C19orf2, CHST11, DYNLL1, IGFBP3, MAX, and VEGFB genes or gene products. [33.] The cell, cell bank, cell culture, or method according to

[32] , wherein the at least one endogenous gene or gene product comprises any two genes or gene products selected from the group consisting of ARHGEF7, ARHGEF17, BNIP1, C19orf2, CHST11, DYNLL1, IGFBP3, MAX, and VEGFB genes or gene products. [34.] The cell, cell bank, cell culture, or method according to any one of [1] to

[29] , wherein the at least one endogenous gene or gene product comprises DYNLL1, IGFBP3, and / or the MAX gene or gene product. [35.] The cell, cell bank, cell culture, or method according to

[34] , wherein the at least one endogenous gene or gene product comprises the DYNLL1 gene or gene product. [36.] The cell, cell bank, cell culture, or method according to

[35] , wherein the modification comprises a heterozygous DYNLL1 mutation. [37.] The cell, cell bank, cell culture, or method according to

[34] , wherein the at least one endogenous gene or gene product comprises the DYNLL1 and MAX genes or gene products. [38.] The cell, cell bank, cell culture, or method according to

[37] , wherein the modification comprises a heterozygous DYNLL1 mutation and a MAX mutation. [39.] The cell, cell bank, cell culture, or method according to any one of [1] to

[29] , wherein the at least one endogenous gene or gene product comprises DYNLL1 and a second gene or gene product selected from the group consisting of ANGPT1, ARHGEF7, ARHGEF17, BID, BNIP1, C19orf2, CHST11, CUL1, DAP3, FIS1, HMGB2, IGFBP3, NRG1, TNFRSF10C, TNFSF12, MAL, MAX, and VEGFB. [40.] A cell, cell bank, cell culture, or method according to any one of [1] to

[29] , wherein the at least one endogenous gene or gene product comprises DYNLL1 and a second gene or gene product selected from the group consisting of ARHGEF7, ARHGEF17, BNIP1, C19orf2, CHST11, IGFBP3, MAX, and VEGFB. [41.] A cell, cell bank, cell culture, or method according to any one of [1] to

[29] , wherein the at least one endogenous gene or gene product comprises DYNLL1 and ANGPT1, ARHGEF7, ARHGEF17, BID, BNIP1, C19orf2, CHST11, CUL1, DAP3, FIS1, HMGB2, IGFBP3, NRG1, TNFRSF10C, TNFSF12, MAL, MAX, and VEGFB. [42.] The cell, cell bank, cell culture, or method according to any one of [1] to

[29] , wherein the at least one endogenous gene or gene product comprises DYNLL1 and one or more genes or gene products selected from the group consisting of ANGPT1, ARHGEF7, ARHGEF17, BID, BNIP1, C19orf2, CHST11, CUL1, DAP3, FIS1, HMGB2, IGFBP3, NRG1, TNFRSF10C, TNFSF12, MAL, MAX, and VEGFB. [43.] The cell, cell bank, cell culture, or method according to any one of [1] to

[29] , wherein the at least one endogenous gene or gene product comprises DYNLL1 and one or more genes or gene products selected from the group consisting of ARHGEF7, ARHGEF17, BNIP1, C19orf2, CHST11, IGFBP3, MAX, and VEGFB. [44.] The cell, cell bank, cell culture or method according to any one of [1] to

[29] , wherein the at least one endogenous gene or gene product comprises DYNLL1 and ARHGEF7. [45.] A cell, cell bank, cell culture, or method according to any one of [1] to

[29] , wherein the at least one endogenous gene or gene product comprises DYNLL1 and ARHGEF17. [46.] A cell, cell bank, cell culture, or method according to any one of [1] to

[29] , wherein the at least one endogenous gene or gene product comprises DYNLL1 and BNIP1. [47.] A cell, cell bank, cell culture, or method according to any one of [1] to

[29] , wherein the at least one endogenous gene or gene product comprises DYNLL1 and C19orf2. [48.] A cell, cell bank, cell culture, or method according to any one of [1] to

[29] , wherein the at least one endogenous gene or gene product comprises DYNLL1 and CHST11. [49.] A cell, cell bank, cell culture, or method according to any one of [1] to

[29] , wherein the at least one endogenous gene or gene product comprises DYNLL1 and IGFBP3. [50.] A cell, cell bank, cell culture, or method according to any one of [1] to

[29] , wherein the at least one endogenous gene or gene product comprises DYNLL1 and MAX. [51.] A cell, cell bank, cell culture, or method according to any one of [1] to

[29] , wherein the at least one endogenous gene or gene product comprises DYNLL1 and VEGFB. [52.] The cells, cell bank, cell culture, or method according to any one of [1] to

[51] , wherein the cells are mammalian cells. [53.] The cells, cell bank, cell culture, or method according to any one of [1] to

[51] , wherein the cells are insect cells. [54.] The cells, cell bank, cell culture, or method according to any one of [1] to

[51] , wherein the cells are HEK293 cells, HEK293-derived cells, CHO cells, CHO-derived cells, HeLa cells, SF-9 cells, BHK cells, Vero cells, CAP cells, or PerC6 cells. [55.] The cells, cell bank, cell culture, or method according to any one of [1] to

[51] , wherein the cells are HEK293 cells. [56.] The cell, cell bank, cell culture, or method according to any one of [1] to

[55] , wherein the cell culture is a suspension culture. [57.] The cell, cell bank, cell culture, or method according to any one of

[12] to

[51] , wherein the adenovirus helper function comprises at least one of the adenovirus E4 gene, E2a gene, and VA gene. [58.] The cells, cell bank, cell culture, or method according to any one of

[12] to

[51] , wherein the adenovirus helper function comprises the adenovirus E4 gene, the E2a gene, and the VA gene. [59.] The cell, cell bank, cell culture, or method according to

[58] , wherein the polynucleotide encoding the adenovirus helper function comprises pAD delta F6 or helper #5. [60.] The cell, cell bank, cell culture, or method according to

[58] , wherein the polynucleotide encoding the adenovirus helper function comprises pHRC#7 or pHRC#8. [61.] The method according to any one of [7] to

[60] , wherein the cell culture is maintained under conditions that enable the generation of the rAAV particles, or the cells are cultured for a period of about 2 to 10 days, about 2 to 15 days, or about 5 to 14 days. [62.] The method according to any one of [7] to

[60] , wherein the cell culture is maintained under conditions that enable the generation of the rAAV particles, or the cells are cultured for a period of about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, or about 7 days. [63.] The method according to any one of [7] to

[60] , wherein the cell culture is maintained under conditions that enable the generation of the rAAV particles, or the cells are cultured for a period of about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, or about 7 days. [64.] The method according to

[63] , wherein the cell culture is maintained under conditions that enable the generation of the rAAV particles, or the cells are cultured, for a period of about 5 days. [65.] The method according to any one of [7] to

[64] , further comprising recovering the rAAV particles. [66.] The method according to any one of [7] to

[65] , wherein the method produces rAAV particles measured as GC / ml in greater quantities than the reference method using cells that do not contain the modification. [67.] The method according to any one of [7] to

[65] , wherein the cell culture produces at least 10%, at least 20%, at least 30%, at least 50%, at least 75%, or at least 100% more rAAV particles as measured as GC / ml than a reference method using cells that do not contain the modification. [68.] The cell culture or method according to any one of [5] to

[67] , wherein the cell culture has a volume of approximately 50 liters to approximately 20,000 liters. [69.] The cell culture or method according to any one of [5] to

[67] , wherein the cell culture has a volume of about 200 liters to about 2,000 liters. [70.] The rAAV is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, and AAV16, AAV.rh8, AAV .rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu32, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B, AAV2.5, AAV2 A cell, cell bank, cell culture, or method described in any one of [1] to

[69] , comprising the capsid protein of the tYF, 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 serotypes. [71.] A cell, cell bank, cell culture, or method according to any one of [1] to

[69] , wherein the rAAV comprises the capsid proteins of serotypes AAV8, AAV9, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu32, and AAV.hu37. [72.] The cell, cell bank, cell culture, or method according to any one of [1] to

[69] , wherein the rAAV comprises a capsid protein of the AAV8 or AAV9 serotype. [73.] The cell, cell bank, cell culture, or method according to any one of [1] to

[72] , wherein the rAAV comprises a genome encoding a polypeptide or a double-stranded RNA molecule. [74.] The cell, cell bank, cell culture, or method according to

[73] , wherein the genome encodes a polypeptide. [75.] Cells, cell banks, cell cultures, or methods according to

[73] , wherein the genome encodes anti-VEGF Fab, anti-kallikrein antibody, anti-TNF antibody, microdystrophin, minidystrophin, iduronidase (IDUA), iduronate 2-sulfatase (IDS), low-density lipoprotein receptor (LDLR), tripeptidyl peptidase 1 (TPP1), or a non-membrane-bound splice variant of VEGF receptor 1 (sFlt-1). [76.] The genome contains gamma-sarcoglycan, Rab escort protein 1 (REP1 / CHM), retinoid isomerohydrolase (RPE65), cyclic nucleotide-gated channel alpha-3 (CNGA3), cyclic nucleotide-gated channel beta-3 (CNGB3), aromatic L-amino acid decarboxylase (AADC), lysosome-associated membrane protein 2 isoform B (LAMP2B), factor VIII, factor IX, retinitis pigmentosa GTPase modulator (RPGR), retinosuxin (RS1), sarcoplasmic reticulum calcium ATPase (SERCA2a), aflibercept, battenin (CLN3), transmembrane ER protein (CLN6), glutathione Minic acid decarboxylase (GAD), glial cell-derived neurotrophic factor (GDNF), aquaporin 1 (AQP1), dystrophin, myotubularin 1 (MTM1), follistatin (FST), glucose-6-phosphatase (G6Pase), apolipoprotein A2 (APOA2), uridine diphosphate glucuronosyltransferase 1A1 (UGT1A1), arylsulfatase B (ARSB), N-acetyl-alpha-glucosaminidase (NAGLU), alpha- Glucosidase (GAA), alpha-galactosidase (GLA), beta-galactosidase (GLB1), lipoprotein lipase (LPL), alpha-1 antitrypsin (AAT), phosphodiesterase 6B (PDE6B), ornithine carbamoyltransferase 9OTC), survival motor neuron (SMN1), survival motor neuron (SMN2), neurturin (NRTN), neurotrophin-3 (NT-3 / NTF3), porphobilinogen deaminase (PB) Cells, cell banks, cell cultures, or methods described in

[73] that encode GD), nerve growth factor (NGF), mitochondrial encoded NADH:ubiquinone oxidoreductate zecore subunit 4 (MT-ND4), protective protein cathepsin A (PPCA), dysferrin, MER proto-oncogene, tyrosine kinase (MERTK), cystic fibrosis transmembrane conductance regulator (CFTR), or tumor necrosis factor receptor (TNFR)-immunoglobulin (IgG1) Fc fusion. [77.] The cell, cell bank, cell culture, or method according to

[73] , wherein the genome encodes dystrophin or microdystrophin. [78.] The cell, cell bank, cell culture, or method according to

[73] , wherein the packaged genome encodes a microRNA.

[0022] 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]

[0023] [Figure 1] Functional relationships of candidate genes obtained from primary screening. [Figure 2-1] Secondary titer validation after siRNA translocation to reduce target gene activity. Titers of AAV8 TG-A, AAV8 TG-B, or AAV9 TG-D particles generated by HEK293 cells in 24-DWP after siRNA translocation were measured using helper #5 with appropriate cis and trans plasmids. [Figure 2-2] See the explanation in Figure 2-1. [Figure 3] AAV titers after siRNA translocation that reduce the activity of two target genes. Titers of AAV9 TG-D, AAV8 TG-A, and AAV8 TG-B particles generated by HEK293 cells in 24-well plates were quantified after siRNA translocation using helper #5 with appropriate cis and trans plasmids. Relative titers are shown in grayscale. [Figure 4A] Host cell manipulation by IGFBP3 knockout. Workflow for single-cell cloning of IGFBP3-KO cells. [Figure 4B]Host cell manipulation by IGFBP3 knockout. AAV titer generated by IGFBP3-KO candidate clones after translocation with helper / cis / trans plasmid combinations of helper #5 / TG-A / AAV8 or helper #5 / TG-D / AAV9. [Figure 5-1] Host cell manipulation by IGFBP3 knockout. (A) Validation of IGFBP3 protein titer levels in clones with higher AAV generation. Results from anti-vinculin and anti-IGFBP3 Western blots are shown. Ctrl is the control cell sample. [Figure 5-2] Host cell manipulation by IGFBP3 knockout. (B~C) AAV titer generated by selected IGFBP3-KO candidate clones after translocation with either (B) helper #5 / TG-A / AAV8 or (C) helper #5 / TG-D / AAV9 helper / cis / trans plasmid combinations. [Figure 6A] Host cell manipulation by DYNLL1 knockout. Western blot verification of protein expression levels in parental cells after CRISPER-Cas9 DYNLL1-sgRNA-C1 / C2 treatment. [Figure 6B] Host cell manipulation by DYNLL1 knockout. AAV titer generated by DYNLL1-KO candidate clones. AAV titer after translocation is shown for helper / cis / trans plasmid combinations of helper #5 / TG-A / AAV8. [Figure 6C] Host cell manipulation by DYNLL1 knockout. AAV titer generated by a selected IGFBP3-KO candidate clone after transduction with a helper / cis / trans plasmid combination of helper #5 / TG-D / AAV9. Wt: wild-type control parental cell. [Figure 7-1]Host cell manipulation by DYNLL1 knockout. Titer validation of selected clones after translocation with (A) pHRC#7 (helper + AAV8-trans) plasmid and TG-A cis plasmid, (B) helper #5, TG-D cis and AAV9 trans plasmids, or (C) pHRC#7 (helper + AAV8-trans) plasmid and TG-B cis plasmid. The test clones in the panel, from left to right, are control, 1C1, 4D9, 2C6, 4A8, 3B7, 4D5, and 4A2. [Figure 7-2] See the explanation in Figure 7-1. [Figure 8] Host cell manipulation by knockout of DYNLL1 and MAX. AAV titers generated by DYNLL1 / MAX KO candidate clones. Titers obtained after translocation with pHRC#7 (helper + AAV8-trans) plasmid and TG-A cis plasmid are shown. [Figure 9-1] Host cell manipulation by knockout of DYNLL1 and MAX. (A) Titers generated by selected clones after translocation with pHRC#7 (helper + AAV8-trans) plasmid and TG-A cis plasmid, (B) pHRC#8 (helper + AAV9-trans) plasmid and TG-C cis plasmid, (C) Helper #5, TG-D cis, and AAV9 trans plasmids, and (D) pHRC#7 (helper + AAV8-trans) plasmid and TG-B cis plasmid are shown. The test clones in the panel, from left to right, are HEK293 parental control, 4A2 (DYNLL1+ / -) control, 4A2-3D3, 4A2-3B11, 4A2-3E6, 4A2-1H7, 4A2-4F12, 4A2-2C12, 4A2-3C3, and 4A2-2H7. [Figure 9-2] See the explanation in Figure 9-1. [Modes for carrying out the invention]

[0024] Detailed explanation In one embodiment, the disclosure provides recombinant cells suitable for generating recombinant viruses (e.g., AAV) or polypeptides (e.g., antibodies), wherein the cells include at least one modification that reduces or eliminates the activity of at least one endogenous gene or gene product in an apoptotic signaling pathway. In some embodiments, the at least one endogenous gene or gene product in an apoptotic signaling pathway is selected from the group consisting of ANGPT1, ARHGEF7, ARHGEF17, BID, BNIP1, C19orf2, CHST11, CUL1, DAP3, DYNLL1, FIS1, HMGB2, IGFBP3, NRG1, TNFRSF10C, TNFSF12, MAL, MAX, and VEGFB genes or gene products. In some embodiments, at least one endogenous gene or gene product in the apoptosis signaling pathway is selected from the group consisting of ARHGEF7, ARHGEF17, BNIP1, C19orf2, CHST11, DYNLL1, IGFBP3, MAX, and VEGFB genes or gene products. In some embodiments, at least one endogenous gene or gene product in the apoptosis signaling pathway is selected from the group consisting of DYNLL1 and MAX genes or gene products. In some embodiments, at least one endogenous gene or gene product in the apoptosis signaling pathway includes the DYNLL1 gene or gene product and a second gene or gene product selected from the group consisting of ANGPT1, ARHGEF7, ARHGEF17, BID, BNIP1, C19orf2, CHST11, CUL1, DAP3, FIS1, HMGB2, IGFBP3, NRG1, TNFRSF10C, TNFSF12, MAL, MAX, and VEGFB.In some embodiments, at least one endogenous gene or gene product in the apoptotic signaling pathway comprises the DYNLL1 gene or gene product and one or more genes or gene products selected from the group consisting of ANGPT1, ARHGEF7, ARHGEF17, BID, BNIP1, C19orf2, CHST11, CUL1, DAP3, FIS1, HMGB2, IGFBP3, NRG1, TNFRSF10C, TNFSF12, MAL, MAX, and VEGFB. In some embodiments, the cells are HEK293 cells, HEK293-derived cells, CHO cells, CHO-derived cells, HeLa cells, SF-9 cells, BHK cells, Vero cells, CAP cells, or PerC6 cells. In some embodiments, the cells are HEK293 cells. In some embodiments, the cells are HEK293-derived cells. In some embodiments, the cells are recombinant HEK293 cells capable of producing rAAV. In some embodiments, the cells are recombinant HEK293 cells capable of producing recombinant polypeptides (e.g., antibodies). In some embodiments, the cells are HEK293 cells adapted to suspension culture.

[0025] In a further embodiment, this specification provides a cell bank comprising a plurality of cells as described herein, and a cell culture comprising a plurality of cells as described herein. Also provided are methods for generating recombinant viruses (e.g., AAV) or polypeptides (e.g., antibodies) using the cells as described herein, and methods for increasing the generation of recombinant viruses (e.g., AAV) or polypeptides (e.g., antibodies) using the cells as described herein.

[0026] 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.

[0027] The term “HEK293 cells” refers to any cells whose lineage can ultimately be traced back to the original HEK293 cell line represented by ATCC catalog number CRL-1573(trademark), and / or which can be produced when human embryonic kidney cells are transformed with a fragment of adenovirus type 5 DNA as described (Graham et al. (1977) J. Gen. Virol. 36:59-74). In some embodiments, HEK293 cells include modifications that reduce or eliminate the activity of at least one gene or gene product selected from the group consisting of ANGPT1, ARHGEF7, ARHGEF17, BID, BNIP1, C19orf2, CHST11, CUL1, DAP3, DYNLL1, FIS1, HMGB2, IGFBP3, NRG1, TNFRSF10C, TNFSF12, MAL, MAX, and VEGFB genes or gene products. In some embodiments, HEK293 cells include mutations that reduce or eliminate the activity of the DYNLL1 gene or its gene product. In some embodiments, HEK293 cells include mutations that reduce or eliminate the activity of the IGFBP3 gene or its gene product. In some embodiments, HEK293 cells include mutations that reduce or eliminate the activity of the MAX gene or its gene product. In some embodiments, HEK293 cells include mutations that reduce or eliminate the activity of the DYNLL1 and MAX genes or their gene products. In some embodiments, HEK293 cells include mutations that reduce or eliminate the activity of the DYNLL1 gene or its gene product, as well as a second gene or gene product selected from the group consisting of ANGPT1, ARHGEF7, ARHGEF17, BID, BNIP1, C19orf2, CHST11, CUL1, DAP3, FIS1, HMGB2, IGFBP3, NRG1, TNFRSF10C, TNFSF12, MAL, MAX, and VEGFB.In some embodiments, HEK293 cells contain mutations that reduce or eliminate the activity of the DYNLL1 gene or gene product, as well as one or more genes or gene products selected from the group consisting of ANGPT1, ARHGEF7, ARHGEF17, BID, BNIP1, C19orf2, CHST11, CUL1, DAP3, FIS1, HMGB2, IGFBP3, NRG1, TNFRSF10C, TNFSF12, MAL, MAX, and VEGFB. In some embodiments, HEK293 cells are HEK293-derived cells derived from a HEK293 cell starting population by single-cell cloning to select cells having desirable properties (e.g., high levels of recombinant AAV production or high levels of recombinant polypeptide (e.g., antibody) production). In some embodiments, HEK293 cells are HEK293-derived cells derived from a HEK293 cell starting population by single-cell cloning to select cells capable of producing high recombinant AAV titers.

[0028] In some embodiments, HEK293 cells are genetically modified cells by introducing at least one modification that reduces or eliminates the activity of at least one gene or gene product selected from the group consisting of, for example, ANGPT1, ARHGEF7, ARHGEF17, BID, BNIP1, C19orf2, CHST11, CUL1, DAP3, DYNLL1, FIS1, HMGB2, IGFBP3, NRG1, TNFRSF10C, TNFSF12, MAL, MAX, and VEGFB genes or gene products. In some embodiments, HEK293 cells are genetically modified cells by introducing at least one modification that reduces or eliminates the activity of at least one gene or gene product selected from the group consisting of, for example, DYNLL1, IGFBP3, and MAX genes or gene products. In some embodiments, HEK293 cells are genetically modified cells to reduce or eliminate the activity of DYNLL1 and / or MAX genes or gene products. In some embodiments, HEK293 cells are genetically modified to reduce or eliminate the activity of the DYNLL1 gene or gene product, as well as a second gene or gene product selected from the group consisting of ANGPT1, ARHGEF7, ARHGEF17, BID, BNIP1, C19orf2, CHST11, CUL1, DAP3, FIS1, HMGB2, IGFBP3, NRG1, TNFRSF10C, TNFSF12, MAL, MAX, and VEGFB. In some embodiments, HEK293 cells are genetically modified cells that reduce or eliminate the activity of the DYNLL1 gene or gene product, as well as one or more genes or gene products selected from the group consisting of ANGPT1, ARHGEF7, ARHGEF17, BID, BNIP1, C19orf2, CHST11, CUL1, DAP3, FIS1, HMGB2, IGFBP3, NRG1, TNFRSF10C, TNFSF12, MAL, MAX, and VEGFB.In some embodiments, HEK293 cells are genetically modified cells by (1) introducing at least one modification that reduces or eliminates the activity of at least one gene or gene product selected from the group consisting of ANGPT1, ARHGEF7, ARHGEF17, BID, BNIP1, C19orf2, CHST11, CUL1, DAP3, DYNLL1, FIS1, HMGB2, IGFBP3, NRG1, TNFRSF10C, TNFSF12, MAL, MAX, and VEGFB genes or gene products, and (2) transposing one or more polynucleotides encoding at least one of the following: a packaged rAAV genome, an adenovirus helper function necessary for packaging, an AAV rep protein sufficient for packaging, and an AAV cap protein sufficient for packaging. In some embodiments, HEK293 cells are genetically modified cells by (1) introducing at least one modification that reduces or eliminates the activity of at least one gene or gene product selected from the group consisting of ANGPT1, ARHGEF7, ARHGEF17, BID, BNIP1, C19orf2, CHST11, CUL1, DAP3, DYNLL1, FIS1, HMGB2, IGFBP3, NRG1, TNFRSF10C, TNFSF12, MAL, MAX, and VEGFB genes or gene products, and (2) transposing a recombinant polynucleotide encoding a recombinant polypeptide of interest (e.g., an antibody).

[0029] "AAV" is an abbreviation for adeno-associated virus and can be used to mean the virus itself or its variants, derivatives, or pseudotypes. Unless otherwise required, the term encompasses all subtypes, as well as both the natural and recombinant forms. The abbreviation "rAAV" means 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 variants, 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.

[0030] 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.

[0031] 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, including any variants, derivatives, or pseudotypes (e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, or AAV10, or their derivatives / variants / pseudotypes). Such AAV serotypes and derivatives / variants / pseudotypes, as well as methods for generating such serotypes / variants / variants / pseudotypes, are known in the art (see, for example, Asokan et al., Mol.Ther. 20(4):699-708 (2012)).

[0032] 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.

[0033] 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, and AAV16, or their derivatives, modifiers, or pseudotypes. In some embodiments, the rAAV particles have an AAV capsid protein of a serotype of AAV8, AAV9, or their derivatives, modifiers, or pseudotypes.

[0034] The term "antibody" means an immunoglobulin molecule (or group of immunoglobulin molecules) that recognizes and specifically binds to a target (e.g., a protein, polypeptide, peptide, carbohydrate, polynucleotide, lipid, or a combination thereof) through at least one antigen-recognizing site within the variable region of the immunoglobulin molecule. As used herein, the terms "antibody (singular)" and "antibody (plural)" are technical terms and may be used interchangeably herein, and refer to molecules having an antigen-binding site that specifically binds to an antigen.

[0035] Examples of antibodies include monoclonal antibodies, recombinant antibodies, human antibodies, humanized antibodies, resurfaced antibodies, chimeric antibodies, immunoglobulins, synthetic antibodies, tetrameric antibodies containing two heavy chains and two light chain molecules, antibody light chain monomers, antibody heavy chain monomers, antibody light chain dimers, antibody heavy chain dimers, antibody light chain-antibody heavy chain pairs, intrabodies, heteroconjugated antibodies, single-domain antibodies, monovalent antibodies, single-chain antibodies or single-chain Fv(scFv), aphibodies, Fab fragments, F(ab')2 fragments, disulfide-bonded Fv(sdFv), anti-idiotype (anti-Id) antibodies (e.g., anti-anti-Id antibodies), bispecific antibodies, and multispecific antibodies. Antibodies can be immunoglobulin molecules of any type (e.g., IgG, IgE, IgM, IgD, IgA, or IgY), any class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, or IgA2), or any subclass (isotype) of these (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), based on the contents of the heavy chain constant domains, respectively, referred to as alpha, delta, epsilon, gamma, and mu. Different classes of immunoglobulins have different known subunit structures and three-dimensional configurations. Antibodies may be naked or conjugated or fused with other molecules (e.g., toxins, radioisotopes, other polypeptides, etc.).

[0036] The term "antibody fragment" refers to a portion of an intact antibody. "Antigen-binding fragment" refers to a portion of an intact antibody that binds to an antigen. Antigen-binding fragments may contain the antigen-determining variable region of an intact antibody. Examples of antibody fragments, but are not limited to, include Fab, Fab', F(ab')2, Fv fragments, linear antibodies, and single-chain antibodies.

[0037] 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.

[0038] 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.

[0039] 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 approximately 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%.

[0040] 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.

[0041] 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".

[0042] When the term "and / or" is used herein in an expression 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 an expression 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).

[0043] Where embodiments of the Disclosure describe a Markush group or other alternative groups, the Method of the Disclosure encompasses not only the entire group as a whole, but also each individual member of the group, all possible subgroups of a principal group, and even principal groups in which one or more group members are absent. The Method of the Disclosure also anticipates the express exclusion of one or more of the group members in the Method of the Disclosure.

[0044] Recombinant cells In one embodiment, the disclosure provides recombinant cells suitable for generating recombinant viruses (e.g., AAV) or polypeptides (e.g., antibodies), wherein the cells include at least one modification that reduces or eliminates the activity of at least one endogenous gene or gene product in an apoptotic signaling pathway. In some embodiments, the cells are immortalized cells. In some embodiments, the cells are mammalian cells. In some embodiments, the cells are immortalized mammalian cells. In some embodiments, the cells are HEK293 cells, HEK293-derived cells, CHO cells, CHO-derived cells, HeLa cells, SF-9 cells, BHK cells, Vero cells, CAP cells, or PerC6 cells. In some embodiments, the cells are HEK293 cells. In some embodiments, the cells are HEK293-derived cells.

[0045] In some embodiments, the cells are adherent cells. In some embodiments, the cells are suspension cells. In some embodiments, the cells are suspension-adapted HEK293 cells. In some embodiments, the cells are suspension-adapted HEK293-derived cells.

[0046] In some embodiments, at least one endogenous gene or gene product in the apoptosis signaling pathway is selected from the group consisting of ANGPT1, ARHGEF7, ARHGEF17, BID, BNIP1, C19orf2, CHST11, CUL1, DAP3, DYNLL1, FIS1, HMGB2, IGFBP3, NRG1, TNFRSF10C, TNFSF12, MAL, MAX, and VEGFB genes or gene products. In some embodiments, at least one endogenous gene or gene product in the apoptosis signaling pathway is selected from the group consisting of ARHGEF7, ARHGEF17, BNIP1, C19orf2, CHST11, DYNLL1, IGFBP3, MAX, and VEGFB genes or gene products. In some embodiments, at least one endogenous gene or gene product in the apoptosis signaling pathway is selected from the group consisting of DYNLL1, IGFBP3, and MAX genes or gene products. In some embodiments, the endogenous gene or gene product comprises the DYNLL1 and / or MAX gene or gene product. In some embodiments, the endogenous gene or gene product comprises the DYNLL1 gene or gene product and a second gene or gene product selected from the group consisting of ANGPT1, ARHGEF7, ARHGEF17, BID, BNIP1, C19orf2, CHST11, CUL1, DAP3, FIS1, HMGB2, IGFBP3, NRG1, TNFRSF10C, TNFSF12, MAL, MAX, and VEGFB. In some embodiments, at least one endogenous gene or gene product comprises the DYNLL1 gene or gene product and one or more genes or gene products selected from the group consisting of ANGPT1, ARHGEF7, ARHGEF17, BID, BNIP1, C19orf2, CHST11, CUL1, DAP3, FIS1, HMGB2, IGFBP3, NRG1, TNFRSF10C, TNFSF12, MAL, MAX, and VEGFB.

[0047] The human angiopoietin 1 gene (also known as ANGPT1, AGP1, AGPT, AGPT-1, ANG1, and HAE5) encodes a secreted glycoprotein belonging to the angiopoietin family. The angiopoietin 1 protein binds to the TEK / TIE2 receptor and activates it by inducing its dimerization and tyrosine phosphorylation. This plays a crucial role in regulating angiogenesis, endothelial cell survival, proliferation, migration, adhesion, and cell diffusion, as well as in the rearrangement of the actin cytoskeleton and the maintenance of vasoquia. In quiescent blood vessels, ANGPT1 oligomers recruit TEK to intercellular contact and form complexes with TEK molecules from neighboring cells, which leads to the preferential activation of phosphatidylinositol 3 kinase and the AKT1 signaling cascade. In migrating endothelial cells lacking cell-cell adhesion, ANGT1 recruits TEK to contact the extracellular matrix, leading to the formation of adhesion plaque complexes, activation of PTK2 / FAK, and activation of downstream kinases MAPK1 / ERK2 and MAPK3 / ERK1, ultimately stimulating angiogenesis. In some embodiments, the human ANGPT1 gene refers to the gene represented by NCBI gene ID number 284. In some embodiments, the human ANGPT1 gene encodes human ANGPT1 mRNA with NCBI accession number NM_001146. In some embodiments, the human ANGPT1 gene encodes one or more of the following human ANGPT1 mRNA variants: NCBI accession numbers NM_001146.5, NM_001199859.3, NM_001314051.1, and XR_928319.2. In some embodiments, the human ARHGEF7 gene encodes one or more of the following human ARHGEF7 polypeptide isoforms: UniProt accession numbers Q15389-1 and Q15389-2.

[0048] The human Rho guanine nucleotide exchange factor 7 gene (also known as ARHGEF7, P85SPR, COOL1, PIXB, P85, PAK-interaction exchange factor beta, Nbla10314, KIAA0142, beta-PIX, P85COOL1, P50BP, PAK3, P50, Rho guanine nucleotide exchange factor (GEF) 7, SH3 domain-containing proline-rich protein, DKFZp686C12170, DKFZp761K1021, COOL-1, beta-Pix, PAK3BP) encodes a protein belonging to the cytoplasmic protein family that activates the Ras-like family of Rho proteins by exchanging bound GDP for GTP. The ARHGEF7 protein forms a complex with the small GTP-binding protein Rac1, recruiting Rac1 to membrane ruffles and adhesion plaques. For this gene, multiple alternative splicing transcript variants encoding different isoforms have been observed. In some embodiments, the human ARHGEF7 gene refers to the gene represented by NCBI gene ID number 8874. In some embodiments, the human ARHGEF7 gene encodes the human ARHGEF7 mRNA with NCBI accession number NM_003899.5.In some embodiments, the human ARHGEF7 gene encodes one or more of the following human ARHGEF7 mRNA variants: NCBI accession numbers NM_001113511.2, NM_001113512.2, NM_001113513.2, NM_001320851.2, NM_001320852.1, NM_001320853.2, NM_001320854.2, NM_001330597.2, NM_00133 0598.2, NM_001354046.1, NM_001354047.1, NM_001354048.1, NM_001354049.2, NM_001354050.2, N M_001354051.2, NM_001354052.2, NM_001354053.2, NM_001354054.2, NM_001354055.2, NM_001354 056.1, NM_001354057.2, NM_001354058.2, NM_001354059.2, NM_001354060.2, NM_001354061.2, NM _003899.5, NM_145735.3, XM_006719956.3, XM_011521133.2, XM_017020814.1, XM_017020815.1, X M_017020816.1, XM_017020819.1, XM_017020820.1, XM_017020821.2, XM_017020822.1, XM_017020 823.2, XM_017020828.1, XM_017020829.2, XM_024449428.1, XR_001749712.1, and XR_002957481.1. In some embodiments, the human ARHGEF7 gene encodes one or more of the following human ARHGEF7 polypeptide isoforms: UniProt accession numbers Q14155-1, Q14155-2, Q14155-3, Q14155-4, Q14155-5, and Q14155-6.

[0049] The human Rho guanine nucleotide exchange factor (GEF) 17 gene (also known as ARHGEF17, TEM4, RHOGEF17, P164RHOGEF, p164-RhoGEF) encodes a protein belonging to the cytoplasmic protein family that activates the Ras-like family of Rho proteins by exchanging bound GDP for GTP. The ARHGEF7 protein acts as a guanine nucleotide exchange factor (GEF) in RhoA GTPases. For this gene, multiple alternative splicing transcript variants encoding different isoforms have been observed. In some embodiments, the human ARHGEF17 gene refers to the gene represented by NCBI gene ID number 9828. In some embodiments, the human ARHGEF17 gene encodes human ARHGEF17 mRNA with NCBI accession number NM_014786. In some embodiments, the human ARHGEF17 gene encodes one or more of the following human ARHGEF17 mRNA variants: NCBI accession numbers NM_014786.4, XM_017018623.1, XM_017018624.1, XR_001748051.1, XR_001748052.1, and XR_950116.2. In some embodiments, the human ARHGEF17 gene encodes the human ARHGEF7 polypeptide with UniProt accession number Q96PE2.

[0050] The human BH3 interaction domain death agonist gene (also known as BID and FP497) encodes a death agonist that regulates apoptosis by heterodimerizing with the agonist BAX or the antagonist BCL2. BID is a member of the BCL-2 family of cell death regulators and mediates caspase-8 (CASP8)-induced mitochondrial damage. CASP8 cleaves BID, and the COOH-terminal portion moves to the mitochondria, where it induces cytochrome c release. Multiple alternative splicing transcript variants have been found. In some embodiments, the human BID gene refers to the gene represented by NCBI gene ID number 637. In some embodiments, the human BID gene encodes human BID mRNA with NCBI accession number NM_001196. In some embodiments, the human BID gene encodes one or more of the following human BID mRNA variants: NCBI accession numbers NM_001196.3, NM_001244567.1, NM_001244569.1, NM_001244570.1, NM_001244572.1, NM_197966.2, and NM_197967.2. In some embodiments, the human BID gene encodes one or more of the human BID polypeptide isoforms: UniProt accession numbers P55957-1, P55957-2, P55957-3, and P55957-4.

[0051] The human BCL2 / adenovirus E1B 19kDa interacting protein 1 gene (also known as BNIP1, BCL2 interacting protein 1, SEC20, TRG-8, and Nip1) is a member of the BCL2 / adenovirus E1B 19kDa-interacting protein (BNIP) family. BNIP1 interacts with the E1B 19kDa protein, thereby protecting cells from virus-induced cell death. BNIP1 also interacts with the E1B 19kDa-like sequence of BCL2, another apoptotic protective agent. Furthermore, BNIP1 is involved in vesicular transport to the endoplasmic reticulum. In some embodiments, the human BNIP1 gene refers to the gene represented by NCBI gene ID number 662. In some embodiments, the human BNIP1 gene encodes human BNIP1 mRNA with NCBI accession number NM_001205. In some embodiments, the human BNIP1 gene encodes one or more of the following human BNIP1 mRNA variants: NCBI accession numbers NM_001205.3, NM_013978.3, NM_013979.3, NM_013980.3, XM_011534638.1, and XM_011534639.1. In some embodiments, the human BNIP1 gene encodes one or more of the human BNIP1 polypeptide isoforms: UniProt accession numbers Q12981-1, Q12981-2, Q12981-3, and Q12981-4.

[0052] The human chromosome 19 open reading frame 2 gene (also known as C19orf2, URI1, RMP, URI, NNX3, and PPP1R19) is a member of the prefoldin family of molecular chaperones. The C19orf2 protein functions as a scaffolding protein, partly through interaction with the RNA polymerase II subunit RPB5, and is involved in ubiquitination and transcription. C19orf2 may be involved in several malignancies, including ovarian cancer and hepatocellular carcinoma. In some embodiments, the human C19orf2 gene refers to the gene represented by NCBI gene ID number 8725. In some embodiments, the human C19orf2 gene encodes human C19orf2 mRNA with NCBI accession number NM_003796. In some embodiments, the human C19orf2 gene encodes one or more of the following human C19orf2 mRNA variants: NCBI accession numbers NM_001252641.2, NM_003796.3, NR_045557.1, XM_005259362.2, XM_005259363.4, XM_011527435.2, and XM_024451751.1. In some embodiments, the human C19orf2 gene encodes one or more of the human C19orf2 polypeptide isoforms: UniProt accession numbers O94763-1, O94763-2, O94763-3, and O94763-4.

[0053] The human carbohydrate (chondroitin 4) sulfotransferase 11 gene (also known as CHST11, C4ST, C4ST1, OCBMD, C4ST-1, and HSA269537) encodes a protein belonging to the sulfotransferase 2 family. CHST11 is localized to the Golgi membrane and catalyzes the transfer of sulfate to position 4 of the N-acetylgalactosamine (GalNAc) residue of chondroitin. Chondroitin sulfate constitutes the main proteoglycan present in cartilage and is distributed on the surface of many cells and extracellular matrix. A chromosomal translocation, t(12;14)(q23;q32), associated with CHST11 and IgH has been reported in patients with B-cell chronic lymphocytic leukemia. In some embodiments, the human CHST11 gene refers to the gene represented by NCBI gene ID number 50515. In some embodiments, the human CHST11 gene encodes human CHST11 mRNA with NCBI accession number NM_018413. In some embodiments, the human CHST11 gene encodes one or more of the following human CHST11 mRNA variants: NCBI accession numbers NM_001173982.2, NM_018413.6, and XM_017019369.1. In some embodiments, the human CHST11 gene encodes one or more of the human CHST11 polypeptide isoforms: UniProt accession numbers Q9NPF2-1 and Q9NPF2-2.

[0054] The human cullin1 gene (also known as CUL1) encodes a protein expected to enable ubiquitin protein ligase binding activity and ubiquitin-protein transferase activity. CUL1 is involved in SCF-dependent proteasome ubiquitin-dependent protein catabolism and protein ubiquitination. CUL1 is located in the cell membrane and is part of the Parkin-FBXW7-Cul1 ubiquitin ligase complex and the SCF ubiquitin ligase complex. In some embodiments, the human CUL1 gene refers to the gene represented by NCBI gene ID number 8454. In some embodiments, the human CUL1 gene encodes human CUL1 mRNA with NCBI accession number NM_003592. In some embodiments, the human CUL1 gene encodes one or more of the following human CUL1 mRNA variants: NCBI accession numbers NM_001370660.1, NM_001370661.1, NM_001370662.1, NM_001370663.1, NM_001370664.1, and NM_003592.3. In some embodiments, the human CUL1 gene encodes the human CUL1 polypeptide with UniProt accession number Q13616.

[0055] The three human cell death-related protein genes (also known as DAP3, DAP-3, S29mt, MRPS29, MRP-S29, and bMRP-10) encode mitrobosome 28S subunit proteins that are also involved in the apoptotic pathway initiated by tumor necrosis factor alpha, Fas ligand, and gamma interferon. DAP3 may bind to ATP / GTP and may be a functional partner of mitrobosome protein S27. In some embodiments, the human DAP3 gene refers to the gene represented by NCBI gene ID number 7818. In some embodiments, the human DAP3 gene encodes human DAP3 mRNA with NCBI accession number NM_004632. In some embodiments, the human DAP3 gene is the following human DAP3 Encodes one or more mRNA variants: NCBI accession numbers NM_001199849.1, NM_001199850.1, NM_001199851.1, NM_004632.4, NM_033657.2, XM_005245480.2, XM_005245481.2, XM_01700 2289.1, XM_017002290.1, XM_017002291.1, XM_017002292.1, XM_017002293.1, XM_017002294.1, XM_017002295.1, XM_024449697.1, XM_024449698.1, and XM_024449700.1. In some embodiments, the human DAP3 gene encodes one or more human DAP3 polypeptide isoforms: UniProt accession numbers P51398-1, P51398-2, and P51398-3.

[0056] The human dynein light chain LC8-1 gene (also known as DYNLL1, LC8, PIN, DLC1, DLC8, LC8a, DNCL1, hdlc1, and DNCLC1) encodes a cytoplasmic dynein component. Cytoplasmic dynein is a large enzyme complex with a molecular weight of approximately 1,200 kD. Dynein consists of two force-generating heads, primarily formed from the dynein heavy chain, and a stalk that binds the heads to a basal domain (containing a variety of auxiliary intermediate chains). This complex is involved in intracellular transport and motility. DYNLL1 is a light chain and exists as part of this complex, but it also physically interacts with neural nitric oxide synthase, inhibiting its activity. DYNLL1 binding destabilizes the neural nitric oxide synthase dimer, a conformation necessary for its activity, and can regulate many biological processes through its effects on nitric oxide synthase activity. In some embodiments, the human DYNLL1 gene refers to the gene represented by NCBI gene ID number 8655. In some embodiments, the human DYNLL1 gene encodes human DYNLL1 mRNA with NCBI accession number NM_003746. In some embodiments, the human DYNLL1 gene encodes one or more of the following human DYNLL1 mRNA variants: NCBI accession numbers NM_001037494.2, NM_001037495.2, and NM_003746.3. In some embodiments, the human DYNLL1 gene encodes human DYNLL1 polypeptide with UniProt accession number P63167.

[0057] The human fission 1 (mitochondrial outer membrane) homolog (S. cerevisiae) gene (also known as FIS1, TTC11, and CGI-135) encodes a protein involved in several processes, including calcium-mediated signaling using intracellular calcium sources, intracellular calcium ion homeostasis, and mitochondrial organization. FIS1 acts upstream or within mitochondrial morphogenesis. FIS1 is located in mitochondria and peroxisomes. FIS1 is an essential component of the mitochondrial outer membrane and also an essential component of the peroxisomal membrane. In some embodiments, the human FIS1 gene refers to the gene represented by NCBI gene ID number 51024. In some embodiments, the human FIS1 gene encodes human FIS1 mRNA with NCBI accession number NM_016068. In some embodiments, the human FIS1 gene encodes human FIS1 polypeptide with UniProt accession number Q9Y3D6.

[0058] The human high-mobility group box 2 gene (also known as HMGB2 and HMG2) encodes a member of the non-histone chromosome high-mobility group protein family. Proteins in this family are chromatin-associative and ubiquitously distributed within the nucleus of higher eukaryotic cells. In vitro studies have demonstrated that HMGB2 can efficiently bend DNA and form DNA circles. These studies suggest a role in promoting cooperative interactions between cis-acting proteins by enhancing DNA flexibility. HMGB2 has also been reported to be involved in DNA double-strand break repair and the final ligation step in the DNA end-joining process of V(D)J recombination. In some embodiments, the human HMGB2 gene refers to the gene represented by NCBI gene ID number 3148. In some embodiments, the human HMGB2 gene encodes human HMGB2 mRNA with NCBI accession number NM_002129. In some embodiments, the human HMGB2 gene encodes one or more of the following human HMGB2 mRNA variants: NCBI accession numbers NM_001130688.1, NM_001130689.1, and NM_002129.4. In some embodiments, the human HMGB2 gene encodes the human HMGB2 polypeptide with UniProt accession number P26583.

[0059] The human insulin-like growth factor-binding protein 3 gene (also known as IGFBP3, IBP3, and BP-53) is a member of the insulin-like growth factor-binding protein (IGFBP) family and encodes a protein having an IGFBP domain and a thyroglobulin type I domain. IGFBP3 forms a ternary complex with insulin-like growth factor acid-unstable subunits (IGFALS) and insulin-like growth factor (IGF) I or II. IGFBP3 also exhibits IGF-independent antiproliferative and apoptotic activity mediated by its receptor TMEM219 / IGFBP-3R. In some embodiments, the human IGFBP3 gene refers to the gene represented by NCBI gene ID number 3486. In some embodiments, the human IGFBP3 gene encodes human IGFBP3 mRNA with NCBI accession number NM_001013398. In some embodiments, the human IGFBP3 gene encodes one or more of the following human IGFBP3 mRNA variants: NCBI accession numbers NM_000598.5 and NM_001013398.2. In some embodiments, the human IGFBP3 gene encodes one or more of the human IGFBP3 polypeptide isoforms: UniProt accession numbers P17936-1 and P17936-2.

[0060] The human neuregulin 1 gene (also known as NRG1, GGF, HGL, HRG, NDF, ARIA, GGF2, HRG1, HRGA, SMDF, MST131, MSTP131, and NRG1-IT2) encodes a membrane glycoprotein that mediates intercellular signaling and plays a critical role in the growth and development of multiple organ systems. This gene generates various distinct isoforms through the use of alternative promoters and splicing. These isoforms are expressed in a tissue-specific manner, exhibit significantly different structures, and are classified as types I, II, III, IV, V, and VI. Dysregulation of NRG1 has been associated with diseases such as cancer, schizophrenia, and bipolar disorder (BPD). NRG1 consists of an NH2-terminal ECD (extracellular structural domain), a transmembrane structural domain, and a highly conserved COOH-terminal ICD (intracellular structural domain). Ectopic expression of NRG1 leads to NRG1-ICD-dependent apoptosis. In some embodiments, the human NRG1 gene refers to the gene represented by NCBI gene ID number 3084. In some embodiments, the human NRG1 gene encodes human NRG1 mRNA with NCBI accession number NM_004495.In some embodiments, the human NRG1 gene encodes one or more of the following human NRG1 mRNA variants: NCBI accession numbers NM_001159995.3, NM_001159996.2, NM_001159999.3, NM_001160001.3, NM_001160002.2, NM_001160004.3, NM_001160005.1, NM_001160007.2, NM_001160008.2, NM_001322197.2, NM_001322201.2, NM_001322202.2, NM_001322205.2, NM_001322206.2, NM_001322207.2 , NM_004495.4, NM_013956.5, NM_013957.5, NM_013958.3, NM_013959.3, NM_0 13960.5, NM_013962.2, NM_013964.5, XM_005273486.3, XM_005273487.3, XM_0 06716335.3, XM_011544512.2, XM_017013365.2, XM_017013366.2, XM_017013 367.1, XM_017013368.2, XM_017013369.2, XM_017013370.1, XM_017013371.2, XM_017013372.2, and XM_024447143.1. In some embodiments, the human NRG1 gene encodes one or more of the human NRG1 polypeptide isoforms: UniProt accession numbers Q02297-1, Q02297-2, Q02297-3, Q02297-4, Q02297-5, Q02297-6, Q02297-7, Q02297-8, Q02297-9, Q02297-10, and Q02297-11.

[0061] The human tumor necrosis factor receptor superfamily, member 10c, a decoy gene without an intracellular domain (also known as TNFRSF10C, LIT, DCR1, TRID, CD263, TRAILR3, TRAIL-R3, and DCR1-TNFR), encodes a member of the TNF receptor superfamily. TNFRSF10C contains an extracellular TRAIL-binding domain and a transmembrane domain, but lacks a cytoplasmic death domain. TNFRSF10C is not capable of inducing apoptosis and is thought to function as an antagonist receptor protecting cells from TRAIL-induced apoptosis. TNFRSF10C has been found to be a p53 regulatory DNA damage-inducing gene. TNFRSF10C expression was detected in many normal tissues but not in most cancer cell lines. This explains why cancer cells are specifically sensitive to the apoptosis-inducing activity of TRAIL. In some embodiments, the human TNFRSF10C gene refers to the gene represented by NCBI gene ID number 8794. In some embodiments, the human TNFRSF10C gene encodes human TNFRSF10C mRNA with NCBI accession number NM_003841. In some embodiments, the human TNFRSF10C gene encodes human TNFRSF10C polypeptide with UniProt accession number O14798.

[0062] The human tumor necrosis factor (ligand) superfamily, consisting of 12 member genes (also known as TNFSF12, APO3L, DR3LG, TWEAK, and TNLG4A), encodes cytokines belonging to the tumor necrosis factor (TNF) ligand family. TNFSF12 is a ligand for the FN14 / TWEAKR receptor. TNFSF12 has signaling functions that overlap with TNF but exhibits a broader tissue distribution. TNFSF12 exists in both membrane-bound and secretory forms and can induce apoptosis via multiple cell death pathways in a cell type-specific manner. TNFSF12 is also known to promote endothelial cell proliferation and migration and act as a regulator of angiogenesis. In some embodiments, the human TNFSF12 gene refers to the gene represented by NCBI gene ID number 8742. In some embodiments, the human TNFSF12 gene encodes human TNFSF12 mRNA with NCBI accession number NM_003809. In some embodiments, the human TNFSF12 gene encodes one or more of the following human TNFSF12 mRNA variants: NCBI accession numbers NM_003809.3 and NR_037146.2. In some embodiments, the human TNFSF12 gene encodes one or more of the human TNFSF12 polypeptide isoforms: UniProt accession numbers O43508-1 and O43508-2.

[0063] The human MAL, T cell differentiation protein gene (also known as MAL, MVP17, and VIP17), encodes a highly hydrophobic complex membrane protein belonging to the MAL family of proteolipids. MAL is localized in the endoplasmic reticulum of T cells and is a candidate linker protein in T cell signaling. Furthermore, this proteolipid is localized in compact myelin of nervous system cells and is associated with myelin biosynthesis and / or function. MAL is involved in the formation, stabilization, and maintenance of sphingoglycolipid-rich membrane microdomains. Downregulation of the MAL gene is associated with various human epithelial malignancies. In some embodiments, the human MAL gene refers to the gene represented by NCBI gene ID number 4118. In some embodiments, the human MAL gene encodes human MAL mRNA with NCBI accession number NM_002371. In some embodiments, the human MAL gene encodes one or more of the following human MAL mRNA variants: NCBI accession numbers NM_002371.4, NM_022438.2, NM_022439.2, and NM_022440.2. In some embodiments, the human MAL gene encodes one or more of the following human MAL polypeptide isoforms: UniProt accession numbers P21145-1, P21145-2, P21145-3, and P21145-4.

[0064] The human MYC-related factor X gene (also known as MAX and bHLHd4) encodes a member of the basic helix-loop-helix leucine zipper (bHLHZ) family of transcription factors. MAX can form homodimers and heterodimers with other family members, including Mad, Mxi1, and Myc. Myc is an oncoprotein involved in cell proliferation, differentiation, and apoptosis. Homodimers and heterodimers compete for a common DNA target site (E-box), and rearrangements between these dimeric forms result in a complex transcriptional regulatory system. Mutations in MAX have been reported to be associated with hereditary pheochromocytoma. In some embodiments, the human MAX gene refers to the gene represented by NCBI gene ID number 4149. In some embodiments, the human MAX gene encodes human MAX mRNA with NCBI accession number NM_002382. In some embodiments, the human MAX gene is the following human MAX Encodes one or more mRNA variants: NCBI accession numbers NM_001271068.1, NM_001271069.1, NM_001320415.2, NM_002382.5, NM_145112.3, NM_145113.3, NM_145114.2, NM_197957.3, NR_07 3137.1, NR_073138.1, XM_011536773.3, XM_017021312.2, XM_017021313.1, XR_001750326.2, XR_001750327.2, XR_002957553.1, XR_943450.3, XR_943451.3, and XR_943452.3. In some embodiments, the human MAX gene encodes one or more of the human MAX polypeptide isoforms: UniProt accession numbers P61244-1, P61244-2, P61244-3, P61244-4, P61244-5, and P61244-6.

[0065] The human vascular endothelial growth factor B gene (also known as VEGFB, VRF, and VEGFL) encodes a member of the PDGF (platelet-derived growth factor) / VEGF (vascular endothelial growth factor) family. Members of the VEGF family regulate angiogenesis and are involved in endothelial cell physiology. VEGFB is a ligand for VEGFR-1 (vascular endothelial growth factor receptor 1) and NRP-1 (neuropilin-1). By binding to VEGFR-1, VEGF-B can regulate angiogenesis, redox reactions, and apoptosis. VEGF-B is a potent survival factor for various cell types by inhibiting apoptosis through the repression of the BH3 protein and other apoptosis / cell death-related genes. In some embodiments, the human VEGFB gene refers to the gene represented by NCBI gene ID number 7423. In some embodiments, the human VEGFB gene encodes human VEGFB mRNA with NCBI accession number NM_003377. In some embodiments, the human VEGFB gene encodes one or more of the following human VEGFB mRNA variants: NCBI accession numbers NM_001243733.2 and NM_003377.5. In some embodiments, the human VEGFB gene encodes one or more of the following human VEGFB polypeptide isoforms: UniProt accession numbers P49765-1 and P49765-2.

[0066] In some embodiments, at least one endogenous gene or gene product in the apoptotic signaling pathway is neither Bax (NCBI gene ID number 581) nor Bak (NCBI gene ID number 578).

[0067] In some embodiments, the cells include modifications that reduce or eliminate the activity of at least one endogenous gene or gene product selected from the group consisting of ANGPT1, ARHGEF7, ARHGEF17, BID, BNIP1, C19orf2, CHST11, CUL1, DAP3, DYNLL1, FIS1, HMGB2, IGFBP3, NRG1, TNFRSF10C, TNFSF12, MAL, MAX, and VEGFB genes or gene products. In some embodiments, the cells include modifications that reduce or eliminate the activity of the ANGPT1 gene or gene product. In some embodiments, the cells include modifications that reduce or eliminate the activity of the ARHGEF7 gene or gene product. In some embodiments, the cells include modifications that reduce or eliminate the activity of the ARHGEF17 gene or gene product. In some embodiments, the cells include modifications that reduce or eliminate the activity of the BID gene or gene product. In some embodiments, the cells include modifications that reduce or eliminate the activity of the BNIP1 gene or gene product. In some embodiments, the cells include modifications that reduce or eliminate the activity of the C19orf2 gene or gene product. In some embodiments, the cells include modifications that reduce or eliminate the activity of the CHST11 gene or gene product. In some embodiments, the cells include modifications that reduce or eliminate the activity of the CUL1 gene or gene product. In some embodiments, the cells include modifications that reduce or eliminate the activity of the DAP3 gene or gene product. In some embodiments, the cells include modifications that reduce or eliminate the activity of the DYNLL1 gene or gene product. In some embodiments, the cells include modifications that reduce or eliminate the activity of the FIS1 gene or gene product. In some embodiments, the cells include modifications that reduce or eliminate the activity of the HMGB2 gene or gene product. In some embodiments, the cells include modifications that reduce or eliminate the activity of the IGFBP3 gene or gene product. In some embodiments, the cells include modifications that reduce or eliminate the activity of the NRG1 gene or gene product.In some embodiments, the cells include modifications that reduce or eliminate the activity of the TNFRSF10C gene or its gene product. In some embodiments, the cells include modifications that reduce or eliminate the activity of the TNFSF12 gene or its gene product. In some embodiments, the cells include modifications that reduce or eliminate the activity of the MAL gene or its gene product. In some embodiments, the cells include modifications that reduce or eliminate the activity of the MAX gene or its gene product. In some embodiments, the cells include modifications that reduce or eliminate the activity of the VEGFB gene or its gene product. In some embodiments, the cells are HEK293 cells. In some embodiments, the cells are HEK293-derived cells. In some embodiments, the cells are suspension-adapted HEK293 cells. In some embodiments, the cells are suspension-adapted HEK293-derived cells.

[0068] In some embodiments, the cells include modifications that reduce or eliminate the activity of at least two endogenous genes or gene products selected from the group consisting of ANGPT1, ARHGEF7, ARHGEF17, BID, BNIP1, C19orf2, CHST11, CUL1, DAP3, DYNLL1, FIS1, HMGB2, IGFBP3, NRG1, TNFRSF10C, TNFSF12, MAL, MAX, and VEGFB genes or gene products. In some embodiments, the cells include a first modification that reduces or eliminates the activity of the ARHGEF7 gene or gene product, and a second modification that reduces or eliminates the activity of a second endogenous gene or gene product selected from the group consisting of ARHGEF17, BNIP1, C19orf2, CHST11, DYNLL1, IGFBP3, MAX, and VEGFB genes or gene products. In some embodiments, the cells include a first modification that reduces or eliminates the activity of the ARHGEF17 gene or gene product, and a second modification that reduces or eliminates the activity of a second endogenous gene or gene product selected from the group consisting of ARHGEF7, BNIP1, C19orf2, CHST11, DYNLL1, IGFBP3, MAX, and VEGFB genes or gene products. In some embodiments, the cells include a first modification that reduces or eliminates the activity of the BNIP1 gene or gene product, and a second modification that reduces or eliminates the activity of a second endogenous gene or gene product selected from the group consisting of ARHGEF7, ARHGEF17, C19orf2, CHST11, DYNLL1, IGFBP3, MAX, and VEGFB genes or gene products.In some embodiments, the cells include a first modification that reduces or eliminates the activity of the C19orf2 gene or gene product, and a second modification that reduces or eliminates the activity of a second endogenous gene or gene product selected from the group consisting of ARHGEF7, ARHGEF17, BNIP1, CHST11, DYNLL1, IGFBP3, MAX, and VEGFB genes or gene products. In some embodiments, the cells include a first modification that reduces or eliminates the activity of the CHST11 gene or gene product, and a second modification that reduces or eliminates the activity of a second endogenous gene or gene product selected from the group consisting of ARHGEF7, ARHGEF17, BNIP1, C19orf2, DYNLL1, IGFBP3, MAX, and VEGFB genes or gene products. In some embodiments, the cells include a first modification that reduces or eliminates the activity of the DYNLL1 gene or gene product, and a second modification that reduces or eliminates the activity of a second endogenous gene or gene product selected from the group consisting of ARHGEF7, ARHGEF17, BNIP1, C19orf2, CHST11, IGFBP3, MAX, and VEGFB genes or gene products. In some embodiments, the cells include a first modification that reduces or eliminates the activity of the DYNLL1 gene or gene product, and a second modification that reduces or eliminates the activity of the MAX gene or gene product. In some embodiments, the cells include a first modification that reduces or eliminates the activity of the DYNLL1 gene or gene product, and a second modification that reduces or eliminates the activity of the ANGPT1 gene or gene product. In some embodiments, the cells include a first modification that reduces or eliminates the activity of the DYNLL1 gene or gene product, and a second modification that reduces or eliminates the activity of the ARHGEF7 gene or gene product. In some embodiments, the cells include a first modification that reduces or eliminates the activity of the DYNLL1 gene or gene product, and a second modification that reduces or eliminates the activity of the ARHGEF17 gene or gene product. In some embodiments, the cells include a first modification that reduces or eliminates the activity of the DYNLL1 gene or gene product, and a second modification that reduces or eliminates the activity of the BID gene or gene product.In some embodiments, the cells include a first modification that reduces or eliminates the activity of the DYNLL1 gene or gene product, and a second modification that reduces or eliminates the activity of the BNIP1 gene or gene product. In some embodiments, the cells include a first modification that reduces or eliminates the activity of the DYNLL1 gene or gene product, and a second modification that reduces or eliminates the activity of the C19orf2 gene or gene product. In some embodiments, the cells include a first modification that reduces or eliminates the activity of the DYNLL1 gene or gene product, and a second modification that reduces or eliminates the activity of the CHST11 gene or gene product. In some embodiments, the cells include a first modification that reduces or eliminates the activity of the DYNLL1 gene or gene product, and a second modification that reduces or eliminates the activity of the CUL1 gene or gene product. In some embodiments, the cells include a first modification that reduces or eliminates the activity of the DYNLL1 gene or gene product, and a second modification that reduces or eliminates the activity of the DAP3 gene or gene product. In some embodiments, the cells include a first modification that reduces or eliminates the activity of the DYNLL1 gene or gene product, and a second modification that reduces or eliminates the activity of the FIS1 gene or gene product. In some embodiments, the cells include a first modification that reduces or eliminates the activity of the DYNLL1 gene or gene product, and a second modification that reduces or eliminates the activity of the HMGB2 gene or gene product. In some embodiments, the cells include a first modification that reduces or eliminates the activity of the DYNLL1 gene or gene product, and a second modification that reduces or eliminates the activity of the IGFBP3 gene or gene product. In some embodiments, the cells include a first modification that reduces or eliminates the activity of the DYNLL1 gene or gene product, and a second modification that reduces or eliminates the activity of the NRG1 gene or gene product. In some embodiments, the cells include a first modification that reduces or eliminates the activity of the DYNLL1 gene or gene product, and a second modification that reduces or eliminates the activity of the TNFRSF10C gene or gene product.In some embodiments, the cells include a first modification that reduces or eliminates the activity of the DYNLL1 gene or gene product, and a second modification that reduces or eliminates the activity of the TNFSF12 gene or gene product. In some embodiments, the cells include a first modification that reduces or eliminates the activity of the DYNLL1 gene or gene product, and a second modification that reduces or eliminates the activity of the MAL gene or gene product. In some embodiments, the cells include a first modification that reduces or eliminates the activity of the DYNLL1 gene or gene product, and a second modification that reduces or eliminates the activity of the VEGFB gene or gene product. In some embodiments, the cells include a first modification that reduces or eliminates the activity of the IGFBP3 gene or gene product, and a second modification that reduces or eliminates the activity of a second endogenous gene or gene product selected from the group consisting of ARHGEF7, ARHGEF17, BNIP1, C19orf2, CHST11, DYNLL1, MAX, and the VEGFB gene or gene product. In some embodiments, the cells include a first modification that reduces or eliminates the activity of the MAX gene or gene product, and a second modification that reduces or eliminates the activity of a second endogenous gene or gene product selected from the group consisting of ARHGEF7, ARHGEF17, BNIP1, C19orf2, CHST11, DYNLL1, IGFBP3, and the VEGFB gene or gene product. In some embodiments, the cells include a first modification that reduces or eliminates the activity of the MAX gene or gene product, and a second modification that reduces or eliminates the activity of the DYNLL1 gene or gene product. In some embodiments, the cells include a first modification that reduces or eliminates the activity of the VEGFB gene or gene product, and a second modification that reduces or eliminates the activity of a second endogenous gene or gene product selected from the group consisting of ARHGEF7, ARHGEF17, BNIP1, C19orf2, CHST11, DYNLL1, IGFBP3, and the MAX gene or gene product. In some embodiments, the cells are HEK293 cells. In some embodiments, the cells are HEK293-derived cells. In some embodiments, the cells are suspension-adapted HEK293 cells.In some embodiments, the cells are suspension-adapted HEK293-derived cells.

[0069] In some embodiments, the cells include modifications that reduce or eliminate the activity of at least three endogenous genes or gene products selected from the group consisting of ANGPT1, ARHGEF7, ARHGEF17, BID, BNIP1, C19orf2, CHST11, CUL1, DAP3, DYNLL1, FIS1, HMGB2, IGFBP3, NRG1, TNFRSF10C, TNFSF12, MAL, MAX, and VEGFB genes or gene products. In some embodiments, the cells include modifications that reduce or eliminate the activity of the DYNLL1 and MAX genes or gene products, as well as a third gene or gene product selected from the group consisting of ANGPT1, ARHGEF7, ARHGEF17, BID, BNIP1, C19orf2, CHST11, CUL1, DAP3, FIS1, HMGB2, IGFBP3, NRG1, TNFRSF10C, TNFSF12, MAL, and VEGFB genes or gene products. In some embodiments, the cells include modifications that reduce or eliminate the activity of the DYNLL1 and MAX genes or gene products, as well as a third gene or gene product selected from the group consisting of ARHGEF7, ARHGEF17, BNIP1, C19orf2, CHST11, IGFBP3, and VEGFB genes or gene products. In some embodiments, the cells are HEK293 cells. In some embodiments, the cells are HEK293-derived cells. In some embodiments, the cells are suspension-adapted HEK293 cells. In some embodiments, the cells are suspension-adapted HEK293-derived cells.

[0070] In some embodiments, the cells include modifications that reduce or eliminate the activity of at least four, five, or six endogenous genes or gene products selected from the group consisting of ANGPT1, ARHGEF7, ARHGEF17, BID, BNIP1, C19orf2, CHST11, CUL1, DAP3, DYNLL1, FIS1, HMGB2, IGFBP3, NRG1, TNFRSF10C, TNFSF12, MAL, MAX, and VEGFB genes or gene products. In some embodiments, the cells include modifications that reduce or eliminate the activity of the DYNLL1 and MAX genes or gene products, as well as at least third and fourth genes or gene products selected from the group consisting of ANGPT1, ARHGEF7, ARHGEF17, BID, BNIP1, C19orf2, CHST11, CUL1, DAP3, FIS1, HMGB2, IGFBP3, NRG1, TNFRSF10C, TNFSF12, MAL, and VEGFB genes or gene products. In some embodiments, the cells include modifications that reduce or eliminate the activity of the DYNLL1 and MAX genes or gene products, as well as at least third and fourth genes or gene products selected from the group consisting of ARHGEF7, ARHGEF17, BNIP1, C19orf2, CHST11, IGFBP3, and VEGFB genes or gene products. In some embodiments, the cells are HEK293 cells. In some embodiments, the cells are HEK293-derived cells. In some embodiments, the cells are suspension-adapted HEK293 cells. In some embodiments, the cells are suspension-adapted HEK293-derived cells.

[0071] In some embodiments, modifications that reduce or eliminate the activity of an endogenous gene or gene product are gene mutations. In some embodiments, the mutation is a loss-of-function mutation. A “loss-of-function mutation” in a gene refers to a gene manipulation or mutation (e.g., substitution, deletion, insertion, duplication, frameshift, or translocation) that reduces or eliminates one or more functions of the corresponding gene product. In some embodiments, a loss-of-function mutation is a null mutation (e.g., a deletion that removes part or all of the coding sequence) that removes one or more functions of the corresponding gene product. In some embodiments, the mutation is a missense mutation. In some embodiments, the mutation is a nonsense mutation. In some embodiments, the mutation is a frameshift mutation. In some embodiments, the mutation is a deletion. In some embodiments, mutations that reduce or eliminate the activity of an endogenous gene or gene product are present in all copies of the genome. In some embodiments, mutations that reduce or eliminate the activity of an endogenous gene or gene product are not present in all copies of the genome.

[0072] Those skilled in the art recognize that eukaryotic cells (e.g., mammalian cells such as HEK293 cells) contain multiple copies of genes. Consequently, modifications that reduce or eliminate the activity of endogenous genes or gene products may be mutations (one or more) that affect one copy, multiple copies, or all copies of the gene. In one embodiment, the mutation is a heterozygous mutation that does not affect all copies of the gene. In one embodiment, the mutation is a heterozygous mutation that affects one copy of the gene. In one embodiment, the mutation is a heterozygous mutation that affects two copies of the gene. In one embodiment, the mutation is a heterozygous mutation that affects all but one copy of the gene. In one embodiment, the mutation is a homozygous mutation that affects all copies of the gene. In some embodiments, the mutation is a heterozygous mutation (e.g., deletion) in an endogenous gene selected from the group consisting of ARHGEF7, ARHGEF17, BNIP1, C19orf2, CHST11, DYNLL1, IGFBP3, MAX, and VEGFB. In some embodiments, the mutation is a heterozygous mutation (e.g., deletion) in DYNLL1. In some embodiments, the mutation is a heterozygous mutation (e.g., deletion) in MAX. In some embodiments, the mutation is a heterozygous mutation in MAX. In some embodiments, the mutation is a homozygous mutation (e.g., deletion) in an endogenous gene selected from the group consisting of ARHGEF7, ARHGEF17, BNIP1, C19orf2, CHST11, DYNLL1, IGFBP3, MAX, and VEGFB. In some embodiments, the mutation is a homozygous mutation (e.g., deletion) in DYNLL1. In some embodiments, the mutation is a homozygous mutation (e.g., deletion) in MAX.

[0073] Those skilled in the art are aware of techniques that can be used to introduce mutations (e.g., loss-of-function mutations) into the genetic material of cells (e.g., HEK293 cells). Such techniques include, but are not limited to, zinc finger nuclease techniques, CRISPR / Cas9 techniques, and TALEN techniques for introducing targeted mutations, as well as chemical and insertional mutagenesis with selection for identifying random mutations. Using any technique known to those skilled in the art, modifications (e.g., loss-of-function mutations) that reduce or eliminate the activity of endogenous genes or gene products selected from the group consisting of ARHGEF7, ARHGEF17, BNIP1, C19orf2, CHST11, DYNLL1, IGFBP3, MAX, and VEGFB genes or gene products can be introduced.

[0074] In some embodiments, modifications that reduce or eliminate the activity of endogenous genes or gene products are inhibitory nucleic acids capable of reducing or eliminating the activity of genes or gene products. Those skilled in the art are aware of inhibitory nucleic acid-based technologies that can be used to reduce or eliminate the activity of endogenous genes or gene products in cells (e.g., HEK293 cells). In some embodiments, inhibitory nucleic acids include small interfering RNA (siRNA), microRNA (miRNA), or short hairpin RNA (shRNA). In some embodiments, inhibitory nucleic acids include antisense RNA. In some embodiments, inhibitory nucleic acids include morpholino oligomers. Tools for designing inhibitory nucleic acids that can target specific genes are well known in the art. Sequences of inhibitory nucleic acid molecules targeting target genes are readily available to those skilled in the art, for example, from The Genetic Perturbation Platform (formerly RNA interference (RNAi) Platform) (accessible at portals.broadinstitute.org / gpp / public / ). Further sources of information and materials include commercial services such as Invitrogen. Those skilled in the art understand that inhibitory nucleic acid molecules can be introduced into cells by direct translocation or expression from a vector. Using any technique known to those skilled in the art, modifications (e.g., inhibitory nucleic acids) can be introduced that reduce or eliminate the activity of endogenous genes or gene products selected from the group consisting of ARHGEF7, ARHGEF17, BNIP1, C19orf2, CHST11, DYNLL1, IGFBP3, MAX, and VEGFB genes or gene products.

[0075] In some embodiments, modifications that reduce or remove the activity of an endogenous gene or gene product remove the mRNA transcript encoded by the gene. In some embodiments, modifications that reduce or remove the activity of an endogenous gene or gene product reduce the level of mRNA encoded by the gene by at least about 50%, 60%, 70%, 80%, 90%, or 95%.

[0076] In some embodiments, modifications that reduce or remove the activity of an endogenous gene or gene product remove the polypeptide encoded by the gene. In some embodiments, modifications that reduce or remove the activity of an endogenous gene or gene product reduce the level of the protein encoded by the gene by at least about 50%, 60%, 70%, 80%, 90%, or 95%.

[0077] In some embodiments, modifications that reduce or eliminate the activity of an endogenous gene or gene product are missense mutations that reduce the activity of the protein encoded by the gene by at least about 50%, 60%, 70%, 80%, 90%, or 95%.

[0078] In some embodiments, the cells described herein further comprise one or more polynucleotides encoding a recombinant virus. Recombinant virus particle generation 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). 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).

[0079] In some embodiments, the cells described herein further comprise one or more polynucleotides encoding recombinant AAV. In some embodiments, the cells described herein further comprise one or more polynucleotides encoding at least one of the following: a packaged rAAV genome, an adenovirus helper function required for packaging, an AAV rep protein sufficient for packaging, and an AAV cap protein sufficient for packaging. In some embodiments, the cells described herein further comprise one or more polynucleotides encoding a packaged rAAV genome, an adenovirus helper function required for packaging, an AAV rep protein sufficient for packaging, and an AAV cap protein sufficient for packaging.

[0080] In some embodiments, the cells described herein further comprise one or more polynucleotides encoding a recombinant polypeptide. In some embodiments, the recombinant polypeptide is an antibody or antibody fragment. In some embodiments, the antibody is a chimeric, human, or humanized antibody. In some embodiments, the antibody is an IgG antibody. In some embodiments, the recombinant polypeptide is a fusion protein. In some embodiments, the fusion protein comprises an antibody or a fragment thereof. In some embodiments, the fusion protein comprises an Fc region. In some embodiments, the fusion protein comprises an antigen-binding antibody fragment.

[0081] Cell bank In one embodiment, the disclosure provides a cell bank comprising a plurality of cells as described herein. In some embodiments, the cell bank comprises a plurality of cryopreserved cells. In some embodiments, the cells are suitable for recombinant virus (e.g., recombinant AAV) production. In some embodiments, the cells are suitable for recombinant polypeptide production. In some embodiments, the cells are HEK293 cells. In some embodiments, the cells are HEK293-derived cells. In some embodiments, the cells are suspension-adapted HEK293 cells. In some embodiments, the cells are suspension-adapted HEK293-derived cells.

[0082] In one embodiment, the Disclosure provides a method for constructing a cell bank, comprising cryopreserving a composition comprising a plurality of cells as described herein. In some embodiments, the cells are suitable for recombinant virus (e.g., recombinant AAV) production. In some embodiments, the cells are suitable for recombinant polypeptide production. In some embodiments, the cells are HEK293 cells. In some embodiments, the cells are HEK293-derived cells. In some embodiments, the cells are suspension-adapted HEK293 cells. In some embodiments, the cells are suspension-adapted HEK293-derived cells.

[0083] cell culture In one embodiment, this disclosure provides a cell culture comprising a plurality of cells as described herein. In some embodiments, the cell culture comprises the plurality of cells as described herein and a suitable medium. In some embodiments, the cell culture is a suspension culture. In some embodiments, the cells are capable of producing recombinant viruses (e.g., recombinant AAV). In some embodiments, the cells comprise one or more polynucleotides encoding at least one of the following: a packaged rAAV genome, an adenovirus helper function required for packaging, an AAV rep protein sufficient for packaging, and an AAV cap protein sufficient for packaging. In some embodiments, the cells comprise one or more polynucleotides encoding a packaged rAAV genome, an adenovirus helper function required for packaging, an AAV rep protein sufficient for packaging, and an AAV cap protein sufficient for packaging. In some embodiments, the cells are capable of producing recombinant polypeptides (e.g., antibodies). In some embodiments, the cells comprise one or more polynucleotides encoding an antibody or an antigen-binding antibody fragment. In some embodiments, the cells are HEK293 cells. In some embodiments, the cells are HEK293-derived cells. In some embodiments, the cells are suspension-adapted HEK293 cells. In some embodiments, the cells are suspension-adapted HEK293-derived cells.

[0084] Method for generating recombinant virus particles In one embodiment, the disclosure provides a method for generating recombinant virus (e.g., recombinant adeno-associated virus (rAAV)) particles within a host cell as described herein. In some embodiments, the method further includes recovering the virus (e.g., rAAV) particles.

[0085] In some embodiments, the Disclosure provides a method for generating recombinant viral particles (e.g., rAAV), comprising: (a) preparing a cell culture comprising a plurality of cells described herein that are suitable for the generation of recombinant viral particles; (b) transtransferring one or more polynucleotides containing genes necessary for the generation of recombinant viral particles into the cells; and (c) maintaining the cell culture comprising the transtransferred cells under conditions that enable the generation of recombinant viral particles. Transtransfer-based recombinant viral particle generation systems are known to those skilled in the art. See, for example, Reiser et al., Gene Ther 7(11):910-3(2000); Dull et al., J Virol.72(11):8463-8471(1998); Hoffmann et al., PNAS 97(11)6108-6113(2000); Milian et al., Vaccine 35(26):3423-3430 (2017) (each of these is incorporated herein by reference in whole). 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).

[0086] In some embodiments, the Disclosure provides a method for generating rAAV particles, comprising (a) preparing a cell culture comprising a plurality of cells described herein that are capable of generating rAAV, and (b) maintaining the cell culture under conditions that enable the generation of rAAV particles. In some embodiments, the cells capable of generating rAAV particles are transfected with one or more polynucleotides encoding at least one of the following: a packaged rAAV genome, an adenovirus helper function required for packaging, an AAV rep protein sufficient for packaging, and an AAV cap protein sufficient for packaging. In some embodiments, the cells capable of generating rAAV are transfected with one or more polynucleotides encoding a packaged rAAV genome, an adenovirus helper function required for packaging, an AAV rep protein sufficient for packaging, and an AAV cap protein sufficient for packaging. In some embodiments, the adenovirus helper function comprises at least one of the adenovirus E4 gene, the E2a gene, and the VA gene. In some embodiments, the adenovirus helper function comprises the adenovirus E4 gene, the E2a gene, and the VA gene. In some embodiments, the polynucleotide encoding the adenovirus helper function comprises pAD delta F6. In some embodiments, the polynucleotide encoding the adenovirus helper function comprises a helper plasmid (e.g., Helper #5) disclosed in WO2023 / 060113 (the whole thereof is incorporated herein by reference). In some embodiments, the polynucleotide encoding the adenovirus helper function also encodes the AAV rep protein and the AAV cap protein. In some embodiments, the polynucleotide encoding the adenovirus helper function, the AAV rep protein, and the AAV cap protein comprises a helper / rep / cap plasmid, e.g., pHRC#7 and pHRD#8 disclosed in PCT / US2024 / 023368 (filed April 5, 2024, the whole thereof is incorporated herein by reference).

[0087] In some embodiments, the disclosure provides a method for generating rAAV particles, comprising culturing cells described herein that are capable of generating rAAV particles under conditions that enable the generation of rAAV particles. In some embodiments, the cells capable of generating rAAV particles are transfected with one or more polynucleotides encoding at least one of the following: a packaged rAAV genome, an adenovirus helper function required for packaging, an AAV rep protein sufficient for packaging, and an AAV cap protein sufficient for packaging. In some embodiments, the cells capable of generating rAAV are transfected with one or more polynucleotides encoding a packaged rAAV genome, an adenovirus helper function required for packaging, an AAV rep protein sufficient for packaging, and an AAV cap protein sufficient for packaging. In some embodiments, the adenovirus helper function comprises at least one of the adenovirus E4 gene, the E2a gene, and the VA gene. In some embodiments, the adenovirus helper function comprises the adenovirus E4 gene, the E2a gene, and the VA gene. In some embodiments, the polynucleotide encoding the adenovirus helper function comprises pAD delta F6. In some embodiments, the polynucleotide encoding the adenovirus helper function comprises a helper plasmid (e.g., Helper #5) disclosed in WO2023 / 060113 (the whole thereof is incorporated herein by reference). In some embodiments, the polynucleotide encoding the adenovirus helper function also encodes the AAV rep protein and the AAV cap protein. In some embodiments, the polynucleotide encoding the adenovirus helper function, the AAV rep protein, and the AAV cap protein comprises a helper / rep / cap plasmid, e.g., pHRC#7 and pHRD#8 disclosed in PCT / US2024 / 023368 (filed April 5, 2024, the whole thereof is incorporated herein by reference).

[0088] In some embodiments, the Disclosure provides a method for increasing the production of rAAV particles, comprising (a) preparing a cell culture comprising a plurality of cells described herein that are capable of producing rAAV, and (b) maintaining the cell culture under conditions that enable the production of rAAV particles. In some embodiments, the cells capable of producing rAAV particles are transfected with one or more polynucleotides encoding at least one of the following: a packaged rAAV genome, an adenovirus helper function required for packaging, an AAV rep protein sufficient for packaging, and an AAV cap protein sufficient for packaging. In some embodiments, the cells capable of producing rAAV are transfected with one or more polynucleotides encoding a packaged rAAV genome, an adenovirus helper function required for packaging, an AAV rep protein sufficient for packaging, and an AAV cap protein sufficient for packaging. In some embodiments, the adenovirus helper function comprises at least one of the adenovirus E4 gene, the E2a gene, and the VA gene. In some embodiments, the adenovirus helper function comprises the adenovirus E4 gene, the E2a gene, and the VA gene. In some embodiments, the polynucleotide encoding the adenovirus helper function comprises pAD delta F6. In some embodiments, the polynucleotide encoding the adenovirus helper function comprises a helper plasmid (e.g., Helper #5) disclosed in WO2023 / 060113 (the whole thereof is incorporated herein by reference). In some embodiments, the polynucleotide encoding the adenovirus helper function also encodes the AAV rep protein and the AAV cap protein.In some embodiments, polynucleotides encoding adenovirus helper function, AAV rep protein, and AAV cap protein include helper / rep / cap plasmids, e.g., pHRC#7 and pHRD#8 disclosed in PCT / US2024 / 023368 (filed April 5, 2024, the entirety of which is incorporated herein by reference).

[0089] In some embodiments, the disclosure provides a method for increasing the production of rAAV particles, comprising culturing a plurality of cells described herein that are capable of producing rAAV particles under conditions that enable the production of rAAV particles. In some embodiments, the cells capable of producing rAAV particles are transfected with one or more polynucleotides encoding at least one of the following: a packaged rAAV genome, an adenovirus helper function required for packaging, an AAV rep protein sufficient for packaging, and an AAV cap protein sufficient for packaging. In some embodiments, the cells capable of producing rAAV are transfected with one or more polynucleotides encoding a packaged rAAV genome, an adenovirus helper function required for packaging, an AAV rep protein sufficient for packaging, and an AAV cap protein sufficient for packaging. In some embodiments, the adenovirus helper function comprises at least one of the adenovirus E4 gene, the E2a gene, and the VA gene. In some embodiments, the adenovirus helper function comprises the adenovirus E4 gene, the E2a gene, and the VA gene. In some embodiments, the polynucleotide encoding the adenovirus helper function comprises pAD delta F6. In some embodiments, the polynucleotide encoding the adenovirus helper function comprises a helper plasmid (e.g., Helper #5) disclosed in WO2023 / 060113 (the whole thereof is incorporated herein by reference). In some embodiments, the polynucleotide encoding the adenovirus helper function also encodes the AAV rep protein and the AAV cap protein. In some embodiments, the polynucleotide encoding the adenovirus helper function, the AAV rep protein, and the AAV cap protein comprises a helper / rep / cap plasmid, e.g., pHRC#7 and pHRD#8 disclosed in PCT / US2024 / 023368 (filed April 5, 2024, the whole thereof is incorporated herein by reference).

[0090] In some embodiments, the Disclosure provides a method for generating rAAV particles, comprising: (a) preparing a cell culture comprising a plurality of cells; (b) introducing into the cells one or more polynucleotides encoding at least one of the following: (i) a packaged rAAV genome; (ii) an adenovirus helper function required for packaging; (iii) an AAV rep protein sufficient for packaging; and (iv) an AAV cap protein sufficient for packaging; and (c) maintaining the cell culture under conditions that enable the generation of rAAV particles. In some embodiments, the Method comprises introducing into the cells one or more polynucleotides encoding at least one of the following: (i) a packaged rAAV genome; (ii) an adenovirus helper function required for packaging; (iii) an AAV rep protein sufficient for packaging; and (iv) an AAV cap protein sufficient for packaging. In some embodiments, the introduction of one or more polynucleotides into the cells is carried out by transfusion. In some embodiments, the adenovirus helper function comprises at least one of the adenovirus E4 gene, E2a gene, and VA gene. In some embodiments, the adenovirus helper function comprises the adenovirus E4 gene, the E2a gene, and the VA gene. In some embodiments, the polynucleotide encoding the adenovirus helper function comprises pAD delta F6. In some embodiments, the polynucleotide encoding the adenovirus helper function comprises a helper plasmid (e.g., Helper #5) disclosed in WO2023 / 060113 (the whole thereof is incorporated herein by reference). In some embodiments, the polynucleotide encoding the adenovirus helper function also encodes the AAV rep protein and the AAV cap protein.In some embodiments, polynucleotides encoding adenovirus helper function, AAV rep protein, and AAV cap protein include helper / rep / cap plasmids, e.g., pHRC#7 and pHRD#8 disclosed in PCT / US2024 / 023368 (filed April 5, 2024, the entirety of which is incorporated herein by reference).

[0091] In some embodiments, the Disclosure provides a method for increasing the production of rAAV particles, comprising: (a) preparing a cell culture comprising a plurality of cells; (b) introducing into the cells one or more polynucleotides encoding at least one of (i) a packaged rAAV genome, (ii) an adenovirus helper function required for packaging, (iii) an AAV rep protein sufficient for packaging, and (iv) an AAV cap protein sufficient for packaging; and (c) maintaining the cell culture under conditions that enable the production of rAAV particles. In some embodiments, the Method comprises introducing into the cells one or more polynucleotides encoding (i) a packaged rAAV genome, (ii) an adenovirus helper function required for packaging, (iii) an AAV rep protein sufficient for packaging, and (iv) an AAV cap protein sufficient for packaging. In some embodiments, the introduction of one or more polynucleotides into the cells is carried out by transfusion. In some embodiments, the adenovirus helper function comprises at least one of the adenovirus E4 gene, E2a gene, and VA gene. In some embodiments, the adenovirus helper function comprises the adenovirus E4 gene, the E2a gene, and the VA gene. In some embodiments, the polynucleotide encoding the adenovirus helper function comprises pAD delta F6. In some embodiments, the polynucleotide encoding the adenovirus helper function comprises a helper plasmid (e.g., Helper #5) disclosed in WO2023 / 060113 (the whole thereof is incorporated herein by reference). In some embodiments, the polynucleotide encoding the adenovirus helper function also encodes the AAV rep protein and the AAV cap protein.In some embodiments, polynucleotides encoding adenovirus helper function, AAV rep protein, and AAV cap protein include helper / rep / cap plasmids, e.g., pHRC#7 and pHRD#8 disclosed in PCT / US2024 / 023368 (filed April 5, 2024, the entirety of which is incorporated herein by reference).

[0092] In some embodiments, maintaining a cell culture or culturing cells under conditions that allow for the generation of rAAV particles lasts for about 2 to 10 days, about 2 to 15 days, or about 5 to 14 days. In some embodiments, maintaining a cell culture or culturing cells under conditions that allow for the generation of rAAV particles lasts for about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, or about 7 days. In some embodiments, maintaining a cell culture or culturing cells under conditions that allow for the generation of rAAV particles lasts for about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, or about 7 days. In some embodiments, maintaining a cell culture or culturing cells under conditions that allow for the generation of rAAV particles lasts for about 5 days.

[0093] In some embodiments, the cells are mammalian cells. In some embodiments, the cells are insect cells. In some embodiments, the cells are HEK293 cells, HEK-derived cells, CHO cells, CHO-derived cells, HeLa cells, SF-9 cells, BHK cells, Vero cells, or PerC6 cells. In some embodiments, the cells are HEK293 cells. In some embodiments, the cells are HEK293-derived cells. In some embodiments, the cells are suspension-adapted HEK293 cells. In some embodiments, the cells are suspension-adapted HEK293-derived cells.

[0094] In some embodiments, the cell culture is a suspension culture or an adherent culture.

[0095] In some embodiments, the cell culture has a volume of approximately 50 liters to approximately 20,000 liters. In some embodiments, the cell culture has a volume of approximately 100 liters to approximately 5,000 liters. In some embodiments, the cell culture has a volume of approximately 100 liters to approximately 2,500 liters. In some embodiments, the cell culture has a volume of approximately 100 liters to approximately 1,500 liters. In some embodiments, the cell culture has a volume of approximately 200 liters to approximately 5,000 liters. In some embodiments, the cell culture has a volume of approximately 200 liters to approximately 2,500 liters. In some embodiments, the cell culture has a volume of approximately 200 liters to approximately 1,500 liters. In some embodiments, the cell culture has a volume of approximately 250 liters to approximately 5,000 liters. In some embodiments, the cell culture has a volume of approximately 250 liters to approximately 2,500 liters. In some embodiments, the cell culture has a volume of approximately 250 liters to approximately 1,500 liters.

[0096] In some embodiments, this method further includes recovering rAAV particles.

[0097] In some embodiments, the method described herein produces more rAAV particles measured as GC / ml than the reference method. In some embodiments, the reference method uses host cells that do not contain at least one modification that reduces or removes the activity of at least one endogenous gene or gene product in the apoptotic signaling pathway. In some embodiments, the reference method uses HEK293 host cells that do not contain at least one modification that reduces or removes the activity of at least one endogenous gene or gene product in the apoptotic signaling pathway. In some embodiments, the method described herein produces at least about 10% more rAAV particles measured as GC / ml than the reference method. In some embodiments, the method described herein produces at least about 20% more rAAV particles measured as GC / ml than the reference method. In some embodiments, the method described herein produces at least about 30% more rAAV particles measured as GC / ml than the reference method. In some embodiments, the method described herein produces at least about 40% more rAAV particles measured as GC / ml than the reference method. In some embodiments, the method described herein produces at least about 50% more rAAV particles measured as GC / ml than the reference method. In some embodiments, the method described herein produces at least about 70% more rAAV particles measured as GC / ml than the reference method. In some embodiments, the method described herein produces at least about 90% more rAAV particles measured as GC / ml than the reference method. In some embodiments, the method described herein produces at least about twice as many rAAV particles measured as GC / ml as the reference method. In some embodiments, this method produces at least about three times more rAAV particles measured as GC / ml than the reference method. In some embodiments, this method produces at least about four times more rAAV particles measured as GC / ml than the reference method.

[0098] In some embodiments, this method generates a population of rAAV particles containing more complete capsids than the reference method. In some embodiments, the reference method uses host cells that do not contain at least one modification that reduces or eliminates the activity of at least one endogenous gene or gene product in the apoptotic signaling pathway. In some embodiments, the reference method uses HEK293 host cells that do not contain at least one modification that reduces or eliminates the activity of at least one endogenous gene or gene product in the apoptotic signaling pathway.

[0099] 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.hu32, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m 8, AAV.PHP.B, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, or AAV.HSC16 serotype capsid proteins. In some embodiments, rAAV particles contain AAV8, AAV9, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu32, or AAV.hu37 serotype capsid proteins. In some embodiments, the rAAV particles contain the AAV8 serotype capsid protein. In some embodiments, the rAAV particles contain the AAV9 serotype capsid protein.

[0100] In some embodiments, the rAAV particle contains a transgene encoding a gene product. In some embodiments, the gene product is a polypeptide or a double-stranded RNA molecule. In some embodiments, the gene product is a polypeptide. In some embodiments, the transgene encodes an antibody or its antigen-binding fragment, a fusion protein, an Fc fusion polypeptide, an immunoadhesin, an immunoglobulin, a modified protein, a protein fragment, or an enzyme. In some embodiments, the transgene contains a regulatory element responsively bound to a polynucleotide encoding the gene product.

[0101] In some embodiments, the gene product is anti-VEGF Fab, anti-kallikrein antibody, anti-TNF antibody, microdystrophin, minidystrophin, iduronidase (IDUA), iduronate 2-sulfatase (IDS), low-density lipoprotein receptor (LDLR), tripeptidyl peptidase 1 (TPP1), or a non-membrane-bound splice variant of VEGF receptor 1 (sFlt-1).In some embodiments, the gene products include gamma-sarcoglycan, Rab escort protein 1 (REP1 / CHM), retinoid isomerohydrolase (RPE65), cyclic nucleotide-dependent channel alpha-3 (CNGA3), cyclic nucleotide-dependent 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 modulator (RPGR), and retinooxygenase. Calcium ATPase (RS1), sarcoplasmic reticulum calcium ATPase (SERCA2a), aflibercept, battenin (CLN3), transmembrane ER protein (CLN6), glutamate decarboxylase (GAD), glial cell-derived neurotrophic factor (GDNF), aquaporin 1 (AQP1), dystrophin, myotubularin 1 (MTM1), follistatin (FST), glucose-6-phosphatase (G6Pase), apolipoprotein A2 (APOA2), uridine diphosphate glucuronosyltransferase 1A1 (U GT1A1), 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), neuruturin (N These include RTN, neurotrophin-3 (NT-3 / NTF3), porphobilinogen deaminase (PBGD), nerve growth factor (NGF), mitochondrial-encoded NADH:ubiquinone oxidoreductase core subunit 4 (MT-ND4), protective protein cathepsin A (PPCA), dyspherin, MER proto-oncogene, tyrosine kinase (MERTK), cystic fibrosis transmembrane conductance regulator (CFTR), or tumor necrosis factor receptor (TNFR)-immunoglobulin (IgG1)Fc fusion. In some embodiments, the gene product is dystrophin or microdystrophin.In some embodiments, the gene product is microRNA.

[0102] In some embodiments, the method described herein increases the generation 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 method described herein is the same as the quality of rAAV particles or compositions produced by a reference method using host cells that do not contain at least one modification that reduces or removes the activity of at least one endogenous gene or gene product in an apoptotic signaling pathway. In some embodiments, the quality of rAAV particles or compositions containing the same produced by the methods described herein is better than the quality of rAAV particles or compositions produced by a reference method using host cells that do not contain at least one modification that reduces or eliminates the activity of at least one endogenous gene or gene product in an apoptotic signaling pathway.

[0103] In the art, numerous cell culture-based systems for the generation of rAAV particles are known, and any of these can be used to carry out the methods described herein. An rAAV-generating culture for generating rAAV virus particles requires (1) a suitable host cell (including human cell lines such as HeLa, A549, or HEK293 cells and their derivatives (HEK293T cells, HEK293F cells), or mammalian cell lines such as Vero, CHO cells, or CHO-derived cells); (2) a suitable helper virus function (provided by wild-type or mutant adenovirus (e.g., temperature-sensitive adenovirus), herpesvirus, baculovirus, or a plasmid construct providing helper function); (3) AAV rep and cap genes and gene products; (4) a transgene flanked by the AAV ITR sequence (e.g., a therapeutic transgene); and (5) a suitable medium and medium components to support rAAV generation.

[0104] 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 an inverse terminal repeat (ITR)) into cells. The expression "adenovirus helper function" means a set of 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 virus (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 described 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.

[0105] 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 packaged rAAV genome are introduced into cells by transduction using one or more polynucleotides, for example, a vector. In some embodiments, the method described 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 packaged rAAV genome). 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.hu32, 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, AAV.hu32, and AAV.hu37. In some embodiments, the vector encoding the packaged rAAV genome 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.hu32, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B, It is from AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, or AAV.HSC16, or another AAV serotype.

[0106] 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 method described herein, a first plasmid vector encoding the rAAV genome containing a target gene adjacent to an AAV reverse terminal repeat (ITR), a second vector encoding the AAV rep and cap genes, and a third vector encoding the helper gene can be used. In some embodiments, a mixture of the three vectors can be simultaneously translocated into cells.

[0107] In some embodiments, a combination of translocation and infection is used by using a viral vector in conjunction with a plasmid vector.

[0108] 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.

[0109] 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, A AV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu32, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B, AAV2.5, AAV2tYF, A These are from AV3B, 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 derived 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.hu32, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B, AAV2.5, AAV2tYF These are from AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, or AAV.HSC16, or other AAV serotypes (e.g., hybrid serotypes having sequences from two or more serotypes). In some embodiments, the AAV rep and cap genes for rAAV particle generation are derived from different serotypes. For example, the rep gene is derived from AAV2, while the cap gene is derived from AAV9.

[0110] Any suitable culture medium known in the art can be used to generate recombinant viral particles (e.g., rAAV particles) according to the method 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 method herein uses 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.

[0111] 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).

[0112] Any cell or cell line known in the art to produce recombinant virus particles (e.g., rAAV particles) can be used in any one of the methods described herein. In some embodiments, the methods for producing or increasing recombinant virus particles (e.g., rAAV particles) described herein 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 described herein use mammalian cells. In some embodiments, the methods described herein use insect cells, such as SF-9 cells. In some embodiments, the methods described herein use cells adapted for growth in suspension culture. In some embodiments, the methods described herein use HEK293 cells adapted for growth in suspension culture.

[0113] In some embodiments, the cell cultures described herein are suspension cultures. In some embodiments, the large-scale suspension cell cultures described herein contain HEK293 cells adapted for growth in suspension culture. In some embodiments, the cell cultures described herein contain serum-free medium, animal component-free medium, or chemically defined medium. In some embodiments, the cell cultures described herein contain serum-free medium. In some embodiments, suspension-adapted cells are cultured in a shaking flask, spinner flask, cell bag, or bioreactor.

[0114] In some embodiments, the cell cultures described herein include serum-free media, animal component-free media, or chemically defined media. In some embodiments, the cell cultures described herein include serum-free media.

[0115] In some embodiments, the large-scale suspension cell cultures described herein include high-density cell cultures. In some embodiments, the culture has a total cell density between approximately 1 × 10⁻⁶ cells / ml and approximately 30 × 10⁻⁶ cells / ml. In some embodiments, more than approximately 50% of the cells are viable cells. In some embodiments, the cells are HeLa cells, HEK293 cells, HEK293-derived cells (e.g., HEK293T cells, HEK293F cells), Vero cells, or SF-9 cells. In further embodiments, the cells are HEK293 cells.

[0116] The methods described herein can be used to produce rAAV particles containing capsid proteins from any AAV capsid serotype. In some embodiments, the rAAV particles may be AAV1, AAV2, rAAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu32, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.P It contains capsid proteins from AAV capsid serotypes selected 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 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.hu32, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B, A This includes capsid proteins that are derivatives, modifiers, or pseudotypes of 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 capsid proteins.

[0117] 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.

[0118] 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.hu32, 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, AAV.hu32, and AAV.hu37.

[0119] 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.

[0120] 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.

[0121] In some embodiments, the rAAV particles contain capsid proteins having at least 80% identity to the VP1, VP2, and / or VP3 sequences of AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu32, AAV.hu37, AAV.PHB, or AAV.7m8 capsid proteins, for example, 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, etc., i.e., up to 100% identity. In some embodiments, rAAV particles contain AAV capsid proteins with high sequence homology to AAV8 or AAV9, such as AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu32, and AAV.hu37, with at least 80% identity to the VP1, VP2, and / or VP3 sequences, e.g., 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, i.e., capsid proteins with up to 100% identity.

[0122] 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.

[0123] rAAV particles The method provided is suitable for use in generating any isolated recombinant AAV particles. Therefore, rAAV can be any serotype, variant, 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.hu32, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PH P.B, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, or AAV.HSC16, or other rAAV particles, or a combination of two or more of these.

[0124] 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.hu32, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B, AAV2.5 It has a capsid protein from an AAV serotype selected from AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, or AAV.HSC16, or its 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.hu32, AAV.hu37, AAV.Anc80, rAAV.Anc80L65, AAV.7m8, AAV.PHP.B, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.H The capsid protein contains a VP1, VP2, and / or VP3 sequence of an AAV capsid serotype selected from SC3, 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 that is at least 80% identical, e.g., 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, etc., i.e., up to 100% identical.

[0125] 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.hu32, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B, AAV2.5, This includes capsid proteins 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, or AAV.HSC16, or their derivatives, modifiers, or capsid proteins from AAV capsid serotypes selected from 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.hu32, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HS It contains a capsid protein that is at least 80% identical to the VP1, VP2, and / or VP3 sequences of an AAV capsid serotype selected from C3, 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, 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.

[0126] 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., AAVPHP.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).

[0127] 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, and 9,193,9 This includes AAV capsids disclosed in any of the following: Patent Nos. 56, 9458517, and 9,587,282; U.S. Patent Publication Nos. 2015 / 0374803, 2015 / 0126588, 2017 / 0067908, 2013 / 0224836, 2016 / 0215024, 2017 / 0051257; and International Patent Application Nos. PCT / US2015 / 034799 and PCT / EP2015 / 053335. In some embodiments, the rAAV particles have a capsid protein that is at least 80% identical to the VP1, VP2, and / or VP3 sequences of the AAV capsid disclosed in any of the following patents and patent applications (each of which is incorporated herein by reference in whole): for example, 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, etc., i.e., up to 100% identical: U.S. Patent No. 7,282,199, No. 7,906,111, No. 8,524,446, No. 8, U.S. Patent Applications No. 999,678, 8,628,966, 8,927,514, 8,734,809, U.S. Patent No. 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. 2015 / Patent applications No. 0374803, No. 2015 / 0126588, No. 2017 / 0067908, No. 2013 / 0224836, No. 2016 / 0215024, No. 2017 / 0051257, and International Patent Application Numbers PCT / US2015 / 034799 and PCT / EP2015 / 053335.

[0128] In some embodiments, rAAV particles are used in international patent applications published as WO2003 / 052051 (see, for example, SEQ ID NO. 2), WO2005 / 033321 (see, for example, SEQ ID NOs. 123 and 88), WO03 / 042397 (see, for example, SEQ ID NOs. 2, 81, 85, and 97), WO2006 / 068888 (see, for example, SEQ ID NOs. 1 and 3-6), WO2006 / 110689 (see, for example, SEQ ID NOs. 5-38), and WO2009 / 104 The capsid protein is disclosed in Patent No. 964 (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 Publication Nos. WO2003 / 052051 (see, e.g., SEQ ID NO. 2), WO2005 / 033321 (see, e.g., SEQ ID NOs. 123 and 88), WO03 / 0423 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).

[0129] 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, 9458,517, and 9,587,282, U.S. Patent Application Publications 2015 / 0374803 and 2015 / 0126. Patent applications No. 588, 2017 / 0067908, 2013 / 0224836, 2016 / 0215024, 2017 / 0051257, International Patent Application Nos. PCT / US2015 / 034799, PCT / EP2015 / 053335, WO2003 / 052051, WO2005 / 033321, WO03 / 042397, WO2006 / 068888, WO2006 / 110689, WO2009 / 104964, WO2010 / 127097, and WO2015 / 191508, as well as U.S. Patent Application Publication No. 20150023924.

[0130] The provided method is suitable for use in generating recombinant AAV encoding a transgene. In certain embodiments, the transgenes are those shown in Tables 2A-2C. In some embodiments, the rAAV genome comprises a vector containing the following components: (1) an AAV reverse terminal repeat adjacent to the expression cassette, (2) regulatory elements, e.g., (a) a promoter / enhancer, (b) a polyA signal, and (c) an intron (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) an AAV reverse terminal repeat adjacent to the expression cassette, (2) regulatory elements, e.g., (a) a promoter / enhancer, (b) a polyA signal, and (c) an intron (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 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) optionally introns;(3) for example, Fab or full-length anti-VEGF (e.g., sevacizumab, ranibizumab, bevacizumab, and brolucizumab), anti-EpoR (e.g., LKA-651), anti-ALK1 (e.g., askrinbakumab), anti-C5 (e.g., tesidorumab, clobarimab, and eculizumab), anti-CD105 (e.g., carotuximab), anti-CC1Q (e.g., ANX-007), anti-TNFα (e.g., adalimumab, infliximab, and golimumab) 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);Optionally, a heavy chain such as 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 a modified Fc thereof) and anti-VEGF (e.g., sevacizumab, ranibizumab, bevacizumab, and brolucizumab), anti-EpoR (e.g., LKA-651), anti-ALK1 (e.g., askrinbakumab), anti-C5 (e.g., tesidorumab and eculizumab), anti-CD105 or anti-ENG (e.g., carotuximab), anti-CC1Q (e.g., ANX-0) may be selected. 07) Anti-TNFα (e.g., adalimumab, infliximab, and golimumab), 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 mAb (e.g., etrolizumab), anti-SOST mAb (e.g., romosozumab), anti-pKal nucleotide sequences encoding the light chain of mAbs (e.g., lanadermab), 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) (where the heavy chain (Fab and optionally the Fc region) and light chain are separated by autocleaved furin(F) / F2A or a flexible linker to ensure the expression of equal amounts of heavy and light chain polypeptides).

[0131] (Table 2A) TIFF2026516074000002.tif140149TIFF2026516074000003.tif206149TIFF2026516074000004.tif225149TIFF2026516074000005.tif222149TIFF2026516074000006.tif125149

[0132] (Table 2B) TIFF2026516074000007.tif79146TIFF2026516074000008.tif223147TIFF2026516074000009.tif218147

[0133] (Table 2C) TIFF2026516074000010.tif212146TIFF2026516074000011.tif193146TIFF2026516074000012.tif79146

[0134] In some embodiments, the rAAV particles are rAAV viral vectors encoding an anti-VEGF antibody (e.g., Fab). In certain embodiments, the rAAV particles are rAAV8-based viral vectors encoding an anti-VEGF antibody (e.g., 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 of VEGF receptor 1 (sFlt-1).In some embodiments, rAAV particles contain gamma-sarcoglycan, Rab escort protein 1 (REP1 / CHM), retinoid isomerohydrolase (RPE65), cyclic nucleotide-dependent channel alpha-3 (CNGA3), cyclic nucleotide-dependent 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 modulator (RPGR), and retinoskin (RS1). , 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 (UG T1A1), arylsulfatase B (ARSB), N-acetyl-alpha-glucosaminidase (NAGLU), alpha-glucosidase (GAA), alpha-galactosidase (GLA), beta-galactosidase (GLB1), lipoprotein lipase (LPL), alpha-1-antitrypsin (AAT), phosphodiesterase 6B (PDE6B), ornithine carbamoyltransferase 9OTC), survival motor neuron (SMN1), survival motor neuron (SMN2), neuronurulin (NRTN), neuron The rAAV viral vector encodes trophin-3 (NT-3 / NTF3), porphobilinogen deaminase (PBGD), nerve growth factor (NGF), mitochondrial-encoded NADH:ubiquinone oxidoreductase core subunit 4 (MT-ND4), protective protein cathepsin A (PPCA), dyspherin, MER proto-oncogene tyrosine kinase (MERTK), cystic fibrosis transmembrane conductance regulator (CFTR), or tumor necrosis factor receptor (TNFR)-immunoglobulin (IgG1)Fc fusion.

[0135] 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)).

[0136] In additional embodiments, the rAAV particles contain a capsid comprising a capsid protein that is a chimeric of two or more AAV capsid serotypes. In some embodiments, the capsid protein is AAV1, 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.hu32, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.P It is a chimera of two or more AAV capsid proteins from an AAV serotype selected 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, or AAV.HSC16.

[0137] 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)).

[0138] 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.

[0139] 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.

[0140] 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.

[0141] In additional embodiments, the rAAV particles include a mosaic capsid. The mosaic AAV particles consist of a mixture of viral capsid proteins derived from different serotypes of AAV. In some embodiments, the rAAV particles include AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, and AAV16, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu32, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP It contains a mosaic capsid containing a capsid protein of a serotype selected from .B, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, and AAV.HSC16. In some embodiments, 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.

[0142] 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, and AAV16, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu32, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, It contains a capsid composed of capsid proteins derived from 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, 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 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)).

[0143] In additional embodiments, the rAAV particles contain a capsid comprising a capsid protein that is a chimeric of two or more AAV capsid serotypes. In some embodiments, the rAAV particles contain the AAV8 capsid protein and AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, and AAV16, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu32, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B, It contains an AAV capsid protein that is a chimera with one or more AAV capsid proteins from AAV 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, the rAAV particles contain an AAV capsid protein that is a chimera of the AAV8 capsid protein and one or more AAV capsid proteins from AAV serotypes selected from AAV1, AAV2, AAV5, AAV6, AAV7, AAV9, AAV10, rAAVrh10, AAVrh.8, AAVrh.10, AAVrh.37, AAVrh.20, and AAVrh.74.In some embodiments, rAAV particles contain the AAV9 capsid protein and AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, and AAV16, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu32, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B It contains an AAV capsid protein that is a chimera with a capsid protein of one or more 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, the rAAV particles contain an AAV capsid protein that is a chimeric of the AAV9 capsid protein and the capsid protein of one or more AAV capsid serotypes selected from AAV1, AAV2, AAV3, AAV4, AAV5, AA6, AAV7, AAV8, AAV9, AAVrh.8, AAVrh.10, AAVrh.37, AAVrh.20, and AAVrh.74.

[0144] Method for isolating rAAV particles In some embodiments, the Disclosure provides a method for producing isolated recombinant adeno-associated virus (rAAV) particles, 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 as described herein comprises (a) isolating rAAV particles from a feed containing impurities (e.g., an rAAV-producing culture), and (b) formulating the isolated rAAV particles to produce a formulation.

[0145] In some embodiments, the Disclosure further provides a method for generating pharmaceutical unit doses of formulations comprising isolated recombinant adeno-associated virus (rAAV) particles, comprising isolating rAAV particles from a feed containing impurities (e.g., an rAAV-producing culture) and formulation the isolated rAAV particles.

[0146] 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, hydroxyapatite chromatography, sterile filtration, or any combination(s) of these. In some embodiments, the downstream processing includes at least two, at least three, at least four, at least five, or at least six of the following: collection of cell cultures, clarification of the collected cell cultures (e.g., by centrifugation or deep filtration), tangential flow filtration, affinity chromatography, anion exchange chromatography, cation exchange chromatography, size exclusion chromatography, hydrophobic interaction chromatography, hydroxyapatite chromatography, and sterile filtration. In some embodiments, the downstream process includes collecting the cell culture, clarifying the collected cell culture (e.g., by deep filtration), sterile filtration, tangential flow filtration, affinity chromatography, and anion exchange chromatography. In some embodiments, the downstream process includes clarifying the collected cell culture, sterile filtration, tangential flow filtration, affinity chromatography, and anion exchange chromatography. In some embodiments, the downstream process includes clarifying the collected cell culture by deep filtration, sterile filtration, tangential flow filtration, affinity chromatography, and anion exchange chromatography. In some embodiments, the clarification of the collected cell culture includes sterile filtration. In some embodiments, the downstream process does not include centrifugation. In some embodiments, the rAAV particles contain the AAV8 serotype capsid protein. In some embodiments, the rAAV particles contain the AAV9 serotype capsid protein.

[0147] In some embodiments, a method for isolating rAAV particles according to the method described 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 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 described 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, the method for isolating rAAV particles described herein includes clarification of the collected cell culture by deep filtration, 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 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 AAV8 serotype capsid protein. In some embodiments, the rAAV particles contain the AAV9 serotype capsid protein.

[0148] 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 (including, for example, human cell lines such as HeLa, A549, or HEK293 cells and their derivatives (HEK293T cells, HEK293F cells), mammalian cell lines such as Vero, or, in the case of baculovirus-producing systems, insect cell lines such as SF-9); (2) a suitable helper virus function (provided by wild-type or mutant adenovirus (e.g., temperature-sensitive adenovirus), herpesvirus, 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. In some embodiments, the suitable helper virus function is provided by recombinant polynucleotides or plasmids as described herein. Suitable media known in the art can be used for the production of rAAV vectors. Such media include, but are not limited to, modified Eagle medium (MEM), Dulbecco's modified Eagle medium (DMEM), and media produced by Hyclone Laboratories and JRH, including Sf-900 II SFM medium as described in U.S. Patent No. 6,723,551 (which is incorporated herein by reference in its entirety).

[0149] 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).

[0150] 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.

[0151] 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.

[0152] 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 of about 0.1 mM to 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.

[0153] In some embodiments, rAAV particles are generated as disclosed in WO2020 / 033842 (which is incorporated herein by reference in its entirety).

[0154] Recombinant AAV particles can be collected from rAAV-producing cultures by collecting the product culture containing host cells, or by collecting the consumed medium from the product culture, provided that the cells are cultured under conditions known in the art to induce the release of rAAV particles from intact host cells into the culture medium. Recombinant AAV particles can also be collected from rAAV-producing cultures by lysing the host cells of the product culture. Suitable methods for lysing cells are also known in the art, and include, for example, multiple freeze / thaw cycles, sonication, microfluidization, and treatment with chemicals (e.g., surfactants and / or proteases).

[0155] 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.

[0156] 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.

[0157] 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 comprising one or more different filtration media. In some embodiments, the filter train comprises one deep filtration medium. In some embodiments, the filter train comprises one or more deep filtration media. In some embodiments, the filter train comprises two deep filtration media. In some embodiments, the filter train comprises one sterile filtration medium. In some embodiments, the filter train comprises 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 comprises Clarisolve® 20MS, Millistak+® C0HC, and sterile-grade filter 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 flocculent to the collected cell culture. In some embodiments, the collected cell culture is not pretreated before contact with the deep filter.

[0158] In some embodiments, the collected culture is clarified by filtration, as disclosed in WO2019 / 212921 (which is incorporated herein by reference in its entirety).

[0159] In some embodiments, rAAV-producing culture collections are treated with a nuclease (e.g., Bensonase®) or endonuclease (e.g., endonuclease from Serratia marcescens) to digest the high molecular weight DNA present in the producing culture. Nuclease or endonuclease digestion can be routinely carried out under standard conditions known in the art. For example, nuclease digestion is carried out for 30 minutes to several hours with a final concentration of 1 to 2.5 units / mL of Bensonase® at temperatures ranging from ambient temperature to 37°C.

[0160] 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.

[0161] 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 about 3 to about 10 diafiltration volumes of buffer. In some embodiments, the diafiltration includes using about 5 diafiltration volumes of buffer. Those skilled in the art will also recognize that TFF may be used at any step of the purification process when 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 described herein includes the use of TFF for buffer exchange.

[0162] 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 include, but are not limited to, AVB Sepharose®, POROS® CaptureSelect® AAVX affinity resin, POROS® CaptureSelect® AAV9 affinity resin, and POROS® CaptureSelect® AAV8 affinity resin. In some embodiments, the affinity chromatography media is POROS® CaptureSelect® AAV9 affinity resin. In some embodiments, the affinity chromatography media is POROS® CaptureSelect® AAV8 affinity resin. In some embodiments, the affinity chromatography medium is POROS® CaptureSelect® AAVX affinity resin.

[0163] 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.

[0164] 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).

[0165] 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).

[0166] 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).

[0167] In additional embodiments, the disclosure provides compositions comprising isolated rAAV particles produced according to the method described herein. In some embodiments, the compositions are pharmaceutical compositions comprising a pharmaceutically acceptable carrier.

[0168] 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).

[0169] 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 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 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, and AAV16, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu32, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B, A The rAAV particles contain capsid proteins from AAV capsid 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 AAV capsid serotype is AAV8. In some embodiments, the AAV capsid serotype is AAV9.

[0170] Method for generating recombinant polypeptides In one embodiment, the present disclosure provides a method for expressing recombinant polypeptides in eukaryotic host cells as described herein. In some embodiments, the method further includes recovering the polypeptides.

[0171] In some embodiments, the Disclosure provides a method for producing recombinant polypeptides, comprising (a) preparing a cell culture comprising a plurality of cells as described herein, each comprising one or more polynucleotides encoding a recombinant polypeptide, and (b) maintaining the cell culture under conditions that enable the production of recombinant polypeptides. In some embodiments, the recombinant polypeptide is an antibody or antibody fragment. In some embodiments, the antibody is a chimeric, human, or humanized antibody. In some embodiments, the antibody is an IgG antibody. In some embodiments, the recombinant polypeptide is a fusion protein. In some embodiments, the fusion protein comprises an antibody or a fragment thereof. In some embodiments, the fusion protein comprises an Fc region. In some embodiments, the fusion protein comprises an antigen-binding antibody fragment. In some embodiments, the cells are HEK293 cells. In some embodiments, the cells are HEK293-derived cells. In some embodiments, the cells are suspension-adapted HEK293 cells. In some embodiments, the cells are suspension-adapted HEK293-derived cells. In some embodiments, the cell culture is a suspension culture or an adherent culture. In some embodiments, the cell culture is a suspension culture. In some embodiments, the cell culture has a volume ranging from approximately 50 liters to approximately 20,000 liters.

[0172] In some embodiments, the Disclosure provides a method for generating rAAV particles, comprising culturing cells as described herein, wherein the cells comprise one or more polynucleotides encoding a recombinant polypeptide. In some embodiments, the recombinant polypeptide is an antibody or antibody fragment. In some embodiments, the antibody is a chimeric, human, or humanized antibody. In some embodiments, the antibody is an IgG antibody. In some embodiments, the recombinant polypeptide is a fusion protein. In some embodiments, the fusion protein comprises an antibody or a fragment thereof. In some embodiments, the fusion protein comprises an Fc region. In some embodiments, the fusion protein comprises an antigen-binding antibody fragment. In some embodiments, the cells are HEK293 cells. In some embodiments, the cells are HEK293-derived cells. In some embodiments, the cells are suspension-adapted HEK293 cells. In some embodiments, the cells are suspension-adapted HEK293-derived cells.

[0173] In some embodiments, the Disclosure provides a method for generating rAAV particles, comprising: (a) preparing a cell culture comprising a plurality of cells; (b) introducing one or more polynucleotides encoding a recombinant polypeptide into the cells; and (c) maintaining the cell culture under conditions that enable the generation of the recombinant polypeptide. In some embodiments, the recombinant polypeptide is an antibody or antibody fragment. In some embodiments, the antibody is a chimeric, human, or humanized antibody. In some embodiments, the antibody is an IgG antibody. In some embodiments, the recombinant polypeptide is a fusion protein. In some embodiments, the fusion protein comprises an antibody or a fragment thereof. In some embodiments, the fusion protein comprises an Fc region. In some embodiments, the fusion protein comprises an antigen-binding antibody fragment. In some embodiments, the cells are HEK293 cells. In some embodiments, the cells are HEK293-derived cells. In some embodiments, the cells are suspension-adapted HEK293 cells. In some embodiments, the cells are suspension-adapted HEK293-derived cells. In some embodiments, the cell culture is a suspension culture or an adherent culture. In some embodiments, the cell culture is a suspension culture. In some embodiments, the cell culture has a volume ranging from approximately 50 liters to approximately 20,000 liters. [Examples]

[0174] Example 1. Identification of target genes for modification As disclosed in international patent application publication number WO2023 / 24392 (which is incorporated herein by reference in its entirety), adding a caspase inhibitor to recombinant AAV-producing cell cultures increases rAAV yield. To identify candidate genes to be modified in host cells to improve AAV productivity, a screening was performed using an array RNAi library targeting apoptotic signaling pathway genes.

[0175] To systematically understand the gene profile of the HEK293 cell line and the cellular factors that regulate AAV production, a high-throughput RNAi screening was performed using a library targeting apoptotic signaling pathways. In the initial screening, the contributions of 923 human genes were evaluated using 2769 unique siRNAs (i.e., 3 per gene) from Invitrogen's Silencer® library. Briefly, on day 1, HEK293 cells in 96-well plates were seeded at approximately 40-50% confluence into collagen-I pre-coated 24-well plates, and a single siRNA capable of downregulating mRNA expression by a specific target gene was reverse-transferred. Approximately 24 hours later, the cells were transfused with combinations of three plasmids (i.e., helper, cis, and trans plasmids) to generate rAAV particles. On day 4, the cell suspension from each well was dissolved in AAV-MAX® lysis buffer, and the titer of the generated rAAV particles was quantified using ddPCR. This screening used the helper #5 helper plasmid (see WO2023 / 060113) and cis and trans plasmids encoding AAV8 TG-A recombinant AAV particles. The initial screening yielded evaluable results for 845 target genes via biological replicates, based on which 267 genes were selected for further validation. Similar to the initial screening, the validation screening used translocation-mediated RNAi to downregulate mRNA produced by target genes in adherent HEK293 cells, followed by translocation with combinations of the three plasmids (i.e., helper #5, cis, and trans plasmids) to generate rAAV particles. The titers of AAV8 TG-A, AAV9 TG-D, and AAV8 TG-B recombinant AAV particles were independently measured in the validation screening. This unbiased screening approach identified 137 genes in which downregulation increased recombinant AAV production (reflected by increased titer). Genetic ontology analysis of these candidate genes that affect titer revealed functional classifications related to several signaling pathways. Figure 1.More than half of the identified genes are linked to pathways involved in the immune response to viruses.

[0176] To further confirm the results from the primary screening and to identify siRNAs targeting genes that have a specific effect on titer improvement without significantly affecting cell viability during long-term cell culture, a lentiviral-based shRNA assay was established. Lentiviral delivery of RNAi vectors offers the advantages of permanent integration of the RNAi vector into the host cell genome, high transduction efficiency, and relatively low cost, making this approach suitable for further testing in suspension cultures. Two separate lentiviral shRNA vectors were generated for each of the 137 genes identified by the primary screening. Secondary testing was performed in 24-well deep-well plates using HEK293 suspension cultures. rAAV particles were generated by triple transduction using helper #5 helper plasmid (see WO2023 / 060113). The titers of recombinant AAV particles AAV8 TG-A, AAV8 TG-B, and AAV9 TG-D were independently measured. Figure 2. rAAV titers were quantified by ddPCR as GC per 1 ml of suspension culture. In all cases, no significant difference in rAAV titers was observed between cells infected with the scrambled shRNA control vector and control cells without lentivirus infection. This indicates that the host cells fully recovered after lentivirus infection and scale-up. The 19 genes that yielded the highest rAAV titers after downregulation are listed in the table below. Most genes (15 / 19) after shRNA-mediated knockdown showed similar titer improvement percentages with all three different rAAV particles tested. However, VEGFB knockdown significantly improved the titers of AAV8 TG-B and AAV9 TG-D; FIS1 knockdown increased the titers of AAV8 TG-B and AAV9 TG-D but decreased the titer of AAV8 TG-A; BNIP1 knockdown significantly improved only the titer of AAV8 TG-A; and CUL1 knockdown significantly improved the titers of AAV8 TG-B and AAV9 TG-D but decreased the titer of AAV8 TG-A.This data is consistent with the understanding that most of the genes in the table have a universal effect on titer improvement, while some genes indirectly influence titer through other signaling pathways that may be transgene-specific.

[0177] (Table 3) Absolute and relative rAAV titers of target genes after lentiviral-based shRNA silencing in 24-well deep-well suspension cultures. Absolute titer is GC per 1 ml of suspension culture. Relative titer of non-target control is 1. TIFF2026516074000013.tif148147

[0178] The effect of simultaneous reduction in the activity of two target genes on rAAV titer was quantified. Briefly, on day 1, HEK293 cells were seeded in collagen-I pre-coated 24-well plates at approximately 40-50% confluence. Specific siRNAs targeting two of the top nine candidate genes were reverse-simultaneously transfused using equal amounts of siRNA for each gene. Approximately 24 hours later, rAAV particles were generated by transfusing cells with combinations of three plasmids (i.e., helper, cis, and trans plasmids). On day 4, the cell suspension from each well was dissolved in AAV-MAX® lysis buffer, and the titer of the generated rAAV particles was quantified using ddPCR. This screening used the helper #5 helper plasmid (see WO2023 / 060113) and cis and trans plasmids encoding AAV8 TG-A, AAV8 TG-B, or AAV9 TG-D recombinant AAV particles. The results are shown in Figure 3.

[0179] Example 2. Host cell manipulation by IGFBP3 knockout We selected the gene that most significantly affects rAAV titer (e.g., IGFBP3) and performed CRISPR / Cas9-mediated knockout editing. Figure 4A. Briefly, on day 1, HEK293 cells were seeded in collagen I pre-coated 24-well plates at approximately 40-50% confluence, and specific sgRNAs targeting the Cas9 protein and IGFBP3 gene were reverse-simultaneously transfused. Approximately 48 hours later, cells were harvested from two wells, and editing efficiency was evaluated by PCR and Western blotting. On day 4, the remaining cells were trypsin-treated and resuspended in 96-well plates for single-cell cloning. Approximately 14-18 days later, the emerging clones were transferred to 24-well plates. After 7-10 days, viable clones were transferred to 24-deep-well plates to produce enough cells for quantitative analysis of IGFPBP3 expression and measurement of AAV production by Western blotting. AAV production was measured by transtransferring cells with a combination of three plasmids (i.e., a helper plasmid, a cis plasmid encoding the transgene, and a transplasmid encoding rep / cap) to generate AAV8 TG-A, AAV8 TG-B, or AAV9 TG-D rAAV particles. 72 hours after triple transtransfer, the cell suspension of each clone was dissolved in AAV-MAX® lysis buffer, and the titer of the generated AAV particles was quantified using ddPCR.

[0180] After transfusion with Cas9 protein and IGFBP3-specific sgRNA, 40.8% (170 / 416) of the clones in the 96-well plate successfully expanded to the 24-well plate. This is only about half the cloning efficiency (>80%) after treatment with random control sgRNA. 94.1% (160 / 170) of the clones in the 24-well plate survived after transfer to the 24-well deep-well plate. Figure 4B shows the AAV titers generated by candidate clones after transfusion with helper / cis / trans plasmid combinations of helper #5 / TG-A / AAV8 or helper #5 / TG-D / AAV9. Most candidate clones obtained titers equal to or lower than those of the control cells.

[0181] As shown in Figure 5A, most clones were assessed to have either a + / + wild-type or + / - heterozygous mutant IGFBP3 genotype based on IGFBP3 expression detected by Western blotting. Further testing failed to identify any clones that could yield significantly higher AAV yields than control cells. Figures 5B and C.

[0182] Example 3. Host cell manipulation by DYNLL1 knockout. Candidate DYNLL1-KO clones were generated substantially as described above. The efficiency of two DYNLL1-specific sgRNAs (C1 and C2) was tested by Western blotting on cells simultaneously transfused with Cas9 protein and DYNLL1-specific sgRNA (Figure 6A). 49% (357 / 744) of the clones in 96 wells successfully expanded to 24 wells, but this was still only about half the cloning efficiency after treatment with random control sgRNA (>80%). AAV generation in candidate clones was measured after transfusion with helper / cis / trans plasmid combinations: helper #5 / TG-A / AAV8 (Figure 6B) and helper #5 / TG-D / AAV9 (Figure 6C). Most candidate clones showed higher AAV titers than the parental control clones. Clones with titers similar to or better than the parental control clones were selected as candidate clones for further evaluation. The DYNLL1 genotype of candidate clones was evaluated by sequencing and ICE analysis. DYNLL1+ / - heterozygous clones showed higher AAV titers than DYNLL1+ / +WT or DYNLL1- / -KO clones. DYNLL1- / -KO clones exhibited significantly slower growth rates compared to wild-type and heterozygous clones. While not bound by any specific theory, this slower growth rate of - / -KO clones may explain why these clones did not show a significant improvement in AAV production. DYNLL1- / -KO clones showed significantly slower growth rates compared to DYNLL1+ / - heterozygotes (28-30 hours vs. 22-24 hours in terms of doubling time). Despite slower doubling times, AAV generation by DYNLL1- / -KO clones remained comparable to that of DYNLL1+ / - heterozygotes, with DYNLL1- / -KO clones achieving approximately 80-90% of the titer achieved by DYNLL1+ / - heterozygotes. Previous experiments (data not shown) compared the effect of viable cell density (VCD) on AAV generation, finding that cultures with a VCD of 6.5E6 during translocation yielded 30-40% higher AAV titers than cultures with a VCD of 5E6 during translocation.When cultures of DYNLL1- / - KO clone cells with a doubling time of approximately 29 hours were seeded at the same density as cultures containing the corresponding DYNLL1+ / - or DYNLL1+ / + cells, the VCD may not have reached 6.5e6 during translocation. Therefore, the reason for the lower AAV titer in DYNLL1- / - clones compared to DYNLL1+ / - clones can be explained by the slower doubling time.

[0183] Figure 7 shows the AAV titers obtained in 1C1, 4D9, 2C6, 4A8, 3B7, 4D5, and 4A2 DYNLL1+ / - clones after transtransfection with (i) the pHRC#7(helper+AAV8-trans) plasmid and the TG-A cis plasmid, (ii) the helper#5, TG-D cis, and AAV9 trans plasmids, and (iii) the pHRC#7(helper+AAV8-trans) plasmid and the TG-B cis plasmid. The pHRC#7 helper / rep / cap plasmid is disclosed in PCT / US2024 / 023368 (filed April 5, 2024). The titers increased similarly for all three transgenes tested in 1C1, 4D9, 2C6, 4A8, 3B7, 4D5, and 4A2 clones.

[0184] 35% of the clones selected after transfusion with Cas-9 / sgRNA-C1 were identified as DYNLL1- / -, while only 1% of the clones selected after transfusion with Cas-9 / sgRNA-C1 clones were identified as DYNLL1- / -.

[0185] Example 4. Host cell manipulation by knockout of DYNLL1 and MAX Candidate DYNLL1 / MAX double KO clones were generated using 4A2 and 4D5 DYNLL1+ / - cells as a starting point, essentially as described above. Cas-9 protein and MAX-specific sgRNA were transfused into 4A2 or 4D5 cells. MAX sgRNA KO efficiency was evaluated by sequencing of samples collected one day after transfusion. In 96-well 4A2-derived clones, 65.8% (252 / 383) successfully expanded to 24-well cells, while in 96-well 4D5-derived clones, only 4.2% (16 / 384) successfully expanded to 24-well cells. AAV generation in candidate clones was measured after transfusion with pHRC#7 (helper + AAV8-trans) plasmid and TG-A cis plasmid. Figure 8. The majority of candidate clones showed higher AAV titers than the parental control clones. Clones with titers similar to or better than the parent control clone were selected as candidate clones for further evaluation.

[0186] Figure 9 shows the AAV titers obtained after transtransferring (i) pHRC#7 (helper + AAV8-trans) plasmid and TG-A cis plasmid, (ii) pHRC#8 (helper + AAV8-trans) plasmid and TG-C cis plasmid, (iii) helper #5, TG-D cis, and AAV9 trans plasmids, and (iv) pHRC#8 (helper + AAV8-trans) plasmid and TG-B cis plasmid in 4A2-3D3, 4A2-3B11, 4A2-3E6, 4A2-1H7, 4A2-4F12, 4A2-2C12, 4A2-3C3, and 4A2-2H7 DYNLL1+ / - / MAX KO clones with (i) pHRC#7 (helper + AAV8-trans) plasmid and TG-A cis plasmid, (ii) pHRC#8 (helper + AAV8-trans) plasmid and TG-C cis plasmid, (iii) helper #5, TG-D cis, and AAV9 trans plasmids, and (iv) pHRC#8 (helper + AAV8-trans) plasmid and TG-B cis plasmid. The titers were similarly increased in all four transgenes tested with the 4A2-3D3, 4A2-3B11, 4A2-3E6, 4A2-1H7, 4A2-4F12, 4A2-2C12, 4A2-3C3, and 4A2-2H7 clones. The pHRC#7 and pHRD#8 helper / rep / cap plasmids are disclosed in PCT / US2024 / 023368 (filed April 5, 2024).

[0187] Example 5. Host cell manipulation through knockout of ANGPT1, ARHGEF7, ARHGEF17, BID, BNIP1, C19ORF2, CHST11, CUL1, DAP3, DYNLL1, FIS1, HMGB2, IGFBP3, NRG1, TNFRSF10C, TNFSF12, MAL, MAX, and / or VEGFB. We selected the gene that most significantly affects rAAV titer and performed CRISPR / Cas9-mediated knockout editing. Briefly, on day 1, HEK293 cells were seeded in collagen I pre-coated 24-well plates at approximately 40-50% confluence, and Cas9 protein and specific sgRNAs targeting candidate genes were reverse-simultaneously transfused. Approximately 48 hours later, cells from two wells were harvested, and editing efficiency was evaluated by PCR and Western blotting. On day 4, the remaining cells were trypsin-treated and resuspended in 9-well plates for single-cell cloning. Approximately 14-18 days later, the clones were transferred to 24-well plates. After Sanger sequencing and protein level detection, several positive clones that specifically knocked out the candidate genes were selected and transferred to 24-deep-well plates. After scaling up to a shaking flask, cells are transfused with a combination of three plasmids (i.e., helper, cis, and trans plasmids) to generate AAV8 TG-A, AAV8 TG-B, or AAV9 TG-D rAAV particles. 72 hours after triple transfusion, the cell suspension of each clone is dissolved in AAV-MAX® lysis buffer, and the titer of the generated AAV particles is quantified using ddPCR.

[0188] 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.

[0189] 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. Recombinant cells capable of producing rAAV, wherein the cells include at least one modification that reduces or removes the activity of at least one endogenous gene or gene product in an apoptotic signaling pathway.

2. The cell according to claim 1, wherein the cell comprises at least two modifications that reduce or remove the activity of at least two genes or gene products in an apoptotic signaling pathway.

3. A cell bank comprising a plurality of cells, wherein each cell comprises at least one modification that reduces or removes the activity of at least one endogenous gene or gene product in an apoptotic signaling pathway.

4. The cell bank according to claim 3, wherein the cells include at least two modifications that reduce or remove the activity of at least two genes or gene products in an apoptotic signaling pathway.

5. A cell culture comprising a plurality of cells capable of producing rAAV, wherein the cells include at least one modification that reduces or removes the activity of at least one endogenous gene or gene product in an apoptotic signaling pathway.

6. The cell culture according to claim 5, wherein the cells include at least two modifications that reduce or remove the activity of at least two genes or gene products in an apoptotic signaling pathway.

7. A method for generating rAAV particles, a) Prepare a cell culture containing multiple cells capable of producing rAAV, b) Maintaining the cell culture under conditions that enable the generation of the rAAV particles. Includes, The cells include at least one modification that reduces or removes the activity of at least one endogenous gene or gene product in the apoptotic signaling pathway. The aforementioned method.

8. A method for generating rAAV particles, comprising culturing cells capable of generating rAAV particles under conditions that enable the generation of the rAAV particles, wherein the cells include at least one modification that reduces or removes the activity of at least one endogenous gene or gene product in an apoptotic signaling pathway.

9. A method for increasing the generation of rAAV particles, a) Prepare a cell culture containing multiple cells capable of producing rAAV, b) Maintaining the cell culture under conditions that enable the generation of the rAAV particles. Includes, The cells include at least one modification that reduces or removes the activity of at least one endogenous gene or gene product in the apoptotic signaling pathway. The aforementioned method.

10. A method for increasing the production of rAAV particles, comprising culturing cells capable of producing rAAV particles under conditions that enable the production of the rAAV particles, wherein the cells include at least one modification that reduces or removes the activity of at least one endogenous gene or gene product in an apoptotic signaling pathway.

11. The method according to any one of claims 7 to 10, wherein the cells include at least two modifications that reduce or remove the activity of at least two genes or gene products in an apoptotic signaling pathway.

12. To the cells capable of generating rAAV, the following: a) Packaged rAAV genome, 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 any one of claims 1 to 11, wherein one or more polynucleotides encoding at least one of the following are transfused.

13. To the cells capable of generating rAAV, the following: a) Packaged rAAV genome, 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 any one of claims 1 to 12, wherein one or more polynucleotides encoding are transfected.

14. A method for generating rAAV particles, a) Prepare a cell culture containing multiple cells, b) To the cells: i. The packaged rAAV genome, ii. Adenovirus helper function necessary for packaging, iii. Sufficient AAV rep protein for packaging, and iv. Sufficient AAV cap protein for packaging Introducing one or more polynucleotides that encode at least one of the following, c) Maintaining the cell culture under conditions that enable the generation of the rAAV particles. Includes, The cells include at least one modification that reduces or removes the activity of at least one endogenous gene or gene product in the apoptotic signaling pathway. The aforementioned method.

15. A method for increasing the generation of rAAV particles, a) Prepare a cell culture containing multiple cells, b) To the cells: i. The packaged rAAV genome, ii. Adenovirus helper function necessary for packaging, iii. Sufficient AAV rep protein for packaging, and iv. Sufficient AAV cap protein for packaging Introducing one or more polynucleotides that encode at least one of the following, c) Maintaining the cell culture under conditions that enable the generation of the rAAV particles. Includes, The cells include at least one modification that reduces or removes the activity of at least one endogenous gene or gene product in the apoptotic signaling pathway. The aforementioned method.

16. The method according to claim 14 or 15, wherein the cells include at least two modifications that reduce or eliminate the activity of at least two genes or gene products in an apoptotic signaling pathway.

17. To the aforementioned cells, the following: i. The packaged rAAV genome, ii. Adenovirus helper function necessary for packaging, iii. Sufficient AAV rep protein for packaging, and iv. Sufficient AAV cap protein for packaging The method according to any one of claims 14 to 16, comprising introducing one or more polynucleotides encoding a

18. The method according to any one of claims 14 to 17, wherein the introduction of one or more polynucleotides into the cells is carried out by transfusion.

19. The cell, cell bank, cell culture, or method according to any one of claims 1 to 18, wherein the modification includes a mutation in the gene.

20. The cell, cell bank, cell culture, or method according to claim 19, wherein the gene modification includes a missense mutation, a nonsense mutation, or a frameshift mutation.

21. The cell, cell bank, cell culture, or method according to claim 19, wherein the gene modification includes a deletion.

22. The cell, cell bank, cell culture, or method according to any one of claims 19 to 21, wherein the mutation is a heterozygous mutation.

23. The cell, cell bank, cell culture, or method according to claim 22, wherein the heterozygous mutation affects one copy of the gene.

24. The cell, cell bank, cell culture, or method according to claim 22, wherein the heterozygous mutation affects more than one copy of the gene.

25. The cell, cell bank, cell culture, or method according to claim 22, wherein the heterozygous mutation affects one, two, or three copies of the gene.

26. The cell, cell bank, cell culture, or method according to any one of claims 19 to 21, wherein the mutation is a homozygous mutation.

27. The cell, cell bank, cell culture, or method according to any one of claims 1 to 22, wherein the modification comprises an inhibitory nucleic acid molecule capable of reducing or removing the activity of the gene or gene product.

28. The cell, cell bank, cell culture, or method according to claim 27, wherein the modification includes antisense RNA.

29. The cell, cell bank, cell culture, or method according to claim 27, wherein the modification comprises a small interfering RNA (siRNA), a microRNA (miRNA), or a short hairpin RNA (shRNA).

30. A cell, cell bank, cell culture, or method according to any one of claims 1 to 29, wherein the at least one endogenous gene or gene product in the apoptotic signaling pathway is selected from the group consisting of ANGPT1, ARHGEF7, ARHGEF17, BID, BNIP1, C19orf2, CHST11, CUL1, DAP3, DYNLL1, FIS1, HMGB2, IGFBP3, NRG1, TNFRSF10C, TNFSF12, MAL, MAX, and VEGFB genes or gene products.

31. The cell, cell bank, cell culture, or method according to claim 30, wherein the at least one endogenous gene or gene product comprises any two genes or gene products selected from the group consisting of ANGPT1, ARHGEF7, ARHGEF17, BID, BNIP1, C19orf2, CHST11, CUL1, DAP3, DYNLL1, FIS1, HMGB2, IGFBP3, NRG1, TNFRSF10C, TNFSF12, MAL, MAX, and VEGFB genes or gene products.

32. The cell, cell bank, cell culture, or method according to any one of claims 1 to 29, wherein the at least one endogenous gene or gene product is selected from the group consisting of ARHGEF7, ARHGEF17, BNIP1, C19orf2, CHST11, DYNLL1, IGFBP3, MAX, and VEGFB genes or gene products.

33. The cell, cell bank, cell culture, or method according to claim 32, wherein the at least one endogenous gene or gene product comprises any two genes or gene products selected from the group consisting of ARHGEF7, ARHGEF17, BNIP1, C19orf2, CHST11, DYNLL1, IGFBP3, MAX, and VEGFB genes or gene products.

34. The cell, cell bank, cell culture, or method according to any one of claims 1 to 29, wherein the at least one endogenous gene or gene product comprises DYNLL1, IGFBP3, and / or the MAX gene or gene product.

35. The cell, cell bank, cell culture, or method according to claim 34, wherein the at least one endogenous gene or gene product comprises the DYNLL1 gene or gene product.

36. The cell, cell bank, cell culture, or method according to claim 35, wherein the modification includes a heterozygous DYNLL1 mutation.

37. The cell, cell bank, cell culture, or method according to claim 34, wherein the at least one endogenous gene or gene product comprises the DYNLL1 and MAX genes or gene products.

38. The cell, cell bank, cell culture, or method according to claim 37, wherein the modification includes a heterozygous DYNLL1 mutation and a MAX mutation.

39. The cell, cell bank, cell culture, or method according to any one of claims 1 to 29, wherein the at least one endogenous gene or gene product comprises DYNLL1 and a second gene or gene product selected from the group consisting of ANGPT1, ARHGEF7, ARHGEF17, BID, BNIP1, C19orf2, CHST11, CUL1, DAP3, FIS1, HMGB2, IGFBP3, NRG1, TNFRSF10C, TNFSF12, MAL, MAX, and VEGFB.

40. The cell, cell bank, cell culture, or method according to any one of claims 1 to 29, wherein the at least one endogenous gene or gene product comprises DYNLL1 and a second gene or gene product selected from the group consisting of ARHGEF7, ARHGEF17, BNIP1, C19orf2, CHST11, IGFBP3, MAX, and VEGFB.

41. The cell, cell bank, cell culture, or method according to any one of claims 1 to 29, wherein the at least one endogenous gene or gene product comprises DYNLL1 and ANGPT1, ARHGEF7, ARHGEF17, BID, BNIP1, C19orf2, CHST11, CUL1, DAP3, FIS1, HMGB2, IGFBP3, NRG1, TNFRSF10C, TNFSF12, MAL, MAX, and VEGFB.

42. The cell, cell bank, cell culture, or method according to any one of claims 1 to 29, wherein the at least one endogenous gene or gene product comprises DYNLL1 and one or more genes or gene products selected from the group consisting of ANGPT1, ARHGEF7, ARHGEF17, BID, BNIP1, C19orf2, CHST11, CUL1, DAP3, FIS1, HMGB2, IGFBP3, NRG1, TNFRSF10C, TNFSF12, MAL, MAX, and VEGFB.

43. The cell, cell bank, cell culture, or method according to any one of claims 1 to 29, wherein the at least one endogenous gene or gene product comprises DYNLL1 and one or more genes or gene products selected from the group consisting of ARHGEF7, ARHGEF17, BNIP1, C19orf2, CHST11, IGFBP3, MAX, and VEGFB.

44. The cell, cell bank, cell culture, or method according to any one of claims 1 to 29, wherein the at least one endogenous gene or gene product comprises DYNLL1 and ARHGEF7.

45. The cell, cell bank, cell culture, or method according to any one of claims 1 to 29, wherein the at least one endogenous gene or gene product comprises DYNLL1 and ARHGEF17.

46. The cell, cell bank, cell culture, or method according to any one of claims 1 to 29, wherein the at least one endogenous gene or gene product comprises DYNLL1 and BNIP1.

47. The cell, cell bank, cell culture, or method according to any one of claims 1 to 29, wherein the at least one endogenous gene or gene product comprises DYNLL1 and C19orf2.

48. The cell, cell bank, cell culture, or method according to any one of claims 1 to 29, wherein the at least one endogenous gene or gene product comprises DYNLL1 and CHST11.

49. The cell, cell bank, cell culture, or method according to any one of claims 1 to 29, wherein the at least one endogenous gene or gene product comprises DYNLL1 and IGFBP3.

50. The cell, cell bank, cell culture, or method according to any one of claims 1 to 29, wherein the at least one endogenous gene or gene product comprises DYNLL1 and MAX.

51. The cell, cell bank, cell culture, or method according to any one of claims 1 to 29, wherein the at least one endogenous gene or gene product comprises DYNLL1 and VEGFB.

52. The cell, cell bank, cell culture, or method according to any one of claims 1 to 51, wherein the cell is a mammalian cell.

53. The cell, cell bank, cell culture, or method according to any one of claims 1 to 51, wherein the cell is an insect cell.

54. The cell, cell bank, cell culture, or method according to any one of claims 1 to 51, wherein the cells are HEK293 cells, HEK293-derived cells, CHO cells, CHO-derived cells, HeLa cells, SF-9 cells, BHK cells, Vero cells, CAP cells, or PerC6 cells.

55. The cell, cell bank, cell culture, or method according to any one of claims 1 to 51, wherein the cell is HEK293 cell.

56. The cell, cell bank, cell culture, or method according to any one of claims 1 to 55, wherein the cell culture is a suspension culture.

57. The cell, cell bank, cell culture, or method according to any one of claims 12 to 51, wherein the adenovirus helper function comprises at least one of the adenovirus E4 gene, E2a gene, and VA gene.

58. The cell, cell bank, cell culture, or method according to any one of claims 12 to 51, wherein the adenovirus helper function comprises the adenovirus E4 gene, the E2a gene, and the VA gene.

59. The cell, cell bank, cell culture, or method according to claim 58, wherein the polynucleotide encoding the adenovirus helper function comprises pAD delta F6 or helper #5.

60. The cell, cell bank, cell culture, or method according to claim 58, wherein the polynucleotide encoding the adenovirus helper function comprises pHRC#7 or pHRC#8.

61. The method according to any one of claims 7 to 60, wherein the cell culture is maintained or the cells are cultured under conditions that enable the generation of the rAAV particles for a period of about 2 to 10 days, about 2 to 15 days, or about 5 to 14 days.

62. The method according to any one of claims 7 to 60, wherein the cell culture is maintained under conditions that enable the generation of the rAAV particles, or the cells are cultured for a period of about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, or about 7 days.

63. The method according to any one of claims 7 to 60, wherein the cell culture is maintained under conditions that enable the generation of the rAAV particles, or the cells are cultured for a period of about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, or about 7 days.

64. The method according to claim 63, wherein the cell culture is maintained under conditions that enable the generation of the rAAV particles, or the cells are cultured, for a period of about 5 days.

65. The method according to any one of claims 7 to 64, further comprising recovering the rAAV particles.

66. The method according to any one of claims 7 to 65, wherein the method produces more rAAV particles measured as GC / ml than a reference method using cells that do not contain the modification.

67. The method according to any one of claims 7 to 65, wherein the cell culture produces at least 10%, at least 20%, at least 30%, at least 50%, at least 75%, or at least 100% more rAAV particles, as measured as GC / ml, than a reference method using cells that do not contain the modification.

68. The cell culture or method according to any one of claims 5 to 67, wherein the cell culture has a volume of about 50 liters to about 20,000 liters.

69. The cell culture or method according to any one of claims 5 to 67, wherein the cell culture has a volume of about 200 liters to about 2,000 liters.

70. The rAAV is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, and AAV16, AAV. rh8, AAV. rh10, AAV. rh20, AAV. rh39, AAV. Rh74, AAV. RHM4-1, AAV. hu32, AAV. hu37, AAV. Anc80, AAV. Anc80L65, AAV. 7m8, AAV. PHP. A cell, cell bank, cell culture, or method according to any one of claims 1 to 69, comprising capsid proteins of serotypes 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.

71. The cell, cell bank, cell culture, or method according to any one of claims 1 to 69, wherein the rAAV comprises the capsid proteins of serotypes AAV8, AAV9, AAV. rh10, AAV. rh20, AAV. rh39, AAV. Rh74, AAV. RHM4-1, AAV. hu32, and AAV. hu37.

72. The cell, cell bank, cell culture, or method according to any one of claims 1 to 69, wherein the rAAV comprises a capsid protein of the AAV8 or AAV9 serotype.

73. The cell, cell bank, cell culture, or method according to any one of claims 1 to 72, wherein the rAAV comprises a genome encoding a polypeptide or a double-stranded RNA molecule.

74. The cell, cell bank, cell culture, or method according to claim 73, wherein the genome encodes a polypeptide.

75. The cell, cell bank, cell culture, or method according to claim 73, wherein the genome encodes an anti-VEGF Fab, an anti-kallikrein antibody, an anti-TNF antibody, a microdystrophin, a minidystrophin, an isulonidase (IDUA), an iduronate 2-sulfatase (IDS), a low-density lipoprotein receptor (LDLR), a tripeptidyl peptidase 1 (TPP1), or a non-membrane-bound splice variant of VEGF receptor 1 (sFlt-1).

76. The genome contains gamma-sarcoglycan, Rab escort protein 1 (REP1 / CHM), retinoid isomerohydrolase (RPE65), cyclic nucleotide-gated channel alpha-3 (CNGA3), cyclic nucleotide-gated channel beta-3 (CNGB3), aromatic L-amino acid decarboxylase (AADC), lysosome-associated membrane protein 2 isoform B (LAMP2B), factor VIII, factor IX, retinitis pigmentosa GTPase modulator (RPGR), retinosuxin (RS1), sarcoplasmic reticulum calcium ATPase (SERCA2a), aflibercept, battenin (CLN3), transmembrane ER protein (CLN6), and glutathione. Minic acid decarboxylase (GAD), glial cell-derived neurotrophic factor (GDNF), aquaporin 1 (AQP1), dystrophin, myotubularin 1 (MTM1), follistatin (FST), glucose-6-phosphatase (G6Pase), apolipoprotein A2 (APOA2), uridine diphosphate glucuronosyltransferase 1A1 (UGT1A1), arylsulfatase B (ARSB), N-acetyl-alpha-glucosaminidase (NAGLU), alpha -Glucosidase (GAA), alpha-galactosidase (GLA), beta-galactosidase (GLB1), lipoprotein lipase (LPL), alpha-1-antitrypsin (AAT), phosphodiesterase 6B (PDE6B), ornithine carbamoyltransferase 9OTC), survival motor neuron (SMN1), survival motor neuron (SMN2), neurturin (NRTN), neurotrophin-3 (NT-3 / NTF3), porphobilinogen deaminase (P A cell, cell bank, cell culture, or method according to claim 73, encoding BGD), nerve growth factor (NGF), mitochondrial encoded NADH:ubiquinone oxidoreductate zecoa subunit 4 (MT-ND4), protective protein cathepsin A (PPCA), dysferrin, MER proto-oncogene tyrosine kinase (MERTK), cystic fibrosis transmembrane conductance regulator (CFTR), or tumor necrosis factor receptor (TNFR)-immunoglobulin (IgG1) Fc fusion.

77. The cell, cell bank, cell culture, or method according to claim 73, wherein the genome encodes dystrophin or microdystrophin.

78. The cell, cell bank, cell culture, or method according to claim 73, wherein the packaged genome encodes microRNA.

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