Culture medium composition

Abstract

The present invention provides: a culture medium composition which contains an iron ionophore, and which is for promoting the production of viruses each carrying a target gene or for improving the proportion of viruses each carrying the target gene with respect to all of produced viruses in the culturing of mammalian cells having the target gene introduced therein and having virus production capability; and others.

Description

Culture medium composition

[0001] The present invention relates to a culture medium composition, etc., for promoting the production of viruses carrying the target gene or increasing the proportion of viruses carrying the target gene among all viruses produced, during the culture of virus-producing cells into which a target gene has been introduced, including an iron ionophore.

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

[0003] When designing and optimizing culture media for virus-producing cells, the components of the media are a crucial consideration. The components in the media affect cell growth and proliferation.

[0004] In addition, in the case of culture media for virus-producing cells, the components contained in the medium also affect the virus-producing ability of the cells. Furthermore, when producing viruses using cells into which a gene that produces a cancer-suppressing protein has been introduced, the components contained in the medium also affect the proportion of viruses carrying that gene among the total viruses produced. However, satisfactory results have not always been obtained with the components known to date. For example, when producing adeno-associated viruses using cells into which a gene has been introduced, the proportion of adeno-associated viruses carrying that gene among the adeno-associated viruses is said to be around 5% to 30% (Non-Patent Literature 1).

[0005] On the other hand, in gene therapy media, including culture media for virus-producing cells, ferric citrate is often used as an iron source. However, Non-Patent Document 2, for example, states that ammonium ferric citrate is an important factor that hinders transfection.

[0006] Furthermore, transferrin, a protein involved in iron transport, is often used in culture media as a substitute for an iron source. However, since transferrin is an animal-derived protein that may contain exogenous factors such as pathogens, there is a need for substitutes that can replace transferrin. As such substitutes, for example, 2-hydroxypyridine-N-oxide (oxide), a chelating agent, can be used when growing cells (Patent Document 1) or when transfecting cells (Patent Documents 1 and 2), as disclosed in each document.

[0007] Furthermore, hinokitiol is known as an iron ionophore that has the ability to bind to iron ions and increase the membrane permeability of iron ions (Non-Patent Literature 3). However, the relationship between iron ionophores and the production of viruses carrying target genes is unknown.

[0008] Special table 2003-508046 publication Special table 2005-506048 publication

[0009] Molecular Therapy Methods & Clinical Development; 2021 June 11; Vol. 21; p. 642-655Biotechnology Progress, 2020 May; 36(3):e2954. ; doi: 10.1002 / btpr. 2954. Epub 2019 Dec 30Science; 12 May 2017; Vol. 356, Issue 6338; p. 608-616; doi: 10.1126 / science. aah3862

[0010] The present invention aims to provide a culture medium composition, etc., for improving the amount of virus carrying a target gene produced or the ratio of viruses carrying the target gene to the total number of viruses produced when producing a virus carrying a target gene using virus-producing cells.

[0011] As a result of diligent research into the above-mentioned problems, the inventors discovered that when hinokitiol 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.

[0012] In other words, the present invention is as follows:

[0013] [1] A culture medium composition for promoting the production of a virus carrying a target gene during the culture of a virus-producing mammalian cell into which a target gene has been introduced, the target gene containing an iron ionophore. [2] The culture medium composition according to [1], wherein the iron ionophore is hinokitiol. [3] The culture medium composition according to [1] or [2], wherein the mammalian cell is selected from the group consisting of HEK293 cells, MDCK cells, Vero cells, A549 cells, PerC6 cells, and HeLa cells. [4] The culture medium composition according to [1] or [2], wherein the mammalian cell is HEK293 cells. [5] The culture medium composition according to any one of [1] to [4], wherein the virus-producing mammalian cell is a mammalian cell that produces adeno-associated virus. [6] The culture medium composition according to any one of [1] to [5], wherein the concentration of the iron ionophore in the culture medium composition is 10 nM to 5,000 nM. [7] A culture medium additive for promoting the production of viruses carrying the target gene during the culture of virus-producing mammalian cells into which the target gene has been introduced, and which contain an iron ionophore. [8] Use of an iron ionophore in the manufacture of a culture medium composition for promoting the production of viruses carrying the target gene during the culture of virus-producing mammalian cells into which the target gene has been introduced. [9] A method for promoting the production of viruses carrying the target gene, comprising the step of culturing virus-producing mammalian cells into which the target gene has been introduced, in a culture medium containing an iron ionophore.

[10] A culture medium composition for increasing the ratio of viruses carrying the target gene among all viruses produced during the culture of virus-producing mammalian cells into which the target gene has been introduced, and which contain an iron ionophore.

[0014]

[11] The culture medium composition according to

[10] , wherein the iron ionophore is hinokitiol.

[12] The culture medium composition according to

[10] or

[11] , wherein the mammalian cell is a cell selected from the group consisting of HEK293 cells, MDCK cells, Vero cells, A549 cells, PerC6 cells, and HeLa cells.

[13] The culture medium composition according to

[10] or

[11] , wherein the mammalian cell is a HEK293 cell.

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

[10] to

[13] , wherein the virus-producing mammalian cell is a mammalian cell that produces adeno-associated virus or lentivirus.

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

[10] to

[14] , wherein the concentration of the iron ionophore in the culture medium composition is 10 nM to 5,000 nM.

[16] A culture medium additive for increasing the proportion of viruses carrying the target gene among all viruses produced during the culture of virus-producing mammalian cells into which the target gene has been introduced, including an iron ionophore.

[17] Use of an iron ionophore in the production of a culture medium composition for increasing the proportion of viruses carrying the target gene among all viruses produced during the culture of virus-producing mammalian cells into which the target gene has been introduced.

[18] A method for increasing the proportion of viruses carrying the target gene among all viruses produced during the culture, comprising the step of culturing virus-producing mammalian cells into which the target gene has been introduced, in a culture medium containing an iron ionophore.

[19] A method for producing viruses carrying the target gene, comprising the step of culturing virus-producing mammalian cells into which the target gene has been introduced, in a culture medium containing an iron ionophore.

[20] A method for producing a virus carrying a target gene, comprising the step of culturing a virus-producing mammalian cell into which the target gene has been introduced in a culture medium containing an iron ionophore, the method for promoting the production of a virus carrying the target gene, or for improving the ratio of viruses carrying the target gene among the total viruses produced during the culture of a virus-producing mammalian cell into which the target gene has been introduced.

[0015] The present invention makes it possible to improve the amount of viruses carrying the target gene produced or the ratio of viruses carrying the target gene to the total number of viruses produced when producing viruses carrying the target gene using virus-producing mammalian cells.

[0016] Figure 1 shows the results of verifying the effect of hinokitiol 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 hinokitiol added being set to 100%. The horizontal axis, "HyClone HyCell TransFx-H liquid medium," represents the case where AAV8 was produced using a medium without hinokitiol, while "+50 nM hinokitiol," "+100 nM hinokitiol," "+500 nM hinokitiol," "+1000 nM hinokitiol," and "+1500 nM hinokitiol" represent the cases where AAV8 was produced using a medium to which hinokitiol was added to final concentrations of 50 nM, 100 nM, 500 nM, 1000 nM, and 1500 nM, respectively. Figure 2 shows the results of verifying the effect of hinokitiol or 2-hydroxypyridine-N-oxide (HOPO) on AAV8 genome titer in Example 2. 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 either hinokitiol or HOPO set to 100%. The horizontal axis, "Viral Production Medium," represents the case when AAV8 is produced using a medium without the addition of either hinokitiol or HOPO, while "+10 μM HOPO," "+25 μM HOPO," "+50 μM HOPO," and "+500 nM Hinokitiol" represent the cases when AAV8 is produced using a medium with HOPO added to a final concentration of 10 μM, 25 μM, and 50 μM, respectively, and hinokitiol added to a final concentration of 500 nM. Figure 3 shows the results of the investigation into the effect of hinokitiol or HOPO on the full / empty capsid ratio (full capsid ratio) in Example 2. The vertical axis represents the relative full capsid ratio, with the full capsid ratio obtained when AAV8 is produced using a medium without the addition of either hinokitiol or HOPO set to 100%.The horizontal axis, "Viral Production Medium," represents the case where AAV8 was produced using a medium without the addition of either hinokitiol or HOPO. "+10 μM HOPO," "+25 μM HOPO," "+50 μM HOPO," and "+500 nM hinokitiol" represent the cases where AAV8 was produced using a medium with HOPO added to a final concentration of 10 μM, 25 μM, and 50 μM, respectively, and hinokitiol added to a final concentration of 500 nM. Figure 4 shows the results of verifying the effect of hinokitiol on AAV8 genome titer in Example 3. The vertical axis represents the relative AAV8 genome titer, with the AAV8 genome titer obtained when AAV8 was produced using a medium without hinokitiol added set to 100%. The horizontal axis, "Proprietary Development Medium," represents the case where AAV8 was produced using a medium without added hinokitiol, while "+500nM Hinokitiol" represents the case where AAV8 was produced using a medium to which hinokitiol was added to a final concentration of 500nM. Figure 5 shows the results of verifying the effect of hinokitiol on the ratio of virus copy number to p24 capsid protein amount in Example 4. The vertical axis represents the virus copy number per 1 μg of p24 capsid protein. The horizontal axis, "Control," represents the case where lentivirus was produced using a medium without added hinokitiol (0 nM), while "500nM Hinokitiol" and "1000nM Hinokitiol" represent the cases where lentivirus was produced using a medium to which hinokitiol was added to a final concentration of 500nM and 1000nM, respectively.

[0017] 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 viruses carrying a target gene during the culture of virus-producing mammalian cells into which a target gene has been introduced, and which contain an iron ionophore.

[0018] In this invention, "iron ionophore" refers to a substance that has the function of increasing the permeability of iron ions in biological membranes, for example, a substance that binds to iron ions and passes through biological membranes. More specifically, examples of iron ionophores include hinokitiol (also called "β-thujaplisin"), α-thujaplisin, γ-thujaplisin, etc., with hinokitiol being preferred. Iron ionophores may be used individually or in combination of two or more.

[0019] Iron ionophores such as hinokitiol may also exist in the form of salts, but in this invention, these salts are also referred to as iron ionophores.

[0020] Examples of salts of iron ionophores include metal salts, ammonium salts, organic amine addition salts, and amino acid addition salts.

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

[0022] Furthermore, iron ionophores 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 iron ionophores.

[0023] The iron ionophore used in culture medium composition 1 of the present invention may be synthesized by a method known to the present day, or it may be obtained as a commercially available product.

[0024] The concentration of iron ionophore in the culture medium composition 1 of the present invention is not particularly limited as long as the effects of the present invention are achieved, but is usually 10 nM to 5,000 nM, preferably 20 nM to 4,000 nM, more preferably 30 nM to 3,000 nM, even more preferably 40 nM to 2,000 nM, and particularly preferably 50 nM to 1,500 nM.

[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, AAVs, and lentiviruses are preferred, AAVs and lentiviruses are more preferred, and AAVs are even 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 mammalian cells that produce the virus are not particularly limited as long as they can produce a virus carrying the desired target gene, and cells from humans, monkeys, rodents, etc., can be used. Specifically, examples include HEK293 cells, MDCK cells, Vero cells, A549 cells, PerC6 cells, HeLa cells, CV-1 cells, LLC-MK2 cells, MDBK cells, WI-38 cells, MRC5 cells (human fibroblasts), BHK21 cells, etc., and HEK293 cells, MDCK cells, Vero cells, A549 cells, PerC6 cells, HeLa cells, etc., are preferred, and all 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 mammalian 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 vessel. 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 mammalian cells that produce the target gene. Specifically, this means (a) improving the full capsid ratio regardless of the increase or decrease in the number of capsids (number of viruses), 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; proteins such as albumin and transferrin; peptides such as glycylglycylglycine and soy peptide; serum; vitamins such as choline, vitamin A, B vitamins (thiamine, pyridoxine, cyanocobalamin, biotin, pantothenic acid, nicotinamide, etc.), vitamin C, and vitamin E; fatty acids such as oleic acid, arachidonic acid, and linoleic acid; lipids such as cholesterol; inorganic salts such as sodium chloride, calcium chloride, and sodium dihydrogen phosphate; trace elements such as zinc and selenium; buffering agents such as N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES) and N-[tris(hydroxymethyl)methyl]glycine (Tricine); antibiotics such as amphotericin B, kanamycin, gentamicin, streptomycin, and penicillin; Type I collagen, Type II collagen. Examples of these components include cell adhesion factors and extracellular matrix components such as collagen, fibronectin, laminin, poly-L-lysine, and poly-D-lysine; cytokines and growth factors such as interleukins, fibroblast growth factor (FGF), hepatocyte growth factor (HGF), transforming growth factor (TGF)-α, transforming growth factor (TGF)-β, vascular endothelial growth factor (VEGF), and activin A; and hormones such as dexamethasone, hydrocortisone, estradiol, progesterone, glucagon, and insulin. Appropriate components can be selected and used depending on the type of cells to be cultured 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] In addition, the culture medium composition 1 of the present invention may contain serum or may be serum-free. The serum is not particularly limited as long as it is an animal-derived serum and does not inhibit cell growth. Preferably, it is a mammalian-derived serum (such as fetal bovine serum, human serum, etc.), more preferably fetal bovine serum. The concentration of the serum may be within a concentration range known per se. The culture medium composition 1 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 iron ionophore to the basal medium. Also, because it is simple, such an embodiment is preferable. Further, when the iron ionophore is in a solution state in the form of the culture medium additive 1 of the present invention described below, 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, more preferably 1 / 1,000 amount of the culture medium additive 1 of the present invention to the basal medium.

[0037] The basal medium refers to a medium containing a carbon source, a nitrogen source, inorganic salts, etc. essential for cell culture, and is not particularly limited as long as the effects of the present invention are achieved, and may be appropriately selected according to 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). 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.

[0039] When the culture medium composition 1 of the present invention contains serum and / or the above other components, the culture medium composition 1 of the present invention can be produced by appropriately adding an iron ionophore, serum and / or the above other components to the above basal medium and mixing them as they are, or by other production methods common in the field of culture medium compositions. The order of adding these components and the like is not particularly limited and can be appropriately set according to the purpose of use and the like.

[0040] The culture medium composition 1 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 freeze-dried powder, and diluted with a solvent such as water at the time of use, or dissolved or dispersed in a solvent such as water and used.

[0041] By culturing mammalian cells that produce a virus into which a target gene has been introduced in the culture medium composition 1 of the present invention, the production of the virus carrying the target gene can be promoted.

[0042] The present invention also provides the use of an iron ionophore in the production of a culture medium composition for promoting the production of a virus carrying a target gene during the culture of mammalian cells that produce a virus into which the target gene has been introduced. Each term and the like are as described above.

[0043] 2. Culture Medium Additive 1 The present invention also provides a culture medium additive for promoting the production of a virus carrying a target gene during the culture of mammalian cells that produce a virus into which the target gene has been introduced, which contains an iron ionophore (hereinafter, also referred to as "the culture medium additive 1 of the present invention").

[0044] Regarding the "iron ionophore", "target gene", "virus", "mammalian cells that produce a virus", "culture", and "promotion of the production of a virus carrying a target gene", as well as the method for introducing the target gene into mammalian cells that produce a virus, they are as described above for the culture medium composition 1 of the present invention.

[0045] The concentration of iron ionophore in the culture medium additive 1 of the present invention is not particularly limited as long as the effects of the present invention are achieved. However, the iron ionophore 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 10 nM to 5,000 nM, preferably 20 nM to 4,000 nM, more preferably 30 nM to 3,000 nM, even more preferably 40 nM to 2,000 nM, and particularly preferably 50 nM to 1,500 nM. For example, the concentration of iron ionophore 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 the effects of the present invention are achieved. However, it is usually 0.1 μM to 1,000 mM, preferably 0.3 μM to 300 mM, more preferably 1 μM to 100 mM, even more preferably 3 μM to 30 mM, and particularly preferably 5 μM to 5 mM.

[0046] The "basic culture medium" is as described above for culture medium composition 1 of the present invention.

[0047] 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.

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

[0049] If the culture medium additive 1 of the present invention is an iron ionophore itself, it can be used as is. If it contains the other components mentioned above, it can be manufactured by appropriately adding the other components to the iron ionophore and mixing them together, or by other manufacturing methods common in the field of culture medium additives 1. 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.

[0050] 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 mammalian cells that produce viruses are cultured in a culture medium that does not contain iron ionophores to increase the number of cells to a certain extent, and if necessary the number of cells is adjusted, a target gene is introduced into the cells, and the cells are continued to be cultured in the same culture medium, by adding it to the culture medium before (for example, several hours before), during introduction, or after introduction (for example, several hours after) the introduction of the target gene into the mammalian cells that produce viruses. For example, if the culture medium additive 1 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 1 to a basal culture medium or a culture medium that does not contain iron ionophores.

[0051] By culturing virus-producing mammalian 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 iron ionophore, the production of a virus carrying the target gene can be promoted.

[0052] 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 mammalian cells into which a target gene has been introduced in a culture medium containing an iron ionophore.

[0053] The terms "iron ionophore," "target gene," "virus," "mammalian cell producing the virus," "culture," and "promotion of virus production carrying the target gene," as well as the method for introducing the target gene into mammalian cells producing the virus, are as described above for Culture Medium Composition 1 of the present invention. Furthermore, "culture medium containing an iron ionophore" refers to a culture medium having the same composition as Culture Medium Composition 1 of the present invention.

[0054] The culture of virus-producing mammalian cells into which the target gene has been introduced can be carried out under the same culture conditions as for normal mammalian cells. For example, humidity 95%, 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.

[0055] Furthermore, in the acceleration method of the present invention, the culture of virus-producing mammalian cells into which the target gene has been introduced can be carried out by any of the following methods: (1) Culture virus-producing mammalian cells in a medium containing iron ionophores to increase the 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 virus-producing mammalian cells in a medium without iron ionophores to increase the cells to a certain extent, adjust the number of cells as necessary, then replace the medium with a medium containing iron ionophores, 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 virus-producing mammalian cells in a medium without iron ionophores to increase the 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 iron ionophore is added to the culture medium before (e.g., several hours before) introducing the target gene into the virus-producing mammalian cells, during the introduction, or after (e.g., several hours later).

[0056] 4. Culture Medium Composition 2 The present invention also provides a culture medium composition (hereinafter also referred to as "Culture Medium Composition 2 of the present invention") for increasing the ratio of viruses carrying the target gene among all viruses produced during the culture of virus-producing mammalian cells into which the target gene has been introduced, and which contain an iron ionophore.

[0057] The "iron ionophore," "target gene," "virus," "virus-producing mammalian cell," and "culture," as well as the method for introducing the target gene into the virus-producing mammalian cell, are as described above for Culture Medium Composition 1 of the present invention.

[0058] "Increasing the proportion of viruses carrying the target gene among all viruses produced" means increasing the proportion of viruses (capsids) carrying the target gene among all viruses (capsids) produced during the culture of mammalian cells that produce the virus into which the target gene has been introduced.

[0059] The concentration of iron ionophore in the culture medium composition 2 of the present invention is not particularly limited as long as the effects of the present invention are achieved, but is usually 10 nM to 5,000 nM, preferably 20 nM to 4,000 nM, more preferably 30 nM to 3,000 nM, even more preferably 40 nM to 2,000 nM, and particularly preferably 50 nM to 1,500 nM.

[0060] 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.

[0061] The manufacturing method, form, and method of use of culture medium composition 2 are the same as those described above for culture medium composition 1 of the present invention.

[0062] By culturing virus-producing mammalian cells into which the target gene has been introduced in the culture medium composition 2 of the present invention, the ratio of viruses carrying the target gene among the total viruses produced can be improved.

[0063] The present invention also provides the use of iron ionophores in the production of a culture medium composition for increasing the proportion of viruses carrying the target gene among all viruses produced during the culture of virus-producing mammalian cells into which the target gene has been introduced. The terms and other details are as explained above.

[0064] 5. Culture medium additive 2 The present invention also provides a culture medium additive (hereinafter also referred to as "culture medium additive 2 of the present invention") for increasing the ratio of viruses carrying the target gene among all viruses produced during the culture of virus-producing mammalian cells into which the target gene has been introduced, and which contain an iron ionophore.

[0065] The "iron ionophore," "target gene," "virus," "virus-producing mammalian cell," and "culture," as well as the method for introducing the target gene into the virus-producing mammalian cell, are as described above for Culture Medium Composition 1 of the present invention, and the "increase in the ratio of viruses carrying the target gene among all viruses produced" is as described above for Culture Medium Composition 2 of the present invention.

[0066] The concentration of iron ionophore in the culture medium additive 2 of the present invention is not particularly limited as long as the effects of the present invention are achieved. However, the iron ionophore is included in the culture medium additive 2 such that the final concentration in the basal medium when the culture medium additive 2 is added to the basal medium is usually in the range of 10 nM to 5,000 nM, preferably 20 nM to 4,000 nM, more preferably 30 nM to 3,000 nM, even more preferably 40 nM to 2,000 nM, and particularly preferably 50 nM to 1,500 nM. For example, the concentration of iron ionophore in the culture medium additive 2 when the culture medium additive 2 of the present invention is in solution is also not particularly limited as long as the effects of the present invention are achieved. However, it is usually 0.1 μM to 1,000 mM, preferably 0.3 μM to 300 mM, more preferably 1 μM to 100 mM, even more preferably 3 μM to 30 mM, and particularly preferably 5 μM to 5 mM.

[0067] The "basic culture medium" is as described above for culture medium composition 1 of the present invention.

[0068] 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.

[0069] The manufacturing method, form, and usage method of the culture medium additive 2 of the present invention are the same as those described above for the culture medium additive 1 of the present invention.

[0070] By adding the culture medium additive 2 of the present invention to a basal culture medium and culturing virus-producing mammalian cells into which the target gene has been introduced, the ratio of viruses carrying the target gene among the total viruses produced can be improved.

[0071] 6. Method for increasing the proportion of viruses carrying the target gene The present invention also provides a method for increasing the proportion of viruses carrying the target gene among all viruses produced during the culture, comprising the step of culturing virus-producing mammalian cells into which the target gene has been introduced in a culture medium containing an iron ionophore.

[0072] The terms "target gene," "virus," "virus-producing mammalian cell," "culture," and "iron ionophore," as well as the method for introducing the target gene into virus-producing mammalian cells, are as described above for Culture Medium Composition 1 of the present invention; the terms "improving the ratio of viruses carrying the target gene among all produced viruses" are as described above for Culture Medium Composition 2 of the present invention; and the method of "culture" is as described above for the acceleration method of the present invention. Furthermore, "culture medium containing an iron ionophore" refers to a culture medium having the same composition as Culture Medium Composition 2 of the present invention.

[0073] 7. 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 mammalian cells into which the target gene has been introduced in a culture medium containing an iron ionophore.

[0074] The terms "iron ionophore," "target gene," "virus," "virus-producing mammalian cell," and "culture," as well as the method for introducing the target gene into the virus-producing mammalian cell, 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 iron ionophore" means a culture medium having the same composition as Culture Medium Composition 1 or Culture Medium Composition 2 of the present invention.

[0075] In the production method of the present invention, after culturing virus-producing mammalian cells into which the target gene has been introduced in a culture medium containing an iron ionophore, the virus carrying the target gene can be purified by a method known to the present day. For example, the cultured cells can be recovered from the obtained culture medium, lysated, 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.

[0076] The manufacturing method of the present invention can promote the production of viruses carrying the target gene, or improve the ratio of viruses carrying the target gene among the total viruses produced during the culture of virus-producing mammalian cells into which the target gene has been introduced. In other words, the present invention also provides a method for producing viruses carrying the target gene, which includes the step of culturing virus-producing mammalian cells into which the target gene has been introduced in a culture medium containing an iron ionophore, thereby promoting the production of viruses carrying the target gene, or improving the ratio of viruses carrying the target gene among the total viruses produced during the culture of virus-producing mammalian cells into which the target gene has been introduced.

[0077] 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.

[0078] In Example 1 below, the AAV production ability of human embryonic kidney cells 293 (HEK293) induced by hinokitiol was evaluated. The reagents, cells, and culture medium used are as follows. • Hinokitiol: Hinokitiol (Fujifilm Wako Pure Chemical Industries, Ltd.: 085-06251) • 2-Hydroxypyridine-N-oxide (HOPO): 2-Hydroxypyridine-N-oxide (Tokyo Chemical Industries, Ltd.: H0672) • HEK293: Viral Production Cells 2.0 (Thermo Fisher Scientific: A49784) • Culture medium for HEK293: HyClone HyCell TransFx-H liquid medium (Cytiva: SH30939.02) or Viral Production Medium (Thermo Fisher Scientific GmbH: A4817901)

[0079] Example 1: AAV production of HEK293 by hinokitiol 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 0°C, and passed through two passages. Subsequently, 0.6 × 10⁶ samples were collected in either a control medium (30 mL of HyClone HyCell TransFx-H liquid medium to which GlutaMAX™ Supplement was added to a final concentration of 4 mM), or a medium (30 mL of HyClone HyCell TransFx-H liquid medium to which GlutaMAX™ Supplement was added to a final concentration of 4 mM, and hinokitiol was added to final concentrations of 50, 100, 500, 1,000, and 1,500 nM). 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. Subsequently, the number of viable cells was 3 × 10⁶. ６ Each medium was diluted with fresh cells to a concentration of cells / mL, and 3 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 the 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 7500 Fast Real-Time PCR System (Thermo Fisher Scientific: 4357362).

[0080] Figure 1 shows the results of a series study investigating the effect of hinokitiol on AAV production by HEK293 (relative AAV8 genome titer). An improvement in AAV8 genome titer was confirmed with the addition of hinokitiol.

[0081] Example 2: AAV production of HEK293 by hinokitiol or HOPO 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 was seeded in this medium and cultured at 37°C and 8% CO2. ２The cultures were stirred and incubated in an incubator set to 300 μM, and passed through 3 passages. Subsequently, 0.6 × 10⁶ samples were collected in 30 mL Viral Production Medium with GlutaMAX™ Supplement added to a final concentration of 4 mM (control), 30 mL Viral Production Medium with GlutaMAX™ Supplement added to a final concentration of 4 mM and HOPO added to final concentrations of 10, 25, and 50 μM (comparison), or 30 mL Viral Production Medium with GlutaMAX™ Supplement added to a final concentration of 4 mM and hinokitiol added to a final concentration of 500 nM. 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. Subsequently, the number of viable cells was 3 × 10⁶. ６ Each medium was diluted with fresh cells to a concentration of cells / mL, and 3 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 the 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 7500 Fast Real-Time PCR System (Thermo Fisher Scientific: 4357362). In addition, the number of viral particles was measured using Simple Plex AAV8 Cartridge (protein simple: SPCKB-OT-007970). From this, the full / empty capsid ratio (full capsid ratio) was calculated.

[0082] The results of a series of verifications of the effects of hinokitiol or HOPO on AAV production in HEK293 are shown in FIGS. 2 and 3. The AAV8 genome titer (relative) is shown in FIG. 2, and the full capsid ratio (relative) is shown in FIG. 3. The addition of hinokitiol confirmed an improvement in the AAV8 genome titer and the full capsid ratio. On the other hand, when the chelating agent HOPO was added, the full capsid ratio decreased and the AAV8 genome titer also decreased.

[0083] Example 3: AAV production in HEK293 by adding hinokitiol using a suspension culture system. Using a 125 mL Erlenmeyer flask (VIALAMO: SEF125V), 30 mL of Viral Production Medium (Thermo Fisher Scientific: A4817901) was supplemented with GlutaMAX (trademark) Supplement (Thermo Fisher Scientific: 35050061) at a final concentration of 4 mM, and Viral Production Cells 2.0 were seeded. The cells were cultured with stirring in an incubator set at 37°C and 8% CO ２ and subcultured twice. Then, after subculturing five times in 30 mL of a self-developed medium (a medium based on "Basal 10" (Ajinomoto Health Supply Co., Ltd., CELLiST ＴＭ Basal media BASAL 10)) or a medium obtained by adding hinokitiol to the self-developed medium at a final concentration of 500 nM, the cells were seeded at a cell density of 0.4×10 ６ cells / mL and cultured with stirring for 3 days. On the third day, the viable cell count was measured using a viable cell autoanalyzer Vi-CELl BLU (Beckman). Then, when the viable cell count reached 3×10 ６Each medium was diluted with fresh cells to a concentration of cells / mL, and 3 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 the 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 7500 Fast Real-Time PCR System (Thermo Fisher Scientific: 4357362).

[0084] Figure 4 shows the results of a series study investigating the effect of hinokitiol on AAV production by HEK293 (relative AAV8 genome titer). An improvement in AAV8 genome titer was confirmed with the addition of hinokitiol.

[0085] Example 4: Lentivirus production of HEK293 by hinokitiol addition using a suspension culture system. In a 125 mL Erlenmeyer flask (VIOLAMO: SEF125V), 30 mL of LV-MAX Production Medium (Thermo Fisher Scientific: A3583401) was seeded with Viral Production Cells (Thermo Fisher Scientific: A35347), and cultured at 37°C and 8% CO2. ２ The culture was stirred and incubated in an incubator set to 5 passages. Afterwards, 3.5 × 10⁶ cells were transferred to 30 mL of LV-MAX Production Medium. ６ Cells were seeded at a cell density of cells / mL and cultured with agitation for one day. The number of viable cells was measured the following day using a hemocytometer. Subsequently, the number of viable cells was 4.7 × 10⁶. ６The culture medium was diluted to a cells / mL ratio using fresh medium, and 3.4 mL was seeded onto a 6-well suspension culture plate (Sumitomo Bakelite Co., Ltd.: MS-8006R). 0.2 mL of LV-MAX Supplement (Thermo Fisher Scientific: A35348) was added. Furthermore, hinokitiol was added to final concentrations of 0, 500, and 1000 nM. Subsequently, using the cationic lipid-based LV-MAX Transfer Kit (Thermo Fisher Scientific: A35348), pLVSIN-AcGFP1-N1 Vector (Takara Bio Inc.: 6187) and ViraPower were added. ＴＭ Lentivial Packaging Mix, Japan only (Thermo Fisher Scientific: JPG0035) was introduced. Six hours after transfection, 0.16 mL of LV-MAX Enhancer (Thermo Fisher Scientific: A35348) was added. After two days of incubation, the culture medium from each well was collected, centrifuged at 1300 × g at room temperature for 15 minutes, filtered through a 0.45 μm filter, and used as the lentivirus solution. RNA was purified from lentivirus solution using the Lenti-XTM qRT-PCR Titation Kit (Takara Bio Inc.: 631235), and the viral copy number (copies / mL) was measured using the QuantStudio 3 Real-Time PCR System (Thermo Fisher Scientific: QS3-96S). The amount of p24 capsid protein was also measured using the Lenti-XTM p24 Rapid Titer Kit (Takara Bio Inc.: 632200). The ratio of viral copy number to p24 capsid protein was then calculated from these results.

[0086] Figure 5 shows the results of a two-stage study investigating the effect of hinokitiol on lentivirus production by HEK293. The addition of 500 nM hinokitiol resulted in an improvement in the ratio of virus copy number to p24 capsid protein amount, indicating an increase in the proportion of viruses carrying the target gene among all viruses produced.

[0087] The present invention makes it possible to improve the amount of viruses carrying the target gene produced or the ratio of viruses carrying the target gene to the total number of viruses produced when producing viruses carrying the target gene using virus-producing mammalian cells.

[0088] This application is based on Japanese Patent Application No. 2024-167263 (filing date: September 26, 2024), 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 mammalian cells into which a target gene has been introduced, including an iron ionophore.

2. The culture medium composition according to claim 1, wherein the iron ionophore is hinokitiol.

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

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

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

6. The culture medium composition according to any one of claims 1 to 5, wherein the concentration of iron ionophores in the culture medium composition is 10 nM to 5,000 nM.

7. A culture medium additive for promoting the production of viruses carrying the target gene during the culture of virus-producing mammalian cells into which the target gene has been introduced, including iron ionophores.

8. Use of iron ionophores in the preparation of a culture medium composition for promoting the production of a virus carrying the target gene during the culture of virus-producing mammalian cells into which the target gene has been introduced.

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

10. A culture medium composition for increasing the proportion of viruses carrying the target gene among all viruses produced during the culture of virus-producing mammalian cells into which the target gene has been introduced, including iron ionophores.

11. The culture medium composition according to claim 10, wherein the iron ionophore is hinokitiol.

12. The culture medium composition according to claim 10, wherein the mammalian cells are cells selected from the group consisting of HEK293 cells, MDCK cells, Vero cells, A549 cells, PerC6 cells, and HeLa cells.

13. The culture medium composition according to claim 10, wherein the mammalian cells are HEK293 cells.

14. The culture medium composition according to claim 10, wherein the mammalian cells that produce the virus are mammalian cells that produce adeno-associated viruses or lentiviruses.

15. The culture medium composition according to any one of claims 10 to 14, wherein the concentration of iron ionophores in the culture medium composition is 10 nM to 5,000 nM.

16. A culture medium additive for increasing the proportion of viruses carrying the target gene among all viruses produced during the culture of virus-producing mammalian cells into which the target gene has been introduced, including iron ionophores.

17. Use of iron ionophores in the preparation of culture medium compositions for increasing the proportion of viruses carrying the target gene among all viruses produced during the culture of virus-producing mammalian cells into which the target gene has been introduced.

18. A method for increasing the proportion of viruses carrying the target gene among all viruses produced during the culture, comprising the step of culturing virus-producing mammalian cells into which the target gene has been introduced in a culture medium containing an iron ionophore.

19. A method for producing a virus carrying a target gene, comprising the step of culturing virus-producing mammalian cells into which the target gene has been introduced in a culture medium containing an iron ionophore.

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