Medium composition

Abstract

The present invention provides, for example, a medium composition for promoting the production of a virus carrying a target gene during culturing of a virus-producing cell into which the target gene has been introduced, the medium composition comprising one or more selected from the group consisting of guanosine or a salt thereof, hypoxanthine or a salt thereof, cytidine or a salt thereof, and uridine or a salt thereof.

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, during culture of virus-producing cells into which the target gene has been introduced.

[0002] Rapid and stable production technologies for viruses and the like are indispensable to meet the needs for human gene therapy, vaccine production, and the like. 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 virus-producing cell culture, the components of the medium are an important issue. The components contained in the medium affect the growth and proliferation of cells.

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

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

[0006] International Publication No. WO 2025 / 053165 International Publication No. WO 2025 / 053166

[0007] Molecular Therapy Methods & Clinical Development; 2021 June 11; Vol. 21; p. 642-655

[0008] 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 the target gene using virus-producing cells.

[0009] As a result of diligent research into the above-mentioned problems, the inventors discovered that when guanosine, hypoxanthine, cytidine, or uridine 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.

[0010] In other words, the present invention is as follows:

[0011] [1] A culture medium composition for promoting the production of a virus carrying a target gene during the culture of a virus-producing cell into which a target gene has been introduced, comprising one or more selected from the group consisting of guanosine or a salt thereof, hypoxanthine or a salt thereof, cytidine or a salt thereof, and uridine or a salt thereof. [2] The culture medium composition according to [1], wherein one or more selected from the group consisting of guanosine or a salt thereof, hypoxanthine or a salt thereof, cytidine or a salt thereof, and uridine or a salt thereof is guanosine or a salt thereof. [3] The culture medium composition according to [1] or [2], wherein the cells are cells 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] above, wherein the concentrations of guanosine or a salt thereof, hypoxanthine or a salt thereof, cytidine or a salt thereof, and uridine or a salt thereof in the culture medium composition are 3 μM to 150 μM, 1 μM to 500 μM, 3 μM to 500 μM, and 20 μM to 500 μM, respectively.

[0012] [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 one or more selected from the group consisting of guanosine or a salt thereof, hypoxanthine or a salt thereof, cytidine or a salt thereof, and uridine or a salt thereof. [8] The use of one or more selected from the group consisting of guanosine or a salt thereof, hypoxanthine or a salt thereof, cytidine or a salt thereof, and uridine or a salt thereof, 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 comprising one or more selected from the group consisting of guanosine or a salt thereof, hypoxanthine or a salt thereof, cytidine or a salt thereof, and uridine or a salt thereof.

[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 one or more selected from the group consisting of guanosine or a salt thereof, hypoxanthine or a salt thereof, cytidine or a salt thereof, and uridine or a salt thereof.

[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 one or more selected from the group consisting of guanosine or a salt thereof, hypoxanthine or a salt thereof, cytidine or a salt thereof, and uridine or a salt thereof, thereby promoting the production of a virus carrying a target gene.

[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 investigating the effect of guanosine, hypoxanthine, cytidine, or uridine 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 the addition of guanosine, hypoxanthine, cytidine, or uridine set to 100%. The numbers on the horizontal axis represent the results when AAV8 is produced using a medium to which guanosine, hypoxanthine, cytidine, or uridine is added so that the final concentration is the respective value (unit: μM). Figure 2 shows the results of investigating the effect of guanosine, hypoxanthine, cytidine, or uridine on the full / empty capsid ratio (full capsid ratio) in Example 1. The vertical axis represents the relative full capsid ratio, with the full capsid ratio obtained when AAV8 is produced using a medium without any of guanosine, hypoxanthine, cytidine, or uridine being set to 100%. The numbers on the horizontal axis represent the results when AAV8 is produced using a medium with guanosine, hypoxanthine, cytidine, or uridine added to achieve the respective final concentrations (in μM).

[0015] 1. Culture Medium Composition The present invention provides a culture medium composition for promoting the production of viruses carrying a target gene during the culture of virus-producing cells into which a target gene has been introduced (hereinafter also referred to as "the culture medium composition of the present invention"). This composition comprises one or more selected from the group consisting of guanosine or a salt thereof, hypoxanthine or a salt thereof, cytidine or a salt thereof, and uridine or a salt thereof.

[0016] Examples of salts of guanosine, hypoxanthine, and cytidine include acid addition salts and metal salts, respectively.

[0017] Examples of acid addition salts include inorganic acid salts such as hydrochloride, sulfate, hydrobromide, nitrate, and phosphate, and organic acid salts such as acetate, mesylate, succinate, maleate, fumarate, citrate, and tartrate. Examples of metal salts include alkali metal salts such as sodium salt and potassium salt, alkaline earth metal salts such as magnesium salt and calcium salt, aluminum salt, and zinc salt. Among these, sodium salt is preferred. Furthermore, free forms of guanosine or its salts, hypoxanthine or its salts, and cytidine or its salts are more preferred.

[0018] When obtaining salts of guanosine, hypoxanthine, or cytidine, if the salts are obtained, they can be purified directly. If they are obtained in free form, the guanosine, hypoxanthine, or cytidine can be dissolved or suspended in a suitable solvent, and then isolated and purified by adding an acid or base.

[0019] Furthermore, guanosine or its salts, hypoxanthine or its salts, and cytidine or its salts may also exist in the form of adducts with water or various solvents, but in the present invention, these adducts are also included and referred to as guanosine or its salts, hypoxanthine or its salts, and cytidine or its salts, respectively.

[0020] The guanosine or its salts, hypoxanthine or its salts, and cytidine or its salts used in the culture medium composition of the present invention may be synthesized by methods known to the present day, but they can also be obtained as commercially available products.

[0021] The concentrations of guanosine or its salt, hypoxanthine or its salt, and cytidine or its salt in the culture medium composition of the present invention are not particularly limited as long as the effects of the present invention are achieved, but are usually 0.1 μM to 3,000 μM each. Furthermore, the following concentrations are preferred for each: - Guanosine or its salt: 0.3 μM to 1,000 μM, more preferably 1 μM to 500 μM, even more preferably 2 μM to 300 μM, particularly preferably 3 μM to 150 μM - Hypoxanthine or its salt: 0.3 μM to 1,500 μM, more preferably 0.5 μM to 1,000 μM, even more preferably 0.7 μM to 700 μM, particularly preferably 1 μM to 500 μM - Cytidine or its salt: 0.3 μM to 2,000 μM, more preferably 0.5 μM to 1,500 μM, even more preferably 1 μM to 1,000 μM, particularly preferably 3 μM to 500 μM

[0022] Examples of uridine salts include metal salts. Examples of metal salts include those similar to the metal salts exemplified above for guanosine salts, hypoxanthine salts, and cytidine salts. Among these, sodium salts are preferred. Furthermore, free uridine is more preferred as the uridine or its salt.

[0023] When obtaining a salt of uridine, if uridine is obtained in salt form, it can be purified directly. If it is obtained in free form, uridine can be dissolved or suspended in a suitable solvent, isolated and purified by adding a base.

[0024] Furthermore, uridine or its salts may also exist in the form of adducts with water or various solvents, but in this invention, these adducts are also included and referred to as uridine or its salts.

[0025] The uridine or its salt used in the culture medium composition of the present invention may be synthesized by methods known to the present day, or it may be available as a commercially available product.

[0026] The concentration of uridine or its salt 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 0.1 μM to 3,000 μM, preferably 1 μM to 2,000 μM, more preferably 3 μM to 1,500 μM, even more preferably 10 μM to 1,000 μM, and particularly preferably 20 μM to 500 μM.

[0027] In one embodiment, one or more selected from the group consisting of guanosine or a salt thereof, hypoxanthine or a salt thereof, cytidine or a salt thereof, and uridine or a salt thereof is guanosine or a salt thereof. Also in one embodiment, one or more selected from the group consisting of guanosine or a salt thereof, hypoxanthine or a salt thereof, cytidine or a salt thereof, and uridine or a salt thereof is one or more selected from guanosine or a salt thereof and uridine or a salt thereof. Also in one embodiment, one or more selected from the group consisting of guanosine or a salt thereof, hypoxanthine or a salt thereof, cytidine or a salt thereof, and uridine or a salt thereof is one or more selected from guanosine or a salt thereof, cytidine or a salt thereof, and uridine or a salt thereof.

[0028] 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.

[0029] 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.

[0030] 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.

[0031] 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.

[0032] 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.

[0033] 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.

[0034] 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.

[0035] "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.

[0036] The culture medium composition 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.), and vitamin B Examples of components include vitamins such as 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, 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.

[0037] The other components may be included in the culture medium composition 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.

[0038] Furthermore, the culture medium composition 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.

[0039] The culture medium composition of the present invention can be prepared, for example, by directly adding one or more selected from the group consisting of guanosine or a salt thereof, hypoxanthine or a salt thereof, cytidine or a salt thereof, and uridine or a salt thereof to a basal culture medium, and this embodiment is preferred because it is simple. Alternatively, if one or more selected from the group consisting of guanosine or a salt thereof, hypoxanthine or a salt thereof, cytidine or a salt thereof, and uridine or a salt thereof are in solution form in the form of the culture medium additive of the present invention described later, the culture medium composition of the present invention can be obtained by adding 1 / 10,000 to 1 / 5, preferably 1 / 5,000 to 1 / 8, more preferably 1 / 1,000 of the culture medium additive of the present invention to a basal culture medium.

[0040] 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.

[0041] 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). This may include commercially available products such as HEK293 (Fujifilm Wako Pure Chemical Corporation: 551-34231), EX-CELL® 293 (Merck: 14571C), FreeStyle® 293 Expression Medium (Thermo Fisher Scientific: 12338018), CDM4HEK293 (cytiva: SH30858.02), and Expi293® Expression Medium (Thermo Fisher Scientific: A1435101), or products currently under development.

[0042] When the medium composition of the present invention contains serum and / or the other components described above, the medium composition of the present invention can be produced by appropriately adding one or more selected from the group consisting of guanosine or a salt thereof, hypoxanthine or a salt thereof, cytidine or a salt thereof, and uridine or a salt thereof, as well as serum and / or the other components described above to the basal medium, followed by mixing as it is, or by any other production method common in the art of medium compositions. There are no particular restrictions on the order of adding these respective components, and the order can be appropriately set according to the purpose of use and the like.

[0043] The medium composition of the present invention may be provided in a liquid state, or may be prepared in a concentrated state higher than the concentration at the time of use, or in a solid state such as a lyophilized powder, and then diluted with a solvent such as water, or dissolved or dispersed in a solvent such as water when used.

[0044] Culturing virus-producing cells into which a target gene has been introduced in the medium composition of the present invention can promote the production of a virus carrying the target gene.

[0045] The present invention also provides use of one or more selected from the group consisting of guanosine or a salt thereof, hypoxanthine or a salt thereof, cytidine or a salt thereof, and uridine or a salt thereof 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.

[0046] 2. Medium Additive The present invention also provides a medium additive for promoting production of a virus carrying a target gene during culturing of virus-producing cells into which the target gene has been introduced, which contains one or more selected from the group consisting of guanosine or a salt thereof, hypoxanthine or a salt thereof, cytidine or a salt thereof, and uridine or a salt thereof (hereinafter also referred to as "the medium additive of the present invention").

[0047] The terms "guanosine or a salt thereof," "hypoxanthine or a salt thereof," "cytidine or a salt thereof," "uridine or a salt thereof," "one or more selected from the group consisting of guanosine or a salt thereof, hypoxanthine or a salt thereof, cytidine or a salt thereof, and uridine or a salt thereof," "target gene," "virus," "virus-producing cell," "culture," and "promotion of virus production carrying the target gene," as well as the method for introducing the target gene into virus-producing cell, are as described above for the culture medium composition of the present invention.

[0048] In this specification, the term "culture medium additive" includes feed medium, which is a culture medium that is added to the culture medium at some point after the start of cultivation for purposes such as nutrient supply.

[0049] The concentrations of guanosine or its salts, hypoxanthine or its salts, cytidine or its salts, and uridine or its salts in the culture medium additive of the present invention are not particularly limited as long as the effects of the present invention are achieved. However, guanosine or its salts, hypoxanthine or its salts, cytidine or its salts, and uridine or its salts are included in the culture medium additive such that the final concentration in the basal medium when the culture medium additive is used by adding it to the basal medium is usually in the range of 0.1 μM to 3,000 μM. Furthermore, it is preferable that each is included in the culture medium additive such that the final concentration is within the following concentration range. • Guanosine or its salts: 0.3 μM to 1,000 μM, more preferably 1 μM to 500 μM, even more preferably 2 μM to 300 μM, particularly preferably 3 μM to 150 μM • Hypoxanthine or its salts: 0.3 μM to 1,500 μM, more preferably 0.5 μM to 1,000 μM, even more preferably 0.7 μM to 700 μM, particularly preferably 1 μM to 500 μM • Cytidine or its salts: 0.3 μM to 2,000 μM, more preferably 0.5 μM to 1,500 μM, even more preferably 1 μM to 1,000 μM, particularly preferably 3 μM to 500 μM • Uridine or its salts: 1 μM to 2,000 μM, more preferably 3 μM to 1,500 μM, even more preferably 10 μM to 1,000 μM, particularly preferably 20 μM to 500 μM

[0050] For example, when the culture medium additive of the present invention is in solution form, the concentrations of guanosine or its salt, hypoxanthine or its salt, cytidine or its salt, and uridine or its salt in the culture medium additive are not particularly limited as long as the effects of the present invention are achieved, but are usually 1 μM to 1,000 mM each. Furthermore, the following concentrations are preferable for each. • Guanosine or its salts: 3 μM to 300 mM, more preferably 10 μM to 250 mM, even more preferably 20 μM to 200 mM, particularly preferably 30 μM to 100 mM • Hypoxanthine or its salts: 3 μM to 500 mM, more preferably 5 μM to 400 mM, even more preferably 7 μM to 350 mM, particularly preferably 10 μM to 300 mM • Cytidine or its salts: 3 μM to 700 mM, more preferably 5 μM to 500 mM, even more preferably 10 μM to 400 mM, particularly preferably 30 μM to 300 mM • Uridine or its salts: 10 μM to 800 mM, more preferably 30 μM to 700 mM, even more preferably 100 μM to 600 mM, particularly preferably 200 μM to 500 mM

[0051] The "basic culture medium" is as described above for the culture medium composition of the present invention.

[0052] The culture medium additive 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 commonly added to culture media. The above-mentioned other components may be included individually or in combination of two or more.

[0053] The content of the above-mentioned other components in the culture medium additive 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.

[0054] The culture medium additive of the present invention can be used as is if it is one or more selected from the group consisting of guanosine or its salts, hypoxanthine or its salts, cytidine or its salts, and uridine or its salts. If it contains the above other components, it can be manufactured by appropriately adding the above other components to one or more selected from the group consisting of guanosine or its salts, hypoxanthine or its salts, cytidine or its salts, and uridine or its salts, and mixing them as is, 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 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.

[0055] The culture medium additive of the present invention can usually be used by adding it to a basal culture medium. Furthermore, the culture medium additive of the present invention can also be used, for example, when culturing virus-producing cells in a medium that does not contain one or more selected from the group consisting of guanosine or its salts, hypoxanthine or its salts, cytidine or its salts, and uridine or its salts 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 culture 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 of the present invention is a solution, it can be used by adding 1 / 10,000 to 1 / 5, preferably 1 / 5,000 to 1 / 8, more preferably 1 / 1,000 of the culture medium additive to a basal culture medium or a medium that does not contain one or more selected from the group consisting of guanosine or its salts, hypoxanthine or its salts, cytidine or its salts, and uridine or its salts. Furthermore, for example, if the culture medium additive of the present invention is a feed medium, the culture medium additive is added to the medium at some point after the start of cultivation, in an amount of 1 / 100 to 1 / 5, preferably 1 / 50 to 1 / 7, and more preferably 1 / 20 to 1 / 10.

[0056] By adding the culture medium additive of the present invention to a basal medium or a medium that does not contain one or more selected from the group consisting of guanosine or its salts, hypoxanthine or its salts, cytidine or its salts, and uridine or its salts, and culturing virus-producing cells into which the target gene has been introduced, the production of a virus carrying the target gene can be promoted.

[0057] 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 one or more selected from the group consisting of guanosine or a salt thereof, hypoxanthine or a salt thereof, cytidine or a salt thereof, and uridine or a salt thereof.

[0058] The terms "guanosine or a salt thereof," "hypoxanthine or a salt thereof," "cytidine or a salt thereof," "uridine or a salt thereof," "one or more selected from the group consisting of guanosine or a salt thereof, hypoxanthine or a salt thereof, cytidine or a salt thereof, and uridine or a salt thereof," "target gene," "virus," "virus-producing cell," "culture," and "promotion of virus production carrying the target gene," as well as the method for introducing the target gene into virus-producing cell, are as described above for the culture medium composition of the present invention. Furthermore, "a culture medium containing one or more selected from the group consisting of guanosine or a salt thereof, hypoxanthine or a salt thereof, cytidine or a salt thereof, and uridine or a salt thereof" means a culture medium having the same composition as the culture medium composition of the present invention.

[0059] 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 ２Culture 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.

[0060] 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) The virus-producing cells are cultured in a medium containing one or more selected from the group consisting of guanosine or a salt thereof, hypoxanthine or a salt thereof, cytidine or a salt thereof, and uridine or a salt thereof to increase the cells to a certain extent, and the number of cells is adjusted as necessary, after which the target gene is introduced into the cells and the cells are continued to be cultured in the same medium. (2) The virus-producing cells are cultured in a medium that does not contain one or more selected from the group consisting of guanosine or a salt thereof, hypoxanthine or a salt thereof, cytidine or a salt thereof, and uridine or a salt thereof to increase the cells to a certain extent, and the number of cells is adjusted as necessary, after which the medium is replaced with a medium containing one or more selected from the group consisting of guanosine or a salt thereof, hypoxanthine or a salt thereof, cytidine or a salt thereof, and uridine or a salt thereof, and then the target gene is introduced into the cells and the cells are continued to be cultured in the same medium (the order of medium replacement and target gene introduction may be reversed). (3) Cells that produce the virus are cultured in a medium that does not contain one or more selected from the group consisting of guanosine or a salt thereof, hypoxanthine or a salt thereof, cytidine or a salt thereof, and uridine or a salt thereof, and the cells are grown to a certain extent, and the number of cells is adjusted as necessary, and the target gene is introduced into the cells and the cells are continued to be cultured in the same medium. Here, one or more selected from the group consisting of guanosine or a salt thereof, hypoxanthine or a salt thereof, cytidine or a salt thereof, and uridine or a salt thereof are added to the medium before introducing the target gene into the virus-producing cells (for example, several hours before), during introduction, or after introduction (for example, several hours later).

[0061] 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 "the method of production 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 one or more selected from the group consisting of guanosine or a salt thereof, hypoxanthine or a salt thereof, cytidine or a salt thereof, and uridine or a salt thereof.

[0062] The terms "guanosine or a salt thereof," "hypoxanthine or a salt thereof," "cytidine or a salt thereof," "uridine or a salt thereof," "one or more selected from the group consisting of guanosine or a salt thereof, hypoxanthine or a salt thereof, cytidine or a salt thereof, and uridine or a salt thereof," "target gene," "virus," "virus-producing cell," and "culture," as well as the method for introducing the target gene into the virus-producing cell, are as described above for the culture medium composition of the present invention, and the method for "culture" is as described above for the acceleration method of the present invention. Furthermore, "a culture medium containing one or more selected from the group consisting of guanosine or a salt thereof, hypoxanthine or a salt thereof, cytidine or a salt thereof, and uridine or a salt thereof" means a culture medium having the same composition as the culture medium composition of the present invention.

[0063] In the production method of the present invention, virus-producing cells into which the target gene has been introduced can be cultured in a culture medium containing one or more selected from the group consisting of guanosine or a salt thereof, hypoxanthine or a salt thereof, cytidine or a salt thereof, and uridine or a salt thereof. After that, the virus carrying the target gene can be purified by a method known to the present invention. For example, the cultured cells can be recovered from the obtained culture medium and lysed, and then the obtained 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.

[0064] The production method 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 one or more selected from the group consisting of guanosine or a salt thereof, hypoxanthine or a salt thereof, cytidine or a salt thereof, and uridine or a salt thereof, thereby promoting the production of a virus carrying the target gene.

[0065] 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.

[0066] In Example 1 below, the AAV production ability of human embryonic kidney cells 293 (HEK293) induced by guanosine, hypoxanthine, cytidine, or uridine was evaluated. The reagents, cells, and culture medium used are as follows. • Guanosine: Guanosine (Tokyo Chemical Industries, Ltd.: C0171) • Hypoxanthine: Hypoxanthine (Fujifilm Wako Pure Chemical Industries, Ltd.: 088-03402) • Cytidine: Cytidine (Tokyo Chemical Industries, Ltd.: C0522) • Uridine: Uridine (Yamasa Soy Sauce Co., Ltd.) • HEK293: Viral Production Cells 2.0 (Thermo Fisher Scientific: A49784) • Culture medium for HEK293: FreeStyle™ F17 Expression Medium (Thermo Fisher Scientific: A1383501)

[0067] Example 1: AAV production of HEK293 by addition of guanosine, hypoxanthine, cytidine, or uridine 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 was seeded in this medium and cultured at 37°C and 8% CO2. ２ The cultures were stirred and cultured in an incubator set to 300 μM, and passed through 3 passages. Subsequently, 0.3 × 10⁻¹⁴ samples were fed into 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 guanosine, hypoxanthine, cytidine, or uridine added to a final concentration of 3–500 μM. ６Cells 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 3.0 ± 0.5 × 10⁶. ６ After 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). In addition, the number of viral particles was measured using Simple Plex AAV8 Cartridge (protein simple Inc.: SPCKB-OT-007970). From this, the full / empty capsid ratio (full capsid ratio) was calculated.

[0068] Figures 1 and 2 show the results of a series of studies investigating the effects of guanosine, hypoxanthine, cytidine, and uridine on AAV production in HEK293. Figure 1 shows the relative AAV8 genome titer, and Figure 2 shows the relative full-capsid ratio. The addition of guanosine, hypoxanthine, cytidine, and uridine resulted in an improvement in AAV8 genome titer. Furthermore, the addition of guanosine, as well as specific concentrations of hypoxanthine, cytidine, and uridine, resulted in an improvement in the full-capsid ratio.

[0069] 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.

[0070] This application is based on Japanese Patent Application No. 2025-055082 (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 a target gene during the culture of virus-producing cells into which a target gene has been introduced, comprising one or more selected from the group consisting of guanosine or a salt thereof, hypoxanthine or a salt thereof, cytidine or a salt thereof, and uridine or a salt thereof.

2. The culture medium composition according to claim 1, wherein one or more selected from the group consisting of guanosine or a salt thereof, hypoxanthine or a salt thereof, cytidine or a salt thereof, and uridine or a salt thereof is guanosine 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 concentrations of guanosine or a salt thereof, hypoxanthine or a salt thereof, cytidine or a salt thereof, and uridine or a salt thereof in the culture medium composition are 3 μM to 150 μM, 1 μM to 500 μM, 3 μM to 500 μM, and 20 μM to 500 μM, respectively.

7. A culture medium additive 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, comprising one or more selected from the group consisting of guanosine or a salt thereof, hypoxanthine or a salt thereof, cytidine or a salt thereof, and uridine or a salt thereof.

8. 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, the use of one or more substances selected from the group consisting of guanosine or a salt thereof, hypoxanthine or a salt thereof, cytidine or a salt thereof, and uridine or a salt thereof.

9. A method for promoting the production of 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 one or more selected from the group consisting of guanosine or a salt thereof, hypoxanthine or a salt thereof, cytidine or a salt thereof, and uridine or a salt thereof.

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 one or more selected from the group consisting of guanosine or a salt thereof, hypoxanthine or a salt thereof, cytidine or a salt thereof, and uridine or a salt thereof.

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 one or more selected from the group consisting of guanosine or a salt thereof, hypoxanthine or a salt thereof, cytidine or a salt thereof, and uridine or a salt thereof, thereby promoting the production of a virus carrying a target gene.

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