Method of virus vector production

Abstract

To provide methods for increasing the amount of virus vectors produced in a virus vector producing process using cultured cells.SOLUTION: Provided herein is a method for producing a virus vector, comprising culturing a cell comprising nucleic acids necessary for formation of virus particles of the virus vector and a nucleic acid to be delivered by the virus vector under conditions that activate protein kinase C (PKC), where the nucleic acids necessary for virus particle production are operably linked to a promoter comprising a binding site of at least one transcription factor selected from SP1, CEBP, AP1, NF-κB and YY1.SELECTED DRAWING: Figure 33

Description

[Technical field]

[0001] The present disclosure relates to methods for producing viral vectors. [Background technology]

[0002] Gene therapy is a field that is expected to grow in recent years. In gene therapy, various vectors are used to deliver the target gene to cells. Viral vectors are one type, and retroviral vectors, lentiviral vectors, adenoviral vectors, adeno-associated viral vectors, etc. are selected depending on the purpose.

[0003] Viral vectors are produced using cultured cells, similar to biological products (biopharmaceuticals) such as antibodies. Usually, nucleic acids required for the production of viral particles and nucleic acids of a gene of interest are expressed in cultured cells to produce a viral vector containing the gene of interest, and the viral vector released into the culture medium is then collected. Therefore, the amount of viral vector produced depends on the protein production capacity of the cells, the culture volume, and the culture time. If the protein production capacity of the cells can be increased, it is expected that the production amount of viral vectors can be increased while suppressing production costs. Summary of the Invention [Problem to be solved by the invention]

[0004] An object of the present disclosure is to increase the yield of viral vectors. [Means for solving the problem]

[0005] The inventors have demonstrated that activating PKC can increase the transcriptional activity of promoters such as the CMV promoter, and have discovered a new method for activating PKC, which can increase the production of viral vectors in cultured cells.

[0006] Thus, in one aspect, the disclosure provides a method for producing a viral vector, comprising culturing a cell containing nucleic acid necessary for production of viral particles of the viral vector and a nucleic acid to be delivered by the viral vector under conditions that activate PKC; The method is provided, wherein the nucleic acid necessary for the production of viral particles is operably linked to a promoter that contains a binding site for at least one transcription factor selected from SP1, CEBP, AP1, NF-κB and YY1.

[0007] In one aspect, the disclosure provides a kit for producing a viral vector, comprising a nucleic acid encoding a PKC activator or an activated form of PKC, and nucleic acids necessary for producing viral particles of the viral vector, The kit is provided, in which the nucleic acid required for production of viral particles is operably linked to a promoter that contains a binding site for at least one transcription factor selected from SP1, CEBP, AP1, NF-κB, and YY1.

[0008] In certain aspects, the disclosure provides a composition for enhancing production of a viral vector comprising a PKC activator or a nucleic acid encoding an activated form of PKC. Effect of the Invention

[0009] The present disclosure makes it possible to increase the production yield of viral vectors. [Brief description of the drawings]

[0010] [Figure 1] FIG. 1 shows luciferase activity in HEK293A cells constitutively expressing pRL-CMV (Renilla luciferase) in the presence of Compound X or any of Compounds #1 to #7, and in the presence or absence of a PKC inhibitor. [Diagram 2] 1 shows the change in luciferase activity in HEK293A cells constitutively expressing pRL-CMV (Renilla Luciferase) in the presence or absence of Compound X. [Diagram 3] FIG. 1 shows luciferase activity in HEK293A cells constitutively expressing pRL-CMV (Renilla luciferase) in the presence of compound X or any of PKC activators #1 to #4, and in the presence or absence of a PKC inhibitor. [Figure 4] FIG. 1 shows luciferase activity in HEK293A cells constitutively expressing pRL-CMV (Renilla luciferase) in the presence of compound X or any of PKC activators #5 to #6, and in the presence or absence of a PKC inhibitor. [Diagram 5] FIG. 1 shows luciferase activity in HEK293A cells transfected with pRL-CAG or pRL-EF1 in the presence of Compound X or any of Compounds #1 to #5 and in the presence or absence of a PKC inhibitor.

[0011] [Figure 6] FIG. 1 shows luciferase activity in HEK293A cells transfected with pRL-CAG in the presence of Compound X, Compounds #6 to #7, and PKC activators #1 to #6, and in the presence or absence of a PKC inhibitor. [Figure 7] FIG. 1 shows luciferase activity in HEK293A cells transfected with pRL-EF1 in the presence of any one of Compound X, Compounds #6 to #7, and PKC activators #1 to #6, and in the presence or absence of a PKC inhibitor. [Figure 8] FIG. 1 shows the phosphorylation state of PKC substrate proteins in HEK293A cells in the presence of any one of Compound X, Compounds #1 to #7, PKC activators #1 to #6, Compound SC, and Compound SB, and in the presence or absence of a PKC inhibitor. [Figure 9] 1 shows luciferase activity in HEK293A cells constitutively expressing pRL-CMV (Renilla Luciferase) in the presence of Compound X and / or Compound SC and in the presence or absence of a PKC inhibitor. [Figure 10]1 shows luciferase activity in HEK293A cells constitutively expressing pRL-CMV (Renilla Luciferase) in the presence of Compound X, Compound SC and / or SB.

[0012] [Figure 11] FIG. 1 shows luciferase activity in HEK293A cells constitutively expressing pRL-CMV (Renilla luciferase) in the presence of Compound X and any one of Compounds #1 to #7, and in the presence or absence of Compound SB or Compound SB+SC. [Figure 12] FIG. 1 shows luciferase activity in HEK293A cells constitutively expressing pRL-CMV (Renilla luciferase) in the presence of compound X and any one of PKC activators #1 to #6, and in the presence or absence of compound SB or compound SB+SC. [Figure 13] 1 shows luciferase activity in HEK293A cells constitutively expressing pRL-CMV (Renilla luciferase) in the presence of Compound X or Compound X + Compound SC, and in the presence of various histone deacetylase inhibitors. [Figure 14] FIG. 1 shows the results of transfecting HEK293A cells with an expression construct in which mouse IgG antibody heavy chain cDNA is linked under the control of a CMV promoter and an expression construct in which mouse IgG antibody light chain cDNA is linked under the control of a CMV promoter, culturing the cells in a medium containing compound X, compounds #1 to #5, or PKC activators #1 to #6, and in the presence or absence of compound SB+SC, and measuring the concentration of mouse IgG antibody in the medium. [Figure 15] HEK293A cells that constitutively express human proinsulin under the control of a CMV promoter were cultured in a medium containing compound X, compounds #1 to #5, PKC activator #1 to #3, #5 or #6, and in the presence or absence of compounds SB+SC, and the concentration of human proinsulin in the medium was measured.

[0013] [Figure 16] HEK293A cells were transfected with an expression construct in which human leptin cDNA was linked under the control of a CMV promoter, and cultured in a medium containing either compound X, compounds #1 to #5, or PKC activators #1 to #6, or in the presence or absence of compound SB+SC, and the concentration of human leptin in the medium was measured. [Figure 17] As in Figures 14 to 16, cells transiently or constitutively expressing mouse IgG antibody heavy and light chains, human proinsulin, or human leptin were cultured in medium containing Compound X, Compound #6, or Compound #7, and in the presence or absence of Compound SB+SC, and the concentrations of each protein in the medium were measured. [Figure 18] HEK293A cells that constitutively express mouse IgG antibody heavy and light chains, human proinsulin, or human leptin under the control of a CMV promoter were cultured in the presence or absence of Compound X or Compound X+SB, and the concentrations of each protein in the medium were measured over a period of 3 or 4 days. [Figure 19] FIG. 1 is a schematic diagram of an expression construct in which a CMV promoter, a protein cDNA (XXX), an IRES, and an activated PKC cDNA are linked. [Figure 20] The expression construct in which a CMV promoter, Rluc cDNA, IRES and activated PKC cDNA were linked was transfected into HEK293A cells, and luciferase activity was measured.

[0014] [Figure 21] The expression construct comprising a CMV promoter, Rluc cDNA, IRES or P2A, and activated PKC cDNA was transfected into HEK293A cells, and luciferase activity was measured. [Figure 22]An expression construct linking a CMV promoter, mouse IgG antibody heavy chain cDNA, IRES, and activated PKC cDNA, and an expression construct linking a CMV promoter, mouse IgG antibody light chain cDNA, IRES, and activated PKC cDNA were transfected into HEK293A cells, and the IgG antibody concentration in the medium was measured over a period of 4 days. [Figure 23] An expression construct linking a CMV promoter, human leptin cDNA, IRES and activated PKC cDNA was transfected into HEK293A cells, and the leptin concentration in the medium was measured over a period of 4 days. [Figure 24] 1 shows luciferase activity in HEK293A cells transfected with pRL-CMV in the presence of naphthalenesulfonamide derivatives and in the presence or absence of Compound X. [Diagram 25] FIG. 1 shows a schematic diagram of an expression construct in which a CMV promoter, Rluc cDNA, an IRES, and PML (wild-type or defective-type) cDNA are linked.

[0015] [Figure 26] 1 shows luciferase activity in the presence or absence of compound X in HEK293A cells transfected with an expression construct containing a CMV promoter, Rluc cDNA, an IRES, and PML (wild-type or deleted) cDNA. [Figure 27] Luciferase activity in HEK293A cells transfected with an expression construct containing a CMV promoter, Rluc cDNA, IRES and PMLΔ9 or PML-Ring cDNA in the presence or absence of Compound X, SB, Compound SC or a combination thereof is shown. [Figure 28] The amino acid sequence of the RING region of PML was analyzed by BLAST analysis, and a phylogenetic tree between genes with homologous amino acid sequences is shown. [Figure 29]Schematic diagram of an expression construct in which a CMV promoter, Rluc cDNA, and cDNAs of an IRES and RING region are linked. [Diagram 30] The figure shows luciferase activity in the presence or absence of compound X in HEK293A cells transfected with an expression construct comprising a CMV promoter, Rluc cDNA, and cDNAs for IRES and RING regions.

[0016] [Diagram 31] Schematic diagrams of an expression construct linking a CMV promoter, Rluc cDNA, IRES, and PML (with stop codon introduced) cDNA, and an expression construct linking a CMV promoter, Rluc cDNA, and PML (wild-type or deleted-type) cDNA are shown. [Diagram 32] The luciferase activity in the presence or absence of compound X is shown in HEK293A cells transfected with an expression construct linking a CMV promoter, Rluc cDNA, IRES, and PML (with a stop codon introduced) and an expression construct linking a CMV promoter, Rluc cDNA, and PML (wild-type or deleted-type) cDNA. [Diagram 33] A retroviral vector into which a GFP gene has been inserted was produced in Plat-E cells in the presence of compound X, compound SC and / or SB, and then infected into HEK293A cells, and the number of GFP-positive cells was measured. [Diagram 34] The lentiviral vector into which the GFP gene has been inserted was produced in Lenti-X293T cells in the presence of compound X, compound SC and / or SB, and then infected into HEK293A cells, and the number of GFP-positive cells was measured. [Diagram 35] The adenovirus vector into which the GFP gene has been inserted was produced in Lenti-X293T cells in the presence of compound X, compound SC and / or SB, and then infected into HEK293A cells, and the number of GFP-positive cells was measured.

[0017] [Diagram 36] An adeno-associated virus vector into which a GFP gene has been inserted was produced in Lenti-X293T cells in the presence of compound X, compound SC, and / or SB, and then infected into HEK293A cells, and the number of GFP-positive cells was measured. [Figure 37] Retroviral vectors into which the GFP gene was inserted were produced in Plat-E cells in the presence of compound X, compounds #1 to #4, #6, or #7, or PKC activators #1 to #6, or in the presence or absence of a PKC inhibitor, and then infected into HEK293A cells, and the number of GFP-positive cells was measured. [Figure 38] A retroviral vector into which a GFP gene has been inserted was produced in Plat-E cells in the presence of compound X, compound SC, SB and / or a PKC inhibitor, and then infected into HEK293A cells, followed by measuring the number of GFP-positive cells. [Figure 39] A lentiviral vector into which a GFP gene has been inserted was produced in Plat-E cells in the presence of compound X, compound SC, SB and / or a PKC inhibitor, and infected into HEK293A cells, and the number of GFP-positive cells was measured. [Diagram 40] An adenovirus vector into which a GFP gene has been inserted was produced in Plat-E cells in the presence of compound X, compound SC, SB and / or a PKC inhibitor, and then infected into HEK293A cells, followed by counting the number of GFP-positive cells.

[0018] [Diagram 41] An adeno-associated virus vector into which a GFP gene has been inserted was produced in Plat-E cells in the presence of compound X, compound SC, SB and / or a PKC inhibitor, and then infected into HEK293A cells, followed by measuring the number of GFP-positive cells. [Diagram 42] 1 shows the binding sites of transcription factors in the CMV promoter, CAG promoter, and EF1 promoter used in the Examples. [Diagram 43]The binding sites of transcription factors in the promoter contained in the pAd / DEST / CMV vector are shown. [Diagram 44] The binding sites of transcription factors in the promoter of the adenovirus genes in HEK293 cells are shown. Adenovirus type 5 nt 1-4355 (including E1 genes: E1A and E1B) is integrated into HKE293 cells. [Diagram 45] The binding sites of transcription factors in the promoter contained in the pAAV-2 / 9n vector are shown. The REP upstream region is a 258-nt region from the f1ori sequence of pAAV-2 / 9n to the upstream of the REP gene. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0019] Unless otherwise specifically defined, terms used in this disclosure have the meanings commonly understood by those skilled in the art of organic chemistry, medicine, pharmacology, molecular biology, microbiology, etc. Below, definitions of some terms used in this disclosure are provided, but these definitions take precedence over the common understanding in this disclosure.

[0020] The method for producing a viral vector disclosed herein utilizes a genetic engineering technique in which a nucleic acid required for the production of a viral vector is introduced into a cultured cell, and the viral vector is produced and collected. Such genetic engineering techniques are well known in the art and can be carried out in accordance with the methods described in the literature (e.g., Molecular Cloning, T. Maniatis et al., CSH Laboratory (1983), DNA Cloning, DM. Glover, IRL PRESS (1985)).

[0021] By "viral vector" is meant a vector in which a viral genome that has been engineered to contain a nucleic acid to be delivered to a cell in vitro, ex vivo, or in vivo is packaged within the viral particle.

[0022] Various viral vectors are known that are based on wild-type viruses, and any viral vector can be used.Known viral vectors include, for example, retroviral vector, lentiviral vector, adenoviral vector, adeno-associated viral vector, vaccinia viral vector, poxvirus vector, herpes simplex viral vector, murine leukemia viral vector, and hybrid viral vector.In some embodiments, the viral vector is a retroviral vector, lentiviral vector, adenoviral vector, or adeno-associated viral vector, preferably a retroviral vector.

[0023] Generally, viral vectors are produced by culturing cells containing the nucleic acid required for the production of the viral vector, i.e., the nucleic acid required for the production of viral particles of the viral vector and the nucleic acid to be delivered by the viral vector. The nucleic acid required for the production of viral particles of the viral vector encodes the protein or RNA of the virus from which the viral vector is derived, and may be a natural nucleic acid or may be modified according to the purpose of the viral vector.

[0024] The nucleic acid required for the production of viral particles of the viral vector can be a nucleic acid encoding a protein required for the production of viral particles of the viral vector. The protein required for the production of viral particles of the viral vector varies depending on the virus from which the viral vector is derived and is well known in the art.

[0025] For example, proteins required for the production of retroviral vector viral particles include Gag, Pol and envelope proteins (VSV-G, MLV, etc.). Proteins required for the production of lentiviral vector viral particles include Gag, Pol, Rev and envelope proteins (VSV-G, MLV, etc.).

[0026] Adenovirus vectors are composed of many proteins, and for example, proteins necessary for the production of virus particles of adenovirus vectors are proteins encoded in the E1A, E1B, E2A, E2B, E3, E4, L1, L2, L3, L4 or L5 regions of the adenovirus genome. It is also known that RNAs such as virus-associated RNA (VA RNA) are also necessary for the production of virus particles in adenovirus. Many serotypes of adenovirus are known, and for example, adenovirus vectors derived from human adenovirus type 2 or human adenovirus type 5, or these adenovirus vectors with E1 gene and / or E3 gene deleted, particularly adenovirus vectors derived from human adenovirus type 5 with E1 gene and E3 gene deleted, can be used.

[0027] Adeno-associated virus vectors are vectors based on adeno-associated viruses that require a helper virus such as adenovirus for viral replication, and proteins necessary for the production of viral particles include, for example, proteins encoded in the E1A, E1B, E2A and E4 regions of the adenovirus genome, and proteins encoded in the Rep and Cap regions of the adeno-associated virus genome. It is also known that RNAs such as virus-associated RNA (VA RNA) of helper viruses are also necessary for the production of viral particles. There are many known serotypes of adeno-associated viruses, and adeno-associated virus vectors derived from, for example, AAV2, AAV5 or AAV9 adeno-associated viruses can be used.

[0028] The nucleic acid required for the production of at least one virus particle is operably linked to a promoter that contains a binding site for at least one transcription factor selected from SP1, CEBP, AP1, NF-κB, and YY1. As long as the nucleic acid required for the production of at least one virus particle is operably linked to the promoter, the nucleic acid required for the production of other virus particles may or may not be operably linked to the promoter. Two or more, for example, all, of the nucleic acids required for the production of virus particles may be operably linked to the promoter, and in this case, the type of promoter linked to each nucleic acid may be the same or different. One nucleic acid required for the production of virus particles may be operably linked to one promoter, and two or more of the nucleic acids may be operably linked to one promoter.

[0029] In the present disclosure, the promoter comprises a binding site for at least one transcription factor selected from SP1, CEBP, AP1, NF-κB and YY1. That is, the promoter comprises one of these binding sites, two, three or four of these binding sites in any combination, or all of these binding sites. The promoter may further comprise a binding site for other transcription factors, for example CREB. In an embodiment, the promoter comprises binding sites for SP1, CEBP, AP1, NF-κB and YY1. In an embodiment, the promoter comprises binding sites for SP1, CEBP, AP1, NF-κB, YY1 and CREB. Examples of promoters that may be used include the CMV promoter, the CAG promoter and the EF1 promoter, preferably the CMV promoter and the EF1 promoter, particularly preferably the CMV promoter. The promoter may be a promoter derived from a virus. For example, the adenovirus E1A promoter, E1B promoter, E2A promoter, E2B promoter, E4 promoter, major late promoter or IX promoter, and the adeno-associated virus p5 promoter, p19 promoter or p40 promoter may be used. These transcription factors, binding sites and promoters are well known in the art and can be used in accordance with methods described in the literature.

[0030] In the present disclosure, "operably linked" means that regulatory sequence elements, such as a promoter, an internal ribosome entry site (IRES), and a 2A self-cleaving peptide (2A peptide) sequence, and a nucleic acid necessary for producing a viral vector are linked in a manner that allows gene expression, and the 3' end of each DNA may be directly linked to the 5' end of the downstream DNA, or any DNA sequence may be present between them.

[0031] The nucleic acid to be delivered by the viral vector may be a nucleic acid containing a nucleotide sequence encoding a desired protein or nucleic acid. The nucleic acid to be delivered by the viral vector is not limited as long as it is not unacceptably toxic to the cultured cells used. For example, the nucleic acid may be a nucleic acid for gene therapy. The nucleic acid may contain a marker gene that allows the selection of cells into which the gene has been introduced, such as a drug resistance gene or a fluorescent protein gene. The nucleic acid may be produced based on the sequence information of the nucleic acid, for example, by conventional DNA synthesis or amplification by RT-PCR. The promoter to which the nucleic acid to be delivered by the viral vector is linked is not limited, and for example, the nucleic acid may be operably linked to a promoter that contains a binding site for at least one transcription factor selected from SP1, CEBP, AP1, NF-κB, and YY1.

[0032] An expression construct can be prepared by incorporating a nucleic acid required for the production of a viral vector into an expression vector. The expression vector used here can be appropriately selected depending on the viral vector to be produced, the cultured cells to be used, etc., and includes plasmids, viral vectors, etc. Examples include plasmid vectors such as pQCXIN, pcDNA, pLVSIN, psPAX2, pAd, pAAV, pAdDeltaF6, pKCR, pCDM8, pGL2, pcDNA3.1, pRc / RSV, and pRc / CMV, and viral vectors such as retroviral vectors, lentiviral vectors, adenoviral vectors, adeno-associated viral vectors, vaccinia viral vectors, pox viral vectors, herpes simplex viral vectors, murine leukemia viral vectors, and hybrid viral vectors. The expression construct may contain factors such as a selection marker gene and a terminator. The expression construct containing a nucleic acid to be delivered by a viral vector may contain a factor that enables packaging of the nucleic acid into a viral particle. An expression construct containing all the nucleic acids necessary for the production of a viral vector may be used, or a combination of expression constructs containing the nucleic acids necessary for the production of one or more viral vectors may be used. For example, when producing an adeno-associated viral vector, a helper plasmid containing a part of an adenovirus-derived gene (E4, E2A, VA RNA, etc.) may be used.

[0033] A cell containing a nucleic acid required for the production of a viral vector can be prepared by transforming a host cell with an expression construct. All nucleic acids required for the production of a viral vector may be introduced into a host cell into which a part of the nucleic acid required for the production of a viral vector has already been introduced, and the remaining nucleic acids required for the production of a viral vector may be introduced. A nucleic acid to be delivered by a viral vector and, in some cases, the remaining nucleic acids required for the production of a viral particle may be introduced into a host cell (packaging cell) into which a part or all of the nucleic acids required for the production of a viral particle have already been introduced. The host cell is typically an animal cell, and examples of the host cell include HEK293A cells, HEK293T cells, CHO cells, COS cells, Vero cells, HeLa cells, L929 cells, BALB / c3T3 cells, C127 cells, and NIH3T3 cells. Examples of packaging cells include cells into which Gag and / or Pol and / or envelope proteins of a retrovirus have been introduced, and cells into which the E1A and / or E1B regions of an adenovirus have been introduced. Commercially available cells suitable for producing viral vectors may be used, for example, Plat-E cells (Cosmo Bio, #RV-101) and Lenti-X293T cells (Takara, #Z2180N).

[0034] The expression construct can be introduced into the host cell by a conventional method suitable for the host cell and expression vector, such as the calcium phosphate method, the DEAE-dextran method, the electroporation method, and the lipofection method.

[0035] Cells may be transformed to transiently produce a viral vector, or may be transformed to have the ability to continuously produce a virus and be made into a cell line. In the latter case, the cell line may be used as a master cell bank (MCB) or working cell bank (WCB).

[0036] The transformed cells are cultured under conditions that activate PKC, thereby enabling efficient production of viral vectors. Culture conditions such as medium, culture time, and culture temperature suitable for culturing each cell and each viral vector are well known to those skilled in the art and may be appropriately selected. For example, under conditions that activate PKC, cells are cultured for 1 hour or more, 2 hours or more, 4 hours or more, 6 hours or more, 8 hours or more, 12 hours or more, 18 hours or more, 24 hours or more, 36 hours or more, 48 hours or more, and within 72 hours, within 60 hours, within 48 hours, within 36 hours, within 30 hours, or within 24 hours, for example, 24 hours to 72 hours, 36 hours to 60 hours, 42 hours to 54 hours, 45 hours to 51 hours, 46 hours to 50 hours, 47 hours to 49 hours, or about 48 hours, and then the culture supernatant containing the viral vector is collected.

[0037] The viral vector may be used as the supernatant, or as a filtrate obtained by filtering the supernatant, or as a viral vector diluted, concentrated or purified by a known method, and may be stored by an appropriate method, for example, frozen, until use. Purification methods include ultracentrifugation, salting out, ion exchange chromatography, adsorption chromatography, affinity chromatography, gel filtration chromatography, etc. Commercially available concentration reagents, such as Retro-X Concentrator (TaKaRa #631455) and Lenti-X Concentrator (TaKaRa #631231), may also be used.

[0038] In one embodiment, the condition for activating PKC is culturing cells in the presence of a PKC activator. PKC is a type of protein kinase that phosphorylates the hydroxyl groups of serine and threonine residues of substrate proteins, and more than 10 types of isozymes are known. Isozymes are classified into three subfamilies, conventional (α, βI, βII, γ), novel (δ, ε, θ, η), and atypical (ζ, Mζ, ι / λ), depending on their structure, activation mechanism, and physiological activity. In the present disclosure, PKC is preferably conventional PKC isozyme or novel PKC isozyme, particularly preferably PKCα or PKCδ.

[0039] In the present disclosure, "PKC activator" refers to a substance that enhances the kinase activity of PKC. Two or more PKC activators may be used in combination.

[0040] In one embodiment, the PKC activator is a compound of formula (I): [ka] {In the formula, R1 is H, halogen, -OH, C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, C1-6 alkoxy, C6-14 aryl or -OC(O)R3, wherein C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, C1-6 alkoxy or aryl is optionally substituted by 1 to 3 halogens, which may be the same or different; R2 is C6-12 alkyl, C6-12 alkenyl, C6-12 alkynyl or C6-12 alkoxy, wherein the C6-12 alkyl, C6-12 alkenyl, C6-12 alkynyl or C6-12 alkoxy is optionally substituted by 1 to 3 halogens, which may be the same or different; R3 is C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, amino or C6-14 aryl, wherein C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl or C6-14 aryl are optionally substituted by 1 to 3 halogens which may be the same or different; Formula (II): [ka] {In the formula, R4 is H, halogen, -OH, C1-18 alkyl, C2-18 alkenyl, C2-18 alkynyl, C1-18 alkoxy or -OC(O)R6, wherein C1-18 alkyl, C2-18 alkenyl, C2-18 alkynyl or C1-18 alkoxy is optionally substituted by 1 to 3 identical or different halogens; R5 is C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl or amino, wherein C1-6 alkyl, C2-6 alkenyl or C2-6 alkynyl is optionally substituted by 1 to 3 identical or different halogens; R6 is C1-18 alkyl, C2-18 alkenyl, C2-18 alkynyl or amino, wherein the C1-18 alkyl, C2-18 alkenyl or C2-18 alkynyl is optionally substituted by 1 to 3 halogens which may be the same or different; or Formula (III): [ka] {In the formula, R7 is H, C1-18 alkyl, C2-18 alkenyl, C2-18 alkynyl or -C(O)R9, wherein C1-18 alkyl, C2-18 alkenyl or C2-18 alkynyl is optionally substituted by 1 to 3 halogens, which may be the same or different; R8 is H, C1-18 alkyl, C2-18 alkenyl, C2-18 alkynyl or -C(O)R9, wherein C1-18 alkyl, C2-18 alkenyl or C2-18 alkynyl is optionally substituted by 1 to 3 halogens, which may be the same or different; R9 is C1-18 alkyl, C2-18 alkenyl, C2-18 alkynyl or amino, wherein the C1-18 alkyl, C2-18 alkenyl or C2-18 alkynyl is optionally substituted with 1 to 3 halogens which may be the same or different. or an ester, salt or solvate thereof may be used.

[0041] In this disclosure, the term "halogen" means an atom selected from fluorine, chlorine, bromine and iodine. In this disclosure, the term "alkyl" refers to a saturated, straight or branched chain hydrocarbon group. In this disclosure, the term "alkenyl" means a straight or branched chain hydrocarbon containing one or more double bonds. In this disclosure, the term "alkynyl" means a straight or branched chain hydrocarbon containing one or more triple bonds. As used herein, the term "alkoxy" refers to --O-alkyl, where alkyl is as defined in this disclosure.

[0042] In this disclosure, the term "aryl" refers to a monovalent aromatic carbocyclic group of 6 to 14 carbon atoms having a single ring (e.g., phenyl) or multiple condensed rings (e.g., naphthyl or anthryl). Aryl typically includes phenyl and naphthyl. In this disclosure, the term "amino" refers to the group --NH.

[0043] In the present disclosure, "ester" refers to an ester that can be hydrolyzed in vivo or in vitro, including those that are easily decomposed to release the parent compound or its salt. Suitable ester groups include, for example, those derived from aliphatic carboxylic acids, particularly alkanoic acids, alkenoic acids, cycloalkanoic acids, and alkanedioic acids, where each alkyl or alkenyl group has, for example, 6 or less carbon atoms. Specific examples of esters include formates, acetates, propionates, butyrates, acrylates, and ethylsuccinates.

[0044] In the present disclosure, "salt" can be a salt of a compound with an inorganic or organic acid.Preferred salts are salts with inorganic acids, such as hydrochloric acid, hydrobromic acid, phosphoric acid or sulfuric acid, or salts with organic carboxylic or sulfonic acids, such as acetic acid, trifluoroacetic acid, propionic acid, maleic acid, fumaric acid, malic acid, citric acid, tartaric acid, lactic acid, benzoic acid or methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, toluenesulfonic acid, naphthalenesulfonic acid or naphthalenedisulfonic acid.

[0045] Salts may also be salts with conventional bases, such as alkali metal salts (e.g. sodium or potassium salts), alkaline earth metal salts (e.g. calcium or magnesium salts), or ammonium salts, especially sodium salts, derived from ammonia or organic amines (e.g. diethylamine, triethylamine, ethyldiisopropylamine, procaine, dibenzylamine, N-methylmorpholine, dihydroabietylamine, methylpiperidine, L-arginine, creatine, choline, L-lysine, ethylenediamine, benzathine, ethanolamine, meglumine or tromethamine).

[0046] In the present disclosure, a "solvate" refers to a compound that, in the solid or liquid state, forms a complex by coordination with a solvent molecule. The preferred solvate is a hydrate.

[0047] In one embodiment, in formula (I): R1 is H or -OC(O)R3; R2 is C6-12 alkyl or C6-12 alkenyl; R3 is C1-6 alkyl or C6-14 aryl.

[0048] In one embodiment, in formula (I): R1 is H or -OC(O)R3; R2 is nonyl or 1,3-nonadienyl; R3 is methyl or phenyl.

[0049] In certain embodiments, the compound of formula (I) is Compound X, Compound #1, or Compound #2, described below.

[0050] In one embodiment, in formula (II): R4 is H or -OC(O)R6; R5 is C1-6 alkyl or C2-6 alkenyl; R6 is C1-18 alkyl.

[0051] In one embodiment, in formula (II): R4 is H or -OC(O)R6; R5 is methyl, propyl, sec-butyl or butenyl; R6 is propyl, nonyl or tridecyl.

[0052] In certain embodiments, the compound of formula (II) is Compound #3, Compound #4, Compound #5, TPA, phorbol 12,13-dibutyrate or prostratin, particularly Compound #3, Compound #4 or Compound #5, described below.

[0053] In one embodiment, in formula (III): R7 is H or -C(O)R9; R8 is H or -C(O)R9; R9 is C1-18 alkyl or C2-18 alkenyl.

[0054] In one embodiment, in formula (III): R7 is H or -C(O)R9; R8 is H or -C(O)R9; R9 is pentadecyl or butenyl.

[0055] In certain embodiments, the compound of formula (III) is compound #6, compound #7 or ingenol 3-angelate, particularly compound #6 or compound #7, described below.

[0056] The compounds of formulae (I) to (III) or their esters, salts or solvates may be added to the culture medium at a concentration of, for example, 0.1 to 10 μg / ml, 0.1 to 1 μg / ml, 0.1 to 1000 ng / ml, 2 to 500 ng / ml or 5 to 200 ng / ml.

[0057] In some embodiments, the PKC activator is a compound of formula (I) or an ester, salt, or solvate thereof, which may be added to the culture medium at a concentration of, for example, 0.1-10 μg / ml, 0.1-1 μg / ml, 0.1-1000 ng / ml, 20-500 ng / ml, or 50-200 ng / ml.

[0058] In one embodiment, the PKC activator is a compound selected from Compound X and Compounds #1 to #7 below, or an ester, salt, or solvate thereof. [ka]

[0059] Compound X and compounds #1 to #7 may be obtained by chemical synthesis or extracted from plants containing them. For example, compound X may be extracted from Lowdaphne Stringbush, compounds #1 and #2 from Lilac Daphne, compounds #3 to #5 from Croton, and compounds #6 to #7 from Cape Euphorbia. Plant processed products containing these compounds, such as plant extracts or extracts, may also be used.

[0060] In certain embodiments, the PKC activator is compound X or an ester, salt, or solvate thereof.

[0061] Known PKC activators may be used. Known PKC activators include 12-O-tetradecanoylphorbol 13-acetate (TPA, also called phorbol 12-myristate 13-acetate (PMA)), prostratin, bryostatin 1, bryostatin 2, FR236924, (-)-indolactam V, PEP005, phorbol 12,13-dibutyrate, 1-oleoyl-2-acetyl-sn-glycerol, 1-O-hexadecyl-2-O-arachidonyl, and 1-O-hexadecyl-2-O-arachidonyl. Examples of PKC activators include, but are not limited to, 1,2-dioctanoyl-sn-glycerol, 1,2-dioctanoyl-sn-glycerol, PIP2, resiniferatoxin, phorbol 12,13-dihexanoate, mezerein, ingenol 3-angelate, RHC-80267, DCP-LA, lipoxin A4, (2S,5S)-(E,E)-8-(5-(4-(trifluoromethyl)phenyl)-2,4-pentadienoylamino)benzolactam, and the like. In some embodiments, the PKC activator is TPA, prostratin, (-)-indolactam V, phorbol 12,13-dibutyrate, ingenol 3-angelate, or (2S,5S)-(E,E)-8-(5-(4-(trifluoromethyl)phenyl)-2,4-pentadienoylamino)benzolactam. Known PKC activators may be used as appropriate according to methods known in the art, for example, methods recommended by the manufacturer.

[0062] In one embodiment, the PKC activator is a compound selected from compound X, compounds #1-#7, TPA, prostratin, (-)-indolactam V, phorbol 12,13-dibutyrate, ingenol 3-angelate, and (2S,5S)-(E,E)-8-(5-(4-(trifluoromethyl)phenyl)-2,4-pentadienoylamino)benzolactam, or an ester, salt, or solvate thereof.

[0063] In one embodiment, the cell further comprises a nucleic acid encoding a peptide comprising a Pro region or a RING region. The nucleic acid encoding a peptide comprising a Pro region or a RING region may be included in the same expression construct as the nucleic acid required for the production of viral particles, or may be included in a separate expression construct. For example, an expression construct may be used that comprises a promoter, a nucleic acid required for the production of viral particles, and a nucleic acid encoding a peptide comprising a Pro region or a RING region, all of which are operably linked. In one embodiment, the promoter, the nucleic acid required for the production of viral particles, and the nucleic acid encoding a peptide comprising a Pro region or a RING region are operably linked in this order.

[0064] The nucleic acid encoding a peptide comprising a Pro region or a RING region may or may not be translated into a peptide. If it is to be translated, an IRES or 2A peptide sequence may be inserted between the nucleic acid required for the production of viral particles and the nucleic acid encoding the peptide comprising a Pro region or a RING region. Details of the IRES and 2A peptide sequence are described below. For example, an expression construct may be used that includes a promoter, a nucleic acid required for the production of viral particles, an IRES or 2A peptide sequence, and a nucleic acid encoding a peptide comprising a Pro region or a RING region, all of which are operably linked. In one embodiment, the promoter, the nucleic acid required for the production of viral particles, the IRES or 2A peptide sequence, and the nucleic acid encoding the peptide comprising a Pro region or a RING region are operably linked in this order.

[0065] In the present disclosure, the Pro or RING region may be from any species, for example, mouse, rat, hamster, rabbit, cat, dog, cow, pig, sheep, monkey, human, etc., particularly from human. Preferably, the Pro or RING region is from the same species as the cell used.

[0066] Pro regions refer to regions with proline-rich amino acid sequences that mediate specific interactions with functional domains such as WW and SH3 domains. Pro regions include regions with short repetitive proline-rich sequences, tandemly repeated proline-rich sequences, non-repetitive proline-rich sequences, and hydroxyproline-rich sequences. Pro regions can be found in a variety of proteins, including, but not limited to, nuclear proteins, transcription factors, integral membrane proteins such as transporters, channels, and receptors, globular proteins, hormones, neuropeptides, mucins, immunoglobulins, and extracellular matrix proteins.

[0067] The Pro region may be derived from any protein. Examples of proteins that contain the Pro region include PML, ARHGEF1, aggrecan-1, RALGDS, DGKK, SPATA21, rabfilin-3A, TEAD3, SPPL2B, and FLJ43093.

[0068] RING domains, also called RING finger domains, bind a pair of zinc atoms and mediate protein-protein interactions. RING domains generally have the following consensus sequence: C-X2-C-X9-39-C-X1-3-H-X2-3-C-X2-C-X4-48-C-X2-C {wherein C is a cysteine ​​residue, H is a histidine residue, and X is any amino acid residue.} These cysteine ​​and histidine residues are necessary for forming the structure through binding with the zinc atom and are highly conserved.

[0069] The RING region can be derived from any protein.Proteins that contain RING region include, for example, TRIM13, LONRF3, TRIM47, RNF135, TRIM10, TRIM72, TRIM60, TRIM39, TRIM4, TRIM43B, TRIM43, TRIM25, TRIM26, TRIM31, HTLF, BRCA1, TRIM50, TRIM21, SSA1, TRIM5d, TRIM22, KIAA0182, TRIM65, RAG1, BFAR, Pex10, RNF8, RING2, COPI, TRIM2, TRIM3, SH3RF2, PML and TRIM56.In some embodiments, the RING region is the RING region of PML, TRIM3, TRIM56, COPI, Pex10, BRCA1 or HTLF.

[0070] In one embodiment, the Pro region or RING region is derived from promyelocytic leukemia protein (PML). PML is required for the assembly of intranuclear structures called PML bodies. PML bodies have diverse functions and are suggested to be involved in a wide range of intracellular processes. Multiple isoforms of human PML are known, and the N-terminal side has the same amino acid sequence in all isoforms.

[0071] The amino acid sequence and nucleotide sequence of human wild-type (WT) PML isoform 5 (Gene ID: 5371, NCBI Reference Sequence: NP_150247.2) are shown in SEQ ID NOs: 7 and 8. The amino acid sequence consists of 560 amino acids, and has the regions shown in FIG. 25. Positions 1 to 45 (SEQ ID NO: 9) of the amino acid sequence of SEQ ID NO: 7 are the Pro region, and positions 46 to 105 (SEQ ID NO: 10) are the RING region. Positions 1 to 135 (SEQ ID NO: 11) of the nucleotide sequence of SEQ ID NO: 8 code for the Pro region, and positions 136 to 315 (SEQ ID NO: 12) code for the RING region.

[0072] The Pro region may be a region of a protein that matches the region from position 1 to position 45 of SEQ ID NO: 7 when the amino acid sequence of the protein and the amino acid sequence of SEQ ID NO: 7 are aligned in an optimal state (state in which amino acid identity is maximized). In one embodiment, the Pro region comprises or consists of an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 9. In one embodiment, the Pro region comprises the amino acid sequence of SEQ ID NO: 9. In one embodiment, the Pro region consists of the amino acid sequence of SEQ ID NO: 9. In one embodiment, the Pro region is encoded by a nucleotide sequence having at least 90% identity with or including the nucleotide sequence of SEQ ID NO: 11. In one embodiment, the Pro region is encoded by a nucleotide sequence comprising the nucleotide sequence of SEQ ID NO: 11. In one embodiment, the Pro region is encoded by the nucleotide sequence of SEQ ID NO: 11.

[0073] The RING region may be a region of a protein that corresponds to a region from position 46 to position 105 of SEQ ID NO: 7 when the amino acid sequence of the protein and the amino acid sequence of SEQ ID NO: 7 are optimally aligned. In one embodiment, the RING region comprises or consists of an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 10. In one embodiment, the RING region comprises the amino acid sequence of SEQ ID NO: 10. In one embodiment, the RING region consists of the amino acid sequence of SEQ ID NO: 10. In one embodiment, the RING region is encoded by a nucleotide sequence having at least 90% identity with or including the nucleotide sequence of SEQ ID NO: 12. In one embodiment, the RING region is encoded by a nucleotide sequence comprising the nucleotide sequence of SEQ ID NO: 12. In one embodiment, the RING region is encoded by the nucleotide sequence of SEQ ID NO: 12.

[0074] In the present disclosure, the identity of a nucleotide sequence or an amino acid sequence refers to the degree of sequence similarity between nucleic acids or proteins, and is determined by comparing two sequences that are aligned in an optimal state (maximum nucleotide or amino acid identity) across the region of the sequence to be compared. The numerical value (%) of sequence identity is calculated by determining the identical nucleotides or amino acids present in both sequences, determining the number of matching sites, and then dividing the number of matching sites by the total number of nucleotides or amino acids in the region of the sequence to be compared, and multiplying the obtained numerical value by 100. Algorithms for obtaining optimal alignment and sequence identity include various algorithms (e.g., BLAST algorithm, FASTA algorithm, etc.) that are commonly available to those skilled in the art. Sequence identity can be determined using sequence analysis software such as BLAST, FASTA, etc.

[0075] In one embodiment, the condition for activating PKC is to express activated PKC in a cell. "Activated PKC" refers to a PKC mutant that constitutively exhibits kinase activity. Activated PKC may be a PKC lacking an N-terminal regulatory region (Molecular and Cellular Biology 19(2):1313-24, 1999). Activated PKC may further have a mutation that enhances kinase activity (PNAS 115(24):E5497-E5505. 2018). Examples of activated PKC include PKCδ-CA (SEQ ID NO: 1) and PKCαCA-M489V (SEQ ID NO: 2).

[0076] The kinase activity of a PKC mutant can be measured by various methods known in the art. For example, a method of overexpressing a PKC mutant in cultured cells and detecting the phosphorylation level of the substrate by Western blot using a phosphorylated substrate-specific antibody (e.g., THE JOURNAL OF BIOLOGICAL CHEMISTRY, Vol. 279, No. 27, pp. 27986-27993, 2004), an ELISA method (e.g., Cell Death and Differentiation (2015) 22, 2078-2086), or a method of evaluating the incorporation of a phosphate group into a substrate in vitro using 32P-gamma-ATP (e.g., THE JOURNAL OF BIOLOGICAL CHEMISTRY, Vol. 262, No. 20, pp. 9569-9573, 1987) can be used. Various kits for measuring kinase activity may be used, such as the PKC kinase activity kit (Enzo Life Science, #ADI-EKS-420A).

[0077] In one embodiment, cells expressing a PKC mutant are cultured in the presence and absence of a PKC inhibitor, and the amount of phosphorylated protein is measured by an immunological method using an antibody that specifically recognizes the phosphorylated protein. If the amount of phosphorylated protein is reduced by the PKC inhibitor, the PKC mutant can be determined to be an activated PKC. Examples of the immunological method include flow cytometry analysis, radioisotope immunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), western blotting, and immunohistochemical staining.

[0078] For example, an expression construct containing a nucleic acid encoding an activated form of PKC may be used, and a protein required for viral particle production and an activated form of PKC may be expressed in a cell using an expression construct containing a promoter, a nucleic acid required for viral particle production, an IRES or 2A peptide sequence, and a nucleic acid encoding an activated form of PKC, which are operably linked together. In one embodiment, the promoter, the nucleic acid required for viral particle production, the IRES or 2A peptide sequence, and the nucleic acid encoding an activated form of PKC are operably linked in this order.

[0079] By expressing proteins required for viral particle production and activated PKC from a single expression construct using IRES, it is possible to efficiently produce the proteins required for viral particle production. IRES is an RNA region that can recruit eukaryotic ribosomes to mRNA, allowing cap-independent initiation of translation as part of the process of protein synthesis. Many IRES have been identified in viral and eukaryotic genomes, and synthetic IRES have also been developed.

[0080] For example, IRES can be derived from various viruses, including enteroviruses (e.g., human papillomavirus 1, human coxsackievirus B); rhinoviruses (e.g., human rhinovirus); hepatoviruses (hepatitis A virus); cardioviruses (encephalomyocarditis virus ECMV and Theiler's encephalomyelitis virus); aphthoviruses (foot and mouth disease virus, equine rhinitis A virus, equine rhinitis B virus); pestiviruses (e.g., bovine viral diarrhea virus and hog cholera virus; hepaciviruses (e.g., hepatitis C virus) and GB virus B). Alternatively, the IRES can be derived from viruses of the Retroviridae family, such as members of the lentivirus family (e.g., Simian Immunodeficiency Virus and Human Immunodeficiency Virus 1); BLV-HTLV retroviruses (e.g., Human T-Lymphotropic Virus Type 1); and mammalian C-type retrovirus family (e.g., Moloney Murine Leukemia Virus, Friend Murine Leukemia Virus, Harvey Murine Sarcoma Virus, Avian Reticuloendotheliosis Virus, Murine Leukemia Virus (envRNA), Rous Sarcoma Virus). IRESs derived from eukaryotic mRNAs include, for example, the IRESs of BiP, Drosophila antennapedia (exons d and e), c-myc, and the X-linked inhibitor of apoptosis (XIAP) gene. Also, various synthetic IRES have been developed, see, e.g., De Gregorio et al. (1999) EMBO J. 75:4865-74; Owens et al. (2001) PNAS 4:1471-6; and Venkatesan et al. (2001) Molecular and Cellular Biology 21:2826-37. For additional IRES known in the art, see, e.g., rangueil.inserm.fr / IRESdatabase. In one embodiment, an IRES from the encephalomyocarditis virus ECMV is used.

[0081] The 2A peptide sequence induces ribosome skipping during protein translation. When the 2A peptide sequence is present in the amino acid sequence of a protein, the protein is translated as two polypeptides cleaved at the C-terminus of the 2A peptide sequence. Examples of 2A peptides include those described in Kim, JH, et al., PLoS One. 6(4), e18556 (2011), and examples of known 2A peptides include P2A peptide (SEQ ID NO: 3: (GSG)ATNFSLLKQAGDVEENPGP), T2A peptide (SEQ ID NO: 4: (GSG)EGRGSLLTCGDVEENPGP), E2A peptide (SEQ ID NO: 5: (GSG)QCTNYALLKLAGDVESNPGP), and F2A peptide (SEQ ID NO: 6: (GSG)VKQTLNFDLLKLAGDVESNPGP) (GSG at the N-terminus may or may not be present in each sequence).

[0082] When PKC is activated, a calmodulin inhibitor may be further added to the medium. Calmodulin is an acidic protein that functions as a calcium sensor and regulates intracellular calcium levels. As the calmodulin inhibitor, a compound represented by the formula (IV) [ka] {In the formula, n is an integer from 1 to 8; R is C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, C1-6 alkoxy, C6-14 aryl, amino, hydroxy, COOH or COOR', where R' is C1-6 alkyl. or an ester, salt or solvate thereof may be used.

[0083] In one embodiment, in formula (IV), n is an integer from 4 to 6; R is C1-6 alkyl, C6-14 aryl or amino.

[0084] In one embodiment, in formula (IV), n is an integer of 4 or 6, R is methyl, phenyl or amino.

[0085] Compounds of formula (IV) include, for example, SC-9, SC-10, and W-7. Compounds of formula (IV) may be used at a concentration of, for example, 0.5 to 100 μg / ml, 1 to 50 μg / ml, or 2 to 10 μg / ml.

[0086] Known calmodulin inhibitors, such as W-7, calmidazolium, bisindolylmaleimide I, trifluoperazine, ruthenium red, ophiobolin A, CaM kinase II (290-309), E6 berbamine, mastoparan, compound 48 / 80, phenoxybenzamine, W-7 isomer, polystes mastoparan, A-7, fluphenazine-N-2-chloroethane, W-13, W-13 isomer, CGS 9343B, W-5 isomer, W-12, N-(5-aminopentyl)-5-chloro-2-naphthalenesulfonamide, and W-5, may be used as appropriate according to methods known in the art, such as those recommended by the manufacturer. Two or more calmodulin inhibitors may be used in combination.

[0087] When activating PKC, a histone deacetylase inhibitor may be further added to the medium. Histone deacetylase is an enzyme that deacetylates histones, which are major components of chromatin structure, and plays an important role in gene transcription control. Known histone deacetylase inhibitors, such as trichostatin A, M344, butyrate, phenylbutyrate, apicidin, valproic acid, BML-210, depudecin, romidepsin (FK-228), HC toxin, oxamflatin, scriptaid, splitomycin, suberoyl bis-hydroxamic acid, vorinostat, dacinostat (LAQ-824), panobinostat (LBH-589), belinosutate (PXD- 101), phenyl acetate, IF2357, FK-228, entinostat (MS-275), mocetinostat (MGCD0103) or tacedinaline (CI994), preferably sodium butyrate, valproic acid, trichostatin A, vorinostat, apicidin, entinostat or tacedinaline, particularly preferably sodium butyrate, may be used as appropriate according to methods well known in the art, for example, methods recommended by the manufacturer. For example, sodium butyrate may be used at a concentration of 0.1 to 10 mM, 0.5 to 5 mM or 1 to 3 mM. Two or more histone deacetylase inhibitors may be used in combination.

[0088] In one embodiment, a kit that can be used in the method of the present disclosure is also provided. Each component included in the kit can be provided separately or, if possible, mixed, dissolved in water or a suitable buffer, or lyophilized, and contained in a suitable container. Suitable containers include bottles, vials, test tubes, tubes, plates, etc. The containers can be made of a variety of materials, such as glass, plastic, metal, etc. The containers can have labels. The kits can further include other components that are desirable from a commercial and user standpoint, such as documents (e.g., written or storage media, etc.) that include instructions for use.

[0089] In one embodiment, a composition for enhancing production of a viral vector comprising a PKC activator or a nucleic acid encoding an activated form of PKC is also provided, the composition comprising a PKC activator or a nucleic acid encoding an activated form of PKC together with a suitable excipient and may be used according to the methods of the present disclosure.

[0090] For example, the following embodiment is provided. [1] A method for producing a viral vector, comprising culturing a cell containing a nucleic acid required for producing viral particles of the viral vector and a nucleic acid to be delivered by the viral vector under conditions that activate protein kinase C (PKC), The method, wherein the nucleic acid required for the production of viral particles is operably linked to a promoter containing a binding site for at least one transcription factor selected from SP1, CEBP, AP1, NF-κB and YY1. [2] The method according to claim 1, which comprises a step of introducing into a cell a nucleic acid required for production of viral particles of the viral vector and / or a nucleic acid to be delivered by the viral vector. [3] The method according to claim 1 or 2, further comprising a step of recovering the viral vector. [4] The method according to any one of items 1 to 3, wherein the viral vector is a retroviral vector, a lentiviral vector, an adenoviral vector, or an adeno-associated viral vector. [5] The method according to any one of items 1 to 4, wherein the viral vector is a retroviral vector. [6] The method according to claim 5, wherein the nucleic acid required for the production of viral particles encodes at least one protein selected from Gag, Pol and envelope proteins. [7] The method according to any one of items 1 to 4, wherein the viral vector is a lentiviral vector. [8] The method according to claim 7, wherein the nucleic acid required for the production of viral particles encodes at least one protein selected from Gag, Pol, Tat, Rev and envelope proteins. [9] The method according to any one of items 1 to 4, wherein the viral vector is an adenoviral vector.

[10] The method according to claim 9, wherein the nucleic acid required for the production of viral particles encodes at least one protein selected from proteins encoded in the E1A, E1B, E2A, E2B, E3, E4, L1, L2, L3, L4 and L5 regions of the adenoviral genome or VA RNA.

[0091]

[11] The method according to any one of items 1 to 4, wherein the viral vector is an adeno-associated viral vector.

[12] The method according to claim 11, wherein the nucleic acid necessary for the production of viral particles encodes at least one protein selected from proteins encoded in the E1A, E1B, E2A and E4 regions of the adenovirus genome and the Rep and Cap regions of the adeno-associated virus genome or VA RNA.

[13] The method according to any one of items 1 to 12, wherein all nucleic acids necessary for producing viral particles are operably linked to the promoter.

[14] The method according to any one of items 1 to 13, wherein the nucleic acid to be delivered by the viral vector is operably linked to a promoter containing a binding site for at least one transcription factor selected from SP1, CEBP, AP1, NF-κB, and YY1.

[15] The method according to any one of items 1 to 14, wherein the promoter comprises binding sites for SP1, CEBP, AP1, NF-κB and YY1.

[16] The method according to any one of items 1 to 15, wherein the promoter further comprises a binding site for CREB.

[17] The method according to any one of items 1 to 16, wherein the promoter is a CMV promoter, a CAG promoter or an EF1 promoter.

[18] The method according to any one of items 1 to 17, wherein the promoter is a CMV promoter or an EF1 promoter.

[19] The method according to any one of items 1 to 18, wherein the promoter is a CMV promoter.

[20] The method according to any one of items 1 to 14, wherein the promoter is a viral promoter.

[0092]

[21] The method according to claim 20, wherein the virus-derived promoter is an adenovirus E1A promoter, E1B promoter, E2A promoter, E2B promoter, E4 promoter, major late promoter or IX promoter, or an adeno-associated virus p5 promoter, p19 promoter or p40 promoter.

[22] The method according to any one of items 1 to 21, wherein PKC is activated by culturing the cells in the presence of a PKC activator.

[23] The method according to claim 22, wherein the PKC activator is a compound of formula (I), formula (II) or formula (III) or an ester, salt or solvate thereof.

[24] The method of claim 22 or 23, wherein the PKC activator is a compound of formula (I) or an ester, salt or solvate thereof.

[25] The method of claim 24, wherein R1 is H or -OC(O)R3, R2 is C6-12 alkyl or C6-12 alkenyl, and R3 is C1-6 alkyl or C6-14 aryl.

[26] The method of claim 24 or 25, wherein R1 is H or -OC(O)R3, R2 is nonyl or 1,3-nonadienyl, and R3 is methyl or phenyl.

[27] The method of claim 22 or 23, wherein the PKC activator is a compound of formula (II) or an ester, salt or solvate thereof.

[28] The method of claim 27, wherein R4 is H or -OC(O)R6, R5 is C1-6 alkyl or C2-6 alkenyl, and R6 is C1-18 alkyl.

[29] The method of claim 25 or 28, wherein R4 is H or -OC(O)R6, R5 is methyl, propyl, sec-butyl or butenyl, and R6 is propyl, nonyl or tridecyl.

[30] The method of claim 22 or 23, wherein the PKC activator is a compound of formula (III) or an ester, salt or solvate thereof:

[0093]

[31] The method of claim 30, wherein R7 is H or -C(O)R9, R8 is H or -C(O)R9, and R9 is C1-18 alkyl or C2-18 alkenyl.

[32] The method of claim 30 or 31, wherein R7 is H or -C(O)R9, R8 is H or -C(O)R9, and R9 is pentadecyl or butenyl.

[33] The method according to item 22, wherein the PKC activator is a compound selected from compound X, compounds #1 to #7, TPA, prostratin, (-)-indolactam V, phorbol 12,13-dibutyrate, ingenol 3-angelate, and (2S,5S)-(E,E)-8-(5-(4-(trifluoromethyl)phenyl)-2,4-pentadienoylamino)benzolactam, or an ester, salt, or solvate thereof.

[34] The method according to item 33, wherein the PKC activator is a compound selected from compound X and compounds #1 to #7, or an ester, salt or solvate thereof.

[35] The method of claim 31 or 34, wherein the PKC activator is compound X or an ester, salt or solvate thereof.

[36] The method according to any one of items 1 to 35, wherein the cell further contains a nucleic acid encoding a peptide comprising a Pro region or a RING region.

[37] The method according to any one of items 1 to 36, wherein the nucleic acid required for the production of viral particles is linked to a nucleic acid encoding a peptide including a Pro region or a RING region.

[38] The method of claim 36 or 37, wherein the cells further contain a nucleic acid encoding a peptide comprising a RING region.

[39] The RING domain is C-X2-C-X9-39-C-X1-3-H-X2-3-C-X2-C-X4-48-C-X2-C {wherein C is a cysteine ​​residue, H is a histidine residue, and X is any amino acid residue.} 39. The method of claim 38, comprising the amino acid sequence of

[40] The method of claim 38, wherein the RING region is a RING region of TRIM13, LONRF3, TRIM47, RNF135, TRIM10, TRIM72, TRIM60, TRIM39, TRIM4, TRIM43B, TRIM43, TRIM25, TRIM26, TRIM31, HTLF, BRCA1, TRIM50, TRIM21, SSA1, TRIM5d, TRIM22, KIAA0182, TRIM65, RAG1, BFAR, Pex10, RNF8, RING2, COPI, TRIM2, TRIM3, SH3RF2, PML, or TRIM56.

[0094]

[41] The method according to any one of items 38 to 40, wherein the RING region is a RING region of PML, TRIM3, TRIM56, COPI, Pex10, BRCA1 or HTLF.

[42] The method according to any one of items 38 to 41, wherein the RING region is a PML RING region.

[43] The method according to any one of items 38 to 42, wherein the RING region consists of an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO:10.

[44] The method according to any one of items 38 to 43, wherein the RING region is encoded by a nucleotide sequence having at least 90% identity to the nucleotide sequence of SEQ ID NO:12.

[45] The method of claim 36 or 37, wherein the cells further contain a nucleic acid encoding a peptide comprising a Pro region.

[46] The method according to paragraph 45, wherein the Pro region is the Pro region of PML, ARHGEF1, aggrecan-1, RALGDS, DGKK, SPATA21, rabfilin-3A, TEAD3, SPPL2B or FLJ43093.

[47] The method of claim 45 or 46, wherein the Pro region is the Pro region of PML.

[48] ​​The method according to any one of items 45 to 47, wherein the Pro region consists of an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO:9.

[49] The method according to any one of items 45 to 48, wherein the nucleic acid encoding a peptide comprising a Pro region is a nucleotide sequence having at least 90% identity to the nucleotide sequence of SEQ ID NO: 11.

[50] The method of claim 36 or 37, wherein the cells further contain a nucleic acid encoding a peptide comprising a Pro region and a RING region.

[0095]

[51] The method according to any one of items 36 to 50, wherein the cell contains an expression construct comprising, operably linked thereto, a promoter, a nucleic acid required for production of viral particles, and a nucleic acid encoding a peptide comprising a Pro region or a RING region.

[52] The method according to claim 51, wherein a promoter, a nucleic acid necessary for production of viral particles, and a nucleic acid encoding a peptide comprising a Pro region or a RING region are operably linked in this order.

[53] The method of claim 51 or 52, wherein the expression construct further comprises a nucleic acid encoding an internal ribosome entry site or a 2A peptide sequence.

[54] The method according to any one of items 51 to 53, wherein a promoter, a nucleic acid necessary for production of viral particles, an internal ribosome entry site or a 2A peptide sequence, and a nucleic acid encoding a peptide comprising a Pro region or a RING region are operably linked in this order.

[55] The method according to any one of items 1 to 21, wherein PKC is activated by expressing an activated form of PKC in the cell.

[56] The method of claim 55, wherein the cell contains an expression construct comprising a nucleic acid encoding an activated form of PKC.

[57] The method of claim 55, wherein the cell contains an expression construct comprising, operably linked, a promoter, a nucleic acid required for production of viral particles, an internal ribosome entry site or a 2A peptide sequence, and a nucleic acid encoding an activated form of PKC.

[58] The method according to item 57, wherein a promoter, a nucleic acid necessary for production of viral particles, an internal ribosome entry site or a 2A peptide sequence, and a nucleic acid encoding an activated PKC are operably linked in this order.

[59] The method according to any one of items 55 to 58, wherein the activated PKC is PKCδ-CA or PKCαCA-M489V.

[60] The method according to any one of items 1 to 59, wherein the cells are cultured in the presence of a calmodulin inhibitor.

[0096]

[61] The method according to claim 60, wherein the calmodulin inhibitor is a compound of formula (IV) or an ester, salt or solvate thereof:

[62] The method of claim 60 or 61, wherein the calmodulin inhibitor is SC-9, SC-10 or W-7.

[63] The method according to any one of items 60 to 62, wherein the calmodulin inhibitor is SC-10.

[64] The method according to any one of items 1 to 63, wherein the cells are cultured in the presence of a histone deacetylase inhibitor.

[65] The method of claim 64, wherein the histone deacetylase inhibitor is sodium butyrate, valproic acid, trichostatin A, vorinostat, apicidin, entinostat, or tacedinaline.

[66] The method of claim 64 or 65, wherein the histone deacetylase inhibitor is sodium butyrate.

[67] The method according to any one of items 1 to 66, wherein the cell is a packaging cell.

[68] The method according to any one of items 1 to 67, wherein the cells are Plat-E cells or Lenti-X293T cells.

[69] A kit for producing a viral vector, comprising a nucleic acid encoding a protein kinase C (PKC) activator or an activated form of PKC, and a nucleic acid necessary for producing viral particles of the viral vector, A kit, in which the nucleic acid necessary for the production of viral particles is operably linked to a promoter containing a binding site for at least one transcription factor selected from SP1, CEBP, AP1, NF-κB and YY1.

[70] A composition for enhancing production of a viral vector, comprising a PKC activator or a nucleic acid encoding an activated form of PKC.

[0097] All documents cited herein are hereby incorporated by reference. The present invention will be described in more detail below with reference to the following examples, but the present invention is not limited to these examples. The above descriptions are all non-limiting, and the present invention is defined in the appended claims, and various modifications are possible without departing from the technical spirit of the present invention. EXAMPLES

[0098] construct For measuring luciferase activity using the CMV promoter, pRL-CMV (Promega, #E2261) was used. The CAG promoter was derived from the pCAGGS vector, and the EF1 promoter was derived from the pEFBOS vector. Constructs were created by replacing the CMV region of pRL-CMV with the CAG promoter. Constructs expressing mouse IgG antibodies (heavy and light chains), human proinsulin, and human leptin were created by inserting these cDNAs downstream of the CMV promoter of the pcDNA vector. The binding sites of transcription factors in these promoters are shown in Figure 42.

[0099] cell HEK293A cells (human embryonic kidney cells) were cultured in DMEM medium containing 10% FCS and 1% penicillin / streptomycin under conditions of 37°C, 5% CO2, and 90% humidity. HEK293A cell lines that constitutively express mouse IgG antibody, human proinsulin, and leptin were transfected with constructs in which these cDNAs were inserted downstream of the CMV promoter, and then single clones were formed by long-term culture. Expression of the target protein from each clone was then confirmed using ELISA, and cell lines were established. These cell lines were also cultured under the same conditions.

[0100] Transfection HEK293A cells were seeded in a 24-well plate at 1.0×105 cells / well and cultured for 24 hours. Unless otherwise stated, 50 ng of expression construct, 1 μl Plus Reagent, and 1 μl Lipofectamine LTX per well were prepared in 50 μl Opti-MEM and added to each well of the 24-well plate where the cells were cultured, and then transfection was performed by culturing at 37°C for 24 hours.

[0101] Luciferase assay 24 hours after transfection, the medium was removed from the wells, and the medium containing each compound was added and cultured for another 24 hours. After that, the medium was removed, the cells were washed with 500 μl of PBS, and then lysed with 50 μl of 1x Glo Lysis buffer (Promega #E2661). Of this, 5 μl was used for luciferase activity measurement, and 5 μl was used for protein concentration measurement. Luciferase activity was quantified by detecting the luminescence of coelenterazine h (FUJIFILM #035-22991), and the protein concentration was quantified using the BCA method, and each luciferase activity was calculated as the Rluc / BCA value. A multi-label reader 2030 ARVOTM X (Perkin Elmer) was used for luminescence detection and BCA measurement.

[0102] Western blot HEK293A cells were seeded in a 6-well plate at 5.0 x 105 cells / well and cultured for 24 hours. After that, each compound group was treated by changing the medium, and after 3 hours, the cells were washed with 2 ml of PBS and then lysed using 200 μl of RIPA buffer. The lysed cells were disrupted using ultrasonic treatment and centrifuged (20,000 g, 15 min, 4°C), the supernatant was collected, and the protein concentration was quantified. The lysates were combined to an equal volume, 1x SDS sample buffer was added, and the mixture was heated at 95°C for 3 min to be used as a sample for SDS-PAGE (5 μg of protein per lane). SDS-PAGE gels were SuperSepTM Ace 5-20%, 17-well (Wako) gels, and each gel contained 1 μg of protein. Electrophoresis was performed under conditions of 500V, 40mA, 35 minutes. Then, using a blocking device (ATTO), the gel was transferred to a PVDF membrane under conditions of 500V, 100mA, 60 minutes per gel. The transferred membrane was permeated with blocking solution (3%BSA / TBS-T) at room temperature for 30 minutes, and the primary antibody reaction was performed overnight at 4°C under conditions of 3000-fold dilution of the primary antibody (Phospho-PKC Substrate Motif [(R / K)XpSX(R / K)] MultiMabTM Rabbit mAb mix, Cell Signaling Technology #6967)) in the blocking solution. Then, the membrane was washed three times with TBS-T, and the secondary antibody reaction was performed at room temperature for 3 hours under conditions of 5000-fold dilution of HRP-labeled secondary antibody (Anti-Rabbit IgG, HRP-Linked Whole Ab Donkey, Cytiva #NA934-1ML) in TBS-T. The membrane was then washed three times with TBST and detection was performed using Chemi-Lumi One (Nacalai #07880-70) and ImageQuant LAS4010.

[0103] ELISA method Each secretory protein expressing cell was seeded in a 24-well plate at 1.0×104 cells / well and cultured for 24 hours. After that, each compound group was treated by changing the medium at 1ml / well, and 120μl of the medium was collected every 24 hours and stored in a refrigerator. After that, the concentration of the target protein in the medium was measured using each ELISA kit (mouse IgG, Betyl Lab #E99-131, human proinsulin: Mercodia #10-1118-01, human leptin: Proteintech #KE00095). A multi-label reader 2030 ARVOTM X (Perkin Elmer) was used for the measurement.

[0104] compound The compounds in the table below were used. [Table 1]

[0105] Test 1: Identification of compounds that enhance the transcriptional activity of the CMV promoter HEK293T cells were transfected with a plasmid containing a luciferase gene downstream of the CMV promoter at 20 ng per well of a 24-well plate, and 24 hours later, the medium was replaced with one containing 30 μg / ml of plant extract. After 24 hours, CMV promoter activity was measured using a luciferase assay. Approximately 1,000 types of plant extracts were used in this study. From a unique plant (herbal medicine) extract library, five plant extracts that dramatically enhance the transcriptional activity of the CMV promoter and the compounds responsible for this activity (8 types listed below) were identified. [ka]

[0106] Test 2: The activity of the compound is inhibited by inhibitors of protein kinase C (PKC) HEK293A cells constitutively expressing pRL-CMV were cultured for 24 hours in medium containing compound X or compounds #1 to #7 at the concentrations shown in Figure 1 in the presence or absence of a PKC inhibitor, and luciferase activity was measured. The results are shown in Figure 1. The transcriptional activity of the CMV promoter was enhanced in the presence of the compounds, and this enhancement was inhibited by the PKC inhibitor.

[0107] HEK293A cells that constitutively express pRL-CMV were cultured in medium containing compound X (100 ng / ml) for 24 hours, and luciferase activity was measured over time. The results are shown in Figure 2. Enhancement of the transcriptional activity of the CMV promoter by compound X was observed from 1 hour after the addition of compound X.

[0108] Test 3: CMV promoter activity is also enhanced by known PKC activators HEK293A cells constitutively expressing pRL-CMV were cultured for 24 hours in medium containing compound X or PKC activators #1 to #4 at the concentrations shown in Figure 3, in the presence or absence of a PKC inhibitor, and luciferase activity was measured. The results are shown in Figure 3. The transcriptional activity of the CMV promoter was also enhanced by known PKC activators.

[0109] Compound X, compounds #1 to #7, and PKC activators #1 to #4 belong to the terpene group, which are collectively called diterpenes. They are classified into the following three types based on their skeletons. [ka]

[0110] Test 4: CMV promoter activity is enhanced by PKC activators other than diterpenes HEK293A cells constitutively expressing pRL-CMV were cultured for 24 hours in medium containing compound X or PKC activators #5 to #6 at the concentrations shown in Figure 4, in the presence or absence of a PKC inhibitor (Ro-318425), and luciferase activity was measured. The results are shown in Figure 4. The transcriptional activity of the CMV promoter was also enhanced by PKC activators other than diterpenes.

[0111] Test 5: Compounds also enhance the activity of the CAG and EF1 promoters HEK293A cells were transfected with pCAG-Rluc or pEF1-Rluc and cultured in a medium containing compound X, compounds #1 to #7, or PKC activators #1 to #6 at the concentrations shown in Figures 5 to 7, and the transcriptional activity of the CAG promoter derived from the pCAGGS vector and the EF1 promoter derived from the pEFBOS vector was measured. The results are shown in Figures 5 to 7. The transcriptional activity of the CAG promoter and the EF1 promoter was enhanced in the presence of the compounds or PKC activators, but the enhancement was inhibited by a PKC inhibitor. These promoters have binding sites for transcription factors such as SP1, CEBP, AP1, NF-κB, and YY1. Therefore, it is suggested that these compounds and PKC activators activate these promoters through a common transcription factor.

[0112] Test 6: Compounds activate PKC HEK293A cells were cultured for 3 hours in medium containing compound X, compounds #1-#7, PKC activators #1-#6, compound SC or sodium butyrate (SB) at the same concentrations as in tests 2-5, in the presence or absence of a PKC inhibitor (3 μM). The cells were lysed and Western blots were performed using an antibody that specifically recognizes proteins phosphorylated by PKC. The results are shown in Figure 8. Compound X, compounds #1-#7 and PKC activators #1-#6 activated PKC. PKC activation by compound SC and sodium butyrate was not confirmed.

[0113] Study 7: Identification of novel CMV promoter activators HEK293A cells constitutively expressing pRL-CMV were cultured in a medium containing compound X and / or compound SC at the concentrations shown in Figure 9 in the presence or absence of a PKC inhibitor for 24 hours, and luciferase activity was measured. The results are shown in Figure 9. The transcriptional activity of the CMV promoter was also enhanced by compound SC, but this enhancement was not inhibited by the PKC inhibitor. The combined use of compound X and compound SC further enhanced the transcriptional activity.

[0114] Study 8: The transcriptional activity of the CMV promoter was further enhanced by the combined use of a PKC activator, compound SC, and a histone deacetylase inhibitor HEK293A cells constitutively expressing pRL-CMV were cultured for 24 hours in medium containing compound X, compound SC and / or sodium butyrate (SB) at the concentrations shown in Figure 10, and luciferase activity was measured. The results are shown in Figure 10. The transcriptional activity of the CMV promoter was highest when compound X, compound SC and sodium butyrate were used in combination. Similar results were obtained with compounds #1-#7 and PKC activators #1-#6 (Figures 11 and 12). Similar results were obtained when histone deacetylase inhibitors valproic acid, trichostatin A (TSA), vorinostat (SAHA), apicidin, entinostat (MS-275) or tacedinarin (CI994) were used instead of sodium butyrate (Figure 13).

[0115] Test 9: Increasing protein production with PKC activators The cDNA of mouse IgG antibody heavy chain, mouse IgG antibody light chain, human proinsulin or human leptin was inserted downstream of the CMV promoter of the pcDNA vector to prepare an expression construct. The mouse IgG antibody and human leptin constructs were transfected into HEK293A cells, respectively. The human proinsulin construct was transfected into HEK293A cells to prepare HEK293A cells that constitutively express human proinsulin. The cells were cultured for 24 hours in a medium containing compound X, compounds #1 to #7, and PKC activators #1 to #6 at the concentrations shown in Figures 14 to 17, in the presence or absence of compound SC or compound SC + sodium butyrate (SB), and the concentration of each protein in the medium was measured by ELISA. The results are shown in Figures 14 to 17. Furthermore, for compound X, HEK293A cells constitutively expressing mouse IgG antibody heavy and light chains, human proinsulin, or human leptin were used, and protein concentrations were measured every 24 hours for 4 days in the presence or absence of sodium butyrate (SB). The results are shown in Figure 18. The production of IgG antibody, proinsulin, and leptin was enhanced by these compounds. The production of IgG antibody and proinsulin was further enhanced when these compounds were combined with compound SC and sodium butyrate. The production of leptin was most enhanced when these compounds were combined with compound SC, and was reduced in the presence of SB, but was higher than when cultured in a medium without any of the substances.

[0116] Test 10: Activation of the CMV promoter by engineered PKC activation PKC was genetically activated to examine the transcriptional activity of the CMV promoter. An expression construct was used in which the cDNA of the protein (XXX), an IRES, and the cDNA of activated PKC were linked downstream of the CMV promoter. As activated PKC, the 334-695 amino acid region of human PKCδ (PKCδ-CA) and the constitutively activated form in which the 489th methionine in the 326-672 amino acid region of human PKCα was changed to valine (PKCαCA-M489V) were used. In addition, Rluc, mouse IgG antibody (heavy chain, light chain), and human leptin genes were inserted into XXX. Schematic diagrams of these constructs are shown in Figure 19.

[0117] An expression construct was prepared by inserting Rluc cDNA, IRES, and PKCδ-CA or PKCαCA-M489V cDNA downstream of the CMV promoter of a pcDNA vector. A control construct lacking PKC was also prepared. These constructs were transfected into HEK293A cells at concentrations of 6.25 to 200 ng / well, cultured for 24 hours, and luciferase activity was measured. The results are shown in Figure 20. Luciferase activity was high in the PKC group. This result indicates that the transcriptional activity of the CMV promoter is enhanced by positive feedback. Similar results were obtained with an expression construct that used the P2A peptide sequence, a 2A self-cleaving peptide sequence, instead of the IRES (Figure 21).

[0118] Mouse IgG antibody heavy or light chain cDNA, IRES, and PKCδ-CA or PKCαCA-M489V cDNA were inserted downstream of the CMV promoter of the pcDNA vector to create an expression construct. A control construct lacking PKC was also created. The IgG antibody heavy and light chain constructs were combined and transfected into HEK293A cells at a concentration of 100 ng / well, cultured for 24 hours, and the concentration of IgG antibody in the medium was measured by ELISA every 24 hours for 4 days. The results are shown in Figure 22. In the PKC group, IgG antibody production was enhanced. This result indicates that genetic engineering PKC activation can activate the CMV promoter and enhance protein production.

[0119] Human leptin cDNA, IRES, and PKCαCA-M489V cDNA were inserted downstream of the CMV promoter of the pcDNA vector to prepare an expression construct. A control construct lacking PKC was also prepared. These constructs were transfected into HEK293A cells at a concentration of 100 ng / well, cultured for 24 hours, and the concentration of leptin in the medium was measured over 4 days by ELISA. The results are shown in Figure 23. In the PKC group, leptin production was enhanced. This result indicates that genetically engineered PKC activation can activate the CMV promoter and enhance protein production.

[0120] Test 11: Search for target proteins of compound SC The synergistic effect of naphthalenesulfonamide derivatives having the following structure, including compound SC, with compound X was examined. [Table 2]

[0121] HEK293A cells constitutively expressing pRL-CMV were cultured for 24 hours in the presence or absence of compound X (100 ng / ml) in a medium containing any of the naphthalenesulfonamide derivatives at the concentrations shown in Figure 24, and luciferase activity was measured. The results are shown in Figure 24. The transcription activity of the CMV promoter did not change with the naphthalenesulfonamide derivative alone, but was enhanced when the naphthalenesulfonamide derivative was used in combination with compound X. The naphthalenesulfonamide derivative W-7 is used as a calmodulin inhibitor, and compounds SC and SC-9 also act as calmodulin inhibitors, and are thought to show a synergistic effect with compound X.

[0122] Study 12: PKC activators and engineered PML expression enhance the transcriptional activity of the CMV promoter An expression construct was prepared by inserting Rluc cDNA, IRES, and wild-type PML or deletion mutant PML (PMLΔ1-10 or PML-Ring) cDNA downstream of the CMV promoter of a pcDNA vector (FIG. 25). The constructs of wild-type PML and PMLΔ1-10 were transfected into HEK293A cells, which were cultured for 24 hours in the presence or absence of compound X, and luciferase activity was measured. The results are shown in FIG. 26. In cells into which wild-type PML, PMLΔ1, and PMLΔ6-10 had been introduced, luciferase activity in the presence of compound X was higher than that in control (-) cells. This result suggests that PML further enhances the transcriptional activity of the CMV promoter enhanced by compound X, and that the Pro region including positions 1 to 46 of PML is responsible for this activity. Furthermore, in cells into which wild-type PML, PMLΔ1, and PMLΔ6 to 10 had been introduced, luciferase activity was higher than that in control (-) cells, even in the absence of compound X.

[0123] In addition, constructs containing PMLΔ9 or PML-Ring were transfected into HEK293A cells, and the cells were cultured for 24 hours in the presence or absence of compound X, SB, compound SC, or a combination thereof, and luciferase activity was measured. The results are shown in Figure 27. In cells into which PMLΔ9 or PML-Ring was introduced, luciferase activity was higher under all conditions compared to control (Mock) cells. This result suggests that the RING region containing positions 47 to 106 of PML also further enhances the transcriptional activity of the CMV promoter enhanced by compound X. In addition, in cells into which PMLΔ9 or PML-Ring was introduced, luciferase activity was higher than that of control (-) cells, even in the absence of compound X.

[0124] Test 13: Expression of PKC activators and peptides containing the RING domain enhances the transcriptional activity of the CMV promoter The RING region has a common sequence among various proteins. The amino acid sequence of the RING region of PML was analyzed by BLAST, and the proteins shown in FIG. 28 were identified as having a similar sequence. In the RING regions of these proteins, some cysteine ​​and histidine residues were highly conserved. Several proteins with different degrees of similarity to the RING region of PML were selected and used in the following analysis.

[0125] The cDNA of Rluc, IRES, and the cDNA of the RING region of PML, TRIM3, TRIM56, COPI, Pex10, BRCA1, or HTLF were inserted downstream of the CMV promoter of the pcDNA vector to prepare an expression construct (Figure 29). These expression constructs were transfected into HEK293A cells, cultured for 24 hours in the presence or absence of compound X, and luciferase activity was measured. The results are shown in Figure 30. In the cells into which the RING region was introduced, the luciferase activity in the presence of compound X was higher than that in the control (-) cells. This result suggests that the RING region of various proteins further enhances the transcriptional activity of the CMV promoter enhanced by compound X. In addition, in the cells into which the RING region was introduced, the luciferase activity was higher than that in the control (-) cells, even in the absence of compound X.

[0126] Study 14: Enhancement of CMV promoter transcriptional activity by PML depends on mRNA expression An expression construct was prepared by inserting Rluc cDNA, IRES, and wild-type PML or deletion mutant PML (PMLΔ9, PMLΔ9 with a stop codon introduced at position 2, or PMLΔ9 with a stop codon introduced at position 7) downstream of the CMV promoter of a pcDNA vector (Figure 31, left). In addition, an expression construct was prepared by inserting Rluc cDNA and wild-type PML or deletion mutant PML (PMLΔ9, PMLΔ10, or PML-RING) cDNA downstream of the CMV promoter of a pcDNA vector (Figure 31, right). When these constructs are introduced into cells, PML mRNA is transcribed but not translated into protein. These constructs were transfected into HEK293A cells, cultured for 24 hours in the presence or absence of compound X, and luciferase activity was measured. The results are shown in Figure 32. All constructs showed higher luciferase activity than the control in the presence and absence of compound X. This result suggests that PML-induced enhancement of the CMV promoter transcriptional activity depends on the expression of mRNA encoding the Pro or RING domain of PML, rather than on the expression of PML protein.

[0127] Test 15: Viral Vector Production Materials and Methods construct A retroviral vector was prepared by inserting the GFP gene into the multiple cloning site of the pQCXIN vector (TaKaRa #631514), and pcDNA-VSV-G (vesicular stomatitis virus G glycoprotein) was used as the vector to express the envelope protein. A lentiviral vector was prepared by inserting the GFP gene into the multiple cloning site of the pLVSIN-CMV Pur vector (TaKaRa #6183). In addition, pcDNA-VSV-G was used as a vector expressing the envelope protein, and psPAX2 (Addgene #12260) was used as a vector expressing the structural genes (gag, pol) required for virus production. The adenovirus vector was prepared by inserting GFP into the pAd / DEST / CMV / V5-DEST Gateway vector (Invitrogen #V49320), which was then treated with the restriction enzyme PacI, purified, and used. The adeno-associated virus vectors used were pAAV-CMV-PI-EGFP-WPRE-bGH (Addgene #105530), pAAV-2 / 9n (Addgene #112865), and pAdDeltaF6 (Addgene #112867).

[0128] The promoters of each construct are shown in the table below and in Figures 43 to 45. [Table 1] *Ad5(ΔE1,ΔE3) contains the E2A, E2B, E4, L1, L2, L3, L4 and L5 regions of the adenovirus genome.

[0129] cell Plat-E cell line (Cosmobio #RV-101) and Lenti-X293T cell line (TaKaRa #Z2180N) were cultured in DMEM medium containing 10% FCS and 1% penicillin / streptomycin under conditions of 37°C, 5% CO2, and 90% humidity.

[0130] Transfection Plat-E cells or Lenti-X293T cells were seeded in a 6-well plate at 2.0 x 106 cells / well and cultured for 24 hours. 2-5 μg of construct per well and 12 μl-PEI-Max were adjusted with 100 μl of DMEM, added to each well of the 6-well plate where the cells were cultured, and cultured at 37°C for 24 hours to perform transfection.

[0131] Collection and preparation of virus fluid 24 hours after transfection, the medium was removed from the wells, and medium containing each concentration of compound was added and cultured for another 48 hours. The medium was then collected and passed through a syringe filter (pore size 0.45 μm), after which an appropriate amount of Retro-X Concentrator (TaKaRa #631455) was added to the medium for retrovirus and adeno-associated virus, and an appropriate amount of Lenti-X Concentrator (TaKaRa #631231) was added to the medium for lentivirus and adenovirus, and the wells were left to stand overnight at 4°C. Then, the wells were centrifuged (1,500g, 45 minutes, 4°C), and the precipitated fraction was suspended in the virus medium to obtain a concentrated virus solution.

[0132] Counting virus-infected cells HEK293A cells were seeded in a 24-well plate at 1.0×105 cells / well and cultured for 24 hours. The concentrated virus solution was then treated by changing the medium, and the virus was infected for 48 hours. Then, when changing the medium, nuclear DNA was stained by treating with Hoechst 33258 (Wako #343-07961). After leaving the cells in a CO2 incubator for 30 minutes or more, GFP and Hoechst 33258 images were photographed using an all-in-one fluorescence microscope (Keyence BZ-X810), and the proportion of virus-infected cells was then measured using (number of GFP-positive cells / number of Hoechst 33258-positive cells).

[0133] result (1) Enhancement of retroviral vector production by CMV promoter-activating compounds Plat-E cells were seeded in a 6-well plate at 2.0×106 cells / well and cultured for 24 hours, and then transfected with 1.5μg of a construct in which a GFP gene was inserted into the multicloning site of the pQCXIN vector and 0.4μg of pcDNA-VSV-G per well. After 24 hours, the medium was replaced and each compound (compound X: 100ng / ml, sodium butyrate (SB): 2mM, compound SC: 5μg / ml) was added and cultured for 48 hours. The virus was then collected and concentrated from the medium and suspended in 400μl of DMEM medium containing 10% FCS and 1% penicillin / streptomycin. The suspension was used to infect HEK293A cells in the undiluted form, and after 48 hours, nuclear DNA was stained by treatment with Hoechst 33258 and photographed. The ratio of virus-infected cells was measured by measuring (GFP-positive cell count / Hoechst 33258-positive cell count). A dramatic increase in the number of GFP-positive cells (virus-infected cell count) was confirmed by treatment with compound X, or treatment with compounds X and SB, or treatment with compounds X, SB, and compound SC (FIG. 33).

[0134] (2) Enhancement of lentiviral vector production by PKC activators Lenti-X293T cells were seeded in a 6-well plate at 2.0×106 cells / well, and cultured for 24 hours. Then, 2μg of a construct in which GFP was inserted into the multicloning site of the pLVSIN-CMVPur vector, 0.6μg of pcDNA-VSV-G, and 1.6μg of psPAN2 were transfected per well. After 24 hours, the medium was replaced, and each compound (compound X: 100ng / ml, sodium butyrate (SB): 2mM, compound SC: 5μg / ml) was added and cultured for 48 hours. Then, the virus was collected and concentrated from the medium, and suspended in 400μl of DMEM medium containing 10% FCS and 1% penicillin / streptomycin. The suspension was diluted 100-fold and then infected into HEK293A cells. After 48 hours, nuclear DNA was stained by treatment with Hoechst 33258 and photographed. The ratio of virus-infected cells was measured by measuring (GFP-positive cell count / Hoechst 33258-positive cell count). An increase in the number of GFP-positive cells (virus-infected cell count) was confirmed by treatment with compound X, or treatment with compounds X and SB, or treatment with compounds X, SB, and compound SC (FIG. 34).

[0135] (3) Enhancement of adenovirus vector production by CMV promoter-activating compounds Lenti-X293T cells were seeded in a 6-well plate at 2.0×106 cells / well, and cultured for 24 hours. Then, 2μg of a vector in which GFP was inserted into the PacI-treated pAd / DEST / CMV / V5-DEST Gateway vector was transfected per well. After 24 hours, the medium was replaced, and each compound (compound X: 100ng / ml, sodium butyrate (SB): 2mM, compound SC: 5μg / ml) was added and cultured for 48 hours. Then, the virus was collected and concentrated from the medium, and suspended in 400μl of DMEM medium containing 10% FCS and 1% penicillin / streptomycin. The suspension was diluted 3-fold and then infected into HEK293A cells. After 48 hours, nuclear DNA was stained by treatment with Hoechst 33258, and photographed. The ratio of virus-infected cells was measured by measuring (number of GFP-positive cells / number of Hoechst 33258-positive cells). An increase in the number of GFP-positive cells (number of virus-infected cells) was confirmed by treatment with compound X, or treatment with compounds X and SB, or treatment with compounds X, SB, and compound SC (FIG. 35).

[0136] (4) Enhancement of adeno-associated virus vector production by CMV promoter activating compounds Lenti-X293T cells were seeded in a 6-well plate at 2.0×106 cells / well, cultured for 24 hours, and then transfected with 1.5 μg of pAAV-CMV-PI-EGFP-WPRE-bGH, 1.5 μg of pAAV-2 / 9n, and 1.5 μg of pAdDeltaF6 per well. After 24 hours, the medium was replaced, and each compound (compound X: 100 ng / ml, sodium butyrate (SB): 2 mM, compound SC: 5 μg / ml) was added and cultured for 48 hours. The virus was then collected and concentrated from the medium, and suspended in 400 μl of DMEM medium containing 10% FCS and 1% penicillin / streptomycin. The suspension was diluted 3-fold and then infected into HEK293A cells, and 48 hours later, nuclear DNA was stained by treatment with Hoechst 33258 and photographed. The ratio of virus-infected cells was measured by measuring (number of GFP-positive cells / number of Hoechst 33258-positive cells). An increase in the number of GFP-positive cells (number of virus-infected cells) was confirmed by treatment with compound X, or treatment with compounds X and SB, or treatment with compounds X, SB, and compound SC (FIG. 36).

[0137] (5) Changes in enhancement of retroviral vector production by CMV promoter activating compounds and PKC inhibitors As in (1), a retroviral vector was transfected using Plat-E cells, and after 24 hours, the medium was replaced and each active compound was added in the presence or absence of PKC inhibitor Ro-318425 at 3 μM, and the cells were cultured for 48 hours. The virus was then collected and concentrated from the medium and suspended in 400 μl of DMEM medium containing 10% FCS and 1% penicillin / streptomycin. The suspension was used to infect HEK293A cells in the original form, and after 48 hours, nuclear DNA was stained by treating with Hoechst 33258 and photographed. The ratio of virus-infected cells was measured by measuring (number of GFP-positive cells / number of Hoechst 33258-positive cells). A dramatic increase in the number of GFP-positive cells (number of virus-infected cells) by the active compound group and suppression by the PKC inhibitor were confirmed (Figure 37).

[0138] (6) Changes in enhanced viral vector production by CMV promoter activating compounds and PKC inhibitors As in (5), Plat-E cells were transfected with retrovirus vectors, lentivirus vectors, adenovirus vectors, and adeno-associated virus vectors. After 24 hours, the medium was replaced and each active compound was added in the presence or absence of PKC inhibitor Ro-318425 at 3 μM, and the cells were cultured for 48 hours. The viruses were then collected and concentrated from the medium and suspended in 400 μl of DMEM medium containing 10% FCS and 1% penicillin / streptomycin. The suspension was used to infect HEK293A cells with the retrovirus as a stock solution, the lentivirus diluted 100-fold, the adenovirus vector and the adeno-associated virus vector diluted 3-fold, and the cells were infected with HEK293A cells. After 48 hours, the nuclear DNA was stained by treating with Hoechst 33258 and photographed. The percentage of virus-infected cells was measured by measuring (number of GFP-positive cells / number of Hoechst 33258-positive cells). A dramatic increase in the number of GFP-positive cells (number of virus-infected cells) was observed with the activating compound group, while the PKC inhibitor suppressed this increase (FIGS. 38 to 41). [Industrial Applicability]

[0139] INDUSTRIAL APPLICABILITY The present disclosure makes it possible to increase the production yield of viral vectors, and therefore can be used in the production of biopharmaceuticals.

Claims

1. A method for producing a viral vector, comprising culturing cells containing nucleic acids necessary for the production of viral particles of the viral vector and nucleic acids to be delivered by the viral vector under conditions that activate protein kinase C (PKC), A method in which nucleic acids necessary for the production of viral particles are operably linked to a promoter that includes a binding site for at least one transcription factor selected from SP1, CEBP, AP1, NF-κB, and YY1.

2. The method according to claim 1, wherein the viral vector is a retroviral vector, a lentiviral vector, an adenovirus vector, or an adeno-associated virus vector.

3. The method according to claim 1, wherein the viral vector is a retroviral vector.

4. The method according to claim 1, wherein the nucleic acid to be delivered by the viral vector is operably linked to a promoter that includes a binding site for at least one transcription factor selected from SP1, CEBP, AP1, NF-κB, and YY1.

5. The method according to claim 1, wherein PKC is activated by culturing the cells in the presence of a PKC activator.

6. The PKC activator is given by formula (I): 【Chemistry 1】 {During the ceremony, R １ is H, halogen, -OH, C １－６ alkyl, C ２－６ alkenyl, C ２－６ alkynyl, C １－６ alkoxy, C ６－１４ aryl or -OC(O)R ３ wherein C １－６ alkyl, C ２－６ alkenyl, C ２－６ alkynyl, C １－６ alkoxy or aryl may be the same or different and may be substituted by one to three halogen atoms, R ２ C ６－１２ Alkyl, C ６－１２ Alkenil, C ６－１２ Alkinyl or C ６－１２ It is an alkoxy, and here, C ６－１２ Alkyl, C ６－１２ Alkenil, C ６－１２ Alkinyl or C ６－１２ The alkoxy may be substituted with one to three identical or different halogens. R ３ C １－６ Alkyl, C ２－６ Alkenil, C ２－６ Alkinyl, amino, or C ６－１４ It is aryl, and here C １－６ Alkyl, C ２－６ Alkenil, C ２－６ Alkinyl or C ６－１４ The aryl group may be substituted with one to three identical or different halogens. Formula (II): 【Chemistry 2】 {During the ceremony, R ４ H, halogen, -OH, C １－１８ Alkyl, C ２－１８ Alkenil, C ２－１８ Alkinyl, C １－１８ Alkoxy or -OC(O)R ６ And here, C １－１８ Alkyl, C ２－１８ Alkenil, C ２－１８ Alkinyl or C １－１８ The alkoxy may be substituted with one to three identical or different halogens. R ５ C １－６ Alkyl, C ２－６ Alkenil, C ２－６ It is an alkynyl or amino, where C １－６ Alkyl, C ２－６ Alkenyl or C ２－６ Alkynyl may be substituted with one to three identical or different halogens. R ６ C １－１８ Alkyl, C ２－１８ Alkenil, C ２－１８ It is an alkynyl or amino, where C １－１８ Alkyl, C ２－１８ Alkenyl or C ２－１８ The alkynyl may be substituted with one to three identical or different halogens. or Formula (III): 【Transformation 3】 {During the ceremony, R ７ H, C １－１８ Alkyl, C ２－１８ Alkenil, C ２－１８ Alkinyl or -C(O)R ９ And here, C １－１８ Alkyl, C ２－１８ Alkenyl or C ２－１８ Alkynyl may be substituted with one to three identical or different halogens. R ８ H, C １－１８ Alkyl, C ２－１８ Alkenil, C ２－１８ Alkinyl or -C(O)R ９ And here, C １－１８ Alkyl, C ２－１８ Alkenyl or C ２－１８ Alkynyl may be substituted with one to three identical or different halogens. R ９ C １－１８ Alkyl, C ２－１８ Alkenil, C ２－１８ It is an alkynyl or amino, where C １－１８ Alkyl, C ２－１８ Alkenyl or C ２－１８ The alkynyl may be substituted with one to three identical or different halogens. The method according to claim 5, wherein the compound or its ester, salt, or solvate.

7. PKC activator, 【Chemistry 4】 The method according to claim 5, wherein the compound is selected from TPA, prostratin, (-)-indolactam V, phorbol 12,13-dibutyrate, ingenol 3-angelate, and (2S,5S)-(E,E)-8-(5-(4-(trifluoromethyl)phenyl)-2,4-pentadienoylamino)benzolactam or an ester, salt, or solvate thereof.

8. The method according to claim 7, wherein the PKC activator is a compound selected from compound X and compounds #1 to #7, or an ester, salt, or solvate thereof.

9. The method according to claim 5, wherein the nucleic acid necessary for the production of viral particles is linked to a nucleic acid encoding a peptide including a Pro region or a RING region.

10. The method according to claim 1, wherein PKC is activated by expressing activated PKC in the cells.

11. The method according to any one of claims 1 to 10, wherein the cells are cultured in the presence of a calmodulin inhibitor.

12. The method according to any one of claims 1 to 10, wherein the cells are cultured in the presence of a histone deacetylase inhibitor.

13. The method according to any one of claims 1 to 10, wherein the cells are cultured in the presence of a calmodulin inhibitor and a histone deacetylase inhibitor.

14. The method according to any one of claims 1 to 10, wherein the cells are packaging cells.

15. A kit for producing a viral vector, comprising a protein kinase C (PKC) activator or nucleic acid encoding activated PKC, and nucleic acid necessary for the production of viral particles of a viral vector, A kit in which nucleic acids necessary for the production of viral particles are operably linked to a promoter containing a binding site for at least one transcription factor selected from SP1, CEBP, AP1, NF-κB, and YY1.

16. The kit according to claim 15, comprising a PKC activator.

17. A composition for enhancing the production of viral vectors, comprising a protein kinase C (PKC) activator or a nucleic acid encoding activated PKC.

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