WO2020171222A1 - Procédé d'intégration d'un gène étranger long dans une région sûre d'une cellule souche pluripotente humaine et lui permettant de fonctionner normalement dans cette dernière - Google Patents

Procédé d'intégration d'un gène étranger long dans une région sûre d'une cellule souche pluripotente humaine et lui permettant de fonctionner normalement dans cette dernière Download PDF

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WO2020171222A1
WO2020171222A1 PCT/JP2020/007164 JP2020007164W WO2020171222A1 WO 2020171222 A1 WO2020171222 A1 WO 2020171222A1 JP 2020007164 W JP2020007164 W JP 2020007164W WO 2020171222 A1 WO2020171222 A1 WO 2020171222A1
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cells
gene
cell
differentiation
promoter
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健一郎 小戝
薫 三井
佳菜子 井手
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国立大学法人 鹿児島大学
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    • 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
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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Definitions

  • the present invention relates to the field of efficiently and comprehensively removing tumorigenic cells from human pluripotent stem cells after differentiation induction.
  • hPSCs human pluripotent stem cells
  • hESCs human embryonic stem cells
  • hiPSCs human induced pluripotent stem cells
  • hPSCs have high plasticity to form teratomas, and that gene mutation cells that lead to carcinogenesis may also occur in culture due to the instability of the chromosome, and results similar to those in clinical studies using hematopoietic stem cells are obtained. It may also occur in regenerative medicine using hPSC. Therefore, in addition to the conventional strategy of "reducing" the mixture of remaining undifferentiated cells (tumor-causing cells), such as improving the hPSC culture method and improving the differentiation-inducing method, these tumorigenic cells are "direct” and “directly”. There is a demand for new technology development that specifically and safely kills and removes.
  • non-viral DNA delivery method in which plasmid vector is introduced by methods such as electroporation and lipofection, and infection transfer method using virus vector are mainly used.
  • the efficiency of gene transfer into hPSC is higher in the method using a viral vector than in the non-viral DNA delivery method.
  • Lentivirus vectors and retrovirus vectors are extremely suitable for establishing long-term stable expression cells because they incorporate a foreign (target) gene into the hPSC genome.
  • TC-LV tumorigenic cell targeting lentiviral vector
  • Patent Document 1 Non-Patent Document 1
  • Patent Document 4 a tumorigenic cell targeting lentiviral vector (TC-LV) platform containing a reporter gene and a suicide gene and having a promoter region in a recombination cassette
  • the safe harbor region on the genome that is, it is known that gene insertion does not cause phenotypic changes in cells. More probably, "Since the chromosome part to be introduced into the gene has been determined to be the same, the effects of the chromosome structure and epigetics are also the same, so the expression of the introduced gene is stable and high ( It is likely that it will not be suppressed easily.) Inserting a foreign gene, a reporter and a suicide gene, that is, developing a technology to knock in a foreign target gene to a specific chromosomal site called the so-called safe harbor region. Was needed.
  • Genome editing technology that was performed using zinc finger nuclease or TALEN has dramatically developed in recent years with the advent of CRISPR/Cas9. However, even when CRISPR/Cas9 is used, gene editing efficiency is low in hPSC as compared with differentiated cells such as 293T cells and K562 cells. Most studies of gene modification using genome editing technology have been carried out by gene knockout by a non-homologous end joining (NHEJ) mechanism. Using genome editing technology, a foreign (target) gene can be identified by homologous recombination. There are very few reports of knocking in at the chromosomal site (Non-Patent Document 5). Ruan, J et al.
  • Non-Patent Document 6 In primate pluripotent stem cells, CRISPR/Cas9-mediated genome editing using rhesus iPS cells has been reported, but only expression of the reporter gene was confirmed (Non-Patent Document 7). Therefore, there has been no report to date of incorporating a foreign (target) gene having a long nucleotide sequence into hPSC and expressing not only a marker gene but also a gene that functions in a cell such as a suicide gene.
  • the labeling and removal methods of undifferentiated cells remaining in the differentiated cells that is, the tumorigenesis-causing cells have problems in efficiency and simplicity. Workable methods for identification and removal are still under development. So far, the present inventors have used the lentivirus vector to efficiently search and identify a promoter that can specifically function in undifferentiated cells and to demonstrate the same, and to selectively visualize undifferentiated cells.
  • the TC-LV method which enables identification of suicide genes that reliably and selectively kills tumorigenic cells when required by high-throughput analysis (that is, comprehensive analysis with orders of magnitude higher efficiency).
  • HPSCs into which a foreign (target) gene having a long base sequence has been introduced will be introduced into an important part of the genome and inhibit or lose important normal molecule functions, or overactivate genes that lead to carcinogenesis.
  • a stable expression pattern at an expression level expected by a foreign (target) gene having a long nucleotide sequence is added to the optimally identified genomic site.
  • the present invention efficiently and simply removes undifferentiated cells remaining in differentiated cells, that is, tumorigenic cells when hPSCs are induced to differentiate into target cells without affecting the traits of hPSCs.
  • the purpose is to provide a method.
  • the present invention selectively visualizes/identifies undifferentiated cells, which are represented by optimal candidate killing gene units found by comprehensive analysis by genetic recombination using TC-LV platform technology, and selects when necessary.
  • the sequence having a suicide gene that reliably kills tumorigenic cells is safely and efficiently inserted into the genome-safe harbor region of hPSC, and the normal function of the inserted hPSCs. It is intended to provide a method for efficiently and comprehensively removing tumorigenic cells remaining after differentiation into target cells without inhibiting the genes that maintain the cells and without the risk of carcinogenesis due to gene insertion.
  • this technique not only kills tumor-causing cells, but also safely and efficiently inserts a foreign (target) gene and a long gene unit such as its expression control promoter into the genome safe harbor region of hPSC. It imparts the proper expression of the foreign (target) gene and the ability to exert the expected function without inhibiting the gene that maintains the normal function of hPSC and without the risk of carcinogenesis due to gene insertion.
  • the present invention is also aimed at this new technology.
  • the present inventors efficiently insert a tumorigenic cell targeting sequence, which is a foreign (target) gene having a long base sequence, into the safe harbor region in the hPSC genome by homologous recombination via CRISPR/Cas9. I succeeded in doing it for the first time.
  • a tumorigenic cell targeting sequence which is a foreign (target) gene having a long base sequence
  • CRISPR/Cas9 for example, it was reported that a marker gene such as a fluorescent protein or ⁇ hCD19 was introduced into rhesus monkey iPSC (Hong, SG et al. 2017), supra), but no example of introduction into hPSC has been reported.
  • the safe harbor region has been inferred and reported as a region that does not cause phenotypic changes in cells due to gene insertion and that the introduced gene is less susceptible to expression suppression.
  • TC-LV lentiviral vector
  • the present invention relates to: (1) hPSC containing a promoter having strong activity in human cells and a foreign gene functionally linked to the promoter in a safe harbor region in the genome.
  • the remaining undifferentiated cells and/or existing tumorigenic cells that are contained in hPSCs after differentiation induction treatment are selectively treated with the foreign gene.
  • a method of expressing A method comprising culturing a cell group obtained by subjecting the cells according to (1) to (5) to differentiation induction treatment.
  • the level of the drug to be contacted is such that the survival rate of the differentiated cells after the drug treatment is three times or more higher than the survival rate of the undifferentiated cells and/or tumorigenic cells after the drug treatment, The method according to (11).
  • (13) To selectively damage remaining undifferentiated cells and/or existing tumorigenic cells contained in hPSCs after differentiation induction treatment (cell group obtained by the differentiation induction treatment of hPSCs)
  • the level of a drug corresponding to the suicide gene which is brought into contact with hPSC after differentiation induction treatment, wherein among the cells according to (4) or (5) after differentiation induction treatment, undifferentiated cells and/or tumorigenesis
  • Differentiation-inducing treatment of the obtained suicide gene-introduced hPSC Contacting each of the obtained differentiation-inducing treated cells and each of the suicide gene-introduced hPSCs that have not been differentiation-inducing treated with a plurality of levels of the drug,
  • the level of the drug having a high cytotoxic effect in the cells that have not been subjected to the differentiation induction treatment is higher than that in the cells that have been subjected to the differentiation induction treatment, and it is highly likely that the undifferentiated cells and/or the tumorigenic cells are damaged. , And determining as a level that is likely not to damage the differentiated cells.
  • a method for selectively damaging remaining undifferentiated cells and/or existing tumorigenic cells contained in hPSCs after differentiation induction treatment An undifferentiated cell and/or a tumor contained in the cell group by contacting a cell obtained by subjecting the cell according to (4) or (5) to differentiation induction treatment, with a drug corresponding to the suicide gene Including selectively damaging the cells that cause aging,
  • the method, wherein the level of the drug to be contacted is the level determined in (13) or (14).
  • a cell group of hPSCs containing the herpesvirus-derived thymidine kinase gene in the safe harbor region of the genome (differentiation induction treatment of hPSCs containing the herpesvirus-derived thymidine kinase gene in the safe harbor region of the genome is performed.
  • a group of cells thus obtained which selectively damages the undifferentiated cells remaining in the cells and/or the tumorigenic cells present.
  • the cells obtained by subjecting the cells according to (4) or (5) to differentiation induction treatment are brought into contact with 0.01 to 1 ⁇ g/mL ganciclovir to give undifferentiated cells and/or Alternatively, a method comprising selectively damaging tumorigenic cells.
  • a method for selectively injuring remaining undifferentiated cells and/or existing tumorigenic cells contained in hPSCs after differentiation induction treatment (cell group obtained by the differentiation induction treatment of hPSCs) And Among the cells according to (4) or (5) after the differentiation induction treatment, the method according to (13) or (14) has a high possibility of damaging undifferentiated cells and/or tumorigenic cells. Determining the level of said drug, which is likely not to damage the differentiated cells, Differentiating the cells according to (4) or (5).
  • a method comprising selectively injuring remaining undifferentiated cells and existing undifferentiated cells and/or tumorigenic cells by contacting the differentiation-inducing treated cells with the drug at the level.
  • a structure contained in the donor used for homologous recombination in the AAVS1 region The structures of a donor vector in which a CA promoter, a Survivin (Surv) promoter, and a Nanog promoter are inserted into a basal donor vector and a recombination cassette (RC) of the basal donor vector are shown in order from the top.
  • RC utilizes a site-specific recombination system involved in the entry of ⁇ phage into the Escherichia coli chromosome and carries out an exchange reaction of a DNA sequence sandwiched between modified att sequences between vectors (James, L. et al. (2000). ) Genome Res. 10: 1788-1795).
  • the RC part has a DNA sequence (att R sequence) that interacts specifically with the recombination at both ends, and has a chloramphenicol resistance gene (CMR) and an Escherichia coli suicide gene ccdB between them.
  • CMR chloramphenicol resistance gene
  • LA a sequence having homology with the flanking sequence on the left side of the double-strand break site in the AAVS1 region
  • SA-2A-Puro expression cassette for puromycin selection (SA; splicing acceptor sequence, 2A: self-processing peptide) Sequence
  • Puro puromycin resistance gene
  • pA poly A sequence
  • Venus Venus expression region under the control of each promoter
  • HSVtk herpes simplex virus thymidine kinase (which causes cytotoxicity by phosphorylating GCV).
  • Expression region, RA a sequence having homology to the flanking sequence on the right side of the double-strand break site in the AAVS1 region.
  • CA CA promoter inserted in RC.
  • the same abbreviations as in FIG. 1 are used for LA, SA-2A-Puro, pA, Venus, HSVtk, RA.
  • B Genomic DNA was extracted from the resulting puromycin-resistant clones and subjected to PCR screening for homologous recombination. Since the reverse primer was placed inside the knock-in gene, it was reflected in the 1539 bp PCR amplicon when HDR was successful.
  • VH-doner, pSurv. VH-doner, pNanog. 6 is a photograph showing Venus expression in a puromycin-resistant clone obtained by homologous recombination via CRISPR/Cas9 using VH-doner. pCA. Although Venus expression was observed in hESC (CA.VH) into which VH-doner was introduced, pSurv. VH-doner and pNanog. In hESCs (Surv. VH, Nanog.
  • FIG. 2 is a photograph showing Venus expression observed by fluorescence microscopy (scale bar, 200 ⁇ m) in the following cells: (a) undifferentiated KhES1 having CA-Venus-2A-HSVtk (CA.VH) in the AAVS1 region and KhES3 clone; (b) KhES3-CA. Embryoid bodies (EB) derived from VH#2 clone; (c) KhES3-CA. VH#2 clone.
  • Non-specific GCV-dependent cytotoxicity independent of HSVtk was observed in undifferentiated wild-type KhES1 at GCV concentrations of 10 and 100 ⁇ g/ml, and in undifferentiated wild-type KhES3 at 100 ⁇ g/ml GCV concentration.
  • Undifferentiated CA VH clones showed no viability over 0.01 ⁇ g/ml GCV containing medium.
  • Undifferentiated Surv Among the VH clones, the KhES1-derived clone showed HSVtk/GCV-dependent cytotoxicity in 1 ⁇ g/ml GCV-containing medium and in the KhES3-derived clone at 10 ⁇ g/ml GCV-containing medium.
  • Undifferentiated Nanog is a type of HSVtk was observed in undifferentiated wild-type KhES1 at GCV concentrations of 10 and 100 ⁇ g/ml, and in undifferentiated wild-type KhES3 at 100 ⁇ g/ml GCV concentration.
  • FIG. 6 is a diagram showing HSVtk/GCV-dependent cytotoxicity and specificity of VH and WT differentiated cells. Non-specific GCV-dependent cytotoxicity independent of HSVtk was observed at a GCV concentration of 100 ⁇ g/ml in both differentiated wild-type KhES1 and KhES3.
  • HSVtk/GCV-dependent cytotoxicity was associated with differentiated CA. It was lower in VH clones and there was no apparent cytotoxicity at GCV concentrations up to 1 ⁇ g/ml. Differentiated Surv. VH and Nanog. In the VH clone, no GCV-dependent cytotoxicity was observed, and nonspecific GCV-dependent cytotoxicity was observed at a GCV concentration of 100 ⁇ g/ml as in the wild type. *P ⁇ 0.005 and **P ⁇ 0.001 (versus GCV). KhES3-CA. It is a figure which shows that a VH clone does not show the teratoma formation effect after in vivo GCV administration.
  • pluripotent stem cell means a cell having pluripotency and self-renewal ability.
  • pluripotency is synonymous with pluripotency and means a state of cells capable of differentiating into cells of multiple lineages by differentiation.
  • pluripotency refers to a state capable of differentiating into all types of cells constituting a living body (totipotency), a state capable of differentiating into all types of cells excluding extraembryonic tissues (differentiation).
  • the “pluripotent stem cells” in the present specification include stem cells, embryonic stem (ES) cells, cloned embryo-derived embryonic stem cells (“ntES cells”) obtained by nuclear transfer, and germline stem cells (“GS cells”). ), embryonic germ cells (“EG cells”), and induced pluripotent stem (iPS) cells, neural stem cells, hematopoietic stem cells, mesenchymal stem cells, liver stem cells, pancreatic stem cells, skin stem cells, muscle stem cells, or germ stem cells including.
  • stem cells include stem cells, embryonic stem (ES) cells, cloned embryo-derived embryonic stem cells (“ntES cells”) obtained by nuclear transfer, and germline stem cells (“GS cells”).
  • GS cells germline stem cells
  • EG cells embryonic germ cells
  • iPS induced pluripotent stem
  • Whether or not a cell is a pluripotent stem cell is determined, for example, when a test cell forms an embryoid body in an in vitro culture system or after culturing under differentiation-inducing conditions (differentiation-inducing treatment). When differentiated into a desired cell, the cell can be determined to be a pluripotent stem cell.
  • differentiation refers to a phenomenon in which a pluripotent cell divides to give rise to a daughter cell having a specific functional or morphological characteristic.
  • differentiation treatment and “differentiation induction treatment” have the same meaning and mean a treatment for inducing stem cells into differentiated cells. Differentiation of cells has been reported to be induced by various methods. Pluripotent cells can be differentiated by a differentiation-inducing treatment using a differentiation-inducing substance or the like, depending on the type of cells to be differentiated.
  • differentiation-inducing substance or the like depending on the type of cells to be differentiated.
  • differentiated cell is meant a daughter cell that has specialized functional or morphological characteristics resulting from differentiation. Differentiated cells are usually stable, their proliferative capacity is low, and differentiation to another type of cell occurs exceptionally.
  • the "undifferentiated cell” means an undifferentiated cell, and is usually synonymous with a stem cell, but the undifferentiated cell in the present specification does not necessarily have to have the above-mentioned stem cell characteristics completely. Rather, it means a cell having a higher (self) proliferation ability than a differentiated cell (for example, a proliferation rate of 2 times or more, 3 times or more, 5 times or more, 10 times or more). Particularly preferably, the "undifferentiated cell” is a "tumorigenic cell".
  • Whether or not a cell is an undifferentiated cell is determined by, for example, determining a cell in which c-Myc is activated, or a cell having a high expression level of telomerase reverse transcriptase and/or survivin as an undifferentiated cell. You can
  • tumor-causing cell is an undifferentiated cell that has not differentiated despite being subjected to a differentiation treatment for inducing differentiation into a stem cell, and is a cell capable of generating a tumor cell.
  • Means Tumor-causing cells are not only cells that maintain the same properties as pre-differentiated stem cells (pluripotent stem cells), but also have properties that are different from undifferentiated cells before differentiation. It also includes cells that have not been grown, such as oligopotent cells (cells that can differentiate only into several types of cells), and cells having tumorigenicity (eg, cancer stem cells).
  • “capable of generating tumor cells” does not necessarily mean that 100% of tumor cells are generated, but means that the ability to generate tumor cells is higher than that of differentiated cells. Whether or not a certain cell has tumorigenicity is determined by, for example, determining a cell having c-Myc activation, or a cell having a high expression level of telomerase reverse transcriptase and/or survivin as a cell having tumorigenicity. Can be determined.
  • undifferentiated cells expressed by “undifferentiated cells remaining after differentiation treatment”, “residual undifferentiated cells”, “undifferentiated cells in cells after differentiation treatment” and the like, particularly differentiation to stem cells
  • the undifferentiated cells remaining after the induction treatment can be “tumor-causing cells”.
  • the "promoter having a strong activity in human cells” is not particularly limited as long as it is a promoter that exerts a strong activity in human cells, and even if it is derived from a human cell, a cell or virus other than human It may be derived.
  • a promoter include a CA promoter (cytomegalovirus enhancer/b-actin promoter with b-actin intron), a survivin promoter, a CMV (cytomegalovirus PGV) promoter, a CBA (chicken betaphalogeric porcine kine), a CBA (chicken beta vulgaris CG), a CBA (chicken beta vulgaris CG), and a CBA (chicken beta LVK) promoter.
  • CA promoter cytomegalovirus enhancer/b-actin promoter with b-actin intron
  • CMV cytomegalovirus PGV
  • CBA chicken betaphalogeric porcine kine
  • CBA chicken beta vulgaris CG
  • LTR promoter LTR promoter, RSV (respiratory syncy virus) promoter, EF1 ⁇ promoter, TERT promoter, E3 promoter, HSV (herpes)
  • the simplex virus) promoter, Nanog promoter, and Rex1 promoter may be mentioned.
  • the promoter in the present application may consist of a part of these promoter sequences as long as it can exhibit the activity as a promoter.
  • a gene is operably linked to a promoter is synonymous with “it is operably linked”, the gene is located downstream of the promoter, and the promoter is It means that the promoter and the gene are linked so that the expression of the downstream gene becomes possible by exerting the expression.
  • Formal gene is a gene that is artificially inserted into hPSC and has a desired function, and includes, for example, a suicide gene, a toxic gene, and a marker gene.
  • “Suicide gene” means a gene that causes toxicity to cells having the gene by undergoing an activation process. Typically, the suicide gene encodes an enzyme that converts an inactive drug into a toxic substance. In addition, the suicide gene is used together with a drug that is converted into a compound that exerts cytotoxicity by its gene product (hereinafter, may be referred to as “drug corresponding to suicide gene” or “prodrug” in the present specification). .. Prodrugs corresponding to each suicide gene are already well known. Particularly preferably, the suicide gene converts a prodrug into a cell growth inhibitor such as a nucleic acid synthesis inhibitor or an antimetabolite. In the present specification, the term prodrug means a prodrug corresponding to a suicide gene with which it is used, unless otherwise required.
  • Examples of combinations of drug-dependent suicide genes and prodrugs (drugs) include herpes simplex virus thymidine kinase, ganciclovir or acyclovir, Varicella Zoster virus thymidine kinase, and 6-methoxypurine arabinonucleoside (Huber et al., 1991, Proc . Natl . Acad . Sci . USA 88 :8039), E.I. coli and cytosine deaminase, fluorouracil (Mullen et al., 1992, Proc Natl Acad Sci USA 89: doi 33), E.
  • the combination of the suicide gene and the drug includes a combination of herpesvirus-derived thymidine kinase gene and ganciclovir, and a combination of cytosine deaminase gene and 5-fluorouracil.
  • thymidine kinase such as HSV-tk
  • GCV ganciclovir
  • HSV-tk thymidine kinase
  • AZT azidothymidine
  • Triphosphoric acid can be generated.
  • cytosine deaminase CD
  • 5-fluorotoxin 5-fluorotoxin
  • 5-FC is metabolized by CD.
  • the toxic substance 5-fluorouricyl (5-FU) can be generated.
  • varicella virus thymidine kinase is used as the drug-dependent suicide gene and 6-methoxypurine arabinonucleoside (Ara-M) is used as the non-toxic prodrug
  • Ara-M is metabolized by varicella virus thymidine kinase and is a toxic substance.
  • Certain 6-methoxypurine arabinonucleosides Ara-MMP
  • the drug-dependent suicide gene of the present invention may be a gene encoding a protein that converts a non-toxic protein into a toxic substance by adding a drug.
  • the drug-dependent suicide gene of the present invention may encode a protein that does not exhibit toxicity with a monomer but exhibits toxicity by forming a multimer (for example, a dimer).
  • the "drug that can exert toxicity by the drug-dependent suicide gene” means a substance that converts the drug-dependent suicide gene into a toxic substance.
  • caspases such as Caspase 9 and Caspase 3 are used as drug-dependent suicide genes
  • Dimerizer when caspases such as Caspase 9 and Caspase 3 are used as drug, caspase-9 dimer, which is a toxic substance by dimerization of caspases by Dimerizer, is used. Can be induced to induce apoptosis.
  • the drug-dependent suicide gene that exerts toxicity by becoming such a multimer for example, dimer
  • Fas receptor Fas receptor and FADD.
  • the “marker gene” means a gene encoding a marker substance capable of detecting or measuring cells into which the gene has been introduced.
  • the marker gene green fluorescent protein (GFP), EGFP (enhanced GFP) or Venus whose labeling ability is further improved by changing the amino acid sequence of GFP, or red fluorescent protein (RFP) exhibiting another fluorescent wavelength, blue Fluorescent protein (BFP), yellow fluorescent protein (YFP), red, green, blue, or yellow fluorescent protein such as mKate2; genes encoding ⁇ -glucuronidase, ⁇ -galactosidase, luciferase, and dihydrofolate reductase. Can be used, and preferably mKate2 or Venus.
  • label means to identify the presence or position of a target cell temporarily or for a long period of time depending on the purpose of use, and to measure the target cell number (may be the amount or ratio). Or, it means that the target cell can be distinguished from other cells.
  • identify means to temporarily or long-term identify the presence or position of a target cell, and “identify” to temporarily or long-term purpose. It is meant to distinguish cells from other cells and tissues.
  • the label in the present specification is not particularly limited as long as it can identify the presence or position of the target cell, or can distinguish the target cell from other cells, and cells other than the target cell are completely stained. It doesn't need not be done.
  • the undifferentiated cells can be distinguished to the extent that the undifferentiated cells can be distinguished from the differentiated cells in the cells. It does not prevent the differentiated cells from being (weakly) stained as long as they are stained.
  • Toxic gene means a gene having the ability to cause toxicity to the introduced cells, and induces any apoptosis-inducing gene (Bax, p53, DP5, PL-3, reaper, hid), cell death. It may be a gene or a gene that suppresses tumor cells. Alternatively, a gene that promotes differentiation induction may be used instead of the “toxic gene”.
  • a sequence capable of simultaneously expressing two genes with one promoter means a sequence capable of translating two kinds of proteins or peptides by activation of one promoter. means.
  • Such a sequence is a sequence having a self-cleaving activity, which causes two genes to be expressed together by the activation of one promoter and is translated as two proteins or peptides linked via the sequence, It may be a sequence in which the two proteins or peptides bound via the sequence are self-cleaving in the region of the sequence, resulting in the translation of the two separated proteins or peptides.
  • a sequence capable of expressing two genes at the same time by one promoter attracts ribosomes and initiates a second translation from the middle of mRNA, causing two different genes from one mRNA to be bicistronic. May be a sequence to be translated into.
  • Examples of the former include “2A sequence”, and examples of the latter include “IRES (internal ribosomal entry site) sequence”.
  • the 2A sequence includes the 2A sequence derived from foot-and-mouth disease virus and the 2A sequence derived from equine rhinitis A virus (Furler, S et al. (2001) Gene Ther. 8,864-73; and Hasegawa et al. 2007) See Stem Cells 25, 1707-12).
  • Recombination cassette means a region in which a gene can be recombined by a recombinase (recombinase), and includes a nucleic acid sequence recognized by the recombinase and a foreign gene insertion site sandwiched between the nucleic acid sequences.
  • the recombination cassette examples include LR recombinations (bacteriophage ⁇ integrase and excisionase, and Escherichia coli integration host factor protein) in which recombination occurs between an attL-terminal DNA fragment and an attR-containing donor vector, And recombinations between the attB-terminal DNA fragment and the attP-containing donor vector, such as BP recombinations (bacteriophage ⁇ integrase and E. coli integration host factor protein) such as ⁇ phage recombinations (Landy, A (1989) Ann. Rev. Biochem.
  • the “safe harbor region” means a region that does not affect cell function, survival, morphology, properties, etc. even when a foreign gene is introduced. Rosa 26, AAVS1 and H11 are known as such regions.
  • the gene can be introduced into the safe harbor region by using a donor vector targeting these regions in the homologous recombination method. (Cells into which the transgene has been introduced)
  • the present invention relates to a hPSC containing a promoter having strong activity in human cells and a foreign gene (eg, suicide gene) operably linked to the promoter in a safe harbor region in the genome.
  • a promoter having a strong activity in human cells and a foreign gene (eg, a suicide gene) functionally linked to the promoter” (hereinafter, sometimes referred to as “transgene”) is a label.
  • a nucleic acid (molecule) containing a gene or suicide gene and a promoter which may be an expression cassette in which the marker gene or the suicide gene is functionally linked to the promoter, or one promoter
  • the expression cassette may further have a recombination cassette containing a promoter region therein.
  • the present invention provides a hPSC containing a promoter having strong activity in human cells and a foreign gene (for example, a suicide gene) functionally linked to the promoter in a safe harbor region in the genome (hereinafter, referred to as “the book”).
  • hPSC a promoter having a strong activity in human cells in a safe harbor region in the genome of hPSC by a homologous recombination method, and a foreign gene functionally linked to the promoter ( For example, a method comprising introducing a suicide gene).
  • the homologous recombination method means homologous recombination utilizing a genome editing technique, and a site-specific nuclease or a combination of a guide RNA and a nuclease is used to form two DNAs at a target site of genomic DNA. It is a method of introducing a strand break and inserting a donor vector into a target site through homologous recombination which is a repair mechanism of cells.
  • Site-specific nucleases include ZFN (Zinc-Finger Nuclease) and TALEN (Transcription Actor-Like Effector Nuclease).
  • ZFN and TALEN are artificial restriction enzymes composed of a DNA-binding domain and a DNA-cleaving domain, and cleave DNA by a dimer.
  • the DNA binding domain usually contains 3 to 6 zinc fingers modified to recognize any 3 base pair DNA sequence, allowing recognition of 9 to 18 base pairs.
  • TALEN utilizes a DNA binding domain that recognizes 15 to 20 bases, which is a modified TAL effector (TALE) secreted by a plant pathogenic bacterium belonging to the genus Xanthomonas.
  • TALE modified TAL effector
  • CRISPR/Cas9 is a method in which a guide RNA and a nuclease are combined.
  • a gene can be introduced into a target site of a genome by introducing Cas9 nuclease (Cas9) responsible for DNA double-strand break, a guide RNA for orienting Cas9 to a target site of 20 bases on a chromosome and a donor vector into a cell.
  • Cas9 nuclease Cas9 nuclease
  • the gene introduction by the homologous recombination method via CRISPR/Cas9 can be carried out by cotransfecting a target cell with a plasmid containing Cas9, a plasmid encoding guide RNA, and a plasmid containing the transgene.
  • the guide RNA is designed to recognize a nucleotide sequence in or near the safe harbor region.
  • the transfection method can be appropriately selected from the methods applicable to hPSC, and can be performed using, for example, FuGENE HD transfection reagent.
  • the composition ratio of each plasmid at the time of transfection be such that the molar ratio is equal (1:1:1).
  • the present invention relates to the present hPSCs after the differentiation inducing treatment, and cells obtained by the differentiation inducing treatment of the present hPSCs (hereinafter, referred to as “differentiation-inducing cells”).
  • Pluripotent cells can be differentiated by a differentiation-inducing treatment using a differentiation-inducing substance or the like, depending on the type of cells to be differentiated.
  • neural stem cells for example, JP 2002-291469 A
  • pancreatic stem-like cells for example, JP 2004-121165 A
  • hematopoietic cells for example, Table 2003-505006
  • differentiation into activated neurons eg STEM CELLS. (2009); 27(4):806-811
  • cardiomyocytes eg Circulation Research. (2012); 111:344-358.
  • the induction method is known.
  • the hPSCs after differentiation induction have a 100% probability of becoming target differentiated cells, but cells that remain undifferentiated cells without actually being differentiated, or cells other than the target differentiated cells, For example, it may include cells that are transformed into tumorigenic cells. Therefore, in the present specification, the “differentiation-inducing cell” does not necessarily mean a differentiated cell, and may be a cell other than the differentiated cell as long as it is a cell obtained as a result of the differentiation-inducing treatment.
  • the present invention relates to a method for selectively expressing a foreign gene in undifferentiated cells and tumorigenic cells contained in differentiation-inducing cells, the method comprising culturing the cells.
  • the present invention is a method for selectively injuring undifferentiated cells and tumorigenic cells contained in differentiation-inducing cells, comprising a differentiation-inducing cell or a cell group consisting of differentiation-inducing cells ( Hereinafter, collectively referred to as "differentiation-inducing cell group”), selectively injuring undifferentiated cells and/or tumorigenic cells contained in the cell group by contacting with a prodrug Regarding
  • the method may include a step of obtaining the differentiation-inducing cells by subjecting the hPSCs to a differentiation-inducing treatment before the step of injuring.
  • the method (as a further preceding step) is, by a homologous recombination method, a safe harbor region in the genome of hPSC, a promoter having a strong activity in the human cell, and a foreign molecule functionally linked to the promoter. It may include a step of introducing a gene (for example, a suicide gene) to obtain the hPSC of the present invention.
  • the contact of the prodrug with the differentiation-inducing cell population can be carried out by adding an appropriate amount of the prodrug to the culture of the differentiation-inducing cell population. Since a promoter having a strong activity is employed in the present invention, the amount of the prodrug can be appropriately controlled so that toxicity to undifferentiated cells and/or tumorigenic cells and non-toxicity to differentiated cells are compatible. Very important.
  • the appropriate amount is an amount by which the final level of the prodrug after addition to the cell culture is such that toxicity to undifferentiated cells and/or tumorigenic cells is stronger than that to differentiated cells.
  • the level means a unit that numerically represents the abundance, and typically represents the concentration, but it may be another unit such as a petri dish or an absolute amount per well, It may be a measurement value itself when the abundance is measured, or a value converted from the measurement value to another numerical value.
  • the level of prodrug damages the majority of undifferentiated cells and/or tumorigenic cells, eg, half or more, 60% or more, 70% or more, 80% or more, 90% or more, or 95% or more.
  • the prodrug level is such that the survival rate of undifferentiated cells and/or tumorigenic cells is 3 times or more, 4 times or more, 5 times or more, 6 times or more, 7 times or more, 8 times or more than that of differentiated cells.
  • the level may be higher than double, higher than 9 times, or higher than 10 times.
  • Appropriate amounts of such prodrugs can be determined by the methods for determining drug levels described below.
  • the appropriate amount of prodrug may also be at a level determined by the methods for determining drug levels described below.
  • the suicide gene is the herpesvirus-derived thymidine kinase gene
  • the appropriate amount of the corresponding prodrug ganciclovir may be 0.01-1 ⁇ g/mL.
  • the present invention relates to a proliferative cell that is highly likely to damage undifferentiated cells and/or tumorigenic cells among differentiation-inducing cells, and that does not damage differentiated cells.
  • a method for determining the level of a drug which comprises contacting a differentiation-inducing cell and the differentiation-untreated hPSC with a plurality of levels of the prodrug, and comparing the differentiation-inducing cell with the prodrug.
  • the level of the drug which has a high cytotoxic effect in cells that have not been subjected to differentiation-inducing treatment, is defined as "it is highly likely to damage undifferentiated cells and/or tumorigenic cells and not damage differentiated cells. High level".
  • the above-mentioned method may include subjecting the present hPSCs to differentiation induction treatment before contacting the prodrug with cells. Further, as a pre-step thereof, it is possible to obtain the hPSC of the present invention by introducing into the safe harbor region of the hPSC genome a suicide gene functionally linked to the promoter that causes the strong expression, by a homologous recombination method. You can leave.
  • the cells that have not been subjected to the differentiation-inducing treatment have a higher cytotoxic effect, and preferably the survival rate of undifferentiated cells and/or tumorigenic cells is higher than the survival rate of differentiated cells. It may mean 3 times or more, 4 times or more, 5 times or more, 6 times or more, 7 times or more, 8 times or more, 9 times or more, or 10 times or more.
  • the method of the present application may include all steps from the determination of an appropriate amount of a prodrug to the selective injury of undifferentiated cells and/or tumorigenic cells. That is, the present invention is a method for selectively damaging undifferentiated cells and/or tumorigenic cells contained in hPSCs after differentiation induction treatment, wherein the hPSCs after differentiation induction treatment are obtained by the above method.
  • determining the level of the drug which has a high possibility of damaging undifferentiated cells and/or tumorigenic cells and is not likely to damage the differentiated cells, and, if necessary, Differentiation-inducing treatment of the present hPSCs, and contacting the differentiation-inducing cells with a determined level of a prodrug to selectively damage undifferentiated cells and/or tumorigenic cells Including the method.
  • the present invention may be a method for removing undifferentiated cells and/or tumorigenic cells derived from the differentiation-inducing cells transplanted into the body of a patient.
  • the method comprises the step of administering to a patient transplanted with differentiation-inducing cells a prodrug capable of exerting toxicity of the suicide gene, which is derived from differentiation-inducing cells transplanted in a patient. It may be a method of killing or removing differentiated cells and/or tumorigenic cells. Alternatively, the method is derived from a differentiation-inducing cell transplanted into a patient, which comprises the step of transplanting the differentiation-inducing cell into a patient, and the step of administering to the patient a prodrug capable of exerting toxicity of the suicide gene. It may be a method of killing or removing undifferentiated cells and/or tumorigenic cells.
  • the dose of the drug capable of exerting toxicity by the suicide gene can be appropriately selected according to the type of each suicide gene, but preferably the final prodrug of the undifferentiated cell and/or the tumorigenic cell is selected.
  • the level is the amount at which toxicity to undifferentiated cells and/or tumorigenic cells is stronger than toxicity to differentiated cells.
  • Administration can be systemic, but local administration is preferred.
  • KhES-1 and KhES-3 hESCs (Suemori, H et al. (2006) Biochem Biophys Res Commun 345:926-932.) were provided by Kyoto University. hESCs were seeded on 60 mm dishes (Corning Japan) coated with Matrigel or human recombinant Laminin 521 and cultured in mTeSR1 medium (Stem Cells Technologies, Vancouver, Canada).
  • hESCs were dispersed in single cells using Accutase® (Innovative Cell Technologies, San Diego, Calif.), followed by the required amount of hESCs, a new Rho-related kinase inhibitor Y-27632 (10 ⁇ M; The cells were suspended in mTeSR1 containing Wako, Japan) and seeded on a 60 mm dish coated with Matrigel or human recombinant Laminin 521. The medium was exchanged every day from the next day, and the cells were passaged every 4 days. (Construction of plasmid for homologous recombination into AAVS1 region) To test the feasibility of this system, pPS (Ide, K et al.
  • HSVtk-pA a donor plasmid having a CA promoter, a Survivin promoter, or a Nanog promoter upstream of Venus-2A-HSVtk was prepared (FIG. 1).
  • a plasmid expressing Cas9 under the control of CA promoter was prepared.
  • Each plasmid was constructed as follows. pFlag-Venus-2A and pT-HSVtk were constructed in a previous experiment (Ide, K et al. (2017) supra). HSVtk obtained by cutting pT-HSVtk with EcoRI/BamHI was inserted into pFlag-Venus-2A cut with SmaI to obtain pFLAG-VH. Next, in order to insert a SwaI site between Venus-2A and HSVtk to allow easy replacement with other reporter gene or suicide gene, the following primers were used to PCR the Venus-2A-HSVtk gene.
  • RC-Venus-2A-HSVtk-pA was removed from this plasmid by NdeI/SacII digestion, and the pSA-2A-Puro-pA donor containing the AAVS1 homologous region digested with SalI (Addgene 22075; F, Beard C et al. Nat Biotechnol. 2009;27:851-7) to obtain pRC-VH-donor.
  • the final donor plasmid pCA-VH donor was generated using a LR-clonase (Thermo Fisher Scientific) recombination reaction between pPS (including promoter) and pRC-VH donor.
  • pPS and pRC-VH donors were mixed with the LR-ClonaseTM II enzyme mixture, incubated for 1 hour at 25°C, and then reacted with proteinase K for 10 minutes at 37°C.
  • E. coli was transformed with this mixture and grown on LB plates containing ampicillin. Plasmid DNA was extracted from the colonies that appeared the next day, examined for the correct donor plasmid, amplified and purified.
  • pSurvivin-VH-donor or pNanog-VH-donor was made by a similar method.
  • the CMV of pCMV-Cas9 (Addgene 41815; Mali, P, Yang et al., Science 2013; 339:823-826.) was digested with XbaI/SpeI- and SalI/EcoRI containing CA from pCA-EGFP. By replacing, pCA-Cas9 was constructed. (Establishment of homologous recombination hPSC) Forty-eight hours before transfection, 2.0 ⁇ 10 6 hESCs were seeded on a 100 mm dish. Cells were treated with pCA-Cas9, pgRNA-T2 (Addgene 41818; Mali, P, Yang et al.
  • QuantiFast SYBR QRT-PCR using Green PCR (Qiagen, Japan) was performed on the Rotor-Gene RG-3000 (Qiagen). Relative mRNA expression levels were determined by the comparative Ct method and expression levels of individual genes were normalized to the levels of the reference gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH). QRT-PCR analysis was performed at an annealing temperature of 60° C. using the following primer set.
  • GAPDH 5'-CCATCACCATCTTCCCAGGAG-3' (SEQ ID NO: 5) and 5'-TGTCATACCAGGAAATGAGCTT-3' (SEQ ID NO: 6)
  • Venus 5'-GGGGCACAAGCTGGAGTACAACTAC-3' (SEQ ID NO: 7) and 5'-GAACTCCAGCAGGACCATGTGAT-3' (SEQ ID NO: 8)
  • HSVtk 5'-AGCAAGAAGCCACGGAAGTC-3' (SEQ ID NO: 9), and 5'-GCACCCGCCAGTAAGTCATC-3' (SEQ ID NO: 10) (In vitro analysis of undifferentiated cell-specific GCV-dependent cell killing effect) As previously reported (Suemiri, H et al.
  • Undifferentiated hESCs (CA.VH, Survivin.VH and Nanog.VH clones) were seeded on mitomycin C-treated mouse embryo fibroblasts in ES medium consisting of 1:1 mixture. After colony formation, 1 mg/mL Collagenase IV (Thermo Fisher Scientific), 0.25% Trypsin (Thermo Fisher Scientific), 1 mM CaCl 2 (Nacalai Tesque) (20% KnockouterFercimerTM ReactorTM) and 20% KnockoutOuter(TM) ReactorSerpicTM.
  • Collagenase IV Thermo Fisher Scientific
  • Trypsin Thermo Fisher Scientific
  • 1 mM CaCl 2 (Nacalai Tesque) (20% KnockouterFercimerTM ReactorTM) and 20% KnockoutOuter(TM) ReactorSerpicTM.
  • the cells were detached by treatment with, and suspended in hES medium supplemented with 10 ⁇ M Y-27632 containing no bFGF, and then transferred to an ultra-low binding plate (Corning, New York, NY, USA), and transferred to embryoid bodies (EBs). ) Was generated. After culturing in suspension for 8 days, EBs were transferred to gelatin-coated plates and cultured for an additional 20 or 48 days. Subsequently, the differentiated cells were seeded in a gelatin-coated 96-well culture plate at 2.0 ⁇ 10 4 cells/well and further added with 0, 0.01, 0.1, 1, 10 or 100 ⁇ g/mL GCV. Cultured for 7 days. Cell viability was determined by WST-8 assay using Cell Count Reagent SF® (Nacalai Tesque).
  • VH clones were seeded at 1.0 ⁇ 10 4 cells/well on Matrigel-coated 96-well culture plates under undifferentiated conditions, followed by 0, 0.01, 0.1, 1, 10 or 100 ⁇ g/mL. After culturing in GCV for 7 days, WST-8 assay was performed to determine cell viability.
  • hESCs (4.0 ⁇ 10 6 cells) were suspended in 100 ⁇ L of PBS containing 30% Matrigel and subcutaneously injected into the dorsal abdomen of 6-week-old female scid (severe combined immunodeficiency) mice (CLEA Japan, Tokyo, Japan). From the first day after the injection, the mice were intraperitoneally injected with either PBS or GCV (50 mg/kg) twice a day for 7 consecutive days. From the 4th week after the start of the GCV treatment for a further 4 weeks, the size of teratoma formed was measured twice a week, after which the mice were euthanized for histological analysis.
  • the transfection efficiency of the EGFP expression plasmid was 76.32% for KhES3 and 25.0% for KhES1.
  • the VH-donor should increase the ratio of homologous recombination to random integration as the puromycin resistance gene is only expressed from an active promoter located upstream of the vector integration site.
  • a total of 128 puromycin-resistant colonies were obtained from KhES3, and PCR analysis confirmed that 20% of the puromycin-resistant colonies were inserted into the AAVS1 locus by homologous recombination. In KhES1, only 2 colonies were correctly inserted (Fig. 2b, Table 1).
  • VH Exactly homologous recombination clones were expanded for further experiments and the resulting cells were cloned into CA. It was named VH. Similarly, 4 and 2 colonies obtained from cells transfected with pSurvivin-VH-donor and pNanog-VH-donor, respectively, were isolated from Survivin. VH and Nanog. It was named VH.
  • HSVtk and Venus mRNA expression levels in differentiated cells were examined and all differentiated clones showed undifferentiated CA. It was found to express the same level of both genes as VH (Fig. 5c, d). Survivin. VH and Nanog. The same analysis was performed for VH, and the expression of HSVtk and the expression of Venus were confirmed by real-time PCR. (HSVtk-specific/GCV-dependent cytotoxicity specific to undifferentiated cells) The above-mentioned real-time PCR experiment confirmed that HSV-tk was expressed in all cells. Therefore, undifferentiated and differentiated CA. VH, Survivin. VH and Nanog. HSVtk-specific/GCV-dependent cytotoxicity in VH was verified.
  • Cytotoxicity due to non-specific (ie, HSVtk-independent) GCV results in undifferentiated KhES3-CA. VH was observed at a GCV concentration of 100 ⁇ g/mL, and the undifferentiated KhES1-CA. VH was observed at GCV concentrations of 10 and 100 ⁇ g/mL. Undifferentiated KhES3-CA. HSVtk-specific/GCV-dependent cytotoxicity in VH cells was induced at lower GCV concentrations (0.01, 0.1, 1 and 10 ⁇ g/mL). On the other hand, this cytotoxicity was not induced in wild-type KhES3 cells at GCV concentrations below 10 ⁇ g/mL.
  • HSVtk-specific/GCV-dependent cytotoxicity was associated with undifferentiated KhES1-CA.
  • KhES3-CA Stronger than VH, most cells died even at extremely low GCV concentrations of 0.01 ⁇ g/mL.
  • non-specific GCV cytotoxicity was stronger in wild type KhES1 than in wild type KhES3 (FIG. 6).
  • Each undifferentiated CA VH clones showed no viability over 0.01 ⁇ g/ml GCV containing medium.
  • GCV-dependent cytotoxicity was higher in KhES1 than in KhES3.
  • HSVtk/GCV-dependent cytotoxicity was associated with differentiated CA. It was lower in VH clones with no apparent cytotoxicity at GCV concentrations up to 1 ⁇ g/ml *P ⁇ 0.005 and **P ⁇ 0.001 (versus GCV).
  • HSVtk cytotoxicity is associated with reduced cell proliferative activity
  • cytotoxicity in differentiated cells was tested. In the differentiated clones after differentiation, non-specific cytotoxicity due to GCV was not observed at any concentration, and HSVtk-specific/GCV-dependent cytotoxicity was observed at GCV concentrations of 0.01, 0.1, and 1 ⁇ g/mL. Was not observed (Fig. 7). Therefore, HSVtk-specific/GCV-dependent cytotoxicity is associated with KhES3-CA. VH and KhES1-CA. Observed in both VHs, differentiation reduced this cytotoxicity. Survivin. VH and Nanog. VH is a CA.
  • HSVtk-specific/GCV-dependent cytotoxicity was weaker than that of VH, resulting in a differentiated state and further decreased HSVtk cytotoxicity. As a result, HSVtk-specific/GCV-dependent cytotoxicity was not observed. It seems to be.
  • VH and wild-type KhES3 were subcutaneously transplanted into immunodeficient mice (NOD-scid mice) and then transplanted into two groups in which teratoma formation experiments were performed.
  • PBS phosphate buffered saline
  • significant and continuous proliferation of teratomas did not differ significantly between the two groups (Fig. 8a, b).
  • GCV phosphate buffered saline
  • KhES3-CA Teratoma formation in VH-transplanted mice was more strongly inhibited compared to wild-type KhES3-transplanted control mice. This dose of GCV (50 mg/kg) has been confirmed to be safe in our previous studies (Ide, K et al.

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Abstract

La présente invention a pour but d'introduire de manière sûre et efficace un gène cytotoxique dans le génome d'une cellule souche pluripotente humaine, puis de permettre au gène d'exercer sa fonction au point que des cellules indifférenciées, restant après la différenciation en cellules cibles, peuvent être éliminées de manière efficace et exhaustive. Par recombinaison homologue à l'aide d'une technologie d'édition de génome, une séquence de gène étranger (cible) a été introduite avec succès et efficacement dans une région de port sûr du génome hPSC pour la première fois. En outre, les constatations suivantes ont été obtenues, à savoir : La Région AAVS1 étant définie comme une région de port sûr est susceptible de présenter un niveau d'expression génique inférieur par rapport à un transfert de gène aléatoire à l'aide, par exemple, d'un vecteur lentiviral; dans les cellules recombinées homologues ainsi obtenues, un promoteur est nécessaire pour obtenir une forte capacité d'expression; et une toxicité sélective pour des cellules indifférenciées peut être obtenue en employant, en tant que gène fonctionnel, un gène suicide en corrélation avec l'activité de croissance cellulaire et en régulant la concentration d'un promédicament correspondant à cette dernière. Par conséquent, la présente invention fournit avec succès un mécanisme grâce auquel un gène est introduit en toute sécurité dans une région de port sûr d'un génome par recombinaison homologue et, après l'induction de la différenciation, des cellules indifférenciées seules peuvent être éliminées de manière exhaustive et sélective à partir des cellules différenciées.
PCT/JP2020/007164 2019-02-22 2020-02-21 Procédé d'intégration d'un gène étranger long dans une région sûre d'une cellule souche pluripotente humaine et lui permettant de fonctionner normalement dans cette dernière WO2020171222A1 (fr)

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WO2024162452A1 (fr) * 2023-02-02 2024-08-08 国立研究開発法人国立がん研究センター Composition pharmaceutique pour le traitement d'un cancer testé positif au hpv ou d'un gène de changement pathologique du cancer pré-testé positif au hpv, et vecteur d'adénovirus recombinant

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WO2015107888A1 (fr) * 2014-01-14 2015-07-23 国立大学法人鹿児島大学 Nouveau procédé d'étiquetage d'une cellule responsable de la cancérisation dans des cellules souches, et méthode thérapeutique
JP2018531612A (ja) * 2015-11-04 2018-11-01 フェイト セラピューティクス，インコーポレイテッド 万能性細胞のゲノム改変

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WO2015107888A1 (fr) * 2014-01-14 2015-07-23 国立大学法人鹿児島大学 Nouveau procédé d'étiquetage d'une cellule responsable de la cancérisation dans des cellules souches, et méthode thérapeutique
JP2018531612A (ja) * 2015-11-04 2018-11-01 フェイト セラピューティクス，インコーポレイテッド 万能性細胞のゲノム改変

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Publication number Priority date Publication date Assignee Title
WO2024162452A1 (fr) * 2023-02-02 2024-08-08 国立研究開発法人国立がん研究センター Composition pharmaceutique pour le traitement d'un cancer testé positif au hpv ou d'un gène de changement pathologique du cancer pré-testé positif au hpv, et vecteur d'adénovirus recombinant

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