Viral vector production inducer

Abstract

An agent for inducing viral vector production, said agent containing a cell proliferation inhibitor a, characterized in that the cell proliferation inhibitor a contains a compound that inhibits the progression in the G2 or M phase of the cell cycle; and a method for producing a viral vector and a method for inducing the production of a viral vector, each method using the aforesaid agent. A method for producing a viral vector and a method for inducing the production of a viral vector, each method comprising a step for introducing a nucleic acid into cells and a step for adding a cell proliferation inhibitor A to the cells, characterized in that the cell proliferation inhibitor A is added from 6 hours before to 6 hours after with reference to the time of the introduction of the nucleic acid.

Description

[Technical Field]

[0001] This invention relates to a viral vector production inducer. [Background technology]

[0002] Viral vectors are widely used in gene therapy. A viral vector is a vector in which the genes of a wild virus are replaced with foreign genes (such as genes that express fluorescent proteins or genes that express therapeutic proteins). One method for producing viral vectors involves creating viral vector-producing cells by introducing nucleic acids that express the viral vector into cells, and then purifying the viral vector from the cell supernatant or cell suspension. However, challenges include insufficient viral vector productivity in these viral vector-producing cells. One known method to address this problem is to use histone deacetylase (HDAC) inhibitors as production inducers (Patent Document 1). However, when the amount (titer) of viral vector production in viral vector-producing cells is already high, this method does not appear to further increase the amount, making it difficult to consider it a useful method. Furthermore, nocodazole-DMSO has been disclosed as an AAV production enhancer (Patent Document 2), but the concentration of nocodazole-DMSO, the timing of its addition, the type of solubilizer, and the type of nucleic acid introduced into the cells have not been investigated at all. Moreover, nocodazole is known to reduce the productivity of virus-like particles (Non-Patent Document 1).

[0003] Therefore, viral vector production inducers containing compounds that inhibit progression in the G2 or M phase of the cell cycle to improve the productivity of viral vectors, as well as methods for producing viral vectors and inducing viral vector production using these, and methods for producing viral vectors and inducing viral vector production to improve the productivity of viral vectors, are completely unknown, and there is a strong demand for the provision of such materials. [Prior art documents] [Patent Documents]

[0004] [Patent Document 1] Special Publication No. 2020-524498 [Patent Document 2] International Publication No. 2020 / 172624 [Non-patent literature]

[0005] [Non-Patent Document 1] Applied. Microbiology and Biotechnology 2015 99:9935-9949 [Overview of the Initiative] [Problems that the invention aims to solve]

[0006] The present invention aims to solve the aforementioned problems in the conventional era and achieve the following objectives. Specifically, the present invention aims to provide a viral vector production inducer containing a compound that inhibits progression in the G2 or M phase of the cell cycle, which improves the productivity of viral vectors, as well as a method for producing viral vectors and a method for inducing viral vector production using these, and a method for producing viral vectors and a method for inducing viral vector production which improves the productivity of viral vectors. [Means for solving the problem]

[0007] As a result of diligent research conducted by the present inventors to achieve the above objective, we have found that we can provide a viral vector production inducer that includes a cell proliferation inhibitor a, wherein the cell proliferation inhibitor a includes a compound that inhibits progression in the G2 or M phase of the cell cycle, thereby improving the productivity of viral vectors, and a method for producing viral vectors and a method for inducing viral vector production using these, which includes the steps of introducing nucleic acids into cells and adding cell proliferation inhibitor A to the cells, wherein the cell proliferation inhibitor A is added from 6 hours before to 6 hours after the introduction of the nucleic acids, thereby improving the productivity of viral vectors, and we have found that we can provide a method for producing viral vectors and a method for inducing viral vector production that includes the steps of introducing nucleic acids into cells and adding cell proliferation inhibitor A to the cells, wherein the cell proliferation inhibitor A is added from 6 hours before to 6 hours after the introduction of nucleic acids, thereby improving the productivity of viral vectors.

[0008] The present invention is based on the aforementioned findings by the inventors, and the means for solving the aforementioned problems are as follows: <1> The viral vector production inducer is characterized by comprising a cell proliferation inhibitor a, wherein the cell proliferation inhibitor a contains a compound that inhibits progression in the G2 or M phase of the cell cycle. <2> The aforementioned <1> This method for producing a viral vector is characterized by using a viral vector production inducer described above. <3> A method for producing a viral vector, comprising the steps of introducing nucleic acids into cells and adding a cell proliferation inhibitor A to the cells, characterized in that the cell proliferation inhibitor A is added 6 hours before to 6 hours after the introduction of the nucleic acids. <4> The aforementioned <1> This method for inducing viral vector production is characterized by using the viral vector production inducer described in [reference]. <5> The method for inducing viral vector production includes the steps of introducing nucleic acids into cells and adding a cell proliferation inhibitor A to the cells, characterized in that the cell proliferation inhibitor A is added 6 hours before to 6 hours after the introduction of the nucleic acids.

Advantages of the Invention

[0009] According to the present invention, the above-mentioned various problems in the prior art can be solved, the above object can be achieved, a virus vector production inducer containing a compound that inhibits progression in the G2 or M phase of the cell cycle and improves the productivity of the virus vector, and a method for producing a virus vector and a method for inducing the production of a virus vector using these can be provided.

Brief Description of the Drawings

[0010] [Figure 1] Figure 1 is a diagram showing the effect of the final concentration of nocodazole on the titer of the adeno-associated virus vector in Example 1. [Figure 2] Figure 2 is a diagram showing the effect of the timing of adding cell growth inhibitor A on the titer of the adeno-associated virus vector in Example 2. [Figure 3] Figure 3 is a diagram (part 1) showing the effect of adding various cell growth inhibitors A on the titer of the adeno-associated virus vector in Example 3. [Figure 4] Figure 4 is a diagram (part 2) showing the effect of adding various cell growth inhibitors A on the titer of the adeno-associated virus vector in Example 3. [Figure 5] Figure 5 is a diagram (part 3) showing the effect of adding various cell growth inhibitors A on the titer of the adeno-associated virus vector in Example 3. [Figure 6] Figure 6 is a diagram (part 4) showing the effect of adding various cell growth inhibitors A on the titer of the adeno-associated virus vector in Example 3. [Figure 7] Figure 7 is a diagram (part 5) showing the effect of adding various cell growth inhibitors A on the titer of the adeno-associated virus vector in Example 3. [Figure 8] Figure 8 is a diagram showing the effect of adding sodium valproate on the titer of the adeno-associated virus vector in Comparative Example 1. [Figure 9]Figure 9 shows the effect of the presence or absence of cell proliferation inhibitor A on the titer of the adeno-associated virus vector prepared using a bioreactor in Example 4. [Figure 10] Figure 10 is a diagram (part 1) showing the effect of the use of cell proliferation inhibitor A and linear covalent closed DNA on the titer of the adeno-associated virus vector in Example 5. [Figure 11] Figure 11 is a diagram (part 2) showing the effect of the use of cell proliferation inhibitor A and linear covalent closed DNA on the titer of the adeno-associated virus vector in Example 5. [Figure 12] Figure 12 shows the vector map of pAAV-Venus_telRL_ara_TelN in manufacturing example 2-2. [Figure 13] Figure 13 shows the vector map of pRC2-mi342_telRL_ara_TelN in manufacturing example 2-3. [Figure 14] Figure 14 shows the vector map of pHelper_telRL_ara_TelN in manufacturing example 2-4. [Figure 15] Figure 15 shows the evaluation results of DNA electrophoresis in production example 2-6. [Figure 16] Figure 16 shows the evaluation results of DNA electrophoresis in production example 2-7. [Modes for carrying out the invention]

[0011] (Viral vector production inducer) The aforementioned viral vector production inducer comprises a cell proliferation inhibitor a and may further contain other components.

[0012] <Viral vector> There are no particular restrictions on the aforementioned viral vectors, and they can be appropriately selected depending on the purpose. Examples include adeno-associated virus (AAV) vectors, adenovirus vectors, retrovirus vectors, lentivirus vectors, herpesvirus vectors, poliovirus vectors, papillomavirus vectors, vaccinia virus vectors, poxvirus vectors, Simian virus vectors, and Sendai virus vectors. Among these, adeno-associated virus (AAV) vectors are preferred due to their low pathogenicity.

[0013] Examples of serotypes of the aforementioned AAV include AAV1 (type 1 AAV), AAV2 (type 2 AAV), AAV3 (type 3 AAV), AAV4 (type 4 AAV), AAV5 (type 5 AAV), AAV6 (type 6 AAV), AAV7 (type 7 AAV), AAV8 (type 8 AAV), AAV9 (type 9 AAV), AAV10 (type 10 AAV), AAV11 (type 11 AAV), AAV12 (type 12 AAV), AAV13 (type 13 AAV), AAV14 (type 14 AAV), and their variants, but there are no particular restrictions, and they can be appropriately selected depending on the purpose. There are no particular restrictions on the aforementioned modified organisms, and they can be appropriately selected according to the purpose. For example, AAV modified by genetic engineering (from wild-type AAV) to improve the tissue specificity (directivity of cells to infect) of target cells can be used.

[0014] <Cell proliferation inhibitor a> The cell proliferation inhibitor a comprises a compound that inhibits progression during the G2 or M phase of the cell cycle. This compound may be used alone or in combination of two or more compounds. There are no particular restrictions on the compounds that inhibit progression during the G2 or M phase of the cell cycle, and they can be appropriately selected depending on the purpose. Examples include compounds that inhibit microtubule polymerization and compounds that stabilize microtubules. Among these, compounds that inhibit microtubule polymerization are preferred in terms of their effect in inducing viral vector production.

[0015] There are no particular limitations on the compounds that inhibit microtubule polymerization, and they can be appropriately selected depending on the purpose. Examples include benzimidazole compounds, vinca alkaloid compounds, and colchicine derivatives. The benzimidazole compound is not particularly limited as long as it is a compound having a benzimidazole ring, and can be appropriately selected depending on the purpose. Examples include nocodazole, albendazole, mebendazole, thiabendazole, fenbendazole, triclabendazole, flubendazole, oxybendazole, parbendazole, oxfendazole, lycobendazole, or salts thereof. Among these, nocodazole, albendazole, mebendazole, thiabendazole, fenbendazole, triclabendazole, flubendazole, oxybendazole, parbendazole, or salts thereof are preferred. The vinca alkaloid compound is not particularly limited and can be appropriately selected depending on the purpose. Examples include vinblastine, vincristine, vindesine, vinorelbine, vinflunin, or derivatives of the above compounds or salts thereof. Among these, vinblastine or its salts are preferred. The colchicine derivative is not particularly limited and can be appropriately selected depending on the purpose. Examples include colchicine, colsemid (demecolsin), allocolchicine, thiocolchicine, or salts thereof. Among these, colsemid or its salts are preferred. Among these, nocodazole, albendazole, mebendazole, oxybendazole, vinblastine, colsemid, or salts thereof are preferred in terms of their effect in inducing viral vector production. As the compound that inhibits microtubule polymerization, salt forms can be preferably used, and examples of such salt forms include hydrochloride salts and sulfates. Specific examples of such salt forms include vinblastine sulfate and vincristine sulfate.

[0016] There are no particular restrictions on the compounds used to stabilize the microtubules, and they can be appropriately selected depending on the purpose. Examples include taxane compounds. The aforementioned taxane-based compound is not particularly limited as long as it is a compound having a taxane ring skeleton, and can be appropriately selected depending on the purpose. Examples include paclitaxel and docetaxel.

[0017] <Other ingredients> The aforementioned other components are not particularly limited and can be appropriately selected depending on the purpose. Examples include solubilizers, surfactants, colorants, stabilizers, and pH adjusters. These may be used individually or in combination of two or more. Examples of the solubilizing agents include water, acidic aqueous solutions, alcohol compounds, dimethyl sulfoxide (DMSO), dimethylformamide (DMF), dimethylacetamide (DMA), hexamethylphosphate triamide (HMPA), acetonitrile, acetone, dioxane, and tetrahydrofuran (THF). The acidic aqueous solution is not particularly limited and can be appropriately selected depending on the purpose. Examples include aqueous solutions of acidic compounds such as hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, acetic acid, monofluoroacetic acid, difluoroacetic acid, trifluoroacetic acid, formic acid, oxalic acid, malonic acid, and succinic acid. The acidic compound may be used alone or in a mixture of two or more. The concentration of the acidic aqueous solution is greater than 0 for the concentration of the acidic compound and less than the maximum solubility inherent to the acidic compound. Specifically, the concentration of the acidic compound is greater than 0 w / w% and less than or equal to the saturated solution concentration of the acidic compound at 20°C and normal pressure w / w%. The aforementioned alcohol-based compound is not particularly limited as long as it is a compound having a hydroxyl group, and can be appropriately selected depending on the purpose. Examples include methanol, ethanol, propanol, isopropanol, n-butyl alcohol, isobutyl alcohol, sec-butyl alcohol, and tert-butyl alcohol. Among these, water, hydrochloric acid, formic acid aqueous solution, ethanol, or DMSO are preferred in terms of their viral vector production induction effect and compound solubility.

[0018] (Method for producing viral vectors 1) A first aspect of the method for producing the viral vector is a method using a viral vector production inducer containing the cell proliferation inhibitor a.

[0019] There are no particular limitations on the method of using the aforementioned viral vector production inducer, and it can be appropriately selected depending on the purpose. For example, one method is to add the viral vector production inducer to viral vector-producing cells.

[0020] There are no particular restrictions on the viral vector-producing cells, and they can be appropriately selected depending on the purpose. For example, cells into which nucleic acids for expressing a viral vector have been introduced (transfected) can be used. There are no particular restrictions on the cells mentioned above, and they can be appropriately selected depending on the purpose. Examples include mammalian cells and insect cells. Among these, mammalian cells are preferred.

[0021] There are no particular restrictions on the mammalian cells mentioned above, and they can be appropriately selected depending on the purpose. Examples include human-derived cells, mouse-derived cells, hamster-derived cells, rat-derived cells, dog-derived cells, monkey-derived cells, and kangaroo-derived cells. Among these, HEK293 cells, CHO cells, Hela cells, Vero cells, BHK cells, COS cells, MDCK cells, 3T3 cells, HepG2 cells, A549 cells, C2C12 cells, C6 cells, HT-1080 cells, Huh-7 cells, H9C2 cells, HCT116 cells, HT-29 cells, K562 cells, LNCaP cells, Jurkat cells, L6 cells, USO2 cells, or PC12 cells are preferred, with HEK293 cells being more preferred, in terms of viral vector yield and the safety of the produced viral vectors. The aforementioned cells include cell lines derived from parent cells. For example, for HEK293 cells, HEK293T cells, FreeStyle® 293F cells, and Viral Production Cells 2.0, which are derived from HEK293 cells, can be used.

[0022] There are no particular restrictions on the insect cells mentioned above, and they can be appropriately selected depending on the purpose. Examples include moth-derived cells and fly-derived cells. Among these, Sf-9 cells, Sf-21 cells, High five cells, Tni cells, or S2 cells are preferred. The aforementioned cells include cell lines derived from parent cells.

[0023] There are no particular restrictions on the cell culture method; it can be appropriately selected according to the purpose, and either adherent culture or suspension culture can be used. Among these methods, suspension culture is preferred due to its yield per unit volume of culture medium and ease of operation.

[0024] The aforementioned "nucleic acid" can also be called "polynucleotide," and examples include deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), but DNA is preferred. The DNA may be single-stranded, double-stranded, or triple-stranded, but double-stranded DNA is preferred. There are no particular restrictions on the structure of the double-stranded DNA, and it can be appropriately selected depending on the purpose, and examples include circular plasmid DNA, linear plasmid DNA, ring-open plasmid DNA, circular DNA, linear DNA, and linear covalent closed DNA. Among these, circular plasmid DNA, circular DNA, and linear covalent closed DNA are preferred, and linear covalent closed DNA is more preferred in terms of gene transfer efficiency. The aforementioned "Linear Covalently Closed DNA" refers to a linear double-stranded DNA with a hairpin-shaped closed end structure, and can also be abbreviated as "LCC DNA".

[0025] There are no particular restrictions on the method of introducing the nucleic acid, and it can be appropriately selected depending on the purpose. Examples include methods using cationic polymers such as polyethyleneimine (PEI), methods using liposomes, methods using calcium phosphate, methods using branched organic compounds such as dendrimers, methods using diethylaminoethyl (DEAE)-dextran, electroporation, particle gun method, microinjection method, magnetofection method, and methods using viral vectors. Among these methods, the method using cationic polymers is preferred due to its high introduction efficiency, low reagent cost, and the fact that it does not require special equipment.

[0026] There are no particular restrictions on the timing of adding the viral vector production inducer, and it can be appropriately selected depending on the purpose. However, in terms of simplicity and improving the viral vector production induction effect, it is preferable to add the agent 6 hours to 6 hours after the introduction of the nucleic acid, more preferably 4 hours to 4 hours after, even more preferably 5 minutes to 4 hours after, even more preferably 5 minutes to 2 hours after, and most preferably simultaneously to 2 hours after. There are no particular restrictions on the number of times the aforementioned viral vector production inducer can be added, and it can be appropriately selected depending on the purpose. For example, the entire amount can be added at once, or it can be added in multiple installments.

[0027] There are no particular restrictions on the method of introducing the nucleic acid; the transfection reagent and nucleic acid can be added after forming a complex, or the transfection reagent and nucleic acid can be added separately. Furthermore, the entire amount of the transfection reagent and nucleic acid can be added at once, or they can be added in multiple installments. When introducing the nucleic acid, if an introduction reagent (transfection reagent) is used during the introduction of the nucleic acid, it refers to the time when the introduction reagent is added, and if an electric current, pressure, etc. is applied during the introduction of the nucleic acid, it refers to the time when an electric current, pressure, etc. is applied. If the introduction reagent is added in multiple doses, the term "time of adding the introduction reagent" refers to the first addition.

[0028] There are no particular restrictions on the lower limit of the final concentration of the cell proliferation inhibitor a, and it can be appropriately selected depending on the purpose. However, from the standpoint of improving the viral vector production induction effect, a concentration of 1 nM or higher is preferred, and further, in the following order, 10 nM or higher, 100 nM or higher, 200 nM or higher, 250 nM or higher, 300 nM or higher, and 400 nM or higher are more preferred. There are no particular restrictions on the upper limit of the final concentration of the cell proliferation inhibitor a, and it can be appropriately selected depending on the purpose. However, from the viewpoint of improving the viral vector production induction effect, a concentration of 100 μM or less is preferred, and further, in the following order, 10 μM or less, 5 μM or less, 2.5 μM or less, 1 μM or less, 800 nM or less, and 600 nM or less are more preferred.

[0029] When the cell proliferation inhibitor a is nocodazole, there are no particular restrictions on the final concentration of nocodazole, and it can be appropriately selected depending on the purpose. However, from the viewpoint of improving the viral vector production induction effect, a concentration of 100 nM to 5 μM is preferred, 250 nM to 2 μM is more preferred, 300 nM to 1 μM is even more preferred, and 400 nM to 800 nM is particularly preferred.

[0030] When the cell proliferation inhibitor a is albendazole, there are no particular restrictions on the final concentration of albendazole, and it can be appropriately selected depending on the purpose. However, from the standpoint of improving the viral vector production induction effect, a concentration of 500 nM to 20 μM is preferred, and 1 μM to 10 μM is more preferred.

[0031] When the cell proliferation inhibitor a is oxybendazole, there are no particular restrictions on the final concentration of oxybendazole, and it can be appropriately selected depending on the purpose. However, from the viewpoint of improving the viral vector production induction effect, a concentration of 500 nM to 200 μM is preferred, 1 μM to 150 μM is more preferred, and 10 μM to 100 μM is even more preferred.

[0032] When the cell proliferation inhibitor a is mebendazole, there are no particular restrictions on the final concentration of mebendazole, and it can be appropriately selected depending on the purpose. However, from the viewpoint of improving the viral vector production induction effect, a concentration of 500 nM to 200 μM is preferred, 1 μM to 150 μM is more preferred, and 10 μM to 100 μM is even more preferred.

[0033] When the cell proliferation inhibitor a is vinblastine, there are no particular restrictions on the final concentration of vinblastine, and it can be appropriately selected depending on the purpose. However, from the viewpoint of improving the viral vector production induction effect, a concentration of 10 nM to 10 μM is preferred, 50 nM to 10 μM is more preferred, and 100 nM to 5 μM is even more preferred.

[0034] When the cell proliferation inhibitor a is colsemid, there are no particular restrictions on the final concentration of colsemid, and it can be appropriately selected depending on the purpose. However, from the viewpoint of improving the viral vector production induction effect, a concentration of 10 nM to 10 μM is preferred, 50 nM to 10 μM is more preferred, and 100 nM to 5 μM is even more preferred.

[0035] There are no particular restrictions on the amount of viral vector obtained by the viral vector production method 1 described above, and it can be appropriately selected according to the purpose. However, the titer of the viral vector compared to the titer of the viral vector when the viral vector production inducer is not used is preferably 1.1 times or more, more preferably 1.2 times or more, even more preferably 1.5 times or more, even more preferably 2 times or more, and the higher the ratio, the better. The titer of the aforementioned viral vector is determined by quantitative PCR (Quantstudio3, SYBR Green method) as described below.

[0036] The quantitative PCR described above uses a sample consisting of 12.5 μL of PowerUp® SYBR® Green Master Mix (Thermo Fisher Scientific), 0.125 μL of forward primer (50 μM, SEQ ID NO: 1), 0.125 μL of reverse primer (50 μM, SEQ ID NO: 2), 11.25 μL of Milli-Q® water, and 1 μL of standard or a 5000-fold diluted sample (total 25 μL). PCR was performed on the above sample using QuantStudio3 (Thermo Fisher Scientific) by repeating the following cycles 30 times: 94°C / 15 seconds (thermal denaturation), 60°C / 30 seconds (annealing), and 72°C / 30 seconds (extension reaction). If the viral vector is the adeno-associated virus (AAV) vector, the standard used is a pAAV-MCS Expression Vector (0.67 μg / μL, TE solution, Cell Biolabs) linearized by digesting it in PvuII (Takara Bio Inc.) at 37°C for 2 hours.

[0037] There are no particular restrictions on the titer of the viral vector when the aforementioned viral vector production inducer is not used, and it can be appropriately selected depending on the purpose. However, in order to improve the viral vector production induction effect, 1 × 10 13 A vector genome of 1 / L or more is preferred, and 2 × 10 13 More preferably, vector genome / L or higher, 5 × 10 13 A vector genome of 1 / L or higher is even more preferable, and 8 × 10 13 More preferably, vector genome / L or higher, 1 × 10 14 A vector genome ratio of 1 / L or higher is particularly preferred.

[0038] (Method for producing viral vectors 2) A second aspect of the method for producing the viral vector includes the steps of introducing nucleic acid into cells (transfection) and adding a cell proliferation inhibitor A described later to the cells, and may further include other steps.

[0039] <The process of introducing nucleic acids into cells (transfection)> There are no particular restrictions on the cells mentioned above, and they can be appropriately selected depending on the purpose. Examples include mammalian cells and insect cells. Among these, mammalian cells are preferred.

[0040] There are no particular restrictions on the mammalian cells mentioned above, and they can be appropriately selected depending on the purpose. Examples include human-derived cells, mouse-derived cells, hamster-derived cells, rat-derived cells, dog-derived cells, monkey-derived cells, and kangaroo-derived cells. Among these, HEK293 cells, CHO cells, Hela cells, Vero cells, BHK cells, COS cells, MDCK cells, 3T3 cells, HepG2 cells, A549 cells, C2C12 cells, C6 cells, HT-1080 cells, Huh-7 cells, H9C2 cells, HCT116 cells, HT-29 cells, K562 cells, LNCaP cells, Jurkat cells, L6 cells, USO2 cells, or PC12 cells are preferred, with HEK293 cells being more preferred, in terms of viral vector yield and the safety of the produced viral vectors. The aforementioned cells include cell lines derived from parent cells. For example, for HEK293 cells, HEK293T cells, FreeStyle® 293F cells, and Viral Production Cells 2.0, which are derived from HEK293 cells, can be used.

[0041] There are no particular restrictions on the insect cells mentioned above, and they can be appropriately selected depending on the purpose. Examples include moth-derived cells and fly-derived cells. Among these, Sf-9 cells, Sf-21 cells, High five cells, Tni cells, or S2 cells are preferred. The aforementioned cells include cell lines derived from parent cells.

[0042] There are no particular restrictions on the cell culture method; it can be appropriately selected according to the purpose, and either adherent culture or suspension culture can be used. Among these methods, suspension culture is preferred due to its yield per unit volume of culture medium and ease of operation.

[0043] The aforementioned "nucleic acid" can also be called "polynucleotide," and examples include deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), but DNA is preferred. The DNA may be single-stranded, double-stranded, or triple-stranded, but double-stranded DNA is preferred. There are no particular restrictions on the structure of the double-stranded DNA, and it can be appropriately selected depending on the purpose, and examples include circular plasmid DNA, linear plasmid DNA, ring-open plasmid DNA, circular DNA, linear DNA, and linear covalent closed DNA. Among these, circular plasmid DNA, circular DNA, and linear covalent closed DNA are preferred, and linear covalent closed DNA is more preferred in terms of gene transfer efficiency. The aforementioned "Linear Covalently Closed DNA" refers to a linear double-stranded DNA with a hairpin-shaped closed end structure, and can also be abbreviated as "LCC DNA".

[0044] There are no particular restrictions on the method of introducing the nucleic acid, and it can be appropriately selected according to the purpose. Examples include methods using cationic polymers such as polyethyleneimine (PEI), methods using liposomes, methods using calcium phosphate, methods using branched organic compounds such as dendrimers, methods using diethylaminoethyl (DEAE)-dextran, electroporation, particle gun methods, microinjection, magnetofection, and methods using viral vectors. Among these methods, the method using cationic polymers is preferred due to its high introduction efficiency, low reagent cost, and the fact that it does not require special equipment.

[0045] <Step of adding cell proliferation inhibitor A to cells> The cell proliferation inhibitor A is not particularly limited and can be appropriately selected depending on the purpose. Examples include compounds that inhibit the progression of the cell cycle. The compound may be used alone or in combination of two or more. The aforementioned cell proliferation inhibitor A does not contain any compounds that induce cell death, such as sodium valproate. Examples of compounds that inhibit the progression of the cell cycle include compounds that inhibit progression in the G0 phase of the cell cycle, compounds that inhibit progression in the G1 phase of the cell cycle, compounds that inhibit progression in the S phase of the cell cycle, and compounds that inhibit progression in the G2 or M phase of the cell cycle (corresponding to the cell proliferation inhibitor a). Among these, compounds that inhibit progression during the G2 or M phase of the cell cycle are preferred because they enhance the viral vector production induction effect. The compounds that inhibit progression during the G2 or M phase of the cell cycle are as described above in (Viral Vector Production Inducers). In the step of adding cell proliferation inhibitor A to the cells, other components may be added together with the cell proliferation inhibitor A. These other components are as described above in (Viral vector production inducer).

[0046] The timing for adding the cell proliferation inhibitor A is not particularly limited, as long as it is between 6 hours and 6 hours after the introduction of the nucleic acid, and can be appropriately selected depending on the purpose. However, for simplicity and to improve the viral vector production induction effect, it is more preferable to add the inhibitor between 4 hours and 4 hours after the introduction of the nucleic acid, even more preferable to add it between 5 minutes and 4 hours after, even more preferable to add it between 5 minutes and 2 hours after, and particularly preferable to add it simultaneously and 2 hours after. There are no particular restrictions on the number of times the cell proliferation inhibitor A can be added, and it can be appropriately selected depending on the purpose. For example, the entire amount can be added at once, or it can be added in multiple installments.

[0047] There are no particular restrictions on the method of introducing the nucleic acid; the transfection reagent and nucleic acid can be added after forming a complex, or the transfection reagent and nucleic acid can be added separately. Furthermore, the entire amount of the transfection reagent and nucleic acid can be added at once, or they can be added in multiple installments. When introducing the nucleic acid, if an introduction reagent (transfection reagent) is used during the introduction of the nucleic acid, it refers to the time when the introduction reagent is added, and if an electric current, pressure, etc. is applied during the introduction of the nucleic acid, it refers to the time when an electric current, pressure, etc. is applied. If the introduction reagent is added in multiple doses, the term "time of adding the introduction reagent" refers to the first addition.

[0048] There are no particular restrictions on the lower limit of the final concentration of the cell proliferation inhibitor A added, and it can be appropriately selected depending on the purpose. However, from the standpoint of improving the viral vector production induction effect, a concentration of 1 nM or higher is preferred, and further, in the following order, concentrations of 10 nM or higher, 100 nM or higher, 200 nM or higher, 250 nM or higher, 300 nM or higher, and 400 nM or higher are more preferred. There are no particular restrictions on the upper limit of the final concentration of the cell proliferation inhibitor A added, and it can be appropriately selected depending on the purpose. However, from the standpoint of improving the viral vector production induction effect, a concentration of 100 μM or less is preferred, and further, in the following order, 10 μM or less, 5 μM or less, 2.5 μM or less, 1 μM or less, 800 nM or less, and 600 nM or less are more preferred.

[0049] When the cell proliferation inhibitor A is nocodazole, there are no particular restrictions on the final concentration of nocodazole, and it can be appropriately selected depending on the purpose. However, from the viewpoint of improving the viral vector production induction effect, a concentration of 100 nM to 5 μM is preferred, 250 nM to 2 μM is more preferred, 300 nM to 1 μM is even more preferred, and 400 nM to 800 nM is particularly preferred.

[0050] When the cell proliferation inhibitor A is albendazole, there are no particular restrictions on the final concentration of albendazole, and it can be appropriately selected depending on the purpose. However, from the standpoint of improving the viral vector production induction effect, a concentration of 500 nM to 20 μM is preferred, and 1 μM to 10 μM is more preferred.

[0051] When the cell proliferation inhibitor A is oxybendazole, there are no particular restrictions on the final concentration of oxybendazole, and it can be appropriately selected depending on the purpose. However, from the viewpoint of improving the viral vector production induction effect, a concentration of 500 nM to 200 μM is preferred, 1 μM to 150 μM is more preferred, and 10 μM to 100 μM is even more preferred.

[0052] When the cell proliferation inhibitor A is mebendazole, there are no particular restrictions on the final concentration of mebendazole, and it can be appropriately selected depending on the purpose. However, from the viewpoint of improving the viral vector production induction effect, a concentration of 500 nM to 200 μM is preferred, 1 μM to 150 μM is more preferred, and 10 μM to 100 μM is even more preferred.

[0053] When the cell proliferation inhibitor A is vinblastine, there are no particular restrictions on the final concentration of vinblastine, and it can be appropriately selected depending on the purpose. However, from the viewpoint of improving the viral vector production induction effect, a concentration of 10 nM to 10 μM is preferred, 50 nM to 10 μM is more preferred, and 100 nM to 5 μM is even more preferred.

[0054] When the cell proliferation inhibitor A is colsemid, there are no particular restrictions on the final concentration of colsemid, and it can be appropriately selected depending on the purpose. However, from the viewpoint of improving the viral vector production induction effect, a concentration of 10 nM to 10 μM is preferred, 50 nM to 10 μM is more preferred, and 100 nM to 5 μM is even more preferred.

[0055] <Other processes> The other steps are not particularly limited and can be appropriately selected according to the purpose. For example, a cell culture step before the step of introducing nucleic acid into the cells, a cell culture step after the step of adding cell growth inhibitor A to the cells, and the like can be mentioned.

[0056] -Cell culture step before the step of introducing nucleic acid into the cells- The cell culture step before the step of introducing nucleic acid into the cells is not particularly limited and can be appropriately selected according to the purpose. For example, a method of culturing cells at 37 °C in the presence of 8% CO2, adjusting the number of cells, and then seeding them in a culture vessel can be mentioned.

[0057] The number of cells seeded in the culture vessel is not particularly limited and can be appropriately selected according to the purpose. However, from the viewpoint of improving the virus vector production induction effect, it is preferably 1×10 5 cells / mL or more and 1×10 9 cells / mL or less, more preferably 3×10 5 cells / mL or more and 1×10 8 cells / mL or less, and even more preferably 5×10 5 cells / mL or more and 1×10 7 cells / mL or less. The volume of the medium when seeding in the culture vessel is not particularly limited and can be appropriately selected according to the purpose. However, from the viewpoint of improving the virus vector production induction effect, it is preferably 0.1 mL or more and 1000 L or less, more preferably 1 mL or more and 100 L or less, even more preferably 3 mL or more and 10 L or less, and particularly preferably 5 mL or more and 1 L or less.

[0058] -Cell culture step after the step of adding cell growth inhibitor A to the cells- The cell culture step after the step of adding cell growth inhibitor A to the cells is not particularly limited and can be appropriately selected according to the purpose. For example, a method of culturing the cells after adding cell growth inhibitor A at 37 °C in the presence of 8% CO2 and extracting the virus vector from the cell culture solution containing the cells after culturing can be mentioned.

[0059] There are no particular restrictions on the number of culture days, and it can be appropriately selected depending on the purpose. However, from the standpoint of improving the viral vector production induction effect, it is preferable to have a culture period of 1 to 7 days, more preferably 2 to 6 days, and even more preferably 3 to 5 days.

[0060] There are no particular restrictions on the amount of viral vector obtained by the viral vector production method 2 described above, and it can be appropriately selected according to the purpose. However, the titer of the viral vector compared to the titer of the viral vector when the cell proliferation inhibitor A is not added is preferably 1.1 times or more, more preferably 1.2 times or more, even more preferably 1.5 times or more, even more preferably 2 times or more, and the higher the ratio, the better. The titer of the aforementioned viral vector is determined by quantitative PCR. The quantitative PCR method described above is as described in (Method 1 for producing viral vectors).

[0061] When the cell proliferation inhibitor A is not used, there are no particular restrictions on the titer of the viral vector, and it can be appropriately selected depending on the purpose. However, in order to improve the viral vector production induction effect, 1 × 10⁻⁶ 13 A vector genome of 1 / L or more is preferred, and 2 × 10 13 More preferably, vector genome / L or higher, 5 × 10 13 A vector genome of 1 / L or higher is even more preferable, and 8 × 10 13 More preferably, vector genome / L or higher, 1 × 10 14 A vector genome ratio of 1 / L or higher is particularly preferred.

[0062] (Method 1 for inducing viral vector production) A first aspect of the method for inducing the production of the viral vector is a method using a viral vector production inducer containing the cell proliferation inhibitor a.

[0063] There are no particular restrictions on the amount of viral vector obtained by the viral vector production induction method 1 described above, and it can be appropriately selected according to the purpose. However, the titer of the viral vector compared to the titer of the viral vector when the viral vector production induction agent is not used is preferably 1.1 times or more, more preferably 1.2 times or more, even more preferably 1.5 times or more, even more preferably 2 times or more, and the higher the ratio, the better. The titer of the aforementioned viral vector is determined by quantitative PCR. The quantitative PCR method described above is as described in (Method 1 for producing viral vectors).

[0064] There are no particular restrictions on the titer of the viral vector when the aforementioned viral vector production inducer is not used, and it can be appropriately selected depending on the purpose. However, in order to improve the viral vector production induction effect, 1 × 10 13 A vector genome of 1 / L or more is preferred, and 2 × 10 13 More preferably, vector genome / L or higher, 5 × 10 13 A vector genome of 1 / L or higher is even more preferable, and 8 × 10 13 More preferably, vector genome / L or higher, 1 × 10 14 A vector genome ratio of 1 / L or higher is particularly preferred.

[0065] The method using the aforementioned viral vector production inducer is as described above in (Method 1 for producing viral vectors).

[0066] (Method 2 for inducing viral vector production) A second embodiment of the induction of viral vector production includes the steps of introducing nucleic acids into cells and adding cell proliferation inhibitor A to the cells, and may further include other steps. The steps of introducing nucleic acids into the cells, adding cell proliferation inhibitor A to the cells, and the other steps are as described above in (Method 2 for producing a viral vector).

[0067] There are no particular restrictions on the amount of viral vector obtained by the viral vector production induction method 2 described above, and it can be appropriately selected according to the purpose. However, the titer of the viral vector compared to the titer of the viral vector when the cell proliferation inhibitor A is not added is preferably 1.1 times or more, more preferably 1.2 times or more, even more preferably 1.5 times or more, even more preferably 2 times or more, and the higher the ratio, the better. The titer of the aforementioned viral vector is determined by quantitative PCR. The quantitative PCR method described above is as described in (Method 1 for producing viral vectors).

[0068] When the cell proliferation inhibitor A is not used, there are no particular restrictions on the titer of the viral vector, and it can be appropriately selected depending on the purpose. However, in order to improve the viral vector production induction effect, 1 × 10⁻⁶ 13 A vector genome of 1 / L or more is preferred, and 2 × 10 13 More preferably, vector genome / L or higher, 5 × 10 13 A vector genome of 1 / L or higher is even more preferable, and 8 × 10 13 More preferably, vector genome / L or higher, 1 × 10 14 A vector genome ratio of 1 / L or higher is particularly preferred. [Examples]

[0069] The following describes embodiments of the present invention, but the present invention is not limited in any way to these embodiments.

[0070] <Cell culture> Suspension HEK293 cells (Thermo Fisher Scientific; FreeStyle® 293F cells) were seeded in culture vessels and cultured at 37°C in the presence of 8% CO2, with subculturing every 3 to 4 days.

[0071] <Nucleic acid> <<Production Example 1: Double-stranded circular plasmid DNA>> pRC2-mi342 (packaging plasmid DNA) and pHelper (helper plasmid DNA) were purchased from Takara Bio Inc. (AAVpro® Helper Free System). pAAV-Venus (vector plasmid DNA) was manufactured by replacing the nucleic acid sequence encoding green fluorescent protein (GFP) in pAAV-GFP Control Plasmid (Cell Biolabs) with the nucleic acid sequence encoding the fluorescent protein (Venus) (SEQ ID NO: 3).

[0072] <<Production example 2: Linear covalently closed DNA>> RC2_LCC_DNA, Helper_LCC_DNA, and Venus_LCC_DNA were prepared as follows.

[0073] In the following manufacturing examples 2-2, 2-3, and 2-4, the double-stranded circular plasmid DNA used for the transformation of E. coli was prepared by introducing the constructed vector into E. coli DH5α competent cells (9057, Takara Bio Inc.), culturing the resulting transformants, and amplifying the resulting DNA. Plasmid preparation from double-stranded circular plasmid DNA-carrying strains was performed using the FastGene Plasmid Mini Kit (Nippon Genetics Co., Ltd.).

[0074] <Manufacturing Example 2-1: Preparation of various genes used in vector preparation> The nucleic acid sequence (SEQ ID NO: 4) encoding Escherichia virus N15-derived protelomerase (TelN protelomerase), used in vector construction, was prepared by PCR using synthetic DNA as a template. This synthetic DNA is produced by methods such as synthesizing multiple oligonucleotides designed to overlap sequences, and then extending them to the desired chain length using DNA ligase or DNA polymerase after annealing. It can also be obtained by using artificial gene synthesis services provided by various companies.

[0075] The pair of nucleic acid sequences (telRL sequence) recognized by protelomerase used in vector construction were synthesized as a nucleic acid fragment (SEQ ID NO: 5).

[0076] The promoter-controlled AraC gene (SEQ ID NO: 6) used in vector construction was prepared by PCR using synthetic DNA as a template. This synthetic DNA is produced by methods such as synthesizing multiple oligonucleotides designed to overlap sequences, and then extending them to the desired chain length using DNA ligase or DNA polymerase after annealing. It can also be obtained by using artificial gene synthesis services provided by various companies.

[0077] The arabinose-derived promoter used in the construction of the vector was totally synthesized from a nucleic acid fragment (SEQ ID NO: 7).

[0078] The nucleic acid sequence encoding the fluorescent protein (Venus) used in vector construction (SEQ ID NO: 3) was prepared by PCR using synthetic DNA as a template. This synthetic DNA is produced by methods such as synthesizing multiple oligonucleotides designed to overlap sequences, and then extending them to the desired chain length using DNA ligase or DNA polymerase after annealing. It can also be obtained by using artificial gene synthesis services provided by various companies.

[0079] The nucleic acid sequence encoding TelN protelomerase was prepared by PCR using primer 1 (sequence number 8 forward primer) and primer 2 (sequence number 9 reverse primer), the promoter-controlled AraC gene was prepared using primer 3 (sequence number 10 forward primer) and primer 4 (sequence number 11 reverse primer), and the nucleic acid sequence encoding the fluorescent protein (Venus) was prepared using primer 5 (sequence number 12 forward primer) and primer 6 (sequence number 13 reverse primer).

[0080] For the PCR described above, Prime STAR MAX DNA Polymerase (Takara Bio Inc.) was used, and the reaction conditions were carried out according to the method described in the attached manual.

[0081] <Manufacturing Example 2-2: Construction of a vector containing the target gene> Nucleic acid fragments were prepared by PCR using primer 5 (sequence number 12) and primer 6 (sequence number 13) to obtain the nucleic acid sequence encoding the fluorescent protein (Venus) (sequence number 3). These fragments were then replaced with the nucleic acid sequence encoding the green fluorescent protein (GFP) from pAAV-GFP Control Plasmid (CELL BioLABs) to construct pAAV-Venus.

[0082] Next, a nucleic acid fragment (SEQ ID NO: 5) of the pair of nucleic acid sequences (telRL sequence) recognized by protelomerase was prepared by total synthesis and inserted into the PciI and KasI sites of pAAV-Venus to construct pAAV-Venus_telRL.

[0083] Next, nucleic acid fragments were prepared from the nucleic acid sequence encoding TelN protelomerase (SEQ ID NO: 4) by PCR using primer 1 (SEQ ID NO: 8) and primer 2 (SEQ ID NO: 9), from the promoter-controlled AraC gene (SEQ ID NO: 6) by PCR using primer 3 (SEQ ID NO: 10) and primer 4 (SEQ ID NO: 11), and from the nucleic acid fragment of the arabinose-inducible promoter (SEQ ID NO: 7) by total synthesis. Each of these was then inserted into the PsiI site of pAAV-Venus_telRL to construct pAAV-Venus_telRL_ara_TelN (Figure 12).

[0084] The pAAV-Venus_telRL_ara_TelN vector is a vector for producing linear covalent closed DNA, and is designed so that TelN protelomerase is expressed under the control of an arabinose-inducible promoter. Furthermore, the telRL sequence is used as the pair of nucleic acid sequences recognized by the protelomerase, and the nucleic acid sequence encoding the fluorescent protein (Venus) is located between the telRL sequences as the nucleic acid sequence encoding the target protein (target gene).

[0085] Figure 12 shows telRL, a pair of nucleic acid sequences (telRL sequence) recognized by protelomerase. Figure 12 shows ITR, a terminal inversion sequence. Figure 12 shows CMV Promotor, a promoter derived from cytomegalovirus. Figure 12 shows Venus, a nucleic acid sequence encoding the fluorescent protein (Venus). Figure 12 shows PolyA, a sequence involved in the polyadenylation of messenger RNA. Figure 12 shows AraC, a promoter-regulated AraC gene sequence. Figure 12 shows Arabinose-inducible Promoter, a nucleic acid sequence of the arabinose-inducible promoter. Figure 12 shows TelN, a nucleic acid sequence encoding TelN protelomerase. Figure 12 shows ampR, a nucleic acid sequence encoding the antibiotic resistance protein (AmpR).

[0086] <Manufacturing Example 2-3: Construction of a vector containing a packaging gene> A nucleic acid fragment (SEQ ID NO: 5) of a pair of nucleic acid sequences (telRL sequence) recognized by protelomerase was prepared by total synthesis and inserted into the EcoRV site and SnaBI site of pRC2-mi342 (Takara Bio Inc.) to construct pRC2-mi342_telRL. Next, nucleic acid fragments were prepared from the nucleic acid sequence encoding TelN protelomerase (SEQ ID NO: 4) by PCR using primer 1 (SEQ ID NO: 8) and primer 2 (SEQ ID NO: 9). Nucleic acid fragments were also prepared from the promoter-controlled AraC gene (SEQ ID NO: 6) by PCR using primer 3 (SEQ ID NO: 10) and primer 4 (SEQ ID NO: 11). Nucleic acid fragments of the arabinose-inducible promoter (SEQ ID NO: 7) were prepared by total synthesis. Each of these was inserted into the SmaI site of pRC2-mi342_telRL to construct pRC2-mi342_telRL_ara_TelN (Figure 13).

[0087] The pRC2-mi342_telRL_ara_TelN vector is a vector for producing linear covalent closed DNA, and is designed so that TelN protelomerase is expressed under the control of an arabinose-inducible promoter. Furthermore, the telRL sequence is used as the pair of nucleic acid sequences recognized by the protelomerase, and the nucleic acid sequence encoding the packaging protein (Rep: SEQ ID NO: 14, Cap: SEQ ID NO: 15) is located between the telRL sequences.

[0088] Figure 13 shows that Rep is the nucleic acid sequence encoding the adeno-associated virus packaging protein Rep. Figure 13 also shows that Cap is the nucleic acid sequence encoding the adeno-associated virus packaging protein Cap.

[0089] <Manufacturing Example 2-4: Construction of a vector containing a helper gene> A nucleic acid fragment (SEQ ID NO: 5) of a pair of nucleic acid sequences (telRL sequence) recognized by protelomerase was prepared by total synthesis and inserted into the BamHI and SalI sites of pHelper (Takara Bio Inc.) to construct pHelper_telRL. Next, nucleic acid fragments were prepared from the nucleic acid sequence encoding TelN protelomerase (SEQ ID NO: 4) by PCR using primer 1 (SEQ ID NO: 8) and primer 2 (SEQ ID NO: 9), from the promoter-controlled AraC gene (SEQ ID NO: 6) by PCR using primer 3 (SEQ ID NO: 10) and primer 4 (SEQ ID NO: 11), and from the nucleic acid fragment of the arabinose-inducible promoter (SEQ ID NO: 7) by total synthesis. Each of these was then inserted into the NdeI site of pHelper_telRL to construct pHelper_telRL_ara_TelN (Figure 14).

[0090] The pHelper_telRL_ara_TelN vector is a vector for producing linear covalent closed DNA, and is designed so that TelN protelomerase is expressed under the control of an arabinose-inducible promoter. Furthermore, the telRL sequence is used as the pair of nucleic acid sequences recognized by the protelomerase, and the nucleic acid sequences encoding helper proteins (E2A: SEQ ID NO: 16, E4: SEQ ID NO: 17, VA: SEQ ID NO: 18) are located between the telRL sequences.

[0091] Figure 14 shows that E2A is the nucleic acid sequence encoding the adenovirus helper protein E2A. Figure 14 shows that E4 is the nucleic acid sequence encoding the adenovirus helper protein E4. Figure 14 shows that VA is the nucleic acid sequence encoding the adenovirus helper protein VA.

[0092] <Manufacturing Example 2-5: Obtaining Transformed E. coli> E. coli was transformed as follows using the linear covalent closed DNA vector pAAV-Venus_telRL_ara_TelN constructed in Production Example 2-2, the linear covalent closed DNA vector pRC2-mi342_telRL_ara_TelN constructed in Production Example 2-3, or the linear covalent closed DNA vector pHelper_telRL_ara_TelN constructed in Production Example 2-4.

[0093] A 25 μL solution of competent cells of Escherichia coli NEB10β strain (New England Biolabs, C3019H) was mixed with a vector solution for producing linear covalent closed DNA containing 100 pg of pAAV-Venus_telRL_ara_TelN, 100 pg of pRC2-mi342_telRL_ara_TelN, or 100 pg of pHelper_telRL_ara_TelN, and the mixture was left to stand on ice for 30 minutes. After standing for 30 minutes, the sample was heat-treated at 42°C for 45 seconds, and then left to stand on ice for 2 minutes.

[0094] After standing for 2 minutes, 225 μL of SOC medium was added, and the E. coli were spread onto LB agar medium (1% tryptone, 0.5% dried yeast extract, 1% sodium chloride, 0.005% carbenicillin disodium (Nacalai Tesque Co., Ltd.)). Strains that grew after static culture at 37°C for 1 day were selected, and E. coli into which the linear covalent closed DNA vector was introduced was obtained.

[0095] <Manufacturing Example 2-6: Culture of Transformed E. coli> The transformed Escherichia coli obtained in Production Example 2-5 was inoculated into 2 mL of Plusgrow II medium (4% Plusgrow II (Nacalai Tesque Co., Ltd.), 0.005% carbenicillin disodium), and incubated at 37°C for 6 hours with shaking to obtain the preculture. 100 μL of pre-culture solution was subpoenaed into 50 mL of Plusgrow II medium (4% Plusgrow II, 0.005% carbenicillin disodium), and the mixture was incubated at 37°C for 16 hours with shaking.

[0096] After 16 hours of shaking incubation, 50 mL of Plusgrow II medium (4% Plusgrow II, 0.005% carbenicillin disodium) and 1 mL of arabinose solution (10% arabinose) were added, and the mixture was incubated at 37°C for 1 hour with shaking. After 1 hour of shaking incubation, the bacterial cells were collected by centrifugation.

[0097] <<Evaluation of linear covalent closed DNA by electrophoresis>> The linear covalent closed DNA prepared from E. coli cells obtained in Production Example 2-6 was evaluated by electrophoresis using the method described below.

[0098] DNA was prepared from E. coli cells using the FastGene Plasmid Mini Kit (Genetics Japan Co., Ltd.). The obtained DNA solution was applied to wells of a 1% agarose gel prepared with TAE buffer and electrophoresis was performed at 100V for 40 minutes. Nucleic acid stains were used for staining, and UV light was used for detection. The results are shown in lanes 3, 5, and 7 of Figure 15. The evaluation results of DNA obtained from E. coli cells cultured in the same manner except that the 1 mL arabinose solution was not added are shown in lanes 2, 4, and 6 of Figure 15. In Figure 15, lane 1 contains a DNA marker (1kb DNA Ladder, 3412A, Takara Bio Inc.), lane 3 contains a sample from pAAV-Venus_telRL_ara_TelN, lane 5 contains a sample from pHelper_telRL_ara_TelN, lane 7 contains a sample from pRC2-mi342_telRL_ara_TelN, lane 2 contains a sample from pAAV-Venus_telRL_ara_TelN without the addition of arabinose solution, lane 4 contains a sample from pHelper_telRL_ara_TelN without the addition of arabinose solution, and lane 6 contains a sample from pRC2-mi342_telRL_ara_TelN without the addition of arabinose solution.

[0099] In the results shown in Figure 15, two bands derived from linear covalent closed DNA were observed in E. coli cells from culture medium supplemented with arabinose (lanes 3, 5, and 7) (indicated by arrows in Figure 15: a band derived from linear covalent closed DNA containing a nucleic acid sequence encoding the target protein, a band derived from linear covalent closed DNA containing a nucleic acid sequence encoding a packaging protein, or a band derived from linear covalent closed DNA containing a nucleic acid sequence encoding a helper protein, and a band derived from linear covalent closed DNA containing a nucleic acid sequence encoding TelN protelomerase). In contrast, no bands derived from linear covalent closed DNA were observed in E. coli cells from culture medium without arabinose supplementation (lanes 2, 4, and 6), and only a band derived from double-stranded circular plasmid DNA was observed. This suggests that by introducing a vector into E. coli containing a nucleic acid sequence encoding protelomerase, a pair of nucleic acid sequences recognized by the protelomerase, and a nucleic acid sequence located between the pair of nucleic acid sequences that encodes a protein, TelN protelomerase cleaved and rejoined the telRL sequences recognized by TelN protelomerase, and linear covalent closed DNA was produced that contains a nucleic acid sequence encoding a target protein located between the telRL sequences, a nucleic acid sequence encoding a packaging protein located between the telRL sequences, or a nucleic acid sequence encoding a helper protein located between the telRL sequences.

[0100] <Manufacturing Example 2-7: Purification of Linear Covalent Closed DNA> The linear covalent closed DNA obtained in Production Example 2-6 was treated with the restriction enzyme BstZ17I (New England Biolabs), and the linear covalent closed DNA was purified using the NucleoSpin Gel and PCR Clean-up Kit (Machley-Nagel). As a result, the linear covalent closed DNA and the vector for producing linear covalent closed DNA containing the nucleic acid sequence encoding TelN protelomerase were cleaved at their BstZ17I recognition sites. Next, the DNA was treated with Exonuclease (New England Biolabs) and purified into linear covalent closed DNA using the NucleoSpin Gel and PCR Clean-up Kit (Machley-Nagel). This process degraded nucleotides from the ends of the single-stranded or double-stranded DNA.

[0101] <<Evaluation of linear covalent closed DNA by electrophoresis>> The linear covalent closed DNA obtained in Production Example 2-7 was evaluated by electrophoresis using the method described below. The obtained DNA solution was applied to the wells of a 1% agarose gel prepared with TAE buffer, and electrophoresis was performed at 100V for 40 minutes. Nucleic acid stains were used for staining, and UV light was used for detection. The results are shown in Figure 16 (lanes 2, 3, and 4).

[0102] Here, a linear covalent closed DNA having a nucleic acid sequence encoding the target protein located between telRL sequences is denoted as Venus_LCC_DNA, a linear covalent closed DNA having a nucleic acid sequence encoding the packaging protein located between telRL sequences is denoted as RC2_LCC_DNA, and a linear covalent closed DNA having a nucleic acid sequence encoding the helper protein located between telRL sequences is denoted as Helper_LCC_DNA.

[0103] In Figure 16, lane 1 contains the DNA marker (1kb DNA Ladder, Takara Bio Inc., 3412A), lane 2 contains Venus_LCC_DNA, lane 3 contains Helper_LCC_DNA, and lane 4 contains RC2_LCC_DNA.

[0104] In the results shown in Figure 16, bands derived from linear covalent closed DNA containing nucleic acid sequences encoding proteins located between telRL sequences were observed, whereas bands derived from linear covalent closed DNA containing nucleic acid sequences encoding TelN protelomerase and from vectors for producing linear covalent closed DNA were not observed. This suggests that the restriction enzyme BstZ17I cleaved the nucleic acid sequence encoding TelN protelomerase, and the exonuclease degraded nucleotides from the ends of single-stranded or double-stranded DNA. In other words, it suggests that the ends of the linear covalent closed DNA obtained in Production Example 2-6 are indeed closed.

[0105] <Example 1: Effect of the final concentration of the cell proliferation inhibitor A (including the cell proliferation inhibitor a) on the titer of the adeno-associated virus vector> This Example 1 is an example that corresponds to any of the above-mentioned viral vector production method 1, viral vector production method 2, viral vector production induction method 1, and viral vector production induction method 2. On the day nucleic acids are introduced into the cells, the number of suspension HEK293 cells (Thermo Fisher Scientific; FreeStyle® 293F cells) is reduced to 1 × 10⁻¹⁶ 6 The seeds were adjusted to 5 mL of culture medium and seeded into 50 mL centrifuge tubes with filter caps. pRC2-mi342 (1 μg / μL, 3 μL, packaging plasmid DNA), pAAV-Venus (1 μg / μL, 1 μL, vector plasmid DNA), pHelper (1 μg / μL, 2 μL, helper plasmid DNA), and polyethyleneimine (PEI) PEI MAX (1 μg / μL, 12 μL, PolySciences) were mixed in Opti-MEM (Thermo Fisher Scientific) and allowed to stand to prepare a double-stranded circular plasmid DNA-PEI mixture. The nucleic acid was introduced into cells by adding the aforementioned double-stranded circular plasmid DNA-PEI mixture to the cell culture medium. Subsequently, AAV2 was produced by culturing the cells for 4 days at 37°C in the presence of 8% CO2. In addition to nocodazole (Cayman Chemical) as the cell proliferation inhibitor A, DMSO was used as a solubilizer to adjust the concentrations to 0 nM, 25 μM, 50 μM, and 200 μM. Four hours after nucleic acid introduction into the cells, 1% (50 μL) of nocodazole was added to 5 mL of cell culture medium to achieve final concentrations of 0 nM, 250 nM, 500 nM, and 2 μM.

[0106] The extraction of adeno-associated virus vectors from the cell culture medium was performed as follows. Four days after introducing nucleic acids into cells, the supernatant and cells were separated. The obtained supernatant was enriched with PEG8000 (Sigma-Aldrich) to concentrate the adeno-associated virus vector. Triton X-100 (Sigma-Aldrich) was added to the residue to lyse the cells, and then Kaneka Endonuclease (Kaneka Corporation) was added to degrade the nucleic acids. EDTA (Nippon Gene Co., Ltd.) was added, and the mixture was centrifuged to obtain the adeno-associated virus vector (supernatant-derived sample). The obtained cells were lysed with Triton X-100 (Sigma-Aldrich), and then the nucleic acids were degraded with Kaneka Endonuclease (Kaneka Corporation). EDTA (Nippon Gene Co., Ltd.) was added, and the cells were centrifuged to obtain an adeno-associated virus vector (cell-derived sample).

[0107] The adeno-associated virus vector was quantified as follows. The titers of adeno-associated virus vectors contained in supernatant-derived samples and cell-derived samples were measured by quantitative PCR (QuantStudio3, SYBR-Green method) using forward primers (SEQ ID NO: 1) and reverse primers (SEQ ID NO: 2), as described below.

[0108] The quantitative PCR used a sample consisting of 12.5 μL of PowerUp® SYBR® Green Master Mix (A25742, Thermo Fisher Scientific), 0.125 μL of forward primer (50 μM, SEQ ID NO: 1), 0.125 μL of reverse primer (50 μM, SEQ ID NO: 2), 11.25 μL of Milli-Q® water, and 1 μL of standard or a 5000-fold diluted sample (total 25 μL). PCR was performed on the above sample using QuantStudio3 (Thermo Fisher Scientific) by repeating the following cycles 30 times: 94°C / 15 seconds (thermal denaturation), 60°C / 30 seconds (annealing), and 72°C / 30 seconds (extension reaction). The above standard was prepared by linearizing a pAAV-MCS Expression Vector (0.67 μg / μL, TE solution, Cell Biolabs) by digesting it in PvuII (1243A, Takara Bio Inc.) at 37°C for 2 hours.

[0109] Figure 1 shows the effect of the final concentration of nocodazole, a cell proliferation inhibitor A, on the titer of adeno-associated virus vectors. Figure 1 shows the total titer of adeno-associated virus vectors contained in cell-derived samples and supernatant-derived samples. "0 nM" in Figure 1 indicates that the final concentration of nocodazole was 0 nM, and only 50 μL of DMSO was added 4 hours after nucleic acid was introduced into the cells. The results shown in Figure 1 indicate that adding nocodazole, a cell proliferation inhibitor A, to the cell culture medium to a final concentration of 2 μM from 250 nM improves the titer of the adeno-associated virus vector.

[0110] <Example 2: Effect of the timing of adding the cell proliferation inhibitor A (containing the cell proliferation inhibitor a) on the titer of the adeno-associated virus vector> This Example 2 is an example that corresponds to any of the above-mentioned viral vector production method 1, viral vector production method 2, viral vector production induction method 1, and viral vector production induction method 2 (when cell proliferation inhibitor A is added 4 hours before introducing nucleic acid into cells, and 1 minute, 2 hours, and 4 hours after introducing nucleic acid into cells, it corresponds to viral vector production method 2 and viral vector production induction method 2). Nucleic acids were introduced into cells using the same method as in Example 1. Adeno-associated virus vectors were prepared under different conditions, with a constant final concentration of cell proliferation inhibitor A, and their titers were measured. Nocodazole was used as cell proliferation inhibitor A, and DMSO was used as a solubilizer to adjust the concentration to 50 μM. 1% of nocodazole (50 μL) was added to 5 mL of cell culture medium at 24 hours, 20 hours, and 4 hours before introducing nucleic acids into cells, and at 1 minute, 2 hours, 4 hours, and 24 hours after introducing nucleic acids into cells, so that the final concentration of nocodazole was 500 nM.

[0111] Figure 2 shows the effect of the timing of adding cell proliferation inhibitor A on the titer of the adeno-associated virus vector. Figure 2 shows the titer of the adeno-associated virus vector contained in the cell-derived sample. In Figure 2, "DMSO" indicates that only 50 μL of DMSO was added 1 minute after nucleic acid was introduced into the cells. "1m" indicates that cell proliferation inhibitor A was added 1 minute after nucleic acid was introduced into the cells, "4h" indicates that it was added 4 hours after nucleic acid was introduced into the cells, and "-4h" indicates that cell proliferation inhibitor A was added 4 hours before nucleic acid was introduced into the cells. The results shown in Figure 2 indicate that adding cell proliferation inhibitor A to the cell culture medium from 24 hours before to 4 hours after introducing nucleic acids into cells improves the titer of the adeno-associated virus vector.

[0112] <Example 3: Comparison of various cell proliferation inhibitors A (including the aforementioned cell proliferation inhibitor a)> This third example is an example that corresponds to any of the above-mentioned methods for producing a viral vector (method 1), methods for producing a viral vector (method 2), methods for inducing viral vector production (method 1), and methods for inducing viral vector production (method 2). Nucleic acids were introduced into cells using the same method as in Example 1, and adeno-associated virus vectors were prepared under different conditions using different types of cell proliferation inhibitor A and solubilizers, and their titers were measured. Each cell proliferation inhibitor A was adjusted with a solubilizer to a concentration 100 times the target final concentration, as in Example 1, and 1% (50 μL) was added to 5 mL of cell culture medium 4 hours after nucleic acids were introduced into the cells.

[0113] If the cell proliferation inhibitor A is nocodazole, DMSO is used as a solubilizer and it is added to the cell culture medium so that the final concentration of nocodazole is 500 nM. If the cell proliferation inhibitor A is fenbendazole (Fujifilm Wako Pure Chemical Industries, Ltd.), albendazole (Tokyo Chemical Industries, Ltd.), or mebendazole (Tokyo Chemical Industries, Ltd.), DMSO is used as a solubilizer and it is added to the cell culture medium so that the final concentrations of cell proliferation inhibitor A are 50 μM, 5 μM, and 500 nM. If the cell proliferation inhibitor A is vinblastine sulfate (Fujifilm Wako Pure Chemical Industries, Ltd.), colsemid (Cayman Chemical), paclitaxel (Fujifilm Wako Pure Chemical Industries, Ltd.), or docetaxel (LKT Labs), DMSO is used as a solubilizer and it is added to the cell culture medium so that the final concentrations of cell proliferation inhibitor A are 5 μM, 500 nM, and 100 nM. When the cell proliferation inhibitor A is aphydicolin (Fujifilm Wako Pure Chemical Industries, Ltd.) or MG-132 (Fujifilm Wako Pure Chemical Industries, Ltd.), DMSO is used as a solubilizer, and the inhibitor A is added to the cell culture medium so that the final concentrations of the inhibitor A are 100 nM, 10 nM, and 1 nM. When the cell proliferation inhibitor A is genistein (Fujifilm Wako Pure Chemical Industries, Ltd.) or roscovitine (Fujifilm Wako Pure Chemical Industries, Ltd.), DMSO is used as a solubilizer, and the inhibitor A is added to the cell culture medium so that the final concentrations of the inhibitor A are 10 μM, 500 nM, and 100 nM. When the cell proliferation inhibitor A is thymidine (Tokyo Chemical Industries, Ltd.), water is used as a solubilizer, and the inhibitor is added to the cell culture medium so that the final concentrations of thymidine are 10 mM, 1 mM, and 0.1 mM.

[0114] Figures 3 to 7 show the effects of the addition of various cell proliferation inhibitors A on the titer of adeno-associated virus vectors. Figures 3 to 7 show the titer of adeno-associated virus vectors contained in cell-derived samples. In Figures 3 to 7, "w / o" indicates that no cell proliferation inhibitor A or solubilizer was added. "DMSO" indicates that only 50 μL of DMSO was added 4 hours after nucleic acid was introduced into the cells. "NDZ" indicates that nocodazole was added as cell proliferation inhibitor A. "VPA" indicates that sodium valproate was added as cell proliferation inhibitor A.

[0115] The results shown in Figures 3 to 7 indicate that the titer of adeno-associated virus vectors is improved not only by nocodazole, but also by using compounds that inhibit progression in the G2 or M phase of the cell cycle as cell proliferation inhibitor A, such as benzimidazole compounds like albendazole, mebendazole, and fenbendazole, vinca alkaloid compounds like vinblastine, colchicine derivatives like colsemid, and taxane compounds like paclitaxel and docetaxel.

[0116] <Comparative Example 1: VPA (Sodium Valproate)> Nucleic acids were introduced into cells using the same method as in Example 1, and adeno-associated virus vectors were prepared and their titers measured under conditions in which sodium valproate (VPA, Tokyo Chemical Industry Co., Ltd.), a viral vector production inducer that is not a cell proliferation inhibitor A, was added. Sodium valproate was added to the cell culture medium using water as a solubilizer to achieve final concentrations of 1 mM, 2.5 mM, 5 mM, 10 mM, and 12.5 mM. As a control, nocodazole (Cayman Chemical), a cell proliferation inhibitor A, was used. Nocodazole was added using DMSO as a solubilizer to achieve a final concentration of 500 nM.

[0117] Figure 8 shows a comparison of the effects of adding cell proliferation inhibitor A or VPA (sodium valproate) on the titer of adeno-associated virus vectors. Figure 8 shows the titer of adeno-associated virus vectors contained in cell-derived samples. In Figure 8, "w / o" indicates that cell proliferation inhibitor A and a solubilizer were not added. "NDZ" indicates that nocodazole was added as cell proliferation inhibitor A. "VPA" indicates that sodium valproate was added. The results in Figure 8 show that viral vector production inducers containing cell proliferation inhibitor A are more effective in improving the titer of adeno-associated virus vectors than viral vector production inducers containing sodium valproate (HDAC inhibitor).

[0118] <Example 4: Production of adeno-associated virus vectors using a bioreactor> This embodiment 4 is an embodiment that corresponds to any of the above-mentioned methods for producing a viral vector 1, method for producing a viral vector 2, method for inducing viral vector production 1, and method for inducing viral vector production 2. On the day nucleic acids are introduced into the cells, the number of suspended HEK293 cells is 1 × 10⁻⁶. 6 The cells were adjusted to cells / mL (400mL of culture medium) and seeded in a bioreactor (Animal cell culture device BCP, 1L volume, Able Corporation). pRC2-mi342 (1 μg / μL, 240 μL, packaging plasmid DNA), pAAV-Venus (1 μg / μL, 80 μL, vector plasmid DNA), pHelper (1 μg / μL, 160 μL, helper plasmid DNA), and polyethyleneimine (PEI) PEI MAX (1 μg / μL, 960 μL, PolySciences) were mixed in Opti-MEM (Thermo Fisher Scientific) and allowed to stand to prepare a double-stranded circular plasmid DNA-PEI mixture. The nucleic acid was introduced into cells by adding the aforementioned double-stranded circular plasmid DNA-PEI mixture to the cell culture medium. Subsequently, AAV2 was produced by culturing the cells for 4 days at 37°C in the presence of 8% CO2.

[0119] Four days after introducing nucleic acids into cells, the supernatant and cells were separated. The obtained supernatant was enriched with PEG8000 (Sigma-Aldrich) to concentrate the adeno-associated virus vector. Triton X-100 (Sigma-Aldrich) was added to the residue to lyse the cells, and then Kaneka endonuclease (Kaneka Corporation) was added to degrade the DNA. EDTA (Nippon Gene Co., Ltd.) was added, and the mixture was centrifuged to obtain the adeno-associated virus vector (supernatant-derived sample). The obtained cells were lysed with Triton X-100 (Sigma-Aldrich), and then the nucleic acids were degraded with Kaneka Endonuclease (Kaneka Corporation). EDTA (Nippon Gene Co., Ltd.) was added, and the cells were centrifuged to obtain an adeno-associated virus vector (cell-derived sample).

[0120] The adeno-associated virus vector was quantified using the same method as in Example 1.

[0121] Figure 9 shows the effect of the presence or absence of nocodazole as cell proliferation inhibitor A on the titer of adeno-associated virus vectors prepared using a bioreactor. Figure 9 shows the total titer of adeno-associated virus vectors contained in cell-derived samples and supernatant-derived samples. In Figure 9, "w / o" indicates that no cell proliferation inhibitor A or solubilizer was added, and "NDZ 500nM" indicates that 4 hours after introducing nucleic acid into cells, nocodazole as cell proliferation inhibitor A and DMSO as a solubilizer were adjusted to 50 μM, and then added at a concentration of 1% (4 mL) to 400 mL of cell culture medium so that the final concentration of nocodazole was 500 nM. The results shown in Figure 9 demonstrate that the cell proliferation inhibitor A increased the titer of the adeno-associated virus vector in Examples 1 to 3, and this was reproduced on a bioreactor scale. This indicates that adeno-associated virus vectors can be manufactured and their production induced using cell proliferation inhibitor A.

[0122] <Example 5: Preparation of an adeno-associated virus vector using cell proliferation inhibitor A (containing the cell proliferation inhibitor a) and LCC DNA> This Example 5 is an example that corresponds to any of the above-mentioned methods for producing a viral vector (Method 1), producing a viral vector (Method 2), inducing viral vector production (Method 1), and inducing viral vector production (Method 2). On the day nucleic acids are introduced into the cells, the number of suspended HEK293 cells is 1 × 10⁻⁶. 6 The seeds were adjusted to 5 mL of culture medium and seeded into 50 mL centrifuge tubes with filter caps.

[0123] Under conditions where double-stranded circular plasmid DNA was used as the nucleic acid to be introduced into cells, pRC2-mi342 (1 μg / μL, 4 μL, packaging plasmid DNA), pAAV-Venus (1 μg / μL, 1 μL, vector plasmid DNA), pHelper (1 μg / μL, 1 μL, helper plasmid DNA), and polyethyleneimine (PEI) PEI MAX (1 μg / μL, 12 μL, PolySciences) were mixed in Opti-MEM (Thermo Fisher Scientific) and allowed to stand to prepare a double-stranded circular plasmid DNA-PEI mixture. The nucleic acid was introduced into cells by adding the above double-stranded circular plasmid DNA-PEI mixture to the cell culture medium. Subsequently, AAV2 was produced by culturing for 4 days at 37°C in the presence of 8% CO2. Under conditions where linear covalent closed DNA was used as the nucleic acid to be introduced into cells, RC2_LCC_DNA (1 μg / μL, 4 μL, packaging plasmid DNA), Venus_LCC_DNA (1 μg / μL, 1 μL, vector plasmid DNA), Helper_LCC_DNA (1 μg / μL, 1 μL, helper plasmid DNA), and polyethyleneimine (PEI) PEI MAX (1 μg / μL, 12 μL, PolySciences) were mixed in Opti-MEM (Thermo Fisher Scientific) and allowed to stand to prepare an LCC DNA-PEI mixture. The nucleic acid was introduced into the cells by adding the LCC DNA-PEI mixture to the cell culture medium. Subsequently, AAV2 was produced by culturing for 4 days at 37°C in the presence of 8% CO2. The adeno-associated virus vector was extracted and quantified using the same method as in Example 1.

[0124] Figure 10 shows the titer of the adeno-associated virus vector contained in the cell-derived sample. In Figure 10, "pDNA" indicates that double-stranded circular plasmid DNA was used as the nucleic acid introduced into the cells, "LCC DNA" indicates that linear covalent closed DNA was used as the nucleic acid introduced into the cells, "w / o" indicates that no cell proliferation inhibitor A or solubilizer was added, and "NDZ 500nM" indicates that, one minute after introducing the nucleic acid into the cells, nocodazole was added as cell proliferation inhibitor A and DMSO as a solubilizer in the same manner as in Example 2, so that the final concentration of nocodazole was 500nM. Under conditions using double-stranded circular plasmid DNA, the titer of the adeno-associated virus vector increased eightfold with cell proliferation inhibitor A, while under conditions using linear covalent closed DNA, it increased fourteenfold. This indicates that the effect of cell proliferation inhibitor A was significantly enhanced under conditions using linear covalent closed DNA. In other words, it is preferable to use both cell proliferation inhibitor A and linear covalent closed DNA for the production of adeno-associated virus vectors.

[0125] Figure 11 shows the total titer of adeno-associated virus vectors obtained from cell-derived samples under conditions where LCC DNA was used as the nucleic acid introduced into cells. In Figure 11, "w / o" indicates that no cell proliferation inhibitor A or solubilizer was added, and "DMSO" indicates that only 50 μL of DMSO was added 1 minute after nucleic acid was introduced into the cells. "NDZ" indicates that nocodazole was added as cell proliferation inhibitor A and DMSO as a solubilizer 1 minute after nucleic acid was introduced into the cells, in the same manner as in Example 2, so that the final concentration of nocodazole was 500 nM. When cell proliferation inhibitor A was oxybendazole (Tokyo Chemical Industries, Ltd.), DMSO was used as a solubilizer, and oxybendazole was added to the cell culture medium to achieve four different final concentrations of oxybendazole: 50 μM, 5 μM, 500 nM, and 50 nM. The titer of the adeno-associated virus vector increased not only under conditions using nocodazole but also oxybendazole, indicating that nocodazole is not the only cell proliferation inhibitor A that is preferable for the production of adeno-associated virus vectors when used with linear covalent closed DNA.

[0126] <Example 6: Preparation of an adeno-associated virus vector using cell proliferation inhibitor A (containing the aforementioned cell proliferation inhibitor a) and a solubilizer other than DMSO> This embodiment 6 is an embodiment that corresponds to any of the above-mentioned methods for producing a viral vector 1, methods for producing a viral vector 2, methods for inducing viral vector production 1, and methods for inducing viral vector production 2. On the day nucleic acids are introduced into the cells, the number of suspended HEK293 cells is 1 × 10⁻⁶. 6 The seeds were adjusted to 5 mL of culture medium and seeded into 50 mL centrifuge tubes with filter caps.

[0127] pRC2-mi342 (1 μg / μL, 4 μL, packaging plasmid DNA), pAAV-Venus (1 μg / μL, 1 μL, vector plasmid DNA), pHelper (1 μg / μL, 1 μL, helper plasmid DNA), and polyethyleneimine (PEI) PEI MAX (1 μg / μL, 12 μL, PolySciences) were mixed in Opti-MEM (Thermo Fisher Scientific) and allowed to stand to prepare a double-stranded circular plasmid DNA-PEI mixture. The nucleic acid was introduced into the cells by adding the aforementioned double-stranded circular plasmid DNA-PEI mixture to the cell culture medium. Subsequently, the cells were cultured for 4 days at 37°C in the presence of 8% CO2 to produce or induce AAV2 production. Nocodazole (Cayman Chemical) was added as the cell proliferation inhibitor A, and hydrochloric acid, formic acid aqueous solution, or ethanol was used as a solubilizer. One minute after the nucleic acid was introduced into the cells, the nocodazole was added to the cell culture medium so that the final concentration was 500 nM. The adeno-associated virus vector was extracted and quantified using the same method as in Example 1.

[0128] Examples of embodiments of the present invention include the following: <1> The viral vector production inducer is characterized by comprising a cell proliferation inhibitor a, wherein the cell proliferation inhibitor a contains a compound that inhibits progression in the G2 or M phase of the cell cycle. <2> The compound that inhibits progression in the G2 or M phase of the cell cycle includes a compound that inhibits microtubule polymerization or a compound that stabilizes microtubules. <1> It is a viral vector production inducer as described above. <3> The compound that inhibits progression in the G2 or M phase of the cell cycle includes a benzimidazole compound, a vinca alkaloid compound, or a colchicine derivative. <1> or <2> It is a viral vector production inducer as described above. <4> The compound that inhibits progression in the G2 or M phase of the cell cycle comprises at least one selected from nocodazole, albendazole, mebendazole, vinblastine, colsemid, thiabendazole, fenbendazole, triclabendazole, flubendazole, oxybendazole, parbendazole, paclitaxel, and docetaxel. <1> from <3> It is a viral vector production inducer as described in any of the following. <5> The solubilizing agent comprises water, hydrochloric acid, formic acid aqueous solution, ethanol, or DMSO. <1> from <4> It is a viral vector production inducer as described in any of the following. <6> The viral vector is an adeno-associated virus vector. <1> from <5> It is a viral vector production inducer as described in any of the following. <7> The aforementioned <1> from <6> This method is characterized by using a viral vector production inducer described in any of the following. <8> A method for producing a viral vector, comprising the steps of introducing nucleic acids into cells and adding a cell proliferation inhibitor A to the cells, characterized in that the cell proliferation inhibitor A is added 6 hours before to 6 hours after the introduction of the nucleic acids. <9> In the step of adding cell proliferation inhibitor A to the cells, the cell proliferation inhibitor A and a solubilizer such as water, hydrochloric acid, formic acid aqueous solution, ethanol, or DMSO are added. <8> This is a method for producing a viral vector as described above. <10> The viral vector is an adeno-associated virus vector. <8> or <9> This is a method for producing a viral vector as described above. <11> The nucleic acid is a linear covalent closed DNA, <8> from <10> This is a method for producing a viral vector as described in any of the following. <12> The aforementioned <1> from <6> This method for inducing viral vector production is characterized by using a viral vector production inducer described in any of the above. <13> The method for inducing viral vector production includes the steps of introducing nucleic acids into cells and adding a cell proliferation inhibitor A to the cells, characterized in that the cell proliferation inhibitor A is added 6 hours before to 6 hours after the introduction of the nucleic acids. <14> In the step of adding cell proliferation inhibitor A to the cells, the cell proliferation inhibitor A and a solubilizer such as water, hydrochloric acid, formic acid aqueous solution, ethanol, or DMSO are added. <13> This is a method for inducing viral vector production as described above. <15> The viral vector is an adeno-associated virus vector. <13> or <14> This is a method for inducing the production of the viral vector described in [reference]. <16> The nucleic acid is a linear covalent closed DNA. <13> from <15> This is a method for inducing the production of a viral vector as described in any of the following.

[0129] This international application claims priority based on Japanese Patent Application No. 2021-162665, filed on 1 October 2021, and the entire contents of Japanese Patent Application No. 2021-162665 are incorporated herein by reference.

Claims

1. Adeno-associated virus vector production inducer, The adeno-associated virus vector production inducer comprises a compound that inhibits microtubule polymerization or stabilizes microtubules. The adeno-associated virus vector production inducer is intended to be added to cells into which linear covalent closed DNA is introduced. The compound is selected from the group consisting of benzimidazole compounds, vinca alkaloid compounds, colchicine derivatives, and taxane compounds. The benzimidazole compound is selected from the group consisting of nocodazole, albendazole, mebendazole, thiabendazole, fenbendazole, triclabendazole, flubendazole, oxybendazole, parbendazole, oxfendazole, lycobendazole, and salts thereof. The vinca alkaloid compound is selected from the group consisting of vinblastine, vincristine, vindesine, vinorelbine, vinflunin, and salts thereof. The colchicine derivative is selected from the group consisting of colchicine, colsemid, allocolchicine, thiocolchicine, and salts thereof. The taxane compound is paclitaxel or docetaxel, the adeno-associated virus vector production inducer.

2. The adeno-associated virus vector production inducer according to claim 1, comprising water, hydrochloric acid, aqueous formic acid solution, ethanol, and / or DMSO as a solubilizing agent.

3. The adeno-associated virus vector production inducer according to claim 1, wherein the linear covalent closed DNA comprises a nucleic acid sequence encoding a target protein and / or a terminal inversion sequence.

4. The adeno-associated virus vector production inducer according to claim 1, wherein linear covalent closed DNA encoding a packaging protein and / or a helper protein is further introduced into the cell.

5. A method for producing an adeno-associated virus vector, The process of introducing linear covalent closed DNA into cells, Addition step, adding a compound that inhibits microtubule polymerization or stabilizes microtubules to the cells, and A culture step in which the cells after the introduction step and the addition step are cultured. Includes, The compound is selected from the group consisting of benzimidazole compounds, vinca alkaloid compounds, colchicine derivatives, and taxane compounds. The benzimidazole compound is selected from the group consisting of nocodazole, albendazole, mebendazole, thiabendazole, fenbendazole, triclabendazole, flubendazole, oxybendazole, parbendazole, oxfendazole, lycobendazole, and salts thereof. The vinca alkaloid compound is selected from the group consisting of vinblastine, vincristine, vindesine, vinorelbine, vinflunin, and salts thereof. The colchicine derivative is selected from the group consisting of colchicine, colsemid, allocolchicine, thiocolchicine, and salts thereof. The method wherein the taxane compound is paclitaxel or docetaxel.

6. The method according to claim 5, wherein the addition step is performed from 6 hours before to 6 hours after the introduction step.

7. The method according to claim 5, wherein the addition step further involves adding water, hydrochloric acid, aqueous formic acid solution, ethanol, and / or DMSO as a solubilizing agent to the cells.

8. The method according to claim 5, wherein the linear covalent closed DNA comprises a nucleic acid sequence encoding a target protein and / or a terminal inversion sequence.

9. The method according to claim 5, wherein the introduction step further introduces linear covalent closed DNA encoding a packaging protein and / or a helper protein into the cells.

10. A method for inducing the production of an adeno-associated virus vector, Addition step, in which a compound that inhibits microtubule polymerization or stabilizes microtubules is added to cells into which linear covalent closed DNA is introduced, and The addition step and the culture step of culturing the cells after introduction. Includes, The compound is selected from the group consisting of benzimidazole compounds, vinca alkaloid compounds, colchicine derivatives, and taxane compounds. The benzimidazole compound is selected from the group consisting of nocodazole, albendazole, mebendazole, thiabendazole, fenbendazole, triclabendazole, flubendazole, oxybendazole, parbendazole, oxfendazole, lycobendazole, and salts thereof. The vinca alkaloid compound is selected from the group consisting of vinblastine, vincristine, vindesine, vinorelbine, vinflunin, and salts thereof. The colchicine derivative is selected from the group consisting of colchicine, colsemid, allocolchicine, thiocolchicine, and salts thereof. The method wherein the taxane compound is paclitaxel or docetaxel.

11. The method according to claim 10, wherein the addition step is performed from six hours before to six hours after the introduction.

12. The method according to claim 10, wherein the linear covalent closed DNA comprises a nucleic acid sequence encoding a target protein and / or a terminal inversion sequence.

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