Viral vector-producing cells with improved vector production capacity, methods for producing the same, and methods for selecting the same.

Abstract

The present invention aims to provide viral vector-producing cells with improved viral vector production capabilities, a method for producing the same, a kit containing the same, a method for selecting viral vector-producing cells, and the like. [Solution] Viral vector-producing cells have improved viral vector production capacity, characterized in that the expression of proteins derived from viral vector-producing cells is regulated, specifically, the expression of the proteins is regulated compared to viral vector-producing cells or HEK293 cells (deposit number ATCC CRL1573) before the regulation of such protein expression.

Description

[Technical Field]

[0001] The present invention relates to viral vector-producing cells and methods for producing the same, in particular to viral vector-producing cells with improved vector production capacity, methods for producing the same, methods for selecting the same, and kits containing the same. [Background technology]

[0002] In the medical field, including gene therapy, the use of virus-derived gene transfer vectors (hereinafter referred to as viral vectors) is common as a method for introducing genes into mammalian cells, including human cells. Viral vectors are vectors created by modifying naturally occurring viruses using genetic engineering technology, allowing for the transfer of desired genes or other elements into a target. Technological development in this field has been progressing in recent years. Well-known viruses that serve as the source of viral vectors include enveloped viruses such as retroviruses, lentiviruses, Sendai viruses, and herpesviruses, as well as non-enveloped viruses such as adenoviruses and adeno-associated viruses (AAVs). In particular, AAV is considered promising as a gene transfer vector for gene therapy because it can infect a wide range of cell types, including human cells, can infect non-dividing cells that have completed differentiation, has no pathogenicity to humans, thus reducing concerns about side effects, and its viral particles are physicochemically stable.

[0003] Non-enveloped viral vectors such as adenoviruses and AAVs exhibit desirable characteristics for gene delivery, including broad tissue and cell affinity, large expression cassette capacity, and high transduction efficiency. The production of viral vectors requires engineered cell lines capable of complementing the functions removed from the viral genome. For the pharmaceutical development and commercial production of viral vectors, it is crucial to use cells that are scalable and capable of producing viral vectors of sufficient quality (safety) and purity.

[0004] For example, HEK293 cells are a cell line established by transforming human fetal kidney cells with the adenovirus E1 gene, and are used for various purposes such as recombinant protein production, recombinant adenovirus production, and as a host for amplification. However, their productivity has remained a challenge. On the other hand, HEK293T cells, which express the SV40 Large T antigen, have improved productivity and the ability to produce viral vectors, but there are concerns about their clinical use due to the risk of carcinogenicity / immunogenicity (Non-Patent Literature 1). Regarding the production of AAV vectors, efforts have been made to obtain high-titer AAV vector solutions using cells into which miRNA has been artificially introduced (Patent Document 1). However, the production volume of AAV vectors remains insufficient, and there is a need to develop manufacturing methods that can achieve higher productivity. [Prior art documents] [Patent Documents]

[0005] [Patent Document 1] Patent No. 6093358 [Non-patent literature]

[0006] [Non-Patent Document 1] Dahae Hailey Bae et al., (2020) Mol Ther Methods Clin Dev. 2020 Sep 11; 18: 631-638 [Overview of the project] [Problems that the invention aims to solve]

[0007] The object of this invention is to provide viral vector-producing cells with improved viral vector production capacity, a method for producing the same, a method for selecting the same, and a kit containing the same. [Means for solving the problem]

[0008] In view of the above problems, the inventors compared the protein expression profiles of viral vector-producing cells exhibiting high viral vector productivity (hereinafter also referred to as high viral vector-producing cells or simply high-productivity cells) and viral vector-producing cells exhibiting low productivity (hereinafter also referred to as low viral vector-producing cells or simply low-productivity cells), and searched for proteins with discrepancies in expression levels between the samples. As a result, they found several proteins whose expression levels were increased and several proteins whose expression levels were decreased in high viral vector-producing cells. These proteins were designated as candidate proteins to be regulated in viral vector-producing cells, and by promoting or inhibiting their expression, or suppressing or inhibiting it, they succeeded in obtaining viral vector-producing cells with high viral vector production capacity, thus completing the present invention. In other words, the present invention is as follows:

[0009] [1] A viral vector-producing cell having improved vector production capacity, characterized in that the expression of viral vector-producing cell-derived proteins in the cell is regulated. [2] The viral vector-producing cell described in [1] above, wherein the expression of the protein derived from the viral vector-producing cell is regulated compared to the viral vector-producing cell before the regulation of the protein expression. [3] The viral vector-producing cells described in [1] above, wherein the expression of proteins derived from the viral vector-producing cells is regulated compared to HEK293 cells (deposit number ATCC CRL1573). [4] The viral vector-producing cell according to [1] or [2] above, wherein the expression of the protein derived from the viral vector-producing cell is increased compared to the viral vector-producing cell before the expression of the protein was regulated. [5] The viral vector-producing cell described in [4] above, wherein the expression of the protein derived from the viral vector-producing cell is increased by 1.5 times or more at the time of viral vector production compared to the viral vector-producing cell before the expression of the protein was regulated. [6] The viral vector-producing cells described in [1] or [3] above, wherein the expression of proteins derived from the viral vector-producing cells is increased compared to HEK293 cells (deposit number ATCC CRL1573). [7] The viral vector-producing cells described in [6] above, wherein the expression of proteins derived from the viral vector-producing cells is increased by 1.5 times or more compared to HEK293 cells (deposit number ATCC CRL1573) at the time of viral vector production. [8] The viral vector-producing cell according to any one of [4] to [7] above, wherein the increase in the expression of the protein derived from the viral vector-producing cell is due to the addition of an additive to the cell. [9] The viral vector-producing cell according to any one of [4] to [7] above, wherein the increase in the expression of the protein derived from the viral vector-producing cell is due to the introduction of an exogenous plasmid into the viral vector-producing cell.

[10] The viral vector-producing cell according to any one of [4] to [9] above, wherein the protein derived from the viral vector-producing cell is at least one selected from the group consisting of PIK3C2A, UBE2C, ASNS, NIFK, PDCD4, DDX47, GTPBP4, FTSJ3, DDX49, GNL2, NAT10, UBL5, SERBP1, and NOVA2.

[0010]

[11] The viral vector-producing cell according to any one of [4] to

[10] above, wherein the protein derived from the viral vector-producing cell is at least one selected from the group consisting of PIK3C2A, UBE2C, ASNS, NIFK, GTPBP4, FTSJ3, DDX49, GNL2, SERBP1, and NOVA2.

[12] The viral vector-producing cell according to any one of [4] to

[11] above, wherein the protein derived from the viral vector-producing cell is NIFK and NOVA2 or SERBP1 and NOVA2.

[13] The viral vector-producing cell according to [1] or [2] above, wherein the expression of the protein derived from the viral vector-producing cell is reduced compared to the viral vector-producing cell before the expression of the protein was regulated.

[14] The viral vector-producing cell described in

[13] above, wherein the expression of the protein derived from the viral vector-producing cell is reduced by 10% or more at the time of viral vector production compared to the viral vector-producing cell before the expression of the protein was regulated.

[15] The viral vector-producing cells described in [1] or [3] above, wherein the expression of proteins derived from the viral vector-producing cells is reduced compared to HEK293 cells (deposit number ATCC CRL1573).

[16] The viral vector-producing cells described in

[15] above, wherein the expression of proteins derived from the viral vector-producing cells is reduced by more than 10% at the time of viral vector production compared to HEK293 cells (deposit number ATCC CRL1573).

[17] A viral vector-producing cell according to any one of

[13] to

[16] above, wherein the decrease in the expression of the protein derived from the viral vector-producing cell is due to the addition of an additive to the cell.

[18] The viral vector-producing cell according to any one of

[13] to

[16] above, wherein the reduction in the expression of the protein derived from the viral vector-producing cell is due to the introduction of an exogenous plasmid into the viral vector-producing cell.

[19] The viral vector-producing cell according to any one of

[13] to

[18] above, wherein the protein derived from the viral vector-producing cell is at least one selected from the group consisting of ISG15, CD44, LMNA, ACP2, and MRE11.

[20] The viral vector-producing cell according to any one of

[13] to

[19] above, wherein the protein derived from the viral vector-producing cell is at least one selected from the group consisting of ISG15, CD44, and MRE11.

[0011]

[21] A viral vector-producing cell derived from an animal cell, as described in any of [1] to

[20] above.

[22] A virus vector-producing cell according to any one of [1] to

[21] above, which is derived from a human cell.

[23] The virus vector-producing cell according to

[22] above, wherein the human cell is a HEK293 cell.

[24] The virus vector-producing cell according to any one of [1] to

[23] above, wherein the virus vector is derived from a virus belonging to the family Parvoviridae or Retroviridae.

[25] The virus vector-producing cell according to any one of [1] to

[23] above, wherein the virus vector is an adeno-associated virus vector.

[26] A method for producing a virus vector-producing cell with improved vector-producing ability, which comprises regulating the expression of a protein derived from the virus vector-producing cell in the virus vector-producing cell.

[27] The method for producing a virus vector-producing cell according to

[26] above, wherein the regulation is carried out by comparing with the virus vector-producing cell before the expression of the protein is regulated.

[28] The method for producing a virus vector-producing cell according to

[26] above, wherein the regulation is carried out by comparing with HEK293 cells (deposit number ATCC CRL1573).

[29] The method for producing a virus vector-producing cell according to

[26] or

[27] above, wherein the regulation is to increase the expression of the protein derived from the virus vector-producing cell as compared with the virus vector-producing cell before the expression of the protein is regulated.

[30] The method for producing a virus vector-producing cell according to

[29] above, wherein the regulation is to increase the expression of the protein derived from the virus vector-producing cell by 1.5 times or more as compared with the virus vector-producing cell before the expression of the protein is regulated during virus vector production.

[0012]

[31] The method for producing a virus vector-producing cell according to

[26] or

[28] above, wherein the regulation is to increase the expression of the protein derived from the virus vector-producing cell as compared with HEK293 cells (deposit number ATCC CRL1573).

[32] The method for producing viral vector-producing cells according to

[31] , wherein the regulation increases the expression of the protein derived from the viral vector-producing cells by 1.5 times or more compared to HEK293 cells (deposit number ATCC CRL1573) at the time of viral vector production.

[33] A method for producing viral vector-producing cells according to any one of

[29] to

[32] above, wherein the increase in the expression of proteins derived from the viral vector-producing cells is due to the addition of an additive to the cells.

[34] A method for producing viral vector-producing cells according to any one of

[29] to

[32] above, wherein the increase in the expression of the protein derived from the viral vector-producing cells is due to the introduction of an exogenous plasmid into the viral vector-producing cells.

[35] A method for producing a viral vector-producing cell according to any one of the above

[29] to

[34] , wherein the protein derived from the viral vector-producing cell is at least one selected from the group consisting of PIK3C2A, UBE2C, ASNS, NIFK, PDCD4, DDX47, GTPBP4, FTSJ3, DDX49, GNL2, NAT10, UBL5, SERBP1, and NOVA2.

[36] A method for producing a viral vector-producing cell according to any one of the above

[29] to

[35] , wherein the protein derived from the viral vector-producing cell is at least one selected from the group consisting of PIK3C2A, UBE2C, ASNS, NIFK, GTPBP4, FTSJ3, DDX49, GNL2, SERBP1, and NOVA2.

[37] A method for producing a viral vector-producing cell according to any one of

[29] to

[36] above, wherein the protein derived from the viral vector-producing cell is NIFK and NOVA2 or SERBP1 and NOVA2.

[38] The method for producing viral vector-producing cells according to

[26] or

[27] , wherein the regulation reduces the expression of the protein derived from the viral vector-producing cells compared to the viral vector-producing cells before the regulation of the protein expression.

[39] The method for producing viral vector-producing cells according to

[38] , wherein the regulation reduces the expression of the protein derived from the viral vector-producing cells by 10% or more at the time of viral vector production compared to the viral vector-producing cells before the regulation of the protein expression.

[40] A method for producing viral vector-producing cells according to

[26] or

[28] above, wherein the reduction in the expression of proteins derived from the viral vector-producing cells is reduced compared to HEK293 cells (deposit number ATCC CRL1573).

[0013]

[41] The method for producing viral vector-producing cells according to

[40] , wherein the regulation reduces the expression of proteins derived from the viral vector-producing cells by 10% or more compared to HEK293 cells (deposit number ATCC CRL1573) at the time of viral vector production.

[42] The method for producing viral vector-producing cells according to any one of

[26] to

[41] above, wherein the reduction in the expression of proteins derived from the viral vector-producing cells is due to the addition of an additive to the cells.

[43] A method for producing viral vector-producing cells according to any one of

[26] to

[41] above, wherein the reduction in the expression of proteins derived from the viral vector-producing cells is due to the introduction of an exogenous plasmid into the viral vector-producing cells.

[44] A method for producing a viral vector-producing cell according to any one of

[26] to

[43] above, wherein the protein derived from the viral vector-producing cell is at least one selected from the group consisting of ISG15, CD44, LMNA, ACP2, and MRE11.

[45] A method for producing a viral vector-producing cell according to any one of

[26] to

[44] above, wherein the protein derived from the viral vector-producing cell is at least one selected from the group consisting of ISG15, CD44, and MRE11.

[46] A method for producing a viral vector-producing cell according to any of the above

[26] to

[45] , which is derived from animal cells.

[47] A method for producing a virus vector-producing cell derived from human cells, as described in any of

[26] to

[46] above.

[48] ​​The method for producing viral vector-producing cells according to

[47] above, wherein the human cells are HEK293 cells.

[49] A method for producing a viral vector-producing cell according to any one of

[26] to

[48] above, wherein the viral vector is derived from a virus belonging to the family Parvoviridae or Retroviridae.

[50] A method for producing a viral vector-producing cell according to any one of

[26] to

[48] above, wherein the viral vector is an adeno-associated virus vector.

[0014]

[51] A method for enhancing viral vector production in viral vector-producing cells, characterized by regulating the expression of viral vector-derived proteins in viral vector-producing cells.

[52] A method for enhancing the production of viral vectors in viral vector-producing cells according to

[51] , wherein the regulation is performed compared to viral vector-producing cells before the regulation of protein expression.

[53] A method for enhancing the production of viral vectors in the viral vector-producing cells described in

[51] above, wherein the regulation is performed in comparison to HEK293 cells (deposit number ATCC CRL1573).

[54] A method for enhancing the production of viral vectors in viral vector-producing cells according to

[51] or

[52] , wherein the regulation increases the expression of the protein derived from the viral vector-producing cells compared to the viral vector-producing cells before the expression of the protein was regulated.

[55] A method for enhancing the amount of viral vector produced in viral vector-producing cells according to

[54] above, wherein the regulation increases the expression of the protein derived from the viral vector-producing cells by 1.5 times or more at the time of viral vector production compared to viral vector-producing cells before the expression of the protein was regulated.

[56] A method for enhancing viral vector production in viral vector-producing cells according to

[51] or

[53] , wherein the regulation involves increasing the expression of the protein derived from the viral vector-producing cells compared to HEK293 cells (deposit number ATCC CRL1573).

[57] A method for enhancing the amount of viral vector produced in viral vector-producing cells according to

[56] above, wherein the regulation increases the expression of the protein derived from the viral vector-producing cells by 1.5 times or more compared to HEK293 cells (deposit number ATCC CRL1573) at the time of viral vector production.

[58] A method for increasing the amount of viral vector produced in viral vector-producing cells according to any one of

[54] to

[57] above, wherein the increase in the expression of the protein derived from the viral vector-producing cells is due to the addition of an additive to the cells.

[59] A method for increasing the amount of viral vector produced in viral vector-producing cells according to any one of

[54] to

[57] above, wherein the increase in the expression of the protein derived from the viral vector-producing cells is due to the introduction of an exogenous plasmid into the viral vector-producing cells.

[60] A method for enhancing the production of viral vectors in viral vector-producing cells according to any one of the above

[54] to

[59] , wherein the protein derived from the viral vector-producing cells is at least one selected from the group consisting of PIK3C2A, UBE2C, ASNS, NIFK, PDCD4, DDX47, GTPBP4, FTSJ3, DDX49, GNL2, NAT10, UBL5, SERBP1, and NOVA2.

[0015]

[61] A method for enhancing the amount of viral vector produced in viral vector-producing cells according to any one of

[54] to

[60] above, wherein the protein derived from the viral vector-producing cells is at least one selected from the group consisting of PIK3C2A, UBE2C, ASNS, NIFK, GTPBP4, FTSJ3, DDX49, GNL2, SERBP1, and NOVA2.

[62] A method for enhancing the amount of viral vector produced in a viral vector-producing cell according to any one of

[54] to

[61] above, wherein the protein derived from the viral vector-producing cell is NIFK and NOVA2 or SERBP1 and NOVA2.

[63] A method for increasing the production of viral vectors in viral vector-producing cells according to

[51] or

[52] , wherein the regulation reduces the expression of the protein derived from the viral vector-producing cells compared to the viral vector-producing cells before the regulation of the protein expression.

[64] A method for increasing the amount of viral vector produced in viral vector-producing cells according to

[63] above, wherein the regulation reduces the expression of the protein derived from the viral vector-producing cells by 10% or more at the time of viral vector production compared to viral vector-producing cells before the expression of the protein was regulated.

[65] A method for enhancing viral vector production in viral vector-producing cells according to

[51] or

[53] above, wherein the decrease in the expression of proteins derived from the viral vector-producing cells is reduced compared to HEK293 cells (deposit number ATCC CRL1573).

[66] A method for enhancing the amount of viral vector produced in viral vector-producing cells as described in

[65] above, wherein the regulation reduces the expression of the protein derived from the viral vector-producing cells by 10% or more compared to HEK293 cells (deposit number ATCC CRL1573) during viral vector production.

[67] A method for increasing the amount of viral vector produced in viral vector-producing cells according to any one of

[63] to

[66] above, wherein the decrease in the expression of proteins derived from the viral vector-producing cells is due to the addition of an additive to the cells.

[68] A method for increasing the amount of viral vector produced in viral vector-producing cells according to any one of

[63] to

[66] above, wherein the reduction in the expression of the protein derived from the viral vector-producing cells is due to the introduction of an exogenous plasmid into the viral vector-producing cells.

[69] A method for enhancing the production of viral vectors in viral vector-producing cells according to any one of

[63] to

[68] above, wherein the protein derived from the viral vector-producing cells is at least one selected from the group consisting of ISG15, CD44, LMNA, ACP2, and MRE11.

[70] A method for enhancing the amount of viral vector produced in a viral vector-producing cell according to any one of the above

[63] to

[69] , wherein the protein derived from the viral vector-producing cell is at least one selected from the group consisting of ISG15, CD44, and MRE11.

[0016]

[71] A method for enhancing the production of viral vectors in viral vector-producing cells derived from animal cells, as described in any of

[51] to

[70] above.

[72] A method for enhancing the production of viral vectors in viral vector-producing cells derived from human cells, as described in any of

[51] to

[71] above.

[73] A method for enhancing the production of viral vectors in the viral vector-producing cells described in

[72] above, wherein the human cells are HEK293 cells.

[74] A method for enhancing the production of a viral vector in a viral vector-producing cell according to any one of

[51] to

[73] above, wherein the viral vector is derived from a virus belonging to the family Parvoviridae or Retroviridae.

[75] A method for enhancing the production of a viral vector in a viral vector-producing cell according to any of

[51] to

[74] above, wherein the viral vector is an adeno-associated viral vector.

[76] A kit for producing a viral vector, comprising viral vector-producing cells described in any of [1] to

[25] above or viral vector-producing cells produced by the manufacturing method described in any of

[26] to

[50] above.

[77] A method for selecting viral vector-producing cells with improved viral vector production capacity, comprising the step of selecting cells in which the expression of viral vector-derived proteins in viral vector-producing cells is regulated.

[78] A method for selecting viral vector-producing cells according to

[77] , comprising the step of selecting cells in which the expression of a viral vector-derived protein in the viral vector-producing cells is regulated compared to viral vector-producing cells before the expression of the protein was regulated.

[79] A method for selecting viral vector-producing cells according to

[77] above, comprising the step of selecting cells in which the expression of viral vector-derived proteins in viral vector-producing cells is regulated compared to HEK293 cells (deposit number ATCC CRL1573).

[80] A method for selecting viral vector-producing cells according to

[77] or

[78] , wherein the regulation is increased compared to viral vector-producing cells before the regulation of protein expression.

[0017]

[81] A method for selecting viral vector-producing cells according to

[77] or

[79] above, wherein the regulation is increased compared to HEK293 cells (deposit number ATCC CRL1573).

[82] The method for selecting viral vector-producing cells according to

[77] or

[78] , wherein the regulation is performed such that the expression of the protein derived from the viral vector-producing cells is reduced compared to the viral vector-producing cells before the regulation of the protein expression.

[83] The method for selecting viral vector-producing cells according to

[77] or

[79] above, wherein the regulation is performed such that the expression of the protein derived from the viral vector-producing cells is reduced compared to HEK293 cells (deposit number ATCC CRL1573).

[84] A method for selecting a viral vector-producing cell according to

[82] or

[83] above, wherein the protein derived from the viral vector-producing cell is CD44. [Effects of the Invention]

[0018] The viral vector-producing cells of this invention have a high viral vector production capacity. Therefore, by using the viral vector-producing cells of this invention as packaging cells, it becomes possible to produce a larger amount of viral vectors. Increasing the amount that can be produced at once makes it possible to reduce the number of production lots, thereby reducing variations in quality between lots. [Brief explanation of the drawing]

[0019] [Figure 1] Figure 1 is a graph evaluating the gene transfer efficiency of cell fractions of AAV2 vectors produced by cells transiently expressing PIK3C2A, NIFK, or UBE2C during AAV2 vector production (expressed as GFP positivity rate). [Figure 2] Figure 2 is a graph evaluating the gene transfer efficiency of cell fractions of AAV2 vectors produced by cells transiently expressing PIK3C2A, UBE2C, DDX47, SERBP1, or NIFK during AAV2 vector production (expressed as the percentage of GFP positivity compared to the control group). [Figure 3] Figure 3 is a graph evaluating the gene transfer efficiency when AAV2 vectors produced by cells transiently expressing NOVA2, ASNS, FTSJ3, PIK3C2A, GTPBP4, DDX49, GNL2, UBL5, DNMT1, UBE2C, SERBP1, NAT10, or NIFK were extracted with a surfactant during AAV2 vector production (expressed as the percentage of GFP positivity compared to the control group). [Figure 4] Figure 4 is a graph evaluating the gene transfer efficiency of the supernatant fraction of AAV1 vector produced by cells transiently expressing UBE2C, ASNS, PIK3C2A, PDCD4, or NIFK during AAV1 vector production (expressed as the percentage of GFP positivity compared to the control group). [Figure 5]Figure 5 is a graph evaluating the gene transfer efficiency when AAV1 vectors produced by cells transiently expressing NOVA2, DDX49, NAT10, FTSJ3, UBL5, GTPBP4, SERBP1, or GNL2 during AAV1 vector production were extracted with a surfactant (expressed as the percentage of GFP positivity compared to the control group). [Figure 6] Figure 6 is a graph evaluating the gene transfer efficiency when AAV6 vectors produced by cells transiently expressing NIFK or PIK3C2A during AAV6 vector production were extracted with a surfactant (expressed as the percentage of GFP positivity compared to the control group). [Figure 7] Figure 7 is a graph evaluating the gene transfer efficiency of the cell fraction of AAV2 vector produced by cells in which MRE11 or ISG15 was knocked down during AAV2 vector production (expressed as GFP positivity rate). [Figure 8] Figure 8 is a graph evaluating the gene transfer efficiency of the cell fraction of AAV2 vector produced by cells in which MRE11 or ISG15 was knocked down during AAV2 vector production (expressed as the percentage of GFP positivity compared to the control group). [Figure 9] Figure 9 is a graph evaluating the gene transfer efficiency when AAV2 vectors produced by cells in which ISG15, LMNA, MRE11, CD44, or APC2 were knocked down during AAV2 vector production were extracted with a surfactant (expressed as the percentage of GFP positivity compared to the control group). [Figure 10] Figure 10 is a graph evaluating the gene transfer efficiency when AAV1 vectors produced by cells in which CD44, MRE11, or ISG15 were knocked down during AAV1 vector production were extracted with a surfactant (expressed as the percentage of GFP positivity compared to the control group). [Figure 11] Figure 11 is a graph evaluating the gene transfer efficiency when AAV6 vectors produced by cells in which ISG15, CD44, or MRE11 were knocked down during AAV6 vector production were extracted with a surfactant (expressed as the percentage of GFP positivity compared to the control group). [Figure 12]Figure 12 is a graph evaluating the total amount of AAV vector genome when AAV2 vectors produced by cells transiently expressing PIK3C2A or UBE2C during AAV2 vector production were sampled separately from the cell fraction and the supernatant fraction (expressed as AAV vector production per unit area). [Figure 13] Figure 13 is a graph evaluating the total amount of AAV vector genome when AAV2 vectors produced by cells transiently expressing PIK3C2A, UBE2C, or DDX47 during AAV2 vector production were sampled separately from the cell fraction and the supernatant fraction (expressed as the ratio of AAV vector production per unit area compared to the control group). [Figure 14] Figure 14 is a graph evaluating the amount of AAV vector genome when AAV2 vectors produced by cells transiently expressing NOVA2, ASNS, FTSJ3, PIK3C2A, GTPBP4, DDX49, GNL2, UBL5, DNMT1, UBE2C, or SERBP1 during AAV2 vector production were extracted with a surfactant (expressed as the ratio of AAV vector production per unit area compared to the control group). [Figure 15] Figure 15 is a graph evaluating the total AAV vector genome volume when AAV1 vectors produced by cells transiently expressing UBE2C, ASNS, PIK3C2A, or PDCD4 during AAV1 vector production were sampled separately from the cell fraction and the supernatant fraction (expressed as the ratio of AAV vector production per unit area compared to the control group). [Figure 16] Figure 16 is a graph evaluating the amount of AAV vector genome when AAV1 vectors produced by cells transiently expressing NOVA2, DDX49, NAT10, FTSJ3, UBL5, GTPBP4, or SERBP1 during AAV1 vector production were extracted with a surfactant (expressed as the ratio of AAV vector production per unit area compared to the control group). [Figure 17]Figure 17 is a graph evaluating the amount of AAV genome when AAV6 vectors produced by cells transiently expressing NIFK or PIK3C2A during AAV6 vector production were extracted with a surfactant (expressed as the ratio of AAV vector production per unit area compared to the control group). [Figure 18] Figure 18 is a graph evaluating the total amount of AAV vector genome when AAV2 vectors produced by cells in which MRE11 or ISG15 were knocked down during AAV2 vector production were sampled separately from the cell fraction and the supernatant fraction (expressed as AAV vector production per unit area). [Figure 19] Figure 19 is a graph evaluating the total amount of AAV vector genome when AAV2 vectors produced by cells in which MRE11 or ISG15 were knocked down during AAV2 vector production were sampled separately from the cell fraction and the supernatant fraction (expressed as the ratio of AAV vector production per unit area compared to the control group). [Figure 20] Figure 20 is a graph evaluating the amount of AAV vector genome when AAV2 vectors produced by cells in which ISG15, LMNA, MRE11, CD44, or APC2 were knocked down during AAV2 vector production were extracted with a surfactant (expressed as the ratio of AAV vector production per unit area compared to the control group). [Figure 21] Figure 21 is a graph evaluating the amount of AAV vector genome when AAV1 vectors produced by cells in which CD44, MRE11, or ISG15 were knocked down during AAV1 vector production were extracted with a surfactant (expressed as the ratio of AAV vector production per unit area compared to the control group). [Figure 22] Figure 22 is a graph evaluating the amount of AAV vector genome when AAV6 vectors produced by cells in which ISG15, CD44, or MRE11 were knocked down during AAV6 vector production were extracted with a surfactant (expressed as the ratio of AAV vector production per unit area compared to the control group). [Figure 23]Figure 23 is a graph evaluating the gene transfer efficiency when AAV2 vectors produced by cells transiently expressing NOVA2 alone, NIFK alone, or both NOVA2 and NIFK simultaneously were extracted with a surfactant during AAV2 vector production (expressed as the ratio of AAV vector production per unit area compared to the control group). [Figure 24] Figure 24 is a graph evaluating the gene transfer efficiency when AAV2 vectors produced by cells transiently expressing NOVA2 alone, SERBP1 alone, or both NOVA2 and SERBP1 simultaneously were extracted with a surfactant during AAV2 vector production (expressed as the ratio of AAV vector production per unit area compared to the control group). [Figure 25] Figure 25 is a graph comparing the expression levels of CD44 on the cell surface of clone A, which was recovered by single-cell cloning using CD44 as a surface marker, with those of the cell population before selection. [Figure 26] Figure 26 is a graph evaluating the gene transfer efficiency when AAV2 vectors were produced using clone A, recovered by single-cell cloning with CD44 as a surface marker, and the cell population before selection (expressed as the percentage of GFP positivity relative to the control group (cell population before selection)). [Figure 27] Figure 27 is a graph evaluating the amount of AAV vector genome produced when clone A, recovered by single-cell cloning using CD44 as a surface marker, and the cell population before selection were used to produce the AAV2 vector (expressed as the ratio of AAV vector production per unit area compared to the control group (cell population before selection)). [Modes for carrying out the invention]

[0020] The present invention will be described below. Unless otherwise specified, terms used herein have the meanings commonly used in the art.

[0021] 1. Viral vector-producing cells and methods for producing the same Cells capable of producing viral vectors (hereinafter referred to as virus-producing cells, virus-producing cells, producing cells, or production cells in this specification) are mainly known to be of two types: 1) transient producing cells and 2) constitutive producing cells. Taking retrovirus-producing cells as an example, transient producing cells are cells that acquire the ability to produce retroviruses by transiently introducing a nucleic acid construct containing nucleic acids encoding proteins necessary for viral vector production (i.e., viral particle formation) and a nucleic acid construct containing nucleic acids supplying RNA to be encapsulated in viral particles into any cell. Constitutive producing cells are cells that have already had nucleic acids encoding proteins essential for viral particle formation introduced into their chromosomes and that constitutively possess the ability to produce the said proteins, and then acquire the ability to produce retroviruses by introducing a retrovirus vector plasmid or retrovirus vector into the cell. The viral vector-producing cells of the present invention are preferably cells whose ability to produce viral vectors is enhanced by the regulation of the expression of proteins derived from the viral vector-producing cells compared to the expression of the proteins in viral vector-producing cells before the regulation of protein expression (hereinafter also referred to as pre-regulation cells), and more preferably cells whose ability to produce viral vectors is enhanced by the regulation of the expression of proteins derived from the viral vector-producing cells compared to the expression of HEK293 cells (deposit number ATCC CRL-1573). They may be transient-producing cells or homeostatic-producing cells, but transient-producing cells are preferred. In the present invention, "improved vector production capacity" means that the ability to produce vectors (in this case, viral vectors) is improved compared to cells before regulation. The degree of improvement in production capacity is not particularly limited as long as the difference is significant. "Vector production capacity" may be qualitative or quantitative. For example, a qualitative improvement in vector production capacity means that the ability to produce highly infectious viral vectors (viral particles) is improved compared to cells before regulation, and a quantitative improvement means that the amount of viral vectors (viral particles) produced is increased compared to cells before regulation. Qualitative improvement can be evaluated, for example, by measuring the infectivity (gene transfer efficiency) of the produced viral vectors (viral particles) to cells. The infectivity of the produced viral vectors (viral particles) to cells can also be expressed as infectivity titer. Preferably, the gene transfer efficiency of viral vectors produced in the viral vector-producing cells of the present invention is increased compared to the gene transfer efficiency of viral vectors produced in cells before regulation, and more preferably, it is increased compared to the gene transfer efficiency of viral vectors produced in HEK293 cells (deposit number ATCC CRL1573). The gene transfer efficiency of the viral vector produced in the viral vector-producing cells of the present invention is preferably 1.01 times or more, more preferably 1.02 times or more, even more preferably 1.05 times or more, particularly preferably 1.1 times or more, and most preferably 1.15 times or more, compared to the gene transfer efficiency of the viral vector produced in the cells before regulation. Furthermore, the gene transfer efficiency of the viral vector produced in the viral vector-producing cells of the present invention is preferably 1.01 times or more, more preferably 1.02 times or more, even more preferably 1.05 times or more, particularly preferably 1.1 times or more, and most preferably 1.15 times or more, compared to the gene transfer efficiency of the viral vector produced in HEK293 cells (deposit number ATCC CRL1573). The quantitative improvement can be evaluated by measuring the number of genomes (genome titer) obtained from the viral vector (viral particles) produced from the viral vector-producing cells.The genomic titer of the viral vector produced by the viral vector-producing cells of the present invention is preferably increased compared to the genomic titer of the viral vector produced by the cells before regulation, and more preferably increased compared to the genomic titer of the viral vector produced by HEK293 cells (deposit number ATCC CRL1573). The genomic titer of the viral vector produced by the viral vector-producing cells of the present invention is preferably increased by 1.01 times or more, more preferably by 1.02 times or more, even more preferably by 1.05 times or more, particularly preferably by 1.1 times or more, and most preferably by 1.15 times or more, compared to the genomic titer of the viral vector produced by the cells before regulation. Furthermore, the genomic titer of the viral vector produced by the viral vector-producing cells of the present invention is preferably increased by 1.01 times or more, more preferably by 1.02 times or more, even more preferably by 1.05 times or more, particularly preferably by 1.1 times or more, and most preferably by 1.15 times or more, compared to the genomic titer of the viral vector produced by HEK293 cells (deposit number ATCC CRL1573). As an example of a method for measuring genome titer, one method involves testing the copy number of the viral genome in a viral vector-containing sample, after nucleic acid impurities have been degraded by nuclease treatment followed by protease treatment, using the PCR method. As an example of a method for measuring the gene transfer efficiency of a viral vector, one method involves infecting appropriate target cells with a series of dilutions of the viral vector-containing sample, detecting the expression of the introduced gene, changes in cell shape (cytopathy), and measuring the copy number of the provirus introduced into the cells. More specifically, the methods implemented in the examples described later are mentioned.

[0022] In the present invention, examples of viruses from which viral vectors are derived include, but are not limited to, retroviruses, lentiviruses, Sendai viruses, and enveloped viruses such as herpesviruses, as well as non-enveloped viruses such as adenoviruses and AAVs (hereinafter referred to as non-enveloped viruses). The envelope is formed when a virus buddings by penetrating membranes such as the nucleus, endoplasmic reticulum, Golgi apparatus, plasma membrane, and cell membrane, and usually contains host-derived proteins or viral proteins expressed on the host cell membrane, playing an important role in infecting target cells. Specifically, examples include DNA viruses such as adenovirus, parvovirus, papovavirus, and human papillomavirus, and RNA viruses such as rotavirus, coxsackievirus, enterovirus, sapovirus, norovirus, poliovirus, echovirus, hepatitis A virus, hepatitis E virus, rhinovirus, and astrovirus. More preferably, viruses of the retroviridae family, adenoviridae family, and parvoviridae family, with adeno-associated viruses (AAV) of the parvoviridae family being particularly preferred. AAV has an icosahedral outer shell (capsid) without an envelope and a single linear single-stranded DNA inside. The capsid has three capsid proteins (VP1, VP2, and VP3). In this specification, AAV includes wild-type viruses and their derivatives, and includes all serotypes and clades unless otherwise specified. While there are various reports on AAV serotypes, at least 15 serotypes of AAV that infect humans are known: AAV1, AAV2, AAV3a, AAV3b, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAVrh.10, AAV11, AAV12, and AAV13.

[0023] "Virus particles" refer to particles composed of a capsid protein shell. Furthermore, in this invention, "virus particles" include not only those containing a viral genome (nucleic acid shape), but also hollow particles, which are virus-like particles composed only of capsid protein and do not contain a viral genome.

[0024] In the present invention, the term "viral vector" includes both the meaning of the viral particle and the meaning of the viral genome (nucleic acid shape) contained within the viral particle. For example, in the case of AAV, a recombinant AAV (rAAV) vector means either an rAAV particle or viral genome DNA present within the rAAV particle.

[0025] In the present invention, there are no particular restrictions on the cells from which the viral vector-producing cells are derived, as long as the desired virus can proliferate. Preferably, these are packaging cells into which a portion of the genes necessary for viral vector production have been introduced, and which alone do not produce viral vectors. They are eukaryotic cells, and preferably include HEK293 cells, HEK293T cells, HEK293F cells, HEK293FT cells, G3T-hi cells, Sf9 cells, commercially available virus-producing cell lines, AAV293 cells, etc., which have high transfection efficiency. HEK293 cells are preferred. Furthermore, for example, the HEK293 cells etc. constitutively express adenovirus E1 protein, but cells may also be modified to transiently or constitutively express one or more of the proteins necessary for rAAV.

[0026] As an example, elements necessary for rAAV production include (A) AAV-derived Rep protein and Cap protein, and (B) adenovirus-derived elements, such as E1a, E1b, E2, E4, and VARNA genes. There are no limitations on the form of these nucleic acids, and they can be introduced into cells by being loaded onto plasmids or viral vectors as one or more nucleic acid constructs capable of supplying each element to the cells in use.

[0027] The viral vector-producing cells of the present invention preferably have regulated protein expression compared to that in cells before regulation, and more preferably regulated compared to that in HEK293 cells (deposit number ATCC CRL1573). The inventors have found that such regulation of protein expression improves the vector-producing capacity of the cells. In the present invention, "protein expression is regulated" means "protein expression is increased" or "protein expression is decreased," preferably "at the time of viral vector production, it is increased by 1.5 times or more compared to that in cells before regulation" or "at the time of viral vector production, it is decreased by 10% or more compared to that in cells before regulation," and more preferably "at the time of viral vector production, it is increased by 1.5 times or more compared to that in HEK293 cells (deposit number ATCC CRL1573)" or "at the time of viral vector production, it is decreased by 10% or more compared to that in HEK293 cells (deposit number ATCC CRL1573)." Furthermore, "protein expression is regulated" may also mean "protein activity is regulated." Furthermore, there may be two or more proteins whose expression is regulated.

[0028] The protein whose expression should be regulated in order to improve vector production capacity is not particularly limited as long as the desired effect is obtained. Regulation of expression means an increase of 1.5 times or more, preferably 2.0 times or more, 3.0 times or more, 4.0 times or more, more preferably 5.0 times or more, 6.0 times or more, 7.0 times or more, even more preferably 8.0 times or more, 9.0 times or more, most preferably 10.0 times or more, compared to the expression in cells before regulation, or a decrease of 10% or more, preferably 20% or more, more preferably 30% or more. Alternatively, regulation of expression may mean an increase of 1.5 times or more, preferably 2.0 times or more, 3.0 times or more, 4.0 times or more, more preferably 5.0 times or more, 6.0 times or more, 7.0 times or more, even more preferably 8.0 times or more, 9.0 times or more, most preferably 10.0 times or more, compared to the expression in HEK293 cells (deposit number ATCC CRL1573), or a decrease of 10% or more, preferably 20% or more, more preferably 30% or more.

[0029] The following proteins are examples of proteins whose expression is increased during viral vector production compared to pre-regulation cells or HEK293 cells (deposit number ATCC CRL1573) (see also Table 1 below). phosphatidylinositol 4-phosphate 3-kinase C2 domain-containing subunit alpha Protein FAM162A Ubiquitin-conjugating enzyme E2 C Guanine nucleotide-binding protein-like 3 Asparagine synthetase [glutamine-hydrolyzing] MKI67 FHA domain-interacting nucleolar phosphoprotein Programmed cell death protein 4 Probable ATP-dependent RNA helicase DDX47 Nucleolar GTP-binding protein 1 pre-rRNA processing protein FTSJ3 Probable ATP-dependent RNA helicase ddx49 Nucleolar GTP-binding protein 2 RNA cytidine acetyltransferase Translation machinery-associated protein 7 ubiquitin-like protein 5 DNA (cytosine-5)-methyltransferase 1 Plasminogen activator inhibitor 1 RNA-binding protein RNA-binding protein Nova-2 DNA-directed RNA polymerase II subunit RPB1

[0030] The following proteins are examples of proteins whose expression is reduced during viral vector production compared to pre-regulation cells or HEK293 cells (deposit number ATCC CRL1573) (see also Table 2 below). Ubiquitin-like protein ISG15 CD44 antigen Prelamin-A / C Lysosomal acid phosphatase hydroxymethylglutaryl-CoA synthase, cytoplasmic Double-strand break repair protein MRE11

[0031] During viral vector production, proteins whose expression is increased compared to pre-regulation cells or HEK293 cells (deposit number ATCC CRL1573) are preferably phosphatidylinositol 4-phosphate 3-kinase C2 domain-containing subunit alpha (PIK3C2A), MKI67 FHA domain-interacting nucleolar phosphoprotein (NIFK), Ubiquitin-conjugating enzyme E2 C (UBE2C), Asparagine synthetase [glutamine-hydrolyzing] (ASNS), Nucleolar GTP-binding protein 1 (GTPBP4), pre-rRNA processing protein FTSJ3 (FTSJ3), Probable ATP-dependent RNA helicase ddx49 (DDX49), Nucleolar GTP-binding protein 2 (GNL2), Plasminogen activator inhibitor 1 RNA-binding protein (SERBP1), and RNA-binding protein Nova-2 (NOVA2) is one example. More preferably, NIFK, FTSJ3, DDX49, SERBP1, and NOVA2 are mentioned. Even more preferably, FTSJ3 and NOVA2 are mentioned. PIK3C2A is a protein belonging to the PI3 kinase family, and is involved in signal transduction in cell proliferation, intracellular protein transport, and other processes. NIFK is a protein whose function is largely unknown, but it binds to RNA and contributes to mitosis and cell cycle acceleration. UBE2C is known as an enzyme that receives ubiquitin from E1 and catalyzes its covalent bonding to other proteins. ASNS is an asparagine synthase that synthesizes L-asparagine and L-glutamate from L-aspartate and L-glutamine in an ATP-dependent manner. GTPBP4 is a type of GTPase that is known to be highly expressed in various cancer cells and proliferating cells. FTSJ3 is a type of methyltransferase that has recently been reported to be involved in 2'-O-methylation of RNA, which is a molecular marker used by the cell's innate immune system to distinguish between endogenous and exogenous mRNA. DDX49 is an RNA helicase that has been suggested to be involved in viral infections, but its detailed function remains unclear. GNL2 is a GTPase involved in the transport and maturation of the 60S ribosomal subunit, and has also been reported to have cell proliferation-promoting effects. SERBP1 has been suggested to be involved in regulating mRNA stability. NOVA2 binds to single-stranded RNA and is suggested to be involved in RNA splicing and metabolism in nerve cells and other cells.

[0032] Proteins whose expression is reduced during viral vector production compared to those expressed in pre-regulation cells or HEK293 cells (deposit number ATCC CRL1573) are preferably Ubiquitin-like protein ISG15 (ISG15), Double-strand break repair protein MRE11 (MRE11), and CD44 antigen (CD44). ISG15 is a ubiquitin-like molecule that is thought to play an important role in the innate immune response to viral infections. MRE11 is a component of the MRN complex, which plays a major role in double-strand break repair, recombination, and telomere maintenance. CD44 is a protein known as a hyaluronic acid receptor, and is involved in cell aggregation and the maintenance of the surrounding matrix.

[0033] During viral vector production, the ability to produce viral vectors is enhanced by the regulation of the expression of two or more proteins compared to the expression in unregulated cells or HEK293 cells (deposit number ATCC CRL1573). Examples of combinations of two or more proteins include RNA-binding protein Nova-2 (NOVA2) and MKI67 FHA domain-interacting nucleolar phosphoprotein (NIFK), and RNA-binding protein Nova-2 (NOVA2) and Plasminogen activator inhibitor 1 RNA-binding protein (SERBP1).

[0034] The method for regulating protein expression is not particularly limited as long as it can achieve the desired effect, namely increasing or decreasing the expression of the protein derived from viral vector-producing cells compared to the expression in cells before regulation or in HEK293 cells (deposit number ATCC CRL1573), preferably by 1.5 times or more, or decreasing it by 10% or more. Examples include inducing protein expression in viral vector-producing cells by adding an additive (Aspect 1), suppressing (inhibiting) protein expression in viral vector-producing cells by adding an additive (Aspect 2), increasing the expression level by introducing an exogenous plasmid that induces protein expression into viral vector-producing cells (Aspect 3), and decreasing the expression level by introducing an exogenous plasmid that suppresses (inhibits) protein expression into viral vector-producing cells (Aspect 4). Methods for measuring protein expression levels include, but are not limited to, Western blotting and ELISA.

[0035] Examples of additives used to induce protein expression in viral vector-producing cells in Embodiment 1 include proteins whose expression is desired to be enhanced within the viral vector-producing cells, or genes encoding such proteins (e.g., mRNA). Examples of such genes include at least one of the proteins listed in Table 1. Gene introduction can be carried out by methods commonly used in this field for nucleic acid introduction (e.g., calcium phosphate method, lipofection method, DEAE dextran method, polyethyleneimine method, electroporation method, introduction by retroviral vector). Embodiment 3 details the case in which an exogenous plasmid, which is a gene encoding a protein whose expression is desired to be enhanced within the viral vector-producing cells, is used as the additive. Furthermore, the additive may be an activator capable of inducing the expression of the protein (e.g., a low-molecular-weight compound that acts allosterically to enhance enzyme activity).

[0036] The additive used in Embodiment 2 to suppress (inhibit) protein expression in viral vector-producing cells includes those that can suppress the intracellular expression of proteins whose expression reduction in viral vector-producing cells is desired. Specifically, this includes nucleic acids (e.g., antisense nucleic acids, siRNA, shRNA) that can suppress (inhibit) the expression of such proteins. For example, nucleic acids that suppress (inhibit) the expression of at least one of the proteins listed in Table 2 are included. The introduction of nucleic acids can be carried out by methods commonly used in this field for nucleic acid introduction (e.g., calcium phosphate method, lipofection method, DEAE dextran method, polyethyleneimine method, electroporation method, introduction by retroviral vector). Embodiment 4 will detail the case in which an exogenous plasmid, which is a nucleic acid capable of suppressing (inhibiting) the expression of proteins whose expression reduction in viral vector-producing cells is desired, is used as the additive. Furthermore, the additive may be an inhibitor (e.g., a small molecule compound, a protein) that reduces the expression of the protein.

[0037] The exogenous plasmid used in Embodiment 3 to induce protein expression (hereinafter also referred to as the exogenous induction plasmid) is a plasmid capable of expressing, for example, at least one of the proteins listed in Table 1 to viral vector-producing cells, preferably at least one selected from the group consisting of PIK3C2A, UBE2C, ASNS, NIFK, PDCD4, DDX47, GTPBP4, FTSJ3, DDX49, GNL2, NAT10, UBL5, SERBP1, and NOVA2, more preferably at least one selected from the group consisting of PIK3C2A, NIFK, UBE2C, ASNS, GTPBP4, FTSJ3, DDX49, GNL2, SERBP1, and NOVA2. It may also be a plasmid capable of expressing a combination of NIFK and NOVA2 or SERBP1 and NOVA2. Each plasmid can be prepared based on the genetic information of each protein listed in Table 1.

[0038] The exogenous plasmid used in Embodiment 4 to suppress (inhibit) the expression of a protein (hereinafter referred to as the exogenous suppressor plasmid) is a plasmid capable of suppressing (inhibiting) the expression of at least one of the exogenous proteins listed in Table 2, preferably at least one selected from the group consisting of ISG15, CD44, LMNA, ACP2, and MRE11, and more preferably at least one selected from the group consisting of ISG15, MRE11, and CD44, in viral vector-producing cells. Suppression (inhibition) of expression may be carried out by knocking out or knocking down a gene. Gene knockdown refers to reducing the transcription amount of a specific gene or inhibiting translation from a specific gene. Unlike gene knockout, which destroys the gene itself, knockdown significantly weakens the function of a gene but does not completely eliminate it. Methods for knocking out a gene include methods known in the art, such as gene targeting, which involves disrupting the gene using a targeting vector that induces homologous recombination at an arbitrary position in the target gene, or methods for disrupting the gene and rendering it non-functional by inserting a trap vector (a reporter gene without a promoter) at an arbitrary position in the target gene. Methods for knocking down a gene include methods known in the art, such as antisense methods, which involve introducing RNA equivalent to the antisense strand of mRNA into cells, and RNAi methods, which use siRNA, shRNA, microRNA, etc. For example, a method for knocking down the expression of ISG15 is the introduction of siRNA prepared based on the genetic information of Uniprot ID: P05161. A method for knocking down the expression of MRE11 is the introduction of siRNA prepared based on the genetic information of Uniprot ID: P05161. A method for knocking down the expression of CD44 is the introduction of siRNA prepared based on the genetic information of Uniprot ID: P16070. Knockdown efficiency can be confirmed by measuring changes in mRNA levels and protein expression levels.Methods for measuring mRNA levels include, but are not limited to, real-time quantitative PCR (qPCR) and Northern blotting. Methods for measuring protein expression levels include, but are not limited to, Western blotting and ELISA. In the present invention, the knockdown efficiency is not particularly limited as long as viral vector productivity can be improved in the obtained viral vector-producing cells, but for example, it may be an efficiency of about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99%.

[0039] Exogenous induction plasmids and exogenous suppression plasmids are collectively referred to as exogenous regulatory plasmids.

[0040] Methods for introducing the plasmid include transient or permanent introduction methods. The plasmid can be incorporated into the same plasmid as the plasmid used for viral vector production, or into a separate plasmid; however, separate plasmids are preferred. There are no particular limitations on the transient introduction method; known transient introduction methods such as the calcium phosphate method, lipofection method, DEAE dextran method, polyethyleneimine method, and electroporation method can be used. Commercially available reagents may also be used. There are no particular limitations on the method for constitutively introducing plasmids into cells. Known constitutive introduction methods, such as those using retroviral vectors, or methods similar to transient plasmid introduction methods, followed by selecting cells in which plasmids have been incorporated into the chromosome, can be used. In the method using retroviral vectors, commercially available reagents may be used.

[0041] The viral vector-producing cells of the present invention can be produced in the same manner as commonly practiced in the art, except that it includes a step of regulating the expression of proteins derived from the viral vector-producing cells compared to the expression in pre-regulation cells or HEK293 cells (deposit number ATCC CRL1573). The viral vector-producing cells of the present invention can be manufactured by introducing nucleic acids to be encapsulated in viral particles into cells capable of producing viral vectors, and by regulating the expression of proteins derived from the viral vector-producing cells compared to the expression in the cells before regulation or in HEK293 cells (deposit number ATCC CRL1573). The nucleic acids encapsulated in the viral particles, for example in the case of AAV, consist of an ITR sequence derived from AAV and nucleic acids that are desired to be loaded into the AAV vector. Examples of nucleic acids desired to be loaded into the AAV vector include nucleic acids encoding any foreign gene, such as polypeptides (enzymes, growth factors, cytokines, receptors, structural proteins, etc.), antisense RNA, ribozymes, decoys, and RNA that causes RNA interference. Appropriate promoters, enhancers, terminators, and other transcriptional regulatory elements may be inserted into the nucleic acids to control the expression of the foreign gene. The nucleic acids encapsulated in the viral particles can be introduced into cells in the form of plasmids. The step of regulating the expression of a protein derived from a viral vector-producing cell compared to its expression in the cell before regulation or in HEK293 cells (deposit number ATCC CRL1573) is a step aimed at enhancing or reducing the expression of the protein, and examples of methods for regulating the expression of the above protein include the methods described in Embodiments 1 to 4. The viral vector-producing cells thus obtained are cells with enhanced ability to produce viral vectors. Therefore, the present invention provides a method for producing viral vector-producing cells with improved viral vector production ability, and this method is also a method for improving the viral vector production ability of viral vector-producing cells. In other words, the present invention provides a method for enhancing the production of viral vectors in viral vector-producing cells, which includes regulating the expression of proteins derived from viral vector-producing cells compared to their expression in unregulated cells or in HEK293 cells (deposit number ATCC CRL1573). In cells into which a portion of the genes necessary for the production of a viral vector, which is capable of replicating a desired virus, can be introduced, the viral production capacity of the viral vector-producing cells can be improved by regulating (enhancing or reducing) the expression of proteins derived from the viral vector-producing cells compared to the expression in the cells before regulation or in HEK293 cells (deposit number ATCC CRL1573) (wherein the definitions of each term are as described above). Furthermore, the present invention provides a method for selecting viral vector-producing cells with improved viral vector production capacity, which includes a step of selecting cells in which the expression of proteins derived from viral vector-producing cells is regulated compared to the expression in cells before regulation or in HEK293 cells (deposit number ATCC CRL1573). In cells into which a portion of the genes necessary for the production of a viral vector, preferably a portion of the genes required for the proliferation of a desired virus, cells in which the expression of proteins derived from viral vector-producing cells is regulated (enhanced or reduced) compared to the expression in pre-regulation cells or HEK293 cells (deposit number ATCC CRL1573) are viral vector-producing cells with improved viral vector production capacity. The present invention makes it possible to select such viral vector-producing cells (wherein the definitions of each term are as described above). For example, by selecting cells from a certain cell population in which the expression of CD44 antigen is reduced compared to the expression in pre-regulation cells or HEK293 cells (deposit number ATCC CRL1573), viral vector-producing cells with improved viral vector production capacity can be obtained.

[0042] 2. Method for producing viral vectors The present invention relates to a method for producing a viral vector, and more particularly to a method for producing it efficiently. The method for producing a viral vector of the present invention can be carried out in the same manner as commonly practiced in the art, except that the viral vector-producing cells of the present invention, i.e., viral vector-derived cells, are preferably regulated compared to the expression in cells before regulation, more preferably regulated compared to the expression in HEK293 cells (deposit number ATCC CRL1573), and viral vector-producing cells with improved vector production capacity are used as packaging cells. The production of the viral vector of the present invention is carried out by culturing viral vector-producing cells obtained by a method that includes a step of transiently or constitutively introducing nucleic acids that supply elements essential for viral particle formation into the viral vector-producing cells of the present invention. The method for transiently or constitutively introducing nucleic acids is not particularly limited, and for example, the known transient or constitutive introduction method described above as a plasmid introduction method may be used.

[0043] Furthermore, the cells can be cultured under known culture conditions. For example, culture at a temperature of 30-37°C, humidity of 95% RH, and CO2 concentration of 5-10% (v / v) is an example, but it is not limited to these. If proliferation of viral vector-producing cells and production of viral particles can be achieved, the culture may be carried out at temperatures, humidity, and CO2 concentrations outside the above range. The culture period is not particularly limited, for example, 12-150 hours, preferably 48-120 hours. The culture medium used for culturing viral vector-producing cells can be any medium that contains the components necessary for cell culture, such as basic synthetic media such as DMEM, IMDM, and DMEM:F-12, or, if necessary, these basic synthetic media to which fetal bovine serum, growth factors, peptides, or amino acids have been added. The recovery of viral vectors from viral vector-producing cells can be carried out using methods commonly employed in this field. These include mechanical stirring, sonication, freeze-thaw cycles, solution extraction, chemical methods that appropriately adjust the pH and salt concentration of the extraction solution, and combinations thereof.

[0044] The viral vectors produced using the viral vector-producing cells of the present invention are not particularly limited, and examples include recombinant viral vectors derived from existing serotypes or newly obtained wild-type viruses. In the case of AAV, a recombinant AAV vector based on the desired serotype of AAV can be created by selecting appropriate materials, such as nucleic acids encoding the Rep protein or the Cap protein, when producing the cells of the present invention.

[0045] 3. Kit (a kit for manufacturing viral vectors) The present invention provides a kit for producing a viral vector, comprising viral vector-producing cells of the present invention or viral vector-producing cells obtained by a method for producing viral vector-producing cells of the present invention. The kit may include nucleic acids that supply the proteins necessary for viral particle formation of the viral vector (e.g., in the case of AAV; nucleic acids encoding the Rep protein, nucleic acids encoding the Cap protein, and nucleic acids encoding adenovirus-derived proteins). Furthermore, a kit for carrying out the production of viral vector-producing cells and the production of viral vectors in a series of steps according to the present invention includes (i) viral vector-producing cells before regulation, (ii) exogenous regulatory plasmid (exogenous induction plasmid or exogenous suppression plasmid), and (iii) nucleic acid that supplies proteins necessary for viral particle formation of the viral vector. Exogenous regulatory plasmid is introduced into viral vector-producing cells before regulation to obtain viral vector-producing cells with enhanced viral vector production capacity, and a viral vector is produced using these cells.

[0046] The present invention will be described in detail below with reference to examples, but the present invention is not limited in any way. Unless otherwise specified, the reagents and materials used are commercially available or can be prepared according to known literature, etc. Furthermore, those skilled in the art will understand that any substitute having similar effects and functions is acceptable. [Examples]

[0047] Example 1 (1) Preparation of viral vector-producing cell pellets and non-viral vector-producing cell pellets HEK293 cells (deposit number ATCC CRL1573) suspended in DMEM (Sigma-A) containing 10% FBS (Gibco) and 1% MEM non-essential amino acid solution (100x) (Nacalai Tesque), and HEK293 T cells (Takara Bio, product number 632273) suspended in DMEM containing 10% FBS were seeded in T225 flasks (Corning). Subsequently, the cells were cultured in a 37°C CO2 incubator until approximately 70-80% confluence was confirmed. Plasmids (i) to (iii) were transfected into HEK293 cells and HEK293T cells from the AAV vector-producing group. (i) Plasmids encoding the Rep and Cap proteins of AAV2 (Takara Bio Inc., pRC2-mi342 Vector) (ii) Plasmid containing the E2A, VA, and E4 sequences of adenovirus (Takara Bio, pHelper Vector) (iii) Plasmid containing an expression cassette for the fluorescent protein GFP between the two ITRs of AAV2 (CELL BIOLABS, pAAV-GFP) For the control group of HEK293 cells and HEK293T cells, only the same amount of transfection reagent and DMEM as in the AAV vector production group was added. Cells were cultured in a 37°C CO2 incubator. 24 hours after transfection, the culture medium in the HEK293 cell culture flasks was changed to DMEM containing 2% FBS and 1% MEM non-essential amino acid solution (100×), and the culture medium in the HEK293T cell culture flasks was changed to DMEM containing 2% FBS. Cells were cultured in a 37°C CO2 incubator, and 48 hours after transfection, 1 / 80 volume of 0.5 M EDTA (pH 8.0) (Sigma-A) was added. After standing at room temperature for 10 minutes, the detached cells were collected in a centrifuge tube. After centrifugation at 400 × g, 4°C for 5 minutes, the supernatant was removed, and the cell pellet was washed with DPBS (WAKO). 2 × 10 6 The cell suspension was transferred to a tube, centrifuged at 400×g at 4°C for 5 minutes, the supernatant was removed, and the cell pellet was stored at -80°C until use.

[0048] (2) Identification of host cell proteins by LC-MS / MS measurement Example 1-(1) 2×10 6 100 μL of Lysis buffer (8M Urea (Fujifilm Wako Pure Chemical Industries), 50 mM TEAB (Thermo), pH 8.0) was added to a cell pellet consisting of cells. Endonuclease (Kaneka Corporation) was added to reduce viscosity, and the mixture was centrifuged at 16,000 × g at 4°C for 10 minutes. The supernatant was collected. The total protein content in the collected solution was then measured using the Pierce BCA Protein Assay Kit (Thermo Corporation) according to the manufacturer's protocol. Based on the measurement results, 100 μL of a solution with a total protein concentration of 1 μg / μL was prepared, and 5 μL of 200 mM TCEP (Thermo Corporation) was added, and the mixture was reacted at 55°C for 1 hour. 5 μL of 375 mM iodoacetaminde (Thermo) was added and the mixture was reacted at room temperature for 30 minutes in the dark. 600 μL of acetone (Kanto Chemical) was added and the mixture was allowed to stand overnight at -30°C to precipitate the protein. After centrifugation at 8,000 × g at 4°C for 10 minutes, the supernatant was removed and the pellet was dried. 1 mL of 50 mM TEAB was added and the mixture was dissolved. 2.5 μg of trypsin (Promega) was added and the mixture was digested overnight at 37°C. After adjusting the pH of the solution to approximately 3 to complete the digestion reaction, samples equivalent to approximately 50 μg of total protein were separated and prepared in 100 μL of 100 mM TEAB. Samples for LC-MS / MS analysis were prepared using a TMT-10plex (Thermo) according to the manufacturer's protocol. LC-MS / MS was performed using a system connecting an Easy-nLC1200 (Thermo) and an Orbitrap Eclipse (Thermo), and analysis was performed using the Orbitrap Eclipse. Proteome Discoverer 2.4 (Thermo) was used to identify and quantitatively analyze the TMT-labeled samples. Four different cell lysate samples (AAV vector-producing HEK293 cells, control HEK293 cells, AAV vector-producing HEK293T cells, and control HEK293T cells) were subjected to LC-MS / MS analysis. The procedures from Example 1-(1) to Example 1-(2) were performed independently twice, and three MS analyses were performed on the samples from each batch. From the identified host cell-derived proteins, proteins that satisfied p<0.05 and whose abundance in HEK293 cells or HEK293T cells fluctuated by 1.5 times or more during viral vector production compared to non-production conditions, and whose abundance differed by 1.5 times or more between HEK293 cells and HEK293T cells during viral vector production, were extracted. Proteins that met the criteria are listed in Tables 1 and 2. These proteins were ranked as follows based on their relative abundance in HEK293 cells and HEK293T cells during AAV vector production. AAV vector-producing HEK293T cells / AAV vector-producing HEK293 cells or AAV vector-producing HEK293 cells / AAV vector-producing HEK293T cells were classified as A if the ratio was 10.0 times or more, B if it was 3.0 times or more but less than 10.0 times, and C if it was 1.5 times or more but less than 3.0 times. In Table 1, the protein abundance in HEK293T cells during AAV vector production is increased compared to HEK293 cells during AAV vector production, and in Table 2, the protein abundance in HEK293T cells during AAV vector production is decreased compared to HEK293 cells during AAV vector production.

[0049] [Table 1]

[0050] [Table 2]

[0051] Example 2 (1) Transient expression of PIK3C2A, UBE2C, ASNS, NIFK, PDCD4, DDX47, GTPBP4, FTSJ3, DDX49, GNL2, NAT10, UBL5, SERBP1 and NOVA2 in production cells Table 1 lists the following names: phosphatidylinositol 4-phosphate 3-kinase C2 domain-containing subunit alpha as PIK3C2A, ubiquitin-conjugating enzyme E2 C as UBE2C, asparagine synthetase [glutamine-hydrolyzing] as ASNS, MKI67 FHA domain-interacting nucleolar phosphoprotein as NIFK, programmed cell death protein 4 as PDCD4, probable ATP-dependent RNA helicase DDX47 as DDX47, nucleolar GTP-binding protein 1 as GTPBP4, pre-rRNA processing protein FTSJ3 as FTSJ3, probable ATP-dependent RNA helicase ddx49 as DDX49, nucleolar GTP-binding protein 2 as GNL2, RNA cytidine acetyltransferase as NAT10, ubiquitin-like protein 5 as UBL5, and plasmagen activator The inhibitor 1 RNA-binding protein is denoted as SERBP1, and the RNA-binding protein Nova-2 is denoted as NOVA2. HEK293 cells (Agilent, product number 240073), suspended in DMEM containing 10% FBS, were seeded into a 6-well cell culture plate (Corning). The cells were then cultured in a 37°C CO2 incubator until approximately 70-80% confluence was confirmed. The cell culture supernatant was removed and replaced with serum-free DMEM, and plasmids (i) to (iv) were transfected. Control cells were similarly transfected with plasmids (ii) to (iv) and an empty vector. (i) Plasmid encoding the relevant factor (Vector Builder, pRP-Neo-CAG-hPIK3C2A Vector or pRP-Neo-CAG-hUBE2C Vector or pRP-Neo-CAG-hASNS Vector or pRP-Neo-CAG-hNIFK Vector or pRP-Neo-CAG-hPDCD4 Vector or pRP-Neo-CAG-hDDX47 Vector or pRP-Neo-CAG-hGTPBP4 Vector or pRP-Neo-CAG-hFTSJ3 Vector or pRP-Neo-CAG-hDDX49 Vector or pRP-Neo-CAG-hGNL2 Vector or pRP-Neo-CAG-hNAT10 Vector or pRP-Neo-CAG-hUBL5 Vector or pRP-Neo-CAG-hSERBP1 Vector or pRP-Neo-CAG-hNOVA2 Vector)

[0052] [Table 3]

[0053] (ii) pRC2-mi342 Vector or pRC1 Vector (Takara Bio Inc.) or pRC6 Vector (Takara Bio Inc.)

[0054] [Table 4]

[0055] (iii) pHelper Vector (iv) pAAV-GFP After transfection, the cells were incubated in a 37°C CO2 incubator for 3 days.

[0056] (2) Sampling of the supernatant fraction AAV vector After the culture in Example 2-(1) was completed, a portion of the culture medium was sampled and centrifuged (1,800 × g, 10 min, 4°C). The supernatant after centrifugation was collected and designated as the "AAV vector supernatant fraction sample". After sampling, 0.5 M EDTA (pH 8.0) was added to the culture medium remaining in the well plate at 1 / 80th of the culture medium volume and mixed thoroughly. After reacting at room temperature for 10 minutes, the cells were detached. The detached cells were collected in solution and centrifuged (1,800 × g, 10 min, 4°C). After centrifugation, the supernatant was removed to obtain a cell pellet.

[0057] (3) Sampling of cell fraction AAV vector 80 μL of Extraction Solution A (AAVpro® Extraction Solution, Takara Bio Inc.) was added to the cell pellet from Example 2-(2), and the cells were suspended by vortexing for 15 seconds. After standing at room temperature for 5 minutes, the cells were suspended again by vortexing for another 15 seconds. The cell suspension was centrifuged (9,000 × g, 10 min, 4°C). The cells were then resuspended by vortexing for another 15 seconds. After standing at room temperature for 5 minutes, the cells were suspended again by vortexing for another 15 seconds. After centrifugation (9,000 × g, 10 min, 4°C), the supernatant was collected, and 8 μL of Extraction Solution B (AAVpro® Extraction Solution, Takara Bio Inc.) was added and mixed well to obtain the "AAV vector cell fraction sample". (4) Sampling of AAV vectors using surfactants As a sampling method for the AAV vector different from that used in Examples 2-(2) and (3), sampling was performed using a surfactant. After the completion of culture in Example 2-(1), 1 / 1000 volume of Tween20 (Sigma-A) was added to the culture medium, and then 2 mM MgCl2 was added to reach the final concentration, and Endonuculease (KANEKA) was added to reach a final concentration of 50 U / mL, and the mixture was thoroughly mixed. The cells were lysed by incubation in a 37°C CO2 incubator for 2 hours, and then the culture medium was transferred to a centrifuge tube and centrifuged at 1,800 × g for 10 minutes. The supernatant after centrifugation was designated as "AAV vector surfactant-extracted sample (supernatant fraction + cell fraction)".

[0058] Example 3 (1) Knockdown of MRE11, ISG15, CD44, LMNA, and ACP2 in production cells In Table 2, Ubiquitin-like protein ISG15 is denoted as ISG15, Double-strand break repair protein MRE11 as MRE11, CD44 antigen as CD44, Prelamin-A / C as LMNA, and Lysosomal acid phosphatase as ACP2. HEK293 cells (Agilent, product number 240073), suspended in DMEM containing 10% FBS, were seeded into a 6-well cell culture plate. The cells were then cultured in a 37°C CO2 incubator until approximately 70-80% confluence was achieved. The culture supernatant was removed, and the medium was replaced with serum-free DMEM. Lipofectamine 3000 (Invitrogen) was used to transfect cells with (i) siRNA and (ii)-(iv) plasmids. Control cells were similarly transfected with plasmids (ii)-(iv). (i) siRNA for suppressing the expression of the relevant factor (Sigma-America, si-MRE11 or si-ISG15 or si-CD44) (Thermo-America, LMNA Silencer or ACP2 Silencer)

[0059] [Table 5]

[0060] (ii) pRC2-mi342 Vector or pRC1 Vector or pRC6 Vector [Table 6]

[0061] (iii) pHelper Vector (iv) pAAV-GFP After transfection, the cells were incubated in a 37°C CO2 incubator for 3 days.

[0062] (2) Sampling of the supernatant fraction AAV vector After the culture in Example 3-(1) was completed, a portion of the culture medium was sampled and centrifuged (1,800 × g, 10 min, 4°C). The supernatant after centrifugation was collected and designated as the "AAV vector supernatant fraction sample". After sampling, 0.5 M EDTA (pH 8.0) was added to the culture medium remaining in the well plate at 1 / 80th of the culture medium volume and mixed thoroughly. After reacting at room temperature for 10 minutes, the cells were detached. The detached cells were collected in solution and centrifuged (1,800 × g, 10 min, 4°C). The supernatant after centrifugation was removed to obtain a cell pellet.

[0063] (3) Sampling of cell fraction AAV vector 80 μL of Extraction Solution A was added to the cell pellet from Example 3-(2), and the cells were suspended by vortexing for 15 seconds. After standing at room temperature for 5 minutes, the cells were vortexed again for another 15 seconds. The cell suspension was centrifuged (9,000 × g, 10 min, 4°C). The cells were then resuspended by vortexing for another 15 seconds. After standing at room temperature for 5 minutes, the cells were vortexed again for another 15 seconds. After centrifugation (9,000 × g, 10 min, 4°C), the supernatant was collected, and 8 μL of Extraction Solution B was added and mixed well to obtain the "AAV vector cell fraction sample". (4) Sampling of AAV vectors using surfactants As a sampling method for the AAV vector different from that used in Examples 3-(2) and (3), sampling was performed using a surfactant. After the completion of culture in Example 3-(1), 1 / 1000 volume of Tween 20 was added to the culture medium, and then 2 mM MgCl2 was added to reach the final concentration, and Endonuculease (KANEKA Corporation) was added to reach a final concentration of 50 U / mL, and the mixture was thoroughly mixed. The cells were lysed by incubation in a 37°C CO2 incubator for 2 hours, and then the culture medium was transferred to a centrifuge tube and centrifuged at 1,800 × g for 10 minutes. The supernatant after centrifugation was designated as "AAV vector surfactant-extracted sample (supernatant fraction + cell fraction)".

[0064] Example 4 Analysis of mRNA levels of the relevant factor mRNA extraction was performed on the cultured cells from Example 2-(1) and Example 3-(1) using the High Pure RNA Isolation Kit (Roche) according to the manufacturer's protocol. A portion of the RNA solution cleaned up using the column provided with the kit was taken, and its absorbance was measured using NanoDrop (Thermo). Based on the measurement results, the RNA concentrations between samples were equalized, and cDNA was synthesized using the iScript AdV cDNA kit for RT-qPCR (BIO-RAD) according to the manufacturer's protocol. Real-time PCR was performed using the prepared cDNA solution as a template with THUNDERBIRD SYBR qPCR Mix (TOYOBO). The real-time PCR reaction was performed using the CFX96Touch real-time PCR analysis system (BIO-RAD). The amount of GAPDH mRNA was measured for each sample, and the measurement results for PIK3C2A, UBE2C, ASNS, NIFK, PDCD4, DDX47, GTPBP4, FTSJ3, DDX49, GNL2, NAT10, UBL5, SERBP1, MRE11, ISG15, CD44, LMNA, and ACP2 were normalized. Table 7 shows the ratio in each sample when the mRNA amount of the relevant factor in the control group (cells before regulation) is set to 1. Table 8 shows the ratio in each sample for the GAPDH mRNA amounts of PIK3C2A, UBE2C, ASNS, NIFK, PDCD4, DDX47, GTPBP4, DDX49, GNL2, NAT10, UBL5, SERBP1, ISG15, and LMNA, when the mRNA amount of the relevant factor in HEK293 cells (deposit number ATCC CRL1573) under normal conditions (when not producing AAV vector) is set to 1.

[0065] [Table 7]

[0066] [Table 8]

[0067] As shown in Table 7, in Example 2-(1), cells transfected with expression plasmids for PIK3C2A, UBE2C, ASNS, NIFK, PDCD4, DDX47, GTPBP4, FTSJ3, DDX49, GNL2, NAT10, UBL5, or SERBP1 showed improved expression levels of the respective factors compared to the control group (cells before regulation). In addition, in Example 3-(1), cells into which siRNAs targeting MRE11, ISG15, CD44, LMNA, or ACP2 were introduced showed suppression of the respective factor expression levels compared to the control group (cells before regulation). As shown in Table 8, compared to the normal state (when AAV vector was not produced) in HEK293 cells (deposit number ATCC CRL1573), an increase or decrease in the expression level of the factor in question was observed.

[0068] Example 5 Evaluation of gene transfer efficiency of AAV vectors HEK293 cells (Agilent, product number 240073) suspended in DMEM containing 10% FBS, or HeLa cells (ATCC, product number CCL-2) suspended in EMEM (WAKO) containing 10% FBS, were seeded into a 24-well cell culture plate (Corning). The cells were then cultured in a 37°C CO2 incubator until approximately 70-80% confluence was achieved. To each well, the following samples were added: AAV vector cell fraction samples prepared in Example 2-(3) and Example 3-(3) for AAV2 vector, AAV vector supernatant fraction samples prepared in Example 2-(2) for AAV1 vector, and AAV vector surfactant extract samples (supernatant fraction + cell fraction) prepared in Example 2-(4) and Example 3-(4) for AAV2, AAV1, and AAV6 vectors. Since AAV2 vector is mainly present in the cell fraction and AAV1 vector is mainly present in the supernatant fraction, the sample fraction used was changed depending on the serotype of the AAV vector. After culturing in a 37°C CO2 incubator for 3 days, the samples were subjected to flow cytometry analysis, and the GFP positivity rate in each sample was measured, and the ratio of transduction units (TU) to the control group was calculated. The results are shown in Figures 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, and 11. As shown in Figures 1, 2, 3, 4, 5, and 6, transient expression of PIK3C2A, UBE2C, ASNS, NIFK, PDCD4, DDX47, GTPBP4, FTSJ3, DDX49, GNL2, NAT10, UBL5, DNMT1, SERBP1, and NOVA2 during AAV vector production resulted in the production of AAV vectors with greater infectivity compared to the control group. Furthermore, as shown in Figures 7, 8, 9, 10, and 11, knockdown of MRE11, ISG15, CD44, LMNA, and ACP2 during AAV vector production also resulted in the production of AAV vectors with greater infectivity compared to the control group. Furthermore, these results indicate that the gene transfer efficiency of AAV vectors can be improved in viral vector-producing cells by regulating the expression of any of the genes ranked A through C, as listed in Tables 1 and 2.

[0069] Example 6 (1) Genome extraction from AAV vectors For the AAV vector supernatant fraction samples prepared in Example 2-(2) and Example 3-(2), the AAV vector cell fraction samples prepared in Example 2-(3) and Example 3-(3), and the AAV vector surfactant extraction samples (supernatant fraction + cell fraction) prepared in Example 2-(4) and Example 3-(4), DNaseI (Takara Bio Inc.) was added and reacted at 37°C for 15 minutes to remove free genome and plasmid. Then, heat treatment was performed at 95°C for 10 minutes to inactivate DNaseI. 1 / 10 volume of Proteinase K (QIAGEN) and an equal volume of Buffer AL (QIAGEN) were added, and after standing at 56°C for 10 minutes, an equal volume of ethanol (Kanto Chemical Co., Inc.) was added, and using the DNeasy Blood&Tissue Kit (QIAGEN), the AAV vector genome extract was cleaned up according to the manufacturer's protocol. (2) Genome titer measurement by real-time PCR Using the AAV vector genome extract prepared in Example 6-(1) as a template, real-time PCR was performed using THUNDERBIRD SYBR qPCR Mix. The standard for the calibration curve was a linearized pAAV-GFP solution serially diluted to 2×10 7 , 2×10 6 , 2×10 5 , 2×10 4 , 2×10 3 , 2×10 2 copies / μL. Primers that amplify the promoter region of the AAV vector genome were used. The real-time PCR reaction was performed using a CFX96 Touch real-time PCR analysis system (BIO-RAD). Quantification of the AAV vector genome was performed for the AAV vector supernatant fraction samples and the AAV vector cell fraction samples, and from the total value of the supernatant fraction and the cell fraction, the amount of AAV vector genome per unit culture bottom area in Example 2 and Example 3 (viral genome / cm 2 , hereinafter vg / cm 2The amount of AAV vector genome per unit culture substrate area (vg / cm³) was calculated and obtained as a percentage of the control group. In addition, the AAV vector genome was quantified in the AAV vector surfactant extraction sample (supernatant fraction + cell fraction), and the amount of AAV vector genome per unit culture substrate area (vg / cm³) in Examples 2 and 3 was calculated. 2 The ratio (vg / ratio) was calculated and the proportion relative to the control group was obtained. These results are shown in Figures 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, and 22. As shown in Figures 12, 13, 14, 15, 16, and 17, transient expression of PIK3C2A, UBE2C, ASNS, NIFK, PDCD4, DDX47, GTPBP4, FTSJ3, DDX49, GNL2, NAT10, UBL5, DNMT1, SERBP1, and NOVA2 during AAV vector production resulted in higher AAV vector genome titers compared to the control group. Furthermore, as shown in Figures 18, 19, 20, 21, and 22, knockdown of MRE11, ISG15, CD44, LMNA, and ACP2 during AAV vector production resulted in higher AAV vector genome titers per unit culture substrate area compared to the control group. Furthermore, these results indicate that the genomic titer of the AAV vector can be improved in viral vector-producing cells by regulating the expression of any of the genes ranked A through C, as listed in Tables 1 and 2.

[0070] Example 7 (1) Transient expression of NIFK, SERBP1, and NOVA2 individually or in combination in production cells HEK293 cells (Agilent, product number 240073), suspended in DMEM containing 10% FBS, were seeded into a 6-well cell culture plate (Corning). The cells were then cultured in a 37°C CO2 incubator until approximately 70-80% confluence was confirmed. The cell culture supernatant was removed and replaced with serum-free DMEM, and plasmids (i) to (iv) were transfected. Control cells were similarly transfected with plasmids (ii) to (iv) and an empty vector. (i) Plasmid encoding the relevant factor (Vector Builder, pRP-Neo-CAG-hNIFK Vector, pRP-Neo-CAG-hSERBP1 Vector, or pRP-Neo-CAG-hNOVA2 Vector)

[0071] [Table 9]

[0072] (ii) pRC2-mi342 Vector (iii) pHelper Vector (iv) pAAV-GFP After transfection, the cells were incubated in a 37°C CO2 incubator for 3 days.

[0073] (2) Sampling of AAV vectors using surfactants After the culture in Example 7-(1) was completed, 1 / 1000 volume of Tween20 (Sigma-A) was added to the culture medium, and then 2 mM MgCl2 was added to reach the final concentration, and Endonuculease (KANEKA) was added to reach a final concentration of 50 U / mL, and the mixture was thoroughly stirred. The cells were lysed by incubating in a 37°C CO2 incubator for 2 hours, and then the culture medium was transferred to a centrifuge tube and centrifuged at 1,800 × g for 10 minutes. The supernatant after centrifugation was designated as "AAV vector surfactant extraction sample (supernatant fraction + cell fraction)".

[0074] (3) Evaluation of gene transfer efficiency of AAV vectors HeLa cells suspended in EMEM containing 10% FBS were seeded into a 24-well cell culture plate. The cells were then cultured in a 37°C CO2 incubator until approximately 70-80% confluence was achieved. The AAV vector surfactant extract sample (supernatant fraction + cell fraction) prepared in Example 7-(1) was added to each well, and after incubation in a 37°C CO2 incubator for 3 days, the samples were subjected to flow cytometry analysis to measure the GFP positivity rate in each sample and calculate the ratio of gene transfer units (TUs) to the control group. The results are shown in Figures 23 and 24. As shown in Figure 23, simultaneous transient expression of NOVA2 and NIFK during AAV vector production resulted in the production of AAV vectors with higher infectivity compared to the control group and transient expression of NOVA2 or NIFK alone. Furthermore, as shown in Figure 24, simultaneous transient expression of NOVA2 and SERBP1 during AAV vector production also resulted in the production of AAV vectors with higher infectivity compared to the control group and transient expression of NOVA2 or SERBP1 alone. Furthermore, these results indicate that the gene transfer efficiency of AAV vectors can be improved in viral vector-producing cells by regulating the expression of multiple genes selected from ranks A to C listed in Tables 1 and 2.

[0075] Example 8 (1) Selection of CD44-low expression cells Tables 1 and 2 show that by selecting proteins with different expression levels in HEK293 cells and HEK293T cells as selection markers, it is possible to obtain production cells with higher viral vector production capacity. As an example, we decided to select production cells using CD44 as the selection marker. 1 x 10 6A pellet of HEK293 cells (deposit number ATCC CRL1573) prepared to form cells was suspended in 196 μL of 0.5% BSA (Sigma-A) and 2 mM EDTA-containing DPBS. Then, 4 μL of APC-labeled anti-CD44 antibody (Miltenyi Biotec) was added and thoroughly mixed, and the mixture was allowed to stand for 10 minutes at 4°C in the dark. 1 mL of DMEM containing 10% FBS and 1% MEM non-essential amino acid solution (100×) was added, and the mixture was centrifuged at 300× g at 4°C for 10 minutes, after which the supernatant was removed. The cell pellet was suspended in DMEM containing 10% FBS and 1% MEM non-essential amino acid solution (100×), and then subjected to the AS ONE Cell Picking System (AS ONE). Single cells with low APC brightness values ​​were collected into a 384 plate. The recovered clone (clone A) was expanded and cultured in DMEM containing 10% FBS and 1% MEM non-essential amino acid solution (100×) and used in subsequent tests.

[0076] (2) Measurement of CD44 expression level of recovered clones To measure the expression level of CD44 on the cell surface of clone A obtained in Example 8-(1), 5 × 10⁶ samples were taken from clone A and the pre-selection cell population, HEK293 cells (deposit number ATCC CRL1573). 5 Cell pellets were prepared. The cell pellet was suspended in 98 μL of DPBS containing 0.5% BSA and 2 mM EDTA, then 2 μL of APC-labeled anti-CD44 antibody was added and mixed thoroughly. The mixture was then allowed to stand for 10 minutes at 4°C in the dark. 1 mL of DPBS containing 0.5% BSA and 2 mM EDTA was added, and the mixture was centrifuged at 300 × g at 4°C for 10 minutes. The supernatant was removed. The resulting cell pellet was suspended in 200 μL of DPBS containing 0.5% BSA and 2 mM EDTA and subjected to flow cytometry analysis. APC fluorescence intensity histograms were obtained for each sample, and the results are shown in Figure 25. As shown in Figure 25, the expression level of CD44 on the cell surface of clone A was found to be lower compared to the HEK293 cell population (deposit number ATCC CRL1573) before selection.

[0077] (3) Production of AAV vectors using recovered clones To evaluate the AAV vector production ability of clone A obtained in Example 8-(1), AAV vector production tests were performed on clone A and the pre-selection cell population, HEK293 cells (deposit number ATCC CRL1573), as described below. To evaluate the AAV vector production capacity of clone A suspended in DMEM containing 10% FBS and 1% MEM non-essential amino acid solution (100×), clone A and the pre-selection cell population HEK293 cells (deposit number ATCC CRL1573) were seeded in a 6-well cell culture plate. The cells were then cultured in a 37°C CO2 incubator until approximately 70-80% confluence was confirmed. The cell culture supernatant was removed and replaced with serum-free DMEM, after which plasmids (i) to (iii) were transfected. (i)pRC2-mi342 Vector (ii) pHelper Vector (iii) pAAV-GFP After transfection, the cells were incubated in a 37°C CO2 incubator for 3 days.

[0078] (4) Sampling of AAV vectors using surfactants After the completion of the culture in Example 8-(3), 1 / 1000 volume of Tween 20 was added to the culture medium, and then 2 mM MgCl2 was added to reach the final concentration, and Endonuculease was added to reach a final concentration of 50 U / mL, and the mixture was thoroughly stirred. The cells were lysed by incubation in a 37°C CO2 incubator for 2 hours, and then the culture medium was transferred to a centrifuge tube and centrifuged at 1,800 × g for 10 minutes. The supernatant after centrifugation was designated as "AAV vector surfactant extraction sample (supernatant fraction + cell fraction)".

[0079] (5) Evaluation of gene transfer efficiency of AAV vectors HeLa cells suspended in EMEM containing 10% FBS were seeded into a 24-well cell culture plate. The cells were then cultured in a 37°C CO2 incubator until approximately 70-80% confluence was achieved. The AAV vector surfactant extract sample (supernatant fraction + cell fraction) prepared in Example 8-(4) was added to each well and cultured in a 37°C CO2 incubator for 3 days. Flow cytometry analysis was then performed to measure the GFP positivity rate in each sample, and the proportion of gene transfer units (TUs) relative to the original cell population, HEK293 cells (deposit number ATCC CRL1573), was calculated. The results are shown in Figure 26. As shown in Figure 26, clone A, a CD44-low-expressing clone, produced an AAV vector that retained more infectivity compared to the HEK293 cell population (deposit number ATCC CRL1573) before selection. Furthermore, these results indicate that, in viral vector-producing cells, it is possible to select cells with higher AAV vector production capacity than the cell population before selection by using the proteins listed in Tables 1 and 2 as selection markers.

[0080] (6) Genome extraction of AAV vectors To the AAV vector surfactant-extracted sample (supernatant fraction + cell fraction) prepared in Example 8-(4), DNaseI was added and the mixture was reacted at 37°C for 15 minutes to remove free genome and plasmid. Subsequently, 500 mM EDTA in an amount equal to 1 / 10 of the DNaseI reaction solution was added to stop the DNaseI reaction. One-tenth the volume of Proteinase K and an equal volume of Buffer AL were added to the DNaseI reaction solution, and the mixture was allowed to stand at 56°C for 10 minutes. Then, an equal volume of ethanol was added to the DNaseI reaction solution, and the AAV vector genome extract was cleaned up using the DNeasy Blood & Tissue Kit according to the manufacturer's protocol.

[0081] (7) Genomic titer measurement by digital PCR The AAV vector genome extract prepared in Example 8-(6) was used as a template for ddPCR. TM Digital droplet PCR was performed using Supermix for Probes (No dUTP) (BIO-RAD). Primers were used to amplify the ITR and GFP regions of the AAV vector genome. A FAM-labeled probe was used for the ITR region and a HEX-labeled probe for the GFP region. The instrument used was the QX200 AutoDG Droplet Digital PCR system (BIO-RAD). For AAV vector surfactant-extracted samples (supernatant fraction + cell fraction), the AAV vector genome was quantified from the number of droplets positive for both the ITR region and the GFP region, and the amount of AAV vector genome per unit culture substrate area (vg / cm²) in Example 8-(3) was determined. 2 The ratio was calculated and is shown in Figure 27 as the proportion to the cell population HEK293 cells (deposit number ATCC CRL1573). As shown in Figure 27, clone A, a CD44-low-expressing clone, yielded a higher AAV vector genome titer compared to the HEK293 cell population (deposit number ATCC CRL1573) used before selection. Furthermore, these results indicate that, in viral vector-producing cells, it is possible to select cells with higher AAV vector production capacity than the cell population before selection by using the proteins listed in Tables 1 and 2 as selection markers. [Industrial applicability]

[0082] The viral vector-producing cells obtained in this invention have improved ability to produce viral vectors, particularly AAV vectors. Therefore, by using the viral vector-producing cells of this invention as packaging cells, it is possible to safely improve the productivity of said viral vectors. This application is based on Japanese Patent Application No. 2020-217338, filed on 25 December 2020, the contents of which are all incorporated herein by reference.

Claims

1. A viral vector-producing cell having improved vector production capacity, characterized in that the expression of viral vector-derived proteins in the cell is regulated.

2. The viral vector-producing cell according to claim 1, wherein the expression of a protein derived from the viral vector-producing cell is regulated compared to the viral vector-producing cell before the regulation of the protein expression.

3. The viral vector-producing cell according to claim 1, wherein the expression of proteins derived from the viral vector-producing cell is regulated compared to HEK293 cells (deposit number ATCC CRL1573).

4. The viral vector-producing cell according to claim 1 or 2, wherein the expression of the protein derived from the viral vector-producing cell is increased compared to the viral vector-producing cell before the expression of the protein was regulated.

5. The viral vector-producing cell according to claim 4, wherein the expression of the protein derived from the viral vector-producing cell is increased by 1.5 times or more at the time of viral vector production compared to the viral vector-producing cell before the expression of the protein was regulated.

6. The viral vector-producing cell according to claim 1 or 3, wherein the expression of the protein derived from the viral vector-producing cell is increased compared to HEK293 cells (deposit number ATCC CRL1573).

7. The viral vector-producing cell according to claim 6, wherein the expression of the protein derived from the viral vector-producing cell is increased by 1.5 times or more compared to HEK293 cells (deposit number ATCC CRL1573) at the time of viral vector production.

8. The viral vector-producing cell according to any one of claims 4 to 7, wherein the increase in the expression of the protein derived from the viral vector-producing cell is due to the addition of an additive to the cell.

9. The viral vector-producing cell according to any one of claims 4 to 7, wherein the increase in the expression of the protein derived from the viral vector-producing cell is due to the introduction of an exogenous plasmid into the viral vector-producing cell.

10. The viral vector-producing cell according to any one of claims 4 to 9, wherein the protein derived from the viral vector-producing cell is at least one selected from the group consisting of PIK3C2A, UBE2C, ASNS, NIFK, PDCD4, DDX47, GTPBP4, FTSJ3, DDX49, GNL2, NAT10, UBL5, SERBP1, and NOVA2.

11. The viral vector-producing cell according to claim 1 or 2, wherein the expression of the protein derived from the viral vector-producing cell is reduced compared to the viral vector-producing cell before the expression of the protein was regulated.

12. The viral vector-producing cell according to claim 11, wherein the expression of the protein derived from the viral vector-producing cell is reduced by 10% or more at the time of viral vector production compared to the viral vector-producing cell before the expression of the protein was regulated.

13. The viral vector-producing cell according to claim 1 or 3, wherein the expression of the protein derived from the viral vector-producing cell is reduced compared to HEK293 cells (deposit number ATCC CRL1573).

14. The viral vector-producing cell according to claim 13, wherein the expression of the protein derived from the viral vector-producing cell is reduced by 10% or more at the time of viral vector production compared to HEK293 cells (deposit number ATCC CRL1573).

15. The viral vector-producing cell according to any one of claims 11 to 14, wherein the decrease in the expression of the protein derived from the viral vector-producing cell is due to the addition of an additive to the cell.

16. The viral vector-producing cell according to any one of claims 11 to 14, wherein the decrease in the expression of the protein derived from the viral vector-producing cell is due to the introduction of an exogenous plasmid into the viral vector-producing cell.

17. The viral vector-producing cell according to any one of claims 11 to 16, wherein the protein derived from the viral vector-producing cell is at least one selected from the group consisting of ISG15, CD44, LMNA, ACP2, and MRE11.

18. A virus vector-producing cell according to any one of claims 1 to 17, which is derived from human cells.

19. The virus vector producing cell according to claim 18, wherein the human cell is a HEK293 cell.

20. The viral vector-producing cell according to any one of claims 1 to 19, wherein the viral vector is an adeno-associated virus vector.

21. A method for producing viral vector-producing cells with improved vector-producing ability, characterized by regulating the expression of proteins derived from the viral vector-producing cells in the viral vector-producing cells.

22. The method for producing viral vector-producing cells according to claim 21, wherein the regulation is performed by regulating the expression of the protein compared to viral vector-producing cells before the regulation.

23. A method for producing viral vector-producing cells according to claim 21, wherein the adjustment is made by comparing with HEK293 cells (deposit number ATCC CRL1573).

24. The method for producing viral vector-producing cells according to claim 21 or 22, wherein the regulation increases the expression of the protein derived from the viral vector-producing cells compared to the viral vector-producing cells before the regulation of the protein expression.

25. The method for producing viral vector-producing cells according to claim 24, wherein the regulation increases the expression of the protein derived from the viral vector-producing cells by 1.5 times or more at the time of viral vector production compared to viral vector-producing cells before the regulation of the protein expression.

26. The method for producing viral vector-producing cells according to claim 21 or 23, wherein the regulation increases the expression of the protein derived from the viral vector-producing cells compared to HEK293 cells (deposit number ATCC CRL1573).

27. The method for producing viral vector-producing cells according to claim 26, wherein the regulation increases the expression of the protein derived from the viral vector-producing cells by 1.5 times or more compared to HEK293 cells (deposit number ATCC CRL1573) at the time of viral vector production.

28. A method for producing viral vector-producing cells according to any one of claims 24 to 27, wherein the increase in the expression of the protein derived from the viral vector-producing cells is due to the addition of an additive to the cells.

29. A method for producing viral vector-producing cells according to any one of claims 24 to 27, wherein the increase in the expression of the protein derived from the viral vector-producing cells is achieved by introducing an exogenous plasmid into the viral vector-producing cells.

30. A method for producing a viral vector-producing cell according to any one of claims 24 to 29, wherein the protein derived from the viral vector-producing cell is at least one selected from the group consisting of PIK3C2A, UBE2C, ASNS, NIFK, PDCD4, DDX47, GTPBP4, FTSJ3, DDX49, GNL2, NAT10, UBL5, SERBP1, and NOVA2.

31. The method for producing viral vector-producing cells according to claim 21 or 22, wherein the regulation reduces the expression of the protein derived from the viral vector-producing cells compared to the viral vector-producing cells before the regulation of the protein expression.

32. The method for producing viral vector-producing cells according to claim 31, wherein the regulation reduces the expression of the protein derived from the viral vector-producing cells by 10% or more at the time of viral vector production compared to viral vector-producing cells before the regulation of the protein expression.

33. A method for producing viral vector-producing cells according to claim 21 or 23, wherein the decrease in the expression of the protein derived from the viral vector-producing cells is reduced compared to HEK293 cells (deposit number ATCC CRL1573).

34. The method for producing viral vector-producing cells according to claim 33, wherein the regulation reduces the expression of the protein derived from the viral vector-producing cells by 10% or more compared to HEK293 cells (deposit number ATCC CRL1573) at the time of viral vector production.

35. A method for producing viral vector-producing cells according to any one of claims 21 to 34, wherein the decrease in the expression of the protein derived from the viral vector-producing cells is due to the addition of an additive to the cells.

36. A method for producing viral vector-producing cells according to any one of claims 21 to 34, wherein the reduction in the expression of the protein derived from the viral vector-producing cells is due to the introduction of an exogenous plasmid into the viral vector-producing cells.

37. A method for producing a viral vector-producing cell according to any one of claims 21 to 36, wherein the protein derived from the viral vector-producing cell is at least one selected from the group consisting of ISG15, CD44, LMNA, ACP2, and MRE11.

38. A method for producing a virus vector-producing cell according to any one of claims 21 to 37, which is derived from human cells.

39. The method for producing viral vector-producing cells according to claim 38, wherein the human cells are HEK293 cells.

40. A method for producing a viral vector-producing cell according to any one of claims 21 to 39, wherein the viral vector is an adeno-associated virus vector.

41. A method for enhancing viral vector production in viral vector-producing cells, characterized by regulating the expression of viral vector-derived proteins in viral vector-producing cells.

42. A method for enhancing the production of a viral vector in a viral vector-producing cell according to claim 41, wherein the regulation is performed compared to a viral vector-producing cell before the regulation of the expression of the protein.

43. A method for enhancing the production of viral vectors in viral vector-producing cells according to claim 41, wherein the regulation is performed in comparison to HEK293 cells (deposit number ATCC CRL1573).

44. A method for enhancing the production of viral vectors in viral vector-producing cells according to claim 41 or 42, wherein the regulation involves increasing the expression of a protein derived from the viral vector-producing cells compared to viral vector-producing cells before the expression of the protein was regulated.

45. A method for enhancing the production of viral vectors in viral vector-producing cells according to claim 41 or 43, wherein the regulation involves increasing the expression of proteins derived from the viral vector-producing cells compared to HEK293 cells (deposit number ATCC CRL1573).

46. A method for enhancing the amount of viral vector produced in viral vector-producing cells according to claim 44 or 45, wherein the increase in the expression of the protein derived from the viral vector-producing cells is due to the addition of an additive to the cells.

47. A method for enhancing the amount of viral vector produced in viral vector-producing cells according to claim 44 or 45, wherein the increase in the expression of the protein derived from the viral vector-producing cells is achieved by introducing an exogenous plasmid into the viral vector-producing cells.

48. A method for enhancing viral vector production in viral vector-producing cells according to claim 41 or 42, wherein the regulation reduces the expression of the protein derived from the viral vector-producing cells compared to the viral vector-producing cells before the regulation of the protein expression.

49. A method for enhancing viral vector production in viral vector-producing cells according to claim 41 or 43, wherein the decrease in the expression of the protein derived from the viral vector-producing cells is reduced compared to HEK293 cells (deposit number ATCC CRL1573).

50. A method for increasing the amount of viral vector produced in viral vector-producing cells according to claim 48 or 49, wherein the decrease in the expression of the protein derived from the viral vector-producing cells is due to the addition of an additive to the cells.

51. A method for increasing the amount of viral vector produced in viral vector-producing cells according to claim 48 or 49, wherein the decrease in the expression of the protein derived from the viral vector-producing cells is due to the introduction of an exogenous plasmid into the viral vector-producing cells.

52. Viral vector producing cell according to any one of claims 1 to 20 A kit for producing a viral vector, comprising viral vector-producing cells produced by the manufacturing method described in any one of claims 21 to 40.

53. A method for selecting viral vector-producing cells with improved viral vector production capacity, comprising the step of selecting cells in which the expression of viral vector-derived proteins in viral vector-producing cells is regulated.

54. A method for selecting viral vector-producing cells according to claim 53, comprising the step of selecting cells in which the expression of a protein derived from the viral vector-producing cell is regulated compared to viral vector-producing cells before the expression of the protein was regulated.

55. A method for selecting viral vector-producing cells according to claim 53, comprising the step of selecting cells in which the expression of viral vector-derived proteins in viral vector-producing cells is regulated compared to HEK293 cells (deposit number ATCC CRL1573).

56. The method for selecting viral vector-producing cells according to claim 53 or 54, wherein the regulation is performed such that the expression of the protein is increased compared to the viral vector-producing cells before the regulation.

57. A method for selecting viral vector-producing cells according to claim 53 or 55, wherein the regulation is increased compared to HEK293 cells (deposit number ATCC CRL1573).

58. The method for selecting viral vector-producing cells according to claim 53 or 54, wherein the regulation is performed such that the expression of the protein derived from the viral vector-producing cells is reduced compared to the viral vector-producing cells before the regulation of the protein expression.

59. The method for selecting viral vector-producing cells according to claim 53 or 55, wherein the regulation is performed such that the expression of the protein derived from the viral vector-producing cells is reduced compared to HEK293 cells (deposit number ATCC CRL1573).

60. The method for selecting a viral vector-producing cell according to claim 58 or 59, wherein the protein derived from the viral vector-producing cell is CD44.

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