WO2016056438A1 - Procédé de production de cellules de type myocardique, et composition destinée à être utilisée dans ledit procédé pour la production de cellules de type myocardique - Google Patents

Procédé de production de cellules de type myocardique, et composition destinée à être utilisée dans ledit procédé pour la production de cellules de type myocardique Download PDF

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WO2016056438A1
WO2016056438A1 PCT/JP2015/077664 JP2015077664W WO2016056438A1 WO 2016056438 A1 WO2016056438 A1 WO 2016056438A1 JP 2015077664 W JP2015077664 W JP 2015077664W WO 2016056438 A1 WO2016056438 A1 WO 2016056438A1
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mef2c
tbx5
gata4
cells
gene
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真樹 家田
和享 宮本
井上　誠
孝史 弘中
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学校法人 慶應義塾
株式会社Ｉｄファーマ
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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
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • 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

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  • the present invention relates to a method for efficiently producing myocardial cells and a composition for producing myocardial cells used therefor.
  • Sendai virus vectors can replicate the genome in mononuclear cells, but cannot form infectious virus particles, and are considered to be highly safe.
  • Patent Document 1 and Non-Patent Documents 1 and 2 disclose a technique for inducing differentiation from fibroblasts into which three factors (Gata4, Mef2c, and Tbx5) have been gene-transferred by a retroviral vector into cardiomyocyte-like cells.
  • three factors Ga4, Mef2c, and Tbx5
  • a retroviral vector into cardiomyocyte-like cells.
  • gene transfer using a retroviral vector has been pointed out to be cancerous because the transgene is integrated into the host chromosome (nucleus-derived chromosome).
  • the present invention has been made to solve the above-mentioned problems, and an object of the present invention is to provide a method capable of efficiently producing highly safe myocardial cells and a composition for producing myocardial cells used therein. It is to provide.
  • the method for producing cardiomyocyte-like cells of the present invention has at least one reprogramming factor selected from Gata4, Mef2c, Tbx5, Mesp1, and Myocd polypeptide, and a nucleic acid encoding the reprogramming factor in the genome. It is characterized in that it is introduced into fibroblasts using one or more Sendai virus vectors.
  • the reprogramming factor can be configured to be three of Gata4, Mef2c, and Tbx5.
  • the reprogramming factor can be configured to be five of Gata4, Mef2c, Tbx5, Mesp1, and Myocd.
  • a microRNA can be introduced into the fibroblast.
  • it can be configured such that two or more of the reprogramming factors are inserted in series in the Sendai virus vector.
  • Gata4, Mef2c, and Tbx5 can be configured to be adjacent in the order of Gata4, Mef2c, and Tbx5 from the upstream side toward the downstream side.
  • Mesp1 and Myocd can be configured to be adjacent to each other in the order of Mesp1 and Myocd from the upstream side toward the downstream side.
  • the fibroblast can be configured to be derived from a human.
  • the composition for producing cardiomyocyte-like cells of the present invention is a Sendai virus vector in which one or more reprogramming factors selected from Gata4, Mef2c, Tbx5, Mesp1, and Myocd polypeptides are retained on the viral genome. It is characterized by including. (10) In (9), the reprogramming factor can be configured to be three of Gata4, Mef2c, and Tbx5. In (11) or (9), the reprogramming factor can be configured to be five of Gata4, Mef2c, Tbx5, Mesp1, and Myocd. (12) Further, in (9) to (11), a microRNA (miRNA) can be included.
  • miRNA microRNA
  • two or more of the reprogramming factors may be configured to be inserted in series in the Sendai virus vector.
  • Gata4, Mef2c, and Tbx5 can be configured to adjoin G4, Mef2c, and Tbx5 in this order from the upstream side toward the downstream side.
  • Mesp1 and Myocd can be configured to be adjacent in the order of Mesp1 and Myocd from the upstream side toward the downstream side.
  • FACS analysis for expression of cardiac troponin T (cTnT). It is the fluorescence-microscope image 4 weeks after gene introduction
  • cardiomyocyte-like cells mean cardiomyocytes directly generated from fibroblasts using the method of the present invention, and are also referred to as “induced cardiomyocytes”.
  • the “cardiomyocyte” means a cell expressing at least cardiac troponin (cTnT) or ⁇ MHC.
  • one or more of Gata4, Mef2c, Tbx5, Mesp1, and Myocd polypeptides are referred to as “reprogramming factors”.
  • fibroblasts into which one or more reprogramming factors are introduced are directly reprogrammed into differentiated cardiomyocytes without becoming stem cells.
  • the method for producing cardiomyocyte-like cells of this embodiment has one or more reprogramming factors selected from Gata4, Mef2c, Tbx5, Mesp1, and Myocd polypeptide, and a nucleic acid encoding the reprogramming factor polypeptide in the genome.
  • One or more Sendai virus vectors are introduced into fibroblasts.
  • a factor gene into fibroblasts that is, introduction of a polynucleotide encoding these polypeptides or a polynucleotide having a base sequence complementary to the base sequence is, for example, a retrovirus vector or an adenovirus vector.
  • a retrovirus vector or an adenovirus vector.
  • the reprogramming factor is preferably introduced into fibroblasts by one or more recombinant Sendai virus vectors having a nucleic acid encoding the reprogramming factor polypeptide in the genome.
  • the vector is preferably a viral vector.
  • the viral vector is a vector that has a genomic nucleic acid derived from the virus and can express the gene by incorporating a transgene into the nucleic acid.
  • the Sendai virus vector is a non-chromosomal viral vector, and since the vector is expressed in the cytoplasm, there is no risk that the transgene is integrated into the host chromosome (nucleus-derived chromosome). Therefore, the safety is high, and the vector can be removed from the introduced cell after the purpose is achieved.
  • the Sendai virus vector is an infectious virus particle, a virus core, a complex of virus genome and virus protein, or a complex composed of non-infectious virus particles, which is introduced into a cell.
  • a complex with the ability to express the gene carried by is included.
  • a ribonucleoprotein virus core part
  • NP, P, and L proteins Sendai virus genome
  • NP, P, and L proteins Sendai virus genome
  • the introduction into the cells may be appropriately performed using a transfection reagent or the like.
  • RNP ribonucleoprotein
  • Sendai virus is one of the Mononegavirales viruses belonging to the family of Paramyxoviridae (Paramixoviridae; Paramyxovirus, Morbilillirus, Rubulavirus, and Pneumovirus genus).
  • RNA of antisense strand to sense strand is included as genome.
  • Negative strand RNA is also called negative strand RNA.
  • Paramyxoviridae viruses other than Sendai virus include Newcastle disease virus (Newcastle disease virus), Mumps virus (Measles virus), measles virus (Measles virus), and RS virus (Respiris virus).
  • Sendai virus SeV
  • human parainfluenza virus-1 HPIV-1
  • human parainfluenza virus-3 HPIV-3
  • phocine distemper virus PDV
  • candinvirdin candinvirdin (individualVirdin).
  • Peste-des-petits-luminances virus PDPR
  • measles virus MV
  • renderpest virus RSV
  • Hendra virus Hendra
  • Nipah virus Nipah virus
  • accession number of the base sequence database of each gene of Sendai virus is M29343, M30202, M30203, M30204, M51333, M55563, M69046, X17218 for the NP gene, and M30202, M30203, M30204, M55556, M69046 for the P gene.
  • NP gene also referred to as N gene
  • CDV AF014953; DMV, X75961; HPIV-1, D01070; HPIV-2, M55320; HPIV-3, D10025 Mupera, X85128; Mumps, D86172; MV, K01711; NDV, AF064091; PDPR, X74443; PDV, X75717; RPV, X68311; SeV, X00087; SV5, M81442; and Tupaia, AF07780, V, X DMV, Z47758; HPIV-1, M74081; HPIV-3, X04721; HPIV-4a, M55975; HPIV-4b, M55976; umps, D86173; MV, M89920; NDV, M20302; PDV, X75960; RPV, X68311; SeV, M
  • HPIV-3 D00047; MV, ABO16162; RPV, X68311; SeV, AB005796; and Tupaia, AF0797780, CDV for the M gene, M12669; DMV Z30087; HPIV-1, S38067; HPIV-2, M62734; HPIV-4a, D10241; HPIV-4b, D10242; Mumps, D86171; MV, AB012948; NDV, AF089819; PDPR, Z47977; PDV, X75717; RPV, M34018; SeV, U31956; and SV5, M32248, F
  • HPIV-3 AB011322; HPIV-4A, M34033; HPIV-4B, AB006954; Mumps, X99040; MV, K01711; NDV, AF204872; PDPR, Z81358; PDV, Z36979; RPV, AF132433; SeV, U06433; -5, S76876.
  • a plurality of strains are known for each virus, and there are genes having sequences other than those exemplified above depending on the strain.
  • a Sendai virus vector having a viral gene derived from any of these genes is useful as the vector of this embodiment.
  • the Sendai virus vector of this embodiment is 90% or more, preferably 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identical to the coding sequence of any of the above viral genes.
  • the Sendai virus vector of the present embodiment is, for example, 90% or more, preferably 95% or more, 96% or more, 97% or more, 98% or more with the amino acid sequence encoded by the coding sequence of any of the above viral genes. Or a nucleotide sequence encoding an amino acid sequence having 99% or more identity.
  • the Sendai virus vector of the present embodiment is, for example, within 10 amino acids, preferably within 9, within 8, within 7, within 6 within the amino acid sequence encoded by any of the above viral gene coding sequences. It includes a base sequence encoding an amino acid sequence in which 5 amino acids, 4 amino acids, 3 amino acids, 2 amino acids, 2 amino acids, or 1 amino acid is substituted, inserted, deleted, and / or added.
  • sequences to which database accession numbers such as base sequences and amino acid sequences described in this specification are referred to, for example, the sequences on the filing date and priority date of the present application, and the filing date and priority date of the present application. It is possible to specify as a sequence at any point of time, preferably as a sequence as of the filing date of the present application. The sequence at each time point can be specified by referring to the revision history of the database.
  • the Sendai virus vector used in the present embodiment may be a derivative, and examples of the derivative include a virus whose virus gene has been modified, a virus that has been chemically modified, and the like so as not to impair the gene transfer ability by the virus. included.
  • Sendai virus may also be derived from natural strains, wild strains, mutant strains, laboratory passage strains, and artificially constructed strains.
  • An example is Z strain (disclosed in Medical Journal of Osaka University Vol. 6, No. 1, March 1955 p1-15). That is, as long as the target function can be achieved, the virus may be a virus vector having the same structure as a virus isolated from nature, or a virus artificially modified by genetic recombination. For example, any gene possessed by the wild-type virus may be mutated or defective. It is also possible to use incomplete viruses such as DI particles (disclosed in J. Virol. 68: 8413-8417, 1994).
  • a virus having a mutation or deletion in at least one gene encoding a viral envelope protein or outer shell protein can be preferably used.
  • a viral vector is, for example, a viral vector that can replicate the genome in infected cells but cannot form infectious viral particles.
  • a transmission ability-deficient virus vector is highly safe because there is no concern of spreading infection around it.
  • a viral vector that does not contain at least one gene encoding an envelope protein or spike protein such as F and / or HN, or a combination thereof can be used (International Publication No. WO00 / 70055, International Publication No. WO00). / 70070, Li, H.-O. et al., J. Virol. 74 (14) 6564-6659 (2000)).
  • proteins necessary for genome replication are encoded in genomic RNA
  • the genome can be amplified in infected cells.
  • a defective virus for example, a defective gene product or a protein capable of complementing it is supplied exogenously in virus-producing cells (International Publication WO00 / 70055, International Publication WO00 / 70070, Li, H.-O. et al., J. Virol. 74 (14) 6564-6695 (2000)).
  • VLP non-infectious viral particle
  • the viral vector is recovered as RNP (for example, RNP comprising N, L, P protein, and genomic RNA)
  • the vector can be produced without complementing the envelope protein.
  • Sendai virus vectors in the present embodiment include, for example, mutations of G69E, T116A, and A183S in the M protein, mutations of A262T, G264, and K461G in the HN protein, an L511F mutation in the P protein, and L
  • the protein may be an F gene deletion-type Sendai virus vector (for example, Z strain) having N1197S and K1795E mutations, and a mutation of TS 7, TS 12, TS 13, TS 14, or TS 15 is further introduced into this vector. Vectors are more preferred.
  • SeV18 + / TS ⁇ F (WO 2010/008054, WO 2003/025570) and SeV (PM) / TS ⁇ F, and further, mutations of TS 7, TS 12, TS 13, TS 14, or TS 15 were introduced into these. Examples include, but are not limited to, vectors.
  • T ⁇ F has mutations of G69E, T116A, and A183S in the M protein, mutations of A262T, G264, and K461G in the HN protein, L511F mutation in the P protein, and N1197S and K1795E mutations in the L protein, Deletion of the F gene.
  • reconstitution of a recombinant Sendai virus vector having a reprogramming factor can be performed using a known method. Specifically, (a) a cell encoding Sendai virus genomic RNA (minus strand) or its complementary strand (plus strand) and a virus protein (N, P, and L) necessary for virus particle formation. And (b) a step of recovering the culture supernatant containing the produced virus.
  • the viral protein necessary for particle formation may be expressed from the transcribed viral genomic RNA, or may be supplied to trans from other than the genomic RNA. For example, expression plasmids encoding N, P, and L proteins can be introduced into cells and supplied.
  • the viral gene can be separately expressed in virus-producing cells to complement particle formation.
  • a vector in which DNA encoding the protein or genomic RNA is linked downstream of an appropriate promoter that functions in the host cell is introduced into the host cell.
  • the transcribed genomic RNA is replicated in the presence of viral proteins to form infectious viral particles.
  • the defective virus or another viral protein capable of complementing its function can be expressed in the virus-producing cell.
  • Sendai virus can be produced using the following known methods (International Publication WO97 / 16539; International Publication WO97 / 16538; International Publication WO00 / 70055; International Publication WO00 / 70070). International Publication No. WO01 / 18223; International Publication No. WO03 / 025570; International Publication No. WO2005 / 071092; International Publication No. WO2006 / 137517; International Publication No. WO2007 / 083644; International Publication No. WO2008 / 007581; Hasan, M. K. et. al., J. Gen. Virol.78: 2813-2820, 1997, Kato, A.
  • the Sendai virus genome consists of NP (nucleocapsid) gene, P (phospho) gene, M (matrix) gene, F (fusion) gene, and HN (hemagglutinin / neuraminidase in this order from 3 ′ end to 5 ′ end. ) Gene and L (large) gene.
  • Sendai virus can sufficiently function as a vector if it has the NP gene, P gene, and L gene, and replicates the genome in the cell (in this embodiment, Gata4, Mef2c, Tbx5). , Mesp1, and Myocd) can be expressed. Since Sendai virus has minus-strand RNA in the genome, the 3 'side of the genome is upstream and the 5' side is downstream.
  • the gene mounted on the Sendai virus vector can be inserted between any of the above genes, for example, between the P gene and the M gene. Even when inserted in other places, only the overall expression level changes, and there is a possibility that the induction efficiency of myocardial cells is different, but the target cell induction itself can be performed in the same manner.
  • Gata4, Mef2c, Tbx5, Mesp1, and Myocd are incorporated. That is, only one of Gata4, Mef2c, Tbx5, Mesp1, and Myocd may be inserted between the P gene and the M gene, or two or more may be inserted. Two or more genes may be inserted at positions separated from each other via other genes, or may be inserted adjacent to each other.
  • the Gata4 gene, the Mef2c gene, and the Tbx5 gene are immediately adjacent to the Sendai virus P gene in this order, that is, immediately downstream of the P gene (immediately 5 'to the minus-strand RNA genome). And incorporated.
  • no other transcription unit for example, a transcription unit encoding a gene encoding a protein is included between the P gene and the Gata4 gene.
  • the Gata4 gene, the Mef2c gene, and the Tbx5 gene are arranged in this order on the Sendai virus genome, the Gata4 gene is arranged on the 3 ′ side most among the three genes, and the Tbx5 gene is arranged on the most 5 ′ side. .
  • five genes namely, the Gata4 gene, the Mef2c gene, the Tbx5 gene, the Mesp1 gene, and the Myocd gene are in this order immediately after the Sendai virus P gene, that is, immediately downstream of the P gene (negative strand RNA). It may be incorporated adjacent to the 5 ′ side of the genome).
  • two genes that is, the Mesp1 gene and the Myocd gene are integrated in this order immediately after the Sendai virus P gene, that is, immediately downstream of the P gene (immediately 5 'to the minus-strand RNA genome). May be.
  • the Sendai virus vector containing one or more of Gata4, Mef2c, Tbx5, Mesp1, and Myocd can be used alone for gene transfer in the production of the cardiomyocyte-like cell of this embodiment. It can also be used in reprogramming to further introduce (miRNA).
  • miR133 is used as the microRNA (miRNA)
  • Snail1 which is a cell fate control factor of fibroblasts, suppresses the characteristics of fibroblasts and promotes myocardial induction (Muraoka).
  • N Yamakawa H, Miyamoto K, Ieda M.
  • MiR-133 promotes cardiographic reprogramming by directing repressing Snai1 and silencing firs.
  • the microRNA can be inserted into the Sendai virus vector containing the Gata4, Mef2c, and Tbx5 genes, or the Sendai virus vector containing the Mesp1 and Myocd genes. Also good.
  • a desired vector such as a plasmid, viral vector, non-viral vector (for example, liposome) can be used as the vector.
  • viral vectors include adenovirus vectors and retrovirus vectors, but are not limited thereto. More preferably, the microRNA is inserted into a liposome and introduced into a fibroblast by a lipofection method.
  • micro RNA (miR133) is shown below (Muraoka N, Yamakawa H, Miyamoto K, Ieda M. MiR-133 promoted birefringable bidirective. : 1565-81.214.).
  • the fibroblast into which the gene and / or polypeptide of each factor is introduced may be isolated from humans or non-human vertebrates such as mice, rats, and pigs. It may be a subcultured cell.
  • fibroblasts for example, fetal fibroblasts, tail tip-derived fibroblasts, cardiac fibroblasts, foreskin fibroblasts, skin fibroblasts, lung fibroblasts and the like can be used.
  • the method for producing cardiomyocyte-like cells uses one or more Sendai virus vectors having in their genome a nucleic acid encoding one or more reprogramming factors selected from Gata4, Mef2c, Tbx5, Mesp1, and Myocd polypeptides. It has the process which introduce
  • the culture of fibroblasts is not particularly limited as long as it is suitable, and is usually performed at a temperature in the range of 25 ° C. to 37 ° C. and under 5% CO 2 .
  • the culture of fibroblasts into which at least one reprogramming factor selected from Gata4, Mef2c, Tbx5, Mesp1, and Myocd polypeptide in this embodiment has been introduced is 1 to 4 weeks after gene transfer (virus infection), or It is preferable to carry out over this.
  • culturing in a serum-free medium as described later during this period differentiation induction into myocardial cells is efficiently performed, and beating myocardial cells can be efficiently produced.
  • the serum-free medium is preferably added with cytokines as described below.
  • culture may be performed in a medium containing serum for 1 to 2 weeks after gene transfer.
  • the serum is not particularly limited, and for example, fetal bovine serum (FBS), calf serum (NCS), horse serum and the like can be used.
  • Examples of the medium to which serum is added include Dulbecco's modified Eagle medium (DMEM), Iskov modified Dulbecco medium (IMDM), DMEM / M199 medium, and the like, but are not limited thereto. .
  • DMEM Dulbecco's modified Eagle medium
  • IMDM Iskov modified Dulbecco medium
  • M199 medium examples of the medium to which serum is added.
  • the medium used in the method for producing cardiac muscle-like cells of the present embodiment is a medium that does not contain serum, that is, a serum-free medium.
  • a serum-free medium for example, StemPro34 serum-free medium (manufactured by Gibco) can be used, but is not limited thereto.
  • the serum-free medium preferably contains at least either a cytokine or a small molecule compound.
  • VEGF vascular endothelial growth factor
  • FGF2 basic fibroblast growth factor
  • FGF10 fibroblast growth factor-10
  • JAK inhibitor JAK inhibitor
  • IWP4 IWP4
  • the myocardial cells can be confirmed by, for example, confirming whether or not the cells are beating with a microscope, or expressing a myocardial specific marker gene by a known method such as FACS method, RT-PCR method, immunostaining method, microarray assay method, etc. It can be performed by checking whether or not it is done, but is not limited to these methods.
  • myocardial specific marker gene examples include, but are not limited to, Myh6, cTnT, RyR2, HCN4, Actc1, Gja1, Col1a2, and the like.
  • composition for producing myocardial cells of the present embodiment is a Sendai virus vector having a nucleic acid encoding one or more reprogramming factors selected from Gata4, Mef2c, Tbx5, Mesp1, and Myocd polypeptide in the genome, or Fibroblasts into which one or more reprogramming factors selected from Gata4, Mef2c, Tbx5, Mesp1, and Myocd polypeptides have been introduced.
  • the composition of this embodiment may further include a salt, a buffer, a stabilizer, a cell membrane and / or cell wall preserving compound, a nutrient medium, a biocide, a medium such as deionized water, and the like.
  • mice Female ICR mice (CLEA Japan, Inc.) 7 to 10 weeks old were mated with ⁇ MHC-GFP transgenic mice (male). The day when fertilization was confirmed was defined as day 0 of pregnancy, and fetuses were removed from ICR mice that became pregnant 12 days after the confirmation of pregnancy.
  • the heart was removed from the fetus, and the heart was projected with fluorescence with an inverted microscope (manufactured by Olympus, IX71) to select a fetus that emits GFP fluorescence. From the selected fetus, extremities and parenchymal organs such as the head 1/2 to 2/3, lung, liver, kidney, intestinal tract and the like were removed.
  • the remaining trunk tissue was washed with PBS (-) (manufactured by WAKO, 045-29795) to sufficiently remove blood cell components, and the tissue was sheared as finely as possible with sterilized surgical scissors.
  • a medium for MEF (10% FBS / DMEM / PSA) shown in Table 1 and seeded in a 10 cm tissue culture dish (Thermo Scientific, 172958). (For 2 to 3 fetuses, one 10cm dish) Thereafter, the cell precipitate was cultured under the conditions of 37 ° C. and 5% CO 2 . The next day, the medium was changed to a new medium for MEF, and thereafter the medium was changed every 3 to 4 days.
  • a medium for MEF 10% FBS / DMEM / PSA
  • Migrated cells were collected with 0.25% trypsin-EDTA (Gibco, 25200-072), and filtered using a 40 ⁇ m cell strainer (BD, REF352340) to obtain mouse embryonic fibroblasts (MEF).
  • BD 40 ⁇ m cell strainer
  • MEF mouse embryonic fibroblasts
  • the MEF isolated in Experiment 1 was seeded (0.5 ⁇ 10 5 cells / well) in a 12-well cell culture multiwell plate (manufactured by FALCON, 353043) and allowed to stand overnight. Thereafter, the medium in the well was removed by aspiration, and the medium was replaced with a retrovirus mixed solution obtained by mixing the same amount of each gene retrovirus solution (Gata4, Mef2c, Tbx5) at 37 ° C. under 5% CO 2 condition. Then, it was left to stand overnight and cultured to infect the virus (infection).
  • a retrovirus mixed solution obtained by mixing the same amount of each gene retrovirus solution (Gata4, Mef2c, Tbx5) at 37 ° C. under 5% CO 2 condition. Then, it was left to stand overnight and cultured to infect the virus (infection).
  • the purified fragment was digested with Not I and separated by agarose gel electrophoresis, and then a band of about 1.4 kbp was cut out and purified with a QIAquick Gel Extraction kit.
  • the pSeV18 + / TS ⁇ F plasmid having a transgene insertion site (NotI site) upstream of the NP gene was digested with NotI and purified with the QIAquick PCR purification kit, then treated with alkaline phosphatase, and again into the QIAquick PCR purification kit. And purified.
  • the purified Gata4 fragment was ligated to the Not I site of the pSeV18 + / TS ⁇ F plasmid, cloned into E. coli and cloned, and a clone with the correct nucleotide sequence was selected by sequencing to obtain the pSeV18 + Gata4 / TS ⁇ F plasmid.
  • the Sendai virus prepared from the transcription product of the pSeV18 + Gata4 / TS ⁇ F plasmid is referred to as SeV18 + Gata4 / TS ⁇ F (hereinafter also referred to as Sendaivirus ⁇ F Gata4 vector, SeV-G).
  • the obtained Sendai virus solution was rapidly frozen in liquid nitrogen and stored at ⁇ 80 ° C.
  • Mef2c_A12G_N (5′-CTATGGGGAGAAAGAAGATTCAGATTACG-3 ′ (SEQ ID NO: 3)
  • Mef2c_A12G_C (5′-CGTAATCTGAATCTTCTTTCTCCCCATAG-3 ′ (SEQ ID NO: 4)) as primers for the 12th mutagenesis, and the 348th mutagenesis
  • Mef2c_A348G_N 5′-GAAGAAAAATACAAGAAAATTAATGAAG-3 ′ (SEQ ID NO: 5)
  • Mef2c_A348G_C (5′-CTTCATTAATTTTCTTGTATTTTTCTTC-3 ′ (SEQ ID NO: 6)) were used as primers.
  • Mef2cmut gene with mutations introduced in two places as described above as a template
  • Mef2c-N (5'-attGCGGCCGCGACGACACTATGGGGAGAAAAAAGATTC-3 '(SEQ ID NO: 7)
  • Mef2c-C (5'-attGCGGCCGCGATGAACTTTCCGCGCTAAGTTTTTCTTGCTCG (SEQ ID NO: 8)) as a primer and KOD-Plus-Ver.
  • the amplified fragment was purified with the QIAquick PCR purification kit.
  • the purified fragment was digested with Not I and separated by agarose gel electrophoresis, and then a band of about 1.4 kbp was cut out and purified with a QIAquick Gel Extraction kit.
  • the pSeV18 + / TS ⁇ F plasmid having a transgene insertion site (NotI site) upstream of the NP gene is digested with Not I and purified with the QIAquick PCR purification kit, then treated with alkaline phosphatase, and again treated with the QIAquick PCR purification kit. It refine
  • the purified Mef2c fragment was ligated to the Not I site of the pSeV18 + / TS ⁇ F plasmid, cloned into E.
  • the Sendai virus prepared from the transcription product of the pSeV18 + Mef2c / TS ⁇ F plasmid is referred to as SeV18 + Mef2c / TS ⁇ F (hereinafter also referred to as Sendaivirus ⁇ F Mef2c vector, SeV-M).
  • the obtained Sendai virus solution was rapidly frozen in liquid nitrogen and stored at ⁇ 80 ° C.
  • the amplified fragment was purified with the QIAquick PCR purification kit.
  • the purified fragment was digested with Not I and separated by agarose gel electrophoresis, and then a band of about 1.6 kbp was cut out and purified with a QIAquick Gel Extraction kit.
  • the pSeV18 + / TS ⁇ F plasmid having a transgene insertion site (NotI site) upstream of the NP gene is digested with NotI and purified with the QIAquick PCR purification kit, then treated with alkaline phosphatase, and again treated with the QIAquick PCR purification kit. It refine
  • the purified Tbx5 fragment was ligated to the Not I site of the pSeV18 + / TS ⁇ F plasmid, cloned into E.
  • SeV18 + Tbx5 / TS ⁇ F A Sendai virus produced from the transcription product of the pSeV18 + Tbx5 / TS ⁇ F plasmid is referred to as SeV18 + Tbx5 / TS ⁇ F (hereinafter also referred to as Sendaivirus ⁇ F Tbx5 vector, SeV-T).
  • SeV18 + Tbx5 / TS ⁇ F Sendaivirus ⁇ F Tbx5 vector, SeV-T.
  • the obtained Sendai virus solution was rapidly frozen in liquid nitrogen and stored at ⁇ 80 ° C.
  • PCR is performed under the conditions of 94 ° C for 2 minutes, 30 cycles of 98 ° C for 10 seconds, 55 ° C for 30 seconds, 68 ° C for 1.5 minutes), 68 ° C for 5 minutes, 4 ° C ⁇
  • the Gata4 gene fragment (about 1.5 kbp) was purified with the QIAquick PCR purification kit.
  • the Mef2c gene fragment was prepared by PCR using the Mef2c gene mounted on the pSeV18 + Mef2c / TS ⁇ F plasmid as a template.
  • Gata4_Mef2c_N (5′-GAAAAACTTAGGGTGAAAGTTCATCCACGTACACTTGTAATGGGGAGAAAGAAGATTCAG-3 ′ (SEQ ID NO: 13)) and Mef2c_Tbx5_C (5′-CTTTCACCCTAAGTTTTTCTTACTACGGTCATGT TC-TC: V PCR is performed under the conditions of 94 ° C for 2 minutes, 30 cycles of 98 ° C for 10 seconds, 55 ° C for 30 seconds, 68 ° C for 1.5 minutes), 68 ° C for 5 minutes, 4 ° C ⁇
  • the obtained Mef2c gene fragment (about 1.4 kbp) was purified with the QIAquick PCR purification kit.
  • the Tbx5 gene fragment was prepared by PCR using the Tbx5 gene mounted on pSeV18 + Tbx5 / TS ⁇ F DNA as a template.
  • Gata4-Mef2c gene fragment was prepared by PCR using the Gata4 gene fragment and Mef2c gene fragment prepared by the above-described PCR as templates.
  • SeVF6 and Mef2c_A348G_C (5'-CTTCATTAATTTTCTTGTATTTTTCTTC-3 '(SEQ ID NO: 17)) as primers, KOD-Plus-Ver.
  • PCR was performed under the conditions of 94 ° C for 2 minutes, 98 ° C for 10 seconds, 55 ° C for 30 seconds, 68 ° C for 2 minutes, 30 cycles, 68 ° C for 5 minutes, 4 ° C ⁇
  • the Gata4-Mef2c gene fragment (about 1.9 kbp) was purified using the QIAquick PCR purification kit.
  • the Mef2c-Tbx5 gene fragment was prepared by PCR using the Mef2c gene fragment and the Tbx5 gene fragment as a template.
  • Mef2c_A348G_N (5'-GAAGAAAAATACAAGAAAATTAATGAAG-3 '(SEQ ID NO: 18)) and SeVR199 as primers
  • KOD-Plus-Ver. 2 was used, and PCR was performed under the conditions of 30 cycles of 94 ° C. for 2 minutes, (98 ° C. for 10 seconds, 55 ° C. for 30 seconds, 68 ° C. for 3 minutes), 68 ° C. for 5 minutes, and 4 ° C. for ⁇
  • the Mef2c-Tbx5 gene fragment (about 2.7 kbp) was purified using the QIAquick PCR purification kit.
  • PCR was performed using the Gata4-Mef2c gene fragment and the Mef2c-Tbx5 gene fragment as a template.
  • SeVF15 (5'-AAAACATGTATGGGATATGT-3 '(SEQ ID NO: 19)
  • SeVR150 5'-AATGTATCGAAGGTGCTCAA-3' (SEQ ID NO: 20)
  • PCR is carried out under the conditions of 94 ° C for 2 minutes, 30 cycles of 98 ° C for 10 seconds, 55 ° C for 30 seconds, 68 ° C for 4.5 minutes, 68 ° C for 5 minutes, 4 ° C for ⁇
  • the Gata4-Mef2c-Tbx5 gene fragment (about 4.5 kbp) was purified using the QIAquick PCR purification kit.
  • this purified fragment was digested with Not I and separated by agarose gel electrophoresis, and then the Gata4-Mef2c-Tbx5 gene fragment was purified with QIAquick Gel Extraction kit.
  • a pSeV (PM) / TS ⁇ F plasmid having a transgene insertion site (NotI site) between P gene and M gene was digested with Not I and purified with QIAquick PCR purification kit, and then treated with alkaline phosphatase, It was purified again using the QIAquick PCR purification kit.
  • the purified Gata4-Mef2c-Tbx5 gene fragment was ligated to the Not I site of the pSeV (PM) / TS ⁇ F plasmid, transformed into E. coli, cloned, and a clone with the correct nucleotide sequence was selected by sequencing.
  • pSeV (PM) Gata4-Mef2c-Tbx5 / TS ⁇ F plasmid was obtained.
  • the Sendai virus prepared from the transcription product of pSeV (PM) Gata4-Mef2c-Tbx5 / TS ⁇ F plasmid is referred to as SeV (PM) Gata4-Mef2c-Tbx5 / TS ⁇ F (hereinafter referred to as Sendaivirus ⁇ F Gata4-Mef2c-Tbx5 ).
  • the obtained Sendai virus solution was rapidly frozen in liquid nitrogen and stored at ⁇ 80 ° C.
  • the MEF isolated in Experiment 1 was seeded (0.5 ⁇ 10 5 cells / well) in a 12-well cell culture multiwell plate (manufactured by FALCON, 353043) and allowed to stand overnight. Thereafter, the medium in the well was removed by aspiration, and the medium was replaced with a DMEM / Sendai virus mixture. The cells were statically cultured overnight at 37 ° C. and 5% CO 2 to infect the virus (infection). The resulting products are referred to as “SeV-G / M / T-MEF” and “SeV-GMT-MEF”, respectively.
  • [Evaluation 1] FACS analysis method
  • Mouse embryonic fibroblasts (MEF) were cultured in SeV-G / M / T, SeV-GMT Sendai virus solution, and retrovirus solution (mixture of Gata4, Mef2c, Tbx5), and after 1 week, The medium was aspirated. Thereafter, the plate was washed with PBS ( ⁇ ), 0.5 mL of 0.25% trypsin-EDTA was added to each well, and the plate was allowed to stand at 37 ° C. under 5% CO 2 for 5 minutes.
  • the cells were neutralized with 1 mL of a medium for MEF (10% FBS / DMEM / PSA) shown in Table 1, and the cells were collected in a 15 mL tube (Corning, 430791).
  • a medium for MEF 10% FBS / DMEM / PSA
  • the collected cells were centrifuged at 4 ° C. (1500 rpm, 5 minutes). After aspirating the supernatant, 350 ⁇ L of the FACS enforcement solution (5% FBS / PBS) shown in Table 5 was added and mixed thoroughly. This suspension was filtered with a 5 mL polystyrene round tube with a cell strainer cap (FALCON, REF 353335) to obtain a sample for FACS.
  • FACS enforcement solution 5% FBS / PBS
  • GFP positive cells and cTnT positive cells in the above samples were measured using flow cytometry (Nippon Becton Dickinson, FACS Calibur).
  • FIG. 1 is a FACS analysis for the expression of cardiac troponin T (cTnT).
  • A is a figure which shows the result of a control experiment.
  • B shows the results of culturing mouse fetal fibroblasts (MEF) in a Sendai virus solution of SeV-G / M / T (mixture of Gata4, Mef2c, Tbx5).
  • C shows the results of culturing mouse fetal fibroblasts (MEF) in a Sendai virus solution of SeV-GMT.
  • (D) is the result of culturing mouse fetal fibroblasts (MEF) in a retrovirus solution (mixture of Gata4, Mef2c, Tbx5) for comparison. Compared with (b), the number of cTnT positive cells and the cTnT intensity are increased in (c).
  • FIG. 2 (a) shows the results of the control experiment.
  • B is a fluorescence microscope image 4 weeks after virus infection of mouse fetal fibroblasts (MEF) with a Sendi virus solution of SeV-GMT.
  • C is a fluorescence microscopic image 4 weeks after virus infection of mouse fetal fibroblasts (MEF) with a retrovirus solution (mixture of Gata4, Mef2c, Tbx5) for comparison.
  • cardiomyocyte-like cells can be induced by introduction of GMT3 gene with a retroviral vector (WO2011 / 139688), but myocardial protein positive (cTnT, ⁇ -) can also be obtained with Sendai virus vector (SeV-GMT). Actinin), a unique striated structure appeared, and the induction of myocardial cells was confirmed.
  • FIG. 3 is a fluorescence microscope image showing the result of introduction of the reprogramming factor. Gata4 expressing cells (green) and Mef2c expressing cells (red) are shown, respectively. DAPI (blue) indicates the nucleus of all cells. The merge image shows the overlay of each image.
  • A is a fluorescence microscope image showing introduction of each factor of GMT into cells by a retroviral vector for comparison.
  • B is a fluorescence microscope image showing introduction of G, M, and T factors into cells by a Sendi virus solution of SeV-G / M / T.
  • C) and (d) are fluorescence microscopic images showing introduction of G, M, and T factors into cells by a SeV-GMT Sendai virus solution.
  • (c) and (d) confirm the introduction of G, M, and T factors into more cells.
  • the merge image of (b) is compared with the merge images of (c) and (d)
  • the cell and the Mef2c-expressing cell match, and the gene transfer efficiency is improved.
  • the factor introduction efficiency into the cells is improved by changing the cell density and the passage number.
  • FIG. 4 is a fluorescence microscope image one week after mouse embryo fibroblasts (MEF) were infected with SeV-GMT Sendai virus solution. Compared to FIG. 2 (b), it was confirmed that a myocardial protein-positive (cTnT, ⁇ -Actinin), a unique striated structure appeared, and myocardial-like cells were induced within 1 week after GMT3 gene transfer. did it.
  • MEF mouse embryo fibroblasts
  • the GMT3 gene When the GMT3 gene is introduced with a Sendai virus vector, it is not clear why the myocardial cells are induced earlier than when introduced with a retrovirus, but the Sendai virus vector is a non-chromosomal viral vector. In addition, since the vector is expressed in the cytoplasm and the transgene is not integrated into the host chromosome (nucleus-derived chromosome), the time required for myocardial induction is expected to be short.
  • FIG. 5 (a) is a graph showing changes in the number of beating myocardial cells over time.
  • GMT Sendai virus vector
  • (B) is a graph showing the number of beating myocardial cells as of 19 days after infection. Compared with the myocardial cells in which the GMT gene was introduced by the conventional retroviral vector, the number of pulsating cells of the myocardial cells induced by the Sendai virus vector (SeV-GMT) in the present invention increased by 40 to 80 times. I can confirm that.
  • FIG. 6 is a FACS analysis on the expression of cardiac troponin T (cTnT) as a result of in vitro induction from human dermal fibroblasts.
  • A is a figure which shows the result of a control experiment.
  • B shows the result of culturing cells into which Gata4, Mef2c, and Tbx5 were introduced with a Sendi virus solution of SeV-GMT.
  • C shows the result of culturing cells in which miR133 was further introduced in addition to the introduction of Gata4, Mef2c, and Tbx5 by SeV-GMT.
  • (D) shows the result of culturing cells into which Mesp1 and Myocd were further introduced in addition to the introduction of Gata4, Mef2c, and Tbx5 by SeV-GMT.
  • (E) is the result of culturing cells into which miR133 was further introduced in addition to (d).
  • the number of cTnT positive cells and cTnT intensity by introducing Mesp1, Myocd, and miR133 in addition to Gata4, Mef2c, and Tbx5 Can be confirmed to increase.
  • FIG. 7 is a fluorescence microscopic image when cells in which Mesp1, Myocd, and miR133 were further introduced into human skin fibroblasts in vitro in addition to the introduction of Gata4, Mef2c, and Tbx5 by SeV-GMT in vitro.
  • Myocardial protein positive ( ⁇ -actinin) a unique striated structure appeared, and induction of myocardial cells was confirmed.
  • FIG. 8 is a diagram showing a modification of the present invention.
  • MEFs mouse fetal fibroblasts
  • SeV-GMT Sendai virus solution and introducing Gata4, Mef2c, and Tbx5
  • MOI multipleplicity of infection
  • the change in the number of heartbeat-like cells Since it can be confirmed that the number of beating cells increases depending on the seeding density of fibroblasts and the virus infection concentration, the above cells can be cultured while changing the seeding density of fibroblasts or MOI. good.
  • FIG. 9 is a diagram showing a modification of the present invention.
  • MEF mouse fetal fibroblasts
  • FIG. 10 is a diagram showing a modification of the present invention. Beating when using serum-free and serum media when introducing mouse 4, fief, and Tbx5 genes into mouse fetal fibroblasts (MEF) with SeV-GMT Sendai virus solution It is a comparison figure which shows the number of myocardial cells.
  • (A) is the case where FFV medium (serum-free medium) shown in Table 4 is used, and (b) is the case where fetal bovine serum (FBS) is added to Dulbecco's modified Eagle medium (DMEM) (serum) Medium).
  • FFV medium serum-free medium
  • DMEM Dulbecco's modified Eagle medium
  • the use of serum-free medium significantly increased the number of beating myocardial cells as compared to the case of using serum medium.
  • Production of the myocardial cells of the present invention may be performed using a conventional serum medium, but may also be performed using a serum-free medium.

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Abstract

Le problème abordé par la présente invention est de pourvoir à un procédé et à un vecteur recombiné capables de produire efficacement des cellules de type myocardique. La solution selon l'invention porte sur un procédé de production de cellules de type myocardique, le procédé étant caractérisé par l'utilisation d'un ou de plusieurs vecteurs du virus Sendai pour introduire dans des fibroblastes un ou plusieurs facteurs de reprogrammation choisis parmi les polypeptides Gata4, Mef2c, Tbx5, Mesp1, et Myocd, le ou les vecteurs du virus Sendai ayant dans leur génome un acide nucléique qui code pour ledit ou lesdits facteurs de reprogrammation. Une composition pour la production de cellules de type myocardique, la composition comprenant un vecteur du virus Sendai ayant un génome viral qui contient un ou plusieurs facteurs de programmation choisis parmi les polypeptides Gata4, Mef2c, Tbx5, Mesp1, et Myocd est en outre décrite.
PCT/JP2015/077664 2014-10-06 2015-09-30 Procédé de production de cellules de type myocardique, et composition destinée à être utilisée dans ledit procédé pour la production de cellules de type myocardique WO2016056438A1 (fr)

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WO2019151097A1 (fr) * 2018-01-30 2019-08-08 株式会社片岡製作所 Procédé de production de cardiomyocytes
JPWO2019151097A1 (ja) * 2018-01-30 2021-01-14 株式会社片岡製作所 心筋細胞の製造方法
US11834679B2 (en) 2018-01-30 2023-12-05 Kataoka Corporation Method for producing cardiomyocytes

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