WO2024075819A1 - Procédé de production de vecteur viral utilisant une cellule - Google Patents

Procédé de production de vecteur viral utilisant une cellule Download PDF

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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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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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    • 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

Le but de la présente invention est de procurer, dans le cadre de la production de vecteurs viraux, un procédé permettant de produire efficacement un vecteur viral présentant un titre infectieux élevé. La présente invention concerne un procédé de production de vecteurs viraux comprenant une étape de culture de cellules productrices de vecteurs viraux à une température comprise entre 30,0 et 35,9 ºC. En l'occurrence, avec le procédé selon la présente invention qui est caractérisé en ce que lesdites cellules productrices de vecteurs viraux sont cultivées à une température comprise entre 36,0 et 38,0 ºC avant de commencer une étape de production de vecteurs viraux, il est possible de produire très efficacement un vecteur viral présentant un titre infectieux élevé.
PCT/JP2023/036412 2022-10-07 2023-10-05 Procédé de production de vecteur viral utilisant une cellule WO2024075819A1 (fr)

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