Method for producing viral vector

Abstract

The present invention addresses the problem of providing a method for producing a viral vector, with which the production amount of the viral vector can be improved. The present invention provides a method for producing a viral vector, the method comprising culturing producer cells or packaging cells in a culture solution that contains the producer cells or the packaging cells at a cell density of 1 × 106 cells / mL or more and also contains a histone deacetylase inhibitor.

Description

Method for producing viral vectors

[0001] The present invention relates to a method for producing a viral vector, which comprises culturing producer cells or packaging cells under predetermined conditions.

[0002] Adeno-associated virus (AAV) is a linear, single-stranded DNA virus belonging to the Parvoviridae family. The wild-type AAV genome contains a replication regulatory gene (Rep gene) and a capsid structural gene (Cap gene), and is flanked by inverted terminal repeats (ITRs) for viral replication and packaging. AAV vectors are capable of gene transfer into both proliferating and non-proliferating cells, and are particularly capable of long-term expression in non-dividing cells. AAV is also considered non-pathogenic and has low immunogenicity. For these reasons, AAV vectors are increasingly being used clinically as gene therapy vectors. Generally, AAV vectors for gene therapy contain a therapeutic gene sandwiched between ITRs. A method for producing viral vectors such as AAV vectors involves culturing producer cells or packaging cells to produce viral vectors.

[0003] Patent Document 1 describes the production of recombinant AAV (rAAV) particles by culturing cells capable of producing rAAV particles in the presence of a histone deacetylase (HDAC) inhibitor. Non-Patent Document 1 shows that the titer of rAAV8 genome is improved by using M344 (a selective HDAC inhibitor) when producing rAAV using HEK293 cells.

[0004] Special Publication No. 2021-533757

[0005] Joseph M. Scarrott et al. , Biotechnol. J. 2023:18:2200450

[0006] Currently, gene therapy drugs using AAV vectors have been commercialized, and there is a demand for the development of a production method that improves the production yield of viral vectors such as AAV vectors. An object of the present invention is to provide a method for producing a viral vector that can improve the production yield of the viral vector.

[0007] As a result of extensive research into solving the above problems, the present inventors have found that ６ It has been found that the production yield of viral vectors can be improved by culturing producer cells or packaging cells at a cell density of 1000 cells / mL or more in the presence of a histone deacetylase inhibitor. The present invention was completed based on this finding.

[0008] According to the present invention, the following inventions are provided: <1> 1 × 10 ６ A method for producing a viral vector, comprising culturing producer cells or packaging cells in a culture medium containing the producer cells or packaging cells at a cell density of 100 cells / mL or more and a histone deacetylase inhibitor. <2> The production method according to <1>, wherein the histone deacetylase inhibitor is a compound having a benzamide structure or hydroxamic acid. <3> The production method according to <1>, wherein the histone deacetylase inhibitor is a compound represented by formula (1) or formula (2). R １ -CONH-R ２ (1) In formula (1), R １ and R ２ is an aromatic group which may have a substituent, and R １ and R ２ The other is an optionally substituted amino group, an optionally substituted alkyl group, an optionally substituted alkenyl group, an optionally substituted aromatic group, or an optionally substituted alkoxy group: R ３ -CONH-OH (2) In formula (2), R ３ is an optionally substituted alkyl group or an optionally substituted alkenyl group. <4> The production method according to <1>, wherein the histone deacetylase inhibitor is a compound represented by formula (1A), formula (1B), or formula (2A). In formula (1A), R １０ represents a halogen atom, a heterocyclic group which may have a substituent, an alkyl group having 1 to 6 carbon atoms which may have a substituent, or an amino group which may have a substituent, and R ２ is an optionally substituted amino group, an optionally substituted alkyl group, or an optionally substituted aromatic group: In formula (1B), R ２０ represents an amino group, and R １ is an alkyl group which may have a substituent, or an aromatic group which may have a substituent: R ３１ -R ３０ -CONH-OH (2A) In formula (2A), R ３０ is -(CH ２ ) ｎ - or CH=CH-, n is an integer of 2 to 8, R ３１ represents an aromatic group which may have a substituent, or CONH—OH. １０ But bromine, Methyl, or CH ３ <6> The method according to <1>, wherein the histone deacetylase inhibitor is any one of the following compounds: <7> The production method according to any one of <1> to <6>, which comprises perfusion culturing the cells. <8> The production method according to any one of <1> to <6>, wherein the virus is an adeno-associated virus. <9> The production method according to <8>, wherein the producer cell or packaging cell is a cell having a Rep gene, a Cap gene, and a helper gene integrated into its chromosome. <10> The production method according to any one of <1> to <6>, wherein the cell is an animal cell. <11> The production method according to any one of <1> to <6>, wherein the content of the histone deacetylase inhibitor in the culture medium is 50 μmol / L or less. <12> The production method according to any one of <1> to <6>, wherein the culture is carried out for 24 hours or more in a culture medium containing a histone deacetylase inhibitor. <13> The production method according to any one of <1> to <6>, wherein the cell density is 30×10 ６<14> The method according to any one of <1> to <6>, wherein the culture is a suspension culture.

[0009] According to the present invention, the production yield of viral vectors can be improved.

[0010] Figure 1 shows the titer-improving effect of compounds. Figure 2 shows the results of measuring ddPCR titers in high-density cells. Figure 3 shows the results of measuring cell density in a reactor. Figure 4 shows the results of measuring AAV titers in cell culture fluid continuously separated for 7 days. Figure 5 shows the results of high-performance chromatography measurements. Figure 6 shows the cell culture density and the titer-improving effect of compounds. Figure 7 shows the titer-improving effect of compounds at different concentrations.

[0011] An example of an embodiment of the present disclosure will be described below. However, the present disclosure is not limited to the following embodiment in any way, and can be implemented with appropriate modifications within the scope of the object of the present disclosure. In this specification, a numerical range indicated using "to" means a range that includes the numerical values ​​written before and after "to" as the minimum and maximum values, respectively.

[0012] <Explanation of Terms> A promoter refers to a DNA control region / sequence capable of binding RNA polymerase and involved in initiating transcription of a downstream coding or non-coding sequence.

[0013] A vector is a DNA or RNA molecule used to artificially transport a foreign gene into another cell. A vector used to express a gene is called an expression vector. When a vector containing a foreign gene to be introduced is introduced into a cell, the foreign gene is replicated and / or expressed within the cell. Vectors include episomal (e.g., plasmid) vectors and non-episomal vectors. Vectors can be introduced into host cells by methods such as transfection, transduction, cell fusion, and lipofection.

[0014] As used herein, a gene refers to a nucleic acid molecule that encodes a polypeptide or RNA, and includes, for example, nucleic acid molecules such as cDNA and genomic DNA.

[0015] The method for producing the viral vector of the present invention is to ６ The method comprises culturing producer cells or packaging cells in a culture medium containing the producer cells or packaging cells at a cell density of 1000 cells / mL or more and a histone deacetylase inhibitor.

[0016] In the present invention, in producer cells or packaging cells in which a gene or a part thereof required for the expression of a viral vector has been inserted into the chromosome of a host cell, the activity of histone deacetylase (HDAC) expressed in the cells is inhibited, thereby enhancing the acetylation of histones and inducing changes in chromatin structure, and it is presumed that this makes it possible to improve the titer and full rate (the ratio of the amount of capsids containing the complete gene to the total amount of capsids) of the viral vector.

[0017] The histone deacetylase inhibitor is not particularly limited as long as it is a substance that has the effect of inhibiting histone deacetylase, but is preferably a compound having a benzamide structure or hydroxamic acid.

[0018] Preferred examples of histone deacetylase inhibitors are compounds represented by formula (1) or formula (2). １ -CONH-R ２ (1) In formula (1), R １ and R ２ is an aromatic group which may have a substituent, and R １ and R ２ The other is an optionally substituted amino group, an optionally substituted alkyl group, an optionally substituted alkenyl group, an optionally substituted aromatic group, or an optionally substituted alkoxy group: R ３ -CONH-OH (2) In formula (2), R ３ represents an alkyl group which may have a substituent, or an alkenyl group which may have a substituent.

[0019] More preferred examples of histone deacetylase inhibitors include compounds represented by formula (1A), formula (1B) or formula (2A). In formula (1A), R １０ represents a halogen atom, a heterocyclic group which may have a substituent, an alkyl group having 1 to 6 carbon atoms which may have a substituent, or an amino group which may have a substituent, and R ２ is an optionally substituted amino group, an optionally substituted alkyl group, or an optionally substituted aromatic group: In formula (1B), R ２０ represents an amino group, and R １ is an alkyl group which may have a substituent, or an aromatic group which may have a substituent: R ３１ -R ３０ -CONH-OH (2A) In formula (2A), R ３０ is -(CH ２ ) ｎ - or CH=CH-, n is an integer of 2 to 8, R ３１ represents an aromatic group which may have a substituent, or CONH-OH.

[0020] As the aromatic group, a phenyl group is preferred. As the alkyl group, an alkyl group having 1 to 8 carbon atoms is preferred, and an alkyl group having 1 to 6 carbon atoms is more preferred. The alkyl group may be linear or branched. As the alkenyl group, an alkenyl group having 2 to 8 carbon atoms is preferred, an alkenyl group having 2 to 6 carbon atoms is more preferred, and an alkenyl group having 2 to 4 carbon atoms is even more preferred. As the alkoxy group, an alkoxy group having 1 to 8 carbon atoms is preferred, and an alkoxy group having 1 to 6 carbon atoms is more preferred. The alkoxy group may be linear or branched. As the halogen, fluorine, chlorine, bromine, or iodine is preferred.

[0021] Substituents include halogen (for example, fluorine, chlorine, bromine, or iodine), an alkyl group having 1 to 6 carbon atoms, -NH ２ , -CONHC ６ H ５ , -CONHC ６ H ５ (NH ２ ), -NHCOCH３ ,-NHCOC ６ H ５ (CH ３ ), —CONHOH, a pyridyl group, an aromatic group substituted with —CONHOH (for example, a phenyl group substituted with —CONHOH), an optionally substituted heterocyclic group, (* indicates the bonding position).

[0022] Examples of the optionally substituted heterocyclic group include: (* indicates the bonding position).

[0023] Particularly preferably, in formula (1A), R １０ is bromine, Methyl, or CH ３ It represents —CO—NH—.

[0024] Specific examples of histone deacetylase inhibitors include any of the following compounds:

[0025] Specific preferred examples of the histone deacetylase inhibitor include UF010, CI-994, and resminostat. The content of the histone deacetylase inhibitor in the culture medium is preferably 50 μmol / L or less, more preferably 0.01 μmol / L to 50 μmol / L, more preferably 0.05 μmol / L to 50 μmol / L, even more preferably 0.1 μmol / L to 50 μmol / L, even more preferably 1 μmol / L to 20 μmol / L, even more preferably 1 μmol / L to 10 μmol / L, even more preferably 3 μmol / L to 10 μmol / L, and particularly preferably 5 μmol / L to 10 μmol / L.

[0026] In the present invention, the cells are cultured in a culture medium containing a histone deacetylase inhibitor for preferably 24 hours or more, more preferably 24 hours to 240 hours, even more preferably 24 hours to 120 hours, and particularly preferably 48 hours to 96 hours. There is no particular upper limit to the period during which the histone deacetylase inhibitor is contained in the medium, but it is generally 240 hours or less, preferably 120 hours or less, and particularly preferably 96 hours or less.

[0027] When a doxycycline-inducible promoter is used to express an exogenous recombinant nucleic acid, the medium may contain doxycycline. The concentration of doxycycline is preferably 5 μg / mL or less, more preferably 1 μg / mL or less, even more preferably 700 ng / mL or less, and particularly preferably 500 ng / mL or less. The addition time of doxycycline is preferably 1 hour or more, more preferably 6 hours or more, even more preferably 12 hours or more, even more preferably 24 hours or more, and even more preferably 48 hours or more.

[0028] In the present invention, a medium is preferably supplied during culture, and the supplied medium may contain doxycycline and a histone deacetylase inhibitor. The supplied medium refers to a medium added during cell culture. The culture method using the supplied medium is not particularly limited, but perfusion culture is preferred. That is, the method according to the present invention preferably includes perfusion culture of cells.

[0029] The period during which doxycycline is allowed to act (treatment time) is preferably 1 hour or more, more preferably 6 hours or more, even more preferably 12 hours or more, even more preferably 24 hours or more, and even more preferably 48 hours or more.

[0030] In the present invention, producer cells or packaging cells are used.

[0031] Producer cells are cells that contain all of the genes necessary for virus production. Preferably, producer cells are cells in which all of the necessary genes, including the gene of interest (GOI) encapsulated in the viral vector, have been integrated into their chromosomes.

[0032] Packaging cells are cells that have a portion of the genes necessary for virus production. Preferably, the packaging cells used are cells in which a portion of the genes necessary for virus production has been integrated into the chromosome. Virus can be produced by transfecting the remaining genes necessary for virus production into packaging cells that have a portion of the genes necessary for virus production.

[0033] Examples of viruses include adeno-associated viruses, lentiviruses, baculoviruses, and retroviruses, with adeno-associated viruses being preferred.

[0034] Adeno-associated virus (AAV) refers to a small, replication-incompetent, non-enveloped virus of the Parvoviridae and Dependoparvovirus families, containing a single-stranded DNA consisting of approximately 4,700 bases. There are over 100 serotypes of AAV, and it is known that the host range and viral characteristics differ depending on the serotype. Serotype 2 (AAV2) is one of the serotypes that has been widely studied for a long time and is known to have a very wide host range. Serotype 1 (AAV1), serotype 5 (AAV5), and serotype 6 (AAV6) are serotypes with higher tissue tropism. AAV1 is said to have high gene transfer efficiency in muscles, liver, respiratory tract, central nervous system, etc.; AAV5 is said to have high gene transfer efficiency in the central nervous system, liver, retina, etc.; and AAV6 is said to have high gene transfer efficiency in heart, muscles, liver, etc. In the present invention, serotype 2 or serotype 5 is preferably used. Serotype 5 is particularly preferred.

[0035] The adeno-associated virus gene refers to a gene composed of one or more nucleic acid sequences derived from one or more adeno-associated virus serotypes. The adeno-associated virus gene is preferably a gene involved in AAV replication and packaging, and a gene encoding an AAV structural protein.

[0036] AAV is a non-enveloped virus that grows in the presence of a helper virus such as adenovirus or herpesvirus. When preparing AAV for gene therapy or nucleic acid transfer, traditionally, adenovirus is co-infected into host cells to allow AAV replication. In addition, the gene responsible for the adenovirus helper function has been identified, and a plasmid incorporating this gene has also been used. For example, a plasmid containing Rep gene and Cap gene, an adenovirus helper plasmid, and a plasmid containing a therapeutic or preventive gene can be simultaneously transfected into cells to be packaged into recombinant AAV (rAAV).

[0037] The Rep and Cap genes encode proteins involved in virion replication and packaging. In the wild type, the Rep gene is expressed from the P5 and P19 promoters. The Cap region drives the expression of VP1, VP2, and VP3. The promoter naturally retained by the Cap gene can be the P40 promoter.

[0038] The adeno-associated virus genes (such as the Rep gene and the Cap gene) may be wild-type genes, but genes that have been modified by base substitution, deletion, insertion, or addition, etc., may also be used, as long as they exhibit their original functions.

[0039] When wild-type adeno-associated virus genes (such as the Rep gene and the Cap gene) are modified by base substitution, deletion, insertion, or addition, the number of modified bases is preferably 1 to 20, more preferably 1 to 10, and even more preferably 1 to 3. The base sequence of the modified adeno-associated virus gene preferably exhibits 85% or more sequence identity, more preferably 90% or more sequence identity, even more preferably 95% or more sequence identity, and even more preferably 98% or more sequence identity to the base sequence of the wild-type adeno-associated virus gene.

[0040] When the Rep gene and the Cap gene are introduced into a cell, a vector containing the Rep gene and the Cap gene can be introduced into the cell. The arrangement and orientation of the Rep gene and the Cap gene in the vector are not particularly limited, but preferably, the orientations of the Rep gene and the Cap gene are different.

[0041] The Rep gene refers to a region of the AAV genome that encodes viral replication proteins collectively required for replication of the viral genome, or functional homologs thereof, such as the human herpesvirus 6 (HHV-6) Rep gene (known to mediate AAV-2 DNA replication), as known to those skilled in the art. Thus, the coding region of the Rep gene includes at least the genes encoding AAV REP78 and REP68 (long-form REP proteins) and REP52 and REP40 (short-form REP proteins), or functional homologs thereof. The coding region of the Rep gene used in the present invention may be derived from any AAV serotype, but is preferably derived from AAV2. Those derived from AAV2 include REP78 and REP68, REP52 and REP40, and ITRs.

[0042] The Cap gene refers to a region in the AAV genome that encodes viral capsid proteins known to those skilled in the art. Examples of these capsid proteins are AAV capsid proteins VP1, VP2, and VP3. The Cap gene used in the present invention may be derived from any AAV serotype, but is preferably derived from AAV2 or AAV5. AAV5 is particularly preferred.

[0043] As a vector containing the Rep gene and the Cap gene, for example, a plasmid, a nucleic acid sequence derived from a virus, an artificially designed nucleic acid, etc. can be used, and a plasmid is preferred.

[0044] In the present invention, a gene of interest (GOI) may be introduced into a cell. The GOI is preferably introduced in a state where it is sandwiched between ITRs. The GOI is preferably a therapeutic or preventive gene.

[0045] Therapeutic or prophylactic genes may be, but are not limited to, genes that are incomplete or missing in the genome of target cells, or genes that encode non-native proteins with a desired biological or therapeutic effect (e.g., antiviral function). Specific examples of therapeutic or prophylactic genes include genes used to treat or prevent inflammatory diseases, autoimmune diseases, chronic and infectious diseases (including disorders such as AIDS, cancer, nervous system diseases, cardiovascular diseases, and hypercholesterolemia), various blood diseases such as anemia and hemophilia, and genetic defects (e.g., cystic fibrosis, Gaucher disease, adenosine deaminase (ADA) deficiency, emphysema, etc.).

[0046] The therapeutic or preventive gene may also be some antisense oligonucleotides (e.g., short oligonucleotides complementary to sequences around the translation start site (AUG codon) of mRNA) that are useful in antisense therapy for cancer and viral diseases.

[0047] The GOI may be linked to a promoter for expressing the GOI. The promoter for expressing the GOI is not particularly limited, but examples include a cytomegalovirus-derived promoter (optionally containing an enhancer), an SV40 early promoter, a human elongation factor-1α (EF-1α) promoter, a human ubiquitin C promoter, a Rous sarcoma virus LTR promoter (a retrovirus), a dihydrofolate reductase promoter, a β-actin promoter, and a phosphoglycerate kinase (PGK) promoter. The promoters for expressing the GOI and the gene are preferably flanked by ITR sequences.

[0048] In the present invention, preferably, a viral helper gene derived from adenovirus is introduced into a cell. The viral helper gene is a non-adeno-associated virus gene that enables replication and packaging of the adeno-associated virus. As the viral helper gene, a gene derived from a virus other than the adeno-associated virus is used. Specific examples of the viral helper gene include a viral helper gene derived from an adenovirus or a herpesvirus, and preferably, the viral helper gene is derived from an adenovirus.

[0049] Examples of adenovirus-derived viral helper genes include E1A, E1B, E2A, E4, and VA-RNA. In host cells having all or a part of the E1 region, the regions of the adenovirus genome necessary for the AAV genome to replicate and be packaged into capsids to form viral virions are the E2A region, E4 region, and VA-RNA region. Regarding the function of the E4 region, the E4 34 kDa protein encoded by open reading frame 6 (E4ORF6) of the E4 region is required for AAV replication. Preferably, the viral helper genes are the E2 gene, the E4 gene, and the VA-RNA gene. More preferably, the viral helper genes are the E2 gene and the E4 gene. The VA-RNA gene is preferably the VA-RNAI gene.

[0050] The adenovirus-derived viral helper genes (E1A, E1B, E2A, E4, VA-RNA, etc.) may be wild-type genes, but genes that have been modified by base substitution, deletion, insertion, addition, or the like may also be used, as long as they exhibit their inherent functions.

[0051] When a wild-type viral helper gene is modified by base substitution, deletion, insertion, addition, or the like, the number of bases to be modified is preferably 1 to 20, more preferably 1 to 10, and even more preferably 1 to 3. The base sequence of the modified viral helper gene exhibits preferably 85% or more sequence identity, more preferably 90% or more sequence identity, even more preferably 95% or more sequence identity, and even more preferably 98% or more sequence identity to the base sequence of the wild-type viral helper gene.

[0052] When a viral helper gene is introduced into a cell, a vector containing the viral helper gene can be introduced into the cell.

[0053] As a vector containing a viral helper gene, for example, a plasmid, a virus-derived sequence, an artificially designed nucleic acid, etc. can be used, and a plasmid is preferred.

[0054] The viral helper gene is preferably under the control of a promoter, and may be under the control of a promoter whose expression can be regulated.

[0055] As the expression-regulatable promoter, a promoter whose expression can be turned on and off depending on the presence or absence of a stimulus that induces condition-specific expression can be used. Examples of stimuli that induce condition-specific expression include, but are not limited to, chemical stimuli and physical stimuli. Chemical stimuli include endogenous hormones, stress responses, lactose, tetracycline or its derivatives (e.g., doxycycline), cumic acid, rapamycin, FKCsA, abscisic acid (ABA), tamoxifen / Cre-loxP (a system using a Cre promoter modified so that activation can be induced by tamoxifen), and riboswitches. Physical stimuli include blue light, heat, and the like.

[0056] Among the above, the expression-regulatable promoter is preferably a promoter whose expression can be regulated by a drug. The drug is preferably a tetracycline drug, more preferably doxycycline. That is, the stimulus that induces condition-specific expression may be a medium containing doxycycline.

[0057] Specific examples of promoters whose expression can be regulated include tetracycline-responsive promoters, RU486-inducible promoters, ecdysone-inducible promoters, rapamycin-inducible promoters, and metallothionein promoters. Specific examples of tetracycline-responsive promoters include the Tet on / off system (TET Systems).

[0058] The promoter capable of regulating expression may be a promoter of the Tet on / off system, which is a promoter whose expression can be regulated by tetracycline, or may be a Tet on system.

[0059] In the Tet-on system, the promoter additionally contains at least one Tet operon. The Tet operon (tetracycline-controlled transcriptional activation) can be used to reversibly switch transcription on or off in the presence of the antibiotic tetracycline or one of its derivatives (e.g., doxycycline). The Tet repressor protein present in the cell blocks expression by binding to the Tet operator sequence introduced into the promoter. Therefore, when the Tet repressor is bound to the Tet operator sequence, no gene expression is observed. Upon addition of tetracycline or doxycycline, the Tet repressor is sequestered, allowing promoter activity and turning on gene expression. Tet operon systems are widely available, such as the Tet operon used in the pcDNA™4 / TO mammalian expression vector available from Invitroge. In the Tet-on system, the Tet operator sequence may be located upstream or downstream of the promoter whose expression is to be regulated. In order to reduce expression leakage when expression is turned off, it is preferable that the Tet operator sequence be located downstream of the promoter whose expression can be regulated.

[0060] Specific examples of promoters include, but are not limited to, a cytomegalovirus-derived promoter (CMV promoter) (optionally containing an enhancer), an SV40 early promoter, a human elongation factor-1α (EF-1α) promoter, a human ubiquitin C promoter, a retroviral Rous sarcoma virus (RSV) LTR promoter, a dihydrofolate reductase promoter, a β-actin promoter, and a phosphoglycerate kinase (PGK) promoter.

[0061] An exogenous recombinant nucleic acid refers to a recombinant nucleic acid introduced into a cell from outside the cell. The exogenous recombinant nucleic acid can be introduced into a cell in the form of a vector containing the exogenous recombinant nucleic acid by methods such as transfection, transduction, or lipofection.

[0062] Transfection refers to the introduction of nucleic acid into a eukaryotic cell across its membrane by chemical (e.g., using reagents such as calcium phosphate or polyethyleneimine), mechanical (e.g., electroporation), or physical means (e.g., bioballistic delivery).

[0063] Transduction refers to the introduction of nucleic acids into a cell via a virus-derived vector across the membrane of a eukaryotic cell.

[0064] Lipofection refers to the process of forming a complex between a vector and a positively charged lipid or the like through electrical interaction, and then introducing nucleic acid into cells through endocytosis or membrane fusion.

[0065] Preferably, in animal cells, the exogenous recombinant nucleic acid is expressed in a constitutive expression system. A constitutive expression system is a method that allows expression for more than one month. By inserting a plasmid into a chromosome, introducing an episomal vector, or introducing an exogenous nucleic acid using an artificial chromosome, it is possible to maintain expression of the target gene for a long period of time. In this case, if a drug resistance gene is introduced into the plasmid, drug selection becomes possible.

[0066] The recombinant nucleic acid is not particularly limited as long as it is a sequence that can express a virus. Preferably, it is a sequence that expresses an adeno-associated virus. As described above, known sequences that express an adeno-associated virus include sequences derived from helper viruses and sequences derived from adeno-associated viruses, but there is no limitation to these as long as they can express an adeno-associated virus. Helper virus-derived components include, but are not limited to, E1A, E1B, E2A, E4, and / or VA-RNA. Known adeno-associated virus-derived sequences include, but are not limited to, Rep and Cap.

[0067] In the present invention, it is preferred that at least one of the exogenous recombinant nucleic acids contains a promoter controlled by RNA polymerase III. In the present invention, the exogenous recombinant nucleic acid is preferably a nucleic acid encoding E2A, E4, VA-RNA, Rep, or Cap.

[0068] It is preferred that at least one of the exogenous recombinant nucleic acids contains a nucleic acid sequence that induces condition-specific expression. The promoter induced by the nucleic acid sequence that induces condition-specific expression is not particularly limited, but is preferably a doxycycline-inducible TET promoter.

[0069] In the present invention, it is preferable to use producer cells or packaging cells in which the Rep gene, Cap gene, and helper gene are integrated into the chromosome, it is more preferable to use producer cells in which the Rep gene, Cap gene, helper gene, and GOI are integrated into the chromosome, it is even more preferable, and it is particularly preferable to use producer cells in which the Rep gene, Cap gene, helper gene, and therapeutic or preventive gene are integrated into the chromosome.

[0070] The cells in the present invention are preferably animal cells. While the animal cells are not particularly limited, mammalian cells or insect cells are preferred, and mammalian cells are more preferred. Examples of mammalian cells include, but are not particularly limited to, human cells, mouse cells, rat cells, monkey cells, and hamster cells. Preferably, human cells can be used. Examples of cells include mouse myeloma (NS0) cell lines, Chinese hamster ovary (CHO) cell lines, HT1080, H9, HepG2, MCF7, MDBK Jurkat, NIH3T3, PC12, BHK (baby hamster kidney cells), VERO, SP2 / 0, YB2 / 0, YO, C127, L cells, COS (e.g., COS1 and COS7), QC1-3, HEK293 (human embryonic kidney cells), VERO, PER. Examples of suitable cell lines include C6, HeLa, EB1, EB2, EB3, oncolytic, or hybridoma cell lines. Preferably, the cells are HEK293 cells or CHO cells, more preferably HEK293 cells. The cells of the present invention are preferably cells adapted to growth in suspension culture. Preferably, the cells are HEK293 cells or CHO cells adapted to growth in suspension culture, more preferably HEK293 cells adapted to growth in suspension culture.

[0071] The method of the present invention involves culturing producer cells or packaging cells in a medium. The cell culture can be carried out under normal conditions for cell culture. The cell density in the viral vector production process is 1 x 10 ６ cells / mL or more, more preferably 10 x 10 ６ cells / mL or more, more preferably 20 x 10 ６ cells / mL or more, more preferably 30 x 10 ６ cells / mL or more, more preferably 50 x 10 ６ cells / mL or more, more preferably 70 x 10 ６ cells / mL or more, 100 x 10 ６ The upper limit of the cell density is not particularly limited, but generally, it is 1000 × 10 ６ cells / mL or less.

[0072] In the present invention, a proliferation step of growing cells may be carried out before the viral vector production (production) step.

[0073] For example, prior to the manufacturing process, cells were cultured at 1×10 in a medium without doxycycline and histone deacetylase inhibitors. ４ cells / mL or more (more preferably 1 x 10 ５ cells / mL or more, more preferably 1 x 10 ６ cells / mL or more, more preferably 10 x 10 ６ cells / mL or more, more preferably 30 x 10 ６ cells / mL or more, particularly preferably 100 x 10 ６ Alternatively, a growth step may be performed in which the cells are cultured until the cell density reaches a concentration of 10 ...

[0074] The temperature for cell culture is not particularly limited, and is set at a temperature at which the cells can survive. The culture temperature is generally 25°C to 45°C, preferably 30°C to 42°C, more preferably 35°C to 40°C, and an example is 37°C. ２ The concentration is generally 3 to 10% CO ２ and preferably 5 to 10% CO ２ As an example, 8% CO ２ is.

[0075] The culture can be batch culture, fed-batch culture, perfusion culture, or shake culture. At least a part of the virus production process is preferably perfusion culture. The culture is preferably in a suspension state, and suspension culture is preferred.

[0076] The culture vessel may be, but is not limited to, a flask or a bioreactor.

[0077] The culture scale is not particularly limited, and cells can be cultured in any volume of medium, for example, 1 mL to 5000 L of medium. The lower limit is preferably 1 L, more preferably 10 L, even more preferably 50 L, and particularly preferably 100 L. The upper limit is preferably 5000 L, more preferably 2500 L, more preferably 1000 L, and particularly preferably 500 L.

[0078] Cultivation may be carried out with shaking agitation. The agitation speed when shaking agitation is generally 50 rpm to 200 rpm, preferably 80 to 150 rpm. Agitation culture may be rotation agitation culture using a propeller or the like in a reactor. The agitation speed when rotating agitation is generally 50 rpm to 200 rpm, preferably 80 to 150 rpm. Agitation may also be performed by wave-type shaking agitation or by up-and-down movement of an agitator blade, but is not particularly limited thereto.

[0079] Examples of media include, but are not limited to, Expi293 Expression Medium (Thermo Fisher Scientific, A1435101), Dulbecco's Modified Eagle's Medium (DMEM) containing 10% (vol / vol) fetal bovine serum (FBS), serum-free UltraCULTURE™ Medium (Lonza), STEMPRO™-34 SFM Medium, human endothelial-SFM, GIBCO® FREESTYLE™ 293 Expression Medium, CD CHOAGT Medium, CHO-S-SFM Medium, GIBCO® FREESTYLE™ CHO Expression Medium, CD OPTICHO™ Medium, CD CHO Medium, CD These include DG44 medium, SF-900™ medium, Expi293™ Expression medium, 293 SFM medium, BALANCD® HEK293 medium, and any derivatives or variations thereof. In certain non-limiting embodiments, the high density culture medium may be CD FORTICHO™ Medium, CD CHOAGT Medium, CHO-S-SFM Medium, GIBCO® FREESTYLE™ CHO Expression Medium, CD OPTICHO™ Medium, C DCHO Medium, CD DG44 Medium, GIBCO® FREESTYLE™ 293 Expression Medium, Expi293™ Expression Medium, LV-MAX™ Production Medium, FREESTYLE™ F17 Expression Medium, DYNAMIS™ Medium, BALANCD® HEK293 Medium, or similar media, or variations thereof.

[0080] The method for producing a viral vector according to the present invention may include recovering the virus. The viral vector produced in the cells that produce the viral vector remains within the cells or is released into the culture supernatant.

[0081] When recovering a viral vector from cells, a sample containing the viral vector can be prepared, for example, by disrupting the cells with ultrasound, by disrupting the cells by freezing and thawing, or by contacting the cells with an acidic solution or a surfactant. The sample prepared in this manner may be used as is, or may be purified as desired. When recovering a viral vector from a culture supernatant, the culture supernatant may be used as is as a sample containing the viral vector, or may be purified as desired.

[0082] The purification method for purifying a viral vector is not particularly limited, but examples thereof include CsCl gradient ultracentrifugation, chromatography, and ultrafiltration. By using the above purification methods, the viral vector can be purified from a sample containing the viral vector. Furthermore, if desired, the sample purified as described above can be purified by adding MgCl ２ Benzonase may be added to the reaction mixture to digest gDNA (genomic DNA) and remaining plasmids.

[0083] The titer of the viral vector produced by the method of the present invention can be measured by a conventional method known to those skilled in the art. For example, the cell culture medium after culturing is collected, the cells are disrupted by freezing and thawing, and the supernatant is collected by centrifugation. ２ The viral vector-containing sample obtained as described above can be used as a ddPCR (Droplet Digital PCR) sample, and the titer of the viral vector can be measured by ddPCR.

[0084] Furthermore, the capsid particle titer (vp / mL) can be measured using an ELISA kit, and the Full rate can be calculated from the ratio to the genome titer (vg / mL) calculated by ddPCR. Alternatively, the sample solution after virus extraction and purification can be analyzed by HPLC measurement, isoelectric focusing capillary electrophoresis, or the like, and the Full / Empty ratio can be determined by analysis within the same measurement system.

[0085] In one example of the present invention, by culturing in the presence of a histone deacetylase inhibitor, the production amount of a viral vector such as AAV can be improved by preferably 1.5 times or more, more preferably 2 times or more, and even more preferably 3 times or more, compared to when a histone deacetylase inhibitor is not used.

[0086] In one example of the present invention, in the virus produced in the culture step, the ratio of the amount of capsids containing complete genes to the total amount of capsids (full rate) is preferably 5% or more, more preferably 10% or more, even more preferably 20% or more, even more preferably 25% or more, and particularly preferably 30% or more.

[0087] The present invention will be explained in more detail with reference to the following examples, but the present invention is not limited to these examples.

[0088] [Materials and Methods] <Cell Maintenance Culture> Plasmids (SEQ ID NO: 1 and SEQ ID NO: 2) were introduced into HEK293 cells (Thermo Fisher Scientific, A49784) adapted to growth in suspension culture, and a stable expression line (AAV producer cell) in which Sequence 1 (comprising a reverse tetracycline-controlled transactivator (rtTA) gene, Rep gene, Cap gene, E2 gene, E4 gene, and a GFP gene (as a GOI) sandwiched between ITR sequences, with the Rep gene, Cap gene, E2 gene, and E4 gene being under the control of a promoter with a tetracycline response element (TRE) sequence) was integrated into the cell chromosome was cultured in BalanCD HEK293 medium (FUJIFILM Irvine Scientific, 551-34231). The cell density was 2 × 10 ６ ~30 x 10 ６ The cells were suspended at 125 mL / mL and maintained in a 125 mL shake flask. The culture was maintained at 37°C, 8% CO with constant agitation at 120 rpm. ２ The mixture was incubated under the conditions.

[0089] <Perfusion culture> 0.5 × 10 cells were cultured in 280 mL of BalanCD HEK293 medium (FUJIFILM Irvine Scientific, 551-34231). ６ AAV producer cells suspended at 100 cells / mL were seeded into a 0.5 L reactor (Biott). Perfusion culture was performed using BalanCD HEK293 medium (FUJIFILM Irvine Scientific, 551-34231) and BalanCD HEK293 Feed (FUJIFILM Irvine Scientific, 550-34245).

[0090] <Plasmid Construction> The entire sequences of the plasmids used are shown in SEQ ID NOs: 1 to 5 in the sequence listing. The sequences were produced by total synthesis.

[0091]

[0092] <AAV titer measurement> 72 hours after gene transfection, 37°C, 8% CO ２ The HEK cell culture medium was collected under the conditions, and the cells were disrupted by freezing and thawing at −80°C. The supernatant was then collected by centrifugation at 13,800 × g and diluted with MgCl at a final concentration of 2 mmol / L. ２25 U / mL Benzonase was added and the reaction was carried out at 37°C for 2 hours to digest gDNA (genomic DNA) and remaining plasmid. After the reaction, the sample was incubated at 95°C for 15 minutes, 70°C for 3 minutes, 40°C for 3 minutes, and 20°C for 3 minutes to inactivate the Benzonase, and this was used as a ddPCR (Droplet Digital PCR) sample. The ddPCR sample was diluted 50-fold with TE buffer (pH 8.0, containing 0.05% Pluronic F-68 and 10 μg / mL calf thymus DNA). ddPCRtm Supermix for Probes (no dUTP) (BIORAD) 10 μL, diluted ddPCR sample 2 μL, primers (SEQ ID NO: 3, SEQ ID NO: 4, final concentration 900 nmol / L) and probe (SEQ ID NO: 5, final concentration 250 nmol / L), nuclease-free water (Thermo Fisher Scientific, 10977015) were mixed in a tube on ice to adjust the total volume to 22 μL, and ddPCR was performed. In ddPCR, droplets were formed from the prepared solution using a droplet formation device, and 40 cycles of denaturation, annealing, and extension were performed at 95 ° C for 10 minutes (preheating), 94 ° C for 30 seconds (denaturation), and then 55 ° C for 60 seconds (annealing and extension), followed by incubation at 98 ° C for 10 minutes. Measurement was performed using a droplet reader, and the AAV genome titer (vg / mL) was calculated.

[0093] Experiment 1: <Method> AAV producer cells were cultured at 1 x 10 ６ The cells were seeded onto a 24-well plate at 1000 cells / mL, and doxycyclin (final concentration: 0.5 μg / mL) and Epigenetics Screening Library (CAYMAN, 11076) were added at final concentrations of 2.5 μmol / L and 5.0 μmol / L. After 72 hours of treatment, the cells were harvested and AAV titer was measured.

[0094] <Results> Compounds that improve potency compared to the potency when DMSO was added were selected, and the results of potency measurement are shown in Figure 1, and a list of compounds is shown in Table 2. Screening results revealed that HDAC inhibitors improve potency.

[0095]

[0096] Experiment 2: <Method> AAV producer cells were cultured at 30 x 10 ６ After culturing at 1000 cells / mL, 24 mL of culture medium was transferred to a 125 mL flask. Doxycyclin (final concentration 5 μg / mL) and CI-994 (CAYMAN, 11076) were added to the cell culture medium at a final concentration of 7.5 μmol / L. Every other day, 140 μL of BalanCD HEK293 Feed (FUJIFILM Irvine Scientific, 550-34245) and 10 μL of 45 w / v% D(+)-glucose solution (FUJIFILM Wako Pure Chemical Industries, 079-05511) were added per mL of cell culture medium to maintain the cells. After drug addition, cells were harvested after 72 hours of treatment, and AAV titer was measured.

[0097] <Results> The results of titration are shown in Figure 2. The titer of the sample not treated with CI-994 (CI-994 conc: 0 µM) was 1.1 x 10 １２ The titer of the CI-994-treated sample (CI-994 conc: 7.5 μM) was 2.2 × 10 １２ vg / mL, it was revealed that there is a titer-improving effect on high-density cell culture medium.

[0098] The HDAC inhibitors listed in Table 2 other than CI-994 also show similar potency-improving effects.

[0099] Experiment 3: <Method> As in Experiment 2, AAV producer cells were cultured at 30 x 10 ６ The cells were cultured at a concentration of 30 × 10 cells / mL or more, and then aliquoted into flasks for AAV production studies. 30% of the total cell culture volume was aliquoted from the reactor every 24 hours and re-diluted with fresh medium to obtain 30 × 10 cells / mL for 7 days. ６While maintaining a cell density of ≥ 100 cells / mL, 24 mL of the aliquot cell culture medium was transferred to a 125 mL flask, and doxycyclin (final concentration 5 μg / mL) and CI-994 (CAYMAN, 11076) were added to the cell culture medium at a final concentration of 7.5 μmol / L. Every other day, 140 μL BalanCD HEK293 Feed (FUJIFILM Irvine Scientific, 550-34245) and 10 μL 45 w / v% D(+)-glucose solution (FUJIFILM Wako Pure Chemical, 079-05511) were added per mL of cell culture medium to maintain the cells. After drug addition, the cells were harvested after 72 hours of treatment, and AAV titer measurements were performed.

[0100] <Results> The change in cell density in the reactor is shown in Figure 3, and the titer measurement results of AAV produced in the cell culture medium aliquoted every 24 hours are shown in Figure 4. Under conditions in which AAV was continuously produced every 24 hours in a separate culture vessel while maintaining a high cell density in the reactor, the CI-994-treated sample (+CI-994) showed a higher titer than the CI-994-untreated sample (-CI-994), confirming that the HDAC inhibitor has a titer-improving effect.

[0101] Experiment 4: <Method> AAV was extracted and purified from the Day 7 sample in Experiment 3 using the AAVpro Purification Kit (Takara Bio, 6675). The purified sample solution was subjected to high-performance liquid chromatography to measure the area values ​​of the peaks of AAV empty and full particles, and the full rate (the ratio of the amount of capsid containing the complete gene to the total amount of capsid) was calculated. An anion exchange column was used as the separation column, and a fluorescence detector (excitation: 280 nm, detection: 348 nm) was used for detection.

[0102] <Results> The measurement results of the CI-994 untreated sample (-CI-994) and the CI-994 treated sample (+CI-994) obtained using high performance liquid chromatography are shown in Figure 5. The peak areas were measured, and the area ratios of the peaks representing full particles and empty particles were calculated. The results are shown in Table 3. It was revealed that CI-994 treatment had an effect of improving the full rate.

[0103]

[0104] Experiment 5: <Method> AAV producer cells were cultured at 0.2 x 10 ６ cells / mL, 1.0×10 ６ cells / mL, 6.0×10 ６ cells / mL, 24×10 ６ The cells are seeded at 125 mL flasks at 1000 cells / mL. After treatment with doxycyclin (final concentration: 0.5 μg / mL) for 72 hours, the cells are harvested and AAV titer is measured.

[0105] <Results> The titer of AAV producer cells decreases as the cell density increases. ６ cells / mL to 1.0 x 10 ６ When the cell density is increased to 1.0 x 10 cells / mL, the titer is reduced to about 1 / 5. ６ When an HDAC inhibitor was added to produce viruses at a cell density of 0.2 × 10 cells / mL or more, the decrease in titer was suppressed compared to when no HDAC inhibitor was added. ６ Using the titer in cells / mL (without HDAC inhibitor) as a standard, a titer equivalent to or higher than that can be obtained.

[0106] Experiment 6: <Method> AAV producer cells were cultured at 0.2 x 10 ６ cells / mL, 1.0×10 ６ cells / mL, 6.0×10 ６ cells / mL, 24×10 ６ cells / mL, 30×10 ６The cells were seeded at 0.2 × 10 cells / mL in a 125 mL flask, and treated with doxycyclin (final concentration 0.5 μg / mL) alone for 72 hours. The cells were then collected and subjected to AAV titer measurement. ６ cells / mL, 1.0×10 ６ cells / mL, 8.0×10 ６ cells / mL, 30×10 ６ The cells were seeded at 1000 cells / mL into a 125 mL flask, and treated with doxycyclin (final concentration 0.5 μg / mL) and CI-994 (final concentration 7.5 μg / mL) for 72 hours. After that, the cells were collected and the AAV titer was measured.

[0107] <Results> The titer per cell of AAV producer cells decreased as the cell density increased (Figure 6). ６ cells / mL to 1.0 x 10 ６ When the cell density was adjusted to 1.0 × 10 cells / mL, the titer was reduced to about 1 / 5, but ６ When CI-994 was added to produce viruses at a cell density of 0.2 × 10 cells / mL or more, the decrease in titer was suppressed, and the decrease in titer was 0.2 × 10 ６ The titer obtained was at the same level as the titer in cells / mL (without CI-994 treatment) as a standard.

[0108] Experiment 7: <Method> AAV producer cells were cultured at 1 x 10 ６ Doxycyclin (final concentration: 0.5 μg / mL) and various HDAC inhibitors (CI-994, Scriptaid, Resminostat, CBHA) were added at final concentrations of 1.3 μmol / L, 2.5 μmol / L, 5 μmol / L, and 10 μmol / L. After 96 hours of treatment, the cells were harvested and AAV titer was measured.

[0109] <Results> Compounds that improve the potency compared to the potency when DMSO was added were selected, and the results of the potency measurement are shown in Figure 7. A particularly high potency-improving effect was confirmed when the concentration of each compound was 5 µmol / L or higher.

[0110] [Discussion] In this technology, in a strain (AAV producer cell) in which a gene required for AAV vector expression has been inserted into the chromosome of a host cell, the activity of histone deacetylase (HDAC) expressed in the cell is inhibited, thereby enhancing histone acetylation and inducing changes in chromatin structure, which is presumably contributing to the improvement of AAV titer and full rate. Histone acetylation is generally known to disaggregate chromatin structure, which is thought to improve the expression of the inserted gene required for AAV production and improve titer. Furthermore, it is presumed that disaggregation of chromatin structure also contributes to the improvement of GOI replication in the cell encapsulated in the AAV vector, leading to the improvement of full particles.

[0111]

[0112]

[0113] SEQ ID NO: 3: GGAACCCCTA GTGATGGAGT T SEQ ID NO: 4: CGGCCTCAGT GAGCGA SEQ ID NO: 5: CACTCCCTCT CTGCGCGCTC G

Claims

1. 1 × 10 ６ A method for producing a viral vector, comprising culturing producer cells or packaging cells in a culture solution containing producer cells or packaging cells having a cell density of 1 × 10 or more cells / mL and a histone deacetylase inhibitor.

2. The production method according to claim 1, wherein the histone deacetylase inhibitor is a compound having a benzamide structure or a hydroxamic acid.

3. The production method according to claim 1, wherein the histone deacetylase inhibitor is a compound represented by formula (1) or formula (2). R １ -CONH-R ２ (1) In formula (1), R １ and R ２ one of which is an aromatic group which may have a substituent, and the other of R １ and R ２ is an amino group which may have a substituent, an alkyl group which may have a substituent, an alkenyl group which may have a substituent, an aromatic group which may have a substituent, or an alkoxy group which may have a substituent: R ３ -CONH-OH (2) In formula (2), R ３ is an alkyl group which may have a substituent, or an alkenyl group which may have a substituent.

4. The production method according to claim 1, wherein the histone deacetylase inhibitor is a compound represented by formula (1A), formula (1B) or formula (2A). In formula (1A), R １０ represents a halogen, an optionally substituted heterocyclic group, an optionally substituted alkyl group having 1 to 6 carbon atoms, or an optionally substituted amino group, and R ２ is an optionally substituted amino group, an optionally substituted alkyl group, or an optionally substituted aromatic group: In formula (1B), R ２０ represents an amino group, and R １ is an optionally substituted alkyl group, or an optionally substituted aromatic group: R ３１ -R ３０ -CONH-OH (2A) In formula (2A), R ３０ represents -(CH ２ ) ｎ - or CH=CH-, n represents an integer of 2 to 8, and R ３１ is an optionally substituted aromatic group, or CONH-OH.

5. In formula (1A), R １０ is bromine, methyl, or CH ３ -CO-NH- as claimed in claim 1 of the manufacturing method.

6. The production method according to claim 1, wherein the histone deacetylase inhibitor is any of the following compounds.

7. The production method according to any one of claims 1 to 6, which comprises perfusing and culturing cells.

8. The production method according to any one of claims 1 to 6, wherein the virus is an adeno-associated virus.

9. The production method according to claim 8, wherein the producer cell or the packaging cell is a cell in which the Rep gene, the Cap gene, and the helper gene are integrated into the chromosome.

10. The production method according to any one of claims 1 to 6, wherein the cells are animal cells.

11. The production method according to any one of claims 1 to 6, wherein the content of the histone deacetylase inhibitor in the culture solution is 50 μmol / L or less.

12. The production method according to any one of claims 1 to 6, wherein the cells are cultured for 24 hours or more in a culture solution containing a histone deacetylase inhibitor.

13. The production method according to any one of claims 1 to 6, wherein the cell density is 30 × 10 ６ cells / mL or more.

14. The production method according to any one of claims 1 to 6, wherein the culture is a suspension culture.

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