Culture medium composition

Abstract

The present invention provides: a culture medium composition that contains an aconitic acid decarboxylase 1 inhibitor and into which a gene of interest has been introduced, the culture medium being for, when culturing cells that produce a virus, promoting the production of a virus loaded with the abovementioned gene; a culture medium composition for culturing cells that produce a virus or virus-like particles; and the like.

Description

Medium composition

[0001] The present invention relates to a medium composition and the like for promoting production of a virus carrying a target gene when culturing virus-producing cells into which the target gene has been introduced, and for culturing cells that produce viruses or virus-like particles.

[0002] In order to meet the needs for human gene therapy, vaccine production, and the like, rapid and stable production technology for viruses and the like is indispensable. In particular, culture of cells that produce viruses or the like (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. Components contained in the medium affect cell growth and proliferation.

[0004] In addition, in the case of a medium for culturing virus-producing cells, components contained in the medium also affect the virus production ability within the cells. Furthermore, for example, when producing a virus using cells into which a gene encoding a cancer-suppressing protein has been introduced, components contained in the medium also affect the proportion of viruses carrying the gene among all produced viruses. However, conventionally known components have not necessarily yielded satisfactory results. For example, when producing adeno-associated virus using gene-introduced cells, the proportion of adeno-associated virus carrying the target gene among the produced adeno-associated virus is said to be about 5% to 30% (Non-Patent Document 1).

[0005] On the other hand, there have been proposed a medium composition for promoting production of a virus carrying a target gene when culturing virus-producing cells into which the target gene has been introduced, the medium composition comprising a pyruvate dehydrogenase kinase inhibitor (Patent Document 1), and a medium additive for promoting production of a virus carrying a target gene when culturing mammalian virus-producing cells into which the target gene has been introduced, the medium additive comprising β-alanine (Patent Document 2), and the like.

[0006] Furthermore, aconitate decarboxylase 1 is an enzyme that catalyzes the production of itaconic acid from cis-aconitate, and itaconic acid inhibits the conversion of succinate to fumarate in the TCA cycle (Non-Patent Literature 2). Therefore, aconitate decarboxylase 1 inhibitors suppress the inhibition of the conversion of succinate to fumarate in the TCA cycle. In addition, it has been reported that citraconic acid inhibits itaconic acid production by competitive inhibition by aconitate decarboxylase 1 in macrophage-like cells, thereby inhibiting the catalytic activity of itaconic acid (Non-Patent Literature 3).

[0007] International Publication No. 2025 / 053165, International Publication No. 2025 / 053166

[0008] Molecular Therapy Methods & Clinical Development; 2021 June 11; Vol. 21; p. 642-655Frontiers in Chemistry;2021 May 12;9:669308. doi:10.3389 / fchem. 2021.669308 Nature Metabolism; 2022 May; 4(5): p. 534-546

[0009] The present invention aims to provide a culture medium composition, etc., for improving the production volume of viruses carrying a target gene when producing viruses carrying a target gene using virus-producing cells, and for promoting the production of viruses or virus-like particles when producing viruses or virus-like particles using cells.

[0010] As a result of diligent research into the above-mentioned problems, the inventors discovered that when citraconic acid is added to human embryonic kidney cells 293 (HEK293), the ability to produce adeno-associated viruses carrying the target gene is improved. Based on this finding, further research led to the completion of the present invention. That is, the present invention is as follows.

[0011] [1] A culture medium composition for promoting the production of a virus carrying the target gene during the culture of virus-producing cells into which the target gene has been introduced, comprising an aconitate decarboxylase 1 inhibitor. [2] The culture medium composition according to [1], wherein the aconitate decarboxylase 1 inhibitor is citraconic acid or a salt thereof. [3] The culture medium composition according to [1] or [2], 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 culture medium composition according to [1] or [2], wherein the cells are HEK293 cells. [5] The culture medium composition according to any one of [1] to [4], wherein the virus-producing cells are cells that produce adeno-associated virus. [6] The culture medium composition according to any one of [1] to [5], wherein the concentration of the aconitate decarboxylase 1 inhibitor in the culture medium composition is 10 μM to 10 mM. [7] A culture medium additive for promoting the production of a virus carrying the target gene during the culture of virus-producing cells into which the target gene has been introduced, comprising an aconitate decarboxylase 1 inhibitor. [8] Use of an aconitate decarboxylase 1 inhibitor in the manufacture of a culture medium composition for promoting the production of a virus carrying the target gene for the culture of virus-producing cells into which the target gene has been introduced. [9] A method for promoting the production of a virus carrying the target gene, comprising the step of culturing virus-producing cells into which the target gene has been introduced in a culture medium containing an aconitate decarboxylase 1 inhibitor.

[10] A method for producing a virus carrying the target gene, comprising the step of culturing virus-producing cells into which the target gene has been introduced in a culture medium containing an aconitate decarboxylase 1 inhibitor.

[11] A method for producing a virus carrying the target gene, which promotes the production of a virus carrying the target gene, comprising the step of culturing virus-producing cells into which the target gene has been introduced in a culture medium containing an aconitate decarboxylase 1 inhibitor.

[0012]

[12] A culture medium composition for cells that produce viruses or virus-like particles, comprising an aconitate decarboxylase 1 inhibitor.

[13] The culture medium composition according to

[12] , wherein the aconitate decarboxylase 1 inhibitor is citraconic acid or a salt thereof.

[14] The culture medium composition according to

[12] or

[13] , 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.

[15] The culture medium composition according to

[12] or

[13] , wherein the cells are HEK293 cells.

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

[12] to

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

[17] A culture medium composition according to any one of

[12] to

[16] above, wherein the concentration of aconitate decarboxylase 1 inhibitor in the culture medium composition is 10 μM to 10 mM.

[18] A culture medium additive for culturing cells that produce viruses or virus-like particles, comprising an aconitate decarboxylase 1 inhibitor.

[19] Use of an aconitate decarboxylase 1 inhibitor in the manufacture of a culture medium composition for culturing cells that produce viruses or virus-like particles.

[20] A method for culturing cells that produce viruses or virus-like particles, comprising the step of culturing the cells in a culture medium containing an aconitate decarboxylase 1 inhibitor.

[21] A method for producing viruses or virus-like particles, comprising the step of culturing the cells that produce viruses or virus-like particles in a culture medium containing an aconitate decarboxylase 1 inhibitor.

[0013] The present invention makes it possible to improve the production volume of viruses carrying a target gene when producing viruses carrying the target gene using virus-producing cells.

[0014] Figure 1 shows the results of verifying the effect of citraconic acid on AAV8 genome titer in Example 1. The vertical axis represents the relative AAV8 genome titer, with the AAV8 genome titer obtained when AAV8 is produced using a medium without added citraconic acid set to 100%. The horizontal axis, "FreeStyle F17 Expression Medium," represents the case when AAV8 is produced using a medium without added citraconic acid, while "+0.1 mM citraconic acid," "+0.3 mM citraconic acid," and "+1 mM citraconic acid" represent the cases when AAV8 is produced using a medium to which citraconic acid was added to final concentrations of 0.1 mM, 0.3 mM, and 1 mM, respectively. Figure 2 shows the results of verifying the effect of citraconic acid on the number of AAV8 capsids (number of virus particles) in Example 1. The vertical axis represents the relative number of capsids, with the number of AAV8 capsids obtained when AAV8 is produced using a medium without added citraconic acid being set to 100%. The horizontal axis, "FreeStyle F17 Expression Medium," represents the case when AAV8 is produced using a medium without added citraconic acid, while "+0.1 mM citraconic acid," "+0.3 mM citraconic acid," and "+1 mM citraconic acid" represent the cases when AAV8 is produced using a medium to which citraconic acid is added to final concentrations of 0.1 mM, 0.3 mM, and 1 mM, respectively.

[0015] 1. Culture Medium Composition 1 The present invention provides a culture medium composition (hereinafter also referred to as "Culture Medium Composition 1 of the present invention") for promoting the production of a virus carrying a target gene during the culture of virus-producing cells into which a target gene has been introduced, and which contains an aconitate decarboxylase 1 inhibitor.

[0016] Aconitate decarboxylase 1 (hereinafter also referred to as "ACOD1") is an enzyme that catalyzes the following reaction, and the itaconic acid it produces inhibits the conversion of succinate to fumarate in the TCA cycle.

[0017]

[0018] Examples of ACOD1 inhibitors include citraconic acid or its salts.

[0019] Examples of salts of citraconic acid include metal salts, ammonium salts, organic amine addition salts, and amino acid addition salts.

[0020] 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, and zinc salts. Examples of ammonium salts include ammonium salts, diisopropylammonium salts, and tetramethylammonium salts. Examples of organic amine addition salts include morpholine and piperidine addition salts. Among these, sodium salts or potassium salts are preferred, with sodium salts being more preferred. Furthermore, as citraconic acid or its salts, free citraconic acid is more preferred.

[0021] When obtaining a salt of citraconic acid, if citraconic acid is obtained in salt form, it can be purified directly. If it is obtained in free form, citraconic acid can be dissolved or suspended in a suitable solvent, isolated and purified by adding a base.

[0022] Furthermore, citraconic acid or its salts may also exist in the form of solvates such as hydrates, but in this invention, these solvates are also referred to as citraconic acid.

[0023] The ACOD1 inhibitor used in the culture medium composition of the present invention may be synthesized by a method known to the present day, or it may be available as a commercially available product.

[0024] The concentration of the ACOD1 inhibitor in the culture medium composition of the present invention is not particularly limited as long as it achieves the effects of the present invention, but is usually 3 μM to 30 mM, preferably 5 μM to 20 mM, more preferably 10 μM to 10 mM, even more preferably 20 μM to 5 mM, and particularly preferably 50 μM to 2 mM.

[0025] In this specification, "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 is transcribed into an inhibitory polynucleotide (e.g., siRNA, miRNA, shRNA, etc.), and is preferably intended for the treatment of diseases in animals, including humans.

[0026] 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, with AAVs being 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 chimeras thereof, with AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAV8 and AAV9 being more preferred, and AAV2 and AAV8 being even more preferred.

[0027] The cells that produce the virus are not particularly limited as long as they can produce a virus carrying the desired target gene, and mammalian cells such as human, monkey, and rodent cells, as well as insect cells, can be used. Specifically, examples include mammalian cells such as 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), and BHK21 cells, and insect cells such as Sf9 cells. Preferably, HEK293 cells, MDCK cells, Vero cells, A549 cells, PerC6 cells, HeLa cells, and insect cells are used, all of which are commercially available. When the virus is an adenovirus or AAV, HEK293 cells, Vero cells, A549 cells, etc. are more preferably used, and HEK293 cells are even more preferably used.

[0028] The method for introducing the target gene into virus-producing cells is not particularly limited and can be carried out 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)), and calcium phosphate; 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 target gene can be prepared by methods well known to those skilled in the art, or it can be obtained as a commercially available product.

[0029] The culture may be either adherent culture or suspension culture, but suspension culture is preferred. In this specification, "adherent culture" means culturing cells by adhering them to a culture substrate, and specifically means a method of proliferation in which cells are adhered to the surface of the culture substrate and also adhere to each other. Examples of culture substrates include, but are not limited to, multi-well plates, culture dishes, petri dishes, culture flasks, microcarriers, and hollow fibers. The culture may be static culture on the substrate. In this specification, "suspension culture" means a cell culture method performed in a state in which cells do not adhere to the culture substrate. Suspension culture may or may not involve external pressure or vibration to the liquid medium, or shaking or rotation in the liquid medium.

[0030] The incubators used for culturing are not particularly limited as long as they are capable of culturing the target cells, but 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.

[0031] The culture vessel may be cell-adherent or non-cell-adherent, and the appropriate choice depends on the purpose. Cell-adherent culture vessels may be coated with any cell-supporting substrate, such as an extracellular matrix (ECM), to improve cell adhesion to the vessel surface. The cell-supporting substrate may be any substance intended for cell adhesion.

[0032] "Promoting the production of viruses carrying the target gene" refers to increasing the genomic titer of the target gene in the total viruses produced during the culture of virus-producing cells into which the target gene has been introduced. Specifically, this means (a) improving the full capsid ratio regardless of the increase or decrease in the number of capsids (number of virus particles), resulting in an overall increase in genomic titer, or (b) increasing the number of capsids regardless of the level of the full capsid ratio, resulting in an overall increase in genomic titer.

[0033] The culture medium composition 1 of the present invention may further contain other components that are favorable for virus production, as long as they do not impair the effects of the present invention. Other components include, for example, sugars such as glucose, fructose, sucrose, and maltose; amino acids such as glycine, L-arginine, L-glutamine, L-histidine, L-isoleucine, L-lysine, L-methionine, L-phenylalanine, L-serine, and L-tryptophan; proteins such as albumin and transferrin; peptides such as glycylglycylglycine and soy peptide; choline, vitamin A, B vitamins (thiamine, pyridoxine, cyanocobalamin, biotin, pantothenic acid, nicotinamide, folic acid, myo-inositol, etc.), vitamin C, and Examples of other components include vitamins such as vitamin E; fatty acids other than citraconic acid, such as oleic acid, arachidonic acid, and linoleic acid; lipids such as cholesterol; inorganic salts such as sodium chloride, potassium chloride, calcium chloride, and sodium dihydrogen phosphate; trace elements such as zinc, selenium, magnesium, and iron; buffering agents such as sodium bicarbonate, 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; cell adhesion factors and extracellular matrix components such as Type I collagen, Type II collagen, fibronectin, laminin, poly-L-lysine, and poly-D-lysine. Appropriate components can be selected and used depending on the type of cells to be cultured and the type of virus produced. The other components mentioned above may be included individually or in combination of two or more.

[0034] The other components may be included in the culture medium composition 1 of the present invention at a concentration of, for example, 0.001% to 99.9% by weight in total, preferably 0.01% to 99% by weight, and more preferably 0.1% to 95% by weight.

[0035] Furthermore, the culture medium composition 1 of the present invention may contain or be serum-free. The serum is not particularly limited as long as it is derived from an animal and does not inhibit cell proliferation, but is preferably derived from a mammal (e.g., fetal bovine serum, human serum, etc.), and more preferably human serum. The concentration of the serum may be within a known concentration range. The culture medium composition of the present invention is preferably serum-free.

[0036] The culture medium composition 1 of the present invention can be prepared, for example, by directly adding an ACOD1 inhibitor to a basal culture medium, and this embodiment is preferred because it is simple. Furthermore, if the ACOD1 inhibitor is in solution form in the form of the culture medium additive 1 of the present invention described later, the culture medium composition 1 of the present invention can be obtained by adding 1 / 10,000 to 1 / 5, preferably 1 / 5,000 to 1 / 8, and more preferably 1 / 1,000 of the culture medium additive 1 of the present invention to a basal culture medium.

[0037] The basal culture medium refers to a culture medium containing a carbon source, nitrogen source, and inorganic salts essential for cell culture, and is not particularly limited as long as it achieves the effects of the present invention; it can be appropriately selected depending on the cells to be cultured.

[0038] Examples of basal 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), and alpha-modified Eagle's Minimum Essential Medium. Examples include 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 mixed media of these mediums.The basal medium may be prepared by a method known to the present day, or by using Viral Production Medium (Thermo Fisher Scientific: A4817901), HyClone HyCell TransFx-H transmission media (Cytiva: SH30939.01), HyClone HyCell TransFx-H liquid medium (Cytiva: SH30939.02), or BalanCD(R). HEK293 (Fujifilm Wako Pure Chemical Corporation: 551-34231), EX-CELL® 293 (Merck: 14571C), FreeStyle® 293 Expression Medium (Thermo Fisher Scientific: 12338018), FreeStyle® F17 Expression Medium (Thermo Fisher Scientific: A1383501), CDM4HEK293 (cytiva: SH30858.02), Expi293® Expression Medium (Thermo Fisher This may be a commercially available product such as Scientific (A1435101), or a product currently under development.

[0039] If the culture medium composition 1 of the present invention contains serum and / or the other components mentioned above, the culture medium composition 1 of the present invention can be produced by appropriately adding the ACOD1 inhibitor and serum and / or the other components mentioned above to the basal medium and mixing them as is, or by other production methods common in the field of culture medium compositions. The order in which these components are added is not particularly limited and can be set as appropriate according to the intended use, etc.

[0040] The culture medium composition 1 of the present invention may be provided in liquid form, or it 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.

[0041] In the medium composition 1 of the present invention, culturing virus-producing cells into which a target gene has been introduced can promote the production of a virus carrying the target gene.

[0042] The present invention also provides use of an ACOD1 inhibitor in the production of a medium composition for promoting production of a virus carrying a target gene, for culturing virus-producing cells into which the target gene has been introduced. All terms and the like are as explained above.

[0043] 2. Medium additive 1 The present invention also provides a medium additive for promoting production of a virus carrying a target gene when culturing virus-producing cells into which the target gene has been introduced, which comprises an aconitate decarboxylase 1 inhibitor (hereinafter also referred to as "the medium additive 1 of the present invention").

[0044] The "aconitate decarboxylase 1 inhibitor", "target gene", "virus", "virus-producing cells", "culturing", "promotion of production of a virus carrying a target gene", and the method for introducing a target gene into virus-producing cells are as described above for the medium additive 1 of the present invention.

[0045] As used herein, the term "medium additive" is a concept that also includes feed medium, which is a medium supplemented to the medium for purposes such as nutrient supplementation at any time point after the start of culture.

[0046] The concentration of the ACOD1 inhibitor in the culture medium additive 1 of the present invention is not particularly limited as long as it achieves the effects of the present invention. However, the ACOD1 inhibitor is included 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 usually in the range of 3 μM to 30 mM, preferably 5 μM to 20 mM, more preferably 10 μM to 10 mM, even more preferably 20 μM to 5 mM, and particularly preferably 50 μM to 2 mM. For example, the concentration of the ACOD1 inhibitor in the culture medium additive 1 when the culture medium additive 1 of the present invention is in solution form is also not particularly limited as long as it achieves the effects of the present invention. However, it is usually in the range of 30 μM to 3,000 mM, preferably 50 μM to 2,000 mM, more preferably 100 μM to 1,000 mM, even more preferably 200 μM to 500 mM, and particularly preferably 500 μM to 200 mM.

[0047] The "basic culture medium" is as described above for culture medium composition 1 of the present invention.

[0048] The culture medium additive 1 of the present invention may contain other components that are favorable for virus production, as long as they do not impair the effects of the present invention. Examples of other components include nutrients, vitamins, minerals, amino acids, sugars, etc., which are normally added to culture media. The above-mentioned other components may be included individually or in combination of two or more.

[0049] 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.

[0050] If the culture medium additive 1 of the present invention is the ACOD1 inhibitor itself, it can be used as is. If it contains the other components mentioned above, it can be manufactured by adding the other components to the ACOD1 inhibitor as appropriate and mixing them together, or by other manufacturing methods common in the field of culture medium additives. The order in which these components are added is not particularly limited and can be set as appropriate according to the purpose of use. Furthermore, the culture medium additive 1 of the present invention may be in solution form, or it may be obtained as a solid powder by freeze-drying the solution or the like.

[0051] The culture medium additive 1 of the present invention can normally be used by adding it to a basal culture medium. Furthermore, the culture medium additive 1 of the present invention can also be used, for example, when culturing virus-producing cells in a medium that does not contain an ACOD1 inhibitor to increase the cells to a certain extent, adjusting the number of cells as necessary, introducing a target gene into the cells, and continuing to cultivate them in the same medium, by adding it to the medium before (for example, several hours before), during introduction, or after introduction (for example, several hours after) introducing the target gene into the virus-producing cells. For example, if the culture medium additive 1 of the present invention is a solution, it is used by adding 1 / 10,000 to 1 / 5, preferably 1 / 5,000 to 1 / 8, more preferably 1 / 1,000 to 1 / 10 of the culture medium additive 1 to a basal culture medium or a medium that does not contain an ACOD1 inhibitor. Furthermore, if the culture medium additive 1 of the present invention is a feed medium, it is used by replenishing the medium with 1 / 100 to 1 / 5, preferably 1 / 50 to 1 / 7, more preferably 1 / 20 to 1 / 10 of the culture medium additive 1 at any point after the start of cultivation.

[0052] By culturing virus-producing cells into which the target gene has been introduced in a culture medium in which the culture medium additive 1 of the present invention has been added to a basal culture medium or a culture medium that does not contain the above-mentioned ACOD1 inhibitor, the production of a virus carrying the target gene can be promoted.

[0053] 3. Method for promoting the production of a virus carrying a target gene The present invention also 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"), which includes the step of culturing virus-producing cells into which a target gene has been introduced in a culture medium containing an aconitate decarboxylase 1 inhibitor.

[0054] The terms "aconitate decarboxylase 1 inhibitor," "target gene," "virus," "virus-producing cells," "culture," and "promotion of virus production carrying the target gene," as well as the method for introducing the target gene into virus-producing cells, are as described above for Culture Medium Composition 1 of the present invention. Furthermore, "culture medium containing aconitate decarboxylase 1 inhibitor" means a culture medium having the same composition as Culture Medium Composition 1 of the present invention.

[0055] Cells that produce the virus into which the target gene has been introduced can be cultured under the same conditions as when culturing normal cells. For example, 95% humidity, CO2 ２ Culture at a concentration of 5% to 10% (v / v) is exemplified, but is not limited to these conditions. Culture can be carried out, for example, at 30°C to 37°C, but may be carried out at temperatures outside the above range as long as the proliferation of the desired cells and the production of the virus into which the target gene has been introduced can be achieved. The culture period is not particularly limited, but 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 acceleration method of the present invention, the culture of virus-producing cells into which the target gene has been introduced can be carried out by any of the following methods: (1) Culture the virus-producing cells in a medium containing an ACOD1 inhibitor to increase the number of cells to a certain extent, adjust the number of cells as necessary, introduce the target gene into the cells, and continue to culture them in the same medium. (2) Culture the virus-producing cells in a medium that does not contain an ACOD1 inhibitor to increase the number of cells to a certain extent, adjust the number of cells as necessary, then replace the medium with a medium containing an ACOD1 inhibitor, introduce the target gene into the cells, and continue to culture them in the same medium (the order of medium replacement and introduction of the target gene may be reversed). (3) Culture the virus-producing cells in a medium that does not contain an ACOD1 inhibitor to increase the number of cells to a certain extent, adjust the number of cells as necessary, introduce the target gene into the cells, and continue to culture them in the same medium. Here, the ACOD1 inhibitor is added to the culture medium before introducing the target gene into the virus-producing cells (for example, several hours before), during the introduction, or after the introduction (for example, several hours later).

[0057] 4. Method for producing a virus carrying a target gene The present invention also provides a method for producing a virus carrying a target gene (hereinafter also referred to as "method 1 of the present invention"), which includes the step of culturing virus-producing cells into which a target gene has been introduced in a culture medium containing an aconitate decarboxylase 1 inhibitor.

[0058] The terms "aconitate decarboxylase 1 inhibitor," "target gene," "virus," "virus-producing cells," and "culture," as well as the method for introducing the target gene into virus-producing cells, are as described above for Culture Medium Composition 1 of the present invention, and the method for "culture" is as described above for the acceleration method of the present invention. Furthermore, "culture medium containing aconitate decarboxylase 1 inhibitor" means a culture medium having the same composition as Culture Medium Composition 1 of the present invention.

[0059] In the production method 1 of the present invention, after culturing virus-producing cells into which the target gene has been introduced in a culture medium containing an aconitate decarboxylase 1 inhibitor, the virus carrying the target gene can be purified by a method known to the present day. For example, the cultured cells can be collected from the obtained culture medium, lysed, and then the resulting cell lysate containing the virus can be subjected to appropriate steps such as filter filtration, ultracentrifugation, chromatography, and ultrafiltration to purify the virus carrying the target gene.

[0060] The production method 1 of the present invention can promote the production of a virus carrying the target gene. That is, the present invention also provides a method for producing a virus carrying the target gene, which includes the step of culturing virus-producing cells into which the target gene has been introduced in a culture medium containing an aconitate decarboxylase 1 inhibitor, thereby promoting the production of a virus carrying the target gene.

[0061] 5. Culture Medium Composition 2 The present invention also provides a culture medium composition for culturing cells that produce viruses or virus-like particles, comprising an aconitate decarboxylase 1 inhibitor (hereinafter also referred to as "Culture Medium Composition 2 of the present invention").

[0062] The terms "aconitate decarboxylase 1 inhibitor," "virus," and "culture" are as described above for Culture Medium Composition 1 of the present invention.

[0063] Virus-like particles refer to molecules that resemble viruses but do not contain genetic material and are not infectious.

[0064] Viruses are preferred as the virus or virus-like particle.

[0065] 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.

[0066] The culture medium composition 2 of the present invention may contain serum and / or other components in the same amounts as described above for the culture medium composition 1 of the present invention, as long as the effects of the present invention are not impaired, unless otherwise specifically stated below.

[0067] The concentration of the aconitate decarboxylase 1 inhibitor in the culture medium composition 2 of the present invention, as well as the manufacturing method, form, and method of use of the culture medium composition 2, are as described above for the culture medium composition 1 of the present invention.

[0068] The present invention also provides the use of an aconitate decarboxylase 1 inhibitor in the preparation of culture medium compositions for cells that produce viruses or virus-like particles. The terms and conditions are as described above.

[0069] 6. Culture Medium Additive 2 The present invention also provides a culture medium additive for culturing viruses or virus-like particles of cells, comprising an aconitate decarboxylase 1 inhibitor (hereinafter also referred to as "Culture Medium Additive 2 of the present invention").

[0070] Regarding "aconitate decarboxylase 1 inhibitor," "virus," and "culture," they are as described above for Culture Medium Composition 1 of the present invention; regarding "culture medium additive," they are as described above for Culture Medium Additive 1 of the present invention; and regarding "virus-like particles," they are as described above for Culture Medium Composition 2 of the present invention.

[0071] Viruses are preferred as the virus or virus-like particle.

[0072] 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.

[0073] The culture medium additive 2 of the present invention may contain other components similar to those described above for the culture medium additive 1 of the present invention, in the same amounts as described above for the culture medium additive 1 of the present invention, as long as the effects of the present invention are not impaired.

[0074] The concentration of the aconitate decarboxylase 1 inhibitor in the culture medium additive 2 of the present invention, as well as the manufacturing method, form, and method of use of the culture medium additive 2, are as described above for the culture medium additive 1 of the present invention.

[0075] 7. Cell Culture Method The present invention also provides a cell culture method (hereinafter also referred to as "the cell culture method of the present invention") which includes the step of culturing cells that produce viruses or virus-like particles in a culture medium containing an aconitate decarboxylase 1 inhibitor.

[0076] The terms "aconitate decarboxylase 1 inhibitor," "virus," and "culture" are as described above for Culture Medium Composition 1 of the present invention, and the terms "virus-like particles" are as described above for Culture Medium Composition 2 of the present invention. Furthermore, "culture medium containing aconitate decarboxylase 1 inhibitor" means a culture medium having the same composition as Culture Medium Composition 1 of the present invention.

[0077] Viruses are preferred as the virus or virus-like particle.

[0078] 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.

[0079] Cells that produce viruses or virus-like particles can be cultured under the same conditions as those used for culturing normal cells. For example, 95% humidity, CO2 ２ Culture at a concentration of 5% to 10% (v / v) is exemplified, but is not limited to these conditions. Culture can be carried out, for example, at 30°C to 37°C, but may be carried out at temperatures outside this range as long as the desired cell proliferation and production of viruses or virus-like particles can be achieved. The culture period is not particularly limited, but 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, at any point during culture, an aconitate decarboxylase 1 inhibitor may be added and the culture may be continued.

[0080] 8. Method for producing viruses or virus-like particles The present invention also provides a method for producing viruses or virus-like particles (hereinafter also referred to as "method 2 of the present invention"), which includes the step of culturing cells that produce viruses or virus-like particles in a culture medium containing an aconitate decarboxylase 1 inhibitor.

[0081] Regarding "aconitate decarboxylase 1 inhibitor," "virus," and "culture," these are as described above for the culture medium additive 1 of the present invention; regarding "virus-like particles," these are as described above for the culture medium composition 2 of the present invention; and regarding the "culture" method, these are as described above for the culture method of the present invention. Furthermore, "culture medium containing aconitate decarboxylase 1 inhibitor" means a culture medium having the same composition as the culture medium composition 1 of the present invention.

[0082] Viruses are preferred as the virus or virus-like particle.

[0083] 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.

[0084] In the production method 2 of the present invention, after culturing cells that produce viruses or virus-like particles in a culture medium containing an aconitate decarboxylase 1 inhibitor, the viruses or virus-like particles can be purified by a method known to the present day. For example, the cultured cells can be collected from the resulting culture medium, lysated, and then the resulting cell lysate containing the viruses or virus-like particles can be subjected to appropriate steps such as filter filtration, ultracentrifugation, chromatography, or ultrafiltration to purify the viruses or virus-like particles.

[0085] Next, the present invention will be specifically described with reference to examples, but the present invention is not limited in any way by these examples.

[0086] In Example 1 below, the AAV production ability of human embryonic kidney cells 293 (HEK293) induced by citraconic acid was evaluated. The reagents, cells, and culture medium used were as follows: • Citraconic acid: Citraconic acid (Tokyo Chemical Industries, Ltd.: C0363) • HEK293: Viral Production Cells 2.0 (Thermo Fisher Scientific, A49784) • HEK293 culture medium: FreeStyle™ F17 Expression Medium (Thermo Fisher Scientific, A1383501)

[0087] Example 1: AAV production of HEK293 by citraconic acid addition using a suspension culture system. In a 125 mL Erlenmeyer flask (VIOLAMO, SEF125V), 30 mL of Viral Production Medium (Thermo Fisher Scientific: A4817901) was mixed with GlutaMAX™ Supplement (Thermo Fisher Scientific: 35050061) to a final concentration of 4 mM. Viral Production Cells 2.0 were seeded in this medium and cultured at 37°C and 8% CO2. ２ The cultures were stirred and cultured in an incubator set to 30 mL and passed through 3 passages. Subsequently, 0.3 × 10¹⁶ samples were collected in either 30 mL of FreeStyle® F17 Expression Medium with GlutaMAX® Supplement added to a final concentration of 4 mM, or 30 mL of FreeStyle® F17 Expression Medium with GlutaMAX® Supplement added to a final concentration of 4 mM and citraconic acid added to a final concentration of 0.1, 0.3, or 1 mM. ６ Cells were seeded at a cell density of cells / mL and cultured with agitation for 3 days. On the 3rd day, the number of viable cells was measured using a Vi-CELL BLU (Becman) autoanalyzer, and the number of viable cells was 2.8 ± 0.1 × 10⁶. ６After confirming the cells / mL ratio, 4 mL was seeded onto a 6-well suspension culture plate (Sumitomo Bakelite Co., Ltd.: MS-8006R). Subsequently, AAVpro® Packaging Plasmid (AAV8) (Takara Bio Inc.: 6681) and pAAV-ZsGreen1 Vector (Takara Bio Inc.: 6231) were introduced using a cationic lipid-based AAV-MAX Transfection Kit (Thermo Fisher Scientific: A50515). After 3 days of culture, AAVpro® Transfection Kit (for Real Time PCR) Ver. Using product 2 (Takara Bio Inc.: 6233), the AAV8 vector was extracted, and the AAV8 genome titer was measured using the QuantStudio® 3 real-time PCR system (Thermo Fisher Scientific: QS3-96S). The number of virus particles was measured using Simple Plex AAV8 Cartridge (protein simple Inc.: SPCKB-OT-007970).

[0088] Figures 1 and 2 show the results of a series of studies investigating the effect of citraconic acid on AAV production by HEK293. Figure 1 shows the relative AAV8 genome titer, and Figure 2 shows the relative AAV8 capsid number. The addition of citraconic acid resulted in an improvement in AAV8 genome titer and an increase in the AAV8 capsid number.

[0089] The present invention makes it possible to improve the production volume of viruses carrying a target gene when producing viruses carrying the target gene using virus-producing cells.

[0090] This application is based on Japanese Patent Application No. 2025-055084 (filing date: March 28, 2025), the contents of which are fully incorporated herein.

Claims

1. A culture medium composition for promoting the production of a virus carrying the target gene during the culture of virus-producing cells into which the target gene has been introduced, comprising an aconitate decarboxylase 1 inhibitor.

2. The culture medium composition according to claim 1, wherein the aconitate decarboxylase 1 inhibitor is citraconic acid or a salt thereof.

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

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

6. The culture medium composition according to any one of claims 1 to 5, wherein the concentration of the aconitate decarboxylase 1 inhibitor in the culture medium composition is 10 μM to 10 mM.

7. A culture medium additive containing an aconitate decarboxylase 1 inhibitor, for promoting the production of viruses carrying the target gene during the culture of virus-producing cells into which the target gene has been introduced.

8. Use of an aconitate decarboxylase 1 inhibitor in the preparation of a culture medium composition for promoting the production of a virus carrying the target gene, for the culture of virus-producing cells into which the target gene has been introduced.

9. A method for promoting the production of a virus carrying the target gene, comprising the step of culturing virus-producing cells into which the target gene has been introduced in a culture medium containing an aconitate decarboxylase 1 inhibitor.

10. A method for producing a virus carrying a target gene, comprising the step of culturing virus-producing cells into which the target gene has been introduced in a culture medium containing an aconitate decarboxylase 1 inhibitor.

11. A method for producing a virus carrying a target gene, comprising the step of culturing virus-producing cells into which the target gene has been introduced in a culture medium containing an aconitate decarboxylase 1 inhibitor, thereby promoting the production of a virus carrying the target gene.

12. A culture medium composition for cells that produce viruses or virus-like particles, comprising an aconitate decarboxylase 1 inhibitor.

13. The culture medium composition according to claim 12, wherein the aconitate decarboxylase 1 inhibitor is citraconic acid or a salt thereof.

14. The culture medium composition according to claim 12, 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.

15. The culture medium composition according to claim 12, wherein the cells are HEK293 cells.

16. The culture medium composition according to claim 12, wherein the cells that produce viruses or virus-like particles are cells that produce adeno-associated viruses.

17. The culture medium composition according to any one of claims 12 to 16, wherein the concentration of the aconitate decarboxylase 1 inhibitor in the culture medium composition is 10 μM to 10 mM.

18. A culture medium additive for cells that produce viruses or virus-like particles, containing an aconitate decarboxylase 1 inhibitor.

19. Use of aconitate decarboxylase 1 inhibitors in the preparation of culture medium compositions for cells that produce viruses or virus-like particles.

20. A method for culturing cells that produce viruses or virus-like particles in a culture medium containing an aconitate decarboxylase 1 inhibitor.

21. A method for producing a virus or virus-like particles, comprising the step of culturing cells that produce a virus or virus-like particles in a culture medium containing an aconitate decarboxylase 1 inhibitor.

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