WO2013146480A1 - Procédé d'introduction de gène - Google Patents

Procédé d'introduction de gène Download PDF

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WO2013146480A1
WO2013146480A1 PCT/JP2013/057858 JP2013057858W WO2013146480A1 WO 2013146480 A1 WO2013146480 A1 WO 2013146480A1 JP 2013057858 W JP2013057858 W JP 2013057858W WO 2013146480 A1 WO2013146480 A1 WO 2013146480A1
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cells
cell
gene
cell population
vector
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Japanese (ja)
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和久 新村
敬章 片山
広文 吉岡
高蔵　晃
峰野　純一
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タカラバイオ株式会社
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/18—Antivirals for RNA viruses for HIV
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/0018—Culture media for cell or tissue culture
    • 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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/065—Modulators of histone acetylation
    • 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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/30—Hormones
    • C12N2501/38—Hormones with nuclear receptors
    • C12N2501/385—Hormones with nuclear receptors of the family of the retinoic acid recptor, e.g. RAR, RXR; Peroxisome proliferator-activated receptor [PPAR]

Definitions

  • the present invention relates to a method for introducing a foreign gene into a target cell and a medium for introducing the foreign gene.
  • Retroviral vectors can stably incorporate a target foreign gene into the chromosomal DNA of a target cell. Therefore, gene transfer using a retroviral vector is a preferable gene transfer means for gene therapy in which long-term gene expression is desired.
  • An object of the present invention is to develop a medium used for gene introduction into target cells using a viral vector, and to provide a method for producing a cell population into which a gene has been introduced using a viral vector.
  • the present inventors use a medium containing retinoic acids and histone deacetylase (hereinafter sometimes referred to as histone deacetylase) inhibitors as active ingredients. As a result, it was found that a high gene transfer efficiency can be obtained by introducing a gene into a target cell using a viral vector, and the present invention has been completed.
  • histone deacetylase histone deacetylase
  • the present invention provides: [1] A method for introducing a foreign gene into a target cell using a viral vector, wherein at least the target cell is present in a medium containing retinoic acids and histone deacetylase inhibitor as active ingredients at the time of introduction of the foreign gene.
  • a method comprising the steps of: [2] The method according to [1], wherein the target cell is a T cell and / or a progenitor cell that can differentiate into a T cell, [3] The method according to any one of [1] or [2], wherein the viral vector is a retroviral vector, [4] The method according to any one of [1] to [3], wherein the histone deacetylase inhibitor is at least one substance selected from trichostatin A, sodium butyrate, or valproic acid, [5] A method for producing a transduced cell population, comprising a step of introducing a foreign gene into a target cell by the method according to any one of [1] to [4], [6] A transduced cell population obtained by the method according to [5], [7] The cell population according to [6] for use in medicine, [8] The cell population according to [6], which is used for production of a medicine, [9] A medicine containing the cell population according to [6] as an active ingredient,
  • the present invention discloses a medium suitable for transduction of target cells using a viral vector.
  • the medium is a basic medium prepared by mixing components necessary for cell culture, and contains retinoic acids and histone deacetylase inhibitor as active ingredients.
  • retinoic acid is also called vitamin A acid, which is either all-trans-retinoic acid in which all double bonds in the chain are trans or 9-cis-retinoic acid in which the 9-position has a cis structure. Good. Other retinoic acid isomers and retinoic acid derivatives can also be used in the present invention. The above-mentioned retinoic acid, retinoic acid isomers and retinoic acid derivatives or their salts are generically referred to herein as retinoic acids. Furthermore, in the present invention, the retinoic acid to be used may be one kind or a combination of plural kinds.
  • the concentration of retinoic acid used in the present invention in the medium is not particularly limited as long as it is a concentration that acts as an active ingredient.
  • ATRA all-trans-retinoic acid
  • it is preferably 1 nM to 10 ⁇ M, more preferably 5 nM to 200 nM, and particularly preferably 10 to 100 nM.
  • the “histone deacetylase inhibitor” is not limited as long as it has a histone deacetylase inhibitory activity.
  • Aliphatic acids such as sodium butyrate, butyrate, phenyl Butyrate, valproic acid, salts and derivatives thereof, etc.
  • hydroxamic acids such as trichostatin A, oxamflatin, suberoylanilide, salts and derivatives thereof, (3) cyclic peptides such as Trapoxin, apicidin, FK228, salts and derivatives thereof, and the like (4) benzamide, salts and derivatives thereof can be used.
  • one kind of histone deacetylase inhibitor may be used, or a plurality of kinds may be used in combination.
  • histone deacetylase inhibitors sodium butyrate (hereinafter referred to as NaB), trichostatin A (hereinafter referred to as TSA) having a wide isoform inhibition range of histone deacetylase. ) Or valproic acid (hereinafter referred to as VPA) is preferably used.
  • NaB sodium butyrate
  • TSA trichostatin A
  • VPA valproic acid
  • the concentration of the histone deacetylase inhibitor used in the present invention in the medium may be a concentration that acts as an active ingredient, and is not particularly limited.
  • TSA for example, preferably 10 nM to 50 ⁇ M. More preferably, it is 20 nM to 10 ⁇ M, and particularly preferably 100 nM to 3 ⁇ M.
  • NaB and VPA for example, it is preferably 1 nM to 50 mM, more preferably 1 mM to 10 mM.
  • the components of the basic medium include energy sources such as amino acids, sugars, and organic acids, vitamins, buffer components for adjusting pH, inorganic salts, and the like. Further, it may contain a pH indicator such as phenol red.
  • a basic medium a known medium containing no serum, for example, DMEM, IMDM, Ham F12 medium or the like may be used, and these can be obtained as commercial products from Invitrogen, Sigma, and the like.
  • Commercially available media such as Opti-ProSFM, VP-SFM, 293SFMII (all manufactured by Invitrogen), HyQ SFM4 MegaVir (manufactured by High Clone) can also be used.
  • serum-added medium may be used, use of a serum-free medium is preferable in order to prevent contamination with unknown viruses derived from serum.
  • serum-free medium it contains serum albumin highly purified from human blood (for example, serum albumin preparations approved as pharmaceuticals), highly purified serum albumin derived from animals, or recombinant serum albumin A serum-free medium is preferably used (Japanese Patent Laid-Open No. 2007-105033).
  • the gene introduction method of the present invention is characterized in that a medium containing the above components is used for culturing target cells.
  • the medium is used at the time of gene transfer using a viral vector, that is, when the target cell is brought into contact with the viral vector.
  • a retrovirus is a generic term for RNA viruses belonging to the Retroviridae family whose genome is composed of RNA and has a life cycle that converts the RNA into DNA in an infected cell.
  • Oncoretrovirus and lentivirus including.
  • An example of the oncoretrovirus is Moloney murine leukemia virus (MMLV).
  • Examples of the lentivirus include human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus (SIV).
  • the viral vector used in the present invention is not particularly limited.
  • retrovirus vectors including oncovirus vectors, lentivirus vectors and their modifications
  • adenovirus vectors adeno-associated virus vectors
  • simian virus vectors vaccinia virus vectors
  • Sendai virus vectors and the like can be mentioned.
  • a retroviral vector that is, a recombinant retroviral vector
  • a replication-defective retrovirus vector in which unlimited infection and gene transfer are prevented is preferably used in the present invention.
  • Known replication-defective retrovirus vectors include MFG vector and ⁇ -SGC vector (International Publication No.
  • retroviral vectors such as pLXIN (Clontech), pDON-AI-2 (Takara Bio), lentiviral vectors [human immunodeficiency virus (HIV) -derived vectors, simian immunodeficiency virus (SIV) -derived Vector etc.] or a vector obtained by modifying these (for example, pseudo-type vector).
  • the vector is non-pathogenic because it lacks replication ability so that it cannot replicate in infected cells.
  • These vectors can infect vertebrate cells, particularly host cells such as mammalian cells, and can stably incorporate a foreign gene carried in the vector into its chromosomal DNA.
  • a foreign gene to be introduced into a target cell can be used by being mounted in a recombinant retroviral vector under the control of an appropriate promoter, for example, an LTR promoter or a foreign promoter present in a retroviral vector.
  • an appropriate promoter for example, an LTR promoter or a foreign promoter present in a retroviral vector.
  • other regulatory elements cooperating with a promoter and transcription initiation site for example, an enhancer sequence, terminator sequence, and intron sequence may be present in the vector.
  • the foreign gene introduced into the target cell may be natural or artificially generated, or a DNA molecule having a different origin is bound by a known means such as ligation. Also good.
  • any gene desired to be introduced into the cell can be selected.
  • exogenous genes include enzymes encoding proteins and proteins related to the disease to be treated, intracellular antibodies (see, for example, WO94 / 02610 pamphlet), T cell receptor genes, growth factors. , Antisense RNA, RNA causing RNA interference, ribozyme, false primer (see, for example, International Publication No. 90/13641 pamphlet) and the like can be used.
  • a CD4 positive T cell exhibiting an anti-HIV effect can be obtained by introducing a gene expressing MazF, which is a sequence-specific RNase, into a CD4 positive T cell as a foreign gene (for example, International Publication No.
  • a gene that confers sensitivity to a specific drug for example, a thymidine kinase gene, can be introduced into cells to confer sensitivity to the drug.
  • the retroviral vector used in the present invention may contain an appropriate marker gene that enables selection of the transfected cell.
  • marker genes include drug resistance genes that confer resistance to antibiotics on cells, reporter genes that can distinguish cells introduced by enzymatic activity and fluorescence, and cell surface marker genes that are localized on the cell surface. Is available.
  • the neomycin phosphotransferase gene is used as a marker gene, the introduced cell can be isolated and purified after confirming resistance to G418 as an index.
  • LNGFR Low Affinity Nerve Growth Factor Receptor
  • the transfected cell can be isolated and purified by using an anti-LNGFR antibody.
  • retroviral vectors examples include MFG vector (ATCC No. 68754), ⁇ -SGC vector (ATCC No. 68755), LXSN vector [BioTechniques, Vol. 7, 980-990]. Page (1989)], pDON-5, pDON-AI-2, pMEI-5 retroviral vectors and pLVSIN-CMV lentiviral vectors manufactured by Takara Bio Inc., Retro-X Q vector series, Lenti-X manufactured by Clontech There are vectors such as vector series.
  • vectors are known packaging cell lines such as PG13 (ATCC CRL-10686), PA317 (ATCC CRL-9078), GP + E-86 (ATCC CRL-9642), GP + envAm12 (ATCC CRL-9964), [Proceedings of the National Academy of Sciences of the United States of America (Proc. Natl. Acad. Sci. USA), vol. 85, 6460-6464 (1988) It can be prepared by using a cell line such as ⁇ CRIP as described in “Year)”.
  • 293T cells ATCC CRL-11268), G3T-hi cells (manufactured by Takara Bio Inc.), etc. are transiently transfected with a packaging plasmid that expresses the structural protein of the recombinant virus and a retrovirus vector plasmid. It can also be prepared by collecting the culture supernatant.
  • a retrovirus-binding substance may be further used.
  • the retrovirus-binding substance used in the present invention is not particularly limited as long as it has a binding affinity for retrovirus.
  • fibronectin for example, fibronectin, fibroblast growth factor, type V collagen, polylysine, DEAE-dextran, And at least one substance selected from these fragments. It is also possible to enhance the binding property to the virus by chemically modifying these substances (for example, Patent Document 2).
  • Fibronectin and fragments thereof are suitable for gene introduction by retrovirus as a substance having both retrovirus binding property and cell binding property. Fibronectin and fragments thereof are described, for example, in Journal of Biochemistry (J. Bio Chem.), Vol. 256, page 7277 (1981), Journal of Cell Biology (J. Cell Biol.), 102, 449 (1986), Journal of Cell Biology, 105, 489 (1987), in a substantially pure form from naturally occurring materials. Can be manufactured. Moreover, it can also be produced using recombinant DNA technology by the method described in US Pat. No. 5,198,423.
  • fibronectin fragments containing a heparin-II region that is a retrovirus binding site for example, recombinant polypeptides such as fibronectin fragments CH-296, H-271, H-296, and CH-271, and methods for obtaining them are described in detail in US Pat. No. 5,198,423.
  • H-296 has a VLA-4 binding domain polypeptide
  • CH-271 has a VLA-5 binding domain peptide
  • CH-296 has both. [Nature Medicine, Vol. 2, pp. 876-882 (1996)].
  • CH-296 is commercially available under the trade name of RetroNectin, RetroNectin.
  • the culture vessel used in the present invention is not particularly limited, and examples thereof include a cell culture bag, a cell culture plate, a cell culture petri dish, a cell culture test tube, and a cell culture flask.
  • the material of the culture vessel is not particularly limited, and for example, a plastic or glass culture vessel can be used in the present invention.
  • a cell culture bag particularly a gas permeable cell culture bag, is suitable for the present invention.
  • the retrovirus-binding substance is immobilized on the inner surface of the culture vessel, the present invention is not particularly limited.
  • the culture vessel material include polystyrene, polyethylene, cycloolefin resin, and fluororesin.
  • the method for immobilizing the retrovirus-binding substance to the culture vessel can be appropriately selected depending on the type of the substance and the type of culture vessel to be used.
  • the retrovirus-binding substance is a polypeptide
  • it can be immobilized by physical adsorption on the surface of the culture vessel.
  • the retrovirus-binding substance may be immobilized on the culture vessel by covalent bonding using a crosslinking agent or the like.
  • stem cells stem cells: hematopoietic stem cells, mesenchymal stem cells, embryonic stem cells, etc.), hematopoietic cells, mononuclear cells (peripheral blood mononuclear cells, umbilical cord blood mononuclear cells, etc.), embryonic cells, primal germ -Cells (primary germ cell), oocytes, oocytes, ova, spermatocytes, sperm, erythroid progenitor cells, lymphocyte mother cells, mature blood cells, lymphocytes, B cells, T cells, NK cells, fibers Blast cells, neuroblast cells, nerve cells, endothelial cells, vascular endothelial cells, hepatocytes, myoblasts, skeletal muscle cells, smooth muscle cells, cancer cells, myeloma cells, leukemia cells and the like can be used.
  • stem cells hematopoietic stem cells, mesenchymal stem cells, embryonic stem cells, etc.
  • Hematopoietic cells obtained from blood or bone marrow are relatively easy to obtain, and since their culture and maintenance techniques have been established, they are suitable for utilizing the method of the present invention.
  • pluripotent stem cells hematopoietic stem cells, mesenchymal stem cells, etc.
  • progenitor cells are suitable as target cells.
  • immune system cells such as CD4-positive T cells and their progenitor cells are suitable as target cells.
  • preliminary stimulation also referred to as initial stimulation
  • the preliminary stimulation step is usually performed by culturing the target cell or a cell population (PBMC or the like) containing the target cell in the presence of a CD3 ligand.
  • the CD3 ligand is not particularly limited as long as it is a substance having binding activity to CD3, and examples thereof include anti-CD3 antibody, ConA, PHA, PMA + ionomycin and the like.
  • an anti-CD3 monoclonal antibody such as OKT3 [Science, Vol. 206, pp. 347-349 (1979)] is used in the present invention.
  • fibronectin, a fragment thereof or a mixture thereof may be added in addition to the CD3 ligand to perform preliminary stimulation. Fibronectin and fragments thereof useful for culturing T cells or their progenitor cells are described in, for example, International Publication No. 2008/111506.
  • costimulatory factors such as cytokines (eg, IL-2) and CD28 ligand may be added to the medium.
  • CD3 ligand and “fibronectin, a fragment thereof or a mixture thereof” are dissolved and coexisted in a medium, and an appropriate solid phase, for example, cell culture equipment (open system, petri dish, flask, bag, etc.) Or a closed system), or may be immobilized on a cell culture carrier such as a bead, a membrane or a slide glass.
  • cell culture equipment open system, petri dish, flask, bag, etc.
  • a closed system a cell culture carrier
  • a cell culture carrier such as a bead, a membrane or a slide glass.
  • the gene introduction step using a viral vector that is, the step of bringing the target cell into contact with the viral vector is performed by bringing the target cell together with the viral vector in a medium containing the retinoic acid and histone deacetylase inhibitor. It is carried out by culturing.
  • This step can be performed under normal cell culture conditions. For example, culture at a humidity of 95% and a CO 2 concentration of 5% is exemplified, but the present invention is not limited to such conditions.
  • This step can be performed, for example, at 30 to 37 ° C., but may be performed at a temperature other than the above range as long as gene introduction into a desired target cell by a viral vector can be achieved.
  • the step is not limited, and is performed, for example, for 2 hours to 3 days, preferably 3 hours to 2 days, and more preferably 4 to 24 hours.
  • the present invention provides a method for producing a transduced cell population.
  • the cell population produced by the present invention is useful for the treatment of various diseases in which the foreign gene is therapeutically effective because it has a high content of cells carrying the desired foreign gene. That is, the cell population produced by the present invention can have various diseases such as cancer, leukemia, malignant tumor, hepatitis, or infectious diseases (for example, influenza, tuberculosis, It can be used for treatment or prevention of HIV (Human Immunodeficiency Virus) infection, AIDS, MRSA infection, VRE infection, or deep mycosis).
  • HIV Human Immunodeficiency Virus
  • the present invention provides a medicament (therapeutic agent or preventive agent) for the above-mentioned diseases containing the above cell population as an active ingredient.
  • the pharmaceutical is in accordance with a known method in the pharmaceutical field, for example, with a cell population prepared by the method of the present invention as an active ingredient, and a known organic or inorganic carrier, excipient, stabilizer, etc. suitable for parenteral administration. They can be mixed and prepared as drops or injections.
  • the content of the cell population of the present invention in the therapeutic agent, the dose of the therapeutic agent, and various conditions relating to the therapeutic agent can be determined as appropriate.
  • gene therapy using CD4 positive T cells as target cells can be performed by the following procedure.
  • a material containing CD4-positive T cells such as bone marrow tissue, peripheral blood, umbilical cord blood, etc. is collected from a donor. These materials can be used as they are for the gene transfer operation, but usually the mononuclear cell fraction is prepared by a method such as density gradient centrifugation.
  • purification of cells using CD4 molecule as an index, removal of CD8 positive T cells and / or monocytes, and culture operation for expanding the number of CD4 positive T cells may be performed.
  • These cell populations are subjected to appropriate pre-stimulation (for example, stimulation with CD3 ligand, CD28 ligand, or IL-2) as necessary, and then a recombinant retro loaded with the target gene by the method of the present invention. Infect with a viral vector.
  • the gene-transferred cells thus obtained can be transplanted into a recipient by intravenous administration, for example.
  • the recipient is preferably the donor itself, but allogeneic transplants can also be performed.
  • hematopoietic stem cells that complement genes that are deficient or abnormal in patients, such as gene therapy for ADA deficiency and Gaucher disease.
  • a drug resistance gene may be introduced into hematopoietic stem cells in order to alleviate the damage of hematopoietic cells caused by chemotherapeutic agents used for the treatment of cancer and leukemia.
  • a gene therapy method for cancer there is a method in which a specific cytotoxic activity against cancer cells expressing the antigen is given to lymphocytes by introducing a gene encoding a T cell receptor that recognizes a tumor antigen.
  • a nucleic acid molecule single-strand specific endoribonuclease, antisense nucleic acid, ribozyme that interferes with HIV replication or gene expression on T cells such as CD4 positive T cells infected with HIV that causes AIDS. Etc.
  • International Publication No. 2007/020873 pamphlet, Human Gene Therapy, Vol. 22, pp. 35-43 (2011). for example, International Publication No. 2007/020873 pamphlet, Human Gene Therapy, Vol. 22, pp. 35-43 (2011).
  • DON-ZsGreen retroviral vector pZsGreen Vector (Clontech) was cleaved with restriction enzymes BamHI and EcoRI (Takara Bio), agarose gel electrophoresis was performed, and the sequence encoding green fluorescent protein ZsGreen was obtained. An approximately 0.7 kbp fragment was recovered. The recovered fragment was blunt-ended using DNA Blunting Kit (Takara Bio) and then inserted into pDON-AI DNA (Takara Bio) to obtain a recombinant retrovirus vector plasmid pDON-ZsGreen. Next, an ecotropic DON-ZsGreen virus was prepared using the plasmid and Retrovirus Packaging Kit Eco (manufactured by Takara Bio Inc.).
  • GaLV retrovirus packaging cells PG13 This was then infected with GaLV retrovirus packaging cells PG13.
  • a high-titer virus-producing cell was cloned from the infected cell to establish a retrovirus vector-producing cell line PG13 / DON-ZsGreen.
  • a GaLV / DON-ZsGreen virus solution was obtained by a conventional method in a medium containing 5 mM NaB (hereinafter referred to as DON-ZsGreen retrovirus vector).
  • retronectin / anti-CD3 antibody-immobilized plate Fibronectin fragment CH-296 (RetroNectin (registered trademark), manufactured by Takara Bio Inc.), hereinafter referred to as retronectin), 25 ⁇ g / mL, and anti-CD3 antibody (OKT3, Janssen) Pharma Co., Ltd.) was dissolved in ACD-A (manufactured by Terumo) so as to be 5 ⁇ g / mL. This lysate was added to a 6-well plate for cell culture (manufactured by BD Falcon) at a volume of 1 mL / well and left at 37 ° C. for 5 hours.
  • ACD-A manufactured by Terumo
  • a virus dilution solution was prepared by diluting the virus solution obtained in Example 1 (1) 10-fold. At this time, for the dilution, ACD-A was adjusted to a final concentration of 5%, and human serum albumin “Albuminer 25%” (manufactured by CSL Behring) was physiologically adjusted so that the final concentration of albumin was 2%. What was added to the salt solution (made by Terumo) was used. Next, 0.5 mL of retronectin previously diluted with ACD-A to a final concentration of 20 ⁇ g / mL is added to each well of a 24-well non-treatment plate (BD Falcon) and treated at 4 ° C. overnight. The retronectin solution was removed from the plate.
  • BD Falcon 24-well non-treatment plate
  • a washing operation of adding 0.5 mL of ACD-A to each well and removing it was performed twice. 1 mL of the virus dilution was added to each well of the plate and centrifuged (32 ° C., 2000 ⁇ g, 2 hours). After centrifugation, the virus dilution supernatant was removed from each well, and human serum albumin “Albuminer 25%” was added to physiological saline so that the final concentration of albumin was 1.5%. Washed twice. The plate thus obtained was used as a virus-binding plate.
  • PBMC Human peripheral blood mononuclear cells
  • GT-T503 containing 0.6% autologous plasma, 600 IU / mL IL-2 (manufactured by NOVARTIS), 0.2% human serum albumin “Albuminer 25%”, 0.5 ⁇ g / mL fungizone (manufactured by Bristol Myers)
  • the suspension was suspended in a medium (hereinafter referred to as culture medium), and added to the retronectin / anti-CD3 antibody-immobilized plate prepared in Example 1 (2) so as to be 6.7 mL / well (start of PBMC stimulation).
  • the plate was cultured in a CO 2 incubator at 37 ° C. for 4 days (until day 4 after the start of PBMC stimulation).
  • Modified Cell (hereinafter the same). These plates were further cultured in a CO 2 incubator (37 ° C., humidity 95%, CO 2 concentration 5%) for 1 day (until day 5 after the start of PBMC stimulation). With respect to the plate after culture, 0.90 mL of cell suspension in each well was seeded in each well of a 6-well plate for cell culture, and further 3.6 mL of culture medium was added to each well for 5-fold dilution. Thereafter, the cells were cultured in a CO 2 incubator (37 ° C., humidity 95%, CO 2 concentration 5%) for 1 day (up to the sixth day after the start of PBMC stimulation).
  • Flow cytometry analysis was performed according to the instrument instruction using a BD FACSCanto II flow cytometer (Becton Dickinson).
  • GT% gene transfer efficiency
  • FSC forward scattered light
  • SSC side scattered light
  • Y-axis SSC
  • the cell population in the gate is first measured for isotype control (negative control) on a histogram of APC-Cy7 and ZsGreen detection parameters (x-axis: APC-Cy7 fluorescence intensity, y-axis: ZsGreen fluorescence intensity).
  • the gate region of a cell population expressing CD8 and ZsGreen was set, and then the ratio (%) of cells in the gate region of the subject cells was measured to determine gene transfer efficiency.
  • Table 2 shows the measurement results of gene transfer efficiency in CD8 positive cells. “MFI” shown in Table 2 indicates the mean fluorescence intensity (Mean Fluorescence Intensity) of the ZsGreen-expressing cell population.
  • a GaLV / MS-MA24-siTCR virus solution was obtained by a conventional method in a medium containing 5 mM NaB (hereinafter referred to as MS-MA24-siTCR retrovirus vector).
  • virus binding plate was prepared in the same manner as in Example 1 (3).
  • the virus solution used here is a solution obtained by diluting the virus solution obtained in Example 1 (1) 10 times and the virus stock solution obtained in Example 2 (1).
  • Example 2 (3) Gene transfer operation 1 mL (0.5 ⁇ 10 6 cells) of cell suspension stimulated for 4 days in Example 2 (3) was added to each well on the virus-binding plate prepared in Example 2 (2). Were added) / well. Thereafter, each additive was added to the cell suspension in each well so that the final concentration was as shown in Table 3.
  • CM and NGMC were prepared as negative controls, and each cell solution was further cultured for 4 hours in a CO 2 incubator (37 ° C., humidity 95%, CO 2 concentration 5%). Thereafter, the culture was continued until the 10th day after the start of PBMC stimulation in the same manner as in Example 1 (5).
  • Flow cytometry analysis of gene transfer efficiency was performed in the same procedure as in Example 1 (6) using a BD FACSCanto II flow cytometer.
  • the expression rate of ZsGreen is first gated on the target cell population on the two-parameter histogram (X-axis: FSC, y-axis: SSC) of FSC and SSC.
  • the cell population in the gate is expressed on the histogram of ZsGreen detection parameters (x axis: cell number, y axis: ZsGreen fluorescence intensity), and first, ZsGreen is expressed using the measurement result of isotype control (negative control) as an index.
  • the gate area of the cell population was set, and then the ratio (%) of the cells in the gate area of the subject cells was measured.
  • Table 4 shows the measurement results of ZsGreen expression gene transfer efficiency.
  • the expression rate of TCR was determined as follows. In order to examine the gene transfer efficiency by retrovirus, cells cultured for up to 7 days in Example 2 (4) were analyzed with MAGE-A4 tetramer-PE (Ludwig) and Human CD8-FITC (Becton Dickinson). The percentage of cells that were CD8 positive and tetramer positive was measured using a flow cytometer. Specifically, 0.5 ⁇ 10 6 cells after infection culture were transferred to an Eppendorf tube, and the cells were precipitated by centrifugation (4 ° C., 5000 ⁇ rpm, 2 minutes).
  • the precipitated cells were suspended in 950 ⁇ L of 0.5% BSA / PBS, and the cells were precipitated again by centrifugation (4 ° C., 5000 ⁇ rpm, 2 minutes). After removing the supernatant again, the suspension was suspended in a mixed solution obtained by adding 8 ⁇ L of 0.5% BSA / PBS to 1 ⁇ L of MAGE-A4 tetramer-PE, and reacted at 4 ° C. for 30 minutes. Thereafter, 1 ⁇ L of Human CD8-FITC was added and reacted at 4 ° C. for 15 minutes.
  • the target cell population is gated on the two-parameter histogram (x axis: FSC, y axis: SSC) of FSC and SSC.
  • the cell population in the gate is shown on the histogram of FITC and PE detection parameters (x axis: FITC, y axis: shows tetramer-PE fluorescence intensity).
  • the measurement result of isotype control (negative control) is used as an index for CD8.
  • the gate region of the positive and tetramer positive cell population was set, and then the percentage of cells in the gate region among the CD8 positive cells of the subject was measured to determine the expression rate of TCR.
  • Table 4 shows the measurement results of TCR gene transfer efficiency.
  • surface shows the ratio of the tetramer positive cell in CD8 positive cell.
  • Flow cytometry analysis (cell phenotype) To examine the cell phenotype of the MS-MA24-siTCR gene-introduced cell suspension cultured until the 10th day after the start of PBMC stimulation, the expression of cell surface proteins involved in differentiation was examined by the following procedure. First, the cell suspension for 0.5 ⁇ 10 6 cells was transferred to an Eppendorf tube, and the cells were precipitated by centrifugation (4 ° C., 5000 ⁇ rpm, 2 minutes). After removing the supernatant, the precipitated cells were suspended in 950 ⁇ L of 0.5% BSA / PBS, and the cells were precipitated again by centrifugation (4 ° C., 5000 ⁇ rpm, 2 minutes).
  • Flow cytometry analysis was performed according to the instrument instructions using a BD FACSCanto II flow cytometer.
  • the target cell population is gated on the two-parameter histogram of FSC and SSC (x axis: FSC, y axis: SSC).
  • the CD8-positive cell rate is determined based on the cell population in the gate of PECy7, APCCy7 detection parameter histogram (x-axis: PEcy7 fluorescence intensity, y-axis: APCCy7 fluorescence intensity) cell population expressing PECy7, APCCy7
  • the gate region was set, and then the ratio (%) of the cells in the gate region of the subject cells was measured.
  • the cell phenotype is the cell population in the gate defined by FSC and SSC.
  • PE FITC detection parameter histogram (x axis: PE fluorescence intensity, y axis: FITC fluorescence intensity) PE, FITC
  • the gate region of the cell population to be expressed was set, and then the ratio (%) of the cells in the gate region of the subject cells was measured.
  • CCR7 + / CD45RA + is known as a naive T cell
  • CCR7 + / CD45RA ⁇ is known as a central memory T cell
  • CCR7 ⁇ / CD45RA ⁇ is known as an effector memory T cell phenotype.
  • virus binding plate was prepared in the same manner as in Example 1 (3).
  • the virus diluted solution used here is a solution obtained by diluting the virus solution obtained in Example 1 (1) 20 times with a diluting solution.
  • Example 3 (2) Initial stimulation culture of human peripheral blood mononuclear cells for gene transfer A PBMC cell solution was prepared in the same manner as in Example 1 (4), and the retronectin / anti-CD3 antibody immobilized plate prepared in Example 3 (1) Then, it was added to the anti-CD3 antibody-immobilized plate at 6.7 mL / well (stimulation of PBMC). The plate was cultured in a CO 2 incubator at 37 ° C. for 4 days (until day 4 after the start of PBMC stimulation).
  • Example 3 (4) Gene transfer operation 1 mL (0.5 ⁇ 10 6 cells) of cell suspension stimulated for 4 days in Example 3 (3) was added to each virus-binding plate prepared in Example 3 (2). Were added) / well. Thereafter, ATRA and / or NaB (manufactured by Wako Pure Chemical Industries, Ltd.) described in Example 1 (5) are dissolved in PBS (manufactured by Gibco) as each additive in the cell suspension of each well and adjusted to 500 mM. A 0.22 ⁇ m filter (manufactured by Millipore) was added to the filtered solution under the conditions shown in Table 6.
  • PBS manufactured by Gibco
  • CM and NGMC were prepared as negative controls, and each cell solution was further cultured for 4 hours in a CO 2 incubator (37 ° C., humidity 95%, CO 2 concentration 5%). Thereafter, the culture was continued until the 10th day after the start of PBMC stimulation in the same manner as in Example 1 (5).
  • Flow cytometry analysis was performed according to the instrument instructions using a BD FACSCanto II flow cytometer.
  • the target cell population is gated on the two-parameter histogram of FSC and SSC (x axis: FSC, y axis: SSC).
  • the CD4 and CD8 positive cell ratios express PECy7 and APCCy7 on the histogram of PECy7 and APCCy7 detection parameters (x axis: PECy7 fluorescence intensity, y axis: APCCy7 fluorescence intensity).
  • the gate region of the cell population to be determined was set, and then the proportion (%) of the cells in the gate region of the subject cells was measured.
  • the cell phenotype is the cell population in the gate defined by FSC and SSC, PE, APC detection parameter histogram (x axis: PE fluorescence intensity, y axis: APC fluorescence intensity), PE, APC
  • the gate region of the cell population to be expressed was set, and then the ratio (%) of the cells in the gate region of the subject cells was measured.
  • the expression rate of ZsGreen is gated on the target cell population on the two-parameter histogram of FSC and SSC (x axis: FSC, y axis: SSC).
  • the cell population in the gate is expressed on the histogram of ZsGreen detection parameters (x axis: cell number, y axis: ZsGreen fluorescence intensity), and first, ZsGreen is expressed using the measurement result of isotype control (negative control) as an index.
  • the gate area of the cell population was set, and then the ratio (%) of the cells in the gate area of the subject cells was measured.
  • Table 7 shows the measurement results of gene transfer efficiency.
  • a method for producing a cell population comprising efficient gene transfer by virus, high expression of a transgene, and a high ratio of cells having a desired phenotype.
  • gene introduction efficiency and expression after introduction can be controlled, and a desired gene-introduced cell can be prepared with a small amount of vector, production cost can be reduced, and the target gene can be efficiently functioned. Demonstrate etc. can be realized.
  • the present invention is particularly useful in fields such as medicine, cell engineering, genetic engineering, and developmental engineering.

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Abstract

La présente invention concerne : un milieu de culture qui contient un composé acide rétinoïque et une substance inhibitrice d'histone désacétylase en tant que principes actifs et qui peut être utilisé pour l'introduction d'un gène dans une cellule cible à l'aide d'un vecteur viral ; un procédé d'introduction d'un gène dans une cellule cible à l'aide d'un vecteur viral, le milieu de culture étant utilisé dans l'introduction du gène ; et un procédé de production d'une masse cellulaire dans laquelle un gène a été introduit à l'aide d'un vecteur viral, le milieu de culture étant utilisé.
PCT/JP2013/057858 2012-03-29 2013-03-19 Procédé d'introduction de gène WO2013146480A1 (fr)

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JP2002527101A (ja) * 1998-10-16 2002-08-27 ノバルティス アクチエンゲゼルシャフト ヒストンデアセチラーゼ阻害剤による造血幹細胞への遺伝子形質導入の改良及び自己再生の促進
JP2004535809A (ja) * 2001-07-06 2004-12-02 アーンスティテュ ナシオナール ド ラ サント エ ド ラ ルシェルシェ メディカル（イー．エンヌ．エス．ウー．エール．エンム．） 新規なヒト肝臓癌細胞株、この細胞を取得する方法およびその使用
JP2005519620A (ja) * 2002-03-12 2005-07-07 アーク・セラピューティックス・リミテッド 遺伝子操作したバキュロウィルスおよびその使用
WO2009048024A1 (fr) * 2007-10-09 2009-04-16 Hiroshima University Vecteur d'amplification poussée d'un gène cible de façon extrachromosomique et son utilisation
WO2011024791A1 (fr) * 2009-08-25 2011-03-03 タカラバイオ株式会社 Procédé de fabrication d'une population de lymphocytes t en présence d'acide rétinoïque
JP2011526786A (ja) * 2008-06-30 2011-10-20 セントコア・オーソ・バイオテツク・インコーポレーテツド 多能性幹細胞の分化

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JP2002527101A (ja) * 1998-10-16 2002-08-27 ノバルティス アクチエンゲゼルシャフト ヒストンデアセチラーゼ阻害剤による造血幹細胞への遺伝子形質導入の改良及び自己再生の促進
WO2001096532A2 (fr) * 2000-06-15 2001-12-20 Tanja Dominko Cellules multipotentes de mammifere
JP2004535809A (ja) * 2001-07-06 2004-12-02 アーンスティテュ ナシオナール ド ラ サント エ ド ラ ルシェルシェ メディカル（イー．エンヌ．エス．ウー．エール．エンム．） 新規なヒト肝臓癌細胞株、この細胞を取得する方法およびその使用
JP2005519620A (ja) * 2002-03-12 2005-07-07 アーク・セラピューティックス・リミテッド 遺伝子操作したバキュロウィルスおよびその使用
WO2009048024A1 (fr) * 2007-10-09 2009-04-16 Hiroshima University Vecteur d'amplification poussée d'un gène cible de façon extrachromosomique et son utilisation
JP2011526786A (ja) * 2008-06-30 2011-10-20 セントコア・オーソ・バイオテツク・インコーポレーテツド 多能性幹細胞の分化
WO2011024791A1 (fr) * 2009-08-25 2011-03-03 タカラバイオ株式会社 Procédé de fabrication d'une population de lymphocytes t en présence d'acide rétinoïque

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