Culture medium composition

Abstract

The present invention provides, for example: a culture medium composition for promoting the production of viruses each carrying a target gene in the culturing of cells capable of producing viruses each having the target gene introduced therein, the composition containing a pyruvate dehydrogenase kinase inhibitor; and a culture medium composition for promoting the production of viruses or virus-like particles in the culturing of cells capable of producing the viruses or the virus-like particles.

Description

Medium Composition

[0001] The present invention relates to a medium composition and the like for promoting production of a virus carrying a gene of interest when culturing virus-producing cells into which a gene of interest has been introduced, the virus-producing cells containing the gene of interest, and for promoting production of the virus or virus-like particle when culturing virus- or virus-like particle-producing cells, the medium composition comprising a pyruvate dehydrogenase kinase inhibitor.

[0002] To meet the needs for human gene therapy, vaccine production, etc., rapid and stable production technologies for viruses are essential. In particular, the cultivation of cells that produce viruses (hereinafter also referred to as virus-producing cells) is extremely important.

[0003] When designing and optimizing a medium for culturing virus-producing cells, the components of the medium are an important issue. The components contained in the medium affect the growth and proliferation of the cells. For example, Patent Document 1 discloses that the culture efficiency of animal cells is improved by culturing the cells in a medium containing a pyruvate dehydrogenase kinase inhibitor.

[0004] In addition, in the case of media for culturing virus-producing cells, the components contained in the media also affect the virus production ability within the cells. Furthermore, when producing viruses using cells transfected with a gene that produces a cancer-suppressing protein, the components contained in the media also affect the proportion of viruses carrying that gene among all the viruses produced. However, the components known to date have not necessarily produced satisfactory results. For example, when producing adeno-associated viruses using cells transfected with a gene, the proportion of adeno-associated viruses carrying that gene among all the adeno-associated viruses is said to be approximately 5% to 30% (Non-Patent Document 1).

[0005] WO 2002 / 04598

[0006] Molecular Therapy Methods & Clinical Development; 2021 June 11; Vol. 21; p. 642-655

[0007] An object of the present invention is to provide a medium composition and the like for improving the production amount of a virus carrying a gene of interest when the virus is produced using virus-producing cells, and for promoting the production of a virus or virus-like particle when the virus or virus-like particle is produced using cells.

[0008] As a result of extensive research into the above-mentioned problems, the present inventors have found that the addition of sodium dichloroacetate, a pyruvate dehydrogenase kinase inhibitor, to human embryonic kidney 293 (HEK293) cells improves their ability to produce adeno-associated viruses carrying a gene of interest. Based on this finding, further research has led to the completion of the present invention.

[0009] [1] A medium composition for promoting virus production carrying a gene of interest during the culture of virus-producing cells into which the gene of interest has been introduced, the medium composition comprising a pyruvate dehydrogenase kinase inhibitor. [2] The medium composition according to [1] above, wherein the pyruvate dehydrogenase kinase inhibitor is dichloroacetic acid or a salt thereof. [3] The medium composition according to [1] or [2] above, wherein the cells are selected from the group consisting of HEK293 cells, MDCK cells, Vero cells, A549 cells, PerC6 cells, HeLa cells, and insect cells. [4] The medium composition according to [1] or [2] above, wherein the cells are HEK293 cells. [5] The medium composition according to any of [1] to [4] above, wherein the virus-producing cells are adeno-associated virus-producing cells. [6] The medium composition according to any of [1] to [5] above, wherein the concentration of the pyruvate dehydrogenase kinase inhibitor in the medium composition is 0.1 mM to 1,000 mM. [7] A medium additive comprising a pyruvate dehydrogenase kinase inhibitor for promoting production of a virus carrying a gene of interest during the cultivation of virus-producing cells into which the gene of interest has been introduced. [8] Use of a pyruvate dehydrogenase kinase inhibitor in the manufacture of a medium composition for promoting production of a virus carrying a gene of interest during the cultivation of virus-producing cells into which the gene of interest has been introduced. [9] A method for promoting production of a virus carrying a gene of interest, comprising the step of culturing virus-producing cells into which the gene of interest has been introduced, in a medium containing a pyruvate dehydrogenase kinase inhibitor.

[10] A method for producing a virus carrying a gene of interest, comprising the step of culturing virus-producing cells into which the gene of interest has been introduced, in a medium containing a pyruvate dehydrogenase kinase inhibitor.

[11] A medium composition comprising a pyruvate dehydrogenase kinase inhibitor for promoting production of a virus or virus-like particle during the cultivation of virus- or virus-like particle-producing cells.

[12] The medium composition according to

[11] above, wherein the pyruvate dehydrogenase kinase inhibitor is dichloroacetic acid or a salt thereof.

[13] The medium composition according to

[11] or

[12] above, wherein the cells are selected from the group consisting of HEK293 cells, MDCK cells, Vero cells, A549 cells, PerC6 cells, HeLa cells, and insect cells.

[14] The medium composition according to

[11] or

[12] above, wherein the cells are HEK293 cells.

[15] The medium composition according to any of

[11] to

[14] above, wherein the cells that produce viruses or virus-like particles are cells that produce adeno-associated viruses.

[16] The medium composition according to any of

[11] to

[15] above, wherein the concentration of a pyruvate dehydrogenase kinase inhibitor in the medium composition is 0.1 mM to 1,000 mM.

[17] A medium additive for promoting the production of viruses or virus-like particles during the culture of cells that produce the viruses or virus-like particles, comprising a pyruvate dehydrogenase kinase inhibitor.

[18] Use of a pyruvate dehydrogenase kinase inhibitor in the manufacture of a medium composition for promoting the production of a virus or virus-like particle during the cultivation of cells that produce the virus or virus-like particle.

[19] A method for promoting the production of a virus or virus-like particle, comprising the step of culturing cells that produce the virus or virus-like particle in a medium containing a pyruvate dehydrogenase kinase inhibitor.

[20] A method for producing a virus or virus-like particle, comprising the step of culturing cells that produce the virus or virus-like particle in a medium containing a pyruvate dehydrogenase kinase inhibitor.

[0010] According to the present invention, when a virus carrying a gene of interest is produced using virus-producing cells, it is possible to improve the production amount of the virus carrying the gene of interest.

[0011] FIG. 1 shows the results of examining the effect of sodium dichloroacetate on the proliferation of HEK293 in Example 1. VCD means viable cell density, and the vertical axis represents the viable cell density (×10 ６The horizontal axis represents the result of using a medium without sodium dichloroacetate, and the horizontal axis represents the result of using a medium supplemented with sodium dichloroacetate to a final concentration of 5 mM. FIG. 2 shows the results of examining 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 is set to 100%. "Viral Production Medium" on the horizontal axis represents the case where AAV8 was produced using a medium not supplemented with sodium dichloroacetate, and "Viral Production Medium + 5 mM DCA" represents the case where AAV8 was produced using a medium supplemented with sodium dichloroacetate to a final concentration of 5 mM. Figure 3 shows the results of examining 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 was produced using a medium not supplemented with sodium dichloroacetate was set to 100%. On the horizontal axis, "Viral Production Medium" represents the case where AAV8 was produced using a medium to which sodium dichloroacetate was not added, and "Viral Production Medium + 5 mM DCA" represents the case where AAV8 was produced using a medium to which sodium dichloroacetate was added to a final concentration of 5 mM.

[0012] 1. Medium Composition 1 The present invention provides a medium composition (hereinafter also referred to as "medium composition 1 of the present invention") for promoting virus production carrying a gene of interest during the cultivation of virus-producing cells into which the gene of interest has been introduced, the medium composition comprising a pyruvate dehydrogenase kinase inhibitor.

[0013] Pyruvate dehydrogenase (PDH) kinase is an enzyme that phosphorylates and inactivates PDH, and regulates its activity in an antagonistic manner to 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, which is involved in regulating PDH activity.

[0014] Various compounds are known as PDH kinase inhibitors, such as dichloroacetic acid or a salt thereof, AZD7545, VER-246608, PDHK-IN-5, and Leelamin, with dichloroacetic acid or a salt thereof being preferred.

[0015] Examples of salts of dichloroacetic acid include metal salts, ammonium salts, and organic amine addition salts.

[0016] 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 salts, tetramethylammonium salts, etc. Examples of organic amine addition salts include addition salts of morpholine, piperidine, etc. Among these, sodium salts or potassium salts are preferred, and sodium salts are more preferred.

[0017] When a salt of dichloroacetic acid is to be obtained, if dichloroacetic acid is obtained in the form of a salt, it can be purified as is. Alternatively, if dichloroacetic acid is obtained in the free form, it can be isolated and purified by dissolving or suspending dichloroacetic acid in an appropriate solvent and adding an acid or a base.

[0018] Dichloroacetic acid or a salt thereof may exist in the form of a solvate such as a hydrate, and in the present invention, these solvates are also included in the term dichloroacetic acid or a salt thereof.

[0019] The PDH kinase inhibitor used in medium composition 1 of the present invention may be synthesized by a method known per se, or may be available as a commercially available product.

[0020] The concentration of the PDH kinase inhibitor in medium composition 1 of the present invention is not particularly limited as long as the effects of the present invention are exhibited, but is usually 0.1 mM to 1,000 mM, preferably 0.3 mM to 300 mM, more preferably 0.5 mM to 100 mM, and even more preferably 1 mM to 20 mM.

[0021] As used herein, the term "target gene" refers to a heterologous polynucleotide that can be introduced into a cell or organism via a viral vector or the like, and may be any polynucleotide, such as a gene encoding a polypeptide or protein, or a polynucleotide that can be transcribed into an inhibitory polynucleotide (e.g., siRNA, miRNA, shRNA, etc.), and is preferably intended for the treatment of a disease in an animal, including a human.

[0022] Examples of viruses include adenoviruses, adeno-associated viruses (AAVs), retroviruses, lentiviruses, and Sendai viruses, as well as orthomyxoviruses, paramyxoviruses, reoviruses, picornaviruses, flaviviruses, arenaviruses, herpesviruses, and poxviruses, and recombinant viruses thereof. Among these, adenoviruses and AAVs are preferred, and AAVs are more preferred. Examples of AAV include AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVDJ, AAVDJ8, AAV-B1, AAVM41, AAVrhlO, AAVrh74, and chimeras thereof, with AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAV8, and AAV9 being more preferred, and AAV2 and AAV8 being even more preferred.

[0023] The cells that produce the virus are not particularly limited as long as they are capable of producing a virus carrying the desired gene of interest, and include mammalian cells such as human, monkey, and rodent cells, insect cells, and the like. Specific examples include HEK293 cells, MDCK cells, Vero cells, A549 cells, PerC6 cells, HeLa cells, CV-1 cells, LLC-MK2 cells, MDBK cells, WI-38 cells, MRC5 cells (human fibroblasts), BHK21 cells, insect cells, and the like. Preferred examples include HEK293 cells, MDCK cells, Vero cells, A549 cells, PerC6 cells, HeLa cells, insect cells, and the like, all of which are commercially available. When the virus is adenovirus or AAV, HEK293 cells, Vero cells, A549 cells, and the like are more preferably used, and HEK293 cells are even more preferably used.

[0024] The method for introducing a gene of interest into virus-producing cells is not particularly limited and can be performed using gene transfer methods well known to those skilled in the art. More specifically, examples include chemical methods using transfection reagents such as cationic lipids, cationic polymers (e.g., polyetherimide (PEI)), calcium phosphate, etc.; physical methods such as lipofection, electroporation, microinjection, sonoporation, and laser irradiation; and infection methods (biological methods) using viral vectors (e.g., adenovirus vectors, adeno-associated virus vectors, retrovirus vectors, lentivirus vectors, and Sendai virus vectors). Chemical methods using transfection reagents are preferred, and chemical methods using cationic lipids or cationic polymers are more preferred. The gene of interest can be prepared by methods well known to those skilled in the art, but it can also be obtained as a commercially available product.

[0025] The culture may be either adherent culture or suspension culture, but suspension culture is preferred. As used herein, "adherent culture" refers to culturing cells by adhering them to a culture substrate, specifically a method of growing cells while adhering to the surface of the culture substrate and while adhering cells to each other. Examples of culture substrates include, but are not limited to, multiwell plates, culture dishes, petri dishes, culture flasks, microcarriers, hollow fibers, and the like. The culture may be static culture on a substrate. As used herein, "suspension culture" refers to a cell culture method in which cells are not adhered to the culture vessel. Suspension culture may or may not involve external pressure or vibration on the liquid medium, or shaking or rotation in the liquid medium.

[0026] The culture vessel used for culturing is not particularly limited as long as it is capable of culturing the target cells, and examples include flasks, tissue culture flasks, dishes, Petri dishes, tissue culture dishes, multi-dishes, microplates, microwell plates, multi-plates, multi-well plates, microslides, chamber slides, petri dishes, tubes, trays, culture bags, roller bottles, bioreactors, etc.

[0027] The culture vessel may be cell-adhesive or cell-non-adhesive, and is appropriately selected depending on the purpose. A cell-adhesive culture vessel may be coated with any cell-supporting substrate, such as an extracellular matrix (ECM), for the purpose of improving adhesion of cells to the surface of the culture vessel. The cell-supporting substrate may be any substance intended for cell adhesion.

[0028] "Promoting the production of viruses carrying a gene of interest" refers to increasing the genome titer of the gene of interest in all viruses produced during the culture of virus-producing mammalian cells into which the gene of interest has been introduced. That is, it refers to (a) improving the full capsid ratio regardless of whether the capsid number (virus number) increases or decreases, thereby increasing the overall genome titer, or (b) increasing the capsid number regardless of whether the full capsid ratio is high or low, thereby increasing the overall genome titer.

[0029] Medium composition 1 of the present invention may further contain other components that are favorable for virus production, as long as the effects of the present invention are not impaired. Other components include, for example, sugars such as glucose, fructose, sucrose, and maltose; amino acids; proteins such as albumin and transferrin; peptides such as glycylglycylglycine and soybean peptide; serum; vitamins such as choline, vitamin A, B vitamins (thiamine, pyridoxine, cyanocobalamin, biotin, pantothenic acid, nicotinamide, etc.), vitamin C, and vitamin E; fatty acids such as oleic acid, arachidonic acid, and linoleic acid; lipids such as cholesterol; inorganic salts such as sodium chloride, calcium chloride, and sodium dihydrogen phosphate; trace elements such as zinc and selenium; buffers such as N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES) and N-[tris(hydroxymethyl)methyl]glycine (Tricine); antibiotics such as amphotericin B, kanamycin, gentamicin, streptomycin, and penicillin; Type I collagen, Type II collagen, and the like. Examples of other components include cell adhesion factors and extracellular matrix components such as collagen, fibronectin, laminin, poly-L-lysine, and poly-D-lysine; cytokines and growth factors such as interleukin, fibroblast growth factor (FGF), hepatocyte growth factor (HGF), transforming growth factor (TGF)-α, transforming growth factor (TGF)-β, vascular endothelial growth factor (VEGF), and activin A; and hormones such as dexamethasone, hydrocortisone, estradiol, progesterone, glucagon, and insulin. Appropriate components can be selected and used depending on the type of cells to be cultured, the type of virus to be produced, and the like. The above-mentioned other components may be contained alone or in combination of two or more.

[0030] The other components can be contained in the medium composition 1 of the present invention at a concentration of, for example, a total amount of 0.001% by weight to 99.9% by weight, preferably 0.01% by weight to 99% by weight, and more preferably 0.1% by weight to 95% by weight.

[0031] Furthermore, medium composition 1 of the present invention may contain serum or may be serum-free. The serum is not particularly limited as long as it is derived from an animal and does not inhibit cell growth, but preferred examples include mammalian serum (e.g., fetal bovine serum, human serum, etc.), and more preferred is fetal bovine serum. The serum concentration may be within a known concentration range. Medium composition 1 of the present invention is preferably serum-free.

[0032] Furthermore, medium composition 1 of the present invention may or may not further contain β-alanine.

[0033] When medium composition 1 of the present invention contains β-alanine, the concentration of β-alanine in medium composition 1 of the present invention is generally 0.1 μM to 800 μM, preferably 1 μM to 700 μM, more preferably 3 μM to 600 μM, even more preferably 10 μM to 500 μM, and particularly preferably 30 μM to 400 μM.

[0034] The medium composition 1 of the present invention can be prepared, for example, by adding the PDH kinase inhibitor directly to a basal medium, and this embodiment is preferred because of its simplicity. Furthermore, when the PDH kinase inhibitor is in the form of a solution in the form of the medium additive 1 of the present invention described below, the medium composition 1 of the present invention can be obtained by adding, for example, 1 / 120 to 1 / 5, preferably 1 / 110 to 1 / 8, more preferably 1 / 100 of the amount of the medium additive 1 of the present invention to a basal medium.

[0035] The basal medium refers to a medium containing carbon sources, nitrogen sources, inorganic salts, etc., which are essential for cell culture. There are no particular limitations on the medium as long as it can achieve the effects of the present invention, and it may be selected appropriately depending on the cells to be cultured.

[0036] Examples of basal media include Dulbecco's Modified Eagle's Medium (DMEM), Ham's Nutrient Mixture F12, DMEM / F12 medium, McCoy's 5A medium, Minimum Essential Medium (MEM), Eagle's Minimum Essential Medium (EMEM), and alpha Modified Eagle's Minimum Essential Medium (alpha Modified Eagle's Minimum Essential Medium). Examples of the medium include αMEM, Roswell Park Memorial Institute (RPMI) 1640 medium, Iscove's Modified Dulbecco's Medium (IMDM), MCDB131 medium, William's Medium E, Fischer's Medium, and a mixture of these media. The basal medium may be prepared by a method known per se, or may be prepared using Viral Production Medium (Thermo Fisher Scientific: A4817901), HyClone HyCell TransFx-H transfection media (Cytiva: SH30939.01), BalanCD® HEK293 (Fujifilm Wako Pure Chemical Industries, Ltd.: 551-34231), EX-CELL® 293 (Merck: 14571C), FreeStyle™ 293 Expression Medium (Thermo Fisher Scientific: The medium may be commercially available, such as PEG-400 (available from Thermo Fisher Scientific: 12338018), CDM4HEK293 (available from Cytiva: SH30858.02), or Expi293™ Expression Medium (available from Thermo Fisher Scientific: A1435101), or may be one currently under development.

[0037] When medium composition 1 of the present invention contains the above-mentioned other components, serum, and / or β-alanine, it can be produced by adding a PDH kinase inhibitor and the above-mentioned other components, serum, and / or β-alanine to the above-mentioned basal medium as appropriate and mixing them directly, or by other production methods commonly used in the field of medium compositions. The order in which these components are added is not particularly limited and can be determined appropriately depending on the intended use, etc.

[0038] The medium composition 1 of the present invention may be provided in a liquid state, or may be prepared in a more concentrated state than the concentration at the time of use, or in a solid state such as a freeze-dried powder, and then diluted with a solvent such as water, or dissolved or dispersed in a solvent such as water, at the time of use.

[0039] By culturing virus-producing cells into which a target gene has been introduced in medium composition 1 of the present invention, the production of viruses carrying the target gene can be promoted regardless of whether or not the medium composition has an effect of promoting the proliferation of the cells. In this sense, medium composition 1 of the present invention can also be called a "medium composition for promoting the production of viruses carrying the target gene, regardless of whether or not the medium composition has an effect of promoting the proliferation of virus-producing cells into which a target gene has been introduced."

[0040] The present invention also provides use of a pyruvate dehydrogenase kinase inhibitor in the production of a medium composition for promoting virus production carrying a gene of interest during the cultivation of virus-producing cells into which the gene of interest has been introduced. The respective terms, etc., are as explained above.

[0041] 2. Medium Additive 1 The present invention also provides a medium additive for promoting virus production carrying a gene of interest during the cultivation of virus-producing cells into which the gene of interest has been introduced (hereinafter also referred to as "medium additive 1 of the present invention"), the medium additive comprising a pyruvate dehydrogenase kinase inhibitor.

[0042] The "pyruvate dehydrogenase kinase inhibitor," "target gene," "virus," "virus-producing cells," "culture," and "promotion of production of virus carrying a target gene," as well as the method for introducing a target gene into virus-producing cells, are as described above for medium composition 1 of the present invention.

[0043] The concentration of the PDH kinase inhibitor in medium additive 1 of the present invention is not particularly limited as long as the effects of the present invention are exhibited, but when medium additive 1 is added to a basal medium for use, the final concentration of the PDH kinase inhibitor in the basal medium is typically in the range of 0.1 mM to 1,000 mM, preferably 0.3 mM to 300 mM, more preferably 0.5 mM to 100 mM, and even more preferably 1 mM to 20 mM. For example, when medium additive 1 of the present invention is in the form of a solution, the concentration of the PDH kinase inhibitor in medium additive 1 is not particularly limited as long as the effects of the present invention are exhibited, but is typically 1 mM to 10,000 mM, preferably 3 mM to 3,000 mM, more preferably 10 mM to 2,000 mM, and even more preferably 20 mM to 1,000 mM.

[0044] The "basal medium" is as explained above for medium composition 1 of the present invention.

[0045] The medium additive 1 of the present invention may contain other components in addition to the PDH kinase inhibitor, as long as the effects of the present invention are not impaired. Examples of other components include nutrients, vitamins, minerals, amino acids, sugars, etc., which are typically added to culture media. The above-mentioned other components may be contained alone or in combination of two or more.

[0046] Furthermore, the medium additive 1 of the present invention may or may not contain β-alanine.

[0047] "β-alanine" is as explained above for medium composition 1 of the present invention.

[0048] The content of the above-mentioned other components in the culture medium additive 1 of the present invention is not particularly limited, but the total amount is preferably 0.000001 to 99.99% by weight, more preferably 0.00001 to 99.9% by weight, even more preferably 0.00001 to 99% by weight, even more preferably 0.0001 to 95% by weight, and particularly preferably 0.0001 to 90% by weight.

[0049] When the other component is β-alanine, β-alanine is contained in medium additive 1 so that the final concentration in the basal medium when medium additive 1 is added to the basal medium is typically 0.1 μM to 800 μM, preferably 1 μM to 700 μM, more preferably 3 μM to 600 μM, even more preferably 10 μM to 500 μM, and particularly preferably 30 μM to 400 μM. For example, when medium additive 1 of the present invention is in the form of a solution, the concentration of β-alanine in medium additive 1 is typically 1 μM to 500 mM, preferably 10 μM to 475 mM, more preferably 30 μM to 450 mM, even more preferably 100 μM to 425 mM, and particularly preferably 300 μM to 400 mM.

[0050] When medium additive 1 of the present invention is a PDH kinase inhibitor itself, it can be used as is. When it contains other components, etc., it can be produced by appropriately adding the other components, etc. to the PDH kinase inhibitor and mixing them as is, or by other production methods commonly used in the field of medium additive 1. The order in which these components are added is not particularly limited and can be appropriately determined depending on the purpose of use, etc. Furthermore, medium additive 1 of the present invention may be in the form of a solution, or may be obtained by freeze-drying the solution or other methods to form a solid powder.

[0051] Culture medium additive 1 of the present invention can usually be used by adding it to a basal medium. Furthermore, in cases where, for example, virus-producing mammalian cells are cultured in a medium that does not contain a pyruvate dehydrogenase kinase inhibitor to grow the cells to a certain extent, the cell number is adjusted as necessary, a gene of interest is introduced into the cells, and the cells are subsequently cultured in the same medium, culture medium additive 1 of the present invention can also be added to the medium before (e.g., several hours before), during, or after (e.g., several hours after) the gene of interest is introduced into the virus-producing mammalian cells. For example, when culture medium additive 1 of the present invention is in the form of a solution, 1 / 120 to 1 / 5, preferably 1 / 110 to 1 / 8, and more preferably 1 / 100 of the amount of culture medium additive 1 is added to a basal medium or the above-mentioned medium that does not contain a pyruvate dehydrogenase kinase inhibitor.

[0052] By culturing virus-producing cells into which a gene of interest has been introduced in a basal medium or a medium not containing a pyruvate dehydrogenase kinase inhibitor, medium additive 1 of the present invention can promote the production of viruses carrying the gene of interest, regardless of whether or not it has an effect of promoting the growth of the cells. In this sense, medium additive 1 of the present invention can also be called "a medium additive for promoting the production of viruses carrying the gene of interest, regardless of whether or not it has an effect of promoting the growth of virus-producing cells into which the gene of interest has been introduced."

[0053] 3. Method for promoting production of a virus carrying a gene of interest The present invention also provides a method for promoting production of a virus carrying a gene of interest (hereinafter also referred to as "promotion method 1 of the present invention"), which comprises the step of culturing virus-producing cells into which a gene of interest has been introduced, in a medium containing a pyruvate dehydrogenase kinase inhibitor.

[0054] The "pyruvate dehydrogenase kinase inhibitor," "target gene," "virus," "virus-producing cells," "culture," and "promotion of production of virus carrying a target gene," as well as the method for introducing a target gene into virus-producing cells, are as explained above for medium composition 1 of the present invention. Furthermore, "medium containing a pyruvate dehydrogenase kinase inhibitor" refers to a medium having the same composition as medium composition 1 of the present invention.

[0055] The cells into which the target gene has been introduced and which produce the virus can be cultured under the same conditions as those used to culture normal animal cells, such as 95% humidity and CO ２ An example of such a culture is at a concentration of 5% to 10% (v / v), but the culture is not limited to these conditions. Culture can be carried out at, for example, 30°C to 37°C, but may be carried out at a temperature outside the above range as long as the desired cell growth and virus production into which the target gene has been introduced are achieved. The culture period is not particularly limited, and is usually 1 hour to 14 days, preferably 5 hours to 7 days, more preferably 12 hours to 150 hours, and even more preferably 48 hours to 120 hours.

[0056] Furthermore, in the promotion method 1 of the present invention, the culture of virus-producing cells into which a gene of interest has been introduced can be carried out by any of the following methods: (1) Virus-producing cells are cultured in a medium containing a pyruvate dehydrogenase kinase inhibitor to grow the cells to a certain extent, and the cell number is adjusted if necessary, and then the gene of interest is introduced into the cells, and the cells are subsequently cultured in the same medium; (2) Virus-producing cells are cultured in a medium not containing a pyruvate dehydrogenase kinase inhibitor to grow the cells to a certain extent, and the cell number is adjusted if necessary, and the medium is replaced with a medium containing a pyruvate dehydrogenase kinase inhibitor, and then the gene of interest is introduced into the cells, and the cells are subsequently cultured in the same medium (the order of medium replacement and gene of interest introduction can be reversed); (3) Virus-producing cells are cultured in a medium not containing a pyruvate dehydrogenase kinase inhibitor to grow the cells to a certain extent, and the cell number is adjusted if necessary, and then the gene of interest is introduced into the cells, and the cells are subsequently cultured in the same medium. Here, a pyruvate dehydrogenase kinase inhibitor is added to the medium before (e.g., several hours before), during, or after (e.g., several hours after) the gene of interest is introduced into the virus-producing cells.

[0057] Promotion method 1 of the present invention can promote the production of a virus carrying a target gene, regardless of whether or not it has the effect of promoting the growth of virus-producing cells into which the target gene has been introduced.

[0058] 4. Method for Producing a Virus Carrying a Gene of Interest The present invention also provides a method for producing a virus carrying a gene of interest (hereinafter also referred to as "Production Method 1 of the present invention"), which comprises the step of culturing virus-producing cells into which a gene of interest has been introduced, in a medium containing a pyruvate dehydrogenase kinase inhibitor.

[0059] The "pyruvate dehydrogenase kinase inhibitor," "target gene," "virus," "virus-producing cells," and "culture," as well as the method for introducing the target gene into the virus-producing cells, are as explained above for medium composition 1 of the present invention, and the "culture" method is as explained above for promotion method 1 of the present invention. Furthermore, "medium containing a pyruvate dehydrogenase kinase inhibitor" refers to a medium having the same composition as medium composition 1 of the present invention.

[0060] In Production Method 1 of the present invention, virus-producing cells into which a gene of interest has been introduced are cultured in a medium containing a pyruvate dehydrogenase kinase inhibitor, and the virus carrying the gene of interest can then be purified by a method known per se. For example, the cultured cells are recovered from the resulting culture medium and disrupted, and the resulting cell lysate containing the virus is then subjected to appropriate steps such as filtration, ultracentrifugation, chromatography, and ultrafiltration, thereby purifying the virus carrying the gene of interest.

[0061] The manufacturing method 1 of the present invention can promote the production of a virus carrying a target gene, regardless of whether or not it has the effect of promoting the growth of virus-producing cells into which the target gene has been introduced.

[0062] 5. Medium Composition 2 The present invention also provides a medium composition for promoting production of a virus or virus-like particle during cultivation of cells that produce the virus or virus-like particle, the medium composition comprising a pyruvate dehydrogenase kinase inhibitor (hereinafter also referred to as "medium composition 2 of the present invention").

[0063] The "pyruvate dehydrogenase kinase inhibitor," "virus," and "culture" are as explained above for medium composition 1 of the present invention.

[0064] Virus-like particles are molecules that resemble viruses but do not contain genetic material and are not infectious.

[0065] The virus or virus-like particle is preferably a virus.

[0066] The cells that produce viruses or virus-like particles are the same as the "virus-producing cells" explained above for medium composition 1 of the present invention.

[0067] Medium composition 2 of the present invention may contain other components, serum and / or β-alanine in the same amounts as those described above for medium composition 1 of the present invention, as long as the effects of the present invention are not impaired.

[0068] The concentration of the pyruvate dehydrogenase kinase inhibitor in medium composition 2 of the present invention and the method of producing, form and method of using the medium composition are as explained above for medium composition 1 of the present invention.

[0069] By culturing cells that produce viruses or virus-like particles in medium composition 2 of the present invention, it is possible to promote the production of the viruses or virus-like particles without promoting the proliferation of the cells. In this sense, medium composition 2 of the present invention can also be called "a medium composition for promoting the production of viruses or virus-like particles, regardless of whether it has an effect of promoting the proliferation of cells that produce viruses or virus-like particles."

[0070] The present invention also provides use of a pyruvate dehydrogenase kinase inhibitor in the manufacture of a medium composition for promoting the production of a virus or virus-like particle during the cultivation of cells that produce the virus or virus-like particle.

[0071] 6. Medium Additive 2 The present invention also provides a medium additive for promoting the production of viruses or virus-like particles during the cultivation of cells that produce the viruses or virus-like particles, the medium additive comprising a pyruvate dehydrogenase kinase inhibitor (hereinafter also referred to as "medium additive 2 of the present invention").

[0072] The "pyruvate dehydrogenase kinase inhibitor," "virus," and "culture" are as explained above for medium composition 1 of the present invention, and the "virus-like particle" is as explained above for medium composition 2 of the present invention.

[0073] The virus or virus-like particle is preferably a virus.

[0074] The cells that produce viruses or virus-like particles are the same as the "virus-producing cells" explained above for medium composition 1 of the present invention.

[0075] Culture medium additive 2 of the present invention may contain components other than the PDH kinase inhibitor, as described above for culture medium additive 1 of the present invention, in the same amounts as described above for culture medium additive 1 of the present invention, as long as the effects of the present invention are not impaired.

[0076] The concentration of the PDH kinase inhibitor in medium additive 2 of the present invention and the method of producing, form and method of using the medium additive are as explained above for medium additive 1 of the present invention.

[0077] By adding medium additive 2 of the present invention to a basal medium and culturing cells that produce viruses or virus-like particles, it is possible to promote the production of the viruses or virus-like particles without promoting the proliferation of the cells. In this sense, medium additive 2 of the present invention can also be called "a medium additive for promoting the production of viruses or virus-like particles, regardless of whether it has an effect of promoting the proliferation of cells that produce viruses or virus-like particles."

[0078] The "basal medium" is as explained above for medium composition 1 of the present invention.

[0079] 7. Method for Promoting Virus or Virus-Like Particle Production The present invention also provides a method for promoting virus or virus-like particle production (hereinafter also referred to as "promoting method 2 of the present invention"), which comprises the step of culturing cells that produce the virus or virus-like particles in a medium containing a pyruvate dehydrogenase kinase inhibitor.

[0080] The "pyruvate dehydrogenase kinase inhibitor," "virus," and "culture" are as explained above for medium composition 1 of the present invention, and the "virus-like particle" is as explained above for medium composition 2 of the present invention. Furthermore, "medium containing a pyruvate dehydrogenase kinase inhibitor" refers to a medium having the same composition as medium composition 1 of the present invention.

[0081] The virus or virus-like particle is preferably a virus.

[0082] The cells that produce viruses or virus-like particles are the same as the "virus-producing cells" explained above for medium composition 1 of the present invention.

[0083] The cells that produce viruses or virus-like particles can be cultured under the same conditions as those used to culture ordinary animal cells, such as 95% humidity and CO ２ An example of such a culture is at a concentration of 5% to 10% (v / v), but the culture is not limited to these conditions. Culture can be carried out at, for example, 30°C to 37°C, but may be carried out at a temperature outside the above range as long as the desired cell growth and virus or virus-like particle production are achieved. The culture period is not particularly limited and is usually 1 hour to 14 days, preferably 5 hours to 7 days, more preferably 12 hours to 150 hours, and even more preferably 48 hours to 120 hours. Furthermore, a PDH kinase inhibitor may be added at any point during the culture period, and the culture may be continued.

[0084] Culturing method 2 of the present invention can promote the production of viruses or virus-like particles without promoting the proliferation of the viruses or virus-like particles.

[0085] 8. Method for Producing Virus or Virus-Like Particles The present invention also provides a method for producing the virus or virus-like particles (hereinafter also referred to as "Production Method 2 of the present invention"), which comprises the step of culturing cells that produce the virus or virus-like particles in a medium containing a pyruvate dehydrogenase kinase inhibitor.

[0086] The "pyruvate dehydrogenase kinase inhibitor," "virus," and "culture" are as explained above for medium composition 1 of the present invention, the "virus-like particle" is as explained above for medium composition 2 of the present invention, and the "culture" method is as explained above for promotion method 2 of the present invention. Furthermore, "medium containing a pyruvate dehydrogenase kinase inhibitor" means a medium having the same composition as medium composition 1 of the present invention.

[0087] The virus or virus-like particle is preferably a virus.

[0088] The cells that produce viruses or virus-like particles are the same as the "virus-producing cells" explained above for medium composition 1 of the present invention.

[0089] In Production Method 2 of the present invention, cells that produce viruses or virus-like particles are cultured in a medium containing a pyruvate dehydrogenase kinase inhibitor, and the viruses or virus-like particles can then be purified by a method known per se. For example, the cultured cells are recovered from the resulting culture medium and disrupted, and the resulting cell lysate containing the viruses or virus-like particles is then subjected to an appropriate step such as filter filtration, ultracentrifugation, chromatography, or ultrafiltration, thereby purifying the viruses or virus-like particles.

[0090] Production method 2 of the present invention can promote the production of viruses or virus-like particles without promoting the growth of cells that produce the viruses or virus-like particles.

[0091] Next, the present invention will be described in more detail with reference to examples, but the present invention is not limited to these examples in any way.

[0092] In the following Example 1, the ability of human embryonic kidney cell 293 (HEK293) to produce AAV using sodium dichloroacetate was evaluated. The reagents, cells, and media used were as follows: Sodium dichloroacetate: Sodium dichloroacetate (Fujifilm Wako Pure Chemical Industries, Ltd., 326-87772) HEK293: Viral Production Cells 2.0 (Thermo Fisher Scientific, A49784) HEK293 medium: Viral Production Medium (Thermo Fisher Scientific: A4817901)

[0093] Example 1: AAV production in HEK293 cells using a suspension culture system with the addition of sodium dichloroacetate Viral Production Cells 2.0 were seeded in 30 mL of Viral Production Medium (VIOLAMO, SEF125V) containing GlutaMAX™ Supplement (Thermo Fisher Scientific: 35050061) added to a final concentration of 4 mM. The cells were incubated at 37°C and 8% CO. ２ The cells were then cultured with agitation in an incubator set at 4°C for two passages. Subsequently, 0.6 x 10 cells were cultured in a medium prepared by adding GlutaMAX™ Supplement to 30 mL of Viral Production Medium to a final concentration of 4 mM, or in a medium prepared by adding GlutaMAX™ Supplement to 30 mL of Viral Production Medium to a final concentration of 4 mM and sodium dichloroacetate to a final concentration of 5 mM. ６ The cells were seeded at a cell density of 100 cells / mL and cultured with stirring for 3 days. On the third day, the number of viable cells was measured using a viable cell autoanalyzer, Vi-CELL BLU (manufactured by Becman). Then, when the number of viable cells was 3 x 10 ６The cells were diluted with fresh medium to a concentration of 1000 cells / mL, and 3 mL of the solution was seeded into a 6-well suspension culture plate (Sumitomo Bakelite Co., Ltd.: MS-8006R). Then, using a cationic lipid-based AAV-MAX Transfection Kit (Thermo Fisher Scientific: A50515), AAVpro® Packaging Plasmid (AAV8) (Takara Bio Inc.: 6681) and pAAV-ZsGreen1 Vector (Takara Bio Inc.: 6231) were transfected. After 3 days of culture, the cells were diluted with fresh medium to a concentration of 1000 cells / mL, and 3 mL of the solution was seeded into a 6-well suspension culture plate (Sumitomo Bakelite Co., Ltd.: MS-8006R). Then, using a cationic lipid-based AAV-MAX Transfection Kit (Thermo Fisher Scientific: A50515), the cells were transfected with AAVpro® Packaging Plasmid (AAV8) (Takara Bio Inc.: 6681) and pAAV-ZsGreen1 Vector (Takara Bio Inc.: 6231). After 3 days of culture, the cells were titrated using AAVpro® Titration Kit (for Real Time PCR) Ver. AAV8 vector was extracted using PCR kit No. 2 (Takara Bio Inc.: 6233), and the AAV8 genome titer was measured using a 7500 Fast Real-Time PCR System (Thermo Fisher Scientific: 4357362). Furthermore, the number of virus particles was measured using an AAV-8 Titration ELISA Kit (PROGEN Biotechnik: PRAAV8). From this, the full / empty capsid ratio (full capsid ratio) was calculated.

[0094] The effects of sodium dichloroacetate on HEK293 proliferation and AAV production were examined in series, and the results are shown in Figures 1 to 3. The viable cell density before dilution on day 3 after seeding is shown in Figure 1, the AAV8 genome titer (relative) is shown in Figure 2, and the full capsid ratio (relative) is shown in Figure 3. Although the addition of sodium dichloroacetate slightly inhibited cell proliferation, it was confirmed that the AAV8 genome titer and the full capsid ratio were improved.

[0095] Example 2: Adenovirus production in HEK293 cells using a suspension culture system with the addition of sodium dichloroacetate In the following example, the adenovirus production ability of human embryonic kidney cells 293 (HEK293) using sodium dichloroacetate can be evaluated. The reagents, cells, and media used are as follows: Sodium dichloroacetate: sodium dichloroacetate (Fujifilm Wako Pure Chemical Industries, Ltd., 326-87772) HEK293: suspension-type HEK293 2. sus (ATCC: CRL-1573.3) Adenovirus: Human adenovirus 32 (ATCC V R-625 ＴＭ ) HEK293 medium: 293 SFM II (Thermo Fisher Scientific), CDM4HEK293 (Cytiva)

[0096] (1) After thawing the frozen cells in an incubator set to approximately 37°C, 10 mL of glutamine-containing 293 SFM II was placed in a 15 mL centrifuge tube, and 1 mL of the thawed cell solution was added and suspended. (2) The cells were centrifuged (1000 rpm, 190 x g, 24°C) for 5 minutes using a centrifuge (AX-310, Tomy Seiko Co., Ltd.) and the supernatant was removed. (3) 1 mL of subculture medium was added to the 15 mL centrifuge tube and the cells were resuspended. The entire volume was transferred to a 50 mL tube, and 29 mL of glutamine-containing 293 SFM II was added and suspended. 5 mL of the cell suspension was placed in a petri dish and heated with CO ２ Incubator (Set temperature: 37°C, CO ２ (4) After culturing, collect the culture medium and transfer it to a 15 mL centrifuge tube. (5) Centrifuge for 5 minutes (1000 rpm, 190 × g, 24 °C) and remove the supernatant. (6) Add 1 mL of glutamine-containing 293 SFM II to the 15 mL centrifuge tube and resuspend. Count the cell number using a hemocytometer and count 3 × 10 cells. ５ Add glutamine-containing 293 SFM II to a concentration of 100 cells / mL. Add 5 mL of the adjusted cell suspension to a petri dish and incubate in CO ２ Cultivate in an incubator with shaking (50 rpm) for 3 to 4 days. (7) After culturing, repeat steps (4) to (6) once. (8) CO２ After culturing in an incubator for 3 to 4 days, collect the culture medium and transfer it to a 15 mL centrifuge tube. (9) Centrifuge for 5 minutes (1000 rpm, 190 × g, 24 °C) and remove the supernatant. (10) Add 1 mL of fresh culture medium to the 15 mL centrifuge tube and resuspend. Count the cell number using a hemocytometer and confirm that the number is 3 × 10 ５ Add 5 mL of the adjusted cell suspension to two dishes and incubate in CO ２ The cells were cultured in an incubator with shaking for 3 to 4 days. (11) After culturing, steps (8) to (10) were repeated three times. (12) After culturing for 3 days, the number of cells was counted (Figure 1) and 1 x 10 ５ The virus solution was thawed from frozen storage and diluted to 1 x 10 cells / mL with CDM4HEK293 with or without sodium dichloroacetate, and 5 mL was added to each petri dish. ５ For cells / mL, the virus solution is added to a petri dish so that the MOI is 0.1.

[0097] The addition of sodium dichloroacetate can be confirmed to increase the titer of adenovirus.

[0098] According to the present invention, when a virus carrying a gene of interest is produced using virus-producing cells, it is possible to improve the production amount of the virus carrying the gene of interest.

[0099] This application is based on patent application No. 2023-143946 filed in Japan (filing date: September 5, 2023), the contents of which are incorporated in their entirety herein.

Claims

1. A medium composition for promoting production of a virus carrying a target gene during the cultivation of a virus-producing cell into which the target gene has been introduced, the medium composition comprising a pyruvate dehydrogenase kinase inhibitor.

2. The medium composition according to claim 1, wherein the pyruvate dehydrogenase kinase inhibitor is dichloroacetic acid or a salt thereof.

3. The medium composition of claim 1, wherein the cells are selected from the group consisting of HEK293 cells, MDCK cells, Vero cells, A549 cells, PerC6 cells, HeLa cells, and insect cells.

4. The medium composition according to claim 1, wherein the cells are HEK293 cells.

5. The medium composition according to claim 1, wherein the virus-producing cells are adeno-associated virus-producing cells.

6. The medium composition according to any one of claims 1 to 5, wherein the concentration of the pyruvate dehydrogenase kinase inhibitor in the medium composition is 0.1 mM to 1,000 mM.

7. A medium additive for promoting virus production carrying a target gene during the cultivation of virus-producing cells into which the target gene has been introduced, the medium additive comprising a pyruvate dehydrogenase kinase inhibitor.

8. Use of a pyruvate dehydrogenase kinase inhibitor in the manufacture of a medium composition for promoting production of a virus carrying a target gene during the cultivation of a virus-producing cell into which the target gene has been introduced.

9. A method for promoting production of a virus carrying a target gene, comprising the step of culturing a virus-producing cell into which a target gene has been introduced in a medium containing a pyruvate dehydrogenase kinase inhibitor.

10. A method for producing a virus carrying a gene of interest, comprising the step of culturing virus-producing cells into which a gene of interest has been introduced in a medium containing a pyruvate dehydrogenase kinase inhibitor.

11. A medium composition for promoting production of a virus or virus-like particle during cultivation of a cell producing the virus or virus-like particle, comprising a pyruvate dehydrogenase kinase inhibitor.

12. The medium composition according to claim 11, wherein the pyruvate dehydrogenase kinase inhibitor is dichloroacetic acid or a salt thereof.

13. The medium composition of claim 11, wherein the cells are selected from the group consisting of HEK293 cells, MDCK cells, Vero cells, A549 cells, PerC6 cells, HeLa cells, and insect cells.

14. The medium composition of claim 11, wherein the cells are HEK293 cells.

15. The medium composition of claim 11, wherein the cells that produce viruses or virus-like particles are cells that produce adeno-associated viruses.

16. The medium composition according to any one of claims 11 to 15, wherein the concentration of the pyruvate dehydrogenase kinase inhibitor in the medium composition is 0.1 mM to 1,000 mM.

17. A medium additive for promoting production of a virus or virus-like particle during cultivation of a cell which produces the virus or virus-like particle, comprising a pyruvate dehydrogenase kinase inhibitor.

18. Use of a pyruvate dehydrogenase kinase inhibitor in the manufacture of a medium composition for promoting the production of a virus or virus-like particle during the cultivation of a cell that produces the virus or virus-like particle.

19. A method for promoting the production of a virus or virus-like particle, comprising the step of culturing a cell that produces the virus or virus-like particle in a medium containing a pyruvate dehydrogenase kinase inhibitor.

20. A method for producing the virus or virus-like particle, comprising the step of culturing a cell that produces the virus or virus-like particle in a medium containing a pyruvate dehydrogenase kinase inhibitor.

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