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KR-20260066111-A - Culture medium composition

KR20260066111AKR 20260066111 AKR20260066111 AKR 20260066111AKR-20260066111-A

Abstract

The present invention provides a culture medium composition for promoting the production of a virus loaded with a target gene in the culture of a cell producing a virus into which a target gene has been introduced, comprising a pyruvate dehydrogenase kinase inhibitor, and a culture medium composition for promoting the production of a virus or a virus-like particle in the culture of a cell producing a virus or a virus-like particle.

Inventors

  • 히구치, 다쿠야

Assignees

  • 아지노모토 가부시키가이샤

Dates

Publication Date
20260512
Application Date
20240904
Priority Date
20230905

Claims (20)

  1. A culture medium composition for promoting virus production loaded with said target gene, wherein the target gene is introduced into a virus-producing cell containing a pyruvate dehydrogenase kinase inhibitor.
  2. In paragraph 1, A culture medium composition in which the pyruvate dehydrogenase kinase inhibitor is dichloroacetic acid or its salt.
  3. In paragraph 1, A culture medium composition in which the cell is a cell selected from the group consisting of HEK293 cells, MDCK cells, Vero cells, A549 cells, PerC6 cells, HeLa cells and insect cells.
  4. In paragraph 1, A culture medium composition in which the cells are HEK293 cells.
  5. In paragraph 1, A culture medium composition in which the virus-producing cell is a cell that produces adeno-associated virus.
  6. In any one of paragraphs 1 through 5, A culture medium composition in which the concentration of a pyruvate dehydrogenase kinase inhibitor in the culture medium composition is 0.1 mM to 1,000 mM.
  7. A culture medium additive for promoting the production of a virus loaded with a target gene, wherein the target gene is introduced into a cell producing the virus, the cell containing a pyruvate dehydrogenase kinase inhibitor.
  8. Use of a pyruvate dehydrogenase kinase inhibitor in the preparation of a culture medium composition for promoting virus production loaded with said target gene, in the culture of a virus-producing cell into which a target gene has been introduced.
  9. A method for promoting the production of a virus carrying said target gene, comprising the process of culturing a virus-producing cell having said target gene introduced in a medium containing a pyruvate dehydrogenase kinase inhibitor.
  10. A method for producing a virus carrying said target gene, comprising the process of culturing a cell that produces a virus into which the target gene has been introduced in a medium containing a pyruvate dehydrogenase kinase inhibitor.
  11. A culture medium composition for promoting the production of a virus or virus-like particle during the culture of a cell that produces a virus or virus-like particle, comprising a pyruvate dehydrogenase kinase inhibitor.
  12. In Paragraph 11, A culture medium composition in which the pyruvate dehydrogenase kinase inhibitor is dichloroacetic acid or its salt.
  13. In Paragraph 11, A culture medium composition in which the cell is a cell selected from the group consisting of HEK293 cells, MDCK cells, Vero cells, A549 cells, PerC6 cells, HeLa cells and insect cells.
  14. In Paragraph 11, A culture medium composition in which the cells are HEK293 cells.
  15. In Paragraph 11, A culture medium composition in which a cell that produces a virus or virus-like particle is a cell that produces an adeno-associated virus.
  16. In any one of paragraphs 11 through 15, A culture medium composition in which the concentration of a pyruvate dehydrogenase kinase inhibitor in the culture medium composition is 0.1 mM to 1,000 mM.
  17. A culture medium additive for promoting the production of a virus or virus-like particle during the culture of a cell that produces the virus or virus-like particle, comprising a pyruvate dehydrogenase kinase inhibitor.
  18. Use of a pyruvate dehydrogenase kinase inhibitor in the preparation of a medium composition for promoting the production of said virus or virus-like particles during the culture of cells that produce said virus or virus-like particles.
  19. A method for promoting the production of said virus or virus-like particles, comprising the process of culturing cells that produce said virus or virus-like particles in a medium containing a pyruvate dehydrogenase kinase inhibitor.
  20. A method for producing said virus or virus-like particle, comprising the process of culturing a cell that produces a virus or virus-like particle in a medium containing a pyruvate dehydrogenase kinase inhibitor.

Description

Culture medium composition The present invention relates to a culture medium composition, etc., for promoting the production of a virus carrying said target gene in the culture of a cell producing a virus into which a target gene has been introduced, comprising a pyruvate dehydrogenase kinase inhibitor, and also for promoting the production of said virus or virus-like particles in the culture of a cell producing said virus or virus-like particles. To meet the demands for human gene therapy and vaccine manufacturing, rapid and stable production technology for viruses and similar substances is indispensable. In particular, the cultivation of cells that produce viruses (hereinafter also referred to as virus-producing cells) is of extreme importance. When designing and optimizing a culture medium for virus-producing cells, the composition of the medium becomes an important issue. The components included in the medium affect the growth and proliferation of cells. For example, Patent Document 1 discloses that the culture efficiency of said cells is improved by culturing animal cells in a medium containing a pyruvate dehydrogenase kinase inhibitor. Additionally, in the case of a culture medium for virus-producing cells, the components included in the medium also affect the virus-producing ability within the cell. Furthermore, for example, when producing viruses using cells into which a gene that produces a protein that suppresses cancer has been introduced, the components included in the medium also affect the proportion of viruses carrying the said gene among the total viruses produced. However, satisfactory results are not necessarily obtained with the components known so far, and for example, when producing adeno-associated viruses using cells into which the gene has been introduced, the proportion of adeno-associated viruses carrying the said gene among the said adeno-associated viruses is said to be about 5% to 30% (Non-patent Literature 1). Figure 1 shows the results of verifying the effect of sodium dichloroacetate on the proliferation of HEK293 in Example 1. VCD represents live cell density, and the vertical axis represents live cell density (× 10⁶ cells/mL). The horizontal axis “Viral Production medium” indicates the case where a medium without added sodium dichloroacetate was used, and “Viral Production medium + 5 mM DCA” indicates the case where a medium with added sodium dichloroacetate to a final concentration of 5 mM was used. Figure 2 shows the results of verifying the effect of sodium dichloroacetate on the AAV8 genome titer in Example 1. The vertical axis represents the relative AAV8 genome titer when the AAV8 genome titer obtained when AAV8 is produced using a medium without sodium dichloroacetate added is set to 100%. The horizontal axis “virus production medium” represents the case where AAV8 is produced using a medium without sodium dichloroacetate added, and “virus production medium + 5 mM DCA” represents the case where AAV8 is produced using a medium with sodium dichloroacetate added to a final concentration of 5 mM. Figure 3 shows the results of verifying the effect of sodium dichloroacetate on the full/empty capsid ratio (full capsid ratio) in Example 1. The vertical axis represents the relative full capsid ratio when the full capsid ratio obtained when AAV8 is produced using a medium without sodium dichloroacetate added is set to 100%. The horizontal axis, "virus production medium," represents the case where AAV8 is produced using a medium without sodium dichloroacetate added, and "virus production medium + 5 mM DCA" represents the case where AAV8 is produced using a medium with sodium dichloroacetate added to a final concentration of 5 mM. 1. Culture medium composition 1 The present invention provides a culture medium composition for promoting the production of a virus loaded with a target gene, wherein the target gene is introduced into a cell that produces a virus, and the culture medium composition 1 of the present invention comprises a pyruvate dehydrogenase kinase inhibitor. Pyruvate dehydrogenase (PDH) kinase is an enzyme that phosphorylates PDH to inactivate it and antagonistically regulates the activity of PDH phosphatase. PDH kinase inhibitors are organic molecules that prevent PDH kinase from phosphorylating specific serine residues on the E1 (alpha subunit) of the PDH complex involved in regulating PDH activity. As PDH kinase inhibitors, various compounds are known, such as dichloroacetic acid or its salts, AZD7545, VER-246608, PDHK-IN-5, and lylamin, but dichloroacetic acid or its salts are preferred. Examples of salts of dichloroacetic acid include metal salts, ammonium salts, and organic amine addition salts. Examples of metal salts include alkali metal salts such as sodium salts and potassium salts, alkaline earth metal salts such as magnesium salts and calcium salts, aluminum salts, zinc salts, etc. Examples of ammonium salts include ammonium salts, diisopropylammonium sa