WO2024075819A1 - Virus vector production method employing cell - Google Patents

Virus vector production method employing cell Download PDF

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Publication number
WO2024075819A1
WO2024075819A1 PCT/JP2023/036412 JP2023036412W WO2024075819A1 WO 2024075819 A1 WO2024075819 A1 WO 2024075819A1 JP 2023036412 W JP2023036412 W JP 2023036412W WO 2024075819 A1 WO2024075819 A1 WO 2024075819A1
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viral vector
cells
producing
aav2
viral
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PCT/JP2023/036412
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French (fr)
Japanese (ja)
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俊介 椎名
惇一 長谷川
桃梨 杉本
泰広 川野
敬太 清水
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Ａｇｃ株式会社
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Publication of WO2024075819A1 publication Critical patent/WO2024075819A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C12N15/864—Parvoviral vectors, e.g. parvovirus, densovirus

Definitions

  • the present invention relates to a method for efficiently producing a viral vector using cells.
  • Biopharmaceuticals are medicines manufactured by utilizing the ability of microorganisms or cells to produce proteins (hormones, enzymes, antibodies, etc.) through the application of recombinant gene technology and cell culture technology, and are becoming the mainstream of medicine in recent years.
  • the gene for a target protein such as an antibody is introduced into a cell (preferably a mammalian cell), and the cell produces the target protein.
  • Methods for gene transfer can be broadly divided into two categories: methods using retroviruses, lentiviruses, or adeno-associated virus vectors, and physicochemical methods such as lipofection, electroporation, and microinjection.
  • viral vector a virus-derived gene transfer vector
  • a viral vector is a vector that is made by modifying a naturally occurring virus using recombinant gene technology so that a desired gene or the like can be transferred to a target, and technological development has progressed in recent years.
  • viruses from which viral vectors are derived include enveloped viruses such as retroviruses, lentiviruses, Sendai viruses, and herpes viruses, as well as non-enveloped viruses such as adenoviruses and adeno-associated viruses (AAV).
  • AAV is considered promising as a gene transfer vector for use in gene therapy because it can infect cells of a wide range of species, including humans, and can also infect non-dividing cells that have completed differentiation; it is not pathogenic to humans, so there is little risk of side effects; and the viral particles are physicochemically stable.
  • AAV is generally produced by introducing plasmid DNA required for AAV production into mammalian cells such as human embryonic kidney 293 (HEK293 cells).
  • HEK293 cells human embryonic kidney 293
  • quality issues such as the packaging rate of the AAV genome into the AAV capsid and fragmentation of the AAV genome packaged within the capsid.
  • Non-enveloped viral vectors such as adenovirus and AAV exhibit broad tissue and cell tropism, capacity to accommodate large expression cassettes, and high transduction efficiency, providing desirable characteristics for gene delivery.
  • Patent Document 1 Non-Patent Documents 1 and 2
  • further improvements are required in methods for efficiently producing viral vectors with high infectious titers.
  • the objective of the present invention is to provide a method for efficiently producing a viral vector with a high infectious titer.
  • the present inventors have found that, in the production of viral vectors using human-derived cells, the steps of pre-culturing the cells and transfection of a transformation plasmid to prepare viral vector-producing cells are carried out under temperature conditions of around 37°C, which is generally preferred for culturing human-derived cells, but that in the step of making the viral vector-producing cells produce viral vectors, a temperature condition of around 35°C, which is slightly lower than the generally used temperature, is deliberately applied, thereby producing unexpected effects such as (1) improved survival rate of viral vector-producing cells after transfection, (2) continued proliferation of viral vector-producing cells, (3) improved viral vector productivity in viral vector-producing cells, (4) no adverse effect on the proportion of complete genomes packaged in the viral vector, (5) an increase in the proportion of viral particles in which the viral genome is packaged, and (6) an increase in the infectious titer of the obtained viral particles.
  • the present inventors have found that the effects of (1) to (6) are further enhanced when an organic solvent such as ethanol is added to the medium after the temperature shift in addition to the above-mentioned temperature shift, and have completed the present invention by conducting further research based on this finding. That is, the present invention is as follows.
  • a method for producing a viral vector comprising a step of culturing a viral vector-producing cell at 30.0 to 35.9°C to produce a viral vector, wherein the viral vector-producing cell is cultured at a temperature of 36.0 to 38.0°C before the start of the step of producing the viral vector.
  • the step of causing the viral vector-producing cells to produce the viral vector is carried out in a medium containing an organic solvent.
  • the organic solvent is an alcohol or dimethyl sulfoxide.
  • a method for improving the production efficiency of a viral vector having an improved infectious titer comprising a step of culturing viral vector-producing cells at 30.0 to 35.9°C to produce a viral vector, wherein the viral vector-producing cells are cultured at a temperature of 36.0 to 38.0°C before the start of the step of producing the viral vector.
  • the method according to [4], wherein the step of causing the viral vector-producing cells to produce the viral vector is carried out in a medium containing an organic solvent.
  • the organic solvent is an alcohol or dimethyl sulfoxide.
  • a method for improving the ratio of viral particles containing a complete viral genome to all viral particles produced in production of a viral vector using viral vector-producing cells comprising the step of culturing viral vector-producing cells at 30.0 to 35.9°C to produce a viral vector, wherein the viral vector-producing cells are cultured at a temperature of 36.0 to 38.0°C prior to the start of the step of producing the viral vector.
  • the step of causing the viral vector-producing cells to produce the viral vector is carried out in a medium containing an organic solvent.
  • the organic solvent is an alcohol or dimethyl sulfoxide.
  • the present invention makes it possible to produce highly infectious viral vectors with extremely high efficiency.
  • FIG. 1 shows the total production amount of viral vectors when the entire process from preparation of viral vector-producing cells to production of viral vectors by the viral vector-producing cells was carried out at 35° C.
  • A A diagram showing the influence of culture temperature and addition of ethanol during viral vector production on viral vector productivity by producer cells
  • B A diagram showing the influence of culture temperature and addition of ethanol during viral vector production on the proportion of complete genomes packaged in viral vectors in producer cells
  • C A diagram showing the influence of culture temperature and addition of ethanol during viral vector production on the full virus rate in producer cells.
  • A A diagram showing the influence of culture temperature and addition of ethanol during viral vector production on the proliferation of producer cells
  • B A diagram showing the influence of culture temperature and addition of ethanol during viral vector production on the viability of producer cells.
  • A A diagram showing the influence of culture temperature and addition of ethanol during viral vector production on viral vector productivity by producer cells.
  • FIG. 1 shows the total production amount of viral vectors when the culture temperature during viral vector production was shifted to 33° C.
  • virus includes not only naturally occurring viruses, but also recombinant virus particles that have been modified based on naturally occurring viruses, etc., to remove pathogenicity and to include a region for introducing a foreign gene.
  • recombinant virus particles are also called virus vectors.
  • naturally occurring viruses and recombinant virus particles may be collectively referred to as viruses.
  • viral vector refers to a virus particle that has been modified based on a natural virus or the like to remove pathogenicity and to have a region for introducing a foreign gene.
  • viral vector refers not only to a virus vector that contains a viral genome (nucleic acid form), but also to a hollow particle that is a virus-like particle that does not contain a viral genome. It is preferable to obtain a "viral vector” that contains a viral genome (nucleic acid form) inside the particle.
  • Viral vectors can be used for genetic engineering (i.e., "cloning vectors") for the introduction/transfer of nucleic acids into cells and for transcription or translation of the inserted nucleic acid in cells.
  • An "expression vector” is a specialized vector that contains a gene or nucleic acid sequence with regulatory regions necessary for expression in a host cell.
  • a vector nucleic acid sequence generally contains at least an origin of replication for propagation in a cell and optionally additional elements such as heterologous polynucleotide sequences, expression control elements (e.g., promoters, enhancers), introns, ITR(s), selection markers (e.g., antibiotic resistance), polyadenylation signals.
  • Viral vectors are derived from or based on one or more nucleic acid elements comprising a viral genome.
  • cells for transfection refers to cells that can be transfected with a nucleic acid to prepare cells capable of producing a viral vector.
  • Cells for transfection are known to those skilled in the art and are not particularly limited. Examples of such cells include, but are not limited to, HEK293 cells, HEK293T cells, HEK293F cells, HEK293FT cells, G3T-hi cells, Sf9 cells, commercially available cell lines for virus production, and AAV293 cells.
  • transfection cells include Expi293F (ThermoFisher), Viral Production Cells 2.0 (VPC2.0; ThermoFisher), FreeStyle 293-F (ThermoFisher), 293-F (ThermoFisher), 293-H (ThermoFisher), HEK293 (ATCC), HEK293T (ATCC), 293E (ATCC), AAV-293 (Agilent), AAVPro293T (Clontech), etc.
  • Preferred are human-derived HEK293 cells such as Expi293F, VPC2.0, 293-F, HEK293, and 293T.
  • the transfection cells may be human-derived cells.
  • HEK293 cells and the like can be suitably used as AAV vector producing cells because they constitutively express the adenovirus E1 protein.
  • Such cells modified to transiently or constitutively express one or more proteins necessary for constructing a viral vector can be used in the present invention.
  • viral vector producing cell refers to a cell that is prepared by transfecting a nucleic acid encoding a protein required for the production of a desired viral vector and an optional exogenous nucleic acid into a transfection cell, and that can produce a desired viral vector in the cell.
  • the preparation of such a viral vector producing cell is known per se, and in brief, it can be prepared by introducing one or more nucleic acids encoding a viral packaging protein and a helper protein into a cell using a transfection reagent.
  • an AAV vector producing cell can be prepared by transfecting a vector plasmid in which a gene of interest has been substituted between ITRs (inverted terminal repeats), a packaging plasmid expressing REP and CAP, and a helper plasmid expressing E2A, E4, and VA into HEK293 cells (cells expressing E1A and E1B).
  • the method of transfecting various nucleic acids one or more nucleic acids encoding a viral packaging protein and a helper protein required for the preparation of a viral vector producing cell may also be a method known per se.
  • transfection reagents include, but are not limited to, one or more compounds and/or compositions including cationic polymers such as polyethyleneimine (PEI), polymers of positively charged amino acids such as polylysine and polyarginine, positively charged dendrimers and fragmented dendrimers, cationic ⁇ -cyclodextrin-containing polymers (CD polymers), DEAE-dextran, and the like.
  • PEI polyethyleneimine
  • CD polymers cationic ⁇ -cyclodextrin-containing polymers
  • DEAE-dextran DEAE-dextran
  • the present invention provides a method for producing a viral vector , comprising the step of culturing viral vector-producing cells at 30.0 to 35.9°C to produce a viral vector, characterized in that the viral vector-producing cells are cultured at a temperature of 36.0 to 38.0°C before the start of the step of producing the viral vector (hereinafter, sometimes referred to as the "method for producing the viral vector of the present invention").
  • the key to the viral vector production method of the present invention is culturing viral vector-producing cells in two specific temperature zones (a first temperature zone and a second temperature zone).
  • the viral vector-producing cells before producing a viral vector are cultured in a temperature range of 36.0 to 38.0°C (the "first temperature range").
  • the viral vector producing cells cultured in the first temperature zone may be cells for transfection at a stage prior to the introduction of a gene encoding a viral vector.
  • the method for producing a viral vector of the present invention is characterized in that the viral vector producing cells or the transfection cells that are the source of the viral vector producing cells are cultured in the first temperature zone before the viral vector producing cells are allowed to produce a viral vector.
  • the first temperature zone may typically be 36.0 to 38.0°C.
  • the lower limit of the first temperature zone may typically be 36.0°C or higher, preferably 36.1°C or higher, 36.2°C or higher, 36.3°C or higher, 36.4°C or higher, 36.5°C or higher, 36.6°C or higher, 36.7°C or higher, or 36.8°C or higher, but is not limited thereto.
  • the upper limit of the first temperature zone may typically be 38.0°C or lower, preferably 37.9°C or lower, 37.8°C or lower, 37.7°C or lower, 37.6°C or lower, 37.5°C or lower, 37.4°C or lower, 37.3°C or lower, or 37.2°C or lower, but is not limited thereto.
  • the first temperature zone is typically 36.0 to 38.0°C, preferably 36.1 to 37.9°C, 36.2 to 37.8°C, 36.3 to 37.7°C, 36.4 to 37.6°C, 36.5 to 37.5°C, 36.6 to 37.4°C, 36.7 to 37.3°C, or 36.8 to 37.2°C, but is not limited thereto.
  • steps a and b maintaining and growing the cells for transfection
  • steps b preparing a viral vector-producing cell by transfecting a nucleic acid into a cell for transfection
  • steps c A step of maintaining and growing the viral vector-producing cells.
  • the first temperature zone may be applied to at least one of the steps before the viral vector is produced in the viral vector-producing cells.
  • the first temperature zone may be applied to any one of these steps [(step a), (step b), or (step c)], any two of these steps [(step a) to (step b), (step a) and (step c), (step b) to (step c), preferably two consecutive steps, (step a) to (step b), and (step b) to (step c)], or all three of these steps [(step a) to (step c)].
  • the culture of the cells for transfection or the cells producing a viral vector in the step of applying the first temperature zone can be performed under culture conditions known per se, except for the culture temperature.
  • the culture can be performed under a humidity of 95% RH and a CO2 concentration of 5 to 10% (v/v), but is not limited thereto.
  • the medium that can be used is not particularly limited as long as the cells for transfection or the cells producing a viral vector can survive. Examples of such a medium include, but are not limited to, basic synthetic media such as DMEM, IMDM, and DMEM:F-12.
  • additives known per se fetal bovine serum, growth factors, peptides, amino acids
  • the culture form of the cells is not particularly limited, and may be either suspension culture or adhesion culture.
  • the culture time is not particularly limited, and may be, for example, usually 1 hour or more, preferably 2 hours or more, 4 hours or more, 8 hours or more, 12 hours or more, 16 hours or more, 24 hours or more, 30 hours or more, 36 hours or more, 42 hours or more, 48 hours or more, 54 hours or more, 60 hours or more, 66 hours or more, 72 hours or more, 4 days or more, or 5 days or more, but is not limited thereto.
  • the culture time may be usually 10 days or less, and may be, for example, 9 days or less, 8 days or less, 7 days or less, or 6 days or less, but is not limited thereto. In one embodiment, the culture time is 1 hour to 10 days, preferably 12 hours to 8 days, 24 hours to 6 days, but is not limited thereto.
  • the second temperature zone (30.0 to 35.9°C) is applied in the step of causing viral vector-producing cells that have been cultured in the first temperature zone to produce a viral vector.
  • the second temperature range may be typically 30.0 to 35.9°C.
  • the lower limit of the second temperature range may be typically 30.0°C or higher, preferably 30.5°C or higher, 31.0°C or higher, 31.5°C or higher, 32.0°C or higher, 32.5°C or higher, 33.0°C or higher, 33.5°C or higher, 34.0°C or higher, or 34.5°C or higher, but is not limited thereto.
  • the upper limit of the second temperature range may be typically 35.9°C or lower, preferably 35.8°C or lower, 35.7°C or lower, 35.6°C or lower, 35.5°C or lower, 35.4°C or lower, 35.3°C or lower, 35.2°C or lower, 35.1°C or lower, or 35.0°C or lower, but is not limited thereto.
  • the second temperature zone may be, but is not limited to, typically 30.0 to 35.9°C, 31.5 to 35.8°C, 32.0 to 35.7°C, 32.5 to 35.6°C, 33.0 to 35.5°C, 33.5 to 35.5°C, 34.0 to 35.5°C, 34.5 to 35.5°C, or 35.0 to 35.5°C.
  • culture conditions known per se can be used, except for the culture temperature, as long as the viral vector-producing cells can survive and produce the viral vector. Such conditions are the same as those described for the first temperature range.
  • the second temperature zone is characterized by being lower than the first temperature zone.
  • viral vector-producing cells are cultured at 37°C, which is considered to be the optimal temperature, to produce the viral vector.
  • the viral vector-producing cells are caused to produce the viral vector at a temperature zone lower than this temperature.
  • the method for producing a viral vector of the present invention may include, but is not limited to, the following embodiments.
  • the viral vectors produced using the viral vector production method of the present invention are not particularly limited, but examples include viral vectors derived from enveloped viruses such as retroviruses, lentiviruses, Sendai viruses, and herpes viruses, or non-enveloped viruses such as adenoviruses and adeno-associated viruses (AAV) (hereinafter referred to as non-enveloped viruses).
  • enveloped viruses such as retroviruses, lentiviruses, Sendai viruses, and herpes viruses
  • non-enveloped viruses such as adenoviruses and adeno-associated viruses (AAV) (hereinafter referred to as non-enveloped viruses).
  • AAV adeno-associated viruses
  • the viral vector may be a viral vector derived from a DNA virus such as adenovirus, parvovirus, papovavirus, or papillomavirus.
  • the viral vector may be a viral vector derived from an RNA virus such as rotavirus, coxsackievirus, enterovirus, sapovirus, norovirus, poliovirus, echovirus, hepatitis A virus, hepatitis E virus, rhinovirus, or astrovirus.
  • the viral vector may be a viral vector derived from a virus of the Retroviridae family, a virus of the Adenoviridae family, or a virus of the Parvoviridae family.
  • the viral vector may be an adeno-associated virus (AAV) of the Parvoviridae family.
  • AAV adeno-associated virus
  • AAV has a non-enveloped icosahedral outer shell (capsid) with a single linear single-stranded DNA inside.
  • the capsid has three capsid proteins (VP1, VP2, and VP3).
  • AAV includes wild-type viruses and their derivatives, and includes all serotypes and clades unless otherwise specified.
  • AAV serotypes There are various reports on AAV serotypes, but at least 15 types of AAV serotypes that infect humans are known: AAV1, AAV2, AAV3A, AAV3b, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAVrh.10, AAV11, AAV12, and AAV13.
  • the cell culture in the first temperature zone and/or the second temperature zone is carried out in a medium containing an organic solvent.
  • a medium containing an organic solvent is used in the step of culturing viral vector-producing cells in the second temperature zone (i.e., 30.0 to 35.9°C) and producing a viral vector.
  • Such organic solvents include alcohols or dimethyl sulfoxide (DMSO), and are preferably alcohols.
  • DMSO dimethyl sulfoxide
  • Alcohol is a substance in which the hydrogen atoms of a hydrocarbon have been replaced with hydroxyl groups, and in this specification they are collectively referred to as alcohols.
  • the alcohol is preferably a lower alcohol having 1 to 6 carbon atoms, such as at least one selected from the group consisting of methanol, ethanol, isopropanol, and glycerol (glycerin).
  • DMSO is commercially available.
  • the amount of organic solvent added to the medium is not particularly limited as long as the desired effect of the present invention is obtained.
  • the organic solvent when the organic solvent is an alcohol (e.g., ethanol), the organic solvent can be added to the entire medium composition at a concentration of typically 2 V/V% or less. If the concentration is below this upper limit, the cells will not die and the effects of the present invention (i.e., the effect of improving the production amount of viral genome produced in the cells and/or the integrity of the viral genome, etc.) can be obtained. There is no particular limit to the lower limit of the amount of organic solvent added to the total reaction mixture as long as the effects of the present invention can be obtained, but it is a concentration of more than 0 V/V%.
  • the organic solvent is an alcohol (e.g., ethanol)
  • the organic solvent can be added to the entire medium composition at a concentration of typically 2 V/V% or less. If the concentration is below this upper limit, the cells will not die and the effects of the present invention (i.e., the effect of improving the production amount of viral genome produced in the cells and/or the integrity of the viral genome, etc.) can be obtained.
  • the organic solvent is used in an amount of 0.05 V/V% or more, 0.06 V/V% or more, 0.07 V/V% or more, 0.08 V/V% or more, 0.09 V/V% or more, 0.1 V/V% or more, 0.11 V/V% or more, 0.12 V/V% or more, 0.13 V/V% or more, 0.14 V/V% or more, 0.15 V/V% or more, 0.16 V/V% or more, 0.17 V/V% or more, 0.18 V/V% or more, 0.19 V/V% or more, or 0.2 V/V% or more, based on the total amount of the reaction mixture.
  • the amount of the organic solvent added is usually 0.05 to 2 V/V%, preferably 0.1 to 1.5 V/V%, 0.2 to 1 V/V%, or 0.2 to 0.56 V/V%, but is not limited thereto.
  • the amount of organic solvent used may be changed as appropriate depending on the organic solvent, cell type, culture conditions, etc.
  • the organic solvent is ethanol, which may be added before, at the start of, or within 24 hours (e.g., within 30 minutes, within 1 hour, within 2 hours, within 4 hours, within 6 hours, within 8 hours, within 10 hours, within 12 hours, within 16 hours, or within 20 hours) after the start of the process of culturing the viral vector-producing cells in the second temperature zone (i.e., 30.0 to 35.9°C) and producing the viral vector.
  • the second temperature zone i.e., 30.0 to 35.9°C
  • the organic solvent may be added prior to, at the start of, or within 24 hours after the start of (step a) (e.g., within 30 minutes, within 1 hour, within 2 hours, within 4 hours, within 6 hours, within 8 hours, within 10 hours, within 12 hours, within 16 hours, or within 20 hours, etc.).
  • the organic solvent can be added prior to, at the start of, or within 24 hours after the start of (step b) (e.g., within 30 minutes, within 1 hour, within 2 hours, within 4 hours, within 6 hours, within 8 hours, within 10 hours, within 12 hours, within 16 hours, or within 20 hours, etc.).
  • the organic solvent can be added prior to, at the start of, or within 24 hours after the start of (step c) (e.g., within 30 minutes, within 1 hour, within 2 hours, within 4 hours, within 6 hours, within 8 hours, within 10 hours, within 12 hours, within 16 hours, or within 20 hours, etc.).
  • the present invention also provides a method for improving the production efficiency of a viral vector with an improved infectious titer, which comprises a step of culturing viral vector-producing cells at 30.0 to 35.9°C, characterized in that the viral vector-producing cells are cultured at a temperature of 36.0 to 38.0°C before the start of the step of producing the viral vector (hereinafter, sometimes referred to as the "method for improving the production efficiency of a viral vector of the present invention").
  • the various conditions used in the method for improving the production efficiency of a viral vector of the present invention are the same as those explained in the method for producing a viral vector of the present invention.
  • the viral vector prepared by the method for improving viral vector production efficiency of the present invention is characterized by having an improved infectious titer.
  • "improved infectious titer” means that the infectious titer is improved compared to the infectious titer of a viral vector produced by viral vector-producing cells that have been consistently cultured at about 37°C (e.g., 36.0 to 38.0°C) before and after viral vector production (in other words, viral vector-producing cells that have not experienced a temperature shift from the first temperature zone to the second temperature zone).
  • a method for improving the ratio of viral particles containing a complete viral genome to all viral particles produced in the production of a viral vector using viral vector-producing cells also provides a method for improving the ratio of viral particles containing a complete viral genome (also referred to as the Full rate) to all viral particles produced in the production of a viral vector using viral vector-producing cells, which comprises the steps of culturing viral vector-producing cells at 30.0 to 35.9°C to produce a viral vector, characterized in that the viral vector-producing cells are cultured at a temperature of 36.0 to 38.0°C before the start of the step of producing the viral vector (hereinafter, this method may be referred to as the "method of the present invention for improving the Full rate of viral particles").
  • the various conditions used in the method of the present invention for improving the full rate of virus particles are the same as those explained in the method of the present invention for producing a virus vector.
  • the improvement in "the ratio of virus particles containing a complete viral genome to all virus particles produced” refers to an improvement compared to the said ratio obtained when using virus vector-producing cells that have been consistently cultured at about 37°C (e.g., 36.0 to 38.0°C) before and after production of the virus vector (in other words, virus vector-producing cells that have not experienced a temperature shift from the first temperature zone to the second temperature zone).
  • AAV2 was extracted from the cell pellet using a commercially available AAV extraction kit (Takara, AAVpro Extraction Solution) according to the kit's instructions, and the extract was frozen at -80°C.
  • the AAV2 extracted from the culture supernatant and the producing cells was quantified by droplet digital PCR (ddPCR) method according to the following procedure.
  • DNase was added to the culture supernatant and the AAV2 extract from the producing cells to digest the nucleic acid present outside the AAV2 particles in the measurement sample, and then the activity of DNase was stopped.
  • the capsid protein of AAV2 was decomposed with Proteinase K, the AAV genome embedded in the AAV2 particles was extracted, and then Proteinase K was inactivated to obtain an AAV2 genome extract.
  • the obtained AAV2 genome extract was subjected to 2D-ddPCR using two types of primers that amplify the base sequence of the ITR (Inverted Terminal Repeat) region and the GFP region of the AAV2 genome, and the production amount of AAV2 in the sample was absolutely quantified.
  • the percentage of AAV2 containing the complete AAV2 genome was calculated for AAV2 produced under each condition from the percentage of droplets that detected both the ITR region and the GFP region.
  • Figure 2(A) shows the relative AAV2 concentration in the cell extract when AAV2 was produced at 35°C compared to when the temperature after transfection was 37°C
  • Figure 2(B) shows the percentage of AAV2 containing the complete AAV2 genome.
  • AAV2 particles particles containing the AAV2 genome and all particles not containing the AAV2 genome extracted from the culture supernatant and the producing cells were measured using a commercially available AAV2 ELISA kit (PROGENE, AAV2 Titration ELISA) according to the kit's procedure.
  • the percentage of AAV2 particles containing the complete AAV2 genome was calculated from the total number of AAV2 particles quantified by ELISA and the quantitative value of particles containing the complete AAV2 genome obtained by ddPCR.
  • the relative value of the Full rate of AAV2 in the cell extract when AAV2 was produced at 35°C compared to when AAV2 was produced at 37°C after transfection is shown in Figure 2(C).
  • Example 2 Examination of the effect of culture temperature on viral vector production 2 The viral vector-producing cells were allowed to produce the viral vector under the same conditions as in Example 1, except that all steps from preparation of the viral vector-producing cells to production of the viral vector were carried out at 35° C., and the amount of the produced AAV viral vector was measured. The results are shown in FIG.
  • Example 3 Examination of the effect of adding an organic solvent (ethanol) during viral vector production 1 The procedure up to the point where the temperature was changed after transfection to produce AAV2 was the same as in Example 1. Approximately 4 hours after transfection, 0.56 v/v% filtered ethanol was added to the culture medium volume, and AAV2 was produced for 3 days.
  • FIG. 4(A) shows the relative concentration of AAV2 in the cell extract when ethanol was added at 35°C, compared to the control condition (37°C and no ethanol was added) during AAV2 production
  • FIG. 4(B) shows the ratio of AAV2 in the cell extract that contains the complete genome. Furthermore, FIG.
  • FIG. 4(C) shows the relative value of the full rate of AAV2 contained in the cell extract under each production condition compared to the control condition.
  • FIG. 5(A) and FIG. 5(B) show the transition of cell density and cell viability during AAV2 production (after transfection) under each production condition. Note that all analyses were performed in the same manner as in Example 1.
  • AAV2 extracted from viral vector-producing cells was added to cultured HeLa cells at the same genome titer (vg), and the amount of AAV2 required per number of cells expressing GFP protein derived from the added AAV2 (transduction unit: TU) three days later was calculated.
  • the results are shown in Table 1 below.
  • Example 4 Examination of the effect of adding an organic solvent (ethanol) during viral vector production 2
  • the same procedure as in the culture of the viral vector producing cells in Example 1 was used to grow the cells in an Erlenmeyer flask, and the resulting cell culture solution was collected aseptically, and then the cell pellet was collected by centrifugation.
  • the cell pellet was resuspended in fresh synthetic medium to about half the cell concentration suitable for AAV2 production, and transferred to a 250 mL culture tank, and then aerated and cultured with stirring (37°C, pH 7.2 ⁇ 0.2, dissolved oxygen >40%). After 20 to 28 hours, the culture tank was transfected with a plasmid required for AAV2 production using the same procedure as in the transfection in Example 1.
  • Example 5 Examination of organic solvents 1 Viral vectors were produced and analyzed in the same manner as in Example 3, except that the organic solvent was changed from ethanol to glycerol, methanol, and isopropanol, and the temperature when the viral vector was produced in the viral vector-producing cells was 37°C. The results of measurement of the amount of AAV2 produced in the producing cells 3 days after transfection and the integrity of the genome in the produced AAV2 particles are shown in Table 2.
  • Example 6 Examination of organic solvents 2 Viral vectors were produced and analyzed in the same manner as in Example 3, except that the organic solvent was changed from ethanol to dimethyl sulfoxide (DMSO) and the temperature for producing the viral vector in the viral vector-producing cells was 37°C. The results of AAV2 productivity in the culture supernatant and cells 3 days after transfection are shown in Table 3.
  • DMSO dimethyl sulfoxide
  • DMSO improved the genome integrity and genome titer (productivity) in the produced AAV2.
  • DMSO was shown to have favorable effects on VCD and viability.
  • Example 7 Study of AAV serotypes Viral vectors were produced and analyzed in the same manner as in Example 3, except that the plasmid encoding the Cap protein of AAV2 was changed to a plasmid encoding the Cap protein of AAV1 or AAV6, and the temperature during production of the viral vector in the viral vector-producing cells was set to 37° C. The results regarding the productivity of AAV1 and AAV6 in the culture supernatant and cells 3 days after transfection are shown in Table 4.
  • Example 8 Examination of the lower limit of the second temperature range The procedure up to the operation of changing the temperature after transfection and producing AAV2 was performed in the same manner as in Example 1, and about 4 hours after transfection, 0.56 v/v% filtered ethanol was added to the culture solution volume, and AAV2 was produced for 3 days.
  • Figure 7 shows the relative value of the total production amount of viral vectors when ethanol was added at 33°C compared to the condition (control condition) where ethanol was not added at 37°C during AAV2 production.
  • a viral vector having a high infectious titer can be produced very efficiently, and therefore the present invention is extremely useful in the fields of drug discovery and research.
  • This application is based on Japanese Patent Application No. 2022-162794 (filing date: October 7, 2022) filed in Japan, the contents of which are incorporated in their entirety herein.

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Abstract

The purpose of the present invention is to provide, in virus vector production, a method for efficiently producing a virus vector having a high infectious titer. Provided is a virus vector production method including a step for culturing virus vector-producing cells at 30.0-35.9ºC, and, here, with the method according to the present invention that is characterized in that said virus vector-producing cells are cultured at a temperature of 36.0-38.0ºC before starting a virus vector production step, it is possible to very efficiently produce a virus vector having a high infectious titer.

Description

細胞を用いたウイルスベクターの生産方法Method for producing viral vectors using cells
　本発明は、細胞を用いたウイルスベクターの効率的な生産方法に関する。 The present invention relates to a method for efficiently producing a viral vector using cells.
　バイオ医薬品とは、遺伝子組換え技術や細胞培養技術等を応用して、微生物や細胞が持つタンパク質（ホルモン、酵素、抗体等）等を作る力を利用して製造される医薬品であり、近年の医薬の主流となりつつある。抗体等の目的とするタンパク質の遺伝子を細胞（好ましくは哺乳動物細胞）に導入し、その細胞が目的とするタンパク質を産生する。
　遺伝子導入の方法としては、レトロウイルスやレンチウイルス、アデノ随伴ウイルスベクターを用いた方法と、リポフェクション法、エレクトロポレーション法、マイクロインジェクション法など物理化学的方法の2つに大別される。
　一方、遺伝子治療等の医療分野において、ヒトを含む哺乳動物細胞に遺伝子を導入する方法としては、ウイルス由来の遺伝子導入用のベクター（以下、ウイルスベクター）を用いる方法が一般的である。ウイルスベクターとは、遺伝子組み換え技術を用いて天然由来のウイルスを改変し、所望の遺伝子等を標的に移入することができるようにしたベクターのことで、近年技術開発が進んでいる。
　ウイルスベクターの由来となるウイルスとしては、レトロウイルスやレンチウイルス、センダイウイルス、ならびにヘルペスウイルス等のエンベロープを持つウイルス、アデノウイルス、アデノ随伴ウイルス（以下、AAV）等のエンベロープを持たないウイルス（以下、非エンベロープウイルス）がよく知られている。
　特にAAVはヒトを含む広範な種の細胞に感染可能で、分化を終えた非分裂細胞にも感染すること、ヒトに対する病原性がないため副作用の心配が低いこと、ウイルス粒子が物理化学的に安定であること等から、遺伝子治療法に用いる遺伝子導入用のベクターとして有望視されている。
　一般的にAAVは、ヒト胎児腎細胞293（HEK293細胞）等の哺乳類細胞にAAV産生に必要なプラスミドDNAを導入する事で生産されるが、生産性の低さに加え、AAVのカプシド内へのAAVゲノムのパッケージング率、カプシド内にパッケージングされたAAVゲノムの断片化など、品質面での課題があることが知られている。 Biopharmaceuticals are medicines manufactured by utilizing the ability of microorganisms or cells to produce proteins (hormones, enzymes, antibodies, etc.) through the application of recombinant gene technology and cell culture technology, and are becoming the mainstream of medicine in recent years. The gene for a target protein such as an antibody is introduced into a cell (preferably a mammalian cell), and the cell produces the target protein.
Methods for gene transfer can be broadly divided into two categories: methods using retroviruses, lentiviruses, or adeno-associated virus vectors, and physicochemical methods such as lipofection, electroporation, and microinjection.
On the other hand, in the medical field such as gene therapy, a method for introducing genes into mammalian cells, including human cells, generally uses a virus-derived gene transfer vector (hereinafter, viral vector). A viral vector is a vector that is made by modifying a naturally occurring virus using recombinant gene technology so that a desired gene or the like can be transferred to a target, and technological development has progressed in recent years.
Well-known viruses from which viral vectors are derived include enveloped viruses such as retroviruses, lentiviruses, Sendai viruses, and herpes viruses, as well as non-enveloped viruses such as adenoviruses and adeno-associated viruses (AAV).
In particular, AAV is considered promising as a gene transfer vector for use in gene therapy because it can infect cells of a wide range of species, including humans, and can also infect non-dividing cells that have completed differentiation; it is not pathogenic to humans, so there is little risk of side effects; and the viral particles are physicochemically stable.
AAV is generally produced by introducing plasmid DNA required for AAV production into mammalian cells such as human embryonic kidney 293 (HEK293 cells). However, in addition to low productivity, there are known quality issues, such as the packaging rate of the AAV genome into the AAV capsid and fragmentation of the AAV genome packaged within the capsid.
　アデノウイルスやAAV等の非エンベロープウイルスベクターは、広い組織親和性および細胞親和性、大型の発現カセットの収容能力および高い形質導入効率を示し、遺伝子送達のための望ましい特徴を有している。 Non-enveloped viral vectors such as adenovirus and AAV exhibit broad tissue and cell tropism, capacity to accommodate large expression cassettes, and high transduction efficiency, providing desirable characteristics for gene delivery.
　バイオ医薬品やウイルスベクター医薬の開発および商業上の製造のためには、高効率で細胞をトランスフェクションすることや細胞内で目的物の生産量を増加させることが重要である。トランスフェクション効率や遺伝子の転写活性を高めるために増強剤（エンハンサー）を添加することが知られている（特許文献1、非特許文献1及び2）。
　しかしながら、感染力価の高いウイルスベクターを効率よく製造する方法には更なる改良が求められている。 For the development and commercial production of biopharmaceuticals and viral vector medicines, it is important to transfect cells with high efficiency and increase the production of the target substance in the cells. It is known that enhancers can be added to increase the transfection efficiency and gene transcription activity (Patent Document 1, Non-Patent Documents 1 and 2).
However, further improvements are required in methods for efficiently producing viral vectors with high infectious titers.
特表２０２０－５２４４９８号公報JP 2020-524498 A
　本発明は、ウイルスベクターの製造において、感染力価の高いウイルスベクターを効率よく製造する方法を提供することを課題とする。 The objective of the present invention is to provide a method for efficiently producing a viral vector with a high infectious titer.
　本発明者らは、上記課題に対して鋭意検討した結果、ヒト由来細胞を用いたウイルスベクターの生産において、当該細胞の前培養やウイルスベクター産生細胞を調製するための形質転換用プラスミドのトランスフェクションまでの工程は通常用いられるヒト由来細胞を培養するうえで好ましいとされる37℃付近の温度条件下で行うが、ウイルスベクター産生細胞にウイルスベクターを生産させる工程においては、敢えて通常用いられる温度よりも若干低い35℃付近の温度条件を適用することにより、(1)トランスフェクション後のウイルスベクター産生細胞の生存率が向上すること、(2)ウイルスベクター産生細胞の増殖が継続すること、(3)ウイルスベクター産生細胞内のウイルスベクター生産性が向上すること、(4)ウイルスベクター内にパッケージングされたゲノム完全体の割合に悪影響はないこと、(5)ウイルスゲノムがパッケージングされたウイルス粒子の割合が増加すること、(6)得られたウイルス粒子の感染力価が高まること、等の予想外の効果があることを見出した。加えて、本発明者らは、上述の温度シフトに加えて、温度シフト後の培地にエタノールなどの有機溶媒を添加すると、(1)～(6)の効果がさらに高まることを見出し、かかる知見に基づいてさらに研究を進めることによって本発明を完成するに至った。
　すなわち、本発明は以下の通りである。 As a result of intensive research into the above-mentioned problems, the present inventors have found that, in the production of viral vectors using human-derived cells, the steps of pre-culturing the cells and transfection of a transformation plasmid to prepare viral vector-producing cells are carried out under temperature conditions of around 37°C, which is generally preferred for culturing human-derived cells, but that in the step of making the viral vector-producing cells produce viral vectors, a temperature condition of around 35°C, which is slightly lower than the generally used temperature, is deliberately applied, thereby producing unexpected effects such as (1) improved survival rate of viral vector-producing cells after transfection, (2) continued proliferation of viral vector-producing cells, (3) improved viral vector productivity in viral vector-producing cells, (4) no adverse effect on the proportion of complete genomes packaged in the viral vector, (5) an increase in the proportion of viral particles in which the viral genome is packaged, and (6) an increase in the infectious titer of the obtained viral particles. In addition, the present inventors have found that the effects of (1) to (6) are further enhanced when an organic solvent such as ethanol is added to the medium after the temperature shift in addition to the above-mentioned temperature shift, and have completed the present invention by conducting further research based on this finding.
That is, the present invention is as follows.
[1]
　ウイルスベクター産生細胞を30.0～35.9℃で培養し、ウイルスベクターを産生させる工程を含む、ウイルスベクターを生産する方法であって、ここで、当該ウイルスベクター産生細胞が、ウイルスベクターを産生させる工程の開始前に、36.0～38.0℃の温度下で培養されていることを特徴とする、方法。
[2]
　前記ウイルスベクター産生細胞にウイルスベクターを産生させる工程が、有機溶媒を含む培地で行われることを特徴とする、[1]記載の方法。
[3]
　有機溶媒が、アルコール類又はジメチルスルホキシドである、[2]記載の方法。
[4]
　ウイルスベクター産生細胞を30.0～35.9℃で培養し、ウイルスベクターを産生させる工程を含む、感染力価が向上したウイルスベクターの生産効率を向上させる方法であって、ここで、当該ウイルスベクター産生細胞が、ウイルスベクターを産生させる工程の開始前に、36.0～38.0℃の温度下で培養されていることを特徴とする、方法。
[5]
　前記ウイルスベクター産生細胞にウイルスベクターを産生させる工程が、有機溶媒を含む培地で行われることを特徴とする、[4]記載の方法。
[6]
　有機溶媒が、アルコール類又はジメチルスルホキシドである、[5]記載の方法。
[7]
　ウイルスベクター産生細胞を30.0～35.9℃で培養し、ウイルスベクターを産生させる工程を含む、ウイルスベクター産生細胞を用いたウイルスベクターの製造において、製造される全ウイルス粒子に対してのウイルスゲノムの完全体を含むウイルス粒子の割合を向上させる方法であって、ここで、当該ウイルスベクター産生細胞が、ウイルスベクターを産生させる工程の開始前に、36.0～38.0℃の温度下で培養されていることを特徴とする、方法。
[8]
　前記ウイルスベクター産生細胞にウイルスベクターを産生させる工程が、有機溶媒を含む培地で行われることを特徴とする、[7]記載の方法。
[9]
　有機溶媒が、アルコール類又はジメチルスルホキシドである、[8]記載の方法。 [1]
A method for producing a viral vector, comprising a step of culturing a viral vector-producing cell at 30.0 to 35.9°C to produce a viral vector, wherein the viral vector-producing cell is cultured at a temperature of 36.0 to 38.0°C before the start of the step of producing the viral vector.
[2]
The method according to [1], wherein the step of causing the viral vector-producing cells to produce the viral vector is carried out in a medium containing an organic solvent.
[3]
The method according to [2], wherein the organic solvent is an alcohol or dimethyl sulfoxide.
[Four]
A method for improving the production efficiency of a viral vector having an improved infectious titer, comprising a step of culturing viral vector-producing cells at 30.0 to 35.9°C to produce a viral vector, wherein the viral vector-producing cells are cultured at a temperature of 36.0 to 38.0°C before the start of the step of producing the viral vector.
[Five]
The method according to [4], wherein the step of causing the viral vector-producing cells to produce the viral vector is carried out in a medium containing an organic solvent.
[6]
The method according to [5], wherein the organic solvent is an alcohol or dimethyl sulfoxide.
[7]
A method for improving the ratio of viral particles containing a complete viral genome to all viral particles produced in production of a viral vector using viral vector-producing cells, the method comprising the step of culturing viral vector-producing cells at 30.0 to 35.9°C to produce a viral vector, wherein the viral vector-producing cells are cultured at a temperature of 36.0 to 38.0°C prior to the start of the step of producing the viral vector.
[8]
The method according to [7], wherein the step of causing the viral vector-producing cells to produce the viral vector is carried out in a medium containing an organic solvent.
[9]
The method according to [8], wherein the organic solvent is an alcohol or dimethyl sulfoxide.
　本発明によれば、感染力価の高いウイルスベクターを非常に効率よく生産することができる。 The present invention makes it possible to produce highly infectious viral vectors with extremely high efficiency.
(A)ウイルスベクターの生産時の培養温度が産生細胞の増殖に与える影響を示す図である。(B)ウイルスベクターの生産時の培養温度が産生細胞の生存率に与える影響を示す図である。(A) A diagram showing the effect of culture temperature during viral vector production on the proliferation of producer cells (B) A diagram showing the effect of culture temperature during viral vector production on the viability of producer cells. (A)ウイルスベクターの生産時の培養温度が産生細胞によるウイルスベクターの生産性に与える影響を示す図である。(B)ウイルスベクターの生産時の培養温度が産生細胞におけるウイルスベクター内にパッケージングされたゲノム完全体の割合に与える影響を示す図である。(C)ウイルスベクターの生産時の培養温度が産生細胞におけるウイルスのFull率に与える影響を示す図である。(A) A diagram showing the effect of culture temperature during viral vector production on viral vector productivity by producer cells, (B) A diagram showing the effect of culture temperature during viral vector production on the proportion of complete genomes packaged within viral vectors in producer cells, and (C) A diagram showing the effect of culture temperature during viral vector production on the full virus rate in producer cells. ウイルスベクター産生細胞の調製からウイルスベクター産生細胞によるウイルスベクターの産生までを一貫して35℃で行ったときの、ウイルスベクターの総生産量を示す図である。FIG. 1 shows the total production amount of viral vectors when the entire process from preparation of viral vector-producing cells to production of viral vectors by the viral vector-producing cells was carried out at 35° C. (A)ウイルスベクターの生産時の培養温度およびエタノールの添加が産生細胞によるウイルスベクターの生産性に与える影響を示す図である。(B)ウイルスベクターの生産時の培養温度およびエタノールの添加が産生細胞におけるウイルスベクター内にパッケージングされたゲノム完全体の割合に与える影響を示す図である。(C)ウイルスベクターの生産時の培養温度およびエタノールの添加が産生細胞におけるウイルスのFull率に与える影響を示す図である。(A) A diagram showing the influence of culture temperature and addition of ethanol during viral vector production on viral vector productivity by producer cells, (B) A diagram showing the influence of culture temperature and addition of ethanol during viral vector production on the proportion of complete genomes packaged in viral vectors in producer cells, and (C) A diagram showing the influence of culture temperature and addition of ethanol during viral vector production on the full virus rate in producer cells. (A)ウイルスベクターの生産時の培養温度およびエタノールの添加が産生細胞の増殖に与える影響を示す図である。(B)ウイルスベクターの生産時の培養温度およびエタノールの添加が産生細胞の生存率に与える影響を示す図である。(A) A diagram showing the influence of culture temperature and addition of ethanol during viral vector production on the proliferation of producer cells (B) A diagram showing the influence of culture temperature and addition of ethanol during viral vector production on the viability of producer cells. (A)ウイルスベクターの生産時の培養温度およびエタノールの添加が産生細胞によるウイルスベクターの生産性に与える影響を示す図である。(B)ウイルスベクターの生産時の培養温度およびエタノールの添加が産生細胞におけるウイルスベクター内にパッケージングされたゲノム完全体の割合に与える影響を示す図である。(C)ウイルスベクターの生産時の培養温度およびエタノールの添加が産生細胞におけるウイルスのFull率に与える影響を示す図である。(A) A diagram showing the influence of culture temperature and addition of ethanol during viral vector production on viral vector productivity by producer cells. (B) A diagram showing the influence of culture temperature and addition of ethanol during viral vector production on the proportion of complete genomes packaged in viral vectors in producer cells. (C) A diagram showing the influence of culture temperature and addition of ethanol during viral vector production on the full virus rate in producer cells. ウイルスベクターの生産時の培養温度を33℃にシフトさせたときの、ウイルスベクターの総生産量を示す図である。FIG. 1 shows the total production amount of viral vectors when the culture temperature during viral vector production was shifted to 33° C.
（定義）
ウイルス
　本明細書において、「ウイルス」とは、天然のウイルスに加え、天然のウイルス等を基に病原性を取り除き、外来遺伝子を導入するための領域を備えるように改変された組換えウイルス粒子が包含される。かかる組換えウイルス粒子をウイルスベクターとも称する。本発明では、天然のウイルス、組換えウイルス粒子（ウイルスベクター）をウイルスと総称する場合がある。 (Definition)
In this specification, the term " virus " includes not only naturally occurring viruses, but also recombinant virus particles that have been modified based on naturally occurring viruses, etc., to remove pathogenicity and to include a region for introducing a foreign gene. Such recombinant virus particles are also called virus vectors. In the present invention, naturally occurring viruses and recombinant virus particles (virus vectors) may be collectively referred to as viruses.
ウイルスベクター
　本明細書において、「ウイルスベクター」とは、天然のウイルス等を基に病原性を取り除き、外来遺伝子を導入するための領域を備えるように改変されたウイルス粒子を意味する。さらに本発明では、「ウイルスベクター」は、ウイルスゲノム（核酸形状）を含むものだけでなく、ウイルスゲノムを含んでいないウイルス様の粒子である中空粒子も意味する。「ウイルスベクター」は、粒子の内部にウイルスゲノム（核酸形状）を含むものを得ることが好ましい。
　ウイルスベクターは、核酸の細胞への導入／移入、及び細胞内に挿入された核酸の転写又は翻訳のための、遺伝子操作（すなわち、「クローニングベクター」）に使用できる。「発現ベクター」は、宿主細胞内の発現に必要な調節領域を持つ遺伝子又は核酸配列を含む、特殊ベクターである。ベクター核酸配列は、一般に、少なくとも細胞内での増殖のための複製起点と、任意選択で異種ポリヌクレオチド配列、発現制御要素（例えば、プロモーター、エンハンサー）、イントロン、ITR(複数可)、選択マーカー（例えば、抗生物質耐性）、ポリアデニル化シグナルのような追加要素を含む。
　ウイルスベクターは、ウイルスゲノムを含む1つ又は複数の核酸要素に由来するか、又は基づく。 Viral vector : In this specification, the term "viral vector" refers to a virus particle that has been modified based on a natural virus or the like to remove pathogenicity and to have a region for introducing a foreign gene. Furthermore, in the present invention, the term "viral vector" refers not only to a virus vector that contains a viral genome (nucleic acid form), but also to a hollow particle that is a virus-like particle that does not contain a viral genome. It is preferable to obtain a "viral vector" that contains a viral genome (nucleic acid form) inside the particle.
Viral vectors can be used for genetic engineering (i.e., "cloning vectors") for the introduction/transfer of nucleic acids into cells and for transcription or translation of the inserted nucleic acid in cells. An "expression vector" is a specialized vector that contains a gene or nucleic acid sequence with regulatory regions necessary for expression in a host cell. A vector nucleic acid sequence generally contains at least an origin of replication for propagation in a cell and optionally additional elements such as heterologous polynucleotide sequences, expression control elements (e.g., promoters, enhancers), introns, ITR(s), selection markers (e.g., antibiotic resistance), polyadenylation signals.
Viral vectors are derived from or based on one or more nucleic acid elements comprising a viral genome.
トランスフェクション用細胞
　本明細書において、「トランスフェクション用細胞」とは、核酸をトランスフェクションすることでウイルスベクターを産生できる細胞を調製することができる細胞を意味する。トランスフェクション用細胞は当業者に自体公知であり、特に限定されない。かかる細胞としては、例えば、HEK293細胞やHEK293T細胞、HEK293F細胞、HEK293FT細胞、G3T-hi細胞、Sf9細胞、市販のウイルス産生用細胞株、AAV293細胞等が例示されるが、これらに限定されない。市販のトランスフェクション用細胞としては、Expi293F（ThermoFisher社製）、Viral Production Cells 2.0（VPC2.0; ThermoFisher社製）、FreeStyle 293-F (ThermoFishere社製)、293-F (ThermoFishere社製)、293-H (ThermoFishere社製)、HEK293 (ATCC)、HEK293T (ATCC) 、293E (ATCC)、AAV-293 (Agilent社製)、AAVPro293T (Clontech社製) 等が挙げられる。好ましくはヒト由来HEK293細胞であるExpi293F、VPC2.0、293-F、HEK293、293T等が挙げられる。一態様において、トランスフェクション用細胞は、ヒト由来の細胞であり得る。また、例えば、HEK293細胞等はアデノウイルスE1タンパク質を恒常的に発現するため、AAVベクターの産生細胞として好適に用いることができる。このような、ウイルスベクターの構成に必要なタンパク質の1つ又は複数を一過性又は恒常的に発現するように改変した細胞を本発明において使用することができる。 As used herein, the term "cells for transfection " refers to cells that can be transfected with a nucleic acid to prepare cells capable of producing a viral vector. Cells for transfection are known to those skilled in the art and are not particularly limited. Examples of such cells include, but are not limited to, HEK293 cells, HEK293T cells, HEK293F cells, HEK293FT cells, G3T-hi cells, Sf9 cells, commercially available cell lines for virus production, and AAV293 cells. Commercially available transfection cells include Expi293F (ThermoFisher), Viral Production Cells 2.0 (VPC2.0; ThermoFisher), FreeStyle 293-F (ThermoFisher), 293-F (ThermoFisher), 293-H (ThermoFisher), HEK293 (ATCC), HEK293T (ATCC), 293E (ATCC), AAV-293 (Agilent), AAVPro293T (Clontech), etc. Preferred are human-derived HEK293 cells such as Expi293F, VPC2.0, 293-F, HEK293, and 293T. In one embodiment, the transfection cells may be human-derived cells. Furthermore, for example, HEK293 cells and the like can be suitably used as AAV vector producing cells because they constitutively express the adenovirus E1 protein. Such cells modified to transiently or constitutively express one or more proteins necessary for constructing a viral vector can be used in the present invention.
ウイルスベクター産生細胞
　本明細書において、「ウイルスベクター産生細胞」とは、所望のウイルスベクターの生産に必要なタンパク質をコードする核酸と任意で外来性の核酸をトランスフェクション用細胞にトランスフェクションすることにより調製され、細胞内において所望のウイルスベクターを産生することができる細胞を意味する。かようなウイルスベクター産生細胞の調製は自体公知であり、簡潔には、ウイルスパッケージングタンパク質とヘルパータンパク質とをコードする1つ又は複数の核酸を、トランスフェクション試薬を用いて細胞に導入することにより調製することができる。例えば、AAVベクター産生細胞は、ITR（Inverted terminal repeat）の間を目的遺伝子と置換したベクタープラスミドと、REP及びCAPを発現するパッケージングプラスミドと、E2A、E4、VAを発現するヘルパープラスミドとをHEK293細胞（E1A及びE1Bを発現している細胞）にトランスフェクションすることで調製することができる。また、ウイルスベクター産生細胞の調製に必要となる各種核酸（ウイルスパッケージングタンパク質とヘルパータンパク質とをコードする１つ又は複数の核酸）をトランスフェクトする方法もまた、自体公知の方法を用いればよい。好適なトランスフェクション試薬としては、ポリエチレンイミン(PEI)などのカチオン性ポリマー、ポリリジン及びポリアルギニンなどの正に荷電したアミノ酸のポリマー、正に荷電したデンドリマー及び分裂したデンドリマー、カチオン性β-シクロデキストリン含有ポリマー(CDポリマー)、DEAE-デキストランなどを含む、１つ以上の化合物及び/又は組成物が例示されるが、これらに限定されない。 Viral vector producing cell In this specification, the term "viral vector producing cell" refers to a cell that is prepared by transfecting a nucleic acid encoding a protein required for the production of a desired viral vector and an optional exogenous nucleic acid into a transfection cell, and that can produce a desired viral vector in the cell. The preparation of such a viral vector producing cell is known per se, and in brief, it can be prepared by introducing one or more nucleic acids encoding a viral packaging protein and a helper protein into a cell using a transfection reagent. For example, an AAV vector producing cell can be prepared by transfecting a vector plasmid in which a gene of interest has been substituted between ITRs (inverted terminal repeats), a packaging plasmid expressing REP and CAP, and a helper plasmid expressing E2A, E4, and VA into HEK293 cells (cells expressing E1A and E1B). In addition, the method of transfecting various nucleic acids (one or more nucleic acids encoding a viral packaging protein and a helper protein) required for the preparation of a viral vector producing cell may also be a method known per se. Examples of suitable transfection reagents include, but are not limited to, one or more compounds and/or compositions including cationic polymers such as polyethyleneimine (PEI), polymers of positively charged amino acids such as polylysine and polyarginine, positively charged dendrimers and fragmented dendrimers, cationic β-cyclodextrin-containing polymers (CD polymers), DEAE-dextran, and the like.
1．ウイルスベクターを生産する方法
　本発明は、ウイルスベクター産生細胞を30.0～35.9℃で培養し、ウイルスベクターを産生させる工程を含む、ウイルスベクターを生産する方法であって、ここで、当該ウイルスベクター産生細胞が、ウイルスベクターを産生させる工程の開始前に、36.0～38.0℃の温度下で培養されていることを特徴とする、方法（以下、「本発明のウイルスベクターの生産方法」と称することがある）を提供する。 1. Method for Producing a Viral Vector The present invention provides a method for producing a viral vector , comprising the step of culturing viral vector-producing cells at 30.0 to 35.9°C to produce a viral vector, characterized in that the viral vector-producing cells are cultured at a temperature of 36.0 to 38.0°C before the start of the step of producing the viral vector (hereinafter, sometimes referred to as the "method for producing the viral vector of the present invention").
　本発明のウイルスベクターの生産方法における要諦は、ウイルスベクター産生細胞を2つの特定の温度帯（第1の温度帯と第2の温度帯）で培養する点にある。 The key to the viral vector production method of the present invention is culturing viral vector-producing cells in two specific temperature zones (a first temperature zone and a second temperature zone).
[第１の温度帯]
　本発明のウイルスベクターの生産方法において、ウイルスベクターを産生させる前のウイルスベクター産生細胞は、36.0～38.0℃の温度帯（「第１の温度帯」）で培養されることを特徴とする。 [First temperature zone]
In the method for producing a viral vector of the present invention, the viral vector-producing cells before producing a viral vector are cultured in a temperature range of 36.0 to 38.0°C (the "first temperature range").
　尚、第1の温度帯で培養されるウイルスベクター産生細胞は、ウイルスベクターをコードする遺伝子が導入される前の段階にある、トランスフェクション用細胞であってもよい。換言すれば、本発明のウイルスベクターの生産方法においては、ウイルスベクター産生細胞にウイルスベクターを産生させる前に、当該ウイルスベクター産生細胞又はその起源となるトランスフェクション用細胞が、第1の温度帯で培養されていることを特徴とする。 The viral vector producing cells cultured in the first temperature zone may be cells for transfection at a stage prior to the introduction of a gene encoding a viral vector. In other words, the method for producing a viral vector of the present invention is characterized in that the viral vector producing cells or the transfection cells that are the source of the viral vector producing cells are cultured in the first temperature zone before the viral vector producing cells are allowed to produce a viral vector.
　第1の温度帯は、通常36.0～38.0℃であり得る。一態様において、第1の温度帯の下限は通常36.0℃以上、好ましくは36.1℃以上、36.2℃以上、36.3℃以上、36.4℃以上、36.5℃以上、36.6℃以上、36.7℃以上、又は36.8℃以上であり得るが、これらに限定されない。また、一態様において、第1の温度帯の上限は、通常38.0℃以下、好ましくは、37.9℃以下、37.8℃以下、37.7℃以下、37.6℃以下、37.5℃以下、37.4℃以下、37.3℃以下、又は37.2℃以下であり得るが、これらに限定されない。一態様において、第1の温度帯は、通常36.0～38.0℃、好ましくは、36.1～37.9℃、36.2～37.8℃、36.3～37.7℃、36.4～37.6℃、36.5～37.5℃、36.6～37.4℃、36.7～37.3℃、又は36.8～37.2℃であり得るが、これらに限定されない。 The first temperature zone may typically be 36.0 to 38.0°C. In one embodiment, the lower limit of the first temperature zone may typically be 36.0°C or higher, preferably 36.1°C or higher, 36.2°C or higher, 36.3°C or higher, 36.4°C or higher, 36.5°C or higher, 36.6°C or higher, 36.7°C or higher, or 36.8°C or higher, but is not limited thereto. Also, in one embodiment, the upper limit of the first temperature zone may typically be 38.0°C or lower, preferably 37.9°C or lower, 37.8°C or lower, 37.7°C or lower, 37.6°C or lower, 37.5°C or lower, 37.4°C or lower, 37.3°C or lower, or 37.2°C or lower, but is not limited thereto. In one embodiment, the first temperature zone is typically 36.0 to 38.0°C, preferably 36.1 to 37.9°C, 36.2 to 37.8°C, 36.3 to 37.7°C, 36.4 to 37.6°C, 36.5 to 37.5°C, 36.6 to 37.4°C, 36.7 to 37.3°C, or 36.8 to 37.2°C, but is not limited thereto.
　ここで、第1の温度帯が適用され得る工程の具体例としては、次のような工程が例示され得る:
(工程a)トランスフェクション用細胞を維持・増殖させる工程;
(工程b)トランスフェクション用細胞に核酸をトランスフェクトすることによりウイルスベクター産生細胞を調製する工程;
(工程c)ウイルスベクター産生細胞を維持・増殖させる工程。 Here, specific examples of processes to which the first temperature zone can be applied include the following processes:
(step a) maintaining and growing the cells for transfection;
(step b) preparing a viral vector-producing cell by transfecting a nucleic acid into a cell for transfection;
(Step c) A step of maintaining and growing the viral vector-producing cells.
　尚、本発明において、第1の温度帯は、ウイルスベクター産生細胞にウイルスベクターを産生させる前の工程の少なくとも1つの工程に適用すればよい。例えば、ウイルスベクター産生細胞にウイルスベクターを産生させる前の工程として、(工程a)～(工程c)を実施する場合、これらの工程のいずれか1つ［（工程a）、（工程b）又は（工程c）］、いずれか2つ［（工程a）～（工程b）、（工程a）と（工程c）、（工程b）～（工程c）、好ましくは連続する２つの工程である(工程a)～（工程b)、及び（工程b）～（工程c）］、又は3つ全ての工程［（工程a）～（工程c）］に第1の温度帯を適用することができる。 In the present invention, the first temperature zone may be applied to at least one of the steps before the viral vector is produced in the viral vector-producing cells. For example, when (step a) to (step c) are performed as the steps before the viral vector is produced in the viral vector-producing cells, the first temperature zone may be applied to any one of these steps [(step a), (step b), or (step c)], any two of these steps [(step a) to (step b), (step a) and (step c), (step b) to (step c), preferably two consecutive steps, (step a) to (step b), and (step b) to (step c)], or all three of these steps [(step a) to (step c)].
　また、第1の温度帯を適用する工程におけるトランスフェクション用細胞やウイルスベクター産生細胞の培養は、培養温度以外の点では、自体公知の培養条件を用いることができる。例えば、湿度95%RH、CO2濃度5～10%(v/v)での培養が例示されるが、これらに限定されない。また、使用し得る培地も、トランスフェクション用細胞やウイルスベクター産生細胞が生存可能であれば特に限定されない。かかる培地としては、例えば、DMEM、IMDM、DMEM:F-12等の基本合成培地が挙げられるが、これらに限定されない。また、必要に応じてこれらの基本合成培地に対して、自体公知の添加物（ウシ胎児血清、成長因子類、ペプチド類、アミノ酸類）を添加してもよい。また、自体公知の培地成分を適宜改変して用いてもよい。また、細胞の培養形態も特に限定されず、浮遊培養でもよく接着培養でもあってもよい。また、培養時間も特に限定されず、例えば、通常1時間以上であってよく、好ましくは、2時間以上、4時間以上、8時間以上、12時間以上、16時間以上、24時間以上、30時間以上、36時間以上、42時間以上、48時間以上、54時間以上、60時間以上、66時間以上、72時間以上、4日以上、又は5日以上であってよく、これらに限定されない。また、培養時間は、通常10日以下であってよく、例えば、9日以下、8日以下、7日以下、又は6日以下であってよいが、これらに限定されない。一態様において、培養時間は、1時間～10日であり、好ましくは12時間～8日、24時間～6日であるが、これらに限定されない。 In addition, the culture of the cells for transfection or the cells producing a viral vector in the step of applying the first temperature zone can be performed under culture conditions known per se, except for the culture temperature. For example, the culture can be performed under a humidity of 95% RH and a CO2 concentration of 5 to 10% (v/v), but is not limited thereto. In addition, the medium that can be used is not particularly limited as long as the cells for transfection or the cells producing a viral vector can survive. Examples of such a medium include, but are not limited to, basic synthetic media such as DMEM, IMDM, and DMEM:F-12. In addition, additives known per se (fetal bovine serum, growth factors, peptides, amino acids) may be added to these basic synthetic media as necessary. In addition, known medium components may be appropriately modified and used. In addition, the culture form of the cells is not particularly limited, and may be either suspension culture or adhesion culture. The culture time is not particularly limited, and may be, for example, usually 1 hour or more, preferably 2 hours or more, 4 hours or more, 8 hours or more, 12 hours or more, 16 hours or more, 24 hours or more, 30 hours or more, 36 hours or more, 42 hours or more, 48 hours or more, 54 hours or more, 60 hours or more, 66 hours or more, 72 hours or more, 4 days or more, or 5 days or more, but is not limited thereto. The culture time may be usually 10 days or less, and may be, for example, 9 days or less, 8 days or less, 7 days or less, or 6 days or less, but is not limited thereto. In one embodiment, the culture time is 1 hour to 10 days, preferably 12 hours to 8 days, 24 hours to 6 days, but is not limited thereto.
[第2の温度帯]
　本発明のウイルスベクターの生産方法では、第1の温度帯での培養を経験したウイルスベクター産生細胞にウイルスベクターを産生させる工程において、第2の温度帯(30.0～35.9℃)を適用する。 [Second temperature zone]
In the method for producing a viral vector of the present invention, the second temperature zone (30.0 to 35.9°C) is applied in the step of causing viral vector-producing cells that have been cultured in the first temperature zone to produce a viral vector.
　第2の温度帯は、通常30.0～35.9℃であり得る。一態様において、第2の温度帯の下限は、通常30.0℃以上、好ましくは、30.5℃以上、31.0℃以上、31.5℃以上、32.0℃以上、32.5℃以上、33.0℃以上、33.5℃以上、34.0℃以上、又は34.5℃以上であり得るが、これらに限定されない。また、一態様において、第2の温度帯の上限は、通常35.9℃以下、好ましくは、35.8℃以下、35.7℃以下、35.6℃以下、35.5℃以下、35.4℃以下、35.3℃以下、35.2℃以下、35.1℃以下、又は35.0℃以下であり得るが、これらに限定されない。一態様において、第2の温度帯は、通常30.0～35.9℃、31.5～35.8℃、32.0～35.7℃、32.5～35.6℃、33.0～35.5℃、33.5～35.5℃、34.0～35.5℃、34.5～35.5℃、又は35.0～35.5℃であり得るが、これらに限定されない。 The second temperature range may be typically 30.0 to 35.9°C. In one embodiment, the lower limit of the second temperature range may be typically 30.0°C or higher, preferably 30.5°C or higher, 31.0°C or higher, 31.5°C or higher, 32.0°C or higher, 32.5°C or higher, 33.0°C or higher, 33.5°C or higher, 34.0°C or higher, or 34.5°C or higher, but is not limited thereto. In one embodiment, the upper limit of the second temperature range may be typically 35.9°C or lower, preferably 35.8°C or lower, 35.7°C or lower, 35.6°C or lower, 35.5°C or lower, 35.4°C or lower, 35.3°C or lower, 35.2°C or lower, 35.1°C or lower, or 35.0°C or lower, but is not limited thereto. In one embodiment, the second temperature zone may be, but is not limited to, typically 30.0 to 35.9°C, 31.5 to 35.8°C, 32.0 to 35.7°C, 32.5 to 35.6°C, 33.0 to 35.5°C, 33.5 to 35.5°C, 34.0 to 35.5°C, 34.5 to 35.5°C, or 35.0 to 35.5°C.
　第2の温度帯でのウイルスベクター産生細胞の培養は、ウイルスベクター産生細胞が生存し、且つ、ウイルスベクターを産生できる限り、培養温度以外は、自体公知の培養条件を用いることができる。かかる条件は、第1の温度帯において説明したものと同様である。 When culturing the viral vector-producing cells in the second temperature range, culture conditions known per se can be used, except for the culture temperature, as long as the viral vector-producing cells can survive and produce the viral vector. Such conditions are the same as those described for the first temperature range.
　本発明のウイルスベクターの生産方法において、第2の温度帯は、第1の温度帯よりも低いことを特徴とする。一般的なウイルスベクターの製造においては、ウイルスベクター産生細胞を至適温度と考えられる37℃で培養し、ウイルスベクターを生産させる。しかし、本発明においては、かかる温度よりも低い温度帯でウイルスベクター産生細胞にウイルスベクターを生産させることを特徴とする。 In the method for producing a viral vector of the present invention, the second temperature zone is characterized by being lower than the first temperature zone. In general viral vector production, viral vector-producing cells are cultured at 37°C, which is considered to be the optimal temperature, to produce the viral vector. However, in the present invention, the viral vector-producing cells are caused to produce the viral vector at a temperature zone lower than this temperature.
　第2の温度帯を適用してウイルスベクター産生細胞を培養し、ウイルスベクターを産生させる工程を(工程d)として示すとき、本発明のウイルスベクターの生産方法は、次のような実施態様を含み得るがこれらに限定されない。 When the step of culturing viral vector-producing cells by applying the second temperature zone to produce a viral vector is shown as (step d), the method for producing a viral vector of the present invention may include, but is not limited to, the following embodiments.
(実施態様1)
第1の温度帯:(工程a)～(工程c)
第2の温度帯:(工程d) (Embodiment 1)
First temperature zone: (step a) to (step c)
Second temperature zone: (step d)
(実施態様2)
第1の温度帯:(工程b)～(工程c)
第2の温度帯:(工程d) (Embodiment 2)
First temperature zone: (process b) to (process c)
Second temperature zone: (step d)
(実施態様3)
第1の温度帯:(工程c)
第2の温度帯:(工程d) (Embodiment 3)
First temperature zone: (step c)
Second temperature zone: (step d)
(実施態様4)
第1の温度帯:(工程a)～(工程b)
第2の温度帯：(工程d) (Embodiment 4)
First temperature zone: (step a) to (step b)
Second temperature zone: (step d)
(実施態様5)
第1の温度帯:(工程b)
第2の温度帯：(工程d) (Embodiment 5)
First temperature zone: (step b)
Second temperature zone: (step d)
（実施形態6）
第１の温度帯:（工程a）
第２の温度帯:（工程d） (Embodiment 6)
First temperature zone: (step a)
Second temperature zone: (step d)
　本発明のウイルスベクターの生産方法を用いて生産されるウイルスベクターとしては、特に限定されないが、レトロウイルス、レンチウイルス、センダイウイルス、及びヘルペスウイルス等のエンベロープを持つウイルス、又は、アデノウイルス、アデノ随伴ウイルス(AAV)等のエンベロープを持たないウイルス（以下、非エンベロープウイルス）に由来するウイルスベクターが例示される。 The viral vectors produced using the viral vector production method of the present invention are not particularly limited, but examples include viral vectors derived from enveloped viruses such as retroviruses, lentiviruses, Sendai viruses, and herpes viruses, or non-enveloped viruses such as adenoviruses and adeno-associated viruses (AAV) (hereinafter referred to as non-enveloped viruses).
　一態様において、ウイルスベクターは、アデノウイルス、パルボウイルス、パポバウイルス、パピローマウイルス等のDNAウイルスに由来するウイルスベクターであってよい。また、別の一態様において、ウイルスベクターは、ロタウイルス、コクサッキーウイルス、エンテロウイルス、サポウイルス、ノロウイルス、ポリオウイルス、エコーウイルス、A型肝炎ウイルス、E型肝炎ウイルス、ライノウイルス、アストロウイルス等のRNAウイルスに由来するウイルスベクターであってもよい。本発明の好ましい一態様において、ウイルスベクターはレトロウイルス科のウイルス、アデノウイルス科のウイルス、又は、パルボウイルス科のウイルスに由来するウイルスベクターであり得る。また、本発明のより好ましい一態様において、ウイルスベクターは、パルボウイルス科のアデノ随伴ウイルス（AAV）であり得る。 In one embodiment, the viral vector may be a viral vector derived from a DNA virus such as adenovirus, parvovirus, papovavirus, or papillomavirus. In another embodiment, the viral vector may be a viral vector derived from an RNA virus such as rotavirus, coxsackievirus, enterovirus, sapovirus, norovirus, poliovirus, echovirus, hepatitis A virus, hepatitis E virus, rhinovirus, or astrovirus. In a preferred embodiment of the present invention, the viral vector may be a viral vector derived from a virus of the Retroviridae family, a virus of the Adenoviridae family, or a virus of the Parvoviridae family. In a more preferred embodiment of the present invention, the viral vector may be an adeno-associated virus (AAV) of the Parvoviridae family.
　AAVはエンベロープを持たない正20面体の外殻（キャプシド）とその内部に1本の線状一本鎖DNAを有する。キャプシドは3つのキャプシドタンパク質（VP1、VP2、及びVP3）を有する。本明細書において、AAVは野生型ウイルス及びその派生物を含み、特に記載する場合を除き全ての血清型及びクレードを含む。AAVの血清型については種々の報告があるが、ヒトに感染するAAVの血清型としては、少なくともAAV1、AAV2、AAV3A、AAV3b、AAV4、AAV5、AAV6、AAV7、AAV8、AAV9、AAV10、AAVrh.10、AAV11、AAV12及びAAV13の15種類が知られている。 AAV has a non-enveloped icosahedral outer shell (capsid) with a single linear single-stranded DNA inside. The capsid has three capsid proteins (VP1, VP2, and VP3). In this specification, AAV includes wild-type viruses and their derivatives, and includes all serotypes and clades unless otherwise specified. There are various reports on AAV serotypes, but at least 15 types of AAV serotypes that infect humans are known: AAV1, AAV2, AAV3A, AAV3b, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAVrh.10, AAV11, AAV12, and AAV13.
　本発明のウイルスベクターの生産方法の好ましい一態様において、本発明のウイルスベクターの生産方法では、第1の温度帯及び/又は第2の温度帯を適用した細胞培養が、有機溶媒を含む培地で行われることを特徴とする。好ましい一態様において、ウイルスベクター産生細胞を第2の温度帯(即ち、30.0～35.9℃)で培養し、ウイルスベクターを産生させる工程において、有機溶媒を含む培地が用いられる。 In a preferred embodiment of the method for producing a viral vector of the present invention, the cell culture in the first temperature zone and/or the second temperature zone is carried out in a medium containing an organic solvent. In a preferred embodiment, a medium containing an organic solvent is used in the step of culturing viral vector-producing cells in the second temperature zone (i.e., 30.0 to 35.9°C) and producing a viral vector.
　かかる有機溶媒としては、アルコール類又はジメチルスルホキシド（DMSO）が挙げられ、好ましくはアルコール類である。 Such organic solvents include alcohols or dimethyl sulfoxide (DMSO), and are preferably alcohols.
　アルコールとは、炭化水素の水素原子をヒドロキシ基に置き換えた物質で本願明細書ではそれらを総称してアルコール類とする。アルコール類としては好ましくは炭素数1～6の低級アルコールであり、例えば、メタノール、エタノール、イソプロパノールおよびグリセロール(グリセリン)からなる群から選ばれる少なくとも1種である。DMSOは市販されている。 Alcohol is a substance in which the hydrogen atoms of a hydrocarbon have been replaced with hydroxyl groups, and in this specification they are collectively referred to as alcohols. The alcohol is preferably a lower alcohol having 1 to 6 carbon atoms, such as at least one selected from the group consisting of methanol, ethanol, isopropanol, and glycerol (glycerin). DMSO is commercially available.
　培地に添加される有機溶媒の量は、本発明の所望の効果が得られる限り特に限定されない。 The amount of organic solvent added to the medium is not particularly limited as long as the desired effect of the present invention is obtained.
　一態様において、有機溶媒がアルコール類（例、エタノール）である場合は、有機溶媒は、培地組成物全体に対して、通常2V/V％以下の濃度となるように添加することができる。当該上限値以下であれば、細胞が死滅することなく、本発明の効果（即ち、細胞内で生産されるウイルスゲノムの産生量及び／又はウイルスゲノムの完全性を向上させる効果等）を得ることができる。有機溶媒の反応混合物全量における添加量の下限は本発明の効果が得られる限り特に限定されないが、0V/V%を超える濃度である。好ましくは、前記反応混合物全量に対して、0.05V/V%以上、0.06V/V%以上、0.07V/V%以上、0.08V/V%以上、0.09V/V%以上、0.1V/V%以上、0.11V/V%以上、0.12V/V%以上、0.13V/V%以上、0.14V/V%以上、0.15V/V%以上、0.16V/V%以上、0.17V/V%以上、0.18V/V%以上、0.19V/V%以上、又は0.2V/V%以上となるよう使用される。一態様において、有機溶媒の添加量は、通常0.05～2V/V%、好ましくは0.1～1.5V/V%、0.2～1V/V%、0.2～0.56V/V%であり得るが、これらに限定されない。尚、有機溶媒の使用量は、有機溶媒や細胞の種類、培養条件等に応じて適宜変更してもよい。 In one embodiment, when the organic solvent is an alcohol (e.g., ethanol), the organic solvent can be added to the entire medium composition at a concentration of typically 2 V/V% or less. If the concentration is below this upper limit, the cells will not die and the effects of the present invention (i.e., the effect of improving the production amount of viral genome produced in the cells and/or the integrity of the viral genome, etc.) can be obtained. There is no particular limit to the lower limit of the amount of organic solvent added to the total reaction mixture as long as the effects of the present invention can be obtained, but it is a concentration of more than 0 V/V%. Preferably, the organic solvent is used in an amount of 0.05 V/V% or more, 0.06 V/V% or more, 0.07 V/V% or more, 0.08 V/V% or more, 0.09 V/V% or more, 0.1 V/V% or more, 0.11 V/V% or more, 0.12 V/V% or more, 0.13 V/V% or more, 0.14 V/V% or more, 0.15 V/V% or more, 0.16 V/V% or more, 0.17 V/V% or more, 0.18 V/V% or more, 0.19 V/V% or more, or 0.2 V/V% or more, based on the total amount of the reaction mixture. In one embodiment, the amount of the organic solvent added is usually 0.05 to 2 V/V%, preferably 0.1 to 1.5 V/V%, 0.2 to 1 V/V%, or 0.2 to 0.56 V/V%, but is not limited thereto. The amount of organic solvent used may be changed as appropriate depending on the organic solvent, cell type, culture conditions, etc.
　一態様において、有機溶媒はエタノールであり、ウイルスベクター産生細胞を第2の温度帯(即ち、30.0～35.9℃)で培養し、ウイルスベクターを産生させる工程の開始前、開始時点又は開始後24時間以内（例、30分以内、1時間以内、2時間以内、4時間以内、6時間以内、8時間以内、10時間以内、12時間以内、16時間以内、又は20時間以内など）に添加され得る。 In one embodiment, the organic solvent is ethanol, which may be added before, at the start of, or within 24 hours (e.g., within 30 minutes, within 1 hour, within 2 hours, within 4 hours, within 6 hours, within 8 hours, within 10 hours, within 12 hours, within 16 hours, or within 20 hours) after the start of the process of culturing the viral vector-producing cells in the second temperature zone (i.e., 30.0 to 35.9°C) and producing the viral vector.
　或いは、別の一態様において、有機溶媒は、(工程a)の開始前、開始時点又は開始後24時間以内（例、30分以内、1時間以内、2時間以内、4時間以内、6時間以内、8時間以内、10時間以内、12時間以内、16時間以内、又は20時間以内など）に添加され得る。 Alternatively, in another embodiment, the organic solvent may be added prior to, at the start of, or within 24 hours after the start of (step a) (e.g., within 30 minutes, within 1 hour, within 2 hours, within 4 hours, within 6 hours, within 8 hours, within 10 hours, within 12 hours, within 16 hours, or within 20 hours, etc.).
　或いは、別の一態様において、有機溶媒は、(工程b)の開始前、開始時点又は開始後24時間以内（例、30分以内、1時間以内、2時間以内、4時間以内、6時間以内、8時間以内、10時間以内、12時間以内、16時間以内、又は20時間以内など）に添加され得る。 Alternatively, in another embodiment, the organic solvent can be added prior to, at the start of, or within 24 hours after the start of (step b) (e.g., within 30 minutes, within 1 hour, within 2 hours, within 4 hours, within 6 hours, within 8 hours, within 10 hours, within 12 hours, within 16 hours, or within 20 hours, etc.).
　或いは、別の一態様において、有機溶媒は、(工程c)の開始前、開始時点又は開始後24時間以内（例、30分以内、1時間以内、2時間以内、4時間以内、6時間以内、8時間以内、10時間以内、12時間以内、16時間以内、又は20時間以内など）に添加され得る。 Alternatively, in another embodiment, the organic solvent can be added prior to, at the start of, or within 24 hours after the start of (step c) (e.g., within 30 minutes, within 1 hour, within 2 hours, within 4 hours, within 6 hours, within 8 hours, within 10 hours, within 12 hours, within 16 hours, or within 20 hours, etc.).
2．感染力価が向上したウイルスベクターの生産効率を向上させる方法
　本発明はまた、ウイルスベクター産生細胞を30.0～35.9℃で培養する工程を含む、感染力価が向上したウイルスベクターの生産効率を向上させる方法であって、ここで、当該ウイルスベクター産生細胞が、ウイルスベクターを産生させる工程の開始前に、36.0～38.0℃の温度下で培養されていることを特徴とする、方法（以下、「本発明のウイルスベクターの生産効率向上方法」と称することがある）を提供する。 2. Method for improving the production efficiency of a viral vector with an improved infectious titer The present invention also provides a method for improving the production efficiency of a viral vector with an improved infectious titer, which comprises a step of culturing viral vector-producing cells at 30.0 to 35.9°C, characterized in that the viral vector-producing cells are cultured at a temperature of 36.0 to 38.0°C before the start of the step of producing the viral vector (hereinafter, sometimes referred to as the "method for improving the production efficiency of a viral vector of the present invention").
　本発明のウイルスベクターの生産効率向上方法において用いられる各種条件等は、本発明のウイルスベクターの生産方法において説明したものと同様である。
　本発明のウイルスベクターの生産効率向上方法において調製されるウイルスベクターは、感染力価が向上していることを特徴とする。ここで、「感染力価が向上した」とは、ウイルスベクターを産生させる前後において一貫して約37℃（例えば、36.0～38.0℃）で培養されたウイルスベクター産生細胞が（換言すれば、第1の温度帯から第2の温度帯への温度シフトを経験していないウイルスベクター産生細胞が）産生したウイルスベクターの感染力価と比較して、感染力価が向上していることを意味するものとする。 The various conditions used in the method for improving the production efficiency of a viral vector of the present invention are the same as those explained in the method for producing a viral vector of the present invention.
The viral vector prepared by the method for improving viral vector production efficiency of the present invention is characterized by having an improved infectious titer. Here, "improved infectious titer" means that the infectious titer is improved compared to the infectious titer of a viral vector produced by viral vector-producing cells that have been consistently cultured at about 37°C (e.g., 36.0 to 38.0°C) before and after viral vector production (in other words, viral vector-producing cells that have not experienced a temperature shift from the first temperature zone to the second temperature zone).
3．ウイルスベクター産生細胞を用いたウイルスベクターの製造において、製造される全ウイルス粒子に対してのウイルスゲノムの完全体を含むウイルス粒子の割合を向上させる方法
　本発明はまた、ウイルスベクター産生細胞を30.0～35.9℃で培養し、ウイルスベクターを産生させる工程を含む、ウイルスベクター産生細胞を用いたウイルスベクターの製造において、製造される全ウイルス粒子に対してのウイルスゲノムの完全体を含むウイルス粒子の割合（Ｆｕｌｌ率とも称する）を向上させる方法であって、ここで、当該ウイルスベクター産生細胞が、ウイルスベクターを産生させる工程の開始前に、36.0～38.0℃の温度下で培養されていることを特徴とする、方法（以下、「本発明のウイルス粒子のFull率を向上させる方法」と称することがある）を提供する。 3. A method for improving the ratio of viral particles containing a complete viral genome to all viral particles produced in the production of a viral vector using viral vector-producing cells The present invention also provides a method for improving the ratio of viral particles containing a complete viral genome ( also referred to as the Full rate) to all viral particles produced in the production of a viral vector using viral vector-producing cells, which comprises the steps of culturing viral vector-producing cells at 30.0 to 35.9°C to produce a viral vector, characterized in that the viral vector-producing cells are cultured at a temperature of 36.0 to 38.0°C before the start of the step of producing the viral vector (hereinafter, this method may be referred to as the "method of the present invention for improving the Full rate of viral particles").
　本発明のウイルス粒子のFull率を向上させる方法において用いられる各種条件等は、本発明のウイルスベクターの生産方法において説明したものと同様である。
　尚、本発明のウイルス粒子のFull率を向上させる方法における「製造される全ウイルス粒子に対してのウイルスゲノムの完全体を含むウイルス粒子の割合」の向上とは、ウイルスベクターを産生させる前後において一貫して約37℃（例えば、36.0～38.0℃）で培養されたウイルスベクター産生細胞（換言すれば、第1の温度帯から第2の温度帯への温度シフトを経験していないウイルスベクター産生細胞）を用いた場合に得られる当該割合と比較しての向上を意味するものとする。 The various conditions used in the method of the present invention for improving the full rate of virus particles are the same as those explained in the method of the present invention for producing a virus vector.
In the method of the present invention for improving the full rate of virus particles, the improvement in "the ratio of virus particles containing a complete viral genome to all virus particles produced" refers to an improvement compared to the said ratio obtained when using virus vector-producing cells that have been consistently cultured at about 37°C (e.g., 36.0 to 38.0°C) before and after production of the virus vector (in other words, virus vector-producing cells that have not experienced a temperature shift from the first temperature zone to the second temperature zone).
　以下の実施例において本発明を更に具体的に説明するが、本発明はこれらの例によってなんら限定されるものではない。 The present invention will be explained in more detail in the following examples, but the present invention is not limited to these examples.
[実施例1]ウイルスベクターの生産に対して培養温度が与える影響の検討1
　ヒト由来細胞によるAAVベクターの生産時の培養温度が、生産されるAAVベクターに対して与える影響を検討した。具体的な試験手順を以下に示す。 [Example 1] Examination of the effect of culture temperature on viral vector production 1
We investigated the effect of the culture temperature during AAV vector production using human-derived cells on the AAV vector produced. The specific test procedure is as follows.
1.ヒト由来細胞によるウイルスベクターの生産
　浮遊化したHEK293細胞を、浮遊細胞用の完全合成培地(GMEP社、HE400AZ）を入れた三角フラスコにて、3～4日間振とう培養（37℃、8%CO2）し、2.2～3.5x106 Viable Cells/mLまで増殖させた。得られた細胞培養液を無菌的に回収した後、遠心分離により細胞ペレットを回収した。細胞ペレットをAAV2生産に適した細胞濃度の半分程度となるように、フレッシュな完全合成培地で再懸濁し、三角フラスコに移した後、振とう培養した(37℃、8%CO2)。22～26時間後、GFPを発現するAAV2生産用の以下(i)～(iii)のプラスミドを、トランスフェクション試薬(Polyplus社、FectoVIR-AAV)と共に培養液に添加し、トランスフェクションした。
(i)AAV2のRepタンパク質及びCapタンパク質をコードするプラスミド(タカラバイオ社、ｐRC2-mi342 Vector)
(ii)アデノウイルスのE2A配列、VA配列、E4配列を含むプラスミド(タカラバイオ社、pHelper Vector)
(iii)AAV2の2つのITRの間に蛍光タンパク質GFPの発現カセットを含むプラスミド(CELL BIOLABS社、pAAV-GFP)
　トランスフェクション後は、培養温度を35℃に下げ、3日間AAV2を生産させた。 1. Production of viral vectors in human-derived cells Suspended HEK293 cells were cultured with shaking (37°C, 8% CO2 ) for 3-4 days in an Erlenmeyer flask containing a complete synthetic medium for suspension cells (GMEP, HE400AZ) and grown to 2.2-3.5x106 viable cells/mL. The resulting cell culture medium was collected aseptically and the cell pellet was collected by centrifugation. The cell pellet was resuspended in fresh complete synthetic medium to about half the cell concentration suitable for AAV2 production, transferred to an Erlenmeyer flask, and cultured with shaking (37°C, 8% CO2 ). After 22-26 hours, the following plasmids (i) to (iii) for AAV2 production expressing GFP were added to the culture medium together with a transfection reagent (Polyplus, FectoVIR-AAV) for transfection.
(i) A plasmid encoding the Rep and Cap proteins of AAV2 (Takara Bio, pRC2-mi342 Vector)
(ii) A plasmid containing the adenovirus E2A, VA, and E4 sequences (Takara Bio, pHelper Vector)
(iii) A plasmid containing an expression cassette for the fluorescent protein GFP between the two ITRs of AAV2 (CELL BIOLABS, pAAV-GFP).
After transfection, the culture temperature was lowered to 35°C and AAV2 was produced for 3 days.
2.細胞密度及び生存率測定
　細胞培養時、及びAAV2生産時の培養液中の細胞密度、及び細胞生存率は、NucleoCounter(chemometec社、NC-3000)を用いて測定した。各条件におけるAAV2生産時(トランスフェクション後)の細胞密度、及び細胞生存率の推移を図１(A)及び(B)に示す。 2. Measurement of cell density and viability The cell density and cell viability in the culture medium during cell culture and AAV2 production were measured using a NucleoCounter (Chemometec, NC-3000). The changes in cell density and cell viability during AAV2 production (after transfection) under each condition are shown in Figure 1 (A) and (B).
3.ウイルスベクター抽出
　トランスフェクションから3日後、AAV2を生産したHEK293細胞培養液を採取し、遠心分離により培養上清と細胞ペレットを分離後、培養上清を-80℃にて凍結した。細胞ペレットは、市販のＡＡＶ抽出キット(Takara社、AAVpro Extraction Solution)を用い、キットの手順に従ってAAV2を抽出後、抽出液を-80℃にて凍結した。 3. Viral Vector Extraction Three days after transfection, the HEK293 cell culture medium that produced AAV2 was collected, and the culture supernatant and cell pellet were separated by centrifugation, and the culture supernatant was frozen at -80°C. AAV2 was extracted from the cell pellet using a commercially available AAV extraction kit (Takara, AAVpro Extraction Solution) according to the kit's instructions, and the extract was frozen at -80°C.
4.ウイルスベクター定量
　培養上清中、及び産生細胞から抽出したAAV2を、以下に示す手順により、Dropret Digital PCR(ddPCR)法で定量した。培養上清、及び産生細胞からのAAV2抽出液にDNaseを添加して、測定サンプル中のAAV2粒子外に存在する核酸を消化した後、DNaseの活性を停止した。続けてProteinase Kにて、AAV2のカプシドタンパク質を分解し、AAV2粒子内に包埋されているAAVゲノムを抽出した後、Proteinase Kを不活化させてAAV2ゲノム抽出液を得た。得られたAAV2ゲノム抽出液に対して、AAV2ゲノムのITR(Inverted Terminal Repeat)領域、及びGFP領域の塩基配列を増幅する2種類のプライマーを用いた2D-ddPCRを行い、サンプル中のAAV2の産生量を絶対定量した。また、ITR領域、及びGFP領域の両方を検出したドロップレットの割合から、各条件で産生したAAV2について、AAV2ゲノムの完全体を含むAAV2の割合を算出した。トランスフェクション後の温度を、37℃でAAV2を生産させた時に対して、35℃でAAV2を生産させた際の、細胞抽出液中のAAV2の濃度の相対値を図2(A)に、AAV2ゲノムの完全体を含むAAV2の割合を図2(B)に示す。 4. Quantification of viral vectors The AAV2 extracted from the culture supernatant and the producing cells was quantified by droplet digital PCR (ddPCR) method according to the following procedure. DNase was added to the culture supernatant and the AAV2 extract from the producing cells to digest the nucleic acid present outside the AAV2 particles in the measurement sample, and then the activity of DNase was stopped. Subsequently, the capsid protein of AAV2 was decomposed with Proteinase K, the AAV genome embedded in the AAV2 particles was extracted, and then Proteinase K was inactivated to obtain an AAV2 genome extract. The obtained AAV2 genome extract was subjected to 2D-ddPCR using two types of primers that amplify the base sequence of the ITR (Inverted Terminal Repeat) region and the GFP region of the AAV2 genome, and the production amount of AAV2 in the sample was absolutely quantified. In addition, the percentage of AAV2 containing the complete AAV2 genome was calculated for AAV2 produced under each condition from the percentage of droplets that detected both the ITR region and the GFP region. Figure 2(A) shows the relative AAV2 concentration in the cell extract when AAV2 was produced at 35°C compared to when the temperature after transfection was 37°C, and Figure 2(B) shows the percentage of AAV2 containing the complete AAV2 genome.
5.ウイルスベクター由来粒子数の定量
　培養上清中、及び産生細胞から抽出したAAV2の粒子（AAV2ゲノムを含む粒子と、含まない粒子全体）は、市販のAAV2 ELISAキット(PROGENE社、AAV2 Titration ELISA)を用いて、キットの手順に従って測定した。ELISA法により定量したAAV2の全粒子数と、ddPCRから得られたAAV2ゲノムの完全体を含む粒子の定量値から、AAV2ゲノムの完全体を含むAAV2粒子の割合(Full率に相当）を算出した。トランスフェクション後37℃でAAV2を生産させた場合に対する35℃でAAV2を生産させた場合の、細胞抽出液中のAAV2のFull率の相対値を図2(C)に示す。 5. Quantification of the number of particles derived from the viral vector The AAV2 particles (particles containing the AAV2 genome and all particles not containing the AAV2 genome) extracted from the culture supernatant and the producing cells were measured using a commercially available AAV2 ELISA kit (PROGENE, AAV2 Titration ELISA) according to the kit's procedure. The percentage of AAV2 particles containing the complete AAV2 genome (equivalent to the Full rate) was calculated from the total number of AAV2 particles quantified by ELISA and the quantitative value of particles containing the complete AAV2 genome obtained by ddPCR. The relative value of the Full rate of AAV2 in the cell extract when AAV2 was produced at 35°C compared to when AAV2 was produced at 37°C after transfection is shown in Figure 2(C).
　図1(A)及び(B)の結果から、AAV2生産時の温度を35℃に下げることによって、コントロール条件の37℃と比べて、細胞の活性低下が抑制されており、トランスフェクション後48～72時間の生存率が向上した。また、細胞の増殖も継続していた。また、図2(A)、(B)及び(C)の結果から、生産時の温度を35℃に下げることによって、コントロール条件(37℃)と比べてAAV2の生産性が向上していた。温度を下げたことによって、AAV2内にパッケージングされたゲノムの完全体の割合への影響は無く、一方で、ゲノムがパッケージングされたAAV2粒子の割合は、コントロール条件と比べて増加した。図1(A)及び(B)、並びに、図2(A)、(B)及び(C)の結果から、ウイルスベクター生産時に適切な温度範囲で培養することにより、細胞の生存や増殖に必要な活性をより向上させ、且つ、AAV2の生産に必要な代謝活性を向上させることができた。 　The results in Figures 1(A) and (B) show that lowering the temperature during AAV2 production to 35°C suppressed the decrease in cell activity compared to the control condition of 37°C, and improved the survival rate 48 to 72 hours after transfection. Cell proliferation also continued. The results in Figures 2(A), (B), and (C) show that lowering the temperature during production to 35°C improved AAV2 productivity compared to the control condition (37°C). Lowering the temperature did not affect the proportion of complete genomes packaged within AAV2, while the proportion of AAV2 particles with genomes packaged increased compared to the control condition. The results in Figures 1(A) and (B) and Figures 2(A), (B), and (C) show that culturing at an appropriate temperature range during viral vector production further improved the activities required for cell survival and proliferation, and also improved the metabolic activity required for AAV2 production.
[実施例2]ウイルスベクターの生産に対して培養温度が与える影響の検討2
　ウイルスベクター産生細胞の調製からウイルスベクターの生産までを一貫して35℃で行う以外は実施例1と同様の条件を用いてウイルスベクター産生細胞にウイルスベクターを産生させ、生産されたAAVウイルスベクターの量を測定した。結果を図3に示す。 [Example 2] Examination of the effect of culture temperature on viral vector production 2
The viral vector-producing cells were allowed to produce the viral vector under the same conditions as in Example 1, except that all steps from preparation of the viral vector-producing cells to production of the viral vector were carried out at 35° C., and the amount of the produced AAV viral vector was measured. The results are shown in FIG.
　図3に示される通り、ウイルスベクター産生細胞の調製からウイルスベクター産生細胞によるウイルスベクターの産生までの工程を一貫して35℃で行った場合、コントロール条件（一貫して37℃）と比べてウイルスベクターの総生産量は低下した。 As shown in Figure 3, when the entire process from preparation of viral vector-producing cells to production of viral vectors by the viral vector-producing cells was consistently performed at 35°C, the total amount of viral vector produced was reduced compared to the control condition (consistently at 37°C).
　以上の結果から、ウイルスベクター産生細胞によるウイルスベクターの産生効率を高めるためには、当該細胞に2つの温度帯を経験させることが必要であることが示された。 These results show that in order to increase the efficiency of viral vector production by viral vector-producing cells, it is necessary for the cells to experience two temperature zones.
[実施例3]ウイルスベクター産生時の有機溶媒(エタノール)の添加の影響の検討1
　トランスフェクション後に温度を変えてAAV2の生産を行う操作までを、実施例1と同様の手順で行い、トランスフェクションから約4時間後、培養液量に対して0.56v/v%のろ過済みエタノールを添加し、3日間AAV2を生産させた。
　AAV2生産時に37℃、且つエタノールを添加しなかった条件(コントロール条件）に対して、35℃でエタノールを添加した際の、細胞抽出液中のAAV2の濃度の相対値を図4(A)に、細胞抽出液中に含まれるAAV2のうち、完全体のゲノムを含む割合を図4(B)に示す。更に、各生産条件において細胞抽出液中に含まれるAAV2のFull率のコントロール条件に対する相対値を図4(C)に示す。また、各生産条件における、AAV2生産時（トランスフェクション後）の細胞密度、及び細胞生存率の推移を図5(A)及び図5(B)に示す。尚、いずれの分析も実施例1と同様の手順で行った。 [Example 3] Examination of the effect of adding an organic solvent (ethanol) during viral vector production 1
The procedure up to the point where the temperature was changed after transfection to produce AAV2 was the same as in Example 1. Approximately 4 hours after transfection, 0.56 v/v% filtered ethanol was added to the culture medium volume, and AAV2 was produced for 3 days.
FIG. 4(A) shows the relative concentration of AAV2 in the cell extract when ethanol was added at 35°C, compared to the control condition (37°C and no ethanol was added) during AAV2 production, and FIG. 4(B) shows the ratio of AAV2 in the cell extract that contains the complete genome. Furthermore, FIG. 4(C) shows the relative value of the full rate of AAV2 contained in the cell extract under each production condition compared to the control condition. In addition, FIG. 5(A) and FIG. 5(B) show the transition of cell density and cell viability during AAV2 production (after transfection) under each production condition. Note that all analyses were performed in the same manner as in Example 1.
　図4(A)、(B)及び(C)の結果から、生産時の温度を35℃に下げ、更に0.56v/v%のエタノールを添加することによって、AAV2の生産性、AAV2内にパッケージングされたゲノム完全体の割合、ゲノムがパッケージングされたAAV2粒子の割合のいずれも温度を下げただけの時以上に向上した。また、図5(A)及び(B)の結果から、生産時の温度を35℃に下げ、更に0.56v/v%のエタノールを添加することによって、更に細胞の生存率が向上した。図4(A)、(B)及び(C)並びに図5(A)及び(B)の結果から、ウイルスベクター生産時に適切な温度範囲で培養することに加えてエタノールを添加したことで、細胞の生存や増殖に必要な活性をより向上させ、且つ、AAV2の生産に必要な代謝活性を向上させることができた。 The results of Figures 4(A), (B), and (C) show that lowering the temperature during production to 35°C and adding 0.56 v/v% ethanol improved AAV2 productivity, the percentage of complete genomes packaged within AAV2, and the percentage of AAV2 particles with genomes packaged, more than when the temperature was lowered alone. Furthermore, the results of Figures 5(A) and (B) show that lowering the temperature during production to 35°C and adding 0.56 v/v% ethanol further improved cell viability. The results of Figures 4(A), (B), and (C) and Figures 5(A) and (B) show that adding ethanol in addition to culturing at an appropriate temperature range during viral vector production further improved the activities required for cell survival and proliferation, and also improved the metabolic activity required for AAV2 production.
　また、ウイルスベクター産生細胞から抽出したAAV2を、培養したHeLa細胞にゲノムタイター(vg)を揃えて添加し、3日後の細胞のうち、添加したAAV2に由来するGFPタンパク質を発現している細胞数あたり(Transduction unit : TU)に必要なAAV2の量（vg/TU:感染力価）を算出した。結果を以下の表1に示す。 In addition, AAV2 extracted from viral vector-producing cells was added to cultured HeLa cells at the same genome titer (vg), and the amount of AAV2 required per number of cells expressing GFP protein derived from the added AAV2 (transduction unit: TU) three days later was calculated. The results are shown in Table 1 below.
　表１の結果から、AAV2生産時の温度を37℃から35℃に下げることで、形質導入細胞を得るために必要なゲノムタイターの値が低下した。これは、温度を下げることで、生産されたAAV2の細胞感染性（感染力価）が向上しており、品質の高いAAV2ウイルスベクターが生産できたことを示す。 The results in Table 1 show that lowering the temperature during AAV2 production from 37°C to 35°C reduced the genome titer required to obtain transduced cells. This indicates that lowering the temperature improved the cell infectivity (infectious titer) of the produced AAV2, enabling the production of high-quality AAV2 viral vectors.
[実施例4]ウイルスベクター産生時の有機溶媒(エタノール)の添加の影響の検討2
　実施例1におけるウイルスベクター産生細胞の培養と同様の手順で、三角フラスコにて細胞を増殖させて得られた細胞培養液を無菌的に回収した後、遠心分離により細胞ペレットを回収した。細胞ペレットをAAV2生産に適した細胞濃度の半分程度となるように、フレッシュな完全合成培地で再懸濁し、250mL容量の培養槽に移送したのち、通気、撹拌培養した(37℃、pH7.2±0.2、溶存酸素>40%）。20～28時間後、実施例1におけるトランスフェクションと同様の手順で、培養槽にAAV2生産に必要なプラスミドをトランスフェクションした。トランスフェクション後は、一部の培養槽は、培養温度を35℃に下げて、更にそのうちの一部の培養槽については、トランスフェクションから約4時間後に、培養液量に対して、0.56v/v%のろ過済みエタノールを添加し、3日間AAV2を生産させた。AAV2生産時に37℃、且つエタノールを添加しなかった条件(コントロール条件)に対して、各生産条件における、細胞抽出液中のAAV2の濃度の相対値を図6(A)に、各生産条件において細胞抽出液中に含まれるAAV2のうち、完全体のゲノムを含む割合を図6(B)に示す。更に、各生産条件において細胞抽出液中に含まれるAAV2のFull率のコントロール条件に対する相対値を図6(C)に示す。いずれの分析も実施例1と同様の手順で分析を行った。 [Example 4] Examination of the effect of adding an organic solvent (ethanol) during viral vector production 2
The same procedure as in the culture of the viral vector producing cells in Example 1 was used to grow the cells in an Erlenmeyer flask, and the resulting cell culture solution was collected aseptically, and then the cell pellet was collected by centrifugation. The cell pellet was resuspended in fresh synthetic medium to about half the cell concentration suitable for AAV2 production, and transferred to a 250 mL culture tank, and then aerated and cultured with stirring (37°C, pH 7.2±0.2, dissolved oxygen >40%). After 20 to 28 hours, the culture tank was transfected with a plasmid required for AAV2 production using the same procedure as in the transfection in Example 1. After transfection, the culture temperature of some culture tanks was lowered to 35°C, and for some of the culture tanks, 0.56 v/v% filtered ethanol was added to the culture solution volume about 4 hours after transfection, and AAV2 was produced for 3 days. The relative values of the AAV2 concentration in the cell extract under each production condition, compared to the condition (control condition) where AAV2 was produced at 37°C and ethanol was not added, are shown in Figure 6(A), and the proportion of AAV2 containing a complete genome in the cell extract under each production condition is shown in Figure 6(B). Furthermore, the relative values of the full rate of AAV2 contained in the cell extract under each production condition compared to the control condition are shown in Figure 6(C). All analyses were performed using the same procedure as in Example 1.
　図6(A)、(B)及び(C)の結果から、培養槽を用いたAAV2の生産においても、生産時の温度を35℃に下げ、更に0.56v/v%のエタノールを添加することにより、AAV2の生産性、AAV2内にパッケージングされたゲノム完全体の割合、ゲノムがパッケージングされたAAV2粒子の割合のいずれについてもコントロール条件よりも向上した。 The results in Figures 6(A), (B), and (C) show that, even in the production of AAV2 using a culture tank, lowering the production temperature to 35°C and adding 0.56 v/v% ethanol improved AAV2 productivity, the percentage of complete genomes packaged within AAV2, and the percentage of AAV2 particles with genomes packaged, compared to the control conditions.
[実施例5]有機溶媒の検討1
　有機溶媒をエタノールからグリセロール、メタノール、イソプロパノールに変更したこと、及び、ウイルスベクター産生細胞にウイルスベクターを産生させる際の温度を37℃としたこと以外は、実施例3と同様の手順でウイルスベクターの産生、及び分析を行った際の、トランスフェクション3日後の産生細胞中のAAV2の産生量、及び産生したAAV2粒子中のゲノムの完全性に関する測定結果を表2に示す。 [Example 5] Examination of organic solvents 1
Viral vectors were produced and analyzed in the same manner as in Example 3, except that the organic solvent was changed from ethanol to glycerol, methanol, and isopropanol, and the temperature when the viral vector was produced in the viral vector-producing cells was 37°C. The results of measurement of the amount of AAV2 produced in the producing cells 3 days after transfection and the integrity of the genome in the produced AAV2 particles are shown in Table 2.
　表2に示される通り、エタノール以外のアルコール類であっても、産生されたAAV2内のゲノム完全性の向上、及びゲノムタイター（生産性）の向上に効果があることが分かった。 As shown in Table 2, it was found that alcohols other than ethanol were also effective in improving the genome integrity and genome titer (productivity) of the produced AAV2.
[実施例6]有機溶媒の検討2
　有機溶媒をエタノールからジメチルスルホキシド(DMSO)に変更したこと、及び、ウイルスベクター産生細胞にウイルスベクターを産生させる際の温度を37℃としたこと以外は、実施例3と同様の手順でウイルスベクターの産生、及び分析を行った際の、トランスフェクション3日後の培養上清、及び、細胞中のAAV2の生産性に関する結果を表3に示す。 [Example 6] Examination of organic solvents 2
Viral vectors were produced and analyzed in the same manner as in Example 3, except that the organic solvent was changed from ethanol to dimethyl sulfoxide (DMSO) and the temperature for producing the viral vector in the viral vector-producing cells was 37°C. The results of AAV2 productivity in the culture supernatant and cells 3 days after transfection are shown in Table 3.
　表3に示される通り、DMSOは産生されたAAV2内のゲノム完全性の向上及びゲノムタイター（生産性）を向上させた。加えて、DMSOは、VCDやViabilityに関しても好ましい効果を有することが示された。 As shown in Table 3, DMSO improved the genome integrity and genome titer (productivity) in the produced AAV2. In addition, DMSO was shown to have favorable effects on VCD and viability.
[実施例7]AAVセロタイプの検討
　AAV2のCapタンパク質をコードするプラスミドを、AAV1又はAAV6のCapタンパク質をコードするプラスミドに変更したこと、及び、ウイルスベクター産生細胞にウイルスベクターを産生させる際の温度を37℃としたこと以外は、実施例3と同様の手順でウイルスベクターの産生及び分析を行った。トランスフェクション3日後の培養上清、及び細胞中のAAV1及びAAV6の生産性に関する結果を表4に示す。 [Example 7] Study of AAV serotypes Viral vectors were produced and analyzed in the same manner as in Example 3, except that the plasmid encoding the Cap protein of AAV2 was changed to a plasmid encoding the Cap protein of AAV1 or AAV6, and the temperature during production of the viral vector in the viral vector-producing cells was set to 37° C. The results regarding the productivity of AAV1 and AAV6 in the culture supernatant and cells 3 days after transfection are shown in Table 4.
　表4に示される通り、エタノールの添加は、様々なセロタイプのAAVに対して、生産性とゲノム完全性の向上に効果があることが示された。 As shown in Table 4, the addition of ethanol was shown to be effective in improving the productivity and genome integrity of various AAV serotypes.
[実施例8]第2の温度帯の下限の検討
　トランスフェクション後に温度を変えてAAV2の生産を行う操作までを、実施例1と同様の手順で行い、トランスフェクションから約4時間後、培養液量に対して0.56v/v%のろ過済みエタノールを添加し、3日間AAV2を生産させた。AAV2生産時に37℃、且つエタノールを添加しなかった条件(コントロール条件）に対して、33℃でエタノールを添加した際の、ウイルスベクターの総生産量の相対値を図7に示す。 [Example 8] Examination of the lower limit of the second temperature range The procedure up to the operation of changing the temperature after transfection and producing AAV2 was performed in the same manner as in Example 1, and about 4 hours after transfection, 0.56 v/v% filtered ethanol was added to the culture solution volume, and AAV2 was produced for 3 days. Figure 7 shows the relative value of the total production amount of viral vectors when ethanol was added at 33°C compared to the condition (control condition) where ethanol was not added at 37°C during AAV2 production.
　図7に示される通り、ウイルスベクター産生細胞にウイルスベクターを生産させる際の温度を33℃としても、ウイルスベクターの総生産量を増加させることができることが示された。 As shown in Figure 7, it was demonstrated that the total production amount of viral vectors can be increased even when the temperature for producing viral vectors in viral vector-producing cells is set to 33°C.
　本発明によれば、感染力価の高いウイルスベクターを非常に効率よく生産することができる。従って、本発明は創薬や試験研究の分野において極めて有用である。
　本出願は、日本で出願された特願２０２２－１６２７９４（出願日：２０２２年１０月７日）を基礎としておりその内容は本明細書に全て包含されるものである。 According to the present invention, a viral vector having a high infectious titer can be produced very efficiently, and therefore the present invention is extremely useful in the fields of drug discovery and research.
This application is based on Japanese Patent Application No. 2022-162794 (filing date: October 7, 2022) filed in Japan, the contents of which are incorporated in their entirety herein.

Claims (9)

  1. 　ウイルスベクター産生細胞を30.0～35.9℃で培養し、ウイルスベクターを産生させる工程を含む、ウイルスベクターを生産する方法であって、ここで、当該ウイルスベクター産生細胞が、ウイルスベクターを産生させる工程の開始前に、36.0～38.0℃の温度下で培養されていることを特徴とする、方法。 A method for producing a viral vector, comprising a step of culturing viral vector-producing cells at 30.0-35.9°C to produce a viral vector, characterized in that the viral vector-producing cells are cultured at a temperature of 36.0-38.0°C before the start of the step of producing the viral vector.
  2. 　前記ウイルスベクター産生細胞にウイルスベクターを産生させる工程が、有機溶媒を含む培地で行われることを特徴とする、請求項1記載の方法。 The method according to claim 1, characterized in that the step of causing the viral vector-producing cells to produce the viral vector is carried out in a medium containing an organic solvent.
  3. 　有機溶媒が、アルコール類又はジメチルスルホキシドである、請求項2記載の方法。 The method according to claim 2, wherein the organic solvent is an alcohol or dimethyl sulfoxide.
  4. 　ウイルスベクター産生細胞を30.0～35.9℃で培養し、ウイルスベクターを産生させる工程を含む、感染力価が向上したウイルスベクターの生産効率を向上させる方法であって、ここで、当該ウイルスベクター産生細胞が、ウイルスベクターを産生させる工程の開始前に、36.0～38.0℃の温度下で培養されていることを特徴とする、方法。 A method for improving the production efficiency of a viral vector with improved infectious titer, comprising a step of culturing viral vector-producing cells at 30.0-35.9°C to produce a viral vector, wherein the viral vector-producing cells are cultured at a temperature of 36.0-38.0°C before the start of the step of producing the viral vector.
  5. 　前記ウイルスベクター産生細胞にウイルスベクターを産生させる工程が、有機溶媒を含む培地で行われることを特徴とする、請求項4記載の方法。 The method according to claim 4, characterized in that the step of causing the viral vector-producing cells to produce the viral vector is carried out in a medium containing an organic solvent.
  6. 　有機溶媒が、アルコール類又はジメチルスルホキシドである、請求項5記載の方法。 The method according to claim 5, wherein the organic solvent is an alcohol or dimethyl sulfoxide.
  7. 　ウイルスベクター産生細胞を30.0～35.9℃で培養し、ウイルスベクターを産生させる工程を含む、ウイルスベクター産生細胞を用いたウイルスベクターの製造において、製造される全ウイルス粒子に対してのウイルスゲノムの完全体を含むウイルス粒子の割合を向上させる方法であって、ここで、当該ウイルスベクター産生細胞が、ウイルスベクターを産生させる工程の開始前に、36.0～38.0℃の温度下で培養されていることを特徴とする、方法。 A method for improving the ratio of virus particles containing complete viral genomes to all virus particles produced in the production of a viral vector using viral vector-producing cells, the method including a step of culturing viral vector-producing cells at 30.0-35.9°C to produce a viral vector, characterized in that the viral vector-producing cells are cultured at a temperature of 36.0-38.0°C before the start of the step of producing the viral vector.
  8. 　前記ウイルスベクター産生細胞にウイルスベクターを産生させる工程が、有機溶媒を含む培地で行われることを特徴とする、請求項7記載の方法。 The method according to claim 7, characterized in that the step of causing the viral vector-producing cells to produce the viral vector is carried out in a medium containing an organic solvent.
  9. 　有機溶媒が、アルコール類又はジメチルスルホキシドである、請求項8記載の方法。 The method according to claim 8, wherein the organic solvent is an alcohol or dimethyl sulfoxide.
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