Medium composition

Abstract

The present invention provides: a culture medium composition which contains a pyruvate dehydrogenase kinase inhibitor and is used for promoting the production of a virus having a target gene mounted thereon when culturing a virus-producing cell into which the target gene has been introduced; a culture medium composition for promoting the production of a virus or a virus-like particle when culturing a cell that produces the virus or the virus-like particle; and the like.

Description

Technical Field

[0001] The present invention relates to a culture medium composition comprising a pyruvate dehydrogenase kinase inhibitor and for promoting the production of a virus carrying the target gene during the culture of a virus-producing cell in which the target gene has been introduced, and 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. Background Technology

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

[0003] When designing / optimizing culture media for virus production cell culture, the composition of the media becomes an important issue. The components contained in the culture medium can affect cell growth and proliferation. For example, Patent Document 1 discloses improving the culture efficiency of animal cells by culturing them in a culture medium containing a pyruvate dehydrogenase kinase inhibitor.

[0004] Furthermore, in the case of culture media used for virus production cells, the components contained in the culture medium can also affect the virus production capacity within the cells. Moreover, for example, when using cells incorporating a gene that generates a cancer-inhibiting protein to produce the virus, the components contained in the culture medium can also affect the proportion of viruses carrying that gene among all the produced viruses. However, with the currently known components, unsatisfactory results have not been obtained. It has been reported, for example, that when using cells incorporating the gene to produce adeno-associated virus (AAV), the proportion of AAV carrying that gene among the AAV is approximately 5% to 30% (Non-Patent Literature 1).

[0005] Existing technical documents

[0006] Patent documents

[0007] Patent Document 1: International Publication No. 2002 / 04598

[0008] Non-patent literature

[0009] Non-patent literature 1: Molecular Therapy Methods & Clinical Development; 2021 June 11; Vol.21; p.642-655 Summary of the Invention

[0010] The problem that the invention aims to solve

[0011] The present invention aims to provide a culture medium composition for increasing the production of a virus carrying a target gene when using virus-producing cells to produce the virus, and a culture medium composition for promoting the production of the virus or virus-like particles when using cells to produce the virus or virus-like particles.

[0012] Methods for solving problems

[0013] The inventors conducted in-depth research on the above-mentioned issues and found that when sodium dichloroacetate, as a pyruvate dehydrogenase kinase inhibitor, was added to human embryonic kidney cells 293 (HEK293), the ability to produce adeno-associated viruses carrying the target gene was improved. Based on this insight, further research was conducted, and the present invention was completed.

[0014] That is, the present invention is as follows.

[0015] [1] A culture medium composition comprising a pyruvate dehydrogenase kinase inhibitor for promoting the production of a virus carrying the target gene during the culture of a virus-producing cell in which the target gene has been introduced.

[0016] [2] According to the culture medium composition described in [1] above, wherein the pyruvate dehydrogenase kinase inhibitor is dichloroacetic acid or a salt thereof.

[0017] [3] The culture medium composition according to [1] or [2] above, wherein the cells are selected from HEK293 cells, MDCK cells, Vero cells, A549 cells, PerC6 cells, HeLa cells and insect cells.

[0018] [4] The culture medium composition according to [1] or [2] above, wherein the cells are HEK293 cells.

[0019] [5] The culture medium composition according to any one of [1] to [4] above, wherein the cell that produces the virus is a cell that produces adeno-associated virus.

[0020] [6] The culture medium composition according to any one of [1] to [5] above, wherein the concentration of the pyruvate dehydrogenase kinase inhibitor in the culture medium composition is 0.1 mM to 1000 mM.

[0021] [7] A culture medium additive comprising a pyruvate dehydrogenase kinase inhibitor for promoting the production of a virus carrying the target gene during the culture of a virus-producing cell in which the target gene has been introduced.

[0022] [8] Use of pyruvate dehydrogenase kinase inhibitor in the manufacture of a culture medium composition for promoting the production of a virus carrying the target gene during the culture of a virus-producing cell in which the target gene has been introduced.

[0023] [9] A method for promoting the production of a virus carrying a target gene, the method comprising: culturing cells carrying the target gene in a culture medium containing a pyruvate dehydrogenase kinase inhibitor.

[0024]

[10] A method for manufacturing a virus carrying a target gene, the method comprising: culturing a virus-producing cell incorporating the target gene in a culture medium containing a pyruvate dehydrogenase kinase inhibitor.

[0025]

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

[0026]

[12] According to the culture medium composition described above

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

[0027]

[13] The culture medium composition according to

[11] or

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

[0028]

[14] The culture medium composition according to

[11] or

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

[0029]

[15] The culture medium composition according to any one of

[11] to

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

[0030]

[16] The culture medium composition according to any one of

[11] to

[15] above, wherein the concentration of the pyruvate dehydrogenase kinase inhibitor in the culture medium composition is 0.1 mM to 1000 mM.

[0031]

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

[0032]

[18] Use of pyruvate dehydrogenase kinase inhibitors in the manufacture of culture medium compositions for promoting the production of viruses or virus-like particles during the culture of cells that produce viruses or virus-like particles.

[0033]

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

[0034]

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

[0035] The effects of the invention

[0036] This invention enables the production of viruses carrying the target gene in the context of using virus production cells to generate viruses carrying the target gene, thereby increasing the production quantity of such viruses. Attached Figure Description

[0037] Figure 1 The results obtained in Example 1, verifying the effect of sodium dichloroacetate on the proliferation of HEK293, are shown. VCD refers to viable cell density, with the vertical axis representing viable cell density (×10). 6 Cells / mL). The horizontal axis “Viral Production Medium” indicates the use of medium without sodium dichloroacetate, and “Viral Production Medium + 5mM DCA” indicates the use of medium with sodium dichloroacetate added to achieve a final concentration of 5mM.

[0038] Figure 2 The results obtained in Example 1 verifying the effect of sodium dichloroacetate on AAV8 genome titer are shown. The vertical axis represents the relative AAV8 genome titer when AAV8 was generated using a medium without added sodium dichloroacetate, with the titer set to 100%. The horizontal axis, "Virus Production Medium," represents the case where AAV8 was generated using a medium without added sodium dichloroacetate, and "Virus Production Medium + 5 mM DCA" represents the case where AAV8 was generated using a medium with added sodium dichloroacetate at a final concentration of 5 mM.

[0039] Figure 3The results obtained in Example 1, verifying the effect of sodium dichloroacetate on the intact / empty capsid ratio (intact capsid ratio), are shown. The vertical axis represents the relative intact capsid ratio when AAV8 was generated using a medium without added sodium dichloroacetate, with the intact capsid ratio set to 100%. The horizontal axis, "Virus Production Medium," represents the case where AAV8 was generated using a medium without added sodium dichloroacetate, and "Virus Production Medium + 5 mM DCA" represents the case where AAV8 was generated using a medium with added sodium dichloroacetate at a final concentration of 5 mM. Detailed Implementation

[0040] 1. Culture medium composition 1

[0041] The present invention provides a culture medium composition (hereinafter also referred to as "culture medium composition 1 of the present invention") comprising a pyruvate dehydrogenase kinase inhibitor for promoting the production of a virus carrying the target gene during the culture of virus-producing cells in which the target gene has been introduced.

[0042] Pyruvate dehydrogenase (PDH) kinase is an enzyme that phosphorylates and inactivates PDH, and it is an enzyme that antagonizes PDH phosphatase in regulating its activity. PDH kinase inhibitors are organic molecules that prevent PDH kinase from phosphorylating specific serine residues on the E1 (α subunit) of the PDH complex involved in regulating PDH activity.

[0043] As PDH kinase inhibitors, various compounds are known, such as dichloroacetic acid or its salts, AZD7545, VER-246608, PDHK-IN-5, and leelamine, with dichloroacetic acid or its salts being preferred.

[0044] Salts of dichloroacetic acid include: metal salts, ammonium salts, and organic amine addition salts.

[0045] Examples of metal salts include: alkali metal salts such as sodium and potassium salts, alkaline earth metal salts such as magnesium and calcium salts, aluminum salts, and zinc salts. Examples of ammonium salts include: ammonium salts, diisopropylammonium salts, and tetramethylammonium salts. Examples of organic amine addition salts include: addition salts of morpholine and piperidine. Sodium or potassium salts are preferred, with sodium salts being more preferred.

[0046] When you want to obtain a salt of dichloroacetic acid, you can directly purify it when it is obtained in salt form. Alternatively, when it is obtained in free form, you can dissolve or suspend dichloroacetic acid in a suitable solvent and add acid or alkali for separation and purification.

[0047] In addition, dichloroacetic acid or its salts sometimes exist in the form of solvates such as hydrates, but in this invention, the term dichloroacetic acid or its salts will be used to refer to all such solvates.

[0048] The PDH kinase inhibitor used in the culture medium composition 1 of the present invention can be synthesized by methods known to the public or obtained in the form of commercially available products.

[0049] The concentration of the PDH kinase inhibitor in the culture medium composition 1 of the present invention is not particularly limited as long as it can exert the effect of the present invention. It is usually 0.1mM to 1000mM, preferably 0.3mM to 300mM, more preferably 0.5mM to 100mM, and even more preferably 1mM to 20mM.

[0050] In this specification, "target gene" refers to a heterologous polynucleotide that can be introduced into cells or organisms using a viral vector or the like. For example, it can be any polynucleotide such as a gene encoding a polypeptide or protein, or a polynucleotide that can be transcribed into a repressive polynucleotide (e.g., siRNA, miRNA, shRNA, etc.), preferably for the purpose of treating diseases in animals, including humans.

[0051] Examples of viruses include: adenoviruses, adeno-associated viruses (AAVs), retroviruses, lentiviruses, and Sendai viruses, as well as orthomyxoviruses, paramyxoviruses, reoviruses, parvoviruses, flaviviruses, arenaviruses, herpesviruses, and poxviruses, and their recombinant viruses, among which adenoviruses and AAVs are preferred, and AAVs are more preferred. Examples of AAVs include: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVDJ, AAVDJ8, AAV-B1, AAVM41, AAVrh10, AAVrh74, and their chimeras, among which AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAV8, and AAV9 are more preferred, and AAV2 and AAV8 are even more preferred.

[0052] The cells that produce the virus are not particularly limited as long as they can produce a virus carrying the desired target gene. Mammalian cells such as human, monkey, and rodent cells, as well as insect cells, can be used. 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, and insect cells. Preferred cell types include: HEK293 cells, MDCK cells, Vero cells, A549 cells, PerC6 cells, HeLa cells, and insect cells. All of these are commercially available. When the virus is adenovirus or AAV, HEK293 cells, Vero cells, and A549 cells are preferred, with HEK293 cells being the most preferred.

[0053] There are no particular limitations on the method for introducing the target gene into virus-producing cells, and gene introduction methods known to those skilled in the art can be used. 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 liposome transfection, electroporation, microinjection, acoustic perforation, and laser irradiation; and infection methods (biological methods) using viral vectors (e.g., adenovirus vectors, adeno-associated virus vectors, retrovirus vectors, lentivirus vectors, Sendai virus vectors), with chemical methods using transfection reagents being preferred, and chemical methods using cationic lipids or cationic polymers being more preferred. The target gene can also be prepared by methods known to those skilled in the art, but it can also be obtained in the form of commercially available products.

[0054] The culture can be either adherent culture or suspension culture, preferably suspension culture. In this specification, "adherent culture" refers to culturing cells by attaching them to a culture medium, specifically a method of allowing cells to adhere to the surface of the culture medium, adhere to each other, and proliferate. Examples of culture media include, but are not limited to, multi-well plates, culture dishes, culture flasks, microcarriers, hollow fibers, etc. The culture can be static culture on a substrate. In this specification, "suspension culture" refers to a cell culture method in which cells are in a non-adherent state relative to the culture container. Suspension culture may or may not be accompanied by external pressure, vibration, or shaking / rotation of the liquid culture medium.

[0055] The culture container used for culturing can be any container that can be used to culture the cells being cultured; there are no particular limitations. Examples include: flasks, tissue culture flasks, culture trays, petri dishes, tissue culture dishes, multi-well culture dishes, microplates, microplates, multi-well plates, multi-well plates, miniature slides, chamber slides, culture dishes, test tubes, trays, culture bags, roller flasks, bioreactors, etc.

[0056] Culture containers can be either cell-adherent or cell-non-adherent, and the appropriate choice can be made depending on the purpose. For cell-adherent culture containers, any cell support matrix, such as extracellular matrix (ECM), can be coated to improve the adhesion between the surface of the culture container and the cells. The cell support matrix can be any material intended for cell adhesion.

[0057] "Promoting the production of viruses carrying the target gene" refers to increasing the genomic titer of the target gene in all viruses produced during the culture of mammalian cells carrying the target gene. Specifically, it refers to a state where: (a) the proportion of intact capsids increases regardless of changes in the number of capsids (viruses), resulting in an overall increase in genomic titer; or (b) the number of capsids increases regardless of the proportion of intact capsids, resulting in an overall increase in genomic titer.

[0058] The culture medium composition 1 of the present invention may further contain other components suitable for virus production, provided that the effects of the present invention are not impaired. Examples of such other components include: sugars such as glucose, fructose, sucrose, and maltose; amino acids; proteins such as albumin and transferrin; peptides such as glycylglycylglycine and soybean peptides; 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); amphotericin B and kanamycin. Antibiotics such as gentamicin, streptomycin, and penicillin; cell adhesion factors and extracellular matrix components such as type I collagen, type II collagen, fibronectin, laminin, poly-L-lysine, and poly-D-lysine; cytokines and growth factors such as interleukins, 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; hormones such as dexamethasone, hydrocortisone, estradiol, progesterone, glucagon, and insulin. Appropriate components can be selected and used depending on the cells to be cultured and the type of virus to be produced. Other components may be included individually or in combination of two or more.

[0059] In the culture medium composition 1 of the present invention, for example, the other components may be contained in a concentration of 0.001% to 99.9% by weight, preferably 0.01% to 99% by weight, and more preferably 0.1% to 95% by weight, based on the total amount.

[0060] Furthermore, the culture medium composition 1 of the present invention may contain serum or be serum-free. As for the serum, it can be animal-derived serum, as long as it does not inhibit cell proliferation; there are no particular limitations. Mammal-derived serum (e.g., fetal bovine serum, human serum, etc.) is preferred, and fetal bovine serum is more preferred. The concentration of the serum is only required to be within a range known to the public. The culture medium composition 1 of the present invention is preferably serum-free.

[0061] In addition, the culture medium composition 1 of the present invention may further contain β-alanine, or may not contain it.

[0062] When the culture medium composition 1 of the present invention contains β-alanine, the concentration of β-alanine in the culture 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, further preferably 10 μM to 500 μM, and particularly preferably 30 μM to 400 μM.

[0063] The culture medium composition 1 of the present invention can be prepared, for example, by directly adding a PDH kinase inhibitor to a basal culture medium, and this method is preferred due to its simplicity. Furthermore, when the PDH kinase inhibitor is in solution form as described later in the culture medium additive 1 of the present invention, the culture 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, and more preferably 1 / 100 of the culture medium additive 1 of the present invention to a basal culture medium.

[0064] The basal culture medium refers to a culture medium containing carbon sources, nitrogen sources, and inorganic salts necessary for cell culture. It is not particularly limited as long as it can achieve the effects of this invention, and can be appropriately selected according to the cells being cultured.

[0065] Examples of basic culture media include: Dulbecco's Modified Eagle's Medium (DMEM), Ham's Nutrient Mixture F12, DMEM / F12, McCoy's 5A medium, Minimum Essential Medium (MEM), Eagle's Minimum Essential Medium (EMEM), alpha Modified Eagle's Minimum Essential Medium (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 mixtures of these media. The basal culture medium can be prepared by methods known to the public, or it can be a commercially available medium such as virus production medium (Thermo Fisher Scientific: A4817901), HyClone HyCell TransFx-H transfection medium (Cytiva: SH30939.01), BalanCD® HEK293 (Fujifilm Wako Pure Chemical Industries, Ltd.: 551-34231), EX-CELL (registered trademark) 293 (Merck: 14571C), FreeStyle (trademark) 293 Expression Medium (Thermo Fisher Scientific: 12338018), CDM4HEK293 (Cytiva: SH30858.02), Expi293 (trademark) expression medium (Thermo Fisher Scientific: A1435101), or a medium currently under development.

[0066] When the culture medium composition 1 of the present invention contains the other components described above, serum and / or β-alanine, it can be manufactured by directly mixing the PDH kinase inhibitor, the other components described above, serum and / or β-alanine appropriately with the above-mentioned basal culture medium, or by other conventional manufacturing methods in the field of culture medium compositions. There are no particular limitations on the order in which these components are added, etc., and they can be appropriately determined according to the intended use, etc.

[0067] The culture medium composition 1 of the present invention can be provided in a liquid state. Alternatively, it can be prepared in a solid state such as a powder obtained by freeze-drying, in a state that is more concentrated than the concentration at the time of use, and used by diluting it with a solvent such as water, or dissolving or dispersing it in a solvent such as water.

[0068] By culturing virus-producing cells incorporating the target gene in the culture medium composition 1 of the present invention, the production of the virus carrying the target gene can be promoted regardless of whether it promotes the proliferation of the cells. In this sense, the culture medium composition 1 of the present invention can also be considered as "a culture medium composition for promoting the production of virus carrying the target gene, regardless of whether it promotes the proliferation of virus-producing cells incorporating the target gene".

[0069] Furthermore, this invention provides the use of a pyruvate dehydrogenase kinase inhibitor in the manufacture of a culture medium composition for promoting the production of a virus carrying the target gene during the culture of virus-producing cells incorporating the target gene. Regarding the various terms, etc., as described above.

[0070] 2. Culture medium additive 1

[0071] In addition, the present invention provides a culture medium additive (hereinafter also referred to as "culture medium additive 1 of the present invention"), which contains a pyruvate dehydrogenase kinase inhibitor for promoting the production of a virus carrying the target gene during the culture of a virus-producing cell in which the target gene has been introduced.

[0072] Regarding "pyruvate dehydrogenase kinase inhibitor", "target gene", "virus", "virus-producing cell", "culture" and "promoting the production of virus carrying the target gene", and the method of introducing the target gene into virus-producing cell, as described above for the culture medium composition 1 of the present invention.

[0073] The concentration of the PDH kinase inhibitor in the culture medium additive 1 of the present invention is not particularly limited as long as it can achieve the effects of the present invention. Specifically, the PDH kinase inhibitor is included in the culture medium additive 1 such that when the culture medium additive 1 is added to the basal culture medium, the final concentration in the basal culture medium is typically in the range of 0.1 mM to 1000 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 the culture medium additive 1 of the present invention is in solution form, the concentration of the PDH kinase inhibitor in the culture medium additive 1 is not particularly limited as long as it can achieve the effects of the present invention. Typically, it is 1 mM to 10000 mM, preferably 3 mM to 3000 mM, more preferably 10 mM to 2000 mM, and even more preferably 20 mM to 1000 mM.

[0074] Regarding "basal culture medium", it is as described above in the description of culture medium composition 1 of the present invention.

[0075] The culture medium additive 1 of the present invention may contain components other than PDH kinase inhibitors, provided that the effects of the present invention are not impaired. Examples of such other components include nutrients, vitamins, minerals, amino acids, sugars, etc., which are commonly added to culture media. These other components may be present individually or in combination of two or more.

[0076] In addition, the culture medium additive 1 of the present invention may or may not contain β-alanine.

[0077] Regarding "β-alanine", it is as described above in the description of culture medium composition 1 of the present invention.

[0078] The content of the other components mentioned above in the culture medium additive 1 of the present invention is not particularly limited, but is preferably 0.000001 wt% to 99.99 wt% in total amount in the culture medium additive 1 of the present invention, more preferably 0.00001 wt% to 99.9 wt%, further preferably 0.00001 wt% to 99 wt%, even more preferably 0.0001 wt% to 95 wt%, and particularly preferably 0.0001 wt% to 90 wt%.

[0079] When other components are β-alanine, the β-alanine is contained in the culture medium additive 1 such that the final concentration in the basal medium when the culture 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, further preferably 10 μM to 500 μM, and particularly preferably 30 μM to 400 μM. For example, when the culture medium additive 1 of the present invention is in solution form, the concentration of β-alanine in the culture medium additive 1 is typically 1 μM to 500 mM, preferably 10 μM to 475 mM, more preferably 30 μM to 450 mM, further preferably 100 μM to 425 mM, and particularly preferably 300 μM to 400 mM.

[0080] Regarding the culture medium additive 1 of the present invention, if it is a PDH kinase inhibitor itself, it can be used directly. If it contains the other components mentioned above, it can be manufactured by appropriately adding the other components to the PDH kinase inhibitor and mixing them directly, or by other conventional manufacturing methods in the field of culture medium additive 1. There are no particular restrictions on the order in which these components are added, and they can be appropriately set according to the intended use. Furthermore, the culture medium additive 1 of the present invention can be formulated as a solution, or the solution can be freeze-dried or otherwise formulated into a solid powder.

[0081] The culture medium additive 1 of the present invention can generally be added to a basal culture medium for use. Furthermore, for example, when culturing virus-producing mammalian cells in a culture medium without pyruvate dehydrogenase kinase inhibitors to allow the cells to proliferate to a certain extent, adjusting the cell number as needed, then introducing the target gene into the cells and continuing to culture in the same medium, the culture medium additive 1 can be added to the aforementioned culture medium before (e.g., several hours before) introducing the target gene into the virus-producing mammalian cells, during the introduction, or after the introduction (e.g., several hours later). For example, when the culture medium additive 1 of the present invention is a solution, it can be added to a basal culture medium or the aforementioned culture medium without pyruvate dehydrogenase kinase inhibitors in an amount of 1 / 120 to 1 / 5, preferably 1 / 110 to 1 / 8, more preferably 1 / 100.

[0082] By culturing virus-producing cells carrying the target gene in a medium obtained by adding the culture medium additive 1 of the present invention to a basal medium or a medium that does not contain a pyruvate dehydrogenase kinase inhibitor, the production of the virus carrying the target gene is promoted regardless of whether it promotes the proliferation of the cells. In this sense, the culture medium additive 1 of the present invention can also be considered as "a culture medium additive for promoting the production of the virus carrying the target gene, regardless of whether it promotes the proliferation of virus-producing cells carrying the target gene."

[0083] 3. Methods to promote the production of viruses carrying the target gene

[0084] In addition, the present invention provides a method for promoting the production of a virus carrying a target gene (hereinafter also referred to as "the promotion method of the present invention"), the method comprising: culturing cells carrying the target gene and producing the virus in a culture medium containing a pyruvate dehydrogenase kinase inhibitor.

[0085] Regarding "pyruvate dehydrogenase kinase inhibitor," "target gene," "virus," "virus-producing cells," "culture," and "promotion of virus production carrying the target gene," as well as the method of introducing the target gene into virus-producing cells, as described above for the culture medium composition 1 of the present invention. Furthermore, "culture medium containing a pyruvate dehydrogenase kinase inhibitor" refers to a culture medium having the same composition as culture medium composition 1 of the present invention.

[0086] The culture of virus-producing cells infused with the target gene can be carried out under the usual culture conditions for culturing animal cells. For example, culture at 95% humidity and 5% to 10% (v / v) CO2 concentration can be used, but it is not limited to these conditions. Culture can also be carried out at, for example, 30°C to 37°C, but it can also be carried out at temperatures outside the range mentioned above, within which the desired cell proliferation and the production of the virus infused with the target gene can be achieved. The culture period is not particularly limited, but is typically 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.

[0087] In addition, the culture of virus-producing cells in which the target gene has been introduced in the promotion method 1 of the present invention can also be carried out by any of the following methods.

[0088] (1) The virus-producing cells are cultured in a medium containing a pyruvate dehydrogenase kinase inhibitor to allow the cells to proliferate to a certain extent, and the cell number is adjusted as needed. The target gene is then introduced into the cells and cultured in the medium.

[0089] (2) The virus-producing cells are cultured in a medium that does not contain pyruvate dehydrogenase kinase inhibitors to allow the cells to proliferate to a certain extent and the cell number is adjusted as needed. Then the medium is replaced with a medium containing pyruvate dehydrogenase kinase inhibitors. The target gene is then introduced into the cells and cultured in the medium again (the order of medium replacement and target gene introduction can also be reversed).

[0090] (3) The virus-producing cells are cultured in a medium that does not contain pyruvate dehydrogenase kinase inhibitor to allow the cells to proliferate to a certain extent, and the cell number is adjusted as needed. The target gene is then introduced into the cells, and the cells are cultured in the same medium. Here, the pyruvate dehydrogenase kinase inhibitor is added to the above-mentioned medium before (e.g., several hours before) the target gene is introduced into the virus-producing cells, during the introduction, or after the introduction (e.g., several hours later).

[0091] The promotion method 1 of the present invention can promote the production of viruses carrying the target gene, regardless of whether it promotes the proliferation of cells that have been introduced with the target gene.

[0092] 4. Methods for manufacturing viruses carrying the target gene

[0093] In addition, the present invention provides a method for manufacturing a virus carrying a target gene (hereinafter also referred to as "manufacturing method 1 of the present invention"), the method comprising: culturing a virus-producing cell incorporating the target gene in a culture medium containing a pyruvate dehydrogenase kinase inhibitor.

[0094] Regarding "pyruvate dehydrogenase kinase inhibitor," "target gene," "virus," "virus-producing cell," and "culture," as well as the method of introducing the target gene into the virus-producing cell, the description of the culture medium composition 1 of the present invention is as described above. Regarding the method of "culture," the description of the promotion method 1 of the present invention is as described above. Furthermore, "culture medium containing pyruvate dehydrogenase kinase inhibitor" refers to a culture medium having the same composition as the culture medium composition 1 of the present invention.

[0095] In manufacturing method 1 of the present invention, after culturing cells containing the target gene that produce the virus in a culture medium containing a pyruvate dehydrogenase kinase inhibitor, the virus carrying the target gene can be purified using methods known herein. For example, the virus carrying the target gene can be purified by recovering the cultured cells from the obtained culture medium and lysing them, and then subjecting the resulting cell lysate containing the virus to a process such as filtration, ultracentrifugation, chromatography, and ultrafiltration.

[0096] The manufacturing method 1 of the present invention can promote the production of viruses carrying the target gene, regardless of whether it promotes the proliferation of cells that have been introduced with the target gene.

[0097] 5. Culture medium composition 2

[0098] In addition, the present invention provides a culture medium composition (hereinafter also referred to as "culture medium composition 2 of the present invention") containing a pyruvate dehydrogenase kinase inhibitor for promoting the production of a virus or virus-like particle during the culture of cells that produce a virus or virus-like particle.

[0099] Regarding "pyruvate dehydrogenase kinase inhibitor", "virus" and "culture", as described above for the culture medium composition 1 of the present invention.

[0100] Virus-like particles are molecules that are similar to viruses but do not contain genetic material and are not infectious.

[0101] As a virus or virus-like particle, a virus is preferred.

[0102] The cells that produce viruses or virus-like particles are the same as the "virus-producing cells" described above for culture medium composition 1 of the present invention.

[0103] The culture medium composition 2 of the present invention may contain the same other components, serum and / or β-alanine as described above for the culture medium composition 1 of the present invention, as long as it does not impair the effects of the present invention.

[0104] Regarding the concentration of the pyruvate dehydrogenase kinase inhibitor in the culture medium composition 2 of the present invention, and the manufacturing method / form / usage method of the culture medium composition, as described above for the culture medium composition 1 of the present invention.

[0105] By culturing cells that produce viruses or virus-like particles in the culture medium composition 2 of the present invention, the production of viruses or virus-like particles can be promoted regardless of whether the proliferation of the cells is promoted. In this sense, the culture medium composition 2 of the present invention can also be considered as "a culture medium composition for promoting the production of viruses or virus-like particles that is independent of the proliferation-promoting effect of cells that produce viruses or virus-like particles".

[0106] Furthermore, this invention provides the use of a pyruvate dehydrogenase kinase inhibitor in the manufacture of a culture medium composition for promoting the production of a virus or virus-like particle during the culture of cells that produce such a virus or virus-like particle. Regarding the various terms, etc., as described above.

[0107] 6. Culture medium additive 2

[0108] In addition, the present invention provides a culture medium additive (hereinafter also referred to as "culture medium additive 2 of the present invention"), which contains a pyruvate dehydrogenase kinase inhibitor for promoting the production of a virus or virus-like particle during the culture of cells that produce a virus or virus-like particle.

[0109] Regarding "pyruvate dehydrogenase kinase inhibitor", "virus" and "culture", as described above in the description of culture medium composition 1 of the present invention, and regarding "virus-like particles", as described above in the description of culture medium composition 2 of the present invention.

[0110] As a virus or virus-like particle, a virus is preferred.

[0111] The cells that produce viruses or virus-like particles are the same as the "virus-producing cells" described above for culture medium composition 1 of the present invention.

[0112] The culture medium additive 2 of the present invention may contain the same components as described above for culture medium additive 1 of the present invention, except for the PDH kinase inhibitor, as long as it does not impair the effect of the present invention.

[0113] The concentration of the PDH kinase inhibitor in the culture medium additive 2 of the present invention, as well as the manufacturing method / form / usage method of the culture medium additive, are as described above in the description of the culture medium additive 1 of the present invention.

[0114] By adding the culture medium additive 2 of the present invention to the basal culture medium to culture cells that produce viruses or virus-like particles, the production of viruses or virus-like particles can be promoted regardless of whether it promotes the proliferation of the cells. In this sense, the culture medium additive 2 of the present invention can also be considered as "a culture medium additive used to promote the production of viruses or virus-like particles, regardless of whether it promotes the proliferation of cells that produce viruses or virus-like particles".

[0115] Regarding "basal culture medium", it is as described above in the description of culture medium composition 1 of the present invention.

[0116] 7. Methods to promote the production of viruses or virus-like particles

[0117] In addition, the present invention provides a method for promoting the production of a virus or virus-like particles (hereinafter also referred to as "promotion method 2 of the present invention"), the method comprising: a step of culturing cells that produce the virus or virus-like particles in a culture medium containing a pyruvate dehydrogenase kinase inhibitor.

[0118] Regarding "pyruvate dehydrogenase kinase inhibitor," "virus," and "culture," as described above for culture medium composition 1 of the present invention, and regarding "virus-like particles," as described above for culture medium composition 2 of the present invention, furthermore, "culture medium containing pyruvate dehydrogenase kinase inhibitor" refers to a culture medium having the same composition as culture medium composition 1 of the present invention.

[0119] As a virus or virus-like particle, a virus is preferred.

[0120] The cells that produce viruses or virus-like particles are the same as the "virus-producing cells" described above for culture medium composition 1 of the present invention.

[0121] The culture of cells that produce viruses or virus-like particles can be carried out under the usual culture conditions for culturing animal cells. For example, culture at 95% humidity and 5% to 10% (v / v) CO2 concentration can be exemplified, but such conditions are not limited. Culture can be carried out at, for example, 30°C to 37°C, but can also be carried out at temperatures outside the ranges described above, within which the desired cell proliferation and the production of viruses or virus-like particles can be achieved. The culture period is not particularly limited, typically 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 can be added at any point during culture to continue the culture.

[0122] The culture method 2 of the present invention can promote the production of the virus or virus-like particles, regardless of whether it promotes the proliferation of the virus or virus-like particles.

[0123] 8. Methods for manufacturing viruses or virus-like particles

[0124] In addition, the present invention provides a method for manufacturing a virus or virus-like particle (hereinafter also referred to as "manufacturing method 2 of the present invention"), the method comprising: culturing cells that produce the virus or virus-like particle in a culture medium containing a pyruvate dehydrogenase kinase inhibitor.

[0125] Regarding "pyruvate dehydrogenase kinase inhibitor," "virus," and "culture," as described above in the description of culture medium composition 1 of the present invention; regarding "virus-like particles," as described above in the description of culture medium composition 2 of the present invention; and regarding the method of "culture," as described above in the description of promotion method 2 of the present invention. Furthermore, "culture medium containing pyruvate dehydrogenase kinase inhibitor" refers to a culture medium having the same composition as culture medium composition 1 of the present invention.

[0126] As a virus or virus-like particle, a virus is preferred.

[0127] The cells that produce viruses or virus-like particles are the same as the "virus-producing cells" described above for culture medium composition 1 of the present invention.

[0128] In manufacturing method 2 of the present invention, after culturing cells that produce viruses or virus-like particles in a culture medium containing a pyruvate dehydrogenase kinase inhibitor, the viruses or virus-like particles can be purified using methods known herein. For example, the virus or virus-like particles can be purified by recovering and lysing the cultured cells from the obtained culture medium, and then subjecting the resulting cell lysate containing viruses or virus-like particles to a process such as filtration, ultracentrifugation, chromatography, or ultrafiltration.

[0129] The manufacturing method 2 of the present invention can promote the production of the virus or virus-like particles regardless of whether it promotes the proliferation of cells that produce the virus or virus-like particles.

[0130] The present invention will now be specifically described through examples, but the present invention is not limited by these examples.

[0131] Example

[0132] In Example 1 below, the AAV production capacity of human embryonic kidney cells 293 (HEK293) based on sodium dichloroacetate was evaluated. The reagents, cells, and culture media used are described below.

[0133] ·Sodium dichloroacetate: Sodium dichloroacetate (Fujifilm Wako Pure Chemical Industries, Ltd., 326-87772)

[0134] HEK293: Virus-Producing Cells 2.0 (Thermo Fisher Scientific, A49784)

[0135] • HEK293 culture medium: Virus production medium (Thermo Fisher Scientific: A4817901)

[0136] Example 1: AAV generation based on HEK293 with added sodium dichloroacetate in a suspension culture system

[0137] Using a 125 mL Erlenmeyer flask (VIOLAMO, SEF125V), GlutaMAX (trademark) supplement (Thermo Fisher Scientific: 35050061) was added to 30 mL of virus production medium to achieve a final concentration of 4 mM. Two cells of virus production were then inoculated into the resulting medium and cultured with stirring in an incubator set at 37°C and 8% CO2. Two passages were performed. Then, the cells were cultured in the following medium at a concentration of 0.6 × 10⁻⁶. 6 Cells were seeded at a density of [number] cells / mL and cultured with stirring for 3 days. The culture medium was either GlutaMAX (trademark) supplement added to 30 mL of virus production medium to achieve a final concentration of 4 mM, or GlutaMAX (trademark) supplement added to 30 mL of virus production medium to achieve a final concentration of 4 mM and sodium dichloroacetate added to 30 mL of virus production medium to achieve a final concentration of 5 mM. On day 3, the viable cell count was determined using a Vi-CELL BLU (Becman) automated viable cell analyzer. Then, to achieve a viable cell count of 3 × 10⁶ cells / mL... 6 Diluted in fresh culture medium at cell / mL ratios, 3 mL was inoculated into 6-well suspension plates (Sumitomo Bakelite: MS-8006R). Gene transfer was then performed using the AAV-MAX transfection kit (Thermo Fisher Scientific: A50515) with a cationic lipid matrix to the AAVpro (trademark) packaging plasmid (AAV8) (Takara Bio: 6681) and the pAAV-ZsGreen1 vector (Takara Bio: 6231). After 3 days of culture, the AAV8 vector was extracted using the AAVpro (trademark) titration kit (for RealTime PCR) Ver.2 (Takara Bio: 6233), and the AAV8 genome titer was determined using a 7500 Fast real-time PCR system (Thermo Fisher Scientific: 4357362). Viral particle counts were also determined using the AAV-8 titer ELISA kit (PROGEN Biotechnik: PRAAV8). This led to the calculation of the intact / empty capsid ratio (intact capsid ratio).

[0138] The effects of sodium dichloroacetate on the proliferation of HEK293 and AAV production were verified through a series of experiments, and the results are shown below. Figures 1-3 The pre-dilution viable cell density on day 3 after inoculation is shown in [the figure]. Figure 1The AAV8 genome titer (relative) is shown in... Figure 2 And show the complete capsid ratio (relative) in Figure 3 It was confirmed that the addition of sodium dichloroacetate, although slightly inhibiting cell proliferation, increased AAV8 genome titer and the proportion of intact capsids.

[0139] Example 2: Adenovirus production based on sodium dichloroacetate supplemented with HEK293 in a suspension culture system

[0140] In the following examples, the adenovirus production capacity of human embryonic kidney cells 293 (HEK293) based on sodium dichloroacetate can be evaluated. The reagents, cells, and culture media used are described below.

[0141] ·Sodium dichloroacetate: Sodium dichloroacetate (Fujifilm Wako Pure Chemical Industries, Ltd., 326-87772)

[0142] HEK293: Suspended HEK293 2.sus (ATCC: CRL-1573.3)

[0143] • Adenovirus: Human adenovirus 32 (ATCC V R-625) TM )

[0144] • HEK293 culture medium: 293 SFM II (Thermo Fisher Scientific), CDM4HEK293 (Cytiva)

[0145] (1) After thawing the frozen cells in a constant temperature bath set to about 37°C, add 10 mL of 293 SFM II containing glutamine to a 15 mL centrifuge tube and add 1 mL of the thawed cell solution to suspend them.

[0146] (2) The supernatant was removed by centrifugation for 5 minutes (1000 rpm, 190 × g, 24 °C) using a centrifuge (AX-310, TOMY SEIKO).

[0147] (3) Add 1 mL of subculture medium to a 15 mL centrifuge tube for resuscitation, then add the entire volume to a 50 mL test tube, add 29 mL of 293 SFM II containing glutamine, and suspend the cells. Add 5 mL of the cell suspension to a culture dish and incubate with a CO2 incubator (temperature set: 37℃, CO2 concentration: 5%, model: MCO-170AICUV-PJ, Panasonic Healthcare) for 2-3 days with shaking (50 rpm).

[0148] (4) After incubation, the culture medium is recovered and transferred to a 15mL centrifuge tube.

[0149] (5) Centrifuge for 5 minutes (1000 rpm, 190 × g, 24 ° C) to remove the supernatant.

[0150] (6) Add 1 mL of 293 SFM II containing glutamine to a 15 mL centrifuge tube for resuscitation. Measure the cell count using a hemocytometer to ensure a count of 3 × 10⁻⁶ cells / mL. 5 293 SFM II containing glutamine was added at a rate of cells / mL. 5 mL of the adjusted cell suspension was added to a culture dish, and the cells were incubated in a CO2 incubator with shaking (50 rpm) for 3–4 days.

[0151] (7) After culturing, repeat (4) to (6) once.

[0152] (8) After culturing in a CO2 incubator for 3 to 4 days, the culture medium was recovered and transferred to a 15 mL centrifuge tube.

[0153] (9) Centrifuge for 5 minutes (1000 rpm, 190 × g, 24 ° C) to remove the supernatant.

[0154] (10) Resuspend the cells in a 15mL centrifuge tube by adding 1mL of fresh culture medium. Measure the cell count using a hemocytometer to ensure a count of 3×10⁻⁶ cells / mL. 5 Add the culture medium at a rate of cells / mL. Add 5 mL of the adjusted cell suspension to two culture dishes and incubate with shaking in a CO2 incubator for 3-4 days.

[0155] (11) After culturing, repeat (8)~(10) three times.

[0156] (12) After 3 days of culture, the cell number was measured. Figure 1 Adjusted to 1×10 using CDM4HEK293 with or without sodium dichloroacetate. 5 Add 5 mL of virus solution per culture dish, at a concentration of cells / mL. Thaw the frozen virus solution and add it to a concentration of 1 × 10⁻⁶ cells / mL. 5 Virus solution was added to the culture dish in a manner that achieved an MOI of 0.1 per cell / mL.

[0157] It can be confirmed that the adenovirus titer increases with the addition of sodium dichloroacetate.

[0158] Industrial applicability

[0159] This invention enables the production of viruses carrying target genes by using virus-producing cells to generate viruses carrying target genes, thereby increasing the production quantity of such viruses.

[0160] This application is based on Japanese Special Application 2023-143946 (application date: September 5, 2023), the entire contents of which are contained in this specification.

Claims

1. A culture medium composition comprising a pyruvate dehydrogenase kinase inhibitor for use in promoting production of a virus carrying a target gene when a virus-producing cell into which the target gene is introduced is cultured.

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

3. The culture medium composition according to claim 1, wherein 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. The culture medium composition according to claim 1, wherein the cell is a HEK293 cell.

5. The culture medium composition according to claim 1, wherein the virus-producing cell is an adeno-associated virus-producing cell.

6. The culture medium composition according to any one of claims 1 to 5, wherein the concentration of the pyruvate dehydrogenase kinase inhibitor in the culture medium composition is from 0.1 mM to 1000 mM.

7. A culture medium additive comprising a pyruvate dehydrogenase kinase inhibitor for use in promoting production of a virus carrying a target gene when a virus-producing cell into which the target gene is introduced is cultured.

8. Use of a pyruvate dehydrogenase kinase inhibitor in the production of a culture medium composition for use in promoting production of a virus carrying a target gene when a virus-producing cell into which the target gene is introduced is cultured.

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

10. A method for producing a virus carrying a target gene, the method comprising: a step of culturing a virus-producing cell into which the target gene is introduced in a culture medium comprising a pyruvate dehydrogenase kinase inhibitor.

11. A culture medium composition comprising a pyruvate dehydrogenase kinase inhibitor for use in promoting production of a virus or a virus-like particle when a virus- or virus-like particle-producing cell is cultured.

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

13. The culture medium composition according to claim 11, wherein 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. The culture medium composition according to claim 11, wherein the cell is a HEK293 cell.

15. The culture medium composition according to claim 11, wherein the virus- or virus-like particle-producing cell is an adeno-associated virus-producing cell.

16. The culture medium composition according to any one of claims 11 to 15, wherein the concentration of the pyruvate dehydrogenase kinase inhibitor in the culture medium composition is from 0.1 mM to 1000 mM. ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ 17. A culture medium additive comprising a pyruvate dehydrogenase kinase inhibitor for use in promoting production of a virus or virus-like particle in culture of cells producing the virus or virus-like particle.

18. Use of a pyruvate dehydrogenase kinase inhibitor in the manufacture of a culture medium composition for promoting production of a virus or virus-like particle in culture of cells producing the virus or virus-like particle.

19. A method of promoting production of a virus or virus-like particle, the method comprising: a step of culturing cells producing the virus or virus-like particle in a culture medium comprising a pyruvate dehydrogenase kinase inhibitor.

20. A method of manufacturing a virus or virus-like particle, the method comprising: a step of culturing cells producing the virus or virus-like particle in a culture medium comprising a pyruvate dehydrogenase kinase inhibitor.

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